Dual metalloprotease inhibitors. 6. Incorporation of bicyclic and substituted monocyclic azepinones as dipeptide surrogates in angiotensin-converting enzyme/neutral endopeptidase inhibitors.

Article abstract of DOI:10.1021/jm950677a

A series of substituted monocyclic and bicyclic azepinones were incorporated as dipeptide surrogates in mercaptoacetyl dipeptides with the desire to generate a single compound which would potently inhibit both angiotensin-converting enzyme (ACE) and neutr

Full text of DOI:10.1021/jm950677a

494
J . Med. Chem. 1996, 39, 494-502
Du a l Meta llop r otea se In h ibitor s. 6. In cor p or a tion of Bicyclic a n d Su bstitu ted
Mon ocyclic Azep in on es a s Dip ep tid e Su r r oga tes in An gioten sin -Con ver tin g
En zym e/Neu tr a l En d op ep tid a se In h ibitor s
J effrey A. Robl,* Maria P. Cimarusti, Ligaya M. Simpkins, Baerbel Brown, Denis E. Ryono, J . Eileen Bird,
Magdi M. Asaad, Thomas R. Schaeffer, and Nick C. Trippodo
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New J ersey 08543-4000
Received September 14, 1995X
A series of substituted monocyclic and bicyclic azepinones were incorporated as dipeptide
surrogates in mercaptoacetyl dipeptides with the desire to generate a single compound which
would potently inhibit both angiotensin-converting enzyme (ACE) and neutral endopeptidase
(NEP). Many of these compounds displayed excellent potency against both enzymes. Two of
the most potent compounds, monocyclic azepinone 2n and bicyclic azepinone 3q, demonstrated
a high level of activity versus ACE and NEP both in vitro and in vivo.
In tr od u ction
) 30 nM vs ACE), was found to be marginally active
versus NEP in vitro (IC50 ) 400 nM). The related
mercaptopropanoyl analog 1b possessed excellent in
vitro potency against both enzymes but, unlike its
mercaptoacetyl analog 1a , was poorly active in vivo.4
In an effort to increase both the in vitro and in vivo
potency of 1 toward ACE and NEP, the Ala-Pro portion
of the molecule was replaced with a variety of confor-
mationally restricted dipeptide mimetic surrogates.3b-e
One of the most potent of these compounds was benza-
zepinone-based mercaptoacetyl 4 (BMS-182657). This
compound possessed excellent activity versus ACE and
NEP in vitro and in addition demonstrated potent
inhibition of both metalloproteases in vivo.3b,5
Angiotensin-converting enzyme (ACE), a zinc-con-
taining carboxydipeptidase, is largely responsible for the
conversion of the inactive decapeptide angiotensin I (AI)
to the biologically active octapeptide angiotensin II (AII).
AII, a main component of the renin-angiotensin-
aldosterone system (RAS), serves to raise blood pressure
by both increasing vascular resistance and triggering
the production of the sodium-retaining steroid aldos-
terone. Thus, inhibition of ACE has been an actively
pursued target in the drug industry. Over 2 decades of
research has established the benefits of ACE inhibitors
in the treatment of essential human hypertension and
congestive heart failure.
In this present study, we examined the effect of
conformational restraint on the Ala-Pro portion of
compound 1a utilizing a series of monocyclic and bicyclic
dipeptide surrogates, leading to inhibitors represented
by 2 and 3, respectively. Conceptually, bicyclic com-
pounds of type 3 arise via incorporation of an ethyl
linker between the alanine methyl group and the
5-position of the proline ring. Combining this cycliza-
tion with a ring-opened proline gives the monocyclic
derivatives 2. Although both types of dipeptide sur-
rogates (mono- and bicyclic azepinones) had been previ-
ously exploited for use as conformationally restricted
surrogates of Ala-Pro in the ACE inhibitor enalapril,6
past synthetic methodologies allowed access to only a
few simple dipeptide surrogates. Recently, we have
developed new synthetic methods for the synthesis of
both the 7,5-fused (m ) 0) and the 7,6-fused (m ) 1)
bicyclic azepinone nuclei7 found in 3 as well as methods
for the generation of a wide variety of substituted
monocyclic azepinones8 (as found in 2) in homochiral
form. Incorporation of these azepinones as conforma-
tionally restricted mimetics of mercaptoacetyl dipeptides
and the biological evaluation of these compounds versus
both ACE and NEP are the basis of this report.
Neutral endopeptidase (E.C. 3.4.24.11, NEP), a zinc-
containing endopeptidase found in high concentration
within the brush border region of the kidney, is in large
part responsible for the degradation and inactivation
of atrial natriuretic peptide (ANP). ANP is a 28-amino
acid peptide hormone produced by the heart in response
to atrial distention and plays a role in modulating
cardiovascular homeostasis. Through interaction with
its biological receptor, ANP upregulates cGMP produc-
tion, which in turn leads to vasodilatation, natriuresis,
diuresis, and inhibition of aldosterone formation. Be-
cause the hormonal actions of AII and ANP are func-
tionally opposed, it is expected that attenuation of AII
with concurent potentiation of ANP will lead to a
beneficial synergistic effect by lowering vascular resis-
tance and inhibiting the RAS. Indeed, recent studies
have shown that coadministration of selective ACE and
NEP inhibitors is more effective in animal models of
hypertension and congestive heart failure than treat-
ment with either enzyme inhibitor given alone.1 On the
basis of this premise, a number of groups, including our
own, have been active in the pursuit of a dual-acting
ACE/NEP inhibitor.2
In a recent series of articles, we outlined the rationale,
design, and synthesis of a class of sulfhydryl-containing
compounds which inhibited ACE and NEP both in vitro
and in vivo.3 In our initial disclosure, we identified
mercaptoacyl dipeptides 1a ,b as early leads.3a Com-
pound 1a , originally developed as an ACE inhibitor (IC50
Ch em istr y
Construction of mercaptoacetyl-containing inhibitors
2 and 3 is depicted in Scheme 1. BOP reagent-mediated
coupling of the requisite amine dipeptide surrogates
5a -q (where R2 and R3 could be joined to form an
additional ring) with (S)-R-(acetylthio)-2-benzenepro-
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0022-2623/96/1839-0494$12.00/0 © 1996 American Chemical Society

Dual Metalloprotease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 495
tion of the mixture provided the corresponding C-7
cyclopentyl-substituted azepinone 7m as a single dias-
tereomer, confirming the stereochemical homogeneity
of the lactam ring at the C-7 center.
The allyl functionality at the C-7 position of the
azepinone nucleus of 7i provided an excellent handle
for subsequent alkyl group modification. Palladium-
catalyzed cyclopropanation10 of 7i provided the corre-
sponding methylenecyclopropyl analog 7k in nearly
quantitative yield (Scheme 4). Hydrazinolysis of 7k
provided 5k cleanly. Hydrogenation of 5k under forcing
conditions occurred regioselectively at the least hindered
face of the cyclopropyl group11 to give the C-7 isobutyl
analog 5l. In this case removal of the protecting group
prior to hydrogenation was necessary in order to prevent
reduction of the phthalimido group. Additionally, re-
duction of the C-7 allyl substituent cleanly afforded the
propyl-substituted azepinone 7g. This method for the
generation of 7g (where PG is phthalimido and R is
methyl) is complimentary to the previously described
procedure for the synthesis of this C-7-substituted
azepinone (where PG is Boc and R is ethyl).8b The
hydroxyl isostere of 7g, azepinone 7j, was generated by
the procedure outlined in Scheme 5. Due to the
relatively base labile nature of the phthalimido group,
compound 7i was first treated with hydrazine and the
intermediate amine 5i was reprotected as its Boc
derivative. Oxidative cleavage of 15 with NaIO4 in the
presence of catalytic OsO4 afforded aldehyde 16 which
cleanly underwent reduction to the corresponding al-
cohol 7j. Subsequent removal of the Boc group with HCl
in dioxane gave amine 5j.
F igu r e 1.
Sch em e 1
panoic acid9 gave 6a -q in generally good yield and
excellent diastereomeric purity. Saponification of the
thioacetate and alkyl ester groups under strictly oxygen-
free conditions followed by acidification afforded the
desired inhibitors 2 or 3.
Amines 5a -q were generated from the corresponding
N-protected azepinone esters 7a -q (Table 1) by treat-
ment with either hydrazine monohydrate in methanol
(protecting group (PG) ) phthalimido) or anhydrous
hydrogen chloride in dioxane (PG ) Boc). Azepinones
7a -d ,i,n were synthesized using methodology previ-
ously developed in this laboratory.8a Generation of the
methyl- and propyl-substituted azepinones 7e-h fol-
lowed our recent literature procedures8b as did the
synthesis of bicyclic azepinones 7p ,q.7 The remaining
surrogates were constructed following the chemistry
outlined in Schemes 2-5.
Discu ssion
Compounds 2 and 3 were tested for their ability to
inhibit both ACE and NEP in vitro, and the data are
listed in Table 2. Inhibition of ACE activity in vivo was
determined from plots of percent maximal inhibition of
the AI-induced pressor response versus dose after
intravenous (iv) administration in the conscious nor-
motensive rat. The ED50 values correspond to the dose
required to effect 50% inhibition of the pressor response.
For comparative purposes, data for compounds 1a and
4 are also listed in Table 2.
Compound 7o was synthesized utilizing methodology
similar to that developed for the generation of 7n . Thus,
(S)-6-hydroxy-2-phthalimidohexanoic acid (8) was coupled
to (S)-2-aminobutyric acid (9) to give an intermediate
dipeptidyl alcohol (Scheme 2). Subsequent Swern oxi-
dation afforded aldehyde 10 which was subjected to
acid-catalyzed cyclization to give bicyclic lactam 11 in
72% yield and 94% diastereomeric purity. The assigned
stereochemistry at the newly formed stereocenter was
confirmed by the observance of a strong NOE signal
between the bridgehead hydrogen and the hydrogen R
to the phthalimido-protected nitrogen. Treatment of
diastereomerically pure 11 with allyltrimethylsilane in
the presence of the Lewis acid catalyst TiCl4 gave crude
acid 12a which was esterified to give 12b as a single
diastereomer. Subsequent hydrogenation of 12b over
palladium cleanly afforded the highly substituted aze-
pinone 7o. In close analogy, 3-(trimethylsilyl)cyclopen-
tene was added to bicyclic lactam 138a in the presence
of SnCl4 to give, after esterification, compound 14 as a
2:1 mixture (atom labeled with an asterisk in structure
14) of diastereomers (Scheme 3). Catalytic hydrogena-
Compound 2a , the simplest of the conformationally
restricted ACE/NEP inhibitors (R1 ) R2 ) R3 ) H),
exhibited a 5-fold gain in in vitro inhibitory potency
versus NEP as compared to the Ala-Pro analog 1a .
Unfortunately this modification led to a 14-fold diminu-
tion in ACE activity in vivo despite similar potency
versus ACE in vitro. In addition a dramatic loss in
duration of activity for 2a was observed in the AI
pressor assay (t1/2 ≈ <10 min for 2a versus t1/2 ≈ 45
min for 1a at a comparative dose of 0.5 µmol/kg iv).
Introduction of a methyl substituent on the acetic acid
side chain of 2a to give 2b resulted in essentially no
change in activity versus ACE or NEP both in vitro and
in vivo. Larger substituents at this position (benzyl
analog 2c and isopropyl analog 2d ) afforded compounds
with greatly diminished activity against one or both
enzymes. In contrast, the presence of substituents at
the C-7 position of the monocyclic azepinone ring
resulted in a subset of inhibitors with greater affinity
for the target enzymes. Introduction of a methyl (2e)
or propyl (2g) group in the S configuration at the C-7

496 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Robl et al.
Ta ble 1. Protected Monocyclic and Bicyclic Azepinones 7 Utilized in the Synthesis of Amines 5
protected
azepinones
R
R1
R2
R3
PG removed
7a
7b
7c
7d
7e
7f
Et
H
H
H
H
H
H
H
H
H
H
Boca
Phtb
Pht
Pht
Boc
Me
Me
Me
Et
methyl
benzyl
isopropyl
H
methyl
H
Et
methyl
H
Boc
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
Me, Et
Et
propyl
H
allyl
hydroxyethyl
methylcyclopropyl
isobutyl
cyclopentyl
propyl
H
H
H
H
H
H
H
H
Pht, Boc
Boc
Pht
Boc
Pht
propyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
Pht
Pht
Pht
Pht
Pht
methyl
ethyl
propyl
H
H
-CH2CH2-
-CH2CH2CH2-
a
b
Boc ) (tert-butyloxy)carbonyl. Pht ) phthalimido.
Sch em e 2
Sch em e 4
2h indicates that the preferred stereochemistry at C-7
with respect to NEP is S. The corresponding R isomers
of 2e,g were 3- and 11-fold less potent versus NEP
in vitro. Surprisingly ACE seemed relatively insensitive
to both the nature and stereochemical orientation of the
substituent at this position.
Sch em e 3
We next explored a series of monocyclic based inhibi-
tors which varied with respect to steric bulk at the R1
position. Allyl-substituted azepinone 2i was slightly
less potent than its dihydro analog 2g versus NEP,
whereas the hydroxyethyl analog 2j was somewhat more
potent. Both the allyl and the hydroxyethyl analogs
displayed activity comparable to 2g against ACE both
in vitro and in vivo. NEP proved to be exquisitely
sensitive to the steric nature of the R1 substituent. The
addition of a single methylene group in 2g, giving 2k ,
resulted in a 3.5-fold loss in NEP inhibitory potency.
More surprisingly, replacement of the propyl group in
2g with isobutyl, a one-carbon addition, decreased the
inhibitors affinity for NEP by 40-fold. Introduction of
the sterically demanding cyclopentyl group in 2m had
a similar effect on NEP inhibitory potency. In sharp
contrast, in vitro activity versus ACE did not vary more
than 3-fold among the C-7-substituted azepinones stud-
position resulted in an additional 2-fold enhancement
in both ACE and NEP activity in vitro relative to 2a .
More importantly, the activities of these compounds in
the AI pressor assay were dramatically enhanced (8-
14-fold) by this modification. The reason for this boost
in in vivo activity is unclear. It may be that the
presence of alkyl substituents at this position of the
molecule confers greater metabolic stability to the
inhibitors. A comparison of 2e with 2f and of 2g with

Dual Metalloprotease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 497
Sch em e 5
Ta ble 2. Inhibition of ACE and NEP for Compounds 2 and 3
ACE AI
pressord ED50
IC50 (nM)
yield
no.a
R1
R2
R3
NEPb ACEc (µmol/kg iv) from 7 (%) [R]D (deg) (c, solvent)
formulae
1a
4
400
6
82
92
30
12
22
26
0.06
0.02
0.84
1.22
NTg
NT
0.11
NT
0.06
NT
0.04
0.08
NT
NT
NT
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
H
H
H
H
H
H
H
H
H
H
83
67
65
75
37
37
50
75
55
18
66
28
36
30
31
76
80
+8.0 (0.6, CH3OH)
-39.1 (0.6, CHCl3)
-64.2 (0.5, CHCl3)
-90.4 (0.8, CH3OH) C20H28N2O4S
-28.7 (0.3, CH3OH) C18H24N2O4S
+16.8 (0.3, CH3OH) C18H24N2O4S‚0.1H2O
-51.2 (0.7, CHCl3)
+7.6 (0.8, CHCl3)
-48.5 (0.6, CHCl3)
-41.9 (1.0, CDCl3)
-78.9 (1.0, CDCl3)
-40.1 (1.0, CDCl3)
-78.1 (1.0, CDCl3)
C17H22N2O4S
methyl
benzyl
C18H24N2O4Sf
32 479
isopropyl 1000 907
C24H28N2O4S‚0.3H2O
methyl
H
propyl
H
allyl
hydroxyethyl
H
49
149
40
466
64
8
16
11
14
7
25
23
10
17
5
methyl H
H
propyl
H
H
H
H
H
H
H
H
H
C20H28N2O4S‚0.3H2Oh
C20H28N2O4S‚0.1H2O
C20H26N2O4S‚0.2H2O
C19H26N2O5S‚0.5H2O
C21H28N2O4S‚0.5H2O
C21H30N2O4S
31
methylcyclopropyl H
140
1655
1653
17
86
18
isobutyl
cyclopentyl
propyl
H
H
H
H
C22H30N2O4S
methyl
ethyl
0.12
NT
0.06
0.12
-51.6 (0.5, CH2Cl2) C21H30N2O4S
-57.7 (0.4, CH2Cl2) C22H32N2O4S‚0.2EtOAc
-107.9 (0.6, CHCl3) C19H24N2O4S
propyl
25
5
11
3p (n ) 0)
3q (n ) 1)
25
-31.0 (0.8, CHCl3)
C20H26N2O4S‚0.45H2Oi
a
b
All spectral data were consistent with the assigned structures. Compounds were assayed against purified rat kidney neutral
endopeptidase using a fluorometric assay with dansyl-Gly-Phe-Arg as the substrate. c Compounds were assayed against angiotensin-
d
converting enzyme isolated from rabbit lung extract using hippuryl-L-histidyl-L-leucine (HHL) as the substrate. Represents dose required
for 50% inhibition of the Al pressor response in normotensive rats. See the Experimental Section for a description of this assay. e Analyzed
for C, H, N, and S. Results were within (0.4% of theory unless otherwise noted. f Anal. Calcd: S, 8.80. Found: S, 8.36. Not tested.
g
h
i
Anal. Calcd: S, 8.06. Found: S, 7.63. Anal. Calcd: N, 7.03. Found: N, 6.57.
ied. In combination with other studies we have done
with mercaptoacetyl-containing inhibitors, these results
underscore our belief that NEP is much more sensitive
than ACE with respect to the nature of the ligands
within their binding subsites.
bicyclic lactam 3q was found to be comparable in
potency to 2n both in vitro and in vivo (iv administra-
tion). The related 7,5-fused analog 3p was 2-fold more
active than either 3q or 2n as an ACE inhibitor in vivo,
but this increase in activity was not accompanied by an
improved duration of activity in the AI pressor assay
(t1/2 ≈ 30 min for 3p versus t1/2 ≈ 60 min for 3q at a
comparable dose of 0.5 µmol/kg iv). We have previously
found that in various bicyclic fused inhibitors, an
R-configured hydrogen at the bridgehead center is
optimal for good inhibitory potency versus NEP.12 The
excellent in vitro potency of 3p ,q against both NEP and
ACE supports this statement. In contrast, optimal
activity versus NEP in the monocyclic class of com-
pounds could only be obtained when the related hydro-
gen at the C-7 position was in the â-configuration. It
is suspected that the presence of the substituent at the
C-7 position serves to effect the overall conformation of
the monocyclic azepinone ring, positioning the C-3
amine, the C-2 carbonyl group, and the acetic acid side
chain in an optimum orientation for binding similar to
that attained by the bicyclic class of inhibitors. Thus
when the alkyl substituent in 2 is R (hydrogen is â),
the monocyclic seven-membered lactam ring is believed
to adopt a conformation similar to that of the highly
active 7,6(5)-fused bicyclic class of inhibitors 3.
In the case of 2a , introduction of a methyl group on
the acetic acid side chain to give 2b had little effect on
inhibitory potency versus either enzyme. In contrast,
the C-7 propyl, R-methyl side chain analog 2n exhibited
2 times the potency against both ACE and NEP in vitro
as compared to its desmethyl counterpart 2g. Inhibitor
2n was found to be the most potent of the monocyclic
based ACE/NEP inhibitors in this study, and its in vitro
activity compared favorably with that of the previous
lead benzazepinone 4. Although the ED50 of 2n is 6-fold
less than that of 4, its duration of activity in this assay
is longer (t1/2 ≈ 50 min for 2n versus t1/2 ≈ 20 min for 4)
at a comparable dose of 0.5 µmol/kg iv. The ethyl analog
of 2n , azepinone 2o, was 5-fold less active with respect
to both ACE and NEP in vitro.
The potent activity observed by the fused bicyclic
lactams 3p ,q underscores our previous findings3d that
greater in vitro inhibitory potency versus both metal-
loproteases, especially NEP, can be more easily attained
with conformationally constrained bicyclic mimetics as
opposed to their monocyclic counterparts. The 7,6-fused

498 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Robl et al.
Ch a r t 2. Inhibition of the AI Pressor Response after
Ch a r t 1. ACE/NEP Inhibitors in the 1K DOCA Rats at
100 µmol/kg iv
Oral Administration at 5 µmol/kg in Rats
Compounds which displayed acceptable iv activity in
the AI pressor assay and possessed reasonable potency
versus NEP in vitro were tested in the 1K DOCA salt
hypertensive rat assay. Selective NEP inhibitors have
been shown to lower mean arterial pressure in this
assay, whereas selective ACE inhibitors (i.e., captopril)
are usually ineffective. Compounds were given as bolus
injections at 100 µmol/kg (≈40 mpk) iv, and mean
arterial pressure (MAP) was measured over a 24 h
period. The data in Chart 1 represent the compiled
area-over-the-curve (AOC) during this time period.
Dual inhibitor 3q was essentially equipotent to the
selective NEP inhibitors SQ 28,60313 and candoxatri-
lat.14 Monocyclic based inhibitors 2i,g,n were somewhat
less effective but still showed a significant antihyper-
tensive effect in this assay. Benzazepinone-based in-
hibitor 4 was the most potent compound in this com-
parison, displaying activity only slightly better than that
of SQ 28,603 and 3q. Since the antihypertensive
activity of selective NEP inhibitors is not potentiated
by the addition of ACE inhibitors in this assay,1a the
ability of the dual-acting inhibitors to lower MAP is
attributed to their intrinsic ability to singularly inhibit
NEP in vivo.
or with appropriately substituted monocyclic azepino-
nes. Both the bicyclic inhibitor 3q and the monocyclic
inhibitor 2n exhibited high levels of activity in vitro and
in vivo versus ACE and NEP. In the AI pressor assay,
compound 2n also demonstrated oral activity that was
slightly superior to that of captopril, a clinically proven
ACE inhibitor. A more detailed pharmacological evalu-
ation of these conformationally restrained monocyclic
and bicyclic mercaptoacetyl dipeptides and their related
analogs will be presented in the near future.
Exp er im en ta l Section
All reactions were carried out under a static atmosphere of
argon, and the mixtures were stirred magnetically unless
otherwise noted. All reagents used were of commercial quality
and obtained from Aldrich Chemical Co. or Sigma Chemical
Co. DMF was obtained from American Burdick and J ackson
and used without purification. Dry CH2Cl2 was obtained by
distillation from CaH2 under nitrogen. 3-(Trimethylsilyl)-
cyclopentene was purchased from Pfaltz & Bauer, Inc. Melting
points were obtained on a Hoover Unimelt melting point
apparatus and are uncorrected. Infrared spectra were re-
corded on a Mattson Sirius 100-FTIR spectrophotometer. 1H
(400 MHz) and 13C (100 MHz) NMR spectra were recorded on
a J EOL GSX400 spectrometer using Me4Si as an internal
standard. Optical rotations were measured in a 1 dm cell on
a Perkin-Elmer 241 polarimeter, and c is expressed in g/100
mL. All flash chromatographic separations were performed
using E. Merck silica gel 60 (particle size, 0.040-0.063 mm).
Reactions were monitored by TLC using 0.25 mm E. Merck
silica gel plates (60 F254) and were visualized with UV light or
5% phosphomolybdic acid in 95% EtOH.
Based on their potent activity against ACE and NEP
both in vitro and in vivo, compounds 2n and 3q were
evaluated orally in the AI pressor assay at a compara-
tive dose of 5 µmol/kg (≈2 mpk) (Chart 2). Bicyclic
lactam 3q was less effective than captopril with respect
to both potency and duration of action. In contrast,
monocyclic azepinone 2n exhibited activity in this assay
which was slightly superior to that of captopril.
Gen er a l P r oced u r e for th e Con ver sion of P h th a l-
im id o-P r otected Azep in on es 7 to Th eir Cor r esp on d in g
Am in es 5. (3S)-tr a n s-3-Am in o-7-(cyclop r op ylm eth yl)-
h exa h yd r o-2-oxo-1H-a zep in e-1-a cetic Acid , Meth yl Ester
(5k ). A solution of phthalimido-protected azepinone 7k (697
mg, 1.81 mmol) in methanol (8 mL) at room temperature was
treated with hydrazine monohydrate (92 µL, 95 mg, 1.90
mmol). After 2 days of stirring, the reaction mixture was
filtered to remove the phthalhydrazide byproduct, and the solid
was washed well with methanol. The filtrate was concen-
trated, and the residue was triturated with CH2Cl2 to precipi-
tate out additional byproduct. Filtration followed by removal
of the solvent provided pure amine 5k (441 mg, 96% of theory)
as a white foam which was used without further purification:
Con clu sion
In previous studies we have demonstrated that re-
placement of the dipeptide portion of mercaptoacetyl
dipeptides with suitably constrained peptidomimetics
can dramatically enhance the ability of these compounds
to act as dual ACE and NEP inhibitors both in vitro
and in vivo. In this work we have concentrated on the
utilization of non-benzo-fused azepinones as conforma-
tionally restrained mimetics of Ala-Pro, applying new
chemical methods developed in this laboratory for their
preparation. Structure-activity relationships within
these classes of compounds clearly demonstrate that
potent in vitro activity versus both ACE and NEP can
be obtained with 7,5- or 7,6-fused bicyclic azepinones
1
TLC Rf 0.25 (1:9 MeOH:CH2Cl2); H NMR (CDCl3) δ 0.09 (m,
2H), 0.49 (m, 2H), 0.64 (m, 1H), 1.50 (m, 2H), 1.71-2.05 (m,
6H), 3.47 (m, 1H), 3.53 (d, J ) 9.4 Hz, 1H), 3.74 (s, 3H), 3.76
(d, J ) 17 Hz, 1H), 4.66 (d, J ) 17 Hz, 1H); 13C NMR (CDCl3)
δ 176.75, 170.36, 61.74, 54.37, 52.70, 52.06, 37.23, 33.38, 31.04,
21.66, 8.70, 4.58.

Dual Metalloprotease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 499
1
(3S)-tr a n s-3-Am in oh exa h yd r o-7-(2-m et h ylp r op yl)-2-
oxo-1H-a zep in e-1-a cetic Acid , Meth yl Ester (5l). A solu-
tion of amine 5k (405 mg, 1.59 mmol) in HOAc (10 mL) and
MeOH (3 mL) was charged with PtO2 on carbon (30% by
weight, 122 mg) and subsequently hydrogenated (45 psi) on a
Parr apparatus at 50 °C. An additional 60 and 30 mg of PtO2
on carbon were added after 60 and 104 h, respectively. After
a total of 5 days, the solution was passed through a polycar-
bonate filter and the filtrate was concentrated to give an oil.
The oil was dissolved in CH2Cl2 and subsequently washed with
saturated NaHCO3. Drying of the solution (Na2SO4) followed
by removal of the solvent gave crude 5l (120 mg, 29%) as an
oil: TLC Rf 0.30 (1:9 MeOH:CH2Cl2); 1H NMR (CDCl3) δ 0.90
(d, J ) 6.0 Hz, 3H), 0.94 (d, J ) 6.4 Hz, 3H), 1.25 (m, 1H),
1.56-2.06 (m, 8H), 3.4 (m, 1H), 3.68 (d, J ) 17.1 Hz, 1H), 3.73
(s, 3H), 4.12 (d, J ) 13 Hz, 1H), 4.62 (d, J ) 17.1 Hz, 1H),
5.26-5.30 (s, 2H).
Gen er a l P r oced u r e for th e Cou p lin g of Am in oa zep i-
n on es 5 w ith (S)-2-(Acetylth io)ben zen ep r op ion ic Acid .
[rS-(rR*,2R*,3R*)]-3-[[2-(Acetylth io)-1-oxo-3-p h en ylp r o-
pyl]am in o]h exah ydr o-r-(1-m eth yleth yl)-2-oxo-1H-azepin e-
1-a cetic Acid , Meth yl Ester (6d ). A solution of (S)-2-
(acetylthio)benzenepropionic acid9 (463 mg, 2.06 mmol) and
free amine 5d (416 mg, 1.72 mmol) in CH2Cl2 (20 mL) was
cooled in an ice bath and treated with TEA (260 µL, 189 mg,
1.86 mmol) followed by (benzotriazol-1-yloxy)tris(dimethylami-
no)phosphonium hexafluorophosphate (BOP reagent; 770 mg,
1.74 mmol). The mixture was stirred at 0 °C for 1 h and then
at room temperature for 2 h. The solvent was removed by
rotary evaporation, and the residue was dissolved in EtOAc
and washed successively with 0.5 N HCl, H2O, 50% saturated
NaHCO3, and brine. After drying (Na2SO4), the organic layer
was filtered and concentrated in vacuo. Flash chromatography
(1:3 EtOAc:hexanes as eluant) afforded diastereomerically
pure 6d (743 mg, 96%) as an oil: TLC Rf 0.62 (1: EtOAc:
hexanes); 1H NMR (CDCl3) δ 0.78 (d, J ) 6.8 Hz, 3H), 0.96 (d,
J ) 6.0 Hz, 3H), 1.11-1.44 (m, 2H), 1.70-2.23 (m, 6H), 2.32
(s, 3H), 3.00 (dd, J ) 7.7, 1.41 Hz, 1H), 3.29 (m, 2H), 3.56 (d,
J ) 14.9 Hz, 1H), 3.70 (s, 3H), 4.30 (pseudo-t, 1H), 4.52 (dd, J
) 6.2, 10.9 Hz, 1H), 4.91 (d, J ) 10.7 Hz, 1H), 7.15-7.31 (m,
5H), 7.43 (d, J ) 5.6 Hz, 1H); 13C NMR (CDCl3) δ 194.55,
173.03, 171.25, 169.07, 137.60, 129.20, 128.39, 126.82, 61.56,
52.73, 51.90, 48.16, 43.49, 36.82, 31.22, 30.39, 27.41, 27.37,
19.47, 18.64.
TLC Rf 0.29 (1:1 EtOAc:hexanes); H NMR (CDCl3) δ 0.99 (t,
J ) 7.2 Hz), 1.38 (m, 2H), 1.81-2.17 (m, 8H), 2.74 (m, 1H),
3.43 (m, 1H), 3.71 (s, 3H), 3.73 (d, J ) 17.2 Hz, 1H), 4.66 (d,
J ) 17.2 Hz, 1H), 5.06 (dd, J ) 1.9, 12.6 Hz, 1H), 7.70 (m,
2H), 7.82 (m, 2H); IR (CHCl3 film) 1751, 1715, 1649, 1387,
1208, 719 cm-1
.
(3S)-tr a n s-3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-
h exa h yd r o-7-(2-h yd r oxyet h yl)-2-oxo-1H -a zep in e-1-a ce-
tic Acid , Meth yl Ester (7j). An ice-chilled solution of
aldehyde 16 (309 mg, 0.90 mmol) in MeOH (5 mL) was treated
portionwise with NaBH4 (68 mg, 1.80 mmol). After 1.5 h, the
reaction was quenched with H2O; the mixture was warmed to
room temperature and then partitioned between EtOAc and
1 N HCl. The aqueous layer was extracted twice with
additional EtOAc, and the pooled organic extracts were washed
with saturated NaHCO3 and brine and then dried (MgSO4),
filtered, and concentrated. Purification by flash chromatog-
raphy (2:1 EtOAc:hexanes followed by EtOAc as eluant)
provided azepinone 7j (292 mg, 94%) as a clear oil: TLC Rf
1
0.30 (EtOAc); H NMR (CDCl3) δ 1.43 (s, 9H), 1.84-2.08 (m,
8H), 3.74 (m, 6H), 3.94 (d, J ) 17.1 Hz, 1H), 4.32 (m, 1H),
4.51 (d, J ) 17.1 Hz, 1H), 5.91 (s, 1H); 13C NMR (CDCl3) δ
173.21, 170.28, 155.10, 79.85, 59.12, 57.59, 53.96, 52.67, 52.12,
34.21, 31.93, 30.87, 28.39, 21.68.
(3S)-tr a n s-7-(Cyclopr opylm eth yl)-3-(2,3-dih ydr o-1,3-di-
oxo-1H-isoin d ol-2-yl)h exa h yd r o-2-oxo-1H-a zep in e-1-a ce-
tic Acid , Meth yl Ester (7k ). Following the procedure of
Suda,10 a solution of alkene 7i (700 mg, 1.89 mmol) in CH2Cl2
(5 mL) at 0 °C was added to a solution of diazomethane in
Et2O followed by Pd(OAc)2 (7 mg, 0.03 mmol). The reaction
mixture bubbled vigorously and gradually turned clear. After
10 min, an additional 25 mL of ethereal diazomethane was
added and the mixture was stirred for an additional 50 min
at 0 °C. The solution was filtered through Celite and concen-
trated by rotary evaporation. Flash chromatographic purifica-
tion of the resulting oil (1:2 EtOAc:hexanes as eluant) provided
azepinone 7k (717 mg, 99%) as a white foam: TLC Rf 0.30
1
(1:1 hexanes:acetone); H NMR (CDCl3) δ 0.16 (m, 2H), 0.52
(m, 2H), 0.70 (m, 1H), 1.25-2.18 (m, 6H), 2.72 (m, 1H), 3.62
(m, 1H), 3.72 (s, 3H), 3.78 (d, J ) 17.6 Hz, 1H), 4.74 (d, J )
17.6 Hz, 1H), 5.01 (d, J ) 13.2 Hz, 1H), 7.68 (m, 2H), 7.83 (m,
2H); 13C NMR (CDCl3) δ 170.65, 170.71, 160.85, 133.85, 131.91,
123.33, 61.79, 55.09, 52.67, 52.09, 36.51, 31.13, 28.77, 22.69,
8.78, 4.55, 4.46.
Gen er a l P r oced u r e for th e Sa p on ifica tion of 6 to
In h ibitor s 2 or 3. [rS-(rR*,3R*)]-Hexa h yd r o-3-[(2-m er -
ca p to-1-oxo-3-p h en ylp r op yl)a m in o]-r-(1-m eth yleth yl)-2-
oxo-1H-a zep in e-1-a cetic Acid (2d ). A solution of 6d (658
mg, 1.47 mmol) in CH3OH (10 mL) was purged with argon for
30 min and then cooled to 0 °C and treated with 1 N NaOH
(5.9 mL, previously purged with argon for 30 min). Bubbling
of argon through the solution was maintained throughout the
addition and length of the reaction. After stirring at 0 °C for
3 h and at room temperature for 3 h, the mixture was acidified
with 5% KHSO4 and extracted with EtOAc. The EtOAc
extract was washed with H2O and brine and then dried (Na2-
SO4), filtered, and concentrated to give a solid. Trituration of
the solid with 1:3 EtOAc:Et2O (30 mL) gave pure 2d (451 mg,
78%): TLC Rf 0.72 (5:95 HOAc:EtOAc); mp 217-219 °C; [R]D
(3S)-tr a n s-7-Cyclop en tylh exa h yd r o-2-oxo-3-p h th a lim -
id o)-1H-a zep in e-1-a cetic Acid , Meth yl Ester (7m ).
A
mixture of compound 14 (2:1 mixture, 700 mg, 1.89 mmol) and
Pd on carbon (10% by wt, 150 mg) in MeOH (5 mL) was
hydrogenated (balloon) at room temperature for 5 h. The
reaction mixture was filtered through Celite and concentrated
to give diastereomerically pure 7m (594 mg, 97%) as a white
1
foam: TLC Rf 0.30 (1:1 hexanes:acetone); H NMR (CDCl3) δ
1.21 (m, 1H), 1.25 (m, 1H), 1.57 (s, 3H), 1.68 (m, 3H), 1.70-
2.19 (m, 5H), 2.73 (m, 2H), 3.14 (dt, J ) 11.1 Hz, 1H), 3.55 (d,
J ) 17.1 Hz, 1H), 3.72 (s, 3H), 4.89 (d, J ) 17.1 Hz, 1H), 5.15
(d, J ) 12.6 Hz, 1H), 7.69 (m, 2H), 7.82 (m, 2H); IR (CH2Cl2
film) 2924, 2868, 1751, 1715, 1653, 1468, 1387 cm-1
.
[3S-(1R*,3R,7â)]-Hexa h yd r o-r-eth yl-2-oxo-3-p h th a lim -
id o-7-p r op yl-1H-a zep in e-1-a cetic Acid , Meth yl Ester (7o).
A mixture of compound 12b (580 mg, 1.46 mmol) and Pd on
carbon (10%, 60 mg) in EtOAc (10 mL) was hydrogenated
(balloon) at room temperature for 3.5 h. The reaction mixture
was filtered through Celite and concentrated to give pure 7o
(558 mg, 95%) as an oil: TLC Rf 0.52 (1:9 EtOAc:CH2Cl2); 1H
NMR (CDCl3) δ 0.95 (t, J ) 7.2 Hz, 3H), 1.01 (t, J ) 7.0 Hz,
3H), 1.31 (m, 1H), 1.43 (m, 1H), 1.70 (m, 2H), 1.80-2.11 (m,
6H), 2.19 (m, 1H), 2.67 (m, 1H), 3.42 (m, 1H), 3.70 (s, 3H),
4.90 (dd, J ) 3.8, 5.1 Hz, 1H), 5.07 (d, J ) 8.8 Hz, 1H), 7.69
(m, 2H), 7.82 (m, 2H); 13C NMR (CDCl3) δ 171.99, 170.64,
168.56, 134.08, 132.40, 123.54, 62.45, 57.41, 55.50, 52.13,
34.26, 29.55, 29.27, 22.72, 22.66, 20.78, 14.21, 11.45.
1
) -90.4° (c 0.5, CH3OH); H NMR (CDCl3/CD3OD) δ 0.79 (d,
J ) 6.8 Hz, 3H), 1.01 (d, J ) 6.4 Hz, 3H), 1.45 (m, 2H), 1.71-
2.26 (m, 5H), 3.08 (dd, J ) 7.3, 13.7 Hz, 1H), 3.24 (dd, J )
6.8, 13.7 Hz, 1H), 3.37 (m, 1H), 3.61 (m, 2H), 4.56 (dd, J )
6.4, 9.4 Hz, 1H), 4.80 (d, J ) 10.7 Hz, 1H), 7.15-7.40 (m, 5H),
7.80 (d, J ) 6.0 Hz, 1H); 13C NMR (CDCl3/CD3OD) δ 173.60,
172.63, 171.83, 137.79, 129.47, 128.61, 127.12, 62.41, 52.98,
44.60, 44.12, 41.71, 31.34, 27.57, 27.40, 27.22, 19.67, 18.78.
Anal. Calcd for C20H28N2O4S: C, 61.20; H, 7.19; N, 7.14; S,
8.17. Found: C, 61.26; H, 7.27; N, 7.14; S, 8.03.
(3S)-tr a n s-H exa h yd r o-3-p h t h a lim id o-2-oxo-7-p r op yl-
1H-a zep in e-1-a cetic Acid , Meth yl Ester (7g). A solution
of 7i (767 mg, 2.07 mmol) in methanol (10 mL) and EtOAc (10
mL) was hydrogenated (balloon) over palladium on carbon
(10%, 62 mg) at room temperature for 1 h. The solution was
filtered through Celite, and the solvent was removed by rotary
evaporation to give pure 7g (771 mg, 100%) as a white foam:
[S-(R*,R*)]-r-[[2-(2,3-Dih yd r o-1,3-d ioxo-1H-isoin d ol-2-
yl)-1,6-dioxoh exyl]am in o]bu tan oic Acid, Eth yl Ester (10).
A solution of L-2-aminobutyric acid, ethyl ester hydrochloride
(9; 9.70 g, 48.0 mmol) and 4-methylmorpholine (NMM; 6.44
mL, 5.92 g, 58.5 mmol) in DMF (100 mL) was treated with

500 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Robl et al.
acid 88a (9.48 g, 34.1 mmol) and 1-hydroxybenzotriazole
hydrate (HOBT‚xH2O; 4.74 g, 35.1 mmol). The mixture was
cooled in an ice bath and subsequently treated with 1-ethyl-
3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDAC;
7.84 g, 40.9 mmol). After 1 h at 0 °C and 2 h at room
temperature, the mixture was partitioned between H2O and
EtOAc. The EtOAc extract was washed successively with H2O,
0.5 N HCl, H2O, 50% saturated aqueous NaHCO3, and brine
and then dried (Na2SO4), filtered, and concentrated to give the
intermediate dipeptide alcohol (12.50 g, 94%) as a white foam
which was used directly in the next reaction: TLC Rf 0.10 (1:1
134.61, 134.57, 133.62, 124.09, 119.04, 58.06, 57.43, 55.80,
37.47, 29.86, 29.56, 23.02, 15.62.
A solution of 12a in MeOH (15 mL) and CH2Cl2 (5 mL) was
treated with excess ethereal diazomethane in Et2O for 10 min
at 0 °C. The excess diazomethane was destroyed by the
addition of HOAc, and the solvent was removed by rotary
evaporation. Flash chromatography (5:95 EtOAc:CH2Cl2 as
eluant) provided pure methyl ester 12b (179 mg, 30% fron 11)
1
as an oil: TLC Rf 0.52 (1:9 EtOAc:CH2Cl2); H NMR (CDCl3)
δ 0.95 (t, J ) 7.4 Hz, 3H), 1.69-2.05 (m, 7H), 2.56 (m, 1H),
2.68 (m, 1H), 2.93 (m, 1H), 3.51 (m, 1H), 3.70 (s, 3H), 4.86 (m,
1H), 5.06 (d, J ) 10.5 Hz, 1H), 5.13 (d, J ) 10.0 Hz, 1H), 5.22
(d, J ) 17.0 Hz, 1H), 5.75 (m, 1H), 7.70 (m, 2H), 7.82 (m, 2H);
13C NMR (CDCl3) δ 171.58, 170.31, 168.21, 133.96, 133.82,
132.05, 123.27, 118.07, 62.38, 57.19, 55.22, 51.88, 36.41, 29.29,
28.86, 22.40, 22.33, 11.15; IR (KBr) 1738, 1715, 1649, 1387,
719 cm-1; HRMS (FAB) calcd for C22H27N2O5 (M + H)+
399.1905, found 399.1920.
1
EtOAc:hexane); H NMR (CDCl3) δ 0.88 (t, J ) 7.4 Hz, 3H),
1.23 (t, J ) 7.0 Hz, 3H), 1.40 (m, 2H), 1.55-1.80 (m, 3H), 1.91
(m, 1H), 2.24 (m, 1H), 2.46 (m, 1H), 3.62 (m, 2H), 4.18 (m,
2H), 4.56 (m, 1H), 4.87 (dd, J ) 5.5, 10.8 Hz, 1H), 6.82 (d, J )
6.4 Hz, 1H), 7.76 (m, 2H), 7.82 (m, 2H); IR (CH2Cl2 film) 1717,
1670, 1539, 1385, 721 cm-1; HRMS (FAB) calcd for C20H27N2O6
(M + H)+ 391.1869, found 391.1860.
A -78 °C solution of oxalyl chloride (4.70 mL, 6.84 g, 54.9
mmol) in CH2Cl2 (114 mL) was treated dropwise with a
solution of dry DMSO (5.75 mL, 6.33 g, 81.0 mmol) in CH2Cl2
(6 mL). After 10 min, a solution of the above alcohol (12.0 g,
30.7 mmol) in CH2Cl2 (50 mL) was added dropwise to the above
mixture. Fifteen minutes after the addition, TEA (25 mL) was
added and the mixture was stirred at -78 °C for 10 min and
then warmed to 0 °C. The mixture was partitioned between
EtOAc/Et2O and H2O. The organic layer was washed succes-
sively with 1 N HCl and brine and then dried (Na2SO4), filtered
and concentrated. Flash chromatograph (1:1 EtOAc:hexanes
as eluant) afforded aldehyde 10 (9.00 g, 75% from 8) as a white
solid: [R]D + 17.0° (c 0.3, CH2Cl2); mp 125-127 °C; TLC Rf
(3S)-tr a n s-7-(2-Cyclop en t en -1-yl)h exa h yd r o-2-oxo-3-
p h th a lim id o-1H-a zep in e-1-Acetic Acid (14). A solution of
bicyclic lactam 138a (800 mg, 2.55 mmol) and 3-(trimethylsi-
lyl)cyclopentene (2.85 g, 20.4 mmol) in CH2Cl2 (60 mL) was
treated dropwise with SnCl4 (1.0 M in CH2Cl2, 5.1 mmol). After
stirring at room temperature for 18 h, the reaction was
quenched by the addition of H2O and the mixture subsequently
extracted three times with EtOAc. The combined organic
extracts was washed with brine, dried (Na2SO4), filtered, and
concentrated to give an oil. Flash chromatography (1:1 EtOAc:
hexanes followed by 2:98 HOAc:EtOAc as eluant) provided the
slightly impure carboxylic acid (788 mg, 81%) as a foam: TLC
Rf 0.55 (5.95 HOAc:EtOAc).
1
0.34 (1: EtOAc:hexanes); H NMR (CDCl3) δ 0.89 (t, J ) 7.4
The acid (768 mg, 2.01 mmol) was dissolved in MeOH (7
mL) and Et2O (10 mL) and treated with an excess of ethereal
diazomethane for 10 min. The excess CH2N2 was destroyed
by the addition of HOAc, and the solvent was removed by
rotary evaporation. Flash chromatography (1:2 EtOAc:hex-
anes as eluant) provided compound 14 (640 mg, 65% from 13)
as a white foam (2:1 mixture of diastereomers at the asterisk
as determined by NMR): TLC Rf 0.60 (2% HOAc in EtOAc);
1H NMR (CDCl3) δ 1.61 (m, 1H), 1.90-2.20 (m, 5H), 2.43 (m,
1H), 2.82 (m, 1H), 3.20 (m, 2H), 3.46 (d, J ) 17.6 Hz, 1H),
3.59 (d, J ) 17.6 Hz, 1H), 3.72 (s, 3H), 4.88 (dd, J ) 17, 16.9
Hz, 1H), 5.15 (t, J ) 10.6 Hz, 1H), 5.7 (m, 1H), 5.92 (m, 1H),
7.69 (m, 2H), 7.84 (m, 2H);
Hz, 3H), 1.25 (t, J ) 7.2 Hz, 3H), 1.57-1.77 (m, 3H), 1.90 (m,
1H), 2.24 (m, 1H), 2.32 (m, 1H), 2.52 (m, 2H), 4.15 (m, 2H),
4.54 (m, 1H), 4.83 (dd, J ) 5.6, 10.8 Hz, 1H), 6.73 (d, J ) 6.8
Hz, 1H), 7.75 (m, 2H), 7.87 (m, 2H), 9.76 (s, 1H); 13C NMR
(CDCl3) δ 201.43, 172.03, 168.17, 168.00, 134.45, 131.51,
123.71, 61.45, 54.22, 53.53, 42.93, 28.22, 25.42, 18.77, 14.08,
9.33; IR (KBr) 3289, 1734, 1659, 1549, 1385, 723 cm-1; HRMS
(FAB) calcd for C20H25N2O6 (M + H)+ 389.1713, found 389.1705.
Anal. Calcd for C20H24N2O6: C, 61.85; H, 6.23; N, 7.21.
Found: C, 61.70; H, 6.13; N, 7.16.
[3S-(3R,6â,9a R)]-Tetr a h yd r o-3-eth yl-2,5-d ioxo-6-p h th a l-
im id ooxa zolo[3,2-a ]a zep in e-2,5(3H,6H)-d ion e (11). A so-
lution of aldehyde 10 (8.70 g, 22.4 mmol) and TFA (66 mL) in
CHCl3 (700 mL) was refluxed under argon for 6 days. The
solvent was removed by rotary evaporation, and the residue
was azeotroped twice with CH2Cl2. Flash chromatography (5:
95 acetone:CH2Cl2 as eluant) afforded bicyclic lactam 11 (5.61
g, 73%) as a white solid in 94% diastereomeric purity as
determined by NMR. Diastereomerically pure material was
obtained by recrystallization from EtOAc/CH2Cl2: [R]D +103.4°
(c 0.59, CH2Cl2); mp 185-190 °C dec; TLC Rf 0.51 (5:95
13C NMR (CDCl3) δ 168.35, 133.88,
133.79, 133.21, 132.09, 130.91, 123.36, 65.94, 54.89, 53.62,
53.24, 52.15, 47.46, 31.76, 29.43, 28.51, 28.36, 27.59, 22.35;
IR (CH2
Cl2 film) 2922, 1777, 1751, 1715, 1653, 1468, 1385
cm-1
.
(3S)-tr a n s-3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-
h exah ydr o-2-oxo-7-(2-pr open yl)-1H-azepin e-1-acetic Acid,
Meth yl Ester (15). A solution of azepinone 7i (609 mg, 2.45
mmol) in methanol (5 mL) at room temperature was treated
with hydrazine monohydrate (133 µL, 137 mg, 2.74 mmol).
After stirring at room temperature for 64 h, the reaction
mixture was filtered to remove the precipitated byproduct, and
the solid was washed well with methanol. The filtrate was
concentrated, and the residue was triturated with CH2Cl2 to
precipitate out additional byproduct. Filtration followed by
removal of the solvent provided the crude amine as a pale
yellow oil. The oil was redissolved in CH2Cl2 (10 mL) and
treated sequentially with TEA (510 µL, 370 mg, 3.65 mmol)
and di-tert-butyl dicarbonate (Boc2O; 642 mg, 2.94 mmol). After
16 h at room temperature, the mixture was charged with
additional TEA (170 µL) and Boc2O (160 mg), and the reaction
was continued for an additional 16 h. The mixture was diluted
with CH2Cl2, subsequently washed with H2O, and then dried
(MgSO4), filtered, and concentrated in vacuo. Flash chroma-
tography (1:6 EtOAc:hexane followed by 1:4 EtOAc:hexanes
as eluant) gave compound 15 (651 mg, 78%) as a clear oil: TLC
1
acetone:CH2Cl2); H NMR (CDCl3) δ 0.86 (t, J ) 7.5 Hz, 3H),
1.67-2.48 (m, 8H), 2.78 (m, 1H), 4.55 (dd, J ) 2.1, 6.0 Hz,
1H), 4.77 (dd, J ) 1.7, 12.4 Hz, 1H), 5.81 (d, J ) 9.8 Hz, 1H),
7.73 (m, 2H), 7.86 (m, 2H); 13C NMR (CDCl3) δ 170.79, 167.96,
166.25, 134.23, 123.61, 90.43, 57.74, 55.15, 35.73, 28.62, 24.59,
22.32, 7.91; IR (KBr) 1800, 1719, 1659, 1391, 1364, 1211, 1057,
721 cm-1
.
[3S-(1R*,3R,7â)]-Hexa h yd r o-r-eth yl-2-oxo-3-p h th a lim -
id o-7-(2-p r op en yl)-1H-a zep in e-1-a cetic Acid , Meth yl Es-
ter (12b). Neat TiCl4 (520 µL, 897 mg, 4.73 mmol) was added
to a mixture of 11 (515 mg, 1.50 mmol) and allyltrimethylsi-
lane (2.0 mL, 12.2 mmol) in CH2Cl2 (25 mL) at 0 °C. After 28
h, the reaction was quenched with H2O and the solution
extracted with EtOAc. The EtOAc extract was washed with
brine, dried (Na2SO4), filtered, and stripped. Flash chroma-
tography (EtOAc followed by 1:99 HOAc:EtOAc as eluant) of
the residue provided the impure allyl-substituted azepinone
12a (R ) H; 319 mg) as an oil: TLC Rf 0.66 (2:98 HOAc:
EtOAc); 1H NMR (CDCl3) δ 0.97 (t, J ) 7.6 Hz, 3H), 1.75-
2.12 (m, 7H), 2.54-2.76 (m, 2H), 2.89 (m, 1H), 3.49 (m, 1H),
4.75 (m, 1H), 5.05 (d, J ) 10.5 Hz, 1H), 5.14 (d, J ) 10.0 Hz,
1H), 5.23 (d, J ) 16.8 Hz, 1H), 5.73 (m, 1H), 7.71 (m, 2H),
7.84 (m, 2H); 13C NMR (CDCl3) δ 176.89, 170.98, 168.94,
1
Rf 0.70 (1:9 MeOH:CH2Cl2); H NMR (CDCl3) δ 1.44 (s, 9H),
1.85-2.08 (m, 6H), 2.60 (m, 2H), 3.41 (m, 1H), 3.68 (d, J ) 17
Hz, 1H), 3.74 (s, 3H), 4.37 (m, 1H), 4.60 (d, J ) 17 Hz, 1H),
5.10 (d, J ) 11 Hz, 1H), 5.18 (d, J ) 1.8 Hz, 1H), 5.70 (m, 1H),
5.89 (d, J ) 6.4 Hz, 1H); 13C NMR (CDCl3) δ 173.56, 170.01,
155.10, 79.52, 61.56, 53.88, 52.55, 52.09, 31.76, 30.23, 28.39,
21.16.

Dual Metalloprotease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 501
(3S)-tr a n s-3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-
h exah ydr o-2-oxo-7-(2-oxoeth yl)-1H-azepin e-1-acetic Acid,
Meth yl Ester (16). A solution of osmium tetraoxide (2.5%
by wt in t-BuOH, 400 µL, 0.98 mmol) was added to a solution
of compound 15 (554 mg, 1.63 mL) and NaIO4 (731 mg, 3.42
mmol) in a 1:1 mixture of H2O and dioxane (6 mL). After
stirring at room temperature for 16 h, the mixture was filtered
and concentrated in vacuo. The residue was directly flash
chromatographed (1:4 EtOAc:hexanes as eluant) to give alde-
hyde 16 (309 mg, 55%) as a clear oil: TLC Rf 0.10 (1:3 EtOAc:
hexanes); 1H NMR (CDCl3) δ 1.44 (s, 9H), 1.51-2.08 (m, 6H),
3.04 (dd, J ) 2.9, 6.4 Hz, 1H), 3.28 (dd, J ) 2.9, 6.4 Hz, 1H),
3.34 (m, 1H), 3.73 (s, 3H), 4.11-4.26 (m, 1H), 4.29 (d, J ) 2.94
Hz, 2H), 5.91 (s, 1H), 9.77 (s, 1H)); 13C NMR (CDCl3) δ 198.70,
173.80, 170.04, 91.15, 79.95, 69.99, 65.68, 62.55, 54.40, 54.08,
52.67, 52.15, 46.85, 31.88, 31.44, 28.39, 22.08.
P u r ifica tion of Ra t Kid n ey Neu tr a l En d op ep tid a se.
Neutral endopeptidase was solubilized and purified from rat
kidney membrane fractions using, with slight modification, the
procedure described by Almenoff and Orlowski.15 The enzyme
was solubilized from rat kidney membrane fractions with a
1% sodium deoxycholate solution in 50 mM Tris-HCl buffer
(pH 7.6) and released from the membrane fragments by
incubation at 37 °C for 90 min with papain (40 µg/mg of
protein) and 5 mM dithiothreitol. Papain was completely
separated from kidney metallopeptidases by gel filtration using
Sephacryl S-200. Aminopeptidase activity, which constituted
a major portion of kidney metallopeptidase activity, was
effectively removed by hydrophobic chromatography using
phenyl sepharose. Fractions containing endopeptidase activity
were pooled, concentrated by ultrafiltration, dialyzed against
50 mM Tris-HCl buffer (pH 7.6), and stored at 4 °C.
In Vitr o In h ib it ion of P u r ified R a t Kid n ey Neu t r a l
En d op ep tid a se. This assay is based on the cleavage of the
fluorescent dansylated tripeptide dansyl-Gly-Phe-Arg to form
the strongly fluorescent dansyl-Gly which is extracted from
the acidified mixture by ethyl acetate. Incubation mixtures
employed for the assay contained (in mM) the substrate
dansyl-Gly-Phe-Arg, 0.5; Tris-HCl buffer (pH 7.5), 50; enzyme
preparation in 0.05% Triton X-100; and varied concentrations
of inhibitor solution or water to a final volume of 0.25 mL.
Inhibitor solutions were preincubated for 10 min with the
enzyme peparation and buffer before the reaction was initiated
by the addition of the substrate. Incubation was carried out
at 37 °C for 30 min, and the reaction was stopped by the
addition of 0.25 mL of 1 N HCl and by extraction of the dansyl-
Gly product into 1.5 mL of ethyl acetate. Relative fluorescent
intensity (RFI) of the ethyl acetate extract was measured using
a Perkin-Elmer LS-5B luminescence spectrometer at the
excitation and emission wavelengths of 342 and 510 nm,
respectively, corrected for the RFI of a zero-time control and
compared with assays lacking inhibitor.
In Vitr o In h ib it ion of R a b b it Lu n g An giot en sin -
Con ver tin g En zym e. Compounds were evaluated for their
ACE inhibitory potencies using the spectrophotometric assay
of Cushman and Cheung.16 In the assay, rabbit lung acetone
powder extract was used as the source of ACE, and the rate
of hippuric acid formation from the substrate hippuryl-L-
histidyl-L-leucine (HHL) was monitored. Each 250 µL assay
mixture contained the following at the indicated final concen-
tration (in mM): phosphate buffer (pH 8.3), 100; NaCl, 300;
HHL, 5; 100 µL of rabbit lung acetone powder extract (1:20
dilution); and 50 µL of varied concentrations of compound
solution or vehicle. Following 30 min of incubation at 37 °C,
the reaction was terminated by the addition of 250 µL of 1 N
HCl followed by 1.5 mL of ethyl acetate. After mixing and
centrifuging, a 1.0 mL aliquot of the ethyl acetate layer was
removed and evaporated to dryness. Hippuric acid was
reconstituted in 1 mL of water, and its concentration was
determined spectrophotometrically at 228 nm.
Approximately 2 weeks later, aortic blood pressure was
monitored directly in the conscious rats. Four rats were used
for each assay. Pressor responses to intravenous injections
of AI (310 ng/kg) were obtained before (control) and at 2-30
min intervals up to 6 h after intravenous or oral administra-
tion of test compounds. Compounds were administered at
various doses in 5% NaHCO3 (intravenous) or 0.25% agar
(oral). Inhibition (percent change from control) of the AI
pressor response was determined at each interval. A dose-
response curve was constructed using the time point after each
dose showing maximum inhibition. The median effective dose
(ED50), which represents the dose required to effect 50%
inhibition of the AI-induced pressor response, was determined
by interpolation.
1-Kid n ey DOCA Sa lt Ra t Assa y. This low-renin model
of hypertension was used to evaluate the ability of the
compounds to lower MAP in vivo. The procedure described is
a modification of that reported by Sybertz et al.18 Male
Sprague-Dawley rats (150 g) wre anesthetized with sodium
pentobarbital (50 mg/kg ip), and the left kidney was ligated
with nonabsorbable suture and removed. An indwelling
catheter was implanted a short distance into the abdominal
aorta terminating below the origin of the renal ateries. The
abdominal muscle was then closed, a 100 mg pellet of deoxy-
corticosterone acetate (DOCA) was placed subcutaneously, and
the abdominal skin was closed using stainless steel clips. The
rats were individually housed and placed on 0.9% NaCl/0.2%
KCl drinking water and regular rat chow diet. After a
minimum of 2-3 weeks, a sustained hypertension of 165-175
mmHg was established. Each rat was placed in a harness
which was secured to one end of a tightly wound spring. The
other end of the spring was passed through the top of the cage
containing the rat and attached to the lower end of a feed-
through miniature swivel (King Chatham). The steel spring
served as a protective cover for the catheter within it that
connected the aterial cannula to the swivel connector. The
upper portion of the swivel was provided with two connec-
tions: one to a Cobe transducer through a rotary fluid switch
and the other to a pressurized (13 lb/in.2) reservoir that
provided a slow flow (0.03-0.06 mL/min) of saline into the
aterial cannula to maintain patency.
The animals (N ) 6-10) were dosed sid intravenously with
the appropriate compound (100 µmol/kg) in 1 mL/kg 2.5%
NaHCO3 or with vehicle (1 mL/kg). Blood pressure was
measured every 5 min over a 10 s period. Six such sets of
data were averaged to give a mean value representing a 30
min sample. Measurements were continued over a 24 h
period. The data were plotted as blood pressure (mmHg)
versus time or, as in the case of Chart 1, are represented as
the cumulative AOC over the 24 h period of study.
Ack n ow led gm en t. We are grateful for the technical
assistance provided by Maxine Fox, Mary Giancarli,
Balkrushna Panchal, Yolanda Pan, and Hong Sun
Cheung. In addition we thank Edward Petrillo for his
support of this work.
Refer en ces
(1) (a) Seymour, A. A.; Swerdel, J . N.; Abboa-Offei, B. Antihyper-
tensive activity during inhibition of neutral endopeptidase and
angiotensin converting enzyme. J . Cardiovasc. Pharmacol.
1991, 17, 456-465. (b) Krulan, C.; Ghai, R. D.; Lappe, R. W.;
Webb, R. L. Blood pressure effects during chronic inhibition of
angiotensin converting enzyme (ACE) and neutral endopeptidase
(NEP) in the telemetered spontaneously hypertensive rat (SHR).
FABEB J . 1993, 7, A247. (c) Pham, I.; Gonzalez, W.; El Amrani,
A.-I. K.; Fournie-Zaluski, M.-C.; Philippe, M.; Laboulandine, I.;
Roques, B. P.; Michel, J .-B. Effects of converting enzyme
inhibitor and neutral endopeptidase inhibitor on blood pressure
and renal function in experimental hypertension. J . Pharmacol.
Exp. Ther. 1993, 265, 1339-1347. (d) Trippodo, N. C.; Fox, M.;
Natarajan, V.; Panchal, B. C.; Dorso, C. R.; Asaad, M. M.
Combined inhibition of neutral endopeptidase and angiotensin
converting enzyme in cardiomyopathic hamsters with compen-
sated heart failure. J . Pharmacol. Exp. Ther. 1993, 267, 108-
116. (e) Seymour, A. A.; Asaad, M. M.; Lanoce, V. M.; Langen-
bacher, K. M.; Fennell, S. A.; Rogers, W. L. Systemic
In Vivo Biologica l Eva lu a tion : AI P r essor Resp on se
Assa y. The procedure described is a modification of that
reported by Rubin et al.17 Sprague-Dawley male rats (220-
240 g) were anesthetized and implanted with abdominal aortic
and vena caval catheters. The rats were individually housed
and placed on regular rat chow diet and water ad libitum.

502 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Robl et al.
M.; Bird, J . E.; Trippodo, N. C.; Petrillo, E. W.; Karanewsky, D.
S. Dual metalloprotease inhibitors. IV. Utilization of thiaz-
epines and thiazines as constrained peptidomimetic surrogates
in mercaptoacyl dipeptides. BioMed. Chem. Lett. 1994, 4, 2193-
2198.
hemodynamics, renal function and hormonal levels during
inhibition of neutral endopeptidase 3.4.24.11 and angiotensin-
converting enzyme in conscious dogs with pacing-induced heart
failure. J . Pharmacol. Exp. Ther. 1993, 266, 872-883.
(2) (a) Gros, C.; Noel, N.; Souque, A.; Schwartz, J .-C.; Danvy, D.;
Plaquevent, J .-C.; Duhamel, L.; Duhamel, P.; Lecomte, J .-M.;
Bralet, J . Mixed inhibitors of angiotensin-converting enzyme (EC
3.4.15.1) and enkephalinase (EC 3.4.24.11): Rational design,
properties, and potential cardiovascular applications of glycopril
and alatriopril. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4210-
4214. (b) Fournie-Zaluski, M. C. Inhibition conjointe de l’enzyme
de conversion et de l’endopeptidase neutre: Une nouvelle voie
de recherche dans le domaine de l’hypertension. (Dual inhibition
of angiotensin-converting enzyme and neutral endopeptidase:
a new research pathway in the field of hypertension.) C. R.
Seances Soc. Biol. Ses Fil. 1992, 186, 612-625. (c) Roques, B.
P. Zinc metallopeptidases: Active site structure and design of
selective and mixed inhibitors: New approaches in the search
for analgesics and anti-hypertensives. Biochem. Soc. Trans.
1993, 21, 678-685. (d) Flynn, G. A.; Beight, D. W.; Mehdi, S.;
Koehl, J . R.; Giroux, E. L.; French, J . F.; Hake, P. W.; Dage, R.
C. Application of a conformationally restricted Phe-Leu dipeptide
mimetic to the design of a combined inhibitor of angiotensin
I-converting enzyme and neutral endopeptidase 24.11 J . Med.
Chem. 1993, 36, 2420-2423. (e) Stanton, J . L.; Sperbeck, D.
M.; Trapani, A. J .; Cote, D.; Sakane, Y.; Berry, C. J .; Ghai, R.
D. Heterocyclic lactam derivatives as dual angiotensin convert-
ing enzyme and neutral endopeptidase 24.11 inhibitors. J . Med.
Chem. 1993, 36, 3829-3833. (f) French, J . F.; Flynn, G. A.;
Giroux, E. L.; Mehdi, S.; Anderson, B.; Beach, D. C.; Koehl, J .
R.; Dage, R. C. Characterization of a dual inhibitor of angiotensin
I-converting enzyme and neutral endopeptidase. J . Pharmacol.
Exp. Ther. 1993, 268, 180-186. (g) Fournie-Zaluski, M.-C.;
Coric, P.; Turcaud, S.; Rousselet, N.; Gonzalez, W.; Barbe, B.;
Pham, I.; J ullian, N.; Michel, J .-B.; Roques, B. P. New dual
inhibitors of neutral endopeptidase and angiotensin-converting
enzyme: Rational design, bioavailability, and pharmacological
responses in experimental hypertension. J . Med. Chem. 1994,
37, 1070-1083. (h) Gomez-Monterrey, I.; Beaumont, A.; Nem-
ecek, P.; Roques, B. P.; Fournie-Zaluski, M.-C. New thiol
inhibitors of neutral endopeptidase EC 3.4.24.11: Synthesis and
enzyme active-site recognition. J . Med. Chem. 1994, 37, 1865-
1873. (i) DeLombaert, S.; Tan, J .; Stamford, L. J .; Sakane, Y.;
Berry, C.; Ghai, R. D. Dual inhibition of neutral endopeptidase
and angiotensin-converting enzyme by N-phosphonomethyl and
N-carboxyalkyl dipeptides. Biomed. Chem. Lett. 1994, 4, 2715-
2720. (j) Bralet, J .; Marie, C.; Mossiat, C.; Lecomte, J .-M.; Gros,
C.; Schwartz, J .-C. Effect of Alatriopril, a mixed inhibitor of
atriopeptidase and angiotensin I-converting enzyme, on cardiac
hypertrophy and hormonal responses in rats with myocardial
infarction. Comparison with captopril. J . Pharmacol. Exp. Ther.
1994, 270, 8-14. (k) Bhagwat, S. S.; Fink, C. A.; Gude, C.; Chan,
K.; Qiao, Y.; Sakane, Y.; Berry, C.; Ghai, R. D. 4-Substituted
proline derivatives that inhibit angiotensin converting enzyme
and neutral endopeptidase 24.11. Biomed. Chem. Lett. 1994,
4, 2673-2676. (l) Vera, W. G.; Fournie-Zaluski, M.-C.; Pham,
I.; Laboulandine, I.; Roques, B.-P.; Michel, J .-P. Hypotensive and
natriuretic effects of RB 105, a new dual inhibitor of angiotensin
converting enzyme and neutral endopeptidase in hypertensive
rats. J . Pharmacol. Exp. Ther. 1995, 272, 343-351. (m)
Bhagwat, S. S.; Fink, C. A.; Gude, C.; Chan, K.; Qiao, Y.; Sakane,
Y.; Berry, C.; Ghai, R. D. R-Mercaptoacyl dipeptides that inhibit
angiotensin converting enzyme and neutral endopeptidase 24.11.
Biomed. Chem. Lett. 1995, 5, 735-738.
(4) In virtually all cases we have examined, the mercaptopropanoyl-
containing conformationally restricted inhibitors are signifi-
cantly less potent as ACE inhibitors in vivo (AI pressor) than
their mercaptoacetyl counterparts, despite similar in vitro
potencies versus ACE. Numerous examples have been cited in
this laboratory (see refs 3a,b,e). For this reason we have
concentrated our efforts in this work on the mercaptoacetyl class
of inhibitors.
(5) Trippodo, N. C.; Robl, J . A.; Asaad, M. M.; Bird, J . E.; Panchal,
B. C.; Schaeffer, T. R.; Fox, M.; Giancarli, M. R.; Cheung, H. S.
Cardiovascular effects of the novel dual inhibitor of neutral
endopeptidase and angiotensin converting enzyme BMS-182657
in experimental hypertension and heart failure. J . Pharmacol.
Exp. Ther. 1995, 275, 745-752.
(6) (a) Thorsett, E. D.; Harris, E. E.; Aster, S. D.; Peterson, E. R.;
Snyder, J . P.; Springer, J . P.; Hirshfield, J .; Tristram, E. W.;
Patchett, A. A.; Ulm, E. H.; Vassil, T. C. Conformationally
restricted inhibitors of angiotensin converting enzyme: Synthe-
sis and computations. J . Med. Chem. 1986, 29, 251-260. (b)
Thorsett, E. D. Conformationally restricted inhibitors of angio-
tensin converting enzyme. Actual. Chim. Ther. 1986, 13, 257-
268. (c) Yanagisawa, H.; Ishihara, S.; Ando, A.; Kanazaki, T.;
Miyamoto, S.; Koike, H.; Iijima, Y.; Oizumi, K.; Matsushita, Y.;
Hata, T. Angiotensin-converting enzyme inhibitors. 2. Perhy-
droazepin-2-one derivatives. J . Med. Chem. 1988, 31, 422-428.
(d) Thorsett, E. D.; Harris, E. E.; Aster, S. D.; Peterson, E. R.;
Tristram, E. W.; Synder, J . P.; Springer, J . P.; Patchett, A. A.
Conformationally restricted inhibitors of angiotensin converting
enzyme. Pept.: Struct. Funct., Proc. Am. Pept. Symp., 8th 1983,
555-558.
(7) Robl, J . R. Peptidomimetic synthesis: Utilization of N-acylimin-
ium ion cyclization chemistry in the generation of 7,6- and 7,5-
fused bicyclic lactams. Tetrahedron Lett. 1994, 35, 393-396.
(8) (a) Robl, J . A.; Cimarusti, M. P.; Simpkins, L. M.; Weller, H. N.;
Pan, Y. Y.; Malley, M.; DiMarco, J . D. Peptidomimetic synthe-
sis: A novel, highly stereoselective route to substituted Fre-
idinger lactams. J . Am. Chem. Soc. 1994, 116, 2348-2355. (b)
Robl, J . R.; Cimarusti, M. P. A synthetic route for the generation
of C-7 substituted azepinones. Tetrahedron Lett. 1994, 35,
1393-1396.
(9) Strijtveen, B.; Kellogg, R. M. Synthesis of (racemization prone)
optically active thiols by S
N2 substitution using cesium thiocar-
boxylates. J . Org. Chem. 1986, 51, 3664-3671.
(10) Suda, M. Cyclopropanation of terminal olefins using diaz-
omethane/palladium(II) acetate. Synthesis 1981, 714.
(11) Wong, H. N. C.; Hon, M.-Y.; Tse, C.-W.; Yip, Y.-C.; Tanko, J .;
Hudlicky, T. Use of cyclopropanes and their derivitives in organic
synthesis. Chem. Rev. 1989, 89, 165-198.
(12) Slusarchyk, W. A.; Robl, J . A.; Taunk, P. C.; Asaad, M. M.; Bird,
J . E.; DiMarco, J .; Pan, Y. Dual metalloprotease inhibitors V.
Utilization of bicyclic azepinonethiazolidines and azepinonetet-
rahydrothiazines in constrained peptidomimetics of mercaptoa-
cyl dipeptides. Bioorg. Med. Chem. Lett. 1995, 5, 753-758.
(13) (a) Seymour, A. A. The pharmacology of SQ 28,603, an inhibitor
of neutral endopeptidase 3.4.24.11. Cardiovasc. Drug Rev. 1991,
9, 285-298. (b) Seymour, A. A.; Norman, J . A.; Asaad, M. M.;
Fennell, S. A.; Little, D. K.; Kratunis, V. J .; Rogers, W. L.
Antihypertensive and renal activity of SQ 28,603, an inhibitor
of neutral endopeptidase. J . Cardiovasc. Pharmacol. 1991, 17,
296-304.
(14) Northridge, D. B.; Alabaster, C. T.; Connell, J . M. C.; Dilly, S.
G.; Lever, A. F.; J ardine, A. G.; Barclay, P. L.; Dargie, H. J .;
Findlay, I. N.; Samuels, G. M. R. Effects of UK 69578: A novel
atriopeptidase inhibitor. Lancet 1989, ii, 591-593.
(15) Almenoff, J .; Orlowski, M. Membrane-bound kidney neutral
metalloendopeptidase: Interaction with synthetic substrates,
Neutral Peptides, and Inhibitors. Biochemistry 1983, 22, 590-
599.
(3) (a) Delaney, N. G.; Barrish, J . C.; Neubeck, R.; Natarajan, S. I.;
Rovnyak, G. C.; Huber, G.; Murugesan, N.; Girotra, R.; Sieber-
McMaster, E.; Robl., J . A.; Asaad, M.; Cheung, H. S.; Bird, E.;
Waldron, T.; Petrillo, E. W. Mercaptoacyl dipeptides as dual
inhibitors of angiotensin converting enzyme and neutral en-
dopeptidase. Preliminary structure-activity studies. BioMed.
Chem. Lett. 1994, 4, 1783-1788. (b) Robl, J . A.; Simpkins, L.
M.; Stevenson, J .; Sun, C. Q.; Murugesan, N.; Barrish, J . C.;
Asaad, M. M.; Bird, J . E.; Schaeffer, T. R.; Trippodo, N. R.;
Petrillo, E. W.; Karanewsky, D. S. Dual metalloprotease inhibi-
tors. I. Constrained peptidomimetics of mercaptoacyl dipep-
tides. BioMed. Chem. Lett. 1994, 4, 1789-1794. (c) Robl, J . A.;
Simpkins, L. M.; Sulsky, R.; Sieber-McMaster, E.; Stevenson,
J .; Kelly, Y. F.; Sun, C. Q.; Misra, R. N.; Ryono, D. E.; Asaad,
M. M.; Bird, J . E.; Trippodo, N. C.; Karanewsky, D. S. Dual
metalloprotease inhibitors. II. Effect of substitution and ster-
eochemistry on benzazepinone based mercaptoacetyls. BioMed.
Chem. Lett. 1994, 4, 1795-1800. (d) Robl, J . A.; Sun, C. Q.;
Simpkins, L. M.; Ryono, D. E.; Barrish, J . C.; Karanewsky, D.
S.; Asaad, M. M.; Schaeffer, T. R.; Trippodo, N. C. Dual
metalloprotease inhibitors. III. Utilization of bicyclic and
monocyclic diazepinone based mercaptoacetyls. BioMed. Chem.
Lett. 1994, 4, 2055-2060. (e) Das, J .; Robl, J . A.; Reid, J . A.;
Sun, C.-Q.; Misra, R. N.; Brown, B. R.; Ryono, D. E.; Asaad, M.
(16) Cushman, D. W.; Cheung, H. S. Spectrophotometric assay and
properties of the angiotensin-converting enzyme of rabbit lung.
Biochem. Pharmacol. 1971, 20, 1637-1648.
(17) Rubin, B.; Laffan, R. J .; Kotler, D. G.; O’Keefe, E. H.; Demaio,
D. A.; Goldberg, M. E. SQ 14,225 (D-3-Mercapto-2-methylpro-
panoyl-L-proline)), a novel orally active inhibitor of angiotensin
converting enzyme. J . Pharmacol. Exp. Ther. 1978, 204, 271.
(18) Sybertz, E. J .; Chiu, P. J . S.; Vemulapalli, S.; Pitts, B.; Foster,
C. J .; Watkins, R. W.; Barnett, A.; Haslanger, M. F. SCH 39370,
a neutral metalloendopeptidase inhibitor, potentiates biological
responses to atrial natriuretic factor and lowers blood pressure
in desoxycorticosterone acetate-sodium hypertensive rats. J .
Pharmacol. Exp. Ther. 1989, 250, 624-631.
J M950677A