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Original Article

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME NEW SUCCINIMIDE, 2-

IMINOTHIAZOLINE AND OXAZINE DERIVATIVES BASED BENZOPYRONE AS

ANTICONVULSANT AGENTS

SOHAIR L. EL-ANSARY1,2, GHANEYA S. HASSAN1, DOAA E. ABDEL RAHMAN1, NAHLA A. FARAG3,

MOHAMMED I. HAMED2,*, MARAWAN A. BASET4

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt, 2Department of

Pharmaceutical Chemistry, Faculty of Pharmacy, Misr University for Science and Technology, 6th October City, Egypt, 3Department of

Pharmaceutical Chemistry, Faculty of Pharmacy, Misr International University, Cairo, P. O. Box 1, Holiopolis, Egypt, 4Department of

Pharmacology and Toxicology, National Research Centre, Dokki, Cairo, Egypt

Email: [email protected]

Received: 17 Jan 2016 Revised and Accepted: 18 Feb 2016

ABSTRACT

Objective: The objective of the present study was to synthesize novel benzopyrone derivatives with potential and safer anticonvulsant activity.

Methods: New benzopyrone derivatives have been synthesized and characterized by spectral and elemental analysis. These compounds tested for anticonvulsant activity using the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens (phase 1), which are the most widely employed seizure models for early identification of new anticonvulsant agents. Phase 2 including, neurotoxicity screening and quantitative determination of the median effective dose (ED50), median lethal dose (LD50) and protective index (PI) for the active compounds from phase 1.

Results: Compound 12b possessed potent anticonvulsant activity with ED50 values of 94.75 and 70.7 mg/kg in the MES and scPTZ screens respectively, and had LD50 value of 2546 mg/kg after intraperitoneal injection to mice, which provide compound 12b with a wide protective index of 26.87 and 36.01 for MES and scPTZ screens respectively compared to the reference drug Phenobarbital with PI of 12.16 and 20.08, respectively. In addition, compound 12b exhibited mild neurotoxicity at the maximum administrated dose (200 mg/kg).

Conclusion: Compound 12b possessed broad spectrum activity for the treatment of all types of seizures, with a wide protective index compared to Phenobarbital. Consequently, compound 12b can be selected as a new bio candidate lead for further study.

Keywords: Benzopyrone, Succinimide, 2-Iminothiazoline, Oxazine; Anticonvulsant.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

According to the International League Against Epilepsy (ILAE), epilepsy is defined as a brain disorder characterized by an enduring predisposition to generate epileptic seizures and by the neurobiological, cognitive, psychological and social consequences of this condition [1]. World health organization (WHO) estimated that approximately 50 million people currently live with epilepsy worldwide [2]. Many patients have seizures that are resistant to available medical therapies. Despite the introduction of new anticonvulsant drugs, all currently approved antiepileptic drugs have a dose-related toxicity and idiosyncratic side effects. As a result, intensive research efforts aim to find new, more effective and safer antiepileptic drugs.

Surveying the literature reveals that, benzopyran-2-one derivatives are promising anticonvulsant candidates. Scoparone [3], osthole [4], esuprone [5] and 7-isopentenyloxycoumarin [6] have been found to possess moderate to strong anticonvulsant properties. In addition, succinimides [7] and 2-iminothiazolines [8, 9] ring systems have shown an anticonvulsant effect in pharmacological screening. Thus, the purpose of this work was to synthesize new derivatives of 3,4-cyclopentene-8-methyl-2H-1-benzopyran-2-one 4hybridized at 7-position with succinimides 7a-c and 2-iminothiazoline 9a,b, through oxy acetamido linker (fig. 1). The anticonvulsant activity of new compounds 7a-c and 9a, b was evaluated.

Pyranobenzoxazines derivatives showed anticonvulsant activity in the preliminary pharmacological screening (fig. 2). Compounds 1 and 3 showed anticonvulsant activity against seizures induced by strychnine sulphate [10] while compound 2 showed significant anticonvulsant activity at 100 mg/kg in MES screen (50 % protection) [11].

NR

R1

O

O

7 a RR1=CH2CH2 b RR1=CH=CH c RR1=C6H4

N

S

H3C

N

R

9 a R=C2H5, R1=CH b R=C6H5, R1=OCH3

O

HN

O O O

CH3

Benzopyran-2-one (4)Linker

Heterocyclic ring systems

Fig. 1: Design strategy and structures of the target compounds

Consequently, the present work deals with the synthesis of novel derivatives of aryl pyrans benzoxazines 12 a, b (Scheme 2) aiming to produce potent and selective anticonvulsant candidates.

MATERIALS AND METHODS

Chemistry

Melting points were determined by an open capillary tube method using Stuart SMP10 melting point apparatus and were uncorrected. Microanalysis was carried out at The Regional Center for Mycology and Biotechnology, Al-Azhar University. Infrared Spectra were

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 8, Issue 4, 2016

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Int J Pharm Pharm Sci, Vol 8, Issue 4, 222-228

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recorded as potassium bromide discs on Shimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan) and expressed in wave number (KBr) (cm-1). 1H-NMRspectra were recorded in δ scale given in ppm and performed on a JEOL ECA 300, 400 MHz spectrometer using CDCl3 or DMSO as stated, using TMS as an internal standard at Cairo University.

O

CH3

O

N

O

CH3

O

CH3

O

N

O

H3C

1 3

O O

2

O

N

Br

Fig. 2: Linear and angular aryl pyranobenzoxazines with

anticonvulsant activity

Mass spectra were performed as EI at 70eV on Hewlett Packard Varian (Varian, Polo, USA) and Shimadzu Gas Chromatograph Mass spectrometer-QP 1000 EX and TSQ quantum (Thermo Electron Corporation) instrument prepared with a triple, quadruple mass detector (Thermo Finnigan) and an ESI source. TLC was carried out using Macherey-Nagel AlugramSil G/UV254 silica gel plates with fluorescent indicator UV254 and chloroform/methanol 9.5:0.5 as the eluting system and the spots were visualized at 366, 254 nm by UV Vilber Lourmat 77202 (Vilber, Marne La Vallee, France). Compounds 4 and 5 were prepared according to reported procedure [12, 13].

General procedure for the synthesis of (3,4-cyclopentene-8-methyl-

2-oxo-2H-1-benzopyran-7-yloxy) acetic acid hydrazide (6)

A solution of compound 5 (3.02 g, 0.01 mol) and hydrazine hydrate 99 % (1.00 g, 0.98 ml, 0.02 mol) in absolute ethanol (10 ml) was refluxed for 2 h. The reaction mixture was cooled and the precipitate 6 was filtered and dried (yield 87 %). The product was crystallized from 10 % acetic acid, mp above 300 °C. 1H-NMR (300 MHz)(DMSO-d6) δ: 2.08-2.13 (m, 2H, CH2 cyclopentene), 2.28 (s, 3H, CH3), 2.74 (t, 2H, J=7.1 Hz, CH2 cyclopentene), 3.05 (t, 2H, J=7.2 Hz, CH2 cyclopentene), 4.32 (s, 2H, NH2), 4.78 (s, 2H, OCH2), 6.99 (d, 1H, J=8.4 Hz, H-6 Ar), 7.41 (d, 1H, J=8.1 Hz, H-5 Ar), 10.21 (s, 1H, NH). IR (KBr) cm-1: 3460, 3417 (NH, NH2), 3045 (CH Ar), 2960, 2920 (CH aliphatic), 1728, 1689 (2 C=O), 1651, 1629, 1606, 1577 (NH, C=C). MS m/z: 288 (M+), 0.78 %. Anal. Calcd for C15H16N2O4; C, 62.49; H, 5.59; N, 9.72. Found: C, 62.63; H, 5.64; N, 9.87.

General procedure for the synthesis of compounds 7a-c

To a solution of the acid hydrazide derivative 6 (2.88 g, 0.01 mol) in glacial acetic acid (15 ml), cyclic acid anhydrides (0.01 mol), namely, succinic, maleic and phthalic anhydride, was added and the mixture was heated under reflux for 9 h. The solvent was concentrated then the mixture was poured onto ice-water, the precipitated product was filtered, dried and crystallized from isopropanol.

2-[(3,4-Cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-

yl)oxy]-N-(2,5-dioxopyrrolidin-1-yl) acetamide (7a)

Yield 76 %, mp 283-286 °C.1H-NMR (300 MHz) (DMSO-d6) δ: 2.08-2.20 (m, 2H, CH2 cyclopentene), 2.29 (s, 3H, CH3), 2.74 (t, 2H, J=7.2 Hz, CH2 cyclopentene), 2.80 (s, 4H, 2CH2 pyrrolidine), 3.06 (t, 2H, J=6.8 Hz, CH2 cyclopentene), 4.91 (s, 2H, OCH2), 7.02 (d, 1H, J=8.7 Hz, H-6 Ar), 7.43 (d, 1H, J=8.7 Hz, H-5 Ar), 10.69 (s, 1H, NH, D2O exchangable). IR (KBr) cm-1: 3344 (NH), 3095 (CH Ar), 2958, 2914 (CH aliphatic), 1735, 1722, 1707 (4 C=O), 1649, 1629, 1608, 1577 (NH, C=C). MS m/z: 370 (M+), 2.17 %. Anal. Calcd for C19H18N2O6; C, 61.62; H, 4.90; N, 7.56. Found: C, 61.84; H, 4.94; N, 7.67.

2-[(3,4-Cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]-

N-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetamide (7b)

Yield 79 %, mp 245-247 °C.1H-NMR (300 MHz) (DMSO-d6) δ: 2.09-2.16 (m, 2H, CH2 cyclopentene), 2.30 (s, 3H, CH3), 2.75 (t, 2H, J=6.8 Hz, CH2 cyclopentene), 3.06 (t, 2H, J=7.1 Hz, CH2 cyclopentene), 4.93

(s, 2H, OCH2), 7.01 (d, 1H, J=8.7 Hz, H-6 Ar), 7.20 (s, 2H, CH=CH pyrrole), 7.44 (d, 1H, J=8.7 Hz, H-5 Ar), 10.66 (s, 1H, NH, D2O exchangable). IR (KBr) cm-1: 3344 (NH), 3076 (CH Ar), 2958, 2929 (CH aliphatic), 1730, 1725, 1705 (4 C=O), 1631, 1610, 1575, 1550 (NH, C=C). MS m/z: 368 (M+), 8.05 %. Anal. Calcd for C19H16N2O6; C, 61.95; H, 4.38; N, 7.61. Found: C, 62.12; H, 4.45; N, 7.74.

2-[(3,4-Cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-yl)

oxy]-N-(1,3-dioxoisoindolin-2-yl) acetamide (7c)

Yield 81 %, mp 290-291 °C.1H-NMR (300 MHz) (DMSO-d6) δ: 2.09-2.14 (m, 2H, CH2 cyclopentene), 2.32 (s, 3H, CH3), 2.75 (t, 2H, J=7.4 Hz, CH2 cyclopentene), 3.07 (t, 2H, J=7.4 Hz, CH2 cyclopentene), 5.00 (s, 2H, OCH2), 7.06 (d, 1H, J=9 Hz, H-6 Ar), 7.46 (d, 1H, J=8.7 Hz, H-5 Ar), 7.93-8.00 (m, 4H, Ar-H), 10.92 (s, 1H, NH, D2O exchangable). IR (KBr) cm-1: 3363 (NH), 3095, 3066 (CH Ar), 2968, 2958 (CH aliphatic), 1788, 1735, 1712, 1678 (4 C=O), 1649, 1631, 1610, 1577 (NH, C=C). MS m/z: 418 (M+), 35.15 %. Anal. Calcd for C23H18N2O6; C, 66.40; H, 4.34; N, 6.70. Found: C, 66.17; H, 4.36; N, 6.82.

General procedure for the synthesis of compounds (8a, b)

To a solution of the acid hydrazide derivative 6 (2.88 g, 0.01 mol) in absolute ethanol (40 ml), the appropriately substituted isothiocyanate (0.01 mol) was added and the mixture was refluxed while stirring for 12 h. the separated solid was filtered, washed with ether and dried. The crude product was crystallized from ethanol.

1-(3,4-Cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-

yloxy)acetyl-4-ethyl thiosemicarbazide (8a)

Yield 80 %, mp 211-212 °C. 1H-NMR (300 MHz) (DMSO-d6) δ: 1.07 (t, 3H, J=7.2 Hz, CH2CH3), 2.08-2.13 (m, 2H, CH2 cyclopentene), 2.29 (s, 3H, CH3), 2.74 (t, 2H, J=7.2 Hz, CH2 cyclopentene), 3.05 (t, 2H, J=6.9 Hz, CH2 cyclopentene), 3.46 (q, 2H, J=6.6 Hz, CH2CH3), 4.74 (s, 2H, OCH2), 6.99 (d, 1H, J=8.4 Hz, H-6 Ar), 7.41 (d, 1H, J=8.7 Hz, H-5 Ar), 7.96 (s, 1H, NH, exchanged with D2O), 9.20 (s, 1H, NH, exchanged with D2O), 9.96 (s, 1H, NH, exchanged with D2O). IR (KBr) cm-1: 3416, 3324, 3238 (3 NH), 3065 (CH Ar), 2965, 2925 (CH aliphatic), 1713, 1666 (2 C=O), 1607, 1574, 1539 (NH, C=C), 1264 (C=S). MS m/z: 375 (M+), 0.45 %. Anal. Calcd for C18H21N3O4S; C, 57.58; H, 5.64; N, 11.19. Found: C, 57.76; H, 5.71; N, 11.34.

1-(3,4-Cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-

yloxy)acetyl-4-phenyl thiosemicarbazide (8b)

Yield 85 %, mp 255-256 °C. 1H-NMR (300 MHz) (DMSO-d6) δ: 2.05-2.12 (m, 2H, CH2 cyclopentene), 2.27 (s, 3H, CH3), 2.70 (t, 2H, J=7.2 Hz, CH2 cyclopentene), 2.99 (t, 2H, J=7.8 Hz, CH2 cyclopentene), 4.78 (s, 2H, OCH2), 5.17 (s, 2H, 2NH), 6.98 (d, 1H, J=8.4 Hz, H-6 Ar), 6.99 (d, 1H, J=8.7 Hz, H-5 Ar), 7.32-7.53 (m, 5H, Ar-H), 10.21 (s, 1H, NH). IR (KBr) cm-1: 3446, 3417, 3147 (3 NH), 3037 (CH Ar), 2920 (CH aliphatic), 1726, 1674 (2 C=O), 1651, 1606, 1606, 1571 (NH, C=C), 1282 (C=S). MS m/z: 423 (M+), 0.58 %. Anal. Calcd for C22H21N3O4S; C, 62.40; H, 5.00; N, 9.92. Found: C, 62.57; H, 5.13; N, 9.97.

General procedure for the synthesis of compounds (9a,b)

A mixture of acyl thiosemicarbazide derivative 8a,b (1 mmol) and methyl phenacyl bromide (0.32 g, 1.5 mmol) and anhydrous sodium acetate (0.1 g, 1.2 mmol) in absolute ethanol (10 ml) was refluxed for 24 h. The solvent was distilled under vacuum, and the residue was extracted with chloroform. The extract was washed with water, filtered over anhydrous sodium sulfate and evaporated under vacuum. The solid obtained was crystallized from glacial acetic acid.

N-[2-Ethylimino-4-(4-methylphenyl)thiazol-2H-3-yl]-2-(3,4-

cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-yloxy)

acetamide (9a)

Yield 67 %, mp 219-221 °C. 1H-NMR (300 MHz) (CDCl3) δ: 1.32 (t, 3H, J=7.2 Hz, CH2CH3), 2.13-2.23 (m, 2H, CH2 cyclopentene), 2.30 (s, 3H, CH3 at C8), 2.41 (s, 3H, CH3 at C4′), 2.88 (t, 2H, J=7.4 Hz, CH2 cyclopentene), 3.02 (t, 2H, J=7.8 Hz, CH2 cyclopentene), 4.08 (q, 2H, J=7.2 Hz, CH2CH3), 5.31 (s, 2H, OCH2), 7.05-7.08 (m, 3H, H-3′,5′ Ar and C5-H thiazoline), 7.23-7.27 (m, 2H, H-5 and H-6 Ar), 7.96 (d, 2H, J=8.1 Hz, H-2′,6′ Ar), 10.11 (s, 1H, NH, D2O exchangable). IR (KBr) cm-1: 3446 (NH), 3064, 3032 (CH Ar), 2983, 2968 (CH aliphatic),

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1705, 1680 (2 C=O), 1610, 1606, 1573, 1550 (C=N, NH, C=C). MS m/z: 489(M+), 1.00 %. Anal. Calcd for C27H27N3O4S; C, 66.24; H, 5.56; N, 8.58. Found: C, 66.43; H, 5.62; N, 8.67.

N-[4-(4-Methylphenyl)-2-phenyliminothiazol-2H-3-yl]-2-(3,4-

cyclopentene-8-methyl-2-oxo-2H-1-benzopyran-7-yloxy)

acetamide (9b)

Yield 71 %, mp 219-221 °C. 1H-NMR (300 MHz) (DMSO-d6) δ: 2.07-2.12 (m, 2H, CH2 cyclopentene), 2.26 (s, 3H, CH3 at C8), 2.40 (s, 3H, CH3 at C4′), 2.74 (t, 2H, J=7.2 Hz, CH2 cyclopentene), 3.04 (t, 2H, J=7.4 Hz, CH2 cyclopentene), 5.34 (s, 2H, OCH2), 6.98 (d, 1H, J=8.7 Hz, H-6 Ar), 7.02 (s, 1H, C5-H thiazoline), 7.29-7.99 (m, 10H, H-5 Ar, Ar-H), 9.77 (s, 1H, NH, D2O exchangable). IR (KBr) cm-1: 3444 (NH), 3053, 3026 (CH Ar), 2960, 2933 (CH aliphatic), 1693, 1680 (2 C=O), 1610, 1600, 1571, 1560 (C=N, NH, C=C). MS m/z: 537(M+), 0.83 %. Anal. Calcd for C31H27N3O4S; C, 69.25; H, 5.06; N, 7.82. Found: C, 69.42; H, 5.17; N, 7.98.

Synthesis of 3,4-cyclopentene-7-hydroxy-8-methyl-6-nitro-2H-

1-benzopyran-2-one (10)

A solution of nitric acid (6.3 ml, 0.1 mol) in sulfuric acid (6.5 ml) was added to a stirred solution of compound 4 (21.6 g, 0.1 mol) in sulfuric acid (38.6 ml) at such a rate as to keep the temperature below 5 (ice-salt bath cooling). The reaction mixture was poured into a stirred ice-water mixture, and a yellow precipitate was collected by filtration and dried to yield 24.0 g (92 %). The product was crystallized from ethanol, mp 194-195 °C. 1H-NMR (300 MHz)(DMSO-d6) δ: 2.06-2.16 (m, 2H, CH2 cyclopentene), 2.25 (s, 3H, CH3), 2.76 (t, 2H, J=7.1 Hz, CH2 cyclopentene), 3.08 (t, 2H, J=7.2 Hz, CH2 cyclopentene), 8.05 (s, 1H, H-5 Ar), 10.94 (s, 1H, OH, exchanged with D2O). IR (KBr) cm-1: 3210 (OH), 3078 (CH Ar), 2957, 2854 (CH aliphatic), 1737 (C=O), 1620 (C=C), 1530, 1380 (NO2). MS m/z: 261 (M+), 100 %. Anal. Calcd for C13H11NO5; C, 59.77; H, 4.24; N, 5.36. Found: C, 59.89; H, 4.31; N, 5.42.

Synthesis of 6-amino-3, 4-cyclopentene-7-hydroxy-8-methyl-

2H-1-benzopyran-2-one (11)

A solution of sodium dithionite (7 g, 0.04 mol) in water (30 ml) was quickly added to a solution of the nitro compound 10 (2.61 g, 0.01 mol) in 30 % ammonium hydroxide solution (20 ml) and the reaction mixture was refluxed for 15 min. After cooling, the crude product was filtered off, washed and dried to yield 1.73 g (75 %). The product was crystallized from isopropanol, mp 222-223 °C. 1H-NMR (300 MHz)(DMSO-d6) δ: 2.02-2.12 (m, 2H, CH2

cyclopentene), 2.20 (s, 3H, CH3), 2.69 (t, 2H, J=7.1 Hz, CH2

cyclopentene), 2.94 (t, 2H, J=7.4 Hz, CH2 cyclopentene), 6.16 (s, 2H, NH2, exchanged with D2O), 6.60 (s, 1H, H-5 Ar), 10.20 (s, 1H, OH, exchanged with D2O). IR (KBr) cm-1: 3375, 3315 (NH2), 3238 (OH), 2957 (CH aliphatic), 1664 (C=O), 1577, 1503 (NH, C=C). MS m/z: 231 (M+), 100 %. Anal. Calcd for C13H13NO3; C, 67.52; H, 5.67; N, 6.06. Found: C, 67.76; H, 5.74; N, 6.19.

General procedure for the synthesis of compounds (12a, b)

To a solution of the amino derivative 11 (2.31 g, 0.01 mol) and sodium ethoxide (0.01 mol) in absolute ethanol (50 ml), the appropriate phenacyl bromide derivative (0.015 mol) was added and the solution was heated under reflux for 2 h. The reaction mixture was filtered, and the filtrate was concentrated then left to cool. The formed precipitate was filtered, washed, and dried. The crude product was crystallized from isopropanol.

6,7-Cyclopentene-10-methyl-3-phenyl-2,8-dihydropyrano[5,6-

g]-1,4-benzoxazin-8-one (12a)

Yield 75 %, mp 230-231 °C. 1H-NMR (400 MHz) (CDCl3) δ: 2.15-2.25 (m, 2H, CH2 cyclopentene), 2.39 (s, 3H, CH3), 2.84 (t, 2H, J=9.3 Hz, CH2 cyclopentene), 3.09 (t, 2H, J=9.3 Hz, CH2 cyclopentene), 5.22 (s, 2H, CH2 oxazine), 7.35 (s, 1H, H-5 Ar), 7.50 (d, 2H, J=6 Hz, H-2′,6′ Ar), 7.78 (t, 1H, J=3.2 Hz, H-4′ Ar), 7.95 (d, 2H, J=6 Hz, H-3′,5′ Ar). IR (KBr) cm-1: 3058 (CH Ar), 2922, 2857 (CH aliphatic), 1716 (C=O), 1621, 1571 (C=N, C=C). MS m/z: 331 (M+), 64.07 %. Anal. Calcd for C21H17NO3; C, 76.12; H, 5.17; N, 4.23. Found: C, 76.29; H, 5.21; N, 4.29.

6,7-Cyclopentene-10-methyl-3-(4-methylphenyl)-2,8-dihydro-

pyrano-[5,6-g]-1,4-benzoxazin-8-one (12b)

Yield 83 %, mp 210-211 °C. 1H-NMR (300 MHz) (CDCl3) δ: 2.19-2.26 (m, 2H, CH2 cyclopentene), 2.34 (s, 3H, CH3), 2.43 (s, 3H, CH3), 2.92 (t, 2H, J=7.1 Hz, CH2 cyclopentene), 3.07 (t, 2H, J=7.1 Hz, CH2 cyclopentene), 5.16 (s, 2H, CH2 oxazine), 7.30 (d, 2H, J=8.1 Hz, H-3′,5′ Ar), 7.35 (s, 1H, H-5 Ar), 7.81 (d, 2H, J=7.8 Hz, H-2′,6′ Ar). IR (KBr) cm-1: 3050 (CH Ar), 2922 (CH aliphatic), 1715 (C=O), 1610 (C=N, C=C). MS m/z: 345 (M+), 100 %. Anal. Calcd for C22H19NO3; C, 76.50; H, 5.54; N, 4.06. Found: C, 76.68; H, 5.61; N, 4.17.

Anticonvulsant screening

All the compounds prepared herein were evaluated for their potential in vivo anticonvulsant activity against scPTZ and MES-induced seizures in mice. The tested compounds were suspended in water and 1 % Tween 80 and administered to animals at a dose of 100 mg/kg ip. Standard drug used was Phenobarbital sodium at the dose of 30 mg/kg.

Adult albino mice weighing 20–25 g of both sexes (obtained from the animal house colony in the National Research) were used throughout this study. Animals were housed in groups of 6 and were allowed free access to food pellets (vit mix 1 %, mineral mix 4 %, corn oil 10 %, sucrose 20 %, cellulose 0.2 %, casein (95 %pure) 10.5 %, starch 54.3 %) and water except for the short time that animals were removed from their cages for testing. All behavioral experiments were conducted during the period between 10:00 and 13:00 with normal room light (12 h regular light/dark cycle) and temperature (22±18 OC). All Procedures involving animals and their care were performed after the Ethics Committee of the National Research Centre and in accordance with the recommendations for the proper care and use of laboratory animals, “Canadian Council on Animal Care Guidelines, 1984”. Additionally, all efforts were made to minimize animals suffering and to use only the number of animals necessary to produce reliable data.

Subcutaneous pentylenetetrazole (scPTZ)-induced seizures test

The tested compounds or the reference drug were given ip. to groups of 6 mice. Another group of 6 mice serves as a control. Sixty min after intraperitoneal administration, a dose of 85 mg/kg pentylenetetrazol (PTZ) was injected subcutaneously in a loose fold of skin on the back of the neck. Each animal is placed into an individual plastic cage for observation lasting 30 min. The incidence of tonic-clonic convulsions lasting for at least 5 seconds was recorded [14, 15]. Animal devoid of generalized convulsions were considered to be protected, and the results were represented as percentage protection, table 1. Besides that, the onset and duration of seizures were recorded, and statistics was done using chi-squared test with the aid of Graph pad Prism software, version 6 (inc., San Diego, USA).

Maximal electroshock seizure (MES) test

The procedure was carried out as described by Krall et al., [16] and Kitano et al., [17]. Electroshock was applied via ear-lip electrodes and generated by a stimulator (deliver an alternating 60 HZ current by Ugo Basile ECT Unit (Pulse generator 57800-001), the stimulus duration was 2.5 seconds, and the end point was tonic hind limb extension [18]. The maximum electroshock was determined. The drugs were administered orally 60 min before the test. The control animals were administered the vehicle. The mean threshold current for electroshock-induced tonic hind limb extensor seizure was calculated for each drug. The maximal seizures typically consist of a short period of initial tonic flexion and a prolonged period of tonic extension (especially of the hind limbs) followed by terminal clonus. The typical seizure lasts approximately 22 seconds failure to extend the hind limbs to an angle with trunk greater than 90o is defined as protection, table 1. The mean convulsion threshold of compounds under investigation as well as the standard error was calculated using chi-squared test with the aid of Graph pad Prism software, version 6 (inc., San Diego, USA).

Quantitative studies (phase 2)

The most potent anticonvulsant compound 12b, from phase 1, were subjected to phase 2, which include neurotoxicity screen and

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quantitative determination of median effective dose (ED50), median lethal dose (LD50) and protective index (PI), as shown in table 3.

Determination of the median effective dose (ED50)

Anticonvulsant activity was expressed in terms of the median effective dose (ED50), that is, the dose of drug required to produce the biological responses in 50 % of animals [19]. Compound 12b, that gave the highest protection at a dose of 100 mg/kg were studied at different doses (50, 100 and 200 mg/kg) to calculate the ED50 which was determined by log-linear regression analysis from the dose response curves to compare with ED50 of the reference drug Phenobarbital (10, 20 and 30 mg/kg), table 3.

Determination of the neurotoxicity (Rotarod test)

The neurotoxicity was assessed by rotarod test [20]. Prior to the experimental, male albino mice were placed on 3-centimeter rod (Ugo-Basile Accele. ROTA-ROD for mice, 7650) rotating at 6 rpm, in two training sessions that last 10 and 15 min respectively. The candidate under investigation was injected ip. (200 mg/kg in 1 % Tween 80), while Phenobarbital tested at 30 mg/kg. One hour later, the animals were again tested on the rotarod to assess the locomotor coordination and neurological deficit (e. g. ataxia, sedation, hyper-excitability), which are reflected by the inability of the animal to maintain equilibrium on the rod after the administration of the selected candidate, table 3. The end point for minimal neurotoxicity assessment was reflected by the inability of mice to maintain their equilibrium on accelerating rotarod in each of the five trials.

Determination of the median lethal dose (LD50)

Male albino mice weight 20-25 g was divided into groups each of 8 animals. Preliminary experiments were done to determine the minimal dose that kills all animals (LD100), and the maximal dose that fails to kill any animal. Several doses at equal logarithmic intervals were chosen in between these two doses, each dose was injected in a group of eight animals, the number of dead animals in each group after 24 h was recorded and LD50 was calculated

according to the following formula using Spearman Karber method [21-23], Table 4.

M = Xk+1/2 d–dr/N

M = Log LD50, Xk = Log dose causing 100 % mortality, D = Logarithmic interval of doses, R = Sum of the number of dead animals at each of the individual dose levels, N = Number of animals at each of the dose level.

RESULTS AND DISCUSSION

Chemistry

The general methods for the synthesis of target compounds 7a-c-9a,b, and 12a,b are depicted in Schemes 1-2. The key intermediate 6 was synthesized from 3, 4-cyclopentene-7-hydroxy-8-methyl-2H-1-benzopyran-2-one 4 through etherification with ethyl chloroacetate to give the ether derivative5, followed by hydrazinolysis.

The target imides 7a-c was obtained by condensation of the acid hydrazide 6 with the appropriate acid anhydrides, succinic, maleic and phthalic anhydride in glacial acetic acid, respectively. Acyl thiosemicarbazides 8a, b were prepared by refluxing solution of the acid hydrazide 6 in absolute ethanol with different isothiocyanates. While, 2-iminothiazoline derivatives 9a, b were prepared by refluxing acylthiosemicarbazides8a,b with methyl phenacyl bromide and anhydrous sodium acetate in absolute ethanol.

The typical method for nitration of 3, 4-cyclopentene-7-hydroxy-8-methyl-2H-1-benzopyran-2-one 4 involves the use of nitric acid and sulfuric acid, so-called ''mixed acid''. Sulfuric acid acts as a catalyst as well as an absorbent for water. 6-Nitrobenzopyran-2-one 10 was reduced to the corresponding amino compound 11 using sodium dithionite in ammonia for 15 min. Pyranobenzoxazin-8-one derivatives 12a,b were synthesized in good yield by reaction of the amino compound 11 with the appropriate phenacyl bromide derivatives in the presence of absolute ethanol and sodium ethoxide. Both the analytical and spectral data (IR, 1H-NMR, MS) of all the newly synthesized compounds were in full agreement with the proposed structures.

O

HN

O

H2N

O

HN

O

NH

NH

S

R O

HN

O

NS

N

CH3

R

8 a R=C2H5 b R=C6H5

6

9 a R=C2H5 b R=C6H5

O

HN

NR

R1

O

O

7 a RR1=CH2CH2 b RR1=CH=CH c RR1=C6H4

Oc

O O

CH3

O O

CH3

O

O O

CH3

CH3

O

d

OHO O

CH3

O

O

Oa

H3C

4

O

CH3

O

b

5

e

Scheme 1: Reagents and conditions: (a) ethyl acetoacetate, dry acetone, reflux; (b) hydrazine hydrate 99 %, absolute ethanol, reflux; (c)

acid anhydrides, glacial acetic acid, reflux; (d) isothiocyanates, absolute ethanol, reflux; (e) methylphenacyl bromide, anhydrous sodium

acetate, absolute ethanol, reflux

Hamed et al.


226

O O

CH3

HO O O

CH3

HO O O

CH3

O2N H2N

O O

CH3

N

O

R

HO

a

10 11 12 a R=H b R=CH3

b c

4

Scheme 2: Reagents and conditions: (a) HNO3/H2SO4; (b) NaS2O4; (c) phenacyl bromide derivatives, sodium ethoxide, absolute ethanol, reflux

Biological evaluation

Preliminary anticonvulsant screening (phase 1)

The initial evaluation (phase 1) of anticonvulsant activity of the synthesized compounds included the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) screens, which are the most widely employed seizure models for early identification of new anticonvulsant drugs. scPTZ test represents a valid model for generalized myoclonic seizures and also generalized seizures of the absence (petit mal) type and identify compounds that elevate seizure threshold [24]. On the other hand, the MES test is considered to be a predictor of likely therapeutic efficacy against generalized tonic-clonic seizures and identify clinical candidates that prevent seizure spread [25]. In the preliminary evaluation, the anticonvulsant activity was estimated at a dose of 100 mg/kg and the results are summarized in table 1.

The results of MES screen revealed that:

- Cyclization of the flexible thiosemicarbazide derivatives 7a,b into 2-iminothiazoline derivatives 8a,b, exhibited non-significant anticonvulsant activity. In addition cyclic imides 6a-c didn’t show activity.

- Alkylation, the aryl group of pyranobenzoxazin-8-one 12b, gave potent anticonvulsant activity than the unsubstituted derivative 12a, which showed no significant activity, table 1. In addition, compound 12b showed potent significantly anticonvulsant activity in both MES and scPTZ screens compared to the previously reported linear

pyrano-benzoxazine derivative 1 due to alkylation of the aryl group. Also, compound 12b showed promising activity than angular pyrano-benzoxazine derivative 2, table 2.

Concerning scPTZ screen, three parameters were considered, percentage protection for animals devoid of generalized seizures, beside that the onset and duration of clonic seizures were recorded for the unprotected animals. Compound 12b exhibited promising anticonvulsant activity (66.67 % protection). Moreover, compound 12b delay the onset and decrease the time of clonic convulsion for the unprotected animals.

Quantitative studies (phase 2)

Based on the previous results from the preliminary study, compound 12b was selected for quantification of the pharmacological parameters, median effective dose (ED50), median lethal dose (LD50) and protective index (PI). Results of the quantitative and neurotoxicity screens, along with the data on the Phenobarbital, are reported in table 3.

Compound 12b possessed broad spectrum activity with ED50 values of 94.75 and 70.7 mg/kg in the MES and scPTZ screens respectively, and had LD50 value of 2546 mg/kg after intraperitoneal injection to mice, which provide compound 12b with a wide protective index of 26.87 and 36.01 for MES and scPTZ screens respectively compared to Phenobarbital with PI of 12.16 and 20.08, respectively. In addition, compound 12b showed mild motor impairment at the maximum administrated dose (200 mg/kg).

Table 1: Anticonvulsant activity of compounds using maximal electroshocks (MES) and subcutaneous pentylenetetrazole (scPTZ)-induced

convulsion in mice

Compound

number

MES scPTZ

Mean convulsion

threshold (Am±S. E)*

%

Potency**

%

Protection***

Mean onset of clonic

convulsion (min.)*

Mean duration of clonic

convulsion* (sec.)

control 3.2±0.37 43.2 0 2.8±0.84 36±0.052 Phenobarbital 7.4±0.40a ----- 83.3 8.4±0.19a 3.6±0.028a 7a 3.2±0.20b 43.2 0 3.0±0.45b 31.2±0.040b 7b 3.8±0.37b 51.4 0 3.4±0.49b 30±0.028b 7c 3.3±0.21b 44.6 0 2.9±0.67b 28.8±0.036b 8a 3.2±0.20b 43.2 0 3.1±0.50b 29.4±0.042b 8b 3.4±0.24b 45.9 0 2.7±0.42b 30.6±0.032b 9a 3.6±0.24b 48.6 16.67 3.1±0.42b 30.6±0.032b 9b 3.4±0.40b 45.9 16.67 2.8±0.46b 31.8±0.040b 12a 3.2±0.20b 43.2 0 3.2±0.52b 31.8±0.041b 12b 6.0±0.68a 81.1 66.67 10.2±0.85a 11.4 ±0.028a

* Values represent the mean±standard error of 6 animals for each group, (a) Values are statistically significant (p<0.05) from the control group by using one-way ANOVA (followed by Tukey’s Multiple Comparison tests)., (b) Values are statistically non-significant (p<0.05) from the Phenobarbital group., ** %Potency ( %potency from Phenobarbital)., *** %Protection (number of animals devoid of convulsion/number of animals used).

Table 2: Comparison of the activity of the new active compound 12b to previously reported leads using maximal electroshock (MES) and

subcutaneous pentylenetetrazole (scPTZ) screens

Compound number MES %protection scPTZ %protection

1[10] --- --- 2[11] 50 --- 12b (this work) 81 66.7

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Table 3: Quantitative studies for anticonvulsant activity (test drug administered ip.)

PI LD50 mg/kg (mmol/kg) Neurotoxicity

Stability time

(Ratio)

ED50 Compound number

scPTZ MES scPTZ mg/kg (mmol/kg) MES mg/kg (mmol/kg)

36.01 26.87 2546 (7.37) 4.783±0.1797 (2/6) 70.7 (0.21) 94.75 (0.27) 12b 20.08 12.16 265

(1.14) 4.167±0.5426 (1/6) 13.2a

(0.057) 21.8a

(0.09) Phenobarbital

Median effective dose (ED50) determined in maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens.

Values in neurotoxicity screen represent stability time/min, the mean±standard error of 6 animals for each group; statistics was done by one-way ANOVA. (Ratio=animals exhibited neurotoxicity/protected animals). Protective index (PI) = [median lethal dose (LD50)/median effective dose (ED50)]. (a) Data from reference [26].

Table 4: Median lethal dose (LD50) for the selected active compound 12b and phenobarbital using Spearman Karber method [21-23]

Compound number Log dose Dose mice Compound number Log dose Dose mice

12b 3.30 1995 0/8 Phenobarbital 2.27 186 0/8 3.35 2238 2/8 2.32 209 1/8 3.40 2500 4/8 2.37 234 2/8 3.45 2818 5/8 2.42 263 4/8 3.50 3160 8/8 2.47 295 5/8

M=3.50+(0.5*0.05)-((0.05*19)/8)= 3.406 LD50 of 12b = antilog of M = 2546 mg/kg

2.52 333 8/8 M=2.52+(0.5*0.05)-((0.05*20)/8)= 2.42 LD50 of Phenobarbital = antilog of M = 265 mg/kg

CONCLUSION

In summary, Compound 12b possessed broad spectrum activity for treatment of all types of seizures induced by MES and scPTZ, with ED50 values of 94.75 and 70.7 mg/kg respectively, and had LD50 value of 2546 mg/kg after intraperitoneal injection to mice, which provide compound 12b with a wide protective index of 26.87 and 36.01 for MES and scPTZ screens respectively compared to Phenobarbital with PI of 12.16 and 20.08, respectively. In addition, compound 12b exhibited mild neurotoxicity at the maximum administrated dose (200 mg/kg). Consequently, compound 12b can be selected as a new bio candidate lead for further study.

CONFLICT OF INTERESTS

Declare none

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Der Pharma Chemica, 2014, 6(6):169-191 (http://derpharmachemica.com/archive.html)

ISSN 0975-413X CODEN (USA): PCHHAX

169 www.scholarsresearchlibrary.com

New furobenzopyrones: Synthesis, antimicrobial and photochemotherapeutic evaluation, QSAR and molecular docking studies

Sohair L. El-Ansary1,2, Mohammed M. Hussein1,2, Doaa E. Abdel Rahman1* and

Mohammed I. A.-L. Hamed2

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo,

Egypt 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr University for Science and Technology, 6th

October City, Egypt _____________________________________________________________________________________________

ABSTRACT The synthesis of some new linear furobenzopyrone 3a-h, 7a,b and angular furobenzopyrone derivatives 9a-s and 12a,b were described on the base of being monofunctional compounds to decrease possible toxicity. All the prepared compounds were evaluated for their antimicrobial and photosensitizing activities. Compounds 2e, 4 and 7b were found to have good antimicrobial activity while only compound 2e exhibited higher photosensitizing activity than xanthotoxin. In addition, photosensitizing activity increased upon increasing time of radiation and concentration of substance. Quantitative structure–activity relationship (QSAR) study was applied to find a correlation between the photosensitizing activities of the newly synthesized furobenzopyrone derivatives and their physicochemical parameter. Furthermore, docking study was undertaken to gain insight into the possible binding mode of these compounds with the binding site of the DNA gyrase (topoisomerase II) enzyme which is responsible for resolving topological problems which arise during the various processes of DNA. Keywords Furobenzopyrones, Antimicrobial, Photochemotherapeutic, QSAR study, Docking study. _____________________________________________________________________________________________

INTRODUCTION

Furobenzopyrones are an important class of photosensitizing drugs used in combination with radiation in the interval of UV-A (320-400 nm) (PUVA) for treatment of some skin diseases such as psoriasis, vitiligo, mycosis and eczema [1-4]. These compounds are derivatives of psoralen (linear furobenzopyrones), or angelicin (its angular isomers) [5]. Psoralen tricyclic moiety constitutes the basic chromophore from which drugs employed in this therapy were developed in particular, xanthotoxin (8-methoxypsoralen), 5-bergapten (5-methoxypsoralen) and to a lesser extent trioxsalen (4,5′,8-trimethylpsoralen, TMP) [1]. The biological activity of psoralens is primarily due to intercalation between two base pairs of DNA. The process is believed to involve three major steps: a) non-covalent interactive binding to DNA helix, b) formation of monoaddition product between DNA base and psoralen upon long wavelength ultraviolet irradiation, c) absorption of a second photon by some of the monoadducts to form diadducts, result in interstrand cross linkage [6]. Therefore, 2,3 (furan side) and 5,6 (pyrone) double bonds of the linear furobenzopyrones are the two photoreactive sites responsible for the DNA photobinding and for the biological activity [7]. Linear furobenzopyrones are reported to induce bifunctional photodamage to the DNA of the cutaneous cells in a selective way, thus inhibiting DNA functions and as a consequence, cell proliferation [8]. The photodamages consist of the products of photocycloaddition between one molecule of psoralen and two pyrimidine bases (biadduct) [7]. From the biological point of view, cross linkage provokes more pronounced biological consequences, but repair of interstrand cross linkage is less effective than repair of the monofunctional adduct [9]. Skin phototoxicity is strictly connected with

Doaa E. Abdel Rahman et al Der Pharma Chemica, 2014, 6 (6):169-191 _____________________________________________________________________________


the bifunctional lesions in DNA which seems to be the main cause of skin cancer. On the other hand, monoadducts are reported to lack skin phototoxicity [10]. Nowadays, most of researches are devoted to develop new photochemotherapeutic compounds endowed with photo-antiproliferative activity and lower skin phototoxicity. DNA monofunctional furobenzopyrones such as carbmethoxypsoralen [11], carbethoxypsoralen [12], pyridopsoralen [12], benzo- and tetrahydrobenzopsoralen [13,14] and phenylpsoralen [15] analogues were designed and synthesized in order to prevent DNA interstrand cross linking formation, maintain the photosensitizing activity and consequently lack skin phototoxicity. Intercalation complex between furobenzopyrones and nucleic acid revealed that, the C5 methyl of thymidine and the C5 substitution of the furobenzopyrone are in close proximity. Thus, presence of methyl group in this position could lead to steric crowding not present in the demethyl case. The results reported about TMP and psoralen added further support to this interpretation (TMP showed ≈ 98% furan addition, while psoralen lacking methyl at 5-position, showed nearly ≈ 20% pyrone addition) [16]. Enforced by these informations, we were encouraged to design and synthesize new linear furobenzopyrone derivatives (3a-h and 7a,b) with variety of peripheral substituents that may produce monofunctional adduct with nucleic acid, therefore inhibiting the genotoxicity. Moreover, linear furobenzopyrones were reported to be more phototoxic than angular furobenzopyrones [17]. Therefore, new angular furobenzopyrone derivatives (9a-s and 12a,b) were also synthesized in order to optimize the biological activity. All the newly synthesized compounds were evaluated for the antimicrobial and photosensitizing activities. In addition, quantitative structure–activity relationship (QSAR) study was also performed for understanding and validating the photosensitizing activities. Furthermore, attempt to elucidate a molecular target for the antimicrobial activity was achieved via molecular docking of the prepared compounds in the active site of DNA gyrase enzyme using Molecular Operating Environment (MOE).


1.1. Chemistry Melting points were determined by open capillary tube method using Electrothermal 9100 melting point apparatus MFB-595-010M (Gallen Kamp, London, England) and were uncorrected. Microanalyses were carried out at The Regional Center for Mycology and Biotechnology, Al-Azhar University. Infrared Spectra were recorded as potassium bromide discs on Schimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan) and expressed in wave number (cm-1). The 1H-NMR spectra were recorded on Varian Mercury VX-300 NMR spectrometer at 300 MHz and *JEOL-ECA500 NMR spectrometer at 500 MHz in dimethylsulphoxide (DMSO-d6) or deuterated chloroform (CDCl3). Chemical shifts are quoted in δ as parts per million (ppm) downfield from tetramethylsilane (TMS) as internal standard and J values are reported in Hz. Mass spectra were performed as EI at 70eV on Hewlett Packard Varian (Varian, Polo, USA) and Shimadzu Gas Chromatograph Mass spectrometer-QP 1000 EX. TLC were carried out using Art.DC-Plastikfolien, Kieselgel 60 F254 sheets (Merck, Darmstadt, Germany), the developing solvents was chloroform/methanol 9.5:0.5 and the spots were visualized at 366, 254 nm by UV Vilber Lourmat 77202 (Vilber, Marne La Vallee, France). Starting compounds 3,8-disubstituted-7-hydroxy-4-methyl-2H-1-benzopyran-2-one (1a-d) [18] and phenacyl bromide derivatives [19] were prepared according to reported procedures. 2.1.1. General Procedure for synthesis of 3-ethyl-4-methyl-8-substituted-7-((un)substituted phenacyloxy)-2H-1-benzopyran-2-ones (2a-h) (Scheme 1). A solution of compound 1a,b (0.01 mol) and appropriate phenacyl bromide derivative (0.015 mol) in acetone (50 ml) was refluxed in presence of anhydrous potassium carbonate (2.76 g, 0.02 mol) for 24 h. The solution was filtered and the remaining residue was washed with acetone. The combined filtrates and washings were distilled under reduced pressure. The product was crystallized from isopropanol. 2.1.1.1. 4,8-Dimethyl-3-ethyl-7-phenacyloxy-2H-1-benzopyran-2-one (2a) Yield 83%. mp 189-190 °C. IR (KBr) cm-1: 3062 (CH Ar), 2964, 2870 (CH aliphatic), 1708, 1697 (2 C=O), 1600, 1577, 1500 (C=C). 1H-NMR (DMSO-d6) δ: 1.04 (t, 3H, CH2CH3), 2.27 (s, 3H, CH3 at C4), 2.37 (s, 3H, CH3 at C8), 2.51 (q, 2H, CH2CH3), 5.77 (s, 2H, OCH2), 6.98 (d, 1H, J=8.7 Hz, H-6 Ar), 7.49-7.71 (m, 4H, H-5 Ar, H-3′,4′,5′ Ar), 8.03 (d, 2H, J=7.5 Hz, H-2′,6′ Ar). MS (m/z) %: 336 (M+) 5.48%. Anal. Calcd. for C21H20O4 (336.38): C, 74.98; H, 5.99. Found: C, 75.04; H, 6.12.



2.1.1.2. 4,8-Dimethyl-3-ethyl-7-(4-methylphenacyloxy)-2H-1-benzopyran-2-one (2b) Yield 92%. mp 181-183 °C. IR (KBr) cm-1: 3020 (CH Ar), 2964, 2872 (CH aliphatic), 1700, 1695 (2 C=O), 1606, 1571, 1554 (C=C). 1H-NMR (CDCl3) δ: 1.13 (t, 3H, CH2CH3), 2.31 (s, 3H, CH3 at C4), 2.37 (s, 3H, CH3 at C8), 2.45 (s, 3H, CH3 at C4′), 2.67 (q, 2H, CH2CH3), 5.35 (s, 2H, OCH2), 6.70 (d, 1H, J=8.7 Hz, H-6 Ar), 6.89 (d, 1H, J=9.3 Hz, H-5 Ar), 7.91 (d, 2H, J=7.8 Hz, H-3′,5′ Ar), 8.21 (d, 2H, J=8.4 Hz, H-2′,6′ Ar). MS (m/z) %: 350 (M+) 13.20%. Anal. Calcd. for C22H22O4 (350.41): C, 75.41; H, 6.33. Found: C, 75.47; H, 6.38.

O

CH3

RO

3a-h

CH3

O

X1 X2

O

CH3

R

HO O O

CH3

RO O

O

X1

X2

2a-h

O

CH3

OO

H3COOCH3

1a R=CH3 b R=H

4

(i) (ii)

(iii)

CH3 CH3

CH3

a R=CH3, X1=X2=Hb R=CH3, X1=CH3, X2=Hc R=CH3, X1=OCH3, X2=Hd R=CH3, X1=Br, X2=He R=CH3, X1=X2=OCH3f R=H, X1=X2=Hg R=H, X1=CH3, X2=Hh R=H, X1=OCH3, X2=H

Scheme 1. Reagents and conditions: (i ) Appropriate phenacyl bromide derivative, K2CO3, dry acetone, reflux 24 h, (ii ) Ethanolic KOH, reflux 18 h, (iii ) 3,4-Dimethoxyphenacyl bromide, K2CO3, dryacetone, reflux 18 h.

R=H

2.1.1.3. 4,8-Dimethyl-3-ethyl-7-(4-methoxyphenacyloxy)-2H-1-benzopyran-2-one (2c) Yield 72%. mp 191-193 °C. IR (KBr) cm-1: 3055 (CH Ar), 2922, 2839 (CH aliphatic), 1710, 1695 (2 C=O), 1602, 1598, 1566, 1512 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 2.26 (s, 3H, CH3 at C4), 2.37 (s, 3H, CH3 C8), 2.53 (q, 2H, CH2CH3), 3.86 (s, 3H, OCH3), 5.69 (s, 2H, OCH2), 6.94 (d, 1H, J=9.0 Hz, H-6 Ar), 7.09 (d, 2H, J=8.7 Hz, H-3′,5′ Ar), 7.55 (d, 1H, J=8.7 Hz, H-5 Ar), 8.01 (d, 2H, J=9 Hz, H-2′,6′ Ar). MS (m/z) %: 366 (M+) 32.41%. Anal. Calcd. for C22H22O5 (366.41): C, 72.12; H, 6.05. Found: C, 72.08; H, 6.13. 2.1.1.4. 7-(4-Bromophenacyloxy)-4,8-dimethyl-3-ethyl-2H-1-benzopyran-2-one (2d) Yield 79%. mp 203-206 °C. IR (KBr) cm-1: 3098 (CH Ar), 2977, 2860 (CH aliphatic), 1710, 1701 (2 C=O), 1604, 1585 (C=C). 1H-NMR (CDCl3) δ: 1.14 (t, 3H, CH2CH3), 2.30 (s, 3H, CH3 at C4), 2.36 (s, 3H, CH3 at C8), 2.68 (q, 2H, CH2CH3), 5.31 (s, 2H, OCH2), 6.85 (d, 1H, J=8.7 Hz, H-6 Ar), 7.38 (d, 1H, J=8.7 Hz, H-5 Ar), 7.88 (d, 2H, J=8.7 Hz, H-3′,5′ Ar), 8.20 (d, 2H, J=8.7 Hz, H-2′,6′ Ar). MS (m/z) %: 415 (M+) 11.04%, 417 (M++2) 10.28%. Anal. Calcd. for C21H19BrO4 (415.28): C, 60.74; H, 4.61. Found: C, 60.81; H, 4.68. 2.1.1.5. 7-(3,4-Dimethoxyphenacyloxy)-4,8-dimethyl-3-ethyl-2H-1-benzopyran-2-one (2e) Yield 68%. mp 215-217 °C. IR (KBr) cm-1: 3020 (CH Ar), 2922, 2846 (CH aliphatic), 1705, 1700 (2 C=O), 1604, 1581, 1550 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 2.27 (s, 3H, CH3 at C4), 2.36 (s, 3H, CH3 at C8), 2.56 (q, 2H, CH2CH3), 3.82 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 5.70 (s, 2H, OCH2), 6.94 (d, 1H, J=9.0 Hz, H-6 Ar), 7.12 (d, 1H, J=8.7 Hz, H-5′ Ar), 7.48 (s, 1H, H-2′ Ar), 7.55 (d, 1H, J=8.7 Hz, H-5 Ar), 7.72 (d, 1H, J=9 Hz, H-6′ Ar). MS (m/z) %: 396 (M+) 13.18%. Anal. Calcd. for C23H24O6 (396.43): C, 69.68; H, 6.10. Found: C, 69.73; H, 6.16. 2.1.1.6. 3-Ethyl-4-methyl-7-phenacyloxy-2H-1-benzopyran-2-one (2f) Yield 85%. mp 169-170 °C. IR (KBr) cm-1: 3060 (CH Ar), 2964, 2850 (CH aliphatic), 1708, 1697 (2 C=O), 1600, 1577, 1544 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 2.38 (s, 3H, CH3), 2.55 (q, 2H, CH2CH3), 5.73 (s, 2H, OCH2), 7.01 (d, 1H, J=8.7 Hz, H-6 Ar), 7.04 (s, 1H, H-8 Ar), 7.56-7.71 (m, 4H, H-5 Ar, H-3′,4′,5′ Ar), 8.04 (d, 2H, J=7.5 Hz, H-2′,6′ Ar). MS (m/z) %: 322 (M+) 0.90%. Anal. Calcd. for C20H18O4 (322.35): C, 74.52; H, 5.63. Found: C, 74.60; H, 5.67.



2.1.1.7. 3-Ethyl-4-methyl-7-(4-methylphenacyloxy)-2H-1-benzopyran-2-one (2g) Yield 94%. mp 138-141 °C. IR (KBr) cm-1: 3070 (CH Ar), 2970, 2875 (CH aliphatic), 1712, 1666 (2 C=O), 1606, 1571, 1554, 1502 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 2.38 (s, 3H, CH3 at C4), 2.40 (s, 3H, CH3 at C4′), 2.55 (q, 2H, CH2CH3), 5.68 (s, 2H, OCH2), 6.99 (d, 1H, J=8.7 Hz, H-6 Ar), 7.02 (s, 1H, H-8 Ar), 7.38 (d, 2H, J=8.1 Hz, H-3′,5′ Ar), 7.69 (d, 1H, J=8.7 Hz, H-5 Ar), 7.93 (d, 2H, J=8.4 Hz, H-2′,6′ Ar). MS (m/z) %: 336 (M+) 10.68%. Anal. Calcd. for C21H20O4 (336.38): C, 74.98; H, 5.99. Found: C, 75.06; H, 6.03. 2.1.1.8. 3-Ethyl-7-(4-methoxyphenacyloxy)-4-methyl-2H-1-benzopyran-2-one (2h) Yield 60%. mp 136-137 °C. IR (KBr) cm-1: 3060 (CH Ar), 2958, 2872 (CH aliphatic), 1708, 1695 (2 C=O), 1610, 1602, 1562, 1508 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 2.37 (s, 3H, CH3), 2.54 (q, 2H, CH2CH3), 3.86 (s, 3H, OCH3), 5.65 (s, 2H, OCH2), 6.98 (d, 1H, J=8.7 Hz, H-6 Ar), 7.00 (s, 1H, H-8 Ar), 7.09 (d, 2H, J=8.7 Hz, H-3′,5′ Ar), 7.68 (d, 1H, J=9.0 Hz, H-5 Ar), 8.01 (d, 2H, J=9.0 Hz, H-2′,6′ Ar). MS (m/z) %: 352 (M+) 23.35%. Anal. Calcd. for C21H20O5 (352.38): C, 71.58; H, 5.72. Found: C, 71.61; H, 5.80. 2.1.2. General Procedure for synthesis of 6-ethyl-5-methyl-9-substituted-3-((un)substituted phenyl)-7H-furo[3,2-g]benzopyran-7-ones (3a-h) (Scheme 1). Compound 2a-h (0.01 mol) was added to solution of 2% potassium hydroxide in absolute ethanol (50 ml) and the mixture was refluxed for 18 h. Solution was concentrated and acidified with a cold solution of 10% HCl. The precipitated product was filtered, washed and dried. Product was crystallized from isopropanol. 2.1.2.1. 5,9-Dimethyl-6-ethyl-3-phenyl-7H-furo[3,2-g]benzopyran-7-one (3a) Yield 85%. mp 228-229 °C. IR (KBr) cm-1: 3055 (CH Ar), 2974, 2870 (CH aliphatic), 1701 (C=O), 1593 (C=C). 1H-NMR *(CDCl3) δ: 1.18 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3 at C5), 2.63 (s, 3H, CH3 at C9), 2.70 (q, 2H, CH2CH3), 7.42-7.83 (m, 5H, Ar-H), 8.01 (s, 1H, H-4 Ar), 8.19 (s, 1H, H-2 Ar). MS (m/z) %: 318 (M+) 100%. Anal. Calcd. for C21H18O3 (318.37): C, 79.22; H, 5.70. Found: C, 79.31; H, 5.69. 2.1.2.2. 5,9-Dimethyl-6-ethyl-3-(4-methylphenyl)-7H-furo[3,2-g]benzopyran-7-one (3b) Yield 80%. mp 294-296 °C. IR (KBr) cm-1: 3099 (CH Ar), 2924, 2860 (CH aliphatic), 1705 (C=O), 1608, 1593, 1571 (C=C). 1H-NMR (CDCl3) δ: 1.18 (t, 3H, CH2CH3), 2.45 (s, 3H, CH3 at C5), 2.49 (s, 3H, CH3 at C9), 2.64 (s, 3H, CH3 at C4′), 2.74 (q, 2H, CH2CH3), 7.34 (d, 2H, J=7.8 Hz, H-3′,5′ Ar), 7.54 (d, 2H, J=7.5 Hz, H-2′,6′ Ar), 7.80 (s, 1H, H-4 Ar), 7.83 (s, 1H, H-2 Ar). MS (m/z) %: 332 (M+) 1.08%. Anal. Calcd. for C22H20O3 (332.39): C, 79.50; H, 6.06. Found: C, 79.58; H, 6.12. 2.1.2.3. 5,9-Dimethyl-6-ethyl-3-(4-methoxyphenyl)-7H-furo[3,2-g]benzopyran-7-one (3c) Yield 60%. mp 188-190 °C. IR (KBr) cm-1: 3082 (CH Ar), 2926, 2852 (CH aliphatic), 1714 (C=O), 1606, 1577 (C=C). 1H-NMR (CDCl3) δ: 1.19 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3 at C5), 2.65 (s, 3H, CH3 at C9), 2.75 (q, 2H, CH2CH3), 3.89 (s, 3H, OCH3), 7.06 (d, 2H, J=8.1 Hz, H-3′,5′ Ar), 7.60 (d, 2H, J=8.1 Hz, H-2′,6′ Ar), 7.77 (s, 1H, H-4 Ar), 7.80 (s, 1H, H-2 Ar). MS (m/z) %: 348 (M+) 100%. Anal. Calcd. for C22H20O4 (348.39): C, 75.84; H, 5.79. Found: C, 75.90; H, 5.82. 2.1.2.4. 3-(4-Bromophenyl)-5,9-dimethyl-6-ethyl-7H-furo[3,2-g]benzopyran-7-one (3d) Yield 75%. mp 287-289 °C. IR (KBr) cm-1: 3060 (CH Ar), 2958, 2870 (CH aliphatic), 1701 (C=O), 1569, 1558 (C=C). 1H-NMR (CDCl3) δ: 1.16 (t, 3H, CH2CH3), 2.38 (s, 3H, CH3 at C5), 2.50 (s, 3H, CH3 at C9), 2.73 (q, 2H, CH2CH3), 7.35 (s, 1H, H-4 Ar), 7.54 (d, 2H, J=8.4 Hz, H-2′,6′ Ar), 7.61 (d, 2H, J=9.0 Hz, H-3′,5′ Ar), 7.73 (s, 1H, H-2 Ar). MS (m/z) %: 397 (M+) 0.90%, 399 (M++2) 1.03%. Anal. Calcd. for C21H17BrO3 (397.26): C, 63.49; H, 4.31. Found: C, 63.53; H, 4.37. 2.1.2.5. 3-(3,4-Dimethoxyphenyl)-5,9-dimethyl-6-ethyl-7H-furo[3,2-g]benzopyran-7-one (3e) Yield 57%. mp 158-160 °C. IR (KBr) cm-1: 3080 (CH Ar), 2947, 2841 (CH aliphatic), 1705 (C=O), 1593, 1512 (C=C). 1H-NMR (CDCl3) δ: 1.04 (t, 3H, CH2CH3), 2.27 (s, 3H, CH3 at C5), 2.37 (s, 3H, CH3 at C9), 2.53 (q, 2H, CH2CH3), 3.83 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.06 (d, 1H, J=8.1 Hz, H-5′ Ar), 7.13 (s, 1H, H-2′ Ar), 7.30 (d, 1H, J=9.0 Hz, H-6′ Ar), 7.79 (s, 1H, H-4 Ar), 7.82 (s, 1H, H-2 Ar). MS (m/z) %: 378 (M+) 6.12%. Anal. Calcd. for C23H22O5 (378.42): C, 73.00; H, 5.86. Found: C, 73.09; H, 5.91. 2.1.2.6. 6-Ethyl-5-methyl-3-phenyl-7H-furo[3,2-g]benzopyran-7-one (3f) Yield 79%. mp 154-155 °C. IR (KBr) cm-1: 3059 (CH Ar), 2970, 2873 (CH aliphatic), 1712 (C=O), 1577 (C=C). 1H-NMR (CDCl3) δ: 1.19 (t, 3H, CH2CH3), 2.52 (s, 3H, CH3), 2.75 (q, 2H, CH2CH3), 7.41-7.84 (m, 6H, H-9 Ar, Ar-H), 8.00 (s, 1H, H-4 Ar), 8.03 (s, 1H, H-2 Ar). MS (m/z) %: 304 (M+) 14.64%. Anal. Calcd. for C20H16O3 (304.34): C, 78.93; H, 5.30. Found: C, 78.79; H, 5.28.



2.1.2.7. 6-Ethyl-5-methyl-3-(4-methylphenyl)-7H-furo[3,2-g]benzopyran-7-one (3g) Yield 74%. mp 171-173 °C. IR (KBr) cm-1: 3028 (CH Ar), 2966, 2870 (CH aliphatic), 1701 (C=O), 1604, 1581, 1512 (C=C). 1H-NMR (CDCl3) δ: 1.16 (t, 3H, CH2CH3), 2.48 (s, 3H, CH3 at C5), 2.54 (q, 2H, CH2CH3), 2.70 (s, 3H, CH3 at C4′), 7.29 (d, 2H, J=7.8 Hz, H-3′,5′ Ar), 7.52 (d, 2H, J=8.4 Hz, H-2′,6′ Ar), 7.67 (s, 1H, H-9 Ar), 7.80 (s, 1H, H-4 Ar), 7.83 (s, 1H, H-2 Ar). MS (m/z) %: 318 (M+) 16.69%. Anal. Calcd. for C21H18O3 (318.37): C, 79.22; H, 5.70. Found: C, 79.26; H, 5.79. 2.1.2.8. 6-Ethyl-3-(4-methoxyphenyl)-5-methyl-7H-furo[3,2-g]benzopyran-7-one (3h) Yield 56%. mp 155-158 °C. IR (KBr) cm-1: 3074 (CH Ar), 2966, 2873 (CH aliphatic), 1710 (C=O), 1597, 1573, 1508 (C=C). 1H-NMR (CDCl3) δ: 1.03 (t, 3H, CH2CH3), 2.39 (s, 3H, CH3), 2.50 (q, 2H, CH2CH3), 3.85 (s, 3H, OCH3), 7.07 (d, 2H, J=8.4 Hz, H-3′,5′ Ar), 7.59 (d, 2H, J=8.4 Hz, H-2′,6′ Ar), 7.63 (s, 1H, H-9 Ar), 7.78 (s, 1H, H-4 Ar), 7.81 (s, 1H, H-2 Ar). MS (m/z) %: 334 (M+) 0.59%. Anal. Calcd. for C21H18O4 (334.37): C, 75.43; H, 5.43. Found: C, 75.90; H, 5.44. 2.1.3. Synthesis of 3-(3,4-dimethoxyphenyl)-6-ethyl-7-methyl-5H-furo[2,3-h]benzopyran-5-one (4) (Scheme 1). Previous procedure adopted for synthesis of compounds 2a-h was applied on reacting 3-ethyl-7-hydroxy-4-methyl-2H-1-benzopyran-2-one 1b and 3,4-dimethoxyphenacyl bromide except that reaction was proceeded for 18 h instead of 24 h. Product was crystallized from isopropanol. Yield 53%. mp 187-189 °C. IR (KBr) cm-1: 3082 (CH Ar), 2966, 2873 (CH aliphatic), 1716 (C=O), 1610, 1595, 1517, 1508 (C=C). 1H-NMR (DMSO-d6) δ: 1.02 (t, 3H, CH2CH3), 2.37 (s, 3H, CH3), 2.55 (q, 2H, CH2CH3), 3.79 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.09-7.17 (m, 3H, H-2′,5′,6′ Ar ), 7.55 (s, 1H, H-2 Ar), 7.75 (d, 1H, J=9.0 Hz, H-9 Ar), 7.83 (d, 1H, J=8.4 Hz, H-8 Ar). MS (m/z) %: 364 (M+) 0.14%. Anal. Calcd. for C22H20O5 (364.39): C, 72.51; H, 5.53. Found: C, 72.58; H, 5.54.

O

CH3

R

CH3

HO O

CH3

R

CH3

OO O

1a R = C2H5 c R = CH3

R = C2H5 , CH3

OO O

H3C CH3

R

CH3

HH

OO O

H3C CH3

R

CH3

O

CH3

R

CH3

HO O

H2C

O

CH3

R

CH3

HO O

H3C

5a R = C2H5 b R = CH3

6a R = C2H56 b R = CH3

7a R = C2H5 b R = CH3

(i) (ii,iii) (iv,v)

(vi) (vii)

(viii)

Scheme 2. Reagents and conditions: (i ) Cinnamyl chloride, K2CO3, dry acetone, reflux 24 h, (ii ) Claisenrearrangement: N,N-diethylaniline, 185 oC, 3 h, (iii ) 10% HCl, (iv ) 5% NaOH extract, (v ) 10% HCl, (vi ) Ether extract, (vii ) 48% HBr, glacial acetic acid, reflux 8 h, (viii ) DDQ, dry benzene, refluxr 8 h.

2.1.4. General Procedure for synthesis of 7-cinnamyloxy-4,8-dimethyl-3-substituted-2H-1-benzopyran-2-ones (5a,b) (Scheme 2). A mixture of compound 1a,c (0.01 mol) and cinnamyl chloride (1.52 g, 0.01 mol) was refluxed in acetone (50 ml) in presence of anhydrous potassium carbonate (2.76 g, 0.02 mol) for 24 h. Acetone was distilled off and the residue washed with water and dried. Product was crystallized from isopropanol to give 5a,b. 2.1.4.1. 7-Cinnamyloxy-4,8-dimethyl-3-ethyl-2H-1-benzopyran-2-one (5a) Yield 67%. mp 131-133 °C. IR (KBr) cm-1: 3024 (CH Ar), 2962, 2860 (CH aliphatic), 1705 (C=O), 1604, 1577, 1550 (C=C). 1H-NMR (CDCl3) δ: 1.15 (t, 3H, CH2CH3), 1.59 (s, 3H, CH3 at C4), 2.39 (s, 3H, CH3 at C8), 2.68 (q, 2H, CH2CH3), 4.80 (d, 2H, J=6.0 Hz, OCH2CH=CH), 6.40-6.47 (m, 1H, OCH2-CH=CH), 6.76 (d, 1H, J= 16.2 Hz, OCH2-CH=CH), 6.88 (d, 1H, J=9.3 Hz, H-6 Ar), 7.30-7.44 (m, 6H, H-5 Ar, Ar-H). MS (m/z) %: 334 (M+) 0.53%. Anal. Calcd. for C22H22O3 (334.41): C, 79.02; H, 6.63. Found: C, 79.13; H, 6.68. 2.1.4.2. 7-Cinnamyloxy-3,4,8-trimethyl-2H-1-benzopyran-2-one (5b) Yield 72%. mp 139-140 °C. IR (KBr) cm-1: 3024 (CH Ar), 2999, 2868 (CH aliphatic), 1697 (C=O), 1608, 1593, 1573 (C=C). 1H-NMR (CDCl3) δ: 1.57 (s, 3H, CH3 at C4), 2.20 (s, 3H, CH3 at C3), 2.37 (s, 3H, CH3 at C8), 4.80 (d,



2H, J=5.7 Hz, OCH2CH=CH), 6.40-6.46 (m, 1H, OCH2CH=CH), 6.73 (d, 1H, J=16.6 Hz, OCH2CH=CH), 6.88 (d, 1H, J=8.7 Hz, H-6 Ar), 7.32-7.44 (m, 6H, H-5 Ar, Ar-H). MS (m/z) %: 320 (M+) 1.01%. Anal. Calcd. for C21H20O3 (320.38): C, 78.73; H, 6.29. Found: C, 78.90; H, 6.36. 2.1.5. General Procedure for synthesis of 4,8-dimethyl-7-hydroxy-6-(1-phenyl-(1 or 2)-propenyl)-3-substituted -2H-1-benzopyran-2-ones (6a,b) (Scheme 2). The cinnamyloxy derivative 5a,b (0.003 mol) in N,N-diethylaniline (5 ml) was refluxed at 185 oC for 3 h. The reaction mixture was poured onto cold 10% HCl and the separated solid was filtered off. Solid product was dissolved in ether and extracted with 5% NaOH then ether layer was separated from alkali aqueous layer. The alkali soluble fraction was acidified with 10% HCl and the separated product was filtered, purified by column chromatography using silica gel as stationary phase and chloroform as mobile phase. Subsequent crystallization was carried out from benzene:petroleum ether (1:1) to give 6a,b. 2.1.5.1. 4,8-Dimethyl-3-ethyl-7-hydroxy-6-(1-phenyl-1-propenyl)-2H-1-benzopyran-2-one (6a) Yield 35%. mp 201-203 °C. IR (KBr) cm-1: 3329 (OH), 3064 (CH Ar), 2970, 2872 (CH aliphatic), 1700 (C=O), 1591, 1577, 1554 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 1.59 (d, 3H, J=6.9 Hz, CH-CH3), 2.23 (s, 3H, CH3 at C4), 2.31 (s, 3H, CH3 at C8), 2.53 (q, 2H, CH2CH3), 6.40 (q, 1H, CHCH3), 7.17-7.29 (m, 6H, H-5 Ar, Ar-H), 9.04 (s, 1H, OH exch. D2O). MS (m/z) %: 334 (M+) 100%. Anal. Calcd. for C22H22O3 (334.41): C, 79.02; H, 6.63. Found: C, 79.10; H, 6.67. 2.1.5.2. 7-Hydroxy-6-(1-phenyl-2-propenyl)-3,4,8-trimethyl-2H-1-benzopyran-2-one (6b) Yield 36%. mp 209-210 °C. IR (KBr) cm-1: 3157 (OH), 3076 (CH Ar), 2926, 2852 (CH aliphatic), 1685 (C=O), 1604, 1583, 1571, 1504 (C=C). 1H-NMR (DMSO-d6) δ: 1.98 (s, 3H, CH3 at C4), 2.18 (s, 3H, CH3 at C3), 2.30 (s, 3H, CH3 at C8), 4.89 (d, 1H, J=15.6 Hz, CHCH=CH2), 5.12-5.18 (m, 2H, CHCH=CH2), 6.39-6.50 (m, 1H, CHCH=CH2), 7.14-7.35 (m, 6H, H-5 Ar, Ar-H), 9.34 (s, 1H, OH exch. D2O). MS (m/z) %: 320 (M+) 100%. Anal. Calcd. for C21H20O3 (320.38): C, 78.73; H, 6.29. Found: C, 78.76; H, 6.41. 2.1.6. General Procedure for synthesis of 2-phenyl-6-substituted-3,5,9-trimethyl-7H-furo[3,2-g]benzopyran-7-ones (7a,b) (Scheme 2) Following procedure 1 or 2: Procedure 1: The ether layer from the previous extraction was evaporated and the residue was purified by column chromatography using silica gel as stationary phase and chloroform as mobile phase to give 7a,b. Procedure 2: The rearranged product 6a,b (0.003 mol) was refluxed in mixture of glacial acetic acid (12 ml) and HBr (48%, 8 ml) for 8 h. The reaction mixture was poured onto crushed ice and the separated product was dried and dehydrogenated directly through refluxing with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.5 g) in dry benzene (15 ml) on boiling water bath for 8 h. The mixture was filtered while hot and benzene was concentrated. On standing, crystals of the same furobenzopyrones 7a,b separated out and were purified by column chromatography using silica gel as stationary phase and chloroform as mobile phase. 2.1.6.1. 6-Ethyl-2-phenyl-3,5,9-trimethyl-7H-furo[3,2-g]benzopyran-7-one (7a) Yield 40%. mp 180-183 °C. IR (KBr) cm-1: 3060 (CH Ar), 2956, 2854 (CH aliphatic), 1701 (C=O), 1593, 1577, 1560 (C=C). 1H-NMR (CDCl3) δ: 1.18 (t, 3H, CH2CH3), 2.44 (s, 3H, CH3 at C5), 2.57 (s, 3H, CH3 at C3), 2.63 (s, 3H, CH3 at C9), 2.73 (q, 2H, CH2CH3), 7.40-7.54 (m, 5H, Ar-H), 7.57 (s, 1H, H-4 Ar). MS (m/z) %: 332 (M+) 96.34%. Anal. Calcd. for C22H20O3 (332.39): C, 79.50; H, 6.06. Found: C, 79.64; H, 6.15. 2.1.6.2. 2-Phenyl-3,5,6,9-tetramethyl-7H-furo[3,2-g]benzopyran-7-one (7b) Yield 43%. mp 234-236 °C. IR (KBr) cm-1: 3084 (CH Ar), 2920, 2855 (CH aliphatic), 1701 (C=O), 1593, 1573 (C=C). 1H-NMR (CDCl3) δ: 2.24 (s, 3H, CH3 at C5), 2.42 (s, 3H, CH3 at C6), 2.57 (s, 3H, CH3 at C3), 2.63 (s, 3H, CH3 at C9), 7.40-7.54 (m, 5H, Ar-H), 7.57 (s, 1H, H-4 Ar). MS (m/z) %: 318 (M+) 100%. Anal. Calcd. for C21H18O3 (318.37): C, 79.22; H, 5.70. Found: C, 79.27; H, 5.73. 8-Acetyl-7-hydroxy-4-methyl-3-substituted-2H-1-benzopyran-2-one (8a,c) (Scheme 3) were repared as reported in literature [20]. 2.1.7. General Procedure for synthesis of 8-benzoyl-7-hydroxy-4-methyl-3-substituted-2H-1-benzopyran-2-one (8b,d) (Scheme 3). A mixture of compound 1b,d (0.05 mol) and benzoyl chloride (10.52 g, 8.7 ml, 0.075 mol) in pyridine (5 ml) was refluxed in an oil bath at 165 oC for 2 h, then poured onto crushed ice with stirring. The separated product was filtered, washed with sodium bicarbonate solution, then with water and dried. The separated solid was mixed with



anhydrous aluminium chloride (20 g, 0.15 mol) and heated in an oil bath at 165 oC for 2 h. The reaction mixture was cooled and treated with 10% HCl then filtered. The product was dissolved in 5% NaOH, filtered and reprecipitated by acidification with 10% HCl. The separated solid was collected, washed with water and crystallized from isopropanol giving 8b,d.

O

CH3R

OO

R1O

X1

9a-s

X2

O

CH3CH3

O OO

CH3CH3

O OO

CH3CH3

O

O R

O

R

O

CH3R

HO O O

CH3R

HO O

O R11b R1=C2H5 d R1=CH3 8a R=C2H5, R1=CH3

b R=C2H5, R1=C6H5 c R=CH3, R1=CH3 d R=CH3, R1=C6H5

10a R=CH3 b R=C6H5

O R

R=CH3

O

CH3

OO

OH

a R=C2H5, R1=CH3, X1=X2=Hb R=C2H5, R1=CH3, X1=CH3, X2=Hc R=C2H5, R1=CH3, X1=OCH3, X2=Hd R=C2H5, R1=CH3, X1=X2= OCH3e R=C2H5, R1=CH3, X1=Br, X2=Hf R=C2H5, R1=C6H5, X1=X2=Hg R=C2H5, R1=C6H5, X1=CH3, X2=Hh R=C2H5, R1=C6H5, X1=OCH3, X2=Hi R=C2H5, R1=C6H5, X1=X2=OCH3j R=R1=CH3, X1=X2=Hk R=R1=CH3, X1=CH3, X2=Hl R=R1=CH3, X1=OCH3, X2=Hm R=R1=CH3, X1=X2=OCH3n R=R1=CH3, X1=Br, X2=Ho R=CH3, R1=C6H5, X1=X2=Hp R=CH3, R1=C6H5, X1=CH3, X2=Hq R=CH3, R1=C6H5, X1=OCH3, X2=H r R=CH3, R1=C6H5, X1=X2=OCH3 s R=CH3, R1=C6H5, X1=Br, X2=H

11a R=CH3 b R=C6H5

12a R=CH3 b R=C6H5

(i ) or (ii )

(iii,iv,v )

(viii,ix )(x )

(vi )(vii )

Scheme 3. Reagents and conditions: (i ) Acetic anhydride, reflux 1h, (ii ) Benzoyl chloride, pyridine, 165 oC, 2 h, (iii ) Fries rearrangement: AlCl3,165 oC,2h, (iv ) 5% NaOH, (v ) 10% HCl, (vi ) Appropriate phenacylbromide derivative,K2CO3,dryacetone,reflux24h, (vii ) Ethylchloroacetate,K2CO3,dryacetone,reflux24h, (viii ) 5% KOH,methanol/watermixture

(1:1 ), reflux 30 min., (ix ) 10% HCl, (x ) Sodium acetate, acetic anhydride, reflux 1 h. 2.1.7.1. 8-Benzoyl-3-ethyl-7-hydroxy-4-methyl-2H-1-benzopyran-2-one (8b) Yield 51%. mp 197-200 °C. IR (KBr) cm-1: 3246 (OH), 3047 (CH Ar), 2970, 2868 (CH aliphatic), 1685, 1668 (2 C=O), 1595, 1577 (C=C). 1H-NMR *(DMSO-d6) δ: 0.97 (t, 3H, CH2CH3), 2.35 (s, 3H, CH3), 2.46 (q, 2H, CH2CH3), 6.91 (d, 1H, J=9.2 Hz, H-6 Ar), 7.49 (t, 2H, H-3′,5′ Ar), 7.63 (t, 1H, H-4′ Ar), 7.70 (d, 1H, J=9.2 Hz, H-5 Ar), 7.72 (d, 2H, J=9.2 Hz, H-2′,6′ Ar), 10.80 (s, 1H, OH exch. D2O). MS (m/z) %: 308 (M+) 72.07%. Anal. Calcd. for C19H16O4 (308.33): C, 74.01; H, 5.23. Found: C, 74.08; H, 5.29. 2.1.7.2. 8-Benzoyl-3,4-dimethyl-7-hydroxy-2H-1-benzopyran-2-one (8d) Yield 56%. mp 230-234 °C. IR (KBr) cm-1: 3421 (OH), 3062 (CH Ar), 2970, 2877 (CH aliphatic), 1708, 1693 (2 C=O), 1604, 1529, 1512 (C=C). 1H-NMR (DMSO-d6) δ: 2.02 (s, 3H, CH3 at C4), 2.38 (s, 3H, CH3 at C3), 6.95 (d, 1H, J=8.7 Hz, H-6 Ar), 7.55 (t, 2H, H-3′,5′ Ar), 7.66 (t, 1H, H-4′ Ar), 7.76 (d, 3H, J=8.4 Hz, H-5 Ar, H-2′,6′ Ar), 10.75 (s, 1H, OH exch. D2O). MS (m/z) %: 294 (M+) 54.82%. Anal. Calcd. for C18H14O4 (294.30): C, 73.46; H, 4.79. Found: C, 73.52; H, 4.82. 2.1.8. General Procedure for synthesis of 3,6-disubstituted-2-((un)substituted benzoyl)-7-methyl-5H-furo [2,3-h]benzopyran-5-ones (9a-s) (Scheme 3). A mixture of compound 8a-d (0.01 mol) and appropriate phenacyl bromide derivative (0.015 mol) in acetone (50 ml) was refluxed in presence of anhydrous potassium carbonate (2.76 g, 0.02 mol) with stirring for 24 h. The solution was filtered and the remaining residue was washed with acetone. The combined filtrates and washings were distilled under reduced pressure to give 9a-s. The solid product was crystallized from isopropanol. 2.1.8.1. 2-Benzoyl-3,7-dimethyl-6-ethyl-5H-furo[2,3-h]benzopyran-5-one (9a) Yield 74%. mp 221-223 °C. IR (KBr) cm-1: 3070 (CH Ar), 2968, 2875 (CH aliphatic), 1703 (2 C=O), 1598, 1575, 1548 (C=C). 1H-NMR (DMSO-d6) δ: 1.09 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3 at C7), 2.63 (q, 2H, CH2CH3), 2.80 (s, 3H, CH3 at C3), 7.60-8.00 (m, 7H, H-8,9 Ar, Ar-H). MS (m/z) %: 346 (M+) 6.68%. Anal. Calcd. for C22H18O4 (346.38): C, 76.29; H, 5.24. Found: C, 76.61; H, 5.40. 2.1.8.2. 3,7-Dimethyl-6-ethyl-2-(4-methylbenzoyl)-5H-furo[2,3-h]benzopyran-5-one (9b) Yield 65%. mp 235-236 °C. IR (KBr) cm-1: 3079 (CH Ar), 2955, 2845 (CH aliphatic), 1706 (2 C=O), 1601, 1510 (C=C). 1H-NMR (DMSO-d6) δ: 1.08 (t, 3H, CH2CH3), 2.42 (s, 3H, CH3 at C7), 2.50 (s, 3H, CH3 at C3), 2.62 (q, 2H,



CH2CH3), 2.77 (s, 3H, CH3 at C4′), 7.39 (d, 1H, J=7.8 Hz, H-9 Ar), 7.63 (d, 1H, J=9.0 Hz, H-8 Ar), 7.88-7.92 (m, 4H, H-2′,3′,5′,6′ Ar). MS (m/z) %: 360 (M+) 23.75%. Anal. Calcd. for C23H20O4 (360.40): C, 76.65; H, 5.59. Found: C, 76.93; H, 5.74. 2.1.8.3. 3,7-Dimethyl-6-ethyl-2-(4-methoxybenzoyl)-5H-furo[2,3-h]benzopyran-5-one (9c) Yield 68%. mp 230-232 °C. IR (KBr) cm-1: 3078 (CH Ar), 2966, 2839 (CH aliphatic), 1708 (2 C=O), 1597, 1570, 1546, 1508 (C=C). 1H-NMR (CDCl3) δ: 1.19 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3 at C7), 2.75 (q, 2H, CH2CH3), 2.95 (s, 3H, CH3 at C3), 3.92 (s, 3H, OCH3), 7.02 (d, 2H, J=8.7 Hz, H-3′,5′ Ar), 7.43 (d, 1H, J=9.0 Hz, H-9 Ar), 7.68 (d, 1H, J=9.0 Hz, H-8 Ar), 8.11 (d, 2H, J=8.1 Hz, H-2′,6′ Ar). MS (m/z) %: 376 (M+) 63.53%. Anal. Calcd. for C23H20O5 (376.40): C, 73.39; H, 5.36. Found: C, 73.58; H, 5.52. 2.1.8.4. 2-(3,4-Dimethoxybenzoyl)-3,7-dimethyl-6-ethyl-5H-furo[2,3-h]benzopyran-5-one (9d) Yield 61%. mp 252-255 °C. IR (KBr) cm-1: 3086 (CH Ar), 2966, 2850 (CH aliphatic), 1701 (2 C=O), 1593, 1550, 1516 (C=C). 1H-NMR (CDCl3) δ: 1.19 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3 at C7), 2.72 (q, 2H, CH2CH3), 2.92 (s, 3H, CH3 at C3), 3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.98 (d, 1H, J=8.7 Hz, H-5′ Ar), 7.44 (d, 1H, J=8.7 Hz, H-9 Ar), 7.63 (s, 1H, H-2′ Ar), 7.69 (d, 1H, J=9.0 Hz, H-8 Ar), 7.84 (d, 1H, J=8.7 Hz, H-6′ Ar). MS (m/z) %: 406 (M+) 34.78%. Anal. Calcd. for C24H22O6 (406.43): C, 70.92; H, 5.46. Found: C, 70.97; H, 5.48. 2.1.8.5. 2-(4-Bromobenzoyl)-3,7-dimethyl-6-ethyl-5H-furo[2,3-h]benzopyran-5-one (9e) Yield 65%. mp 234-238 °C. IR (KBr) cm-1: 3068 (CH Ar), 2964, 2870 (CH aliphatic), 1707 (2 C=O), 1602, 1583, 1562, 1546 (C=C). 1H-NMR (DMSO-d6) δ: 1.06 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3 at C7), 2.64 (q, 2H, CH2CH3), 2.81 (s, 3H, CH3 at C3), 7.63 (d, 1H, J=9.3 Hz, H-9 Ar), 7.80 (d, 1H, J=8.4 Hz, H-8 Ar), 7.92 (d, 2H, J=3.9 Hz, H-3′,5′ Ar), 7.95 (d, 2H, J=3.3 Hz, H-2′,6′ Ar). MS (m/z) %: 425 (M+) 6.90%. Anal. Calcd. for C22H17BrO4 (425.27): C, 62.13; H, 4.03. Found: C, 62.08; H, 4.17. 2.1.8.6. 2-Benzoyl-6-ethyl-7-methyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9f) Yield 80%. mp 233-235 °C. IR (KBr) cm-1: 3057 (CH Ar), 2966, 2873 (CH aliphatic), 1714 (2 C=O), 1600, 1577, 1544 (C=C). 1H-NMR (CDCl3) δ: 1.13 (t, 3H, CH2CH3), 2.50 (s, 3H, CH3), 2.67 (q, 2H, CH2CH3), 7.29-7.79 (m, 12H, Ar-H). MS (m/z) %: 408 (M+) 30.20%. Anal. Calcd. for C27H20O4 (408.45): C, 79.40; H, 4.94. Found: C, 79.68; H, 5.21. 2.1.8.7. 6-Ethyl-7-methyl-2-(4-methylbenzoyl)-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9g) Yield 67%. mp 213-216 °C. IR (KBr) cm-1: 3034 (CH Ar), 2968, 2873 (CH aliphatic), 1714 (2 C=O), 1602, 1544 (C=C). 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, CH2CH3), 2.33 (s, 3H, CH3 at C7), 2.49 (s, 3H, CH3 at C4′), 2.54 (q, 2H, CH2CH3), 7.22 (d, 1H, J=7.8 Hz, H-9 Ar), 7.33-7.53 (m, 5H, H-3′,5′ Ar, H-3′',4′′,5′′ Ar), 7.69 (d, 1H, J=8.4 Hz, H-8 Ar), 7.77 (d, 2H, J=8.7 Hz, H-2′′,6′′ Ar), 7.99 (d, 2H, J=8.7 Hz, H-2′,6′ Ar). MS (m/z) %: 422 (M+) 82.96%. Anal. Calcd. for C28H22O4 (422.47): C, 79.60; H, 5.25. Found: C, 79.54; H, 5.29. 2.1.8.8. 6-Ethyl-2-(4-methoxybenzoyl)-7-methyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9h) Yield 69%. mp 218-220 °C. IR (KBr) cm-1: 3090 (CH Ar), 2962, 2839 (CH aliphatic), 1710 (2 C=O), 1600, 1575, 1544, 1508 (C=C). 1H-NMR (CDCl3) δ: 1.13 (t, 3H, CH2CH3), 2.49 (s, 3H, CH3), 2.67 (q, 2H, CH2CH3), 3.83 (s, 3H, OCH3), 6.79 (d, 3H, J=9.3 Hz, H-9 Ar, H-3′,5′ Ar), 7.35 (t, 1H, H-4′′ Ar), 7.50 (t, 2H, H-3′′,5′′ Ar), 7.55 (d, 1H, J=8.7 Hz, H-8 Ar), 7.74 (d, 2H, J=9.3 Hz, H-2′′,6′′ Ar), 7.83 (d, 2H, J=9.0 Hz, H-2′,6′ Ar). MS (m/z) %: 438 (M+) 99.13%. Anal. Calcd. for C28H22O5 (438.47): C, 76.70; H, 5.06. Found: C, 76.79; H, 5.11. 2.1.8.9. 2-(3,4-Dimethoxybenzoyl)-6-ethyl-7-methyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9i) Yield 59%. mp 215-218 °C. IR (KBr) cm-1: 3090 (CH Ar), 2962, 2843 (CH aliphatic), 1720 (2 C=O), 1593, 1581, 1554, 1512 (C=C). 1H-NMR (CDCl3) δ: 1.13 (t, 3H, CH2CH3), 2.49 (s, 3H, CH3), 2.67 (q, 2H, CH2CH3), 3.85 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.73 (d, 2H, J=8.4 Hz, H-9 Ar, H-5′ Ar), 7.34-7.57 (m, 6H, H-8 Ar, H-2′,6′ Ar, H-3′′,4′′,5′′ Ar), 7.74 (d, 2H, J=8.7 Hz, H-2′′,6′′ Ar). MS (m/z) %: 468 (M+) 95.11%. Anal. Calcd. for C29H24O6 (468.50): C, 74.35; H, 5.16. Found: C, 74.42; H, 5.22. 2.1.8.10. 2-Benzoyl-3,6,7-trimethyl-5H-furo[2,3-h]benzopyran-5-one (9j) Yield 75%. mp 154-156 °C. IR (KBr) cm-1: 3066 (CH Ar), 2922, 2860 (CH aliphatic), 1701 (2 C=O), 1597, 1550 (C=C). 1H-NMR (DMSO-d6) δ: 2.14 (s, 3H, CH3 at C7), 2.63 (s, 3H, CH3 at C6), 2.80 (s, 3H, CH3 at C3), 7.75 (m, 4H, H-9 Ar, H-3′,4′,5′ Ar), 7.93 (d, 1H, J=8.7 Hz, H-8 Ar), 7.99 (d, 2H, J=7.2 Hz, H-2′,6′ Ar). MS (m/z) %: 332 (M+) 92.23%. Anal. Calcd. for C21H16O4 (332.35): C, 75.89; H, 4.85. Found: C, 76.14; H, 5.02.



2.1.8.11. 2-(4-Methylbenzoyl)-3,6,7-trimethyl-5H-furo[2,3-h]benzopyran-5-one (9k) Yield 64%. mp 243-246 °C. IR (KBr) cm-1: 3080 (CH Ar), 2922, 2854 (CH aliphatic), 1705 (2 C=O),1602, 1566, 1548 (C=C). 1H-NMR (DMSO-d6) δ: 2.15 (s, 3H, CH3 at C7), 2.43 (s, 3H, CH3 at C6), 2.50 (s, 3H, CH3 at C3), 2.79 (s, 3H, CH3 at C4′), 7.41 (d, 1H, J=8.7 Hz, H-9 Ar), 7.66 (d, 1H, J=9.0 Hz, H-8 Ar), 7.91 (d, 2H, J=4.8 Hz, H-3′,5′ Ar), 7.94 (d, 2H, J=5.7 Hz, H-2′,6′ Ar). MS (m/z) %: 346 (M+) 0.97%. Anal. Calcd. for C22H18O4 (346.38): C, 76.29; H, 5.24. Found: C, 76.38; H, 5.42. 2.1.8.12. 2-(4-Methoxybenzoyl)-3,6,7-trimethyl-5H-furo[2,3-h]benzopyran-5-one (9l) Yield 70%. mp 238-241 °C. IR (KBr) cm-1: 3070 (CH Ar), 2966, 2873 (CH aliphatic), 1708 (2 C=O), 1597, 1566, 1548 (C=C). 1H-NMR (CDCl3) δ: 2.15 (s, 3H, CH3 at C7), 2.43 (s, 3H, CH3 at C6), 2.79 (s, 3H, CH3 at C3), 3.91 (s, 3H, OCH3), 7.03 (d, 2H, J=9.0 Hz, H-3′,5′ Ar), 7.44 (d, 1H, J=9.3 Hz, H-9 Ar), 7.69 (d, 1H, J=9.0 Hz, H-8 Ar), 8.12 (d, 2H, J=8.7 Hz, H-2′,6′ Ar). MS (m/z) %: 362 (M+) 5.17%. Anal. Calcd. for C22H18O5 (362.38): C, 72.92; H, 5.01. Found: C, 73.15; H, 4.97. 2.1.8.13. 2-(3,4-Dimethoxybenzoyl)-3,6,7-trimethyl-5H-furo[2,3-h]benzopyran-5-one (9m) Yield 64%. mp 176-178 °C. IR (KBr) cm-1: 3085 (CH Ar), 2931, 2839 (CH aliphatic), 1712 (2 C=O), 1593, 1512 (C=C). 1H-NMR (CDCl3) δ: 2.19 (s, 3H, CH3 at C7), 2.37 (s, 3H, CH3 at C6), 2.92 (s, 3H, CH3 at C3), 3.90 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 7.00 (d, 1H, J=9.0 Hz, H-5′ Ar), 7.43-7.68 (m, 4H, H-8,9 Ar, H-2′,6′ Ar). MS (m/z) %: 392 (M+) 51.74%. Anal. Calcd. for C23H20O6 (392.40): C, 70.40; H, 5.14. Found: C, 70.38; H, 5.19. 2.1.8.14. 2-(4-Bromobenzoyl)-3,6,7-trimethyl-5H-furo[2,3-h]benzopyran-5-one (9n) Yield 73%. mp 252-254 °C. IR (KBr) cm-1: 3085 (CH Ar), 2924, 2858 (CH aliphatic), 1714 (2 C=O), 1604, 1581, 1548 (C=C). 1H-NMR (DMSO-d6) δ: 2.13 (s, 3H, CH3 at C7), 2.45 (s, 3H, CH3 at C6), 2.79 (s, 3H, CH3 at C3), 7.62 (d, 1H, J=8.7 Hz, H-9 Ar), 7.80 (d, 1H, J=8.7 Hz, H-8 Ar), 7.91 (d, 2H, J=4.8 Hz, H-3′,5′ Ar), 7.93 (d, 2H, J=5.4 Hz, H-2′,6′ Ar). MS (m/z) %: 411 (M+) 4.78%, 413 (M++2) 4.09%. Anal. Calcd. for C21H15BrO4 (411.25): C, 61.33; H, 3.68. Found: C, 61.41; H, 3.76. 2.1.8.15. 2-Benzoyl-6,7-dimethyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9o) Yield 77%. mp 127-129 °C. IR (KBr) cm-1: 3057 (CH Ar), 2924, 2860 (CH aliphatic), 1716 (2 C=O), 1597, 1579, 1554 (C=C). 1H-NMR (CDCl3) δ: 2.19 (s, 3H, CH3 at C7), 2.37 (s, 3H, CH3 at C6), 7.29-7.58 (m, 10H, H-8,9 Ar, H-3′,4′,5′, H-2′′,3′′,4′′,5′′,6′′ Ar), 7.80 (d, 2H, J=6.9 Hz, H-2′,6′ Ar). MS (m/z) %: 394 (M+) 71.43%. Anal. Calcd. for C26H18O4 (394.42): C, 79.17; H, 4.60. Found: C, 79.22; H, 4.63. 2.1.8.16. 6,7-Dimethyl-2-(4-methylbenzoyl)-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9p) Yield 69%. mp 230-232 °C. IR (KBr) cm-1: 3040 (CH Ar), 2919, 2850 (CH aliphatic), 1714 (2 C=O), 1604, 1544 (C=C).1H-NMR (DMSO-d6) δ: 2.09 (s, 3H, CH3 at C7), 2.34 (s, 3H, CH3 at C6), 2.48 (s, 3H, CH3 at C4′), 7.22 (d, 3H, J=8.4 Hz, H-9 Ar, H-3′,5′ Ar), 7.33-7.53 (m, 3H, H-3′′,4′′,5′′ Ar), 7.70 (d, 1H, J=8.1 Hz, H-8 Ar), 7.77 (d, 2H, J=9.0 Hz, H-2′′,6′′ Ar), 7.98 (d, 2H, J=8.7 Hz, H-2′,6′ Ar). MS (m/z) %: 408 (M+) 45.99%. Anal. Calcd. for C27H20O4 (408.45): C, 79.40; H, 4.94. Found: C, 79.46; H, 5.08. 2.1.8.17. 6,7-Dimethyl-2-(4-methoxybenzoyl)-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9q) Yield 72%. mp 224-226 °C. IR (KBr) cm-1: 3055 (CH Ar), 2966, 2839 (CH aliphatic), 1714 (2 C=O), 1600, 1573, 1544, 1508 (C=C). 1H-NMR (CDCl3) δ: 2.20 (s, 3H, CH3 at C7), 2.48 (s, 3H, CH3 at C6), 3.83 (s, 3H, OCH3), 6.79 (d, 3H, J=8.7 Hz, H-9 Ar, H-3′,5′ Ar), 7.33-7.49 (m, 3H, H-3′′,4′′,5′′ Ar), 7.51 (d, 1H, J=8.7 Hz, H-8 Ar), 7.73 (d, 2H, J=9.3 Hz, H-2′′,6′′ Ar), 7.83 (d, 2H, J=8.7 Hz, H-2′,6′ Ar). MS (m/z) %: 424 (M+) 79.00%. Anal. Calcd. for C27H20O5 (424.44): C, 76.40; H, 4.75. Found: C, 76.53; H, 4.72. 2.1.8.18. 2-(3,4-Dimethoxybenzoyl)-6,7-dimethyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9r) Yield 68%. mp 150-153 °C. IR (KBr) cm-1: 3059 (CH Ar), 2935, 2839 (CH aliphatic), 1716 (2 C=O), 1597, 1512 (C=C). 1H-NMR (CDCl3) δ: 2.20 (s, 3H, CH3 at C7), 2.48 (s, 3H, CH3 at C6), 3.85 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.72-7.75 (m, 10H, Ar-H). MS (m/z) %: 454 (M+) 45.73%. Anal. Calcd. for C28H22O6 (454.47): C, 74.00; H, 4.88. Found: C, 74.08; H, 4.91. 2.1.8.19. 2-(4-Bromobenzoyl)-6,7-dimethyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (9s) Yield 75%. mp 258-262 °C. IR (KBr) cm-1: 3060 (CH Ar), 2924, 2855 (CH aliphatic), 1720 (2 C=O), 1610, 1585, 1540 (C=C). 1H-NMR (CDCl3) δ: 2.20 (s, 3H, CH3 at C7), 2.49 (s, 3H, CH3 at C6), 7.26-7.76 (m, 11H, Ar-H). MS (m/z) %: 473 (M+) 100%. Anal. Calcd. for C26H17BrO4 (473.31): C, 65.98; H, 3.62. Found: C, 66.04; H, 3.59.



2.1.9. General Procedure for synthesis of ethyl (8-acyl-3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetates (10a,b) (Scheme 3). A mixture of compound 8c,d (0.01 mol) and ethyl chloroacetate (2.4 ml, 0.02 mol) in acetone (50 ml) in presence of anhydrous potassium carbonate (2.76 g, 0.02 mol) was refluxed while stirring for 24 h. The solvent was distilled off and the residue was washed with water, filtered and dried to give 10a,b. Product was crystallized from isopropanol. 2.1.9.1. Ethyl (8-acetyl-3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetate (10a) Yield 91%. mp 152-154 °C. IR (KBr) cm-1: 3091 (CH Ar), 2984, 2927 (CH aliphatic), 1746, 1705 (3 C=O), 1604, 1498 (C=C). 1H-NMR (DMSO-d6) δ: 1.20 (t, 3H, CH2CH3), 2.07 (s, 3H, CH3 at C4), 2.37 (s, 3H, CH3 at C3), 2.54 (s, 3H, COCH3), 4.16 (q, 2H, CH2CH3), 5.01 (s, 2H, OCH2CO), 7.08 (d, 1H, J=9.3 Hz, H-6 Ar), 7.78 (d, 1H, J=9.0 Hz, H-5 Ar). MS (m/z) %: 318 (M+) 68.33%. Anal. Calcd. for C17H18O6 (318.32): C, 64.14; H, 5.70. Found: C, 64.31; H, 5.78. 2.1.9.2. Ethyl (8-benzoyl-3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetate (10b) Yield 72%. mp 160-161 °C. IR (KBr) cm-1: 3057 (CH Ar), 2987, 2870 (CH aliphatic), 1757, 1735, 1712 (3 C=O), 1597, 1531 (C=C). 1H-NMR (CDCl3) δ: 1.22 (t, 3H, CH2CH3), 2.15 (s, 3H, CH3 at C4), 2.39 (s, 3H, CH3 at C3), 4.18 (q, 2H, CH2CH3), 4.64 (s, 2H, OCH2CO), 6.79 (d, 1H, J=9.0 Hz, H-6 Ar), 7.41-7.91 (m, 4H, H-5 Ar, H-3′,4′,5′ Ar), 7.62 (d, 2H, J=7.2 Hz, H-2′,6′ Ar). MS (m/z) %: 380 (M+) 53.06%. Anal. Calcd. for C22H20O6 (380.39): C, 69.46; H, 5.30. Found: C, 69.53; H, 5.27. 2.1.10. General Procedure for synthesis of (8-acyl-3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetic acid (11a,b) (Scheme 3). A solution of 10a,b (0.01 mol) and potassium hydroxide (2.52 g, 0.045 mol) in methanol/water mixture (1:1) (50 ml) was refluxed for 30 min. The solution was acidified with 10% HCl and the precipitated solid was filtered, washed and dried. The obtained solid was crystallized from ethyl acetate to give 11a,b. 2.1.10.1. (8-Acetyl-3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetic acid (11a) Yield 73%. mp 252-253 °C. IR (KBr) cm-1: 3059 (CH Ar), 2929, 2860 (CH aliphatic), 2800, 2560 (carboxylic OH), 1766, 1710, 1674 (3 C=O), 1600, 1500 (C=C). 1H-NMR (DMSO-d6) δ: 2.07 (s, 3H, CH3 at C4), 2.27 (s, 3H, CH3 at C3), 2.53 (s, 3H, COCH3), 4.90 (s, 2H, CH2), 7.06 (d, 1H, J=8.7 Hz, H-6 Ar), 7.78 (d, 1H, J=9.3 Hz, H-5 Ar), 13.20 (s, 1H, OH exch. D2O). MS (m/z) %: 290 (M+) 31.17%. Anal. Calcd. for C15H14O6 (290.27): C, 62.07; H, 4.86. Found: C, 62.08; H, 4.94. 2.1.10.2. (8-Benzoyl-3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetic acid (11b) Yield 61%. mp 200-202 °C. IR (KBr) cm-1: 3088 (CH Ar), 2968, 2850 (CH aliphatic), 2586, 2493 (carboxylic OH), 1720, 1710, 1689 (3 C=O), 1583, 1508 (C=C). 1H-NMR (DMSO-d6) δ: 2.04 (s, 3H, CH3 at C4), 2.40 (s, 3H, CH3 at C3), 4.80 (s, 2H, CH2), 7.09 (d, 1H, J=8.7 Hz, H-6 Ar), 7.50-7.89 (m, 6H, H-5 Ar, Ar-H), 13.19 (s, 1H, OH exch. D2O). MS (m/z) %: 352 (M+) 12.38%. Anal. Calcd. for C20H16O6 (352.34): C, 68.18; H, 4.58. Found: C, 68.14; H, 4.63. 2.1.11. General Procedure for synthesis of 6,7-dimethyl-3-substituted-5H-furo[2,3-h]benzopyran-5-ones (12a,b) (Scheme 3). A mixture of 11a,b (0.02 mol) and anhydrous sodium acetate (1.64 g, 0.02 mol) in acetic anhydride (35 ml) was refluxed for 1 h, water was added and the mixture was refluxed for 10 min., diluted with water and extracted with ethyl acetate. The organic layer was washed with sodium bicarbonate solution (50 ml). The ethyl acetate was evaporated and the residue was crystallized from isopropanol giving 12a,b. 2.1.11.1. 3,6,7-Trimethyl-5H-furo[2,3-h]benzopyran-5-one (12a) Yield 70%. mp 244-246 °C. IR (KBr) cm-1: 3101 (CH Ar), 2927, 2868 (CH aliphatic), 1701 (C=O), 1613, 1527 (C=C). 1H-NMR (CDCl3) δ: 2.25 (s, 3H, CH3 at C7), 2.47 (s, 3H, CH3 at C6), 2.55 (s, 3H, CH3 at C3), 7.36 (d, 1H, J=9.0 Hz, H-9 Ar), 7.41 (s, 1H, H-2 Ar), 7.49 (d, 1H, J=9.0 Hz, H-8 Ar). MS (m/z) %: 228 (M+) 100%. Anal. Calcd. for C14H12O3 (228.24): C, 73.67; H, 5.30. Found: C, 73.69; H, 5.33. 2.1.11.2. 6,7-Dimethyl-3-phenyl-5H-furo[2,3-h]benzopyran-5-one (12b) Yield 65%. mp 190-191 °C. IR (KBr) cm-1: 3095 (CH Ar), 2924, 2856 (CH aliphatic), 1701 (C=O), 1601 (C=C). 1H-NMR (CDCl3) δ: 2.22 (s, 3H, CH3 at C7), 2.48 (s, 3H, CH3 at C6), 7.39-7.75 (m, 8H, Ar-H). MS (m/z) %: 290 (M+) 100%. Anal. Calcd. for C19H14O3 (290.31): C, 78.61; H, 4.86. Found: C, 78.82; H, 4.91.



1.2. Antimicrobial and photosensitizing screening All the synthesized compounds were screened for their antimicrobial and photosensitizing activities by the paper disc diffusion method [21] and compared with xanthotoxin as reference compound. The tested organism used was Bacillus Subtilus. In the preliminary test, employing strong condition (high concentration of the substance) was used for selecting the active compounds, even weakly active, from the inactive ones. In another test, only the active compounds were tested to determine the effect of radiation time (exposure to UV-A) and concentration on their photosensitizing activity. Pre-experimental preparations a) Nutrient agar medium: 0.3% of the beef extract, 0.5% of peptone, 0.1% of dipotassium hydrogen phosphate and 1.5% agar b) Broth culture of the organism: slant agar seeded with the tested organism (Bacillus Subtilus) and incubated overnight. c) Paper disc: Whatman no. 1 filter paper disc (5 mm) were sterilized and impregnated with different concentrations of the tested compounds (compounds were dissolved in dimethylformamide, DMF), and allowed to dry overnight. Two concentrations were prepared for the tested compounds. EXPERIMENTAL 0.02 ml of the prepared broth culture was added carefully in the sterile petri dishes then 10 ml of the liquefied nutrient agar medium were added, allowed to be mixed uniformly and solidified. The impregnated discs were arranged uniformly on the solidified agar layer. Each plate contained disc impregnated with DMF (neglect effect of the solvent) and another disc impregnated with xanthotoxin as reference compound. Two groups of plates were used, one as test plates, incubated in the dark at 37 oC for 3 h before irradiation (to allow for diffusion of the tested compounds through the agar layer) and the duplicate plates were left in the incubator overnight as control to determine the antimicrobial activity. Covers were removed from plates of first group (tested petri dishes) and exposed to UV lamp (365 nm) for 20 min. After irradiation, plates were reincubated in the dark at 37 oC overnight and examined for photosensitizing activity (antimicrobial and photosensitizing activities were determined by measuring the produced inhibition zones). Effect of increasing time of UV-A radiation and concentration on photosensitizing activity for active benzopyrone and furobenzopyrone derivatives The experiment was repeated using the selected active compounds to study the effect of radiation time and concentration on the photosensitizing activity. Two groups of discs were prepared. One group of discs was impregnated with 0.01 ml (each disc contained 0.5 mg of the tested compounds) and the other group was impregnated with 0.02 ml (each disc contained 1 mg of the tested compounds). 1.3. QSAR 2.3.1. Computational method All the computational works were performed on Molecular Operating Environment software (MOE version 2008.10.2) [22]. The structures of 17 compounds (14 new compound in addition to 3 puplished compound [23], Figure 1) used as training set and structures of 3 compounds used as test set (2 new compounds in addition to 1 published compound [23], Figure 1) were sketched using molecular builder of MOE and each structure was subjected to energy minimization up to 0.01 Kcal/mol Å using the MMFF94x force field. Optimization methods were used followed by conformational search of each energy-minimized structure. The most stable conformer of each structure was selected and saved into database to generate the common descriptors. QuaSAR descriptor module of MOE was used to calculate descriptors for each molecule. The probability density functions used are Gaussian. The RMSD tolerance was set to 0.5 Å. Regression analysis was performed using photosensitizing activity after radiation for fourty min. as dependent factor and the calculated descriptors as predictable variables.



O

CH3

CH3

OO

13a R=CH3, X1=Br, X2=H b R=CH3, X1=X2=OCH3 c R=H, X1=Br, X2=H

OOR

CH3

CH3

OO

X1

X2

14

Figure 1: Structure of published compounds used in QSAR study

In this study, the pool of descriptors was optimized using principal components analysis (PCA). The optimization started with the reduction in the number of molecular descriptors by the determination of the highly inter-correlated descriptor pairs and only one from each pair was selected then the descriptors with insignificant variance through the data set were also rejected. QSAR model was then constructed after ensuring reasonable correlation of photosensitizing activity with the individual descriptors and minimum inter-correlation among the descriptors used in the derived model. The quality of the model was assessed using the statistical parameter r2. 2.3.2. Molecular descriptors AM1_dipole: The dipole moment calculated using the AM1 Hamiltonian [MOPAC]. The magnitude of the dipole moment does not depend on the absolute orientation in space. logP: Log of the octanol/water partition coefficient (including implicit hydrogens). This property is calculated from a linear atom type model. logS: Log of the aqueous solubility (mol/l). This property is calculated from an atom contribution linear atom type model. SMR: Molecular refractivity (including implicit hydrogens). This property is an atomic contribution model that assumes the correct protonation state (washed structures). TPSA: Polar surface area (Å2) calculated using group contributions to approximate the polar surface area from connection table information only. 2.3.3. Model evaluation: Evaluation of the model and its trial on test set (3 compounds) was used for further assessment of predictivity for the produced model. The predictive ability of the model was expressed by the predictive r2 value (r2

pred.). 1.4. Docking study 2.4.1. Docking procedure Docking studies of active antimicrobial compounds were performed by Molecular Operating Environment software (MOE version 2008.10.2) [22]. The program operated under “Window XP” operating system installed on an Intel Pentium IV PC with a 2.8 MHz processor and 512 RAM. All minimizations were performed with MOE until a RMSD gradient of 0.05 Kcal mol-1 Ǻ-1 with MMFF94 force field and the partial charges were automatically calculated. The score function, dock function (S, Kcal/mol) developed by MOE program was used for evaluation of the binding affinity of the ligand. 2.4.1.1. Preparation of the target DNA gyrase The X-ray crystal structure of the enzyme with benzopyrone ligand (PDB code 1AJ6) [24] was obtained from the protein data bank in PDB formate. The enzyme was prepared for docking. (i) 3D protonation for the amino acid side chain and novobiocin. (ii) Isolation of the active site, fixation to be dealt with as rigid structure and recognition of amino acids. (iii) Creation of dummies around active site. (iv) Studying the interactions of the ligand (novobiocin) with the amino acids of the active site. 2.4.1.2. Preparation of compounds for docking The 3D structures of the synthesized compounds were built using MOE and subjected to the following procedure: (i) 3D protonation of the structures. (ii) Running conformational analysis using systemic search. (iii) Selecting the least energetic conformer. (iv) Applying the same docking protocol used with novobiocin.



2.4.1.3. Docking running Prior to docking of benzopyrone and furobenzopyrone derivatives, redocking of the native ligand bound in the topoisomerase II active site was performed to validate the docking protocol. The generated most stable conformer of each compound was virtually docked into the predefined active site of topoisomerase II. The developed docked models were energetically minimized and then used to predict the interaction of the ligand with the amino acids in the active site of the enzyme.


1.5. Chemistry The target compounds 3a-h, 7a,b, 9a-s and 12a,b were synthesized as depicted in Schemes 1-3. The starting compounds 3,8-disubstituted-7-hydroxy-4-methyl-2H-1-benzopyran-2-one 1a-d [18] and phenacyl bromide derivatives [19] were prepared according to the previously reported procedures. Refluxing 7-hydroxy-2H-1-benzopyran-2-one 1a,b with appropriate phenacyl bromide derivative in dry acetone containing anhydrous potassium carbonate yielded ether derivatives 2a-h, Scheme 1. This mild reaction condition was adopted to avoid any probability for the opening of the sensitive pyrone ring [25]. Complete reaction was achieved with negative ferric chloride test. Cyclization of ether derivatives 2a-h to furo[3,2-g]benzopyran-7-ones 3a-h were achieved through reflux with alcoholic potassium hydroxide followed by subsequent acidification, Scheme 1. Attempts for etherification of 3-ethyl-7-hydroxy-4-methyl-2H-1-benzopyran-2-one 1b with 3,4-dimethoxyphenacyl bromide under previous conditions gave the angular furo[2,3-h]benzopyran-5-one 4 in one step reaction, Scheme 1. Trials to decrease reaction time to obtain the ether derivative gave same product. Cinnamylation of 7-hydroxybenzopyran-2-one 1a,c with cinnamyl chloride through reflux in dry acetone in presence of anhydrous potassium carbonate yielded cinnamyl ethers 5a,b, Scheme 2. These ether derivatives 5a,b gave negative ferric chloride test. Claisen rearrangement of 7-cinnamyloxybenzopyrones 5a,b was achieved through refluxing with N,N-diethylaniline and gave mixture of products, the open rearranged product 6a,b and the cyclized furobenzopyran-7-one 7a,b, Scheme 2. Use of N,N-diethylaniline [26] was preferred than N,N-dimethylaniline [27] due to reduction in reaction time and lower temperature. Separation of product was obtained through alkalinization and extraction with ether. The open rearranged product 6a,b were achieved from aquous alkaline solution after acidification with hydrochloric acid. Compounds 6a,b gave positive result with ferric chloride test. 1H-NMR spectra proved different position of double bond of propenyl group that either 1-phenyl-1-propenyl 6a or 1-phenyl-2-propenyl 6b. Compound 6a spectrum showed presence of doublet signal at 1.59 ppm corresponding to CH3-CH=C and quartet at 6.40 ppm corresponding to CH3-CH=C. On the other hand, spectrum for compound 6b showed presence of doublet signal at 4.89 ppm and two multiplet at 5.12-5.18 and 6.39-6.50 ppm assigned to CH2=CH-CH, CH2=CH-CH and CH2=CH-CH, respectively. Furo[3,2-g]benzopyran-7-ones 7a,b were obtained by two different procedures, Scheme 2. First, the ether layer from the previous extraction was evaporated to achieve the cyclized furobenzopyran-7-ones 7a,b. Second, the rearranged product 6a,b underwent cyclization by refluxing in a mixture of glacial acetic acid and hydrobromic acid. Cyclization mechanism was presumably via cyclopropane intermediate to give dihydrofurobenzopyrone derivatives, Figure 2 [28]. Dihydrofurobenzopyran-7-ones were directly dehydrogenated without further purification by refluxing with DDQ in dry benzene to achieve the target compounds 7a,b. 8-Acetyl-7-hydroxy derivatives 8a,c were obtained according to reported procedure [20]. Reaction of 7-hydroxybenzopyrones 1b,d with benzoyl chloride yielded 7-benzoyloxy derivative which were subjected to Fries rearrangement by fusion with anhydrous aluminium chloride to obtain 8-benzoyl-7-hydroxy derivatives 8b,d, Scheme 3. The rearrangement occurs either to C6 or C8, however, the majority of rearrangements take place to the 8-position as it was stabilized by the pyrone ring [29]. Positive ferric chloride test indicated free phenolic OH group. Refluxing 8-acyl-7-hydroxybenzopyrones 8a-d with different phenacyl bromide derivatives in presence of anhydrous potassium carbonate in dry acetone afforded furo[2,3-h]benzopyran-5-ones 9a-s, Scheme 3. Condensation and cyclization took place in one step reaction. Ferric chloride test gave negative result.



Refluxing 7-hydroxycoumarins 8c,d with ethyl chloroacetate in dry acetone in presence of anhydrous potassium carbonate yielded the required ether derivatives 10a,b, Scheme 3. Use of anhydrous potassium carbonate [30] is preferred than metal alcoholate [31] to keep the benzopyrone nucleus intact. Saponification of esters 10a,b with methanolic potassium hydroxide, followed by acidification gave compounds 11a,b, Scheme 3. Use of methanolic potassium hydroxide [32] decreased reaction time than use of sodium ethoxide in ethanol or sodium hydroxide [33,34].

O O O

CH3

R1

CH3

CH3

Ph

O O

CH3

R1

CH3

OPh

H3C

-2H+

O O

CH3

R1

CH3

OPh

H3C

O

CH3

R

CH3

O O

H2C

O

CH3

R

CH3

HO O

H3C

6a R = C2H5

6b R = CH3

HH

H

7a R = C2H5 b R = CH3

Figure 2: Mechanism of cyclization for open rearranged products 6a,b

Table 1: Preliminary screenining of tested compounds for antimicrobial and photosensitizing activities

Cpd. No. Control Test Cpd. No. Control Test DMF --- --- 8c *6 10

Xanthotoxin 9 12 8d --- --- 2a --- --- 9a --- --- 2b --- --- 9b --- 7 2c --- --- 9c --- --- 2d 7 7 9d --- --- 2e 15 17 9e --- --- 2f --- --- 9f --- --- 2g --- --- 9g --- --- 2h --- --- 9h --- --- 3a --- --- 9i --- --- 3b --- --- 9j --- --- 3c --- --- 9k *6 10 3d 8 8 9l --- --- 3e --- 6 9m --- --- 3f 8 10 9n --- --- 3g --- --- 9o --- 8 3h --- --- 9p --- 6 4 11 11 9q --- ---

5a --- 8 9r --- --- 5b --- --- 9s 7 7 6a --- 8 10a --- --- 6b --- --- 10b --- --- 7a --- --- 11a *6 6 7b 13 13 11b 9 9 8a --- --- 12a 7 10 8b *6 7 12b 8 10

Control (disc contains 0.01 ml of the screened, the reference compound). Test (disc contains 0.01 ml of the screened, the reference compound and time of radiation is 20 min.).

* Non significant antimicrobial agents. Cyclization of benzopyronoxyacetic acid derivatives 11a,b to the corresponding angular furobenzopyrones 12a,b were performed using acetic anhydride in presence of anhydrous sodium acetate, Scheme 3.



All the new synthesized compounds were characterized by spectral and elemental analyses which were in full agreement with the proposed structures. 1.6. Antimicrobial and photosensitizing screening Screening of antimicrobial and photosensitizing activities for all the synthesized benzopyrone and furobenzopyrone analogues (linear and angular) were performed by paper disc diffusion method [21] using xanthotoxin as reference compound. The tested organism used was Bacillus Subtilus. 3.2.1. Antimicrobial activity The Antimicrobial activity was compared with that of xanthotoxin ''diameter of inhibition zone before UV-A radiation '', Table 1. Benzopyrone derivatives 2d,e and 11b showed antimicrobial activity, from which compound 2e showed higher activity even than xanthotoxin. Linear furobenzopyrone analogues 3d,f and 7b exhibited antimicrobial activity. Compound 7b, in particular, had good activity higher than xanthotoxin. Angular furobenzopyrone analogues 4, 9s and 12a,b showed antimicrobial activity. Compounds 4 had reasonable antimicrobial activity better than xanthotoxin. Compounds 8b,c, 11a and 9k had non significant antimicrobial activity. The rest of the screened compounds showed no antimicrobial activity. 3.2.2. Photosensitizing activity The photosensitizing activity was compared with that of xanthotoxin ''diameter of inhibition zone after UV-A radiation '', when the disc contains 0.01 ml of the reference or screened compounds and the time of radiation was 20 min, Table 1. Benzopyrone derivatives 2e, 5a, 6a and 8b,c showed photosensitizing activity. Linear furobenzopyrones 3e,f exhibited photosensitizing activity. Angular furobenzopyrones 9b,k,o,p and 12a,b proved to be active as photosensitizing. The rest of the screened compounds were devoid of photosensitizing activity. Increasing the time of UV-A radiation up to 40 min. instead of 20 min. and using the same concentration of the screened, reference compounds, Table 2, Figure 3. Photosensitizing activity for benzopyrone derivatives 2d,e, 5a, 6a and 8b,c was affected by increasing time of radiation. Compounds 11a,b showed no change in photosensitizing activity. Photosensitizing activity for linear furobenzopyrone analogues 3d-f was directly proportional with time of radiation to UV-A. Otherwise, compound 7b exhibited no photosensitizing activity. Photosensitizing activity for angular furobenzopyrone analogues 9b,k,o,p,s and 12a,b was directly proportional with time of radiation to UV-A. However, compound 4 had no photosensitizing activity.

Table 2: Effect of increasing time of radiation and concentration on photosensitizing activity for the active compounds

Cpd. No. Control Test •Test ••Test

DMF ---- ---- ---- ---- 2d 7 7 15 21 2e 15 17 23 27 3d 8 8 12 18 3e ---- 6 10 16 3f 8 10 17 22 4 11 11 11 14

5a ---- 8 10 12 6a ---- 8 9 10 7b 13 13 13 18 8b 6 7 10 10 8c 6 10 13 15 9b ---- 7 8 12 9k 6 10 11 14 9o ---- 8 10 12 9p ---- 6 11 12 9s 7 7 9 15

11a 6 6 6 9 11b 9 9 9 13 12a 7 10 12 14 12b 8 10 13 17

Xanthotoxin 9 12 15 19 Control (disc contains 0.01 ml of the screened, the reference compound).

Test (disc contains 0.01 ml of the screened, the reference compound and time of radiation is 20 min). •Test (disc contains 0.01 ml of the screened, the reference compound and time of radiation is 40 min.). ••Test (disc contains 0.02 ml of the screened, the reference compound and time of radiation is 20 min.).



Figure 3: The bar diagram showing antimicrobial, photosensitizing activities and effect of increasing time of radiation and concentration on photosensitizing activity of the screened compounds and their comparison to solvent DMF and reference standard xanthotoxin

Increasing the concentration of the reference or screened compounds up to 1 mg/ml instead of 0.5 mg/ml and using the same period of UV-A radiation, 20 min., Table 2, Figure 3, results showed a direct correlation between photosensitizing activity and concentration for most of the compounds. 1.7. QSAR Study In an attempt to correlate the photosensitizing activity with the structure conformation of the synthesized benzopyrone, linear and angular furobenzopyrone derivatives, QSAR study was undertaken. Descriptors of the molecular modeling software, Molecular Operating Environment (MOE version 2008.10.2) were used [22]. The structural descriptors used in the generation of these models include: The dipole moment calculated using the AM1 Hamiltonian (AM1_dipole), Log of the octanol/water partition coefficient (logP), Log of the aqueous solubility (logS), Molecular refractivity (SMR) and Polar surface area (TPSA) as shown in Table 3.

Table 3: The molecular descriptor values of the training set compounds

Cpd. No. Descriptors

AM1_dipole LogP (o/w) LogS SMR TPSA 2d 7.1672 5.2590 -7.0341 10.3666 52.6000 2e 8.0418 4.1597 -6.0444 10.9071 71.0600 3d 3.7779 6.2240 -8.7561 10.2607 39.4400 3e 6.9207 4.7800 -7.5155 10.5898 65.7400 3f 5.8849 5.1690 -7.5053 9.0170 39.4400 5a 5.8238 5.5500 -6.1181 10.0670 35.5300 6a 6.0687 5.4560 -6.2327 9.9846 46.5300 8b 5.6644 4.0710 -5.3444 8.6501 63.6000 9b 2.9581 5.1220 -7.9390 10.4082 56.5100 9k 2.6750 4.6470 -7.4238 9.9466 56.5100 9o 2.5574 6.0110 -9.2158 11.5428 56.5100 9s 5.4731 6.8090 -10.3062 12.3127 56.5100

12a 3.6649 2.9860 -4.7242 6.4854 39.4400 12b 3.7244 4.6180 -6.9921 8.5553 39.4400 13a 7.2331 7.7840 -6.5188 9.9050 52.6000 13b 7.9229 3.6847 -5.5292 10.4453 71.0600 13c 7.2405 4.5270 -6.3584 9.4312 52.6000

The observed photosensitizing activity "expressed in the term of produced inhibition zones after UV-A irradiation for 40 min." together with the predicted activities (Pred. activity) for the training set compounds. The best derived QSAR linear model for the 17 compounds (14 new compounds in addition to 3 published compounds [23], Figure 1) was presented by the following estimated equation: Photosensitizing activity = 12.92794 + 1.34495 x AM1_dipole - 8.60214 x logP (o/w) –1.97840 x logS + 3.80798 x SMR - 0.31966 x TPSA r2 = 0.6153



From the equation, photosensitizing activity was positively correlated with AM1_dipole, SMR and negatively correlated with LogP (o/w), LogS and TPSA. The high coefficient value of logP (o/w), SMR and the comparatively lower value of LogS, AM1_dipole and TPSA suggested that the decrease in partition coefficient and increase in molecular refractivity lead to enhancement of activity. This was in good agreement with the obtained experimental data, where in case of most active compounds 2e and 13b showed slight increase in molecular refractivity value accompanied by decrease in partition coefficient value leading to increase in activity. The observed activities (Obs. activity) together with the predicted activities (Pred. activity) for the tested compounds calculated using multi-linear regression (MLR) are listed in Table 4. All compounds showed very good results with Z-scores not exceed the value of 2.5 indicating excellent predictive ability of the model.

Table 4: The observed photosensitizing activity (Obs. activity) "expressed in the term of produced inhibition zones after UV-A irradiation for 40 min." together with the predicted activity (Pred. activity) for the training set compounds

Cpd. No. Obs. Activity Pred. Activity Residual Z-score

2d 15 13.9069 1.0932 0.4188 2e 23 18.7382 4.2618 1.6327 3d 12 8.2576 3.7424 1.4337 3e 10 15.2975 -5.2575 2.0295 3f 17 12.9560 4.0440 1.5493 5a 10 12.1004 -2.1004 0.8047 6a 9 9.6752 -0.6752 0.2587 8b 10 8.7096 1.2904 0.4943 9b 8 10.1234 -2.1234 0.8135 9k 11 11.0512 -0.0512 0.0196 9o 10 8.7832 1.2168 0.4661 9s 9 10.9296 -1.9295 0.7392

12a 12 13.8643 -1.8643 0.7142 12b 13 12.0126 0.9874 0.3783 13a 12 15.3041 -3.3041 1.2658 13b 22 19.8868 2.1132 0.8095 13c 14 15.4034 -1.4034 0.5377

The Z score method was adopted for the detection of outliers. Z score can be defined as absolute difference between the value of the model and the activity field, divided by the square root of the mean square error of the data set. Any compound which shows a value of Z score higher than 2.5, during generation of a particular QSAR model, is considered as outlier [35]. No outliers were observed in this model as showed in Table 4, indicating that, molecular descriptors used for the training set were good. The observed activity was plotted against their predicted values (calculated by MLR) with a value of r2 found to be 0.6153, Table 4, Figure 3.

Figure 3: Correlation plot of observed and predicted activities of the training set for QSAR model, r2 = 0.6153 Fraction of the Variance (r2): Represent the goodness of fit. The value of r2 may vary between 0 and 1, when multiplied by 100 gives explained variance in biological activity, where 1 means a perfect model explaining 100% of the variance in the data, and 0 means a model without any explanatory power. It has already been suggested that



the only QSAR model having r2 > 0.6 will be considered for validation [36]. Value of r2 for this QSAR model is 0.6153. 3.3.1. Model evaluation: The true predictive power of a QSAR model was determined by comparing the predicted and observed activities of the test set compounds (2 new compounds in addition to 1 published compound [23], Figure 1) that not used in the QSAR model development of training set. The observed activities were plotted against their predicted values, Table 5, Figure 4.

Table 5: The observed photosensitizing activity (Obs. activity) "expressed in the term of produced inhibition zones after UV-A

irradiation for 40 min." together with the predicted activity (Pred. activity) for the test set

Cpd. No. Obs. Activity Pred. Activity Residual 8c 13 12.3304 0.6696 9b 11 10.2702 0.7298 14 14 14.3391 -0.3391

Figure 4: Correlation plot of observed and predicted activities of the test set for QSAR model, r2pred = 0.9690

The predictive ability of the model was expressed by the predictive r2 value (r2

pred.), the value of r2pred is 0.9690, it

was calculated by the following Equation [37]:

where y pred(test) and y test are the respective predicted and observed activities of the test set compounds and ӯ training is the observed mean activity of the training set compounds. 1.8. Docking study DNA topoisomerases are ubiquitous enzymes responsible for controlling the topological state of DNA in cells. They are charged with the task of resolving topological problems which arise during the various processes of DNA including transcription, recombination, replication and chromosome partitioning during cell division. The mechanism of these enzymes involves DNA cleavage and DNA strand passage through the break, followed by religation of the cleaved DNA. From a medical point of view, topoisomerases are important targets for a large variety of antitumor as well as antibacterial compounds [38]. Topoisomerase inhibitors are often divided into, according to which type of enzyme it inhibits, topoisomerase I and II inhibitors. Type I enzymes, which cleave a single strand of DNA during the course of the reaction and type II enzymes, which cleave both strands. In addition, there are two subclasses of type II topoisomerase, type IIA and type IIB [39]. Type IIA topoisomerases include the enzymes DNA gyrase, eukaryotic topoisomerase II (topo II) and bacterial topoisomerase IV (topo IV). Type IIB topoisomerases, comprise a single family member, topoisomerase VI (topo VI). DNA gyrase is essential to the cell due to its unique ability to introduce negative supercoils into DNA and so involved during replication and transcription. Gyrase is present in prokaryotes and some eukaryotes, but not present in humans. This makes gyrase a good target for antibiotics. The enzyme consists of two subunits, A and B. The A



protein is responsible for DNA cleavage and rejoining, whereas the B protein contains the ATP-binding site. There are two classes of antibiotics that inhibit gyrase [24]. The aminocoumarins, including novobiocin, work by competitive inhibition of energy transduction of DNA gyrase by binding to the ATPase active site located on the GyrB subunit. The quinolones, including nalidixic acid and ciprofloxacin, act by interfering with the DNA breaking-rejoining step on the A subunit. The gyrase B subunit contains N-terminal domain which includes the site of ATP hydrolysis and C-terminal domain which interacts with the A subunit and probably DNA. The crystal structure of this N-terminal fragment complexed with novobiocin showed that, residues that make contact with bounded coumarin derivatives in the active site of DNA gyrase are Thr165, Gly146, Arg136, Ile78, Gly77, Arg76, Asp73 and Asn46 amino acids [24,40]. The binding affinity of the ligand was evaluated with energy score (S, Kcal/mol). The compound which revealed the highest binding affinity, minimum dock score, is the one forming the most stable ligand-enzyme complex. Length of the hydrogen bond and arene cation interaction were also used to assess the binding models. The results of docking studies; dock score, involved DNA gyrase active site amino acid interacting ligand moieties and hydrogen bond length for each compound and ligand are listed in Table 6, Figures 6-9.

Table 6: Docking results

Cpd. No.

Energy score S (Kcal/mol) Binding amino acid Interacting function group Hydrogen bond

length Ǻ

Novobiocin -13.6636

Thr165(through water molecule) Gly77(through water molecule) Gly77(through water molecule) Arg76(cation-arene) Arg76(through water molecule) Asp73(through water molecule) Asp73 Asn46 Val43(through water molecule)

CO carbamate CO carbamate CO benzopyrone Benzene of benzopyrone CO amide CO carbamate NH2 carbamate OH pyrane NH2 carbamate

2.02 2.02 1.82

2.04 2.02 1.91 2.05 2.30

2d -9.4339

Thr165(through water molecule) Gly77 Gly77(through water molecule) Arg76(cation-arene) Asp73(through water molecule)

CO acyloxy CO acyloxy CO acyloxy Benzene of benzopyrone CO acyloxy

1.93 2.17 1.93

1.93

2e -12.0553

Thr165 Thr165(through water molecule) Gly77(through water molecule) Arg76(through water molecule) Asp73(through water molecule) Val43(through water molecule)

3′-OCH3

3′-OCH3

3′-OCH3

CO benzopyrone 3′-OCH3

4′-OCH3

3.12 1.64 1.64 2.21 1.64 2.78

3d -10.2129

Thr165 Thr165(through water molecule) Gly77(through water molecule) Asp73(through water molecule)

CO benzopyrone CO benzopyrone CO benzopyrone CO benzopyrone

2.95 1.52 1.52 1.52

3f -9.7639 Val120(through water molecule) Arg76(cation-arene) Asn46(through water molecule)

CO benzopyrone Phenyl at C3 CO benzopyrone

2.16

2.16

4 -10.9973

Thr165 Thr165(through water molecule) Gly77(through water molecule) Asp73(through water molecule)

3′-OCH3

3′-OCH3

3′-OCH3

3′-OCH3

3.24 1.44 1.44 1.44

7b -11.0759

Thr165 Thr165(through water molecule) Gly77(through water molecule) Arg76(cation-arene) Arg76(cation-arene) Asp73(through water molecule)

CO benzopyrone CO benzopyrone CO benzopyrone Benzene of benzopyrone Furan ring CO benzopyrone

2.91 1.44 1.44

1.44

9s -9.4329 Thr165(through water molecule) Gly77(through water molecule) Asp73(through water molecule)

CO benzopyrone CO benzopyrone CO benzopyrone

1.85 1.85 1.85

11b -11.8855

Arg76 Arg76 Arg76(through water molecule) Arg76(cation-arene) Gly50

CO benzoyl O ether linker CO carboxylic acid Benzene of benzopyrone CO benzopyrone

1.88 3.28 3.52

1.90

12a -8.5277 Gly77 CO benzopyrone 1.92

12b -9. 4829 Arg76 Arg76(through water molecule)

CO benzopyrone CO benzopyrone

1.97 2.81



Analysis of the docking results revealed that: i- The novobiocin-DNA gyrase complex was precisely reproduced by the docking procedure as demonstrated by low root mean square deviation, rmsd (0.6204) and dock score (-13.6636 Kcal/mol, Table 6), i.e. the docking protocol was valid. Novobiocin nearly fits in the active site forming various hydrogen bonding interactions with the active site residues: CO carbamate with Thr165, Gly77 and Asp73 (2.02 Å) through water molecule, CO benzopyrone with Gly77 (1.82 Å) through water molecule, CO amide with Arg76 (2.04 Å) through water molecule, NH2 carbamate with Asp73 (1.91 Å) and Val43 (2.30 Å) through water molecule and OH pyrane with Asn46 (2.05 Å). Also novobiocin formed arene cation interaction of benzene of benzopyrone with Arg76, Figure 6. ii-

Figure 6: 2D interactions of Novobiocin on the active site of Topoisomerase II iii- Dock scores for significantly active antimicrobial compounds (2d,e, 11b, 3d,f, 4, 7b, 9s, 12a,b) were found to have dock score in the range (-12.0553 to -8.5277 Kcal/mol). A significant correlation between dock scores and antimicrobial activity (diameters of inhibition zones produced before UV-A radiation) of compounds was observed. Benzopyrone derivatives 2d,e and 11b, which had antimicrobial activity (inhibition zones were 7, 15 and 9 mm, respectively), showed strong binding affinity with the active site of the DNA gyrase enzyme (dock score, -9.4339, -12.0553 and -11.8855 Kcal/mol, respectively). Linear furobenzopyrone derivatives 3d,f and 7b, which have antimicrobial activity (inhibition zones were 8, 8 and 13 mm, respectively), showed strong binding affinity with the active site of the enzyme (dock score, -10.2129, -9.7639 and -11.0759 Kcal/mol, respectively). Angular furobenzopyrone derivatives 4, 9s and 12a,b, which have potential antimicrobial activity (inhibition zones were 11, 7, 7 and 8 mm, respectively), showed strong binding affinity with the active site of the enzyme (dock score, -10.9973, -9.4329, -8.5277 and -9.4829 Kcal/mol, respectively). The highest negative dock score among all tested compounds was estimated for the derivative 2e (with dimethoxy substitution) that exhibited higher antimicrobial activity than xanthotoxin. This result was attributed to envolvement of 3′,4′-dimethoxy groups in hydrogen bonding with amino acids in active site of the enzyme. iv- Inspection of the binding mode also demonstrated that all compounds showed one to six hydrogen bonds and arene cation interaction with the enzyme active site residue. Thr165, Val120, Gly77, Arg76, Asp73, Gly50, Asn46 and Val43 are the amino acid residues involved in this interaction, Table 6, Figures 7-9.

The benzopyrone 2e with low energy score (-12.0553 Kcal/mol), the most active compound mediated six strong hydrogen bonds with Thr165 (3.12 Å), Thr165 through water molecule (1.64 Å), Gly77 through water molecule (1.64 Å) and Asp73 through water molecule (1.64 Å) with 3′-methoxy group, Val43 through water molecule (2.78 Å) with 4′-methoxy group and Arg76 (1.95 Å) and through water molecule (2.21 Å) with CO benzopyrone, Figure 7.



Figure 7: 2D interactions of 2e on the active site of Topoisomerase II The angular furobenzopyrone 4 with energy score (-10.9973Kcal/mol), the most active compound, mediated four strong hydrogen bonds with Thr165 (3.24 Å) and through water molecule (1.44 Å), Gly77 through water molecule (1.44 Å) and Asp73 through water molecule (1.44 Å) with 3′-methoxy group, Figure 8.

Figure 8: 2D interactions of 4 on the active site of Topoisomerase II The linear furobenzopyrone 7b with energy score equal -11.0759 Kcal/mol showed four strong hydrogen bonds with Thr165 (2.91 Å) and through water molecule (1.44 Å), Gly77 through water molecule (1.44 Å) and Asp73 through water molecule (1.44 Å) with CO benzopyrone, in addition, two cation arene were mediated through Arg76 with benzene ring of benzopyrone and furan ring, Figure 9.

Figure 9: 2D interactions of 7b on the active site of Topoisomerase II



CONCLUSION

According to photosensitizing activity of tested compounds, it was apparent from the results that: For benzopyrone derivatives: 4-bromophenacyloxy substituent had strong photosensitizing activity after increasing exposure time (40 min.) for UV-A radiation. In addition, 7-cinnamyloxy derivatives and its rearranged product with ethyl group at C3 had photosensitizing activity, but were completely abolished by replacing ethyl with methyl group. For linear furobenzopyrones: C3 substitution with 4-methylphenyl or 4-methoxyphenyl substituents completely abolished the photosensitizing activity. On the other hand, 3,4-dimethoxyphenyl substituents showed photosensitizing activity after UV-A radiation for 20 min. and increased after exposure for 40 min. Photosensitizing activity of 4-bromophenacyloxy substituent increased by increasing exposure time for UV-A radiation. Moreover, substitution at C2 and C3 with aromatic and alkyl groups, respectively, produced compounds devoid of photosensitizing activity. For angular furobenzopyrones: substitution at C3 with methyl, phenyl and substituted phenyl moieties, respectively, led to descending order of photosensitizing activity. Briefly, photosensitizing activity is time dependant (needed long exposure time for UV-A light to exhibit their activity) and directly proportional with concentration for most compounds. Physicochemical properties are dependant factors for activity as expressed from QSAR study. Concerning the Antimicrobial activity, presence of 3,4-dimethoxyphenyl substituent at C7 of benzopyrone or C3 of angular furobenzopyrone, exhibited good antimicrobial activity. This was attributed to possible hydrogen bonding with amino acid residues in the active site of DNA gyrase enzyme as showed by molecular docking. Acknowledgment Authors are thankful to Mohmoud A.-A. F. Khalil, Department of Microbiology, Faculty of Pharmacy, Misr University for Science and Technology for carrying out antimicrobial and photosensitizing screening.

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Int. J. Pharm. Med. & Bio. Sc. 2014 Dalia H Soliman et al., 2014

SYNTHESIS OF NOVEL STEROIDAL17-TRIAZOLYL DERIVATIVES VIACU(I)-CATALYZED AZIDE-ALKYNE

CYCLOADDITION AND THEIR EVALUATIONAS POTENTIAL PROGESTATIONAL AGENTS

Magda M F Ismail1, Dalia H Soliman1*, Nehad M A Eldydamony2,G A Abdel Jaleel3, Adel F Youssef 2,4

Research Paper

Many progestins have been developed for use in contraception, Hormone Replacement Therapy(HRT), menopausal hormone therapy and treatment of gynecological diseases. Progestins arealso highly efficacious in decreasing the occurrence of endometrial hyperplasia and carcinomacaused by unopposed estrogens. Click reaction was adapted for the condensation of the terminalethynyl group of norethindroneenanthate (NET-EN)3 and levonorgestrel (LNG)5 with substitutedphenyl azides2a-c to prepare 1, 4-disubstituted-1, 2, 3-triazole derivatives 4a-c and 6a-c. Theassigned structures were confirmed by spectroscopic and element microanalytical data.Biological screening of the prepared compounds was undertaken to evaluate possibleprogestational activity on rat uterus in vivo and ex vivo on the isolated rat uterus. Thehistopathologic changes of the uterus associated with the new compounds and the referencedrugs NET-EN and LNG encompass a variety of morphologic features that were interpreted. Allthe prepared compounds conserve their progestational activity, significantly much higher thanvehicle. Compound 4c showed activity parameters higher than NET-EN, while 6b matched thosedisplayed by LNG. Compound 4b displayed 200 fold the uterolytic effect of NET-EN. On the otherhand, LNG derivatives showed uterolytic effect much weaker than the parent drug. Molecularmodeling studies were performed using MOE software (V.10.2010) to further corroborate thebiological assay results acquired for this new group of compounds and gain insight into theirplausible mode of interaction(s) within the progesterone receptor.

Keywords: Click reaction, Steroidal azoles, Progestational activity, Uterolytic effect,Molecular modeling, Copper catalyzed azide-alkyne cycloaddition

*Corresponding Author: Dalia H [email protected]

INTRODUCTIONProgesterone (Prog), the natural agonist of

ISSN 2278 – 5221 www.ijpmbs.comVol. 3, No. 3, July 2014

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Int. J. Pharm. Med. & Bio. Sc. 2014

1 Department of Pharmaceutical chemistry, Faculty of Pharmacy (Girls), Al-Azhar University.2 Department of Medicinal Chemistry, College of Pharmacy and Pharmaceutical Industry, MUST.3 Departmentof Pharmacology, National Research Center.4 Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University.

Progesterone Receptor (PR), is critical for femalereproductive function (Djerassi, 1995). Increased

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Prog levels in plasma are responsible for the lackof ovulation during pregnancy. Effective inhibitoryeffect of Prog is the basis of oral contraceptives,which comprise also synthetic progesteroneanalogues called progestins. Progestins are aclass of synthetic compounds structurally distinct,but functionally similar to Prog, with longerbiological half-lives (Speroff and Darney, 1996).Synthetic steroidal progestins are widely used astherapeutic agents such as contraceptives,combination Hormone Replacement Therapy(HRT), and a variety of other therapeuticapplications such as treatment of gynecologicaldisorders and in cancer therapy (Collins, 1994;Mishell, 1996; De Ziegler and Fanchin, 2000;Schiff, 1982; Lundgren, 1992; Schneider andJackisch, 1998).

However, a number of side-effects have beenreported with the clinical use of progestins suchas their effect on bone density, blood pressure,immune function, neurological effects and evenminor effects as mood swings, weight gain, hotflushes and loss of libido (Goodman andGilman’s, 2006; Mueck and Sitruk, 2011).Recently, considerable interest has been focusedon steroidal azoles; the azole moiety often showssome special biological activity when introducedto biologically active compounds (Levine et al.,2012; Conner et al., 2012; Corrales, 2011; Kadar,2011; Banday, 2010). Although a number oftriazolyl derivatives have been reported, steroidscontaining this kind of structural moiety havereceived less attention from both synthetic andpharmacological aspects.

The aforementioned findings about theimportance of steroidal azoles coupled with thereported enhancement in progestational activityand selectivity by larger chemical moieties

introduced at the 17 position of the steroidbackbone (Wanga et al., 2008), prompted us toplan and synthesize novel steroidal 17-triazolesvia an in situ one-pot “click chemistry” approach(Kolb and Sharpless, 2003; Kolb et al., 2001). Thepremier example of click reaction is Huisgen 1,3-dipolar cycloaddition of azides and terminalalkynes yielding 1,2,3-triazoles (Tornoe et al.,2002). Cu(I)-catalyzed azide-alkyne 1, 3-dipolarcycloaddition (CuAAC) regioselectively produces1,4-disubstituted-1H-1,2,3-triazoles (Rostovtsevet al., 2002). This reaction has found numerousapplications in many aspects of drug discovery(Meldal and Tornoe, 2008; Hein et al., 2008, Bocket al., 2006). Thus, our scope of investigation wasto boost the progestational activity throughenhancement of anticipated physicochemicalbinding forces (HBD, HBA, lipophilicity, PolarSurface Area (PSA) and ionic interaction) and tostudy in silico the binding mode of the newcompounds to the PR. These potentialities werechallenged via attachment ofnorethendroneenanthate and levonorgestrelterminal acetylene to functionalized phenyl moietythrough 1,2,3-triazole ring linker.m-orp-Nitrophenyl group was prepared to probe thestrong electron withdrawing character on thebinding mode of the new steroid–triazole-substituted phenyl molecule to PR. Following thesame objective acetic acid moiety was added assubstituent on the benzene ring to reveal theimpact of added bulkiness and enhancement offlexibility of the molecule on the binding to thereceptor. The presence of the carboxyl affordsthe potential of formation of a water soluble saltwith the suitably selected counter base.

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MATERIALS AND METHODSChemistryGeneral MethodsAll solvents were commercially available. NMRspectra were recorded on a Varian Mercury VX-300 NMR spectrophotometer 300 MHz. DMSO-d6 was used as a solvent, the chemical shift (scale) are reported in (ppm) relative to (TMS) asinternal standard. IR spectra were obtained usingpotassium bromide disc on a Schimadzu 435 IRspectrophotometer. Melting points were obtainedon Griffin apparatus in open capillary tubes andare uncorrected. Column chromatography wasperformed on silica gel (60-120 mesh, E. Merck).The azides employed in the synthesis of the newcompounds were successfully prepared, 2-(4-azidophenyl)acetic acid (2a), Yield (%): 83,m.p.(0C): 90–92; IR (KBr): 2800-3400 (OH), 2119(N3), 1695 (CO-OH) (Chamni, 2011; Settimo,1979); 1-azido-3-nitrobenzene (2b), Yield (%): 65;m.p.(0C): 54–56; IR (KBr): 2125 (N3), 1525,1351(NO2) (Yang, 2011; Appl, 1959); and 1-azido-4-nitrobenzene (2c), Yield (%): 80; m.p.(0C): 71-74; IR (KBr): 2124 (N3), 1525, 1351(NO2) (Smith,1937; Adamo, 1958).

4-[4-(17-Heptanoyloxy-3-oxo-19-norandrost-4-ene-17-yl)-1H-1, 2, 3-triazol-1-yl] phenylacetic acid (4a)Norethindroneenanthate 3 (3 mmol, 1.23 g) and4-azidophenylacetic acid 2a (3 mmol, 0.53 g)were suspended in a 1:1 mixture of water andtert-butyl alcohol (12 mL). Freshly preparedsodium ascorbate (0.7 mmol, 0.14 g) was added,followed by copper (II) sulphate pentahydrate (0.28mmol, 0.07 g). The heterogenous mixture wasstirred 24 h, (monitored by TLC). The reactionmixture was diluted with water (50 mL), cooled inice, the residue was purif ied by column

chromatography over 60-120 silica gel usingEtOAc : hexane : MeOH mixture (4:6:1). A brownsticky mass was obtained after evaporation ofsolvent in 34% yield. IR (KBr):3400 (OH), 1712(CO-OR), 1700 (CO-OH), 1656 (CO), 1HNMR(CDCl3): 7.08(d, 2H, Ph-H2,Ph-H6), 7.2 (d, 2H,Ph-H3,Ph-H5), 7.9 (s, 1H, triazole-H), MS (m/z,%):Calcd. for C35H45N3O5, 587.75, found 587 (M+,1.16), 458 (M+ - C7H13O2, 4.28), 410 (M+ -C8H7N3O2, 1.48), 385 (M+ - C10H8N3O2, 6.66), 256(M+ -C17H21N3O4, 6.02), Anal. (%) :Calcd.: C,71.52; H, 7.72; N, 7.15. Found: C, 71.73; H, 7.89;N, 7.41.

17-[1-(3-Nitrophenyl)-1H-1, 2, 3-triazol-4-yl]-3-oxo-19-norandrost-4-ene-17-ylheptanoate(4b)The titled compound was prepared following theprocedure adapted for 4a synthesis, usingNorethindroneenanthate3 (3 mmol, 1.23 g) and1-azido-3-nitrobenzene 2b (9 mmol, 1.48 g). Theheterogenous mixture was stirred vigorously for96 h, (monitored by TLC). The residue waspurified by column chromatography on silica gelusing hexane. A brown powder was obtained afterevaporation of solvent in 29% yield. M.P. = 66-68oC, IR (KBr) :1830 (CO-OR), 1670 (CO), 1534,1342 (NO2), 1HNMR(CDCl3): 0.7 (t, 3H, CH3-heptanoate), 7.58 (d, 1H, Ph-H6, J = 8.4), 7.65-7.71 (m, 1H, Ph-H5), 7.85 (s, 1H, triazole-H), 8.00(s, 1H, Ph-H2), 8.03 (d, 1H, Ph-H4, J = 8.4), MS(m/z,%): Calcd for C33H42N4O5, 574.7, found 575(M+ 1, 0.01), 385 (M+ - C8H5N4O2, 0.04), 445 (M+

- C7H13O2, 0.02), Anal. (%) :Calcd.: C, 68.97; H,7.37; N, 9.75. Found: C, 69.13; H, 7.40; N, 10.11.

17-[1-(4-Nitrophenyl)-1H-1, 2, 3-triazol-4-yl]-3-oxo-19-norandrost-4-ene-17-ylheptanoate(4c)The titled compound was obtained following theprocedure described under 4a, using

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Norethindroneenanthate 3 (3 mmol, 1.23 g) and1-azido-4-nitrobenzene 2c (9 mmol, 1.48 g). Theresidue was purified by column chromatographyon silica gel using EtOAc: hexane mixture (2:8).A brown sticky mass was obtained afterevaporation of solvent in 58% yield. IR (KBr) :1734(CO-OR), 1666 (CO), 1524, 1344 (NO 2),1HNMR(CDCl3): 0.7 (t, 3H, CH3- heptanoate),5.65 (s, 1H, ethylene), 8.22 (d, 2H, Ph-H2, Ph-H6,J = 9), 8.43 (d, 2H, Ph-H3, Ph-H5, J = 9), 8.82 (s,1H, triazole-H), MS (m/z,%): Calcd. forC33H42N4O5, 574.7, found 576 (M+ 2, 0.02), 411(M+ - C6H4N4O2, 4.63), 256 (M+ - C15H18 N4O4,0.06), Anal. (%) :Calcd.: C, 68.97; H, 7.37; N, 9.75.Found: C, 69.13; H, 7.49, N, 10.14.

4-[4-(18, 19-dinor-18-ethyl-17-OH-3-oxo-androst-4-ene-17-yl)-1H-1, 2, 3-triazolyl]phenyl acetic acid (6a)

Levonorgestrel5 (3 mmol, 0.93 g) and 4-azidobenzoic acid 2a (3 mmol, 0.53 g) weresuspended in a 1:1 mixture of water and tert-butylalcohol (12 mL). Freshly prepared sodiumascorbate (0.7 mmol, 0.14 g) was added, followedby copper (II) sulphate pentahydrate (0.28 mmol,0.07 g). The heterogenous mixture was stirredfor 24 h, (monitored by TLC). The reaction mixturewas diluted with water (50 mL), cooled in ice, theresidue was purified by column chromatographyon silica gel using EtOAc: hexane mixture (7:3)in 27.29 % yield.IR (Kbr):3324 (OH), 1700 (CO-OH), 1654 (CO), 1HNMR(CDCl3): 0.96 (t, 3H,CH2CH3), 1.2 (q, 2H, CH2CH3), 5.65 (s, 1H,ethylene), 7.38 (d, 2H, PhH2, PhH6), 7.78 (d, 2H,PhH3, PhH5), 8.56 (s, 1H, triazole-H), MS (m/z,%):Calcd. for C29H35N3O4, 489.26, found 488 (M- 1,0.99), 312 (M+ - C8H7N3O2, 2.02), Anal. (%):Calcd.: C, 71.14; H, 7.21; N, 8.58. Found: C,

71.39; H, 7.15, N, 8.87.

17-hydroxy-17-[1-(3-Nitrophenyl)-1H-1, 2,3-triazol-4-yl]-18,19-dinor-18-ethylandrost-4-ene-3-one(6b)Levonorgestrel5 (3 mmol, 0.93 g) and 1-azido-3-nitrobenzene 2b (3 mmol, 0.49 g) weresuspended in a 1:1 mixture of water and tert-butylalcohol (12 mL). Freshly prepared sodiumascorbate (0.7 mmol, 0.14 g) was added, followedby copper (II) sulphate pentahydrate (0.28 mmol,0.07 g). The heterogenous mixture was stirredvigorosly for 50 h, (monitored by TLC). Thereaction mixture was diluted with water (50 mL),cooled in ice, the residue was purified by columnchromatography over 60-120 silica gel usingEtOAc: hexane mixture (1:9). Yellow crystals wereobtained after evaporation of solvent in 28.19%yield. M.P. : 120-122 oC, IR (KBr):3345 (OH),1651(CO), 1450, 1369 (NO2), 1HNMR(CDCl3): 0.98 (t, 3 H, CH2CH3), 1.4 (q, 2H, CH2CH3), 5.25(s, 1H, OH), 5.71 (s, 1 H, ethylene), 7.61 (d, 1 H,PhH6), 7.61-7.71(m, 1H, PhH5), 7.69 (s, 1 H,triazole-H), 7.86 (s, 1 H, PhH2), 8.03 (d, 1H,PhH4), MS (m/z,%): Calcd. for C27H32N4O4,476.57, found 476 (M+, 0.99), 287 (M+ - C8H8N3O2,1.41), Analysis (%) : Calcd.: C, 68.05; H, 6.77; N,11.76. Found: C, 68.14; H, 6.83, N, 12.19.

17-hydroxy-17-[1-(4-Nitrophenyl)-1H-1, 2, 3-triazol-4-yl]-18,19-dinor-18-ethylandrost-4-ene-3-one(6c)Levonorgestrel 5 (3 mmol, 0.93 g) and 1-azido-4-nitrobenzene 2c (3 mmol, 0.49 g) were reactedas previously mentioned under the preparationof 6a. The residue was purified by columnchromatography on silica gel using EtOAc:hexane mixture (2:8). An Orange powder wasobtained after evaporation of solvent in 60 % yield.M.P. :115-117 oC, IR (KBr):3350 (OH), 1652(CO),

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1515, 1340 (NO2), 1HNMR(CDCl3): 0.97 (t, 3H,CH2CH3), 1.4 (q, 2H, CH2CH3), 5.30 (s, 1H, OH),5.71 (s, 1H, ethylene), 7.37 (d, 2H, PhH2, PhH6, J= 9 Hz), 8.27 (d, 2H, PhH3, PhH5, J = 9 Hz), 8.4(s, 1H, triazole-H), MS (m/z,%): Calcd. forC27H32N4O4, 476.57, found 477 (M+ 1, 12.31), 164(C6H4N4O2, 12.22), Anal. (%) :Calcd.: C, 68.05;H, 6.77; N, 11.76. Found: C, 68.21; H, 6.72, N,11.94.

In-vivo Progestational Agonist ActivityThe assessment of the progestational of the newlysynthesized compounds were performed at thepharmacology department, National ResearchCentre, Cairo, Egypt.

Animals: Adult female Wistar rats (weighing 120-140 g) were housed 6 per cage at 20-22°C roomtemperature under controlled conditions of light,with free access to rat chow and tap water. Ratsshowing regular estrous cycle length (4–5 days).The phases of estrous cycle were determinedby observing the vaginal smear in the morningaccording to the reported procedure (Cooper etal., 1993) . Only those rats showing at least twoconsecutive 4-day estrous cycles were used.

Drugs and Treatments: Rats received s.c.injections of test compounds daily, for 8 days.Norethindroneenanthate 3 and its synthesizedtriazole derivatives 4a-c were dissolved in DMSOat a concentration of 0.018 mg/animal/day (Pagetand Saranes, 1964). Rats received s.c. injectionsof 1 mL daily. Levonorgestrel 5 and itssynthesized triazole derivatives 4a-c weredissolved in DMSO at a concentration of 0.1 mg/animal/day (Muhn et al., 1995). Rats received s.c.injections of 1 mL daily. For all experiments thetreatment was started when the animals were inestrus phase (Murthy et al., 1997). Initial bodyweight before treatment and final body weight at

the time of sacrifice were recorded. 24 h afterthe final dose, rats were killed, and their uteri werecarefully excised, trimmed of extraneous tissue,blotted filter paper to remove excess fluid,weighed on electronic balance, fixed, and stained.Paraffin sections of fixed uteri were evaluated forendometrial gland. Morphometric measurementswere calculated using LiecaQwin 500 ImageAnalyzer in Pathology Department, NationalResearch Centre. The thickness of endometrium,myometrium and the uterine epithelial cell heightswere measured using an objective lens ofmagnification 10, and eye lens 10 the totalmagnification was 100. Ten fields were chosenin each specimen and the mean values wererecorded.

Ex-vivo Uterine Relaxant EffectAnimals: non-pregnant female Wistar ratsweighing 120-140 g were used in this study. Theanimals were obtained from the animal house;National Research Centre, Giza, Egypt. Standardlaboratory food and water are provided ad libitum.Animal procedures are performed in accordancewith the Ethics Committee of the NationalResearch Centre and followed therecommendations of the National Institute ofHealth’s Guide for Care and Use of LaboratoryAnimals (National Institute of Health, 1985).

The animals in the estrus stage (as detectedby vaginal smears) were sacrif iced. Theabdomens were opened and the two uterine hornsfrom each rat were exposed avoiding stretchingof uterine smooth muscles. One horn from eachrat was freed from its surrounding fat andmesenteric attachments. A myometrial strip oflength about 3 cm was cut longitudinally andtransferred to a petri dish containing Krebssolution (chemical composition pH and temp. gas

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bubbling). The strips were placed in automaticbath organs of 10 mL capacity in accordance withthe reported procedure (Alvarez et al., 1988; Dalyet al., 1981). The Krebs- Henseleit buffer (NaCl –118 mM; KCl – 4.7 mM; CaCl2 – 2.5 mM; MgSO4

– 1.6 mM; NaHCO3 – 24.3 mM; KH2PO4– 1.18mM; glucose 5.6 mM) was used as an incubationenvironment (Coruzzi et al., 1988). The incubationof strips was conducted at the temperature of 37°C, loaded with 1 g and the oxygen and carbondioxide gas mixture (95% O2 and 5% CO2) wasadded so that its pH remained within 7.3-7.5. Thewhole preparation was allowed to equilibrate for30 minutes according to the method described(Calixto et al., 1991)1X10–4M concentration ofacetylcholine (Ach) was added to the bath andcontractile activity was then measured using anisometric force transducer (Grass Model 7EPolygraph, USA). The bath solution was drainedcompletely and washed two to three times andfilled with fresh solution. Isotonic contractions ofthe uterine muscle with different test drugconcentrations were recorded, andconcentration—response curves wereconstructed. The test drug additions werecumulative.

STATISTICAL ANALYSISThe data were expressed as mean± SEM andanalyzed using SPSS statistical software. Oneway analysis of variance (ANOVA) was used toassess the variation of the means among thetreatments. If the variation was greater thanexpected by chance alone, multiple comparisontests was performed to compare each treatmentgroup with the control and standard groups.Significance was established when the p valuewas less than 0.05. PRISM, versions 5.0

(GraphPad, Inc., San Diego, CA), was used fordetermination of IC50 value for inhibition usingnonlinear regression.

Molecular ModelingDocking and molecular modeling studies werecarried out at the Department of MedicinalChemistry, Faculty of Pharmacy, Assiut University,Assiut, Egypt.

Software and HardwareAll the molecular modeling calculations anddocking simulation studies were performed usingMolecular Operating Environment (MOE®, 2010)version 10.2010, Chemical Computing GroupInc., Montreal, Canada. The computationalsoftware operated under “Windows XP” installedon an Intel Pentium IV PC with a 1.6 GHzprocessor and 512 MB memory.

Target Compounds OptimizationThe target compounds were constructed into a3D model using the builder interface of the MOEprogram. The stereo chemical configurations ofthe steroidal nucleus were constrained as innorethindrone. After checking their structures andthe formal charges on atoms by 2D depiction,the following steps were carried out:

• The target compounds were subjected to aconformational search.

• All conformers were subjected to energyminimization, all the minimizations wereperformed with MOE until a RMSD gradient of0.01 Kcal/mole and RMS distance of 0.1 Å withMMFF94X force-field and the partial chargeswere automatically calculated.

• The obtained database was then saved asMDB file to be used in the docking calculations.

Optimization of the Enzymes Active Site

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The three-dimensional structure of progesteronereceptor complexed with norethindrone (PDB Id:1SQN) was obtained from the Protein Data Bankthrough the internet.

Hydrogen atoms were added to the systemwith their standard geometry

• The atoms connection and type were checkedfor any errors with automatic correction.

• Selection of the receptor and its atomspotential were fixed.

• MOE Alpha Site Finder was used for the activesite search in the enzyme structure using alldefault items. Dummy atoms were createdfrom the obtained alpha Spheres.

Docking of the Target Molecules to the PRActive SiteDocking of the conformation database of thetarget compounds was done using MOE-Docksoftware. The following methodology wasgenerally applied:

• The enzyme active site file was loaded andthe Dock tool was initiated. The programspecifications were adjusted to:

• Dummy atoms as the docking site.

• Triangle matcher as the placementmethodology to be used.

• London dG as Scoring methodology to be usedand was adjusted to its default values.

• The MDB file of the ligand to be docked wasloaded and Dock calculations were runautomatically.

• The obtained poses were studied and theposes showed best ligand-receptorinteractions were selected and stored forenergy calculations

RESULTS AND DISCUSSIONChemistryThe development of copper (I)-catalyzed ‘triazoleclick chemistry’ by Sharpless and co-workershas led to synthesis of highly functional moleculesfrom simple building blocks, namely azides andan alkynes which in turn has led to manyinteresting applications (Kolb and Sharpless,2003; Kolb et al., 2001; Tornoe et al., 2002;Rostovtsevet al., 2002).

The attractive characteristics of this reactionare its excellent regiospecificity, reliability, mildconditions, good yields and the biocomptabilityof the generated 1,2,3- triazole nucleus(Rostovtsev et al., 2002). We herein, report thesynthesis of novel steroid-triazoles, -[1-(substitutedphenyl)-1H-1,2,3-triazol-4-yl]-3-oxo-19-nor-androst-4-ene-17-yl heptanoates (4a-c)and 17-hydroxy-17-[1-(substitutedphenyl)-1H-1,2,3-triazol-4-yl]-18,19-dinor-18-ethylandrost-4-ene-3-one (6a-c) outlined in schemes (1). Thetargeted sterod-triazoles 4a-c and 6a-c wereprepared according to the reported procedure(Rostovtsevet al., 2002), where norethindroneenanthate (NET-EN) 3 or Levonorgestrel (LNG)5 andthe aryl azides 2a-c were reacted at ambienttemperature in the presence of CuSO4 as a pre-catalyst and sodium ascorbate using t-BuOH/H2Oas a solvent. The reaction time ranged from 24-96 h was monitored by TLC.

It is worthy to comment on the noticed-difference in the reaction time and yields of thesteroid-triazoles synthesized by the “clickreaction” between them- and p-nitrophenylazides2b and 2c with NET-EN3 and LNG 5 (scheme 1).Table 1 reveals the evidence that 1-(m-

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2a-c

(i)

O

H

H H

O

H

R1

3, 5

O

H

H H

O

H

R1

N

NN

R

(ii)

Reagents and conditions:(i) NaNO2,HCl,NaN3,0oC, (ii); CuSO4.5H2O, ascorbicacid or sodium ascorbate,H2O/t-Bu OH 1:1, rt, 24-96 hrs.as specified in experiment

Scheme (1)

a; R =b; R =c; R =

p-CH2COOHm-NO2p-NO2

R

N3

R

NH2

1a-c

R2

R2

4a-c , 6a-c

3, 4: R1=COC6H13; R2=H5, 6: R1= H; R2=CH3

Scheme 1: Reagents and Conditions (i) NaNO2, HCl, NaN3, 0oC (ii) CuSO4, 5H2O, AscorbicAcid or Sodium Ascorbate, H2Ot-Bu OH 1:1 rt, 24-96 hrs. as Specified in Experiment

Entry NO2(x Value)(Hansch and Taft, 1991) Reaction Time (hr) Yield(%)

4 b 0.71(x=m) 96 29

6 b 0.71(x=m) 50 28

4 c 0.78 (x=p) 24 58

6 c 0.78 (x=p) 24 60

Table 1: Effect of the Position Of The Nitro Group In The Nitro- Phenyl Azideson The Yield and Time of Click Reaction with NET-EN and LNG

Figure 1(a): p-nitrophenylazide2csatbilizes the negative charge on N1,(b) m-nitrophenylazide2bfacilitates electron delocalization over the azide group

N-

N+

O-O

N+N

N-

N

N- N

N N-O

O

-O

O-

-O

O-

N+N N+N N+-N

(a)

(b)

N

NOO

NN + -

--

N-

NO--O

N+N

+

+

+

+

+++ + +

6 6


Int. J . Pharm. Med. & Bio. Sc. 2014 Dalia H Soliman et al., 2014

nitrophenyl)-1H-1,2,3-triazole derivatives 4b and6b needed more than double the time to givepractically half the percentage yield of 1-(p-nitrophenyl)-1H-1,2,3-triazole analogues 4c and6c.

The evident differences may be attributed tothe strong electron-withdrawing character of theNO2 group expressed by its m- and p- valuesTable 1. Proximity of the positive charge in theresonating structure of p-nitrophenylazide 2cFigure (1a) might stabilize the negative charge ofthe directly attached nucleophilic nitrogen N1 ofthe azide group rendering it more localized andavailable for coordination with Cu(I)-acetylidein therate limiting step in the mechanism of CuAAC(Fomin et al., 1999). On the other hand the m-nitro resonating structures 2b Figure (1-b) arelacking intimacy between opposite charges thatallows more facile delocalization of the negativecharge over the N1 and hence, impedes smoothcoordination with Cu(I) acetylide.

On the other side, 4b took longer time oncarrying the “click reaction” than 6b (96 h, 50 h,respectively), that can be attributed to the bulkyeffect of the enanthate ester moiety geminal tothe alkyne center hindring the attack by the azidegroup. It was also observed that the yield of 4cand 6c increased five folds on carrying thereaction under N2 using freshly prepared sodiumascorbate.

The IR spectra of 4a-c revealed 4-ene-3-onecarbonyl stretchings in the range of 1656-1670cm–1, and the ester carbonyls at 1712-1830 cm–

1 in addition of a third carbonyl stretching band at1700 cm-1 assigned to the free carboxyl in 4a.Two peaks at 1524, 1344 cm-1 attributed to NO2

group were shown by 4b,c wile 4a showed abroad band at 3400 cm -1 corresponding to

carboxylic OH. 1HNMR spectra of 4a-c revealeda singlet at 7.9, 7.85 and 8.82 ppm attributed tothe triazole 5-H proton. Compound 4a showedthe benzene protons as two doublets one at 7.2ppm (J=8.7) assigned to (3-H, 5-H) and a secondat 7.08 ppm (J=8.9) assigned to (2-H, 6- H)protons. Compound 4c benzene protons showedthe same pattern as 4a with more downfieldshifted doublets of (3-H, 5-H) at 8.43 ppm (J=7.9)and (2-H, 6-H) at 8.22 ppm (J= 7.9). The m- NO2

derivative 4b showed the benzene2-H proton assinglet at 8.0 ppm, 4-H as doublet at 8.03 (J=8.1),5-H as multiplet at 7.65-7.7 ppm and 6-H asdoublet at 7.58 ppm (J=9.3). Members of theseries 4a-c showed the pattern concerning thesteroid nucleus; ring A, - methyne CO-CHproton at 5.65 ppm, a triplet integrated by twoprotons assigned for the -methylenic CH2-COat 2.7, 2.49 and 3.0 ppm. A triplet in the range of0.9-0.7 ppm assigned to the CH3 of theheptanoate radical and a broad multiplet thatoccupy the range 0.8-2.45 integrated by 31protons assigned collectively to twelve CH2

groups, four CH groups and 19-CH3 group.Compound 4a showed an additional signal at 3.4ppm integrated by two protons assigned to themethylenic CH2COOH.

The IR spectra of 6a-c revealed a commonpattern: strong stretching band at 1651-1654 cm-

1 attributed to 4-ene-3-one carbonyls and a broadband in the range 3324- 3350 cm-1 assigned to17-OH stretching. In addition to the assignedabsorption bands compounds 6b,c showed thetwo prominent peaks of NO2 group at 1517, 1344cm-1 while 6a showed a carbonyl stretching bandat 1700 cm-1 assigned to the COOH group.1HNMR spectra of 6a-crevealed a singlet at 8.56,7.69 and 8.4 ppm attributed to the triazole 5-Hproton. Compound 6a showed the benzene p-

67



substituted pair of doublets one assigned to (3-Hand 5-H) protons at 7.78 ppm (J= 8.9), and asecond assigned to (2-H and 6-H) protons at 7.38pm (J=8.7). Compound 6-c showed the samearomatic pattern as 6a with more downfield shifteddoublet of (3H- and 5-H) protons at 8.27 ppm(J=9) and (2-H and 6-H) doublet at 7.37 ppm(J=9.3). The m- NO2 derivative 6b showed thebenzene 6-H as doublet at 7.61 ppm (J=8.1), 5-Has multiplet at 7.61-7.71 ppm, 2-H as singlet at7.86 ppm and 4-H as doublet at 8.03 ppm (J =8.4).Members of the 6a-c series showed the patternconcerning the steroid nucleus; ring A, a singletat 5.65, 5.71or 5.71 ppm assigned to the -methyne CO-CH proton, triplet at 2.8, 2.6 and2.65 ppm assigned to methylenic - CH2-CO inthe three derivatives. Methylenic and methylprotons of C13-Et protons appeared as quartetat 1.19-1.20 ppm and triplet at 0.88-0.98 ppm,respectively. A multiplet integrated by 18 protonsin the range 1.20 - 2.59 ppm was collectivelyassigned to seven CH2 and four CH of the steroidnucleus. Compound 6a showed a singlet at 3.48ppm assigned to the methylenic CH2-COOHintegrated by two protons. All members of 4a-cand 6a-c series showed molecular ion peaks andelemental analyses complying with the calculatedvalues of the target structures.

PROGESTATIONAL ACTIVITYIn-vivo Progestationalagonist ActivityThe progestational activity of the newlysynthesized compounds and the parent steroidswas assessed on rats by evaluation of the effecton body and uterine weights, histopathologicchanges of the endometrium, myometrium andepithelial cell height illustrated in Table 2. In allrats, there was an observed increase in the fivemeasured parameters compared to the control

group. The most pronounced effect was elicitedby norethindrone enanthate derivative 4c whichshowed a significant increase ranging from 1.3up to 2.3 times in all parameters compared tothe control. In case of LNG derivatives, the bestresult was elicited by 6b which showed asignificant increase in all the five parameterscompared to control rats ranging from 1.3 up to 3times. Most of the values of LNG derivatives werematching with those shown by Levonorgestrelitself. No signs of toxicity were observed for allexperimental groups following treatment with alltest compounds during the experiment.

The histopathological sections obtained fromthe treated uteri showed that the weight gain ofthe uterus was significantly attributed to anincrease in both endometrial and myometrialwidths. An impressive increase in epithelial cellheight was also observed for all thesecompounds, Table 2. Endometrial uterine cross-sections of the rats exposed to the most effectivenorethindroneenanthate analogue 4c representedby Figure (2b) demonstrated an evident increaseof the uterus diameter and lumen with healthymyometrium and elongation of uterus epithelium,compared to the control groups Figure 2a. As forthe Levonorgestrel analogues, compound 6b wasthe most effective of this series, it showed wideendometrium and an increase in epithelial glandsas shown in Figure 2c.

Ex-vivo Uterine Relaxant Effect:It is well known that compounds possessingprogestational activity should suppress themyometrial rhythmic contractions (Fomin et al.,1999; Papka et al., 1999; Dutta and Sanyal, 1969).Therefore, an in vitro study was carried out toevaluate the uterolytic effect of the newlysynthesized steroidal derivatives. Norethindrone

68



Table 2: Changes Induced By The Prepared Derivatives on Body Weight and Uterus of Ratsa

Entry Daily Dose %Body Relative Uterus Endometrial Myometrial Epithelial Cell(mg/day) Weight Weight (kg) Width (µm) Width (µm) Height (µm)

Vehicle 20%DMSO 22±2 1.5±0.03 151±6 121±3 58±1

3(NET-EN) 0.018 28±1 1.7±0.02 300±2* 225±7* 67±1*

4a 0.018 36±2 1.75±0.10 316±1.5* 239±4 * 71±2*

4b 0.018 34±2 1.73±0.02 310±4* 229±1* 69±0.8*

4c 0.018 49±2* b 2.2±0.08 * 347±24* 241±3* 78±1* b

5(LNG) 0.100 49±4* 2.0±0.10* 402±5* 264±7* 73±1*

6a 0.100 36±3 1.8±0.10 341±28* 251±8* 71±2*

6b 0.100 50±4* 2.1±0.20* 453±45* 325±21*c 76±1*

6c 0.100 41±3* 1.9± 0.04 392±14* 256±3* 72±1

Note: **Significantly different from control group at P < 0.05.

Figure 2(a): A Light Micrograph Of The Uterus Of Control Rat Showing Normal Architecture(B)A Light Micrograph Of The Uterus Of Rat Treated With

4c. (C ) A Light Micrograph Of The Uterus Of Rat Treated With 6b

6 9


Int. J . Pharm. Med. & Bio. Sc. 2014 Dalia H Soliman et al., 2014

enanthate derivatives induced marked decreasein uterine contraction, that was proportional to theirincreased concentrations. The IC50 values of thetested compounds on Ach-induced contractionswere shown in Table 3. Norethindrone enanthatederivative 4b (IC50=11 µMol) showed the mostpotent relaxant effect at concentration 200 timesless than norethindrone enanthate (IC50 = 2.3mMol). While, Levonorgestrel derivativespossessed relaxant activity less than that

observed for Levonorgestrel. It can be concludedthat the prepared norethindrone enanthatederivatives possess potent progestational activityin addition to an evident uterolytic effect whichmay be of value in certain cases of threateningabortion.

MOLECULAR MODELINGMolecular Operating Environment (MOE®, 2010)version 10.2010 as a flexible docking program

Table 3: Potency of Uterine Relaxation (IC50) Values of the Tested Compounds

O

H

H H

O

H

R2R1

R3

70



enables us to predict favorable protein-ligandcomplex structures with reasonable accuracy andspeed. Norethindrone, levonorgestrel and theprepared ligands 4a-c and 6a-c were docked intoprogesterone (PDB code: 1SQN) active siteusing X-ray crystal structure data for Prog tofurther corroborate the biological assays resultsacquired for the new group of compounds andgain insights into their plausible mode of

interaction(s) within the PR. Insights into the PRbinding site, revealed that the Ligand BindingDomain (LBD) contains 11-helices (H1, H3-H12)and two short -sheets, organized into threelayers. Within the ligand binding cavity, the Gln725and the Arg766 residues anchor the C3 ketonefunction via hydrogen bonds and make contactwith water molecule. Most of the remaining PR-ligand interactions were hydrophobic as steroids

Figure 3: Netdocked Into The Active Site Of Pr; (a) And (b) Are2 Dand 3d Ligand-receptor Interactions (Hydrogen Bonds Are Illustratedas Dotted Purple Lines; O Atoms Are Colored Red, N Blue and C Gray)

71



Table 4: Ligand/ PR interaction groups, CLogPand PSA calculated values of the docked molecules

are completely buried into a mainly hydrophobicLigand Binding Proteins (LBP) inside globularprotein structure (Alvarez et al., 2011; Petit-Topinet al., 2009; Wanga et al., 2008).

The target compounds were constructed intoa 3D model using the builder interface of the MOEprogram. The stereo chemical configurations of

the steroidal nucleus in the ligands wereconstrained as in norethindrone (NET). Afterrunning the conformational search all conformerswere subjected to energy minimization by MOEuntil a RMSD gradient of 0.01 Kcal/mole and RMSdistance of 0.1 Å with MMFF94X force-field andthe partial charges were automatically calculated.

72



The PR receptor was prepared for dockingstudies by adding hydrogen atoms to the systemwith their standard geometry and checked for anyerrors with automatic correction then selectionof the receptor and its atoms potential were fixed.MOE Alpha Site Finder was used for the active

site search in the enzyme structure using alldefault items. Dummy atoms were created fromthe obtained alpha spheres. Docking of the targetmolecules to the PR active site was then followedand LondondG scoring methodology wasadjusted to its default values and Dock

Figure 4: 2D Representation of Docking of Compound:(a) 4a and (b) 6a into the PR Active Site

73



Figure 5: 2D Representation of Docking of Compound:(a) 4b and (b) 6b into the PR Active Site

calculations were run automatically. Posesshowed best ligand-receptor interactions wereselected and stored for energy calculations.

Examination of the bindings between thesynthesized ligands and the receptor revealedthat the active site amino acids are Leu-715, Cys-

74



891, Thr-894, Tyr-890, Leu-797, Leu -887, Met-756, Met-801, Met-759, Phe-778, Arg-766, Gln-725, Leu-721, Leu-718, Trp-755, Asn-719, Met-909 and Phe-905. As observed in other PR/steroidcomplexes, a hydrogen bond was observedbetween oxygen atom of C3 carbonyl of the

steroid A-ring of norethindrone and the side chainof Gln725 and Arg766. Phe778 made van derWaals contact with the steroid A-ring while Asn719and Try890 with Ring D substituents. Most of theremaining PR-ligand interactions were

Figure 6: 2D Representation of Docking of Compound:(a) 4c and (b) 6c into the PR Active Site

75



hydrophobic, but some polar interactions involvingthe D-ring may be responsible for molecularrecognition and increased affinity, Figure 3.

The intermolecular docking interactions of theparent drugs 3 and 5 and the synthesizedderivatives 4a-c and 6a-c are listed in Table 4.

In our work, we are interested to explore theeffects of the planned 17-molecular extensiontactic, as well as the electronic effect on thearomatic nucleus on the binding interactions withPR. All derivatives showed enhanced hydrophobicand van der Waals interactions binding mode toPR relative to the reference hormones. In caseof compounds 4a and 6a, the introduction ofCOOH group flipped the orientation inside theactive site so COOH is oriented towards Arg766and Gln 725 Figure 4. On the other handm- andp- NO2 substituted compounds 4b, 4c, 6b and6C showed equal placement and orientation inthe active site as the references 3 and 5, howeverdifferently affected dG values Figures 5 and 6.

CONCLUSIONOur docking scores of dG are directly correlatedwith the measured in vivo and ex vivoprogestational activity parameters. Under ourexperimental conditions, the synthesizedcompounds displayed on the µM dose level eithercomparative or more potent progestational actionthan the reference drugs. Compound 4b wasdistinguished by the displayed uterine relaxationpotential at much lower IC50 relative to thereference. Revealed progestational activityenhancement can be correlated with the visualbinding hydrophobic and van der Waals modesof interactions of the prepared compounds at theactive site of PR or due to increased lipohilicityindicated by CLog P values. These modificationsare expected to modify in some way the ADMET

requirements which needs more detailedinvestigations. The two compounds 4a and 6aoffer steroidal derivatives carrying a free aromaticcarboxyl group pave the way to explore thepreparation of water soluble salt with a suitablebase. This study may provide some insights intothe development of novel potent steroid-17a-functionalized progestational lead molecules.

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List of Abbreviations

Acetylcholine =Ach

ADMET = Absorption, Distribution,Metabolism, Elimination, Toxicity

Arg = Arginine

Asn = Asparagine

BW = Body weight

CUAAC = Copper-catalyzed azide-alkynecycloaddition

DBD = DNA-binding domain

Gln = Glutamine

Gly = Glycine

His = Histidine

HRT= Hormone Replacement Therapy

LBD= Ligand Binding Domain

LBP = Ligand Binding Pocket

APPENDIX.: SUPPLEMENTARY DATA

Supplementary data associated with this article can be found in the online version.at

Leu=Leucine

LNG = Levonorgestrel

Lys = Lysine

Met = Methionine

MOE = ®Molecular Operating Environment

NET = Norethindrone

NET-EN = Norethindrone enanthate

PDB = Protein Data Bank

PR= Progesterone Receptor

Prog = Progesterone

PSA = Polar surface area

QSAR = Quantitative structure–activityrelationship

RMS = Root mean square

SAR = Structure–activity relationship

Digest Journal of Nanomaterials and Biostructures Vol. 7, No. 2, April - June 2012, p. 537 - 553

DOPAMINE D2 RECEPTOR ANTAGONIST ACTIVITY AND MOLECULAR

MODELING OF CERTAIN NEW CYCLOHEXANE DERIVED

ARYLCARBOXAMIDES STRUCTURALLY RELATED TO

METOCLOPRAMIDE

MOHAMED N. ABOUL-ENEINa*, AIDA A. EL-AZZOUNYa, MOHAMED I. ATTIAa,b, YOUSREYA A. MAKLADc, MOHAMED ABD EL-HAMID ISMAILd, NASSER M.S. ISMAILd, WALAA H.A. ABDEL-HAMIDe

aMedicinal and Pharmaceutical Chemistry Department (Medicinal Chemistry group), Pharmaceutical and Drug Industries Research Division, National Research Centre, 12622, Dokki, Giza, Egypt bDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud

University, Riyadh 11451, Saudi ArabiacMedicinal and Pharmaceutical Chemistry Department (Pharmacology group), Pharmaceutical and Drug Industries Research Division, National Research Centre, 12622, Dokki, Giza, EgyptdPharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, EgypteDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr University for Science & Technology, 6th of October City, Egypt

A series of certain new N-{[1-(4-aralkyl/ethylpiperazine-1-yl)cyclohexyl]methyl}arylcarboxamides 1a-t structurally related to the antiemetic Metoclopramide (I) was synthesized starting from cyclohexanone, N-aralkyl and/or ethylpiperazine, and KCN in the presence of conc. HCl to furnish the carbonitrile derivatives 3a-d. Subsequent reduction of 3a-d produced the respective amines 4a-d

which were elaborated to the desired arylcarboxamides 1a-t through amide coupling reactions. The target compounds 1a-t were evaluated for their dopamine D2 receptor antagonistic activity in vivo by measuring their ability to inhibit apomorphine-induced chewing “Zwansgnagen” in rats. Compound 1h (ED50 = 5.94 �mol/kg) is the most active congener being nearly 2-fold more potent than the previously reported cyclohexane-based dopamine D2 receptor antagonist II (ED50 = 11.66 �mol/kg). Molecular simulation study including fitting to dopamine D2 receptor antagonists 3D-pharmacophore model using Discovery Studio 2.5 programs showed high-fit values. The experimental dopamine D2

receptor antagonistic activity of compounds 1a-t was consistent with the molecular modeling study.

(Received February 6, 2012; Accepted April 7, 2012)

Keywords: Metoclopramide analogues; Arylcarboxamides; 1,1-Disubstituted cyclohexanes; Antiemitcs; D2-Receptor antagonists.

��

Metoclopramide (I), the parent arylcarboxamide in the orthopramides family, is clinically used as a gastroprokinetic agent (stimulant of upper gastrointestinal motility) as well as an antiemetic.1 This gastroprokinetic activity is ascribed to the release of acetylcholine upon stimulation of 5-HT4 receptors whereas the antiemetic activity is attributed to the antagonistic

*Corresponding author: [email protected]

538

activity at both 5-HT3 serotoninergic and D2 dopaminergic receptors in the chemoreceptor trigger zone (CTZ) in the central nervous system (CNS). Orthopramides possess three common structural elements required for binding to the receptor site: an aromatic moiety, carbonyl function or its bioisosteric group, and a basic nitrogen atom.2-4 Amongst benzamide derivatives, the cyclohexane derivative II was originally reported as Metoclopramide analogue.5 The weak affinity and lack of selectivity of Metoclopramide for dopaminergic and serotoninergic receptors can be explained by the large number of permissible conformers due to the flexibility of its amino chain.6 Accordingly, the intense interest for studying certain molecular modifications of Metoclopramide implies; change in the substituents of the aromatic ring, structural variations in the amine moiety to obtain a conformationally restricted amino side chain, and increasing the lipophilicity via inclusion of the vicinal carbon atom of the basic nitrogen atom into a cyclohexane ring. This concept will be addressed through the synthesis and biological evaluation of new cyclohexane derivedarylcarboxamides 1a-t as potential dopamine D2 receptor antagonists structurally related to Metoclopramide (I).

Fig. 1. Structures of Metoclopramide (I), compound II, and target compounds 1a-t.

��

2.1. Chemistry

All melting points were determined using Electrothermal Capillary melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded as thin film (for oils) in NaCl discs or as KBr pellets (for solids) with JASCO FT/IR-6100 spectrometer and values are represented in cm–1. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were carried out on Jeol ECA 500 MHz spectrometer using TMS as internal standard and chemical shift values were recorded in ppm on � scale. The 1H NMR data were represented as follows: chemical shifts, multiplicity (s. singlet, d. doublet, dd. doublet of doublet, t. triplet, m. multiplet, br. broad), number of protons, and type of protons. The 13C NMR data were represented as chemical shifts and type of carbons. Mass spectral data were obtained with electron impact (EI) ionization technique at 70 eV and chemical ionization (CI/CH4) from a Finnigan Mat SSQ-7000 Spectrometer. Elemental analyses were carried out in Microanalytical Unit, National Research Centre and Cairo University. Silica gel TLC (thin layer chromatography) cards from Merck (silica gel precoated aluminum cards with fluorescent indicator at 254 nm) were used for thin layer chromatography. Visualization was performed by illumination with UV light source (254 nm). Column chromatography was carried out on silica gel 60 (0.063-0.200 mm) obtained from Merck.

2.1.1. General procedure for the synthesis of N-aralkylpiperazines 2b-d

Piperazine dihydrochloride monohydrate (3.54 g, 20.0 mmol) was added to a stirred warmed (65°C) solution of piperazine hexahydrate (3.89 g, 20.0 mmol) in absolute ethanol (9.87 mL). Appropriate aralkyl chloride (20.0 mmol) was added to the reaction mixture dropwise during 5 min with vigorous stirring. Separation of white needles was observed immediately and stirring was further continued for 25 min at 65 °C, then cooling at 0 °C for 30 min. The precipitated piperzine dihydrochloride was filtered off and washed with cold absolute ethanol (10 mL). The combined filtrate and washings were dried (Na2SO4), filtered and evaporated under reduced pressure to afford the respective piperazine derivatives 2b-d as monohydrochloride salts in 66-95% yields.

O

NH

N

NR

NNH

H2N

O

C2H5

C2H5

OMe

Cl

H2N

O

OMe

NH

NC2H5

C2H5

Cl

1a-tMetoclopramide (I)

R3

R2

R1

II

R4

539

2.1.1.1. 1-Benzylpiperazine (2b)7

White solid, m.p. 164 oC of monohydrochloride salt, yield 4.1g (95%). 2.1.1.2. 1-(3,4,5-Trimethoxybenzyl)piperazine (2c)

Buff solid, m.p. 230 oC of dihydrochloride salt (Lit.8 210-211), yield 4.8 g (80%). 2.1.1.3. 1-Benzhydrylpiperazine (2d)

Yellowish white solid, m.p. 70-72 oC of base (Lit. 9 70-72), yield 3.8 g (66%). ��General procedure for the Synthesis of [1-(4-aralkyl/ethylpiperazin-1-yl)cyclo-

hexyl]acetonitriles 3a-d

Ethylpiperazine 2a and/or the appropriate N-aralkylpiperazine 2b-d as monohydrochloride (100 mmol) were mixed carefully with conc. HCl (0.96 mL, 26.0 mmol) and pH of the reaction mixture was adjusted to 3-4. Cyclohexanone (0.81 mL, 100.0 mmol) was added to the resulting solution followed by addition of potassium cyanide (0.65 g, 100.0 mmol) in H2O (1.72 mL). The reaction mixture was stirred for 2 h at room temperature then was allowed to stand overnight. The reaction mixture was basified (10% NaOH) and the formed precipitate was filtered off and washed with water (10 mL) to afford the corresponding carbonitrile derivatives 3a-d in 50-82% yields. The crude 3a-d were used in the next step without further purification. Analytical samples of 3a, 3c, and 3d were obtained after recrystallisation from isopropanol.

2.1.2.1. [1-(4-Ethylpiperazin-1-yl)cyclohexyl]acetonitrile (3a)

Yellow solid, m.p. 76-78 oC, yield 5.4 g (50%). IR (KBr, cm-1) exhibited bands at 2217 (CN), 3742, 643. 1H NMR (CDCl3) �: 1.08-1.84 (m, 15H, 5 x CH2) cyclohexyl, CH2-CH3, CH2-CH3), 2.09-2.86 (m, 8H, (4 x CH2) piperazine). 13C NMR (CDCl3) �: 11.8 (CH3), 21.9, 24.7, 33.6, 46.2, 51.8, 52.7, 60.5 (6 x CH2), Cq), 116.6 (CN). MS (EI) m/z (%): 221.2 (13.7, M+), 114 (25), 109.1 (10.3), 71 (100). Anal. Calcd. for C14H25N3: C, 71.44; H, 10.71; N, 17.85. Found: C, 71.64; H, 10.53; N, 17.75.

2.1.2.2. [1-(4-Benzylpiperazin-1-yl)cyclohexyl]acetonitrile (3b)10

White solid, m.p. 94 oC, yield 9.2 g (81.4%). 2.1.2.3. {1-[4-(3,4,5-Trimethoxybenzyl)piperazin-1-yl]cyclohexyl}acetonitrile (3c)

Yellowish white solid, m.p. 95 oC, yield 11 g (75%). IR (KBr, cm-1) exhibited bands at 2221(CN), 2931, 2826, 1004.1H NMR (CDCl3) �: 1.49-1.73 (m, 10H, (5 x CH2) cyclohexyl), 2.46-2.66 (m, 8H, (4 x CH2) piperazine), 3.50 (s, 2H, CH2-C6H5), 3.81 (s, 3H, OCH3) , 3.82 (s, 6H, 2 OCH3), 6.54 (s, 2H, Har.).

13C NMR (CDCl3) �: 22.0, 22.8, 24.9, 33.9, 46.6, 46.7, 60.8 (6 x CH2), Cq), 53.2, 56.1 (OCH3), 105.7 (CHar.) 122.6 (CN), 129.2, 135.9, 153.1 (Car.). MS (EI) m/z (%): 373 (8.9, M+), 265 (21), 182.2 (38), 181 (100). Anal. Calcd. for C22H33N3O3: C, 68.19; H, 8.58; N, 10.84. Found: C, 67.94; H, 8.82; N, 10.66.

2.1.2.4. [1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]acetonitrile (3d)

Yellow solid, m.p. 134-136 oC, yield 11.7 g (82%). IR (KBr, cm-1) exhibited bands at 2096 (CN), 3425, 1443, 703. 1H NMR (CDCl3) �: 1.62-2.14 (m, 10H, (5 x CH2) cyclohexyl), 2.25-2.33 (m, 8H, (4 x CH2), piperazine), 4.20 (s, 1H, CH), 7.23-7.42 (m, 10H, Har.).

13C NMR (CDCl3) �: 22.2, 25.0, 27.0, 46.9, 52.0, 53.4 (5 x CH2), Cq), 126.9 (CN), 128.0, 128.09, 128.5 (CHar.), 142.8 (Car.). MS (EI) m/z (%): 354 (0.2, M+), 167 (100), 152 (26.5). Anal. Calcd. for C25H31N3: C, 80.39; H, 8.37; N, 11.25. Found: C, 80.22; H, 8.64; N, 11.46.

�� General procedure for the synthesis of N-{[1-(4-aralkyl/ethylpiperazin-1-

yl)cyclohexyl]- methyl}arylcarboxamides 1a-l A solution of anhydrous aluminum chloride (2.1 g, 16.0 mmol) in dry THF (5 mL) was

added dropwise to a stirred suspension of LiAlH4 (1.9 g, 49.0 mmol) in dry THF (100 mL) at 0 °C. A solution of the appropriate carbonitrile 3a-d (11.0 mmol) in dry THF (15 mL) was added dropwise to the cooled (0 °C) reaction mixture and stirring was continued for 24 h at room temperature. The reaction was quenched by a slow addition of saturated sodium sulfate solution at 0-5 °C. The formed precipitate was filtered off and washed with THF (10 mL) and ethyl acetate (25 mL). The combined filtrate and washings were dried (Na2SO4), filtered and evaporated under reduced pressure to afford 1-[1-(4-aralkyl/ethylpiperazin-1-yl)cyclohexyl] methanamines 4a-d in 75-85% yields as pale yellow viscous oils which were solidified upon storage. The crude 4a-d

were pure enough to be used in the next step without further purification. A solution of the appropriate acyl chloride 5a-c (4.86 mmol) in benzene (20 mL) was added dropwise to a stirred solution of 4a-d (4.42 mmol) and triethylamine (0.04 mL) in benzene (60 mL). The reaction

540

mixture was refluxed for 5 h, cooled to room temperature, the formed precipitate was filtered off and washed with benzene (10 mL). The combined filtrate and washings were dried (Na2SO4), filtered and evaporated under reduced pressure to afford the respective arylcarboxamides 1a-l as brown viscous oils. The obtained amides were purified through their dihydrochloride salts which were recrystallised from isopropanol to furnish pure 1a-l.

2.1.3.1. 1-[1-(4-Ethylpiperazin-1-yl)cyclohexyl]methanamine (4a)

Pale yellow viscous oil, yield 2.1 g (87.5%). IR (KBr, cm-1) exhibited bands at 3745, 3677 (NH2), 1458, 612.1H NMR (DMSO-d6) �: 1.18 (t, 3H, J = 6.9, CH3), 1.21-1.58 (m, 10H, (5 x CH2) cyclohexyl), 3.05 (s, 4H, CH2-CH3 and CH2-NH2), 3.11-3.46 (m, 8H, (4 x CH2) piperazine) 8.09 (br. s, 2H, NH2).13C NMR (DMSO-d6) �: 9.2 (CH3), 21.9, 26.3, 28.5, 42.3, 45.1, 47.8, 50.9, 50.0 (7 x CH2), Cq). MS (EI) m/z (%): 225.2(0.9, M+), 195.2 (100), 113.1 (6), 83.1 (10.1).

2.1.3.2. 1-[1-(4-Benzylpiperazin-1-yl)cyclohexyl]methanamine (4b)

Pale yellow viscous oil, yield 2.6 g (86.6%). IR (KBr, cm-1) exhibited bands at 3061, 3027 (NH2), 14531, 741. 1H NMR (CDCl3) �: 1.19-1.93 (m, 10H, (5 x CH2) cyclohexyl), 2.06-2.08 (m, 4H, (2 x CH2) piperazine), 2.47-2.93 (m, 6H, (2 x CH2) piperazine, CH2-C6H5), 3.47 (d, 2H, J = 5 Hz, CH2-NH) 7.26-7.27 (m, 5H, Har.).

13C NMR (CDCl3) �: 26.2, 28.4, 29.8, 37.7, 52.4, 52.7, 56.1, 66.7 (7 x CH2), Cq), 123.0, 131.2, 132.2, (CHar.), 133.3 (Car.).

2.1.3.3. 1-{1-[4-(3,4,5-Trimethoxybenzyl)piperazin-1-yl]cyclohexylmethanamine (4c)

Pale yellow viscous oil, yield 3.8 g (91%). IR (KBr, cm-1) exhibited bands at 3060, 3029 (NH2), 750. 1H NMR (CDCl3) �: 1.16-1.43 (m, 10H, (5 x CH2) cyclohexyl), 2.33-2.74 (m, 8H, (4 x CH2) piperazine), 3.52 (s, 2H, CH2-NH2), 3.73 (s, 2H, CH2-C6H5), 3.76 (s, 3H, OCH3), 3.76 (s, 2H, 2 OCH3), 6.48 (s, 2H, Har.).�

13C NMR (CDCl3) �: 25.8, 26.2, 29.4, 29.5, 35.6, 45.7, 53.4, 54.5 (7 x CH2), Cq), 58.0, 60.8 (OCH3), 106.0 (CHar.), 133.8, 136.8, 153.0 (Car.). MS (EI) m/z (%): 378.4 (1.8, M+ +1), 167 (100), 181 (99), 196 (7).

2.1.3.4. 1-[1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]methanamine (4d)

Pale viscous oil, yield 2.7 g (67.5%). IR (KBr, cm-1) exhibited bands at 3060, 3029 (NH2). 1H NMR (CDCl3) �: 1.31-1.36 (m, 10H, (5 x CH2) cyclohexyl), 2.08 (br.s, 4H, and (2x

CH2) piperazine), 3.57-3.65 (m, 6H, CH2-NH2, (2x CH2) piperazine), 5.50 (s, 1H, CH-(C6H5)2), 7.37-7.45 (m, 10H, Har.).

13C NMR (CDCl3) �: 24.3, 25.6, 30.1, 35.6, 48.8, 51.8, 64.6 (6 x CH2), Cq), 127.3, 127.5, 128.6 (CHar.), 142.7 (Car.). MS (EI) m/z (%): 363.3 (0.23, M+), 333.2 (100), 126.2 (81), 112.1 (47.99).

2.1.3.5. N-{[1-(4-Ethylpiperazin-1-yl)cyclohexyl]methyl}benzamide (1a)

White solid, m.p. 218 °C (dihydrochloride salt), yield 0.58 g (39.9%). IR (KBr, cm-1)exhibited bands at 3064 (NH), 2813, 1644 (C=O). 1H NMR (CDCl3) �: 1.04 (t, 3H, J = 6.9 Hz, CH2-CH3), 1.39-1.59 (m, 10H, (5 x CH2) cyclohexyl), 2.37 (q, 2H, J = 6.9 Hz, CH2-CH3), 2.40-2.67 (m, 8H, (4 x CH2) piperazine), 3.49 (d, 2H, J = 9.5 Hz, CH2-NH), 7.39-7.74 (m, 5H, Har.), 7.04 (br. s, 1H, NH). 13C NMR (CDCl3) �: 12.0 (CH3-CH2), 22.31, 25.98, 29.56, 40.7, 44.35, 52.36, 54.38, 58.05 (7 x CH2), Cq), 126.8, 128.6, 131.3 (CHar.), 134.8 (Car.), 167.1 (C=O). MS (EI) m/z (%): 329.2 (0.05, M+), 195.1 (100), 105.05 (13.74), 77.05 (9.87).

2.1.3.6. N-{[1-(4-Ethylpiperazin-1-yl)cyclohexyl]methyl}-4-chlorobenzamide (1b)

White solid, m.p. 230 °C (dihydrochloride salt), yield 0.48 g (30%). IR (KBr, cm-1) exhibited bands at 3254 (NH), 2930, 1641 (C=O). 1H NMR (CDCl3) �: 1.08 (t, 3H, J = 6.9 Hz, -CH2-CH3), 1.41-1.61 (m, 10H, 5 x CH2) cyclohexyl), 1.88 (br. s, 4H, (2 x CH2) piperazine), 2.38 (q, 2H, J = 6.9 Hz, CH2-CH3), 2.69 (br. s, 4H, (2 x CH2) piperazine), 3.51 (d, 2H, J = 5 Hz, CH2-NH) 7.06 (br. s,1H, NH), 7.40 (d, 2H, J = 10 Hz, Har.), 7.70 (d, 2H, J = 10 Hz, Har.).

13C NMR (CDCl3) �: 12.0 (CH2-CH3), 22.4, 25.9, 29.6, 40.8, 44.3, 52.4, 54.4, 58.1 (7 x CH2), Cq), 128.3, 128.9 (CHar.), 133.2, 137.5 (Car.), 166.2 (C=O). MS (EI) m/z (%): 364.2 (0.04, M+ + 1), 195.2 (100), 111.05 (10.59), 84.1 (21.52). Anal. Calcd. for C20H30ClN3O.2HCl: C, 54.99; H, 7.38; N, 9.62. Found: C, 54.57; H, 7.76; N, 9.65.

2.1.3.7. N-{[1-(4-Ethylpiperazin-1-yl)cyclohexyl]methyl}-4-nitrobenzamide (1c)

Buff solid, m.p. 210 °C (dihydrochloride salt), yield 0.49 g (30%). IR (KBr, cm-1) exhibited bands at 3393 (NH), 2696, 1650 (C=O). 1H NMR (CDCl3) �: 1.07 (t, 3H, J = 6.9, CH2-CH3), 1.40-1.96 (m, 10H, (5 x CH2) cyclohexyl), 2.38 (q, 2H, J = 6.9, CH2-CH3), 2.68 (br. s, 8H, (4 x CH2) piperazine), 7.22 (br. s, 1H, NH), 7.91 (d, 2H, J = 7.7 Hz, Har.), 8.27 (d, 2H, J = 7.7 Hz, Har.).

13C NMR (CDCl3) �: 12.0 (CH3-CH2), 22.3, 25.8, 29.5, 44.3, 48.9, 52.3, 54.5, 58.0 (7 x CH2),

541

Cq), 123.9, 128.0 (CHar.), 140.4, 149.5 (Car.), 165.0 (C=O). MS (EI) m/z (%): 374.2 (0.02, M+), 195.2 (100), 104.05 (4.59), 84.1 (14.58). Anal. Calcd. for C20H30N4O3.2HCl: C, 53.69; H, 7.21; N, 12.52. Found: C, 54.11; H, 7.59; N, 12.43.

2.1.3.8. N-{[1-(4-Benzylpiperazin-1-yl)cyclohexyl]methyl}benzamide (1d)

Buff solid, m.p. 224 °C (dihydrochloride salt), yield 0.64 g (37.1%). IR (KBr, cm-1) exhibited bands at 3265 (NH), 2447, 1673 (C=O), 719.

1H NMR (CDCL3) �: 1.14-1.62 (m, 10H, (5 x CH2) cyclohexyl), 2.49 (br. s, 4H, (2 x CH2) piperazine), 2.67 (br. s, 4H, (2 x CH2) piperazine), 3.50 (s, 2H, CH2-C6H5), 3.53 (d, 2H, J = 5 Hz, CH2-NH), 7.23-7.51 (m, 10H, Har.), 7.6 (s, 1H, NH). 13C NMR (CDCl3) �: 21.4, 25.9, 28.9, 33.9, 44.2, 48.9, 54.6, 63.2 (7 x CH2), Cq), 126.9, 128.2, 128.3, 129.1, (CHar., Car.), 167.1 (C=O). MS (EI) m/z (%): 391.9 (0.52, M+), 168 (63), 167 (43), 114 (100). Anal. Calcd. for C25H33N3O.2HCl: C, 64.65; H, 7.60; N, 9.05. Found: C, 64.35; H, 7.45; N, 8.98.

2.1.3.9. N-{[1-(4-Benzylpiperazin-1-yl)cyclohexyl]methyl}-4-chlorobenzamide (1e)

White solid, m.p. 226 °C (dihydrochloride salt), yield 1.27 g (67.8%). IR (KBr, cm-1) exhibited bands at 3296 (NH), 2872, 929, 1664 (C=O). 1H NMR (CDCl3 �: 1.39-1.60 (m, 10H, (5 x CH2) cyclohexyl), 2.46 (br. s, 4H, (2 x CH2) piperazine), 2.67 (br. s, 4H, (2 x CH2) piperazine), 3.51 (s, 4H, CH2-NH, CH2-C6H5), 7.07 (s, 1H, NH), 7.25-7.29 (m, 5H, Har.), 7.4 (d, 2H, J = 8.4 Hz, Har.), 7.7 (d, 2H, J = 8.4 Hz, Har.).

13C NMR (CDCl3) �: 22.3, 25.9, 29.5, 44.4, 53.1, 54.6, 58.0, 63.14 (7 x CH2), Cq), 127.0, 127.2, 128.2, 128.8, 129.3 (CHar.), 133.2, 137.5, 137.8 (Car.) , 166.0 (C=O). MS (EI) m/z (%): 423 (4.3, M+-2), 257.7 (100), 111 (25), 91 (43.3). Anal. Calcd. for C25H32ClN4O.2HCl: C, 60.18; H, 6.87; N, 8.42. Found: C, 60.49; H, 6.65; N, 8.45.

2.1.3.10. N-{[1-(4-Benzylpiperazin-1-yl )cyclohexyl]methyl}-4-nitrobenzamide (1f)

White solid, m.p. 245 °C (dihydrochloride salt), yield 0.51 g (22.6%). IR (KBr, cm-1) exhibited bands at 3250 (NH), 2948, 751, 1658 (C=O). 1H NMR (CDCl3) �: 1.22-1.60 (m, 10H, (5 x CH2) cyclohexyl), 2.00 (s, 2H,CH2 piperazine), 2.45 (br. s, 2H, CH2 piperazine) 2.66 (s, 4H, (2 x CH2) piperazine), 3.52 (d, 2H, J = 4 Hz, CH2-NH), 7.26 (s, 6H, Har., NH), 7.9 (d, 2H, J = 8 Hz, Har.), 8.26 (d, 2H, J = 8 Hz, Har.).

13C NMR (CDCl3) �: 25.9, 28.7, 29.6, 40.8, 44.3, 54.5, 58.0, 60.4 (7 x CH2), Cq), 123.0, 128.0, 128.3, 129.3, 137.5, (CHar.), 140.4 ,149.5, 165.0 (Car.), 171.1 (C=O). MS (EI) m/z (%): 434 (5, M+-2), 141.8 (39), 139.9 (46), 91 (100). Anal. Calcd. for C25H32N4O.2HCl: C, 58.94; H, 6.73; N, 11.00. Found: C, 58.67; H, 7.01; N, 10.85.

2.1.3.11. N-({1-[4-(3,4,5-Trimethoxybenzyl)piperazin-1-

yl]cyclohexyl}methyl)benzamide (1g)

White solid, m.p. 200 °C (dihydrochloride salt), yield 1.62 g (76.1%). IR (KBr, cm-1) exhibited bands at 3297 (NH), 2498, 1668 (C=O), 754. 1H NMR (CDCl3) �: 1.16-1.93 (m, 10H, (5 x CH2) cyclohexyl), 2.65-2.79 (br. s, 8H, (4 x CH2) piperazine), 3.39 (d, 2H, J = 5 Hz, CH2-NH), 3.48 (s, 2H, CH2-C6H5), 3.79 (s, 3H, OCH3), 3.81 (s, 6H, 2OCH3), 6.50 (s, 2H, Har.), 7.35-7.73 (m, 5H, Har.), 7.97 (s, 1H, J = 8 Hz, NH). 13C NMR (CDCl3) �: 22.2, 25.6, 29.6, 40.8, 44.3, 53.0, 54.6, 60.8 (7 x CH2, Cq), 56.13, 58.0 (OCH3), 105.7, 127.0, 128.4, 129.5 (CHar.), 132.9, 133.0, 133.8, 153.1 (Car.), 167.1 (C=O). MS (EI) m/z (%): 482 (0.16, M+), 209 (61), 104 (100), 76 (35). Anal. Calcd. for C28H39N3O4.2HCl: C, 60.64; H, 7.45; N, 7.58. Found: C, 60.93; H, 7.58; N, 7.47.

2.1.3.12. N-({1-[4-(3,4,5-Trimethoxybenzyl)piperazin-1-yl]cyclohexyl}methyl)-4-

chlorobenzamide (1h)

White solid, m.p. 200 °C (dihydrochloride salt), yield 0.85 g (37.1%). IR (KBr, cm-1) exhibited bands at 3027 (NH), 2809, 1627 (C=O), 750. 1H NMR (CDCl3) �: 1.10-1.66 (m, 10H, (5 x CH2) cyclohexyl), 2.04-2.58 (m, 8H, (4 x CH2) piperazine), 3.34 (d, 2H, J = 10 Hz, CH2-NH), 3.68 (s, 3H, OCH3), 3.72 (s, 8H, 2OCH3, CH2-C6H5), 6.46 (s, 2H, Har.), 7.06 (br.s, 1H, NH), 7.29 (d, 2H, J = 5 Hz, Har.), 7.62 (d, 2H, J = 5 Hz, Har.).

13C NMR (CDCl3) �: 22.1, 25.8, 28.8, 30.3, 44.2, 48.8, 53.3, 60.8 (7 x CH2, Cq), 56.4, 58.0 (OCH3), 105.8, 128.6, 128.7 (CHar.), 133.4, 134.1, 135.6, 136.9, 153.1 (Car.), 169.1 (C=O). MS (EI) m/z (%): 518 (0.34, M+ + 2), 348 (29.9), 181 (100), 139 (26.5). Anal. Calcd. for C28H38ClN3O4.2HCl: C, 57.10; H, 6.85; N, 7.13. Found: C, 56.88; H, 7.08; N, 6.91.


nitrobenzamide (1i)

Buff solid, m.p. 100 °C (dihydrochloride salt), yield 1.39 g (52.6%). IR (KBr, cm-1) exhibited bands at 3369, 3225 (NH2), 2442, 1597 (C=O), 623. 1H NMR (CDCl3) �: 1.23-1.48 (m,

542

10H, (5 x CH2) cyclohexyl), 1.93-2.26 (m, 8H, (4 x CH2) piperazine), 3.84 (s, 2H, CH2-C6H5), 3.89 (d, 2H, J = 4 Hz, CH2-NH), 3.96 (s, 3H, OCH3), 3.96 (s, 6H, 2OCH3) 6.94 (br.s, 1H, NH), 7.24 (d, 2H, J = 5 Hz, Har.), 8.19 (d, 2H, J = 5 Hz, Har.), 8.27-8.29 (m, 2H, Har.).

13C NMR (CDCl3) �: 25.9, 26.3, 29.0, 48.9, 51.6, 60.8, 63.4, 63.5 (7 x CH2, Cq), 53.6, 56.1, (OCH3), 105.9, 113.7, 119.4 (CHar.), 131.6, 134.0, 136.8, 151.1, 153.0 (Car.), 167.2 (C=O). MS (EI) m/z (%): 526 (0.01, M+), 123 (57), 84 (100), 78 (47). Anal. Calcd. for C28H38N4O6.2HCl: C, 56.09; H, 6.72; N, 9.34. Found: C, 56.29; H, 6.62; N, 9.56.

2.1.3.14. N-{[1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]methyl}benzamide (1j)

White solid, m.p. 222 °C (dihydrochloride salt), yield 0.83 g (40%). IR (KBr, cm-1) exhibited bands at 3058.55 (NH), 1663 (C=O), 699. 1H NMR (CDCl3) �: 1.28-1.63 (m, 10H, (5 x CH2) cyclohexyl), 2.44-2.69 (m, 8H, (4 x CH2) piperazine), 3.55 (s, 2H, CH2-NH), 4.22 (s, 1H, CH), 7.18-7.46 (m, 15H, Har.), 7.79 (br.s, 1H, NH). 13C NMR (CDCl3) �: 22.4, 26.0, 29.4, 40.6, 44.6, 48.9, 53.4, (6 x CH2, Cq), 126.9, 127.0, 128.0, 128.5, 128.6, 128.7 (CHar.), 142.4, 142.7 (Car.), 167.1 (C=O). MS (EI) m/z (%): 467.64 (0.42, M+), 333 (93.2), 167 (100), 105 (51.5). Anal. Calcd. for C31H37N3O.2HCl: C, 68.88; H, 7.27; N, 7.77. Found: C, 68.54; H, 7.47; N, 8.01.

2.1.3.15. N-{[1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]methyl}-4-chlorobenzamide

(1k)

White solid, m.p. 218 °C (dihydrochloride salt), yield 1.27 g (56.9%). IR (KBr, cm-1) exhibited bands at 3409 (NH), 1649.8 (C=O), 1018, 954. 1H NMR (CDCl3) �: 1.12-1.97 (m, 10H, (5 x CH2) cyclohexyl), 2.23-2.87 (m, 8H, (4 x CH2) piperazine), 3.68 (d, 2H, J = 10 Hz, CH2-NH), 4.24 (s, 1H, CH ), 7.16-7.19 (m, 11H, Har., NH), 7.27 (d, 2H, J = 5 Hz, Har.), 7.44 (d, 2H, J = 5 Hz, Har.).

13C NMR (CDCl3) �: 26.4, 29.1, 30.4, 49.3, 52.4, 53.5, 63.4 (6 x CH2, Cq), 126.9, 127.0, 128.0, 128.5, 128.6 (CHar.), 128.7, 142.4, 142.7 (Car.), 167.1 (C=O). MS (EI) m/z (%): 502.35 (0.04, M+), 334 (100), 167 (33.26). Anal. Calcd. for C31H36ClN3O.2HCl: C, 64.75; H, 6.66; N, 7.31. Found: C, 64.85; H, 6.91; N, 7.64.

2.1.3.16. N-{[1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]methyl}-4-nitrobenzamide (1l)

Buff solid, m.p. 222 °C (dihydrochloride salt), yield 2.12 g (93.3%). IR (KBr, cm-1)exhibited bands at 3037 (NH), 1650 (C=O), 698. 1H NMR (CDCl3) �: 1.09-1.94 (m, 10H, (5 x CH2) cyclohexyl), 2.26-2.42 (m, 8H, (4 x CH2) piperazine), 3.60 (br.s, 2H, CH2-NH), 4.27 (s, 1H, CH ), 7.16-7.48 (m, 14H, Har.), 8.20 (br.s, 1H, NH). 13C NMR (CDCl3) �: 25.7, 25.9, 34.0, 45.5, 49.1, 51.5, 52.1 (6 x CH2, Cq), 63.5 (CH), 126.9, 127.0, 128.0, 128.5, 128.6, (CHar.) 128.7, 42.4, 142.7 (Car.), 167.8 (C=O). MS (EI) m/z (%): 512.5 (0.6, M+), 167 (100), 150 (35.4). Anal. Calcd. for C31H36N4O3.2HCl: C, 63.59; H, 6.54; N, 9.54. Found: C, 63.25; H, 6.47; N, 9.84.

2.1.4. General procedure for synthesis of 4-amino-N-{[1-(4-aralkyl/ethylpiperazin-1-

yl)cyclohexyl]methyl}benzamides 1m-p

A solution of the appropriate arylcarboxamide 1c, 1f, 1i, and 1l (2.62 mmol) in 250 mL ethanol (95%) was hydrogenated at room temp and normal pressure for 48 h, using (120 mg) of 10% Pd/C for 1c.HCl and Raney nickel for 1f, 1i, and 1l. The catalyst was filtered off, and ethanol was evaporated under vacuum to afford the corresponding amines 1m-p as viscous oils in 33-78.7% yields.

2.1.4.1. N-{[1-(4-Ethylpiperazin-1-yl)cyclohexyl]methyl}-4-aminobenzamide (1m)

Yellow viscous oil, yield 0.30 g (33.3%). IR (KBr, cm-1) exhibited bands at 3751 and 3422 (NH2), 2927, 1636 (C=O), 1604 (NH bending). 1H NMR (CDCl3) �: 1.07 (t, 3H, J = 6.9 Hz, CH2-CH3), 1.42-1.58 (m, 10H, (5 x CH2) cyclohexyl), 2.39 (q, 2H, J = 6.9 Hz, CH2-CH3), 2.42-2.69 (m, 8H, (4 x CH2) piperazine), 3.47 (d, 2H, J = 3.4, CH2-NH), 4.01 (s, 1H, NH2), 6.63 (d, 2H, J = 8.4 Hz, Har.), 6.84 (br. s, 1H, NH), 7.58 (d, 2H, J = 8.4 Hz, Har.).

13C NMR (CDCl3) �: 11.9 (CH3-CH2), 22.9, 26.01, 29.6, 40.6, 44.2, 52.3, 54.3, 58.1 (7 x CH2), Cq), 114.2, 124.3 (CHar.), 128.6, 149.5 (Car.), 167.0 (C=O). MS (EI) m/z (%): 344.25 (0.35, M+), 265.1 (35), 181.05 (100). Anal. Calcd. for C20H32N4O: C, 69.73; H, 9.36; N, 16.26. Found: C, 69.84; H, 9.49; N, 15.99.

2.1.4.2. N-{[1-(4-Benzylpiperazin-1-yl)cyclohexyl]methyl}-4-aminobenzamide (1n)

Colourless viscous oil, yield 0.84 g (78.7%). IR (KBr, cm-1) exhibited bands at 3342 and 3219 (NH2), 2927, 1636 (C=O), 1604 (NH bending), 810. 1H NMR (CDCl3) �: 1.42-1.59 (m, 10H, (5 x CH2) cyclohexyl), 2.46 (s, 4H, (2 x CH2) piperazine), 2.67 (s, 4H, 2 x CH2) piperazine), 3.50 (d, 2H, J = 8.4 Hz, CH2-NH) 3.97 (br. s, 2H, , CH2-C6H5), 6.7 (d, 2H, J = 8.4 Hz, Har), 7.25-7.30 (m, 6H, Har., NH), 7.6 (d, 2H, J = 8.4 Hz, Har).

13C NMR (CDCl3) �: 22.36, 25.85, 26.03, 29.61,

543

44.41, 48.87, 54.71, 63.21 (7 x CH2), Cq), 114.2, 124.5, 125.6, 127.1, 128.3 (CHar.), 128.6, 137.9, 149.4 (Car.) 167.0 (C=O). MS (EI) m/z (%): 406.1 (0.08, M+), 257 (100), 114.05 (58). Anal. Calcd. for C25H34N4O: C, 73.85; H, 8.43; N, 13.78. Found: C, 73.65; H, 8.75; N, 13.98.


aminobenzamide (1o)

Yellow viscous oil, yield 0.52 g (40%). IR (KBr, cm-1) exhibited bands at 3369 and 3225 (NH2), 2850, 1636 (C=O), 1604 (NH bending), 847. 1H NMR (CDCl3) �: 0.79-.1.51 (m, 10H, (5 x CH2) cyclohexyl), 1.97-2.93 (m, 8H, (4 x CH2) piperazine), 3.38 (s, 4H, CH2-NH and CH2-C6H5), 3.78 (s, 3H, OCH3), 3.81 (s, 6H, 2 OCH3), 6.49 (s, 2H, Har.), 6.55 (d, 2H, J = 10 , Har.), 7.16 (d, 2H, J = 10 , Har.), 7.51 (s, 1H, NH). 13C NMR (CDCl3) �: 22.2, 22.8, 26.0, 31.9, 44.2, 51.2, 53.0, 60.8 (7 x CH2), Cq), 54.5, 56.1 (OCH3), 105.8, 113.7, 124.6 (CHar.), 129.3, 131.5, 133.5, 148.6, 153.1 (Car.), 170.9 (C=O). MS (EI) m/z (%): 495.5 (0.22, M+ -1), 181 (22.84), 114 (100). Anal. Calcd. for C28H40N4O4: C, 67.71; H, 8.12; N, 11.28. Found: C, 67.91; H, 8.46; N, 11.68.

2.1.4.4. N-{[1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]methyl}-4-aminobenzamide

(1p)

Colourless viscous oil, yield 0.56 g (52.6%). IR (KBr, cm-1) exhibited bands at 3059 and 3027 (NH2), 3223 (NH), 1605 (C=O), 920. 1H NMR (CDCl3) �: 1.20-.1.45 (m, 10H, (5 x CH2) cyclohexyl), 2.30-2.88 (m, 8H, (4 x CH2) piperazine), 3.56 (s, 2H, CH2-NH), 4.21 (s, 1H, CH), 6.53 (d, 2H, J = 10 , Har.), 6.94 (br.s, 1H, NH) 7.24-7.43 (m, 10H, Har.), 7.60 (d, 2H, J = 10 , Har). 13C NMR (CDCl3) �: 22.8, 29.0, 44.6, 52.4, 53.6, 57.9 (6 x CH2), Cq),128.01, 128.09, 128.5, 128.6, 138.1 (CHar.), 142.3, 148.7, 150.0 (Car.), 170.9 (C=O). MS (EI) m/z (%): 482.45 (0.05, M+), 334 (41.7), 167 (100). Anal. Calcd. for C31H38N4O: C, 77.14; H, 7.94; N, 11.61. Found: C, 77.54; H, 7.65; N, 11.45.

2.1.5. Synthesis of methyl 2-methoxy-4-[(phenylcarbonyl)amino]benzoate (7)�

Anhydrous K2CO3 (29.14 g, 211.0 mmol) was added to a stirred solution of 4-benzamido-2-hyroxybenzoic acid 6 (20.3 g, 79.0 mmol) in acetone (150 mL) and stirring was continued for 5 min at RT, then dimethyl sulphate (19.93 mL, 210.0 mmol) was added dropwise. The reaction mixture was stirred for 1 h at RT then stirring was continued for 18 h at 45 °C. The reaction mixture was filtered, 80% of the filtrate was evaporated under normal pressure, the evaporated solvents were replaced with water, evaporate 40% of this water, cooling (0-5 °C), then add ammonia to adjust pH 9.5-10 and the reaction mixture was stirred for 2 h at 0-5 °C. The precipitated solid was filtered off to afford 13.5 g (60%) of 7 m.p. 148 °C.

2.1.6.�Synthesis of�methyl 5-chloro-2-methoxy-4-[(phenylcarbonyl)amino]benzoate (8)

Compound 7 (6 g, 20.0 mmol) was mixed with glacial acetic acid (10.6 mL, 175 mmol), conc. HCl (2.84 mL, 20.0 mmol) and distilled H2O (3.16 mL). The reaction mixture was cooled to 20 °C, and then potassium chlorate was added in four successive portions at 10 min intervals. During addition, The white color of the reaction mixture changed to canary yellow with evolution of chlorine gas. The temperature of the reaction mixture must be kept between 30-35 °C to avoid loss of chlorine gas. After complete addition, the reaction mixture was stirred at RT for 7 h, then water was added (13.2 mL) and stirring was continued for another 1 h at RT to help complete precipitation of the chlorinated compound 7. The precipitate was filtered off, washed with water several times till neutral to afford 4.9 g (71%) of 8 as buff solid, m.p. 106 °C.

2.1.7. Synthesis of�5-chloro-2-methoxy-4-[(phenylcarbonyl)amino]benzoic acid (9)

To a stirred solution of 8 (4.0 g, 12 mmol) a solution of 1.7 N lithium hydroxide (13 mL) in THF (13.4 mL) was added. The reaction mixture was stirred overnight at RT, then was evaporated under reduced pressure. The residue was dissolved in H2O (20 mL) and extracted with diethyl ether (2 x 15 mL). The aqueous layer was acidified with conc. HCl under cooling, extracted with ethyl acetate (3 x 15 mL), the organic layer was dried (Na2SO4) and evaporated under reduced pressure to afford 3.20 g (84.2%) of 9 as yellowish white solid m.p. 170 °C.

2.1.8. General procedure for synthesis of 4-amino-N-{[1-(4-aralkyl/ethylpiperazin-1-

yl)- cyclohexyl]methyl}-5-chloro-2-methoxybenzamides 1q-t

To a stirred solution of 9 (1.31 g, 4.30 mmol) in CH2Cl2 (10 m), EDCI.HCl (1.3g, 6.79 mmol) was added, then a solution of the appropriate amine 4a-d (4.30 mmol) in CH2Cl2 (5 mL) was added to the reaction mixture. The reaction mixture was stirred overnight at RT, washed with water (2 x 20 mL) then with 10% NaHCO3 (2 x 15 mL). The organic layer was separated, dried

544

(Na2SO4) and evaporated under vacuum to afford the corresponding benzamides N-{[1-(4-aralkyl/ethylpiperazin-1-yl)cyclohexyl]methyl}-5-chloro-2-methoxy-4-[(phenylcarbonyl)- amino]benzamides 10a-d in 44-91% yields which were used in the subsequent hydrolysis step without further purification. A suspension of the appropriate benzamide 10a-d (20.0 mmol) with 10% NaOH (40 mL) was heated to reflux for 24 h. The reaction mixture was cooled, extracted with CH2Cl2 (2 x 25 mL). The organic layer was separated, dried (Na2SO4) and evaporated under reduced pressure to give crude 1q-t as brown viscous oils which were purified through column chromatography to afford the respective pure target compounds 1q-t in 50-62.5% yields.

2.1.8.1. 5-Chloro-N-{[1-(4-ethylpiperazin-1-yl)cyclohexyl]methyl}-2-methoxy-4-

[(phenylcarbonyl)amino]benzamide (10a)

Pale yellow viscous oil, yield 1.98 g (91%). IR (KBr, cm-1) exhibited bands at 3205 (NH), 1644 (C=O), 1532 (C=O), 678. 1H NMR (CDCl3) �: 1.04-1.55 (m, 13H, CH3 and (5 x CH2) cyclohexyl), 2.15-2.39 (m, 10H, CH2-CH3 and (4 x CH2) piperazine), 3,84 (s, 3H, OCH3,), 7.26-7.88 (m, 7H, Har.), 8.54 (s, 1H, CH2-NH), 9.29 (s, 1H, NH-C=O). 13C NMR (CDCl3) �: 14.8 (CH3-CH2), 22.1, 27.0, 35.5, 35.8, 44.6, 45.3, 48.0, 54.2 (7 x CH2, Cq), 56.1 (OCH3), 103.9, 114.0, 126.9, 129.1, 132.7 (CHar.), 134.2, 136.9, 154.7 (Car.), 164.8 (C=O), 165.5 (C=O). MS (EI) m/z (%): 512.7 (0.05, M+), 195 (34.5), 105 (50.7), 58 (100).

2.1.8.2. N-{[1-(4-Benzylpiperazin-1-yl)cyclohexyl]methyl}-5-chloro-2-methoxy-4-

[(phenylß carbonyl)amino]benzamide (10b) Pale yellow viscous oil, yield 1.6 g (65%). IR (KBr, cm-1) exhibited bands at 3391.21

(NH), 1686 (C=O), 1645 (C=O), 699. 1H NMR (CDCl3) �: 1.42-1.61 (m, 10H, (5 x CH2) cyclohexyl), 2.48 (br.s, 4H, (2 x CH2) piperazine), 2.69 (br. s, 4H, (2 x CH2) piperazine), 3.55 (s, 2H, CH2-C6H5), 3.57 (d, 2H, J = 5, CH2-NH), 3.92 (s, 3H, OCH3), 7.25-7.30 (m, 10H, Har.,), 7.53-7.62 (m, 2H, Har.), 8.48 (s, 1H, CH2-NH), 8.63 (s, 1H, NH-C6H5).

13C NMR (CDCl3) �: 25.9, 29.0, 29.7, 44.4, 53.1, 56.5, 57.8, 63.1 (7 x CH2), Cq), 54.1(OCH3), 103.8, 114.0, 127.0, 127.1, 132.2, 157.1, 128.2, 128.4 (CHar.), 129.2, 129.3, 129.4, 129.5, 132.6, 132.7(Car.), 165.5 (C=O), 165.6 (C=O). MS (EI) m/z (%): 579 (0.71, M+ + 4), 175 (4), 91 (15), 63 (100).

2.1.8.3. 5-Chloro-2-methoxy-4-[(phenylcarbonyl)amino]-N-({1-[4-(3,4,5-trimethoxy-

benzyl)piperazin-1-yl]cyclohexyl}methyl)benzamide (10c)

Brown viscous oil, yield 1.1 g (44%). IR (KBr, cm-1) exhibited bands at 3409 (NH), 1628 (C=O), 1592 (C=O), 843. 1H NMR (CDCl3) �: 1.16-1.89 (m, 10H, (5 x CH2) cyclohexyl), 2.48-2.60 (m, 8H, (4 x CH2) piperazine), 3.42 (s, 4H, CH2-NH, CH2-C6H5), 3.81 (s, 3H, OCH3), 3.81 (s, 9H, 3OCH3), 6.54 (s, 2H, Har), 7.25-8.35 (m, 7H, Har.,), 8.50 (s, 1H, NH-CH2), 8.64 (br. s, 1H, NH-C6H5).

13C NMR (CDCl3) �: 25.96, 26.1, 29.4, 48.9, 53.2, 53.3, 60.9, 63.4 (7 x CH2), Cq), 56.1 (4 x OCH3), 105.7, 127.1, 114.7, 128.5, 129.1, 132.2, (CHar.), 105.7, 153.2, 132.7, 133.9 134.0, 153.1, 136.9, 157.7 (Car.), 163.6 (C=O), 166.8 (C=O). MS (EI) m/z (%): 663.5 (0.95, M+ -2), 181.1 (37.6), 167.1 (34.3), 57 (100).

2.1.8.4. N-{[1-(4-Benzhydrylpiperazin-1-yl)cyclohexyl]methyl}-5-chloro-2-methoxy-4-

[(phenylcarbonyl)amino]benzamide (10d)

Pale yellow viscous oil, yield 2.15 g (77%). IR (KBr,cm-1) exhibited bands at 3060 (NH), 1600 (C=O), 700. 1H NMR (CDCl3) �: 1.14-1.84 (m, 10H, (5 x CH2) cyclohexyl), 2.14-2.53 (m, 8H, (4 x CH2) piperazine), 2.79 (s, 2H, CH2-NH), 3.6 (s, 3H, OCH3), 4.29 (s, 1H, CH), 7.31-7.40 (m, 15H, Har.), 7.73 (br.s, 2H, Har.), 7.97 (br.s, 1H, CH2-NH), 8.04 (br.s, 1H, NH-C6H5).

13C NMR (CDCl3) �: 22.3, 25.3, 25.8, 27.0, 44.8, 48.3, 49.2 (6 x CH2, Cq), 52.5 (OCH3), 127.1, 127.7, 127.9, 128.0, 128.6, 128.7, 128.8, 129.4, (CHar.), 131.0, 134.9, 131.9 136.7, 137.5, 142.3 (Car.), 161.3 (C=O), 166.4 (C=O). MS (EI) m/z (%): 649.2 (2.1, M+ -2), 167 (100), 152 (43.5).

2.1.8.5. 4-Amino-5-chloro-N-{[1-(4-ethylpiperazin-1-yl)cyclohexyl]methyl}-2-

methoxybenz- amide (1q)

Colourless viscous oil, yield 5.0 g (62.5%). IR (KBr, cm-1) exhibited bands at 3785 and 3657 (NH2), 2929, 1634 (C=O). 1H NMR (CDCl3) �: 1.04 (t, 3H, J = 6.9 Hz, CH2-CH3), 1.15-1.54 (m, 10H, (5 x CH2) cyclohexyl), 2.36 (q, 2H, J = 6.9 Hz, CH2-CH3), 2.65 (br.s, 8H, (4 x CH2) piperazine), 3.47 (d, 2H, J = 5, CH2-NH), 3.82 (s, 3H, OCH3), 4.35 (s, 2H, NH2), 6.28 (s, 1H, Har.), 7.94 (s, 1H, Har.), 8.09 (s, 1H, NH). 13C NMR (CDCl3) �: 11.83 (CH3-CH2), 22.1, 26.0, 29.6, 44.1, 48.5, 52.3, 56.2, 57.8 (7 x CH2) Cq), 53.9 (OCH3), 97.9, 111.3, 112.4, 132.9, 146.8, 157.6 (CHar.,

545

Car.) 164.6 (C=O). MS (EI) m/z (%): 408.45 (0.29, M+), 195 (100), 58 (67.95). Anal. Calcd. for C21H33ClN4O2: C, 61.67; H, 8.13; N, 13.70. Found: C, 61.29; H, 7.89; N, 13.45.

2.1.8.6. 4-Amino-N-{[1-(4-benzylpiperazin-1-yl)cyclohexyl]methyl}-5-chloro-2-

methoxy

benzamide (1r)

Colourless viscous oil, yield 4.70 g (50%). IR (KBr, cm-1) exhibited bands at 3059 and 3026 (NH2), 2854, 1644 (C=O), 848. 1H NMR (CDCl3) �: 1.07-1.88 (m, 10H, (5 x CH2) cyclohexyl), 2.19-2.67 (m, 8H, (4 x CH2) piperazine), 3.53 (s, 4H, CH2-NH and CH2-C6H5), 3.72 (s, 3H, OCH3), 4.21 (s, 2H, NH2), 7.25-7.34 (m, 8H, Har., NH). 13C NMR (CDCl3) �: 22.8, 26.0, 29.0, 29.4, 32.0, 48.9, 53.9, 63.9 (7 x CH2), Cq), 55.7 (OCH3), 98.2, 110.7, 116.5, 128.4, 129.2, 137.7, 138.1, 142.6, 144.8, 153.3 (CHar., Car.), 166.7 (C=O). MS (EI) m/z (%): 472 (0.6, M++1), 153 (47.3), 84 (43.9). Anal. Calcd. for C26H35ClN4O2: C, 66.30; H, 7.49; N, 11.89. Found: C, 65.95; H, 7.75; N, 11.64.

2.1.8.7. 4-Amino-5-chloro-2-methoxy-N-({1-[4-(3,4,5-trimethoxybenzyl)piperazin-1-

yl]cyclohexyl}methyl)benzamide (1s)

Colourless viscous oil, yield 3.88 g (40%). IR (KBr, cm-1) exhibited bands at 3854 and 3746 (NH2), 2851, 1629 (C=O), 891.1H NMR (CDCl3) �: 0.84-2.01 (m, 10H, (5 x CH2) cyclohexyl), 2.40 (br.s, 8H, (4 x CH2) piperazine), 3.42 (s, 4H, CH2-NH and CH2-C6H5), 3.70 (s, 3H, OCH3), 3.70 (s, 9H, OCH3) 6.23 (s, 1H, Har.), 6.52 (br. s, 3H, Har.), 7.10 (s, 1H, NH). 13C NMR (CDCl3) �: 14.2, 22.7, 29.7, 32.0, 41.9, 47.1, 52.9 (7 x CH2), 53.3 (Cq), 55.7, 56.1,60.9, 63.2 (4 x OCH3), 98.2, 105.7, 110.7, 116.3, 129.1, 133.6, 137.0, 144.9, 153.1, 155.3 (CHar., Car.), 166.9 (C=O). MS (EI) m/z (%): 562 (0.2, M++1), 399 (27.2), 181 (56.5). Anal. Calcd. for C29H41ClN3O5: C, 62.07; H, 7.36; N, 9.98. Found: C, 61.87; H, 7.76; N, 10.08.

2.1.8.8. 4-Amino-N-{[1-(4-benzhydrylpiperazin-1-yl)cyclohexyl]methyl}-5-chloro-2-

methoxy- benzamide (1t)

Colourless viscous oil, yield 7.60 g (70%). IR (KBr, cm-1) exhibited bands at 3333, 3203 (NH2), 1619 (C=O), 1249, 752. 1H NMR (DMSO-d6) �: 1.10-1.96 (m, 10H, (5 x CH2) cyclohexyl), 2.24-2.72 (m, 8H, (4 x CH2) piperazine), 3.29 (br.s, 2H, CH2-NH), 3.75 (s, 3H, OCH3), 4.21-4.42 (m, 3H, CH, NH2), 6.23 (s, 2H, Har.), 7.17-7.41 (m, 10H, Har.), 8.09 (br.s, 1H,NH). 13C NMR (DMSO-d6) �: 26.0, 28.4, 30.4, 44.7, 47.3, 49.1, 52.2 (6 x CH2, Cq), 55.7 (OCH3), 98.2, 110.7, 116.4, 127.2, 127.9, 128.6, 128.7, 142.3, 144.9, 155.3 (CHar., Car.), 166.8 (C=O). MS (EI) m/z (%): 549 (0.34, M++2), 251.2 (4.6), 181 (100), 167 (84.5). Anal. Calcd. for C32H39ClN4O2: C, 70.25; H, 7.18; N, 10.24. Found: C, 69.92; H, 7.58; N, 10.54.

2.2. Biological evaluation

Adult male albino rats weighing 200-300 g were used in this study. The animals were purchased from Animal House colony of National Research Centre, Cairo, Egypt and were housed under standardized conditions (room temperature 23±2°C, relative humidity 55±5%, 12h-

light/12h-dark cycle) .They had free access to tap water and were feeded with commercially available standard rat chow throughout the whole experimental period. All animal procedures were performed after the Ethics Committee of the National Research Centre and in accordance with the recommendations for the proper care and use of laboratory animals "Canadian Council on Animal Care Guidelines, 1984." Tween-80 (Polyoxyethylene-sorbitan monooleate, Sigma USA), apomorphine hydrochloride (Research Biochemicals Inc., Wayland, USA), and Metoclopramide hydrochloride (CID Company, Giza, Egypt).

2.2.1. Dopamine D2 receptor antagonistic activity (Zwangsnagen test)

Groups of 6 rats each were placed individually in cages having shavings of wood on the floor and an observation window and allowed to habituate for 15 minutes before injection of drugs. A series of doses ranging from 1.5 to 20 mg/kg of the test compounds 1a-t was investigated. Each dose was suspended in tween-80 (7% aqueous solution) as vehicle and administered subcutaneously. A minimum of 4 dose levels per compound and 6 rats per dose were used. One hour later, 0.5% solution of apomorphine hydrochloride (1.25 mg/kg) in saline was injected intravenously, such that the injected solution does not exceed 2 mL/kg. After 5, 10, 20 min., theanimals were observed for 1 min. The presence or absence of chewing movement (Zwangsnagen) or compulsory gnawing as well as severity of chewing were noted. The absence of chewing movement 5, 10 or 20 minutes after apomorphine hydrochloride injection is indicative of

546

dopamine D2 receptor antagonistic activity and hence antiemetic activity.11 The studied biological activity of the tested compounds was compared with that of Metoclopramide hydrochloride used as reference standard. The ED50 of the most potent compounds were calculated according to the method of Litchfield Wilcoxon.12

2.3. Molecular modeling

Pharmacophore was produced using the Discovery Studio 2.5 software. (Accelrys Inc., San Diego, CA, USA).

2.3.1. Generation of dopamine D2 receptor antagonists pharmacophore

The pharmacophore modeling method has been widely used in lead discovery and optimization as a key tool of computer aided drug design. A hypothesis was formulated using generation common feature pharmacophore model protocol in Discovery studio 2.5. The lead compounds (I-X), which were reported to have dopamine D2 receptor antagonistic activity (Figure 2), were used to generate common feature pharmacophore for the dopamine D2 receptor antagonists.13 A set of conformational models of each structure of the lead compounds was performed and used to generate the common feature hypotheses, where ten hypotheses were generated.14

�

�

��3.1. Chemistry

The target compounds 1a-l were synthesized as outlined in Scheme 1. Thus, cyclohexanone was allowed to react via Strecker synthesis with N-ethyl and/or aralkylpiperzine 2a-d and KCN in the presence of concentrated HCl to produce the carbonitrile derivatives 3a-d. Subsequently, the nitrile functionality of 3a-d was subjected to reduction using LiAlH4/AlCl3

reducing mixture15 in dry THF to yield the corresponding amines 4a-d. Compounds 4a-d were then reacted with the appropriate acyl chloride 5a-c in the presence of triethylamine to yield the respective target compounds 1a-l in moderate yields.

Compound Nr. R R1

1a C2H5 H 1b C2H5 Cl 1c C2H5 NO2

1d CH2-C6H5 H 1e CH2-C6H5 Cl 1f CH2-C6H5 NO2

1g CH2-C6H2(OCH3)3 H 1h CH2-C6H2(OCH3)3 Cl 1i CH2-C6H2(OCH3)3 NO2

1j CH(C6H5)2 H 1k CH(C6H5)2 Cl 1l CH(C6H5)2 NO2

Scheme 1: Synthesis of the target compounds 1a-l.

O

NH

N

+

R NNC

NR

NH2N

NR

NNH

NR

R1

O

2a-d 3a-d 4a-d 1a-l

a: R = C2H5

b: R = CH2-C6H5

c: R = CH2-C6H2(OCH3)3

d: R = CH(C6H5)2

i ii iii

547

Reagents and conditions: i) KCN, conc. HCl, water, RT, 18 h; ii) LiAlH4/AlCl3, THF, RT, 18 h; iii) appropriate 5a-c, triethylamine, benzene, reflux, 18 h.

Nitro functionality of compounds 1c, 1f, 1i, and 1l was reduced using 10% Pd/C (for 1c) or Raney nickel (for 1f, 1i, and 1l) and molecular hydrogen under normal pressure and room temperature to give the respective amines 1m-p (Scheme 2).

Scheme 2: Synthesis of the target compounds 1m-p.

Reagents and conditions: i) 10% Pd/C (for compound 1c) or Raney nickel (for compounds 1f, 1i, and 1l), H2, RT, 18 h.

The acid 9, which was required to prepare the Metoclopramide analogues 1q-t, was prepared as depicted in Scheme 3. Thus, N-benzoyl-4-aminosalicylic acid 6 was methylated at both phenolic OH and carboxylic acid functionalities using dimethyl sulfate in acetone at 45 °C for 24 hours to give the corresponding methoxybenzoic acid methyl ester 7. Subsequent chlorination of 7 using KClO3 in the presence of concentrated hydrochloric acid at room temperature for 24 h furnished the chlorinated compound 8. The ester functionality of 8 was hydrolyzed by LiOH in THF at room temperature to furnish N-benzoyl-4-aminobenzoic acid derivative 9.

Scheme 3: Synthesis of compound 9. Reagents and conditions: i) (CH3)2SO4, anhyd. K2CO3, 45 °C, 24 h; ii) KClO3/HCl, RT, 24 h;

iii) LiOH/THF, RT, 24 h.

Metoclopramide analogues 1q-t were synthesized as illustrated in Scheme 4. Thus, the benzoic acid derivative 9 was coupled with the appropriate amine 4a-d using ethyl-3-(3-

NNH

NR

O2N

O

i

NNH

NR

H2N

O

1c, 1f, and 1l 1m-p

1c, 1m: R = C2H5

1f, 1n: R = CH2-C6H5

1i, 1o: R = CH2-C6H2(OCH3)3

1l, 1p: R = CH(C6H5)2

HN

ii

HN

i

C

O

C

O

HN C

O

iii

COOH

OH

COOCH3

OCH3

HN C

O

COOCH3

OCH3

Cl

COOH

OCH3

Cl

6 7 8

9

548

dimethylaminopropyl)carbodiimide hydrochloride (EDCI.HCl) in DCM at room temperature to furnish the respective amides 9a-d. Subsequently, the target compounds 1q-t were obtained viarefluxing the N-benzoyl derivatives 9a-d in 10% aqueous NaOH solution (Scheme 4).

Scheme 4: Synthesis of the target compounds 1q-t. Reagents and conditions: i) EDCI.HCl, DCM, RT, 18 h; ii) NaOH, H2O, reflux, 18 h.

The spectral data of the newly synthesized compounds in the present investigation were in accordance with their assigned structures.

3.2. In vivo dopamine D2 receptor antagonistic activity

The newly synthesized compounds 1a-t were evaluated for their dopamine D2 receptor antagonistic activity in vivo by measuring their ability to inhibit apomorphine-induced chewing “Zwangsnagen” in rats.11 This test measures the inhibition of compulsive stereotyped hyperactivity behavior induced by apomorphine through its stimulation of central dopamine D2 receptors in rats.16 The dopamine D2 receptor antagonistic activity of 1a-t and ED50 values of the selected candidates with potent activity are displayed in Table 1.

Metoclopramide (I) is a relatively weak serotonin-3 (5-HT3) as well as dopamine D2

receptor antagonist. Metoclopramide is one of the most effective agents used intravenously in a high dose to alleviate cisplatin-induced nausea and vomiting.17 Nevertheless, its clinical usefulness is restricted due to its extrapyramidal side effects. Accordingly, Metoclopramide is a ready target for extensive molecular modification to enhance some of its desirable effects and attenuate or abolish side effects. Certain molecular modifications were examined previously in our research group which implied structural variation in the amide side chain through incorporating cyclohexyl moiety in the �-position to the amidic nitrogen to give compound

II.5 Insertion of cyclohexyl moiety increased the lipophilicity of compound II which plays a remarkable role in distribution and binding of drugs to their targets in vivo. Further molecular modifications of compound II were achieved, to improve its dopamine D2 receptor antagonistic profile, through the synthesis of the target compounds 1a-t.

HN C

O

COOH

OCH3

Cl

9 4a-d

+

NH2N

N RN

NH

N RO

OCH3NH

OCl

NNH

N RO

OCH3H2N

Cl

1q-t

10a-d

a: R = C2H5

b: R = CH2C6H5

c: R = CH2C6H2(OCH3)3

d: R = CH(C6H5)2

1q: R = C2H5

1r: R = CH2C6H5

1s: R = CH2C6H2(OCH3)3

1t: R = CH(C6H5)2

i

ii

549

Table 1: Fit values and dopamine D2 receptor antagonistic activity of compounds 1a-t.

Compd. No. Dose mg/kg (�mol)* % Inhibition ED50 mg/kg (95% confidence limit)

Fit value

1a 20 (49.70) 66.6 ND 3.00 1b 20 (45.78) 100 7.0 (13.4-3.82) 4.34 1c 10 (22.35) 100 4.0 (7.49-2.69) 4.67 1d 20 (43.06) 16.6 ND 3.21 1e 20 (40.09) 50 ND 3.90 1f 20 (39.26) 66.6 ND 3.39 1g 10 (18.03) 100 4.5 (7.72-2.79) 3.97 1h 10 (16.98) 100 3.5 (6.99-1.89) 4.86 1i 10 (16.68) 50 ND 4.14 1j 10 (18.49) 83.3 ND 4.81 1k 20 (34.78) 83.3 ND 4.81 1l 10 (17.08) 66.6 ND 3.89

1m 10 (29.03) 100 4.5 (8.42-2.77) 4.46 1n 20 (49.19) 50 ND 2.98 1o 20 (40.29) 50 ND 3.11 1p 20 (49.19) 66.6 ND 3.19 1q 10 (24.45) 100 4.0 (6.57-2.44) 4.69 1r 20 (42.46) 83.3 10.0 (17.09-5.35) 4.10 1s 10 (17.82) 100 4.0 (7.10-3.52) 4.80 1t 10 (18.28) 83.3 5.50 (10.85-2.93) 4.19 II - - 4.9 (6.03-3.99)** 4.01

Metoclopramide hydrochloride

5.5 (16.36) 100 1.0 (1.87-0.69) 4.78

* The smallest dose which gives the best % inhibition. ** Data from Reference 5. ND: Not determined

The Beecham group18 documented that selectivity of action of Metoclopramide could be achieved through restriction of the conformational freedom of its basic (diethylamino)ethyl side chain. Accordingly, in the present study this basic moiety was replaced with a substituted heteroalicyclic piperazine ring bearing ethyl (compounds 1a-c, 1m, and 1q), benzyl (1d-f, 1n, and 1r), trimethoxybenzyl (1g-i, 1o, and 1s), or benzhydryl (1j-l, 1p, and 1t) substituents. Furthermore, the influence of benzoyl group substituents in 1a-t on their dopamine D2 receptor antagonistic activity was examined while retaining the N-substituted piperazine moiety.

The benzamide derivative 1a showed 66.6% inhibition against apomorphine-induced chewing in rats at a dose level of 49.70 �mol/kg. Furthermore, substitution of the benzoyl moiety of 1a with an electron withdrawing group like chlorine improves its inhibition activity as in 1b

which displayed 100% inhibition at a dose level of 45.78 �mol/kg. Whereas, the 4-nitro analogue 1c showed the best activity of the N-ethylpiperazine congeners with 100% inhibition at a dose level of 22.35 �mol/kg. Reduction of the nitro group of 1c yielded 1m which produced 100% inhibition at a dose level of 29.03 �mol/kg. The N-ethylpiperazine analogue of Metoclopramide, compound 1q, exhibited 100% inhibition at a dose level of 24.45 �mol/kg.

On the other hand, replacing the N-ethyl group by N-benzyl group furnished compounds 1d-f, 1n, and 1r. N-Benzyl analogue of 1a, compound 1d, exhibited only 16.6% inhibition at a dose level of 43.06 �mol/kg, while the 4-chloro congener, compound 1e, showed 50% inhibition at a dose level of 40.09 �mol/kg. Similarly, the nitro derivative 1f displayed 66.6% inhibition at a dose level of 39.26 �mol/kg. Whereas, its respective amino candidate, compound 1n, exhibited slightly weaker dopamine D2 receptor antagonistic activity (50% inhibition) at a dose level of 49.19 �mol/kg. Compound 1r showed the highest activity in the N-benzyl derivatives with 83.3% inhibition at a dose level of 42.46 �mol/kg.

550

A sizable number of antiemetics incorporate trimethoxybenzyl moiety in their structures.12

Therfore, N-trimethoxybenzyl derivatives 1g-i, 1o, and 1s were synthesized and screened for their dopamine D2 receptor antagonistic potential. Trimethoxybenzyl analogue of 1a, compound 1g, displayed 100 % inhibition of apomorphine-induced chewing in rats at a dose level of 18.03 �mol/kg. Moreover, the 4-chloro derivative 1h emerged as the most potent dopamine D2 receptor antagonist of all the synthesized compounds 1a-t. It exhibited 100% inhibition at a dose level of 16.98 �mol/kg being nearly equipotent with the reference standard, Metoclopramide hydrochloride, that displayed 100 % inhibition at a dose level of 16.36 �mol/kg. Surprisingly, the nitro derivative 1i and its respective amino derivative 1o showed only 50% inhibition at dose levels of 16.68 and 40.29 �mol/kg, respectively. Furthermore, the N-trimethoxybenzylpiperazine analogue of Metoclopramide, compound 1s, exhibited 100% inhibition at a dose level of 17.82 �mol/kg.

In the N-benzhydrylpiperazine derivatives 1j-l, 1p, and 1t, compounds 1j, 1k, and 1t

displayed 83.3% inhibition of apomorphine-induced chewing in rats at dose levels of 18.49, 34.78, and 18.28 �mol/kg, respectively. Whereas, both 1l and 1p exhibited only 66.6% inhibition at dose levels of 17.08 and 49.19 �mol/kg, respectively.

The most active candidates in the synthesized compounds bearing N-ethyl, benzyl, trimethoxybenzyl, and benzhydrylpiperazine moieties were subjected to quantitative estimation (median effective dose, ED50) and the results are depicted in Table 1. Variation of the substituents at both piperazine and benzoyl moieties influence their D2-receptor antagonistic activity as indicated by their inhibition of apomorphine-induced chewing in rats in the following decreasing order: 1h > 1s > 1g > 1c > 1q > 1t > 1m > 1b > 1r.

Fig. 2: Chemical structures of potent dopamine D2 receptor antagonists.

3.3. Validation of the generated pharmacophore

Ten hypotheses were generated and the one ranked number 4 was chosen as the valid ideal hypothesis (Figure 3 of the supplementary material) based on the following: (a) the hypothesis

NH

NO

C2H5

C2H5

H2N

Cl

OCH3

Metoclopramide (I)

F

NOH

Cl

O

F

N

O

N

NCH3

Haloperidol (III)

F

N

O

NNH

O

NH

OH

Cl NH

OH

BrH3CO

NH

NOH

SMe

H3CO

NH

N OH

Cl

F

N

O

N

NCH3

O

H2N

O

OMe

NH

NC2H5

C2H5

Cl

(II)

(IV) (V)

(VII)

(VI)

(VIII) (IX)

(X)

551

showed full mapping of all its features without any steric clashes together with high fit values with the training set (compounds I-X, e.g., see Figure 4 of the supplementary material), (b) retrospectively, the simulated fit values of test set compounds (1a-t) with hypothesis 4 were more consistent with the experimental results than other hypotheses, (c) the database search study for examining the affinity of such hypothesis with the molecular structures of MiniMaybridge databases revealed that only 17 hits have been retrieved from the databases (2000 compounds).19

Such a low number of the recognized database molecules by generated hypothesis may give an additional advantage and selectivity to our hypothesis.14 Such an ideal hypothesis encompassed five features namely; positive ionizable (PI), hydrogen bonding acceptor (HBA), ring aromatic (RA) and two hydrophobic features (HY1 and HY2). Herein, the constraint distances and angles between the essential features existed in the generated hypothesis is reported as shown Table 2 and in Fig. 3 of the supplementary material.

(A) (B)

Fig. 3: (A and B) Constraint distances and angles of dopamine D2 receptor antagonists. The chemical features coloured light blue, green, red and orange represent hydrophobic features (HY), hydrogen bonding acceptor (HBA), positive ionisable (PI), and ring aromatic (RA), respectively!"

Table 2: The constraint distances and angles between the features of the generated dopamine D2 receptor antagonists pharmacophore model.

Dimensions Features of D2-receptor antagonists Constraint distances (Å) between features HY2-HBA, 9.34; PI-HBA, 4.18; HY1-HY2,

14.06; HY2-PI,5.46; HY2-RA, 11.07; PI-RA, 5.67; RA-HBA, 3.60

Constraint angles between features

HY2-PI-HBA, 11.69; PI-HBA-RA, 39.22; PI-RA vector, 75.78; RA-HBA vector, 79.22; HY2-PI-RA, 167.31

Structures of the test set of arylcarboxamides 1a-t were built using the Discovery Studio software, and their conformational models were generated in the energy range of 20 kcal/mol above the estimated global energy minima. Fitting of each tested compound was performed using best fit during the compare/fit process. Different mappings for all the conformers of each compound of the test set to the hypothesis were visualized and the fit values of the best-fitting conformers are shown in Table 1 and in Fig. 4 of the supplementary material.

552

(A) (B)

(C) (D)

(E) (F)

Fig. 4: (A–F) Mapping of dopamine D2 receptor antagonist pharmacophore with lead compound VIII (E), III (F) and test set compounds 1c (A), 1h (B), 1s (C) and 1m (D),

respectively.

��

The synthesis of certain new N-{[1-(4-aralkyl/ethylpiperazine-1-yl)cyclohexyl]methyl}arylcarboxamides 1a-t is reported. Dopamine D2-receptor antagonistic activity of 1a-t was determined using apomorphine-induced chewing “Zwangsnagen” test in rats. Compound 1h is the most active congener in all the synthesized compounds 1a-t displaying ED50

of 3.5 mg/kg (5.94 �mol/kg) being nearly 2-fold more potent than the previously reported cyclohexane-containing dopamine D2 receptor antagonist II (ED50 = 4.90 mg/kg, 11.66 �mol/kg). Whereas, 1h exhibited 100% inhibition at a dose level of 16.98 �mol/kg being nearly equipotent with the reference standard, Metoclopramide hydrochloride, that showed 100 % inhibition at a dose level of 16.36 �mol/kg. Additionally, Molecular simulation study including fitting to dopamine D2 receptor antagonists 3D-pharmacophore model using Discovery Studio 2.5 programs showed high-fit values. The experimental dopamine D2 receptor antagonistic activity of compounds 1a-t was consistent with the molecular modeling study.

��

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Int. J. Mol. Sci. 2014, 15, 16911-16935; doi:10.3390/ijms150916911

International Journal of

Molecular Sciences ISSN 1422-0067

www.mdpi.com/journal/ijms

Article

Anticonvulsant Profiles of Certain New 6-Aryl-9-substituted-6,9-diazaspiro-[4.5]decane-8,10-diones and 1-Aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones

Mohamed N. Aboul-Enein 1,*, Aida A. El-Azzouny 1, Mohamed I. Attia 1,2, Yousreya A. Maklad 3,

Mona E. Aboutabl 3, Fatma Ragab 4 and Walaa H. A. Abd El-Hamid 5

1 Medicinal and Pharmaceutical Chemistry Department (Medicinal Chemistry Group),

Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki,

Giza 12622, Egypt; E-Mails: [email protected] (A.A.E.-A.); [email protected] (M.I.A.) 2 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P. O. Box 2457,

Riyadh 11451, Saudi Arabia 3 Medicinal and Pharmaceutical Chemistry Department (Pharmacology Group) Pharmaceutical and

Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt;

E-Mails: [email protected] (Y.A.M.); [email protected] (M.E.A.) 4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,

Cairo 11562, Egypt; E-Mail: [email protected] 5 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr University for Science &

Technology, 6th of October City 12566, Egypt; E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +2-012-216-8624; Fax: +2-023-337-0931.

Received: 19 August 2014; in revised form: 10 September 2014 / Accepted: 12 September 2014 /

Published: 23 September 2014

Abstract: Synthesis and anticonvulsant potential of certain new 6-aryl-9-substituted-6,9-

diazaspiro[4.5]decane-8,10-diones (6a–l) and 1-aryl-4-substituted-1,4-diazaspiro[5.5]

undecane-3,5-diones (6m–x) are reported. The intermediates 1-[(aryl)(cyanomethyl)amino]

cycloalkanecarboxamides (3a–f) were prepared via adopting Strecker synthesis on the

proper cycloalkanone followed by partial hydrolysis of the obtained nitrile functionality

and subsequent N-cyanomethylation. Compounds 3a–f were subjected to complete nitrile

hydrolysis to give the respective carboxylic acid derivatives 4a–f which were cyclized

under mild conditions to give the spiro compounds 5a–f. Ultimately, compounds 5a–f were

alkylated or aralkylated to give the target compounds 6a–i and 6m–u. On the other hand,

compounds 6j–l and 6v–x were synthesized from the intermediates 5a–f through

OPEN ACCESS

Int. J. Mol. Sci. 2014, 15 16912

alkylation, dehydration and finally tetrazole ring formation. Anticonvulsant screening of

the target compounds 6a–x revealed that compound 6g showed an ED50 of 0.0043 mmol/kg

in the scPTZ screen, being about 14 and 214 fold more potent than the reference drugs,

Phenobarbital (ED50 = 0.06 mmol/kg) and Ethosuximide (ED50 = 0.92 mmol/kg), respectively.

Compound 6e exhibited an ED50 of 0.019 mmol/kg, being about 1.8 fold more potent than

that of the reference drug, Diphenylhydantoin (ED50 = 0.034 mmol/kg) in the MES screen.

Interestingly, all the test compounds 6a–x did not show any minimal motor impairment at

the maximum administered dose in the neurotoxicity screen.

Keywords: cycloalkanones; Strecker synthesis; alkylation; spiro compounds;

tetrazole; anticonvulsant

1. Introduction

Epilepsy is a group of neurological disorders characterized by excessive abnormal bioelectrical

functions of the brain leading to recurrent unprovoked seizures [1,2]. It affects about 1% of the global

population with the majority of cases being in the developing countries [3]. Estimates suggest that

approximately 20%–30% of patients are not adequately controlled by the available antiepileptic

medications [4,5]. Furthermore, the clinically used antiepileptics display serious side effects such as ataxia,

hepatotoxicity, gingival hyperplasia and megaloblastic anaemia [6–8]. Therefore, there is a substantial

need for novel, more effective and more selective antiepileptic agents with lesser side effects.

Diketopiperazines (DKPs) are the smallest cyclic peptides known, commonly biosynthesized from

amino acids by a large variety of organisms [9]. They are privileged structures for the discovery of new

lead compounds. They display attractive chemical characteristics, such as resistance to proteolysis,

mimicking of peptidic pharmacophoric groups, conformational rigidity and donor as well as acceptor

groups for hydrogen bonding which might influence interactions with biological targets [10].

DKPs include 2,3-DKPs, 2,5-DKPs and 2,6-DKPs (3-aza-glutarimides). Although various methods

and synthetic protocols are reported for the synthesis of 2,6-DKPs, there is a paucity of information on

their induced biological profiles, including anticonvulsant, antiviral and anticancer activities [2,11–13].

Incorporation of lipophilic moieties in the scaffold of new bioactive chemical entities could

improve their anticonvulsant potential. Accordingly, cyclohexane and/or cyclopentane moieties were

embedded in the skeleton of the new 2,6-DKP derivatives 6a–x aiming to enhance their anticonvulsant

activity. On the other hand, the tetrazole moiety is a bioisostere of carboxylic acid functionality and it

is an integrated part in the construction of certain anticonvulsants [14,15]. Therefore, compounds 6j–l

and 6v–x, bearing a tetrazole moiety, were synthesized and screened for their anticonvulsant potential.

Our research group has previously reported the synthesis and anticonvulsant activity of certain

1-alkyl-1,4-diazaspiro[4.5]decane and [5.5]undecane-3,5-diones [16] as ring expanded hydantoins

which are one of the well known classical families of anticonvulsants. As an extension of this study,

we describe herein the synthesis and anticonvulsant profile of certain new 6-aryl-9-substituted-6,9-

diazaspiro[4.5]decane-8,10-diones (6a–l) and 1-aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones

(6m–x) aiming to get new anticonvulsant biocandidates.

Int. J. Mol. Sci. 2014, 15 16913

2. Results and Discussion

2.1. Chemistry

Syntheses of the target compounds 6a–x and their intermediates are depicted in Schemes 1–3.

Thus, cyclopentanone and/or cyclohexanone were allowed to react with the appropriate commercially

available aniline derivative and potassium cyanide in glacial acetic acid under Strecker synthesis

conditions to give the respective nitrile derivatives 1a–f. The nitrile group of compounds 1a–f was

subjected to hydrolysis under acidic conditions using sulfuric acid at ambient temperature to yield the

amide derivatives 2a–f. Subsequently, cyanomethylation of the secondary amine moiety of compounds

2a–f was successfully achieved using potassium cyanide, paraformaldehyde and formaldehyde to

furnish the corresponding compounds 3a–f (Scheme 1).

Scheme 1. Synthesis of compounds 1–3a–f. Reagents and conditions: (i) KCN, glacial

acetic acid, RT, 24 h; (ii) Conc. H2SO4, RT, 48 h; (iii) KCN, formaldehyde 37% solution,

paraformaldehyde, 60 °C-RT, 3–18 h.

NH2

+i ii iii

R1

(CH2)n

O

(CH2)n (CH2)n

HN CN

R1

HN

NH2

R1

O

(CH2)n

N

NH2

R1

ONC

1a–f 2a–f 3a–f

1–3 n R1

a 0 H

b 0 4-CH3

c 0 4-OCH3

d 1 H

e 1 4-CH3

f 1 4-OCH3

The target compounds 6a–i and 6m–u as well as their intermediates 4a–f and 5a–f were obtained as

portrayed in Scheme 2. Thus, the nitrile moiety in compounds 3a–f was hydrolysed via reflux in

sodium hydroxide solution to yield the corresponding carboxylic acid derivatives 4a–f. Cyclization of

the latter compounds 4a–f was successfully realized using ethylenediamine in 4 N HCl solution to give

the respective spiro compounds 5a–f according to our previously developed procedure [16]. The imide

functionality of compounds 5a–f was alkylated under phase transfer catalysis conditions using the

appropriate alkyl/aralkyl halide to give the target compounds 6a–i and 6m–u.

Int. J. Mol. Sci. 2014, 15 16914

Scheme 2. Synthesis of compounds 4a–f, 5a–f, 6a–i and 6m–u.

(CH2)n

N

NH2

R1

ONC

(CH2)n

i ii iii

NH

NR1

O

O

(CH2)n

N

NR1

O

O

R2

(CH2)n

N

NH2

R1

OHOOC

3a–f 4a–f 5a–f 6a–i, 6m–u

3-5 n R1

a 0 H

b 0 4-CH3

c 0 4-OCH3

d 1 H

e 1 4-CH3

f 1 4-OCH3

6 n R1 R2

a 0 H -CH2COOCH3

b 0 H -CH2Ph

c 0 H -CH2CH2Ph

d 0 4-CH3 -CH2COOCH3

e 0 4-CH3 -CH2Ph

f 0 4-CH3 -CH2CH2Ph

g 0 4-OCH3 -CH2COOCH3

h 0 4-OCH3 -CH2Ph

i 0 4-OCH3 -CH2CH2Ph

m 1 H -CH2COOCH3

n 1 H -CH2Ph

o 1 H -CH2CH2Ph

p 1 4-CH3 -CH2COOCH3

q 1 4-CH3 -CH2Ph

r 1 4-CH3 -CH2CH2Ph

s 1 4-OCH3 -CH2COOCH3

t 1 4-OCH3 -CH2Ph

u 1 4-OCH3 -CH2CH2Ph

Reagent and conditions: (i) NaOH, reflux, 24 h;(ii) Ethylenediamine, 4N HCl, reflux, 24 h;(iii) BrCH2COOCH3 or ClCH2C6H5 or BrCH2CH2C6H5,acetone, tetrabutylammoniumbromide, reflux, 7 h.

The synthesis of the intermediates 7a–f and 8a–f as well as the target compounds 6j–l and 6v–x

were successfully achieved as illustrated in Scheme 3. Synthesis of compounds 6j–l and 6v–x was

commenced with the reaction of compounds 5a–f with chloroacetamide to give the corresponding

compounds 7a–f. Dehydration of compounds 7a–f using trifluoroacetic anhydride furnished the

respective penultimate cyanomethyl derivatives 8a–f. Elaboration of the cyano group of compounds

8a–f to the tetrazolyl moiety was acquired using sodium azide in the presence of aluminium chloride to

yield the desired compounds 6j–l and 6v–x.

Int. J. Mol. Sci. 2014, 15 16915

Scheme 3. Synthesis of compounds 7a–f, 8a–f, 6j–l and 6v–x. Reagents and conditions:

(i) Acetone, K2CO3, tetrabutylammonium bromide, reflux 7 h; (ii) Triflouroacetic anhydride,

THF, cooling, 0–5 °C, 2 h, ammonium bicarbonate; (iii) NaN3, AlCl3, cooling then reflux 24 h.

+ iiiCl

(CH2)n

N

NR1

O

O

(CH2)n

N

NR1

O

O

CN

5a-f

7a-f 8a-f

iii

(CH2)n

N

NR1

O

O

N N

NHN

NH2

ONH2

O

6j-l, 6v-x

Compound Nr. n R1

6j, 7a, 8a 0 H

6k, 7b, 8b 0 4-CH3

6l, 7c, 8c 0 4-OCH3

6v, 7d, 8d 1 H

6w, 7e, 8e 1 4-CH3

6x, 7f, 8f 1 4-OCH3

2.2. Anticonvulsant Activity

The test compounds 6a–x were subjected to preliminary anticonvulsant evaluation (Phase I screening)

according to the protocol given by the Epilepsy Section of the National Institute of Neurological

Disorders and Stroke (NINIDS) using the standard procedure adopted by the Antiepileptic Drug

Development (ADD) program [17]. Those include the ‘gold standard’ screens, namely subcutaneous

Pentylenetetrazole (scPTZ) screen and the maximal electroshock seizure (MES) screen. The former

screen identifies compounds that elevate seizure threshold while the latter one measures the ability of

the test compound to prevent seizure spread. Compounds exhibited 100% protection against

induced seizures, were subjected to median effective dose (ED50) estimation and minimal motor

impairment (neurotoxicity) evaluation.

It has been indicated that PTZ-induced seizures can be prevented by drugs that reduce T-type Ca2+

currents such as Ethosuximide and also by drugs that enhance gamma amino butyric acid type A (GABAA)

receptor-mediated inhibitory neurotransmission such as Phenobarbital [18].

The results of the initial anticonvulsant screening of the test compounds 6a–x are given in Table 1.

The evaluation indicated that, all the compounds were effective in scPTZ screen while most of

them were effective in MES screen. scPTZ screen showed that, compound 6g (R1 = 4-OCH3 and

R2 = -CH2COOCH3) was the most potent congener in the cyclopentane series 6a–l, displaying 100%

protection against PTZ-induced seizure at dose level of 0.0086 mmol/kg as compared with

Phenobarbital (0.13 mmol/kg) and Ethosuximide (1.06 mmol) which were used as reference standards.

Meanwhile, compound 6b (R1 = H, R2 = CH2-Ph) and compound 6d (R1 = 4-CH3, R2 = -CH2COOCH3)

exerted equal anticonvulsant activity (100% protection) at a dose level of 0.018 mmol/kg. Moreover,

Int. J. Mol. Sci. 2014, 15 16916

all compounds of the cyclopentane series 6a–l were more potent than the reference drugs as they

showed the same anti-seizure profile (100% protection) at lower doses on molecular bases (Table 1).

The different congeners of this series showed anticonvulsant potential in the following decreasing order:

6g > 6b = 6d > 6i > 6a > 6e = 6f > 6k > 6c > 6j > 6l > 6h

Table 1. Anticonvulsant potential (scPTZ and MES screens) of compounds 6a–x as well as

the reference standards, Phenobarbital, Ethosuximide and Diphenylhydantoin in adult male

albino mice.

Compound Nr. Dose (mmol/kg) * % Protection

scPTZ MES

6a 0.0280 100 50 6b 0.0180 100 50 6c 0.0570 100 60 6d 0.0180 100 50 6e 0.0320 100 100 6f 0.0320 100 60 6g 0.0086 100 60 6h 0.1300 100 60 6i 0.0230 100 33 6j 0.0600 100 20 6k 0.0350 100 40 6l 0.0780 100 0

6m 0.0360 100 33 6n 0.1400 100 66 6o 0.2700 100 50 6p 0.1400 100 0 6q 0.0690 100 80 6r 0.0310 100 0 6s 0.0350 100 0 6t 0.0310 100 0 6u 0.2500 83.3 33 6v 0.0470 100 60 6w 0.0560 100 40 6x 0.0320 100 60

Phenobarbital 0.1300 100 - Ethosuximide 1.0600 100 -

Diphenylhydantoin 0.1600 - 100

* The minimal dose which exhibits the maximum anticonvulsant activity; The dash (-) indicates the absence

of anticonvulsant activity at the tested dose level.

Regarding the cyclohexane series 6m-x, compounds 6r (R1 = 4-CH3, R2 = -CH2CH2Ph) and 6t

(R1 = 4-OCH3, R2 = -CH2Ph) exhibited the highest anticonvulsant potential with 100% protection

against PTZ-induced seizures in mice at the same dose level of 0.031 mmol/kg. Meanwhile,

compound 6o (R1 = H, R2 = -CH2CH2Ph) and compound 6u (R1 = 4-OCH3, R2 = -CH2CH2Ph) require

high doses to achieve the 100% protection (0.27 and 0.25 mmol/kg, respectively).

Int. J. Mol. Sci. 2014, 15 16917

The different congeners of the cyclohexane series 6m–x showed a decrease in the anticonvulsant

potential in the following decreasing order:

6r = 6t > 6x > 6s > 6m > 6v > 6w > 6q > 6n = 6p > 6u > 6o

Concerning the MES test, the dose which exerted 100% anticonvulsant protection in the scPTZ

screening has been selected. In this screening test, all of the compounds showed protection in half or

more of the tested mice after 0.5 h post administration except compounds 6i, 6j, 6k, 6m, 6u and 6w.

On the other hand, compounds 6l, 6p, 6r, 6s and 6t were devoid from anticonvulsant activity.

Meanwhile, 6e (R1 = 4-CH3, R2 = -CH2Ph) exhibited 100% protection at dose level of 0.032 mmol/kg

being more potent than the reference drug, Diphenylhydantoin, which exerted the same protection at a

dose level of 0.16 mmol/kg. It is worthwhile to mention that, compound 6e displayed 100% protection

against both scPTZ and MES-induced seizures in mice.

Compounds showed 100% protection in scPTZ and/or MES screens, were subjected to median

effective dose (ED50) estimation as well as to minimal motor impairment (neurotoxicity) evaluation.

Table 2 summarizes ED50 of the selected test compounds along with their neurotoxicity evaluation.

Compound 6g gave an ED50 of 0.0043 mmol/kg ≡ 1.5 mg/kg in the scPTZ screen being about 14 and

214 fold more potent than the reference drugs, Phenobarbital (ED50 = 0.06 mmol/kg ≡ 13.2 mg/kg) and

Ethosuximide (ED50 = 0.92 mmol/kg ≡ 130 mg/kg), respectively. In the MES screen, only compound 6e

showed 100% protection against induced seizures with ED50 of 0.019 mmol/kg ≡ 7.0 mg/kg being

about 1.8 fold more potent than that of the reference drug, Diphenylhydantoin (ED50 = 0.034 mmol/kg ≡

9.5 mg/kg [19]). Interestingly, all the test compounds did not show any minimal motor impairment at

the maximum administered dose in the neurotoxicity screen.

Table 2. Median effective dose (ED50, mg/kg) of compounds 6a–t and 6v–x exhibiting

100% protection against scPTZ-induced seizers and their neurotoxicity in adult male albino

mice using Phenobarbital and Ethosuximide as reference standards.

Compound Nr. ED50 (Confidence Limits) Neurotoxicity *

6a 4.5 (6.85–2.96) 0/6 6b 2.5 (3.56–1.76) 0/6 6c 11.5 (13.66–9.68) 0/6 6d 2.4 (4.10–1.40) 0/6

6e ** 6.0 (7.95–4.53) 0/6 6f 2.5 (2.84–2.20) 0/6 6g 1.5 (2.40–0.94) 0/6 6h 19.0 (25.24–14.30) 0/6 6i 4.2 (6.82–2.59) 0/6 6j 13.5 (15.38–11.85) 0/6 6k 6.5 (8.44–4.27) 0/6 6l 16.5 (19.65–13.85) 0/6

6m 4.0 (6.92–2.31) 0/6 6n 25.0 (33.35–18.74) 0/6 6o 35.0 (66.42–18.44) 0/6 6p 24.0 (32.24–17.87) 0/6 6q 10.0 (13.67–7.32) 0/6

Int. J. Mol. Sci. 2014, 15 16918

Table 2. Cont.

Compound Nr. ED50 (Confidence Limits) Neurotoxicity *

6r 4.0 (6.14–2.60) 0/6

6s 6.0 (8.79–4.09) 0/6 6t 6.0 (8.44–4.27) 0/6 6v 6.5 (11.89–3.55) 0/6 6w 12.0 (15.42–9.34) 0/6 6x 6.0 (11.00–3.27) 0/6

Phenobarbital 13.2 (15.90–6.80) ND Ethosuximide 130.0 (111–150) ND

* Rotarod test: number of animals exhibiting neurotoxicity/number of animals tested; ** ED50 in MES

screen = 7.0 mg/kg; ND: not determined.

3. Experimental Section

3.1. Chemistry

All melting points were determined using Electrothermal Capillary melting point apparatus and

are uncorrected. Infrared (IR) spectra were recorded as thin film (for oils) in NaCl discs or as KBr

pellets (for solids) with JASCO FT/IR-6100 spectrometer and values are represented in cm−1. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were carried out on Jeol ECA 500 MHz

spectrometer using TMS as internal standard and chemical shift values were recorded in ppm on δ scale.

The 1H-NMR data were represented as follows: chemical shifts, multiplicity (s. singlet, d. doublet, t. triplet,

m. multiplet, br. broad), number of protons, and type of protons. The 13C-NMR data were represented

as chemical shifts and type of carbons. Mass spectral data were obtained with electron impact (EI)

ionization technique at 70 eV from a Finnigan Mat SSQ-7000 Spectrometer. Elemental analyses were

carried out in Microanalytical Units at National Research Centre and Cairo University. Silica gel TLC

(thin layer chromatography) cards from Merck (silica gel precoated aluminum cards with fluorescent

indicator at 254 nm) were used for thin layer chromatography. Visualization was performed by

illumination with UV light source (254 nm). Column chromatography was carried out on silica gel

60 (0.063–0.200 mm) obtained from Merck.

3.1.1. General Procedure for the Synthesis of 1-(Arylamino)cycloalkanecarbonitriles (1a–f)

A solution of potassium cyanide (9.75 g, 0.15 mol) in water (25 mL) was added drop-wise to a

solution of cycloalkanone (0.15 mol) and the appropriate aniline derivative (0.15 mol) in glacial acetic acid

(75 mL). The reaction mixture was stirred mechanically at room temperature for 24 h. The precipitated

product was filtered off, washed with water, dried and recrystallized from petroleum ether (40–60 °C) to

afford 1a–f. The spectral data of compounds 1a–f were consistent with the published ones (cited below).

1-(Phenylamino)cyclopentanecarbonitrile (1a) [20]. Yield: 92%; white solid, m.p. 60 °C.

1-[(4-Methylphenyl)amino]cyclopentanecarbonitrile (1b) [21]. Yield: 89%; buff solid, m.p. 56 °C.

1-[(4-Methoxyphenyl)amino]cyclopentanecarbonitrile (1c) [22]. Yield: 80%; brown solid, m.p. 132 °C.

Int. J. Mol. Sci. 2014, 15 16919

1-(Phenylamino)cyclohexanecarbonitrile (1d) [23]. Yield: 75%; white solid, m.p. 74–76 °C.

1-[(4-Methylphenyl)amino]cyclohexanecarbonitrile (1e) [24]. Yield: 75%; yellowish white solid,

m.p. 76–78 °C.

1-[(4-Methoxyphenyl)amino]cyclohexanecarbonitrile (1f) [24]. Yield: 84.5%; buff solid, m.p. 76 °C.

3.1.2. General Procedure for the Synthesis of 1-(Arylamino)cycloakanecarboxamides (2a–f)

The appropriate nitrile derivative 1a–f (0.125 mol) was dissolved in cold concentrated sulfuric acid

(20 mL). After remaining at room temperature for 48 h, the reaction mixture was poured over crushed

ice and rendered alkaline with 25% ammonium hydroxide solution. The precipitated amide was

filtered off, washed with water, dried and recrystallized from ethanol to give 2a–f. The spectral data of

compounds 2a–f were consistent with the published ones (cited below).

1-(Phenylamino)cyclopentanecarboxamide (2a) [25]. Yield: 90%; white solid, m.p. 166 °C.

1-[(4-Methylphenyl)amino]cyclopentanecarboxamide (2b) [26]. Yield: 85%; buff solid, m.p. 120 °C.

1-[(4-Methoxyphenyl)amino]cyclopentanecarboxamide (2c) [27]. Yield: 50%; buff solid, m.p.

90–93 °C.

1-(Phenylamino)cyclohexanecarboxamide (2d) [24]. Yield: 85%; white solid, m.p. 148 °C.

1-[(4-Methylphenyl)amino]cyclohexanecarboxamide (2e) [27]. Yield: 85%; white solid, m.p. 154 °C.

1-[(4-Methoxyphenyl)amino]cyclohexanecarboxamide (2f) [27]. Yield: 75%; buff solid, m.p. 110 °C.

3.1.3. General Procedure for the Synthesis of 1-[(Aryl)(cyanomethyl)amino]cycloalkanecarboxamides

(3a–f)

Paraformaldehyde (1.52 g, 0.05 mol) was added to a solution of the appropriate

1-(arylamino)cycloakanecarboxamides (2a–f) (0.05 mol) in glacial acetic acid (30 mL). A solution of

potassium cyanide (3.9 g, 0.06 mol) was added drop-wise to the stirred and cooled (15 °C)

reaction mixture. The temperature was raised gradually to 45 °C over 30 min and was maintained at

50–60 °C for 3 h. After cooling to 35 °C, a 37% formaldehyde solution (5 mL) was added and the

reaction mixture was stirred at room temperature for 18 h. Water (30 mL) was added, the reaction

mixture was cooled and neutralized with 10% sodium carbonate solution. The precipitated product was

extracted with CH2Cl2 (3 × 50 mL), washed with water (2 × 30 mL), dried (Na2SO4) and evaporated

under vacuum to give the anticipated compounds 3a–f. The crude 3a–f were pure enough to be used

in the following step without any further purification. The spectral data of compounds 3a–f were

consistent with the published ones (cited below).

1-[(Cyanomethyl)(phenyl)amino]cyclopentanecarboxamide (3a) [16]. Yield: 78%; pale yellow

viscous oil.

Int. J. Mol. Sci. 2014, 15 16920

1-[(Cyanomethyl)(4-methylphenyl)amino]cyclopentanecarboxamide (3b) [16]. Yield: 86.6%; pale

yellow viscous oil.

1-[(Cyanomethyl)(4-methoxyphenyl)amino]cyclopentanecarboxamide (3c) [16]. Yield: 80%; pale

yellow viscous oil.

1-[(Cyanomethyl)(phenyl)amino]cyclohexanecarboxamide (3d) [16]. Yield: 85%; yellowish white

solid, m.p. 135 °C.

1-[(Cyanomethyl)(4-methylphenyl)amino]cyclohexanecarboxamide (3e) [16]. Yield: 95%; buff

solid, m.p. 83 °C.

1-[(Cyanomethyl)(4-methoxyphenyl)amino]cyclohexanecarboxamide (3f) [16]. Yield: 97%; buff

solid, m.p. 103 °C.

3.1.4. General Procedure for the Synthesis of [(Aryl)(1-carbamoylcycloalkyl)amino]acetic

Acids (4a–f)

A mixture of the appropriate cyanomethyl derivative 3a–f (0.01 mol) and NaOH (0.48 g, 0.012 mol) in

50% aqueous ethanol (25 mL) was stirred under reflux for 18 h, utill complete evolution of ammonia

was ceased. The ethanol was removed by evaporation under vacuum. The residue was extracted with

ethyl acetate (2 × 15 mL) and the aqueous layer was acidified with 2 N HCl. The acidic layer was

extracted with ethyl acetate (3 × 15 mL), dried (Na2SO4) and evaporated under reduced pressure to

yield compounds 4a–f. The crude 4a–f were pure enough to be used in the following step without any

further purification. The spectral data of compounds 4a–f were consistent with the published ones

(cited below).

[(1-Carbamoylcyclopentyl)(phenyl)amino]acetic acid (4a) [16]. Yield: 85%; white solid, m.p.

120–121 °C.

[(1-Carbamoylcyclopentyl)(4-methylphenyl)amino]acetic acid (4b) [16]. Yield: 80%; yellowish

white solid, m.p. 118 °C.

[(1-Carbamoylcyclopentyl)(4-methoxyphenyl)amino]acetic acid (4c) [16]. Yield: 70%; buff solid,

m.p. 105 °C.

[(1-Carbamoylcyclohexyl)(phenyl)amino]acetic acid (4d) [16]. Yield: 70%; white solid, m.p. 186 °C.

[(1-Carbamoylcyclohexyl)(4-methylphenyl)amino]acetic acid (4e) [16]. Yield: 80%; buff solid,

m.p. 188 °C.

[(1-Carbamoylcyclohexyl)(4-methoxyphenyl)amino]acetic acid (4f) [16]. Yield: 70%; buff solid,

m.p. 163 °C.

Int. J. Mol. Sci. 2014, 15 16921

3.1.5. General Procedure for the Synthesis of 6-Aryl-6,9-diazaspiro-[4.5]decane-8,10-diones (5a–c)

and 1-Aryl-1,4-diazaspiro[5.5]undecane-3,5-diones (5d–f)

4 N HCl (40 mL, 0.16 mol) was added to a solution of the appropriate carboxylic acid derivative 4a–f

(0.01 mol) and ethylenediamine (3.61 g, 0.06 mol) in dioxan (60 mL). The reaction mixture was

refluxed under stirring for 18 h. The solvent was evaporated in vacuo and the residue was neutralized

(pH 6–7) with 5% NaHCO3 solution utill no effervescence occured, extracted with CH2Cl2 (3 × 20 mL),

dried (Na2SO4) and the organic layer was evaporated under reduced pressure to give compounds 5a–f.

The crude 5a–f were pure enough to be used in the following step without any further purification.

The spectral data of compounds 5a–f were consistent with the published ones (cited below).

6-Phenyl-6,9-diazaspiro[4.5]decane-8,10-dione (5a) [16]. Yield: 50%; white solid, m.p.

73–74 °C.

6-(4-Methylphenyl)-6,9-diazaspiro[4.5]decane-8,10-dione (5b) [16]. Yield: 60%; white solid, m.p.

88 °C.

6-(4-Methoxyphenyl)-6,9-diazaspiro[4.5]decane-8,10-dione (5c) [16]. Yield: 50%; buff solid m.p.

60 °C.

1-Phenyl-1,4-diazaspiro[5.5]undecane-3,5-dione (5d) [16]. Yield: 80%; white solid, m.p. 162 °C.

1-(4-Methylphenyl)-1,4-diazaspiro[5.5]undecane-3,5-dione (5e) [16]. Yield: 85%; white solid, m.p.

183 °C.

1-(4-Methoxyphenyl)-1,4-diazaspiro[5.5]undecane-3,5-dione (5f) [16]. Yield: 60%; buff solid, m.p.

110 °C.

3.1.6. General Procedure for the Synthesis of 6-Aryl-9-substituted-6,9-diazaspiro-[4.5]decane-8,10-

diones (6a–i) and 1-Aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones (6m–u)

To a mixture of the appropriate diketopiperazine derivative 5a–f (0.01 mol) in acetone (100 mL),

was added the proper alkylating agent (0.07 mol), namely methyl bromoacetate, benzyl chloride or

phenethylbromide in the presence of K2CO3 (1.38 g, 0.01 mol) and a catalytic amount of

tetrabutylammoniun bromide (0.32 g, 0.001 mol) as a phase transfer catalyst. The reaction mixture was

heated under reflux for 7 h, cooled to room temperature, filtered and the filtrate was evaporated under

vacuum. The residue was purified using column chromatography (chloroform:ethyl acetate, 9:1) to

furnish the target compounds 6a–i and 6m–u.

Methyl 2-(8,10-dioxo-6-phenyl-6,9-diazaspiro[4.5]decane-9-yl)acetate (6a). Yield: 65%; yellow

viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1752 (carbonyl ester),

1720, 1685 (imide carbonyls), 609, 557; 1H-NMR (CDCl3) δ ppm 1.80 (br.s, 4H, 2 × CH2, cyclopentyl),

2.00–2.37 (m, 4H, 2 × CH2, cyclopentyl), 3.76 (s, 3H, COOCH3), 4.32 (s, 2H, O=C-CH2-N), 4.58

(s, 2H, N-CH2-COO), 7.02–7.32 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 25.08, 36.25 (4 × CH2,

cyclopentyl), 40.12 (CH2-COOCH3), 52.59, 56.59 (O=C-CH2-N, COOCH3), 69.65 (Cq), 124.82,

Int. J. Mol. Sci. 2014, 15 16922

128.48, 129.32 (CHar.), 148.71 (Car.), 169.91, 170.11, 176.18 (3 × C=O); MS (EI) m/z (%): 316.2

([M]+, 17), 91 (100), 172.2 (90); Anal. Calcd for C17H20N2O4 (316.35): C, 64.54%; H, 6.37%; N, 8.86%.

Found: C, 64.51%; H, 6.15%; N, 8.66%.

9-Benzyl-6-phenyl-6,9-diazaspiro[4.5]decane-8,10-dione (6b). Yield: 60%; Yellow viscous oil; IR

(KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1678 (imide carbonyls), 604,

555; 1H-NMR (CDCl3) δ ppm 1.79–2.30 (m, 8H, 4 × CH2, cyclopentyl), 4.27 (s, 2H, O=C-CH2-N),

5.10 (s, 2H, CH2-C6H5), 6.85–6.86 (m, 2H, Har.), 7.05–7.17 (m, 3H, Har.), 7.30–7.34 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 25.50, 36.70 (4 × CH2, cyclopentyl), 42.6 (CH2-C6H5), 56.91 (O=C-CH2-N),

67.27 (Cq), 124.71, 126.96, 127.49, 128.21, 128.40, 129.42 (CHar.), 136.81, 148.76 (2 × Car.), 170.33,

176.00 (2 × C=O); MS (EI) m/z (%): 334.3 ([M]+, 15), 91 (100), 77.1 (40); Anal. Calcd. for

C21H22N2O2 (334.41): C, 75.42%; H, 6.63%; N, 8.38%. Found: C, 75.32%; H, 6.61%; N, 8.17%.

9-Phenethyl-6-phenyl-6,9-diazaspiro[4.5]decane-8,10-dione (6c). Yield: 71.5%; yellow viscous oil;

IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1677 (carbonyl imides),

575, 500; 1H-NMR (CDCl3) δ ppm 1.77 (br.s, 4H, 2 × CH2, cyclopentyl), 1.96 (s, 2H, CH2, cyclopentyl),

2.25 (s, 2H, CH2, cyclopentyl), 2.85 (t, 2H, J = 7.5 Hz, CH2-C6H5) , 4.09 (t, 2H, J = 7.5 Hz,

CH2-CH2-C6H5), 4.24 (s, 2H, O=C-CH2-N), 6.95–7.12 (m, 3H, Har.), 7.26–7.32 (m, 7H, Har.); 13C-NMR (CDCl3) δ ppm 25.05, 33.92 (4 × CH2, cyclopentyl), 36.59, 40.53 (CH2-C6H5, CH2-CH2-C6H5),

56.92 (O=C-CH2-N), 63.71 (Cq), 124.53, 124.62, 126.52, 128.45, 128.61, 129.05 (CHar.), 138.27, 148.82

(2 × Car.), 170.03, 175.97 (2 × C=O); MS (EI) m/z (%): 348.23 ([M]+, 22), 91 (100), 172.1 (65), 229 (65),

Anal. Calcd. for C22H24N2O2 (348.44): C, 75.38%; H, 6.94%; N, 8.04%. Found: C, 75.41%; H, 6.91%;

N, 8.23%.

Methyl 2-(8,10-dioxo-6-(4-methylphenyl)-6,9-diazaspiro[4.5]decane-9-yl)acetate (6d). Yield: 79%;

yellow viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1752

(carbonyl ester), 1722, 1686 (imide carbonyls), 607, 564; 1H-NMR (CDCl3) δ ppm 1.73–1.77 (m, 8H,

4 × CH2, cyclopentyl), 2.29 (s, 3H, CH3), 3.75 (s, 3H, COOCH3), 4.23 (s, 2H, O=C-CH2-N), 4.65

(s, 2H, CH2-COOCH3), 6.91 (d, 2H, J = 8.6 Hz, Har), 7.05 (d, 2H, J = 8.6 Hz, Har.); 13C-NMR (CDCl3)

δ ppm 21.31 (CH3), 25.62, 36.85 (4 × CH2, cyclopentyl), 40.16 (CH2-COOCH3), 52.53, 56.89

(O=C-CH2-N, COOCH3), 69.81 (Cq), 124.90, 130.57, (CHar.), 134.56, 146.22 (2 × Car.), 170.17,

176.01 (2 × C=O); MS (EI) m/z (%): 330.24 ([M]+, 24), 105.1(100), 186.2 (53); Anal. Calcd. for


9-Benzyl-6-(4-methylphenyl))-6,9-diazaspiro[4.5]decane-8,10-dione (6e). Yield: 90%; colourless

viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1678 (imide

carbonyls), 634, 582; 1H-NMR (CDCl3) δ ppm 1.75–1.91 (m, 8H, 4 × CH2, cyclopentyl), 2.23 (s, 3H,

CH3), 4.20 (s, 2H, O=C-CH2-N), 5.01 (s, 2H, CH2-C6H5), 6.71 (d, 2H, J = 8.6 Hz, Har.), 7.25 (d, 2H,

J = 8.6 Hz, Har.), 7.27–7.33 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 20.85 (CH3), 25.12, 36.71 (4 × CH2,

cyclopentyl), 42.72 (CH2-C6H5), 56.89 (O=C-CH2-N), 69.97 (Cq), 124.98, 127.57, 128.50, 128.94,

129.76 (CHar.), 134.57, 136.92, 146.27 (3 × Car.), 170.59, 176.19 (2 × C=O); MS (EI) m/z (%): 364.26

([M]+, 28), 91(100), 105 (98); Anal. Calcd. for C22H24N2O3 (364.44): C, 72.50%; H, 6.64%; N, 7.69%.

Found: C, 72.43%; H, 6.75%; N, 7.81%.

Int. J. Mol. Sci. 2014, 15 16923

6-(4-Methylphenyl)-9-phenethyl-6,9-diazaspiro[4.5]decane-8,10-dione (6f). Yield: 71.2%; yellow

viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1678 (imide

carbonyls), 646, 606; 1H-NMR (CDCl3) δ ppm 1.73–2.17 (m, 8H, 4 × CH2, cyclopentyl),

2.20–2.27 (m, 3H, CH3), 2.84 (t, J = 7.7 Hz, 2H, CH2-C6H5), 4.04 (t, 2H, J = 7.7 Hz, CH2-CH2), 4.16

(s, 2H, O=C-CH2-N), 7.21 (d, 2H, J = 6.7 Hz, Har.), 7.24 (d, 2H, J = 6.7 Hz, Har.), 7.25–7.26 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 20.83 (CH3), 25.09, 34.08, (4 × CH2, cyclopentyl), 36.59, 40.58

(CH2-C6H5, CH2-CH2-C6H5), 56.90 (O=C-CH2-N), 69.88 (Cq), 124.63, 126.56, 128.68, 128.68, 129.13

(CHar.), 134.40, 138.39, 146.30 (3 × Car.), 170.30, 176.18 (2 × C=O); MS (EI) m/z (%): 362.2 ([M]+, 15),

81(100); Anal. Calcd. for C23H26N2O2 (362.46): C, 76.21%; H, 7.23%; N, 7.73%. Found: C, 76.02%;

H, 7.15%; N, 7.89%.

Methyl 2-(6-(4-methoxyphenyl)-8,10-dioxo-6,9-diazaspiro[4.5]decan-9-yl)acetate (6g). Yield: 71.4%;

yellow viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands 1752 (carbonyl

ester), 1720, 1685 (imide carbonyls), 609, 557; 1H-NMR (CDCl3) δ ppm 1.79–1.82 (m, 4H, 2 × CH2,

cyclopentyl), 2.25–2.28 (m, 4H, 2 × CH2, cyclopentyl), 3.82 (s, 6H, COOCH3, OCH3), 4.25 (s, 2H,

O=C-CH2-N), 4.61 (s, 2H, CH2-COOCH3), 6.83 (d, 2H, J = 9.0 Hz, Har.), 7.12 (d, 2H, J = 9.0 Hz, Har.); 13C-NMR (CDCl3) δ ppm 24.95, 36.24 (4 × CH2, cyclopentyl), 40.19 (CH2-COOCH3), 51.97, 52.44,

55.45 (O=C-CH2-N, COOCH3, OCH3), 70.68 (Cq), 114.40, 114.99 (CHar.), 133.40, 157.42, (2 × Car.),

169.69, 170.59, 176.19 (3 × C=O); MS (EI) m/z (%): 346.23 ([M]+, 17), 121.14 (100), 77.1 (29); Anal.

Calcd. for C18H22N2O5 (346.38): C, 62.42%; H, 6.40%; N, 8.09%. Found: C, 62.22%; H, 6.35%; N, 8.22%.

9-Benzyl-6-(4-methoxyphenyl)-6,9-diazaspiro[4.5]decane-8,10-dione (6h). Yield: 90%; yellow

viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1678

(imide carbonyls), 634, 582; 1H-NMR (CDCl3) δ ppm 1.78 (br.s, 4H, 2 × CH2, cyclopentyl), 1.92–2.21

(m, 4H, 2 × CH2, cyclopentyl), 3.78 (s, 3H, OCH3), 4.17 (s, 2H, O=C-CH2-N), 5.51 (s, 2H, CH2-C6H5),

6.67 (d, 2H, J = 8.5 Hz, Har.), 6.75 (d, 2H, J = 8.5 Hz, Har.), 7.28–7.39 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 25.58, 36.40 (4 × CH2, cyclopentyl), 39.0 (CH2-C6H5), 42.63 (O=C-CH2-N),

55.48 (OCH3), 73.15 (Cq), 114.27, 114.96, 126.50, 127.54, 128.18 (CHar.), 135.47, 136.78, 141.35

(3 × Car.), 170.43, 172.47 (2 × C=O); MS (EI) m/z (%): 364.2 ([M]+, 28), 91(100), 121 (85);

Anal. Calcd. for C22H24N2O3 (364.44): C, 72.50%; H, 6.64%; N, 7.96%. Found: C, 72.33%; H, 6.46%;

N, 7.79%.

6-(4-Methoxyphenyl)-9-phenethyl-6,9-diazaspiro[4.5]decane-8,10-dione (6i). Yield: 90%; yellow

viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands 1722, 1687 (imide

carbonyls), 634, 582; 1H-NMR (CDCl3) δ ppm 1.64–1.83 (m, 8H, 4 × CH2, cyclopentyl), 2.79 (t, 3H,

J = 8.0 Hz, CH2-CH2-C6H5)), 3.68 (s, 2H, CH2-CH2-C6H5), 3.99 (s, 3H, OCH3), 4.02 (s, 2H, O=C-CH2-N),

6.68 (d, 2H, J = 9.0 Hz, Har.), 6.79 (d, 2H, J = 9.0 Hz, Har.), 7.28–7.39 (m, 5H, Har.); 13C-NMR (CDCl3) δ

ppm 24.94, 36.40 (4 × CH2, cyclopentyl), 39.08, 42.63 (CH2-C6H5, O=C-CH2-N), 44.75 (CH2-CH2-C6H5),

55.41 (OCH3), 73.15 (Cq), 114.27, 114.96, 126.50, 128.17, 129.00 (CHar.), 135.47, 136.78, 141.35

(3 × Car.), 170.43, 172.47 (2 × C=O); MS (EI) m/z (%): 378.24 ([M]+, 40), 121 (100); Anal. Calcd. for


Int. J. Mol. Sci. 2014, 15 16924

Methyl 2-(3,5-dioxo-1-phenyl-1,4-diazaspiro[5.5]undecane-4-yl)acetate (6m). Yield: 78%; white

solid m.p. 118 °C; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at

1760 (carbonyl ester), 1726, 1675 (imide carbonyls), 633, 588; 1H-NMR (CDCl3) δ ppm 1.41–2.00

(m, 10H, 5 × CH2, cyclohexyl), 3.72 (s, 3H, COOCH3), 4.11 (s, 2H, O=C-CH2-N), 4.56 (s, 2H,

CH2-COOCH3), 7.12–7.25 (m, 5H, CHar.); 13C-NMR (CDCl3) δ ppm 20.46, 25.52, 31.48 (5 × CH2,

cyclohexyl), 40.19 (CH2-COOCH3), 59.11 (O=C-CH2-N), 60.59 (Cq), 126.01, 127.28, 129.36 (CHar.),

147.95 (Car.), 168.46, 170.56, 176.27 (3 × C=O); MS (EI) m/z (%): 330.1 ([M]+, 80), 186.2 (100),

91.1 (49); Anal. Calcd. for C18H22N2O4: C, 65.44%; H, 6.71%; N, 8.48%. Found: C, 65.52%; H, 6.68%;

N, 8.31%.

4-Benzyl-1-phenyl-1,4-diazaspiro[5.5]undecane-3,5-dione (6n). Yield: 80%; colourless viscous oil; IR

(KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1678 (imide carbonyls), 634, 582; 1H-NMR (CDCl3) δ ppm 1.48–1.97 (m, 10H, 5 × CH2, cyclohexyl), 4.11 (s, 2H, O=C-CH2-N), 5.03 (s, 2H,

CH2-C6H5), 6.86 (s, 2H, CHar.), 6.86–7.13 (m, 5H, CHar.), 7.38–7.39 (m, 5H, CHar.); 13C-NMR (CDCl3)

δ ppm 20.53, 25.63, 31.57 (5 × CH2, cyclohexyl)), 42.66, 55.37 (CH2-C6H5, O=C-CH2-N), 60.71 (Cq),

125.84, 127.08, 128.52, 129.17, 129.31, 129.76 (CHar.), 136.94, 148.06 (2 × Car.), 170.94, 176.42

(2 × C=O); MS (EI) m/z (%): 348.2 ([M]+, 100), 186.2 (70), 91.1 (58); Anal. Calcd. for C22H24N2O2

(348.44): C, 75.83%; H, 6.94%; N, 8.04%. Found: C, 75.78%; H, 6.88%; N, 8.27%.

4-Phenethyl-1-phenyl-1,4-diazaspiro[5.5]undecane-3,5-dione (6o). Yield: 64.5%; buff solid, m.p.

138 °C; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1720, 1681 (imide

carbonyls), 592, 555; 1H-NMR (CDCl3) δ ppm 1.48–1.96 (m, 10H, 5 × CH2, cyclohexyl), 2.87 (t, 2H, J =

7.6 Hz, CH2-C6H5), 4.08 (s, 2H, CH2CH2-C6H5), 4.12 (s, 2H, O=C-CH2-N), 6.99–7.00 (m, 2H, Har.),

7.23–7.29 (m, 8H, Har.); 13C-NMR (CDCl3) δ ppm 20.57, 25.63, 31.56 (5 × CH2, cyclohexyl), 34.12, 40.61

(CH2-C6H5, CH2-CH2-C6H5), 55.36 (O=C-CH2-N), 60.48 (Cq), 125.78, 126.61, 126.92, 128.56,

129.12, 129.40 (CHar.), 138.39, 148.20 (2 × Car.), 170.74, 176.56 (2 × C=O); MS (EI) m/z (%): 362.2

([M]+, 100), 243.1 (90), 186.1 (70), 91.1 (48); Anal. Calcd. for C23H26N2O2 (362.46): C, 76.21%;

H, 7.23%; N, 7.73%. Found: C, 76.41%; H, 7.42%; N, 7.91%.

Methyl 2-(3,5-dioxo-1-(4-methylphenyl)-1,4-diazaspiro[5.5]undecan-4-yl)acetate (6p). Yield: 75%;

white solid, m.p. 156–158 °C; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at

1752 (carbonyl ester), 1726, 1675 (imide carbonyls), 619, 523; 1H-NMR (CDCl3) δ ppm 1.49–1.98

(m, 10H, 5 × CH2, cyclohexyl), 2.28 (s, 3H, CH3), 3.77 (s, 3H, COOCH3), 3.79 (s, 2H, O=C-CH2-N),

4.58 (s, 2H, CH2-COOCH3), 7.07 (s, 4H, Har.); 13C-NMR (CDCl3) δ ppm 20.94, 21.15, 31.53

(5 × CH2, cyclohexyl), 24.35 (CH3), 40.19 (CH2-COOCH3), 52.48, 54.98 (O=C-CH2-N,

CH2COOCH3), 60.79 (Cq), 127.08, 129.99 (CHar.), 135.81, 145.32 (2 × Car.), 168.48, 170.64, 176.30

(3 × C=O); MS (EI) m/z (%): 344.2 ([M]+, 50), 200.1 (100), 105(37), 91.1(29); Anal. Calcd. for


4-Benzyl-1-(4-methylphenyl)-1,4-diazaspiro[5.5]undecane-3,5-dione (6q). Yield: 70%; white solid

m.p. 110 °C; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1717, 1673 (imide

carbonyls), 615, 516; 1H-NMR (CDCl3) δ ppm 1.47–1.87 (m, 10H, 5 × CH2, cyclohexyl), 1.92 (s, 3H,

CH3), 4.07 (s, 2H, O=C-CH2-N), 5.07 (s, 2H, CH2-C6H5), 6.92 (d, 2H, J = 7.5 Hz, Har.), 7.30 (d, 2H,

Int. J. Mol. Sci. 2014, 15 16925

J = 7.5 Hz, Har.), 7.38-7.40 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 20.57, 20.89, 31.60 (5 × CH2,

cyclohexyl), 25.62 (CH3), 42.66 (CH2-C6H5), 55.31 (O=C-CH2-N), 60.91 (Cq), 126.91, 127.61,

128.49, 128.68, 129.19 (CHar.), 129.90 130.16, 145.41 (3 × Car.), 170.99, 176.41 (2 × C=O); MS (EI)

m/z (%): 362.3 ([M]+, 84), 91.1(100), 200.2 (93); Anal. Calcd. for C23H26N2O2 (362.46): C, 76.21%;

H, 7.23%; N, 7.73%. Found: C, 76.31%; H, 7.19%; N, 7.75%.

1-(4-Methylphenyl)-4-phenethyl-1,4-diazaspiro[5.5]undecane-3,5-dione (6r). Yield: 59%; white

solid, m.p. 92 °C; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1719, 1674

(imide carbonyls), 597, 559; 1H-NMR (CDCl3) δ ppm 1.48–1.94 (m, 10H, 5 × CH2, cyclohexyl),

2.25 (s, 3H, CH3), 2.87 (t, J = 7.7 Hz, 2H, CH2-C6H5), 4.04 (s, 2H, CH2-CH2-C6H5), 4.06 (s, 2H,

O=C-CH2-N), 7.04 (d, 2H, J = 8.4 Hz, Har.), 7.28 (d, 2H, J = 8.4 Hz, Har.), 7.29–7.30 (m, 5H, Har.);

13C-NMR (CDCl3) δ ppm 20.61, 20.91, 25.63 (5 × CH2, cyclohexyl), 31.58 (CH3), 34.12, 40.58

(CH2-C6H5, CH2-CH2-C6H5), 55.32 (O=C-CH2-NH2), 60.63 (Cq), 126.58, 126.72, 128.53, 129.10,

129.97 (CHar.), 135.53, 138.44, 145.56 (3 × Car.), 170.81, 176.58 (2 × C=O); MS (EI) m/z (%): 376.25

([M]+, 70), 257.23 (100); Anal. Calcd. for C24H28N2O2 (376.49): C, 76.56%; H, 7.50%; N, 7.44%.

Found: C, 76.33%; H, 7.75%; N, 7.29%.

Methyl 2-(1-(4-methoxyphenyl)-3,5-dioxo-1,4-diazaspiro[5.5]undecan-4-yl)acetate (6s). Yield:

(53.5%); yellow viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1752

(carbonyl ester), 1626, 1675 (imide carbonyls), 619, 523; 1H-NMR (CDCl3) δ ppm 1.07–1.47 (m, 10H,

5 × CH2, cyclohexyl), 3.61 (s, 3H, COOCH3), 3.73 (s, 3H, OCH3), 4.22 (s, 2H, O=C-CH2-N), 4.64

(s, 2H, CH2-COOCH3), 6.75 (d, 2H, J = 8.6 Hz, Har.), 7.04 (d, 2H, J = 8.6 Hz, Har.); 13C-NMR (CDCl3) δ

ppm 22.88, 25.51, 32.94 (5 × CH2, cyclohexyl), 37.70 (CH2COOCH3), 51.84, 52.10 (O=C-CH2-N,

CH2COOCH3), 55.24 (OCH3), 67.64 (Cq), 113.38, 127.76 (CHar.), 141.72, 156.29 (2 × Car.), 168.2,

170.13, 178.70 (3 × C=O); MS (EI) m/z (%): 360 ([M]+, 0.5), 218.2 (100), 77 (5); Anal. Calcd. for


1-(4-Methoxyphenyl)-4-benzyl-1,4-diazaspiro[5.5]undecane-3,5-dione (6t). Yield: 63%; yellow

viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibited bands at 1725, 1675

(imide carbonyls), 615, 516; 1H-NMR (CDCl3) δ ppm 1.41-1.90 (m, 10H, 5 × CH2, cyclohexyl), 2.28

(s, 3H, OCH3), 4.16 (O=C-CH2-N), 4.94 (s, 2H, CH2-C6H5), 6.67 (d, 2H, J = 7.6 Hz, Har.), 6.90 (d, 2H,

J = 7.6 Hz, Har.), 7.23–7.29 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 20.80, 25.59, 36.66 (5 × CH2,

cyclohexyl), 42.66 (CH2-C6H5), 56.84, 59.07 (O=C-CH2-N, OCH3), 69.94 (Cq), 114.35, 124.97,

127.53, 128.46, 128.87 (CHar.), 129.782, 136.89, 146.25 (3 × Car.), 170.62, 176.18 (2 × C=O); MS (EI)

m/z (%): 378.4 ([M]+, 7), 91.12 (100); Anal. Calcd. for C23H26N2O3 (378.46): C, 72.99%; H, 6.92%;

N, 7.40%. Found: C, 72.75%; H, 6.78%; N, 7.52%.

1-(4-Methoxyphenyl)-4-phenethyl-1,4-diazaspiro[5.5]undecane-3,5-dione (6u). Yield: 66.5%;

yellow viscous oil; IR (KBr, ν, cm−1) absence of NH band at 3100 and exhibit bands at 1725, 1685

(imide carbonyls), 671, 538; 1H-NMR (CDCl3) δ ppm 1.23–2.05 (m, 10H, 5 × CH2, cyclohexyl), 2.81

(s, 2H, CH2-C6H5), 3.72 (s, 3H, OCH3), 3.77(s, 2H, CH2-CH2-C6H5), 4.20 (s, 2H, O=C-CH2-N),

6.80–6.81 (m, 5H, Har.), 7.13 (s, 4H, Har.); 13C-NMR (CDCl3) δ ppm 22.78, 22.99, 32.11 (5 × CH2,

cyclohexyl), 32.78, 38.02 (CH2-C6H5, CH2-CH2-C6H5), 55.52, 55.70 (O=C-CH2-N, OCH3), 68.20 (Cq),

Int. J. Mol. Sci. 2014, 15 16926

113.66, 114.60, 117.64, 127.65, 128.23 (CHar.), 138.23, 140.11, 157.74 (3 × Car.), 170.22, 176.11

(2 × C=O); MS (EI) m/z (%): 392.39 ([M]+, 14), 105 (60); Anal. Calcd. for C24H28N2O3 (392.49):

C, 73.44%; H, 7.19%; N, 7.14%. Found: C, 73.59%; H, 7.15%; N, 7.24%.

3.1.7. General Procedure for the Synthesis of 2-(6-Aryl-8,10-dioxo-6,9-diazaspiro[4.5]decan-9-

yl)acetamides (7a–c) and 2-(1-Aryl-3,5-dioxo-1,4-diazaspiro[5.5]undecan-4-yl)acetamides (7d–f)

Chloroacetamide (6.55 g, 0.07 mol) was added to a cold solution of the appropriate cyclized

compound 5a–f in acetone (100 mL) in the presence of K2CO3 (1.38 g, 0.01 mol) and a catalytic

amount of tetrabutylammoniun bromide (0.32 g, 0.001 mol) as a phase transfer catalyst. The reaction

mixture was heated under reflux for 7 h. The reaction mixture was filtered off and acetone was

evaporated under reduced pressure to give compounds 7a–f. The crude 7a–f were purified via

recrystallization from ethanol.

2-(8,10-Dioxo-6-phenyl-6,9-diazaspiro[4.5]decan-9-yl)acetamide (7a). Yield: 95%; yellowish white

solid m.p. 104 °C; IR (KBr, ν, cm−1) exhibited bands at 3383.14, 3180.62 (NH2), 1674.21, 1647.21, 1614

(3 × C=O); 1H-NMR (CDCl3) δ ppm 1.75–1.96 (m, 6H, 3 × CH2, cyclopentyl), 2.35 (br.s, 2H,

CH2-cyclopentyl), 4.05, 4.44 (2 × s, 4H, O=C-CH2-N, CH2-C=O), 5.97 (s, 2H, NH2), 6.51 (s, 3H, Har.),

7.01–7.24 (m, 2H, Har.); 13C-NMR (CDCl3) δ ppm 25.23, 36.73 (4 × CH2, cyclopentyl), 41.36 (CH2-C=O),

56.67 (O=C-CH2-N), 69.83 (Cq), 124.83, 129.30, 131.03 (CHar.), 148.58 (Car.), 169.08, 169.20, 176.02

(3 × C=O); MS (EI) m/z (%): 301.26 ([M]+, 7), 77.11 (100); Anal. Calcd. for C16H19N3O3 (301.34):

C, 63.77%; H, 6.36%; N, 13.94%. Found: C, 63.79%; H, 6.35%; N, 13.92%.

2-(8,10-Dioxo-6-(4-methylphenyl)-6,9-diazaspiro[4.5]decan-9-yl)acetamide (7b). Yield: 98%;

yellowish white solid m.p. 120 °C; IR (KBr, ν, cm−1) exhibited bands at 3383.14, 3197.98 (NH2),

1658.78, 1645.28, 1620.21 (3 × C=O); 1H-NMR (CDCl3) δ ppm 1.31-1.93 (m, 8H, 4 × CH2,

cyclopentyl), 3.73 (s, 3H, CH3), 3.99, 4.48 (2s, 4H, O=C-CH2-N, CH2-C=O), 6.14 (s, 2H, NH2),

6.78 (d, 2H, J = 8.7 Hz, Har.), 7.18 (d, 2H, J = 8.6 Hz, Har.); 13C-NMR (CDCl3) δ ppm 20.55, 31.52

(4 × CH2, cyclopentyl), 25.51 (CH3), 41.37 (CH2-C=O), 55.45 (O=C-CH2-N), 61.26 (Cq), 114.48,

128.67 (CHar.), 140.59, 157.7 (2 × Car.), 169.56, 171.05, 176.44 (3 × C=O); MS (EI) m/z (%): 317.28

([M + 2]+, 16), 121.16 (100); Anal. Calcd. for C17H21N3O3 (315.37): C, 64.74%; H, 6.71%; N, 13.32%.

Found: C, 64.77%; H, 6.69%; N, 13.34%.

2-(8,10-Dioxo-6-(4-methoxyphenyl)-6,9-diazaspiro[4.5]decane-9-yl)acetamide (7c). Yield: 98%;


1658.78, 1639.49, 1616.35 (3 × C=O); 1H-NMR (CDCl3) δ ppm 1.02–1.47 (m, 4H, 2 × CH2,

cyclopentyl), 1.68–2.26 (m, 4H, 2 × CH2, cyclopentyl), 3.77 (s, 3H, OCH3), 4.10, 4.13 (2 × s, 4H,

O=C-CH2-N, CH2-C=O-N), 6.41 (s, 2H, NH2), 6.80 (d, 2H, J = 7.5 Hz, Har.), 7.05 (d, 2H, J = 7.5 Hz,

Har.); 13C-NMR (CDCl3) δ ppm 23.87, 36.40 (4 × CH2, cyclopentyl), 42.05 (CH2-C=O), 55.66

(O=C-CH2-N, OCH3), 70.27 (Cq), 114.35, 126.88 (CHar.), 141.71, 157.07 (2 × Car.), 169.26, 169.29,

176.12 (3 × C=O); MS (EI) m/z (%): 331.3 ([M]+, 0.44), 67.17 (100); Anal. Calcd. for C17H21N3O4

(331.37): C, 67.62%; H, 6.39%; N, 12.68%. Found: C, 67.61%; H, 6.37%; N, 12.65%.

Int. J. Mol. Sci. 2014, 15 16927

2-(3,5-Dioxo-1-phenyl-1,4-diazaspiro[5.5]undecan-4-yl)acetamide (7d). Yield: 97%; yellowish

white solid m.p. 110 °C; IR (KBr, ν, cm−1) exhibited bands at 3385.07, 3188.3 (NH2), 1670, 1647.2,

1618.2 (3 × C=O); 1H-NMR (CDCl3) δ ppm 1.18–2.01 (m, 10H, 5 × CH2, cyclohexyl), 4.07, 4.47 (2 × s,

4H, O=C-CH2-N, CH2-C=O), 6.66 (s, 2H, NH2), 7.08-7.13 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm of

24.03, 25.07, 33.15 (5 × CH2, cyclohexyl), 53.85, 54.93 (CH2-C=O, O=C-CH2-N), 58.92 (Cq), 127.34,

129.07, 129.3 (CHar.), 147.97 (Car.), 169.45, 169.78, 171.27 (3 × C=O); MS (EI) m/z (%): 315.26

([M]+, 4.7), 58.17 (51), 100.2 (100); Anal. Calcd. for C17H21N3O3 (315.37): C, 64.74%; H, 6.71%;

N, 13.32%. Found: C, 64.77%; H, 6.73%; N, 13.35%.

2-(3,5-Dioxo-1-(4-methylphenyl)-1,4-diazaspiro[5.5]undecan-4-yl)acetamide (7e). Yield: 94%;


1662.6, 1647.2, 1614.4 (3 × C=O); 1H-NMR (CDCl3) δ ppm of 1.32–1.57 (m, 10H, 5 × CH2,

cyclohexyl), 2.18 (s, 3H, CH3) 3.94, 4.50 (2 × s, 4H, O=C-CH2-N, CH2-C=O), 5.97, 6.58 (2 × s, 2H,

NH2), 6.98–7.29 (m, 4H, Har.); 13C-NMR (CDCl3) δ ppm 20.35, 22.86, 23.49 (5 × CH2, cyclohexyl),

25.44 (CH3), 41.36 (CH2-C=O), 53.58 (O=C-CH2-N), 60.58 (Cq), 127.16, 135.49 (CHar.), 129.41,

145.36 (2 × Car.), 169.0, 169.50, 176.29 (3 × C=O); MS (EI) m/z (%): 329.32 ([M]+, 50), 257.28 (40),

100.16 (100), 142.18 (63); ); Anal. Calcd. for C18H23N3O3 (329.39): C, 65.63%; H, 7.04%; N, 12.76%.

Found: C, 65.66%; H, 7.12%; N, 12.78%.

2-(3,5-Dioxo-1-(4-methoxyphenyl)-1,4-diazaspiro[5.5]undecan-4-yl)acetamide (7f). Yield: 97%; buff

solid, m.p. 94 °C; IR (KBr, ν, cm−1) exhibited bands at 3383.14, 3186.4 (NH2), 1678.07, 1670.35, 1654.92

(3 × C=O); 1H-NMR (CDCl3) δ ppm 1.80–2.38 (m, 10H, 5 × CH2, cyclohexyl), 4.03 (s, 3H, OCH3), 4.28,

4.51 (2s, 4H, O=C-CH2-N, CH2-C=O), 7.01 (d, 2H, J = 8.4 Hz, Har.), 7.08 (d, 2H, J = 8.4 Hz, Har.) 7.26

(s, 2H, NH2); 13C-NMR (CDCl3) δ ppm 21.25, 25.19, 36.51 (5 × CH2, cyclohexyl), 41.50, 42.14

(CH2-C=O, O=C-CH2-N), 56.39 (OCH3), 70.43 (Cq), 124.64, 129.81 (CHar.), 135.25, 145.33 (2 × Car.),

169.04, 169.76, 175.66 (3 × C=O); MS (EI) m/z (%): 315.28 ([M-OCH3]+, 13), 105.14 (100),

287.31(10); Anal. Calcd. for C18H23N3O4 (345.39): C, 62.59%; H, 6.71%; N, 12.17%. Found: C, 62.57%;

H, 6.74%; N, 12.19%.

3.1.8. General Procedure for the Synthesis of 2-(6-Aryl-8,10-dioxo-6-phenyl-6,9-diazaspiro[4.5]decan-

9-yl)acetonitriles (8a–c) and 2-(1-Aryl-3,5-dioxo-1,4-diazaspiro[5.5]undecan-4-yl)acetonitriles (8d–f)

Trifluroacetic anhydride (6.61 g, 0.03 mol) was added to a solution of the appropriate amide 7a–f

(0.02 mol) in THF (40 mL) at 0–5 °C. The reaction mixture was stirred at room temperature for 2 h

(monitored by TLC). Ammonium bicarbonate (12.43 g, 0.16 mol) was added portion-wise during

5–10 min. and the reaction mixture was stirred at room temperature for a further 45 min., concentrated

under vacuum, washed with water (2 × 20 mL) and extracted with ethyl acetate (3 × 30 mL). The

organic layer was dried (Na2SO4) and evaporated under reduced pressure to afford compounds 8a–f.

The crude compounds 8a–f were purified using column chromatography (chloroform:ethyl acetate, 9:1).

2-(8,10-Dioxo-6-phenyl-6,9-diazaspiro[4.5]decan-9-yl)acetonitrile (8a). Yield: 97%; yellowish

white solid m.p.70 °C; IR (KBr, ν, cm−1) absence of amidic NH2 and exhibited bands at 2848.86 (CN),

1743.65, 1687.71 (2 × C=O); 1H-NMR (CDCl3) δ ppm 1.20–2.30 (m, 8H, 4 × CH2, cyclopentyl), 4.25,

Int. J. Mol. Sci. 2014, 15 16928

4.64 (2 × s, 4H, O=C-CH2-N and CH2-CN), 6.68–7.27 (m, 5H, Har.); 13C-NMR (CDCl3) δ ppm 25.18,

26.33 (4 × CH2, cyclopentyl), 29.54 (CH2-CN), 56.61 (O=C-CH2-N), 69.70 (Cq), 114.36 (CN), 124.60,

124.91, 129.27 (CHar.), 148.29 (Car.), 169.36, 175.26 (2 × C=O); MS (EI) m/z (%): 283.25 ([M]+, 25),

91.09 (100), 243.23 (15); Anal. Calcd. for C16H17N3O2 (283.33): C, 67.83%; H, 6.05%; N, 14.83%.

Found: C, 67.85%; H, 6.15%; N, 14.82%.

2-(8,10-Dioxo-6-(4-methylphenyl)-6,9-diazaspiro[4.5]decan-9-yl)acetonitrile (8b). Yield: 90%;

yellowish white solid m.p. 82 °C; IR (KBr, ν, cm−1) absence of amidic NH2 and exhibited bands at

2226.71 (CN), 1735.93, 1689.64 (2 × C=O); 1H-NMR (CDCl3) δ ppm 1.26–1.79 (m, 8H, 4 × CH2,

cyclopentyl), 2.34 (s, 3H, CH3), 4.09 (s, 2H, O=C-CH2-N), 4.71 (s, 2H, CH2-CN), 6.80 (d, 2H,

J = 8.6 Hz, Har.), 6.90 (d, 2H, J = 8.6 Hz, Har.); 13C-NMR (CDCl3) δ ppm 20.51, 29.69 (2 × CH2,

cyclopentyl), 26.26 (CH3), 29.54 (CH2-CN), 54.87 (O=C-CH2-N), 61.33 (Cq), 114.46 (CN), 127.97,

128.69 (CHar.), 140.16, 157.65 (2 × Car.), 170.66, 175.48 (2 × C=O); MS (EI) m/z (%): 299.26

([M + 2]+, 16), 121.15 (100); Anal. Calcd. for C17H19N3O2 (297.35): C, 68.67%; H, 6.44%; N, 14.13%.

Found: C, 68.68%; H, 6.42%; N, 14.15%.

2-(8,10-Dioxo-6-(4-methoxyphenyl)-6,9-diazaspiro[4.5]decane-9-yl)acetonitrile (8c). Yield: 77%;

yellow viscous oil; IR (KBr, ν, cm−1) absence of amidic NH2 and exhibited bands at 2310.70 (CN),

1743.65, 1629.85 (2 × C=O); 1H-NMR (CDCl3) δ ppm 1.27–1.55 (m, 4H, 2 × CH2, cyclopentyl),

1.79–2.21 (m, 4H, 2 × CH2, cyclopentyl), 3.77 (s, 3H, OCH3), 4.13 (s, 2H, O=C-CH2-N), 4.78 (s, 2H,

CH2-CN), 6.89 (d, 2H, J = 7.5 Hz, Har.), 6.97 (d, 2H, J = 7.5 Hz, Har.); 13C-NMR (CDCl3) δ ppm

26.19, 38.30 (2 × CH2, cyclopentyl), 29.06 (CH2-CN), 55.44 (O=C-CH2-N), 56.16 (OCH3), 65.11

(Cq), 114.62 (CN), 127.98, 131.32 (CHar.), 142.62, 157.30 (2 × Car.), 169.54, 175.16 (2 × C=O); MS

(EI) m/z (%): 312.43 ([M − 1]+, 4), 57.15 (100); Anal. Calcd. for C17H19N3O3 (313.35): C, 65.16%;

H, 6.11%; N, 13.41%. Found: C, 65.14%; H, 6.13%; N, 13.42%.

2-(3,5-Dioxo-1-phenyl-1,4-diazaspiro[5.5]undecan-4-yl)acetonitrile (8d). Yield: 90%; yellow

viscous oil; IR (KBr, ν, cm−1) absence of amidic NH2 and exhibited bands at 2254.79 (CN), 1735.9,

1705.07 (2 × C=O); 1H-NMR (CDCl3) δ ppm 1.18 (s, 4H, 2 × CH2, cyclohexyl), 1.45–1.79 (m, 6H, 3 ×

CH2, cyclohexyl), 4.09 (s, 2H, O=C-CH2-N), 4.69 (s, 2H, CH2-CN), 6.93-7.24 (m, 5H, Har.); 13C-NMR

(CDCl3) δ ppm 25.01, 29.07, 29.25 (5 × CH2, cyclohexyl), 29.44 (CH2-CN), 54.22 (O=C-CH2-N),

60.85 (Cq), 114.29 (CN), 128.95, 129.33, 129.64 (CHar.), 147.36 (Car.), 169.76, 175.30 (2 × C=O); MS

(EI) m/z (%): 297.28 ([M]+, 6), 257.2 (4), 77.14 (100); Anal. Calcd. for C17H19N3O2 (297.35):

C, 68.67%; H, 6.44%; N, 14.13%. Found: C, 68.69%; H, 6.46%; N, 14.11%.

2-(3,5-Dioxo-1-(4-methylphenyl)-1,4-diazaspiro[5.5]undecan-4-yl)acetonitrile (8e). Yield: 94.5%;

yellowish white solid m.p. 120–122 °C; IR (KBr, ν, cm−1) absence of amidic NH2 and exhibited bands

at 1888.31 (CN), 1741.72, 1989.6 (2 × C=O); 1H-NMR (CDCl3) δ ppm 1.28 (s, 2H, CH2, cyclohexyl),

1.45–2.01 (m, 8H, 4 × CH2, cyclohexyl), 2.32 (s, 3H, CH3), 4.16 (s, 2H, O=C-CH2-N ), 4.74 (s, 2H,

CH2-CN), 6.91 (d, 2H, J = 7.0 Hz, Har.), 7.15 (d, 2H, J = 7.0 Hz, Har.); 13C-NMR (CDCl3) δ ppm 22.70

(CH3), 23.06, 24.76, 25.36 (5 × CH2, cyclohexyl), 31.33 (CH2-CN), 54.92 (O=C-CH2-N), 60.98 (Cq),

114.23 (CN), 129.77, 130.24 (CHar.), 136.09, 144.74 (2 × Car.), 169.89, 175.37 (2 × C=O); MS (EI)

Int. J. Mol. Sci. 2014, 15 16929

m/z (%): 311.27 ([M]+, 16), 91.15 (100); Anal. Calcd. for C18H21N3O2 (311.38): C, 69.43%; H, 6.80%;

N, 13.49%. Found: C, 69.44%; H, 6.81%; N, 13.47%.

2-(3,5-Dioxo-1-(4-methoxyphenyl)-1,4-diazaspiro[5.5]undecan-4-yl)acetonitrile (8f). Yield: 75%;

brown viscous oil; IR (KBr, ν, cm−1) absence of amidic NH2 and exhibited bands at 2260.57 (CN),

1687.71, 1676.14 (2 × C=O); 1H-NMR (CDCl3) δ ppm 1.76–2.06 (m, 10H, 5 × CH2, cyclohexyl), 3.75

(s, 3H, OCH3), 4.27 (s, 2H, O=C-CH2-N), 4.69 (s, 2H, CH2-CN), 6.91 (d, 2H, J = 7.8 Hzs, Har.), 7.04

(d, 2H, J = 7.8 Hz, Har.); 13C-NMR (CDCl3) δ ppm 20.41, 26.23, 31.40 (5 × CH2, cyclohexyl), 29.75

(CH2-CN), 54.86 (O=C-CH2-N), 61.37 (OCH3) 68.26 (Cq), 114.72 (CN), 127.82, 128.09 (CHar.),

140.19, 157.88 (2 × Car.), 170.13, 175.43 (2 × C=O); MS (EI) m/z (%): 327.23 ([M]+, 85), 121.11

(100), 287.22 (20); Anal. Calcd. for C18H21N3O3 (327.38): C, 66.04%; H, 6.47%; N, 12.84%. Found:

C, 66.13%; H, 6.49%; N, 12.85%.

3.1.9. General Procedure for the Synthesis of 6-Aryl-9-(1H-tetrazol-5-yl)methyl)-6,9-diazaspiro[4.5]

decane-8,10-diones (6j–l) and 1-Aryl-4-((1H-tetrazol-5-yl)methyl)-1,4-diazaspiro[5.5]undecane-3,5-

diones (6v–x)

Anhydrous AlCl3 (13.3 g, 0.1 mol) was added to a cold dry THF (200 mL) under stirring during 10 min.

Thereafter, NaN3 (28.9 g, 0.45 mol) was added portion-wise through 10 min. The appropriate

penultimate nitrile derivative 8a–f was added and the reaction mixture was stirred under refluxed for 24 h.

After cooling, the reaction mixture was filtered and the filtrate was evaporated under vacuum.

The crude residues were purified through column chromatography (chloroform:ethyl acetate, 9:1) to

give the ultimate respective compounds 6j–l and 6v–x.

6-Phenyl-9-((1H-tetrazol-5-yl)methyl)-6,9-diazaspiro[4.5]decane-8,10-dione (6j). Yield: 71%;

colorless viscous oil; IR (KBr, ν, cm−1) exhibited band at 3419 (NH) and disappearance of CN band; 1H-NMR (CDCl3) δ ppm 1.11–2.23 (m, 8H, 4 × CH2, cyclopentyl), 4.22 (s, 2H, O=C-CH2-N), 5.14

(s, 2H, N-CH2-tetrazole)), 6.75–7.07 (m, 5H, Har.), 7.37 (s, 1H, NH); 13C-NMR (CDCl3) δ ppm 25.15,

29.77 (4 × CH2, cyclopentyl), 38.86 (N-CH2-tetrazole), 56.70 (O=C-CH2-N), 69.98 (Cq), 124.92,

129.42, 132.50 (CHar.), 148.48 (Car.), 153.55 (C=N-tetrazole), 170.55, 176.06 (2 × C=O); MS (EI) m/z

(%): 326.43 ([M]+, 33), 327.27 (100); Anal. Calcd. for C16H18N6O2 (326.35): C, 58.88%; H, 5.56%;

N, 25.75%. Found: C, 58.66%; H, 5.51%; N, 25.72%.

6-(4-Methylphenyl)-9-((1H-tetrazol-5-yl)methyl)-6,9-diazaspiro[4.5]decane-8,10-dione (6k). Yield:

61%; yellow viscous oil; IR (KBr, ν, cm−1) exhibited band at 3419 (NH) and disappearance of CN band; 1H-NMR (CDCl3) δ ppm 1.47 (s, 3H, CH3), 1.51–1.56 (m, 4H, 2 × CH2, cyclopentyl), 1.77 (br.s, 4H,

2 × CH2 cyclopentyl), 3.91 (s, 2H, O=C-CH2-N), 4.10 (s, 2H, N-CH2-tetrazole), 5.21 (s, 1H, NH), 6.83

(d, 2H, J = 6.0 Hz, Har.), 7.12 (d, 2H, J = 6.0 Hz, Har.); 13C-NMR (CDCl3) δ ppm 25.38 (CH3), 30.02,

34.84 (4 × CH2, cyclopentyl), 50.98 (N-CH2-tetrazole), 55.59 (O=C-CH2-N), 60.89 (Cq), 125.36,

128.92 (CHar.), 140.92, 151.94 (2 × Car.), 157.55(C=N-tetrazole), 168.93, 170.71 (2 × C=O); MS (EI)

m/z (%): 340.29 ([M]+, 0.5), 121.14 (100); Anal. Calcd. for C17H20N6O2 (340.38): C, 59.99%; H, 5.92%;

N, 24.69%. Found: C, 59.74%; H, 5.82%; N, 24.67%.

Int. J. Mol. Sci. 2014, 15 16930

6-(4-Methoxyphenyl)-9-((1H-tetrazol-5-yl)methyl)-6,9-diazaspiro[4.5]decane-8,10-dione (6l). Yield:

63.3%; yellow viscous oil; IR (KBr, ν, cm−1) exhibited band at 3421.72 (NH) and disappearance of CN

band; 1H-NMR (CDCl3) δ ppm 1.47 (br.s, 6H, 3 × CH2, cyclopentyl), 2.19 (br.s, 2H, CH2 cyclopentyl),

3.73 (s, 3H, OCH3), 3.80 (s, 2H, O=C-CH2-N), 4.21 (s, 2H, N-CH2-tetrazole)), 6.64 (s, 1H, NH), 6.99

(d, 2H, J = 6.0 Hz, Har.), 7.25 (d, 2H, J = 6.0 Hz, Har.); 13C-NMR (CDCl3) δ ppm 23.08, 30.43 (4 × CH2,

cyclopentyl), 51.01 (N-CH2-tetrazole), 55.83 (O=C-CH2-N), 59.45 (OCH3), 61.02 (Cq), 128.49,

132.02 (CHar.), 140.0, 152.0 (2 × Car.), 159.96 (C=N-tetrazole), 160.01, 169.1 (2 × C=O); Anal. Calcd.

for C17H20N6O3 (356.38): C, 57.29%; H, 5.66%; N, 23.58%. Found: C, 57.11%; H, 5.64%; N, 23.38%.

1-Phenyl-4-((1H-tetrazol-5-yl)methyl)-1,4-diazaspiro[5.5]undecane-3,5-dione (6v). Yield: 75%;

yellow viscous oil; IR (KBr, ν, cm−1) exhibited band at 3400 (NH) and disappearance of CN band; 1H-NMR (CDCl3) δ ppm 0.87 (br.s, 2H, CH2 cyclohexyl), 1.18–1.98 (m, 8H, 4 × CH2, cyclohexyl),

4.09 (s, 2H, O=C-CH2-N), 5.03 (s, 2H, N-CH2-tetrazole), 7.05–7.13 (m, 5H, Har and 1H, NH); 13C-NMR (CDCl3) δ ppm 13.58, 25.37, 31.36 (5 × CH2, cyclohexyl), 55.01 (N-CH2-tetrazole), 58.88

(O=C-CH2-N), 60.77 (Cq), 125.93, 127.15, 129.38 (CHar.), 147.86 (Car.), 153.74 (C=N-tetrazole),

170.57, 175.98 (2 × C=O); MS (EI) m/z (%): 340.29 ([M]+, 3), 77.13 (100); Anal. Calcd. for


1-(4-Methylphenyl)-4-((1H-tetrazol-5-yl)methyl)-1,4-diazaspiro[5.5]undecane-3,5-dione (6w). Yield:

61%; yellow viscous oil; IR (KBr, ν, cm−1) exhibited band at 3419.79 (NH) and disappearance of CN band; 1H-NMR (CDCl3) δ ppm 1.47 (s, 3H, CH3), 1.51–1.59 (m, 6H, 3 × CH2, cyclohexyl), 2.14

(br.s, 4H, 2 × CH2 cyclohexyl), 4.05 (s, 2H, O=C-CH2-N ), 4.30 (s, 2H, N-CH2-tetrazole), 6.65 (s, 1H,

NH), 7.16 (d, 2H, J = 6.0 Hz, Har.), 7.19 (d, 2H, J = 6.0 Hz, Har.); 13C-NMR (CDCl3) δ ppm 22.66,

31.50, 34.94 (5 × CH2, cyclohexyl), 29.74 (CH3), 45.801 (N-CH2-tetrazole), 50.95 (O=C-CH2-N),

60.73 (Cq), 127.83, 130.13 (CHar.), 140.10, 145.92 (2 × Car.), 151.94 (C=N-tetrazole), 168.82, 170.42

(2 × C=O); MS (EI) m/z (%): 354.7 ([M]+, 3), 105.1 (100); Anal. Calcd. for C18H22N6O2 (354.41):

C, 61.00%; H, 6.26%; N, 23.71%. Found: C, 61.15%; H, 6.22%; N, 23.61%.

1-(4-Methoxyphenyl)-4-((1H-tetrazol-5-yl)methyl)-1,4-diazaspiro[5.5]undecane-3,5-dione (6x).

Yield: 66%; buff solid, m.p 73 °C; IR (KBr, ν, cm−1) exhibited band at 3419 (NH) and disappearance

of CN band; 1H-NMR (CDCl3) δ ppm 1.18–1.88 (m, 10H, 5 × CH2, cyclohexyl), 3.75 (OCH3), 4.20

(s, 2H, O=C-CH2-N), 5.29 (s, 2H, N-CH2-tetrazole), 6.73 (d, 2H, J = 7.0 Hz, Har.), 6.95 (d, 2H, J = 7.0 Hz,

Har.), 7.19 (s, 1H, NH); 13C-NMR (CDCl3) δ ppm 20.71, 25.04, 32.60 (5 × CH2, cyclohexyl), 36.59

(N-CH2-tetrazole), 41.32 (O=C-CH2-N), 56.50 (OCH3), 70.07 (Cq), 127.79, 129.88 (CHar.), 134.86,

145.77 (2 × Car.), 153.48 (C=N-tetrazole), 170.73, 176.09 (2 × C=O); MS (EI) m/z (%): 340.3

([M-OCH3]+, 5), 105.17 (100); Anal. Calcd. for C18H22N6O3 (370.41): C, 58.37%; H, 5.99%; N, 22.69%.

Found: C, 58.32%; H, 5.79%; N, 22.54%.

Int. J. Mol. Sci. 2014, 15 16931

3.2. Anticonvulsant Activity

3.2.1. Materials

Animals: The anticonvulsant activity of the target compounds 6a–x was tested on Swiss strain adult

male albino mice weighing 19–25 g. Animals were obtained from the Animals House Colony of the

National Research Centre, Cairo, Egypt. Animals were housed in polypropylene cages under the

standard conditions of light (12 h light/dark cycle) and temperature (23 ± 2 °C), and were allowed free

access to water and maintained on a daily standard schedule of laboratory diet. Procedures involving

animals and their care were performed after the Ethics Committee of the National Research Centre and

in accordance with the recommendations for the proper care and use of laboratory animals, “Canadian

Council on Animal Care Guidelines, 1984”. Additionally, all efforts were made to minimize animals

suffering and to use only the number of animals necessary to produce reliable data.

Drugs and Chemicals: Phenobarbital (Memphis Co. for Pharm & Chem. Ind., Cairo, Egypt),

Ethosuximide (Pfizer Co., Giza, Egypt), Diphenylhydantoin (Nasr Co., Giza, Egypt), Tween 80 and

Pentylenetetrazole (Sigma, St. Loius, MO, USA) were used. Ethosuximide, Phenobarbital and

Pentylenetetrazole (PTZ) were dissolved in physiologic saline solution, Diphenylhydantoin was

dissolved in saline that was alkalinized slightly with 0.1 mmol potassium hydroxide. Reference drugs

and tested compounds were administered intraperitoneally (i.p) in volumes of 0.1 mL/10 g of mice

body weight.

3.2.2. Methods

After 7 days of adaptation to laboratory conditions, the animals were randomly assigned to control,

reference and tested experimental groups consisting of 6 mice. Each mouse was used only once and all

tests were performed between 09:00 a.m. and 04:00 p.m. All the tested compounds were suspended in

7% Tween 80 as a vehicle.

Subcutaneous Pentyleneteterazole (scPTZ)-induced Seizures Test [28]: A PTZ dose of 85 mg/kg

administered subcutaneously to mice causes seizures in more than 97% of the animals. This is called the

convulsive dose 97 (CD97). The control experiments were performed using the solvent alone. The other

groups each received individually the reference drugs Ethosuximide (150 mg/kg ≡ 1.06 mmol/kg) [29]

and/or Phenobarbital (30 mg/kg ≡ 0.13 mmol/kg) [30] or one of the test compounds in graded doses,

6a–l (1.5–50 mg/kg), 6m–x (6–100 mg/kg). Thirty minutes later Pentylenetetrazole was administered

subcutaneously in a loose fold of skin on the back of the neck in a dose of 85 mg/kg. Each animal was

observed for 30 min after PTZ administration, failure to observe even a threshold seizure (a single

episode of clonic spasms of at least 5 s duration) was defined as protection [31]

Maximal Electroshock Seizure (MES) Test [32]: Animals were randomly assigned to groups of

6 mice each. The first group served as the control group. The second group received

Diphenylhydantoin (45 mg/kg) as a reference drug and the other groups of animals received the test

compounds individually by intraperitoneal injection with the dose which induces 100% protection in

the Pentylenetetrazole test. Thirty minutes later electroconvulsions were induced by a current

(fixed current intensity of 25 mA, 0.2 s stimulus duration) delivered via ear-clip electrodes by a Rodent

Shocker generator (constant-current stimulator Type 221, Hugo Sachs Elektronik, Freiburg, Germany).

Int. J. Mol. Sci. 2014, 15 16932

The maximal seizures typically consist of a short period of initial tonic flexion and a prolonged

period of tonic extension (especially of the hind limbs) followed by terminal clonus. The typical

seizure lasts approximately 22 s. Failure to extend the hind limbs to an angle with trunk greater than

90° is defined as protection [33].

Neurotoxicity [34]: This test is designed to detect minimal neurological deficit. In this test, the

animals were trained to maintain equilibrium on a rotating 1-inch-diameter knurled plastic rod at a

speed of 6 rev/min for at least 1 min in each of three trials using a rotarod device (UGO Basile, 47600,

Varese, Italy). Only animals that fulfill this criterion were included in the experiment. The selected

trained animals were classified into control and experimental groups. The animals in the experimental

groups were given the reference drug or one of the test compounds via i.p. route at doses which

exerted 100% protection in the PTZ test; meanwhile, the control group received the vehicle.

Thirty minutes later, the mice were placed again on the rotating rod and the neurotoxicity was

indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min.

3.2.3. Determination of the ED50

Anticonvulsant activity of the test compounds was expressed in term of median effective dose

(ED50) that is, the dose of drug required to produce the required biological response in 50% of animals.

For determination of the ED50, groups of 8 mice were given a range of i.p. doses of the test compound

until at least three points were established in the range of 15%–84% seizure protection. From the plot

of these data, the respective ED50 value and the confidence limits were calculated [18].

4. Conclusions

The anticonvulsant potential of certain new 6-aryl-9-substituted-6,9-diazaspiro[4.5]decane-8,10-diones

(6a–l) and 1-aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones (6m–x) was described. The title

compounds 6a–x showed good anticonvulsant activity especially in the scPTZ screen. Compound 6g

displayed an ED50 of 0.0043 mmol/kg in the scPTZ screen being about 14 and 214 fold more potent

than the reference drugs, Phenobarbital and Ethosuximide, respectively. Compound 6e exhibited

an ED50 of 0.019 mmol/kg being about 1.8 fold more potent than that of the reference drug,

Diphenylhydantoin in the MES screen. None of the test compounds exhibited any minimal motor

impairment at the maximum administered dose in the neurotoxicity screen.

Acknowledgments

The authors thank the National Research Centre, Dokki, Giza, Egypt, for the support of this

research through project No. 10010302 (2013–2016). The authors would like to extend their sincere

appreciation to the Deanship of Scientific Research at King Saud University for its funding of this

research through the Research Group Project No. RGP-VPP-196.

Int. J. Mol. Sci. 2014, 15 16933

Author Contributions

Mohamed N. Aboul-Enein, Aida A. El-Azzouny, Mohamed I. Attia and Fatma Ragab conceived the

study, designed the work, contributed in the strategy of the chemistry part, performed interpretation of

the analytical data of the prepared compounds, prepared the manuscript and revised it for publication.

Yousreya A. Maklad designed the pharmacological part, contributed in performing pharmacology

experiments and revised the manuscript. Mona E. Aboutabl participated in conducting pharmacology

experiments. Walaa H. A. Abdel-Hamid synthesized all compounds and participated in conducting

pharmacology experiments.

Conflicts of Interests

The authors declare no conflict of interest.

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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/3.0/).

SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL COUMARIN DERIVATIVES AS POTENTIAL ANTIMICROBIALs AGENTS

Original Article

KAMILIA M. AMINa, SAHAR M. ABOU-SERIa, RANA M. ABDELNABYb, HEBA S. RATEBb,d, MAHMOUD A. F. KHALILc, MOHAMED M. HUSSEINa,b

aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, Egypt, bPharmaceutical Chemistry Department, Faculty of Pharmacy, Misr University for Science and Technology, Al-Motamayez District, 6th of October City,

Egypt, cMicrobiology Department, Faculty of Pharmacy, Misr University for Science and Technology, Al-Motamayez District, 6th of October City, Egypt, dDepartment of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al-Madinah Al-

munawara, 30001, Kingdom of Saudi Arabia. Email: [email protected]

Received: 09 Jan 2016 Revised and Accepted: 11 Feb 2016

ABSTRACT

Objective: Synthesize new series of 7-hydroxy-4-methylcoumarin and 7-alkoxy-4-methylcoumarin derivatives featuring thiosemicarbazone or thiazolidin-4-one moieties and to evaluate their antimicrobial activity against two strains of Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), two Gram-negative bacteria (Escherichia Coli and Pseudomonas aeruginosa), and Candida albicans.

Methods: Preparation of the new coumarin derivatives was done by adopting Pechmann condensation and attaching different isothiocyanates to give coumarin-thiosemicarbazone hybrids. Thiosemicarbazones were cyclized into thiazolidine-4-ones using chloroacetic acid or diethyl bromo malonate.

Results: Compounds VIb, Xb, XIVb, and XVc gave the highest inhibition zones (>20 mm) against Staphylococcus aureus. Their MIC (minimum inhibitory concentration) values ranging from 0.19-0.36 µg/ml were better than the reference drug tobramycin with MIC= 2µg/ml.

Conclusion: The newly synthesized compounds with the 7-hydroxyl group showed better antimicrobial activity than those with the 7-alkoxy groups.

Keywords: Coumarin, Thiosemicarbazones, Thiazolidin-4-ones, Antimicrobial activity

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Coumarins are a class of naturally occurring compounds, found in variable levels throughout the plant kingdom. Some important coumarins were isolated from microorganisms such as novobiocin 1 from Streptomyces species. They are used by plants as pesticides to protect themselves from predators [1, 2]. Applications of coumarins range from additives in food, perfumes, and cosmetics, to the preparation of insecticides, optical brighteners, and tunable laser dyes [3]. Today coumarins are very important in the pharmaceutical field due to their wide occurrence, and versatile pharmacological activity associated with low toxicity profile such as antimicrobial, anticoagulant, antioxidant, and anticancer activities [1-4]. Coumarin itself was reported to have an immunostimulatory activity on macrophages and other cells of the immune system. This results in the use of coumarin in chronic infections such as chronic brucellosis, mycoplasmosis, toxoplasmosis, and Q fever [1].

Novobiocin 1 and clorobiocin 2 are DNA-gyrase inhibitors having a strong activity against Gram-positive bacteria especially methicillin-resistant strains of Staphylococcus aureus (MRSA) [fig. 1]. But due to limitations regarding solubility, toxicity, and development of resistance, efforts were dedicated to designing an effective, orally bioavailable antimicrobial agents bearing coumarin nucleus [4]. Over the past decades, thiosemicarbazones attracted researchers for thorough investigation due to their diverse biological activity. They were known to have antiviral [5], antibacterial [6], anti-tuberculosis [7], anti-Trypanosoma cruzi [8] and antineoplastic activities [9]. This wide range of pharmacological activities was attributed to the strong chelating ability of thiosemicarbazones ligand to biologically important metals like iron, copper, nickel, and to their reductive capacities [10]. In 2011, Patil et al. reported the synthesis of new coumarin-8-yl-thiosemicarbazones 3, 4 that possessed potential antibacterial activity against S. aureus, S. typhi, and E. coli [11]. Also,

thiosemicarbazones act as key intermediates in the preparation of important compounds that in turn have a potential antimicrobial activity such as thiazolidin-4-one derivatives. Thiazolidine-4-one derivative 5 possessed comparable activity to ampicillin and chloramphenicol at a concentration 25 µg/ml [12].

Also, 4-methylcoumarin-thiazolidine-4-one hybrids 6 and 7 were reported to exhibit good antimicrobial activity; the former compound had comparable activity to ciprofloxacin and griseofulvin at 10 µg/ml [13, 14] and the later possessed potent antifungal activity with MIC value of 0.10 µg/ml [15] (fig. 1).

Thus, the purpose of this work was to study the effect of hybridizing 7-hydroxy-4-methylcoumarin and their 7-alkoxy analogs with different N4-substituted thiosemicarbazone that were cyclized into the C5-substituted-thiazolidine-4-one ring (fig. 2). The antimicrobial activity of new compounds VI-XVII was evaluated.


Starting materials and reagents were purchased from Sigma-Aldrich and were used without further purification. Melting points were determined using Electrothermal capillary melting point apparatus 9100 and were uncorrected. IR spectra were recorded on a Shimadzu FT-IR Affinity-1 Spectrophotometer, using KBr discs at MUST University. 1H-NMR and 13C-NMR spectra were recorded in δ scale given in ppm and performed on a JEOL ECA 300, 400 MHz spectrometer using CDCl3 or DMSO as stated, using TMS as an internal standard at Cairo University.

Mass spectra were performed on Shimadzu Qp-2010 plus (70 eV) spectrometer at Cairo and Azhar University. Elemental analysis was performed at Azhar University. The microorganisms were purchased from Microbiological Resources Centre (MIRCEN), Faculty of agriculture, Ain-Shams University.

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 8, Issue 4, 2016

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Synthesis of 8-Acetyl-7-alkoxy-4-methylcoumarin (IIIa-c): General Procedures: The 7-hydroxy Compound II (2.18 g., 0.010 mol) was stirred in dry acetone with anhydrous K2CO3 (1.5 g., 0.011 mol) for one hour, then the appropriate alkyl halide (ethyl iodide for IIIa, allyl bromide for IIIb, butyl bromide for IIIc) (0.050 mol) was added to the solution. The reaction mixture was refluxed for 8 h,

concentrated and poured onto ice cold water. The solid formed was filtered and recrystallized from ethanol.

8-Acetyl-7-ethoxy-4-methylcoumarin (IIIa): Yield: 98%; m. p: 123-124 ᵒC; IR (ṽ max, cm-1): 3084 (CH, Ar), 2980 (CH, aliphatic), 1728 (CH3-C-C=O), 1705 (C=O, α–pyrone), 1598 (C=C, Ar).

O O

CH3

NS

O (7)

F

Cl

NN

S

NO

(5) (6)

O O

CH3

OHN

ONS

N

O

OCH3

O

OH

O

HN

O

OH

CH3

CH3

R1

O

O

OHO

O

R2

H3CO

N

CH3

Novobiocin (1): R1= CH3, R2= NH2

Clorobiocin (2): R1= Cl, R2=

O O

N

HO

CH3

NHC

HN

S

R1

(3), R1= Cl(4), R1= NO2

Fig. 1: Some reported lead antimicrobials having the main pharmacophores under investigation

Fig. 2: Design strategy for the new compounds VI-XVII

8-Acetyl-7-allyloxy-4-methylchromen-2-one (IIIb): Yield: 97%; m. p: 118-120 ᵒC; IR (ṽ max, cm-1): 3088 (CH, Ar), 2991 (CH, aliphatic), 1724 (CH3-C-C=O), 1703 (C=O, α–pyrone), 1598 (C=C, Ar and allyl); MS (m/z): 258.

8-Acetyl-7-butoxy-4-methylcoumarin (IIIc): Yield: 97%; m. p: 89-90 ᵒC; IR (ṽ max, cm-1): 3084 (CH, Ar), 2987 (CH, aliphatic), 1716 (CH3-C-C=O), 1703 (C=O, α–pyrone), 1597 (C=C, Ar).

Synthesis of 8-Acetyl-7-substituted-4-methylcoumarin-hydrazones (IV and Va-Vc): General Procedures: 8-Acetyl-7-substituted-4-methylcoumarins II and IIIa-c (0.010 mol) were dissolved in 25 ml ethanol, poured onto hydrazine hydrate 99% (0.55 ml, 0.011 mole) and heated under reflux for 2 h. Light yellow crystals of the hydrazones were separated, collected by filtration and washed with water.

8-Acetyl-7-hydroxy-4-methylcoumarin-hydrazone (IV): Yield= 68%; m. p: 205-208 ᵒC; IR (ṽ max, cm-1): 3468 (OH), 3381 and 3381 (NH2), 2926 (CH, aliphatic), 1697 (C=O, α–pyrone), 1558 (C=C, Ar).

8-Acetyl-7-ethoxy-4-methylcoumarin-hydrazone (Va): Yield= 92%; m. p: 138-140 ᵒC; IR (ṽ max, cm-1): 3412 and 3234 (NH2), 3051 (CH, Ar), 2981 (CH, aliphatic), 1708 (C=O, α–pyrone), 1597 (C=C, Ar).

8-Acetyl-7-allyloxy-4-methylcoumarin-hydrazone (Vb): Yield= 99%; m. p: 84-86 ᵒC; IR (ṽ max, cm-1): 3379 and 3226 (NH2), 3088 (CH, Ar), 2968 (CH, aliphatic), 1724 (C=O, α–pyrone), 1597 (C=C, Ar); MS (m/z):247.

8-Acetyl-7-butoxy-4-methylcoumarin-hydrazone (Vc): Yield= 60%; m. p: 146-148 ᵒC; IR (ṽ max, cm-1): 3390 (NH), 3084 (CH, Ar), 2987 (CH, aliphatic), 1707 (C=O, α–pyrone), 1622 (C=N, imine),

Abdelnaby et al.


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1597 (C=C, Ar); Anal. Calc: C, 66.65; H, 6.99; N, 9.72; Found: C, 66.91; H, 7.12; N, 9.89.

Synthesis of thiosemicarbazones (VI-IX): General Procedures: The hydrazones IV and Va-Vc (0.005 mol) were dissolved in the minimal amount of dimethyl formamide diluted with 20 ml ethanol then the appropriate isothiocyanate derivative (0.005 mol) was added. The solution was refluxed for 8 h then diluted with iced cold water. A crystalline solid was separated, collected, and recrystallize from ethanol.

8-Acetyl-7-hydroxy-4-methylcoumarin-4-benzyl thiosemicarbazone (VIa): Yield= 74.9%; m. p: 220-222 ᵒC; IR (ṽ max, cm-1): 3466 (OH), 3406 and 3292 (2NH), 3040 (CH, Ar), 1720 (C=O, α–pyrone), 1616 (C=N imine), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.54 (s, 3H, N=C-CH3), 2.45 (s, 3H,-C=C-CH3), 4.56 (d, 2H,-CH2-Ph, J=4 Hz), 6.16 (s, 1H, 3-H), 6.94 (d, 1H, 6-H, J= 8 Hz), 7.29-7.32 (m, 5H, Ar), 7.52 (d, 1H, 5-H, J=8 Hz), 6.12 (s, 1H, OH, D2O exchangeable), 9.42 and 7.97 (s, 2H, 2NH, D2O exchangeable); MS (m/z): 381, 383; Anal calcd. C, 62.97; H, 5.02; N, 11.02; found: C, 63.14; H, 5.09; N, 11.17.

8-Acetyl-7-hydroxy-4-methylcoumarin-4-benzoyl thiosemicarbazone (VIb): Yield= 73%; m. p: 220-223 ᵒC; IR (ṽ max, cm-1): 3547 (OH), 3234 and 3473 (2NH), 3120 (CH, Ar), 1728 and 1662 (2C=O), 1635 (C=N imine), 1598 (C=C, Ar); 1HNMR (300 MHz, CDCl3): δ= 2.4 (s, 3H, N=C-CH3), 2.7 (s, 3H, C4-CH3), 6.15 (s,1H, H-3 of coumarin), 7.03 (d, 1H, C6-H of coumarin, J= 9 Hz), 7.59 (t, t, 2H, C3-H, C5-H of phenyl, J=6 Hz), 7.68 (d, d, 2H, C2-H,C6-H of phenyl, J=6 Hz), 7.93 (d, 1H, C6-H of coumarin), 9.29 and 13.85 (s, 2H, 2NH, D2O exchangeable); MS (m/z): 395; Anal calcd. C, 60.75; H, 4.33; N, 10.63; found: C, 60.89; H, 4.38; N, 10.79.

8-Acetyl-7-ethoxy-4-methylcoumarin-4-cyclohexyl thiosemicarbazone (VIIa): Yield= 92%; m. p: 190-191 ᵒC; IR (ṽ max, cm-1): 3244 and 3142 (2NH), 3014 (CH, Ar), 2931 (CH, aliphatic), 1728 (C=O), 1629 (C=N imine), 1597 (C=C, Ar); 1HNMR (300 MHz, CDCl3): δ= 1.47 (t, 3H, CH3-, J=6 Hz), 2.31 (s,3H, N=C-CH3), 2.37-2.43 (m, 6H,C3-2H,C4-2H, and C5-2H of cyclohexyl), 2.71 (s, 3H, C4-CH3), 2.82-2.89 (m, 4H, C2-2H, C6-2H of cyclohexyl), 3.22 (m, 1H, C1-H of cyclohexyl), 4.23 (q, 2H,-CH2-O, J=6 Hz), 5.99 (s,1H, NH, D2O exchangeable), 6.17 (s,1H, C3-H), 6.94 (d,1H, C6-H, J=9 Hz), 7.64 (d,1H, C5-H, J=9); MS (m/z): 401; Anal calcd. C, 62.82; H, 6.78; N, 10.47; found: C, 63.04; H, 6.86; N, 10.61.

8-Acetyl-7-ethoxy-4-methylcoumarin-4-phenyl thiosemicarbazone (VIIb): Yield= 98%; m. p: 110-112 ᵒC; IR (ṽ max, cm-1): 3444 and 3460 (2NH), 3055 (CH, Ar), 2981 (CH, aliphatic), 1734 (C=O), 1597 (C=C, Ar), 1174 (C-O ether); 1HNMR (300 MHz, CDCl3): δ= 1.45 (t, 3H, CH3-, J=6 Hz), 2.36 (s, 3H, N=C-CH3), 2.42,(s, 3H, 4-CH3), 4.22 (m, 2H,-CH2-O, J=6 Hz), 6.20 (s, 1H, C3-H), 6.95 (d, 1H, C6-H, J=9 Hz), 7.32 (t, 1H, C4-H of phenyl), 7.37 (d, 2H, C2-H, C6-H of phenyl, J=9 Hz), 7.65 (d, 2H, C3-H, C5-H of phenyl, J=9 Hz), 7.69 (d,1H, C5-H), 9.84 (s,1H, NH, D2O exchangeable), 10.46 (s,1H, OH, D2O exchangeable); 13CNMR (400 MHz, CDCl3): δ= 14.6 (CH3-), 18.6 (CH3-), 23.2 (CH3-), 32.6 (C5 of thiazolidin-4-one), 64.5 (-CH2-O), 107.9 (C3 of coumarin), 108.3 (C8 of coumarin), 112.0 (C6 of coumarin), 117.3 (C10 of coumarin), 125.0 (C2, C6 of phenyl), 127.3 (C4 of phenyl), 128.2 (C3, C5 of phenyl), 129.1 (C5 of coumarin), 134.5 (C1 of phenyl), 151.9 (C9 of coumarin), 152.1 (C4 of coumarin), 157.4 (C7 of coumarin), 159.2 (C2 of coumarin), 160.3 (C2 of thiazolidin-4-one), 161.1 (-C=N-), 171.5 (-C=O of thiazolidin-4-one); MS (m/z): 395; Anal calcd. C, 63.78; H, 5.35; N, 10.63; found: C, 63.97; H, 5.42; N, 10.88.

8-Acetyl-7-ethoxy-4-methylcoumarin-4-(4-methoxyphenyl) thiosemicarbazone (VIIc): Yield= 99%; m. p: 206-208 ᵒC; IR (ṽ max, cm-1): 3319 and 3278 (2NH), 3062 (CH, Ar), 2837-2981 (CH, aliphatic), 1722 (C=O), 1595 (C=C, Ar), 1182 (C-O ether); 1HNMR (300 MHz, CDCl3): δ= 1.45 (t, 3H, CH3-, J=8 Hz), 2.35 (s, 3H, CH3-), 2.45 (s, 3H, C4-CH3), 3.83 (s, 3H, CH3-O-Ph), 4.17-4.24 (m, 2H,-CH2-O, J=8 Hz), 6.20 (s, 1H, 3-H), 6.88-6.97 (m, 4H, Ar), 7.48 (d, 1H, C6-H, J=8 Hz), 7.67 (d,1H, C5-H, J=8 Hz), 8.27 and 9.19 (s,2H, 2NH, D2O exchangeable); MS (m/z): 426; Anal calcd. C, 62.10; H, 5.45; N, 9.88; found: C, 62.42; H, 5.51; N, 10.03.

8-Acetyl-7-allyloxy-4-methylcoumarin-4-cyclohexyl thiosemicarbazone (VIIIa): Yield= 96%; m. p: 206-208 ᵒC; IR (ṽ

max, cm-1): 3354 and 3244 (2NH), 3053 (CH, Ar), 2962 (CH, aliphatic), 1724 (C=O), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.59 (s, 3H, N=C-CH3), 1.88-1.92 (m, C3-H, C4-H, and C5-H of cyclohexyl), 2.11-2.17 (m C2-H, C6-H of cyclohexyl), 2.37 (m, 1H, C1-H of cyclohexyl), 2.42 (s, 3H, C4-CH3), 4.77 (d, 2H,–CH2-O, J= 8 Hz), 5.23 and 5.31 (d, d, 2H, CH2=, J=8 Hz), 5.96-6.00 (m, 1H, =CH–), 6.12 (s, 1H, C3-H), 6.92 (d, 1H, C6-H, J=8 Hz), 7.53 (d, 1H, C5-H, J=8 Hz); MS (m/z): 413; Anal calcd. C, 63.90; H, 5.92; N, 10.16; found: C, 64.08; H, 5.97; N, 10.31.

8-Acetyl-7-allyloxy-4-methylcoumarin-4-benzyl thiosemicarbazone (VIIIb): Yield= 84%; m. p: 102-104ᵒC; IR (ṽ max, cm-1): 3419 and 3367 (2NH), 3084 (CH, Ar), 2980 (CH, aliphatic), 1732 (C=O), 1598 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 2.26 (s, 3H, N=C-CH3), 2.43 (s, 3H, C4-CH3), 4.50 (d, 2H,-CH2-O-, J= 8 Hz), 4.76 (d, 2H, Ph-CH2-), 5.33 and 5.36 (d, d, 2H, CH2=, J=8 Hz), 5.95-6.04 (m, 1H, =CH–), 6.20 (s, 1H, C3-H), 6.95 (d, 1H, C6-H, J=8 Hz), 7.25-7.40 (m, 5H, Ar), 7.54 (d, 1H, C5-H, J=8 Hz), 8.2 and 8.8 (s, 2H, 2NH, D2O exchangeable); 13CNMR (400 MHz, CDCl3): δ= 18.7 (CH3-), 23.5 (-CH3), 48.4 (-CH2-ph), 69.7 (-CH2-O-), 109.0 (C3 of coumarin), 110.0 (C8 of coumarin), 112.3 (C6 of coumarin), 114.7 (CH2=), 118.7 (C10 of coumarin), 125.7 (C4 of phenyl), 127.4 (C2, C6 of phenyl), 127.9 (C3, C5 of phenyl), 128.7 (C5 of coumarin), 131.6 (=CH-), 137.5 (C1 of phenyl), 142.9 (C9 of coumarin), 151.1 (C4 of coumarin), 152.1 (-C=N-), 157.1 (C2 of coumarin), 159.8 (C7 of coumarin), 177.6 (-C=S); MS (m/z): 421; Anal calcd: C, 65.54; H, 5.5; N, 9. 97; found: C, 65.73; H, 5.54; N, 10.08.

8-Acetyl-7-allyloxy-4-methylcoumarin-4-(4-methoxyphenyl) thiosemicarbazone (VIIIc): Yield= 95%; m. p: 186-187 ᵒC; IR (ṽ max, cm-1): 3319 and 3278 (2NH), 3032, (CH, Ar), 2970 (CH, aliphatic), 1720 (C=O), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 2.29 (s, 3H,-N=C-CH3), 2.43 (s, 3H, C4-CH3), 3.81 (s, 3H, CH3-O-Ph-), 4.69 (d, 2H,-CH2-O-, J= 8 Hz), 5.37 (d, 2H, CH2=, J=8 Hz), 6.02 (m, 1H, =CH–), 6.18 (s, 1H, C3-H), 6.88-6.97 (m, 4H, phenyl), 7.47-7.51 (d, 2H, C5-H, C6-H, J=8 Hz), 8.8 and 9.10 (s, 2H, 2NH, D2O exchangeable); MS (m/z): 437, 438 (M+1); Anal calcd. C 63.14; H 5.30; N 9.60; found: C 63.29; H 5.32; N 9.67.

8-Acetyl-7-butoxy-4-methylcoumarin-4-ethyl thiosemicarbazone (IXa): Yield= 99%; m. p: 170-169 ᵒC; IR (ṽ max, cm-1): 3448 and 3220 (2NH), 2954 (CH, aliphatic), 1732 (C=O), 1598 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 0.98 (t, 3H, CH3-, J= 8 Hz), 1.25 (t, 3H, CH3-, J= 8 Hz), 1.47 (m, 2H,-CH2-, J= 8 Hz), 1.78 (m, 2H,-CH2-, J= 8 Hz), 2.22 (s, 3H,-N=C-CH3), 2.43 (s, 3H, C4-CH3), 3.72 (q, 2H,-CH2-N-, J= 8 Hz), 4.09 (t, 2H,-CH2-O-, J= 8 Hz), 6.17 (s, 1H, C3-H), 6.92 (d,1H, C6-H), 7.58 (d, H, C5-H, J= 8 Hz), 8.64 (s, 1H, NH, D2O exchangeable); MS (m/z): 375; Anal calcd. C, 60.78, H, 6.71, N, 11.19; found: C, 60.91; H, 6.82; N, 11.31.

8-Acetyl-7-butoxy-4-methylcoumarin-4-benzyl thiosemicarbazone (IXb): Yield= 99%; m. p: 140-142 ᵒC; IR (ṽ max, cm-1): 3419 and 3253 (2NH), 3088 (CH, Ar), 2960 (CH, aliphatic), 1732 (C=O), 1602 (C=N imine), 1550 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 0.96 (t, 3H, CH3-, J= 8 Hz), 1.44 (m, 2H,-CH2-, J= 8 Hz), 1.74 (m, 2H,-CH2-, J= 8 Hz), 2.24 (s, 3H, N=C-CH3), 2.40 (s, 3H, C4-CH3), 4.05 (t, 2H,-CH2-O, J= 8 Hz), 4.92 (s, 2H,-CH2-ph), 6.15 (s, 1H, C3-H), 6.88 (d,1H, C6-H, J= 8 Hz), 7.26-7.37 (5H, phenyl), 7.55 (d, H, C5-H, J= 8 Hz), 8.79 (s, 1H, NH, D2O exchangeable) MS (m/z): 437; Anal calcd. C, 65.88; H, 6.22; N, 9.60; found: C, 65.98; H, 6.28; N, 9.72.

Synthesis of thiazolidine-4-ones (X-XIII): General procedures: The thiosemicarbazones VI-IX (0.005 mol) were reacted with chloroacetic acid (0.00505 mol, 0.618 g) in freshly fused sodium acetate (0.00505 mol, 0.414 g) and 30 ml ethanol. The solution was refluxed for 8 h, concentrated, and diluted with ice cold water. A crystalline solid was separated, collected, and recrystallized from ethanol.

3-Benzyl-2-{[1-(7-hydroxy-4-methylcoumarin-8-yl)-ethylidene] -hydrazono}-thiazolidin-4-one (Xa): Yield= 67%; m. p: 144-146 ᵒC; IR (ṽ max, cm-1): 3444 (OH), 3089 (CH, Ar), 2924 (CH, aliphatic), 1720 and1687 (2C=O), 1629 (C=N imine), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.56 (s, 3H, 4-CH3), 2.46 (s, 3H, N=C-CH3), 2.85 (s, 2H, S-CH2-CO), 4.57 (s, 2H, Benzyl CH2), 6.16 (s, 1H, C3-H), 6.95 (d,1H, C6-H), 7.35 (m, 5H, Ar), 7.62 (d, 2H, 4, C5-H, J= 8 Hz); MS

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(m/z): 421; Anal calcd. C 62.69; H 4.54; N 9.97; found: C 62.78; H 4.51, N 10.08.

3-Benzoyl-2-{[1-(7-hydroxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one (Xb): Yield= 75%; m. p: 128-130 ᵒC; IR (ṽ max, cm-1): 3446 (OH), 3066 (CH, Ar), 2980 (CH, aliphatic), 1728 and1670 (2C=O), 1624 (C=N imine), 1598 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 2.44 (s, 3H, 4-CH3), 2.84 (s, 3H, N=C-CH3), 3.83 (s, 2H, S-CH2-CO), 6.19 (s, 1H, C3-H), 6.95 (d,1H, C6-H, J=8 Hz), 7.50-7.62 (m, 3H, C3, C4, C5-H of phenyl), 7.70 (d,1H, C5-H, J=8 Hz); MS (m/z): 435; Anal calcd: C 60.68; H 3.93; N 9.65; found: C, 60.74; H, 3.96; N, 9.77.

3-Cyclohexyl-2-{[1-(7-ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one (XIa): Yield= 56%; m. p: 246-247 ᵒC; IR (ṽ max, cm-1): 3059 (CH, Ar), 2978 (CH, aliphatic), 1716 (C=O), 1620 (C=N imine), 1597 (C=C, Ar); 1HNMR (400 MHz, DMSO): δ= 1.08 (t, 3H, CH3-CH2-O), 2.07 (s, 3H, 4-CH3), 2.16 (s, 3H, N=C-CH3), 2.27 (t, 4H, 2andC6-H of cyclohexyl), 3.65 (m, 1H, C1-H of cyclohexyl), 3.68 (s, 2H, S-CH2-CO), 3.93 (t, 2H, CH3-CH2-O, J= 8 Hz), 5.98 (s, 1H, C3-H), 6.91 (d,1H, C6-H, J=8 Hz), 7.52 (d, 2H, 4, C5-H, J=8 Hz); MS (m/z): 441; Anal calcd: C, 62.56; H, 6.16; N, 9.52; found: C, 62.67; H, 6.30; N, 9.61.

2-{[1-(7-Ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-phenyl-thiazolidin-4-one (XIb): Yield= 89%; m. p: 257-260 ᵒC; IR (ṽ max, cm-1): 3064 (CH, Ar), 2980 (CH, aliphatic), 1732 and 1720 (2C=O), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.32 (t, 3H, CH3-CH2-O, J=8 Hz), 2.23 (s, 3H, C4-CH3), 2.39 (s, 3H, N=C-CH3), 3.92 (m, 4H, S-CH2-CO and CH3-CH2-O), 6.10 (s, 1H, C3-H), 6.71 (d,1H, C6-H, J=8 Hz), 6.95 (d, 2H, C2, C6-H of phenyl, J= Hz), 7.11 (m, 3H, C3,4,C5-H of phenyl), 7.41 (d, 2H, 4, C5-H, J=8 Hz); MS (m/z): 435; Anal calcd. C, 63.43; H, 4.86; N, 9.65; found: C 63.65; H 4.92; N 9.78.

2-{[1-(7-Ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-(4-methoxy-phenyl)-thiazolidin-4-one (XIc): Yield= 89%; m. p: 170-172 ᵒC; IR (ṽ max, cm-1): 2980 (CH, aliphatic), 1718 (C=O), 1624 (C=N imine), 1598 (C=C); 1HNMR (400 MHz, CDCl3): δ= 1.34 (t, 3H, CH3-CH2-O, J=8 Hz), 2.19 (s, 3H, 4-CH3), 2.41 (s, 3H, N=C-CH3), 3.75 (s, 3H, CH3-O-Ph), 3.88 (s, 2H, S-CH2-CO), 4.05 (q, 2H, CH3-CH2-O), 6.10 (s, 1H, C3-H), 6.87 (d,1H, C6-H, J=8 Hz), 6.89-7.39 (m, 5H, Ar), 7.55 (d, 2H, 4, C5-H, J=8 Hz),; MS (m/z): 465; Anal calcd: C, 64.13; H, 5.16; N, 9.35; found: C, 64.28; H, 5.20; N, 9.43.

3-Cyclohexyl-2-{[1-(7-allyloxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one (XIIa): Yield= 91%; m. p: 114-116 ᵒC; IR (ṽ max, cm-1): 3080 (CH, Ar and allyl), 2962 (CH, aliphatic), 1720 (C=O), 1618 (C=N imine), 1598 (C=C, Ar, allyl); 1HNMR (400 MHz, CDCl3): δ= 2.10 (s, 3H, C4-CH3), 2.17 (s, 3H, N=C-CH3), 3.45 (s, 2H, S-CH2-C=O), 3.74-3.82 (m, 1H, C1-H of cyclohexyl), 4.40 (d, 2H, CH2=CH-CH2-O-, J= 8 Hz), 5.01-5.15 (d, d, 2H, CH2=CH-CH2-O-, J=8 Hz), 5.72-5.76 (m, 1H, CH2=CH–CH2-O-), 6.69 (d, 1H, C6-H, J=8 Hz), 7.31 (d, 1H, C5-H, J=8 Hz); MS (m/z): 453; Anal calcd.: C, 63.56; H, 6.00; N, 9. 26; found, C, 63.78; H, 6.13; N, 9.49.

3-Benzyl-2-{[1-(7-allyloxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one (XIIb): Yield= 81%; m. p: 182-184 ᵒC; IR (ṽ max, cm-1): 3005-3086 (CH, Ar and allyl), 2981 (CH, aliphatic), 1728 and 1710 (2C=O), 1622 (C=N imine), 1600 (C=C, Ar, allyl); 1HNMR (400 MHz, CDCl3): δ= 2.35 (s, 3H, C4-CH3), 2.41 (s, 3H, N=C-CH3), 3.77 (s, 2H, S-CH2-C=O), 4.56 (d, 2H, CH2=CH-CH2-O-, J= 8 Hz), 4.68 (d, 2H, Ph-CH2-NH-), 5.31 and 5.37 (d, d, 2H, CH2=CH-CH2-O-, J=8 Hz), 5.92-6.05 (m, 1H, CH2=CH–CH2-O-), 6.16 (s, 1H, C3-H), 6.88-7.56 (m, 5H, Ar); MS (m/z): 461; Anal calcd. C, 65.06; H, 5.02; N, 9.10; found: C, 65.24; H, 5.11; N, 9.31.

3-(4-Methoxy-phenyl)-2-{[1-(7-allyloxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one (XIIc): Yield= 97%; m. p: 200-202 ᵒC; IR (ṽ max, cm-1): 3057 (CH, Ar and allyl), 2966 (CH, aliphatic), 1724 (C=O), 1616 (C=N imine), 1597 (C=C, Ar, allyl); 1HNMR (400 MHz, CDCl3): δ= 2.3 (s, 3H, C4-CH3), 2.40 (s, 3H, N=C-CH3), 3.74 (s, 3H, methoxy), 3.89 (s, 2H, S-CH2-C=O), 4.50 (d, 2H, CH2=CH-CH2-O-, J=8 Hz), 5.20-5.31 (d, d, 2H, CH2=CH-CH2-O-), 5.85-5.94 (m, 1H, CH2=CH–CH2), 6.09 (s, 1H, C3-H), 6.75 (d, 2H, C3 and C5 of phenyl, J= 8 Hz), 6.72 (d, 1H, C6-H, J=8 Hz), 6.85 (d, 2H, C2 and C6

of phenyl, J= 8 Hz), 7.42 (d, 1H, C5-H, J=8 Hz); MS (m/z): 477; Anal calcd. C, 62.88; H, 4.85; N, 8.80; found: C, 63.01; H, 4.89; N, 8.92.

2-{[1-(7-Butoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-ethyl-thiazolidin-4-one (XIIIa): Yield= 99%; m. p: 201-203 ᵒC; IR (ṽ max, cm-1): 3080 (CH, Ar), 2953 (CH, aliphatic), 1737 and 1722 (2C=O), 1571 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 0.96 (t, 3H, CH3-(CH2)3-O, J= 8 Hz), 1.34 (t, 3H, CH3-CH2-N, J= 8 Hz), 1.48 (m, 2H, CH3-CH2-(CH2)2-O, J= 8 Hz), 1.79 (m, 2H, CH3-CH2-CH2-CH2-O, J= 8 Hz), 2.36 (s, 3H, C4-CH3), 2.41 (s, 3H,-N=C-CH3), 3.73 (s, 2H, S-CH2-C=O), 3.94 (t,2H, CH3-CH2-N-, J= 8 Hz), 4.09 (t, 2H, CH3-CH2-CH2-CH2-O, J= 8 Hz), 6.14 (s, 1H, C3-H), 6.90 (d,1H, C6-H, J= 8 Hz), 7.54 (d, H, C5-H, J= 8 Hz); MS (m/z): 415; Anal calcd. C, 60.70; H, 6.06; N, 10.11; found: C, 60.37; H, 5.38; N, 10.69.

3-Benzyl-2-{[1-(7-butoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one (XIIIb): Yield= 91%; m. p: 180-182 ᵒC; IR (ṽ max, cm-1): 3082 (CH, Ar), 2939 (CH, aliphatic), 1720 (C=O), 1589 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 0.91 (t, 3H, CH3-(CH2)3-O, J= 8 Hz), 1.39 (m, 2H, CH3-CH2-(CH2)2-O, J= 8 Hz), 1.68 (m, 2H, CH3-CH2-CH2-CH2-O, J= 8 Hz), 2.39 (s, 3H, C4-CH3), 2.43 (s, 3H, N=C-CH3), 3.76 (s, 2H, S-CH2-C=O), 3.91-3.95 (t, 2H, CH3-CH2-CH2-CH2-O, J= 8 Hz), 4.54 (s, 2H, CH2-N-), 6.12 (s, 1H, C3-H), 6.90 (d,1H, C6-H, J= 8 Hz), 7.05 (m, 3H, C2,6,4 of phenyl), 7.15 (t, 2H, C3,5 of phenyl), 7.55 (d, H, C5-H); MS (m/z): 474; Anal calcd. C, 65.39; H, 5.70; N, 8.80; found: C, 65.62; H, 5.78; N, 8.91.

Synthesis of thiazolidine-4-ones (XIV-XVII): General procedures: The thiosemicarbazones V-VIII (0.005 mol) were reacted with diethyl bromo malonate (0.00505 mol, 1.207 g) in freshly fused sodium acetate (0.00505 mol, 0.41400 g) and 30 ml ethanol. The solution was refluxed for 8 h, concentrated, and diluted with ice cold water. A crystalline solid was separated, collected, and recrystallized from ethanol.

3-Benzyl-2-{[1-(7-hydroxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one-5-carboxylic acid ethyl ester (XIVa): Yield= 91%; m. p: 137 ᵒC; IR (ṽ max, cm-1): 3088 (CH, Ar), 2981 (CH, aliphatic), 1737 and 1724 (2C=O), 1595 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 2.37 (s, 3H, C4-CH3), 2.45 (t, 3H, CH3-), 2.79 (s, 3H,-N=C-CH3), 4.42-4.91 (m, 4H, 2CH2-of benzyl and ethyl), 5.1 (s, 2H, S-CH2-C=O), 6.30 (s, 1H, C3-H), 6.95 (d,1H, C6-H, J= 8 Hz), 7.28-7.75 (m, 6H, Ar); MS (m/z): 493; Anal calcd. C, 60.84; H, 4.70; N, 8.51; found: C, 60.47; H, 4.89; N, 8.91.

3-Benzoyl-2-{[1-(7-hydroxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one-5-carboxylic acid ethyl ester (XIVb): Yield= 94%; m. p: 135 ᵒC; IR (ṽ max, cm-1): 3066 (CH, Ar), 2933 (CH, aliphatic), 1732 and 1724 (2C=O), 1598 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 2.44 (t, 3H, CH3-), 2.84 (s, 3H, C4-CH3), 2.98 (s, 3H, N=C-CH3), 4.23-4.39 (m, 4H, 2CH2-of benzyl and ethyl), 6.19 (s, 1H, C3-H), 6.94 (d,1H, C6-H, J= 8 Hz), 7.54-8.09 (m, 6H, Ar), 7.61 (d, H, C5-H); MS (m/z): 508; Anal calcd. C, 59.16; H, 4.17; N, 8.28; found: C, 58.73; H, 4.37; N, 8.63.

3-Cyclohexyl-2-{[1-(7-ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one-5-carboxylic acid ethyl ester (XVa): Yield= 85%; m. p: 247-248 ᵒC; IR (ṽ max, cm-1): 3055 (CH, Ar), 2976 (CH, aliphatic), 1720 (C=O), 1598 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.22-1.36 (t, 6H, 2CH3-), 2.00-2.43 (m, 10H, cyclohexyl), 2.28 (s, 3H, C4-CH3), 2.44 (s, 3H,-N=C-CH3), 4.01 (t, 2H,-CH2-O), 4.12-4.28 (m, 3H, S-CH-C=O of thiazolidine and-CH2-O of ethyl ester), 6.21 (s, 1H, C3-H), 6.99 (d,1H, C6-H), 7.63 (d, H, C5-H); MS (m/z): 514; Anal calcd. C, 60.80; H, 6.08; N, 8.18; found: C, 60.55; H, 6.37; N, 8.61.

2-{[1-(7-Ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-4-oxo-3-phenyl-thiazolidine-5-carboxylic acid ethyl ester (XVb): Yield= 86%; m. p: 196-198 ᵒC; IR (ṽ max, cm-1): 2983 (CH, aliphatic), 1745 and 1730 (2C=O), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.23-1.38 (m, 6H, 2CH3-), 2.21 (s, 3H, C4-CH3), 2.41 (s, 3H,-N=C-CH3), 3.85-3.91 (m, 2H,-O-CH2-), 4.19-4.41 (m, 2H,-O-CH2-), 4.67 (s, 1H, S-CH-C=O), 6.11 (s, 1H, C3-H), 6.68 (s, 1H, C6-H), 7.05-7.15 (m, 2H, C3-H, C5-H of phenyl), 7.39 (m, 2H, C2-H, C6-H of phenyl), 7.54 (s, 1H, C5-H); MS (m/z): 507; Anal calcd. C, 61.53; H, 4.96; N, 8.28; found: C, 61.32; H, 5.19; N, 8.73.

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2-{[1-(7-Ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-(4-methoxy-phenyl)-thiazolidin-4-one-5-carboxylic acid ethyl ester (XVc): Yield= 75%; m. p: 217-218 ᵒC; IR (ṽ max, cm-1): 3062 (CH, Ar), 2981 (CH, aliphatic), 1716 (C=O), 1595 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 1.18-1.35 (m, 6H, 2CH3-), 2.28 (s, 3H, C4-CH3), 2.41 (s, 3H, N=C-CH3), 3.31 (s, 3H,-O-CH3), 3.73 (q, 2H,-O-CH2-), 4.25 (q, 2H, CO-O-CH2-), 4.63 (s, 1H, S-CH-C=O), 6.24 (s, 1H, C3-H), 6.85 (d, 2H, C3-H, C5-H of phenyl), 6.90 (s, 1H, C6-H), 7.42 (d, 2H, C3-H, C5-H of phenyl), 7.75 (s, 1H, C5-H); MS (m/z): 537; Anal calcd. C, 60.32; H, 5.06; N, 7.82; found: C, 59.95; H, 5.30; N, 8.19.

2-{[1-(7-Allyloxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-cyclohexyl-4-oxo-thiazolidine-5-carboxylic acid ethyl ester (XVIa): Yield= 80%; m. p: 80-82 ᵒC; IR (ṽ max, cm-1): 3055 (CH, Ar and allyl), 2962 (CH, aliphatic), 1737 and1724 (2C=O), 1620 (C=N imine), 1597 (C=C, Ar, allyl); 1HNMR (400 MHz, CDCl3): δ= 1.19-1.42 (m, 9H, C3-2H, C4-2H, C5-2H of cyclohexyl and-CH3 of ethyl), 1.77-1.87 (m, 4H, C2-2H, C6-2H of cyclohexyl), 2.34 (s, 3H, C4-CH3), 2.42 (s, 3H, N=C-CH3), 3.40 (s, 1H, C1-H of cyclohexyl), 4.06-4.09 (m, 2H,-CH2-O), 4.56 (s, 1H, S-CH-C=O), 4.67 (d, 2H,-CH2-O), 5.22-5.42 (m, 2H, CH2=), 5.97-6.01 (m, 1H, =CH-), 6.15 (s, 1H, C3-H), 6.91 (d, 1H, C-6, J=8 Hz), 7.55 (d, 1H, C5-H, J=8 Hz); MS (m/z): 523; Anal calcd. C, 61.70; H, 5.94; N, 7.99; found C, 61.94; H, 6.01; N, 8.14.

2-{[1-(7-Allyloxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-4-methoxyphenyl-thiazolidin-4-one-5-carboxylic acid ethyl ester (XVIc): Yield= 87%; m. p: 188 ᵒC; IR (ṽ max, cm-1): 3066 (CH, Ar and allyl), 2968 (CH, aliphatic), 1730 and 1718 (C=O), 1598 (C=C, Ar, allyl); 1HNMR (400 MHz, CDCl3): δ= 2.35 (s, 3H, C4-CH3), 2.44 (s, 3H, N=C-CH3), 3.83 (s, 3H, CH3-O-Ph), 4.05-4.12 (m, 3H,-CH2-O and S-CH-C=O), 4.72 (d, 2H,-CH2-O), 5.35-5.40 (d, d, 2H, CH2=), 5.99-6.04 (m, 1H, =CH–), 6.17 (s, 1H, C3-H), 6.92-6.98 (m, 3H, C3-H, C5-H of phenyl and C6-H), 7.49 (d, 2H, C2 and C6 of phenyl, J= 8 Hz), 7.66 (d, 1H, C5-H, J=8 Hz); MS (m/z): 550; Anal calcd. C, 61.19; H, 4.95; N, 7.65; found: C, 61.09; H, 5.21; N, 8.08.

2-{[1-(7-Butoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-ethyl-thiazolidin-4-one-5-carboxylic acid ethyl ester (XVIIa): Yield= 85%; m. p: 224-225 ᵒC; IR (ṽ max, cm-1): 2958 (CH, aliphatic), 1732 (C=O), 1627 (C=N, imine), 1593 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 0.96 (t, 3H, CH3-, J= 8 Hz), 1.35-1.38 (m, 3H, CH3-), 1.43-1.52 (m, 2H,-CH2-), 1.75-1.82 (m, 2H,-CH2-), 2.37 (s, 3H, C4-CH3), 2.40 (s, 3H, N=C-CH3), 3.50-3.53 (m, 2H,-CH2-N), 4.01 (t, 2H,-CH2-O), 4.06-H, J= 8 Hz), 7.54 (d, 1H, C5-H, J= 8 Hz); MS (m/z): 485, 487; Anal calcd. C, 59.12; H, 6.00; N, 8.62; found: C, 58.75; H, 6.26; N, 9.03. 4.10 (m, 2H,-CH2-O), 4.18 (s, 2H, S-CH-C=O), 6.13 (s, 1H, C3-H), 6.90 (d,1H, C6-

3-Benzyl-2-{[1-(7-butoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-one-5-carboxylic acid ethyl ester (XVIIb): Yield= 93%; m. p: charring; IR (ṽ max, cm-1): 3032 (CH, Ar), 2958 (CH, aliphatic), 1724 (C=O), 1627 (C=N, imine), 1597 (C=C, Ar); 1HNMR (400 MHz, CDCl3): δ= 0.95 (t, 3H, CH3-, J= 8 Hz), 1.39-1.48 (m, 2H,-CH2-), 1.75-1.79 (m, 2H,-CH2-), 2.32 (s, 3H, C4-CH3), 2.40 (s, 3H, N=C-CH3), 3.74 (t, 2H,-CH2-O, J= 8 Hz), 4.04-4.07 (m, 3H,-CH2-N-and S-CH-), 5.09 (s, 2H, CH2-), 6.12 (s, 1H, C3-H), 6.88 (d,1H, C6-H, J= 8 Hz), 7.30-7.37 (m, 3H, C2-H,C6-H, and C4-H of phenyl), 7.48 (t, 2H, C3-H, C5-H of phenyl), 7.53 (d, H, C5-H); MS (m/z): 549; Anal calcd. C, 63.37; H, 5.68; N, 7.65; found: C, 62.97; H, 5.89; N, 7.94.

Antimicrobial activity

Sensitivity test

The agar disc plate method using Hi-Media agar medium was employed to study the antimicrobial activity of the synthesized compounds with tobramycin as the reference drug. The prepared compounds were examined against two strains of Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 14579), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922) and Candida albicans ATCC 10231). Each test compound (50 mg) was dissolved in DMSO (dimethyl sulphoxide) (0.5 ml, 100 mg/ml), which was used as a sample solution. 6 mm discs were impregnated with 100 mg/ml solution of the test compound were placed on the solidified nutrient

agar medium that had been inoculated with the respective microorganism and the Petri dishes were subsequently incubated at 37 ᵒC for 48 h. Tobramycin was used as reference drugs and DMSO as a negative control. Zones of inhibition produced by each compound were measured in millimetres [16].

Minimum inhibitory concentration test (MIC)

The agar cup plate method using Hi-Media agar medium was employed to study the antibacterial activity against Staphylococcus aureus. Each test compound (50 mg) was dissolved in dimethyl sulphoxide (100 mg/ml), which was used as a stock solution to carry out two-fold dilution technique. The sample size for all the compounds was fixed at 0.1 ml. Using a sterilized cork borer, cups were scooped out of Agar medium contained in a Petri dish which was previously inoculated with the microorganisms. The test compound solution (0.1 ml) was added to the cups, and the Petri dishes were subsequently incubated at 37 ᵒC for 48 h. MIC was defined as the lowest compound concentration preventing visible bacterial growth [16].


Chemistry

The starting compound 7-hydroxy-4-methylcoumarin (I) was prepared as reported in the literature via Pechmann-Duisburg reaction [17]. Then the 8-acetylcoumarin derivative (II) was prepared by acetylation of the 7-hydroxy group with acetic anhydride [18] followed by Fries rearrangement using anhydrous AlCl3 [19]. To study the effect of alkylation of the 7-hydroxyl group on antimicrobial activity, the 7-ethoxy (IIIa), 7-allyloxy (IIIb), and 7-butyloxy (IIIc) derivatives were prepared using the appropriate alkyl halide in dry acetone [20]. The 7-hydroxy (II) and the 7-alkoxy derivatives (IIIa-IIIc) were then treated with hydrazine hydrate to yield the hydrazones (IV and Va-Vc) [21, 22], that reacted with different isothiocyanates to give coumarin-thiosemicarbazones (VI-IX) in good yields [23]. Coumarin-thiazolidine-4-ones (X-XIII) were formed by cyclizing the thiosemicarbazone (VI-IX) with chloroacetic acid, freshly fused sodium acetate in absolute ethanol, while the thiazolidin-4-one-5-carboxylix acid ethyl ester derivatives (XIV-XVII) were prepared from intermediates (VI-IX) through cyclization with diethyl bromo malonate in refluxing absolute ethanol in the presence of fused sodium acetate [24].

The synthetic pathways are outlined in scheme 1 and 2. The structures of the synthesized compounds were confirmed by spectral data and elemental analysis, and they were in full agreement with the proposed structures.

O O

OCH3

RO

CH3

O O

NNH2CH3

OH

CH3

O O

NNH2CH3

RO

CH3

O O

N CH3

RO

CH3

NHNH

SR1

IIIa, R= EthylIIIb, R= AllylIIIc, R= ButylO O

OCH3

OH

CH3

Va, R= EthylVb, R= AllylVc, R= ButylO O

N CH3

OH

CH3

NHHN

S

VII, R= Ethyl , a; R1= cyclohexyl, b; R1= phenyl, c; R1= p.methoxyphenyl VIII, R= Allyl, a; R1= cyclohexyl, b; R1= benzyl, c; R1= p.methoxyphenyl IX, R= Butyl , a; R1= ethyl, b; R1= benzyl

O OOH

CH3

(I) (II)

e

e

a, b c

d d

VI, a; R1= benzyl, b; R1= benzoyl

R1

IV

Scheme 1: Reagent and conditions: a) acetic anhydride, reflux; b) Aluminum Chloride, fusion, 2 h, from 120 to 175 ᵒC; c)

appropriate Alkyl halide, anhydrous K2CO3 in dry acetone, and reflux; d) Hydrazine hydrate, ethanol, and reflux; e)

appropriate isothiocyanate derivatives, ethanol, and reflux

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O O

N CH3

RO

CH3

NNR1

S

OO O

N CH3

RO

CH3

NHNH

SR1

O O

N CH3

RO

CH3

NNR1

SC2H5OOC

Xa,b; R= H XIa-c; R= EthylXIIa-c; R= AllylXIIIa, b; R= Butyl

XIVa, b; R= HXVa-c; R= EthylXVIa, c; R= AllylXVIIa, b; R= Butyl

O

VIa, b; R= H, VIIa-c; R= EthylVIIIa-c; R= AllylIXa, b; R= Butyl

f

g

Scheme 2: Reagent and conditions: f) Chloroacetic acid and fused sodium acetate, ethanol, and reflux; g) diethyl bromo

malonate and fused sodium acetate, ethanol, and reflux

Antimicrobial activity

The results (table 1) showed that compounds VIb, Xb, XIVb, XVa and XVc possessed strong inhibitory activity against S. aureus compared to the reference leads listed in table 2. While, compounds VIIa, VIIIa, IXa, IXb, and XIVa have moderate activity compared to the reference compound tobramycin. Compounds XIII and XIVb have fair to moderate activity against B. subtilis. The synthesized compounds had no activity on the Gram-negative strains used. The active compounds Xb, XIVb, XVa, and XVc showed activity against C. albicans beside the antibacterial activity which was better than lead 7 that had only antifungal activity with MIC value of 0.1µg/ml.

Compounds VIb, Xb, XIVb, and XVc, showed better inhibitory activity in terms of lower MIC values (0.19-0.36 µg/ml) than the lead drug bearing the same nucleus novobiocin 1 (MIC= 1 µg/ml). Novobiocin is known for its potent inhibitory activity against S. aureus especially methicillin-resistant strains through DNA-gyrase inhibition. Thus, the newly synthesized compounds represent promising antibacterial agents, especially against this strain.

By comparing the minimum inhibitory concentration (MIC) (table 3) of the active compounds VIb, Xb, XIVb, and XVc to tobramycin (MIC= 2 µg/ml) and novobiocin (MIC= 1 µg/ml), they gave better values ranging from 0.195-0.390 µg/ml; which were also better values reported for lead 5 (MIC= 25 µg/ml) and lead 6 (MIC= 10 µg/ml).

Among the thiosemicarbazone series VI-IX, the 7-hydroxy derivative VIb with benzoyl group at N4 of thiosemicarbazone was the most active with a zone of inhibition value of 35 mm and MIC value of 0.195µg/ml. This result showed the importance of the free hydroxyl group at this position. On the other hand, the 7-alkoxy derivatives VIIa, VIIIa, and IXa were moderately active when compared to their aromatic analogs VIIb, VIIc, VIIIb, and VIIIc.

In thiazolidine-4-one series X-XIII, compound Xb had stronger activity against S. aureus with MIC value of 0.390 µg/ml and showed weak activity against C. albicans, which may be attributed to the presence of thiazolidine-4-one ring when compared to its precursor compound VIb. Also, compound XIIIa showed slight activity against B. subtilis. While, in the thiazolidine-4-one-5-carboxylic acid ethyl ester series XIV-XVII, compounds XIVb, XVa, XVc had strong antibacterial activity when compared to their analogs (Xb), (VIIa, XIa), and (VIIc, XIc) respectively. Compound XIVa possessed moderate activity over compound VIa, and Xa which may be attributed to the 5-carboxylic acid ethyl ester on the thiazolidine-4-one ring. In summary, it was evidenced that the presence of free 7-hydroxyl group on 4-methycoumarin ring was very important for activity. The hybridization with thiosemicarbazone substituted with benzoyl moiety as in compound VIb or thiazolidine-4-one-5-carboxylic acid ethyl ester as in compound XIVb gave rise to promising antimicrobial agents.

Table 1: The mean* of zone of inhibition (mm) of the active compounds against gram-positive bacteria

O O

N CH3

RO

CH3

HN

HN

SR1

VI-IX

O O

N CH3

RO

CH3

NNR1

SO

R2X-XVII

Compound number R R1 R2 S. aureus B. subtilis C. albicans Tobramycin 21 mm ND ND VIb H Benzoyl 35 mm - - VIIa Ethyl Cyclohexyl 10 mm - - VIIIa Allyl Cyclohexyl 14 mm - - IXa Butyl Ethyl 14 mm ND ND IXb Butyl Benzyl 10 mm ND ND Xb H Benzoyl H 30 mm - 8 mm XIIIa Butyl Ethyl H - 8 mm - XIVa H Benzyl COOC2H5 13 mm - - XIVb H Benzoyl COOC2H5 30 mm 12 mm 14 mm XVa Ethyl Cyclohexyl COOC2H5 19 mm - 9 mm XVc Ethyl P. methoxyphenyl COOC2H5 22 mm - 11 mm

*Presented are the mean of 3 separate experiments; Errors are in the range±10% of the reported values. Inactive: inhibition zone<5 mm; slightly active: inhibition zone = 5-10 mm; moderately active: inhibition zone = 10-15 mm; highly active: inhibition zone>15 mm, **ND; not determined, *** S. aureus: Staphylococcus aureus, B. subtilis: Bacillus subtilis, C. albicans: Candida albicans

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Table 2: Comparison of the activity of the new active compounds to previously reported leads against Staphylococcus aureus

Compound number Zone of inhibition (mm) % inhibition*

S

N

O NHN

N

S

O O

O

8 [25]

15 mm 71%

9 [25]

S

N

O NHN

N

S

O O

23 mm 109%

O

HN

S N

O

O O O

10 [26]

18 mm 85%

O

HN

S N

O

O O O

11 [26]

H3CO

18 mm 85%

VIb (this work) 35 mm 166% Xb (this work) 30 mm 142% XIVb (this work) 30 mm 142% XVc (this work) 22 mm 104%

* % Inhibition= zone of inhibition of compounds in mm/mm of tobramycin*100 [25]

Table 3: The mean* MIC (µg/ml) values of the active compounds VIb, Xb, XIVb, and XVc and the comparison of these values to the reported MIC values of compounds having similar pharmacophores

Compound number Minimum inhibitory concentration MIC (µg/ml) Tobramycin 2 Novobiocin 1 [27] 5 25 [12] 6 10 [13, 14] 7 0.1 [15] VIb 0.195* Xb 0.390* XIVb 0.195* XVc 0.390*

* Presented are the mean of 3 separate experiments; errors are in the range±10% of the reported values. MIC mean values for compounds with a zone of inhibition>20 mm was determined.

CONCLUSION

The novel series of 4-methylcoumarin bearing thiosemicarbazone moiety (VI-IX) and the series having thiazolidine-4-one (X-XVII) were synthesized, and their antimicrobial activity was evaluated. These novel coumarin derivatives showed potential activity against Gram-positive Staphylococcus aureus especially these with a free 7-hydroxy group (VIb, Xb, XIVb); that emphasize the importance of this position for antibacterial activity and compounds XIVa, XVa, and XVc with 5-carboxylic acid ethyl ester group on thiazolodin-4-one ring showed enhanced potency than their parent compounds. Hence, it can be concluded that these novel compounds are potential antibacterial agents better than the reference compounds and

represent promising leads for further optimization and clinical studies.

CONFLICT OF INTERESTS

Declared none

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