Microwave assisted one-pot synthesis of highly potent novel isoniazid analogues

Article abstract of DOI:10.1016/j.bmcl.2011.01.122

A series of novel isoniazid (INH) analogues were synthesized by microwave assisted one pot reaction of INH, various benzaldehydes and dimedone in water with catalytic amount of DBSA. The synthesized compounds were evaluated for their anti-TB activity agai

Full text of DOI:10.1016/j.bmcl.2011.01.122

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2125–2128
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave assisted one-pot synthesis of highly potent novel isoniazid
analogues
⇑
Thimmappa H. Manjashetty, Perumal Yogeeswari, Dharmarajan Sriram
Medicinal Chemistry and Antimycobacterial Research Laboratory, Department of Pharmacy, Birla Institute of Technology and 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:
A series of novel isoniazid (INH) analogues were synthesized by microwave assisted one pot reaction of
INH, various benzaldehydes and dimedone in water with catalytic amount of DBSA. The synthesized
compounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37Rv (MTB)
and multi-drug resistant Mycobacterium tuberculosis (MDR-TB). Among the 29 compounds, compound
N-[9-[2-(benzyloxy)phenyl]-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-10(1H)-acridinyl]
Received 30 November 2010
Revised 27 January 2011
Accepted 28 January 2011
Available online 2 February 2011
isonicotinamide (12) inhibited MTB with MIC of <0.17
l
M and MDR-TB with MIC of 0.69
lM.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Isoniazid derivative
Anti-TB activity
Antimycobacterial activity
MDR-TB
Tuberculosis (TB) is the leading cause of mortality among all
infectious diseases worldwide and is responsible for over two
million deaths annually.1 The major concerns for current TB treat-
ment are its latency, co-infection with HIV, poor patient compliance,
and drug resistance issues caused by the emergence of MDR-TB and
the recent advent of extensively drug resistant tuberculosis (XDR-
TB).2 It is estimated that between 2002 and 2020, approximately 1
billion people will be newly infected, more than 150 million people
will get sick, and 36 million will die of TB if new disease prevention
and treatment measures are not developed.3 Hence, there is an ur-
gent need to develop novel, highly effective, and fast acting anti-TB
drugs with low toxicity profiles and performing activity against both
drug-sensitive and drug resistant MTB.
Isoniazid (INH) has become the single most researched antitu-
bercular agent which is not effective against MDR-TB. Several re-
cent 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, particularly the (hetero)aromatic derivatives, had been
carried out and had demonstrated good activities by the methods
then available ( Fig. 1).4–8 The important relationship between
serum levels of INH and the emergence of INH-resistant cultures
in patients with pulmonary tuberculosis became evident in careful
studies done some time ago.9 These studies stressed the
importance of early intensive chemotherapy, with an emphasis
on initial peak serum concentrations of INH, rather than on con-
centrations measured three or more hours after the dose. Further,
the improved therapeutic outcome of certain treatment regimens
was attributed to the higher early peak serum concentrations of
INH that they permitted, rather than to the period that minimum
inhibitory concentrations (MIC) of INH were maintained in the ser-
um.10 Serum concentrations are influenced by a number of factors,
but among the most important of these is the enzymatic acetyla-
tion of INH by N-acetyl transferase (NAT). This represents a major
metabolic pathway for INH in human beings. Acetylation greatly
reduces the therapeutic activity of the drug. NAT is also present
in mycobacterial pathogens. Resistance to INH in mycobacteria
can thus be related to increased expression of NAT.11
Chemical modification of the hydrazine unit of INH with a
functional group that blocks acetylation, while maintaining strong
anti-TB action, has the potential to improve clinical outcomes and
reduce the emergence in patients of acquired INH resistance. The
goal of our study was to investigate such chemical modification.
In the present study we incorporated the INH moiety in the deca-
hydroacridine scaffold and evaluated for in vitro MTB and MDR-TB
activities.
The synthetic procedure (Scheme 1) consists of the microwave
assisted reaction of 5,5-dimethylcyclohexane-1,3-dione (dime-
done) with the various benzaldehydes and the INH in water with
catalytic amount p-dodecylbenezenesulfonic acid (DBSA)12
(Table 1). The selection of reaction conditions (in particularly the
solvent and the catalyst) appears to be the most critical. Although
today’s environmental consciousness imposes the use of water as a
⇑
Corresponding author. Tel.: +91 4066303506.
E-mail
addresses:
dsriram@bits-hyderabad.ac.in,
drdsriram@yahoo.com
(D. Sriram).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.122

2126
T. H. Manjashetty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2125–2128
H3C
O
N
N
NNH
HO
N
HO
CH3
CF3
N
F3 C
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
H
N
F
N
S
N
CONHN
O
N
H
N
H
MIC: 0.45 µM
MIC: 0.0004 µM
Figure 1. Potential reported isoniazid analogues.
R
O
O
O
CHO
O
CONHNH2
R
DBSA
N
2
+
+
Water
HN
O
N
MWI
10-16 minutes
1-29
N
Scheme 1. Synthetic protocol of the compounds.
solvent on both industrial and academic chemists, organic solvents
are still used instead of water for that most organic substances are
insoluble in water and many reactive substrates, reagents and cat-
alysts are decomposed or deactivated by water. DBSA is a Brønsted
acid-surfactant-combined catalyst, and the surfactant used in
water can make organic materials soluble or form colloidal disper-
sions, and the Brønsted acid is stable in water, so it can solve the
two drawbacks of the reactions in water. DBSA has been used in
a number of organic reactions as a good catalyst. The effect of elec-
tron and the nature of substituents on the aromatic ring did not
show expected strong effects in terms of yields under these reac-
tion conditions. Benzaldehyde containing electron-withdrawing
groups (such as nitro, halide groups) or electron-donating groups
(such as hydroxy, alkoxyl groups) were employed and they were
found to react well to give the corresponding N-[9-(aryl)-3,3,6,6-
tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-10(1H)-acridi-
nyl]isonicotinamides in good to excellent yields (65–96%). Use of
10 mol % DBSA in refluxing water is sufficient to push the reaction
forward. The yields are, in general, very high regardless of the
structural variations in benzaldehyde. In this synthetic protocol
we have utilized microwave irradiation, which when compared
to conventional reflux (6 h), took just 10–16 min. The purity of
compounds 1–29 was checked by TLC and elemental analyses.
Both analytical and spectral data (1H NMR, 13C NMR, and mass
spectra) of all the synthesized compounds were in full agreement
with the proposed structures.13 The 1H NMR spectra of compounds
1–29 show singlets at 0.80–0.84 and 0.90–0.95 ppm which are as-
signed to the protons of the four methyl groups in positions 3 and
6, quadrates for the CH2 group protons of the cyclohexane rings in
the range 1.80–2.21 ppm, a singlet for the methine proton at 5.24–
5.40 ppm, and signals for the aromatic protons in the range 6.80–
8.72 ppm. Lipophilicity of the synthesized derivatives and that of
the parent compound, INH, are expressed in terms of their log P
values (Table 1). These values were computed with a routine

T. H. Manjashetty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2125–2128
2127
Table 1
Physical constants, in vitro anti-tubercular and cytotoxicity activities
O
Ar
O
N
HN
O
N
No.
Ar
Yield (%)
Mp (°C)
C Log Pa
CC50 in
l
Mb
MIC in
l
Mc
MDR-TB
MTB
6.66
3.22
6.44
1
2
3
4
5
6
7
8
Phenyl
2-Methylphenyl
3-Methylphenyl
4-Methylphenyl
91
82
70
94
77
71
70
68
71
89
96
65
75
78
67
76
89
68
68
86
67
79
67
66
65
79
74
67
75
146–148
142–144
151–154
82–84
182–184
218–221
252–254
132–136
98–100
148–150
178–180
96–98
146–148
134–136
182–184
152–154
126–128
84–86
4.32
4.77
4.82
4.82
3.45
3.65
3.65
3.84
4.24
4.49
5.75
5.61
6.01
6.01
3.50
3.62
5.20
5.20
5.20
5.03
5.18
5.18
5.18
4.46
4.46
4.46
3.98
4.06
4.06
À0.66
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.69
1.35
0.69
—
—
6.44
2-Hydroxyphenyl
3-Hydroxyphenyl
4-Hydroxyphenyl
2-Methoxyphenyl
4-Methoxyphenyl
4-Dimethylaminophenyl
4-Isopropylphenyl
2-Benzyloxyphenyl
3-Benzyloxyphenyl
4-Benzyloxyphenyl
4-Hydroxy-3-methoxyphenyl
2,3,4-Trimethoxyphenyl
2-Trifluoromethylphenyl
3-Trifluoromethylphenyl
4-Trifluoromethylphenyl
4-Chlorophenyl
2-Bromophenyl
3-Bromophenyl
4-Bromophenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2-Nitrophenyl
3-Nitrophenyl
>12.87
>12.87
12.87
12.51
12.51
6.10
1.52
<0.17
0.17
0.17
3.02
11.16
0.36
0.18
0.36
0.79
0.18
0.73
0.73
0.41
0.41
0.20
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Isoniazid
>108.56
>108.56
>108.56
—
—
>116.26
>116.26
>116.26
>124.00
>113.95
<113.95
<113.95
>128.18
>128.18
>128.18
<121.46
<121.46
<121.46
>455.73
1.45
1.45
0.74
1.54
2.84
2.84
2.84
1.59
0.82
0.20
1.51
3.03
3.03
45.57
110–112
94–96
172–174
174–178
120–122
102–104
90–92
106–108
234–236
120–122
90–92
0.19
0.77
0.77
0.73
4-Nitrophenyl
a
b
c
Calculated with ChemBioDraw Ultra 11.0.
Cytotocity in VERO cell line.
Minimum inhibitory concentration.
method called calculated log P (C log P) using ChemBioDraw Ultra
11.0 software.
0.73
l
M); 10 compounds ( 12–14, 17–19, 21, 24–27) were more
potent with MIC less than 0.73
lM and two compounds (22–23)
The compounds 1–29 were screened for their in vitro antimyco-
bacterial activity against log-phase cultures of MTB and MDR-TB in
Middlebrook 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.14 The MIC is defined as the minimum concen-
tration 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
first phase of screening, all the compounds showed in vitro activity
were equally active. Compound N-[9-[2-(benzyloxy)phenyl]-
3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-10(1H)-
acridinyl]isonicotinamide (12) was found to be the most promising
compound with MIC of <0.17 lM. Compound 12 was found to be
more than four times active as parent drug INH. The enhanced
activity might be due to its hydrophobicity, C log P of this
compound 12 was 5.61 whereas parent compound INH is À0.66.
With respect to structure anti-TB activity, the substituent in the
9th position dictates the activity. Electron donating group like
hydroxyl, methyl and methoxyl groups are detrimental; among al-
kyl substituents isopropyl (11) showed better activity than methyl
(2–4) group. Among alkoxy/aryloxy substituents benzyloxy group
showed maximum activity and 2-benzyloxy group was found to
against MTB with MIC ranging from <0.17 to >12.87
compounds (12–14, 17–29) inhibited MTB with less than 1
When compared to standard first line drug INH (MIC of
lM. Sixteen
l
M.

2128
T. H. Manjashetty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2125–2128
be more potent than 3 and 4-benzyloxy group. Electron withdraw-
ing groups like halogen and nitro group showed better activity. The
compounds which showed activity against MTB with less than
Acknowledgements
The authors are thankful to the Indian Council of Medical Re-
search (58/34/2008-BMS), Government of India, for their financial
assistance.
1 lM were further screened against MDR-TB. The MDR-TB clinical
isolate was obtained from Tuberculosis Research Center, Chennai,
India, and was resistant to INH, rifampicin, ethambutol and oflox-
acin. The screened compounds inhibited MDR-TB with MIC ranging
References and notes
from 0.20 to 2.84
more potent than INH (MIC of 45.57
rophenyl substituent showed very good activity with MIC of
0.20 M and was found to be 227 times more potent than INH
l
M and all the 16 screened compounds were
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octahydroacridin-10(9H)-yl)isonicotinamide (12): 1H NMR (300 MHz, CDCl3):
d = 0.82 (s, 6H, 2 Â CH3), 0.95 (s, 6H, 2 Â CH3), 1.89 (d, J = 16.0 Hz, 2H, HAB, H-
4a, H-5a), 2.10 (d, J = 16.0 Hz, 2H, HAB, H-4e, H-5e), 2.28 (dd, J Gem = 9.8 Hz,
4H, HAB, 2 Â CH2, H-2, H-7), 5.01 (s, 2H, CH2 of benzyloxy), 5.34 (s, 1H, H-9),
6.8–6.93 (m, 4H, ArH of phenyl), 7.42–7.60 (m, 5H, ArH of benzyl), 8.1–8.79 (m,
4H, ArH of pyridyl), 8.79 (s, 1H, NH, D2O exchangeable). 13C NMR (75 MHz,
CDCl3): d = 27.2, 32.8, 50.5, 51.2, 71.1, 111.9, 112.2, 121.7, 128.9, 136.7, 140.8,
145.7, 149.7, 158.8, 194.1. Anal. Calcd for C36H37N3O4: C, 75.11; H, 6.48; N,
7.30. Found: C, 75.31; H, 6.40; N, 7.28.
lM). Compounds with 4-fluo-
l
against MDR-TB.
Some of the compounds were further examined for toxicity
(CC50
62.5
)
l
in
a mammalian Vero cell line at concentrations of
g/ml.15 After 72 h of exposure of tested compounds, viability
was assessed on the basis of cellular conversion of MTT into a for-
mazan product using the Promega Cell Titer 96 non-radioactive
cell proliferation assay and reported in Table 1. Compounds with
bromo and nitro substituted phenyl at 9th position showed toxic-
ity at 62.5 mg/ml (56–72%). Compound 18 showed maximum
selectivity index (CC50/MIC) of >645. These results are important
as nitrophenyl substituted analog 27 was more active and at the
same time it was cytotoxic. Certainly, the development of any
new compounds for TB would depend on an excellent safety profile
since it would need to be used for several months in combination
with other antitubercular agents.
Screening of the antimycobacterial activity of these novel series
identified novel INH analogue with high activity toward MDR-TB,
with MIC values between 0.2 and 2.84 lM. In conclusion, it has
been shown that the potency, selectivity, and low cytotoxicity of
these compounds make them valid leads for synthesizing new
compounds that possess better activity. Further structure-activity
and mechanistic studies should prove fruitful.
14. 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.
15. Gundersen, L. L.; Meyer, J. N.; Spilsberg, B. J. Med. Chem. 2002, 45, 1383.