Synthesis and structure-activity relationships of a new class of 1-oxacephem-based human chymase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00488-1

1-Oxacephem derivatives were synthesized and evaluated as a novel series of chymase inhibitors. Structure-activity relationship studies of 1-oxacephems led to compound 34, which exhibited 6 nM inhibition of human chymase and high selectivity for human chymase compared to other serine enzymes. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00488-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2397±2401
Synthesis and Structure±Activity Relationships of a New Class of
1-Oxacephem-Based Human Chymase Inhibitors
Yasunori Aoyama,a,* Masaaki Uenaka,a Toshiro Konoike,b,* Yasuyoshi Iso,a
Yasuhiro Nishitani,a Akiko Kanda,a Noriyuki Nayac and Masatoshi Nakajimac
aShionogi Research Laboratories, Shionogi & Co., Ltd, Fukushima-ku, Osaka 553-0002, Japan
bShionogi Research Laboratories, Shionogi & Co., Ltd, Amagasaki, Hyogo 660-0813, Japan
cShionogi Research Laboratories, Shionogi & Co., Ltd, Futaba-cho, Toyonaka, Osaka 561-0825, Japan
Received 29 June 2000; accepted 9 August 2000
AbstractÐ1-Oxacephem derivatives were synthesized and evaluated as a novel series of chymase inhibitors. Structure±activity
relationship studies of 1-oxacephems led to compound 34, which exhibited 6 nM inhibition of human chymase and high selectivity
for human chymase compared to other serine enzymes. # 2000 Elsevier Science Ltd. All rights reserved.
Chemistry
Introduction
Human chymase is a chymotrypsin-like serine protease
that is stored in the secretory granules of mast cells.1
Although the physiological and pathological roles of
chymase have not been fully elucidated, this enzyme has
been shown to convert angiotensin I to angiotensin II
with greater eciency than angiotensin I converting
enzyme.2 Chymase has also been shown to participate in
histamine release from mast cells,3 activation of pre-
cursor interleukin-1b,4 and cleavage of type I procolla-
gen5 and progelatinase B.6 Thus, chymase is speculated
to play an important role in cardiovascular diseases and
chronic in¯ammation following ®brosis such as cardiac,
renal, and pulmonary ®brosis.7 Chymase inhibitors8 are
thought to be potentially useful as tools for elucidating
the physiological function of chymase and therapeutic
agents.
1-Oxacephem derivatives were prepared as shown in
Schemes 1 and 2. Amine 2 was prepared by a procedure
established in our laboratories.11 First, 7b-substituted 1-
oxacephems 3±13 were obtained by treatment with
amine 2 and a variety of acid chlorides prepared from the
corresponding acids by a usual method (oxalyl chloride,
DMF), in the presence of pyridine. Next, 4-substituents
15±25 were prepared in the following way. Deprotection
(AlCl3, anisole, CH2Cl2 99%) of diphenylmethylester 1
provided compound 14. Esteri®cation of Na salt of 14
with a variety of alkyl halides and amidation of mixed
anhydride prepared from 14 with three amines gave 4-
substituted 1-oxacephem esters 15, 16, 20±25 and amides
17±19, respectively. Next, 30-substituents 27±32 were
obtained by the following procedures. Reduction of 21
(Mg, CH2Cl2±AcOH) gave exomethylene 26 (47%) and
3-methyl 27 (32%). Protection of phenol 26 (CCl3COCl,
pyridine, CH2Cl2), dichlorination of exomethylene (Cl2,
CCl4, hn) followed by dehydrochlorination and depro-
tection (NaHCO3, H2O, 100% from 26) provided
chloromethyl 28.12 30-Substituted 1-oxacephems 29±32
were obtained by treatment with 28 and the corre-
sponding thiols or tetrazol in the presence of i-Pr2NEt.
The most potent compound 34 was prepared from 11 by
the same procedures described above (Scheme 2).
Screening of the Shionogi compound collection led to
identi®cation of the 1-oxacephem derivative 19 as a
chymase inhibitor (IC50 0.25 mM, Fig. 1), which was
prepared for a project on Latamoxef,10 an antibacterial
agent developed at our company. This is the ®rst report
of b-lactam compounds inhibiting human chymase. We
describe herein the structure±activity relationships by
chemical modi®cations at the 30-, 4- and 7b-positions of
the 1-oxacephem nucleus.
Results and Discussion
*Corresponding authors. Tel.: +81-6-6458-5861; fax: +81-6-6458-0987;
e-mail: yasunori.aoyama@shionogi.co.jp (Y. Aoyama); e-mail: toshiro.
konoike@shionogi.co.jp (T. Konoike).
First, we examined the substituent e€ects on the phenyl
group at 7b-position as shown in Table 1.13 In the para-
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00488-1

2398
Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2397±2401
Among the methoxy-substituted benzamide derivatives,
ortho-substituent 11 was more potent than the corre-
sponding para- 9 and meta-substituents 10. Substituents
at the ortho-position, such as chlorine 12, methoxy-
carbonyl 13 and hydrogen (unsubstituted) 8 decreased
the activity moderately. ortho-Methoxy 11 gave the highest
IC50 value (0.55 mM) among the benzamide derivatives.
Next, a substituent at the 4-position was optimized for
the 7b-para-hydroxyphenylacetamide as shown in Table
2. Ester derivatives 1, 15 and 16 were more potent than
amide derivatives 17±19. Especially, esters 1 and 16 dis-
played 44- and 110-fold increase compared to the corre-
sponding amides 17 and 18, respectively. Because 4-
benzyl ester 16 was the most active, we prepared a variety
of substituted benzyl esters. Introduction of a methyl
group into meta-position 21 in 4-benzyl ester derivative
resulted in a threefold increase of potency over that of
16, while introduction into the para- and ortho-position
20 and 22 led to a mild decrease. With regard to meta-
Figure 1.
substituted phenylacetamide derivatives, unsubstituted
compound 3 and hydrophilic substituents 4 and 5
showed approximately the same activity as the lead
compound 1. Hydrophobic substituents such as bro-
mine 6 and phenyl 7 remarkably decreased potency. The
hydroxy group 1 appears optimal (IC50 0.25 mM).
Scheme 1. Reagents and conditions: (a) XCOCl, pyridine, CH2Cl2; (b) AlCl3, anisole, CH2Cl2, 99%; (c) (i) sodium 2-ethylhexanoate, MeOH±
EtOAc; (ii) RCH2Br or MeI, DMF; (d) (i) t-BuCOCl, Et3N, THF; (ii) NMM, HNRR0; (e) Mg, CH2Cl2±AcOH, 26 (47%), 27 (32%); (f) (i)
CCl3COCl, pyridine, CH2Cl2; (ii) Cl2, CCl4, hn; (iii) NaHCO3, H2O, 100%; (g) ZH, i-Pr2NEt, MeCN.
Scheme 2. Reagents and conditions: (a) AlCl3, anisole, CH2Cl2; (b) (i) sodium 2-ethylhexanoate, MeOH±EtOAc; (ii) BrCH2C6H4-3-Me, DMF; (c)
Mg, CH2Cl2±AcOH; (d) (i) Cl2, CCl4, hn; (ii) NaHCO3, H2O; (e) 5-mercapto-1-tetrazoleacetic acid, Et3N, MeCN.

Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2397±2401
2399
Table 1. Modi®cations at 7b-position
Compound
X
IC50 (mM)
Compound
X
IC50 (mM)
1
3
4
5
6
7
CH2C6H4-4-OH
CH2C6H5
CH2C6H4-4-OMe
CH2C6H4-4-NMe2
CH2C6H4-4-Br
CH2C6H4-4-Ph
0.25
0.30
0.30
0.41
6.80
>10
8
9
10
11
12
13
C6H5
1.40
>10
7.31
0.55
1.70
3.50
C6H4-4-OMe
C6H4-3-OMe
C6H4-2-OMe
C6H4-2-C1
C6H4-2-CO2Me
Table 2. Modi®cations at 4-position
Compound
Y
IC50 (mM)
Compound
Y
IC50 (mM)
1
OCHPh2
OMe
OCH2Ph
NHCHPh2
NHCH2Ph
N(CH2)4
0.25
1.60
0.14
11.0
15.6
>1 0
20
21
22
23
24
25
OCH2C6H4-4-Me
OCH2C6H4-3-Me
OCH2C6H4-2-Me
OCH2C6H4-3-Br
OCH2C6H4-3-CF3
OCH2C6H4-3-CO2Me
0.17
0.05
0.43
0.07
0.05
1.65
15
16
17
18
19
substituents, methyl substituent 21 gave approximately
the same activity as bromine 23 and tri¯uoromethyl 24.
Methoxycarbonyl group 25 led to a 33-fold loss of
potency versus 21. meta-Methyl 21 showed the best
result (IC50 0.05 mM) among the 4-substituted deriva-
tives. 4-Carboxylate as shown in latamoxef (Fig. 1) is
necessary for expression of the antibacterial activity, but
also dramatically decreases the potency against human
chymase. On the other hand, 4-benzyl ester increased
the activity against human chymase and led to loss of
antibacterial activity (data not shown).
Table 3. Modi®cations at 30-position
Compound
Z
IC50 Compound
(mM)
Z
IC50
(mM)
21
27
0.05
>10
30
31
32
0.08
0.44
4.00
Finally, in order to enhance the potency of 21, we
modi®ed the 30-substituents (Table 3). Compound 21
was more active than compounds 27, 28, 31 and 32, but
roughly showed the same potency as compounds 29 and
30. 30-Thiotetrazoleacetic acid 29 was designed and pre-
pared in order to improve water solubility and conse-
quently facilitate in vivo evaluation.
H
28
29
Cl
0.15
0.07
In order to investigate the inhibitory mechanism of 1-
oxacephems against human chymase, we performed a
series of kinetic studies.14 Judging from the results of
kinetic studies and reports concerning b-lactam inhibitors
of human leukocyte elastase,15,16 we assumed that the
inhibition mechanism of 1-oxacephems against human
chymase was as presented in Figure 2 which shows that
the active site serine residue (serine 195) in the enzyme
approaches the b-lactam ring of 1-oxacephem, followed
by generation of an acyl-enzyme.
Table 2 shows that amide derivatives 17 and 18 were less
potent than 4-ester derivatives 1 and 16. Two reasons
can be considered for this: (1) a more electron-
withdrawing character of the ester group than the amide
group may facilitate the nucleophilic attack of active
serine 195 to b-lactam carbonyl and (2) 4-electron-rich
amide carbonyl or amide-NH may prevent hydrogen

2400
Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2397±2401
Figure 2. Proposed mechanism for inhibition of human chymase by 1-oxacephem.
Table 4. Selectivity of 34 as an inhibitor of human chymase com-
pared to other serine proteases
3. He, S.; Gaca, M. D. A.; McEuen, A. R.; Walls, A. F. J.
Pharmacol. Exp. Ther. 1999, 291, 517.
4. Mizutani, H.; Schechter, N.; Lazarus, G.; Black, R. A.;
Kupper, T. S. J. Exp. Med. 1991, 174, 821.
Enzyme
IC50 (mM)
Enzyme
IC50 (mM)
5. Ko€ord, M. W.; Schwartz, L. B.; Schechter, N. M.; Yager,
D. R.; Diegelmann, R. F.; Graham, M. F. J. Biol. Chem. 1997,
272, 7127.
6. Fang, K. C.; Raymond, W. W.; Blount, J. L.; Caughey, G.
H. J. Biol. Chem. 1997, 272, 25628.
Chymase
0.006
0.16
0.23
0.43
Trypsin
Elastase
Plasmin
0.44
>10
>10
a-Chymotrypsin
Cathepsin G
Thrombin
7. (a) Shiota, M.; Fukamizu, A.; Takai, S.; Okunishi, H.;
Murakami, K.; Miyazaki, M. J. Hypertens. 1997, 15, 431. (b)
Hamada, H.; Terai, M.; Kimura, H.; Hirano, K.; Oana, S.;
Niimi, H. Am. J. Respir. Crit. Care. Med. 1999, 160. 1303. (c)
Takai, S.; Yuda, A.; Jin, D.; Nishimoto, M.; Sakaguchi, M.;
Sasaki, S.; Miyazaki, M. FEBS Lett. 2000, 467, 141.
8. (a) Hayashi, Y.; Iijima, K.; Katada, J.; Kiso, Y. Bioorg.
Med. Chem. Lett. 2000, 10, 199. (b) Groutas, W. C.; Schech-
ter, N. M.; He, S.; Yu, H.; Huang, P.; Tu, J. Bioorg. Med.
Chem. Lett. 1999, 9, 2199. (c) Iijima, K.; Katada, J.; Hayashi,
Y. Bioorg. Med. Chem. Lett. 1999, 9, 413. (d) Fukami, H.;
Okunishi, H.; Miyazaki, M. Curr. Pharm. Des. 1998, 4. 439
and references therein.
abstraction of serine 195 by histidine 57 or the approach
of serine 195 to b-lactam carbonyl.17 Figure 2 depicts
the mechanism at work when the 30-substituent Z is a
good leaving group. According to Table 3, when 30-
substituent Z was not the leaving group (hydrogen, 27),
no activity was observed. This result supports the pro-
posed mechanism.
Considering the match±mismatch between 30, 4 and 7b-
substituents, several hybrid compounds were prepared
based on the results presented in Tables 1±3. For-
tunately, we found hybrid compound 34 to possess the
highest potency (IC50 6 nM) and to be 40-fold more
active than lead compound 1 (Scheme 2). In compound
34, the combination of substituents at 30, 4 and 7 posi-
tions may match synergetically. Enzymatic work
showed that 34 was an extremely selective inhibitor,
causing weak or no inhibition of several other serine
proteases (Table 4).18 In addition, the Na salt of 34
possessed water solubility (500 mg/mL). Consequently,
it was considered to be suitable for in vivo evaluation.
9. Lead compound 1 was prepared as follows: (a) (i) dihydro-
pyrane, TsOH, CH2Cl2; (ii) NaOH, acetone, 94% from 35; (b)
(i) POCl3, CH2Cl2; (ii) 2, pyridine; (iii) HCl±acetone, 76%
from 36.
10. Narisada, M.; Yoshida, T.; Onoue, H.; Ohtani, M.;
Okada, T.; Tsuji, T.; Kikkawa, I.; Haga, N.; Satoh, H.; Itani,
H.; Nagata, W. J. Med. Chem. 1979, 22, 757.
11. Uyeo, S.; Kikkawa, I.; Hamashima, Y.; Ona, H.; Nishi-
tani, Y.; Okada, K.; Kubota, T.; Ishikura, K.; Ide, Y.;
Nakano, K.; Nagata, W. J. Am. Chem. Soc. 1979, 101, 4403.
12. Yoshioka, M.; Tsuji, T.; Uyeo, S.; Yamamoto, S.; Aoki,
T.; Nishitani, Y.; Mori, S.; Satoh, H.; Hamada, Y.; Ishitobi,
H.; Nagata, W. Tetrahedron Lett. 1980, 351.
Conclusion
We have described here the synthesis of 1-oxacephem
derivatives and their inhibitory activities against human
chymase. We found that compound 34 possesses high
potency and selectivity against human chymase.
13. The human chymase assay was performed as follows: ®rst,
human chymase was puri®ed according to the method of
Takai (ref, Takai, S.; Siota, N.; Sakaguchi, M.; Muraguchi,
H.; Matsumura, E.; Miyazaki, M. Clin. Chim. Acta 1997,
265, 13). The puri®ed chymase was preincubated with test
compounds dissolved in DMSO at 37 ꢀC for 30 min in 0.1 M
Tris±HCl (pH 8.0) containing 1.8M NaCl, after then the chy-
mase reaction was started by adding succinyl-Ala-Ala-Pro-Phe-
p-nitroanilides (Sigma Chemical Co.). The change of absorbance
was measured at 405nm after 2 h incubation at 37ꢀC. The IC50
value was calculated from the inhibition of p-nitroaniline for-
mation at each concentration of the test compound.
Acknowledgement
We wish to thank Prof. M. Miyazaki, Osaka Medical
College, for the kind gift of human chymase.
14. Detailed data will be reported elsewhere.
References and Notes
15. Mascaretti, O. A.; Boschetti, C. E.; Danelon, G. O.; Mata,
E. G.; Roveri, O. A. Curr. Med. Chem. 1995, 1, 441 and
references therein.
16. See ref 8d with respect to the catalytic triad (Ser-195, His-
57, Asp-102) in human chymase.
1. (a) Caughey, G. H. J. Respir. Crit. Care Med. 1994, 150,
S138. (b) Wang, Z.; Walter, M.; Selwood, T.; Rubin, H.;
Schechter, N. M. Biol. Chem. 1998, 379, 167.
2. Ihara, M.; Urata, H.; Kinoshita, A.; Suzumiya, J.; Sasa-
guri, M.; Kikuchi, M.; Ideishi, M.; Arakawa, K. Hypertension
1999, 33, 1399.
17. Cephalosporin ester and amide sulfones were tested to
determine the structure±activity relationships for inhibition of

Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2397±2401
2401
human leukocyte elastase (ref, Finke, P. E.; Ashe, B. M.;
Knight, W. B.; Maycock, A. L.; Navia, M. A.; Shah, S. K.;
Thompson, K. R.; Underwood, D. J.; Weston, H.; Zimmer-
man, M.; Doherty, J. B. J. Med. Chem. 1990, 33, 2522).
18. The inhibitory e€ects of compound 34 on the enzymatic
activities of seven serine proteases were evaluated using the
puri®ed enzymes and chromogenic substrates. The enzymes
and substrates used here were as follows: N-succinyl-Ala-Ala-
Pro-Phe-pNA (Bachem) for bovine pancreatic a-chymotrypsin
(Sigma) and human cathepsin G (Wako); chromozyme TH
(Boehringer Mannheim) for human thrombin (Sigma); N-suc-
cinyl-Ala-Ala-Phe-Arg-pNA (Bachem) for bovine pancreatic
trypsin (Sigma); N-succinyl-Ala-Ala-Val-pNA (Bachem) for
human neutrophil elastase (Athens Research and Technology,
Inc.); chromozym PL (Boehringer Mannheim) for human
plasmin (Sigma). The assay bu€er used here was as follows:
50 mM Tris±HCl (pH=8.0) containing 2 mM CaCl2 for a-
chymotrypsin, trypsin and elastase; 50 mM Tris±HCl (pH=
7.5) containing 2 mM CaCl2 for cathepsin G; 50 mM Tris±HCl
(pH=7.5) containing 50 mM NaCl for plasmin; 0.1 M Tris±
HCl (pH=8.0) containing 10 mM CaCl2 and 0.1 M NaCl for
thrombin.