Thiol-based angiotensin-converting enzyme 2 inhibitors: P1′ modifications for the exploration of the S1′ subsite

Article abstract of DOI:10.1016/j.bmcl.2008.01.046

Explorations of the S^1′ subsite of ACE2 via modifications of the P^1′ methylene biphenyl moiety of thiol-based metalloprotease inhibitors led to improvements in ACE2 selectivity versus ACE and NEP, while maintaining potent ACE2 inhibi

Full text of DOI:10.1016/j.bmcl.2008.01.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1681–1687
Thiol-based angiotensin-converting enzyme 2 inhibitors:
P1 modifications for the exploration of the S1 subsite
0
0
David N. Deaton,a,* Kevin P. Graham,b Jeffrey W. Grossb and Aaron B. Millerc
aDepartment of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
bMolecular Discovery Research, Screening & Compound Profiling, GlaxoSmithKline, Upper Providence, PA 19426, USA
cMolecular Discovery Research, Computational and Structural Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
Received 29 November 2007; revised 10 January 2008; accepted 14 January 2008
Available online 18 January 2008
0
0
Abstract—Explorations of the S1 subsite of ACE2 via modifications of the P1 methylene biphenyl moiety of thiol-based metallo-
protease inhibitors led to improvements in ACE2 selectivity versus ACE and NEP, while maintaining potent ACE2 inhibition.
Ó 2008 Elsevier Ltd. All rights reserved.
Dampening of the renin-angiotensin signaling cascade
(RAS) has proven invaluable in the treatment of cardio-
vascular disease. Drugs that inhibit the M2 family dica-
rboxymetallopeptidase angiotensin-converting enzyme
(ACE, EC 3.4.15.1) or the A1 family aspartic protease
renin (EC 3.4.23.15), as well as those that antagonize
the 7-transmembrane G protein-coupled angiotensin
receptor II (AT2), improve hypertension. Dual ACE
and M13 metalloprotease neutral endopeptidase (nepri-
lysin, NEP, EC 3.4.24.11) inhibition has also been ex-
plored for the treatment of high blood pressure.
Recently, a new member of the RAS, the M2 family
monocarboxymetallopeptidase angiotensin-converting
enzyme 2 (ACE2), was identified.1,2 It is a membrane-
associated and secreted metalloprotease, expressed in
heart, kidney, testes, intestine, and lung, with highest
homology to ACE. ACE2 has been implicated in cardio-
vascular disease, kidney disease, obesity, and lung dis-
ease.3–5
tility,6 angiotensin II-induced hypertension,7 and heart
failure.8 Furthermore, male ACE2 (ꢀ/Y) mice, but not
female ACE2 (ꢀ/ꢀ) mice, also develop glomerulosclero-
sis of the kidneys,9 but ACE2’s exact role in the renin-
angiotensin system (RAS) still needs to be clarified. In
addition, ACE2 (ꢀ/ꢀ) mice are resistant to weight gain
on a high fat diet.10 Finally, ACE2 is the primary recep-
tor for the severe acute respiratory syndrome (SARS)
corona virus’s entry into cells.11 ACE2 (ꢀ/ꢀ) mice resist
SARS corona virus infection.12 Because of limited space,
the authors refer the interested reader to a recent review
by Hamming et al. for a more comprehensive summary
of the physiology and pathology of ACE2.13
With the many putative roles of ACE2, small molecule
modulators of this enzyme’s activity could be utilized
to help further define the physiological functions of this
enzyme. Although small molecule inducers of proteases
are difficult to discover, ACE2 activators could provide
further insight into the functions of ACE2 and the sub-
strates it processes. Similarly, inhibitors could also shed
more light on the roles of ACE2 and the proteins/pep-
tides it activates/degrades.
Angiotensin I and the AT1 & AT2 receptor agonist
angiotensin II are substrates for ACE2, which converts
them into angiotensin (1-9) and the mas receptor agonist
angiotensin (1-7), respectively, revealing a role for ACE2
in RAS regulation. Moreover, ACE2 (ꢀ/ꢀ) mice studies
have revealed roles for this protease in cardiac contrac-
Efforts to discover ACE2 inhibitors have recently been
disclosed. Millennium Pharmaceutical researchers re-
ported the discovery of a reversible, subnanomolar
ACE2 inhibitor MLN-4760 (IC50 = 0.44 nM), derived
from a micromolar lead.14 In addition, Dyax researchers
identified micromolar peptide inhibitors of ACE2 using
a phage display library approach, including the 29 ami-
no acid cyclic peptide DX600 (IC50 = 10 nM).15
Keywords: Angiotensin-converting enzyme 2; Metalloproteases;
Protease inhibitors; Thiols.
*
Corresponding author. Tel.: +1 919 483 6270; fax: +1 919 315
0430; e-mail: david.n.deaton@gsk.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.046

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D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1681–1687
Furthermore, a pharmacophore-based virtual screening
approach of commercial databases identified
several micromolar ACE2 inhibitors. The best inhibitor
was 4S-16659 (IC50 = 62,000 nM).16 Finally, researchers
at the University of Florida also utilized a virtual
screening exercise of the NCI database to identify
hydroxybenzotriazole ester via the carbodiimide, to pro-
duce the fully protected amides. Then, hydrolysis of the
thioacetate and methyl ester with lithium hydroxide
afforded the thiol acids 1a–1t.19
Amine hydrochlorides 2a–2g, 2l, and 2o were commer-
cially available. The remaining amine hydrochlorides
2h–2k, 2m–2n, and 2p–2t were prepared as depicted in
Scheme 2. The commercially available amino acids 4a–
4b were converted to their corresponding methyl esters
with thionyl chloride and methanol at reflux. Then,
the resulting amine hydrochlorides were protected as
the tert-butyl carbamates 5a–5b with di-tert-butyl dicar-
bonate. A Suzuki cross coupling of the o-bromide 5a
(X = Br) with phenyl boronic acid afforded the o-biphe-
nyl derivative 6a. Acid catalyzed cleavage of the carba-
mate gave the o-biphenyl amine hydrochloride 2h. The
m-phenol 5b (X = OH) was converted into the triflate
with trifluoromethanesulfonic anhydride, and then cou-
pled via the Suzuki protocol to phenyl boronic acid,
providing the fully protected m-biphenyl derivative 6b.
Subsequent hydrolysis of the amine masking group gave
the m-biphenyl amine hydrochloride 2i. The o-bromide
5a (X = Br) was converted into the o-phenol 5c
(X = OH) via palladium-mediated transformation of
the bromide into a boronate ester, and then oxidation
of the boronate to the phenol with hydrogen peroxide.
Employing a modified Ullmann ether synthesis proto-
col, the o-phenol 5c and m-phenol 5b were coupled with
phenyl boronic acid to afford the o- and m-diaryl ethers
N-(2-aminoethyl)-1
(IC50 = 57,000 nM) as an irreversible inhibitor of
ACE2.17
aziridine-ethanamine
(NAAE)
Cl
L
O
R
Y
P
S
Cl
O
H
T
Y
P
G H2N
E
H
Y
P
C
C
S
Y
D
D
G
G
N
N
HO
W
W
K
P
G
N
O
OH
MLN-4760
DX600
H
N
S
O
N
NH2
N
H
N
N
HO
O
Cl
4S-16659
NAAE
Recently, employing a directed screen of a set of metal-
loprotease inhibitors from the GlaxoSmithKline com-
pound collection, GSK researchers reported the
identification of a known ACE/NEP inhibitor as a po-
tent inhibitor of the zinc metalloprotease ACE2.18
Structure/activity studies of the S1 subsite of ACE2 led
to the identification of P1 modified thiol acid 1a as a
more potent ACE2 inhibitor (Ki App = 1.5 nM) with im-
proved selectivity versus ACE (Ki App = 490 nM) and
NEP (Ki App = 27 nM). It also inhibits the M14 metallo-
protease carboxypeptidase A1 (CpA, EC 3.4.17.1,
Ki App= 11,000 nM). Speculating that selectivity could
be0 improved by exploiting potential differences in the
t-Bu
O
X
X
a, b
OH
O
H2N
4a-b
O
N
H
5a-d
O
O
O
S1 subsites of ACE, ACE2, and NEP, the structure–
c, or
d, c
e, f,
or f
g
0
activity relationships of the P1 position of inhibitor 1a
were explored, with the goal of maintaining potency
and reducing ACE and NEP inhibitory activity.
Ar
Ph
O
Ph
O
O
O
O
O
O
O
H
t-Bu
NH
t-Bu
NH
t-Bu
NH
N
HS
OH
O
O
O
O
O
O
O
1a
6a-b
6c-d
6e-k
h
h
h
R
O
HCl*H2N
The thiol analogs 1a–1t were prepared as depicted in
Scheme 1. The amine hydrochlorides 2a–2t were coupled
to the acid 3, after its in situ activation to the aza-
O
2h-k, 2m-n, & 2p-t
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, "#, 95–99%;
(b) Boc2O, i-Pr2NEt, CH2Cl2, 0 °C to rt, 97–99%; (c) X = Br or OTf,
PhB(OH)2, Pd(PPh3)4, K2CO3, PhMe, 90 °C, 41–57%; (d) X = OH,
Tf2O, pyridine, CH2Cl2, 0 °C to rt, 88%; (e) X = Br, PdCl2(dppf),
bis(pinacolato)diborane, KOAc, DMF, 80 °C; 30% H2O2 (aq), MeOH,
O
O
O
H
N
OH
HCl∗H2N
+
a, b
HS
OH
1a-t
R
S
O
2a-t
3
O
O
R
˚
44%; (f) X = OH, Cu(OAc)2, PhB(OH)2, pyridine, powdered 4 A
Scheme 1. Reagents and conditions: (a) EDC, HOAt, i-Pr2NEt,
CH2Cl2, rt, 39–76%; (b) LiOHÆH2O, THF, H2O, rt, 56–94%.
molecular sieves, CH2Cl2, rt, 70–96%; (g) X = OH, ArCH2Br, K2CO3,
acetone, rt, 71–99%; (h) HCl, MeOH, 91–98%.

D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1681–1687
1683
6c and 6d. Acidic uncloaking of the tert-butyl carbonyl
protecting group, then produced the phenyl ethers 2j
and 2k. Finally, utilizing the Williamson ether synthesis,
the o-phenol 5c, the m-phenol 5b, and the commercially
available p-phenol 5d were alkylated with various benzyl
bromides to give the fully masked benzyl aryl ethers 6e–
6k. Then, unmasking of the carbamates afforded the
amine hydrochlorides 2m–2n and 2p–2t.
Although the large biphenyl derivatives were fairly
0
rigid, they were tolerated in the S1 subsite of ACE2.
0
Thus, bulkier P1 groups were explored. The o-, m-,
and p-phenoxy phenylalanine derivatives 1j (Ki
0.90 nM), 1k (Ki App = 5.8 nM), and 1l (Ki
=
=
App
App
2.2 nM) retained good potency for ACE2 inhibition
and >100-fold selectivity versus ACE (1j Ki
=
App
5000 nM, 1k Ki App = 1100 nM, 1l Ki App = 250 nM)
and CpA (1j Ki App = 2300 nM, 1k Ki App = 14,000 nM,
0
The structure/activity relationships of P1 analogs are de-
1l K
= 8900 nM), despite altered branching termini.
i
App
0
picted in Table 1. Complete removal of the P1 substituent
as in analog 1b resulted in a large decrease in ACE2
(Ki App = 90 nM) and NEP (Ki App = 5100 nM) inhibitory
In contrast, their NEP selectivity differed. The o-phenyl-
oxy analog 1j (Ki App = 180 nM) was 200-fold selective
versus NEP, while the m-phenoxy analog 1k (Ki App
79 nM) and the p-phenoxy analog 1l (Ki App = 10 nM)
=
activity. The potential reduction in van der Waals interac-
0
tions from the absence of a P1 side chain as well as the
entropic gain from increased rotational freedom of the
acid could account for this decrease in potency. In
contrast, this change had little affect on ACE
(Ki App = 250 nM) or CpA (Ki App = 35,000 nM). The
alanine-derived analog 1c (ACE2 Ki App = 14 nM, NEP
Ki App = 950 nM) decreases rotational freedom and
recovers some of0 the ACE2 inhibitory activity lost upon
removal of the P1 group, while not dramatically changing
the activity versus ACE (Ki App = 180 nM) or CpA
(Ki App = 11,000 nM). The phenylalanine and tyrosine
derivatives 1d (Ki App = 1.5 nM) and 1e (Ki App = 1.3 nM)
were equipotent ACE2 inhibitors to the methylene
were 14-fold and 5-fold NEP selective, respectively.
Surprisingly, the extended o-, m-, and p-benzyloxy phen-
ylalanine derivatives 1m (Ki App = 6.0 nM), 1n (Ki App
=
2.4 nM), and 01o (Ki App = 2.7 nM) were also well toler-
ated in the S1 subsite of ACE2. In addition, they main-
tained good selectivity versus ACE (1m Ki
=
App
770 nM, 1n Ki App = 750 nM, 1o Ki App = 570 nM) and
CpA (1m Ki App = 1600 nM, 1n Ki App = 5000 nM, 1o
Ki App = 4600 nM). In contrast to the phenyloxy ana-
logs, the p-benzyl analog 1o (Ki App = 300 nM) was quite
selective versus NEP (110-fold), while the o-benzyloxy
analog 1m (Ki App = 120 nM) and the m-benzyloxy ana-
log 1n (Ki App = 37 nM) were less selective (20-fold and
15-fold, respectively).
p-biphenyl analog 1a with similar ACE (1d Ki
570 nM, 1e Ki App = 410 nM) and NEP (1d Ki App
=
=
App
35nM, 1e Ki App = 75 nM) activities as well. In contrast
to 1a, the benzyl derivatives 1d and 1e were >10-fold
more potent inhibitors of CpA (1d Ki App = 530 nM, 1e
Both the methylene o-phenoxyphenyl derivative 1j and
the methylene p-benzyloxyphenyl analog 1o are potent
and selective ACE2 inhibitors, but the latter inhibitor is
readily derived from the cheap natural amino acid tyro-
sine. Therefore, fluorinated derivatives of inhibitor 1o
were prepared with the goal of maintaining potency and
selectivity, while blocking potential metabolic sites of
the naked phenyl ring. It was hoped that electron with-
drawing groups would not only sterically impede metab-
olism, but also electronically deactivate the aryl ring to
Ki App = 350 nM). Since many good CpA substrates
0
contain P1 phenylalanine residues, this increase in
CpA inhibition for 1d and 1e is not surprising.
0
It was decided to explore other bulky P1 substituents
with the goal of further improving on the potency
and/or selectivity0 of analog 1a, since the bulky methy-
lene biphenyl P1 moiety is readily accommodated by
0
the ACE2 enzyme, implying a large S1 subsite.
Although the a-methylene naphthyl and the b-methy-
lene naphthyl derivatives 1f (Ki App = 2.2 nM) and 1g
(Ki App = 2.4 nM) were also potent ACE2 inhibitors
oxidation. The mono- and difluoro analogs 1p (Ki App =
2.5 nM), 1q (Ki App = 2.0 nM), and 1r (Ki App = 0.85 nM)
were potent ACE2 inhibitors with good selectivity. The
3,4-difluorobenzyl tyrosine derivative 1r was the most
with >100-fold selectivity versus ACE (1f Ki
1,800 nM, 1g Ki App = 310 nM) and CpA (1f Ki App
=
=
selective analog with >750-fold separation in K
s
App
i
App
between ACE2 and ACE (Ki App = 1800 nM), NEP
(Ki App = 640 nM), and CpA (Ki App = 3300 nM). In
contrast to the ACE2 inhibition of the fluoro derivatives
1p–1r, the larger trifluoromethyl derivatives 1s (Ki
3600 nM, 1g Ki App = 7200 nM), they still retained sub-
stantial inhibitory activity against NEP (1f Ki
12 nM, 1g Ki App = 46 nM).
=
App
App = 35 nM) and 1t (Ki App = 84 nM) were substantially
0
In contrast to the p-phenyl phenylalanine derivative 1a,
the o-phenyl phenylalanine analog 1h (Ki App = 37 nM)
was an order of magnitude less potent ACE2 inhibitor.
less potent ACE2 inhibitors. Likely, these larger P1
0
substituents clash with the residues that make up the S1
subsite.
This substitution also reduced ACE (Ki
=
App
20,000 nM) and NEP (Ki App = 190 nM) inhibition,
while not altering CpA potency (Ki App = 9900 nM). In
contrast to 1h, the methylene m-biphenyl derivative 1i
(Ki App = 2.2 nM) was an equipotent ACE2 inhibitor
to the p-derivative 1a, despite branching from the phenyl
moiety at a different angle, but it offered no clear
selectivity advantages over ACE (Ki App = 1000 nM),
NEP (Ki App = 19 nM), or CpA (Ki App = 5000 nM)
versus 1a.
In vitro drug metabolism and pharmacokinetic assays
were performed to help predict in vivo oral bioavailabil-
ities and pharmacokinetics of the thiols. Although these
inhibitors conform to Lipinski’s Rule of 5, the Madin-
Darby canine kidney (MDCK) assay revealed that this
inhibitor class had poor to moderate passive cell perme-
abilities (PAPP = 5–47 nm/s).20 Since many dipeptides
are absorbed by active transport mechanisms, represen-
tative thiol-based inhibitors were dosed orally in the rat

1684
D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1681–1687
Table 1. Inhibition of human ACE2, ACE, NEP, and CpA
O
H
N
HS
OH
O
1a-t
R
#
R
ACE2
a
Ki App (nM)
ACE
Ki App (nM)
NEP
Ki App (nM)
CpA
Ki App (nM)
b
c
d
1a
1.5
490
27
11,000
Ph
1b
1c
H
Me
90
14
250
180
5100
950
35,000
11,000
1d
1e
1.5
1.3
570
410
35
75
530
350
OH
1f
2.2
1800
12
3600
1g
1h
2.4
37
310
46
7200
9900
Ph
20,000
190
Ph
1i
2.2
0.90
5.8
2.2
6.0
1000
5000
1100
250
19
180
79
5000
2300
O
Ph
1j
O
1k
1l
14,000
8900
Ph
Ph
10
O
Ph
O
1m
770
120
1600
O
Ph
1n
1o
2.4
2.7
750
570
37
5000
4600
Ph
300
O

D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1681–1687
1685
Table 1 (continued)
#
R
ACE2
Ki App (nM)
ACE
Ki App (nM)
NEP
Ki App (nM)
CpA
Ki App (nM)
a
b
c
d
F
F
1p
2.5
4000
180
16,000
O
O
1q
1r
1s
1t
2.0
0.85
35
950
1800
75
640
1900
3300
F
F
O
O
F
CF3
2600
1500
4600
CF3
84
25,000
11,000
11,000
O
CF3
a Inhibition of recombinant human ACE2 activity in a fluorescence assay using 0.4 nM ACE2, 30 lM MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-
OH as substrate in 1 lM Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The Ki App values are means
of at least two inhibition assays.
b Inhibition of recombinant human ACE activity in a fluorescence assay using 0.5 nM ACE, 10 lM MCA-Ala-Ser-Asp-Lys-Dap(DNP)-OH as
substrate in 1 lM Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The Ki App values are means of at
least two inhibition assays.
c Inhibition of recombinant human NEP activity in a fluorescence assay using 0.15 nM NEP, 2 lM FAM-Gly-Pro-Leu-Gly-Leu-Phe-Ala-Arg-
Lys(TAMRA)-NH2 as substrate in 1 lM Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The Ki App
values are means of at least two inhibition assays.
d Inhibition of recombinant human CpA activity in a fluorescence assay using 37 nM CpA, 30 lM Abz-Gly-Gly-Nph-OH as substrate in 1 lM
Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The Ki App values are means of at least two inhibition
assays.
despite their poor predicted cell permeation, but they
had limited absorption (F < 10%) in agreement with
the MDCK assay.
A model21,22 of the thiol 1r docked into the active site of
ACE2 based on the recent Millennium X-ray crystal
structure23 is shown in Figure 1. It provides insight into
0
the P1 SAR of the thiol series. The terminal carboxylate
of the inhibitor accepts hydrogen bonds from the imida-
zoles of 345His and 505His and forms a salt bridge to the
guanidine of 273Arg. Also, the amide nitrogen of the
inhibitor donates a hydrogen atom to the carbonyl of
346Pro, while the amide carbonyl accepts a hydrogen
atom from 515Tyr. Moreover, in addition to its normal
ACE2 ligands, the imidazole nitrogens of 374His and
378His and the carboxylate of 402Glu, the active site zinc
coordinates the thiol of the inhibitor. Furthermore, the
P1 sec-butyl group of the inhibitor forms van der Waals
Figure 1. A model of the thiol compound 1r bound to the active site of
ACE2 based on the X-ray co-crystal structure of MLN-4760 bound to
interactions with the S1 pocket composed of 347Thr,
0
ACE2 (PDB code 1R4L). The ACE2 carbons are colored cyan with
inhibitor 1r carbons colored green. The semi-transparent gray surface
represents the molecular surface, while hydrogen bonds are depicted as
yellow dashed lines. Several residues were removed for visual clarity.
This figure was generated using PYMOL version 1.0 (Delano
Scientific, www.pymol.org).
504Phe, 510Tyr, and 514Arg. The greasy P1 3,4-dif-
luorobenzyltyrosine side chain of 1r forms significant
0
lipophilic interactions with the quite large S1 channel
composed of the lengthwise canal between the two sub-
domains, including residues 274Phe, 445Thr, 406Glu,

1686
D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1681–1687
Acknowledgments
The authors thank Andrea Epperly, Chuck Poole, and
Jo Salisbury for the pharmacokinetic studies.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
01.046.
References and notes
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Circ. Res. 2000, 87, e1.
2. Tipnis, S. R.; Hooper, N. M.; Hyde, R.; Karran, E.;
Christie, G.; Turner, A. J. J. Biol. Chem. 2000, 275, 33238.
3. Danilczyk, U.; Penninger, J. M. Circ. Res. 2006, 98, 463.
4. Kuba, K.; Imai, Y.; Penninger, J. M. Curr. Opin. Pharm.
2006, 6, 271.
Figure 2. A comparison of the models of the thiol compounds 1j
(carbons colored in green) and 1r (carbons colored in magenta) bound
to the active site of ACE2 (carbons colored in cyan) based on the X-ray
co-crystal structure (PDB code 1R4L). The semi-transparent gray
surface represents the molecular surface, while hydrogen bonds are
depicted as yellow dashed lines. Several residues were removed for
visual clarity. This figure was generated using PYMOL version 1.00.
5. Tallant, E. A.; Ferrario, C. M.; Gallagher, P. E. Future
Cardiol. 2006, 2, 335.
409Ser, 370Leu, 371Thr, 276Thr and extending over to
441Lys and 442Gln. Although several residues in this
pocket are hydrophilic, most of the polar groups are
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Kozieradzki, I.; Scanga, S. E.; Oliveira-dos-Santos, A. J.;
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either involved in hydrogen bonds with other enzyme
0
residues or oriented away from the S1 pocket, maintain-
ing the lipophilic nature of this subsite. Based on this
0
model, the 3,4-difluorobenzyl portion of the P1 substitu-
ent is hypothesized to oc0cupy a different part of this
large pocket than the P1 side chain of the carboxyl
inhibitor MLN-4760 co-crystallized in 1R4L.
In contrast, as shown in Figure 2, a model21,22 of the
thiol 1j docked into 0the active site of ACE2 revealed that
the o-phenyloxy P1 group occupies a different part of
0
the S1 subsite than the 3,4-difluorobenzyl substituent
0
of analog 1r. The o-phenyloxy P1 moiety fits into a por-
0
tion of the S1 subsite composed of 360Met, 346Pro,
362Thr, 271Trp, 368Asp, 371Thr, 127Tyr, 144Leu, 149Asn,
363Lys, and 269Asp, and the disulfide pair of 344Cys
0
and 361Cys. Thus, the o-phenyloxy P1 moiety of 1j binds
0
similarly to the P1 side chain of the Millennium car-
boxyl inhibitor MLN-4760 co-crystallized in 1R4L.
The other interactions of inhibitor 1j are similar to those
for analog 10r. Thus, these two models help explain the
12. Kuba, K.; Imai, Y.; Rao, S.; Gao, H.; Guo, F.; Guan, B.;
Huan, Y.; Yang, P.; Zhang, Y.; Deng, W.; Bao, L.; Zhang,
B.; Liu, G.; Wang, Z.; Chappell, M.; Liu, Y.; Zheng, D.;
Leibbrandt, A.; Wada, T.; Slutsky, A. S.; Liu, D.; Qin, C.;
Jiang, C.; Penninger, J. M. Nat. Med. 2005, 11, 875.
13. Hamming, I.; Cooper, M. E.; Haagmans, B. L.; Hooper,
N. M.; Korstanje, R.; Osterhaus, A. D. M. E.; Timens,
W.; Tumer, A. J.; Navis, G.; van Goor, H. J. Pathol. 2007,
212, 1.
14. Dales, N. A.; Gould, A. E.; Brown, J. A.; Calderwood, E.
F.; Guan, B.; Minor, C. A.; Gavin, J. M.; Hales, P.;
Kaushik, V. K.; Stewart, M.; Tummino, P. J.; Vickers, C.
S.; Ocain, T. D.; Patane, M. A. J. Am. Chem. Soc. 2002,
124, 11852.
0
divergent P1 SAR, since the very large, forked S1 sub-
site can tolerate substituents with different branching
points.
0
In summary, variation of substituents at the P1 position
in a series of a-thiol amide-based inhibitors of ACE2 re-
sulted in the discovery of potent inhibitors with good
ACE and NEP selectivity. Inhibitors containing p-meth-
0
ylene aryl tyrosine P1 moieties like 1o, 1p, and 1r were
some of the more selective ACE2 inhibitors. In addition,
o-phenyloxy phenylalanine analog 1j was also a potent
and selective ACE2 inhibitor. These analogs may prove
useful in further defining the roles ACE2 plays in the
RAS cascade.
15. Huang, L.; Sexton, D. J.; Skogerson, K.; Devlin, M.;
Smith, R.; Sanyal, I.; Parry, T.; Kent, R.; Enright, J.; Wu,

D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1681–1687
1687
Q.; Conley, G.; DeOliveira, D.; Morganelli, L.; Ducar,
M.; Wescott, C. R.; Ladner, R. C. J. Biol. Chem. 2003,
278, 15532.
tions, both the thiols and their corresponding disulfides
showed enzyme inhibitory activity. Presumably, the disul-
fides were reduced to their corresponding thiols by TCEP
during the pre-incubation period, before substrates were
added. In contrast, if the assays were performed without
TCEP, the disulfides were completely inactive, while the
potencies of the thiols were attenuated, probably because
of partial aerobic oxidation to their corresponding disul-
fides during the pre-incubation period.
16. Rella, M.; Rushworth, C. A.; Guy, J. L.; Turner, A. J.;
Langer, T.; Jackson, R. M. J. Chem. Inf. Model 2006, 46,
708.
17. Huentelman, M. J.; Zubcevic, J.; Hernandez Prada, J. A.;
Xiao, X.; Dimitrov, D. S.; Raizada, M. K.; Ostrov, D. A.
Hypertension 2004, 44, 903.
18. Deaton, D. N.; Gao, E. N.; Graham, K. P.; Gross, J. W.;
Miller, A. B.; Strelow, J. M. Bioorg. Med. Chem. Lett.
2008, 18, 732.
20. Irvine, J. D.; Takahashi, L.; Lockhart, K.; Cheong, J.;
Tolan, J. W.; Selick, H. E.; Grove, J. R. J. Pharm. Sci.
1999, 88, 28.
19. Hydrolyses were performed under an argon atmosphere.
When hydrolyses were carried out with reactions open to
the atmosphere, varying amounts of disulfide products
were isolated, likely from aerobic oxidation. The enzyme
assays contain a reducing agent, Tris-(2-chloroethyl)-
phosphate (TCEP), to prevent oxidation of the thiols to
disulfides during the assays. Dilutions of 10 mM stock
solutions of the thiols 1a–1t to final assay concentrations
were done with 50% aqueous acetonitrile just prior to
protease inhibition studies. Under these standard condi-
21. Lambert, M. H. In Practical Application of Computer-
Aided Drug Design; Charifson, P. S., Ed.; Marcel Dekker:
New York, NY, 1997; p 243.
22. The ‘grow’ algorithm within the MVP program was used
to dock the inhibitor into the active site beginning from
the carboxylic acid group.
23. Towler, P.; Staker, B.; Prasad, S. G.; Menon, S.; Tang, J.;
Parsons, T.; Ryan, D.; Fisher, M.; Williams, D.; Dales, N.
A.; Patane, M. A.; Pantoliano, M. W. J. Biol. Chem. 2004,
279, 17996.