Synthesis and antimycobacterial activity of 4-(5-substituted-1,3,4-oxadiazol-2-yl)pyridines

Article abstract of DOI:10.1016/j.bmc.2007.05.053

4-(5-Substituted-1,3,4-oxadiazol-2-yl)pyridine derivatives 1-12 were synthesized and evaluated for their in vitro antimycobacterial activity. Some compounds showed an interesting activity against Mycobacterium tuberculosis H₃₇Rv and five clinic

Full text of DOI:10.1016/j.bmc.2007.05.053

Bioorganic & Medicinal Chemistry 15 (2007) 5502–5508
Synthesis and antimycobacterial activity of
4-(5-substituted-1,3,4-oxadiazol-2-yl)pyridines
a,
b
a
*
´
´
Gabriel Navarrete-Vazquez, Gloria Marıa Molina-Salinas, Zetel Vahi Duarte-Fajardo,
b
a
b
´
Javier Vargas-Villarreal, Samuel Estrada-Soto, Francisco Gonzalez-Salazar,
a
b
´
´
Emanuel Hernandez-Nu´nez and Salvador Said-Fernandez
˜
aFacultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos 62210, Mexico
bDivisio´n de Biolog´ıa Celular y Molecular, Centro de Investigacio´n Biome´dica del Noreste, IMSS, Monterrey,
Nuevo Leo´n 64720, Mexico
Received 18 April 2007; revised 16 May 2007; accepted 18 May 2007
Available online 25 May 2007
Abstract—4-(5-Substituted-1,3,4-oxadiazol-2-yl)pyridine derivatives 1–12 were synthesized and evaluated for their in vitro antimy-
cobacterial activity. Some compounds showed an interesting activity against Mycobacterium tuberculosis H37Rv and five clinical iso-
lates (drug-sensitive and -resistant strains). Compound 4 [4-(5-pentadecyl-1,3,4-oxadiazol-2-yl)pyridine] was 10 times more active
than isoniazid, 20 times more active than streptomycin, and 28 times more potent than ethambutol against drug-resistant strain
CIBIN 112. Compound 5 [4-(5-heptadecyl-1,3,4-oxadiazol-2-yl)pyridine] showed the same behavior as compound 4. Both of the
above structures bear a high lipophilic chain bonded to the 5-position of the oxadiazole moiety. This fact implies that there exists
a contribution of lipophilicity, which could facilitate the entrance of these molecules through lipid-enriched bacterial cell membrane.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(INH), rifampicin (RIF), ethambutol (ETH), streptomy-
cin (STR), and pyrazinamide (PYR).3
Tuberculosis (TB) is one of the most common infectious
diseases known by the mankind. About 32% of the
world’s population is infected by Mycobacterium tuber-
culosis, the main causal agent of TB. Every year,
approximately 8 million of the infected people develop
active TB, and 2 million die.1 The World Health Orga-
nization estimates that about 30 million people will be
infected by M. tuberculosis within the next 20 years.2
The incidence of TB infection has steadily risen in the
last decade. The reemergence of TB infection has been
further complicated by an increase in the prevalence of
drug-resistant TB cases. Current control efforts are
severely hampered due to M. tuberculosis being a leading
opportunistic infection in patients with acquired
immune deficiency syndrome and the spreading of mul-
tidrug-resistant strains (MDR-MTB). Problems in the
chemotherapy of tuberculosis arise when patients devel-
op bacterial resistance to the first-line drugs: isoniazid
The ever-increasing drug resistance, toxicity, and side ef-
fects of currently used antituberculosis drugs, and the
absence of their bactericidal activity highlight the need
for new, safer, and more effective antimycobacterial
compounds. Since no effective vaccine is available, the
major strategy to combat the spreading of TB is the
chemotherapy.4
New chemical entities with novel mechanisms of action
will most likely possess activity against MDR-MTB.
There are two sources of these new chemical entities.
The first one is the extraordinary diversity provided by
natural product extraction, biological evaluation, and
structural elucidation. The second one comes from
synthetic compounds made through drug design. Some
natural and synthetic scaffolds have been tested as
antimycobacterial drugs,5–9 and the research for novel
vaccines is in progress.10 Three critical reviews
have been published recently, and they may give an out-
look on the latest research developments on antimyco-
bacterial substances, either of synthetic or natural
products.11–13
Keywords: Mycobacterium tuberculosis; 1,3,4-Oxadiazoles; Multidrug-
resistant strain.
*
Corresponding author. Tel./fax: +52 777 3297089; e-mail: gabriel_
navarrete@uaem.mx
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.053

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 15 (2007) 5502–5508
5503
However, these alone will not provide the breakthrough
that is needed. The key to improving therapy is to develop
new agents with potent sterilizing activity that will lead
to a shortening of the duration of chemotherapy.13
in the last step. Solid compounds were purified by recrys-
tallization. The structure of the purified compounds was
established by spectroscopic and spectrometric data.
One of the most effective first-line anti-TB drugs is INH.
Many analogues featuring the structure of INH have
been synthesized and tested as antimycobacterials. In a
critical review published recently, the existence of more
than 3000 compounds based on the INH core was
reported, about 66% of them being hydrazones.11
3. Results and discussion
Following the Microplate Alamar Blue Assay
(MABA),34 compounds 1–12 were tested in vitro for
their antimycobacterial activity against M. tuberculosis
strains H37Rv (ATCC 27294), and two drug-sensitive
and three drug-resistant clinical isolates. All the active
compounds were further analyzed for intrinsic cytotox-
icity in mammalian cells from the VERO line. The
results are summarized in Table 1.
It has been reported that conversion of INH to oxadiazoles
produces the corresponding 5-substituted 3H-1,3,4-oxa-
diazol-2-thione and 3H-1,3,4,-oxadiazol-2-one and their
3-alkyl or aralkyl derivatives, characterized by their high
activity against M. tuberculosis strain H37Rv.14,15 1,3,4-
Oxadiazoles conform to an important class of heterocy-
clic compounds with a wide range of biological activities
such as antiviral,16 tyrosinase inhibitors,17 antimicro-
bial,18,19 cathepsin K inhibitors,20 fungicidal,19,21 and
antineoplastic properties.22 Accordingly, their synthesis
and transformations have been a focus of interest for
a long time.
Results revealed that compounds 4 and 5 exhibited high
antimycobacterial activity. Among others, these struc-
tures were found to be the most potent compounds with
MIC’s 0.35 and 0.65 lM, respectively, showing similar
activity as INH (0.44 lM) against M. tuberculosis strain
H37Rv. Compounds 6, 8, 11 and 12 showed biological
activity against this strain in the range of 3.76–
8.97 lM. The remaining compounds did not show
important activity against this strain.
Here, we report the synthesis of some 4-(5-substituted-
1,3,4-oxadiazol-2-yl)pyridine derivatives and their
in vitro activity on two first-line drug-sensitive and three
drug-resistant M. tuberculosis clinical isolates and the
H37 Rv strain.
Compounds were also tested against five clinical isolates
of M. tuberculosis (Table 2). Compounds 4 and 5 were
as active as INH and also were the most active for the ser-
ies, showing nanomolar activities against CIBIN 687
strain. In particular, compound 4 was 10-fold more active
than INH and as active as STR against CIBIN 650 strain.
Compound 5 was six times more potent than INH against
this strain. Against CIBIN 687 strain, compounds 1, 6, 8,
10–12 showed MIC’s ranging between 3.53 and 6.39 lM.
Compound 12 showed moderate activity against drug-
sensitive clinical isolates, with IC50’s < 9 lM. This com-
pound has been reported before and showed good activity
against different strains of bacteria and fungi.19
2. Chemistry
The common synthetic route to 1,3,4-oxadiazoles involves
cyclization of diacylhydrazines with a variety of anhy-
drous reagents such as thionyl chloride,19,23 phosphorus
pentoxide,24 phosphorus oxychloride,25 polyphosphoric
acid,26 and sulfuric acid.27 They have also been prepared
by oxidation of acylhydrazones with different oxidizing
agents.28–30 One-pot syntheses of 1,3,4-oxadiazoles from
acid hydrazides and hydrazine with an acid chloride,31 as
well as from hydrazines with carboxylic acids,32 have also
been reported. The sequence followed in the present study
is shown in Scheme 1. Compounds 1 and 2 were prepared
using acetic and trifluoroacetic acid, respectively, and
INH.Acatalyticamountofsulfuricacidwasaddedtopro-
mote the dehydration and intramolecular cyclocondensa-
tion via microwave irradiation with low yields (<35%).
Compounds 3–7 and 9 were obtained by treatment of
INHwith acyl chloridesinDMF, throughone-potN-acyl-
ation and cyclodehydration.
Interestingly, compound 7, substituted with a 4-nitro phe-
nyl moiety, did not show significant activity against any
M. tuberculosis strains (MIC = 29.85 lM), whereas its
regioisomeric compound 8 (2-nitrophenyl-substituted)
showed MICs ranging from 3.76 to 7.46 lM. The pres-
ence of additional nitro group in compound 9 (3,5-dini-
trophenyl-substituted) resulted in a 2-fold less potency
against CIBIN 687 strain, but a 2-fold improvement in
activity against CIBIN 112 strain compared to compound
8. The bioactivity of these compounds could be related to
their reduced amino forms, which were also reported pre-
viously with antibacterial and antifungal activity.19
Reaction of INH and different aldehydes in presence
of sodium metabisulfite and dimethoxyethane afforded
N1-(arylmethylene)isonicotinohydrazides 13–16, which
were used immediately in a subsequent step without
purification. Oxidation of N1-(arylmethylene)ison-
icotinohydrazides 13–16 with potassium permanganate
in a mixture of acetone and water (5:1)33 under micro-
wave irradiation yielded compounds 8 and 10–12. Com-
pounds 8 and 10 were obtained with low yields due to
decomposition and superoxidation subproducts formed
All compounds were more active than INH, RIF, and
STR against MDR-MTB CIBIN 234 strain. Com-
pounds 6, 8, 11, and 12 showed anti-TB activity against
H37Rv in the range of 3.76–8.93 lM. The remaining
compounds showed an activity similar to that of INH
against INH-resistant strains and higher than that of
INH in the sensitive clinical isolates.
Anti-TB activity of compounds 6, 8, 10, and 12 was in
the range of 7.06–8.97 lM against CIBIN 650 strain.

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 15 (2007) 5502–5508
5504
O
O
N
N
N
O
N
R
Cl
DMF
R
OH
NH2
R
R
O
N
O
H2SO4
μw irradiation
H
N
N
N
INH
1: R = CH3
2: R = CF3
3: R = CH2Cl
O
4: R = C15H32
5: R = C17H35
6: R = C6H5
Na2S2O5
Ar
H
μw irradiation
7: R = 4-NO2C6H4
9: R = 3,5-(NO2)2C6H3
N
N
O
Ar
KMnO4
N
Ar
O
N
Acetone-water
μw irradiation
H
H
N
N
8: Ar = 2-NO2C6H4
13: Ar = 2-NO2C6H4
10: Ar = 3,4-(CH3O)2C6H3
11: Ar = 4-N(CH3)2C6H4
12: Ar = 4-Pyridyl
14: Ar = 3,4-(CH3O)2C6H3
15: Ar = 4-N(CH3)2C6H4
16: Ar = 4-Pyridyl
Scheme 1. Synthetic pathway of 4-(5-substituted-1,3,4-oxadiazol-2-yl)pyridine derivatives (1–12).
Table 1. Physicochemical data and in vitro antimycobacterial activity of 1–12 against M. tuberculosis H37Rv and two drug-sensitive and three drug-
resistant clinical isolates
N
N
R
O
N
Compound
R
MW Mp (ꢁC)
ClogP
H37Rv
49.69
M. tuberculosis clinical isolates MIC (lM)
IC50 VERO
cells (lM)
CIBIN CIBIN CIBIN CIBIN CIBIN
112
687
650
675
234
1
ACH3
ACF3
ACH2Cl
AC15H31
AC17H35
AC6H5
4-NO2C6H4
2-NO2C6H4
3,5-(NO2)2C6H3
161
215
195
357
385
223
268
268
313
239.9–241.2
257.2–258.5
nd
0.70 0.6
6.21
49.69
37.21
41.03
0.09
0.16
8.97
29.85
7.46
25.54
7.06
30.04
8.93
0.91
0.10
nd
49.69
37.21
41.03
11.19
10.37
35.87
29.85
29.85
25.54
28.25
30.04
35.71
29.19
6.87
49.69 49.69
37.21 37.21
41.03 41.03
nd
nd
2
1.71 0.98 37.21
1.22 0.62 41.03
37.21
41.03
0.70
0.65
4.48
29.85
3.73
6.39
3.53
3.76
4.46
0.91
nd
3
64.6
95.5
86.7
67.8
nd
4
121.1–122.2
112.2–113.0
152.2–155.136
203.5–205.437
66.9–68.7
225.7–226.8
217.6–219.2
204.9–206.8
235.2–236.3
—
8.14 0.60
9.20 0.60
2.89 0.62
0.35
0.65
8.97
22.38
20.75
2.80
2.59
5
6
35.87 35.87
29.85 29.85
29.85 29.85
25.54 12.77
28.25 14.12
30.04 30.04
35.71 35.71
58.38 29.19
55.02 55.02
7
2.85 0.62 29.85
2.38 0.62 7.46
8
32.4
77.1
250.2
50.4
92.41
39.8
>100
>100
>100
9
10
2.65 0.63 25.54
3.13 0.63 14.12
3,4,5-(CH3)3OC6H3 283
11
12
4-N(CH3)2C6H3
4-Pyridyl
266
224
137
581
822
204
3.30 0.63
1.63 0.62
3.76
8.93
0.44
0.86
0.07
9.80
Isoniazid
Streptomycin
Rifampicin
Ethambutol
—
—
—
—
ꢀ0.89 0.24
ꢀ3.20 1.04
0.49 0.74
—
—
nd
nd
0.94
121.51
nd
3.79
78.31
—
ꢀ0.05 0.44
nd
nd
MIC, minimal inhibitory concentration; nd, not determined.
Table 2. Profile of susceptibility of clinical isolated of M. tuberculosis
and propyl-1,3,4-oxadiazole-2-yl)pyridines showed a
low tuberculostatic in vitro effect.35 Apparently, it is
necessary to increase the steric hindrance at position 5
of oxadiazole moiety to improve the biological activity
of these derivatives. It also implies that lipophilicity
plays an important role in the bioactivity of these 4-(5-
substituted-1,3,4-oxadiazol-2-yl)pyridines.
Clinical isolate
Drug-resistance profile
CIBIN 687
CIBIN 650
CIBIN 675
CIBIN 234
CIBIN 112
Sensitivea
Sensitivea
STR, INH,
STR, INH, RIF, PYR
STR, INH, ETH
a Of all first-line antituberculosis drugs.
Compound 4 showed 10 and 20 times more potency than
INH and STR, respectively, against the INH-resistant
M. tuberculosis CIBIN 112 strain. This compound was
27 times more active than ETH. Compound 5 showed
the same behavior as 4 against this strain. These com-
pounds bear a highly lipophilic chain bonded to the 5-po-
sition of oxadiazole moiety. When we compared the
It is interesting to note that these 5-arylsubstituted com-
pounds possessed moderate activity, whereas 5-low alkyl
and haloalkyl (ACH3, ACF3, and ACH2Cl) substituted
derivatives did not show significant activity. It was
reported that 5-low alkyl homologues (methyl, ethyl,

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 15 (2007) 5502–5508
5505
values of ClogP of INH (ꢀ0.89 0.24) and synthesized
Table 3. Toxicity of compounds 4 and 5
compounds 4 and 5 (8.14 0.60 and 9.20 0.60, respec-
tively), we realized that there exists a contribution of lipo-
philicity, which could facilitate the entrance of these
molecules through lipid-enriched bacterial membrane,
which is formed by long chain fatty acids. Moreover,
b-ketoacyl-acyl carrier protein synthase III (FabH) cata-
lyzes a two-step reaction that initiates the pathway offatty
acid biosynthesis in plants and bacteria. FabH catalyzes
extension of lauroyl, myristoyl, and palmitoyl groups
from which cell wall mycolic acids of the bacterium are
formed.38
PBMC
IC50 (lg/mL)
4
5
Isoniazid
Adherent cells
Non-adherent cells
125.21
15.84
312.18
45.41
>100a
87.08
PBMC, peripheral blood mononuclear cells.
a Isoniazid treated adherent cells (100 lg/mL) survive more than 95%.
terial activity. The high bioactivity of compounds 4 and
5 makes them suitable hits for additional in vitro and
in vivo evaluations, in order to develop new antimyco-
bacterial drugs or prodrugs with potential use in the
tuberculosis treatment. Further studies in this area are
in progress in our laboratory.
Another raised hypothesis explores the possibility that
compounds 1–3, 6–8, and 11 and 12 could be acting as
INH prodrugs.12 None of them showed more potency
than INH against the three drug-resistant M. tuberculosis
clinical isolates. Although oxadiazole nucleus is very
stable to acid hydrolysis, it has been reported that it
may be chemically hydrolyzed with an strong base and
heating,39,40 leading thus to the generation of acyl-
INH, which are very likely to be completely hydrolyzed
to INH. According to Scior and Garces-Eisele12 the
pharmacological role of INH derivatives (isonicotinoyl
hydrazones, hydrazides, and amides) must be considered
as bio-reversible prodrugs of INH or isonicotinic acid.
Worse activities showed by these kinds of structures
can be explained by the compounds with a structural
gain of stability against prodrug hydrolysis. The best
activity could be related to decomposition products
formed in situ, inferring membrane toxicity. Such mem-
brane toxicity can only be of true help in an in vitro
study, since they would be undesired for the host cells
of any patient, too.
5. Experimental
Melting points were determined on a EZ-Melt MPA120
automated melting point apparatus from Stanford Re-
search Systems and are uncorrected. Reactions were
monitored by TLC on 0.2 mm precoated silica gel 60
F254 plates (E. Merck). 1H NMR and 13C NMR spectra
were measured with a Varian EM-390 (300 and
75.5 MHz) spectrometer. Chemical shifts are given in
ppm relative to tetramethylsilane (Me4Si, d = 0) in
CDCl3; J values are given in Hz. The following abbrevi-
ations are used: s, singlet; d, doublet; q, quartet; dd,
doublet of doublet; t, triplet; m, multiplet; br s, broad
signal. MS were recorded on a JEOL JMS-SX102A
spectrometer by electron impact (EI). Reactions under
microwave irradiation were performed in a domestic
microwave oven, Samsung MW1446WC, 1000 W. The
ClogP values were obtained using ACD/labs software
v.4.5.
On the other hand, compounds 4 and 5 could not be
considered as INH prodrugs, because INH derivatives
cannot be expected to overcome INH-resistance, as the
molecular action mechanism is identical.
5.1. Synthesis of 4-(5-substituted-1,3,4-oxadiazol-2-
yl)pyridines
All active compounds were examined for cytotoxicity
(IC50) in a mammalian VERO cell line. All of them
showed moderate toxicity levels, with IC50’s > 33 lM,
and selective indexes (IC50/MIC) ranging from 2- to 6-
fold (Table 1).
5.1.1. General method of synthesis of derivatives 1 and 2.
Isoniazid (0.0036 mol), 1.6 equiv of CH3COOH or
CF3COOH, and 1 drop of concentrated H2SO4 were
mixed and introduced in an open Erlenmeyer flask.
The mixture was irradiated in a household microwave
oven for 120 s. TLC was used to monitor the reaction.
After irradiation, the cooled mixture was neutralized
with saturated NaHCO3 solution, and the crude com-
pound was extracted with AcOEt. The solvent was
removed under vacuum, and the resulting solid was iso-
lated by filtration through a fritted 60 mL glass funnel
packed with Al2O3, basic type, and then crystallized.
We also evaluated the toxicity of most promissory com-
pounds (4 and 5) on peripheral blood mononuclear
human cells (PBMC; Table 3). Compounds 4 and 5
showed IC50 similar to INH on adherent cells, and both
compounds showed IC50 6- and 2-fold more toxic, respec-
tively, on non-adherent cells. These results are important
because PBMC are the mainly human cells implicated on
the immune response versus mycobacterial infection.41–43
5.1.1.1. 4-(5-Methyl-1,3,4-oxadiazol-2-yl)pyridine (1).
Recrystallized from methanol. Yield 0.176 g (30%) of
1
white solid. Mp 239.9–241.2 ꢁC. H NMR (300 MHz,
4. Conclusion
CDCl3) d 2.50 (s, 3H, CH3), 8.00–8.02 (m, 2H, H-3, H-
5), 9.01–9.03 (m, 2H, H-2, H-6) ppm; 13C NMR
(75.5 MHz, CDCl3) d 20.50 (CH3), 117.94 (C-3, C-5),
132.03 (C-4), 151.20 (C-2, C-6), 152.21 (C-20), 164.70 (C-
50) ppm; MS: m/z (% rel. int.) 161 (M+, 100), 146 (78),
HRMS: Calcd for C8H7N3O: 161.0589. Found: 161.0595.
A series of 4-(5-substituted-1,3,4-oxadiazol-2-yl)pyri-
dine derivatives were synthesized and tested against
M. tuberculosis drug-sensitive and drug-resistant strains.
The present results highlight the importance of lipophil-
icity of these compounds to present good antimycobac-

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 15 (2007) 5502–5508
5506
5.1.1.2. 4-[5-(Trifluoromethyl)-1,3,4-oxadiazol-2-yl]pyr-
50), 8.09 (dd, 2H, H-3, H-5), 8.29–8.34 (m, 2H, H-20,
H-60), 8.93 (m, 2H, H-2, H-6) ppm; 13C NMR
(75.5 MHz, CDCl3) d 118.41 (C-3, C-5), 127.54 (C-300,
C-500), 127.79 (C-200, C-600), 127.69 (C-100), 132.60 (C-4),
133.53 (C-400), 150.96 (C-2, C-6), 159.37 (C-20), 166.61
(C-50) ppm; MS: m/z (% rel. int.) 223 (M+, 100); HRMS:
Calcd for C13H9N3O: 223.0746. Found: 223.0750.
idine (2). Recrystallized from ethanol. Yield 0.247 g
(32%) of pale yellow solid. Mp 257.2–258.5 ꢁC. 1H
NMR (300 MHz, CDCl3) d 8.27–8.29 (m, 2H, H-3, H-
5), 9.02–9.04 (m, 2H, H-2, H-6) ppm; 13C NMR
(75.5 MHz, CDCl3) d 118.08 (q, CF3, J = 285.2 Hz),
119.18 (C-3, C-5), 131.97 (C-4), 138.30 (q, C-50,
J = 45.2 Hz) 152.72 (C-2, C-6), 163.37 (C-20) ppm;
MS: m/z (% rel. int.) 215 (M+, 100), 196 (20); HRMS:
Calcd for C8H4F3N3O: 215.0306. Found: 215.0312.
5.1.2.5. 4-[5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl]pyri-
dine (7). Recrystallized from MeOH–ethyl acetate. Yield
0.261 g (27%) of pale yellow solid. Mp 203.5–205.4 ꢁC.
1H NMR (300 MHz, CDCl3) d 8.08–8.10 (m, 2H, H-3,
H-5), 8.38–8.44 (m, 4H, H-2, H-3, H-5, H-6), 8.90–
8.92 (m, 2H, H-2, H-6) ppm; 13C NMR (75.5 MHz,
CDCl3) d 118.45 (C-3, C-5), 126.41 (C-200, C-600),
126.64 (C-300, C-500), 132.40 (C-4), 133.70 (C-100), 150.83
(C-2, C-6), 151.21 (C-400), 159.37 (C-20), 165.85 (C-50)
ppm; MS: m/z (% rel. int.) 268 (M+, 100); HRMS: Calcd
for C13H8N4O3: 268.0596. Found: 268.0584.
5.1.2. General method of synthesis of derivatives 3–7 and
9. A mixture of INH (0.0036 mol) and 1.1 equiv of
appropriate acyl chloride in 10 mL of DMF was heated
to reflux for 3–4.5 h. TLC was used to monitor the reac-
tion. After cooling, the mixture was neutralized with sat-
urated NaHCO3 solution and the precipitate formed
was filtered by suction. The crude product was purified
by recrystallization from adequate solvent.
5.1.2.1. 4-[5-(Chloromethyl)-1,3,4-oxadiazol-2-yl]pyri-
dine (3). Recrystallized from methanol. Yield 0.582 g
5.1.2.6. 4-[5-(2,4-Dinitrophenyl)-1,3,4-oxadiazol-2-yl]-
pyridine (9). Recrystallized from methanol. Yield 0.225 g
(20%) of brown solid. Mp 225.7–226.8 ꢁC. 1H NMR
(300 MHz, CDCl3) d 8.09 (dd, 2H, H-3, H-5), 8.91
(dd, 2H, H-2, H-6), 9.00 (t, 1H, H-400, J = 2.2 Hz), 9.57
(d,2H, H-200, H-600 J = 2.0, J = 2.2 Hz) ppm; 13C NMR
(75.5 MHz, CDCl3) d 118.41 (C-3, C-5), 119.37 (C-400),
128.50 (C-200, C-600), 128.97 (C-300), 132.60 (C-4), 150.96
(C-2, C-6), 151.76 (C-300, C-500), 159.30 (C-20), 168.19
(C-50) ppm; MS: m/z (% rel. int.) 313 (M+, 100); HRMS:
Calcd for C13H7N5O5: 313.0447. Found: 313.0455.
1
(83%) of yellow crystals. H NMR (300 MHz, CDCl3)
d 4.51 (s, 2H, CH2), 8.22 (dd, 2H, H-3, H-5), 8.94 (dd,
2H, H-2, H-6) ppm; 13C NMR (75.5 MHz, CDCl3) d
34.11 (CH2), 118.41 (C-3, C-5), 133.60 (C-4), 146.29
(C-50), 151.14 (C-2, C-6), 160.56 (C-20) ppm; MS: m/z
(% rel. int.) 313 (M+, 98), 248 (100); HRMS: Calcd for
C8H6ClN3O: 195.0199. Found: 195.0210.
5.1.2.2. 4-(5-Pentadecyl-1,3,4-oxadiazol-2-yl)pyridine
(4). Recrystallized from methanol. Yield 1.19 g (93%)
of white solid. Mp 121.1–122.2 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 0.90 (t, 3H, CH3), 1.24–1.36
(m, 24H, H-20, H-30, H-40, H-50 H-60), 1.52–1.60 (m,
2H, CH2), 2.37–2.41 (m, 2H, CH2), 8.00–8.02 (m, 2H,
H-3, H-5), 8.93–8.96 (m, 2H, H-2, H-6) ppm; 13C
NMR (75.5 MHz, DMSO-d6) d 14.22 (CH3), 22.68 (C-
1400), 29.39, 29.36, 29.56, 29.48, 29.78, 32.22, 117.90
(C-3, C-5), 131.73 (C-4), 151.17 (C-2, C-6), 161.03 (C-
20), 170.18 (C-50) ppm; MS: m/z (% rel. int.) 357 (M+,
100), 328 (10), 217 (30), 174 (80), 161 (80); HRMS:
Calcd for C22H35N3O: 357.2780. Found: 357.2792.
5.1.3. General method of synthesis of derivatives 8 and
10–12. A mixture of INH (0.0036 mol) and adequate
aldehyde (0.0039 mmol) was dissolved in dimethoxye-
thane (10 mL). Then, 1 equiv of sodium metabisulfite
was added and the mixture placed in a open Erlenmeyer
flask. The mixture was then subjected to microwave irra-
diation at 1000 W for 60 s. After complete conversion as
indicated by TLC, the reaction mixture was cooled and
the precipitated solids were filtered off to yield
N1-(arylmethylene)isonicotinohydrazides 13–16, which
were used immediately in a subsequent step without
purification. Oxidation of N1-(arylmethylene)ison-
icotinohydrazides.29 A mixture of 13–16 and potassium
permanganate (3 equiv) was dissolved in a mixture of
acetone/water (10:2), and then transferred to a open
Erlenmeyer flask. The mixture was then subjected to
irradiation at 1000 W. After complete conversion as
indicated by TLC, the solvent was removed in vacuo
and the aqueous layer was extracted with ethyl acetate
(3· 15 mL), washed with water (3· 20 mL), and dried
over anhydrous Na2SO4. The solvent was evaporated
in vacuo and the precipitated solids were recrystallized
from an appropriate solvent.
5.1.2.3. 4-(5-Heptadecyl-1,3,4-oxadiazol-2-yl)pyridine
(5). Recrystallized from EtOH. Yield 0.845 g (61%) of
1
white flakes. Mp 112.2–113.1 ꢁC. H NMR (300 MHz,
DMSO-d6) d 0.90 (m, 3H, CH3), 1.21–1.35 (m, 28H,
H-20, H-30, H-40, H-50 H-60), 1.53–1.60 (m, 2H,
CH2), 2.37–2.41 (m, 2H, CH2), 8.01 (dd, 2H, H-3,
H-5), 8.94 (dd, 2H, H-2, H-6) ppm; 13C NMR
(75.5 MHz, DMSO-d6) d 14.24 (CH3), 22.58 (CH2),
29.22, 29.36, 29.39, 29.54, 29.56, 29.48, 29.78, 32.22,
117.94 (C-3, C-5), 131.70 (C-4), 151.20 (C-2, C-6),
163.03 (C-20), 170.48 (C-50) ppm; MS: m/z (% rel.
int.) 385 (M+, 100), 356 (10), 174 (80), 161 (70);
HRMS: Calcd for C24H39N3O: 385.3093. Found:
385.3099.
5.1.3.1. 4-[5-(2-Nitrophenyl)-1,3,4-oxadiazol-2-yl]pyri-
dine (8). Recrystallized from methanol. Yield 0.289 g
1
(30%) of pale yellow solid. Mp 66.9–68.7 ꢁC. H NMR
5.1.2.4. 4-(5-Phenyl-1,3,4-oxadiazol-2-yl)pyridine (6).
Recrystallized from ethyl acetate. Yield 0.273 g (34%) of
pale yellow solid. Mp 152.2–155.1 ꢁC. 1H NMR
(300 MHz, CDCl3) d 7.28–7.49 (m, 3H, H-30, H-40, H-
(300 MHz, CDCl3) d 7.52–7.56 (m, 1H, H-400), 7.71–
7.81 (m, 1H, H-500), 8.00–8.03 (m, 1H, H-600), 8.08–8.11
(m, 2H, H-3, H-5), 8.23–8.25 (m, 1H, H-300), 8.90–8.92
(m, 2H, H-2, H-6) ppm; 13C NMR (75.5 MHz, CDCl3)

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 15 (2007) 5502–5508
5507
d 118.41 (C-3, C-5), 124.70 (C-100) 126.41 (C-600), 127.39
(C-300), 129.98 (C-4), 131.57 (C-400), 133.83 (C-500), 145.39
(C-200), 150.96 (C-2, C-6), 159.37 (C-20), 160.68 (C-50)
ppm; MS: m/z (% rel. int.) 268 (M+, 100); HRMS: Calcd
for C13H8N4O3: 268.0596. Found: 268.0604.
for the assay was prepared by diluting a logarithmically
growing culture to match the McFarland 1 turbidity
standard and then further diluting this to 1:50 with Mid-
dlebrook 7H9 broth to obtain a concentration of 6 · 106
colony forming units/mL. The working suspension was
prepared just before inoculation. The antibacterial activ-
ity of compounds against M. tuberculosis strains was
tested using the modified MABA. The concentrations
for compounds ranged from 8.000 to 0.016 lg/mL.
5.1.3.2. 4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-
2-yl]pyridine (10). Recrystallized from methanol. Yield
0.141 g (13%) of white solid. Mp 217.6–219.2 ꢁC. 1H
NMR (300 MHz, CDCl3) d 3.81 (s, 3H, 4-CH3O) 3.84
(d, 6H, 3-CH3O, 5-CH3O), 7.66 (d, 2H, H-200, H-600),
8.08–8.11 (M, 2H, H-3, H-5), 8.90–8.92 (m, 2H, H-2,
H-6) ppm; 13C NMR (75.5 MHz, CDCl3) d 58.28 (3,
5-(CH3)2), 60.39 (4-CH3O), 104.60 (C-200, C-600), 118.41
(C-3, C-5), 121.33 (C-100), 132.60 (C-4), 140.89 (C-400),
151.86 (C-2, C-6), 155.23 (C-300, C-500), 158.37 (C-20),
161.88 (C-50) ppm; MS: m/z (% rel. int.) 313 (M+,
100); HRMS: Calcd for C16H15N3O4: 313.1063. Found:
313.1070.
5.2.2. Toxicity on VERO cell line.44 The VERO cells
(ATCC Cat. No. CCL-81) were gently suspended in
Dulbecco’s modified Eagle’s medium (Gibco, Grand
Island, NY) and their concentration adjusted at
1.5 · 105 cells/mL in medium with 10% (v/v) bovine fetal
serum (FBS), and distributed in 200 lL aliquots into
each of the 96 wells of a sterile flat-bottomed Microtest
II microassay plate and incubated at 36.5 ꢁC in 5% CO2
atmosphere for 24 h. The spent medium in each well was
replaced with 200 lL of fresh supplemented DMEM
plus 10% FBS. Each microplate was divided in 9-well
rows that were added (in triplicate) with 5 lL of each
of the tested compounds (4000 lg of each compound/
mL was dissolved in 100% DMSO, Sigma). From these
stock solutions, a two-step serial dilution (100–0.390 lg/
mL) was prepared. In addition, a control untreated cell
culture, that was incubated in fresh supplemented
DMEM plus 2.5% DMSO, was included in each plate
(in triplicate) as a 100% viability internal control. These
preparations were re-incubated for 24 h. The cells were
trypsinized adding 100 lL of 0.25% trypsin–EDTA to
each well. The cells from each of the above preparations
were counted with a hemacytometer, and 50 lL of each
of these was transferred to a new 1.5 mL capacity and
added with 5 lL of 5% trypan blue dissolved in isotonic
buffered saline phosphate, pH 7.4. Twenty microliters of
this suspension was smeared on a slide and covered with
a cover slide. The percentage of blue cells (dead) from a
total of 100 was determined with the aid of a micro-
scope. The number of dead cells in each well was cor-
rected with respect to the mean of dead cells
percentage determined in the 100% viability controls.
The percentage of mortality was calculated by dividing
the corrected number of dead cells by the total number
of cells in each preparation · 100. The compound dose
producing 50% of dead cells (IC50) and 95% confidence
limits were calculated applying a Probit analysis with the
aid of the Statistical Package for Social Science (SPSS
for Windows, Standard Version 10.0). Results regarding
each compound and four antituberculosis drugs against
each cell line were reported as means standard error
(SE) of the three independent experiments.
5.1.3.3. 4-[5-(4-N,N-dimethylaminophenyl)-1,3,4-oxa-
diazol-2-yl]pyridine (11). Recrystallized from ethanol.
Yield 0.766 g (80%) of yellow crystals. Mp 204.9–
1
206.8 ꢁC. H NMR (300 MHz, CDCl3) d 3.20 (s, 6H,
(CH3)2N) 7.09–7.11 (m, 2H, H-300, H-500), 7.85–7.89
(m, 2H, H-200 ,H-600), 8.08–8.10 (m, 2H, H-30, H-50),
8.92–8.94 (m, 2H, H-2, H-6) ppm; 13C NMR
(75.5 MHz, CDCl3) d 40.44 ((CH3)2N), 114.30 (C-300,
C-500), 118.41 (C-3, C-5), 123.64 (C-100), 126.23 (C-200,
C-600), 132.60 (C-4), 150.96 (C-2, C-6), 153.10 (C-400),
159.34 (C-20), 166.65 (C-50) ppm; MS: m/z (% rel. int.)
266 (M+, 100); HRMS: Calcd for C15H14N4O:
266.1168. Found: 266.1176.
5.1.3.4. 4-(5-Pyridyl-1,3,4-oxadiazol-2-yl)pyridine (12).
Recrystallized from ethanol. Yield 0.677 g (84%) of
white crystals. Mp 235.2–236.3 ꢁC. 1H NMR
(300 MHz, CDCl3) d 8.09 (m, 4H, H-3, H-5, H-300, H-
500), 8.91 (m, 4H, H-200, H-600, H-2 ,H-6) ppm; 13C
NMR (75.5 MHz, CDCl3) d 118.41 (C-3, C-5, C-300, C-
500), 132.60 (c-4), 150.96 (C-2, C-6, C-200, C-600), 159.37
(C-20, C-50) ppm; MS: m/z (% rel. int.) 224 (M+, 100);
HRMS: Calcd for C12H8N4O: 224.2183. Found:
224.2196.
5.2. Biological assays
5.2.1. Anti-Mycobacterium tuberculosis assay.34 The fol-
lowing strains were used in the present study: M. tuber-
culosis H37Rv (ATTC 27294), which is sensitive to all
five first-line antituberculosis drugs (STR, INH, RIF,
ETH, and PYR), and five clinical isolates (Table 2) from
patients bearing advanced pulmonary tuberculosis: two
sensitive to all first-line drugs and three having different
drug-resistance profiles. These were isolated, identified,
and characterized in the Mycobacteriology laboratory
5.2.3. Toxicity on peripheral blood mononuclear human
cells. Blood was drawn from one healthy volunteer and
peripheral blood mononuclear cells (PBMC) were iso-
lated at room temperature following the method
described by Boyum.45 The PBMC were suspended
and incubated in RPMI 1640 medium added with 10%
FBS, cellular density was adjusted to 1 · 106 cells/mL,
and 100 lL of this suspension was placed in each well
of 96-well flat-bottomed sterile culture plates. On the
other hand, compounds were dissolved in DMSO
´
of the Centro de Investigacion Biomedica del Noreste,
Instituto Mexicano del Seguro Social (Monterrey, NL,
´
´
Mexico). All of these were cultured at 37 ꢁC and 5%
CO2 atmosphere in Middlebrook 7H9 broth supple-
mented with 0.2% glycerol and 10% OADC enrichment
(Oleic acid-Albumin-Dextrose-Catalase) until the loga-
rithmic phase of growth was reached. The inoculum

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G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 15 (2007) 5502–5508
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FBS to obtain a final concentration ranging from 20
to 100 lg/mL of each compound and a final concentra-
tion of 2.5% of DMSO. One hundred microliters from
these solutions was tested by quintuplicate assay, except
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Acknowledgments
This work was supported in part by grant from PROM-
EP-SEP, UAEMOR-PTC-131 (GNV). ZVDF acknowl-
edges the fellowship awarded by CONACyT to carry
out graduate studies. We also thank Juan Carlos Barb-
osa-Ordaz from Facultad de Farmacia, UAEM, for his
technical assistance. We also express our thanks to Dr.
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