1-Oxacephem-based human chymase inhibitors: Discovery of stable inhibitors in human plasma

Article abstract of DOI:10.1016/S0960-894X(00)00489-3

1-Oxacephem derivatives were evaluated as a novel series of chymase inhibitors. The structure-activity relationship studies of 1-oxacephems led to compounds 15, which exhibited 27 nM inhibition of human chymase and improvement of stability in human plasma (t(1/2) 1.5 h). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00489-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2403±2406
1-Oxacephem-Based Human Chymase Inhibitors:
Discovery of Stable Inhibitors in Human Plasma
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 evaluated as a novel series of chymase inhibitors. The structure±activity relationship stu-
dies of 1-oxacephems led to compounds 15, which exhibited 27 nM inhibition of human chymase and improvement of stability in
human plasma (t1/2 1.5 h). # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Chemistry
In the preceding paper,1 we reported a novel series of 1-
oxacephem-based human chymase inhibitors as poten-
tial therapeutic agents for cardiovascular diseases and
chronic in¯ammation following ®brosis such as cardiac,
renal, and pulmonary ®brosis.2 1-Oxacephem derivative
1 exhibited potent and selective inhibition against
human chymase. However, in vivo evaluation of candi-
date 1 (IC50 6 nM) was limited by its lability in human
plasma (t1/2<10 min). The results of in vivo assay of
non-peptidyl chymase inhibitors3 have been scarcely
reported, and one reason is their lability in human
plasma. In order to ®nd a drug candidate, it is essential
to enhance the stability of the active compound in
human plasma. We thus studied to improve stability in
human plasma while maintaining high activity against
human chymase.
1-Oxacephem derivatives 1 and 7±13 were prepared by
the same synthetic route as described in the preceding
paper.1 1-Oxacephems 14±19 bearing 4-carboxy-
phenylmethyl ester at the 4-position were prepared
starting from 3 (Scheme 1). Amine 31 was treated with a
variety of benzoyl chlorides 2a±f to give 7b-amides 4a±f.
4-Diphenylmethyl esters 4a±f were deprotected by AlCl3
and anisole to yield acids 5a±f. Compounds 6a±f were
obtained by esteri®cation of Na salt of 5a±f with
diphenylmethyl 4-bromomethylbenzoate, which was
prepared by treatment with 4-bromomethylbenzoic acid
and diphenyldiazomethane. Deprotection of diphenyl-
methyl esters 6a±f (TFA, anisole, CH2Cl2) provided 1-
oxacephem derivatives 14±19.
Results and Discussion
Two paths may be suggested for the destruction of 1-
oxacephems in human plasma. One is a simple hydro-
lysis of the 4-ester, and the other is cleavage of the b-
lactam ring shown in Figure 1. We assumed the latter
was the major destruction path, because our preliminary
investigation showed that no carboxylic acid was detected
when oxacephem ester was treated in human plasma.
Figure 1 shows that a nucleophile such as the hydroxy
group in a serine residue in various proteins in human
plasma attacks the b-lactam carbonyl of 1-oxacephem,
leading to cleavage of the b-lactam ring and suggests
inhibition against human chymase and destruction in
*Corresponding author Tel.: +81-6-6458-5861; fax: +81-6-6458-0987;
e-mail: yasunori.aoyama@shionogi.co.jp, toshiro.konoike@shionogi.
co.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00489-3

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Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2403±2406
Scheme 1. Reagents and conditions: (a) pyridine, CH2Cl2; (b) AlCl3, anisole, CH2Cl2; (c) (i) NaHCO3, H2O±MeOH; (ii) BrCH2C6H4-4-CO2CHPh2,
DMF; (d) TFA, anisole, CH2Cl2.
Figure 1. Proposed mechanism for destruction of 1-oxacephems in human plasma.
Table 1. E€ects of modi®cations at the 30-, 4- and 7b-positions
Compound
X
Y
Z
Stability (%)a
IC50 (mM)
1
7
8
9
10
11
12
13
14
C6H4-2-OMe
CH2C6H4-4-OH
CH2C6H4-4-OH
CH2C6H4-4-OH
CH2C6H4-4-OH
CH2C6H4-4-OH
CH2C6H4-4-NMe2
C6H4-2-OMe
OCH2C6H4-3-Me
OCH2C6H4-3-Me
OCH2C6H4-3-Me
OCH2C6H4-3-Me
OCH2C6H4-3-Me
OCHPh2
OCHPh2
OCHPh2
OCH2C6H4-4-CO2H
A
A
B
C
H
B
B
B
B
11
5
0.006
0.072
0.050
0.077
>10
0.25
0.41
0.55
0.019
10
13
59
40
53
73
26
C6H42-OMe
aThe percent of test compounds remaining in human plasma after 30 min was observed.
human plasma via the same mechanism as cleavage of
b-lactam ring. Accordingly, increase of potency is con-
sidered to be inversely proportional to improvement of
stability in human plasma. In considering this problem,
we investigated the enhancement of stability without
drastic decrease of the potency by modi®cations at the
30-, 4- and 7b-positions (Tables 1 and 2).4,5
First, we investigated the substituent e€ect at the 30-
position of oxacephem as shown in Table 1. When sub-
stituent Z was not the leaving group, the stability
improved (10 versus 7, 8 and 9). This result is in accor-
dance with our proposed destruction mechanism of 1-
oxacephems in human plasma (Fig. 1). However, com-
pound 10 showed no signi®cant inhibition against

Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2403±2406
2405
human chymase. Consequently, modi®cations at the 30-
position did not lead to enhancement of stability with
maintenance of the potency, and we guessed that mod-
i®cations at 30-position were not suitable for achieving
our purpose.
A model for the inhibition of compound 15 of human
chymase, using atomic coordinates from the published
X-ray structure of the enzyme,6 is shown in Figure 2.7,8
In this model, the inhibitor 15 is bound to the active site
of the enzyme with hydroxy of Ser195 and the resulting
hemiketal oxygen is in the oxyanion hole formed by the
backbone amide NH of Ser195 and Gly193. The benz-
amide substituent at the 7b-position is fully enclosed by
the residues of the S1 pocket and capped with the side
chain of Phe191. The benzylester substituent at the 4-
position occupies the hydrophobic prime site region
Next, 4-substituents were evaluated. 4-Ester 1 was much
less stable in human plasma than the corresponding 4-
sodium carboxylate. The electronegative e€ect of the 4-
ester group has been known to increase reactivity at the
b-lactam carbonyl group and we think this is the reason
for the poor stability of compound 1. The 4-ester group
is needed to increase the potency as shown in the pre-
ceding report.1 We next investigated a variety of 4-esters
and found that the diphenylmethyl ester group enhanced
stability (8 versus 11, 12 and 13). However, these com-
pounds 11, 12 and 13 displayed 40±90-fold less potency
than benzyl ester 1 presumably owing to steric hin-
drance. Further investigation showed 4-para-carboxy-
phenylmethyl ester 14 to be e€ective for improvement of
stability, although this ester was not as good as diphe-
nylmethyl ester as a stabilizing group. Compound 14
was only threefold less potent than compound 1.
Table 3. Selectivity of 15 as an inhibitor of human chymase com-
pared to other serine proteases
Enzyme
IC50 (mM)
Enzyme
IC50 (mM)
Chymase
0.027
0.16
0.28
3.6
Trypsin
Elastase
Plasmin
2.1
9.5
>10
a-Chymotrypsin
Cathepsin G
Thrombin
Finally, a substituent at the 7b-position was optimized
for 4-para-carboxyphenylmethyl ester oxacephems
(Table 2). In order to enhance the stability of 14, we
modi®ed ortho-alkoxy substituents. Ethoxy derivative
15 possessed good potency and stability in human
plasma (t1/2 1.5 h). Because there are only minor di€er-
ences between compounds 14±19 as regards the inhibi-
tory activity against human chymase, we supposed that
ortho-substituents especially play an important role in
stability in human plasma.
Additional enzymatic work showed that compound 15
was a selective inhibitor, displaying weak or no inhibi-
tion of several other serine proteases (Table 3).4
Table 2. Modi®cation at 7b-position
Compound
R
Stability (%)a
IC50 (mM)
14
15
16
17
18
19
OMe
OEt
OPr-n
OPr-iso
OBu-iso
OPh
26
87 (1.5 h)b
0.019
0.027
0.024
0.037
0.026
0.057
26
42
39
63
aThe percent of test compounds remaining in human plasma after
30 min was observed.
bThe number in parentheses represents the half-life period.
Figure 2. Docking model of inhibitor 15 into human chymase. (a)
Surface representation. (b) Schematic drawing.

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Y. Aoyama et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2403±2406
(S10) of the enzyme. The terminal carboxylic acid elec-
trostatically interacts with the side chain of Lys40. This
model accounts for the potency of compound 15 against
human chymase.
3. (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.
4. See ref 1 for the enzyme assay.
Conclusion
5. The stability assay in human plasma was performed as fol-
lows. Test compounds dissolved in DMSO were added at
20 mg/mL to human plasma and incubated at 37 ꢀC. Aliquots
were removed at intervals of 30 min, quenched with threefold
volume of AcOH:MeOH (1:1) or AcOH:MeCN (1:1), and
analyzed by reverse-phase HPLC after removing proteins. The
disappearance of compound 15 was then monitored for the
half-life period and the results ®tted a ®rst-order decay curve
from which the reported t1/2 value was derived.
We described here the discovery of 1-oxacephem-based
human chymase inhibitors with both high inhibitory
activities and moderate stability in human plasma. As a
compromise between potency and stability, 4-para-car-
boxyphenylmethyl ester was found to be optimum.
Compound 15 was found to possess high potency
against human chymase (IC50 27 nM) and stability in
human plasma (t1/2 1.5 h), and accordingly, is a prom-
ising candidate for in vivo assay.
6. Pereira, P. J. B.; Wang, Z. M.; Rubin, H.; Huber, R.; Bode,
W.; Schechter, N. M.; Strobl, S. J. Mol. Biol. 1999, 286, 163.
(PDB code, 1pjp)
7. The docking study for the binding of cephalosporin ester to
human leukocyte elastase was performed at Merck (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.; Wes-
ton, H.; Zimmerman, M.; Doherty, J. B. J. Med. Chem. 1990,
33, 2522).
Acknowledgements
We wish to thank Prof. M. Miyazaki, Osaka Medical
College, for the kind gift of human chymase.
8. Molecular modeling was performed using the SYBYL6.6
software package. The three-dimensional model of compound
15 was constructed based on the published X-ray structure of
an oxacephem compound (ref, Shiro, M.; Nakai, H.; Onoue,
H.; Narisada, M. Acta Cryst. 1980, B36, 3137). The model of
inhibitor 15 was manually docked into the active site of
human chymase on the basis of the structure±activity rela-
tionships of oxacephem derivatives against human chymase
and the docking study for the binding of cephalosporin ester
to human leukocyte elastase.7 The resulting hemiketal inter-
mediate (human chymase±15 complex) was energy-minimized
using the Tripos force ®eld.
References and Notes
1. Aoyama, Y.; Uenaka, M.; Konoike, T.; Iso, Y.; Nishitani,
Y.; Kanda, A.; Naya, N.; Nakajima, M. Bioorg. Med. Chem.
Lett. 2000, 10, 2397.
2. (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.