1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 2: Lead optimization affording selective, orally bioavailable compounds with potent Anti-HIV activity

Article abstract of DOI:10.1016/S0960-894X(01)00545-5

Investigations of the structure-activity relationships of 1,3,4-trisubstituted pyrrolidine human CCR5 receptor antagonists afforded orally bioavailable compounds with the ability to inhibit HIV replication in vitro.

Full text of DOI:10.1016/S0960-894X(01)00545-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2741–2745
1,3,4-Trisubstituted Pyrrolidine CCR5 Receptor Antagonists.
Part 2: Lead Optimization Affording Selective, Orally
Bioavailable Compounds with Potent Anti-HIV Activity
Jeffrey J. Hale,a,* Richard J. Budhu,a Edward B. Holson,a Paul E. Finke,a Bryan Oates,a
Sander G. Mills,a MalcolmMacCoss, Sandra L. Gould,b Julie A. DeMartino,b
a
c
Martin S. Springer,b Salvatore Siciliano,b Lorraine Malkowitz,b WilliamA. Schleif,
Daria Hazuda,c Michael Miller,c Joseph Kessler,c Renee Danzeisen,c Karen Holmes,c
Janet Lineberger,c Anthony Carella,c Gwen Carverc and Emilio Eminic
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology Research, Merck Research Laboratories, Rahway, NJ 07065, USA
cDepartment of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 8 June 2001; accepted 3 August 2001
Abstract—Investigations of the structure–activity relationships of 1,3,4-trisubstituted pyrrolidine human CCR5 receptor antago-
nists afforded orally bioavailable compounds with the ability to inhibit HIV replication in vitro. # 2001 Elsevier Science Ltd. All
rights reserved.
The inhibition of viral entry into host cells has recently
emerged as an attractive new target for human immu-
nodeficiency virus (HIV) therapy. Agents that interfere
with the interaction of the HIV envelope glycoprotein
gp120 and the cellular receptor CD4 as well as agents
that inhibit the function of the HIV fusion peptide gp41
have been described.1 A Phase II clinical trial with the
entry inhibitor T-20 has provided an important proof-
of-concept experiment in humans as it was demon-
strated that this compound could successfully block
HIV entry in vivo.2 The b-chemokine receptor CCR5
has been identified as the major co-receptor required for
affinity for the CCR5 receptor and potent anti-HIV
activity.8À11 Our efforts to incorporate some of the
favorable pharmacophoric elements from the acyclic
compounds into the pyrrolidines has resulted in the
identification of a series of selective, orally bioavailable
pyrrolidine analogues possessing the ability to inhibit
HIV replication in vitro.
the entry of HIV-1 into monocytes, macrophages and
5
primary T-cells,3,4 and reports fromTakeda
and
Schering–Plough6 describing small molecule human
CCR5 receptor antagonists that inhibit HIV replication
have recently appeared.
In a previous communication,7 we described the dis-
covery of 1,3,4-trisubstituted pyrrolidines (exemplified
by 1) as a novel lead class of CCR5 receptor antago-
nists. Investigations of acyclic 2-aryl-4-(piperidin-1-
yl)butanamine CCR5 antagonists has led to the identi-
fication of several analogues (such as 2–4) with good
*Corresponding author. Fax: +1-732-594-5966; e-mail: jeffrey_hale@
merck.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00545-5

2742
J. J. Hale et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2741–2745
The synthetic chemistry used to prepare the majority of
the pyrrolidine analogues in this work has been pre-
viously described.7 Analogues with structural elements
that were incompatible with the catalytic hydrogenation
employed in the earlier synthetic route were prepared as
shown in Scheme 1. Three analogues (41–43) that were
used to probe the SAR of the C3 phenyl ring of the
pyrrolidines were prepared by adding a palladium-cata-
lyzed coupling step to the previously described route.
The syntheses of the 4-(N-alkyl-N-carbamoyl) amino-
piperidine subunits used to prepare analogues 46–63
have been reported.10
was found to offer little apparent advantage while trun-
cation (12–13) afforded less potent compounds. Other
alterations of the functionality linking the pendant
phenyl ring to the pyrrolidine nitrogen (14–16) led to
the most potent analogue of the series, benzamide 15.
The phenyl sulfonamide substituent of the acyclic
antagonists was determined to be metabolically labile
when incubated with rat liver microsomes;11 while simi-
lar experiments were not carried out with these initial
analogues, they did provide a flexible starting point for
further investigations.
A series of substituted benzamide derivatives based on
15 was prepared and the CCR5 binding affinities of
The discovery that several distinct piperidine subunits
could impart significant CCR5 binding affinity to the
acyclic series of compounds8À11 prompted the incor-
poration of these same subunits into the pyrrolidine
scaffold. Racemic pyrrolidine analogues containing the
structurally simple 4-phenylpiperidine subunit9 were
among the first targeted and the phenylsulfonamide 7
was prepared (Table 1). Compound 7 was found to be
of comparable potency to the initial pyrrolidine lead
compounds in an assay to measure the ability of test
compounds to displace ligand [125I]-MIP-1a fromthe
CCR5 receptor expressed on Chinese Hamster Ovary
(CHO) cell membranes,7 but a result obtained with one
of the intermediates used in its preparation provided a
point of divirgence fromthe acyclic antagonists. While
the N-phenylsulfonyl substituent in the acyclic com-
pounds (such as 2–4) was absolutely required for CCR5
activity,8 it quickly became apparent that this was not
so with the pyrrolidines as N-benzyl analogue 8 was
only 2-fold less potent than phenylsulfonamide 7. Fol-
lowing up on this observation, chain extension (9–11)
Table 1. Inhibition of [125I]-MIP-1a bindinga by various N1 analo-
gues
Compound
R
CCR5
IC50 (nM)
7
8
9
10
11b
12
13
14
15
16
PhSO2–
PhCH2–
PhCH2CH2–
PhCH2CH2CH2–
Ph(CH3)CH–
Ph–
152Æ16
255Æ40
200Æ17
>1000
480Æ120
>1000
H–
980Æ580
610Æ170
67Æ7
PhNHCO–
PhCO–
PhCH2CO–
155Æ10
aDisplacement of 125I-labeled MIP-1a fromthe CCR5 receptor
expressed on CHO cell membranes. Data are reported as meanÆSD
for n=3 determinations. See ref 7 for assay protocol.
b1:1 Mixture of racemic diastereomers.
Table 2. Inhibition of [125I]-MIP-1a binding by N1 benzamide ana-
loguesa
Compound
R
CCR5 IC50 (nM)
15
17
18
19
20
21
22
23
24
25
26
27
H
2-Cl
3-Cl
4-Cl
2-MeO
3-MeO
4-MeO
2-CF3
2-F
67Æ7
52Æ3
94Æ14
750Æ100
140Æ11
140Æ11
>1000
97Æ17
96Æ5
Scheme 1. Reagents: (a) RCOCl, DIEA or TEA, CH2Cl2; (b) RCO2H,
EDC or BOP-Cl, DIEA or TEA, CH2Cl2; (c) TBAF, THF, 0 ꢀC to rt;
(d) (COCl)2, DMSO, DIEA, CH2Cl2, À78 to 0 ꢀC; (e) 4-substituted
piperidine, NaB(OAc)3H, TEA, CH2Cl2; (f) PhB(OH)2, Na2CO3,
(Ph3P)4Pd, EtOH/toluene, reflux (R=–Ph, 41, 44%); (g) (CH3)4Sn,
(Ph3P)3PdCl2, DMF, 100 ꢀC (R=–Me, 42, 61%); (h) CO, TEA,
(Ph3P)4Pd, MeOH, reflux (R=–CO2CH3, 43, 38%).
2-Ph
2-Br
2-CO2CH3
89Æ8
48Æ5
32Æ3
aDisplacement of 125I-labeled MIP-1a fromthe CCR5 receptor
expressed on CHO cell membranes. Data are reported as meanÆSD
for n=3 determinations. See ref 7 for assay protocol.

J. J. Hale et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2741–2745
2743
these compounds are shown in Table 2. Substitution at
the 2-position of the benzamide was found to be toler-
ated and electron-withdrawing groups seemed to be
favored over electron-donating groups (17–22). Exchan-
ging other functional groups for the 2-chloro substituent
of 17 was generally deleterious with the methyl ester 27
being the only new compound with greater CCR5 affinity
than 15. All of the six possible dichlorobenzamide ana-
logues of 15 were prepared and all were less potent than
the 2-chloro analogue 17 (data not shown).
compounds containing various N1 and C3 pharmaco-
phores identified fromthe previously examined (4-phe-
nyl)piperidine analogues (Table 5). In addition to
determining the [125I]-MIP-1a binding affinity for this
set of compounds, selected analogues were also exam-
ined for their ability to inhibit a single HIV infection
cycle in a CCR5-expressing HeLa Magi cell line.12 The
potency trends observed in the [125I]-MIP-1a binding
assay with the less active (4-phenyl)piperidine analogues
(8, 15, 17, 28, 33, and 34) approximately carried over to
analogues 46–57, while the more potent analogues 58–
63 appeared to diverge fromthe expected result. Grati-
fyingly, the antiinfectivity activity of these compounds
as determined by their CIC90 values in the HeLa Magi
cells followed the predicted trends with (2-chloro)-
benzoyl analogue 60 being the most active.13
Since substitution of the benzamide phenyl ring of 15
did not generally afford compounds with enhanced
CCR5 affinity, the nature of the amide substituent itself
was examined (Table 3). The 1-naphthoyl analogue 28
was about 3-fold more potent than benzamide 15;
interestingly, the corresponding 2-naphthoyl analogue
was at least 50-fold less active (data not shown). The
introduction of a basic pyridine ring (29) gave a less
active compound, but other neutral heterocycles, such
as furan and thiophene, were found to be suitable
phenyl substitutes (30–32). An aromatic group was not
required for activity (33–35), while cyclic substituents
seemed to be favored over an acyclic one (cf. 34 and 36).
The pharmacokinetics and the ability of analogues 54
and 60 to inhibit HIV replication in normal human
donor PMBCs14 were examined (Table 6). While being a
relatively weak antiviral compound, 54 did exhibit the
cross-species oral bioavailability that was lacking in
acyclic analogue 4. Analogue 60 demonstrated that
potent anti-HIV activity could be obtained with the
1,3,4-trisubstituted pyrrolidine compounds. Both 54
and 60 were found to bind in a highly selective manner
to CCR5 as compared to other chemokine receptors in
competition assays similar to that used to examine the
inhibiton of [125I]-MIP-1-a binding to CCR5. Both 54
and 60 were determined to have IC50 values greater than
1 mM for the CCR1, CCR2, CCR3, CCR4, CXCR3,
and CXCR4 receptors. Additionally, 60 was found to be
a full antagonist of CCR5.15
A series of N1-benzyl pyrrolidine compounds was pre-
pared in order to the examine the SAR of the C3 aro-
matic ring (Table 4). Substitution with a variety of
functional groups at the 3-position of the phenyl ring
was tolerated, but was sensitive to the steric require-
ments of the substituent as evidenced by the loss in
potency seen with any group larger than methyl or
fluoro. 3-Thienyl (45) was also a suitable phenyl sub-
stitute; these observations paralleled those seen for the
central aromatic ring of the acyclic 2-aryl-4-(piperidin-1-
yl)butanamine antagonists.9 4-(N-Alkyl-N-carbamoyl)-
amino piperidine pharmacophores had been demon-
strated to impart appreciable CCR5 affinity to acyclic
antagonists such as 2–4; the ability to significantly inhibit
HIV replication in vitro was observed of 4.11 Three of these
piperidine subunits were incorporated into pyrrolidine
Investigation of the SAR requirements of 1,3,4-trisub-
stituted pyrrolidine CCR5 receptor antagonists led to
Table 4. Inhibition of [125I]-MIP-1a binding by C3 aryl analoguesa
Table 3. Inhibition of [125I]-MIP-1a binding by N1 amide analoguesa
Compound
R
CCR5
IC50 (nM)
8
H
2-Cl
3-Cl
4-Cl
3-Br
3-Ph
255Æ40
>1000
37
38
39
40
41
42
43
44
Compound
R
CCR5
IC50 (nM)
810Æ350
>1000
15
28
29
30
31
32
33
34
35
36
PhCO–
1-Naphthoyl
Nicotinoyl
2-Furanoyl
3-Furanoyl
2-Thienoyl
Cyclohexanoyl
Cyclopentanoyl
Cyclobutanoyl
2-(Ethyl)butanoyl
67Æ7
19Æ2
260Æ34
55Æ5
33Æ3
46Æ4
34Æ3
19Æ2
48Æ5
57Æ6
>1000
>1000
3-CH3
3-CO2CH3
3-F
390Æ40
>1000
150Æ15
45
220Æ35
aDisplacement of 125I-labeled MIP-1a fromthe CCR5 receptor
expressed on CHO cell membranes. Data are reported as meanÆSD
for n=3 determinations. See ref 7 for assay protocol.
aDisplacement of 125I-labeled MIP-1a fromthe CCR5 receptor
expressed on CHO cell membranes. Data are reported as meanÆSD
for n=3 determinations. See ref 7 for assay protocol.

2744
J. J. Hale et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2741–2745
Table 5. Inhibition of [125I]-MIP-1a binding by analogues with var-
the identification of novel elements in these compounds,
which are diverse fromthose found in acyclic antago-
nists such as 4. Potent anti-HIV activity was achieved in
several analogues; compound 60 was determined to be
ious piperidine pharmacophoresa
of comparable potency to 4 and to the key compounds
5
featured in reports fromTakeda
and Schering–
Plough.6 Cross-species oral bioavailability was demon-
strated in analogue 54. The incorporation of additional
diverse structural elements into the pyrrolidine class of
CCR5 receptor antagonists is in progress and reports of
the further optimization of these compounds will be
forthcoming.
R1
Series
3
1
2
4
Acknowledgements
8
46
52
58
0.1Æ0.04b (100)c
59
PhCH2–
255Æ40 46Æ3
15
67Æ7
17
52Æ3
28
19Æ2
33
34Æ3
34
9Æ1
53
5Æ1
54
The authors would like to thank Dr. G. Kieczykowski,
Mr. J. Leone and Mr. F. Wong for synthetic assistance
and Dr. Gloria Kwei for providing the pharmacokinetic
data for compounds 4, 54 and 60.
47
PhCO–
31Æ5
48
0.2Æ0.02b (11)c
60
(2-Cl)PhCO–
1-Naphthoyl
Cyclohexanoyl
Cyclopentanoyl
12Æ2 2Æ0.2b (50)c 0.8Æ0.5b (1)c
49
55
4Æ0.4
56
4Æ0.4
57
61
0.8Æ0.2b (4)c
62
22Æ3
50
17Æ2
51
References and Notes
0.8Æ0.2b (4)c
63
1. Blair, W. S.; Lin, P.-F.; Meanwell, N. A.; Wallace, O. B.
Drug Development Today 2000, 5, 183.
19Æ 2 15Æ 1
2Æ0.2
0.2Æ0.02b (4)c
2. Kilby, J. M.; Hopkins, S.; Venetta, T. M.; DiMassimo, B.;
Cloud, G. A.; Lee, J. Y.; Alldredge, L.; Hunter, E.; Lambert,
D.; Bolognesi, D.; Matthews, T.; Johnson, M. R.; Nowak,
M. A.; Shaw, G. M.; Saag, M. S. Nat. Med. 1998, 4, 1302.
3. Dragic, T.; Litwin, V.; Allaway, G. P.; Martin, S. R.; Huang,
Y.; Nagashima, K. A.; Cayanan, C.; Maddon, P. J.; Koup,
R. A.; Moore, J. P.; Paxton, W. A. Nature 1996, 381, 667.
4. Deng, H.; Liu, R.; Ellmeier, W.; Choe, S.; Unutmaz, D.;
Burkhart, M.; Di Marzio, P.; Marmon, S.; Sutton, R. E.; Hill,
C. M.; Davis, C. B.; Peiper, S. C.; Schall, T. J.; Littman, D. R.;
Landau, N. R. Nature 1996, 381, 661.
aDisplacement of 125I-labeled MIP-1a fromthe CCR5 receptor
expressed on CHO cell membranes. Data are reported as meanÆSD
for n=3 determinations. See ref 7 for assay protocol.
bData for 3-(S),4-(S) enantiomer.
cInhibition (CIC90) over a 48 h period of a single HIV (BAL) infection
cycle in HeLa Magi cells expressing both CXCR4 and CCR5. See ref
12 for assay protocol.
5. 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.
Table 6. Anti-HIV activity and pharmacokinetics
Compound
6. Baroudy, B. 7th Conference of Retroviruses and Opportu-
nistic Infections, San Francisco, CA, Jan 31–Feb 3, 2000;
Abstr S17.
4
54
60
Anti-HIV activitya
7. Hale, J. J.; Budhu, R. J.; Mills, S. G.; MacCoss, M.; Mal-
kowitz, L.; Gould, S. L.; Springer, M. S. Bioorg. Med. Chem.
Lett. 2001, 11, 1437.
PBMC CIC95 (nM)
13 (BAL)
1500 (YU-2)
31 (BAL)
Rat pharmacokineticsb
8. Dorn, C. P.; Finke, P. E.; Oates, B.; Budhu, R. J.; Mills,
S. G.; MacCoss, M.; Malkowitz, L.; Springer, M. S.; Daugh-
erty, B. L.; Gould, S. L.; DeMartino, J. A.; Siciliano, S. J.;
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, 259.
9. Finke, P. E.; Meurer, L. C.; Oates, B.; Mills, S. G.; Mac-
Coss, M.; Malkowitz, L.; Springer, M. S.; Daugherty, B. L.;
Gould, S. L.; DeMartino, J. A.; Siciliano, S. J.; 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, 265.
10. 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.
11. Caldwell, C. G.; Chen, P.; Donnelly, K. F.; Finke, P. E.;
Shankaran, K.; Meurer, L. C.; Oates, B.; Caldwell, C. G.;
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
14.2
2.2
2.0
29
12.2
0.8
1.0
18
41
1.7
1.2
39
%F
Dog pharmacokineticsc
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
24
20.2
1.0
1.0
34
Ndd
Ndd
Ndd
Ndd
14.1
10.2
<1
%F
aInhibition of HIV expansion in uninfected normal donor PBMCs
over a 7 day period. CIC95 values were determined by comparing
mean viral p24 antigen levels in test cultures with the levels expressed
in control cultures. HIV strain used in the assay is indicated in par-
entheses. See ref 14 for assay protocol.
bAverage data generated after 5 mg/kg po and 1 mg/kg iv doses in n=3
animals/dose.
cAverage data generated after 2 mg/kg po and 0.5 mg/kg iv doses in
n=3 animals/dose.

J. J. Hale et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2741–2745
2745
MacCoss, M.; Mills, S. G.; Malkowitz, L.; Springer, M.
S.; Schlief, W. A.; Carella, A.; Carver, G.; Holmes, K.;
Emini, E. A. American Chemical Society 220th National
Meeting, Washington, DC, Aug 20–25, 2000; Abstr
MEDI-85.
12. Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Still-
mock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski, L.; Schleif,
W.; Blau, C.; Miller, M. D. Science 2000, 287, 646.
13. The discordant abilities of CCR5 monoclonal antibodies
to block ligand binding to the CCR5 receptor and to inhibit
HIV infection has been rationalized to arise fromthe potential
existence of multiple conformational states of CCR5. See: Lee,
B.; Sharron, M.; Blanpain, C.; Doranz, B. J.; Vakili, J.; Setoh,
P.; Berg, E.; Liu, G.; Durell, S. R.; Parmentier, M.; Chang,
C. N.; Price, K.; Tsang, M.; Doms, R. W. J. Biol. Chem. 1999,
274, 9617.
14. Saag, M. S.; Emini, E.; Laskin, O. L.; Douglas, J.; Lapi-
dus, W. I.; Schleif, W. A.; Whitley, R. J.; Hildebrand, C.;
Byrnes, V. W.; Kappes, J. C.; Anderson, K. W.; Massari, F. E.;
Shaw, G. M. New Engl. J. Med. 1993, 329, 1065.
15. Compound 60 at a concentration of 1 mM was found to
elicit 8% of the initial response of MIP-1a at its EC50 (1–
2 nM) in a microphysiometer experiment analogous to that
described in ref 7.