1,3,4 Trisubstituted pyrrolidine CCR5 receptor antagonists bearing 4-aminoheterocycle substituted piperidine side chains

Article abstract of DOI:10.1016/S0960-894X(02)00988-5

A new class of 4-(aminoheterocycle)piperidine derived 1,3,4 trisubstituted pyrrolidine CCR5 antagonists is reported. Compound 4a is shown to have good binding affinity (1.8 nM) and antiviral activity in PBMC's (IC₉₅=50 nM). Compound 4a also has improved PK properties relative to 1.

Full text of DOI:10.1016/S0960-894X(02)00988-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 427–431
1,3,4 Trisubstituted Pyrrolidine CCR5 Receptor Antagonists
Bearing 4-Aminoheterocycle Substituted Piperidine Side Chains
a,
a
a
a
Christopher A. Willoughby, * Keith G. Rosauer, Jeffery J. Hale, Richard J. Budhu,
a
a
a
b
Sander G. Mills, Kevin T. Chapman, Malcolm MacCoss, Lorraine Malkowitz,
b
b
b
b
Martin S. Springer, Sandra L. Gould, Julie A. DeMartino, Salvatore J. Siciliano,
b
c
c
c
Margaret A. Cascieri, Anthony Carella, Gwen Carver, Karen Holmes,
c
c
c
c
William A. Schleif, Renee Danzeisen, Daria Hazuda, Joseph Kessler,
c
c
c
Janet Lineberger, Michael Miller and Emilio A. Emini
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Antiviral Research, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 12 June 2002; accepted 30 October 2002
Abstract—A new class of 4-(aminoheterocycle)piperidine derived 1,3,4 trisubstituted pyrrolidine CCR5 antagonists is reported.
Compound 4a is shown to have good binding affinity (1.8 nM) and antiviral activity in PBMC’s (IC95=50 nM). Compound 4a also
has improved PK properties relative to 1.
#
2002 Elsevier Science Ltd. All rights reserved.
In 1996, it was shown that HIV-1 infection of macro-
phages, monocytes and T-cells is mediated by inter-
action with, in addition to the cell surface molecule
1
CD4, the b-chemokine receptor CCR5. This discovery
initiated an intense research effort directed at the devel-
opment of CCR5 antagonists as potential anti-HIV
2
therapeutic agents. Our own work in this area has led
to the discovery of 1,3,4 trisubstituted pyrrolidine deri-
vatives that have been found to be high affinity CCR5
receptor antagonists with excellent anti-HIV activity in
3
vitro.
Compound 1 was recently reported as an example of
these compounds.3b Although 1 has excellent antiviral
activity, its poor in vivo properties prompted further
optimization. In this paper we wish to report the modi-
fication of the carbamate moiety in 1 to an aromatic
heterocycle with the aim of improving the pharmaco-
kinetic parameters while maintaining the desired antiviral
activity.
The synthesis of these 4-aminoheteroaryl piperidine
derivatives is illustrated in Scheme 1. The 1-Boc-4-ami-
nopiperidine was prepared in two steps from commer-
cially available N-Boc-piperidin-4-one by reductive
amination with benzyl amine followed by hydro-
genolysis of the benzyl group. Arylation of the 4-amino
group was carried out in one of two ways depending on
the reactivity of the aryl halide. For aryl halides known
to undergo electrophilic substitution, such as 2-chloro-
pyrimidine derivatives, simple substitution by heating
the aryl halide and 4-amino-1-Boc-piperidine in iso-
propanol afforded products in reasonable yields. Less
*Corresponding author. Fax: +1-805-480-1337; e-mail: cwilloug@
amgen.com
0
960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00988-5

428
C. A. Willoughby et al. / Bioorg. Med. Chem. Lett. 13 (2003) 427–431
of the Boc group with HCl and reductive alkylation
with the requisite 3-formyl-4-phenyl pyrrolidine deriva-
tives afforded after deprotection, the final pyrrolidine
products.
Based on the assumption that the carbamate carbonyl
in 1 is acting as an H-bond acceptor our initial studies
focused on 2-pyridyl derivatives. Receptor binding and
antiviral activities are shown in Table 1. Data are
5
reported as IC ’s for both binding versus MIP-1a, and
5
0
6
antiviral activity in a single-cycle infection assay. We
were pleased to find that although the activity for this
series was weaker than that for 1, low nano-molar
binding affinity and modest antiviral activity were
observed for this class (antiviral data is reported as
IC ’s since the compounds in this series were of modest
Scheme 1. Reagents and conditions: (a) benzyl amine, NaHB(OAc)
ꢀ
3
,
DCE; (b) Pd/C, H
2
, MeOH; (c) ArCl, DIEA, iPrOH, 90 C (Ar=
2
-chloropyrimidine derivatives); (d) ArX, Pd(OAc)
2
,
BINAP,
NaOtBu, dioxane, 100 C (Ar=phenyl, pyridyl or unreactive pyr-
imidine derivatives); (e) KNTMS , alkyl halide, THF; (f) HCl/MeOH;
g) aldehyde, NaHB(OAc) , DIEA, DCE.
ꢀ
2
5
0
(
3
activity). For example 2-chlorobenzoyl derivative 2a
was 14 nMin the binding assay and blocked 50% of the
viral infection at 300 nM. Simple modifications to the
pyrrolidine N-group such as with the urea derivative 2b
or the cycloalkyl derivatives 2c and 2d, also provided
similar activity. When the pyrroldine substituent was a
cyclohexylcarbonyl group, changing the R group on the
reactive aryl halides were incorporated by palladium
4
mediated coupling according to method of Buchwald.
Alkylation of the nitrogen was then carried out by
deprotonation with potassium hexamethyldisilazide
and reaction with an appropriate alkyl halide. Removal
Table 1. CCR5 binding affinity and antiviral activity of pyrrolidine amides
Compd
R
1
R
2
MIP-1a IC50 (nM)a
HeLa IC50 (nM)b
300
2
2
2
2
2
2
2
2
a
b
c
d
e
f
14
9
10
12
6
300
300
100
11
9
33
g
h
64% inh @ 1000 nM
78% inh @ 1000 nM
a
Values are means of three experiments for displacement of 125-I labeled MIP-1a from CCR5 receptor expressed on cell (CHO) membranes, see ref
5
for assay conditions.
Inhibition over 48 h of the BAL strain of HIV in HeLa Magi cells expressing CCR5 and CXCR4, SD were generally+20% of average, see ref 6 for
b
assay conditions.

C. A. Willoughby et al. / Bioorg. Med. Chem. Lett. 13 (2003) 427–431
429
4
-amino position of the piperidine to an allyl (2e) or a
piperidine series. The data for these compounds are
shown in Table 3. We were gratified to find that these
compounds had low nano-molar binding affinity and
indeed were better inhibitors of viral infection (data
reported as IC ’s since compounds in this series were
cyclopropylmethyl (2f) group improved the antiviral
activity ꢁ3- to 10-fold. Larger groups such as n-hexyl
or benzyl, as in 2g and 2h, resulted in loss of activity.
9
0
We then investegated a series of N-allyl compounds
with different aromatic groups substituted at the 4-ami-
nopiperidine position. The data for these compounds
are shown in Table 2. Moving the pyridine nitrogen
from the 2 position (2e) to the 3 position (2i) gave a
compound with similar antiviral activity but 4-fold
weaker receptor binding affinity. The 4-pyridyl deriva-
tive 2j was weaker still, with a binding affinity of 66 nM
and no appreciable antiviral activity. Similarly, the
phenyl derivative 2l was also unable to block viral
infection and was less potent in the binding assay. Pyr-
imidine derivative 2k was equipotent to 2e in the anti-
viral assay but displayed a 2-fold weaker binding
affinity. Substitution of the 2-pyridine with a tri-
fluoromethyl group resulted in a ꢁ4-fold loss in both
binding and antiviral activity.
more potent). For example, compound 3a was 3.9 nM
at the receptor and blocked 90% of viral infection at
100 nM. This compares to 2d which at the same con-
centration only blocked 50% of the viral infection.
Similarly, compounds 3b and 3c were potent antivirals
both having IC ’s of 33 nM.
9
0
We tested compounds 3a–3c for their in-vivo properties
by dosing them in rats at 0.5 mpk iv and 2 mpk po.
Unfortunately, pyridyl derivatives 3a and 3b had high
clearances (53 and 106 mL/min/kg, respectively) and
poor bioavailabilities (20 and 8%, respectively) resulting
in low plasma levels (Cmax=53 and 19 nM, respec-
tively). Although pyrimidine derivative 3c had a much
lower clearance (17 mg/mL/kg), its bioavailibility was
only 8%. During the course of this optimization we
found that by incorporating a 3-fluorophenyl rather
than phenyl at the 4 position of the pyrrolidne the in
vivo profile was substantially improved. Thus the 20%
bioavailability of 3d was 2.5-fold better than that of 3c
and the clearance was similar at 13 mg/mL/kg. This
resulted in an increased plasma exposure for 3d
(Cmax=140 nMvs 49 nMfor 3c).
At this point in our studies it was shown that incor-
poration of a carboxylic acid at the N-1 position of the
pyrrolidine resulted in improved antiviral activity as
well as better selectivity for the CCR5 receptor versus
3
d
other targets. As a result of this observation, we pre-
pared a series of analogues to probe the effect of acidic
functionality in the 4-(N-propylamino heterocyclic)
To further enhance the binding and antiviral activity of
this series of compounds we prepared a series of ana-
logues with a N-1 cyclobutylmethyl in place of a N-1
cyclohexyl group off the pyrrolidine nitrogen. In pre-
vious work this modification was shown to provide a 10-
Table 2. Modification of the piperidine 4-aryl group
3
d
fold improvement in binding and/or antiviral potency.
Table 3. Effects of incorporation of a carboxylic acid
Compd
Ar
MIP-1a IC50 (nM)a
HeLa IC50 (nM)b
11
2
2
2
2
2
2
e
6
i
23
66
12
42
21
12
n/a
12
Compd
Ar
X
H
MIP-1a IC50 (nM)a
HeLa IC50 (nM)b
100
j
3a
3.9
k
l
3b
3c
3d
H
H
F
5
33
33
33
n/a
37
6.6
1.6
m
a
a
See note a Table 1.
See note b Table 1.
See note a Table 1.
See note b Table 1.
b
b

430
C. A. Willoughby et al. / Bioorg. Med. Chem. Lett. 13 (2003) 427–431
Table 4. Incorporation of cyclobutylmethyl and 3-fluorophenyl substituents
Compd
Ar
R
MIP-1a IC50 (nM)a
HeLa IC50 (nM)b
4
a
b
n-C
3
H
7
–
–
1.8
1.5
11
4
n-C
2
H
5
100
4
4
4
4
4
4
4
c
d
e
f
1.1
1.7
1.2
0.4
0.5
1.2
0.8
11
11
n-C
n-C
n-C
n-C
n-C
n-C
3
3
3
3
3
3
H
7
H
7
H
7
H
7
H
7
H
7
–
–
–
–
–
–
33
33
g
h
i
100
450
100
a
See note a Table 1.
See note b Table 1.
b
The binding and antiviral data for this series of com-
pounds are shown in Table 4 and pharmacokinetic
paramaters for some analogues are shown in Table 5.
increase in antiviral activity, compound 4a having a
binding affinity of 1.8 nMand an IC 90 in the HeLa
assay of 11 nM. Changing the n-propyl group to an
ethyl group at the piperidine-4-amino position resulted
in a 10-fold loss in antiviral activity. In contrast to the
earlier studied pyrrolidine N-1 amide series (2d vs 2f)
the cyclopropylmethyl derivative 4c showed no
improvement in activity versus 4a. Isomeric pyrimidine
derivatives 4d and 4e had similar binding affinity and
antiviral activity. Since the 2-aminopyrimidine deriva-
tives showed the best in vivo profiles (see Table 5) we
examined ring substitution in this series of compounds.
Substitution at the 5 position with either a fluoro (4f) or
a trifluoromethyl (4g) group resulted in ꢁ3-fold
increase in binding affinity versus 4a, however, the anti-
viral activity was 3-fold (4f) and 10-fold (4g) less than
In this series of compounds the substitution of the
cyclobutylmethyl for the cyclohexyl group at the pyrro-
lidine N-1 position resulted in only a modest (3-fold)
Table 5. Pharmacokinetic paramaters for compounds 4a–e, 4g, and
4h dosed 0.5 mpk iv and 2 mpk orally in rats
Compd
Clp (mL/min/kg)
t1/2 (h)
%F
C
max (nM)
4
4
4
4
4
4
4
a
b
c
d
e
28
26
28
81
90
9
1.1
1.1
0.7
0.3
0.9
2.8
1.5
29
33
iv
256
210
iv
28
iv
433
177
a
a
8
iv
a
a
4a. Compounds 4h and 4i, having a 4-trifluoromethyl or
g
h
72
25
a 4-methoxy substituent, respectively, were also potent
in the binding assay but showed decreased antiviral
activity compared to 4a.
13
aiv, tested via iv dose only.

C. A. Willoughby et al. / Bioorg. Med. Chem. Lett. 13 (2003) 427–431
431
We investigated the in vivo properties of some of these
compounds by dosing in rats (Table 5). Compound 4a,
the most interesting antiviral compound, had a moder-
ate clearance rate (28 mg/mL/kg) and good bioavail-
ability of 29%. Although the half life was somewhat
short (1.1 h), the plasma exposure was good, Cmax being
2. (a) Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.;
Sawada, H.; Iizawa, Y.; Shiraishi, M.; Aramaki, Y.; Okonogi,
K.; Ogawa, Y.; Meguro, K.; Fujino, M. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 5698. (b) Tagat, J. R.; Steensma, R. W.;
McCombie, S. W.; Narareno, D. V.; Lin, S.-I.; Neustadt,
B. R.; Cox, K.; Xu, S.; Wojcik, L.; Murray, M. G.; Vantuno,
N.; Baroudy, B. M.; Strizki, J. M. J. Med. Chem. 2001, 44,
2
56 nM. The ethyl derivative 4b had a very similar pro-
3
343 and references therein. (c) Finke, P. E.; Oates, B.; Mills,
file. The isomeric pyrimidine derivative 4d had a much
higher clearance and shorter half life. Plasma exposure
was poor for this compound which had a Cmax of 28
nM. In contrast, the 5-trifluoromethyl derivative 4g had
significantly enhanced PK parameters. The low clear-
ance of 9 mg/mL/kg and the increased bioavailability of
S. G.; MacCoss, M.; Malkowitz, L.; Springer, M. S.; Gould,
S. L.; DeMartino, J. A.; Carella, A.; Carver, G.; Holmes, K.;
Danzeisen, R.; Hazuda, D.; Kessler, J.; Lineberger, J.; Miller,
M.; Schlief, W. A.; Emini, E. A. Bioorg. Med. Chem. Lett.
2001, 11, 2475 and references therein. (d) Armour, D. R.;
Price, D. A.; Stammen, B. L. C.; Wood, A.; Perros, M.;
Edwards, M. P. WO 00/38680, 2000; Chem. Abstr. 2000, 133,
7
2% resulted in good plasma levels, the Cmax of 433 nM
89523. (e) Armour, D. R.; Price, D. A.; Stammen, B. L. C.;
Wood, A.; Perros, M.; Edwards, M. P. WO 00/39125, 2000;
was the best in this series. These enhancements were not
observed with the 4-trifluoromethyl derivative 4h. On
balance, compound 4a had the best profile so we
assayed it, along with the cyclohexyl analogue 3d, for
antiviral activity in a more rigorous 7-day assay in pri-
Chem. Abstr. 2000, 133, 74024.
3
. (a) Hale, J. J.; Budhu, R. J.; Mills, S. G.; MacCoss, M.;
Malkowitz, L.; Gould, S. L.; Springer, M. S. Bioorg. Med.
Chem. Lett. 2001, 11, 1437. (b) Hale, J. J.; Budhu, R. J.; Hol-
son, E. B.; Finke, P. E.; Oates, B.; Mills, S. G.; MacCoss, M.;
Gould, S. L.; DeMartino, J. A.; Springer, M. S.; Siciliano, S.;
Malkowitz, L.; Schlief, W. A.; Hazuda, D.; Miller, M.; Kess-
ler, J.; Danzeisen, R.; Holmes, K.; Lineberger, J.; Carella, A.;
Carver, G.; Emini, E. A. Bioorg. Med. Chem. Lett. 2001, 11,
6
mary blood mononuclear cells. Compound 3d was able
to block 95% of the viral infection (IC ) at 375 nM
9
5
while 4a was equally effective at 50 nM. The activity of
a compares well with that of compound 1 (31 nM) in
the PBMC assay.
4
2
741. (c) Willoughby, C. W.; Berk, S. C.; Rosauer, K. G.;
Delgrado, S.; Chapman, K. T.; Gould, S. L.; Springer, M. S.;
Malkowitz, L.; Schlief, W. A.; Hazuda, D.; Miller, M.; Kess-
ler, J.; Danzeisen, R.; Holmes, K.; Lineberger, J.; Carella, A.;
Carver, G.; Emini, E. A. Bioorg. Med. Chem. Lett. 2001, 11,
In summary, we have developed a new class of 4-(het-
eroarylamino)piperidine derived 1,3,4 trisubstituted
pyrrolidine CCR5 antagonists. Many of these com-
pounds are potent antivirals and have good in vivo
properties.
3
137. (d) Lynch, C. L.; Hale, J. J.; Budhu, R. J.; Gentry, A. L.;
Mills, S. G.; Chapman, K. T.; MacCoss, M.; Malkowitz, L.;
Springer, M. S.; Gould, S.; DeMartino, J. A.; Siciliano, S. J.;
Cascieri, M. A.; Carella, A.; Carver, G.; Holmes, K.; Schleif,
W. A.; Danzeisen, R.; Hazuda, D.; Kessler, J.; Lineberger, J.;
Miller, M.; Emini, E. A. Bioorg. Med. Chem. Lett. 2002, 12,
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