Synthesis of highly potent novel anti-tubercular isoniazid analogues with preliminary pharmacokinetic evaluation

Article abstract of DOI:10.1016/j.bmcl.2012.02.091

Thirty two novel isoniazid analogues were prepared by one-pot three component condensations of isoniazid (INH), 3-mercaptopropionic acid and various aryl/heteroaryl aldehydes. The synthesized compounds were evaluated for their anti-TB activity against Myc

Full text of DOI:10.1016/j.bmcl.2012.02.091

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2764–2767
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of highly potent novel anti-tubercular isoniazid analogues
with preliminary pharmacokinetic evaluation
Addepalli Venkata Ramani, Arumalla Monika, Vadlamani Lakshmi Indira, Gopisetti Karyavardhi,
Jangala Venkatesh, Variam Ullas Jeankumar, Thimmappa H. Manjashetty, Perumal Yogeeswari,
⇑
Dharmarajan Sriram
Medicinal Chemistry & Tuberculosis Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar,
Hyderabad 500 078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty two novel isoniazid analogues were prepared by one-pot three component condensations of iso-
niazid (INH), 3-mercaptopropionic acid and various aryl/heteroaryl aldehydes. The synthesized com-
pounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37Rv (MTB) and
cytotoxicity. Among the compounds, compound N-(2-(4-(benzyloxy) phenyl)-4-oxo-1,3-thiazinan-3-yl)
Received 21 November 2011
Revised 17 February 2012
Accepted 27 February 2012
Available online 3 March 2012
isonicotinamide (17) inhibited MTB with MIC of 0.12 lM and was three times more potent than INH.
The main pharmacokinetic parameters after intravenous administration (10 mg/kg body weight) in male
Keywords:
Wistar rats viz. t , Kel, mean plasma clearance and mean volume of distribution were found to be
½
Isoniazid analogue
Anti-TB activity
Anti-mycobacterial activity
Tuberculosis
1.14 0.20 h, 0.62 0.10 hÀ1
,
22.48 0.16 mL/kg/min and 1.99 0.49 L, respectively. The systemic
absorption was slow after oral administration (50 mg/kg body weight). The peak plasma concentration
was found to be 1.31 0.06 g/mL attained in 3 h. The bioavailability was found to be 16.7%.
Ó 2012 Elsevier Ltd. All rights reserved.
l
Tuberculosis (TB) is the leading cause of mortality among all
infectious diseases worldwide and is responsible for over two mil-
lion deaths annually.1 The number of people who fell ill with TB
dropped to 8.8 million in 2010, including 1.1 million cases among
people with HIV. The number has been falling since 2005.2 The esti-
mated global incidence rate fell to 128 cases per 100,000 popula-
tions in 2010, after peaking in 2002 at 141 cases per 100,000. The
rate is falling but very slowly. The number of people who died from
TB fell to 1.4 million in 2010, including 350,000 people with HIV,
equal to 3800 deaths a day. The TB death rate has fallen by 40% since
1990, and the number of deaths is also declining.2 It is estimated
that between 2002 and 2020, approximately 1 billion people would
be newly infected, more than 150 million people would get sick,
and 36 million would die of TB if new disease prevention and treat-
ment measures are not developed.2 Hence, there is an urgent need
to develop novel, highly effective, and fast acting anti-TB drugs with
low toxicity profiles and performing activity against both drug-sen-
sitive and drug resistant MTB. One of the most effective and widely
used anti-tuberculosis (TB) drugs is isoniazid (INH); a pro-drug
activated via oxidation that forms an adduct with NAD (+) to inhibit
NADH dependent targets of Mycobacterium tuberculosis (MTB), such
as the enoyl-acyl carrier protein reductase (InhA). INH has become
the single most researched anti-tubercularagent which is not effec-
tive against multi-drug resistant TB. Several recent experiments
indicate that the incorporation of hydrophobic moieties into the
framework of INH can enhance penetration of the drug into the
tissues of the mammalian host and into the waxy cell wall of
the bacterium. Previous work on several members of this class, par-
ticularly the (hetero) aromatic derivatives, had been carried out and
had demonstrated good activities by the methods then available
(Fig. 1).3–9 In the present study the INH moiety was incorporated
in the 1,3-thiazinan scaffold and evaluated for in vitro MTB activity,
cytotoxicity studies and preliminary pharmacokinetic studies.
T. Srivastava et al. have earlier reported the dicyclohexyl carbo-
diimide mediated synthesis of 4-thiazolidinones and metathiaza-
nones, by one-pot three component condensation of amine,
bezaldehyde, mercaptopropinoic acid in the ratio of 1:2:3 to give
the desired product in quantitative yield.10 In continuation of our ef-
forts in discovering new leads for antimycobacterial activities, we
tried to extend the above work by replacing the amine with
isoniazid. Though reaction went smoothly for simple amines, our
initial effort to synthesize the isoniazid analogue of metathiazanon-
es resulted in very poor yield as majority of the product was found in
the filtered residue along with dicyclohexyl urea formed as the side
product of the reaction. Purification of the resultant residue by col-
umn chromatography gave the desired product in very low yield.
As previously reported, the reaction involves the addition of
sulfur nucleophile to the imine centre followed by attack of the
⇑
Corresponding author. Tel.: +91 4066303506.
E-mail addresses:
drdsriram@yahoo.com,
dsriram@bits-hyderabad.ac.in
(D. Sriram).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.091

A. V. Ramani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2764–2767
2765
H3C
O
N
N
NNH
N
HO
N
HO
CF3
CH3
N
F3C
N
N
NH
N
O
N
O
MIC: 0.56 µM
MIC: 0.77 µM
N
LL3858 (Sudoterb)
MIC: 0.25 µg/ml.
N
H
N
MIC: 0.0004 µM
H
O
O
N
F
O
O
N
S
O
N
H
N
H
MIC: 0.45 µM
N
HN
O
NH
HN
N
C
H2C
MIC: 0.17 µM
MIC: 0.2 µg/ml.
N
Figure 1. Potential reported isoniazid analogues.
nitrogen on the carboxylic moiety with the expulsion of water giv-
ing the cyclized product. In DCC mediated synthesis of thiazolidi-
nones, carbodiimide enhances the activation of the carboxyl
group of an adduct obtained by the sulfur addition to the imine
thereby facilitating cyclization. Keeping this in mind we decided
to set a trial with 1-ethyl-3-(3-dimethylaminopropyl) carbodiim-
ide (EDC) as the activating carbodiimide for the synthesis of
metathiazanones, as the corresponding urea formed as side prod-
uct during the course of the reaction would be highly water soluble
and can be easily eliminated by simple workup. The reaction not
only gave the desired product in quantitative yield but also with
desired purity. It was observed that as reported in the case of
DCC mediated cyclization, addition of EDC in ice cold conditions
gives better yields as compared with the reaction carried out at
ambient temperature. To explore the scope and limitations of these
conditions a series of 32 aromatic and heterocyclic aldehydes con-
taining electron-withdrawing groups (such as nitro, halogens) or
electron-donating groups (such as methoxy, methyl etc.) were em-
ployed and as apparent from the Table 1, they were found to react
well to give the corresponding N-aryl-4-oxo-1,3-thiazinan-3-yl
isonicotinamide in good to excellent yields (50–95%).
The target compounds were synthesized as illustrated in
Scheme 1. INH was dissolved in THF and the reaction mixture
was cooled to 0 °C, the corresponding aldehyde was then added
drop wise, followed by 3-mercaptopropionic acid and 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide, the reaction mixture was
then slowly warmed to room temperature and stirred at room
temperature for another 5–6 h (monitored by TLC for completion),
it was then quenched with water and worked up to give the
desired metathiazanones in quantitative yield.
and mass spectra) of all the synthesized compounds were in full
agreement with the proposed structures.11 Lipophilicity of the syn-
thesized derivatives and that of the parent compound, INH, are
expressed in terms of their logP values (Table 1). These values
were computed with a routine method called calculated logP
(ClogP) using ChemBioDraw Ultra 11.0 software.
The compounds 1–32 were screened for their in vitro antimyco-
bacterial activity against log-phase cultures of MTB Middle brook
7H11 agar medium supplemented with OADC by agar dilution
method similar to that recommended by the National Committee
for Clinical Laboratory Standards for the determination of MIC in
triplicate.12 The MIC is defined as the minimum concentration of
a compound required to completely inhibit the bacterial growth.
The MIC’s of the synthesized compounds along with the standard
drug INH for comparison are reported in Table 1. In the MTB screen-
ing, all the compounds showed in vitro activity against MTB with
MIC ranging from 0.12 to 41.2
19, 22–30) inhibited MTB with less than 1
to standard first line drug INH (MIC of 0.36
(15–17, 23, 27, 30) were more potent with MIC less than 0.36
Compound N-(2-(4-(benzyloxy) phenyl)-4-oxo-1,3-thiazinan-3-yl)
isonicotinamide (17) inhibited MTB with MIC of 0.12 M and was
l
M. Thirteen compounds (15–17,
M. When compared
M); six compounds
M.
l
l
l
l
three times more potent than INH. The enhanced activity might
be due to its hydrophobicity, ClogP of this compound 17 was
4.25 whereas parent compound INH was À0.66. With respect to
structure anti-TB activity, Compound 1, without any substituent
on the phenyl rings, displayed an MIC of 9.9 lM and serves as the
standard for potency comparison. Introduction of smaller electron
donating groups in the phenyl ring 2–13 not alter the activity
much; MIC ranging from 4.54 to 18.98 lM. Among them only
The purity of compounds 1–32 was checked by TLC and elemen-
4-methoxy group (7) group enhances the activity twice and intro-
duction of hydroxyl group is detrimental to activity. Introduction
tal analysis. Both analytical and spectral data (1H NMR, 13C NMR,

2766
A. V. Ramani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2764–2767
Table 1
Table 2
Physical constants, anti-tubercular activity & cytotoxicity of the compounds
Pharmacokinetic profile of compound 17 in male Wistar rats (n = 4) in comparison
with isoniazid
N
O
O
Parameters Units
Route of administration
Compound 17 Isoniazid
Intravenous Oral
10 50
NH
N
Oral
50
Intravenous
10
Ar
S
Dose
mg/
Sl.
Ar
Yield Mp (°C) ClogP MIC
(%)
HEPG2 cell
line %
cytotoxicity
kgbwt
No.
(lM)
AUC(0–t)
lg h/
5.87 0.24 7.50 0.94 197.09 7.99 684 5.5
mL
Cmax/C0
Tmax
Kel
t1/2
Vd
l
h
1/h
h
L
g/mL 1.31 0.06 5.29 1.41 52.33 1.28 298.2 3.18
1
2
3
4
5
6
7
8
Phenyl
87
83
69
71
89
93
84
81
146–148
111–113
170–172
126–128
151–153
129–131
166–168
176–178
2.56 9.9
3.01 9.56 12.6
3.06 9.56 8.6
3.06 9.56 12.4
2.45 9.08 15.2
2.48 9.08 10.4
2.48 4.54 12.6
10.6
3.00
—
—
—
—
—
2.00
—
—
—
—
—
2-Methyl phenyl
3-Methyl phenyl
4-Methyl phenyl
2-Methoxy phenyl
3-Methoxy phenyl
4-Methoxy phenyl
3,4,5-Trimethoxy
phenyl
0.62 0.10
1.14 0.20
1.99 0.49
22.48 0.16
0.24 0.002
2.88 0.13
0.026 0.002
0.21 0.001
CL
mL/kg/
min
%F
16.7
17.36
1.86 7.76
8.6
AUC(0–t)—Area under the curve till ‘t’ hours.
Cmax—Peak plasma concentration.
C0—Initial plasma concentration (at 0 h).
Tmax—Time taken to attain peak plasma concentration.
Kel—Elimination rate constant.
t1/2—Half life.
Vd—Volume of distribution.
CL—Clearance.
9
4-Hydroxy-3-
75
133–135
1.74 8.7
16.11
methoxy-phenyl
2-Hydroxyl phenyl
3-Hydroxyl phenyl
4-Hydroxyl phenyl
4-Dimethylamino
phenyl
10
11
12
13
72
74
76
80
98–100
107–109
121–123
148–150
1.84 18.98
1.89 9.49
1.89 18.9
2.70 8.78
8.6
8.2
10.7
4.6
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
4-Isopropyl phenyl
2-Benzyloxy phenyl
3-Benzyloxy phenyl
4-Benzyloxy phenyl
2-Nitro phenyl
3-Nitro phenyl
4-Nitro phenyl
2-Chloro phenyl
4-Chloro phenyl
2-Bromo phenyl
4-Bromo phenyl
2-Fluoro phenyl
3-Fluoro phenyl
4-Fluoro phenyl
2-(Trifluoromethyl)
phenyl
3-(Trifluoromethly)
phenyl
4-(Trifluoromethyl)
phenyl
2-Furfuryl
5-Nitro furfur-2-yl
Isoniazid
91
84
62
84
68
71
53
61
65
67
81
91
71
60
74
123–125
181–183
163–165
189–190
169–171
186–188
194–196
155–157
161–163
157–159
142–144
102–104
131–133
183–185
191–193
3.99 2.138 19
2.25 0.24 10.6
4.25 0.24 12.6
%F—Oral bioavailability.
4.25 0.12
8.8
2.22 2.12 28.8
2.30 0.56 28.6
2.30 1.11 32
3.27 1.15 20.6
3.27 0.575 26.8
3.42 0.25 22.2
3.42 0.50 18.4
2.70 0.60 18.6
4.25 0.60 16.4
2.70 0.30 18.6
3.42 0.52 24.2
8.0
7.5
7.0
6.5
6.0
5.5
intravenous
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
oral
MIC
29
30
62
66
197–199
199–201
3.44 0.52 26.4
3.44 0.13 20.6
31
32
70
89
—
172–174
179–181
—
1.74 41.2
1.74 4.48 38.6
À0.66 0.36 10.0
18.6
HS
OH
N
O
NHNH
2
O
N
O
O
S
0
2
4
6
8
10
12
14
NH
Ar
Ar CHO
+
EDC, THF
Time (hours)
N
Figure 2. Graph depicting the pharmacokinetics of compound 17 by both iv and
oral routes and MIC of this compound at 0.05 g/mL.
Scheme 1. Synthetic protocol of the compounds.
l
bulky groups like benzyloxy on the phenyl ring enhances the activ-
ity to 39–92 times with MIC of less than <1 M. The enhanced activ-
ity might be due to its hydrophobicity, ClogP of this compounds
15–17 was 4.25 whereas standard compound 1 is 2.56. Introduction
of electron withdrawing groups like nitro, halogen, and triflourom-
l
method. Briefly, cells (2500/well) were seeded in 96-well plates
and cultured for 24 h, followed by treatment with the compounds
1–32 for another 48 h. Twenty microliters of 5 mg/mL MTT was
added per well and incubated for another 2.5 h at 37 °C. Then the
supernatant fluid was removed and MTT formazan precipitate
ethyl enhances the potency with MIC ranging from 0.13 to 2.12 lM.
The order of activity among the electron withdrawing groups CF3
>Br >F >C l>NO2. Replacement of phenyl with furan ring makes
the compound less active but introduction of nitro group in the
furan ring makes the compound 10 times more active with MIC of
was dissolved in 150 lL of DMSO, shaken mechanically for
15–20 min. The optical density of each well was measured at
570 nm, using a Perkin Elmer Victor 3 microplate spectrophotome-
ter. The inhibitory rates at 20 lM are given in Table 1, all the active
4.48
The safety profile of compounds 1–32 was tested on human can-
cer cell lines HepG2 at the concentration of 25 M using the MTT
l
M.
compounds showed low toxicity in this assay and the most potent
compound 17 exhibited good safety profile with inhibitory rates as
low as 8.8%.
l

A. V. Ramani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2764–2767
2767
The most potent compound 17 was selected and assessed for its
Acknowledgment
basic pharmacokinetic parameters after oral (50 mg/kg body
weight) and intravenous administration (10 mg/kg body weight)
in male Wistar rats.13 The oral formulation constituted of a suspen-
sion of drug in 0.25% sodium CMC and Tween 80. The formulation
for intravenous administration was prepared with 20% propylene
glycol, 10% cremophore ELP, 20% N-Methylpyrrolidone in normal
saline. Plasma samples were analyzed using reverse phase HPLC
(Perkin Elmer LC system equipped with Flexar quaternary pump
along with peltier controlled Flexar auto-sampler and Flexar PDA)
The authors are thankful to the Indian Council of Medical
Research [58/34/2008-BMS], Government of India, for their finan-
cial assistance.
References and notes
1. Valadas, E.; Antunes, F. Eur. J. Radiol. 2005, 55, 154.
2. World Health Organization, 2011/2012 Tuberculosis Global Facts, Geneva,
Switzerland (2011).
3. Manjashetty, T. H.; Yogeeswari, P.; Sriram, D. Bioorg. Med. Chem. Lett. 2011, 21,
2125.
4. Pedro, E.; Silva, A. D.; Ramosa, D. F.; Bonacorso, H. G.; Iglesia, A. I.; Oliveira, M.
R.; Coelho, T.; Navarini, J.; Morbidoni, H. R.; Zanatta, N.; Martins, M. A. P. Int. J.
Antimicrob. Agents 2008, 32, 139.
on Brownlee Analytical C18 column (4.6 Â 150 mm, 3
lm, Perkin
Elmer Corporation, U.S.A) column. The mobile phase used is a mix-
ture of 0.01 M ammonium acetate (pH 4.5) and acetonitrile mixture
(62:38, v/v) delivered at 1 mL/min. Plasma concentration time data
of the analyte were analyzed by non-compartmental method using
WinNonlin Version 5.1 (Pharsight Corporation, Mountain View, CA).
5. Sriram, D.; Yogeeswari, P.; Madhu, P. Bioorg. Med. Chem. Lett. 2005, 15,
4502.
6. Sriram, D.; Yogeeswari, P.; Madhu, P. Bioorg. Med. Chem. Lett. 2006, 16, 876.
7. Hearn, M. J.; Cynamon, M. H. J. Antimicrob. Chemother. 2004, 53, 185.
8. Arora, S. K.; Sinha, N.; Jain, S.; Upadhyaya, R. S.; Jana, G.; Sinha, R. K.; Ajay, S.
WO2004/026828 A1.
9. Rosanna, M.; Rosaria, O.; Francesca, M.; Maria, G. V. Antimicrob. Agents
Chemother. 2002, 46, 294.
The basic pharmacokinetic parameters assessed were half life (t ),
½
elimination rate constant (Kel), mean plasma clearance (CL) and
mean volume of distribution (Vd) as reported in Table 2.14–17 After
a single iv bolus dose of 10 mg/kg body weight, compound 17 had
a t of 1.14 h with moderate clearance (22.48 mL/min/kg) and vol-
½
10. Srivastava, T.; Haq, W.; Katti, S. B. Tetrahedron 2002, 58, 7619.
11. N-(2-(4-(benzyloxy) phenyl)-4-oxo-1,3-thiazinan-3-yl) isonicotinamide (17):
White solid; mp—189–190; Yield (84%); Anal. Calcd for C23H21N3O3S: C, 65.85;
H, 5.05; N, 10.02. Found: C, 65.89; H, 4.99.; N, 9.97. 1H NMR (300 MHz, CDCl): d
2.79–2.87 (m, 4H, CH2CH2), 5.21 (s, 2H, CH2) 5.80 (s, 1H, CH), 6.8–7.01 (m, 4H,
ArH of phenyl), 7.43–7.54 (m, 5H, ArH of benzyl), 7.91–8.87 (m, 4H, ArH of
pyridyl), 8.3 (s, IH, NH, D2O exchangeable). 13C NMR (75 MHz, CDCl3): d 171.1,
163.9, 158.3, 148.9, 141.1, 138.8, 130.3, 128.2, 127.1, 125.2, 124.1, 120.8,
114.12, 70.1, 63.7, 38.2, 31.5 MS m/z: 419.13 [M+].
12. National Committee for Clinical Laboratory Standards. Antimycobacterial
susceptibility testing for Mycobacterium tuberculosis. Proposed standard M24-
T. National Committee for Clinical Laboratory Standards, Villanova, Pa., 1995.
13. Ramani, A. V.; Pinaki, S.; Ramesh, M. Biomed. Chromatogr. 2009, 23, 615.
14. Toutain, P. L.; Bousquet-Melou, A. J. Vet. Pharmacol. Therap. 2004, 27, 415.
15. Toutain, P. L.; Bousquet-Melou, A. J. Vet. Pharmacol. Therap. 2004, 27, 427.
16. Toutain, P. L.; Bousquet-Melou, A. J. Vet. Pharmacol. Therap. 2004, 27, 441.
17. Toutain, P. L.; Bousquet-Melou, A. J. Vet. Pharmacol. Therap. 2004, 27, 455.
ume of distribution (1.99 L). Oral administration of a single dose of
50 mg/kg body weight showed low oral bioavailability (F = 16.7%)
based on 10 h AUC (5.87 lg h/mL) and Cmax (1.31 lg/mL).
The low systemic bioavailability of the compound can be attrib-
uted to its very poor aqueous solubility in the gastrointestinal flu-
ids, therefore low and delayed absorption. The pharmacokinetic
profile was compared with that of the parent compound isoniazid
(Table 2) from which it can be inferred that the compound 17 had a
low systemic exposure and higher clearance values comparatively.
As (Fig. 2) the levels of compound 17 are much above the MIC
values, a dose–response relationship has to be established. Also,
the bioavailability of the compound 17 has to be enhanced by
improvising the oral formulation.