1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 4: Synthesis of N-1 acidic functionality affording analogues with enhanced antiviral activity against HIV

Article abstract of DOI:10.1016/S0960-894X(02)00606-6

A series of α-(pyrrolidin-1-yl)acetic acids is presented as selective and potent antivirals against HIV. Several of the pyrrolidine zwitterions demonstrated reasonable in vitro properties, enhanced antiviral activities and improved pharmacokinetic profiles over pyrrolidine 1.

Full text of DOI:10.1016/S0960-894X(02)00606-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3001–3004
1,3,4-Trisubstituted Pyrrolidine CCR5 Receptor Antagonists. Part
4: Synthesis ofN-1 Acidic Functionality Affording Analogues with
Enhanced Antiviral Activity Against HIV
Christopher L. Lynch,a,* Jeffrey J. Hale,a Richard J. Budhu,a Amy L. Gentry,a
Sander G. Mills,a Kevin T. Chapman,a Malcolm MacCoss,a Lorraine Malkowitz,b
Martin S. Springer,b Sandra L. Gould,b Julie A. DeMartino,b Salvatore J. Siciliano,b
Margaret A. Cascieri,b Anthony Carella,c Gwen Carver,c Karen Holmes,c
William A. Schleif,c Renee Danzeisen,c Daria Hazuda,c Joseph Kessler,c
Janet Lineberger,c Michael Millerc and Emilio A. Eminic
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 5 April 2002; accepted 19 June 2002
Abstract—A series of a-(pyrrolidin-1-yl)acetic acids is presented as selective and potent antivirals against HIV. Several of the pyr-
rolidine zwitterions demonstrated reasonable in vitro properties, enhanced antiviral activities and improved pharmacokinetic pro-
files over pyrrolidine 1.
# 2002 Elsevier Science Ltd. All rights reserved.
The discovery that the b-chemokine receptor CCR5 is a
co-receptor utilized by the human immunodeficiency
virus (HIV) to gain entry into host cells has led to the
pursuit of small molecule antagonists as novel antiviral
agents.1À3 Recent reports from these laboratories have
described 1,3,4-trisubstituted pyrrolidines as potent
inhibitors of viral entry in the HeLa cell anti-infectivity
assay.4 In the preceding paper, the introduction of
acidic functionality into a combinatorial chemistry lead5
increased both the in vitro selectivity and antiviral
activity within this class of CCR5 antagonists, as exem-
plified by tetrazole 1.6 Although 1 was found to possess
desirable in vitro properties (e.g., l-Type Ca+2 channel
IC50 >10 mM), its rat pharmacokinetic (PK) profile was
not acceptable. In an effort to further explore zwitter-
ionic pyrrolidines, this paper will present the synthesis
and SAR of a-(pyrroldin-1-yl)acetic acids as selective
and potent antivirals with improved pharmacokinetics.
The synthesis of the a-(pyrrolidin-1-yl)acids was exe-
cuted through an alkylation of a 3,4-disubstituted pyr-
rolidine intermediate7 with triflates derived from
enantiopure a-hydroxy esters which occurs with clean
SN2 inversion.8 The required esters (see Scheme 1) were
obtained from commercially available chiral a-hydroxy
acids (A)9 or were synthesized through a variety of
routes, including enolate oxidation (B),10 Grignard
addition to an oxalate (C),11 diazotization of cyclopro-
pylalanine (D)12 and an asymmetric reduction of an a-
ketoamide (E).13 In addition, the para-methoxybenzyl
(PMB) ester was prepared in specific examples to
accommodate reducible functionality under catalytic
hydrogenation. In the examples (B and C) employing
*Corresponding author. Fax: +1-908-594-5966; e-mail: chris_lynch@
merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00606-6

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C. L. Lynch et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3001–3004
esters 5. Final cleavage of the benzyl group under cata-
lytic hydrogenation or with warm formic acid for the
PMB-esters provided the desired target compounds.
Derivatives other than carboxylates were also explored
(Scheme 3). Standard chemistry allowed the preparation
of the methyl esters 8 and primary alcohols 10 from
intermediates generated along the synthetic route in
Scheme 2.
The CCR5 receptor affinity and antiviral data for the
compounds are presented in Tables 1, 2, and 4. Initially,
analogues were screened for their ability to displace
[
125I]-labeled MIP-1a from the CCR5 receptor expres-
sed on CHO cell membranes.15 The most potent com-
pounds were further evaluated as antivirals in a HeLa
cell antiinfectivity assay versus the BAL strain of HIV.16
Based on the functionality within 1, the a-(pyrrolidin-1-
yl)cyclohexylacetic acid and its derivatives were pre-
pared to study the initial SAR (Table 1). Several trends
became apparent with the modifications of this scaffold.
This class of pyrrolidines favors the (R)-stereochemistry
at the pyrrolidine center (comparing 11–14 with 15-–18).
In every case, the (S)-stereochemistry led to a loss in both
CCR5 binding and antiviral activity. Although both the
free acid and primary alcohol are tolerated as determined
Scheme 1. Reagents and conditions: (a) 0.5 N KOH, Bu4NI, Bn–Br,
CHCl3; (b) NaHMDS, THF, À78 ꢀC; 2-(Phenylsulfonyl)-3-phenyl-
oxaziridine; (c) Cylcobutylmagnesium bromide, Et2O, À78 ꢀC; (d) 2 N
H2SO4, NaNO2; (e) PMB–Cl, TEA, DMF; (f) Mg–K, (Bromo-
methyl)cyclobutane, THF; (g) (R)-Alpine Borane, THF; (h) KOtBu,
THF, H2O; (i) Bn–Br, TEA, DMF.
racemic a-hydroxy esters, the diastereomers were sepa-
rated by HPLC after cleavage of the TBS ether (see
Scheme 2). Upon separation of the diastereomers, the
assignment of relative stereochemistry was based on the
1
correlation of H NMR data with the products derived
from the enantiomerically pure starting materials.
The general route to the a-(pyrrolidin-1-yl)acids is pre-
sented in Scheme 2. With routes to the hydroxy esters
established, alkylation of 2 with the intermediate tri-
flates was straight forward to give 3. The cleavage of the
silyl ether with tetrabutylammonium fluoride, followed
by a Swern oxidation, gave aldehyde 4. Reductive ami-
nation with several 4-substituted piperidines5 under the
conditions of Abdel-Magid and co-workers14 provided
Scheme 3. (a) TMSCHN2, MeOH, THF; (b) LiAlH4, THF, 0 ꢀC.
Table 1. CCR5 receptor affinity and antiviral activity of a-(pyrroli-
din-1-yl)cyclohexylacetic acid derivatives
No.
R
MIP 1aa (HeLa)b No.
R
MIP 1aa (HeLa)b
11
12
13
14
CH2OH
CO2Bn 3.6Æ1.0 (>300)
1Æ0.2 (300)
1Æ0.3 (NTc)
15 CH2OH
16 CO2Bn
17 CO2Me
1Æ0.4 (NTc)
24Æ10 (>300)
4Æ0.7 (NTc)
5Æ0.6 (>300)
CO2Me
CO2H
0.5Æ0.05 (11)
18
CO2H
aDisplacement of [125I]-labeled MIP-1a from the CCR5 receptor
expressed on CHO cell membranes (IC50, nM). Data are reported as
meanÆSD for n=3 determinations. See ref 15 for assay protocol.
bIC90 (nM) values obtained in the HeLa cell antiinfectivity assay ver-
sus BAL. See ref 16 for assay protocol.
Scheme 2. (a) Triflate, DIEA, CH2Cl2, À78 to 0 ꢀC; (b) TBAF, THF;
(c) (COCl)2, DMSO, TEA, CH2Cl2; (d) Piperidine analogue,
NaB(OAc)3H, CH2Cl2; (e) H2, Pd–C, MeOH (or HCO2H, 50 ꢀC for
PMB esters).
cNT=Not tested.

C. L. Lynch et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3001–3004
3003
Although the (R)-a-(pyrrolidin-1-yl)cyclohexylacetic
acid 14 possessed comparable antiviral activity to pyr-
rolidine 1, the rat pharmacokinetics of 14 were poor
(Table 3). Based on this data, our focus shifted to stra-
tegies for improving the PK parameters. Synthesis of
more compact molecules, and thus minimizing the
functionality of the piperidine, appeared a logical step
for improving the oral bioavailability. Initially, the
4-hydroxy of 14 was removed to provide the 4-(3-phen-
propyl)piperidine analogues 23 and 24. Immediately, it
was observed that neither the MIP 1a nor the HeLa
data was compromised within 23, however the antiviral
activity decreased with 24. More importantly, the PK
profile of 23 improved with a decrease in clearance and
an increase in bioavailability in three species (see Table
3). In order to determine the minimum requirement for
the piperidine side chain and further enhance the PK, a
4-(4-fluorophenyl)piperidine was introduced (25 and
26). Although the oral bioavailability was maintained or
improved slightly with 26, the viral activity dropped
with this truncated side chain.
Table 2. CCR5 receptor affinity and antiviral activity of a-(pyrroli-
din-1-yl)acetic acid analoguesa
R
c-Hex
Ph
i-Pr
CH3
H
14 0.5Æ0.05 (11)
19 0.8Æ0.1 (33)
20 2Æ0.2 (333)
21 3Æ0.5 (333)
22 6Æ1 (3000)
23 0.1Æ0.03 (11)
24 0.7Æ0.03 (111)
25 4Æ0.4 (100)
26 36Æ4 (>100)
aDisplacement of [125I]-labeled MIP-1a from the CCR5 receptor
expressed on CHO cell membranes (IC50, nM). Data are reported as
meanÆSD for n=3 determinations. See ref 15 for assay protocol.
Numbers in parentheses are the IC90 (nM) values obtained in the
HeLa cell antiinfectivity assay versus BAL. See ref 16 for assay
protocol.
Since it was clear from the above data that a cyclohexyl
side chain with the 4-(3-phenpropyl)piperidine was
optimal for both antiviral and PK properties (e.g., 23),
additional cyclohexyl replacements were then investi-
gated (Table 4). The cyclopropane 27 did not show
adequate antiviral activity, which was comparable to
the isopropyl analogue 24. However, analogues 28, 29,
and 31 maintained the activity of 23. An enhancement
in antiviral activity came with introduction of the
cyclobutyl methylene moiety (30). Although the recep-
tor binding data of 30 was similar to 23 and 28, the
antiviral activity displayed a 10-fold increase in potency.
The pharmacokinetics and Ca+2 channel activity of 23
were maintained with this improvement in antiviral
activity of 30 (Table 3).
by the receptor binding assay (11 and 14), the zwitterion
14 displays a 30-fold increase in antiviral activity.
Further studies (Table 2) demonstrated that decreasing
the size of the side chain of the a-(pyrrolidin-1-yl)acetic
acids (14 and 19–22) resulted in a loss of antiviral
activity. Although substituting an isopropyl group for
the cyclohexane (14 and 20) maintained the affinity for
the CCR5 receptor (0.5 vs 2 nM), a drop in the HeLa
assay suggested a large hydrophobic moiety was
required for antiviral activity. Introduction of a simple
aromatic side chain as in 19 led to a 3-fold loss in anti-
viral activity. Removal of the side chain to provide the
acetic acid 22 eliminated all antiviral activity. In gen-
eral, the drop in CCR5 binding with smaller side chains
was not substantial, however the antiviral activity was
highly dependent on the size of the substituent. This
result suggests that measurements of MIP-1a binding
do not rigorously predict antiviral activity.
In summary, the goal of our studies was to improve the
pharmacokinetics of 1 while maintaining selectivity and
antiviral activity. Although (R)-a-(pyrrolidin-1-yl)cy-
clohexylacetic acid 14 maintained both potency against
the BAL strain in the PBMC assay and selectivity
Table 3. PBMC and Ca+2 channel activities17 with the pharmacokinetic profiles of 14, 23, 26, and 30
14
23
26
30
PBMC (BAL) IC95 nM (n) (ref 16)
l-Type Ca+2 Channel IC50 mM (n)
Rat
187 (3)
>10
77 (2)
5.3
>3000 (1)
>10
<8 (2)
2.7
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
%F
Dog
128
15
1.8
0
17.6
3.3
2.1
26
68.3
8.4
1.9
50
26.5
5.9
3.0
29
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
%F
Rhesus
10.9
3.1
9.2
43
13.1
8.6
6.4
34
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
17.1
6.2
6.3
10
14.6
4.2
7.8
84
%F

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C. L. Lynch et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3001–3004
Table 4. CCR5 receptor affinity and antiviral activity of a-(pyrroli-
din-1-yl)acetic acid analoguesa
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R
c-Pr
c-Bu
(c-Pr)CH2
(c-Bu)CH2
c-Pent
27 3Æ0.5 (300)
28 0.1Æ0.03 (11)
29 0.1Æ0.01 (11)
30 0.1Æ0.02 (1.2)
31 0.2Æ0.03 (11)
aDisplacement of [125I]-labeled MIP-1a from the CCR5 receptor
expressed on CHO cell membranes (IC50, nM). Data are reported as
meanÆSD for n=3 determinations. See ref 15 for assay protocol.
Numbers in parentheses are the IC90 (nM) values obtained in the
HeLa cell antiinfectivity assay versus BAL. See ref 16 for assay
protocol.
against the l-type Ca+2 channel (IC50 10 mM; Table 3),
compound 14 lacked oral bioavailability. However, fur-
ther efforts uncovered several improved analogues (23,
26, and 30). Both 23 and 30 possessed potent antiviral
activity in the PBMC assay and improved rat PK prop-
erties over 14. In addition, 23 and 26 demonstrated that
this class of pyrrolidine zwitterions could display rea-
sonable PK profiles in three separate species and anti-
viral activity comparable to tetrazole 1.
10. Davis, F. A.; Haque, M. S.; Ulatowski, T. G.; Towson,
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12. For a related diazotization see: Kolasa, T.; Miller, M. J.
J. Org. Chem. 1987, 52, 4978.
Acknowledgements
13. For a related reduction of ketoesters see: Brown, H. C.;
Pai, G. G. J. Org. Chem. 1985, 50, 1384.
The authors would like to thank Dr. G. Kieczykowski,
Mr. J. Leone and Mr. F. Wong for synthetic assistance.
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yanoff, C. A.; Shah, R. D. J. Org. Chem. 1996, 61, 3849.
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References and Notes
1. Finke, P. E.; Oates, B.; Mills, S. G.; MacCoss, M.; Mal-
kowitz, 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.
2. Panali, A.; Shapiro, S.; Clader, J. W.; Greenlee, W. J.; Cox,