Targeting ACE and ECE with dual acting inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.013

A series of urea analogues related to SA6817 and a GSK phosphonic acid with reported ACE inhibitory activity were prepared and tested for dual ACE and ECE activities. Although excellent ACE and NEP inhibition was achieved, only modest ECE inhibition was o

Full text of DOI:10.1016/j.bmcl.2007.12.013

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1058–1062
Targeting ACE and ECE with dual acting inhibitors
a
a
a
b
Stephen Hanessian,a, Sebastien Guesne, Ludivine Riber, Julien Marin, Alain Benoist,
*
´
´
Philippe Mennecier,c Alain Rupin,c Tony J. Verbeurenc and Guillaume De Nanteuilb
aDepartment of Chemistry, Universite´ de Montre´al, PO Box 6128, Station Centre-Ville, Montre´al, Que., Canada H3C 3J7
bDivision D of Medicinal Chemistry, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France
cDivision of Angiology, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France
Received 8 November 2007; accepted 6 December 2007
Available online 21 December 2007
Abstract—A series of urea analogues related to SA6817 and a GSK phosphonic acid with reported ACE inhibitory activity were
prepared and tested for dual ACE and ECE activities. Although excellent ACE and NEP inhibition was achieved, only modest
ECE inhibition was observed with one analogue.
Ó 2008 Published by Elsevier Ltd.
Cardiovascular disease manifests itself in a number of
clinically relevant ways, affecting a large segment of
the adult population regardless of geographic location.1
The market for a variety of drugs prescribed for two of
the more prevalent indications, namely hypertension2
and hypercholesteremia,3 accounts for several billion
dollars annually. In spite of the availability of highly
effective drugs to lower blood pressure and control levels
of cholesterol in humans, the quest for newer entities
with improved therapeutic profiles and overall better
tolerance is an on-going objective.4 Inhibition of angio-
tensin converting enzyme (ACE) has been a highly suc-
cessful program in the pharmaceutical industry resulting
in a number of effective antihypertensive drugs.5 Elegant
studies relying on structure-based design have laid a
strong foundation for early pioneering efforts in the
development of Captopril6 and several of its variants.7
Another enzyme that has been identified in relation to
hypertension is endothelin converting enzyme
(ECE),8,14a which cleaves big-endothelin into endothe-
lin, a highly vasoconstricting peptide. The absence of
X-ray crystallographic information on ECE has ham-
pered progress in the development of inhibitors with
clinical potential, although the literature is abound with
synthetic compounds showing promising inhibitory
activity.9
A third enzyme, neutral endopeptidase (NEP),10 is also
a potential target in the field of hypertension. However
the clinical development of dual ACE–NEP inhibitors
as well as triple ACE–ECE–NEP inhibitors has been
the subject of many failures, mainly due to unwanted
NEP-inhibition related side-effects such as angiodema.
Thus, the overall benefits of developing NEP inhibitors
are not as evident as with ACE, ECE, or renin11 which is
well known for its hypertensive activity in humans.
The development of a dual acting inhibitor toward ACE
and ECE and selective versus NEP presents a veritable
challenge. Toward this end we selected two known
inhibitors exemplified by SA681712 1 and 2 (GSK)9c,13
(Fig. 1) which are structurally unrelated except for the
presence of Zn binding groups, a hallmark of small mol-
ecule inhibitors of metalloproteases.14 They formed the
basis of our choice toward the synthesis of hybrid struc-
tures with potential dual inhibitory activity against ACE
and ECE. Herein we report the results of our efforts to-
ward this goal.
Our main focus was to prepare a series of substituted
urea congeners of SA6817 1 in which we systematically
varied the hydrophobic substituent R1, and probed the
nature of the amino acid residue R2 (Fig. 1).
The readily available L-isoserine 4 was transformed in
three steps into the N,O-protected ester 5 (Scheme 1).
Removal of the N-Cbz group by hydrogenolysis affor-
ded the corresponding amine which was subjected to
Keywords: SA6817 analogues; Dual ECE–ACE enzyme inhibitors.
*
Corresponding author. Tel.: +1 514 343 6738; fax: +1 514 343
5728; e-mail: stephen.hanessian@umontreal.ca
0960-894X/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.12.013

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1058–1062
1059
the secondary amine, and the latter was treated with
appropriate naphthylimide N-alkanals under reductive
amination conditions to give the adduct exemplified by
the N-ethyl analogue 9. Activation of the amine as the
isocyanate and coupling with L-naphthylalanine fol-
lowed by deprotection gave 10 as a prototype of this
class.
O
N
O
OH
H
N
H
N
O
P
N
CO2H
HO
HO
CO2H
HO2C
N
H
H
O
O
N
1
2
The phosphonic acid hybrid analogues of SA 6817 were
prepared in a standard fashion from aminoalkyl phos-
phonic acid esters 11 and 12 (Scheme 2). Installation
of the N-isobutyl group by reductive amination led to
13 and 14, which were converted to the phosphonic acid
analogues 15 and 16.
(SA 6817)
(GlaxoSmithKline)
OH R1
N
H
N
CO2H
HO2C
O
R2
3
Preliminary enzyme inhibitory studies identified a num-
ber of individual hits toward one or more of the three
target enzymes.15 Of these, we selected those that exhib-
ited measurable IC50 nM inhibition against at least
ACE, ECE, or NEP and combinations thereof. For pur-
poses of comparison, we list the compounds grouped
according to the R1 and R2 variations in Table 1. The
known SA 6817 1 showed potent ACE and NEP inhib-
itory activities at IC50 52 and 4 nM, respectively, while it
was barely active as an ECE inhibitor with an IC50 of
2.5 lM (Table 1, entry 1). Variation of the R2 group
as in analogues 7a–e maintained NEP activity at the ex-
pense of ACE with the exception of 7c and 7e, but the
minimal activity against ECE was lost. Next we varied
the R1, N-alkyl substituent in the naphthylalanine series
(Table 1, entries 8–11 and 14–15, compounds 7f–i, 7l,
and 10). Increasing the bulk of R1 led to good ACE inhi-
bition while eradicating NEP as exemplified by com-
pounds 7h–i and 7l. Surprisingly the naphthylimide
analogue 10 restored some ECE activity at the expense
of ACE (Table 1, entry 15).
Figure 1.
reductive amination with a variety of aldehydes to afford
intermediates 6a–k. The formation of dialkylated prod-
ucts was minimized by addition of Et3N. Conversion of
the secondary amines to the intended ureas was accom-
plished in the presence of carbonyldiimidazole and an
amino acid ester. Removal of the TBS protective group
and hydrolysis with LiOH gave a series of analogues 7a–
k. Alternatively, L-isoserine was converted to the N-al-
kyl analogue 8, then coupled to the amino acid as the
methyl ester, followed by hydrolysis to afford the ana-
logue 7l. Final products were purified by preparative
HPLC and isolated as the free dicarboxylic acids. To ac-
cess a ‘hybrid’ analogue in which the original isobutyl
group was replaced by N-alkyl naphthylimide residues,
we adopted an alternative sequence. The ester group
of choice was trimethylsilylethyl rather than methyl.
Thus, 5 was transesterified, the product converted to
O
N
O
O
O
N
O
OH
TBSO
TMSEO2C
f,l
H
N
N
CO2H
NH
HO2C
k,d,e
d,e
9
10
OTBS
NHCbz
MeO2C
5
OH R1
N
TBSO
R1
NH
f-h
H
N
CO2H
HO2C
f,h
MeO2C
O
7a-l
R2
a-c
6a-k
OH
OH
i,j
NH
NH2
MeO2C
HO2C
4
8
Scheme 1. Typical synthesis of inhibitors. Reagents and conditions: (a) 1 M NaOH, CbzCl, 0 °C to rt, 5 h; (b) TMSCHN2, MeOH, 0 °C; (c) TBSCl,
ImH, DMAP, DCM, rt, 70% (3 steps); (d) H2, Pd/C, MeOH, rt, 2 h, quantitative; (e) MgSO4, DCM, aldehyde, Et3N, NaHB(OAc)3, 5 h, rt, 50–90%;
(f) amino acid, CDI, ImH, THF, rt then reflux; (g) TBAF, AcOH (1:1), THF, rt, 0.5 h; (h) LiOH 1 M, MeOH, 0 °C to rt; (i) fluorene-2-
carboxaldehyde, KOH, H2O/EtOH (1:1, v/v), NaBH4; (j) SOCl2, MeOH; (k) TMSEtOH, Ti(Oi-Pr)4, 80 °C, 75%; (l) TFA/H2O (9:1, v/v), 0 °C to rt,
3 h.

1060
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1058–1062
Table 1. IC50 values for compounds 1, 7a–l, and 10
OH R1
N
H
N
CO2H
HO2C
O
R2
Entry
Compounds
R1
R2
IC50 (nM)
ECE
ACE
52
NEP
1
2
1
2500
4
8
7a
103
16,500
NH
a
4
5
7b
7c
7d
7e
7f
7g
7h
7i
110
35
—
9
3490
10
6
6
60
—
OH
OH
OH
7
23
—
19
5
8
191
—
—
9
—
36
O
10
11
12
13
14
39
—
—
—
O
O
40
—
7j
—
—
90
OH
OH
7k
7l
—
—
1540
150
—
19,000
15
10
1980
600
650
O
N
O
a IC50 were not calculated, but enzymatic inhibitions at 0.1 and 10 lM were obtained (see Supporting Information).

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1058–1062
1061
were found among these analogues, but our desire to
achieve dual acting inhibitors against ACE and ECE
in particular was not realized.
O
P
O
P
O
P
a-d
e-g
H
N
HO
HO
HO
HO
MeO
MeO
N
CO2H
NH2
n=1,2
NH
n
n
O
11, n=1
12, n=2
13, n=1
14, n=2
15, n=1
16, n=2
Acknowledgments
Scheme 2. Synthesis of phosphonic acid analogues. Reagents and
conditions: (a) NaOH 2 N, NaHCO3, Na2CO3, CbzCl, 0 °C to rt; (b)
TMSCHN2, MeOH, 0 °C to rt, 1 h; (c) H2, Pd/C, MeOH, rt, 2 h,
quantitative; (d) MgSO4, iso-butyraldehyde, NaHB(OAc)3, DCM, 1 h;
(e) naphthylalanine methyl ester, CDI, ImH, THF, rt then reflux; (f)
LiOHÆH2O, THF:H2O:MeOH, 0 °C, 2 h, quantitative; (g) TMSI,
DCM, 0 °C to rt, 2 h then CH3CN, H2O, 4 °C, 16 h.
We thank NSERC, FQRNT, and Servier for financial
support.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.12.013.
Lengthening the alkyl chain by one and two methylene
groups resulted in loss of activity against the three en-
zymes.15 Variation of R1 and R2 as in 7j and 7k (Table
1, entries 12 and 13) resulted in loss of ACE and ECE
activity, but maintained NEP. The 15-fold improvement
in going from an N-cyclohexyl to N-cyclopentyl is inex-
plicably odd.
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CO2H
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IC50 (nM)
ECE
ACE
NEP
1
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15
16
1
2
29
—
—
—
2
6

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