New lipophilic isoniazid derivatives and their 1,3,4-oxadiazole analogues: Synthesis, antimycobacterial activity and investigation of their mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2018.04.017

The development of novel drugs is essential for the treatment of tuberculosis and other mycobacterial infections in future. A series of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides was synthesized from isoniazid (INH) and then cyclized to N-alkyl-5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines. All derivatives were characterised spectroscopically. The compounds were screened for their in vitro antimycobacterial activity against susceptible and multidrug-resistant Mycobacterium tuberculosis (Mtb.) and nontuberculous mycobacteria (NTM; M. avium, M. kansasii). The most active carboxamides were substituted by a short n-alkyl, their activity was comparable to INH with minimum inhibitory concentrations (MICs) against Mtb. of 0.5–2 μM. Moreover, they are non-toxic for HepG2, and some of them are highly active against INH-resistant NTM (MICs ≥4 μM). Their cyclization to 1,3,4-oxadiazoles did not increase the activity. The experimentally proved mechanism of action of 2-isonicotinoylhydrazine-1-carboxamides consists of the inhibition of enoyl-ACP reductase (InhA) in a way similar to INH, which is blocking the biosynthesis of mycolic acids. N-Dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine as the most efficacious oxadiazole inhibits growth of both susceptible and drug-resistant Mtb. strains with uniform MIC values of 4–8 μM with no cross-resistance to antitubercular drugs including INH. The mechanism of action is not elucidated but it is different from INH. Obtained results qualify these promising derivatives for further investigation.

Full text of DOI:10.1016/j.ejmech.2018.04.017

Accepted Manuscript
New lipophilic isoniazid derivatives and their 1,3,4-oxadiazole analogues: Synthesis,
antimycobacterial activity and investigation of their mechanism of action
Rudolf Vosátka, Martin Krátký, Markéta Švarcová, Jiří Janoušek, Jiřina Stolaříková,
Jan Madacki, Stanislav Huszár, Katarína Mikušová, Jana Korduláková, František
Trejtnar, Jarmila Vinšová
PII:
S0223-5234(18)30350-7
10.1016/j.ejmech.2018.04.017
EJMECH 10366
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2017
Revised Date: 27 March 2018
Accepted Date: 8 April 2018
Please cite this article as: R. Vosátka, M. Krátký, Marké. Švarcová, Jiří. Janoušek, Jiř. Stolaříková,
J. Madacki, S. Huszár, Katarí. Mikušová, J. Korduláková, Františ. Trejtnar, J. Vinšová, New lipophilic
isoniazid derivatives and their 1,3,4-oxadiazole analogues: Synthesis, antimycobacterial activity and
investigation of their mechanism of action, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.04.017.
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ACCEPTED MANUSCRIPT
New lipophilic isoniazid derivatives and their 1,3,4-oxadiazole analogues: synthesis,
antimycobacterial activity and investigation of their mechanism of action
Rudolf Vosátkaa, Martin Krátkýa, Markéta Švarcováa,b, Jiří Janoušekc, Jiřina Stolaříkovád, Jan
Madackie, Stanislav Huszáre, Katarína Mikušováe, Jana Kordulákováe, František Trejtnarc, Jarmila
Vinšováa,*
a
Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové,
Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Department of Chemistry, Faculty of Science, J. E. Purkinje University, České mládeže 8, 400 96
Ústí nad Labem, Czech Republic
c
Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles
University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
d Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in
Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic
e
Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava,
Mlynská dolina CH-1, Ilkovičova 6, 842 15 Bratislava, Slovakia
* Corresponding author. Heyrovského 1203, 500 05 Hradec Králové, Czech Republic. E-mail
address: jarmila.vinsova@faf.cuni.cz, tel.: +420-495067343, fax: +420-495067166.
Abstract
The development of novel drugs is essential for the treatment of tuberculosis and other
mycobacterial infections in future. A series of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides
was synthesized from isoniazid (INH) and then cyclized to N-alkyl-5-(pyridin-4-yl)-1,3,4-
oxadiazole-2-amines. All derivatives were characterised spectroscopically. The compounds were
screened for their in vitro antimycobacterial activity against susceptible and multidrug-resistant
Mycobacterium tuberculosis (Mtb.) and nontuberculous mycobacteria (NTM; M. avium, M.
kansasii). The most active carboxamides were substituted by a short n-alkyl, their activity was
comparable to INH with minimum inhibitory concentrations (MICs) against Mtb. of 0.5-2 µM.
Moreover, they are non-toxic for HepG2, and some of them are highly active against INH-resistant
NTM (MICs ≥4 µM). Their cyclization to 1,3,4-oxadiazoles did not increase the activity. The
experimentally proved mechanism of action of hydrazine-1-carboxamides consists of the inhibition
of enoyl-ACP-reductase (InhA) in a way similar to INH, which is blocking the biosynthesis of
mycolic acids. N-Dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine as the most efficacious
oxadiazole inhibits growth of both susceptible and drug-resistant Mtb. strains with uniform MIC
values of 4-8 µM with no cross-resistance to antitubercular drugs including INH. The mechanism of
action is not elucidated but it is different from INH. Obtained results qualify these promising
derivatives for further investigation.
Highlights
•2-Isonicotinoylhydrazine-1-carboxamides and 1,3,4-oxadiazoles were synthesised.
•Activity against M. tuberculosis and nontuberculous mycobacteria (MIC ≥0.5 µM).
•Low or no cytotoxicity for HepG2 cells.
•2-Isonicotinoyl-N-methylhydrazine-1-carboxamide targets InhA identical to isoniazid.
•5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-amines inhibit multidrug-resistant strains.

ACCEPTED MANUSCRIPT
Keywords
antimycobacterial activity; isoniazid; 2-isonicotinoylhydrazine-1-carboxamide; Mycobacterium
tuberculosis; 1,3,4-oxadiazole; tuberculosis
Graphical abstract

ACCEPTED MANUSCRIPT
1. Introduction
Genomic revolution optimism in tuberculosis (TB) drug research remains unfulfilled; results did not
bring a new therapeutic intervention and the whole effort returned to the cell screening.1 The
treatment of drug sensitive TB involves isoniazid (INH), rifampicin (RIF), pyrazinamide and
ethambutol (EMB) for the first two months followed generally by 4 months treatment with INH and
RIF. Beside the need to shorten and simplify the therapy of susceptible TB, the treatment of
multidrug- (MDR) and extensively drug-resistant (XDR) TB requires the regimens based on in vitro
drug susceptibility testing results and needs to be treated much longer with other drugs, usually
more toxic and with unpleasant side effects. These drugs should be also compatible with
antiretroviral therapy due to HIV/AIDS co-infection. Thus, the development and implementation of
new drugs overcoming resistance is a major goal for future control of TB.2
Isoniazid (INH, isonicotinic hydrazide, pyridine-4-carbohydrazide) is still one of the most efficient
and useful first line anti-TB drug, which plays the most important role in the treatment of TB all
over the world. In fact it has been used since 1952 as a prodrug, which must be activated by the
multifunctional mycobacterial enzyme catalase-peroxidase (KatG). The main target of INH is the
inhibition of the synthesis of mycolic acids, very specific building blocks in the mycobacterial cell
wall, responsible for its highly lipophilic character. The activated molecule of INH is thought to
suppress mycolic acid biosynthesis by the inhibition of enoyl-ACP reductase (InhA), an enzyme
involved in fatty acid biosynthesis in the presence of NADH or NAD+.3 This makes InhA one of the
best-validated targets for the treatment of TB. Most cases of INH-resistance are mediated by
mutations in katG gene, leading to the inability to activate the drug; the second most common
resistance is caused by mutations in the inhA gene. These two mutations among others are
responsible for approximately 75 % of all cases of Mycobacterium tuberculosis (Mtb.) resistance in
clinical setting.4 For example, the mutation rate responsible for INH resistance is 100 times greater
in comparison with resistance to RIF.5
A long duration of the treatment and other facts have induced an increase of resistance to all anti-
TB drugs and to the key molecule INH. A growing awareness of the increasing drug-resistance and
a great need for therapy shortening together with killing also latent forms of Mtb., lead to the
discovery of more efficient and less toxic treatment regimens. The need to overcome the resistance
to INH led to a huge number of structure modifications.6 Hydrophobic moieties were introduced
into the basic structure of INH to enhance penetration into a highly lipophilic cell wall and avoid
toxicity7. To protect INH molecule against N-arylaminoacetyl transferases at N2 and thus combat
the rise of resistance functionalization of hydrazine group 8,9,10,11, incorporation of one nitrogen12,13
or both nitrogen atoms14 of hydrazide group into another heterocycle ring and other modifications
were performed and then summarized in many review articles.15,16,17,18
We have already described several synthetic modifications of the isoniazid molecule linked with
another active part through a methine bridge19 by conjugation with an aniline group with electron-
withdrawing substituents20, via a carbonyl group21 and by the preparation of a new type of “double”
active molecules based on the fluorinated hydrazides of a benzoic acid scaffold as an isoniazid
isostere22. The current report deals with an introduction of lipophilic aliphatic chains into the
hydrazide part of INH molecule and with a cyclization of these derivatives into the 1,3,4-
oxadiazoles, which have been published as very efficient antimycobacterial agents.23,24,25,26
Hence, an attempt to improve the INH molecule by introducing chemical modifications in its core
structure in order to enhance biological responses against Mtb., to reduce hepatotoxicity, to
circumvent resistance phenomena for both drug-resistant Mtb. and nontuberculous mycobacteria
(NTM) continues to be an interesting scientific challenge. The goal of the new effort in

ACCEPTED MANUSCRIPT
development is to identify compounds that are able to attain the same clinical efficacy as isoniazid
and avoid much of the current resistance to INH by bypassing the requirement for KatG activation
and directly inhibiting InhA.
2. Results and Discussion
2.1. Chemistry
The majority of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides (2b-i, 2k, 2l, 2n, 2p, 2q) were
synthesised from isoniazid 1 and appropriate commercially available isocyanate via a previously
described method21 (Method A; yields 72-92 %). 2-Isonicotinoyl-N-methylhydrazine-1-
carboxamide 2a was obtained from N-succinimidyl N-methylcarbamate as a methyl isocyanate
substitute and isoniazid 1 in presence of N,N-diisopropylethylamine (DIPEA; Method B with 87%
yield). Three carboxamides (2j, 2m, 2o) were prepared from appropriate amines, triphosgene
(bis(trichloromethyl)carbonate) and INH 1 under the nitrogen atmosphere (Method C) with yields
of 65-76 %. The synthetic approach is depicted in Scheme 1.
Scheme 1. Synthesis of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides 2a-q (R: n-alkyl from C1
to C18; DEE: diethyl ether; DIPEA: N,N-diisopropylethylamine; MeCN: acetonitrile; Et3N:
triethylamine; DCM: dichloromethane; (Cl3CO)2CO: triphosgene.
N-Substituted 5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines 3 were obtained by cyclization of the 2-
isonicotinoylhydrazine-1-carboxamides 2 with triphenylphosphine in the presence of 1,2-dibromo-
1,1,2,2-tetrachloroethane in anhydrous solvent21 (Scheme 2). Their yields ranged from 52 to 68 %.
Scheme 2. Synthesis of N-alkyl-5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines
3
(Ph3P:
triphenylphosphine; (BrCl2C)2: 1,2-dibromo-1,1,2,2-tetrachloroethane; MeCN: acetonitrile; Et3N:
triethylamine; THF: tetrahydrofuran)

ACCEPTED MANUSCRIPT
All of the compounds 2-3 (Table 1) were characterized by 1H and 13C NMR, IR spectra and melting
points. Their purity was checked by TLC and elemental analysis.
2.2. Antimycobacterial Activity
Both series, 2-isonicotinoylhydrazine-1-carboxamides 2 and their cyclic analogues 5-(pyridine-4-
yl)-1,3,4-oxadiazole-2-amines 3 were evaluated for their in vitro antimycobacterial activity against
Mtb. 331/88 (H37Rv) and three NTM strains: Mycobacterium avium 330/88 and two strains of
Mycobacterium kansasii: 235/80 and a clinical isolate, 6509/96. The first-line antituberculosis drug
and synthetic precursor, isoniazid (INH), was used as the reference compound (Table 1).
All of the N-alkyl-2-isonicotinoylhydrazine-1-carboxamides 2 and N-alkyl-5-(pyridin-4-yl)-1,3,4-
oxadiazole-2-amines 3 displayed a significant antimycobacterial activity with minimum inhibitory
concentrations (MICs) starting from 0.5 µM; a limited solubility in the testing medium prevents
determination of all exact MIC values of two derivatives (2q, 3n) bearing a long alkyl chain. An
identical complication was present partly for 3l and 3m in the case of NTM.
Mtb. was the most susceptible species with MICs ≥0.5 µM for 2-isonicotinyolhydrazine-1-
carboxamides 2 and ≥4 µM for 5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines 3. Contrarily,
Mycobacterium avium showed the highest rate of tolerance (MIC values ≥32 µM and ≥8 µM for 2
and 3, respectively). Compounds 2 inhibited the growth of M. kansasii at the concentrations of 4
µM and higher, derivatives 3 with MICs ≥8 µM with no significant difference between the
collection and the isolated strain.
Analysing structure-activity relationships in the group of carboxamides 2, the optimal activity for
Mtb. is conferred by a small alkyl (methyl, ethyl, propyl 2a-c, MICs ≤2 µM). Pentyl and butyl (2d-
e) led to a moderately increased MIC of 8 µM. Thus, hydrophilicity (logP <1) modulates anti-TB
properties positively. An additional elongation of the alkyl chain decreases potency up to 125 µM
(octyl and nonyl, 2h-i). N-Decyl-2-isonicotinyolhydrazine-1-carboxamide 2j exhibited the best
suppression of Mtb. cells among derivatives with a long aliphatic chain (16/32 µM), MIC values of
its higher homologues are uniform of 32-62.5 µM. For M. avium, the most potent carboxamides are
those substituted from C11 to C16 alkyls (MICs ≤125 µM) with C12 superiority (2l, 32-62.5 µM).
Contrarily to Mtb., the introduction of shorter alkyls resulted in very low active or inactive
compounds (2a-b, 2d-e, MICs ≥1000 µM), the propyl derivative 2c being an exception (250/500
µM). 2-Isonicotinoyl-N-propylhydrazine-1-carboxamide 2c was also identified to be the most
potent agent against both M. kansasii strains with MIC values of 4-16 µM. Other favourable
substituents are undecyl (2k), tridecyl (2m) and ethyl (2b). Generally, an optimum is associated
with C2-C3 plus eight-membered and longer chains.
In contrast to 2, their cyclized counterparts 3 with C1-C3 alkyls showed only a limited
antimycobacterial action (MICs ≥500 µM) including a complete resistance of M. avium. This strain
tolerates also 2d-e at the concentration of 1000 µM. Butyl, hexyl, heptyl, tridecyl, pentadecyl,
hexadecyl and octadecyl derivatives (3d, 3f-g, 3m, 3o-q) exhibited moderate MIC values (≥125
µM), while N-pentylamine 3e was slightly superior against Mtb. (62.5 µM). The excellent
antimycobacterial properties are connected with ten, eleven and twelve-membered alkyl chains (3j-
k). N-Dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine 3l is the most efficient and selective in the
suppression of Mtb. (4/8 µM, i.e., 8 times higher concentration than obtained for INH) and M.
avium (8/16 µM), while N-decyl derivative 3j blocks both strains of M. kansasii at the lowest
concentration observed (8-16 µM).
Predominantly, the cyclization of the INH-based carboxamides 2 to the more lipophilic oxadiazoles
3 did not improve antimicrobial potency. For Mtb., the majority of carboxamides (bearing the

ACCEPTED MANUSCRIPT
shortest, shorter and the longest alkyls 2a-g, 2m-p) exceeded their analogous oxadiazoles 3, four
pairs are comparable (± one dilution; 2h-k) and there is only one but a very important example of
an improved anti-TB activity: N-dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine 3l, which is 4-8
times more efficient than its counterpart 2l. Oxadiazoles 3h, 3j and 3l share an enhanced action
against M. avium when compared to carboxamides 1 (up to 15.63 times). In the inhibition of M.
kansasii, only decyl derivative 3j improved sharply the activity of 2j (up to 15.63 times), three other
adjacent pairs with even-membered carbon chains produced a comparable inhibition (3f, 3h, 3k).
When compared to parent INH, three derivatives led to an identical in vitro inhibition of Mtb. (2a-
c), overall majority was superior against M. avium (2c, 2g-i, 2k-p, 3h-j, 3l-m, 3o-p). INH, 2c and 3j
exhibited a comparable activity for the clinical isolate of M. kansasii 6509/96, remaining novel
molecules 2 and 3 are inferior. Carboxamides 2b-c, 2e-p as well as oxadiazoles 3f-l and 3o-q
produced MIC values against M. kansasii 235/80 lower than INH.
Table 1. Antimycobacterial activity of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides 2 and N-
alkyl-5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines 3
MIC [µM]
Mtb.
331/88
M. avium
330/88
M. kansasii
235/80
14 d
>1000
1000
M. kansasii
6509/96
14 d
500
1000
Code
R
ClogP
14 d
21 d
1
500
2
14 d
21 d
>1000
>1000
>1000
>1000
500
7 d
1000
500
32
21 d
>1000
>1000
62.5
7 d
500
1000
32
21 d
1000
1000
62.5
-1.03
0.36
-0.69
0.69
-0.20
1.18
0.22
1.60
0.63
2.02
1.05
2.43
1.47
2.86
1.89
3.27
2.30
3.68
2.72
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
2f
0.5
500
1
>1000
>1000
1000
Methyl
Ethyl
62.5
62.5
1000
1000
>1000
1
>1000
1
>1000
250
>1000
8
>1000
16
>1000
8
>1000
8
4
500
500
500
62.5
500
250
250
62.5
250
32
4
Propyl
500
500
1000
1000
1000
125
1000
250
500
125
500
62.5
125
62.5
250
62.5
16
1000
1000
1000
125
1000
250
500
125
500
125
125
125
250
125
16
1000
250
1000
125
500
250
125
125
250
62.5
62.5
62.5
125
125
16
1000
250
1000
125
1000
500
250
250
500
125
125
125
250
250
16
1000
500
1000
125
1000
500
500
250
500
250
125
125
250
250
16
>1000
>1000
>1000
>1000
>1000
500
>1000
>1000
>1000
>1000
>1000
500
8
8
Butyl
Pentyl
Hexyl
Heptyl
Octyl
250
8
500
8
62.5
32
62.5
32
250
32
250
62.5
125
125
62.5
125
62.5
32
500
500
3f
250
250
2g
3g
2h
3h
2i
125
62.5
62.5
62.5
62.5
16
500
500
250
250
62.5
250
125
62.5
32
250
Nonyl
Decyl
125
125
125
32
3i
500
500
2j
3j
4.10
3.14
4.52
3.55
4.94
3.97
5.35
16
16
32
62.5
125
8
32
62.5
32
125
16
32
62.5
32
62.5
125
62.5
125*
32
125
125
62.5
125*
62.5
250
125
250
62.5
125*
62.5
250*
2k
3k
2l
Undecyl
Dodecyl
Tridecyl
32
500
500
8
16
32
32
32
62.5
32
62.5
125*
32
125
125*
62.5
250*
125
16
3l
4
8
8
16
32
62.5
250
125
250
125
250
2m
3m
250
250*
250*
125

ACCEPTED MANUS6C2R.5IPT
4.39
5.77
4.81
6.19
5.22
6.61
6.06
7.44
-0.64
32
250*
32
62.5
250*
62.5
125
62.5
250
250*
250
1
125
250*
125
125
250*
125
32
250*
32
62.5
250*
62.5
250
32
250
62.5
125
32
62.5
250*
62.5
250
62.5
500
250*
500
8
62.5
250*
125
250
62.5
500
250*
500
8
2n
3n
2o
3o
2p
3p
2q
3q
Tetradecyl
Pentadecyl
Hexadecyl
250*
62.5
125
125
32
250
500
62.5
32
125
125
62.5
500
62.5
500
250
250*
125
0.5
250
250
250
250*
250
250
250*
250
8
250*
500
250*
500
250*
250
250*
250
Octadecyl
INH
>250
>250
>250
>250
>250
INH: isoniazid; the best values for each strain are provided in bold. *: at presented concentration grow of strain was observed, at duplex concentration
there was present precipitate and/or turbidity; the determination of exact MIC value was not possible.
The most active compounds 2 (i.e., with MIC against Mtb. ≤1 µM) and the most efficient 1,3,4-
oxadiazole 3l underwent an advanced screening against four MDR-TB strains and one XDR-TB
strain with different resistance patterns (Table 2). MDR strain is a Mtb. strain that is resistant
concomitantly to INH and RIF. XDR involves MDR in addition to resistance to any of the
fluoroquinoles and at least one of the three parenteral second-line drugs (amikacin, kanamycin,
capreomycin).
Although all of the evaluated carboxamides 2 showed a growth inhibition of drug-resistant strains
independently of the resistance pattern, their MIC values are significantly higher than those
obtained for drug-susceptible Mtb. strain H37Rv (up to 125 times). The highest activity was
observed for 2-isonicotinoyl-N-propylhydrazine-1-carboxamide 2c (a uniform value of 16 µM),
followed by N-methyl derivative 2a (32-62.5 µM). These results indicate the cross-resistance to
parent INH 1. The described phenomena is common for the majority of “simple” INH modifications
but not inevitable.16
Importantly, MIC values of N-dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine 3l for both drug-
susceptible and resistant TB are identical (4-8 µM) thus indicating INH-independent mechanism of
action and/or the ability to circumvent the mechanism(s) of INH resistance. Our findings indicate
that 3l does not share any cross-resistance with conventionally used drugs (INH, rifamycines, EMB,
streptomycin, ofloxacin, clofazimine, aminoglycosides) and it may be a prospective and perspective
agent for combating drug-resistant TB. Importantly, the drug-likeness of 3l was checked using
traditional Lipinski´s rule of five. It defines four physicochemical parameter ranges (molecular
weight ≤500, logP ≤5, number of H-bond donors ≤5 and number of H-bond acceptors ≤10) that are
associated with an acceptable aqueous solubility and intestinal permeability required for oral
bioavailability.27 Notwithstanding the presence of C12 alkyl, the oxadiazole fits this rule without any
violation.
Table 2. MIC values of selected carboxamides 2 and oxadiazole 3l against MDR- and XDR-TB
MIC [µM]
Mtb. Praha 131
(XDR-TB)
Mtb.
9449/2006
Mtb.
Praha 1
Mtb.
Praha 4
Mtb.
234/2005
14 d 21 d 14 d 21 d 14 d 21 d
62.5 62.5 62.5 62.5 62.5 62.5
14 d
21 d
62.5
14 d
32
21 d
62.5
62.5
125
16
2a
2b
2c
3l
125 125 125 125
125
16
4
125
16
8
125
16
8
125
16
4
125
16
8
16
16
16
16
4
4
4
8
4
The lowest MIC values are given in bold. Resistance patterns for MDR-TB strains: 9449/2006 is resistant to INH, RIF,
rifabutine, and streptomycin (STM); 234/2005 resistant to INH, RIF, rifabutine, STM, and EMB; Praha 1 resistant to

INH, RIF, rifabutine, STM, EMB, and AcloCfaCziEmPinTe;EPDrahMa 4AreNsisUtaSntCtoRIINPHT, RIF, rifabutine, STM, EMB, ofloxacin
(OFX), and clofazimine. XDR-TB strain Praha 131 is resistant to INH, RIF, rifabutine, STM, EMB, OFX, gentamicin,
and amikacin.
Among 2-substituted 5-(pyridin-4-yl)-1,3,4-oxadiazoles, 2-(4,5-dibromo-1H-pyrrol-2-yl)-5-
(pyridin-4-yl)-1,3,4-oxadiazole (Fig. 1, compound a) showed a growth inhibition of Mtb. H37Rv
with MIC of 3.5 µg/mL (i.e., 8.75 times higher than those determined for INH in the study).
Regrettably, INH-resistant strains were not involved.28 Interestingly, Navarette-Vázquez et al.23
synthesized 5-(pyridin-4-yl)-2-substituted-1,3,4-oxadiazoles that are active against Mtb. H37Rv and
INH-susceptible clinical isolates. However, these derivatives lack amino group. In contrast to here
reported results, the highest anti-TB potential for an INH-susceptible strain was observed in the
presence of 2-pentadecyl and heptadecyl moieties (Fig. 1, structure b; MIC of 0.35 and 0.65 µM,
respectively). The replacement by methyl or substituted methyl reduced dramatically activity, 2-
(substituted)aryls produced intermediate MIC values. Other alkyl chains were not investigated.
Unfortunately, their MIC values against isolates that are, i.a., resistant to INH, are sharply higher
(up to 63.9 times). Authors concluded that an increased steric hindrance at position 2 of the
oxadiazole concomitantly with an escalated lipophilicity is required for the excellent biological
activity. A follow-up study brought an evidence that 2-pentadecyl-5-(pyridin-4-yl)-1,3,4-oxadiazole
(Fig. 1, structure b, n = 14) had a significant therapeutic effect in vivo in a murine model of
progressive pulmonary TB when it is administered in liposomal form.29 1,5-Dimethyl-2-phenyl-4-
({[5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl]methyl}amino)-1,2-dihydro-3H-pyrazol-3-one (Fig. 1,
compound c) exhibited a comparable activity for both INH-susceptible and resistant Mtb. strains.30
Described derivative containing 5-(pyridine-4-yl)-(1,3,4-oxadiazol-2-yl)methylamino moiety can be
considered as a higher homologue of 3. Based on presented data, it seems that the presence of
alkylamino group on the position 2 of 1,3,4-oxadiazole is required for the activity against INH- and
multidrug-resistant Mtb.
Figure 1. Related 2-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-based antimycobacterial agents
2.3. In vitro Cytotoxicity Determination
Cytotoxicity of the tested compounds was measured using the standard hepatic in vitro model,
cancer cell line HepG2. The used CellTiter 96 assay is based on the reduction of tetrazolium dye
MTS in living cells to formazan, which is then determined colorimetrically. The reduction of the
reagent is related to availability of NADH or NADPH. The decline in levels of these metabolically
important compounds in the cell causes that the production of formazan is reduced.
The parameter IC50 allows the quantitative comparison of the toxicity among tested compounds.
IC50 is the inhibitory concentration that reduces viability of the cell population to 50 % of the
maximal viability. The cytotoxicity was determined for the majority of the substances (all
carboxamides 2, oxadiazoles 3j-l; Table 3).
In general, the investigated carboxamides 2 and 1,3,4-oxadiazoles 3 share a substantially low
cytotoxicity at concentrations in which are soluble in the testing medium. Only toxicity course of 3k

ACCEPTED MANUSCRIPT
did allow a valid IC50 value calculation; in other cases, we could not determine exact IC50 values
due to their limited solubility. As expected, a longer alkyl led to a reduced solubility. None of the
derivatives 2-3 was toxic at the concentration of 50 µM.
In conclusion, the substances 2a-f as well as parent isoniazid 1 were found as in vitro certainly non-
toxic compounds in HepG2 cells (IC50 >1000 µM), remaining compounds did not reduced growth
of eukaryotic cells up to the fully soluble concentration. Obviously, the modification of INH 1 to N-
alkyl-2-isonicotinoylhydrazine-1-carboxamides 2 did not enhance cytotoxicity.
We also calculated the selectivity indices (SI) as the ratio of IC50 to MIC, and values higher than 10
indicate rather acceptable toxicity (based on the analogy of the therapeutic index). Table 3 reports
SI higher than 10, SI of 3l and for the compound 3k with the exactly known IC50 value. For Mtb.
331/88, SI of 2a-f indicate a safety and a desired selectivity for tubercular bacilli. This distinct
selectivity is also preserved for the drug-resistant strains in spite of the lower in vitro activity (SI
>31.3). SI of 3k is low (5.4) and for 3l, it is higher than 12.5 and 6.25 after 2 and 3 weeks of
incubation, respectively. Due to a low efficacy of INH analogues with shorter alkyls against M.
avium in vitro and a limited solubility of longer alkyls bearing derivatives, no satisfactory SI was
found. Regarding both strains of M. kansasii, three derivatives were identified as the selective
verifiable: N-ethyl 2b, N-pentyl 2e and especially N-propyl 2c carboxamides.
Table 3. Cytotoxicity of the tested substances 2-3 for HepG2 cells
IC50
[µM]
Range of
concentrations tested
0.5-5000
10-4000
5-5000
SI for Mtb.
331/88
>5000
>2000
>5000
>375
>500
>31.3
ND
SI for MDR-
and XDR-TB
>80
SI for M. kansasii
Code
>5000*
>4000*
>5000*
>3000*
>4000*
>1000*
>500**
>500**
>250**
>250**
>50**
>50**
>100**
>50**
>75**
>50**
>50**
>50**
ND
>64
>312.5
ND
>32
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
3j
>32
>312.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
5-3000
10-4000
0.5-1000
0.5-750
0.5-750
0.5-500
0.5-500
0.5-500
0.5-350
1-300
1-300
0.3-186
0.5-200
0.5-200
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.5-350
ND
5.4 (235/80)/0.7
(6509/96)
ND
>625 (6509/96)/
resistant (235/80)
~172.3
>50**
0.5-250
0.5-100
0.5-5000
5.4
ND
>6.25 (>12.5)
ND
3k
3l
>6.25 (>12.5)
>5000
INH (1)
>5000*
* IC50 is higher than tested concentration range
** measurement at higher concentration was unable due to the precipitation of the tested compound in the cell culture
medium
Only SI higher than 10 and those obtained for 3k and 3l are reported.
2.4. Investigation of Mechanism of Action
The most active oxadiazole 3l and the 2-isonicotinyol-N-methylhydrazine-1-carboxamide 2a as the
carboxamide that showed the lowest MIC values for Mtb., cytotoxicity and the highest selectivity
for Mtb. were subjected for the investigation of mechanism of action. In order to evaluate the enoyl-

ACCEPTED MANUSCRIPT
ACP-reductase InhA as a target of 3l and 2a, we analyzed MICs of these compounds against Mtb.
H37Ra overproducing InhA protein. In principle, the strains overproducing drug target should be
more resistant to the studied inhibitor due to an increased amount of the inhibited protein. Indeed,
analysis of sensitivity of Mtb. H37Ra carrying empty vector pMV261 and Mtb. H37Ra pMV261-
InhA by drop dilution method revealed that compared to the control strain InhA overproducer is a
significantly more resistant to the compound 2a. This result confirms InhA as molecular target of 2a
inside Mtb. cells (Fig. 2A). On the contrary, the sensitivity of InhA overproducer to the compound
3l was identical to the control strain indicating another mode of action of this inhibitor (Fig. 2B).
A.
100
10-1
10-2
10-3
0
M
25
M
50
M
100
M
200 M
2a
B.
100
10-1
10-2
10-3
0
M
1
M
5
M
10
M
25 M
3l
Figure 2. Determination of sensitivity of Mtb. H37Ra pMV261 and Mtb. H37Ra pMV261-InhA to
(A) 2a and (B) 3l by drop dilution method.
The inhibitory effect of 2a on mycolic acids production was verified by 14C metabolic labelling of
Mtb. H37Ra treated with this carboxamide derivative. Isoniazid was used as a control drug that
inhibits InhA protein. The lipids were extracted from the labeled cells by the mixture of chloroform
and methanol and separated by TLC (Fig. 3A). Clearly, the compound 2a inhibited the synthesis of
trehalose monomycolates and trehalose dimycolates similar to INH. Moreover, analysis of fatty and
mycolic acids content of lipid fractions by TLC after their derivatization to corresponding methyl
esters confirmed the inhibition of the synthesis of all types of mycolic acids by 2a, while the
production of short chain fatty acids was not affected (Fig. 3B).

ACCEPTED MANUSCRIPT
Figure 3. TLC analysis of (A) lipids and (B) corresponding methyl esters of fatty (FAME) and
mycolic (MAME) acids isolated from 14C labeled Mtb. H37Ra cells treated with 40 µM final
concentration of 2a and 36.5 µM of INH. Lipids were separated in chloroform: methanol: water
[20:4:0.5] and detected by autoradiography. Different forms of methyl esters were separated in n-
hexane: ethyl acetate [95:5; 3x] and detected by autoradiography. (TDM: trehalose dimycolates;
TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin; α-, methoxy- and
keto- refer to the forms of MAMEs).
14
Next, we studied the mechanism of action of 3l by C metabolic labelling of Mtb. H37Ra treated
with this compound in final concentrations 4 and 8 µM. Tested compound was added when O.D. of
14
the culture reached 0.263 and after 24 h of further cultivation C acetate as a metabolic tracer was
added. Cells were harvested after next 24 h of cultivation. Comprehensive investigation of lipids,
fatty and mycolic acids profiles by TLC using different solvent systems to separate major lipid
components of mycobacterial envelope did not reveal any differences between the control and
treated cultures suggesting that 3l does not target the pathways leading to the synthesis of cell
envelope. The mode of action of this compound thus remains to be elucidated.
Experimental Section
3.1. Synthesis
All chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) or Penta Chemicals
(Prague, Czech Republic) and were used as received. Melting points were determined on a Büchi
melting point B-540 apparatus (BÜCHI, Flawil, Switzerland) in open capillaries and the reported
values are uncorrected. Elemental analyses (C, H, N) were performed with an automatic Fisons EA
1110 CHNS-O CE microanalyzer (FISONS EA 1110, Milano, Italy). Infrared spectra (ATR) were
recorded on a Nicolet 6700 FTIR spectrometer in the range of 400–4000 cm−1. NMR spectra (500
1
13
MHz for H and 126 MHz for C) were measured in DMSO-d6 or CDCl3 at ambient temperature
using a Varian VNMR S500 instrument or a Varian Mercury-Vxbb 300 (300 MHz for 1H and 75.5
13
MHz for C). The chemical shifts (
δ
) are given in ppm, related to tetramethylsilane (TMS) as an
internal standard. The coupling constants (J) are reported in Hz. The reactions and purity of the
products were monitored by thin-layer chromatography using a mixture of dichloromethane and
methanol (2:1, v/v) as the eluent; the plates were coated with 0.2 mm Merck 60 F254 silica gel
(Merck Millipore, Darmstadt, Germany) and were visualised by UV irradiation (254 nm).

ACCEPTED MANUSCRIPT
The calculated logP values (ClogP), which are the logarithms of the partition coefficients for octan-
1-ol/water, were determined using the program CS ChemOffice Ultra version 15.0 (CambridgeSoft,
Cambridge, MA, USA).
3.1.1. General procedure for synthesis of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides 2
Method A
Isoniazid 1 (274.2 mg, 2.0 mmol) was suspended in anhydrous diethyl ether (8 mL) and heated to
initiate boiling of the mixture. The appropriate isocyanate (1.05 of equivalents, i.e., 2.1 mmol) was
added in one volume. The reaction mixture was refluxed for 1 h, then let cool at the room
temperature and stored for 24 hrs at -20 °C. Precipitate was filtered off and recrystallised from ethyl
acetate when necessary.
Method B
Isoniazid 1 (137.0 mg, 1.0 mmol) was mixed with N,N-diisopropylethylamine (DIPEA, 348 µL,
2.0 mmol) and N-succinimidyl N-methylcarbamate (258.2 mg, 1.5 mmol) in acetonitrile (4 mL).
The reaction mixture was stirred at the room temperature for 24 h, formed precipitate was filtered
off and recrystallised from ethyl acetate.
Method C
Method C involved the generation of an appropriate isocyanate in situ. Triphosgene (240 mg, 0.8
mmol) was dissolved in anhydrous dichloromethane (7 mL) under nitrogen atmosphere and the
appropriate amine (2.02 mmol) dissolved in anhydrous dichloromethane (4 mL) was added
dropwise. The mixture was stirred for 30 min at room temperature, then treated with triethylamine
(586 µL, 4.2 mmol) in anhydrous dichloromethane (4 mL). After 30 min, isoniazid 1 (277 mg, 2.02
mmol) was added. The reaction mixture was stirred for 10 hours at room temperature, then
evaporated to dryness, treated with water (15 mL) and extracted with ethyl acetate (3 x 25 mL). The
combined organic phase was dried over anhydrous sodium sulphate, filtered off and evaporated to
dryness to give the final product, which was recrystallised from ethyl acetate when necessary.
3.1.1.1. 2-Isonicotinoyl-N-methylhydrazine-1-carboxamide (2a). Yield 87 % (method B); mp 203-
205 °C (lit.31 224-225 °C). IR (ATR): 3248, 3091, 2928, 1665, 1556, 1532, 1490, 1413, 1305, 1223,
1151, 1064, 999, 848, 754, 670 cm-1. 1H NMR (DMSO, 300 MHz): δ 10.41 (1H, s, NH), 8.74 (2H,
d, J = 5.6 Hz, H2, H6), 8.01 (1H, s, NH), 7.79 (2H, d, J = 5.7 Hz, H3, H5), 6.52 (1H, q, J = 4.6 Hz,
NH), 2.57 (3H, d, J = 4.4 Hz, CH3). 13C NMR (DMSO, 75 MHz): δ 165.06, 158.83, 150.41, 139.98,
121.69, 26.46. Anal. Calcd for C8H10N4O2 (194.19): C, 49.48; H, 5.19; N, 28.85. Found: C, 48.85;
H, 5.81; N, 28.91.
3.1.1.2. N-Ethyl-2-isonicotinoylhydrazine-1-carboxamide (2b). Yield 92 % (method A); mp 214-
216 °C (lit.32 228-229 °C). IR (ATR): 3266, 3080, 2935, 1668, 1529, 1490, 1441, 1407, 1361, 1330,
1307, 1143, 1063, 998, 918, 852, 755, 712, 688 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.39 (1H, s,
NH), 8.74 (2H, d, J = 5.9 Hz, H2, H6), 7.94 (1H, s, NH), 7.79 (2H, d, J = 6.1 Hz, H3, H5), 6.55
(1H, t, J = 5.5 Hz, NH), 3.09-3.02 (2H, m, CH2), 1.01 (3H, t, J = 7.3 Hz, CH3). 13C NMR (DMSO,
126 MHz): δ 164.73, 157.88, 150.19, 139.76, 121.43, 34.04, 15.51. Anal. Calcd for C9H12N4O2
(208.22): C, 51.92; H, 5.81; N, 26.91. Found: C, 52.05; H, 5.98; N, 26.78.

ACCEPTED MANUSCRIPT
3.1.1.3. 2-Isonicotinoyl-N-propylhydrazine-1-carboxamide (2c). Yield 85 % (method A); mp. 182-
183°C. IR (ATR): 3261, 3079, 2928, 1671, 1556, 1532, 1488, 1408, 1385, 1303, 1246, 1148, 1064,
1
997, 916, 851, 757, 669 cm-1. H NMR (DMSO, 500 MHz): δ 10.39 (1H, s, NH), 8.74 (2H, d, J =
5.8 Hz, H2, H6), 7.92 (1H, s, NH), 7.79 (2H, d, J = 6.3 Hz, H3, H5), 6.56 (1H, t, J = 5.5 Hz, NH),
3.02-2.95 (2H, m, C1H2), 1.55-1.45 (2H, m, C2H2), 0.83 (3H, t, J = 7.4 Hz, CH3). 13C NMR
(DMSO, 126 MHz): δ 164.74, 158.23, 150.22, 139.77, 121.50, 41.23, 23.01, 11.23. Anal. Calcd for
C10H14N4O2 (222.24): C, 54.04; H, 6.35; N, 25.21. Found: C, 53.85; H, 6.61; N, 24.96.
3.1.1.4. N-Butyl-2-isonicotinoylhydrazine-1-carboxamide (2d)33. Yield 79 % (method A); mp 175-
177 °C. IR (ATR): 3293, 3089, 2933, 1669, 1555, 1518, 1487, 1436, 1407, 1375, 1292, 1221, 1140,
1063, 999, 902, 852, 757 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.39 (1H, s, NH), 8.74 (2H, d, J =
6.2 Hz, H2, H6), 7.91 (1H, s, NH), 7.88 (2H, d, J = 6.2 Hz, H3, H5), 6.58 (1H, t, J = 5.5 Hz, NH),
3.07-2.97 (2H, m, C1H2), 1.43-1.33 (2H, m, C2H2), 1.32-1.21 (2H, m, C3H2), 0.86 (3H, t, J =
13
7.7 Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.73, 158.00, 150.19, 139.96, 121.43, 40.20,
31.95, 19.41, 13.71. Anal. Calcd for C11H16N4O2 (236.27): C, 55.92; H, 6.83; N, 23.71. Found: C,
55.84; H, 6.60; N, 23.92.
3.1.1.5. 2-Isonicotinoyl-N-pentylhydrazine-1-carboxamide (2e). Yield 91 % (method A); mp 175-
177 °C. IR (ATR): 3293, 3090, 2933, 2860, 1663, 1556, 1518, 1437, 1406, 1376, 1350, 1288, 1219,
1142, 1062, 999, 908, 852, 758, 669 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.40 (1H, s, NH), 8.74
(2H, d, J = 6.0 Hz, H2, H6), 7.92 (1H, s, NH), 7.79 (2H, d, J = 5.8 Hz, H3, H5), 6.55 (1H, t, J =
5.5 Hz, NH), 3.06-2.96 (2H, m, C1H2), 1.47-1.32 (2H, m, C2H2), 1.32-1.16 (4H, m, C3H2, C4H2),
13
0.88 (3H, t, J = 7.6 Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.94, 158.23, 150.41, 139.96,
121.45, 40.48, 29.53, 28.51, 21.91, 13.98. Anal. Calcd for C12H18N4O2 (250.30): C, 57.58; H, 7.25;
N, 22.38. Found: C, 57.83; H, 7.60; N, 22.41.
3.1.1.6. N-Hexyl-2-isonicotinoylhydrazine-1-carboxamide (2f). Yield 86 % (method A); mp 175-
177 °C. IR (ATR): 3286, 3085, 2927, 2858, 1671, 1549, 1516, 1485, 1436, 1406, 1374, 1341, 1300,
1220, 1139, 1064, 998, 913, 851, 756, 719, 695 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.40 (1H, s,
NH), 8.75 (2H, d, J = 6.3 Hz, H2, H6), 7.92 (1H, s, NH), 7.79 (2H, d, J = 6.2 Hz, H3, H5), 6.55
(1H, t, J = 5.6 Hz, NH), 3.06-2.94 (2H, m, C1H2), 1.46-1.32 (2H, m, C2H2), 1.31-1.16 (6H, m, C3H2,
13
C4H2, C5H2), 0.86 (3H, t, J = 6.7 Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.73, 158.12,
150.18, 139.82, 121.37, 40.26, 31.06, 29.81, 25.98, 22.10, 13.94. Anal. Calcd for C13H20N4O2
(264.32): C, 59.07; H, 7.63; N, 21.20. Found: C, 59.24; H, 7.80; N, 22.33.
3.1.1.7. N-Heptyl-2-isonicotinoylhydrazine-1-carboxamide (2g). Yield 83 % (method A); mp 173-
175 °C (lit.21 150-152.5 °C). IR (ATR): 3291, 3080, 2928, 2855, 1671, 1557, 1522, 1508, 1436,
1
1406, 1375, 1340, 1296, 1220, 1142, 1063, 997, 904, 850, 758, 677, 669 cm-1. H NMR (DMSO,
500 MHz): δ 10.40 (1H, s, NH), 8.75 (2H, d, J = 5.8 Hz, H2, H6), 7.92 (1H, s, NH), 7.78 (2H, d, J =
5.7 Hz, H3, H5), 6.57 (1H, t, J = 5.6 Hz, NH), 3.04-2.93 (2H, m, C1H2), 1.46-1.16 (10H, m, C2H2,
13
C3H2, C4H2, C5H2, C6H2), 0.88 (3H, t, J = 6.4 Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.95,
158.03, 150.42, 139.78, 121.66, 40.27, 31.26, 29.85, 28.75, 26.37, 22.11, 13.95. Anal. Calcd for
C14H22N4O2 (278.36): C, 60.41; H, 7.97; N, 20.13. Found: C, 60.68; H, 7.71; N, 20.35.
3.1.1.8. 2-Isonicotinoyl-N-octylhydrazine-1-carboxamide (2h). Yield 82 % (method A); mp 168-
170 °C. IR (ATR): 3292, 3080, 2923, 2855, 1671, 1632, 1557, 1522, 1508, 1487, 1436, 1406, 1375,

ACCEPTED MANUSCRIPT
1
1340, 1295, 1219, 1141, 1063, 997, 907, 850, 758, 721, 674 cm-1. H NMR (DMSO, 500 MHz): δ
10.39 (1H, s, NH), 8.74 (2H, d, J = 5.9 Hz, H2, H6), 7.92 (1H, s, NH), 7.79 (2H, d, J = 5.6 Hz, H3,
H5), 6.57 (1H, t, J = 5.6 Hz, NH), 3.06-2.94 (2H, m, C1H2), 1.47-1.16 (12H, m, C2H2, C3H2, C4H2,
C5H2, C6H2, C7H2), 0.88 (3H, t, J = 7.7 Hz, CH3). 13C NMR (DMSO, 126 MHz): δ 164.93, 158.24,
149.98, 140.02, 121.57, 40.34, 31.52, 30.01, 29.00, 28.93, 26.32, 22.24, 13.97. Anal. Calcd for
C15H24N4O2 (292.38): C, 61.62; H, 8.27; N, 19.16. Found: C, 62.05; H, 8.09; N, 19.17.
3.1.1.9. 2-Isonicotinoyl-N-nonylhydrazine-1-carboxamide (2i). Yield 87 % (method A); mp 168-
170 °C. IR (ATR): 3291, 3082, 2921, 2853, 1671, 1636, 1557, 1517, 1508, 1488, 1472, 1437, 1406,
1
1375, 1340, 1296, 1220, 1135, 1063, 998, 900, 851, 760, 720, 687 cm-1. H NMR (DMSO, 500
MHz): δ 10.39 (1H, s, NH), 8.74 (2H, d, J = 6.5 Hz, H2, H6), 7.92 (1H, s, NH), 7.88 (2H, d, J =
6.5 Hz, H3, H5), 6.56 (1H, t, J = 5.8 Hz, NH), 3.06-2.93 (2H, m, C1H2), 1.50-1.15 (14H, m, C2H2,
C3H2, C4H2, C5H2, C6H2, C7H2, C8H2), 0.83 (3H, t, J = 7.4 Hz, CH3). 13C NMR (DMSO, 126 MHz):
δ 164.83, 158.28, 150.21, 139.78, 121.46, 40.25, 31.42, 29.95, 29.21, 28.92, 28.83, 26.39, 22.18,
14.16. Anal. Calcd for C16H26N4O2 (306.41): C, 62.72; H, 8.55; N, 18.29. Found: C, 62.87; H, 8.83;
N, 17.98.
3.1.1.10. N-Decyl-2-isonicotinoylhydrazine-1-carboxamide (2j). Yield 76 % (method C); mp 168-
169 °C. IR (ATR): 3321, 3061, 2925, 2851, 1683, 1508, 1484, 1436, 1419, 1327, 1278, 1229, 1185,
1055, 1009, 905, 835, 754, 725, 653 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.46 (1H, s, NH), 8.79
(2H, d, J = 6.8 Hz, H2, H6), 7.95 (1H, s, NH), 7.87 (2H, d, J = 6.1 Hz, H3, H5), 6.54 (1H, t, J =
5.6 Hz, NH), 3.04-2.97 (2H, m, C1H2), 1.49-1.17 (16H, m, C2H2, C3H2, C4H2, C5H2, C6H2, C7H2,
13
C8H2, C9H2), 0.85 (3H, t, J = 6.9 Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.95, 158.14,
149.39, 139.65, 121.58, 40.20, 31.42, 29.88, 29.23, 29.15, 28.99, 28.88, 26.47, 22.23, 14.01. Anal.
Calcd for C17H28N4O2 (320.44): C, 63.72; H, 8.81; N, 17.48. Found: C, 63.82; H, 8.61; N, 17.94.
3.1.1.11. 2-Isonicotinoyl-N-undecylhydrazine-1-carboxamide (2k). Yield 80 % (method A); mp
169-170 °C. IR (ATR): 3275, 3083, 2924, 2853, 1671, 1553, 1522, 1487, 1436, 1406, 1375, 1341,
1
1303, 1221, 1133, 1064, 997, 913, 852, 758, 721, 670, 657 cm-1. H NMR (DMSO, 500 MHz): δ
10.39 (1H, s, NH), 8.74 (2H, d, J = 6.4 Hz, H2, H6), 7.91 (1H, s, NH), 7.79 (2H, d, J = 5.9 Hz, H3,
H5), 6.53 (1H, t, J = 5.7 Hz, NH), 3.05-2.95 (2H, m, C1H2), 1.48-1.16 (18H, m, C2H2, C3H2, C4H2,
13
C5H2, C6H2, C7H2, C8H2, C9H2, C10H2), 0.86 (3H, t, J = 6.9 Hz, CH3). C NMR (DMSO, 126
MHz): δ 164.77, 158.21, 150.40, 139.96, 121.64, 40.44, 31.52, 30.04, 29.26, 29.23, 29.18, 29.03,
28.93, 26.50, 22.30, 14.15. Anal. Calcd for C18H30N4O2 (334.46): C, 64.64; H, 9.04; N, 16.75.
Found: C, 64.86; H, 8.89; N, 16.97.
3.1.1.12. N-Dodecyl-2-isonicotinoylhydrazine-1-carboxamide (2l). Yield 83 % (method A); mp
167-169 °C. IR (ATR): 3291, 3088, 2923, 2853, 1663, 1554, 1509, 1486, 1436, 1407, 1376, 1348,
1
1296, 1220, 1136, 1063, 997, 901, 850, 751, 721, 692 cm-1. H NMR (DMSO, 500 MHz): δ 10.38
(1H, s, NH), 8.76 (2H, d, J = 6.4 Hz, H2, H6), 7.90 (1H, s, NH), 7.80 (2H, d, J = 5.6 Hz, H3, H5),
6.52 (1H, t, J = 5.7 Hz, NH), 3.05-2.96 (2H, m, C1H2), 1.47-1.16 (20H, m, C2H2, C3H2, C4H2, C5H2,
13
C6H2, C7H2, C8H2, C9H2, C10H2, C11H2), 0.85 (3H, t, J = 6.6 Hz, CH3). C NMR (DMSO, 126
MHz): δ 164.85, 158.19, 150.37, 139.93, 121.61, 40.29, 31.48, 30.08, 29.25, 29.22, 29.19, 29.15,
29.01, 28.89, 26.48, 22.26, 14.11. Anal. Calcd for C19H32N4O2 (348.49): C, 65.48; H, 9.26; N,
16.08. Found: C, 65.82; H, 8.98; N, 15.95.

ACCEPTED MANUSCRIPT
3.1.1.13. 2-Isonicotinoyl-N-tridecylhydrazine-1-carboxamide (2m). Yield 68 % (method C); mp
167-169 °C. IR (ATR): 3293, 3087, 2922, 2852, 1664, 1555, 1509, 1486, 1436, 1407, 1376, 1348,
1
1297, 1220, 1145, 1063, 997, 901, 850, 757, 721, 687 cm-1. H NMR (DMSO, 300 MHz): δ 10.38
(1H, s, NH), 8.75 (2H, d, J = 6.8 Hz, H2, H6), 7.92 (1H, s, NH), 7.77 (2H, d, J = 5.4 Hz, H3, H5),
6.55 (1H, t, J = 5.7 Hz, NH), 3.05-2.96 (2H, m, C1H2), 1.49-1.17 (22H, m, C2H2, C3H2, C4H2, C5H2,
C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, C12H2), 0.85 (3H, t, J = 6.6 Hz, CH3). 13C NMR (DMSO, 75
MHz): δ 164.93, 158.23, 150.41, 139.97, 121.66, 40.28, 31.52, 30.05, 29.29, 29.25, 29.23, 29.21,
29.18, 29.06, 28.94, 26.51, 22.32, 14.17. Anal. Calcd for C20H34N4O2 (362.52): C, 66.26; H, 9.45;
N, 15.46. Found: C, 66.51; H, 9.34; N, 15.73.
3.1.1.14. 2-Isonicotinoyl-N-tetradecylhydrazine-1-carboxamide (2n). Yield 72 % (method A); mp
167-169 °C. IR (ATR): 3294, 3087, 2922, 2852, 1664, 1632, 1554, 1513, 1484, 1433, 1408, 1377,
1
1349, 1296, 1220, 1145, 1063, 998, 902, 850, 758, 721, 692, 665 cm-1. H NMR (DMSO, 500
MHz): δ 10.37 (1H, s, NH), 8.72 (2H, d, J = 6.9 Hz, H2, H6), 7.90 (1H, s, NH), 7.77 (2H, d, J =
5.6 Hz, H3, H5), 6.53 (1H, t, J = 5.7 Hz, NH), 3.05-2.94 (2H, m, C1H2), 1.50-1.18 (24H, m, C2H2,
C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, ,C12H2, C13H2), 0.88 (3H, t, J = 6.4 Hz,
CH3). 13C NMR (DMSO, 126 MHz): δ 164.90, 158.18, 150.37, 139.96, 121.62, 40.25, 31.47, 30.01,
29.25, 29.23, 29.22, 29.20, 29.17, 29.14, 29.01, 28.90, 26.48, 22.27, 14.12. Anal. Calcd for
C21H36N4O2 (376.55): C, 66.99; H, 9.64; N, 14.88. Found: C, 66.72; H, 9.75; N, 14.52.
3.1.1.15. 2-Isonicotinoyl-N-pentadecylhydrazine-1-carboxamide (2o). Yield 65 % (method C); mp
168-170 °C. IR (ATR): 3319, 3024, 2921, 2849, 1645, 1555, 1481, 1408, 1382, 1338, 1287, 1065,
1005, 919, 846, 724, 672 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.47 (1H, s, NH), 8.73 (2H, d, J =
6.5 Hz, H2, H6), 7.93 (1H, s, NH), 7.74 (2H, d, J = 5.6 Hz, H3, H5), 6.58 (1H,t, J = 6.1 Hz, NH),
3.06-2.94 (2H, m, C1H2), 1.47-1.14 (26H, m, C2H2, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2,
C10H2, C11H2, ,C12H2, C13H2, C14H2), 0.85 (3H, t, J = 6.2 Hz, CH3). 13C NMR (DMSO, 126 MHz): δ
164.83, 158.02, 150.29, 139.89, 121.37, 40.28, 31.50, 30.06, 29.26, 29.24, 29.21, 29.20, 29.16,
29.12, 29.08, 29.03, 28.92, 26.54, 22.97, 14.08. Anal. Calcd for C22H38N4O2 (390.57): C, 67.66; H,
9.81; N, 14.35. Found: C, 67.98; H, 9.65; N, 14.63.
3.1.1.16. N-Hexadecyl-2-isonicotinoylhydrazine-1-carboxamide (2p). Yield 79 % (method A); mp
169-170 °C. IR (ATR): 3314, 3024, 2920, 2849, 1647, 1557, 1508, 1481, 1409, 1338, 1280, 1233,
1065, 994, 846, 755, 724, 661 cm-1. 1H NMR (DMSO, 500 MHz): δ 10.38 (1H, s, NH), 8.72 (2H, d,
J = 6.5 Hz, H2, H6), 7.90 (1H, s, NH), 7.75 (2H, d, J = 5.8 Hz, H3, H5), 6.53 (1H, t, J = 5.9 Hz,
NH), 3.07-2.95 (2H, m, C1H2), 1.48-1.08 (28H, m, C2H2, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2,
C9H2, C10H2, C11H2, ,C12H2, C13H2, C14H2, C15H2), 0.87 (3H, t, J = 6.3 Hz, CH3). 13C NMR (DMSO,
126 MHz): δ 164.68, 157.97, 150.23, 139.68, 121.40, 40.20, 31.46, 30.05, 29.27, 29.25, 29.23,
29.21, 29.19, 29.17, 29.15, 29.12, 29.05, 28.93, 26.48, 22.23, 14.01. Anal. Calcd for C23H40N4O2
(404.60): C, 68.28; H, 9.97; N, 13.85. Found: C, 68.33; H, 9.78; N, 13.99.
3.1.1.17. 2-Isonicotinoyl-N-octadecylhydrazine-1-carboxamide (2q). Yield 71 % (method A); mp
171-172 °C (lit.32 173-175 °C). IR (ATR): 3291, 2920, 2851, 1671, 1557, 1521, 1508, 1487, 1436,
1
1406, 1375, 1340, 1296, 1221, 1136, 1063, 998, 901, 850, 756, 721, 692 cm-1. H NMR (DMSO,
500 MHz): δ 10.39 (1H, s, NH), 8.78 (2H, d, J = 6.6 Hz, H2, H6), 7.93 (1H, s, NH), 7.72 (2H, d, J =
5.5 Hz, H3, H5), 6.54 (1H,t, J = 6.1 Hz, NH), 3.09-2.93 (2H, m, C1H2), 1.47-1.09 (32H, m, C2H2,
C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, ,C12H2, C13H2, C14H2, C15H2, C16H2,

ACCE1P3TED MANUSCRIPT
C17H2), 0.86 (3H, t, J = 6.4 Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.70, 158.07, 150.30,
139.49, 121.57, 40.22, 31.48, 30.05, 29.25, 29.23, 29.22, 29.20, 29.18, 29.17, 29.15, 29.13, 29.11,
29.08, 29.05, 28.92, 26.49, 22.30, 13.99. Anal. Calcd for C25H44N4O2 (432.65): C, 69.40; H, 10.25;
N, 12.95. Found: C, 68.51; H, 10.01; N, 12.76.
3.1.2. Cyclization to 5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines 3
Triphenylphosphine (393.4 mg, 1.5 mmol) was added to the stirred suspension of 1,2-dibromo-
1,1,2,2-tetrachloroethane (271.1 mg, 0.83 mmol) and the appropriate N-alkyl-2-
isonicotinoylhydrazine-1-carboxamide 2 (0.75 mmol) in anhydrous acetonitrile or tetrahydrofuran
(4 mL) at room temperature. The reaction mixture was stirred at room temperature for 10 min and
cooled to 0 °C. Then, triethylamine (459 µL, 3.3 mmol) was added dropwise, and the stirring was
continued for an additional 10 hours. A solid phase was filtered off. The resulting liquid was
evaporated until dryness and dissolved in ethyl acetate. Undissolved solid was filtered off and
recrystallized from acetonitrile, if necessary, giving the pure oxadiazole products 3. Alternatively,
they were purified using column chromatography and the mixture toluene/ethyl acetate 2:1 as the
eluent.
3.1.2.1. N-Methyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3a). Yield 57 %; mp 146-147 °C. IR
(ATR): 3174, 2930, 1651, 1604, 1578, 1493, 1417, 1380, 1057, 1030, 824, 748, 731, 681. 1H NMR
(CDCl3, 500 MHz): δ 8.71 (2H, d, J = 5.9 Hz, H2, H6), 7.74 (2H, d, J = 6.1 Hz, H3, H5), 5.85 (1H,
q, J = 4.8 Hz, NH), 3.12 (3H, d, J = 4.1 Hz, CH3). 13C NMR (CDCl3, 126 MHz): δ 164.66, 156.95,
150.46, 131.49, 119.19, 29.83. Anal. Calcd for C8H8N4O (176.18): C, 54.54; H, 4.58; N, 31.80.
Found: C, 54.32; H, 4.69; N, 31.62.
3.1.2.2. N-Ethyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3b). Yield 66 %; mp 147-148 °C (lit.34
131-132 °C). IR (ATR): 3191, 3139, 2977, 2866, 1654, 1628, 1579, 1494, 1463, 1451, 1421, 1377,
1
1061, 1038, 830, 756, 730, 681. H NMR (CDCl3, 500 MHz): δ 8.72 (2H, d, J = 6.0 Hz, H2, H6),
7.73 (2H, d, J = 6.1 Hz, H3, H5), 5.84 (1H, t, J = 6.0 Hz, NH), 3.55-3.47 (2H, m, CH2), 1.33 (3H, t,
13
J = 7.2 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 163.97, 156.82, 150.46, 131.52, 119.15, 38.49,
14.96. Anal. Calcd for C9H10N4O (190.21): C, 56.83; H, 5.30; N, 29.46. Found: C, 56.77; H, 5.60;
N, 29.49.
3.1.2.3. N-Propyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3c). Yield 58 %; mp 112-113 °C. IR
(ATR): 3212, 2967, 1638, 1614, 1578, 1508, 1486, 1458, 1417, 1394, 1048, 827, 753, 721, 696. 1H
NMR (CDCl3, 500 MHz): δ 8.71 (2H, d, J = 6.1 Hz, H2, H6), 7.78 (2H, d, J = 5.7 Hz, H3, H5),
5.82 (1H, t, J = 5.6 Hz, NH), 3.48-3.36 (2H, m, C1H2), 1.69-1.60 (2H, m, C2H2), 0.92 (3H, t, J = 7.1
13
Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.27, 156.52, 150.38, 131.46, 119.18, 45.86, 22.98,
11.34. Anal. Calcd for C10H12N4O (204.23): C, 58.81; H, 5.92; N, 27.43. Found: C, 58.78; H, 5.73;
N, 27.13.
3.1.2.4. N-Butyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3d). Yield 64 %; mp 106-108°C. IR
1
(ATR): 3240, 2936, 1627, 1606, 1576, 1458, 1422, 1357, 1027, 830, 754, 722, 696 cm-1. H NMR
(CDCl3, 500 MHz): δ 8.70 (2H, d, J = 6.1 Hz, H2, H6), 7.76 (2H, d, J = 5.9 Hz, H3, H5), 5.68 (1H,
t, J = 5.4 Hz, NH), 3.50-3.39 (2H, m, C1H2), 1.58-1.50 (2H, m, C2H2), 1.44-1.29 (2H, m, C3H2),
13
0.91 (3H, t, J = 6.9 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.38, 156.53, 150.32, 131.58,

ACCEPTED MANUSCRIPT
118.96, 43.38, 25.65, 19.20, 13.64. Anal. Calcd for C11H14N4O (218.26): C, 60.53; H, 6.47; N,
25.67. Found: C, 60.85; H, 6.32; N, 25.44.
3.1.2.5. N-Pentyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3e). Yield 52 %; mp 105-106 °C. IR
1
(ATR): 2958, 2930, 2859, 1625, 1605, 1574, 1437, 1414, 1373, 1044, 828, 749, 722, 695 cm-1. H
NMR (CDCl3, 300 MHz): δ 8.74 (2H, d, J = 6.1 Hz, H2, H6), 7.74 (2H, d, J = 5.7 Hz, H3, H5),
5.56 (1H, t, J = 5.8 Hz, NH), 3.49-3.38 (2H, m, C1H2), 1.76-1.63 (2H, m, C2H2), 1.45-1.27 (4H, m,
C3H2, C4H2), 0.91 (3H, t, J = 7.0 Hz, CH3). 13C NMR (CDCl3, 75 MHz): δ 164.12, 156.58, 150.49,
131.68, 119.21, 43.68, 29.25, 28.75, 22.26, 13.94. Anal. Calcd for C12H16N4O (232.29): C, 62.05;
H, 6.94; N, 24.12. Found: C, 62.28; H, 6.67; N, 24.50.
3.1.2.6. N-Hexyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3f). Yield 58 %; mp 105-106 °C. IR
(ATR): 3333, 2953, 2923, 2861, 1611, 1573, 1500, 1480, 1415, 1389, 1057, 1032, 829, 740, 726,
696 cm-1. 1H NMR (CDCl3, 500 MHz): δ 8.71 (2H, d, J = 6.1 Hz, H2, H6), 7.73 (2H, d, J = 6.1 Hz,
H3, H5), 5.70 (1H, t, J = 5.9 Hz, NH), 3.49-3.41 (2H, m, C1H2), 1.74-1.63 (2H, m, C2H2), 1.45-1.25
(6H, m, C3H2, C4H2, C5H2), 0.88 (3H, t, J = 7.2 Hz, CH3). 13C NMR (CDCl3, 126 MHz): δ 164.20,
156.75, 150.41, 131.54, 119.19, 43.66, 31.51, 30.33, 26.29, 22.49, 13.95. Anal. Calcd for
C13H18N4O (246.31): C, 63.39; H, 7.37; N, 22.75. Found: C, 63.54; H, 7.17; N, 22.52.
3.1.2.7. N-Heptyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3g). Yield 61 %; mp 102-103 °C. IR
(ATR): 3333, 2955, 2922, 2857, 1612, 1573, 1500, 1481, 1417, 1392, 1058, 1040, 829, 754, 748,
1
721, 696 cm-1. H NMR (DMSO, 500 MHz): δ 8.75 (2H, d, J = 6.7 Hz, H2, H6), 7.91 (1H, t, J =
6.1 Hz, NH), 7.70 (2H, d, J = 6.5 Hz, H3, H5), 3.33-3.21 (2H, m, C1H2), 1.57-1.48 (2H, m, C2H2),
1.40-1.22 (8H, m, C3H2, C4H2, C5H2, C6H2), 0.84 (3H, t, J = 7.3 Hz, CH3). 13C NMR (DMSO, 126
MHz): δ 164.88, 155.16, 150.35, 131.94, 120.60, 44.74, 31.44, 29.76, 28.63, 26.41, 22.21, 14.12.
Anal. Calcd for C14H20N4O (260.34): C, 64.59; H, 7.74; N, 21.52. Found: C, 64.48; H, 7.92; N,
21.33.
3.1.2.8. N-Octyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3h). Yield 66 %; mp 96-98 °C. IR
(ATR): 3285, 2954, 2922, 2850, 1611, 1573, 1480, 1470, 1417, 1389, 1053, 1034, 829, 741, 730,
1
721, 695 cm-1. H NMR (DMSO, 500 MHz): δ 8.74 (2H, d, J = 6.7 Hz, H2, H6), 8.05 (1H, t, J =
5.8 Hz, NH), 7.64 (2H, d, J = 6.5 Hz, H3, H5), 3.30-3.23 (2H, m, C1H2), 1.59-1.51 (2H, m, C2H2),
13
1.38-1.21 (10H, m, C3H2, C4H2, C5H2, C6H2, C7H2), 0.84 (3H, t, J = 7.1 Hz, CH3). C NMR
(DMSO, 126 MHz): δ 164.68, 155.95, 150.83, 131.63, 118.88, 42.78, 31.40, 29.72, 28.92, 28.85,
26.41, 22.25, 14.09. Anal. Calcd for C15H22N4O (274.37): C, 65.67; H, 8.08; N, 20.42. Found: C,
65.42; H, 8.25; N, 20.35.
3.1.2.9. N-Nonyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3i). Yield 65 %; mp 95-96 °C. IR
1
(ATR): 3334, 2921, 2853, 1613, 1573, 1482, 1415, 1044, 829, 752, 741, 721, 697 cm-1. H NMR
(DMSO, 500 MHz): δ 8.72 (2H, d, J = 6.4 Hz, H2, H6), 8.01 (1H, t, J = 6.1 Hz, NH), 7.63 (2H, d, J
= 6.4 Hz, H3, H5), 3.28-3.19 (2H, m, C1H2), 1.58-1.51 (2H, m, C2H2), 1.35-1.21 (12H, m, C3H2,
13
C4H2, C5H2, C6H2, C7H2, C8H2), 0.83 (3H, t, J = 7.0 Hz, CH3). C NMR (DMSO, 126 MHz): δ
164.32, 155.97, 150.80, 131.52, 118.87, 42.73, 31.26, 29.69, 29.19, 28.96, 28.85, 26.39, 22.16,
13.95. Anal. Calcd for C16H24N4O (288.40): C, 66.64; H, 8.39; N, 19.43. Found: C, 66.39; H, 8.12;
N, 19.55.

ACCEPTED MANUSCRIPT
3.1.2.10. N-Decyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3j). Yield 55 %; mp 97-98 °C. IR
(ATR): 3275, 2954, 2918, 2849, 1613, 1573, 1493, 1471, 1416, 1390, 1058, 1049, 829, 741, 733,
1
720, 695 cm-1. H NMR (DMSO, 500 MHz): δ 8.78 (2H, d, J = 6.4 Hz, H2, H6), 8.05 (1H, t, J =
6.2 Hz, NH), 7.70 (2H, d, J = 6.4 Hz, H3, H5), 3.26-3.19 (2H, m, C1H2), 1.59-1.50 (2H, m, C2H2),
1.38-1.22 (14H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2), 0.82 (3H, t, J = 7.0 Hz, CH3). 13C
NMR (DMSO, 126 MHz): δ 164.37, 156.01, 150.90, 131.63, 118.93, 42.75, 31.48, 29.70, 29.20,
29.14, 28.91, 28.86, 26.36, 22.28, 14.06. Anal. Calcd for C17H26N4O (302.42): C, 67.52; H, 8.67; N,
18.53. Found: C, 67.48; H, 8.98; N, 18.59.
3.1.2.11. 5-(Pyridin-4-yl)-N-undecyl-1,3,4-oxadiazol-2-amine (3k). Yield 63 %; mp 102-103 °C. IR
(ATR): 3335, 2953, 2923, 2859, 1611, 1573, 1501, 1480, 1468, 1413, 1390, 1056, 1031, 828, 740,
725, 696 cm-1. H NMR (DMSO, 500 MHz): δ 8.73 (2H, d, J = 6.5 Hz, H2, H6), 8.03 (1H, t, J =
6.2 Hz, NH), 7.65 (2H, d, J = 6.4 Hz, H3, H5), 3.28-3.20 (2H, m, C1H2), 1.59-1.51 (2H, m, C2H2),
1.39-1.22 (16H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2), 0.83 (3H, t, J = 6.9 Hz,
CH3). 13C NMR (DMSO, 126 MHz): δ 164.33, 155.96, 150.84, 131.44, 118.88, 42.74, 31.02, 29.74,
29.18, 29.14, 29.06, 28.93, 28.86, 25.97, 22.19, 13.99. Anal. Calcd for C18H28N4O (316.45): C,
68.32; H, 8.92; N, 17.71. Found: C, 68.12; H, 8.76; N, 17.65.
3.1.2.12. N-Dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3l). Yield 68 %; mp 105-107 °C. IR
(ATR): 3331, 2955, 2918, 2851, 1612, 1573, 1501, 1481, 1471, 1414, 1389, 1053, 829, 741, 730,
1
719, 697 cm-1. H NMR (DMSO, 500 MHz): δ 8.75 (2H, d, J = 6.5 Hz, H2, H6), 8.03 (1H, t, J =
6.0 Hz, NH), 7.72 (2H, d, J = 6.5 Hz, H3, H5), 3.28-3.19 (2H, m, C1H2), 1.60-1.51 (2H, m, C2H2),
1.38-1.20 (18H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2), 0.84 (3H, t, J = 7.0
13
Hz, CH3). C NMR (DMSO, 126 MHz): δ 164.38, 156.18, 150.78, 131.58, 119.11, 42.82, 31.38,
29.75, 29.21, 29.17, 29.13, 29.08, 28.96, 28.87, 26.23. 22.16, 14.10. Anal. Calcd for C19H30N4O
(330.48): C, 69.05; H, 9.15; N, 16.95. Found: C, 69.14; H, 9.22; N, 16.72.
3.1.2.13. 5-(Pyridin-4-yl)-N-tridecyl-1,3,4-oxadiazol-2-amine (3m). Yield 54 %; mp 105-107 °C.
IR (ATR): 3325, 2955, 2920, 2851, 1613, 1573, 1507, 1481, 1470, 1417, 1388, 1060, 1035, 830,
754, 721, 696 cm-1. 1H NMR (CDCl3, 500 MHz): δ 8.69 (2H, d, J = 5.9 Hz, H2, H6), 7.70 (2H, d, J
= 6.3 Hz, H3, H5), 5.63 (1H, t, J = 5.8 Hz, NH), 3.49-3.41 (2H, m, C1H2), 1.71-1.66 (2H, m, C2H2),
1.44-1.19 (20H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, C12H2), 0.88 (3H, t, J
13
= 6.7 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.11, 156.82, 150.51, 131.55, 119.16, 43.69,
31.87, 29.64, 29.62, 29.59, 29.57, 29.53, 29.49, 29.31, 29.19, 26.65, 22.64, 14.07. Anal. Calcd for
C20H32N4O (344.50): C, 69.73; H, 9.36; N, 16.26. Found: C, 69.55; H, 9.16; N, 16.43.
3.1.2.14. 5-(Pyridin-4-yl)-N-tetradecyl-1,3,4-oxadiazol-2-amine (3n). Yield 59 %; mp 109-111 °C.
IR (ATR): 3275, 2955, 2920, 2850, 1614, 1574, 1472, 1418, 1390, 1059, 830, 741, 732, 720, 697
cm-1. 1H NMR (CDCl3, 500 MHz): δ 8.73 (2H, d, J = 6.4 Hz, H2, H6), 7.75 (2H, d, J = 6.4 Hz, H3,
H5), 5.55 (1H, t, J = 5.8 Hz, NH), 3.50-3.42 (2H, m, C1H2), 1.72-1.66 (2H, m, C2H2) 1.43-1.23
(22H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, C12H2, C13H2), 0.88 (3H, t, J =
13
6.8 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.07, 156.84, 150.51, 131.54, 119.16, 43.70,
31.87, 29.64, 29.62, 29.60, 29.57, 29.56, 29.53, 29.48. 29.30, 29.18, 26.64, 22.64, 14.07. Anal.
Calcd for C21H34N4O (358.53): C, 70.35; H, 9.56; N, 15.63. Found: C, 70.11; H, 9.75; N, 15.83.

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3.1.2.15. N-Pentadecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3o). Yield 60 %; mp 108-109 °C.
IR (ATR): 3327, 2955, 2919, 2850, 1613, 1573, 1481, 1471, 1417, 1390, 1057, 829, 740, 731, 719,
696 cm-1. 1H NMR (CDCl3, 500 MHz): δ 8.73 (2H, d, J = 5.7 Hz, H2, H6), 7.78 (2H, d, J = 6.3 Hz,
H3, H5), 5.50 (1H, t, J = 5.9 Hz, NH), 3.50-3.42 (2H, m, C1H2), 1.73-1.67 (2H, m, C2H2), 1.42-1.21
(24H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, C12H2, C13H2, C14H2), 0.88
13
(3H, t, J = 6.8 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.06, 156.80, 150.35, 131.67, 119.21,
43.71, 31.88, 29.65, 29.64, 29.62, 29.61, 29.59, 29.57, 29.53, 29.49, 29.31, 29.20, 26.64, 22.65,
14.08. Anal. Calcd for C22H36N4O (372.56): C, 70.93; H, 9.74; N, 15.04. Found: C, 70.72; H, 9.53;
N, 15.27.
3.1.2.16. N-Hexadecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3p). Yield 63 %; mp 109-111 °C.
IR (ATR): 3330, 2955, 2918, 2851, 1612, 1572, 1501, 1481, 1471, 1415, 1389, 1052, 1042, 830,
741, 730, 718, 697 cm-1. 1H NMR (CDCl3, 500 MHz): δ 8.71 (2H, d, J = 6.4 Hz, H2, H6), 7.75 (2H,
d, J = 6.6 Hz, H3, H5), 5.49 (1H, t, J = 5.9 Hz, NH), 3.49-3.40 (2H, m, C1H2), 1.73-1.67 (2H, m,
C2H2), 1.45-1.21 (26H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, C12H2, C13H2,
13
C14H2, C15H2), 0.88 (3H, t, J = 6.7 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.07, 156.86,
150.53, 131.53, 119.17, 43.71, 31.89, 29.68, 29.66, 29.63, 29.62, 29.60, 29.59, 29.57, 29.54, 29.50,
29.32, 29.19, 26.65, 22.65, 14.08. Anal. Calcd for C23H38N4O (386.58): C, 71.46; H, 9.91; N, 14.49.
Found: C, 71.59; H, 10.04; N, 14.53.
3.1.2.17. N-Octadecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3q). Yield 65 %; mp 114-115 °C.
IR (ATR): 3293, 2954, 2917, 2850, 1611, 1572, 1507, 1481, 1471, 1414, 1388, 1050, 829, 741,
730, 717, 696 cm-1. 1H NMR (CDCl3, 500 MHz): δ 8.72 (2H, d, J = 6.3 Hz, H2, H6), 7.80 (2H, d, J
= 6.3 Hz, H3, H5), 5.48 (1H, t, J = 5.9 Hz, NH), 3.41-3.32 (2H, m, C1H2), 1.74-1.68 (2H, m, C2H2)
1.45-1.20 (30H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2, C10H2, C11H2, C12H2, C13H2, C14H2,
13
C15H2, C16H2, C17H2), 0.88 (3H, t, J = 6.5 Hz, CH3). C NMR (CDCl3, 126 MHz): δ 164.08,
156.83, 149.84, 131.41, 118.98, 43.73, 31.91, 29.73, 29.69, 29.68, 29.66, 29.64, 29.63, 29.61,
29.59, 29.58, 29.55, 29.50, 29.33, 29.19, 26.66. 22.58, 14.08. Anal. Calcd for C25H42N4O (414.64):
C, 72.42; H, 10.21; N, 13.51. Found: C, 72.64; H, 10.33; N, 13.28.
4.2. Biological Activity
4.2.1. In vitro Antimycobacterial Susceptibility Determination
Both series were evaluated for their in vitro antimycobacterial activity against M. tuberculosis
331/88 (i.e., H37Rv; dilution of this strain was 10-3), Mycobacterium avium 330/88 (resistant to
INH, RIF, ofloxacin (OFX) and EMB; dilution 10-5) and two strains of M. kansasii: 235/80
(dilution 10-4) and the clinically isolated strain 6509/96 (dilution 10-5). The used method is
described in ref. [35]. The following concentrations were used: 1000, 500, 250, 125, 62.5, 32, 16, 8,
4, 2, 1, 0.5 and 0.25 µM. MIC (reported in µM) was the lowest concentration at which the complete
inhibition of mycobacterial growth occurred. Isoniazid (INH) as both a first-line oral
antimycobacterial drug and the synthetic precursor was used as the reference compound.
The most active derivatives (2a-c, 3l) were evaluated under similar conditions against four MDR-
TB strains and one XDR-TB strain (dilution 10-3) with different resistance patterns. All strains were
resistant to INH, RIF, rifabutine, and streptomycin (STM); an additional resistance was present in
some cases: 234/2005 resistant additionally to EMB; Praha 1 resistant additionally to EMB, and
clofazimine; Praha 4 to EMB, OFX, and clofazimine; and Praha 131 with an additional resistance to

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EMB, OFX, gentamicin and amikacin (i.e., XDR-TB strain). The following concentrations were
used: 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, and 1 µM.
4.2.2. Cytotoxicity Evaluation (HepG2 cells)35
All of the carboxamides 2 and the selected 1,3,4-oxadiazoles (3j-l) were tested for their cytotoxicity
in the human hepatocellular liver carcinoma cell line HepG2 (passage 2-4; ECACC, Salisbury, UK)
using a standard colorimetric method that involves measuring a tetrazolium salt reduction
(CellTiter(R) 96 AQueous One Solution Assay, Promega G3580, Fitchburg, USA).
The cells were routinely cultured in Eagle’s minimum essential media supplemented with 10%
foetal bovine serum, 1%
L-glutamine solution, and a non-essential amino acid solution. The
investigated compounds were dissolved in a very small volume of DMSO, and a small volume was
added to the cell culture. The tested compounds were prepared in triplicate at eight incubation
concentrations. The following types of controls were included: determination of 100% viability and
0% viability (the cells treated by 10% DMSO), no cell control, control for the determination of
possible interaction of tested compounds with reagents, control of the setting of incubation medium
and the control of the toxicity of DMSO.
The results are expressed as the inhibitory concentration that reduces cell viability to 50% of the
maximal (control) viability (IC50). IC50 was calculated in each of the tested substances using
GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA) and Microsoft Excel
2010.
4.2.3. Analysis of sensitivity of Mtb. H37Ra strain overproducing InhA to 2a and 3l
InhA protein was overproduced in Mtb. H37Ra using pMV261-InhA construct as already described.
Sensitivity of InhA overproducing strain, as well as a control strain carrying an empty vector to
compounds 2a and 3l were analysed by drop dilution method. Both cultures grown in 7H9 broth
supplemented with albumin-dextrose-catalase and 0.05% Tween 80 were adjusted to O.D.(600) 0.5
and 4 µl aliquots of 100, 10-1, 10-2 and 10-3 dilutions were dropped on 7H11 agar supplemented with
oleic acid-albumin-dextrose-catalase and different concentrations of tested compounds dissolved in
DMSO (2 % final concentration). Plates were incubated for 25 days at 37 °C.
4.2.4. Determination of mode of action of 2a and 3l in Mtb. H37Ra
The mechanism of action of 2a was analyzed by metabolic labeling of Mtb. H37Ra strain with C
14
glucose. Mtb. H37Ra culture was grown in GAS media supplemented by 0.025% tyloxapol at 37 °C
till O.D. (600) reached 0.307. Then tested compound dissolved in DMSO and INH were added in
final concentrations 40 µM and 36.5 µM. The final concentration of DMSO was kept at 1%. 14C
glucose (specific activity 290 mCi/mmol, ARC) in the final concentration of 1 µCi/mL was added
to each of the cultures after 1 h of cultivation with drugs and the cells were cultivated for next 24 h.
Cells harvested from 18 ml cultures were 15 min incubated with 98% ethanol at 70 °C. Samples
were centrifuged for 10 min at 10 000 x g and resulting pellets were delipidated by two 1 h
extractions with chloroform: methanol (2:1, v/v) at 56 °C. These extracts were combined with
ethanol extracts, dried under the stream of N2 and subjected to biphasic Folch washing. The bottom
organic phase was dried and dissolved in chloroform: methanol (2:1) – 0.2 ml per 40 mg of
harvested cells. 10 µL of the lipid extracts were loaded on thin-layer chromatography (TLC) silica
gel plates F254 (Merck) and the lipids were separated in the mixture of chloroform: methanol:
water (20: 4: 0.5) and visualized by autoradiography. Fatty acid methyl esters (FAME) and mycolic

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acids methyl esters (MAME) were prepared from 100 µl of lipid fractions as previously described36.
Dried extracts were dissolved in chloroform/methanol (2:1) and loaded on TLC plates. FAME and
different forms of MAME were separated by three runs in n-hexane: ethyl acetate (95:5) and
detected by autoradiography.
For investigation of the mechanism of action of 3l Mtb. H37Ra culture was grown in 7H9 broth
supplemented with albumin-dextrose-catalase and 0.05% Tween 80. When O.D. (600) reached
0.263, compound 3l was added in final concentrations 4 and 8 µM. After 24 h of cultivation with
shaking (120 rpm) 14C acetate (specific activity 106 mCi/mmol, ARC) in the final concentration of
0.5 µCi/mL was added to each of the cultures and the cells were cultivated for next 24 h. Lipids
were extracted and analyzed as described above. Following solvent systems were used for their
separation: chloroform: methanol: water (20:4:0.5); chloroform: methanol: ammonium hydroxide:
water (65:25:0.5:4) and petroleum ether: ethyl acetate (98:2, three runs). FAME and MAME were
prepared from whole cells and analyzed as described above, alternatively on TLC plates
impregnated in 5% AgNO3 and activated at 100 °C for 1h.
5. Conclusions
In summary, a series of seventeen N-alkyl-2-isonicotinoylhydrazine-1-carboxamides and their
cyclic analogues, N-alkyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amines, was synthesized and evaluated
against drug-susceptible and multidrug-resistant Mycobacterium tuberculosis strains and three
strains of nontuberculous mycobacteria. The most active compounds against Mtb. comparable to
isoniazid were 2-isonicotinoylhydrazine-1-carboxamides with a shorter alkyl. They also were found
to be active against NTM as well as in vitro non-toxic compounds in HepG2 cells. 2-Isonicotinoyl-
N-methylhydrazine-1-carboxamide as the most active and selective anti-TB agent was chosen for an
investigation of mechanism of action. It targets InhA and inhibits the synthesis of mycolic acids
inside Mtb. cells similarly to INH.
N-Decyl and dodecyl alkyls represent optimal substituents for 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-
amine scaffold. N-Dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine affects the growth of both
susceptible, MDR- and XDR-TB strains at an identical concentration with no cross-resistance to
established antimycobacterial drugs. Its mechanism of action is different from those of INH. It was
proved that this oxadiazole is definitely not a prodrug of INH, thus constituting a new potentially
promising group of antimycobacterial agents.
Conflicts of interest
None of the authors has declared any conflict of interest.
Acknowledgments
The work was supported by the Czech Science Foundation [grant number 17-27514Y]; the Charles
University [project SVV 260 401, 260 414]; and Slovak Grant Agency [grant number VEGA
1/0284/15].
We thank to prof. Williams R. Jacobs, Jr. for pMV261-InhA construct and to P. Mašek and D.
Mašek for the language help provided.
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ACCEPTED MANUSCRIPT
Highlights
•2-Isonicotinoylhydrazine-1-carboxamides and 1,3,4-oxadiazoles were synthesised.
•Activity against M. tuberculosis and nontuberculous mycobacteria (MIC ≥0.5 µM).
•Low or no cytotoxicity for HepG2 cells.
•2-Isonicotinoyl-N-methylhydrazine-1-carboxamide targets InhA like isoniazid.
•5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-amines inhibit multidrug-resistant strains.