Synthesis, characterization, and in vitro anti-Mycobacterium tuberculosis activity of terpene Schiff bases

Article abstract of DOI:10.1007/s00044-012-0458-3

A novel series of Schiff bases (3-13) were synthesized by the reaction of isoniazid (INH), nalidixic acid hydrazide, and fenamic acid hydrazides with monoterpenes (citral, camphor, and carvone) to obtain anti-mycobacterial agents in good yield and purity. The structures of the compounds were confirmed on the basis of their elemental analysis and spectral data. The structural modification of INH, nalidixic acid hydrazide, and fenamic acid hydrazides provided lipophilic adaptation of the respective hydrazides. The anti-mycobacterial activity of the synthesized compounds was investigated against four Mycobacterium strains: M. intercellulari (ATCC 35743), M. xenopi (ATCC 14470), M. cheleneo (ATCC 35751), and M. smegmatis (ATCC 35797). Compound 5, N′-[(1E)-2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-ylidene] pyridine-4-carbohydrazide with minimum inhibitory concentration (MIC 12 ± 0.03 μg/mL) was found to be more potent than INH under the in vitro investigation conditions. It was found that there is no evident relation between the anti-tubercular activity of the tested compounds and their lipophilicity. However, lipophilicity has an influence on the activity, but it does not solely determine the anti-tubercular activity of these compounds. All compounds presented lipophilicity higher than that of respective parent hydrazide.

Full text of DOI:10.1007/s00044-012-0458-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4522–4528
DOI 10.1007/s00044-012-0458-3
ORIGINAL RESEARCH
Synthesis, characterization, and in vitro anti-Mycobacterium
tuberculosis activity of terpene Schiff bases
•
Mashooq A. Bhat Mohamed A. Al-Omar
Received: 13 October 2012 / Accepted: 27 December 2012 / Published online: 12 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A novel series of Schiff bases (3–13) were syn-
thesized by the reaction of isoniazid (INH), nalidixic acid
hydrazide, and fenamic acid hydrazides with monoterpenes
(citral, camphor, and carvone) to obtain anti-mycobacterial
agents in good yield and purity. The structures of the com-
pounds were confirmed on the basis of their elemental anal-
ysis and spectral data. The structural modification of INH,
nalidixic acid hydrazide, and fenamic acid hydrazides pro-
vided lipophilic adaptation of the respective hydrazides. The
anti-mycobacterial activity of the synthesized compounds
was investigated against four Mycobacterium strains:
M. intercellulari (ATCC 35743), M. xenopi (ATCC 14470),
M. cheleneo (ATCC 35751), and M. smegmatis (ATCC
35797). Compound 5, N0-[(1E)-2-methyl-5-(prop-1-en-2-yl)
cyclohex-2-en-1-ylidene] pyridine-4-carbohydrazide with
minimum inhibitory concentration (MIC 12 0.03 lg/mL)
was found to be more potent than INH under the in vitro
investigation conditions. It was found that there is no evident
relation between the anti-tubercular activity of the tested
compounds and their lipophilicity. However, lipophilicity
has an influence on the activity, but it does not solely
determine the anti-tubercular activity of these compounds.
All compounds presented lipophilicity higher than that of
respective parent hydrazide.
Introduction
Tuberculosis (TB) is a chronic disease caused by several
species of Mycobacteriae. The incidence of TB is increasing
worldwide, partly due to poverty and inequity and partly due
to the HIV/AIDS pandemic. The problem of clinical treat-
ment has become more acute especially in immuno-com-
promised AIDS patients where the rise in TB incidence and
consequent deaths over the past two decades has escalated by
more than 12 % (Patole et al., 2006). The major issue is the
increase of multidrug-resistant tuberculosis. There is
emerging demand for the development of new anti-tuber-
cular agents effective against pathogens resistant to current
treatment regimens, which is limited to five drugs including
rifampicin, isoniazid (INH), ethambutol, streptomycin, and
pyrazinamide. There are several strategies for the develop-
ment of new anti-mycobacterial drugs. These include design
of analogs of existing agents, broad screening, and target-
directed drug design. In spite of major advances that have
been made in the discovery process, no new drugs have been
introduced in clinic since the discovery of rifampicin (Bur-
man and Jones, 2001). The need for newer compounds
remains urgent due to increasing resistance of Mycobacterial
strains to certain type of currently used anti-mycobacterials.
Therefore, there is an urgent need for anti-TB drugs with
improved properties such as enhanced activity against MDR
strains, reduced toxicity, shortened duration of therapy, rapid
mycobactericidal mechanism of action, the ability to pene-
trate host cells, and exert anti-mycobacterial effects in the
intracellular environment. INH is still maintaining its
importance as a first line drug for treatment of TB. Although
there are many reports on synthesis and anti-TB screening of
a large number of compounds containing isoniazid moiety
(Judge et al., 2012), reports suggest that INH, a prodrug
which is converted into its active form by mycobacterial
Keywords Schiff bases ꢀ Terpenes ꢀ Isoniazid ꢀ
Nalidixic acid hydrazide ꢀ Fenamic acid hydrazide ꢀ
Anti-tubercular activity
M. A. Bhat (&) ꢀ M. A. Al-Omar
Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, P.O. Box. 2457, Riyadh 11451, Kingdom
of Saudi Arabia
e-mail: mashooqbhat@rediffmail.com
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Med Chem Res (2013) 22:4522–4528
4523
catalase-peroxidase, acts on mycobacterial cell wall by
inhibiting the fatty acid synthetase II system to produce long
chain fatty acid precursors for mycolic acid synthesis (Fu and
Shinnick, 2007). Nalidixic acid hydrazide derivatives have
been reported to possess anti-tubercular activities (Aboul-
Fadl et al., 2010). Mefenamic and flufenamic acid are
congeners of diclofenac acid, which is reported to have anti-
tubercular activity (Dutta et al., 2004). Incorporation of
hydrophobic moieties into the framework of INH, nalidixic
acid hydrazide, and fenamic acid hydrazides can enhance
penetration of the drug into tissues of mammalian host and
waxy cell wall of bacterium. This strategy has been used for
augmenting fundamental drug activity (Mohamad et al.,
2004). There are reports that structural-modified drugs have
increased efficacy because of suppression of transformation
(Hearn and Cynamon, 2003). Our rationale has been to
prepare Schiff bases of INH, nalidixic acid hydrazide,
and fenamic acid hydrazides with monoterpenes (citral,
camphor and carvone) with enhanced lipophilicity and to
examine their activity against four Mycobacterium strains:
M. intercellulari (ATCC 35743), M. xenopi (ATCC 14470),
M. cheleneo (ATCC 35751), and M. smegmatis (ATCC
35797).
oxo-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (nali-
dixic acid hydrazide) and fenamic acid hydrazides were
carried out by reported methods (Grover and Kini, 2006;
Aboul-Fadl et al., 2011), respectively.
N0-[(1E, 2E)-3,7-dimethyloct-2-en-1-ylidene] pyridine-4-
carbohydrazide (3) (Hearn et al., 2009)
The pyridine-4-carbohydrazide, (isoniazid) Schiff base was
prepared by reaction of 3,7-dimethylocta-2,6-dienal (cit-
ral), (0.15 g, 1 mmol) with isoniazid (0.14 g, 1 mmol) in
ethanol/H2O (10 mL), initially dissolving the isoniazid in
H2O and adding the respective solution of citral in ethanol.
After stirring for 1–3 h, at room temperature, the resulting
mixture was concentrated under reduced pressure. The
residue purified by washing with cold ethyl alcohol and
ethyl ether, afforded the pure compound. Colorless blocks
of the compound suitable for X-ray determination were
recrystallized from ethanol by slow evaporation of solvent
at room temperature. Compounds 4 and 5 were prepared by
the same method by reaction of isoniazid with camphor and
carvone, respectively.
Yield: (50 %). m. p.: 110–112 °C. FT-IR (v, cm-1):
3200 (NH str.), 3000 (C–H str.), 1636 (C=O str.), 1549
1
(C=N str.). H NMR (500 MHz, DMSO-d6, d ppm): 11.7
(1H, s, –NH, D2O exch.), 7.8–8.7 (4H, m, Ar–H), 6.0 (1H,
s, –CH), 5.0 (1H, s, –CH), 3.38 (1H, s, –CH), 2.17 (4H, s,
2 9 CH2), 1.88 (3H, s, CH3), 1.63 (6H, s, 2 9 CH3). 13C
NMR (125.76 MHz, DMSO-d6, d ppm): 167.2, 161.0,
150.2, 149.4, 149.1, 148.3, 144.6, 141.7, 140.4, 131.8,
123.4, 122.8, 121.5, 121.3, 121.1, 120.9, 26.3, 25.5, 23.9,
17.5, 16.9, 16.7. MS (ESI) m/z = 272.3 [M-1]?. Anal.:
Calcd. for C16H21N3O (273.37): C (70.82), H (7.80), N
(15.49). Found: C (70.62), H (7.83), N (15.43). The single
crystal X-ray of the compound 3 has been reported (Bhat
et al., 2012a).
Chemistry
Experimental
All the solvents were of LR grade and were obtained from
Merck. The elemental analysis (C, H, N) of all compounds
were performed on the CHN Elementar (Analysen systeme
GmbH, Germany) and Vario EL III (Elementar Americas
Corporation) and were within a limit of 0.4 %, of the
theoretical values. The homogeneity of the compounds was
checked by TLC performed on Silica gel G-coated plates
(Merck). Iodine chamber was used for visualization of TLC
spots. The FT-IR spectra were recorded in SHIMADZU
spectrophotometer by dissolving samples in carbon tetra-
chloride (CCl4). Melting points were determined on a
Gallenkamp melting point apparatus, and are uncorrected.
NMR Spectra were scanned in DMSO-d6 on a Bruker
NMR spectrophotometer operating at 500 MHz for 1H and
125.76 MHz for 13C at the Research Center, College of
Pharmacy, King Saud University, Saudi Arabia. Chemical
shifts are expressed in d-values (ppm) relative to TMS as
an internal standard and D2O was added to confirm the
exchangeable protons. Mass spectra were measured on
Agilent Triple Quadrupole 6410 QQQ LC/MS with ESI
(Electrospray ionization) source.
N0-[(1Z)-5-methyl-2-(propan-2-yl)
cyclohexylidene]pyridine-4-carbohydrazide (4)
Yield: (50 %). m. p.: 205–207 °C. FT-IR (v cm-1): 3190
(NH str.), 3000 (C–H str.), 1668 (C=O str.), 1559 (C=N
str.). 1H NMR (500 MHz, DMSO-d6, d ppm): 10.65 (s, 1H,
–NH, D2O exch.), 7.6–8.7 (m, 4H, Ar–H), 0.7–0.9 (s, 9H,
CH3), 1.5 (s, 1H, –CH), 1.7 (s, 6H, –CH2); 13C NMR
(125.76 MHz, DMSO-d6, d ppm): 175.4, 167.1, 164.4,
150.0, 149.2, 142.0, 141.2, 123.2, 121.6, 52.7, 47.6, 43.2,
34.9, 34.2, 32.2, 26.7, 19.2, 11.3; MS (ESI) m/z = 274.5
[M ? 1]?. Anal.: Calcd. for C16H21N3O (273.37): C
(70.82), H (7.80), N (15.49). Found: C (71.00), H (7.77), N
(15.55).
Pyridine-4-carbohydrazide (isoniazid) was purchased
from Aldrich Chemicals. Synthesis of 1-ethyl-7-methyl-4-
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Med Chem Res (2013) 22:4522–4528
N0-[(1E)-2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-
–NH, D2O exch.), 7.5–9.0 (m, 3H, Ar–H), 4.6 (s, 2H,
–CH2), 2.6 (s, 3H, CH3), 2.0 (s, 6H, CH2). 1.4 (s, 1H,
–CH), 1.3 (s, 3H, CH3), 0.7–1.0 (s, 9H, CH3); 13C NMR
(125.76 MHz, DMSO-d6, d ppm): 175.8, 168.7, 163.4,
159.7, 148.1, 135.9, 121.6, 119.3, 111.8, 55.9, 52.1, 47.7,
46.2, 43.3, 34.4, 32.3, 26.7, 24.8, 19.2, 18.5, 15.0, 11.4; MS
(ESI) m/z = 382.2 [M]?. Anal.: Calcd. for C22H28N4O2
(382.4) C (69.45), H (7.42), N (14.73). Found: C (69.65), H
(7.40), N (14.79).
ylidene]pyridine-4-carbohydrazide (5)
Yield: (70 %). m. p.: 140–142 °C. FT-IR (v cm-1): 3250
(NH str.), 3000 (C–H str.), 1655 (C=O str.), 1540 (C=N
str.). 1H NMR (500 MHz, DMSO-d6, d ppm): 10.89 (1H, s,
–NH, D2O exch.), 7.6–8.7 (4H, m, Ar–H), 6.2 (1H, s, CH),
4.6 (2H, s, CH2), 2.3 (1H, s, CH2), 2.2 (4H, s, CH2), 1.7
(6H, s, 2 9 CH3). 13C NMR (125.76 MHz, DMSO-d6, d
ppm): 168.5, 163.7, 162.1, 158.4, 151.1, 150.1, 149.1,
147.4, 142.2, 141.2, 134.9, 133.3, 132.1, 123.0, 121.7,
120.9, 110.1, 30.0, 29.7, 29.3, 29.1, 20.6, 17.8. MS (ESI)
m/z = 269.2 [M]?. Anal.: Calcd. for C16H19N3O (269.34):
C (71.35), H (7.11), N (15.60). Found: C (71.37), H (7.14),
N (15.66).
(17Z)-1-ethyl-1,4-dihydro-7-methyl-N0-(2-methyl-5-
(prop-1-en-2-yl)cyclohex-2-enylidene)-4-oxo-1,
8-naphthyridine-3-carbohydrazide (8)
Yield: (70 %). m.p.: 230–232 °C. Yield: (70 %). m.p.:
230–232 °C. FT-IR (v cm-1): 3300 (NH str.), 3000 (C–H
str.), 1682 (C=O str.), 1533 (C=N str.); 1H NMR
(500 MHz, DMSO-d6, d ppm): 12.93 (1H, s, –NH, D2O
exch.), 7.5–9.0 (3H, m, Ar–H), 6.2 (1H, s, CH), 4.8 (2H, s,
CH2), 4.6 (2H, s, CH2), 2.5 (3H, s, CH3), 2.3 (4H, s,
2 9 CH2), 2.2 (1H, s, CH), 1.8 (3H, s, CH3), 1.7 (3H, s,
CH3), 1.4 (3H, s, CH3). MS (ESI) m/z = 378.2 [M]?.
Anal.: Calcd. for C22H26N4O2 (378.4): C (69.82), H (6.92),
N (14.80). Found: C (69.60), H (0.95), N (14.76).
(17E)-1-ethyl-1,4-dihydro-7-methyl-N0-((Z)-3,
7-dimethylocta-2,6-dienylidene)-4-oxo-1,8-naphthyridine-
3-carbohydrazide (6)
The 1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carbohydrazide, (nalidixic acid hydrazide) Schiff base was
prepared by reaction of citral, 3,7-dimethylocta-2,6-dienal
(0.15 g, 1 mmol) with nalidixic acid hydrazide (0.24 g,
1 mmol) in ethanol (10 mL). After stirring for 1–3 h, at
room temperature, the resulting mixture was concentrated
under reduced pressure. The residue purified by washing
with cold ethyl alcohol and ethyl ether, afforded the pure
compound. Compounds 7 and 8 were prepared by the same
methodbyreactionof1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,
8-naphthyridine-3-carbohydrazide with camphor and carv-
one, respectively.
2-(2,3-dimethylphenylamino)-N0-((Z)-3,7-dimethylocta-
2,6-dienylidene)benzohydrazide (9)
The 2-[(2,3-dimethylphenyl)amino]benzohydrazide,
(mefanimic acid hydrazide) Schiff base was prepared by
reaction of citral, 3,7-dimethylocta-2,6-dienal (0.15 g,
1 mmol) with mefenamic acid hydrazide (0.25 g, 1 mmol)
in ethanol (10 mL). After stirring for 1–3 h, at room tem-
perature, the resulting mixture was concentrated under
reduced pressure. The residue purified by washing with
cold ethyl alcohol and ethyl ether, afforded the pure
compound. Compounds 10 and 11 were prepared by the
same method by reaction of 2-[(2,3-dimethylphenyl)
amino]benzohydrazide with camphor and carvone,
respectively. Colorless blocks of the compound 11 suitable
for X-ray structure determination were recrystallized from
ethanol by slow evaporation of the solvent at room
temperature.
Yield: (60 %). m. p.: 148–150 °C. FT-IR (v cm-1):
3300 (NH str.), 3050 (C–H str.), 1677 (C=O str.), 1527
1
(C=N str.). H NMR (500 MHz, DMSO-d6, d ppm): 12.73
(1H, s, –NH, D2O exch.), 8.25–9.06 (3H, m, Ar–H), 7.51
(1H, s, –CH), 6.0 (1H, s, –CH), 5.1 (1H, s, –CH), 4.6 (2H,
s, –CH2), 2.6 (3H, s, CH3), 2.2 (4H, s, 2 9 CH2), 1.9 (3H,
s, CH3), 1.6 (6H, s, 2 9 CH3), 1.2 (3H, s, CH3); 13C NMR
(125.76 MHz, DMSO-d6, d ppm): 175.5, 163.3, 160.2,
148.4, 148.0, 146.9, 135.8, 131.3, 123.5, 121.6, 119.3,
111.3, 46.1, 32.0, 26.3, 25.6, 25.4, 24.8, 24.0, 17.5, 17.0,
15.0; MS (ESI) m/z = 380.2 [M]?. Anal.: Calcd.
C22H28N4O2 (380.48): C (69.45), H (7.42), N (14.73).
Found: C (69.25), H (7.45), N (14.78).
Yield: (65 %). m. p.: 135–137 °C. FT-IR (v cm-1):
3200 (NH str.), 3000 (C–H str.), 1647 (C=O str.), 1516
1
(C=N str.); H NMR (500 MHz, DMSO-d6, d ppm): 11.51
(8E)-1-ethyl-1,4-dihydro-7-methyl-N0-(1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)-4-oxo-1,
8-naphthyridine-3-carbohydrazide (7)
(1H, s, –NH, D2O exch.), 9.28 (1H, s, –NH, D2O exch.),
6.7–8.3 (7H, m, Ar–H), 7.6 (1H, s, –CH), 5.9 (1H, s, –CH),
5.09 (1H, s, –CH), 2.36 (6H, s, 2 9 CH3), 2.1 (4H, s,
2 9 CH2), 1.2 (9H, s, 3 9 CH3. 13C NMR (125.76 MHz,
DMSO-d6, d ppm): 165.0, 148.4, 148.1, 147.3, 147.0,
146.4, 139.0, 137.6, 132.3, 131.7, 131.3, 129.4, 128.6,
125.8, 125.2, 123.4, 123.3, 122.6, 121.7, 119.8, 116.8,
Yield: (60 %). m. p.: 238–240 °C. FT-IR (v cm-1): 3300
(NH str.), 3050 (C–H str.), 1677 (C=O str.), 1527 (C=N
1
str.); H NMR (500 MHz, DMSO-d6, d ppm): 12.4 (s, 1H,
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Med Chem Res (2013) 22:4522–4528
4525
115.9, 114.0, 32.32, 26.3, 25.6, 25.4, 23.9, 20.2, 17.5, 16.9,
13.4. MS (ESI) m/z = 389.2 [M]?. Anal.: Calcd. for
C25H31N3O (389.53): C (77.08), H (8.02), N (10.79).
Found: C (77.38), H (8.06), N (10.83).
by the same method by reaction of 2-{[2-methyl-(trifluo-
romethyl) phenyl]amino}benzohydrazide with carvone.
Yield: (70 %). m. p.: 160–162 °C. FT-IR (v cm-1):
3300 (NH str.), 3000 (C–H str.), 1634 (C=O str.), 1582
1
(C=N str.); H NMR (500 MHz, DMSO-d6, d ppm): 10.2
(s, 1H, –CONH, D2O exch.), 8.9 9s, 1H, –NH, D2O exch.),
7.0–7.6 (m, 8H, Ar–H), 0.5–1.7 (s, 6H, –CH2), 1.2 (s, 1H,
–CH), 0.9 (s, 9H, CH3); 13C NMR (125.76 MHz, DMSO-
d6, d ppm): 172.4, 163.8, 144.1, 141.1, 131.6, 130.0, 129.7,
124.2, 121.3, 120.1, 119.2, 116.2, 112.9, 52.4, 47.5, 43.2,
34.2, 32.2, 26.7, 19.1, 11.3; MS (ESI) m/z = 431.0 [M]?.
Anal.: Calcd. for C24H26F3N3O (431.4): C (67.12), H
(6.10), N (9.78). Found: C (67.32), H (6.12), N (9.80).
(E)-2-(2,3-dimethylphenylamino)-N0-(2-isopropyl-5-
methylcyclohexylidene)benzohydrazide (10)
Yield: (70 %). m.p.: 200–202 °C. Yield: (70 %). m.p.:
200–202 °C. FT-IR (v cm-1): 3100 (NH str.), 3000 (C–H
str.), 1631 (C=O str.), 1581 (C=N str.); 1H NMR
(500 MHz, DMSO-d6, d ppm): 10.51 (1H, s, –NH, D2O
exch.), 10.25 (1H, s, –NH, D2O exch.), 6.86–9.0 (7H, m,
Ar–H), 2.27 (6H, s, 2 9 CH3), 2.0 (6H, s, 3 9 CH2), 1.9
(2H, s, –CH), 1.09 (9H, s, 3 9 CH3); 13C NMR
(125.76 MHz, DMSO-d6, d ppm): 155.5, 145.2, 139.2,
137.5, 131.8, 128.9, 125.7, 124.8, 119.1, 117.1, 114.2,
47.5, 43.2, 34.4, 32.2, 26.6, 25.0, 20.1, 19.1, 18.4, 17.9,
13.3, 11.3; MS (ESI) m/z = 391.2 [M]?. Anal.: Calcd. for
C25H31N3O (391.5): C (77.08), H (8.02), N (10.79). Found:
C (77.38), H (8.05), N (10.75).
(20E)-2-(3-(trifluoromethyl) phenylamino)-N0-(2-methyl-5-
(prop-1-en-2-yl) cyclohex-2-enylidene)benzohydrazide (13)
Yield: (70 %). m. p.: 170–172 °C. FT-IR (v cm-1): 3200
(NH str.), 3000 (C–H str.), 1673 (C=O str.), 1517 (C=N
1
str.); H NMR (500 MHz, DMSO-d6, d ppm): 10.8 (1H, s,
–NH, CONH, D2O exch.) 8.9 (1H, s, –NH, D2O exch.),
7.0–7.7 (8H, m, Ar–H), 6.1 (1H, s, –CH), 4.7 (2H, s,
–CH2), 2.8 (1H, s, –CH), 2.29 (4H, s, 2 9 CH2), 1.68 (6H,
s, 2 9 CH3). 13C NMR (125.76 MHz, DMSO-d6, d ppm):
170.1, 164.3, 156.0, 147.4, 144.0, 141.4, 134.1, 132.3,
131.6, 129.9, 127.4, 125.2, 124.3, 123.0, 121.3, 120.4,
119.0, 116.3, 114.8, 113.1, 111.2, 109.9, 29.6, 20.3, 17.7
MS (ESI) m/z = 427.0 [M]?. Anal.: Calcd. for
C24H24F3N3O (427.4): C (67.43), H (5.66), N (9.83).
Found: C (67.63), H (5.63), N (9.79).
(E)-2-(2,3-dimethylphenylamino)-N0-(2-methyl-5-(prop-1-
en-2-yl)cyclohex-2-enylidene)benzohydrazide (11)
Yield: (65 %). m. p.: 170–172 °C. FT-IR (v cm-1): 3200
(NH str.), 3000 (C–H str.), 1636 (C=O str.), 1579 (C=N
str.); 1H NMR (500 MHz, DMSO-d6, d ppm): 10.75 (1H, s,
–NH, CONH, D2O exch.), 8.8 (1H, s, –NH, D2O exch.),
6.8–7.6 (7H, m, Ar–H), 6.2 (1H, s, CH), 4.8 (2H, s, –CH2),
2.5 (1H, s, CH), 2.3 (4H, s, 2 9 CH3), 2.27 (6H, s,
2 9 CH3), 1.7 (6H, s, CH3); 13C NMR (125.76 MHz,
DMSO-d6, d ppm): 157.0, 147.5, 145.7, 139.3, 137.6,
134.2, 132.3, 129.5, 125.8, 119.4, 118.0, 114.5, 110.1,
29.9, 20.4, 20.2, 17.8, 13.4. MS (ESI) m/z = 387.0 [M]?.
Anal.: Calcd. for C25H29N3O (387.5): C (77.48), H (7.54),
N (10.84). Found: C (77.18), H (7.51), N (10.88). The
single crystal x-ray of compound 11 has been reported
(Bhat et al., 2012b).
Anti-mycobacterial activity
Anti-mycobacterial activity was performed at the Research
Center, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia. The tested Mycobacterium tuber-
culosis strains are M. intercellulari (ATCC 35743),
M. xenopi (ATCC 14470), M. cheleneoi (ATCC 35751),
and M. smegmatis (ATCC 35797) using Rist and Grosset
proportion method, agar dilution method (Canetti et al.,
1963). The synthesized compounds (3–13) and INH was
dissolved in dimethylsulfoxide (DMSO) at a concentration
of 1 mg/mL. The appropriate aliquot of each solution was
diluted with 10 % molten agar to give concentrations of
100 lg/mL. The agar and the compound solution were
mixed thoroughly and the mixture was poured into Petri-
dishes on a level surface to result in an agar depth of
3–4 mm and allowed to harden. The inocula were prepared
by growing overnight culture in Muller-Hinton broth. The
cultures were diluted 1:100. Tested organisms were
streaked in a radial pattern, and plates were incubated at
35 °C for 48 h to check the growth of the tested strains at
this single concentration. Active compounds were further
(E)-2-(3-(trifluoromethyl) phenylamino)-N0-(2-isopropyl-5-
methylcyclohexylidene) benzohydrazide (12)
The 2-{[2-methyl-3-(trifluoromethyl)phenyl]amino}ben-
zohydrazide, (flufenamic acid hydrazide) Schiff base was
prepared by reaction of camphor, 1,7,7-trimethylbicy-
clo[2.2.1]heptan-2-one (0.15 g, 1 mmol) with flufenamic
acid hydrazide (0.30 g, 1 mmol) in ethanol (10 mL). After
stirring for 1–3 h, at room temperature, the resulting mix-
ture was concentrated under reduced pressure. The residue
purified by washing with cold ethyl alcohol and ethyl ether,
afforded the pure compound. Compound 13 was prepared
123

4526
Med Chem Res (2013) 22:4522–4528
diluted and tested by the same way to determine the min-
imum inhibitory concentration (MIC) of these compounds.
Experiment using the tested strains in a medium free of
investigated compounds was also carried out.
was used as a reference drug and control experiments were
done using a growth media free from drugs or the tested
compounds. Results of the in vitro anti-tubercular activity
of the tested compounds along with the standard
drug for comparison are given in (Table 1). Compound 5
(N0-[(1E)-2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-yli-
dene]pyridine-4-carbohydrazide) presented significant
growth inhibition against all strains mycobacterium with
minimum inhibitory concentration (MIC 12 0.03 lg/
mL). Compounds 4 (N0-[(1Z)-5-methyl-2-(propan-2-yl)cy-
clohexylidene]pyridine-4-carbohydrazide) and compound 6
(17E)-1-ethyl-1,4-dihydro-7-methyl-N0-((Z)-3,7-dimethyl-
octa-2,6-dienylidene)-4-oxo-1,8-naphthyridine-3-carbohy-
drazide also showed growth inhibition against all strains of
Mycobacterium at concentration of 50 lg/mL. The data of
the anti-Mycobacterium screening of compounds 3, 6, 8, 9,
10, 11, 12, and 13 revealed no activity on the tested strains up
to concentration of 100 lg/mL. The active compound 5 was
found to be more potent than first line anti-tubercular drug
INH under the investigation conditions. The lipophilicity is a
well-known physicochemical factor affecting biological
activities, characterizing the distribution process of com-
pound in the human organism, and being a key factor of both
pharmacokinetic and pharmacodynamic properties of drug
molecules (plasma protein binding, blood–brain barrier
(BBB) penetration, and penetration through cell membranes
(Lesyk et al., 2006). Correlation between lipophilicity and
anti-TB was reported, as lipophilicity of drug molecules may
render them more capable of penetrating various biomem-
branes consequently improving their permeation properties
toward microbial cell membranes (Sivakumar et al., 2007).
Lipophilicity of the synthesized compounds expressed in the
term of their C log P values, is shown in (Table 1). It was
found that there is no evident relation between the anti-TB
activity of the tested compounds and their lipophilicity.
Clearly, the lipophilicity has an influence on the activity, but
it does not solely determine the anti-Mycobacterium activity
of these compounds. All compounds presented lipophilicity
higher than that of respective parent hydrazide.
Results and discussion
The synthesized compounds (3–13) were prepared by
condensation reaction between the Isoniazid, hydrazides of
drugs; nalidixic acid, mefenamic acid and flufenamic acid
(1a–d) with respective monoterpenes; citral, camphor, and
carvone (2a–c) in ethanol by stirring for 1–3 h at room
temperature. The resulting mixture was concentrated under
reduced pressure. The residue was purified by washing
with cold ethyl alcohol and ethyl ether to get the pure
compound. The purity of synthesized compounds was
checked by thin layer chromatography (TLC) and ele-
mental analyses, and the structures were identified by
spectral data. The compounds were obtained in good yields
ranging from 50 to 70 % with high purity (Scheme 1).
Compounds 3 and 11 were further recrystallized by slow
evaporation of solvent (ethanol) for single crystal X-ray
spectroscopy. The IR spectra of compounds, exhibited in
each case, a band in the region of 1,686–1,631 cm-1 due to
carbonyl absorption, 1,582–1,516 cm-1 due to C=N
stretching whereas the absorption band of NH function
appeared in the region of 3,300–3,100 cm-1. The 1H NMR
and 13C NMR spectra, the signals of the respective pre-
pared derivatives were verified on the basis of their
chemical shifts, multiplicities, and coupling constants. The
spectra of the compounds showed the characteristic D2O
exchangeable NH protons at d 10.2–12.93 ppm in addition
to the aromatic protons of the phenyl moieties. The mass
spectra of compounds showed the mass peaks of [M?] and
[M-1]?. The elemental analysis results were within
0.4 % of the theoretical values. The lipophilicities of the
synthesized compounds were calculated using Chem Office
6.0 software. ACD/Chem Sketch (freeware) and Chem
Draw Ultra 8.0 version program were used to draw the
chemical structures.
Conclusion
Anti-mycobacterial activity
The Schiff bases of INH, Nalidixic acid hydrazide, and
Fenimic acid hydrazides were readily prepared for evalua-
tion against four Mycobacterium strains; M. intercellulari
(ATCC 35743), M. xenopi (ATCC 14470), M. cheleneo
(ATCC 35751), and M. smegmatis (ATCC 35797) in good
yield and purity. The structural modification of INH, nali-
dixic acid hydrazide, and fenamic acid hydrazides provided
lipophilic adaptation of respective hydrazides. Compound 5
N0-[(1E)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-yli-
dene]pyridine-4-carbohydrazide (MIC 12 0.03 lg/mL)
The synthesized compounds (3–13) were evaluated for
their anti-mycobacterial activity in vitro against four
Mycobacterium strains: M. intercellulari (ATCC35743),
M. xenopi (ATCC 14470), M. cheleneo (ATCC 35751) and
M. smegmatis (ATCC 35797) by agar dilution method
according to the protocol described in the experimental
section similar to that recommended by the National
Committee for Clinical Laboratory Standards (NCCLS) for
the determination of MIC [Wayne, 2000]. Isoniazid (INH)
123

Med Chem Res (2013) 22:4522–4528
4527
CH3
CH3
CH3
O
CH3
CH3
CH3
R
NH
EtOH, Stirring 1- 3 hr.
N
+
R
NH
O
O
NH2
(3, 6, 9)
2a
R= 1a, 1b, 1c
O
CH3
CH3
CH3
CH3
O
R
NH
N
O
EtOH, Stirring 1- 3 hr.
+
NH2
R
NH
H3C
H3C
R= 1a, 1b, 1c, 1d
(4, 7, 10, 12)
2b
CH3
O
O
O
R
NH
CH3
N
+
R
NH
NH2
EtOH, Stirring 1- 3 hr.
R= 1a, 1b, 1c, 1d
H3C
CH2
2c
H3C
(5, 8, 11, 13)
CH2
Me
Me
Me
O
Me
O
Me
R
O
Me
2b
2a
Me
2c
Me
Me
N
Me
Me
Me
Me
H
N
N
N
N
H
N
O
N
N
H
N
1a
Me
O
N
O
Me
3
4
5
Me
Me
Me
Me
O
N
O
Me
Me
Me
Me
Me
N
O
O
N
H
N
N
N
H
H
N
N
Me
N
O
N
N
Me
N
N
N
Me
Me
Me
O
O
Me
Me
6
9
1b
Me
7
8
Me
Me
Me
Me
Me
Me
H
N
H
H
N
Me
N
N
NH
Me
O
H
N
N
Me
NH
Me
O
N
NH
Me
O
Me
Me
1c
Me
Me
10
11
Me
Me
Me
H
F3C
N
H
N
H
N
N
-
Me
NH
O
N
NH
O
Me
1d
CF3
CF3
13
12
Scheme 1 Synthesis of Schiff bases of hydrazide drugs (3–13)
123

4528
Med Chem Res (2013) 22:4522–4528
Bhat MA, Abdel-Aziz HA, Ghabbour HA, Hemamalini M, Fun HK
(2012a) N’-[(1E, 2E)-3,7-dimethyloct-2-en-1-ylidene] pyridine-
4-carbohydrazide. Acta Crystallogr Sect E E68:o1144–o1145.
doi:10.1107/S1600536812009075
Bhat MA, Abdel-Aziz HA, Ghabbour HA, Hemamalini M, Fun HK
(2012b) (E)-2-(2,3-dimethylphenylamino)-N’-(2-methyl-5-(prop-
1-en-2-yl)cyclohex-2-enylidene)benzohydrazide. Acta Crystallogr
Sect E E68:o1135. doi:10.1107/S1600536812009087
Burman WJ, Jones BE (2001) Treatment of HIV-related tuberculosis
in the era of effective antiretroviral therapy. Am J Respir Crit
Care Med 164:7–12
Canetti G, Rist N, Grosset J (1963) Measurement of sensitivity of the
tuberculous bacillus to antibacillary drugs by the method of
proportions. Methodology, resistance criteria, results and inter-
pretation. Rev Tuberc Pneumol (Paris) 27:217–272
Dutta NK, Kumar KA, Mazumdar K, Dastidar SG (2004) In vitro and
in vivo antimycobacterial activity of antiinflammatory drug,
diclofenac sodium. Indian J Exp Biol 42:922–927
Fu LM, Shinnick TM (2007) Understanding the action of INH-
resistant mycobacterium tuberculosis strain using Genechips.
Tuberculosis 87:63–70
Grover G, Kini SG (2006) Synthesis and evaluation of new
quinazolone derivatives of nalidixic acid as potential antibacte-
rial and antifungal agents. Eur J Med Chem 41:256–262. doi:
10.1016/j.ejmech.2005.09.002
Hearn M, Cynamon M (2003) In vitro and in vivo activities of
acylated derivatives of isoniazid against mycobacterium tuber-
culosis. Drug Des Discov 18:103–108
Table 1 Lipophilicity (C log P) and in vitro anti-mycobacterial
activities of compounds (3–13)
Compound C log MIC (lg/ML)b
no.
Pa
M.
intercellulari xenopi cheleneoi smegmatis
M.
M.
M.
3
3.88
2.20
2.80
4.9
[100
50
12 0.03c 12
[100
50
[100
50
[100
50
4
5
12
12
6
[100
50
[100
50
[100
50
[100
50
7
3.22
3.83
7.93
6.25
6.86
6.54
7.15
0.67
8
[100
[100
[100
[100
[100
[100
12.5
[100
[100
[100
[100
[100
[100
12.5
[100
[100
[100
[100
[100
[100
12.5
[100
[100
[100
[100
[100
[100
12.5
9
10
11
12
13
Isoniazid
a
C log P was calculated using software Chem Office 6.0, nalidixic
acid hydrazide (C log P 0.27); mefenamic acid hydrazide (C log P
3.04); flufenamic acid hydrazide (C log P 3.33)
b
Minimal inhibition concentration (MIC) is expressed in lg/mL
c
Value are expressed as mean SEM and analyzed by ANOVA with
P \ 0.05
Hearn M, Cynamon MH, Chen MF, Coppins R, Davis J, Kang HJ,
Noble A, Tu-Sekine B, Terrot MS, Trombino D, Thai M,
Webster ER, Wilson R (2009) Preparation and antitubercular
activities in vitro and in vivo of novel Schiff bases of isoniazid.
Eur J Med Chem 44:4169–4178
was found to be more potent than first line anti-tubercular
drug INH under the investigation conditions.
Judge V, Narasimhan B, Ahuja M (2012) Isoniazid: the magic
molecule. Med Chem Res 21:3940–3957
´
Lesyk R, Zimenkovsky B, Atamanyuk D, Jensen F, Kiec-Kononowicz
K, Gzella A, (2006) Anticancer thiopyrano[2,3-d][1,3]thiazol-2-
ones with norbornane moiety. Synthesis, cytotoxicity, physico-
chemical properties, and computational studies. Bioorg Med
Chem 14: 5230–5240. doi:10.1016/j.bmc.2006.03.053
Acknowledgments The authors are thankful to Deanship of Sci-
entific Research and Research Center, College of Pharmacy, King
Saud University. The authors are also thankful to Mustafa Omer for
carrying out anti-tubercular activity of the compounds.
Mohamad S, Ibrahim P, Sadikun A (2004) Susceptibility of myco-
bacterium tuberculosis to isoniazid and its derivative, 1-isoni-
cotinyl-2-nonanoyl hydrazine: investigation at cellular level.
Tuberculosis 84:56–62
NCCLS (2000) Susceptibility testing of mycobacteria, nocardia, and
other aerobic actinomycetes; tentative standard NCCLS docu-
ment M24-T2 [ISBN 1-56238-423-6], second ed. NCCLS, 960
West Valley Road, Suite 1400, Wayne, pp. 19087–1898
Patole J, Shingnapurkar D, Padhye S, Ratledge C (2006) Schiff base
conjugates of p-aminosalicylic acid as antimycobacterial agents.
Bioorg Med Chem Lett 16:1514–1517
References
Aboul-Fadl T, Bin-Jubair FAS, Aboul-Wafa O (2010) Schiff bases of
indoline-2,3-dione (isatin) derivatives and nalidixic acid carbo-
hydrazide, synthesis, antitubercular activity and pharmacophoric
model building. Eur J Med Chem 45:4578–4586
Aboul-Fadl T, Abdel-Aziz HA, Kadi A, Bari A, Ahmad P, Al-Samani
T, Ng SW (2011) Microwave-assisted one-step synthesis of
fenamic acid hydrazides from the corresponding acids. Mole-
cules 16:3544–3551. doi:10.3390/molecules16053544
Sivakumar PM, Seenivasan SP, Kumar V, Doble M (2007) Synthesis,
antimycobacterial activity evaluation, and QSAR studies of
chalcone derivatives. Bioorg Med Chem Lett 17:1695–1700
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