Development of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as orally active human chymase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.03.085

A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones was designed, synthesized, and evaluated as human chymase inhibitors. From this series, we identified several compounds which were effective, via oral administration, in a mou

Full text of DOI:10.1016/j.bmcl.2007.03.085

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3435–3439
Development of 6-benzyl substituted 4-aminocarbonyl-1,4-
diazepane-2,5-diones as orally active human chymase inhibitors
Hiroshi Maruoka,* Tsuyoshi Muto,* Taisaku Tanaka, Seiichi Imajo, Yoshiaki Tomimori,
Yoshiaki Fukuda and Takashi Nakatsuka
Biomedical Research Laboratories, Daiichi Asubio Pharma Co., Ltd, 1-1-1 Wakayamadai Shimamoto-cho, Mishima-gun,
Osaka 618-8513, Japan
Received 28 November 2006; revised 8 March 2007; accepted 27 March 2007
Available online 30 March 2007
Abstract—A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones was designed, synthesized, and evalu-
ated as human chymase inhibitors. From this series, we identified several compounds which were effective, via oral administration,
in a mouse model of chronic dermatitis.
Ó 2007 Elsevier Ltd. All rights reserved.
In the preceding paper,1 we reported 6-benzyl substi-
tuted 4-arylsulfonyl-1,4-diazepane-2,5-diones as a novel
scaffold for human chymase inhibitors, and the inhibitor
1 (Fig. 1) and its analogues exhibited potent and selec-
tive inhibition of human chymase. However, in the
course of our investigation into the application of these
inhibitors as therapeutic agents, we found that 1 was rel-
atively unstable in aqueous solution at neutral pH (t1/2
,
1.1 h, Table 1) and was hydrolyzed at the 5-position
amide of the 1,4-diazepane ring, to ring-opening prod-
ucts. It seems essential to enhance the compound stabil-
ity in order for it to be a drug candidate, since such a
physicochemical property may hamper pharmaceutical
development. In this report, we describe efforts to iden-
tify potent, stable, and orally active human chymase
inhibitors, the 4-aminocarbonyl-1,4-diazepane-2,5-dione
derivatives (Schemes 1 and 2).
Figure 1. Replacement of electron-withdrawing group on the 4-
position of the 1,4-diazepane-2,5-dione scaffold.
4-position, determines the ability for acylation of the
active site serine residue, as well as instability in
aqueous media. Based on the above considerations,
we attempted to reduce the reactivity of the lactam
carbonyl at the 5-position by changing the substituent
at the 4-position to a less electron-withdrawing group.
In the case of pyrrolidine-5,5-trans-lactam-based cyto-
megalovirus protease inhibitors2 and azetidinone-based
tryptase inhibitors,3 the substituents on the lactam
nitrogen affected the stability of the compounds, with
the following order: CONHMe ꢀ COMe > SO2Me.
Therefore, we selected the aminocarbonyl group as a
less electron-withdrawing group and synthesized related
compounds (Fig. 1).
From a structural viewpoint of compound 1, chymase
may be converted to an inactivated form, the acylated
enzyme, formed by attack from the activated hydroxyl
group of the catalytic Ser195 on the lactam carbonyl at
the 5-position of 1. It is likely that the reactivity of the
lactam carbonyl at the 5-position, which is activated by
the electron-withdrawing arylsulfonyl group at the
Keywords: Chymase; Inhibitor.
*
Corresponding authors. Tel.: +81 75 962 8481; fax: +81 75 962 6448.
(T.M.); e-mail addresses: maruoka.hiroshi.ri@asubio.co.jp; muto.
tsuyoshi.ba@asubio.co.jp
4-Aminocarbonyl-1,4-diazepane-2,5-dione-containing
chymase inhibitors 14–29 listed in Tables 1 and 2 were
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.085

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H. Maruoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3435–3439
Scheme 1. Reagents and conditions: (a) glycine ethyl ester HCl, EDCI HCl, HOBt, Et3N, CH2Cl2, rt, 2 h; (b) 4 M HCl/EtOAc, rt, 40 min; (c) 2,4,6-
(MeO)3PhCHO, NaBH(OAc)3, THF, rt, 1.5 h; (d) aq NaOH, MeOH, 60 °C, 2.5 h; (e) EDCI HCl, HOBt, Et3N, DMF, CH2Cl2, rt, 18 h; (f) H2,
Pt(sulfide)–C, THF, rt, 18 h; (g) BuLi, THF, ꢁ78 °C, 20 min then 4-NO2PhOCOCl (for 7a) or 2-ClPhOCOCl (for 7b), ꢁ78 °C, 1 h; (h) amines, Et3N,
DMAP, DMF, 0 °C, 18 h; (i) 1 M HCl/AcOH, rt, 18 h; (j) trimethylsilyldiazomethane, EtOAc, MeOH, rt, 10 min; (k) optical resolution using chiral
HPLC (CHIRALCEL OD).
prepared from b-amino acid 31 via the racemic 7-mem-
bered lactam rac-6 as a common intermediate (Scheme
1). Condensation of 3 with glycine ethyl ester hydrochlo-
ride, followed by deprotection of the Boc group,
provided amine 4. Introduction of the 2,4,6-trimeth-
oxybenzyl group (TMB) to 4 by reductive alkylation,
and subsequent hydrolysis of the ester moiety, gave ami-
no acid 5. Lactamization of 5, followed by catalytic
hydrogenation, afforded the 7-membered lactam rac-6.
Then, rac-6 was converted to the carbamate rac-74 by
sequential treatment with n-butyllithium, then 4-nitro-
phenylchloroformate or 2-chlorophenylchloroformate.
Coupling reactions of rac-7 with amines furnished com-
pounds 8, which were deprotected to yield compounds
14–29, except for 23 and 27. Compounds 23 and 27 were
prepared by esterification of the corresponding carbox-
ylic acids 22 and 26, respectively, with trimethylsilyldia-
zomethane. For compounds 22–29, the diastereomers
derived from the stereogenic centers of the 6-position
of the diazepane ring and the amine moiety could be
separated by chromatography at the purification step
of 8. The optically active compounds 25c–f and 26c,d
were synthesized in a similar manner from optically pure
7-membered lactams (R)-6 or (S)-6, which were
prepared by optical resolution of rac-6 using a chiral
HPLC column (CHIRALCEL OD).
Table 1 shows the chymase inhibitory activity and
stability6 in aqueous solution at pH 7.0 of compounds
14–21. As expected, the stability of these compounds
was increased compared with compound 1. Among
these, benzylaminocarbonyl derivative 14 showed
Phenylpropylamines 9a–c for the synthesis of 22, 26, and
28 were prepared from aldehydes 10, which were con-
verted to secondary alcohols 11 by sequential manipula-
tion (protection and Grignard reaction) (Scheme 2).
Mitsunobu reaction of 11 using phthalimide, followed
by deprotection, afforded 9a–c. Other phenylpropyl-
amine derivatives 9d–f used for the synthesis of 24, 25,
and 29 were prepared from halobenzoic acid derivatives
12. Palladium-catalyzed arylation of nitropropane5 with
suitably protected 12 afforded a-arylnitropropanes,
which were converted to propiophenones 13 by a Nef
reaction. A two-step sequence of reactions (oxime
formation and reduction) yielded amines 9d–f.
Scheme 2. Reagents and conditions: (a) N,N0-diisopropyl-O-tert-
butylisourea, t-BuOH, CH2Cl2, rt, 16 h; (b) EtMgBr, CuI, Et2O,
ꢁ23 °C, 30 min; (c) BnBr, K2CO3, DMF, rt, 2 h; (d) phthalimide,
DEAD, PPh3, THF, rt, 18 h; (e) hydrazine hydrate, MeOH, reflux, 1 h;
(f) nitropropane, Pd2(dba)3, 2-(di-tert-butylphosphinyl)-20-methylbi-
phenyl, K3PO4, DME, 100 °C, 24 h; (g) aq NaOH, MeOH, rt, 16 h; (h)
KO t-Bu, 1,4-dioxane, then 1 M aq HCl; (i) hydroxylamine HCl,
NaOAc, EtOH, reflux, 3 h; (j) H2, Pd–C, EtOH, rt, 2–8 h.

H. Maruoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3435–3439
3437
prime site amino acids such as Lys40, Phe41, and
Arg143 (Table 2). To elucidate the influence of the rela-
tive configuration derived from the two chiral centers,
the 6-position of the diazepane ring and the 10-position
of the amine moiety, two possible racemic stereoisomers
were synthesized and evaluated.7 As shown in Table 2,
(6R*,10R*)-isomers were generally more potent than
the co0rresponding (6S*,10R*)-isomers. In the series of
(6R*,1 R*)-isomers (left part of Table 2), installation of
a carboxyl group at the para- (22a) or meta-position
(26a) of the phenyl ring resulted in an increase in the
inhibitory activity, but the corresponding methyl esters
(23a, 27a) were less potent. These results indicated that
the carboxyl group may have been involved in the ionic
interaction with Arg143 and/or Lys40 located at the
prime site region. In contrast to our previous findings
with 3-phenylsulfonylquinazoline-2,4-diones,8 introduc-
tion of an additional substituent, such as a hydroxyl
or amino group to 22a or 26a, did not further improve
the potency (cf. 24a, 25a, 28a, and 29a). In the case of
(6S*,10R*)-isomers (right part of Table 2), only com-
pound 25b, which has a 3-amino and a 4-carboxyl
group, showed high inhibitory activity. To examine the
effects of the absolute configurations of the stereogenic
centers of compounds 25a, 25b, and 26a on inhibitory
activity, we synthesized and evaluated the optically ac-
tive compounds 25c–f and 26c,d (Table 3). In all cases,
the (10R)-isomers 25c, 25e, and 26c were found to be
more potent than the corresponding (10S)-isomers. Fur-
thermore, all the active enantiomers 25c, 25e, and 26c
showed high stability in aqueous solution (Table 3),
and selectivity against other serine proteases, including
chymotrypsin and chymotrypsin-type protease cathep-
sin G (Table 4).
Table 1. Inhibitory activity against recombinant human chymase and
stability in aqueous solution of compounds 1 and 14–21
Compound R1
R2
IC50 (l M) Stabilityb (t1/2
)
a
1c
0.034
4.9
>100
1.1
6.8
14c
15c
16c
17d
18d
19d
20d
21c
H CH2Ph
Me CH2Ph
>48
12.9
6.2
H
H
H
H
H
H
CH2CH2Ph 44.5
CH(Me)Ph
CH(Et)Ph
5.3
2.8
6.3
8.0
CH(i-Pr)Ph 3.0
CH(n-Pr)Ph 69.3
7.6
16.5
CHPh2
>100
a For details of the assay conditions, see Ref. 1.
b For details of the assay conditions, see Ref. 6.
c Racemates.
d Mixture of diastereomers.
moderate activity, but N-methylation at the urea moiety
(15) markedly diminished the activity. These results indi-
cate that an intramolecular hydrogen bond between
urea NH and carbonyl oxygen at the 5-position is criti-
cal to fix the conformation of inhibitors to interact with
enzyme. Introduction of an alkyl group at the benzylic
position of compound 14 resulted in retention or slight
enhancement of the inhibitory activity (17–19), whereas
substitution with a longer alkyl (20) or phenyl group
(21), or chain elongation (16), decreased the activity.
These results were confirmed by computer-assisted
docking studies (vide infra) that indicated that the ben-
zylaminocarbonyl moiety of 14 was situated around the
S10 and S20 regions of the enzyme, and the substituent at
the benzylic position interacted with a narrow and
hydrophobic S10 pocket. We next attempted to intro-
duce the substituents on the phenyl ring of compound
18, with the intention of making an interaction with
We investigated the in vivo activity of 25c, 25e, and 26c
in a mouse model of chronic dermatitis,9 in which the
enzymatic activity of chymase is involved in the develop-
ment of skin edema. As shown in Table 5, oral adminis-
tration of each compound at a dose of 10 mg/kg/day
significantly inhibited the dermatitis.
Table 2. Inhibitory activity against recombinant human chymase of compounds 22–29
Compounda
R1
R2
IC50 (lM)
Compounda
R1
R2
IC50 (lM)
b
b
22a
23a
24a
25a
26a
27a
28a
29a
COOH
COOMe
COOH
COOH
H
H
0.47
2.5
22b
23b
24b
25b
26b
27b
28b
29b
COOH
COOMe
COOH
COOH
H
H
5.0
14.2
1.8
H
OH
H
OH
0.46
0.5
NH2
NH2
0.34
4.3
COOH
COOMe
COOH
COOH
0.54
2.6
COOH
COOMe
COOH
COOH
H
OH
H
OH
51.4
4.1
0.41
0.63
NH2
NH2
6.1
a All compounds are racemates, and relative stereochemistry is represented.
b For details of the assay conditions, see Ref. 1.

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H. Maruoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3435–3439
Table 3. Inhibitory activity against recombinant human chymase and stability in aqueous solution of optically active compounds 25c–f and 26c,d
a
Compound
R1
R2
Configuration
10-
IC50 (lM)
Stabilityb (t1/2
)
6-
25c
25d
25e
25f
26c
26d
COOH
COOH
COOH
COOH
H
NH2
NH2
R
S
S
R
R
S
R
S
R
S
R
S
0.30
>10
0.17
>10
0.24
>10
6.7
c
—
6.1
NH2
NH2
c
—
7.4
COOH
COOH
c
—
H
a For details of the assay conditions, see Ref. 1.
b For details of the assay conditions, see Ref. 6.
c Not measured.
Table 4. IC50 valuesa of inhibition of human chymase and other serine
proteinases for compounds 25c, 25e, and 26c
Enzyme
Compound
25c
25e
26c
Human chymase
Bovine a-chymotrypsin
Human cathepsin G
Bovine trypsin
0.30
>100
16.0
0.17
23.0
28.0
>100
>100
0.24
56.0
8.0
>100
>100
>100
>100
Human elastase
a For details of the assay conditions, see Ref. 1.
Table 5. Effect of compounds 25c, 25e, and 26c on ear edema in a
mouse model of chronic dermatitisa
Compound
% inhibitiond
25cb
29.7%**
35.8%**
22.9%*
54.6%**
25eb
26cb
Prednisolonec
a See Ref. 9 and 10 for details.
Figure 2. Docking structure of compound 26c in the active site of
human chymase.
b Each compound was orally administered at 10 mg/kg once a day
during the experimental period.
c Prednisolone was administered intraperitoneally at 10 mg/kg once a
day during the experimental period.
clarify the binding mode of this class of inhibitors,
further studies including the X-ray crystal structure
analysis of chymase with inhibitors are currently under
way and the results will be reported in the near future.
d Inhibition ratio of the ear thickness at 24 h after the sixth painting of
DNFB.
* P < 0.05 versus vehicle (Student’s t test).
** P < 0.01.
In conclusion, we have reported 4-aminocarbonyl-1,4-
diazepane-2,5-diones as being potent, stable and, orally
active human chymase inhibitors. Compounds 25c, 25e,
and 26c were orally active in a mouse model of chronic
dermatitis, in which chymase activity may be involved.
These results indicate that 4-aminocarbonyl-1,4-diaze-
pane-2,5-dione is a promising scaffold for chymase
inhibitors that are possible therapeutic candidates for
diseases such as atopic dermatitis.
A docking structure11 of compound 26c in the active site
of human chymase (Fig. 2) indicated that: (1) the 5-
chloro-2-methoxybenzyl group occupied the S1 pocket;
(2) the 1,4-diazepane ring was located in the proper
position at which the amide-NH at the 1-position
formed a hydrogen bond with the Ser214 backbone
amide carbonyl; (3) the phenylpropylamine moiety was
located in the prime site region of the enzyme, and the
ethyl substituent occupied the hydrophobic S10 pocket
constructed by Lys40, Cys42, and Cys58 side chains;
and (4) the carboxyl group on the phenyl ring electro-
statically interacted with the Arg143 side chain, and this
ionic interaction may have accounted for the increase in
the inhibitory activity of the benzoic acid derivatives. To
Acknowledgments
We are grateful to Drs. H. Fukami and T. Nishihara for
their helpful advice and valuable discussion during this
study.

H. Maruoka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3435–3439
3439
8. Fukami, H.; Imajo, S.; Ito, A.; Kakutani, S.; Shibata, S.;
Sumida, M.; Tanaka, T.; Niwata, S.; Saitoh, M.; Kiso, Y.;
Miyazaki, M.; Okunishi, H.; Urata, H.; Arakawa, K.
Drug Des. Discov. 2000, 17, 69.
9. It has been reported10 that repeated application of 2,4-
dinitrofluorobenzene (DNFB) to mouse ear causes edema,
and intraperitoneal injection of prednisolone (corticoste-
roid) or other chymase inhibitors during the experimental
period inhibits dermatitis, judging by skin thickness.
10. Tomimori, Y.; Muto, T.; Fukami, H.; Saito, K.; Horik-
awa, C.; Tsuruoka, N.; Saito, M.; Sugiura, N.; Yamashi-
ro, K.; Sumida, M.; Kakutani, S.; Fukuda, Y. Lab. Invest.
2002, 82, 794.
References and notes
1. Tanaka, T.; Muto, T.; Maruoka, H.; Imajo, S.; Harukazu,
F.; Tomimori, Y.; Fukuda, Y.; Nakatsuka, T. Bioorg.
Med. Chem. Lett., in press. doi:10.1016/j.bmcl.
2007.03.038.
2. Borthwick, A. D.; Exall, A. M.; Haley, T. M.; Jackson, D.
L.; Mason, A. M.; Weingarten, G. G. Bioorg. Med. Chem.
Lett. 2002, 12, 1719.
3. Sutton, J. C.; Bolton, S. A.; Hartl, K. S.; Huang, M.-H.;
Jacobs, G.; Meng, W.; Ogletree, M. L.; Pi, Z.; Schum-
acher, W. A.; Seiler, S. M.; Slusarchyk, W. A.; Treuner,
U.; Zahler, R.; Zhao, G.; Bisacchi, G. S. Bioorg. Med.
Chem. Lett. 2002, 12, 3229.
4. During our study, we found that 2-chlorophenylcarba-
mate rac-7b was superior to 4-nitrophenylcarbamate
rac-7a, since rac-7b was sufficiently stable during
chromatographic purification and had sufficient reactivity
to amines.
5. Vogl, E. M.; Buchwald, S. L. J. Org. Chem. 2002, 67, 106.
6. Each compound was incubated at 37 °C in 0.1 M phos-
phate buffer (pH 7.0) at a concentration of 10 lM for the
following times: 0, 4, and 8 h. The ratio of the remaining
compounds was measured by HPLC analysis.
7. The relative configuration of 26b was determined by X-ray
crystal structure analysis of the corresponding intermedi-
ate 8. As for the other compounds, the stereochemistry
was tentatively determined by comparison of the chro-
matographic behavior of the separated diastereomers with
that of 26a and 26b. Furthermore, we confirmed the
relative configuration of compound 25e by X-ray crystal
structure analysis.
11. The structure of the human chymase 1KLT determined
˚
at 1.9 A resolution was obtained from PDB, and the co-
crystallized inhibitor (benzylsulfinic acid) was removed
from the crystal structure. The side chain conformation
of His57 was modified to interact with Asp102 and Ser195
(catalytic triad), and the missing side chain coordinates
of Lys109
, ,
Arg137 and Asn245 were generated with
reference to another chymase structure, 1PJP. The
structure of 26c was prepared using MacroModel
(Schrodinger) and was docked into the substrate-binding
site of the enzyme structure using the GOLD (version
3.0) program.12 For the docking run, all variable
parameters had default values, and the docking area
was defined using the OG atom of Ser195 as a center,
˚
with a radius of 15 A. The docking structure of the
compound was optimized using the OPLS2005 force field
implemented in MacroModel.
12. Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245,
43.