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Anti-Tuberculosis Evaluation and Conformational Study ofN-Acylhydrazones Containing the Thiophene Nucleus

Laura N. F. Cardoso1,2, Marcelle L. F. Bispo1, Carlos R. Kaiser2, James L. Wardell1,3,Solange M. S. V. Wardell3, Maria C. S. Lourenço4, Flávio A. F. M. Bezerra4, Rodrigo P. P. Soares5,Marcele N. Rocha5, and Marcus V. N. de Souza1,2

1 Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far Manguinhos, Manguinhos, Rio de Janeiro,Brazil

2 Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Brazil3 Department of Chemistry, University of Aberdeen, Old Aberdeen, Scotland4 Fundação Oswaldo Cruz, Instituto de Pesquisas Clínicas Evandro Chagas, Departamento de Bacteriologia,Manguinhos, Rio de Janeiro, Brazil

5 Fundação Oswaldo Cruz, Centro de Pesquisas René Rachou, Laboratório de Biomarcadores de Diagnóstico eMonitoração, Belo Horizonte, Minas Gerais, Brazil

A series of N-acylhydrazonyl-thienyl derivatives (compounds 2 and 3), mainly of the type 2-(aryl-CH��N–NHCOCH2)-thiene (2: aryl¼ substituted-phenyl; 3: aryl¼heteroaryl) were evaluated againstMycobacterium tuberculosis. Particularly active compound was 3 (heteroaryl¼ 5-nitrothien-2-yl or5-nitrofuran-2-yl) with MIC values of 8.5 and 9.0mM, respectively. Moderately active compounds werecompound 3 (heteroaryl¼pyridin-2-yl) and compound 2 containing aryl¼ 2- or 4-hydroxyphenylgroups, with MIC values between 170 and 408mM. Compound 2 containing OMe, H, F, Cl, Br, CN,and NO2 substituents and compound 3 (heteroaryl¼ furan-2-yl, thien-2-yl, pyrrol-2-yl, imidazol-2-yl,pyridin-3-yl, and pyridin-4-yl) were all inactive. Clearly, there is no correlation of activity with theelectronic effects of the substituents. The activities suggest different modes of biological action of thecompounds having nitro-heteroaryl groups, on the one hand, and the 2-hydroxyphenyl or pyridin-2-ylsubstituents, on the other hand. Compounds having 2- or 4-hydroxyphenyl, 2-hydroxy-5-nitrophenyl,or 4-hydroxy-3-chlorophenyl were less cytotoxic than ethambutol. It is important to notice thatcompound 3 (aryl¼ 5-NO2-furan-2-yl) exhibited a promising therapeutic index (TI¼ 1093.90), with avalue 4.4 less than that of ethambutol. Compounds 2 and 3 exist in DMSO or MeOD solutions asmixtures of EC(O)N/EC��N and ZC(O)N/EC��N conformers.

Keywords: Antimycobacterial activity / Conformational study / N-Acylhydrazones / Thiophene / Tuberculosis

Received: November 5, 2013; Revised: December 10, 2013; Accepted: December 11, 2013

DOI 10.1002/ardp.201300417

Introduction

Since 1993, tuberculosis (TB) has been considered animportant global public health problem by the World HealthOrganization (WHO) with one-third of the world’s populationinfected with Mycobacterium tuberculosis. TB is the second

greatest contributor among infectious diseases to adultmortality, resulting in approximately 1.4 million deathsworldwide in 2010. The most important cause for currentalarms is the emergence of resistant strains to the currenttreatments. There are two types of resistant strains: multi-drug-resistant TB (MDR-TB, resistant to isoniazid and rifampi-cin) and extensively drug-resistant TB (XDR-TB, resistant toisoniazid and rifampicin plus resistant to any fluorquinolone,and to at least one of three injectable second-line anti-TBdrugs). WHO estimates that 650,000 MDR-TB cases occurredin 2010. Furthermore, by the end of 2011, 77 countries andterritories had reported at least one case of XDR-TB. However,

Correspondence: Dr. Marcus V. N. de Souza, Fundação Oswaldo Cruz,Instituto de Tecnologia em Fármacos-Far Manguinhos, Rua SizenandoNabuco, 100, Manguinhos, 21041-250 Rio de Janeiro, Brazil.E-mail: [email protected]: þ55-21-33485050

432 Arch. Pharm. Chem. Life Sci. 2014, 347, 432–448

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

it is estimated that 20% of MDR cases are in fact XDR-TB andthe cure is possible for up to 50–60% of the people affected [1].Considering the high impact of MDR and XDR on TBtreatment, there is an urgent need to develop new anti-TBdrugs.The thiophene nucleus has played an important role

in drug discovery with both natural and syntheticproducts having a wide range of pharmacological activities,such as antiviral [2], anticancer [3], antibacterial [4, 5],antifungal [5, 6], and anti-inflammatory [7]. Our researchgroup has explored the potential of thiophene compoundsas new anti-TB agents and has reported the biologicalactivities of three different series of acetamides contain-ing the thiophene core, as shown in Fig. 1, some ofwhich have exhibited promising activities: particularlysignificant was 2-(phenyl-NH–CO–CH2)thiene, I, with a MICvalue of 115.21mM, and so can be considered as a usefulstarting point for further researches for new anti-TB leadcompounds [8–10].We have recently turned our attention to N-acylhydrazones

containing the thiopheneacetamide moiety; see Fig. 2. Ourinterest in N-acylhydrazone derivatives as antitubercularagents was heightened by the excellent anti-TB activitiesof N-acylhydrazones derivatives of isoniazid (INH), whichexhibited MIC values of between 0.88 and 7.91mM [11–13].

In this study, we report a study on the conformations,anti-TB activities, and cytotoxicities of series of compounds,2-(XYZC6H4–CH��N–NH–CO–CH2)-thiene, 2, and 2-(heteroaryl-CH��N–NH–CO–CH2)-thiene, 3.

Results and discussion

Synthesis and characterizationThe syntheses of 2-(XYZC6H4–CH��N–NH–CO–CH2)-thiene, 2,and 2-(heteroaryl-CH��N–NH–CO–CH2)-thiene (heteroaryl¼furanyl, thienyl, pyrrolyl, imidazolyl, or pyridinyl derivative:3) were achieved from reactions of 2-(H2NNHCOCH2)-thiene, 4,with arenecarbaldehydes as summarized in Scheme 1. Atwo-step reaction was used to prepare 4, as a direct reaction ofthien-2-ylacetyl chloride with hydrazine.hydrate produced thedisubstituted product, thien-2-ylCH2CONHNHCOCH2thiene-2.As shown in Table 1, the compounds studied possessed a widerange of substituents in 2, covering electron-releasing and-withdrawing groups, and different heteroaryl groups in 3.The 1H and 13C NMR spectra of 2 and 3 in either MeOD or

DMSO solutions indicated the presence of two forms, with twosets of signals clearly observed for, at least, the NH, CH, andCH2 atoms in the appropriate NMR spectrum. Specifically, forcompounds containing fluoro substituents in the phenyl ring,two sets of fluorine coupled signals were observed for the

SHN

O

MIC = 111.61 µM

SHN

O

IMIC = 115.21 µM

HN

HN

O OS

S( )10

MIC = 117.92 µM

S N

O

R2

R1

A

R1, R 2 = Me, Et, Pr, But, i-Pr, t-But, CycloPr and Cyclohex

SHN

O

R1

R2

R3

R4

R5

B

R1, R2, R3, R4, R5 = H, CH3, Cl, Br, F, CN, OCH3, CF3, NO2

HN

HN

O OS

S( )n

C

n = 2, 3, 4, 6, 8, 10, 2-cyclohexyldiamine and 2-aminoaniline

Figure 1. Groups of 2-(RNHCOCH2)-thiene derivatives previously evaluated by our research group and the derivatives with the bestactivity [8–10].

Arch. Pharm. Chem. Life Sci. 2014, 347, 432–448 N-Acylhydrazones Containing the Thiophene Nucleus 433

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.archpharm.com

Figure 2. 2-(Aryl-CH��N–NHCOCH2)-thiene derivatives, 2 and 3, designed by molecular hybridization.

S Cl

O

S OCH3

O1

S NHNH2

O4

SHN

O

N Aryl

2 and 3

S N

O

N

SHN

O

N CH3

CH3

CH3

5

6

a

b

c

d

e

Scheme 1. Reagents and conditions: (a) MeOH, r.t., 4 h, 100%; (b) N2H4 · H2O (55%), EtOH, 80°C, 2 h, 75%; (c) EtOH, appropriatealdehyde, r.t., 1–72 h, 52–87%; (d) i: acetone, K2CO3, r.t. 30min. ii: CH3I, 40°C, 23 h, 63%; and (e) acetone, 60°C, 12 h, 94%.

434 L. N. F. Cardoso et al. Arch. Pharm. Chem. Life Sci. 2014, 347, 432–448


Table 1. Data for N 0-[(E/Z)-phenylmethylene]-2-(2-thienyl) acetohydrazide derivatives in solution.

SHN

O

N

X

Y

Z

Compoundd 13C (CH��N) d 1H (NH) d 1H (CH) Conformationa)

ZCONH/ECONH ZCONH/ECONH ZCONH/ECONH ZCONH/ECONH MIC (mM)

3 (2: X¼ Y¼Z¼H) 147.4/143.6 11.63/11.45 8.21/8.02 0.50 Inactivec)

4 (2: X¼ 2-Br; Y¼Z¼H) 145.1/141.5 12.00/11.66 8.56/8.39 1.07b) Inactivec)

5 (2: X¼ 3-Br; Y¼Z¼H) 145.1/141.4 – 8.10/7.91 1.06b) Inactivec)

6 (2: X¼ 4-Br: Y¼Z¼H) 145.6/141.9 – 8.07/7.90 1.14b) Inactivec)

7 (2: X¼ 2-Cl; Y¼Z¼H) 141.5/136.5 – 8.62/8.43 1.06b) Inactivec)

8 (2: X¼ 3-Cl; Y¼Z¼H) 148.3/144.4 – 8.10/7.93 1.05 Inactivec)

9 (2: X¼ 4-Cl; Y¼Z¼H) 145.6/141.8 – 8.11/7.94 1.10b) Inactivec)

10 (2: X¼ 2-F; Y¼Z¼H) 143.5/138.9 8.41/8.21 1.05 Inactivec)

11 (2: X¼ 3-F; Y¼Z¼H) 145.7/142.0 11.74/11.55 8.22/8.02 0.48 Inactivec)

12 (2: X¼ 4-F; Y¼Z¼H) 145.7/142.0 11.65/11.47 8.21/8.02 0.51 Inactivec)

13 (2: X¼ 3-CN; Y¼Z¼H) 144.9/141.2 11.84/11.64 8.25/8.07 0.53 Inactivec)

14 (2: X¼ 4-CN; Y¼Z¼H) 144.6/140.8 11.88/11.69 8.27/8.06 0.42 Inactivec)

15 (2: X¼ 2-NO2; Y¼Z¼H) 148.1/142.4 11.98/11.74 8.61/8.40 0.54 Inactivec)



18 (2: X¼ 2-OMe; Y¼Z¼H) 142.3/138.8 – 8.56/8.37 1.18 Inactivec)



21 (2: X¼ 2-OH; Y¼Z¼H) 147.3/140.8 11.86/11.40 8.41/8.32 1.33 404.722 (2: X¼ 3-OH; Y¼Z¼H) 146.9/143.3 11.58/11.39 8.11/7.93 0.58 Inactivec)

23 (2: X¼ 4-OH; Y¼Z¼H) 147.2/143.4 11.44/11.24 8.09/7.91 0.59 404.724 (2: X¼ Y¼ 2,3-(OH)2; Z¼H 148.0/141.7 11.87 (br)/11.40 8.36/8.31 1.53 Inactivec)

25 (2: X¼ Y¼ 2,4-(OH)2; Z¼H) 148.2/142.1 11.68/11.21 8.27/8.18 0.70 361.926 (2: X¼ Y¼ 2,5-(OH)2; Z¼H) 146.4/140.7 11.76/11.36 8.33/8.25 1.14 Inactivec)

27 (2: X¼ Y¼ 3,4-(OH)2; Z¼H) 147.4/143.8 11.40/11.21 8.00/7.83 0.61 361.928 (2: X¼ 2-OH, Y¼ 4-Me; Z¼H) 147.5/141.7 11.80/11.33 8.36/8.26 1.58 364.529 (2: X¼ 2-OH, Y¼ 5-Me; Z¼H) 147.1/141.0 11.85/11.38 8.36/8.28 1.25 364.530 (2: X¼ 2-OH, Y¼ 3-OMe; Z¼H) 147.1/140.7 11.84/11.41 8.42/8.34 1.13 344.431 (2: X¼ 2-OH, Y¼ 4-OMe; Z¼H) 147.8/141.4 11.76/11.36 8.31/8.21 1.96 172.232 (2: X¼ 2-OH, Y¼ 3-NO2; Z¼H) 146.0/138.5 12.19 (br)/11.66 8.50/8.39 1.60 327.533 (2: X¼ 2-OH, Y¼ 5-NO2; Z¼H) 145.7/142.7 12.04/11.60 8.63/8.53 1.00 81.934 (2: X¼ 4-OH, Y¼ 3-Cl; Z¼H) 142.0/136.8 11.56/11.36 8.07/7.89 0.60 169.635 (2: X¼ Y¼Z¼ 3,4,5-(OH)3 147.6/144.1 11.53/11.15 7.91/7.74 0.65 Inactivec)

36 (3: Aryl¼ furan-2-yl) 149.2/144.1 11.56/11.39 8.10/7.83 0.65 Inactivec)

37 (3: Aryl¼ 5-NO2-furan-2-yl) – 12.02/11.86 8.18/7.96 0.67 9.038 (3: Aryl¼ thien-2-yl) 142.1/138.2 11.57/11.42 8.42/8.19 0.70 Inactivec)

39 (3: Aryl¼ 5-NO2-thien-2-yl) 146.5/140.5 12.00/11.86 8.49/8.19 0.48 8.540 (3: Aryl¼pyrrol-2-yl) 140.0/135.8 11.29/11.12 8.04/7.84 0.70 Inactivec)

41 (3: Aryl¼ imidazol-2yl) 142.3/136.4 11.65/11.44 8.11/7.90 0.58 Inactivec)

42 (4: Aryl¼pyridin-2-yl) 147.1/143.6 11.86/11.7 (br) 8.22/8.06 0.50 409.843 (4: Aryl¼pyridin-3-yl) - 11.80/11.62 8.27/8.05 0.50 Inactivec)

44 (4: Aryl¼pyridin-4-yl) 144.5/140.7 11.90/11.73 8.20/8.00 0.46 Inactivec)

5 Inactivec)

6 386.1EMB 15.3INH 0.46

a) Mean ZCON/ECON ratios calculated from integrations of d 1H (CH��N), d 1H(CH2) and d 1H(NH) signals in DMSO-d6 except whereb) indicates in MeOD solutions at 30°C.c) >100mg/mL.



phenyl carbons. In general, the complexity of the NMR signalsin the aromatic region prevented a complete spectral analysis,but integration of groups of equivalent proton signals was asexpected for each compound.There are, in principle, four possible arrangements about

the C(O)NH��N fragment in acylhydrazones, RCONHN��CHRi:these are obtained from combinations of two conformers,EC(O)NH/ZC(O)NH (anti-periplanar/syn-periplanar), and two geo-metric N��C isomers: EC��N/ZC��N; see Fig. 3. NMR spectraland theoretical studies have previously indicated thatacylhydrazones generally exist mainly or solely as mixturesof EN��C/EC(O)NH and EN��C/ZC(O)NH conformers [14–18]. Theabsence, or very low proportions, of ZC��N geometric isomersis a consequence of steric hindrance [16, 17]. To confirm thatthis also occurred in compounds 2 and 3, the NMR spectra ofcompounds 5 and 6 (see Scheme 1), were also examined. The1H NMR spectrum of 5 in DMSO exhibited two signals for boththe CH2 (4.05 and 3.77ppm) and Me2C (1.93 and 1.86ppm)groups. Dimethyl substitution at the imine fragment elimi-nates the possibility of geometric isomers and hence the twosignal sets indicate EC(O)NH and ZC(O)NH conformers. The 1H NMRspectrum of 6 in DMSO solution did not exhibit duplicatedsignals, indicating that only one form is present in solution, atleast to the extent of NMRdetection. As anticipated, the presenceof themethyl substitution at nitrogen of the C(O)N fragment didin fact result in punitive steric effects. The extra steric hindranceengendered by the methyl substituent on nitrogen preventseasy rotation about the (thienylCH2)-C(O)-N(Me)-(N��CHPh) coreand results in the less hindered rotamer to dominate. In thiscase, the less hindered form would be the ZC(O)NMe conformer.Furthermore, the extra steric hindrance of the methylsubstitution would further disadvantage the ZN��C geometricisomer resulting in formation of just the EN��C/ZC(O)NH isomerand elimination of other isomers, at least to the limits ofNMR detection. In confirmation that conformers of 2 werepresent in solution, variable-temperature 1H NMR spectra of(2: X¼ Y¼ Z¼H) in DMSO solution indicated coalescence ofthe signals of NH (11.62–11.25 to 11.10ppm), CH��N (8.25–8.02to 8.07 ppm), and CH2 (4.19–3.77 to ca. 4.15 ppm) atoms on

heating. At such low temperatures, E$ Z isomerization couldnot occur and so it is concluded that 2 and 3 in solution existas mixtures of EC��N/EC(O)NH and EC��N/ZC(O)N/H forms, which areable to interconvert through rotation of the amide bond.From the studies, in particular by Syakaev et al. [15], the

chemical shifts of the CH��N, CH2, and H(N) atoms in theappropriate NMR spectra for the EN��C/ZC(O)NH conformer insolution are reported to appear downfield of the corre-sponding atoms in the EN��C/EC(O)NH conformer, while thereverse is true for the CH2 and CO atoms. The d 13C(CH��N)and d 1H(CH��N) values for the two forms are listed inTable 1. As these CH atoms are closest to the substituted arylgroups in 2 and 3, they reflect the general differences inelectronic effects of the substituents much more so thanother atoms.Of interest, the differences in the d 1H(CH��N) values for the

EN��C/ZC(O)NH and EN��C/EC(O)NH forms for compound 2 withouta 2-hydroxy substituent are ca. 0.18� 0.02 ppm, while forcompound 2 with a 2-hydroxy substituent the differencesreduce to 0.05–0.09 ppm. In a similar manner, the differencesin the d 1H(CH��N) values for compound 2without a 2-hydroxysubstituent are also ca. 0.18� 0.02 ppm, while for compound2 with a 2-hydroxy substituent the differences increase tobetween 0.40 and 0.53 ppm; see Table 1. From a comparisonof the d 1H(CH��N) values, shown in Table 1, it appears thatthe CH��N proton of the EN��C/EC(O)NH form in the 2-hydroxycontaining compounds is shifted upfield, probably arisingfrom an hydrogen-bonding interaction, not present in theEN��C/ZC(O)NH form. As similar hydrogen bonds, involving theCH��N proton, would occur in both molecular EN��C/ZC(O)NHand EN��C/EC(O)NH forms, and so such differences must occurin dimers or higher aggregates of molecules 2.Values for d 1H(CH2), d

13C(CH2), and d 13C(CO) generallyfall in very close ranges. The ranges, quoted as EN��C/ZC(O)NH:EN��C/EC(O)NH, are 3.72–3.88:4.10–4.26, 33.1–33.6:35.1–35.5,and 170.4–171.5:165.2–166.2 ppm, respectively. Furthermore,the differences in chemical shift values between EN��C/ZC(O)NHand EN��C/EC(O)NH forms are consistent and are ca. 0.41, 1.9,and 5.5 ppm for CH2, CH2, and C��O, respectively.

N

R O

N H

R1 H

N

O R

N H

R1 H

N

R O

N H

H R1

N

O R

N H

H R1

EN=C/ZC(O)NH EN=C/ EC(O)NH ZN=C/ZC(O)NH ZN=C/EC(O)NH

Figure 3. Arrangements of acylhydrazones.



Ratios of EN��C/ZC(O)NH:EN��C/EC(O)NH forms in either DMSO-d6or MeOD solutions at 30°C are listed in Table 1 and extendoverall from 0.48 to 1.96. The values are mean valuesderived from the integrations of the d 1H(CH��N), d 1H(CH2),and d 1H(NH) signals.Some conclusions regarding EN��C/ZC(O)NH:EN��C/EC(O)NH ratios

in DMSO or MeOD solutions at 27°C can be gleaned fromTable 1. These are:(a) Ratios >1 are observed for compound 2 containing ortho-

hydroxy substituents, no matter if other substituentspresent have electron-releasing or -withdrawing effects,an exception being (2: X¼ Y¼ 2,4-(OH)2; Z¼H).

(b) Ratios <1 are generally observed for compound 2containing meta- or para-hydroxy substituents, an excep-tion being (2: X¼Y¼ 2,3-(OH)2; Z¼H).

(c) Ratios <1 are generally observed for compound 2 havingjust nitro, cyano, or methoxy sustituents.

(d) Ratios close to 1 are found for chloro- or bromo-substituted 2: a wider range is realized for fluoroderivatives.

(e) Ratios about 0.5:1 are found for the heteroarylcompound 3.

Crystal structure of (2: X¼ 2-OH; Y¼Z¼H)Characterization of solid (2: X¼ 2-OH; Y¼Z¼H) was carriedout by X-ray crystallography using data collected at 120K [19–23]. The monoclinic space group, P21/c, was assigned. Thesample of (2: X¼ 2-OH; Y¼ Z¼H), an active compound, wasrecrystallized from MeOH solution for the structure determi-nation and was found to exist as the EN��C/EC(O)NH form.Disorder of the planar thiophene ring was found with twodifferent sites for sulfur, S1A and S1B, and arises by rotationsof ca. 180°, about the C1–C2 bond. However, the disorder doesnot affect the angles between the planes of the thienyl ringwith the 2-hydroxyphenyl ring and CH2–CO–NH–N��CHlinker. The atom arrangements of the major componentare shown in Fig. 4a, with the N2-H–O7 strong intramolecular

hydrogen bond drawn as a dashed line. Bond lengths andangles are all in the ranges found for related compounds.Figure 4b shows the comformation of the molecule, drawnwithout hydrogens for reasons of clarity: this illustrates wellthe near 80° angle between the planes of the phenyl andthienyl rings. The EN��C/EC(O)NH form, shown in Fig. 4a, isideally arranged to allow O,N,O chelation, for example, withmetals. This point will be referred to later in the study.Intermolecular hydrogen bonds in crystalline (2: X¼ 2-OH;

Y¼ Z¼H), detected by the PLATON analysis [23], are N1-H1–O12i and C13-H13–O12i hydrogen bonds; symmetry oper-ations: i¼ x, 3/2� y, 1/2þ z. The strong N1-H1–O12 hydrogenbonds, augmented by the weaker C13-H13–O12 bonds,generate C4 chains of molecules, aligned in the direction ofthe c axis. These chains are linked by C1-H1A–p(thienyl ring)

interactions, thereby forming sheets in the bc plane.

Biological activityThe activity against M. tuberculosis H37Rv (ATTC27294) wasassessed using the microplate alamar blue assay (MABA) [19,20]. This nontoxic methodology uses a thermally stablereagent, and shows a good correlation with proportional andBACTEC radiometric methods [21, 22].As shown by the data in Table 1, particularly active

compounds are (3: aryl¼ 5-NO2-thien-2-yl) and (3: aryl¼ 5-NO2-furan-2-yl-yl) with MIC values of 8.5 and 9.0mM, respectively:indeed these compounds exhibit higher activities than thefirst-line anti-TB drug, EMB. In stark contrast, compounds(3: aryl¼ furan-2-yl, thien-2-yl, pyrrol-2-yl, or imidazol-2-yl)are all inactive. Specific nitro-substituted five-memberedring heteroaromatics, e.g., megazol, have been known formany years to possess useful biological activities, mainlyagainst parasites and bacterias [23–27]. The mechanism ofaction of these compounds is based on their ready reductionto radical intermediates using a flavine-based enzyme, presentin the intracellular media. A radical-based mechanismcould also apply with these anti-TB active nitro-heteroarylcompounds.

Figure 4. (a) Atom arrangement and numbering scheme for crystalline (2: X¼ 2-HO, Y¼Z¼H), showing the major orientation of the thienylring, (b) two views of the conformation: hydrogen atoms have been omitted.



The results in Table 1 also indicate that only compound 2,containing a 2-OH or 4-OH groups, in general, was active,albeit only to a moderate extent. While (2: X¼ 2-OH, Y¼H, 4-OMe, 5-Me, 3-OMe, 4-OMe, 3-NO2, and 5-NO2; Z¼H) and (2:X¼ 4-OH, Y¼H, 3-OH, and 3-Cl, Z¼H) were active, with MICvalues of between 81.9 and 404.7mM, compound (2: X¼Y¼2,3-(OH)2 and 2,5-(OH)2, Z¼H) were inactive. The activitydifferences between (3: aryl¼ 5-NO2-thien-2-yl) and (3: aryl¼5-NO2-furan-2-yl-yl), on one hand, and (2: X¼ 2-NO2, 3-NO2

and 2-NO2: Y¼ Z¼H), on the other hand, are striking. Thedifferent effects must stem from the different reductionpotentials of nitrophenyl and the nitro-heteroaryl groups. Ingeneral, compounds containing just H, F, Cl, Br, CN, and OMe,as well as NO2, substituents in the phenyl ring were inactive.As these substituents contain either electron-releasing orelectron-withdrawing effects, it is clear that no correlationexists between biological activities and electron effects. Thelack of activity, in general, of meta-hydroxyl substitutedcompounds, such as (2: X¼ 3-OH, Y¼H, Z¼H) and even (2:X¼Y¼ 2,3-(OH)2 or 2,5-(OH)2, Z¼H), points to a significantnegative influence of ameta-hydroxyl substituent on activities.The activities of compound 2 containing an ortho-hydroxy

group can be grouped together with that of (3: aryl¼pyridin-2-yl). The latter compound, unlike (3: aryl¼pyridin-3-yl)and (3: aryl¼pyridin-4-yl), is active, even if only moderatelyso with a MIC value of 409.8mM. The link is provided bythe abilities to act as O,N,O or O,N,N-tridentate ligands,for example, for vital metal centers; see Fig. 5. Crystalstructure determinations indicate these ortho-hydroxyphenyl-CH��N–NH–C(O)–CH2–R, and pyridin-2-yl-CH��N–NH–C(O)–CH2–Rdo act as ONO and ONN tridentate ligands, respectively. Even,if the major conformer in solution is not the ideal one,rotation about the bond will generate the required conformer.Various ortho-hydroxy-phenyl-CH��N–NH–C(O)–CH2–R, andpyridin-2-yl-CH��N–NH–C(O)–CH2–R compounds have beenreported to form complexes with metals essential for

cellular functions [28–35]. The use of acylhydrazones as ironchelators is a good example of the potential applications ofthese molecules [31–35]. Chelation therapy has demonstratedthat effective iron regulation is antitumor [36] and anti-malarials [37] in humans by depriving cancer cells andmalarial parasites of iron, an essential ingredient forproliferation.As 4-hydroxyphenyl compounds are also mildly active, an

alternate mechamism for action of the 2-hydroxyhenylderivatives could be via a radical route involving quinones.Themechanism of action of quinones is not so clear, but therehave been studies that indicate quinones interact withtopoisomerase II, which is responsible for the DNA beforethe replication process, precluding bacteria growth [38].The precursor, 2-(H2N–NH–CO–CH2)-thiene, 4, was also

tested and found to be slightly active with a MIC value of641mM. Thus the precursor of the 2-(aryl-CH��N–NH–CO–CH2)-thienes, 2 and 3, is more active than many of the 2 and 3compounds synthesized from it. This provides evidence thatthe compounds 2 and 3, or at least many of them, cannot behydrolyzed back to 4 during the testing in any appreciableamount. Compounds 2 and 3 are in general stable compounds,as exemplified by the failed attempts to reduce the C��Ndouble bond.While compound (2: X¼ Y¼ Z¼H) was inactive, its

N-methylated derivative, 6, was found to be moderately sowith a MIC value of 386.1mM. Further study of the biologicalactivities of analogs of 6 is currently underway. As reportedin the earlier discussion of the NMR spectra, compound 6exists in solution in a single EN��C/ZC(O)NMe form.

Cytoxicity assaysA number of the more active compounds were selected forevaluation of their cytotoxicities against the human hepato-ma (HepG2). The cellular viability in the presence and absenceof the test compounds was determined using the Mosman’sMTT (3-(4.5-demethylthylthiazol-2-yl)-2.5-dimethyltetrazoliumbromide; Merck) microcultured tetrazolium assay [39]. Theresults are expressed as the minimum lethal dose that killed50% of the cells (MLD50) inmMand are listed in Table 2. Severalcompounds were found to have promising TIs compared(>50) to first-line anti-TB drugs (EMB and INH). These included(2: X¼ 2-OH; Y¼Z¼H), (2: X¼ 4-OH; Y¼ Z¼H), (2: X¼ 2-OH,Y¼ 5-NO2; Z¼H), (2: X¼ 4-OH, Y¼ 3-Cl; Z¼H), (3: aryl¼ 5-NO2-furan-2-yl), and (3: aryl¼ 5-NO2-thien-2-yl). It is importantto note that compound (3: aryl¼ 5-NO2-furan-2-yl) exhibited apromising TI (1093.90), a value 4.4 less than that of EMB.

Conclusion

The syntheses, conformations in solution, and anti-TB activityof N-acylhydrazone derivatives, mainly of the type 2-(aryl-

MN

O H

HO

HNC

CH2S

MN

O H

HNC

CH2S

N

(a) (b)

Figure 5. 2-(2-Hydroxyphenyl)-CH��N–NHCOCH2)-thiene (a) and2-(2-pyridin-2-yl)-CH��N–NHCOCH2)-thiene (b) drawn in the correctconformations to act as tridentate ligands to metals, M.



CH��N–NHCOCH2)-thiene, are reported. The antimycobacte-rial activities of (3: heteroaryl¼ 5-nitro-furan-2-yl and 5-nitro-thienyl-2-yl) were good, 8.5 and 9.0mM, respectively, whilemoderate activity was established for compound 2 containing

2- and 4-hydroxyphenyl groups, with activities between 170and 407mM. Six derivatives (2: X¼ 2-OH; Y¼ Z¼H), (2: X¼ 4-OH; Y¼ Z¼H), (2: X¼ 2-OH, Y¼ 5-NO2; Z¼H), (2: X¼ 4-OH,Y¼ 3-Cl; Z¼H), (3: aryl¼ 5-NO2-furan-2-yl), and (3: aryl¼ 5-NO2-thien-2-yl) exhibited promising TIs compared to referencedrugs EMB and INH. These results could be a good startingpoint for further study toward new lead compounds againstmulti-drug resistant TB, especially regarding compound (3:aryl¼ 5-NO2-furan-2-yl), which exhibited good activity andwas the less toxic among them. Figure 6 summarizes thefindings.

Experimental

General proceduresMelting points were determined on a Buchi apparatus and areuncorrected. Infrared spectra were recorded on a Thermo NicoletNexus 670 spectrometer as potassium bromide pellets (a finelyground, approximately 1%mixture of a solid sample in KBr is fusedinto a transparent disk using a hydraulic press) and frequenciesare expressed in cm�1. Mass spectra (ESI assay in solution ofammonium chloride) were recorded onMicromass ZQWatersmassspectrometer. NMR spectra were recorded on a Bruker Avance 400operating at 400.00MHz (1H) and 100.0MHz (13C) and BrukerAvance 500 spectrometer operating at 500.00MHz (1H) and125.0MHz (13C), in deuterated dimethylsulfoxide. Chemical shiftsare reported in ppm (d) relative to tetramethylsilane and J-couplingin Hertz (Hz). Proton and carbon spectra were typically obtained atroom temperature. For thin layer chromatography (TLC), platescoated with silica gel were run in chloroform/methanol mixtureand spots were developed in ultraviolet light.

Table 2. Minimal inhibitory concentrations (MIC), minimal lethaldoses (MLD50), and therapeutic indexes (TIs) of ethambutol(EMB), isoniazid (INH) and active N-acylhydrazones containingthe thiophene nucleus.

CompoundMIC(mM)

MLD50

(mM) TIa)

(2: X¼ 2-OH; Y¼Z¼H) 404.7 33,400.8 82.54(2: X¼ 4-OH; Y¼Z¼H) 404.7 40,485.8 100.03(2: X¼ Y¼2,4-(OH)2; Z¼H) 361.9 9072.8 25.07(2: X¼ Y¼3,4-(OH)2; Z¼H) 361.9 1299.2 3.51(2: X¼ 2-OH; Y¼ 4-Me; Z¼H) 364.5 Insol.b)

(2: X¼ 2-OH; Y¼ 5-Me; Z¼H) 364.5 Insol.b)

(2: X¼ 2-OH; Y¼ 3-OMe; Z¼H) 344.4 1021.2 2.97(2: X¼ 2-OH; Y¼ 4-OMe; Z¼H) 172.2 Insol.b)

(2: X¼ 2-OH; Y¼ 3-NO2; Z¼H) 327.5 4775.0 14.6(2: X¼ 2-OH; Y¼ 5-NO2; Z¼H) 81.9 32,750.4 399.9(2: X¼ 4-OH; Y¼ 3-Cl; Z¼H) 169.6 33,653.9 198.4(3: Aryl¼ 5-NO2-furan-2-yl) 9.0 9845.2 1093.9(3: Aryl¼ 5-NO2-thien-2-yl) 8.5 458.8 53.98(3: Aryl¼pyridin-2-yl) 409.8 17.8 0.043EMB 5.3 25,340.2 4781.17INH 0.46 71,649.6 155,760.00

2-(Aryl-CH��N–NH–CO–CH2)thienes derivatives against Mycobacte-rium tuberculosis.a) Therapeutic index, based on the MLD50/MIC ratios.b) Insoluble.

S N

O

N

CH3

SHN

O

N

Z

YX

N

N

N

SHN

O

N

Y

X

R3 NO2

R = O or S

N

NH

5 membersmore activity

SHN

O

N

R1

R2

Br, Cl, F, CN, NO2, OCH3

OH

OH

2 6

33

conformational retention

Figure 6. Structure–activity relationships (SAR) for 2, 3, and 6. Bold boxes indicate substitutions leading to activity, while dashed boxesindicate substitutions resulting in inactive compounds.



Synthesis of methyl thien-2-ylacetate (1)Thien-2-ylacetyl chloride (5.0mL) was added slowly in methanol(30.0mL) at 0°C under N2 atmosphere. The mixture was allowedto warm to room temperature and stirred for 4h. After that, thesolution was concentrated under reduced pressure, and theresidue was poured into 20.0mL of water distillate. The aqueousphase was extracted with CH2Cl2 (3� 10mL). The organic layerswere combined then dried with anhydrous MgSO4 and concen-trated under vacuum to yield methyl 2-thienylacetate as a brownsyrup (5.0mg, 100%). The crude product was used in the next stepwithout further purification.

Yield: 100%; brown syrup; 1H NMR (400MHz, MeOD): d 7.42–7.41 (1H, m, H-5), 6.98–6.96 (2H, m, H-3 and H-4), 3.93 (2H, s, H-10),3.64 (3H, s, H-40). 13C NMR (100MHz; MeOD): d 170.9 (C-20), 134.9(C-2), 126.8 and 126.7 (C-3 and C-4), 124.9 (C-5), 52.2 (C-40), 35.1(C-10). MS/ESI: [MþNa]: 179. IR nmax (cm�1; KBr pellets): 1743(C��O); 1008 (C–O).

Synthesis of 2-(thien-2-yl)acetohydrazide (4)To a solution of methyl 2-thienylacetate (1, 1.0 g, 1.0 equiv.) inethanol (5.0mL) was added N2H4 · H2O (2.8mL, 1.5 equiv., aqueoussolution 55%). After stirring for 1h at 80°C, the resulting mixturewas concentrated under reduced pressure and the residue waspurified by washing with cold EtOH (2� 10mL) and Et2O(2� 10mL), leading to compound 4 as a yellow solid (0.75 g,75%). m.p. 90–91°C (lit. value [40] m.p. 89–90°C).

Yield: 75%; yellow solid; m.p. 90–91°C; (89–90°C) [40].1H NMR (400MHz; MeOD) d: 9.22 (1H, sl, NH), 7.34 (1H, dd,J¼ 1.0 and 5.1 Hz, H-5), 6.94–6.93 (1H, m, H-3), 6.89 (1H, d,JHH¼ 2.5 Hz, H-4), 3.56 (2H, s, H-1). 13C NMR (100MHz; MeOD)d: 168.6 (C��O), 137.5 (C-2), 126.6 and 126.1 (C-3 and C-4), 124.8(C-5), 34.7 (C-1). MS/ESI: [MþNa]: 179. IR nmax (cm

�1; KBr pellets):1643 (C��O); 3286 (N–H).

General procedures for the synthesis of N 0-[(E/Z)-arylmethylene]-2-(thien-2-yl)acetohydrazide derivatives(2 and 3)N0-[(E/Z)-Arylmethylene]-2-(thien-2-yl)acetohydrazide derivatives 2and 3 were prepared by reaction between 2-(thien-2-yl)aceto-hydrazide (4, 0.2 g, 1.0 equiv.) and the appropriate aldehyde(1.2 equiv.) in ethanol (2.0mL). The reaction mixture was stirredfor 1–72h at room temperature. After that, the excess of solventwas concentrated under reduced pressure and the residue waspurified by washing with cold Et2O (2� 10mL), leading to thepure derivatives as solids in 52–87% yields.

N 0-[(E/Z)-Arylmethylene]-2-(thien-2-yl)acetohydrazide(2: X¼Y¼Z¼H)Yield: 0.184 g, 52%; white solid; m.p. 135–137°C. 1H NMR(400MHz; DMSO-d6) d: 11.63 (0.33H, sl, NH, conformer Z), 11.45(0.66H, sl, NH, conformer E), 8.21 (0.33H, s, CH��N, conformer Z),8.02 (0.66H, s, CH��N, conformer E), 7.76–7.67 (2H, m), 7.47–7.36(4H, m), 6.99–6.96 (2H, m), 4.19 (1.33H, s, CH2, conformer E), 3.78(0.66H, s, CH2, conformer Z). 13C NMR (100MHz, DMSO-d6) d: 171.2(CO, conformer Z), 166.0 (CO, conformer E), 147.4 (CH��N,conformer Z), 143.6 (CH��N, conformer E), 137.2 (C-2), 134.6(C-60), 130.5, 130.3, 129.3, (C-70, C-80, C-100 and C-110), 127.5, 127.3,127.2, 126.9 (C-3 and C-4), 125.5 (C-5), 35.3 (CH2, conformer E), 33.2(CH2, conformer Z). MS/ESI: [MþNa]: 267. IR nmax (cm�1; KBrpellets): 3178 (N–H); 1667 (C��O); 1608 (C��N).

N 0-[(E/Z)-(2-Bromophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 2-Br, Y¼Z¼H)Yield: 0.327 g, 70%. Yellow solid; m.p. 166–169°C. 1H NMR(400MHz; MeOD) d: 8.56 (0.52H, s, CH��N, conformer Z), 8.39(0.48H, s, CH��N, conformer E), 8.14 (0.52H, dd, J¼ 7.8 and 1.4Hz,conformer Z), 8.13 (0.48H, dd, J¼ 7.8 and 1.4Hz, conformer E),7.71–7.65 (1H, m), 7.43–7.24 (3H, m), 7.01–6.90 (2H, m) 4.27(0.96H, s, CH2, conformer E), 3.84 (1.04H, s, CH2, conformer Z).13C NMR (100MHz DMSO-d6) d: 171.3 (CO, conformer Z), 165.7(CO, conformer E, 145.1 (CH��N, conformer Z), 141.5 (CH��N,conformer E), 136.54 and 136.50 (C-2 and C-60), 133.1, 132.8, 131.7and 131.5 (C-70, C-90 and C-100), 128.1 and 127.2 (C-80), 126.7, 126.5(C-3 and C-4), 125.1 (C-5), 123.5 and 123.3 (C-110), 35.5 (CH2,conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]: 347.IR nmax (cm

�1; KBr pellets): 3172 (N–H); 1674 (C��O); 1589 (C��N).

N 0-[(E/Z)-(3-Bromophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-Br, Y¼Z¼H)Yield: 0.327 g, 70%. White solid; m.p. 165–170°C. 1H NMR(400MHz; MeOD) d: 8.10 (0.52H, s, CH��N, conformer Z), 8.02(0.52H, br.s, conformer Z), 7.91 (0.96H, br.s, CH��Nþ another aryl-H, conformer E), 7.72–7.67 (1H, m), 7.59–7.53 (1H, m), 7.40–7.28(1H, m), 7.29 (0.52H, dd, J¼ 5.2 and 1.1Hz, conformer Z), 7.25(0.48H, dd, J¼ 5.2 and 1.0Hz, conformer E), 7.01–6.90 (2H,m), 4.26(1.04H, s, CH2, conformer E), 3.84 (0.96H, s, CH2 conformer Z).13C NMR (100MHz; DMSO-d6) d: 171.3 (CO, conformer Z), 165.8(CO, conformer E), 145.1 (CH��N, conformer Z), 141.4 (CH��N,conformer E), 136.6 (C-2), 132.5, 132.3, 130.9, 129.2, 129.0 (C-70,C-80, C-90, C-110), 126.7, 126.6, 126.5, 126.4 (C-3 and C-4), 125.1 (C-5),122.2 and 122.1 (C-100), 35.4 (CH2, conformer E), 33.6 (CH2,conformer Z). MS/ESI: [MþNa]: 345. IR nmax (cm�1; KBr pellets):3182 (N–H); 1670 (C��O); 1604 (C��N).

N 0-[(E/Z)-(4-Bromophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 4-Br, Y¼Z¼H)Yield: 0.327 g, 70%. White solid; m.p. 197°C (dec.). 1H NMR(400MHz; MeOD) d: 8.07 (0.53H, s, CH��N, conformer Z), 7.90(0.47H, s, CH��N, conformer E), 7.71–7.56 (4H, m), 7.29 (0.53H, dd,J¼ 5.1 and 1.0Hz, conformer Z), 7.24 (0.47H, dd, J¼ 5.2 and 1.2Hz,H-5, conformer E), 7.01–6.92 (2H, m), 4.22 (1.06H, s, CH2,conformer E), 3.81 (0.94H, s, CH2, conformer Z). 13C NMR(100MHz; DMSO-d6) d: 171.2 (CO, conformer Z), 165.6 (CO,conformer E), 145.6 (CH��N, conformer Z), 141.9 (CH��N,conformer E), 136.6 (C-2), 133.4 (C-6), 131.8, 130.2, 128.9 and128.7 (C-70, C-80, C-100 and C-110), 126.6, 126.5 and 126.4 (C-3 andC-4) 125.1 (C-5), 123.3 and 123.0 (C-90), 35.4 (CH2, conformer E),33.5 (CH2, conformer Z). MS/ESI: [MþNa]: 345. IR nmax (cm

�1; KBrpellets): 3174 (N–H); 1689 (C��O); 1610 (C��N).

N 0-[(E/Z)-(2-Chlorophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼Cl, Y¼Z¼H)Yield: 0.242g, 60%. White solid; m.p. 168–170°C. 1H NMR (400MHz;MeOD) d: 8.62 (0.51H, s, CH��N; conformer Z), 8.43 (0.49H, s, CH��N,conformer E), 8.17 (0.51H, dd, J¼ 7.6 and 1.6Hz, conformer Z), 8.10–8.08 (0.49H, m, conformer E), 7.46–7.33 (3H, m), 7.30 (0.49H, dd,J¼ 5.2 and 1.1Hz, conformer Z), 7.25 (1H, dd, J¼ 5.1 and 1.0Hz,conformer E), 7.03–6.93 (2H, m), 4.27 (1.02H, s, CH2, conformer E, B),3.84 (0.98H, s, CH2, conformer E). 13C NMR (100MHz; DMSO-d6)d: 171.3 (CO, conformer Z), 165.6 (CO; conformer Z), 142.7 (C-6), 141.5(CH��N; conformer Z), 136.5 (CH��N; conformer Z), 136.50 (C2) 133.1,



132.9 (C-9 andC-7), 131.5, 131.4, 131.2 and 129.9 (C-8 andC-11), 126.8and 127.6 (C-10), 126.8, 126.7, 126.5 (C-3 and C-4), 125.1 (C-5), 35.5(CH2; conformer E), 33.5 (CH2; conformer Z). MS/ESI: [MþNa]: 301.0.IR nmax (cm


N 0-[(E/Z)-(3-Chlorophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-Cl, Y¼Z¼H)Yield: 0.271 g, 67%. White solid; m.p. 156–160°C. 1H NMR(400MHz, DMSO-d6) d: 8.10 (0.51H, s, CH��N, conformer Z), 7.93(0.49H, s, CH��N; conformer E), 7.86 (0.51H, br.s, conformer Z),7.76 (0.49H, br.s, conformer E), 7.69–7.61 (1H, m), 7.42–7.37 (2H,m), 7.29 (0.51H, dd, conformer Z), 7.25 (0.49H, dd, conformer E),7.01–6.93 (2H, m), 4.26 (1.02H, s, CH2, conformer E), 3.84 (0.98H, s,CH2; conformer Z). 13C NMR (125MHz DMSO-d6) d: 174.5 (CO;conformer Z), 169.7 (CO; conformer E), 148.3 (CH��N; conformerZ), 144.4 (CH��N; conformer E), 137.8, 137.6 and 137.2 (C-2 andC-6), 136.0 (C-8), 131.4 and 131.0 (C-9), 128.2, 127.9, 127.7, 127.3and 126.8 (C-3, C-4, C-10 and C11), 126.1 and 125.8 (C-5), 36.5 (CH2;conformer E), 33.5 (CH2; conformer Z). MS/ESI: [MþNa]: 301. IRnmax (cm�1; KBr pellets): 3184 (N–H); 1670 (C��O); 1606 (C��N).

N 0-[(E/Z)-(4-Chlorophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 4-Cl, Y¼Z¼H)Yield: 0.295 g, 73%. White solid; m.p. 179–181°C. 1H NMR(400MHz; MeOH-d4) d: 8.11 (0.52H, s, CH��N, conformer Z), 7.94(0.48H, s, CH��N, conformer E), 7.77 (0.52H, s, conformer Z), 7.73(0.48H, s, conformer E), 7.45–7.41 (2H, t, J¼ 8Hz), 7.29 (0.52H, dd,J¼ 5.1 and 1.1Hz, H-5, conformer Z), 7.24 (0.48H, dd, J¼ 5.2 and1.0Hz, H-5, conformer E), 7.01–6.92 (2H, m), 4.26 (0.96H, s, CH2,conformer E), 3.84 (1.04H, s, CH2, conformer Z). 13C NMR (100MHzDMSO-d6) d: 171.2 (CO, conformer Z), 165.7 (CO, conformer E),145.6 (CH��N; conformer Z), 141.8 (CH��N; conformer E), 136.6 (C-2),134.5 and 134.3 (C-9), 133.1, 130.0, 129.1 and 128.9, 128.7 (C-7, C-8,C-10 and C11), 126.6, 126.5 and 126.4 (C-3 and C-4), 125.1 (C-5), 35.5(CH2, conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]: 301. IRnmax (cm�1; KBr pellets): 3172 (N–H); 1666 (C��O); 1610 (C��N).

N 0-[(E/Z)-(2-Fluorophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 2-F, Y¼Z¼H)Yield: 0.205g, 54%.White solid; m.p. 135–137°C. 1HNMR (400MHz;DMSO-d6) d: 8.41 (0.51H, s, CH��N, conformer Z), 8.21 (0.49H, s,CH��N, conformer E), 8.11 (0.51H, dd, J¼ 1.5, 7.7 and 7.5Hz,conformer Z), 8.02 (0.49H, ddd, J¼ 1.6, 7.7 and 7.5Hz, conformer E),7.47–7.41 (1H, m), 7.30–7.12 (3H, m), 7.01–6.93 (2H, m), 4.27(1.02H, s, CH2, conformer E), 3.84 (0.98H, s, CH2, conformer Z. 13CNMR (100MHz DMSO-d6) d: 174.6 (CO, conformer Z), 169.6 (CO,conformer E), 164.0 (JC,F¼ 249Hz) and 163.9 (JC,F¼ 249Hz) [C7],143.5 (JC,F¼ 5Hz, CH��N, conformer Z), 138.7 (JC,F¼ 5Hz, CH��N,conformer E), 138.4 and 138.0 (C-2), 134.3 (C-9), 133.6 (JC,F¼ 9Hz)and 133.1 (JC,F¼ 9Hz, C-11), 129.0 and 128.7 (C-10), 126.8 (C-3 andC-4), 126.6 (C-5), 123.9 (JC,F¼ 27Hz) and 123.8 (JC,F¼ 27Hz) (C-6),117.6 (JC,F¼ 18Hz) and 116.8 (JC,F¼ 18Hz) (C11), 36.7 (CH2,conformer E), 35.1 (CH2, conformer Z). MS/ESI: [MþNa]: 285. IRnmax (cm


N 0-[(E/Z)-(3-Fluorophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-F, Y¼Z¼H)Yield: 0.315 g, 83%. White solid; m.p. 155–159°C. 1H NMR(400MHz; DMSO-d6) d: 11.74 (0.32H, s, NH, conformer Z), 11.55

(0.68H, s, NH, conformer E), 8.22 (0.32H, s, CH��N, conformer Z),8.02 (0.68H, s, CH��N, conformer E), 7.60–7.45 (3H, m), 7.40–7.36(1H, m), 7.25–7.20 (1H, m), 7.00–6.94 (2H, m), 4.21 (0.64H, s, CH2,conformer E), 3.79 (1.36H, s, CH2, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 171.1 (CO, conformer Z), 165.6 (CO,conformer E), 163.3 (JC,F¼ 249Hz) and 163.0 (JC,F¼ 249Hz) (C8),145.7 (CH��N, conformer Z), 142.0 (CH��N, conformer E), 136.6(JC,F¼ 17Hz, C-6), 130.8 (JC,F¼ 5Hz, C-10), 126.6, 126.5 and 125.1,C-5), 125.1 (C-5), 123.3 (C-11), 116.6 (JC,F¼ 21Hz) and 116.4(JC,F¼ 21Hz) (C-9), 112.8 (JC,F¼ 20Hz) and 112.6 (JC,F¼ 20Hz) (C7),35.5 (CH2 conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]:285. IR nmax (cm�1; KBr pellets): 3182 (N–H); 1668 (C��O);1604 (C��N).

N 0-[(E/Z)-(4-Fluorophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 4-F, Y¼Z¼H)Yield: 0.323 g, 85%. White solid; m.p. 173–175°C. 1H NMR(400MHz; DMSO-d6) d: 11.65 (0.34H, s, NH, conformer Z), 11.47(0.66H, s, NH, conformer E), 8.21 (0.34H, s, CH��N, conformer Z),8.02 (0.66H, s, CH��N, conformer Z), 7.81–7.74 (2H, m), 7.40–7.36(1H, m), 7.32–7.26 (2H, m), 6.98–6.95 (2H, m), 4.19 (1.34H, s,CH2, conformer E), 3.77 (0.66H, s, CH2, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 171.1 (CO, conformer Z), 165.6 (CO,conformer E), 163.2 (d, JCF¼ 300Hz) and 162.0 (d, JCF¼ 300.0Hz)(C-9), 145.7 (CH��N, conformer Z), 142.0 (CH��N, conformer E),136.7 (C-2), 130.8 (d, JCF¼ 3Hz, C-6), 129.2 (d, JCF¼ 810Hz) and129.0 (d, JCF¼ 10Hz) (C-7 and C-11), 126.6, 126.5 and 126.4 (C-3 andC-4), 125.1 (C-5), 115.7 and 116.0 (d, JCF¼ 25Hz) (C-8 and C-10), 35.5(CH2, conformer E), 33.5 (CH2 (d; JCF¼ 260Hz), conformer Z).MS/ESI: [MþNa]: 285. IR nmax (cm

�1; KBr pellets): 3180 (N–H); 1666(C��O); 1612 (C��N).

N 0-[(E/Z)-(3-Cyanophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-CN, Y¼Z¼H)Yield: 0.296 g, 76%. White solid. m.p. 175–177°C. 1H NMR(400MHz; DMSO-d6) d: 11.84 (0.35H, s, NH, conformer Z), 11.64(0.65H, s, NH, conformer E), 8.25 (0.35H, s, CH��N, conformer Z),8.20 (0.65H, s, CH��N, conformer E), 8.11–8.04 (2H, m), 7.90 (1H,br, d, J¼ 7.7Hz), 7.66 (1H, t), 7.41–7.37 (1H, m), 7.00–6.95 (2H, m),4.24 (1.30H, s, CH2, conformer Z), 3.80 (0.70H, s, CH2, conformer E).13C NMR (125MHz; DMSO-d6) d: 171.4 (CO, conformer Z), 165.9(CO, conformer E), 144.9 (CH��N, conformer Z), 141.2 (CH��N,conformer E), 138.6 (C-6), 136.5, 132.7, 130.8 and 13.0 (C-7, C-9 andC-11), 127.6 and 127.4 (C-10), 126.7 and 126.5 (C-3 and C-4), 125.1C-5), 118.6 (C�N), 111.6 (C-8), 35.4 (CH2, conformer E), 33.5 (CH2,conformer Z). MS/ESI: [MþNa]: 292. IR nmax (cm�1; KBr pellets):3182 (N–H), 1670 (C��O), 1604 (C��N).

N 0-[(E/Z)-(4-Cyanophenyl)methylene]-2-(2-thienyl)-acetohydrazide (2: X¼ 4-CN, Y¼Z¼H)Yield: 0.261 g, 67%. White solid. m.p. 187–190°C. 1H NMR(400MHz; DMSO-d6) d: 11.88 (0.30H, s, NH, conformer Z), 11.69(0.70H, s, NH, conformer E), 8.27 (0.30H, s, CH��N, conformer Z),8.06 (0.70H, s, CH��N, conformer E, 7.91–7.86 (5H, m, H-70, H-8,H-100, and H-110), 7.00–6.90 (2H, m, H-3, H-4 and H-5), 4.22(1.40H, s, CH2, conformer E), 3.81 (0.60H, s, CH2, conformer Z).13C NMR (125MHz; DMSO-d6) d: 171.4 (CO, conformer Z), 165.8(CO, conformer E), 144.6 (CH��N, conformer Z), 140.8 (CH��N,conformer E), 136.6 and 135.5 (C-2 and C-6), 133.2, 132.9, 132.7(C-8 and C-10), 131.0, 130.9, 130.7, 130.0 (C-7 and C-11), 126.7 and



126.5 (C-3 and C-4), 125.1 (C-5), 118.4 (C�N), 112.0 (C-9), 35.4 (CH2,conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]: 292. IRnmax (cm�1; KBr pellets): 3182 (N–H), 1670 (C��O), 1597 (C��N).

N 0-[(E/Z)-(2-Nitrophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 2-O2N, Y¼Z¼H)Yield: 0.335 g, 80%. White solid. m.p. 143–145°C. 1H NMR(400MHz DMSO-d6) d: 11.98 (0.35H, s, NH, conformer Z), 11.74(0.65H, s, NH, conformer E), 8.61 (0.35H, s, CH��N, conformer Z),8.40 (0.65H, s, CH��N, conformer E), 8.15–8.08 (2H, m), 7.85–7.75(1H, t, J¼ 7.6Hz), 7.70–7.64 (1H, m), 7.40–7.37 (1H, m), 7.00–6.95(2H, m), 4.20 (1.30H, s, CH2, conformer Z), 3.81 (0.70H, s, CH2,conformer E). 13C NMR (125MHzDMSO-d6) d: 171.4 (CO, conformerZ), 165.9 (CO, conformer), 148.1 (C-7), 142.4 (CH��N, conformer Z),138.5 (CH��N, conformer E), 136.4 (C-2), 133.7, 133.5, 130.6and 130.4 (C-6, C-9 and C-10), 128.7 and 128.1 (C-11), 126.7and 126.6 (C-3 and C-4), 125.1 (C-5), 124.7 and 124.5 (C-8), 35.4(CH2, conformer E), 33.3 (CH2, conformer Z). MS/ESI: [MþNa]: 312.IR nmax (cm

�1; KBr pellets): 3196 (N–H), 1668 (C��O), 1592 (C��N).

N 0-[(E/Z)-(3-Nitrophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-O2N, Y¼Z¼H)Yield: 0.272 g, 65%. White solid. m.p. 190–193°C. 1H NMR(400MHz, DMSO-d6) d: 11.89 (0.38H, s, NH, conformer Z), 11.69(0.62H, s, NH, conformer E), 8.53 (1.0H, br.s) 8.34 (0.38H, s, CH��N,conformer Z), 8.25 (1H, br.d, J¼ 8Hz), 8.19–8.12 (1.62, m, s, CH��N,conformer E and 1 aryl-H), 7.74 (0.38H, t, J¼ 8Hz, conformer Z),7.73 (0.62H, t, J¼ 8Hz, conformer E), 7.40 (0.38H, dd, J¼ 2.5, 4Hz,conformer Z), 7.38 (0.62H, dd, J¼ 1.25Hz, conformer E), 7.00–6.98(2H, m), 4.23 (1.24H, s, CH2, conformer E), 3.82 (0.76H, s, CH2,conformer Z). 13C NMR (125MHz; DMSO-d6) d: 171.3 (CO,conformer Z), 165.9 (CO, conformer E), 148.2 (C-8), 144.5 (CH��N,conformer Z), 140.8 (CH��N, conformer E), 136.5 and 136.0 (C-6and C-2), 133.2, 132.8 and 130.4 (C-9, C-10 and C-11), 126.7, 126.5and 125.4 (C-3 and C-4), 125.2 (C-5), 124.2 and 124.0 (C-7), 121.0,35.4 (CH2, conformer E), 33.6 (CH2, conformer Z). MS/ESI: [MþNa]:312. IR nmax (cm�1; KBr pellets): 3180 (N–H), 1668 (C��O), 1600(C��N).

N 0-[(E/Z)-(4-Nitrophenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 4-O2N, Y¼Z¼H)Yield: 0.364 g, 87%. Yellow solid. m.p. 208–210°C. 1H NMR(400MHz, DMSO-d6) d: 11.93 (0.33H, s, NH, conformer Z), 11.75(0.67H, s, NH, conformer E), 8.31 (0.33H, s, CH��N, conformer Z),8.29 (2H, d, J¼ 8Hz), 8.12 (0.67H, s, CH��N, confomer E), 8.00(1.34H, br.d, J¼ 8Hz, conformer E), 7.96 (0.66H, br.d, J¼ 8Hz,conformer Z), 7.40–7.35 (1H, m), 7.05–6.95 (2H, m), 4.24 (1.34H, s,CH2, conformer E), 3.82 (0.66H, s, CH2, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 171.5 (CO, conformer Z), 166.0 (CO,conformer E), 147.7 (C-9), 144.4 (CH��N, conformer Z), 140.8(CH��N, conformer E), 140.4 (C-6), 136.4 (C-2), 128.0 and 127.8 (C-6),126.7 and 126.6 (C-3 and C-4), 125.2 (C-5), 124.0 (C-8 and C-10), 35.4(CH2, conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]: 312.IR nmax (cm


N 0-[(E/Z)-(2-Methoxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 2-MeO, Y¼Z¼H)Yield: 0.254 g, 64%. Yellow solid. m.p. 136–138°C. 1H NMR(400MHz, DMSO-d6) d: 8.56 (0.54H, s, CH��N, conformer Z), 8.37

(0.46H, s, CH��N, conformer E), 8.01 (0.54H, br.dd, J¼ 8 and 1.5Hz,conformer Z), 8.00 (0.46H, br.dd, J¼ 8 and 1.5Hz, conformer E),7.41–7.37 (1H, m), 7.28 (0.54H, dd, J¼ 5 and 1.2Hz, conformer Z),7.24 (0.46H, dd, J¼ 5 and 1.2Hz, conformer E), 7.05–6.90 (5H, m),4.25 (0.92H, s, CH2, conformer E), 3.88 (3H, s, OMe), 3.82 (1.08H, s,CH2, conformer Z). 13C NMR (125MHz, DMSO-d6) d: 171.0 (CO,conformer Z), 165.4 (CO, conformer E), 157.6 (C-7), 142.3 (CH��N,conformer Z), 138.8 (CH��N, conformer E), 135.6 (C-9), 131.6 and131.3 (C-11). 126.7, 126.6, 125.5 and 125.4 (C-3 and C-4), 125.0 (C-5),122.2 and 120.7 (C-6), 111.8 (C-8), 54.0 (OMe), 35.5 (CH2, conformerE), 33.6 (CH2, conformer Z). MS/ESI: [MþNa]: 297. IR nmax (cm�1;KBr pellets): 3174 (N–H), 1670 (C��O), 1610 (C��N).

N 0-[(E/Z)-(3-Methoxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-MeO, Y¼Z¼H)Yield: 0.222 g, 56%. White solid. m.p. 111–112°C. 1H NMR(400MHz, DMSO-d6) d: 8.10 (0.56H, s, CH��N, conformer Z), 7.92(0.44H, s, CH��N, conformer E), 7.49 (0.56H, s, conformer Z), 7.35–7.25 (4.44H, m), 7.00–6.90 (3H, m), 4.25 (0.88H, s, CH3, conformerE), 3.83 (3H, s, OMe), 3.83 (1.12H, m, CH2, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 171.1 (CO, conformer Z), 165.6 (CO,conformer E), 158.5 (C-8). 146.7 (CH��N, conformer Z), 142.8(CH��N, conformer E), 136.7 and 135.6 (C-2 and C-6), 129.9 (C-10),126.6 (C-3 and C-4), 125.1 (C-5), 119.6, 115.8 and 111.3 (C-7, C-9 andC-11), 55.1 (OMe), 35.5 (CH2, conformer E), 33.6 (CH2, conformer Z).MS/ESI: [MþNa]: 297. IR nmax (cm


N 0-[(E/Z)-(4-Methoxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 4-MeO, Y¼Z¼H)Yield: 0.329 g, 83%. White solid. m.p. 147–150°C. 1H NMR(400MHz, DMSO-d6) d: 8.05 (0.57H, s, CH��N, conformer Z), 7.90(0.43H, s, CH��N, conformer E), 7.71 (1.14H, br.d, J¼ 9Hz,conformer Z), 7.67 (0.86H, br.d, J¼ 9Hz, conformer E), 7.28(0.57H, dd, J¼ 1, 5Hz, conformer Z), 7.24 (0.43H, dd, J¼ 1, 5Hz,conformer E), 7.10–6.90 (4H, m), 4.24 (0.86H, s, CH2, conformer E),3.86 (1.14H, s, CH2, conformer Z), 3.83 (3H, s, OMe). 13C NMR(125MHz; DMSO-d6) d: 170.8 (CO, conformer Z), 165.3 (CO,conformer E), 160.8 and 160.6 (C-9), 146.7 (CH��N, conformer Z),143.0 (CH��N, conformer E), 136.9 and 136.8 (C-2 and C-6), 128.7and 128.4 (C-7 and C-11), 126.8, 126.7, 126.6, 126.5 and 126.3 (C-3and C-4), 125.1, 125.0 (C-5), 114.3 and 114.2 (C-8 and C-10), 55.3(OMe), 35.5 (CH2 conformer E), 33.6 (CH2, conformer Z). MS/ESI:[MþNa]: 297. IR nmax (cm

�1; KBr pellets): 3184 (N–H); 1670 (C��O);1614 (C��N).

N 0-[(E/Z)-(2-Hydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 2-HO, Y¼Z¼H)Yield: 0.218 g, 58%. Yellow solid; m.p. 173–176°C. 1H NMR(400MHz; DMSO-d6) d: 11.86 (0.57H, s, NH, conformer Z), 11.40(0.43H, s, NH, conformer E), 11.04 and 10.06 (OH), 8.41 (0.57H, s,CH��N, conformer Z), 8.32 (0.43H, s, CH��N, conformer E), 7.77(0.43H, dd, J¼ 1.4, 8Hz, conformer E), 7.53 (0.57H, dd, J¼ 1.4, 8Hz,conformer Z), 7.41–7.31 (0.86H, m, conformer E), 7.30–7.20(1.14H, m, conformer Z), 7.01–6.85 (4H, m), 4.16 (0.86H, s, CH2,conformer E), 3.80 (1.14H, s, CH2, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 170.8 (CO, conformer Z), 165.4 (COconformer E), 157.3 and 156.4 (C-7), 147.3 (CH��N, conformerZ), 140.8 (CH��N, conformer E), 136.7 and 136.5 (C-2 and C-9), 131.4and 131.0 (C-11), 129.3, 126.7, 126.5 and 126.4 (C-3 and C-4), 125.2



and 125.1 (C-5), 120.2, 119.4 and 119.3 (C-6 and C-10), 118.6, 116. 3and 116.1 (C-8), 35.2 (CH2, conformer E), 33.6 (CH2, conformer Z).MS/IES: [MþNa]: 283. IR nmax (cm


N 0-[(E/Z)-(3-Hydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 3-HO, Y¼Z¼H)Yield: 0.271 g, 72%. White solid. m.p. 186–190°C. 1H NMR(400MHz, DMSO-d6) d: 11.58 (0.37H, s, NH, confomer Z), 11.39(0.63H, s, NH, conformer E), 9.61, 8.11 (0.37H, s, CH��N,conformer Z), 7.93 (0.63H, s, CH��N, conformer E), 7.39 (0.37H,dd, J¼ 1.28 and 5.2Hz, conformer Z), 7.37 (0.63H, dd, J¼ 1.28 and5.2 Hz, conformer E), 7.26–7.20 (1H, m), 7.15 (1H, br.s), 7.11–7.05(1H, m), 6.99–6.95 (2H, m), 6.83–6.80 (1H, m), 4.17 (1.26H, s, CH2,conformer E), 3.76 (0.74H, s, CH2, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 171.0 (CO, conformer Z), 165.5 (CO,confomer E), 157.6 (C-8), 146.9 (CH��N, conformer Z), 143.3(CH��N, conformer E), 136.7 and 135.4 (C-2 and C-6), 129.9 (C-10),126.7, 126.6, 126.5 and 126.4 (C-3 and C-4), 125.1 and 125.0 (C-5),118.8 and 118.4 (C-11), 117.4 and 117.2 (C-9), 112.6 (C-7), 35.5(CH2; conformer E or B), 33.5 (CH2; conformer Z). MS/ESI: [MþNa]:283. IR nmax (cm�1; KBr pellets): 3207 (N–H); 1647 (C��O); 1612(C��N).

N 0-[(E/Z)-(4-Hydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X¼ 4-HO, Y¼Z¼H)Yield: 0.313 g, 83%. White solid. m.p. 200–203°C. 1H NMR(400MHz, DMSO-d6) d: 11.44 (0.37H, s, NH, conformer Z), 11.24(0.63H, s, NH, conformer E), 8.09 (0.37H, s, CH��N, conformer Z),7.91 (0.63H, s, CH��N, conformer E), 7.54 (1.26H, d, J¼ 9Hz,conformer E), 7.52 (0.74H, d, J¼ 9Hz, conformer Z), 7.37 (0.74H,dd, J¼ 1.2 and 5Hz, conformer Z), 7.35 (1.26H, d, J¼ 1.2 and5Hz, conformer E), 7.00–6.93 (2H, m), 6.84–6.80 (2H, m), 4.15(1.26H, s, CH2, conformer E), 3.74 (0.74H, s, CH2, conformer Z).13C NMR (125MHz; DMSO-d6) d: 170.7 (CO, conformer Z), 165.2(CO, conformer E), 158.4 and 158.2 (C-9), 147.2 (CH��N,conformer Z), 143.4 (CH��N, conformer E), 136.9 (C-2), 128.8,and 128.5 (C-6, C-7 and C-11), 126.7, 126.5, 126.4 and 126.3 (C-3and C-4), 125.2 and 1250 (C-5), 115.7 and 115.6 (C-6, C-11), 35.5(CH2, conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]:283. IR nmax (cm�1; KBr pellets): 3184 (N–H); 1622 (C��O); 1579(C��N).

N 0-[(E/Z)-(2,3-Dihydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X, Y¼ 2,3-(HO)2, Z¼H)Yield: 0.248 g, 62%; m.p. 236–237°C; 1H NMR (400MHz; DMSO-d6):d 11.87 (0.61H, s, NH, conformer Z), 11.40 (0.39H, s, NH, conformerE), 10.85 (OH), 9.20 (OH), 8.36 (0.61H, s, CH��N, conformer Z), 8.31(0.39H, s, CH��N, conformer E), 7.41 (0.61H, dd, J¼ 1.0 and 5.2Hz,conformer Z), 7.37 (0.39H, dd, J¼ 1.5 and 5.0Hz, conformer E),7.19 (0.39H, dd, J¼ 7.9 and 1.3Hz, conformer E), 7.00–6.94 (2.61H,m) 6.72 (0.61H, t, J¼ 8.0Hz, conformer Z), 6.69 (0.39H, t, J¼ 8.0Hz,conformer E), 4.15 (0.78H, s, CH2, conformer E), 3.80 (1.22H, s,CH2, conformer E). 13C NMR (100MHz; DMSO-d6): d 170.7 (CO,conformer Z), 165.4 (CO, conformer E), 148.0, 146.0, 145.6, 145.5and 145.2 (C-7, C-8 and CH��N, conformer Z), 141.7 (CH��N, (C-5),120.7 and 119.9, (C-9), 119.3, 119.2, 118.7 and 117.4 (C-10 andC11), 116.9, and 116.6 (C-9), 35.2 (CH2, conformer E), 33.6 (CH2,conformer Z). MS/ESI: [M�H]: 275. IR nmax (cm

�1, KBr pellets): 3263(N–H); 1656 (C��O); 1581 (C��N).

N 0-[(E/Z)-(2,4-Dihydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X, Y¼ 2,4-(HO)2, Z¼H)Yield: 0.260 g, 65%;m.p. 195–196°C; 1HNMR (400MHz; DMSO-d6): d11.68 (0.65H, s, NH, conformer Z), 11.21 (0.35H, s, NH, conformer E),11.2 (OH), 10.2 (OH), 8.27 (0.65H, s, CH��N, conformer Z), 8.18(0.35H, s, CH��N, conformer E), 7.52 (0.35H, dd, J¼ 5 and 8Hz,conformer E), 7.39 (0.65H, dd, J¼ 2.5 and 4Hz, conformer Z), 7.35(0.35H,m, conformer E), 7.30 (0.65H, d, J¼ 8Hz, conformer Z), 7.00–6.94 (2H, m), 6.34–6.25 (2H, m), 4.11 (1.30H, s, CH2, conformer E),3.76 (0.70H, s, CH2, conformer Z). 13C NMR (100MHz; DMSO-d6):d 171.3 (CO, conformer Z), 165.0 (CO, conformer E), 160.7, 160.4,159.4 and 158.1 (C- and C-9), 148.2 (CH��N, conformer Z), 142.1(CH��N, conformer E), 136.8 and 136.7 (C-2), 131.2 (C-11), 126.7,126.5 and 126.4 (C-3 and C-4), 125.1 and 125.0 (C-5), 111.7, 110.4(C-6), 107.9 and 107.7 (C-10), 102.6 and 102.4 (C-8), 35.2 (CH2,conformer E), 33.6 (CH2, conformer Z). MS/ESI: [M�H]: 275. IR nmax

(cm�1; KBr pellets): 3263 (N–H); 1656 (C��O); 1581 (C��N).

N 0-[(E/Z)-(2,5-Dihydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X, Y¼ 2,5-(HO)2, Z¼H)Yield: 0.300 g, 75%; m.p. 192–193°C; 1H NMR (400MHz; DMSO-d6):d 11.76 (0.53H, s, NH, conformer Z), 11.36 (0.47, s, NH, conformerE), 10.16 (1H, s), 9.35 (1H, s), 8.97 (0.47H, s, H-110, conformer E),8.89 (0.53H, s, H-110, conformer Z), 8.33 (1H, s, CH��N, conformerZ), 8.25 (0.53H, s, CH��N, conformer E), 7.40–7.36 (1H, m), 7.16(0.47H, d, J¼ 2.6Hz, H-5), 7.00–6.95 (2.53H, m), 6.70–6.68 (2H, m),4.15 (0.94H, s, CH2, conformer E), 3.77 (1.06H, s, CH2, conformer Z).13C NMR (100MHz, DMSO-d6): d 170.7 (CO, conformer Z), 165.3(CO, conformer E), 150.1, 150.0, 149.9 and 149.3 (C-7 and C-10),146.4 (CH��N, conformer Z), 140.7 (CH��N, conformer E), 136.8and 136.6 (C-2), 126.7, 126.5 and 126.2 (C-3 and C-4), 125.1 (C-5),120.4, 119.0, 118.7, 117.0, 113.6, 111.1 (C-6, C-8, C-9 and C-11), 35.3(CH2, conformer E), 33.6 (CH2, conformer Z). MS/ESI: [M�]: [M�H]:275. IR nmax (cm�1; KBr pellets): 3302 (N–H); 1664 (C��O); 1581(C��N).

N 0-[(E/Z)-(3,4-Dihydroxyphenyl)methylene]-2-(thien-2-yl)-acetohydrazide (2: X, Y¼ 3,4-(HO)2, Z¼H)Yield: 0.304 g, 76%; m.p. 159–160°C; 1H NMR (400MHz; DMSO-d6):d 11.40 (0.38H, s, NH, conformer Z), 11.21 (0.62H, s, NH, conformerE), 9.40 (1H, s), 9.3 (1H, s), 8.00 (0.38H, s, CH��N, conformer Z), 7.83(0.62H, s, CH��N, conformer E), 7.38 (0.38H, dd, J¼ 1.5 and 5.0Hz,conformer Z), 7.37 (0.62H, dd, J¼ 1.5 and 5.0Hz, conformer E),7.19 (0.62H, d, J¼ 2Hz, conformer E), 7.18 (0.38H, d, J¼ 2Hz,conformer Z), 7.00–6.90 (2H, m), 6.90 (1H, dd, J¼ 2.0 and 8.0Hz),6.76 (1H, dd, J¼ 4.5 and 8Hz) 4.14 (1.24, s, CH2, conformer E), 3.73(0.76H, s, CH2, conformer Z). 13C NMR (100MHz; DMSO-d6): d 170.7(CO, conformer Z), 165.2 (CO, conformer E), 148.0 and 147.8 (C-9),147.4 (CH��N, conformer Z), 145.7 (C-8), 143.8 (CH��N, conformerE), 137.0 and 136.9 (C-2), 126.7, 126.5 and 126.4 (C-3 and C-4), 125.7and 125.6 (C-6), 125.1 and 125.0 (C-5), 121.2 and 121.0 (C-11), 115.6and 115.4 (C-7), 112.7 (C-10), 35.5 (CH2, conformer E), 33.6 (CH2,conformer Z). MS/ESI: [M�]: [M�H]: 275. IR nmax (cm

�1; KBr pellets):3523 (N–H); 1643 (C��O); 1591 (C��N).

N 0-[(E/Z)-(2-Hydroxy-4-methylphenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 2-HO, Y¼ 4-Me, Z¼H)Yield: 0.332 g, 79%; m.p. 179–180°C; 1H NMR (400MHz; DMSO-d6):d 11.80 (0.61H, s, NH, conformer Z), 11.33 (0.39H, s, NH, conformerE), 11.01 (1H, s), 8.36 (0.61H, s, CH��N, conformer Z), 8.26 (0.39H, s,



CH��N, conformer E), 7.61 (0.39h, d, J¼ 6Hz, conformer E), 7.41–7.35 (1.61H, m), 7.00–6.94 (2H, m), 6.75–6.65 (2H, m), 4.15(0.78H, s, CH2, conformer E), 3.78 (1.22H, s, CH2, conformer Z), 2.27and 2.26 (3H, s, Me). 13C NMR (100MHz; DMSO-d6): d 170.6 (CO,conformer Z), 165.3 (CO, conformer E), 157.3 and 156.4 (C-7), 147.5(CH��N, conformer Z), 141.7 (CH��N, conformer E), 141.2 (C-9),136.7 and 136.5 (C-2), 129.4 (C-11), 126.7, 126.6 and 126.5 (C-3 andC-4), 125.2 and 125.1 (C-5), 120.5 and 120.4 (C-10), 117.4 and 116.7(C-8), 116.5 and 115.9 (C-6), 21.2 (Me), 35.2 (CH2, conformer E), 33.6(CH2, conformer Z), 21.2 (Me). MS/ESI: [MþNa]: 298. IR nmax (cm

�1;KBr pellets): 3088 (N–H); 1670 (C��O); 1600 (C��N).

N 0-[(E/Z)-(2-Hydroxy-5-methylphenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 2-HO, Y¼ 5-Me, Z¼H)Yield: 0.309 g, 78%; m.p. 181–182°C; 1H NMR (400MHz; DMSO-d6):d 11.85 (0.56H, s, NH, conformer Z), 11.38 (0.44H, s, NH, conformerE), 10.78 (0.56H, s, OH, conformer Z), 10.78 (0.44H, s, OH,conformer E), 8.36 (0.56H, s, CH��N, conformer Z), 8.28 (0.44H, s,CH��N, conformer E), 7.53 (0.44H, br.s, H-110, conformer A or B),7.46 (0.56H, dd, J¼ 3.1 and 3.2Hz, conformer Z), 7.38 (0.44H, dd,J¼ 1.2 and 5.0Hz, conformer E), 7.33 (0.56H, br.s, conformer Z),7.09 (0.56H, dd, J¼ 1.8 and 8Hz, conformer Z), 7.05 (0.44H, dd,J¼ 1.8 and 8Hz, conformer E), 7.00–6.95 (2H, m), 6.82–6.75 (1H,dd, J¼ 4.5 and 8Hz), 4.17 (1.12H, s, CH2, conformer E), 3.79(0.88H, s, CH2, conformer Z), 2.23 (3H, s, Me). 13C NMR (100MHz;DMSO-d6): d 170.8 (CO, conformer Z), 165.4 (CO, conformer E),155.2 and 154.3 (C-7), 147.1 (CH��N, conformer Z), 141.0 (CH��N,conformer E), 136.8 and 136.6 (C-2), 132.1 and 131.8 (C-9), 129.1,127.95 and 127.91 (C-10 7 C-11), 126.8 and 126.7, 126.6 and 126.4(C-3 and C-4), 125.3 and 125.2 (C-5), 119.8 and 118.3 (C-6), 116.2and 116.1 (C-8), 35.3 (CH2, conformer E), 33.6 (CH2, conformer Z),20.2 and 20.0 (Me). MS/ESI: [M–H]: 273. IR nmax (cm

�1; KBr pellets):3072 (N–H); 1670 (C��O); 1602 (C��N).

N 0-[(E/Z)-(2-Hydroxy-3-methoxyphenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 2-HO, Y¼ 3-MeO, Z¼H)Yield: 0.315 g, 75%; m.p. 137–138°C; 1H NMR (400MHz; DMSO-d6):d 11.84 (0.53H, s, NH, conformer Z), 11.41 (0.47H, s, NH, conformerE), 10.9 and 9.30 (OH), 8.42 (0.53H, s, CH��N, conformer Z), 8.34(0.47H, s, CH��N, conformer E), 7.41–7.39 (0.47H, m, conformer E),7.03–6.94 (3H, m), 6.86–6.81 (1H, m), 4.15 (0.94H, s, CH2,conformer E), 3.82 (1.06H, s, CH2, conformer Z), 3.82 and 3.80(3H, Me). 13C NMR (100MHz; DMSO-d6): d 170.8 (CO, conformer Z),165.4 (CO, conformer E), 148.0 and 147.9 (C-70), 147.1 and 147.0 (C-80), 145.9 (CH��N, conformer Z), 140.7 (CH��N, conformer E), 136.7and 136.5 (C-2), 126.7 and 126.5 (C-3 or C-4), 125.2 and 125.1 (C-5),120.6 (C-110), 119.2 and 119.0 (C-90), 118.9 and 117.8 (C-100), 113.8and 112.9 (C-60) 55.8 (OMe), 35.2 (CH2, conformer E), 33.6 (CH2).MS/ESI: [MþNa]: 303. IR nmax (cm


N 0-[(E/Z)-(2-Hydroxy-4-methoxyphenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 2-HO, Y¼ 4-MeO, Z¼H)Yield: 0.357 g, 85%; m.p. 163–164°C; 1H NMR (400MHz; DMSO-d6):d 11.76 (0.66H, s, NH, conformer Z), 11.36 (1H, s), 11.28 (0.33H, s,NH, conformer E), 8.31 (0.66H, s, CH��N, conformer Z), 8.21(0.33H, s, CH��N, conformer E), 7.63 (0.33H, d, J¼ 8Hz, conformerE), 7.41 (0.66H, d, J¼ 8Hz, conformer Z), 7.40–7.35 (1H, m), 7.00–6.94 (2H, m), 6.52–6.44 (2H, m), 4.13 (0.66H, s, CH2, conformer E),3.77 (1.32H, s, CH2, conformer Z), 3.76 and 3.74 (3H, OMe).

13C NMR (100MHz; DMSO-d6): d 170.4 (CO, conformer Z), 165.1(CO, conformer E), 162.1 and 161.8 (C-9), 159.2 and 157.9 (C-7),147.8 (CH��N, conformer Z), 141.4 (CH��N, conformer E), 136.7and 136.6 (C-2), 131.0 and 128.0 (C-11), 126.7 and 126.4 (C-3 and C-4), 125.1 and 125.0 (C-5), 113.1 and 111.6 (C-6), 106.4 (C-10), 101.1and (C-8), 55.3 and 55.2 (OMe), 35.2 (CH2, conformer E), 33.6 (CH2,conformer Z). MS/ESI: [M�H]: 289. IR nmax (cm

�1; KBr pellets): 3088(N–H); 1668 (C��O); 1604 (C��N).

N 0-[(E/Z)-(2-Hydroxy-3-nitrophenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 2-HO, Y¼ 3-O2N, Z¼H)Yield: 0.292 g, 66%; m.p. 182°C. 1H NMR (400MHz; DMSO-d6):d 12.19 (0.62H, s, NH, conformer Z), 11.66 (0.38H, s, NH, conformerE), 8.50 (1H, s, CH��N, conformer Z), 8.39 (0.38H, s, CH��N,conformer E), 8.17 (0.38H, dd, J¼ 7.7 and 1.3Hz, H-90, conformerE), 8.05 (0.38H, dd, J¼ 8.2 and 1.4Hz, H-110, conformer E), 7.99(0.62H, dd, J¼ 8.2 and 1.4Hz, H-110, conformer Z), 7.91 (0.62H, dd,J¼ 7.7 and 1.3, H-90, conformer Z), 7.42 (0.62H, dd, J¼ 4.6 and1.8Hz, H-100, conformer Z), 7.38 (0.38H, dd, J¼ 5.0 and 1.2Hz, H-100, conformer E), 7.00–6.95 (2H, m, H-3, H-4 and H-5), 4.21(0.76H, s, CH2, conformer E or B), 3.84 (1.24H, s, CH2, conformer Z).13C NMR (100MHz; DMSO-d6): d 171.1 (CO, conformer Z), 165.8(CO, conformer E), 151.4 and 150.7 (C-7, 146.0 (CH��N, conformerZ), 138.5 and 137.5 (C8), 136.8 and 136.4 (C-2), 136.1 (C-11), 134.9and 132.7 (C-9), 126.8, 126.7, 126.5 and 126.3 (C-3 and C-4), 125.3and 125.2 (C-5), 124.7 and 121.6 (C-10), 119.9 and 119.2 (C-9), 35.1(CH2, conformer E), 33.5 (CH2, conformer Z). MS/ESI: [MþNa]: 328.IR nmax (cm


N 0-[(E/Z)-(2-Hydroxy-5-nitrophenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 2-HO, Y¼ 5-O2N, Z¼H)Yield: 0.304 g, 69%; m.p. 226–227°C; 1H NMR (400MHz; DMSO-d6):d 12.04 (0.5H, s, NH, conformer Z), 11.60 (0.5H, s, NH, conformer E),8.63 (0.5H, d, J¼ 2.8Hz) and 8.53 (0.5H, d, J¼ 2.8Hz) (H-110,conformers Z and E), 8.51 (0.5H, s, CH��N, conformer Z), 8.31(0.5H, s, CH��N, conformer E), 8.18 (0.5H, dd, J¼ 2.8 and 6.8Hz)and 8.15 (0.5H, dd, J¼ 2.8 and 6.8Hz) (H-80, conformer Z and E),7.41 (0.5H, dd, J¼ 2.4 and 4Hz) and 7.39 (0.5H, dd, J¼ 1 and 5Hz)(H-90, conformer Z and E), 7.10 (0.5H, d, J¼ 8Hz) and 7.09 (0.5H, d,J¼ 8Hz) (H-5, conformer Z and E), 7.00–6.95 (2H, m, H-3 and H-4),4.20 (1H, s, CH2, conformer E), 3.81 (1H, s, CH2, conformer A or B).13C NMR (100MHz; DMSO-d6): d: 171.1 (CO, conformer Z), 165.8(CO, conformer E), 162.5 and 161.8 (C-7), 143.6 (CH��N, conformerZ), 140.0 (CH��N, conformer E), 139.9 (C-10), 137.7, 136.6 and 136.3(C-2), 126.7, 126.6, 126.6 and 126.3 (C-9, C-3 and C-4), 125.2 and125.1 (C-5), 123.8, 121.4 and 121.2 (C-6 and C-11), 119.8, 117.0 and116.7 (C-8), 35.2 (CH2, conformer E), 33.7 (CH2, conformer Z).MS/ESI: [M�H]: 304.IR nmax (cm


N 0-[(E/Z)-(3-Chloro-4-hydroxyphenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X¼ 3-Cl, Y¼ 4-HO, Z¼H)Yield: 0.243 g, 57%; m.p. 116–117°C; 1H NMR (400MHz; DMSO-d6):d 11.56 (0.38H, s, NH, conformer Z), 11.36 (0.62H, s, NH, conformerE), 8.07 (0.38H, s, CH��N, conformer Z), 7.89 (0.62H, s, CH��N,conformer E), 7.71 (0.62H, d, J¼ 1.9Hz, H-70, conformer E), 7.67(0.62H, d, J¼ 1.9Hz, H-70, conformer Z), 7.52 (0.62H, dd, J¼ 2 and5Hz, H-110, conformer E), 7.49 (0.62H, dd, J¼ 2 and 5Hz, H-110,conformer Z), 7.39 (0.38H, dd, J¼ 1.8 and 4Hz, H-100, conformer Z),7.37 (0.62H, dd, J¼ 1.8 and 4Hz, H-100, conformer E), 7.02 (0.62H,



d, J¼ 8Hz, H-5, conformer E), 7.01 (0.38H, d, J¼ 8Hz, H-5,conformer Z), 7.00–6.94 (2H, m, H-3 and H-4), 4.16 (1.24H, s, CH2,conformer E), 3.75 (0.76H, s, CH2, conformer Z). 13C NMR(100MHz; DMSO-d6): d 170.9 (CO, conformer Z), 165.4 (CO.conformer E), 154.8 and 154.6 (C-9), 145.7 (CH��N, conformer Z),142.0 (CH��N, conformer E), 136.8 (C-2), 128.5 and 128.1 (C-11 andC-6), 127.1, 126.7, 126.6, 126.5 and 126.4 (C-3, C-4 and C-7), 125.1and 125.0 (C-5), 120.4 and 120.2 (C-8), 116.9 (C-10), 35.5 (CH2,conformer E), 33.6 (CH2, conformer Z). MS/ESI: [M�H]: 293. IR nmax


N 0-[(E/Z)-(3,4,5-Trihydroxyphenyl)methylene]-2-(thien-2-yl)acetohydrazide (2: X, Y, Z¼ 3,4,5-(HO)3)Yield: 0.212 g, 50%; m.p. 225 (dec)°C; 1H NMR (400MHz; DMSO-d6):d 11.53 (0.39H, s, NH; conformer Z), 11.15 (0.61H; s, NH, conformerE), 9.10 (2H), 8.57 (1H, s), 7.91 (0.39H, s, CH��N, conformer Z), 7.74(0.61H, s, CH��N; conformer E), 7.37 (1H, dd, J¼ 1.2 and 6Hz), 7.00–6.94 (2H,m), 6.65 (1.22H, s, conformer E), 6.64 (0.78H, s, conformerZ), 4.13 (1.22H, s, CH2, conformer E), 3.72 (0.78H, s, CH2, conformerZ). 13C NMR (100MHz; DMSO-d6): d 170.6 (CO, conformer Z), 165.1(CO, conformer E), 147.6 (CH��N, conformer Z), 146.2 (C-10), 144.1(CH��N, conformer E), 137.0 and 136.9 (C-2 and C-6), 135.8 and135.6 (C-9), 126.7, 126.6 and 126.4 (C-3 and C-4), 125.1 and 125.0(C-5), 124.5 (C-6), 106.3 and 106.3 (C-7 and C-11), 35.5 (CH2,conformer E), 33.5 (CH2, conformer Z). MS/ESI: [M�H]: 291. IR nmax


N 0-[(E/Z)-Furan-2-ylmethylene]-2-(thien-2-yl)-acetohydrazide (3: heteroaryl¼ furan-2-yl)Yield: 0.112 g, 61%. Yellow solid. m.p. 115°C. 1H NMR (400MHz,DMSO-d6) d: 11.56 (0.39H, s, NH, conformer Z), 11.39 (0.61H, s, NH,conformer E), 8.10 (0.39H, s, CH��N, conformer Z), 7.83 (1H, s), 7.90(0.61H, s, CH��N, conformer E), 7.40–7.35 (1H, m), 7.00–6.94(2H, m, H-3, H-4 and H-5), 6.90 (1H, d, J¼ 3.4Hz, H-100), 6.63–6.61(1H, m, H-90), 4.13 (1.22H, s, CH2, conformer E), 3.75 (0.78H, s, CH2,conformer Z). 13C NMR (125MHz; DMSO-d6) d: 170.9 (CO,conformer Z), 165.5 (CO, conformer E), 149.2 (CH��N, conformerZ), 145.1 (CH��N, conformer E), 144.9 (furanyl-H), 136.7 and 136.5(C-2), 133.3 (furanyl-H), 126.6 and 126.5 (C-3 and C-4), 111.5, 113.0,112.1 (furanyl-H), 35.4 (CH2, conformer E), 33.1 (CH2, conformer Z).MS/ESI: [M�H]: 233. IR nmax (cm

�1; KBr pellets): 3209 (N–H), 1680(C��O), 1564 (C��N).

N 0-[(E/Z)-(5-Nitrofuran-2-yl)methylene]-2-(thien-2-yl)-acetohydrazide (3: heteroaryl¼ 5-nitrofuran-2-yl)Yield: 0.130 g, 52%. Yellow solid. m.p. 158–161°C. 1H NMR(400MHz, DMSO-d6) d: 12.02 (0.40H, s, NH, conformer Z), 11.86(0.60H, s, NH, conformer E), 8.73 (s), 8.18 (0.40H, s, CH��N,conformer Z), 7.96 (0.60H, s, CH��N, conformer E), 7.80 (0.60H, d,J¼ 8.0Hz, H-90, conformer E), 7.78 (0.40H, d, J¼ 8.0Hz, H-90,conformer Z), 7.42–7.38 (1H, m), 7.26 (0.60H, d, J¼ 3.9Hz, H-100,conformer E), 7.23 (0.40H, d, J¼ 3.9Hz, H-100, conformer Z), 7.00–6.95 (2H, m, H-3, H-4 and H-5), 4.19 (1.20H, s, CH2, conformer E),3.83 (0.80H, s, CH2, conformer Z). 13C NMR (125MHz; DMSO-d6) d:171.5 (CO, conformer Z), 166.2 (CO, conformer E), 151.7 (furanyl-H), 136.1 (C-2), 134.8 and 131.2 (furanyl-H), 126.9, 126.7 and 126.6(C-2 and C-3), 125.2 (C-5), 115.3, 114.7, 114.5 and 114.4 (furanyl-H),35.4 (CH2, conformer E), 33.2 (CH2, conformer Z). MS/ESI: [M�H]:278. IR nmax (cm�1; KBr pellets): 3122 (N–H), 1678 (C��O), 1593(C��N).

2-(Thien-2-yl)-N0-[(E/Z)-2-thien-2-ylmethylene]-acetohydrazide (3: heteroaryl¼ thien-2-yl)Yield: 0.168 g, 81%. Yellow solid. m.p. 166–170°C. 1H NMR(400MHz, DMSO-d6) d: 11.57 (0.41H, s, NH, conformer Z), 11.42(0.59H, s, NH, conformer E), 8.43 (0.41H, s, CH��N, conformer Z),8.19 (0.59H, s, CH��N, conformer E), 7.66–7.63 (1H, m, H-80), 7.45(0.41H, d, J¼ 3.2Hz, conformer Z), 7.42 (0.59H, d, J¼ 3.2Hz,conformer E), 7.39 (0.41H, dd, J¼ 2.0 and 4.4Hz, conformer Z),7.37 (0.59H, dd, J¼ 1.0 and 5.2Hz, conformer E), 7.12 (1H, m, H-90,7.00–6.94 (2H, m), 4.10 (1.18H, s, CH2, conformer E or B), 3.75(0.82H, s, CH2, conformer Z). 13C NMR (125MHz; DMSO-d6) d: 170.7(CO, conformer Z), 165.4 (CO, conformer E), 142.1 (CH��N,conformer Z), 138.9 and 136.6 (C-20), 138.2 (CH��N, conformerE), 136.8 and 136.6 (C-2), 130.9, 130.2, 128.8, 128.4, 127.82 and117.76 (C-30, C-40 and C-50), 126.6 and 126.4 (C-2), 125.1 and 125.0(C-5), 35.4 (CH2 conformer E), 33.5 (CH2, conformer Z). MS/ESI:[M�H]: 249. IR nmax (cm

�1; KBr pellets): 3201 (N–H), 1656 (C��O),1591 (C��N).

N 0-[(E/Z)-(5-Nitro-2-thienyl)methylene]-2-(2-thienyl)-acetohydrazide (3: heteroaryl¼ 5-nitrothien-2-yl)Yield: 0.148 g, 54%. Yellow solid. m.p. 180–183°C. 1H NMR(400MHz, DMSO-d6) d: 12.00 (0.33H, s, NH, conformer Z), 11.86(0.67H, s, NH, conformer E), 8.49 (0.33H, s, CH��N, conformer Z),8.19 (0.67H, s, CH��N, conformer E), 8.12 (1H, d, J¼ 4.3Hz, H-100),7.55 (0.33H, d, J¼ 4.4Hz, conformer Z), 7.54 (0.67H, d, J¼ 4.4Hz,conformer E), 7.42–7.38 (1H, m, H-90), 7.00–6.95 (2H, m), 4.15(1.34H, s, CH2, conformer E), 3.81 (0.66H, s, CH2, conformer Z).13C NMR (125MHz; DMSO-d6) d: 171.2 (CO, conformer Z), 166.1(CO, conformer E), 150.8 and 150.5 (C-50), 146.5 (CH��N, conformerZ), 146.4 (C-20), 140.5 (CH��N, conformer E), 136.5 and 136.1 (C-2),130.5, 130.3, 129.6, 129.1 (C-30 and C-40), 126.7 and 126.6 (C-3 andC-4) 125.2 and 125.1 (C-5), 35.4 (CH2, conformer E), 33.4 (CH2,conformer Z). MS/ESI: [M�H]: 294. IR nmax (cm

�1; KBr pellets): 3109(N–H), 1672 (C��O), 1583 (C��N).

N 0-[(E/Z)-1H-Pyrrol-2-ylmethylene]-2-(thien-2-yl)-acetohydrazide (3: heteroaryl¼ pyrrol-2-yl)Yield: 0.135 g, 74%. White solid. m.p. 208–210°C. 1H NMR(400MHz, DMSO-d6) d: 11.46 (0.41H, s, H-70, conformer Z), 11.35(0.59H, s, H-70, conformer E), 11.29 (0.41H, s, NH, conformer Z),11.12 (0.59H, sl, NH, conformer E), 8.04 (0.41H, s, CH��N,conformer Z), 7.84 (0.59H, s, CH��N, conformer E), 7.38 (0.41H,dd, J¼ 1.5 and 4.8Hz, conformer Z), 7.35 (0.59H, dd, J¼ 1.5 and4.8Hz, conformer E), 7.00–6.85 (3H, m), 6.45 (0.41H, br.s,conformer Z), 6.42 (0.459H, br.s, conformer E), 6.12 (1H, s, H-90),4.20 (1.18H, s, CH2, conformer E or B), 3.72 (0.82H, s, CH2,conformer Z). 13C NMR (125MHz; DMSO-d6) d: 170.6 (CO,conformer Z), 164.9 (CO, conformer E), 140.0 (CH��N, conformerZ), 137.2 (C-2), 135.8 (CH��N, conformer E), 127.2, 126.6 and 126.4(C-3 and C-4), 124.9 (C-5), 121.8, 113.2, 112.3, 109.1 (pyrrol-H), 35.5(CH2 conformer E), 33.1 (CH2, conformer Z). MS/ESI: [M�H]: 232.IR nmax (cm


N 0-[(E/Z)-1H-Imidazol-2-ylmethylene]-2-(thien-2-yl)acetohydrazide (3: heteroaryl¼ imidazol-2-yl)Yield: 0.138 g, 74%. White solid. m.p. 215°C (dec). 1H NMR(400MHz; DMSO-d6) d: 11.65 (0.38H, s, NH, conformer Z), 11.44(0.62H, s, NH, conformer E), 8.11 (0.38H, s, CH��N, conformer Z),7.90 (0.62H, s, CH��N, conformer E), 7.39 (0.38H, m, conformer Z),



7.36 (0.62H, dd, J¼ 1.0 and 5.2Hz, conformer E), 7.2 (br.s), 7.00–6.94 (2H, m), 4.25 (1.24H, s, CH2, conformer E), 3.78 (0.76H, s, CH2,conformer Z). 13C NMR (125MHz; DMSO-d6) d: 171.2 (CO,conformer Z), 165.5 (CO, conformer E), 142.6 (imidazol-H),142.3 (CH��N, conformer Z), 138.8 (imidazol-H), 136.8 (C-2)136.4 (CH��N, conformer E), 134.5 (imidazol-H), 126.7 and 126.5(C-3 and C-4), 125.0 (C-5), 35.5 (CH2, conformer E), 32.9 (CH2,conformer Z). MS/ESI: [M�H]: 233. IR nmax (cm

�1; KBr pellets): 3228(N–H), 1671 (C��O), 1552 (C��N).

N 0-[(E/Z)-Pyridin-2-ylmethylene]-2-(2-thien-2-yl)-acetohydrazide (3: heteroaryl¼ pyridin-2-yl)Yield: 0.129 g, 64%. Yellow solid. m.p. 124–125°C. 1H NMR(400MHz; DMSO-d6) d: 11.86 (0.33H, sl, NH, conformer Z), 8.61 (1H,d, J¼ 4.1Hz, H-80), 8.22 (0.33H, s, CH��N, conformer Z), 8.06(0.67H, s, CH��N, conformer E), 8.02 (1H, d, J¼ 8.0Hz, H-100), 7.90–7.85 (2H, m), 7.42–7.37 (3H; m), 7.00–6.95 (3H, m), 4.23 (1.34H, s,CH2, conformer E), 3.81 (0.66H, s, CH��N, conformer Z). 13C NMR(125MHz; DMSO-d6) d: 171.3 (CO, conformer Z), 165.8 (CO,conformer E), 153.1 and 153.0 (py-H2), 149.5 (py-H6), 147.1 (CH��N,conformer Z), 143.6 (CH��N, conformer E), 136.8 (C-2), 136.5 (py-H4), 126.7, 126.6 and 126.5 (C-3 and C-4), 125.2 (C-5), 124.4 and124.2 (py-H-5), 119.9 and 119.6 (py-H3), 35.5 (CH2, conformer E),33.4 (CH2, conformer Z). MS/ESI: [M�H]: 249. IR nmax (cm�1; KBrpellets): 3201 (N–H), 1666 (C��O), 1560 (C��N).

N 0-[(E/Z)-Pyridin-3-ylmethylene]-2-(thien-2-yl)-acetohydrazide (3: heteroaryl¼ pyridin-3-yl)Yield: 0.169 g, 84%. Yellow solid. m.p. 132–135°C. 1H NMR(400MHz, DMSO-d6) d: 11.80 (0.33H, s, NH, conformer Z), 11.62(0.67H, s, NH, conformer E), 8.88 (0.67H, s, H-70, conformer E), 8.83(0.33H, s, H-70, conformer Z), 8.60 (1H, d, J¼ 3.7Hz, H-90), 8.27(0.33H, s, CH��N, conformer Z), 8.15 (0.67H, d, J¼ 8.0Hz, H-110,conformer E), 8.10 (0.33H, d, J¼ 8.0Hz, H-110, conformer Z), 8.05(0.67H, s, CH��N, conformer E), 7.50–7.45 (1H, m, H-100), 7.40(0.33H,m, conformer Z), 7.38 (0.67H, br.d, J¼ 5.0Hz, conformer E),7.00–6.94 (2H, m), 4.21 (1.34H, s, CH2, conformer E), 3.79 (0.66H, s,CH2, conformer Z). 13C NMR (125MHz, DMSO-d6) d: 171.3 (CO,conformer Z), 165.7 (CO, conformer E), 150.7 and 150.4 (py-H4),148.7 (py-H3), 148.4 (CH��N, conformer Z), 144.2 (CH��N,conformer E), 140.3 (py-H3), 136.6 (C-2), 133.4 and 130.1 (py-H),126.2 and 126.5 (C-3 and C-4), 125.1 (C-5), 123.9 (py-H5), 35.4 (CH2,conformer E), 33.5 (CH2, conformer Z). MS/ESI: [M�H]: 244. IR nmax

(cm�1; KBr pellets): 3201 (N–H), 1664 (C��O), 1552 (C��N).

N 0-[(E/Z)-Pyridin-4-ylmethylene]-2-(thien-2-yl)-acetohydrazide (3: heteroaryl¼ pyridin-4-yl)Yield: 0.145 g, 72%. Yellow solid. m.p. 172–180°C. 1H NMR(400MHz; DMSO-d6) d: 11.90 (0.31H, s, NH, conformer Z), 11.73(0.69H, s, NH, conformer E), 8.64 (2H, m), 8.20 (0.31H, s, CH��N,conformer Z), 8.00 (0.69H, s, CH��N, conformer E), 7.68 (1.38H, d,J¼ 5.8Hz, H-70 andH-110, conformer E), 7.63 (0.62H, d, J¼ 5.8Hz, H-70 and H-110, conformer Z), 7.40 (0.31H, m, conformer Z), 7.38(0.69H, dd, J¼ 0.8 and 5.2Hz, conformer E), 7.00–6.95 (2H, m), 4.23(1.38H, s, CH2, conformer E), 3.82 (0.31H, s, CH2, conformer Z).13C NMR (125MHz; DMSO-d6) d: 171.5 (CO, conformer Z), 166.0 (CO,conformer E), 150.2 (py-H3 and py-H5), 144.5 (CH��N, conformer Z),141.4 and 141.3 (py-H4), 140.7 (CH��N, conformer E), 136.5 (C-2),126.7 and 126.6 (C-3 and C-4), 125.2 (C-5), 120.9 and 120.8 (py-H2and py-H5), 35.4 (CH2, conformer E), 33.5 (CH2, conformer Z).

MS/ESI: [M�H]: 244. IR nmax (cm�1; KBr pellets): 3201 (N–H), 1664

(C��O), 1552 (C��N).

Synthesis of N 0-(1-methylethylidene)-2-(thien-2-yl)-acetohiydrazide 5A solution of 2-(H2NNHCOCH2)-thiene 4 (0.25 g, 1.0 eq.) in acetone(20.0mL) was heated for 12h at 60°C, and rotary evaporated toleave the desired product as a white solid in 94% yield.

N 0-(1-Methylethylidene)-2-(thien-2-yl)acetohydrazide (5)Yield: 0.333 g, 94%. White solid. m.p.¼ 89–90°C. 1H NMR(400MHz; MeOD) d: 10.21 (1H, s, NH), 7.37–7.35 (1H, m, H-5),6.97–6.93 (2H, m, H-3 and H-4), 4.06 (1H, s, CH2, conformer E), 3.77(1H, s, CH2, conformer Z), 1.93 (3H, d, J¼ 12.0Hz, Me), 1.86 (3H, d,J¼ 4.3Hz, Me). 13C NMR (125MHz; DMSO-d6) d: 171.2 (C-20,conformer Z), 165.4 (C-20, conformer E), 155.8 (CMe2), 150.6 (CMe2),137.2 and 137.1 (C2), 126.6, 126.5, 126.4 and 126.1 (C-3 and C-4),125.0 and 124.8 (C-5), 35.1 (CH2, conformer E), 31.7 (CH2,conformer Z), 25.2 and 17.6 (CMe2, conformer E), 24.9 and 17.1(CMe2, conformer Z). MS/ESI: [MþNa]: 219. IR nmax (cm�1; KBrpellets): 3307 (N–H), 1674 (C��O), 1525 (C��N).

Synthesis of N-methyl-N 0-[(E/Z)-phenylmethylene]-2-(thien-2-yl)acetohydrazide 6To a solution of (2: X¼ Y¼Z¼H) (1 eq., 0.82mmol, 0.20 g) inacetone (10.0mL), potassium carbonate (3.3mmol, 0.45 g) wasadded. The reaction mixture was stirred at room temperature for30min, methyl iodide (3.3mmol, 0.20mL) was added, and thereaction mixture was heated to 40°C for 23h. The mixture wasrotary evaporated and water (20.0mL) was added to the residue.The mixture was extracted with ethyl acetate (3� 10.0mL); thecombined organic phases were dried over anhydrous MgSO4, androtary evaporated to yield a white solid, 63% yield.

N-Methyl-N 0-[(1E)-phenylmethylene]-2-(thien-2-yl)-acetohydrazide (6)Yield: 0.133 g, 63%. White solid. m.p.¼ 72–73°C. 1H NMR(400MHz, DMSO-d6) d: 8.00 (1H, s, CH��N), 7.84–7.82 (2H, m,phenyl-H), 7.47–7.42 (3H, m, phenyl-H), 7.35 (1H, dd, J¼ 5.1 and1.2Hz, H-5), 7.00–6.96 (1H, m), 6.95–6.93 (1H, m, H-3 or H-4), 4.37(2H, s). 13C NMR (125MHz; DMSO-d6) d: 170.7 (CO), 140.6 (C6),137.1 (C2), 134.7, 129.6, 128.7 and 127.1 (C-7, C-8, C-10 and C11),126.6 and 126.5 (C-3 and C-4), 125.1 (C-5), 34.3 (CH2), 27.9 (NMe).MS/ESI: [MþNa]: 282. IR nmax (cm

�1; KBr pellets): 3273 (N–H), 1678(C��O), 1571 (C��N).

Crystal structure determination

Data were collected at 120(2)K with Mo-Ka radiation usingBruker-Nonius Roper CCD camera on k-goniostat diffractom-eter of the NCS Crystallographic Service, based at theUniversity of Southampton. Data collection was carried outunder the control of the program COLLECT [41] and datareduction and unit cell refinement were achieved with theCOLLECT and DENZO programs [42]. The program ORTEP-3for Windows [43] was used in the preparation of the figureand SHELXL-97 [44] and PLATON [45] in the calculation of



molecular geometry. The structure was solved by directmethods using SHELXS-97 and fully refined by means of theprogram SHELXL-97.9 [44]. In the final stages of refinement,hydrogen atoms were introduced into calculated positionsand refined with a riding model.Crystal data collected at 120(2)K, colorless crystal: 0.56mm

� 0.2mm� 0.1mm. Formula: C13H12N2O2S; M¼ 260.31;monoclinic, P21/c; a¼ 10.2594(6) Å, b¼ 13.5329(8) Å, c¼9.2479(8) Å, b¼ 92.727(4)°, Z¼ 4, V¼ 1282.52(11) Å3, 2934independent reflections [R(int)¼ 0.0907], 4532 observedreflections [I> 2s(I)]: parameters refined 201; number ofrestraints 0; R(F) 0.245 (obs data), largest diff. peak 0.245 e/Å3.Atomic coordinates, bond lengths, angles, and thermalparameters have been deposited at the Cambridge Crystallo-graphic Data Centre, deposition number CCDC 954955.Full details of the crystal structure determination in CIF

format have been deposited with the Cambridge Crystallo-graphic Data Centre with deposition number CCDC 954955.Copies can be obtained free of charge on written applicationto CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: þ441223 336033); on request by e-mail to [email protected] by access to http://www.ccdc.cam.ac.uk.

The use of the NCS X-ray crystallographic service at the University ofSouthampton, UK and the valuable assistance of the staff there isgratefully acknowledged. J.L.W. thanks CAPES (Brazil) for financialsupport.

The authors have declared no conflict of interest.

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