Biarylmethoxy nicotinamides as novel and specific inhibitors of mycobacterium tuberculosis

Article abstract of DOI:10.1021/ml4004815

A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of M. tuberculosis (Mtu) H37Rv. The series exhibited tangible structure-activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 μM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action.

Full text of DOI:10.1021/ml4004815

Letter
pubs.acs.org/acsmedchemlett
Biarylmethoxy Nicotinamides As Novel and Specific Inhibitors of
Mycobacterium tuberculosis
Chaitanya Kumar Kedari,‡ Nilanjana Roy Choudhury,† Sreevalli Sharma,† Parvinder Kaur,†
Supreeth Guptha,† Manoranjan Panda,† Kakoli Mukerjee,‡ Vasanthi Ramachandran,†
Balachandra Bandodkar,‡ Sreekanth Ramachandran,† and Subramanyam J. Tantry*,†
†AstraZeneca India Pvt., Ltd., Avishkar, Bellary Road, Bangalore 560024, India
‡Alkem Laboratories, Ltd., Peenya Industrial Estate, Bangalore 560058, India
S
* Supporting Information
ABSTRACT: A whole cell based screening effort on a focused library from corporate
collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors
of M. tuberculosis (Mtu) H37Rv. The series exhibited tangible structure−activity
relationships, and during hit to lead exploration, a cellular potency of 100 nM was
achieved, which is an improvement of >200-fold from the starting point. The series is very
specific to Mtu and noncytotoxic up to 250 μM as measured in the mammalian cell line
THP-1 based cytotoxicity assay. This compound class retains its potency on several drug
sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of
action.
KEYWORDS: MMIC, biarylmethoxy nicotinamides, anti-TB, clinical isolates
molecules such as delamanid, PA824, SQ-109, Moxifloxacin,
uberculosis (TB) caused by Mycobacterium tuberculosis
T(Mtu) is a highly contagious disease and continues to and AZD5847 are in clinical trials.10 Despite these advances
being made, there are significant challenges associated with the
target product profile for new TB-drugs, which involve tackling
the emergence of resistance, shortening of treatment period,
drug−drug interactions, intolerance, and toxicities, which
demand immediate need for new chemical entities for anti-
TB with novel mode of action.11 Various approaches such as
modifying existing scaffolds, target based, and whole-cell based
screening are followed in TB drug discovery. While the target
based approach appears to be lucrative in terms of tracking the
mode of action MOA, translation of enzyme potency into
cellular activity is the major hurdle. Whole-cell based approach,
however, has antimycobacterial activity already embedded, and
the advances made in genomics have provided the necessary
platform to delayer the MOA.12,13 In fact, recent success
involving Bedaquiline targeting mycobacterial ATP synthase
and BTZ targeting cell wall protein DprE1 have originated out
of whole-cell based approaches.
In our quest to identify new and novel scaffold with
antimycobacterial activity, we ran a whole-cell based screen on a
focused library from AstraZeneca compound collection.
Biarylmethoxy nicotinamide is one of the promising series
that emerged out of this screening. The initial hits were weak in
terms of cellular potency, and a subsequent optimization effort
resulted in about 200-fold improvement. Herein, we report the
chemical optimization of biarylmethoxy nicotinamides.
remain the second leading killer of adult population after HIV.
In 2010, there were 8.8 million incident cases of TB and 1.45
million deaths. TB primarily affects the lungs, but it can also
affect other organs such as the central nervous system,
lymphatic system, and circulatory system.1,2 A deadly synergy
between HIV and TB poses new challenges to healthcare
system, causing rapid progression and reactivation of latent-TB.
Reactivation increases the TB disease burden by an estimated
20% in African and Asian countries where HIV prevalence is
high.3,4 A standard regimen involving a combination of four
drugs, Rifampin, Isoniazid, Ethambutol, and Pyrazinamide for
6−8 months developed decades ago5 needs urgent back-up
with better efficacy, reduced toxicity, and effective coverage on
latent tuberculosis infection (LTBI).6 Emergence of resistant
strains, multidrug resistant (MDR-TB) and extensively drug
resistant (XDR-TB) has taken global proportions with
prevalence of MDR-TB ranging from anywhere between 5
and 47% and 9% of the MDR-TB cases leading to XDR-TB. It
is estimated that, by 2014, 84 countries would have at least
reported one XDR-TB case.7,8 Significant strides have been
made in the past two decades in terms of understanding the
biology of this slow-growing pathogen. The complete genome
sequence of MtuH37Rv was solved by Stewart Cole et al.9 This
has facilitated the process of identifying novel chemical classes
through new druggable targets, understanding their biological
role, essentiality, and determination of point mutations
associated with drug resistance.
Received: November 26, 2013
Accepted: March 10, 2014
Published: March 10, 2014
Recently, FDA approved the J&J compound Bedaquiline
(SIRTURO) for MDR-TB therapy. Few other advanced
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
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To understand the structure−activity relationship (SAR), the
scaffold was broadly divided into rings A, B, and C (Figure 1).
hydride (LAH). Debromination was one of the major side
products formed during the synthesis of 6. However,
debromination could be kept at minimum when the reduction
was carried out at 0 °C. This reaction was closely monitored
and was quenched immediately after completion. Suzuki
coupling of intermediate 7 with commercially available boronic
acids following standard protocol resulted in intermediate 8.
We found that the phenolic hydroxyl group does not need
protection during the reaction sequence and can be substituted
readily with various alkyl bromides to give the title compounds
9a−k (Table 1).
A series of amide analogues 15a−h were synthesized (Table
2) to investigate the function of amide on antimycobacterial
activity and aqueous solubility. The route followed to
synthesize these compounds is illustrated in Scheme 3.
Synthesis starts with alkylation of 4-hydroxy-2-methyl bromo-
benzene with 1-bromo-2-methoxy ethane. Quantitative yield
was obtained for intermediate 11 when the coupling was
carried out at 50 °C in the presence of potassium carbonate.
Intermediate 12 was synthesized following Suzuki protocol
using intermediate 11 and 6-(hydroxymethyl)pyridin-3-ylbor-
onic acid in about 36% yield. The biaryl methanol 12 was
subjected to nucleophilic displacement of chloride group on
methyl 2-chloro nicotinate, and the resulting ester intermediate
13 was hydrolyzed using lithium hydroxide in dioxane to result
in 14. Title compounds 15a−h (Table 2) was obtained in
about 30−50% yield by simple amide coupling with the
corresponding amine using commonly employed coupling
agent HATU.
Figure 1. SAR of biarylmethoxy nicotinamides.
Compounds involving ring A and ring B modifications were
synthesized starting from their corresponding boronic acids
using Suzuki coupling protocol. The synthetic route followed is
represented in Scheme 1.
Scheme 1. Synthesis of Biarylmethoxy Nicotinamide: Ring A
a
and B Modifications
The effect of various substitutions on ring A and the optimal
substitutions at the X1 and X2 positions were examined. The
SAR of these modifications is summarized in Table 1. The
results indicate that the presence of 2, 4-disubstituted phenyl
ring is essential for cellular potency. Heterocyclic rings such as
imidazolyl 5f and pyridyl 5g are inactive as compared to their
match pair 5b. A greater than 10-fold improvement in potency
could be achieved by extending 4-methoxy of 5b to two carbon
4-(2-methoxy) ethyl side chain 9a. An interesting observation
could be made out of compounds 9c−e, the S-isomer appears
to be slightly more potent than the R-isomer. Lipophilic, 2-
chloro substituent is an ideal replacement for 2-methyl (9i−k)
and a combination of 2-chloro and 4-(3-fluoro) propyl side
chain drastically improved the potency by about 10-fold,
resulting in the potent compounds of the series, 9j and 9k.
The details of structural modification carried out on ring C
are shown in Table 2. Throughout this study, 4-(2-
methoxyethoxy)-2-methylphenyl (ring A) and pyridyl (ring
B) were kept constant, and modifications were made only on
ring C. Preliminary changes were directed toward under-
standing the requirement of primary amide. In the process,
secondary amide 15a and tertiary amides 15b,c using methyl
amines, dimethyl amines, and 3-hydroxypyrolidine, respectively,
were synthesized. The results indicate that the donor−acceptor
pair of the amide functionality is very crucial for mycobacterial
activity, and the compounds 15b,c that lacks this were inactive.
Other modifications such as sulphonamides 15f and pyrazine
15g are also tolerated albeit with less cellular potency. Ring C
can also be utilized for improving solubility of the series.
Introduction of secondary amide such as 15a increased the
solubility by more than 35-fold without compromising the
cellular potency.
a
Reagents and conditions: (I) Ar−B(OH)2, PdCl2(dppf)−CH2Cl2,
CsF, MeOH, MW at 120 °C, 30 min, yield 55−70%; (II) LiAlH4,
THF, 0−25 °C, 30 min, yield 35−50%; (III) 2-chloronicotinamide,
NaH (60% suspension in paraffin oil), DMF, 100 °C, 2 h, yield 60−
80%.
Optimum results were obtained when the reactions were
performed under microwave conditions using PdCl2(dppf)−
CH2Cl2 and CsF. Reduction of the biaryl carboxylates 3 by
careful addition of lithium aluminum hydride resulted in
intermediate 4. The yield of 4 was moderate and was improved
when the reducing agent was added at 0 °C. Nucleophilic
displacement of commercially available 2-chloro nicotinamide
with 4 using sodium hydride resulted in title compounds 5a−e
(Table 1).
However, the synthetic route mentioned in Scheme 1 was
not feasible for the synthesis of compounds 9a−k (Table 1).
These compounds were synthesized using an alternative
protocol as shown in Scheme 2. Nucleophilic displacement of
2-chloronicotinamide with corresponding alcohol 6 resulted in
intermediate 7. Alcohol 6 was prepared by reducing the
corresponding carboxylate esters 2 with lithium aluminum
Other modifications involving aminoethanol 15d and basic
side chain such as the amino piperidine (15e) followed similar
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Table 1. Ring A Modifications and SAR
Information). The results suggest that the six-membered ring
containing 1,4 linkers are necessary to retain the cellular
potency, and any deviation from this symmetry results in
inactive compounds 20 and 22. The flexible oxymethylene
bridge appears to be equally important for cellular potency. An
attempt to swap hydrogen bond acceptor with donor 17 and
restricting the rotation of flexible methylene bridge by
introducing a methyl group 21 resulted into loss of cellular
potency.
Scheme 2. Synthesis of Alkoxy Nicotinamide: Ring A
Modification at the 2nd and 4th Positions
a
It is important to assess the potential toxicity of these
compounds given the long duration of TB treatment. TB being
mainly an intracellular infection involving human macrophages,
the THP-1 cell line was selected for the in vitro cytotoxicity
studies. Further, THP-1 is also being used as a macrophage
model to examine intracellular growth rates of Mtu.14 The data
in Tables 1 and 2 indicate good selectivity between Mtu and
eukaryotic THP-1 cell line. Some of the best compounds in the
series did not show cytotoxicity up to 250 μM (9i−k), thus
demonstrating excellent selectivity.
Compounds 9b, 9f, 9j, and 9k were tested on a broad
spectrum panel involving gram positive, gram negative, and
eukaryotic pathogens. The results show that these compounds
do not have MIC up to 60 μM, indicating specificity to Mtu
(Table 3). This also suggests that these compounds may be
inhibiting the target, which is specific to Mtu.
a
Reagents and conditions: (I) NaH (60% suspension in paraffin oil),
DMF, 110 °C, 2 h, yield 60−85%; (II) PdCl2 (dppf)−CH2Cl2 (5 mol
%), CsF, MeOH, MW at 120 °C, 30 min, yield 60−70%; (III) R3-Br,
K2CO3, DMF, 110 °C, 2 h, yield 40−79%.
trends, although with marginal decrease in potency (for
solubility data, see Supporting Information Table S7).
In order to understand the requirement of ring B and
oxymethylene linker, key compounds were synthesized, and the
summary of those changes are given in Table S4 (Supporting
The representative potent compounds were profiled for their
in vitro drug metabolism and pharmacokinetics (DMPK)
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Table 2. Ring C Modifications and SAR
Scheme 3. Synthesis of Alkoxypyridine Nicotinamide: Ring
C Modifications
a
a
Reagents and conditions: (I) 1-bromo-2-methoxyethane, K2CO3,
DMF, 50 °C, 30 min, yield 95%; (II) 6-(hydroxymethyl)pyridin-3-
ylboronic acid PdCl2(dppf)−CH2Cl2 (5 mol %), CsCO3, MeOH, MW
at 120 °C, 45 min, yield 36%; (III) methyl, 2-chloronicotinate, NaH
(60% suspension in paraffin oil), DMF, 120 °C, 2 h, yield 54%; (IV)
LiOH, 1,4-dioxane, r.t., 18 h, yield 94%; (V) HATU, DIEA, DMF, r.t.,
2 h, yield 30−50%.
Table 3. Studies on Broad Spectrum Panel
9b MIC
(μM)
9f MIC
(μM)
9j MIC
(μM)
9k MIC
(μM)
species
M. tuberculosis
H37Rv
1.5
1.1
0.1
0.1
E. coli
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
H. influenzae
S. aureus
S. pneumoniae
P. aeruginosa
K. pneumoniae
S. pyogenes
C. albicans
properties (see Supporting Information Table S5). The log D
of these compounds ranged from 2.8 to 3.4. The intrinsic
clearance determined from mouse microsomes showed low to
moderate values. Among the representatives tested for human
plasma protein binding (PPB), 9a had the best free fraction at
4.2%. These compounds were also subjected to screening
against a panel of cytochrome P450 isozymes (CYP) to
understand the possible drug−drug interaction issues. The
most potent compounds 9j and 9k did not inhibitthe majority
of the isozymes tested except CYP2C19 and CYP2C9, which
had an IC50 of 18 and19 μM, respectively. Compound 9a,
which is marginally less potent, did not inhibit any of the
isozymes tested.
These compounds were further tested on a panel of drug
sensitive and single drug resistant clinical isolates (see
Supporting Information Table S6). The compounds 9a, 9j,
and 9k retain their cellular potency against all drug-sensitive
strains and were found to be within acceptable 4-fold variation
in MIC values. In the case of single drug resistant strains, the
compounds retain their potency across all the five strains. The
significance of potency variation of greater than 4-fold with
certain resistant strains will only be confirmed after target link is
established. However, the results indicate this class of molecules
is likely to be effective against a wide range of clinical isolates
and is likely to act through a novel mode of action.
In conclusion, we have shown biarylmethoxy nicotinamides
as an attractive and novel chemotype with antimycobacterial
activity. The medicinal chemistry optimization resulted in
compounds with excellent potency and good selectivity index
toward eukaryotic cell lines. The series exhibited tangible SAR
and was very specific to Mtu. We believe that this class of
molecules could be acting through a novel mode of action and
would be more likely to be effective against a wide range of
clinical isolates. Studies are underway to identify the target and
decipher the mechanism of action of this class of compounds.
In vivo profiling efforts are in progress to optimize
pharmacokinetic properties for demonstrating efficacy in an
animal model.
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Letter
A.; Rogers, J.; Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares,
S.; Sulston, J. E.; Taylor, K.; Whitehead, S.; Barrell, B. G. Deciphering
the biology of Mycobacterium tuberculosis from the complete genome
sequence. Nature 1998, 393, 537−544.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and compound characterizations. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
(10) Zumla, A.; Nahid, P.; Cole, S. T. Advances in the development
of new tuberculosis drugs and treatment regimens. Nature 2013, 12,
388−404.
(11) Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K.
Challenge of new drug discovery for tuberculosis. Nature 2011, 469,
469−490.
(12) Payne, D. J.; Gwynn, N. M.; Holmes, D. J.; Pompliano, D. L.
Drugs for bad bugs: confronting the challenges of antibacterial
discovery. Nat. Drug Discovery Rev. 2007, 6, 29−40.
AUTHOR INFORMATION
Corresponding Author
*(S.J.T.) Tel: + 91 080 23621212. Fax: + 91 080 23611214. E-
mail: subramanyamj.tantry@astrazeneca.com.
■
Author Contributions
(13) Chan, P. F.; Holmes, D. J.; Payne, D. J. Finding the gems using
genomic discovery: antibacterial drug discovery strategies: The
successes and the challenges. Drug Discovery Today 2004, 4, 519−527.
(14) Theus, S. A.; Cave, M. D.; Eisenach, K. D. Activated THP-1
cells: an attractive model for the assessment of intracellular growth
rates of Mycobacterium tuberculosis isolates. Infect. Immunol. 2004, 72,
1169−1173.
S.J.T., M.P., S.R., and V.R. drafted/corrected the manuscript
and participated in the design and execution of this study.
N.R.C. and C.K. performed the chemical syntheses. S.S., P.K.,
and S.G. performed and interpreted microbiological data. K.M.
was the lead biologist. B.B. contributed in scientific discussions.
Funding
Funding was provided by NM4TB consortium.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank NM4TB consortium for cofunding the
project. The authors would also like to thank Dr. T. Balganesh,
Dr. B. Ugarkar, Dr. Balasubramanian, and Dr. V. Sambanda-
murthy for valuable suggestions and encouragement during the
course of project. The authors acknowledge and appreciate Dr.
S. Kavanagh for helping in getting the THP-1 cytotox data for
project compounds and Dr. P. Shirude for thoroughly
reviewing the manuscript. Lastly, the authors acknowledge
excellent commitment by S. Rudrapatna, J. Sandesh, and S.
Menasinakai for timely support in purification and analysis of
samples.
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