The discovery of structurally novel CCR1 antagonists derived from a hydroxyethylene peptide isostere template

Article abstract of DOI:10.1016/j.bmcl.2004.02.020

The present manuscript details the discovery and early fundamental structure-activity relationship studies involving compound 3, a novel hydroxyethylene peptide isostere derived molecule that provides micromolar inhibition of CCL3 binding to its receptor

Full text of DOI:10.1016/j.bmcl.2004.02.020

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2163–2167
The discovery of structurally novel CCR1 antagonists derived
from a hydroxyethylene peptide isostere template
John C. Kath,* Amy P. DiRico, Ronald P. Gladue, William H. Martin, Eric B. McElroy,
Ingrid A. Stock, Laurie A. Tylaska 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 present manuscript details the discovery and early fundamental structure–activity relationship studies involving
compound 3, a novel hydroxyethylene peptide isostere derived molecule that provides micromolar inhibition of CCL3 bindingto its
receptor CCR1. Initial studies established this screeninghit as a legitimate lead for further medicinal chemistry optimization.
Ó 2004 Elsevier Ltd. All rights reserved.
Chemokines are low molecular weight proteins (8–
10 kDa) that are best known for their potent chemotactic
activity.1–3 They exert their functions by interactingwith
7-transmembrane (7TM) G-protein coupled receptors
that are differentially expressed on subsets of leukocytes.
Chemokines are divided into four subclasses (CC, CXC,
CX3C, and C) dependingon the spacingbetween their
N-terminal cysteine residues. The majority of chemo-
kines are Ôinducible proteinsÕ, that are upregulated in
response to inflammatory stimuli suggesting that they
play a pivotal role in the progression of autoimmune
diseases,4–7 allograft rejection,8;9 asthma,10 atherosclero-
sis,11 AIDS,12;13 and cancer.14
TES) have been shown to be expressed at inflammatory
sites in several diseases. Furthermore, neutralizing
antibodies and/or receptor deficient animals have pro-
vided evidence that blockade of CCR1 may be beneficial
for of the treatment of multiple sclerosis, rheumatoid
arthritis, and allograft rejection.15 Recently, small mol-
ecule antagonists of CCR1 have been reported by Berlex
(1)16 and Banyu (2).17 The most well characterized of
these, BX-471 (1) has shown efficacy in animal models of
multiple sclerosis and organ transplant rejection18;19 and
is reported to be in clinical trials.20
All of the known CCR1 antagonists, in addition to the
majority of other small molecule chemokine receptor
antagonists, are structurally related in that most possess
a positively charged nitrogen under physiological con-
ditions.21 This common structural element is typical not
only for small molecule chemokine receptor antagonists
The CC-chemokine receptor-1 (CCR1) is expressed on
monocytes, T cells, immature dendritic cells, eosino-
phils, and in some cases neutrophils. CCR1, as well as its
primary ligands, CCL3 (MIP-1a), and CCL5 (RAN-
Cl
O
F
O
N
O
HN
Cl-
O
N
Cl
NH
+
N
O
NH2
BX471 (1)
2
Cl
Keywords: Chemokine; CCR1.
* Correspondingauthor. Tel.: +1-860-441-3852; fax: +1-860-441-4111; e-mail: john_c_kath@groton.pfizer.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.020

2164
J. C. Kath et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2163–2167
O
O
O
O
2
4
N
H
NHMe
N
H
NHMe
Cl
5
OH
NH
OH
N
Cl
4
3
Renin: IC50 0.041 µM
CCL3 binding: IC50 2.3µM
CCL3 binding: IC50 >32 µM
CCL5 chemotaxis: IC50 >25 µM
CCL3 chemotaxis: IC50 0.84 µM
CCL5 chemotaxis: IC50 0.63 µM
Renin: IC50 >10 µM
Figure 1.
but, in a larger sense, to small molecule G-protein
coupled receptor antagonists. We disclose here the dis-
covery and preliminary structure–activity relationships
of a structurally novel class of chemokine receptor
antagonists.
THP-1 cells at submicromolar levels. Compound 3 was
originally prepared for a renin inhibitor program, where
structurally similar 5-chloro-indole-2-carboxamide
derivatives such as 4 provided optimal activity. In con-
trast, the quinoline-3-carboxamide found in 3 rendered
it inactive against renin. Within the subset of com-
pounds prepared for the renin program, the CCR1
activity of 3 was unique. Compound 4 and other
structurally similar renin inhibitors did not inhibit
CCL3 bindingnor CCL3 and CCL5 induced chemotaxis
of THP-1 cells. Because (a) it was felt that 3 represented
an atypical chemotype for a 7TM antagonist and (b) the
set of hydroxyethylene peptide isostere-derived renin
In the course of screeningour compound file for
inhibitors of 125I–CCL3 bindingto CCR1, compound 3
was identified as a potential lead compound (Fig. 1).
Compound 3, which contains a hydroxyethylene peptide
isostere more commonly found in aspartyl protease
inhibitors, is a 2.3 lM inhibitor of CCL3 bindingand
blocks both CCL3 and CCL5 induced chemotaxis of
3
8
i-iv
i-iv
LHMDS
BOCHN
BOCHN
BOCHN
+
O
O
O
Br
7
O
O
O
5
6
Temperature/Time
-78oC/3h
54%
42%
<1%
10%
-45oC/3h
LHMDS
BOCHN
BOCHN
BOCHN
+
O
O
O
Br
-45oC/3h
O
O
O
11
9
10
16%
29%
i-iv
i-iv
13
12
Scheme 1. Reagents and conditions: (i) H2; Pd/C; EtOH; rt, (ii) TFA, CH2Cl2; 0 °C, (iii) quinoline-3-carboxylic acid; EDCI, CH2Cl2; rt, (iv) MeNH2,
MeOH; rt.

J. C. Kath et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2163–2167
2165
inhibitors in our file was limited to the 2R,4S,5S ste-
reochemistry; our initial exploration of SAR involved
the synthesis of the remainingseven possible stereo-
isomers of 3.
CCR1 and inhibition of CCL3 induced chemotaxis of
THP-1 cells was undertaken. The results from these
assays (perhaps surprisingly) confirmed the superiority
of the original 2R,4S,5S stereochemistry present in the
initial lead 3 (Table 1). Furthermore, the apparent
stereospecificity of these interactions provided initial
evidence that compound 3 was a viable lead.
Compound 3 was synthesized startingwith known lac-
tone 5, which can be prepared by a variety of meth-
ods.22;23 Low temperature alkylation of 5 with methallyl
bromide provided lactone 6 nearly exclusively (see
Scheme 1). Lactone 6 was converted to compound 3 in
83% overall yield by hydrogenation of the alkene,
removal of the Boc protectinggroup, formation of the
N-terminal quinoline-3-carboxamide, and ringopening
of the lactone with methylamine to form the C-terminal
methyl amide. Preparation of the 2S diastereomer of
compound 3 required lactone 7, which was not obtained
in any appreciable amount in the aforementioned
)78 °C alkylation. However, carryingout the alkylation
at a higher temperature ()45 °C) reduced the diastereo-
selectivity thereby allowingadequate quantities of lac-
tone 7 to be obtained after separation from 6 by silica
gel chromatography. Following the identical four steps
required for the synthesis of 3, diastereomer 8 was
obtained in similar yields.
Havinganswered the questions regardingrelative and
absolute stereochemistry, attention was turned toward
other fundamental components of 3. Beginning at the
N-terminal amide, preparation of the corresponding
quinoline-3-sulfonamide 14 spoke to the importance of
N-terminal amide functionality contributingto CCR1
activity (Table 2). At the opposite end, the C-terminal
N-methyl amide was replaced with a N,N-dimethyl
amide 15 and a primary amide 16. While removal of a
hydrogen bond donor in the C-terminal amide com-
pletely abolished activity, the removal of the N-methyl
to provide a primary amide only resulted in a moderate
loss of potency. Also evident early on was that C-2 alkyl
functionality was a critical component contributingto
the activity of these molecules as removal of the C-2
isobutyl group (17) abolished activity. At the C-5 posi-
tion, replacement of the cyclohexylmethyl group with
the considerably smaller and less lipophilic isobutyl
group (18) resulted in a 5-fold loss of potency while the
differentially branched 2-butyl isomer 19 was inactive.
These results suggested that the C-5 position might be a
fruitful area for additional SAR exploration. Indeed,
continuingefforts at the C-5 position next focused on
replacing the original cyclohexylmethyl group with a
benzyl group (20), which provided improvements in
both CCL3 bindingas well as CCL3 induced chemotaxis
relative to 3. That compound 20 was even more potent
when tested for inhibition of CCL5 induced chemotaxis
(IC50: 50 nM) only strengthened our conviction that this
novel, nonbasic series of chemokine receptor antago-
nists had a great deal of potential. As a result, the 5(S)-
benzyl-4(S)-hydroxy-2(R)-alkyl-5-amino-pentanoic acid
template found in 20 was used as a platform for lead
The precursor for the preparation of the two remaining
analogs possessing 4R,5S stereochemistry was lactone 9,
the minor diastereomer formed in the synthesis of 5. The
nonselective alkylation of the lithium enolate of 9 with
methallyl bromide at )45 °C provided lactone diastere-
omers 10 and 11 in a 2:1 ratio. The two diastereomers
were separated by silica gel chromatography and their
relative stereochemistry confirmed by an X-ray crystal
structure of 10. Both 10 and 11 were transformed to the
2S,4R,5S and 2R,4R,5S stereoisomers 12 and 13
employingchemistry used to obtain 3 and 8. Identical
procedures startingwith the enantiomers of 5 and 9 (ent-
5 and ent-9) provided ent-3, ent-8, ent-12, and ent-13.
With this set of eight stereoisomers in hand, compara-
tive testingfor both inhibition of CCL3 bindingto
Table 1.
O
O
4
2
N
H
NHMe
5
OH
N
Compound C2 stereochem.
C4 stereochem.
C5 stereochem.
CCL3 binding24 IC50 (lM)a
CCL3 chemotaxis25 IC50 (lM)a
3
R
S
S
R
S
R
R
S
S
S
S
S
S
S
R
R
R
R
2.3
30
0.77
12.0
8
12
R
R
R
R
S
Inactive
Inactive
15
7.70
13
Inactive
Inactive
Inactive
Inactive
Inactive
ent-3
ent-8
ent-12
ent-13
Inactive
Inactive
Inactive
S
a Compounds deemed inactive provided <50% inhibition at the highest concentration tested (32 lM in the bindingassay and 25 lM in the chemotaxis
assay).

2166
J. C. Kath et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2163–2167
Table 2.
R1
O
X
N
H
NR3R4
OH R2
R4
N
Compound
X
R1
R2
R3
CCL3 bindingIC (lM) CCL3 chemotaxis IC50 (lM)
50
3
CO
H
Me
2.3
0.84
14
15
16
17
18
19
20
SO2
CO
CO
CO
CO
CO
CO
H
Me
Me
H
Inactive
Inactive
8.0
Inactive
Inactive
6.3
Me
H
H
H
Me
Me
Me
Me
Inactive
11.8
Not tested
Not tested
Inactive
0.46
H
H
Inactive
0.65
H
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