HETEROCYCLES

Paper | Regular issue | Vol. 92, No. 3, 2016, pp. 431-469
Received, 1st December, 2015, Accepted, 15th January, 2016, Published online, 20th January, 2016.
DOI: 10.3987/COM-15-13384

■ Synthesis, Molecular Docking and Anti-Human Breast Cancer Activities of Novel Thiazolylacetonitriles and Thiazolylacrylonitriles and Their Derivatives Containing Benzenesulfonylpyrrolidine Moiety

Mahmoud S. Bashandy* and Shimaa M. Abd El-Gilil

Department of Chemistry, Al-Azhar University, Nasr City, Cairo 002, Egypt

Abstract

This article describes the synthesis of some novel sulfonamides having the biologically active, thiazole 3, 8-10, 13, 19, 20, 24, 30, 31, 35-41, pyrazolo[5,1-c][1,2,4]triazine 5, 1H-1,2,4-triazole 6, thiazolo[3,2-a]pyridine 14, chromen-2-one 16, benzo[f]chromen-3-imine 17, benzo[f]chromen-3-one 18, triazolo[4,3-a]pyrimidine 22, pyrazolo[1,5-a]pyrimidine 23, isoxazole 26, 2,4-diaminopyrimidine 27, benzo[4,5]imidazo[1,2-a]pyridine 28, imidazolidine 32 and 1H-benzo[d]imidazolidene 33 moieties, starting with 2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acetonitrile (2), which was prepared from cyclocondensation of phenacyl bromide derivative 1 with 2-cyanoethanethio-amide. The structures of the newly synthesized compounds were confirmed by elemental analysis, IR, 1H NMR, 13C NMR and Ms spectral data. All the compounds were tested in-vitro antihuman breast cancer cell line (MCF7). Compounds 18, 8, 41 and 28 with IC50 values of 48.01, 49.11, 49.27 and 49.78 µM, respectively, exhibited better activity than doxorubicin (DOX) as a reference drug with IC50 value of 68.6 µM. Molecular Operating Environment (MOE) performed virtual screening using molecular docking studies of the synthesized compounds. The results indicated that some synthesized compounds suitable inhibitor against dihydrofolate reductase (DHFR) enzyme (PDB ID: 4DFR) with further modification.

introduction
Thiazole derivatives have attracted a great deal of interest owing to their antibacterial,1 antifungal,2 antiinflammatory,3 and antiviral4-6 activities. They are also useful as antiallergic,7 anthelmintic8 agents and as sedative hypnotics.9 In addition to being used in the pharmaceutical industry, thiazoles also find a wide application in the dye10 and photographic industry.10 Sulfonamides also have been demonstrated to possess antibacterial,11-13 antifungal,14 insulin-releasing,15,16 carbonic anhydrase inhibitory,17-19 hypoglycemic,20 anesthetic,21 anti-inflammatory,22,23 and anti-carcinogenic24,25 activities. In view of these reports and as a continuation of previous work26-30 directed towards the synthesis of substituted heterocycles, incorporating benzenesulfonamide with anticipated biological activities. Therefore, this article reports new and convenient methods for the synthesis of heterocyclic ring systems that are required to medicinal chemistry utilizing 2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acetonitrile (2) as starting material and investigated their antihuman breast cancer activities.

RESULTS AND DISCUSSION
When 2-bromo-1-(4-(pyrrolidin-1-ylsulfonyl)phenyl)ethanone (1) was treated with 2-cyanoethanethio- amide in refluxing ethanol afforded a single product identified as 2-(4-(4-(pyrrolidin- 1-ylsulfonyl)phenyl)thiazol-2-yl)acetonitrile (2) on the basis of elemental analysis and spectral data. Thus, IR spectrum of compound 2 revealed absorption band at ν 2220 cm-1 due to cyano group. 1H NMR spectrum showed singlet signal at δ 4.01 ppm due to active methylene protons and singlet signal at δ 7.52 ppm for H5 of thiazole. 13C NMR spectrum showed signal at δ 18.9 ppm for aliphatic carbon atom of active methylene, two signals at δ 108.1 and 117.5 ppm corresponding to C5 of thiazole and cyano group, respectively besides, the mass spectrum was compatible with the molecular formula C15H15N3O2S2, confirmed the structure 2. The methylene group in thiazolylacetonitrile derivative 2 proved to be highly reactive. Thus, compound 2 underwent coupling with an equimolar amount of 4-chlorobenzenediazonium chloride in ethanol solution containing sodium acetate, at 0-5 oC, to afford a yellow crystals product for which two isomeric structures 3 or 4 seemed possible. However the appearance of NH and cyano absorption bands at ν 3193 and 2222 cm-1, respectively in the IR spectrum of the isolated product, the lack of the signal due to methylene protons, and the appearance of the signal due to the H5 of thiazole at δ 7.40 ppm in the 1H NMR spectrum provided a firm support for structure 3 and ruled out the other possible isomer 4. The structure 3 was also obtained via direct reaction of phenacyl bromide derivative 1 with 2-amino-N'-(4-chlorophenyl)-2-thioxoacetohydrazonoyl cyanide. In the same manner, thiazolylacetonitrile 2 couples with 4-cyano-3-(methylthio)-1H-pyrazole-5-diazonium chloride to afford the pyrazolo[5,1-c][1,2,4]triazine derivative 5 in moderate yield. The latter structure was established on the basis of its elemental analysis and spectral data (Scheme 1). In contrast to their behavior, it has been found that a buffered solution of 1H-1,2,4-triazole-3-diazonium chloride couples smoothly, and in high yield, with compound 2 to afford a product for which two isomeric structures 6 or 7 seemed possible. However, the appearance of NH and cyano absorption bands at ν 3230 and 2231 cm-1, respectively, in the IR spectrum of the isolated product and the absence of any signals corresponding to amino group provided a firm support for structure 6 and ruled out the other possible isomer 7 (Scheme 1). Formation of thiazolylacrylonitrile derivative 8 is assumed to take place via the condensation of the active methylene group in compound 2 with 4-fluorobenzaldehyde. The structure of compound 8 was established on the basis of their elemental analysis and spectral data. Thus, IR spectrum of compound 8 revealed absorption bands at ν 2220 and 1170 cm-1 due to C≡N group and C-F bond, respectively. 1H NMR spectrum showed two singlet signals at δ 7.60 and 8.21 ppm due to H5 of thiazole and proton of CH=C group. 13C NMR spectrum showed two signals at δ 109.2 and 117.3 ppm for C5 of thiazole and C≡N group, respectively. A final evidence for the proposed structure comes from synthesizing compound 8 via reaction of phenacyl bromide derivative 1 with 2-cyano-3-(4-fluorophenyl)prop-2-enethioamide to afford product identical in all aspects (mp, TLC and IR spectrum) with those obtained previously from the reaction of compound 2 with 4-fluorobenzaldehyde as described before (Scheme 1).

Compound 2 was treated with hydrogen sulfide gas in ethanol containing triethylamine as a catalyst at room temperature to afford 2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)ethanethioamide (9), which upon treatment with ethyl 2-chloroacetate in refluxing ethanol contains fused sodium acetate afforded 4-thiazolidinone derivative 10. IR spectrum of compound 10 revealed absorption band at ν 1673 cm-1 due to C=O group. 1H NMR spectrum showed two singlet signals at δ 3.18 and 4.25 ppm due to protons of acyclic and cyclic methylene groups, respectively and a singlet signal of one proton appeared in aromatic region at δ 7.40 ppm corresponding to H5 of thiazole. 13C NMR spectrum showed two signals at δ 35.3 and 41.6 ppm for carbons of acyclic and cyclic methylene groups, respectively and signal at δ 110.7 ppm for C5 of thiazole. The latter compound was also obtained via direct reaction of thiazolylacetonitrile 2 with 2-mercaptoacetic acid in pyridine. Efforts to cyclize compound 10 with 2-(4-fluorobenzylidene)malononitrile to afford enaminonitrile 12 were not successful; instead the product was identified as 5-(4-fluorobenzylidene)-2-((4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)methyl)- thiazol-4(5H)-one (13) on the basis of elemental analysis and spectral data. The structure of 13 was further confirmed by an independent synthesis of 4-thiazolidinone derivative 10 with 4-fluorobenzaldehyde in ethanolic piperidine solution. The formation of 13 from 4-thiazolidinone derivative 10 and 2-(4-fluorobenzylidene)malononitrile can be explained by the addition of an active methylene group of compound 10 at the olefinic bond of benzylidene malononitrile forming the intermediate 11, which undergoes spontaneous elimination of malononitrile to give the final product 13 (Scheme 2).

Reaction of compound 2 with 2-(bis(methylthio)methylene)malononitrile31,32 in N,N-dimethylformamide (DMF) in the presence of anhydrous potassium carbonate led to 5-imino-7-(methylthio)-3-(4-(pyrrol- idin-1-ylsulfonyl)phenyl)-5H-thiazolo[3,2-a]pyridine-6,8-dicarbonitrile (14) (Scheme 3). The elemental analysis and spectral data of the latter structure were in agreement with its assigned structure. Thus, IR spectrum of compound 14 revealed absorption bands at ν 3235, 2220 and 2218 cm-1 due to NH and two cyano groups, respectively. The 1H NMR spectrum showed two singlet signals at δ 2.83 and 7.52 ppm due to SCH3 and H5 of thiazole, and (D2O exchangeable) singlet signal at δ 8.52 ppm due to NH proton. 13C NMR spectrum showed two signals at δ 16.2 and 118.2 ppm for SCH3 and C5 of thiazole, respectively. Besides, the mass spectrum was compatible with the molecular formula C20H17N5O2S3, m/z 455 confirmed structure 14. Our investigation was also extended to study the reaction of compound 2 with o-phenolic aldehydes,33 namely 2-hydroxybenzaldehyde and 2-hydroxy-1-naphthaldehyde. Thus, cyclocondensation of compound 2 with an equimolar amount of 2-hydroxybenzaldehyde in boiling ethanol solution containing piperidine as a catalyst gave the corresponding 3-(4-(4-(pyrrolidin- 1-ylsulfonyl)phenyl)thiazol-2-yl)-2H-chromen-2-one (16). The formation of compound 16 was assumed to occur via the intermediacy of the Knovenagel condensed intermediate 15, intramolecular cyclization via an anticipated Michael-type addition34 of the acidic hydroxyl group to the cyano function, and spontaneous hydrolysis of the imino function into a carbonyl function under the experimental reaction conditions employed. Similar hydrolysis phenomena have been previously reported35,36 (Scheme 3). The latter structure established based on its elemental analysis and spectral data. A final evidence for the proposed structure comes by boiling 2-oxo-2H-chromene-3-carbothioamide with phenacyl bromide derivative 1 in ethanol. However, when compound 2 was treated with 2-hydroxy-1-naphthaldehyde at the same reaction condition, the reaction afforded the isolable benzo[f]chromen-3-imine derivative 17, which when heated with glacial acetic acid in the presence of few drops of hydrogen chloride afforded benzo[f]chromen-3-one derivative 18 (Scheme 3). On the other hand, when compound 2 was allowed to react with triethoxymethane at reflux temperature, afforded 3-ethoxy-2-(4-(4-(pyrrolidin-1-ylsulfonyl)- phenyl)thiazol-2-yl)acrylonitrile (19), which on treatment with dimethylamine in refluxing methanol afforded 3-(dimethylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (20) which confirmed by elemental analysis and spectral data. Thus, IR spectrum revealed an absorption band at ν 2218 cm-1 corresponding to cyano group. 1H NMR spectrum showed three singlet signals at δ 2.99, 7.42 and 7.65 ppm corresponding to (CH3)2N, CH=C and H5 of thiazole, respectively. 13C NMR spectrum showed signal at δ 41.6 ppm for two carbon atoms of (CH3)2N moiety and two signals at δ = 108.3 and 118.8 ppm corresponding to C5 of thiazole and cyano group, respectively. The latter compound was also obtained via direct reaction of thiazolylacetonitrile 2 with dimethylformamide dimethyl acetal (DMF-DMA) in refluxing xylene (Scheme 3).

The behavior of 3-dimethylamino thiazolylacrylonitrile derivative 20 towards some heterocyclic amines are investigated, thus compound 20 reacted with 4H-1,2,4-triazol-3-amine in DMF37 under reflux to afford triazolo[4,3-a]pyrimidine derivative 22. The formation of 22 is assumed to proceed through the addition of exocyclic amino group of aminotriazole to α,β-unsaturated moiety of 20 to yield acyclic intermediate 21, which undergoes intramolecular cyclization with elimination of dimethylamine to afford the final product triazolo[4,3-a]pyrimidine 22 (Scheme 4). In the same manner, compound 20 reacted with 5-amino-3-methyl-1H-pyrazole-4-carboxamide38 in refluxing DMF to give pyrazolo[1,5-a]- pyrimidine derivative 23. The reactivity of compound 20 towards some nitrogen nucleophiles was also investigated. Thus, when compound 20 was treated with hydrazine hydrate in refluxing ethanol, the reaction afforded acyclic product (3-hydrazinothiazolyl)acrylonitrile derivative 24 and ruled out the amino pyrazole derivative 25, due to the fact that the IR spectrum showed an absorption band at ν 2218 cm-1 assignable to cyano group. Its 1H NMR spectrum revealed two singlet signals at δ 6.75 and 7.45 ppm corresponding to CH=C moiety and H5 of thiazole, respectively, and (D2O exchangeable) two singlet signals at δ 5.08 and 10.22 ppm, corresponding to NH2 and NH protons, respectively. The mass spectrum of 24 showed a molecular ion peak at m/z 375. Similarly, compound 20 reacts with hydroxylamine hydrochloride and guanidine hydrochloride in refluxing ethanol containing sodium acetate to afford isoxazole derivative 26 and 2,4-diaminopyrimidine derivative 27, respectively (Scheme 4). Moreover compound 20 reacts smoothly with 2-(1H-benzo[d]imidazol-2-yl)acetonitrile in refluxing dioxane to afford only one isolable product, identified as benzo[4,5]imidazo[1,2-a]pyridine derivative 28. The IR spectrum of 28 showed, two bi-forked characteristic absorption bands at ν 3416 and 3295 cm-1 assignable to amino group and another absorption band at ν 2220 cm-1 for cyano group. 1H NMR spectrum showed (D2O exchangeable) singlet signal at δ 6.81 ppm, corresponding to amino protons, and its mass spectrum was compatible with molecular formula C25H20N6O2S2, (M+: 500) (Scheme 4).

Formation of compound 28 is assumed to proceed via Michael addition of 2-(1H-benzo[d]imidazol-2-yl)- acetonitrile to ylidenic bond in 3-dimethylamino thiazolylacrylonitrile derivative 20 forming an acyclic intermediate, which cyclized by elimination of dimethylamine then nucleophilic attack of the active methylene on the cyano group followed by tautomerization to the final product 28 (Scheme 5).

The base-promoted nucleophilic addition of the thiazolylacetonitrile 2 to an equimolar amount of carbon disulfide in dry DMF in the presence of KOH at room temperature afforded the non-isolable potassium dithiolate salt 29 and converted into (2-cyanothiazolyl)ethanedithioic acid derivative 30 by treatment with 1N hydrogen chloride. However, when compound 29 was treated with two moles of iodomethane afforded [3,3-bis(methylthio)thiazolyl]acrylonitrile derivative 31 (Scheme 6). The IR spectrum of

compound 31 revealed an absorption band at ν 2218 cm-1 corresponding to cyano group, and two bands at ν 1189 and 1150 cm-1 for two (C-S) bonds. 1H NMR spectrum revealed two singlet signals at δ 2.85 and 7.31 ppm for 2SCH3 and H5 of thiazole, respectively. 13C NMR spectrum showed signal at δ 16.9 ppm for two carbon atoms of 2SCH3 moieties and two signals at δ 109.6 and 119.1 ppm corresponding to C5 of thiazole and cyano group, respectively, and its mass spectrum was compatible with molecular formula C18H19N3O2S4, (M+: 437). Cyclization of compound 31 with 1,2-diamine derivatives namely; ethane-1,2-diamine and benzene-1,2-diamine afforded derivatives of imidazolidine 32 and 1H-benzo[d]imidazolidine 33, respectively (Scheme 6).
Additionally, when thiazolylacetonitrile 2 was reacted with isothiocyanatobenzene in dry DMF in the presence of KOH at room temperature afforded the non-isolable potassium salt 34, which acidified with 1N hydrogen chloride to yield the (2-cyano-N-phenylthiazolyl)ethanethioamide derivative 35. Interaction of intermediate 34 with halogenated compounds, namely; iodomethane, ethyl 2-chloroacetate, ethyl 2-bromopropanoate and ethyl 3-bromobutanoate gave acyclic compounds 36-39, respectively (Scheme 7). The IR spectrum of compound 36 revealed an absorption bands at ν 3230, 2222 and 1160 cm-1 corresponding to NH, C≡N and C-S groups, respectively. 1H NMR spectrum revealed two singlet signals at δ 2.72 and 7.32 ppm for protons of SCH3 and H5 of thiazole, respectively, in addition to, D2O exchangeable singlet signal at δ 9.35 ppm due to NH proton. 13C NMR spectrum showed signal at δ 16.3 ppm for aliphatic carbon atom of SCH3 moiety and two signals at δ 111.3 and 115.9 ppm corresponding to C5 of thiazole and cyano group, respectively, and its mass spectrum was compatible with molecular formula C23H22N4O2S3, (M+: 482). IR spectrum of compound 37 revealed an absorption bands at ν 3220, 2220 and 1697 cm-1 corresponding to NH, C≡N and C=O groups, respectively. 1H NMR spectrum revealed a triplet at δ 1.31 ppm for CH3 of ethyl group, a quartet at δ 4.34 ppm due to CH2 of ethyl group, two singlet signals at δ 3.79 and 7.31 ppm for protons of CH2 and H5 of thiazole, respectively, in addition to, D2O exchangeable singlet signal at δ 10.99 ppm due to NH proton. 13C NMR spectrum showed two signals at δ 18.3 and 59.3 ppm for CH3 and CH2 of ethyl group, respectively and three signals at δ 40.2, 110.2 and 121.0 ppm corresponding to CH2, C5 of thiazole and cyano group, respectively, and its mass spectrum was compatible with molecular formula C26H26N4O4S3, (M+: 554). Interaction of compound 36 with aniline in refluxing ethanol afforded [3,3-bis(phenylamino)thiazolyl]acrylonitrile derivative 40, which was established on the basis of elemental analysis and spectral data. However, when compound 37 heated in ethanol in the presence of piperidine as a catalyst, afforded only one isolable product from two proposed structures 4-thiazolidinone 41 and thiophene 42. IR spectrum indicated the disappearance of amino group bands and showed absorption band for cyano group at ν 2220 cm-1. 1H NMR spectrum showed the lack of the signal due to protons of ester group and the appearance of a signal due to the methylene group at δ 4.01 ppm. 13C NMR spectrum showed signals at δ 41.4, 108.8, 120.3 and 173.8 ppm corresponding to the methylene, C5 of thiazole, cyano and carbonyl groups, respectively. These spectral data provided a firm support for structure 41 and ruled out the other possible structure 42 (Scheme 7).

Docking and molecular modeling
Thymidylate synthase and dihydrofolate reductase are among the main targets involved in anticancer and antimicrobial activity.39,40 Molecular modeling study using Molecular Operating Environment (MOE)41 module was performed in order to rationalize the observed anticancer activity of the newly synthesized compounds. Molecular docking studies further help in understanding the mode of action of the compounds through their various interactions with the active sites of dihydrofolate reductase.

DOCKING OF DOXORUBICIN INTO DHFR:
The active site revealed that several molecular interactions were considered responsible for the observed affinity, as the hydroxyl group of tetracene acted as a hydrogen bond donor with the side chain residue Asp 21 (2.65 Å) with a strength of 1.1%. While, the hydroxyl group of acetyl pyran acted as a hydrogen bond acceptor with the side chain residue Ser 59 (3.02 Å) with a strength of 16.2%. However, the amino group acted as a hydrogen bond donor with the side chain residue Glu 30 (3.31 Å) with a strength of 5%. Moreover, there is arene-arene interaction between the phenyl ring of tetracene and the side chain residue Phe 31. In addition to, hydrophobic interactions involving other atoms of the compound and the following amino acid residues: Val 8, Gly 20, Asp 21, Leu 22, Phe 31, Phe 34, Thr 56, Ile 60, Pro 61, Asn 64, Leu 67, Val 115 and Tyr 121, as shown in Figure 1.

Docking simulation study of the synthesized compounds 1, 2, 8, 10, 16, 18, 22, 27, 28, 30 and 41:
MOE docking studies of the inhibitors were performed using dihydrofolate reductase co-crystallized with methotrexate (PDB ID: 4DFR) as a template.

Docking of compound 1 into DHFR:
The active site revealed that one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residues; Thr 56 and Ser 59 (3.63 Å and 3.16 Å, respectively) with a strength of 2.4% and 10.9%, respectively. Besides to, hydrophobic interactions involving the bromine atom, oxygen atom of carbonyl function and other carbons as well the second oxygen atom of SO2 moiety and the following amino acid residues: Ile 16, Leu 22, Phe 31, Ile 60, Pro 61, Val 115 and Tyr 121, as shown in Figure 2.

Docking of compound 2 into DHFR:
The active site revealed the presence of three hydrogen bond interactions as one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the amino acid residues Ser 59 (2.68 Å) with a strength of (84.2%). Moreover, the nitrogen atom in cyano function acted as a hydrogen bond acceptor with the amino acid residues Val 8 and Thr 136 (3.79 Å and 2.80 Å; respectively) with a strength of 1.4% and 56.6%, respectively. In addition to, hydrophobic interactions with the following amino acid residues: Ile 7, Val 8, Ala 9, Ile 16, Gly 17, Asp 21, Leu 22, Glu 30, Phe 34, Thr 56, Phe 134 and Thr 136, as shown in Figure 3.

Docking of compound 8 into DHFR:
The active site revealed that the two oxygen atoms of SO2 moiety acted as a hydrogen bond acceptor with the amino acid residues Asn 64 (2.88 Å and 3.43 Å, respectively) with a strength of 32.6% and 3.1%, respectively. Furthermore, the nitrogen atom in cyano function acted as a hydrogen bond acceptor with the amino acid residue Thr 56 (3.21 Å) with a strength of 15.3%. In addition to, hydrophobic interactions with the following amino acid residues: Val 8, Ala 9, Ile 16, Leu 22, Glu 30, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Val 115 and Tyr 121, as shown in Figure 4.

Docking of compound 10 into DHFR:
The active site revealed the presence of hydrogen bond interactions between one oxygen atom of SO2 moiety as it acted as a hydrogen bond acceptor with the side chain residues Thr 56 and Ser 59 (3.77 Å and 2.81 Å, respectively) with a strength of (1.4% and 59.1%, respectively). Moreover, the oxygen atom of carbonyl function acted as a hydrogen bond acceptor with the amino acid residues Lys 68 and Arg 70 (3.52 Å and 3.2 Å, respectively) with a strength of 3.3% for both. In addition to, hydrophobic interactions among other atoms of the compound with the following amino acid residues: Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Pro 61, Asn 64, Leu 67, Lys 68, Arg 70, Val 115 and Tyr 121, as shown in Figure 5.

Docking of compound 16 into DHFR:
The active site illustrated that the one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residue Arg 70 (3.35 Å) with a strength of 6%. In addition to, hydrophobic interactions among other atoms of the compound with the following amino acid residues: Ile 16, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Lys 68 and Arg 70, as shown in Figure 6.

Docking of compound 18 into DHFR:
The active site illustrated that the one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residue Arg 70 (3.56 Å) with a strength of 3.9%. In addition to, hydrophobic interactions among other atoms of the compound with the following amino acid residues: Ile 16, Gly 20, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Lys 68, Arg 70 and Thr 146, as shown in Figure 7.

Docking of compound 22 into DHFR:
The active site revealed the presence of several molecular interactions in which two oxygen atoms of SO2 moiety acted as a hydrogen bond acceptor with the amino acid residues Thr 56 and Ser 59 (2.81 Å and 3.58 Å, respectively) with a strength of 43.6% and 2.3%, respectively. In addition to, hydrophobic interactions with the following amino acid residues: Ile 16, Asp 21, Leu 22, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Val 115 and Tyr 121, as shown in Figure 8.

Docking of compound 27 into DHFR:
The active site revealed the presence of hydrogen bond interactions as the one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residue Thr 56 (2.86 Å) with a strength of 26%. Furthermore, the nitrogen atom of pyrimidine moiety acted as a hydrogen bond acceptor with the amino acid residue Asn 64 (3.25 Å) with a strength of (1.5%). In addition to, hydrophobic interactions with the following amino acid residues: Ile 16, Asp 21, Leu 22, Phe 31, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Val 115 and Tyr 121, as shown in Figure 9.

Docking of compound 28 into DHFR:
The active site revealed the presence of one hydrogen bond interaction as one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residue; Asn 64 (3.60 Å) with a strength of 1.7%. Besides, hydrogen atoms of amino function acted as hydrogen bond donor with the amino acid residues Gly 20 and Asp 21 (2.94 Å and 2.48 Å, respectively) with a strength of 2.8% and 6.6%, respectively. Moreover, there is arene-arene interaction between phenyl ring and amino acid residue Phe 31. In addition to, hydrophobic interactions involving the other atoms of the compound with the following amino acid residues: Ile 7, Val 8, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Ser 59, Pro 61, Asn 64, Lys 68, Arg 70, Val 115 and Tyr 121, as shown in Figure 10.

Docking of compound 30 into DHFR:
The active site revealed the presence of a hydrogen bond interaction between the nitrogen atom of cyano function as it acted as a hydrogen bond acceptor with the side chain residue Arg 70 (3.21 Å) with a strength of 23.8%. Moreover, there is arene-arene interaction between phenyl ring and amino acid residue Phe 31. In addition to, hydrophobic interactions among other atoms of the compound with the following amino acid residues: Ile 16, Phe 31, Phe 34, Gln 35, Thr 56, Ile 60, Asn 64, Leu 67, Arg 70, Val 115 and Tyr 121, as shown in Figure 11.

Docking of compound 41 into DHFR:
The active site revealed the presence of four hydrogen bond interactions as one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residue; Asn 64 (2.70 Å) with a strength of 31.1%. Moreover, the oxygen atom of carbonyl function acted as hydrogen bond acceptor with the side chain residue; Ala 9 (3.05 Å) with a strength of 7.4%. Besides, the nitrogen atom of cyano function acted as hydrogen bond acceptor with the amino acid residues Thr 56 and Ser 59 (3.50 Å and 3.18 Å; respectively) with a strength of 6.3% and 15.1%, respectively. In addition to, hydrophobic interactions with the following amino acid residues: Val 8, Ala 9, Ile 16, Asp 21, Leu 22, Glu 30, Phe 31, Phe 34, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Val 115 and Tyr 121, as shown in Figure 12.

Docking and molecular modeling
Docking was performed for the compounds 1, 2, 8, 10, 16, 18, 22, 27, 28, 30 and 41 on the dihydrofolate reductase in a trial to predict their mode of action as anticancer drugs. The compounds show several interactions with dihydrofolate reductase enzyme. Particularly noteworthy are the compounds 41, 18, 28, 8, 16 and 10, which suggest that they might exert their action through inhibition of the DHFR enzyme (Table 1). It is clear from the present data that the comparison of the docking score energy for tested compounds that the compounds follows the order 41 > 18 > 28 > 8 > 16 > 10 > 22 > 27 > 2 > 30 > 1 (Chart 1).

IN VITRO ANTICANCER ACTIVITY
The newly synthesized compounds were evaluated for their in-vitro cytotoxicity against human breast cancer cell line (MCF7). Some of the tested compounds were more potent compared with doxorubicin as the reference drug. From the obtained results in Table 2 and Chart 2, observe that compound 18 having benzo[f]chromen-3-one moiety with IC50 value 48.01 µM, (thiazolyl)acrylonitrile 8 with IC50 value 49.11 µM, N-pheny-4-thiazolidinone 41 with IC50 value 49.27 µM and benzo[4,5]imidazo[1,2-a]pyridine-4- carbonitrile 28 with IC50 value 49.78 µM, showed increased activity when compared to doxorubicin with IC50 value 68.6 µM, while compounds 2, 39, 16, 10, 22 and 30 with IC50 values 78.02, 86.32, 86.85, 93.34, 96.04 and 98.39 µM, respectively, were found to be nearly as active as doxorubicin. While the compounds 17, 27, 31, 40, 23, 38, 5, 36, 1, 6, 37, 3, 14, 32, 9, 26, 24 and 20 with IC50 values 110.21, 111, 112.86, 113.32, 117.66, 119.74, 123.21, 125, 132.11, 133.11, 144.82, 147.26, 171.25, 171.3, 173.49, 177.98, 187.52 and 187.65 µM, respectively were less active than doxorubicin. While the remaining compounds 13, 19, 33 and 35 were non-active (NA). It is clear from the present data that the comparison of the IC50 for the synthesized compounds against human breast cancer cell line (MCF7). Chart 2 has showed that, the cell killing potency follows the order 18 > 8 > 41 > 28 > Dox > 2 > 39 > 16 > 10 > 22 > 30 > 17 > 27 > 31 > 40 > 23 > 38 > 5 > 36 > 1 > 6 > 37 > 3 > 14 > 32 > 9 > 26 > 24 > 20 > 13, 19, 33 and 35. These preliminary results of biological screening of the tested compounds could offer an encouraging framework in this field that may lead to the discovery of potent anticancer agent.

Conclusion
This article proved that compounds having benzenesulfonylpyrrolidine moiety attached to different heterocyclic moieties such as benzo[f]chromen-3-one 18, thiazolylacrylonitrile 8, N-pheny-4-thiazolidinone 41 and benzo[4,5]imidazo[1,2-a]pyridine-4-carbonitrile 28, showed a significant cytotoxic activity against human breast cancer cell line (MCF7) compared to the reference drug doxorubicin.

EXPERIMENTAL
Melting points (oC, uncorrected) were determined in open capillaries on a Gallen Kemp melting point apparatus (Sanyo Gallen Kemp, Southborough, UK). IR spectra (KBr) were recorded on FT-IR 5300 spectrometer and Perkin Elmer spectrum RXIFT-IR system (ν, cm-1). Pre-coated silica gel plates (silica gel 0.25 mm, 60 G F 254; Merck, Germany) were used for thin layer chromatography. The NMR spectra in (DMSO-d6) were recorded at 600 MHz on a Varian Gemini NMR spectrometer (δ, ppm). Mass spectra were obtained on GC Ms-QP 1000 EX mass spectrometer at 70 ev. Elemental analyses were performed on Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau, Germany). All compounds were within ± 0.4% of the theoretical values.
2-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acetonitrile (2)
A mixture of phenacyl bromide derivative 1 (3.32 g, 0.01 mol) and 2-cyanoethanethioamide (1.00 g, 0.01 mol) in EtOH (50 mL) was heated under reflux for 1 h. The solvent was concentrated; after cooling, the solid product that formed was collected and recrystallized from EtOH to give 2. White crystals, Yield, 74%; mp 130-131 oC. IR (KBr, cm-1): vmax 3074 (CH aromatic), 2960, 2908 (CH aliphatic), 2220 (C≡N), 1594 (C=N), 1560 (C=C), 1336, 1160 (SO2). 1H NMR (DMSO-d6): δ 1.93 (m, 4H, CH2-CH2 of pyrrolidine), 3.30 (t, 4H, J=9.8 Hz, CH2-N-CH2 of pyrrolidine), 4.01 (s, 2H, CH2), 7.52 (s, 1H, H5 of thiazole), 7.80, 8.05 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system). 13C NMR (DMSO-d6): δ 18.9 (CH2), 22.7 (2C, CH2-CH2 of pyrrolidine), 60.1 (2C, CH2-N-CH2 of pyrrolidine), 108.1 (C5 of thiazole), 117.5 (C≡N), 125.9 (2C), 128.6 (2C), 137.4, 140.5, 160.0, 169.2. MS m/z (%): 334.06 [M++1] (33.19), 333.03 [M+] (53.04), 332.02 (15.47), 262.97 (14.88), 216.04 (14.16), 200.05 (29.81), 199.02 (45.92), 158.96 (13.55), 133.01 (11.85), 89.04 (35.62), 70.06 (100.00), 69.04 (5.23), 63.02 (9.19), 43.06 (11.04), 42.02 (44.71), 41.02 (12.00). Anal. Calcd for C15H15N3O2S2 (333.43): C, 54.03; H, 4.53; N, 12.60; S, 19.23. Found: C, 54.12; H, 4.67; N, 12.74; S, 19.05%.
N'-(4-Chlorophenyl)-4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazole-2-carbohydrazonoyl cyanide (3)
Procedure A: 4-Chlorobenzenediazonium chloride (prepared by adding sodium nitrite (0.69 g, 0.01 mol) to 4-chloroaniline (1.27 g, 0.01 mol) in conc. hydrogen chloride (6 mL) at 0-5 oC under stirring) was added drop wise to a cold solution of thiazolylacetonitrile derivative 2 (3.33 g, 0.01 mol) in EtOH (20 mL) containing sodium acetate (6.56 g, 0.08 mol), the obtained product was collected and recrystallized from EtOH to give 3 (yield 91%).
Procedure B: A mixture of phenacyl bromide derivative 1 (3.32 g, 0.01 mol) and 2-amino-N'- (4-chlorophenyl)-2-thioxoacetohydrazonoyl cyanide (2.38 g, 0.01 mol) in EtOH (50 mL) was heated under reflux for 1 h. The resulting solid was collected to give the compound 3, mp and mixed mp determined with authentic sample, which was obtained in procedure A gave no depression. Yellow crystals, Yield, 87%; mp 150-152 oC. IR (KBr, cm-1): vmax 3193 (NH), 3017 (CH aromatic), 2983, 2914, 2895 (CH aliphatic), 2222 (C≡N), 1610 (C=N), 1582 (C=C), 1345, 1151 (SO2), 740 (C-Cl). 1H NMR (DMSO-d6): δ 1.96 (m, 4H, CH2-CH2 of pyrrolidine), 3.27 (t, 4H, J=10.1 Hz, CH2-N-CH2 of pyrrolidine), 7.23, 7.31 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system of chlorophenyl), 7.40 (s, 1H, H5 of thiazole), 7.91, 8.06 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system of benzenesulfonyl), 12.76 (s, 1H, NH, Discharged with D2O). 13C NMR (DMSO-d6): δ 23.0 (2C, CH2-CH2 of pyrrolidine), 63.4 (2C, CH2-N-CH2 of pyrrolidine), 111.2 (C5 of thiazole), 115.9 (2C), 121.3 (C≡N), 126.7 (2C), 128.1, 130.5 (2C), 133.2 (2C), 135.8, 140.7, 142.5, 145.2, 155.9, 166.0. MS m/z (%): 471.17 [M+] (62.41), 232.06 (15.13), 194.10 (27.25), 193.11 (35.52), 192.06 (7.75), 143.98 (12.08), 131.05 (15.92), 129.08 (19.70), 127.05 (100.00), 125.04 (23.92), 111.04 (10.32), 92.10 (18.65), 88.04 (50.90), 65.07 (8.83), 60.06 (10.42). Anal. Calcd for C21H18ClN5O2S2 (471.98): C, 53.44; H, 3.84; N, 14.84; S, 13.59. Found: C, 53.58; H, 3.72; N, 14.65; S, 13.66%.
General Procedure for Preparation of (5, 6)
To a stirred solution of compound 2 (3.33 g, 0.01 mol) in EtOH (50 mL) containing sodium acetate (3 g) 4-cyano-3-(methylthio)-1H-pyrazole-5-diazonium chloride, and/or 1H-1,2,4-triazole-3-diazonium chloride (prepared by adding sodium nitrite (0.69 g, 0.01 mol) to heterocyclic amines (0.01 mol) in conc. hydrogen chloride (6 mL) at 0-5 oC under stirring) was added drop wise while cooling to 0-5 oC and stirring. The reaction mixture was then left at room temperature for 2 h, and the solid product formed was collected by filtration and recrystallized from the appropriate solvent to give 5 and 6, respectively.
4-Amino-7-(methylthio)-3-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)pyrazolo[5,1-c][1,2,4]- triazine-8-carbonitrile (5)
White crystals, Yield, 53%; mp 202-204 oC (EtOH/benzene). IR (KBr, cm-1): vmax 3440, 3317 (NH2), 3019 (CH aromatic), 2971, 2923 (CH aliphatic), 2217 (C≡N), 1583 (C=N), 1561 (C=C), 1408 (N=N), 1337, 1155 (SO2), 1179 (C-S). 1H NMR (DMSO-d6): δ 1.99 (m, 4H, CH2-CH2 of pyrrolidine), 3.29 (t, 4H, J=9.9 Hz, CH2-N-CH2 of pyrrolidine), 2.55 (s, 3H, CH3), 7.32 (s, 1H, H5 of thiazole), 7.72, 8.30 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system), 8.51 (s, 2H, NH2, Discharged with D2O). MS m/z (%): 498.53 [M+] (80.51), 309.10 (25.47), 245.11 (25.14), 201.09 (40.52), 192.08 (24.64), 191.06 (16.54), 176.06 (71.75), 175.06 (71.69), 174.07 (16.30), 148.06 (19.49), 146.05 (13.45), 122.08 (11.38), 121.05 (15.87), 109.07 (100.00), 104.08 (17.94), 89.06 (36.83), 82.00 (34.88), 80.00 (38.47), 77.06 (51.86), 69.05 (16.35), 43.07 (16.17), 41.07 (21.39). Anal. Calcd for C20H18N8O2S3 (498.60): C, 48.18; H, 3.64; N, 22.47; S, 19.29. Found: C, 48.30; H, 3.59; N, 22.28; S, 19.37%.
2-((1H-1,2,4-Triazol-3-yl)diazenyl)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acetonitrile (6)
Yellow crystals, Yield, 87%; mp 156-158 oC (EtOH). IR (KBr, cm-1): vmax 3230 (NH), 3090, 3085 (CH aromatic), 2973, 2908 (CH aliphatic), 2231 (C≡N), 1613 (C=N), 1592 (C=C), 1453 (N=N), 1341, 1159 (SO2). 1H NMR (DMSO-d6): δ 1.92 (m, 4H, CH2-CH2 of pyrrolidine), 3.27 (t, 4H, J=9.8 Hz, CH2-N-CH2 of pyrrolidine), 4.99 (s, 1H, CH), 7.73 (s, 1H, H5 of thiazole), 7.90, 8.07 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system), 8.33 (s, 1H, H5 of triazole), 10.98 (s, 1H, NH, Discharged with D2O). MS m/z (%): 428.06 [M+] (50.81), 376.03 (23.13), 289.00 (47.17), 287.99 (51.35), 242.00 (27.83), 222.02 (23.93), 198.04 (20.01), 153.03 (41.96), 146.02 (24.78), 144.02 (19.33), 133.01 (33.87), 120.98 (22.64), 119.05 (28.60), 115.03 (36.90), 113.11 (25.85), 105.04 (18.00), 92.99 (27.92), 78.06 (19.58), 77.04 (100.00), 63.02 (68.67), 62.02 (23.33), 55.05 (25.64), 51.02 (42.62), 47.00 (42.76), 44.01 (25.92), 43.06 (81.12). Anal. Calcd for C17H16N8O2S2 (428.49): C, 47.65; H, 3.76; N, 26.15; S, 14.97. Found: C, 47.38; H, 3.58; N, 26.33; S, 15.00%.
3-(4-Fluorophenyl)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (8)
Procedure A: A mixture of compound 2 (3.33 g, 0.01 mol) and 4-fluorobenzaldehyde (1.24 g, 0.01 mol) in EtOH (40 mL) containing few drops of piperidine was refluxed for 2 h. The obtained product that formed was collected by filtration and recrystallized from EtOH to give 8 (yield 72%).
Procedure B: A mixture of phenacyl bromide derivative 1 (3.32 g, 0.01 mol) and 2-cyano-3- (4-fluorophenyl)prop-2-enethioamide (2.06 g, 0.01 mol) in EtOH (40 mL) was refluxed for 2 h. The product obtained was collected and recrystallized. mp and mixed mp determined with authentic sample gave no depression. White crystals, Yield, 88%; mp 162-163 oC. IR (KBr, cm-1): vmax 3101 (CH aromatic), 2931 (CH aliphatic), 2220 (C≡N), 1630 (C=N), 1594 (C=C), 1364, 1181 (SO2), 1170 (C-F). 1H NMR (DMSO-d6): δ 1.88 (m, 4H, CH2-CH2 of pyrrolidine), 3.28 (t, 4H, J=10.5 Hz, CH2-N-CH2 of pyrrolidine), 6.97-7.44 (m, 4H, Ar-H), 7.55, 8.01 (dd, 4H, “each d, each 2H, J=8.5 Hz”, AB system of benzenesulfonyl), 7.60 (s, 1H, H5 of thiazole), 8.21 (s, 1H, CH=C). 13C NMR (DMSO-d6): δ 24.2 (2C, CH2-CH2 of pyrrolidine), 64.5 (2C, CH2-N-CH2 of pyrrolidine), 100.4, 109.2 (C5 of thiazole), 113.1 (2C), 117.3 (C≡N), 124.7 (2C), 129.9 (2C), 133.0 (2C), 135.2, 139.4, 144.3, 154.7, 159.2, 163.4, 169.9. MS m/z (%): 440.10 [M++1] (21.76), 439.09 [M+] (46.31), 438.07 (12.57), 305.03 (22.40), 134.02 (9.01), 133.02 (9.66), 89.04 (64.99), 70.06 (100.00), 63.02 (19.44), 43.06 (21.32), 42.04 (83.04), 41.04 (24.43). Anal. Calcd for C22H18FN3O2S2 (439.53): C, 60.12; H, 4.13; N, 9.56; S, 14.59. Found: C, 60.09; H, 4.11; N, 9.51; S, 14.71%.
2-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)ethanethioamide (9)
To a solution of compound 2 (3.33 g, 0.01 mol) in EtOH (30 mL) triethylamine (1.01 g, 0.01 mol) was added, and the mixture was saturated with H2S for 3 h and then left overnight. The precipitated solid was separated by filtration and purified by recrystallization from EtOH to give 9. Yellow crystals, Yield, 54%; mp 146-148 oC. IR (KBr, cm-1): vmax 3401, 3299 (NH2), 3019 (CH aromatic), 2995, 2957, 2910 (CH aliphatic), 1593 (C=N), 1554 (C=C), 1544, 1276, 1144, 1056 (N-C=S), 1358, 1170 (SO2), 1340 (C=S). 1H NMR (DMSO-d6): δ 1.94 (m, 4H, CH2-CH2 of pyrrolidine), 3.33 (t, 4H, J=10.6 Hz, CH2-N-CH2 of pyrrolidine), 3.51 (s, 2H, CH2), 7.31 (s, 1H, H5 of thiazole), 7.57, 8.11 (dd, 4H, “each d, each 2H, J=8.5 Hz”, AB system), 8.61 (s, 2H, NH2, Discharged with D2O). 13C NMR (DMSO-d6): δ 22.6 (2C, CH2-CH2 of pyrrolidine), 48.4 (CH2), 61.8 (2C, CH2-N-CH2 of pyrrolidine), 113.5 (C5 of thiazole), 126.6 (2C), 128.8 (2C), 137.0, 141.4, 156.5, 171.7, 203.7 (C=S). MS m/z (%): 367.45 [M+] (90.13), 333.05 (14.20), 309.08 (25.31), 269.08 (18.15), 268.09 (19.48), 263.01 (19.65), 241.09 (32.91), 200.05 (30.76), 199.04 (43.35), 191.07 (15.84), 187.14 (18.21), 176.09 (19.94), 175.10 (56.20), 174.07 (17.12), 168.97 (18.68), 159.95 (16.53), 134.06 (33.23), 133.07 (17.71), 127.89 (18.73), 89.06 (41.73), 81.96 (22.21), 79.97 (16.80), 69.07 (100.00), 64.01 (17.16), 49.03 (28.48). Anal. Calcd for C15H17N3O2S3 (367.51): C, 49.02; H, 4.66; N, 11.43; S, 26.17. Found: C, 48.89; H, 4.54; N, 11.40; S, 26.32%.
2-((4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)methyl)thiazol-4(5H)-one (10)
Procedure A: A mixture of compound 9 (3.67 g, 0.01 mol) and ethyl 2-chloroacetate (1.22 g, 0.01 mol) in EtOH (20 mL) containing 1 g of sodium acetate was refluxed for 2 h. The obtained product that formed was collected and recrystallized from EtOH to give 10 (yield 46%).
Procedure B: A mixture of compound 2 (3.33 g, 0.01 mol) and 2-mercaptoacetic acid (0.92 g, 0.01 mol) in pyridine (10 mL) was refluxed for 45 min. The solid obtained was filtered off and recrystallized; mp and mixed mp determined with authentic sample gave no depression. White crystals, Yield, 61%; mp 203-204 oC. IR (KBr, cm-1): vmax 3102, 3056 (CH aromatic), 2977, 2945, 2911 (CH aliphatic), 1673 (C=O), 1611 (C=N), 1586 (C=C), 1371, 1181 (SO2). 1H NMR (DMSO-d6): δ 1.92 (m, 4H, CH2-CH2 of pyrrolidine), 3.31 (t, 4H, J=10.2 Hz, CH2-N-CH2 of pyrrolidine), 3.18 (s, 2H, CH2), 4.25 (s, 2H, CH2 of thiazolidinone), 7.40 (s, 1H, H5 of thiazole), 7.83, 8.26 (dd, 4H, “each d, each 2H, J=8.4 Hz”, AB system). 13C NMR (DMSO-d6): δ 22.1 (2C, CH2-CH2 of pyrrolidine), 35.3 (CH2), 41.6 (CH2 of thiazolidinone), 63.4 (2C, CH2-N-CH2 of pyrrolidine), 110.7 (C5 of thiazole), 125.0 (2C), 129.2 (2C), 138.1, 142.5, 156.9, 167.9, 170.3, 179.2. MS m/z (%): 407.06 [M+] (71.25), 388.15 (18.57), 255.12 (11.19), 254.14 (40.61), 253.11 (75.20), 252.09 (13.91), 239.10 (13.16), 238.07 (10.54), 212.09 (14.90), 211.08 (17.71), 152.09 (18.05), 123.07 (12.19), 120.09 (37.16), 119.11 (49.97), 105.08 (11.00), 72.13 (100.00). Anal. Calcd for C17H17N3O3S3 (407.53): C, 50.10; H, 4.20; N, 10.31; S, 23.60. Found: C, 50.33; H, 4.13; N, 10.22; S, 23.51%.
5-(4-Fluorobenzylidene)-2-((4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)methyl)thiazol-4(5H)-one (13)
Procedure A: A mixture of compound 10 (4.07 g, 0.01 mol) and 2-(4-fluorobenzylidene)malononitrile (1.72 g, 0.01 mol) in EtOH (50 mL) was refluxed for 2 h. The solid obtained was filtered off and recrystallized from AcOH to give 13 (yield 66%).
Procedure B: A mixture of compound 10 (4.07 g, 0.01 mol) and 4-fluorobenzaldehyde (1.24 g, 0.01 mol) in EtOH (20 mL) and few drops of piperidine was refluxed for 3 h. The mixture was cooled and the separated solid was filtered off and recrystallized, mp and mixed mp determined with authentic sample gave no depression. White crystals, Yield, 83%; mp 213-215 oC. IR (KBr, cm-1): vmax 3090 (CH aromatic), 2910, 2899 (CH aliphatic), 1655 (C=O), 1580 (C=N), 1551 (C=C), 1345, 1155 (SO2), 1172 (C-F). 1H NMR (DMSO-d6): δ 1.87 (m, 4H, CH2-CH2 of pyrrolidine), 3.32 (t, 4H, J=11.1 Hz, CH2-N-CH2 of pyrrolidine), 3.15 (s, 2H, CH2), 7.69 (s, 1H, H5 of thiazole), 7.80 (s, 1H, CH=C), 7.10-7.54 (m, 4H, Ar-H), 7.96, 8.52 (dd, 4H, “each d, each 2H, J=8.5 Hz”, AB system of benzenesulfonyl). MS m/z (%): 513.88 [M+] (57.28), 513.12 (42.87), 354.16 (19.89), 351.07 (6.02), 315.11 (6.95), 311.19 (9.84), 310.16 (100.00), 298.14 (16.56), 282.12 (18.52), 254.11 (14.16), 238.10 (30.62), 224.10 (23.21), 216.08 (5.44), 200.08 (29.68), 199.09 (11.64), 193.08 (16.61), 189.11 (10.88), 155.10 (11.41), 97.14 (41.79), 79.09 (31.20), 60.04 (79.33), 56.11 (8.64), 46.07 (16.68), 40.17 (8.84). Anal. Calcd for C24H20FN3O3S3 (513.63): C, 56.12; H, 3.92; N, 8.18; S, 18.73. Found: C, 56.35; H, 3.79; N, 8.09; S, 18.56%.
5-Imino-7-(methylthio)-3-(4-(pyrrolidin-1-ylsulfonyl)phenyl)-5H-thiazolo[3,2-a]pyridine-6,8-dicarb-onitrile (14)
A mixture of compound 2 (3.33 g, 0.01 mol), anhydrous potassium carbonate (1.66 g, 0.012 mol) and 2-(bis(methylthio)methylene)malononitrile (1.70 g, 0.01 mol) in N,N-dimethylformamide (30 mL) was heated at 50-60 oC with stirring until odor of the methane thiol is ceased. After cooling, the reaction mixture poured into crushed ice (100 g) then acidified with 1N hydrogen chloride. The solid product that formed was collected and recrystallized from dioxane to give 14. White crystals, Yield, 62%; mp 203-205 oC. IR (KBr, cm-1): vmax 3235 (NH), 3070 (CH aromatic), 2990 (CH aliphatic), 2220, 2218 (2C≡N), 1644 (C=N), 1568 (C=C), 1337, 1159 (SO2), 1180 (C-S). 1H NMR (DMSO-d6): δ 1.92 (m, 4H, CH2-CH2 of pyrrolidine), 2.83 (s, 3H, SCH3), 3.30 (t, 4H, J=10.6 Hz, CH2-N-CH2 of pyrrolidine), 7.52 (s, 1H, H5 of thiazole), 7.67, 8.10 (dd, 4H, “each d, each 2H, J=8.8 Hz”, AB system), 8.52 (s, 1H, NH, Discharged with D2O). 13C NMR (DMSO-d6): δ 16.2 (CH3), 23.6 (2C, CH2-CH2 of pyrrolidine), 64.7 (2C, CH2-N-CH2 of pyrrolidine), 77.2, 85.6, 114.0 (2C, 2C≡N), 118.2 (C5 of thiazole), 126.7 (2C), 132.0 (2C), 135.7, 136.9, 141.3, 155.8, 160.0, 166.3. MS m/z (%): 455.43 [M+] (75.92), 401.20 (14.46), 308.15 (21.86), 267.13 (6.25), 266.11 (4.76), 206.14 (5.95), 205.12 (4.81), 139.10 (6.16), 131.11 (8.08), 123.12 (7.56), 120.10 (4.16), 118.11 (14.45), 117.10 (10.07), 112.13 (6.69), 91.09 (75.75), 84.11 (100.00), 78.10 (5.70), 77.08 (9.85), 65.08 (10.22). Anal. Calcd for C20H17N5O2S3 (455.58): C, 52.73; H, 3.76; N, 15.37; S, 21.12. Found: C, 52.64; H, 3.56; N, 15.10; S, 21.40%.
3-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)-2H-chromen-2-one (16)
Procedure A: To a mixture of compound 2 (3.33 g, 0.01 mol) and 2-hydroxybenzaldehyde (1.22 g, 0.01 mol) in EtOH (50 mL), a few drops of piperidine was added as a catalyst. The reaction mixture was refluxed for 3 h, and the solid product was collected by filtration and recrystallized from EtOH/benzene to give 16 (yield 90%).
Procedure B: A mixture of compound 1 (3.32 g, 0.01 mol) and 2-oxo-2H-chromene-3-carbothioamide (2.05 g, 0.01 mol) in EtOH (50 mL) was refluxed for 2 h. The solid obtained was filtered off and recrystallized; mp and mixed mp determined with authentic sample gave no depression. Yellowish white crystals, Yield, 85%; mp 245-246 oC. IR (KBr, cm-1): vmax 3100 (CH aromatic), 2953 (CH aliphatic), 1708 (C=O), 1628 (C=N), 1589 (C=C), 1364, 1180 (SO2), 1276, 1037 (C-O-C). 1H NMR (DMSO-d6): δ 1.93 (m, 4H, CH2-CH2 of pyrrolidine), 3.27 (t, 4H, J=9.8 Hz, CH2-N-CH2 of pyrrolidine), 7.00 (s, 1H, H5 of thiazole), 7.17-7.79 (m, 4H, Ar-H), 7.85, 8.17 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system), 9.11 (s, 1H, H4 of coumarin). MS m/z (%): 439.18 [M++1] (25.27), 438.24 [M+] (59.50), 364.59 (39.76), 364.08 (30.74), 358.16 (17.76), 344.40 (19.43), 335.09 (32.38), 330.47 (27.88), 329.12 (19.68), 325.47 (33.45), 315.12 (30.68), 304.21 (59.19), 283.55 (37.76), 269.16 (21.25), 250.13 (19.41), 240.09 (17.59), 233.44 (58.81), 232.14 (33.39), 212.14 (38.16), 204.14 (57.33), 159.11 (51.86), 157.13 (100.00), 145.12 (35.16), 97.12 (81.54), 49.08 (18.58). Anal. Calcd for C22H18N2O4S2 (438.52): C, 60.26; H, 4.14; N, 6.39; S, 14.62. Found: C, 60.01; H, 4.32; N, 6.45; S, 14.70%.
2-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)-3H-benzo[f]chromen-3-imine (17)
To a mixture of compound 2 (3.33 g, 0.01 mol) and 2-hydroxy-1-naphthaldehyde (1.72 g, 0.01 mol) in EtOH (50 mL), a few drops of piperidine was added as catalyst. The reaction mixture was refluxed for 5 h. The isolated product was collected and recrystallized from EtOH to give 17. White solids, Yield, 72%; mp 199-201 oC. IR (KBr, cm-1): vmax 3199 (NH), 3019 (CH aromatic), 2993 (CH aliphatic), 1583 (C=N), 1554 (C=C), 1349, 1170 (SO2), 1247, 1056 (C-O-C). 1H NMR (DMSO-d6): δ 1.93 (m, 4H, CH2-CH2 of pyrrolidine), 3.30 (t, 4H, J=10.0 Hz, CH2-N-CH2 of pyrrolidine), 7.16 (s, 1H, H5 of thiazole), 7.22-8.97 (m, 10H, Ar-H), 9.17 (s, 1H, H1 of benzocoumarin), 9.78 (br, 1H, NH, Discharged with D2O). MS m/z (%): 487.04 [M+] (36.42), 436.36 (65.48), 372.01 (58.90), 222.03 (14.83), 213.84 (37.09), 176.36 (33.76), 158.29 (42.91), 99.26 (16.53), 89.25 (11.52), 81.23 (27.35), 77.63 (72.59), 70.42 (100.00), 44.73 (10.94), 41.83 (9.37). Anal. Calcd for C26H21N3O3S2 (487.59): C, 64.04; H, 4.34; N, 8.62; S, 13.15. Found: C, 63.89; H, 4.12; N, 8.53; S, 13.09%.
2-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)-3H-benzo[f]chromen-3-one (18)
To a mixture of 30 mL AcOH and 3 mL of hydrogen chloride, (4.87 g, 0.01 mol) of compound 17 was added and refluxed for 2 h. The precipitate obtained after cooling was filtered off and recrystallized from EtOH/benzene to give 18. Yellow crystals, Yield, 91%; mp 203-205 oC. IR (KBr, cm-1): vmax 3105, 3065 (CH aromatic), 2973, 2910 (CH aliphatic), 1703 (C=O), 1630 (C=N), 1582 (C=C), 1333, 1179 (SO2), 1208, 1042 (C-O-C). 1H NMR (DMSO-d6): δ 1.87 (m, 4H, CH2-CH2 of pyrrolidine), 3.29 (t, 4H, J=10.0 Hz, CH2-N-CH2 of pyrrolidine), 7.19 (s, 1H, H5 of thiazole), 7.22-8.86 (m, 10H, Ar-H), 9.19 (s, 1H, H1 of benzocoumarin). MS m/z (%): 489.08 [M++1] (32.09), 488.38 [M+] (59.06), 487.76 (12.68), 486.99 (14.32), 471.10 (16.35), 470.04 (42.94), 336.03 (22.59), 193.05 (39.88), 176.95 (23.46), 176.22 (31.85), 165.06 (17.47), 164.03 (16.15), 134.03 (27.78), 133.01 (18.73), 102.06 (13.38), 101.04 (26.18), 91.06 (17.77), 90.07 (18.35), 89.04 (100.00), 75.04 (12.24), 70.07 (69.38), 63.04 (34.79), 43.07 (34.77), 42.04 (92.64), 41.06 (35.88). Anal. Calcd for C26H20N2O4S2 (488.58): C, 63.92; H, 4.13; N, 5.73; S, 13.13. Found: C, 63.76; H, 4.09; N, 5.54; S, 13.07%.
3-Ethoxy-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (19)
A mixture of compound 2 (3.33 g, 0.01 mol) and triethoxymethane (1.48 g, 0.01 mol) in acetic anhydride (1.02 g, 0.01 mol) was refluxed for 3 h. The reaction mixture was concentrated to 10 mL. After cooling, the solid product was collected by filtration and washed with EtOH then recrystallized from benzene to give 19. White crystals, Yield, 61%; mp 174-176 oC. IR (KBr, cm-1): vmax 3107, 3064 (CH aromatic), 2944, 2921, 2904 (CH aliphatic), 2220 (C≡N), 1611 (C=N), 1592 (C=C), 1347, 1155 (SO2), 1254, 1035 (C-O-C). 1H NMR (DMSO-d6): δ 1.26 (t, 3H, J=7.2 Hz, CH3-CH2), 1.85 (m, 4H, CH2-CH2 of pyrrolidine), 3.32 (t, 4H, J=10.5 Hz, CH2-N-CH2 of pyrrolidine), 4.52 (q, 2H, J=7.2 Hz, CH3-CH2), 7.40 (s, 1H, CH=C), 7.73 (s, 1H, H5 of thiazole), 7.96, 8.55 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system). MS m/z (%): 389.03 [M+] (42.02), 333.03 (6.20), 262.97 (5.30), 216.02 (7.67), 200.04 (17.22), 199.03 (24.45), 159.00 (7.30), 134.06 (9.25), 133.02 (9.22), 89.03 (35.71), 70.06 (100.00), 63.02 (8.19), 43.05 (16.98), 42.03 (46.51), 41.05 (14.96). Anal. Calcd for C18H19N3O3S2 (389.49): C, 55.51; H, 4.92; N, 10.79; S, 16.47. Found: C, 55.34; H, 4.75; N, 10.88; S, 16.31%.
3-(Dimethylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (20)
Procedure A: A mixture of compound 19 (3.89 g, 0.01 mol) and dimethylamine (0.45 g, 0.01 mol) in MeOH (40 mL) was refluxed for 3 h. The mixture was then cooled, and the separated solid was collected by filtration and recrystallized from benzene to give 20 (yield 71%).
Procedure B: A mixture of compound 2 (3.33 g, 0.01 mol) and DMF-DMA (1.19 g, 0.01 mol) in dry xylene (30 mL) was refluxed for 3 h. Then the cooled precipitated product was filtered off, washed with light petroleum ether, and recrystallized, mp and mixed mp determined with authentic sample gave no depression. White crystals, Yield, 85%; mp 189-190 oC. IR (KBr, cm-1): vmax 3090, 3038 (CH aromatic), 2991, 2963, 2905 (CH aliphatic), 2218 (C≡N), 1575 (C=N), 1557 (C=C), 1355, 1161 (SO2). 1H NMR (DMSO-d6): δ 1.95 (m, 4H, CH2-CH2 of pyrrolidine), 2.99 (s, 6H, (CH3)2N), 3.40 (t, 4H, J=11.0 Hz, CH2-N-CH2 of pyrrolidine), 7.42 (s, 1H, CH=C), 7.65 (s, 1H, H5 of thiazole), 7.97, 8.63 (dd, 4H, “each d, each 2H, J=8.7 Hz”, AB system). 13C NMR (DMSO-d6): δ 22.7 (2C, CH2-CH2 of pyrrolidine), 41.6 (2C, (CH3)2N), 62.8 (2C, CH2-N-CH2 of pyrrolidine), 83.2, 108.3 (C5 of thiazole), 118.8 (C≡N), 123.3 (2C), 128.0 (2C), 138.4, 143.5, 156.4, 159.9, 166.4. MS m/z (%): 389.15 [M++1] (26.83), 388.12 [M+] (98.13), 387.10 (7.80), 255.11 (16.63), 254.12 (47.16), 253.11 (100.00), 252.11 (18.22), 239.09 (14.46), 238.09 (12.67), 134.05 (11.64), 119.09 (15.71), 89.03 (22.86), 72.10 (17.98), 42.06 (13.32). Anal. Calcd for C18H20N4O2S2 (388.51): C, 55.65; H, 5.19; N, 14.42; S, 16.51. Found: C, 55.44; H, 5.26; N, 14.62; S, 16.46%.
6-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)-[1,2,4]triazolo[4,3-a]pyrimidin-5-amine (22)
A mixture of compound 20 (3.88 g, 0.01 mol) and 4H-1,2,4-triazol-3-amine (0.84 g, 0.01 mol) in N,N-dimethylformamide (30 mL) was refluxed for 3 h. After cooling, the product was collected by filtration then washed with EtOH and recrystallized from dioxane to give 22. White crystals, Yield, 83%; mp 310-312 oC. IR (KBr, cm-1): vmax 3417, 3310 (NH2), 3074 (CH aromatic), 2934, 2901 (CH aliphatic), 1583 (C=N), 1558 (C=C), 1343, 1155 (SO2). 1H NMR (DMSO-d6): δ 1.80 (m, 4H, CH2-CH2 of pyrrolidine), 3.27 (t, 4H, J=11.1 Hz, CH2-N-CH2 of pyrrolidine), 7.61 (br, 2H, NH2, Discharged with D2O), 7.72-8.96 (m, 7H, Ar-H + H5 of thiazole). 13C NMR (DMSO-d6): δ 22.2 (2C, CH2-CH2 of pyrrolidine), 65.4 (2C, CH2-N-CH2 of pyrrolidine), 109.1 (C5 of thiazole), 113.6, 126.3 (2C), 130.9 (2C), 134.9, 138.2, 143.7, 156.8, 157.4, 160.3, 163.4, 170.2. MS m/z (%): 428.82 [M++1] (30.65), 427.02 [M+] (72.76), 305.09 (21.09), 270.22 (17.64), 218.15 (18.72), 188.13 (18.54), 169.07 (25.29), 168.10 (16.04), 135.09 (18.55), 112.13 (21.29), 110.12 (38.84), 100.08 (18.90), 98.11 (27.42), 95.09 (33.95), 86.10 (43.97), 84.08 (100.00), 83.09 (17.05), 77.08 (44.30), 66.08 (14.28), 65.07 (25.35), 59.08 (29.66), 56.09 (74.85), 55.08 (45.89), 54.08 (79.34), 53.08 (26.00), 47.03 (15.46). Anal. Calcd for C18H17N7O2S2 (427.50): C, 50.57; H, 4.01; N, 22.93; S, 15.00. Found: C, 50.46; H, 4.28; N, 22.78; S, 15.19%.
7-Amino-2-methyl-6-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (23)
The same experimental procedure described above for the synthesis of compound 22 has been followed except for using 5-amino-3-methyl-1H-pyrazole-4-carboxamide (1.40 g, 0.01 mol), instead of 4H-1,2,4-triazol-3-amine. White crystals, Yield, 77%; mp 270-271 oC (dioxane). IR (KBr, cm-1): vmax 3418, 3400, 3371, 3218 (2NH2), 3072 (CH aromatic), 2961, 2942 (CH aliphatic), 1651 (C=O), 1613 (C=N), 1571 (C=C), 1352, 1185 (SO2). 1H NMR (DMSO-d6): δ 1.99 (m, 4H, CH2-CH2 of pyrrolidine), 2.52 (s, 3H, CH3), 3.33 (t, 4H, J=10.4 Hz, CH2-N-CH2 of pyrrolidine), 7.31 (s, 1H, H5 of thiazole), 7.55 (s, 2H, CONH2, Discharged with D2O), 7.71 (s, 2H, NH2, Discharged with D2O), 7.97, 8.63 (dd, 4H, “each d, each 2H, J=8.8 Hz”, AB system), 8.12 (s, 1H, H5 of pyrazolopyrimidine). MS m/z (%): 483.21 [M+] (53.48), 465.82 (14.92), 389.17 (24.27), 387.12 (15.64), 374.31 (21.33), 322.43 (12.87), 309.11 (12.77), 308.11 (16.15), 296.11 (23.31), 291.27 (25.65), 264.09 (14.32), 262.25 (28.13), 261.05 (25.47), 258.25 (22.85), 234.11 (14.02), 233.13 (22.46), 216.04 (100.00), 175.07 (21.65), 142.10 (39.31), 109.07 (12.19), 60.01 (42.41), 52.03 (16.12), 47.98 (66.00), 40.16 (65.02). Anal. Calcd for C21H21N7O3S2 (483.57): C, 52.16; H, 4.38; N, 20.28; S, 13.26. Found: C, 52.23; H, 4.16; N, 20.01; S, 13.34%.
3-Hydrazinyl-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (24)
To a solution of the compound 20 (3.88 g, 0.01 mol) in EtOH (20 mL) hydrazine hydrate (99%, 0.50 g, 0.01 mol) was added. The reaction mixture was refluxed for 2 h, then cooled. The solid product formed was filtered off, washed with EtOH, and dried, then recrystallized from EtOH to give 24. White solids, Yield, 56%; mp 212-214 oC. IR (KBr, cm-1): vmax 3440, 3418 (NH2), 3190 (NH), 3013 (CH aromatic), 2972, 2943 (CH aliphatic), 2218 (C≡N), 1630 (C=N), 1595 (C=C), 1336, 1159 (SO2). 1H NMR (DMSO-d6): δ 1.83 (m, 4H, CH2-CH2 of pyrrolidine), 3.40 (t, 4H, J=9.9 Hz, CH2-N-CH2 of pyrrolidine), 5.08 (s, 2H, NH2, Discharged with D2O), 6.75 (s, 1H, CH=C), 7.45 (s, 1H, H5 of thiazole), 7.86, 8.56 (dd, 4H, “each d, each 2H, J=8.4 Hz”, AB system), 10.22 (s, 1H, NH, Discharged with D2O). MS m/z (%): 375.40 [M+] (70.54), 331.42 (51.72), 302.56 (17.43), 263.43 (11.92), 221.82 (22.73), 183.49 (18.43), 128.34 (11.54), 101.69 (47.83), 91.37 (66.90), 80.83 (15.32), 71.34 (100.00), 60.53 (12.43), 51.93 (15.76), 44.73 (17.53), 41.69 (18.34). Anal. Calcd for C16H17N5O2S2 (375.47): C, 51.18; H, 4.56; N, 18.65; S, 17.08. Found: C, 51.09; H, 4.78; N, 18.70; S, 17.31%.
4-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)isoxazol-5-amine (26)
To a mixture of compound 20 (3.88 g, 0.01 mol) and hydroxylamine hydrochloride (1.04 g, 0.015 mol) in EtOH (30 mL), sodium acetate (2.05 g, 0.025 mol) was added. The resulting mixture was refluxed for 3 h and then allowed to cool to room temperature and diluted with water (20 mL). The solid product so formed was collected by filtration, washed with water, and dried, then recrystallized from EtOH/benzene to give 26. White crystals, Yield, 75%; mp 256-258 oC. IR (KBr, cm-1): vmax 3411, 3316 (NH2), 3015 (CH aromatic), 2962 (CH aliphatic), 1635 (C=N), 1580 (C=C), 1349, 1156 (SO2). 1H NMR (DMSO-d6): δ 1.95 (m, 4H, CH2-CH2 of pyrrolidine), 3.32 (t, 4H, J=9.8 Hz, CH2-N-CH2 of pyrrolidine), 6.73 (br, 2H, NH2, Discharged with D2O), 7.21 (s, 1H, H5 of thiazole), 7.88, 8.71 (dd, 4H, “each d, each 2H, J=8.4 Hz”, AB system), 8.23 (s, 1H, H3 of isoxazole). 13C NMR (DMSO-d6): δ 22.5 (2C, CH2-CH2 of pyrrolidine), 64.9 (2C, CH2-N-CH2 of pyrrolidine), 98.3, 112.0 (C5 of thiazole), 125.5 (2C), 129.6 (2C), 138.3, 142.8, 147.3, 150.7, 157.3, 161.7. MS m/z (%): 376.07 [M+] (70.91), 375.07 (30.31), 374.09 (28.46), 333.05 (5.59), 253.07 (8.82), 239.05 (8.14), 216.04 (10.99), 199.04 (44.19), 172.04 (9.88), 159.02 (16.30), 134.04 (30.03), 133.03 (20.59), 127.01 (16.75), 119.08 (14.62), 89.04 (95.58), 70.07 (100.00), 63.03 (30.54), 44.03 (14.40), 43.06 (35.82), 42.05 (84.74), 41.05 (45.09). Anal. Calcd for C16H16N4O3S2 (376.45): C, 51.05; H, 4.28; N, 14.88; S, 17.04. Found: C, 50.89; H, 4.12; N, 14.76; S, 17.23%.
5-(4-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)pyrimidine-2,4-diamine (27)
The same experimental procedure described above for the synthesis of compound 26 has been followed except for using guanidine hydrochloride (0.95 g, 0.01 mol) instead of hydroxylamine hydrochloride. Brown crystals, Yield, 79%; mp 301-303 oC (AcOH). IR (KBr, cm-1): vmax 3400, 3366, 3258, 3187 (2NH2), 3030 (CH aromatic), 2983 (CH aliphatic), 1611 (C=N), 1584 (C=C), 1359, 1161 (SO2). 1H NMR (DMSO-d6): δ 1.81 (m, 4H, CH2-CH2 of pyrrolidine), 3.28 (t, 4H, J=9.8 Hz, CH2-N-CH2 of pyrrolidine), 7.06, 7.37 (2s, 4H, 2NH2, Discharged with D2O), 7.40 (s, 1H, H5 of thiazole), 7.79, 8.83 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system), 8.35 (s, 1H, H6 of pyrimidine). 13C NMR (DMSO-d6): δ 25.0 (2C, CH2-CH2 of pyrrolidine), 60.5 (2C, CH2-N-CH2 of pyrrolidine), 99.7, 110.7 (C5 of thiazole), 123.1 (2C), 130.1 (2C), 135.3, 141.3, 146.4, 149.8, 153.5, 159.4, 169.2. MS m/z (%): 402.17 [M+] (64.21), 360.21 (100.00), 291.17 (19.66), 256.16 (25.83), 241.14 (38.00), 232.12 (21.19), 229.12 (23.06), 216.09 (38.49), 190.09 (24.68), 188.11 (17.26), 181.11 (23.24), 180.12 (32.87), 168.09 (20.48), 155.09 (23.18), 154.09 (21.21), 131.09 (30.79), 127.08 (27.93), 118.08 (20.43), 111.08 (33.46), 101.08 (33.06), 94.10 (34.52), 78.08 (24.21), 73.07 (25.82), 71.10 (47.88), 57.09 (34.58). Anal. Calcd for C17H18N6O2S2 (402.49): C, 50.73; H, 4.51; N, 20.88; S, 15.93. Found: C, 50.65; H, 4.62; N, 20.74; S, 15.81%.
3-Amino-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)benzo[4,5]imidazo[1,2-a]pyridine-4- carbonitrile (28)
A mixture of compound 20 (3.88 g, 0.01 mol) and 2-(1H-benzo[d]imidazol-2-yl)acetonitrile (1.57 g, 0.01 mol) in dioxane (50 mL) was refluxed for 5 h. The solid product that was obtained after cooling was collected by filtration and recrystallized from DMF to give 28. Brown crystals, Yield, 81%; mp 306-307 oC. IR (KBr, cm-1): vmax 3416, 3295 (NH2), 3027 (CH aromatic), 2908 (CH aliphatic), 2220 (C≡N), 1630 (C=N), 1597 (C=C), 1345, 1155 (SO2). 1H NMR (DMSO-d6): δ 1.85 (m, 4H, CH2-CH2 of pyrrolidine), 3.31 (t, 4H, J=10.3 Hz, CH2-N-CH2 of pyrrolidine), 6.81 (s, 2H, NH2, Discharged with D2O), 7.44-8.78 (m, 10H, Ar-H + H5 of thiazole). MS m/z (%): 500.12 [M+] (70.29), 385.08 (15.09), 370.12 (11.52), 338.02 (13.57), 333.06 (19.05), 310.13 (9.12), 263.04 (20.74), 126.20 (22.97), 91.07 (92.84), 65.06 (36.93), 60.04 (12.42), 57.08 (48.03), 51.04 (21.34), 50.03 (21.35), 45.02 (34.30), 44.08 (100.00), 43.09 (57.32). Anal. Calcd for C25H20N6O2S2 (500.60): C, 59.98; H, 4.03; N, 16.79; S, 12.81. Found: C, 59.73; H, 4.12; N, 16.67; S, 12.59%.
2-Cyano-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)ethanedithioic acid (30)
Carbon disulfide (0.76 g, 0.01 mol) was added gradually to a cold solution of thiazolylacetonitrile 2 (3.33 g, 0.01 mol) in N,N-dimethylformamide (20 mL) containing finely ground potassium hydroxide (1.12 g, 0.02 mol). The reaction mixture left at room temperature for an additional 24 h. The reaction mixture was then triturated with cold water (50 mL) and neutralized with 1N hydrogen chloride. The resulting precipitated solid was collected by filtration, washed with water dried and recrystallized from EtOH to give 30. White solids, Yield, 90%; mp 142-144 oC. IR (KBr, cm-1): vmax 3074 (CH aromatic), 2915 (CH aliphatic), 2590 (SH), 2222 (C≡N), 1613 (C=N), 1588 (C=C), 1360, 1172 (SO2), 1344 (C=S). 1H NMR (DMSO-d6): δ 1.61 (s, 1H, SH), 1.80 (m, 4H, CH2-CH2 of pyrrolidine), 3.27 (t, 4H, J=10.4 Hz, CH2-N-CH2 of pyrrolidine), 5.25 (s, 1H, CH), 7.60 (s, 1H, H5 of thiazole), 7.77, 8.86 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system). MS m/z (%): 409.04 [M+] (81.72), 200.08 (7.13), 199.06 (9.89), 159.05 (5.03), 134.06 (9.64), 133.05 (7.90), 89.06 (40.13), 76.02 (11.00), 70.09 (100.00), 63.06 (12.97), 43.10 (22.54), 42.08 (63.34), 41.09 (22.69). Anal. Calcd for C16H15N3O2S4 (409.57): C, 46.92; H, 3.69; N, 10.26; S, 31.32. Found: C, 46.81; H, 3.47; N, 10.10; S, 31.14%.
3,3-Bis(methylthio)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (31)
The same experimental procedure described above for the synthesis of compound 30 has been followed except iodomethane was added (2.84 g, 0.02 mol) before the reaction mixture was left at room temperature. Orange crystals, Yield, 89%; mp 177-179 oC (EtOH). IR (KBr, cm-1): vmax 3105 (CH aromatic), 2990 (CH aliphatic), 2218 (C≡N), 1575 (C=N), 1560 (C=C), 1333, 1153 (SO2), 1189, 1150 (2C-S). 1H NMR (DMSO-d6): δ 1.89 (m, 4H, CH2-CH2 of pyrrolidine), 2.85 (s, 6H, 2SCH3), 3.32 (t, 4H, J=10.4 Hz, CH2-N-CH2 of pyrrolidine), 7.31 (s, 1H, H5 of thiazole), 7.76, 8.56 (dd, 4H, “each d, each 2H, J=8.7 Hz”, AB system). 13C NMR (DMSO-d6): δ 16.9 (2C, 2SCH3), 25.0 (2C, CH2-CH2 of pyrrolidine), 63.7 (2C, CH2-N-CH2 of pyrrolidine), 84.5, 109.6 (C5 of thiazole), 119.1 (C≡N), 124.9 (2C), 128.9 (2C), 133.6, 139.5, 148.4, 156.4, 164.3. MS m/z (%): 437.33 [M+] (61.41), 420.60 (16.69), 339.11 (19.21), 324.15 (12.02), 312.14 (7.56), 309.13 (15.93), 279.14 (17.71), 252.12 (14.32), 244.15 (14.52), 238.15 (100.00), 237.14 (12.17), 226.11 (49.14), 184.10 (9.63), 183.08 (81.36), 153.30 (31.22), 150.09 (21.77), 125.14 (16.95), 123.10 (13.31), 98.13 (21.59), 85.13 (23.36), 84.12 (31.33), 82.11 (35.01), 71.13 (36.77), 69.11 (44.20). Anal. Calcd for C18H19N3O2S4 (437.62): C, 49.40; H, 4.38; N, 9.60; S, 29.31. Found: C, 49.28; H, 4.17; N, 9.55; S, 29.53%.
General Procedure for Preparation of 32 and 33
A mixture of ketene dithioacetal 31 (4.38 g, 0.01 mol) and the appropriate 1,2-diamines namely (ethane-1,2-diamine or benzene-1,2-diamine) (0.01 mol) in EtOH (50 mL) was refluxed for 3 h. The obtained product was collected by filtration and recrystallized from an appropriate solvent to give 32 and 33, respectively.
2-(Imidazolidin-2-ylidene)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acetonitrile (32)
White crystals, Yield, 86%; mp 196-198 oC (EtOH/benzene). IR (KBr, cm-1): vmax 3215, 3199 (2NH), 3010 (CH aromatic), 2962 (CH aliphatic), 2219 (C≡N), 1630 (C=N), 1579 (C=C), 1337, 1159 (SO2). 1H NMR (DMSO-d6): δ 1.90 (m, 4H, CH2-CH2 of pyrrolidine), 3.02 (s, 4H, CH2-CH2 of imidazoline), 3.30 (t, 4H, J=10.0 Hz, CH2-N-CH2 of pyrrolidine), 7.30, 7.43 (2br, 2H, 2NH, Discharged with D2O), 7.60 (s, 1H, H5 of thiazole), 7.80, 8.55 (dd, 4H, “each d, each 2H, J=8.3 Hz”, AB system). 13C NMR (DMSO-d6): δ 24.0 (2C, CH2-CH2 of pyrrolidine), 43.2 (2C, CH2-CH2 of imidazoline), 57.9, 66.0 (2C, CH2-N-CH2 of pyrrolidine), 109.9 (C5 of thiazole), 117.6 (C≡N), 124.8 (2C), 129.4 (2C), 132.7, 137.9, 155.4, 163.2, 169.7. MS m/z (%): 402.12 [M++1] (21.73), 401.08 [M+] (55.91), 268.06 (93.22), 238.05 (33.28), 174.08 (62.85), 163.02 (100.00), 150.03 (80.59), 122.03 (24.12), 119.07 (43.32), 118.07 (30.38), 106.07 (20.63), 105.06 (27.95), 104.05 (49.57), 91.07 (23.05), 90.05 (23.03), 77.06 (37.98), 76.04 (59.92), 75.02 (23.57), 70.07 (97.52), 65.04 (28.20), 63.04 (23.22), 50.03 (18.47), 43.08 (36.99), 42.06 (72.85), 41.05 (38.04). Anal. Calcd for C18H19N5O2S2 (401.51): C, 53.85; H, 4.77; N, 17.44; S, 15.97. Found: C, 53.73; H, 4.58; N, 17.38; S, 15.73%.
2-(1H-Benzo[d]imidazol-2(3H)-ylidene)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)aceto- nitrile (33)
Brown crystals, Yield, 82%; mp >360 oC (dioxane). IR (KBr, cm-1): vmax 3190, 3200 (2NH), 3018 (CH aromatic), 2986, 2915 (CH aliphatic), 2218 (C≡N), 1583 (C=N), 1555 (C=C), 1349, 1182 (SO2). 1H NMR (DMSO-d6): δ 1.92 (m, 4H, CH2-CH2 of pyrrolidine), 3.39 (t, 4H, J=11.1 Hz, CH2-N-CH2 of pyrrolidine), 6.65-8.01 (m, 8H, Ar-H), 7.55 (s, 1H, H5 of thiazole), 9.58, 11.01 (2br, 2H, 2NH, Discharged with D2O). MS m/z (%): 449.43 [M+] (80.75), 402.90 (38.86), 304.02 (11.29), 301.99 (21.50), 217.02 (12.37), 211.02 (16.09), 187.90 (22.28), 185.04 (5.04), 173.04 (9.58), 168.01 (10.70), 151.48 (100.00), 144.04 (5.61), 142.00 (42.90), 138.04 (13.20), 127.06 (10.12), 122.02 (5.18), 120.95 (84.98), 105.05 (19.15), 85.10 (6.96), 75.04 (9.03), 73.11 (27.60), 60.03 (18.77), 44.05 (23.54), 43.06 (87.76), 40.21 (41.74). Anal. Calcd for C22H19N5O2S2 (449.55): C, 58.78; H, 4.26; N, 15.58; S, 14.27. Found: C, 58.65; H, 4.08; N, 15.46; S, 14.13%.
2-Cyano-N-phenyl-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)ethanethioamide (35)
To a cooled suspension of finely grounded potassium hydroxide (0.56 g, 0.01 mol) in dry N,N-dimethylformamide (40 mL), thiazolylacetonitrile 2 (3.33 g, 0.01 mol) and subsequently isothiocyanatobenzene (1.35 g, 0.01 mol) were added. The reaction mixture was stirred overnight at room temperature, and left at room temperature for an additional 24 h. The reaction mixture was then triturated with cold water (50 mL) and neutralized with 1N hydrogen chloride. The resulting precipitated solid was collected by filtration, washed with water, dried and recrystallized from EtOH to give 35. White crystals, Yield, 86%; mp 256-258 oC. IR (KBr, cm-1): vmax 3190 (NH), 3074 (CH aromatic), 2961, 2957 (CH aliphatic), 2217 (C≡N), 1581 (C=N), 1550 (C=C), 1333, 1161 (SO2), 1343 (C=S). 1H NMR (DMSO-d6): δ 1.87 (m, 4H, CH2-CH2 of pyrrolidine), 3.30 (t, 4H, J=9.9 Hz, CH2-N-CH2 of pyrrolidine), 5.10 (s, 1H, CH), 7.11-7.90 (m, 10H, Ar-H + H5 of thiazole) 10.26 (s, 1H, NH, Discharged with D2O). MS m/z (%): 468.07 [M+] (70.11), 216.02 (12.35), 200.03 (23.79), 199.02 (29.16), 135.00 (49.09), 134.03 (7.25), 133.01 (7.61), 94.06 (8.08), 93.04 (95.53), 92.06 (10.74), 91.05 (6.75), 89.03 (38.27), 78.03 (10.40), 77.03 (59.05), 75.95 (80.27), 70.05 (100.00), 66.04 (7.55), 65.02 (8.79), 63.02 (12.21), 51.02 (18.12), 50.01 (7.31), 44.02 (6.61), 43.06 (8.44), 42.03 (20.29), 41.04 (6.65). Anal. Calcd for C22H20N4O2S3 (468.61): C, 56.39; H, 4.30; N, 11.96; S, 20.53. Found: C, 56.24; H, 4.18; N, 11.75; S, 20.48%.
General Procedure for Preparation of (36-39)
To a cooled suspension of finely grounded potassium hydroxide (0.56 g, 0.01 mol) in dry N,N-dimethylformamide (40 mL), thiazolylacetonitrile 2 (3.33 g, 0.01 mol) and subsequently isothiocyanatobenzene (1.35 g, 0.01 mol) were added. The reaction mixture was stirred overnight at room temperature, then treated with the appropriate halo compounds namely (iodomethane, ethyl 2-chloroacetate, ethyl 2-bromopropanoate and ethyl 3-bromobutanoate) (0.01 mol), and left at room temperature for an additional 24 h. The reaction mixture was then triturated with cold water (50 mL) and neutralized with 1N hydrogen chloride. The resulting precipitated solid was collected by filtration, washed with water, dried and recrystallized from an appropriate solvent to give compounds 36-39, respectively.
3-(Methylthio)-3-(phenylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (36)
Yellow crystals, Yield, 88%; mp 269-270 oC (EtOH). IR (KBr, cm-1): vmax 3230 (NH), 3099 (CH aromatic), 2908 (CH aliphatic), 2222 (C≡N), 1611 (C=N), 1576 (C=C), 1335, 1177 (SO2), 1160 (C-S). 1H NMR (DMSO-d6): δ 1.87 (m, 4H, CH2-CH2 of pyrrolidine), 2.72 (s, 3H, SCH3), 3.29 (t, 4H, J=10.3 Hz, CH2-N-CH2 of pyrrolidine), 6.85-7.11 (m, 5H, Ar-H), 7.32 (s, 1H, H5 of thiazole), 7.83, 8.10 (dd, 4H, “each d, each 2H, J=8.7 Hz”, AB system), 9.35 (s, 1H, NH, Discharged with D2O). 13C NMR (DMSO-d6): δ 16.3 (SCH3), 22.8 (2C, CH2-CH2 of pyrrolidine), 60.6 (2C, CH2-N-CH2 of pyrrolidine), 82.5, 111.3 (C5 of thiazole), 115.9 (C≡N), 120.8, 123.2 (2C), 126.2 (2C), 131.6 (2C), 135.8 (2C), 137.3, 140.2, 144.7, 156.4, 166.9, 171.2. MS m/z (%): 482.13 [M+] (72.36), 435.13 (13.59), 301.09 (19.52), 198.04 (15.25), 197.02 (11.27), 134.04 (38.84), 133.02 (14.34), 105.04 (24.23), 102.05 (25.43), 91.05 (12.94), 90.07 (15.07), 89.04 (57.38), 77.03 (100.00), 76.03 (11.76), 70.07 (87.14), 69.03 (18.67), 63.03 (21.60), 51.02 (40.51), 48.00 (14.68), 47.00 (17.01), 44.99 (24.58), 44.02 (23.76), 43.06 (41.68), 42.04 (74.95), 41.06 (44.45). Anal. Calcd for C23H22N4O2S3 (482.64): C, 57.24; H, 4.59; N, 11.61; S, 19.93. Found: C, 57.18; H, 4.48 N, 11.52; S, 20.04%.
Ethyl 2-((2-cyano-1-(phenylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)vinyl)thio)- acetate (37)
White solids, Yield, 79%; mp 257-258 oC (EtOH/benzene). IR (KBr, cm-1): vmax 3220 (NH), 3085 (CH aromatic), 2985, 2964 (CH aliphatic), 2220 (C≡N), 1697 (C=O), 1630 (C=N), 1593 (C=C), 1354, 1161 (SO2), 1288, 1055 (C-O-C), 1182 (C-S). 1H NMR (DMSO-d6): δ 1.31 (t, 3H, J=7.3 Hz, CH3-CH2), 1.91 (m, 4H, CH2-CH2 of pyrrolidine), 3.28 (t, 4H, J=10.7 Hz, CH2-N-CH2 of pyrrolidine), 3.79 (s, 2H, CH2), 4.35 (q, 2H, J=7.3 Hz, CH3-CH2), 6.40-7.22 (m, 5H, Ar-H), 7.31 (s, 1H, H5 of thiazole), 7.80, 8.42 (dd, 4H, “each d, each 2H, J=8.6 Hz”, AB system), 10.99 (s, 1H, NH, Discharged with D2O). 13C NMR (DMSO-d6): δ 18.3 (CH3-CH2), 23.6 (2C, CH2-CH2 of pyrrolidine), 40.2 (CH2), 59.3 (CH3-CH2), 64.5 (2C, CH2-N-CH2 of pyrrolidine), 81.4, 110.2 (C5 of thiazole), 121.0 (C≡N), 123.6, 125.9 (2C), 127.4 (2C), 130.7 (2C), 135.8 (2C), 137.9, 140.8, 143.6, 156.9, 167.3, 169.0, 173.2 (C=O). MS m/z (%): 554.16 [M+] (62.08), 487.11 (20.54), 470.11 (42.31), 193.06 (23.08), 134.03 (30.80), 133.04 (19.00), 102.06 (14.45), 101.07 (14.39), 90.08 (14.50), 89.05 (77.46), 84.07 (25.66), 82.06 (13.60), 77.05 (40.77), 70.07 (90.14), 69.06 (24.43), 68.07 (21.26), 63.04 (23.05), 56.06 (18.79), 55.06 (30.75), 54.05 (17.73), 53.07 (14.17), 51.04 (22.39), 44.02 (63.52), 43.06 (49.48), 42.06 (100.00), 41.06 (70.56). Anal. Calcd for C26H26N4O4S3 (554.70): C, 56.30; H, 4.72; N, 10.10; S, 17.34. Found: C, 56.27; H, 4.69; N, 10.31; S, 17.10%.
Ethyl 2-((2-cyano-1-(phenylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)vinyl)thio)- propanoate (38)
White crystals, Yield, 73%; mp 244-246 oC (EtOH/benzene). IR (KBr, cm-1): vmax 3190 (NH), 3074 (CH aromatic), 2983, 2953 (CH aliphatic), 2217 (C≡N), 1710 (C=O), 1633 (C=N), 1588 (C=C), 1340, 1155 (SO2), 1206, 1021 (C-O-C), 1178 (C-S). 1H NMR (DMSO-d6): δ 1.33 (t, 3H, J=7.2 Hz, CH3-CH2), 1.72 (d, 3H, J=7.9 Hz, CH3-CH), 1.94 (m, 4H, CH2-CH2 of pyrrolidine), 3.31 (t, 4H, J=10.1 Hz, CH2-N-CH2 of pyrrolidine), 3.70 (q, 2H, J=7.2 Hz, CH3-CH2), 4.36 (q, 1H, CH), 6.75-7.31 (m, 5H, Ar-H), 7.45 (s, 1H, H5 of thiazole), 7.84, 8.11 (dd, 4H, “each d, each 2H, J=8.5 Hz”, AB system), 11.62 (s, 1H, NH, Discharged with D2O). MS m/z (%): 567.99 [M+] (81.33), 389.22 (5.73), 365.18 (8.23), 358.37 (6.74), 346.10 (8.72), 341.16 (5.59), 332.39 (6.12), 317.50 (10.41), 305.11 (7.93), 291.56 (9.30), 271.11 (14.20), 269.14 (6.57), 249.13 (9.34), 246.11 (6.96), 244.51 (7.34), 309.24 (100.00), 229.11 (24.15), 224.12 (6.58), 192.11 (11.39), 168.11 (12.54), 162.10 (12.44), 136.09 (7.98), 123.11 (14.38), 94.10 (14.56), 75.06 (18.76), 64.03 (8.80). Anal. Calcd for C27H28N4O4S3 (568.73): C, 57.02; H, 4.96; N, 9.85; S, 16.91. Found: C, 56.89; H, 4.80; N, 9.72; S, 16.78%.
Ethyl 3-((2-cyano-1-(phenylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)vinyl)thio)- butanoate (39)
White solids, Yield, 62%; mp 235-237 oC (EtOH/benzene). IR (KBr, cm-1): vmax 3220 (NH), 3072 (CH aromatic), 2961, 2945 (CH aliphatic), 2220 (C≡N), 1707 (C=O), 1627 (C=N), 1581 (C=C), 1358, 1170 (SO2), 1287, 1053 (C-O-C), 1199 (C-S). 1H NMR (DMSO-d6): δ 1.27-1.38 (m, 6H, 2CH3), 1.99 (m, 4H, CH2-CH2 of pyrrolidine), 2.64-2.99 (m, 2H, CH2CO), 3.27 (t, 4H, J=11.7 Hz, CH2-N-CH2 of pyrrolidine), 3.52-3.83 (m, 1H, CH), 4.61 (q, 2H, J=8.8 Hz, CH2), 6.87-7.27 (m, 5H, Ar-H), 7.69 (s, 1H, H5 of thiazole), 7.85, 8.11 (dd, 4H, “each d, each 2H, J=8.5 Hz”, AB system), 10.32 (s, 1H, NH, Discharged with D2O). MS m/z (%): 581.61 [M+] (41.99), 537.39 (16.76), 534.90 (12.13), 503.27 (10.36), 475.34 (12.59), 465.26 (18.18), 436.37 (28.08), 402.46 (66.80), 369.09 (34.48), 359.28 (17.04), 354.38 (25.55), 346.27 (16.26), 330.30 (100.00), 296.49 (25.68), 282.13 (28.31), 279.23 (14.65), 200.05 (15.28), 175.06 (12.65), 174.07 (11.57), 85.07 (21.90), 81.02 (10.89), 79.98 (25.53). Anal. Calcd for C28H30N4O4S3 (582.76): C, 57.71; H, 5.19; N, 9.61; S, 16.51. Found: C, 57.65; H, 5.05; N, 9.47; S, 16.36%.
3,3-Bis(phenylamino)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)acrylonitrile (40)
A mixture of compound 36 (4.82 g, 0.01 mol) and aniline (0.93 g, 0.01 mol) in EtOH (50 mL) was refluxed for 3 h, after cooling. The solid product which formed was collected by filtration and recrystallized from EtOH/benzene to give 40. White crystals, Yield, 83%; mp 292-294 oC. IR (KBr, cm-1): vmax 3215, 3201 (2NH), 3039 (CH aromatic), 2972 (CH aliphatic), 2222 (C≡N), 1575 (C=N), 1553 (C=C), 1335, 1165 (SO2). 1H NMR (DMSO-d6): δ 1.88 (m, 4H, CH2-CH2 of pyrrolidine), 3.31 (t, 4H, J=10.4 Hz, CH2-N-CH2 of pyrrolidine), 6.42-7.26 (m, 10H, Ar-H), 7.52 (s, 1H, H5 of thiazole), 7.82, 8.15 (dd, 4H, “each d, each 2H, J=8.7 Hz”, AB system), 10.36, 11.27 (2br, 2H, 2NH, Discharged with D2O). 13C NMR (DMSO-d6): δ 22.3 (2C, CH2-CH2 of pyrrolidine), 46.5, 60.9 (2C, CH2-N-CH2 of pyrrolidine), 108.7 (C5 of thiazole), 117.8 (C≡N), 119.5 (2C), 121.4 (2C), 123.7 (2C), 125.3 (2C), 129.2 (2C), 132.6 (2C), 136.7 (2C), 140.3, 144.8, 150.0 (2C), 157.4, 168.9, 172.1. MS m/z (%): 527.29 [M+] (75.09), 392.18 (20.34), 288.34 (34.01), 189.17 (100.00), 164.13 (45.32), 147.11 (38.24), 112.14 (42.31), 93.06 (55.26), 83.08 (61.06), 71.08 (42.37). Anal. Calcd for C28H25N5O2S2 (527.66): C, 63.73; H, 4.78; N, 13.27; S, 12.15. Found: C, 63.54; H, 4.65; N, 13.10; S, 12.03%.
2-(4-Oxo-3-phenylthiazolidin-2-ylidene)-2-(4-(4-(pyrrolidin-1-ylsulfonyl)phenyl)thiazol-2-yl)aceto- nitrile (41)
To a mixture of EtOH (30 mL) and 1 mL of piperidine, 5.54 g (0.01 mol) of compound 37 was added and refluxed for 3 h. The precipitate obtained after cooling was filtered off and recrystallized from dioxane to give 41. White crystals, Yield, 72%; mp 303-305 oC. IR (KBr, cm-1): vmax 3103, 3085 (CH aromatic), 2990, 2937, 2879 (CH aliphatic), 2220 (C≡N), 1669 (C=O), 1635 (C=N), 1598 (C=C), 1339, 1159 (SO2). 1H NMR (DMSO-d6): δ 1.92 (m, 4H, CH2-CH2 of pyrrolidine), 3.33 (t, 4H, J=11.9 Hz, CH2-N-CH2 of pyrrolidine), 4.01 (s, 2H, CH2 of thiazolidinone), 6.99-7.33 (m, 5H, Ar-H), 7.56 (s, 1H, H5 of thiazole), 7.88, 8.52 (dd, 4H, “each d, each 2H, J=8.3 Hz”, AB system). 13C NMR (DMSO-d6): δ 22.9 (2C, CH2-CH2 of pyrrolidine), 41.4 (CH2 of thiazolidinone), 62.9 (2C, CH2-N-CH2 of pyrrolidine), 82.5, 108.8 (C5 of thiazole), 120.3 (C≡N), 124.5 (2C), 129.0 (2C), 133.2, 135.9 (2C), 139.2 (2C), 140.0, 142.5, 147.3, 156.9, 167.2, 169.5, 173.8 (C=O). MS m/z (%): 509.14 [M++1] (31.70), 508.12 [M+] (71.80), 347.12 (17.88), 268.11 (32.93), 174.11 (21.63), 163.08 (42.80), 134.06 (38.28), 119.10 (36.98), 105.09 (19.76), 104.08 (29.84), 91.10 (22.77), 90.10 (17.54), 89.08 (37.69), 77.09 (65.64), 76.08 (31.72), 71.11 (14.43), 70.10 (100.00), 69.10 (24.08), 63.08 (15.03), 55.09 (20.13), 51.07 (21.91), 45.07 (18.09), 44.07 (59.63), 43.11 (48.89), 42.09 (64.15), 41.08 (51.81). Anal. Calcd for C24H20N4O3S3 (508.64): C, 56.67; H, 3.96; N, 11.02; S, 18.91. Found: C, 56.54; H, 3.83; N, 11.14; S, 19.07%.
Docking and molecular modeling calculations
Docking and molecular modeling calculations were carried out in the department of pharmaceutical chemistry, Faculty of pharmacy, Alexandria University.
Materials
All the molecular studies were carried out on an Intel Pentium 1.6 GHz processor, 512 MB memory with windows XP operating system using Molecular Operating Environment (MOE 2005.06; Chemical Computing Group, Montreal, Canada) as the computational software. All the minimizations were performed with MOE until a RMSD gradient of 0.05 K Cal/mol Å with MMFF94X force field and the partial charges were automatically calculated.
General methodology
The coordinates of the X-ray crystal structure of methotrexate (MTX) bound to dihydrofolate reductase (DHFR) enzyme (PDB ID: 4DFR) were obtained from Protein Data Bank (PDB ID: 1BID). Enzyme structures were checked for missing atoms, bonds and contacts. Hydrogen atoms were added to the enzyme structure. Water molecules and bound ligands were manually deleted. The ligand molecules were constructed using the builder molecule and were energy minimized. The active site was generated using the MOE-Alpha site finder. Dummy atoms were created from the obtained alpha spheres. Ligands were docked within the dihydrofolate reductase active sites using the MOE-Dock with simulated annealing used as the search protocol and MMFF94X molecular mechanics force field for 8000 interactions. The lowest energy conformation was selected and subjected to an energy minimization using MMFF94X force field.
Docking on the active site of dihydrofolate reductase (DHFR)
The recent determination of the three dimensional co-crystal structure of dihydrofolate reductase complexed with the potent inhibitor, methotrexate (MTX) (PDB ID: 4DFR) has led to the development of a model for the topography of the binding site of dihydrofolate reductase.
In vitro anticancer screening
The cytotoxicity activity was measured in vitro for the newly synthesized compounds using the Sulfo-Rhodamine-B stain (SRB) assay.42 The in vitro anticancer screening was done by The Regional Center for Mycology and Biotechnology RCMB, Al-Azhar University, Cairo, Egypt. Cells were plated in 96-multiwell micro titer plate (104 cells/well) for 24 h. before treatment with the compound(s) to allow attachment of cell to the wall of the plate. Test compounds were dissolved in DMSO and diluted with saline to the appropriate volume. Different concentrations of the compound under test (0, 1, 2.5, 5, and 10 μg/mL) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the compound(s) for 48 h at 37 oC and in atmosphere of 5% CO2 after 48 h, cells were fixed, washed, and stained for 30 min. with 0.4% (wt/vol) with SRB dissolved in 1% acetic acid. Excess unbound dye was removed by four washes with 1% acetic acid and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve for breast tumor cell after the specified time.42 The molar concentration required for 50% inhibition of cell viability (IC50) was calculated and the results are given in Table 1.

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