Spirodiketopiperazine-based CCR5 antagonists: Improvement of their pharmacokinetic profiles

Article abstract of DOI:10.1016/j.bmcl.2009.11.018

Spirodiketopiperazine-based CCR5 antagonists, showing improved pharmacokinetic profiles without reduction in antagonist activity, were designed and synthesized. We also demonstrate the anti-HIV activity of a representative compound 12, as measured in a p24 assay.

Full text of DOI:10.1016/j.bmcl.2009.11.018

Bioorganic & Medicinal Chemistry Letters 20 (2010) 763–766
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spirodiketopiperazine-based CCR5 antagonists: Improvement of their
pharmacokinetic profiles
a
a
a
a
Rena Nishizawa a, , Toshihiko Nishiyama , Katsuya Hisaichi , Keisuke Hirai , Hiromu Habashita ,
*
Yoshikazu Takaoka c, Hideaki Tada b, Kenji Sagawa d, Shiro Shibayama b, Kenji Maeda e,f
Hiroaki Mitsuya e,f, Hisao Nakai a, Daikichi Fukushima a, Masaaki Toda a
,
a Minase Research Institute, Ono Pharmaceutical Co., Ltd, Shimamoto, Mishima, Osaka 618-8585, Japan
b Tsukuba Research Institute, Ono Pharmaceutical Co., Ltd, Ibaraki 300-424, Japan
c Fukui Research Institute, Ono Pharmaceutical Co., Ltd, Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
d Ono Pharmaceutical Co., Ltd, Kyutaro, Chuoh, Osaka 541-0056, Japan
e Department of Internal Medicine II, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
f Experimental Retrovirology Section, HIV & AIDS Malignancy Branch, NCI, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Spirodiketopiperazine-based CCR5 antagonists, showing improved pharmacokinetic profiles without
reduction in antagonist activity, were designed and synthesized. We also demonstrate the anti-HIV activ-
ity of a representative compound 12, as measured in a p24 assay.
Received 26 September 2009
Revised 4 November 2009
Accepted 6 November 2009
Available online 12 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CCR5
Chemokine
Anti HIV-1
Chemokines, which were initially identified as chemoattrac-
tants in the context of leukocyte trafficking to sites of inflamma-
tion, exert a variety of biological activities by binding to their
receptors on the surface of specific cells. Chemokines are a large
family of small cytokines that selectively control the adhesion, che-
motaxis, and activation of various leukocyte populations and are
known to be involved in the initiation and progress of inflamma-
tion and allergic disease.1 They are classified into two main
groups—CC chemokines and CXC chemokines—based on their con-
served N-terminal cysteine residues. Additionally, CC chemokine
receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) have at-
tracted substantial interest because they form portals of cellular
entry for human immunodeficiency viruses (HIV-1 and HIV-2)
and related simian or feline retroviruses.2 Thus, identifying the
CCR5 receptor antagonist, which inhibits HIV from binding to the
specific receptor, is one of the most promising approaches for the
treatment of AIDS, especially at the early stage of infection.3 Due
to its novel mode of action, the CCR5 antagonist could be one of
the final agents utilized in salvage therapy in combination with
other active antiviral agents. Maraviroc is the only approved
CCR5 receptor antagonist on the market for treating HIV-1 infec-
tion.4 In this study, we report the optimization process to find
4-(4-{[(3S)-1-butyl-3-(cyclohexylmethyl)-2,5-dioxo-1,4,9-triaza-
spiro[5.5]undec-9-yl]methyl}phenoxy)benzoic acid hydrochloride
(12) with the aim of improving the antiviral activity and pharma-
cokinetics of the newly found chemical leads 1a and 1b (Fig. 1).
Compounds1a, 1b, 2a, 2b, 3b and 4a weresynthesizedby the pre-
viously reported solid-phase method5a and compounds 6–12 were
synthesized by the solution-phase method described in Scheme 1.
A mixture of 1N-benzylpiperidin-4-one, n-butylamine, N-Boc-ami-
no acid and 2-(morpholin-1-yl)ethylisocyanide in methanol was
stirred at 55 °C.6 The Boc protecting group of an amino acid was re-
moved by treatment with concentrated HCl without isolation of the
Ugi product,6 cyclization of which, by heating in toluene in the pres-
ence of acetic acid at 80 °C, afforded 13. Removal of the benzyl group
of 13 by catalytic hydrogenation produced the cyclized spirodiketo-
piperazine 14, which was isolated as HCl salt in 60–70% yield in four
steps. Reductive alkylation of compound 14 resulted in a good yield
(50–90%) of the desired products. Synthesis of 5a is described in
Scheme 2. N-Alkylation of 14 with a tosylate, which was prepared
by the tosylation of 4-phenoxyphenylethyl alcohol with polymer-
supported tosyl chloride, afforded 5a. These basic compounds were
isolated as their HCl salts.
Compounds listed in Tables 1–4 were evaluated for their
inhibitory activities against calcium mobilization of human CCR5
* Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: r.nishizawa@ono.co.jp (R. Nishizawa).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.018

764
R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 763–766
*
1a X = H (3RS), R =
CH3
X
O
*
1b X = H (3RS), R =
CH3
O
H3C
N1
5
8
2
7
9
3
6
N
R
*
4
N
H
12 X = CO H (3S), R =
10
11
HCl
2
O
Figure 1. Discovery of an orally active chemical lead.
CH3
O
C
N+
CH3
HO
O
Ph
a - c
N
O
N
Ph
+
+
+
N
Boc
N
N
H
O
NH2
N
H
O
13
CH3
CH3
O
O
d
e
N
Ar
N
HN
HCl
N
N
H
N
HCl
H
O
O
6-12
14
Scheme 1. Typical synthetic route of spirodiketopiperazines. Reagents and conditions: (a) MeOH, 55 °C; (b) concd HCl, 55 °C; (c) AcOH, toluene, 80 °C; (d) H2, Pd(OH)2/C,
EtOH, 55 °C then 4 N HCl/AcOEt (60–70% in four steps); (e) Ar-CHO, NaBH(OAc)3, AcOH, DMF, then 4 N HCl/AcOEt (50–90%).
CH3
OH
O
HCl
a, b
O
N
N
O
N
H
O
5a
Scheme 2. Synthesis of 5a. Reagents and conditions: (a) polystyrene-supported tosylchloride, pyridine, CH2Cl2; (b) 14, iPr2NEt, MeCN, then 4 N HCl/AcOEt (48% in two steps).
overexpressed CHO cells (hCCR5/CHO) stimulated by MIP-1
a
As shown in Table 2, reductive amination of 14 with commer-
cially available benzaldehyde and p-methoxybenzaldehyde
provided 3b and 4a, respectively. Although reduction of the molec-
ular weight was expected to be a promising strategy for improving
oral absorption, this approach resulted in reduced activity.
N-Alkylation of 14 with 4-phenoxyphenylethyltosylate afforded
5a, also with remarkable reduction in activity relative to 1a. As a
result, the 9-N-phenylmethyl group was found to be one of the
structural requirements for antagonist activity. Introduction of a
p-phenoxy substituent into the 9-N-phenylmethyl group of 3b
tended to show increased activity, as illustrated by the increased
potency of 1b relative to 3b.
As shown in Table 3, the effect of chemical modification of the
linker X on activity profiles was investigated. Replacement of the
9-N-4-phenoxyphenylmethyl moiety of 1a with 9-N-4-phenylthio-
phenylmethyl afforded a diphenyl sulfide analogue 6 with less
potent activity. Replacement of the sulfide moiety of 6 with sulfone
(Ca assay).7
We previously reported the discovery of spirodiketopiperazines
1a, 1b, 2a, and 2b (Table 1), as the novel chemical leads of CCR5
antagonist from our combinatorial library targeting G-protein cou-
pled receptors (GPCRs).5 Analogues 1a and 2a tended to show
stronger activity compared with the corresponding 3-isobutyl ana-
logues 1b and 2b, respectively. Although 1a, 1b, 2a, and 2b showed
from potent to moderate antagonist activity in vitro, they were not
expected to display potent activity in vivo because of their unfa-
vorable PK data, including poor area under the concentration-time
curve (AUC), high clearance (CL) and distribution values (Vss), as
shown in Table 5. Accordingly, structural optimization was focused
not only on increased activity but also on the improvement of
these PK values. Our optimization process was initiated with
chemical modification of the N-substituents at the 9-position using
readily available spirodiketopiperazine 14 as a key intermediate.

R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 763–766
765
Table 1
Table 3
Hits from the newly designed G-protein coupled receptor (GPCR) directed library
Effect of chemical modification of a linker X on activity profiles
CH3
CH3
X
O
1
O
HCl
R1 9N
*
(3RS)
R2
N
N
(3R and/or 3S)
*
8
7
6
2
a R2 =
3
b R2 =
3
CH3
9
N
5
4
H3C
N
N
HCl
10
11
H
H
O
O
Compds
R1
IC50 (nM) Ca assay
28
Compds R1
C3 configuration (3R and/or 3S) IC50 (nM) Ca assay
*
*
S
1a
6
(3S)
170
86
*
*
O
O
O
O
S
7
(3S)
*
*
*
1b
2a
94
50
O
1a
(3RS)
28
*
O
*
O
O
Table 4
Effect of the p-substituent of the biphenyl ether residue on activity profiles
*
2b
180
O
CH3
X
O
O
Table 2
Effect of the spirodiketopiperazines 9-N-substituents on activity profiles
(3R and/or 3S)
N
3
N
CH3
N
HCl
H
O
O
HCl
9
Compds
X
IC50
(nM) Ca
assay
C3
Stability in rat liver
microsomes% remaining at
15 min
1
*
(3R and/or 3S)
R2
*
N
configuration
(3R and/or 3S)
8
7
6
2
N
a R2=
b R2=
CH3
R1
3
5
H3C
4
N
8
9
10
11
12
1a
Me–
MeO–
HO–
F–
HO2C– 13
H– 28
79
130
42
(3S)
(3S)
(3S)
(3S)
(3S)
(3RS)
48
34
22
53
22
33
10
11
H
O
Compds R1
C3 configuration IC50 (nM)
92
(3R and/or 3S)
Ca assay
*
3b
4a
(3RS)
900
*
the terminal phenyl moiety of 1a afforded 4-(p-methylphen-
oxy)phenylmethyl and 4-(p-methoxyphenoxy)phenylmethyl ana-
logues 8 and 9, respectively with a tendency of showing slightly
less potent activity relative to 1a, while demethylation of 9 affor-
ded an analogue 10 with a tendency of showing slightly more po-
tent activity relative to the corresponding methoxy analogue 9.
Introduction of a p-fluoro group as an electron-withdrawing group
instead of p-methyl and p-methoxy groups afforded 11 with a
slightly less potent activity relative to 1a. Introduction of a carbox-
ylic acid group as an electron-withdrawing and hydrophilic substi-
tuent as a p-substituent afforded 12 with an increased activity.
Stability of these test compounds in the rat liver microsomes was
investigated but these in vitro data did not indicate a significant
improvement in metabolic stability in rat liver microsomes.9
PK data obtained after single-dose oral administration of the
initial chemical lead 1a and the representative compound 12 to
rats are presented in Table 5. As described previously, the initial
lead 1a showed very poor bioavailability (1.9%). Other PK values
such as the maximum plasma concentration (Cmax), plasma elimi-
nation half-life (T1/2) and AUC were also very poor. The probable
(3RS)
120
H3C
O
1a
1b
(3RS)
(3RS)
28
94
*
O
O
ca.
10,000
5a
(3S)
*
afforded 7, which showed slightly less potent activity relative to
the corresponding ether analogue 1a. For this reason, the following
optimization was focused on the synthetic work of diphenyl ether
derivatives.
Effect of a p-substitution of the predicted metabolic site of the
terminal phenyl moiety of 1a on stability in rat liver microsomes
was investigated.8 As shown in Table 4, introduction of p-methyl
group and p-methoxy group as electron-donating substituents into

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R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 763–766
Table 5
Pharmacokinetic data for compounds 1a and 11 in rats
Compds
30 mg/kg, po
30 mg/kg, iv
T1/2 min
Cmax ng/mL
T1/2 min
AUC ng h/m (0–1)
74.4a
BA%
AUC ng h/mL (0–1)
400
CL mL/min/mL
Vss mL/kg
1a
12
16.7a
7200
103
48.4
1.9
34.1
19.9
11.1
113
16
2542
145
10,532
3091
a
Dose normalized AUC and Cmax to 30 mg/kg.
Cmax, AUC and BA after oral dosing because of its much improved
solubility and Caco2 permeability. However, its oral absorption
process has not yet been elucidated. The significant reduction of
CL and Vss of compound 12 was also considered to be another plau-
sible reason for the increased Cmax, AUC and BA. As such, introduc-
tion of carboxylic acid into the p-position of the terminal phenoxy
moiety was found to be effective not only to block metabolic deac-
tivation but also to improve PK profiles. The representative com-
pound 12 showed more potent activity than 1b in the p24 assay
(with the BAL strain of HIV). Further optimization of compound
12 to improve its activity and PK profile, will be reported in near
future. These findings will contribute further to the development
of CCR5 antagonists for clinical use.
Table 6
Anti-HIV activity of the representative compounds
Compds
Anti-HIV-1 activity in p24 assay
HIV-1Ba-L (R5) IC50 (nM)
1b
12
160
39
60
Zidovudinea
Nelfinavirb
12
a
Zidovudine is reverse transcriptase inhibitor.
Nelfinavir is HIV-1 protease inhibitor.
b
reasons for such poor PK values of 1a were the large clearance
(CL = 113 mL/min/kg) and distribution volumes (Vss = 2542 mL/kg)
which were unfavorable for drugs which show efficacy in the
blood, such as anti-HIV drugs. However, benzoic acid analogue
12 showed significantly improved PK values such as Cmax
(7200 ng/mL), oral exposure (AUC = 10532 ng h/mL) and bioavail-
ability (BA = 34.1%) after its oral dosing. Remarkable improvement
References and notes
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of solubility (26
l
M)9 and Caco2 permeability (26.4 ꢀ 10ꢁ6 cm/s)9
of 12 relative to 1a (solubility: less than 5 lM and Caco2 perme-
ability: not detected) was estimated to be one of the most plausible
reasons. The marked reduction in clearance (CL = 16 mL/min/kg)
and distribution volume (Vss = 145 mL/kg) after intravenous dosing
was considered to be another plausible reason for the improved
AUC and BA. The marked reduction of CL of 12 strongly suggested
in vivo metabolic stabilization, although in vitro studies did not
indicate a significant improvement in metabolic stability.
Furthermore the representative compound 12, PK profiles of
which were significantly improved relative to the initial lead 1b
without reduction of the potent antagonist activity, was investi-
gated for its anti-HIV activity using a launched reverse transcrip-
3. Hoffmann, C.; Mulcahy, F. Overview of Antiretroviral Agents. In HIV Medicine
2006; Hoffmann, C., Rockstroh, J. K., Kamps, B. S., Eds.; Flying: Paris, 2006; p 94.
FlyingPublisher.com.
4. Leonard, J. T.; Roy, K. Curr. Med. Chem. 2006, 13, 91.
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tase inhibitor Zidovudine as
a positive control. Results are
6. Domling, A.; Ugi, I. Angew. Chem., Int. Ed. 2000, 39, 3168.
summarized in Table 6. Compound 12 showed an IC50 value of
39 nM in an anti-HIV-1 p24 assay (using PBMC as the target cells7).
In conclusion, starting with the initial hit compounds 1a and 1b
which showed unfavorable PK profiles, we identified compound 12
which showed significant improvement in bioavailability and oral
exposure (AUC) without reduction in activity. Compound 12 was
produced by introducing a carboxylic acid group into the p-posi-
tion of the terminal phenyl moiety. Although the role of carboxylic
acid is still unclear, compound 12 is thought to show improved
7. (a) Maeda, K.; Yoshimura, K.; Shibayama, S.; Habashita, H.; Tada, H.; Sagawa, K.;
Miyakawa, T.; Aoki, M.; Fukushima, D.; Mitsuya, H. J. Biol. Chem. 2001, 276,
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Shibayama, S.; Sagawa, K.; Fukushima, D.; Moravek, J.; Koyanagi, Y.; Mitsuya, H.
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8. Kalfutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.; Callegari, E.; Henne, K. R.;
Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.; Nakai, Y.; O’Donnell, J. P.; Boer, J.; Harriman,
S. P. Curr. Drug Metabol. 2005, 6, 161.
9. Full details of the experimental will be reported very soon in the full paper
which we are preparing.