Design, synthesis, and biological evaluation of the combinatorial library with a new spirodiketopiperazine scaffold. Discovery of novel potent and selective low-molecular-weight CCR5 antagonists

Article abstract of DOI:10.1021/jm060051s

We previously reported the discovery of several spirodiketopiperazine derivatives as potent CCR5 antagonists with anti-HIV activity. Herein, we describe in detail the identification of these lead compounds using a combinatorial chemistry approach. A novel

Full text of DOI:10.1021/jm060051s

4140
J. Med. Chem. 2006, 49, 4140-4152
Design, Synthesis, and Biological Evaluation of the Combinatorial Library with a New
Spirodiketopiperazine Scaffold. Discovery of Novel Potent and Selective Low-Molecular-Weight
CCR5 Antagonists
Hiromu Habashita,*,† Masaya Kokubo,† Shin-ichi Hamano,† Nobuyuki Hamanaka,† Masaaki Toda,† Shiro Shibayama,‡
Hideaki Tada,‡ Kenji Sagawa,‡ Daikichi Fukushima,‡ Kenji Maeda,§ and Hiroaki Mitsuya§,|
Medicinal Chemistry Research Laboratories, Ono Pharmaceutical Co., Ltd., Osaka 618-8585, Japan, Exploratory Research Laboratories,
Ono Pharmaceutical Co., Ltd., Ibaraki 300-4247, Japan, Department of Internal Medicine II, Kumamoto UniVersity School of Medicine,
Kumamoto 860-0811, Japan, and Experimental RetroVirology Section, HIV & AIDS Malignancy Branch, NCI, National Institutes of Health,
Bethesda, Maryland 20892
ReceiVed January 16, 2006
We previously reported the discovery of several spirodiketopiperazine derivatives as potent CCR5 antagonists
with anti-HIV activity. Herein, we describe in detail the identification of these lead compounds using a
combinatorial chemistry approach. A novel spirodiketopiperazine scaffold was designed on the basis of the
concept of the privileged structure of G-protein-coupled receptors (GPCRs). This new framework was obtained
in acceptable yield with high purity from the readily prepared isonitrile resin through the Ugi reaction,
sequential transformations, and cyclative cleavage. By measuring the inhibitory activity of each compound
in the initial library against the intracellular calcium mobilization stimulated by MIP-1R, several compounds
were found to show modest but selective CCR5 antagonistic activity. After the rapid evaluation of these hit
compounds, several single-digit nanomolar, low-molecular-weight CCR5 antagonists that can potently block
the infectivity and replication of laboratory and clinical strains of HIV as well as those of highly drug-
resistant HIV variants with minimal cytotoxicity have been identified.
Introduction
enthusiastically investigating novel antagonists against chemo-
kine receptors with suitable pharmaceutical properties, and
several subtype-selective antagonists are now in clinical trials.9
Although there are now many approaches to discover GPCR
agonists or antagonists, most of the reported leads of chemokine
receptor antagonists were identified by high-throughput screen-
ing from the company’s historical chemical collection.10 Herein,
we describe our approach to identifying subtype-selective
antagonists through the synthesis of newly designed combina-
torial library targeting chemokine receptors and the rapid
evaluation process from hit to lead using iterative focused
libraries to identify several single-digit nanomolar, low-molec-
ular-weight, and selective CCR5 antagonists.
Design of the Spirodiketopiperazine Scaffold. During the
past decade, many reports have appeared describing combina-
torial libraries targeting GPCRs.11 One promising approach to
identify selective small molecule ligands of GPCRs is the use
of privileged structures.12 Among various reported privileged
structures, the spiropiperidine structure has been seen in various
GPCR ligands, including neurokinin antagonists, growth hor-
mone secretagogues, somatostatin receptor agonists, C5a recep-
tor agonists, melanocortin receptor agonists, and serotonin
receptor antagonists.13
The 1,4,9-triazaspiro[5.5]undecane-2,5-dione (spirodiketo-
piperazine) was an attractive scaffold for us because of the
following three reasons. (1) Along with spiropiperidine, 2,5-
diketopiperazine (DKP) represents an important class of biologi-
cally active natural products and synthetic compounds.14 DKP
is a simple heterocyclic scaffold in which diversity can be
introduced at up to four positions, and can be prepared from
readily available R-amino acids. (2) When we initiated this
project, only one compound with this spirodiketopiperazine
scaffold, a homodimer of 1,2,5-trimethyl-4-aminopiperidine-4-
carboxylic acid, had been known in the chemical database.15
Chemokines are a large family of small cytokines that
selectively control the adhesion, chemotaxis, and activation of
various leukocyte populations and are known to be involved in
the initiation and progress of inflammation and allergic diseases.1
Chemokines, by binding to their receptors on the surface of
specific cells, exert a variety of biological activities. Because
chemokines are classified into two main groups CC and CXC
on the basis of their conserved N-terminal cysteine residues,
their receptors are also categorized into two groups, CCR and
CXCR. Human immunodeficiency virus type 1 (HIV) also
binds to chemokine receptors including CCR5 and CXCR4,
leading to the infection of its target cells by HIV.2,3,4,5 Thus, it
is thought that interference in the binding of chemokines or HIV
to the specific receptor may open a new avenue for the
development of novel class of antiinflammatory drugs, anti-
allergic drugs, immunosuppressants, and/or antiviral drugs for
HIV infection.
In 1991, the first chemokine receptors CXCR1 and CXCR2
were cloned.6,7 Unlike traditional cytokine and interleukin
receptors, these two receptors were found to belong to the
superfamily of seven transmembrane (7TM) G-protein-coupled
receptors (GPCRs). This information encouraged the pharma-
ceutical industry to work toward the discovery of chemokine
receptor antagonists because drugs that target GPCRs make up
greater than 30% of all known marketed medicines.8 Several
pharmaceutical companies and academic institutions have been
* Corresponding author. Tel: +81 75 961 1151. Fax: +81 75 962 9314.
E-mail: habashita@ono.co.jp.
† Medicinal Chemistry Research Laboratories, Ono Pharmaceutical Co.,
Ltd.
‡ Exploratory Research Laboratories, Ono Pharmaceutical Co., Ltd.
§ Kumamoto University School of Medicine.
| National Institutes of Health.
10.1021/jm060051s CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/14/2006

Low-Molecular-Weight CCR5 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4141
Table 1. Yield and Purity of Spirodiketopiperazine Derivatives 1a-g
on Solid-Phase Synthesis
purity yield
compds
R1
R2
Bn
R3
Bn
R4
H
resina
(%)b
(%)c
1a
Me
Bn
A
B
A
B
B
B
B
B
A
B
100.0
100.0
95.7
100.0
100.0
100.0
81.3
41.7
92.4
96.9
62
91
67
88
96
96
15
30
68
99
1b
Bn
Bn
H
1c
1d
1e
1f
Ph(CH2)5 Bn
Ph(CH2)5 Bn
Ph(CH2)5 Ph
Ph(CH2)5 iPr
Bn
iBu
iBu
iBu
H
H
H
H
1g
Bn
Bn
-CH2CH2CH2-
a Resin A is the isonitrile resin prepared from the Rink-amide resin. Resin
B is the isonitrile resin prepared from the aminomethylated resin. b The
crude sample was analyzed by LC-MS/ELSD. Its purity was calculated by
the % area of the corresponding peak to the desired product on ELSD.
c The isolated yield is based upon the loading level of the isonitrile resin.
The loading level was determined by elemental analysis.
Figure 1. Molecular modeling of type-I â-turn (left) and spirodike-
topiperazine (right) showing their structural similarity.
amide decreased, the intensity of the absorption corresponding
to the isonitrile increased. The reaction of the isonitrile resin
with amine, piperidone, and carboxylic acid could also be
monitored by IR measurements. Heating (65 °C) was required
to achieve the complete conversion of the reaction. After the
Ugi reaction, the Boc group was removed with TMSOTf in the
presence of 2,6-lutidine (resin A)20 or with 25% trifluoroacetic
acid in CH2Cl2 (resin B). Cyclative cleavage from the support
using toluene in the presence of acetic acid at 90 °C yielded
the desired spirodiketopiperazine derivatives in high purity.21
The crude purities and isolated yields of 1a-g are shown in
Table 1.22
To expand the diversity at the R1 position, we used both solid-
and solution-phase techniques because of the limited number
of commercially available 1-substituted piperidone derivatives.
In solid-phase synthesis, the use of 1-allyloxycarbonyl-4-
piperidone was examined (Scheme 2).23 1-Allyloxycarbonyl-
4-piperidone was reacted with isonitrile resin in the presence
of propylamine and N-Boc-leucine. After the Ugi reaction, the
allyloxycarbonyl group was removed with Pd(PPh3)4-Bu3SnH-
AcOH in CH2Cl2.24 The secondary amine was reductively
alkylated by the reaction with aldehyde in the presence of
sodium triacetoxyborohydride.25,26 The removal of the Boc
group followed by cyclative cleavage gave the desired spiro-
compounds (2a-g) in high purity (Table 2).
(3) The predicted 3D structure of spirodiketopiperazine sug-
gested that three substituents on this template might have the
similar orientation of three side chains on the type I â-turn
structure of the protein (Figure 1). These reasons prompted us
to develop the synthetic method for spirodiketopiperazine
derivatives, produce a library with this scaffold, and investigate
their biological action toward several GPCRs, especially
chemokine receptors.
Chemistry
To generate a spirodiketopiperazine-based library, we inves-
tigated the use of the Ugi multiple component reaction (MCR)
on the solid phase followed by cyclative cleavage from the
resin.16 Ugi MCR is one of the most versatile methods to
construct diverse dipeptide derivatives by the mixing of an
amine, an amino acid derivative, a carbonyl compound, and an
isonitrile.17 This reaction followed by cyclative cleavage from
the resin is one of the most reliable approaches to synthesize
diketopiperazine derivatives in high purity. An efficient and
reliable synthetic route is shown in Scheme 1.
There are several reports regarding the use of this MCR on
solid support.18,19 Two types of isonitrile resins were readily
prepared from commercially available resins in two steps. Rink-
isonitrile resin (resin A) and methylene-isonitrile resin (resin
B) could be prepared from Rink-amide resin and aminomethy-
lated resin, respectively. The conversion of formamide to
isonitrile on resin was easily monitored by infrared spectroscopy
(IR). As the intensity of the absorption corresponding to the
In the solution phase, desired product 4 was obtained by the
reductive alkylation of secondary amine 3 with the correspond-
ing aldehyde, followed by catch and release purification utilizing
the cation-exchange resin.27
Scheme 1. Solid-Phase Synthesis of Spirodiketopiperazine Derivativesa
a (a) THF-MeOH (1:1), 65 °C, 16 h; (b) TMSOTf, 2,6-lutidine/CH2Cl2, rt, 1 h (resin A); (c) TFA-CH2Cl2 (1:3), rt, 0.5 h (resin B); (d) AcOH/toluene, 90
°C, 24 h.

4142 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Habashita et al.
Scheme 2. Solid-Phase Synthesis of Spirodiketopiperazine Derivatives 2a-ga
a (a) THF-MeOH (1:1), 65 °C, 16 h; (b) Pd(PPh3)4, Bu3SnH, AcOH/CH2Cl2, rt, 4 h; (c) R5-CHO, NaBH(OAc)3, AcOH/DMF, rt, 16 h; (d) TFA-CH2Cl2
(1:3), rt, 0.5 h; (e) AcOH/toluene, 90 °C, 24 h.
Table 2. Yield and Purity of Spirodiketopiperazine Derivatives on
Solid-Phase Synthesis
difference in antagonistic activities between the enantiomers
could be observed, we decided to synthesize the following lead
purity
(%)a
yield
(%)b
evaluation libraries in racemic form. To evaluate their receptor
selectivity, the newly synthesized compounds were also tested
by the binding assay as shown in Tables 4 and 5.
From Hit to Lead. Iterative Evaluation of Individual Sites.
Among more than 15 subtypes of chemokine receptors, CCR5
is one of the most attractive drug targets to treat HIV
infections,30 asthma,31 rheumatoid arthritis,32 and acute/chronic
transplant rejections.33,34 We decided to follow the evaluation
process from hit to lead in CCR5 antagonistic activity.
The racemic forms of compounds 7 and 8 (R1 ) 6-phenyl-
hexyl, R2 ) n-propyl, R3 ) isobutyl) were used as the lead
compounds. Initially, each diversity site (R1, R2, and R3) was
individually evaluated. This iterative evaluation approach was
reported to be useful for the exploration of a relatively large
number of building blocks to select the most suitable fragments
at each site.35
compds
R5
2a
2b
2c
2d
2e
2f
Ph
4-Cl-Ph
100.0
100.0
95.9
98.9
100.0
91.1
86
97
81
81
86
72
89
2,4,6-trimethoxy-Ph
4-methoxy-Ph
PhCH2CH2
2-furanyl
cyclohexyl
2g
99.0
a The crude sample was analyzed by LC-MS/ELSD. Its purity was
calculated by the % area of the corresponding peak to the desired product
on ELSD. b The isolated yield is based upon the loading level of the isonitrile
resin. Its loading level was determined by elemental analysis.
Scheme 3. Solution-Phase Synthesis of Spirodiketopiperazine
Derivatives 4a
1. R1 Library. To explore the optimal structures at the R1
site, we synthesized 80 derivatives by using the solution-phase
synthesis shown in Scheme 3. The two other sites were fixed
(R2 ) n-propyl; R3 ) isobutyl). A list of aldehydes used in
the R1 library (Supporting Information, Figure 2) was selected
among the commercially available reagents by chemical com-
patibility, molecular weight, and structural diversity. By con-
sidering the substructures of compounds 5-8, we enriched the
3- or 4-substituted benzaldehyde derivatives in this evaluation
set.
Upon screening the R1 library, several compounds with
diverse groups (Figure 3) were identified with significantly
improved antagonistic activity.36 These results indicated that the
conversion of the 6-phenylhexyl or the benzyl groups in
compounds 5-8 to the 4-substituted benzyl group led to a
significant improvement in CCR5 antagonistic activity and that
the introduction of diverse long substructures at this position
might be suitable for the improvement of activity. Along with
the preparation of this R1 library, compound 3 was converted
to amides, sulfonamides, and ureas by the reaction with the
corresponding acyl chlorides, sulfonyl chlorides, and isocyan-
ates, respectively. Some of the acylated products showed modest
CCR5 antagonistic activity (data not shown). However, further
derivatization from these acylated analogues was not pursued.
The activity of representative compound 9 was confirmed
through resynthesis and purification (Table 4, compound 9).
a (a) NaBH(OAc)3/DMF-1,2-dichloroethane, rt, 36 h; (b) cation-
exchange resin (BondElute SCX); (c) elution with 10% Et3N in MeOH.
Results and Discussion
Hit Identification. According to the synthetic procedure
shown in Scheme 1, we synthesized an initial library of 576
compounds employing 8 piperidones, 9 amines, and 8 amino
acids (Supporting Information, Figure 1). Each crude member
of this library was analyzed for purity using LC-MS/ELSD. In
539 samples, the purity exceeded 80% on ELSD, and the mass
spectrum of the major peak was consistent with the expected
structure.
The library was tested at 30 µM for inhibition against the
calcium mobilization of several subtypes of human chemokine
receptor-overexpressed CHO cells stimulated by the correspond-
ing endogenous ligand (hMIP-1R/hCCR5, hMCP-1/hCCR2,
hMDC/hCCR4, hSDF-1R/hCXCR4, etc.). At this stage, several
compounds showed greater than 50% inhibitory activity in
hMIP-1R/hCCR5.28 Among them, two compounds (R1 )
benzyl, R2 ) benzyl, R3 ) isobutyl; R1 ) 6-phenylhexyl, R2
) n-propyl, R3 ) isobutyl) were selected, resynthesized in an
optically pure form, purified, and evaluated as representative
hit compounds. Compounds 5-8 were identified as selective
micromolar CCR5 antagonists (Table 3). Because no significant
2. R2 Library. In a manner similar to the R1 library, we
synthesized 80 derivatives of 7 and 8 using the solid-phase
synthesis shown in Scheme 1. The 80 amines were selected

Low-Molecular-Weight CCR5 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4143
Table 3. Hit Identification29
Figure 2. Iterative library for the hit evaluation.
from the chemically compatible and commercially available
amines, taking into account the similarity to n-propylamine or
benzylamine (Supporting Information, Figure 3).
the following R1R2R3 library was synthesized using racemic
amino acid derivatives.
4. R1R2R3 Library. In the final library, all three sites around
the spirodiketopiperazine scaffold were simultaneously exam-
ined (Figure 4). R1R2R3 library consisted of 14 compounds
(R1 × R2 × R3 ) 7 × 1 × 2), which were synthesized by the
solid-phase synthesis shown in Scheme 3. The more potent hits
(compounds 9-14) were selected for resynthesis and purification
and were subsequently re-screened against a panel of chemokine
receptors (Table 5). Biological evaluation of the purified
compounds provided highly selective and potent CCR5 antago-
nists.
Selectivity. Compound 18, which showed the most potent
affinity for CCR5 among the tested compounds 14 - 19, was
selected as a representative compound to evaluate receptor
selectivity. The binding affinity of 18 for a variety of GPCRs
was examined by the binding assay using the respective radio-
isotope-labeled ligand. Compound 18 showed 1000-fold more
potent affinity for CCR5 relative to that of the other GPCRs
and also showed modest affinity for the muscarinic M3 receptor
The screening results of the R2 library is shown in Table 4.
An n-butyl group at the R2 position showed significantly higher
potency than the n-propyl (10 vs 7 and 8) and benzyl groups
(11 vs 5 and 6). The n-butyl group was selected as the optimal
substituent at the R2 position.
3. R3 Library. The R3 site was the next site to be optimized.
Eighty commercially available Boc-protected amino acid deriva-
tives were employed to construct the R3 Library. The other two
sites were fixed (R1 ) 6-phenylhexyl; R2 ) n-propyl). As the
R3 library, the optically pure Boc-protected amino acid was
used when it was commercially available (Supporting Informa-
tion, Figure 4). After the evaluation of the antagonistic activity
of each compound, compounds 12 and 13 with a cyclohexyl-
methyl group at R3 showed comparable activity to 7 (Table 4).
No significant difference in activity was observed between
enantiomers 12/13 and 7/8. On the basis of these observations,

4144 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Table 4. Hits from the Iterative Libraries
Habashita et al.
a NT: not tested.
Figure 3. Representative substructures of the R1 hit compounds.
and sigma receptor (for the muscarinic M3 receptor, IC50
)
and 18 potently inhibited not only the replication of laboratory
and primary R5 HIV-1 strains but also that of various multidrug-
resistant monocyte/macrophage tropic (R5) HIV-1 (see data in
ref 37). All examined compounds were inactive against T cell
2.07 µM; for the sigma receptor, IC50 ) 0.183 µM).
Anti-HIV Activity.37 We next examined in vitro anti-HIV
activities of these potent CCR5 antagonists. Compounds 10, 15,

Low-Molecular-Weight CCR5 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4145
Figure 4. R1R2R3 library components.
Table 5. Hits from the R1R2R3 Library
tropic (X4) HIV-1. These results support the hypothesis that
spirodiketopiperazines such as 18 show potent anti-HIV activity
through their antagonistic effects on CCR5.
were identified. Further optimization of these potential anti-
HIV agents will be reported soon.
Experimental Section
Conclusions
General. All reactions were carried out under a positive pressure
of argon. 4-(1-(9-Fluorenylmethyloxycarbonylamino)-1-(2,4-dimethox-
yphenyl)methyl)phenyloxy-polystyrene (1-2% divinylbenzene) (N-
Through the design and synthesis of a novel spirodiketopip-
erazine scaffold, several potent and selective CCR5 antagonists

4146 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Habashita et al.
Fmoc-Rink resin) and aminomethylpolystyrene (1-2% divinyl-
benzene) (aminomethylated resin) were purchased from Watanabe
Chemicals. Analytical samples were homogeneous as confirmed
by TLC and afforded spectroscopic results consistent with the
assigned structures. Proton nuclear magnetic resonance spectra (1H
NMR) were recorded on a Varian VXR300 spectrometer with
tetramethylsilane as an internal standard. Matrix-assisted laser
desorption ionization-time-of-flight (MALDI-TOF) mass spectra
were obtained on Perseptive Elete. Infrared spectra (IR) were
measured on a Perkin-Elmer FT-IR 1760X spectrometer. Column
chromatography was carried out on silica gel (Merck silica gel 60
(0.063-0.200 mm) or Fuji Silysia FL60D). Thin-layer chromatog-
raphy was performed on silica gel (Merck TLC, silica gel 60 F254).
The solvents and reagents were purchased and used for the reaction
without further purification. The following abbreviations for
solvents and reagents are used: tetrahydrofuran (THF), N,N-
dimethylformamide (DMF), dichloromethane (CH2Cl2), chloroform
(CHCl3), methanol (MeOH), acetic acid (AcOH). For compounds
where analysis was not obtained, HPLC-MS/UV/ELSD analysis
was used, and purity was determined to be >95%.
mg, 1.47 mmol), and N-Boc-phenylalanine (390 mg, 1.47 mmol).
The mixture was shaken for 16 h at 65 °C. After cooling to room
temperature, the mixture was filtered. The resin was successively
washed with THF-MeOH (1:1) (4 mL × 3) and CH2Cl2 (4 mL ×
3). To a mixture of the resin in a 1.5 M solution of 2,6-lutidine in
CH2Cl2 (2 mL) was added a 1.0 M solution of trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in CH2Cl2 (2 mL) at 0 °C.
The mixture was allowed to warm up to room temperature and
then stirred for 30 min. The resin was collected by filtration. To a
mixture of the resin in a 1.5 M solution of 2,6-lutidine in CH2Cl2
(2 mL) was added a 1.0 M solution of TMSOTf in CH2Cl2 (2 mL)
at 0 °C. The mixture was allowed to warm up to room temperature
and then stirred for 30 min. The resin was collected by filtration
and successively washed with CH2Cl2 (4 mL × 3), toluene (4 mL
× 3), and 1.25 M acetic acid in toluene (4 mL). The suspension of
the resin in 1.25 M acetic acid in toluene (4 mL) was shaken for
24 h at 90 °C. After cooling to room temperature, the mixture was
filtered, and the resin was washed with CHCl3-MeOH (1:1) (4
mL × 2). The filtrate and washings were combined and concentrated
under reduced pressure. The residue was purified by column
chromatography over silica gel with a gradient of AcOEt-MeOH
from 1:0 to 10:1 to give the title compound (68.8 mg, 62% yield)
as a white powder. 1H NMR (300 MHz, CDCl3): δ 7.29 (m, 8 H),
7.03 (m, 2 H), 4.84 (m, 1 H), 4.54 (t, J ) 4.5 Hz, 1 H), 4.53 (m,
1 H), 3.38 (m, 1 H), 3.16 (m, 1 H), 3.03 (dd, J ) 13.8, 4.5 Hz, 1
H), 2.90 (m, 2 H), 2.70 (m, 1 H), 2.45 (s, 3 H), 2.06 (m, 1 H),
1.92-1.71 (m, 3 H); IR (KBr): 3448, 2928, 2855, 1682, 1497,
Rink-Isonitrile Resin (Resin A). The N-Fmoc-Rink resin (0.61
mmol/g, 4.0 g, 2.42 mmol) was successively washed with DMF
(40 mL × 2) and 20% piperidine in DMF (40 mL × 1). The resin
was suspended in 20% piperidine in DMF (40 mL), and the mixture
was agitated for 30 min at room temperature. The mixture was
filtered. The resin was washed with DMF (40 mL × 7) and
suspended in DMF (15 mL). To it was added ethyl formate (25
mL). The mixture was stirred under reflux for 6 h. After cooling
to room temperature, the resin was collected by filtration and
washed with DMF (50 mL × 2), CH2Cl2 (50 mL × 4), MeOH (50
mL × 4), and CH2Cl2 (50 mL × 4). Drying under reduced pressure
gave the N-formylated Rink resin (3.00 g, 0.71 mmol/g calculated
by nitrogen content of elementary analysis: N%, 1.00) as a white
resin. IR (KBr): 1692 (CONH) cm-1. The N-formylated Rink resin
(0.71 mmol/g, 3.00 g, 2.13 mmol) was washed with CH2Cl2 (30
mL × 2) and suspended in CH2Cl2 (30 mL). To the mixture were
successively added triethylamine (1.70 mL, 12.2 mmol), CCl4 (1.18
mL, 12.2 mmol), and triphenylphosphine (3.20 g, 12.2 mmol). The
suspension was stirred under reflux for 1 h. After cooling to room
temperature, the mixture was filtered. The resin was successively
washed with CH2Cl2 (30 mL × 4), MeOH (30 mL × 2), and CH2-
Cl2 (30 mL × 4). Drying under reduced pressure gave the Rink-
isonitrile resin (2.56 g, 0.84 mmol/g) as a pale yellow resin. IR
1455, 1430, 1361, 1261, 1206, 1137, 840, 802, 724, 703 cm-1
.
MS (ESI, Pos. 20 V) m/z 755 (2M + H)+, 378 (M + H)+. HRMS
(MALDI-TOF, Pos., Internal Standard PEG) calcd for C23H27-
N3O2‚H: 378.2182. Found: 378.2192.
1,3,9-Tribenzyl-1,4,9-triazaspiro[5.5]undeca-2,5-dione (1b).
Using a procedure identical to that described for the preparation of
1a, the title compound was obtained from 1-benzyl-4-piperidone,
benzylamine, N-Boc-phenylalanine, and Rink-isonitrile resin in 67%
1
yield as a white powder. H NMR (300 MHz, CDCl3): δ 7.36-
7.15 (m, 15 H), 5.95 (m, 1 H), 4.89 (d, J ) 16.2 Hz, 1 H), 4.74 (d,
J ) 16.2 Hz, 1 H), 4.37 (m, 1 H), 3.77 (m, 1 H), 3.42 (dd, J )
13.8, 3.9 Hz, 1 H), 3.12-2.88 (m, 5 H), 2.24 (m, 3 H), 1.83 (m, 1
H), 1.24 (m, 1 H); IR (KBr): 3445, 3063, 3032, 1677, 1570, 1490,
1455, 1417, 1350, 1280, 1258, 1169, 1032, 735, 702, 639 cm-1
.
MS (ESI, Pos. 20 V) m/z 907 (2M + H)+, 454 (M + H)+. HRMS
(MALDI-TOF, Pos., Internal Standard PEG) calcd for C29H31-
N3O2‚H: 454.2495. Found: 454.2495.
(KBr): 2137 cm-1
.
Methylene-Isonitrile Resin (Resin B). The aminomethyl resin
(0.52 mmol/g, 10.0 g, 5.2 mmol) was washed with DMF (40 mL
× 2) and suspended in a mixture of DMF (40 mL) and ethyl formate
(60 mL). The suspension was stirred under reflux for 6 h. After
cooling to room temperature, the resin was filtered and washed
with DMF (50 mL × 2), CH2Cl2 (50 mL × 4), MeOH (50 mL ×
4), and CH2Cl2 (50 mL × 4). The resin was dried under reduced
pressure to give the N-formylated aminomethyl resin (10.0 g, 0.44
mmol/g calculated by nitrogen content of elementary analysis) as
a white resin. IR (KBr): 1682 (CONH) cm-1. The N-formylated
aminomethyl resin (0.44 mmol/g, 10.0 g, 4.4 mmol) was washed
with CH2Cl2 (100 mL × 2) and then suspended in CH2Cl2 (100
mL). To it were successively added triethylamine (3.62 mL, 26.0
mmol), CCl4 (2.51 mL, 26.0 mmol) and triphenylphosphine (6.82
g, 26.0 mmol). The suspension was stirred under reflux for 1 h.
After cooling to room temperature, the mixture was filtered. The
resin was successively washed with CH2Cl2 (100 mL × 4), MeOH
(100 mL × 2), and CH2Cl2 (100 mL × 4). Drying under reduced
pressure gave the methylene-isonitrile resin (9.73 g, 0.45 mmol/g)
1,9-Dibenzyl-3,4-propylene-1,4,9-triazaspiro[5.5]undeca-2,5-
dione (1g). Using a procedure identical to that described for the
preparation of 1a, the title compound was obtained from 1-benzyl-
4-piperidone, benzylamine, N-Boc-proline, and Rink-isonitrile resin
1
in 68% yield as a white powder. H NMR (300 MHz, CDCl3): δ
7.33-7.16 (m, 10H), 5.07 (m, 1H), 4.46 (m, 2H), 3.65 (m, 4H),
3.22 (m, 1H), 2.90 (m, 2H), 2.45 (m, 1H), 2.30-1.78 (m, 8H); IR
(KBr): 3482, 1655, 1496, 1455, 1416, 1363, 1260, 1169, 1034,
981, 763, 740, 701, 641 cm-1. MS (ESI, Pos. 20 V) m/z 404 (M +
H)+. Anal. Calcd for C25H29N3O2‚1.1H2O: C, 70.93; H, 7.43; N,
9.93. Found: C, 70.82; H, 7.37; N, 9.65.
Typical Procedure for the Preparation of Spirodiketopipera-
zine Derivative from Methylene-Isonitrile Resin. 1,3-Dibenzyl-
9-(5-phenylpentyl)-1,4,9-triazaspiro[5.5]undeca-2,5-dione (1c).
The methylene-isonitrile resin (0.45 mmol/g, 500 mg, 0.225 mmol)
was washed with THF-MeOH (1:1) (4 mL × 2). To a suspension
of the resin in THF-MeOH (1:1) (4 mL) were successively added
N-(5-phenylpentyl)-4-piperidone (206 mg, 1.125 mmol), benzyl-
amine (213 mg, 1.125 mmol), and N-Boc-phenylalanine (299 mg,
1.125 mmol). The mixture was shaken for 16 h at 65 °C. After
cooling to room temperature, the mixture was filtered. The collected
resin was washed with THF-MeOH (1:1) (4 mL × 3), and CH2-
Cl2 (4 mL × 3). The resin was then added to 25% TFA in CH2Cl2
(4 mL) at 0 °C. The mixture was allowed to warm up to room
temperature and then stirred for 30 min. After filtration, the resin
was washed with CH2Cl2 (4 mL × 3), toluene (4 mL × 3), and
1.25 M acetic acid in toluene (4 mL). The suspension of the resin
as a pale yellow resin. IR (KBr): 2146 cm-1
.
Typical Procedure for the Preparation of Spirodiketopipera-
zine Derivative from Rink-Isonitrile Resin. 1,3-Dibenzyl-9-
methyl-1,4,9-triazaspiro[5.5]undeca-2,5-dione (1a). The Rink-
isonitrile resin (0.84 mmol/g, 350 mg, 0.294 mmol) was washed
with THF-MeOH (1:1, 4 mL × 2). To a mixture of the above
resin in THF-MeOH (1:1, 4 mL) were successively added
N-methyl-4-piperidone (166 mg, 1.47 mmol), benzylamine (158

Low-Molecular-Weight CCR5 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4147
in 1.25 M acetic acid in toluene was agitated for 24 h at 90 °C.
After cooling to room temperature, the mixture was filtered. The
resin was washed with CHCl3-MeOH (1:1) (4 mL × 2). The filtrate
and washings were concentrated under reduced pressure. The
residue was purified by column chromatography over silica gel with
a gradient of AcOEt-MeOH from 1:0 to 10:1 to give the title
compound (103 mg, 96% yield) as a white powder. 1H NMR (300
MHz, CDCl3): δ 7.25 (m, 15H), 5.95 (m, 1H), 4.80 (m, 2H), 4.43
(m, 1H), 3.29 (m, 5H), 2.82 (m, 2H), 2.58 (m, 3H), 2.41 (m, 1 H),
1.64 (m, 6H), 1.34 (m, 3H), 0.86 (m, 1H); 13C NMR (75.5 MHz,
CDCl3): δ 169.6, 166.6, 142.0, 137.7, 135.3, 130.2, 129.4, 128.9,
128.6, 128.0, 127.3 126.6, 126.1, 58.7, 57.1, 55.8, 49.2, 45.3, 40.4,
35.7, 30.9, 30.2, 30.1, 26.4, 23.9; IR (KBr): 3448, 3239, 3062,
3029, 2938, 2860, 1679, 1496, 1454, 1415, 1361, 1200, 1130, 830,
799, 732, 720, 701 cm-1. MS (ESI, Pos. 20 V) m/z 510 (M + H)+.
HRMS (MALDI-TOF, Pos., Internal Standard PEG) calcd for
C33H39N3O2‚H: 510.3120. Found 510.3103.
(4 mL × 4), and CH2Cl2 (4 mL × 4). The resin was suspended in
25% TFA in CH2Cl2 (4 mL) at 0 °C. The mixture was allowed to
warm up to room temperature and then stirred for 30 min. After
filtration, the resin was rinsed with CH2Cl2 (4 mL × 3), toluene (4
mL × 3), and 1.25 M acetic acid in toluene (4 mL). The resin was
suspended with 1.25 M acetic acid in toluene (4 mL). The
suspension was shaken for 24 h at 90 °C. After cooling to room
temperature, the mixture was filtered. The resin was washed with
CHCl3-MeOH (1:1) (4 mL × 2). The filtrate and washings were
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel with a gradient of AcOEt-
MeOH from 1:0 to 10:1 to give the title compound (89 mg, 97%
1
yield) as a white powder. H NMR (300 MHz, CDCl3): δ 7.29
(m, 4H), 6.59 (m, 1H), 3.96 (m, 1H), 3.50 (s, 2H), 3.33 (m, 2H),
2.85 (m, 2H), 2.57 (m, 1H), 1.75 (m, 10H), 0.93 (m, 9H); 13C NMR
(75.5 MHz, CDCl3): δ 171.1, 168.3, 137.1, 132.7, 130.3, 128.4,
61.9, 60.1, 52.5, 50.2, 49.8, 44.3, 42.7, 32.9, 24.3, 23.4, 23.1, 21.2,
11.4; IR (KBr): 3437, 3205, 3081, 2953, 2869, 1682, 1659, 1490,
1469, 1415, 1364, 1329, 1087, 1068, 1016, 844 cm-1. MS (ESI,
Pos. 20 V) m/z 406 (M + H)+. Anal. Calcd for C22H32N3O2Cl: C,
65.09; H, 7.95; N, 10.35. Found: C, 65.23; H, 8.17; N, 10.20.
9-Benzyl-3-(2-methylpropyl)-1-propyl-1,4,9-triazaspiro[5.5]-
undeca-2,5-dione (2a). Using a procedure identical to that described
for the preparation of 2b, the title compound was obtained from
1-allyloxycarbonyl-4-piperidone, 1-propylamine, N-Boc-leucine,
methylene-isonitrile resin, and benzaldehyde in 86% yield as a white
1-Benzyl-3-(2-methylpropyl)-9-(5-phenylpentyl)-1,4,9-
triazaspiro[5.5]undeca-2,5-dione (1d). Using a procedure identical
to that described for the preparation of 1c, the title compound was
obtained from 1-(5-phenylpentyl)-4-piperidone, benzylamine, N-
Boc-leucine, and methylene-isonitrile resin in 96% yield as a white
powder. 1H NMR (300 MHz, CDCl3): δ 7.25 (m, 10 H), 6.18 (m,
1 H), 4.83 (m, 2 H), 4.12 (m, 1 H), 3.54 (m, 1 H), 3.43 (m, 3 H),
2.94 (m, 2 H), 2.72 (m, 2 H), 2.60 (t, J ) 7.5 Hz, 2 H), 2.37 (m,
1 H), 2.05 (m, 1 H), 1.88-1.60 (m, 7 H), 1.39 (m, 2 H), 1.01 (m,
6 H); 13C NMR (75.5 MHz, CDCl3): δ 169.6, 167.8, 141.7, 137.7,
128.7, 128.3, 127.1, 126.3, 125.9, 58.4, 56.9, 52.6, 49.3, 49.1, 45.2,
43.4, 35.4, 30.6, 30.3, 29.7, 26.1, 24.4, 23.7, 23.3, 21.0; IR (KBr):
3433, 3233, 2956, 2869, 1678, 1496, 1455, 1414, 1361, 1329, 1200,
1131, 830, 799, 720, 700 cm-1. MS (ESI, Pos. 20 V) m/z 951 (2M
+ H)+, 476 (M + H)+. HRMS (MALDI-TOF, Pos., Internal
Standard PEG) calcd for C30H41N3O2‚H: 476.3277. Found 476.3288.
3-(2-Methylpropyl)-1-phenyl-9-(5-phenylpentyl)-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione (1e). Using a procedure identical
to that described for the preparation of 1c, the title compound was
obtained from 1-(5-phenylpentyl)-4-piperidone, aniline, N-Boc-
leucine, and methylene-isonitrile resin in 15% yield as a white
1
powder. H NMR (300 MHz, CDCl3): δ 7.39 (m, 5 H), 6.80 (m,
1 H), 3.96 (m, 1 H), 3.57 (s, 2 H), 3.39 (m, 2 H), 2.88 (m, 2 H),
2.62 (m, 1 H), 1.88 (m, 7 H), 1.57 (m, 3 H), 0.96 (m, 9 H); 13C
NMR (75.5 MHz, CDCl3): δ 171.2, 168.3, 129.1, 128.3, 127.2,
62.6, 60.0, 52.5, 50.2, 49.8, 44.3, 42.7, 32.8, 32.6, 24.2, 23.4, 23.1,
21.2, 11.3; IR (KBr): 3437, 3202, 3063, 2959, 2870, 1677, 1658,
1470, 1418, 1366, 1329, 1266, 1199, 1158, 1068, 742, 698 cm-1
.
MS (ESI, Pos. 20 V) m/z 372 (M + H)+. Anal. Calcd for
C22H33N3O2‚0.67H2O; C, 68.90; H, 9.02; N, 10.96. Found: C,
68.99; H, 8.87; N, 10.88.
3-(2-Methylpropyl)-1-propyl-9-((2,4,6-trimethoxyphenyl)-
methyl)-1,4,9-triazaspiro[5.5]undeca-2,5-dione (2c). Using a
procedure identical to that described for the preparation of 2b, the
title compound was obtained from 1-allyloxycarbonyl-4-piperidone,
1-propylamine, N-Boc-leucine, methylene-isonitrile resin, and 2,4,6-
trimethoxybenzaldehyde in 81% yield as a white powder. 1H NMR
(300 MHz, CDCl3): δ 6.39 (m, 1H), 6.12 (s, 2H), 3.94 (m, 1H),
3.81 (s, 3H), 3.79 (s, 6H), 3.67 (s, 2H), 3.36 (m, 2H), 2.96 (m,
1
powder. H NMR (300 MHz, CDCl3): δ 7.43-6.98 (m, 10 H),
6.22 (m, 1 H), 4.15 (m, 1 H), 3.63 (m, 1 H), 3.47 (m, 2 H), 3.26
(m, 1 H), 2.86 (m, 2 H), 2.57 (m, 3 H), 2.24 (m, 2 H), 2.06 (m, 2
H), 1.65 (m, 6 H), 1.30 (m, 2 H), 0.97 (m, 6 H); 13C NMR (75.5
MHz, CDCl3): δ 170.1, 168.2, 141.7, 135.7, 130.0, 129.6, 129.3,
128.3, 125.9, 118.3, 58.7, 56.8, 52.9, 49.2, 48.7, 41.8, 35.4, 31.4,
30.7, 30.6, 26.0, 24.5, 23.5, 23.1, 21.3; IR (KBr): 3422, 3247, 2956,
2868, 1678, 1493, 1545, 1347, 1200, 1131, 831, 799, 752, 720,
700 cm-1. MS (ESI, Pos. 20 V) m/z 923 (2M + H)+, 462 (M +
H)+. HRMS (MALDI-TOF, Pos., Internal Standard PEG) calcd for
C29H39N3O2‚H: m/z 462.3120. Found: 462.3087.
2H), 2.66 (m, 1H), 1.88 (m, 7H), 1.55 (m, 3H), 0.94 (m, 9H); 13
C
NMR (75.5 MHz, CDCl3): δ 171.2, 168.5, 160.1, 159.7, 90.6, 60.4,
55.7, 55.3, 52.5, 49.7, 49.2, 48.5, 44.2, 42.6, 32.4, 24.3, 23.3, 23.2,
21.1, 11.3; IR (KBr): 3440, 2955, 1671, 1608, 1468, 1417, 1227,
1205, 1151, 1132, 1064, 952 cm-1. MS (ESI, Pos. 20 V) 462 (M
+ H)+, 181. Anal. Calcd for C25H39N3O5‚0.33H2O: C, 64.22; H,
8.55; N, 8.99. Found: C, 64.34; H, 8.65; N, 8.84.
Typical Procedure for the Preparation of Spirodiketopipera-
zine Derivative from 1-Alloc-4-piperidone and Methylene-Iso-
nitrile Resin. 9-(4-Chlorophenylmethyl)-3-(2-methylpropyl)-1-
propyl-1,4,9-triazaspiro[5.5]undeca-2,5-dione (2b). The resin
(0.45 mmol/g, 500 mg, 0.225 mmol) was washed with THF-MeOH
(1:1) (4 mL × 2). To the suspension of the resin in THF-MeOH
(1:1) (4 mL) were added N-allyloxycarbonyl-4-piperidone (206 mg,
1.13 mmol), 1-propylamine (66.5 mg, 1.125 mmol), and N-Boc-
leucine (213 mg, 1.125 mmol). The mixture was shaken for 16 h
at 65 °C. After cooling to room temperature, the mixture was
filtered. The resin was successively washed with THF-MeOH (1:
1) (4 mL × 3) and CH2Cl2 (4 mL × 3). To the suspension of the
resin in CH2Cl2 (4 mL) were successively added acetic acid (135
mg, 2.25 mmol), tetrakis(triphenylphosphine)palladium (0) (52.0
mg, 0.045 mmol), and tributyltinhydride (327 mg, 1.125 mmol).
The mixture was shaken for 4 h at room temperature. After filtration,
the resin was washed with CH2Cl2 (4 mL × 4) and DMF (4 mL ×
4). To the suspension of the resin in 1% acetic acid in DMF (4
mL) were successively added 4-chlorobenzaldehyde (158 mg, 1.125
mmol) and sodium triacetoxyborohydride (238 mg, 1.125 mmol).
The mixture was shaken for 16 h at room temperature and filtered.
The resin was successively washed with MeOH (4 mL × 2), DMF
3-(2-Methylpropyl)-9-(4-methoxyphenymethyl)-1-propyl-1,4,9-
triazaspiro[5.5]undeca-2,5-dione (2d). Using a procedure identical
to that described for the preparation of 2b, the title compound was
obtained from 1-allyloxycarbonyl-4-piperidone, 1-propylamine,
N-Boc-leucine, methylene-isonitrile resin, and 4-methoxybenzal-
1
dehyde in 81% yield as a white powder. H NMR (300 MHz,
CDCl3): δ 7.24 (d, J ) 8.2 Hz, 2H), 6.86 (d, J ) 8.2 Hz, 2H),
6.55 (m, 1H), 3.96 (m, 1H), 3.80 (s, 3H), 3.48 (s, 2H), 3.36 (m,
2H), 2.79 (m, 2H), 2.56 (m, 1H), 1.88 (m, 7H), 1.56 (m, 3H), 0.95
(m, 9H); 13C NMR (75.5 MHz, CDCl3): δ 171.2, 168.4, 158.7,
130.3, 113.6, 62.0, 60.2, 55.2, 52.5, 50.2, 49.7, 44.2, 42.7, 32.8,
24.3, 23.4, 23.1, 21.2, 11.3; IR (KBr): 3435, 3205, 2955, 1667,
1612, 1513, 1468, 1399, 1366, 1244 cm-1. MS (ESI, Pos. 20 V)
m/z 402 (M + H)+. Anal. Calcd for C23H35N3O3: C, 68.80; H,
8.79; N, 10.46. Found: C, 68.69; H, 9.09; N, 10.46.
3-(2-Methylpropyl)-9-(3-phenylpropyl)-1-propyl-1,4,9-triaza-
spiro[5.5]undeca-2,5-dione (2e). Using a procedure identical to
that described for the preparation of 2b, the title compound was
obtained from 1-allyloxycarbonyl-4-piperidone, 1-propylamine,
N-Boc-leucine, methylene-isonitrile resin, and 3-phenylpropanal in

4148 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Habashita et al.
1
86% yield as a white powder. H NMR (300 MHz, CDCl3): δ
was transferred into a 96-well plate. The solvent was removed by
evaporation under reduced pressure with centrifugation. The
residues were dissolved in DMSO (0.438 mL) (10 mM solution
for in vitro biological assay). The overall yield was estimated to
be 40% yield from Rink-isonitrile resin.
7.27 (m, 2H), 7.17 (m, 3H), 6.60 (m, 1H), 3.96 (m, 1H), 3.39 (m,
2H), 2.84 (m, 3H), 2.64 (m, 3H), 2.42 (m, 2H), 2.09 (m, 2H), 1.85
(m, 6H), 1.57 (m, 3H), 0.93 (m, 9H); 13C NMR (75.5 MHz,
CDCl3): δ 171.1, 168.3, 141.9, 128.3, 125.8, 60.1, 57.7, 52.5, 50.3,
49.9, 44.2, 42.7, 33.8, 32.7, 28.6, 24.2, 23.3, 23.2, 21.2, 11.3; IR
(KBr): 3433, 3220, 3065, 2956, 2868, 1677, 1661, 1469, 1417,
1370, 1328, 1262, 1200, 1158, 1124, 1071, 742, 701 cm-1. MS
(ESI, Pos. 20 V) m/z 400 (M + H)+. Anal. Calcd for C24H37N3O2:
C, 72.14; H, 9.33; N, 10.52. Found: C, 71.82; H, 9.42; N, 10.44.
9-(2-Furanylmethyl)-3-(2-methylpropyl)-1-propyl-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione (2f). Using a procedure identical
to that described for the preparation of 2b, the title compound was
obtained from 1-allyloxycarbonyl-4-piperidone, 1-propylamine,
N-Boc-leucine, methylene-isonitrile resin, and 2-furfural in 72%
yield as a white powder. 1H NMR (300 MHz, CDCl3): δ 7.40 (m,
1H), 6.42 (m, 1H), 6.31 (m, 1H), 6.19 (m, 1H), 3.94 (m, 1H), 3.57
(s, 2H), 3.37 (m, 2H), 2.87 (m, 3H), 2.62 (m, 1H), 2.15 (m, 2H),
1.92 (m, 2H), 1.74 (m, 2H), 1.54 (m, 3H), 0.93 (m, 9H); 13C NMR
(75.5 MHz, CDCl3): δ 174.2, 170.9, 168.3, 162.0, 142.3, 110.1,
108.9, 60.0, 54.7, 52.5, 50.1, 49.8, 44.2, 42.7, 32.8, 24.3, 23.3, 23.1,
21.2, 11.3; IR (KBr): 3448, 3207, 3113, 2958, 2871, 2816, 1680,
1655, 1470, 1403, 1364, 1340, 1303, 1150, 1075, 1012, 923, 745,
602 cm-1. MS (ESI, Pos. 20 V) m/z 362 (M + H)+. Anal. Calcd
for C20H31N3O3: C, 66.45; H, 8.64; N, 11.62. Found: C, 66.36;
H, 8.86; N, 11.50.
(3S)-1,9-Dibenzyl-3-(2-methylpropyl)-1,4,9-triazaspiro[5.5]-
undeca-2,5-dione Hydrochloride (5). To an agitated suspension
of Rink-isonitrile resin (1.0 g, 0.56 mmol) in THF-MeOH (10 mL,
1:1) were successively added benzylamine (0.183 mL, 1.68 mmol),
N-Boc-L-leucine (419 mg, 1.68 mmol), and 1-benzyl-4-piperidone
(0.30 mL, 1.68 mmol) at room temperature. The mixture was stirred
at 65 °C for 16 h. After cooling to room temperature, the mixture
was filtered and washed with THF (6 mL × 3), MeOH (6 mL ×
3), and CH2Cl2 (6 mL × 3). The resin was suspended in a 1.5 M
solution of 2,6-lutidine in CH2Cl2 (4 mL) and a 1.0 M solution of
TMSOTf in CH2Cl2 (4 mL) at room temperature. The mixture was
agitated for 30 min at room temperature and then filtered. The resin
was suspended in a 1.5 M solution of 2,6-lutidine in CH2Cl2 (4
mL) and a 1.0 M solution of TMSOTf in CH2Cl2 (4 mL) at room
temperature. The mixture was agitated for 30 min at room
temperature and then filtered. The resin was washed with CH2Cl2
(6 mL × 4), MeOH (6 mL × 4), and CH2Cl2 (6 mL × 5). The
resin was suspended in a 1.2 M solution of AcOH in toluene (6
mL). The mixture was warmed at 90 °C for 20 h. After cooling to
room temperature, the resin was collected by filtration and washed
with MeOH (6 mL × 2). The filtrate and washings were loaded on
cation-exchanged resin (BondElute SCX, Varian, 1.0 g), which was
washed with MeOH (6 mL × 2), H2O (6 mL), and MeOH (6 mL)
prior to use. The resin was washed with MeOH (4 mL × 2). Elution
with 10% Et3N in MeOH (5 mL × 2) was concentrated under
reduced pressure to give an oily residue. This residue was purified
by flash chromatography over silica gel (40 g) with CHCl3-i-PrOH
(20:1) to give a colorless oil, which was dissolved in MeOH (10
mL). Then, 1 N HCl (0.60 mL) was added to it. Concentration
under reduced pressure gave a white powder, which was triturated
with Et2O to yield 5 (72 mg, 28% yield, 95% ee determined by
HPLC) as a white powder. 1H NMR (200 MHz, CD3OD): δ 7.48
(m, 5 H), 7.23 (m, 5 H), 5.06-4.82 (m, 2 H), 4.31 (s, 2 H), 4.17
(dd, J ) 8.0, 4.6 Hz, 1 H), 3.72 (m, 2 H), 3.40 (m, 2 H), 2.52 (m,
2 H), 2.08 (m, 2 H), 2.00-1.60 (m, 3 H), 0.98 (t, J ) 6.0 Hz, 6
H); IR (KBr): 3435, 3231, 3063, 2956, 2550, 1673, 1497, 1456,
1413, 1364, 1327, 1155, 1131, 968, 750, 700 cm-1. MS (APCI,
Pos., 40 V) m/z 420 (M + H)+. Anal. Calcd for C26H33N3O2‚HCl‚
H2O: C, 65.88; H, 7.65; N, 8.86. Found: C, 65.99; H, 7.77; N,
8.83. HPLC (Chiralcel OJ-R 4.6 × 150 mm. 0.5 M NaClO4/CH3-
CN ) 65/35. 0.8 mL/min. UV 210 nm. Temp 40 °C) retention
time, 11.44 min.
9-Cyclohexylmethyl-3-(2-methylpropyl)-1-propyl-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione (2g). Using a procedure identical
to that described for the preparation of 2b, the title compound was
obtained from 1-allyloxycarbonyl-4-piperidone, 1-propylamine,
N-Boc-leucine, methylene-isonitrile resin, and cyclohexylaldehyde
1
in 89% yield as a white powder. H NMR (300 MHz, CDCl3): δ
6.71 (m, 1H), 3.96 (m, 1H), 3.39 (m, 2H), 2.79 (m, 3H), 2.51 (m,
1H), 2.17 (m, 3H), 1.93 (m, 4H), 1.79 (m, 7H), 1.58 (m, 4H), 1.21
(m, 3H), 0.95 (m, 10H); 13C NMR (75.5 MHz, CDCl3): δ 171.3,
168.5, 65.2, 60.3, 52.5, 50.7, 50.3, 44.2, 42.6, 35.3, 32.8, 32.0, 26.8,
26.1, 24.2, 23.4, 23.2, 21.2, 11.3; IR (KBr): 3436, 3203, 3076,
2924, 2871, 2851, 1679, 1468, 1414, 1369, 1329, 1152, 1130, 1066
cm-1. MS (ESI, Pos. 20 V) m/z 378 (M + H)+. Anal. Calcd for
C22H39N3O2: C, 69.98; H, 10.41; N, 11.13. Found: C, 69.83; H,
10.71; N, 11.07.
General Procedure for the Preparation of the Initial, R2, and
R3 Libraries. Rink-isonitrile resin (1.6 g, 0.61 mmol/g resin) was
suspended in a mixture of CHCl3-MeOH (3:1). This suspension
was equally divided into 80 test tubes with a screwed cap. One
test tube contained 20 mg (0.122 mmol) of the resin. The solvent
was removed by evaporation under reduced pressure with centrifu-
gation. To this test tube were successively added 0.122 mL of a
1.0 M solution of the corresponding amine in THF-MeOH (1:1)
(0.122 mmol), 0.122 mL of a 1.0 M solution of the corresponding
N-Boc-amino acid in THF-MeOH (1:1) (0.122 mmol), and 0.122
mL of a 1.0 M solution of the corresponding 1-substituted
4-piperidone in THF-MeOH (1:1) (0.122 mmol) at room temper-
ature. The mixture was warmed at 65 °C for 18 h. After cooling to
room temperature, the mixture was filtered, and the resin was
washed with THF (2 mL × 2), MeOH (2 mL × 2), and CH2Cl2 (2
mL × 3). The resin was suspended in 0.2 mL of CH2Cl2. To this
suspension were successively added a 1.5 M solution of 2,6-lutidine
in CH2Cl2 (0.2 mL) and a 1.0 M solution of TMSOTf in CH2Cl2
(0.2 mL) at room temperature. The mixture was agitated for 30
min at room temperature and then filtered. The resin was washed
with CH2Cl2 (2 mL × 2), MeOH (2 mL × 2), and CH2Cl2 (2 mL
× 3). After the resin was dried under reduced pressure, it was
transferred into a test tube with a screwed cap. The resin was
suspended in a 1.25 M solution of AcOH in toluene (0.2 mL). The
mixture was warmed at 90 °C for 16 h. After cooling to room
temperature, the resin was collected by filtration and washed with
MeOH (1 mL × 2). The filtrate and washings were concentrated
under reduced pressure with centrifugation. The residue was
dissolved in MeOH (0.488 mL). A small amount of this solution
(0.050 mL) was saved for LC-MS analysis. The remaining solution
(3R)-1,9-Dibenzyl-3-(2-methylpropyl)-1,4,9-triazaspiro[5.5]-
undeca-2,5-dione Hydrochloride (6). Using a procedure identical
to that described for the preparation of 5, the title compound (62
mg, 94% ee determined by HPLC) was obtained from Rink-
isonitrile resin, benzylamine, N-Boc-D-leucine, and 1-benzyl-4-
1
piperidone in 24% yield as a white powder. H NMR (200 MHz,
CD3OD): δ 7.48 (m, 5 H), 7.23 (m, 5 H), 4.82 (m, 2 H), 4.31 (s,
2 H), 4.17 (dd, J ) 8.0, 4.6 Hz, 1 H), 3.72 (m, 2 H), 3.40 (m, 2 H),
2.52 (m, 2 H), 2.08 (m, 2 H), 2.00-1.60 (m, 3 H), 0.98 (t, J ) 6.0
Hz, 6 H); IR (KBr): 3434, 3215, 3063, 2956, 2660, 2532, 2419,
2361, 1674, 1496, 1456, 1413, 1364, 1327, 1154, 1131, 1073, 1032,
1005, 968, 929, 750, 733, 700 cm-1. MS (APCI, Pos., 40 V) 420
(M + H) +. Anal. Calcd for C26H33N3O2‚HCl‚H2O: C, 65.88; H,
7.65; N, 8.86. Found: C, 66.18; H, 7.76; N, 8.80. HPLC (Chiralcel
OJ-R 4.6 × 150 mm. 0.5 M NaClO4/CH3CN ) 65/35. 0.8 mL/
min. UV 210 nm. Temp 40 °C) retention time, 9.46 min.
(3S)-3-(2-Methylpropyl)-9-(6-phenylhexyl)-1-propyl-1,4,9-
triazaspiro[5.5]undeca-2,5-dione Hydrochloride (7). Using a
procedure identical to that described for the preparation of 5, the
title compound (69 mg, 91% ee. determined by HPLC) was obtained
from Rink-isonitrile resin, 1-propylamine, N-Boc-L-leucine, and
1-(6-phenylhexyl)-4-piperidone in 26% yield as a white powder.
[R]26D -24.6° (c 0.680, MeOH); 1H NMR (200 MHz, CD3OD): δ
7.18 (m, 5 H), 4.02 (dd, J ) 7.6, 4.8 Hz, 1 H), 3.70 (m, 2 H), 3.56

Low-Molecular-Weight CCR5 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4149
(m, 2 H), 3.39 (m, 2 H), 3.11 (m, 2 H), 2.63 (dd, J ) 7.8, 7.2 Hz,
2 H), 2.48 (m, 2 H), 2.17 (m, 2 H), 1.95-1.50 (m, 9 H), 1.42 (m,
4 H), 1.00-0.89 (m, 9 H); IR (KBr): 3435, 3205, 3082, 3026,
2935, 2870, 2493, 2361, 1674, 1454, 1417, 1370, 1331, 1155, 1070,
1004, 961, 750, 700 cm-1. MS (FAB, Pos., glycerin + m-NBA)
442 (M + H) +, 232, 171, 79. Anal. Calcd for C27H43N3O2‚HCl:
C, 67.83; H, 9.28; N, 8.79. Found: C, 67.56; H, 9.50; N, 8.71.
HPLC (Chiralcel OD-R 4.6 × 250 mm. 0.5 M NaClO4/CH3CN )
55/45. 0.5 mL/min. UV 210 nm. Temp 40 °C) retention time, 23.32
min.
(3R)-3-(2-Methylpropyl)-9-(6-phenylhexyl)-1-propyl-1,4,9-
triazaspiro[5.5]undeca-2,5-dione Hydrochloride (8). Using a
procedure identical to that described for the preparation of 5, the
title compound (63 mg, 89% ee determined by HPLC) was obtained
from Rink-isonitrile resin, 1-propylamine, N-Boc-D-leucine, and
1-(6-phenylhexyl)-4-piperidone in 23% yield as a white powder.
[R]26D +23.0° (c 0.453, MeOH); 1H NMR (200 MHz, CD3OD): δ
7.18 (m, 5 H), 4.02 (dd, J ) 7.6, 4.6 Hz, 1 H), 3.70 (m, 2 H), 3.56
(m, 2 H), 3.39 (m, 2 H), 3.11 (m, 2 H), 2.63 (dd, J ) 7.8, 7.2 Hz,
2 H), 2.48 (m, 2 H), 2.17 (m, 2 H), 1.95-1.50 (m, 9 H), 1.42 (m,
4 H), 1.00-0.89 (m, 9 H); IR (KBr): 3441, 3204, 3082, 3026,
2935, 2870, 2660, 2499, 2413, 2361, 1674, 1455, 1417, 1370, 1330,
1267, 1205, 1154, 1070, 1003, 960, 928, 899, 750, 700 cm-1. MS
(FAB, Pos., glycerin + m-NBA) 442 (M + H) +, 294, 232, 202,
171, 79. Anal. Calcd for C27H43N3O2‚HCl: C, 67.83; H, 9.28; N,
8.79. Found: C, 67.52; H, 9.51; N, 8.70. HPLC (Chiralcel OD-R
4.6 × 250 mm. 0.5 M NaClO4/CH3CN ) 55/45. 0.5 mL/min. UV
210 nm. Temp 40 °C) retention time, 25.17 min.
was washed with MeOH (10 mL), H2O (10 mL), and MeOH (10
mL) prior to use. The resin was washed with MeOH (10 mL × 3).
Elution with 10% Et3N in MeOH (10 mL × 3) was concentrated
under reduced pressure to give a red oil. Purification by column
chromatography over silica gel with CHCl3-MeOH-Et3N (38:2:
1) gave a solid, which was triturated with Et2O to yield the title
compound (189 mg, 38% yield) as a white powder. 1H NMR (200
MHz, CD3OD): δ 3.99 (dd, J ) 7.8, 4.4 Hz, 1 H), 3.50-3.20 (m,
4 H), 3.05-2.85 (m, 2 H), 2.10-1.75 (m, 5 H), 1.75-1.40 (m, 4
H), 1.00-0.85 (m, 9 H); MS (APCI, Pos., 40 V) m/z, 282 (M +
H)+, 159.
General Procedure for the Preparation of the R1 Library.
Secondary amine 3 (40 mg, 0.143 mmol) was dissolved in 1,2-
dichloroethane (3.20 mL) and equally divided into 80 wells of a
96-well plate (0.40 mL). Each well contained 0.00178 mmol of
the starting material. Then, 0.017 mL of a 0.5 M DMF solution of
the corresponding aldehyde (0.00533 mmol) was added to it. To
this mixture was added a 0.33 M DMF solution of sodium
triacetoxyborohydride (0.0211 mL, 0.00697 mmol) at room tem-
perature. The mixture was agitated for 36 h at room temperature,
acidified by the addition of 5% acetic acid in MeOH (0.050 mL),
and then loaded on cation-exchange resin (Waters OASIS MCX
Extraction Plate (30 mg) 60 µm (LP), 1.02 meq/g, 0.0306 mmol/
well), which was washed with MeOH (0.40 mL) prior to use. The
resin was washed with MeOH (0.30 mL × 2). Elution with 10%
Et3N in MeOH (0.30 mL × 2) was collected in a 96-well plate
(1.0 mL). A small amount of this elution (0.60 mL) was saved for
LC-MS analysis. The remaining solution was concentrated under
reduced pressure. The residue was dissolved in 0.064 mL of DMSO
to a concentration of 10 mM (estimated yield: 50%), which was
used for the in vitro biological assay without further purification.
3-(2-Methylpropyl)-1-propyl-1,4,9-triazaspiro[5.5]undeca-2,5-
dione (3). To a stirred solution of 1-allyloxycarbonyl-4-piperidone
(1.83 g, 10.0 mmol) in MeOH (20 mL) were successively added
1-propylamine (1.64 mL, 20.0 mmol), N-Boc-leucine (4.99 g, 20.0
mmol), and benzylisonitrile (1.22 mL, 10.0 mmol) under argon at
room temperature. The mixture was left for 3 days at room
temperature and then diluted with AcOEt (150 mL). The mixture
was washed with H2O, 1 N HCl, H2O, sat. NaHCO3, and sat. NaCl.
The organic layer was dried over MgSO4 and filtered. The filtrate
was concentrated under reduced pressure to give a yellow oil, which
was purified by flash chromatography over silica gel (Fujisilicia
FL-60D, 200 g) with AcOEt-hexane (1:2). The obtained oil was
triturated with hexane and gave the Ugi product (3.84 g, 67% yield)
3-(2-Methylpropyl)-9-(4-phenoxyphenylmethyl)-1-propyl-1,4,9-
triazaspiro[5.5]undeca-2,5-dione Hydrochloride (9). To a solution
of 3 (20.0 mg, 0.071 mmol) in DMF-AcOH (0.80-0.080 mL)
were successively added 4-phenoxybenzaldehyde (0.037 mL, 0.213
mmol) and sodium triacetoxyborohydride (45 mg, 0.213 mmol) at
room temperature. The mixture was stirred for 16 h at room
temperature and then diluted with MeOH (1.0 mL). The mixture
was loaded on cation-exchange resin (Bondesil SCX, 1.0 g, 0.6
mmol), which was washed with MeOH (2.0 mL), H2O (2.0 mL),
and MeOH (2.0 mL) prior to use. The resin was washed with MeOH
(2.0 mL × 2). Elution with 10% Et3N in MeOH (2.0 mL × 3) was
concentrated under reduced pressure to give a colorless oil.
Purification by column chromatography over silica gel with CHCl3-
MeOH (10:1) yielded a colorless oil, which was dissolved in MeOH
(10 mL). The solution was acidified by 1 N HCl (0.10 mL) and
then concentrated under reduced pressure. Trituration with Et2O
gave the title compound (31 mg, 87% yield) as a white powder.
1H NMR (200 MHz, CD3OD): δ 7.55 (m, 2 H), 7.40 (m, 2 H),
7.18 (m, 1 H), 7.05 (m, 4 H), 4.33 (s, 2 H), 4.01 (dd, J ) 7.6, 4.8
Hz, 1 H), 3.79 (m, 2 H), 3.60-3.30 (m, 4 H), 2.46 (m, 2 H), 2.17
(m, 2 H), 1.95-1.40 (m, 5 H), 0.94 (m, 9 H); IR (KBr): 3439,
3220, 3066, 2959, 2872, 2663, 2561, 1672, 1590, 1509, 1489, 1418,
1
as a white powder. H NMR (200 MHz, CD3OD): δ 7.40-7.10
(m, 5 H), 5.96 (m, 1 H), 5.30 (m, 1 H), 5.21 (m, 1 H), 4.58 (m, 2
H), 4.45 (d, J ) 15.0 Hz, 1 H), 4.42 (m, 1 H), 4.19 (d, J ) 15.0
Hz, 1 H), 3.96 (m, 2 H), 3.50 (m, 2 H), 3.25 (m, 2 H), 2.62 (m, 1
H), 2.25 (m, 1 H), 2.00-1.50 (m, 6 H), 1.41 (s, 9 H), 1.23 (m, 1
H), 1.00-0.80 (m, 9 H). The above powder was dissolved in 4 N
HCl in 1,4-dioxane (20 mL) at 0 °C. The mixture was stirred for
3 h at 0 °C. Concentration under reduced pressure gave a solid
residue, which was dissolved in MeOH (10 mL). The mixture was
stirred for 1 h at room temperature, and concentrated under reduced
pressure to give an oily residue. The residue was suspended in 1,2-
dichloroethane (20 mL). N,N-Diisopropylethylamine (4.0 mL) was
added to it. The mixture was refluxed for 16 h. After cooling to
room temperature, the mixture was diluted with AcOEt, and washed
with 1 N HCl, sat. NaHCO3, and sat. NaCl. The organic layer was
dried over MgSO4 and filtered. The filtrate was concentrated under
reduced pressure to give a yellow amorphous substance, which was
purified by column chromatography over silica gel with AcOEt-
hexane (2:3) to yield the cyclic compound (651 mg, 27%) as a
1370, 1330, 1241, 1200, 1172, 1072, 931, 873, 787, 694 cm-1
.
MS (APCI, Pos. 40 V) m/z 464 (M + H)+. Anal. Calcd for
C28H37N3O3‚HCl‚H2O: C, 64.59; H, 7.74; N, 8.07. Found: C,
64.38; H, 7.67; N, 8.07.
1-Butyl-3-(2-methylpropyl)-9-(6-phenylhexyl)-1,4,9-triazaspiro-
[5.5]undeca-2,5-dione Hydrochloride (10). Using a procedure
identical to that described for the preparation of 1c, the title
compound (34 mg) was obtained from 1-(6-phenylhexyl)-4-
piperidone, 1-butylamine, N-Boc-leucine, and methylene-isonitrile
resin in 35% yield as a white powder. Mp 127-133 °C; 1H NMR
(200 MHz, CD3OD): δ 7.30-7.06 (m, 5 H), 4.02 (dd, J ) 7.8,
4.8 Hz, 1 H), 3.70 (m, 2 H), 3.56 (m, 2 H), 3.43 (m, 2 H), 3.11 (m,
2 H), 2.63 (t, J ) 7.8 Hz, 2 H), 2.46 (m, 2 H), 2.18 (m, 2 H),
1.95-1.50 (m, 9 H), 1.50-1.25 (m, 6 H), 0.97 (m, 9 H); IR
(KBr): 3447, 3199, 2934, 2869, 2663, 2502, 2440, 1673, 1455,
1418, 1372, 1329, 1152, 1086, 1003, 962, 750, 700 cm-1. MS
(MALDI-TOF, Pos.) m/z 456 (M + H)+. Anal. Calcd for
1
white powder. H NMR (200 MHz, CD3OD): δ 6.10-5.85 (m, 1
H), 5.31 (m, 1 H), 5.22 (m, 1 H), 4.59 (m, 2 H), 4.16-3.92 (m, 3
H), 3.70-3.40 (m, 4 H), 2.10-1.40 (m, 8 H), 1.30 (m, 1 H), 1.10-
0.80 (m, 9 H); MS (APCI, Pos., 40 V) m/z 316 (M + H)+, 308 (M
- OAllyl)+. To a stirred solution of the above powder in CH2Cl2
(4.0 mL) under argon were successively added AcOH (0.24 mL,
4.2 mmol), tributyltin hydride (0.57 mL, 2.1 mmol), and tetrakis-
(triphenylphosphine)-palladium (0) (40 mg, 0.035 mmol) at room
temperature. The mixture was stirred for 20 min at room temper-
ature and then diluted with MeOH (20 mL). The mixture was loaded
on cation-exchange resin (Bondesil SCX, 5.0 g, 3.0 mmol), which

4150 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Habashita et al.
C28H45N3O2‚HCl‚0.4H2O: C, 67.35; H, 9.45; N, 8.41. Found: C,
67.67; H, 9.39; N, 8.42.
mL × 4), CH2Cl2 (1 mL × 3), MeOH (1 mL × 2), and CH2Cl2 (1
mL × 3). To it was added 50% TFA in CH2Cl2 (2 mL). The mixture
was agitated for 5 min at room temperature and then drained. The
resin was suspended in 50% TFA in CH2Cl2 (2 mL). The mixture
was agitated for 20 min at room temperature and then drained. The
resin was washed with CH2Cl2 (2 mL × 4), MeOH (2 mL × 2),
toluene (2 mL × 4), and 1.25 M AcOH in toluene (2 mL). The
resin was suspended in 1.25 M AcOH in toluene (2.0 mL). The
mixture was agitated for 16 h at 90 °C. After cooling to room
temperature, the mixture was filtered, and the resin was washed
with MeOH (2.0 mL × 2). The filtrate and washings were combined
(6.0 mL). Then, 0.10 mL of this solution was saved for LC-MS
analysis (0.00050 mmol). The remaining solution was concentrated
under reduced pressure. The residue was weighted and dissolved
in DMSO to a concentration of 10 mM for the in vitro biological
assay.
9-Benzyl-1-butyl-3-(2-methylpropyl)-1,4,9-triazaspiro[5.5]-
undeca-2,5-dione Hydrochloride (11). Using a procedure identical
to that described for the preparation of 1c, the title compound (217
mg) was obtained from 1-benzyl-4-piperidone, 1-butylamine, N-
Boc-leucine, and methylene-isonitrile resin in 59% yield as a white
1
powder. H NMR (200 MHz, CD3OD): δ 7.64-7.44 (m, 5 H),
4.36 (s, 2 H), 4.01 (dd, J ) 7.8, 4.8 Hz, 1 H), 3.77 (m, 2 H), 3.55-
3.35 (m, 4 H), 2.60-2.30 (m, 2 H), 2.17 (m, 2 H), 1.95-1.75 (m,
1 H), 1.75-1.60 (m, 2 H), 1.60-1.45 (m, 2 H), 1.45-1.20 (m, 2
H), 1.10-0.80 (m, 9 H); IR (KBr): 3435, 3230, 2957, 2871, 2505,
2454, 1680, 1647, 1459, 1413, 1370, 1326, 1147, 955, 746, 698
cm-1. MS (MALDI-TOF, Pos.) m/z 386 (M + H)+, 91. Anal. Calcd
for C23H35N3O2‚HCl: C, 65.46; H, 8.60; N, 9.96. Found: C, 65.09;
H, 8.63; N, 9.88.
1-Butyl-3-cyclohexylmethyl-9-(4-methoxyphenylmethyl)-1,4,9-
triazaspiro[5.5]undeca-2,5-dione Hydrochloride (14). Using a
procedure identical to that described for the preparation of 2b, the
title compound (17 mg) was obtained from 1-allyloxycarbonyl-4-
piperidone, 1-butylamine, N-Boc-cyclohexylalanine, methylene-
isonitrile resin, and 4-methoxybenzaldehyde in 59% yield as a white
(3R)-3-Cyclohexylmethyl-9-(6-phenylhexyl)-1-propyl-1,4,9-
triazaspiro[5.5]undeca-2,5-dione Hydrochloride (12). Using a
procedure identical to that described for the preparation of 1c, the
title compound (100 mg) was obtained from 1-(6-phenylhexyl)-4-
piperidone, 1-propylamine, N-Boc-D-cyclohexylalanine, and me-
thylene-isonitrile resin in 74% yield as a white powder. Mp 105-
1
1
109 °C; [R]25 +19.5° (c 0.126, MeOH); H NMR (300 MHz,
powder. H NMR (200 MHz, CD3OD): δ 7.47 (d, J ) 8.8 Hz, 2
D
H), 7.03 (d, J ) 8.8 Hz, 2 H), 4.29 (s, 2 H), 4.04 (dd, J ) 7.6, 4.8
Hz, 1 H), 3.83 (s, 3 H), 3.74 (m, 2 H), 3.55-3.35 (m, 4 H), 2.41
(m, 2 H), 2.15 (m, 2 H), 1.85-1.55 (m, 7 H), 1.55-1.42 (m, 3 H),
1.42-1.30 (m, 3 H), 1.30-1.10 (m, 2 H), 1.08-0.80 (m, 5 H); IR
(KBr): 3436, 3221, 2926, 2851, 2666, 2560, 2362, 1672, 1613,
1585, 1517, 1448, 1419, 1373, 1305, 1255, 1182, 1031, 929, 826,
795, 587 cm-1. MS(FAB., Pos., Glycerin + m-NBA) m/z 456 (M
+ H)+, 121. Anal. Calcd for C27H41N3O3‚HCl‚1.5H2O: C, 62.47;
H, 8.74; N, 8.09. Found: C, 62.59; H, 8.35; N, 7.90.
CD3OD): δ 7.29-7.09 (m, 5 H), 4.05 (dd, J ) 7.59, 4.48 Hz, 1
H), 3.85-3.63 (m, 2 H), 3.61-3.46 (m, 2 H), 3.42-3.33 (m, 2 H),
3.19-3.05 (m, 2 H), 2.67-2.59 (m, 2 H), 2.50-2.30 (m, 2 H),
2.29 - 2.08 (m, 2 H), 1.85-1.09 (m, 21 H), 1.08-0.84 (m, 5 H);
IR (KCl): 3410, 2926, 2853, 2514, 2439, 1670, 1450, 1417, 1371,
748, 700 cm-1. Exact mass spectrum (FAB, Pos.) m/z calcd for
C30H48N3O2: 482.3747; found: 482.3752.
(3S)-3-Cyclohexylmethyl-9-(6-phenylhexyl)-1-propyl-1,4,9-
triazaspiro[5.5]undeca-2,5-dione Hydrochloride (13). Using a
procedure identical to that described for the preparation of 1c, the
title compound (100 mg) was obtained from 1-(6-phenylhexyl)-4-
piperidone, 1-propylamine, N-Boc-L-cyclohexylalanine, and meth-
ylene-isonitrile resin in 82% yield as a white powder. [R]25D -18.7°
1-Butyl-3-(2-methylpropyl)-9-(4-phenoxyphenylmethyl)-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione Hydrochloride (15). Using a
procedure identical to that described for the preparation of 2b, the
title compound (16 mg) was obtained from 1-allyloxycarbonyl-4-
piperidone, 1-butylamine, N-Boc-leucine, methylene-isonitrile resin,
and 4-phenoxybenzaldehyde in 48% yield as a white powder. Mp
1
(c 1.092, MeOH); H NMR (300 MHz, CD3OD): δ 7.29-7.09
(m, 5 H), 4.05 (dd, J ) 7.59, 4.48 Hz, 1 H), 3.85-3.63 (m, 2 H),
3.61-3.46 (m, 2 H), 3.42-3.33 (m, 2 H), 3.19 - 3.05 (m, 2 H),
2.67-2.59 (m, 2 H), 2.50-2.30 (m, 2 H), 2.29-2.08 (m, 2 H),
1.85-1.09 (m, 21 H), 1.08-0.84 (m, 5 H); IR (KCl): 3410, 2926,
2853, 2514, 2439, 1670, 1450, 1417, 1371, 748, 700 cm-1. Exact
mass spectrum (FAB, Pos.) m/z calcd. for C30H48N3O2: 482.3747;
found: 482.3752.
1
158-164 °C; H NMR (200 MHz, CD3OD): δ 7.54 (d, J ) 8.8
Hz, 2 H), 7.40 (m, 2 H), 7.18 (m, 1 H), 7.11-7.00 (m, 4 H), 4.33
(s, 2 H), 4.01 (dd, J ) 7.6, 4.8 Hz, 1 H), 3.80 (m, 2 H), 3.60-3.35
(m, 4 H), 2.43 (m, 2 H), 2.18 (m, 2 H), 1.80 (m, 1 H), 1.70 (m, 1
H), 1.54 (m, 2 H), 1.37 (m, 3 H), 1.00-0.90 (m, 9 H); IR (KBr):
3440, 3221, 3066, 2957, 2871, 2559, 1673, 1590, 1509, 1489, 1419,
1371, 1329, 1242, 1172, 873, 693 cm-1. MS (FAB, Pos., Glycerin
+ m-NBA) 478 (M + H)+, 183. Anal. Calcd for C29H39N3O3‚HCl‚
H2O: C, 65.46; H, 7.96; N, 7.90. Found: C, 65.67; H, 7.89; N,
7.83.
General Procedure for the Preparation of the R1R2R3
Library. The methylene-isonitrile resin (1.00 g, 0.94 mmol) was
suspended in mixed solvent THF-MeOH (1:1, 6 mL). To it were
successively added 1-butylamine (0.464 mL, 4.70 mmol), N-Boc-
amino acid (4.70 mmol), and 1-allyloxycarbonyl-4-piperidone
(0.861 g, 4.70 mmol). The mixture was agitated for 16 h at 65 °C
under argon. After cooling to room temperature, the mixture was
drained. The resin was washed with THF (6 mL × 2), MeOH (6
mL × 2 ), and CH2Cl2 (6 mL × 3). An aliquot of this resin was
dried under reduced pressure to measure its IR spectra. IR (KBr):
1714, 1669 cm-1. The resin was suspended in CH2Cl2 (6 mL). To
it were successively added acetic acid (0.323 mL, 5.64 mmol),
tributyltin hydride (0.759 mL, 2.82 mmol), and tetrakis(triph-
enylphosphine)palladium (0) (0.109 g, 0.094 mmol) at room
temperature. The mixture was agitated for 6 h at room temperature.
The mixture was drained. The resin was washed with CH2Cl2 (6
mL × 4), MeOH (6 mL × 2), and CH2Cl2 (6 mL × 3), and dried
under reduced pressure to give a yellow resin. The loading ratio
was determined by a picrate assay to be 0.66 mmol/g resin (81%
conversion). Then, 100 mg of this resin (0.066 mmol) was washed
with DMF (2 mL × 2) and then suspended in DMF (0.2 mL). A
1.0 M solution of the corresponding aldehyde in DMF (0.33 mL,
0.33 mmol) was added. The mixture was agitated for 5 min at room
temperature. Then a 0.33 M solution of sodium triacetoxyborohy-
dride in DMF (1.0 mL, 0.33 mmol) and acetic acid (0.10 mL) were
added to it. The mixture was agitated for 24 h at room temperature.
The mixture was drained, and the resin was washed with DMF (1
1-Butyl-3-cyclohexylmethyl-9-(4-phenoxyphenylmethyl)-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione Hydrochloride (16). Using a
procedure identical to that described for the preparation of 2b, the
title compound (23 mg) was obtained from 1-allyloxycarbonyl-4-
piperidone, 1-butylamine, N-Boc-cyclohexylalanine, methylene-
isonitrile resin, and 4-phenoxybenzaldehyde in 70% yield as a white
1
powder. H NMR (200 MHz, CD3OD): δ 7.74-7.56 (m, 1 H),
7.53 (d, J ) 8.8 Hz, 2 H), 7.40 (m, 2 H), 7.18 (m, 1 H), 7.10-7.00
(m, 3 H), 4.33 (s, 2 H), 4.04 (dd, J ) 7.4, 4.8 Hz, 1 H), 3.80 (m,
2 H), 3.60-3.35 (m, 4 H), 2.43 (m, 2 H), 2.17 (m, 2 H), 1.90-
1.60 (m, 7 H), 1.60-1.45 (m, 2 H), 1.45-1.30 (m, 2 H), 1.30-
1.15 (m, 4 H), 1.10-0.80 (m, 5 H); IR (KBr): 3434, 3210, 3064,
2926, 2851, 2664, 2558, 1672, 1590, 1509, 1489, 1418, 1373, 1241,
1173, 1118, 1072, 1048, 929, 873, 723, 694, 542 cm-1. MS (FAB.,
Pos., Glycerin + m-NBA) m/z 518 (M + H)+, 334, 279, 183. Anal.
Calcd for C32H43N3O3‚HCl‚0.5H2O: C, 68.25; H, 8.05; N, 7.46.
Found: C, 68.23; H, 7.88; N, 6.77.
9-(1,4-Benzodioxane-6-yl)-1-butyl-3-(2-methylpropyl)-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione Hydrochloride (17). Using a
procedure identical to that described for the preparation of 2b, the
title compound (147 mg) was obtained from 1-allyloxycarbonyl-
4-piperidone, 1-butylamine, N-Boc-leucine, methylene-isonitrile
resin, and 6-formyl-1,4-benzodioxane in 31% yield as a white

Low-Molecular-Weight CCR5 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4151
1
(6) Holmes, W. E.; Lee, J.; Kuang, W. J.; Rice, G. C.; Wood, W. I.
Structure and functional expression of a human interleukin-8 receptor.
Science 1991, 253, 1278-1280.
powder. Mp 194-198 °C; H NMR (200 MHz, CD3OD): δ 7.08
(d, J ) 2.2 Hz, 1 H), 7.01 (dd, J ) 8.2, 2.2 Hz, 1 H), 6.93 (d, J )
8.2 Hz, 1 H), 4.27 (s, 4 H), 4.23 (s, 2 H), 4.01 (dd, J ) 7.8, 4.8
Hz, 1 H), 3.72 (m, 2 H), 3.55-3.35 (m, 4 H), 2.43 (m, 2 H), 2.16
(m, 2 H), 1.80 (m, 1 H), 1.67 (m, 2 H), 1.55 (m, 2 H), 1.37 (m, 2
H), 1.00-0.90 (m, 9 H); IR (KBr): 3436, 2957, 2565, 1672, 1591,
1512, 1465, 1419, 1294, 1262, 1152, 1067, 888 cm-1. MS (MALDI-
TOF, Pos.) m/z 444 (M + H)+, 149. Anal. Calcd for C25H37-
N3O4‚HCl: C, 62.55; H, 7.98; N, 8.75. Found: C, 62.40; H, 8.20;
N, 8.66.
9-(1,4-Benzodioxane-6-yl)-1-butyl-3-cyclohexyl-1,4,9-triazaspiro-
[5.5]undeca-2,5-dione Hydrochloride (18). Using a procedure
identical to that described for the preparation of 2b, the title
compound (698 mg) was obtained from 1-allyloxycarbonyl-4-
piperidone, 1-butylamine, N-Boc-cyclohexylalanine, methylene-
isonitrile resin, and 6-formyl-1,4-benzodioxane in 57% yield as a
white powder. Mp 163-169 °C; 1H NMR (200 MHz, CD3OD): δ
7.08 (d, J ) 2.2 Hz, 1 H), 6.99 (dd, J ) 8.0, 2.2 Hz, 1 H), 6.92 (d,
J ) 8.0 Hz, 1 H), 4.27 (s, 4 H), 4.23 (s, 2 H), 4.04 (dd, J ) 7.6,
4.8 Hz, 1 H), 3.74 (m, 2 H), 3.60-3.35 (m, 4 H), 2.43 (m, 2 H),
2.15 (m, 2 H), 1.90-1.60 (m, 7 H), 1.60-1.45 (m, 2 H), 1.45-
1.30 (m, 2 H), 1.30-1.10 (m, 4 H), 1.10-0.80 (m, 5 H); IR
(KBr): 3436, 2926, 2852, 2511, 1675, 1645, 1591, 1511, 1418,
1374, 1294, 1261, 1068, 1050, 930, 888 cm-1. MS (MALDI-TOF,
Pos.) m/z 484 (M + H)+, 149. Anal. Calcd for C28H41N3O4‚HCl‚
0.3H2O: C, 64.00; H, 8.17; N, 8.00. Found: C, 64.00; H, 7.94; N,
7.90.
(7) Murphy, P. M.; Tiffany, H. L. Cloning of complementary DNA
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9-(4-Allyloxyphenylmethyl)-1-butyl-3-cyclohexyl-1,4,9-tri-
azaspiro[5.5]undeca-2,5-dione Hydrochloride (19). Using a
procedure identical to that described for the preparation of 2b, the
title compound (10 mg) was obtained from 1-allyloxycarbonyl-4-
piperidone, 1-butylamine, N-Boc-cyclohexylalanine, methylene-
isonitrile resin, and 4-allyloxybenzaldehyde in 34% yield as a white
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(16) For preparations of diketopiperazine via Ugi MCR see the follow-
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synthesis of diverse 1,4-benzodiazepine-2,5-diones using the Ugi four-
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C.; Cherrier, M.-P. Novel applications of ethyl glyoxalate with the
Ugi MCR. Tetrahedron Lett. 1999, 40, 5296-5299. (c) Hulme, C.;
Ma, L.; Kumar, V. N.; Krolikowski, P. H.; Allen, A. C.; Labaudiniere,
R. Novel applications of resin bound R-amino acids for the synthesis
of benzodiazepines (via Wang resin) and ketopiperazines (via
hydroxymethyl resin). Tetrahedron Lett 2000, 41, 1509-1514. (d)
Golebiowski, A.; Jozwik, J.; Klopfenstein, S. R.; Colson, A.-O.;
Grieb, A. L.; Russell, A. F.; Rastogi, V. L.; Diven, C. F.; Portlock,
D. E.; Chen, J. J. Solid-supported synthesis of putative peptide-turn
mimetics via Ugi reaction for diketopiperazine formation. J. Comb.
Chem. 2002, 4, 584-590. (e) Kennedy, A. L.; Fryer, A. M.; Josey,
J. A. A new resin-bound universal isonitrile for the Ugi 4CC
reaction: preparation and applications to the synthesis of 2,5-
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4, 1167-1170.
1
powder. H NMR (200 MHz, CD3OD): δ 7.46 (d, J ) 8.4 Hz, 2
H), 7.04 (d, J ) 8.4 Hz, 2 H), 6.06 (m, 1 H), 5.41 (m, 1 H), 5.28
(m, 2 H), 4.59 (m, 2 H), 4.28 (s, 2 H), 4.04 (dd, J ) 7.2, 4.8 Hz,
1 H), 3.77 (m, 2 H), 3.55-3.35 (m, 4 H), 2.39 (m, 2 H), 2.16 (m,
2 H), 1.90-1.60 (m, 7 H), 1.60-1.45 (m, 2 H), 1.45-1.30 (m, 2
H), 1.30-1.10 (m, 3 H), 1.10-0.80 (m, 5 H); IR (KBr): 3436,
3202, 2925, 2853, 2661, 2514, 2362, 1669, 1611, 1583, 1515, 1450,
1417, 1372, 1306, 1252, 1183, 998, 838, 796 cm-1. MS (MALDI-
TOF, Pos.) m/z 482 (M + H)+, 147. Anal. Calcd for C29H43N3O3‚
HCl‚0.4H2O: C, 66.30; H, 8.60; N, 8.00. Found: C, 66.45; H, 8.40;
N, 7.61.
Acknowledgment. We thank Mr. Y. Ushio, Mr. T. Inohara,
and Mrs. S. Fujita for their technical assistance and valuable
comments. We also thank Dr. I. C. Choong for the critical
reading of this manuscript.
(17) Domling, A.; Ugi, I. Multicomponent reactions with isocyanides.
Angew. Chem., Int. Ed. 2000, 39, 3168-3210.
(18) Chen, J. J.; Golebiowski, A.; Klopfenstein, S. R.; West, L. The
universal Rink-isonitrile resin: applications in Ugi reactions. Tetra-
hedron Lett. 2002, 43, 4083-4085.
Supporting Information Available: Epimerization study in
Scheme 1 and structural lists of building blocks in R1, R2, and R3
libraries. This material is available free of charge via the Internet
at http://pubs.acs.org.
(19) Hulme, C.; Ma, L.; Cheerier, M.-P.; Romano, J. J.; Morton, G.;
Duquenne, C.; Salvino, J.; Labaudiniere, R. Novel applications of
convertible isonitriles for the synthesis of mono and bicyclic
γ-lactams via a UDC strategy. Tetrahedron Lett. 2000, 41, 1883-
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(21) After the completion of this work, we reinvestigated whether
epimerization occurred with optically pure amino acids during
synthesis, as shown in Scheme 1. In some cases, epimerization could
be observed. As a result of an investigation to avoid this epimeriza-
tion, we found that the removal of acetic acid in the diketopiperazine
formation step completely prevented epimerization. For details, see
the Supporting Information.
(22) In the synthesis of compound 1e, 1,9-bis(5-phenylpentyl)-3-(2-
methylpropyl)-1,4,9-triazaspiro[5.5]undeca-2,5-dione was obtained in
45% yield. This result indicated that 1-(5-phenypentyl)-4-piperidone
might decompose during the reaction to produce 5-phenylpenty-
lamine. For the decomposition of 4-piperidone, see the following:
(a) Shimano, M.; Meyers, A. I. Asymmetric Michael-type additions
of lithium amides to aromatic systems leading to novel â-amino acids.
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JM060051S