Synthesis and biological activity of potent heterocyclic thiol-based inhibitors of endothelin-converting enzyme-1

Article abstract of DOI:10.1016/S0960-894X(02)00683-2

Directed screening of metalloprotease inhibitors identified CGS 30084 (1) as a potent inhibitor of endothelin-converting enzyme-1 (ECE-1) in vitro (IC₅₀=77 nM). Herein we report the syntheses and biological activities of analogues containing modified biphenyl moieties, bearing heterocyclic proximal rings. Compound 20, the thioacetate ethyl ester prodrug derivative of compound 19a, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.

Full text of DOI:10.1016/S0960-894X(02)00683-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3059–3062
Synthesis and Biological Activity of Potent Heterocyclic Thiol-
Based Inhibitors of Endothelin-Converting Enzyme-1
Fariborz Firooznia,* Candido Gude, Kenneth Chan, Jenny Tan, Cynthia A. Fink,
Paula Savage, Michael E. Beil, Charles W. Bruseo, Angelo J. Trapani and Arco Y. Jeng
Metabolic and Cardiovascular Diseases, Novartis Institute for Biomedical Research, Summit, NJ 07901, USA
Received 19 June 2002; accepted 5 August 2002
Abstract—Directed screening of metalloprotease inhibitors identified CGS 30084 (1) as a potent inhibitor of endothelin-converting
enzyme-1 (ECE-1) in vitro (IC50=77 nM). Herein we report the syntheses and biological activities of analogues containing modified
biphenyl moieties, bearing heterocyclic proximal rings. Compound 20, the thioacetate ethyl ester prodrug derivative of compound
19a, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.
# 2002 Elsevier Science Ltd. All rights reserved.
Potent inhibitors of endothelin-1 (ET-1) production are
considered to be attractive potential therapeutic agents
for the treatment of disorders such as cerebral vasos-
pasm, stroke, asthma, and cardiac and renal failure,1ꢀ4
which are linked with elevated ET-1 levels.5 Endothelin-
converting enzyme-1 (ECE-1) catalyzes the post-trans-
lational conversion of big ET-1 to ET-1,6 thus present-
ing a logical target for the design of therapeutic agents
that regulate the production of ET-1 in vivo.7,8
focusing on compounds containing a heteroaryl prox-
imal aromatic ring.
We had previously noted that the introduction of
2-substituted distal aromatic rings, which lead to an
increased twisting angle between the two aryl moieties,
significantly reduced (ca. 5-fold)10 the in vitro potency
of ECE-1 inhibitors. We hoped that the introduction of
a nitrogen atom in place of a proximal CH group in
compounds such as 6a would impart a higher degree of
co-planarity between the two rings, thus improving
potency. The in vitro activity of 6a (IC50=77ꢁ5 nM for
the dl mixture) provided some support for our
hypothesis.
In a prior communication,9 we reported the identifica-
tion of CGS 30084 (1) (Fig. 1, IC50=77 nM) as a start-
ing lead structure. Modifications carried out at the thiol
end of CGS 30084 led to the discovery of compound 2,
which displayed higher potency (IC50=11 nM) as an
ECE-1 inhibitor. More importantly, the corresponding
prodrug (3) was found to be a long-acting orally avail-
able inhibitor of ECE-1 activity in vivo. We then turned
our attention to modifications of the biphenyl portion of
CGS 30084, initially focusing on variations at the distal
aromatic ring.10 These efforts resulted in the identifica-
tion of compound 4 (IC50=120 nM for the dl mixture),
and its prodrug 5, which also possessed a superior in
vivo profile compared to CGS 30084 and its prodrug.10
The present report describes our continued efforts in
further exploring the SAR of the biphenyl moiety,
*Corresponding author. Tel.: +1-908-277-5343; fax: +1-908-277-
2405; e-mail: fariborz.firooznia@pharma.novartis.com
Figure 1. Various derivatives of CGS 30084.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00683-2

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F. Firooznia et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3059–3062
Table 1. Modifications at the distal ring of thiols bearing a proximal
pyridine ring
Compd
Ar
ECE-1 IC50 (nM)a
6a
6b
6c
6d
6e
6f
6g
6h
6i
Ph
2-Furyl
3-Furyl
2-Thienyl
3-Thienyl
77ꢁ5
43ꢁ7
39ꢁ10
49ꢁ13
30ꢁ5
Pyridin-3-yl
2-Methoxy-Ph
3-Amino-Ph
3-Nitro-Ph
3-Acetamido-Ph
41ꢁ2
Figure 2. Comparison of the inhibition of the big ET-1 pressor
response by compounds 19a and CGS 30084 (iv in anesthetized rats),
at 15 min after dosing.
700ꢁ150
64ꢁ4
98ꢁ17
130ꢁ18
6j
aValues are meanꢁSEM.
Reasoning that the incorporation of heteroatoms in 4
was the key contributor to its more favorable pharma-
cokinetic profile,10 we also hoped to discover ECE-1
inhibitors with improved in vivo activity when replacing
the proximal phenyl ring with a pyridine moiety.
Indeed, analogues of 6a showed a significantly greater
inhibition of the big ET-1 pressor response11 upon iv
administration in anesthetized rats when compared with
CGS 30084 (Fig. 2).
reactions with the appropriate arylboronic acids or aryl
stannanes, respectively.
The compounds thus prepared were tested for their
ability to inhibit ECE-1 activity in vitro. The experi-
mental details for the assays utilized have been pre-
viously described.11 The results are summarized in
Table 1.
The target molecules 6a–j were prepared originally as
diastereomeric mixtures, using the synthetic approach
previously described,9 starting from various aryl-sub-
stituted pyridyl alanines 9a–j (Scheme 1). These starting
amino acids were in turn synthesized via the alkylation
of the protected glycine derivative 7 with bromide 8
(accessible through bromination of 2-bromo-5-methyl-
pyridine), followed by Suzuki or Stille cross-coupling
Table 1 shows the SAR for ECE-1 inhibition by thiol
derivatives in which the biphenyl ring of CGS 30084 is
replaced with a proximal pyridine ring, and various
distal aromatic moieties. As expected, all unsubstituted
distal aromatic rings (6a–f) led to improved potency,12
while 2-substitution of the distal ring (6g) resulted in
Scheme 1. Preparation of ECE-1 inhibitors containing proximal pyridine rings.

F. Firooznia et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3059–3062
Table 2. Modifications at the thiol end of compound 6e
3061
shown in Scheme 2. The protected glycine precursors
16a–b were synthesized via alkylation of 7 followed by
Suzuki cross-coupling with 2- or 3-thiopheneboronic
acid. Deprotection of the imines under acidic condi-
tions, followed by acetylation, led to the formation of
the key derivatives 17a–b, which were then resolved
using alcalase, according to previously described pro-
cedures.13 The resulting N-acetyl l-amino acids 18a–b
were deprotected, esterified, and then converted to the
desired inhibitors 19a–b as described in Scheme 1.
Compd
R
R0
ECE-1 IC50 (nM)a
6e
i-Pr
i-Bu
n-Pr
H
n-Bu
H
H
H
n-Pr
H
n-Bu
H
30ꢁ5
14a
14b
14c
14d
14e
14f
14g
9.8ꢁ1.1
27ꢁ2.3
82ꢁ14
24ꢁ2.3
56ꢁ6.8
55ꢁ13
23ꢁ5.8
Compound 19a (IC50=9.5ꢁ2.1 nM) showed a superior
in vivo profile (Fig. 2) when compared with 19b (data
not shown). In anesthetized rats, intravenous, bolus
injection of 19a as its HCl salt at 10 mg/kg inhibited the
big ET-1 (1 nmol/kg, iv)-induced pressor response by
61% at 15 min after dosing (Fig. 2). Under similar con-
ditions, CGS 30084 (administered iv at 10 mg/kg) was
only able to inhibit the big ET-1 pressor response by
40%. The in vivo effect of 19a is dose-dependent, as a
dose of 20 mg/kg blocked the big ET-1-induced increase
in mean arterial pressure (MAP) by 77% at 15 min after
dosing (Fig. 2).
H
MeS(CH2)2
Cyclohexyl-CH2
H
aValues are meanꢁSEM.
significant loss of ECE-1 inhibitory activity. Electronic
effects on the distal ring do not appear to play a sig-
nificant role in potency, although there is a slight pref-
erence noted for electron-donating groups (6h) over
electron-withdrawing substituents (6i–j).
We next evaluated 20, the thioacetate methyl ester pro-
drug of 19a. Conscious rats were dosed with either
vehicle or 20 and 2 h later challenged with big ET-1 at
0.3 nmol/kg iv. The ECE-1 inhibitory effect of 20 is
shown in Figure 3 as the area under the curve (AUC) of
the change in MAP produced during the first 30 min
following the intravenous, bolus injection of big ET-1.
Upon oral administration at doses between 3 and 30
mg/kg, 20 inhibited the big ET-1-induced pressor
response by 65–90% at 2 h post-dosing. There were no
significant differences in the degree of inhibition pro-
duced by the 3 doses of 20.
Encouraged by these results, we chose to further inves-
tigate derivatives of 6e, and focused on the substituents
at the carbon bearing the thiol group. The results of
these investigations are summarized in Table 2. As pre-
viously noted,9 there appears to be a slight preference
for the (S)-stereochemistry at the chiral center bearing
the thiol functionality (see 14b and 14c). A variety of
alkyl chains, both linear and branched, are tolerated,
with the isobutyl group providing the highest in vitro
potency (14a). The presence of a heteroatom in the alkyl
chain (14f) does not appear to have a detrimental effect
on the in vitro ECE-1 inhibitory activity.
Most importantly, 20 showed significantly long dura-
tion of action when inhibiting the effects of ECE-1 in
vivo (Fig. 4). At 0.5, 2, 4, and 8 h after an oral dose of
20 at 3 mg/kg, the pressor effects of big ET-1 were
blocked by 90, 82, 70, and 34%, respectively (p<0.05 at
all time points). However, no significant effect was
observed at 24 h after dosing.
Finally, we chose to synthesize the chiral (l-pyr-
idylalanine) derivatives of 14a and its 2-thienyl ana-
logue, in order to evaluate their biological activity in
vivo. The chiral inhibitors 19a–b were prepared as
Scheme 2. Synthesis of chiral heterocyclic ECE-1 inhibitors.

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F. Firooznia et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3059–3062
References and Notes
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Figure 3. Inhibition of big ET-1 pressor response by 20, prodrug of
19a (po in conscious rats).
9. Fink, C. A.; Moskal, M.; Firooznia, F.; Hoyer, D.;
Symonsbergen, D.; Wei, D.; Qiao, Y.; Savage, P.; Beil, M.;
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11. The big ET-1 pressor response in anesthetized rats is
measured as the change in mean arterial pressure produced by
big ET-1 (1 nmol/kg) administered iv. For more details, see:
Wallace, E. M.; Moliterni, J. A.; Moskal, M. A.; Neubert,
A. D.; Marcopoulos, N.; Stamford, L. B.; Trapani, A. J.;
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1513.
12. As previously described (see ref 10), only the l-stereo-
isomers of the biarylalanines exhibit ECE inhibitory activity.
The compounds prepared as dl mixtures contain 50% inac-
tive diastereomers.
13. (a) Chenevert, R.; Letourneau, M.; Thiboutot, S. Can. J.
Chem. 1990, 68, 960. (b) Tyagi, O. D.; Boll, P. M.; Parmar,
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Figure 4. Duration of action for ECE-1 inhibition by 20.
In conclusion, through variations at the proximal and
distal rings of the biphenyl region of CGS 30084, we
were able to identify 20, a potent, long acting, and
orally available inhibitor of ECE-1 activity in vivo.