B. Desai et al. / Bioorg. Med. Chem. 9 (2001) 1993–1998
1997
20,40-Dichloro phenyl-2, 6-dimethyl-3,5-bis-N-[40-nitro-
phenyl] carbamoyl-1,4-dihydropyridine (4c1). MS: m/z
added to BACTEC 12 broth to achieve a range of
concentrations for determination of minimum inhibi-
tory concentration (MIC, lowest concentration inhi-
biting 99% of the inoculums; MIC value of
Rifampicin is 0.25 g/mL @ 95% Inhibition of H37Rv
strain).
1
582 (M+). H NMR (60 MHz, TFA) d: 2.452 (s, 6H,
2ÂCH3), 5.502 (s,1H,CH), 5.898 (brs, 1H, NH), 8.912
(brs, 2H, 2ÂCONH), 7.012–8.122 (m, 10H, ArH) IR
(KBr, cmÀ1): 3198 (DHP NH), 3355 (amide NH), 1680
(amido C¼O).
M. Tuberculosis H37Rv (ATCC 27294; American type
culture collection, Rockville, MD) was cultured at 37 ꢀC
on a rotary shaker in middle brook 7H9 broth (Difco
laboratories, Detroit, MI) supplemented with 0.2 v/v
glycerol and 0.05% v/v Tween 80 until the culture tur-
bidity achieved an optical density of 0.45–0.55 nm. Bac-
teria were then pelleted by centrifugation, washed twice
and resuspended in one fifth the original volume in
dulbecco’s phosphate-buffered saline [PBS, Irvine Sci-
entific, Santa Ana, (A)]. Large bacterial clumps were
removed by passage through an 8 mm filter (Malgene,
Rochester, NY) and aliquots were frozen at À80 ꢀC.
Culture was thawed and an appropriate dilution per-
formed such that a BACTEC 12 B vial inoculated with
0.1 mL would reach a growth index (GI) of 999 in 5
days. One tenth of the diluted inoculum was used to
inoculate. Four millilitres of fresh BACTEC 12 B broth
containing the test compounds. An additional control
vial was included which received a further 1:100 diluted
inoculum (as well as 50 mL DMSO) for use in calculat-
ing the MIC of Rifampicin, respectively by establishing
procedures.
40 -Methoxy phenyl-2,6-dimethyl-3,5-bis-N-[20 -chloro
phenyl] carbamoyl-1,4-dihydropyridine (4d1). MS: m/z
1
522 (M+). H NMR (300 MHz, CDCl3+DMSO-d6) d:
2.272 (s, 6H, 2ÂCH3), 3.751 (s, 3H, OCH3), 4.840 (s, 1H,
CH), 5.682 (brs, 1H, NH), 8.205 (brs, 2H, 2ÂCONH),
6.825–7.720 (m, 12H, ArH) IR (KBr, cmÀ1): 3215 (DHP
NH), 3352 (amide NH), 1680 (amido C¼O).
30-Nitrophenyl-2,6-dimethyl-3,5-bis-N-[phenyl]
carba-
moyl-1,4-dihydropyridine (4e1). MS: m/z 468 (M+). H
NMR (60 MHz, TFA) d: 2.311 (s, 6H, 2ÂCH3), 5.505
(s,1H,CH), 5.789 (brs, 1H, NH), 8.429 (brs, 2H,
2ÂCONH), 7.228–7.653 (m, 14H, ArH) IR (KBr, cmÀ1):
3210 (DHP NH), 3327 (amide NH), 1701 (amido C¼O).
1
40-N,N-dimethylaminophenyl-2,6-dimethyl-3,5-bis-N-[20-
methoxyphenyl]carbamoyl-1,4-dihydropyridine (4f1). MS:
m/z 526(M +). 1H NMR (60 MHz, TFA) d: 2.308
(s, 6H, 2ÂCH3), 2.689 (s, 3H, SCH3), 3.618 (s, 3H,
OCH3), 5.625 (s, 1H, CH), 6.024 (brs, 1H, NH), 8.385
(brs, 2H, 2ÂCONH), 7.296–7.853 (m, 12H, ArH) IR
(KBr, cmÀ1): 3233 (DHP NH), 3370 (amide NH), 1702
(amido C¼O).
20-Methylthio phenyl-2,6-dimethyl-3,5-bis-N-[20,40-dime-
thyl phenyl] carbamoyl-1,4-dihydropyridine,(4g1). MS:
m/z 525 (M+). 1H NMR (60 MHz,TFA) d: 2.231 (s, 3H,
ArCH3), 2.334 (s, 3H, ArCH3), 2.451 (s, 3H, CH3),
5.542 (s, 1H, CH), 2.823 (s,3H,SCH3), 5.979 (brs, 1H,
NH), 7.239–7.985 (m, 10H, ArH) IR (KBr, cmÀ1):
3200 (DHP NH), 3352 (amide NH), 1690 (amido
C¼O).
Cultures were incubated at 37 ꢀC and the GI deter-
mined daily until control cultures achieved a GI of 999.
Assays were usually completed in 5–8 days. Percent
inhibition was defined as 1À(GI of test sample/GI of
control)Â100. Minimum inhibitory concentration of
compound effecting a reduction in daily change in GI,
which was less than that, observed with a 1:100 diluted
control culture on day the latter reached a GI of at least
30.
20-hydroxy phenyl-2,6-dimethyl-3,5-bis-N-[20,50-dimethyl
phenyl] carbamoyl-1,4-dihydropyridine (4h1). MS: m/z
495 (M+). 1H NMR (60 MHz, TFA) d: 2.189
(s,3H,ArCH3), 2.315 (s, 3H, ArCH3), 2.597 (s, 3H,
CH3), 5.506(s, 1H, CH), 7.110–7.899 (m, 10H, ArH) IR
(KBr, cmÀ1): 3225 (DHP NH), 3358 (amide NH), 1689
(amido C¼O).
Acknowledgements
The authors are very thankful to the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility
(TAACF), funded by the National Institute of Allergy
and Infectious Diseases, a division of the National
Institute of Health, USA, for providing biological
screening results. The authors are thankful to the direc-
tor, CDRI, Lucknow for providing necessary training
and computer facilities. The authors are thankful to Mr.
A. S. Kushwaha for excellent technical assistance. One
of us (BD) is thankful to the state government for
research fellowship.
Biological activity
Antitubercular activity was determined using the mod-
ified BACTEC 460 system in which stock solution of
test compounds were prepared in dimethylsulfoxide
(DMSO) at 1mg/mL and sterilized by passage through
0.22 mm PFTE filters (Millex-FG, Millipore, Bedford,
MA) 50 mL was added to 4 mL radiometric 7H12 broth
(BACTEC 12B, Becton Dickinson Diagnostic Instru-
ment Systems, Sparks, MD) to achieve a final con-
centration of 12.5 mg/mL. Controls received 50 mL
DMSO. Rifampicin (Sigma Chemical Co., St. Louis,
References and Notes
1. Bloom, B. R.; Murray, C. J. L. Science 1992, 257,
1055.
2. WHO report on the Tuberculosis Epidemic stop TB at the
source. Tuberculosis Programme World Health Organization,
Geneva, Switzerland, 1995.
MO) was included as a positive drug control.
Rifampicin was solubilized and diluted in DMSO and