Piperazine-based CCR5 antagonists as HIV-1 inhibitors. I: 2(S)-methyl piperazine as a key pharmacophore element

Article abstract of DOI:10.1016/S0960-894X(01)00381-X

Optimization of the piperidino-piperazines 1 and 2 provided early leads 3 and 4, which showed good activity in the CCR5-RANTES binding assay and in antiviral assays. A systematic study around these structures showed that the 2(S)-methyl piperazine is essential for CCR5 affinity, which is further enhanced by forming the 2,6-dimethyl benzamide of the piperidine.

Full text of DOI:10.1016/S0960-894X(01)00381-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2143–2146
Piperazine-Based CCR5 Antagonists as HIV-1 Inhibitors.
I: 2(S)-Methyl Piperazine as a Key Pharmacophore Element
Jayaram R. Tagat,a,* Stuart W. McCombie,a Ruo W. Steensma,a Sue-Ing Lin,a
Dennis V. Nazareno,a Bahige Baroudy,b Nicole Vantuno,b Serena Xub and Jia Liub
aDepartment of Chemical Research, Schering-Plough Research Institute, 2015 Galloping Hill Road,
K-15-2B-2800 Kenilworth, NJ 07033-1300, USA
bDepartment of Antiviral Therapy, Schering-Plough Research Institute, 2015 Galloping Hill Road,
K-15-4E-4650 Kenilworth, NJ 07033-1300, USA
Received 18 April 2001; accepted 22 May 2001
Abstract—Optimization of the piperidino-piperazines 1 and 2 provided early leads 3 and 4, which showed good activity in the
CCR5–RANTES binding assay and in antiviral assays. A systematic study around these structures showed that the 2(S)-methyl
piperazine is essential for CCR5 affinity, which is further enhanced by forming the 2,6-dimethyl benzamide of the piperidine. # 2001
Elsevier Science Ltd. All rights reserved.
The worldwide AIDS pandemic caused by the type 1
human immunodeficiency virus (HIV-1), with over 30
million infected individuals, continues to be a fatal
medical condition.1 Intensive efforts to combat this ret-
rovirus have led to the adoption of combination ther-
apy, using inhibitors of the viral protease and reverse
transcriptase enzymes. Although effective in the short
term, this approach suffers from patient compliance
issues and incomplete suppression of the virus in the
long term. This has led to the emergence of HIV strains
resistant to the current regimen of drugs.2 Hence, there
is a need for new agents which act at different points in
the viral life cycle.
compromise the immune system in individuals who are
homozygous for a defective genetic sequence for recep-
tor coding. Further, such individuals are significantly
resistant to HIV-1 infection.5 These observations sug-
gest that appropriate, small-molecule antagonists of the
virus–CCR5 interaction should be novel anti-HIV-1
agents.6 During the course of our studies, such a mole-
cule, TAK-779, was described and shown to be an
effective inhibitor of HIV-1 infection of T-cells.7 Groups
from Merck have also disclosed their work in this area.8
In this paper, we describe the identification and ela-
boration of piperidino-piperazine lead compounds to
obtain effective inhibitors of HIV-1 cell entry.
It has been shown recently that binding to specific, cell-
surface co-receptors is an essential process, when HIV-1
gains entry to the CD4+ cells of the immune system.3
HIV-1 utilizes the chemokine receptor CCR5 on mac-
rophages and T-cells, which are its primary targets.
CCR5 is a G-protein coupled, 7-trans-membrane recep-
tor whose endogenous ligands are the chemokines
RANTES, MIP-1a and MIP-1b, which have been
reported to suppress HIV-1 cell entry.4
Three assays were used to evaluate compounds. The
primary assay, also used in initial high-throughput
library screening, measured the ability of compounds to
inhibit 125I-labeled RANTES binding to the CCR5
receptor on membranes.9 Selected compounds were then
evaluated in a viral entry assay,10 in which a pseudo-
type virus bearing a reporter gene for luciferase was
used to infect cells expressing CD4 and CCR5. Finally,
antiviral activity was measured as the ability of com-
pounds to inhibit the growth of primary HIV-1 isolates
in human peripheral blood mononuclear cells.11
Although CCR5 activation is involved in normal cell
trafficking, the lack of functional CCR5 does not
From our compound library, several structural types
inhibited the RANTES–CCR5 interaction. Further
evaluation for inhibition of viral entry at sub-cytotoxic
*Corresponding author. Tel.: +1-908-740-3461; fax: +1-908-740-
7152; e-mail: jayaram.tagat@spcorp.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00381-X

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J. R. Tagat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2143–2146
levels revealed a series of compounds as exemplified by
1 and 2 (Fig. 1), originally prepared for our muscarinic
receptor antagonist program.12
Figure 1.
Accordingly, we synthesized a series of compounds in
which the methylation pattern and stereochemistry, the
diaryl linking unit, and the amide substitution pattern
were varied. The diaryl sulfones were prepared as out-
lined in Scheme 1. In this route, the benzylic chirality
derives from the enantioselective reduction of a pro-
chiral ketone.13 The success of the subsequent SN2 dis-
placement reaction depends upon the presence of an
electron-withdrawing para substituent, to suppress SN1
chemistry at the benzylic center.
Scheme 2. The diketopiperazine route to CCR5 antagonists.
Results for the inhibition of RANTES binding (Ki) and
viral entry (IC50), as well as the M2 receptor affinity
(Ki),12 for selected compounds are shown in Table 1.
Comparison of compounds 1 and 3 clearly shows that
the chirality of the piperazine 2-substituent determines
the selectivity: for effective CCR5 binding, 2(S) stereo-
chemistry is needed.9 In contrast, the 2(R) compounds
such as 1 are M2 antagonists. Further, any reduction in
the level of methylation reduces the CCR5 affinity
(compare compound 3 with 13c–e and compound 4 with
13a–b). The amide substitution pattern also affects
potency: a 2,6-disubstituted aryl ring is very important
(compare compound 2 with 4). The choice of the groups
R3 and R4 is critical: although methyl may be replaced
by isosteric polar groups such as NH2 or Cl (com-
pounds 13f–h), a smaller group (F) diminishes binding
(compound 13i). Finally, compound 14 shows that the
3,4-methylenedioxyphenyl ring can be replaced by 3-
chlorophenyl ring, with only a modest potency loss in
CCR5 binding, but a more significant reduction in the
M2 affinity.
Scheme 1. SN2 displacement route to CCR5 antagonists.
Alternatively, p-bromo a-methylbenzylamine was lithi-
ated and treated with piperonal to obtain the diaryl
methane 9 after deoxygenation of the initial carbinol.
Reaction of 9 with (R)-methyl lactate led to the diketo-
piperazine 10 (Scheme 2). Reduction of the carbonyl
groups in 10 and reductive amination with N-tert-
butoxycarbonyl-4-piperidinone gave 12. Coupling of the
free piperidine from 12 with 2,6-dimethyl benzoic acid
gave the target 4. The mono- and bis-desmethyl
compounds were similarly prepared.14

J. R. Tagat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2143–2146
Table 1. Binding (CCR5/M2) and inhibition of HIV entry data for selected compounds
2145
Compd
X
R1
R2
R3
R4
Ki (nM)
IC50 (nM)
CCR5
M2
3
4
SO2
CH2
CH2
CH2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
CH2
CH3
CH3
H
H
H
CH3
CH3
CH3
H
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NH2
NH2
Cl
28
18
44
485
760
>40
497
>40
1
31
100
100
270
352
>1400
2.5
1.7
ND
ND
ND
ND
ND
2
11
10
129
12
13a
13b
13c
13d
13e
13f
13g
13h
13i
14
352
74
442
>1300
31
CH3
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
32
49
230
45
Cl
F
CH3
F
CH3
Entry data is for JrFL/ADA strains of HIV-1.
2. Boden, D.; Hruley, A.; Zhang, L.; Cao, Y.; Guo, Y.; Jones,
E.; Tsay, J.; Ip, J.; Farthing, C.; Limoli, K.; Parkin, N.; Mar-
kowitz, M. JAMA 1999, 282, 1135.
3. (a) Littman, D. R. Cell 1998, 93, 677. (b) Murphy, P. M.
Nature Immunol. 2001, 2, 116.
For the more potent compounds in this series (3, 4, 13f),
inhibition of the binding at CCR5 correlated well with
inhibition of viral entry. It is important to note that
IC50 values obtained from the entry assay were gen-
erally less than the Ki values determined from the
RANTES binding assay. This probably reflects the dif-
ferences in both the ligand (RANTES vs virus) and the
target (cell membrane vs live cells) used in these
assays.15 Further, compound 4 inhibited the replication
of a primary HIV-1 isolate (US-1) in PBMCs with a
mean IC50 of 8 nM.11 Compound 4 was also shown to
be an antagonist at the CCR5 receptor, from effects on
RANTES induced calcium flux.5a No appreciable bind-
ing was detected at other chemokine receptors (CCR1,
CCR2 and CCR3).
4. Cocchi, F.; DeVico, A. L.; Garzino Demo, A.; Arya, S.;
Gallo, R.; Lusso, P. Science 1995, 270, 1811.
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S. R.; Horuk, R.; MacDonald, M. E.; Stuhlmann, H.; Koup,
R. A.; Landau, N. R. Cell 1996, 86, 367. (b) Samson, M.;
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M.; Saragosti, S.; Lapoumeroulle, C.; Cognaux, J.; Forceille, C.;
Muyldermans, G.; Vehofstede, C.; Butonboy, G.; Georges, M.;
Imai, T.; Rana, S.; Yi, Y.; Smyth, R. J.; Collman, R. G.; Doms,
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Kanzaki, N.; Okamoto, M.; Sawada, H.; Nishimura, O.;
Baba, M.; Fujino, M. J. Med. Chem. 2000, 43, 2049. (b) Baba,
M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.; Sawada, H.;
Shiraishi, M.; Aramaki, Y.; Okanogi, K.; Meguro, K.; Fujino,
M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5698.
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In summary, these results clearly demonstrate that
compounds containing the piperidino-2(S)-methyl
piperazine pharmacophore prevent the entry of HIV-1
into target cells via inhibition of the binding of the virus
to the co-receptor CCR5. The further development of
this series to give potent, orally bioavailable CCR5
antagonists that inhibit HIV replication will be reported
in the near future.
Acknowledgements
We thank the structural chemistry department for mass
spectral data, Dr. Jean Lachowicz of the Pharmacology
department for providing muscarinic antagonist data
and Dr. A. K. Ganguly for discussion.
9. Binding assay: Membrane preparations from CHO cells
expressing CCR5 were incubated with 125I-RANTES in the
presence or absence of compound for 1 h. Compounds were
serially diluted over a range of 0.3–Â3000 nM and tested in
replicates of four. Reaction mixtures were harvested through
glass fiber filters, and washed thoroughly. Total replicate
References and Notes
1. UNAIDS. Report on the Global HIV/AIDS Epidemic 2000.
XIII International AIDS Conference, Durban, South Africa,
2000.

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J. R. Tagat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2143–2146
counts were averaged and IC50 values calculated as the
amount of compound required to inhibit 50% of total 125I-
RANTES binding. The binding affinity constant, Ki, was
determined using prism software analysis. The standard error
was 10% and variability was 2–3 fold from assay-to-assay.
For the compounds 1 and 2 from high-throughput screening,
Ki values are not available.
10. Entry assay: Connor, R. I.; Chen, B. K.; Choe, S.;
Landau, N. R. Virology 1995, 206, 935.
11. Replication assay: Trkola, A.; Ketas, T. J.; Nagashima,
K. A.; Zhao, L.; Cilliers, T.; Morris, L.; Moore, J. P.; Mad-
don, P. J.; Olson, W. C. J. Virol. 2001, 75, 579.
14. All compounds reported in here were fully characterized
by 1H NMR (300 MHz, CDCl3), MS and HRMS. Selected
data is shown below.
Compound 3: 1H NMR d 0.86 (m, 3H), 1.12 (br s, 3H), 1.20
(br m, 2H), 1.25 (br m, 3H), 1.78 (br s, 1H), 2.10 (m, 4H), 2.19
(s, 3H), 2.27 (s, 3H), 2.78 (m, 2H), 2.93 (m, 2H), 3.44 (br d,
1H), 4.10 (m, 1H), 4.86 (br d, 1H), 6.05 (s, 2H), 6.87 (d, J=8.6
Hz, 1H), 7.02 (m, 2H), 7.14 (dd, J=7.3 Hz, 7.3 Hz, 1H), 7.32
(d, J=1.8 Hz, 1H), 7.55 (d, J=8.6 Hz, 3H) and 7.83 (d, J=7.9
Hz, 2H). HRMS calcd for C34H42N3O5S (M+H+) 604.2845.
Found 604.2861.
1
Compound 4: H NMR d 1.38 (d, J=3 Hz, 3H), 1.50 (br s,
12. Kozlowski, J. A.; Lowe, D. B.; Guzik, H. S.; Zhou, G.;
Ruperto, V. B.; Duffy, R. A.; McQuade, R.; Crosby, G., Jr.;
Taylor, L. A.; Billard, W.; Binch, H., III; Lachowicz, J. E.
Bioorg. Med. Chem. Lett. 2000, 10, 2255.
13. (a) Corey, E. J.; Bakshi, R. K.; Shibata, S. J. Am. Chem.
Soc. 1987, 109, 5551. (b) Mathre, D. J.; Jones, T. K.; Xavier,
L. C.; Blacklock, T. J.; Reamer, R. A.; Mohan, J. J.; Turner-
Jones, E. T.; Hoogsteen, K.; Baum, M. W.; Grabowski, E. J. J.
J. Org. Chem. 1991, 56, 751.
3H), 1.6–1.8 (br m, 2H), 1.9–2.2 (br m, 6H), 2.27 (s, 3H), 2.28
(s, 3H), 2.4–3.1 (br m, 6H), 3.2–3.5 (m, 2H), 3.8 (s, 2H), 4.9 (br
d, 1H), 5.8 (s, 2H), 6.6 (s, 1H), 6.65 (d, J=6 Hz, 1H), 6.75
(d, J=6 Hz, 1H), 6.9–7.2 (m, 5H) and 7.45 (br s, 2H).
HRMS calcd for C35H44N3O3 (M+H+) 554.3383. Found
554.3383.
15. Compounds from this series have similar effects on the
activity of other CCR5 ligands such as MIP-1b.