4
4
P.P. Dixit et al. / European Journal of Medicinal Chemistry 107 (2016) 38e47
allowed to stir at room temperature overnight. Progress of the re-
action was monitored by TLC. After completion of reaction, the
contents were poured on to ice cold water, stirred for 15 min. It was
then filtered under suction, dried under high vacuum to get off
white solid product.
3.1.6. N-(3-((2-(5-(2,4-dichlorophenyl)-4,5-dihydroisoxazol-3-yl)
phenoxy)methyl)-5-mercapto-4H-1,2,4-triazol-4-yl)substituted-
amide PDST136-PDST138
2 3
Mixture of triazole PDST121 (0.01 mol) and Na CO (1 mol) in
ethanol was treated drop-wise with an equimolar amount of the
ꢀ
{
3-Benzyl sulfanyl-5-[2-(3-2,3-dichlorophenyl-acryloyl)-phe-
acid/acetyl chloride at 0 C, which was stirred for 30e45 min.
noxymethyl]-[1,2,4]triazol-4-yl}-carbamic acid tert-butyl ester 8.
Progress of the reaction was monitored by TLC. After completion of
reaction, the reaction mixture was washed with water (50 mL),
dried over anhydrous Na SO , filtered and concentrated under
2 4
reduced pressure to afford white product. The precipitate was then
filtered, washed thoroughly with water and crystallized to yield
PDST136-PDST138.
ꢀ
1
Yield 68%; mp ¼ 135e139 C; H NMR (500 MHz, DMSO-d
.46 (s, 9H, CH ), 4.23e4.25 (s, 2H, CH ), 5.23e5.27 (s, 1H, CH
.91e6.95 (d, 1H, ArH), 7.01e7.06 (m, 6H, ArH), 7.10e7.13 (d, 1H
6
)
d1.42-
1
3
2
2
),
6
J ¼ 4 MHz, ArH), 7.17e7.20 (d, 1H J ¼ 4 MHz, ArH), 7.25e7.27 (s, 1H,
ArH), 7.33e7.36 (d, 1H J ¼ 2 MHz, CH]CH), 7.42e7.44 (m, 1H, ArH),
7
8
.68e7.70 (d, 1H, ArH), 8.17e8.21 (d, 1H J ¼ 2 MHz, CH¼CH),
13
.30e8.35 (br, 1H, NH); CNMR (300 MHz, DMSO-d
6
)
d
28.5 (3
3.1.7. N-(3-((2-(5-(2,4-Dichlorophenyl)-4,5-dihydroisoxazol-3-yl)
phenoxy)methyl)-5-((ethoxycarbonyl)methylthio)-4H-1,2,4-triazol-
4-yl)substituted-amide PDST139-PDST141
carbon), 38.2, 62.5, 79.2, 114.8, 121.2, 121.4, 121.7, 126.8, 127.3, 127.7,
27.9, 128.7, 128.8, 129.3, 130.4, 130.8, 131.1, 132.7, 134.8, 135.5,
39.8, 145.3, 148.6, 151.5, 154.6, 161.3, 189.4; HRMS [ESI]: calculated
1
1
A solution of triazoles PDST136-PDST138 (0.01 mol), (0.01 mol)
þ
for C30
H28Cl
2
N
4
O
4
S [M ]: 611.539; Found: 611.524.
of K
2
CO
3
and ethyl 2-bromoacetate (0.01 mol) was prepared. The
ꢀ
reaction was stirred at 40 C for 2 h. Progress of the reaction was
monitored by TLC. After completion of reaction, the reaction
mixture was washed with water (50 mL), dried over anhydrous
3
3
.1.5. Synthesis of PDST121
.1.5.1. Cyclization. {3-Benzyl sulfanyl-5-[2-(3-2,4-dichlorophenyl-
2 4
Na SO , filtered and concentrated under reduced pressure to afford
acryloyl)-phenoxymethyl]-[1,2,4]triazol-4-yl}-carbamic acid tert-
butyl ester 8 (0.0018 mol) and hydroxyamine(0.0036 mol) were
heated in triethylamine (15 mL). As soon as, the solution starts
bumping (10e15 min), heating was stopped. Te reaction mixture
was cooled and poured onto ice-cold water. The product thus ob-
tained was filtered, washed with water and recrystallized from
ethanol to give Boc and benzyl protected PDST121.
white product. The precipitate was then filtered, washed thor-
oughly with water and crystallized to yield PDST139-PDST141.
3.1.8. N-(3-((2-(5-(2,4-Dichlorophenyl)-4,5-dihydroisoxazol-3-yl)
phenoxy)methyl)-5-(substituted-thio)-4H-1,2,4-triazol-4-yl)
substituted-amide PDST142-PDST168
A solution of triazoles PDST136-PDST138 (0.01 mol), (0.01 mol)
of K
2
CO
3
and alkyl halide (0.01 mol) was prepared. The reaction was
ꢀ
stirred at 40 C for 4 h. Progress of the reaction was monitored by
TLC. After completion of reaction, the reaction mixture was washed
3.1.5.2. Debenzylation. One of the Boc and benzyl protected
PDST121 (0.0012 mol) was dissolved in Methanol (10 mL) and 10%
Pd/C (50%wet) catalyst (2 times) was added into this solution. The
resulting mixture was kept in shaker under hydrogen pressure of
2 4
with water (50 mL), dried over anhydrous Na SO , filtered and
concentrated under reduced pressure to afford white product. The
precipitate was then filtered, washed thoroughly with water and
crystallized to yield PDST142-PDST168. (See Supplementary
information)
8
0psi for 4 h. Progress of the reaction was monitored by TLC. After
completion of reaction, reaction mixture was filtered through celite
under suction, washed with methanol. Filtrate and washings were
mixed together and concentrated under reduced pressure, dried
under high vacuum to get colorless oily debenzylated but Boc
protected PDST121 product.
3
3
.2. Pharmacology
.2.1. Strains, growth conditions and reagents
Two mycobacterial strains differed in virulence were used pri-
2
3
.1.5.3. Boc deprotection. One of the Boc protected PDST121
marily in this study: M. smegmatis mc 155 ATCC700084 and
M. tuberculosis H37Rv ATCC27294T. M. tuberculosis strains were
maintained in Middlebrook broth medium. Mycobacteria-specific
Middlebrook 7H9 and 7H10 media were obtained from Difco
(Detroit, MI, USA) supplemented with 0.2% glycerol, 0.05% Tween
80 and 10% ADS supplement. Thioridazine (TZ), verapamil (VP),
isoniazid (INH), rifampicin (RIF), amikacin (AMK), ofloxacin (OFX),
EtBr, phosphate-buffered saline (PBS), and glucose were purchased
from SigmaeAldrich (India). All solutions were prepared in
deionized water, except rifampicin and the PDST101-135, which
were prepared in DMSO. All solutions were prepared on the day of
the experiment.
(
0.0009 mol) was dissolved in dichloromethane (2.5 mL). This so-
ꢀ
lution was cooled to ꢂ10 C. Trifluoro acetic acid (2.5 mL) was
ꢀ
added slowly, drop wise to this solution at ꢂ10 C over a period of
ꢀ
1
0 min. The resulting mixture was allowed to stir at ꢂ10 C for 1 h.
Progress of the reaction was monitored by TLC. After completion of
reaction, the reaction mass was poured onto Petroleum ether
(
50 mL), stirred for 5 min, decanted. This process was repeated
thrice. The thick oily material was then poured onto diethyl ether
50 ml), stirred for 15 min, decanted. This process was also repeated
(
thrice. Finally, the solid obtained was dried under high vacuum to
give PDST121.
5-((2-(5-(2,4-dichlorophenyl)-4,5-dihydroisoxazol-3-yl)phenoxy)
methyl)-4-amino-1,2,4-triazole-3-thiol PDST121. Yield 55%;
3.2.2. Determination of minimum inhibitory concentration
ꢀ
1
mp ¼ 147e152 C; White solid; H NMR (500 MHz, DMSO-d
6
)
For M. smegmatis the determination of the MICs of synthesized
compounds, the efflux inhibitors TZ and VP, and the efflux substrate
ethidium bromide were conducted by the 96-well broth micro-
dilution method. Briefly, M. smegmatis was grown in MB7H9 sup-
d
3.03e3.05 (d, 1H, CH
2
), 3.32e3.34 (d, 1H, CH
2
), 4.55e4.56 (t, 1H,
CH), 4.91 (s, 2H, NH
2
), 5.21 (s, 2H, CH
2
), 6.82e6.85 (m, 2H, ArH),
7
.04e7.05 (d, 1H, ArH), 7.05e7.07 (d, 1H, ArH), 7.15e7.17 (m, 1H,
ArH), 7.21 (s, 1H, ArH), 7.51e7.55 (m, 1H, ArH), 12.5 (s, 1H, SH),;
ꢀ
plemented with 10% OADC at 37 C with shaking until an OD600 of
13
CNMR (300 MHz, DMSO-d
6
)
d
41.8, 62.2, 71.5, 114.5, 117.1, 121.4,
0.8. The inoculum was prepared by adjusting the culture to a
density corresponding to 0.5 McFarland standard diluted to 1:100.
Aliquots of 0.1 mL were transferred to each well of the 96-well plate
containing 0.1 mL of each compound at concentrations prepared
1
27.3,130.0,130.1, 130.5,131.2, 133.9, 134.7, 135.4,151.4,156.4,160.3,
þ
2 5 2
H15Cl N O S [M ]: 435.0324;
167.3; HRMS [ESI]: calculated for C18
Found: 435.0342.