Introduction
Mycobacterium tuberculosis (MTB, M.tuberculosis), the etiological
agent of tuberculosis (TB), is the leading cause of mortality due to
bacterial pathogens, claiming about 2 million lives annually. The field
of anti-tuberculosis drug discovery culminated in the 1960s with the
incorporation of rifampicin and pyrazinamide in the tuberculosis drug
regimen. The use of these two antimicrobials, in combination with
isoniazid, ethambutol and/or streptomycin, represents a landmark in the
treatment of human tuberculosis and resulted in the implementation of
short-course chemotherapy (SCC), reducing the time of treatment from
18 to 6 months [1-3]. Short-course chemotherapy contributed towards
controlling tuberculosis burden for the next 20 years. Nevertheless,
tuberculosis cases started to rise again in the 1990s under the pressure
of the HIV pandemic and the emergence of multidrug-resistant (MDR)
and extremely drug-resistant (XDR) tuberculosis strains. MDR strains are
resistant to at least isoniazid (INH) and rifampicin (RIF), whereas XDR
strains are MDR isolates that are additionally resistant to fluoroquinolones
and to one of the three injectable drugs capreomycin, amikacin and
kanamycin. The emergence and dissemination of MDR and XDR isolates,
estimated to account for more than 400,000 new cases per year, impart
new challenges in tuberculosis control [4]. Indeed, current treatment
of drug-resistant tuberculosis requires 18–36 months and is associated
with an unacceptable rate of treatment failure and relapse. Consequently,
developing new compounds active against MDR and XDR tuberculosis
constitutes a main objective in anti-tuberculosis drug discovery. In
addition, new antimycobacterial agents should ideally contribute to
shorten tuberculosis treatment to 2 months or less [5,6]. Few promising
drug candidates fulfilling these criteria have been discovered in recent
years [7-9]. Mainly, TMC207, which has been shown to be highly active
in proof-of-concept trials, and shows the potential to shorten the duration
of therapy [10,11].
Recently, there are several reports citing pyridine and oxadiazoles as
potential antibacterial and anti-tubercular agent [12-15]. Inspired by the
citations, we decided to design around the heterocycles and evaluate the
antimycobacterial activity of the same. Nonetheless, given the number
of tuberculosis cases and the rate of emergence of drug resistance, more
compounds are clearly needed to combat and have a significant impact
on the control and spread of tuberculosis. Thus in-continuation with
the search of new drug candidate we herein discuss this report about
development of a lead to hit molecule.
Experimental
Chemistry
1
H NMR spectra were recorded on a Bruker Avance 500 MHz instrument
using TMS as internal standard; the chemical shifts (δ) are reported in
ppm and coupling constants (J) are given in Hertz. Signal multiplicities
are represented by s (singlet), d (doublet), t (triplet), ds (double singlet),
dd (double doublet), m (multiplet), and bs (broad singlet). Mass spectra
were recorded on a Finnigan LCQ mass spectrometer. Elemental analysis
was performed on a Heracus CHN-Rapid Analyser. Analysis indicated
by the symbols of the elements of functions was within ± 0.4% of the
theoretical values. The purity of the compounds was checked on silica gel
coated Al plates (Merck) (Table 1).
Table 1: Preliminary Structure Activity Relationship of compounds 5a-h, 6a-h, 7a-h and 8a-h.
The inhibitory activity (MIC50) was determined against M. tuberculosis H37Rv. The cidal activity (MBC90) and cytotoxicity (CC50) were determined after 5 days
of exposure to a single dose of compound. Assays were carried out at least two times. MIC50: Minimum Inhibitory Concentration 50%; MBC90: Minimum
Bactericidal Concentration 90%, CC50: Cyototoxic concentration 50%. n.d.: not determined.
Synthesis of 2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-
(substituted)phenylacetamide 5a-h, 2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(substituted)phenylpropanamide 6a-h, 3-(5-(pyridin-4-yl)- 1,3,4-oxadiazol-2-ylthio)-N-(substituted)phenylpropanamide7a-h and 3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-2-methyl-N-(substituted) phenylpropanamide 8a-h.
To a cool solution of metallic sodium (1 mol) in an absolute ethanol;
5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiol (1 mol) (4) was added with
stirring, at about 15°C. The solution was filtered. The excess of solvent was
removed under suction and cold water was added to get a clear solution.
The solution was again filtered to remove suspended particles. Then
N-substituted-chloroalkylamide (1 mol) was added in small portion at
room temperature with stirring, then stirring was continue between 60 to
65°C for 8 h cooled and extracted with EtOAc. The EtOAc layer was dried
with sodium sulphate and the column purified.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-phenylacetamide(5a). Yield 76%; mp 187°C;1 H NMR (500 MHz, CDCl3 ): δ 3.83 (s, 2H, CH2 ), 7.02-7.51 (m, 5H, ArH), 7.60-7.63 (dd, 2H, J CH=CH=1.8, ArH), 8.10 (br, 1H, NH), 8.67-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 313.41 (M+, 100); Anal. Calcd. for C15H12N4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-benzylacetamide(5b).
Yield 68%; mp 189°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.87 (s, 2H, CH2 ), 4.45 (s, 2H, CH2 ), 7.01-7.15 (m, 5H, ArH), 7.62-7.65 (dd, 2H, J CH=CH=1.8, ArH), 8.13 (br, 1H, NH), 8.62-8.65 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 327.48 (M+, 100); Anal. Calcd. for C16H14N4 O2 S.
N-(2-Chlorobenzyl)-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio) acetamide (5c).
Yield 82%; mp 212°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.89 (s, 2H, CH2 ), 4.48 (s, 2H, CH2 ), 7.05-7.17 (m, 4H, ArH), 7.61-7.64 (dd, 2H, J CH=CH=1.8, ArH), 8.15 (br, 1H, NH), 8.60-8.64 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 362.88 (M+, 100); Anal. Calcd. for C16H13ClN4 O2 S.
N-(4-Chlorobenzyl)-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio) acetamide (5d).
Yield 68%; mp 208°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.84 (s, 2H, CH2 ), 4.43 (s, 2H, CH2 ), 7.03-7.05 (dd, 2H, JCH=CH=2.4, ArH), 7.12-7.14 (dd, 2H, J CH=CH=2.4, ArH), 7.63-7.66 (dd, 2H, J CH=CH=1.8, ArH), 8.13 (br, 1H, NH), 8.63-8.67 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 362.88 (M+, 100); Anal. Calcd. for C16H13ClN4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2-chlorophenyl) acetamide (5e).
Yield 82%; mp 112°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.82 (s, 2H, CH2 ), 6.96-7.53 (m, 4H, ArH), 7.64-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.17 (br, 1H, NH), 8.68-8.71 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 348.90 (M+, 100); Anal. Calcd. for C15H11ClN4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(4-chlorophenyl) acetamide (5f).
Yield 69%; mp 198°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.85 (s, 2H, CH2 ), 7.24-7.27 (dd, 2H, JCH=CH=2.6, ArH), 7.55-7.58 (dd, 2H, JCH=CH=2.6, ArH), 7.62-7.67 (dd, 2H, J CH=CH=1.8, ArH), 8.19 (br, 1H, NH), 8.62-8.67 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 348.90 (M+, 100); Anal. Calcd. for C15H11ClN4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,6-dichlorophenyl) acetamide (5g).
Yield 73%; mp 216°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.86 (s, 2H, CH2 ), 6.94-7.24 (m, 3H, ArH), 7.64-7.67 (dd, 2H, J CH=CH=1.8, ArH), 8.12 (br, 1H, NH), 8.69-8.72 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 383.3 (M+, 100); Anal. Calcd. for C15H10Cl2N4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,4-dichlorophenyl) acetamide (5h).
Yield 81%; mp 227°C; 1 H NMR (500 MHz, CDCl3 ): δ 3.87 (s, 2H, CH2 ), 7.12-7.15 (dd, 2H, JCH=CH=2.2, ArH), 7.24 (s, 1H, ArH), 7.51-7.53 (dd, 2H, J CH=CH=2.2, ArH), 7.64-7.69 (dd, 2H, J CH=CH=1.8, ArH), 8.13 (br, 1H, NH), 8.67-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 383.3 (M+, 100); Anal. Calcd.for C15H10Cl2N4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-phenylpropanamide (6a).
Yield 53%; mp 248°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.53 (d, 3H, CH3 ), 3.60-3.64 (q, 1H, CH), 7.02-7.51 (m, 5H, ArH), 7.62-7.64 (dd, 2H, J CH=CH=1.8, ArH), 8.14 (br, 1H, NH), 8.62-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 327.5 (M+, 100); Anal. Calcd. for C16H14N4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-benzylpropanamide (6b).
Yield 60%; mp 237°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.50 (d, 3H,CH3 ), 3.61-3.65 (q, 1H, CH), 4.45 (s, 2H, CH2 ), 7.01-7.15 (m, 5H, ArH), 7.61-7.64 (dd, 2H, J CH=CH=1.8, ArH), 8.13 (br, 1H, NH), 8.63-8.67 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 341.5 (M+, 100); Anal. Calcd. for C17H16N4 O2 S.
N-(2-Chlorobenzyl)-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio) propanamide (6c).
Yield 68%; mp 227°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.52 (d, 3H, CH3 ), 3.65-3.69 (q, 1H, CH), 4.48 (s, 2H, CH2 ), 7.05-7.17 (m, 4H, ArH), 7.63-7.66 (dd, 2H, J CH=CH=1.8, ArH), 8.16 (br, 1H, NH), 8.62-8.66 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 376.9 (M+, 100); Anal. Calcd. for C17H15ClN4 O2 S.
N-(4-Chlorobenzyl)-2-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio) propanamide (6d)
Yield 56%; mp 208°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.54 (d, 3H, CH3 ),3.63-3.67 (q, 1H, CH), 4.43 (s, 2H, CH2 ), 7.03-7.05 (dd, 2H, JCH=CH=2.4, ArH), 7.12-7.14 (dd, 2H, JCH=CH=2.4, ArH), 7.64-7.67 (dd, 2H, J CH=CH=1.8, ArH), 8.12 (br, 1H, NH), 8.65-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 376.9 (M+, 100); Anal. Calcd. for C17H15ClN4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2-chlorophenyl) propanamide(6e).
Yield 63%; mp 215°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.55 (d, 3H, CH3 ), 3.63-3.66 (q, 1H, CH), 6.96-7.53 (m, 4H, ArH), 7.62-7.67 (dd, 2H, J CH=CH=1.8, ArH), 8.14 (br, 1H, NH), 8.68-8.73 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 362.9 (M+, 100); Anal. Calcd. for C16H13ClN4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(4-chlorophenyl) propanamide (6f).
Yield 84%; mp 235°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.52 (d, 3H, CH3 ), 3.62-3.65 (q, 1H, CH),7.24-7.27 (dd, 2H, JCH=CH=2.6, ArH), 7.55-7.58 (dd, 2H, JCH=CH=2.6, ArH), 7.65-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.10 (br, 1H, NH), 8.67-8.71 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 362.9 (M+, 100); Anal. Calcd. for C16H13ClN4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,6-dichlorophenyl) propanamide (6g).
Yield 80%; mp 242°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.57 (d, 3H, CH3 ), 3.63-3.67 (q, 1H, CH), 6.94-7.24 (m, 3H, ArH), 7.63-7.66 (dd, 2H, J CH=CH=1.8, ArH), 8.11 (br, 1H, NH), 8.65-8.68 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 397.3 (M+, 100); Anal. Calcd. for C16H12Cl2 N4 O2 S.
2-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,4-dichlorophenyl) propanamide (6h).
Yield 64%; mp 236°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.56 (d, 3H, CH3 ), 3.60-3.63 (q, 1H, CH), 7.12-7.15 (dd, 2H, JCH=CH=2.2, ArH), 7.24 (s, 1H, ArH), 7.51-7.53 (dd, 2H, JCH=CH=2.2, ArH), 7.60-7.63 (dd, 2H, J CH=CH=1.8, ArH), 8.10 (br, 1H, NH), 8.63-8.68 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 397.3 (M+, 100); Anal. Calcd.for C16H12Cl2 N4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-phenylpropanamide (7a).
Yield 68%; mp 203°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.63-2.67 (tt, 2H, J CH=CH=2.7, ArH), 3.32-3.36 (tt, 2H, J CH=CH=2.7, ArH), 7.02-7.51 (m, 5H, ArH), 7.61-7.64 (dd, 2H, J CH=CH=1.8, ArH), 8.15 (br, 1H, NH), 8.60-8.76 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 327.4 (M+, 100); Anal. Calcd. for C16H14N4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-benzylpropanamide (7b).
Yield 63%; mp 218°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.61-2.64 (tt, 2H, J CH=CH=2.7, ArH), 3.31-3.36 (tt, 2H, J CH=CH=2.7, ArH), 4.45 (s, 2H, CH2 ), 7.01-7.15 (m, 5H, ArH), 7.62-7.66 (dd, 2H, J CH=CH=1.8, ArH), 8.16 (br, 1H, NH), 8.67-8.73 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 341.5 (M+, 100); Anal. Calcd. for C17H16N4 O2 S.
N-(2-Chlorobenzyl)-3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio) propanamide (7c).
Yield 60%; mp 213°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.62-2.65 (tt, 2H, J CH=CH=2.7, ArH), 3.30-3.35 (tt, 2H, J CH=CH=2.7, ArH), 4.48 (s, 2H, CH2 ), 7.05-7.17 (m, 4H, ArH), 7.62-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.12 (br, 1H,NH), 8.63-8.68 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 376.9 (M+, 100); Anal. Calcd. for C17H15ClN4 O2 S.
N-(4-Chlorobenzyl)-3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio) propanamide (7d).
Yield 59%; mp 217°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.64-2.68 (tt, 2H, J CH=CH=2.7, ArH), 3.34-3.38 (tt, 2H, J CH=CH=2.7, ArH),4.43 (s, 2H, CH2 ), 7.03-7.05 (dd, 2H, JCH=CH=2.4, ArH), 7.12-7.14 (dd, 2H, JCH=CH=2.4, ArH), 7.62-7.69 (dd, 2H, J CH=CH=1.8, ArH), 8.19 (br, 1H, NH), 8.67-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 376.9 (M+, 100); Anal. Calcd. for C17H15ClN4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2-chlorophenyl) propanamide (7e).
Yield 63%; mp 203°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.62-2.65 (tt, 2H, J CH=CH=2.7, ArH), 3.33-3.37 (tt, 2H, J CH=CH=2.7, ArH), 6.96-7.53 (m, 4H, ArH), 7.66-7.73 (dd, 2H, J CH=CH=1.8, ArH), 8.12 (br, 1H, NH), 8.63-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 362.9 (M+1, 100); Anal. Calcd. for C16H13ClN4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(4-chlorophenyl) propanamide(7f).
Yield 64%; mp 211°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.62-2.66 (tt, 2H, J CH=CH=2.7, ArH), 3.35-3.38 (tt, 2H, J CH=CH=2.7, ArH), 7.24-7.27 (dd, 2H, J CH=CH=2.6, ArH), 7.55-7.58 (dd, 2H, JCH=CH=2.6, ArH), 7.65-7.69 (dd, 2H,
J CH=CH=1.8, ArH), 8.18 (br, 1H, NH), 8.66-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 362.9 (M+1, 100); Anal. Calcd. for C16H13ClN4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,6-dichlorophenyl) propanamide (7g).
Yield 60%; mp 207°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.61-2.66 (tt, 2H, J CH=CH=2.7, ArH), 3.32-3.35 (tt, 2H, J CH=CH=2.7, ArH), 6.94-7.24 (m, 3H, ArH), 7.64-7.69 (dd, 2H, J CH=CH=1.8, ArH), 8.12 (br, 1H, NH), 8.65-8.72 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 397.3 (M+1, 100); Anal. Calcd. for C16H12Cl2 N4 O2< S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,4-dichlorophenyl) propanamide(7h).
Yield 61%; mp 209°C; 1 H NMR (500 MHz, CDCl3 ): δ 2.64-2.67 (tt, 2H, J CH=CH=2.7, ArH), 3.30-3.33 (tt, 2H, J CH=CH=2.7, ArH), 7.12-7.15 (dd, 2H, JCH=CH=2.2, ArH), 7.24 (s, 1H, ArH), 7.51-7.53 (dd, 2H, JCH=CH=2.2, ArH), 7.60-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.17 (br, 1H, NH), 8.68-8.73 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 397.3 (M+1, 100); Anal. Calcd. for C16H12Cl2 N4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-2-methyl-Nphenylpropanamide (8a).
Yield 62%; mp 205°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.25 (d, 3H, CH3 ), 2.88-2.92 (m, 1H, CH), 3.21-3.34 (d, 2H, CH2 ), 7.02-7.51 (m, 5H, ArH), 7.63-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.12 (br, 1H, NH), 8.68-8.77 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 341.4 (M+, 100); Anal. Calcd. for C17H16N4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-benzyl-2- methylpropanamide (8b).
Yield 71%; mp187°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.21 (d, 3H, CH3 ), 2.81-2.85 (m, 1H, CH), 3.20-3.31 (d, 2H, CH2 ), 4.45 (s, 2H, CH2 ), 7.01- 7.15 (m, 5H, ArH), 7.66-7.75 (dd, 2H, J CH=CH=1.8, ArH), 8.10 (br, 1H, NH), 8.67-8.76 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 355.5 (M+, 100); Anal. Calcd. for C18H18N4 O2 S.
N-(2-Chlorobenzyl)-3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-2- methylpropanamide (8c).
Yield 72%; mp186°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.26 (d, 3H, CH3 ), 2.84-2.89 (m, 1H, CH), 3.21-3.30 (d, 2H, CH2 ), 4.48 (s, 2H, CH2 ), 7.05- 7.17 (m, 4H, ArH), 7.63-7.69 (dd, 2H, J CH=CH=1.8, ArH), 8.17 (br, 1H, NH), 8.64-8.71 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 390.9 (M+1, 100); Anal. Calcd. for C18H17ClN4 O2 S.
N-(4-Chlorobenzyl)-3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-2- methylpropanamide (8d).
Yield 73%; mp176°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.22 (d, 3H, CH3 ), 2.83-2.88 (m, 1H, CH), 3.22-3.31 (d, 2H, CH2 ), 4.43 (s, 2H, CH2 ), 7.03-7.05 (dd, 2H, JCH=CH=2.4, ArH), 7.12-7.14 (dd, 2H, JCH=CH=2.4, ArH), 7.62-7.67 (dd, 2H, J CH=CH=1.8, ArH), 8.16 (br, 1H, NH), 8.63-8.70 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 390.9 (M+1, 100); Anal. Calcd. for C18H17ClN4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2-chlorophenyl)-2- methylpropanamide (8e).
Yield 77%; mp 208°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.24 (d, 3H, CH3 ), 2.84-2.87 (m, 1H, CH), 3.23-3.35 (d, 2H, CH2 ), 6.96-7.53 (m, 4H, ArH), 7.62-7.69 (dd, 2H, J CH=CH=1.8, ArH), 8.13 (br, 1H, NH), 8.65-8.71 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 376.9 (M+1, 100); Anal. Calcd. for C17H15ClN4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(4-chlorophenyl)-2- methylpropanamide (8f).
Yield 73%; mp185°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.27 (d, 3H, CH3 ), 2.82-2.86 (m, 1H, CH), 3.24-3.36 (d, 2H, CH2 ), 7.24-7.27 (dd, 2H, J CH=CH=2.6, ArH), 7.55-7.58 (dd, 2H, JCH=CH=2.6, ArH), 7.61-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.14 (br, 1H, NH), 8.64-8.73 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 376.9 (M+1, 100); Anal. Calcd. for C17H15ClN4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,6-dichlorophenyl)-2- methylpropanamide (8g).
Yield 71%; mp 174°C; 1 H NMR (500 MHz, CDCl3 ): δ 1.20 (d, 3H, CH3 ), 2.83-2.90 (m, 1H, CH), 3.23-3.37 (d, 2H, CH2 ), 6.94-7.24 (m, 3H, ArH), 7.62-7.68 (dd, 2H, J CH=CH=1.8, ArH), 8.17 (br, 1H, NH), 8.63-8.75 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 411.3 (M+1, 100); Anal. Calcd. for C17H14Cl2N4 O2 S.
3-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)-N-(2,4-dichlorophenyl)-2- methylpropanamide (8h).
Yield 78%; mp180°C; 1 H NMR (500 MHz, CDCl3): δ 1.23 (d, 3H, CH3 ), 2.80-2.85 (m, 1H, CH), 3.24-3.38 (d, 2H, CH2 ), 7.12-7.15 (dd, 2H, JCH=CH=2.2, ArH), 7.24 (s, 1H, ArH), 7.51-7.53 (dd, 2H, JCH=CH=2.2, ArH), 7.62-7.70 (dd, 2H, J CH=CH=1.8, ArH), 8.18 (br, 1H, NH), 8.61-8.73 (dd, 2H, J CH=CH=1.8, ArH); MS m/z (%) 411.3 (M+1, 100); Anal. Calcd. for C17H14Cl2N4 O2 S.
Antimycobacterial activity
Strains and growth conditions: M. tuberculosis H37Rv (ATCC, cat.
no. 27294), derivative strains and clinical isolates were maintained in
Middlebrook 7H9 broth medium supplemented with 0.2% glycerol, 0.05%
Tween 80 and 10% ADS supplement. Culture media were supplemented
with hygromycin (50 μg ml−1) or kanamycin (20 μg ml−1) when required.
High-throughput cell-based screen: M. bovis BCG was cultured to an
OD600 of 0.5–0.6 in complete 7H9 broth medium. In preparation for 1536-
well dispensing, the culture was diluted to an OD600 of 0.01 using complete
7H9 media. A volume of 4 μl of complete 7H9 media was dispensed into
a white, solid bottom 1536-well plate using a custom Bottle Valve liquid
dispenser (GNF). A volume of 100 nl of test compound in DMSO (1 mM)
was then transferred into each assay plates using a custom 1536 Pintool
(GNF). Diluted culture (4 μl) was subsequently added to the assay plates
using a Bottle Valve liquid dispenser (final OD600 in 8 μl is 0.005). The plates
were incubated at 37°C for 48 h. Growth was assessed by measuring ATP
levels using the BacTiter-Glo Microbial Cell Viability Assay (Promega).
Luminescence was measured using a ViewLux plate reader.
MIC50 determination: MIC50 were determined as previously described,
with slight modifications [17]. Briefly, compounds dissolved in 90%
DMSO were twofold serial-diluted in duplicates and spotted by mosquito
HTS (TTP LabTech) to 384-well clear plates, resulting in 10 dilutions
of each compound. A volume of 50 μl of M. tuberculosis culture (final
OD600 of 0.02) was added to each well, and the assay plates were incubated
at 37°C for 5 days. OD600 values were recorded using a SpectraMax M2
spectrophotometer, and MIC50 curves were plotted using GraphPad Prism
5 software. Under the assay setting, MIC50 values, which fall in the linear
part of the inhibition curve, are more robust and reproducible than
MIC90. Therefore, only MIC50 values are reported. Clinical isolates
used in drug susceptibility testing were strain typed by IS6110 analysis
as described [18].
Cytotoxicity: Cytotoxicity was tested against cell lines HepG2
(ATCC, cat. no. HB-8065) and BHK21 (ATCC, cat. no.CCL-10) in
96-well microplates. The cells were seeded at a density of 105 cells per
well, incubated at 37°C for 24 h and exposed to twofold serial-diluted
compounds for 3 days. Cell viability was monitored using the Cell
Proliferation Kit II (Invitrogen).
Determination of intracellular ATP levels: The intracellular ATP
level was quantified as previously described [19]. Briefly, 25 μl of M.
tuberculosis culture was mixed with an equal volume of freshly prepared
BacTiter-Glo reagent in white 384 flat-bottom plates and incubated in the
dark for 5 min. Luminescence was measured using a Tecan Safire2 plate
reader.
Drug preparation: Unless specified, all the compounds were
obtained from Sigma and were prepared in sterile de-ionized water. The
experimental compounds were prepared in dimethyl sulphoxide (Sigma)
for in vitro drug susceptibility testing.
Results and Discussion
Chemistry
In our attempt to synthesise cost effective drug, oxadiazole was identified
as better target, easy and cheaper to synthesis. The synthetic route was
followed as reported in (Figure-1)[16]. In brief, the ester to hydrazide
chemotransformation was carried out by using hydrazine hydrate in
ethanol at reflux condition. The hydrazide was then transformed to
thiosemicarbazate by usual CS2
, KOH and ethanol method. The resultant
solid was then cyclised using proton donar (sulphuric acid) at temperature
ranging from 0 to 5°C. The pyridinyl-oxadiazole “parent” then reacted
with various chloro-substituted compounds for further analog synthesis.
These reactions led us to the thio substituted array of compounds 5, 6, 7
and 8.
Antitubercular activity
A cellular screen was developed to identify mycobacterial growth
inhibitors. The screen was carried out against Mycobacterium bovis (M.
bovis) BCG using intracellular ATP content as a surrogate marker of
bacillary growth. Compound hits with confirmed activity against M.
tuberculosis were chemically clustered to identify any emerging SAR.
Our attention was drawn to a cluster of pyridinyl-oxadiazole compound.
One of the lead 4, (synthesized at our laboratory for another program,
unpublished) with an MIC50 ranging from 0.21 μM (Scheme 1). The
compound was bactericidal with cytotoxic profile within acceptable
range. Therefore, a lead optimization programme was initiated with the
goal of achieving potent antitubercular activity.
Figure 1: Route of synthesis for the compounds 5a-h, 6a-h, 7a-h and 8a-h.
The program of chemo-transformation initiated with compound 5a.
An increase in the length of alkyl chain with amide linkage intact (5a)
has shown improved potency. Another substitution with Ar =Benzyl, 5b
showed similar pharmacological activity. To achieve better activity, we
used phenyl and benzyl group with chloro substitution. In case of 5e and
5f, improved activity was observed as reported. Similar attempt with the
benzyl substitution (5c and 5d) was disappointing with no further increase
in activity. Assuming, chlorine has vital role to play in the orientation
and receptor bindings, 5g and 5h were synthesised but failed to reflect
potentiation.
Close look at SAR revealed that “small changes make big difference”,
thus wondering if one carbon elongation or branching will embark
any betterment in inhibition, further set of series 6a-h, 7a-h and 8a-h
were synthesized and tested. In case of branched series 6a-h it was not
surprising to note either equipotent or diminished action. This may owe
to the addition of bulk to the 3D structure of molecules. Only compound
6f has shown slight improved activity. The attempt of chain elongation,
7a-h disclosed different SAR. Except for dichloro compounds 7g and 7h, all showed increased potency. Molecule 7f has emerged as hit with almost
equipotent to isoniazide.
When synthesised branched analogs of 7 series i.e. 8a-h series, on
contrary to our expectations, none of the compounds was as promising as
7f. Thus we decided to further evaluate 7f to compare with standard drug
isoniazid (INH).
First we compared 7f with INH for their susceptibilities on 18 clinical
isolates table-2 of MTB (M. tuberculosis), out of which 16 were pan
susceptible and 2 were mono-rifampin resistant isolates. We are happy
to report that our compound 7f have been shown almost equipotent to
that of INH. Having seen its potential, we decided to evaluate 7f against
9 multi drug resistant (MDR) and 2 poly-drug resistant MTB strains
(Table-3). Gladly, compound has shown promising activity against almost
all the resistant strains. The compound 7f is now under further evaluation
stage, which shall be shortly communicated.
Conclusion
Keeping a widespread use of future antimycobacterials, we aimed
to synthesis a cheaper but better agent for today’s XDR and MDR
tuberculosis. In order to do so, we have zeroed at pyridinyl-oxadiazole,
which gave us really tractable small molecules. The evaluation of the
synthesised series revealed a potent compound 7f which was comparable
with Isoniazid against H37Rv. The next step of evaluation surprised us with
the effectiveness of 7f against 25 different isolates. The newer compound
has shown promising anti-XDR and anti-MDR tuberculosis activity.
Further attempts to study the toxophore of the compound are on and
communicated short as the outcomes are available.
Table 2: 7f drug susceptibilities for MTB (pan-susceptible and monorifampin resistant) clinical isolates.
Compound 7f and Isoniazid drug susceptibilities were determined on 16
pan-susceptible and 2 mono-rifampin resistant (asterisk) clinical isolates.
Table 3: 7f drug susceptibilities for MTB (MDR and poly-resistant) clinical
isolates.
The 7f susceptibilities were also tested on 9 multi-drug resistant (MDR)
and 2 poly-resistant MTB strains. (b) Twenty of the twenty-five sensitive
and resistant clinical isolates tested were previously determined to
be genetically distinct by IS6110 genotyping. I, isoniazid; R, rifampin;
S, streptomycin; EM, ethambutol; ET, ethionamide; K, kanamycin; P,
pyrazinamide; Cl, ciprofloxacin; CA, capreomycin.
Acknowledgment
We sincerely thank to Dr. Mahendra Shiradkar for all his support in
the activity and characterization.