Use of a Conformational-Switching Mechanism to Modulate Exposed Polarity: Discovery of CCR2 Antagonist BMS-741672

Article abstract of DOI:10.1021/acsmedchemlett.8b00439

We encountered a dilemma in the course of studying a series of antagonists of the G-protein coupled receptor CC chemokine receptor-2 (CCR2): compounds with polar C3 side chains exhibited good ion channel selectivity but poor oral bioavailability, whereas

Full text of DOI:10.1021/acsmedchemlett.8b00439

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Letter
Use of a conformational switching mechanism to modulate
exposed polarity: discovery of CCR2 antagonist BMS-741672
Michael G. Yang, Zili Xiao, Robert Joseph Cherney, Andrew Joseph Tebben, Douglas G. Batt,
Gregory D. Brown, Jing Chen, Mary Ellen Cvijic, Marta Dabros, John V. Duncia, Michael A.
Galella, Daniel S Gardner, Purnima Khandelwal, Soo S Ko, Mary F Malley, Ruowei Mo, Jian Pang,
Anne V Rose, Joseph B Santella, Hong Shi, Anurag S. Srivastava, Sarah C. Traeger, Bei Wang,
Songmei Xu, Rulin Zhao, Joel C Barrish, Sandhya Mandlekar, Qihong Zhao, and Percy H Carter
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00439 • Publication Date (Web): 16 Jan 2019
Downloaded from http://pubs.acs.org on January 17, 2019
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Use of a conformational switching mechanism to modulate exposed
polarity: discovery of CCR2 antagonist BMS-741672
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Michael G. Yang, Zili Xiao, Robert J. Cherney, Andrew J. Tebben, Douglas G. Batt, Gregory D.
Brown, Jing Chen, Mary Ellen Cvijic, Marta Dabros, John V. Duncia, Michael Galella, Daniel S.
Gardner, Purnima Khandelwal, Soo S. Ko, Mary F. Malley, Ruowei Mo, Jian Pang, Anne V. Rose,
Joseph B. Santella, III, Hong Shi, Anurag Srivastava, Sarah C. Traeger, Bei Wang, Songmei Xu, Rulin
Zhao, Joel C. Barrish, Sandhya Mandlekar, Qihong Zhao and Percy H. Carter*
Bristol-Myers Squibb Company, Research & Development, Rt. 206 & Province Line Rd., Princeton, NJ 08543 (USA)
KEYWORDS chemokine, G protein-coupled receptor, conformational analysis, hydrogen bonding, oral bioavailability, ion
channels.
ABSTRACT: We encountered a dilemma in the course of studying a series of antagonists of the G-Protein Coupled Receptor CC
Chemokine Receptor-2 (CCR2): compounds with polar C3 sidechains exhibited good ion channel selectivity but poor oral
bioavailability, whereas compounds with lipophilic C3 sidechains exhibited good oral bioavailability in pre-clinical species but
poor ion channel selectivity. Attempts to solve this through the direct modulation of physicochemical properties failed. However,
the installation of a protonation-dependent conformational switching mechanism resolved the problem, as it enabled a highly
selective and relatively polar molecule to access a small population of a conformer with lower polar surface area and higher
membrane permeability. Optimization of the overall properties in this series yielded the CCR2 antagonist BMS-741672 (7), which
embodied properties suitable for study in human clinical trials.
Drug discovery efforts are frequently characterized by
challenges in obtaining compounds with the right combination
of biological activity, off-target selectivity, and oral
bioavailability. Our studies on a series of antagonists of the
G-protein coupled receptor CCR2 have uncovered similar
issues, as have many other programs at different
institutions.1,2,3 Although each research program is unique,
general correlations between physicochemical properties and
drug candidate attrition have now been posited.4 Herein we
focused on the development of small molecule CCR2
antagonists, as highlighted by the two representative
molecules below (Figure 1).
N
N
O
O
CF3
CF3
H
N
H
N
N
N
O2S
N
O
N
1
2
describe the discovery of
a
protonation-dependent
conformational switching mechanism that enabled the
discovery of small molecules with both sufficient polarity to
CCR2 Bnd IC50 = 0.7 nM
CCR5 Bnd IC50 = 2.4 nM
hERG = 83% Inhib @ 10 uM
CCR2 Bnd IC50 = 1.8 nM
CCR5 Bnd IC50 > 1,000 nM
hERG = 4% Inhib @ 10 uM
rat PK = 1 F% (PO)
engender selectivity and the ability to adopt
a more
rat PK = 68 F% (PO)
hydrophobic conformation to facilitate oral bioavailability.
This approach enabled us to identify a CCR2 antagonist
suitable for clinical studies and may be valuable for other
scientists engaged in drug design.
Figure 1. Compounds with similar CCR2 binding affinity but
strikingly different overall profiles.
CCR2 is a G protein-coupled receptor that plays a central
role in the biology of classical “inflammatory” monocytes,5
mediating their emigration from the bone marrow and
extravasation into inflamed tissues,6 wherein they differentiate
into either monocyte-derived macrophages or monocyte-
Compound 1, a potent CCR2/CCR5 dual antagonist,
exhibited good oral bioavailability in the rat, as well as mouse,
monkey, and dog.13 Although it was selective over hERG, as
assessed by a >2,000-fold difference in CCR2 binding IC50
(0.7 nM) and its hERG patch clamp IC50 (1,800 nM), our
subsequent detailed in vivo electrophysiology studies
derived dendritic cells.7
Unlike their tissue-resident
counterparts, CCR2+ macrophages promote inflammation
through the secretion of cytokines and other inflammatory
mediators.8 Consistent with this paradigm, gene knockout or
knockdown studies have implicated CCR2 as playing a
contributory role in numerous pre-clinical analogs of human
diseases, including models of diabetes, atherosclerosis,
autoimmune disease, and cancer.9,10,11,12 Our own efforts
suggested
that
the
molecule
did
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Table 1. Key in vitro data for analogs of 1 and 2.a
1
2
3
4
5
N
N
O
O
CF3
CF3
H
H
N
N
N
N
X
X
6
O
N
N
7
2, 3, 4
1, 5, 6, 7
8
9
CCR2 Binding
(IC50, nM)
CCR5 Binding
(IC50, nM)
hERG p.c.
PAMPA perm.
(nm/sec)
HLM
Cmpd
X
10
11
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(%Inhibition)
(rate, %rem.)
0.00, 100%
0.05, 85%
0.00, 100%
0.00, 100%
0.00, 99%
0.13, 58%
0.01, 95%
2
3
4
1
5
6
7
CH2SO2CH3
NHSO2CH3
N(Me)SO2CH3
CH2CH2CH3
CH2SO2CH3
NHSO2CH3
NHC(O)CH3
1.8  0.8 (12)
1.9  0.8 (10)
1.4  0.6 (8)
0.7  0.3 (23)
0.9  0.3 (5)
0.4  0.20 (5)
1.1  0.7 (18)
1636  760 (6)
3469  906 (3)
1570 (1)
4%, 10 M
45%, 10 M
7%, 10 M
83%, 10 M
16%, 30 M
74%, 10 M
33%, 10 M
94  58 (10)
593 (1)
65  43 (3)
529  157 (9)
14 (1)
2.4  1.0 (200)
190 (1)
287  196 (2)
780b
394  340 (2)
443  114 (8)
aBoth binding values are reported as mean  SD (n). CCR2 binding was measured through blockade of MCP-1 (CCL2) binding to
human peripheral blood mononuclear cells. CCR5 binding was assessed through antagonism of MIP-1 binding to HT1080 cells stably
expressing CCR5. Inhibition of hERG was assessed in a manual patch clamp assay, and is expressed as %inhibition at a single test
concentration. PAMPA permeability (reported in nanometers/second) was assessed at RT and a pH of 7.4; data are reported as the average
of a single experiment performed in triplicate (standard deviation <10% mean) or as the average of multiple runs (with standard deviation
across those experiments). The metabolism in human liver microsomes at a substrate concentration of 3 M is shown as rate of
disappearance (nmol/min/mg) and % parent remaining (10 min). bThe CCR5 binding for this lead compound 7 was also assessed in native
T-cells, wherein it exhibited a binding value of 23.6  12 nM
not have a sufficient safety margin for advancement. In
contrast, the CCR2-selective antagonist 2 exhibited high
selectivity over hERG, which was confirmed in vivo, but did
not have good oral bioavailability in the rat, mouse, or
monkey.14 In order to improve oral bioavailability and ion
channel selectivity in our tri-substituted cyclohexyl series, we
engaged in extensive structure-activity relationship (SAR)
studies of the exocyclic amine, cyclohexyl C3-sidechain, and
benzamide region.15 Generally speaking, our efforts were
permeability (Table 1).
It was surprising to us that
sulfonamide 6 had a PAMPA Pc that was similar to that of the
n-propyl analog 1, despite the marked difference in their
polarity (cLogP of 1.0 vs. 4.7). We synthesized a range of
compounds that contained different sidechain motifs and
studied the PAMPA permeability of those that exhibited
appropriate CCR2 affinity. A selection of these compounds is
shown in Figure 2. A clear structure-property relationship was
evident: those compounds that contained an –N(H)R motif as
the linking unit on the C3-sidechain (Fig. 2, blue data series)
exhibited higher PAMPA values than those that did not (Fig.
2, red data series). This trend held largely independent of
cLogP, although there were exceptions at the high and low end
of the cLogP range (as expected).
aimed at introducing polarity into analogs of
1 and
hydrophobicity into analogs of 2, in an attempt to find a
compound with more balanced properties.16 A particularly
noteworthy result emerged from the replacement of the
proximal methylene of sulfone 2 with an amine to give
sulfonamide 3 (Table 1). Although the sulfonamide exhibited
similar CCR2 and CCR5 binding affinities to the sulfone, it
had lower hERG selectivity and, to our surprise, higher
PAMPA permeability, making it closer to the n-propyl series
(cf. 1 - 3, Table 1). In contrast, the N-methyl sulfonamide 4,
which was ostensibly more lipophilic than 3, had good hERG
selectivity and poor PAMPA permeability, like the polar
sulfone 2. The differences in permeability and metabolism
between compounds 2 – 4 were confirmed in vivo. Relative to
sulfone 2, sulfonamide 3 exhibited higher oral bioavailability
in mouse (35 vs 16 F%) despite having higher clearance (258
vs. 70 mL/min/kg). In contrast, N-Me sulfonamide 4
exhibited poor mouse pharmacokinetics, like 2 (IV Cl = 67 vs.
70 mL/min/kg; PO F% 11 vs. 16).
Relative to their sulfone counterparts, both sulfonamides
exhibited worse metabolic stability (Table 1, cf. 3 and 2; 6 and
5). Intriguingly, the N-Me analog 4, which had a new
metabolic soft spot, exhibited superior metabolic stability to
the parent 3 in both human and rodent liver microsomes.
Analysis of the in vitro metabolites indicated that the
dominant metabolic pathway for 2 and 3 was identical:
oxidative demethylation of the tertiary amine.
remarkably, the simple acetamide did not exhibit this
metabolic
Quite
The replacement of the sulfone sidechain with
a
sulfonamide was also studied in the context of the quinazoline
capping group. The sulfonamide 6 retained the CCR2-
selectivity of the sulfone 5, but exhibited improved PAMPA
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Scheme 1. Synthesis of BMS-741672 (7) a
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2
3
O
Boc
Boc
N
a - d
N
NHCbz
N
NHCbz
84%
4
O
O
5
6
7
8
S.1
S.2
e
93%
BocHN
O
BocHN
O
O
f, g
N
NHCbz
N
NHCbz
9
87%
N
H
HO
O
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S.4
S.3
N
O
O
CF3
H
N
h - l
86%
N
N
Figure 2. Shown is a plot of cLogP vs. PAMPA (normalized to
reference standard 1) for a series of analogs from the tri-
substituted cyclohexyl series. All of these compounds are analogs
of 1/5/6, in that they contain the N-isopropyl, N-methyl-amine
motif, the -lactam linker, and the quinazoline capping group.
H
N
N
7
a Reagents & Conditions: (a) 1 atm H2, Pd/C, EtOAc; (b) Cbz-
Met-OH, TBTU, iPr2NEt, MeCN; (c) MeI, 48 h; (d) Cs2CO3,
DMSO; (e) LiOH, THF/H2O; (f) EDC, HOBt, CH2Cl2, then NH3;
(g) PhI(OAc)2, MeCN/H2O, then Ac2O, iPr2NEt; (h) TFA,
CH2Cl2; (i) acetone, NaBH3CN, MeOH; (j) CH2O, NaB(OAc)3H,
CH2Cl2; (k) 1 atm H2, Pd/C, MeOH; (l) 4-Cl,6-CF3-quinazoline,
iPr2NEt, iPrOH.
liability, and proved to be one of the most effective
replacements of the sulfonamide that we identified in this
survey. Indeed, acetamide 7 retained potent CCR2 binding
activity while exhibiting high hERG selectivity, PAMPA
permeability, and in vitro metabolic stability (Table 1).17
Furthermore, compound 7 (BMS-741672) had improved
mouse oral bioavailability (28 F%) and IV half-life (38
mL/min/kg) relative to 2, 3, and 4, so we advanced it into
additional studies (see below and ref. 17).
The optimized first generation synthesis of compound 7 is
shown below in Scheme 1. Our previously described18 key
intermediate S.1 was subjected to a four step sequence19 to
install the -lactam. Hydrolytic opening of S.2 to give acid
S.3 was followed by coupling with ammonia to yield the
carboxamide, which was subjected to Hoffman rearrangement
(including trapping with acetic anhydride) to yield acetamide
S.4. Conversion of S.4 to the key compound BMS-741672 (7)
followed a simple sequence consisting of acid-mediated Boc
removal, double reductive amination, hydrogenolysis of the
Cbz, and coupling with 4-chloro-6-trifluoromethylquinazoline.
This route served to provide material for all pre-clinical
studies, and was an important precursor to our recently
reported second generation route.20
In the context of investigating the optimal formulation of
compound 7 to support clinical trials, we conducted a number
of small molecule crystal studies with various salt forms. The
conformation of the molecule depended on whether the
compound was studied as a salt or free base. Specifically,
when studied as an amine salt, we found that the lactam of 7
was axial while the acetamide and amine moieties were
equatorially disposed (Fig. 3A, orange, Lactam Axial
DiEquatorial, “LADE”). This conformation matched that of
the receptor-bound,21 protonated 1 (Fig. 3C, blue), as well as
Figure 3. X-ray crystal structures of compounds 1, 6 and 7. (A)
The bis-benzenesulfonic acid (omitted for clarity) salt of 7
(orange) exists in the chair configuration with -lactam disposed
axially. The free-base of 7 (blue) exists in the chair configuration
with the -lactam disposed equatorially. (B) Hydrogen bonding
network in the 7 free base crystal structure. (C) The X-ray
structures of the free-base of 1 (red) and TFA (omitted for clarity)
salt of 6 (orange) overlaid with the CCR2-bound conformation of
the protonated form of 1 (blue) extracted from the 1/CCR2
complex crystal structure (pdb id 5T1A). All exist in the LADE
chair conformation. (D) Hydrogen bonding network in the 6 TFA
salt crystal structure.
the
TFA
salt
of
compound
6
(Fig.
3C,
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orange; see also Fig. 3D). The free-base of compound 1 was
Table 2. Ab initio calculated relative energies and polar
surface areas.
1
2
3
4
5
6
7
8
also shown to exhibit this LADE conformation (Fig. 3C, red).
However, the crystalline form of the free base of 7 displayed a
very different conformation, in which the cyclohexyl chair
flipped such that the lactam was equatorial, while the
acetamide and amino moieties were diaxially disposed (Fig.
3A, blue, Lactam Equatorial DiAxial, “LEDA”). Note that the
acetamide bridges the tertiary amine and quinazoline NH via
intramolecular H-bonds in this LEDA conformation (Fig. 3B).
The pH-dependent conformational switching of 7 that was
observed in the solid state was further confirmed in solution
studies via 1H-NMR spectroscopy (see Supporting
Information).
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Protonated
Free Base
The aforementioned crystal structures were coupled with ab
initio calculations to enable a detailed understanding of the
protonation dependent conformational biases within these
systems. When the tertiary amine is protonated, the LADE
chair conformation is lower in energy for every substituent in
the third position (Table 2, Protonated; see Supporting
Information for additional details). However, in the free-base
form, the conformational preferences vary depending on the
nature of the C3-substituent. The polar moieties in compounds
6 (sulfonamide) and 7 (acetamide) are able to engage the
hydrogen bonding network observed in the compound 7 free-
base crystal structure (Fig. 3B). These hydrogen bonds
stabilize the LEDA conformation and overcome the
unfavorable steric clashes inherent to the diaxial disposition of
the two substituents. Indeed, calculations predict the LEDA
conformation to be favored by 4.8 and 5.4 kcal/mol for
compounds 6 and 7, respectively (Table 2, Free Base).
Compound 5 shows a smaller calculated bias (0.6 kcal/mol)
toward the LEDA conformation because, while the sulfone
still hydrogen bonds the aminoquinazoline NH, the hydrogen
bond to the tertiary amine is not present in the structure. The
n-propyl group in compound 1 is unable to stabilize the LEDA
conformation through any hydrogen bonding and the
calculated conformational preference is LADE in both the
protonated and free base species.
LEDA
Ea
1.7
LEDA
PSAb PSAb
PSAb PSAb
Ea
3.6
Cpd
1
5
6
7
a
267
270
270
272
266
310
299
282
247
252
255
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242
288
282
264
0.9
-0.6
-4.8
-5.4
1.5
1.9
E = ELEDA – ELADE (kcal/mol). Representative LEDA and
LADE conformations for compound 1 are illustrated above the
table. The minimized conformations for compounds 1, 5, 6 and 7
are shown in SI Figure 1. bPolar surface area, PSA (Å2), is
computed from the 3D conformations.
nors, lowers PSA, and desolvates the acetamide moiety. The
small relative concentrations of the non-protonated, LEDA
form can readily enter the lipid environment, driving the
equilibrium toward this species. The compound then exits the
lipid environment to enter either the aqueous environment of
the lumen or the bloodstream. Once the compound exits the
membrane into the circulation, oral bioavailability has been
secured. In this environment, the equilibrium population will
again favor the protonated LADE conformation, which is the
conformation required for CCR2 binding.21 In this
conformation, the polar sidechain is exposed and the
selectivity for the molecule relative to ion channels is
therefore high. Thus, the model suggests that compound 7
largely exists in protonated LADE in aqueous environments,
but that a small equilibrium population of unprotonated LEDA
is sufficient to act as a shuttle between the intestine and the
bloodstream by enabling 7 to enter the lipid environment and
thereby traverse the intestinal wall.
The above observations led us to propose a model of pH-
dependent conformational flipping to enable oral
bioavailability, as shown in Figure 4. The progression begins
with the tertiary amine predominately in the protonated form22
within the slightly acidic to neutral environment of the
intestinal lumen (measured pKa=9.5, calculated population
98%). In this state, compound 7 is predicted to be almost
entirely LADE as it is calculated to be 1.9 kcal/mol lower in
energy than LEDA (see Supporting Information for detailed
discussion of the ab initio calculations). However, within the
small non-protonated subpopulation, the conformational
stabilities are reversed with the LEDA conformation being 5.4
kcal/mol lower in energy. Its relative stability arises from two
intramolecular hydrogen bonds, which masks two hydrogen
bond do-
Changes in pH and ion content have previously been shown
to enable “molecular switching” in certain pre-disposed
systems, including six-membered carbo- and heterocycles.23,24
Furthermore, intramolecular hydrogen-bonding is known to
mask exposed polarity in peptides and can sometimes be
correlated with enhanced membrane permeability and even
oral bioavailability.25,26,27,28,29 The solution to our problem
required that this masking was context dependent, so that the
polar form of the molecule was the one predominantly
available.
Based
on
the
structure-activity
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of 30 mg/kg. Taken together, the pre-clinical studies with
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2
BMS-741672 enabled its advancement into human clinical
trials.
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ASSOCIATED CONTENT
Supporting Information. Synthetic procedures and complete
characterization data for the compounds described, including
structural studies on different forms of compound 7. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
* (P.H.C.) Tel.: 609-252-4144. E-mail: percy.carter@bms.com
Author Contributions
The manuscript was written through contributions of all authors.
Funding Sources
This work was funded by Bristol-Myers Squibb Company.
ACKNOWLEDGMENT
We acknowledge our many colleagues in the CCR2 Discovery
Working Group, as well as the Department of Discovery
Synthesis at the Biocon-BMS R&D Center (Bangalore, India).
We also thank Dr. Nicholas A. Meanwell for helpful comments
on the manuscript.
ABBREVIATIONS
CCR2, CC chemokine receptor 2; CCR5, CC chemokine receptor
5; LADE, Lactam Axial DiEquatorial, LEDA, Lactam Equatorial
DiAxial.
Figure 4. Working model for the equilibrium populations of
acetamide 7 in both high-dielectric aqueous and low-dielectric
lipid environments. Percentages on the horizontal arrows are
Boltzmann populations calculated from the relative ab initio
conformational energies, denoted beneath each structure. The
Boltzmann populations on the vertical arrows are calculated using
the Henderson-Hasselbalch equation using an amine pKa of 9.5
and buffer pH of 7.3.
REFERENCES
1 Carter, P. H. Progress in the discovery of CC chemokine receptor
2 antagonists, 2009 - 2012. Expert Opin. Ther. Pat. 2013, 23 (5), 549
– 568.
relationships relative to CCR2 binding, hERG functional
activity, and membrane permeability, the conformational
switching mechanism (Figure 4) appeared to remain intact
within this trisubstituted cyclohexane series so long as three
elements remained in place: a secondary or tertiary amine at
C1, an amide or sulfonamide directly linked to C3, and the
Friedinger lactam at C4. Modification of these groups (e.g.
homologation of the acetamide, or replacement of the lactam
with a glycinamide) disrupted the SAR trends. However, with
these groups in place, optimized molecules for the inhibition
of CCR2 could be obtained. Acetamide 7 (BMS-741672) was
one such molecule. Specifically, BMS-741672 was found to
have 51% oral bioavailability in rat with a t1/2 of 5.1 h (IV),
and 46% oral bioavailability in Cynomolgus monkey with a
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Michael G. Yang, Zili Xiao, Robert J. Cherney, Andrew J. Tebben, Douglas G. Batt, Gregory D.
Brown, Jing Chen, Mary Ellen Cvijic, Marta Dabros, John V. Duncia, Michael Galella, Daniel S. Gard-
ner, Purnima Khandelwal, Soo S. Ko, Mary F. Malley, Ruowei Mo, Jian Pang, Anne V. Rose, Joseph B.
Santella, III, Hong Shi, Anurag Srivastava, Sarah Traeger, Bei Wang, Songmei Xu, Rulin Zhao, Joel C.
Barrish, Sandhya Mandlekar, Qihong Zhao and Percy H. Carter*
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Research and Development, Bristol-Myers Squibb Company, Princeton, NJ 0843, United States.
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