Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2

Article abstract of DOI:10.1016/S0960-894X(01)00249-9

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.

Full text of DOI:10.1016/S0960-894X(01)00249-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1445–1450
Discovery of Potent and Selective Phenylalanine
Derived CCR3 Receptor Antagonists. Part 2
Dashyant Dhanak,* Lisa T. Christmann, Michael G. Darcy, Richard M. Keenan,
Steven D. Knight, Judithann Lee, Lance H. Ridgers, Henry M. Sarau, Dinubhai H. Shah,
John R. White and Lily Zhang
SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA19426-0989, USA
Received 21 November 2000; accepted 6 April 2001
Abstract—Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based
leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists. # 2001
Elsevier Science Ltd. All rights reserved.
The orchestrated infiltration of specific pro-
inflammatory leukocytes to sites of inflammation is
recognized as one of the hallmarks of a robust immune
response.1 Recently, several CC chemokines have been
shown to be key players in this response2 and amongst
these, eotaxin and eotaxin-2 have been reported to be
important mediators for the selective recruitment of
eosinophils into the lungs of patients suffering from
allergic diseases such as asthma.3 Both of these proteins
elicit their biological response by binding to and acti-
vating a cell surface, seven-transmembrane spanning G-
protein coupled receptor designated CCR3.4 Under
pathophysiological conditions, the recruitment and
subsequent activation of eosinophils results in the
release of cytotoxic and other mediators which ulti-
mately lead to the clinically observed manifestations of
inflammatory disease.5 Approaches towards the in vivo
blockade of CCR3 have included the evaluation of
neutralizing antibodies raised against eotaxin,6 modified
chemokines,7 and also small molecule antagonists.8 Our
interest in this area has been focused on the last of
these approaches, that is, the development of selec-
tive, small molecule CCR3 antagonists as antiin-
flammatory agents. We have recently reported the
discovery and initial SARs of a series of highly
selective and potent phenylalanine derived CCR3
antagonists.9
A prototypical member of this class of compound, 1,
was highly effective in blocking both the binding and
functional activity of a number of physiologically rele-
vant CCR3 agonists such as eotaxin, eotaxin-2, and
MCP-4. The presence of a metabolically labile ester
functionality, however, precluded the evaluation of 1 in
in vivo models of inflammatory disease and we sought
to replace the undesired ester moiety with a more stable
isostere. We report herein the successful realization of
this goal and describe the SAR of this novel class of
CCR3 antagonists.
Chemistry10
The compounds described in this paper were prepared
using the procedures outlined in Schemes 1–7 which are
based on previously reported protocols. Compounds 2–
5 and 8–15 (Table 1) were synthesized by the routes in
Schemes 1–4 and the tetrazoles 6 and 7 obtained using
the chemistry reported by Liskamp11a and Moltzen.11b
*Corresponding author. Fax: +1-610-917-4157; e-mail: dash_
dhanak-1@sbphrd.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00249-9

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D. Dhanak et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1445–1450
Scheme 1.12 Reagents: (a) N,O-Dimethylhydroxylamine hydrochloride, EDCI, HOBT, NMM, DMF; (b) DIBAL-H, THF, 0 ꢀC; (c) p-toluene-
sulfonyl isocyanate, K2CO3, MeOH; (d) ammonia, glyoxal (40%), DMF; (e) aq KOH, EtI, DMF.
Scheme 2.13 Reagents: (a) (COCl)2, cat. DMF, CH2Cl2; (b) 2-trimethylsilyl-1,2,3-triazole, sulfolane, 140–150 ꢀC; (c) H2NNHCHO, EDCI, HOBT,
NMM, DMF; (d) POCl3, 100–105 ꢀC; (e) hydrazine, EtOH, reflux; (f) 1-naphthoic acid, EDCI, HOBT, NMM, DMF.
Scheme 3.14 Reagents: (a) P2S5, Na2CO3, THF; (b) BrCH2CH(OEt)2, HCl, molecular sieves (4 A), DMF; (c) BrCH2(CO)CO2Et, EtOH; (d) 4 N HCl
in dioxane; (e) 1-naphthoic acid, NMM, HOBT, EDCI, DMF; (f) NaOH, MeOH:H2O; (g) NHR, DIEA, HOBT, EDCI, DMF.
Scheme 4.15 Reagents: (a) NH2(CHR)CH2OH, EDCI, HOBT, NMM, DMF; (b) CH3O2CNSO2NEt3, DMA; (c) BrCCl3, DBU, CH2Cl2.
Scheme 5. Reagents: (a) NH2CH(CH3)CO2R, EDCI, HOBT, NMM, DMF; (b) 60% TFA in CH2Cl2; (c) NH2R0, EDCI, HOBT, NMM, DMF.
Compounds 17–22 (Table 2) were prepared as shown in
Scheme 5, with the exception of 21 where the final
amide formation was accomplished using the active
anhydride method. Scheme 6 details the synthesis of the
compounds in Table 3 (24–27) and the compounds of
Table 4 (28–35) were prepared in a parallel synthesis
approach as illustrated in Scheme 7.
alternatives (e.g., 2, 4, 6, and 8) was surprising. Never-
theless, the dihydrooxazole 9 did retain some receptor
affinity and was found to be a potent functional
antagonist as evidenced by its ability to block the
eotaxin induced intracellular calcium mobilization in
primary human eosinophils (IC50=600 nM).17 In view
of the stereoelectronic similarity between the hetero-
cycles and an ester group, the general lack of activity of
the ester mimetics suggested a more subtle role for the
ester moiety that the heterocycles were somehow unable
to mimic. The exact nature of this role remains unclear
but does not appear to involve an irreversible transacyl-
ation of a critical residue in the CCR3.9
Our initial efforts were directed towards simple primary
and secondary amides as ester replacements but were
unsuccessful and generally resulted in large losses in
receptor binding affinity.9 This prompted us to investi-
gate a variety of heterocycles that have previously been
reported to function as effective ester bioisosteres (Table
1).16 The almost complete loss of CCR3 affinity seen
upon replacing the ester group with simple heterocyclic
As shown in Table 1, introduction of an ester group into
some of the inactive templates resulted in the restoration

D. Dhanak et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1445–1450
1447
Scheme 6. Reagents: (a) NHR1(CHR2)CON(R3)Ph, EDCI, HOBT, NMM, DMF or isobutyl chloroformate, NMM, THF, ꢂ15 to 25 ꢀC; (b) 4 N
HCl in dioxane; (c) 1-naphthoic acid, EDCI, HOBT, NMM, DMF.
simple modifications to the ester in 12, that is the cor-
responding amides 13–15, led to a loss of affinity.
We reasoned that because of conformational constraints
inherent in the heterocycle, both the attenuation in the
activity of the esters 11 and 12 and the lack of activity of
Scheme 7. Reagents: (a) 4 N HCl in dioxane; (b) P-EDC,
the corresponding amides could be related to an inabil-
R2CH2CH(NHBOC)CO2H, DCE/DMF; (c) 4 N HCl in dioxane; (d)
ity of these analogues to access important binding
1-naphthoic acid, EDCI, HOBT, NMM, DMF.
pockets or residues in the receptor. This led initially to
the preparation of acyclic derivatives of 10 such as the
of submicromolar receptor affinity. The effect was par-
ticularly marked for the oxazole 11 and thiazole 12 sys-
tems, although these compounds were still ꢁ100-fold
less potent than 1. Consistent with this apparent striking
preference for an ester moiety in this part of the molecule,
dehydroserine 16 and alanines 17 and 18 in which the
conformational restrictions of the cyclic system were
relieved. Encouragingly, as shown in Table 2, these less
constrained analogues either retained the activity of or
were substantially more potent than their corresponding
Table 1.
Compound
R
IC50 (nM)18
22,500
Compound
R
IC50 (nM)
563
(ꢃ)-2
9
(ꢃ)-3
(ꢃ)-4
>33,000
>33,000
10
11
213
500
(ꢃ)-5
(ꢃ)-6
(ꢃ)-7
8
17,000
>33,000
>33,000
32,000
12
13
14
15
663
18,000
7700
12,500

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D. Dhanak et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1445–1450
cyclic analogues 11 and 12. Interestingly, the alanine
derivatives showed a marked preference for the natural,
l-stereochemistry at both asymmetric centers, con-
sistent with a high degree of enantiospecificity in the
receptor and antagonist interaction (17 and 18, Table
2). As previously observed, the nature of the alkyl group
of the ester did not greatly affect the CCR3 affinity and
even the tert-butyl ester 19 was a highly potent eotaxin
antagonist.
interactions and, with appropriate substitution, could
be useful in modulating the physicochemical properties
of the antagonist. N-Methylation of the central amide
bond (26) appeared to be more important and may be
related to changes in the geometry around the amide
linkage.19 However, the effect was not observed for the
benzamide where N-methylation did not affect the
activity (27).
Importantly, amides such as 23 and 26 successfully
demonstrated the feasibility of identifying nonester
CCR3 antagonists and warranted additional investiga-
tion with the objective of further improving the receptor
affinity. Given the synthetic amenability of 23, we chose
to accomplish this optimization using solution-phase
parallel synthesis (Scheme 7). The most optimal approach
in practice was to retain the N-acyl residue and vary only
the amino acid core and the benzamide group.
Although the compounds in Table 2 suggested that the
phenylalanine ester could be replaced with an amide,
the presence of an additional ester moiety in example 18
still precluded in vivo evaluation. We again considered
amides as simple ester replacements in these more flex-
ible systems and in this case, found them to retain rea-
sonable CCR3 affinity (20, 21, and 23, Table 2). In
general, secondary amides were preferred over tertiary
amides (20 and 23) and the presence of an aromatic
group was found to be particularly favorable (21 and
23).
A small, explorative library with these variables was
prepared for assay versus the CCR3 and representative
compounds are shown in Table 4. A number of CCR3
antagonists, significantly more potent than 23, were
identified (Table 4) and the SAR around the molecule
further delineated. The nature of the amino acid side
chain was clearly important for good affinity. A sub-
stituted phenyl group was preferred over both cyclo-
hexyl or 4-thiazoyl (28 vs 29 and 30, respectively). As in
the related ester series,9 a range of phenyl substituents
was associated with good receptor affinity (28, 32, and
34). Although the 4-pyridyl derivative 38 was devoid of
activity, weak affinity was restored when this group was
combined with a different benzamide (39). The parallel
synthesis approach allowed for the rapid identification
of substituents that in combination led to additive, and
sometimes surprising, increases in potency. For exam-
ple, although the activity seemed insensitive to changes
in the benzamide part of the molecule (Table 4), the
combination of an unsubstituted phenylbenzamide with
a 4-chlorophenylalanine moiety was dramatic and pro-
vided a compound, 36, whose CCR3 affinity was com-
parable to that of 1.
As shown in Table 3, replacement of the alanine methyl
group in 23 with either hydrogen (24) or phenyl (25) did
not markedly affect the activity, suggesting that this
position was not involved in making critical receptor
Table 2.
Compound
R
IC50 (nM)
350
16
17
383
18
19
20
21
3
20
A survey of alternative N-acyl groups was also carried out
and quickly led to the identification of potent naphthoyl
Table 3.
666a
300a
22
23
1125a
190
Compound
R1
R2
R3
IC50 (nM)
23
24
25
26
27
H
H
H
Me
H
Me
H
Ph
Me
Me
H
H
H
H
Me
190
400
116
65
600a
aMixture of four diastereomers.
aMixture of four diastereomers.

D. Dhanak et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1445–1450
1449
Table 4.
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23
28
29
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31
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33
34
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37
38
39
4-NO2-Ph
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190
45
4-OMe-Ph
4-OMe-Ph
4-OMe-Ph
4-Cl-Ph
2-OMe-Ph
4-OMe-Ph
2-OMe-Ph
4-CN-Ph
Ph
Cyclohexyl
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4-CN-Ph
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4-OMe-Ph
4-OMe-Ph
4-Cl-Ph
700
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800
125
90
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5
4-Cl-Ph
4-Pyridyl
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4-OMe-Ph
4-Ph-Ph
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surrogates as exemplified by 40. Clearly, our limited
survey suggests that there is considerable scope for fur-
ther optimization to generate compounds with
improved CCR3 affinity and different physicochemical
properties. However, we concluded that the profile of 36
was suitable for evaluation in functional assays and
determined its ability to inhibit the eotaxin induced
chemotaxis of primary human eosinophils derived from
allergic individuals.17 In this assay, and consistent with
its CCR3 affinity determined in the binding assay, 36
was found to be a potent inhibitor of eosinophil che-
motaxis (IC50=15 nM). In contrast, in the same assay,
36 had no effect on the C5a induced eosinophil chemo-
taxis. Taken together with the previously reported abil-
ity of related compounds to block the functional
responses mediated by eotaxin, MCP-3 or MCP-4, the
compounds reported herein appear to be acting via
CCR3 antagonism.9
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Highly potent CCR3 antagonists have been developed
from a series of phenylalanine ester-based leads.
Although classical heterocyclic ester mimetics proved to
be ineffective in the present study, conformationally less
constrained derivatives gave more promising results.
Two-dimensional, solution-phase parallel synthesis
optimization was utilized to allow for rapid improve-
ment in the receptor affinity and highly potent, func-
tional CCR3 antagonists have been identified. The
hydrolytic stability of these novel antagonists should
allow for their in vivo evaluation and the results of these
studies will be reported in due course.

1450
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18. The IC50 values reported are the mean of duplicate
samples from at least two determinations.17
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