Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 1: Discovery and SAR study of 4-pyrazolylpiperidine side chains

Article abstract of DOI:10.1016/j.bmcl.2003.12.004

Replacement of the flexible connecting chains between the piperidine moiety and an aromatic group in previous CCR5 antagonists with heterocycles, such as pyrazole and isoxazole, provided potent CCR5 antagonists with excellent anti-HIV-1 activity in vitro. SAR studies revealed optimal placement of an unsubstituted nitrogen atom in the heterocycle to be meta to the bond connected to the 4-position of piperidine. Truncation of a benzyl group to a phenyl group afforded compounds with dramatically improved oral bioavailability, albeit with reduced activity.

Full text of DOI:10.1016/j.bmcl.2003.12.004

Bioorganic & Medicinal Chemistry Letters 14 (2004) 935–939
Antagonists of human CCR5 receptor containing
4-(pyrazolyl)piperidine side chains. Part 1:
Discovery and SAR study of 4-pyrazolylpiperidine side chains
Dong-Ming Shen,a,* Min Shu,a Sander G. Mills,a Kevin T. Chapman,a
Lorraine Malkowitz,b Martin S. Springer,b Sandra L. Gould,b Julie A. DeMartino,b
Salvatore J. Siciliano,b Gloria Y. Kwei,c Anthony Carella,d Gwen Carver,d
Karen Holmes,d William A. Schleif,d Renee Danzeisen,d Daria Hazuda,d Joseph Kessler,d
Janet Lineberger,d Michael D. Millerd and Emilio A. Eminid
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
dDepartment of Antiviral Research, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 2 September 2003; revised 24 November 2003; accepted 2 December 2003
Dedicated to the fond memory of our colleague and friend Dr. Christopher A. Willoughby
Abstract—Replacement of the flexible connecting chains between the piperidine moiety and an aromatic group in previous CCR5
antagonists with heterocycles, such as pyrazole and isoxazole, provided potent CCR5 antagonists with excellent anti-HIV-1 activity
in vitro. SAR studies revealed optimal placement of an unsubstituted nitrogen atom in the heterocycle to be meta to the bond
connected to the 4-position of piperidine. Truncation of a benzyl group to a phenyl group afforded compounds with dramatically
improved oral bioavailability, albeit with reduced activity.
# 2004 Elsevier Ltd. All rights reserved.
CCR5 is a chemokine receptor from the family of seven-
transmembrane G-protein coupled receptors. It is the
primary co-receptor with CD4 for macrophage tropic
HIV-1 virus strains’ entry into host cells.1,2 HIV-1
strains using CCR5 for cell entry, now named R5 vari-
ants, are believed to be primarily responsible for the
transmission of HIV-1 between individuals. They are
usually present throughout the course of HIV-1 infec-
tion, both before and after the onset of AIDS. Persons
homozygous for a 32-base pair deletion in their CCR5
gene do not express functional CCR5 at the cell surface.
These individuals are highly resistant to HIV-1 infec-
tion.3 Heterozygous individuals, while susceptible to
HIV-1 infections, showed delayed progression to AIDS
compared to infected homozygous persons.4,5 These
observations highlight the central role CCR5 receptor
plays in the establishment and perpetuation of HIV-1
infection in vivo. Individuals without cell surface CCR5
receptor appear to be physiologically normal. Thus,
CCR5 antagonists have become a potential target for the
treatment and prevention of HIV-1 infection.6ꢀ8 Other
reports have also suggested a linkage between the
CCR5Á32 deletion and age of onset of multiple sclerosis.9
Previous reports from our laboratories disclosed the
discovery and SAR studies on several series of CCR5
antagonists including 113 and 2.10 They all have piper-
idines with a flexible side chain at the 4-position
Keywords: CCR5 antagonists; HIV-1; Antiviral; Pyrazole.
* Corresponding author. Tel.: +1-732-594-7303; fax: +1-732-594-
8080; e-mail: dongming_shen@merck.com
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.004

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D.-M. Shen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 935–939
Scheme 1. Reagents: (a) O,N-dimethylhydroxylamine hydrochloride, HOBt, EDC, DIEA, DMF, 97%; (b) CH3MgBr (2.25 equiv), ether, 0 ꢁC,
100%; (c) methyl phenylacetate, NaH, THF, rt, 66%; (d) ethylhydrazine oxalate, MeOH, 50 or 60 ꢁC; (e) 10% TFA in DCM, anisole or HCl in
MeOH, rt, 58% 6a and 23% 6b (two steps); (f) NaBH(OAc)3, DIEA, 1,2-dichloroethane; (g) Pd/C, H2, MeOH, 68% (two steps).
connected to an aromatic group.10ꢀ13 To improve anti-
viral activity and in vivo properties, we wished to con-
strain this side chain by replacing the flexible linking
group with an aromatic moiety. Our interest in pyrazole
chemistry14 led us to initiate these studies with this
heterocycle.15,16
hydrazines. Isomeric pairs of 12 were prepared from
separated benzyl precursor and coupled to give 13 and
14. Among the two isomeric products, 13 and 14, the
more active isomer 13 showed similar activities as 8a in
both CCR5 binding and HeLa assays (Table 2). In
this series, phenylethyl is more active than benzyl
based on comparison of 13a/b and 13c/d. Among
substitutions on the pyrazole nitrogen, the antiviral
activity decreases in the order of Et, Me, n-Pr, and H.
Some isomeric pyrazoles 13 and 14 have similar binding
activities (see 13b and 14b). However, in the antiviral
assay, 13 was much more active than 14.
The synthesis of an initial group of pyrazolyl piperidines
is illustrated by the preparation of 8a in Scheme 1. Also
shown are other pyrazolylpiperidine intermediates 6b–h
used for the similar preparation of 8b–h. The Boc
precursors to 6a and 6b can be separated by RP-HPLC
(acetonitrile and water with 0.1% trifluoroacetic acid)
or silica gel chromatography (methylene chloride and
acetonitrile). The ratio of 6a/6b varies from 2 to 3. Their
structures were established by NOE difference or
NOESY experiments. Ratios of isomeric mixtures of 8e/
Parallel investigation of the SAR of the alkyl group on
the pyrazole ring of compound 8a showed a similar
Table 1. CCR5 binding affinity and antiviral activity of pyrazole
compounds 8a–h
1
f and 8g/h were determined by H NMR. Compounds
6c–h were prepared from appropriate b-diketones and
1-benzyl-4-hydrazinopiperidine followed by debenzyl-
ation using ammonium formate and Pd(OH)2. The
diketone precursors themselves were either commer-
cially available (for 6c and 6d) or prepared according to
a literature method from 2-butanone and an ester.17 The
synthesis of the aldehyde
previously.13
7 has been reported
After compounds were tested for their activity in dis-
placing [125I]-labeled MIP-1a from the CCR5 receptor
expressed on CHO cell membranes,19 the more potent
compounds were further evaluated as antivirals in a cell
culture HIV-1 infectivity assay using HeLa cells (here-
after referred to as HeLa assay).20 Table 1 shows these
results for pyrazoles 8a–h. Among these compounds,
compound 8a emerged as a new lead structure with
potent CCR5 binding and in vitro antiviral activity.
Compd (ratio)
R=
CCR5 IC50 (nM)a,b
HeLa IC90, (nM)e
n=
8a
8b
8c
8d
1.2,c 2.1
76
65% Inh. @ 2 mM
79% Inh. @ 2 mM
0.4, 0.4
>300
NDd
NDd
Me
Me
0
0
8e and 8f (3:2)
Et
8g Et
8g and 8h (1:1)
Et
1
2
78
82% Inh. @ 2 mM
>300
NDd
The dramatic difference in the antiviral activities of 8a
and 8f demonstrated the importance of the correct
placement of the nitrogen atoms on the pyrazole ring.
These results prompted us to prepare compounds 13
and 14 having the alternative benzyl placement from
compound 8a (Scheme 2). When alkylhydrazines were
used in the cyclization of 11 to 12, the major isomer was
12b by about 4 to 5-fold over 12a. We believe that steric
factors overrode the usual preference of aldehyde
bonding to the unsubstituted nitrogen of alkyl
2
105
>300
a IC50’s reported are averages of triplicate measurements whose
standard errors were normally <15% in a given assay. Assay to
assay variability was within ꢂ2-fold based on a standard compound.
b Unless otherwise noted, data from a GP120-membrane-based assay.
See ref 19 for assay protocol.
c Displacement of [125I]-labeled MIP-1a from the CCR5 receptor
expressed on CHO cell membranes. See ref 12 footnote 20 for assay
protocol.
d ND=Not determined.
e See ref 20 for assay conditions.

D.-M. Shen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 935–939
937
.
Scheme 2. Reagents: (a) triphenylphosphine, toluene, reflux, 70–90%; (b) N-benzylpiperidone, KN(TMS)2, toluene, 40–62%; (c) BH3 THF (3
equiv), THF; (d) CrO3, H2SO4, H2O, 25% (two steps); (e) methyl formate, KO-t-Bu, THF, 60%; (f) RNHNH2, MeOH, 50 ꢁC; (g) semi-preparative
HPLC separation; (h) ammonium formate, Pd(OH)2, MeOH, 60 ꢁC, for three steps: 98% (R¼H), 22–39% (12a), 50–76% (12b); (i) NaBH(OAc)3,
DIEA, 1,2-dichloroethane; (j) Pd/C, H2, MeOH, 50–89% (two steps).
trend, except here the parent compound 8i was equip-
otent with the ethyl compound 8a (Table 3). Bulkier and
polar groups were not well tolerated at this position.
Table 3. CCR5 binding affinity and antiviral activity of pyrazoles
The marked difference in antiviral activity of the
isomeric compounds 8a/8b and 13a/14a might be due to
a steric effect of the ethyl group or preferred location of
the unsubstituted nitrogen atom in 8a and 13a. To
distinguish these two possibilities, isoxazole analogues
were prepared (Scheme 3). The isomeric isoxazoles 15a
Compd
R=
CCR5 IC50 (nM)a
HeLa IC90, (nM)
1
and 15b were assigned using H NMR.18 Even though
they have similar CCR5 receptor binding activities,
compound 16 was about 10-fold more active than
isomer 17 in the antiviral assay (Table 4). This result
suggests that placement of the unsubstituted nitrogen
atom meta to the bond connected to the piperidine
provides optimal antiviral activity.
8a
8i
8j
8k
8l
8m
8n
8o
Et
H
Me
n-Pr
2.1
2.2
4.2
4.6
3.0
4.8
9.0
33
0.4, 0.4
0.4
3.7
1.2
11
33
n-Bu
CH2CF3
CH2CO2Et
CH2CO2H
11, 33
ND
Compounds 8a, 13b, and 16 all showed excellent anti-
viral activity in a more rigorous 7-day in vitro viral
infectivity assay using peripheral blood mononuclear
cells (PBMC),21 with IC95’s of <8 nM against the BAL-
1 strain of HIV-1. Therefore, they are more potent
antivirals than the compounds with flexible connecting
groups between phenyl and piperidine rings such as 1.
However, these compounds showed poor pharmacoki-
netic profiles in the rat (Table 5). Truncation of the
benzyl group in 8a to a phenyl group (18a) dramati-
cally improved its pharmacokinetic profile in the rat,
but unfortunately at the expense of a considerable loss
a See footnote bof Table 1 .
Table 2. CCR5 binding affinity and antiviral activity of pyrazole
compounds 13 and 14 with alternative benzyl placement
Scheme 3. Reagents: (a) hydroxylamine hydrochloride, DIEA, EtOH,
50 ꢁC; (b) TFA, anisole, DCM, rt; (c) RP-HPLC separation, 18% each
15a and 15b (three steps); (d) NaBH(OAc)3, DIEA, 1,2-dichloro-
ethane; (e) Pd/C, H2, MeOH, 67% (16) and 92% (17).
Compd
R=
CCR5 IC50 (nM)a
HeLa IC90, (nM)
n=
13a
13b
13c
13d
13e
13f
Et
Et
H
H
Me
n-Pr
1
2
1
2
2
2
3.0
0.6
4.8
3.7, 10
0.4
100
33
3.7
11
1.2
1.0b
0.6b
Table 4. CCR5 binding affinity and antiviral activity versus location
of the key nitrogen atom in the heterocycle
14a
14b
14e
14f
Et
Et
Me
n-Pr
1
2
2
2
20
>300
300
Compd
CCR5 IC50 (nM)a
HeLa IC90, (nM)
2.3
23b
9.0b
ND
ND
16
17
0.8
1.2
0.4
3.7
a See footnote bof Table 1 .
b See footnote c of Table 1.
a See footnote c of Table 1.

938
D.-M. Shen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 935–939
Table 5. Pharmacokinetic parameters of CCR5 antagonists in the rat
Table 7. CCR5 binding affinity and antiviral activity of metabolically
blocked benzyl pyrazoles
.
Compd
Clp, mL/min/kg
AUCNpo, mM hr/dose
t1/2, h
F,%
8a
64
79
67
3.8
72
0.02
0.01
0.03
1.7
0.82
ND
ND
1.5
4.1
2.6
5.9
21
13ba
16a
18a
18ja
0.01
0.45
3.2
a Dosed as a mixture of five compounds. Parameters for 18a from
mixture dosing were similar to those reported for single compound
dosing. For 8a, AUCNpo and %F were about 3- and 2-fold higher in
mixture dosing, respectively.
Compd
R=
CCR5 IC50 (nM)a
HeLa IC90 (nM)
R0,R0=
19a
19b
19c
19d
19e
19f
19g
H
H
Et
H
Et
H
Et
F2
¼O
2.1
41
23
11.5
6.9
4.4
11
300
ND
ND
ND
300
Table 6. CCR5 binding affinity and antiviral activity in the truncated
and elongated pyrazole series
¼O
Me2
Me2
–CH2CH2—
–CH2CH2—
>300
ND
a See footnote c of Table 1.
13a, and 16 which were potent binders to the CCR5 recep-
tor. These compounds also showed improved antiviral
activity in vitro against HIV-1. SAR studies indicated the
optimal placement of the unsubstituted nitrogen atom in
these heterocycles to be meta to the bond connected to the
piperidine ring for the best antiviral activity. Truncation of
the benzyl group to phenyl group led to compound 18a
which had excellent oral bioavailability. Further studies of
these and related series leading to compounds with both
oral bioavailability and potent antiviral activity are
reported in the following paper.
Compd
Ar=
CCR5 IC50 (nM)a HeLa IC90 (IC50, nM)
R= X=
18a
18b
Ph
4-CNPh
18c 4-H2NCH2Ph Et
18d 4-CNPh
18e 1-Naphthyl
Et
Et
H
H
H
H
H
F
22
16
112
>300 (100)
>300 (33)
ND
>300 (100)
>300
ND
H
H
7.5b
7.5b
18f 1-Naphthyl Et
16b
18g 2,4-Cl2Ph
18h 4-ClPh
18i 4-MeOPh
H
H
H
F
F
F
9b
4b
>300
>300
12b
>300
11
18j PhCH2CH2 Et
F
2.2b
Acknowledgements
a See footnote bof Table 1 .
b See footnote c of Table 1.
The authors would like to thank Dr. Scott Berk for a
generous sample of intermediate 7, Dr. Jeffrey J. Hale
for informing us about better conditions for the
separation of the Boc precursors of 6a and 6b on silica
gel, Dr. Gerard R. Kieczykowski, Mr. Joseph F. Leone
and Mr. Frederick Wong for synthetic assistance, and
Drs. Charles G. Caldwell and Paul E. Finke for reviewing
the manuscript.
of binding and antiviral activities (Table 6). A survey of
several different aryl groups failed to improve the anti-
viral potency significantly in this series. In addition,
elongation of the benzyl group in 8a to phenethyl in 18j
did not offer any advantage over 8a in its pharma-
cokinetics. It also resulted in a considerable loss of
antiviral activity in 18j.
References and notes
The dramatic difference in pharmacokinetic profiles of
8a and 18a prompted us to block the benzylic methylene
position of 8a to prevent metabolism at this site (Table
7). Some of these compounds retained good binding
affinity, such as the gem-difluoro compound 19a, gem-
dimethyl compound 19e, and cyclopropane compound
19f. However, their antiviral activities were greatly
diminished compared to the parent compound 8a. In
addition, compounds 19a, 19e, and 19f showed no
significant improvements in clearance or oral AUC
when dosed as mixtures in the rat compared to 8a (data
not shown).
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