SYNTHESIS AND DOCKING STUDIES OF 2-AMINOTHIAZOLE-5-AROMATIC CARBOXAMIDE DERIVATIVES

SYNTHESIS AND DOCKING STUDIES OF 2-AMINOTHIAZOLE-5-AROMATIC CARBOXAMIDE DERIVATIVES

K. Hemalatha*¹, Nandakishore Agarwal ¹ and G. Buchappa ²

Pharmaceutical Chemistry Department, Nimra College of Pharmacy ¹, Ibrahimpatnam, Vijayawada, Andhra Pradesh

NATCO Research Centre ², Sanathnagar, Hyderabad, Andhra Pradesh, India

ABSTRACT

A  novel  N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-pipear azinyl]-2-methyl-4-pyrimidinyl] amino]-5-thiazolecarboxamide derivatives (6a-6d) and (8a-8b) were synthesized  by coupling 2-[(6-chloro-2-methylpyrimidin-4ylamino]-N-(2-chloro-6-methylphenyl) thiazole-5-Carboxamide(3) with Piperazine derivatives(4,7) in butanol and DIPEA (Diisopropylethylamine) at 120ºC for 4-5hrs. All the synthesized compounds were characterized physically and elucidated by Infrared spectroscopy, Mass spectroscopy, ¹H-NMR spectroscopic techniques.  All the synthesized compounds were subjected to Molecular docking studies by using 2GQG as its PDB ID.  Among   all the compounds, 6a, 6b, 6d, 8c and 8b exhibit anticancer activity.

2-amino-thiazole-5-carboxamide derivatives, Piperazine derivatives, Docking and 2GQG, anti-cancer activity

INTRODUCTION: Molecules containing a thiazole amine-moiety exhibit interesting biological activities depending on the substitution pattern at the thiazole ring ¹. 2-Aminothiazole nucleus is a potential pharmacophore for a broad spectrum of activities, comprising of antibacterial ², anti-fungal ³, antitubercular ⁴, anti-HIV ⁵, pesticidal ⁶, anti-inflammatory ⁷, antiprotozoal ⁸, hypertension, ⁹ schizophrenia ¹⁰ etc.

Aminothiazoles are known to be ligands of estrogen receptors ¹¹ as well as a novel class of adenosine receptor antagonists ¹² whereas other analogues are used as fungicides, inhibiting in vivo growth of Xanthomonas and as an ingredient of herbicides or as schistosomicidal and anthelmintic drugs ¹³. It is reported that 2-aminothiazole-5-aromatic carboxamides are useful as kinase inhibitors of protein tyrosine kinase and p38 kinase, intermediates and crystalline forms thereof ¹⁴.

Docking is a method which predicts the preferred orientation of one molecule to a second whenbound to each other to form a stable complex ¹⁵.

Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using for example scoring functions.

By  considering the above kinase inhibitor activities of 2-aminothiazole-5-aromatic caroboxamides, we have synthesized  some new  2-aminothiazole-5-aromatic carboxamide derivatives containing pyrimidine, piperazine ring extensions and check their  anti-cancer activity through Molecular docking studies  by using 2GQG as their PDB ID  and Dasatinib as a reference.

SCHEME FOR PREPARATION OF 6a-6d AND 8a-8b

8a-8b

TABLE 1: LIST OF SUBSTITUTED GROUPS IN PLACE OF R₁, R, R₂

EXPERIMENTAL:

Chemistry: Melting points were determined on open capillaries using a cintex melting point apparatus. T.L.C analyses were performed on pre-coated silicagel (E-Merck Kieselgel 60F254) plates and visualization was done by exposing to iodine vapour. Solvents were purified by standard procedures before use. IR Spectra were recorded in KBr on Perkin-Elmer Spectrum. 1H-NMR spectrum was recorded in DMSO-d₆ with TMS as internal standard on a Bruker spectrometer at 400 MHz.(chemical shifts in δ ppm). Mass spectra were scanned on a Varian MATCH-7 and Jeol JMSD-300 mass spectrometer at 70ev. All the chemicals used in this work were purchased from Aldrich chemicals. Docking studies were performed   at NATCO Research Centre, Hyderabad.

General procedure for the preparation of compounds:

1. Synthesis of N-(2-chloro-6-methyl phenyl)(6-chloro-2-methylpyrimidine-4-ylamino)- 1,3- thiazole carboxamide(3): To  a  stirring  solution  of 2-Amino-N-(2-chloro-6-methylphenyl)-1,3-thia-zole- 5-carboxamide (1) (5gr, 18.67mmol) and 4,6-dichloro-2 methylpyrimidine (2) (3.65gr, 22.4mmol) was added in Tetrahydrofuran  (65ml)  and  stirred  for  15min.

To this a 30% wt solution of sodium-t-butoxide  in Tetrahydrofuran (21gr,65.36mmol) was added  slowly  with  cooling  to  keep  the temperature  at  10-20ºC. The  mixture  was  stirred  for  1.5hr  and  cooled to 0-5^oC. Then 21.5ml  of  2N HCl  was  added  slowly  and the mixture was collected by vacuum filtration. Then the compound  was  washed  with  water  (15ml)  and  dried to  give  6.63g (86.4%)  of  cream  coloured  solid,  M.P  282-286.5^oC.

2. Synthesis of N-(2-chloro-6-methylphenyl)-2-{[6-(piperazin-1-yl)-2-methylpyrimidin-4-yl] amino} - 1, 3 -thiazole- 5 - carboxamide (7): To N-(2-chloro-6-methylphenyl)(6-chloro-2-methyl pyrimidine-4-ylamino)-1,3- thiazole carboxamide (3) (5gr, 0.01mol) in n-butanol (40ml), piperazine (5) (5.45gr, 0.06mol) was added. Slowly followed by  addition of Diisopropylethylamine (4.42ml, 0.02 mol). The reaction  mixture   was heated at  120^oC for 4-5hr. Then,  the  mixture  was cooled  slowly to room temperature. The  obtained solid  was collected by  vacuum  filtration, washed  with n-butanol and dried to give 8.1gr (81.2%) of cream  coloured solid, M.P 256-260^oC.
3. General procedure for synthesis of compounds (6a-6d): To a mixture of N-(2-chloro-6-methyl phenyl)(6-chloro-2-methylpyrimidine-4-ylamino)- 1,3- thiazole carboxamide (3) (2gr, 0.005mol) in  40ml  of  n-Butanol, 1.766ml (0.01mol) of DIPEA (Diisopyopylethyl amine) was added. To this,  reaction  mixture piperazine  derivative (4) (4gr, 0.025mol) was added. Slowly the slurry was  heated  at  120^oC  for  4-5hr  then  cooled slowly  to  room  temperature. The  obtained  solid  was  collected  by  vacuum  filtration, washed  with  2ml of n-Butanol  and   dried. The  product  obtained  was  dissolved  in   hot  40ml of ethanol: water mixture (80:20) , and  the  solution  was  clarified  by  filtration. The  hot solution  was  slowly  diluted  with  7ml  of  water  and cooled  slowly  to  room temperature. The solid  was  collected  by  vacuum   filtrattion  and   washed   with  2ml   of   50%    ethanol- water   and   dried.
4. Synthesis of N-(2-chloro-methyl-phenyl)-2-(6-(4-methyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole-5-carboxamide (6a): Compound 6a was synthesized by reacting N-(2-chloro-6-methyl phenyl)(6-chloro-2-methylpyrimidine-4-ylamino)- 1,3- thiazole carboxamide(3) with N-methyl piperazine. The compound 6a  obtained  as  white  solid  with  74.5% yield. IR ((KBr cm^-1): 3193.7cm^-1 (NH-streching),  3059.1cm^-1(Ar- CH),  1622cm^-1(C=O,amide streching), 1579cm^-1(Ar- C=C streching), 1292cm^-1(Ar-C-Nstreching,2^o Amine), 765cm^-1(C-Cl streching). Mass Spectra (m/z) M+, found  458.3, Calculated 458. ¹H-NMR (400MHz, DMSO-d₆): 11.479(1H,s)(CONH), 9.879 (1H,s)(NH), 8.220(1H,s)(ArH), 7.2377(3H,m)(ArH), 6.051(1H,s)(ArH), 3.513(4H,t)(2CH₂),   2.407 (3H,s) (CH₃), 2.356-2.368(4H,t)(2CH₂),  2.212-2.239 (6H,s) (2CH₃).
5. Synthesis of N-(2-chloro-methyl-phenyl)-2-(6-(4-ethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole-5-carboxamide (6b): Compound 6b was synthesized by reacting N-(2-chloro-6-methyl phenyl)(6-chloro-2-methylpyrimidine-4-ylamino)- 1,3- thiazole carboxamide(3) with N-ethyl piperazine. IR ((KBr  cm ^-1  ): 3215.7cm^-1 (NH-streching), 2947.1cm^-1(Ar- CH), 1628.8cm^-1(C=O,amide streching), 1577cm^-1(Ar- C=C streching), 1289.3cm^-1(Ar-C-Nstreching,2^o Amine), 766cm^-1(C-Cl streching). Mass Spectra(m/z) M+1, found  473.3 , Calculated 472: ¹H NMR (400MHz, DMSO-d₆): 11.465(1H,s)(CONH), 9.873(1H,s) (NH),   8.215(1H,s)(Ar-H), 7.236-7.407(3H,m) (ArH), 6.052 (1H,s)(ArH), 3.512(4H,t) (2CH₂), 2.406 (7H,m) (CH₃,2CH₂), 2.3332.369 (2H,q) (CH₂),     2.237 (3H,s)(CH₃), 1.013-1.048(3H,t) (CH₃).
6. Synthesis   of     N-(2-chloro-methyl-phenyl) - 2 - (6-(4-phenyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole – 5 -carboxamide (6c): Compound 6c was synthesized by reacting N-(2-chloro-6-methyl phenyl)(6-chloro-2-methyl pyrimidine-4-ylamino)- 1,3- thiazole carboxamide(3) with N-phenyl piperazine.The compound obtained as cream colour solid with 75.31% yield. IR ((KBr cm^-1): 3188cm^-1 (NH-streching), 2846cm^-1(Ar- CH), 1620.2cm^-1(C=O,amide streching),1578.5cm^-1(Ar- C=C streching), 1293.7cm^-1(Ar-C-Nstreching,2^o Amine), 766.7cm^-1(C-Cl streching). Mass Spectra(m/z) M+1, found 520.1, Calculated 519. ¹H NMR (400MHz, DMSO-d₆): 11.525(1H,s)(CONH), 9.889(1H,s)(NH), 8.234(1H,s)(Ar-H), 6.798-7.412(8H,m)(ArH), 6.121 (1H,s)(ArH),  3.688(4H,t) (2CH₂), 3.240(4H,t)(2CH₂), 2.439(3H,s)(CH₃), 2.246 (3H,s)(CH₃).
7. Synthesis of N-(2-chloro-methyl-phenyl)-2-(6-(3,5-dimethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole-5-carboxamide (6d): Compound 6d was synthesized by reacting N-(2-chloro-6-methyl phenyl)(6-chloro-2-methylpyrimidine-4-ylamino)- 1,3- thiazole carboxamide(3) with 2,6-dimethyl piperazine.The compound  obtained  as  cream  coloured  solid  with  95%  yield. IR ((KBr  cm ^-1): 3227cm^-1 (NH-streching),  2862 cm^-1, 2959cm^-1(Ar- CH), 1620.2cm^-1(C=O,amide streching), 1578.5cm^-1(Ar- C=C streching),  1293.7cm^-1(Ar-C-Nstreching, 2^o Amine),  766.7cm^-1(C-Cl streching). Mass Spectra (m/z) M+1, found 473.1, Calculated 472. ¹H NMR (400MHz,DMSO-d₆): 11.369 (1H,s)(CONH), 9.866(1H,s)(NH), 8.212(1H,s) (Ar-H), 7.235-7.407(3H,m)(ArH), 6.047(2H,s)(Ar-H), 4.078 (2H,s)(2CH), 2.29-2.319,2.69(4H,t)(2CH₂), 2.40 (3H,s)(CH₃), 2.237(3H,s)(CH₃),  1.015-1.03 (6H,d) (2CH₃).
8. General procedure for Synthesis of compounds  (8a-8b): To mixture of compound N-(2-chloro-6-methylphenyl)-2-{[6-(piperazin-1-yl)-2-methyl pyrimidin-4-yl] amino}-1, 3-thiazole-5-carboxamide (7) (2gr, 0.005mol) in 40ml of n-butanol, DIPEA (0.52ml, 0.0045mol) was added. To this reaction mixture (0.52ml, 0.0045mol) of alkyl or aryl  chloride  was added. Slowly the slurry was heated at 120^oC for 4-5hr then cooled slowly to room temperature. The obtained solid was collected by vacuum filtration, washed with 2ml of n-Butanol and  dried. The product obtained was dissolved in hot 40ml of ethanol: water mixture (80:20) , and the solution was clarified by filtration. The hot solution was slowly diluted with 7ml of water and cooled slowly to room temperature. The solid was colected by vacuum filtrattion and washed with 2ml of 50% ethanol- water and dried.
9. Synthesis of N-(2-chloro-methyl-phenyl)-2-(6-(4-benzyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole-5-carboxamide (8a): Compound 8a was synthesized by reacting N-(2-chloro-6-methylphenyl)-2-{[6-(piperazin-1-yl)-2-methylpyrimidin-4-yl] amino}-1,3-thiazole-5-carboxamide (7) with benzyl chloride. The compound obtained as cream coloured solid with 83.3% yield.  IR ((KBr cm^-1): 3189cm^-1 (NH-streching), 2813 cm^-1, 2937.8cm^-1(Ar- CH),  1624cm^-1(C=O,amide streching), 1577cm^-1(Ar- C=C streching), 1293.4cm^-1(Ar-C-Nstreching, 2^o Amine), 764.6cm^-1(C-Cl streching). Mass Spectra (m/z) M+, found 534.2, Calculated 534. ¹H-NMR (400MHz,DMSO-d₆): 11.447(1H,s)(CONH), 9.873 (1H,s)(NH), 8.216(1H,s)(ArH), 7.2547.408 (8H,m) (ArH), 6.028(1H,s)(Ar-H), 3.453-3.516(6H,m) (3CH₂), 2.402-2.766(7H, m)(CH₃), 2.238(3H,s) (CH₃).
10. Synthesis of N-(2-chloro-methyl-phenyl)-2-(6-(4-propane-2-yl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole-5-carboxamide (8b): Compound 8b was synthesized by reacting N-(2-chloro-6-methylphenyl)-2-{[6-(piperazin-1-yl)-2-methylpyrimidin-4-yl] amino}-1, 3-thiazole-5-carboxamide (7) with isopropyl chloride. The compound obtained as brick colour solid with 54.65 yield. IR ((KBr cm^-1): 3208.7cm^-1 (NH-streching), 2813 cm^-1, 2937.8cm^-1(Ar- CH), 1624.2cm^-1(C=O,amide streching), 1580.1cm^-1(Ar- C=C streching), 1291.8cm^-1(Ar-C-Nstreching,2º Amine), 761.8cm^-1(C-Cl streching). Mass Spectra (m/z) M+1, found 486.2, Calculated 485: ¹H-NMR (400MHz,DMSO-d₆):  11.503(1H,s)(CONH),  9.905 (1H,s)(NH), 8.249(1H,s)(ArH), 7.2587.410 (3H,m) (ArH), 6.117(1H,s)(ArH), 3.685(2H,t)(CH2), 3.5313.567(2H,t)(CH₂), 3.663.398(1H,m)(CH), 3.099(4H,m)(2CH₂),    2.434(3H,s)(CH₃),   2.242 (3H,s)(CH₃),    1.234-1.251(6H,d)(2C?CCH₃).

Table-2 physical characterization of synthesized compounds (6a-6d) & (8a-8b)

 Docking Studies: The docking studies were carried out for the synthesized compounds to know the activity of the compounds for its anti-cancer effects. The various steps for the docking process is as follows:

Library of Compounds: A chemical library or compound library is a collection of stored chemicals usually used ultimately in high throughput screening. The chemical library can consist in simple terms of a series of stored chemicals. Once the target is identified, it is then is docked with the designed library of compounds the library prepared was

TABLE- 3 DESIGNS AND DOCKING STUDIES OF 2-AMINOTHIAZOLE-5-AROMATIC CARBOXAMIDE DERIVATIVES (6a-6b)

Here, the docking is carried out between the designed set of molecules and the protein 2GQG by using the auto dock software; the various interactions are shown in the results.DOCKING: Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hence docking plays an important role in the rational design of drugs.Given the biological and pharmaceutical significance of molecular docking, considerable efforts have been directed towards improving the methods used to predict docking.

TABLE 4: DESIGNS AND DOCKING STUDIES OF 2-AMINOTHIAZOLE-5-AROMATIC CARBOXAMIDE DERIVATIVES (8a-8b)

Title: X-ray crystal structure of dasatinib (BMS- 354825) bound to activated ABL kinase Domain.Docking Report:

• PDB id: 2GQG.
• Chain : B
• Classification: Transferases.
• Software: AUTODOCK VINA.
• Binding Site Prediction: Grid based approach.

Parameter;

1. Center_X = 45.092
2. Center_Y = 21.198
3. Center_Z = -44.022
4. Size_X = 18
5. Size_Y = 20
6. Size_Z= 18.

The docking was carried out by grid based approach for the 2GQG molecule and the interaction sites and the binding distances from the molecule was found to be at a distance of 45.092 from x-axis, 21.918 from Y-axis and -44.022 from z-axis from the center and at a distance of 18, 20, 18 from the x, y, z axis respectively from the sides.

TABLE 5: THE INTERACTIONS OF THE VARIOUS MOLECULES WITH THE RECEPTOR AND ITS INTERACTION RESIDUES ALONG WITH THE DISTANCES

Therefore, from the above docking studies the interaction with the various residues and the binding of the molecule with its active pharmacological sites can depict a valid action and thus the synthesized molecules can show a good pharmacological action than the reference (Dasatinib).

RESULT & DISCUSSION: The compounds were synthesized by coupling N-(2-chloro-6-methyl phenyl)(6-chloro-2-methylpyrimidine-4-ylamino)- 1,3- thiazole carboxamide  with various piperazine derivatives. All  the  synthesized   compounds  were physically characterized   by  determining their physical state,   M.P,  solubility  and it  is tabulated (table 1) . The structures of   the compounds were established by means of IR, MASS, ¹H-NMR spectra.  The compounds (6a-6d) & (8a-8b) were evaluated for anti-cancer activity by docking studies using Dasatinib as a reference compound, and the results were tabulated   (table 5). Among   all the compounds   6a, 6b, 6d, 8a and 8b can show a good pharmacological action i.e., anti-cancer activity more than the reference drug.

CONCLUSION: The   results   shows (table 5) that compounds 6a, 6b, 6d, 8a and 8b is having considerable anti-cancer activity than reference compound (Dasatinib).

Thus. the present investigation offers the synthesis of newer compounds with anti-cancer activity having 2-aminothiazole as a basic nucleus

 ACKNOWLEDGEMENT: We offer our deep sense of   gratitude towards Dr. K. Durga Prasad, NATCO Research Centre, Hyderabad, for their valuable guidance in completion of this work.

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   How to cite this article:

   Hemalatha K, Agarwal N and Buchappa G: Synthesis and Docking Studies of 2-Aminothiazole-5-Aromatic Carboxamide Derivatives. Int J Pharm Sci Res. 2013; 4(2); 829-835

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