Spirodiketopiperazine-based CCR5 antagonists: Lead optimization from biologically active metabolite

Article abstract of DOI:10.1016/j.bmcl.2006.10.084

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from β-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1α to h

Full text of DOI:10.1016/j.bmcl.2006.10.084

Bioorganic & Medicinal Chemistry Letters 17 (2007) 727–731
Spirodiketopiperazine-based CCR5 antagonists: Lead optimization
from biologically active metabolite
a,
a
a
a
*
Rena Nishizawa, Toshihiko Nishiyama, Katsuya Hisaichi, Naoki Matsunaga,
a
a
a
a
Chiaki Minamoto, Hiromu Habashita, Yoshikazu Takaoka, Masaaki Toda,
b
b
b
b
Shiro Shibayama, Hideaki Tada, Kenji Sagawa, Daikichi Fukushima,
c
c,d
Kenji Maeda and Hiroaki Mitsuya
a
Medicinal Chemistry Research Laboratories, Ono Pharmaceutical Co., Ltd, Osaka 618-8585, Japan
b
Exploratory Research Laboratories, Ono Pharmaceutical Co., Ltd, Ibaraki 300-424, Japan
Department of Internal Medicine II, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
c
d
Experimental Retrovirology Section, HIV & AIDS Malignancy Branch, NCI, National Institutes of Health,
Bethesda, Maryland 20892, USA
Received 17 August 2006; revised 23 October 2006; accepted 26 October 2006
Available online 1 November 2006
Abstract—Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds
derived from b-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of
MIP-1a to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.
Ó 2006 Elsevier Ltd. All rights reserved.
Millions of people in the world are still suffering from ac-
1
quired immune deficiency syndrome (AIDS). Although
G protein-coupled receptors (GPCRs), which greater
than 30% of all known marketed medicines modulate
5
the highly active antiretroviral therapy (HAART), a
cocktail of protease and reverse transcriptase inhibitors,
has been useful for many patients, several issues still
remain for anti-HIV therapy: a gradual spread of drug-
resistant strains, severe adverse effects, expensive thera-
the function of. After these reports, many pharmaceu-
tical companies and academic institutions have been
enthusiastically investigating novel antagonists against
6
CCR5 with suitable pharmaceutical properties.
2
peutic cost, etc. These issues require new anti-HIV drugs
to have a different mode of action from conventional
drugs.
We previously reported the identification of several spi-
rodiketopiperazine derivatives, for example, 1 (Fig. 1),
from a combinatorial library targeting chemokine recep-
7
tors. Compound 1 not only selectively inhibited the
Agents inhibiting HIV entry into target cells are one of
3
the most promising approaches to treat AIDS. A num-
binding of macrophage inflammatory protein (MIP)-1a
to human CCR5 receptor, but also potently blocked
the infectivity and replication of laboratory and clinical
strains of HIV as well as those of highly drug-resistant
ber of potential sites for therapeutic intervention
become accessible during the narrow window between
virus attachment and the subsequent fusion of viral
envelope with the cell membrane. In 1996, it was
revealed that one of the C–C chemokine receptor 5
8
HIV variants with minimal cytotoxicity. Although
(
and that the endogenous ligand showed anti-HIV-1
CCR5) is utilized by HIV-1 as an essential co-receptor
4
activity in vitro. CCR5 belongs to the superfamily of
Keywords: CCR5; HIV-1; Active metabolite.
*
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962
314; e-mail: r.nishizawa@ono.co.jp
9
Figure 1. The structure of lead compound 1.
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.084

728
R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 727–731
O
N
OH
(b)
NH
O
N
O
OH
a)
OH
(c)
(
Unchanged
form (1)
(b)(c)
(a)
Figure 2. Metabolites of the compound 1. Three main metabolites were identified on LC-MS after incubation with human liver microsome (5.0 mg
protein/ml) for 1.5 h.
7
compound 1 showed potent activity in vitro, oxidative
metabolism in liver microsomes resulted in low bioavail-
ability in rodents.
aldehyde by the reported solid-phase synthesis. The
compounds 9–11 and 13 were synthesized from the
corresponding amino acid derivatives by the identical
procedure to the synthesis of compound 4 shown in
Scheme 1. The mixture of 1-benzylpiperidone, butyl-
amine, N-Boc-b-hydroxy-D-leucine, and 2-(4-morpholi-
After the incubation with human liver microsomes,
metabolites of 1 were purified by HPLC. The three ma-
jor isolated metabolites (a, b, c in Fig. 2) were analyzed
1
0
nyl)ethylisocyanide
in methanol was stirred at
1
11
by LC-MS and H NMR, and found to be compounds
9
hydroxylated on the n-butyl and/or the i-butyl group.
55 °C. The enantiomerically pure b-hydroxy-a-amino
acids were prepared according to the reported method
through Sharpless asymmetric epoxidation from the cor-
Additionally, we evaluated the in vitro antagonistic
activity of each isolated metabolite. Fortunately, the
samples of the peaks (b) and (c) showed significant
antagonistic activities (data not shown). This informa-
tion prompted us to try the introduction of hydroxyl
group on side chains to improve the in vitro activity as
well as pharmaceutical properties. Herein, we describe
the preliminary structure–activity relationship (SAR)
of the hydroxylated-spirodiketopiperazines and the
unexpected improvement on the activities, especially
in vitro anti-HIV activities.
1
2
responding allyl alcohol. The Boc protecting group of
amino acid was removed by the treatment of concentrat-
ed HCl without isolation of the Ugi product. Cyclization
of the obtained crude product by heating in toluene in
the presence of acetic acid at 80 °C followed by the
removal of the benzyl group by catalytic hydrogenation
afforded the cyclized spirodiketopiperazine, and com-
pound 3 was isolated as a HCl salt in acceptable yield.
Reductive alkylation of compound 3 resulted in desired
product 4 in high yields.
The compounds 1, 5, 7, 8, and 12 were synthesized from
the N-alloc-4-piperidone, the corresponding amine, the
corresponding N-Boc-amino acid, and 4-phenoxybenz-
The compounds listed in Tables 1–3 were evaluated for
their inhibitory activities against calcium mobilization
of human CCR5 overexpressed CHO cell (hCCR5/
O
HO
OH
+
(a) - (c)
O
Ph
+
Ph
N
OH
N
O
+
+
C N
Boc N
H
N
NH2
N
N
O
H
O
2
O
(
d)
(
e)
O
O
N
OH
N
OH
HN
N
N
N
O
H
O
H
HCl
HCl
3
4
Scheme 1. Typical synthetic route for spirodiketopiperazines. Reagents and condition: (a) MeOH, 55 °C; (b) concd HCl, 55 °C; (c) AcOH/toluene,
8
0 °C; (d) H
2
, Pd(OH)
2
3
/C, EtOH, 55 °C then 4 N HCl/AcOEt (60–70% in four steps); (e) 4-phenoxybenzaldehyde, NaBH(OAc) , AcOH, DMF then
4
N HCl/AcOEt (80%).

R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 727–731
Table 1. Activity of the compounds 5 and 6
729
Compound
Structure
IC50
Binding assay (nM)
Ca assay (nM)
O
O
O
1
8.3
94
N
N
HCl
HCl
N
O
H
HO
O
5
6
Not tested
Ca. 10,000
N
N
N
N
O
H
O
O
28
79
N
N
HCl
O
H
OH
Table 2. Activity of the stereoisomers 4 and 7–11
Compound
Structure
IC50
Binding assay (nM)
Ca assay (nM)
O
O
7
8
4
9
1
1
R form
S form
29
130
N
N
N
N
N
N
N
HCl
N
O
H
O
O
O
O
O
O
11
3.5
24
68
53
84
N
N
HCl
HCl
HCl
O
H
O
0
(3R,1 R) form
33
N
OH
1'
N 3
H
O
O
0
(3R,1 S) form
210
400
150
N
OH
N
H
O
O
0
0 (3S,1 R) form
N
OH
OH
N
H
HCl
HCl
O
O
0
1 (3S,1 S) form
N
N
H
O
CHO) stimulated by MIP-1a (Ca assay) and for their
The compound 5, the proposed structure of metabolite
(a), was synthesized from the 3-hydroxy-1-butylamine
and N-Boc-leucine as a mixture containing the same
1
25
CCR5 binding affinity by the inhibition of I-MIP-1a
binding to hCCR5/CHO (binding assay).
8
,13

730
R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 727–731
Table 3. Antagonistic activity and anti-HIV activity in compounds 12 and 13
Compound Structure
Binding assay IC50 (nM) Ca assay IC50 (nM) Anti-HIV IC50 (nM) MAGI assay IC50 (nM)
O
O
1
2
3
N
6.1
1.1
28
53
31
337
6.0
N
N
HCl
O
H
O
O
1
N
OH
0.6
N
N
HCl
O
H
quantity of all possible four isomers. The compound 6,
the proposed structure of metabolite (b), was prepared
from the N-Boc-aspartic acid derivative in racemic
form. Compounds 5 and 6 were evaluated for their
activities and the results are summarized in Table 1.
Whereas the compound 5 showed significant decrease
of activity in calcium mobilization assay, the compound
group is still unclear, two hypotheses have been made
to explain the increase in activity. One is the formation
of a new hydrogen bond between the hydroxyl group
and CCR5. The other is restricting the conformation of
the compound to favorably orient the side chains.
Further investigations are in progress.
1
4
6
1
showed a comparable activity to the parent compound
1
Introduction of the hydroxyl group could not improve
the bioavailability in rodent. However, the introduction
of a hydrophilic moiety on the molecule showed some
favorable pharmaceutical properties. Further optimi-
zation of these compounds to improve their oral absorp-
tion and metabolic stability which are necessary to
provide CCR5 antagonists suitable for clinical use will
be discussed in our future reports.
5
.
1
7
Since the proposed structure of metabolite (c) had two
chiral centers, we synthesized all four possible enantio-
mers 4 and 9–11 in optically pure form from the corre-
sponding b-hydroxylated-leucine to evaluate their
biological activities (Table 2). Whereas there was no
remarkable difference between the activities of the two
enantiomers, 7 and 8, in lead compound 1, there was a
significant difference among the hydroxylated stereoiso-
References and notes
0
mers (4 versus 9–11). Compound 4 having 3R,1 R-config-
uration exhibited approximately 10-fold more potency
than the other isomers 9–11. This result indicates that
introducing hydroxyl group on the side chain at the 3-po-
sition of diketopiperazine ring could lead to a significant
improvement in the interaction with the receptor. Based
on the observation of this unexpectedly improved antag-
onistic activity of compound 4, we applied this informa-
tion to compound 12 which showed more potent anti-
1. AIDS Epidemic Update 2005 reported by the Joint United
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HIV activity than compound 1. It was found that the
0
(
3R,1 R)-hydroxyl compound 13 exhibited strong inhibi-
tory activities in both binding and Ca assays. Further-
more, investigating anti-HIV activity in the next step,
compound 13 showed 6 nM of IC value in anti-HIV as-
9
0
+
say (CCR5 MAGI cell anti-infectivity single cycle assay
a
8
versus the BAL strain of HIV, ), which was 50-fold
1
stronger than non-hydroxyl analogue 12.
6
5
. Muller, G. Drug Discov. Today 2003, 8, 681.
6
. (a) Kazmierski, W.; Bifulco, N.; Yang, H.; Boone, L.;
DeAnda, F.; Watson, C.; Kenakin, T. Bioorg. Med. Chem.
2003, 11, 2663; (b) Maeda, K.; Nakata, H.; Ogata, H.;
Koh, Y.; Miyakawa, T.; Mitsuya, H. Curr. Opin. Phar-
macol. 2004, 4, 447; (c) Palani, A.; Tagat, J. R. J. Med.
Chem. 2006, 49, 2851.
. Habashita, H.; Kokubo, M.; Hamano, S.; Hamanaka, N.;
Toda, M.; Shibayama, S.; Tada, H.; Sagawa, K.; Fuku-
shima, D.; Maeda, K.; Mitsuya, H. J. Med. Chem. 2006,
We also evaluated the oral bioavailability of compounds
and 13 in rat. Unfortunately, the bioavailability of
both compounds was less than 1% (data not shown).
Further assessment and optimization are required to
identify promising clinical candidates with acceptable
pharmaceutical profile.
4
7
In conclusion, using metabolite data of lead compound
1, we discovered the excellent enhancement on the activ-
ity in binding and anti-HIV assays by the introduction of
4
9, 4140.
8
. (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,
a b-hydroxyl group. Although the role of the hydroxyl

R. Nishizawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 727–731
731
3
5194; (b) Maeda, K.; Nakata, H.; Koh, Y.; Miyakawa,
13. All IC50 values represent the average of two or more
determinations, and the standard deviations were no
greater than 30% from the mean.
T.; Ogata, H.; Takaoka, Y.; Shibayama, S.; Sagawa, K.;
Fukushima, D.; Moravek, J.; Koyanagi, Y.; Mitsuya, H.
J. Virol. 2004, 78, 8654.
14. Compound 6 was prepared by the reaction of methylmag-
nesium bromide with the benzyl ester derivative, which
was obtained from the N-alloc-piperidone, butylamine, N-
Boc-c-benzyl-aspartic acid, and 4-phenoxybenzaldehyde
according to the solid-phase synthesis reported in Ref. 7.
15. Compounds described herein showed selective CCR5
antagonistic activities over other chemokine receptors,
for example, CCR1 and CXCR4.
16. Lack of correlation between anti-chemokine activity and
anti-HIV activity has been reported by other researchers.
See Ref. (a) Shankaran, K.; Donnelly, K. L.; Shah, S. K.;
Guthikonda, R. N.; MacCoss, M.; Mills, S. G.; Gould, S.
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Bioorg. Med. Chem. Lett. 2004, 14, 3419; (b) Wood, A.;
Armour, D. Prog. Med. Chem. 2005, 43, 239.
1
9
.
H NMR spectra of metabolites (a) and (c) indicated to be
a single compound, respectively. However, it was difficult
to determine the stereochemistry at each asymmetric
center. As for the metabolite (c), the H NMR data were
identical to those of the compounds 4 and 11, which
indicates that this metabolite has anti-configuration.
1
1
0. We initially used several isocyanides, for example, benzy-
lisocyanide, for this reaction to yield the desired product.
However, most of isocyanides were difficult to be handled
because of the offensive odor. It was found that a commer-
cially available isocyanide, 2-(4-morpholinyl)ethylisocya-
nide, is not malodorous, and we decided to employ this
reagent for the further derivatization.
1. Domling, A.; Ugi, I. Angew. Chem. Int. Ed. 2000, 39, 3168.
2. (a) Caldwell, C. G.; Bondy, S. S. Synthesis 1990, 34; (b)
Nagamitsu, T.; Sunazuka, T.; Tanaka, H.; Omura, S.;
Sprengeler, P. A.; Smith, A. B., III J. Am. Chem. Soc.
1
1
17. For example, whereas compound 12 inhibited CYP 3A4 at
9.3 lM of IC , compound 13 did not show any significant
1
996, 118, 3584, Compound 13 was synthesized from the
5
0
b-hydroxy-cyclohexylalanine, which was obtained from
the 3-cyclohexyl-allyl alcohol by an identical procedure to
that reported in Ref. 12a.
inhibition at 30 lM. Furthermore, compound 13 showed
an improved aqueous solubility (pH 6.8) compared to
compound 12 (12, <0.2 lg/ml; 13, 2.7 lg/ml).