Degradation-promoters of cellular inhibitor of apoptosis protein 1 based on bestatin and actinonin

Article abstract of DOI:10.1016/j.bmc.2008.02.024

A series of hybrid compounds of bestatin (1) and actinonin (3), which promote degradation of cellular inhibitor of apoptosis protein 1 (cIAP1), were designed and synthesized. Structure-activity relationship studies indicated that absolute configuration, hydrophobicity at the α-position of the internal amide carbonyl group, and the presence of a small substituent at the α-position of the ester group are important factors for the expression of potent cIAP1 degradation-promoting activity. HAB-5A (30b) showed the most potent activity (IC₅₀ = 0.53 μM) among the compounds prepared.

Full text of DOI:10.1016/j.bmc.2008.02.024

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4685–4698
Degradation-promoters of cellular inhibitor of apoptosis
protein 1 based on bestatin and actinonin
Shinichi Sato,a Masashi Tetsuhashi,a Keiko Sekine,a,b Hiroyuki Miyachi,a
Mikihiko Naito,a Yuichi Hashimotoa and Hiroshi Aoyamaa,*
aInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bPharmaceutical Research Laboratories, Research & Development Group, Nippon Kayaku Co., Ltd,
3-31-12, Shimo, Kita-ku, Tokyo 115-8588, Japan
Received 12 December 2007; revised 2 February 2008; accepted 8 February 2008
Available online 13 February 2008
Abstract—A series of hybrid compounds of bestatin (1) and actinonin (3), which promote degradation of cellular inhibitor of apop-
tosis protein 1 (cIAP1), were designed and synthesized. Structure–activity relationship studies indicated that absolute configuration,
hydrophobicity at the a-position of the internal amide carbonyl group, and the presence of a small substituent at the a-position of
the ester group are important factors for the expression of potent cIAP1 degradation-promoting activity. HAB-5A (30b) showed the
most potent activity (IC50 = 0.53 lM) among the compounds prepared.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
caspases via their BIR domains.11,17 So far, at least eight
kinds of human IAPs, neuronal apoptosis inhibitory
protein (NAIP),18 cellular inhibitor of apoptosis protein
1 (cIAP1),19 cellular inhibitor of apoptosis protein 2
(cIAP2),19 x-linked inhibitor of apoptosis protein
(XIAP),20 Livin,21 apollon,22 ILP-2,23 and survivin,24
have been identified.
Programmed cell death, often called apoptosis, is re-
quired for normal embryonic development, growth,
differentiation, and homeostasis of multicellular organ-
isms.1–4 Apoptosis can be triggered by distinct extracel-
lular and intracellular stimuli, and it can involve the
activation of a unique class of cysteine proteases known
as caspases.5–8 The function of caspases is regulated by
another set of proteins called inhibitor of apoptosis pro-
teins (IAPs).9–11
cIAP1 was first found as a binding factor to TNF recep-
tor associated factor-1 (TRAF-1) and TNF receptor
associated factor-2 (TRAF-2) in the signaling pathway
mediated by tumor necrosis factor 2 (TNFR2).19 In this
pathway, cIAP1 directly inhibits the activity of caspase-
3, caspase-7, and caspase-9.25,26 cIAP1 is highly ex-
pressed in various organs, such as kidney, small intes-
tine, and lung, and one of the factors causing
treatment resistance in cancer patients has been sug-
gested to be the apoptosis-inhibiting activity of cIAP1
in these organs. Thus, inhibition of cIAP1 function is re-
garded as a potential therapeutic target.
IAPs are members of a group of intracellular survival
proteins, first identified in baculoviruses, whose function
is to keep the host cells alive while the virus continues to
replicate.12,13 IAPs have two distinctive motifs, that is,
one to three copies of baculovirus IAP repeat (BIR) do-
mains, which are zinc-binding domains of about 80 ami-
no acid residues, and a second zinc-binding motif known
as really interesting new gene (RING) domain, which
exhibits E3-ubiquitin-ligase activity.14–16 Several human
IAPs have been shown to bind directly to, and to inhibit,
Generally, inhibition of apoptosis by XIAP, a potent
anti-apoptosis mediator in mammals, is overcome by
Smac, an apoptosis stimulator, which binds to XIAP.
Stimulation depends on the relative concentrations of
Smac and XIAP, as a high concentration of XIAP can
protect cells against expression of Smac.27–30 However,
cIAPs inhibit the inhibitory effect of Smac on XIAP.31,32
Keywords: Bestatin; Actinonin; cIAP1; Structural development;
Inhibitor.
*
Corresponding author. Tel.: +81 03 5841 7848; fax: +81 03 5841
8495; e-mail: aoyama@iam.u-tokyo.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.024

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S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
moral and cell-mediated immune responses and inhibi-
tion of aminopeptidases.42 We recently found that
bestatin ester derivatives, such as MeBS (2), possess po-
tent cIAP1 degradation-promoting activity based on
auto-ubiquitination followed by proteasome-dependent
digestion.43 We also found that actinonin (3, Fig. 1),44
an antibiotic and an inhibitor of leucine aminopeptidase
and aminopeptidase N,45 possesses similar cIAP1 degra-
dation-promoting activity.
Figure 1. Structures of bestatin (1), MeBS (2), and actinonin (3).
Thus, cIAPs have become one of the molecular targets
for drugs to treat cancer. Recently, some Smac mimics
were reported to possess potent inhibitory activity not
only against XIAP-mediated inhibition of caspase activ-
ity, but also against cIAPs-mediated inhibition of Smac
activity.33–37
Based on these previous studies, bestatin (1) and acti-
nonin (3) were expected to be suitable lead compounds
for the development of compounds with more potent
cIAP1 degradation-promoting activity. Here, we exam-
ined the structure–activity relationships of bestatin es-
ters and hybrid compounds based on bestatin (1) and
actinonin (3) structures.
In addition, it was reported that Smac and Smac mimics
enhance auto-ubiquitination of cIAP1 and cIAP2,38,39
and this might provide the basis for a novel anti-tumor
therapy. However, some problems, such as low mem-
brane permeability and insufficient in vivo stability of
these compounds, still remain.
2. Results and discussion
2.1. Synthesis
Bestatin methyl ester analogs 5, 9, 14, and 18a–c were syn-
thesized as shown in Scheme 1. Briefly, condensation of
(2R,3R)-3-[N-(benzyloxycarbonyl)amino]-2-hydroxy-4-
phenylbutanoic acid (4) and L-leucine methyl ester, fol-
lowed by deprotection of the Boc group with TFA gave
EpiMeBS (5). Wittig reaction of phenylacetaldehyde (6)
and methyl (triphenylphosphoranylidene)acetate, fol-
Bestatin (1, Fig. 1), N-[(2S,3R)-3-amino-2-hydroxy-4-
phenylbutanoyl]-L-leucine, was isolated from Strepto-
myces olivoreticulithe in 1976,40 and shown to be a po-
tent competitive inhibitor of aminopeptidase B and
leucine aminopeptidase.41 Bestatin (1) also possesses
immunomodulatory effects through stimulation of hu-
Scheme 1. Reagents and conditions: (a) L-leucine methyl ester hydrochloride, EDCI, HOBt, CH2Cl2, rt; (b) TFA, CH2Cl2, rt; (c)
Ph3P@CHCOOCH3, toluene, reflux; (d) K2OsO4, AD-mix-b, tert-butanol–H2O, 0 ꢁC–rt; (e) TFA, H2O, 60 ꢁC; (f) L-leucine methyl ester
hydrochloride, EDCI, HOBt, CH2Cl2, rt; (g) BrCH2COOEt, K2CO3, acetone, rt; (h) NaH, DMF, 0 ꢁC; (i) (Boc)2O, Et3N, DMAP, CH2Cl2, rt; (j)
3.73 N NaOH aq, MeOH–H2O, rt; (k) L-leucine methyl ester hydrochloride, EDCI, HOBt, CH2Cl2, rt; (l) TFA, CH2Cl2, rt; (m) NaNO2, 6 N HCl,
0 ꢁC; (n) KOH, EtOH, 0 ꢁC; (o) 2 N HCl, rt; (p) L-leucine methyl ester hydrochloride, EDCI, HOBt, CH2Cl2, rt; (q) RNH2, CH2Cl2, 70 ꢁC; (r) NaN3,
NH4Cl, THF–H2O, rt; (s) H2, Pd/C, MeOH, rt.

S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4687
lowed by dihydroxylation with osmium reagent, hydroly-
sis, and condensation with leucine methyl ester gave
DioMeBS (9). Application of Williamson ether synthesis
using 2-cyanophenol (10) and ethyl bromoacetate, fol-
lowed by cyclization reaction, afforded the benzofuran
intermediate 12. Subsequent protection of the amino
group with Boc group, hydrolysis of the ethyl ester, con-
densation with leucine methyl ester, and deprotection of
the amino group afforded FurMeBS (14). Intermediate
17 for the synthesis of compounds 18a–c was prepared
by the treatment of D-serine (15) with sodium nitrate,
potassium hydroxide, and aqueous HCl, followed by con-
densation of the product with leucine methyl ester. The
intermediate 17 was treated with sodium azide, benzyl-
amine, or phenethylamine to afford priAMeBS (18a),
BAMeBS (18b), or PAMeBS (18c), respectively. In the
case of the preparation of 18a, hydrogenation under a
hydrogen atmosphere using Pd/C catalyst was needed.
the presence of Pd/C afforded the (2S,5R)-configured
product, HAB-5RL [(2S,5R)-25a], HAB-2RL [(2S,5R)-
25b], and HAB-BRL [(2S,5R)-25c], respectively. The
configurational isomer of (2S,5R)-25a, HAB-5SL
[(2S,5S)-25a], was prepared by the similar method using
an enantiomer of 20a as a starting material.
The dealkylated derivative of 25 at the a-position of the
internal amide carbonyl group (HAB-0L, 25d) was pre-
pared from succinic anhydride (26) in three steps
(Scheme 3). Derivatives modified at the a-position of es-
ter carbonyl group of (2S,5R)-25a were similarly pre-
pared from intermediate 22a using methyl ester
derivatives of glycine (HAB-5G: 30a), L-alanine (HAB-
5A: 30b), L-valine (HAB-5V: 30c), L-tert-leucine
(HAB-5TL: 30d), and L-phenylalanine (HAB-5F: 30e),
instead of L-leucine methyl ester (Scheme 4).
HAB-A0L (36a), bearing an amino group at the a-posi-
tion of the internal amide carbonyl group of 25d, and its
N-butylated derivative [HAB-A4L (36b)] and N,N-dibu-
tylated derivative [HAB-A44L (36c)] were prepared
from D-aspartic acid (31) (Scheme 5). As shown in the
scheme, benzyl N-Boc-D-aspartate (32), prepared from
D-aspartic acid (31) by the general protective method
in two steps, was condensed with leucine methyl ester.
The product was hydrogenated with hydrogen gas over
Pd/C and then condensed with tert-butyldimethylsilyl-
oxyamine to afford the O-silylated hydroxamic acid
35a. The product was treated with trifluoroacetic acid,
followed by deprotection of the tert-butyldimethylsilyl
group using trifluoroacetic acid, to give HAB-A0L
Hybrid compounds of bestatin and actinonin, 25a–d and
30a–e, were prepared from the corresponding acid chlo-
rides based on Evans’s asymmetric alkylation method
(Schemes 2–4). As shown in Scheme 2, (S)-N-n-alka-
noyl-4-benzyloxazolidinone 20a–c, prepared from n-
alkanoyl chloride 19a–c and 4-(S)-benzyloxazolidinone,
was treated with benzyl 2-bromoacetate, followed by the
removal of the chiral auxiliary to afford the chiral benzyl
ester intermediate 22a–c. The product was condensed
with leucine methyl ester, then hydrogenated with
hydrogen gas over Pd/C, and condensed with benzyloxy-
amine to afford the benzylated hydroxamic acid 24a–c.
Finally, hydrogenation of 24a–c with hydrogen gas in
Scheme 2. Reagents and conditions: (a) (S)-N-n-alkanoyl-4-benzyloxazolidinone, n-BuLi, THF, ꢀ78 ꢁC–rt; (b) BrCH2COOBn, LiHMDS, THF,
ꢀ78 to 0 ꢁC; (c) 30% H2O2 aq, LiOH, THF–H2O, 0 ꢁC–rt; (d) L-leucine methyl ester hydrochloride, EDCI, HOBt, DIPEA, CH2Cl2, rt; (e) H2, Pd/C,
EtOAc, rt; (f) BnONH2HCl, EDCI, DIPEA, CH2Cl2, rt; (g) H2, Pd/C, MeOH, rt.
Scheme 3. Reagents and conditions: (h) L-leucine methyl ester hydrochloride, DMAP, Et3N, CH2Cl2, rt; (i) BnONH2HCl, EDCI, DIPEA, CH2Cl2,
rt; (j) H2, Pd/C, MeOH, rt.

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S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
Scheme 4. Reagents and conditions: (k) L-amino acid methyl ester hydrochloride, EDCI, HOBt, DIPEA, CH2Cl2, rt; (l) H2, Pd/C, EtOAc, rt; (m)
BnONH2HCl, EDCI, DIPEA, CH2Cl2, rt; (n) H2, Pd/C, MeOH, rt.
Scheme 5. Reagents and conditions: (a) BnOH, p-TsOHH2O, benzene, reflux; (b) 20% NaOH aq, acetone–H2O, 13 ꢁC; (c) (Boc)2O, Et3N, acetone–
H2O, rt; (d) L-leucine methyl ester hydrochloride, EDCI, HOBt, DIPEA, CH2Cl2, rt; (e) H2, Pd/C, EtOAc, rt; (f) TBSONH2, EDCI CH2Cl2, rt; (g)
BnONH2HCl, EDCI, DIPEA, CH2Cl2, rt; (h) TFA, CH2Cl2, 0 ꢁC; (i) n-butylaldehyde (1 equiv or 2 equiv), NaBH(OAc)3, AcOH, THF, rt; (j) H2, Pd/
C, MeOH, rt.
(36a). On the other hand, condensation with benzyloxy-
amine instead of tert-butyldimethylsilyloxyamine at step
‘g’ in Scheme 5 afforded the O-benzylated hydroxamic
acid 35b. After deprotection of the Boc group with tri-
fluoroacetic acid, the resulting amino derivative 37 was
treated with 1 equiv or 2 equiv of n-butylaldehyde for
reductive amide alkylation using sodium triacetoxy-
borohydride, and then hydrogenated over Pd/C to give
HAB-A4L (36b) and HAB-A44L (36c).
2.2. Biological evaluation
In our previous studies, various esters derived from best-
atin (1) were found to enhance CH11 (anti-Fas antibody
which has been established to act as an Fas agonist)46-in-
duced apoptosis more efficiently than bestatin (1).43 In
particular, the methyl ester derivative (MeBS, 2) exhibited
potent enhancing activity on CH11-induced apoptosis of
HT1080 cells (Fig. 2). On the basis of this information,
Figure 2. HT1080 cells were treated with 30 lM bestatin (1) or MeBS
(2) in the presence (+) or absence (ꢀ) of 100 ng/mL CH1146 for 24 h.
and the results are summarized in Figure 3 and Table 1.
The diastereomer of MeBS (EpiMeBS: 5), the diol com-
pound (DioMeBS: 9), the benzofuran-type compound
(FurMeBS: 14), and compounds debenzylated at the 3-
position (priAMeBS: 18a; BAMeBS: 18b; PAMeBS:
18c) possessed no or very weak cIAP1 degradation-pro-
moting activity, even though they have an ester moiety,
our preliminary efforts for the structural optimization of
MeBS (2) analogs were focused on altering the b-amino-
alcohol moiety, that is, compounds 5, 9, 14, 18a–c, and
(2S,5R)-25a (Fig. 3). The cIAP1 degradation-promoting
activity of these analogs was evaluated by using human
fibrosarcoma HT-1080 cells stably expressing FLAG-
tagged cIAP1, and treated with the prepared compounds,

S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4689
Figure 3. The effects of MeBS (2) derivatives on cIAP1 degradation.
except for 5, which exhibited moderate activity (Fig. 3). In
contrast, the hybrid compound [HAB-5RL: (2S,5R)-25a]
composed of the L-leucine part of MeBS (2) and the
hydroxamic acid part of actinonin (3) exhibited potent
cIAP1 degradation-promoting activity with an IC50 value
of 1.06 lM, which is comparable with that of MeBS
(IC50 = 0.69 lM) (Table 1). This result suggested that a
putative zinc-binding structure, such as a b-aminoalcohol
moiety and/or a hydroxamic acid moiety, as well as an es-
ter moiety, is essential for potent cIAP1 degradation-pro-
moting activity. In addition, the putative zinc-binding
structure seemed to be critically recognized by the target
molecule(s), because the stereoisomer of MeBS (Epi-
MeBS: 5) showed lower activity than MeBS (2) (Fig. 3).
The down-regulation of cIAP1 caused by MeBS (2) and
some of the prepared derivatives can plausibly be attrib-
uted to the enhancement of cIAP1 degradation in the
cells, because incubation of the cells with the compounds
for only 3 h was sufficient for manifestation of the degra-
dation-promoting activity. We suggest that these com-
pounds promote degradation, analogously with Yang’s
and Eugene’s conclusion that Smac and Smac mimics en-
hanced auto-ubiquitination of cIAP1.38,39
On the basis of the initial screening results mentioned
above, further structural modification of HAB-5RL
[(2S,5R)-25a] was conducted. First, we evaluated the ef-
fects on the cIAP1 degradation-promoting activity of
various substituents at the a-position of the internal
amide carbonyl group (Table 2). The stereoisomer
[HAB-5SL: (2S,5S)-25a] showed lower activity
(IC50 = 12.1 lM, Table 2, entry 2) than (2S,5R)-25a
(IC50 = 1.06 lM, Table 2, entry 1). The non-substituted
compound [HAB-0L: 25d], and the compound with an
ethyl [HAB-2L: (2S,5R)-25b] or a benzyl [HAB-BL:
(2S,5R)-25c] group at the corresponding position also
exhibited lower activity (IC50 values of 19.9–45.2 lM,
Table 2, entries 3–5) than (2S,5R)-25a (Table 2, entry
1). The activity decreased in the order of: R = n-C5H11
Table 1. cIAP1 production-inhibiting activity of MeBS (2) and HAB-
5RL [(2S,5R)-25a]
a
Compound
IC50 (lM)
0.69
[HAB-5RL:
(2S,5R)-25a] > R = H
(HAB-0L:
25d) > R = C2H5 (HAB-2L: 25b) > R = CH2Ph (HAB-
BL: 25c). These results indicated that derivatives with
the R configuration at the a-position of the internal
amide carbonyl group are more effective than the corre-
sponding derivatives with the S configuration. Addition-
ally, the expression of potent cIAP1 degradation-
promoting activity seems to require a long/flexible alkyl
chain, such as an n-pentyl group, at the a-position of the
1.06
a IC50 is the concentration that decreases cIAP1 production to 50%.

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S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
Table 2. cIAP1 production-inhibiting activity of compounds 25a–d
Table 4. cIAP1 production-inhibiting activity of compounds 36a–c
a
Entry
Compound
Config.
R
IC50
(lM)
Entry
Compound
R
R1
IC50 (lM)
a
1
2
3
4
5
HAB-5RL [(2S,5R)-25a]
HAB-5SL [(2S,5S)-25a]
HAB-2L (25b)
R
S
n-C5H11
n-C5H11
C2H5
1.06
12.1
24.1
45.2
19.9
1
2
3
HAB-A0L (36a)
HAB-A4L (36b)
HAB-A44L (38c)
H
H
N.A.
4.69
9.20
H
n-C4H9
n-C4H9
n-C4H9
R
R
HAB-BL (25c)
HAB-0L (25d)
CH2Ph
H
a IC50 is the concentration that decreases cIAP1 production to 50%.
a IC50 is the concentration that decreases cIAP1 production to 50%.
5F: 30e) > R = i-C3H7 (HAB-5V: 30c) > R = H (HAB-
5G: 30a) ꢁ R = t-C4H9 (HAB-5TL: 30d). The potent
activity (IC50 values of 0.53–1.25 lM) of HAB-5A
(30b), HAB-5V (30c), and HAB-5F (30e), in contrast
to HAB-5TL (30d) and HAB-5G (30a) (IC50 values of
>20 lM and 1.54 lM, respectively), implies that the
presence of a hydrogen atom (small substituent) on the
b-carbon at the a-position of ester carbonyl group is
necessary for potent cIAP1 degradation-promoting
activity.
internal amide carbonyl group. Introduction of spheri-
cal/rigid groups, such as a benzyl group (HAB-BL:
25c), decreased the activity.
Next, we examined the effect on cIAP1 degradation-pro-
moting activity of substituents at the a-position of the es-
ter carbonyl group (Table 3). The compound with a tert-
butyl group (HAB-5TL: 30d) was almost inactive (Table
4, entry 4). In contrast, the unsubstituted compound
(HAB-5G: 30a), and the compound with a methyl
(HAB-5A: 30b), an isopropyl (HAB-5V: 30c), or a benzyl
(HAB-5F: 30e) group exhibited potent cIAP1 degrada-
tion-promoting activity (Table 4, entries 1–3, and 5).
HAB-5A (30b) showed the most potent activity
(IC50 = 0.53 lM) among the prepared compounds,
including the lead compound (2S,5R)-25a. As shown in
Figure 4, HAB-5A (30b) showed cIAP1 degradation-pro-
moting activity with the potency comparable to that of
MeBS (2) as far as that measured by Western blot analysis
(Fig. 4, panel a). The degradation-promoting activity elic-
ited by HAB-5A (30b) seems to be based on auto-ubiqui-
tination followed by proteasome-dependent digestion, as
that elicited by MeBS (2) does, because addition of pro-
teasome inhibitor MG13247 caused accumulation of
cIAP1 and its ubiquitinated form (Fig. 4, panel b). As ex-
pected, HAB-5A (30b) showed more potent enhancing
activity than that of MeBS (2) on apoptosis induced by
actinomycin D, cisplatin, etoposide, or camptothecin
(Fig. 4, panels c and d).
As regards the alkyl chain at the a-position of the inter-
nal amide carbonyl group, we introduced a hetero
atom(s), and examined the effect on cIAP1 degrada-
tion-promoting activity (Table 4). Although the non-
substituted amino compound (HAB-A0L: 36a) was
inactive (Table 4, entry 1), the compound with a
mono-n-butylamino group (HAB-A4L: 36b), which
can be regarded as an aza-analog of HAB-5RL
[(2S,5R)-25a] (Table 2), exhibited moderate cIAP1 deg-
radation-promoting activity (IC50 = 4.89 lM, Table 4,
entry 2), though the activity was lower than that of
HAB-5RL [(2S,5R)-25a]. Further introduction of an n-
butyl group, that is, the compound with a di-n-butylami-
no group (HAB-A44L: 36c), caused a decrease of the
activity (IC50 = 9.20 lM, Table 4, entry 2). These results
suggest that the introduction of hetero atom(s) at the a-
position of the internal amide carbonyl group is permis-
sible, but decreases the activity.
3. Conclusions
The activity of the derivatives of 30 decreased in the or-
der of: R = CH3 (HAB-5A: 30b) > R = CH2Ph (HAB-
We have developed a number of potent cIAP1 degrada-
tion-promoting compounds. Among the compounds
prepared, HAB-5A (30b) exhibited the most potent
activity for promotion of cIAP1 degradation, with an
IC50 value of nanomolar order (IC50 = 0.53 lM). Fur-
ther structure–activity relationship studies based on
HAB-5A (30b) and a search for compounds targeting
other IAPs are in progress.
Table 3. cIAP1 production-inhibiting activity of compounds 30a–e
a
Entry
Compound
R
IC50 (lM)
4. Experimental
4.1. Cell culture
1
2
3
4
5
HAB-5G (30a)
HAB-5A (30b)
HAB-5V (30c)
HAB-5TL (30d)
HAB-5F (30e)
H
CH3
1.54
0.53
1.25
>20
0.84
i-C3H7
t-C4H9
CH2Ph
Human fibrosarcoma HT-1080 cells stably expressing
FLAG-tagged cIAP1 were cultured in RPMI 1640 con-
a IC50 is the concentration that decreases cIAP1 production to 50%.

S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4691
Figure 4. The effects of HAB-5A (30b) on cIAP1 degradation and apoptosis. (a) Western blot analysis of FLAG-tagged cIAP1 extracted from
HT1080 cells stably expressing FLAG-tagged cIAP1. (b) Inhibition of cIAP-1 degradation-promoting activity of HAB-5A (30b)/MeBS (2) by
proteasome inhibitor, MG132.47 (c) Effects of HAB-5A (30b) and MeBS (2) on actinomycin- and cisplatin-induced cell apoptosis. (d) Effects of HAB-
5A (30b) and MeBS (2) on etoposide- and camptothecin-induced cell apoptosis.
taining 10% heat-inactivated fetal bovine serum (FBS)
and antibiotic–antimycotic mixture (Nacalai) at 37 ꢁC
in a humidified atmosphere of 5% CO2 in air.
the lowest, by HPLC equipped with chiral column
[CHIRALPAK AD-H: eluted with a mixture of hexane
and isopropanol (1:2) containing 0.1% v/v TFA].
4.2. cIAP1 production inhibition
4.3.1.1. N-[(2R,3R)-3-Amino-2-hydroxy-4-phenylbuta-
noyl]-L-leucine methyl ester (EpiMeBS: 5). To a solution
of compound 448 (50.0 mg, 0.109 mmol) in dry CH2Cl2
(4 mL) were added L-leucine methyl ester hydrochloride
(21.9 mg, 0.120 mmol), HOBt (16.3 mg, 0.120 mmol),
and EDC (25.2 mg, 0.131 mmol). The reaction mixture
was stirred at rt for 2 h and quenched with water. The or-
ganic layer was collected, dried over MgSO4, and concen-
trated under reduced pressure. The intermediate was
obtained after column chromatography (AcOEt/
Hex = 1:1) (28.5 mg, 62%). To a solution of the intermedi-
ate (28.5 mg, 0.068 mmol) in dry CH2Cl2 (4 mL) was
added TFA (0.4 mL). The reaction mixture was stirred
at rt for 60 min and concentrated under reduced pressure.
Pure 5 was obtained by means of PLC (CH2Cl2/
MeOH = 7:1) (17.4 mg, 80%). 1H NMR (500 MHz,
CD3OD) d: 7.88 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 7.7 Hz,
1H), 7.75–7.24 (m, 5H), 4.49 (m, 1H), 4.38 (d,
J = 3.0 Hz, 1H), 3.77 (m, 1H), 3.70 (s, 3H), 3.10 (dd,
J = 14.5, 4.3 Hz, 1H), 2.88 (dd, J = 14.5, 10.7 Hz, 1H),
1.75–1.67 (m, 3H), 0.96 (d, J = 6.0 Hz, 3H), 0.93 (d,
HT1080 cells stably expressing FLAG-tagged cIAP1
(5 · 105) in six-well plates were treated with serial dilu-
tions of compound for 3 h. The cells were harvested and
lysed with lysis buffer (1% SDS, 10% glycerol, 0.1 M Tris,
pH 7.4). Cell lysates containing equal amounts of protein
were separated by 4–20% gradient polyacrylamide gel
electrophoresis, transferred to PVDF membranes, and
Western-blotted using appropriate antibodies (anti-
FLAG and anti-action antibodies). Protein bands were
detected using Enhanced Chemiluminescense detection
kits and the quantity of protein was determined with im-
age analysis software (MetaMorph).
4.3. Chemistry
4.3.1. General. 1H NMR (500 MHz) spectra were re-
corded on a JEOL JNM-a500 spectrometer. Mass spec-
tra were obtained on JEOL JMA-HX 110 spectrometer
with m-nitrobenzyl alcohol. Flash column chromatogra-
phy was performed on silica gel 60 Kanto Kagaku (40–
100 lm). Optical purity of optically active compounds
prepared were confirmed to be more than 97% ee at
J = 6.0 Hz, 3H); HRMS (FAB) calcd for C17H27N2O4
22
D
323.1971; found: 323.1949 (M+H)+; ½aꢂ ꢀ1.71 (c 0.70
in MeOH).

4692
S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4.3.1.2. (E)-Methyl 4-phenylbut-2-enoate (7). To a
4.3.1.6. Ethyl 3-aminobenzofuran-2-carboxylate (12).51
To a solution of 11 (500 mg, 2.44 mmol) in DMF was
added NaH (64.5 mg, 2.68 mmol) at 0 ꢁC, and the mixture
was stirred for 1 h and quenched with water. Pure 12 was
obtained by extraction with Et2O and column chromatog-
raphy (AcOEt/Hex = 3:7) (307 mg, 61%). 1H NMR
(500 MHz, CDCl3) d: 7.58–7.25 (m, 4H), 4.46 (q,
J = 6.8 Hz, 2H), 1.45 (t, J = 6.8 Hz, 3H).
solution of phenyl acetaldehyde (500 mg, 4.2 mmol) in
toluene (50 mL) was added (triphenylphosphoranylid-
ene)acetic acid methyl ester (1.4 g, 4.2 mmol). The reac-
tion mixture was stirred at reflux for 3 h. Pure 7 was
obtained by means of column chromatography (AcOEt/
Hex = 1:2) (667 mg, 91%). 1H NMR (500 MHz, CDCl3)
d: 7.28–7.12 (m, 5H), 7.06 (dt, J = 15.4, 6.8 Hz, 1H),
5.79 (d, J = 15.4 Hz, 1H), 3.67 (s, 3H), 3.47 (d,
J = 6.8 Hz, 2H).
4.3.1.7. 3-(N-tert-Butoxycarbonyl)aminobenzofuran-2-
carboxylic acid (13). To a solution of 12 (157 mg,
0.77 mmol) in CH2Cl2 (20 mL) were added Boc2O
(250 mg, 1.15 mmol), DMAP (93.4 mg, 0.77 mmol),
and NEt3 (2 mL). The mixture was stirred for 2.5 h at
rt and quenched with 1 N HCl. The organic layer was
collected, dried over MgSO4, and concentrated under re-
duced pressure. The residue was dissolved in MeOH/
H2O (4 mL:2 mL), and 15% v/v NaOH aq (1 mL) was
added. Stirring was continued for 1.5 h at rt, then
CH2Cl2 was added. The aqueous layer was acidified with
1 N HCl. Pure 13 was obtained by extraction with
4.3.1.3. Methyl (2S,3R)-2,3-dihydroxy-4-phenylbut-
anoate (8). To a solution of 7 (667 mg, 3.8 mmol) in
tBuOH/H2O (20 mL:20 mL) were added AD-mix-b
(5.3 g), CH3SO2NH2 (360 mg, 3.8 mmol), and K2SOsO4
(210 mg, 0.57 mmol) at 0 ꢁC. The reaction mixture was
stirred at 0 ꢁC for 50 min and then stirred at rt for 3 h.
The reaction mixture was quenched with NaSO3 (5.3 g)
and water, and the product was extracted with AcOEt.
The extract was dried over MgSO4. Pure 8 was obtained
by means of column chromatography (AcOEt/
Hex = 1:2) (583 mg, 73%). 1H NMR (500 MHz, CDCl3)
d: 7.34–7.25 (m, 5H), 4.17 (tdd, J = 7.3, 8.1, 1.7 Hz, 1H),
4.08 (dd, J = 5.1, 1.7 Hz, 1H), 3.80 (s, 3H), 3.05 (d,
J = 5.1 Hz, 1H), 2.96 (dd, J = 13.7, 7.3 Hz, 1H), 2.95
1
AcOEt (187 mg, two steps, 88%). H NMR (500 MHz,
CDCl3) d: 7.60–7.26 (m, 4H), 1.40 (s, 9H).
4.3.1.8. N-(3-Aminobenzofuran-2-carbonyl)-L-leucine
methyl ester (FurMeBS: 14). To a solution of 13
(10.0 mg, 0.036 mmol) in dry CH2Cl2 (1 mL) were added
HOBt (6.8 mg, 0.050 mmol), EDCI (10.3 mg,
0.037 mmol), and L-leucine methyl ester hydrochloride
(7.2 mg, 0.040 mmol). The reaction mixture was stirred
at rt for 1 h and quenched with water. The organic layer
was collected, dried over MgSO4, and concentrated un-
der reduced pressure. The intermediate was dissolved in
dry CH2Cl2 (2 mL), TFA (0.2 mL) was added, and the
mixture was stirred for 1 h. Pure 14 was obtained by
PLC (AcOEt/Hex = 1:2) (56 mg, two steps, 56%). 1H
NMR (500 MHz, CDCl3) d: 7.54–7.23 (m, 4H), 6.50
(d, J = 8.1 Hz, 1H), 4.84 (m, 1H), 3.78 (s, 3H), 1.79–
1.68 (m, 3H), 1.00 (d, J = 4.3 Hz, 3H), 0.99 (d,
J = 4.3 Hz, 3H); HRMS (FAB) calcd for C16H21N2O4
305.1501; found: 305.1530 (M+H)+.
(dd, J = 13.7, 7.3 Hz, 1H), 2.00 (d, J = 8.1 Hz, 1H); MS
24
(FAB) 211 (M+H)+; ½aꢂD +38.55 (c 1.01 in CHCl3).
4.3.1.4. N-[(2S,3R)-2,3-Dihydroxy-4-phenylbutanoyl]-
L-leucine methyl ester (DioMeBS: 9). Compound 8
(50.0 mg, 0.24 mmol) was dissolved in a mixture of H2O
(10 mL) and TFA (6 mL). The reaction mixture was stir-
red at 60 ꢁC for 12 h. The pure intermediate {the corre-
24
sponding carboxylic acid, ½aꢂ +28.20 (c 0.58 in
D
MeOH)}49,50 was obtained by chromatography eluted
with AcOEt (30.0 mg, 64%). To a solution of the interme-
diate (28.0 mg, 0.14 mmol) in dry THF (15 mL) were
added L-leucine methyl ester hydrochloride (28.6 mg,
0.16 mmol), HOBt (27.0 mg, 0.20 mmol), EDCI
(41.0 mg, 0.21 mmol), and DIPEA (29.0 lL, 0.17 mmol)
under an argon atmosphere. The reaction mixture was
stirred at rt for 90 min and quenched with NH4Cl aq.
The organic layer was collected, dried over MgSO4, and
concentrated under reduced pressure. Pure 9 was ob-
tained by means of column chromatography (AcOEt/
Hex = 1:2) (46.1 mg, 100%). 1H NMR (500 MHz, CDCl3)
d: 7.79 (m, 1H), 7.45 (m, 1H), 7.33–7.23 (m, 3H), 7.07 (d,
J = 8.1 Hz, 1H), 4.66 (m, 1H), 4.26 (m, 1H), 4.15 (d,
J = 3.4 Hz, 1H), 3.75 (s, 3H), 2.99 (dd, J = 13.7, 4.3 Hz,
1H), 2.77 (dd, J = 13.7, 9.8 Hz, 1H), 1.72–1.58 (m, 3H),
4.3.1.9. (S)-Oxirane-2-carboxylic acid (16).52 D-Ser-
ine (2.0 g, 19.0 mmol) was dissolved in 6 N HCl at
0 ꢁC, NaNO2 was added, and the mixture was stirred
at 0 ꢁC for 6 h, then extracted with Et2O, dried over
MgSO4, and concentrated under reduced pressure. The
residue was dissolved in EtOH (5 mL), KOH (1.26 g,
22.5 mmol) was added, and the mixture was stirred at
0 ꢁC for 3 h then at rt for 13 h. The precipitate was re-
moved by filtration and recrystallized (AcOEt/MeOH).
The crystalline potassium salt was dissolved in water
(20 mL), then 2 N HCl was added, and the mixture
was stirred for 30 min at rt. Pure 16 was obtained by
extraction with AcOEt and concentration (403 mg, three
steps, 24%). 1H NMR (500 MHz, CDCl3) d: 3.49 (t,
J = 3.4 Hz, 1H), 3.03–3.00 (m, 2H).
0.94 (d, J = 6.0 Hz, 6H); HRMS (FAB) calcd for
24
D
C17H26NO5 324.1811; found: 324.1851 (M+H)+; ½aꢂ
ꢀ29.89 (c 0.51 in MeOH).
4.3.1.5. Ethyl 2-(2-cyanophenoxy)acetate (11).51 2-
Cyanophenol (10) (1.00 g, 8.40 mmol), ethyl 2-bromoace-
tate (1.8 g, 12.0 mmol) and K2CO3 (1.5 g, 10.9 mmol)
were dissolved in acetone (100 mL). The reaction mixture
was stirred at rt for 64 h. K2CO3 was removed by filtra-
tion, and pure 11 was obtained by concentration of the fil-
trate (1.59 g, 77%). 1H NMR (500 MHz, CDCl3) d: 7.61–
6.84 (m, 4H), 4.77 (s, 2H), 4.25 (q, J = 7.3 Hz, 2H), 1.29 (t,
J = 7.3 Hz, 3H); MS (FAB) 206 (M+H)+.
4.3.1.10. N-[(S)-Oxirane-2-carbonyl]-L-leucine methyl
ester (17). To a solution of 16 (403 mg, 4.58 mmol) in
CH2Cl2 (30 mL) were added HOBt (862 mg, 6.38 mmol),
EDCI (1.32 g, 6.89 mmol), and L-leucine methyl ester
hydrochloride (862 mg, 4.74 mmol). The reaction mixture

S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4693
was stirred at rt for 30 min and quenched with water. The
organic layer was collected, dried over MgSO4, and con-
centrated under reduced pressure. Pure 17 was obtained
by column chromatography (AcOEt/Hex = 1:3)
at rt, quenched with sat. NH4Cl aq and satd NaHCO3
aq. Extraction with AcOEt and concentration under re-
duced pressure afforded the product. Pure 20a was ob-
tained by column chromatography (AcOEt/Hex = 1:6)
(14.25 g, 95%). 1H NMR (500 MHz,CDCl3) d: 7.35–
7.20 (m, 5H), 4.67 (m, 1H), 4.17 (m, 2H), 3.30 (dd,
J = 13.3, 3.4 Hz, 1H), 3.00–2.86 (m, 2H), 2.77 (dd,
J = 13.3, 9.8 Hz, 1H), 1.69 (m, 2H), 1.40–1.31 (m, 6H),
0.90 (t, J = 6.9 Hz, 3H); MS (FAB) 290 (M+H)+.
1
(617 mg, 63%). H NMR (500 MHz, CDCl3) d: 6.48 (d,
J = 8.1 Hz, 1H), 4.46 (m, 1H), 3.74 (s, 3H), 3.46 (dd,
J = 4.7, 2.6 Hz, 1H), 3.00 (dd, J = 5.6, 4.7 Hz, 1H), 2.75
(dd, J = 5.6, 2.6 Hz, 1H), 1.64 (m, 1H), 1.57–1.49 (m,
2H), 0.91 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H);
MS (FAB) 216 (M+H)+.
4.3.1.15. (S)-4-Benzyl-3-butyryloxazolidin-2-one (20b).
This compound was prepared from 19b according to the
general procedure (91%). H NMR (500 MHz,CDCl3)
d: 7.39–7.25 (m, 5H), 4.72 (m, 1H), 4.25–4.19 (m, 2H),
3.35 (dd, J = 13.7, 3.4 Hz, 1H), 3.04–2.89 (m, 2H), 2.81
(dd, J = 13.7, 10.7 Hz, 1H), 1.78 (qdd, J = 7.3, 7.3,
2.6 Hz, 2H), 1.05 (t, J = 7.3 Hz, 3H); MS (FAB) 248
(M+H)+.
4.3.1.11. N-[(S)-3-Amino-2-hydroxypropanoyl]-L-leu-
cine methyl ester (priAMeBS: 18a). To a solution of
NaN3 (29.0 mg, 0.45 mmol) and NH4Cl (6.8 mg,
0.44 mmol) in H2O (2 mL) was added a solution of 17
(47.8 mg, 0.22 mmol) in THF (4 mL), and the mixture
was stirred for 24 h at rt. The intermediate azide was ob-
tained by column chromatography (AcOEt/Hex = 1:1)
(34.0 mg, 59%). This intermediate (34.0 mg, 0.13 mmol)
was dissolved in MeOH (15 mL), then 10% Pd–C
(3.4 mg) was added, and the mixture was stirred under
H2 gas for 1 h. Pure 18a was obtained by filtration of
1
4.3.1.16. Benzyl 4-[(S)-4-benzyl-2-oxooxazolidin-3-yl]-
3-pentyl-4-oxobutanoate (21a). General procedure. To a
solution of 20a (3.5 g, 12.1 mmol) in THF (70 mL) was
added LiHMDS (1.0 M in THF) (14.6 mL, 14.6 mmol)
at ꢀ78 ꢁC, and the mixture was stirred for 20 min at
0 ꢁC. To this solution was added dropwise benzyl bro-
moacetate (3.59 g, 15.7 mmol) in THF (50 mL) over
90 min at ꢀ78 ꢁC. The reaction mixture was stirred for
30 min at 0 ꢁC and for 17 h at rt, then quenched with
sat. NH4Cl aq and satd NaHCO3 aq. Extraction with
AcOEt and concentration under reduced pressure affor-
ded the product. Pure 21a was obtained by means of col-
umn chromatography (AcOEt/Hex = 1:5) (4.35 g, 82%).
1H NMR (500 MHz, CDCl3) d: 7.27–7.13 (m, 10H), 5.02
(s, 2H), 4.54 (m, 1H), 4.19–4.02 (m, 2H), 3.16 (dd,
J = 13.3, 3.4 Hz, 1H), 2.89 (dd, J = 16.7, 10.7 Hz, 1H),
2.53 (dd, J = 16.7, 3.9 Hz, 1H), 2.40 (dd, J = 13.3,
9.8 Hz, 1H), 1.38 (m, 1H), 1.27–1.18 (m, 6H), 0.79 (t,
J = 6.9 Hz, 3H).
1
Pd–C (27.2 mg, 89%). H NMR (500 MHz, CDCl3) d:
7.49 (d, J = 8.1 Hz, 1H), 4.61 (m, 1H), 4.02 (m, 1H),
3.73 (s, 3H), 3.07 (dd, J = 12.8, 5.1 Hz, 1H), 2.98 (dd,
J = 12.8, 5.1 Hz, 1H), 1.70–1.56 (m, 3H), 0.94–0.92 (m,
6H); HRMS (FAB) calcd for C18H29N2O4 233.1501;
found: 233.1507 (M+H)+.
4.3.1.12. N-[(S)-2-Hydroxy-3-phenethylaminopropa-
noyl]-L-leucine methyl ester (PAMeBS: 18c). General pro-
cedure. To a solution of 17 (50.0 mg, 0.23 mmol) in
CH2Cl2 (50 mL) was added dropwise 2-phenylethylamine
(280 mg, 2.31 mmol) over 30 min. The reaction mixture
was stirred for 1 h at rt, quenched with water, and ex-
tracted with AcOEt. The extract was concentrated under
reduced pressure. Pure 18c was obtained by means of col-
umn chromatography (CH2Cl2/MeOH = 20:1, contain-
ing 1% NEt3) (7.0 mg, 36%). 1H NMR (500 MHz,
CDCl3) d: 7.73 (d, J = 8.1 Hz, 1H), 7.32–7.18 (m, 5H),
4.59 (m, 1H), 3.98 (dd, J = 6.8, 6.0 Hz, 1H), 3.73 (s,
3H), 3.00 (dd, J = 12.4, 6.0 Hz, 1H), 2.99–2.88 (m, 3H),
2.81–2.77 (m, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.92 (d,
J = 6.4 Hz, 3H); HRMS (FAB) calcd for C18H29N2O4
337.2127; found: 337.2111 (M+H)+.
4.3.1.17. Benzyl 4-[(S)-4-benzyl-2-oxooxazolidin-3-yl]-
3-benzyl-4-oxobutanoate (21c). This compound was pre-
pared from 19c according to the general procedure (two
1
steps, 44%). H NMR (500 MHz, CDCl3) d: 7.37–7.20
(m, 15H), 5.06 (s, 2H), 4.57 (m, 1H), 4.48 (m, 1H),
4.03 (dd, J = 8.6, 2.1 Hz, 1H), 3.91 (dd, J = 8.6,
8.1 Hz, 1H), 3.19 (dd, J = 13.3, 3.0 Hz, 1H), 3.05–2.97
(m, 2H), 2.65 (dd, J = 13.3, 9.4 Hz, 1H), 2.53–2.44 (m,
2H); MS (FAB) 458 (M+H)+.
4.3.1.13. N-[(S)-3-Benzylamino-2-hydroxypropanoyl]-
L-leucine methyl ester (BAMeBS: 18b). This compound
was prepared from 17 according to the general procedure
(9%). 1H NMR (500 MHz, CDCl3) d: 7.75 (d, J = 8.6 Hz,
1H), 7.35–7.28 (m, 5H), 4.60 (m, 1H), 4.08 (dd, J = 6.0,
6.4 Hz, 1H), 3.83 (s, 2H), 3.73 (s, 3H), 3.05–2.96 (m,
2H), 1.67–1.58 (m, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.91
(d, J = 6.4 Hz, 3H); HRMS (FAB) calcd for
C17H27N2O4 323.1971; found: 323.2006 (M+H)+.
4.3.1.18. (R)-3-Benzyloxycarbonyl-2-pentylpropanoic
acid (22a). General procedure. To a solution of 21a
(4.35 g, 9.95 mmol) in THF/H2O (72 mL:18 mL)
were added 30% H2O2 aq (6.0 mL) and a solution of
LiOHÆH2O (600 mg) in H2O (24 mL). The mixture was
stirred for 2 h at 0 ꢁC and 2 h at rt, then acidified with
2 N HCl. Extraction with AcOEt and concentration un-
der reduced pressure afforded the product. Pure 22a was
obtained by means of column chromatography (AcOEt/
Hex = 1:5) (1.8 g, 65%). 1H NMR (500 MHz, CDCl3) d:
7.38–7.30 (m, 5H), 5.13 (d, J = 4.3 Hz, 2H), 2.90 (m,
1H), 2.77 (dd, J = 16.7, 9.4 Hz, 1H), 2.51 (dd, J = 16.7,
5.1 Hz, 1H), 1.68 (m, 1H), 1.55 (m, 1H), 1.35–1.26 (m,
6H), 0.87 (t, J = 6.8 Hz, 3H).
4.3.1.14. (S)-4-Benzyl-3-heptanoyloxazolidin-2-one
(20a). General procedure.53 (S)-Benzyl-2-oxazolinone
(10 g, 56.5 mmol) was dissolved in THF (70 mL) under
an argon atmosphere, n-BuLi (1.6 M in hexane)
(35.3 mL, 56.5 mmol) was added, and the mixture was
stirred for 1 h at ꢀ78 ꢁC. To the mixture was added 19a
(8.0 mL, 51.7 mmol), and the whole was stirred for 2 h

4694
S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4.3.1.19. (R)-3-Benzyloxycarbonyl-2-benzylpropanoic
0.5 N HCl. The organic layer was collected, dried over
MgSO4, and concentrated under reduced pressure. Pure
24a was obtained by means of column chromatography
acid (22c). This compound was prepared from 21c accord-
ing to the general procedure (89%). 1H NMR (500 MHz,
CDCl3) d: 7.36–7.15 (m, 10H), 5.09 (s, 2H), 3.22–3.12 (m,
2H), 2.79 (dd, J = 11.3, 8.6 Hz, 1H), 2.70 (dd, J = 17.1,
9.0 Hz, 1H), 2.46 (dd, J = 17.1, 4.7 Hz, 1H); MS (FAB)
299 (M+H)+.
1
(AcOEt/Hex = 1:3 to 1:1) (245 mg, two steps, 70%). H
NMR (500 MHz, CDCl3) d: 7.39–7.35 (m, 5H), 6.07 (s,
1H), 4.87 (s, 2H), 4.58 (m, 1H), 3.72 (s, 3H), 2.69 (m,
1H), 2.37 (m, 1H), 2.21 (m, 1H), 1.66–1.55 (m, 4H), 1.42
(m, 1H), 1.32–1.26 (m, 6H), 0.94 (d, J = 6.0 Hz, 3H),
0.93 (d, J = 6.0 Hz, 3H), 0.87 (t, J = 6.8 Hz, 3H).
4.3.1.20. N-[(R)-3-Benzyloxycarbonyl-2-pentylpropa-
noyl]-L-leucine methyl ester (23a). General procedure.
To a solution of compound 22a (273 mg, 0.98 mmol)
in dry CH2Cl2 (20 mL) were added L-leucine methyl
ester hydrochloride (214 mg, 1.18 mmol), HOBt
(147 mg, 1.09 mmol), EDCI (245 mg, 1.28 mmol),
and DIPEA (186 lL, 1.09 mmol) under an argon
atmosphere. The reaction mixture was stirred at rt
for 2 h and quenched with 0.5 N HCl. The organic
layer was collected, dried over MgSO4, and concen-
trated under reduced pressure. Pure 23a was obtained
by means of column chromatography (AcOEt/
Hex = 1:3) (339 mg, 85%). 1H NMR (500 MHz,
CDCl3) d: 7.45–7.30 (m, 5H), 6.08 (d, J = 8.1 Hz,
1H), 5.12 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.4 Hz,
1H), 4.62 (m, 1H), 3.74 (s, 3H), 2.79 (dd, J = 16.7,
9.4 Hz, 1H), 2.64 (m, 1H), 2.45 (dd, J = 16.7, 5.1 Hz,
1H), 1.66–1.51 (m, 3H), 1.38–1.36 (m, 2H), 1.33–1.26
(m, 6H), 0.97–0.90 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H);
MS (FAB) 406 (M+H)+.
4.3.1.24. N-[(R)-3-O-Benzyloxycarbamoyl-2-ethylprop-
anoyl]-L-leucine methyl ester (24b). This compound was
prepared from 23b according to the general procedure
1
(two steps 13%). H NMR (500 MHz, CDCl3) d: 7.47–
7.35(m, 5H), 6.58 (d, J = 8.1 Hz, 1H), 4.88 (s, 2H), 4.59
(m, 1H), 3.72 (s, 3H), 2.63 (m, 1H), 2.38 (m, 1H), 2.20
(m, 1H), 1.67–1.49 (m, 5H), 0.96–0.91 (m, 9H); MS
(FAB) 379 (M+H)+.
4.3.1.25. N-[(R)-3-O-Benzyloxycarbamoyl-2-benzyl-
propanoyl]-L-leucine methyl ester (24c). This compound
was prepared from 23c according to the general proce-
1
dure (two steps 82%). H NMR (DMSO-d6) d: 10.98
(s, 1H), 8.30 (d, J = 7.7 Hz, 1H), 7.36–7.16 (m, 10H),
4.70 (s, 2H), 4.25 (m, 1H), 3.56 (s, 3H), 3.03 (dd,
J = 7.3, 6.8 Hz, 1H), 2.96 (dd, J = 13.7, 7.3 Hz, 1H),
2.51 (m, 1H), 2.20 (dd, J = 15.0, 8.1 Hz, 1H), 1.90 (dd,
J = 15.0, 6.0 Hz, 1H), 1.62–1.50 (m, 2H), 1.45 (m, 1H),
0.85 (d, J = 6.4 Hz, 3H), 0.79 (d, J = 6.4 Hz, 3H); MS
(FAB) 441 (M+H)+.
4.3.1.21. N-[(R)-3-Benzyloxycarbonyl-2-ethylpropa-
noyl]-L-leucine methyl ester (23b). This compound was
prepared from 20b according to the general procedure
(three steps, 32%). 1H NMR (500 MHz, CDCl3) d: 7.38–
7.30 (m, 5H), 6.00 (d, J = 8.1 Hz, 1H), 5.11 (d,
J = 12.0 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 4.62 (m,
1H), 3.72 (s, 3H), 2.80 (dd, J = 16.7, 9.4 Hz, 1H), 2.58
(m, 1H), 2.46 (dd, J = 16.7, 4.3 Hz, 1H), 1.72–1.58 (m,
3H), 1.55–1.46 (m, 2H), 0.96–0.90 (m, 9H); MS (FAB)
364 (M+H)+.
4.3.1.26. N-[(R)-3-Hydroxycarbamoyl-2-pentylpropa-
noyl]-L-leucine methyl ester (HAB-5RL: 25a). General
procedure. To a solution of 24a (210 mg, 0.84 mmol) in
MeOH (20 mL) was added 10% Pd–C (20 mg), and the
mixture was stirred under H2 gas for 40 min. Pure 25a
was obtained by removal of Pd–C by filtration
(160 mg, 97%). 1H NMR (500 MHz, DMSO-d6) d:
10.36 (s, 1H), 8.67 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H),
4.23 (m, 1H), 3.32 (s, 3H), 2.62 (m, 1H), 2.09 (m, 1H),
1.98(m, 1H), 1.67–1.51 (m, 2H), 1.48–1.36 (m, 2H),
1.27–1.12 (m, 7H), 0.88–0.79 (m, 9H); HRMS (FAB)
calcd for C16H30N2NaO5 353.2052; found: 353.2040
(M+Na)+.
4.3.1.22. N-[(R)-3-Benzyloxycarbonyl-2-benzylpropa-
noyl]-L-leucine methyl ester (23c). This compound was
prepared from 22c according to the general procedure
1
(57%). H NMR (500 MHz, CDCl3) d: 7.36–7.16 (m,
10H), 5.88 (d, J = 8.6 Hz, 1H), 5.07 (s, 2H), 4.55 (m,
1H), 3.67 (s, 3H), 2.99 (dd, J = 13.3, 7.5 Hz, 1H), 2.91
(m, 1H), 2.82 (dd, J = 17.1, 9.4 Hz, 1H), 2.72 (dd,
J = 13.3, 7.5 Hz, 1H), 2.46 (dd, J = 17.1, 4.0 Hz, 1H),
1.59–1.52 (m, 2H), 1.45 (m, 1H), 0.89–0.88 (m, 6H);
MS (FAB) 426 (M+H)+.
4.3.1.27. N-[(R)-3-Hydroxycarbamoyl-2-ethylpropa-
noyl]-L-leucine methyl ester (HAB-2L: 25b). This com-
pound was prepared from 24b according to the general
procedure (87%). 1H NMR (500 MHz, DMSO-d6) d:
10.35 (s, 1H), 8.66 (s, 1H), 8.21 (d, J = 8.1 Hz, 1H),
4.23 (m, 1H), 3.59 (s, 1H), 2.59 (m, 1H), 2.12 (dd,
J = 14.5, 6.4 Hz, 1H), 1.98 (dd, J = 14.5, 7.7 Hz, 1H),
1.65–1.35 (m, 5H), 0.87 (d, J = 6.4 Hz, 3H), 0.87 (d,
J = 6.4 Hz, 3H), 0.83–0.81 (m, 6H); HRMS (FAB) calcd
for C13H25N2O5 289.1763; found: 289.1768 (M+H)+.
4.3.1.23. N-[(R)-3-O-Benzyloxycarbamoyl-2-pentyl-
propanoyl]-L-leucine methyl ester (24a). General proce-
dure. To a solution of 23a (339 mg, 0.84 mmol) in
AcOEt (25 mL) was added 10% Pd–C (30 mg), and the
mixture was stirred under H2 gas for 1 h. The intermediate
was obtained by removal of the Pd–C by filtration, and
dissolved in CH2Cl2 (20 mL). To a solution of the inter-
mediate were added O-benzylhydroxylamine hydrochlo-
ride (152 mg, 0.95 mmol), HOBt (147 mg, 1.09 mmol),
EDCI (183 mg, 0.95 mmol), and DIPEA (113 lL,
0.87 mmol) under an argon atmosphere. The reaction
mixture was stirred for 2 h at rt and quenched with
4.3.1.28. N-[(R)-3-Hydroxycarbamoyl-2-benzylpropa-
nyl]-L-leucine methyl ester (HAB-BL: 25c). This com-
pound was prepared from 24c according to the general
procedure (97%). 1H NMR (500 MHz, DMSO-d6) d:
10.33 (s, 1H), 8.93 (s, 1H), 8.26 (d, J = 7.7 Hz, 1H),
7.35–7.15 (m, 5H), 4.21 (m, 1H), 3.54 (s, 3H), 3.00
(dd, J = 7.3, 6.8 Hz, 1H), 2.86 (dd, J = 13.7, 7.3 Hz,

S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4695
1H), 2.52 (m, 1H), 2.19 (dd, J = 15.0, 7.7 Hz, 1H), 1.90
(dd, J = 15.0, 6.8 Hz, 1H), 1.59–1.49 (m, 2H), 1.44 (m,
1H), 0.86 (d, J = 6.4 Hz, 3H), 0.80 (d, J = 6.4 Hz, 3H);
HRMS (FAB) calcd for C18H27N2O5 351.1920; found:
351.1870 (M+H)+.
1H), 1.64 (m, 1H), 1.40 (m, 1H), 1.32 (d, J = 7.3 Hz,
3H), 1.33–1.25 (m, 6H), 0.86 (t, J = 6.8 Hz, 3H); MS
(FAB) 364 (M+H)+.
4.3.1.33. N-[(R)-3-Benzyloxycarbonyl-2-pentylpropa-
noyl]-L-valine methyl ester (28c). This compound was
prepared from 22a according to the general procedure
4.3.1.29. N-[N-Benzyloxysuccinamyl]-L-leucine methyl
ester (27). To a solution of 26 (1.0 g, 10.0 mmol) in
CH2Cl2 (200 mL) were added L-leucine methyl ester
hydrochloride (1.50 g, 8.26 mmol), DMAP (1.02 g,
8.36 mmol), and NEt3 (1.15 mL). The mixture was stirred
for 17 h at rt, then the reaction was quenched with 10%
NaHCO3 aq. The aqueous layer was acidified with 2 N
HCl and extracted with AcOEt. Concentration under re-
duced pressure afforded the pure intermediate (1.68 g,
83%). To a solution of intermediate (320 mg, 1.31 mmol)
were added O-benzylhydroxylamine hydrochloride
(250 mg, 1.57 mmol), EDCI (300 mg, 1.47 mmol), and
DIPEA (222 lL, 1.31 mmol) under an argon atmosphere.
The reaction mixture was stirred for 1 h at rt and
quenched with 0.5 N HCl. The organic layer was col-
lected, dried over MgSO4, and concentrated under re-
duced pressure. Pure 27 was obtained by means of
column chromatography (CH2Cl2/MeOH = 15:1) and
recrystallization (AcOEt/hexane) at ꢀ78 ꢁC (281 mg,
1
(92%). H NMR (500 MHz, CDCl3) d: 7.36–7.30 (m,
5H), 6.12 (d, J = 8.6 Hz, 1H), 5.10 (s, 2H), 4.55 (m,
1H), 3.73 (s, 3H), 2.80 (dd, J = 16.7, 9.4 Hz, 1H), 2.64
(m, 1H), 2.46 (dd, J = 16.7, 3.4 Hz, 1H), 2.14 (m, 1H),
1.65 (m, 1H), 1.40 (m, 1H), 1.31–1.24 (m, 6H), 0.92–
0.84 (m, 9H); MS (FAB) 392 (M+H)+.
4.3.1.34. N-[(R)-3-Benzyloxycarbonyl-2-pentylpropa-
noyl]-L-tert-leucine methyl ester (28d). This compound
was prepared from 22a according to the general proce-
1
dure (100%). H NMR (500 MHz, CDCl3) d: 7.37–7.31
(m, 5H), 6.19 (d, J = 16.7, 9.4 Hz, 1H), 5.10 (s, 2H),
4.45 (d, J = 9.4 Hz, 1H), 3.71(s, 3H), 2.80 (dd,
J = 16.7, 9.4 Hz, 1H), 2.64 (m, 1H), 2.46 (dd, J = 16.7,
3.9 Hz, 1H), 1.64 (m, 1H), 1.39 (m, 1H), 1.29–1.22 (m,
6H), 0.95 (s, 9H), 0.86(t, J = 6.8 Hz, 3H); MS (FAB)
406 (M+H)+.
1
67%). H NMR (500 MHz, DMSO-d6) d: 11.0 (s, 1H),
4.3.1.35. N-[(R)-3-Benzyloxycarbonyl-2-pentylpropa-
nyl]-L-phenylalanine methyl ester (28e). This compound
was prepared from 22a according to the general proce-
8.22 (d, J = 7.7 Hz, 1H), 7.38–7.35 (m, 5H), 4.75 (s, 2H),
4.26 (m, 1H), 3.62 (s, 3H), 2.44–2.33 (m, 3H), 2.18 (m,
1H), 1.61(m, 1H), 1.51 (m, 1H), 1.46 (m, 1H), 0.88 (d,
J = 6.4 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H).
1
dure (100%). H NMR (500 MHz, CDCl3) d: 7.37–7.13
(m, 10H), 6.09 (d, J = 7.7 Hz, 1H), 5.11 (d,
J = 12.4 Hz, 1H), 5.06 (d, J = 12.4 Hz, 1H), 4.89 (dt,
J = 7.7, 6.0 Hz, 1H), 3.70 (s, 3H), 3.09 (dd, J = 14.1,
6.0 Hz, 1H), 3.05 (dd, J = 14.1, 6.0 Hz, 1H), 2.43 (dd,
J = 16.7, 9.0 Hz, 1H), 2.58 (m, 1H), 2.43 (dd, J = 16.7,
4.7 Hz, 1H), 1.59 (m, 1H), 1.38 (m, 1H), 1.27–1.22 (m,
6H), 0.84 (t, J = 6.8 Hz, 3H); MS (FAB) 440 (M+H)+.
4.3.1.30. N-[N-Hydroxysuccinamiyl]-L-leucine methyl
ester (HAB-0L: 25d). To a solution of 27 (81.0 mg,
0.23 mmol) in MeOH (14 mL) was added 10% Pd–C
(7.0 mg), and the mixture was stirred under H2 gas for
40 min. Pure 25d was obtained by the removal of Pd–
C by filtration (59.0 mg, 98%). 1H NMR (500 MHz,
DMSO-d6) d: 10.36 (s, 1H), 8.68 (s, 1H), 8.24 (d,
J = 6.8 Hz, 1H), 4.25 (m, 1H), 3.60 (s, 3H), 2.38–2.31
(m, 3H), 1.63 (m, 1H), 1.60 (m, 1H), 1.54–1.42 (m,
2H), 0.87 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H);
HRMS (FAB) calcd for C11H21N2O5 261.1450; found:
261.1458 (M+H)+.
4.3.1.36. N-[(R)-3-O-Benzyloxycarbamoyl-2-pentyl-
propanoyl]glycine methyl ester (29a). This compound
was prepared from 28a according to the general proce-
1
dure (two steps 74%). H NMR (500 MHz, DMSO-d6)
d: 11.00 (s, 1H), 8.36 (t, J = 6.0 Hz, 1H), 7.37–7.34 (m,
5H), 4.75 (s, 2H), 3.83 (dd, J = 17.1, 6.0 Hz, 1H), 3.73
(dd, J = 17.1, 6.0 Hz, 1H), 3.59 (s, 3H), 2.66 (m, 1H),
2.15 (dd, J = 14.5, 6.8 Hz, 1H), 1.98 (dd, J = 14.5,
8.1 Hz, 1H), 1.38 (m, 1H), 1.26–1.16 (m, 7H), 0.84 (t,
J = 6.8 Hz, 3H); MS (FAB) 365 (M+H)+, 242
(MꢀBnONH)+.
4.3.1.31. N-[(R)-3-Benzyloxycarbonyl-2-pentylpropa-
noyl]glycine methyl ester (28a). This compound was pre-
pared from 22a according to the general procedure
1
(76%). H NMR (500 MHz, CDCl3) d: 7.37–7.31 (m,
5H), 6.13 (m, 1H), 5.13 (d, J = 12.4 Hz, 1H), 5.08 (d,
J = 12.4 Hz, 1H), 4.07 (dd, J = 18.4, 5.6 Hz, 1H), 3.92
(dd, J = 18.4, 4.7 Hz, 1H), 3.74 (s, 3H), 2.79 (dd,
J = 16.7, 9.4 Hz, 1H), 2.64 (m, 1H), 2.47 (dd, J = 16.7,
4.3 Hz, 1H), 1.66 (m, 1H), 1.41 (m, 1H), 1.32–1.23 (m,
6H), 0.86 (t, J = 6.8 Hz, 3H); MS (FAB) 350 (M+H)+.
4.3.1.37. N-[(R)-3-O-Benzyloxycarbamoyl-2-pentyl-
propanoyl]-L-alanine methyl ester (29b). This compound
was prepared from 28b according to the general proce-
1
dure (two steps 68%). H NMR (500 MHz, DMSO-d6)
d: 10.99 (s, 1H), 8.31 (d, J = 6.8 Hz, 1H), 7.37–7.33 (m,
5H), 4.74 (s, 2H), 4.21 (qd, J = 7.3, 6.8 Hz, 1H), 3.58 (s,
3H), 2.64 (m, 1H), 2.14 (dd, J = 14.5, 7.3 Hz, 1H), 1.98
(dd, J = 14.5, 7.3 Hz, 1H), 1.38 (m, 1H), 1.28–1.16 (m,
6H), 1.24 (d, J = 7.3 Hz, 3H), 0.85 (t, J = 6.8 Hz, 3H);
MS (FAB) 379 (M+H)+, 256 (MꢀBnONH)+.
4.3.1.32. N-[(R)-3-Benzyloxycarbonyl-2-pentylpropa-
noyl]-L-alanine methyl ester (28b). This compound was
prepared from 22a according to the general procedure
1
(96%). H NMR (500 MHz, CDCl3) d: 7.37–7.31 (m,
5H), 6.11 (d, J = 7.3 Hz, 1H), 5.12 (d, J = 12.4 Hz,
1H), 5.08 (d, J = 12.4 Hz, 1H), 4.57 (quant,
J = 7.3 Hz, 1H), 3.74 (s, 3H), 2.79 (dd, J = 16.7,
9.4 Hz, 1H), 2.59 (m, 1H), 2.45 (dd, J = 1.67, 4.7 Hz,
4.3.1.38. N-[(R)-3-O-Benzyloxycarbamoyl-2-pentyl-
propanoyl]-L-valine methyl ester (29c). This compound
was prepared from 28c according to the general proce-

4696
S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
1
dure (two steps, 78%). H NMR (500 MHz, CDCl3) d:
7.37–7.36 (m, 5H), 6.24 (s, 1H), 4.88 (s, 2H), 4.51 (m,
1H), 3.73 (s, 3H), 2.75 (m, 1H), 2.40 (m, 1H), 2.23–
2.16 (m, 2H), 1.60 (m, 1H), 1.41(m, 1H), 1.29–1.24 (m,
6H), 0.93 (dd, J = 12.4, 6.8 Hz, 6H), 0.87 (t,
J = 6.8 Hz, 3H); MS (FAB) 407 (M+H)+, 284
(MꢀBnONH)+.
4.3.1.44. N-[(R)-3-Hydroxycarbamoyl-2-pentylpropa-
noyl]-L-tert-leucine methyl ester (HAB-5TL: 30d). This
compound was prepared from 29d according to the
general procedure (97%). 1H NMR (500 MHz,
DMSO-d6) d: 10.35 (s, 1H), 8.67 (s, 1H), 8.03 (d,
J = 8.1 Hz, 1H), 4.10 (d, J = 8.1 Hz, 1H), 3.58 (s,
3H), 2.83 (m, 1H), 2.12 (dd, J = 14.5, 6.8 Hz, 1H),
1.99 (dd, J = 14.5, 7.7 Hz, 1H), 1.36 (m, 1H), 1.29–
1.12 (m, 7H), 0.93 (s, 9H), 0.83 (t, J = 6.8 Hz, 3H);
HRMS (FAB) calcd for C16H31N2O5 331.2233; found:
331.2247 (M+H)+.
4.3.1.39. N-[(R)-3-O-Benzyloxycarbamoyl-2-pentyl-
propanoyl]-L-tert-leucine methyl ester (29d). This com-
pound was prepared from 28d according to the general
procedure (two steps, 78%). 1H NMR (500 MHz, CDCl3)
d: 7.38–7.36 (m, 5H), 6.32 (s, 1H), 4.87 (s, 2H), 4.41 (d,
J = 7.7 Hz, 1H), 3.72 (s, 3H), 2.75 (m, 1H), 2.39 (m,
1H), 2.22 (m, 1H), 1.59 (m, 1H), 1.40 (m, 1H), 1.29–1.23
(m, 6H), 0.98 (s, 9H), 0.87 (t, J = 6.8 Hz, 3H); MS
(FAB) 421 (M+H)+, 298 (MꢀBnONH)+.
4.3.1.45. N-[(R)-3-Hydroxycarbamoyl-2-pentylpropa-
noyl]-L-phenylalanine methyl ester (HAB-5F: 30e). This
compound was prepared from 29e according to the gen-
1
eral procedure (93%). H NMR (500 MHz, DMSO-d6)
d: 10.35 (s, 1H), 8.68 (s, 1H), 8.42 (d, J = 7.3 Hz, 1H),
7.27–7.17 (m, 5H), 4.43 (ddd, J = 9.0, 7.3, 6.0 Hz, 1H),
3.54 (s, 3H), 3.00 (dd, J = 13.7, 5.6 Hz, 1H), 2.90 (dd,
J = 13.7, 9.4 Hz, 1H), 2.62 (m, 1H), 2.48 (m, 1H), 1.91
(m, 1H), 1.33 (m, 1H), 1.22–1.13 (m, 7H), 0.83 (t,
J = 6.8 Hz, 3H); HRMS (FAB) calcd for C19H29N2O5
365.2076; found: 365.2044 (M+H)+.
4.3.1.40. N-[(R)-3-O-Benzyloxycarbamoyl-2-pentyl-
propanoyl]-L-phenylalanine methyl ester (29e). This com-
pound was prepared from 28e according to the general
procedure (two steps, 60%). 1H NMR (500 MHz,
CDCl3) d: 7.38–7.13 (m, 10H), 6.17 (s, 1H), 4.87 (s,
2H), 4.83 (m, 1H), 3.70 (s, 3H), 3.09 (d, J = 6.0 Hz,
2H), 2.64 (m, 1H), 2.32 (m, 1H), 2.15 (m, 1H), 1.56
(m, 1H), 1.38 (m, 1H), 1.26–1.21 (m, 6H), 0.85 (t,
J = 6.8 Hz, 3H); MS (FAB) 455 (M+H)+, 332
(MꢀBnONH)+.
4.3.1.46. (R)-2-tert-Butoxycarbonylaminosuccinic acid
4-benzyl ester (32). To a solution of 15 (6.66 g, 50 mmol)
in benzene (250 mL) were added p-toluenesulfonic acid
monohydrate (10.5 g, 55 mmol) and benzyl alcohol
(35 mL, 339 mmol). The mixture was stirred at reflux
with continuous removal of generated H2O for 3.5 h.
Benzene was removed under reduced pressure and the
residue was dissolved in acetone:H2O (62 mL:47 mL).
To this solution was added 20% NaOH aq (15 mL),
and the mixture was stirred at 13 ꢁC for 30 min. Then
NEt3 (10 mL) and Boc2O (16 g, 73 mmol) were added,
and the whole was stirred for 2 h at rt. Acetone was re-
moved under reduced pressure, and the aqueous layer
was acidified with 2 N HCl. Pure 32 was obtained by
extraction with AcOEt and concentrated under reduced
pressure (7.73 g, three steps, 48%). 1H NMR (500 MHz,
CDCl3) d: 7.37–7.26 (m, 5H), 5.55 (d, J = 8.1 Hz, 1H),
5.16 (d, J = 12.4 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H),
4.63 (m, 1H), 3.07 (dd, J = 17.1, 3.8 Hz, 1H), 2.89 (dd,
J = 17.1, 5.1 Hz, 1H), 1.45 (s, 9H).
4.3.1.41. N-[(R)-3-Hydroxycarbamoyl-2-pentylpropa-
noyl]glycine methyl ester (HAB-5G: 30a). This com-
pound was prepared from 29a according to the general
1
procedure (100%). H NMR (500 MHz, DMSO-d6) d:
10.37 (s, 1H), 8.69 (s, 1H), 8.41 (m, 1H), 3.84 (dd,
J = 17.1, 6.4 Hz, 1H), 3.71 (dd, J = 17.1, 5.6 Hz, 1H),
3.60 (s, 3H), 2.64 (m, 1H), 2.14 (dd, J = 14.5, 6.4 Hz,
1H), 1.98 (dd, J = 14.5, 8.1 Hz, 1H), 1.40 (m, 1H),
1.27–1.16 (m, 7H), 0.84 (t, J = 6.8 Hz, 3H); HRMS
(FAB) calcd for C12H23N2O5 275.1607; found:
275.1588 (M+H)+.
4.3.1.42. N-[(R)-3-Hydroxycarbamoyl-2-pentylpropa-
noyl]-L-alanine methyl ester (HAB-5A: 30b). This com-
pound was prepared from 29b according to the general
procedure (99%). 1H NMR (500 MHz, DMSO-d6) d:
10.35 (s, 1H), 8.70 (s, 1H), 8.29 (d, J = 6.7 Hz, 1H),
4.20 (td, J = 7.3, 6.7 Hz, 1H), 3.58 (s, 3H), 2.63 (m,
1H), 2.12 (dd, J = 14.6, 6.7 Hz, 1H), 1.96 (dd, J = 14.6,
7.9 Hz, 1H), 1.39–1.21 (m, 8H), 1.24 (d, J = 7.3 Hz,
3H), 0.84 (t, J = 6.7 Hz, 3H); HRMS (FAB) calcd for
C13H25N2O5 289.1763; found: 289.1758 (M+H)+.
4.3.1.47. N-[(R)-3-Benzyloxycarbonyl-2-tert-butoxy-
carbonylaminopropanoyl]-L-leucine methyl ester (33). To
a solution of compound 32 (6.60 g, 20.4 mmol) in dry
CH2Cl2 (100 mL) were added L-leucine methyl ester
hydrochloride (4.08 g, 22.5 mmol), HOBt (3.04 g,
22.5 mmol), and EDCI (4.70 g, 34.5 mmol), under an ar-
gon atmosphere. The reaction mixture was stirred at rt
for 2 h and quenched with 0.5 N HCl. The organic layer
was collected, dried over MgSO4, and concentrated un-
der reduced pressure. The intermediate was obtained by
means of column chromatography (AcOEt/Hex = 1:4 to
1:1) (4.76 g, 52%). 1H NMR (500 MHz, CDCl3) d: 7.36–
7.32 (m, 5H), 6.86 (d, J = 6.8 Hz, 1H), 5.67 (d,
J = 7.3 Hz, 1H), 5.16 (d, J = 12.0 Hz, 1H), 5.11 (d,
J = 12.0 Hz, 1H), 4.59–4.55 (m, 2H), 3.71 (s, 3H), 3.01
(dd, J = 17.1, 3.8 Hz, 1H), 2.74 (dd, J = 17.1, 6.0 Hz,
1H), 1.66–1.52 (m, 3H), 1.46 (s, 9H), 0.91 (d,
J = 6.0Hz, 6H); MS (FAB) 451 (M+H)+.
4.3.1.43. N-[(R)-3-Hydroxycarbamoyl-2-pentylpropa-
noyl]-L-valine methyl ester (HAB-5V: 30c). This com-
pound was prepared from 29c according to the general
1
procedure (100%). H NMR (500 MHz, DMSO-d6) d:
10.34 (s, 1H), 8.67 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H),
4.09 (dd, J = 7.7, 6.4 Hz, 1H), 3.58 (s, 3H), 2.75 (m,
1H), 2.13 (dd, J = 14.5, 6.8 Hz, 1H), 2.03–1.96 (m,
2H), 1.37 (m, 1H), 1.29–1.14 (m, 7H), 0.88 (d,
J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H), 0.84 (t,
J = 6.8 Hz, 3H); HRMS (FAB) calcd for C15H29N2O5
317.2076; found: 317.2073 (M+H)+.

S. Sato et al. / Bioorg. Med. Chem. 16 (2008) 4685–4698
4697
4.3.1.48. N-[(R)-3-O-Benzyloxycarbamoyl-2-tert-but-
oxycarbonylaminopropanoyl]-L-leucine methyl ester
4.3.1.52. N-[(R)-3-O-Benzyloxycarbamoyl-2-dibutyla-
minopropanoyl]-L-leucine methyl ester (38b). Compound
35b (100 mg, 0.215 mmol) was dissolved in TFA/
CH2Cl2 (0.5 mL:5 mL) and the solution was stirred
for 1 h at rt. The TFA was removed under reduced
pressure, and the residue was dissolved in THF
(5 mL). To this solution were added n-butylaldehyde
(35b). General procedure. To a solution of 33 (1.2 g,
2.66 mmol) in AcOEt (50 mL) was added 10% Pd–C
(120 mg). The mixture was stirred under H2 gas for
1 h. The intermediate was obtained by the removal of
Pd–C by filtration, and dissolved in CH2Cl2 (30 mL).
To this solution were added O-benzylhydroxylamine
hydrochloride (467 mg, 2.93 mmol), HOBt (395 mg,
2.92 mmol), EDCI (612 mg, 3.19 mmol), and DIPEA
(226 lL, 1.33 mmol) under an argon atmosphere. The
reaction mixture was stirred for 2 h at rt and quenched
with 0.5 N HCl. The organic layer was collected, dried
over MgSO4, and concentrated under reduced pressure.
Pure 35b was obtained by column chromatography
(48.5 lL,
0.538 mmol),
NaBH(OAc)3
(137 mg,
0.646 mmol), and AcOH (26 lL), and the mixture
was stirred for 30 min at rt. Pure 38b was obtained
by means of column chromatography (AcOEt/
Hex = 1:1 to AcOEt/Net3 = 50:1) (49 mg, two steps
48%). 1H NMR (500 MHz, DMSO-d6) d: 11.0 (s,
1H), 7.88 (d, J = 8.1 Hz, 1H), 7.39–7.33 (m, 5H),
4.75 (s, 2H), 4.31 (td, J = 8.6, 4.7 Hz, 1H), 3.79 (dd,
J = 7.7, 5.6 Hz, 1H), 3.60 (s, 3H), 2.35 (t, J = 6.8 Hz,
4H), 2.30 (dd, J = 14.5, 7.7 Hz, 1H), 2.15 (dd,
J = 14.5, 5.6 Hz, 1H), 1.59–1.50 (m, 3H), 1.41–1.36
(m, 4H), 1.29–1.23 (m, 4H), 0.88–0.83 (m, 12H); MS
(FAB) 478 (M+H)+.
1
(AcOEt/Hex = 2:3 to 3:2) (758 mg, two steps, 61%). H
NMR (500 MHz, CDCl3) d: 7.41–7.35 (m, 5H), 4.89
(s, 2H), 4.52 (m, 1H), 4.45 (m, 1H), 3.71 (s, 3H), 2.67
(m, 1H), 2.46 (m, 1H), 1.67–1.56 (m, 3H), 1.49 (s, 9H),
0.94–0.90 (m, 6H).
4.3.1.53. N-[(R)-3-Hydroxycarbamoyl-2-butylamino-
propanoyl]-L-leucine methyl ester (HAB-A4L: 36b). Gen-
4.3.1.49. N-[(R)-3-O-tert-Butyldimethylsilyloxycarba-
moyl-2-tert-butoxycarbonylaminopropanoyl]-L-leucine
methyl ester (35a). This compound was prepared from
33 according to the general procedure (two steps 29%).
1H NMR (500 MHz, CDCl3) d: 4.51 (m, 1H), 4.43 (m,
1H), 3.72 (s, 3H), 2.72 (m, 1H), 2.47 (m, 1H), 1.68–
1.55 (m, 3H), 1.45 (s, 9H), 0.95 (s, 9H), 0.17 (s, 6H);
MS (FAB) 490 (M+H)+.
eral procedure. To
a solution of 38a (23.7 mg,
0.056 mmol) in MeOH (4 mL) was added 10% Pd–C
(2.4 mg) and the mixture was stirred under H2 gas for
40 min. Pure 36b was obtained by the removal of Pd–C
by filtration (18.6 mg, 100%). 1H NMR (500 MHz,
DMSO-d6) d: 10.63 (s, 1H), 8.91 (m, 2H), 4.26 (m, 1H),
3.63 (m, 1H), 3.62 (s, 3H), 2.86–2.67 (m, 2H), 2.52–2.42
(m, 2H), 1.65–1.47 (m, 5H), 1.32–1.27 (m, 2H), 0.89 (d,
J = 6.4 Hz, 3H), 0.87 (t, J = 7.7 Hz, 3H), 0.83 (d,
J = 6.4 Hz, 3H); HRMS (FAB) calcd for C15H30N3O5
332.2185; found: 332.2207 (M+H)+.
4.3.1.50. N-[(R)-3-Hydroxycarbamoyl-2-aminopropa-
noyl]-L-leucine methyl ester (HAB-A0L: 36a). 35a
(50 mg, 0.102 mmol) was dissolved in TFA/CH2Cl2
(2 mL:2 mL) and the solution was stirred for 5 h at
0 ꢁC. The TFA was removed under reduced pressure,
and pure 36a was obtained by recrystallization
(AcOEt/Hex) (38.7 mg, 100%). 1H NMR (500 MHz,
DMSO-d6) d: 10.63 (s, 1H), 8.91 (m, 2H), 4.26 (m,
1H), 3.63 (m, 1H), 3.62 (s, 3H), 2.86–2.67 (m, 2H),
2.52–2.42 (m, 2H), 1.65–1.47 (m, 5H), 1.32–1.27 (m,
2H), 0.89 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 7.7 Hz, 3H),
0.83 (d, J = 6.4 Hz, 3H); HRMS (FAB) calcd for
C11H22N3O5 276.1559; found: 276.1561 (M+H)+.
4.3.1.54. N-[(R)-3-Hydroxycarbamoyl-2-dibutylamino-
propanyl]-L-leucine methyl ester (HAB-A4L: 36c). This
compound was prepared from 38b according to the gen-
1
eral procedure (99%). H NMR (500 MHz, DMSO-d6)
d: 10.34 (s, 1H), 8.65 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H),
4.30 (m, 1H), 3.78 (m, 1H), 3.60 (s, 3H), 2.36 (t,
J = 6.8 Hz, 4H), 2.31 (dd, J = 15.0, 7.3 Hz, 1H), 2.13
(dd, J = 15.0, 5.6 Hz, 1H), 1.58–1.50 (m, 3H), 1.42–1.36
(m, 4H), 1.29–1.22 (m, 4H), 0.88–0.83 (m, 12H); HRMS
(FAB) calcd for C19H38N3O5 388.2811; found: 388.2791
(M+H)+.
4.3.1.51. N-[(R)-3-O-Benzyloxycarbamoyl-2-butylami-
nopropanyl]-L-leucine methyl ester (38a). 35b (62 mg,
0.133 mmol) was dissolved in TFA/CH2Cl2 (0.5 mL:
5 mL) and the solution was stirred for 1 h at rt. The
TFA was removed under reduced pressure, and the res-
idue was dissolved in THF (5 mL). To this solution
were added n-butylaldehyde (13.2 lL, 0.146 mmol),
NaBH(OAc)3 (42.4 mg, 0.200 mmol), and AcOH (8
lL), and the mixture was stirred for 30 min at rt. Pure
38a was obtained by means of column chromatography
(AcOEt/Hex = 1:1 to AcOEt/Net3 = 50:1) (39.1 mg, two
Acknowledgments
The authors are grateful to Prof. Aya Tanatani (Ochan-
omizu University) for her helpful discussion. The work
described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry
of Education, Culture, Sports, Science and Technology,
Japan, and the Japan Society for the promotion of
Science.
1
steps, 70%). H NMR (500 MHz, DMSO-d6) d: 11.37
(s, 1H), 7.38–7.36 (m, 5H), 4.78 (d, 2H), 4.29 (m,
1H), 4.15 (m, 1H), 3.71 (s, 3H), 2.84 (m, 2H), 2.59
(d, J = 5.6 Hz, 1H), 2.49 (m, 1H), 1.68–1.48 (m, 5H),
1.31 (dd, J = 15.0, 7.3 Hz, 2H), 0.88 (t, J = 7.7 Hz,
3H), 0.87(d, J = 6.4 Hz, 3H), 0.81 (d, J = 6.4 Hz, 3H);
MS (FAB) 422 (M+H)+.
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