Design and synthesis of potent thiol-based inhibitors of endothelin converting enzyme-1

Article abstract of DOI:10.1016/S0960-894X(00)00403-0

Through directed screening of compounds prepared as metalloprotease inhibitors a compound, CGS 30084, that had potent endothelin converting enzyme-1 (ECE-1) in vitro inhibitory activity (IC₅₀=77nM) was identified. Herein we report the synthesis and optimization of ECE-1 inhibitory activity of additional analogues from this lead. Compound 3c, the thioacetate methyl ester derivative of compound 4c, was found to be a long acting inhibitor of ECE-1 activity in rats after oral administration. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00403-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2037±2039
Design and Synthesis of Potent Thiol-Based Inhibitors of
Endothelin Converting Enzyme-1
Cynthia A. Fink,* Michael Moskal, Fariborz Firooznia, Denton Hoyer,
David Symonsbergen, Dongchu Wei, Ying Qiao, Paula Savage, Michael E. Beil,
Angelo J. Trapani and Arco Y. Jeng
Metabolic and Cardiovascular Diseases, Novartis Institute for Biomedical Research, Summit, NJ 07901, USA
Received 25 April 2000; accepted 7 July 2000
AbstractÐThrough directed screening of compounds prepared as metalloprotease inhibitors a compound, CGS 30084, that had
potent endothelin converting enzyme-1 (ECE-1) in vitro inhibitory activity (IC50=77 nM) was identi®ed. Herein we report the
synthesis and optimization of ECE-1 inhibitory activity of additional analogues from this lead. Compound 3c, the thioacetate
methyl ester derivative of compound 4c, was found to be a long acting inhibitor of ECE-1 activity in rats after oral administration.
# 2000 Elsevier Science Ltd. All rights reserved.
Endothelin-1 (ET-1), a 21-amino acid peptide originally
isolated from porcine aortic endothelial cells, is one of
the most potent vasoconstrictors known.1 Abnormally
high plasma and tissue levels of ET-1 have been detected
in a number of disease states, including hypertension,
cerebral vasospasm, asthma, congestive heart failure,
and chronic and acute renal failure.2 5 Modulation of
the biological e€ects of this peptide has been the target
of much recent e€ort in the pharmaceutical commu-
nity.6 Two approaches have been investigated to block
the biological actions of ET-1: antagonism of the ET-1
G-protein coupled receptors ETA and ETB and inhibi-
tion of endothelin converting enzyme-1 (ECE-1), a zinc-
containing metalloprotease distributed in the vascular
endothelium and in brain tissue. ECE-1 is responsible
for catalyzing the post-translational conversion of big
ET-1, a 38-amino acid precursor to ET-1.7,8 We chose
the latter approach in our e€orts to ®nd means to inhi-
bit the detrimental e€ects of elevated ET-1. A directed
screening of historical compounds originally prepared
as zinc-metalloprotease inhibitors led to the identi®cation
of CGS 30084. We then initiated a chemistry program
using this compound as our starting point and strived to
further improve its ECE-1 inhibitory activity in vitro. In
this paper we will describe modi®cations carried out at
the thiol end of the molecule, and the biological activity
of the resulting compounds. Modi®cations made to
other portions of CGS 30084 will be the subject of
future publications.
The target molecules were prepared as outlined in
Scheme 1. Treatment of 4-phenyl-phenyl alanine ester
with N-BOC or N-Cbz-protected cycloleucine, 1-[3-(di-
methylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDCI), and 1-hydroxybenzotriazole (HOBt) or 1-hy-
droxy-7-azabenzotriazole (HOAt) followed by removal
of the N-BOC or N-Cbz groups a€orded the amine
hydrochlorides or free bases 2, respectively. Subsequent
treatment of these amines (2) with a variety of a-bromo
acids (6), EDCI, and HOAt provided the a-bromo
dipeptides.9 Treatment then with the potassium salt of
thio acetic acid a€orded the thioacetates 3. The esters
were hydrolyzed by treatment with 1 N sodium
hydroxide or lithium hydroxide in degassed methanol to
give the free thiol acids 4. The required a-bromoacids
could be prepared in good yield from the appropriate
*Corresponding author. Tel.: +1-908-277-7532; fax: +1-908-277-2405;
e-mail: cynthia.®nk@pharma.novartis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00403-0

2038
C. A. Fink et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2037±2039
Scheme 1. (a) EDCI, HOBt or HOAt, Et3N, CH2Cl2, CbzNH-cycloleucine or BOCNH-cycloleucine, rt (>95%); (b) HCl (gas) or 10% Pd/C (95±99%);
(c) EDCI, HOAt, Et3N, 6, CH2Cl2 (41±93%); (d) AcSK (20±90%); (e) 1 N LiOH or NaOH, methanol (65±98%); (f) 48% HBr, KBr, NaNO2 (56±92%).
amino acids via diazotization with sodium nitrite and
treatment with hydrobromic acid and potassium bro-
mide. The free thiol dipeptides prepared are shown in
Table 1. These compounds were tested for their ability
to inhibit human ECE-1 activity in vitro. The experi-
mental details for the assay have been reported pre-
viously.10 As can be seen from Table 1, the isopropyl
group of CGS 30084 can be replaced with other n-alkyl
and branched alkyl groups leading to compounds with
similar or improved ECE-1 inhibitory activity (e.g.,
compounds 4c and 4g). The simple methylene com-
pound (4a) had very little ECE-1 inhibitory activity. It
appears that the (S)-stereochemistry at the center bear-
ing the thiol group is preferred for ECE-1 inhibitory
activity in vitro (compare, for example, compounds 4c
and 4d). The introduction of a heteroatom in the alkyl
chain, as can be seen in comparing compounds 4c and
4m, does not diminish ECE-1 inhibition. The free
hydroxyl analogue of 4m, compound 4l, was however
less potent in vitro. The only aryl containing analogue
synthesized, compound 4p, was less active than many of
the alkyl and branched alkyl analogues prepared. We
selected one of the most potent derivatives, compound
Table 1. In vitro activity for the a-thiol derivatives
Compound
R
R0
ECE IC50, (nM)a
CGS 30084
-CH(Me)2
H
-Me
-Propyl
H
-Butyl
H
H
H
H
H
-Propyl
77b
26%
62%
11 (0)
240 (30)
54 (8)
170 (40)
19 (3)
210 (20)
97 (20)
120 (15)
410 (80)
200 (20)
11 (0.9)
140 (20)
40 (5)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
H
-Butyl
H
-CH2CH(Me)2
-CH2CH(Me)2
H
-CH(Me)(Et), (R)
-CH(Me)(Et), (S)
H
-CH2OH
-CH2OMe
-CH(OMe)Me, (S)
-(CH2)2SMe
-CH2Ph
H
H
-CH(Me)(Et), (R)
H
H
H
H
H
-Me
280 (85)
510 (100)
-Me
aStandard deviation is given in parentheses (%=% inhibition of
activity at 1 mM).
Figure 1. Inhibition of big ET-1 pressor response by compound 3c in
conscious rats. 30 mgEq/kg dose of 3c is the molar equivalent of a 30
mg/kg dose of compound 4c.
bOnly n=1 for this IC50 measurement.

C. A. Fink et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2037±2039
2039
4c, for additional in vivo studies. The methyl ester,
thioacetyl prodrug of this compound, analogue 3c, was
evaluated in vivo in rats for its ability to inhibit the
pressor response produced by big ET-1 administration.11
The results are expressed in Fig. 1 as the area under the
curve for the change in mean arterial pressure produced
by big ET-1 (0.3 nmol/kg, iv) injected at 2, 8, and 24 h.
As can be seen in Fig. 1, compound 3c is a long-acting
orally active inhibitor of ECE-1 activity in vivo. It inhi-
bits big ET-1 pressor response by 75% at 8 h and 30%
at 24 h after dosing.
Chemistry Sta€ (Summit) for providing the physico-
chemical data on the compounds presented.
References and Notes
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9. Triethylamine was omitted as the base when the free base
amines (2) were used in the coupling reaction to provide 3.
10. 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.
The further evaluation of these inhibitors in vivo, as well
as the synthesis and activity of other thiol containing
ECE-1 inhibitors will be presented in future disclosures.
11. From previous experience in pro®ling thiol-containing
metalloprotease inhibitors, the ester thioacetate prodrugs in
general gave better in vivo results as compared to the car-
boxylic acid thiols; see Fink, C. A.; Carlson, J. E.; McTaggart,
P. A.; Qiao, Y.; Webb, R.; Chatelain, R.; Jeng, A. Y.;
Trapani, A. J. J. Med. Chem. 1996, 39, 3158.
Acknowledgements
The authors wish to thank Dr. Stephane De Lombaert
for his helpful discussions and constant encouragement
throughout this work and the Novartis Analytical