Development of a highly water-soluble peptide-based human neutrophil elastase inhibitor; AE-3763 for treatment of acute organ injury

Article abstract of DOI:10.1016/j.bmc.2009.09.020

A series of peptide-based transition-state human neutrophil elastase (HNE) inhibitors with N-terminal acidic moieties were synthesized and their inhibitory activity against HNE was evaluated both in vitro and in vivo. Our results show that compounds conta

Full text of DOI:10.1016/j.bmc.2009.09.020

Bioorganic & Medicinal Chemistry 17 (2009) 7477–7486
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a highly water-soluble peptide-based human neutrophil
elastase inhibitor; AE-3763 for treatment of acute organ injury
*
Yasunao Inoue , Tomoki Omodani, Ryotaro Shiratake, Hiroshi Okazaki, Akemi Kuromiya,
Taeko Kubo, Fuminori Sato
Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd, Enoki-cho 33-94, Suita, Osaka 564-0053, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of peptide-based transition-state human neutrophil elastase (HNE) inhibitors with N-terminal
acidic moieties were synthesized and their inhibitory activity against HNE was evaluated both in vitro
and in vivo. Our results show that compounds containing cyclic amide bridged acidic moieties at the
N-terminal have not only improved water solubility but also high in vivo potency. Among these com-
pounds, AE-3763 showed remarkable efficacy in hamster models of elastase-induced lung hemorrhage
and lipopolysaccharide (LPS)-induced lung injury as well as in a mouse model of LPS/galactosamine-
induced acute multiple organ dysfunctions. The water solubility of AE-3763 (>1000 mg/ml in H2O)
was also far superior to that of any of the other compounds synthesized. Thus, it is believed that AE-
3763 would be useful for treatment of HNE-associated respiratory disorders, such as acute respiratory
distress syndrome (ARDS), acute lung injury (ALI), and acute exacerbation of chronic obstructive pulmon-
ary disease (COPD).
Received 12 August 2009
Revised 10 September 2009
Accepted 11 September 2009
Available online 16 September 2009
Keywords:
HNE inhibitor
AE-3763
Trifluoromethylketone
Structure–activity relationship (SAR)
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
acute destructive diseases requires the use of parenteral drugs as
patients are usually in a critical condition. Therefore, high solubil-
Human neutrophil elastase (HNE, EC 3.4.21.37), a neutral serine
protease with broad substrate specificity, is a major constituent in
the azurophilic granules of human neutrophils. It is one of the
many proteolytic enzymes released to combat invading foreign
bodies during inflammation.1,2 HNE has potent catalytic activity
to hydrolyze a variety of extracellular matrix proteins, including
elastin, which plays a major role in lung elasticity and proteolytic
resistance. Under physiological conditions, organs are protected
from HNE elastolytic activity by endogenous inhibitors, such as
ity and good stability in aqueous solution are essential physico-
chemical properties required for any potential HNE inhibitor. We
have previously reported peptide-based carboxylic acid-containing
transition-state inhibitors of HNE and have shown that the pres-
ence of both a benzene ring and an amide bond at the N-terminal
position are preferred for good HNE inhibitory activity.5 On the ba-
sis of these findings, exchange of the lipophilic benzene ring with a
hydrophilic ring was further studied with the aim of improving
compounds water solubility. In this study, we first replaced the
aromatic group at the N-terminal of the parent compound 14a5
with various heterocyclic aliphatic rings having an acidic residue
with hydrophilic five- or six-membered cyclic amides as water-sol-
uble group (WG). Next, we examined the effects of substitution of
the P10 carbonyl-activating group (CAG) at the C-terminal and eval-
uated various combination of WG and CAG to optimize compounds
efficacy/solubility balance. Herein, we describe the SAR of the syn-
thesized compounds and the development of a highly water-solu-
ble HNE inhibitor 14v (AE-3763) from the lead compound 14a
(Fig. 1).
a1-protease inhibitor, a2-macrogloblin, and secretory leukocyte
protease inhibitor. However, in the course of a pathological condi-
tion, such as emphysema, cystic fibrosis, chronic obstructive pul-
monary disease (COPD), acute respiratory distress syndrome
(ARDS), or acute lung injury (ALI), the balance between HNE and
its endogenous inhibitors is displaced in favor of the enzyme.3 Such
unbalance leads to massive infiltration of neutrophils into the
lungs and other organs and subsequently tissue injury.
Although numerous HNE inhibitors, including peptidic and non-
peptidic compounds, have been reported with some studied at
clinical level, sivelestat4 is so far the only marketed agent (Japan
and Korea only) for the treatment of ALI associated with systemic
inflammatory response syndrome (SIRS). Generally, treatment of
2. Chemistry
Synthesis of the key intermediates 5 and 8 is illustrated in
Schemes 1 and 2, respectively. The cyanohydrin 16 was converted
to the tripeptides 5 in five steps as shown in Scheme 1. In brief, the
* Corresponding author. Tel.: +81 6 6337 5903; fax: +81 6 6337 6010.
E-mail address: yasunao-inoue@ds-pharma.co.jp (Y. Inoue).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.020

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Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
HCl/MeOH and then condensed with Z-Val-Pro-OH to afford the
Z-tripeptides 7. Deprotection of the Z-group in 7 in a usual manner
afforded the tripeptides 8. Similar tripeptides, that is, 9a (2S,3S-iso-
mer),9 9b (RS-mixture at 2- and 3-position)10 and 10,6,11 were also
synthesized by the reported methods (Fig. 2).
Scheme 3 shows the final three steps in the synthesis of the tar-
get compounds 14a–w. The synthetic methods and physical data
for the half esters 11b–o have already been described previously.9
After coupling of the tripeptides (5, 8, 9a, 9b, and 10) and the half
esters 11a–o, 11u12 using EDC, oxidation of the secondary hydro-
xyl group with Dess–Martin periodinane reagent (DMP) yielded
the ketones 13a–w. Finally, TFA cleavage of the tert-butyl ester
provided the target compounds 14a–w.
3. Results and discussion
Figure 1. Lead compound (14a) to 14v (AE-3763).
Firstly, several N-terminal acidic compounds appended with a
benzoxazole ketone moiety as P10 CAG were synthesized, and the
in vivo efficacy of each compound was evaluated by iv administra-
tion in a lung hemorrhage hamster model produced by treatment
with HNE. As shown in Table 1, replacement of the benzene ring
of the starting compound 14a with a six-membered heterocyclic
amide (compound 14b) decreased the in vitro inhibitory activity
for HNE, however, iv bolus administration of the same compound
resulted in an increase in the in vivo efficacy. Furthermore, water
solubility of compound 14b (>100 mg/ml at pH 7) was superior
to that of compound 14a.
Based on these results, various compounds containing a cyclic
amide moiety (14c–o) were synthesized, and both their in vitro
inhibitory activity for HNE and in vivo efficacy in the hamster mod-
el were evaluated. Most compounds showed in vitro inhibitory
activity and in vivo efficacy similar to those of compound 14b.
For example, the position of carbonyl group in the cyclic amide
moiety was tolerable (14b vs 14c, 14h vs 14i, 14j vs 14k, 14m vs
14n). Decrease of ring size in the heterocyclic amides from 6 to 5
(14e vs 14i) or deletion of the carbonyl group (14h and 14i vs
14l) did not affect the in vitro inhibitory activity. These results sug-
gest that the cyclic amide portion does not bind to human neutro-
phil elastase tightly, although similar physicochemical properties
may contribute to favorable in vivo efficacy.
Scheme 1. Reagents: (a) HCl, EtOH; (b) PhCH2NH2, LiAlH4; (c) H2, Pd(OH)2-C; (d) Z-
As drug infusion is considered to be the most favorable route of
administration for patients with acute respiratory disorders, we
examined the in vivo efficacy of compounds that showed over
60% inhibition at 10 mg/kg by iv bolus administration (14b, 14d,
14h–I, and 14l–o) following iv infusion for over 70 min in the lung
hemorrhage hamster model produced by treatment with HNE. As
expected, some compounds showed better efficacy by iv infusion
than by iv bolus administration, especially compounds 14b and
14m, which exhibited prominent efficacy at a low dose (over 50%
inhibition at 1 mg/kg/h).
Val-Pro-OH, EDC; (e) H2, Pd(OH)2-C.
Scheme 2. Reagents: (a) MeOH, HCl; (b) Z-Val-Pro-OH, EDC; (c) H2, Pd(OH)2-C.
cyanohydrin 1 was directly transformed into the ethyl ester 2 by
treatment with dry ethanolic hydrogen chloride followed by
in situ hydrolysis of the intermediate imidate hydrochloride.
Aminolysis7 of the ethyl ester 2 gave the amide 3, which was
hydrogenated and then condensed with Z-Val-Pro-OH to give the
Z-tripeptides 4. Deprotection of the Z-group in 4 in a usual manner
afforded the tripeptides 5.
The
tides 8 in three steps as shown in Scheme 2. First, the diastereo-
meric mixture of -hydroxyl-b-amino acid 6 was esterified with
a
-hydroxyl-b-amino acid 68 was converted to the tripep-
a
Figure 2. Tripeptides 9a, 9b, and 10.

Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
7479
Scheme 3. Reagents: (a) EDC; (b) DMP, t-BuOH; (c) TFA.
Next, the effects of substitution of P10 CAG, which had been re-
ported10,13 as an important group in the development of various
serine protease inhibitors, were examined (Table 2). Compounds
in vivo efficacy was determined following iv infusion to the earlier
mentioned hamster models. The in vitro inhibitory activity of the
diketoester derivatives 14r and 14s for HNE was superior to that
of other compounds, although their in vivo efficacy was not much
different from that of other compounds. This discrepancy is prob-
ably due to the fact the diketoester moiety is metabolically less sta-
ble. On the other hand, deletion of the acetic acid moiety in 14t led
to a loss of inhibitory activity against HNE (compound 14u, Table
2). This indicates that the N-terminal carboxylic acid is indispens-
able not only for increasing water solubility but also for HNE inhib-
itory activity. In addition, conversion of the stereochemistry at the
P1 position in 14v (AE-3763) gave complete loss of in vitro
inhibitory activity (compound 14w); presumably because the con-
formational requirement of the isopropyl group at the P1 position
in AE-3763 for binding to HNE is crucial to the inhibitory activity.
Preliminary study on stability has indicated that a compound
having a trifluoromethylketone group was more stable than that
3763 dose-dependently prevented hemorrhage when given intra-
venously by infusion (ED50: 0.42 mg/kg/h) or by bolus injection
(1.2 mg/kg). With regard to the toxicity of AE-3763 in mice, the re-
sults of a preliminary study have shown no overt toxic effect even
at the high dose of 300 mg/kg, iv. These results suggest that AE-
3763 would be of therapeutic value in the treatment of excess
HNE-associated destructive acute diseases, such as ARDS, septic
shock, and acute organ injury.
4. Conclusion
A series of peptide-based transition-state inhibitors of HNE with
a carboxylic acid group at the N-terminal were synthesized and
their pharmacological activity and solubility in water were evalu-
ated. Marked in vitro and in vivo HNE inhibitory activities as well
as high water solubility were observed in several compounds pos-
sessing a carboxylic acid group with an aliphatic cyclic amide moi-
ety at the N-terminal (e.g., compound 14b). Optimization of the
cyclic amide part at the N-terminal and then the P10 CAG at the
C-terminal led to the discovery of AE-3763, which exhibited potent
in vitro inhibitory activity against HNE as well as extremely high
solubility and stability in water. Pharmacological and toxicological
evaluation of in vivo AE-3763 revealed that this compound would
be therapeutically useful in the treatment of destructive acute dis-
eases associated with excess HNE.
having
a-ketobenzoxazole group. In the case of AE-3763, which
has a trifluoromethylketone at the C-terminal, generation of the
epimer was less than 1% after 24 h incubation in phosphate buffer
solution (pH 5.5) at 25 °C. In addition, it has also been reported that
placement of a large lipophilic group in the P10 position increases
the in vitro inhibitory activity, although small P10 substituents
are preferable to large lipophilic groups in order to maintain good
aqueous solubility.14 In fact, the water solubility of AE-3763
(>1000 mg/ml in H2O) was far superior to that of other compounds.
Thus, AE-3763, which is characterized by high solubility and sta-
bility in water, was chosen as candidate for further therapeutic
evaluation in the treatment of other destructive acute diseases
associated with excess HNE.
Edema and leukocytes infiltration into the lung, induced by LPS
administration in hamsters, were significantly inhibited by infu-
sion of AE-3763 (Fig. 3). The lowest doses of AE-3763 that pro-
duced statistically significant inhibition were 3 mg/kg/h (lung
edema), and 1.5 mg/kg/h (leukocyte infiltration). These inhibitory
effects were also observed when AE-3763 administration was
started after the establishment of the disease. Furthermore, AE-
3763 significantly improved survival rate by 24 h in a mouse
model of fatal shock associated with multiple organ dysfunction
5. Experimental
5.1. Chemistry
5.1.1. General methods
Unless otherwise noted, reagents were obtained from commer-
cial suppliers and used without further purification. Melting points
were determined on a cover glass with an electrothermal melting
point apparatus and are given as uncorrected values. 1H NMR spec-
tra were recorded on a JNMLA300 (JEOL, 300 MHz) and chemical
shifts are expressed in ppm downfield from internal TMS. Mass
spectra were recorded with electron spray ionization (ESI) or atmo-
spheric pressure chemical ionization (APCI) and liquid secondary
ion (LSI) on LTQ Orbitrap Discovery (Thermo Fisher Scientific) or
M-1000 (Hitachi). Optical rotation was measured with P-1020
(JASCO). Silica gel column chromatography was performed on a Sil-
ica Gel 60 (70–230 mesh, Merck). Reactions requiring anhydrous
induced by LPS/D-galactosamine administration (Fig. 4). In the lung
hemorrhage hamster model produced by treatment with HNE, AE-

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Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
Table 1
Effects of modification in the N-terminal WG (R2) on HNE inhibitory activity
Compda
IC50 (nM)
% Inhibition after iv administrationc
Infusion (mg/kg/h)
b
Bolus (mg/kg)
10
30
56
3
10
3
1
14a
14b
14c
14d
14e
14f
7.6
24
27
14
33
27
43
42
35
47
23
17
62
38
66
47
70
34
48
33
67
72
42
59
68
67
97
94
92
24
34
39
38
93
62
50
56
38
33
14g
14h
14i
14
32
37
40
20
22
20
91
81
78
53
44
57
ꢀ7
39
14j
14k
14l
90
90
31
73
25
55
14m

Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
7481
Table 1 (continued)
Compda
IC50 (nM)
% Inhibition after iv administrationc
b
Bolus (mg/kg)
10
Infusion (mg/kg/h)
3
30
3
10
95
1
14n
30
62
88
57
76
48
41
14o
70
44
87
34
a
All compounds had more than 90% of the diastereomers in the S-configuration at the P1 position.
Inhibition of HNE-catalyzed hydrolysis of the synthetic substrate Suc-Ala-Pro-Ala-MCA.2
Inhibition of HNE-induced lung hemorrhage in hamsters. See the Section 5 for further details.4
b
c
Table 2
Effects of modification in the N-terminal WG (R2) and the C-terminal CAG (R1) on HNE inhibitory activity
Compda
R1
IC50 (nM)
% Inhibition after iv administrationc
Infusion (mg/kg/h)
b
10
90
3
1
14p
21
78
32
14q
20
89
94
94
79
47
43
48
7.6
31
32
14r
6.6
14s
6.6
14t
28
ꢀ1.2
14u
1000
29
14v (AE-3763)
97
84
45
14w
R-Configuration of 14v at P1 position
>1000
a
All compounds except for 14t, 14u, and 14w had more than 90% of diastereomers in the S-configuration at the P1 position.
b,cSee footnotes in Table 1.

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Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
Lung edema
Infiltration of leukocytes into the lung
400
300
200
100
0
(8)
##
(8)
##
0.6
0.5
(8)
(8)
(8)
(7)
(7)
(8)
*
(8)
*
*
*
(7)
**
(8)
(8)
normal LPS 0.75
1.5
3
6
normal LPS
0.3
1
3
10
AE-3763 infusion (mg/kg/hr)
AE-3763 infusion (mg/kg/hr)
Figure 3. Effects of AE-3763 on LPS-induced lung injury in hamsters. The results are shown as means SEM (n = 7–8) ##p <0.01, significantly different from the control group
(edema: Student’s t-test; total cell count: Welch test) *p <0.05, **p <0.01, significantly different from LPS group (Dunnett’s multiple comparison test). See Section 5 for further
details.
AE-3763 or Saline
LPS/GalN
5.1.3. (1S)-3-Benzylamino-1-benzyloxycarbonylamino-1-isopro-
pyl-2-hydroxy-3-oxopropane (3)
AE-3763 100 mg/kg x 6
AE-3763 10 mg/kg x 6
Control
A suspension containing lithium aluminum hydride7 (1.2 g) and
THF (200 ml) was stirred under reflux for 1.5 h. The reaction solu-
tion was then cooled to room temperature, and thereto was added
dropwise benzylamine (17.3 g). To this mixture was added drop-
wise THF (150 ml) containing compound 2 (10.0 g), obtained in
the above step, and the new mixture was stirred at room temper-
ature for 12 h. Water was carefully added to the reaction solution,
and the mixture was extracted with AcOEt. The extract was dried
over MgSO4, and the solvent was removed by evaporation under
reduced pressure. The residue was purified by silica gel column
chromatography [solvent; n-hexane–AcOEt (1:1)] to give the de-
sired 3 (9.0 g, 75%) as colorless oil: MS (LSI, positive) m/z 371
[(M+H)+]; 1H NMR (300 MHz, CDCl3, d): 0.97 (3H, d), 1.04 (3H, d),
2.28 (1H, m), 3.45 (1H, br t), 4.29–4.49 (4H, m), 5.02 (2H, dd),
5.13 (1H, d), 5.43 (1H, d), 7.07 (1H, m), 7.21–7.38 (10H, m).
100
80
60
40
20
0
p<
p<0.01 vs control
NS vs control
0
4
8
12
24
time (hr)
Figure 4. Effects of AE-3763 on LPS/galactosamine-induced shock in mice Log-Rank
test was used to test statistical differences in survival rate between the control
group and each AE-3763-treated group. See Section 5 for further details.
5.1.4. N-Benzyloxycarbonyl-
hydroxy-1-isopropyl-3-oxopropyl]-
L
-valyl-N-[(1S)-3-benzylamino-2-
conditions were performed under argon atmosphere. Solutions
were evaporated under reduced pressure on a rotary evaporator.
Synthetic yields of all compounds were not optimized. The follow-
ing abbreviations are used: NaCl, sodium chloride; HCl, hydrochlo-
ric acid; MgSO4, anhydrous magnesium sulfate; NaHCO3, sodium
hydrogen carbonate; K2CO3, potassium carbonate; THF, tetrahy-
drofuran; DMF, N,N-dimethylformamide; CH2Cl2, methylene chlo-
ride; CHCl3, chloroform; DMSO, dimethyl sulfoxide; MeOH,
methanol; EtOH, ethanol; AcOEt, ethyl acetate; EDC, 1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide hydrochloride; CDCl3,
chloroform-d; TFA, trifluoroacetic acid; Et3N, triethylamine.
L-prolinamide (4)
To EtOH (200 ml) containing compound 3 (9.0 g), obtained in the
above step, was added a catalytic amount of 20% palladium hydrox-
ide, and the mixture was subjected to hydrogenolysis at room tem-
perature. The catalyst was removed by filtration, and the filtrate was
concentrated under reduced pressure. To the remaining oil was
added CH2Cl2 (200 ml) and then N-(benzyloxycarbonyl)-L-valyl-L-
proline (10.2 g) and EDC (5.6 g). The mixture was stirred at room
temperature for 15 h and concentrated under reduced pressure at
room temperature. To the residue was added AcOEt, and the mix-
ture was washed successively with 1.0 N HCl, saturated NaHCO3,
and brine, and dried over MgSO4. The solvent was removed by evap-
oration under reduced pressure, and the residue was purified by sil-
ica gel column chromatography [solvent; CHCl3–MeOH (100:1)] to
give the desired 4 (4.6 g, 33%) as colorless oil: MS (LSI, positive)
m/z 567 [(M+H)+]; 1H NMR (300 MHz, CDCl3, d): 0.79–1.06 (12H,
m), 1.77–2.35 (5H, m), 3.56 (1H, m), 3.73 (1H, m), 4.23–4.45 (4H,
m), 5.06 (1H, d), 5.11 (1H, d), 5.48 (1H, br t), 7.17–7.42 (11H, m).
5.1.2. (3S)-3-Benzyloxycarbonylamino-2-hydroxy-4-methylbu-
tyric acid ethyl ester (2)
To (3S)-3-benzyloxycarbonylamino-2-hydroxy-4-methylpen-
tanenitrile 1 (62.0 g) was added 30% HCl in EtOH (500 ml) at
0 °C, and the mixture was stirred at room temperature for
15 h. The reaction mixture was then concentrated, and to the
resulting residue was added THF (250 ml) and water (250 ml),
and the mixture was stirred at room temperature for 30 min.
The reaction mixture was concentrated, poured into water and
extracted with AcOEt. The combined organic layer was washed
with saturated NaHCO3, brine, dried over MgSO4, and evaporated
in vacuo. The residue was purified by silica gel column chroma-
tography [solvent; n-hexane–AcOEt (7:3)] to give the desired 2
(40.0 g, 55%) as colorless oil: MS (LSI, positive) m/z 310
[(M+H)+]; 1H NMR (300 MHz, CDCl3, d): 0.92–1.05 (6H, m),
1.21–1.30 (3H, m), 1.84–1.93 (1H, m), 3.01–3.10 (1H, m), 3.76–
3.90 (1H, m), 4.14–4.36 (3H, m), 4.96–5.16 (3H, m), 7.26–7.38
(5H, m).
5.1.5.
L-Valyl-N-[(1S)-(3-benzylamino-1-isopropyl-2-hydroxy-3-
oxo-propyl)-
L
-prolinamide (5)
To a solution of compound 4 (4.6 g), obtained in the above step,
in EtOH (100 ml) was added a catalytic amount of 20% palladium
hydroxide on carbon, and the mixture was stirred at room temper-
ature under hydrogen current for 2 h. The catalyst was removed by
filtration, and the filtrate was concentrated under reduced pressure
to give the desired 5 (3.5 g, quant.) as colorless oil. MS (LSI, posi-
tive) m/z 433 [(M+H)+]. This product was used as starting com-
pound in the next step without further purification.

Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
7483
5.1.6. N-Benzyloxycarbonyl-
oxy-1-isopropyl-3-oxopropyl]-
L
-valyl-N-[(1S)-3-methoxy-2-hydr-
-prolinamide (7)
tracted with AcOEt. The extract was washed with brine and dried
over MgSO4. The solvent was removed by evaporation under re-
duced pressure, and the precipitated crystals were washed with
diethyl ether to give the desired compound (1.4 g, 60%) as colorless
crystals: Mp 192–194 °C ; MS (LSI, positive) m/z 261 [(M+H)+]; 1H
NMR (300 MHz, DMSO-d6, d): 4.59 (2H, s), 5.20 (2H, s), 5.62 (1H,
d), 7.37 (5H, m), 7.65 (1H, d), 11.4 (1H, s).
L
To (3S)-3-amino-2-hydroxy-4-methylpentanoic acid hydro-
chloride salt 6 (1.0 g) was added 10% HCl in MeOH (30 ml) at
0 °C, and the mixture was stirred at room temperature for 16.5 h.
The reaction mixture was then concentrated under reduced pres-
sure. To the remaining oil was added pyridine (20 ml) and then
N-(benzyloxycarbonyl)-L-valyl-L-proline (1.9 g) and EDC (1.6 g).
5.1.9.2. Benzyl tert-butyl 2,20-(2,4-dioxopyrimidine-1,3(2H,4H)-
diyl)diacetate. To anhydrous DMF (10 ml) containing benzyl (2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (1.0 g) obtained in
the above step was gradually added sodium hydride (purity; 60%,
0.18 g) under ice-cooling, and the mixture was stirred under ice-
cooling for 15 min. To this mixture was added tert-butyl bromo-
acetate (0.9 g), and the mixture was stirred at room temperature
for 1 h. Saturated aqueous ammonia chloride solution was added
to the reaction mixture, and the mixture was extracted with AcOEt.
The extract was washed with brine dried over MgSO4, and the sol-
vent was removed by evaporation under reduced pressure. The res-
idue was purified by silica gel column chromatography [solvent; n-
hexane–AcOEt (2:1?1:1)] to give the desired compound (1.2 g,
84%) as colorless oil: MS (LSI, positive) m/z 375 [(M+H)+]; 1H
NMR (300 MHz, CDCl3, d): 1.46 (9H, s), 4.51 (2H, s), 4.58 (2H, s),
5.21 (2H, s), 5.81 (1H, d), 7.10 (1H, d), 7.36 (5H, m).
The mixture was stirred at room temperature for 15 h and concen-
trated under reduced pressure at room temperature. To the residue
was added AcOEt, and the mixture was washed successively with
10% HCl, saturated NaHCO3, and brine, and dried over MgSO4.
The solvent was removed by evaporation under reduced pressure,
and the residue was purified by silica gel column chromatography
[solvent; CHCl3–MeOH (50:1?20:1)] to give the desired 7 (0.64 g,
24%) as foam: MS (LSI, positive) m/z 492 [(M+H)+]; 1H NMR
(300 MHz, CDCl3, d): 0.89–1.03 (12H, m), 1.81–2.35 (6H, m),
3.38–4.57 (10H, m), 5.02–5.13 (2H, m), 5.61 (0.5H, d), 5.74 (0.5H,
d), 6.82 (0.5H, d), 6.99 (0.5H, d), 7.34 (5H, m).
5.1.7.
oxo-propyl)-
L
-Valyl-N-[(1S)-(3-benzylamino-1-isopropyl-2-hydroxy-3-
-prolinamide (8)
L
To a solution of compound 7 (0.3 g), obtained in the above step,
in AcOEt (30 ml) was added a catalytic amount of 20% palladium
hydroxide on carbon, and the mixture was stirred at room temper-
ature under hydrogen current for 4 h. The catalyst was removed by
filtration, and the filtrate was concentrated under reduced pressure
to give the desired 8 (0.19 g, 90%) as colorless oil: MS (LSI, positive)
m/z 358 [(M+H)+]. This product was used as starting compound in
the next step without further purification.
5.1.9.3.
[3-(2-tert-Butoxy-2-oxoethyl)-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl]acetic acid (11b). To AcOEt (20 ml) contain-
ing benzyl tert-butyl 2,20-(2,4-dioxopyrimidine-1,3(2H,4H)-
diyl)diacetate (1.2 g) obtained in the above step was added 20%
palladium hydroxide on carbon (50 mg), and the mixture was stir-
red at room temperature under hydrogen atmosphere for 1 h. The
catalyst was removed by filtration, and the filtrate was concen-
trated under reduced pressure to give the desired 11b (0.7 g,
77%) as colorless powder: MS (LSI, positive) m/z 285 [(M+H)+];
1H NMR (300 MHz, CDCl3, d): 1.46 (9H, s), 4.51 (2H, s), 4.59 (2H,
s), 5.86 (1H, d), 7.17 (1H, d).
5.1.8. Typical procedure for the synthesis of half esters: [4-(2-
tert-butoxy-2-oxoethoxy)phenoxy]acetic acid (11a)
5.1.8.1. Benzyl tert-butyl 2,20-[benzene-1,4-diylbis(oxy)]diace-
tate. To anhydrous DMF (200 ml) containing tert-butyl(4-
hydroxyphenoxy)acetate15 (26.0 g) were added benzyl bromoace-
tate (32.0 g) and K2CO3 (32.0 g) at room temperature, and the mix-
ture was stirred at room temperature for 15 h. The reaction
solution was then poured into water, and the mixture was ex-
tracted with AcOEt. The extract was washed with brine and dried
over MgSO4. The solvent was removed by evaporation under re-
duced pressure, and the residue was purified by silica gel column
chromatography [solvent; n-hexane–AcOEt (8:1)] to give the de-
sired compound (40.4 g, 94%) as colorless oil: MS (LSI, positive)
m/z 373 [(M+H)+].
5.1.10. Typical procedure for the synthesis of half esters: [3-(2-
tert-butoxy-2-oxoethyl)-2-oxoimidazolidin-1-yl]acetic acid
(11l)
5.1.10.1. Di-tert-Butyl 2,20-(2-oxoimidazolidine-1,3-diyl)diace-
tate. To anhydrous DMF (200 ml) containing 2-oxo-imidazoli-
dine (10.0 g) and tert-butyl bromoacetate (50.7 g) was added
lithium tert-butoxide (20.5 g) under ice-cooling, and the mixture
was stirred for 1 h at room temperature. The reaction mixture
was poured into ice, and the precipitated solids were washed
with water and air-dried overnight. Crude solid was crystallized
from AcOEt to give the desired compound (26.0 g, 69%) as white
crystals: Mp 100–102 °C; MS (LSI, positive) m/z 315 [(M+H)+];
1H NMR (300 MHz, CDCl3, d): 1.46 (18H, s), 3.54 (4H, s), 3.89
(4H, s).
5.1.8.2.
[4-(2-tert-Butoxy-2-oxoethoxy)phenoxy]acetic acid
(11a). To AcOEt (150 ml) containing benzyl tert-butyl 2,20-[ben-
zene-1,4-diylbis(oxy)]diacetate (40.4 g) obtained in the above
step was added 20% palladium hydroxide on carbon (4.04 g),
and the mixture was stirred at room temperature under hydrogen
atmosphere for 1 h. The catalyst was removed by filtration, and
the filtrate was concentrated under reduced pressure to give
the desired 11a (30.0 g, 98%) as colorless powder: Mp 95–97 °C;
MS (LSI, positive) m/z 283 [(M+H)+]; 1H NMR (300 MHz, DMSO-
d6, d): 1.42 (9H, s), 4.56 (2H, S), 4.59 (2H, s), 6.83 (4H, s), 12.93
(1H, s).
5.1.10.2.
[3-(2-tert-Butoxy-2-oxoethyl)-2-oxoimidazolidin-1-
yl]acetic acid (11l). To a mixture of EtOH and water in equal vol-
ume (20 ml) containing di-tert-butyl 2,20-(2-oxoimidazolidine-1,3-
diyl)diacetate (1.14 g) obtained in the above step was added potas-
sium hydroxide (0.27 g), and the mixture was stirred at 70 °C for
5 h. EtOH was removed by evaporation under reduced pressure,
and to the residue was added aqueous saturated NaHCO3. The
aqueous layer was washed with AcOEt, acidified with 10% HCl,
and extracted with AcOEt. The extract was dried over MgSO4,
and the solvent was removed by evaporation under reduced pres-
sure. The precipitated crystals were recrystallized from AcOEt to
give the desired 11l (0.30 g, 32%) as colorless crystals: Mp 112–
113 °C; MS (LSI, positive) m/z 259 [(M+H)+]; 1H NMR (300 MHz,
5.1.9. Typical procedure for the synthesis of half esters: [3-(2-tert-
butoxy-2-oxoethyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]-
acetic acid (11b)
5.1.9.1. Benzyl (2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ace-
tate. To anhydrous DMF (10 ml) containing 2,4-dioxopyrimidine
(1.0 g) were added benzyl bromoacetate (2.5 g) and K2CO3 (2.5 g),
and the mixture was stirred at room temperature for 15 h. The
reaction solution was poured into water, and the mixture was ex-

7484
Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
CDCl3, d): 1.46 (9H, s), 3.53 (4H, s), 3.89 (2H, s), 4.01 (2H, s), 7.11
(1H, br s).
5.1.11.5. N-{[3-(Carboxymethyl)-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]acetyl}- -valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-
methyl-1-oxobutan-2-yl]- -prolinamide (14b). Compound 14b
L
L
5.1.11. Typical procedure for the synthesis of AE-3763 (14v)
5.1.11.1. N-{[3-(2-tert-Butoxy-2-oxoethyl)-2-oxoimidazolidin-
(12.0 g, 61.2%) was prepared from 10 and 11b as white powder:
HRMS (ESI, negative) m/z calcd for C30H35O9N6 [(MꢀH)ꢀ]
623.2460, found 623.2458; 1H NMR (300 MHz, DMSO-d6, d):
0.85–1.05 (12H, m), 1.70–2.05 (5H, m), 2.38 (1H, m), 3.55 (1H,
m), 3.64 (1H, m), 4.32 (1H, dd), 4.40–4.55 (5H, m), 5.28 (1H, dd),
5.75 (1H, dd), 7.55 (1H, dd), 7.64 (2H, m), 7.90 (1H, d), 8.01 (1H,
d), 8.45 (2H, m), 12.9 (1H, s).
1-yl]acetyl}-
methylpentan-3-yl]-
containing the half ester 11l (46.4 g) obtained in the above step
and -valyl-N-[(2S,3S)-1,1,1-trifluoro-2-hydroxy-4-methylpentan-
3-yl]- -prolinamide hydrochloride salt 9a (72.0 g) were added EDC
L-valyl-N-[(2S,3S)-1,1,1-trifluoro-2-hydroxy-4-
L-prolinamide (12v). To CH2Cl2 (1000 ml)
L
L
(41.4 g) and Et3N (38.5 g), and the mixture was stirred at room tem-
perature for 15 h and concentrated under reduced pressure at room
temperature. To the residue was added AcOEt, and the mixture was
washed successively with 1.0 N HCl, saturated NaHCO3, and brine,
and dried over MgSO4. The solvent was removed by evaporation un-
der reduced pressure, and the residue was purified by silica gel col-
umn chromatography [solvent; CHCl3–MeOH (50:1?30:1)] to give
thedesired12v(79.5 g, 73%)as colorlesspowder:MS(APCI, positive)
m/z 608 [(M+H)+]; 1H NMR (300 MHz, DMSO-d6, d): 0.77–0.89 (12H,
m), 1.40 (9H, s), 1.66–2.11 (6H, m), 3.34 (4H, m), 3.53 (1H, m), 3.67–
3.80(6H, m), 3.99(1H, ddd), 4.27–4.35(2H, m), 6.37(1H, d), 7.69(1H,
d), 8.07 (1H, d).
5.1.11.6. N-{[3-(Carboxymethyl)-2,6-dioxo-3,6-dihydropyrimi-
din-1(2H)-yl]acetyl}-
L-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-
methyl-1-oxobutan-2-yl]-
L-prolinamide (14c). Compound 14c
(0.92 g, 41.6%) was prepared from 10 and 11c as white powder:
HRMS (ESI, negative) m/z calcd for C30H35O9N6 [(MꢀH)ꢀ]
623.2460, found 623.2462; 1H NMR (300 MHz, DMSO-d6, d):
0.82–1.01 (12H, m), 1.71–2.05 (5H, m), 2.38 (1H, m), 3.53 (1H,
m), 3.62 (1H, m), 4.29 (1H, dd), 4.41–4.52 (5H, m), 5.27 (1H, dd),
5.75 (1H, d), 7.55 (1H, ddd), 7.65 (1H, ddd), 7.72 (1H, d), 7.90
(1H, d), 8.02 (1H, d), 8.35 (1H, d), 8.45 (1H, d), 13.1 (1H, br s).
5.1.11.7. N-{[3-(Carboxymethyl)-5-methyl-2,4-dioxo-3,4-dihy-
5.1.11.2. N-{[3-(2-tert-Butoxy-2-oxoethyl)-2-oxoimidazolidin-1-
dropyrimidin-1(2H)-yl]acetyl}-
L-valyl-N-[(2S)-1-(1,3-benzoxa-
yl]acetyl}-L-valyl-N-[(3S)-1,1,1-trifluoro-4-methyl-2-oxopentan-
zol-2-yl)-3-methyl-1-oxobutan-2-yl]-
L
-prolinamide
3-yl]- -prolinamide (13v). To CH2Cl2 (1000 ml) containing com-
L
(14d). Compound 14d (2.0 g, 68.1%) was prepared from 10 and
11d as white powder: HRMS (ESI, negative) m/z calcd for
C31H37O9N6 [(MꢀH)ꢀ] 637.2617, found 637.2628; 1H NMR
(300 MHz, DMSO-d6, d): 0.86–1.01 (12H, m), 1.68–2.04 (5H, m),
1.81 (3H, s), 2.38 (1H, m), 3.36–3.68 (6H, m), 4.31 (1H, dd), 4.50
(1H, dd), 5.28 (1H, dd), 7.53–7.68 (3H, m), 7.90 (1H, d), 8.02 (1H,
d), 8.45 (2H, m), 12.9 (1H, br s).
pound 12v (79.5 g), obtained in the above step, was added Dess–Mar-
tin reagent (112.3 g), and the mixture was stirred at room
temperature for 4 h and concentrated under reduced pressure. To
the residue was added AcOEt, and the mixture was washed succes-
sively with saturated aqueous sodium thiosulfate solution, saturated
NaHCO3, and brine, and dried over MgSO4. The solvent was removed
by evaporation under reduced pressure, and the residue was purified
by silica gel column chromatography [solvent; CHCl3–MeOH (100:3)]
to give the desired 13v (67.2 g, 85%) as colorless foam: MS (APCI, po-
sitive) m/z 606 [(M+H)+]; 1H NMR (300 MHz, CDCl3, d): 0.83–1.02
(12H, m), 1.47 (9H, s), 1.89–2.32 (6H, m), 3.48–4.06 (10H, m), 4.58
(1H, dd), 4.64 (1H, dd), 4.84 (1H, dd), 7.35 (1H, d), 7.72 (1H, d).
5.1.11.8. N-{[3-(Carboxymethyl)-2,6-dioxotetrahydropyrimi-
din-1(2H)-yl]acetyl}-L-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-
methyl-1-oxobutan-2-yl]-
L-prolinamide (14e). Compound 14e
(1.7 g, 52.1%) was prepared from 10 and 11e as white powder:
HRMS (ESI, negative) m/z calcd for C30H37O9N6 [(MꢀH)ꢀ]
625.2617, found 625.2624; 1H NMR (300 MHz, DMSO-d6, d):
0.82–1.00 (12H, m), 1.70–2.04 (5H, m), 2.38 (1H, m), 2.70 (2H, t),
3.46 (2H, t), 3.50 (1H, m), 3.63 (1H, m), 4.07 (2H, s), 4.25 (2H, s),
4.29 (1H, m), 4.51 (1H, dd), 5.27 (1H, dd), 7.55 (1H, dd), 7.65 (1H,
dd), 7.90 (1H, d), 8.02 (1H, d), 8.19 (1H, d), 8.44 (1H, d), 12.8 (1H, s).
5.1.11.3. N-{[3-(Carboxymethyl)-2-oxoimidazolidin-1-yl]acetyl}-
L-valyl-N-[(3S)-1,1,1-trifluoro-4-methyl-2-oxopentan-3-yl]-L-pro-
linamide (AE-3763, 14v). To CH2Cl2 (200 ml) containing compound
13v (34.9 g), obtained in the above step, was added TFA (200 ml) at
room temperature, and the mixture was stirred at room temperature
for 1 h and concentrated under reduced pressure. To the residue was
added diisopropyl ether, and the precipitated crystals were collected
by filtration, and recrystallized from AcOEt to give AE-3763 (14v)
(14.0 g, 44%) as colorless crystals. According to high performance li-
quid chromatography, the purity of the product was 98.71% and the
5.1.11.9. N-{[4-(Carboxymethyl)-6-methyl-3,5-dioxo-4,5-dihy-
dro-1,2,4-triazin-2(3H)-yl]acetyl}-
xazol-2-yl)-3-methyl-1-oxobutan-2-yl]-
L
-valyl-N-[(2S)-1-(1,3-benzo-
-prolinamide
L
(14f). Compound 14f (1.25 g, 45.7%) was prepared from 10 and
11f as white powder: HRMS (ESI, negative) m/z calcd for
C30H36O9N7 [(MꢀH)ꢀ] 638.2569, found 623.2586; 1H NMR
(300 MHz, DMSO-d6, d): 0.85–1.01 (12H, m), 1.71–2.06 (5H, m),
2.15 (3H, s), 2.36–2.42 (1H, m), 3.48–3.66 (2H, m), 4.31 (1H, dd),
4.47 (2H, s), 4.52 (1H, dd), 4.60 (2H, s), 5.28 (1H, dd), 7.53–7.68
(2H, m), 7.96 (2H, dd), 8.46 (2H, m), 13.1 (1H, br s).
percentage of isomer was 0.78%: Mp 177–178 °C; ½a D20
ꢁ : ꢀ63.0 (c 1.0,
CHCl3); HRMS (ESI, negative) m/z calcd for C23H33O7N5F3 [(MꢀH)ꢀ]
548.2327, found 548.2329; 1H NMR (300 MHz, CDCl3, d): 0.85–1.02
(12H, m), 1.90–2.31 (6H, m), 3.51 (4H, m), 3.68 (1H, m), 3.81–4.13
(5H, m), 4.58 (2H, m), 4.92 (1H, dd), 7.45 (2H, m).
The same synthetic routes were used to synthesize compounds
14a–x (Tables 1 and 2).
5.1.11.10. N-{[4-(Carboxymethyl)-3,5-dioxo-4,5-dihydro-1,2,4-
triazin-2(3H)-yl]acetyl}-
3-methyl-1-oxobutan-2-yl]-
L
-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-
-prolinamide (14g). Compound
5.1.11.4. N-{[4-(Carboxymethoxy)phenoxy]acetyl}-
L-valyl-N-
L
benzoxazol-2-yl)-3-methyl-1-oxobutan-2-yl]- -prolinamide
L
14g (0.50 g, 44.5%) was prepared from 10 and 11g as white pow-
der: HRMS (ESI, negative) m/z calcd for C29H34O9N7 [(MꢀH)ꢀ]
624.2413, found 624.2413; 1H NMR (300 MHz, DMSO-d6, d):
0.83–1.00 (12H, m), 1.71–2.09 (5H, m), 2.39 (1H, m), 3.34–3.56
(14H, m), 3.69 (1H, m), 4.33 (1H, dd), 4.50 (1H, m), 5.29 (1H, m),
7.55 (1H, dd), 7.65 (1H, dd), 7.90 (1H, d), 8.02 (1H, d), 8.26 (1H,
m), 8.43 (2H, m).
(14a). Compound 14a (3.2 g, 40.6%) was prepared from 10 and
11a as white powder: HRMS (ESI, negative) m/z calcd for
C32H37O9N4 [(MꢀH)ꢀ] 621.2555, found 621.2565; 1H NMR
(300 MHz, CDCl3, d): 0.90–1.13 (12H, m), 1.99–2.26 (5H, m),
2.50 (1H, m), 3.71 (1H, m), 3.94 (1H, m), 4.45–4.68 (6H, m),
5.61 (1H, dd), 6.77–6.87 (4H, m), 7.25 (1H, d), 7.47 (1H, ddd),
7.56 (1H, ddd), 7.66 (2H, d), 7.92 (1H, d).

Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
7485
5.1.11.11. N-{[3-(Carboxymethyl)-2,4-dioxoimidazolidin-1-
yl]acetyl}- -valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-methyl-1-
oxobutan-2-yl]- -prolinamide (14h). Compound 14h (1.80 g,
65.8%) was prepared from 10 and 11h as white powder: HRMS
(ESI, negative) m/z calcd for C29H35O9N6 [(MꢀH)ꢀ] 611.2460, found
611.2466; 1H NMR (300 MHz, DMSO-d6, d): 0.83–1.01 (12H, m),
1.72–2.09 (5H, m), 2.36–2.42 (1H, m), 3.51–3.74 (2H, m), 4.05–
4.14 (6H, m), 4.34 (1H, t), 4.52 (1H, dd), 5.29 (1H, dd), 7.53–7.69
(2H, m), 7.96 (2H, dd), 8.37 (1H, d), 8.44 (1H, d), 13.0 (1H, br s).
ative) m/z calcd for
C
30H37O9N6 [(MꢀH)ꢀ] 625.2617, found
L
625.2619; 1H NMR (300 MHz, CDCl3, d): 0.82–1.13 (12H, m),
1.90–2.73 (6H, m), 3.56–3.90 (6H, m), 4.13–4.36 (4H, m), 4.54–
4.69 (2H, m), 5.64 (1H, dd), 7.39 (1H, br d), 7.47 (1H, t), 7.55 (1H,
t), 7.66 (1H, d), 7.74 (1H, br d), 7.91 (1H, d).
L
5.1.11.18. N-{[4-(Carboxymethyl)piperazin-1-yl]acetyl}-
N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-methyl-1-oxobutan-2-yl]-
L-valyl-
L
-
prolinamide (14o). Compound 14o (0.82 g, 20.6%) was prepared
from 10 and 11o as white powder: HRMS (ESI, negative) m/z calcd
for C30H41O7N6 [(MꢀH)ꢀ] 597.3037, found 597.3038; 1H NMR
(300 MHz, DMSO-d6, d): 0.84–1.00 (12H, m), 1.71–2.06 (5H, m),
2.39 (1H, m), 3.29–4.03 (14H, m), 4.37 (1H, dd), 4.51 (1H, m),
5.28 (1H, dd), 7.56 (1H, dd), 7.66 (1H, dd), 7.91 (1H, d), 8.02 (1H,
d), 8.47 (1H, m), 8.69 (1H, m).
5.1.11.12. N-{[3-(Carboxymethyl)-2,5-dioxoimidazolidin-1-
yl]acetyl}-
oxobutan-2-yl]-
L
-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-methyl-1-
-prolinamide (14i). Compound 14i (1.24 g,
L
47.2%) was prepared from 10 and 11i as white powder: HRMS
(ESI, negative) m/z calcd for C29H35O9N6 [(MꢀH)ꢀ] 611.2460, found
611.2475; 1H NMR (300 MHz, DMSO-d6, d): 0.84–1.01 (12H, m),
1.72–2.04 (5H, m), 2.35–2.40 (1H, m), 3.49–3.69 (2H, m), 4.04–
4.12 (6H, m), 4.31 (1H, t), 4.52 (1H, dd), 5.26–5.30 (1H, m), 7.53–
7.68 (2H, m), 7.96 (2H, dd), 8.42 (2H, m), 13.0 (1H, br s).
5.1.11.19. N-{[3-(Carboxymethyl)-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]acetyl}-
L
-valyl-N-[(3S)-1-(benzylamino)-4-methyl-
1,2-dioxopentan-3-yl]-
L-prolinamide (14p). Compound 14p
(3.0 g, 58.0%) was prepared from 5 and 11b as white powder:
HRMS (ESI, negative) m/z calcd for C31H39O9N6 [(MꢀH)ꢀ]
639.2773, found 639.2775; 1H NMR (300 MHz, CDCl3, d): 0.75–
1.06 (12H, m), 1.88–2.50 (6H, m), 3.65 (1H, m), 3.78 (1H, m),
4.35–4.69 (7H, m), 4.94 (1H, d), 5.82 (1H, d), 7.15 (1H, d), 7.18–
7.40 (5H, m), 8.07 (1H, br s).
5.1.11.13. N-{[3-(Carboxymethyl)-5,5-dimethyl-2,4-dioxoimi-
dazolidin-1-yl]acetyl}-
3-methyl-1-oxobutan-2-yl]-
L
-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-
-prolinamide (14j). Compound 14j
L
(1.27 g, 31.0%) was prepared from 10 and 11j as white powder:
HRMS (ESI, negative) m/z calcd for C31H39O9N6 [(MꢀH)ꢀ]
639.2773, found 639.2789; 1H NMR (300 MHz, DMSO-d6, d):
0.83–1.01 (12H, m), 1.29 (6H, s), 1.72–2.02 (4H, m), 2.33–2.44
(1H, m), 3.50–3.72 (2H, m), 3.99 (2H, s), 4.09 (2H, s), 4.35 (1H,
dd), 4.50 (1H, dd), 5.27 (1H, dd), 7.53–7.68 (2H, m), 7.96 (2H,
dd), 8.22 (1H, d), 8.44 (1H, d), 13.0 (1H, br s).
5.1.11.20. N-{[4-(Carboxymethyl)-2,3-dioxopiperazin-1-yl]ace-
tyl}-
tan-3-yl]-
L
-valyl-N-[(3S)-1-(benzylamino)-4-methyl-1,2-dioxopen-
-prolinamide (14q). Compound 14q (0.65 g, 41%) was
L
prepared from 5 and 11n as white powder: HRMS (ESI, negative)
m/z calcd for C31H41O9N6 [(MꢀH)ꢀ] 641.2930, found 641.2947;
1H NMR (300 MHz, DMSO-d6, d): 0.73–1.00 (12H, m), 1.67–2.30
(6H, m), 4.00–4.20 (5H, m), 4.23–4.42 (4H, m), 4.49 (1H, m), 4.95
and 5.01 (1H, each dd), 8.16 (1H, d), 8.32 (1H, d), 9.24 (1H, t).
5.1.11.14. N-{[3-(Carboxymethyl)-4,4-dimethyl-2,5-dioxoimi-
dazolidin-1-yl]acetyl}-
3-methyl-1-oxobutan-2-yl]-
L
-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-
-prolinamide (14k). Compound
L
14k (1.04 g, 32.1%) was prepared from 10 and 11k as white pow-
der: HRMS (ESI, negative) m/z calcd for C31H39O9N6 [(MꢀH)ꢀ]
639.2773, found 639.2781; 1H NMR (300 MHz, DMSO-d6, d):
0.84–1.01 (12H, m), 1.31 (6H, s), 1.71–2.07 (5H, m), 2.34–2.42
(1H, m), 3.49–3.65 (2H, m), 4.04–4.06 (4H, m), 4.35 (1H, dd),
4.52 (1H, dd), 5.28 (1H, dd), 7.53–7.68 (2H, m), 7.96 (2H, dd),
8.36 (1H, d), 8.45 (1H, d), 12.8 (1H, br s).
5.1.11.21. N-{[3-(Carboxymethyl)-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]acetyl}-L-valyl-N-[(3S)-1-methoxy-4-methyl-1,2-
dioxopentan-3-yl]- -prolinamide (14r). Compound 14r (43 mg,
L
16.3%) was prepared from 8 and 11b as white powder: MS (APCI,
positive) m/z 566 [(M+H)+]; 1H NMR (300 MHz, DMSO-d6, d):
0.81–0.92 (12H, m), 1.69–2.27 (7H, m), 3.77 (3H, s), 4.31–4.61
(7H, m), 5.74 (1H, d), 7.66 (1H, d), 8.38–8.48 (2H, m).
5.1.11.15. N-{[3-(Carboxymethyl)-2-oxoimidazolidin-1-yl]ace-
tyl}-
L-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-methyl-1-oxob-
5.1.11.22. N-{[4-(Carboxymethyl)-2,3-dioxopiperazin-1-yl]ace-
utan-2-yl]-L-prolinamide (14l). Compound 14l (0.3 g, 41.3%) was
tyl}-L-valyl-N-[(3S)-1-methoxy-4-methyl-1,2-dioxopentan-3-
prepared from 10 and 11l as white powder: HRMS (ESI, negative)
m/z calcd for C29H37O8N6 [(MꢀH)ꢀ] 597.2667, found 597.2668;
1H NMR (300 MHz, CDCl3, d): 0.82–1.13 (12H, m), 1.88–2.28 (5H,
m), 2.50 (1H, m), 3.25–3.74 (5H, m), 3.82–4.12 (5H, m), 4.54–
4.71 (2H, m), 5.65 (1H, m), 7.35–7.59 (4H, m), 7.66 (1H, d), 7.91
(1H, d).
yl]- -prolinamide (14s). Compound 14s (30 mg, 5.2%) was pre-
L
pared from 8 and 11n as white powder: MS (APCI, positive) m/z
568 [(M+H)+]; 1H NMR (300 MHz, CDCl3, d): 0.81–1.38(12H, m),
1.86–3.00 (7H, m), 3.53–3.95 (9H, m), 4.03–4.35 (4H, m), 4.47–
4.69 (2H, m), 5.02 (1H, m).
5.1.11.23. N-{[3-(Carboxymethyl)-2,4-dioxo-3,4-dihydropyrimi-
5.1.11.16. N-{[4-(Carboxymethyl)-2,5-dioxopiperazin-1-yl]ace-
din-1(2H)-yl]acetyl}-L-valyl-N-[(3R,S)1,1,1-trifluoro-4-methyl-
tyl}-
L-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-methyl-1-oxob-
2-oxopentan-3-yl]- -prolinamide (14t). Compound 14t (1.25 g,
L
utan-2-yl]-
L
-prolinamide (14m). Compound 14m (1.93 g, 70.0%)
62.3%) was prepared from 9b and 11b as white powder: HRMS
(ESI, negative) m/z calcd for C24H31O8N5F3 [(MꢀH)ꢀ] 574.2119,
found 574.2130; 1H NMR (300 MHz, DMSO-d6, d): 0.79–0.95
(12H, m), 1.69–2.23 (8H, m), 3.53–3.72 (2H, m), 4.24–4.64 (6H,
m), 5.72–8.63 (5.5H, m), 12.9 (1H, br s).
was prepared from 10 and 11m as white powder: HRMS (ESI, neg-
ative) m/z calcd for C30H37O9N6 [(MꢀH)ꢀ] 625.2617, found
625.2613; 1H NMR (300 MHz, CDCl3, d): 0.86–1.11 (12H, m),
1.91–2.25 (5H, m), 2.51 (1H, m), 3.19 (1H, br s), 3.69 (1H, m),
3.83 (1H, m), 4.00–4.31 (8H, m), 4.55–4.69 (2H, m), 5.65 (1H,
dd), 7.36–7.59 (3H, m), 7.66 (2H, d), 7.91 (1H, d).
5.1.11.24. N-[(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)ace-
tyl]-L-valyl-N-[(3R,S)-1,1,1-trifluoro-4-methyl-2-oxopentan-3-
5.1.11.17. N-{[4-(Carboxymethyl)-2,3-dioxopiperazin-1-yl]ace-
yl]- -prolinamide (14u). Compound 14u (0.18 g, 8.4%) was pre-
L
tyl}-
utan-2-yl]-
was prepared from 10 and 11n as white powder: HRMS (ESI, neg-
L
-valyl-N-[(2S)-1-(1,3-benzoxazol-2-yl)-3-methyl-1-oxob-
pared from 9b and 11u as white powder: HRMS (ESI, negative)
m/z calcd for C22H29O6N5F3 [(MꢀH)ꢀ] 516.2064, found 516.2072;
1H NMR (300 MHz, DMSO-d6, d): 0.79–0.94 (12H, m), 1.69–2.25
L-prolinamide (14n). Compound 14n (0.7 g, 30.0%)

7486
Y. Inoue et al. / Bioorg. Med. Chem. 17 (2009) 7477–7486
(6H, m), 3.56–3.69 (2H, m), 4.01–4.64 (5H, m), 5.54 (1H, d), 6.70–
8.62 (2.5H, m), 7.54 (1H, d), 11.2 (1H, s).
maintained up to 6 h after LPS treatment. BALF was then collected,
and total leukocytes in BALF were counted.
5.1.11.25. N-{[3-(Carboxymethyl)-2-oxoimidazolidin-1-yl]ace-
5.2.4. AE-3763 effects on
-Galactosamine shock was induced in C3H/HeN mice (6–7 w,
22–25 g) by administration of -galactosamine (GalN) and LPS
(20 mg/mouse and 0.3 g/mouse, respectively) into the tail vein.
Immediately after LPS/GalN administration, AE-3763 (10 or
100 mg/kg) was administrated intraperitoneally six times at 2 h
interval. Control animals received the vehicle (PBS) instead of
AE-3763. Animal’s survival rate was observed up to 24 h after
shock induction.
D-galactosamine-induced shock
tyl}-
L-valyl-N-[(3R)-1,1,1-trifluoro-4-methyl-2-oxopentan-3-
D
yl]- -prolinamide (14w). Compound 14w (0.25 g, 14.4%) was pre-
L
D
pared from 9b and 11l as white powder. This compound was puri-
fied by MCI GEL CHP20P (Mitsubishi Chemical Co.) column
chromatography [solvent; MeCN–H2O (0:100?30:70)] to isolate
from diastereomeric mixture: HRMS (ESI, negative) m/z calcd for
C23H33O7N5F3 [(MꢀH)ꢀ] 548.2332, found 548.2328; 1H NMR
(300 MHz, CDCl3, d): 0.86–1.06 (12H, m), 1.88–2.40 (6H, m),
3.47–4.02 (10H, m), 4.57 (1H, dd), 4.68 (1H, dd), 4.88 (1H, dd),
7.33 (1H, d), 7.71 (1H, d).
l
Acknowledgment
5.2. Biological study
We are grateful to Ms. M. Honma for recording MS spectra.
5.2.1. Compounds inhibitory activity for HNE
Supplementary data
HNE (0.2 unit/ml) was preincubated with each test-compound
in the assay solution (100 nM HEPES, pH 7.5 containing 1 M NaCl
and 0.001% Brij-35) at 37 °C for 4 min. The reaction was started
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.09.020.
by addition of
a
substrate, Suc(OMe)-Ala-Ala-Pro-Val-pNA
(0.5 mM), for HNE. Hydrolysis of the substrate to pNA was contin-
uously measured spectrophotometrically by monitoring absor-
bance at 404 nm. IC50 value was estimated using non-linear
regression of data to a logistic function.
References and notes
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model
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cheal instillation of HNE (29 lg). One hour after HNE instillation,
the bronchoalveolar lavage fluid (BALF) was collected, centri-
fuged, and absorbance of the supernatant at 414 nm was mea-
sured. Hemorrhage in BALF was expressed as absorbance at
414 nm.
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5.2.2.2. (b) Continuous intravenous infusion. Each test-com-
pound was infused (for over 70 min) into the femoral vein of
the animal model starting 10 min before intratracheal instillation
of HNE (29 lg) and finishing at the end of the experimental per-
iod. One hour after HNE instillation, BALF was collected, centri-
fuged, and absorbance of the supernatant at 414 nm was
measured. Hemorrhage in BALF was expressed as absorbance
at 414 nm.
5.2.3. AE-3763 effects in LPS-induced lung injury model
5.2.3.1. (a) Lung edema. LPS (50 lg/kg) was administrated intrat-
racheally to the animal model. AE-3763 infusion into the jugular
vein was started immediately after LPS treatment and continued
up to 6 h after LPS treatment. The wet weight of the whole lung
was measured 6 h after LPS treatment and corrected for each indi-
vidual animal based on body weight.
5.2.3.2. (b) Leukocyte infiltration. LPS (50 lg/kg) was adminis-
trated intratracheally to the animal model. AE-3763 infusion into
the jugular vein was started immediately after LPS treatment and