Discovery and Optimization of Potent, Selective, and in Vivo Efficacious 2-Aryl Benzimidazole BCATm Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00389

To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.

Full text of DOI:10.1021/acsmedchemlett.5b00389

Letter
pubs.acs.org/acsmedchemlett
Discovery and Optimization of Potent, Selective, and in Vivo
Efficacious 2‑Aryl Benzimidazole BCATm Inhibitors
,†
†,⊥
†
†
‡
Hongfeng Deng,* Jingye Zhou, Flora Sundersingh, Jeffrey A. Messer, Donald O. Somers,
§
†
‡
†
Myriam Ajakane, Christopher C. Arico-Muendel, Arthur Beljean, Svetlana L. Belyanskaya,
‡
∥
§
§
†
‡
Ryan Bingham, Emily Blazensky, Anne-Benedicte Boullay, Eric Boursier, Jing Chai, Paul Carter,
‡
§
†
§
‡
∥
Chun-Wa Chung, Alain Daugan, Yun Ding, Kenny Herry, Clare Hobbs, Eric Humphries,
†
§
§
‡
,§
Christopher Kollmann, Van Loc Nguyen, Edwige Nicodeme, Sarah E. Smith, Nerina Dodic,*
and Nicolas Ancellin*
,§
†
Platform of Technology and Science, GlaxoSmithKline, 830 Winter Street, Waltham, Massachusetts 02451, United States
Medicines Research Centre, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, U.K.
‡
§
∥
Centre de Recherche, GlaxoSmithKline, Les Ulis, 25,27 Avenue du Queb
Chemistry Department, Northeastern University, Boston, Massachusetts 02115, United States
S Supporting Information
́
ec, 91140 Villebon sur Yvette, France
*
ABSTRACT: To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity
screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both
biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-
(
5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with
much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via
multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched
chain amino acids as a consequence of BCATm inhibition.
KEYWORDS: BCATm, branched chain amino acids (BCAAs), DNA Encoded Library, ELT, 2-aryl benzimidazole
ecent studies suggest branched chain aminotransferases
(BCAAs) leucine, isoleucine, and valine to their respective α-
keto acids, which are further converted to succinyl-CoA and
acetyl-CoA and participate in the tricarboxylic acid cycle and
R
(
BCATs) as a potential target for metabolic disorders.
BCATs catalyze transamination of branched chain amino acids
1
glycolysis. There are two isozymes, mitochondrial (BCATm)
and cytosolic (BCATc), and the human BCAT isozymes are
Scheme 1. Structure of DNA Encoded Benzimidazole
Library
2
5
8% identical in amino acid sequence. While BCATm is
expressed in many peripheral tissues, BCATc is largely
1
restricted to the central nervous system. A recent mouse
BCATm knockout study demonstrated that lack of this enzyme
increased the mice’s energy expenditure, associated with a futile
protein turnover cycle, and protected them from obesity when
Received: October 5, 2015
Accepted: February 8, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00389
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ACS Medicinal Chemistry Letters
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Table 1. Benzimidazole 2-Position SAR
Figure 1. Result of BCATm screen of the DNA encoded
benzimidazole library and the generic structures of the two BCATm
hit series I and II, where BB1, BB2, and BB3 refer to cycle 1, 2, and 3
building blocks, respectively. Compounds on line 1 and line 2 share
common BB1 and BB3 as indicated in their corresponding frames with
BB2 as a variable component. The size of a dot is proportional to the
DNA tag copy number. Library members with a single copy were
removed to simplify visualization. An enlarged Figure 1 can be found
in the SI section.
Scheme 2. Synthesis of 1,2,5-Substituted Benzimidazole
a
Analogues
Scheme 1, the library utilized 65 monoprotected diamines at
cycle 1, formed the benzimidazole ring with 922 aldehydes at
cycle 2, then following amine deprotection was reacted with
a
Reagents and conditions: (a) tert-butyl (3-aminocyclo-hexyl)-
carbamate (mixture of diastereomers), DIEA, EtOH, 85 °C, 6 h; (b)
2
2
carboxylic acid, HATU, DIEA, AcCN; (d) (i) 5-bromothiophene-2-
carboxylic acid, HOBT, EDCI, TEA, DCM; (ii) TFA/DCM; (e) 4-
fluoro-N-methyl-3-nitrobenzamide, DIEA, EtOH, 85 °C, 6 h.
1
960 building blocks at cycle 3 to afford the final library of 117
-(methylthio)benzaldehyde, Na S O , 1,4-dioxane/H O (4:1), 80 °C,
2 2 4 2
million compounds. Selection for the diamines, the aldehydes,
and the cycle 3 building blocks was based on availability, drug-
like properties, and reactivity. The details of the library
synthesis will be the subject of a different publication in the
near future.
4 h; (c) (i) TFA/DCM, overnight; (ii) 5-bromothiophene-2-
The screening of this library suggested two preferred
subscaffolds I and II for BCATm as illustrated by a Spotfire
cube view in Figure 1. The most promising BCATm hit from
this library screening is exemplified by compound 1 (I, R2 = 2-
(methylthio)phenyl, Figure 1; also in Scheme 2) which
exhibited human BCATm inhibition in both biochemical and
cellular assays (pIC , 6.6 and 6.8, respectively) with about 5-
3
subjected to a high fat diet. To further validate BCATm as a
therapeutic target for the intervention of metabolic disorders, a
suitable small molecular inhibitor with potency, selectivity, and
in vivo activity is highly desirable. To develop such inhibitors,
we utilized various screening technologies including HTS,
fragment-based screen, and DNA Encoded Library Technology
50
4
−6
(
ELT).
Herein we report the discovery of a novel BCATm
fold selectivity over BCATc (pIC , 5.9). Mouse pharmacoki-
50
7
−12
hit series from ELT
and subsequent structure−activity
netic (PK) study suggested that 1 has a short half-life of 1.4 h
and low oral bioavailability (10%). To improve potency and PK
properties we conducted SAR studies around three different
moieties of this benzimidazole-based scaffold including the
benzimidazole 2-position (R2), the bromothiophene moiety,
and the benzimidazole 5-position where DNA was originally
attached. Since the subscaffold II is very similar to the
subscaffold I, no exemplars were made for this series.
relationship studies, which ultimately led to the discovery of a
not only highly potent but also highly selective and in vivo
efficacious compound (8b) that raised serum BCAAs levels
after oral administration to mice.
In pursuit of novel small molecule inhibitors of BCATm, we
screened the chemically biotinylated human BCATm protein
construct against our more than 14 billion DNA encoded
compounds. Several hit series were identified and one of
them was from a three-cycle benzimidazole based novel library
that contains 117 million distinct compounds. As shown in
1
3
A representative synthesis for the proposed modifications is
illustrated in Scheme 2. Briefly, fluoro replacement of 4-fluoro-
3-nitrobenzamide (2) with 1,3-cyclohexyldiamine followed by
B
DOI: 10.1021/acsmedchemlett.5b00389
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Table 2. SAR for the Bromothiophene Moiety
Table 3. SAR at Benzimidazole 5-Position
We began our structural optimization by addressing the
potentially labile 2-(methylthio)phenyl group. As shown in
Table 1, at the benzimidazole 2-postion, replacement of this
group with unsubstituted phenyl (8a) led to increased BCATm
inhibitory activity. We then further converted the phenyl group
into 2-pyridinyl (8b), 3-pyridinyl (8d), and 4-pyridinyl (8e)
groups in a hope to further decrease lipophilicity while
maintaining adequate potency. The 2-pyridinyl analogue
showed similar activity to the phenyl analogue in both
enzymatic and cellular assays, whereas both the 3-pyridinyl
and 4-pyridinyl analogues showed decreased potency suggest-
ing that polar interactions in this region are poorly tolerated.
Several five-membered heteroaryl rings including thiophene,
thiazole, and pyrazole were also explored. While the 2- or 3-
thiophene analogues (8f, 8g) achieved similar potency as the
phenyl analogue in both biochemical and cellular assays, the
two thiazole analogues (8h, 8i) showed moderately reduced
activity. Similar to the 4-pyridinyl analogue, the pyrazole
compound (8j) had much decreased enzymatic activity,
confirming the early observation that polarity is not tolerated
here. It is worth mentioning that for all of the compounds in
Tables 1−3 the 1,3-cyclohexyldiamine is in the cis-config-
uration. Switching it to the trans-configuration rendered the
in situ nitro reduction and simultaneous cyclization with an
aldehyde under sodium dithionite (Na S O ) conditions led to
2
2
4
14
the 2-aryl benzimidazole intermediate (4), which after Boc
deprotection was acylated by an appropriate carboxylic acid to
provide the desired product 1 and its analogues. HPLC
purification yielded two diastereomers (each is a racemic
mixture of two enantiomers) with the cis-isomer being the
more active configuration. Alternatively, the disynthon N-(3-
aminocyclohexyl)-5-bromothiophene-2-carboxamide (6, cis-
confirmation) was synthesized first followed by fluoro
replacement of 4-fluoro-3-nitrobenzamide and cyclization with
an appropriate aldehyde to afford the desired compound 1 and
its analogues.
compounds almost inactive.
We next explored SAR around the 5-bromothiophene-2-
carboxamide moiety including introducing bromo replace-
ments, thiophene ring replacements, and modifications of the
linker amide bond. Two series of compounds with either
carboxamide (9A) or N-methyl carboxamide (9B) at the
benzimidazole 5-position were simultaneously prepared for this
purpose as shown in Table 2. In the 9A series, cyano (9Aa) and
chloro (9Ab) replacements of bromo group led to moderately
decreased activity in both enzymatic and cellular activities, and
the methyl analogue (9Ac) produced >10-fold activity decrease
highlighting the importance of the bromo substituent. Likewise,
N-methylation of the amide linkage (9Ad) or conversion to a
sulfonamide (9Ae) significantly abolished activity. In the 9B
series, additional heteroaryl rings as thiophene replacements
All of the synthesized compounds were evaluated for
BCATm inhibition in a biochemical assay, and some selected
4
,5
compounds were further tested in a cellular assay. Both
enzymatic and cellular activities are summarized in Tables 1−3.
C
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Table 4. PK Data for Representative Compounds
mice IV 1 mg/kg
mice PO 5 mg/kg
Cmpd # BCATm pIC50 BCATc pIC50 hCell pIC50 Clb (mL/min/kg) t1/2 (h) Vss (L/kg) AUC0‑inf (ng·h/mL) Cmax (ng/mL) F% mice PPB (%)
1
8
6.6
7.3
5.9
5.4
7.2
7.0
23
1.4
3.7
1.3
0.9
338
129
10
28
99.5
97.1
b
4.0
5809
1648
Figure 2. Views of X-ray crystal structure of BCATm in complex with 8b (in cyan) and cofactor PLP (in yellow).
Figure 3. Overlay of the X-ray structures of 8b, 11, and 12.
Figure 5. Acute dosing of 8b at 30, 100, and 300 mg/kg P.O. followed
by refeeding of amino acid mix after 6 h: quantification of BCAAs
serum level at 1 h postfeeding.
azol-5-yl (9Bd) analogues were all much less active than the
thiophene ring identified from the original library screen.
Lastly we explored substitution effect at the benzimidazole 5-
position where the original DNA linker was attached. As shown
in Table 3, the methyl amide (8b) and primary amide (8c)
have similar enzymatic activity, but the latter showed a
significant decrease in cellular activity. Removal of the
carboxamide moiety (10a) led to more than 1 log loss of
enzymatic activity, and its cellular activity is undetectable.
Converting the carboxamide to N,N-dimethyl amide (10b)
rendered the compound almost inactive. An effort to ensure the
optimal substitution at this position prompted the synthesis of
the cyclopropyl amide (10c) and isopropyl amide (10d)
analogues, but neither exceeded the activity of 8b. Therefore,
the methyl amide group seems the best fit for this position.
Numerous compounds with potent enzymatic and cellular
activities were progressed to BCATc selectivity and pharmaco-
kinetic (PK) studies. The compound 8b was found to inhibit
BCATc with pIC50 = 5.4 (n = 2), which is about 100-fold
selective for BCATm over BCATc, a significant improvement
Figure 4. Acute dosing of 8b at 300 mg/kg followed by refeeding the
mice with amino acid mix after 30 min: quantification of BCAAs serum
level at 1 h postfeeding.
were explored. Switching the thiophene ring to thiazole (9Ba)
led to almost 2 log loss of activity. Similarly 5-bromo-N-methyl
pyrrol-2-yl (9Bb), 4-bromo-pyrro-2yl (9Bc), and 3-chloroisox-
D
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ACS Medicinal Chemistry Letters
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compared to the initial hit, 1 (pIC , BCATm/BCATc, 6.6/
the initial findings and demonstrated dose-dependent enhance-
ment of all three BCAA levels relative to serine and vehicle
controls. These experiments demonstrated that the BCATm
inhibitor 8b effectively raised the levels of BCAAs in an acute
animal model over an extended duration.
5
0
5.9). Meanwhile, 8b also demonstrated a superior PK profile
relative to 1 as shown in Table 4. Compound 8b has decreased
clearance (4.0 mL/min/kg vs 23 mL/min/kg) and significantly
increased oral bioavailability (28% vs 10%) and lower plasma
protein binding (97.1% vs 99.5%). As a consequence, a longer
T1/2 and much increased exposure and Cmax were achieved by
In summary, affinity screening of a 117 million DNA
Encoded Library identified a novel benzimidazole-based
BCATm hit series, which was followed by an extensive SAR
study that led to the discovery of compound 8b, a potent and
selective BCATm inhibitor with reasonable PK properties. The
X-ray crystal structure of BCATm in a complex with 8b
revealed that the compound binds to BCATm at a site that is
proximate to the catalytic residue lysine 202 and cofactor PLP
binding site. Finally, compound 8b demonstrated oral efficacy
in a mouse PD model, where it successfully raised the levels of
all three BCAAs. Compound 8b is thus a valuable
pharmacological tool for investigating the biological roles of
BCATm in metabolic diseases.
8b. Compound 8b was further found to inhibit mouse BCATm
with pIC = 5.9 (n = 4) in the cellular assay. Therefore, 8b was
50
selected as a candidate for in vivo studies aimed at investigating
the pharmacology of BCATm inhibition in mice.
A high-resolution (2.0 Å) X-ray crystal structure of BCATm
in complex with compound 8b was obtained as shown in Figure
2. In this structure, compound 8b binds to the active site of
BCATm proximal to the pyridoxal-5′-phosphate (PLP)
cofactor, which is covalently linked to Lys202 (Figure 2a).
The ligand adopts a T-shaped conformation directing the 2-
pyridyl toward the PLP, while the benzimidazole ring itself sits
perfectly sandwiched between Phe30 and Ala314 forming
extensive aromatic contacts with both (Figure 2b). The methyl
amide at the benzimidazole 5-position makes H-bonds from its
carbonyl to the basic nitrogen of Lys79 and to water from its
NH group (Figure 2c). Increasing the size of the amide
substituent from methyl to cyclopropyl (10c) or isopropyl
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00389.
(
10d) decreases affinity due to the restricted space at this
Experimental for the synthesis of compounds, X-ray
crystal structure of BCATm/PLP in complex with
inhibitor 8b, the X-ray data summary table for 8b, and
enlarged Figure 1 (PDF)
position. The cis-1,3-diaminocyclohexyl group at the benzimi-
dazole 1-position directs the 5-bromo-thiophene ring deep into
a hydrophobic pocket formed by Met241, Gln224, Val182, and
Cys318 (Figure 2a). The amide linker further anchors this
trajectory by forming an H-bond between its carbonyl oxygen
and the backbone NH of Thr240, and a water-bridged
interaction between the amide NH and Tyr173OH. The
thiophene sulfur atom lies on the side of the ring next to the
amide oxygen. Exchanging the bromine on the thiophene for
the chlorine (9Ab) or methyl (9Ac) group results in reduced
activity thus bromine here provides the best lipophilic contact
with the highly hydrophobic pocket.
Accession Codes
The PDB code for 8b is 5HNE
AUTHOR INFORMATION
Corresponding Authors
*Phone: 1-7817954302. E-mail: hongfeng.x.deng@gsk.com.
*Phone: 33-169296035. E-mail: nerina.5.dodic@gsk.com.
*Phone: 33-169296068. E-mail: nicolasancellin@free.fr.
■
A comparison of binding modes between 8b and the recently
published ELT series 11 (compound 15e in ref 4, PDP code
Present Address
⊥
(
J.Z.) Lilly China Research and Development Center
5CR5) as well as the HTS series 12 (compound 66 in ref 5,
(
LCRDC), Eli Lilly and Company, 338 Jia Li Lue Road,
PDP code 5BWX) revealed that the two ELT series bind to
BCATm in a similar orientation, while they only partially
overlap with the HTS series (Figure 3). The key interactions in
Shanghai 201203, P. R. China.
Notes
All studies were conducted in accordance with the GSK Policy
on the Care, Welfare and Treatment of Laboratory Animals and
were reviewed by the Institutional Animal Care and Use
Committee either at GSK or by the ethical review process at
the institution where the work was performed.
The authors declare no competing financial interest.
8b including the extensive aromatic contacts with both Phe30
and Ala314 (Figure 2b) and hydrogen bond interactions with
both Lys79 and a water (Figure 2c) are missing in both 11 and
1
2, which could account for the reduced potency of 11 and lack
of selectivity of 12. The crystallographic analysis also confirms
the outcome of the SAR work and suggests that the
requirements for good binding were well met by the structural
diversity of the benzimidazole library.
ABBREVIATIONS
■
BCAAs, branched chain amino acids; BCATm, mitochondrial
branched-chain aminotransferase; BCATc, cytosolic branched-
chain aminotransferase; ELT, Encoded Library Technology;
NTC, no-target control; PLP, pyridoxal 5′-phosphate
To test whether compound 8b can increase the level of
circulating branched chain amino acids, mice were given either
the 300 mg/kg compound P.O. or vehicle after 5 h of fasting.
After 30 min, the mice were refed with amino acid mixture (1.5
g/kg/10 mL, P.O.) followed by blood and tissue sampling at 1
h postfeeding to quantify amino acid levels. As shown in Figure
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F
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ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX