Synthesis and in vitro and in vivo antimycobacterial activity of isonicotinoyl hydrazones

Article abstract of DOI:10.1016/j.bmcl.2005.07.011

The purpose of this study was to prepare various isoniazid derivatives by introducing the isoniazid pharmacophore into several molecules and screening for antimycobacterial activity. Ortho-hydroxy acetophenone reacts with isoniazid to form acid hydrazones. The C-Mannich bases of the above acid hydrazones were prepared by reacting them with formaldehyde and various secondary amines. The synthesized compounds were screened against M. tuberculosis H₃₇R _v using the alamar blue susceptibility test. The synthesized compounds inhibit Mycobacterium tuberculosis strain H₃₇R_v with minimum inhibitory concentrations ranging from 0.56 to 4.61 μM. Compound N′-{1-[2-hydroxy-3-(piperazin-1-ylmethyl)phenyl]ethylidene} isonicotinohydrazide 8 was found to be the most active compound with an MIC of 0.56 μM, and was more potent than isoniazid (MIC of 2.04 μM). After 10 days of treatment, compound 8 decreased the bacterial load in murine lung tissue by 3.7-log 10 as compared to controls, which was equipotent to isoniazid. The results demonstrate the potential and importance of developing new isoniazid derivatives against mycobacterial infections.

Full text of DOI:10.1016/j.bmcl.2005.07.011

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4502–4505
Synthesis and in vitro and in vivo antimycobacterial activity
of isonicotinoyl hydrazones
Dharmarajan Sriram,* Perumal Yogeeswari and Kasinathan Madhu
Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science, Pilani 33031, India
Received 16 February 2005; revised 5 July 2005; accepted 7 July 2005
Available online 22 August 2005
Abstract—The purpose of this study was to prepare various isoniazid derivatives by introducing the isoniazid pharmacophore into
several molecules and screening for antimycobacterial activity. Ortho-hydroxy acetophenone reacts with isoniazid to form acid
hydrazones. The C-Mannich bases of the above acid hydrazones were prepared by reacting them with formaldehyde and various
secondary amines. The synthesized compounds were screened against M. tuberculosis H37Rv using the alamar blue susceptibility test.
The synthesized compounds inhibit Mycobacterium tuberculosis strain H37Rv with minimum inhibitory concentrations ranging from
0.56 to 4.61 lM. Compound N0-{1-[2-hydroxy-3-(piperazin-1-ylmethyl)phenyl]ethylidene}isonicotinohydrazide 8 was found to be
the most active compound with an MIC of 0.56 lM, and was more potent than isoniazid (MIC of 2.04 lM). After 10 days of treat-
ment, compound 8 decreased the bacterial load in murine lung tissue by 3.7-log10 as compared to controls, which was equipotent to
isoniazid. The results demonstrate the potential and importance of developing new isoniazid derivatives against mycobacterial
infections.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
mechanism of resistance to INH is related to katG
mutations and deletions, and secondly to chromosomal
mutations in inhA and kasA.7 Antibacterial resistance
to a drug can be counteracted by designed new deriva-
tives.8 Further, pharmacokinetic properties and cellular
permeability of a drug can be modulated by derivatiza-
tion to bioreversible forms of this drug, namely hydra-
zones.9 Preparation of Mannich bases of isonicotinoyl
hydrazone have improved the lipid solubility.10 Here,
we report the synthesis and preliminary antimycobacte-
rial activity data of newer isonocotinoyl hydrazones.
Tuberculosis (TB) is one of the most common infectious
diseases known to man. About 32% of the worldÕs pop-
ulation (1.9 billion people) is infected with TB. Every
year, approximately 8 million of these infected people
develop active TB, and almost 2 million of them die
from the disease,1 a life lost to TB every 15 s. The inci-
dence of TB infection has steadily risen in the last dec-
ade and this increase can be attributed to a similar
increase in human immunodeficiency virus (HIV) infec-
tion.2 The association of TB and HIV infections is so
dramatic that, in some cases, nearly two-thirds of the
patients diagnosed with TB are also HIV-1 seroposi-
tive.3 Furthermore, numerous studies have shown that
TB is a cofactor in the progression of HIV infection.4
The reemergence of TB infection is further complicated
by an increase in cases, which are resistant to conven-
tional antitubercular drug therapy.5 On the other hand,
in spite of toxicity on repeated dosing, isoniazid (INH) is
still considered to be a first line drug for chemotherapy
of tuberculosis.6 Recently, it was suggested that the
2. Chemistry
Isonicotinylhydrazones 1–15 described in this study is
shown in Table 1, and a reaction sequence for the prep-
aration is outlined in Figure 1. 2-Hydroxy acetophenone
reacts with isoniazid in the presence of glacial acetic acid
in ethanolic medium refluxed for 9 h to form acid
hydrazones (yield: 72.6%, mp: 242 °C),14 which in turn,
reacts with formaldehyde and appropriate secondary
amines in a microwave oven at an intensity of 80% with
30 s/cycle. The number of cycles in turn depended on the
completion of the reaction, which was checked by TLC.
The reaction timing varied from 1.5–3 min to form tar-
geted compounds (55–64% yields). The purity of the
Keywords: Isoniazide derivatives; Antimycobacterial.
*
Corresponding author. Tel.: +91 1596 244684; fax: +91 1596
244183; e-mail: dsriram@bits-pilani.ac.in
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.011

D. Sriram et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4502–4505
Table 1. Physical data and antimycobacterial activity of synthesized compounds
4503
CH3
NNHCO
C
N
OH
CH2
R
logPa
MICb (lM)
IC50 (lM)
c
Compound
R
Molecular formula
Yield (%)
Mp (°C)
1
2
3
–N(CH3)2
–N(C2H5)2
–N(C4H9)2
C17H20N4O2
C19H24N4O2
C23H32N4O2
56
62
52
234
213
208
2.32
2.75
5.29
2.49
2.29
3.93
>20.00
>18.35
>15.76
4
C20H24N4O2
62
186
3.83
4.43
>17.73
N
N
5
6
C19H22N4O2
C19H22N4O3
C27H24N4O2
C19H23N5O2
C20H21N5O2
C29H28N4O2
58
55
61
50
54
46
194
219
208
183
194
190
3.36
2.05
6.16
2.61
2.02
6.07
4.61
2.20
3.57
0.56
8.61
3.36
>18.47
>17.63
>14.32
>17.68
>17.20
>13.46
N
O
7
–N(C6H5)2
8
N
N
NH
9
NCH3
10
–N(CH2C6H5)2
O
N
F
COOH
11
12
C32H31N6O5F
64
237
1.18
1.30
>10.44
N
N
O
F
COOH
C32H32N6O5F2
63
230
1.42
1.26
>10.10
N
N
N
F
C2H5
CH3
13
14
N
N
N
N
N
N
C24H26N6O2
C26H29N5O2
61
57
51
62
4.87
6.01
0.90
0.88
>14.52
>14.09
N
CH2
15
F
C25H26N5O2F
—
55
—
57
—
3.12
0.87
2.04
>13.97
>45.57
Isoniazid
—
ꢀ0.58
a logP was calculated using online www.logp.com site.
b MIC = minimum inhibitory concentration.
c IC50 = Cytotoxicity represented as inhibitory concentration in 50% of cell line.
compounds was checked by TLC and elemental analy-
ses, and the compounds of this study were identified
by spectral data. In general, IR spectra15 showed C@N
(azomethine) peak at 1640 cmꢀ1 and CH2 (Mannich
methylene) peak at 2860 and 2840 cmꢀ1. In the 1H
NMR spectra, the signals of the respective protons of
the prepared derivatives were verified on the basis of
their chemical shifts, multiplicities, and coupling
constants. The spectra of all the compounds showed a
singlet at d 4.8–5.1 ppm corresponding to –NCH2N–
group. The elemental analysis results were within
0.4% of the theoretical values. Lipophilicity of the
synthesized derivatives 1–15 and the parent compound,
INH, is expressed in terms of their logP values. These
values were computed with a routine method called cal-
culated logP (ClogP) using Alchemy software.
3. Antimycobacterial activity
The synthesized compounds 1–15 were tested for their
antimycobacterial activity in vitro against Mycobacteri-
um tuberculosis H37Rv using the microplate alamar
blue assay method11 in duplicate and MICs of the com-
pounds were reported in Table 1. MIC is defined as the
minimum concentration of compound required to give
90% inhibition of bacterial growth. Results show that
compounds 1–15 exhibited excellent antimycobacterial

4504
D. Sriram et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4502–4505
CH3
CH3
N
C O
C
NNHCO
OH
O
OH
CH3COOH
C2H5OH
H2NHN C
N
+
HCHO
R
R'
HN
Microwave
CH3
N
C
NNHCO
OH
R
CH2 N
R'
Figure 1. Synthetic protocol of the compounds.
ing lung homogenates on day 28 (Table 2). Compound 8
decreased the bacterial load by 3.7-log10 while INH de-
creased counts by 3.4-log10. Statistical analysis (Stu-
dentÕs t-test) suggested that compound 8 and INH
were equipotent.
Table 2. In vivo activity data of 8 and isoniazid against Mycobacte-
rium tuberculosis in mice
Compound
Lungs (log CFU SEM)a
Control
8 (25 mg/kg)
Isoniazid (25 mg/kg)
9.78 0.12
6.08 0.19
6.38 0.18
a CFU = colony forming units; SEM = standard error in mean.
4. Conclusion
It is conceivable that these derivatives showing antimy-
cobacterial activity can be further modified to exhibit
better potency than the standard drugs. These results
need to be refined in terms of degradation kinetic mea-
surements and stability studies of the synthesized
derivatives.
activity. The lipophilicity of the synthesized compounds
increased remarkably compared with the parent drug,
INH. This may render them more capable of penetrating
various biomembranes12, consequently improving their
permeation properties through mycobacterial cell mem-
branes. All the synthesized compounds inhibit M. tuber-
culosis with MIC ranging from 0.56 to 4.61 lM. Six
compounds (8, 11–15) were more potent than INH
and compound 8 (MIC 0.56 lM) emerged as the most
potent derivative, being three times more effective than
INH (MIC 2.04 lM) in vitro.
Acknowledgment
The authors are thankful to Dr. S. Ananthan from the
Southern Research Institute, Birmingham, AL, USA,
for the in vitro evaluation of antimycobacterial activity.
All the compounds were further examined for toxicity
(IC50) in a mammalian cell line, VERO cells by the
TAACF. The compounds were non-toxic as represented
in Table 1 and the selectivity index (IC50/MIC) for the
most active compound 8 was more than 30.
References and notes
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Dye, C. Lancet 2003, 362, 887.
Subsequently, compound 8 was tested for efficacy
against MTB at a dose of 25 mg/kg in mouse model.13
Briefly, 30 inbred female AKR mice (as this strain is
used in the previous report as mentioned in Ref. 13),
weighing 18–20 g were infected iv via the lateral tail vein
with 107 CFU of M. tuberculosis H37Rv. They were
divided into three groups of 10 mice each after 2 days.
One group received daily for 10 days the aqueous sus-
pension of the test compound 8 by intraperitoneal route
at a dose of 25 mg/kg body weight. The second group re-
ceived INH at 25 mg/kg body weights for 10 days by ip
route, whereas the third group served as the control
receiving no drug. The control groups did not receive
sham injections. Bacterial counts were measured by plat-
2. Currie, C. S.; Williams, B. G.; Cheng, R. C.; Dye, C.
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4505
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14. Characterization data for N0-[1-(2-hydroxyphenyl)ethy-
lidene]pyridohydrazide: IR (KBr): 3210, 2820, 1640,
1628 cmꢀ1; 1H NMR, d (ppm): 2.18 (s, 3H, CH3), 6.9–8.77
(m, 8H, Ar-H), 9.9 (s, 1H, OH, D2O exchangeable), 10.85 (s,
1H, CONH, D2O exchangeable); calcd for C14H13N3O2: C,
65.87; H, 5.13; N, 16.46. Found: C, 65.66; H, 5.19, N, 16.42.
15. Characterization data for compound 8 IR (KBr): 3210,
3010, 2858, 2840, 2820, 1680, 1636, 1506, 1236, 1125 cmꢀ1
;
1H NMR, d (ppm): 2.26 (s, 3H, CH3), 2.5–2.65 (m, 8H,
–N(CH2CH2)2N–), 6.7–8.6 (m, 8H, Ar-H), 9.96 (s, 1H,
OH, D2O exchangeable), 10.88 (s, 1H, CONH, D2O
exchangeable); calcd for C19H23N5O2: C, 64.57; H, 6.56;
N, 19.82. Found: C, 64.66; H, 6.49, N, 18.72.