Piperazine-based CCR5 antagonists as HIV-1 inhibitors. III: Synthesis, antiviral and pharmacokinetic profiles of symmetrical heteroaryl carboxamides

Article abstract of DOI:10.1016/S0960-894X(02)00918-6

The unsymmetrical nicotinamide-N-oxide moiety in compound 1 was replaced with symmetrical isonicotinamides as well as 4,6-dimethyl pyrimidine-5-carboxamides. Compound 16 from the latter set reduced the number of rotamers, improved potency of inhibiting UIV entry, slightly diminished the affinity for the muscarine receptors and showed very good oral absorption.

Full text of DOI:10.1016/S0960-894X(02)00918-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 567–571
Piperazine-Based CCR5 Antagonists as HIV-1 Inhibitors.
III: Synthesis, Antiviraland Pharmacokinetic Profiels of
y
SymmetricalHeteroarylCarboxamides
Stuart W. McCombie, Jayaram R. Tagat,* Susan F. Vice, Sue-Ing Lin, Ruo Steensma,
Anandan Palani, Bernard R. Neustadt, Bahige M. Baroudy, Julie M. Strizki,
Michael Endres, Kathleen Cox, Niya Dan and Chuan-Chu Chou
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-2B-2800, Kenilworth, NJ 07033, USA
Received 21 August 2002; accepted 20 September 2002
Abstract—The unsymmetrical nicotinamide-N-oxide moiety in compound 1 was replaced with symmetrical isonicotinamides as well
as 4,6-dimethyl pyrimidine-5-carboxamides. Compound 16 from the latter set reduced the number of rotamers, improved potency
of inhibiting UIV entry, slightly diminished the affinity for the muscarine receptors and showed very good oral absorption.
# 2002 Elsevier Science Ltd. All rights reserved.
The discovery of the chemokine receptor CCR5 as an
essential co-receptor for the type 1Human Immunodefi-
ciency Virus (HIV-1) to dock and gain entry into CD4+
macrophages and T-cells has stimulated wide-spread
programs to discover small molecule antagonists to
block the HIV-1-CCR5 interaction.2 We have recently
disclosed our initial efforts in this regard leading to the
discovery of a prototypical piperazine-based CCR5
antagonist 1.3 Sch-350634 (1) had excellent oral bioa-
vailability in rodents and in primates, as well as good
antiviral activity (CCR5 Ki=7 nM; IC50=2–20 nM vs
HIV isolates in cell-based assays). However, the moder-
observation of cardiovascular effects (at 10 mg/kg, po)
and CNS and GI effects (at 30 mg/kg, po) in rats.
Another interesting feature of 1 was the observation of
four rotational isomers (rotamers) for this compound
under chiral conditions.4 The hindered rotations about
the N–CO and the CO–pyridyl bonds in 1 result in four
diastereomeric rotamers for this structure, which due to
their relatively slow equilibration are observable on
HPLC (Fig. 2) using a chiral mobile phase.5
The lower energy N–CO bond rotation due to the ter-
tiary amide is quite common and leads to a rapidly
equilibrating pair of rotamers. The hindered rotation
ate affinity of
(M1=350 nM; M2=250 nM) was a concern.
1
for the muscarinic receptors
The nicotinamide derivative 2 had better receptor selec-
tivity (Ki: CCR5=2 nM; M2=2500 nM) and potency
(IC50=0.2–2.0 nM vs HIV isolates) than 1. Since the
nicotinamide 2 was rapidly oxidized to its N-oxide 1 in
vivo, 1 was selected for ancillary studies (Fig. 1).
The prevalence of M1 receptors in the intestine and M2
receptors in the heart and the forebrain coupled with
the very high oral blood levels exhibited by 1 led to the
yFor a preliminary account of this work, see ref 1.
*Corresponding author. Tel.: +1-908-740-3461; fax: +1-908-740-
7441; e-mail: jayaram.tagat@spcorp.com
Figure 1. Unsymmetrical heteroaryl carboxamides.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00918-6

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S. W. McCombie et al. / Bioorg. Med. Chem. Lett. 13 (2003) 567–571
Figure 2. HPLC chromatogram of compound 1.
about the CO–pyridyl bond requires more energy and
leads to a slowly equilibrating pair of rotamers due to
the unsymmetrical nature of the pyridine moiety (Fig.
3). Experimentally, the higher energy rotamers equili-
brated to a steady state mixture of ꢀ2:1 in 5 h at 37 ꢁC,
18 h at 25 ꢁC, and 4 days at 10 ꢁC.
Tertiary amides bearing an aryl/heteroaryl ring with a
C2 axis of symmetry would lead to a degenerate pair of
rotamers for the CO–Aryl bond rotation. In practice,
this should result in the observation of a single pair of
rapidly equilibrating rotamers for such compounds.
Scheme 1. Synthesis of isonicotinic acids and their N-oxides.
yield=80–90%) gave the isonicotinamides and their
N-oxides listed in Table 1.
We decided to explore the effect of replacing the
unsymmetrical nicotinamide-N-oxide in 1 with symme-
trical hetroaryl amides in order to minimize the number
of rotamers and possibly affect the receptor selectivity
(CCR5 vs M2) in the process. As polar amides are nee-
ded to obtain high blood levels orally, we focused our
attention on two classes of symmetrical amides: iso-
nicotinamides and pyrimidine carboxamides.
As Table 1 indicates,9 desirable levels of potency,
receptor selectivity (CCR5/M2) and oral blood levels10
in the rat were not observed together for any single
compound in this set and so the isonicotinamides were
not pursued further.
It is well known that the direct condensation of the
diketo-ester 8 with nucleophiles such as the amidines
results in deacylation whereas using the O-alkylated
derivative 9 furnishes the desired heterocycle.11 Thus,
the reaction of 8 with methyl triflate in the presence of
Cs2CO3 in acetonitrile gave exclusively the O-methyl-
ated product 9. Proton NMR analysis revealed no evi-
dence of C-alkylation but ꢀ20% unreacted diketo-ester
8 remained even when excess methyl triflate was used.
Condensation of 9 with amidines under standard con-
ditions formed the pyrimidine esters 10. We have found
that the hydrolysis of the tert-butyl ester 10b avoided the
loss of material due to water solubility that occurred with
the saponification of the ethyl ester 10a, and generally gave
higher yields of the pyrimidine carboxylic acids 11. The
piperidino-piperazine cores 12, prepared as described pre-
viously,3 were then coupled to the newly made pyrimidine
carboxylic acids under standard conditions (Scheme 2).
Deletion of one or both the methyl groups from the
nicotinamide-N-oxide moiety in 1 resulted in loss of
binding at CCR5 (Ki >30 nM). Based on our published
SAR, we decided to keep the two substituents flanking
the carbonyl group as methyl groups or chlorine
atoms.6 The requisite isonicotinic acids 4 and 6 and
their corresponding N-oxides 5 and 7 were prepared as
described in Scheme 1.7 Coupling of the acids 4–7 with
the piperazino-piperidine 12 (R=CF3; see Scheme 2)
under standard conditions (EDCI/HOBt/CH2Cl2/Et3N;
Several core amines 12 were capped with the parent
4,6-dimethyl pyrimidine-5-carboxamide (e.g., 13–16).
While many para-substituents (R) were tolerated, the
p-CF3 derivative (16) had optimal binding at CCR5
(Ki=3 nM), slightly reduced affinity for the muscarinic
receptors (Ki: M1=575; M2=456 nM) compared to 1
and very good oral absorption as well (Table 2). Here
Figure 3. Rotamers of compound 1.

S. W. McCombie et al. / Bioorg. Med. Chem. Lett. 13 (2003) 567–571
569
Table 1. Data for isonicotinamides and their N-oxides8
again, deletion of the methyl group(s) on the pyrimidine
ring diminished activity as did N-oxidation (N-oxide of
16 is not a significant metabolite, unlike 1 is of 2).
As expected, compound 16 exhibits only the two rota-
mers characteristic of a tertiary amide due to hindered
rotation about the N–CO bond. The atropisomers aris-
ing from hindered rotation about the CO–pyrimidine
ring are now degenerate and are not observable on
chiral HPLC (Fig. 4). The amide rotamers seen on
HPLC12 can be isolated and equilibrate rapidly under
physiological conditions (<2 h at 37 ꢁC).
Ar
Ki (nM)a
CCR5
HIV Entryb
IC50 (nM)
Rat PKc
M2
AUC0ꢂ6h
(ng/mlꢃh)
0.7
2031
0.3
450
In keeping with the design of C2 symmetric amides, we
then turned our attention to the synthesis of 2-sub-
stituted-4,6-dimethyl pyrimidine-5-carboxamides. The
requisite acids 11 (X ¼ H) were prepared by employing
the appropriate amidines as shown in Scheme 2 and
coupled to several piperazino-piperidines in the usual
manner. Table 3 summarizes data for selected pyr-
imidine carboxamides of general structure 17.
2.7
5.23
18
348
0.45
ND
0.90
1810
ND
1678
255
6760
aKi (nM).6
bIC50 (nM) for inhibiting HIV (ADA) entry into cells.9
cAUC0ꢂ6h (ng h/mL; oral dose=10 mg/kg).10
Table 2. Data for parent pyrimidine carboxamides8
Compd
R
Ki (nM)a
Entryb (nM)
Rat PKc
CCR5
M2
AUC0ꢂ6 h
13
14
15
16
I
CN
CF3O
CF3
11
20
11
3
169
164
68
ND
1.8
1.2
0.5
ND
6740
8100
6210
456
aKi (nM).6
bIC50 (nM) for inhibiting HIV (ADA) entry into cells.9
cAUC (ng h/mL; oral dose=10 mg/kg).10
Scheme 2. Synthesis of pyrimidine carboxamides.
Table 3. Data for 2-substituted pyrimidine amides (17)8
Compd
X
Ki (nM)a
CCR5 M2
Entryb (nM)
Rat PKc
AUC0ꢂ6 h
16
18
19
20
21
22
H
CH3
CF3
Ph
CH3O
NH2
3
10
10
12
11
38
456
985
613
1225
202
1100
0.5
1.45
0.1
1.8
ND
ND
6210
5520
1970
1290
1970
ND
aKi (nM).6
bIC50 (nM) for inhibiting HIV (ADA) entry into cells.9
cAUC (ng h/mL; oral dose=10mg/kg).10
Figure 4. HPLC chromatogram of compound 16.

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S. W. McCombie et al. / Bioorg. Med. Chem. Lett. 13 (2003) 567–571
the blood brain barrier (AUCbrain/AUCplasma=1.7). The
lower blood levels after oral administration in rats and
monkeys shown by the pyrimidine carboxamide 16
compared to the nicotinamide N-oxide 1,3 coupled with
the modest improvement in the CCR5/M2 ratio (152 vs
35) helped 16 to show statistically significant improve-
ment over 1 in its ancillary pharmacological profile. The
mean BP, heart rate and ECG in rats were clean after
oral administration of 16 up to a dose of 30mg/kg and
there were no negative effects on CNS related behavior at
the lowest tested dose of 3 mg/kg. However, some effects
at higher doses were noted in the gastrointestinal empty-
ing and transit experiments, implicating the effect of 16 on
the muscarinic receptors. Overall, although the new pyr-
imidine carboxamide 16 was a significant improvement
over the nicotinamide-N-oxide 1, its ancillary pharmacol-
ogy profile was not as favorable as the leading compound
Sch-351125 (Sch-C) from the related but distinct piper-
idino-piperidine series.14 The oximino-piperidinio-piper-
idine Sch-351125 cleared the hurdle of toxicology and was
advanced to the clinic as a possible treatment for HIV
infection. Our studies to further improve the CCR5/Mus-
carinic receptor selectivity as well as the broad-spectrum
potency of structures like 16 while retaining their desirable
pharmacokinetics will be reported in due course.
The 2-substituent (X) appeared to affect binding at both
CCR5 and M2 receptors (e.g., 18, 20, and 22) initially.
However, this effect turned out to be very inconsistent.
As can be seen from Table 3, none of the 2-substituted-
4,6-dimethyl pyrimidine-5-carboxamides improved the
overall profile of the parent compound 16.13 Hence, 16
was selected for complete antiviral, metabolic and
pharmacokinetic characterization (Tables 4 and 5).3,8
In summary, the symmetrical 4,5-dimethyl-5-pyrimidine
carboxamide 16 eliminated the pair of rotamers
observed due to very slow rotation about the CO-het-
eroaryl ring in compounds such as 1. The only two
rotamers seen for 16 are characteristic of all tertiary
amides and equilibrate to a steady state (1:1) fairly
rapidly. Compound 16 satisfied our criteria for strong
binding to CCR5 and inhibition of HIV entry as well as
very good oral absorption in rodents and primates. It
also inhibited a small panel of HIV isolates of U.S ori-
gin, although it was less effective against clades from
other geographical areas. There was neither inhibition
nor induction of liver enzymes with this compound. The
compound was shown to be a true antagonist of the
CCR5 receptor in RANTES induced calcium flux assay.9
Compounds 16, 2 and 1 inhibited the MIP-1b (0.1 nM)
induced migration of recombinant mouse pro-B cell line
Ba/F3 that expresses human CCR5 with IC50 values of
0.01, 0.03 and 0.1 nM, respectively.9 Compound 16 is
92% protein bound (1 is 84% protein bound) and crosses
Acknowledgements
We thank Dr. Jean Lachowitz (CNS Pharmacology) for
the muscarinic antagonist data and Drs. William
Greenlee and John Clader for many useful discussions.
Drs. Ashit Ganguly, John Piwinski and Gregory Reyes
are thanked for their support and encouragement.
Thanks are also due to our colleagues in the depart-
ments of Physical and Analytical Chemistry and Dis-
covery Support for their help in the characterization of
the key compounds described herein.
Table 4. Pharmacokinetic profile of compound 16
Species
iv administrationa
Oral administrationb
AUCd
T1/2 (h)
Cmax
AUC
F (%)e
c
References and Notes
Rat
Monkey
22,700
4710
22
8
1490
340
22,000
2370
97
50
1. McCombie, S.; Vice, S.; Tagat, J.; Lin, S.-I.; Palani, A.;
Neustadt, B.; Baroudy, B.; Strizki, J.; Endres, M.; Cox, K.;
Dan, N.; Chou, C.-C. Abstracts of Papers, 223rd ACS
National Meeting, Orlando, FL, April 7–11, 2002; American
Chemical Society: Washington, DC, 2002; MEDI 159.
2. (a) Shiraishi, M.; Aramaki, Y.; Imoto, H.; Nishikawa, Y.;
Kanzaki, N.; Okamoto, M.; Sawada, H.; Nishimura, O.;
Baba, M.; Fujino, M. J. Med. Chem. 2000, 43, 2049. (b)
Lynch, C. L.; Gentry, A. L.; Hale, J. J.; Mills, S. G.; MacCoss,
M.; Malkowitz, L.; Springer, M. S.; Gould, S. L.; Demartino,
J. A.; Siciliano, S. J.; Cascieri, M. A.; Doss, G.; Carella, A.;
Carver, G.; Holmes, K.; Schlief, W. A.; Danzeisen, R.;
Hazuda, D.; Kessler, J.; Lineberger, J.; Miller, M.; Emini,
E. A. Bioorg. Med. Chem. Lett. 2002, 12, 677. (c) Maeda, K.;
Yoshimura, K.; Shibayama, S.; Habashita, H.; Tada, H.;
Sagawa, K.; Miyakawa, T.; Aoki, M.; Fukushima, D.; Mit-
suya, H. J. Biol. Chem. 2001, 276, 35194.
aiv dose=1 mg/kg.
bOral dose=10 mg/kg (rat) and 2 mg/kg (monkey).
cCmax=ng/mL.
dAUC0-24 h (ng/mL h).
eBioavailability (%F). See ref 3 for details.
Table 5. Inhibition of HIV isolates by 16 in PBMCa
Virus
Clade/Origin
IC50 (nM)
IC90 (nM)
US-1
B/USA
B/USA
B/USA
B/USA
B/USA
B/USA
B/USA
B/USA
G/Nigeria
O/Cameroon
0.7
7
4
17
3
24
ASM 108 (wt)
ASM 108 (hz)
301657 (wt)
301657 (hz)
Bal (hz)
YU-2 (wt)
YU-2 (hz)
JV 1083
0.2
3.2
26.8
6.4
2.3
1.3
166
200
>1000
60
8
3. Tagat, J. R.; Steensma, R. W.; McCombie, S. W.; Nazar-
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.
100
>300
>1000
BCF01
aPeripheral blood mononuclear cells. See ref 9.
4. (a) For other reports on the observation of rotamers, see:

S. W. McCombie et al. / Bioorg. Med. Chem. Lett. 13 (2003) 567–571
571
Bastiaansen, L. A. M.; Kanters, J. A.; van der Steen, F. H.; de
Graaf, J. A. C.; Buck, H. M. J. Chem. Soc., Chem. Commun.
1986, 536. (b) Ikeura, Y.; Ishichi, Y.; Tanaka, T.; Fujishima,
A.; Murabayashi, M.; Kawada, M.; Ishimaru, T.; Kamo, I.;
Doi, T.; Natsugari, H. J. Med. Chem. 1998, 41, 4232. (c)
Clayden, J.; Johnson, P.; Pink, J. H.; Helliwell, M. J. Org.
Chem. 2000, 65, 7033. (d) Ates, A.; Curran, D. P. J. Am.
Chem. Soc. 2001, 123, 5130.
5. HPLC conditions for comp. 1: Column: YMC pro, C18,
3 mm, 150 ꢃ 4.6 mm ID. Mobile phase: 30% ACN: 70%
25 mM potassium phosphate buffer, pH 6.4+20 mM b-cyclo-
dextrin. Flow rate: 1.0 mL/min. Detection: 220 nm. Injection
vol.: 10 uL. Sample concn: ꢀ0.3 mg/mL in water. Column
temperature: 20 ꢁC.
than 0.5 log. The differences between the observed IC50 values
in the Entry assay and the Ki values in the binding assay may
reflect the difference between the ligand (RANTES versus
HIV) and the target (cell membrane versus live U-87 cells).
9. For details of all the assays used in this study, see:
Strizki, J. M.; Xu, S.; Wagner, N. E.; Wojcik, L.; Liu, J.;
Endres, M.; Palani, A.; Shapiro, S.; Clader, J. W.; Green-
lee, W. J.; Tagat, J. R.; McCombie, S.; Cox, K.; Fawzi,
A. B.; Chou, C.-C.; Pugliese-Sivo, C.; Davies, L.; Moreno,
M. E.; Ho, D. D.; Trkola, A.; Stoddart, C. A.; Moore,
J. P.; Reyes, G. R.; Baroudy, B. M. Proc. Nat. Acad. Sci.
U.S.A. 2001, 98, 12718.
10. Cox, K. A.; Dunn-Meynell, K.; Korfmacher, W. A.;
Broske, L.; Nomeir, A. A.; Lin, C. C.; Cayen, M. N.; Barr,
W. H. Drug Discov. Today 1999, 4, 232.
11. Brederecek, H.; Gompper, R.; Herlinger, H. Chem. Ber.
1958, 91, 2832.
6. Tagat, J. R.; McCombie, S. W.; Steensma, R. W.; Lin, S.-I.;
Nazareno, D. V.; Baroudy, B.; Vantuno, N.; Xu, S.; Liu, J.
Bioorg. Med. Chem. Lett. 2001, 11, 2143.
7. (a) Gosl, R.; Meuwsen, A. Chem. Ber. 1959, 92, 2521. (b)
Okamoto, T.; Hirobe, M.; Ohsawa, A. Chem. Pharm. Bull.
1966, 14, 518. (c) Cale, A. D., Jr.; Gero, T. W.; Walker, K. R.;
Lo, Y. S.; Welstead, W. J., Jr.; Jacques, L. W.; Johnson, A. F.;
Leonard, C. A.; Nolan, J. C.; Johnson, D. N. J. Med. Chem.
1989, 32, 2178.
8. For the CCR5-RANTES binding assay, the standard error
was 10% and variability was 2- to 3-fold from assay to assay.
For details of the muscarinic receptor binding assay, see:
Lachowicz, J. E.; Lowe, D.; Duffy, R. A.; Ruperto, V.; Taylor,
L. A.; Guzik, H.; Brown, J.; Berger, J. G.; Tice, M.; McQuade,
R.; Kozlowski, J.; Clader, J.; Strader, C. D.; Murgolo, N. Life
Sci. 1999, 64, 535. The viral Entry assay refers to a single-cycle
HIV replication assay adapted from: Connor, R. I.; Chen,
B. K.; Choe, S.; Landau, N. R. Virology 1995, 206, 935 Typi-
cally, the 95% confidence limit for the IC50 values for inhibit-
ing HIV entry was within 1 log and the R2 variance values
between 0.9 and 1.0. Intra-experimental IC50 values varied less
12. HPLC Conditions for compound 16: Column: Chiralcel
OD-R, 25 cm ꢃ 4.6 mm ID. Mobile phase: 40:10:50, ACN:
IPA: 50 mM potassium phosphate buffer, pH 7.8. Flow rate:
0.6 mL/min. Detection: 220 nm. Injection vol.: 10 uL. Column
temp: 10 ꢁC.
13. All new compounds reported herein were fully character-
ized by NMR, MS and HRMS. Spectral data for the key
compound 16: 1H NMR (300 MHz, CDCl3) d 0.93 (s, 3H),
1.14 (d, J=6 Hz, 3H), 1.16–1.27 (m, 1H), 1.28 (d, J=6 Hz,
3H), 1.4 (m, 1H), 1.8 (br-t, J=9 Hz, 1H), 2.0 (br-t, J=9 Hz,
1H), 2.3–2.44 (m, 5H), 2.45 (s, 3H), 2.48 (s, 3H), 2.5–2.65 (m,
1H), 3.0 (m, 2H), 3.4 (dd, J=14, 7 Hz, 2H), 4.0 (br-s, 1H), 4.22
(br-d, J=9 Hz, 1H), 7.53 (dd, J=7, 3 Hz, 4H), 8.94 (s, 1H).
FABMS (MH+): 504 (100%). HRMS calcd for C27H37N5OF3
(MH+): 504.2937; Found 504.2950.
14. Palani, A.; Shapiro, S.; Clader, J. W.; Greenlee, W. J.;
Cox, K.; Strizki, J.; Endres, M.; Baroudy, B. M. J. Med.
Chem. 2001, 44, 3339.