INTRODUCTION:

Tuberculosis (TB), a contagious, airborne communicable disease is responsible for the highest number of deaths among all infectious disease¹ .Every year two million people die of tuberculosis, caused by Mycobacterium tuberculosis. Roughly one third of the world’s population is infected and more and more bacterial strains have developed resistance to drugs.

Further the association of tuberculosis and HIV infection is so dramatic that, in some cases, nearly two-thirds of the patients diagnosed with TB are also HIV-1 seropositive². 4. However this problem has become serious as M. tuberculosis developed resistance against both the firstline and also the second line of drugs³.The increasing incidence of MDR and XDR tuberculosis worldwide highlight the urgent need to search for newer anti tuberculosis drugs . Such factors forced the scientists across the globe to search for newer molecules that can be used as lead compounds for the development of newer antitubercular drugs with better therapeutic effects.

Benzothiazole nucleus is a constituent of many bioactive heterocyclic compounds that are ofwide interest because of their diverse biological and clinical applications. The substituted benzothiazole skeleton is a frequently encountered heterocycle in medicinal chemistry literature with applications including antimicrobial, antisecretory, anticancer, antiHIV, antihypertensive,antitumor, anthelmintic, antidiabetic, antioxidant, antifungal, analgesic and antiinflammatory and anti protozoal activities^4,5,6.. Similarly pyrimidine and their derivatives posses anti bacterial, antitubercular and anti fungal activities^7,8. Hence in the present work we have decided to explore the therapeutic advantages of pyrimidines moiety in combination with benzothiazole.

The present study was undertaken to investigate the opportunities by which the enzyme dihydrofolate reductase[DHFR]^9,10,11 a key enzyme in the folate cycle, a promising drug target for treatment of mycobacterial infections offers development of new TBdrugs. Inhibition of the folate cycle leads to interruption of supply of thymidine and thus to inhibit DNA biosynthesis and cell proliferation. These observations at our in-house antitubercular programme gave the impetus to synthesize new analogues of benzothiazole derivatives, docked by using Glide¹² against dihydrofolate reductase enzyme(DHFR) (PDB ID 1DG7) for the better understanding of specific interactions of the inhibitors with DHFR.

MATERIALS AND METHODS:

All the chemicals were obtained from S.D. Fine Chem. and Loba Cheme and the substituted aldehydes from Himedia. The scheme of the synthesis is presented in Scheme. The structure of the newly synthesized compounds was established by various analytical techniques such as IR, 1H NMR and MASS spectral studies. Melting points were determined in open capillary tubes and are uncorrected. Purity of the compounds was checked by pre-coated TLC plate-GF254 (Merck). The IR spectra of the compounds were recorded ABB BOMEM FTIR, 1H NMR spectra were recorded on a Bruker Avance 500 MHz instrument using TMS as internal standard; the chemical shifts (d) are reported in ppm and coupling constants (J) are given in Hertz and the Mass spectra of the compounds were recorded on GCMS QP 5000 Shimadzu at Indian Institute of Technology, Chennai. Elemental analysis was performed on a Heracus CHN-Rapid Analyser.

Chemical Synthesis

STEP I

Preparation of 2-amino benzothiazole (I) :^13-15

Aromatic amines-aniline (0.01mol),potassium thiocyanate (0.01 mol) in glacial acetic acid was are taken in a beaker .To this bromine(0.01 mol) is added drop by drop and stirred well by maintaining temperature below 10⁰c until it dissolves. Then it is refluxed for about 2-3 hrs. It was dissolved in hot water and neutralized with aqueous ammonia (25%)Then resulting solid was separated and recrystalised using ethanol. The purity of product was established by single spot on T.L.C. plate .Solvent system used was Ethanol: Benzene (4:6). The percentage of yield was found to be 85%w/w

STEP II: Preparation of 4-[2-(1,3- benzothiazol-2ylamino)ethyl phenol(II):^16-18

2- Amino benzothiazole (1.5 gm, 0.02mol) and 4 ml of dry benzene were placed in a 100 ml round bottomed flask fitted with water condenser .To this p- hydroxyl phenyl ethyl chloride (0.02 mol) was added drop wise .The reaction mixture was refluxed on water bath at 80⁰ c for 4 hrs , after completion of reaction , excess of p- hydroxy phenyl ethyl chloride was removed by distillation under reduced pressure. The resulting solid residue was washed with aqueous solution of sodium bicarbonate (50ml) followed by ice cold water. The resulting yellow colour product obtained was recrystallised using ethanol. The purity of product was established by single spot on T.L.C. plate .Solvent system used was Ethanol: Ethyl acetate (4:6). The percentage of yield was found to be 80%w/w.

STEP III: Preparation of N-(2-{4-[2-(1,3-benzothiazol-2-ylamino)ethyl]phenoxy}pyrimidin-4-yl)acetamide

(III)^19-21:

A mixture of compound 2- chloro 4- amino pyrimidine (0.01 mol) and acetic anhydride (5 ml) was heated under reﬂux for 4 h. The reaction mixture was cooled to room temperature. The formed solid N-(2-chloropyrimidin-4-yl)acetamide was ﬁltered, dried and crystallized from ethanol¹⁹.

N-(2-chloropyrimidin-4-yl)acetamide (0.01 mol) and 4-[2-(1,3- benzimidazol-2ylamino)ethyl phenol(0.0mol) and to this a solution of sodium methoxide (0.001ml), absolute methanol(10ml) was added .Immediately sodium chloride separated. The reaction mixture was warmed on a water bath for 30 mts, cooled and diluted with water. The compound N-(2-{4-[2-(1,3-benzimidazol-2-ylamino)ethyl] phenoxy}pyrimidin-4-yl)acetamide was filtered and recrystalised from methanol. The purity of product was established by single spot on T.L.C. plate .Solvent system used was Ethyl acetate: Ethanol (6:4) . The percentage of yield was found to be 65 %w/w.

STEP IV: Preparation of N-(2-{4-[(4-aminopyrimidin-2-yl)oxy]phenyl}ethyl)-1,3-benzothiazol-2-amine(IV)²²:

Dissolve N-(2-{4-[2-(1,3-benzothiazol-2-ylamino)ethyl] phenoxy}pyrimidin-4-yl)acetamide in 30 ml of boiling ethanol in a 500 ml round bottomed flask equipped with a reflux condenser .Add 12 ml of con hydrochloric acid down the condenser in small portions to the boiling solution .Reflux for 30 -40 mts and pour the mixture into 100 ml of ice water and add with vigourous stirring ,5%sodium hydroxide solution until it is alkaline .The compound

N-(2-{4-[(4-aminopyrimidin-2-yl)oxy]phenyl}ethyl)-1,3-benzthiazol-2-amine crystallises out which is filtered off .

STEP V: Preparation of N-{2-[4-({4-[(e)-ethylideneamino]pyrimidin-2-yl}oxy)phenyl]ethyl}-1,3-benzothiazol-2-amine (BTZ₁ )^23,24

The above obtained compound N-(2-{4-[(4-aminopyrimidin-2-yl)oxy]phenyl}ethyl)-1,3-benzothiazol-2-amine was dissolved in ethanol (95%,50ml) and different aromatic substituted aldehydes (0.01mol) was added to it. Add 2-3 drops of glacial acetic acid and the contents was refluxed on waterbath for 3-4 hrs. Alcohol was distilled and poured into ice cold water untill schiff bases N-{2-[4-({4-[(e)-ethylideneamino]pyrimidin-2-yl}oxy)phenyl]ethyl}-1,3-benzothiazol-2-amine commences to crystallized out. The crude product was recrystallised using ethanol. The purity of product was established by single spot on T.L.C. plate .Solvent system used was Ethyl acetate: Ethanol: chloroform (4:3:3). The percentage of yield was found to be 70-80 %w/w.

The compounds of this series (BTZ_2-13) are prepared in same manner with different substituted aldehydes.

The 2-aminosubsituted benzothiazoles were reacted with p- hydroxyl phenyl ethyl chloride to form corresponding 4-[2-benzothiazolo-2ylamino)ethyl phenol derivatives (a). Meanwhile, the amino group of 2- chloro 4- amino pyrimidine was protected by acetylationand made to react with above obtained derivatives(a) to form compounds like N-(2-{4-[2-(1,3-benzothiazolo-2-ylamino)ethyl]phenoxy} pyrimidin-4-yl)acetamideNow the acetylated amino group of pyrimidine was made free by hydrolysisreaction The free amino group of pyrimidine in above obtained compounds have been treated with different aromatic substituted aldehydes to form corresponding Schiff^`s base derivatives consisting of pyrimidines and benzothiazoles .

COMPOUND BTZ₁: Yield 83 %, mp 280 °C; IR (ν_max, cm^-1): NH str (3058cm^-1); Ar.CHstr (2971 cm^-1); Aliph.CH Str (2926 cm^-1); C=N (1682 cm^-1); C-C(1542 cm^-1); C=C(1445 cm^-1); C-N(1334 cm^-1); C-O-C(1293 cm^-1); C-S(1253 cm^-1); Ar CH bending (768 cm^-1); Alip.CH bending (692 cm^-1) ¹H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 3.39(CH2,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.5 – 8.12(BTZCH,4H); 7.37.6(SBArCH,5H); 7.9 (pyrimidinylCH, 2H); 8.1(N=CH,1H):MS(%):451.14(72%); 149.51(100%); Anal. Calcd for C₂₆H₂₁N₅OS (451.2): C, 69.16; H, 4.69; N,15.51%. Found: C, 69.04; H, 4.52; N,15.62%.

COMPOUND BTZ₂: Yield 80 %, mp 275 °C; IR (ν_max, cm^-1):NH str (3313cm^-1); Ar.CHstr (3092cm^-1); Aliph.CH Str (2923 cm^-1); C=N (1684cm^-1); C-C(1589cm^-1); C=C(1546 cm^-1); C-N(1469 cm^-1); C-O-C(1299 cm^-1); C-S(1262 cm^-1); Ar CH bending(690 cm^-1); Alip.CH bending(633 cm^-1); C-Cl str(758 cm^-1): ¹H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.2 – 7.7(BTZ CH,4 H); 7.2 -7.6(SB-Ar CH,4H); 7.9(pyrimidinyl CH,2H); 8.1(N=CH,1H) MS(%):485.10(68%); 149.59(100%); Anal. Calcd for C₂₆H₂₀Cl N₅OS (485.9): C, 64.26; H,4.15; N,14.41 %. Found: , 64.34; H,4.05; N,14.24 %.

COMPOUND BTZ₃: Yield 81 %, mp 284 °C; IR (ν_max, cm^-1): NH str (3305cm^-1); Ar.CHstr (3070cm^-1); Aliph.CH Str (2829 cm^-1); C=N (1654cm^-1); C-C(1593cm^-1); C=C(1565cm^-1); C-N(1447 cm^-1); C-O-C(1268 cm^-1); C-S(1254 cm^-1); Ar CH bending(710 cm^-1); Alip.CH bending(645 cm^-1); C-Cl str(753 cm^-1):¹H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.2– 7.4(BTZ CH,4 H); 7.3-7.6(SB-Ar CH,4H); 7.9 (pyrimidinyl CH,2H); 8.1 (N=CH,1H): MS(%):485.10(68%); 149.59(100%); Anal. Calcd for C₂₆H₂₀Cl N₅OS(485.9): C, 64.26; H,4.15; N,14.41% Found: C, 64.12; H,4.14; N,14.22%

COMPOUND BTZ₄: Yield 68 %, mp 269 °C; IR (ν_max, cm^-1): OH Str (3384cm^-1)NH str (3067cm^-1); Ar.CHstr (2992 cm^-1); Aliph.CH Str (2958 cm^-1); C=N (1695 cm^-1); C-C(1535 cm^-1); C=C(1449 cm^-1); C-N(1356 cm^-1); C-O-C(1298 cm^-1); C-S(1265cm^-1); Ar CH bending(779 cm^-1); Alip.CH bending(698 cm^-1): ¹H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 7.-7.5(SB-Ar CH,4H); 7.9 (pyrimidinyl CH,2H); 8.1(N=CH,1H); (OH,1H):MS(%):468.12(74):149.59(100%):Anal. Calcd for C₂₆H₂₁N₅O ₂S(469) : C, 64.26; H,4.69; N,15. 51 %. Found: C, 64.43; H,4.54; N, 14.45 %.

COMPOUND BTZ₅: Yield 71 %, mp 275 °C; IR (ν_max, cm^-1): NH str (3063cm); Ar.CHstr (2983 cm^-1); Aliph.CH Str (2954cm^-1); C=N (1697 cm^-1); C-C(1581 cm^-1); C=C(1444 cm^-1); C-N(1317cm^-1); C-O-C(1295cm^-1); C-S(1253cm^-1); Ar CH bending(763 cm^-1); Alip.CH b2.35(CH₃,3H); 2.78(CH₂,2H); ¹H NMR (500.1 MHz, CDCl₃): 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 7.2-7.8(SB-Ar CH,4H); 7.1(pyrimidinyl CH,2H); 8.0(N=CH,1H)bending(694cm^-1)MS(%):481.15(61%); 149.51(100%); Anal. Calcd for C₂₇H₂₃N₅O₂S(465.5) : C, 69.65; H,4.98; N,15.04 %. Found: C, 69.69; H,4.78; N,15.12 %.

COMPOUND BTZ₆: Yield 72 %, mp 270 °C; IR (ν_max, cm^-1): NH str (3028cm^-1); Ar.CHstr (2963 cm^-1); Aliph.CH Str (2946 cm^-1); C=N (1652 cm^-1); C-C(1561 cm^-1); C=C(1445 cm^-1); C-N(1342 cm^-1); C-O-C(1295 cm^-1); C-S(1278 cm^-1); Ar CH bending(788 cm^-1); Alip.CH bending(672 cm^-1) : ¹H NMR (500.1 MHz, CDCl₃): .35(CH₃,3H); 2.78(CH₂,2H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); ); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 7.1-7.5(SB-Ar CH,4H); 7.9(pyrimidinyl CH,2H); 8.1(N=CH,1H): MS(%): 465.16 (58%); 149.55(100); Anal. Calcd for C₂₇H₂₃N₅O₂S(465.5) : C, 69.65; H,4.98; N,15.04 %. Found: C, 69.69; H,4.78; N,15.12 %.

COMPOUND BTZ₇: Yield 74 %, mp 282 °C; IR (ν_max, cm^-1): NH str (3017cm^-1); Ar.CHstr (2973 cm^-1); Aliph.CH Str (2937 cm^-1); C=N (1691 cm^-1); C-C(1542 cm^-1); C=C(1427 cm^-1); C-N(1314 cm^-1); C-O-C(1285 cm^-1); C-S(1234 cm^-1); Ar CH bending(776cm^-1); Alip.CH bending(679 cm^-1) : ¹H NMR (500.1 MHz, CDCl₃): 2.35(CH₃,6H); 2.78(CH₂,2H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 7-7.3(SB-Ar CH,4H); 7.9(pyrimidinyl CH,2H); 8.1 (N=CH,1H)MS(%):479.17(65%); 149.56(100%) Anal. Calcd for C₂₈H₂₅N₅OS : C,70.12; H,5.2; N,14.60 %. Found: C,70.06; H,5.4; N,14.55 %.

COMPOUND BTZ₈: Yield 77 %, mp 295 °C; IR (ν_max, cm^-1): NH str (3041cm); Ar.CHstr (2935cm^-1); Aliph.CH Str (2860 cm^-1); C=N (1621 cm^-1); C-C(1587 cm^-1); C=C(1445 cm^-1); C-N(1391 cm^-1); C-O-C(1291 cm^-1); C-S(1263 cm^-1); Ar CH bending (762cm^-1); Alip.CH bending(622 cm^-1); Ar-NO₂-1552 cm^-11H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 7.9-8.2 (SB-Ar CH,4H); 7.9 (pyrimidinyl CH,2H); 8.1(N=CH,1H) :MS(%):496.13(55%); 149.40(100%); Anal. Calcd for C₂₆H₂₀N₆O₃S: C,62.89; H,4.06; N, 16.93 %. Found: C,62.66; H,4.12; N, 16.75 %.

COMPOUND BTZ₉: Yield 73 %, mp 278 °C; IR (ν_max, cm^-1): NH str (3393cm); Ar.CHstr (2973cm^-1); Aliph.CH Str (2805 cm^-1); C=N (1682 cm^-1); -C(1520cm^-1); C=C(1473 cm^-1); C-N(1354 cm^-1); C-O-C(1267 cm^-1); C-S(1242 cm^-1); Ar CH bending(757cm^-1); Alip.CH bending(692 cm^-1) ¹H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 3.39(CH₂,2H) 4.00; (Ar C-NHandNH₂,3 H); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 6.5-7.4(SB-Ar CH,4H); 7.9(pyrimidinyl CH,2H); 8.1(N=CH,1H) MS(%):466.15(71%); 149.49(100%); Anal. Calcd for C₂₆H₂₂N₆OS (466.5): C, 66.93; H,4.75; N,18.01 %. Found: C, 66.82; H4.77,; N,18.12 %.

COMPOUND BTZ₁₀: Yield 75 %, mp 298 °C; IR (ν_max, cm^-1): NH str (3393cm); Ar.CHstr (2973cm^-1); Aliph.CH Str (2805 cm^-1); C=N (1682 cm^-1); C-C(1520cm^-1); C=C(1473 cm^-1); ter(CH₃)₂-1384cm^-1); C-N(1354 cm^-1); C-O-C(1267 cm^-1); C-S(1242 cm^-1); Ar CH bending(757cm^-1); Alip.CH bending(692 cm^-1) ¹H NMR (500.1 MHz, CDCl₃): 2.78(CH₂,2H); 2.85(N(CH₃)2,6H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); ); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZ CH,4 H); 6.6-7.4(SB-Ar CH,4H); 7.9(pyrimidinyl CH,2H); 8.1(N=CH,1H) MS(%):494.18(66%); 149.49(100%); Anal. Calcd for C₂₈H₂₆N₆O₂S (494.6): C, 67.99; H,5.30; N, 16.99 %. Found: C, 67.88; H 5.34; N,16.87

COMPOUND BTZ₁₁: Yield 79 %, mp 246 °C; IR (ν_max, cm^-1): NH str (3031cm^-1); Ar.CHstr (2956 cm^-1); Aliph.CH Str (2947 cm^-1); C=N (1652 cm^-1); C-C(1549 cm^-1); C=C(1454 cm^-1); C-N(1346 cm^-1); C-O-C(1273 cm^-1); C-O(1216 cm^-1); C-S(1254 cm^-1); Ar CH bending(775 cm^-1); Alip.CH bending(682 cm^-1) ¹H NMR (500.1 MHz,CDCl₃): 2.78(CH₂,2H); 3.73(OCH₃,3H); 3.39(CH₂,2H)4.00; (Ar C-NH,1 H); 6.68-6.95(Ar CH,4H); 7.3 – 7.7(BTZCH,4 H); 6.8-7.5(SB-Ar CH,4H); 7.1(pyrimidinyl CH,2H); 8.1 (N=CH,1H) MS(%):481.15(63%); 149.78(100%); Anal. Calcd for C₂₇H₂₃N₅O₂S (481.5) C,67.34; H,4.81; N,14.54 %. Found: C, 67.12; H, 4.75; N, 14.53 %.

Glide docking studies

Crystal structure of DHFR (PDB code: 1DG7) was used for the study. Structure-based docking studies were carried out using GLIDE version 3.0 .The protein 3D structure was downloaded from the protein databank (PDB) , the solvent molecules in the protein were removed and hydrogen atoms were added to the protein using Cerius² module .

The hydrophobic binding pocket of Mycobacterium DHFR is made up of key residues Ile5, Trp6, Asp27, Gln28, Phe31, Arg32, Arg60, Ile94 and Tyr100. Glide fitness scores were compared with observed activity for all molecules which were found to correlate well with the biological activities It was also observed that hydrogen bond interactions play a major role in deciding the fitness score of the molecule. A better understanding of the interactions is obtained by viewing the molecules in the active site. The most energetically favorable conformation for Molecule BTZ_I in stero view of the DHFR complex is shown in Figure1.

Table1: DHFR inhibition studies.

S.NO	COMPOUND CODE(TestSample)	R	DHFR	NADPH	DHF	O.D.	Status
1.	BTZ₁	H	+	+	+	0.2	Moderate Active
2.	BTZ₂	2-Cl	+	+	+	0.4	Moderate Active
3.	BTZ₃	4-Cl	+	+	+	0.3	Moderate active
4.	BTZ₄	2-OH	+	+	+	0.5	Active
5.	BTZ₅	2-CH3	+	+	+	0.6	Active
6.	BTZ₆	4-CH₃	+	+	+	0.5	Active
7.	BTZ₇	3,4-(CH₃₎₂	+	+	+	0.3	Moderate active
8.	BTZ₈	4-NO₂	+	+	+	0.3	Moderate active
9.	BTZ₉	4-NH2	+	+	+	0.3	Moderate active
10.	BTZ₁₀	4-N-(CH₃)₂	+	+	+	0.5	Active
11.	BTZ₁₁	OCH₃	+	+	+	0.4	Moderate active
12.	Methotrexate (std)		+	+	+	0.7	Active
13.	Blank		+	+	+	0.1	Inactive
14.	DHFR enzyme		+	-	+	0.8	Active

Molecule BTZ₁ shows three H-bond interactions: ASP-19 (ie ) between nitrogen on the side chain amino group of BTZ_I with side chain carbonyl group of ASP-19(CO^…..H-N, 2.102Å) and .Nitrogen in pyrimidine group of BTZ_I with side chain carboxylic group of Leu 24(COOH^…..N, 2.454 Å). The glide docking score of molecule BTZ_I is -5.71.

Enzyme inhibition studies: DHFR inhibitory assay

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme playing a key role in thymidine synthesis. Blockage of the DHFR enzyme causes cell death as a result of DNA synthesis inhibition. For this reason, DHFR is considered an excellent target for designing antitubercular drugs. The assay^25-27 is based on the ability of Dihydrofolate reductase to catalyze the reversible NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid. The reaction progress is monitored by the decrease in absorbance at 340 nm.

Procedure:

All the synthesized substituted benzothiazole compounds were evaluated for DHFR inhibitory activity by Spectrophotometric assay using Dihydrofolate Reductase Assay Kit. The spectrophotometer was set at 340 nm .Assay Buffer 1X was added to the test micro centrifuge tube followed by addition of DHFR enzyme .The synthesized compounds BTZ_(I-XIII)was also added to the appropriate tube, mixed well to which 6 µl of NADPH solution and 5 ml of dihydrofolic acid were added .The contents of the tube were transferred to a 1 ml quartz cuvette , mixed and immediately insert the cuvette into the spectrophotometer. The kinetics program was started immediately. The decrease in optical density (DOD) obtained were recorded as absorbance at 340 nm . Results were tabulated and compared with standard methotrexate drug.

Procedure for antitubercular screening using Alamar blue assay method²⁸:

Mycobacterium tuberculosis H₃₇R_v maintained on L- J medium (Lowenstein –Jensen medium).Stock solutions of newly synthesized compounds were prepared in dimethyl sulfoxide, filtered, sterilized and were added to 450µl of Middle Brook 7 H9 TB broth in 1.5ml sterile microcentrifuge tubes to achieve final concentrations of 500,250,100 µg/ml. Colonies from four week old sub cultures were transferred to the tubes containing 0.85%saline, thoroughly vortex mixed and the suspension was allowed to stand for five minutes. 50µl of the supernatant culture were inoculated into all the tubes containing different concentrations of newly synthesized compounds and standard drugs. The tubes were mixed well and incubated at 37⁰C without shaking. On the seventh day, 25µl of Alamar blue solution was added to the first control tube (C₁). The color changed from blue to pink and therefore the dye was added to all tubes and observed for six hours. Isoniazid and streptomycin was used as standard and Blank (without sample) kept as control.Blue color in the tube indicated sensitivity of M. tuberculosis to the newly synthesized compounds and pink color indicated resistance of M. tuberculosis to them.

Table 2: Screening for antitubercular activity by Alamar blue assay method.

S.NO.	COMPOUND CODE	CONCENTRATION (µg/ml)
S.NO.	COMPOUND CODE	500	250	100
1.	BTZ₁	B	B	B
2.	BTZ₂	B	B	B
3.	BTZ₃	B	B	B
4.	BTZ₄	B	B	B
5.	BTZ₅	B	B	B
6.	BTZ₆	B	B	B
7.	BTZ₇	B	B	B
8.	BTZ₈	B	B	B
9.	BTZ₉	B	B	B
10.	BTZ₁₀	B	B	B
11.	BTZ₁₁	B	B	B
12.	Isoniazid	-	-	-
13.	Streptomycin	-	-	-
14.	Blank	P	P	P
15.	Control	P	P	P

B: Blue (Sensitive):P: Pink (Resistant)

II. Minimum Inhibitory Concentration method:

In-vitro studies: Anti Tb activity was performed as follows: 2,700μl of 7H9 broth supplemented with ADC in 250 tubes was inoculated with 10 μl of bacterial culture (Mycobacterium tuberculosis H₃₇Rv strain) 300 μl of each drug at 5 different concentrations namely 100 μg/mL, 10 μg/mL, 1 μg/mL, 0.1 μg/mL and 0.01 μg/mL in duplicate was added to each tube and incubated for five weeks. The presence of mycobacterial growth is observed depending upon the turbidity, when monitored under light .The results were tabulated and compared with the standard isoniazid drug.

Table 3: MIC Method

S.NO	COMPOUND ID CODE	R	MIC VALUE in (µg/ml)
	BTZ₁	H	1
	BTZ₂	2-Cl	0.1
	BTZ₃	4-Cl	1
	BTZ₄	2-OH	0.01
	BTZ₅	2-CH3	0.01
	BTZ₆	4-CH₃	0.01
	BTZ₇	3,4-(CH₃₎₂	0.1
	BTZ₈	4-NO₂	0.1
	BTZ₉	4-NH2	0.1
	BTZ₁₀	4-N-(CH₃)₂	0.01
	BTZ₁₁	OCH₃	0.1
	ISONIAZID	-	0.01

ACKNOWLEDGEMENTS:

The authors thanks to Indian Institute of Technology, Chennai and Madras Medical College for providing all the facilities to carry out the research work.

REFERENCES:

1. Thompson NP, Caplin ME, Hamilton MI, Gillespie SH,Clarke SW, Burroughs AK, McIntyre N, Anti-tuberculosis medication and the liver: dangers and recommendations in management, Eur Respir J. 8;1995.:1384–88.

2. Lawn SD, Zumla AL, Tuberculosis, Lancet, 378;2011.:57–72.

3. Ormerod LP, Horsfield N ,Short-course antituberculous chemotherapy for pulmonary and pleural disease: 5 years' experience in clinical practice, British Journal of Diseases of the Chest , 81 (3);1987.: 268–71.

4. Oldrich pyleta , Vera Klimesova, Effect of substitution on the antimycobacterial activity of 2-(substituted benzyl) sulfanyl benzimidazoles, benzoxazoles, benzothiazoles-a QSAR study, Chemical Pharmaceutical bulletin, 59(2),2011.:179-184.

5. Sigh .N ,Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused – 2 – amino pyrimidines as antitubercular and antidiabetic agents, Bio organic and Medicinal chemistry Letters,21(15);2011.:4404-08.

6. Gangjee A , Queener SF , Design, synthesis, and biological evaluation of 2,4-diamino-5-methyl-6-substituted-pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors.,J. Med. Chem,47;2004.: 3689-92.

7. A. Gangjee, E.Elzein , and S.F. Queener., J. Med. Chem., 41;1409-16:1998

8. Arpana Rana, Siddiqui N, Khan SA. Benzothiazoles: A new profile of biological activities.2007;49(1):10-17

9. Hawser S, Lociuro S, Islam K, Dihydrofolate reductase inhibitors as antitubercular agent.:2006.

10. El-Hamamsy MH, Smith AW, Thompson AS, Threadgill MD.Structure-based design, synthesis and preliminary evaluation of selective inhibitors of dihydrofolate reductase from, Bioorg. Med. Chem ,15; 2007.: 4552-76.

11. Li R, Sirawaraporn R, Chitnumsub P , Sirawaraporn W, Wooden J, Athappilly F , Turley S,Three-dimensional structure of M. tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs, J. Mol. Biol,295, 2000.: 307-323.

12. Glide, Version 9.1. Schrodinger, L.L.C., New York, 2010.

13. J. Jeng Li, Chem. pharm. Res, 4;317-32:2014.

14. R. Deshmuhh , A.S. Thakur, Int. J. Research in Pharmacy and Chemistry., 1(3),329-33(2011).

15. V.S .Velingkar, and N.S. Kolhe, Indian drugs., 47(9);2010.

16. Shashikant R. Pattan, Synthesis and pharmacological screening of some 1,4 dihydropyridine and their derivatives for anti convulsant activity, Indian J. Chem, sec ,47 B;2008.: 626-629.

17. Francis GE, Denise E. Richard,The mechanism of reaction between Di-(2-chloroethyl) sulphone (mustard gas sulphone) aminoacids, Biochem. J. 1947: 142-14 .

18. Arvind Sharma, Sonia Dhiman,Sandeep arora, Prem Chand ,Abhinav Kapoor ,Synthesis of Colon Specific N,N-Bis –(2- chloroethyl)Ailine Polyphosphazene copolymer conjugates .Int.J.Pharm sci.,2( 4): 63-67.

19. Vogels Practical Organic Chemistry ,Furniss, Hannford, Rogers, Smith, Tatchell:4 th edition. Pg.753.

20. Gopal M, Gurupadayya BM, Vaidya VP,Synthesis and biological activities of fluoro benzthiazoles ,India. J. of Het.Chem;2005.

21. Basavaraja KM, Patil VM, and Agasimundin YS, Synthesis and nucleophilic displacement reactions of biologically active 2,4- dichlorobenzofuro[3,2-d} pyrimidine., Indian,J. Het. Chem,15; 2006.: 313-18.

22. Vogels Practical Organic Chemistry, Furniss, Hannford, Rogers, Smith. Tatchell: 4 th edition,pg 685.

23. Pattan SR, Dighe NS, Nirmal SA, Synthesis and biological evaluation of some substituted amino Thiazole derivatives, Asian J. research Chem,2(2);2009.: 196-201.

24. Salvi V, Shwetha Sharma, Chirag Sharma, Talesara GL, Synthesis of phthalimido or succinimido-(2-aryl-4-oxo-3-[{2-oxo-2(phenothiazine-10)-yl)ethyl}_1,3-thiazolidine-5-yl]ethanoate and their anti microbial activities, Indian. J . Chem; 1583-89.

25. Burchall J, Hitchings G. Inhibitor binding analysis of dihydrofolate reductases from various species. Mol Pharmacol , 1;1965.:126-136.

26. Donkor IO, Li H, Queener SF, Synthesis and DHFR inhibitory activity of a series of 6-substituted-2,4-diaminothieno[2,3-d]pyrimidines, Eur. J. Med. Chem38;2003.:605-11.

27. Brigitte C. Widemann, Frank M. Balis and Peter C. Adamson, Dihydrofolate Reductase Enzyme Inhibition Assay for Plasma Methotrexate Determination Using a 96-Well Microplate Reader , Clinical Chemistry,45(2);1999.: 223-228.

28. Ashtekar DR, Costa-Perira R, Nagrajan K, Vishvanathan N, Bhatt AD, In-vitro and in -vivo activities of the nitroimidazole CGI 17341 against Mycobacterium tuberculosis. Antimicrob. Agents Chemother,37;1963:183-186.

Received on 16.08.2012 Modified on 05.09.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1136-1142