Synthesis and bioevaluation of some new isoniazid derivatives

Article abstract of DOI:10.1016/j.bmc.2013.06.013

The aim of the study was to synthesize some new compounds with potential anti-tuberculosis activity, containing isoniazid and α,β-unsaturated thiocinnamamide-like thioamides as precursors. The obtained derivatives were evaluated regarding their biological

Full text of DOI:10.1016/j.bmc.2013.06.013

Bioorganic & Medicinal Chemistry 21 (2013) 5355–5361
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioevaluation of some new isoniazid derivatives
Lilia Matei a,b, Coralia Bleotu a, Ion Baciu b, Constantin Draghici c, Petre Ionita b, Anca Paun b,
Mariana Carmen Chifiriuc d, Adriana Sbarcea e, Irina Zarafu b,
⇑
a Stefan S. Nicolau Institute of Virology, 285 M. Bravu Ave., Bucharest 030304, Romania
b University of Bucharest, Department of Organic Chemistry, Biochemistry and Catalysis, 90-92 Panduri, Bucharest 050663, Romania
c C.D. Nenitzescu Institute of Organic Chemistry, 202B Spl. Independentei, Bucharest 060023, Romania
d University of Bucharest, Faculty of Biology, 3 Portocalelor, Bucharest 060101, Romania
e Prof. Dr. D. Gerota Emergency Hospital, 50 Ferdinand Ave., Bucharest 021392, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of the study was to synthesize some new compounds with potential anti-tuberculosis activity,
Received 11 April 2013
Revised 30 May 2013
Accepted 6 June 2013
Available online 15 June 2013
containing isoniazid and a,b-unsaturated thiocinnamamide-like thioamides as precursors. The obtained
derivatives were evaluated regarding their biological activity (antioxidant and antibacterial), as well as
their influence on the eukaryotic cell cycle. The results suggested that the newly obtained derivatives
of isoniazid exhibited different biological activities, depending on their structure; thus, the most active
compound in terms of anti-oxidant and anti-Mycobacterium tuberculosis effects proved to be the isonicot-
inic acid N0-(1-amino-1-mercapto-3-phenyl-propen-1-yl)-hydrazide. This compound also increased the
expression of NAT1 and NAT2 genes, which are implicated in the metabolism of the isoniazid, demon-
strating that it could be rapidly metabolized, and thus well tolerated. The largest spectrum of antibacte-
rial activity (excluding M. tuberculosis) was noticed for the isonicotinic acid N0-[1-amino-1-mercapto-
3-(p-chloro-phenyl)-propen-1-yl]-hydrazide, which was also the most cytotoxic, especially at high con-
centrations, although not significantly affecting the cellular cycle phases. The obtained results showed
that the new derivatives could represent potential candidates for the treatment of M. tuberculosis infec-
tions, but further research is needed in order to improve their pharmacological properties, by increasing
their antimicrobial activity and reducing the risk of side-effects.
Keywords:
Isoniazid
Thioamides
Synthesis
Biological evaluation
Antitubercular activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
However, new anti-tubercular drugs with new mechanisms of
action have not been developed in the last years.7 The resurgence
Tuberculosis is an infectious disease with a large spectrum of
manifestations caused by Mycobacterium tuberculosis.1 Today,
tuberculosis is among the top five causes of global mortality.2
According to the World Health Organization, one third of people
are infected with M. tuberculosis.3 In 2011 there were 8.7 million
new tuberculosis cases, including 1.1 million cases among people
with HIV, and 1.4 million people died from tuberculosis, including
half a million women and 430,000 people with HIV.4
At present, the treatment of tuberculosis is based on two groups
of drugs. The first-line drugs (isoniazid (INH), rifampin, pyrazina-
mide (PZA), ethambutol (EMB) and streptomycin) have a greater
efficacy and acceptable toxicity, while the second-line ones (kana-
mycin, amikacin, capreomycin, cycloserine (CS), ethionamide
(ETH), para-aminosalicylic acid (PAS), and fluoroquinolones (FQ))
of tuberculosis and the emergence of drug resistant M. tuberculosis
isolates, in the recent years, have renewed the interest in the
development of new effective anti-tubercular drugs6,8 (e.g., deriva-
tives of thiadiazole,2,8 pyrazine,9 ethionamide10).
INH is the most frequently prescribed antibiotic in the treatment
of tuberculosis.11 Although it is being used for more than 60 years
for tuberculosis treatment,12 its mechanisms of action are question-
able.13 INH is a prodrug that is activated in vivo by KatG (mycobac-
terial catalase–oxidase).13,14 INH-derived reactive species inhibit
the synthesis of cell wall lipids and of nucleic acids13 and also inter-
fere with bacterial respiratory metabolism.15 INH derivatives have
proved good anti-mycobacterial activity,6,16–20 some of them being
more active than the prodrug they derived from.11,16,20 So, INH
derivatives might be useful in increasing the effectiveness of
standard drug regiments in the therapy of M. tuberculosis
infections and may serve as promising compounds for future anti-
mycobacterial drug development.6,16,17
are usually characterized by
a lower efficacy or a greater
toxicity.1,5,6
The drug toxicity is another problem in tuberculosis treatment.
INH is metabolized by acetylation and dehydrazination and the
N-acetylhydrazine metabolite is believed to be responsible for
⇑
Corresponding author. Tel.: +40 0214100204.
E-mail address: zarafuirina@yahoo.fr (I. Zarafu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.013

5356
L. Matei et al. / Bioorg. Med. Chem. 21 (2013) 5355–5361
the hepatotoxic effects seen in treated patients. In eukaryotic cells,
N-acetyltransferases (NAT) are involved in the biotransformation
of aromatic amines and hydrazines by transferring the acetyl group
from acetyl coenzyme A to the free amino group of the parent com-
pound, the N-acetylation of the parent amines being considered a
detoxification step.21,22 NATs are cytosolic enzymes found in many
tissues, with distinct tissue distribution and substrate specificity.
In humans, two forms with high level of homology are known, that
is, NAT1 and NAT2. While NAT1 has a ubiquitous tissue distribu-
tion and its expression has been demonstrated to be also related
to cancers, NAT2 activity has been described in liver, colon and
intestinal epithelium.22 The rate of acetylation is genetically deter-
mined and has not been shown to significantly alter the effective-
ness of INH. Slow acetylation may lead to the accumulation of high
drug concentrations, with an increased risk of toxicity. Although
the implication of NAT gene polymorphism in drug metabolism
was intensively studied, to date, the modulation of NATs expres-
sion by different drugs was less evaluated.
between INH and thioamides 1–5 (thioamides obtained previously
by Pappalardo method23,24 Fig. 1A). Several methods, using differ-
ent solvents, were used, like dimethyl sulfoxide, tetrahydrofuran,
ethanol, but the best results were obtained using a mixture of eth-
anol and water, and adding a drop of hydrochloric acid. Starting
materials were added in equimolar quantity, refluxed for 1–2 h,
and then left for 8 days at room temperature. Product formation
(mixture Z/E) was monitored by thin layer chromatography on sil-
ica gel. The Rf values obtained are presented in Table 1. The most
polar compounds have been found to be 7, 8 and 9, followed by
compounds 10 and 6. Table 1 compiles also the yields of the syn-
thesis and the melting points of new derivatives.
The chemical structure of the newly synthesized compounds
was confirmed by 1H and 13C NMR spectral analysis and by ele-
mental analysis. The 1H NMR spectra showed two singlets at
10.06–10.10 ppm and 9.52–9.56 ppm, which represent the two
hydrazide protons (NH–NH) that are deuterable. The 13C NMR
spectra also showed the C(SH)(NH2) signals at 76.25–76.59 ppm.
The complete interpretation of 1H and 13C NMR spectra and the
elemental analyses of new compounds are presented in the exper-
imental part.
The aim of this article is to present a series of newly synthesized
INH derivatives and the preliminary evaluation of their biological
activity.
2. Results and discussion
2.2. Biological evaluation
2.1. Synthesis and structural characterization
2.2.1. Antioxidant activity of INH derivatives
One of the most important properties of the biological active
substances is their antioxidant activity, which reflects their capac-
ity to protect the organism from the damage caused by free radi-
cals. In literature data, several methods are used to measure the
Starting from INH we have synthesized five new derivatives,
containing moieties of thiocinnamamide-like thioamides. All the
compounds were synthesized by
a simple coupling reaction
Figure 1. Synthesis of thioamides 1–5 (A) and of the new isoniazid derivatives 6–10 (B).

L. Matei et al. / Bioorg. Med. Chem. 21 (2013) 5355–5361
5357
Table 1
In order to establish the spectrum of the antibacterial activity of
the novel INH derivatives, they were also tested on other non-
tuberculosis bacterial strains isolated from clinical specimens, that
is: Gram-positive (Streptococcus hominis, Staphylococcus aureus,
Enterococcus faecalis) and Gram-negative (Escherichia coli, Klebsiella
pneumoniae, Proteus mirabilis, Citrobacter koseri, Morganella morg-
anni, Acinetobacter baumanii, Pseudomonas aeruginosa) using an
adapted diffusion technique. Interestingly, the most active com-
pound proved to be the compound 7, substituted with a chlorine
atom in para-position (active against all tested bacterial strains),
followed by the compounds 6 and 9. All new synthesized deriva-
tives proved to be more active on the tested strains than INH and
the thioamides they derived from, excepting compound 10, that
was less active than the compound 5 (Table 3).
Results of the physic-chemical characterization of the obtained compounds 6–10
a
Compd
Yield (%)
Mp (°C)
Rf
TAC (%)
INH
1
2
3
4
5
6
7
8
—
171–173
144–145
192–193
195–197
173–174
175–176
120–123
198.6–201
186–188
169–170
161–164
0.29
0.65
0.80
0.86
0.79
0.70
0.58
0.65
0.63
0.61
0.59
90.00
35.71
15.71
25.71
60.00
37.85
88.23
25.73
12.50
13.97
25.73
44
43
47
52
41
74
75
80
65
60
9
10
a
AcOEt:CH2Cl2:CH3OH = 5:3:2.
2.2.3. Cytotoxicity and effects on cell cycle
total antioxidant activity (TAC). In the present study we have used
the DPPH method, based on the properties of a stable free radical
2,2-diphenyl-1-picrylhydrazyl (DPPH).25 TAC values (%) were cal-
culated using the Eq. 1:
All tested compounds were further tested for their cytotoxicity
and the apoptotic effect on HCT-8 cell line. The discrimination be-
tween intact and apoptotic cells was performed by flow cytometry
using FITC-labeled annexin-V and propidium iodide. The most
toxic compounds proved to be the thioamides 1 and 2 (at the
TAC ð%Þ ¼ Absini ꢀ Abs30 min=Absini ꢁ 100
ð1Þ
tested concentration of 50
lg/mL) and the new derivative 7 (at
where Absini = the initial absorption of DPPH radical (measured at
517 nm), Abs30 min = the absorbance recorded at the same wave-
length after 30 min.
the tested concentration of 25
lg/mL) (see Supplementary data
for more information details).
Occurrence of apoptosis for these three compounds was also
observed in cell cycle analysis, indicated by the presence of one
A comparative study of the antioxidant capacity of both precur-
sors, thioamides and INH, as well as of the products of the coupling
reaction showed that the newly synthesized compounds 6 and 7
exhibited a better antioxidant activity than their precursors, that
is, the compounds 1 and 2, respectively (Table 1). As it could be no-
ticed, INH presented the highest antioxidant activity (90%), fol-
lowed by the compounds 6 (88.23%) and 4 (60%). All the other
tested derivatives exhibited a weaker antioxidant activity with
percentages of inhibition lower than 40%.
sub-G0/G1 (left) peak. At lower concentrations (25
lg/mL for com-
pounds 1 and 2 and 12.5 g/mL for compound 7), these com-
l
pounds also affected cell cycle. Compound 1 increased S and G2/
M phases and decreased G0/G1 phase, effect that was not observed
for its derivative, compound 6, which only slightly affected the cell
cycle. Compound 2 also determined the increase of S and G2/M
phases and the decrease of G0/G1 phase, but its derivative, com-
pound 7, although more toxic, determined a small decrease of S
phase and an increase of G2/M phase. Compound 3 increased G2/
M phase and decreased G0/G1 and S phases, although its deriva-
tive, compound 8, increased G0/G1 phase and decreased S and
G2/M phases. Compound 9 did not significantly affect the cell cy-
cle, while compound 4 increased G2/M and decreased S phases.
Compound 5 slightly increased G0/G1 phase and decreased S
phase, and compound 10 increased both G0/G1 and G2/M phases
and decreased S phase (see also Supplementary data).
2.2.2. Antimicrobial activity
In order to discover new agents potentially useful in the
treatment of tuberculosis,
a,b-unsaturated thioamides combina-
tions of INH were synthesized and tested for their in vitro
anti-tubercular activity. The tested compounds were assayed in
serial binary dilutions ranging from 25 to 0.012
pound 6 exhibited the best anti-Mycobacterium activity, with a
Minimal Inhibition Concentration (MIC) of 0.391 g/mL, followed
by the compounds 8 and 10 (MIC = 0.781 g/mL). The compound
7 exhibited the lowest inhibitory activity (MIC = 6.25 g/mL). This
lg/mL. The com-
l
l
2.2.4. Influence of new INH derivatives on NATs expression
NAT protein is essential in the INH metabolization, being critical
for the control of the magnitude of the side-effects exhibited by
this drug. NAT protein is also essential for the survival of Mycobac-
terium bovis BCG inside macrophages,32 being implicated in the
degradation of cholesterol, which seems to be essential for intra-
cellular mycobacterial survival. It is not yet demonstrated if its
expression is correlated with other stress response genes in myco-
bacteria. So far, mycobacterial NAT genes have been identified in
the slow growing mycobacteria including M. tuberculosis and also
in the non-pathogenic model strain Mycobacterium bovis BCG.33
In the present study we have investigated the influence of new
INH derivatives on the expression of NAT genes in the eukaryotic
cells, as an additional marker for their cytotoxicity. The obtained
results varied for the tested compounds. INH significantly de-
creased the expression of NAT1 and NAT2 genes (Fig. 2). A similar
behavior with that of INH was noticed for the compounds 1, 3, 4
and 9, in case of NAT1 and for the compounds 2, 3, 4, 5 and 9 in
case of NAT2 gene. The rest of the tested compounds induced the
expression of NAT1 and NAT2 genes with different intensities. Con-
cerning the compound 6, which exhibited the best antimicrobial
activity, it induced a slight increase of NAT1 and NAT2 genes, sug-
gesting a better rate of metabolization and thus, a lower risk for
l
result is surprising, knowing that the chlorine substituents are gen-
erally expected to improve the antimicrobial activity.19,26
Although all tested compounds displayed an anti-tubercular
activity lower than that of INH (MIC = 0.098 lg/mL) (Table 2), their
activity is comparable to other INH derivatives reported in the
literature,27–31 Excepting the compound 7, all other synthesized
compounds displayed MICs below the 6.25 lg/mL value,
postulated by the Global Program for the Discovery of New Anti-
Tuberculosis Drugs as the upper threshold for the evaluation of
new anti-M. tuberculosis agents.27
Table 2
In vitro activity of INH derivatives against M. tuberculosis
Compd
MIC (lg/mL)
INH
6
7
0.098
0.391
6.25
8
9
10
0.781
1.563
0.781

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L. Matei et al. / Bioorg. Med. Chem. 21 (2013) 5355–5361
Table 3
The antimicrobial evaluation of the new compounds 6–10 and their precursors 1–5 expressed as bacterial growth inhibition (+), or not (ꢀ)
Compound
INH
1
2
3
4
5
6
7
8
9
10
Micrococcaceae
Streptococcus hominis 1813
Staphylococcus aureus
ꢀ
ꢀ
+
+
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+
+
+
+
+
+
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+
Enterobacteriaceae
Enterobacter cloacae 1845
Citrobacter koseri 1742
Morganella morganni 2810
Proteus mirabilis 1751
Escherichia coli 1777
+
+
+
+/ꢀ
+/ꢀ
ꢀ
+/ꢀ
+/ꢀ
ꢀ
+/ꢀ
+/ꢀ
ꢀ
+
+
+
+
+
+
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+
+
+
+
+
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+/ꢀ
+/ꢀ
ꢀ
+/ꢀ
+/ꢀ
ꢀ
+/ꢀ
ꢀ
+/ꢀ
+
ꢀ
ꢀ
ꢀ
+/ꢀ
ꢀ
+
+
+/ꢀ
ꢀ
+/ꢀ
ꢀ
+
ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
Klebsiella pneumoniae 1756
ꢀ
ꢀ
Pseudomonadaceae
Pseudomonas aeruginosa ATCC 27853
Pseudomonas aeruginosa 165
Pseudomonas aeruginosa 1144
Pseudomonas putida 160
ꢀ
+/ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
+
+
+
+
+
+
+
ꢀ
+
+
+
+/ꢀ
+
+
ꢀ
ꢀ
+/ꢀ
+/ꢀ
+
+/ꢀ
+/ꢀ
+/ꢀ
+
ꢀ
+/ꢀ
+
+
+/ꢀ
+
+
+
+
+
+
+
Enterococcaceae
Enterococcus faecalis 2920
ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+
+/ꢀ
+
+/ꢀ
+/ꢀ
+/ꢀ
Moraxellaceae
Acinetobacter baumanii 122
Acinetobacter baumanii 125
Acinetobacter baumanii 135
Acinetobacter baumanii 136
Acinetobacter baumanii 192
Acinetobacter baumanii 247
ꢀ
+
+/ꢀ
ꢀ
+/ꢀ
ꢀ
+/ꢀ
ꢀ
+/ꢀ
ꢀ
+
+
+
+/ꢀ
+/ꢀ
+
+
+
+
+
+
+
+
+
+/ꢀ
+/ꢀ
+
+/ꢀ
+/ꢀ
+
+/ꢀ
+/ꢀ
+
ꢀ
+/ꢀ
+
+
+
+
+
+/ꢀ
+
+
+
+
+
+
+
+
+/ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
ꢀ
+/ꢀ
+/ꢀ
ꢀ
+/ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+/ꢀ
ꢀ
+/ꢀ
+/ꢀ
Figure 2. The influence of the new isoniazid derivatives 6–10 and their precursors 1–5 on the expression of NAT genes in the eukaryotic cells.
side effects that could be induced by the accumulation of the com-
pound in high concentrations within the host. A similar behavior
was also noticed for the compounds 8 and 10, also exhibiting a
good MIC on Mycobacterium tuberculosis. These results suggest that
these compounds could be better tolerated than INH in the human
host.
it could be rapidly metabolized, and thus well tolerated within
the human host. The largest spectrum of antibacterial activity
was noticed for the compound 7 that was also the most cytotoxic
one. The obtained results showed that the obtained compounds
could represent potential candidates for the development of new
anti-M. tuberculosis agents. However, more research is needed to
improve their pharmacological properties, by increasing their anti-
microbial activity and by reducing the risk of side-effects.
3. Conclusions
The synthesis of the new INH derivatives was performed with
good yields and their structure was confirmed by physico-chemical
analyses. In relation to the biological studies, the obtained results
showed that the newly obtained derivatives of INH exhibit differ-
ent biological activities, depending on their structure. The most ac-
tive compound in terms of anti-oxidant and anti-Mycobacterium
tuberculosis effects proved to be the compound 6. This compound
also increased the expression of NAT1 and NAT2 genes, which
are implicated in the metabolism of the INH, demonstrating that
4. Materials and methods
4.1. Apparatus and chemicals
Starting materials (chemicals, TLC plates) and solvents were
purchased from Sigma–Aldrich and Chimopar and used as
received. 1H and 13C NMR spectra were recorded on a Varian
Inova-300 spectrometer (at selected temperatures, in deuterated
solvent DMSO-d6, isotopic purity 99.9%); UV–vis spectra were

L. Matei et al. / Bioorg. Med. Chem. 21 (2013) 5355–5361
5359
recorded on a UVD-3500 spectrometer, in methanol at ambient
temperature. Melting points were determined with a Böethius
apparatus and device Krüss. For biological tests the tested com-
pounds were initially dissolved into DMSO to prepare a concen-
trated stock solution. Following additional dilutions were
prepared in culture medium.
1H NMR (DMSO-d6, T = 303 K, d ppm, J Hz): 10.08 (br s, 1H, deu-
terable); 9.56 (br s, 1H, deuterable); 9.30 (br s, 1H, deuterable);
8.74 (d, 2H, H-1, H-5, 5.9); 7.71 (d, 2H, H-2, H-4, 5.9); 7.65 (d,
1H, H-9, 15.6); 7.54 (d, 2H, H-11, H-15, 8.3); 7.35 (d, 2H, H-12,
H-14, 8.3); 7.04 (d, 1H, H-8, 15.6); 4.57 (br s, 2H, H-N, deuterable);
2.47 (s, 3H, H-16).
13C NMR (DMSO-d6, T = 303 K, d ppm): 163.95 (C-6); 150.21 (C-
1, C-5); 141.06 (C-9); 140.28 (C-3); 137.16 (C-13); 133.98 (C-10);
129.78 (C-8); 128.91 (C-11, C-15); 127.53 (C-12, C-14); 121.02
(C-2, C-4); 76.56 (C-7); 21.05 (C-16).
4.2. INH derivatives synthesis
Thioamides 1–5 were synthesized using the Pappalardo meth-
od.23,24 Reaction mechanism is shown in Figure 1A.
Rf (silicagel, AcOEt:CH2Cl2:CH3OH = 5:3:2): 0.63.
The isonicotinoyl hydrazide derivatives were prepared by reac-
tion between the appropriate thiocinnamamide-like thioamides
1–5 (1.0 equiv) with INH (1.0 equiv).
To 1 mmol (0.137 g) of INH dissolved in 3.5 mL water was
added 1 mmol of appropriate thioamide dissolved in 5 mL ethanol
4.2.4. Isonicotinic acid N0-[1-amino-1-mercapto-3-(p-methoxy-
phenyl)-propen-1-yl]-hydrazide (9)
Yield: 65%.
Elemental analysis: C16H18N4O2S, M = 330; calcd: C = 58.18,
H = 5.45, N = 16.97, S = 9.70; found: C = 58.24, H = 5.43, N = 16.95,
S = 9.87.
and 10 lL of hydrochloric acid (37%). The mixture was refluxed for
1–2 h and then stirred for 8 days at room temperature. The prod-
ucts were separated on preparative TLC using silica gel plates or
on chromatographic columns filled with silica gel and the mixture
AcOEt:CH2Cl2:CH3OH = 5:3:2 as eluent.
Purities of the synthesized compounds were checked by thin
layer chromatography on silica gel.
1H NMR (DMSO-d6, T = 303 K, d ppm, J Hz): 10.06 (br s, 1H, deu-
terable); 9.52 (br s, 1H, deuterable); 9.23 (br s, 1H, deuterable);
8.71 (d, 2H, H-1, H-5, 6.0); 7.72 (d, 2H, H-2, H-4, 6.0); 7.77 (d,
1H, H-9, 15.3); 7.51 (d, 2H, H-11, H-15, 8.6); 6.9 (d, 2H, H-12, H-
14, 8.6); 6.77 (d, 1H, H-8, 15.3); 4.52 (br s, 2H, H-N, deuterable);
3.84 (s, 3H, H-16).
13C NMR (DMSO-d6, T = 303 K, d ppm): 163.87 (C-6); 161.57 (C-
13); 150.26 (C-1, C-5); 141.23 (C-9); 140.65 (C-3); 133.31 (C-10);
129.55 (C-8); 129.49 (C-11, C-15); 115.51 (C-12, C-14); 121.12
(C-2, C-4); 76.43 (C-7); 55.42 (C-16).
4.2.1. Isonicotinic acid N0-(1-amino-1-mercapto-3-phenyl-
propen-1-yl)-hydrazide (6)
Yield: 74%.
Elemental analysis: C15H16N4OS, M = 300; calcd: C = 60.00,
H = 5.33, N = 18.67, S = 10.67; found: C = 59.94, H = 5.36,
N = 18.72, S = 10.56.
Rf (silicagel, AcOEt:CH2Cl2:CH3OH = 5:3:2): 0.61.
4.2.5. Isonicotinic acid N0-[1-amino-1-mercapto-3-(p-ethoxy-
phenyl)-propen-1-yl]-hydrazide (10)
1H NMR (DMSO-d6, T = 303 K, d ppm, J Hz): 10.08 (br s, 1H, deu-
terable); 9.53 (br s, 1H, deuterable); 9.25 (br s, 1H, deuterable);
8.71 (d, 2H, H-1, H-5, 6.1); 7.73 (d, 2H, H-2, H-4, 6.1); 7.65 (d,
1H, H-9, 15.6); 7.59 (dd, 2H, H-11, H-15, 1.3, 8.2); 7.41 (dd, 2H,
H-12, H-14, 7.5, 8.2); 7.34 (tt, 1H, H-13, 1.3, 7.5); 7.01 (d, 1H, H-
8, 15.6); 4.53 (br s, 2H, H-N, deuterable).
Yield: 60%.
Elemental analysis: C17H20N4O2S, M = 344; calcd: C = 59.30,
H = 5.81, N = 16.28, S = 9.30; found: C = 59.27, H = 5.78, N = 16.24,
S = 9.36
1H NMR (DMSO-d6, T = 303 K, d ppm, J Hz): 10.07 (br s, 1H, deu-
terable); 9.53 (br s, 1H, deuterable); 9.24 (br s, 1H, deuterable);
8.69 (d, 2H, H-1, H-5, 5.8); 7.67 (d, 2H, H-2, H-4, 5.8); 7.76 (d,
1H, H-9, 15.23); 7.50 (d, 2H, H-11, H-15, 8.59); 6.89 (d, 2H, H-12,
H-14, 8.59); 6.76 (d, 1H, H-8, 15.23); 4.55 (br s, 2H, H-N, deuter-
able); 4.07 (q, 2H, H-16, 7.03), 1.43 (t, 3H, H-17, 7.03).
13C NMR (DMSO-d6, T = 303 K, d ppm): 163.89 (C-6); 159.25 (C-
13); 150.18 (C-1, C-5); 143.94 (C-9); 140.52 (C-3); 132.56 (C-10);
129.40 (C-8); 129.10 (C-11, C-15); 115.98 (C-12, C-14); 121.31
(C-2, C-4); 76.25 (C-7); 63.68 (C-16); 14.73 (C-17).
Rf (silicagel, AcOEt:CH2Cl2:CH3OH = 5:3:2): 0.59.
13C NMR (DMSO-d6, T = 303 K, d ppm): 163.80 (C-6); 150.10 (C-
1, C-5); 141.02 (C-9); 140.18 (C-3); 134.69 (C-10); 129.69 (C-8);
128.96 (C-11, C-15); 128.59 (C-13); 127.74 (C-12, C-14); 120.90
(C-2, C-4); 76.51 (C-7).
Rf (silicagel, AcOEt:CH2Cl2:CH3OH = 5:3:2): 0.58.
4.2.2. Isonicotinic acid N0-[1-amino-1-mercapto-3-(p-chloro-
phenyl)-propen-1-yl]-hydrazide (7)
Yield: 75%.
Elemental analysis: C15H15ClN4OS, M = 334.5; calcd: C = 53.81,
H = 4.48, Cl = 10.61, N = 16.74, S = 9.57; found: C = 53.77, H = 4.51,
Cl = 10.67, N = 16.71, S = 9.59.
4.3. Antioxidant activity of INH derivatives
1H NMR (DMSO-d6, T = 303 K, d ppm, J Hz): 10.11 (br s, 1H, deu-
terable); 9.54 (br s, 1H, deuterable); 9.25 (br s, 1H, deuterable);
8.73 (d, 2H, H-1, H-5, 5.9); 7.74 (d, 2H, H-2, H-4, 5.9); 7.63 (d,
1H, H-9, 15.4); 7.58 (d, 2H, H-11, H-15, 8.1); 7.42 (d, 2H, H-12,
H-14, 8.1); 6.98 (d, 1H, H-8, 15.4); 4.54 (br s, 2H, H-N, deuterable).
13C NMR (DMSO-d6, T = 303 K, d ppm): 163.96 (C-6); 150.17 (C-
1, C-5); 140.58 (C-9); 140.25 (C-3); 134.42 (C-13); 134.02 (C-10);
129.52 (C-8); 128.65 (C-11, C-15); 127.56 (C-12, C-14); 120.98
(C-2, C-4); 76.59 (C-7).
A DPPH solution in methanol was prepared at a 2 ꢁ 10ꢀ4 M con-
centration. INH, thioamides 1–5 and new derivatives 6–10 were
dissolved also in methanol at a 0.1 mg/mL concentration. DPPH
solution (1.8 mL) was mixed with the solution of each compound
(0.2 mL) and kept in dark for 30 min. The absorbance of solutions
was measured at 517 nm. The blank mixture was obtained from
1.8 mL DPPH solution and 0.2 mL methanol. TAC values were cal-
culated according to Eq. 1.
Rf (silicagel, AcOEt:CH2Cl2:CH3OH = 5:3:2): 0.65.
4.4. Antibacterial activity
4.2.3. Isonicotinic acid N0-[1-amino-1-mercapto-3-(p-tolyl)-
propen-1-yl]-hydrazide (8)
In vitro antimicrobial tests were carried out by an adapted
agar-disk diffusion technique using a bacterial suspension of 0.5
McFarland density obtained from 24 h cultures. The antimicrobial
activity of the newly synthesized compounds was determined
against the following strains: of Escherichia Enterobacter cloacae,
Yield: 80%.
Elemental analysis: C16H18N4OS, M = 314; calcd: C = 61.15,
H = 5.73, N = 17.83, S = 10.19; found: C = 61.28, H = 5.78,
N = 17.86, S = 10.06.

5360
L. Matei et al. / Bioorg. Med. Chem. 21 (2013) 5355–5361
Citrobacter koseri, Morganella morganii, Proteus mirabilis, Acineto-
bacter baumanii, Pseudomonas aeruginosa, Peudomonas putida,
Streptococcus hominis, Enterococcus faecalis and Staphylococcus
4.9. Cell cycle analysis
The cells were harvested after treatment with 50 lg/mL of INH
aureus. The solutions of tested compounds (5
lL of 1 mg/mL
and compounds 1–10, 25
lg/mL for compounds 1 and 2, and
solution) were spotted on the solid medium previously seeded
with the microbial inocula. The inoculated plates were incubated
for 24 h at 37 °C.
12.5 g/mL for compound 7, for 24 h, washed in a cold solution
of PBS (pH 7.5), then fixed in cold 70% ethanol and stored at
l
ꢀ20 °C overnight. The samples were then centrifuged, washed with
PBS and then re-suspended in 100 lL PBS, treated with RNase A
4.5. Antimycobacterial activity
(1 mg/mL) and labeled with propidium iodide (100 lg/mL), incu-
bated in the dark at room temperature for 30 min prior measure-
ment. DNA content of cells was quantified on a Beckman Coulter
EPICS XL flow cytometer and analyzed using FlowJo 8.8.6 software
(Ashland, Oregon, USA).
The antimycobacterial activities of the new synthesized com-
pounds were assessed against M. tuberculosis clinical strain using
the micro plate Alamar Blue assay (MABA) according to Franzblau
et al.34 Briefly, 200
lL of sterile deionized water was added to all
outer-perimeter wells of sterile 96 well plates to minimize evapo-
ration of the medium in the test wells during incubation. A serial
dilution of the new synthesized compounds 6–10 and INH were
4.10. Quantitative RT-PCR for NAT1 and 2 expressions
Total RNA was extracted with Trizol Reagent (Invitrogen, USA)
according to the manufacturer’ protocol from cells treated with
INH and compounds 1–10 at non-toxic concentrations for 24 h.
made directly in 96 well on the plate in a volume of 50
Middlebrook 7H9 broth. The final drug concentrations tested ran-
ged from 25 to 0.012 g/mL.
An inoculum of M. tuberculosis (50
Plates were covered and sealed with parafilm and incubated at
37 °C for 8 days. After this time, 10 L of a freshly prepared 1:1
lL of the
l
For each sample, 2 lg of total RNA was used for reverse transcrip-
lL) was added in the wells.
tion with High Capacity cDNA Reverse Transcription Kit with
RNAse inhibitor (Applied Biosystem), and 50 ng cDNA from each
sample was used in real time PCR reaction. Real Time PCR was per-
formed on an ABI 7300 Real Time PCR System using pre-validated
Taqman Gene Expression Assays kits (Applied Biosystems): NAT1
(Hs00265080_s1) and NAT2 (Hs01854954_s1). As endogenous
control was used human beta actin. Each experiment was per-
formed three times. Results were analyzed with RQ study software
(Applied Biosystems). The DDCT method was used to compare the
relative expression levels.
l
mixture of Alamar Blue (0.1 mg/mL) reagent and 10% tween 80
was added to the plate and incubated at 37 °C for another 24 h. A
blue color in the well was interpreted as no bacterial growth,
and a pink color was scored as growth. The Minimal Inhibition
Concentration (MIC) was defined as the lowest drug concentration,
which prevented a color change from blue to pink.
4.6. Cell culture
Acknowledgments
The eukaryotic cell culture used in our assays was represented
by HCT-8 (CCL-224) cells. The cells were maintained as an adher-
ent culture in Dulbecco’s Modified Essential Medium (DMEM)
(Sigma, USA) supplemented with 10% heat-inactivated fetal bovine
serum (Sigma, USA) at 37 °C, 5% CO2, in a humid atmosphere.
This work was partially supported by a Grant of the Romanian
National Authority for Scientific Research, CNCS—UEFISCDI, project
number PN-II-RU-TE 13/2011.
Supplementary data
4.7. Trypan blue exclusion assay
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.013.
The cytotoxicity of new INH derivatives (6–10) was determined
in HCT8 cells using Trypan Blue Exclusion method. A number of
3 ꢁ 105 HCT8 cells were seeded in 3.5-cm diameter wells and trea-
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