Novel CCR1 antagonists with improved metabolic stability

Article abstract of DOI:10.1016/j.bmcl.2004.02.022

The synthesis, biological activity, and pharmacokinetic profile of novel CCR1 antagonists are described.

Full text of DOI:10.1016/j.bmcl.2004.02.022

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2175–2179
Novel CCR1 antagonists with improved metabolic stability
Matthew F. Brown,* Mike Avery, William H. Brissette, J. H. Chang, Kevin Colizza,
Maryrose Conklyn, Amy P. DiRico, Ronald P. Gladue, John C. Kath, Suzanne S. Krueger,
Paul D. Lira, Brett M. Lillie, Greg D. Lundquist, Erin N. Mairs, Eric B. McElroy,
Molly A. McGlynn, Timothy J. Paradis, Christopher S. Poss, Michelle I. Rossulek,
Richard M. Shepard, Jeff Sims, Timothy J. Strelevitz, Susan Truesdell,
Laurie A. Tylaska, Kwansik Yoon and Deye Zheng
Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
Received 8 January 2004; revised 4 February 2004; accepted 5 February 2004
Abstract—The synthesis, biological activity, and pharmacokinetic profile of novel CCR1 antagonists are described.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Chemokines represent a growing family of proteins that
have been shown to play an important role in leukocyte
activation and migration.1–3 The chemokines CCL3
(MIP-1a, macrophage inflammatory protein-1a) and
CCL5 (RANTES, regulated on activation normal T-cell
expressed) bind to their shared receptor, CCR1, which is
expressed on a number of cell types including mono-
cytes, immature dendritic cells, and T cells. Blocking the
action of CCR1 signaling may be useful for the treat-
ment of diseases in which these cells play a role including
autoimmune diseases and organ transplant rejection.4;5
the N-terminal, C-5 and C-2 substituents, a variety of
analogues with much improved potency in both a 125I-
CCL3 binding assay8 as well as a CCL3 induced che-
motaxis assay9 were identified (e.g. 6a). While efforts
were ongoing to optimize potency in this series, parallel
efforts were ongoing to optimize pharmacokinetic
parameters. For example, in vitro oxidative metabolic
stability was evaluated by measuring the rate of drug
consumption in human liver microsomes (HLM) thus
providing intrinsic clearance values (Clint). These values
provide the medicinal chemist with a powerful tool for
addressing metabolic issues. 10 Early on in the program,
the majority of analogues in this series were rapidly
metabolized by HLM, suggesting the potential for
Recently we describe a novel series of CCR1 antagonists
derived from compound 1.6;7 Through optimization of
Keywords: CCR1; Chemokine; Microsome.
* Corresponding author. Tel.: +1-860-441-3522; fax: +1-860-686-1103; e-mail: matthew_f_brown@groton.pfizer.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.022

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M. F. Brown et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2175–2179
Scheme 1. Reagents and conditions: (a) LHMDS, THF, R2OTf or R2Br, )78 ꢁC; (b) LDA, THF, ketone, )78 ꢁC; (c) Burgess reagent, benzene,
reflux; H2 35 psi, 10% Pd/C, THF/MeOH; (d) TFA, CH2Cl2; (e) quinoxalyl chloride, TEA, CH2Cl2; (f) amine, MeOH, rt; (g) amine, HOAc, dioxane,
reflux; (h) HFÁpyridine, benzene; (i) TFA, neat.
unacceptably high hepatic clearance in humans. Herein
we further discuss the SARof this series in the context
of simultaneously improving HLM stability.
and deprotected the trifluoroacetate to give the final
products 6.
As described previously, low nanomolar potency in this
series was achieved with a variety of analogues con-
taining lipophilic C-2 substituents.7 However, the
majority of these compounds were found to undergo
rapid metabolism when incubated with HLM. Experi-
ments conducted to elucidate the site(s) of metabolism
suggested that the C-2 alkyl substituent was particularly
prone to oxidative metabolism. For example, incubation
of compound 6a in HLM produced a hydroxylated
compound as the major metabolite.11 In order to pro-
duce the metabolite in quantities sufficient for structure
determination and potency evaluation, microbial bio-
conversion of 6a was explored. Incubation of 6a with
Streptomyces griseolus (strain ATCC 11796) for 5 days
provided milligram quantities of the metabolite whose
structure was determined to be 6b, demonstrating that
oxidative metabolism of the C-2 sidechain had indeed
occurred.12 Not surprisingly, 6b was found to be con-
siderably less potent than the parent compound 6a due
to the reduced lipophilicity of the C-2 sidechain. How-
ever, we were encouraged by the significant improve-
ment in HLM stability of 6b, suggesting that the
incorporation of C-2 substituents designed to block
metabolism and/or provide reduced lipophilicity could
lead to drug candidates with improved pharmacokinetic
profiles. Indeed, replacement of the hydroxyl group in
6b with a blocking fluorine provided an analogue with a
lipophilic C-2 substituent that displayed excellent
potency and metabolic stability (6c).
Compounds were prepared as depicted in Scheme 1.
Alkylation of lactone 26;7 was accomplished by enolate
formation (LHMDS or LDA, THF) followed by addi-
tion of an alkyl bromide or triflate providing a separable
mixture of diastereomers 3a/b wherein 3b was the major
component. Removal of the Boc protecting group of 3b
followed by reacting the resulting amine with quinoxalyl
chloride provided lactone 5, which was then treated with
the required primary or secondary amine to give the
target analogue 6 with (2R,4S,5S) stereochemistry pre-
viously reported to provide the greatest potency.6
Alternatively, reacting the enolate of lactone 2 with a
cyclic ketone provided the hydroxy intermediate 4 as a
mixture of diastereomers, which was readily converted
to a separable mixture of diastereomers 3a/b (Burgess
reagent; 35 psi H2, Pd/C) then converted to target ana-
logues 6 as described above. For analogues containing
cycloalkanol C-2 substituents (i.e., 6f,g), intermediate 4
was directly converted to the final compounds by re-
moval of the Boc protecting group, coupling the resul-
tant amine with quinoxalyl chloride followed by lactone
ring-opening (NH3,MeOH). The fluorinated analogues
6c,l–q were prepared by reacting the olefin 3c with
HFÁpyridine to install both the methine fluorine as well
as remove the Boc protecting group. Reacting the
resulting amine with quinoxalyl chloride providing 5.
Tertiary hydroxyl analogues 6i–k were prepared by
treating 3c with neat TFA, which converted the olefin to
a trifluoroacetate and also removed the Boc protecting
group. As before, reacting with quinoxalyl chloride gave
the desired intermediate 5, which when treated with the
required amine simultaneously opened the lactone ring
Now that we had demonstrated the ability to produce a
potent, microsome stable compound, we attempted to
apply this Ômetabolic blockingÕ strategy to other potent

M. F. Brown et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2175–2179
2177
Table 1.
Compound
R1
R2
R3
CCL3 binding
IC50 (lM)
CCL3 chemo-
taxis IC50 (lM)
Clint (mL/
min/kg)
ClogP
6a
6b
Phenyl
Phenyl
–NH2
–NH2
0.028
0.58
0.002
1.18
202
4.9
4.1
1.9
6c
6d
Phenyl
Phenyl
–NH2
–NH2
0.009
0.008
0.013
0.005
8.0
3.4
4.2
242
6e
6f
Phenyl
–NH2
0.020
0.022
35
3.6
Phenyl
Phenyl
Phenyl
–NH2
–NH2
–NH2
0.056
0.054
3.46
0.068
0.12
6.4
47.7
NT
2.5
3.1
1.8
6g
6h
12.50
6i
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
Phenyl
–NH2
0.052
0.046
>25
0.68
0.065
>25
16.5
6.0
2.0
2.0
2.0
4.1
5.5
4.2
4.6
3.0
6j
–NH2
6k
6l
–NH2
NT
NT
299
836
647
3.6
–NHEt
–NHBn
–Pyrrolidino
0.075
0.85
0.13
6m
6n
6o
6p
Phenyl
0.44
Phenyl
2.32
0.74
Phenyl
4-N-Methyl
piperazino
5.60
7.05
Phenyl
–NHOH
0.027
0.031
(continued on next page)

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M. F. Brown et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2175–2179
Table 1. (continued)
Compound
R1
R2
R3
CCL3 binding
IC50 (lM)
CCL3 chemo-
taxis IC50 (lM)
Clint (mL/
min/kg)
ClogP
3.0
6q
6r
Phenyl
–NHNH2
0.007
0.007
0.001
0.008
32.7
6.1
Phenyl
–NHOH
3.2
analogues lacking microsome stability. For example,
compound 6d, which contains a C-2 cyclohexyl group,
was one of the more potent analogues in the program. In
this case, we did not perform experiments to determine
the metabolic site(s), but thought it reasonable to
assume that the 4-position of the cyclohexyl ring could
be the liability. We then prepared a number of ana-
logues to probe the feasibility of blocking potential
metabolism at this site. The 4,4-difluoro analogue 6e
maintained good potency, but showed only a modest
improvement in microsome stability. In contrast, the
analogous compound containing a tertiary alcohol did
show a significant improvement in microsomal stability
(6f). The reason for the enhanced stability of 6f versus 6e
was not entirely clear, however it may have been due to
the reduced lipophilicity of 6f relative to 6e (ClogP: 2.5
vs 3.6).13 Removal of the blocking fluorine atoms in 6f
to give 6g resulted in diminished microsome stability,
which could be explained by the greater polarity of 6f
versus 6g (ClogP 2.5 vs 3.1) or perhaps the difluoro
substitution is indeed blocking metabolism in this case.
Incorporation of heteroatoms at the 4-position of the
cyclohexyl ring provided analogues with greatly reduced
potency (6h), in line with SARsuggesting the require-
ment for lipophilic substituents at this position.
region of the molecule was quite sensitive to minor
structural changes. For example, while the 3-fluoro-
benzyl analogue 6j showed good potency, the closely
related 4-fluorobenzyl analogue 6k was inactive.
One final area of focus involved exploration of the C-
terminal SARusing compound 6c as the starting point.
Substituted amides (6l–o) were generally less potent or
offered no pharmacokinetic advantage when compared
to the primary amide 6c. However, the hydroxamic acid
6p and hydrazide 6q maintained potency and 6p also
displayed acceptable microsomal stability. Based on this
finding, a number of additional hydroxamic acids were
prepared, some of which had promising profiles (e.g. 6r)
(Table 1).
With a number of potent, HLM stable compounds in
hand, additional information was necessary for further
advancement toward a potential development candi-
date. Pharmacokinetic studies conducted in dogs and
monkeys indicated that these compounds generally had
moderate volumes of distribution (V dss) and plasma
clearances (Clp), resulting in elimination half-lives in
vivo in the range of 0.9–1.7 h (Table 2). The hydroxamic
acid analogues (compounds 6p and 6r) were found to
undergo glucuronidation, resulting in their higher
clearance rates.14 For the series, oral bioavailability was
often low (<10%), which may reflect on generally poor
aqueous solubility. Indeed, the compound with the
highest aqueous solubility (6j) also provided the highest
oral bioavailability for this set of analogues. The HLM
stable compounds in Table 2 were also evaluated for
their ability to inhibit the upregulation of CD11b on
monocytes induced by CCL3 in human whole blood.15
Compounds in this series retained good activity in this
assay as all the analogues in Table 2 provided IC50
values less than 0.5 lM.
Another area of focus involved follow-up to the hydroxy
metabolite 6b. While this analogue was significantly less
potent than the des-hydroxy analogue 6a, we felt it
worthwhile to reexamine the SARaround this com-
pound due to its promising metabolic stability. Explo-
ration of the C-5 position revealed that a number of
halobenzyl C-5 substituents imparted a significant
improvement in potency. Most notably, the 3-fluoro-
benzyl analogue 6j was shown to be >10-fold more
potent than the desfluoro analogue 6b and also retained
excellent HLM stability. Interestingly, the SARin this
Table 2.
Compound
Species
(%) F
Clp (mL/min/kg)
V dss (L/kg)
T1=2 (h)
Aqueous solubility (lg/mL)
6c
Dog
Monkey
Dog
1.6
2.9
12
18
13
18
28
29
38
33
1.2
1.2
1.0
1.3
1.2
1.5
1.3
1.8
1.0
1.2
1.4
0.92
1.7
1.1
0.5
1.1
ND
6j
49
157
21
Monkey
Dog
9.5
3.4
6p
6r
Monkey
Dog
Monkey
6.5
NT
<1.1
12

M. F. Brown et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2175–2179
2179
9. Cole, K.; Strick, C.; Paradis, T.; Ogborne, K. T.;
Loetscher, M.; Gladue, R. P.; Lin, W.; Boyd, J. G.;
Moser, B.; Wood, D. E.; Sahagan, B. G.; Neote, K.
J. Exp. Med. 1998, 187, 2009.
10. Obach, R. S. Curr. Opin. Drug Discov. Dev. 2001, 4, 36.
11. As determined by mass spectral analysis.
12. 6b was prepared by bioconversion of 6a using Streptomy-
ces griseolus strain ATCC 11796 following the procedures
outlined in: Smith, R. V.; Rosazza, J. P. Microbial
Transformations of Bioactive Compounds; Rosazza, J. P.,
Ed.; CRC: Boca Raton, FL, 1982; Vol. 2, pp 1–42. The
metabolite was extracted from the broth with ethyl acetate
following pH adjustment to 3.5 and partially purified
using a Sep-Pakꢂ C-18 cartridge. Final purification was
achieved by HPLC on an Inertsil C8 column
(250 · 200 mm) with a 65:35 H20/CH3CN mobile phase.
1H NMRand mass spectral data was used to assign the
structure.
13. Calculated with ClogP version 4.0 from BioByte.
14. Glucuronidation would not have been detectable in vitro
in liver microsomes under the experimental conditions
employed.
15. Conklyn, M. J.; Neote, K.; Showell, H. J. Cytokine 1996,
8, 762.
16. Gladue, R. P.; Tylaska, L. A.; Brissette, W. H.;
Lira, P. D.; Kath, J. C.; Poss, C. S.; Brown, M. F.;
Paradis, T. J.; Conklyn, M. J.; Ogborne, K. T.; McGlynn,
M. A.; Lillie, B. M.; DiRico, A. P.; Mairs, E. N.; McElroy,
E. B.; Martin, W. H.; Stock, I. A.; Shepard, R. M.;
Showell, H. J.; Neote, K. S. J. Biol. Chem. 2003, 278,
40473.
In summary, a number of potent CCR1 antagonists with
improved HLM stablility were described. Analogues
were designed to inhibit metabolism either by reducing
lipophilicity or by addition of moieties to block
metabolism. Additional studies with compound 6j have
revealed a high degree of selectivity for CCR1 versus a
broad panel of receptors (including other chemokine
receptors), as well as a high degree of selectivity for the
human receptor.16
References and notes
1. Proudfoot, A. E. I. Nat. Rev. Immunol. 2002, 2, 106.
2. Gerard, C.; Rollins, B. J. Nat. Immunol. 2001, 2, 108.
3. Luster, A. D. New. Eng. J. Med. 1998, 338, 436.
4. Saeki, T.; Naya, A. Curr. Pharm. Des. 2003, 9, 1201.
5. Horuk, R. Methods 2003, 29, 369.
6. Kath, J. C.; Dirico, A. P.; Gladue, R. P.; Martin, W. H.;
McElroy, E. B.; Stock, I. A.; Tylaska, L. A.; Zheng, D.
Bioorg. Med. Chem. Lett. 2004, preceding manuscript #1.
doi:10.1016/j.bmcl.2004.02.020.
7. Kath, J. C.; Brissette, W. H.; Brown, M. F.; Conklyn, M.;
DiRico, A. P.; Dorff, P.; Gladue, R. P.; Lillie, B. M.; Lira,
P. D.; Mairs, E. N.; Martin, W. H.; McElroy, E. B.;
McGlynn, M. A.; Paradis, T. J.; Poss, C. S.; Stock, I. A.;
Tylaska, L. A.; Zheng, D. Bioorg. Med. Chem. Lett. 2004,
preceding manuscript #2. doi:10.1016/j.bmcl.2004.02.021.
8. Neote, K.; DeGregorio, D.; Mak, J. Y.; Horuk, R.; Schall,
T. J. Cell 1993, 72, 415.