Repurposing of a drug scaffold: Identification of novel sila analogues of rimonabant as potent antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2016.07.009

The structural similarity between an MmpL3 inhibitor BM212, and a cannabinoid receptor modulator rimonabant, prompted us to investigate the anti-tubercular activity of rimonabant and its analogues. Further optimization, particularly through incorporation of silicon into the scaffold, resulted in new compounds with significant improvement in anti-tubercular activity against Mycobacterium tuberculosis (H37Rv). The sila analogue 18a was found to be the most potent antimycobacterial compound (MIC, 31?ng/mL) from this series with an excellent selectivity index.

Full text of DOI:10.1016/j.ejmech.2016.07.009

Accepted Manuscript
Repurposing of a drug scaffold: Identification of novel sila analogues of rimonabant as
potent antitubercular agents
Remya Ramesh, Rahul D. Shingare, Vinod Kumar, Amitesh Anand, Swetha B,
Sridhar Veeraraghavan, Srikant Viswanadha, Ramesh Ummanni, Rajesh Gokhale, D.
Srinivasa Reddy
PII:
S0223-5234(16)30557-8
10.1016/j.ejmech.2016.07.009
EJMECH 8727
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2016
Revised Date: 5 July 2016
Accepted Date: 7 July 2016
Please cite this article as: R. Ramesh, R.D. Shingare, V. Kumar, A. Anand, S. B, S. Veeraraghavan,
S. Viswanadha, R. Ummanni, R. Gokhale, D. Srinivasa Reddy, Repurposing of a drug scaffold:
Identification of novel sila analogues of rimonabant as potent antitubercular agents, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.009.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Repurposing of a drug scaffold: Identification of novel sila
analogues of rimonabant as potent antitubercular agents
Remya Ramesh,†¥ Rahul D. Shingare,†¥ Vinod Kumar,† Amitesh Anand,∥¥ Swetha B,‡ Sridhar
Veeraraghavan,§ Srikant Viswanadha,§ Ramesh Ummanni,‡¥ Rajesh Gokhale,∥¥ D. Srinivasa
Reddy*†¥
†CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India
∥CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi, 110025, India
‡ CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500007, India
¥Academy of Scientific and Innovative Research (AcSIR), New Delhi - 110 025, India
§ Incozen Therapeutics Pvt. Ltd., Alexandria Knowledge Park, Turkapally, Rangareddy, 500078,
India
*Corresponding author: Dr. D. Srinivasa Reddy; E mail ID: ds.reddy@ncl.res.in
ABSTRACT: The structural similarity between an MmpL3 inhibitor BM212, and a cannabinoid
receptor modulator rimonabant, prompted us to investigate the anti-tubercular activity of
rimonabant and its analogues. Further optimization, particularly through incorporation of silicon
into the scaffold, resulted in new compounds with significant improvement in anti-tubercular
activity against Mycobacterium tuberculosis (H37Rv). The sila analogue 18a was found to be the
most potent antimycobacterial compound (MIC, 31 ng/mL) from this series with an excellent
selectivity index.
KEYWORDS. Drug repurposing, Tuberculosis, Rimonabant, BM212, sila analogue, MmpL3
inhibitor.

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1. Introduction
Drug discovery has become an increasingly tough endeavor. The average cost of bringing
a drug into market is estimated to be about US$ 2600 million with a timeline of up to 15 years
[1]. Identification of entirely novel compounds with unknown pharmacokinetic and safety profile
poses more risk apart from being expensive and time consuming. The discovery of new
indications for an existing drug termed “drug repurposing or drug reprofiling” is a lower risk
strategy with increased probability of success within a short span of time. According to a recent
report, 24 drugs had been remarketed for new uses and more than 15 are currently in the various
developmental stages [2-4]. Also, molecules that failed in the clinic for a particular indication
can be successfully repurposed for treatment of other conditions. Thalidomide, the most
controversial drug of all time, is now used for pain relief in certain cancers and leprosy [5].
Pfizer’s blockbuster drug sildenafil citrate (Viagra®), which was originally intended for
hypertension was serendipitously repurposed after the Phase I clinical trials for erectile
dysfunction [6].
N
N
NH
N
O
N
N
N
Cl
Cl
Rimonabant ( )
Cl
Cl
1
BM212 ( )
2
Cl
MmpL3 inhibitor
CB1 receptor modulator
R3
R1
X
N
R2
N
Ar2
N
Ar1
Our design
Ar1, Ar2 = aryl
R1, R2, R3 = alkyl or H
X = CH2 or CO
R2 and R3 together can
form a ring
WO 2014/181357 A1
1
2
Overlay of (yellow) and (pink)

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Figure 1. Design of diarylpyrazoles (rimonabant analogues) towards antitubercular agents
Tuberculosis (TB) is an infectious disease caused by various strains of mycobacteria; the
most common one being Mycobacterium tuberculosis (Mtb). Almost one-third of the total world
population is asymptomatically infected by Mtb and it is the second leading cause of death due to
an infectious agent [7]. Medications are known to treat TB; however, the response rate is slow
with development of antibiotic resistance posing a serious threat. In view of these challenges,
there is a need to develop new drug candidates with novel mechanisms for treating tuberculosis.
The pre-clinical candidate BM212, a 1,5-aryl substituted pyrrole was reported to be active
against Mtb with a minimum inhibitory concentration (MIC) of 0.7-1.5 µg/mL [8]. BM212
belongs to the MmpL3 class of inhibitors and blocks the transport of mycolic acids that are
essential for the development of cell wall of mycobacteria thereby inhibiting their growth [9].
MmpL3 is a relatively new therapeutic target and other lead compounds such as SQ109, AU-
1235, NITD-304 are also known to act on this transporter protein [10-14]. To identify new
antitubercular agents with novel scaffolds that have already been tested in humans, a scaffold
hopping technique was adopted using BM212. Towards this direction, an anti-obesity drug
rimonabant (2) [15] attracted our attention. An overlay of BM212 (1) and rimonabant (2)
suggested close structural similarities between the two (see Figure 1). Rimonabant acts by
blocking the cannabinoid receptor-1 (CB1). This receptor is expressed mainly in the central and
peripheral nervous system and is involved in controlling food consumption, mood, and anxiety
related disorders [16, 17]. An advantage with the rimonabant scaffold is that it can penetrate
blood-brain-barrier (BBB) and may be useful in developing agents for the treatment of brain
tuberculosis. The objective of the current study was to repurpose the rimonabant scaffold
towards the development of agents active against Mtb [18].
2. Results and discussion
2.1 Synthesis
Initial screening of rimonabant demonstrated moderate potency (MIC = 25 µg/mL, Mtb)
and gave us encouragement to synthesize more analogues. Analogues of rimonabant with
varying structural features were synthesized and evaluated for their activity against Mtb. All the
synthetic details are outlined in Schemes 1- 5. Compound 3 was synthesized according to the
literature protocol and was coupled with several amines to give a series of amides (4a-4k) [19].

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Amide 4l was prepared from the corresponding acid chloride employing procedures known in
the literature [20]. The ester group in 4k was hydrolyzed to obtain the acid 4m. The hydrazide 4n
was obtained from the corresponding ester 6 and hydrazine hydrate [21]. Because of our group’s
continued interest in organosilicon molecules [22, 23], we synthesized a few silicon analogues
(4c and 4d) as well. The bioisosteric replacement of carbon with silicon has gained attention in
recent times [24-29]. The silaamide 4c was converted to thioamide 5 by treating with
Lawesson’s reagent in THF (Scheme 1). Towards slightly more polar analogues, the known ester
6 was subjected to benzylic bromination using N-bromosuccinimide (NBS) and the bromo
derivative was converted to alcohol in the presence of AgNO3 and water. Hydroxylester 7 was
hydrolyzed to obtain the acid 8 [30]. Carboxylic acid 8 was then coupled to the silicon amines A
and B using EDC.HCl and HOBt to obtain amides 9a and 9b, respectively (Scheme 2). Alcohol
10 was prepared from ester 6 by following the literature procedures and was subjected to
alkylation using benzyl bromide and NaH as base to obtain the ether 11 (Scheme 3) [31].
Alcohol was converted into its mesylate and displaced with various amines to generate a library
of rimonabant amine analogues 12a- 12g (Scheme 3).

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Scheme 1. Syntheses of amides and thioamide

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O
N
O
HO
OEt
OEt
i) NBS, CCl4
N
ii) AgNO3
N
N
Cl
Cl
acetone/ water
Cl
Cl
7
Cl
Cl
6
O
N
HO
OH
amine (A or B)
KOH
N
EtOH, H2O
EDC.HCl, HOBt
DIPEA, CH2Cl2
Cl
Cl
Cl
8
O
N
O
HO
N
HO
N
Si
Si
H
N
N
N
Cl
Cl
Cl
Cl
Cl
9a
9b
Cl
NH2HCl
amine B
Si
Si
amine A
NH.HCl
Scheme 2. Synthesis of hydroxymethyl amides

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Scheme 3. Syntheses of ether 11 and amines 12
For the preparation of hydroxymethyl amine, the alcohol 7 was protected as its silyl ether
13 and the ester was reduced using Lithium aluminium hydride (LAH) in THF. The resulting
alcohol was mesylated and displaced with the silicon amine A and finally the protecting group
was removed using TBAF to obtain the hydroxymethyl sila analogue 14. In order to make
compounds with more structural variations, alcohol 10 was converted to its corresponding azide
15. This azide on CuI catalyzed azide-alkyne cycloaddition with ethyl propiolate gave the 1,4 –
substituted triazole ester 16 (Scheme 4). We also wanted to determine the influence of methyl
group of pyrazole moiety on the activity of the compound because the C-4 methyl group is
absent in BM212. Accordingly the alcohol 17 and acid 19 were synthesized [32]. By utilizing the
same strategy adopted for the previous compounds, amines 18a-18e and amides 20a-20e were
prepared (Scheme 5).

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Scheme 4. Synthesis of compounds 14 and 16

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Scheme 5. Synthesis of des-methyl pyrazole derivatives
2.2 Antitubercular activity of compounds
After generating a library of rimonabant analogues with various structural features, our
next task was to check the anti-tubercular activity of the compounds. The MIC of the compounds
against H37Rv was determined in an Alamar- Blue assay (see supplementary data). The MIC
values of selected compounds are given in Table 1. Rimonabant demonstrated moderate activity
with an MIC of 25 µg/mL. Some of the compounds from the amide series (4a, 4i, 4g and 20a,
20c-20e) also showed moderate activity with high MIC values. The thioamide 5, the ether
analogue 11, as well as the triazole 16 were not active. Many of the amines demonstrated good
activity indicating that the basic nitrogen is essential for the desired antitubercular activity. The
silicon amine 12f (MIC 1.17 µg/mL) showed an activity similar to that of BM212. To our
delight, compound 18a showed excellent anti-tubercular activity with an MIC of 0.031 µg/mL,

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more than 25-fold increase in activity compared to the parent compound BM212. Interestingly
this compound lacks the methyl group on pyrazole ring and is structurally more close to BM212.
The compound 18e (MIC 0.39 µg/mL) also showed good activity, but was 12 times less potent
than 18a. We have tested the compounds 18b and 18c in which silapiperidine ring was replaced
with piperidine and piperazine rings, respectively. As expected, both the compounds 18b (MIC
1.56 µg/mL) and 18c (MIC 25 µg/mL) showed inferior activity with respect to corresponding
silicon analogues 18a and 18e. The activity of 18b is similar to that of BM212 which shows that
the replacement of pyrrole to pyrazole in BM212 retains the anti-TB activity and can be
modulated by changing the C3 substituents. The improved activity of silicon analogues may be
explained by the improved cell wall permeability of lipophilic sila compounds. However, we do
not have any experimental evidence with the present set of compounds. The introduction of a
more polar hydroxyl group decreased the activity as seen in compound 14 (MIC 6.25 µg/mL), a
5-fold loss in potency when compared to 12f. The amide 20a showed moderate (MIC 6.25
µg/mL) activity and was the best from the amide series, whereas a similar compound 4c did not
show any activity. The introduction of methyl group on the pyrazole ring seems to be detrimental
for the activity in most of the compounds (20a vs 4c and 18a vs 12f). Based on this current
exercise, we have made a few structure-activity relationship conclusions and they are compiled
in Figure 2.
Table 1. Antitubercular activity of selected compounds
compd
X
R1
Y
R2
MIC# compd
X
R1
Y
R2
MIC#
Cl
Me
CO
25
25
25
H
H
CH2
0.031
2
18a
18b
18c
4b
4g
Cl
Cl
Me
Me
CO
CO
H
H
H
H
CH2
CH2
1.56
25

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Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
Me
Me
Me
Me
Me
Me
CO
CH2
CH2
CH2
CH2
CH2
CH2
25
H
H
H
H
H
H
-
H
H
H
H
H
H
-
CH2
CH2
CO
CO
CO
CO
-
25
0.39
6.25
25
4i
18d
18e
20a
20c
20d
20e
1
12.5
25
12a
12b
12c
12d
12f
12g
14
12.5
6.25
1.17
6.25
6.25
25
25
-
-
0.70
0.3
Cl CH2OH CH2
-
-
-
INH
# MIC values are measured in µg/mL
Figure 2. SAR conclusions (using anti-TB potential) of the current series
2.2 Cytotoxixity and ADME data
We have also carried out cytotoxicity studies on selected compounds against lung
epithelial cells (A549) and human hepatoma cells (HepG2) to determine their selectivity index

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[33]. The potent compounds 12f, 18a and 18e showed excellent selectivity for Mtb over the
cytotoxic effect against A549 and Hep G2 cell lines tested (Table 2). Cytotoxicity of the most
active compounds 12f, 18a, 18e and the standard compound BM212 (1) were also evaluated in
RAW macrophages and THP-1 monocytes (see supplementary data). Cytotoxicity was apparent
at ≥ 3 ꢀM for all the compounds tested and varied in the range of 40-60% across both cell lines.
After this exercise, five best compounds were selected for further profiling. Solubility across
different pH, plasma stability, metabolic stability and plasma protein binding were evaluated
(Table 3). The amide 20a showed poor solubility at all the pH tested. The compound 18e showed
very good solubility at varying pH. The other compounds showed good solubility at lower pH,
but solubility decreased at higher pH. Since these compounds are basic in nature, they are
expected to show good solubility at acidic pH. All the compounds showed good plasma stability
similar to that of BM212. The most potent compound 18a, showed 100% stability in human
plasma. Some compounds are degraded by plasma enzymes; therefore, stability in plasma is vital
for determining efficacy of the parent compound. Metabolic stability of selected compounds was
evaluated using mouse and human liver microsomes after 60 min incubation at 1µM
concentration. Most of the compounds including 18a showed poor metabolic stability in both
mouse and human liver microsomes with respect to BM212. Compound 18b showed moderate
stability in human liver microsomes. However, compound 18e showed very good metabolic
stability in liver microsomes. The compound 18e was the best compound in terms of PK profile
and the overall profile was better than BM212. Compounds demonstrating higher metabolic
stability tend to have an increased half-life translating into superior efficacy in vivo. All the
compounds including BM212 were strongly bound to the plasma proteins. High plasma protein
binding is associated with a lower clearance rate resulting in a greater half-life in vivo compared
to low protein bound compounds. It is generally believed that drugs with low plasma protein
binding are more available and efficacious because of higher free drug concentration. But in an
in vivo setting, several other factors such as metabolism and transport come into play [34, 35].
Table 2. Cytotoxicity effects of the selected compounds
IC50 (µM)
selectivity index
compd
A549
HEP G2
IC50±Std dev
71.00±5.26
A549 HEP G2
IC50±Std dev
91.55±5.16
57.22
44.38
BM212

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88.83±6.37
80.29±3.12
55.82±1.12
54.16±9.64
57.18±5.37
61.94±3.62
58.89±0.52
73.22±3.42
68.33±2.07
>100
3.1
2.9
>3.5
3.1
12a
12c
12d
12f
12g
14
85.05±4.35
57.41±3.39
51.87±3.26
36.32±2.37
30.42±1.21
21.44±2.62
86.83±3.69
58.97±0.53
4.1
4.2
31.86
3.62
4.92
30.51
2.30
2.41
841.30 306.32
18a
18e
20a
84.16
4.85
99.80
4.18
Doxor
ubicin
5.32±3.2
5.34±1.82
-
-
Table 3. In-vitro physchem and ADME data
%
plasma
stability
in
% metabolic stability in
liver microsomes (after 30
min.)
%
human
PPB
compd
solubility (µM) at pH
1.2
2.2
4.5
7.4
10.2
mouse
Human
human
100
45.19 42.21 36.73
40.98 40.58 34.24
44.02 42.47 41.62
43.04 42.28 41.87
44.96 44.65 43.56
43.14 43.20 44.28
26.68
1.05
5.38
10.94
2.39
62.4
9.2
79.8
18.5
2.1
99.23
99.95
99.96
99.93
99.89
99.31
99.99
BM212
12f
99.18
88.12
100.0
91.7
5.31
3.7
14
3.72
1.99
6.3
20.3
60.1
93.1
2.1
18a
18b
18e
11.40
39.76
0.05
1.49
26
38.00
1.28
100.0
89.72
100.0
3.9
0.16
0.18
0.13
20a
3. Conclusion
In summary, we have successfully repurposed the rimonabant scaffold towards the
development of potent antitubercular agents. After the synthesis and screening of several
analogues around the rimonabant scaffold, we have identified the silicon analogue 18a as the
most potent compound against Mtb with an MIC value 31 ng/mL. It was observed that an
increase in lipophilicity improves the potency of the compounds in this series. Based on present
study, we have made a few SAR conclusions with respect to anti-TB potential, en route to the
best compound 18a. An in vitro analysis using ADME assays suggests that it needs further
improvement in PK parameters, in particular, improvement in metabolic stability. However,

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compound 18e (MIC 0.39 µg/mL) showed very good ADME profile. Optimization of the lead to
improve the PK parameters and further biological profiling in in vivo models are in progress.
4. Experimental section
4.1 General
All reagents, starting materials, and solvents (including dry solvents) were obtained from
commercial suppliers and used as such without further purification. Reactions were carried out in
oven-dried glassware under a positive pressure of argon unless otherwise mentioned. Air
sensitive reagents and solutions were transferred via syringe or cannula and were introduced to
the apparatus via rubber septa. Reactions were monitored by thin layer chromatography (TLC)
with 0.25 mm pre-coated silica gel plates (60 F254). Visualization was accomplished with either
UV light, Iodine adsorbed on silica gel or by immersion in ethanolic solution of
phosphomolybdic acid (PMA), p-anisaldehyde or KMnO4 followed by heating with a heat gun
for ~15 sec. Column chromatography was performed on silica gel (100-200 or 230-400 mesh
1
size). Deuterated solvents for NMR spectroscopic analyses were used as received. All H NMR
and 13C NMR spectra were obtained using a 200 MHz, 400 MHz or 500 MHz spectrometer.
Coupling constants were measured in Hertz. All chemical shifts were quoted in ppm, relative to
TMS, using the residual solvent peak as a reference standard. The following abbreviations were
used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad. HRMS (ESI) were recorded on ORBITRAP mass analyser. Chemical nomenclature
was generated using Chem Bio Draw Ultra 14.0. The purity of products was determined by
reverse phase HPLC analysis using Agilent technologies 1200 series; column: ZORBAX Eclipse
XBD-C-18 (4.6 x 250 mm, 5 µ). Flow rate 1.00 mL/min, UV 254 nm; using mobile phases,
Method A: 95/5 CH3OH/H2O for 20 min; Method B: 90/10 CH3CN/H2O for 20 min.
4.2 General procedure for amide coupling
To a solution of the carboxylic acid (1 eq) in dry dichloromethane, was added N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
(EDC.HCl,
1.2
eq),
Hydroxybenzotriazole (1.2 eq) and diisopropylethyl amine (3 eq) at 0°C. Then the amine (1.1 eq)
was added and stirred at RT for 6 h. To the reaction mixture, water was added and the organic
layer was separated, washed with saturated NaHCO3, 1N HCl, dried over Na2SO4 and

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concentrated under reduced pressure. This crude mixture was purified by column
chromatography to give the pure compound.
4.3 General procedure for preparation of amines
To a solution of the alcohol (1 eq) in dry dichloromethane (DCM), was added
triethylamine (2.5 eq) followed by Mesyl chloride (1.1 eq) at 0°C and stirred at the same
temperature for 30 min. To the reaction mixture water was added and the organic layer was
separated, dried over Na2SO4 and concentrated under reduced pressure. This crude product was
immediately taken for the next step. The mesylate was dissolved in dry dimethyl formamide
(DMF), triethylamine (4 eq) and corresponding amine partner (1.2 eq) was added at 0°C and left
to stir at RT for 4h. To the reaction mixture water was added and the organic layer was
separated, the aqueous layer was extracted using dichloromethane, dried over Na2SO4 and
concentrated under reduced pressure. The crude was then purified by column chromatography to
give the required compound.
4.4 Protocol for determination of MICs against H37Rv
The compounds were tested for antitubercular activity through inhibition of growth of the
virulent strain of Mycobacterium tuberculosis H37Rv using Alamar-Blue assay method. MIC
values of the compounds against H37Rv were determined in 7H9-OADC media supplemented
with 0.5% glycerol and 1 mgml−1 tryptone at 37 °C in 96-well microtiter plates using the
colorimetric resazurin microtiter assay, and growth was measured by visual readout. Isoniazid
was used as a positive drug control. Statistically significant differences of individual drugs were
not observed between the triplicates for any of the antimicrobial agents tested.
4.5 Protocol for determination of % inhibition against Mycobacterium smegmatis (mc2155)
Anti-mycobacterial activity of series of compounds synthesized was performed with
Mycobacterium smegmatis strain using turbidometry for growth inhibition. Isolated single
colonies of M. smegmatis mc2155 (ATCC 14468) from 7H10 agar plate were grown overnight in
Middlebrook 7H9 medium (0.47% Middlebrook 7H9 broth base, 10% ADS, 0.2% glycerol, and
0.1% Tween-80) to mid exponential phase at 370 °C. Subsequently, 5 mL of Middlebrook 7H9
broth were inoculated with the overnight grown culture and allowed to grow at 37 °C to early log
phase (OD600 ≈ 0.3). For anti-microbial assay, 98 µL of 1:1000-folds dilution of secondary
culture was dispensed into 96-well microtiter plate. To each well 2 µL of test compound was

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added to attain a final concentration of 6.25, 12.5, 25, 50 and 100 µM, and allowed to grow at 37
ºC for 32 hours. 240 µL of sterile water were added to each well of the peripheral rows of 96-
well plate to minimize media evaporation during assay incubation. Bacterial growth was
assessed after 32 hours of incubation by measuring turbidity at 600 nm OD600 values using
TECAN Infinite 200 PRO™ (Tecan Instruments, Switzerland). Positive controls (Rifampicin
and INH) were included in every assay plates using stock solutions. The results were analyzed as
the percentage of growth inhibition. All experiments were carried out in triplicates and results
were reported as ± SD.
4.6 In vitro metabolic stability [36,37]
10 mM of standard solution was prepared in DMSO. 1 mM of working solution-1 was
prepared in acetonitrile from the standard solution, 50 ꢀM of working solution-2 was prepared in
acetonitrile, water mixture (1:1) from the working solution-1. 98 ꢀL of cocktail mixture (buffer,
microsomes and cofactor) was transferred in a pre labelled micro centrifuge tube, 2 ꢀL of
working solution (50 ꢀM in ACN: Water) was added into the cocktail mixture. Samples were
incubated at 37 °C for 15, 30, 45 and 60 minutes time intervals. After incubation, samples were
terminated with 200 ꢀL of methanol containing internal standard. Then the samples were
Vortexed in a table top vortexer for 5 min and centrifuged at 14000 RPM for 5 minutes.
Supernatant samples were subjected to LC-MS/MS analysis.
4.7 In vitro plasma stability [38]
10 mM of standard solution was prepared in DMSO. 1 mM of working solution-1 was
prepared in Acetonitrile from the standard solution, 50 ꢀM of working solution-2 was prepared
in Acetonitrile, water mixture (1:1) from the working solution-1. 98 ꢀL of test system was
transferred in a pre labelled micro centrifuge tube, 2 ꢀL of working solution (50 ꢀM in ACN:
Water) was added in to the cocktail mixture. Samples were incubated at 37°C for 60 minutes
time intervals. After incubation samples were terminated with 200 ꢀL of methanol containing
internal standard. Samples were Vortexed in a table top vortexer for 5 min and centrifuged at
14000 RPM for 5 minutes, supernatant samples were subjected to LCMS/MS analysis.
4.8 Plasma protein binding (Equilibrium dialysis method) [38]
745 ꢀL of plasma was transferred into 2 mL micro centrifuge tube. To that 5ꢀL of test
compound (150 ꢀM) was added, samples were mixed in the table top vortexer for 2 minutes. 50

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ꢀL plasma (n=2) was transferred in a pre labelled 1.5 mL micro centrifuge tube treated as 0 hour
sample. Remaining 650 ꢀL plasma sample were incubated for 30 minutes at 37 °C in a water
bath, after 30 minute incubation 50 ꢀL plasma (n=2) was removed in a pre labeled 1.5 mL micro
centrifuge tube treated as 0.5 hour sample. 200 ꢀL of plasma sample (n=2) was transferred into
the sample chamber which is indicated by the red ring, RED insert was placed into the base plate
and 350 ꢀL of buffer was transferred into the buffer chamber. Plates were incubated at 37 °C at
approximately 100 RPM on an orbital shaker or 20 RPM on an up-and-down shaker for 4 hours.
50 ꢀL of post dialysis sample from the buffer and the plasma chambers were transferred into pre
labelled micro centrifuged tube. 50 ꢀL of plasma to the buffer samples and an equal volume of
buffer (KH2PO4 Buffer pH 7.4) were added to the collected plasma samples. 150 ꢀL of methanol
containing internal standard (Tolbutamide 250 ng/mL) was added to precipitate the protein and
release compound. Samples were vortexed for 3 minutes in a table top vortexer and centrifuged
for 5 minutes at 14,000 RPM. The supernatant was subjected to LC-MS/MS analysis.
4.9 In vitro solubility
10 mM of standard solution was prepared in DMSO, 5 mM of working solution-1 was
prepared in DMSO from the standard solution. 495 ꢀL of Buffer (1.2, 2.2, 4.5, 7.4 and 10.2 pH)
was transferred in a pre labelled micro centrifuge tube. 5 ꢀL of working solution (5 mM) was
added in to the test system. Samples were vortexed in a table top vortexer for 5 min and
centrifuged at 14000 RPM for 5 minutes; supernatant samples were subjected to HPLC analysis.
The concentration at t=0 is 1µM. Based on the solubility data most of the compounds have ~
1µM solubility at pH 7.4.
4.10 Cytotoxicity assays
Selected compounds were evaluated for their in vitro cytotoxicity against A549 human
lung carcinoma epithelial cells and HepG2 human hepatoma cells. A549 and HepG2 cells used
in this study were purchased from the American Type Culture Collection (ATCC, USA). Cells
were grown in standard Dulbecco’s modified Eagles medium containing 10% FBS in a
humidified atmosphere of 5% CO2 at 37 °C. When cells were sub-confluent in 90 mm dishes,
cells were harvested by trypsinization and seeded in 96 well plates at a density of 7500 cells.
They were further allowed to grow in complete medium under standard conditions. After 18 hrs
to treat cells with test molecules, aliquots of 2 µL of the compound dilutions were added to the

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appropriate microtiter wells containing 198 µL of fresh medium with cells, resulting in the
required final drug concentrations (0.1, 1, 10 and 100 µM as per NCI screening protocol) in
triplicates. As a standard reference drug, Doxorubicin was used in all assay plates and served as
internal control. Plates were incubated further for 48h and assay was terminated by the addition
of 10 µL of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5mg/ml) and
incubated for 60 min at 37 °C. The plates were air-dried and the dye bound to the cells was
subsequently solubilized in 100 µL of DMSO, and the absorbance was red on a multimode
reader (Tecan) at a wavelength of 560 nm, which is directly proportional to cell growth.
Percentage growth was calculated for test wells relative to control wells. The above
determinations were repeated three times and mean values ± SD are reported. The growth
inhibitory effects of the compounds were analyzed by generating dose response curves as a plot
of the percentage surviving cells versus drug concentration. Further selectivity index was
calculated by ratio between cytotoxicity (IC50 in ꢀM) / MIC in ꢀM.
Conflict of interest
No conflict of interest
Acknowledgements
CSIR, New Delhi is acknowledged for the funding through Open Source Drug Discovery
(OSDD) program and NCL-IGIB joint collaborative project (BSC-0124). We thank Prof. Samir
Brahmachari and Open Source Drug Discovery (OSDD) team at CSIR head quarters, in
particular, Dr. Geethavani Rayasam, Dr. Haridas Rode, Dr. Tanjore Balganesh, Dr. Bheemarao
Ugarkar, Dr. Anshu Bhardwaj and Dr. Zakir Thomas for useful discussions in this program. R.R,
and A.A. thank CSIR, New Delhi and R.D. thank UGC, New Delhi for the award of fellowships.
Dr. C.V. Ramana, OSDD coordinator at CSIR-NCL for his support and cooperation. We
acknowledge Miss. Chandanisingh, summer trainee as part of OSDD for her help in initial
experiments.
Abbrevations
MIC, Minimum Inhibitory Concentration; ADME, absorption, distribution, metabolism,
excretion; CB1, Cannabinoid receptor Type 1; Mtb, Mycobacterium tuberculosis; CNS, Central
nervous system; TBAF, Tetrabutylammonium fluoride; INH, Isoniazid; DMPK, Drug

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metabolism and pharmacokinetics; PPB, Plasma protein binding; A549 cells, human alveolar
adenocarcinoma cell line; HepG2 cells , human liver hepatocellular carcinoma cell line.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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Highlights
•
The antitubercular activity of diaryl pyrazoles (rimonabant analogues) was investigated
as part of drug scaffold repurposing.
•
•
•
From SAR analysis, silicon incorporation increased the anti-TB potential of the series.
The sila analogue 18a was the most potent compound (MIC 31 ng/mL, H37Rv).
Compound 18e (MIC 390 ng/mL) was the best in terms of in vitro pharmacokinetics.