Scaffold morphing leading to evolution of 2,4-diaminoquinolines and aminopyrazolopyrimidines as inhibitors of the ATP synthesis pathway

Article abstract of DOI:10.1039/c5md00589b

The success of bedaquiline as an anti-tubercular agent for the treatment of multidrug-resistant tuberculosis has validated the ATP synthesis pathway and in particular ATP synthase as an attractive target. However, limitations associated with its use in the clinic and the drug-drug interactions with rifampicin have prompted research efforts towards identifying alternative ATP synthesis inhibitors with differentiated mechanisms of action. A biochemical assay was employed to screen AstraZeneca's corporate compound collection to identify the inhibitors of mycobacterial ATP synthesis. The high-throughput screening resulted in the identification of 2,4-diaminoquinazolines as inhibitors of the ATP synthesis pathway. A structure-activity relationship for the quinazolines was established and the knowledge was utilized to morph the quinazoline core into quinoline and pyrazolopyrimidine to expand the scope of chemical diversity. The morphed scaffolds exhibited a 10-fold improvement in enzyme potency and over 100-fold improvement in selectivity against inhibition of mammalian mitochondrial ATP synthesis. These novel compounds were bactericidal and demonstrated growth retardation of Mycobacterium tuberculosis in the acute mouse model of tuberculosis infection.

Full text of DOI:10.1039/c5md00589b

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Tantry, V. Shinde, G. Balakrishnan, S. Markad , A. K. Gupta, J. Bhat, A. Narayan , A. Raichurkar, L. Jena, S.
Sharma, N. Kumar, R. Nanduri, S. Bharath, J. Reddy, V. Panduga, P. KR, K. Kandaswamy, P. Kaur, N. Dinesh
,
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Balasubramanian, V. Sambandamurthy, V. Ramachandran, R. Shandil, S. Kavanagh, S. Narayanan, P. Iyer,
K. Mukherjee, V. Hosagrahara, S. Solapure and S. Hameed P, Med. Chem. Commun., 2016, DOI:
1
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DOI: 10.1039/C5MD00589B
Mukherjee, Kakoli; AstraZeneca India Pvt Ltd
Hosagrahara, Vinayak; AstraZeneca India Pvt Ltd
Solapure, Suresh ; AstraZeneca India Pvt Ltd
Hameed P, Shahul; AstraZeneca India Pvt Ltd

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ARTICLE
Scaffold Morphing Led to Evolution of 2,4ꢀDiaminoquinolines and
†
Aminopyrazolopyrimidines As Inhibitors of ATP Synthesis Pathway
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
Subramanyam J. Tantry, Vikas Shinde, Gayathri Balakrishnan, Shankar D.
a
a
a
a
Markad, Amit K. Gupta, Jyothi Bhat, Ashwini Narayan, Anandkumar
Raichurkar, Lalit Kumar Jena, Sreevalli Sharma, Naveen Kumar, Robert
Nanduri, Sowmya Bharath, Jitendar Reddy, Vijender Panduga, Prabhakar K.
R, Karthikeyan Kandaswamy, Parvinder Kaur, Neela Dinesh, Supreeth
Guptha, Ramanatha Saralaya, Manoranjan Panda, Suresh Rudrapatna,
Meenakshi Mallya, Harvey Rubin, Takahiro Yano, Khisi Mdluli, Christopher
Cooper, V Balasubramanian, Vasan K. Sambandamurthy,
DOI: 10.1039/x0xx00000x
a
a
a
a
www.rsc.org/
a
a
a
a
a
a
a
a
a
a
a
a
a
d
d
b
b
a
a
Vasanthi
a
a
c
a
Ramachandran, Radha Shandil, Stefan Kavanagh, Shridhar Narayanan, Pravin
a
a
a
a
Iyer, Kakoli Mukherjee, Vinayak P Hosagrahara, Suresh Solapure, Shahul
a
a
Hameed P, * Sudha Ravishankar. *
The success of bedaquiline as an antiꢀtubercular agent for the treatment of
multiꢀdrug resistant tuberculosis has validated ATP synthesis pathway and in
particular ATP synthase as an attractive target. However, limitations associated
with its use in the clinic and the drugꢀdrug interactions with rifampicin have
prompted research efforts towards identifying alternate ATP synthesis inhibitors
with differentiated mechanism of action. A biochemical assay was employed to
screen AstraZeneca’s corporate compound collection to identify inhibitors of
mycobacterial ATP synthesis. The high throughput screen resulted in the
identification of 2, 4ꢀdiaminoquinazolines as inhibitors of ATP synthesis pathway.
A structure activity relationship for quinazolines was established and the
knowledge was utilized to morph quinazoline core into quinoline and
pyrazolopyrimidine to expand the scope of chemical diversity. The morphed
scaffolds exhibited a 10ꢀfold improvement in the enzyme potency and over 100ꢀ
fold improvement in selectivity against inhibition of mammalian mitochondrial
ATP synthesis. These novel compounds were bactericidal and demonstrated
growth retardation of Mycobacterium tuberculosis in the acute mouse model of
tuberculosis infection.
a
Innovative Medicines, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, India.
Global Alliance for TB Drug Development, 40 Wall Street, 24th Floor. New York, NY 10005, USA.
AstraZeneca, Alderley Park, Mereside, Cheshire, UK.
University of Pennsylvania, 111 Clinical Research Building, 415 Curie Boulevard, Philadelphia PA 19104, USA
b
c
d
Electronic Supplementary Information (ESI) available: [Materials and Methods; Synthesis schemes 1A and 1B; Fig. S1: 3D flexible superimposition of compound 3h, compound 8d
and compound 17a with Bedaquiline, Figs. S2ꢀS3: 2D binding site interaction of inhibitors ꢀ Bedaquiline, compound 3h, 8d, 12 and 17a at the ATPase binding site, Figs. S4ꢀS7: NMR
spectra and HPLC profiles of compounds 8g and 17a], associated with this article can be found in the online version. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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TM
1. Introduction
Sirturo , received expedited approval by the FDA
5
for the treatment of MDRꢀTB. Thus a new TB
Tuberculosis (TB) caused by Mycobacterium
tuberculosis, a highly infectious disease has after a gap of 40 years. Bedaquiline inhibits ATP
drug with a different mode of action got approved
5
,6
plagued mankind for thousands of years. TB still
remains a global pandemic causing around 1.4
synthase and causes depletion in intracellular ATP
levels. This target is essential for the survival of
7
million deaths and 9 million new incidences each bacteria under both replicating and nonꢀreplicating
1
year. The financial burden of TB on developing
countries is estimated to be between one and three
trillion dollars over the next decade. Though the
overall numbers of new cases have decreased by
states, with a six times lower intracellular ATP
levels during the nonꢀreplicating state as compared
to the levels in the actively replicating state.
Maintaining this threshold level of ATP is
absolutely essential to keep the bacteria viable. Any
further depletion would lead to a detrimental effect
4
0% since 1990, the global prevalence of multidrug
resistant (MDR) and extensively drug resistant
XDR) TB has gone up substantially.
2
8
(
on bacterial survival. Although, ATP synthase is
TB can remain dormant for years after infection
highly conserved and of fundamental importance to
both prokaryotes and eukaryotes, bedaquiline is
highly selective towards the mycobacterial enzyme.
This critical information provides the rationale
behind inhibiting a highly conserved target for
and can reactivate once the host become
immunocompromised as in the case of HIV coꢀ
3
infected populations. Considerable progress has
been made in TB drug discovery in the recent past
9
and a number of drug candidates are at different
clinical progression. The unprecedented success
4
stages of clinical trials. Diaryl quinoline (TMC207 and novelty of bedaquiline has triggered several
or bedaquiline, Fig. 1), discovered by Johnson &
groups to investigate this pathway.
Johnson pharmaceuticals and marketed as
Fig. 1. Structures of ATP synthesis pathway inhibitors
Fig. 1 represents the structures of a few leading we screened AstraZeneca’s corporate compound
mycobacterial ATP synthase inhibitors reported in collection by employing a high throughput
1
0ꢀ12
the literature to date.
In order to identify novel
mycobacterial ATP synthesis assay (Myc_ATPS)
scaffolds targeting mycobacterial ATPꢀsynthesis,
which used Mycobacterium smegmatis membrane
2
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1
3
preparation (inverted membrane vesicles ꢀ IMVs)
potent derivatives was obtained following a 4ꢀstep
synthetic protocol starting from corresponding
3ꢀfluoropyridinꢀ2ꢀol (Scheme 1A, supporting
information 1). Similarly, compound 4 with ether
linkage at C2 position was synthesized by
modification of the intermediate 2h (Scheme 1B,
supporting information 1)
to measure ATP synthesis via the oxidative
phosphorylation (OxꢀPhos) process. Compound 3a
2, 4ꢀdiaminoquinazoline, Fig. 2) emerged as one
of the promising hits from this high throughput
screening (HTS) effort. However, this initial hit
3a) was not selective as it inhibited the
mammalian mitochondrial ATP synthesis assay
SMP_ATPS) with equal potency. This report
describes the results of medicinal chemistry
structure activity relationship (SAR) efforts leading
to the evolution of potent mycobacterial ATP
(
(
R
2
R
2
HN
HN
Cl
O
O
O
O
O
O
(a)
(b)
(
N
N
N
R
1
N
Cl
N
X
N
Cl
1
2a-i
3a-i, X = NH
, X = O
4
synthesis
aminopyrazolopyrimidines
diaminoquinolines with about 20ꢀfold and 120ꢀfold
selectivity window respectively.
pathway
inhibitors,
2, 4ꢀ
Scheme 1. Synthesis of 2, 4ꢀdiaminoquinzolines.
Reagents and conditions: (a) R ꢀNH , NaH, DMF,
0 °C, 2 h, yield: 45ꢀ55% and (b) R ꢀNH , Na CO
nꢀBuOH, MW, 170 °C, 1.5 h, yield: 19ꢀ45%.
and
2
2
8
1
2
2
3
,
2.1.2. Quinoline derivatives (8aꢀg)
Condensation of malonic acid with aniline/
substituted anilines 5 followed by treatment with
POCl gave substituted/unsubstituted 2,4ꢀ
3
Fig. 2: Structure of HTS hit (compound 3a)
dichloroquinolines 6. C ꢀsubstitution with R ꢀ
2
1
amines followed by C4ꢀsubstitution with R
under BuchwaldꢀHartwig reaction conditions gave
2
ꢀamines
2. Results and Discussion
compounds 8aꢀg (Scheme 2).
2.1. Chemistry
Cl
X
X
X
X
(a)
(b)
The syntheses of compounds derived from
quinazoline, quinoline and pyrazolopyrimidine
series are described in schemes 1ꢀ4.
NH2
N
Cl
5
6
R2
HN
Cl
X
X
X
X
(c)
2.1.1. Quinazoline derivatives (3aꢀi)
R1
R1
N
N
N
N
H
H
7
8a-g
The required R2 groups were incorporated by
X = H, OCH , F
3
the displacement of 4ꢀchloro functionality of 2,4ꢀ
dichloroꢀ6,7ꢀdimethoxyꢀquinazoline with
Scheme 2. Synthesis of 2, 4ꢀdiaminoquinolines.
corresponding R2ꢀamines to give intermediates 2aꢀi
in good yields. Finally, 2ꢀchloro of intermediates
Reagents and conditions: (a) Malonic acid, POCl
120 °C, 6 h, yield: 60ꢀ65%; (b) Pd (dba)
3
,
,
2
3
2
aꢀi was displaced with R1ꢀamines under XantPhos, NaOtBu, PhCH
microwave conditions to provide compounds 3aꢀi
yield: 35ꢀ40%; (c) Pd (dba)
Scheme 1). While most of the raw materials used PhCH , 120 °C, MW 1 h, yield: 7ꢀ72%.
3
, 120 °C, MW 1 h,
2
3
, XantPhos, NaOtBu,
(
3
as substitutions at R1 and R2 positions were
procured commercially, one of the key intermediate
2.1.3. Pyrazolopyrimidine derivatives (12, 17a,b)
6ꢀ[2ꢀ(dimethylamino)ethoxy]ꢀ5ꢀfluoropyridinꢀ3ꢀ
amine utilized for the synthesis of many of the
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Synthesis of compound 12 is described in
scheme 3a. The condensation of diethyl malonate
and 1Hꢀpyrazolꢀ3ꢀamine with metallic sodium in
ethanol resulted in cyclized 2,4ꢀdihydroxy
pyrazolopyrimidine intermediate 9. Treatment of
intermediate 9 with POCl
3
provided chloro
intermediate 10. Nucleophilic substitution of chloro
intermediate 10 with 6ꢀethoxyꢀ5ꢀfluoropyridinꢀ3ꢀ
amine followed by
a
second nucleophilic
substitution under microwave condition at C2
position resulted in compound 12.
Scheme
aminopyrazolopyrimidines.
conditions: (a) NH NH .H
yield: 33%; (b) R ꢀCOꢀCH
C, 12 h, yield: 48ꢀ55%; (c) POCl
yield: 60ꢀ68%; (d) 1d, K CO , DMF, 75 °C, 2 h,
yield: 22ꢀ33%.
3b.
Synthesis
of
and
Reagents
2
2
2
O, EtOH, 80 °C, 4 h,
ꢀ COOEt, AcOH, 120
, 135 °C, 3 h,
1
2
°
3
2
3
The synthesis of compound 26 with azaindole
core is provided in scheme 4.
OH
OH
Cl
Cl
Cl
NO
2
NO2
NH2
NH2
(a)
(b)
(c)
(d)
Scheme
3a.
Synthesis
of
N
OH
N
OH
N
Cl
N
Cl
Br
N
Cl
18
19
20
21
22
aminopyrazolopyrimidine 12. Reagents and
conditions: (a) Na, EtOH, 85 C, 18 h, yield: 62%;
o
Cl
N
Cl
N
H N
2
H
2
N
(e)
(f)
(g)
o
(b) POCl
3
, 120 C, 3 h, yield: 30%; (c) 6ꢀethoxyꢀ5ꢀ
Cl
o
fluoropyridinꢀ3ꢀamine, NaH, DMF, 75 C, 2 h,
yield: 94%; (d) 2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)
ethanamine, Na CO , DIPEA, nꢀBuOH, MW, 170
23
24
F
F
O
N
Cl
N
N
H
N
HN
2
3
(h)
H
N
o
F
C, 1.25 h, yield: 34%.
F
N
2
5
26
Synthesis of compounds 17a and 17b is
described in scheme 3b. Condensation of 3ꢀ(4ꢀ Scheme 4. Synthesis of azaindole 26. Reagents
fluorophenyl)ꢀ3ꢀoxopropanenitrile 13 with and conditions: (a) HNO , H SO , 0 °C, 0.5 h,
hydrazine hydrate under heating conditions gave 5ꢀ
aminopyrazole derivative 14. The
pyrazolopyrimidinone ring 15 was constructed by 82%; (d) NBS, DMF, 0ꢀ25 °C, 1 h, yield: 47%; (e)
the reaction of 5ꢀaminopyrazole derivative 14 with PhB(OH) , Pd(PPh , K CO , 1,4ꢀdioxane, H O,
various R ꢀsubstituted βꢀketoesters. Treatment of
85 °C, 12 h, yield: 81%; (f) 1ꢀethynylꢀ4ꢀ
fluorobenzene, Pd(Ph) Cl , CuI, TEA, 80 °C, 5 h,
yield: 45%; (g) KO Bu, THF, MW, 100 C, 1 h,
3
2
4
yield: 85%; (b) POCl , 135 °C, 12 h, yield: 72%;
3
4 2
(c) Fe, NH Cl, EtOHꢀH O, (4:1) 80 °C, 5 h, yield:
2
3
)
4
2
3
2
1
compound 15 with POCl
intermediate 16. Nucleophilic substitution of chloro
intermediate 16 with 6ꢀ[2ꢀ(dimethylamino)ethoxy]ꢀ
3
provided chloro
2
2
t
o
t
t
yield: 78%; (h) 1d, BuXPhos, THF, NaO Bu, MW,
5
ꢀfluoropyridinꢀ3ꢀamine (Scheme 1A, supporting 100 °C, 2 h, yield: 63%.
information 1) afforded compounds 17a and 17b
(Scheme 3b).
2.2. Structure Activity Relationship
4
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The initial hit obtained from HTS campaign,
compound 3a (Fig. 2), was less potent in both
enzyme assay (IC50 of 2.1 ꢀM) and antiꢀ
mycobacterial potency (M. tuberculosis minimum
inhibitory concentration (MIC) of 25 ꢀM) as
compared to some of the reported inhibitors of this
pathway. Moreover, it had poor selectivity as it
inhibited mammalian mitochondrial ATP synthesis
group at the C6, C7 positions of quinazoline ring
resulted in a 10ꢀfold improvement in enzyme
potency and selectivity index for compound 3d
(Compound 3c vs 3d, Table 1). Later, we confined
to having dimethoxy group substitution at C6, C7
during the majority of SAR modification due to
potency gains observed and for reasons of ease of
synthesis. Further replacement of pyrazole at C4
position by suitably substituted thiazole and
pyridine ring systems tolerated for potency but the
selectivity index was compromised by 5 fold
(SMP_ATPS) with an IC50 of 2.6 ꢀM. The later
assay was similar to Myc_ATPS assay in principle
but used submitochondrial particles (SMP) from
bovine heart mitochondria as the source of
mitochondrial OxꢀPhos components. Since ATP
synthesis is a ubiquitous pathway and highly
conserved across species, our initial efforts were
focused on understanding the structure activity
relationship (SAR) requirements of quinazoline
series and improving the selectivity index (ratio of
SMP_ATPS IC50 to Myc_ATPS IC50). The
molecular structure of compounds synthesized as
part of this SAR effort along with their enzyme
inhibitory and antiꢀmycobacterial potency have
been listed in Table 1. Simple bioisosteric
replacement of piperidine by pyrrolidine ring at C2
position tolerated for potency but the selectivity
index got worsened for compound 3b (Compound
(compound 3d vs 3f, Table 1). Substituting the
pyran at C2 by pyridine ring system retained the
enzyme potency with improved selectivity
(compound 3f vs 3g, Table 1). However, the
compound 3g lost the MIC completely which may
be due to bacterial permeability. Compound 4 with
an ether linkage at C2 position was found be
inactive in both Myc_ATPS assay and M.
tuberculosis MIC assay (Table 1). This indicated
the requirement of an amino group at C2 position
for mycobacterial ATP synthesis inhibition. The
nitrogen here could be either secondary or tertiary
and indicated that it is probably making a critical
interaction with the enzyme. One of the
explanations for this observed phenomenon could
be the possible quaternization of nitrogen at
physiological pH which is not possible with ether
linkage. Elongation of ethoxy substitution of C4
pyridine side chain with terminal basic group
showed moderate potency with enhanced selectivity
index and an absolute IC50 of >25 µM in
SMP_ATPS assay (Compound 3h vs 3i, Table 1).
This result suggested that an additional base centre
in the molecule may provide handles for selectivity
improvement without compromising on the
potency.
3a vs 3b, Table 1). In the next round of design, we
introduced polarity in the molecules at C2 and C4
position to improve selectivity and potency.
Polarity modification hypothesis around compound
3a resulted in achieving a 15ꢀfold improvement in
Myc_ATPS IC50. Improved enzyme potency for
compounds 3d, 3e, 3g also translated into a 6 to 8ꢀ
fold improvement in MIC but the selectivity index
remained a concern for quinazoline series (Table 1)
except for compounds 3d, 3g and 3i.
Replacement of piperidine at the C2 position
and αꢀmethyl benzylamine at C4 position in
compound 3b with 2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
None of the other attempts made to further
resolve the issue around selectivity was successful
for compounds with 2, 4ꢀdiaminoquinazoline core.
Hence, we decided to expand the chemical scope of
central core ring into 2, 4ꢀdiaminoquinoline and 5ꢀ
phenylpyrazolo[1,5ꢀa]pyrimidine. Majority of the
SAR exploration on the new core was carried out
yl)ethanamine
and
5ꢀtertꢀbutylꢀ1ꢀmethylꢀ1Hꢀ
pyrazolꢀ3ꢀamine respectively yielded compound 3c
with no loss in potency suggesting that there is
enough space for such hydrophobic groups.
Introduction of additional polarity via dimethoxy
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using
6ꢀ(2ꢀ(dimethylamino)ethoxy)ꢀ5ꢀ
position as this
Converting quinazoline core ring into quinoline
by substituting N3 nitrogen with carbon weakened
fluoropyridinꢀ3ꢀamine at R
1
particular group on quinazoline, compound 3i, had the enzyme potency marginally (Match pairs: 3h vs
earlier resulted in an absolute IC value of >25 ꢀM 8a and 3i vs 8b in Table 1). Encouraged with
50
against mammalian mitochondrial ATP synthesis.
expanded chemical diversity of quinoline core with
moderate enzyme potency, we then explored SAR
of quinoline core with various R
improve potency. Replacement
tetrahydropyranyl ethylamine substitution with 2ꢀ
2ꢀchlorophenyl)ethanamine resulted in 60ꢀfold
1
substitutions to
of
(
improvement in enzyme potency and antiꢀ
mycobacterial activity (Match pair: Compound 8b
vs 8c, Table 1). Compound 8c also showed
improved
selectivity
index
(Table
1).
Fig. 3. Generic structures of synthesized
compounds
Table 1: SAR around 2,4ꢀdiaminoquinazoline (3aꢀ3i, 4), 2,4ꢀdiaminoquinoline (8aꢀ8f, 8g) and
aminopyrazolopyrimidine (12, 17aꢀ17b, 26) cores.
M.
Selectivity
Index
R
1
R
2
X
Myc_ATPS
tuberculosis
IC50 (µM) MIC (µM)
a
CN.
3
a
H
2.1
25
2.6
3
b
H
H
2.5
1.7
50
<0.04
4.5
3
c
6.2
3
d
OCH
3
0.1
6.2
52
b
3
e
OCH
OCH
3
3
0.1
0.4
6.2
3.1
ND
3
f
10
6
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3
g
OCH
3
3
0.1
>200
96
3
h
OCH
OCH
OCH
0.6
0.9
25
1.9
25
2.3
3
i
3
3
>28
b
4
>200
ND
8
a
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.8
3.7
6.2
200
3.1
1.6
6.2
3.2
1
8
b
8
c
0.06
0.04
0.06
79
128
44
8
d
8
e
8
f
ꢀ
ꢀ
0.1
0.1
1.6
3.1
51
19
8
1
g
2
ꢀ
ꢀ
ꢀ
21
0.5
0.5
>50
6.2
>1.2
20
1
7a
F
1
7b
12.5
19
O
N
N
*
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2
6
ꢀ
0.1
0.8
13
a
b
Compound number, not determined
The potency improvement observed with
compound 8c suggested that R pocket may be
showed some promise of enzyme potency and M.
tuberculosis MIC. Compound 17a from
pyrazolopyrimdine core demonstrated IC50 and
MIC of 0.5 ꢀM and 6.2 ꢀM respectively.
Replacement of phenyl ring with suitable
hydrophobic group like CF3 at the C5 position of
pyrazolopyrimidine (17b) core was found to be
equipotent as that of 17a. These results indicated
that steric and/or hydrophobic factors could be
mainly contributing to the potency rather than the
planarity of phenyl ring (17b vs 17a, Table 1).
Further scaffold morphing was achieved by
1
hydrophobic in nature and the aromatic ring system
would be a better alternative to an alicyclic ring in
order to improve the potency. Further,
conformational constraining of phenyl ethylamine
group with 3ꢀ(2ꢀchlorophenyl)pyrrolidine at R2
position retained the potency and selectivity
(Compound 8d vs 8c, Table 1). In a similar way,
the
conformational
constraining
of
dimethylaminoethoxy group of R
1
substitution with
Nꢀmethylpyrrolidine ring retained the enzyme
potency but the selectivity index was compromised
by 3ꢀfold (Compound 8d vs 8e, Table 1).
Replacement of oxygen linker of pyridine side
chain with ꢀNH linker in compound 8d retained
enzyme potency but antiꢀmycobacterial potency
and selectivity were compromised by 2 to 3ꢀfold
removing
the
bridgeꢀhead
nitrogen
of
pyrazolopyrimidine and modifying central core into
4ꢀazaindole ring system (Matched pair: 17a vs 26,
Table 1). The resulting compound 26 showed best
MIC in the series with M. tuberculosis MIC of 0.8
ꢀM and Myc_ATPS IC50 of 0.1 ꢀM. Nevertheless,
(
compound 8e vs 8d, Table 1). Modification of
the selectivity index was compromised for
compounds with pyrazolopyrimidine and azaindole
cores compared to those with quinoline core.
Among the different central core ring systems
explored, compounds with quinoline core showed
improved selectivity with compound 8d
demonstrating about 128ꢀfold selectivity index,
best among all the compounds synthesized. Fig. 3
provides a snapshot of the generic structures of the
compounds synthesized in this study towards
understanding the SAR.
dimethoxy substitution of quinoline ring with
difluoro group again retained potency but
selectivity index was compromised by 6 to 7ꢀfold.
Overall, quinoline core demonstrated improved
potency and selectivity compared to quinazoline
core. Compound 8d from quinoline series
displayed the best IC50, MIC, and selectivity index
of 40 nM, 1.6 ꢀM and 128ꢀfold respectively (Table
1
).
In an attempt to further expand chemical scope
of the core ring system, we also morphed the
central quinoline core to 5, 6ꢀfused
2.3. Diaminoquinolines
&
aminopyrazolo
pyrazolopyrimdine ring system. The initial
compound 12 was found to be inactive in both the
biochemical and the antiꢀmycobacterial assay. In
the absence of information on binding of these
compounds at molecular level, we relied on
chemical intuition to progress the series.
Introduction of additional hydrophobic groups like
fluorophenyl ring at the C2 position and a phenyl
ring at the C5 position of pyrazolopyrimidine core
pyrimidines do not damage mycobacterial
membrane
During the SAR exploration, compounds were
tested for potency in the Myc_ATPS assay which
used the inverted membrane vesicles prepared from
M. smegmatis as source of OxꢀPhos components.
To ensure that the inhibition of ATP synthesis and
the consequent M. tuberculosis MIC observed by
8
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these compounds were not due to nonꢀspecific
positive control used in the assay, carbonyl cyanide
perturbation of the mycobacterial membrane, an mꢀchloro phenyl hydrazine (CCCP) had an IC50 of
assay was performed to test their potential to
damage membrane and the results are given in
Table 2. M. bovis Bacillus CalmetteꢀGuerin (BCG)
cells were used as surrogate in this membrane
damage assay for reasons like its closeness to M.
tuberculosis both at the genome and at the cellular
architecture level, translation of M. tuberculosis
MIC’s in BCG and the ease of handling. This assay,
an adaptation from the one reported for S. aureus,
used a fluorophore, 3, 3’ꢀ Diethyloxacarbocyanine
3 ꢀM in the membrane damage assay while its IC50
in the Myc_ATPS assay was also in the same range
(Table 2).
2
1
.4. Modeling studies with compounds 3h, 12 &
7a
Encouraged by the observation that the
inhibition of ATP synthesis is not due to nonꢀ
specific damage to the membrane, we utilized
modeling studies to understand the possible
interaction of these compounds with the ATP
synthesis pathway component. The 2ꢀdimensional
iodide (DiOC ). A red shift in the emission
2
wavelength is exhibited when DiOC gets stacked in
2
14
the bacterial cells.
(2D) tanimoto distance similarity (>50 %) as well
Table 2: Assessment of membrane damage
as the 3ꢀdimensional (3D) shape overlay similarity
potential
of select compounds 3h (diaminoquinazoline core),
8
d
(diaminoquinoline
core)
and
17a
BCG Membrane
Myc_ATPS
(aminopyrazolopyrimidine core) with the crystal
Compound
damage IC50
IC50 (ꢀM)
structure of bedaquiline led us to hypothesize that
these inhibitors may occupy the binding cavity at
the interface of the two cꢀsubunits (chain B and
chain C) and the aꢀsubunit of ATP synthase similar
to that reported for bedaquiline with M.
tuberculosis ATP synthase (Fig. S1, supporting
information 2). In order to predict the possible
mode of binding and to understand the rationale
observed from the structure activity relationship
data in Table 1, we performed docking studies of
these molecules at the active site using the earlier
reported homology model of M. tuberculosis ATP
synthase.
bedaquiline binding site using Glide 6.1 extra
precision(XP) method.
interaction of bedaquiline in the M. tuberculosis
ATP synthase model is shown in Fig. 4, where it
bound the enzyme diagonally at the interface of the
(
ꢀM)
3d
0.13
0.85
0.04
0.13
0.45
0.51
0.13
0.005
4.8
>100
>100
>100
>100
>100
>100
>100
>100
3
8
a
8d
15
8
g
17a
17b
2
6
16
Compounds were docked onto the
Bedaquiline
CCCP
1
7
The binding site
All compounds tested in this assay had an IC50 two cꢀsubunits (chain B and chain C) and the aꢀ
of over 100 ꢀM which is about hundred to thousand
subunit of ATP synthase. These interactions would
fold greater than the IC50 in the Myc_ATPS assay plug the rotation of cꢀsubunits resulting in the
suggesting that these compounds specifically inhibition of ATP synthase and hence ATP
inhibited mycobacterial ATP synthesis unlike the
synthesis (Fig. 4A).
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Fig. 4. Predicted binding mode of inhibitors. A. Bedaquiline (green colored carbon); B. Compound 8d
amaranth pink colored carbon); C. Compound 3h (yellow colored carbon) D. Compound 12 (pink colored
(
carbon); E. Compound 17a (blue colored carbon). Helices in cyan and pink color are chains A and B of cꢀ
subunit respectively whereas helix in green color is aꢀsubunit of Fo component of ATP synthase.
The binding pose analysis of compound 8d at
the active site of M. tuberculosis ATP synthase
revealed that dimethoxyquinoline group of 8d
interacts at the interface of Atpꢀa and Atpꢀc (chainꢀ
The predicted binding mode of compound 12
(Fig. 4D) revealed the Hꢀbond contact of
pyrimidine nitrogen with Tyrꢀ64 of Atpꢀc subunit
(chainꢀB). The loss of Myc_ATPS inhibitory
activity for compound 12 (IC50 = 21 µM) could be
due to the lack of functional group to explore the
hydrophobic pocket (constituting residues from
Atpꢀa and Atpꢀc (chainꢀB) subunits) unlike
bedaquiline where bromoquinoline exploits this
region optimally (Fig. 4C). However, the pyran
and pyridyl ring of the molecule showed similar
binding pattern as noted for compound 3h.
B) subunits through
πꢀπ interaction with Tyrꢀ64
and Hꢀbond interaction with Gluꢀ61 and directs the
fluoropyridin dimethoxyquinolinꢀ4ꢀamine group
towards the interface of Atpꢀa and Atpꢀc (chainꢀC)
subunits. Further, chlorophenyl group of the
molecule nicely fitted into the hydrophobic cavity
between the two Atpꢀc subunits (Fig. 4B). The
occupancy of 8d at the interface of Atpꢀa and Atpꢀc
subunits caused the blockage of Atpꢀc subunit
rotation and thereby its enzyme activity.
The binding pose comparison study of compound
1
7a (Fig. 4E) showed that the addition of para
Similarly, the binding pose study of compound fluoro phenyl group at pyrazole ring in compound
3
h (Fig. 4C) revealed that the dimethoxy 17a allowed the compound to regain its interface
quinazoline group of 3h occupied the cavity
between Atpꢀa and Atpꢀc (chainꢀB) subunits. The
blocking capacity between Atpꢀa and Atpꢀc (chainꢀ
B) subunits and hence regain the inhibitory
amine group attached with pyridyl ring was found potency. Further Hꢀbond contact of pyrimidine
to interact with Argꢀ186 of Atpꢀa subunit, whereas nitrogen with Tyrꢀ64 of Atpꢀc subunit (chainꢀB)
the amine attached with pyran ring made Hꢀbond
contact with Gluꢀ61 of Atpꢀc subunit (chainꢀB)
providing strong anchorage to the molecule and
place the pyran ring towards the hydrophobic space
between the two Atpꢀc subunits.
anchored the ring and directed the attached phenyl
ring to occupy the hydrophobic space between the
two Atpꢀc subunits resulting in ATP synthase
inhibitory activity of compound 17a (IC50 = 0.5
µM) which is comparable to compound 3h. Thus
our docking study not only suggested the plausible
1
0 | J. Name., 2012, 00, 1-3
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binding mode of these derivatives but also
explained the activity variation among them.
diaminoquinoline and aminopyrazolopyrimidine
compounds are equipotent against a range of drug
sensitive M. tuberculosis strains but they are also
equally active against several drug resistant strains
satisfying one of the key requirements of a new TB
drug candidate. However, the absence of cross
resistance against bedaquiline resistant strains
2
.5.
Cross
resistance
and
profile
aminopyrazolo
of
diaminoquinolines
pyrimidines.
Computational studies led us to test
diaminoquinoline and aminopyrazolopyrimidine
derivatives against bedaquiline resistant mutants to
check if they would exhibit the same mechanism of
action as bedaquiline. A select set of compounds
from these subseries were profiled against a panel
of drug sensitive and drug resistant M. tuberculosis
strains which included two of the bedaquiline
resistant strains raised and characterized inꢀhouse.
MICs against all the five drug sensitive and seven
single drug resistant strains were the same as that
of wildꢀtype M. tuberculosis strain with no cross
resistance observed in any of the mutants tested
suggested
that
diaminoquinolines
and
aminopyrazolopyrimidines may not interact with
ATP synthase in the same manner as bedaquiline
despite binding at the same site. Thus, the key
interactions to plug the rotation of the two cꢀ
subunits
for
diaminoquinoline
and
aminopyrazolopyrimidine derivatives could be
different from the residues involved with the
bedaquiline interaction. This needs to be further
validated through characterization of mutants of M.
tuberculosis resistant to diaminoquinoline and
aminopyrazolopyrimidine derivatives.
(Table 3). This data not only indicated that
Table 3: Cross resistance profile of diaminoquinolines and aminopyrazolopyrimidines
MIC ( M)
ꢀ
Inhibitor
Reference
Sensitive strains
Single drug resistant strains
Reference lab
strains
M.tb
M.tb
M.tb
M.tb
M.tb
M.tb
M.tb
M.tb
M.tb
ATCCꢀ strains* 35820 35822 19000 17003 12119 bedaqui bedaqui
R
R
R
R
R
R
R
27294
STR
INH
RIF
EMB
OFX
line
line
(I66M) (A63P)
8
8
1
2
d
6.3
3.1
6.3
3.1
0.2
3.1 ꢀ 12.5 12.5
3.1 ꢀ 12.5 25.0
6.3
3.1
3.1
6.3
3.1
<2
6.3
6.3
6.3
6.3
<2
12.5
25.0
12.5
25
6.3
3.1
6.3
3.1
10
3.1
3.1
g
3.1
12.5
3.1
7a
6
6.3 ꢀ 12.5
3.1 ꢀ 6.3
<2
25
25
<2
12.5
3.1
Bedaqu
iline
<2
<2
>10
Streptom
ycin
0.3
0.3
> 11
0.3
0.3
0.3
0.3
0.3
0.3
(STR)
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Isoniazid
INH)
0.2
0.2
0.2
>15
0.2
0.2
0.2
0.2
0.2
(
Rifampi
cin
0.04
0.04
0.04
0.04
>1.2
0.04
0.04
0.04
9.8
0.04
(RIF)
Ethambu
tol
9.8
0.7
9.8
0.7
9.8
0.7
9.8
0.7
9.8
0.7
>20
0.3
4.9
9.8
0.7
(EMB)
Ofloxaci
n (OFX)
>5.6
0.7
M.tb: M. tuberculosis
MIC tested against four of M. tuberculosis drug sensitive strains: M. tuberculosis 25618, M. tuberculosisꢀ
Erdman, M. tuberculosisꢀBeijing (Eꢀ47/94), M. tuberculosisꢀHarlingen.
*
1
2 | J. Name., 2012, 00, 1-3
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ARTICLE
2
.6. Diaminoquinolines
&
aminopyrazolo
3h
31
23
0
pyrimidines deplete intracellular ATP pool.
2
6
Inhibitors of ATP synthase enzyme or the other
components of ATP synthesis pathway are
expected to deplete the cells off their intracellular
None
ATP pool. In order to test the ability of 2.7. Diaminoquinolines
&
aminopyrazolo
diaminoquinoline and aminopyrazolopyrimidine pyrimidines are bactericidal but exhibit
derivatives in reducing the intracellular ATP pool, different kill rates
we tested a representative set of compounds from
these subseries in the intracellular ATP
During the course of infection, M. tuberculosis
is known to exist in various physiological states
measurement assay. Cell lysates of BCG cultures
such as actively replicating and quiescent nonꢀ
which had been exposed to 5X MIC concentrations
replicating forms. Inhibitors of the energy
of these compounds for 20 hours were used to
production pathway are known to make the
measure the intracellular ATP concentration. While
pathogen highly vulnerable resulting in bactericidal
isoniazid which inhibits FasII system showed no
depletion in ATP levels, bedaquiline used as
positive control exhibited 76% depletion in ATP
levels. Compounds belonging to diaminoquinoline
and aminopyrazolopyrimidines were not as potent
as bedaquiline except for compound 8d in reducing
the cellular ATP levels suggesting a need to
improve their potency further (Table 4).
7,18
effect.
A select set of compounds from the
subseries were tested for their growth inhibitory
properties under aerobic and hypoxic conditions
used as a model for nonꢀreplicating bacilli). All
the compounds tested were bactericidal under both
aerobic and hypoxic conditions with a minimum
bactericidal concentration (MBC) to MIC ratio
reaching a maximum of 15 except for compound
(
Table 4: ATP pool depletion by diamino 8d (Table 5). However, the two scaffolds differed
quinolines & aminopyrazolopyrimidines.
in kill rates as determined by the in vitro kill
kinetics under replicating conditions. Compound 8g
Inhibitor
% ATP
(diaminoquinoline) showed a faster kill rate,
depletion
dependent on both concentration and time whereas
17a (aminopyrazolopyrimidine) seemed to display
a dependence on time for cidality (Figs. 5A and
INH
ꢀ6
76
22
25
5
Bedaquiline
5
B).
8f
8
8
g
a
8
d
68
25
17a
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1
4
Table 5: Bactericidal activity of diamino
quinolines & aminopyrazolopyrimidines
Fig. 5. In vitro survival kinetics of 8g and 17a:
M. tuberculosis cells were exposed to 0–200 M of
ꢀ
compounds 8g (A) and 17a (B) over a period of 7
days. Appropriate dilutions of culture samples were
plated for measuring colony forming units on 7H11
plates on 0 (start of exposure), 3 and 7 days post
compound exposure.
Compound
Aerobic
Hypoxic
M.tb
MIC
M)
M.tb
MBC
M.tb
MBC
(
ꢀ
(
ꢀ
M)
(
ꢀM)
3
d
6.2
3.1
2.2
6.2
2.2
3.1
6.2
7.4
0.2
25
50
2
.8. Diaminoquinolines & amino pyrazolo
3
f
25
19
25
54
38
50
28
1.6
13
3
pyrimidines retard growth of M. tuberculosis in
a mouse model of TB infection
3h
3
e
50
Based on the potency and selectivity for
inhibition of mycobacterial ATP synthesis, 17a and
g were tested for efficacy in an acute model of TB
8d
200
50
50
100
4
8
g
8
infection. Plasma concentrationꢀtime profiles in
infected mice have been shown in Fig. 6A. In the
infected mice, total plasma concentration of 17a
was above MIC for 24 h whereas the same for 8g
was below MIC during the entire dosing interval.
Both the compounds were able to retard the growth
of M. tuberculosis during the course of infection in
mouse lungs unlike the reference drug isoniazid
which exhibited a bactericidal effect by reducing
the bacterial load by more than two logs in the two
week treatment period (Fig. 6B).
8
e
1
7a
Bedaquiline
1
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inhibit M. tuberculosis growth by reducing the
intracellular ATP pool although not as effectively
as bedaquiline. Docking studies implicated the
mode of action to be the inhibition of ATP synthase
enzyme, however, the exact molecular mechanism
of how these compounds deplete the cells off ATP
still needs to be elucidated as no cross resistance
was observed with bedaquiline resistant mutants.
Scaffold morphing efforts resulted in the
identification of multiple cores which enhanced the
scope of SAR expansion for the series. Compounds
Fig. 6. (A) Pharmacokinetic profiles ꢀ 8g and 17a belonging to diaminoquinoline subseries exhibited
were orally administered as a suspension in 0.5% best enzyme inhibition and antiꢀmycobacterial
hydroxypropyl methylcellulose (HPMC) & Tween potency with a fairly good selectivity index among
8
0 at a 10ꢀml/kg dose volume and (B) Log10 lung all the compounds analyzed. Representative
CFU in various groups: untreated early control compounds from the all subseries exhibited
blue circles, filled) after 3 days of infection; bactericidal activity against M. tuberculosis and
(
untreated late control (blue squares, filled) after 2 were able to demonstrate growth retardation of the
weeks of infection; after treatment with isoniazid at bacilli in the lungs of infected mice as compared to
1
0 mg/kg (green symbols) or with 8g (red symbols) the uninhibited control in a murine model of
or 17a (yellow symbols) at 100 or 200 mg/kg once tuberculosis infection.
daily (UID), 6 days per week for 2 weeks.
Additional medicinal chemistry efforts are
needed to further optimize the potency, selectivity
and pharmacokinetic profiles of these lead series to
identify a clinical candidate. Being a novel
chemotype with activity against bedaquiline
resistant mutant, we believe that this class of
compounds has the potential to be developed as an
antiꢀTB drug candidate.
Growth retardation effect by compound 17a was
more pronounced than that of 8g probably due to
its higher plasma concentration. However, the
extent of growth inhibition observed with both
compounds seemed to be maximal as no dose
response effect was seen with the two doses (100
and 200 mg/kg) tested in the study. The growth
inhibition observed with 8g despite its lower
Abbreviations
plasma concentration could be due to the M.tb: M. tuberculosis
accumulation of the compounds in the mouse OxꢀPhos: Oxidative phosphorylation
lungs.
Conclusion
DiOC2: Diethyloxacarbocyanine iodide
CCCP: Carbonyl cyanide mꢀchloro phenyl
hydrazine
Myc_ATPS: Mycobacterial ATP synthesis assay
In conclusion, we report here the discovery of SMP_ATPS: Mitochondrial ATP synthesis assay
diaminoquinoline, aminopyrazolopyrimdines and MIC: Minimum Inhibitory Concentration
azaindoles as novel mycobacterial ATP synthesis MBC: Minimum Bactericidal Concentration
inhibitors with potent M. tuberculosis MIC. Like
bedaquiline, these compounds were found to
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1
6
Author Contributions
References
S.J.T., S.H.P, S.D.M., A.R., C.C., P.I. and M.P.
are responsible for medicinal chemistry design and
analyses. S.J.T., V.S., S.D.M. and L.K.J. are
responsible for synthetic chemistry. S.R. and M.M
are responsible for compound purity. A.K.G and
M.P. designed and performed the modelling.
S.H.P., K.M., M.P., S.K. M.M. and V.B. performed
the hit triage for the high through screen output.
K.M., H.R., T.Y. and K.M. designed IC50
determination experiments. G.B. and J.B.
performed IC50 determination experiments and
analyzed the data. V.R., V.K.S., K.M., V.B. and
S.R. designed the microbiology experiments. A.N.,
S.S., P.K., N.D., S.G., performed microbiology
experiments. V.P.H., S.S., S.N. and R.S. Designed
and Analyzed PKꢀPD experiments. N.K., R.N.,
S.B., J.R., V.P., P.K.R., K.K. and R.S. performed
PKꢀPD experiments. S.R., G.B. and J.B designed,
performed and analyzed ATP pool measurement
assay. S.J.T., S.D.M., A.K.G., S.R. and S.H.P wrote
the manuscript with contributions from all coꢀ
authors.
Acknowledgment
We sincerely thank the compound management
group
AstraZeneca, Waltham, USA), and Global
Discovery Sciences, Global Safety Assessment
AstraZeneca, Alderley Park, UK) for technical
(AstraZeneca
Bangalore),
DMPK
(
(
support. We thank Ms. Anisha Ambady and Ms.
Naina V Mudugal for providing the Bedaquiline
resistant mutants. We acknowledge Sandeep
Ghorpade and Pravin Shirude for scientific
discussions. We also thank Dr. R. Tommasi and Dr.
T. S. Balganesh for their constant support and
encouragement.
Funding Sources
Global Alliance for TB Drug Development.
1
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DOI: 10.1039/C5MD00589B
Scaffold Morphing Led to Evolution of 2, 4-
Diaminoquinolines and Aminopyrazolopyrimidines as
Inhibitors of ATP Synthesis Pathway
a
a
a
a
Subramanyam J. Tantry, Vikas Shinde, Gayathri Balakrishnan, Shankar D. Markad, Amit
a
a
a
a
a
a
K Gupta, Jyothi Bhat, Ashwini Narayan, Anandkumar Raichurkar, Lalit Kumar Jena,
a
a
a
a
Sreevalli Sharma, Naveen Kumar, Robert Nanduri, Sowmya Bharath, Jitendar Reddy,
Vijender Panduga, Prabhakar K. R, Karthikeyan Kandaswamy, Parvinder Kaur, Neela
Dinesh, Supreeth Guptha, Ramanatha Saralaya, Manoranjan Panda, Suresh Rudrapatna,
Meenakshi Mallya, Harvey Rubin, Takahiro Yano, Khisi Mdluli, Christopher Cooper,
Balasubramanian V, Vasan K. Sambandamurthy,
Shandil, Stefan Kavanagh, Shridhar Narayanan, Pravin Iyer, Kakoli Mukherjee, Vinayak
P Hosagrahara, Suresh Solapure, Shahul Hameed P, * Sudha Ravishankar. *
a
a
a
a
a
a
a
a
a
a
d
d
b
b
a
a
a
Vasanthi Ramachandran, Radha
a
c
a
a
a
a
a
a
a
a
Innovative Medicines, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, India.
Global Alliance for TB Drug Development, 40 Wall Street, 24th Floor. New York, NY 10005, USA.
b
c
AstraZeneca, Alderley Park, Mereside, Cheshire, UK.
d
University of Pennsylvania, 111 Clinical Research Building, 415 Curie Boulevard, Philadelphia PA 19104, USA
2
, 4-diaminoquinazolines, 2, 4-diaminoquinolines and aminopyrazolopyrimidines, inhibitors
of mycobacterial ATP synthesis, are novel lead molecules towards discovery and
development of new anti-tubercular agents.