Mercaptoacyl dipeptides as orally active dual inhibitors of angiotensin- converting enzyme and neutral endopeptidase

Article abstract of DOI:10.1021/jm960323z

Dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-I-converting enzyme (ACE, EC 2.4.15.1), have been the focus of much clinical interest for the treatment of hypertension and congestive heart failure. We have previously reported that compound 2 (N-[[1-[(2(S)-mercapto-3-methyl-1-oxobutyl)amino]-1- cyclopentyl]carbonyl]-L-tyrosine) was a potent dual inhibitor in vitro (IC₅₀(ACE) = 7.0 nM, IC₅₀(NEP) = 1.5 nM) (Fink et al. J. Med. Chem. 1995, 38, 5023-5030). This compound was found to have oral activity; however, its duration of effect was short. A series of thioacetate carboxylic acid ester analogs of compound 2 was prepared. Modifications were also made to the tyrosine phenol. These compounds were evaluated for their ability to inhibit plasma ACE activity when administered orally to conscious normotensive rats. Most of the compounds prepared were found to be orally active with longer durations of effect than compound 2. Compound 38 (N[[1-[(2(S)-(acetylthio)- 3-methyl-1-oxobutyl)amino]-1-cyclopentyl]carbonyl]-O-methyl-L-tyrosine ethyl ester), administered at 11.7 mg/kg po, was found to be more efficacious than captopril at 10 mg/kg po. This compound was also found to inhibit plasma NEP activity following oral administration to conscious rats and was more efficacious than acetorphan. Compound 38 was found to lower blood pressure in the aorta-ligated rat and the spontaneously hypertensive rat when administered orally. The synthesis and biological activity of these dual inhibitors are discussed.

Full text of DOI:10.1021/jm960323z

3158
J . Med. Chem. 1996, 39, 3158-3168
Mer ca p toa cyl Dip ep tid es a s Or a lly Active Du a l In h ibitor s of
An gioten sin -Con ver tin g En zym e a n d Neu tr a l En d op ep tid a se
Cynthia A. Fink,* J . Eric Carlson, Patricia A. McTaggart, Ying Qiao, Randy Webb, Ricardo Chatelain,
Arco Y. J eng, and Angelo J . Trapani
Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, New J ersey 07901
Received April 30, 1996X
Dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP, EC 3.4.24.11)
and angiotensin-I-converting enzyme (ACE, EC 2.4.15.1), have been the focus of much clinical
interest for the treatment of hypertension and congestive heart failure. We have previously
reported that compound 2 (N-[[1-[(2(S)-mercapto-3-methyl-1-oxobutyl)amino]-1-cyclopentyl]-
carbonyl]-L-tyrosine) was a potent dual inhibitor in vitro (IC50(ACE) ) 7.0 nM, IC50(NEP) )
1.5 nM) (Fink et al. J . Med. Chem. 1995, 38, 5023-5030). This compound was found to have
oral activity; however, its duration of effect was short. A series of thioacetate carboxylic acid
ester analogs of compound 2 was prepared. Modifications were also made to the tyrosine phenol.
These compounds were evaluated for their ability to inhibit plasma ACE activity when
administered orally to conscious normotensive rats. Most of the compounds prepared were
found to be orally active with longer durations of effect than compound 2. Compound 38 (N-
[[1-[(2(S)-(acetylthio)-3-methyl-1-oxobutyl)amino]-1-cyclopentyl]carbonyl]-O-methyl-L-tyro-
sine ethyl ester), administered at 11.7 mg/kg po, was found to be more efficacious than captopril
at 10 mg/kg po. This compound was also found to inhibit plasma NEP activity following oral
administration to conscious rats and was more efficacious than acetorphan. Compound 38
was found to lower blood pressure in the aorta-ligated rat and the spontaneously hypertensive
rat when administered orally. The synthesis and biological activity of these dual inhibitors
are discussed.
In tr od u ction
plasma renin activity, and the sodium status of the
patient.24 ACE monotherapy is effective in only 40-
50% of the essential hypertensive population and most
efficacious in patients with high renin activity.25,26
Furthermore, the rate of responsiveness for this class
of therapeutic agent can be increased by coadministra-
tion of a diuretic agent.27 Diuretics, however, can evoke
a number of undesired side effects such as activation of
pressor systems, hypokalemia, hyperglycemia, and el-
evation of plasma lipids.28 Selective inhibitors of NEP
have been studied in animal models as well as humans
and have been found to produce significant diuretic and
natriuretic effects without kaliuresis.29 NEP inhibitors
are effective in low-renin animal models of hypertension
in contrast to ACE inhibitors which are ineffective.
Several studies have shown that coadministration of
selective ACE and NEP inhibitors in animal models of
hypertension and CHF has a more beneficial effect over
the administration of the single agents separately.30,31
In recent reports, it has also been demonstrated that
single molecules which possess dual ACE and NEP
inhibitory activity also exhibit these synergistic prop-
erties.32-34 The antihypertensive response that is
achieved with the combination or the single-molecule
dual inhibitor is independent of the plasma renin
activity or the sodium status of the animal model
chosen. These molecules represent a new class of
cardiovascular agents that should be more effective in
a broader range of hypertensive patients and in CHF.
We initially reported our results on a series of
R-mercapto dipeptides which possessed dual ACE and
NEP inhibitory activity.16 One of the best compounds
in this series was the biphenyl analog 1. This compound
The development of a single agent which possesses
the ability to inhibit both of the membrane-bound zinc
metalloproteases, angiotensin converting enzyme (EC
2.4.15.1, ACE) and neutral endopeptidase (EC 3.4.24.11,
NEP), has been the focus of recent drug discovery.1-17
Both of these ectoenzymes are intimately involved in
regulatory systems which modulate blood pressure and
fluid volume homeostasis. ACE is a dipeptidyl carbox-
ypeptidase which is part of the renin-angiotensin-
aldosterone system. This enzyme catalyzes the conver-
sion of the biologically inactive decapeptide angiotensin-I
(AI) to the octapeptide angiotensin-II. Angiotensin-II
is a potent vasoconstrictor, which also promotes the
release of aldosterone, leading to sodium and fluid
retention.
NEP is the major enzyme involved in the metabolic
inactivation of atrial natriuretic factor (ANF).18-20 ANF
is a 28-amino acid polypeptide which is synthesized in
the atrial myocyte and secreted into the circulation in
response to changes in intracardiac pressure. Through
interaction with its receptor, ANF upregulates cGMP
production and elicits a number of biological responses
including diuresis, natriuresis, vasodilation, and reduc-
tion of plasma aldosterone levels.21,22
ACE inhibitors have gained wide acceptance clinically
and are commonly prescribed for the treatment of
hypertension and congestive heart failure (CHF).23 The
magnitude of blood pressure lowering that can be
achieved with ACE inhibitor therapy is dependent upon
several factors such as the severity of hypertension, the
was found to be a potent dual inhibitor (ACE, IC50
62 nM; NEP, IC50 ) 28 nM). Unfortunately, it was
)
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
S0022-2623(96)00323-8 CCC: $12.00 © 1996 American Chemical Society

Mercaptoacyl Dipeptides as ACE and NEP Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3159
Sch em e 1
dual inhibitor (ACE, IC50 ) 7.0 nM; NEP, IC50 ) 1.5
nM).17 It inhibited the AI pressor response in normo-
tensive conscious rats at greater than 50% for more than
6 h after intravenous administration. When adminis-
tered to conscious rats at 10 mg/kg po, 2 was found to
have oral activity with a similar duration of effect as
captopril (Figure 1). The maximum response achieved,
however, was less than that of captopril. To further
improve the efficacy of compound 2, we prepared a
number of carboxylic acid ester-thioester prodrug
derivatives and studied their resulting in vivo activity.
Modifications were also made to the tyrosine residue.
Herein we report our results on a series of mercaptoacyl
spirocyclopentyl-containing dipeptides.
F igu r e 1. Inhibition of AI pressor response in conscious rats
treated with captopril (0) and compound 2 (]) at 10 mg/kg
po.
Ch em istr y
The target R-mercapto spirocyclopentyl-containing
dipeptides were prepared as outlined in Schemes 1 and
2. Synthesis of the dipeptide was initiated by coupling
various R-amino acid esters 3 to Boc-protected cyclo-
leucine by the classical 1,3-dicyclohexylcarbodiimide
(DCC)/1-hydroxybenzotriazole (HOBt) method.35,36 De-
protection of the Boc-protected dipeptides 4a -r with
anhydrous HCl gas in methylene chloride afforded the
amine hydrochloride salts 5. Coupling of the amine
hydrochloride 5 with (S)-2-(acetylthio)-3-methylbutanoic
acid, DCC, 1-hydroxy-7-azabenzotriazole (HOAt), and
triethylamine gave the mercaptoacyl dipeptides 6.37,38
Treatment of the dipeptide 6 (when R ) H) with 1 equiv
of an acid chloride or a chloroformate with triethylamine
yielded the phenolic ester or carbonate 7, respectively.
Treatment of dipeptide 6 with 1 equiv of 1 N sodium
hydroxide in degassed methanol afforded the free thiol
6a . The thiol 6a (R * H) was then reacted with a
variety of acid chlorides and triethylamine to give new
thioesters 8. When R ) H, the free thiol 6a can also be
treated with an acid, DCC, HOAt, and triethylamine
short acting in vivo and inhibited the AI-induced pressor
response in normotensive conscious rats by 50% for only
1 h after intravenous administration. Further modifi-
cation of this compound was undertaken in order to
improve its duration of action. We replaced the amino
acids in this molecule with non-natural amino acids that
would not impair the in vitro potency of the compound
but might improve the metabolic stability and thus its
efficacy. Replacement of the central amino acid with
spiroalkyl amino acids improved the in vivo activity of
these analogs. In a recent communication, we reported
that the cycloleucine analog compound 2 was a potent

3160 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fink et al.
Sch em e 2
made to the tyrosine phenol. We prepared simple
O-alkyl ethers (compounds 34-42), esters (compounds
43-51), and a carbonate (compound 52). In comparing
the tyrosine ethyl ester analog compound 10 to the
O-alkylated tyrosine ethyl ester compounds 35, 36, and
38, it appears that simple alkylation of the phenol
groups provides an enhancement in oral activity. The
esters in general with the exception of compound 47
were not as good as the O-alkyl ethers. Carbonate 52
was less active than compound 10, the tyrosine analog.
We believe that the thioester and carboxylic acid ester
groups are metabolized in vivo to give the free thiocar-
boxylic acids and are thus behaving as prodrugs of
compound 2. For a compound to function as an inhibitor
of ACE, it must contain a functional group that chelates
to the zinc atom which resides in the active site of the
enzyme and a C-terminal carboxylic acid.39 For NEP
inhibition, a zinc-chelating group is also essential. A
C-terminal carboxylic acid is advantageous to NEP
inhibition; however, its exact placement is not as critical
as it is for ACE inhibition.40 To confirm this, several of
the thioester carboxylic acid esters were evaluated for
their in vitro activity against ACE and NEP and found
to have little (1 × 10-6 M) or no activity against either
enzyme.41 The O-alkyl ether group on the tyrosine may
also be metabolically removed, but this is not required
for activity as we have previously reported that the free
thiol carboxylic acid of compound 38 itself is a potent
dual inhibitor (IC50 ) 19 nM (ACE), IC50 ) 2.2 nM
(NEP)).17
A sampling of the different prodrugs prepared was
also investigated for the ability to potentiate exogenous
ANF. The effects of these compounds were studied in
conscious rats after oral administration (Table 3). All
of the compounds evaluated were found to be more
efficacious than acetorphan, the thioacetate analog of
thiorphan, a known NEP inhibitor. Of the compounds
tested in the in vivo ACE and NEP assays, compounds
34 and 38 performed well in both tests. Since we were
concerned with the release of methanol from compound
34 by hydrolysis of the methyl ester in vivo, compound
38, the ethyl ester analog, was selected for further
biological evaluation.
F igu r e 2. Potentiation of infused ANF in conscious rats by
compound 38 at 11.7 mg/kg po (]); vehicle, 20% EtOH/80%
PEG at 0.5 mL/kg po (0).
to yield thioesters 8. A small amount of the phenol-
coupled product is observed which is easily separated
by flash chromatography. The mercaptoacyl dipeptides
(compounds 9-52) prepared are shown in Table 1.
Resu lts a n d Discu ssion
In Vivo ACE a n d NEP In h ibition . The thioester
carboxylic acid esters (compounds 9-52) prepared were
tested for their ability to inhibit the AI pressor response
in normal conscious rats. We studied the effect of these
compounds only after oral administration, since this is
the desired route of administration for an antihyper-
tensive agent. The in vivo effects of these compounds
were compared to those of captopril and benazepril, two
clinically prescribed ACE inhibitors. Several of these
compounds were found to be more efficacious than
compound 2, captopril, and benazepril as can be seen
in Table 2. By simply preparing the thioester carboxylic
acid esters of compound 2, compounds 9-33, significant
improvement in oral activity was observed in some
instances. Among the best compounds were 13, 15, and
27. In an attempt to improve the water solubility of
these derivatives, heteroatoms were added to the acyl
group on the sulfur (compounds 21-24). Unfortunately,
these analogs were not among the longest acting. We
also prepared compounds where modifications were
As can be seen from Figure 2, compound 38 at 11.7
mg/kg po was found to significantly increase exog-
enously administered ANF plasma concentrations, ap-

Mercaptoacyl Dipeptides as ACE and NEP Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3161
Ta ble 1. Physicochemical Properties of R-Mercapto Dipeptides
compd
R
R1
R2
mp, °C
formula
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
Pr
allyl
Bu
hexyl
CH(Me)2
CH2CH(Me)2
(CH2)2CH(Me)2
cyclopentyl
benzyl
Et
Et
Et
Et
Et
Et
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Me
Et
Et
Et
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
tBu
157-158
148-149
119-120
119-120
85-86
C
23H32N2O6S
C24H34N2O6S
C25H36N2O6S
C25H34N2O6S
C26H38N2O6S
C28H42N2O6S
C25H36N2O6S
C26H38N2O6S
C27H40N2O6S
C27H38N2O6S
C29H36N2O6S
C27H40N2O6S
C28H41N3O7S
C25H36N2O7S
C26H39N3O6S
C28H35N3O6S
C28H40N2O6S
C27H40N2O6S
C28H42N2O6S
C28H42N2O6S
C30H44N2O6S
C31H46N2O6S
C30H45N3O7S
C31H47N3O6S
C27H40N2O7S
C24H34N2O6S
C26H38N2O6S
C27H40N2O6S
C26H38N2O7S
C25H36N2O6S
C27H40N2O6S
C29H43N3O7S
C30H38N2O6S
C29H37N3O6S
C26H36N2O7S
C27H38N2O7S
C28H40N2O7S
C27H38N2O8S
C29H40N2O9S
C31H38N2O7S
C29H36N2O7S2
C30H37N3O7S
C30H37N3O7S
C27H38N2O8S
65-66
155-156
148-149
94-95
133-135
66-67
150-151
147-148
129-130
136-137
154-155
130-131
102
CH2N(CH2)4O
CH2OMe
CH2N(Me)2
2-pyridyl
cyclopentyl
Et
Pr
CH(Me)2
cyclopentyl
cyclohexyl
CH2N(CH2)4O
CH2N(CH2)5
CH2OMe
Me
Me
Me
CH2OMe
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
105
125
140
110
143-144
130-131
110-111
118-120
102-103
116
95-100
102-103
114-115
106-107
110-111
107-109
94-95
H
Me
Et
Pr
Me
Me
Me
Me
Me
Me
Bu
CH2CON(Et)2
benzyl
CH2(3-pyridyl)
COMe
COEt
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
102-103
118-119
80-81
COCH(Me)2
COCH2OMe
COCH2CO2Et
CO(phenyl)
CO(2-thienyl)
CO(3-pyridyl)
CO(4-pyridyl)
CO2Et
71-72
121-122
125-126
67-68
85-86
94-95
proximately 250% over controls. We further explored
the in vivo inhibition of ACE and NEP after oral
administration at lower doses as shown in Figures 3 and
4, respectively. Compound 38 exhibits good dose-
response behavior for both the plasma NEP inhibition
and the AI pressor response assays. As can be seen in
Figure 3 compound 38 is approximately equal in its
effect at 3 mg/kg po as captopril (36% inhibition at 10
mg/kg po, 4 h; Table 2) and benazepril (39% inhibition
at 10 mg/kg po, 4 h) given at 10 mg/kg po.
Blood P r essu r e-Low er in g Stu d ies of Com p ou n d
38. The blood pressure-lowering effects of 38 were
further studied in two animal models of hypertension,
a renin-dependent model and a genetic model. In the
aorta-ligated rat, oral administration (3 mg/kg) of
compound 38 significantly lowered mean arterial pres-
sure. At 2 and 4 h, a 15 (p < 0.05) and 17 (p < 0.025)
mmHg lowering of blood pressure was observed.
In the conscious telemetered spontaneously hyper-
tensive rat (SHR), compound 38 given orally at 1, 3, and
10 mg/kg bid lowered mean arterial pressure in a dose-
dependent manner by 16, 21, and 33 mmHg. Benazepril
at the same doses decreased blood pressure by 7, 16,
and 27 mmHg, respectively (Figures 5-7).
In summary, significant enhancement of in vivo
activity was seen for the R-mercapto dipeptides by
preparing the thioester-carboxylic acid esters. These
compounds are most likely acting as prodrugs since
several of these compounds were found to have weak
or no activity in vitro as inhibitors of ACE and NEP.
Compound 38 was identified as a long acting dual ACE
and NEP inhibitor in vivo. This compound was found
to lower blood pressure in two animal models of hyper-
tension. A more detailed pharmacological evaluation
of compound 38 will be presented in the near future.
Exp er im en ta l Section
Ch em istr y. Melting points (mp) were determined on a
Thomas-Hoover or Melt-Temp melting point apparatus and
are uncorrected. Infrared (IR) spectra were recorded on a

3162 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fink et al.
Ta ble 2. Plasma Inhibition of ACE Activity in the Conscious
Rat
inhibition of AI pressor response, %
dose,
compd
mg/kg
1 h
2 h
4 h
6 h
2
2
9
10 (iv)
10 (po)
10 (po)
10 (po)
90 ( 1
48 ( 11
76 ( 2
86 ( 3
74 ( 7
68 ( 3
76 ( 5
91 ( 4
83 ( 1
68 ( 9
59 ( 7 36 ( 13 15 ( 12
72 ( 3
81 ( 2
69 ( 4
75 ( 8
78 ( 3
78 ( 1
85 ( 8
25 ( 4
83 ( 2 47 ( 16
37 ( 2 19 ( 7
67 ( 8 59 ( 10
70 ( 7
61 ( 8
81 ( 6
14 ( 1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
captopril
12.1 (po)
12.0 (po)
12.4 (po)
13.1 (po) 64 ( 12
12.1 (po) 67 ( 14
12.4 (po) 76 ( 10
12.7 (po)
12.7(po)
13.1 (po)
12.8 (po)
13.8 (po)
12.5 (po)
12.8 (po)
13.3 (po)
72 ( 6
16 ( 8
78 ( 2
20 ( 6
60 ( 7
77 ( 2 69 ( 17
77 ( 2 77 ( 4
83 ( 4
58 ( 16 51 ( 15 33 ( 21 29 ( 15
72 ( 2
8 ( 7
54 ( 6 60 ( 12
81 ( 2
70 ( 1
10 ( 6
74 ( 2 54 ( 12
17 ( 9 14 ( 11 19 ( 13
28 ( 5
40 ( 7 28 ( 12
70 ( 7 34 ( 12
F igu r e 3. Dose-response inhibition of AI pressor response 4
h after oral administration of 38 (dotted bar), captopril (open
bar), and benazepril (solid bar).
77 ( 3
63 ( 5 35 ( 10
11 ( 9 8 ( 5
18 ( 4
12 ( 6
13.0 (po) 34 ( 25 33 ( 25 55 ( 13 38 ( 11
12.8 (po) 47 ( 15
13.1 (po) 82 ( 6
13.1 (po) 27 ( 15 16 ( 13 25 ( 10
71 ( 4 56 ( 12 39 ( 14
85 ( 4 76 ( 6 69 ( 18
13.7 (po)
57 ( 9
49 ( 9 38 ( 11 27 ( 14
8 ( 6
14.1 (po) -3 ( 11
14.5 (po)
14.4 (po)
57 ( 7 53 ( 11
65 ( 7 55 ( 6
34 ( 3
50 ( 5
12 ( 5
26 ( 3
13.1 (po) 76 ( 15 75 ( 11 58 ( 17 54 ( 23
11.7 (po)
12.4 (po)
12.3 (po)
11.7 (po)
11.7 (po)
12.8 (po)
13.7 (po)
13.1(po)
13.2(po)
12.8 (po)
13.1 (po)
13.4 (po)
13.5 (po)
14.5 (po)
14.3 (po)
97 ( 3
88 ( 2
90 ( 2
81 ( 1
73 ( 4
75 ( 6
88 ( 3
85 ( 3
99 ( 1
64 ( 4
90 ( 2
76 ( 1
80 ( 3
87 ( 3
73 ( 2
72 ( 3
78 ( 5
70 ( 2
68 ( 3
91 ( 0 76 ( 10 52 ( 15
81 ( 4
70 ( 5
86 ( 7
84 ( 2
97 ( 2
52 ( 7
79 ( 5
65 ( 4
77 ( 4
58 ( 7
73 ( 2 64 ( 12
81 ( 2 64 ( 12
87 ( 6
31 ( 6
63 ( 5
16 ( 5
4 ( 9
50 ( 9 35 ( 11 23 ( 13
F igu r e 4. Plasma NEP inhibition at 4 h after oral adminis-
tration of 38.
73 ( 6
79 ( 6
88 ( 3
75 ( 3 63 ( 12 46 ( 22
79 ( 5
78 ( 6
70 ( 5 61 ( 12
82 ( 1
36 ( 6
73 ( 7
10 ( 5
45 ( 8 30 ( 13
pressure to give a white foam. The foam was purified by flash
chromatography on silica gel, eluting with hexane-ethyl
acetate. The pure fractions were combined and concentrated.
14.4 (po) 29 ( 14 37 ( 12
36 ( 9 26 ( 10
14.3 (po)
14.3 (po)
13.5 (po)
10 (po)
78 ( 6
79 ( 4
24 ( 3
100 ( 0
75 ( 6
75 ( 6
67 ( 6
70 ( 5
60 ( 6
55 ( 9
21 ( 4
1
4a (R ) H, R1 ) Me): white foam (61%); H NMR (CDCl3)
28 ( 3 25 ( 10
δ 1.40 (s, 9H), 1.72 (m, 6H), 2.22 (m, 2H), 3.04 (d, 2H), 3.76 (s,
3H), 4.72 (bs, 1H), 4.80 (q, 1H), 6.78 (d, 2H), 7.02 (d, 2H).
4b (R ) H, R1 ) Et): white foam (62%); 1H NMR (CDCl3) δ
1.22 (t, 3H), 1.43 (s, 9H), 1.69 (m, 6H), 2.17 (m, 2H), 3.00 (d,
2H), 4.11 (q, 2H), 4.70 (m, 2H), 6.69 (d, 2H), 6.95 (d, 2H).
4c (R ) H, R1 ) Pr): white foam (91%); 1H NMR (CDCl3) δ
1.15 (t, 3H), 1.39 (s, 9H), 1.61 (m, 8H), 2.20 (m, 2H), 3.01 (d,
2H), 4.07 (q, 2H), 4.70 (m, 2H), 6.71 (d, 2H), 7.02 (d, 2H).
4d (R ) H, R1 ) allyl): white foam (47%); 1H NMR (DMSO)
δ 1.37 (s, 9H), 1.55 (m, 6H), 1.95 (m, 2H), 2.87 (d, 2H), 4.50
(m, 3H), 5.22 (dd, 2H), 5.80 (dt, 1H), 6.60 (d, 2H), 6.90 (d, 2H),
7.00 (bs, 1H), 7.47 (d, 1H), 9.20 (bs, 1H).
78 ( 1 36 ( 12 13 ( 12
benazepril 10 (po)
100 ( 0 77 ( 11 39 ( 11 16 ( 12
Nicolet 5SXB FTIR spectrometer and are reported in cm-1
.
Proton NMR spectra were recorded on a Bruker AC-250,
Varian XL-300, or Varian XL-400 spectrometer with tetram-
ethylsilane as the internal standard. The chemical shifts (δ)
are reported in parts per million (ppm). Mass spectra were
recorded on a Hewlett-Packard GC/MS 5985 spectrometer
using chemical ionization (DCI) or on a Vestec 201 spectrom-
eter using thermospray technology (TSP). Microanalyses were
carried out at Robertson Laboratory Inc., Madison, NJ . Flash
chromatography under a nitrogen atmosphere on silica gel 60
(0.04-0.06 mm) (Baker) was used for compound purification.
The amino acids used were commercially available or prepared
according to literature procedures. All organic solvents used
were of anhydrous grade. The chemical yields are not opti-
mized.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
4. To a stirred solution of the amino acid ester 3 (1 equiv)
and Boc-cycloleucine (1 equiv) in dichloromethane were added
1,3-dicyclohexylcarbodiimide (1 equiv) and 1-hydroxybenzot-
riazole (1 equiv). The mixture was stirred for 16-24 h, and
then the solid precipitate was removed by filtration. The
organic phase was washed with a saturated solution of sodium
bicarbonate and brine and dried over magnesium sulfate. The
mixture was then filtered and concentrated under reduced
4e (R ) H, R1 ) butyl): white foam (52%); 1H NMR (CDCl3)
δ 0.96 (t, 3H), 1.15 (m, 2H), 1.35 (s, 9H), 1.70 (m, 2H), 1.78
(m, 6H), 2.14 (m, 2H), 3.01 (d, 2H), 4.08 (q, 2H), 4.75 (m, 2H),
6.23 (bs, 1H), 6.72 (d, 2H), 6.95 (d, 2H), 7.02 (bs, 1H).
4f (R ) H, R1 ) hexyl): white foam (68%); 1H NMR (CDCl3)
δ 0.88 (t, 3H), 1.21 (m, 6H), 1.33 (s, 9H), 1.73 (m, 8H), 2.16
(m, 2H), 3.00 (d, 2H), 4.02 (q, 2H), 4.75 (m, 2H), 6.23 (bs, 1H),
6.72 (d, 2H), 6.99 (d, 2H), 7.02 (bs, 1H).
4g (R ) H, R1 ) isopropyl): white foam (55%); 1H NMR
(CDCl3) δ 1.23 (dd, 6H), 1.41 (s, 9H), 1.70 (m, 6H), 2.19 (m,
2H), 3.00 (d, 2H), 4.71 (q, 1H), 4.83 (bs, 1H), 4.95 (m, 1H),
6.49 (bs, 1H), 6.69 (d, 2H), 6.98 (d, 2H).
4h (R ) H, R1) CH2CH(Me)2): white foam (56%); 1H NMR
(CDCl3) δ 0.91 (d, 6H), 1.39 (s, 9H), 1.64 (m, 7H), 2.22 (m, 2H),
3.02 (d, 2H), 4.11 (q, 2H), 4.74 (q, 1H), 5.19 (bs, 1H), 6.69 (d,
2H), 7.01 (d, 2H).

Mercaptoacyl Dipeptides as ACE and NEP Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3163
Ta ble 3. Plasma ANF Concentration in the Conscious Rat (Percent of Control)
percent of control
compd
dose, mg/kg
0.5 h
1 h
2 h
4 h
13
17
34
38
40
41
42
46
50
51
12.4(po)
12.7 (po)
11.7 (po)
11.7 (po)
13.7 (po)
13.1 (po)
13.2 (po)
13.5 (po)
14.3 (po)
14.3 (po)
10 (po)
131 ( 7
178 ( 31
279 ( 30
275 ( 17
159 ( 11
107 ( 7
223 ( 8
203 ( 7
246 ( 6
165 ( 15
209 ( 9
133 ( 15
235 ( 74
337 ( 17
346 ( 54
187 ( 5
178 ( 11
236 ( 58
263 ( 19
250 ( 29
215 ( 9
143 ( 12
260 ( 19
252 ( 23
246 ( 4
183 ( 21
173 ( 1
185 ( 14
251 ( 60
342 ( 16
251 ( 35
264 ( 12
181 ( 11
332 ( 29
223 ( 24
277 ( 7
111 ( 9
267 ( 15
213 ( 18
234 ( 4
193 ( 26
191 ( 9
159 ( 10
106 ( 9
acetorphan
F igu r e 7. Benazepril (0) and compound 38 (]) in the SHR
at 10 mg/kg po × bid.
F igu r e 5. Benazepril (0) and compound 38 (]) in the SHR
at 1 mg/kg po × bid.
4n (R ) Me, R1 ) Et): white foam (70%); 1H NMR (CDCl3)
δ 1.11 (t, 3H), 1.39 (s, 9H), 1.69 (m, 6H), 2.19 (m, 2H), 3.03 (d,
2H), 3.73 (s, 3H), 4.11 (q, 2H), 4.73 (m, 2H), 6.72 (d, 2H), 7.04
(d, 2H).
4o (R ) Me, R1 ) Bu): white foam (64%); 1H NMR (CDCl3)
δ 0.89 (t, 3H), 1.31 (m, 2H), 1.46 (s, 9H), 1.60 (m, 2H), 1.73
(m, 6H), 2.21 (m, 2H), 3.05 (d, 2H), 3.75 (s, 3H), 4.05 (q, 2H),
4.75 (q, 1H), 6.79 (d, 2H), 7.07 (d, 2H).
4p (R ) Me, R1 ) CH2CON(Et)2): white foam (81%); 1H
NMR (CDCl3) δ 1.12 (t, 3H), 1.18 (t, 3H), 1.38 (s, 9H), 1.70 (m,
6H), 2.20 (m, 2H), 3.15 (m, 2H), 3.21 (q, 2H), 3.38 (q, 2H), 3.75
(s, 3H), 4.71 (m, 1H), 4.74 (d, 1H), 4.87 (d, 1H), 6.79 (d, 2H),
7.04 (bs, 1H), 7.18 (d, 2H).
4q (R ) Me, R1 ) benzyl): white foam (66%); 1H NMR
(CDCl3) δ 1.50 (s, 9H), 1.70 (m, 6H), 2.20 (m, 2H), 3.02 (d, 2H),
3.75 (s, 3H), 4.70 (bs, 1H), 4.82 (q, 1H), 5.12 (d, 2H), 6.71 (d,
2H), 6.95 (d, 2H), 7.05 (bs, 1H), 7.32 (m, 5H).
4r (R ) Me, R1 ) CH2(3-pyridyl)): white foam (60%); 1H
NMR (CDCl3) δ 1.40 (s, 9H), 1.78 (m, 6H), 2.20 (m, 2H), 3.02
(d, 2H), 3.77 (s, 3H), 4.78 (bs, 1H), 4.81 (q, 1H), 5.12 (q, 2H),
6.74 (d, 2H), 6.97 (d, 2H), 7.19 (bs, 1H), 7.33 (dd, 1H), 7.63 (d,
1H), 8.56 (d, 1H), 8.60 (dd, 1H).
F igu r e 6. Benazepril (0) and compound 38 (]) in the SHR
at 3 mg/kg po × bid.
4i (R ) H, R1) (CH2)2CH(Me)2): white foam (64%); 1H NMR
(CDCl3) δ 0.87 (d, 6H), 1.39 (s, 9H), 1.70 (m, 9H), 2.19 (m, 2H),
3.00 (d, 2H), 4.09 (q, 2H), 4.72 (q, 1H), 5.12 (bs, 1H), 6.71 (d,
2H), 6.97 (d, 2H).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
6. The purified Boc-protected dipeptide 4 was dissolved in
dichloromethane, and dry HCl gas was bubbled through the
solution for 15 min. The solvent was removed under reduced
pressure to yield a white foam. The compound was used
without further purification. To a solution of the dipeptide
hydrochloride 5 (1 equiv) in dichloromethane was added
triethylamine (1 equiv). The mixture was stirred for 5 min,
and then the (S)-2-(acetylthio)-3-methylbutanoic acid (1 equiv),
1,3-dicyclohexylcarbodiimide (1 equiv), and 1-hydroxy-7-aza-
benzotriazole (1 equiv) were added. The mixture was stirred
for 16 h, and then the solid precipitate was removed by
filtration. The organic phase was washed with a saturated
solution of sodium bicarbonate and brine, dried over magne-
sium sulfate, filtered, and concentrated under reduced pres-
sure to give a white foam. The foam was purified by flash
chromatography on silica gel, eluting with hexane-ethyl
1
4j (R ) H, R1 ) cyclopentyl): white foam (51%); H NMR
(CDCl3) δ 1.35 (s, 9H), 1.70 (m, 14H), 2.19 (m, 2H), 2.98 (d,
2H), 4.69 (q, 1H), 4.89 (bs, 1H), 5.12 (m, 1H), 6.69 (d, 2H),
6.97 (d, 2H).
4k (R ) H, R1 ) benzyl): white foam (48%); 1H NMR
(CDCl3) δ 1.37 (s, 9H), 1.68 (m, 6H), 2.22 (m, 2H), 3.00 (d, 2H),
4.81 (q, 1H), 5.11 (dd, 2H), 6.68 (d, 2H), 6.85 (d, 2H), 7.32 (m,
5H).
4l (R ) Me, R1 ) Me): white foam (66%); 1H NMR (CDCl3)
δ 1.37 (s, 9H), 1.73 (m, 6H), 2.14 (m, 2H), 3.02 (d, 2H), 3.70 (s,
3H), 3.79 (s, 3H), 4.80 (m, 2H), 6.80 (d, 2H), 7.02 (d, 2H).
1
4m (R ) Pr, R1) Et): white foam (78%); H NMR (CDCl3)
δ 0.96 (t, 3H), 1.15 (t, 3H), 1.35 (s, 9H), 1.70 (m, 8H), 2.15 (m,
2H), 2.98 (d, 2H), 3.31 (t, 2H), 4.05 (q, 2H), 4.70 (m, 2H), 6.72
(d, 2H), 6.95 (d, 2H).

3164 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fink et al.
acetate. The pure fractions were combined and concentrated
under reduced pressure.
2.32 (s, 3H), 2.96 (dd, 2H), 3.62 (d, 1H), 4.05 (q, 2H), 4.77 (q,
1H), 6.11 (bs, 1H), 6.30 (s, 1H), 6.69 (d, 2H), 6.97 (d, 2H), 7.09
(d, 1H); MS (CDI, CH4) m/ z 521 (MH+). Anal. (C27H40N2O6S)
C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclopen tyl]car bon yl]-L-tyr osin e m eth yl ester (9): white
solid (55%); mp 157-158 °C; IR (KBr) 3373, 2958, 1748, 1679,
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclopen tyl]car bon yl]-L-tyr osin e cyclopen tyl ester (18):
white solid (44%); mp 133-135 °C; IR (KBr) 3316, 2963, 1736,
1
1657, 1614, 1516, 1227 cm-1; H NMR δ 1.01 (dd, 6H), 1.70
(m, 4H), 1.90 (m, 2H), 2.20 (m, 3H), 2.31 (s, 3H), 3.00 (dd, 2H),
3.62 (d, 1H), 4.69 (q, 1H), 6.36 (bs, 1H), 6.68 (d, 2H), 6.95 (d,
2H), 7.06 (d, 1H); MS (CDI, CH4) m/ z 465 (MH+). Anal.
(C23H32N2O6S) C, H, N.
1681, 1653, 1615, 1515, 1268, 1217, 1163 cm-1 1H NMR
;
(CDCl3) δ 0.95 (dd, 6H), 1.70 (m, 12H), 1.92 (m, 2H), 2.23 (m,
3H), 2.39 (s, 3H), 2.98 (dd, 2H), 3.65 (d, 1H), 4.68 (q, 1H), 5.11
(m, 1H), 6.40 (s, 1H), 6.70 (d, 2H), 6.99 (d, 2H), 7.07 (d, 1H);
MS (CDI, CH4) m/ z 519 (MH+). Anal. (C27H38N2O6S) C, H,
N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclopen tyl]car bon yl]-L-tyr osin e ben zyl ester (19): white
solid (57%); mp 66-67 °C; IR (KBr) 3372, 2962, 1742, 1675,
1655, 1515, 1225 cm-1; 1H NMR δ 0.89 (dd, 6H), 1.65 (m, 4H),
1.89 (m, 2H), 2.21 (m, 3H), 2.39 (s, 3H), 2.98 (dd, 2H), 3.62 (d,
1H), 4.73 (q, 1H), 5.05 (dd, 2H), 6.41 (s, 1H), 6.62 (d, 2H), 6.88
(d, 2H), 7.11 (d, 1H), 7.31 (m, 5H); MS (CDI, CH4) m/ z 541
(MH+). Anal. (C29H36N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl ester (10): white
solid (45%); mp 148-149 °C; IR (KBr) 3380, 2963, 1741, 1682,
1649, 1615, 1515, 1223, 1201, 1173, 1122 cm-1 1H NMR
;
(CDCl3) δ 0.99 (dd, 6H), 1.15 (t, 3H), 1.68 (m, 4H), 1.93 (m,
2H), 2.17 (m, 3H), 2.32 (s, 3H), 3.00 (dd, 2H), 3.64 (d, 1H),
4.11 (q, 2H), 4.69 (q, 1H), 6.29 (s, 1H), 6.37 (s, 1H), 6.70 (d,
2H), 6.96 (d, 2H), 7.11 (d, 1H); MS (CDI, CH4) m/ z 479 (MH+).
Anal. (C24H34N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclopen tyl]car bon yl]-L-tyr osin e pr opyl ester (11): white
solid (48%); mp 119-120 °C; IR (KBr) 3261, 2966, 1741, 1682,
1669, 1650, 1515, 1215, 1200 cm-1; 1H NMR (CDCl3) δ 0.89 (t,
3H), 1.00 (dd, 6H), 1.68 (m, 6H), 1.93 (m, 2H), 2.22 (m, 3H),
2.32 (s, 3H), 2.99 (dd, 2H), 3.64 (d, 1H), 4.01 (q, 2H), 4.72 (q,
1H), 6.39 (bs, 1H), 6.47 (s, 1H), 6.68 (d, 2H), 6.92 (d, 2H), 7.05
(d, 1H); MS (CDI, CH4) m/ z 493 (MH+). Anal. (C25H36N2O6S)
C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-m eth yl-L-tyr osin e m eth yl es-
ter (34): white solid (58%); mp 118-120 °C; IR (KBr) 1756,
1743, 1697, 1688, 1658, 1650, 1512, 1247 cm-1 1H NMR
;
(CDCl3) δ 0.96 (d, 3H), 1.02 (d, 3H), 1.68 (m, 4H), 1.92 (m,
3H), 2.22 (m, 2H), 2.36 (s, 3H), 3.04 (m, 2H), 3.62 (d, 1H), 3.67
(s, 3H), 3.78 (s, 3H), 4.75 (q, 1H), 6.30 (s, 1H), 6.80 (d, 2H),
7.06 (d, 2H), 7.12 (d, 1H); MS (CDI, CH4) m/ z 479 (MH+). Anal.
(C24H34N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-L-tyr osin e a llyl ester (12): white
solid (36%); mp 119-120 °C; IR (KBr) 3361, 2969, 1749, 1675,
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en t yl]ca r b on yl]-O-et h yl-L-t yr osin e et h yl est er
(35): white solid (61%); mp 102-103 °C; IR (KBr) 3290, 1731,
1
1637, 1514, 1229, 1188 cm-1; H NMR δ 1.00 (dd, 6H), 1.69
(m, 4H), 1.92 (m, 2H), 2.22 (m, 3H), 2.32 (s, 3H), 3.00 (dd, 2H),
3.62 (d, 1H), 4.55 (d, 2H), 4.71 (q, 1H), 5.21 (dd, 2H), 5.81 (dt,
1H), 6.32 (bs, 1H), 6.70 (d, 2H), 6.97 (d, 2H), 7.11 (d, 1H); MS
(CDI, CH4) m/ z 491 (MH+). Anal. (C25H34N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl ester (13): white
solid (45%); mp 85-86 °C; IR (KBr) 3334, 2963, 1739, 1682,
1649, 1616, 1515, 1225 cm-1; 1H NMR (CDCl3) δ 0.89 (t, 3H),
1.00 (dd, 6H), 1.33 (m, 2H), 1.60 (m, 6H), 1.90 (m, 2H), 2.20
(m, 3H), 2.32 (s, 3H), 3.00 (dd, 2H), 3.62 (d, 1H), 4.00 (dd, 2H),
4.69 (q, 1H), 6.30 (bs, 2H), 6.70 (d, 2H), 6.94 (d, 2H), 7.08 (d,
1H); MS (CDI, CH4) m/ z 507 (MH+). Anal. (C26H38N2O6S) C,
H, N.
1
1673, 1650, 1512, 1246, 1197 cm-1; H NMR (CDCl3) δ 0.90
(dd, 6H), 1.10 (t, 3H), 1.30 (t, 3H), 1.60 (m, 4H), 1.92 (m, 2H),
2.19 (m, 3H), 2.30 (s, 3H), 3.04 (dd, 2H), 3.59 (d, 1H), 3.89 (q,
2H), 4.05 (q, 2H), 4.62 (q, 1H), 6.22 (s, 1H), 6.70 (d, 2H), 6.99
(m, 3H); MS (CDI, CH4) m/ z 507 (MH+). Anal. (C26H38N2O6S)
C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-p r op yl-L-tyr osin e eth yl ester
(36): white solid (17%); mp 116 °C; IR (KBr) 3300, 2963, 1743,
1695, 1659, 1640, 1610, 1528, 1512, 1243, 1186 cm-1; 1H NMR
(CDCl3) δ 0.99 (m, 9H), 1.19 (t, 3H), 1.75 (m, 8H), 2.22 (m,
3H), 2.34 (s, 3H), 3.02 (m, 2H), 3.64 (d, 1H), 3.85 (t, 2H), 4.10
(q, 2H), 4.70 (q, 1H), 6.30 (s, 1H), 6.78 (d, 2H), 7.04 (m, 3H);
MS (CDI, CH4) m/ z 521 (MH+). Anal. (C27H40N2O6S) C, H,
N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclopen tyl]car bon yl]-L-tyr osin e h exyl ester (14): white
solid (67%); mp 65-66 °C; IR (KBr) 3379, 2960, 1674, 1653,
1615, 1515, 1268, 1224, 1197 cm-1; 1H NMR (CDCl3) δ 0.81 (t,
3H), 1.00 (dd, 6H), 1.30 (m, 6H), 1.70 (m, 6H), 1.92 (m, 2H),
2.23 (m, 3H), 2.32 (s, 3H), 3.00 (dd, 2H), 3.61 (d, 1H), 4.03 (q,
2H), 4.72 (q, 1H), 5.62 (s, 1H), 6.36 (s, 1H), 6.69 (d, 2H), 6.91
(d, 2H), 7.11 (d, 1H); MS (CDI, CH4) m/ z 535 (MH+). Anal.
(C28H42N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-m eth yl-L-tyr osin e eth yl ester
(38): white solid (42%); mp 102-103 °C; IR (KBr) 2962, 1742,
1
1695, 1675, 1652, 1513, 1247, 1102 cm-1; H NMR (CDCl3) δ
0.99 (dd, 6H), 1.21 (t, 3H), 1.70 (m, 4H), 1.90 (m, 2H), 2.25 (m,
3H), 2.36 (s, 3H), 3.02 (m, 2H), 3.62 (d, 1H), 3.80 (s, 3H), 4.08
(q, 2H), 4.72 (q, 1H), 6.30 (bs, 1H), 6.79 (d, 2H), 7.04 (m, 3H);
MS (CDI, CH4) m/ z 493 (MH+). Anal. (C25H36N2O6S) C, H,
N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-L-tyr osin e isop r op yl ester (15):
white solid (36%); mp 155-156 °C; IR (KBr) 3382, 3253, 1736,
1
1516, 1448, 1373, 1218, 1109 cm-1; H NMR (CDCl3) δ 1.00
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-m eth yl-L-tyr osin e bu tyl ester
(39): white solid (36%); mp 114-115 °C; IR (KBr) 1738, 1696,
1657, 1640, 1528, 1245, 1202 cm-1; 1H NMR (CDCl3) δ 0.90 (t,
3H), 1.00 (dd, 6H), 1.30 (m, 2H), 1.64 (m, 6H), 1.95 (m, 2H),
2.20 (m, 3H), 3.00 (dd, 2H), 3.60 (d, 1H), 3.75 (s, 3H), 4.05 (q,
2H), 4.71 (q, 1H), 6.30 (bs, 1H), 6.67 (d, 2H), 7.04 (d, 3H); MS
(CDI, CH4) m/ z 521 (MH+). Anal. (C27H40N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-m eth yl-L-tyr osin e 2-(d ieth y-
la m in o)-2-oxoeth yl ester (40): white solid (19%); mp 106-
107 °C; IR (KBr) 3394, 1768, 1673, 1646, 1573, 1534, 1247,
1148 cm-1; 1H NMR (CDCl3) δ 0.98 (dd, 6H), 1.15 (t, 3H), 1.20
(t, 3H), 1.67 (m, 4H), 1.91 (m, 2H), 2.18 (m, 3H), 2.33 (s, 3H),
3.13 (m, 2H), 3.22 (q, 2H), 3.39 (q, 2H), 3.65 (d, 1H), 3.76 (s,
3H), 4.72 (dd, 2H), 4.82 (m, 1H), 6.34 (s, 1H), 6.78 (d, 2H),
7.06 (d, 1H), 7.15 (d, 2H); MS (CDI, CH4) m/ z 578 (MH+). Anal.
(C29H43N3O7S) C, H, N.
(dd, 6H), 1.15 (dd, 6H), 1.70 (m, 4H), 1.90 (m, 2H), 2.20 (m,
3H), 2.37 (s, 3H), 3.00 (dd, 2H), 3.55 (d, 1H), 4.69 (q, 1H), 4.95
(pentet, 1H), 5.80 (s, 1H), 6.36 (s, 1H), 6.72 (d, 2H), 6.96 (d,
2H), 7.04 (d, 1H); MS (CDI, CH4) m/ z 493 (MH+). Anal.
(C25H36N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-L-tyr osin e isobu tyl ester (16):
white solid (43%); mp 148-149 °C; IR (KBr) 3297, 2963, 1740,
1
1615, 1653, 1447, 1200, 1175 cm-1; H NMR (CDCl3) δ 0.81
(d, 6H), 0.95 (dd, 6H), 1.68 (m, 4H), 1.95 (m, 3H), 2.18 (m, 3H),
2.35 (s, 3H), 2.96 (dd, 2H), 3.62 (d, 1H), 3.82 (dd, 2H), 4.72 (q,
1H), 6.35 (s, 1H), 6.67 (m, 3H), 6.95 (d, 2H), 7.06 (d, 1H); MS
(CDI, CH4) m/ z 507 (MH+). Anal. (C26H38N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-L-tyr osin e 3-m eth ylbu tyl ester
(17): white solid (57%); mp 94-95 °C; IR (KBr) 3413, 2963,
1
1735, 1689, 1515, 1200, 1172 cm-1; H NMR (CDCl3) δ 0.83
(d, 6H), 0.98 (dd, 6H), 1.65 (m, 7H), 1.94 (m, 2H), 2.18 (m, 3H),

Mercaptoacyl Dipeptides as ACE and NEP Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3165
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-m eth yl-L-tyr osin e ben zyl es-
ter (41): white solid (19%); mp 110-111 °C; IR (KBr) 3297,
1741, 1695, 1660, 1640, 1527, 1513, 1248, 1168 cm-1; 1H NMR
(CDCl3) δ 0.98 (dd, 6H), 1.68 (m, 4H), 1.94 (m, 2H), 2.20 (m,
3H), 2.33 (s, 3H), 3.00 (d, 2H), 3.60 (d, 1H), 3.75 (s, 3H), 4.77
(q, 1H), 5.07 (m, 2H), 6.28 (s, 1H), 6.72 (d, 2H), 6.94 (d, 2H),
7.08 (d, 1H), 7.24 (m, 2H), 7.33 (m, 3H); MS (CDI, CH4) m/ z
555 (MH+). Anal. (C30H38N2O6S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-ben zoyl-L-tyr osin e eth yl ester
(48): white solid (53%); mp 121-122 °C; IR (KBr) 3308, 2976,
1744, 1736, 1695, 1679, 1638, 1591, 1535, 1273, 1195, 1165
1
cm-1; H NMR (CDCl3) δ 0.99 (dd, 6H), 1.09 (t, 3H), 1.69 (m,
4H), 1.80 (m, 2H), 2.20 (m, 3H), 2.33 (s, 3H), 3.07 (d, 2H), 3.65
(d, 1H), 4.12 (q, 2H), 4.73 (q, 1H), 6.32 (bs, 1H), 7.10 (d, 2H),
7.20 (m, 3H), 7.49 (t, 2H), 7.60 (t, 1H), 8.19 (d, 2H); MS (CDI,
CH4) m/ z 583 (MH+). Anal. (C31H38N2O7S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en t yl]ca r b on yl]-O-(2-t h ien ylca r b on yl)-L-t yr o-
sin e eth yl ester (49): white solid (52%); mp 125-126 °C; IR
(KBr) 3308, 2960, 1730, 1694, 1674, 1643, 1505, 1273, 1196
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en t yl]ca r b on yl]-O-m et h yl-L-t yr osin e 3-p yr id i-
n ylm eth yl ester (42): white solid (11%); mp 107-109 °C; IR
(KBr) 3340, 1741, 1691, 1674, 1657, 1603, 1513, 1251, 1170
cm-1; 1H NMR (CDCl3) δ 1.00 (dd, 6H), 1.68 (m, 4H), 1.90 (m,
3H), 2.20 (m, 2H), 2.35 (s, 3H), 2.99 (d, 2H), 3.67 (d, 1H), 3.76
(s, 3H), 4.74 (q, 1H), 5.05 (m, 2H), 6.34 (s, 1H), 6.73 (d, 2H),
6.98 (d, 2H), 7.20 (d, 1H), 7.30 (m, 1H), 7.56 (d, 1H), 8.58 (m,
2H); MS (CDI, CH4) m/ z 556 (MH+). Anal. (C29H37N3O6S) C,
H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
7 (R * H). To a stirred solution of the dipeptide 6 (1 equiv)
in dichloromethane were added an acid chloride or a chloro-
formate (1 equiv) and triethylamine (1 equiv). The mixture
was stirred for 5 h and then washed with brine, dried over
magnesium sulfate, filtered, and concentrated under reduced
pressure to give a white foam. The foam was purified by flash
chromatography on silica gel, eluting with hexane-ethyl
acetate. The pure fractions were combined and concentrated
under reduced pressure.
1
cm-1; H NMR (CDCl3) δ 1.00 (dd, 6H), 1.21 (t, 3H), 1.69 (m,
4H), 1.92 (m, 2H), 2.21 (m, 3H), 2.31 (s, 3H), 3.08 (dd, 2H),
3.62 (d, 1H), 4.10 (q, 2H), 4.72 (q, 1H), 6.32 (bs, 1H), 7.15 (m,
5H), 7.62 (d, 1H), 7.92 (d, 1H); MS (CDI, CH4) m/ z 589 (MH+).
Anal. (C29H36N2O7S2) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en t yl]ca r b on yl]-O-(3-p yr id in ylca r b on yl)-L-t y-
r osin e eth yl ester (50): white solid (40%); mp 67-68 °C; IR
(KBr) 3397, 3298, 2964, 1744, 1672, 1652, 1590, 1508, 1276,
1197 cm-1; 1H NMR (CDCl3) δ 0.99 (dd, 6H), 1.17 (t, 3H), 1.69
(m, 4H), 1.87 (m, 2H), 2.20 (m, 3H), 2.32 (s, 3H), 3.10 (d, 2H),
3.65 (d, 1H), 4.12 (q, 2H), 4.75 (q, 1H), 6.31 (bs, 1H), 7.08 (d,
2H), 7.21 (m, 3H), 7.41 (dd, 1H), 8.42 (dt, 1H), 8.81 (d, 1H),
9.39 (s, 1H); MS (CDI, CH4) m/ z 584 (MH+). Anal. (C30H37
-
N3O7S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en t yl]ca r b on yl]-O-(4-p yr id in ylca r b on yl)-L-t y-
r osin e eth yl ester (51): white solid (48%); mp 85-86 °C; IR
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-a cetyl-L-tyr osin e eth yl ester
(43): white solid (68%); mp 94-95 °C; IR (KBr) 3296, 2964,
(KBr) 2920, 1744, 1672, 1508, 1272, 1196 cm-1 1H NMR
;
(CDCl3) δ 1.00 (dd, 6H), 1.19 (t, 3H), 1.68 (m, 4H), 1.89 (m,
2H), 2.20 (m, 3H), 2.32 (s, 3H), 3.09 (dd, 2H), 3.62 (d, 1H),
4.11 (q, 2H), 4.73 (q, 1H), 6.32 (bs, 1H), 7.09 (d, 2H), 7.15 (d,
3H), 8.00 (d, 2H), 8.69 (d, 2H); MS (CDI, CH4) m/ z 584 (MH+).
Anal. (C30H37N3O7S) C, H, N.
1762, 1733, 1673, 1651, 1525, 1508, 1197 cm-1 1H NMR
;
(CDCl3) δ 0.99 (dd, 6H), 1.15 (t, 3H), 1.68 (m, 4H), 1.89 (m,
3H), 2.20 (m, 2H), 2.35 (s, 3H), 2.40 (s, 3H), 3.03 (d, 2H), 3.62
(d, 1H), 4.12 (q, 2H), 4.72 (q, 1H), 6.31 (bs, 1H), 6.91 (d, 2H),
7.12 (m, 3H); MS (CDI, CH4) m/ z 521 (MH+). Anal.
(C26H36N2O7S) C, H, N.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-(eth oxyca r bon yl)-L-tyr osin e
eth yl ester (52): white solid (68%); mp 94-95 °C; IR (KBr)
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-(1-oxop r op yl)-L-tyr osin e eth yl
ester (44): white solid (50%); mp 102-103 °C; IR (KBr) 3255,
3420, 3271, 1749, 1697, 1673, 1647, 1505, 1259, 1216 cm-1
;
1H NMR (CDCl3) δ 1.00 (dd, 6H), 1.20 (t, 3H), 1.30 (t, 3H),
1.70 (m, 4H), 1.87 (m, 2H), 2.20 (m, 3H), 2.30 (s, 3H), 3.07
(dd, 2H), 3.60 (d, 1H), 4.10 (q, 2H), 4.25 (q, 2H), 4.74 (q, 1H),
6.32 (bs, 1H), 7.01 (d, 2H), 7.11 (d, 2H); MS (CDI, CH4) m/ z
551 (MH+). Anal. (C27H38N2O8S) C, H, N.
1760, 1744, 1695, 1659, 1527, 1201, 1141 cm-1 1H NMR
;
(CDCl3) δ 0.99 (dd, 6H), 1.19 (t, 3H), 1.29 (t, 3H), 1.70 (m, 4H),
1.89 (m, 2H), 2.22 (m, 3H), 2.32 (s, 3H), 2.58 (q, 2H), 3.05 (dd,
2H), 3.62 (d, 1H), 4.10 (q, 2H), 4.70 (q, 1H), 6.30 (bs, 1H), 6.94
(d, 2H), 7.05 (d, 1H), 7.10 (d, 2H); MS (CDI, CH4) m/ z 535
(MH+). Anal. (C27H38N2O7S) C, H, N.
Gen er a l P r oced u r e of th e P r ep a r a tion of Com p ou n d s
8. To a stirred solution of the R-thioacetate dipeptide 6 (1
equiv) in degassed methanol was added 1N sodium hydroxide
(1 equiv). The mixture was stirred for 45 min and then
concentrated. The residue was dissolved in ethyl acetate,
washed with brine, dried over magnesium sulfate, and filtered,
and the solvent was removed to give the free thiol 6a which
was used without further purification.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en t yl]ca r b on yl]-O-(1-oxo-2-m et h ylp r op yl)-L-t y-
r osin e eth yl ester (45): white solid (21%); mp 118-119 °C;
1
IR (KBr) 3293, 1757, 1749, 1697, 1660, 1528, 1135 cm-1; H
NMR (DMSO) δ 0.89 (dd, 6H), 1.05 (t, 3H), 1.19 (d, 6H), 1.60
(m, 4H), 1.83 (m, 3H), 2.00 (m, 2H), 2.20 (s, 3H), 2.70 (pentet,
1H), 2.91 (d, 2H), 3.96 (m, 3H), 4.43 (q, 1H), 6.99 (d, 2H), 7.19
(d, 2H), 7.36 (d, 1H), 8.32 (s, 1H); MS (CDI, CH4) m/ z 549
(MH+). Anal. (C28H40N2O7S) C, H, N.
Wh en
R ) H. The residue was dissolved in dichlo-
romethane, and a carboxylic acid (1 equiv), 1,3-dicyclohexyl-
carbodiimide (1 equiv), 1-hydroxy-7-azabenzotriazole (1 equiv),
and triethylamine (1 equiv) were added. The mixture was
stirred overnight, filtered, washed with brine, and dried over
magnesium sulfate. The solution was filtered and concen-
trated under vacuum to give a foam. The foam was purified
by flash chromatography on silica gel, eluting with hexane-
ethyl acetate. The pure fractions were combined and concen-
trated under reduced pressure.
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-(2-m eth oxy-1-oxoeth yl)-L-ty-
r osin e eth yl ester (46): white solid (45%); mp 80-81 °C; IR
(KBr) 3399, 3296, 2962, 1777, 1732, 1673, 1650, 1518, 1197
1
cm-1; H NMR (CDCl3) δ 0.98 (dd, 6H), 1.21 (t, 3H), 1.29 (t,
3H), 1.67 (m, 4H), 1.89 (m, 2H), 2.19 (m, 3H), 2.30 (s, 3H),
3.05 (dd, 2H), 3.51 (s, 3H), 3.63 (d, 1H), 4.11 (q, 2H), 4.22 (s,
2H), 4.72 (q, 1H), 6.31 (bs, 1H), 6.97 (d, 2H), 7.08 (d, 1H), 7.13
(d, 2H); MS (CDI, CH4) m/ z 551 (MH+). Anal. (C27H38N2O8S)
C, H, N.
N-[[1-[[2(S)-(1,1,1-Tr im eth yla cetyl)-3-m eth yl-1-oxobu -
tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl es-
ter (20): white solid (31%); mp 150-151 °C; IR (KBr) 3409,
3262, 2968, 1745, 1660, 1647, 1516, 1225 cm-1 1H NMR
;
N-[[1-[[2(S)-(Acetylth io)-3-m eth yl-1-oxobu tyl]a m in o]-
1-cyclop en tyl]ca r bon yl]-O-(3-eth oxy-1,3-d ioxop r op yl)-L-
tyr osin e eth yl ester (47): white solid (15%); mp 71-72 °C;
(DMSO) δ 0.93 (dd, 6H), 1.08 (t, 3H), 1.28 (s, 9H), 1.70 (m,
4H), 1.85 (m, 3H), 2.11 (m, 2H), 2.73 (d, 2H), 3.97 (m, 3H),
4.35 (q, 1H), 6.62 (d, 2H), 6.88 (d, 2H), 6.98 (d, 2H), 7.23 (d,
1H), 8.27 (bs, 1H), 9.21 (bs, 1H); MS (CDI, CH4) m/ z 521
(MH+). Anal. (C27H40N2O6S) C, H, N.
N-[[1-[[2(S)-[(4-Mor p h olin oa cetyl)th io]-3-m eth yl-1-ox-
obu tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl
ester (21): white solid (24%); mp 147-148 °C; IR (KBr) 3327,
IR (KBr) 3291, 1771, 1741, 1697, 1658, 1530, 1188, 1136 cm-1
;
1H NMR (DMSO) δ 0.79 (t, 6H), 1.05 (t, 3H), 1.21 (t, 3H), 1.70
(m, 4H), 1.87 (m, 3H), 2.12 (m, 2H), 2.25 (s, 3H), 2.94 (d, 2H),
3.71 (s, 2H), 3.96 (m, 3H), 4.20 (q, 2H), 4.40 (q, 1H), 7.00 (d,
2H), 7.22 (d, 2H), 7.39 (d, 1H), 8.31 (s, 1H); MS (CDI, CH4)
m/ z 593 (MH+). Anal. (C29H40N2O9S) C, H, N.

3166 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
1745, 1666, 1649, 1515, 1243, 1116 cm-1 1H NMR (CDCl3) δ
1.00 (dd, 6H), 1.18 (t, 3H), 1.66 (m, 6H), 2.20 (m, 3H), 2.55 (m,
4H), 3.00 (dd, 2H), 3.31 (d, 2H), 3.62 (d, 1H), 3.73 (m, 4H),
4.11 (q, 2H), 4.71 (q, 1H), 5.73 (s, 1H), 6.31 (s, 1H), 6.71 (d,
2H), 6.99 (d, 2H), 7.11 (d, 1H); MS (CDI, CH4) m/ z 564 (MH+).
Anal. (C28H41N3O7S) C, H, N.
Fink et al.
:
2H), 7.18 (d, 1H); MS (CDI, CH4) m/ z 561 (MH+). Anal.
(C27H40N2O6S) C, H, N.
N-[[1-[[2(S)-[(Cycloh exylca r bon yl)th io]-3-m eth yl-1-ox-
obu tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl
ester (30): white solid (13%); mp 110 °C; IR (KBr) 3257, 2932,
1744, 1657, 1644, 1515, 1196 cm-1; 1H NMR (CDCl3) δ 0.89 (t,
3H), 0.98 (dd, 6H), 1.25 (m, 8H), 1.64 (bm, 23H), 2.45 (m, 1H),
2.95 (dd, 2H), 3.64 (d, 1H), 4.05 (t, 2H), 4.74 (q, 1H), 5.54 (bs,
1H), 6.40 (bs, 1H), 6.60 (d, 2H), 6.95 (d, 2H), 7.15 (d, 1H); MS
(CDI, CH4) m/ z 575 (MH+). Anal. (C31H46N2O6S) C, H, N.
N-[[1-[[2(S)-[(4-Mor p h olin oa cetyl)th io]-3-m eth yl-1-ox-
obu tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl
ester (31): white solid (39%); mp 143-144 °C; IR (KBr) 3393,
N-[[1-[[2(S)-[(Meth oxya cetyl)th io]-3-m eth yl-1-oxobu ty-
l]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl ester
(22): white solid (30%); mp 129-130 °C; IR (KBr) 3396, 3259,
1
1746, 1665, 1648, 1615, 1517, 1200 cm-1; H NMR (CDCl3) δ
0.93 (dd, 6H), 1.20 (t, 3H), 1.64 (m, 4H), 1.95 (m, 2H), 2.25 (m,
3H), 3.00 (dd, 2H), 3.49 (s, 3H), 3.68 (d, 1H), 4.05 (m, 4H),
4.70 (q, 1H), 5.55 (s, 1H), 6.32 (bs, 1H), 6.66 (d, 2H), 6.97 (d,
2H), 7.10 (d, 1H); MS (CDI, CH4) m/ z 509 (MH+). Anal.
(C25H36N2O7S) C, H, N.
2961, 1745, 1646, 1515, 1343, 1193 cm-1; H NMR (CDCl3) δ
1
0.90 (t, 3H), 1.00 (dd, 6H), 1.20 (m, 2H), 1.64 (m, 6H), 1.95 (m,
2H), 2.29 (m, 3H), 2.51 (m, 4H), 2.95 (dd, 2H), 3.21 (d, 2H),
3.59 (d, 1H), 3.69 (m, 4H), 4.02 (m, 2H), 4.70 (q, 1H), 6.30 (bs,
1H), 6.65 (d, 2H), 6.97 (m, 3H), 7.10 (d, 1H); MS (CDI, CH4)
m/ z 592 (MH+). Anal. (C30H45N3O7S) C, H, N.
N-[[1-[[2(S)-[(Dim eth yla cetyl)th io]-3-m eth yl-1-oxobu -
tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl es-
ter (23): white solid (20%); mp 136-137 °C; IR (KBr) 3326,
1
1746, 1667, 1643, 1515, 1222, 1195 cm-1; H NMR (CDCl3) δ
0.99 (dd, 6H), 1.20 (t, 3H), 1.70 (m, 4H), 1.90 (m, 2H), 2.22 (m,
3H), 2.30 (s, 6H), 3.01 (dd, 2H), 3.20 (s, 2H), 3.60 (d, 1H), 4.12
(q, 2H), 4.70 (q, 1H), 6.30 (bs, 1H), 6.35 (bs, 1H), 6.68 (d, 2H),
6.95 (d, 2H), 7.10 (d, 1H); MS (CDI, CH4) m/ z 522 (MH+). Anal.
(C26H39N3O6S) C, H, N.
N-[[1-[[2(S)-[(P ip er d in oa cetyl)th io]-3-m eth yl-1-oxobu -
tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl es-
ter (32): white solid (71%); mp 130-131 °C; IR (KBr) 3396,
1744, 1672, 1647, 1628, 1605, 1514, 1213, 1194 cm-1; 1H NMR
(CDCl3) δ 0.92 (t, 3H), 0.99 (dd, 6H), 1.30 (m, 2H), 1.65 (m,
12H), 1.95 (m, 2H), 2.24 (m, 3H), 2.50 (t, 4H), 3.01 (dd, 2H),
3.21 (s, 2H), 3.60 (d, 1H), 4.07 (m, 2H), 4.72 (q, 1H), 5.50 (bs,
1H), 6.30 (bs, 1H), 6.70 (d, 2H), 6.97 (d, 2H), 7.10 (d, 1H); MS
(CDI, CH4) m/ z 590 (MH+). Anal. (C31H47N3O6S) C, H, N.
N-[[1-[[2(S)-[(Meth oxya cetyl)th io]-3-m eth yl-1-oxobu ty-
l]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl ester
N-[[1-[[2(S)-[(2-P yr id in ylca r bon yl)th io]-3-m eth yl-1-ox-
obu tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl
ester (24): white solid (70%); mp 154-155 °C; IR (KBr) 3399,
2969, 1750, 1667, 1633, 1612, 1540, 1225 cm-1 1H NMR
;
(DMSO) δ 0.99 (m, 9H), 1.60 (m, 4H), 1.87 (m, 3H), 2.12 (m,
2H), 2.72 (d, 2H), 3.97 (q, 2H), 4.10 (d, 1H), 4.45 (q, 1H), 6.59
(d, 2H), 6.82 (d, 2H), 7.25 (d, 1H), 7.70 (dd, 1H), 7.79 (d, 1H),
8.05 (t, 1H), 8.47 (bs, 1H), 8.72 (d, 1H), 9.19 (bs, 1H); MS (CDI,
CH4) m/ z 542 (MH+). Anal. (C28H35N3O6S) C, H, N.
N-[[1-[[2(S)-[(Meth oxya cetyl)th io]-3-m eth yl-1-oxobu ty-
l]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e eth yl ester
(25): white solid (32%); mp 130-131 °C; IR (KBr) 1746, 1647,
1549, 1516, 1222 cm-1; 1H NMR (DMSO) δ 0.90 (dd, 6H), 1.20
(t, 3H), 1.73 (bm, 17H), 2.80 (d, 2H), 3.01 (pent, 1H), 3.99 (m,
3H), 4.35 (q, 1H), 6.60 (d, 2H), 6.90 (d, 2H), 7.25 (d, 1H), 8.23
(bs, 1H), 9.21 (bs, 1H); MS (CDI, CH4) m/ z 533 (MH+). Anal.
(C28H40N2O6S) C, H, N.
1
(33): white solid (63%); mp 110-111 °C; H NMR (CDCl3) δ
0.89 (t, 3H), 1.02 (dd, 6H), 1.30 (m, 2H), 1.64 (m, 6H), 1.99 (m,
2H), 2.27 (m, 3H), 3.00 (dd, 2H), 3.47 (s, 3H), 3.65 (d, 1H),
4.05 (m, 2H), 4.08 (s, 2H), 4.73 (q, 1H), 5.81 (s, 1H), 6.30 (bs,
1H), 6.69 (d, 2H), 6.93 (d, 2H), 7.07 (d, 1H); MS (CDI, CH4)
m/ z 537 (MH+). Anal. (C27H40N2O7S) C, H, N.
Wh en
R * H. The residue was dissolved in dichlo-
romethane and triethylamine (1 equiv) and an acid chloride
(1 equiv) were added. The mixture was stirred for 3 h and
then washed with brine and dried over magnesium sulfate.
The solution was filtered and concentrated under vacuum to
give a foam. The foam was purified by flash chromatography
on silica gel, eluting with hexane-ethyl acetate. The pure
fractions were combined and concentrated under reduced
pressure.
N-[[1-[[2(S)-[(Eth ylca r bon yl)th io]-3-m eth yl-1-oxobu ty-
l]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl ester
(26): white solid (13%); mp 102 °C; IR (KBr) 3252, 2962, 1749,
1
1673, 1648, 1615, 1515, 1224, 1196 cm-1; H NMR (CDCl3) δ
0.89 (t, 3H), 1.00 (dd, 6H), 1.15 (t, 3H), 1.30 (m, 2H), 1.64 (m,
6H), 1.95 (m, 2H), 2.24 (m, 3H), 2.60 (q, 2H), 3.00 (dd, 2H),
3.65 (d, 1H), 4.05 (m, 2H), 4.75 (q, 1H), 5.51 (bs, 1H), 6.35 (bs,
1H), 6.70 (d, 2H), 7.00 (d, 2H), 7.13 (d, 1H); MS (CDI, CH4)
m/ z 521 (MH+). Anal. (C27H40N2O6S) C, H, N.
N-[[1-[[2(S)-[(Meth oxya cetyl)th io]-3-m eth yl-1-oxobu ty-
l]am in o]-1-cyclopen tyl]car bon yl]-O-m eth yl-L-tyr osin e eth -
yl ester (37): white solid (70%); mp 95-100 °C; IR (KBr) 3252,
2962, 1740, 1659, 1639, 1605, 1248, 1148 cm-1 1H NMR
;
(CDCl3) δ 1.00 (d, 3H), 1.04 (d, 3H), 1.20 (t, 3H), 1.70 (m, 4H),
1.93 (m, 3H), 2.35 (m, 2H), 3.00 (dd, 2H), 3.44 (s, 3H), 3.67 (d,
1H), 3.76 (s, 3H), 4.10 (m, 4H), 4.70 (q, 1H), 6.27 (bs, 1H), 6.80
(d, 2H), 7.05 (m, 3H); MS (CDI, CH4) m/ z 523 (MH+). Anal.
(C26H38N2O7S) C, H, N.
N-[[1-[[2(S)-[(P r op ylca r bon yl)th io]-3-m eth yl-1-oxobu -
tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl es-
ter (27): white solid (43%); mp 105 °C; IR (KBr) 3256, 2962,
1
1747, 1647, 1614, 1515, 1222, 1196 cm-1; H NMR (CDCl3) δ
0.90 (m, 12H), 1.25 (m, 2H), 1.60 (m, 8H), 1.86 (m, 2H), 2.17
(m, 3H), 2.51 (t, 2H), 2.96 (dd, 2H), 3.63 (d, 1H), 4.00 (m, 2H),
4.68 (q, 1H), 5.60 (bs, 1H), 6.30 (bs, 1H), 6.65 (d, 2H), 6.92 (d,
2H), 7.10 (d, 1H); MS (CDI, CH4) m/ z 535 (MH+). Anal.
(C28H42N2O6S) C, H, N.
Biologica l Tests. In Vivo An gioten sin -I-Con ver tin g
En zym e In h ibition . The in vivo tests were performed in
male, conscious normotensive Sprague-Dawley rats (275-390
g). The rats were anesthetized with methohexital sodium (75
mg/kg, ip) and instrumented with femoral arterial and venous
catheters for direct blood pressure measurements and iv
administration of compounds, respectively. On the following
day, the pressor responses to three challenges of angiotensin-I
(300 ng/kg, iv) were obtained. Test compounds were then
administered iv or po, and the rats were rechallenged with
angiotensin-I at scheduled times thereafter. All responses
obtained after the administration of test compound were
compared to the average of the initial three responses. Any
observed decrease of the pressor response is an indication of
angiotensin-I-converting enzyme inhibition.
N-[[1-[[2(S)-[(Isop r op ylca r b on yl)t h io]-3-m et h yl-1-ox-
obu tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu tyl
ester (28): white solid (33%); mp 125 °C; IR (KBr) 3404, 3255,
2962, 1747, 1647, 1611, 1515, 1225, 1195 cm-1 1H NMR
;
(CDCl3) δ 0.84 (t, 3H), 0.94 (dd, 6H), 1.12 (m, 6H), 1.28 (m,
2H), 1.63 (m, 6H), 1.86 (m, 2H), 2.17 (m, 3H), 2.73 (sept, 1H),
2.98 (dd, 2H), 3.60 (d, 1H), 4.02 (m, 2H), 4.66 (q, 1H), 5.75 (bs,
1H), 6.35 (bs, 1H), 6.35 (s, 2H), 6.65 (d, 2H), 6.92 (d, 2H), 7.10
(d, 1H); MS (CDI, CH4) m/ z 535 (MH+). Anal. (C28H42N2O6S)
C, H, N.
N-[[1-[[2(S)-[(Cyclop en t ylca r b on yl)t h io]-3-m et h yl-1-
oxobu tyl]a m in o]-1-cyclop en tyl]ca r bon yl]-L-tyr osin e bu -
tyl ester (29): white solid (18%); mp 140 °C; IR (KBr) 3260,
2960, 1746, 1646, 1614, 1615, 1515, 1220, 1194 cm-1; 1H NMR
(CDCl3) δ 0.88 (t, 3H), 0.99 (dd, 6H), 1.26 (m, 3H), 1.70 (m,
16H), 2.21 (m, 3H), 2.98 (dd, 3H), 3.65 (d, 1H), 4.04 (m, 2H),
4.70 (q, 1H), 5.30 (bs, 1H), 6.45 (bs, 1H), 6.72 (d, 2H), 7.00 (d,
In Vivo Neu tr a l En d op ep tid a se In h ibition . Male Spra-
gue-Dawley rats (275-390 g) were anesthetized with ket-
amine (150 mg/kg)/acepromazine (10%) and instrumented with
catheters in the femoral artery and vein to obtain blood
samples and infuse ratANF(99-126), respectively. The rats
were tethered with a swivel system and allowed to recover for
24 h before being studied in the conscious, unrestrained state.

Mercaptoacyl Dipeptides as ACE and NEP Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3167
Plasma ANF levels were determined in the presence and
absence of NEP inhibition. On the day of study, all rats were
infused continuously with ANF at 450 ng/kg/min iv for the
entire 5 h of the experiment. Sixty minutes after beginning
the infusion, blood samples for base-line ANF measurements
were obtained (time 0), and the rats were then randomly
divided into groups to be treated with the test compound or
vehicle. Additional blood samples were taken at 30, 60, 120,
180, and 240 min after administration of the test compound.
Plasma ANF concentrations were determined by a specific
radioimmunoassay. The plasma was diluted (50-, 100-, and
200-fold) in buffer containing 19 mM sodium phosphate
monobasic and 81 mM sodium phosphate dibasic (pH 7.4), 50
mM NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.1% NaN3.
One hundred microliters of standard [rANF(99-126)] or
sample was added to 100 µL of rabbit anti-rANF serum and
incubated at 4 °C for 16 h. Ten thousand cpm of [125I]rANF
were then added to the reaction mixture which was incubated
at 4 °C for an additional 24 h. Goat anti-rabbit IgG serum
coupled to paramagnetic particles was added to the reaction
mixture, and bound [125I]rANF was pelleted by exposing the
mixture to an attracting magnetic rack. The supernatant was
decanted, and the pellets were counted in a gamma counter.
All determinations were performed in duplicate.
Blood P r essu r e Deter m in a tion s. Aor tic-Liga ted Ra t.
Male Sprague-Dawley rats (150-200 g) were housed in
pending cages and given tap water and regular pelleted food
ad libitum. They were maintained in an isolated room with a
constant temperature of 74 °F with cycles of light and darkness
lasting for 12 h. A complete aortic ligation between the renal
arteries was performed according to the method of Rojo-Ortega
and Genest.42 Sham-operated animals underwent a similar
surgical procedure by which the peritoneal cavity was opened
but the aorta remained untouched. After surgery, each animal
received a subcutaneous injection of 200 000 U of wycillin
(sterile penicillin procaine suspension; Wyeth Laboratories
Inc., Philadelphia, PA). The elevation in blood pressure
reached a plateau 12 days after aortic ligation. In this time
period, plasma renin activity was significantly increased.
Direct blood pressure measurements were performed in con-
scious rats, mildly restrained in plastic cylinders, instrumented
with an indwelling catheter (PE-50) implanted in the right
carotid artery and connected to a Gould Statham physiological
pressure transducer. This was coupled to a transducer am-
plifer (Gould, model 13-4615-50), and the blood pressure waves
were displayed on a Gould 2400 S recorder. After a period of
stabilization, the compounds were administered orally, and the
changes in blood pressure were monitored for a 4 h period.
Sp on ta n eou sly Hyp er ten sive Ra t. Male spontaneously
hypertensive rats (SHR), 16-18 weeks of age (Tac: N(SHR)-
fBr), were anesthetized with Amytal (sodium amobarbital, 100
mg/kg ip) and implanted with radiotelemetric devices (Data
Sciences International, Minneapolis, MN) to enable continuous
mean arterial pressure and heart rate determinations in
conscious unrestrained rats. After a 10-14 day recovery
period, the chronic effects of compound 38 were examined and
compared to those of the ACE inhibitor benazepril. Separate
groups of rats received twice daily oral administration (gavage)
of either compound 38 or benazepril at doses of 1, 3, and 10
mg/kg (n ) 7/group). Additional groups of control rats received
an identical volume of 3% cornstarch (1 mL/kg of body weight)
administered twice daily by oral gavage. Rats received either
drug or vehicle at 12 h intervals at 6:00 a.m. and 6:00 p.m.
for 14 consecutive days.
Base-line measurements were determined by monitoring
blood pressure continuously over a 3 day period immediately
prior to drug administration. Comparative hemodynamic
effects of compound 38 and benazepril were then evaluated
over the ensuing 14 days. Blood pressure was recorded during
a 1 week recovery period following the 2 week dosing interval.
Data are reported as the group average for the change in mean
arterial pressure.
data, S. Graybill, M. Beil, T. Gerlock, and L. Odorico
for performing the in vivo ACE and NEP experiments,
and M. Wong for the in vitro ACE and NEP assays.
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J M960323Z