CCR5 receptor antagonists: Discovery and SAR study of guanylhydrazone derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.09.052

High throughput screening (HTS) led to the identification of the guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde as a CCR5 receptor antagonist. Initial modifications of the guanylhydrazone series indicated that substitution of the benzyl group at the para-position was well tolerated. Substitution at the 5-position of the central phenyl ring was critical for potency. Replacement of the guanylhydrazone group led to the discovery of a novel series of CCR5 antagonists.

Full text of DOI:10.1016/j.bmcl.2006.09.052

Bioorganic & Medicinal Chemistry Letters 17 (2007) 231–234
CCR5 receptor antagonists: Discovery and SAR study of
guanylhydrazone derivatives
Robert G. Wei,* Damian O. Arnaiz, Yuo-Ling Chou, Dave Davey, Laura Dunning,
Wheeseong Lee, Shou-Fu Lu, James Onuffer, Bin Ye and Gary Phillips
Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
Received 31 August 2006; revised 16 September 2006; accepted 19 September 2006
Available online 1 November 2006
Abstract—High throughput screening (HTS) led to the identification of the guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromo-
benzaldehyde as a CCR5 receptor antagonist. Initial modifications of the guanylhydrazone series indicated that substitution of
the benzyl group at the para-position was well tolerated. Substitution at the 5-position of the central phenyl ring was critical for
potency. Replacement of the guanylhydrazone group led to the discovery of a novel series of CCR5 antagonists.
Ó 2006 Elsevier Ltd. All rights reserved.
Chemokines are a large family of chemotactic proteins
that play an important role in the immune and inflamma-
tory response of various diseases and disorders including
asthma and allergic disease as well as autoimmune dis-
eases such as rheumatoid arthritis (RA). Chemokines
regulate leukocyte activation and recruitment to sites
of inflammation via interaction with a family of GPCRs,
the chemokine receptors. The chemokine receptor CCR5
functions physiologically as a receptor for the leukocyte
chemoattractants RANTES, MIP-1a, and MIP-1b, and
it has also recently been shown to function pathological-
ly as one of the key cell entry co-receptors for HIV-1.1 In
lesions of multiple sclerosis (MS), CCR5 has been detect-
ed on activated myeloid microglial cells and infiltrating T
cells. CCR5 antagonists might, therefore, be useful in
suppressing the chronic inflammatory symptoms of this
disease.
tion to this conclusion,3 we have demonstrated that the
aminoguanidine functional group is not required and
suitable replacements can be identified. Herein, we report
our discovery and initial SAR of guanylhydrazones of 2-
benzyloxybenzaldehyde as CCR5 receptor antagonists.
Potent, orally available, small molecule CCR5 antago-
nists are potential therapeutic agents for the treatment
of chronic inflammatory diseases and HIV.
From high throughput screening of our internal com-
pound library using a 125I-MIP-1a binding assay on a
human CCR5/CD4 transfected HEK293 cell line, 14
compounds with IC50 values of less than 5 lM were
identified. Among these, three compounds were shown
HN
N
NH2
Since the discovery of CCR5 as a co-receptor for HIV-1
cell entry, there has been an increased effort in the phar-
maceutical industry to develop CCR5 antagonists.2 Dur-
ing the course of our work in this area, we became aware
that another group screening for CCR5 antagonists had
identified the same initial lead 1 as our group (Fig. 1).3
This report indicated that the aminoguanidine group of
this compound was required for potency. In contradic-
NH
Br
O
Cl
1
CCR5 binding: IC50 0.84 μM
Ca2+ flux: IC50 2.2 μM
Keywords: CCR5; Chemokine receptor antagonists.
*
Corresponding author. Tel.: +1 510 669 4742; fax: +1 510 669
4310; e-mail: guo_ping__wei@berlex.com
Figure 1. CCR5 antagonist HTS lead 1.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.052

232
R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 231–234
HN
NH2
NH
N
O
Cl
R3
R1
(a), (b)
R2
R3
OH
R1
O
+
R2
3-41
2a
2b
Scheme 1. Reagents and conditions: (a) Cs2CO3, DMF, 60°C, 2 h; (b) aminoguanidine hydrogencarbonate, EtOH, reflux.
Table 1. Optimization of the benzyl group of guanylhydrazones
to be functional antagonists of CCR5. Compound 1, the
guanylhydrazone of 2-benzyloxybenzaldehyde, was val-
idated to be a moderately potent CCR5 antagonist,
exhibiting an IC50 value of 0.84 and 2.2 lM in the bind-
ing and Ca2+ flux assays, respectively. These preliminary
results prompted us to explore lead optimization and to
develop a structure–activity relationship (SAR) for the
series.
HN
NH2
NH
N
Br
O
R2
a
Compound
R2
IC50 (lM)
Guanylhydrazone analogs of our initial lead compound
1 were easily prepared in two steps (Scheme 1). Alkyl-
ation of the phenol (2a) with benzyl chloride or bromide
(2b) using cesium carbonate provided the benzyloxy aryl
ether. Treatment with aminoguanidine hydrogencarbon-
ate led to the formation of guanylhydrazone 3.
1
3
4-Cl
H
0.84
1.9
0.8
0.6
0.48
2
4
4-F
4-Br
5
6
4-CN
3-CN
7
8
4-NO2
3-NO2
2-NO2
0.9
1.7
5.7
1.3
1.6
39
In an attempt to improve the potency of compound 1,
we first explored variations of the benzyl group
(Table 1) with a series of substituted benzyloxy aryl
ether derivatives. The unsubstituted compound 3 was
slightly less potent than compound 1. Different halogen
substitutions had little effect on potency (1, 4, and 5).
Compounds 8 and 9, with nitro groups at the 3- or 4-po-
sition exhibited activity similar to 1, whereas 10 with a
nitro group at the 2-position showed sevenfold lower
activity. Compound 6 with a 4-cyano group exhibited
fourfold higher potency than 7 with a 3-cyano group.
Compounds 11 and 12 with methyl ester groups at the
3- or 4-position were as potent as 1, but 13 with a 3-car-
boxylic acid group was significantly less active. Electron-
donating groups at the 4-position (14, 16, and 17) were
well tolerated. Compound 14 with a 4-methyl group was
threefold more potent than 15 with a 3-methyl group.
Compound 18 with a 4-phenyl substituent was only
twofold less potent than 1. Disubstituted analogs were
prepared and tested. The 2,4-difluoro analog, 20, exhib-
ited fourfold less inhibitory activity than the 4-fluoro
analog, 4. The 3-nitro-6-methoxy analog, 21, exhibited
fivefold less inhibitory activity than the 3-nitro analog,
9. It was interesting that 19 with a fused benzene ring
demonstrated potency comparable to the initial
compound 1. From these results, it was clear that substi-
tution at the 4-position of the benzyl group was general-
ly well tolerated and was more favorable than
substitution at the 3-position.
9
10
11
12
13
14
15
16
17
18
4-CO2Me
3-CO2Me
3-CO2H
4-Me
0.5
1.7
0.7
2.9
1.7
3-Me
4-OMe
4-OBn
4-Ph
2,3-
19
1.1
20
21
2,4-diF
3-NO2-6-OMe
3.7
8.7
a Inhibition of 125I-labeled MIP-1a binding to human CCR5/CD4
transfected HEK-293 cells.
oro substituent was fourfold less active. Electron-
withdrawing groups (Cl, F, NO2, and CN) at the 5-posi-
tion (23–25, and 29) were tolerated. Larger substituents
at the 5-position, methoxycarbonyl group (32) or elec-
tron-donating groups such as amino, diethylamino,
and t-butyl (34, 33, and 28) yielded inactive compounds.
Halogen substitution at the 3- or 4-position (27, 30, and
31) without substitution at the 5-position also resulted in
a loss of inhibitory activity. The 3,5-dichloro substituted
analog, 26, was fourfold less potent than compound 1,
but the 5-bromo-3-methoxy substituted analog, 35,
was inactive. From the SAR study of the aryl ring, it
could be determined that substitution at the 5-position
with bromo, chloro, nitro or cyano group was preferred.
We next examined substitution of the central aryl ring of
the guanylhydrazone template (Table 2). Removal of the
5-bromo substituent from 1 afforded 22, which was
found to be inactive. Compound 23 with a 5-chloro sub-
stituent showed activity similar to 1, but 25 with a 5-flu-
The preparation of ketone guanylhydrazones was also
explored (Table 3). Guanylhydrazones of ketones,

R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 231–234
233
at R1 and R2. These results indicated that the methyl
group substitution was optimal at the R3 position and
the E-configuration was preferred.
Table 2. Optimization of aryl group of guanylhydrazones
HN
NH2
NH
N
It was previously suggested3 that the guanylhydrazone
group was necessary for the activity of this series, as
the corresponding benzaldehyde was inactive. The guan-
ylhydrazone moiety is a basic group and may function
as the positively charged moiety that is present in many
CCR5 antagonists.6 As such, we initially sought to re-
place this group with other similar functional groups.
We then further explored replacement of the guanylhyd-
razone group with tertiary amines (Table 4). We found
that the 4,5-dihydro-1H-imidazolyl guanylhydrazone
42 exhibited the same activity as analog 1, while the cor-
responding N-methyl substituted guanylhydrazone, 43,
resulted in diminished activity by 20-fold. 1,4,5,6-Tetra-
hydropyrimidinyl guanylhydrazone, 44, exhibited two-
fold less potent inhibitory activity than analog 1.
Reductive amination of the benzaldehyde with dimeth-
ylamine afforded compound 45, which demonstrated
similar activity to compound 1. The N,N-diethyl analog
46, and morpholine analog 47 also exhibited activity
R1
O
Cl
R1
IC50 (lM)
a
Compound
1
22
23
24
25
26
27
28
29
30
31
32
33
34
35
5-Br
H
0.84
>30
0.6
5-Cl
5-NO2
5-F
1.2
3.4
3,5-diCl
3-F
3.1
>30
>30
0.6
5-CMe3
5-CN
4-Cl
>30
>30
>30
>30
>30
>30
3-Cl
5-CO2Me
5-NEt2
5-NH2
3-OMe-5-Br
Table 4. Guanylhydrazone replacement
a Inhibition of 125I-labeled MIP-1a binding to human CCR5/CD4
transfected HEK-293 cells.
R
Br
36–41, were obtained as mixtures of E- and Z-isomers.4
When R3 was substituted by a methyl group, the E/Z ra-
tio was around 19:1 by HPLC analysis. Methyl substi-
tuted compound 36 was fourfold more potent than 1.
The E-isomer, 37E5, was 60-fold more potent than the
Z-isomer, 37Z.5 Compound 38 (R2@CN) exhibited the
highest activity in this series with an IC50 value of
0.093 lM. The ethyl substituted compound, 39, showed
twofold reduced potency relative to 36. The n-Bu ana-
log, 40, was 20-fold less active than 36. However, the
i-Bu substituted analog, 41 was inactive. Both com-
pounds 40 and 41, contained additional substitutions
O
Cl
a
Compound
R
IC50 (lM)
HN
NH2
1
0.84
NH
NH
N
N
N
42
43
44
NH
0.75
N
Table 3. Optimization of guanylhydrazones of ketones
HN
NH2
N
NH
14
NH
R3
N
R1
N
O
R2
2.0
NH
N
R1
R2
R3
IC50 (lM)
a
Compound
NH
N
1
Br
Br
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
CN
Cl
F
H
0.84
0.2
36
Me
Me
Me
Me
Et
37Z5
37E5
38
8.5
45
46
NMe2
NEt2
0.6
1.4
1.0
0.14
0.093
0.4
39
40
n-Bu
i-Bu
4.2
47
N
O
41
F
>30
a Inhibition of 125I-labeled MIP-1a binding to human CCR5/CD4
transfected HEK-293 cells.
a Inhibition of 125I-labeled MIP-1a binding to human CCR5/CD4
transfected HEK-293 cells.

234
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similar to compound 1. Based upon these results, it was
clear that the presence of the guanylhydrazone group is
not necessary for inhibitory activity and that this posi-
tion of the molecule is amenable to further optimization
efforts.
In conclusion, modification and optimization of the var-
ious groups of the initial HTS lead compound 1 led to
the discovery of a new series of CCR5 antagonists. It
was found that the guanylhydrazone moiety itself was
not required for CCR5 antagonistic potency as suitable
substitutions could be identified. Further investigations
to improve the potency and explore the PK profile of
novel CCR5 antagonists based upon compound 45 from
this series will be reported in due course.
Acknowledgments
The authors thank Dr. Jerry Dallas for structural elucida-
tion of compound 37E and 37Z using 13C NMR and Dr.
John Bauman for helpful suggestions and discussion.
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´
}
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