N-substituted 2-isonicotinoylhydrazinecarboxamides-new antimycobacterial active molecules

Article abstract of DOI:10.3390/molecules19043851

This report presents a new modification of the isoniazid (INH) structure linked with different anilines via a carbonyl group obtained by two synthetic procedures and with N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation. All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses and were evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4- octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1-2 μM. Among the halogenated derivatives, the best anti-tuberculosis activity was found for 2-isonicotinoyl-N-(2,4,6-trichlorophenyl) hydrazinecarboxamide (MIC = 4 μM). In silico modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl substituents are advantageous for the interactions and affinity to InhA. Most of the hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited significant activity against INH-resistant nontuberculous mycobacteria. gfjh+l;kfldf.

Full text of DOI:10.3390/molecules19043851

Molecules 2014, 19, 3851-3868; doi:10.3390/molecules19043851
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
N-Substituted 2-Isonicotinoylhydrazinecarboxamides — New
Antimycobacterial Active Molecules
Zuzana Rychtarčíková 1, Martin Krátký 1, Martin Gazvoda 2, Markéta Komlóová 1,
Slovenko Polanc 2,*, Marijan Kočevar 2, Jiřina Stolaříková 3 and Jarmila Vinšová 1,*
1
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; E-Mails: rychtarz@faf.cuni.cz (Z.R.);
martin.kratky@faf.cuni.cz (M.K.); komlm3aa@faf.cuni.cz (M.K.)
2
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5,
SI-1000 Ljubljana, Slovenia; E-Mails: martin.gazvoda@fkkt.uni-lj.si (M.G.);
marijan.kocevar@fkkt.uni-lj.si (M.K.)
3
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in
Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic; E-Mail: Jirina.Stolarikova@zu.cz
* Authors to whom correspondence should be addressed; E-Mails: Slovenko.Polanc@fkkt.uni-lj.si (S.P.);
jarmila.vinsova@faf.cuni.cz (J.V.); Tel.: +386-1-24-19-236 (S.P.); Fax: +386-1-24-19-220 (S.P.);
Tel.: +420-495-067-343 (J.V.); Fax: +420-495-067-166 (J.V.).
Received: 28 February 2014; in revised form: 21 March 2014 / Accepted: 24 March 2014 /
Published: 28 March 2014
Abstract: This report presents a new modification of the isoniazid (INH) structure linked
with different anilines via a carbonyl group obtained by two synthetic procedures and with
N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation.
All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses
and were evaluated in vitro for their antimycobacterial activity against Mycobacterium
tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80
and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4-
octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of
INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1–2 μM.
Among the halogenated derivatives, the best anti-tuberculosis activity was found for
2-isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (MIC = 4 μM). In silico
modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl
substituents are advantageous for the interactions and affinity to InhA. Most of the

Molecules 2014, 19
hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited
3852
significant activity against INH-resistant nontuberculous mycobacteria.
Keywords: InhA inhibition; in silico docking; in vitro antimycobacterial activity;
2-isonicotinoylhydrazinecarboxamide; 5-(pyridine-4-yl)-1,3,4-oxadiazol-2-amine; tuberculosis
1. Introduction
Tuberculosis (TB) is one of the most dangerous infectious diseases known. Alarming WHO data [1]
mainly concerning an increasing number of multidrug-resistant (MDR-TB) and extensively
drug-resistant (XDR-TB) forms of TB, have prompted the development of new, potent, fast-acting
anti-tuberculosis drugs. Although a new drug, diarylquinoline bedaquiline (TMC-207), has been
approved recently, the treatment of multidrug-resistant TB and latent forms has still not been
satisfactorily resolved. Treatment of the MDR-TB infection requires shortening the total duration of
therapy, improving potency against resistant strains and reducing the total expenditure and toxicity [2].
Isonicotinic acid hydrazide (INH, 1) represents a unique front-line anti-tuberculosis drug with a
high specificity towards Mycobacterium tuberculosis (Mtb.). Its mechanism of action includes multiple
effects on lipids, glycolysis, biomembranes, proteins and nucleic acid synthesis. In fact, 1 is a prodrug
that has to be activated by a catalase/peroxidase (KatG) inside the mycobacterium to form an
isonicotinoyl radical and/or stable oxidative products. KatG couples the isonicotinic acyl with NADH
to form an isonicotinic acyl-NADH complex. This complex binds tightly to the enoyl-acyl carrier
protein reductase (InhA), thereby blocking the natural enoyl-AcpM substrate and inhibiting the
synthesis of mycolic acid, the main building block of the mycobacterial cell wall [3]. Among others,
these facts are briefly summarised in the review of Vinšová et al. [4]. The discovery of isoniazid was a
major milestone in the chemotherapy of tuberculosis, but the development of INH-resistance has
hampered its wide usage and therapeutic potential. The need to overcome the resistance on INH, which
has been found to correlate with five different genes katG, inhA, ahpC, kasA and ndh [5], led to a huge
number of modifications, as summarised in several review articles [4,6–8].
We have already described a variety of synthetic modifications of INH linked with another active
molecule through a methine bridge [9], by conjugation with an aniline group with electron-withdrawing
substituents [10] and via the preparation of a new type of “double” active molecules based on the
fluorinated hydrazides of a benzoic acid scaffold as an isoniazid isostere [11]. The current report is part
of our on-going research and deals with the modification of the INH structure by introducing a
carbonyl group (in place of previously described methine bridge [9,10]) as the moiety that connects
INH to other molecules. Recently, the thiocarbonyl linker between the 2-(trifluoromethyl)aniline
moiety and INH was shown to contribute to the superior activity of the modified molecule [12].
Furthermore, the conversion of INH to the corresponding 1,3,4-oxadiazoles resulted in compounds
with improved lipophilicity and activity; some of these compounds also exhibited significant activity
against INH-resistant strains [13]. Here, we focused on the activity of compounds that have an INH
linked with different anilines via a carbonyl group and N-substituted-5-(pyridine-4-yl)-1,3,4-oxadiazole-
2-amines prepared by their cyclisation.

Molecules 2014, 19
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2. Results and Discussion
2.1. Chemistry
The most straightforward method to obtain various N-substituted 2-isonicotinoylhydrazine-
carboxamides 3 involves an addition of INH (1) to aryl isocyanates 2a–v. Commercially available
isocyanates were used for the synthesis of the 4-methyl, 4-isopropyl, 4-tert-butyl, 4-n-butyl and
4-methoxy derivatives 3a–d, 3i and N-heptyl derivative 3v without a phenyl ring. To prepare the
products 3e–h and 3k–u, the required aryl isocyanates 2e–h and 2k–u were generated in situ from the
appropriate anilines 4 during treatment with triphosgene in the presence of triethylamine in dry
dichloromethane (DCM; Scheme 1). These reaction intermediates were not isolated. The synthesis,
subsequent isolation and purification gave yields within the range of 67%–97%.
Scheme 1. Synthesis of 2-isonicotinoyl-N-substituted-hydrazinecarboxamides 3.
a R = 4-Me
l R = 2-F,4-Br
m R = 2-Br,4-F
NH2
b
c
d
e
f
g
h
i
R = 4-i-Pr
R = 4-tert-Bu
R = 4-n-Bu
R = 4-n-pentyl
R = 4-n-hexyl
R = 4-n-heptyl
R = 4-n-octyl
R = 4-OMe
R = 4-CF3
n
o
p
q
r
R = 3,4-Cl2
R
R = 3-Cl,4-F
R = 3-CF3,4-Cl
R = 3-CF3,4-Br
R = 3-CF3,4-F
R = 3-F,4-Br
R = 3,5-Cl2
4
s
t
(Cl3CO)2CO
Et3N
DCM
j
k
R = 2,4-Br2
u
R = 2,4,6-Cl3
N
N=C=O
N
+
R
DCM
H
H
N
N
O
N
O
NHNH2
R
H
O
1
2a-2u
3a-3u
N
N
+
O=C=N
H
H
DCM
N
N
O
N
O
NHNH2
H
O
1
2v
3v
Abbreviation: DCM: dichloromethane.
These described products 3 can serve as starting materials for the synthesis of 2,5-disubstituted
1,3,4-oxadiazoles. Namely, we previously developed an efficient procedure for the transformation of
1-acyl-4-substituted semicarbazides into the corresponding 1,3,4-oxadiazoles via in situ formed
diazenes [14]. Some hydrazinecarboxamides 3, because they belong to the same type of compounds
mentioned above, were treated with the mixture of triphenylphosphine and 1,2-dibromo-1,1,2,2-
tetrachloroethane in the presence of triethylamine in dry acetonitrile (MeCN) to generate the expected
N-substituted-5-(pyridine-4-yl)-1,3,4-oxadiazol-2-amines 5, as depicted in Scheme 2, with yields
within the range of 33%–81%.

Molecules 2014, 19
Scheme 2. Synthesis of N-substituted-5-(pyridine-4-yl)-1,3,4-oxadiazol-2-amines 5.
3854
N
Ph3P
(BrCl2C)2
Et3N
N
H
H
H
O
N
N
N
O
N
R
MeCN
R
H
N N
O
5
3
5n R = 3,4-diCl
5s R = 3-F,4-Br
5i R = 4-OMe
5l R = 2-F,4-Br
2.2. Antimycobacterial Activity
Compounds 3 and 5 were evaluated for their in vitro antimycobacterial activity against
M. tuberculosis CNTC 331/88 (H37Rv), M. avium CNTC 330/88, M. kansasii CNTC 235/80 and
M. kansasii 6509/96 (clinically isolated from a patient). The minimal inhibitory concentration (MIC) is
the lowest concentration of the substance at which inhibition of the growth of Mycobacterium species
occurs (Table 1).
None of the synthesised compounds of type 3 exhibited a higher activity than INH (MICs 0.5–1 μM)
for Mtb. The best results were observed for the derivative 3h, which reached an almost equal in vitro
efficacy as isoniazid, with MICs of 1–2 μM. Among the halogenated molecules, the best activity was
found for the 2,4,6-trichloro derivative 3u, which had a MIC of 4 μM, followed by 2,4-dibromo and
3-CF3,4-bromo substituted molecules 3k and 3q (8 μM). These three derivatives also shared the highest
calculated ClogP values (≥2.3) of all halogenated 2-isonicotinoyl-N-phenylhydrazinecarboxamides 3.
When evaluating particular isomeric substitutions of the N-phenyl ring, we found that among the
dichlorinated molecules, the 3,5-dichloro derivative 3t showed a higher activity than the corresponding
3,4- derivative 3n (32/62.5 μM and 125 μM), and for the bromofluoro derivatives, the MICs increased
in the order 2-Br,4-F (3m; 16 μM) < 2-F,4-Br (3l; 32 μM) < 3-F,4-Br (3s; 62.5 μM). The comparison
of the three molecules with a 3-CF3,4-halogen substitution led to the following result: the strongest
activity against Mtb. was with the 3-CF3,4-Br derivative 3q (8 μM), followed by 3-CF3,4-Cl 3p and
3-CF3,4-F 3r (32 μM).
When the N-(4-heptylphenyl) derivative 3g and the N-heptyl molecule 3v, which vary by the
presence of a phenyl ring, were compared they exhibited identical MICs of 16/32 μM despite very
different logP values. The influence of the 4-alkyl length on the activity is interesting: the expanding
length from methyl 3a to n-butyl 3d increased the activity from 62.5 to 4/8 μM; pentyl 3e and hexyl 3f
resulted in a milder activity; and heptyl 3g displayed lower MICs comparable to the isopropyl. Finally,
the octyl substituted molecule 3h was evaluated and was determined to be the most potent compound
in this series. One possible hypothesis that may explain this effect is the similarity of longer
alkyl chain with fatty acids, which are structural fragments of biomembranes.

Molecules 2014, 19
Table 1. Antimycobacterial activity of N-substituted 2-isonicotinoylhydrazinecarboxamides 3
3855
and N-substituted 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amines 5.
N
H
H
N
N
O
N
R
H
O
MIC [μM]
M. kansasii
235/80
14 d
M. tuberculosis
M. avium
M. kansasii
Code
R
331/88
330/88
6509/96
C logP
14 d
62.5
16
8
21 d
62.5
32
14 d
16
21 d
32
7 d
62.5
16
21 d
7 d
14 d 21 d
3a
3b
3c
3d
3e
3f
4-methyl
4-isopropyl
4-tert-butyl
4-n-butyl
4-pentyl
4-hexyl
125
>125 62.5 62.5
125
1.13
1.87
2.34
2.38
2.79
3.21
3.63
4.05
0.51
1.56
2.3
16
62.5
62.5
62.5
62.5
32
32
16
32
8
32
8
16
16
125
125
125
62.5
62.5
500
62.5
32
62.5
125
125
125
125
125
62.5 62.5
62.5 62.5
4
8
62.5
32
125
32
32
32
16
1
62.5
62.5
32
62.5
32
62.5
125
125
125
125
500
32
125
62.5
3g
3h
3i
4-heptyl
4-octyl
32
125
62.5 62.5
250
2
250
32
250
500
>500 125
4-OMe
62.5
62.5
8
62.5
62.5
8
>125 >125 >125 >125 >125 >125
125 125
3j
4-CF3
>125 >125 >125 >125 >125 125
3k
3l
2,4-diBr
2-F,4-Br
2-Br,4-F
3,4-diCl
3-Cl,4-F
3-CF3,4-Cl
3-CF3,4-Br
3-CF3,4-F
3-F,4-Br
3,5-diCl
2,4,6-triCl
>125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125
32
16
125
62.5
32
8
32
1.63
1.63
1.75
1.35
2.12
2.39
1.72
1.63
1.75
2.31
3m
3n
3o
3p
3q
3r
3s
16
125
125
32
>125 >125
125
125
125
125
125
125
>62.5 >125 >125 >125 >125 >125 >125 >125
125
125
62.5
125
125
62.5
125
125
125
125
125
8
>125 >125 >125 >125 >125 125
32
62.5
32
4
32
>125 >125
>125 >125
125
125
125
125
>125 >125 125
125 125 125
125
62.5 62.5
62.5
62.5
4
125 >125
125
125
3t
125
125
125
125
3u
>125 >125 >125 >125 >125 125
3v
-
16
32
32
62.5
32
125
125
32
62.5
125
1.47
5i
5l
4-OMe
2-F,4-Br
3,4-diCl
3-F,4-Br
INH
>125
>125
>125
>125
0.5
>125 >125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125 >125
>125 >125 >125 >125 >125 >125 >125 >125 >125
2.2
3.31
3.44
3.31
−0.6
5n
5s
0.5-1 >250 >250 >250 >250 >250
4
8
8
INH: isoniazid. The best MIC values for each strain are given in bold.

Molecules 2014, 19
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The situation for the atypical mycobacteria was different. Eleven hydrazinecarboxamides showed
significantly lower MICs for M. avium than INH: 3a–g, 3i, 3p, 3t and 3v (range, 16 to 125 μM) with
the superiority of 3a and 3b (MICs of 16–32 μM) indicating that the best substitution pattern for the
phenyl ring is a small 4-alkyl group (3a, 3b) followed by 4-methoxy (3i), and 4-heptyl groups (3g).
Identical MIC values were obtained for 3v, the heptyl derivative without a phenyl ring. In general,
N-substituted 2-isonicotinoyl hydrazinecarboxamides 3, incorporating either alkyl (3a–g, 3v) or
methoxy (3i) groups, exhibited a significantly higher inhibition of M. avium growth than the
halogenated derivatives 3j–u (MICs ≥ 125 μM).
For both strains of M. kansasii, the alkyl substituted hydrazinecarboxamides 3a–g and 3v showed a
higher in vitro potency than the halogenphenyl derivatives 3j–u. Twelve compounds (3a–g, 3n, 3p, 3r,
3t and 3v) inhibited INH-resistant strain 235/80, with MICs ≤ 125 μM, but no molecule produced
lower MIC values than INH for clinical strain 6509/96. Only the 4-isopropyl derivative 3b, the most
active 2-isonicotinoylhydrazinecarboxamide 3 against all nontuberculous strains in this study (MICs
8–32 μM), displayed a similar in vitro efficacy. The most hydrophilic 4-methoxyphenyl 3i and,
surprisingly, the most lipophilic derivative 4-octylphenyl 3h did not exhibit any significant activity.
For the strain 6509/96, the presence of two or three chlorine atoms (3n, 3t, 3u) or a trifluoromethyl
moiety at any position (3j, 3p–r) on the phenyl ring led to better activity among the halogenated
compounds. In general, there is no general and unequivocal correlation of ClogP and antimycobacterial
activity within this series. This finding supports the conclusions of Ventura and Martins [15].
According to the interpretation of Scior and Garcés-Eisele [6], 2-isonicotinoylhydrazinecarbox-
amides 3 act as INH (and then isonicotinic acid) prodrugs that must be activated. None of these
compounds showed superiority over INH against drug-susceptible M. tuberculosis H37Rv.
In contrast, a range of these compounds showed notable in vitro activity at micromolar concentrations
against the INH-resistant strains of M. avium and M. kansasii. In the case of M. avium, due to the
missing catalase/peroxidase enzyme, this observation can be explained on the basis of facilitated
liberation of isonicotinoyl radicale from the less stable hydrazide derivative prodrugs [6].
An examination of the cyclised analogues of the four selected 2-isonicotinoylhydrazinecarboxamides
with different substitution patterns (halogens: 3l, 3n and 3s, and methoxy derivative 3i), i.e.,
N-substituted-5-(pyridine-4-yl)-1,3,4-oxadiazol-2-amines 5i, 5l, 5n and 5s, revealed a complete loss of
efficacy (MICs > 125 μM; Table 1). Having these results in hand, we decided not to further study
additional 5-(pyridine-4-yl)-1,3,4-oxadiazol-2-amines 5 as potential antimycobacterial agents.
In fact, the activity values for our 1,3,4-oxadiazol-2-amines are in sharp contrast with the results
published by Navarrete-Vázques et al. [13] where the 2-position of the oxadiazole ring was occupied
by alkyls, halogenoalkyls and aryls, and the MIC values were within the range of 0.35 to 49.69 μM for
Mtb. H37Rv. Additionally, two other 2-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazoles exhibited a
remarkable inhibition of M. tuberculosis [16,17]. On the other hand, Foks et al. [18] reported
substituted derivatives of 5-(pyridine-4-yl)-1,3,4-oxadiazol-2-amines, which avoid any significant
activity against Mtb. 5-(Pyridine-4-yl)-1,3,4-oxadiazol-2-amine showed mild antimycobacterial
activity; the replacement of the amino group by mercapto decreased the MIC [19]. The reason for the
inactivity of N-(aryl substituted)-5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines 5 at the observed
concentrations when compared to the previously reported derivatives [13,16,17] with other substituents
at position 2, which are based on the hydrocarbon chains or rings, may originate from the changes in

Molecules 2014, 19
3857
the hydrophilic-lipophilic balance due to the presence of an H-N bond as well as possible hydrogen
bridge formations, different steric parameters or because these compounds may be metabolised by
different ways within the cells.
2.3. Molecular Modelling Studies
The enoyl-acyl carrier protein reductase InhA, the target of INH, was further investigated as a
possible target of INH derivatives. Molecular modelling studies were performed to suggest possible
conformations of the novel compounds in the active site of the enzyme and to search for possible
interactions between the ligand and InhA.
In previous studies [20,21], the crystal structure of InhA and its active site were described in detail.
InhA belongs to the family of NADH – dependent dehydrogenase enzymes. Participating in fatty acid
biosynthesis, the structure of InhA is adapted to accommodate its natural substrates, namely, long
chained fatty acids. Adjacent to the NADH binding site is a substrate binding loop (residues 196–219)
forming an oval-shaped cavity with plenty of hydrophobic amino acid residues. One side of the cavity
is widely accessible to solvent. A crystallographic study of a ternary complex of InhA and C16 fatty
acyl substrates revealed that the substrate occupies the substrate binding cavity in a U-shaped
conformation, which is stabilised by surrounding hydrophobic side chains of amino acid residues at
one side of the cavity and by hydrogen bonds with NADH at the other [22].
Molecular modelling studies of N-(4-alkylphenyl) substituted hydrazinecarboxamides (3a–h)
indicate a similar binding of the derivatives to the active site of InhA, as in the previously described
study of Rozwarski et al. [22]. The pyridine moiety of the ligands is positioned near the entrance to the
cavity, and the hydrazinecarboxamide connecting linker is on the top of NADH within hydrogen
bonding distance. Some of the molecules also display one direct hydrogen bond to the protein between
the carboxamide nitrogen and the hydroxyl group of Tyr158. The lipophilic portion of the molecule
point deep into the cavity and is surrounded by the hydrophobic side chains of Ile202, Leu218, Pro193,
Met199, Met161, Met103, Phe149 and Ile215. The longer the alkyl substituent is, the more interactions
are available in the area of the substrate cavity. One of the highest affinities towards the enzyme was
observed in the case of 4-octylphenyl derivative 3h (docking score of −8.6 kJ/mol; Figure 1).
Concerning the docking of variously halogenated derivatives, the conformation of these compounds
was similar to that observed in the 4-alkylphenyl group with a pyridine moiety pointing towards the
entrance of the cavity and a phenyl moiety pointing into the cavity. However, these compounds did not
fill the substrate binding cavity as did the compounds with the longer alkyl substituents. This could
result in the formation of fewer interactions with the hydrophobic residues and therefore lead to a
weaker affinity towards the enzyme. The derivatives containing an oxadiazole moiety displayed
similar conformation with hydrogen bonding to NADH and few hydrophobic interactions with
Met103, Met199, Met161 and Phe149.
According to the observed conformations displayed by various derivatives, longer alkyl substituents
are advantageous in the formation of interactions and therefore in the affinity to InhA. This phenomenon
can be assumed from the structure of the natural substrates – long chain fatty acids. These results
indicate that presented compounds 3 may affect InhA directly without previous necessary activation to
form isonicotinoyl radical.

Molecules 2014, 19
3858
Figure 1. Conformation of compound 3h in the active site of InhA together with NAD and
the interacting hydrophobic residues.
3. Experimental
3.1. General Information
The starting materials for the synthesis of the examined compounds were used as obtained from
commercial sources (Sigma-Aldrich, Prague, Czech Republic, Fluka, Prague, Czech Republic, Penta
Chemicals, Prague, Czech Republic, Fluka Alfa Aesar, Ljubljana, Slovenia, Maybridge Chemical
Company Ltd, Ljubljana, Slovenia). The melting points were determined on a Kofler micro hot stage
or on a Büchi Melting Point machine B-540 apparatus using open capillaries and were uncorrected.
The NMR spectra were recorded at 29 °C on a Varian Mercury-Vxbb 300 (300 MHz for 1H and 75.5 MHz
13
for C; Varian, Inc., Palo Alto, CA, USA) or with a Bruker Avance III 500 spectrometer (1H-NMR
spectra at 500 MHz; 13C-NMR spectra at 125.8 MHz) using deuterated dimethyl sulfoxide (DMSO-d6)
solutions of the samples. Proton spectra are referenced to TMS as the internal standard; the carbon
shifts are given against the central line of the solvent signal (DMSO-d6 at δ = 39.5 ppm; CDCl3 at
δ = 77.1 ppm). The coupling constants (J) are reported in Hz. IR spectra were obtained with a
Perkin–Elmer Spectrum 100, equipped with a Specac Golden Gate Diamond ATR as a solid sample
support, or they were recorded on a Nicolet 6700 FT-IR spectrometer in the range of 400–4,000 cm−1
(ATR). MS spectra were recorded with an Agilent 6224 Accurate Mass TOF LC/MS spectrometer.
Elemental analyses (C, H, N) were performed with a Perkin Elmer 2400 Series II CHNS/O Analyzer.
TLC was performed on Fluka silica-gel TLC-cards or on plates coated with 0.2 mm of Merck 60 F254
silica gel, and the chromatograms were visualised by UV irradiation (254 nm). The purity of
synthesised compounds was checked by TLC, NMR, elemental analysis and for some compounds by
HRMS. 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 12.0
(CambridgeSoft, Cambridge, MA, USA).

Molecules 2014, 19
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3.2. Chemistry
3.2.1. Synthesis of Isonicotinoylhydrazinecarboxamides 3
Method A
Isoniazid (1, 137.1 mg, 1.0 mmol) was suspended in anhydrous dichloromethane (5 mL), followed
by the addition of the appropriate aryl isocyanate (2, 1.0 mmol) in one volume. The reaction mixture
was stirred at room temperature for 30 min. A solid material was filtered off and recrystallised from
boiling ethyl acetate. The reaction was monitored by TLC using the mixture n-hexane/ethyl acetate 3:1
as an eluent.
Method B
Triphosgene (120 mg, 0.4 mmol) was dissolved in anhydrous dichloromethane (5 mL) under argon
atmosphere, and the appropriate aniline (4, 1.01 mmol) in dichloromethane (3 mL) was added
dropwise. This mixture was stirred for 30 min at room temperature and then treated with triethylamine
(293 µL, 2.1 mmol) in dichloromethane (3 mL). After 30 min, isoniazid (1, 138.5 mg, 1.01 mmol) was
added. The reaction mixture was stirred for 30 min at room temperature, evaporated to dryness, treated
with water (10 mL) and extracted with ethyl acetate (3 × 20 mL). The organic phase was dried over
anhydrous sodium sulphate, filtered and evaporated to dryness to give the final product 3, which was
recrystallised from boiling ethyl acetate, if necessary. Some products were not sufficiently soluble to
be extracted by cold ethyl acetate. In these cases, the organic layer of the suspension was filtered off, and
the resulting crystals were recrystallised from boiling ethyl acetate and merged with the crystals obtained
from the solution by evaporation of the solvent. The reaction was monitored by TLC using the mixture
n-hexane/ethyl acetate 3:1 as an eluent.
2-Isonicotinoyl-N-(4-methylphenyl)hydrazinecarboxamide (3a). Method A; yield 97%; mp 227–229 °C;
IR: 3273, 3030, 1673, 1640, 1591, 1516, 1487, 1407, 1339, 1310, 1288, 1234, 1090, 1065, 1020, 993,
1
918, 824, 775, 748, 683, 633 cm−1; H-NMR (500 MHz, DMSO-d6): δ 2.23 (3H, s), 7.07 (2H, d,
J = 8.5 Hz), 7.35 (2H, d, J = 8.5 Hz), 7.82 and 8.78 (4H, AA’XX’), 8.25 (1H, bs), 8.80 (1H, bs), 10.58
13
(1H, bs); C-NMR (125.8 MHz, DMSO-d6): δ 20.3, 118.7, 121.4, 129.0, 130.8, 137.0, 139.6, 150.3,
155.4, 164.9; HRMS (ESI+) for C14H15N4O2 ([M+H]+): calcd. 271.1190; found 271.119. Anal. calcd.
for C14H14N4O2 (270.29): C 62.21, H 5.22, N 20.73; found: C 61.97, H 4.95, N 20.59.
2-Isonicotinoyl-N-[4-(propan-2-yl)phenyl]hydrazinecarboxamide (3b). Method A; yield 93%; mp
230–233 °C; IR: 3283, 3089, 2932, 1692, 1673, 1596, 1516, 1479, 1417, 1347, 1308, 1275, 1237,
1100, 1061, 1019, 1000, 971, 923, 883, 851, 827, 788, 757, 734, 698, 625, 604 cm−1; 1H-NMR (500 MHz,
DMSO-d6): δ 1.18 (6H, d, J = 7.0 Hz), 2.82 (1H, sep, J = 7.0 Hz), 7.14 (2H, d, J = 8.5 Hz), 7.38 (2H,
13
d, J = 8.5 Hz), 7.83 and 8.78 (4H, AA’XX’), 8.26 (1H, bs), 8.82 (1H, bs), 10.59 (1H, bs); C-NMR
(125.8 MHz, DMSO-d6): δ 24.0, 32.8, 118.8, 121.4, 126.3, 137.2, 139.6, 142.0, 150.3, 155.4, 164.9;
HRMS (ESI+) for C16H19N4O2 ([M+H]+): calcd. 299.1503; found 299.1512. Anal. calcd. for
C16H18N4O2 (298.34): C 64.41, H 6.08, N 18.78; found: C 64.36, H 5.98, N 18.81.

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2-Isonicotinoyl-N-[4-(tert-butyl)phenyl]hydrazinecarboxamide (3c). Method A; yield 96%; mp
223.5–226 °C; IR: 3298, 3194, 3075, 2967, 2928, 1691, 1668, 1594, 1549, 1523, 1473, 1412, 1364,
1
1311, 1298, 1276, 1243, 1063, 1019, 1000, 920, 878, 844, 828, 754, 731, 705, 668 cm−1; H-NMR
(300 MHz, DMSO-d6): δ 1.24 (9H, s), 7.26 (2H, d, J = 8.8 Hz), 7.37 (2H, d, J = 8.8 Hz), 7.81 and 8.76
(4H, AA’XX’), 8.25 (1H, bs), 8.83 (1H, bs), 10.58 (1H, bs); 13C-NMR (75.5 MHz, DMSO-d6): δ 31.5,
34.1, 118.7, 121.6, 125.4, 137.1, 139.8, 144.5, 150.5, 155.6, 165.1. Anal. calcd. for C17H20N4O2
(312.37): C 65.37, H 6.45, N 17.94; found: C 65.12, H 6.28, N 17.58.
2-Isonicotinoyl-N-(4-butylphenyl)hydrazinecarboxamide (3d). Method A; yield 91%; mp 181–182.5 °C;
IR: 3277, 3089, 2968, 2932, 2860, 1691, 1673, 1595, 1524, 1484, 1466, 1416, 1377, 1310, 1278, 1241,
1063, 1000, 923, 886, 850, 841, 823, 789, 753, 730, 701, 681, 669 cm−1; 1H-NMR (300 MHz, DMSO-d6):
δ 0.87 (3H, t, J = 7.3 Hz), 1.27 (2H, sextet, J = 7.3 Hz), 1.50 (2H, p, J = 7.6 Hz), 2.49 (2H, t, J = 7.6 Hz),
7.06 (2H, d, J = 8.5 Hz), 7.35 (2H, d, J = 8.5 Hz), 7.81 and 8.76 (4H, AA’XX’), 8.80 (1H, bs), 8.24
13
(1H, bs), 10.57 (1H, bs); C-NMR (75.5 MHz, DMSO-d6): δ 14.0, 21.9, 33.5, 34.4, 119.0, 121.6,
128.6, 136.1, 137.4, 139.8, 150.5, 155.7, 165.1. Anal. calcd. for C17H20N4O2 (312.37): C 65.37, H
6.45, N 17.94; found: C 65.26, H 6.38, N 17.86.
2-Isonicotinoyl-N-(4-pentylphenyl)hydrazinecarboxamide (3e). Method B; yield 81%; mp 195–196.5 °C;
IR: 3277, 3049, 2955, 2922, 2855, 1673, 1637, 1611, 1589, 1562, 1536, 1489, 1467, 1408, 1378, 1341,
1
1304, 1283, 1241, 1068, 1052, 917, 845, 823, 787, 750, 722, 683, 668 cm−1; H-NMR (300 MHz,
DMSO-d6): δ 0.84 (3H, t, J = 6.8 Hz), 1.34–1.18 (4H, m), 1.50 (2H, p, J = 7.4 Hz), 2.48 (2H, t, J = 7.5 Hz),
7.06 (2H, d, J = 8.4 Hz), 7.35 (2H, d, J = 8.4 Hz), 7.81 and 8.76 (4H, AA’XX’), 8.24 (1H, bs), 8.79
(1H, bs), 10.57 (1H, bs); 13C-NMR (75.5 MHz, DMSO-d6): δ 14.1, 22.1, 30.9, 31.0, 34.6, 119.0, 121.6,
128.5, 136.1, 137.4, 139.8, 150.5, 155.6, 165.1. Anal. calcd. for C18H22N4O2 (326.39): C 66.24, H
6.79, N 17.17; found: C 66.12, H 6.53, N 17.02.
2-Isonicotinoyl-N-(4-hexylphenyl)hydrazinecarboxamide (3f). Method B; yield 84%; mp 177–179 °C;
IR: 3271, 3082, 2960, 2929, 2856, 1691, 1672, 1597, 1516, 1482, 1416, 1378, 1340, 1310, 1279, 1239,
1
1063, 1000, 925, 889, 850, 815, 754, 700, 669 cm−1; H-NMR (300 MHz, DMSO-d6): δ 0.84 (3H, t,
J = 7.0 Hz), 1.29–1.21 (6H, m), 1.51 (2H, p, J = 7.4 Hz), 2.48 (2H, t, J = 7.5 Hz), 7.06 (2H, d, J = 8.4 Hz),
7.35 (2H, d, J = 8.4 Hz), 7.81 and 8.76 (4H, AA’XX’), 8.25 (1H, bs), 8.80 (1H, bs), 10.58 (1H, bs);
13C-NMR (75.5 MHz, DMSO-d6): δ 14.2, 22.3, 28.5, 31.3, 31.3, 34.7, 119.0, 121.6, 128.6, 136.1,
137.4, 139.8, 150.5, 155.7, 165.1. Anal. calcd. for C19H24N4O2 (340.42): C 66.04, H 7.11, N 16.46;
found: C 65.95, H 6.84, N 16.22.
2-Isonicotinoyl-N-(4-heptylphenyl)hydrazinecarboxamide (3g). Method B; yield 94%; mp 185.5–186.5 °C;
IR: 3275, 3093, 2959, 2925, 2854, 1691, 1671, 1601, 1515, 1483, 1415, 1378, 1343, 1312, 1278, 1242,
1062, 1001, 924, 887, 851, 821, 782, 754, 733, 698, 670 cm−1; 1H-NMR (300 MHz, DMSO-d6): δ 0.83
(3H, t, J = 6.5 Hz), 1.32–1.18 (8H, m), 1.51 (2H, p, J = 7.2 Hz), 2.48 (2H, t, J = 7.2 Hz), 7.05 (2H, d,
J = 8.1 Hz), 7.34 (2H, d, J = 8.1 Hz), 7.80 and 8.75 (4H, AA’XX’), 8.24 (1H, bs), 8.80 (1H, bs), 10.57
13
(1H, bs); C-NMR (75.5 MHz, DMSO-d6): δ 14.2, 22.3, 28.8, 28.8, 31.3, 31.5, 34.7, 119.0, 121.6,
128.6, 136.1, 137.4, 139.8, 150.5, 155.6, 165.1. Anal. calcd. for C20H26N4O2 (354.45): C 67.77, H
7.39, N 15.81; found: C 67.38, H 7.29, N 15.63.

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2-Isonicotinoyl-N-(4-octylphenyl)hydrazinecarboxamide (3h). Method B; yield 93%; mp 189.5–190.5 °C;
IR: 3276, 3194, 3087, 2922, 2854, 1691, 1671, 1594, 1519, 1479, 1417, 1379, 1340, 1310, 1278, 1241,
1062, 1000, 923, 887, 851, 825, 811, 751, 729, 696, 671, 655 cm−1; 1H-NMR (300 MHz, DMSO-d6): δ
0.84 (3H, t, J = 6.8 Hz), 1.31–1.18 (10H, m), 1.51 (2H, p, J = 6.7 Hz), 2.49 (2H, t, J = 7.4 Hz), 7.06
(2H, d, J = 8.4 Hz), 7.35 (2H, d, J = 8.4 Hz), 7.82 and 8.76 (4H, AA’XX’), 8.25 (1H, bs), 8.80 (1H,
13
bs), 10.58 (1H, bs); C-NMR (75.5 MHz, DMSO-d6): δ 14.4, 22.5, 29.1, 29.1, 29.3, 31.6, 31.7, 34.9,
119.2, 121.9, 128.8, 136.4, 137.6, 140.1, 150.8, 155.9, 165.3. Anal. calcd. for C21H28N4O2 (368.47):
C 68.45, H 7.66, N 15.21; found: C 68.23, H 7.50, N 15.06.
2-Isonicotinoyl-N-(4-methoxyphenyl)hydrazinecarboxamide (3i). Method A; yield 90%; mp 240–242 °C,
IR: 3274, 3090, 2936, 1690, 1672, 1596, 1511, 1479, 1463, 1441, 1418, 1343, 1310, 1298, 1278, 1260,
1234, 1185, 1172, 1111, 1061, 1030, 999, 967, 922, 887, 850, 830, 808, 797, 770, 749, 698, 645, 627 cm−1;
1H-NMR (500 MHz, DMSO-d6): δ 3.71 (3H, s), 6.85 (2H, d, J = 8.5 Hz), 7.36 (2H, d, J = 8.5 Hz), 7.82
and 8.77 (4H, AA’XX‘), 8.22 (1H, bs), 8.73 (1H, bs), 10.57 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6): δ
55.1, 113.8, 120.5, 121.4, 132.5, 139.6, 150.3, 154.5, 155.6, 164.9; HRMS (ESI+) for C14H15N4O3
([M+H]+): calcd. 287.1139; found 287.1139. Anal. calcd. for C14H14N4O3 (286.29): C 58.73, H 4.93, N
19.57; found C 58.48, H 4.63, N 19.53.
2-Isonicotinoyl-N-[4-(trifluoromethyl)phenyl]hydrazinecarboxamide (3j). Method B; yield 67%; mp
207–210 °C; IR: 3115, 3029, 2170, 2113, 1755, 1731, 1674, 1634, 1610, 1554, 1498, 1451, 1428,
1
1335, 1267, 1221, 1110, 1083, 1066, 1015, 911, 879, 865, 840, 757, 726, 637, 613 cm−1; H-NMR
(500 MHz, DMSO-d6): δ 7.62 (2H, d, J = 8.8 Hz), 7.70 (2H, d, J = 8.8 Hz), 7.82 and 8.79 (4H,
AA’XX‘), 8.54 (1H, bs), 9.34 (1H, bs), 10.65 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6): δ 118.3 (q,
J = 2.8 Hz), 121.4, 122.0 (q, J = 31.7 Hz), 124.5 (q, J = 271.1 Hz), 125.9 (q, J = 3.1 Hz), 139.5, 143.4,
150.4, 155.1, 164.9; HRMS (ESI+) for C14H12F3N4O2 ([M+H]+): calcd. 325.0907; found 325.0916.
Anal. calcd. for C14H11F3N4O2 (324.26): C 51.86, H 3.42, N 17.28; found C 51.73, H 3.25, N 17.10.
N-(2,4-Dibromophenyl)-2-isonicotinoylhydrazinecarboxamide (3k). Method B; yield 82%; mp
243–244 °C; IR: 3265, 1680, 1652, 1582, 1556, 1519, 1467, 1405, 1377, 1335, 1291, 1266, 1252,
1092, 1078, 1064, 1039, 919, 861, 834, 787, 751, 731, 683, 636 cm−1; 1H-NMR (500 MHz, DMSO-d6): δ
7.54 (1H, dd, J1 = 8.5 Hz, J2 = 1.3 Hz), 7.81 and 8.78 (4H, AA’XX‘), 7.86 (1H, d, J = 1.3 Hz), 7.96
(1H, d, J = 8.5 Hz), 8.32 (1H, bs), 9.14 (1H, bs), 10.75 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6): δ
114.8, 121.4, 123.3, 131.1, 134.2, 136.5, 139.3, 150.4, 154.8, 164.8; HRMS (ESI+) for C13H11Br2N4O2
[M+H]+): calcd. 412.9244; found 412.9255. Anal. calcd. for C13H10Br2N4O2 (414.05): C 37.71, H 2.43,
N 13.53; found C 37.85, H 2.30, N 13.46.
N-(4-Bromo-2-fluorophenyl)-2-isonicotinoylhydrazinecarboxamide (3l). Method B; yield 96%; mp
235–238 °C; IR: 3298, 1645, 1597, 1554, 1528, 1489, 1408, 1329, 1306, 1248, 1195, 1122, 1093,
1
1069, 1050, 994, 891, 852, 814, 762, 751, 712, 687, 642 cm−1; H-NMR (500 MHz, DMSO-d6): δ
7.33–7.36 (1H, m), 7.55–7.59 (1H, m), 7.81 and 8.78 (4H, AA’XX‘), 7.87–8.02 (1H, m), 8.65 (1H,
bs), 8.86 (1H, bs), 10.72 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6): δ 113.2, 118.5 (d, J = 22.6 Hz),
121.4, 122.2, 126.9 (d, J = 9.9 Hz), 127.5 (d, J = 3.0 Hz), 139.4, 150.4, 152.3 (d, J = 244.6 Hz), 154.8,

Molecules 2014, 19
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164.8; HRMS (ESI+) for C13H11BrFN4O2 ([M+H]+): calcd. 353.0044; found 353.0062. Anal. calcd. for
C13H10BrFN4O2 (353.15): C 44.21, H 2.85, N 15.87; found C 44.09, H 2.80, N 15.71.
N-(2-Bromo-4-fluorophenyl)-2-isonicotinoylhydrazinecarboxamide (3m). Method B; yield 74%; mp
227–229 °C; IR: 3262, 3019, 1670, 1649, 1599, 1554, 1484, 1405, 1389, 1339, 1255, 1189, 1098,
1065, 1032, 923, 903, 854, 845, 814, 785, 750, 672 cm−1; 1H-NMR (500 MHz, DMSO-d6): δ 7.22–7.27
(1H, m), 7.60–7.63 (1H, m), 7.81 and 8.78 (4H, AA’XX‘), 7.87–7.90 (1H, m), 8.28 (1H, s), 8.96 (1H,
13
bs), 10.72 (bs); C-NMR (125.8 MHz, DMSO-d6): δ 115.0 (d, J = 21.8 Hz), 119.1 (d, J = 25.5 Hz),
121.4, 123.9, 133.6 (d, J = 2.4 Hz), 139.4, 143.0, 150.4, 151.1, 154.1 (d, J = 258.5 Hz), 164.8; HRMS
(ESI+) for C13H11BrFN4O2 ([M+H]+): calcd. 353.0044; found 353.0046. Anal. calcd. for C13H10BrFN4O2
(353.15): C 44.21, H 2.85, N 15.87; found C 44.19, H 2.69, N 15.75.
N-(3,4-Dichlorophenyl)-2-isonicotinoylhydrazinecarboxamide (3n). Method B; yield 68%; mp
220–222 °C; IR: 3287, 3081, 2932, 1692, 1666, 1588, 1515, 1472, 1395, 1328, 1295, 1263, 1227,
1
1129, 1064, 1030, 1000, 904, 865, 854, 845, 810, 784, 741, 709, 690, 676, 658, 620 cm−1; H-NMR
(500 MHz, DMSO-d6): δ 7.45–7.47 (1H, m), 7.52 (1H, d, J = 8.5 Hz), 7.82 and 8.79 (4H, AA’XX‘),
7.88 (1H, d, J = 2.0 Hz), 8.59 (1H, bs), 9.25 (1H, bs), 10.64 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6): δ
118.7, 119.8, 121.4, 123.3, 130.5, 130.8, 139.5, 139.9, 150.4, 155.2, 164.9; HRMS (ESI+) for
C13H11Cl2N4O2 ([M+H]+): calcd. 325.0254; found 325.0254. Anal. calcd. for C13H10Cl2N4O2 (325.15):
C 48.02, H 3.10, N 17.23; found C 47.75, H 2.95, N 17.05.
N-(3-Chloro-4-fluorophenyl)-2-isonicotinoylhydrazinecarboxamide (3o). Method B; yield 83%; mp
212–214 °C; IR: 3311, 3183, 3098, 2932, 1689, 1665, 1602, 1539, 1499, 1482, 1406, 1329, 1304,
1287, 1261, 1246, 1209, 1135, 1095, 1066, 1054, 1001, 931, 910, 866, 852, 819, 805, 749, 700, 679,
661, 631, 611 cm−1; 1H-NMR (500 MHz, DMSO-d6): δ 7.30–7.34 (1H, m), 7.39–7.41 (1H, m), 7.77–7.80
(1H, m), 7.82 and 8.78 (4H, AA’XX‘), 8.52 (1H, bs), 9.12 (1H, bs), 10.61 (1H, bs); 13C-NMR
(125.8 MHz, DMSO-d6): δ 116.7 (d, J = 21.6 Hz), 119.0 (d, J = 18.1 Hz), 120.0, 121.5, 136.9 (d,
J = 2.8 Hz), 139.5, 149.6, 150.4, 152.4 (d, J = 241.2 Hz), 155.4, 165.0; HRMS (ESI+) for C13H11ClFN4O2
([M+H]+): calcd. 309.0549; found 309.0549. Anal. calcd. for C13H10ClFN4O2 (308.70): C 50.58, H
3.27, N 18.15; found C 50.48, H 3.00, N 17.98.
N-[4-Chloro-3-(trifluoromethyl)phenyl]-2-isonicotinoylhydrazinecarboxamide (3p). Method B; yield
91%; mp 231–234 °C; IR: 3294, 3188, 3081, 2936, 1692, 1668, 1601, 1515, 1479, 1407, 1319, 1301,
1279, 1259, 1221, 1184, 1127, 1064, 1033, 1008, 908, 889, 877, 846, 824, 804, 736, 696, 666, 609 cm−1;
1H-NMR (500 MHz, DMSO-d6): δ 7.61 (1H, d, J = 9.0 Hz), 7.78–7.84 (3H, m), 8.09 (1H, d, J = 2.5 Hz),
8.70 (1H, bs), 8.78 (2H, AA’XX‘), 9.40 (1H, bs), 10.66 (1H, bs); HRMS (ESI+) for C14H11ClF3N4O2
([M+H]+): calcd. 359.0517; found 359.0521. Anal. calcd. for C14H10ClF3N4O2 (358.70): C 46.88, H
2.81, N 15.62; found C 46.72, H 2.67, N 15.35.
N-[4-Bromo-2-(trifluoromethyl)phenyl]-2-isonicotinoylhydrazinecarboxamide (3q). Method B; yield
68%; mp 197–199 °C; IR: 3266, 1702, 1655, 1584, 1553, 1506, 1482, 1408, 1307, 1286, 1243, 1169,
1
1128, 1108, 1054, 998, 909, 890, 869, 842, 799, 760, 698, 681, 651 cm−1; H-NMR (500 MHz,
DMSO-d6): δ 7.80–7.86 (5H, m), 8.31 (1H, bs), 8.79 (2H, AA’XX‘), 9.05 (1H, bs), 10.75 (1H, bs);

Molecules 2014, 19
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HRMS (ESI+) for C14H11BrF3N4O2 ([M+H]+): calcd. 403.0012; found 403.0018. C14H10BrF3N4O2
(403.15). ¼ EtOAc: C 42.37, H 2.84, N 13.18; found C 42.72, H 3.17, N 13.44.
N-[4-Fluoro-3-(trifluoromethyl)phenyl]-2-isonicotinoylhydrazinecarboxamide (3r). Method B; yield
74%; mp 213–215 °C; IR: 3300, 3194, 3085, 2940, 1694, 1670, 1556, 1506, 1485, 1409, 1324, 1281,
1
1248, 1220, 1166, 1122, 1055, 1002, 911, 885, 850, 821, 749, 708, 653, 611 cm−1; H-NMR
(500 MHz, DMSO-d6): δ 7.40–7.45 (1H, m), 7.74–7.81 (1H, m), 7.83 and 8.79 (4H, AA’XX‘),
7.96–7.99 (1H, m), 8.60 (1H, s), 9.28 (1H, bs), 10.63 (1H, bs); HRMS (ESI+) for C14H11F4N4O2
([M+H]+): calcd. 343.0813; found 343.0829. Anal. calcd. for C14H10F4N4O2 (342.25): C 49.13, H 2.95,
N 16.37; found C 48.97, H 2.72, N 16.03.
N-(4-Bromo-3-fluorophenyl)-2-isonicotinoylhydrazinecarboxamide (3s). Method B; yield 89%; mp
232–234 °C; IR: 3288, 3045, 1675, 1639, 1598, 1555, 1530, 1482, 1417, 1330, 1305, 1270, 1235,
1
1163, 1133, 1092, 1068, 1048, 993, 973, 912, 860, 846, 813, 772, 752, 711, 688, 650 cm−1. H-NMR
(500 MHz, DMSO-d6): δ 7.22–7.25 (1H, m), 7.54–7.58 (1H, m), 7.64 (1H, dd, J1 = 11.5 Hz, J2 = 2.0 Hz),
13
7.81 and 8.78 (4H, AA’XX‘), 8.57 (1H, bs), 9.28 (1H, bs), 10.63 (1H, bs); C-NMR (125.8 MHz,
DMSO-d6): δ 99.1 (d, J = 21.1 Hz), 106.6 (d, J = 27.2 Hz), 116.0, 121.4, 133.1, 139.5, 141.1 (d,
J = 10.4 Hz), 150.4, 155.1, 158.1 (d, J = 241.7 Hz), 164.9; HRMS (ESI+) for C13H11BrFN4O2 ([M+H]+):
calcd. 353.0044; found 353.0033. Anal. calcd. for C13H10BrFN4O2 (353.15): C 44.21, H 2.85, N 15.87;
found 44.10, H 2.62, N 15.64.
N-(3,5-Dichlorophenyl)-2-isonicotinoylhydrazinecarboxamide (3t). Method B; yield 74%; mp
226–229 °C; IR: 3491, 3228, 1682, 1586, 1535, 1471, 1448, 1413, 1344, 1296, 1248, 1226, 1111,
1067, 1021, 996, 928, 904, 841, 833, 802, 757, 704, 667, 624 cm−1; 1H-NMR (500 MHz, DMSO-d6): δ
7.17 (1H, t, J = 2.0 Hz), 7.61 (2H, bs), 7.82 and 8.79 (4H, AA’XX‘), 8.69 (1H, bs), 9.28 (1H, bs),
13
10.64 (1H, bs); C-NMR (125.8 MHz, DMSO-d6): δ 116.7, 121.1, 122.0, 134.0, 140.0, 142.2, 150.3,
155.1, 164.9; HRMS (ESI+) for C13H11Cl2N4O2 ([M+H]+): calcd. 325.0254; found 325.0254. Anal.
calcd. for C13H10Cl2N4O2 (325.15): C 48.02, H 3.10, N 17.23; found C 47.76, H 2.80, N 16.92.
2-Isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (3u). Method B; yield 70%; mp
1
236–238 °C; IR: 3284, 1655, 1576, 1541, 1454, 1322, 1234, 853, 828, 745, 696, 635 cm−1; H-NMR
(500 MHz, DMSO-d6): δ 7.73 (2H, s), 7.95 and 8.83 (4H, AA’XX‘), 8.69 (1H, bs), 8.73 (1H, bs),
13
10.78 (1H, bs); C-NMR (125.8 MHz, DMSO-d6): δ 122.3, 128.1, 131.7, 132.9, 135.2, 141.1, 148.9,
155.3, 164.3; HRMS (ESI+) for C13H10Cl3N4O2 ([M+H]+): calcd. 358.9864; found 358.9869. Anal. calcd.
for C13H9Cl3N4O2 (359.60). 1/10 H2O: C 43.20, H 2.57, N 15.50; found C 42.45, H 2.49, N 15.15.
N-Heptyl-2-isonicotinoylhydrazinecarboxamide (3v). Method A; yield 90%; mp 150–152.5 °C; IR:
3290, 3214, 3096, 2924, 2855, 1671, 1636, 1554, 1522, 1508, 1486, 1436, 1411, 1375, 1339, 1296,
1
1221, 1142, 1063, 997, 904, 849, 758, 722, 677, 663 cm−1. H-NMR (300 MHz, DMSO-d6): δ 0.84
(3H, t, J = 6.9 Hz), 1.27–1.19 (8H, m), 1.38 (2H, p, J = 6.6 Hz), 3.00 (2H, q, J = 6.6 Hz), 6.54 (1H, t,
J = 5.7 Hz), 7.78 and 8.73 (4H, AA’XX’), 7.91 (1H, bs), 10.38 (1H, bs). 13C-NMR (75 MHz,
DMSO-d6): δ 14.2, 22.3, 26.5, 28.7, 30.0, 31.5, 40.5, 121.6, 140.0, 150.4, 158.2, 165.0. Anal. calcd. for
C14H22N4O2 (278.35): C 60.41, H 7.97, N 20.13; found C 60.16, H 7.67, N 19.89.

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3.2.2. General Procedure for the Synthesis of N-Substituted-1,3,4-oxadiazol-2-amines 5
Triphenylphosphine (629.5 mg, 2.4 mmol) and 1,2-dibromo-1,1,2,2-tetrachloroethane (433.8 mg,
1.33 mmol) were added at room temperature to the stirred suspension of the appropriate
isonicotinoylhydrazinecarboxamide (3, 1.2 mmol) in anhydrous acetonitrile (6 mL). The reaction
mixture was stirred at room temperature for 5 min and cooled to 0 °C. Then, triethylamine (733 µL,
5.3 mmol) was added dropwise, and stirring was continued for an additional 30 min. A solid material
was filtered off, washed with water (3 mL) and recrystallised from boiling methanol, giving the
pure product 5.
N-(4-Methoxyphenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (5i) [23]. Yield 48%; mp 233–235 °C
(lit. 231.3–232.3 °C [23]); IR: 2788, 1644, 1598, 1573, 1508, 1439, 1416, 1305, 1253, 1238, 1215,
1176, 1037, 999, 967, 872, 835, 811, 794, 761, 719, 700, 680, 628 cm−1; 1H-NMR (500 MHz, DMSO-d6):
δ 3.74 (3H, s), 6.97 (2H, d, J = 9.0 Hz), 7.54 (2H, d, J = 9.0 Hz), 7.79 and 8.78 (4H, AA’XX‘), 10.65
13
(1H, bs); C-NMR (125.8 MHz, DMSO-d6): δ 55.2, 114.4, 118.9, 119.1, 130.8, 131.5, 150.8, 154.7,
156.0, 160.8; HRMS (ESI+) for C14H13N4O2 ([M+H]+): calcd. 269.1033; found 269.1033. Anal. calcd.
for C14H12N4O2 (268.27): C 62.68, H 4.51, N 20.88; found C 62.37, H 4.20, N 20.76.
N-(4-Bromo-2-fluorophenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (5l). Yield 81%, mp 262–264 °C;
IR: 2940, 1634, 1610, 1568, 1491, 1412, 1309, 1199, 1128, 1054, 997, 886, 825, 700, 680 cm−1;
1H-NMR (500 MHz, DMSO-d6): δ 7.47–7.50 (1H, m), 7.63–7.67 (1H, m), 7.81 and 8.80 (4H,
AA’XX‘), 8.08–8.13 (1H, m), 10.89 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6): δ 114.1 (d, J = 8.6 Hz),
118.9 (d, J = 22.0 Hz), 119.3, 122.0, 125.9 (d, J = 11.2 Hz), 127.8 (d, J = 3.5 Hz), 130.7, 150.8, 152.1
(d, J = 250.5 Hz), 156.9, 160.5; HRMS (ESI+) for C13H9BrFN4O ([M+H]+): calcd. 334.9938; found
334.9936. Anal. calcd. for C13H8BrFN4O (335.13): C 46.59, H 2.41, N 16.72; found C 46.50, H 2.29,
N 16.64.
N-(3,4-Dichlorophenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (5n). Yield 33%; mp 257–259 °C;
IR: 2766, 1635, 1589, 1567, 1475, 1404, 1302, 1249, 1129, 1051, 1001, 870, 819, 723, 703 cm−1;
1H-NMR (500 MHz, DMSO-d6): δ 7.54 (1H, dd, J1 = 8.8 Hz, J2 = 2.5 Hz), 7.62 (1H, d, J = 8.8 Hz),
7.79 and 8.79 (4H, AA’XX‘), 7.93 (1H, d, J = 2.5 Hz), 11.26 (1H, bs); 13C-NMR (125.8 MHz, DMSO-d6):
δ 117.5, 118.4, 119.3, 123.6, 130.6, 131.0, 131.4, 138.4, 150.8, 156.5, 159.9; HRMS (ESI+) for
C13H9Cl2N4O ([M+H]+): calcd. 307.0148; found 307.015. Anal. calcd. for C13H8Cl2N4O (307.13):
C 50.84, H 2.63, N 18.24; found C 50.71, H 2.47, N 18.05.
N-(4-Bromo-3-fluorophenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (5s). Yield 77%; mp 260–262 °C;
IR: 1642, 1592, 1566, 1487, 1417, 1255, 1174, 1054, 1174, 1054, 1002, 822, 750, 720, 703, 682,
1
658 cm−1; H-NMR (500 MHz, DMSO-d6): δ 7.35–7.38 (1H, m), 7.67–7.72 (2H, m), 7.82 and 8.79
13
(4H, AA’XX‘), 11.31 (1H, bs); C-NMR (125.8 MHz, DMSO-d6): δ 99.5 (d, J = 21.1 Hz), 105.4 (d,
J = 27.6 Hz), 115.0 (d, J = 3.0 Hz), 119.3, 130.6, 133.8 (d, J = 1.2 Hz), 139.6 (d, J = 10.4 Hz), 150.9,
156.6, 158.4 (d, J = 242.8 Hz), 160.0; HRMS (ESI+) for C13H9BrFN4O ([M+H]+): calcd. 334.9938;
found 334.9937. Anal. calcd. for C13H8BrFN4O (335.13). ⅕ H2O: C 46.09, H 2.50, N 16.54; found C
45.81, H 2.45, N 16.39.

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3.3. In Vitro Antimycobacterial Evaluation
The in vitro antimycobacterial activity of the prepared compounds was determined against
Mycobacterium tuberculosis My 331/88 (H37Rv; dilution of strain, 10−3), M. avium My 330/88
(resistant to INH, rifampicin, ofloxacin and ethambutol; dilution of strain, 10−5), M. kansasii My
235/80 (dilution of strain, 10−4) and M. kansasii 6509/96 (dilution of strain, 10−4). All strains were
obtained from the Czech National Collection of Type Cultures (CNCTC), with the exception of
M. kansasii 6509/96, which was clinically isolated. The antimycobacterial activity of the compounds
was determined in Šula’s semisynthetic medium (SEVAC, Prague, Czech Republic) via the
micromethod for the determination of the minimum inhibitory concentration (MIC) at 37 °C after
14 and 21 days and after 7, 14 and 21 days for M. kansasii [24]. The tested compounds were added to
the medium in DMSO solutions, and INH was used as a standard in a sterile water solution. The
concentrations of the tested compounds were used as follows: 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1
and 0.5 μM. The same concentrations, over the range of 0.5 to 250 μM, were used for INH. The MIC
(reported in μM) was the lowest concentration at which the complete inhibition of mycobacterial
growth occurred.
3.4. Molecular Docking Studies
The crystal structure of InhA was obtained from the protein data bank (www.pdb.org, pdb code
1ENY), and its energy was minimised using UCSF Chimera software 1.6.2. [25]. The enzyme
molecule was prepared using Autodock Tools 1.5.2. [26]. The hydrogens were added, partial charges
were assigned and grid maps were generated. The ligand structure was created using CS ChemOffice
version 10.0 (CambridgeSoft Cambridge, MA, USA), and its conformation was optimised with the aid
of UCSF Chimera 1.6.2 using Amber force field.
Docking calculations were performed using Autodock 4.2.5.1. A three-dimensional affinity grid
box was designed to include the full active site of InhA. The ligand molecule was kept fully flexible
during the docking calculations, as well as some of the residues of the enzyme. The visualisations of
the enzyme-ligand interactions were prepared using Pymol 1.1r1. [27].
4. Conclusions
The synthesis of new INH derivatives, 2-isonicotinoyl-N-(substituted)hydrazinecarboxamides, was
performed from commercially available or in situ synthesised isocyanates with good yields, and their
structures were confirmed by physico-chemical analyses. The biological test data for Mycobacterium
tuberculosis showed that none of the synthesised compounds exhibited higher activity than INH, but
the N-(4-octylphenyl) derivative 3h reached almost the same in vitro efficacy with MICs 1-2 μM.
Among the halogenated molecules, the best activity was found for the 2,4,6-trichloro derivative 3u,
with a MIC of 4 μM. An incorporation of the alkyl substituent on the carboxamide nitrogen of
2-isonicotinoylhydrazinecarboxamides led to a significantly higher inhibition of the growth of
M. avium and both M. kansasii strains when compared to the halogenated derivatives and INH.
Molecular modelling studies suggested a possible conformation of these novel compounds in the active
site of the enzyme InhA. The 4-alkylphenyl group, the lipophilic portion, may be located in the cavity

Molecules 2014, 19
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surrounded by hydrophobic side chains of amino acids; thus, the longer the alkyl substituent is, the
more interactions are available in this area of the substrate cavity. Based on this hypothesis, derivatives
bearing long alkyl or alkylphenyl connected with isoniazid moiety may bring an additional benefit.
Cyclisation of the selected compounds 3 to the corresponding 1,3,4-oxadiazol-2-amines 5 resulted
in the loss of antimycobacterial efficacy. Although this observation is in contrast with previously
published results, similar compounds of type 5 were not further investigated.
Acknowledgments
This work was financially supported by the Research project IGA NT 13346 (2012). This
publication is a result of the project implementation: “Support of establishment, development, and
mobility of quality research teams at the Charles University”, project number CZ.1.07/2.3.00/30.0022,
supported by The Education for Competitiveness Operational Programme (ECOP) and co-financed by
the European Social Fund and the state budget of the Czech Republic. The publication is co-financed
by the European Social Fund and the state budget of the Czech Republic. Project No.
CZ.1.07/2.3.00/20.0235, the title of the project: TEAB. The Slovenian Research Agency is gratefully
acknowledged for financial support (project P1-0230). This work was also supported with the
infrastructure of the EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia.
Author Contributions
The article is a work of two cooperating groups. Part of this work was done in Slovenia under the
guidance of Prof. Slovenko Polanc and Prof. Marian Kočevar and other part was done in the Czech
Republic under the guidance of Prof. Jarmila Vinšová. Zuzana Rychtarčíková, Martin Gazvoda and
Martin Krátký synthesized and characterized all presented compounds, Markéta Komlóová did
molecular modelling studies, Jiřina Stolaříková made antimycobacterial measurement. J. Vinšová, S.
Polanc and M. Krátký wrote the whole article, made discussion and conclusion.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).