Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors

Article abstract of DOI:10.1021/ja0277226

Angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a recently identified zinc metalloprotease with carboxypeptidase activity that was identified using our genomics platform. We implemented a rational design approach to identify potent and se

Full text of DOI:10.1021/ja0277226

Published on Web 09/12/2002
Substrate-Based Design of the First Class of Angiotensin-Converting
Enzyme-Related Carboxypeptidase (ACE2) Inhibitors
Natalie A. Dales,† Alexandra E. Gould,† James A. Brown,† Emily F. Calderwood,† Bing Guan,†
Charles A. Minor,† James M. Gavin,‡ Paul Hales,‡ Virendar K. Kaushik,§ Michael Stewart,‡
Peter J. Tummino,§ Chad S. Vickers,§ Timothy D. Ocain,† and Michael A. Patane*,†
Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, Massachusetts 02139
Received July 16, 2002
Recent advances in genetic technology have provided new tools
to scientists for target identification. In concert with database mining
and homology searching, we identified a new gene from 5′
sequencing of a cDNA library derived from the ventricle of a failed
human heart. This gene encodes a protein that is highly expressed1
in the following normal human tissues: heart, kidney, colon, small
intestine, and testes. This protein has highest homology to the testes-
specific isoform of angiotensin-converting enzyme (ACE) with 60%
sequence homology and was designated angiotensin-converting
enzyme-related carboxypeptidase, ACE2.2 ACE2 is an 805 amino
acid protein with a transmembrane domain and a single HEXXH
zinc-binding consensus sequence (amino acid positions 374-378).
The catalytic domain of ACE2 is 42% identical to that of ACE. A
series of studies helped classify ACE2 as a metalloprotease that
functions as a carboxypeptidase.3 Potent and selective inhibitors
of ACE2 may be useful pharmacological tools to help understand
its biological relevance and ultimately its potential role in human
disease. Herein we report the design and synthesis of the first potent
and selective ACE2 inhibitors.
In lieu of conducting high throughput screening (HTS), we
applied a rational design approach to develop ACE2 inhibitors on
the basis of our enzyme characterization and substrate studies.
Because ACE2 is a zinc metalloprotease that functions as a
carboxypeptidase, we examined its ability to cleave the C-terminal
residue of 130 known peptide hormones. Of these, ACE2 cleaved
11 peptides, including the endogenous ACE substrate, Ang I (1-
10) to Ang 1-9.4 However, the catalytic activity of ACE2 was not
suppressed by known ACE inhibitors (enalaprilat, lisinopril, cap-
topril), which may be due to the fact that ACE is a peptidyl
dipeptidase and ACE2 is a carboxypeptidase.
To determine the minimal structural features required for binding
to the ACE2 catalytic domain, we examined the substrate screening
data and compared the P5-P1′ sequence alignment of the peptides
that have moderate affinity for ACE2 (Km < 10 uM).3 Our analysis
revealed two useful insights: (1) ACE2 preferentially cleaves Pro-X
terminal peptides (X ) hydrophobic amino acid) with high turnover
rates, and (2) while Ang I, with a His-Leu cleavage site, binds to
the enzyme with low micromolar affinity (Km ) 6.9 uM), it is pro-
teolyzed slowly by ACE2 (Kcat ) 0.034 s-1). Additional experiments
determined that Ang I inhibits ACE2 activity (Ki ) 2.2 uM) and
undergoes less than 2% proteolysis under the assay conditions.
Collectively, these data offered clues to the structural requirements
for ACE2 inhibitors, for example, the necessity for a His or Pro
residue at P1 and maintaining a hydrophobic group at P1′.
For our small molecule design strategy, we conceived nonhy-
drolyzable His-Leu-like molecules bearing a C-terminal carboxyl-
ate and including a centrally located carboxylate to serve as a zinc
coordinating element. We chose the carboxylate as the zinc binding
fragment on the basis of precedence with a known carboxypeptidase
A (CPDA) inhibitor, benzylsuccinic acid.5
The amino dicarboxylate core selected for evaluation is exempli-
fied by 1a, which inhibits ACE2 in the micromolar range, Figure
1. This molecule appears to be capable of occupying both S1 and
S1′ and was amenable to further elaboration.
Prior to exploring substituent effects on the imidazole, we
prepared the four stereoisomers of 1. The R,R isomer (1d) is inactive
(>50 uM); however, the other three isomers are equipotent (1a,
S,S IC50 ) 1.2 µM; 1b, R,S IC50 ) 1.4 µM; 1c, S,R IC50 ) 2.2
µM). Of these, the naturally derived S,S isomer (1a) appeared to
be selective against ACE and CPDA (IC50’s > 50 µM). With the
goal of improving potency and maintaining selectivity, we alkylated
the imidazole core of 1a with groups that may be capable of
occupying S2. A benzyl group at the N-1 position of the imidazole
ring (2) decreased potency 10-fold. However, installing a benzyl
substituent at the imidazole N-3 position6 (3) greatly increased
potency, providing a 24 nM inhibitor of ACE2, which maintained
high selectivity (IC50: ACE >100 uM; CPDA ) 34 uM). Perhaps
the N-3 substituent on the imidazole framework occupies the S2
pocket of ACE2, which could account for the observed potency
enhancements.
We optimized the inhibitor potency and selectivity of 3 by
exploring the putative P2, P1, and P1′ sites. Modifications of the
P1′ hydrophobic residue decreased potency (cf. 4 and 5) and
appeared to diminish selectivity versus CPDA. In the P1 binding
pocket, various substituted aromatic groups were explored,7 and
the imidazole-based scaffold provided superior potency and syn-
thetic flexibility for substituent studies. To explore the presumed
interaction at S2, a variety of substituted benzyl groups were
installed at the imidazole N-3 position. We replaced the N-3 benzyl
group with a cyclohexylmethyl group (6), which reduced ACE2
inhibition by an order of magnitude, while the homologue (7)
yielded a 10 nM inhibitor of ACE2. Many substituents8 were
tolerated, and no significant electronic or steric effects (cf. 8-11)
were observed, providing many low nanomolar inhibitors (Table
1) that retained selectivity (>100 fold) against the related enzymes
tested (ACE and CPDA). A systematic study of regioisomers
revealed a profound preference for meta-substitution (cf. 11-13).
While maintaining a 3-substituent on the phenyl group, a series of
disubstituted derivatives was prepared, and a significant preference
for the 3,5-isomer exists. For example, a second methyl group at
the 4-position of 13 decreased potency (14), but when the additional
* To whom correspondence should be addressed. E-mail: patane@mpi.com.
† Medicinal Chemistry.
‡ Lead Discovery.
§ Biochemistry/Enzymology.
9
11852
J. AM. CHEM. SOC. 2002, 124, 11852-11853
10.1021/ja0277226 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
20 as a mixture of diastereomers.15 The diastereomers are separated
and purified using HPLC and crystallization.
ACE2 is a recently identified zinc metalloprotease with carboxy-
peptidase activity that was discovered using our genomics platform.
As a strategy to circumvent the caveats associated with HTS (i.e.,
expense, time commitment, and low probability for success for this
type of target), we implemented a rational design approach to
identify potent and selective ACE2 inhibitors. To this end, we
successfully designed and synthesized picomolar inhibitors of ACE2
using our understanding of the enzymatic mechanism, the substrate
data, and related protease literature. These inhibitors have low
molecular weights, are synthetically accessible from commercially
available chiral building blocks, and display excellent selectivity
versus related enzymes. Currently, these inhibitors are proving
useful in exploring the physiological relevance of ACE2 in vivo.
Figure 1. Design of ACE2 inhibitors.
Table 1. ACE2 Inhibitorsa
Acknowledgment. The authors thank Ian Parsons for perform-
ing NMR studies and Izumi Takagi, Nelson Troupe, and Ashok
Patil for analytical and preparative chromatographic support.
IC b (µM)
50
1
2
compound
R
R
ACE2
0.30
0.34
ACE
CPDA
Supporting Information Available: Synthetic procedures and
assays protocols (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
4
5
6
Ph
Ph
Me
Ph
2.8
cyclohexyl CH2
CH(CH3)2 0.27
7
8
9
10
11
12
13
14
15
16
cyclohexyl (CH3)2 CH(CH3)2 0.010
>10
12
References
4-NO2Ph
4-ClPh
4-CF3OPh
4-MePh
2-MePh
3-MePh
3,4-diMePh
3,5-diMePh
3,5-diClPh
CH(CH3)2 0.076
CH(CH3)2 0.021
CH(CH3)2 0.052
CH(CH3)2 0.032
CH(CH3)2 0.29
CH(CH3)2 0.0042
CH(CH3)2 0.010
CH(CH3)2 0.0014
>10
(1) Determined by northern blot analysis: Donoghue, M.; Stagliano, N.;
Acton, S., unpublished results.
(2) (a) Donoghue, M.; Hsieh, F.; Baronas, E.; Godbout, K.; Gosselin, M.;
Stagliano, N.; Donovan, M.; Woolf, B.; Robison, K.; Jayaseelan, R.;
Breitbart, R. E.; Acton, S. Circ. Res. 2000, 87, 1e-9e. (b) Tipnis, S. R.;
Hooper, N. M.; Hyde, R.; Karran, E.; Christie, G.; Turner, A. J. J. Biol.
Chem. 2000, 275, 33238.
>10 >10
>10 >10
>10 >10
>10 >10
(3) Vickers, C.; Hales, P.; Kaushik, V.; Dick, L.; Gavin, J.; Tang, J.; Godbout,
K.; Parsons, T.; Baronas, E.; Hsieh, F.; Acton, S.; Patane, M.; Nichols,
A.; Tummino, P. J. Biol. Chem. 2002, 277, 14838.
CH(CH3)2 0.00044 >100
27
(4) Determined by mass spectrometry.
a n g 2, SEM within 50% of IC50 values. b Assay conditions: soluble
human ACE2, porcine, or human testicular ACE, Bovine CPDA.
(5) (a) Byers, L. D.; Wolfenden, R. Biochemistry 1973, 12, 2070. (b) Ondetti,
M. A.; Rubin, B.; Cushman, D. W. Science 1977, 196, 441. (c) Patchett,
A. A.; Harris, E.; Tristram, E. W.; Wyvratt, M. J.; Wu, M. T.; Taub, D.;
Peterson, E. R.; Ikeler, T. J.; tenBroeke, J.; Payne, L. G.; Ondeyka, D.
L.; Thorsett, E. D.; Greenlee, W. J.; Lohr, N. S.; Hoffsommer, R. D.;
Joshua, H.; Ruyle, W. V.; Rothrock, J. W.; Aster, S. D.; Maycock, A. L.;
Robinson, F. M.; Hirschmann, R.; Sweet, C. S.; Ulm, E. H.; Gross, D.
M.; Vassil, T. C.; Stone, C. A. Nature 1980, 288, 280.
Scheme 1. Synthesis of ACE2 Inhibitors
(6) Site of alkylation was confirmed using ROESY NMR.
(7) Data not shown.
(8) Representative data shown.
(9) Configuration confirmed by X-ray crystallography.
(10) R,R IC50 ) 0.072 uM; R,S IC50 ) 8.4 uM; S,R IC50 ) 0.470 uM.
(11) Boger, D. L.; Teramoto, S.; Cai, H. Bioorg. Med. Chem. 1996, 4, 179.
(12) Blankley, C. J.; Hodges, J. C.; Klutchko, S. R.; Himmelsbach, R. J.;
Chucholowski, A.; Connolly, C. J.; Neergaard, S. J.; Van Nieuwenhze,
M. S.; Sebastian, A.; Quin, J., III; Essenburg, A. D.; Cohen, D. M. J.
Med. Chem. 1991, 34, 3248.
(13) (a) N-3 imidazole alkylation was also accomplished using Boc-His(Trit)-
OCH3 and substituted benzyl halides: Anthony, N. J.; Gomez, R. P.;
Schaber, M. D.; Mosser, S. D.; Hamilton, K. A.; O’Neil, T. J.; Koblan,
K. S.; Graham, S. L.; Hartman, G. D.; Shah, D.; Rands, E.; Kohl, N. E.;
Gibbs, J. B.; Oliff, A. I. J. Med. Chem. 1999, 42, 3356. (b) Williams, T.
M.; Bergman, J. M.; Brashear, K.; Breslin, M. J.; Dinsmore, C. J.;
Hutchinson, J. H.; MacTough, S. C.; Stump, C. A.; Wei, D. D.; Zartman,
C. B.; Bogusky, M. J.; Culberson, J. C.; Buser-Doepner, C.; Davide, J.;
Greenberg, I. B.; Hamilton, K. A.; Koblan, K. S.; Kohl, N. E.; Liu, D.;
Lobell, R. B.; Mosser, S. D.; O’Neill, T. J.; Rands, E.; Schaber, M. D.;
Wilson, F.; Senderak, E.; Motzel, S. L.; Gibbs, J. B.; Graham, S. L.;
Heimbrook, D. C.; Hartman, G. D.; Oliff, A. I.; Huff, J. R. J. Med. Chem.
1999, 42, 3779.
methyl group was installed at the 5-position, the potency modestly
increased (15). The 3,5-dichloro analogue (16) inhibits ACE2 in
the picomolar range and has excellent selectivity (>5000-fold)
versus related enzymes.9 All four stereoisomers of 16 were prepared,
and the greatest potency remained within the S,S isomer.10
The preparation of the N-3 substituted imidazole-containing
inhibitors is outlined in Scheme 1. Treatment of (S)-histidine methyl
ester (17) with Boc2O affords the fully protected histidine derivative
18.11 Selective alkylation of the N-3 imidazole nitrogen using the
triflate of a substituted benzyl alcohol (or alkyl alcohol derivative)
provides the N-3 alkylated histidine derivative 19.12,13 Following
Boc deprotection, reductive amination between the histidine deriva-
tive 19 and the â-keto ester furnishes the diester amine.14 Hydrolysis
of this diester yields the desired amino dicarboxylate ACE2 inhibitor
(14) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah,
R. D. J. Org. Chem. 1996, 61, 3849.
(15) With some substituents, minor amounts of epimerization were observed
during ester hydrolysis.
JA0277226
9
J. AM. CHEM. SOC. VOL. 124, NO. 40, 2002 11853