an easy protocol for the domino synthesis of diversely …10.1007... · · 2014-02-242 1....

1

An easy protocol for the domino synthesis of diversely functionalized spirocarbocycles

and their biological evaluation

Selvarangam E. Kiruthikaa, Paramasivan T. Perumal

a*, C. Balachandran

b, S.

Ignacimuthub.

a Organic Chemistry Division, Central Leather Research Institute, CSIR, Adyar, Chennai

600 020, India b

Division of Microbiology, Entomology Research Institute, Loyola College, Chennai 600

034, India

E-mail: [email protected]

Supporting information

Contents Page No.

1. Experimental procedure for synthesis and procedure for

(a) Zone of inhibition

(b) Minimum Inhibitory Concentration

and spectral data for all new compounds 2

2. IR spectrum of compound 4a 12

3. 1H NMR spectrum of compound 4a 13

4. 13

C NMR spectrum of compound 4a 14

5. Mass spectrum of compound 4a 15

6. IR spectrum of compound 6b 16

7. 1H NMR spectrum of compound 6b 17

8. 13

C NMR spectrum of compound 6b 18

9. Mass spectrum of compound 6b 19

10. ORTEP of 4g 20

11. Antimicrobial activity of synthesised compounds using disc diffusion 21

method (Zone of inhibition in mm) (1mg/disc)

12. Minimum inhibitory concentration of synthesised compounds (1mg/disc) 22

2

1. Experimental

2.1 Materials, methods and instruments

Melting points were determined on Gallenkamp melting point apparatus and are uncorrected.

Infrared (IR) spectra were recorded on a Perkin-Elmer FTIR spectrophotometer as KBr

pellets. 1H and

13C NMR spectra were obtained in DMSO-d6 on a JEOL spectrometer at 500

and 125 MHz, respectively. Proton chemical shifts () are relative to tetramethylsilane (TMS,

= 0.00) as internal standard and expressed in parts per million. The number of protons (n)

for a given resonance was indicated as nH. Coupling constants (J) are given in hertz. Spin

multiplicities are given as s (singlet), d (doublet), t (triplet) and m (multiplet). Mass spectra

were recorded on a Thermo Finnigan LCQ Advantage MAX 6000 ESI mass spectrometer

and Perkin-Elmer GC-MS. Elemental analyses were recorded using a Thermo Finnigan

FLASH EA 1112CHN analyzer. All the compounds gave C, H and N analysis within ±0.5%

of the theoretical values. Analytical TLC was performed on precoated plastic sheets of silica

gel G/UV-254 of 0.2 mm thickness (Macherey-Nagel, Germany) using analytical grade

solvents and visualizing with UV light (λ = 254 and 365 nm).

2.2 Experimental procedure for the synthesis of 4a-l:

3-phenyl-5-isoxazolone 1 (1 mmol), aldehhydes 2a-k were stirred in a mixture of EtOH:H2O

(2:1) in the presence of L-Proline (15 mol%) for ten minutes followed by the addition of

alkylidene malononitrile 3a-c (1 mmol) at room temperature for 1 h. The solid precipitated

out was filtered off and purified by recrystallization from methanol to afford product 4a-l as

white crystalline solid.

2.2a Spectral data for compound 4a

White solid. mp: 152-154ºC. max (KBr): 3345, 3224, 2926, 2860, 2207, 1790, 1640, 1593,

1514, 1446, 1393, 1151, 908, 881, 764, 691, 640 cm-1

.1H NMR (DMSO-d6, 500 MHz):

0.74 (q, 1H, J = 9 Hz), 1.25 (m, 1H), 1.40 (m, 1H), 1.62 (m, 1H), 2.07 (m, 1H), 2.17 (m,

3

1H), 2.22 (s, 3H), 3.06 (d, 1H, J = 12.5 Hz), 3.18 (m, 1H), 5.76 (m, 1H), 6.14 (d, 1H, J = 8.5

Hz), 6.60 (bs, 2H, -NH2, D2O exchangeable), 6.86 (d, 1H, J = 7.5 Hz), 7.05 (d, 1H, J = 8 Hz),

7.16 (d, 1H, J = 8.5 Hz), 7.59 (m, 5H). 13

C NMR (DMSO-d6, 125 MHz): 20.6, 21.5, 25.0,

27.4, 31.3, 50.1, 59.1, 82.1, 117.1, 119.2, 125.6, 126.4, 126.7, 128.6, 129.5, 129.6, 130.3,

130.7, 131.5, 132.3, 137.6, 147.6, 164.3, 176.1 MS (m/z): 410 (M+H)+. Anal. Calcd. For

C26H23N3O2 : C, 76.26; H, 5.66; N, 10.26. Found: C, 76.24; H, 5.67; N, 10.27.

2.2b Spectral data for compound 4b


1575, 1512, 1325, 1266, 1157, 906, 763, 685 cm-1

.1H NMR (DMSO-d6, 500 MHz): 1.10 (q,

1H, J = 10.5 Hz), 1.70-.1.75 (m, 1H), 2.26-2.31 (m, 1H), 2.36-2.39 (m, 1H), 3.25 (d, 1H, J =

12.5 Hz), 3.65-3.67 (m, 1H), 5.49-5.51 (m, 1H), 6.95 (bs, 2H, -NH2, D2O exchangeable),

7.21-7.41 (m, 2H), 7.60-7.67 (m, 7H). 13

C NMR (DMSO-d6, 125 MHz): 29.4, 31.1, 40.0,

40.1, 50.0, 60.3, 77.8, 116.9, 118.2, 126.2, 126.8, 129.8, 132.7, 133.3, 133.6, 136.5, 150.1,

164.2, 175.5. MS (m/z): 416 (M+H)+. Anal. Calcd. For C24H18ClN3O2 : C, 69.31; H, 4.36; N,

10.10. Found: C, 69.29; H, 4.37; N, 10.11.

2.2c Spectral data for compound 4c


1641, 1587, 1481, 1446, 1345, 1266, 1009, 880, 833, 767, 692, 642, 567 cm-1

.1H NMR

(DMSO-d6, 500 MHz): 0.78-0.82 (m, 1H), 1.21-1.32 (m, 1H),1.36-1.47 (m, 1H), 1.58-

1.68(m, 1H), 2.01-2.12 (m, 1H), 2.17-2.21 (m, 1H), 3.14-3.19 (m, 2H), 5.73-5.78 (m, 1H),

6.28-6.37 (m, 1H), 6.64 (bs, 2H, -NH2, D2O exchangeable), 6.94 (t, 1H, J = 8.5 Hz), 7.18-

7.25 (m, 2H), 7.56 (d, 2H, J = 7.5 Hz), 7.60 (t, 2H, J = 7.5 Hz), 7.64 (t, 1H, J = 7.5 Hz). 13

C

NMR (DMSO-d6, 125 MHz): 21.5, 25.1, 27.3, 31.4, 49.6, 59.1, 82.2, 115.0, 115.1, 116.1,

116.2, 117.2, 119.4, 126.3, 126.8, 127.9, 129.7, 130.1, 130.8, 132.6, 133.0, 147.6, 164.4,

4

176.1. MS (m/z): 414 (M+H)+. Anal. Calcd. For C25H20FN3O2 : C, 72.63; H, 4.88; N, 10.16.

Found: C, 72.65; H, 4.87; N, 10.15.

2.2d Spectral data for compound 4d


1697, 1614, 1523, 1515, 1422, 1403, 1234, 1105, 1000, 956, 886, 754, 645, 523 cm-1

.1H

NMR (DMSO-d6, 500 MHz): 1.10 (q, 1H, J = 10.5 Hz), 1.70-.1.75 (m, 2H), 2.26-2.31 (m,

2H), 2.36-2.39 (m, 2H), 3.25 (d, 1H, J = 12.5 Hz), 3.65-3.67 (m, 2H), 5.49-5.51 (m, 1H),

6.95 (bs, 2H, -NH2, D2O exchangeable), 7.21-7.41 (m, 2H), 7.60-7.67 (m, 7H). 13

C NMR

(DMSO-d6, 125 MHz): 29.2, 31.0, 40.1, 42.1, 46.0, 48.2, 49.3, 61.9, 76.5, 114.8, 117.3,

121.2, 122.6, 123.4, 128.9, 129.2, 129.6, 132.9, 133.1, 134.5, 135.2, 150.5, 164.3, 175.8. MS

(m/z): 444 (M+H)+. Anal. Calcd. For C26H22ClN3O2 : C, 70.34; H, 5.00; N, 9.47. Found: C,

70.36; H, 4.99; N, 9.46.

2.2e Spectral data for compound 4e


1677, 1604, 1523, 1466, 1408, 1263, 1189, 999, 863, 723, 720, 612 cm-1

.1H NMR (DMSO-

d6, 500 MHz): 0.79 (q, 1H, J = 12.5 Hz), 1.13-.15 (m, 1H), 1.35-1.47 (m, 1H), 1.60-1.69

(m, 1H), 2.08-2.21 (m, 2H), 3.10 (t, 1H, J = 11 Hz), 3.68 (d, 1H, J = 12 Hz), 3.71 (s, 3H),

3.72 (s, 3H), 5.75-5.79 (m, 1H), 6.68 (bs, 2H, -NH2, D2O exchangeable), 6.76 (s, 1H), 6.93

(s, 1H), 7.53-7.59 (m, 5H). 13

C NMR (DMSO-d6, 125 MHz): 21.3, 24.9, 26.7, 32.7, 48.4,

55.5, 55.7, 58.8, 82.0, 109.8, 115.5, 115.8, 117.0, 119.3, 125.6, 126.4, 127.0, 129.4, 130.1,

132.4, 147.9, 148.4, 148.8, 164.3, 176.9. MS (m/z): 534 (M+H)+. Anal. Calcd. For

C27H24BrN3O4 : C, 60.68; H, 4.53; N, 7.86. Found: C, 60.70; H, 4.52; N, 7.85.

2.2f Spectral data for compound 4f

5


1512, 1446, 1255, 1181, 1028, 880, 837, 770, 692, 642, 575 cm-1

.1H NMR (DMSO-d6, 500

MHz): 0.76 (q, 1H, J = 10.5 Hz), 1.21-1.26 (m, 1H), 1.38-1.42 (m, 1H), 1.60-1.69 (m, 1H),

3.12 (t, 1H, J = 10.5 Hz), 3.82 (d, 1H, J = 10.5 Hz), 3.70 (s, 3H), 3.74(s, 3H), 5.81-5.83 (m,

1H), 6.63 (bs, 2H, -NH2, D2O exchangeable), 6.82 (s, 1H), 7.02-7.05 (m, 2H), 7.60-7.69 (m,

5H). 13

C NMR (DMSO-d6, 125 MHz): 29.6, 31.1, 40.3, 50.6, 55.4, 60.6, 77.7, 117.0, 118.0,

126.5, 126.6, 126.8, 129.7, 132.5, 136.8, 148.7, 150.4, 164.4, 175.8 MS (m/z): 442 (M+H)+.

Anal. Calcd. For C26H23N3O4 : C, 70.73; H, 5.25; N, 9.52. Found: C, 70.71; H, 5.26; N, 9.53.

2.2g Spectral data for compound 4g


1519, 1500, 1479, 1466, 1235, 1222, 1148, 946, 905, 723, 620 cm-1


MHz): 0.73 (q, 1H, J = 13.0 Hz), 1.10-1.12 (m, 1H), 1.31-1.36 (m, 1H), 1.54-1.57 (m, 1H),

2.02-2.10 (m, 1H), 2.16-2.20 (m, 1H), 3.28 (t, 1H, J = 13.0 Hz), 4.12 (d, 1H, J = 12.0 Hz),

5.79-5.82 (m, 1H), 6.58 (bs, 2H, -NH2, D2O exchangeable), 6.73 (t, 1H, J = 8 Hz), 6.93 (d,

1H, J = 9 Hz), 7.25-7.28 (m, 3H), 7.40 (t, 3H, J = 7.5 Hz), 7.49 (t, 1H, J = 7.5 Hz), 7.56 (t,

1H, J = 8 Hz), 7.79-7.83 (m, 2H). 13

C NMR (DMSO-d6, 125 MHz): 21.7, 25.3, 26.9, 33.3,

43.4, 59.7, 82.6, 117.5, 119.8, 121.9, 124.1, 125.5, 125.6, 125.7, 126.6, 126.9, 128.9, 129.6,

130.7, 131.7, 132.6, 133.5, 148.5, 164.5, 165.7, 177.0. MS (m/z): 446 (M+H)+. Anal. Calcd.

For C29H23N3O2 : C, 78.18; H, 5.20; N, 9.43. Found: C, 78.20; H, 5.19; N, 9.42.

2.2h Spectral data for compound 4h


1623, 1555, 1500, 1489, 1430, 1198, 907, 850, 763, 642 cm-1


MHz): 0.72 (q, 1H, J = 13.5 Hz), 1.37-1.39 (m, 2H), 1.56-1.65 (m, 1H), 2.05-2.07 (m, 1H),

2.17-2.21 (m, 1H), 3.12 (t, 1H, J = 10.5 Hz), 3.47 (d, 1H, J = 12.5 Hz), 5.77-5.80 (m, 1H),

6

6.09 (d, 1H, J = 8 Hz), 6.47 (t, 1H, J = 7.5 Hz), 6.57 (bs, 2H, -NH2, D2O exchangeable), 6.92

(t, 1H, J = 7.5 Hz), 7.08 (s, 1H), 7.27 (d, 1H, J = 8 Hz), 7.43 (d, 2H, J = 8 Hz), 7.53 (t, 1H, J

= 8 Hz), 7.62-7.64 (m, 2H). 11.18 (bs, 1H, -NH, D2O exchangeable). 13

C NMR (DMSO-d6,

125 MHz): 22.1, 25.6, 27.8, 34.1, 41.8, 60.3, 82.7, 108.9, 111.9, 112.5, 117.4, 117.8, 118.7,

119.5, 121.7, 122.9 126.8, 127.3, 127.9, 129.8, 130.1, 131.2, 132.7, 135.6, 148.3, 165.1,

177.3 MS (m/z): 435 (M+H)+. Anal. Calcd. For C27H22N4O2 : C, 74.64; H, 5.10; N, 12.89.

Found: C, 74.66; H, 5.09; N, 12.88.

2.2i Spectral data for compound 4i


1523, 1510, 1429, 1198, 999, 756, 643 cm-1

.1H NMR (DMSO-d6, 500 MHz): 0.80 (q, 1H, J

= 12 Hz), 1.38-1.45 (m, 2H), 1.60-1.68 (m, 1H), 2.00-2.12 (m, 1H), 2.14-2.24 (m, 1H), 3.13

(d, 1H, J = 12.5 Hz), 3.20-3.27 (m, 1H), 3.59-3.71 (m, 1H), 3.82-3.95 (m, 1H), 5.78-5.80 (m,

1H), 6.26 (d, 1H, J = 7.5 Hz), 6.37 (s, 1H), 6.67 (s, 2H, -NH2, D2O exchangeable), 7.19 (d,

1H, J = 8 Hz), 7.30 (t, 1H, J = 7.5 Hz), 7.33-7.38 (m, 1H), 7.51-7.59 (m, 3H), 7.61-7.70 (m,

2H), 7.82 (d, 1H, J = 8 Hz), 7.88 (d, 1H, J = 8 Hz). 13

C NMR (DMSO-d6, 125 MHz): 21.5,

25.0, 27.4, 31.3, 36.2, 50.6, 59.2, 82.1, 117.1, 19.2, 120.2, 122.3, 124.3, 125.2, 126.5, 126.8,

126.9, 127.0, 127.1, 127.7, 129.6, 130.3, 132.4, 133.0, 140.4, 141.2, 142.6, 143.2, 143.7,

147.6, 164.4, 176.1. MS (m/z): 484 (M+H)+. Anal. Calcd. For C32H25N3O2 : C, 79.48; H,

5.21; N, 8.69. Found: C, 79.50; H, 5.20; N, 8.68.

2.2j Spectral data for compound 4j


1543, 1512, 1489, 1123, 1001, 987, 788 cm-1

.1H NMR (DMSO-d6, 500 MHz): 0.74 (q, 1H,

J = 9 Hz), 0.91 (t, 3H, J = 7 Hz), 1.22 (m, 1H), 1.68-1.72 (m, 1H), 1.89-1.91 (m, 1H), 2.03-

2.05 (m, 1H), 2.52-2.55 (m, 1H), 3.12 (d, 1H, J = 12.5 Hz), 3.16-3.18 (m, 1H), 3.22-3.25(m,

7

2H), 5.70-5.72 (m, 1H), 6.12 (d, 2H, J = 8.5 Hz), 6.73 (s, 2H, -NH2, D2O exchangeable), 6.92

(t, 2H, J = 7.5 Hz), 7.12 (d, 2H, J = 8 Hz), 7.24 (t, 1H, J = 8.5 Hz), 7.49-7.54 (m, 5H). 13

C

NMR (DMSO-d6, 125 MHz): 22.4, 25.3, 29.2, 32.6, 49.8, 56.3, 83.4, 118.1, 118.2, 119.1,

119.5, 119.6, 121.0, 122.1, 123.5, 124.6, 125.3, 126.8, 127.9, 128.4, 129.2, 130.0, 132.6,

133.4, 135.8, 136.9, 140.2, 143.4, 166.1, 177.3. MS (m/z): 513 (M+H)+. Anal. Calcd. For

C33H28N4O2 : C, 77.32; H, 5.51; N, 10.93. Found: C, 77.33; H, 5.49; N, 10.94.

2.2k Spectral data for compound 4k


1522, 1448, 1387, 1163, 951, 862, 703 cm-1

.1H NMR (DMSO-d6, 500 MHz): 0.72 (q, 1H, J

= 10.5 Hz), 1.28-1.30 (m, 1H), 1.42-1.46 (m, 1H), 1.59-1.60 (m, 1H), 2.01-2.03 (m, 1H),

2.18-2.20 (m, 1H), 3.20 (d, 1H, J = 12.5 Hz), 3.22-3.24 (m, 1H), 5.78-5.80 (m, 1H), 6.22 (d,

2H, J = 8 Hz), 6.62 (s, 2H, -NH2, D2O exchangeable), 7.01 (t, 1H, J = 7.5 Hz), 7.12 (t, 1H, J

= 8 Hz), 7.16-7.20 (m, 2H), 7.29-7.35 (m, 2H), 7.40 (d, 1H, J = 7.5 Hz),7.66-7.75 (m, 5H).

13C NMR (DMSO-d6, 125 MHz): 21.7, 25.2, 28.3, 32.4, 51.0, 58.6, 83.4, 112.2, 113.5,

117.8, 118.1, 120.3, 121.2, 122.6, 123.4, 124.2, 127.1, 128.3, 129.4, 130.1, 133.6, 134.5,

136.9, 137.8, 138.2, 139.4, 140.3, 143.1, 144.2, 164.5, 176.3. MS (m/z): 520 (M+H)+. Anal.

Calcd. For C35H25N3O2 : C, 80.90; H, 4.85; N, 8.09. Found: C, 80.92; H, 4.84; N, 8.08.

2.2l Spectral data for compound 4l


1513, 1442, 1398, 1150, 907, 882, 777, 632 cm-1

.1H NMR (DMSO-d6, 500 MHz): 0.69 (q,

1H, J = 12.5 Hz), 1.16-1.17 (m, 1H), 1.46-1.55 (m, 2H), 2.04-2.05 (m, 1H), 2.17-2.21 (m,

1H), 3.17-3.20 (m, 1H), 3.48 (d, 1H, J = 12.5 Hz), 5.76-5.79 (m, 1H), 6.19 (d, 1H, J = 8 Hz),

6.66 (s, 2H, -NH2, D2O exchangeable), 6.72 (t, 1H, J = 8 Hz), 7.18-7.23 (m, 1H), 7.32 (d, 2H,

J = 7 Hz), 7.46 (t, 2H, J = 8 Hz), 7.51 (s, 1H), 7.54 (t, 1H, J = 8 Hz), 7.58-7.63 (m, 1H),

8

7.65-7.71 (m, 2H), 7.82 (d, 1H, J = 9 Hz), 7.91 (d, 2H, J = 7.5 Hz). 13

C NMR (DMSO-d6,

125 MHz): 23.2, 25.8, 27.3, 32.1, 43.6, 61.4, 81.0, 109.2, 112.3, 113.4, 114.5, 114.7, 116.2,

118.9, 119.3, 119.5, 120.1, 120.6, 121.3, 124.5, 125.6, 126.8, 127.1, 127.2, 128.9, 129.2,

132.3, 134.5, 135.6, 136.9, 143.2, 166.2, 177.8. MS (m/z): 575 (M+H)+. Anal. Calcd. For

C33H26N4O4S : C, 68.97; H, 4.56; N, 9.75. Found: C, 68.99; H, 4.55; N, 9.74.

2.3 Experimental procedure for the synthesis of 6a-e

3-(2-oxo-2-phenylethylidene)indolin-2-one1 5 (1 mmol) and alkylidene malononitrile 3a-e (1

mmol) were stirred in a mixture of EtOH:H2O (2:1) in the presence of L-Proline (15 mol%) at

room temperature for 3 h. The solid precipitated out was filtered off and recrystallized from

methanol to afford Product 6a-e as a white solid.

2.3a Spectral data for compound 6a


1667, 1622, 1578, 1462, 1385, 1200, 783, 622 cm-1

.1H NMR (DMSO-d6, 500 MHz): 1.24

(q, 1H, J = 10.0 Hz), 1.78-1.83 (m, 1H), 2.27-2.29 (m, 2H), 3.32-3.38 (m, 1H), 4.03 (d, 1H, J

= 12.0 Hz), 5.36-5.38 (m 1H), 6.30 (bs, 2H, -NH2, D2O exchangeable), 6.70 (d, 1H, J = 7.5

Hz), 6.97 (t, 1H, J = 7.5 Hz), 7.20 (t, 2H, J = 7.5 Hz), 7.43 (t, 2H, J = 8 Hz), 7.59 (t, 1H, J =

7.5 Hz), 7.70 (d, 2H, J = 8 Hz), 10.41 (bs, 1H, -NH, D2O exchangeable). 13

C NMR(DMSO-

d6, 125 MHz): 30.1, 31.2, 42.1, 51.9, 57.2, 75.7, 110.1, 115.8, 117.7, 121.7, 125.1, 128.2,

128.7, 128.9, 129.6, 133.6, 137.4, 137.5, 142.2, 156.5, 175.7, 198.1. MS (m/z): 382 (M+H)+.

Anal. Calcd. For C24H19N3O2 : C, 75.57; H, 5.02; N, 11.02. Found: C, 75.59; H, 5.01; N,

11.01.

2.3b Spectral data for compound 6b


1669, 1633, 1575, 1473, 1395, 1212, 755, 626 cm-1

.1H NMR (DMSO-d6, 500 MHz): 0.96

9

(q, 1H, J = 12.0 Hz), 1.21 (m, 1H), 1.14 (m, 1H), 1.33 (m, 1H), 1.63 (m, 1H), 2.07 (m, 1H),

3.41 (m, 2H), 4.40 (d, 1H, J = 12.5 Hz), 5.42 (s, 2H, -NH2, D2O exchangeable), 5.56 (m,

1H), 6.71 (m, 2H), 7.03 (t, 1H, J = 7.5 Hz), 7.36 (t, 3H, J = 7.5 Hz), 7.50 (t, 1H, J = 8 Hz),

7.80 (d, 1H, J = 8.5 Hz), 10.57 (s, 1H, -NH, D2O exchangeable). 13

C NMR(DMSO-d6, 125

MHz): 14.3, 21.8, 24.9, 27.5, 32.7, 52.0, 54.1, 60.0, 81.0, 110.0, 116.7, 118.2, 121.8, 124.9,

127.3, 128.3, 128.8, 129.6, 131.7, 133.7, 137.7, 143.2, 154.4, 176.0, 200.5. MS (m/z): 396

(M+H)+. Anal. Calcd. For C25H21N3O2 : C, 75.93; H, 5.35; N, 10.63. Found: C, 75.91; H,

5.36; N, 10.64.

2.3c Spectral data for compound 6c


1662, 1620, 1578, 1422, 1396, 1221, 782, 630 cm-1

.1H NMR (DMSO-d6, 500 MHz): 1.26-

1.53 (m, 3H), 1.68-1.80 (m, 2H), 2.00-2.09 (m, 1H), 2.69 (t, 1H, J = 12.5 Hz), 2.88-2.90 (m,

1H), 3.82-3.84 (m, 1H), 4.99 (d, 1H, J = 11.5 Hz), 6.69-6.73 (m, 2H), 7.06 (t, 1H, J = 7.5

Hz), 7.37 (t, 3H, J = 7.5 Hz), 7.53 (t, 1H, J = 7.5 Hz), 7.82 (d, 2H, J = 7.5 Hz), 9.15 (s, 2H, -

NH2, D2O exchangeable), 10.57 (s, 1H, -NH, D2O exchangeable). 13

C NMR(DMSO-d6, 125

MHz): 23.5, 27.9, 28.3, 29.8, 34.5, 49.1, 57.9, 109.6, 111.8, 115.4, 121.8, 125.4, 125.7,

128.3, 128.8, 130.0, 133.9, 137.2, 143.2, 167.1, 174.0, 176.3, 199.5. MS (m/z): 410 (M+H)+.

Anal. Calcd. For C26H23N3O2 : C, 76.26; H, 5.66; N, 10.26. Found: C, 76.28; H, 5.65; N,

10.25.

2.3d Spectral data for compound 6d


1675, 1645, 1589, 1472, 1300, 1211, 751, 622 cm-1

.1H NMR (DMSO-d6, 500 MHz): 1.03-

1.09 (m, 3H), 1.18-1.31 (m, 1H), 1.34-1.47 (m, 1H), 1.52-1.70 (m, 2H), 1.73-1.92 (m, 1H),

2.03-2.13 (m, 1H), 2.20-2.45 (m, 2H), 2.81-2.88 (m, 1H), 4.63-4.66 (m, 1H), 6.20 (bs, 2H, -

10

NH2, D2O exchangeable), 6.76-6.82 (m, 1H), 6.86 (t, 1H, J = 7.5 Hz), 7.13-7.18 (m, 1H),

7.26 (d, 1H, J = 7.5 Hz), 7.43 (t, 2H, J = 8 Hz), 7.51-7.56 (m, 1H), 7.84 (d, 1H, J = 7.5 Hz),

7.93 (d, 1H, J = 7.5 Hz), 10.53 (s, 1H, -NH, D2O exchangeable). 13

C NMR (DMSO-d6, 125

MHz): 19.1, 23.6, 25.5, 26.4, 28.8, 29.4, 31.0, 47.5, 55.7, 77.6, 110.3, 118.0, 120.0, 124.7,

126.6, 128.6, 129.1, 129.2, 129.5, 129.8, 133.6, 138.5, 142.7, 158.0, 165.9, 175.1, 198.7. MS

(m/z): 424 (M+H)+. Anal. Calcd. For C27H25N3O2 : C, 76.57; H, 5.95; N, 9.92. Found: C,

76.59; H, 5.94; N, 9.91.

2.3e Spectral data for compound 6e


1668, 1638, 1571, 1460, 1385, 1210, 725, 620 cm-1

.1H NMR (DMSO-d6, 500 MHz): 2.03-

2.09 (m, 2H), 3.18-3.24 (m, 2H), 4.12 (s, 1H), 6.63 (bs, 2H, -NH2, D2O exchangeable), 6.92-

7.01 (m, 4H), 7.12 (d, 1H, J = 7.5 Hz), 7.19-7.21 (m, 2H), 7.28 (d, 1H, J = 7.5 Hz), 7.30 (t,

2H, J = 7.5 Hz), 7.42-7.44 (m, 1H), 7.63 (d, 1H, J = 7.5 Hz), 7.75 (d, 1H, J = 7.5 Hz), 10.50

(s, 1H, -NH, D2O exchangeable). 13

C NMR (DMSO-d6, 125 MHz): 33.3, 48.2, 56.2, 78.0,

112.1, 113.2, 114.6, 115.2, 120.3, 121.2, 122.4, 123.6, 124.5, 127.8, 128.9, 129.3, 130.1,

132.3, 136.1, 140.3, 143.6, 156.1, 162.1, 176.3, 199.2. MS (m/z): 444 (M+H)+. Anal. Calcd.

For C29H21N3O2 : C, 78.54; H, 4.77; N, 9.47. Found: C, 78.56; H, 4.76; N, 9.46.

2.4 Biological evaluation:

2.4a Antimicrobial activity

Antimicrobial activities were carried out using disc diffusion method.17

Petri plates were

prepared with 20 ml of sterile Mueller Hinton agar (MHA) (Hi-media, Mumbai). The test

cultures were swabbed on the top of the solidified media and allowed to dry for 10 min and a

specific amount of synthesized compound at 1mg/disc was added to each disc separately. The

loaded discs were placed on the surface of the medium and left for 30 min at room

temperature for compound diffusion. Negative control was prepared using respective

11

solvents. Streptomycin was used as positive control against bacteria. Ketoconazole was used

as positive control for fungi. The plates were incubated for 24 h at 37°C for bacteria and for

48 h at 28°C for fungi. Zones of inhibition were recorded in millimetres and the experiment

was repeated twice. Bacterial inocula were prepared by growing cells in Mueller Hinton broth

(MHB) (Himedia) for 24 h at 37°C. The filamentous fungi were grown on Sabouraud

dextrose agar (SDA) slants at 28°C for 10 days and the spores were collected using sterile

doubled distilled water and homogenized. Yeast was grown on Sabouraud dextrose broth

(SDB) at 28°C for 48 h.

2.4b Minimum inhibitory concentration (MIC)

Minimum inhibitory concentration studies of the synthesised compounds were performed

according to the standard reference methods for bacteria,18

for filamentous fungi19

and

yeasts.20

The required concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.62 µg/mL and

7.81µg/mL) of the compound were dissolved in DMSO (2%), and diluted to give serial two-

fold dilutions that were added to each medium in 96 well plates. An inoculum of 100 µL

from each well was inoculated. The antifungal agents Ketoconazole for fungi and

Streptomycin for bacteria were included in the assays as positive controls. For fungi, the

plates were incubated for 48 to 72 hours at 28°C and for bacteria the plates were incubated

for 24 h at 37°C. The MIC for fungi was defined as the lowest extract concentration, showing

no visible fungal growth after incubation time. 5 µL of tested broth was placed on the sterile

MHA plates for bacteria and incubated at respective temperature. The MIC for bacteria was

determined as the lowest concentration of the compound inhibiting the visual growth of the

test cultures on the agar plate.

12

IR spectrum of 4a

4000 400 3500 3000 2500 2000 1500 1000 500

47

0

5

10

15

20

25

30

35

40

45

cm - 1

%T

3345.51cm - 1 1640.01cm - 1

1790.90cm - 1 2926.47cm - 1 3224 .42cm - 1

1593.73cm - 1

2860.49cm - 1

2207.62cm - 1

881.39cm - 1

1446.16cm - 1

1151.11cm - 1

1393.17cm - 1

1514.91cm - 1

908.34cm - 1

764.18cm - 1

691.70cm - 1

640.77cm - 1

13

1H NMR spectrum of 4a

14

13C NMR spectrum of 4a

15

Mass spectrum of 4a

16

IR spectrum of 6b

4000 400 3500 3000 2500 2000 1500 1000 500

42

0

5

10

15

20

25

30

35

40

cm - 1

%T

3433.8 3348 3308.9

3059 2930.2

2863.8 2828.6

2200 1708

1669 1633.8

1575.3

1469.8 1395.7 1446.4

1266.8 1208.2

1001.3 1110.6

1184.8

751.4

485.9

630.37

17

1H NMR spectrum of 6b

18

13C NMR spectrum of 6b

19

Mass Spectrum of 6b

20

ORTEP of 4g

21

Antimicrobial activity of synthesised compounds using disc diffusion method (Zone of

inhibition in mm) (1mg/disc)

-: no activity, C: Streptomycin (standard antibacterial agent) C: Ketoconazole (standard

antifungal agent

Comparison of antimicrobial activity of synthesized compounds with standard drugs

Gram Positive Bacteria Gram Negative Bacteria Fungi

Compound M.

luteus

E.

aerogenes

S.

aureus

S. aureus

(MRSA)

B.

subtilis

S.

typhimurium

K.

pneumoniae

P.

vulgaris

C.

albicans

M.

pachydermatis

4a 9±0.32 10±0.84 - 14±0.55 - 13±0.55 12±0.71 - 9±0.84 -

4b 10±0.71 12±0.71 13±0.71 150.71 11±0.55 10±0.55 13±0.55 14±0.84 - -

4c 13±0.55 14±0.32 14±0.84 12±0.32 15±0.84 - 14±0.71 12±0.32 15±0.71 10±0.84

4d 15±0.32 - 15±0.55 10±0.55 12±0.32 12±0.32 13±0.32 - 12±0.84 -

4e 11±0.63 10±0.63 17±0.32 14±0.32 11±0.63 12±0.71 10±0.84 13±0.55 12±0.71 10±0.55

4f 7±0.63 - - - 9±0.55 7±0.63 7±0.63 - - 11±0.63

4g 9±0.71 9±0.84 11±0.71 10±0.71 - 8±0.32 - - 18±0.63 -

4h 10±0.71 17±0.63 18±0.32 15±0.84 10±0.32 11±0.63 12±0.55 14±0.32 9±0.32 -

4i 8±0.32 11±0.32 - - 8±0.32 9±0.55 - 7±0.55 15±0.32 -

4j 10±0.55 - 9±0.63 11±0.71 11±0.55 11±0.32 8±0.63 8±0.32 16±0.71 -

4k - - 10±0.63 - - 12±0.71 9±0.55 - - 12±0.84

4l - 11±0.71 12±0.84 11±0.32 9±0.55 - 11±0.71 - - 12±0.55

6a 16±0.32 8±0.84 12±0.55 10±0.55 12±0.84 15±0.71 14±0.63 13±0.84 - 10±0.55

6b 18±0.84 16±0.32 14±0.32 9±0.32 16±0.71 17±0.63 17±0.32 18±0.71 - -

6c 14±0.71 15±0.32 16±0.71 9±0.84 10±0.71 10±0.71 14±0.63 10±0.55 12±0.63 -

6d 12±0.32 14±0.71 15±0.32 14±0.32 9±0.32 13±0.55 10±0.32 13±0.32 10±0.32 -

6e 8±0.84 11±0.63 10±0.71 9±0.63 - - 8±0.71 11±0.63 - -

C 26±0.63 22±0.71 14±0.71 30±0.71 22±0.84 18±0.90 20±0.71 30±0.71 28±0.71 26±0.32

0

5

10

15

20

25

30

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 6a 6b 6c 6d 6e C

Zo

ne o

f in

hib

itio

n(m

m)

Tested Compounds

M. luteus

E. aerogenes

S. aureus

S. aureus (MRSA)

B. subtilis

S. typhimurium

K. pneumoniae

P. vulgaris

C. albicans

M.pachydermatis

22

Minimum inhibitory concentration of synthesised compounds (1mg/disc)

Gram Positive Bacteria Gram Negative Bacteria Fungi

compound M.luteus E.

aerogenes S.aureus

S.aureus

MRSA

B.

subtilis

S.

typhimurium

K.

pneumoniae

P.

vulgaris

C.

albicans

4b 500 250 250 125 500 500 250 125 -

4c 250 250 250 500 125 - 250 250 125

4d 125 - 125 500 250 250 250 - 250

4e 500 500 62.5 125 500 500 500 250 250

4h 500 125 62.5 125 500 500 500 125 500

6a 125 250 500 500 250 125 125 250 -

6b 62.5 125 125 500 125 62.5 62.5 62.5 -

6c 250 125 125 500 500 500 250 500 500

6d 250 250 250 125 500 250 500 250 500

C 6.25 25 6.25 6.25 6.25 30 6.25 25 25

-: no activity, C: Streptomycin (standard antibacterial agent) C: Ketoconazole (standard

antifungal agent)

Comparison of MIC values of synthesized compounds with standard drugs

0

50

100

150

200

250

300

350

400

450

500

4b 4c 4d 4e 4h 6a 6b 6c 6d C

MICμ

gm

/ltr

Tested Compounds

M. luteus

E.aerogenes

S. aureus

S. aureus MRSA

B.subtilis

S. typhimurium

K. pneumoniae

P. vulgaris

C. albicans

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