Potent and selective cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.06.032

A novel series of cathepsin K inhibitors derived from Novartis compound I is described. Optimization of the P1, P3, and P1′ units led to the identification of 4-aminophenoxyacetic acid 24b with an IC₅₀ value of 4.8 nM, which possessed an excell

Full text of DOI:10.1016/j.bmc.2006.06.032

Bioorganic & Medicinal Chemistry 14 (2006) 6789–6806
Potent and selective cathepsin K inhibitors
Tsuyoshi Shinozuka,a,* Kousei Shimada,a Satoshi Matsui,a Takahiro Yamane,a
Mayumi Ama,a Takeshi Fukuda,a Motohiko Taki,b Yuko Takeda,b Eri Otsuka,b
Michiko Yamato,b Shin-ichi Mochizuki,c Keiko Ohhatac and Satoru Naitoa
aMedicinal Chemistry Research Laboratories, Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
bLead Discovery Research Laboratories, Sankyo Co. Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
cBiological Research Laboratories, Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 11 May 2006; revised 20 June 2006; accepted 21 June 2006
Available online 13 July 2006
Abstract—A novel series of cathepsin K inhibitors derived from Novartis compound I is described. Optimization of the P1, P3, and
P10 units led to the identification of 4-aminophenoxyacetic acid 24b with an IC50 value of 4.8 nM, which possessed an excellent
selectivity over other human cathepsins and good pharmacokinetic (PK) properties. Oral administration of compound 24b to
ovariectomized (OVX) rats showed a trend toward an improvement of bone mineral density (BMD) in the femur bone.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
inhibit cathepsin K,3 because this compound had been
shown to be a non-covalent and reversible inhibitor.4
Scrutiny of the X-ray crystallographic data of cathepsin
K complexed with inhibitors bearing a benzyloxycar-
bonyl (Cbz) group5,6 revealed that the carbonyl oxygen
of the Cbz group did not interact with the enzyme.
Therefore, we envisaged to convert the Cbz group of
Novartis compound I to a biphenyl group by tethering
the carbonyl oxygen to the benzylic carbon, as shown
in Figure 1. Such P3 sub-structures had not been report-
ed when we started this project.3c
Bone mass is regulated by the continuous equilibrium
between the bone-resorbing process and the bone-form-
ing process. It is known that an imbalance of this
remodeling system causes osteoporosis. In the bone-re-
sorbing process, osteoclasts adhere to the bone surface,
and secrete acid and proteolytic enzymes to resorbe
bone. One of the proteolytic enzymes is a lysosomal cys-
teine protease, cathepsin K,1 which exhibits potent col-
lagenolytic activity against type I collagen, one of the
main constituents of bone matrix. Thus, selective inhibi-
tion of cathepsin K has been expected to provide an effi-
cacious therapeutic treatment for bone diseases. In a
previous paper, we reported biphenylamino acid amides
as novel and potent cathepsin K inhibitors found by
modification of Novartis compound I.2 Herein, we wish
to report on the detailed syntheses and structure–activi-
ty relationships of S-, O-, and NH-linked cathepsin K
inhibitors. In addition, we wish to describe the selectiv-
ity over other human cathepsins, pharmacokinetics, and
in vivo evaluation of several potent compounds.
P3
P2
P1
P1'
OMe
O
H
N
O
N
N
H
H
O
I
OMe
H
N
We selected aryl amine based derivative I as a lead com-
pound from among many kinds of compounds that
X
N
H
O
X= S, O or NH
Keywords: Cathepsin K; Inhibitor; SAR study; OVX rat assay.
*
Figure 1. Modification of compound I to S-, O-, and NH-linked
compounds.
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436
8563; e-mail: sinozu@sankyo.co.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.032

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T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
2. Chemistry
O
H
N
The preparation of S- and O-linked compounds is
shown in Scheme 1. Phenoxycarboxylic acids 3 (X@O)
were prepared by etheration of phenols 1 (X@O) with
N
N
H
H
O
a-bromoesters 2. Phenylsulfanylcarboxylic acids
3
8g
O
(X@S) were prepared in the same manner. Carboxylic
acids 3 were coupled with N-(4-methoxyphenyl)ethane-
1,2-diamine7 under usual reaction conditions (WSCÆHCl
and HOBt in DMF) to provide S- or O-linked com-
pounds 4a–m. As racemic methyl 2-bromo-4-methyl-
pentanoate (2; R2 = i-Bu, R3 = H) was employed in
these reactions, 4a and 4c–f were prepared as racemic
compounds.
H
N
a
N
H
N
H
O
8i
O
H
R
N
N
H
N
H
O
O
The synthesis of N-aryl amino acids 7 and the subsequent
coupling to NH-linked compounds 8 are shown in
Scheme 2. The preparation of N-aryl amino acids 7 was
accomplished by coupling aryl bromides 5 with amino
acids 6 using a copper catalyst.8 Subsequent amidation
of 7 afforded NH-linked compounds 8a–h and 8m–q.
8h: R = OMe
8j: R = OH
b
c
8k: R = 4-morpholinyl
e
N
N
H
CO H
2
N
X
Scheme 3 shows the preparation of NH-linked com-
pounds 8i–l. Cyclohexane 8i was prepared by hydroge-
nation of cyclohexene 8g. Saponification of methyl
ester 8h provided carboxylic acid 8j. Subsequent conden-
sation with morpholine afforded amide 8k. The prepara-
tion of piperidine 8l was achieved by N-alkylation.
Reduction of nitrobenzene 9 (SnCl2Æ2H2O in EtOH)
and N-alkylation (KOH in t-BuOH) provided carboxyl-
ic acid 11. Subsequent amidation of 11 provided 8l.
As racemic 2-bromo-4-methylpentanoic acid9 was
employed in N-alkylation, 8l was prepared as racemic
compound.
11
9: X = NO
2
2
d
10: X = NH
O
H
N
f
N
N
H
N
H
O
8l
Scheme 3. Synthesis of NH-linked compounds 8i–l. Reagents: (a) H2,
Pd(OH)2/C, EtOH, 45%; (b) NaOH, MeOH/H2O, 52%; (c) morpho-
line, WSCÆHCl, HOBt, CH2Cl2, 70%; (d) SnCl2Æ2H2O, EtOH, reflux,
96%; (e) 2-bromo-4-methylpentanoic acid, KOH, t-BuOH, reflux, 42%;
(f) N-(4-methoxyphenyl)ethane-1,2-diamine, WSCÆHCl, HOBt, DMF,
61%.
R2 R3
R2 R3
a
R1
R1
As shown in Scheme 4, the preparation of branched
compounds 17a–l was accomplished using 2-nitroben-
zenesulfonamides as intermediates.10 Commercially
available aminoalcohols were converted to correspond-
ing N-Boc aminoalcohols 13,11 and other N-Boc amino-
alcohols were prepared from the corresponding N-Boc
amino acids 12.12 Mitsunobu reaction13 of N-Boc amin-
oalcohols 13 with 2-nitrobenzenesulfonamide 1414 affor-
ded branched N-Boc ethylenediamines 15, which were
deprotected with PhSH to provide branched ethylenedi-
amines 16. Deprotection of the Boc group and amida-
+
Br
CO2Me
X
CO2H
XH
1
2
3
O
R2 R3
b
R1
H
N
X
N
H
O
4a-m (X = S or O)
Scheme 1. Synthesis of S- and O-linked compounds 4a–m. Reagents
and condition: (a) KOH, t-BuOH, reflux; (b) N-(4-methoxyphenyl)eth-
ane-1,2-diamine, WSCÆHCl, HOBt, DMF.
tion with carboxylic acids
compounds 17a–l.
7 furnished branched
R2 R3
R2 R3
The preparation of compounds 23–29 is shown in
Scheme 5. Mitsunobu reaction13 of N-Boc aminoalcohol
13c with 2-nitrobenzenesulfonamide 18 and subsequent
deprotection provided 20.10 Coupling of 20 with carbox-
ylic acids 7, followed by cleavage of benzyl ether (H2,
Pd/C, and EtOH), provided phenols 22. Alkylation of
22 with tert-butyl bromoacetate afforded esters 23.
Saponification of 23 furnished acids 24. In a similar
manner, ester 25 and nitrile 26 were prepared. Tetrazole
27 was prepared from 26 under usual reaction condi-
tions. The preparation of propanoic acid 29 was
achieved by complete reduction of acrylate 28b, which
was synthesized from phenol 22b.15
a
R1
R1
+
H2N
CO2H
N
CO2H
Br
H
5
6
7
O
R2 R3
N
b
R1
H
N
N
H
H
O
8a-h, 8m-q
Scheme 2. Synthesis of NH-linked compounds 8a–h and 8m–q.
Reagents and condition: (a) CuBrÆSMe2, K2CO3, DMA, 120 ꢁC; (b)
N-(4-methoxyphenyl)ethane-1,2-diamine, WSCÆHCl, HOBt, DMF.

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6791
Scheme 4. Synthesis of branched compounds 17a–l. Reagents: (a) N-methylmorpholine, ethyl chloroformate, THF, then NaBH4, MeOH; (b)
DEAD, PPh3, THF; (c) PhSH, K2CO3, MeCN; (d) HCl, 1,4-dioxane, then 7, WSCÆHCl, HOBt, CH2Cl2. Ns, 2-nitrobenzenesulfonyl.
OBn
BocHN
BocHN
OH
N
a
Y
13c
19: Y = Ns
20: Y = H
b
OBn
3
OR
1
2
NsHN
R
R
H
N
c
N
H
N
H
18
O
3
21: R = Bn
1
2
d
e
f
a: R = i-Bu, R = H
3
22: R = H
1
2
-
b: R = R = -(CH )
3
2 5
23: R = CO t-Bu
2
3
24: R = CO H
2
O
g
h
3
22a
22a
25: R =
O
3
26: R = CH CN
2
i
N
N
3
N
27: R =
N
H
4
O
OR
H
N
O
j
N
N
H
22b
H
O
4
28a: R = Me
4
28b: R = CH Ph
2
O
OH
H
N
O
k
N
N
H
28b
H
O
29
Scheme 5. Synthesis of compounds 23–29. Reagents and conditions: (a) DEAD, PPh3, THF; (b) PhSH, K2CO3, MeCN, 82% (two steps); (c) TFA,
CH2Cl2, then 7, WSCÆHCl, HOBt, DMF, 99% (21a), 84% (21b); (d) H2, Pd/C, EtOH, 87% (22a), 99% (22b); (e) tert-butyl bromoacetate, NaH, DMF/
THF, 81% (23a), 79% (23b); (f) TFA, CH2Cl2, then HCl, 1,4-dioxane, 74% (24a), 95% (24b); (g) ethyl 2-bromo-2-methylpropanoate, NaH, DMF/
THF, 61%; (h) bromoacetonitrile, NaH, DMF/THF, 59%; (i) NaN3, NH4Cl, DMF, 100 ꢁC, 69%; (j) N-methylmorpholine, methylpropiolate or
benzylpropiolate, THF, 64% (28a), 66% (28b); (k) H2, Pd/C, EtOH, 37%.

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T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
Table 2. Inhibitory activities of NH-linked compounds
3. Results and discussion
OMe
R2 R3
R1
H
N
The inhibitory activities of S- and O-linked compounds
against recombinant human cathepsin K are summa-
rized in Table 1. The assays were carried out at pH 3.8
since purified procathepsin K was efficiently activated
at lower pH, such as pH 4.0.16 In addition, the activity
of mature cathepsin K at pH 3.8 was around 40% when
compared to that at pH 6.0 using Cbz-Phe-Arg-AMC as
a substrate (data not shown). Isobutyl and cyclohexyl
groups were employed as the P2 unit, and isobutyl deriv-
atives (4a and 4c–j) were prepared as racemic mixtures.
Compound I indicated an IC50 of 0.09 lM. This IC50
value obtained in our assay was similar to the reported
value (0.07 lM).4 Among the S-linked compounds, iso-
butyl derivative 4a showed 10 times greater potency
than cyclohexyl analogue 4b. Phenyl ethers such as 4c
and 4g exhibited an IC50 value of 3 lM. Although
S-linked compound 4a showed better potency than
O-linked compounds 4c and 4g, further modifications
of the O-linked compounds were performed, because
thio ethers are metabolically unstable in general. Among
isobutyl derivatives, the addition of a phenyl group to
the phenoxy group in compound 4c at the 3- or 4-posi-
tion increased the inhibitory activity by 30 times (4c, 4e,
and 4f), while substitution at the 2-position brought lit-
tle improvement in activity (4d). Similar tendency was
observed among cyclohexyl derivatives (4h–j). Interest-
ingly, alkyl derivatives 4k–m retained good inhibitory
activities, and cyclohexyl derivative 4m was 2-fold more
potent than 4-biphenyl ether 4j.
N
N
H
H
O
Compound R1
R2
R3 IC50 (lM)
a
8a
8b
8c
8d
8e
8f
H
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
i-Bu
11
>20
0.083
0.1
2-Ph
3-Ph
4-Ph
H
H
H
H
H
H
H
H
10
0.04
3-Ph
i-Bu
i-Bu
8g
8i
3-c-Hexenyl
3-c-Hexyl
3-CO2H
0.059
0.06
0.95
3.7
i-Bu
i-Bu
8j
8k
8lc
8m
8n
8o
8p
8q
3-(4-Morpholinecarbonyl) i-Bu
i-Bu
3-(1-Piperidyl)
3-MeO
4-MeO
4.5
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
NIb
>20
2
3-i-PrO
3-i-BuO
3-PhCH2O
0.4
3
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as a substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.
b Not inhibited at 20 lM.
c Racemic compound.
and 8d possessed increased activities compared to the
corresponding biphenyl ethers 4i and 4j. The improve-
ment in activity could be ascribed to a hydrogen bond
between the amino group and the enzyme as mentioned
in the literature.3c In the aryl amine series, 3-biphenyl-
amine derivative 8c displayed better activity than
4-biphenylamine derivative 8d, and the N-(3-biphenyl)-
L-leucyl group was found to be a better P2–P3 substitu-
ent than the 3-biphenylaminocyclohexylcarbonyl group
(8c and 8f). As N-(3-biphenyl)-L-leucine 8f possessed
2-fold better inhibitory activity than compound I, the
replacement of the terminal phenyl group of 8f with
other groups was examined. Cyclohexenyl and cyclohex-
yl groups did not enhance the activity (8g and 8i). This
was unlike the same displacement in aryl ether 4j, which
resulted in more potent 4m. Replacement by more polar
substituents such as a carboxy group reduced the activ-
ity by 15 times (8j). Morpholine amide 8k and piperidine
8l had activities in the micromolar range. Finally, the
incorporation of alkoxy groups was examined. A meth-
oxy group was introduced into the 3- or 4-position of
the benzene ring of 8a, and both compounds 8m and
8n displayed almost no activity. Bulkier alkoxy substitu-
ents increased the activity; however, the most effective
derivative among the alkoxy compounds, 8p, was 10-
fold less potent than 8f.
Then, the inhibitory activities of NH-linked compounds
were examined as indicated in Table 2. Anilines 8a and
8e showed reduced potencies compared to phenyl ethers
4g and 4c, respectively. 2-Biphenylamine 8b did not indi-
cate any inhibitory activities, whereas biphenylamines 8c
Table 1. Inhibitory activities of O- and S-linked compounds
OMe
R2 R3
R1
H
N
X
N
H
O
Compound
X
R1
R2
R3
H
IC50 (lM)
a
I
0.09
1.6
4ab
4b
4cb
4db
4eb
4fb
4g
4h
4i
S
H
H
H
i-Bu
S
–(CH2)5–
i-Bu
i-Bu
20
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
3
1.2
2-Ph
3-Ph
4-Ph
H
i-Bu
i-Bu
0.09
0.1
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
3
2-Ph
3-Ph
4-Ph
4-Et
4-i-Pr
>15
0.32
0.19
0.13
0.2
4j
4k
4l
In the structure–activity relationship study described
above, we found the 3-biphenylamino group was prefer-
able as a P3 substituent. It was expected that the intro-
duction of an alkyl group extending into the S1 pocket
would enhance the inhibitory activity, and we synthe-
sized derivatives with an alkyl side chain in the ethylene-
diamine moiety of compounds 8c and 8f. The effect of
4m
4-c-Hex
0.099
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as a substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.
b Racemic compound.

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6793
Table 4. Variation of P10 substituent: inhibitory activities of NH-
linked compounds
this side chain is summarized in Table 3. First, both ster-
eoisomers of methyl derivatives 17a and 17b were pre-
pared, and (S)-isomer 17a was found to be more
potent. Therefore, derivatives of various alkyl side
chains with the (S)-configuration were examined. The
incorporation of linear alkyl side chains increased inhib-
itory activity more than 10 times, and the best activity in
this series was observed when an ethyl group was incor-
porated (17c). The substitution of a branched alkyl side
chain such as isopropyl and isobutyl groups retained the
potency (17f and 17g), whereas the side chains contain-
ing a phenyl group were less suitable (17h–j). Among the
L-leucine derivatives, ethyl derivative 17l showed excel-
lent inhibitory activity (IC50 = 3.8 nM).
OR3
R1 R2
H
N
N
H
N
H
O
a
IC50 (nM)
Compound
R1
R2
R3
17c
17l
–(CH2)5–
i-Bu
Me
Me
2.7
3.8
H
21b
27
–(CH2)5–
i-Bu
2.8
11
N
N
N
H
H
H
H
H
Next the substituent on the benzene ring in the P10 unit
of 17c and 17l was examined. The result is summarized
in Table 4. As previously reported,4b benzyl derivative
21b retained good inhibitory activity. Unexpectedly,
we found that the replacement of the phenyl group in
compound 21b with a tetrazole group led to moderate
compound 27, which was only 4-fold less potent than
21b. We then synthesized carbonyl derivatives such as
esters and carboxylic acids, although it had been shown
that lipophilic groups are favorable for the S10 pocket.4b
tert-Butyl ester 23a showed a 2-fold increase in the
inhibitory activity compared to tetrazole 27. Dimethyl
derivative 25 and nitrile 26 exhibited moderate activities.
Interestingly, the IC50 value of carboxylic acid derivative
24a was 4.5 nM, which was more potent than ester 23a
and similar to that of methoxy derivative 17l. 3-Biphe-
nylaminocyclohexyl analogues of the above derivatives
were also prepared and evaluated for inhibitory activity.
Compound 24b possessed good inhibitory activity with
an IC50 of 4.8 nM. Acrylates 28a and 28b did not indi-
cate potent activity. Propanoic acid 29 possessed excel-
N
H
O
O
23a
25
i-Bu
6.4
13
O
i-Bu
O
N
26
i-Bu
15
O
24ab
i-Bu
4.5
OH
O
O
23b
24bb
28a
28b
29
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
4.8
4.8
32
O
OH
OMe
O
O
O
O
Ph
32
Table 3. Variation of P1 substituent: inhibitory activities of NH-linked
compounds
OH
29
OMe
R1 R2
H
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.
N
N
H
N
H
R3 R4
O
a
IC50 (nM)
Compound
R1
R2
R3
R4
b 2HCl salt.
8c
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
i-Bu
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
83
10
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
8f
H
Et
82
lent inhibitory activity (IC50 = 2.9 nM), but structural
instability prevented further evaluation.
2.7
3.9
5.6
7.7
7.0
19
n-Pr
n-Bu
i-Pr
i-Bu
Ph
Several potent compounds were selected for further
pharmacological evaluation. Table 5 summarizes in vitro
metabolic stability in rat and human hepatic micro-
somes and pharmacokinetics in rats. Pharmacokinetic
(PK) parameters were measured by orally administering
rats a dose of 50 mg/kg in cremophor/EtOH (2:1). The
metabolic stability of the compounds strongly depended
on the structure of the P10 substituent. Tetrazole 27
showed great metabolic stability, whereas benzyl ether
21b was moderately stable. Although tert-butyl esters
23a and 23b proved to be metabolically unstable, acetic
acids 24a and 24b were stable. These differences in met-
CH2Ph
(CH2)2Ph
H
26
30
H
H
H
40
10
17k
17l
i-Bu
i-Bu
Me
Et
3.8
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 8 lM Cbz-Phe-Arg-AMC as a substrate in 200 mM
NaOAc, 10 mM DTT, 120 mM NaCl, and buffer (pH 3.8). The IC50
values represent the average of at least n = 2.

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T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
Table 5. Metabolic stabilitya and PK parametersb of cathepsin K
inhibitors
Compounds 17c, 17l, 27, 24a, and 24b were selected for
evaluation of selectivity over other human cathepsins S,
B, and L (Table 6). N-(3-Biphenyl)-L-leucine 17l pos-
sessed potent inhibitory activity toward cathepsin S
(IC50 = 4.6 nM), and had poor selectivity toward
cathepsins B and L (10-fold compared to cathepsin K).
The other N-(3-biphenyl)-L-leucine derivatives such as
27 and 24a were also less selective. To the contrary,
cyclohexyl derivative 24b showed greater than 3000-fold
selectivity over cathepsins S, B, and L. In particular, 17c
displayed less than 50% inhibition against cathepsins B
and L at 20 lM. These results suggest that the 3-biphe-
nylaminocyclohexanecarbonyl group is also more suit-
able than the N-(3-biphenyl)-L-leucyl group with regard
to enzyme selectivity. The IC50 value of human cathepsin
K at pH 5.5 was measured with compounds 17l, 24a, and
24b, and all compounds possessed decreased activities
compared to the IC50 values at pH 3.8. In compounds
17c, 17l, and 24b, the IC50 values of rat cathepsin K were
also measured, and these compounds showed the IC50
values of 29, 16, and 58 nM, respectively.
Compound
Metabolic
stability (%)
(rat, human)
Tmax
Cmax
AUC
(lg h/mL)
(h)
(lg/mL)
17cc
17l
31, 75
2, 8
3
2.8
0.57
7.3
1.6
0.4d
0.8
5.0
0
35.6
7.4
4
21b
27
23ac
26c
24ac
23b
24bc
43, 41
98, 100
1, 29
6
93.8
20.3
3.0d
6.7
6
3
28, 35
98, 100
0, 13
3
4
42.9
0
—
4
94, 100
9.2
103.9
a Percent remaining after 30 min of incubation in hepatic microsomes.
b Average of three rats dosed at 50 mg/kg po.
c 2HCl salt.
d Value of 24a.
abolic stability could be ascribed to the polarity of the
P10 substituent. Comparison of methyl ethers 17c and
17l indicated the cyclohexyl group tended to be metabol-
ically more stable than the i-butyl group. Considering
the metabolic stability, tetrazole 27 and acetic acids
24a and 24b were expected to have good PK parameters.
In fact, acetic acids 24a and 24b possessed good PK
parameters, whereas tetrazole 27 did not. Esters 23a
and 23b were not detected in the blood serum at all,
but a small amount of 24a was observed after treatment
with 23a. Interestingly, the derivatives from 3-biphe-
nylaminocyclohexane carboxylic acid tended to have
good PK parameters, whereas the derivatives from N-
(3-biphenyl)-L-leucine had poor PK parameters. When
acetic acid 24a was administered, the Cmax for 24a was
5.0 lg/mL. On the other hand, Cmax for 24b (9.2 lg/
mL) was 2-fold better than that for 24a. Benzyl ether
21b showed good PK parameters in spite of its moderate
metabolic stability. These results suggest that the 3-
biphenylaminocyclohexanecarbonyl group is more suit-
able than the 3-biphenyl-L-leucyl group with regard to
pharmacokinetics.
On the basis of the PK profile, we selected compound
24b to evaluate for the effect on bone mineral density
(BMD). Compound 24b was orally administered to
ovariectomized (OVX) rats at dose of 10 or 50 mg/kg
(bid) in cremophor/EtOH (2:1) for 42 days. As shown
in Figure 2, 24b induced an improvement in the BMD
of the femur bone; however, the improvement was not
statistically significant.
4. Conclusion
Structural modifications of compound I led to the dis-
covery of a new class of cathepsin K inhibitors. A struc-
ture–activity relationship study revealed that the 3-
biphenylamino group and the 4-aminophenoxyacetic
acid group were good substituents for the S3 and S10
pockets, respectively. The most potent compounds,
24a and 24b, showed IC50 values of 4.5 and 4.8 nM,
respectively. Further evaluation indicated compound
Table 6. IC50 values of inhibition of human cathepsins S, B, L, and K (nM)
Compound
17c
17l
4.6
27
24a
6.9
24b
10,000
Human cathepsin Sa,f
Human cathepsin Bb,f
Human cathepsin Lc,f
Human cathepsin Kd,f
Rat cathepsin Ke,f
—
34
—
—
11
—
h
NIi
NIi
2.7
29
48
55
3.8 (19g)
120
120
>20,000
>20,000
h
h
4.5 (31g)
4.8 (27g)
58
h
h
—
16
a Inhibition of recombinant human cathepsin S activity in a fluorescence assay using 20 lM Cbz-Val-Val-Arg-MCA as a substrate in 60 mM
KH2PO4–K2HPO4, 10 mM EDTAÆ2Na, 0.001% Triton X-100, 10 mM DTT, and buffer (pH 6.5).
b Inhibition of recombinant human cathepsin B activity in a fluorescence assay using 10 lM Cbz-Phe-Arg-MCA as a substrate in 340 mM NaOAc,
60 mM AcOH, 4 mM EDTAÆ2Na, 8 mM DTT, and buffer (pH 5.5).
c Inhibition of recombinant human cathepsin L activity in a fluorescence assay using 8 lM Cbz-Phe-Arg-MCA as a substrate in 340 mM NaOAc,
60 mM AcOH, 4 mM EDTAÆ2Na, 8 mM DTT, and buffer (pH 5.5).
d Inhibition of recombinant human cathepsin K activity in a fluorescence assay using 8 lM Cbz-Phe-Arg-AMC as a substrate in 200 mM NaOAc,
10 mM DTT, 120 mM NaCl, and buffer (pH 3.8).
e Inhibition of recombinant rat cathepsin K activity in a fluorescence assay using 8 lM Cbz-Leu-Arg-AMC as a substrate in 200 mM NaOAc and
buffer (pH 3.8).
f The IC50 values represent the average of at least n = 2.
g IC50 values measured at pH 5.5.
h Not measured.
i Not inhibited at 20 lM.

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6795
3332, 2958, 1641, 1515, 1233, 1134, 1035, 814,
750 cmꢀ1; HR-MS found [M+Na]+: 395.1775, calcd
for C21H28N2O2SNa: 395.1769.
5.2.2. N-{2-[(4-Methoxyphenyl)amino]ethyl}-1-(phenyl-
thio)cyclohexanecarboxamide (4b). Yield: 20%; light yel-
1
low solid; mp 87–88 ꢁC; H NMR (400 MHz, CDCl3) d
1.48–1.68 (4H, m), 1.71–1.80 (4H, m), 2.00–2.07 (2H,
m), 3.21 (2H, t, J = 6.6 Hz), 3.49 (2H, q, J = 5.9 Hz),
3.75 (3H, s), 6.55 (2H, d, J = 9.5 Hz), 6.78 (2H, d,
J = 8.8 Hz), 6.85 (1H, br s), 7.20 (2H, t, J = 7.3 Hz),
7.24–7.29 (1H, m), 7.39 (2H, d, J = 6.6 Hz); IR (KBr):
3375, 2934, 1643, 1513, 1255, 1235, 1035, 815,
749 cmꢀ1; HR-MS found [M+H]+: 385.1949, calcd for
C22H29N2O2S: 385.1950.
Figure 2. BMD when compound 24b was administered to an OVX rat
at doses of 10 and 50 mg/kg (bid, n = 7).
24b possessed good metabolic stability and PK parame-
ters, and excellent selectivity to other human cathepsins.
In an OVX rat assay, 24b showed a trend toward an
improvement of BMD in the femur bone.
5.2.3. N-{2-[(4-Methoxyphenyl)amino]ethyl}-4-methyl-2-
phenoxypentanamide (4c). Yield: 8%; brown amorphous
1
substance; H NMR (400 MHz, CDCl3) d 0.91 (3H, t,
J = 6.6 Hz), 0.98 (3H, t, J = 6.6 Hz), 1.75–1.91 (3H,
m), 3.13–3.17 (2H, m), 3.34–3.53 (2H, m), 3.73 (3H, s),
4.56 (1H, dd, J = 4.4, 8.8 Hz), 6.42 (2H, d, J = 8.8 Hz),
6.52–6.57 (1H, m), 6.74 (2H, d, J = 8.8 Hz), 6.90 (2H,
d, J = 8.1 Hz), 7.02 (1H, t, J = 7.3 Hz), 7.26–7.31 (2H,
m); IR (film): 3358, 2957, 1662, 1514, 1236, 1039, 821,
755, 693 cmꢀ1; HR-MS found [M+H]+: 357.2184, calcd
for C21H29N2O3: 357.2178.
5. Experimental
5.1. Materials and methods
Melting points were determined in a Yanaco micromelt-
ing point apparatus and are uncorrected. IR absorption
spectra were recorded on a Jasco FT/IR-830 spectropho-
tometer. NMR spectra were recorded on a VARIAN
Mercury 400 (400 MHz) instrument using tetramethylsi-
lane as an internal standard. Low resolution MS and
HR-MS were recorded on a JEOL JMS-AX505H.
Elemental analyses were performed by Institute of Sci-
ence and Technology, Inc. TLC analysis was performed
on 60F254 plates (Merck 5715). Separation of the com-
pounds by column chromatography was carried out with
silica gel 60 (Merck, 230–400 mesh ASTM).
5.2.4. 2-(Biphenyl-2-yloxy)-N-{2-[(4-methoxyphenyl)ami-
no]ethyl}-4-methylpentanamide (4d). Yield: 5%; brown
1
amorphous substance; H NMR (400 MHz, CDCl3) d
0.91 (6H, dd, J = 2.2, 6.6 Hz), 1.55–1.75 (3H, m), 3.02
(2H, t, J = 5.9 Hz), 3.27 (2H, t, J = 5.9 Hz), 3.74 (3H,
s), 4.63 (1H, dd, J = 4.4, 8.1 Hz), 6.42 (2H, d,
J = 9.5 Hz), 6.74 (2H, d, J = 8.8 Hz), 6.94 (1H, d,
J = 8.1 Hz), 7.10 (1H, t, J = 7.3 Hz), 7.29–7.32 (1H,
m), 7.35 (2H, d, J = 6.6 Hz), 7.40 (2H, t, J = 7.3 Hz),
7.47 (2H, d, J = 8.1 Hz); IR (KBr): 3395, 3231, 3088,
2956, 1649, 1580, 1516, 1482, 1433, 1236, 1134, 1034,
5.2. General procedure for the preparation of amides (4)
817, 755, 702 cmꢀ1
;
455.2314, calcd for C27H32N2O3Na: 455.2311.
HR-MS found [M+Na]+:
A mixture of phenol or thiophenol 1 (0.50 mmol), a-
bromoester 2 (0.50 mmol) and KOH (280 mg, 5.0 mmol)
in t-BuOH (5 mL) was stirred at reflux for 24 h. HCl
(1 M, 10 mL) was added to the cooled reaction mixture.
The mixture was extracted with CH2Cl2 (5· 10 mL). The
combined organic layers were dried (Na2SO4) and con-
centrated to provide crude 3. To residual 3 in DMF
(3 mL) were added N-(4-methoxyphenyl)ethane-1,2-di-
amine7 (83 mg, 0.50 mmol), HOBT (76 mg, 0.50 mmol),
and WSCÆHCl (96 mg, 0.50 mmol). After stirring for
10 h, the reaction mixture was diluted with EtOAc
(10 mL). The mixture was washed with water
(2· 10 mL) and saturated aqueous solution of NaHCO3
(2· 10 mL). The organic layer was dried (Na2SO4), con-
centrated, and purified by column chromatography to
provide amide 4.
5.2.5. 2-(Biphenyl-3-yloxy)-N-{2-[(4-methoxyphenyl)ami-
no]ethyl}-4-methylpentanamide (4e). Yield: 8%; brown
1
amorphous substance; H NMR (400 MHz, CDCl3) d
0.93 (3H, d, J = 6.6 Hz), 0.99 (3H, d, J = 6.6 Hz),
1.78–1.96 (3H, m), 3.14–3.19 (2H, m), 3.40–3.52 (2H,
m), 3.71 (3H, s), 4.65 (1H, dd, J = 4.4, 8.8 Hz), 6.39
(2H, d, J = 8.8 Hz), 6.70 (2H, d, J = 8.8 Hz), 6.87 (1H,
dd, J = 2.9, 8.8 Hz), 7.14 (1H, s), 7.25–7.26 (1H, m),
7.35 (2H, t, J = 8.1 Hz), 7.42 (2H, t, J = 8.1 Hz), 7.55
(2H, d, J = 8.8 Hz); IR (film): 3358, 2956, 1663, 1513,
1477, 1294, 1237, 1199, 1038, 819, 758, 698 cmꢀ1; HR-
MS found [M+Na]+: 455.2324, calcd for C27H32N2O3-
Na: 455.2311.
5.2.1. N-{2-[(4-Methoxyphenyl)amino]ethyl}-4-methyl-2-
(phenylthio)pentanamide (4a). Yield: 45%; brown amor-
phous substance; H NMR (400 MHz, CDCl3) d 0.96
(6H, t, J = 6.6 Hz), 1.58–1.90 (3H, m), 3.09 (2H, t,
J = 5.9 Hz), 3.30–3.50 (2H, m), 3.74 (3H, s), 3.78 (1H,
dd, J = 6.6, 8.8 Hz), 6.46 (2H, d, J = 8.8 Hz), 6.76 (2H,
d, J = 8.8 Hz), 7.21–7.33 (5H, m); IR (KBr): 3387,
5.2.6. 2-(Biphenyl-4-yloxy)-N-{2-[(4-methoxyphenyl)ami-
no]ethyl}-4-methylpentanamide (4f). Yield: 2%; light
brown solid; mp 148–151 ꢁC; 1H NMR (400 MHz,
CDCl3) d 0.94 (3H, d, J = 6.6 Hz), 1.00 (3H, d,
J = 6.6 Hz), 1.76–1.94 (3H, m), 3.17 (2H, t,
J = 5.9 Hz), 3.33–3.60 (2H, m), 3.71 (3H, s), 4.60 (1H,
dd, J = 3.7, 8.1 Hz), 6.42 (2H, d, J = 8.8 Hz), 6.70 (2H,
1

6796
T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
d, J = 8.8 Hz), 6.96 (2H, d, J = 8.8 Hz), 7.40–7.53 (7H,
m); IR (KBr): 3382, 2955, 1649, 1515, 1236, 819, 762,
694 cmꢀ1; HR-MS found [M+Na]+: 455.2325, calcd
for C27H32N2O3Na: 455.2311.
J = 5.9 Hz), 3.73 (3H, s), 6.43 (2H, d, J = 8.8 Hz), 6.66
(1H, t, J = 5.9 Hz), 6.74 (2H, d, J = 8.8 Hz), 6.80 (2H,
d, J = 8.8 Hz), 7.01 (2H, d, J = 8.8 Hz); IR (KBr):
3369, 3331, 2942, 1647, 1510, 1231, 1149, 1040, 970,
815, 711 cmꢀ1; HR-MS found [M+H]+: 397.2488, calcd
for C24H33N2O3: 397.2491.
5.2.7. N-{2-[(4-Methoxyphenyl)amino]ethyl}-1-phenoxy-
cyclohexanecarboxamide (4g). Yield: 36%; white solid;
mp 87–90 ꢁC; 1H NMR (400 MHz, CDCl3) d 1.23–
1.66 (6H, m), 1.92–2.06 (4H, m), 3.17–3.21 (2H, m),
3.41–3.52 (2H, m), 3.73 (3H, s), 6.43 (2H, d,
J = 7.3 Hz), 6.60–6.67 (1H, m), 6.74 (2H, d,
J = 7.3 Hz), 6.89 (2H, d, J = 7.3 Hz), 6.94–7.01 (1H,
m), 7.16–7.26 (2H, m); IR (KBr): 3372, 2942, 1648,
1514, 1235, 1147, 1040, 969, 815, 756, 694 cmꢀ1; HR-
MS found [M+H]+: 369.2177, calcd for C22H29N2O3:
369.2178.
5.2.12.
1-(4-Isopropylphenoxy)-N-{2-[(4-methoxyphe-
nyl)amino]ethyl}cyclohexanecarboxamide (4l). Yield:
14%; brown amorphous substance; 1H NMR
(400 MHz, CDCl3) d 1.20 (6H, d, J = 6.6 Hz), 1.27–
1.66 (6H, m), 1.87–1.95 (2H, m), 2.06 (2H, d,
J = 13.2 Hz), 2.83 (1H, quint., J = 7.3 Hz), 3.19 (2H, t,
J = 5.9 Hz), 3.49 (2H, q, J = 5.1 Hz), 3.73 (3H, s), 6.42
(2H, d, J = 8.8 Hz), 6.66 (1H, t, J = 5.9 Hz), 6.74 (2H,
d, J = 8.8 Hz), 6.80 (2H, d, J = 8.8 Hz), 7.03 (2H, d,
J = 8.8 Hz); IR (KBr): 3366, 2942, 1644, 1511, 1231,
1148, 967, 816, 706 cmꢀ1; HR-MS found [M+H]+:
411.2666, calcd for C25H35N2O3: 411.2648.
5.2.8. 1-(Biphenyl-2-yloxy)-N-{2-[(4-methoxyphenyl)ami-
no]ethyl}cyclohexanecarboxamide (4h). Yield: 44%;
white solid; mp 120–122 ꢁC; 1H NMR (400 MHz,
CDCl3) d 0.99–1.38 (6H, m), 1.83 (2H, dt, J = 2.9,
13.9 Hz), 2.00 (2H, d, J = 13.9 Hz), 3.22 (2H, t,
J = 5.9 Hz), 3.51 (2H, q, J = 5.9 Hz), 3.73 (3H, s), 6.47
(2H, d, J = 8.8 Hz), 6.64 (1H, t, J = 5.9 Hz), 6.75 (2H,
d, J = 9.5 Hz), 6.82 (1H, d, J = 8.1 Hz), 7.03–7.10 (2H,
m), 7.31–7.36 (2H, m), 7.40 (2H, t, J = 7.3 Hz), 7.49
(2H, d, J = 6.6 Hz); IR (KBr): 3318, 2936, 1647, 1515,
1477, 1432, 1235, 1147, 1038, 970, 817, 700 cmꢀ1; HR-
MS found [M+Na]+: 467.2305, calcd for C28H32N2O3-
Na: 467.2310.
5.2.13. 1-(4-Cyclohexylphenoxy)-N-{2-[(4-methoxyphe-
nyl)amino]ethyl}cyclohexanecarboxamide (4m). Yield:
1
15%; white solid; mp 124–126 ꢁC; H NMR (400 MHz,
CDCl3) d 1.21–1.94 (20H, m), 2.06 (2H, d, J = 13.2 Hz),
2.39–2.41 (1H, m), 3.19 (2H, t, J = 5.1 Hz), 3.48 (2H, q,
J = 5.1 Hz), 3.73 (3H, s), 6.41 (2H, d, J = 8.8 Hz), 6.64
(1H, t, J = 5.9 Hz), 6.73 (2H, d, J = 8.8 Hz), 6.79 (2H, d,
J = 8.8 Hz), 7.01 (2H, d, J = 8.1 Hz); IR (KBr): 3373,
3322, 2926, 2852, 1647, 1512, 1232, 1147, 1039, 969,
818 cmꢀ1; HR-MS found [M+]: 450.2881, calcd for
C28H38N2O3: 450.2883.
5.2.9. 1-(Biphenyl-3-yloxy)-N-{2-[(4-methoxyphenyl)ami-
no]ethyl}cyclohexanecarboxamide (4i). Yield: 26%;
brown oil; 1H NMR (400 MHz, CDCl3) d 1.27–1.70
(6H, m), 1.92–2.00 (2H, m), 2.11–2.14 (2H, m), 3.03–
3.16 (2H, m), 3.19 (2H, t, J = 5.2 Hz), 3.61 (2H, q,
J = 5.9 Hz), 3.72 (3H, s), 6.41 (2H, d, J = 8.8 Hz), 6.71
(2H, d, J = 8.8 Hz), 6.83–6.86 (1H, m), 7.15–7.16 (1H,
m), 7.23–7.25 (2H, m), 7.32–7.35 (1H, m), 7.40 (2H, t,
J = 8.1 Hz), 7.54 (2H, d, J = 8.1 Hz); IR (film): 3030,
5.3. 1-Bromo-3-cyclohex-1-en-1-ylbenzene (5g)
To a stirred solution of 1,3-dibromobenzene (1.8 mL,
15 mmol) in THF (50 mL) was added n-BuLi (1.59 M
in hexane, 9.4 mL, 15 mmol) followed by cyclohexanone
(1 mL, 10 mmol) at ꢀ78 ꢁC under N2 atmosphere. After
stirring at ꢀ78 ꢁC for 2 h, saturated aqueous solution of
NH4Cl (50 mL) was added. The mixture was extracted
with EtOAc (2· 50 mL). The combined organic layers
were dried (Na2SO4), concentrated, and purified by
column chromatography (hexane/EtOAc, 3:1) to pro-
vide 1-(3-bromophenyl)cyclohexanol (1.85 g, 73%) as a
2937, 1660, 1513, 1237, 1038, 972, 820, 759, 700 cmꢀ1
;
HR-MS found [M+H]+: 445.2487, calcd for
C28H33N2O3: 445.2491.
5.2.10.
1-(Biphenyl-4-yloxy)-N-{2-[(4-methoxyphenyl)-
1
colorless oil. H NMR (400 MHz, CDCl3) d 1.60–1.81
amino]ethyl}cyclohexanecarboxamide (4j). Yield: 24%;
white solid; mp 157–159 ꢁC; 1H NMR (400 MHz,
CDCl3) d 1.26–1.69 (6H, m), 1.91–1.99 (2H, m), 2.11–
2.14 (2H, m), 2.12 (2H, d, J = 13.9 Hz), 3.20 (2H, t,
J = 5.1 Hz), 3.52 (2H, q, J = 5.1 Hz), 3.71 (3H, s), 6.44
(2H, d, J = 8.1 Hz), 6.67 (1H, t, J = 5.9 Hz), 6.72 (2H,
d, J = 8.8 Hz), 6.94 (2H, d, J = 8.8 Hz), 7.30 (1H, t,
J = 7.3 Hz), 7.38–7.42 (4H, m), 7.49–7.55 (2H, m); IR
(KBr): 3376, 2941, 1648, 1512, 1487, 1233, 1147, 1039,
972, 816, 764, 695 cmꢀ1; HR-MS found [M+Na]+:
467.2312, calcd for C28H32N2O3Na: 467.2311.
(10H, m), 7.21 (1H, t, J = 8.1 Hz), 7.37 (1H, dd,
J = 2.2, 8.8 Hz), 7.42 (1H, dd, J = 2.9, 9.5 Hz), 7.67
(1H, t, J = 2.2 Hz); IR (film): 3405, 2935, 2856, 1593,
1565, 1448, 1259, 1207, 974, 782, 694 cmꢀ1; HR-MS
found [M+]: 254.0309, calcd for C12H15OBr: 254.0306.
A mixture of 1-(3-bromophenyl)cyclohexanol (1.85 g,
7.3 mol) and TsOHÆH2O (126 mg, 0.7 mmol) in toluene
(30 mL) was stirred at reflux for 30 min. The mixture
was concentrated and purified by column chromatogra-
phy (hexane) to provide cyclohexene 5g (1.49 g, 87%) as
a colorless oil. 1H NMR (400 MHz, CDCl3) d 1.62–1.68
(2H, m), 1.73–1.80 (2H, m), 2.18–2.23 (2H, m), 2.34–
2.38 (2H, m), 6.11–6.13 (1H, m), 7.16 (1H, t,
J = 8.1 Hz), 7.29 (1H, d, J = 8.1 Hz), 7.33 (1H, d,
J = 7.3 Hz), 7.51 (1H, s); IR (film): 2929, 1591, 1559,
1476, 1074, 994, 777, 645 cmꢀ1; HR-MS found: [M+]:
236.0199, calcd for C12H13Br: 236.0200.
5.2.11. 1-(4-Ethylphenoxy)-N-{2-[(4-methoxyphenyl)ami-
no]ethyl}cyclohexanecarboxamide (4k). Yield: 22%;
White solid; mp 139–142 ꢁC; 1H NMR (400 MHz,
CDCl3) d 1.19 (3H, t, J = 7.3 Hz), 1.30–1.63 (6H, m),
1.89–1.95 (2H, m), 2.06 (2H, d, J = 13.9 Hz), 2.57 (2H,
q, J = 7.3 Hz), 3.19 (2H, t, J = 5.9 Hz), 3.49 (2H, q,

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6797
5.4. General procedure for the preparation of carboxylic
acids (7)
5.4.7. N-(3-Cyclohex-1-en-1-ylphenyl)-L-leucine (7g).
Yield: 14%; H NMR (400 MHz, CDCl3) d 0.94 (3H,
1
d, J = 6.6 Hz), 1.00 (3H, d, J = 6.6 Hz), 1.61–1.87 (7H,
m), 2.16–2.20 (2H, m), 2.34–2.38 (2H, m), 4.04 (1H,
dd, J = 5.9, 8.8 Hz), 6.06–6.08 (1H, m), 6.51 (1H, dd,
J = 2.2, 8.8 Hz), 6.66 (1H, s), 6.83 (1H, d, J = 7.3 Hz),
7.13 (1H, t, J = 8.1 Hz); IR (KBr): 3295, 2926, 1713,
1605, 1483, 1333, 1197, 790, 692 cmꢀ1; HR-MS found
[M+]: 287.1867, calcd for C18H25O2N: 287.1886.
A mixture of aryl bromide 5 (64.99 mmol), amino acid 6
(64.99 mmol), K2CO3 (13.47 g, 97.49 mmol), and Cu-
BrÆSMe2 (2.67 g, 13.00 mmol) in DMA (200 mL) was
stirred at 120 ꢁC for 3 h under N2 atmosphere. The
cooled reaction mixture was diluted with EtOAc
(200 mL). The mixture was washed with 1 M HCl
(3· 200 mL) and brine (200 mL). The organic layer
was dried (Na2SO4), concentrated, and purified by col-
umn chromatography to provide carboxylic acid 7.
5.4.8. N-[3-(Methoxycarbonyl)phenyl]-L-leucine (7h).
1
Yield: 21%; H NMR (400 MHz, CDCl3) d 0.97 (3H,
d, J = 6.6 Hz), 1.02 (3 H, d, J = 6.6 Hz), 1.66–1.89
(3H, m), 3.89 (3H, s), 4.12–4.16 (1H, m), 6.83 (1H, dd,
J = 1.5, 8.1 Hz), 7.32–7.38 (2H, m), 7.44 (1H, d,
J = 8.1 Hz).
5.4.1. 1-Anilinocyclohexanecarboxylic acid (7a). Yield:
1
17%; mp 142–143 ꢁC; H NMR (400 MHz, CDCl3) d
1.36–1.38 (3H, m), 1.64–1.66 (3H, m), 1.98–1.99 (4H,
m), 6.69 (2H, d, J = 8.8 Hz), 6.89 (1H, t, J = 8.1 Hz),
7.22 (2H, t, J = 8.1 Hz).
5.4.9. 1-[(3-Methoxyphenyl)amino]cyclohexanecarboxylic
1
acid (7m). Yield: 35%; H NMR (400 MHz, CDCl3) d
5.4.2. 1-(Biphenyl-2-ylamino)cyclohexanecarboxylic acid
(7b). Yield: 2%; H NMR (400 MHz, CDCl3) d 0.82–
1.36–1.47 (3H, m), 1.62–1.70 (3H, m), 2.01 (4H, br s),
3.76 (3H, s), 6.27–6.32 (2H, m), 6.45 (1H, dd, J = 2.2,
8.1 Hz), 7.12 (1H, t, J = 8.1 Hz).
1
1.93 (10H, m), 6.63 (1H, d, J = 7.8 Hz), 6.95 (1H,
t, J = 7.8 Hz), 7.18 (1H, d, J = 7.8 Hz), 7.23 (1H, t,
J = 7.8 Hz), 7.41–7.44 (3H, m); 7.50 (2H, t,
J = 7.8 Hz); IR (KBr): 2928, 1700, 1510, 1437, 1319,
1283, 755, 703, cmꢀ1; HR-MS found [M+]: 295.1557,
calcd for C19H21O2N: 295.1573.
5.4.10. 1-[(3-Isopropoxyphenyl)amino]cyclohexanecarb-
oxylic acid (7o). Yield: 18%; 1H NMR (400 MHz,
CDCl3) d 1.31 (6H, d, J = 6.6 Hz), 1.37 (3H, br s),
1.64–1.66 (3H, m), 1.99 (4H, br s), 4.44–4.53 (1H, m),
6.22 (1H, s), 6.25 (1H, d, J = 7.3 Hz), 6.43 (1H, d,
J = 8.1 Hz), 7.10 (1H, t, J = 8.1 Hz).
5.4.3. 1-(Biphenyl-3-ylamino)cyclohexanecarboxylic acid
1
(7c). Yield: 43%; mp 173–174 ꢁC; H NMR (400 MHz,
CDCl3) d 1.40–2.03 (10H, m), 6.67 (1H, ddd, J = 1.0,
2.0, 8.0 Hz), 6.89 (1H, t, J = 2.0 Hz), 7.13 (1H, br d,
J = 8.0 Hz), 7.29 (1H, t, J = 8.0 Hz), 7.36–7.33 (1H,
m), 7.44–7.41 (2H, m), 7.55–7.52 (2H, m); IR (KBr):
3404, 1621, 757, 699 cmꢀ1; MS (EI) m/z: 295 [M+]; Anal.
Calcd for C19H21NO2Æ0.1H2O: C, 76.79; H, 7.19; N,
4.71. Found: C, 76.85; H, 7.12; N, 4.67.
5.4.11.
1-[(3-Isobutoxyphenyl)amino]cyclohexanecarb-
oxylic acid (7p). Yield: 19%; 1H NMR (400 MHz,
CDCl3) d 1.01 (6H, d, J = 6.6 Hz), 1.38 (3H, br s),
1.64–1.66 (3H, m), 2.00–2.08 (5H, m), 3.67 (2H, t,
J = 6.6 Hz), 6.24–6.27 (2H, m), 6.43–6.45 (1H, m), 7.10
(1H, t, J = 8.1 Hz).
5.4.12. 1-{[3-(Benzyloxy)phenyl]amino}cyclohexanecarb-
oxylic acid HCl (7q). Yield: 42%; H NMR (400 MHz,
1
5.4.4. 1-(Biphenyl-4-ylamino)cyclohexanecarboxylic acid
(7d). Yield: 11%; H NMR (400 MHz, CDCl3) d 1.39–
1
DMSO-d6) d 1.32–1.55 (6H, m), 1.81–1.89 (4H, m),
4.95 (2H, s), 6.34–6.47 (2H, m), 7.02 (1H, t,
J = 8.1 Hz), 7.25–7.37 (4H, m); IR (KBr): 2953, 1748,
1615, 1598, 1573, 1456, 1393, 1265, 1159, 1134, 1014,
741, 690, 535, 471 cmꢀ1; MS (FAB) m/z: 326 [M+H]+,
280, 200.
1.49 (2H, m), 1.63–1.70 (2H, m), 1.99–2.04 (6H, m),
6.75 (2H, d, J = 8.1 Hz), 7.29 (1H, t, J = 7.3 Hz), 7.40
(2H, t, J = 8.1 Hz), 7.46 (2H, d, J = 8.8 Hz), 7.53 (2H,
d, J = 6.6 Hz); IR (KBr): 3404, 2934, 1613, 1525, 1489,
828, 761, 695, 612 cmꢀ1
;
295.1567, calcd for C19H21O2N: 295.1572.
HR-MS found [M+]:
5.5. General procedure for the preparation of amides (8)
5.4.5. N-Phenyl-L-leucine (7e). Yield: 17%; mp 171–
172 ꢁC; 1H NMR (400 MHz, CDCl3) d 0.95 (3H, d,
J = 6.6 Hz), 1.01 (3H, d, J = 5.9 Hz), 1.63–1.87 (3H, m),
4.03 (1H, dd, J = 5.1, 8.8 Hz), 6.65 (2H, d, J = 8.1 Hz),
6.80 (1H, t, J = 7.3 Hz), 7.20 (2H, t, J = 7.3 Hz).
A mixture of N-(4-methoxyphenyl)ethane-1,2-diamine7
(0.088 mmol), carboxylic acid 7 (0.088 mmol), HOBt
(18 mg, 0.13 mmol), Et3N (18 lL, 0.13 mmol), and
WSCÆHCl (25 mg, 0.13 mmol) in DMF (3 mL) was stir-
red for 10 h. The reaction mixture was diluted with
EtOAc (10 mL). The mixture was washed with water
(2· 10 mL) and saturated aqueous solution of NaHCO3
(2· 10 mL). The organic layer was dried (Na2SO4), con-
centrated, and purified by column chromatography to
provide amide 8.
5.4.6. N-Biphenyl-3-yl-L-leucine (7f). Yield: 67%; light
1
yellow solid; H NMR (400 MHz, CDCl3) d 0.92 (3H,
d, J = 7.3 Hz), 0.97 (3H, d, J = 6.6 Hz), 1.62–1.89 (3H,
m), 4.06–4.11 (1H, m), 6.58 (1H, d, J = 7.3 Hz), 6.82
(1H, s), 6.97 (1H, d, J = 7.3 Hz), 7.22 (1H, d,
J = 7.3 Hz), 7.26–7.30 (1H, m), 7.37 (2H, t,
J = 8.1 Hz), 7.51 (2H, d, J = 8.1 Hz); IR (KBr): 2957,
1714, 1605, 1481, 1333, 1201, 795, 745, 698 cmꢀ1; HR-
MS found [M+]: 283.1578, calcd for C18H21O2N:
283.1572.
5.5.1. 1-Anilino-N-{2-[(4-methoxyphenyl)amino]ethyl}-
cyclohexanecarboxamide (8a). Yield: 90%; oil; 1H
NMR (400 MHz, CDCl3) d 1.24–2.02 (10H, m), 3.19
(2H, t, J = 6.0 Hz), 3.47 (2H, dt, J = 6.0, 6.0 Hz), 3.73

6798
T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
(3H, s), 3.99 (1H, br s), 6.42 (2H, d, J = 8.9 Hz), 6.60
(2H, dd, J = 1.0, 8.5 Hz), 6.73 (2H, d, J = 8.9 Hz), 6.79
(1H, t, J = 7.4 Hz), 7.13 (2H, dd, J = 7.4, 8.5 Hz), 7.26
(1H, br s); IR (film): 3373, 1653, 1602, 1513, 1237,
753 cmꢀ1; MS (FAB) m/z: 367 [M+]; Anal. Calcd for
C22H29N3O2Æ0.2EtOAc: C, 71.11; H, 8.01; N, 10.91.
Found: C, 71.19; H, 8.33; N, 10.86.
5.5.6. N2-Biphenyl-3-yl-N1-{[(4-methoxyphenyl)amino]-
methyl}-L-leucinamide (8f). Yield: 88%; white amor-
phous substance; H NMR (400 MHz, CDCl3) d 0.94
1
(3H, d, J = 6.6 Hz), 1.01 (3H, d, J = 6.6 Hz), 1.54–1.90
(3H, m), 3.12–3.22 (2H, m), 3.36–3.54 (2H, m), 3.71
(3H, s), 3.81 (1H, d, J = 10.3 Hz), 3.93 (1H, br s), 6.39
(2H, d, J = 8.8 Hz), 6.59 (1H, d, J = 8.1 Hz), 6.70 (2H,
d, J = 8.8 Hz), 6.83 (1H, s), 7.06 (2H, d, J = 7.3 Hz),
7.26 (1H, t, J = 7.3 Hz), 7.33 (1H, t, J = 6.6 Hz), 7.40
(2H, t, J = 6.6 Hz), 7.54 (2H, d, J = 6.6 Hz); IR (KBr):
3354, 2955, 1654, 1605, 1513, 1236, 1037, 821, 758,
700 cmꢀ1; HR-MS found [M+H]+: 432.2653, calcd for
C27H34N3O2: 432.2651.
5.5.2. 1-(Biphenyl-2-ylamino)-N-{2-[(4-methoxyphenyl)-
amino]ethyl}cyclohexanecarboxamide (8b). Yield: 71%;
white solid; mp 113–114 ꢁC; 1H NMR (400 MHz,
CDCl3) d 0.83–1.18 (2H, m), 1.44–1.56 (4H, m), 1.86–
1.89 (4H, m), 3.21 (2H, t, J = 5.9 Hz), 3.47 (2H, q,
J = 5.9 Hz), 3.74 (3H, s), 4.34 (1H, br s), 6.45 (2H, d,
J = 8.8 Hz), 6.54 (1H, d, J = 8.1 Hz), 6.75 (2H, d,
J = 8.8 Hz), 6.85 (1H, t, J = 8.1 Hz), 7.08 (1H, t,
J = 8.1 Hz), 7.16 (1H, d, J = 7.3 Hz), 7.19–7.22 (1H,
m), 7.39–7.42 (2H, m), 7.47–7.51 (2H, m); IR (KBr):
3382, 2929, 1644, 1514, 1235, 1039, 818, 758,
706 cmꢀ1; HR-MS found: [M+H]+: 444.2659, calcd for
C28H34N3O2: 444.2651.
5.5.7.
N2-(3-Cyclohex-1-en-1-ylphenyl)-N-{2-[(4-meth-
oxyphenyl)amino]ethyl}-L-leucinamide (8g). Yield: 74%;
oil; 1H NMR (400 MHz, CDCl3) d 0.92 (3H, d,
J = 5.9 Hz), 0.99 (3H, d, J = 6.6 Hz), 1.51–1.87 (7H,
m), 2.15–2.18 (2H, m), 2.33–2.35 (2H, m), 3.14–3.18
(2H, m), 3.32–3.52 (2H, m), 3.73 (3H, s), 3.74–3.81
(1H, m), 6.07–6.09 (1H, m), 6.38 (2H, d, J = 8.8 Hz),
6.47 (1H, dd, J = 2.2, 8.1 Hz), 6.62 (1H, s), 6.71 (2H,
d, J = 8.8 Hz), 6.86 (1H, d, J = 8.1 Hz), 7.04 (1H, t,
J = 5.9 Hz), 7.13 (1H, t, J = 8.1 Hz); IR (KBr): 3354,
5.5.3. 1-(Biphenyl-3-ylamino)-N-{2-[(4-methoxyphenyl)-
amino]ethyl}cyclohexanecarboxamide (8c). Yield: 74%;
white amorphous substance; 1H NMR (400 MHz,
CDCl3) d 2.04–1.35 (10H, m), 3.18 (2H, t, J = 5.8 Hz),
3.48 (2H, q, J = 5.8 Hz), 3.72 (3H, s), 4.08 (1H, br s),
6.39 (2H, d, J = 8.9 Hz), 6.57 (1H, ddd, J = 1.0, 2.0,
7.8 Hz), 6.70 (2H, d, J = 8.9 Hz), 6.83 (1H, t,
J = 2.0 Hz), 7.03 (1H, br d, J = 7.8 Hz), 7.19 (1H, t,
J = 7.8 Hz), 7.30 (1H, br s), 7.31–7.54 (5H, m); IR
(KBr): 1655, 1600, 1513, 1235, 758 cmꢀ1. MS (FAB)
m/z: 444 [M+H]+; Anal. Calcd for C28H33N3O2Æ0.3H2O:
C, 74.62; H, 7.59; N, 8.94. Found: C, 74.50; H, 7.59; N,
9.04.
2931, 1652, 1603, 1513, 1237, 1038, 821, 755 cmꢀ1
;
HR-MS found [M+]: 435.2866, calcd for C27H37N3O2:
435.2886.
5.5.8. Methyl 3-({(1S)-1-[({2-[(4-methoxyphenyl)ami-
no]ethyl}amino)carbonyl]-3-methylbutyl}amino)benzoate
1
(8h). Yield: 52%; H NMR (400 MHz, CDCl3) d 0.91
(3H, d, J = 6.6 Hz), 0.99 (3H, d, J = 6.6 Hz), 1.53–1.86
(3H, m), 3.18 (2H, t, J = 5.9 Hz), 3.70 (2H, q,
J = 5.9 Hz), 3.73 (3H, s), 3.87 (3H, s), 6.43 (2H, d,
J = 8.8 Hz), 6.73 (2H, d, J = 8.8 Hz), 6.78–6.81 (1H,
m), 7.21–7.31 (2H, m), 7.47 (1H, d, J = 7.3 Hz).
5.5.4. 1-(Biphenyl-4-ylamino)-N-{2-[(4-methoxyphenyl)-
amino]ethyl}cyclohexanecarboxamide (8d). Yield: 98%;
white solid; mp 131–134 ꢁC; 1H NMR (400 MHz,
CDCl3) d 1.34 (2H, br s), 1.64–1.67 (2H, m), 1.98–2.04
(6H, m), 3.20 (2H, t, J = 5.9 Hz), 3.50 (2H, q,
J = 5.9 Hz), 3.71 (3H, s), 4.07 (1H, br s), 6.43 (2H,
d, J = 8.8 Hz), 6.66 (2H, d, J = 8.8 Hz), 6.71 (2H, d,
J = 8.8 Hz), 7.23–7.29 (1H, m), 7.35 (2H, d,
J = 8.8 Hz), 7.39 (2H, t, J = 7.3 Hz), 7.49 (2H, d,
J = 8.1 Hz); IR (KBr): 3375, 2935, 1653, 1611,
5.5.9. 1-[(3-Methoxyphenyl)amino]-N-{2-[(4-methoxyphe-
nyl)amino]ethyl}cyclohexanecarboxamide (8m). Yield:
74%; oil; 1H NMR (400 MHz, CDCl3) d 1.27–1.35
(3H, m), 1.60–1.63 (3H, m), 1.95–1.97 (4H, m), 3.18
(2 H, t, J = 5.9 Hz), 3.44 (2H, q, J = 5.9 Hz), 3.71 (3H,
s), 3.71 (3H, s), 4.03 (1H, br s), 6.17 (1H, d,
J = 2.2 Hz), 6.20 (1H, dd, J = 2.2, 8.1 Hz), 6.35 (1H,
dd, J = 2.2, 8.1 Hz), 6.42 (2H, d, J = 8.8 Hz), 6.72
(2H, t, J = 8.8 Hz), 7.02 (1H, t, J = 8.1 Hz), 7.22–7.26
(1H, m); IR (film): 3374, 2937, 1652, 1602, 1513, 1237,
1164, 1040, 822, 764, 696 cmꢀ1; MS (FAB) m/z: 397
[M+H]+, 273, 242, 220, 204.
1513, 1235, 1038, 821, 764, 698 cmꢀ1
; HR-MS
found [M+H]+: 444.2652, calcd for C28H34N3O2:
444.2651.
5.5.5. N-{2-[(4-Methoxyphenyl)amino]ethyl}-N2-phenyl-
L-leucinamide (8e). Yield: 49%; oil; 1H NMR
(400 MHz, CDCl3) d 0.92 (3H, d, J = 6.3 Hz), 0.99 3H,
(d, J = 6.3 Hz), 1 51–1.57 (1H, m), 1.74–1.86 (2H, m),
3.17 (2H, t, J = 5.9 Hz), 3.34–3.51 (2H, m), 3.73 (3H,
s), 3.74–3.85 (1H, m), 6.40 (2H, d, J = 8.9 Hz), 6.60
(2H, d, J = 7.5 Hz), 6.72 (2H, d, J = 8.9 Hz), 6.82 (1H,
t, J = 7.5 Hz), 7.04 (1H, br s), 7.19 (2H, t, J = 7.5 Hz);
IR (film): 3361, 1654, 1604, 1513, 1237, 754 cmꢀ1; MS
(FAB) m/z: 355 [M+]; Anal. Calcd for C21H29N3O2Æ1-
H2O: C, 67.53; H, 8.37; N, 11.25. Found: C, 67.21; H,
7.93; N, 11.20.
5.5.10. 1-[(4-Methoxyphenyl)amino]-N-{2-[(4-methoxy-
phenyl)amino]ethyl}cyclohexanecarboxamide (8n). Yield:
23%; light yellow solid; mp 119–123 ꢁC; 1H NMR
(400 MHz, CDCl3)
d 1.26–1.31 (3H, m), 1.60–
1.63 (3H, m), 1.86–2.00 (4H, m), 3.20 (2H, t,
J = 5.9 Hz), 3.49 (2H, q, J = 5.9 Hz), 3.70 (3H, s),
3.73 (3H, s), 6.47 (2H, d, J = 8.8 Hz), 6.54 (2H, d,
J = 8.8 Hz), 6.66 (2H, d, J = 8.8 Hz), 6.75 (2H, d,
J = 8.8 Hz), 7.42 (1H, t, J = 5.9 Hz); IR (KBr): 3355,
2948, 1646, 1514, 1237, 1039, 823 cmꢀ1; HR-MS
found [M+H]+: 398.2438, calcd for C23H32N3O3:
398.2443.

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6799
5.5.11. 1-[(3-Isopropoxyphenyl)amino]-N-{2-[(4-methoxy-
phenyl)amino]ethyl}cyclohexanecarboxamide (8o). Yield:
64%; oil; H NMR (400 MHz, CDCl3) d 1.28 (6H, d,
the mixture was extracted with EtOAc (3· 50 mL).
The combined organic layers were dried (Na2SO4), con-
centrated, and purified by column chromatography
(CH2Cl2/MeOH, 20:1) to provide benzoic acid 8j
(0.84 g, 52%) as a dark brown amorphous substance.
1
J = 6.6 Hz), 1.28–1.34 (3H, m), 1.61–1.63 (3H, m),
1.95–2.01 (4H, m), 3.20 (2H, t, J = 5.9 Hz), 3.46 (2H,
q, J = 5.9 Hz), 3.72 (3H, s), 3.97 (1H, br s), 4.43–4.49
(1H, m), 6.16 (1H, s), 6.17 (1H, d, J = 7.3 Hz), 6.34
(1H, d, J = 7.3 Hz), 6.43 (2H, d, J = 8.8 Hz), 6.73 (2H,
d, J = 8.8 Hz), 7.01 (1H, t, J = 7.3 Hz), 7.24–7.26 (1H,
m); IR (film): 3373, 2936, 1653, 1601, 1513, 1237, 822,
757 cmꢀ1; HR-MS found [M+H]+: 426.2736, calcd for
C25H36N3O3: 426.2756.
1H NMR (400 MHz, CD3OD)
d 0.82 (3H, d,
J = 6.6 Hz), 0.89 (3H, d, J = 6.6 Hz), 1.52–1.71 (3H,
m), 3.28–3.33 (2H, m), 3.60–3.67 (2H, m), 3.72 (1H,
dd, J = 6.6, 8.8 Hz), 6.74–6.75 (5H, m), 7.10 (1H, t,
J = 7.3 Hz), 7.16 (1H, s), 7.23 (1H, d, J = 6.6 Hz); IR
(KBr): 3312, 2956, 1606, 1513, 1257, 1031, 758 cmꢀ1
;
HR-MS found: [M+H]+: 400.2237, calcd for
C22H30N3O4: 400.2237.
5.5.12. 1-[(3-Isobutoxyphenyl)amino]-N-{2-[(4-methoxy-
phenyl)amino]ethyl}cyclohexanecarboxamide (8p). Yield:
64%; oil; H NMR (400 MHz, CDCl3) d 0.98 (6H, d,
5.8. N-{2-[(4-Methoxyphenyl)amino]ethyl}-N2-[3-(mor-
pholin-4-ylcarbonyl)phenyl]-L-leucinamide (8k)
1
J = 6.6 Hz), 1.32 (3H, br s), 1.62–1.64 (3H, m), 1.95–
2.06 (5H, m), 3.19 (2H, t, J = 5.1 Hz), 3.45 (2H, q,
J = 5.9 Hz), 3.64 (2H, d, J = 6.6 Hz), 3.72 (3H, s), 3.98
(1H, br s), 6.17–6.18 (2H, m), 6.35 (1H, d, J = 7.3 Hz),
6.41 (2H, d, J = 8.8 Hz), 6.72 (2H, d, J = 8.8 Hz), 7.01
(1H, t, J = 8.1 Hz), 7.21–7.23 (1H, m); IR (film): 3371,
A mixture of benzoic acid 8j (180 mg, 0.45 mmol), mor-
pholine (118 lL, 1.35 mmol), HOBt (91 mg, 0.68 mmol),
and WSCÆHCl (129 mg, 0.68 mmol) in CH2Cl2 (5 mL)
was stirred for 6 h. The reaction mixture was concentrat-
ed and purified by column chromatography (EtOAc) to
provide amide 8k (147 mg, 70%) as a brown amorphous
2935, 1652, 1601, 1513, 1237, 1165, 1039, 821 cmꢀ1
;
HR-MS found [M+H]+: 440.2920, calcd for
C26H38N3O3: 440.2913.
substance. H NMR (400 MHz, CDCl3) d 0.92 (3H, d,
1
J = 5.9 Hz), 0.99 (3H, d, J = 6.6 Hz), 1.57–1.85 (3H,
m), 3.17–3.21 (2H, m), 3.38–3.78 (10H, m), 3.73 (3H,
s), 3.96 (1H, d, J = 3.7 Hz), 6.44 (2H, d, J = 8.8 Hz),
6.61 (1H, dd, J = 2.2, 7.3 Hz), 6.66 (1H, s), 6.72 (2H,
d, J = 8.8 Hz), 6.77 (1H, d, J = 7.3 Hz), 6.92 (1H, t,
J = 6.6 Hz), 7.18 (1H, t, J = 8.8 Hz); IR (KBr): 3340,
2956, 1606, 1514, 1463, 1237, 1115, 1030, 822,
750 cmꢀ1; HR-MS found [M+H]+: 469.2812, calcd for
C26H37N4O4: 469.2815.
5.5.13. 1-{[3-(Benzyloxy)phenyl]amino}-N-{2-[(4-meth-
oxyphenyl)amino]ethyl}cyclohexanecarboxamide
(8q).
1
Yield: 99%; solid; mp 92–96 ꢁC; H NMR (400 MHz,
CDCl3) d 1.31–1.64 (6H, m), 1.90–1.99 (4H, m), 3.19
(2H, t, J = 5.9 Hz), 3.45 (2H, q, J = 5.1 Hz), 3.72 (3H,
s), 3.98 (1H, br s), 4.98 (2H, s), 6.21–6.24 (2H, m),
6.41–6.44 (3H, m), 6.71 (2H, d, J = 8.8 Hz), 7.03 (1H,
t, J = 8.1 Hz), 7.21 (1H, t, J = 5.9 Hz), 7.32–7.38 (4H,
m); IR (film): 3374, 2936, 2858, 1652, 1600, 1513,
1237, 1165, 1038, 821, 734, 697 cmꢀ1; MS (FAB) m/z:
474 [M+H]+, 473.
5.9. 3-Piperidin-1-ylaniline (10)
A mixture of 1-fluoro-3-nitrobenzene (1.41 g, 10 mmol)
and piperidine (5.4 mL, 55 mmol) in DMSO (10 mL)
was stirred at 100 ꢁC for 24 h. The reaction mixture
was cooled, diluted with EtOAc (50 mL), and washed
with water (2· 50 mL). The organic layer was dried
(Na2SO4), concentrated, and purified by column chro-
matography (hexane/EtOAc, 10:1) to provide nitroben-
5.6. N2-(3-Cyclohexylphenyl)-N-{2-[(4-methoxy-
phenyl)amino]ethyl}-L-leucinamide (8i)
A mixture of cyclohexene 8g (17 mg, 39 lmol) and
Pd(OH)2/C (55 mg, 39 lmol) in EtOH (1 mL) was stir-
red under H2 for 4 h. After removal of Pd(OH)2/C by fil-
tration, the filtrate was concentrated. The residue was
purified by PTLC (hexane/EtOAc, 1:1) to provide cyclo-
hexane 8i (8 mg, 45%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d 0.92 (3H, d, J = 6.6 Hz), 0.99
(3H, d, J = 6.6 Hz), 1.19–1.85 (13H, m), 2.38–2.43
(1H, m), 3.13–3.21 (2H, m), 3.35–3.53 (2H, m), 3.72
(3H, s), 3.79 (1H, br s), 6.37 (2H, d, J = 8.8 Hz), 6.41–
6.46 (1H, m), 6.69–6.72 (3H, m), 7.05 (1H, t,
J = 5.9 Hz), 7.12 (1H, t, J = 7.3 Hz); IR (film): 3359,
2927, 1655, 1607, 1513, 1137, 1039, 757 cmꢀ1; HR-MS
found [M+Na]+: 460.2927, calcd for C27H39N3O2Na:
460.2940.
1
zene 9 (2.06 g, 99%). H NMR (400 MHz, CDCl3) d
1.59–1.75 (6H, m), 3.27 (4H, t, J = 5.9 Hz), 7.18
(1H, dd, J = 2.2, 8.1 Hz), 7.34 (1H, t, J = 8.1 Hz),
7.59 (1H, dd, J = 1.5, 8.1 Hz), 7.71 (1H, t,
J = 2.2 Hz). A mixture of nitrobenzene 9 (1.62 g,
7.85 mmol) and SnCl2Æ2H2O (7.08 g, 15.7 mmol) in
EtOH (200 mL) was stirred at reflux for 30 min. The
reaction mixture was cooled, poured into 1 M NaOH
(50 mL), and filtered on a Celite pad. The filtrate
was extracted with EtOAc (2· 50 mL). The combined
organic layers were dried (Na2SO4), concentrated,
and purified by column chromatography (hexane/
EtOAc, 1:1) to provide aniline 10 (1.32 g, 96%). 1H
NMR (400 MHz, CDCl3) d 1.53–1.71 (6H, m), 3.12
(4H, t, J = 5.9 Hz), 3.57 (2H, br s), 6.18 (1H, dd,
J = 2.2, 8.8 Hz), 6.27 (1H, d, J = 2.2 Hz), 6.38 (1H,
dd, J = 2.2, 8.8 Hz), 7.02 (1H, t, J = 8.8 Hz); IR (film):
3348, 2933, 1604, 1500, 1384, 1277, 1208, 1129, 967,
765, 691 cmꢀ1; HR-MS found [M+]: 176.1315, calcd
for C11H16N2: 176.1313.
5.7. 3-({(1S)-1-[({2-[(4-Methoxyphenyl)amino]ethyl}ami-
no)carbonyl]-3-methylbutyl}amino)benzoic acid (8j)
A mixture of ester 8h (1.94 g, 4.7 mmol) and 1 M NaOH
(10 mL) in MeOH (30 mL) was stirred for 24 h. 1 M
HCl (20 mL) was added to the reaction mixture and

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T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
5.10. N-(3-Piperidin-1-ylphenyl)leucine (11)
Anal. Calcd for C11H23NO3: C, 60.80; H, 10.67; N,
6.45. Found: C, 60.58; H, 10.36; N, 6.41.
A mixture of 2-bromo-4-methylpentanoic acid (390 mg,
2.0 mmol), aniline 10 (176 mg, 1.0 mmol), and KOH
(1.12 g, 20 mmol) in t-BuOH (10 mL) was stirred at re-
flux for 24 h. HCl (1 M , 30 mL) was added to the cooled
reaction mixture. The mixture was extracted with EtOAc
(2· 50mL). The combined organic layers were dried
(Na2SO4), concentrated, and purified by column chro-
matography (EtOAc/MeOH, 10:1) to provide carboxylic
5.12.3. tert-Butyl [(1S)-1-(hydroxymethyl)-3-phenylpro-
pyl]carbamate (13j). Yield: 93%; H NMR (400 MHz,
1
CDCl3) d 1.45 (9H, s), 1.74–1.86 (2H, m), 2.22 (1H, br
s), 2.65–2.73 (2H, m), 3.56–3.71 (3H, m), 4.64 (1H, br
s), 7.18–7.30 (5H, m); IR (KBr): 3375, 1686, 1517,
1245, 1175, 744, 700 cmꢀ1
;
Anal. Calcd for
C15H23NO3: C, 67.90; H, 8.74; N, 5.28. Found: C,
67.79; H, 8.33; N, 5.29.
1
acid 11 (123 mg, 42%). H NMR (400 MHz, CD3OD) d
0.95 (3H, d, J = 6.6 Hz), 1.00 (3H, d, J = 6.6 Hz), 1.59–
1.85 (9H, m), 3.22 (4H, t, J = 5.9 Hz), 3.95 (1H, t,
J = 6.6 Hz), 6.38 (1H, d, J = 8.8 Hz), 6.48–6.50 (2H,
m), 7.05–7.09 (1H, m); IR (KBr): 3343, 2954, 1716,
;
291.2074, calcd for C17H27O2N2: 291.2073.
5.13. General procedure for the preparation of substituted
N-Boc diamines (16)
1599, 1468, 1210 cmꢀ1
HR-MS found [M+H]+:
To a stirred solution of N-Boc aminoalcohol 13
(100 mmol), 2-nitrobenzenesulfonamide 1414 (15.40 g,
100 mmol), and PPh3 (26.20 g, 100 mmol) in THF
(250 mL) was added a solution of DEAD (17.40 g,
100 mmol) in THF (100 mL) dropwise over 20 min at
0 ꢁC under N2 atmosphere. After stirring at room tem-
perature for 3 h, the reaction mixture was concentrated.
Et2O (100 mL) was added to the residue and the precip-
itate was removed. The filtrate was concentrated. To the
residual oil in MeCN (100 mL) were added PhSH
(11.02 g, 100 mmol) and K2CO3 (20.74 g, 150 mmol).
After stirring at room temperature for 16 h, the reaction
mixture was diluted with EtOAc (200 mL). The mixture
was washed with water (200 mL) and brine (100 mL).
The organic layer was dried (Na2SO4), concentrated,
and purified by column chromatography to provide
substituted N-Boc diamine 16.
5.11. N-{2-[(4-Methoxyphenyl)amino]ethyl}-N2-(3-piperi-
din-1-ylphenyl)leucinamide (8l)
Compound 8l was prepared according to the procedure
for 8, using appropriate starting materials. Yield: 61%;
oil; 1H NMR (400 MHz, CDCl3) d 0.92 (3H, d,
J = 5.9 Hz), 0.98 (3H, d, J = 6.6 Hz), 1.49–1.85 (9H, m),
3.09–3.18 (6H, m), 3.32–3.52 (2H, m), 3.73–3.77 (1H,
m), 3.73 (3H, s), 6.08 (1H, d, J = 8.1 Hz), 6.15 (1H, s),
6.40 (2H, d, J = 8.8 Hz), 6.72 (2H, d, J = 8.8 Hz), 7.04–
7.08 (2H, m); IR (KBr): 3358, 2933, 1654, 1602, 1514,
1235, 1127, 1038, 821 cmꢀ1; HR-MS found [M+Na]+:
461.2896, calcd for C26H38N4O2Na: 461.2892.
5.12. General procedure for the preparation of substituted
N-Boc aminoalcohols (13) from N-Boc amino acids (12)
5.13.1. tert-Butyl {(1S)-2-[(4-methoxyphenyl)amino]-1-
methylethyl}carbamate (16a). Yield: 73%; white solid;
mp 107–108 ꢁC; 1H NMR (400 MHz, CDCl3) d 1.21
(3H, d, J = 6.7 Hz), 1.45 (9H, s), 3.04 (1H, dd, J = 7.3,
12.3 Hz), 3.12 (1H, dd, J = 4.9, 12.3 Hz), 3.73 (1H, br
s), 3.74 (3H, s), 3.92 (1H, br s), 4.50 (1H, br s), 6.58
(2H, d, J = 8.9 Hz), 6.77 (2H, d, J = 8.9 Hz); IR
(KBr): 3390, 1679, 1515, 1239, 1169, 816 cmꢀ1; MS
(EI) m/z: 280 [M+], 136, 57; Anal. Calcd for
C15H24N2O3: C, 64.26; H, 8.63; N, 9.99. Found: C,
64.20; H, 8.86; N, 9.96.
To a stirred solution of N-Boc amino acid 12 (1.7 mmol)
in THF (10 mL) was added N-methylmorpholine
(0.19 mL, 1.7 mmol) followed by ethyl chloroformate
(0.16 mL, 1.7 mmol) at ꢀ10 ꢁC. After stirring for
20 min, NaBH4 (193 mg, 5.1 mmol) was added. MeOH
(20 mL) was then added to the mixture dropwise over
a period of 20 min at 0 ꢁC. After stirring for 20 min,
the reaction mixture was neutralized with 1 M KHSO4.
The mixture was extracted with EtOAc (3· 50 mL). The
combined organic layers were washed with 1 M KHSO4
(50 mL), saturated aqueous solution of NaHCO3
(50 mL) and H2O (50 mL). The organic layer was dried
(Na2SO4), concentrated, and purified by column chro-
matography to provide N-Boc aminoalcohol 13.
5.13.2. tert-Butyl ((1S)-1-{[(4-methoxyphenyl)amino]-
methyl}propyl)carbamate (16c). Yield: 83%; mp 97–
99 ꢁC; 1H NMR (400 MHz, CDCl3) d 0.97 (3H, t,
J = 7.3 Hz), 1.45 (9H, s), 1.42–1.68 (2H, m), 2.99–3.04
(1H, m), 3.18 (1H, dd, J = 4.4, 11.7 Hz), 3.68–3.78
(2H, m), 3.74 (3H, s), 4.46 (1H, br s), 6.58 (2H, d,
J = 8.8 Hz), 6.77 (2H, d, J = 8.8 Hz); IR (KBr): 3388,
1681, 1517, 1238, 1174, 1041, 816 cmꢀ1; MS (FAB)
m/z: 295 [M+H]+, 294, 239, 136, 57; HR-MS found:
[M+H]+: 295.2023, calcd for C16H27N2O3: 295.2022;
Anal. Calcd for C16H26N2O3: C, 65.28; H, 8.90; N,
9.52. Found: C, 65.16; H, 8.58; N, 9.57.
5.12.1. tert-Butyl [(1S)-1-(hydroxymethyl)butyl]carb-
amate (13d). Yield: 91%; mp 43–45 ꢁC; 1H NMR
(400 MHz, CDCl3) d 0.94 (3H, t, J = 7.1 Hz), 1.33–
1.51 (4H, m), 1.45 (9H, s), 2.38 (1H, br s), 3.51–3.70
(3H, m), 4.59 (1H, br s); IR (KBr): 3355, 1687, 1531,
1176 cmꢀ1; MS (FAB) m/z: 204 [M+H]+, 148, 57.
5.12.2. tert-Butyl [(1S)-1-(hydroxymethyl)pentyl]carb-
amate (13e). Yield: 85%; mp 42–44 ꢁC; 1H NMR
(400 MHz, CDCl3) d 0.90 (3H, t, J = 7.0 Hz), 1.24–
1.54 (6H, m), 1.45 (9H, s), 2.41 (1H, br s), 3.51–3.70
(3H, m), 4.60 (1H, br s); IR (KBr): 3359, 1684, 1531,
1174 cmꢀ1; MS (FAB) m/z: 218 [M+H]+, 162, 118, 57;
5.13.3.
tert-Butyl
((1S)-1-{[(4-methoxyphenyl)ami-
1
no]methyl}butyl)carbamate (16d). Yield: 99%; H NMR
(400 MHz, CDCl3) d 0.94 (3H, t, J = 7.0 Hz), 1.45
(9H, s), 1.19–1.48 (4H, m), 2.98–3.03 (1H, m), 3.17
(1H, dd, J = 4.4, 12.5 Hz), 3.74 (3H, s), 3.76–3.84 (1H,

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6801
m), 4.13–4.23 (1H, m), 4.42–4.48 (1H, m), 6.58 (2H, d,
J = 8.8 Hz), 6.77 (2H, d, J = 8.8 Hz); IR (KBr): 3392,
3381, 2980, 2953, 2872, 1676, 1516 cmꢀ1; MS (FAB)
m/z: 309 [M+H]+, 308, 262, 253, 136, 57; HR-MS found
[M+H]+: 309.2188, calcd for C17H29N2O3: 309.2187.
5.13.9.
tert-Butyl
((1S)-1-{[(4-methoxyphenyl)ami-
no]methyl}-3-phenylpropyl)carbamate (16j). Yield: 53%;
1H NMR (400 MHz, CDCl3) d 1.46 (9H, s), 1.72–1.81
(1H, m), 1.86–1.95 (1H, m), 3.04–3.09 (2H, m), 3.17–
3.21 (2H, m), 3.69 (1H, br s), 3.74 (3H, s), 3.81–3.88
(1H, m), 4.48–4.54 (1H, m), 6.56 (2H, d, J = 8.8 Hz),
6.77 (2H, d, J = 8.8 Hz), 7.17–7.21 (3H, m), 7.27–7.30
(2H, m); IR (KBr): 3395, 3378, 2980, 2950, 2927, 2861,
1679, 1513 cmꢀ1; MS (FAB) m/z: 371 [M+H]+, 370,
315, 136, 57; Anal. Calcd for C22H30N2O3Æ0.32H2O: C,
70.23; H, 8.21; N, 7.45. Found: C, 70.26; H, 7.99; N,
7.53.
5.13.4. tert-Butyl ((1S)-1-{[(4-methoxyphenyl)amino]-
methyl}pentyl)carbamate (16e). Yield: 78%; 1H NMR
(400 MHz, CDCl3) d 0.90 (3H, t, J = 7.0 Hz), 1.30–
1.48 (6H, m), 1.45 (9H, s), 2.97–3.03 (1H, m), 3.18
(1H, dd, J = 4.4, 12.5 Hz), 3.74 (3H, s), 3.75–3.81 (2H,
m), 4.41–4.48 (1H, m), 6.58 (2H, d, J = 8.8 Hz), 6.77
(2H, d, J = 8.8 Hz); IR (KBr): 3389, 2982, 2956, 2934,
2872, 1676, 1516 cmꢀ1; MS (FAB) m/z: 323 [M+H]+,
322, 267, 136, 57; Anal. Calcd for C18H30N2O3Æ0.22H2O:
C, 66.23; H, 9.40; N, 8.58. Found: C, 66.26; H, 9.24; N,
8.73.
5.14. General procedure for the preparation of amides
(17)
A mixture of N-Boc diamine 16 (2.0 mmol) and 2 M
HCl in 1,4-dioxane (6 mL, 12 mmol) was stirred for
30 min. The mixture was concentrated to provide a dia-
mine HCl salt. To the residual solid in CH2Cl2 (20 mL)
were added carboxylic acid (2.0 mmol), HOBt (405 mg,
3.0 mmol), and WSCÆHCl (575 mg, 30 mmol), and the
mixture was stirred for 10 h. The reaction mixture was
diluted with EtOAc (20 mL). The mixture was washed
with water (2· 10 mL) and saturated aqueous solution
of NaHCO3 (2· 10 mL). The organic layer was dried
(Na2SO4), concentrated, and purified by column chro-
matography to give 17.
5.13.5.
no]methyl}-2-methylpropyl)carbamate (16f). Yield: 64%;
1H NMR (400 MHz, CDCl3)
0.95 (3H, d,
tert-Butyl
((1S)-1-{[(4-methoxyphenyl)ami-
d
J = 7.3 Hz), 0.98 (3H, d, J = 6.6 Hz), 1.45 (9H, s),
1.83–1.88 (1H, m), 2.96–3.01 (1H, m), 3.19–3.23 (1H,
m), 3.64–3.73 (2H, m), 3.74 (3H, s), 4.48–4.50 (1H, m),
6.57 (2H, d, J = 8.8 Hz), 6.78 (2H, d, J = 8.8 Hz); IR
(KBr): 3376, 2973, 2957, 2935, 1680, 1518 cmꢀ1; MS
(FAB) m/z: 309 [M+H]+, 308, 253, 136, 57; Anal. Calcd
for C17H28N2O3: C, 66.20; H, 9.15; N, 9.08. Found: C,
65.91; H, 9.08; N, 9.09.
5.14.1. 1-(Biphenyl-3-ylamino)-N-{(1S)-2-[(4-methoxy-
phenyl)amino]-1-methylethyl}cyclohexanecarboxamide
(17a). Yield: 74%; light brown amorphous substance;
5.13.6.
tert-Butyl
((1S)-1-{[(4-methoxyphenyl)ami-
no]methyl}-3-methylbutyl)carbamate (16g). Yield: 76%;
1H NMR (400 MHz, CDCl3) d 0.92–0.95 (6H, m),
1.32–1.38 (2H, m), 1.44 (9H, s), 1.68–1.74 (1H, m),
2.96–3.01 (1H, m), 3.17 (1H, dd, J = 4.0, 12.1 Hz),
3.74 (3H, s), 3.76 (1H, br s), 3.86–3.89 (1H, m), 4.40
(1H, br s), 6.57 (2H, d, J = 8.8 Hz), 6.77 (2H, d,
J = 8.8 Hz); IR (KBr): 3405, 2978, 2953, 2911, 1678,
1516 cmꢀ1; MS (FAB) m/z: 323 [M+H]+, 322, 267,
136, 57; Anal. Calcd for C18H30N2O3Æ0.2H2O: C,
66.31; H, 9.40; N, 8.59. Found: C, 66.30; H, 9.36; N,
8.60.
1H NMR (400 MHz, CDCl3)
d
1.17 (3H, d,
J = 6.6 Hz), 1.32–1.34 (3H, m), 1.60–1.66 (3H, m),
1.92–2.03 (4H, m), 3.03 (1H, dd, J = 7.3, 12.5 Hz),
3.09 (1H, dd, J = 5.1, 12.5 Hz), 3.64 (1H, br s), 3.72
(3H, s), 4.04 (1H, s), 4.20–4.27 (1H, m), 6.44 (2H, d,
J = 8.8 Hz), 6.58 (1H, dd, J = 2.2, 8.1 Hz), 6.71 (2H, d,
J = 8.8 Hz), 6.83–6.84 (1H, m), 7.03 (1H, d,
J = 8.1 Hz), 7.08 (1H, d, J = 8.8 Hz), 7.15 (1H, t,
J = 8.1 Hz), 7.31 (1H, t, J = 7.3 Hz), 7.38 (2H, t,
J = 7.3 Hz), 7.53 (2H, d, J = 7.3 Hz); IR (KBr): 3368,
2933, 2856, 1652, 1601, 1513 cmꢀ1; MS (FAB) m/z:
457 [M+H]+, 250.
5.13.7. tert-Butyl {(1S)-2-[(4-methoxyphenyl)amino]-1-
phenylethyl}carbamate (16h). Yield: 55%; 1H NMR
(400 MHz, CDCl3) d 1.43 (9H, s), 3.40–3.50 (3H, m),
3.74 (3H, s), 4.88–4.96 (1H, m), 5.08–5.16 (1H, m),
6.58 (2H, d, J = 8.8 Hz), 6.77 (2H, d, J = 8.8 Hz),
7.28–7.31 (3H, m), 7.35–7.39 (2H, m); IR (KBr): 3387,
3362, 2976, 2936, 1685, 1517 cmꢀ1; MS (FAB) m/z:
343 [M+H]+, 342, 287, 226, 136, 57.
5.14.2. 1-(Biphenyl-3-ylamino)-N-{(1R)-2-[(4-methoxy-
phenyl)amino]-1-methylethyl}cyclohexanecarboxamide
(17b). Yield: 86%; yellow amorphous substance; 1H
NMR (400 MHz, CDCl3) d 1.17 (3H, d, J = 6.6 Hz),
1.32–1.34 (3H, m), 1.60–1.66 (3H, m), 1.92–2.03 (4H,
m), 3.03 (1H, dd, J = 7.3, 12.5 Hz), 3.09 (1H, dd,
J = 5.1, 12.5 Hz), 3.64 (1H, br s), 3.72 (3H, s), 4.04
(1H, s), 4.20–4.27 (1H, m), 6.44 (2H, d, J = 8.8 Hz),
6.58 (1H, dd, J = 2.2, 8.1 Hz), 6.71 (2H, d, J =
8.8 Hz), 6.83–6.84 (1H, m), 7.03 (1H, d, J = 8.1 Hz),
7.08 (1H, d, J = 8.8 Hz), 7.15 (1H, t, J = 8.1 Hz), 7.31
(1H, t, J = 7.3 Hz), 7.38 (2H, t, J = 7.3 Hz), 7.53 (2H,
d, J = 7.3 Hz); IR (KBr): 3368, 2933, 2856, 1652,
5.13.8. tert-Butyl {(1S)-1-benzyl-2-[(4-methoxyphe-
nyl)amino]ethyl}carbamate (16i). Yield: 53%; H NMR
1
(400 MHz, CDCl3) d 1.41 (9H, s), 2.82–2.90 (2H, m),
3.00–3.04 (1H, m), 3.19 (1H, dd, J = 4.4, 12.5 Hz),
3.63 (1H, br s), 3.74 (3H, s), 4.01–4.10 (1H, m), 4.50–
4.59 (1H, m), 6.54 (2H, d, J = 8.8 Hz), 6.76 (2H, d,
J = 8.8 Hz), 7.19–7.25 (3H, m), 7.31 (2H, t,
J = 7.3 Hz); IR (KBr): 3400, 3378, 2983, 2934, 2905,
1676, 1517 cmꢀ1; MS (FAB) m/z: 357 [M+H]+, 356,
301, 136, 57; Anal. Calcd for C21H28N2O3: C, 70.76;
H, 7.92; N, 7.86. Found: C, 70.60; H, 7.78; N, 7.83.
1601, 1513 cmꢀ1
;
MS (FAB) m/z: 458 [M+H]+,
457, 250; Anal. Calcd for C29H35N3O2Æ0.44H2O: C,
74.89; H, 7.77; N, 9.03. Found: C, 74.89; H, 7.48; N,
8.86.

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T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
5.14.3. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}propyl)cyclohexanecarboxamide
(17c). Yield: 85%; brown amorphous substance; 1H
NMR (400 MHz, CDCl3) d 0.88 (3H, t, J = 7.7 Hz),
1.24–1.48 (5H, m), 1.60–1.66 (3H, m), 1.94–2.08 (4H,
m), 3.02 (1H, dd, J = 8.1, 12.5 Hz), 3.13 (1H, dd,
J = 4.4, 12.5 Hz), 3.66 (1H, br s), 3.72 (3H, s), 4.03–
4.09 (2H, m), 6.43 (2H, d, J = 8.8 Hz), 6.60 (1H, dd,
J = 1.5, 8.1 Hz), 6.72 (2H, d, J = 8.8 Hz), 6.85 (1H, m),
7.01–7.05 (2H, m), 7.13 (1H, t, J = 8.1 Hz), 7.31 (1H,
t, J = 7.3 Hz), 7.37 (2H, t, J = 7.3 Hz), 7.53 (2H, d,
J = 7.3 Hz); IR (KBr): 3366, 2933, 2856, 1651, 1601,
1513 cmꢀ1; MS (FAB) m/z: 472 [M+H]+, 471, 250, 136.
5.14.7. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}-3-methylbutyl)cyclohexanecarbox-
amide (17g). Yield: 88%; light yellow amorphous
substance; 1H NMR (400 MHz, CDCl3) d 0.82 (3H,
d, J = 6.6 Hz), 0.83 (3H, d, J = 6.6 Hz), 1.26–1.39
(5H, m), 1.51–1.65 (4H, m), 1.94–2.02 (4H, m), 2.99
(1H, dd, J = 7.3, 11.7 Hz), 3.11 (1H, dd, J = 4.4,
11.7 Hz), 3.73 (1H, br s), 3.73 (3H, s), 4.04 (1H, s),
4.18–4.25 (1H, m), 6.45 (2H, d, J = 8.8 Hz), 6.59
(1H, dd, J = 1.5, 8.1 Hz), 6.73 (2H, d, J = 8.8 Hz),
6.83 (1H, s), 6.99–7.02 (2H, m), 7.11 (1H, t,
J = 8.1 Hz), 7.30 (1H, t, J = 7.3 Hz), 7.36 (2H, t,
J = 7.3 Hz), 7.52 (2H, d, J = 7.3 Hz); IR (film): 3368,
2934, 2858, 1650, 1601, 1513 cmꢀ1; MS (FAB) m/z:
500 [M+H]+, 499, 250; Anal. Calcd for
C32H41N3O2Æ0.38H2O: C, 75.88; H, 8.31; N, 7.52.
Found: C, 75.90; H, 8.03; N, 8.30.
5.14.4. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}butyl)cyclohexanecarboxamide (17d).
Yield: 72%; amorphous substance; 1H NMR (400 MHz,
CDCl3) d 0.83 (3H, t, J = 7.3 Hz), 1.24–1.70 (10H, m),
1.92–2.07 (4H, m), 3.02 (1H, dd, J = 8.1, 12.5 Hz),
3.12 (1H, dd, J = 4.4, 12.5 Hz), 3.66 (1H, s), 3.72 (3H,
s), 4.05 (1H, s), 4.09–4.18 (1H, m), 6.43 (2H, d,
J = 8.8 Hz), 6.60 (1H, dd, J = 2.2, 8.1 Hz), 6.72 (2H, d,
J = 8.8 Hz), 6.84 (1H, s), 7.01–7.04 (2H, m), 7.12 (1H,
t, J = 8.1 Hz), 7.29–7.32 (1H, m), 7.36 (2H, t,
J = 6.6 Hz), 7.53 (2H, d, J = 6.6 Hz); IR (KBr): 3366,
2932, 2857, 1651, 1601, 1513 cmꢀ1; MS (FAB) m/z:
486 [M+H]+, 485, 250; Anal. Calcd for
C31H39N3O2Æ0.32H2O: C, 75.77; H, 8.13; N, 8.55.
Found: C, 75.76; H, 7.79; N, 8.61.
5.14.8. 1-(Biphenyl-3-ylamino)-N-{(1S)-2-[(4-methoxy-
phenyl)amino]-1-phenylethyl}cyclohexanecarboxamide (17h).
Yield: 96%; white amorphous substance; 1H NMR
(400 MHz, CDCl3) d 1.31–2.05 (10H, m), 3.31–3.41
(3H, m), 3.70 (3H, s), 4.07 (1H, s), 5.19–5.24 (1H,
m), 6.39 (2H, d, J = 8.8 Hz), 6.59 (1H, dd, J = 2.2,
8.1 Hz), 6.68 (2H, d, J = 8.8 Hz), 6.82 (1H, s), 7.04
(1H, d, J = 8.1 Hz), 7.17 (1H, t, J = 7.3 Hz), 7.21–
7.26 (5H, m), 7.30–7.33 (1H, m), 7.37 (2H, t,
J = 7.3 Hz), 7.48 (2H, d, J = 7.3 Hz), 7.64 (1H, d,
J = 8.1 Hz); IR (KBr): 3369, 2934, 1658, 1601,
1513 cmꢀ1; MS (FAB) m/z: 520 [M+H]+, 519, 250.
5.14.5. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}pentyl)cyclohexanecarboxamide (17e).
Yield: 68%; amorphous substance; 1H NMR (400 MHz,
CDCl3) d 0.72 (3H, t, J = 6.6 Hz), 1.11–1.63 (12H, m),
1.88–2.00 (4H, m), 2.95 (1H, dd, J = 8.1, 12.5 Hz),
3.05 (1H, dd, J = 4.4, 12.5 Hz), 3.60 (1H, s), 3.65 (3H,
s), 3.98 (1H, s), 4.02–4.09 (1H, m), 6.37 (2H, d,
J = 8.8 Hz), 6.52 (1H, dd, J = 1.5, 8.1 Hz), 6.65 (2H, d,
J = 8.8 Hz), 6.77 (1H, s), 6.96 (2H, t, J = 7.3 Hz), 7.05
(1H, t, J = 8.1 Hz), 7.21–7.25 (1H, m), 7.29 (2H, t,
J = 7.3 Hz), 7.46 (2H, d, J = 7.3 Hz); IR (KBr): 3366,
3054, 3029, 2931, 1651, 1601, 1574, 1513 cmꢀ1; MS
(FAB) m/z: 500 [M+H]+, 499, 250, 136; Anal. Calcd
for C32H41N3O2Æ0.30H2O: C, 76.09; H, 8.30; N, 8.32.
Found: C, 76.10; H, 8.43; N, 8.30.
5.14.9. N-{(1S)-1-Benzyl-2-[(4-methoxyphenyl)amino]eth-
yl}-1-(biphenyl-3-ylamino)cyclohexanecarboxamide (17i).
Yield: 86%; light yellow amorphous substance; 1H
NMR (400 MHz, CDCl3) d 1.24–2.04 (10H, m),
2.86 (2H, d, J = 6.6 Hz), 3.04 (1H, dd, J = 7.3,
12.5 Hz), 3.16 (1H, dd, J = 4.4, 12.5 Hz), 3.60 (1H,
s), 3.71 (3H, s), 3.99 (1H, s), 4.37–4.47 (1H, m),
6.38 (2H, d, J = 8.8 Hz), 6.52 (1H, d, J = 8.1 Hz),
6.69 (2H, d, J = 8.8 Hz), 6.82 (1H, s), 7.02 (1H,
d, J = 8.1 Hz), 7.08–7.25 (7H, m), 7.29–7.32 (1H,
m), 7.37 (2H, t, J = 6.6 Hz), 7.53 (2H, d,
J = 7.3 Hz); IR (KBr): 3369, 2934, 2956, 1654,
1601, 1513 cmꢀ1
;
MS (FAB) m/z: 534 [M+H]+,
533, 250; Anal. Calcd for C35H39N3O2Æ0.54H2O: C,
77.36; H, 7.43; N, 7.73. Found: C, 77.35; H,
7.51; N, 7.80.
5.14.6. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}-2-methylpropyl)cyclohexanecarboxa-
mide (17f). Yield: 64%; brown amorphous substance; 1H
NMR (400 MHz, CDCl3) d 0.87 (3H, d, J = 6.6 Hz),
0.91 (3H, d, J = 7.3 Hz), 1.22–1.43 (3H, m), 1.58–1.69
(3H, m), 1.83–1.91 (1H, m), 1.94–2.05 (4H, m), 2.98
(1H, dd, J = 8.8, 11.7 Hz), 3.17 (1H, dd, J = 4.4,
11.7 Hz), 3.64 (1H, br s), 3.73 (3H, s), 3.97–4.04 (1H,
m), 4.06 (1H, s), 6.41 (2H, dd, J = 8.8 Hz), 6.61 (1H,
dd, J = 1.5, 8.1 Hz), 6.71 (2H, d, J = 8.8 Hz), 6.85–6.86
(1H, m), 7.01 (1H, d, J = 8.1 Hz), 7.10 (1H, t,
J = 8.1 Hz), 7.14 (1H, d, J = 9.5 Hz), 7.30 (1H, t,
J = 7.3 Hz), 7.36 (2H, t, J = 7.3 Hz), 7.52 (2H, d,
J = 7.3 Hz); IR (KBr): 3368, 2932, 2857, 1654, 1601,
1513 cmꢀ1; MS (FAB) m/z: 486 [M+H]+, 485, 250,
136; Anal. Calcd for C31H39N3O2Æ0.44H2O: C, 75.43;
H, 8.14; N, 8.51. Found: C, 75.50; H, 7.79; N, 8.41.
5.14.10. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}-3-phenylpropyl)cyclohexanecarb-
oxamide (17j). Yield: 97%; amorphous substance; 1H
NMR (400 MHz, CDCl3) d 1.29–2.04 (12H, m),
2.53–2.66 (2H, m), 3.06 (1H, dd, J = 7.3, 12.5 Hz),
3.14 (1H, dd, J = 5.1, 12.5 Hz), 3.60 (1H, s), 3.71 (3H,
s), 4.05 (1H, s), 4.14–4.22 (1H, m), 6.41 (2H, d,
J = 8.8 Hz), 6.61 (1H, d, J = 1.5, 8.1 Hz), 6.70 (2H, d,
J = 8.8 Hz), 6.86 (1H, s), 7.00–7.87 (11H, m),
7.52 (2H, d, J = 7.3 Hz); IR (KBr): 3367, 2935, 2856,
1653, 1602, 1513 cmꢀ1; MS (FAB) m/z: 548 [M+H]+,
547, 250; Anal. Calcd for C36H41N3O2Æ0.84H2O: C,
76.82; H, 7.64; N, 7.47. Found: C, 76.68; H, 7.54; N,
7.61.

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6803
5.14.11. N2-1,10-Biphenyl-3-yl-N1-{(1S)-2-[(4-methoxy-
phenyl)amino]-1-methylethyl}-L-leucinamide (17k). Yield:
98%; white amorphous substance; H NMR (400 MHz,
s), 6.57 (2H, d, J = 8.9 Hz), 6.84 (2H, d, J = 8.9 Hz),
7.28–7.43 (5H, m); IR (KBr): 3375, 1683, 1514, 1245,
1175, 1018, 816 cmꢀ1; MS (FAB) m/z: 370 [M+], 223,
91, 57; Anal. Calcd for C22H30N2O3: C, 71.32; H,
8.16; N, 7.56. Found: C, 71.16; H, 7.93; N, 7.55.
1
CDCl3) d 0.94 (3H, d, J = 6.6 Hz), 1.01 (3H, d,
J = 5.9 Hz), 1.19 (3H, d, J = 6.6 Hz), 1.56–1.91 (3H,
m), 3.00 (1H, dd, J = 7.3, 12.5 Hz), 3.07 (1H, dd,
J = 4.4, 12.5 Hz), 3.52 (1H, br s), 3.70 (3H, s), 3.74–
3.79 (1H, m), 3.93 (1H, d, J = 2.9 Hz), 4.19–4.28 (1H,
m), 6.36 (2H, d, J = 8.8 Hz), 6.58 (1H, d, J = 8.1 Hz),
6.67 (2H, d, J = 8.8 Hz), 6.82–6.84 (2H, m), 7.05 (1H,
d, J = 7.3 Hz), 7.20 (1H, t, J = 7.3 Hz), 7.31–7.37 (3H,
m), 7.52 (2H, d, J = 6.6 Hz); IR (KBr): 3353, 2956,
1650, 1605, 1513, 1323, 1235, 821, 758, 700 cmꢀ1; HR-
MS found [M+H]+: 446.2813, calcd for C28H36N3O2:
446.2807.
5.17. General procedure for the preparation of amides
(21)
Compound 21 was prepared according to the procedure
for 17, using appropriate starting materials.
5.17.1.
N-[(1S)-1-({[4-(Benzyloxy)phenyl]amino}meth-
yl)propyl]-N2-biphenyl-3-yl-L-leucinamide (21a). Yield:
99%; colorless crystals; mp 117–119 ꢁC; 1H NMR
(400 MHz, CDCl3) d 0.93 (3H, t, J = 7.3 Hz), 0.94
(3H, d, J = 6.6 Hz), 1.01 (3H, d, J = 6.6 Hz), 1.39–1.92
(5H, m), 2.96 (1H, dd, J = 7.3, 12.5 Hz), 3.11 (1H, dd,
J = 4.4, 12.5 Hz), 3.40–3.55 (1H, br s), 3.74–3.82 (1H,
m), 3.92–3.95 (1H, m), 4.00–4.12 (1H, m), 4.93 (2H, s),
6.33 (2H, d, J = 9.5 Hz), 6.58 (1H, dd, J = 2.2, 8.1 Hz),
6.72 (2H, d, J = 9.5 Hz), 6.74–6.85 (2H, m), 7.03 (1H,
d, J = 8.1 Hz), 7.18 (1H, t, J = 8.1 Hz), 7.28–7.58
(10H, m); IR (KBr): 3324, 1624, 1511, 1231, 1214,
755 cmꢀ1; Anal. Calcd for C35H41N3O2: C, 78.47; H,
7.71; N, 7.84. Found: C, 78.47; H, 7.52; N, 7.78; MS
(FAB) m/z: 536 [M+H]+, 444, 238.
5.14.12. N2-1,10-Biphenyl-3-yl-N1-((1S)-1-{[(4-methoxy-
phenyl)amino]methyl}propyl)-L-leucinamide (17l). Yield:
1
61%; white solid; mp 99–101 ꢁC; H NMR (400 MHz,
CDCl3) d 0.89 (3H, d, J = 7.3 Hz); 0.90 (3H, d,
J = 5.9 Hz), 0.98 (3H, d, J = 6.6 Hz), 1.37–1.88 (5H,
m), 2.93 (1H, dd, J = 7.3, 12.5 Hz), 3.07 (1H, dd,
J = 4.4, 12.5 Hz), 3.66 (3H, s), 3.72–3.75 (1H, m), 3.89
(1H, br s), 3.97–4.05 (1H, m), 6.31 (2H, d, J = 8.8 Hz),
6.55 (1H, d, J = 8.1 Hz), 6.62 (2H, d, J = 8.8 Hz), 6.73
(1H, d, J = 7.3 Hz), 6.80 (1H, s), 7.00 (1H, d,
J = 7.3 Hz), 7.15 (1H, t, J = 7.3 Hz), 7.25–7.33 (3H,
m), 7.47 (2H, d, J = 7.3 Hz); IR (KBr): 3336, 2961,
1645, 1606, 1514, 1479, 1235, 1038, 821, 759, 699 cmꢀ1
.
5.17.2.
N-[(1S)-1-({[4-(Benzyloxy)phenyl]amino}meth-
yl)propyl]-1-(biphenyl-3-ylamino)cyclohexanecarboxamide
(21b). Yield: 84%; light yellow amorphous substance; 1H
NMR (400 MHz, CDCl3) d 0.88 (3H, t, J = 7.4 Hz),
1.24–1.70 (8H, m), 1.93–2.04 (4H, m), 3.02 (1H, dd,
J = 7.7, 12.1 Hz), 3.12 (1H, dd, J = 4.6, 12.1 Hz), 3.67
(1H, br s), 4.00–4.10 (2H, m), 4.96 (2H, s), 6.42
(2H, d, J = 8.9 Hz), 6.59 (1H, dd, J = 2.0, 7.8 Hz),
6.78 (2H, d, J = 8.9 Hz), 6.85 (1H, br s), 7.01 (1H, d,
J = 7.8 Hz), 7.03 (1H, br s), 7.12 (1H, t, J = 7.8 Hz),
7.27–7.55 (10H, m); IR (KBr): 3367, 1652, 1601,
1511, 1228, 758, 699 cmꢀ1; MS (FAB) m/z: 548
[M+H]+, 250.
5.15. N-[4-(Benzyloxy)phenyl]-2-nitrobenzenesulfonamide
(18)
To a stirred solution of 4-(benzyloxy)aniline hydrochlo-
ride (11.79 g, 50 mmol) and 2-nitrobenzenesulfonyl
chloride (11.08 g, 50 mmol) in CH2Cl2 (150 mL) was
added Et3N (14.6 mL, 105 mmol) dropwise at 0 ꢁC.
The reaction mixture was stirred at room temperature
for 4 h. Water (200 mL) was added to the reaction mix-
ture. The organic layer was washed with 1 M HCl
(100 mL) and saturated aqueous solution of NaHCO3
(100 mL). The organic layer was dried (Na2SO4) and
concentrated. Recrystallization from EtOH (350 mL)
provided 2-nitrobenzenesulfonamide 18 (16.63 g, 87%).
5.18. General procedure for the preparation of phenols
(22)
1
Mp 154–156 ꢁC; H NMR (400 MHz, CDCl3) d 5.00
(2H, s), 6.85 (1H, d, J = 8.9 Hz), 7.09 (1H, d,
J = 8.9 Hz), 7.10 (1H, br s), 7.30–7.40 (5H, m), 7.55
(1H, dt, J = 1.2, 7.8 Hz), 7.68 (1H, dt, J = 1.4, 7.8 Hz),
7.75 (1H, dd, J = 1.4, 7.8 Hz), 7.85 (1H, dd, J = 1.2,
7.8 Hz); IR (KBr): 3312, 1540, 1507, 1166, 1005,
595 cmꢀ1; MS (FAB) m/z: 384 [M+]; Anal. Calcd for
C19H16N2O5S: C, 59.37; H, 4.20; N, 7.29; S, 8.34.
Found: C, 59.21; H, 4.05; N, 7.27; S, 8.33.
A mixture of benzyl ether 21 (3.7 mmol) and Pd/C (10%,
0.40 g, 0.37 mmol) in EtOH (20 mL) was stirred under
H2 at 40 ꢁC for 6 h. After removal of Pd/C by filtration,
the filtrate was concentrated. The residue was purified
by column chromatography to provide phenol 22.
5.18.1. N2-Biphenyl-3-yl-N-((1S)-1-{[(4-hydroxyphenyl)-
amino]methyl}propyl)-L-leucinamide (22a). Yield: 87%;
1H NMR (400 MHz, CDCl3)
d
0.93 (3H, t,
5.16. tert-Butyl [(1S)-1-({[4-(benzyloxy)phenyl]ami-
no}methyl)propyl]carbamate (20)
J = 7.3 Hz), 0.94 (3H, d, J = 6.6 Hz), 1.01 (3H, d,
J = 6.6 Hz), 1.53–1.93 (5H, m), 3.71 (1H, dd, J = 4.4,
9.5 Hz), 3.75–3.82 (1H, m), 3.85 (1H, dd, J = 3.7,
9.5 Hz), 4.91 (1H, br s), 6.47 (2H, d, J = 8.8 Hz), 6.54–
6.62 (1H, m), 6.59 (2H, d, J = 8.8 Hz), 6.79–6.82 (1H,
m), 6.96 (1H, d, J = 8.1 Hz), 7.03 (1H, br d,
J = 9.5 Hz), 7.14 (1H, t, J = 8.1 Hz), 7.28–7.55 (6H,
m); IR (KBr): 2961, 1649, 1605, 1509, 1228 cmꢀ1; MS
(FAB) m/z: 447 [M+H]+, 238.
Compound 20 was prepared according to the procedure
for 16, using appropriate starting materials. Yield: 82%;
mp 108–111 ꢁC; 1H NMR (400 MHz, CDCl3) d 0.97
(3H, t, J = 7.5 Hz), 1.45 (9H, s), 1.40–1.66 (2H, m),
3.01 (1H, dd, J = 7.6, 12.2 Hz), 3.17 (1H, dd, J = 4.6,
12.2 Hz), 3.65–3.80 (2H, m), 4.46 (1H, br s), 4.99 (2H,

6804
T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
5.18.2. 1-(Biphenyl-3-ylamino)-N-((1S)-1-{[(4-hydroxy-
phenyl)amino]methyl}propyl)cyclohexanecarboxamide (22b).
Yield: 99%; 1H NMR (400 MHz, CDCl3) d 0.85
(3H, t, J = 7.4 Hz), 1.23–1.63 (8H, m), 1.95–2.08
(4H, m), 2.98 (1H, dd, J = 7.7, 12.2 Hz), 3.08 (1H,
dd, J = 4.4, 12.2 Hz), 4.00–4.14 (2H, m), 6.34 (2H,
d, J = 8.6 Hz), 6.59 (1H, dd, J = 2.0, 8.0 Hz), 6.67
(2H, d, J = 8.6 Hz), 6.86 (1H, t, J = 2.0 Hz), 7.01
(1H, d, J = 8.0 Hz), 7.12 (1H, br s d, J = 8.9 Hz),
7.12 (1H, t, J = 8.0 Hz), 7.28–7.38 (3H, m), 7.51–
7.53 (2H, m); IR (KBr): 3367, 1647, 1601, 1515,
5.20. General procedure for the preparation of acetic acids
(24)
To a stirred solution of tert-butyl acetate 23 (6.59 mmol)
in CH2Cl2 (25 mL) was added TFA (25 mL) at 0 ꢁC.
After stirring at room temperature for 1 h, the reaction
mixture was concentrated. HCl (4 M) in 1,4-dioxane
(20 mL) was added to the residue. The mixture was con-
centrated, then triturated with Et2O (20 mL) to provide
acetic acid 24.
1241, 1225, 758 cmꢀ1
[M+H]+, 250.
;
MS (FAB) m/z: 458
5.20.1. [4-({(2S)-2-[(N-Biphenyl-3-yl-L-leucyl)amino]bu-
tyl}amino)phenoxy]acetic acid 2HCl (24a). Yield: 74%;
1
colorless crystals; mp 89–91 ꢁC; H NMR (400 MHz,
5.19. General procedure for the preparation of tert-butyl
acetates (23)
CDCl3) d 0.74–0.90 (9H, m), 1.51–1.69 (4H, m), 2.11–
2.22 (1H, m), 3.22–3.27 (1H, m), 3.65–3.72 (2H, m),
4.43–4.50 (2H, m), 4.47 (2H, s), 6.77 (2H, d,
J = 7.4 Hz), 7.34–7.75 (9H, m), 8.03 (1H, br s), 9.52
(1H, m); IR (KBr): 2963, 1739, 1674, 1605, 1554, 1228,
1193 cmꢀ1; MS (FAB) m/z: 560 [M+H]+, 444, 238.
To a stirred solution of phenol 22 (10.4 mmol) in THF/
DMF (100 mL, 4:1) was added NaH (55%, 452 mg,
10.4 mmol) followed by tert-butyl bromoacetate
(1.84 mL, 12.5 mmol) at 0 ꢁC. After stirring at room
temperature for 1.5 h, water (100 mL) was added to
the reaction mixture at 0 ꢁC. The mixture was extracted
with EtOAc (100 mL). The organic layer was washed
with water (100 mL) and brine (100 mL). The organic
layer was dried (Na2SO4), concentrated, and purified
by column chromatography to provide tert-butyl ace-
tate 23.
5.20.2.
(4-{[(2S)-2-({[1-(Biphenyl-3-ylamino)cyclohex-
yl]carbonyl}amino)butyl]amino}phenoxy)acetic acid 2HCl
(24b). Yield: 95%; amorphous substance; 1H NMR
(400 MHz, DMSO-d6) d 0.64 (3H, t, J = 7.4 Hz),
1.14–1.98 (12H, m), 3.07 (1H, dd, J = 5.0, 12.5 Hz),
3.28 (1H, dd, J = 6.5, 12.5 Hz), 3.96–4.01 (1H, br s),
4.68 (2H, s), 6.72–6.73 (1H, br d), 6.95 (2H, d,
J = 9.0 Hz), 6.96–7.10 (2H, m), 7.19 (1H, t,
J = 8.0 Hz), 7.25 (2H, br d, J = 9.0 Hz), 7.31–7.52 (5H,
m), 8.07 (1H, br d, J = 8.7 Hz); IR (KBr): 3343, 1740,
1668, 1605, 1510, 1194, 1177, 1075, 759, 700 cmꢀ1; MS
(FAB) m/z: 516 [M+H]+, 250.
5.19.1. tert-Butyl [4-({(2S)-2-[(N-biphenyl-3-yl-L-leu-
cyl)amino]butyl}amino)phenoxy]acetate (23a). Yield:
81%; amorphous substance; 1H NMR (400 MHz,
CDCl3) d 0.93 (3H, t, J = 7.4 Hz), 0.94 (3H, d,
J = 6.3 Hz), 1.01 (3H, d, J = 6.3 Hz), 1.40–1.50 (1H,
m), 1.48 (9H, s), 1.55–1.66 (2H, m), 1.80–1.91 (2H, m),
2.95 (1H, dd, J = 7.4, 12.5Hz), 3.10 (1H, dd, J = 4.7,
12.5 Hz), 3.74–3.78 (1H, m), 3.91–3.94 (1H, m), 4.00–
4.08 (1H, m), 4.36 (2H, s), 6.30 (2H, d, J = 9.0 Hz),
6.56 (1H, dd, J = 2.2, 7.8 Hz), 6.65 (2H, d, J = 9.0 Hz),
6.74 (1H, d, J = 8.6 Hz), 6.82 (1H, t, J = 2.2 Hz), 7.01
(1H, d, J = 7.8 Hz), 7.15 (1H, t, J = 7.8 Hz), 7.27–7.37
(3H, m), 7.47–7.51 (2H, m); IR (KBr): 2961, 1751,
1652, 1605, 1513, 1217, 1155 cmꢀ1; Anal. Calcd for
C34H45N3O4: C, 72.96; H, 8.10; N, 7.51. Found: C,
72.95; H, 8.00; N, 7.49; MS (FAB) m/z: 560 [M+H]+,
446, 238.
5.21. Ethyl 2-[4-({(2S)-2-[(N-biphenyl-3-yl-L-leucyl)ami-
no]butyl}amino)phenoxy]-2-methylpropanoate (25)
Compound 25 was prepared according to the procedure
for 23, using appropriate starting materials. Yield: 61%;
oil; 1H NMR (400 MHz, CDCl3) d 0.93 (3H, t,
J = 7.4 Hz), 0.94 (3H, d, J = 6.3 Hz), 1.01 (3H, d,
J = 6.3 Hz), 1.27 (3H, t, J = 7.0 Hz), 1.40–1.70 (3H,
m), 1.49 (6H, s), 1.77–1.92 (2H, m), 2.95 (1H, dd,
J = 7.8, 12.1 Hz), 3.10 (1H, dd, J = 4.3, 12.1 Hz), 3.73–
3.82 (1H, m), 3.88–4.10 (3H, m), 4.21 (2H, q,
J = 7.0 Hz), 6.29 (2H, d, J = 8.6 Hz), 6.57 (1H, dd,
J = 2.3, 7.8 Hz), 6.60 (2H, d, J = 8.6 Hz), 6.74–6.83
(2H, m), 6.97–7.03 (1H, m), 7.13 (1H, t, J = 7.8 Hz),
7.25–7.34 (3H, m), 7.46–7.54 (2H, m); IR (KBr): 2961,
1732, 1651, 1607, 1511, 1227, 1140 cmꢀ1; MS (FAB)
m/z: 560 [M+H]+, 444, 238; HR-MS found [M+H]+:
560.3491, calcd for C34H46N3O4: 560.3488.
5.19.2. tert-Butyl (4-{[(2S)-2-({[1-(biphenyl-3-ylamino)cy-
clohexyl]carbonyl}amino)butyl]amino}phenoxy)acetate
(23b). Yield: 79%; amorphous substance; 1H NMR
(400 MHz, CDCl3)
d
0.88 (3H, t, J = 7.4 Hz),
1.24–1.67 (8H, m), 1.97–2.04 (4H, m), 3.01 (1H, dd,
J = 7.8, 12.2 Hz), 3.13 (1H, dd, J = 4.5, 12.2 Hz),
3.72 (1H, br s), 4.00–4.11 (2H, m), 4.40 (2H, s),
6.41 (2H, d, J = 8.9 Hz), 6.59 (1H, dd, J = 2.0,
8.0 Hz), 6.72 (2H, d, J = 8.9 Hz), 6.85 (1H, t,
J = 2.0 Hz), 7.01 (1H, d, J = 8.0 Hz), 7.03 (1H, br d,
J = 8.2 Hz), 7.11 (1H, t, J = 8.0 Hz), 7.29–7.38 (3H,
m), 7.51–7.53 (2H, m); IR (KBr): 3370, 1750, 1653,
1601, 1512, 1217, 1154, 758 cmꢀ1; MS (FAB) m/z:
5.22. N2-Biphenyl-3-yl-N-[(1S)-1-({[4-(cyanometh-
oxy)phenyl]amino}methyl)propyl]-L-leucinamide (26)
Compound 26 was prepared according to the procedure
for 23, using appropriate starting materials. Yield: 59%;
oil; 1H NMR (400 MHz, CDCl3) d 0.94 (3H, t,
J = 7.3 Hz), 0.95 (3H, d, J = 5.9 Hz), 1.02 (3H, d,
J = 5.9 Hz), 1.40–1.68 (3H, m), 1.79–1.92 (2H, m), 2.97
(1H, dd, J = 8.5, 12.5 Hz), 3.13 (1H, dd, J = 3.7,
12.5 Hz), 3.72–3.81 (2H, m), 3.91–3.95 (1H, m), 4.02–
572
[M+H]+,
325,
250; Anal.
Calcd
for
C35H45N3O4Æ0.2H2O: C, 73.06; H, 7.95; N, 7.30.
Found: C, 72.96; H, 7.87; N, 7.25.

T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
6805
4.11 (1H, m), 4.60 (2H, s), 6.34 (2H, J = 8.8 Hz), 6.58
(1H, dd, J = 1.5, 8.1 Hz), 6.72–6.79 (1H, m), 6.74 (2H,
d, J = 8.8 Hz), 6.82–6.85 (1H, m), 7.03–7.07 (1H, m),
7.18 (1H, t, J = 8.1 Hz), 7.30–7.38 (3H, m), 7.48–7.55
(2H, m); IR (KBr): 2962, 1652, 1606, 1512, 1215,
758 cmꢀ1; MS (FAB) m/z: 485 [M+H]+, 446, 238; HR-
MS found [M+H]+: 485.2906, calcd for C30H37N4O2:
485.2917.
5.24.2. Benzyl (2E)-3-(4-{[(2S)-2-({[1-(biphenyl-3-ylami-
no)cyclohexyl]carbonyl}amino)butyl]amino}phenoxy)acry-
late (28b). Yield: 66%; oil; 1H NMR (400 MHz, CDCl3)
d 0.89 (3H, d, J = 7.4 Hz), 1.24–1.66 (8H, m), 1.93–2.04
(4H, m), 2.99–3.04 (1H, m), 3.13–3.16 (1H, m), 4.03–
4.09 (1H, m), 5.17 (2H, s), 5.44 (1H, d, J = 12.1 Hz),
6.41 (2H, d, J = 9.0 Hz), 6.58 (1H, d, J = 7.1 Hz), 6.81
(2H, d, J = 9.0 Hz), 6.85 (1H, s), 7.03 (2H, t,
J = 7.8 Hz), 7.10 (1H, t, J = 7.1 Hz), 7.80–7.86 (8H,
m), 7.51 (2H, d, J = 7.1 Hz), 7.76 (1H, d, J = 12.1 Hz);
IR (KBr): 3374, 2935, 1705, 1645, 1509, 1320, 1287,
5.23. N2-Biphenyl-3-yl-N-[(1S)-1-({[4-(1H-tetrazol-5-
ylmethoxy)phenyl]amino}methyl)propyl]-L-leucinamide
(27)
1208, 1116, 828, 757, 699 cmꢀ1
; HR-MS found
[M+H]+: 618.3337, calcd for C39H44N3O4: 618.3329.
A mixture of nitrile 26 (1.13 g, 2.3 mmol), NaN3
(454 mg), and NH4Cl (374 mg) in DMF (25 mL)
was stirred at 100 ꢁC for 6 h. A saturated aqueous
solution of NH4Cl (50 mL) was added to the
cooled reaction mixture. The mixture was extracted
with EtOAc (5· 50 mL). The combined organic lay-
ers were washed with brine (2· 50 mL). The organic
layer was dried (Na2SO4), concentrated, and puri-
fied by column chromatography (EtOAc) to provide
tetrazole 27 (0.75 g, 69%) as colorless crystals. Mp
154–158 ꢁC; 1H NMR (400 MHz, CDCl3) d 0.93
(3H, t, J = 7.4 Hz), 0.93 (3H, d, J = 6.3 Hz), 1.01
(3H, d, J = 6.3 Hz), 1.36–1.45 (1H, m), 1.56–1.68
(2H, m), 1.77–1.92 (3H, m), 2.86 (1H, dd,
J = 8.2, 12.9 Hz), 3.10 (1H, dd, J = 4.3, 12.9 Hz),
3.80–3.84 (1H, m), 4.04–4.11 (1H, m), 5.21–5.24
(2H, br s), 6.22 (2H, d, J = 8.8 Hz), 6.55–6.59
(1H, m), 6.58 (2H, d, J = 8.8 Hz), 6.79–6.86 (2H,
m), 7.02 (1H, d, J = 7.8 Hz), 7.16 (1H, t,
J = 7.8 Hz), 7.25–7.52 (6H, m); IR (KBr): 2960,
1637, 1605, 1512, 1231, 700 cmꢀ1; Anal. Calcd
for C30H37N7O2: C, 68.29; H, 7.07; N, 18.58.
Found: C, 68.43; H, 6.88; N, 18.35; MS (FAB)
m/z: 528 [M+H]+, 444, 238.
5.25. 3-(4-{[(2S)-2-({[1-(Biphenyl-3-ylamino)cyclohex-
yl]carbonyl}amino)butyl]amino}phenoxy)propanoic acid
(29)
A mixture of acrylate 28b (68 mg, 0.11 mmol) and Pd/C
(10%, 12 mg, 0.011 mmol) in EtOH (5 mL) was stirred
under H2 for 20 h. After removal of Pd/C by filtration,
the filtrate was concentrated. The residue was purified
by PTLC (hexane/EtOAc, 1:1) to provide propanoic
1
acid 29 (22 mg, 37%) as an amorphous substance. H
NMR (400 MHz, CDCl3) d 0.98 (3H, d, J = 7.4 Hz),
1.32–1.65 (8H, m), 1.95–2.04 (4H, m), 2.78 (2H, t,
J = 6.3 Hz), 3.01 (1H, dd, J = 10.1, 12.5 Hz), 3.13 (1H,
dd, J = 4.3, 12.5 Hz), 4.02–4.09 (1H, m), 4.16 (2H, t,
J = 6.3 Hz), 6.42 (2H, d, J = 8.6 Hz), 6.59 (1H, d,
J = 8.2 Hz), 6.72 (2H, d, J = 8.6 Hz), 6.85 (1H, s), 7.02
(1H, d, J = 7.8 Hz), 7.07 (1H, d, J = 7.8 Hz), 7.12 (1H,
t, J = 7.8 Hz), 7.31 (1H, d, J = 7.0 Hz), 7.37 (2H, t,
J = 7.0 Hz), 7.52 (2H, d, J = 7.0 Hz); IR (KBr): 3369,
2933, 1650, 1602, 1513, 1229, 758, 701 cmꢀ1; HR-MS
found [M+H]+: 530.3009, calcd for C32H40N3O4:
530.3027.
References and notes
5.24. General procedure for the preparation of acrylates
(28)
1. For the crystal structure of cathepsin K, see: LaLonde, J.
M.; Zhao, B.; Janson, C. A.; D’Alessio, K. J.; McQueney,
M. S.; Orsini, M. J.; Debouck, C. M.; Smith, W. W.
Biochemistry 1999, 38, 862.
2. Shinozuka, T.; Shimada, K.; Matsui, S.; Yamane, T.;
Ama, M.; Fukuda, T.; Taki, M.; Naito, S. Bioorg. Med.
Chem. Lett. 2006, 16, 1502.
A mixture of phenol 22b (81 mg, 0.18 mmol), N-meth-
ylmorpholine (40 lL, 0.36 mmol), and propiolate
(0.36 mmol) in THF (3 mL) was stirred for 30 h under
N2 atmosphere. The reaction mixture was concentrated.
The residue was purified by PTLC to provide acrylate
28.
3. (a) Tavares, F. X.; Deaton, D. N.; Miller, A. B.; Miller, L.
R.; Wright, L. L.; Zhou, H. Q. J. Med. Chem. 2004, 47,
5049; (b) Catalano, J. G.; Deaton, D. N.; Furfine, E. S.;
Hassell, A. M.; McFadyen, R. B.; Miller, A. B.; Miller, L.
R.; Shewchuk, L. M.; Willard, D. H., Jr.; Wright, L. L.
Bioorg. Med. Chem. Lett. 2004, 14, 275; (c) Robichaud, J.;
Bayly, C.; Oballa, R.; Prasit, P.; Mellon, C.; Falgueyret,
J.-P.; Percival, M. D.; Wesolowski, G.; Rodan, S. B.
Bioorg. Med. Chem. Lett. 2004, 14, 4291; (d) Rydzewski,
R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S.
B.; Bass, K. E.; Wong, D. H. Bioorg. Med. Chem. 2002,
10, 3277; (e) Marquis, R. W.; Ru, Y.; LoCastro, S. M.;
Zeng, J.; Yamashita, D. S.; Oh, H.-J.; Erhard, K. F.;
Davis, L. D.; Tomaszek, T. A.; Tew, D.; Salyers, K.;
Proksch, J.; Ward, K.; Smith, B.; Levy, M.; Cummings,
M. D.; Haltiwanger, R. C.; Trescher, G.; Wang, B.;
Hemling, M. E.; Quinn, C. J.; Cheng, H.-Y.; Lin, F.;
Smith, W. W.; Janson, C. A.; Zhao, B.; McQueney, M. S.;
5.24.1. Methyl (2E)-3-(4-{[(2S)-2-({[1-(biphenyl-3-ylami-
no)cyclohexyl]carbonyl}amino)butyl]amino}phenoxy)acry-
1
late (28a). Yield: 64%; amorphous substance; H NMR
(400 MHz, CDCl3) d 0.89 (3H, d, J = 7.4 Hz), 1.30–1.67
(8H, m), 1.92–2.04 (4H, m), 3.01 (1H, dd, J = 8.2,
12.1 Hz), 3.14 (1H, dd, J = 4.3, 12.1 Hz), 3.70 (3H, s),
4.02–4.08 (1H, m), 5.40 (1H, d, J = 12.1 Hz), 6.40 (2H,
d, J = 9.0 Hz), 6.56 (1H, d, J = 9.0 Hz), 6.80 (2H, d,
J = 8.6 Hz), 6.82 (1H, d, J = 5.9 Hz), 7.01 (2H, t,
J = 5.9 Hz), 7.08 (1H, t, J = 7.8 Hz), 7.28–7.35 (8H,
m), 7.49 (2H, d, J = 8.6 Hz), 7.71 (1H, d, J = 12.1 Hz);
IR (KBr): 3371, 2933, 1709, 1646, 1509, 1210, 1123,
828, 758, 701 cmꢀ1; HR-MS found [M+H]+: 542.3016,
calcd for C33H40N3O4: 542.3019.

6806
T. Shinozuka et al. / Bioorg. Med. Chem. 14 (2006) 6789–6806
D’Alessio, K.; Lee, C.-P.; Marzulli, A.; Dodds, R. A.; Gleason, J. G.; Veber, D. F. Proc. Natl. Acad. Sci. U.S.A.
Blake, S.; Hwang, S.-M.; James, I. E.; Gress, C. J.;
Bradley, B. R.; Lark, M. W.; Gowen, M.; Veber, D. F. J.
Med. Chem. 2001, 44, 1380.
1997, 94, 14249.
6. For the docked structure of I into human cathepsin K, see:
Ref. 4b.
4. (a) Altmann, E.; Renaud, J.; Green, J.; Farley, D.;
Cutting, B.; Jahnke, W. . J. Med. Chem. 2002, 45, 2352;
(b) Altmann, E.; Green, J.; Tintelnot-Blomley, M. .
Bioorg. Med. Chem. Lett. 2003, 13, 1997; (c) Kim, T.-S.;
Hague, A. B.; Lee, T. I.; Lian, B.; Tegley, C. M.; Wang,
X.; Burgess, T. L.; Qian, Y.-X.; Ross, S.; Tagari, P.; Lin,
C.-H.; Mayeda, C.; Dao, J.; Jordan, S.; Mohr, C.;
Cheetham, J.; Viswanadhan, V.; Tasker, A. S. Bioorg.
Med. Chem. Lett. 2004, 14, 87.
7. Fourneau, J.-P.; de Lestrange, M. Y. Bull. Soc. Chim. Fr.
1947, 827.
8. Ma, D.; Zhang, Y.; Yao, J.; Wu, S.; Tao, F. J. Am. Chem.
Soc. 1998, 120, 12459.
9. Kortylewicz, Z. P.; Galardy, R. E. J. Med. Chem. 1990, 33,
263.
10. Fukuyama, T.; Cheung, M.; Kan, T. Synlett 1999, 1301.
11. Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G.
Tetrahedron 1995, 51, 12337.
5. Thompson, S. K.; Halbert, S. M.; Bossard, M. J.;
Tomaszek, T. A.; Levy, M. A.; Zhao, B.; Smith, W. W.;
Abdel-Meguid, S. S.; Janson, C. A.; D’Alessio, K. J.;
McQueney, M. S.; Amegadzie, B. Y.; Hanning, C. R.;
DesJarlais, R. L.; Briand, J.; Sarkar, S. K.; Huddleston,
M. J.; Ijames, C. F.; Carr, S. A.; Garnes, K. T.; Shu, A.;
Heys, J. R.; Bradbeer, J.; Zembryki, D.; Lee-Rykaczewski,
L.; James, I. E.; Lark, M. W.; Drake, F. H.; Gowen, M.;
12. Markidis, T.; Kokotos, G. J. Org. Chem. 2001, 66, 1919.
13. Mitsunobu, O. Synthesis 1981, 1.
14. Burmistrov, K. S.; Burmistrov, S. I.; Malinovskii, M. S.
Chem. Heterocycl. Compd. 1977, 13, 1201.
15. Lagarias, J. C.; Houghten, R. A.; Rapoport, H. J. Am.
Chem. Soc. 1978, 100, 8202.
16. Bro¨mme, D.; Okamoto, K.; Wang, B. B.; Biroc, S. J. Biol.
Chem. 1996, 271, 2126.