Exploration of a new series of CCR5 antagonists: Multi-dimensional optimization of a sub-series containing N-substituted pyrazoles

Article abstract of DOI:10.1016/j.bmcl.2010.06.135

The introduction of N-substituted pyrazoles in a new series of CCR5 antagonists was shown to substantially increase antiviral activity.

Full text of DOI:10.1016/j.bmcl.2010.06.135

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4753–4756
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of a new series of CCR5 antagonists: Multi-dimensional
optimization of a sub-series containing N-substituted pyrazoles
a,
Rémy C. Lemoine a, , Ann C. Petersen , Lina Setti a, Andreas Jekle b, Gabrielle Heilek b, André deRosier b,
*
Changhua Ji b, Pamela Berry c,à, David M. Rotstein a
a Department of Medicinal Chemistry, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
b Department of Viral Diseases, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
c Department of DMPK, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 9430, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The introduction of N-substituted pyrazoles in a new series of CCR5 antagonists was shown to substan-
tially increase antiviral activity.
Received 18 February 2010
Revised 23 June 2010
Accepted 28 June 2010
Available online 1 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CCR5 antagonists
Pyrazoles
CCR5 is one of the many chemokine receptors involved in the
mediation of the inflammation process, In addition to its role as
a receptor on inflammatory leucocytes, CCR5 is also the primary
co-receptor for macrophage-tropic HIV-1. CCR5 has proven to be
an extraordinary target for the pharmaceutical industry, with
demonstrated therapeutic applications in HIV-treatment as well
as potential applications in various chronic or acute inflammatory
diseases.1
We recently described a new series of CCR5 antagonists with
compounds 1 and 3 being two representatives.2 This series con-
tains the four pharmacophores found in most of the series reported
in the literature: a tertiary basic amine, two hydrophobic groups in
the western portion of the molecule (henceforth referred to as the
tail), including the tail hydrophobe 1, and an (hetero)aryl in the
eastern portion of the molecule (the head). We describe herein,
part of our SAR focusing on the head region, specifically the intro-
duction of substituted heteroaryl heads. Early in the project, we
screened a wide range of head acyl and sulfonyl groups. From
the hundreds of compounds that were prepared, several types
stood out, in particular the substituted heteroaryl heads (Fig. 1).
The N-phenyl substituted pyrazole 2 was more than fourfold
more active than 1 in our HIV antiviral assay (Table 1). It was ar-
gued at the time that the increase in activity was lipophilicity-dri-
ven (log D of 2.92 for 2 vs 1.37 for 1) and that 2, ultimately was not
a more efficient ligand than 1 when the lipophilicity increase was
taken into consideration. However, we observed similar increases
in antiviral activity for compounds that contained other substi-
tuted heteroaryl heads, and with similar lipophilicity. In particular,
compound 4 was threefold more potent than compound 3, with
both compounds having virtually equal measured log Ds (Table 1).
Taking into consideration this observation, we decided to ex-
plore the N-substituted pyrazole heads more exhaustively.
Compounds were prepared according to the general procedure
depicted in Scheme 1.
hydrophobe 1
O
H
HN
N
O
N
H
R1
1
2
3
4
R1 =
N
N
N
N
N
* Corresponding author.
N
a
c
d
E-mail address: remy_lemoine@yahoo.com (R.C. Lemoine).
Present address: Discovery Chemistry, Hoffmann-La Roche, Inc., 340 Kingsland
Street, Nutley, NJ 07110, USA.
b
N
à
Present address: DMPK, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ
Figure 1. Structures of representative examples of the original series (1, 3) and
examples of compounds with substituted heads (2, 4).
07110, USA.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.135

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R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4753–4756
Table 1
CO2tBu
CO2tBu
N N
MeO2C
O
O
O
CO2Et or tBu
N N
R-NHNH2
(a)
Antiviral activity and measured lipophilicity compounds 1–4
(b)
Compound
R1
Antivirala (nM)
log Dc
N N
HO2C
OEt or tBu
1
2
3
4
a
b
c
6.2b
<1.5
2.1
1.37
2.92
1.90
1.89
R
CO2Et
CO2Et
d
0.7
(c)
a
b
c
N N
N N
Replication inhibition (IC50) of R5-HIVNLBal in JC53-BL cells.
Values are means of at least two experiments.
High throughput log D determined at pH 7 using the shake flask method.
N
N
N
N
Cl
Scheme 2. De novo synthesis of the pyrazole esters. Reagents and conditions: (a)
MeOH, H2O, AcOH, rt, 24 to 48 h, yields in Figure 2; (b) TMSCHN2, MeOH/CH2Cl2, rt,
2 h, 32% para, >95% meta; (c) H2 (1 psi), 10% Pd/C, MeOH/dioxane, rt, 48 h, 22%.
H
6
HN
N
NHBoc
NHBoc
H
Bn
H
O
N
N
N
Bn
(a)
CO2Et
N N
CO2Et
N N
CO2Et
N N
CO2Et
N N
H
5: X = H, F
7: X = H, F
(a)
(c)
(b)
(d)
X
X
X
(b), (c)
R2
H
R
N
N
N
(d), (e)
O
NHBoc
H
H
HN
Br
N
N
O
O
N
CO2Et
N N
Br
Br
CO2Et
N N
H
H
R1
R1
N
N
2, 9-36
8: X = H, F
X
H
CHO
CHF2
Scheme 1. Synthesis of head- and tail-modified bis-pyrrolidine-based CCR5
antagonists. Reagents and conditions: (a) NaBH(OAc)3, CH2Cl2, rt, 2 h; (b) H2
(1 atm), 20% Pd(OH)2, EtOH, rt, 24 h, >95%; (c) R1CO2H, EDCI, HOBt, Et3N (or DIPEA),
CH2Cl2, rt, overnight, 40–80%; (d) HCl 4 M in dioxane, CH2Cl2, rt, 2 h; (e) R2CO2H,
EDCI, HOBt, Et3N (or DIPEA), CH2Cl2, rt, overnight or R2COCl, pyridine (or Et3N),
CH2Cl2, 0 °C to rt, overnight, 50–80%.
N
Scheme 3. Alkylation of the preformed pyrazole ester. Reagents and conditions:
(a) NaH, DMF, rt, 50 °C or 80 °C, alkylation yields on Figure 2; (b) H2 (1 atm), 10% Pd/
C, EtOH, AcOH, rt, overnight, 53% after recrystallization in MeOH; (c) DAST, Et2O,
CH2Cl2, 0 °C to rt, overnight, 53%; (d) CuI, N,N0-ethylenendiamine, dioxane, reflux,
overnight, 7%.
R1
=
e
f (12%)a
g (24%)
h (48%)
m (43%)
7. The pyrrolidine N-benzyl groups were hydrogenolyzed and the
pyrrolidine nitrogens were acylated with the appropriate acids
R1CO2H (Fig. 2) using standard amidation procedures to give 8.
The tail protecting groups were then hydrolyzed and the primary
amines were finally acylated with R2CO2H, or R2COCl to give com-
pounds 2, 9–36 (Fig. 2).
N N
i (89%)
j
k (87%)
CO2Me
HO2C
l
The pyrazole esters leading to the formation of acids R1CO2H
were either commercially available, like in the case of e, or pre-
pared according to two general procedures, de novo formation of
the N-substituted pyrazole ring or N-alkylation of the pre-formed
pyrazole ester.
The de novo synthesis of the N-substituted pyrazole esters in-
volved reacting ethyl or t-butyldiacetoacetate with the appropriate
N-substituted hydrazines (Scheme 2).
CO2H
CO2Me
F
q (7%)
n (17%)
N
o (10%)
p (66%)
N
N
N
N
N
r (44%)
N
s (70%)
N
t (23%)
N
u (62%)
In the case of i and k, t-butyl diacetoacetate5 was used, leading
to the formation of orthogonally protected diesters, which allowed
subsequent chemoselective hydrolysis of the t-butyl esters. The
hydrazines (free base or hydrochloride salts) used for the prepara-
tion of h, m, n, p, and s, were all commercially available. In the case
of i and k, the products of cyclization with 4- and 3-hydrazinoben-
zoic acid, respectively, were treated with trimethylsilyldiazome-
thane to form the methyl esters (Scheme 2). In the case of p, 3-
chloro-6-hydrazinopyridazine was used and following cyclization,
the chlorine atom was hydrogenolyzed under standard reaction
conditions (Scheme 2). The hydrazines used for the preparation
of pyrazoles f and g were prepared from the corresponding cyclo-
alkylketones according to literature procedures.6
N
N
N
CF3
CHF2
CF3
O
R2
=
A
B
C
D
E
Figure 2. R1 and R2 substituents. aPercentages in parentheses refer to the
un-optimized yields of the corresponding N-substituted pyrazole formation reac-
tion. Pyrazoles R1CO2H where R1 = e was commercially available. Pyrazoles R1CO2H
where R1 = f, g, h, i, j, k, l, m, n, p, and s were prepared by the de novo preparation of
the pyrazole ring. Pyrazoles R1CO2H where R1 = o, q, r, t, and u were prepared by
N-alkylation of the pre-formed pyrazole esters.
The other approach to the synthesis of the pyrazoles involved
N-alkylation of the commercially available 3,5-dimethyl-1H-pyra-
zole-4-carboxylic acid (Scheme 3). We mostly used conditions in
which the pyrazole was deprotonated with sodium hydride in
Aldehydes 5 were prepared according to literature procedures.3
They were treated with template 64 under reductive N-alkylation
reaction conditions to form the orthogonally protected bis-amines

R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4753–4756
4755
Once the N-substituted pyrazoles were prepared, the esters
were generally hydrolyzed under basic conditions (Scheme 4). In
the case of u, basic ester hydrolysis conditions led to the hydrolysis
of the molecule and none of the acid was recovered from the reac-
tion mixture. However, we were able to hydrolyze the ethyl ester
using trimethyltin hydroxide as weak nucleophile (Scheme 4).8 In
the case of i and k, the t-butyl esters were hydrolyzed using triflu-
oroacetic acid (Scheme 4).
Compounds 19 and 21 were prepared by base hydrolysis of the
corresponding methyl esters 18 and 20 (KOH, EtOH/H2O, 40 °C).
The compounds prepared were tested in our R5-HIV-1 antiviral
assay9 and their respective metabolic clearance was measured in
vitro, using human liver microsomes (Table 2).
CO2Et
N N
CO2H
N N
CO2tBu
N N
CO2H
N N
(a)
(b)
R
R
MeO2C
MeO2C
CO2Et
N N
CO2H
N N
(c)
N
N
N
N
CF3
CF3
Scheme 4. Hydrolysis of the pyrazole esters. Reagents and conditions: (a) KOH,
EtOH/H2O, 40 °C, overnight; (b) TFA, Et3SiH, CH2Cl2, rt, 2 h; (c) Me3SnOH,
ClCH2CH2Cl, microwave, 160 °C, 5 h, 37%.
Several SAR trends emerged from the antiviral activity of com-
pounds 9–36.
N-Substitution of the pyrazole with an hydrophobic group led
to a substantial increase in antiviral activity (compounds 2 and
22 compared to 1). The improvement was so substantial that the
tail hydrophobe 1 could be trimmed down and still provide
compounds that retained good antiviral activity (e.g., compounds
10–12 vs 2, 16 vs 17, and 21 vs 22). It is noteworthy to point out
that in our original series bearing unsubstituted heads, we were
not able to decrease the size of the tail hydrophobe 1 without abol-
ishing the activity. Also, the boost in activity was so substantial
that mildly hydrophilic groups could be tolerated in the tail hydro-
phobe 1 (e.g., 23 vs 22, 25 vs 24, 34 vs 33, and 36 vs 35). In our ori-
ginal series with unsubstituted heads, replacement of the optimal
cyclopentyl group with a tetrahydrofuran in hydrophobe 1 led to a
20-fold loss in potency (compound 9 vs 1). These last two observa-
tions showed the superiority of the substituted heads over the
unsubstituted heads, as far as antiviral activity was concerned,
by allowing tail modifications that had hitherto been impossible.
dimethylformamide and then treated with an electrophile such as
an haloalkyl or an haloheteroaryl.
The N-alkylation products most of the time precipitated from
the reaction mixture and were isolated by filtration. Commercially
available chloropyrazine was used for the synthesis of r. In the case
of o, we used the commercially available 2-bromo-5-fluoropyri-
dine. After the N-alkylation reaction, the bromide atom was re-
moved by hydrogenolysis (Scheme 3). In the case of u, we used
3-chloro-6-trifluoromethylpyridazine, which was prepared accord-
ing to literature procedures.7 In the case of t, the electrophile used
was 2-bromo-5-difluoromethylpyridine, that was prepared by
DAST fluorination of the commercially available 6-bromopyri-
dine-3-carbaldehyde (Scheme 3). For the unreactive 5-bromopy-
rimidine, N-alkylation using the sodium hydride conditions did
not yield any product, so for the preparation of q, copper-catalyzed
N-alkylation conditions were used instead (Scheme 3).
Table 2
HIV-1 antiviral activity, c log P, and in vitro human metabolic clearance of compounds 1, 2, and 9–36
a,b
Compound
X
R1
R2
Antiviral
(nM)
c log P
In vitro metabolic clearancec
1
2
9
H
H
H
H
H
H
F
a
e
a
e
e
e
f
g
h
i
i
j
k
l
m
m
m
n
n
o
p
q
r
D
D
E
A
B
C
D
D
D
A
D
D
D
D
A
D
E
6.2
61.5
120
100
8
2.68
4.85
1.33
3.48
4.32
4.61
3.96
4.52
5.08
3.39
4.86
2.40
4.86
2.40
3.79
5.01
3.81
3.92
2.57
3.92
2.69
3.00
1.63
3.00
3.41
3.42
4.11
2.76
3.61
2.26
—
—
17
70
—
—
—
—
—
77
—
0
—
3
213
644
331
512
55
—
137
332
—
909
152
343
—
253
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
7
36
16
60
19
<1.2
24
2
13
11
<1.2
2
12
27
7
15
11
P625
14
7
F
F
H
H
H
H
H
F
H
F
F
F
F
H
F
F
F
F
F
F
F
F
F
D
E
D
D
D
A
D
A
E
D
E
D
E
r
s
s
t
t
u
u
<1.2
5
11
5
43
263
a
b
c
Replication inhibition (IC50) of R5-HIVNL-Bal in JC53-BL cells.
Values are means of at least two experiments.
Hydrolysis rate in lL/min/mg of protein. P35 was considered high.

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R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4753–4756
As far as the pyrazole N-substituent were concerned, an aryl or an
As we improved our understanding of the SAR trends of the
N-substituted pyrazoles, we soon realized that we were limited to
the exploration of a very small region of space as far as multi-dimen-
sional optimization was concerned. Indeed while aryl pyrazole N-
substituents offered a substantial boost in antiviral activity, the best
groups led to compounds with extremely high in vitro human clear-
ance, and projected in vivo clearances higher than hepatic blood
flow. From this set of compounds, 18 and 20 had the lowest meta-
bolic clearance (0 and 3 lL/min/mg of protein, respectively). How-
ever the presence of the carboxylic acid functionality led to an un-
surmountable decrease in intrinsic permeability.
We assumed that by lowering the lipophilicity of the com-
pounds, we would decrease first pass metabolism. We thus focused
on compounds that contained heteroaryl pyrazole N-substituents.
Our efforts to reach the best compromise between optimal activity
and lower c log P in the heteroaryl N-substituted pyrazole series
led to the preparation of 34 (antiviral IC50 = 11 nM, c log P = 2.76).
Unfortunately, 34 also showed low stability in our human liver
heteroaryl group was preferred over a cycloalkyl group (e.g., com-
pounds 13–15 vs 2, 24, or 26). Also, it was observed that electron-
withdrawing groups on the aryl substituent further increased the
antiviral activity. This was more obvious when the tail hydrophobe
1 was un-optimal, such as a methyl group (e.g., compounds 16 and
21 vs 10) as the combined effect of this SAR trend with the optimal
cyclopentyl tail hydrophobe 1 led to compounds with un-compara-
ble antiviral activities below detection levels (e.g., compounds 17
and 22 vs 2). Albeit not as good as an hydrophobic electron-with-
drawing substituent, even the highly hydrophilic electron-with-
drawing carboxylic acid was tolerated (e.g., compounds 18 and
20). The antiviral potency increase brought by the introduction of
N-heteroaryl groups was about 10-fold lower than that of an N-aryl
group (e.g., compounds 24–28, and 30 vs 2). However, electron-
withdrawing hydrophobic groups on the heteroaryl group could,
to a certain extend, modulate the loss in antiviral activity observed
when switching from an aryl to an heteroaryl pyrazole N-substitu-
ent (compound 32 vs 25, 33 vs 24, and 35 vs 27).
microsome assay (253 lL/min/mg of protein). Metabolite
Most of the compounds prepared were more lipophilic than
compound 1, as estimated by their c log Ps. So, it still appeared that
the boost in antiviral activity of this series of N-substituted pyra-
zoles was lipophilicity-driven. However, 18 and 20, both bearing
a carboxylic acid were less lipophilic than 1 and were still very ac-
tive in the antiviral assay. This indicated to us that there was more
to the increase in activity than a simple lipophilicity effect. From
binding inhibition experiments (data not shown), we observed that
the effect of the N-substituted pyrazoles on the binding inhibition
was not as dramatic as it was observed for the antiviral activity
(i.e., activity fold increases were disproportionally higher for the
antiviral assay than for the binding inhibition assay). This observa-
tion could seem to indicate that the N-substituted pyrazoles did
not increase antiviral activity solely via an increase in binding
energy. In order to explain the increase in antiviral potency, we
hypothesized that the N-substituted pyrazole induced a more effi-
cient allosteric rearrangement of the CCR5 receptor which has been
shown to be required for antiviral activity after the binding of the
antagonist.10 From the findings described above, we concluded
that as far as the N-pyrazole substituent was concerned, elec-
tron-poor aromatics were best accommodated in the head CCR5
binding site. We believe that the presence of nitrogen atoms in
the aromatic ring led to a decrease in antiviral activity boost be-
cause they induced a polarization of the electron density of the
ring. Electron density calculations suggested that substitution with
an electron-withdrawing substituent delocalized some of the neg-
ative partial charge from the nitrogen atoms to the substituent.
This led to an overall ‘softer’ electrostatic charge distribution in
the aromatic ring, thus counter-balancing the detrimental effect
of the nitrogen atoms.
identification failed to attribute specific oxidative pathways to
the N-substituted pyrazole heads. We believe that instead of
contributing directly to the metabolic instability, such heads gen-
erated molecules that were better recognized by the cytochrome
P450 enzymes.
Unable to associate potent antiviral activity with good meta-
bolic stability we abandoned our work on the pyrazole sub-series.
We described herein a sub-series of CCR5 antagonists in which
the use of N-substituted pyrazole heads substantially increased
antiviral activity. SAR analysis showed that the best pyrazole sub-
stituents for activity were electron-poor aromatic systems. Such
groups, however, provided metabolically unstable compounds.
References and notes
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