Piperazine-based CCR5 antagonists as HIV-1 inhibitors. II. Discovery of 1-[(2,4-dimethyl-3-pyridinyl)carbonyl]-4methyl-4-[3(S)-methyl-4-[1(S)-[4- (trifluoromethyl) phenyl]ethyl]-1-piperazinyl]piperidine N1-oxide (Sch-350634), an orally bioavailable, poten

Article abstract of DOI:10.1021/jm0155401

Truncation of the original piperidino-2(S)-methyl piperazine lead structure 2, from a family of muscarinic antagonists, gave compound 8 which has improved selectivity for the HIV-1 co-receptor CCR5 over muscarinic receptors. Further optimization for pharm

Full text of DOI:10.1021/jm0155401

J . Med. Chem. 2001, 44, 3343-3346
3343
GPCRs has been an active area of research over the past
decade, and the importance of chemokines and their
receptors in viral disease processes is now well-
recognized.4 Consequently, the goal of discovering an
orally active, potent, and selective small molecule CCR5
antagonist has stimulated vigorous research programs
at several pharmaceutical companies.5 We have recently
disclosed studies in which the initial hits from our
database of privileged structures that also have mus-
carinic M2 antagonist activity were optimized with
regard to CCR5 binding activity to afford piperazine-
and piperidine-based CCR5 antagonists.6 In this paper,
we describe the design, synthesis, and biological activity
of Sch-350634 (1), an orally bioavailable CCR5 antago-
nist that inhibits the replication of HIV-1 via blockade
of its entry into cells.
P ip er a zin e-Ba sed CCR5 An ta gon ists a s
HIV-1 In h ibitor s. II. Discover y of
1-[(2,4-Dim eth yl-3-p yr id in yl)ca r bon yl]-4-
m eth yl-4-[3(S)-m eth yl-4-[1(S)-[4-(tr iflu or o-
m eth yl)p h en yl]eth yl]-1-p ip er a zin yl]-
p ip er id in e N1-Oxid e (Sch -350634), a n
Or a lly Bioa va ila ble, P oten t CCR5
An ta gon ist
J ayaram R. Tagat,*,† Ruo W. Steensma,†
Stuart W. McCombie,† Dennis V. Nazareno,†
Sue-Ing Lin,† Bernard R. Neustadt,† Kathleen Cox,#
Serena Xu,‡ Lisa Wojcik,‡ Michael G. Murray,‡
Nicole Vantuno,‡ Bahige M. Baroudy,‡ and
J ulie M. Strizki‡
Departments of Chemical Research, Drug Metabolism &
Pharmacokinetics, and Antiviral Therapy,
Schering-Plough Research Institute, K-15-2B-2800,
2015 Galloping Hill Road, Kenilworth, New J ersey 07033
Received J une 7, 2001
Abstr a ct: Truncation of the original piperidino-2(S)-methyl
piperazine lead structure 2, from a family of muscarinic
antagonists, gave compound 8 which has improved selectivity
for the HIV-1 co-receptor CCR5 over muscarinic receptors.
Further optimization for pharmacokinetic properties afforded
Sch-350634 (1), a prototypical piperazine-based CCR5 antago-
nist, which is a potent inhibitor of HIV-1 entry and replication
in PBMCs. The title compound (1) has excellent oral bioavail-
ability in rat, dog, and monkey.
F igu r e 1.
Our preliminary studies showed that 2(S)-methyl
piperazine and the 2,6-dimethyl benzamide moiety in
structure 2 (Ki: CCR5 ) 18 nM; M2 ) 760 nM) are
essential to enhance affinity for CCR5 over the musca-
rinic receptors (particularly M2).7 Although compound
2 satisfied preliminary in vitro criteria, we were aware
that the presence of a methylenedioxyphenyl ring in this
structure could be a metabolic liability (Figure 1). In
our earlier work, CCR5 and M2 receptor binding data
for compound 3 (Ki: CCR5 ) 45 nM; M2 ) 1400 nM)
indicated that substitution on the left side of the
molecule was tolerated. We then set out to define the
minimum structural elements that were needed for a
CCR5 antagonist of this structural class, with selectivity
over binding at muscarinic M2 receptor,8 good potency
in anti-viral assays,9 and good oral absorption in rat10
as our preliminary overall criteria.
Ch em ist r y. As shown in Scheme 1, elaboration of
commercially available R-(S)-methyl benzylamine (4) via
halogenation and SN2 displacement of an activated (R)-
methyl lactate gave the diketo piperazine 6, which was
processed to the target 8. Compound 8 exhibits strong
affinity for CCR5 with a significant decrease in the
binding at the M2 muscarinic receptor (CCR5: Ki ) 20
nM; M2: 32% inhibition at 1 µM). This result suggested
that truncation on the left side to simplify the structure
and reduce the molecular weight can provide piperidino-
piperazines, which are more selective ligands for CCR5.
Replacing the benzylic carbon in 8 with groups such as
In tr od u ction . Human immunodeficiency virus type
1 (HIV-1) infection, which eventually leads to the
acquired immunodeficiency syndrome (AIDS), continues
unabated as an epidemic of major proportions in sub-
Saharan Africa and Asia.1 In the Western Hemisphere,
the emergence of viral strains resistant to combinations
of antiretroviral drugs still makes HIV-1 a primary
healthcare issue.2 The recent discovery of CCR5, a co-
receptor essential for HIV recognition and entry into
CD4+ macrophages and T-cells, was therefore received
with excitement in the scientific community as a
potential new target for antiviral therapy.3 The expecta-
tion was that suitable chemical agents which bind to
CCR5 would prevent HIV from entering the CD4+
target cells.3a Further impetus to this approach came
from studies in which individuals homozygous for a 32
base pair deletion in the gene encoding CCR5 and thus
lacking functional receptors were shown to be resistant
to HIV-1 infection; heterozygous individuals, who lack
one of the two allelles needed for CCR5 expression, show
delayed disease progression following HIV infection.3a
CCR5 is a chemokine receptor belonging to the super
family of G-protein coupled receptors (GPCRs). The
design and synthesis of small molecule ligands for
* To whom correspondence should be addressed. Tel: (908)-740-
3461. Fax: (908)-740-7152. E-mail: jayaram.tagat@spcorp.com.
† Chemical Research.
#
Drug Metabolism & Pharmacokinetics.
‡ Antiviral Therapy.
10.1021/jm0155401 CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/07/2001

3344 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Sch em e 1. Diketopiperazine Route
Letters
Sch em e 2. SN2 Displacement Route
amides, carbamates, or sulfonamides resulted in loss of
binding at CCR5, as did attempts to replace the amide
group on the right side with alkyl groups, sulfonamides,
or carbamates. Thus, the structure 8 became a starting
point for further refinement of structure-activity-
pharmacokinetic relationships.
The iodine substituent in 7 served as a functional
handle to introduce less labile groups at the para-
position via standard methodology.11 Alternatively,
targets such as 16 and 17 containing electron-with-
drawing para-substituents that are not readily acces-
sible from the intermediate 7 were prepared by a
route which is enantio- and diastereo-selective (Scheme
2).12 In this route, it is noteworthy that a modified
Strecker reaction (14 f 15) served to introduce an alkyl
or aryl group at the 4-position of the piperidine.13
Standard reductive amination introduces a hydrogen
atom.14
Ta ble 1. Methylation Patterns Determine CCR5 Affinity
CCR5 binding
HIV-1 entry
compd
R1
R2
R3
Ki (nM)a
IC50 (nM)b
Resu lts a n d Discu ssion . In the truncated structure
8, as in compounds 2 and 3, the (S)-methyl group in
the piperazine ring is essential for effective binding at
CCR5 (Table 1). Introduction of a methyl group at the
4-position of the piperidine (compounds 8b, 8d , and 8e)
results in a 3-7-fold improvement in the binding
potency (Ki), with a proportional increase in the anti-
viral potency (IC50) in the functional assay. The methyl
group was found to be optimal at the ring junction (R3:
CH3 > H > Et, i-Pr).
In a related study, we prepared the other three
diastereomers arising from the two chiral centers. The
(S,S)-diastereomer depicted (8) was about 8 times more
potent in binding to CCR5 than the (R,S)-diastereomer
(Ki ) 125 nM), whereas the (S,R) and (R,R) diastereo-
mers were inactive (Ki > 1 µM), again underscoring the
importance of the 2(S)-methyl piperazine in the phar-
macophore.
8a
8b
8c
8
8d
8e
H
H
H
CH3
H
H
H
CH3
CH3
CH3
CH3
H
CH3
H
H
CH3
CH3
440
62
30
20
8
NDc
NDc
NDc
7
2.7
1
CH3
5
a
See Supporting Information for details. Standard error was
b
10%, and assay-to-assay variability was 2-3-fold. Concentration
required to inhibit by 50% the entry of HIV-1 reporter virus (J rFl)
into U-87 cells. For IC50 values, the 95% confidence limit was
within 1 log and the intra-assay variation was less than 0.5 log.
c ND ) not determined.
The bromo-derivative 9 (Ki ) 23 nM; IC50 ) 6 nM),
which was prepared in a manner analagous to 8, was
equipotent to the latter. However, the des-halo com-
pound 10 (Ki ) 275 nM) was significantly less active,
and the ortho- and meta-substituted derivatives were
5-6 times less active than 8 and 9, indicating the

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3345
requirement of a para-substituent of medium size in the
pharmacophore.
With the in vitro structure-activity relationships in
the new, truncated piperidino-piperazine series well-
established, we turned our attention to pharmacokinetic
(PK) characterization of our CCR5 antagonists. Follow-
ing oral administration of compounds (10 mg/kg) to rats,
their plasma concentrations over a 6 h period were
determined.10 The benzamides exhibited only modest
blood levels in the rat model following oral administra-
tion due to oxidative metabolism in the benzamide
portion. Consequently, the two methyl groups were
replaced with heteroatoms (Cl, OH, NH2). The in vitro
and rat PK data for representative new amides are
presented in Table 2.
F igu r e 2.
Pharmacokinetic data for 18 (administered as the
amorphous hydrochloride salt in 0.4% methyl cellulose)
in beagle dogs and in cynomolgus monkeys are sum-
marized in Table 3.
Ta ble 2. Effect of Varying the Amide on Oral Blood Levels in
Rat (10 mg/kg)
Ta ble 3. Pharmacokinetic Profile of Compound 18
iv administrationa
oral administrationb
c
d
c
d
species
Co
AUC0-24h
Cmax
AUC0-24h
BA (%)e
doge
346
390
1609
996
226
281
1583
2742
33e
monkeye
27.5e
a
b
CCR5 data
Ki IC50
(nM)a (nM)b
rat PK (0-6 h)
Intravenous (iv) dose ) 1 mg/kg. Oral dose ) 3 mg/kg (dog)
or 10 mg/kg (monkey). c Co and Cmax are in ng/mL. AUC is in
d
AUC
(ng/mL h)
ng/mL h. e Bioavailability (BA) is dose-corrected.
compd
R1
R2
CH3 CH3
Cl NH2
CH3 OH
CH3 CH3
R3
8e
8f
8g
16
16a
16b
17
17a
17b
I
I
I
5
12
7
1
5
5
3
5
15
1
2
1
0.4
1.2
3.2
0.5
0.8
4
822
1984
2806
922
1872
2543
NDc
447
Following oral administration of 18 to rats, analysis
of plasma samples showed that a single (M+16) me-
tabolite is rapidly produced in amounts that could
exceed the remaining parent compound. This reflects a
high level of absorption, followed by first-pass oxidation.
A similar situation was later found in both dogs and
monkeys.
CF3
CF3
CF3
Cl
NH2
CH3 OH
SO2CH3 CH3 CH3
SO2CH3 Cl NH2
SO2CH3 CH3 OH
384
Surmising that the rapid metabolism of 18 may be
due to oxidation at the pyridyl nitrogen, we prepared
the corresponding nicotinamide-N-oxide (1). Hindered
rotation about the two bonds that constitute the unsym-
metrical tertiary amide on the right side results in the
observation of four rotational isomers (rotamers) under
achiral conditions for compound 1.16
Compound 1, which has good affinity for CCR5
binding and good potency in the entry assay, showed
almost a 3-fold improvement in oral blood level in rats,
relative to 18 (Figure 3).
a
Standard error was 10%, and assay-to-assay variability was
b
2-3-fold. Concentration required to inhibit by 50% the entry of
HIV-1 reporter virus (ADA) into U-87 cells. For IC50 values, the
95% confidence limit was within 1 log and the intra-assay variation
was less than 0.5 log. c ND ) not determined.
Replacing the lipophilic benzamide with the polar
anthranilamides and salicylamides significantly im-
proved oral blood levels, expressed as the area under
the curve (AUC) of concentration vs time plots. While
several para-substituents satisfied the CCR5 binding
criteria, the trifluoromethyl group was optimal based
on PK parameters.
The concept of introducing heteroatoms in the amide
portion was extrapolated by replacing the 2,6-dimethyl
benzamide with the 2,4-dimethyl nicotinamide (Figure
2). The combination of a para-trifluoromethyl substitu-
ent on the left and 2,4-dimethyl nicotinamide on the
right side of the piperidino-piperazine core afforded a
compound (18) that satisfied all our preliminary criteria
in terms of its anti-viral properties and good oral blood
levels in rat. Additionally, 18 has reduced affinity for
the M2 muscarinic receptor (Ki ) 2500 nM) and does
not inhibit the liver enzymes 3A4 and 2D6. Compound
18 also has excellent potency in anti-viral assays: It
inhibits HIV entry in a single cycle infectivity assay with
an IC50 ) 0.5 nM (HIV-1/ADA) and the replication of
several primary HIV-1 isolates in peripheral blood
mononuclear cells (PBMC), with IC50 values in the 0.2-
2.0 nM range.15
F igu r e 3.
Detailed pharmacokinetic evaluation in beagle dogs
and cynomolgus monkeys (Table 4) clearly demon-
strated that compound 1 (amorphous HCl salt) is
capable of achieving and sustaining high blood levels

3346 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Ta ble 4. Pharmacokinetic Profile of the Title Compound (1)
Letters
(2) Pomerantz, R. J . Primary HIV-1 Resistance. J . Am. Med. Assoc.
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(3) (a) Hunt, S. W., III; LaRosa, G. J . Chemokine receptors as HIV
co-receptors: targets for therapeutic intervention in AIDS. Annu.
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N. A.; Wallace, O. B. HIV-1 entry: an expanding portal for drug
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iv administrationa
oral administrationb
d
c
d
species
AUC0-24h
T1/2 (h) Cmax
AUC0-24h
BA (%)e
doge
3240
2610
6
4
690
1420
6290
15400
65e
59e
monkeye
(4) (a) Patchett, A. A.; Nargund, R. P. Privileged structures: an
update. Annu. Rep. Med. Chem. 2000, 35, 289-298. (b) Murphy,
P. M. Viral exploitation and subversion of the immune system
through chemokine mimicry. Nature Immun. 2001, 2, 116-122.
(5) (a) Shiraishi, M.; Aramaki, Y.; Imoto, H.; Nishikawa, Y.; Kan-
zaki, N.; Okamoto, M.; Sawada, H.; Nishimura, O.; Baba, M.;
Fujino, M. Discovery of novel, potent, and selective small-
molecule CCR5 antagonists as anti-HIV-1 agents: Synthesis and
biological evaluation of anilide derivatives with a quarternary
ammonium moiety. J . Med. Chem. 2000, 43, 2049-2063. (b)
Hale, J . J .; Budhu, R. J .; Mills, S. G.; MacCoss, M.; Malkowitz,
L.; Siciliano, S.; Gould, S. L.; DeMartino, J . A.; Springer, M. S.
1,3,4-Trisubstituted pyrrolidine CCR5 Antagonists. Part 1:
Discovery of the pyrrolidine scaffold and determination of its
stereochemical requirements. Bioorg. Med. Chem. Lett. 2001, 11,
1437-1440.
(6) A preliminary account of this work has been presented: Tagat,
J .; Nazareno, D.; Vice, S.; Lin, S.; Steensma, R.; Miller, M.;
Bauer, A.; McCombie, S.; Palani. A.; J osien, H.; Clader, J .;
Neustadt, B.; Greenlee. W.; Ganguly, A.; Piwinski, J .; Chan, T.
M.; Evans, A.; Dan, N.; Baroudy, B.; Endres, M.; Strizki, J .;
Vantuno, N.; Cox, K.; Broske, L.; Zhang, X. Discovery of potent,
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221st National Meeting of the American Chemical Society, San
Diego, CA, April 1-5, 2001; American Chemical Society: Wash-
ington, DC, 2001; MEDI 26.
a
b
Intravenous (iv) dose ) 1 mg/kg. Oral dose ) 3 mg/kg (dog)
c
d
or 10 mg/kg (monkey). Cmax is in ng/mL. AUC is in ng/mL h.
e Bioavailability (BA) is dose-corrected.
following oral administration. The major route of excre-
tion is through the urine in rats and through the bile
in dogs and in monkeys. The major metabolite arises
via oxidative cleavage of the CH3CHCH2 region of the
chiral piperazine. The reduction of the N-oxide (1) back
to the nicotinamide 18 was not observed.
Compound 1 shows 30-50-fold selectivity for CCR5
over the M1 (Ki ) 350 nM) and the M2 (Ki ) 250 nM)
muscarinic receptors. It has no appreciable affinity for
other related receptors of current interest.17 There is
neither inhibition nor induction of the liver enzymes
with this compound. In the PBMC based assay, com-
pound 1 inhibited the replication of HIV-1 isolates with
IC50 values in the 2-20 nM range.15 Importantly,
compounds 1 and 18 also bind to primate CCR5,
suggesting the possibility of evaluation in primate-based
models of the disease.
In conclusion, truncation of the original piperidino-
2(S)-methyl piperazine lead structure 2 gave compound
8 with reduced molecular weight and improved selectiv-
ity for the HIV co-receptor CCR5 over the muscarinic
M2 receptor. Optimization of this pharmacophore for
pharmacokinetic properties afforded the title compound
(1), a prototypical piperazine-based CCR5 antagonist,
which is a potent inhibitor of HIV-1 entry and replica-
tion and has excellent oral bioavailability in rat, dog,
and monkey. Starting with potent muscarinic antago-
nists with weak affinity for CCR5,7 we have designed
structure 1, which is a potent, orally absorbed CCR5
antagonist with modest affinity for the muscarinic M2
receptor. The further improvement of such structures
toward greater selectivity for CCR5 will be reported in
due course.
(7) Tagat, J . R.; McCombie, S. W.; Steensma, R. W.; Lin, S.-I.;
Nazareno, D. V.; Baroudy, B.; Vantuno, N.; Xu, S.; Liu, J .
Piperazine-based CCR5 antagonists as HIV-1 Inhibitors. I: 2(S)-
methyl piperazine as a key pharmacophore element. Bioorg.
Med. Chem. Lett. 2001, 11, 2143-2146.
(8) CCR5-RANTES binding assay: see ref 7. Muscarinic receptor
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G.; Ruperto, V. B.; Duffy, R. A.; McQuade, R.; Crosby, G.; Taylor,
L. A.; Billard, W.; Lachowicz, J . E. Diphenyl sulfoxides as
selective antagonists of the muscarinic M2 receptor. Bioorg. Med.
Chem. Lett. 2000, 10, 2255-2257.
(9) For details of RANTES binding, HIV-1 entry, and replication
assays, see the Supporting Information.
(10) Cox, K. A.; Dunn-Meynell, K.; Korfmacher, W. A.; Broske, L.;
Nomeir, A. A.; Lin, C. C.; Cayen, M. N.; Barr, W. H. A novel in
vivo procedure for rapid pharmacokinetic screening of discovery
compounds in rat. Drug Discovery Today, 1999, 4 (5), 232-237.
(11) The conversion of 7 to targets bearing diverse para-substituents
will be published elsewhere.
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borane reductions of ketones catalyzed by chiral oxaborolidines.
J . Am. Chem. Soc. 1987, 109, 5551-5553. (b) J ones, T. K.;
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P.; Turner-J ones, E. T.; Reamer, R. A.; Roberts, F. E.; Grabowski,
E. J . J . An asymmetric synthesis of MK-0417. Observations on
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3849-3862.
Ack n ow led gm en t. We thank Dr. J ean Lachowicz
(CNS Pharmacology) for the muscarinic antagonist data
and the physical and analytical chemistry department
for spectral and analytical data of our compounds. Drs.
Anandan Palani, J ohn Clader, and William Greenlee
are thanked for many helpful discussions. We are
grateful to Drs. Ashit Ganguly, J ohn Piwinski, and
Gregory Reyes for their strong support of this project.
(15) Primary HIV-1 isolates included: US-1, 92US715, BAL, ASM57,
J RFL, J rCSF, QZ4589, 302056, SF162, DJ 258, 92RW026,
94ZW103, CM235, and BZ162. The cytotoxicity (CC50) of 1 and
18 is >40 µM in PBMC cultures using MTS cell titer 96 protocol.
(16) Studies of the physicochemical phenomenon of rotamers will be
reported in due course.
(17) Compounds 1 and 18 (1-5 µM) show 0-20% binding at CCR1-
3, CCR7, NK1-3, CB1-2, H1, H3, NPY, D1-2, A2a, nociceptin,
opioid, and thrombin receptors.
Su p p or tin g In for m a tion Ava ila ble: Description of an-
tiviral assays and experimental procedures and spectral data
for the preparation and characterization of compounds 1, 8,
and 18. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(1) UNAIDS. Report on the Global HIV/ AIDS Epidemic; XIII
International AIDS Conference, Durban, South Africa, 2000.
J M0155401