Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2013 , Vol 17 , Num 2
Design and synthesis of some new heterocyclic benzylidene hydrazide derivatives for their antileishmanial activity
Ritesh Bhole1, Prashant Patil2, Pritam Agale2, Sanjay Wate2
1Department of Pharm Chemistry and Drug Discovery, Pad. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune (MS), India
2Department of Pharm Chemistry and Drug Discovery, Sharad Pawar College of Pharmacy,Wanadongri, Nagpur (MS), India
DOI : 10.12991/201317381

Summary

Benziliden hidrazit artığı taşıyan bileşiklerin Leishmania donovani’ye karşı yüksek etkinlik gösterdiği bilindiğinden ilgili yapı Leishmania donovani’ye karşı geliştirilen antiprotozoal ilaçların tasarımında önemli bir yere sahiptir. Ayrıca, 5-nitrotiyofen-2-ilbenzilidenhidrazit yapılı bileşiklerin düşük IC50 değerlerine sahip bileşikler olduğu bilinmektedir. Bu bilgiden hareketle Leishmania donovani’ye karşı kullanılacak bileşiklerin taşıması gereken yapısal ve fizikokimyasal özellikleri araştırılmış ve ilgili özellikleri taşıyacak olan yeni bileşiklerin tasarımı kantitatif yapı etki ilişkisi (QSAR) yöntemi ve çeşitli moleküler modelleme sistemleri kullanılarak gerçekleştirilmiştir. Yaptığımız çalışma kapsamında Leishmania donovani’ye karşı geliştirilen bazı bileşiklerin üç boyutlu kantitatif yapı etki ilişkisi (3-DQSAR) VLife MDS, etkileşme çalışmaları ise Schrodinger moleküler modelleme arayüzü kullanılarak yapılmıştır (q2 = 0.9849, pred_r2 = 0.6770 kNN analizi ile). Docking çalışmaları sonucunda, tasarlanan bileşikler ile Leishmania donovani’nin aktif bağlanma bölgesi arasında önemli etkileşmeler tespit edilmiştir. Tasarlanan bileşikler sentezlenmiş ve Leishmania donovani’ye karşı antiprotozoal etkinlikleri taranmıştır. Sentezlenen seriden üç bileşiğin antiprotozoal etki taraması esnasında standart olarak kullanılan ilaç etken maddelerinden daha etkili olduğu tespit edilmiştir. Elde edilen sonuçlar, yapı etki ilişkisi açısından incelendiğinde sübstitüsyonun biyolojik etkiyi etkileyen önemli bir parametre olduğu tespit edilmiştir.

Introduction

Diseases caused by protozoan parasites are responsible for considerable morbidity and mortality, especially in developing countries. The most prevalent parasitic disease is malaria, but leishmaniasis is also considered to be a genuine emerging disease, afflicting worldwide over 12 million people in 88 countries with an annual incidence of about 2 million[1]. Leishmaniasis is defined as a cluster of vector-borne diseases with diverse clinical manifestations, caused by the obligate intracellular protozoan parasite of the genus Leishmania[2]. Its manifestations include three broad groups of disorders: visceral leishmaniasis, cutaneous leishmaniasis, and mucocutaneous leishmaniasis[3].

The treatment of leishmaniasis is far from satisfactory. Since the 1940s, the pentavalent antimony compounds sodium stibogluconate (Pentostam, Glaxo Wellcome, UK) and meglumine antimoniate (Glucantime, Rhone-Poulenc Rorer, France) have been the mainstays of antileishmanial therapy[4,5]. These drugs present high toxicity besides requiring parenteral administration for extended periods, especially in cases of visceral leishmaniasis. Moreover, in recent years, widespread resistance to pentavalent antimonial agents has been observed, especially in cases of Leishmania/HIV co-infection[6]. These agents have been improved with the advent of new formulations or dosage regimens but there is an obvious need for new drugs with structures and mechanisms of action different from those of drugs in use to date with better potency and toxicity profiles[7,8].

The techniques of quantitative structure activity relationship and docking are valuable molecular modeling tools for drug design. In the present manuscript, we report 3D QSAR model developed along with docking studies of Leishmania donavani inhibitors. Quantitative structure activity relationship (QSAR) searches information relating chemical structure to biological and other activites by developing a QSAR model[9,10]. Molecular docking describes the generation, manipulation or representation of three-dimensional structures of molecules and associated physicochemical properties. It is the process by which the two molecules are fit together in complementary fashions in 3D space and design the molecules rationally. QSAR studies were done on VLife MDS, while docking calculations were done using Schrodinger GLIDE.

Though both of these softwares Viz, V-Life Science and Schrodinger can perform QSAR and Molecular Docking Studies we use V-Life Sci for QSAR and Schrodinger for Molecular Docking Studies based on its accuracy and precision based on reported studies[11].

Results

3D-QSAR
3-DQSAR study was performed on a series of 23 compounds of Leishmania donavani inhibitors using V-Life MDS software Version 3.5.15

Statistical results 3-DQSAR analysis showed that QSAR model has good internal as well as external predictability (Table 1). For 3D QSAR a kNN–MFA with stepwise forward backward variable selection method was used resulted in several statistically significant models, of which the corresponding best model is reported herein. The model selection criterion is the value of q2, the internal predictive ability of the model, and that of pred_r2, the ability of the model to predict the activity of external test set. For activity against Leishmania donavani, model was found to be statistically most significant, especially with respect to the internal predictive ability (q2 = 0.9849) of the model. As the cross-validated correlation coefficient (q) is used as a measure of reliability of prediction, the correlation coefficient suggests that our model is reliable and accurate. The predicted versus the experimental selectivity values for the training and test sets are depicted in (Figure 1). The value of pred_r2 was obtained for the test set and gave better results, with a value of 0.6770. Thus, the developed model displays good predictivity in regular cross validation.

TABLE 1: Statistical results of 3D QSAR studies by kNN method


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FIGURE 1: .

3D QSAR studies helped to find out the importance of electronegative with bulkier groups at these positions. The electrostatic data point generated was E_203 (-1.3257 to -1.1251), E_864 (-0.7001 to -0.0473) and H_104 (0.1069,0.1220) (Figure 2). It was found that the electronegative groups like alkoxy groups with increase in bulk were essential for potent Leishmania donavani inhibition activity and accordingly the substitutions were carried out for designing of NCEs.


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FIGURE 2: .

Docking Study
The docking study was performed of the compounds predicted from 3D QSAR in the active site of the protein with side chain flexibility. The docking study revealed hydrogen bond interactions of molecules with different active site residues present in catalytic pocket and specific pocket. Docking pose of compound 4a showed hydrogen bond interactions of aliphatic chain with receptor active site (Figure 3). All selected interacted with leishmania donavani receptor out of which compound 4a showed highest Docking score (GLIDE Score), H Bond energy and affinity towards receptors (Table 3).


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FIGURE 3: .

TABLE 2: Structure, Experimental data and Predicted Activity of Benzohydrazides used in Training and Test Set using (SA-kNN method) model 1

TABLE 3: XP docking of compounds (4.a-4.e) with 2WUU receptor

Chemistry
The synthesis of the intermediate and target compounds were performed by the reaction illustrated in Scheme 1. Compound 2a-2e namely substituted methylbenzoate was synthesized in excellent yield by esterification of substituted benzoic acid with methnol. The structures of the compounds 3a-3e were confirmed on the basis of IR spectra which showed the presence of characteristic absorption peaks at 1520-1500 (C=C vibrations), 1610-1500 (C-O stretching), 890-850 (benzene 1,4 -disubstituted), which confirms esterification. The intermediate (2a-2e) undergoes nucleophilic substitution reaction in presence hydrazine hydrate to form an intermediate substituted benzohydrazide (3a-3e). The structures of the reaction products were confirmed by IR, which showed characteristic peak at 1620-1600 (C-O stretching), 3180-3160 (N-H stretching), 1650-1620 (C=N stretching) confirms amination. The final step was carried by condensing intermediate (3a-3e) with 5- nitrothiophene aldehyde resulting in the formation of substituted N-[(5-nitrothiophene-2yl)methylidene]-benzohydrazide (4a-4e). The IR spectra showed bands at 3215 – 3230 (N-H stretching) and 1309 – 1348 (C-S stretching).


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SCHEME 1: Synthetic approach to obtain the library of compounds.

Biological evaluation
The five predicted compounds from 3D QSAR and confirmed from docking were tested, in vitro, against L. donovani promastigote forms at concentrations ranging from 10 to 0.01 μg/mL using well plates and RPMI 1640 medium supplemented with 10% fetal calf serum at 26°, as described in section 5. The observed IC50 values are summarized in (Table 4). The results obtained show that synthesized and tested compounds (4a), (4b) and (4c) exhibited very promising activities when compared with the standard drug pentamidine, while (4d) and (4e) showed moderate antileishmanial activity (Figure 4).

TABLE 4: Predicted and actual activity of compounds 4a-4e


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FIGURE 4: .

The predicted IC50 values from QSAR studies were compared with the actual IC50 values the results showed that compounds (4c), (4d) and (4e) exhibited comparable IC50 values to that of predicted IC50 values, while (4a) and (4b) showed variation from predicted values (Figure 4).

Conclusion

In conclusion, a series of novel 5-nitroheterocyclic benzohydrazide derivatives were designed and synthesized. 3D QSAR models have good statistical significance and high predictivity. The developed 3D QSAR models revealed the importance of different physicochemical properties of compounds in the Leishmania donovani inhibition. It was also found that descriptors like electrostatic and hydrophobic contributes significantly in the activity while, steric descriptor aris contributing negatively in the activity. Docking study revealed important interactions of compounds in the active binding site. The aliphatic chain present in the compounds showed good affinity towards the active site residues. Designed compounds showed good predictive activity and GLIDE score. The compounds, (4c), (4d) and (4e), exhibit the antileishmanial activity as expected from the QSAR studies. The compound (4a), (4b) and (4c) had shown highest antileishmanial activity while other compounds (4d) and (4e) showed moderate antimicrobial activity. The activity was compared with pentamidine as standard drug.

EXPERIMENTAL WORK
Hardware and software
All molecular modeling studies (3D) were performed using the Molecular Design Suite (VLife MDS software package, version 3.5; from VLife Sciences, Pune, India), conformational analysis was carried out using Schrodinger molecular modeling user interface implemented Dell Desktop Computers with a Dual core processor of Intel and Windows operating system[12].

Data set
A data set comprising 23 compounds belonging to to 5- nitrothiophen- 2-yl-benzylidene hydrazide derivatives as Leishmania donovani inhibitors were taken from the literature[13]. While preparing the data set, compounds whose pharmacological screening was performed by same experimental protocol and conditions were considered. The chemical structures and pIC50 values for the complete set of compounds are listed in (Table 2).

Structure conformation generation
Structures of compounds were sketched using the 2D structure draw application and converted to 3D structures. All the structures were minimized and optimized with the Merck Molecular Force Field (MMFF) method taking the root mean square gradient (RMS) of 0.01 kcal/mol A° and the iteration limit to 10,000. All the structures were ionized at neutral pH 7. Conformers for each structure were generated using ConfGen by applying OPLS-2005 force field method and least energy conformer was selected for further study and all the compounds were aligned by template based method.

3D QSAR
In the present study, (7.48650 to 31.8668) × (-16.7361 to 0.3877) × (-8.4230 to 7.30490) A° grid at the interval of 2.00 was generated around the aligned compounds. The steric, electrostatic and hydrophobic interaction energies are computed at the lattice points of the grid using a methyl probe of charge +1 of gasteiger- marsili type. These interaction energy values are considered for relationship generation and utilized as descriptors to decide nearness between molecules. The QSAR models were developed using Stepwise (SW) Forward – Backward, Simulated (SA) Annealing and Genetic Algorithm (GA) variable selection method with pIC50 activity field as dependent variable and physico-chemical descriptors as independent variable having cross-correlation limit of 0.9,0.7 and 1.0 for model 1, model 2 and model 3 respectively. Selection of test and training set was done by sphere exclusion method having `dissimilarity value of 4.2, 5.3and 4.9 for model 1, model 2 and model 3 respectively. Variance cut off point was 0.0.numbers of maximum and minimum neighbors were 5 and 2 respectively.

Flexible Docking
For docking study, co-crystallized structure of Leishmania donavani (PDB id 2WUU)[14] was taken from Brookhaven Protein Data Bank (www.rcsb.org) and prepared by using the Protein preparation wizard removing water and cofactors from the protein, optimizing hydrogen bonding and deleting the ligand present in crystal structure[15]. The binding site shows a high degree of flexibility, which poses a big challenge to investigate possible binding modes of a given ligand. So these side chain residues which are close enough to the active ligand and interacting with it were considered as flexible during the docking. Because of the stochastic nature of the docking search algorithm, we have employed multiple runs (10 runs) for each ligand protein setup to ensure convergence to the lowest-energy solution and reranking the poses found afterwards. The most promising poses returned when the docking run terminates was further analyzed in the pose organizer.

Synthesis of Designed Compounds
The synthesis of the intermediate and target compounds were performed by the reaction illustrated in Scheme 1. Compound 2a – 2e namely, substituted methyl benzoate was synthesized in excellent yield by esterification of compound 1a – 1e. Reaction of 2a – 2e with hydrazine hydrate gives compounds 3a –3e. Condensation of product 3a – 3e with nitrothiophene aldehyde affords compounds from 4a-4e.

Chemicals were obtained from Alfa Aesar (UK), Loba Chemie/ S.D. Fine-Chem. /E. Merck. Melting points (m.p.) were detected with open capillaries using Thermonik Precision Melting point cum Boiling point apparatus (C-PMB-2, Mumbai, India) and are uncorrected. IR spectra (KBr) were recorded on FTIR-8400s spectrophotometer (Shimadzu, Japan). HNMR was obtained using a BRUKER AVANCE II 400 Spectrophotometer using CDCl3.All chemical shift values were recorded as d (ppm). The purity of compounds was checked by thin layer chromatography (Merck, silica gel, HF, type 60, 0.25 mm). The elemental analysis was performed at RTM Nagpur University, India. Elemental analyses on 4a-4e for C. H, N were within 0.4% of theoretical values.

Synthesis procedure
Synthesis of Compound 2a – 2e
The mixture of substituted benzoic acid (1a-1e) (1 mol) and methanol (30mol) in presence of sulphuric acid was refluxed for 4-6 hr; excess solvent was removed under vacuum. The solid crystals separated were filtered, dried and recrystallized from ethanol.

2a: Yield: 83%, mp 195– 197°C, Rf : 0.44 [ethanol: benzene (1:1)], IR (KBr): cm-1 1506 (C=C vibrations), 1609 (C-O stretching), 884 (benzene 1, 4 -disubstituted), 1H-NMR (DMSO-d6): δ2.4 (s, 3H, decoxy,CH3), 1.39 (m, 2H, decoxy,OCH2), 1.53 (m, 2H, decoxy,CH2), 3.66 (m, 2H, decoxy,CH2), 2.48 (m, 2H, decoxy,CH2), 3.28 (m, 2H, decoxy,CH2), 1.57 (m, 2H, decoxy,CH2), 3.41 (m, 2H, decoxy,CH2), 1.2 (m, 2H, decoxy,CH2), 1.32 (m, 2H, decoxy,CH2), 7.31-7.32 (d, H12 and H14), 7.63 (d, H11 and H15), EIMS (m/z): 292 (M+).

2b: Yield: 69%, mp 44 - 46°C, Rf : 0.52 [ethanol: benzene (1:1)], IR (KBr): cm-1 1514 (C=C vibrations), 1502 (C-O stretching), 884 (benzene 1, 4 -disubstituted), ), 1H-NMR (DMSO-d6): δ 3.78 (s, 3H, octyl,CH3), 2.47(m, 2H, octyl,CH2), 1.55 (m, 2H, octyl,CH2), 1.21 (m, 2H, octyl,CH2), 2.45(m, 2H, octyl,CH2), 1.34(m, 2H, octyl,CH2),3.24 (m, 2H, octyl,CH2), 2.44 (m, 2H, octyl,CH2), 6.97-6.64 (d, H12 and H14), 7.77–7.0 (d, H11 and H15), EIMS (m/z): 248 (M+).

2c: Yield: 83%, mp 122– 124°C, Rf : 0.57 [ethanol: benzene (1:1)], IR (KBr): cm-1 1506 (C=C vibrations), 1609 (C-O stretching), 884 (benzene 1, 4 -disubstituted), 1H-NMR (DMSO-d6): δ 3.71 (s, 3H, heptyl,CH3) , 3.01 (m, 2H, heptyl,CH2), 2.12 (m, 2H, heptyl,CH2), 1.32(m, 2H, heptyl,CH2), 1.53(m, 2H, heptyl,CH2), 1.35(m, 2H, heptyl,CH2), 2.74(m, 2H, heptyl,CH2), 6.90-6.68 (d, H12 and H14), 7.8–7.15 (d, H11 and H15), EIMS (m/z): 234 (M+).

2d: Yield: 71%, mp 44 - 46°C, Rf : 0.6 [ethanol: benzene (1:1)], IR (KBr): cm-1 1514 (C=C vibrations), 1502 (C-O stretching), 880 (benzene 1,4 -disubstituted), 1H-NMR (DMSO-d6): δ 3.78 (s, 3H, octoxy,OCH2), 2.47(m, 2H, octoxy,CH2), 1.55 (m, 2H, octoxy,CH2), 1.21 (m, 2H, octoxy,CH2), 2.45(m, 2H, octoxy,CH2), 1.34(m, 2H, octoxy,CH2),3.24 (m, 2H, octoxy,CH2), 2.44 (m, 2H, octoxy,CH2), 6.99-6.78 (d, H12 and H14), 7.7–7.10 (d, H11 and H15), EIMS (m/z): 264 (M+).

2e: Yield: 57%, mp: 52 - 54°C, Rf : 0.63 [ethanol: benzene(1:1)], IR (KBr): cm-1 1610 (C-O stretching), 1516 (C=C vibrations), 875 (benzene 1,4 -disubstituted), 1H-NMR (DMSO-d6): δ 2.68 (s, 3H, hexyl,CH3), 3.28 (m, 2H, hexyl,CH2), 1.94 (m, 2H, hexyl,CH2), 2.47 (m, 2H, hexyl,CH2), 3.87 (m, 2H, hexyl,CH2), 1.34 (m, 2H, hexyl,CH2), 6.7-6.58 (d, H12 and H14), 7.54–7.23 (d, H11 and H15), EIMS (m/z): 220 (M+).

Synthesis of Compound 3a – 3e
The mixture of (2a-2e) (0.02mol) and hydrazine hydrate (0.6 mol) was refluxed for 12 hr. The excess solvent was removed under vacuum and the reaction mixture was cooled at 4-5°C. The solid crystals separated were filtered, washed with cold water, dried and recrystallized from ethanol.

3a: Yield: 74.91%, mp 198 – 200°C, Rf : 0.48 (ethyl acetate), IR (KBr): cm-1 3178 (N-H stretching), 1648 (C=N stretching), 1506 (C=C vibrations), 1609 (C-O stretching), 1328 (aromatic –CH stretching), 1H-NMR (DMSO-d6): δ2.4 (s, 3H, decoxy,CH3), 1.39 (m, 2H, decoxy,CH2), 1.53 (m, 2H, decoxy,CH2), 3.66 (m, 2H, decoxy,CH2), 2.48 (m, 2H, decoxy,CH2), 3.28 (m, 2H, decoxy,CH2), 1.57 (m, 2H, decoxy,CH2), 3.41 (m, 2H, decoxy,CH2), 1.2 (m, 2H, decoxy,CH2), 1.32 (m, 2H, decoxy,CH2), 7.31-7.32 (d, H12 and H14), 7.63 (d, H11 and H15), 9.6 (d,H8), EIMS (m/z): 292 (M+).

3b: Yield: 81.91%. mp 195 – 197°C, Rf : 0.4 [ethanol: benzene( 1:1)], IR (KBr): cm-1 1506 (C=C vibrations), 1328 (aromatic –CH stretching), 3180 (N-H stretching), 1609 (C-O stretching), 1635 (C=N stretching), 2856 (CH3 – O stretching), 1H-NMR (DMSO-d6): δ 3.78 (s, 3H, octyl,CH3), 2.47(m, 2H, octyl,OCH2), 1.55 (m, 2H, octyl,CH2), 1.21 (m, 2H, octyl,CH2), 2.45(m, 2H, octyl,CH2), 1.34(m, 2H, octyl,CH2),3.24 (m, 2H, octyl,CH2), 2.44 (m, 2H, octyl,CH2), 6.97-6.64 (d, H12 and H14), 7.77–7.0 (d, H11 and H15), 9.6 (d,H8), EIMS (m/z): 264(M+).

3c: Yield: 61%. mp 189 – 191°C, Rf : 0.39 (ethanol: benzene). IR (KBr): cm-1 1335 (aromatic –CH stretching), 1655 (C=N stretching), 1611 (C-O stretching), 1524 (C=C vibrations), 3174 (N-H stretching), 1H-NMR (DMSO-d6): δ 3.71 (s, 3H, heptyl,CH3) , 3.01 (m, 2H, heptyl,CH2), 2.12 (m, 2H, heptyl,CH2), 1.32(m, 2H, heptyl,CH2), 1.53(m, 2H, heptyl,CH2), 1.35(m, 2H, heptyl,CH2), 2.74(m, 2H, heptyl,CH2), 6.90-6.68 (d, H12 and H14), 7.8–7.15 (d, H11 and H15), 9.3 (d, H8), EIMS (m/z): 250 (M+).

3d: Yield: 59%, mp: 173 – 175°C, Rf : 0.52 [ethanol: benzene(1:1)], IR (KBr): cm-1 1335 (aromatic –CH stretching), 2844 (CH3–O stretching), 1639 (C=N stretching), 3178 (N-H stretching), 1524 (C=C vibrations), 1605 (C-O stretching), 1H-NMR (DMSO-d6): δ 3.78 (s, 3H, octoxy,OCH2), 2.47(m, 2H, octoxy,CH2), 1.55 (m, 2H, octoxy,CH2), 1.21 (m, 2H, octoxy,CH2), 2.45(m, 2H, octoxy,CH2), 1.34(m, 2H, octoxy,CH2),3.24 (m, 2H, octoxy,CH2), 2.44 (m, 2H, octoxy,CH2), 6.99-6.78 (d, H12 and H14), 7.7–7.10 (d, H11 and H15), 9.34 (d,H8), EIMS (m/z): 280 (M+).

3e: Yield: 91%, mp: 201 – 203°C, Rf : 0.68 [ethanol: benzene(1:1)], IR (KBr): cm-1 2830 (CH3 – O stretching), 1520 (C=C vibrations), 3178 (N-H stretching), 1616 (C-O stretching), 1640 (C=N stretching), 1341 (aromatic –CH stretching), 1H-NMR (DMSOd6): δ 2.68 (s, 3H, hexyl,CH3), 3.28 (m, 2H, hexyl,CH2), 1.94 (m, 2H, hexyl,CH2), 2.47 (m, 2H, hexyl,CH2), 3.87 (m, 2H, hexyl,CH2), 1.34 (m, 2H, hexyl,CH2), 6.7-6.58 (d, H12 and H14), 7.54–7.23 (d, H11 and H15), 9.68 (d,H8), EIMS (m/z): 236 (M+).

Synthesis of Compound 4a – 4e
The solution of compound (3a-3e) (0.02 mol) and nitrothiophene aldehyde (0.02 mol) was prepared in water: ethanol (2:5) and refluxed with time ranging from 15min to 1hr. The solid crystals separated were filtered, dried and recrystallized from ethanol.

4a: Yield: 93%, mp: 176 – 178°C, Rf : 0.56 [ethanol: benzene (1:1)], IR (KBr): cm-1 1555 (aromatic –C-NO2), 1647 (C=N stretching), 3165 (N-H stretching), 1482 (C=C vibrations), 1616 (C-O stretching), 1H-NMR (DMSO-d6): δ2.4 (s, 3H, decoxy,CH3), 1.39 (m, 2H, decoxy,CH2), 1.53 (m, 2H, decoxy,CH2), 3.66 (m, 2H, decoxy,CH2), 2.48 (m, 2H, decoxy,CH2), 3.28 (m, 2H, decoxy,CH2), 1.57 (m, 2H, decoxy,CH2), 3.41 (m, 2H, decoxy,CH2), 1.2 (m, 2H, decoxy,CH2), 1.32 (m, 2H, decoxy,CH2), 7.4–7.42 (d, 2H, H12 and H14), 7.55–7.56 (d, 1H, H4), 7.7 (d, 2H, H11 and H15), 8.11– 8.12 (d, 1H, H3), 8.66 (s, 1H, H6), 12.17 (s, 1H, H8); EIMS (m/z): 431 (M+).

Anal. C22H29N3O4S: C (60.23%) H (5.78%) N (8.34%)

4b: Yield: 81%, mp : 193-195°C Rf : 0.68 [ethanol: benzene(1:1)], IR (KBr): cm-1 2830 (CH3 – O stretching), 1520 (C=C vibrations), 1600 (C-O stretching), 1341 (aromatic –CH stretching), 1652 (C=N stretching), 3170 (N-H stretching), 1534 (aromatic –C-NO2 ), 1H-NMR (DMSO-d6): δ 3.78 (s, 3H, octyl,CH3), 2.47(m, 2H, octyl,OCH2), 1.55 (m, 2H, octyl,CH2), 1.21 (m, 2H, octyl,CH2), 2.45(m, 2H, octyl,CH2), 1.34(m, 2H, octyl,CH2),3.24 (m, 2H, octyl,CH2), 2.44 (m, 2H, octyl,CH2), 7.04–7.07 (d, 2H, H12 and H14), 7.52–7.54 (d, 1H, H4),7.87–7.90 (d, 2H, H11 and H15), 8.09-8.10 (d, 1H,H3), 8.66 (s, 1H, H6), 12.08 (d, 1H, H8); EIMS (m/z): 387 (M+).

Anal. C20H25N3O3S: C (61.23%) H (6.50%) N (9.84%)

4c: Yield: 64%, mp: 216 – 218°C, Rf : 0.53 [ethanol: benzene(1:1)], IR (KBr): cm-1 1547(aromatic –C-NO2), 3175 (N-H stretching), 1615 (C=N stretching), 1531 (C=C vibrations), 1600 (C-O stretching), 1333 (aromatic –CH stretching), 1H-NMR (DMSO-d6): δ 3.71 (s, 3H, heptyl,CH3) , 3.01 (m, 2H, heptyl,CH2), 2.12 (m, 2H, heptyl,CH2), 1.32(m, 2H, heptyl,CH2), 1.53(m, 2H, heptyl,CH2), 1.35(m, 2H, heptyl,CH2), 2.74(m, 2H, heptyl,CH2), 6.90-6.68 (d, H12 and H14), 7.8–7.15 (d, H11 and H15), 8.11–8.12 (d, 1H, H3), 8.66 (s, 1H, H6), 12.17 (s, 1H, H8); EIMS (m/z): 373 (M+).

Anal. C19H23N3O3S: C (61.10%) H (6.21%) N (11.25%)

4d: Yield: 55%, mp: 182– 184°C, Rf : 0.6 [ethanol: benzene (1:1)], IR (KBr): cm-1 1547 (aromatic –C-NO2), 1438 (C=C vibrations), 3180 (N-H stretching), 1334 (C-O stretching, alcohol), 1637 (C=N stretching), 1H-NMR (DMSO-d6): δ 3.78 (s, 3H, octoxy,OCH2), 2.47(m, 2H, octoxy,CH2), 1.55 (m, 2H, octoxy,CH2), 1.21 (m, 2H, octoxy,CH2), 2.45(m, 2H, octoxy,CH2), 1.34(m, 2H, octoxy,CH2),3.24 (m, 2H, octoxy,CH2), 2.44 (m, 2H, octoxy,CH2), 6.99-6.78 (d, H12 and H14), 7.7–7.10 (d, H11 and H15), 8.09-8.10 (d, 1H,H3), 8.66 (s, 1H, H6), 12.08 (d, 1H, H8); EIMS (m/z): 403 (M+).

Anal. C20H25N3O4S: C (59.53%) H (6.25%) N (10.41%)

4e: Yield: 92%, mp: 208 – 210°C, Rf : 0.59 [ethanol: benzene (1:1)], IR (KBr): cm-1 1578(aromatic –C-NO2), 2830 (CH3 – O stretching), 1566 (C=C vibrations), 3178 (N-H stretching), 1648 (C=N stretching), 1H-NMR (DMSO-d6): δ 2.68 (s, 3H, hexyl,CH3), 3.28 (m, 2H, hexyl,CH2), 1.94 (m, 2H, hexyl,CH2), 2.47 (m, 2H, hexyl,CH2), 3.87 (m, 2H, hexyl,CH2), 1.34 (m, 2H, hexyl,CH2), 6.7-6.58 (d, H12 and H14), 7.54–7.23 (d, H11 and H15), 8.11–8.12 (d, 1H, H3), 8.66 (s, 1H, H6), 12.17 (s, 1H, H8); EIMS (m/z): 359 (M+).

Anal. C18H21N3O3S: C (60.15%) H (5.89%) N (11.69%)

ANTILEISHMANIAL ASSAYS[13]
Antileishmanial activity of the compounds was tested in vitro against a culture of L. donovani promastigotes. The parasites were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco Chem Co.) at 26°C. A 3-day-old culture was diluted to 5*105 promastigotes/mL. Samples were tested at concentrations from 50 to 3.1μg/mL. Drug dilutions were prepared directly in cell suspension in 96-well plates and were incubated at 26°C for 48 h and growth of Leishmania promastigotes was determined by Alamar Blue assay. Standard fluorescence was measured on a Fluostar Galaxy plate reader at excitation wavelength of 544 nm and emission wavelength of 590 nm. Pentamidine were used as the standard antileishmanial agents. Percentual growth was calculated and plotted versus test concentration for computing the IC50values.

ACKNOWLEGMENT
Authors wish to thanks Sharad Pawar College of Pharmacy, University of Nagpur,India for their valuable help in QSAR studies

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