Identification of a novel class of quinoline-oxadiazole hybrids as anti-tuberculosis agents

Article abstract of DOI:10.1016/j.bmcl.2015.11.057

A series of novel quinoline-oxadiazole hybrid compounds was designed based on stepwise rational modification of the lead molecules reported previously, in order to enhance bioactivity and improve druglikeness. The hybrid compounds synthesized were screened for biological activity against Mycobacterium tuberculosis H₃₇Rv and for cytotoxicity in HepG2 cell line. Several of the hits exhibited good to excellent anti-tuberculosis activity and selectivity, especially compounds 12m, 12o and 12p, showed minimum inhibitory concentration values <0.5 μM and selectivity index >500. The results of this study open up a promising avenue that may lead to the discovery of a new class of anti-tuberculosis agents.

Full text of DOI:10.1016/j.bmcl.2015.11.057

Accepted Manuscript
Identification of a novel class of Quinoline-Oxadiazole hybrids as anti-tuber-
culosis agents
Puneet P. Jain, Mariam S. Degani, Archana Raju, Aarti Anantram, Madhav
Seervi, Sadhana Sathaye, Muktikanta Ray, M.G.R. Rajan
PII:
S0960-894X(15)30265-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.11.057
BMCL 23313
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 May 2015
29 October 2015
17 November 2015
Please cite this article as: Jain, P.P., Degani, M.S., Raju, A., Anantram, A., Seervi, M., Sathaye, S., Ray, M., Rajan,
M.G.R., Identification of a novel class of Quinoline-Oxadiazole hybrids as anti-tuberculosis agents, Bioorganic &
Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.057
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Identification of a novel class of Quinoline-Oxadiazole hybrids as anti-
tuberculosis agents
a
a,
a
a
a
Puneet P. Jain , Mariam S. Degani *, Archana Raju , Aarti Anantram , Madhav Seervi ,
a
b
b
Sadhana Sathaye , Muktikanta Ray , M. G. R. Rajan
a
Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019, India
Radiation Medicine Centre, Tata Memorial Hospital, Parel, Mumbai 400012, India
b
*
Corresponding author:
Prof. (Mrs.) Mariam S. Degani, Tel.: +91-22-3361-2213; Fax: +91-22-3361-1020; E-mail:
ms.degani@ictmumbai.edu.in
Abstract:
A series of novel quinoline-oxadiazole hybrid compounds was designed based on stepwise
rational modification of the lead molecules reported previously, in order to enhance
bioactivity and improve druglikeness. The hybrid compounds synthesized were screened for
biological activity against Mycobacterium tuberculosis H Rv and for cytotoxicity in HepG2
37
cell line. Several of the hits exhibited good to excellent anti-tuberculosis activity and
selectivity, especially compounds 12m, 12o and 12p, showed minimum inhibitory
concentration values < 0.5 µM and selectivity index >500. The results of this study open up a
promising avenue that may lead to the discovery of a new class of anti-tuberculosis agents.
Keywords:
Mycobacterium tuberculosis; Isosterism; Oxadiazole; Quinoline; Bedaquiline.
1

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), is estimated to
rd
cause latent infection in 1/3 of the world’s population, with 5-10% lifetime risk of
reactivation. It is one of the leading causes of mortality, especially in low- and middle-
income countries, with approximately 1.5 million deaths and 9 million new TB cases reported
in 2013. Moreover, every fourth death among HIV positive cases is a consequence of TB.
Even though a sustained decline in the TB death rate has been observed since 1990, there is
an alarming increase in the number of multidrug-resistant tuberculosis (MDR-TB) cases since
2
009. An estimated 480,000 new MDR-TB cases were recorded in 2013, covering virtually
every single country surveyed in the world. Such forms of TB do not respond to the standard
six monthly treatment with first-line anti-TB drugs and can take two years or more to treat
1
,2
with more toxic and less effective second-line drugs. This along with recent emergence of
extensively drug resistant TB (XDR-TB) and totally drug resistant TB (TDR-TB), are raising
3
serious questions about the effectiveness of the existing therapies in the near future. The
challenge in controlling this serious and deadly form of TB is reinforcing the need for
4
discovery and development of new anti-TB agents.
During the last decade, persistent efforts in this direction have led to several lead molecules
progressing to clinical trials. Such efforts were successful, when after 40 years, a new drug,
bedaquiline (I), from the diarylquinoline class, with a novel mechanism of action, was
5
approved by U.S. Food and Drug Administration (US-FDA) for treatment of MDR-TB.
Bedaquiline demonstrates potent anti-TB activity with a minimum inhibitory concentration
(MIC) of 0.1 μM. It is also active against variety of strains which are resistant to several of
6
the first-line drugs. Because of this success, many research groups initiated active work on
modifications of the quinoline scaffold, towards the design of promising anti-tuberculosis
7
–10
agents (Figure 1).
In 2009, Kozikowski et al, rationally designed isoxazole-quinoline hybrid compounds using
molecular hybridization of mefloquine and an isoxazole containing hit obtained from a high
throughput screening (HTS). This highly active series of compounds exhibited potent activity
against both active as well as latent forms of tubercle bacilli, with the most active compound,
II, showing MIC of 0.2 μM. Although the molecules are reported to be metabolically
8
unstable, it is no doubt an attractive lead scaffold for rational drug designing.
2

Figure 1: Recently reported quinoline based potent anti-TB agents
In the present work, we report the design of novel Quinoline-Oxadiazole hybrid (QOH)
compounds by step wise rational modification of the lead molecule, II, to obtain a series of
1
9 compounds with potent activity against Mtb H Rv (Figure 2).
37
Earlier reports indicate Oxadiazoles, due to their electronic properties, impart metabolic
1
1
stability and enhanced bioavailability to the designed molecules. It is also known to
12
improve the binding affinity and the bioactivity on replacement with isoxazole. Also, due to
its higher developability score, it can be preferred over the isoxazole ring during drug
1
3
design. Thus, to improve the druglikeness of the designed molecules, isooxazole ring of II
was isosterically replaced by 1,2,4-oxadiazole ring to get intermediate design, IV. The
metabolically unstable ethyl ester functionality was replaced with diverse aryl, heteroaryl and
aliphatic substitution (R) to check the effect of electronic and hydrophobic parameters on the
biological activity.
Likewise, the quinoline scaffold is regarded as a privileged structure due to its frequent
appearance in bioactive substances displaying diverse pharmacological activities including
1
4
anti-TB activity. In addition, newly approved anti-TB drug, bedaquiline (I) and molecules
from our earlier work (III) suggest that 2-methoxy-3-substituted quinolines tend to show
1
0
promising anti-TB activity. Accordingly, the design IV, with substitution on 4-position of
quinoline ring was shifted to 3-position of 2-methoxy quinoline giving the proposed QOH
design (V).
3

Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
1
5
Prior to synthesis, an in silico ADME/Tox prediction was performed using QikProp , a
program that predicts several significant physical descriptors and relevant pharmaceutical
properties of organic molecules, to check the druglikeness of the QOH compounds. The
predicted properties calculated were partition coefficient, Lipinski’s rule of five, human oral
absorption, CNS activity and gut-blood barrier permeability. All the QOH compounds passed
the ADME/Tox analysis (supplementary information), indicating that the designed molecules
have drug-like properties. In addition, an in silico site of metabolism (SoM) prediction of
1
6,17
representative QOH compounds was performed using MetaPrint2D.
The result suggests
that QOH compounds must be metabolically more stable in comparison to II.
The QOH compounds, 12a-12s were synthesized according to Scheme 1. The starting
material, 2-chloro-3-formylquinoline (5) was synthesized by reacting acetanilide (4) with
freshly prepared Vilsmeier-Haack reagent (3) under Meth-Cohn quinoline synthesis
18
conditions. 3 was prepared in-situ by reacting N,N-dimethylformamide (1) with excess of
phosphorous oxychloride (2). Methanolysis of 5, by refluxing in methanolic potassium
1
9
hydroxide solution led to synthesis of 2-methoxy-3-formylquinoline (6) in good yield.
Subsequently, 3-(2-methoxyquinolin-3-yl)acrylonitrile (7) was synthesized by Knoevenagel
condensation of 6 and cyanoacetic acid in toluene, in the presence of catalytic amount of
4

2
0,21
ammonium acetate and pyridine as base.
Treatment of 7 with hydroxylamine
hydrochloride by refluxing in 90:10 MeOH/H O and using sodium bicarbonate as the base
2
2
2
resulted in amidoxime (8), one of the key intermediates. The other key intermediates, N-
acylbenzotriazoles (10a-s) were synthesized by the reaction of corresponding acids (9a-s),
2
3
1
H-benzotriazole and thionyl chloride in a one-pot fashion.
Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12a-
s).Reagents and Conditions: (a) Vilsmeier-Haack Reagent (3) in POCl , 80°C, 16.5h; (b)
3
MeOH, KOH, reflux, 3-4h; (c) Cyanoacetic acid, ammonium acetate, pyridine, toluene,
Dean-Stark trap, reflux, 44h; (d) NH OH, NaHCO , 90:10 MeOH/H O, reflux, 8-12h; (e)
2
3
2
SOCl , CH Cl , RT, 2h; (f) N-acylbenzotriazole (10a-s), EtOH, TEA, RT, 30min; (g) TEA,
2
2
2
EtOH/n-BuOH, reflux, 8-12h.
5

QOHs (12a-s) were synthesized by refluxing corresponding 10 and 8 in either ethanol or
butanol, employing triethylamine as the mild base. The reaction proceeds via carbamate
intermediate (11), which was synthesized at room temperature. These carbamate
intermediates (11a-s) were isolated. When subjected to refluxing alcohol in presence of
2
4
triethylamine, these intermediates led to corresponding QOHs. Structures of all QOH
compounds (12a-s) were characterized by FTIR, NMR and HRMS analysis. The analytical
results were in agreement with the proposed structures.
All 19 QOH compounds (12a-s) were screened for in vitro anti-TB activity against
Mycobacterium tuberculosis H Rv by employing the Resazurin Microplate Assay (REMA)
3
7
2
5,26
with drug concentrations from 50 µg/mL to 0.1 µg/mL (Table 1).
Isoniazid was used as
the standard drug. The compounds show good to excellent activity, especially the aliphatic or
alkyl linked aromatic derivatives (12l - 12p) with MIC 0.4 – 1.05 µM, showing in vitro
bioactivity greater than isoniazid.
The compounds (12a-s) were also screened for in vitro cytotoxicity evaluation against HepG2
cell line, employing sulforhodamine B (SRB) assay using drug concentration from 10 µg/mL
to 80 µg/mL. The serial microdilution technique was optimized for the toxicity screening of
27
compounds to determine 50% cytostatic concentration (CC ). Adriamycin was used as the
50
standard drug. All the compounds (12a-s) showed CC values >200 µM. Compound 12m
5
0
displayed the highest SI of >610, indicating that this compound is very selective in its action
against Mtb, exhibiting substantially low cytotoxicity towards human cells.
Table 1: Biological activity of QOH compounds
a
b
MIC (µM)
CC (µM)
50
Mol. ID
R
SI
(n = 3)
(n = 3)
1
2a
2b
2c
2d
phenyl
12.3
>240
>19.5
>5.75
>1.57
>21.2
1
4-methylphenyl
4-methoxyphenyl
4-bromophenyl
40.0
>140
9.19
>230
>220
>195
1
1
6

1
2e
2f
4-fluorophenyl
2-methylphenyl
2-methoxyphenyl
2-bromophenyl
3-methylphenyl
3-methoxyphenyl
3-chlorophenyl
methyl
5.61
72.8
6.96
16.8
110
>230
>230
>220
>195
>230
>220
>220
>300
>270
>220
>230
>215
>225
>240
>250
-
>41.0
>3.16
>31.6
>11.6
>2.09
>2.10
>2.0
>285
>610
>330
>520
>535
>35.5
>1.60
>9.12
-
1
1
1
2g
2h
1
1
2i
2j
105
1
2k
2l
2m
>110
1.05
0.44
0.67
0.44
0.40
6.33
>150
27.4
0.73
-
1
1
methoxymethyl
phenoxymethyl
benzyl
1
2n
2o
2p
2q
1
1
1
4-methoxybenzyl
(E) styryl
1
2r
3-pyridyl
1
2s
2-furo
INH
-
ADR
-
127
-
*
n = number of repetitions; INH = Isoniazid; ADR = Adriamycin
a
MIC was determined with a drug concentration from 50µg/mL to 0.1µg/mL.
b
CC values is the concentration required to reduce the cell viability by 50%, also termed as
50
5
0% cytostatic concentration. CC were not evaluated for concentrations >80µg/mL due to
50
low solubility of QOH compounds in DMSO at higher concentrations.
A representative QOH derivative, 12m, with the highest SI was subjected to metabolic
2
8–30
stability study in rat liver microsomes.
The disappearance of test compound was
monitored over a period of 60 minutes. Diazepam was used an internal standard (IS). Drug to
IS ratio were calculated to assess the metabolic stability of 12m at different time intervals.
The resultant depletion profile (Figure 3) suggests that the compound 12m is 99%
metabolically stable up to a period of 60 minutes. Thus, in accordance with the results of the
7

in silico SOM prediction, the microsomal stability study shows that the QOH derivatives are
metabolically more stable in comparison to II.
Figure 3: Depletion profile of compound 12m in rat liver microsomes.
The biological results of QOH derivatives aided us to hypothesize a structure activity
relationship (SAR). It was interesting to note that the highly active and selective aliphatic or
alkyl linked aromatic derivatives (12l – 12p), shared a common methylene spacer at 5’-
position of 1,2,4-oxadiazole ring that differentiates them from the lesser active molecules.
Also, the molecule with highest selectivity, 12m showed a great degree of side chain
structural similarity with the lead molecule II (Figure 4), demonstrating that such similarity
improves the activity. The high to moderate activity and selectivity of ligands with alkyl,
alkoxy or alkene linked aromatic ring (12n - 12q), supports the previous statement and
suggests that a 1 to 2 atom flexible spacer in 5’-side chain improves the activity. Amongst
aromatic derivatives, halo substitution at p- and o- position (12d, 12e and 12h) seemed to
contribute significantly to the activity. Replacement of aromatic substitution with a
heteroaromatic ring (12r and 12s) decreased the activity.
8

Figure 4: Structure Activity Relationship (SAR) of QOH molecules
In conclusion, the present work reflects the successful design and synthesis of a novel series
of QOH compounds as potential anti-tuberculosis agents via step by step rational
modification of the lead molecule (II) reported previously. In vitro biological results reveal
that several of the QOH compounds exhibit significant anti-TB activity and selectivity.
Amongst the 19 QOH compounds, the hit molecule 12m exhibits the highest activity with
MIC of 0.4µM and SI of >610, which is comparable to the lead molecule (II). The SAR
results suggest that a 1 to 2 atom flexible spacer in the side chain at 5’-position and a side
chain structural similarity with the lead molecule (II) is crucial for enhancement of activity.
These results suggest that QOH scaffold may serve as a promising anti-TB hit/lead for further
drug optimization. Further investigations on this series are ongoing in our laboratory with a
view to design a potential clinical candidate.
Acknowledgements:
Puneet Jain and Aarti Anantram are thankful to University Grants Commission, India and
Archana Raju is thankful to Indian Council of Medical Research, India for financial support.
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List of Figures and Tables
Figure 1: Recently reported quinoline based potent anti-TB agents
Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
Figure 3: Rat liver microsomal stability study of compound 1
Figure 4: Structure Activity Relationship (SAR) of QOH molecules
Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12a-s).
Table 1: Biological activity of QOH compounds
1
1

Figures:
Figure 1: Recently reported quinoline based potent anti-TB agents
Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
1
2

Figure 3: Depletion profile of compound 12m in rat liver microsomes.
Figure 4: Structure Activity Relationship (SAR) of QOH molecules
1
3

Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12a-
s).Reagents and Conditions: (a) Vilsmeier-Haack Reagent (3) in POCl , 80°C, 16.5h; (b)
3
MeOH, KOH, reflux, 3-4h; (c) Cyanoacetic acid, ammonium acetate, pyridine, toluene,
Dean-Stark trap, reflux, 44h; (d) NH OH, NaHCO , 90:10 MeOH/H O, reflux, 8-12h; (e)
2
3
2
SOCl , CH Cl , RT, 2h; (f) N-acylbenzotriazole (10a-s), EtOH, TEA, RT, 30min; (g) TEA,
2
2
2
EtOH/n-BuOH, reflux, 8-12h.
1
4

Table 1: Biological activity of QOH compounds
a
b
MIC (µM)
CC (µM)
50
Mol. ID
R
SI
(n = 3)
(n = 3)
1
2a
2b
2c
2d
2e
2f
phenyl
12.3
>240
>19.5
>5.75
>1.57
>21.2
>41.0
>3.16
>31.6
>11.6
>2.09
>2.10
>2.0
1
4-methylphenyl
4-methoxyphenyl
4-bromophenyl
4-fluorophenyl
2-methylphenyl
2-methoxyphenyl
2-bromophenyl
3-methylphenyl
3-methoxyphenyl
3-chlorophenyl
methyl
40.0
>140
9.19
5.61
72.8
6.96
16.8
110
>230
>220
>195
>230
>230
>220
>195
>230
>220
>220
>300
>270
>220
>230
>215
>225
>240
>250
-
1
1
1
1
1
1
2g
2h
1
1
2i
2j
105
1
2k
2l
2m
>110
1.05
0.44
0.67
0.44
0.40
6.33
>150
27.4
0.73
-
1
>285
>610
>330
>520
>535
>35.5
>1.60
>9.12
-
1
methoxymethyl
phenoxymethyl
benzyl
1
2n
2o
2p
2q
1
1
1
4-methoxybenzyl
(E) styryl
1
2r
3-pyridyl
1
2s
2-furo
INH
-
ADR
-
127
-
*
n = number of repetitions; INH = Isoniazid; ADR = Adriamycin
a
MIC was determined with a drug concentration from 50µg/mL to 0.1µg/mL.
1
5

b
CC values is the concentration required to reduce the cell viability by 50%, also termed as
50
5
0% cytostatic concentration. CC were not evaluated for concentrations >80µg/mL due to
50
low solubility of QOH compounds in DMSO at higher concentrations.
1
6

Graphical abstract
1
7