Design, synthesis, and progress toward optimization of potent small molecule antagonists of CC chemokine receptor 8 (CCR8)

Article abstract of DOI:10.1021/jm050965z

Activation of CCR8 by its ligand CCL1 may play an important role in diseases such as asthma, multiple sclerosis, and cancer. The study of small molecule CCR8 antagonists will help establish the validation of these hypotheses. We report the design, synthesis, and progress toward optimization of potent small molecule CCR8 antagonists identified from a high-throughput screen. These analogues exhibit good potency in binding and chemotaxis assays, show good selectivity versus the hERG channel, and have good eADME (early absorption, distribution, metabolism, and excretion) profiles.

Full text of DOI:10.1021/jm050965z

© Copyright 2006 by the American Chemical Society
Volume 49, Number 9
May 4, 2006
Letters
Design, Synthesis, and Progress toward
Optimization of Potent Small Molecule
Antagonists of CC Chemokine Receptor 8
(CCR8)
Figure 1. CCR8 screening lead.
Shomir Ghosh,*,† Amy Elder,† Jianping Guo,† Ukti Mani,†
Michael Patane,† Kenneth Carson,† Qing Ye,†
identified as specific human CCR8 antagonists and agonists,
respectively.2
Robert Bennett,† Shannon Chi,† Tracy Jenkins,† Bing Guan,†
Roland Kolbeck,‡ Sean Smith,‡ Cheng Zhang,‡
CCR8 is expressed on a variety of cell types including
monocytes and endothelial cells and is selectively up-regulated
upon activation of T-helper-2 (Th2) cells.3 Th2 cells are the
main source of the cytokines IL-4, IL-5, and IL-13, which are
major mediators of inflammation, airway hyperreactivity, and
mucus hypersecretion in bronchial asthma.4
CCR8 is also expressed on phagocytic macrophages and
activated microglial cells in the human central nervous system
and is found in active demyelinating multiple sclerosis (MS)
lesions, in progressive multifocalleukoencephalopathy (PML),
and in cerebral ischemia.5 Furthermore, endothelial-derived
spindle cells of human Kaposi sarcoma biopsies express CCR8.6
The CCR8 ligand, CCL1, has antiapoptotic activity in adult
T-cell leukemia (ATL), caused by human T-cell leukemia virus
type 1 (HTLV-1).7
Because of the expression pattern of CCR8, activation of the
receptor by CCL1 may play an important role in various diseases
including allergic asthma, multiple sclerosis, and cancer. Our
research was directed toward identifying small molecule an-
tagonists to understand the role of CCR8-CCL1 interactions
in these diseases and to provide novel therapeutics for their
treatment. Reports in the literature describing small molecule
CCR8 antagonists are limited to a few published patent
applications.8 In this Letter, we describe the design, synthesis,
and progress toward optimization of CCR8 antagonists.
Screening of our compound collection in a human CCR8
binding assay led to the discovery of biphenyl ether amine 1
(Figure 1) with a Ki of 2.6 µM as the initial lead. For SAR
exploration, the lead structure was deconstructed into three basic
fragments (Figure 2). We investigated all three fragments
including (1) substituted piperidines and other amines, (2) the
linker between the biphenyl ether and the amine, and (3)
substituents on rings A and B along with biphenyl ether
replacements. To achieve efficient optimization, our screening
Gregory LaRosa,‡ Bruce Jaffee,‡ Hua Yang,§ Priya Eddy,§
Chuang Lu,§ Vinita Uttamsingh,§ Robert Horlick,‡
Geraldine Harriman,† and Daniel Flynn†
Department of Medicinal Chemistry, Department of Pharmacology,
and Drug Safety and Disposition, Millennium Pharmaceuticals,
40 Landsdowne Street, Cambridge, Massachusetts 02139
ReceiVed September 27, 2005
Abstract: Activation of CCR8 by its ligand CCL1 may play an
important role in diseases such as asthma, multiple sclerosis, and cancer.
The study of small molecule CCR8 antagonists will help establish the
validation of these hypotheses. We report the design, synthesis, and
progress toward optimization of potent small molecule CCR8 antago-
nists identified from a high-throughput screen. These analogues exhibit
good potency in binding and chemotaxis assays, show good selectivity
versus the hERG channel, and have good eADME (early absorption,
distribution, metabolism, and excretion) profiles.
Chemokines are chemotactic cytokines that regulate develop-
ment, activation, and recruitment of leukocytes through binding
and activation of seven transmembrane G-protein-coupled
receptors (GPCRs). On the basis of the spacing of conserved
cysteines at the N-terminus, chemokines are classified into CXC,
CC, C, and CX3C subgroups.1 Most chemokine receptors are
activated by multiple chemokines. CCR8 is an exception because
it has only one known natural ligand, CCL1 (also known as
I-309). In addition, pox-viral chemokine MC148 and human
herpes virus 8 (HHV-8) derived chemokine vMIP I have been
* To whom correspondence should be addressed. Phone: (617)-551-
3634. Fax: (617)-444-1483. E-mail: sghosh@mpi.com.
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
§ Drug Safety and Disposition.
10.1021/jm050965z CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/11/2006

2670 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Letters
Scheme 2. Synthesis of 4-Amino-4-carboxy Substituted
Piperidine Biaryl Ethers 17b and 17ca
Figure 2. Chemistry strategy to address SAR.
strategy involved parallel evaluation of various properties in
addition to potency, including in vitro selectivity and safety
parameters. This allowed for early identification of issues such
as hERG binding and metabolic stability.
The synthetic routes used to prepare analogues for SAR
exploration are described in Schemes 1 and 2. Reductive
amination of 3-phenoxybenzaldehyde 2a and 4-carboxyethyl-
piperidine (Na(OAc)3BH, 1% AcOH, 1,2-dichloroethane) readily
provided our initial lead 1. Hydrolysis of 1 produced the
corresponding acid 3 (LiOH, EtOH/THF), which upon treatment
with ethylamine in the presence of EDCI and HOBT yielded
the bioisostere 4 (Table 1). To access substituted biaryl ethers,
2b,c, 3-formylphenylboronic acid was treated with appropriately
substituted phenols in the presence of copper acetate (Scheme
1).9 Most reproducible yields for the biaryl ether synthesis were
obtained when reactions were run open to air. Reductive
amination of 2a-c with 4N-Boc-aminopiperidine followed by
removal of the Boc group with 4 M HCl/dioxane provided biaryl
ether amines as dihydrochloride salts 5a-c. Acylation of the
dihydrochloride salts with various acid chlorides in the presence
of diisopropylethylamine or with acids in the presence of EDCI
and HOBT produced analogues 6a-d (Table 1). Alternatively,
acylation with commercially available N-Boc-4-phenylpiperi-
dine-4-carboxylic acid (7) or N-Boc-3-phenylpyrrolidine-3-
carboxylic acid (8)10 in the presence of EDCI and HOBT and
subsequent removal of the Boc protecting group provided biaryl
ether amines 9b, 9c, and 10c (Scheme 1). Alkylation of 9b and
9c with ethyl bromide in the presence of triethylamine yielded
11b and 11c, respectively. Alkylation of 9c and 10c with ethyl
2-bromoisobutyrate using potassium carbonate as base followed
by hydrolysis of the corresponding esters with 6 N HCl gave
piperidine 12c and racemic pyrrolidine 13c, respectively.
Compounds 17b and 17c, derivatives of 9b and 11c, were
prepared from commercially available piperidine 14 (Scheme
2). Esterification of 14 with TMS-diazomethane followed by
removal of the Boc protecting group provided piperidine 15.
Reductive amination of 2b with 15 followed by removal of the
a Reagents and conditions: (a) TMSCH2N2, toluene/MeOH, room temp,
2 h; (b) 4 M HCl/dioxane, room temp, 2 h; (c) Na(OAc)3BH, 1% AcOH,
ClCH2CH2Cl, room temp, 18 h; (d) Et2NH, DMF, room temp, 4 h; (e) Et3N,
CH2Cl2, room temp, 18 h; (f) 1.0 M NaOH(aq)/MeOH, 80 °C, 2 h.
Fmoc protecting group with diethylamine in DMF yielded the
biaryl ether amine 16b. Acylation of 16b with N-Boc-4-
phenylpiperidine-4-carbonyl chloride (7a) followed by hydroly-
sis of the methyl ester and removal of the Boc group provided
17b. 17c was prepared in a similar manner from 2c using 7b.
Our screening strategy was designed to identify compounds
with good overall properties for evaluation in in vivo models
as proof of concept studies for targeting CCR8. We tested all
analogues in the primary assay (hCCR8 binding assay). On the
basis of primary assay potency, selected compounds were tested
in functional assays (hCCR8 chemotaxis), in a selectivity panel
(other chemokine receptors and GPCR’s), in early absorption,
distribution, metabolism, and excretion (eADME) assays11 such
as P450 inhibition, permeability, and metabolic stability, and
in safety-related assays such as hERG channel binding. Ana-
logues with good overall in vitro properties were then profiled
for their pharmacokinetics (PK) properties in two animal species
(rodent and non-rodent).
On the basis of our SAR plan (Figure 2), a survey of the
pharmacophoric elements of the lead 1 revealed that the
3-biphenyl ether benzylamine moiety was required for binding
activity. Acid 3 readily prepared from 1 showed loss in activity
Scheme 1. General Synthesis of 4-Amino Substituted Biaryl Ether Piperidines and Pyrrolidinesa
a Reagents and conditions: (a) Cu(OAc)2, Et3N, 4 Å molecular sieves, 1,2-dichoroethane, room temp, 18 h; (b) Na(OAc)3BH, 1% AcOH, 1,2-dichloroethane,
room temp, 18 h; (c) 4 M HCl/dioxane, room temp, 2 h; (d) R2COCl, DIEA, CH2Cl2, room temp, 18 h; (e) EDCI, HOBT, NMM, DMF, room temp, 18 h;
(f) CH3CH2Br, Et3N, CH2Cl2, room temp, 18 h; (g) Br(CH3)2CCO2Et, K2CO3, DMF, room temp, 18 h; (h) 6 N HCl, reflux, 20 h.

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2671
Table 1. Binding Activity (Ki) and Chemotaxis Inhibition of Biaryl Ether Piperidines
a Displacement of 125I-I309 from human CCR8 expressed on L1.2 cells. b Values represent the mean of n ) 2.
(Ki > 30 µM). The ethylamide 4 and reversed amide 6a showed
decreased potency. The phenylacetylamide 6b was slightly more
potent than the initial lead 1. An increase in binding potency
was observed with substitution on the A ring of the biphenyl
ether. Thus, 6c, with a 2-methoxy substituent in the A ring
inhibited hCCR8 binding with Ki ) 0.25 µM. 6c also blocked
chemotaxis of CCR8 L1.2 transfected cells induced by I-309
with IC50 ) 0.055 µM. In general, for this series of compounds,
binding potency was within 3-fold of chemotaxis inhibition
potency (see Table 1). Investigation of A and B ring substitution
showed that the 2-position of the A ring, particularly methoxy
and chloro groups, and the benzylic piperidine in the 3-position
of the B ring provided optimal binding activity. Further
piperidine modifications led to the spiro cyclohexyl analogue
6d, which was equipotent to the unsubstituted benzylamide
analogue 6c. Incorporation of a spiro piperidine moiety, e.g.,
9b, improved potency in the whole-cell binding assay and the
chemotaxis assay. The ethyl substituted piperidine 11b, which
was easily accessed from 9b, gained a 4- to 6-fold improvement
in potency. Further investigations revealed that the piperidine
nitrogen could be substituted without loss of potency (additional
data not shown). Thus, substitution on the piperidine nitrogen
provided a handle to optimize other properties of the compounds.
While optimizing for binding potency, we investigated
selectivity against other receptors and eADME11 properties,
including P450 CYP inhibition, metabolic stability, and perme-
ability. In general, compounds of this class showed at least 300-
fold selectivity vs GPCRs including chemokine receptors.12 They
did not significantly inhibit P450 isozymes including CYP 3A4
(IC50 > 10 µM). Caco-2 transport measurements revealed that
our preferred compounds are highly permeable (e.g., 9b Papp,
×106 cm/s): A to B is 9, B to A is 20, ratio is 2.2). Metabolic
stability of selected molecules was assessed by calculating in
vitro hepatic clearance from microsomal incubation studies.
Compounds of this class exhibited low in vitro hepatic clearance
rates in human microsomal studies but higher rates in rat and
dog (e.g., 9b Clh ) 0.6 L/h/kg (human), 3.1 L/h/kg (rat), and
1.4 L/h/kg (dog)). Some of the more potent CCR8 antagonists
also potently inhibited [3H]dofetilide binding to the hERG
channel expressed in HEK293 cells (e.g., 9b Ki ) 0.8 µM).
Our approach to dial out hERG activity was to introduce polar
substituents in the molecule without disrupting target potency.
Thus, substitution of the spiro piperidine 9b with a carboxylic
acid bearing group resulted in a potent compound, 12c, with a
10-fold improvement in hERG window over binding Ki.
Furthermore, introducing a carboxylic acid group in the 4
position of the aminopiperidine resulted in a substantial decrease
in hERG binding, e.g., 17b (hCCR8 Ki ) 0.06 ( 0.003 µM
(n ) 2), hERG Ki > 10 µM) and 17c (hCCR8 Ki ) 0.023 (
0.005 µM (n ) 2), hERG Ki > 10 µM). Introduction of polar
functionality into the molecule did not appreciably affect the
permeability of the compound as measured by the Caco2
transport assay (e.g., 17c Papp, ×106 cm/s): A to B is 25, B to
A is 83, ratio is 3.3). The spiro pyrrolidine version of 12c was
the most potent hCCR8 binder of the series ((()-13c Ki ) 0.006
µM).13 Additionally, 13c showed weaker hERG binding potency
(Ki > 10 µM) than 12c.
Applying our screening paradigm, we evaluated PK properties
for selected compounds. The oral bioavailabilities of these
compounds are low to moderate with high clearance rates, e.g.,
9b [rat (10 mpk po, 1 mpk iv, F ) 17%, t1/2 ) 0.7 h, and Cl )
34 L/h/kg) and dog (10 mpk po, 1 mpk iv, F ) 20%, t1/2 ) 11
h, and Cl ) 4 L/h/kg)]. The high in vivo clearance rates in rats
and dogs for 9b can be attributed at least partly to P450
metabolism in the liver (based on the in vitro microsomal
incubation studies). We are currently investigating the metabolic

2672 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Letters
receptor CCR8 is preferentially expressed in Th2 but not Th1 cells.
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fate of our preferred compounds and other clearance mechanisms
to understand the high clearance rates and help resolve low oral
exposure.
(4) Wills-Karp, M. Immunologic basis of antigen-induced airway hyper-
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(5) Trebst, C.; Staugaitis, S. M.; Kivisakk, P.; Mahad, D.; Cathcart, M.
K.; Tucky, B.; Wei, T.; Rani, M. R.; Horuk, R.; Aldape, K. D.; Pardo,
C. A.; Lucchinetti, C. F.; Lassmann, H.; Ransohoff, R. M. CC
chemokine receptor 8 in the central nervous system is associated
with phagocytic macrophages. Am. J. Pathol. 2003, 162 (2), 427-
438.
In summary, we have identified potent small molecule CCR8
antagonists. Our screening paradigm allowed for parallel evalu-
ation of various properties in addition to potency including in
vitro selectivity and safety. This approach helped for rapid and
efficient optimization of the series. Compounds in this series
potently block chemotaxis and possess good overall profiles
including good hERG selectivity. We are currently focused on
improving PK properties of this series while maintaining good
potency, selectivity, and safety profiles.
(6) Haque, N. S.; Fallon, J. T.; Taubman, M. B.; Harpel, P. C. The
chemokine receptor CCR8 mediates human endothelial cell chemo-
taxis induced by I-309 and Kaposi sarcoma herpes virus-encoded
vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned
medium. Blood 2001, 97 (1), 39-45.
(7) Ruckes, T.; Saul, D.; Van Snick, J.; Hermine, O.; Grassmann, R.
Autocrine antiapoptotic stimulation of cultured adult T-cell leukemia
cells by overexpression of the chemokine I-309. Blood 2001, 98 (4),
1150-1159.
(8) Patent applications: WO2004032856, WO2004073619, WO2004-
074438, WO2004058736, WO2004058709, US20040209948, and
US20040224978.
(9) Evans, D. A.; Katz, J. L.; West, T. R. Synthesis of diaryl ethers
through the copper-promoted arylation of phenols with arylboronic
acids. An expedient synthesis of thyroxine. Tetrahedron. Lett. 1998,
39 (19), 2937-2940.
Acknowledgment. The authors thank Nanda Gulavita,
Marjorie Solomon, and May Zhu for analytical support and Tim
Ocain for helpful suggestions with the manuscript.
Supporting Information Available: Description of binding,
chemotaxis assays, and synthesis procedures including spectral data
for 1-17. This material is available free of charge via the Internet
at http://pubs.acs.org.
(10) (a) Hagen, S. E.; Domagala, J. M.; Heifetz, C. L.; Sanchez, J. P.;
Solomon, M. New quinolone antibacterial agents. Synthesis and
biological activity of 7-(3,3- or 3,4-disubstituted-1-pyrrolidinyl)-
quinoline-3-carboxylic acids. J. Med. Chem. 1990, 33 (2), 849-854.
(b) Padwa, A.; Chen, Y.-C.; Dent, W.; Nimmesgem, H. Synthetic
application of cyanoaminosilanes as azomethine ylide equivalents.
J. Org. Chem. 1985, 50 (21), 4006-4014.
(11) eADME assays: in vitro assays utilized in the hit to lead or lead
optimization phase to study pharmacokinetic properties of compounds
prior to testing in vivo.
(12) For example, for 12c: % inhibition @ 1 µM 5HT2A is 14%, R1 )
4%, â1 ) 10%, â2 ) 15%, % inhibition @ 10 µM CCR3 is 30%.
See Supporting Information for additional data for 12c and 17c.
(13) All spiro pyrrolidines were tested as racemic mixtures. Separation
of the enantiomers of 13c and profiling of the eutomer are in progress.
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JM050965Z