Discovery of a potent and orally bioavailable CCR2 and CCR5 dual antagonist

Article abstract of DOI:10.1021/ml900009d

This report describes the discovery of a potent, orally bioavailable CC chemokine receptor 2 (CCR2) antagonist which, while optimized for CCR2 potency, also had potent CC chemokine receptor 5 (CCR5) activity.

Full text of DOI:10.1021/ml900009d

pubs.acs.org/acsmedchemlett
Discovery of a Potent and Orally Bioavailable CCR2 and
CCR5 Dual Antagonist
Alexander Pasternak,* Stephen D. Goble, Mary Struthers, Pasquale P. Vicario, Julia M. Ayala,
Jerry Di Salvo, Ruth Kilburn, Thomas Wisniewski, Julie A. DeMartino, Sander G. Mills, and
Lihu Yang
Merck Research Laboratories, Rahway, New Jersey 07065
ABSTRACT This report describes the discovery of a potent, orally bioavailable CC
chemokine receptor 2 (CCR2) antagonist which, while optimized for CCR2
potency, also had potent CC chemokine receptor 5 (CCR5) activity.
KEYWORDS CCR2, CCR5, chemokine, dual antagonist
onocyte chemoattractant protein-1 (MCP-1, CCL2),
included within the CC class of chemokines,1 medi-
ates chemotaxis of monocytes to inflammatory
related series of CCR2 antagonists (2) dramatically de-
creased IKr binding affinity when compared to our standard
4-fluorophenylpiperidine-based analogues.17 We applied
this observation to compound 1, giving target analogue 3
(Figure 1).
M
sites via interactions with its receptor, CCR2.2 CCR2, a
member of the G-protein-coupled seven-transmembrane
receptor superfamily, is most abundantly expressed on
monocytes. The CCL2/CCR2 axis has been implicated in
various autoimmune or inflammation associated dis-
eases3-12 including rheumatoid arthritis (RA),5 multiple
sclerosis,6 atherosclerosis,7 chronic obstructive pulmonary
disease (COPD),8 asthma,9 diabetes/obesity,10,11 and neuro-
pathic pain.12 A number of small molecule CCR2 antagonists
have already been described by our group13-17 and by
others.18,19 Several small molecule CCR2 antagonists and a
humanized monoclonal antibody targeting CCL2 have been
advanced to clinical trials for treatment of RA, multiple
sclerosis, atherosclerosis, COPD, pain, and diabetes.18-22
While the outcomes from the earliest trials for CCR2 block-
ade in RA have so far been disappointing,21,22 a trial in
patients at high risk for cardiovascular disease having ele-
vated high-sensitivity C-reactive protein (CRP) levels with
Compound 3 was prepared in an analogous fashion to
that described previously for its close analogue 2.17 CCR2
binding affinities were determined by measuring inhibition
of 125I-MCP-1 binding to the endogenous CCR-2 receptor on
human monocyte whole cells.13 IKr binding data was ob-
tained as described previously.26 While the IKr binding
affinity for analogue 3 was successfully reduced by 600-fold
(IKr IC50 = 21 μM), the CCR2 binding affinity was also
reduced, albeit by only 7-fold to 29 nM. In an effort retrieve
the lost CCR2 potency, we decided to incorporate beneficial
modifications found in other series explored within our
program. In particular, we have observed that substitution
of an oxygen atom in place of carbon at the 4-position of the
7-trifluoromethyltetrahydroisoquinoline subunit of 1 can
improve potency. We decided to incorporate this feature,
and the synthesis of the corresponding target compound 17
is described in Scheme 1. Commercially available fluoride 4
was treated with potassium tert-butoxide to give the ether
5.27 Reduction with hydrogen and catalytic Raney Ni pro-
vided the corresponding benzyl amine 6. Commercially
available ketoacid 7 was protected as its tert-butyl ester28/
dimethylacetal in two steps. Deprotonation of 8, followed by
addition of 2-iodopropane, gave 9. Treatment with a 5:1
mixture of 4 M HCl in dioxane and water removed both
protecting groups to afford ketoacid 10. Conversion to the
corresponding acid chloride, followed by coupling with ben-
zyl alcohol, provided the benzyl ester 11. The ester was
resolved using preparative chiral HPLC (Chiralcel OD column,
Chiral Technologies, Inc., 15% IPA/hexanes). The first peak
to elute was identified as having the desired S absolute
Millenium's CCL2 monoclonal antibody (MLN-120223
)
demonstrated lowering of CRP, consistent with a beneficial
anti-inflammatory effect. This communication describes
the discovery and detailed characteristics of 3-[(3R,4S)-1-
((1R,3S)-3-isopropyl-3-{[6-(trifluoromethyl)-2H-1,3-benzox-
azin-3(4H)-yl]carbonyl}cyclopentyl)-3-methylpiperidin-4-yl]-
benzoic acid, a potent and orally bioavailable CCR2 antag-
onist that was selected as a clinical candidate.
We have previously described CCR2 antagonists based
upon an aminocyclopentane carboxamide scaffold typified
by 1.15,16 One drawback of compound 1 and its analogues
was their high binding affinity at the outward delayed
rectifier potassium channel (IKr). Blockade of IKr is associated
with QTc prolongation in vivo, carrying the potential to
induce cardiac arrhythmias.24,25 Therefore, we set out to
modify 1 to minimize the IKr inhibition, while retaining CCR2
potency. We previously reported that incorporation of a
carboxyphenyl group onto the piperidine 4-position in a
Received Date: December 5, 2009
Accepted Date: December 14, 2009
Published on Web Date: December 28, 2009
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Scheme 1. Synthesis of Benzoxazine Analogue 17a
Figure 1. Leads 1 and 2, and derived target analogue 3.
stereochemistry by elaboration to a known pure CCR2
analogue. Hydrogenolysis of the benzyl ester with hydrogen
(1 atm) and Pd/C in methanol afforded (S)-ketoacid 10.
Conversion of (S)-10 to acid chloride (S)-12, followed by
addition of amine 6 and triethylamine, provided amide 13.
Reduction of the ketone with sodium borohydride was
followed by formation of the corresponding acetate ester.
Cleavage of the tert-butyl ether to the phenol 14 was achieved
by treatment with 4 M HCl in dioxane. Then, addition of an
excess of paraformaldehyde and catalytic TsOH in toluene,
followed by azeotropic removal of the generated water under
reflux, provided the cyclized benzoxazine product15. A more
direct approach where cyclization was attempted in the
presence of the cyclopentanone failed to give any product.
Hydrolysis of the ester group in 15, followed by Swern
oxidation, gave ketoamide 16. Reductive amination with
ethyl 3-piperidin-4-yl benzoate17 gave a mixture (∼1:1) of
cis- and trans-isomers which were separable by preparative
TLC. The higher eluting compound was presumed to be the
cis-isomer on the basis of the CCR2 binding affinity of both
esters (higher eluting IC50 7.2 nM, lower eluting 41% inhibi-
tion at 1 μM). We have previously reported that, of the
4 possible 3-amino-1-carboxamide-cyclopentane stereo-
isomers, the cis-(3R,1S)-isomer alone has potent activity on
CCR2.15 We have never observed an exception to this
stereochemistry preference. Hydrolysis of the benzoate ester
using excess lithium hydroxide gave the target analogue 17.
As anticipated, benzoxazine analogue 17 was more po-
tent compared to 3 in our binding assay (CCR2 IC50 = 7 nM).
In addition, 17 demonstrated weak binding on the IKr
channel (IC50 = 7.9 μM), corresponding to a >1000-fold
selectivity window. In a functional assay measuring inhi-
bition of MCP-1 mediated chemotaxis of monocytes13 17
was a potent functional antagonist (IC50 of 0.5 nM). In
addition, compound 17 was orally bioavailable in rats (F =
21%), beagles (82%), and rhesus monkeys (F = 60%).
Encouraged, we decided to explore the possibility of
further potency enhancements. In particular, we knew from
earlier work that incorporation of a trans-(3R)-methyl group
into the 4-arylpiperidine subunit can improve potency.14,15
a (a) KO-t-Bu (1.5 equiv), THF, 0 °C-rt; (b) H2, Raney Ni, EtOH, 37% aq
NH4OH; (c) H2SO4, MgSO4, t-BuOH, DCM; (d) TMOF, TsOH, MeOH,
DCM; (e) LDA, THF; 2-iodopropane, -78 °C-rt; (f) 4 M HCl/dioxane,
H2O (5:1); (g) oxalyl chloride, DMF (cat.), DCM; BnOH, Et3N, DCM,
DMAP; (h) chiral HPLC, Chiralcel OD column, 15% IPA/hexanes, faster
eluting peak; (i) H2, Pd/C, MeOH, 1 atm; (j) oxalyl chloride, DMF (cat.),
DCM; (k) Et3N (2 equiv), 6, DCM; (l) NaBH4, MeOH, 0 °C-rt; (m) Ac2O,
Et3N, DMAP (cat.); (n) 4 M HCl/dioxane, rt; (o) paraformaldehyde (∼1 g/
3 mmol substrate), TsOH, toluene, Dean-Stark trap, reflux 3.5 h; (p)
LiOH, EtOH, H2O; (q) oxalyl chloride, DMSO, substrate, Et3N, -78
°C-rt; (r) ethyl 3-piperidin-4-ylbenzoate,17 NaB(OAc)3H, 4 Å molecular
sieves, 3-5 days; (s) LiOH (10 equiv), EtOH, water, 50 °C, 1.5 h.
We decided to install the corresponding trans-4-(3-carboxy-
phenyl)-3-methylpiperidine 21 (Scheme 2) to determine if
the same potency enhancement would apply in this series.
The synthesis began with Boc protected ethyl 3-piperidin-
4-ylbenzoate 18.17,29 Oxidation with ruthenium(IV) oxide
and sodium periodate proceeded over 11 days to afford the
lactam 19.30 Deprotonation, followed by treatment with
iodomethane, gave the trans-3-methylpiperidinone 20. Re-
moval of the Boc group, followed by reduction with borane-
dimethylsulfide complex, gave the corresponding piperidine
21. Difficulties with the purification of 21 following the
reduction step led us to protect the amine (Boc), whereupon
purification was readily accomplished. The Boc group was
then removed to provide clean 21 as a mixture of two trans-
isomers. Reductive alkylation with ketone 16 afforded a
mixture of all four possible diastereomeric products
(∼5:5:6:6 ratio, 80% total yield). The four isomers were
separated by preparative chiral HPLC using a Chiralcel OD
column (Chiral Technologies, Inc.). The third (18%) and
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Scheme 2. Synthesis of Analogues 22 and 23a
Table 1. CCR2 Binding and Functional Data and IKr Binding Data
for Analogues 1, 3, 17, 22, and 23a
IC50 (nM)
analogue
CCR2
chemotaxis
IKr
1
4 (3)
29 (1)
7 (16)
4 (5)
2 (2)
49 (2)
21,000 (2)
7,900 (2)
33,000 (2)
7,300 (2)
3
17
22
23
0.5 (2)
0.3 (3)
2 (2)
6 (3)
a Numbers in parentheses represent numbers of determinations.
Standard deviations, when calculable, are always less than 25% of the
measured value.
difficult to measure at room temperature, but was greater
than 9 h. Analogue 22 is a potent inhibitor of MCP-1
mediated chemotaxis of monocytes (IC50 = 0.3 nM). An in
vitro human whole blood shape change assay32 measuring
MCP-1-induced changes in monocyte cell shape was used to
measure the potency of compound 22 to functionally antag-
onize CCR2 on monocytes in whole blood. In this assay
compound 22 has an IC50 of 15 nM with a preincubation of
30 min. Interestingly, when 22 was preincubated in whole
blood for 24 h, the IC50 improved to 0.1 nM, a shift in over 2
orders ofmagnitude. This is likelydueto an underestimation of
potency under standard 30 min preincubation conditions as a
result of a combination of the slow receptor kinetics and the
high level of plasma protein binding. Reversible binding to
plasma proteins in vitro was 98, 98, 97, and 97% in rat, dog,
monkey, and human, respectively (at 0.1 μM). When the
compound was allowed ample time to come to equilibrium
with the receptor on monocytes during the overnight incuba-
tion before whole blood stimulation with MCP-1, the com-
pound's true potency could be determined.
The 3-methyl group in 22 appears to confer improved
selectivity against the IKr channel (IKr/CCR2 > 8,000). Com-
pound 22 displayed potent activity against the closely related
CCR5 receptor. However, the potency of 22 at CCR5 was at least
2.5-fold less than that observed for CCR2 and the compound
exhibited much faster disassociation kinetics at CCR5.33 No
immune system deficits have been described in a group of
individuals deficient for CCR5 (CCR5 delta32 homozygous
individuals). Therefore, CCR5 blockade by 22 is unlikely to be
a treatment liability. Interestingly, the IC50 of 22 on murine
CCR5 is >10 μM. Counterscreening against a panel of 136
receptors, enzymes, and ion channels (MDS Pharma Services)
established 22 to be remarkably selective for CCR2. In addition
to the known CCR5 activity, 22 displayed weak activity against
the M2 and M4 subtypes of the muscarinic receptors (IC50 g10
μM for both). Compound 22 did not inhibit any of five human
CYP marker enzymes assayed (>100 μM on CYP3A4,
CYP2C9, CYP2D6, CYP1A2, and CYP2C19). As shown in
Table 2, 22 had excellent oral bioavailability in rats (F =
48%), dogs (F = 63%), and monkeys (F = 66%).
a (a) Ruthenium(IV)oxide hydrate (0.2 equiv), NaIO4 (3.2 equiv), CHCl3/
H2O, 11 days at rt;31 (b) KHMDS, THF; MeI, -78 °C; (c) 4 M HCl in
dioxane; (d) BH3 DMS, THF, rt; 0.5 M HCl/EtOH, 50 °C, 4 h; (e) Boc2O,
DIEA, DCM, DMAP (cat.); (f) 4 M HCl in dioxane; (g) 17, NaB(OAc)3H, 4 Å
mol sieves powder, DIEA, 4 days, rt; (h) preparative chiral HPLC
(Chiralcel OD column, 8% EtOH/hexanes, cis-peaks were third and
fourth to elute); (i) LiOH, EtOH, H2O, rt, 22 h.
fourth (17%) peaks to elute were determined to be the
desired cis-cyclopentane isomers. Hydrolysis of the esters
collected from the third and fourth peaks afforded the target
acids 22 and 23. We initially assigned the piperidine stereo-
chemistries for 22 and 23 (as shown in Scheme 2) on the
basis of their potencies; by analogy to an earlier series we
knew that while both trans-isomers maintain potency, the
(3R,4S)-4-aryl-3-methylpiperidine stereochemistry is opti-
mal.14 We later definitively confirmed the tentatively as-
signed stereochemistry of 22 by application of an alternative
synthetic route to the (4R)-carboxyphenyl-(3S)-methyl iso-
mer of piperidine intermediate 21, where the stereo-
chemistry was established though use of a chiral starting
material of known absolute stereochemistry.31 Similarly, the
cis-cyclopentane stereochemistry was confirmed by a later
process synthesis using chiral starting materials of known
absolute stereochemistry.
The CCR2 binding, functional, and IKr binding data for
compounds 22, 23, as well as analogues 1, 3, and 17, are
presented in Table 1. While analogues 22 and 23 are both
potent, analogue 22 appears slightly more potent than both
23 and 17. Compound 22 exhibited similar binding potency
for mouse CCR2 (IC50 = 4 nM). Direct equilibrium binding
3
The in vivo efficacy of compound 22 was evaluated in an ex
vivo rhesus whole blood shape change assay in which 22 was
dosed at 2 mg/kg orally. Comparison of the plasma levels
with the inhibition of monocyte shape change produced a
experiments using H-labeled 22 and monocytes demon-
strate that 22 has a KD for the receptor of 0.7 nM. 3H-22 had a
very slow association and disassociation at the receptor
giving a receptor disassociation time (T1/2) which was
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Table 2. Pharmacokinetic Properties of Compound 22a
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SUPPORTING INFORMATION AVAILABLE Experimental
details for the synthesis and characterization of CCR2 antagonists
3, 17, and 22. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Phone: (732) 594-3847. Fax: (732) 594-9556. E-mail:
alexander_pasternak@merck.com.
ABBREVIATIONS CCR2, CC chemokine receptor 2; CCL2,
CC chemokine ligand 2; CCR5, CC chemokine receptor 5;
TLC, thin layer chromatography.
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They also devised a synthesis of the cis-(3R,1S)-cyclopentane
core also using starting materials of known absolute stereo-
chemistry. This improved synthesis will be described else-
where.
(32) Wisniewski, T.; Bayne, E.; Flanagan, J.; Shao, Q.; Wnek, R.;
Matheravidathu, S.; Fischer, P.; Forrest, M. J.; Peterson, L.;
Song, X.; Yang, L.; DeMartino, J. A.; Struthers, M. Assessment
r
2009 American Chemical Society
18
DOI: 10.1021/ml900009d ACS Med. Chem. Lett. 2010, 1, 14–18
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