Design, synthesis and biological activity of novel non-peptidyl endothelin converting enzyme inhibitors, 1-phenyl-tetrazole-formazan analogues

Article abstract of DOI:10.1016/S0960-894X(02)00149-X

A novel non-peptidyl endothelin converting enzyme inhibitor was obtained through a pharmacophore analysis of known inhibitors and three-dimensional structure database search. Analogues of the new inhibitor were designed using the structure-activity relationship of known inhibitors and synthesized. In anesthetized rats, intraperitoneal administration of the analogues suppressed the pressor responses induced by big endothelin-1.

Full text of DOI:10.1016/S0960-894X(02)00149-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1275–1278
Design, Synthesis and Biological Activity of Novel Non-Peptidyl
Endothelin Converting Enzyme Inhibitors,
1-Phenyl-tetrazole-formazan Analogues
Kazuto Yamazaki, Hirohiko Hasegawa, Kayo Umekawa, Yasuyuki Ueki,
Naohito Ohashi and Masaharu Kanaoka*
Research Division, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98 Kasugadenaka, Konohona-ku, Osaka 554-0022, Japan
Received 29 November 2001; accepted 21 February 2002
Abstract—A novel non-peptidyl endothelin converting enzyme inhibitor was obtained through a pharmacophore analysis of known
inhibitors and three-dimensional structure database search. Analogues of the new inhibitor were designed using the structure–
activity relationship of known inhibitors and synthesized. In anesthetized rats, intraperitoneal administration of the analogues
suppressed the pressor responses induced by big endothelin-1. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
To develop an oral drug for chronic diseases, we set out
to find a new non-peptidyl small molecule lead. We have
identified as new leads novel 1-phenyl-tetrazole-for-
mazan analogues through pharmacophore analysis by
superposition of the binding features of 1 and 2 fol-
lowed by a combination of 3-dimensional structure
database search using the pharmacophore and incor-
poration of some substituents consisted of 2. Intraperi-
toneal administration of these leads in anesthetized rats
suppressed the pressor responses induced by big ET-1,
and the potency was consistent with that anticipated
from in vitro activity.
Since endothelin-1 (ET-1) was discovered in 1988 as a
21 amino acid peptide causing a potent and continuous
blood vessel contraction, many studies have been con-
ducted to uncover its mode of action.1 ET-1 is bio-
synthesized by cleavage of the latent precursor big ET-1
at Trp21-Val22 bond by endothelin converting enzyme
(ECE), a zinc metalloprotease.2 ET-1 receptor antago-
nists or ECE inhibitors are expected to be effective
against such diseases as hypertension, cardiovascular
diseases and renal failure.3
Phosphoramidon (1, Fig. 1), a potent inhibitor of neu-
tral endopeptidase (NEP), inhibits ECE with IC50 of
690 nM.4 Some peptide-mimetic analogues of 1 are also
reported as ECE inhibitors.5 SM-19712 (2, Fig. 1) inhi-
bits ECE with IC50 of 42 nM, 16-fold more potent than
1.6 SM-19712 was expected to be an orally available
drug for chronic diseases since it is a non-peptidyl small
molecule. In fact orally administered 2 suppressed the
pressor response induced by big ET-1 in anesthetized
rats.6 However, the in vivo potency of 2 was not
consistent with that anticipated from in vitro activity,
probably because of strong binding to the plasma
proteins.6
Pharmacophore Analysis
To superpose molecular structures for pharmacophore
analysis, conformational coordinates of the compounds
in solution were determined. Conformational analysis
*Corresponding author. Tel.: +81-6-6466-5194; fax: +81-6-6466-
5484; e-mail: kanaoka@sumitomopharm.co.jp
Figure 1. Chemical structures of phosphoramidon (1) and SM-19712 (2).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00149-X

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K. Yamazaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1275–1278
of 2 was performed with the Macromodel, version 4.5.7
The Monte Carlo search was employed to identify the
local energy minimum conformations. At each Monte
Carlo step, the geometry was optimized using AMBER*
force field7 with a distance-dependent dielectric constant
in aqueous solution of GB/SA model,7 and with a 0.001
kcal/mol energy gradient convergence criterion. In
3000 Monte Carlo steps, two lowest energy conforma-
tions and 22 independent low-energy ones within 10
kcal/mol were obtained (Fig. 2).
hydrogen bond features were defined as projection
points at the position of 300 nm to the lone pair direc-
tion, in order to simulate the actual environment better.
The phosphorous and carboxyl groups of 3 and the
sulfonamide group of 2 were treated as deprotonated.
Twelve features were assigned to 2 and nine to 3 (Fig.
3).
The best superposition between the features of 2 and 3
was searched exhaustively using all the conformations
with the DISCO module of the SYBYL, version 6.3.11
Consequently two independent superpositions were
obtained. In one of the two, seven features were super-
posed within 0.11 nm r.m.s. (Fig. 4), and in the other
eight features were within 0.14 nm r.m.s. (not shown).
Since the molecular shapes in the former superposition
were more similar, the pharmacophore model was
adopted for the molecular design of new lead.
On the other hand, 1 had a large number of low-energy
conformations. Fortunately, some crystal structures of
the derivatives of 1 complexed with thermolysin (TL),
one of the zinc protease family members, were repor-
ted.8 C terminal domain of ECE, consisted of 304 amino
acids and containing the active site, has about 20%
sequence identity to TL.9 Therefore we assumed that
active conformation of 1 on ECE was similar to that on
TL. To simplify the superposition analysis, a model
molecule 3 (Fig. 3) was actually used in place of 1. In 3
the sugar moiety of 1 was replaced with a benzyl group
and the leucyl residue with a phenylalanyl, replacements
justified by the structure–activity relationship infor-
mation of 1 against ECE.10 The crystal structure coor-
dinates in TL were used for the main chain and the beta
carbons of 3. Coordinates of other atoms were opti-
mized locally under the same conditions as that used for
conformational analysis of 2.
Molecular Design
The superposition model suggested that the nitrogen
atom of pyrazole of 2 would interact with the zinc atom
of ECE. It also suggested that the phenyl rings adjacent
to pyrazole and sulfonyl were at P10 and P20 sites
respectively in the active site of ECE.
To design a novel ECE inhibitor, a three-dimensional
structure database was searched with the pharmacophore
information using the ISIS, version 2.2.12 The three
substructures mentioned above in the relative arrange-
ment shown in Figure 5 was used as a query, and the
ACD database containing commercially available com-
pounds12 was searched. The tolerance level in the search
was set at 0.03 nm. Seventeen compounds were found as
hits by the search. Only S13946-7 (10a, Salor Inc.)
Three enzyme binding features, hydrogen bond accep-
tor, hydrogen bond donor and hydrophobic center,
were assigned manually to both 2 and 3 using the
DISCO module of the SYBYL, version 6.3.11 The
Figure 2. 24 independent low-energy conformations of 2.
Figure 4. The superposition model of compounds 2 and 3.
Figure 5. The query for the three-dimensional structure database
search.
Figure 3. Enzyme binding features assigned to compounds 2 and 3.

K. Yamazaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1275–1278
1277
among the hits was consistent with the structure–activity
relationship of 2 (in-house data).
The compound identified by the database search, 10a,
showed an ECE inhibitory activity (IC50=10 mM).
The p-chloro substituted compound 10b showed less
potency, but the alkyl substituted analogues 10c–i
showed more potent activities than 10a. Among them
10e showed the highest activity (IC50=2.6 mM).
Although 10e is 2 orders of magnitude less potent than
2, it is about 1/3 as potent as 1, thus we consider it a
good lead for optimization.6
It was suggested that the phenyl ring attached to the
diazo group of 10a was equivalent to the phenyl ring
attached to the sulfonyl of 2. The phenyl ring of 2 was
substituted with a chlorine at the p-position. Thus,
compounds 10b–i were designed by substituting the
p-position of the phenyl ring of 10a.
Intraperitoneal administration (10 mg/kg ip) of 10e in
anesthetized rats suppressed the pressor responses
induced by big ET-1.6 Actually, the plasma level of
ET-1 was lowered to 42.9ꢀ4.1% of that observed by
intraperitoneal administration of saline (n=3).
Chemistry
The compound 10a was obtained from Salor Inc. (Catalog
Code. S13946-7). Derivatives of 10a (10b–i) were syn-
thesized as outlined in Scheme 1. The common inter-
mediate, 9, was prepared by known methods.13 The
starting material 5 was commercially available. In the
last process, the objective compounds were obtained by
the azo-coupling of 9 with the corresponding p-substituted
phenyldiazonium chloride.
Protein binding rate of 10e in rat serum was determined
as 99%, identical to that of 2, though protein binding at
this high potency level could not be determined pre-
cisely. A minute difference of the plasma protein bind-
ing could have a large effect on ECE inhibitory activity.
The structures of 10b–i were verified by LC–MS and
NMR spectroscopy. The geometrical isomerism of these
compounds was not determined. However, it was
known that the main product is cis–trans form because
of making intramolecular hydrogen bonding.13
The proposed pharmacophore model suggests that 2
binds at P10 and P20 in the active site of ECE and the
methyl group at 3-position of pyrazole is directed
toward P1 and P2. It is known that the replacement of
this methyl group with a phenylalkoxylcarbonylmethyl
group leads to more potent activity.14 On the other
hand, it was reported that the benzyloxycarbamate (Z)
group at P2 site of 1 was suitable for ECE inhibitory
activity.15 This correspondence at P2 site verifies further
the pharmacophore model. We will report the struc-
ture–activity relationship of 2 in more detail in another
article.
Results and Discussion
The inhibitory activities of 10a–i against ECE in the
solubilized fraction from rat lung membrane were
shown in Table 1.6
Conformational profile of 10a was analyzed with the same
conditions as 2, giving six independent conformations
Table 1. In vitro assay results for compounds 10a–10i
Compd
R
In vitro assaya
IC50 (mM)
1
2
0.69
0.042
10.0
37%b
8.4
3.8
2.6
3.4
2.7
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
Cl
Me
Et
nPr
iPr
sBu
tBu
Scheme 1. Synthesis of compounds 10a–10i. Reagents, conditions and
yields: (a) Pb(OAc)2, KOH, H2O, 100 ꢁC, 5 min, 80%; (b) NaN3,
NH4Cl, DMF, 80 ꢁC, 2.5 h, 66%; (c) NaNO2, HCl, H2O, 0 ꢁC, 1 h;
(d) Zn, AcOH, H2O, 10ꢁC, 30 min, 28% (two steps); (e) PhCHO, EtOH,
rt, 10 min, 70%; (f) p-R-PhN2Cl, pyridine, EtOH, rt, 5 min, 50–96%.
3.8
3.6
Cyclohexyl
an=2.
b% inhibition at 10 mM.

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K. Yamazaki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1275–1278
Acknowledgements
The authors thank Ms. Masako Sumitomo and Yuka
Yamana for the enzyme assay.
References and Notes
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Figure 6. Superposition model of 2 and 10a.
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with minimum energy. One of the minimum energy
conformations with a potential energy 3.9 kcal/mol
higher than the lowest was similar to 2 in the topology
of three rings used as the query in the three-dimensional
structure database search (Fig. 6). None of the for-
mazane analogues has a negative charge fragment that
corresponds to the sulfonamide in 2. On the other hand,
the phenyl ring next to hydrazone of 10a corresponds to
no fragment in 2 but to the side chain of phenylalanine
in 3. These results suggest that the absence of the nega-
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Conclusions
New non-peptidyl ECE inhibitors were found through a
pharmacophore analysis of two known ECE inhibitors
and a rational design based on the analysis. Intraper-
itoneal administration to anesthetized rats of one of
these inhibitors suppressed the pressor responses
induced by big ET-1. The inhibitors found in this study
apparently lacked a negative ionizable fragment that
can be a zinc chelater. Only a few other ECE inhibitors
without a negative ionizable fragment are known to
date.16 This study demonstrated that rational drug
design of metalloprotease inhibitors without a chelater
is relevant for lead discovery.