Synthesis and biological activity of novel potent endothelin-converting enzyme-1 inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00657-0

Through directed screening of metalloprotease inhibitors, CGS 30084 (1) has been identified as a potent endothelin-converting enzyme-1 (ECE-1) inhibitor in vitro (IC₅₀ = 77 nM). Herein we report the syntheses and biological activities of analogues derived from this lead, based on modifications of the biphenyl moiety. Compound 10, the thioacetate methyl ester prodrug derivative of compound 6m, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.

Full text of DOI:10.1016/S0960-894X(00)00657-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 375±378
Synthesis and Biological Activity of Novel Potent
Endothelin-Converting Enzyme-1 Inhibitors
Fariborz Firooznia,* Candido Gude, Kenneth Chan, Cynthia A. Fink, Ying Qiao,
Yoshitaka Satoh, Nicholas Marcopoulos, 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 7 August 2000; accepted 20 November 2000
AbstractÐThrough directed screening of metalloprotease inhibitors, CGS 30084 (1) has been identi®ed as a potent endothelin-
converting enzyme-1 (ECE-1) inhibitor in vitro (IC50=77 nM). Herein we report the syntheses and biological activities of analogues
derived from this lead, based on modi®cations of the biphenyl moiety. Compound 10, the thioacetate methyl ester prodrug deri-
vative of compound 6m, was found to be an orally active and potent inhibitor of ECE-1 activity in rats. # 2001 Elsevier Science
Ltd. All rights reserved.
The overproduction of endothelin-1 (ET-1), one of the
most potent vasoconstrictive peptides characterized thus
far,1 has been associated with a variety of disorders such
as cerebral vasospasm, stroke, asthma, and cardiac and
renal failure.2 5 The discovery of potent inhibitors of
ET-1 biosynthesis is thus envisaged as an attractive
potential therapeutic approach for the treatment of dis-
eases linked with elevated ET-1 levels.6 Endothelin-
converting enzyme-1 (ECE-1), a zinc metalloprotease,
catalyzes the post-translational conversion of big ET-1
to ET-1,7 thus presenting a logical target for the design
of therapeutic agents that regulate the production of
ET-1 in vivo.8,9
modi®cations of the distal ring were also explored. In all
cases, the thiol end substituents were kept the same (S-
isopropyl) as CGS 30084.
The target molecules 6 were prepared as previously
described,10 starting from various arylalanines
2
(Scheme 1). The synthesis of target molecules (6g±v)
containing 4-aryl-substituted phenylalanines presented a
signi®cant synthetic challenge. At the time we began our
endeavor, only one reliable approach to 4-aryl-sub-
stituted phenylalanines existed (Scheme 2), which
allowed rapid access to a diverse set of such deriva-
tives.11 This methodology, which utilizes the Pd-cata-
lyzed cross-coupling reactions of tyrosine tri¯ates (e.g.,
7) with organometallic reagents had a limited scope,
however, due to the limited availability of highly func-
tionalized organoboron and organotin reagents from
We have previously reported the identi®cation of CGS
30084 (1) (Fig. 1, IC50=77 nM) as an attractive starting
point for the design of potent ECE-1 inhibitors.10 As
described in our earlier report, various modi®cations
carried out at the thiol end of CGS 30084 a€orded the
highly potent (IC50=11 nM) ECE-1 inhibitor 1a, and its
prodrug 1b (Fig. 1), a long-acting orally active inhibitor
of ECE-1 activity in vivo. The current report describes
the modi®cations carried out at the biphenyl portion of
CGS 30084 in order to further explore the SAR of ECE-
1 inhibition, and to improve the in vivo pro®le of CGS
30084 by changing its physicochemical properties (vide
infra). Aside from spatial alternatives to para-biphenyl,
*Corresponding author. Tel.: +1-908-277-5343; fax: +1-908-277-
2405; e-mail: fariborz.®rooznia@pharma.novartis.com
Figure 1. Previously described ECE-1 inhibitors.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00657-0

376
F. Firooznia et al. / Bioorg. Med. Chem. Lett. 11 (2001) 375±378
commercial sources at the time. We have developed an
alternative more versatile procedure for the synthesis of
4-aryl-phenylalanines 2g±v, employing the Pd-catalyzed
cross-coupling reaction of the boronophenylalanine
reagent 9 with aryl halides or tri¯ates (Scheme 2).12,13
In this manner, we were able to incorporate a large
number of diverse 4-aryl-phenylalanines into our target
molecules.
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 reported
previously.14 The results are summarized in Tables 1
and 2.
Table 1 summarizes our results in exploring spatial
alternatives for the para-biphenyl moiety in CGS 30084.
Scheme 1. (a) BOCNH-cycloleucine (EDCI, HOBt) or (DCC, HOAt), Et3N, CH2Cl2, rt; (b) HCl (gas), CH2Cl2; (c) 48% HBr, KBr, NaNO2; (d) 4
(EDCI, HOBt) or (DCC, HOAt), Et3N, CH2Cl2, rt; (e) AcSK, THF; (f) 1 N NaOH, MeOH. * Stereochemistry at the chiral center bearing the
biphenyl.
Scheme 2. (a) BOCNH-cycloleucine, HOBt, EDCI, Et3N, CH2Cl2, rt; (b) Tf2O, pyridine; (c) ArSnMe3, PdCl2(dppf), LiCl, dioxane, re¯ux, or
ArB(OH)2, PdCl2(dppf), DME, K3PO4, re¯ux; (d) HCl (g), CH2Cl2; (e) Ar-X (X=Cl, Br, I, OTf), PdCl2(dppf), K3PO4, DME, re¯ux. *Stereo-
chemistry at the chiral center bearing the biphenyl.

F. Firooznia et al. / Bioorg. Med. Chem. Lett. 11 (2001) 375±378
377
The size and orientation of the biphenyl substituent
appear to be very important for ECE-1 inhibitory
activity. Neither m-biphenyl nor o-biphenyl showed
inhibitory activity, suggesting a `narrow' binding pocket
for the biphenyl group. Even a slightly larger distal ring
(6f) proved detrimental for ECE-1 inhibitory activity. It
appears, however, that some minimal `size' is required
at this position in order to achieve potent ECE-1 inhi-
bition, as the monophenyl derivative 6a is virtually
inactive. The naphthyl derivatives, especially 6e, o€er a
possible replacement for the biphenyl group, without
signi®cant loss of ECE-1 inhibitory activity.
Table 2 summarizes the results of our investigation of
the SAR of the distal ring of the biphenyl moiety in
CGS 30084. The absolute stereochemistry of the chiral
center bearing the biphenyl moiety is very important for
ECE-1 inhibitory activity. While the compound bearing
the l-biphenylalanine stereoisomer (CGS 30084) is a
potent ECE-1 inhibitor, the compound bearing d-
biphenylalanine (6g) has virtually no activity towards
ECE-1.15 The distal phenyl ring can be replaced with a
thiophene, without signi®cant loss of ECE-1 inhibitory
activity (6h vs CGS 30084). Furthermore, the distal ring
of the biphenylalanine moiety tolerates basic or non-
basic heteroatoms at the 2- and 3-position (6h±k and
6m), while the presence of a nitrogen at the 4-position of
the distal ring (6l) signi®cantly reduces the ECE-1 inhi-
bitory activity. It appears that the presence of a het-
eroatom is slightly better tolerated at the 3-position
compared to the 2-position (see 6k and 6m vs 6j). Elec-
tron-withdrawing groups at the 4-position of the distal
ring appear to have a detrimental e€ect towards ECE-1
inhibitory potency (see 6r and 6s vs CGS 30084). In
addition, there appears to be a `size limit' to the sub-
stituents allowed at the 4-position of the distal ring,
with the inhibitory activity decreasing from 4-¯uoro (6s)
to 4-chloro (6p) to 4-tri¯uoromethyl (6r) to 4-methoxy
(6q) derivatives. Substitutions at the 2-position of the
distal ring are preferred to those at the 3-position (e.g.
6o and 6t vs 6n). In general, none of the various elec-
tron-donating or electron-withdrawing substitutions on
the distal ring provided a signi®cant improvement of in
vitro ECE-1 inhibitory activity over CGS 30084.
Table 1. Spatial alternatives for the p-biphenyl group in CGS 30084
Compds
ECE-1 Inhibition
IC50 (nM)a
R
CGS 30084
77b
26%
39%
0%
320 (Æ80)
180 (Æ3)
490 (Æ40)
p-Biphenyl
Ph
6a
6b
6c
6d
6e
6f
m-Biphenyl
o-Biphenyl
1-Naphthyl
2-Naphthyl
4-Cyclohexyl-Ph
aStandard deviation is given in parentheses.
bObtained from ref 10 (%=% inhibition of ECE-1 activity at 1 mM).
Even though we were not able to signi®cantly improve
the in vitro potency of CGS 30084, we were intrigued by
compounds 6k and 6m (dl mixtures), which provided
alternatives with virtually equal potency,15 and possibly
di€erent pharmacokinetic properties, due to di€erent
solubility pro®les.16 We therefore selected compound
6m and its thioacetate methyl ester prodrug 10 (Figs. 2
and 3)10 for additional in vivo studies. First, compound
6m was evaluated in comparison with CGS 30084 for its
ability to inhibit the pressor response produced by big
ET-1 in anesthetized rats (Fig. 2),14 measured as the
change in mean arterial pressure produced by big ET-1
(1 nmol/kg) administered iv. Upon iv administration at
10 and 20 mg/kg, compound 6m inhibited the big ET-1
pressor response by 56 and 73%, respectively, after 15
min. Under similar conditions, CGS 30084 (adminis-
tered iv at 10 mg/kg) was only able to inhibit the big
Table 2. Modi®cations at the distal ring of the biphenylalanine
moiety in CGS 30084
Compds
ECE-1 Inhibition
IC50 (nM)a
*
Stereochemistry
Ar
CGS 30084
77b
18%
130 (Æ20)
300 (Æ60)
220 (Æ40)
150 (Æ10)
22%
120 (Æ20)
990 (Æ10)
350 (Æ30)
810 (Æ180)
49%
l
d
Ph
Ph
3-Thienyl
2-Furyl
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
l
l
dl
dl
dl
dl
dl
dl
l
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
Pyrimidin-3,5-yl
3,5-Dimethoxy-Ph
2,3-Dimethoxy-Ph
4-Cl-Ph
4-Methoxy-Ph
4-CF3-Ph
4-Fluoro-Ph
2-Methoxy-Ph
3-CF3-Ph
l
54%
l
650 (Æ130)
370 (Æ160)
650 (Æ200)
560 (Æ40)
l
l
6u
6v
l
l
3-Cl,4-F-Ph
aStandard deviation is given in parentheses.
bObtained from ref 10 (%=% inhibition of ECE-1 activity at 1 mM).
Figure 2. Comparison of the inhibition of big ET-1 pressor response
by compounds 6m and CGS 30084 (iv in anesthetized rats).

378
F. Firooznia et al. / Bioorg. Med. Chem. Lett. 11 (2001) 375±378
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Figure 3. Inhibition of big ET-1 pressor response by compound 10,
prodrug of compound 6m (po in conscious rats).
ET-1 pressor response by 40%. These results clearly
indicate a superior in vivo pro®le for 6m compared to
CGS 30084, especially considering that 6m contains
50% inactive d-arylalanine stereoisomer.
We next evaluated compound 10, the thioacetate methyl
ester prodrug of 6m. Conscious rats were dosed with
either vehicle or compound 10 and 2 h later challenged
with big ET-1 at 1 nmol/kg iv. Figure 3 shows the
change in mean arterial pressure produced by big ET-1
for 30 min following its administration. Upon po (20
mg Eq/kg) administration, compound 10 inhibited the
big ET-1 pressor response14 by 45% at 2 h post dosing.
In contrast, the corresponding prodrug of CGS 30084
was inactive under similar conditions after 30 min. In
conclusion, through exploration of the biphenyl region
of CGS 30084, we have discovered compound 10, a
potent, orally active inhibitor of ECE-1 activity in vivo.
13. Due to cost issues, only dl-mixtures of the
boronophenylalanine derivative 9 were prepared at ®rst, and
used for the synthesis of the target compounds. It was envi-
saged that all interesting compounds would be synthesized in
optically pure form at a later date, if necessary.
14. Wallace, E. M.; Moliterni, J. A.; Moskal, M. A.; Neubert,
A. D.; Marcopoulos, N.; Stamford, L. B.; Trapani, A. J.;
Savage, P.; Chou, M.; Jeng, A. Y. J. Med. Chem. 1998, 41,
1513.
15. Similar results were observed with d-enantiomers of
several other biphenylalanine derivatives (data not shown).
16. Compound 6m was 10 times more soluble than CGS
30084 (0.02 vs 0.002 mg/mL) at pH 1.0. At pH 6.8 CGS 30084
was more soluble (ca. 0.2 vs 0.05 mg/mL) than 6m.
References and Notes
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