Quinolinyl pyrimidines: Potent inhibitors of NDH-2 as a novel class of anti-TB agents

Article abstract of DOI:10.1021/ml300134b

NDH-2 is an essential respiratory enzyme in Mycobacterium tuberculosis (Mtb), which plays an important role in the physiology of Mtb. Herein, we present a target-based effort to identify a new structural class of inhibitors for NDH-2. High-throughput screening of the AstraZeneca corporate collection resulted in the identification of quinolinyl pyrimidines as the most promising class of NDH-2 inhibitors. Structure-activity relationship studies showed improved enzyme inhibition (IC₅₀) against the NDH-2 target, which in turn translated into cellular activity against Mtb. Thus, the compounds in this class show a good correlation between enzyme inhibition and cellular potency. Furthermore, early ADME profiling of the best compounds showed promising results and highlighted the quinolinyl pyrimidine class as a potential lead for further development.

Full text of DOI:10.1021/ml300134b

Letter
pubs.acs.org/acsmedchemlett
Quinolinyl Pyrimidines: Potent Inhibitors of NDH-2 as a Novel Class
of Anti-TB Agents
Pravin S. Shirude,* Beena Paul, Nilanjana Roy Choudhury, Chaitanya Kedari, Balachandra Bandodkar,
and Bheemarao G. Ugarkar
Department of Medicinal Chemistry, AstraZeneca India Pvt. Ltd., Avishkar, Bellary Road, Bangalore-560024, India
S
* Supporting Information
ABSTRACT: NDH-2 is an essential respiratory enzyme in
Mycobacterium tuberculosis (Mtb), which plays an important
role in the physiology of Mtb. Herein, we present a target-
based effort to identify a new structural class of inhibitors for
NDH-2. High-throughput screening of the AstraZeneca
corporate collection resulted in the identification of quinolinyl
pyrimidines as the most promising class of NDH-2 inhibitors. Structure−activity relationship studies showed improved enzyme
inhibition (IC50) against the NDH-2 target, which in turn translated into cellular activity against Mtb. Thus, the compounds in
this class show a good correlation between enzyme inhibition and cellular potency. Furthermore, early ADME profiling of the
best compounds showed promising results and highlighted the quinolinyl pyrimidine class as a potential lead for further
development.
KEYWORDS: antituberculosis, respiratory chain, NDH-2, quinolinyl pyrimidines
he contributory driving force of tuberculosis (TB) is
that this enzyme is not found in the mammalian mitochondria. The
TMycobacterium tuberculosis (Mtb), a leading killer world- fundamental role of NDH-2 in Mtb respiration is supported by a
wide range of biochemical18 and transcriptional studies.19 The Mtb
genome contains two copies of ndh genes (ndh and ndhA). The Mtb
NDH-2 and NDH-2A share 67% sequence identity. A strain of Mtb
in which ndh has been disrupted by transposon mutagenesis is
nonviable;17,20 however, a ndhA deletion mutant of Mtb can be
easily isolated.17,21 The effect of ndh deletion mutant lacking NDH-2
on bacterial growth under various culture conditions and on animal
infection has been reported.18
Phenothiazines (a class of antipsychotic drugs) are reported to
be inhibitors of NDH-2.17 This class of compounds is known to
inhibit the growth of bacteria in vitro as well as bacteria inside
macrophages. Thus, NDH-2 appears to be a druggable target.17
However, phenothiazines cannot be used as antibacterial agents
as they exhibit potent CNS activities at doses much lower than
those required for antibacterial activity.22
Therefore, we embarked upon finding a new scaffold through
screening of a library of 100K compounds selected from the AZ
corporate collection using Mtb NDH-2 enzyme assay in high-
throughput screening (HTS) format. The enzyme assay mix
contained 1 nM purified Mtb NDH-2 protein (Supporting
Information), 100 mM NaCl, 0.008% Brij-35, and 300 μM
NADH in 50 mM Hepes-NaOH buffer, pH 7.5. The reaction was
started by addition of 50 μM menadione, and the reaction mix
was incubated at 37 °C for 1 h. A decrease in the absorbance at
340 nm as a result of NADH oxidation during the assay was
monitored using an UV spectrophotometer. During screening,
wide that currently infects one-third of the human population.1
The World Health Organization (WHO) reports 2 million
deaths every year.2−4 Only 2−23% of individuals infected with
Mtb carry lifetime risk of developing an active disease.2−4 The
risk, however, radically increases if the carrier's immune system is
suppressed. In spite of a 6 month long, four drug combination
therapy (rifampicin, isoniazid, pyrazinamide, and ethambutol)
for the treatment of TB, multiple drug resistant (MDR) TB with
resistance to isoniazid and rifampicin is reported in 5−10% of
cases.2−4 Novel drugs for TB are essential to reduce the duration
of treatment of drug-sensitive as well as drug-resistant TB, to
impact the rate of transmission of this disease. Novel and shorter
treatment regimens could improve compliance and reduce the
emergence of drug resistance.
Because Mtb can exist in both actively replicating and
nonreplicating phases, it is essential for a novel anti-TB agent
to have activity against both of these populations. Respiration, being
an essential pathway in any physiological state, could be a potentially
high value target.5 The well-known example is ATP synthase, which
is the target of TMC-207, a clinical candidate that has comparative
killing efficiency against replicating and nonreplicating Mtb.5
TMC-207 is currently in phase II clinical studies in patients with
multidrug-resistant TB.6 Similarly, type II NADH-dehydrogenase
(NDH-2) is an essential respiratory enzyme in Mtb having a
significant role in the physiology of Mtb. The enzyme has been
characterized in many species like Saccharomyces cerevisiae,7
Escherichia coli,8,9 Bacillus subtilis,10 Methyloccocus capsulatus,11
Corynebacterium glutamicum,12,13 Acidianus ambicalens,14,15 and
Sulfolobus metallicus.16 It is composed of a single polypeptide chain,
comprising a flavin as an exclusive cofactor.17 It is worth mentioning
Received: May 30, 2012
Accepted: August 13, 2012
© XXXX American Chemical Society
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a
Table 1. In Vitro Evaluation of Compounds
Figure 1. SAR for quinolinyl pyrimidines (HAR, heteroaryl; Pyrd,
pyridine; and Pyr, pyrimidine).
Figure 2. Scatter plot of pIC50 vs pMIC.
compounds were incubated with the enzyme in the reaction mixture
prior to the addition of menadione. Thus, the screening of the 100K
library at a concentration of 20 μM resulted in 1685 hits, which had
>50% inhibition that were selected for concentration−response
studies. After the structures were removed with undesirable features,
frequent hitters, and reactive clusters, hit evaluation analysis gave five
clusters as inhibitors of NDH-2. These five clusters had IC50 values
ranging from 0.6 to 50 μM and a minimum inhibitory concentration
(MIC) between 8 and >32 μg/mL. Compounds that had the best
activity belonged to the quinolinyl-pyrimidine class. In this report,
we described our efforts to understand the structure−activity
relationship (SAR) of this scaffold and improve the biochemical
potency against the target enzyme, which in turn translates into
good cellular potency. We have also built the in vitro DMPK
properties of this scaffold to identify molecules suitable for efficacy
studies in an animal model of TB.
Our initial SAR strategy was to identify the key pharmaco-
phore required for activity. The scaffold has two main areas of
opportunity, namely, the quinoline left-hand side (LHS), and the
pyrimidine right-hand side (RHS) to explore the SAR and
structure−property relationship (SPR) (Figure 1). The results of
our studies indicate that either one or both of the primary amines
of quinoline and pyrimidine ring are critical for NDH-2 enzyme
activity as shown in Table 1, compounds 13a−j (IC50 = 0.04−0.6
μM). However, when one or both amine functions were
removed, the enzyme potency was weaker or completely lost
(compounds 14−16, IC50 = 2 to >100 μM). These results
suggest that quinolinyl pyrimidine with two primary amines is a
key pharmacophore, which may have critical hydrogen-bonding
interactions with NDH-2 target protein. To reduce the
hydrophobic requirement of the scaffold, we introduced a
number of different substituents at various positions of the
scaffold as summarized in Table 1. On the pyrimidine ring, a
monosubstituted phenyl ring (R2) was much better (compounds
13a−i) than hetero aryl groups like pyridine (13j) or pyrazole
(13o), which in turn are better than simple aliphatic groups such
as ethyl or isopropyl group (13k−m). On the quinoline ring, a
a
< and >, end points not determined.
phenyl ring (R1) with a 4-fluoro (13a) or 2-methoxy substituent
(13i) showed better potency than the unsubstituted quinoline
group (13n). This indicated the possibility of strong hydro-
phobic interactions with the protein. The potent enzyme activity
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The synthetic strategy involved a convergent approach in which
the two main fragments, namely, aminoquinoline and amino
pyrimidines, were separately synthesized and condensed. Sub-
stituted quinoline-4,6-diamine (8) was synthesized from commer-
cially available starting materials as described in Scheme 1.
Condensation of substituted ketone (1) with diethyl carbonate
(2) gave the corresponding oxopropanoates (3), which on further
treatment with 4-nitroaniline gave the uncyclised products (4).
These were cyclized in Dowtherm A to the substituted
6-nitroquinolin-4-ol derivatives (5). This was followed by conversion
to the 4-chloro (6)and4-azido(7) derivatives by treating with POCl3
and sodium azide, respectively. The 4-azido derivative was reduced to
give the quinoline-4,6-diamines (8).23 The key hydroxy-pyrimidine
(11) derivatives were synthesized using a modified literature
procedure by treating guanidine hydrochloride (9) with various
β-ketoesters (10) in the presence of sodium hydride. The
resulting hydroxy-pyrimidines were converted to their correspond-
ing triflates, tosylates, mesylates, or chlorides (12). Triflates were
prepared by treating the hydroxyl derivative with trifluorometha-
nesulfonic anhydride, trifluoromethanesulfonyl chloride, or N-
phenylbis (trifluoromethanesulfonimide). Chloro derivatives were
prepared by treatment with POCl3, PCl5, or other chlorinating
agents. Pyrimidin-4-yl-quinoline-diamine (13) was synthesized by
nucleophilic substitution or coupling reaction of the activated
pyrimidines and quinolinediamines in presence of catalytic HCl.
In conclusion, we have discovered a new class of compounds that
target the NDH-2 enzyme with promising attributes of synthetic
accessibility, good SAR, and antitubercular activity. This class has
good in vitro DMPK properties, which can be further improved
through further analogue generation and systematic medicinal
chemistry evaluation. With new antiTB agents desperately needed,
we believe that the quinolinyl pyrimidine class provides interesting
potential for further optimization. Further improvements to this
series will be communicated in due course.
Table 2. Representative Compounds
compd
AZ log D
13a
13b
13c
13d
13e
3.0
0.043
1.91
6
3.18
2.16
0.081
2.88
2450
ND
<4
3.06
0.053
1.14
12
3.06
0.050
2.19
15
Mtb NDH-2 IC50 (μM)
Mtb MIC (μM)
0.096
<0.81
70
solubility (μM)
PPB (% free)
0.8
1.44
ND
2.49
ND
>32
>32
ND
ND
>32
>32
Clint (μL/min/mg)
Sce MIC (μg/mL)
RBC MLC (μg/mL)
13.4
>32
>32
>32
>32
>32
>32
a
ND, not determined; < and >, end points not determined.
(40−600 nM) of these compounds was translated into cellular
potency (<0.35−4 μg/mL), while compounds with weak
enzyme activity showed poor cellular potency. The relationship
between the NDH-2 IC50 and the MIC against Mtb is shown
in Figure 2. This excellent correlation between IC50 and
MIC suggests that these compounds inhibit the NDH-2 target
in Mtb.
The potent compounds in the series were profiled for
physicochemical properties, in vitro plasma protein binding,
and mouse microsomal intrinsic clearance (Clint). Solubility in the
presence of 2% (v/v) DMSO was in the range of 6 μM and 2 mM.
The significant improvement of solubility for compound 13c may be
due to lower log D. The protein binding of the series was generally
on the higher side with percent free fraction between 1 and 3. The
mouse Clint was normally <4 and 13.4 μL/min/mg. The MIC data
against S. cerevisiae (Sce) suggest that the compounds in the series
are selective for bacteria and inactive on eukaryotes. Furthermore,
none of these compounds showed any membrane disruption activity
[minimum lysis concentration (MLC)] in the red blood cell (RBC)
hemolysis assay (Table 2). Representative advanced compounds
13a−e are shown in Table 2 and Figure 3.
Figure 3. Representative compounds.
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Scheme 1. Synthesis of Quinolinyl Pyrimidines
a
Reagents: (a) NaH, DMF, reflux, 16 h. (b) 4-Nitroaniline, n-butanol, 4 Å sieves, conc., HCl reflux, 48 h. (c) Dowtherm, 250 °C, 2 h.
(d) POCl3, 110 °C, 4 h. (e) NaN3, NMP, 60 °C, 18 h. (f) SnCl2, ethylacetate−ethanol (5:1) reflux, 2 h. (g) NaH, DMF, reflux, 16 h. (h) PCl5,
POCl3, reflux, 16 h. (i) N-Phenyltrifluoromethanesulphonamide, triethylamine, 1 h. (j) 4 mol equiv of HCl (4 M in 1,4-dioxane), NMP,
80 °C, 10 h.
ASSOCIATED CONTENT
REFERENCES
■
■
S
(1) Butler, D. New fronts in an old war. Nature 2000, 406, 670−672.
(2) Maher, D.; Raviglione, M. C. In Tuberculosis and Nontuberculous
Mycobacterial Infections, 4th ed.; Schlossberg, D., Ed.; Saunders:
Philadelphia, 1999; pp 104−115.
* Supporting Information
Full experimental details for compounds synthesized and
descriptions of biological assays. This material is available free
of charge via the Internet at http://pubs.acs.org.
(3) WHO. Global Tuberculosis Control, WHO Report, 2009; http://
www.who.int/tb/publications/global_report/2009/en/index.html.
(4) Glaziou, P.; Floyd, K.; Korenromp, E. L.; Sismanidis, C.;
Bierrenbach, A. L.; Williams, B. G.; Atun, R.; Raviglione, M. Lives
saved by tuberculosis control and prospects for achieving the 2015
global target for reducing tuberculosis mortality. Bull. World Health
Organ. 2011, 89, 573−582.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +91 080 23621212. Fax: +91 080 23621214. E-mail:
pravin.shirude@astrazeneca.com.
Author Contributions
(5) Andries, K.; Verhasselt, P.; Guillemont, J.; Gohlmann, H. W. H.;
̈
Neefs, J.; Winkler, H.; van Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.;
Williams, P.; de Chaffoy, D.; Huitric, E.; Hoffner, S.; Cambau, E.; Truffot-
Pernot, C.; Lounis, N.; Jarlier, V. A diarylquinoline drug active on the ATP
synthase of Mycobacterium tuberculosis. Science 2005, 307, 223−227.
(6) http://www.tballiance.org/downloads/Pipeline/TBA-Pipeline-
April-2012.pdf.
(7) De Vries, S.; Grivell, L. A. Purification and characterization of a
rotenone-insensitive NADH: Q6 oxidoreductase from mitochondria of
Saccharomyces cerevisiae. Eur. J. Biochem. 1988, 176, 377−384.
(8) Young, I. G.; Poulis, M. I. Conjugal transfer of cloning vectors
derived from ColE1. Gene 1978, 4, 175−179.
(9) Bjorklof, K.; Zickermann, V.; Finel, M. Purification of the 45 kDa,
membrane bound NADH dehydrogenase of Escherichia coli (NDH-2)
and analysis of its interaction with ubiquinone analogues. FEBS Lett.
2000, 467, 105−110.
(10) Bergsma, J.; Van Dongen, M. B.; Konings, W. N. Purification and
characterization of NADH dehydrogenase from Bacillus subtilis. Eur. J.
Biochem. 1982, 128, 151−157.
P.S.S. drafted the manuscript and participated in the design and
execution of this study. B.P. performed and interpreted enzyme
data. N.R.C. and C.K. performed the chemical syntheses. B.B.
and B.G.U. contributed in scientific discussions.
Funding
Funding was provided by NM4TB consortium.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We deeply acknowledge Reetobrata for assay development and
preparation of protein for screening. We thank the analytical
support provided by Suresh Rudrapatna, Sandesh Jatheendra-
nath, and Sreenivasaiah, Menasinakai. Our sincere thanks to the
Microbiology and DMPK teams for technical support in the
various assays. We convey thanks to Vasan Sambandamurthy,
Suresh Solapure, and Kakoli Mukherjee for proofreading of this
manuscript. We also thank Tanjore Balganesh for his constant
inspiration, encouragement, and support during this study.
(11) Cook, S. A.; Shiemke, A. K. Evidence that a type-2
NADH:quinone oxidoreductase mediates electron transfer to partic-
ulate methane monooxygenase in Methylococcus capsulatus. Arch.
Biochem. Biophys. 2002, 398, 32−40.
D
dx.doi.org/10.1021/ml300134b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(12) Matsushita, K.; Otofuji, A.; Iwahashi, M.; Toyama, H.; Adachi, O.
NADH dehydrogenase of Corynebacterium glutamicum. Purification of
an NADH dehydrogenase II homolog able to oxidize NADPH. FEMS
Microbiol. Lett. 2001, 204, 271−276.
(13) Nantapong, N.; Kugimiya, Y.; Toyama, H.; Adachi, O.;
Matsushita, K. Effect of NADH dehydrogenase-disruption and over-
expression on respiration-related metabolism in Corynebacterium
glutamicum KY9714. Appl. Microbiol. Biotechnol. 2004, 66, 187−193.
(14) Gomes, C. M.; Teixeira, M. The NADH oxidase from the
thermoacidophilic archaea Acidianus ambivalens: Isolation and phys-
icochemical characterisation. Biochem. Biophys. Res. Commun. 1998, 243,
412−415.
(15) Bandeiras, T. M.; Salgueiro, C.; Kletzin, A.; Gomes, C. M.;
Teixeira, M. Acidianus ambivalens type-II NADH dehydrogenase:
Genetic characterisation and identification of the flavin moiety as FMN.
FEBS Lett. 2002, 531, 273−277.
(16) Bandeiras, T. M.; Salgueiro, C. A.; Huber, H.; Gomes, C. M.;
Teixeira, M. The respiratory chain of the thermophilic archaeon
Sulfolobus metallicus: studies on the type-II NADH dehydrogenase.
Biochim. Biophys. Acta 2003, 1557, 13−19.
(17) Yano, T.; Li, L.-S.; Weinstein, E.; The, J.-S.; Rubin, H. Steady-state
kinetics and inhibitory action of antitubercular phenothiazines on
Mycobacterium tuberculosis type-II NADH-menaquinone oxidoreductase
(NDH-2). J. Biol. Chem. 2006, 281, 11456−11463.
(18) Weinstein, E. A.; Yano, T.; Li, L. S.; Avarbock, D.; Avarbock, A.;
Helm, D.; McColm, A. A.; Duncan, K.; Lonsdale, J. T.; Rubin, H.
Inhibitors of type II NADH:menaquinone oxidoreductase represent a
class of antitubercular drugs. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
4548−4553.
(19) Boshoff, H. I.; Myers, T. G.; Copp, B. R.; McNeil, M. R.; Wilson,
M. A.; Barry, C. E., III The transcriptional responses of Mycobacterium
tuberculosis to inhibitors of metabolism: Novel insights into drug
mechanisms of action. J. Biol. Chem. 2004, 279, 40174−40184.
(20) Sassetti, C. M.; Rubin, E. J. Genetic requirements for
mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 12989−12994.
(21) McAdam, R. A.; Quan, S.; Smith, D. A.; Bardarov, S.; Betts, J. C.;
Cook, F. C.; Hooker, E. U.; Lewis, A. P.; Woollard, P.; Everett, M. J.;
Lukey, P. T.; Bancroft, G. J.; Jacobs, W. R., Jr.; Duncan, K.
Characterization of a Mycobacterium tuberculosis H37Rv transposon
library reveals insertions in 351 ORFs and mutants with altered
virulence. Microbiology 2002, 148, 2975−2986.
(22) Amaral, L.; Kristiansen, J. E. Phenothiazines: an alternative to
conventional therapy for the initial management of suspected multidrug
resistant tuberculosis. A call for studies. Int. J. Antimicrob. Agents 2000,
14, 173−176.
(23) Chaudhari, B.; Chapdelaine, M.; Hostetler, G.; Kemp, L.; Mc-
Cauley, J. N-type calcium channel antagonists for the treatment of pain.
PCT publication No. WO02/36586 A1, 2002.
E
dx.doi.org/10.1021/ml300134b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX