Discovery of orally available spirodiketopiperazine-based CCR5 antagonists

Article abstract of DOI:10.1016/j.bmc.2010.05.057

Using the previously reported novel spirodiketopiperazine scaffold, the design and synthesis of orally available CCR5 antagonists was undertaken. Compounds possessing a carboxylic acid function in the appropriate position showed improved oral exposure (AU

Full text of DOI:10.1016/j.bmc.2010.05.057

Bioorganic & Medicinal Chemistry 18 (2010) 5208–5223
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of orally available spirodiketopiperazine-based CCR5 antagonists
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 b, 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:
Using the previously reported novel spirodiketopiperazine scaffold, the design and synthesis of orally
available CCR5 antagonists was undertaken. Compounds possessing a carboxylic acid function in the
appropriate position showed improved oral exposure (AUC) relative to the initial chemical leads without
reduction in the antagonist activity. The optimized compound 40 was found to show potent anti-HIV
activity. Full details of structure–activity relationship (SAR) study are presented.
Received 29 March 2010
Revised 18 May 2010
Accepted 19 May 2010
Available online 25 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CCR5
Chemokine
Anti HIV
1. Introduction
PK data, large clearance (CL) and tissue distribution values (Vss)
were assumed to be one of the plausible reasons for poor AUC.
Despite a tremendous effort to develop antiretroviral therapy,
millions of people in the world are still suffering from Acquired Im-
muneDeficiencySyndrome(AIDS).1 Althoughseveral classesofanti-
retroviral medications are currently available, challenges remain
with anti-HIV therapies due to a gradual spread of drug-resistant
strains, severe side effects, and expensive therapeutic costs among
others. Hence there is a growing need for novel treatments for AIDS
patients who are treatment-experienced and have become resistant
to one or multiple classes of antiretroviral agents.2 These issues
require new anti-HIV drugs to have a different mode of action from
conventional drugs. CCR5 antagonists work by inhibiting HIV entry
into target cells and are one of the most promising approaches to
treat AIDS because of their ability to inhibit a previously untargeted
step in the HIV-1 replication cycle.3 Maraviroc is currently the only
approved CCR5 antagonist on the market.4
Herein we report full details of the discovery of a new chemical
lead 40 with an improved PK profile.
2. Results and discussion
2.1. Chemistry
As shown in Schemes 1 and 2, compounds 1a, 1b, 2–13, 15–18,
21–23, 25, 26, and 28 were efficiently synthesized using solid phase
synthesis.5 The Ugi four-component condensation using the Rink-
isonitrile resin is outlined in Scheme 1a. A mixture of 1-N-allyloxy-
carbonyl piperidone 41a, an optional alkyl amine 42, an optional
N-Boc-amino acid 43, and the Rink-isonitrile resin 44 in THF/MeOH
(1:1) was shaken at 65 °C to afford 45. Palladium catalyzed removal
of the N-allyloxycarbonyl moiety of 45 was followed by reductive
amination with an optional aryl aldehyde to produce 46. Removal
of the Boc protecting group of 46 followed by heating under acidic
conditions resulted in cyclization accompanied by removal of the
supporting-resin to afford spirodiketopiperazines 1a, 1b, 2–13,
15–18, 23, 25, 26, and 28. Compound 14 was prepared by alkaline
hydrolysis of 15. 2-Butynylamine 42f was prepared from 2-butynyl
alcohol as described in Scheme 1b. O-Methanesulfonylation of 47
followed by the substitution reaction with potassium phthalimide
We previously reported the discovery of spirodiketopiperazine
derivatives 1a and 1b (Fig. 1), as structurally novel CCR5 antagonist
leads generated from a combinatorial library targeting GPCRs.5,7
However, it was unfortunate that both 1a and 1b showed poor oral
exposure (AUC) in rodents (Table 7). Based on the analysis of their
* Corresponding author. Tel.: +81 75 962 9314; fax: +81 75 961 1151.
E-mail address: r.nishizawa@ono.co.jp (R. Nishizawa).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.057

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5209
CH3
X
O
O
N1
5
8
2
7
9
N
3
6
R
ClH
4
N
H
10
11
O
*
1a X = H (3RS) R =
1b X = H (3RS) R =
*
40 X = CO2H (3S) R =
*
CH3
H3C
Figure 1. Molecular design of orally active chemical leads.
O
O
R3
R3
R2
O
R2
O
R3
2
N
N
Ar
R2
b, c
a
(a)
C
N+ Pol
HO
+
+
+
Boc
R1
N
O
N
Boc
N
Boc
R1
N
N
NH2
N
H
H
H
O
N
Pol
N
Pol
H
H
41
42
43
44
45
46
a R1 = alloc
a R2 = n-Bu
b R2 = n-Pr
c R2 = i-Bu
a (2RS) R3 = c-Hexylmethyl
b (2RS) R3 = i-Bu
c (2R) R3 = i-Bu
d R2 = (3-OTBS)- d (2S) R3 = i-Bu
n-Bu
e (2RS) R3 = t-Butylmethyl
e R2 = 2-Butenyl f (2RS) R3 =n-Pr
f R2 = 2-Butynyl
g R2 = Bn
g (2RS) R3 = c-Pentylmethyl
h (2RS) R3 = Methoxymethyl
i
j
(2RS) R3 = Benzyloxycarbonylmethyl
(2RS) R3 = 3-c-Hexylpropyl
R2
O
Ar
N
d, e
f
3
N
R3
15
14
N
H
O
1a, 1b, 2-13, 15-18, 23, 25, 26 and 28
O
g, h
OH
i
N
ClH
NH2
(b)
O
47
48
42f
Scheme 1. Solid phase synthesis of spirodiketopiperazines 1a, 1b, 2–18, 23, 25, 26, and 28. Reagents and conditions: (a) THF/MeOH (1:1), 65 °C; (b) Pd(PPh3)4, Bu3SnH, AcOH/
CH2Cl2, rt; (c) Ar-CHO, NaBH(OAc)3, AcOH/DMF; (d) TFA/CH2Cl2 (1:1); (e) AcOH/toluene, 90 °C and then 4 N HCl/AcOEt; (f) 2 N NaOH, MeOH; (g) MsCl, Et3N, CH2Cl2; (h)
potassium phthalimide, DMF, 70 °C; (i) NH2NH2ꢀH2O, EtOH, then concd HCl, reflux.
CH3
O
O
R3
b
a
N
N+ Pol
R3
2
N
R1
N
O
C
+
+
+
R1
N
R3
*
N
H
Boc
HO
Boc
R1
N
N
N
H
NH2
H
O
N
Pol
O
O
H
41
b R1 = Cbz
c R1 = 6-Phenylhexyl
43
42a
44
49
21 and 22
a (2RS) R3 = c-Hexylmethyl
b (2RS) R3 = i-Bu
Scheme 2. Synthesis of 21 and 22. Reagents and conditions: (a) THF/MeOH (1:1), 65 °C; (b) TFA/CH2Cl2 (1:1); (c) AcOH/toluene, 90 °C and then 4 N HCl/AcOEt.
afforded48, deprotectionofwhichwithhydrazinehydrateproduced
42f in good yield. As described in Scheme 2, compounds 21 and 22
were also synthesized from 1-Cbz-piperidone 41b and 1-(6-phenyl-
hexyl)-4-piperidone 41c, respectively, by the solid phase synthesis.
Solution phase synthesis of compounds 24, 27, and 29–40 was
carried out by reductive alkylation of 52a and 52b with an optional
aryl aldehyde, respectively, as described in Scheme 3. Compounds
52a and 52b were prepared by solution phase synthesis. The Ugi

5210
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
CH3
CH3
C
+
N+
O
O
Ph
HO
a - c
O +
+
N
N
Ph
2
NH2
N
3
N
Boc
N
H
O
N
H
O
41d
42a
43a (2RS)
43k (2S)
50
51a (3RS)
51b (3S)
CH3
CH3
O
O
N
Ar
N
d
e
3
HN
3
N
N
H
HCl
N
HCl
H
O
O
52a (3RS)
52b (3S)
24, 27, 29-39 and 40
Scheme 3. Solution phase synthesis of spirodiketopiperazines, 24, 27, and 29–40. Reagents and conditions: (a) MeOH, 55 °C; (b) concd HCl, 55 °C; (c) AcOH/toluene, 80 °C;
(d) H2, Pd(OH)2/C, EtOH, 50 °C, then 4 N HCl/AcOEt (50–90% in four steps); (e) Ar-CHO, NaBH(OAc)3, AcOH, DMF and then 4 N HCl/AcOEt (50–90%).
CO2H
CHO
a, b
O
O
53
54
CHO
CHO
O
HO
c
O
O
55
56
O
MeO2C
CHO
HO2C
f
d, e
CHO
HN
OH
O
O
59
57
58
CO2Me
CO2Me
CHO
O2N
MsHN
MsHN
g, h
i, b
O
O
O
60
61
62
H2NO2S
CHO
H2NO2S
d
OH
O
64
63
Scheme 4. Synthesis of aryl aldehydes 54, 56, 58, 59, 62, and 64. Reagents and conditions: (a) i-BuOCOCl, Et3N, THF then NaBH4, H2O; (b) MnO2, DME; (c) BBr3, CH2Cl2;
(d) 4-fluorobenzaldehyde, K2CO3, DMA, 150 °C; (e) NaOH, MeOH; (f) EDC, HOBt, MeNH2, DMF; (g) H2, 5% Pd/C, AcOEt; (h) MsCl, pyridine, THF; (i) DIBAL, THF.
four-component condensation of 1-benzyl-4-piperidone 41d,
n-butylamine 42a, N-Boc-1-cyclohexylalanine 43a and 2-(4-mor-
pholinyl)-ethylisonitrile 50 afforded spirodiketopiperazine 51a.
Catalytic hydrogenation of 51a followed by the acid treatment pro-
vided 52a as a hydrochloride salt. Compound 52b was prepared by
the same procedures as described above using 43k instead of 43a
as one of the starting materials. Reductive alkylation of 52b with
an optional aldehyde produced 24, 27, and 29–40, respectively.
Commercially unavailable aryl aldehydes 54, 56, 58, 59, 62, and
64 were prepared as outlined in Scheme 4.
ported 4-phenoxyphenylethyl tosylate, which was prepared by
O-tosylation of 4-phenoxyphenylethanol 65 with polystyrene-sup-
ported tosyl chloride in pyridine, followed by the treatment with
hydrogen chloride afforded 19.
Asdescribed in Scheme6.9-N-phenyl analog 20was prepared by the
substitution reaction of ethyl 4-fluorobenzoate with 52a in the pres-
ence of potassium carbonate in DMSO followed by the acid treatment.
2.2. CCR5 antagonist activity
Synthesis of 9-N-(4-phenoxy)phenylethyl analog 19 is de-
scribed in Scheme 5. 9-N-Alkylation of 52b with polymer-sup-
Compounds listed in Tables 1–6 were evaluated for their
inhibitory activities against calcium mobilization of human CCR5

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5211
CH3
OH
a, b
O
O
N
N
O
N
HCl
H
O
65
19
Scheme 5. Synthesis of 19. Reagents and conditions: (a) polystyrene-supported tosyl chloride, pyridine, CH2Cl2; (b) 52b, i-Pr2NEt, MeCN and then 4 N HCl/AcOEt (48% in two
steps).
CH3
simple heterocyclic scaffold in which diversity can be introduced
at up to four positions, and can be prepared from readily available
a
-amino acids. (2) Very few compounds with this spirodiketopip-
O
erazine scaffold have been reported at the starting point of this
project. (3) The predicted 3D structure of spirodiketopiperazine
suggested that utilization of three substituents on this template
might have a similar orientation to three side chains on the type
I b-turn structure of the protein.
a
(3RS)
O
O
N
52a
3
N
N
H
HCl
20
O
H3C
Table 2
Effect of the 3-substituent on activity profiles
Scheme 6. Synthesis of 20. Reagents and conditions: ethyl 4-fluorobenzoate,
K2CO3, DMSO and then 4 N HCl/AcOEt (27%).
CH3
O
Table 1
Effect of the 1-N-substituent on activity profiles
O
N
1
N
3
R
O
N
H
HCl
O
R
O
O
Compds
R
C3
IC50
T1/2 in rat
liver
N
1
(3RS)
CH3
Configuration (nM)
(R and/or S)
N
3
Ca
assay
microsomesa
(min)
N
HCl
H
H3C
CH3
8
9
(3S)
84
7.1
NTb
12
Compds
R
IC50 (nM) Ca
assay
T1/2 in rat liver microsomesa
(min)
*
*
CH3
CH3
H3C
H3C
(3R)
(3RS)
130
79
2
3
500
330
NTb
*
*
CH3
CH3
15
CH3
10
CH3
OH
*
*
CH3
CH3
11
12
(3RS)
(3RS)
50
52
22
20
4
>3000
12
H3C
H3C
*
*
*
5
6
69
54
14
15
*
*
O
13
14
(3RS)
320
16
CH3
H3C
NTb
CO2H
O
(3RS)
>3000
*
*
*
7
300
94
NTb
12
15
16
(3RS)
(3RS)
590
NTb
23
O
1b
H3C
*
a
The data show T1/2 after incubating with the 0.2 mg/mL rat liver microsomes.
NT: not tested.
>3000
b
*
1a
1b
(3RS)
(3RS)
28
94
24
12
overexpressed CHO cells (hCCR5/CHO) stimulated with MIP-1
and with IC50 values as the mean of two experiments.6
a
*
*
CH3
As described in our previous paper,5 the 1,4,9-triazaspi-
ro[5.5]undecane-2,5-dione (spirodiketopiperazine) is an attractive
scaffold to use to identify small molecule ligands of GPCRs because
of the following three reasons. (1) This spirodiketopiperazine is a
CH3
a
The data show T1/2 after incubating with the 0.2 mg/mL rat liver microsomes.
NT: not tested.
b

5212
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
Table 3
Effect of the 9-N-substituent on activity profiles
CH3
O
1
HCl
N
8
7
2
9
3
R2
R1
N
6
5
4
(3R and/or 3S)
N
10
11
H
O
b
Compds
R1
R2
C3 Configuration
(3R and/or 3S)
IC50 (nM)
Ca assay
T1/2 in rat liver
Calculated pKa
microsomesa (min)
*
*
CH3
17
(3RS)
900
NTc
7.54
H3C
*
*
*
*
18
19
(3RS)
(3S)
120
8.3
7.53
7.51
OMe
O
>3000
NTc
*
O
NTc
4.12
H3C
O
20
(3RS)
>3000
*
O
*
21
22
(3RS)
(3RS)
3000
270
NTc
NTc
2.52
8.24
O
*
*
*
*
CH3
H3C
*
*
1a
1b
(3RS)
(3RS)
28
94
24
12
7.15
7.18
O
O
*
CH3
H3C
a
b
c
The data show T1/2 after incubating with the 0.2 mg/mL rat liver microsomes.
Calculated by ADMET predictor (ver. 4.0).
NT: not tested.
Compounds 1a and 1b blocked the infectivity and replication of
i-butyl, 3-hydroxybutyl, 2-butenyl, 2-butynyl and benzyl residues
afforded 2–7, respectively. 1-N-(n-Propyl) analog 2 exhibited less
potentactivityrelative to 1b. Analog3 possessinga branched1-N-al-
kyl residue, showed less potent activity relative to 1b. 1-N-(3-
Hydroxybutyl) analog 4 had a remarkable reduction in its antagonist
activity. Thus, a hydrophilic OH group was found to be unacceptable
at this site. Compounds 5 and 6 possessing 1-N-(2-butenyl) and 1-N-
(2-butynyl) residues, showed nearly equipotent activity as 1b. 1-N-
Benzyl analog 7 showed significantly reduced potency. As a result,
shortening and branching of the 1-N-(n-butyl) residue as illustrated
by 2 and 3 negatively impacted the activity. After substantial chem-
ical modification, lipophilic groups such as n-butyl, 2-butenyl and 2-
butynyl residues, which are relatively linear and have a limited
length (C4), were found to be the most optimal 1-N-substituent.
The stability in rat liver microsomes of compounds 1b and 3–6
was investigated to estimate their metabolic stability. All the ana-
logs tested were not as metabolically stable as 1b. Although this
hydrophobic 1-N-residue was considered to be one of sites that were
laboratory and clinical strains of HIV as well as highly drug-resis-
tant HIV variants with minimal cytotoxicity.6a Despite their prom-
ising activity profiles, both 1a and 1b had very poor AUCs after oral
dosing in rat. Pharmacokinetic analysis after iv dosing indicated
that they exhibited rapid clearance (CL) and a large distribution
volume (Vss). A large clearance value (CL) after iv dosing suggests
that they are metabolically unstable which may explain their poor
AUC and bioavailability (BA). Moreover, the large distribution vol-
ume (Vss) of analogs 1a and 1b after iv dosing is unfavorable as
anti-HIV-1 drugs exert their effects in the blood. Thus, our syn-
thetic effort was focused on increasing oral exposure (AUC), reduc-
tion of in vivo clearance (CL) and distribution volume (Vss). The
optimization of chemical leads 1a and 1b was initiated by evaluat-
ing each of the diversity sites as described in Tables 1–6.
Optimization of the 1-N-alkyl residue was carried out with the
aim of further increasing the in vitro activities and metabolic stabil-
ity (Table 1). Replacement of the n-butyl residue of 1b with n-propyl,

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5213
Table 4
Effect of the 9-N-substituent with heteroaryl moieties on activity profiles
CH3
O
(3R and/or 3S)
N
3
R
N 9
N
H
O
Compds
R
C3 Configuration (3R and/or 3S)
(3RS)
IC50 (nM) Ca assay
21
Stability in rat liver microsomesa remaining%
10
cLog Pb
O
23
6.16
6.16
N
N
*
N
24
(3S)
71
29
*
CH3
N
25
26
(3RS)
(3RS)
35
23
6.05
5.49
*
H3C
H
N
160
NTc
*
N
CH3
O
27
28
1a
(3S)
450
570
28
NTc
NTc
33
5.55
6.00
7.66
*
*
N
S
(3RS)
(3RS)
N
O
*
a
b
c
The data show the remaining% 15 min after incubating with the 0.5 mg/mL rat liver microsomes.
Calculated by ADMET predictor (ver. 4.0).
NT: not tested.
metabolically vulnerable, it seemed to be difficult to improve meta-
bolic stability only by chemical modification of this moiety.
structure of the 9-N-substituent on the piperidine moiety of the lead
structures 1a and 1b, compounds listed in Table 3 were synthesized
and evaluated. To confirm the structural requirement of the
9-N-arylalkyl moiety, compounds 17 to 22 were synthesized and
evaluated. 9-N-Phenylmethyl analogs 17 and 18 tended to show
reduced activities relative to 1b and 1a, respectively. Replacement
of the 9-N-{(4-phenoxy)phenylmethyl} residue with 9-N-{(4-phen-
oxy)phenylethyl} afforded 19 with a remarkable reduction in activ-
ity. Based on this result, the 9-N-phenylmethyl moiety was found to
be required for the antagonist activity. The less basic aniline type
analog 20 (calculated pKa 4.12) and non-basic analog 21 (calculated
pKa 2.52) also showed remarkable reduction in activity. Recovery of
the antagonist activity for 9-N-{6-(phenyl)hexyl} analog 22 relative
to that of 17 strongly suggested another interaction site with the
receptor. As a result, the 9-N-{(4-phenoxy)phenylmethyl} moiety
was concluded to be the optimal 9-N-substituent.
To identify an optimal C3-substituent, synthesis and evaluation
of analogs 8–16 was carried out using the easily available optically
active S-form or racemic RS-form as the starting amino acids. This
was possible because there were no significant differences in antag-
onist activities between the enantiomers 8 (3S) and 9 (3R), both of
which showed nearly equipotent activities relative to 1b. Analogs
10–12 possessing lipophilic C3-substituents exhibited equipotent
activities relative to 1b. Analogs 13–15 possessing relatively hydro-
philic substituents such as methoxymethyl, carboxymethyl, and
benzyloxycarbonylmethyl tended to show reduced activities. Cyclo-
hexylethyl analog 16, which is a C2 homolog of 1a, exhibited a
remarkable decrease in its activity. Based on these results, only lipo-
philic C3-substituentsare acceptable for the receptor pocket as illus-
trated by 1a, 1b and 8–16 while their length is strictly limited as
illustrated by the result of 16. Analogs 8–12, which showed nearly
equipotent activities relative to 1a and 1b, did not show any
improvement in stability in liver microsomes as shown in Table 2.
Further structural optimization of the 9-N-substituent was ex-
plored to provide additional increases in activity and metabolic sta-
bility. Results are summarized in Tables 3–5. To identify the optimal
The effects of the 9-N-substitution with heteroarylmethyl moie-
ties on the activity profiles were investigated. Results are summa-
rized in Table 4. Analogs 23–28 possess lower cLog P values
relative to 1a. 9-N-{2-(phenoxy)pyridine-5-ylmethyl} analog 23
exhibited nearly equipotent in vitro activities relative to 1a although
it showed less stability in rat liver microsomes. Accordingly, the

5214
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
Table 5
agonist-like activity in the calcium assay. 9-N-(2-Phenylimidazol-4-
ylmethyl) analog 26, 9-N-(2-phenyl-5-methyloxazole-4-ylmethyl)
analog 27, and 9-N-(2-phenyl-4-thiazolylmethyl) analog 28 exhib-
ited weaker antagonist activity than 1a. Unfortunately, their stabil-
ities in rat liver microsomes were not improved using this approach.
As described in Table 5, the SAR of the linker X which connects
the two phenyl moieties of the 9-N-{4-phenoxyphenylmethyl}
moiety of 1a was investigated. Results are summarized in Table
5. Replacement of the ether oxygen of 1a with a sulfur atom affor-
ded the corresponding sulfide analog 29 with less potent activity
and an increased stability in rat liver microsomes. The correspond-
ing sulfone analog 30 exhibited less potent activity relative to 1a.
Replacement of the ether oxygen of 1a with a carbonyl provided
31 which also had slightly less potent activity relative to 1a.
Replacement of the ether oxygen of 1a with an amide moiety affor-
ded 32 with slightly less potent activity. Accordingly, an ether oxy-
gen was selected as the best linker to connect the two phenyls of
the 9-N-{4-(phenoxy)phenylmethyl} residue.
Effect of chemical modification of the linker X on activity profiles
CH3
X
O
N
(3R and/or 3S)
3
9
N
N
HCl
H
O
Compds
X
C3
IC50
(nM) Ca
assay
Stability in rat liver
microsomesa
remaining%
Configuration
(3R and/or 3S)
S
29
30
(3S)
(3S)
170
86
38
19
*
*
O
O
S
*
*
The structure activity relationship (SAR) study described in
Tables 3–5 strongly suggests that concurrent conversion of the
activity and stability in rat liver microsomes by chemical modifica-
tion of the 9-N-substituent from 9-N-[4-phenoxy]phenylmethyl]
moiety is difficult. Accordingly, stepwise optimizations of this cite
which had one of the most promising activity profiles was at-
tempted to improve its PK profiles as described in Tables 6 and 7.
The effects of a p-substitution at the predicted metabolic site8 of
the terminal phenyl moiety of 1a on the stability in rat liver micro-
somes were investigated. As shown in Table 6, introduction of
p-methyl and p-methoxy groups as electron-donating substituents
into the terminal phenyl moiety of 1a afforded 4-(p-methylphen-
oxy)phenylmethyl and 4-(p-methoxyphenoxy)phenylmethyl ana-
logs 34 and 35, respectively. Both had a tendency to be slightly
less potent relative to 1a, while demethylation of 35 afforded the
analog 36 which had slightly more potent activity relative to the
corresponding methoxy analog 35. Introduction of a p-fluoro group
as an electron-withdrawing group instead of utilizing the electron-
donating p-methyl and p-methoxy groups afforded 33 with slightly
less potent activity relative to 1a. Introduction of N-methyl amino-
carbonyl, methanesulfonylamino, aminosulfonyl and hydroxycar-
bonyl groups as the electron-withdrawing and hydrophilic
p-substituent afforded 37, 38, 39, and 40, respectively, with nearly
equipotent in vitro activities. The stability of these test compounds
in the rat liver microsomes was investigated but in vitro data did
not indicate a significant improvement in their metabolic stability.
However, analogs possessing hydrophilic substituents are pre-
dicted to have the potential of improved physicochemical proper-
ties such as solubility. PK studies of analogs 36, 37, and 40, which
possess relatively good in vitro potency and hydrophilic substitu-
ents such as hydroxyl, N-methyl aminocarbonyl and hydroxycar-
bonyl residues, were carried out.9
O
31
(3S)
81
23
*
*
*
O
32
1a
(3S)
57
28
29
33
N
H
*
O
(3RS)
*
*
a
The data show the remaining% 15 min after incubating with the 0.5 mg/mL rat
liver microsomes.
Table 6
Effect of the p-substituent of the biphenyl ether residue on activity profiles
CH3
X
O
O
N
(3RS and/orS)
3
N
N
HCl
H
O
Compds
X
C3
IC50
Stability in rat
cLog Pb
Configuration (nM)
(3R and/or
3S)
liver
Ca
assay
microsomesa
remaining%
33
34
35
36
37
38
39
40
F–
(3S)
(3S)
(3S)
(3S)
92
79
130
42
33
40
53
48
34
22
4
15
24
22
7.80
8.16
7.58
6.99
6.38
6.47
5.82
5.13
Me–
MeO–
HO–
MeHNC(O)– (3S)
MeSO2HN–
H2NO2S–
HO2C–
(3S)
(3S)
(3S)
2.3. Pharmacokinetics
28
13
PK data obtained after single-dose oral administration of the
initial chemical leads 1a, 1b and the representative compounds
36, 37, 40 to rats, are presented in Table 7. As described previously,
the initial leads 1a and 1b showed very poor bioavailability (1.9%
and 1.3%, respectively). Other PK values such as the maximum
1a
H
(3RS)
28
33
7.66
a
The data show the remaining% 15 min after incubating with the 0.5 mg/mL rat
liver microsomes.
b
Calculated by ADMET predictor (ver. 4.0).
plasma concentration (Cmax), plasma elimination half-life (T1/2
)
and AUC were also very poor. The probable reasons for such poor
PK values for 1a and 1b were the large clearance (CL = 113 and
137 mL/min/kg, respectively) and the large distribution volumes
(Vss = 2542 and 2349 mL/kg, respectively) which are unfavorable
for compounds such as anti-HIV drugs which require transport in
the blood to achieve efficacy.
benzylic phenyl of 1a could be replaced by a more hydrophilic pyri-
dine moiety without loss of the in vitro activities. 9-N-{4-(pyrid-3-
yl)phenylmethyl} analog 24 exhibited slightly less potent activity
relative to 1a. Five-membered heteroarylmethyl analogs 25–28
were also synthesized and evaluated. Unexpectedly, 9-N-(3,5-di-
methyl-1-phenylpyrazol-4-ylmethyl) analog 25 was found to show

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5215
Table 7
Pharmacokinetic data for 1a, 1b, 36, 37, and 40 in rat (n = 3)
Compds
30 mg/kg, po
3 mg/kg, iv
CL (mL/min/mL)
a
Cmax (ng/mL)
T1/2 (min)
AUCa (ng h/mL)
BA (%)
AUC (ng)
T1/2 (min)
Vss (mL/kg)
1a
1b
36
37
4
16.7
100
100
1900
7200
103
75.7
205
72.7
48.4
74.4
290
195
5453
10,532
1.9
400
19.9
13
113
137
NDb
NDb
16
2542
2349
NDb
NDb
145
1.3
372
NDb
NDb
3091
NDb
NDb
34.1
NDb
NDb
11.1
a
Cmax and AUC are normalized to a dose of 30 mg/kg.
ND: not determined.
b
With the expectation of improved PK profiles, analogs 36, 37, and
(the BAL strain of HIV6). Further optimization of 40 to improve its
activity and PK profile to provide CCR5 antagonists suitable for
clinical use is ongoing.
40 possessing hydrophilic substituents were investigated for their
pharmacokinetics. N-Methyl carboxy amide analog 37 tended to
show improved PK values in its Cmax and oral AUC relative to 1a
and 1b, while the phenol analog 36 did not. Interestingly, benzoic
acid analog 40 showed significantly improved PK values in its Cmax
(7200 ng/mL), oral exposure (AUC = 10,532 ng h/mL) and bioavail-
ability (BA = 34%) after oral dosing. The marked reduction in
4. Experimental
4.1. Chemistry
clearance (CL = 16 mL/min/kg) and distribution volume (Vss
=
4.1.1. General methods
145 mL/kg) after iv dosing was considered to be the most plausible
reason for the improved AUC and BA. Additionally, remarkable
improvement of solubility and Caco-2 permeability of 40 relative
to 1a and 1b was estimated to be other possible reasons as shown
in Table 8. The marked reduction of the CL of 40 strongly suggested
in vivo metabolic stabilization, though in vitro studies did not indi-
cate a significant improvement in metabolic stability.
Analytical samples were homogeneous as confirmed by thin
layer chromatography (TLC), and afforded spectroscopic results con-
sistent with the assigned structures. Proton nuclear magnetic reso-
nance spectra (1H NMR) were recorded on a Varian Gemini-200 or
a MERCURY-300 spectrometer with tetramethylsilane as an internal
standard. The chemical shift values d are reported in ppm and cou-
pling constants (J) in Hertz (Hz). Fast atom bombardment (FAB)
and electron ionization (EI) mass spectra were obtained with a JEOL
JMS-700 spectrometer. Matrix-assisted laser desorption ionization-
time of flight (MALDI-TOF) mass spectra were obtained on Percep-
tive Voyager Elete. Atmospheric pressure chemical ionization (APCI)
massspectraweredeterminedbyHitachiM-1200Hspectrometer. IR
spectra were measured on a JASCO FTIR-430 spectrometer. Elemen-
tal analyses were performed with a Perkin–ElmerPE2400 series II
CHNS/O Analyzer and were only indicated as the elements within
0.4% of thetheoreticalvaluesunless otherwisenoted. Columnchro-
matography was carried out on silica gel [Merck Silica Gel 60 (0.063–
0.200 mm), Fuji Silysia BW235 or Fuji Silysia FL60D]. TLC was per-
formed on silica gel (Merck TLC, Silica Gel 60 F254).
2.4. Anti-HIV activity
The anti-HIV activities of compounds 39 and 40, both potent
CCR5 antagonists, were evaluated in the p24 assay. Results are
shown in Table 9 and compared with 1b. Compounds 39 and 40
were found to show more potent anti-HIV activity in the p24 assay
relative to 1b. Especially 40, which has the most favorable PK pro-
files among the compounds tested, 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 strains.6 Compound 40 was inactive against T cell tropic
(X4) HIV-1. These results support the hypothesis that spirodiketo-
piperazines such as 1b, 39, and 40 possess potent anti-HIV activi-
ties through their antagonist effects on CCR5.
4.1.2. A typical procedure for the Ugi four-component conden-
sation using Rink-isonitrile resin. (1a, 1b, 2–13, 15–18, 23, 25,
26, and 28)
3. Conclusions
4.1.2.1. 1-Butyl-3-(cyclohexylmethyl)-9-(4-phenoxybenzyl)-1,4,9-
In conclusion, starting with the initial hit compounds 1a and 1b,
which showed unfavorable PK profiles, we identified 40 which
showed significant improvement in its bioavailability (BA) and oral
exposure (AUC) without reduction of its antagonist activity by
introducing a carboxylic acid function into the p-position of the
terminal phenyl moiety. Although the role of the carboxylic acid
function is still unclear, significant reduction of CL and Vss was con-
sidered to be the most plausible reason for the increased Cmax, AUC
and BA of 40. Its improved physicochemical properties, through
the increase of hydrophilicity, were thought to contribute to the
improved Cmax, AUC and BA after oral dosing. N-Methyl aminocar-
bonyl analog 37 also tended to show improved PK values including
Cmax and AUC after its oral dosing. As such, introduction of hydro-
philic hydroxycarbonyl and N-methyl aminocarbonyl functions
into the p-position of the terminal phenoxy moiety was found to
be effective not only to block the predicted metabolic deactivation
but also to improve PK profiles. Compound 40 showed more potent
activity than 1b, one of the initial hit compounds, in the p24 assay
triazaspiro[5.5]undecane-2,5-dione
hydrochloride (1a). The
Rink-isonitrile resin 44 (0.45 mmol/g, 500 mg, 0.23 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 added N-allyloxycarbonyl-4-piperi-
done 41a (206 mg, 1.13 mmol), n-butylamine 42a (82 mg, 1.13
mmol) and N-Boc-dl-cyclohexylalanine 43a (305 mg, 1.13 mmol).
The mixture was shaken for 16 h at 65 °C. After cooling to room
temperature, the resin was collected by filtration and successively
washed with THF/MeOH (1:1, 4 mL ꢁ 3) and then CH2Cl2 (4 mL ꢁ
3). To a suspension of the resin in CH2Cl2 (4 mL) were successively
added acetic acid (135 mg, 2.25 mmol), tetrakis(triphenylphos-
phine)palladium(0) (52.0 mg, 0.045 mmol) and tributyl tin hydride
(327 mg, 1.13 mmol). The mixture was shaken for 4 h at room tem-
perature. The resin was collected by filtration and successively
washed with CH2Cl2 (4 mL ꢁ 4) and DMF (4 mL ꢁ 4). To a suspension
of the resin in 1% acetic acid in DMF (4 mL) were successively added
4-phenoxybenzaldehyde (223 mg, 1.13 mmol) and then sodium
triacetoxyborohydride (238 mg, 1.13 mmol). The mixture was

5216
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
Table 8
shaken for 6 h at room temperature. The resin was collected by filtra-
tion and successively washed with MeOH (4 mL ꢁ 2), DMF
(4 mL ꢁ 4), and then 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. The resin was
collected by filtration and rinsed with CH2Cl2 (4 mL ꢁ 3), toluene
(4 mL ꢁ 3), and 1.25 M acetic acid in toluene (4 mL). The resin was
suspended in 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
resin was collected by filtration and washed with CHCl3/MeOH (1:1,
4 mL). The combined filtrate and washings were evaporated. The
resulting residue was purified by column chromatography on silica
gel with a gradient of AcOEt/MeOH (from 1:0 to 10:1). The resulting
residue after evaporation was treated with 4 N HCl in AcOEt and then
washed with Et2O to afford the title compound 1a (23 mg, 70% yield)
as a white powder. TLC Rf 0.73 (CHCl3/MeOH, 10:1); 1H NMR
(200 MHz, CD3OD) d 7.74–7.56 (m, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.40
(m, 2H), 7.18 (m, 1H), 7.10–7.00 (m, 3H), 4.33 (s, 2H), 4.04 (dd,
J = 7.4, 4.8 Hz, 1H), 3.80 (m, 2H), 3.60–3.35 (m, 4H), 2.43 (m, 2H),
2.17 (m, 2H), 1.90–1.60 (m, 7H), 1.60–1.45 (m, 2H), 1.45–1.30 (m,
2H), 1.30–1.15 (m, 4H), 1.10–0.80 (m, 5H); IR (KBr) 3434, 3210,
3064, 2926, 2851, 2664, 2558, 1672, 1590, 1509, 1489, 1418, 1373,
1241, 1173, 1118, 1072, 1048 cmꢂ1; MS (FAB, Pos) m/z 518 (M+H)+;
Elemental Anal. Calcd for C32H43N3O3ꢀHClꢀ0.5H2O: C, 68.25; H, 8.05;
N, 7.46. Found: C, 68.23; H, 7.88; N, 6.77.
In vitro pharmacokinetic data for 1a, 1b, 36, 37, and 40
Compds
Caco-2 papp (ꢁ10ꢂ6 cm/s)
Solubility (
l
M)
cLog Pa
A to B
B to A
1a
1b
36
37
40
0.15
1.51
0.33
3.51
11.0
0.27
2.23
0.14
3.91
16.8
<5
14
<5
6
7.66
6.46
6.99
6.38
5.13
29
a
Calculated by ADMET predictor (ver. 4.0).
MS (APCI, Pos) m/z 478 (M+H)+; Elemental Anal. Calcd for
C
29H39N3O3ꢀHCl: C, 67.75; H, 7.84; N, 8.17. Found: C, 67.35; H,
7.91; N, 8.06.
4.1.2.5. 1-(3-Hydroxybutyl)-3-isobutyl-9-(4-phenoxybenzyl)-
1,4,9-triazaspiro[5.5]undecane-2,5-dione hydrochloride (4). The
title compound was synthesized in 31% yield according to the same
procedures as described for the preparation of 1a using 3-t-buty-
ldimethylsilylhydroxybutylamine 42d and N-Boc-dl-leucine 43b.
TLC Rf 0.49 (CHCl3/MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 7.54
(d, J = 8.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.18 (t, J = 7.5 Hz, 1H),
7.04 (m, 4H), 4.33 (s, 2H), 4.02 (m, 1H), 3.80 (m, 3H), 3.51 (m, 4H),
2.46 (m, 2H), 2.19 (m, 2H), 1.85–1.57 (m, 5H), 1.17 (d, J = 6.0 Hz,
3H), 0.94 (d, J = 9.0 Hz, 6H); IR (KBr) 3405, 2960, 1675, 1590, 1510,
1489, 1421, 1242, 1172, 1048 cmꢂ1; MS (APCI, Pos) m/z 494
(M+H)+; HRMS Calcd 494.3019, Obsd 494.3025.
4.1.2.2. 1-Butyl-3-isobutyl-9-(4-phenoxybenzyl)-1,4,9-triazaspi-
ro[5.5]undecane-2,5-dione hydrochloride (1b). The title com-
pound was synthesized in 48% yield according to the same
procedures as described for the preparation of 1a using N-Boc-dl-
leucine for 43b. TLC Rf 0.63 (CHCl3/MeOH, 10:1); 1H NMR
(200 MHz, CD3OD) d 7.54 (d, J = 8.8 Hz, 2H), 7.40 (m, 2H), 7.18 (m,
1H), 7.11–7.00 (m, 4H), 4.33 (s, 2H), 4.01 (dd, J = 7.6, 4.8 Hz, 1H),
3.80 (m, 2H), 3.60–3.35 (m, 4H), 2.43 (m, 2H), 2.18 (m, 2H), 1.80
(m, 1H), 1.70 (m, 1H), 1.54 (m, 2H), 1.37 (m, 3H), 1.00–0.90 (m,
9H); IR (KBr) 3440, 3221, 3066, 2957, 2871, 2559, 1673, 1590,
1509, 1489, 1419, 1371, 1329, 1242, 1172 cmꢂ1; MS (FAB. Pos., Glyc-
erin+m-NBA) m/z 478 (M+H)+, 183 (base peak); Elemental Anal.
Calcd for C29H39N3O3ꢀHClꢀH2O: C, 65.46; H, 7.96; N, 7.90. Found: C,
65.67; H, 7.89; N, 7.83.
4.1.2.6. 1-[(2E)-2-Butenyl]-3-isobutyl-9-(4-phenoxybenzyl)-
1,4,9-triazaspiro[5.5]undecane-2,5-dione hydrochloride (5). The
title compound was synthesized in 29% yield according to the same
procedures as described for the preparation of 1a using 2-butenyl-
amine 42e and N-Boc-dl-leucine 43b. TLC Rf 0.32 (CHCl3/MeOH,
20:1); 1H NMR (300 MHz, CD3OD) d 7.52 (d, J = 8.7 Hz, 2H),
7.44–7.35 (m, 2H), 7.22–7.14 (m, 1H), 7.06 (d, J = 8.7 Hz, 2H),
7.10–7.00 (m, 2H), 5.75–5.60 (m, 1H), 5.52–5.38 (m, 1H), 4.33 (s,
2H), 4.15–3.93 (m, 2H), 4.03 (dd, J = 7.8, 4.5 Hz, 1H), 3.88–3.66 (m,
2H), 3.55–3.42 (m, 2H), 2.52–2.35 (m, 2H), 2.28–2.08 (m, 2H),
1.90–1.57 (m, 3H), 1.65 (dd, J = 6.3, 1.5 Hz, 3H), 0.95 (d, J = 6.6 Hz,
3H), 0.94 (d, J = 6.6 Hz, 3H); IR (KBr) 2955, 2512, 1673, 1590, 1509,
1490, 1417, 1241 cmꢂ1; MS (APCI, Pos) m/z 476 (M+H)+; Elemental
Anal. Calcd for C29H37N3O3ꢀHCl: C, 68.02; H, 7.48; N, 8.21.Found: C,
66.1; H, 7.51; N, 8.00.
4.1.2.3. 3-Isobutyl-9-(4-phenoxybenzyl)-1-propyl-1,4,9-triazaspi-
ro[5.5]undecane-2,5-dione hydrochloride (2). The title com-
pound was synthesized in 87% yield according to the same
procedures as described for the preparation of 1a using n-propyl-
amine 42b and N-Boc-dl-leucine 43b. TLC Rf 0.61 (CHCl3/MeOH,
10:1); 1H NMR (200 MHz, CD3OD) d 7.55 (m, 2H), 7.40 (m, 2H),
7.18 (m, 1H), 7.05 (m, 4H), 4.33 (s, 2H), 4.01 (dd, J = 7.6, 4.8 Hz,
1H), 3.79 (m, 2H), 3.60–3.30 (m, 4H), 2.46 (m, 2H), 2.17 (m, 2H),
1.95–1.40 (m, 5H), 0.94 (m, 9H); IR (KBr) 3439, 3220, 3066, 2959,
2872, 2663, 2561, 1672, 1590, 1509, 1489, 1418, 1370, 1330, 1241,
1200, 1172, 1072 cmꢂ1; MS (APCI, Pos) m/z 464 (M+H)+; Elemental
Anal. Calcd for C28H37N3O3ꢀHClꢀH2O: C, 64.59; H, 7.74; N, 8.07.
Found: C, 64.38; H, 7.67; N, 8.07.
4.1.2.7. 1-(2-Butynyl)-3-isobutyl-9-(4-phenoxybenzyl)-1,4,9-tri-
azaspiro[5.5]undecane-2,5-dione hydrochloride (6). The title
compound was synthesized in 23% yield according to the same
procedures as described for the preparation of 1a using 2-butynyl-
amine hydrochloride 42f and N-Boc-dl-leucine 43b. TLC Rf 0.70
(CHCl3/MeOH, 10:1); 1H NMR (300 MHz, CD3OD)
d 7.51 (d,
J = 8.7 Hz, 2H), 7.39 (dd, J = 8.7, 7.2 Hz, 2H), 7.18 (t, J = 7.2 Hz,
1H), 7.09–7.00 (m, 4H), 4.33 (br s, 2H), 4.28–4.10 (m, 2H), 4.05
Table 9
4.1.2.4. 1,3-Diisobutyl-9-(4-phenoxybenzyl)-1,4,9-triazaspi-
ro[5.5]undecane-2,5-dione hydrochloride (3). The title com-
pound was synthesized in 62% yield according to the same
procedures as described for the preparation of 1a using i-butylamine
42c and N-Boc-dl-leucine 43b. TLC Rf 0.50 (CHCl3/MeOH, 10:1); 1H
NMR (300 MHz, CD3OD) d 7.50 (d, J = 8.7 Hz, 2H), 7.39 (dd, J = 8.7,
7.5 Hz, 2H), 7.17 (t, J = 7.5 Hz, 1H), 7.13–7.04 (m, 4H), 4.28 (s, 2H),
4.04 (dd, J = 8.1, 4.2 Hz, 1H), 3.81–3.54 (m, 2H), 3.52–3.21 (m, 4H),
2.46–2.11 (m, 4H), 2.00–1.57 (m, 4H), 0.94 (d, J = 6.3 Hz, 6H), 0.90
(d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H); IR (KBr) 2958, 2516,
Anti-HIV activity of representative compounds
Compds
Mean IC50 + SD (nM)
Anti-HIV-1 activity in p24 assay HV-1Ba-L (R5) CCR5 Ca assay
1b
39
40
160 40
10
39 34.4
94
28
13
—
Zidovudinea
Nelfinavirb
7
12
4
8
—
a
Zidovudine is a reverse transcriptase inhibitor.
Nerfinavir is a HIV-1 protease inhibitor.
b
1676, 1590, 1510, 1489, 1410, 1242, 1199, 1172, 1097, 1071 cmꢂ1
;

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5217
(dd, J = 7.8, 4.5 Hz, 1H), 3.86–3.70 (m, 2H), 3.56–3.43 (m, 2H), 2.59–
2.40 (m, 2H), 2.34–2.15 (m, 2H), 1.89–1.57 (m, 6H), 0.94 (d,
J = 6.6 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H); IR (KBr) 3432, 2956, 1675,
1590, 1509, 1489, 1413, 1242, 1173 cmꢂ1; MS (APCI, Pos) m/z
474 (M+H)+; HRMS Calcd 474.2757, Obsd 474.2753.
procedures as described for the preparation of 1a using N-Boc-dl-
norvaline 43f. TLC Rf 0.36 (CHCl3/MeOH, 10:1); 1H NMR (300 MHz,
CD3OD) d 7.51 (d, J = 8.7 Hz, 2H), 7.39 (dd, J = 8.7, 7.5 Hz, 2H), 7.18
(t, J = 7.5 Hz, 1H), 7.10–7.00 (m, 4H), 4.33 (s, 2H), 4.04 (dd, J = 5.7,
4.5 Hz, 1H), 3.93–3.66 (m, 2H), 3.55–3.31 (m, 4H), 2.47–2.09 (m,
4H), 1.92–1.68 (m, 2H), 1.61–1.21 (m, 6H), 1.01–0.90 (m, 6H); IR
(KBr) 3436, 2958, 2872, 2550, 1673, 1590, 1509, 1489, 1419, 1243,
1172, 1071, 954, 873, 787, 693 cmꢂ1; MS (APCI, Pos) m/z 464
(M+H)+; HRMS Calcd 464.2913, Obsd 464.2915.
4.1.2.8. 1-Benzyl-3-isobutyl-9-(4-phenoxybenzyl)-1,4,9-triaza-
spiro[5.5]undecane-2,5-dione hydrochloride (7). The title com-
pound was synthesized in 50% yield according to the same
procedures as described for the preparation of 1a using benzyl-
amine 42g and N-Boc-dl-leucine 43b. TLC Rf 0.66 (CHCl3/MeOH,
10:1); 1H NMR (200 MHz, CD3OD) d 7.50 (d, J = 8.4 Hz, 2H), 7.45–
7.12 (m, 8H), 7.10–6.98 (m, 4H), 4.82 (m, 2H), 4.29 (s, 2H), 4.18
(dd, J = 8.0, 4.6 Hz, 1H), 3.73 (m, 2H), 3.42 (m, 2H), 2.65–2.30 (m,
2H), 2.20–2.05 (m, 2H), 2.00–1.60 (m, 3H), 0.98 (d, J = 6.2 Hz,
6H); IR (KBr) 3405, 3194, 3063, 2954, 2871, 2661, 2508, 2462,
1681, 1614, 1589, 1511, 1488, 1470, 1455, 1412, 1361, 1331,
1307, 1240, 1200, 1176, 1155, 1130, 1069 cmꢂ1; MS (MALDI, Pos)
m/z 512 (M+H)+; Elemental Anal. Calcd for C32H37N3O3ꢀClꢀH: C,
70.12; H, 6.99; N, 7.67. Found: C, 69.64; H, 7.03; N, 7.63.
4.1.2.13. 1-Butyl-3-(cyclopentylmethyl)-9-(4-phenoxybenzyl)-1,4,9-
triazaspiro[5.5]undecane-2,5-dione hydrochloride (12). The title
compound was synthesized in 53% yield according to the same pro-
ceduresasdescribedforthepreparationof 1ausingN-Boc-cyclopne-
tyl-dl-alanine 43g. TLC Rf 0.66 (CHCl3/MeOH, 10:1); 1H NMR
(300 MHz, CD3OD) d 7.52 (d, J = 8.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H),
7.18 (t, J = 7.5 Hz, 1H), 7.05 (m, 4H), 4.34 (s, 2H), 4.00 (t, J = 6.0 Hz,
1H), 3.82 (m, 2H), 3.49 (m, 2H), 3.39 (m, 2H), 2.37 (m, 2H), 2.17 (m,
2H), 1.96 (m, 1H), 1.81 (m, 4H), 1.58 (m, 6H), 1.38 (m, 2H), 1.17 (m,
2H), 0.95 (t, J = 7.0 Hz, 3H); IR (KBr) 3433, 3199, 2953, 2870, 2499,
1681, 1589, 1510, 1488, 1419, 1373, 1336, 1240, 1199, 1174, 1114,
1070, 1049 cmꢂ1; MS (APCI, Pos) m/z 504 (M+H)+; HRMS Calcd
504.3226, Obsd 504.3231.
4.1.2.9. (3S)-1-Butyl-3-isobutyl-9-(4-phenoxybenzyl)-1,4,9-tri-
azaspiro[5.5]undecane-2,5-dione hydrochloride (8). The title
compound was synthesized in 13% yield according to the same
procedures as described for the preparation of 1a using N-Boc-l-
4.1.2.14. 1-Butyl-3-(methoxymethyl)-9-(4-phenoxybenzyl)-1,4,9-
triazaspiro[5.5]undecane-2,5-dione hydrochloride (13). The title
compound was synthesized in 59% yield according to the same pro-
cedures as described for the preparation of 1a using N-Boc-O-
methyl-dl-serine 43h. TLC Rf 0.48 (CHCl3/MeOH, 10:1); 1H NMR
(300 MHz, CD3OD) d 7.51 (d, J = 8.7 Hz, 2H), 7.39 (dd, J = 8.7, 7.2 Hz,
2H), 7.17 (t, J = 7.2 Hz, 1H), 7.09–6.99 (m, 4H), 4.30 (s, 2H), 4.07 (t,
J = 3.0 Hz, 1H), 3.91 (m, 1H), 3.77 (dd, J = 9.0, 3.0 Hz, 1H), 3.67 (m,
1H), 3.58–3.39 (m, 4H), 3.31 (s, 3H), 3.26 (m, 1H), 2.48–2.13 (m,
4H), 1.65 (m, 1H), 1.53–1.28 (m, 3H), 0.95 (t, J = 7.5 Hz, 3H); IR
(KBr) 3424, 2931, 2551, 1662, 1590, 1509, 1489, 1423, 1371, 1242,
1198, 1116, 1075 cmꢂ1; MS (APCI, Pos) m/z 466 (M+H)+; Elemental
Anal. Calcd for C27H35N3O4ꢀClꢀH: C, 64.59; H, 7.23; N, 8.37. Found:
C, 63.45; H, 7.12; N, 8.21.
leucine 43c. ½a 2D2
ꢂ2.11 (c 0.95, MeOH);TLC Rf 0.29 (CHCl3/MeOH,
ꢃ
10:1); 1H NMR (300 MHz, CD3OD) d 7.54 (d, J = 8.7 Hz, 2H), 7.42–
7.36 (m, 2H), 7.18 (m, 1H), 7.05 (d, J = 8.7 Hz, 2H), 7.05–7.02 (m,
2H), 4.33 (s, 2H), 3.98 (dd, J = 8.1, 4.5 Hz, 1H), 3.86–3.72 (m, 2H),
3.53–3.37 (m, 4H), 2.47–2.36 (m, 2H), 2.24–2.12 (m, 2H), 1.80–
1.30 (m, 7H), 0.95 (t, J = 7.2 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.93
(d, J = 6.3 Hz, 3H); IR (KBr) 3445, 2956, 2565, 1676, 1590, 1509,
1489, 1418, 1329, 1242, 1172, 1073, 1049 cmꢂ1; MS (APCI, Pos)
m/z 478 (M+H)+; HRMS Calcd 478.307, Obsd 478.3069.
4.1.2.10. (3R)-1-Butyl-3-isobutyl-9-(4-phenoxybenzyl)-1,4,9-tri-
azaspiro[5.5]undecane-2,5-dione hydrochloride (9). The title
compound was synthesized in 14% yield according to the same
procedures as described for the preparation of 1a using N-Boc-d-
leucine 43d. ½a 2D2
ꢃ
+2.25 (c 1.15, MeOH); TLC Rf 0.29 (CHCl3/MeOH,
10:1); 1H NMR (300 MHz, CD3OD) d 7.54 (d, J = 8.7 Hz, 2H), 7.42–
7.36 (m, 2H), 7.18 (m, 1H), 7.05 (d, J = 8.7 Hz, 2H), 7.05–7.02 (m,
2H), 4.32 (s, 2H), 4.01 (dd, J = 7.8, 4.8 Hz, 1H), 3.85–3.72 (m, 2H),
3.50–3.39 (m, 4H), 2.52–2.38 (m, 2H), 2.24–2.11 (m, 2H), 1.84–
1.20 (m, 7H), 0.95 (t, J = 7.2 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.93
(d, J = 6.3 Hz, 3H); IR (KBr) 3444, 2957, 2565, 1676, 1590, 1509,
1489, 1418, 1242, 1172 cmꢂ1; MS (APCI, Pos) m/z 478 (M+H)+;
HRMS Calcd 478.307, Obsd 478.3069.
4.1.2.15. Benzyl [1-butyl-2,5-dioxo-9-(4-phenoxybenzyl)-1,4,9-
triazaspiro[5.5]undec-3-yl]acetate hydrochloride (15). The title
compound was synthesized in 36% yield according to the same
procedures as described for the preparation of 1a using N-Boc-dl-
aspartic acid b-benzyl ester 43i. TLC Rf 0.74 (CHCl3/MeOH, 9:1);
1H NMR (300 MHz, CD3OD) d 7.52 (d, J = 7.0 Hz, 2H), 7.40 (t,
J = 7.5 Hz, 2H), 7.33 (m, 5H), 7.18 (t, J = 7.5 Hz, 1H), 7.05 (m, 4H),
5.12 (s, 2H), 4.33 (s, 2H), 4.31 (m, 1H), 3.88 (m, 1H), 3.66 (m,
1H), 3.50–3.35 (m, 4H), 3.08 (dd, J = 17.7, 4.8 Hz, 1H), 2.86 (dd,
J = 17.7, 3.0 Hz, 1H), 2.34 (m, 2H), 2.25 (m, 2H), 1.50 (m, 2H),
1.36 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H); IR (KBr) 3735, 3412, 1736,
1675, 1489, 1423, 1242, 1175 cmꢂ1; MS (APCI, Pos) m/z 570
(M+H)+; HRMS Calcd 570.2968, Obsd 570.297.
4.1.2.11. (3S)-1-Butyl-3-neopentyl-9-(4-phenoxybenzyl)-1,4,9-
triazaspiro[5.5]undecane-2,5-dione hydrochloride (10). The ti-
tle compound was synthesized in 51% yield according to the same
procedures as described for the preparation of 1a using N-Boc-dl-t-
butylalanine 43e. TLC Rf 0.52 (CHCl3/MeOH, 20:1); 1H NMR
(300 MHz, CD3OD) d 7.52 (d, J = 9.0 Hz, 2H), 7.40 (t, J = 7.5 Hz,
2H), 7.18 (t, J = 7.5 Hz, 1H), 7.04 (m, 4H), 4.33 (s, 2H), 4.01 (dd,
J = 7.2, 3.3 Hz, 1H), 3.82 (m, 1H), 3.71 (m, 1H), 3.50 (m, 2H), 3.43
(m, 2H), 2.38 (m, 2H), 2.24 (m, 2H), 2.00 (dd, J = 14.0, 3.3 Hz, 1H),
1.55 (dd, J = 14.0, 7.2 Hz, 1H), 1.50 (m, 2H), 1.36 (m, 2H), 0.99 (s,
9H), 0.95 (t, J = 7.0 Hz, 3H); IR (KBr) 2957, 2506, 1678, 1590,
1510, 1489, 1419, 1370, 1285, 1243, 1174, 1115 cmꢂ1; MS (APCI,
Pos) m/z 492 (M+H)+; HRMS Calcd 492.3226, Obsd 492.3226.
4.1.2.16. 1-Butyl-3-(3-cyclohexylpropyl)-9-(4-phenoxybenzyl)-1,4,9-
triazaspiro[5.5]undecane-2,5-dione hydrochloride (16). The title
compound was synthesized in 39% yield according to the same pro-
cedures as described for the preparation of 1a using N-Boc-dl-3-
cyclohexylpropylalanine 43j. TLC Rf 0.76 (CHCl3/MeOH, 10:1); 1H
NMR (300 MHz, CD3OD) d 7.53–7.49 (m, 2H), 7.42–7.36 (m, 2H),
7.18 (m, 1H), 7.10–7.02 (m, 4H), 4.32 (s, 2H), 4.04 (t, J = 4.8 Hz, 1H),
3.87 (m, 1H), 3.71 (m, 1H), 3.56–3.40 (m, 3H), 3.35 (m, 1H), 2.48–
2.12 (m, 4H), 1.86–1.10 (m, 19H), 0.95 (t, J = 7.5 Hz, 3H), 0.95 (m,
2H); IR (KBr) 2923, 2564, 1679, 1591, 1510, 1490, 1245,
1172 cmꢂ1; MS (APCI, Pos) m/z 546 (M+H)+; HRMS Calcd 546.3696,
Obsd 546.3703.
4.1.2.12. 1-Butyl-9-(4-phenoxybenzyl)-3-propyl-1,4,9-triazaspi-
ro[5.5]undecane-2,5-dione hydrochloride (11). The title com-
pound was synthesized in 52% yield according to the same

5218
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
4.1.2.17. 9-Benzyl-1-butyl-3-isobutyl-1,4,9-triazaspiro[5.5]unde-
cane-2,5-dione hydrochloride (17). The title compound was syn-
thesized in 59% yield according to the same procedures as
described for the preparation of 1a using N-Boc-dl-leucine 43b and
benzaldehyde for 4-phenoxybenzaldehyde. TLC Rf 0.54 (CHCl3/
MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 7.64–7.44 (m, 5H), 4.36
(s, 2H), 4.01 (dd, J = 7.8, 4.8 Hz, H), 3.77 (m, 2H), 3.55–3.35 (m, 4H),
2.60–2.30 (m, 2H), 2.17 (m, 2H), 1.95–1.75 (m, 1H), 1.75–1.60 (m,
2H), 1.60–1.45 (m, 2H), 1.45–1.20 (m, 2H), 1.10–0.80 (m, 9H); IR
(KBr) 3435, 3230, 2957, 2871, 2505, 2454, 1680, 1647, 1459, 1413,
1370, 1326, 1147 cmꢂ1; MS (MALDI, Pos) m/z 386 (M+H)+, 91;
Elemental Anal. Calcd for C23H35N3O2ꢀHCl: C, 65.46; H, 8.6; N, 9.96.
Found: C, 65.09; H, 8.63; N, 9.88.
1.60–1.45 (m, 3H), 1.45–1.30 (m, 3H), 1.30–1.10 (m, 3H),
1.10–0.80 (m, 5H); IR (KBr) 3410, 2927, 2854, 2699, 2574, 1781,
1671, 1644, 1554, 1448, 1421, 1373, 1349, 1308, 1257, 1178,
1096, 1052, 1001 cmꢂ1; MS (FAB, Pos) m/z 492 (M+H)+, 336;
Elemental Anal. Calcd for C29H41N5O2ꢀ2HCl: C, 61.69; H, 7.68; N,
12.4. Found: C, 55.71; H, 7.32; N, 10.71.
4.1.2.22.
1-Butyl-3-(cyclohexylmethyl)-9-[(2-phenyl-1,3-thia-
zol-4-yl)methyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione
hydrochloride (28). The title compound was synthesized in 51%
yield according to the same procedures as described for the prep-
aration of 1a using 2-phenyl-1,3-thiazole-4-carboxaldehyde for 4-
(3-pyridyl)benzaldehyde. TLC Rf 0.62 (CHCl3/MeOH, 10:1); 1H NMR
(300 MHz, CD3OD) d 8.03–8.00 (m, 2H), 7.87 (s, 1H), 7.52–7.49 (m,
3H), 4.54 (s, 2H), 4.04 (dd, J = 7.6, 4.8 Hz, 1H), 4.04–3.87 (m, 2H),
3.70–3.58 (m, 2H), 3.51–3.39 (m, 2H), 2.58–2.38 (m, 2H), 2.26–
2.13 (m, 2H), 1.7 8–1.43 (m, 9H), 1.40–1.15 (m, 6H), 1.10–0.90
(m, 5H); IR (KBr) 3426, 3298, 3190, 3128, 3086, 2991, 2955,
2927, 2854, 2653, 2512, 2452, 1678, 1645, 1481, 1462, 1450,
1415, 1371, 1351, 1336, 1314, 1146, 1003 cmꢂ1; MS (APCI, Pos)
m/z 509 (M+H)+; HRMS Calcd 509.295, Obsd 509.295.
4.1.2.18. 1-Butyl-3-(cyclohexylmethyl)-9-(4-methoxybenzyl)-
1,4,9-triazaspiro[5.5]undecane-2,5-dionehydrochloride (18). The
title compound was synthesized in 59% yield according to the same
procedures as described for the preparation of 1a using 4-methoxy-
benzaldehyde for 4-phenoxybenzaldehyde. TLC Rf 0.63 (CHCl3/
MeOH, 10:1); 1H NMR (200 MHz, CD3OD) d 7.47 (d, J = 8.8 Hz, 2H),
7.03 (d, J = 8.8 Hz, 2H), 4.29 (s, 2H), 4.04 (dd, J = 7.6, 4.8 Hz, 1H),
3.83 (s, 3H), 3.74 (m, 2H), 3.55–3.35 (m, 4H), 2.41 (m, 2H), 2.15 (m,
2H), 1.85–1.55 (m, 7H), 1.55–1.42 (m, 3H), 1.42–1.30 (m, 3H),
1.30–1.10 (m, 2H), 1.08–0.80 (m, 5H); IR (KBr) 3436, 3221, 2926,
2851, 2666, 2560, 2362, 1672, 1613, 1585, 1517, 1448, 1419, 1373,
1305, 1255, 1182, 1031 cmꢂ1; MS (FAB, Pos) m/z 456 (M+H)+, 121;
Elemental Anal. Calcd for C27H41N3O3ꢀHClꢀ1.5H2O: C, 62.47; H,
8.74; N, 8.09. Found: C, 62.59; H, 8.35; N, 7.90.
4.1.3. [1-Butyl-2,5-dioxo-9-(4-phenoxybenzyl)-1,4,9-triazaspi
ro[5.5]undec-3-yl]acetic acid hydrochloride (14)
To a stirred solution of the compound 15 (173 mg, 0.303 mmol)
in MeOH (5 mL) was added 2 M NaOH (2 mL). After being stirred
for 3 h at room temperature, the reaction mixture was acidified
with 2 M HCl and then extracted with AcOEt. The organic layer
was washed with water, brine, dried over Na2SO4 and concentrated
in vacuo. The resulting residue was triturated with Et2O to yield
the title compound (127 mg, 56%) as a white powder. TLC Rf 0.51
(CHCl3/MeOH/AcOH, 20:4:1); 1H NMR (300 MHz, CD3OD) d 7.55–
7.53 (m, 2H), 7.42–7.36 (m, 2H), 7.20–7.15 (m, 1H), 7.07–7.02
(m, 4H), 4.33 (s, 2H), 4.27 (t, J = 4.5 Hz, 1H), 3.96–3.90 (m, 1H),
3.72–3.66 (m, 1H), 3.54–3.38 (m, 4H), 2.97 (dd, J = 18.0, 4.8 Hz,
1H), 2.79 (dd, J = 18.0, 4.8 Hz, 1H), 2.50–2.36 (m, 3H), 2.27–2.16
(m, 1H), 1.62–1.48 (m, 2H), 1.41–1.30 (m, 2H), 0.94 (t, J = 7.2 Hz,
3H); IR (KBr) 3485, 3214, 2960, 2717, 2579, 1664, 1589, 1419,
1241, 1076, 1051, 1005 cmꢂ1; MS (APCI, Neg) m/z 478 (M-H)ꢂ;
HRMS Calcd 480.2498, Obsd 480.2499.
4.1.2.19. 1-Butyl-3-(cyclohexylmethyl)-9-[(6-phenoxypyridin-3-
yl)methyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione dihydro-
chloride (23). The title compound was synthesized in 15% yield
according to the same procedures as described for 1a using 6-
phenoxynicotinaldehyde for 4-phenoxybenzaldehyde. TLC Rf 0.67
(CHCl3/MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 8.31 (s, 1H),
8.07 (d, J = 8.3 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.5 Hz,
1H), 7.14 (d, J = 7.5 Hz, 2H), 7.06 (d, J = 8.3 Hz, 1H), 4.39 (s, 2H),
4.04 (dd, J = 7.8, 4.6 Hz, 1H), 3.90–3. 76 (m, 2H), 3.52–3.38 (m,
4H), 2.58–2.36 (m, 2H), 2.25–2.11 (m, 2H), 1.80–1.42 (m, 10H),
1.42–1.17 (m, 5H), 1.05–0.85 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR
(KBr) 3449, 2926, 2564, 1671, 1479, 1260, 1200 cmꢂ1; MS (APCI,
Pos) m/z 519 (M+H)+; HRMS Calcd 519.3335, Obsd 519.3337.
4.1.4. Benzyl 1-butyl-3-(cyclohexylmethyl)-2,5-dioxo-1,4,9-
triazaspiro[5.5]undecane-9-carboxylate (21)
4.1.2.20. 1-Butyl-3-(cyclohexylmethyl)-9-[(3,5-dimethyl-1-phe-
nyl-1H-pyrazol-4-yl)methyl]-1,4,9-triazaspiro[5.5]undecane-
2,5-dione dihydrochloride (25). The title compound was synthe-
sized in 33% yield according to the same procedures as described
for the preparation of 1a using 3,5-dimethyl-1-phenylpyrazole-4-
carboxaldehyde for 4-phenoxybenzaldehyde. TLC Rf 0.35 (CHCl3/
MeOH, 20:1); 1H NMR (300 MHz, CD3OD) d 7.63–7.48 (m, 5H),
4.33 (s, 2H), 4.05 (dd, J = 7.8, 4.5 Hz, 1H), 3.95–3.74 (m, 2H),
3.67–3.56 (m, 2H), 3.48 (m, 2H), 2.72–2.58 (m, 2H), 2.45 (s, 3H),
2.41 (s, 3H), 2.30–2.07 (m, 2H), 1.84–1.10 (m, 15 H), 1.02–0.92
(m, 2H), 0.96 (t, J = 7.2 Hz, 3H); IR (KBr) 3426, 2926, 1670,
To a suspension of the Rink-isonitrile resin 44 (0.75 mmol/g,
10.0 g, 7.5 mmol) resin in THF/MeOH (1:1, 200 mL) were added
N-benzyloxycarbonyl-4-piperidone 41b (5.24 g, 22.5 mmol),
n-butylamine 42a (2.22 mL, 22.5 mmol) and N-Boc-dl-cyclohexyl-
alanine 43a (6.50 g, 22.5 mmol). The mixture was shaken for 16 h
at 65 °C. After cooling to room temperature, the resin was collected
by filtration and successively washed with THF (80 mL ꢁ 3), MeOH
(80 mL ꢁ 3) and then CH2Cl2 (80 mL ꢁ 3). The resin was treated
with 50% trifluoroacetic in CH2Cl2 (100 mL). The mixture was sha-
ken for 30 min at room temperature. The resin was collected by fil-
tration and successively washed with CH2Cl2 (70 mL ꢁ 4) and
toluene (100 mL). The resin was suspended in 1.25 M acetic acid
in toluene (100 mL). The suspension was shaken for 18 h at
90 °C. After cooling to room temperature, the resin was collected
by filtration and washed with CHCl3/MeOH (1:1, 100 mL ꢁ 3).
The combined filtrate and washings were evaporated. The resulting
residue was purified by column chromatography on silica gel with
a gradient of CHCl3/MeOH (40:1) to afford the title compound 21
(209 mg, 5.9% yield) as a white powder. TLC Rf 0.46 (CHCl3/MeOH,
20:1); 1H NMR (300 MHz, CDCl3) d 7.40–7.29 (m, 5H), 5.98 (br s,
1H), 5.15 (s, 2H), 4.14 (br s, 2H), 4.00 (m, 1H), 3.65 (br s, 1H),
3.43 (br s, 1H), 3.26 (m, 2H), 2.03–1.81 (m, 4H), 1.80–1.60 (m,
5H), 1.60–1.10 (m, 10H), 1.10–0.85 (m, 5H); IR (KBr) 3449, 2925,
1421 cmꢂ1
;
MS (APCI, Pos) m/z 520 (M+H)+; HRMS Calcd
520.3652, Obsd 520.3651.
4.1.2.21. 1-Butyl-3-(cyclohexylmethyl)-9-[(2-phenyl-1H-imida-
zol-4-yl)methyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dionedihy-
drochloride (26). The title compound was synthesized in 77%
yield according to the same procedures as described for the prep-
aration of 1a using 2-phenyl-1H-imidazole-4-carboxaldehyde for
4-phenoxybenzaldehyde. TLC Rf 0.25 (CHCl3/MeOH, 10:1); 1H
NMR (200 MHz, CD3OD) d 8.04–7.92 (m, 3H), 7.74–7.62 (m, 3H),
4.58 (s, 2H), 4.05 (dd, J = 7.4, 4.8 Hz, 1H), 3.88 (m, 2H), 3.65 (m,
2H), 3.50 (m, 2H), 2.68 (m, 2H), 2.19 (m, 2H), 1.90–1.60 (m, 6H),

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5219
2852, 1675, 1418, 1352, 1281, 1239, 1179, 1153, 1105, 1018 cmꢂ1
;
4.1.6.2. (3RS)-9-Benzyl-1-butyl-3-cyclohexylmethyl-1,4,9-tri-
azaspiro[5.5]undecane-2,5-dione hydrochloride (52a). Com-
pound 52a was prepared from N-Boc-dl-cyclohexylalanine 43a
according to the same procedure as described for the preparation
of 52b.
MS (APCI, Pos) m/z 470 (M+H)+.
4.1.5. 1-Butyl-3-isobutyl-9-(6-phenylhexyl)-1,4,9-triazaspi
ro[5.5]undecane-2,5-dione hydrochloride (22)
The Rink-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
1-(6-phenylhexyl)-4-piperidone 41c (292 mg, 1.125 mmol),
n-butylamine 42a (82 mg, 1.125 mmol), and N-Boc-dl-leucine
43b (260 mg, 1.125 mmol). The mixture was shaken for 16 h at
65 °C. After cooling to room temperature, the mixture was filtrated.
The collected resin was washed with THF/MeOH (1:1) (4 mL ꢁ 3),
and CH2Cl2 (4 mL ꢁ 3). The resin was then added 25% TFA in CH2Cl2
(4 mL) at 0 °C. The mixture was allowed 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 in 1.25 M acetic acid
in toluene was agitated for 24 h at 90 °C. After cooling to room
temperature, the mixture was filtrated. The resin was washed with
CHCl3/MeOH (1:1) (4 mL ꢁ 2). The filtrate and washings were con-
centrated under reduced pressure. The residue was purified by col-
umn chromatography over silica gel with a gradient of AcOEt/
MeOH from 1:0 to 10:1 to give the title compound (35% yield) as
TLC Rf 0.65 (CHCl3/MeOH/NH3 aq, 20:5:1); 1H NMR (CD3OD) d
4.00 (dd, J = 7.8, 4.5 Hz, 1H), 3.46–3.24 (m, 4H), 3.03–2.92 (m, 4H),
2.08–1.08 (m, 19H), 1.05–0.84 (m, 5H).
4.1.7. General procedure for the preparation of 1-butyl-3-(cyclo
hexylmethyl)-1,4,9-triazaspiro[5.5]undecane-2,5-dione 9-N-
aryl analogs
4.1.7.1. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[(6-phenylpyridin-
3-yl)methyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione dihydro-
chloride (24). To a stirred solution of 52b (100 mg, 0.27 mol), 4-
(3-pyridyl)benzaldehyde (59 mg, 0.32 mmol) and 1 drop of acetic
acid in DMF (2 mL) was added sodium triacetoxyborohydride
(114 mg, 0.54 mmol). After being stirred overnight, the reaction
mixture was evaporated. The resulting residue was purified by col-
umn chromatography on silica gel (AcOEt/MeOH from 1:0 to 10:1)
and treated with 4 N HCl in AcOEt (2 mL) to give the title com-
pound in 50% yield. TLC Rf 0.50 (CHCl3/MeOH, 10:1); 1H NMR
(300 MHz, CD3OD) d 9.14 (m, 1H), 8.75 (m, 1H), 8.36 (m, 1H),
8.02–7.99 (m, 2H), 7.68–7.62 (m, 3H), 4.63 (s, 2H), 4.05 (dd,
J = 7.5, 4.5 Hz, 1H), 4.02–3.94 (m, 2H), 3.64–3.42 (m, 4H), 2.72–
2.56 (m, 2H), 2.25–2.06 (m, 2H), 1.80–1.10 (m, 15H), 1.00–0.86
(m, 5H); IR (KBr) 3408, 3017, 2925, 2852, 2648, 2494, 2426,
1681, 1666, 1636, 1605, 1452, 1427, 1387, 1374, 1347, 1331,
1314, 1276 cmꢂ1; MS (APCI, Pos) m/z 503 (M+H)+; HRMS Calcd
503.3386, Obsd 503.3394.
a
white powder. TLC Rf 0.62 (CHCl3/MeOH, 10:1); 1H NMR
(200 MHz, CD3OD) d 7.30–7.06 (m, 5H), 4.02 (dd, J = 7.8, 4.8 Hz,
1H), 3.70 (m, 2H), 3.56 (m, 2H), 3.43 (m, 2H), 3.11 (m, 2H), 2.63
(t, J = 7.8 Hz, 2H), 2.46 (m, 2H), 2.18 (m, 2H), 1.95–1.50 (m, 9H),
1.50–1.25 (m, 6H), 0.97 (m, 9H); IR (KBr) 3447, 3199, 2934, 2869,
2663, 2502, 2440, 1673, 1455, 1418, 1372, 1329, 1152, 1086,
1003 cmꢂ1; MS (MALDI, Pos) m/z 456 (M+H)+; Elemental Anal.
Calcd for C28H45N3O2ꢀHClꢀ0.4H2O: C, 67.35; H, 9.45; N, 8.41. Found:
C, 67.67; H, 9.39; N, 8.42.
4.1.7.2. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[(5-methyl-2-phe-
nyl-1,3-oxazol-4-yl)methyl]-1,4,9-triazaspiro[5.5]undecane-
2,5-dione hydrochloride (27). The title compound was synthe-
sized in 76% yield according to the same procedures as described
for the preparation of 24 using 5-methyl-2-phenyl-1,3-oxazole-4-
carboxaldehyde for 4-(3-pyridyl)benzaldehyde. TLC Rf 0.48
(CHCl3/MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 8.04–8.00 (m,
2H), 7.51–7.49 (m, 3H), 4.34 (s, 2H), 4.04 (dd, J = 7.8, 4.8 Hz, 1H),
3.98–3.82 (m, 2H), 3.70–3.60 (m, 2H), 3.44–3.38 (m, 2H), 2.52 (s,
3H), 2.50–2.36 (m, 2H), 2.28–2.12 (m, 2H), 1.80–1.12 (m, 15H),
1.00–0.86 (m, 5H); IR (KBr) 3407, 3182, 3131, 3084, 2956, 2926,
2855, 2658, 2554, 2443, 1681, 1664, 1652, 1559, 1485, 1471,
1450, 1413, 1372, 1337, 1313, 1287, 1146, 1096, 1082, 1068,
1050 cmꢂ1; MS (APCI, Pos) m/z 507 (M+H)+; Elemental Anal. Calcd
for C30H42N4O3ꢀHCl: C, 66.34; H, 7.98; N, 10.32. Found: C, 66.06; H,
8.17; N, 10.05.
4.1.6. A typical procedure for the solution phase Ugi four-
component condensation
4.1.6.1. (3S)-9-Benzyl-1-butyl-3-cyclohexylmethyl-1,4,9-triaza-
spiro[5.5]undecane-2,5-dione hydrochloride (52b). To a stirred
solution of 1-benzyl-4-piperidone 41d (49 g, 260 mmol), n-butyl-
amine 42a (19 g, 260 mmol) and N-Boc-l-cyclohexylalanine 43k
(80 g, 260 mmol) in MeOH (1.3 L) was added 2-(4-morpholinyl)-
ethylisonitrile 50 (36 g, 260 mmol). After being stirred at 55 °C
overnight, the reaction mixture was treated with concd HCl
(260 L) with cooling. The reaction mixture was stirred at 55 °C
for another 2 h, evaporated, treated with Na2CO3 and extracted
with AcOEt. The combined organic layers were washed with brine,
dried (Na2SO4) and evaporated to give deprotected Ugi product as a
yellow oil, which was dissolved in AcOH/toluene (1.25 M, 1.3 L)
and stirred at 80 °C for 1 h. The reaction mixture was cooled to
room temperature, diluted with AcOEt and washed twice with a
small amount of water. The organic layer was washed with aque-
ous NaHCO3, brine, dried (Na2SO4) and evaporated to afford N-ben-
zylpiperidinodiketopiperazine 51b as an oil (91.3 g). This
compound was used to next step without further purification. Deb-
enzylation was carried out by the catalytic hydrogenation at an
atmospheric pressure of the resulting oily product 51b (91.3 g,
210 mmol) in EtOH (1.2 L) in the presence of 20% Pd(OH)2/C
(15 g) for 3 h at 50 °C. Catalyst was removed by filtration through
a pad of Celite. The filtrate was treated with 4 N HCl in AcOEt
(130 mL) and evaporated. The resulting powder was washed with
t-butyl methyl ether to afford 52b as a white powder (70 g, 89%
4.1.7.3.
(3S)-1-Butyl-3-(cyclohexylmethyl)-9-[4-(phenylsulfa-
hydro-
nyl)benzyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione
chloride (29). The title compound was synthesized in 49% yield
according to the same procedures as described for the preparation
of 24 using 4-phenylsulfanyl-benzaldehyde for 4-(3-pyridyl)benz-
aldehyde. TLC Rf 0.74 (CHCl3/MeOH, 10:1); 1H NMR (300 MHz,
CD3OD) d 7.50–7.37 (m, 7H), 7.29 (d, J = 8.4 Hz, 2H), 4.31 (s, 2H),
4.03 (dd, J = 7.5, 7.8 Hz, 1H), 3.84–3.70 (m, 2H), 3.50–3.32 (m,
4H), 2.56–2.38 (m, 2H), 2.24–2.05 (m, 2H), 1.81–1.06 (m, 15H),
1.02–0.84 (m, 5H); IR (KBr) 3425, 3207, 3073, 2957, 2924, 2872,
2850, 2656, 2549, 2436, 1679, 1647, 1601, 1582, 1494, 1472,
1445, 1419, 1373, 1340, 1333, 1312, 1288, 1270, 1146, 1116,
1096, 1081, 1049 cmꢂ1; MS (APCI, Pos) m/z 534 (M+H)+; HRMS
Calcd 534.3154, Obsd 534.3159.
yield in four steps). ½a D18
ꢃ
ꢂ37.5 (c 1.04, MeOH); TLC Rf 0.08
(CHCl3/MeOH/AcOH, 9:1:0.1); 1H NMR (CD3OD)
d
4.05 (dd,
J = 7.8, 4.8 Hz, 1H), 3.84–3.68 (m, 2H), 3.46–3.34 (m, 4H), 2.40–
2.04 (m, 4H), 1.83–1.46 (m, 10H), 1.39 (sextet, J = 7.5 Hz, 2H),
1.33–1.15 (m, 3H), 1.05–0.86 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H).
4.1.7.4. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[4-(phenylsulfonyl)-
benzyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione hydrochloride
(30). The title compound was synthesized in 57% yield according

5220
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
to the same procedures as described for the preparation of 24 using
4-(benzenesulfonyl)benzaldehyde for 4-(3-pyridyl)benzaldehyde.
TLC Rf 0.77 (AcOEt/MeOH, 9:1); 1H NMR (300 MHz, CD3OD) d
8.08 (d, J = 8.4 Hz, 2H), 8.02–7.96 (m, 2H), 7.80 (d, J = 8.4 Hz, 2H),
7.70–7.55 (m, 3H), 4.43 (s, 2H), 4.02 (dd, J = 7.8, 4.8 Hz, 1H),
3.89–3.73 (m, 2H), 3.49–3.34 (m, 4H), 2.48–2.33 (m, 2H),
2.23–2.04 (m, 2H), 1.82–1.14 (m, 15H), 1.03–0.85 (m, 5H); IR
(KBr) 3362, 3202, 3065, 2925, 2851, 2516, 2420, 1672, 1476,
2.49–2.30 (m, 2H), 2.33 (s, 3H), 2.30–2.08 (m, 2H), 1.82–1.10 (m,
15H), 1.05–0.85 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR (KBr) 3368,
3206, 3066, 3033, 2925, 2851, 2661, 2514, 2427, 1670, 1657,
1604, 1502, 1478, 1467, 1449, 1420, 1366, 1347, 1331, 1315,
1243, 1210, 1172, 1113, 1101 cmꢂ1; MS (APCI, Pos) m/z 532
(M+H)+; HRMS Calcd 532.3539, Obsd 532.3541.
4.1.7.9. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[4-(4-methoxy-
phenoxy)benzyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione
hydrochloride (35). The title compound was synthesized in 70%
yield according to the same procedures as described for the prep-
aration of 24 using 4-(4-methoxyphenoxy)benzaldehyde for 4-(3-
pyridyl)benzaldehyde. TLC Rf 0.67 (AcOEt); 1H NMR (300 MHz,
CD3OD) d 7.49 (d, J = 8.4 Hz, 2H), 7.02–6.92 (m, 6H), 4.31 (s, 2H),
4.03 (dd, J = 7.5, 4.5 Hz, 1H), 3.86–3.69 (m, 2H), 3.79 (s, 3H),
3.54–3.30 (m, 4H), 2.50–2.30 (m, 2H), 2.28–2.06 (m, 2H), 1.83–
1.10 (m, 15H), 1.05–0.83 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR (KBr)
3417, 3204, 3069, 2925, 2851, 2656, 2496, 2426, 1681, 1615,
1500, 1475, 1464, 1447, 1420, 1374, 1345, 1315, 1300, 1230,
1199, 1173, 1113, 1098, 1035 cmꢂ1; MS (APCI, Pos) m/z 548
(M+H)+; HRMS Calcd 548.3488, Obsd 548.3481.
1469, 1447, 1416, 1372, 1346, 1309, 1156, 1106, 1071 cmꢂ1
;
MS (APCI, Pos) m/z 566 (M+H)+; HRMS Calcd 566.3053, Obsd
566.3055.
4.1.7.5. (3S)-9-(4-Benzoylbenzyl)-1-butyl-3-(cyclohexylmethyl)-
1,4,9-triazaspiro[5.5]undecane-2,5-dione hydrochloride (31). The
title compound was synthesized in 43% yield according to the same
procedures as described for the preparation of 24 using 4-benzoyl-
benzaldehyde for 4-(3-pyridyl)benzaldehyde. TLC Rf 0.68 (CHCl3/
MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 7.87 (d, J = 8.4 Hz, 2H),
7.82–7.74 (m, 4H), 7.67 (t, J = 8.4 Hz, 1H), 7.57–7.51 (m, 2H), 4.48
(s, 2H), 4.04 (dd, J = 7.8, 4.8 Hz, 1H), 3.84–3.78 (m, 2H), 3.58–3.38
(m, 4H), 2.58–2.40 (m, 2H), 2.30–2.10 (m, 2H), 1.82–1.14 (m, 15H),
1.02–0.86 (m, 5H); IR (KBr) 3434, 3370, 3209, 3061, 2925, 2851,
2659, 2517, 2422, 1660, 1612, 1598, 1577, 1469, 1447, 1418, 1372,
1347, 1317, 1278, 1179, 1148, 1115, 1097, 1074 cmꢂ1; MS (APCI,
Pos) m/z 530 (M+H)+; HRMS Calcd 530.3383, Obsd 530.3375.
4.1.7.10. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[4-(4-hydroxyphen-
oxy) benzyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione hydrochlo-
ride (36). The title compound was synthesized in 45% yield
according to the same procedures as described for the preparation
of 24 using 4-(4-hydroxyphenoxy)benzaldehyde for 4-(3-pyri-
dyl)benzaldehyde. TLC Rf 0.54 (CHCl3/MeOH, 10:1); 1H NMR
(300 MHz, CD3OD) d 7.47 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz,
2H), 6.88 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 4.30 (s, 2H),
4.03 (dd, J = 7.5, 4.5 Hz, 1H), 3.83–3.72 (m, 2H), 3.49–3.34 (m,
4H), 2.38 (m, 2H), 2.23–2.10 (m, 2H), 1.78–1.16 (m, 15H), 1.02–
0.92 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR (KBr) 3363, 3199, 2924,
2851, 2670, 2566, 1674, 1638, 1503, 1468, 1448, 1420, 1373,
1346, 1315, 1227, 1196, 1172 cmꢂ1; MS (APCI, Pos) m/z 534
(M+H)+; HRMS Calcd 534.3332, Obsd 534.3333.
4.1.7.6. 4-{[(3S)-1-Butyl-3-(cyclohexylmethyl)-2,5-dioxo-1,4,9-
triazaspiro[5.5]undec-9-yl]methyl}-N-phenylbenzamide hydro-
chloride (32). The title compound was synthesized in 38% yield
according to the same procedures as described for the preparation
of 24 using 4-(phenylcarbamoyl)benzaldehyde for 4-(3-pyri-
dyl)benzaldehyde. TLC Rf 0.25 (CHCl3/MeOH, 10:1); 1H NMR
(300 MHz, CD3OD) d 8.07 (d, J = 8.1 Hz, 2H), 7.73–7.67 (m, 2H),
7.71 (d, J = 8.1 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.17 (t, J = 7.5 Hz,
1H), 4.45 (s, 2H), 4.05 (dd, J = 7.8, 4.8 Hz, 1H), 3.92–3.72 (m, 2H),
3.58–3.36 (m, 4H), 2.50–2.08 (m, 4H), 1.84–1.08 (m, 15H), 0.96
(t, J = 7.8 Hz, 3H), 0.96 (m, 2H); IR (KBr) 3362, 3250, 3061, 2925,
2851, 2662, 2552, 2424, 1669, 1599, 1575, 1540, 1509, 1498,
1493, 1469, 1442, 1420, 1372, 1321, 1261, 1147, 1113, 1098,
4.1.7.11.
4-(4-{[(3S)-1-Butyl-3-(cyclohexylmethyl)-2,5-dioxo-
1,4,9-triazaspiro[5.5]undec-9-yl]methyl}phenoxy)-N-methylb-
enzamide hydrochloride (37). The title compound was synthe-
sized in 68% yield according to the same procedures as described
for the preparation of 24 using 4-(4-N-methylaminocarbonylphen-
oxy)benzaldehyde for 4-(3-pyridyl)benzaldehyde. TLC Rf 0.25
1076 cmꢂ1
;
MS (APCI, Pos) m/z 545 (M+H)+; HRMS Calcd
545.3492, Obsd 545.3499.
4.1.7.7. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[4-(4-fluorophen-
oxy)benzyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione hydro-
chloride (33). The title compound was synthesized in 87% yield
according to the same manner as described for the preparation of
24 using 4-(4-fluorophenoxy)benzaldehyde for 4-(3-pyridyl)benz-
aldehyde. TLC Rf 0.53 (CHCl3/MeOH, 20:1); 1H NMR (300 MHz,
CD3OD) d 7.53 (d, J = 8.7 Hz, 2H), 7.18–7.00 (m, 6H), 4.33 (s, 2H),
4.04 (dd, J = 7.5, 4.5 Hz, 1H), 3.87–3.69 (m, 2H), 3.55–3.32 (m,
4H), 2.52–2.32 (m, 2H), 2.28–2.08 (m, 2H), 1.83–1.12 (m, 15H),
1.06–0.83 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR (KBr) 3432, 3194,
3134, 3071, 2923, 2852, 2656, 2553, 2426, 1678, 1655, 1648,
1614, 1498, 1473, 1446, 1420, 1375, 1332, 1314, 1250, 1214,
1192, 1173, 1146 cmꢂ1; MS (APCI, Pos) m/z 536 (M+H)+; HRMS
Calcd 536.3288, Obsd 536.3287.
(AcOEt/MeOH, 10:1); 1H NMR (300 MHz, CD3OD)
d 7.85 (d,
J = 8.7 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H),
7.08 (d, J = 8.7 Hz, 2H), 4.37 (s, 2H), 4.05 (dd, J = 7.5, 4.5 Hz, 1H),
3.90–3.68 (m, 2H), 3.58–3.36 (m, 4H), 2.92 (s, 3H), 2.58–2.36 (m,
2H), 2.28–2.06 (m, 2H), 1.84–1.10 (m, 15H), 1.06–0.84 (m, 2H),
0.96 (t, J = 7.2 Hz, 3H); IR (KBr) 3263, 2927, 2547, 1674, 1600,
1499, 1417, 1314, 1245, 1175, 1112, 1049, 1006 cmꢂ1; MS (APCI,
Pos) m/z 575 (M+H)+; Elemental Anal. Calcd for C34H46N4O4ꢀHCl:
C, 66.81; H, 7.75; N, 9.17. Found: C, 64.8; H, 7.98; N, 8.92.
4.1.7.12. N-[4-(4-{[(3S)-1-Butyl-3-(cyclohexylmethyl)-2,5-dioxo-
1,4,9-triazaspiro[5.5]undec-9-yl]methyl}phenoxy)phenyl]meth-
anesulfonamide hydrochloride (38). The title compound was
synthesized in 47% yield according to the same procedures as de-
scribed for the preparation of 24 using 4-(4-methansulfonylamino-
phenoxy)benzaldehyde for 4-(3-pyridyl)benzaldehyde. TLC Rf 0.42
4.1.7.8. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[4-(4-methylphen-
oxy)benzyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione hydro-
chloride (34). The title compound was synthesized in 58% yield
according to the same procedures as described for the preparation
of 24 using 4-(4-methylphenoxy)benzaldehyde for 4-(3-pyri-
dyl)benzaldehyde. TLC Rf 0.71 (AcOEt); 1H NMR (300 MHz, CD3OD)
d 7.50 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.7 Hz,
2H), 6.92 (d, J = 8.7 Hz, 2H), 4.32 (s, 2H), 4.04 (dd, J = 7.5, 4.5 Hz,
1H), 3.87–3.69 (m, 2H), 3.55–3.42 (m, 2H), 3.42–3.34 (m, 2H),
(CHCl3/MeOH, 10:1); 1H NMR (300 MHz, CD3OD)
d 7.53 (d,
J = 9.0 Hz, 2H), 7.29 (d, J = 9.0 Hz, 2H), 7.08–7.00 (m, 4H), 4.33 (s,
2H), 4.03 (dd, J = 7.5, 4.8 Hz, 1H), 3.85–3.72 (m, 2H), 3.54–3.36
(m, 4H), 2.95 (s, 3H), 2.48–2.34 (m, 2H), 2.25–2.08 (m, 2H), 1.80–
1.14 (m, 15H), 0.98–0.88 (m, 5H); IR (KBr) 3361, 3237, 3108,
3057, 2925, 2851, 2663, 2580, 1677, 1632, 1502, 1477, 1449,
1420, 1402, 1389, 1330, 1302, 1276, 1254, 1248, 1218, 1171,

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5221
1150, 1109 cmꢂ1; MS (APCI, Pos) m/z 611 (M+H)+; HRMS Calcd.
611.3267, Obsd 611.3267.
ture was cooled to room temperature, diluted with H2O and
extracted with t-butyl methyl ether. The combined organic layers
were washed with brine, dried (MgSO4) and evaporated. The
resulting residue was purified by column chromatography on silica
gel (hexane/AcOEt, from 4:1 to 3:1). The resulting residue after
evaporation was treated with 4 N HCl in AcOEt (2 mL) and washed
with t-butyl methyl ether to afford the title compound 20 (67 mg,
26% yield). TLC Rf 0.27 (hexane/AcOEt, 2:1); 1H NMR (300 MHz,
CD3OD) d 8.13 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 4.37 (q,
J = 7.2 Hz, 2H), 4.31–4.15 (m, 2H), 4.07 (dd, J = 7.5, 4.5 Hz, 1H),
3.85–3.75 (m, 2H), 3.47–3.38 (m, 2H), 2.67–2.50 (m, 2H), 2.30–
2.12 (m, 2H), 1.85–1.46 (m, 10H), 1.44–1.19 (m, 5H), 1.38 (t,
J = 7.2 Hz, 3H), 1.05–0.88 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR (KBr)
3214, 3085, 2959, 2927, 2851, 2630, 2463, 2401, 1726, 1675,
1658, 1608, 1469, 1459, 1448, 1417, 1407, 1374, 1315, 1307,
1277, 1188, 1177, 1146, 1109, 1020, 1006 cmꢂ1; MS (APCI, Pos)
m/z 484 (M+H)+; HRMS Calcd 484.3175, Obsd 484.317.
4.1.7.13. 4-(4-{[(3S )-1-Butyl-3-(cyclohexylmethyl)-2,5-dioxo-
1,4,9-triazaspiro[5.5]undec-9-yl]methyl}phenoxy)benzenesul-
fonamide hydrochloride (39). The title compound was synthe-
sized in 40% yield according to the same procedures as described
for the preparation of 24 using 4-(4-aminosulfonylphenoxy)benz-
aldehyde for 4-(3-pyridyl)benzaldehyde. TLC Rf 0.33 (CHCl3/MeOH,
10:1); 1H NMR (300 MHz, DMSO-d6) d (300 MHz,) 11.03 (br s, 1H),
8.42 (br s, 1H), 7.82 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.33
(br s, 2H), 7.16 (d, J = 8.7 Hz, 4H), 4.38–4.23 (m, 2H), 3.91 (m, 1H),
3.61–3.23 (m, 6H), 2.58–2.30 (m, 2H), 2.18–1.91 (m, 2H), 1.76–1.00
(m, 15H), 0.98–0.71 (m, 5H); MS (APCI, Pos) m/z (APCI, Pos) 597
(M+H)+.
4.1.7.14.
4-(4-{[(3S)-1-Butyl-3-(cyclohexylmethyl)-2,5-dioxo-
1,4,9-triazaspiro[5.5]undec-9-yl]methyl}phenoxy)benzoic acid
hydrochloride (40). The title compound was synthesized in 60%
yield according to the same procedures as described for the prep-
aration of 24 using 4-(4-formylphenoxy)benzoic acid for 4-(3-pyr-
4.1.10. 2-Butynylamine hydrochloride (42f)
4.1.10.1. 2-Butynylphthalimide (48). To a stirred solution of
2-butynylalchol 47 (5,0 g, 71.3 mmol) in triethylamine (14.9 mL
107 mmol) and CH2Cl2 (210 mL) was slowly added methanesulfo-
nyl chloride (6.1 mL, 78.5 mmol) at 0 °C under argon atmosphere.
After being stirred for 1 h, the reaction mixture was quenched with
water and extracted with AcOEt. The organic layer was washed
with 1 N HCl, saturated NaHCO3 aq, brine, dried over Na2SO4, and
evaporated. To a stirred solution of the resulting residue in DMF
(80 mL) was added potassium phthalimide (11.4 g, 61.5 mmol).
After being stirred for 1.5 h at 70 °C, the reaction mixture was
poured into water (400 mL) and extracted with AcOEt. Precipitates
were removed by filtration and washed with diethyl ether. The fil-
trate was extracted with AcOEt, and the organic layer was washed
with 1 N NaOH and brine, dried over Na2SO4 and evaporated. The
resulting solid was triturated with diethyl ether and dried in vacuo
to yield 48 (10.4 g, 73% in two steps). TLC Rf 0.56 (hexane/AcOEt,
2:1); 1H NMR (200 MHz, DMSO-d6) d 7.90–7.77 (m, 4H), 4.30 (q,
J = 2.4 Hz, 2H), 1.74 (t, J = 2.4 Hz, 3H).
idyl)benzaldehyde. ½a D25
ꢂ27.7 (c 1.03, MeOH); TLC Rf 0.37 (CHCl3/
ꢃ
MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 8.03 (d, J = 8.7 Hz, 2H),
7.63 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz,
2H), 4.37 (s, 2H), 4.04 (dd, J = 7.5, 4.5 Hz, 1H), 3.90–3.70 (m, 2H),
3.56–3.35 (m, 4H), 2.59–2.38 (m, 2H), 2.27–2.05 (m, 2H), 1.83–
1.08 (m, 15H), 1.05–0.83 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); IR (KBr)
2925, 1673, 1598, 1502, 1417, 1243, 1160 cmꢂ1; MS (APCI, Pos)
m/z 562 (M+H)+; Elemental Anal. Calcd for C33H43N3O5ꢀHCl: C,
66.26; H, 7.41; N, 7.02. Found: C, 64.8; H, 7.64; N, 6.98.
4.1.8. (3S)-1-Butyl-3-(cyclohexylmethyl)-9-[2-(4-phenoxyphe
nyl)ethyl]-1,4,9-triazaspiro[5.5]undecane-2,5-dione hydrochlo
ride (19)
A solution of 4-phenoxyphenethyl alcohol 65 (214 mg, 1 mmol)
in pyridine/CH2Cl2 (1:1, 4 mL) was added to chlorosulfonated poly-
styrene resin (305 mg, 0.5 mmol). The mixture was shaken for 5 h
at room temperature. The resin was collected by filtration and suc-
cessively washed with CH2Cl2 (4 mL), DMF (4 mL), DMF/H2O (3:1,
4 mL), THF (4 mL), CH2Cl2 (4 mL) and CH3CN (4 mL). To a suspen-
sion of the resin in MeCN (5 mL) were added N,N-diisopropylamine
(271 mg, 2.1 mmol) and then compound 52b (112 mg, 0.3 mmol).
The mixture was shaken for 18 h at 70 °C. After cooling to room
temperature, the resin was collected by filtration and washed with
MeCN. The combined filtrate and washings were evaporated. The
resulting residue was purified by column chromatography on silica
gel AcOEt/MeOH (from 1:0 to 10:1). The resulting residue after
evaporation was treated with 4 N HCl in AcOEt (2 mL) and then
washed with Et2O to afford 19 (81 mg, 48% yield) as a powder.
TLC Rf 0.54 (AcOEt/MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d
7.37–7.29 (m, 4H), 7.11(t, J = 7.2 Hz, 1H), 6.97–6.95 (m, 4H), 4.06
(d, J = 7.5, 4.5 Hz, 1H), 3.88–3.77 (m, 2H), 3.65 (m, 2H), 3.46–3.36
(m, 4H), 3.13–3.07 (m, 2H), 2.48 (m, 2H), 2.28–2.14 (m, 2H),
1.80–1.21(m, 15H), 0.98 (t, J = 7.0 Hz, 3H), 0.99–0.91 (m, 2H); IR
(KBr) 3364, 3195, 3065, 2924, 2851, 2661, 2525, 2421, 1671,
1589, 1508, 1489, 1470, 1449, 1418, 1373, 1347, 1333, 1317,
1238, 1200, 1169, 1148, 1073 cmꢂ1; MS (APCI, Pos) m/z 532
(M+H)+; HRMS Calcd 532.3539, Obsd 532.3536.
4.1.10.2. 2-Butynylamine hydrochloride (42f). To a stirred sus-
pension of 48 (10.3 g, 51.7 mmol) in EtOH (600 mL) was added
hydrazine hydrate (7.6 mL, 155 mmol). After being stirred for
1.5 h at 100 °C, the mixture was cooled to room temperature,
quenched with concd HCl (25 mL) and evaporated. The resulting
residue was treated with 5 N NaOH and extracted with CH2Cl2
repeatedly. The combined organic layers were washed with brine,
dried over Na2SO4. After the addition of 4 N HCl/AcOEt (20 mL), the
solution was evaporated. The resulting solid was washed with
diethyl ether to yield 42f (5.25 g, 96%). TLC Rf 0.23 (CHCl3/MeOH/
AcOH, 20:4:1); 1H NMR (200 MHz, DMSO-d6) d 8.70–8.15 (br,
3H), 3.62 (br, 2H), 1.83 (t, J = 2.4 Hz, 3H).
4.1.11. 4-Benzoylbenzaldehyde (54)
To a stirred solution of 4-benzoylbenzencalboxylic acid 53
(590 mg, 2.61 mmol) in triethylamine (0.44 mL, 3.13 mmol) and
THF (7 mL) was added i-butylchloroformate (0.40 mL, 3.13 mmol)
at ꢂ78 °C. After being stirred at room temperature for 30 min, tri-
ethylamine hydrochloride salt was removed by filtration. The fil-
trate was added to the suspension of sodium borohydride
(296 mg, 7.83 mmol) in water at 0 °C. After being stirred at room
temperature for overnight, the mixture was concentrated. The
resulting residue was extracted with AcOEt. The organic layer
was washed with water, brine, dried over Na2SO4 and evaporated.
To a stirred solution of the resulting residue in DME (20 mL) was
added MnO2 (682 mg, 7.84 mmol). After being stirred at 100 °C
for overnight, additional MnO2 (2.02 g, 23.2 mmol) was added to
the solution. The reaction mixture was stirred at 110 °C for 4 h
4.1.9. Ethyl 4-[1-butyl-3-(cyclohexylmethyl)-2,5-dioxo-1,4,9-
triazaspiro[5.5]undec-9-yl]benzoate hydrochloride (20)
To a solution of compound 52a (186 mg, 0.501 mmol) in MeCN
(2.5 mL) were added ethyl 4-fluorobenzoate (164 mg, 0.975 mmol)
and then K2CO3 (141 mg, 1.02 mmol). After being stirred for 12 h at
100 °C, the reaction mixture was treated with DMSO (0.5 mL). Stir-
ring was continued for another 12 h at 140 °C. The reaction mix-

5222
R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
and then cooled to room temperature. MnO2 was removed by fil-
tration through the pad of Celite, and the filtrate was evaporated.
The resulting residue was purified by column chromatography on
silica gel to yield 54 (277 mg, 51%). TLC Rf 0.35 (hexane/AcOH,
4:1); 1H NMR (300 MHz, CD3OD) d 10.14 (s, 1H), 7.94–7.92 (m,
2H), 7.83–7.80 (m, 2H), 7.67–7.61 (m, 1H), 7.54–7.49 (m, 2H).
2H), 7.26 (s, 1H), 7.05 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H),
3.91 (s, 3H), 3.04 (s, 3H).
4.1.15.2.
4-(4-Methansulfonylaminophenoxy)benzaldehyde
(62). To
a
stirred solution of 61 (29 g, 90.3 mmol) in THF
(300 mL) was added 1.0 M diisobutyl aluminum hydride/n-hexane
solution (270 mL, 270 mmol) at 0 °C. After being stirred for 2 h, the
reaction mixture was treated with additional diisobutyl aluminum
hydride (1.0 M, 90 mL, 90 mmol) and stirred for 20 min at 0 °C. The
reaction mixture was quenched with saturated Na2SO4 aq and the
resulting precipitates were removed by filtration. The filtrate was
evaporated and the resulting residue was dissolved in CH2Cl2/
DME (150:150). MnO2 (40 g, 482 mmol) was added to the solution
The reaction mixture was stirred for overnight and then filtered
through a pad of Celite and the filtrate was concentrated in vacuo
to yield 62 (26.0 g, 99%). TLC Rf 0.46 (hexane/AcOEt); 1H NMR
(300 MHz, CDCl3) d 9.92 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.28 (d,
J = 8.7 Hz, 2H), 7.25 (s, 1H), 7.10–7.05 (m, 4H), 3.05 (s, 3H).
4.1.12. 4-(4-Hydroxyphenoxy)benzaldehyde (56)
To a stirred solution of 55 (3.69 g, 16.1 mmol) in CH2Cl2 (80 mL)
was slowly added 1.0 M boron tribromide/CH2Cl2 solution (35 mL,
0.35 mmol) at 0 °C. After being stirred for 2 h, the reaction mixture
was quenched with water and extracted with diethyl ether. The or-
ganic layer was washed with water, brine, dried over Na2SO4 and
evaporated. The resulting residue was purified by column chroma-
tography on silica gel to yield the title compound 56 (3.06 g, 86%).
TLC Rf 0.28 (hexane/AcOEt, 2:1); 1H NMR (300 MHz, CDCl3) d 9.90
(s, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H), 6.98 (d,
J = 9.0 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 5.47 (br s, 1H). MS (APCI,
Pos) 257 (M+H)+.
4.1.16. 4-(4-Aminosulfonylphenoxy)benzaldehyde (64)
4.1.13. 4-(4-Formylphenoxy)benzoic acid (58)
A suspension of 63 (4.33 g, 25 mmol), 4-fluorobenzaldehyde
(2.68 mL, 25 mmol), and K2CO3 (6.9 g, 50 mmol) in DMA (20 mL)
was stirring under reflux for 2 h. After being cooled to room tem-
perature, the mixture was quenched with water and extracted
with AcOEt. The organic layer was washed with water, brine, dried
over Na2SO4 and evaporated. The resulting solid was recrystallized
from AcOEt/hexane to yield the title compound 64 (1.22 g, 18%).
TLC Rf (CHCl3/MeOH, 10:1); 1H NMR (300 MHz, CD3OD) d 9.93 (s,
1H), 7.96 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.7 Hz, 2H), 7.21 (d,
J = 8.7 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H); MS (APCI, Neg) 276 (MꢂH)ꢂ.
A mixture of methyl 4-hydroxybezoate 57 (25.2 g, 16.5 mmol),
4-fluorobenzaldehyde (18.6 g, 14.9 mmol) and K2CO3 in DMF
(150 mL) was stirring under reflux for 2 h. After being cooled to
room temperature, the reaction mixture was quenched with water
and extracted with AcOEt. The organic layer was washed with
water, brine, dried over MgSO4 and evaporated. The resulting solid
was triturated with n-hexane and dried to yield the methyl ester of
58 (27.8 g, 72%). To a stirred solution of the methyl ester in meth-
anol (400 mL) was added 2 N NaOH (108 mL, 54 mmol). After being
stirred for 2 h at 50 °C, the reaction mixture was cooled to room
temperature and quenched with 2 M HCl (110 mL) and water
(200 mL). The precipitates were collected by filtration and dried
to yield 58 (23.4 g, 89%). TLC Rf 0.20 (hexane/AcOEt, 1:1); 1H
NMR d (300 MHz, CDCl3) 9.98 (s, 1H), 8.16 (d, J = 8.7 Hz, 2H), 7.92
(d, J = 8.7 Hz, 2H), 7.17 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 8.7 Hz, 2H).
4.2. Biology
4.2.1. Stability study of liver microsomes
The test substance (5
195 L of 50% acetonitrile in water to make a 250
of the test substance.
Phosphate buffer (0.1 mol/L, 245
l
L:10 mmol/L in DMSO) was diluted with
l
l
mol/L solution
4.1.14. 4-(4-N-Methylaminocarbonylphenoxy)benzaldehyde
(59)
lL) containing 0.2 mg/mL or
0.5 mg/mL rat liver microsomes and NADPH-Co-factor* was added
to a reaction container, pre-warmed to 37 °C in a water bath, and
incubated for 5 min. The reaction was initiated with the addition
To a stirred solution of compound 58 (9.8 g, 40.5 mmol) in DMF
were added HOBt (6.56 g, 48.6 mmol), EDC hydrochloride (9.32 g,
48.6 mmol) and a solution of methylamine in THF (2.0 M, 41 mL,
81 mmol). After being stirred for 1.5 h, the reaction mixture was
poured into water (500 mL), acidified with 2 N HCl (10 mL) and ex-
tracted with AcOEt. The organic layer was washed with saturated
NaHCO3 aq, brine, dried over Na2SO4, and evaporated. The resulting
solid was triturated with t-butyl methyl ether to yield 59 (9.23 g,
89%). TLC Rf 0.55 (CHCl3/MeOH = 10:1); 1H NMR d (300 MHz,
CDCl3) 9.95 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 9.0 Hz, 2H),
7.13–7.10 (m, 4H), 3.03 (d, J = 4.8 Hz, 3H); MS (APCI, Pos) 256
(M+H)+.
of 5
0.05% DMSO, final concentration of 5
was taken from the mixture immediately after the start of the reac-
tion, and transferred to 180 L of acetonitrile containing the inter-
nal standard (warfarin) to terminate the reaction. A 20 L aliquot
of the mixture was stirred with 180 L of 50% acetonitrile on a
lL of the test substance solution (in 0.975% acetonitrile with
l
mol/L). A 20 L aliquot
l
l
l
l
plate with a filter for deproteinization, and filtered by suction.
The filtrate was used as the standard sample.
After a 15 min incubation of the above mixture, a 20
was transferred to 180 L of acetonitrile containing the internal
standard (warfarin) to terminate the reaction. A 20 L aliquot of
the mixture was stirred with 180 L of 50% acetonitrile on a plate
lL aliquot
l
l
4.1.15. 4-(4-Methansulfonylaminophenoxy)benzaldehyde (62)
4.1.15.1. Methyl-4-(4-methansulfonylaminophenoxy)benzoate
(61). A suspension of 60 (25.5 g, 93.5 mmol) and 5% Pd–C (1.3 g)
in AcOEt (300 mL) was stirred overnight under hydrogen atmo-
sphere. The reaction mixture was filtered through a pad of Celite
and the filtrate was evaporated. To the resulting residue in THF
(300 mL) and pyridine (22.4 mL, 277 mmol) was added methane-
sulfonyl chloride (10.7 mL, 139 mmol) at 0 °C. After being stirred
for overnight at room temperature, the reaction mixture was
quenched with water and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4 and evaporated. The
resulting solid was triturated with t-butyl methyl ether to yield
61 (29.0 g, 98%). TLC Rf 0.5 (hexane/AcOEt, 1:1); 1H NMR
(300 MHz, CD3OD) d 8.01 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 9.0 Hz,
l
with a filter for deproteinization, and filter by suction. The filtrate
was used as the reaction sample.
*NADPH-Co-factor: Dilute solutions A and B in the NADPH-
regenerating system (BD-Bioscience) 20- and 100-fold, respec-
tively, with 0.1 mol/L phosphate buffer (NADP+ 2.6 mmol/L).
4.2.1.1. Determination of test substance concentration and data
processing. One microliter of aliquot was injected into an LC–MS/
MS system. The percent residue (%) is calculated by dividing the
peak area ratio (i.e., test substance/I.S.) for the reaction sample
by the peak area ratio for the standard sample and multiplying
by 100.

R. Nishizawa et al. / Bioorg. Med. Chem. 18 (2010) 5208–5223
5223
4.2.2. Primary screening of solubility
ter (w/v). Test compounds (3 mg/kg) were dosed intravenously to
the fasted male rats (n = 3). Test compounds (30 mg/kg) were
dosed orally to the fasted male rats (n = 3). After dosing, blood
The test substance (10 mmol/L DMSO) was diluted with aceto-
nitrile. Acetonitrile containing the internal standard (warfarin)
was added to prepare samples for calibration curves at 0.1, 0.4,
samples (250 lL) were collected from the jugular vein using a hep-
and 2
The test substance (5
495 L of JP Solution 2. The solution was stirred at room tempera-
ture for 5 h, and transferred onto a filter plate, and filtered by cen-
trifugation at 3000 rpm, 24 °C for 15 min. The filtrate (20 L) was
l
mol/L.
arinized syringe at the selected time points (iv: pre-dosing, 2, 5, 15,
and 30 min; po: 1, 2, 4, 6, and 8 h, respectively). The blood samples
were ice-chilled and then centrifuged at 12,000 rpm for 2 min at
room temperature to obtain plasma, which was preserved at
ꢂ70 °C in a freezer. The AUC, Cmax, Tmax, T1/2, Vss, and CL were ob-
tained by measuring the time course of the plasma concentration
of the test compounds. Bioavailability (BA) was calculated accord-
ing to the following equation:
l
L:10 mmol/L DMSO) was added to
l
l
diluted with acetonitrile, and additional acetonitrile containing
the internal standard to prepare a sample solution.
The sample solution (1
tem for quantification (range: 0.1–2
determined as 50 times of the observed value. The solubility was
lL) was injected into an LC–MS/MS sys-
lmol/L). The solubility was
BA ð%Þ ¼ ðAUCpo=DpoÞ=ðAUCiv=DivÞ ꢁ 100
reported as <5
side the range.
lmol/L or 100 lmol/L if the observed value is out-
References and notes
1. Hoffmann, C.; Mulcahy, F. Overview of Antiretroviral Agents. In HIV Medicine
2006; Hoffmann, C., Rockstroh, J. K., Kamps, B. S., Eds.; Flying Publisher
(FlyingPublisher.com): Paris, 2006; pp 94.
2. (a) Schwarz, M. K.; Wells, T. N. C. Nat. Rev. Drug Disc. 2002, 1, 347; (b) Gerard, C.;
Rollins, B. J. Nat. Immunol. 2001, 2, 108; (c) Pirotte, B.; Tullio, P.; Nguyen, Q. A.;
Somwes, F.; Fraikin, P.; Florence, X.; Wahl, P.; Hansen, J. B.; Lebrun, P. J. Med.
Chem. 2010, 53, 147.
3. (a) Feng, Y.; Broder, C. C.; Kennedy, P. E.; Berger, E. A. Science 1996, 272, 872; (b)
Deng, H. K.; Liu, R.; Ellmeier, W.; Choe, S.; Unutmaz, D.; Burkhart, M.; Marzio, P.
D.; Marmon, S.; Sutton, R. E.; Hill, C. M.; Davis, C. B.; Peiper, S. C.; Schall, T. J.;
Littman, D. R.; Landau, N. R. Nature 1996, 381, 661; (c) Dragic, T.; Litwin, V.;
Allaway, G. P.; Martin, S. R.; Huang, Y.; Nagashima, K. A.; Cayanan, C.; Maddon, P.
J.; Koup, R. A.; Moore, J. P.; Paxton, W. A. Nature 1996, 381, 667; (d) Alkhatib, G.;
Combadiere, C.; Broder, C. C.; Feng, Y.; Kennedy, P. E.; Murphy, P. M.; Berger, E. A.
Science 1996, 272, 1955; (e) Berger, E. A.; Murphy, P. M.; Farber, J. M. Annu. Rev.
Immunol. 1999, 17, 657; (f) Caldwell, D. J.; Evans, J. D. Expert Opin. Pharmacother.
2008, 9, 3231.
4. (a) Leonard, J. T.; Roy, K. Curr. Med. Chem. 2006, 13, 91; (b) Price, D. A.; Armour,
D.; Groot, M.; Leishman, D.; Napier, C.; Perros, M.; Stammen, B. L.; Wood, A.
Bioorg. Med. Chem. Lett. 2006, 16, 4633.
5. Habashita, H.; Kokubo, M.; Hamano, S.; Hamanaka, N.; Toda, M.; Shibayama, S.;
Tada, H.; Sagawa, K.; Fukushima, D.; Maeda, K.; Mitsuya, H. J. Med. Chem. 2006,
49, 4140.
6. (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,
35194; (b) Maeda, K.; Nakata, H.; Koh, Y.; Miyakawa, T.; Ogata, H.; Takaoka, Y.;
Shibayama, S.; Sagawa, K.; Fukushima, D.; Moravek, J.; Koyanagi, Y.; Mitsuya, H.
J. Virol. 2004, 78, 8654.
7. (a) Nishizawa, R.; Nishiyama, T.; Hisaichi, K.; Matsunaga, N.; Minamoto, C.;
Habashita, H.; Takaoka, Y.; Toda, M.; Shibayama, S.; Tada, H.; Sagawa, K.;
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4.2.3. Caco-2 membrane permeability assay
Caco-2 cells were grown on a 12-well Costar Transwell plate
(with a collagen-coated fine porous polycarbonate membrane) un-
til a confluent monolayer is formed. Buffer for permeability assay
was prepared using 10 mM HEPES and Hank’s balanced salt solu-
tion containing 15 mM glucose with the pH being adjusted at
7.3–7.5. A test substance solution was prepared at 10 mM the as-
say buffer. The test substance solution was added to the apical side
of Caco-2 cell monolayer at a final concentration of 10
then the plate was incubated in a humidified incubator (5% CO2,
37 °C). Two hours later, 200- and 50 L aliquots were taken from
lM, and
l
the receiver chambers. The measurements should be performed
in duplicate. The concentration of test substance in the samples
was determined by LC–MS/MS.
The apparent permeability coefficient (Papp) was calculated
according to the following equation:
Papp ¼ ðdCr=dtÞ ꢁ Vr=ðA ꢁ C0Þ
dCr/dt: slope of compound accumulation in the receiver compart-
ment over time (lM/s)
Vr: volume of the receiver compartment (cm3)
A: area of the cell monolayer (1.13 cm2 for 12-well transwell
plate)
C0: initial normality of the buffer (
lM)
4.2.4. Pharmacokinetic (PK) studies
Single dose pharmacokinetics was studied in rats. Formulation
for intravenous injection was prepared using SWI (sterile water
for injection) containing 30% HP-b-CD (w/v). Formulation for oral
dosing was prepared using SWI containing 1% sucrose fatty acid es-
9. Further PK studies of 38 and 39 were not carried out because of the poor AUC
after their oral cassette dosing.