1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 3: Polar functionality and its effect on anti-HIV-1 activity

Article abstract of DOI:10.1016/S0960-894X(02)00605-4

Incorporation of acidic functional groups into a lead CCR5 antagonist identified from a targeted combinatorial library resulted in compounds with enhanced anti-HIV-1 activity and attenuated L-type calcium channel affinity.

Full text of DOI:10.1016/S0960-894X(02)00605-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2997–3000
1,3,4-Trisubstituted Pyrrolidine CCR5 Receptor
Antagonists. Part 3: Polar Functionality and Its Effect on
Anti-HIV-1 Activity
Jeffrey J. Hale,a,* Richard J. Budhu,a Sander G. Mills,a Malcolm MacCoss,a
Sandra L. Gould,b Julie A. DeMartino,b Martin S. Springer,b Salvatore J. Siciliano,b
Lorraine Malkowitz,b William A. Schleif,c 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 A. 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 5 April 2002; accepted 19 June 2002
Abstract—Incorporation of acidic functional groups into a lead CCR5 antagonist identified from a targeted combinatorial library
resulted in compounds with enhanced anti-HIV-1 activity and attenuated L-type calcium channel affinity.
# 2002 Elsevier Science Ltd. All rights reserved.
The b-chemokine receptor CCR5 has been identified as
the major co-receptor required by HIV-1 for the entry
into monocytes, macrophages and primary T-cells.1,2
Reports from Takeda,3 Schering-Plough,4 and Merck5
regarding potent, small molecule human CCR5 receptor
antagonists possessing the ability to inhibit the replica-
tion of HIV-1 in vitro have appeared. CCR5 receptor
antagonists represent a potentially new therapy for the
treatment of HIV-1 infection and clinical data support-
ing their use is anxiously awaited.
compound was orally bioavailable in the rat, its high
rate of clearance and short half life suggested to us that
1 (or close analogues of 1) might possess metabolically
labile functionality that would negatively impact its
long term prospects.
We recently disclosed the details of some initial struc-
ture–activity relationships (SARs) developed for a series
of 1,3,4-trisubstituted pyrrolidine CCR5 receptor
antagonists exemplified by 1.6 Compound 1 has excel-
lent affinity for the human CCR5 receptor, is selective
for CCR5 over other chemokine receptors and was
demonstrated to have the ability to potently inhibit the
entry of HIV-1 into peripheral blood mononuclear cells
(PBMCs) via the antagonism of CCR5. While this
*Corresponding author. Fax: +1-732-594-5966; e-mail: jeffrey_hale@
merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00605-4

2998
J. J. Hale et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2997–3000
Table 1. Inhibition of [125I]-MIP-1a bindinga by pyridine analogues
of 2
Compd
R
CCR5 IC50 (nM)
2
7
8
9
Ph-
0.3
7
6
12
2-Pyridyl
3-Pyridyl
4-Pyridyl
aDisplacement of [125I]-labeled MIP-1a from the CCR5 receptor
expressed on CHO cell membranes. Data are reported as mean for n=
three determinations. SD were generally Æ20% of the average. See ref
8 for assay protocol.
poration of appropriate acidic functional groups into 2
can have a dramatic effect on the anti-HIV-1 properties
of these molecules.
Scheme 1. Reagents and conditions: (a) (CH3)3SOI, NaH, DMSO,
50 ꢀC (53%); (b) trimethylsilylacetylene, LiHMDS, LiClO4, THF,
then K2CO3, MeOH (94%); (c) 1 M HCl, MeOH; (d) 1-cyclo-
hexylmethylene-3-(R)-formyl-4-(S)-phenyl
NaB(OAc)3H, CH2Cl2 (65%, two steps); (e) Ar-Br, cat. Pd(OAc)2, cat.
Ph3P, NaOAc, DMF, 60 ꢀC; (f) H2, 10% Pd/C, MeOH (40–65%, two
pyrrolidine,
DIEA,
We initially envisioned preparing a series of analogues
of 2 possessing a varied array of functional groups in
place of the pendant phenyl ring (Scheme 1). In practice,
commercially available N-BOC-4-piperidinone was
reacted with dimethyloxosulfonium methylide to pro-
vide epoxide 3. Epoxide ring opening with lithio tri-
methylsilylacetylene followed by treatment of the
quenched reaction mixture with potassium carbonate in
methanol afforded terminal alkyne 4 in high yield. Acid
deprotection of 4 followed by reductive amination gave
5, which was a versatile, late-stage intermediate for the
preparation of analogues of 2. Palladium catalyzed
cross-coupling of 5 with aryl halides followed by alkyne
reduction provided the analogues in Tables 1–3.
A targeted combinatorial chemistry library based in
part on the 1,3,4-trisubstituted pyrrolidine scaffold was
designed with the goal of identifying synergisms among
known and novel CCR5 pharmacophores in these
molecules.7 This led to the identification of a new pyr-
rolidine lead analogue (2) which has excellent affinity
for CCR5, but more modest anti-HIV-1 activity than 1.
While we thought that 2 might also have appreciable
ion channel affinity (a common characteristic of many
lipophilic, basic ligands for G-protein coupled recep-
tors), its structural simplicity relative to 1 made it an
attractive starting point for further investigations. We
wish to describe herein our discovery that the incor-
All analogues were tested for their ability to displace
125I]-MIP-1a from the CCR5 receptor expressed on
[
Table 2. Inhibition of [125I]-MIP-1a bindinga and anti-HIV-1 infectivityb activity of carboxy analogues of 2
Compd
R
CCR5 IC50 (nM)
HeLa IC90 (nM)
2
-H
2-CO2H
3-CO2H
4-CO2H
0.3
100
3
10
6
200
>1000
>1000
1000
300
10
11
12
13
14
15
3-CH2CO2H
4-CH2CO2H
4-(CH2)2CO2H
5
2
100
40
aDisplacement of [125I]-labeled MIP-1a from the CCR5 receptor expressed on CHO cell membranes. Data are reported as mean for n= three
determinations. SD were generally Æ20% of the average. See ref 8 for assay protocol.
bInhibition (CIC90) over a 48 h period of a single HIV-1 (BAL) infection cycle in HeLa Magi cells expressing both CXCR4 and CCR5. See ref 9 for
assay protocol.

J. J. Hale et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2997–3000
2999
Table 3. Inhibition of [125I]-MIP-1a bindinga and anti-HIV-1 infectivityb activity of tetrazole analogues of 2
Compd
R
n
CCR5 IC50 (nM)
HeLa IC90 (nM)
2
0.3
0.2
1
0.2
0.4
200
4
100
100
>300
16
17
18
19
-H
-H
1-CH3
2-CH3
0
1
0
0
aDisplacement of [125I]-labeled MIP-1a from the CCR5 receptor expressed on CHO cell membranes. Data are reported as meanÆSD for n= three
determinations. SD were generally Æ20% of the average. See ref 8 for assay protocol.
bInhibition (CIC90) over a 48 h period of a single HIV-1 (BAL) infection cycle in HeLa Magi cells expressing both CXCR4 and CCR5. See ref 9 for
assay protocol.
Chinese Hamster Ovary (CHO) cell membranes.8 The
results obtained for a series of analogues in which the
pendant phenyl ring of 2 was replaced with a pyridine is
summarized in Table 1. None of the pyridine isomers had
CCR5 affinity comparable to 2 and their was no distinct
preference for the position of the nitrogen in pyridine
ring.
marker for potential off-target activities, then zwitter-
ionic compounds analogous to 16 could provide benefits
beyond enhanced antiviral potency. Unfortunately, the
rat pharmacokinetic profile 16 was found to be far from
desirable; the high rate of clearance (Clp=61 mL/min/
kg), short half-life (t1/2=20 min) and neglible oral
bioavailablity precluded further the pursuit of 16 as a
candidate for further development.
More promising results were obtained with carboxylate
derivatives of 2 (Table 2). Based on the results of the
CCR5 binding assay, carboxy substitution at the 3- or
4-position of the phenyl ring was clearly favored over
that at the 2-position (compare 11 and 12 to 10).
Moreover, homologation of these analogues was
potency enhancing, affording analogues (13–15) with
single digit nanomolar IC50’s in the MIP-1a binding
assay and potency comparable to or exceeding lead ana-
logue 2 in their ability to inhibit a single HIV-1 infection
cycle in a CCR5-expressing HeLa Magi cell line.9
In conclusion, while it is not clear how the acidic tetra-
zole contributes to the enhanced antiviral activity of 16
(one possible explanation is that the acidic or zwitter-
ionic nature of this compound can inhibit or limit the
accessibility of receptor states necessary for fusion),
determining the effect of the incorporation of comparable
functionality into other CCR5 antagonist templates was
viewed to be worthwhile based on the results reported
herein. Efforts in this area have been initiated and reports
on these pursuits will follow in due course.
The SAR developed for piperidine pharmacophores as
part of the investigations into 2-aryl-4-(piperidin-1-
yl)butanamine CCR5 antagonists suggested that the
pendant phenyl ring of these current compounds would
tolerate substitution with more sterically demanding
groups.5 Replacing the carboxy group of 12 with the
bioisosteric C-linked tetrazole afforded an analogue (16)
that had subnanomolar affinity for CCR5 in the ligand
competition assay and was 50-fold more potent than 2
in the HeLa cell assay Table 3). The loss in anti-infec-
tivity potency seen in the HeLa cell assay with homo-
logueue 17 and N-methyl regioisomers 18 and 19
demonstrates that the acidic nature of the tetrazole is an
important factor in blocking the HIV-1 fusion process
from occuring. The discordance seen here between
compound activities in the ligand competition and anti-
infectivity assays indicates that care must be taken in
interpreting the results of the former to be predictive of
a compound’s potential as an anti-HIV-1 agent.10
References and Notes
1. 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.
2. 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.
3. 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.
4. Tagat, J. R.; Steensma, R. W.; McCombie, S. W.; Narar-
eno, D. V.; Lin, S.-I.; Neustadt, B. R.; Cox, K.; Xu, S.; Woj-
cik, L.; Murray, M. G.; Vantuno, N.; Baroudy, B. M.; Strizki,
J. M. J. Med. Chem. 2001, 44, 3343, and references therein.
5. 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, and
references therein.
Compound 16 was found to have significantly atte-
nuated affinity for the L-type calcium channel as com-
pared to 2.11 If this property can be considered to be a

3000
J. J. Hale et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2997–3000
6. Hale, J. J.; Budhu, R. J.; Holson, 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.; Kessler, J.; Danzeisen, R.; Holmes,
K.; Lineberger, J.; Carella, A.; Carver, G.; Emini, E. A.
Bioorg. Med. Chem. Lett. 2001, 11, 2741.
7. 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.; Kessler, J.; Dan-
zeisen, R.; Holmes, K.; Lineberger, J.; Carella, A.; Carver, G.;
Emini, E. A. Bioorg. Med. Chem. Lett. 2001, 11, 3137.
8. 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.
9. 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.
10. The discordant abilities of CCR5 monoclonal antibodies
to block ligand binding to the CCR5 receptor and to inhibit
HIV-1 infection has been rationalized to arise from the
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.
11. 16: IC50 >10 mM, 2: IC50=590 nM. For assay protocol:
Reynolds, I. J.; Snowman, A. M.; Snyder, S. H. J. Pharm.
Exp. Therap. 1986, 237, 731.