Design and synthesis of potent, selective inhibitors of endothelin- converting enzyme

Article abstract of DOI:10.1021/jm970787c

Endothelin-1 is the most potent peptidic vasoconstrictor discovered to date. The final step of posttranslational processing of this peptide is the conversion of its precursor by endothelin-converting enzyme-1 (ECE-1), a metalloprotease which displays high

Full text of DOI:10.1021/jm970787c

J . Med. Chem. 1998, 41, 1513-1523
1513
Design a n d Syn th esis of P oten t, Selective In h ibitor s of En d oth elin -Con ver tin g
En zym e
Eli M. Wallace,* J ohn A. Moliterni, Michael A. Moskal, Alan D. Neubert, Nicholas Marcopulos,
Lisa B. Stamford, Angelo J . Trapani, Paula Savage, Mary Chou, and Arco Y. J eng
Metabolic and Cardiovascular Diseases Research, Novartis Pharmaceuticals Corporation, 556 Morris Avenue,
Summit, New J ersey 07901
Received November 18, 1997
Endothelin-1 is the most potent peptidic vasoconstrictor discovered to date. The final step of
posttranslational processing of this peptide is the conversion of its precursor by endothelin-
converting enzyme-1 (ECE-1), a metalloprotease which displays high amino acid sequence
identity with neutral endopeptidase 24.11 (NEP) especially at the catalytic center. A series of
potent and selective arylacetylene-containing ECE-1 inhibitors have been prepared. (S,S)-3-
Cyclohexyl-2-[[5-(2,4-difluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]amino]propion-
ic acid (47), an arylacetylene amino phosphonate dipeptide, was found to inhibit ECE-1 and
NEP with IC50 values of 14 nM and 2 µM, respectively. Similarly, (S)-[[1-[(2-biphenyl-4-ylethyl)-
carbamoyl]-4-(2-fluorophenyl)but-3-ynyl]amino]methyl]phosphonic acid (56), an arylacetylene
amino phosphonate amide, had IC50’s of 33 nM and 6.5 µM for ECE-1 and NEP, respectively.
Slight modification of the aryl moiety was found to have dramatic effects on ECE-1/NEP
selectivity. The 2-fluoro dipeptide analogue, (S,S)-2-[[5-(2-fluorophenyl)-2-[(phosphonomethyl)-
amino]pent-4-ynoyl]amino]-4-methylpentanoic acid (40), showed a 72-fold selectivity for ECE-1
over NEP, while the 3-fluoro dipeptide analogue, (S,S)-2-[[5-(3-fluorophenyl)-2-[(phosphonom-
ethyl)amino]pent-4-ynoyl]amino]-4-methylpentanoic acid (22), was equipotent for ECE-1 and
NEP. Several of these inhibitors were shown to be potent in blocking ET-1 production in vivo
as demonstrated by the big ET-1-induced pressor response in rats. These potent inhibitors
are the most selective for ECE-1 reported to date and are envisaged to have a variety of
therapeutic applications.
In tr od u ction
Endothelin-1 (ET-1), a 21-amino acid peptide, is the
most potent vasoconstrictor known.1 Elevated plasma
levels of ET-1 have been found in several disease states
including asthma, hypertension, cerebral vasospasm,
congestive heart failure, and chronic and acute renal
failure.2 For these reasons, blockade of the ET-1 system
could have therapeutic utility in these areas.
ET-1 is biosynthesized from its inactive 38-amino acid
precursor, big ET-1, by specific proteolytic cleavage. The
enzyme responsible for this last step of posttranslational
processing of big ET-1 is a membrane-bound zinc
metalloprotease, endothelin-converting enzyme-1 (ECE-
1) (Scheme 1).3 It is expected that inhibition of ECE-1
will suppress the production of ET-1 and thus its
pathophysiological effects.4
Inhibitors of ECE-1 have been reported, including
phosphoramidon 1 and amino phosphonates 2.5 How-
ever, all of the ECE-1 inhibitors reported to date have
neutral endopeptidase 24.11 (NEP) inhibitory activity
as well.6 The fact that ECE-1 inhibitors are also
inhibitors of NEP is not surprising since NEP is known
to be a membrane-bound zinc metalloprotease and
shares 37% amino acid sequence identity with ECE-1.7
NEP cleaves a variety of substrates including atrial
It is believed that ECE-1 and NEP share a high degree
natriuretic factor (ANF) and bradykinin, a bronchocon-
of homology in their active sites.3,8
strictor.9,10 ANF is a 28-amino acid peptide whose
biological effects include sodium excretion and vasore-
* Address for correspondence: Department of Medicinal Chemistry,
Glaxo Wellcome Research and Development, Five Moore Dr., Research
laxation.11 Potentiation of ANF levels has been an
Triangle Park, NC 27709.
active area of research for treatment of hypertension
S0022-2623(97)00787-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/08/1998

1514 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Wallace et al.
Sch em e 1. Proteolytic Cleavage of Big ET-1 to ET-1 by ECE-1
Sch em e 2. Arylacetylene Amino Phosphonates
and congestive heart failure. Inhibition of NEP has
been one of the major targets in raising ANF levels as
such inhibitors have shown similar effects to ANF
infusion in animal models.12 In addition, ECE-1/NEP
inhibition, which is believed to combine elevating ANF
levels with lowering of ET-1 levels, has shown positive
effects in several animal models of vascular patho-
physiology.4,5b,c,13
While little progress has been made in the design and
development of selective ECE-1 inhibitors, there re-
mains a need for such agents. Selective ECE-1 inhibi-
tors have potential application in respiratory diseases
since treatment with a dual ECE-1/NEP inhibitor is
likely to increase the levels of bradykinin. Also, ET-1
has been shown to be a substrate for NEP,14 so that
inhibitors of NEP may raise ET-1 levels. Finally,
without selective ECE-1 inhibitors, the involvement of
overproduction of ET-1 in various disease states could
never be definitively validated. All in vivo studies on
dual inhibitors would be complicated by the fact that
the observed response could be due to ECE-1 or NEP
inhibition, or both. For these reasons and the potential
therapeutic benefit of blocking ET-1 biosynthesis, re-
search efforts were focused on the design of potent and
selective ECE-1 inhibitors.
Screens of NEP inhibitors for ECE-1 activity identi-
fied CGS 26303, a potent nonpeptidic NEP inhibitor
with moderate ECE-1 inhibitory activity.5a Previous
studies on optimization of the ECE-1 inhibitory activity
of CGS 26303 have shown that alterations to the core
of CGS 26303 result in significantly weaker inhibitors.
These studies also showed that a dramatic increase in
ECE-1 inhibitory activity, while maintaining potent
NEP inhibition, can be achieved by introduction of
arylethyl side chains at the P1 binding subsite (see CGS
31447).5d However, these ECE-1 inhibitors with P1 side
chains lack the desired in vivo profile. Subsequent
optimization of the P1′ binding subsite of CGS 31447
has implied that selectivity for ECE-1 over NEP could
be obtained.5f Recent efforts have focused on decreasing
NEP inhibition while maintaining potent ECE-1 inhibi-
tion. In the search for selective inhibitors, arylacety-
lenes were investigated as potential novel biphenyl
surrogates (Scheme 2).
cemic derivatives 14 and 15 is outlined in Scheme 3.
(S)-2-[(tert-Butoxycarbonyl)amino]pent-4-ynoic acid (5)
was prepared by enzymatic resolution of 3 with alcalase.
This gave 96% yield (based on 50% recovery) of (S)-N-
Boc-propargylglycine (5) having 86% ee which could be
crystallized with (R)-(-)-methylbenzylamine to optical
purity. Amide formation was followed by Pd(0)-cata-
lyzed cross-coupling with 2-iodoanisole to give the
arylacetylene amino amide 7.16 Amide 7 was then
converted to the tetrazole amino phosphonate 15 with
slight modifications of previously reported proce-
dures.5a,12d The Mosher amide17 derivative 17 was
prepared and compared to the Mosher amide of the
racemate to show that the Pd(0)-catalyzed cross-
coupling reaction proceeded without epimerization of the
asymmetric center (Scheme 3).
The leucine dipeptides 22-25 were synthesized from
(S)-N-Boc-propargylglycine (5) using standard methods
as outlined in Scheme 4.
2-Fluoro- and 2,4-difluoroarylacetylene amino phos-
phonate amides, dipeptides, and tripeptides 38-69 were
prepared by the procedure outlined in Scheme 5. Ra-
cemic 2-[(tert-butoxycarbonyl)amino]pent-4-ynoic acid
methyl ester (3)15 was subjected to Pd(0) cross-coupling
with 2,4-difluoroiodobenzene to afford the arylacetylene
amino ester in 85% yield.16 Enzymatic resolution with
alcalase gave the (S)-amino acid 27 in 96% yield (based
on 50%) along with the (R)-amino ester 28. The
enantiomeric excess of 27 was determined to be 96%
by examination and comparison of the derived Mosher
amides 29 and 30 by 19F NMR. Removal of the BOC
group and alkylation of the primary amine as previously
described5a,12d were followed by selective hydroylsis of
the ester with NaOH to give the dimethyl phosphonate
carboxylic acid 36. Coupling of acid 36 with (L)-leucine
methyl ester hydrochloride was accomplished with BOP
reagent. Deprotection yielded the arylacetylene amino
phosphonate 41.
Ch em istr y
Target compounds 12-15 could be prepared from
racemic propargylglycine which was prepared as its
2-tert-butoxycarbonylamino methyl ester 3 following
literature procedures.15 Alternatively, both propargyl-
glycine and N-acetylpropargylglycine are commerically
available in achiral form. The route for chiral, nonra-

Potent, Selective Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1515
Sch em e 3. Synthesis of Tetrazole Analoguesa
a
(a) Alcalase, 0.2% NaHCO3, MeCN; (b) 3-aminopropionitrile, EDCI, HOBt, Et3N, DMF; (c) PdCl2(dppf), CuI, ArI, Et3N, DMF; (d)
TMSN3, Ph3P, DIAD, MeCN; (e) HCO2H; (f) (CH2O)n, EtOAc; (PhO)2POH, PhMe; (g) DBU, MeCN; (h) NaHCO3(aq), MeCN; (i) (S)-(-)-
R-methoxy-R-(trifluoromethyl)phenylacetyl chloride, Et3N, CH2Cl2.
Sch em e 4. Synthesis of Leucine Dipeptide Analoguesa
a
(a) L-Leucine methyl ester hydrochloride, EDCI, HOBt, Et3N, DMF; (b) PdCl2(dppf), CuI, ArI, Et3N, DMF; (c) HCO2H.
Resu lts
In Vitr o Activity. In the search for more potent and
Interestingly, the o-methoxy derivative 15 was a poor
ECE-1 inhibitor. Finally, arylacetylene seemed to be
of optimal length in the S1′ binding pocket of ECE-1, as
the tolyl derivative 13 resulted in a dramatic loss of
activity.
In search of greater ECE-1 selectivity, attention was
turned to replacement of the tetrazole moiety. Prepara-
tion of the carboxylic acid 38 led to a selective ECE-1
inhibitor (Table 2). A slight reduction in ECE-1 inhibi-
tion was observed when compared to 14. However, NEP
activity was decreased dramatically from 14 nM for 14
to 11 µM for 38. The 2,4-difluoroarylacetylene carboxy-
late 39 was also a selective ECE-1 inhibitor (Table 2).
selective ECE-1 inhibitors, arylacetylene analogues of
CGS 26303 were investigated. Direct replacement of
the P1′ biphenyl moiety of CGS 26303 with phenylacety-
lene 12 led to a nearly equipotent ECE-1 inhibitor and
a weaker NEP inhibitor (see Table 1). This inhibitor
was the first nonbiaryl amino phosphonate to show
potent ECE-1 inhibition.5c In addition, while 12 was
still more potent for NEP than ECE-1, it was 85-fold
less potent than CGS 26303 for NEP. Encouraged by
these results, we focused our efforts on optimization of
arylacetylene amino phosphonates.
Examination of the aryl ring of 12 was undertaken
first (Table 1). This study showed that placement of
an o-fluoro substituent on the aryl ring resulted in an
increase in ECE-1 inhibitory activity (see 14). A similar
increase in NEP inhibitory activity was also observed.
Biaryl dipeptide amino phosphonates were shown to
be potent NEP inhibitors, but they have not been shown
to inhibit ECE-1.12d Dipeptides of the arylacetylene
amino phosphonates were examined to see if they
behaved similarly to the biaryl series. Several dipep-

1516 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Wallace et al.
Sch em e 5. Synthesis of Amide, Dipeptide, and Tripeptide Analoguesa
a
(a) PdCl2(dppf), CuI, ArI, Et3N, DMF; (b) alcalase, 0.2% NaHCO3, MeCN; (c) TMSCHN2; (d) HCO2H; (e) (R)-(-)-R-methoxy-R-
(trifluoromethyl)phenylacetyl chloride, Et3N; (f) (MeO)2OPCH2OSO2CF3, DIPEA; (g) 1 N NaOH; (h) L-leucine methyl ester hydrochloride,
BOP, NMM, DMF; (i) 1 N NaOH; TMSBr, DIPEA, CH2Cl2.
Ta ble 1. Tetrazole Arylacetylene Amino Phosphonatesa
derivative 40. This illustrated that a minute structural
modification could have a dramatic effect on potency and
selectivity. The unsubstituted analogue 23 fell between
40 and 22 with IC50 values of 510 and 948 nM for ECE-1
and NEP, respectively. Interestingly, the 2-chloro and
2-methoxy derivatives 24 and 25 were not tolerated by
either enzyme.
Good potency and selectivity were also obtained with
a series of amides of arylacetylene amino phosphonates
IC50 (nM)
IC50 NEP/
compd
Ar
*
rhECE-1
NEP
IC50 ECE
(Table 4). The phenethyl and tryptamine derivatives
52-55 showed potent inhibitory activity and selectivity
(IC50 values for ECE-1 inhibition ranged from 76 to 230
nM, while selectivity for ECE-1 over NEP ranged from
83- to 136-fold). The biphenethyl amides showed an
increase in ECE-1 inhibitory activity as compared to the
phenethyl derivatives (compare 56 and 57 to 54 and 55).
Both 1- and 2-naphthylethyl amides were well-tolerated
(62 and 63), indicating a large lipophillic binding pocket.
Interestingly, in the 1-naphthylethyl series, the 2,4-
difluoro derivative 62 was found to be almost 4-fold
more potent than the 2-fluoro analogue 61 in ECE-1
inhibition (34 and 130 nM, respectively). While the
arylethyl amides seemed to be optimal for ECE-1
inhibitory activity, the arylpropyl derivative 58 was still
significantly active, but the arylbutyl 59 was inactive
(IC50 of 150 nM and 0% inhibition at 1 µM, respectively).
The arylpropyl amide 58 was much more potent in NEP
inhibition than the arylethyl amide derivative 55 (IC50
values of 640 and 15 000 nM, respectively). This
differential inhibition of ECE-1 and NEP was significant
and has yet to be fully explored. Finally, the complete
loss of activity of tertiary amide 60 shows the impor-
tance of the hydrogen-bonding role of the secondary
amide 55 in the putative ECE-1 active site.
12
13
14
15
Ph
R,S 590 ( 170
R,S 45% at 1 µM ND
170 ( 7
0.3
ND
0.1
ND
4-Me-C6H4
2-F-C6H4
2-MeO-C6H4
S
S
150 ( 6
0% at 1 µM
14 ( 1
ND
a
The IC50 values are expressed in mean ( SEM (n ) 3). rhECE-
1, recombinant human ECE-1.
tides were prepared and found to be potent and selective
ECE-1 inhibitors (Table 2). For example, leucine de-
rivative 48 was found to be a highly potent and selective
ECE-1 inhibitor (IC50 of 28 nM for ECE-1 and 6.3 µM
for NEP), as were derivatives 41 and 47. In addition
to dipeptides, two tripeptides were prepared (Table 2).
These were also selective ECE-1 inhibitors. For ex-
ample, 49 exhibited excellent potency and selectivity
with IC50 values of 8 and 5800 nM for ECE-1 and NEP,
respectively.
A study of the leucine arylacetylene amino phospho-
nate dipeptides showed the sensitivity of ECE-1 and
NEP to aryl substitution (Table 3). The 2-fluoro and
2,4-difluoro derivatives 40 and 41 were shown to be
potent and selective inhibitors of ECE-1. Preparation
of the 3-fluoro derivative 22 led to a 3-fold weaker
ECE-1 inhibitor. Strikingly, the 3-fluoro derivative 22
was 21-fold more active against NEP than the 2-fluoro

Potent, Selective Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1517
Ta ble 2. Carboxylic and Peptidic Arylacetylene Amino Phosphonatesa
IC50 (nM)
compd
X
R
rhECE-1
NEP
IC50 NEP/IC50 ECE
38
39
40
41
42
43
44
45
OH
OH
H
F
H
F
H
H
H
H
240 ( 42
170 ( 48
120 ( 3
51 ( 1
11000 ( 280
26000 ( 160
8600 ( 150
6700 ( 72
4060 ( 130
6300 ( 210
1200 ( 50
250 ( 7
46
153
72
131
18
57
21
19
Leu-OH
Leu-OH
Met-OH
Val-OH
Phe-OH
230 ( 35
110 ( 15
56 ( 5
13 ( 1
46
47
48
H
F
25 ( 4
14 ( 2
28 ( 1
1300 ( 67
2070 ( 13
6300 ( 210
52
148
225
H
49
50
Leu Ala-OH
F
F
8 ( 2
82 ( 8
5800 ( 190
730 ( 15
725
9
a
The IC50 values are expressed in mean ( SEM (n ) 3).
Ta ble 3. Leucine Arylacetylene Amino Phosphonate
of the arylacetylene derivatives is that the binding of
these inhibitors in the P1′ subsite alters the overall
binding mode of the inhibitor so that the enzyme will
tolerate acids, amides, and peptides in the P2′ subsite.
This effect of the P1′ subsite of ECE-1 is enhanced by
the 2-fluorophenyl derivative as seen in Table 3.
Dipeptidesa
No ECE-1/NEP selectivity was observed for the tet-
razole analogues 12 and 14 (Table 1). Perhaps the P2′
tetrazole has a stronger influence on the binding mode,
and this effect overshadows any effect the arylacetylenes
may have in the P1′ binding subsite. A definative
explanation and understanding awaits further struc-
tural information on the ECE-1 and NEP active sites.
IC50 (nM)
IC50 NEP/
IC50 ECE
compd
Ar
rhECE-1
NEP
40
41
22
23
24
25
2-F-C6H4
120 ( 3
8600 ( 150
6700 ( 72
407 ( 11
948 ( 31
b
72
2,4-diF-C6H4 51 ( 1
131
1.0
1.9
ND
ND
3-F-C6H4
Ph
400 ( 22
510 ( 54
In Vivo Activity. The ability of selected compounds
to inhibit the increase in mean arterial pressure (MAP)
produced by big ET-1 in vivo is shown in Figure 1.
Intravenous bolus injection of 1 nmol/kg big ET-1 in
anesthetized, ganglion-blocked rats treated with vehicle
resulted in an 86 ( 4 mmHg rise in MAP. Pretreatment
of the animals with compound 14,18 48, 41, or 49, at 10
mg/kg iv, was associated with an 85 ( 4%, 56 ( 4%, 51
( 11%, and 64 ( 3% inhibition of the big ET-1 pressor
response, respectively. By comparison, the administra-
tion of phosphoramidon at 15 and 30 mg/kg iv caused a
59 ( 9% and 79 ( 2% inhibition of the response to big
ET-1 (data not shown).
2-Cl-C6H4
2-MeO-C6H4
4300 ( 380
0% at 1 µM
c
a
b
The IC50 values are expressed in mean ( SEM (n ) 3). 38%
at 10 µM. c 12% at 10 µM.
From these studies, it appears that the selectivity
between ECE-1 and NEP is a function of both the P1′
and P2′ binding subsites. Interestingly, P1′ biaryl acids,
amides, and peptides do not inhibit ECE-1,5d,12d whereas
the arylacetylene analogues (Tables 2 and 4) were found
to be potent and selective ECE-1 inhibitors suggesting
that a combination of the P1′ and P2′ binding sites may
play a role. One explanation for the observed selectivity

1518 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Ta ble 4. Amide Arylacetylene Amino Phosphonatesa
Wallace et al.
IC50 (nM)
IC50 NEP/
IC50 ECE
compd
X
R
rhECE-1
NEP
51
52
53
54
55
56
57
58
59
60
CH2Ph
H
H
F
H
F
H
F
250 ( 85
76 ( 6
111 ( 17
230 ( 58
110 ( 3
33 ( 8
28 ( 4
150 ( 23
0% at 1 µM
3% at 1 µM
55000 ( 3800
10000 ( 120
9700 ( 120
19000 ( 720
15000 ( 100
6500 ( 110
2500 ( 32
640 ( 4
220
132
87
tryptamine
tryptamine
(CH2)2Ph
(CH2)2Ph
(CH2)2PhPh
(CH2)2PhPh
(CH2)3Ph
83
136
197
89
4
ND
ND
F
H
(CH2)4Ph
ND
ND
61
62
63
64
65
66
67
68
69
(CH2)2-1-Nap
(CH2)2-1-Nap
(CH2)2-2-Nap
(CH2)2CHMe2
(CH2)2pBr-C6H4
(CH2)2pBr-C6H4
(CH2)2(3,4-diMeOC6H3)
(CH2)2-2-pyridinyl
H
F
F
F
H
F
F
F
130 ( 3
34 ( 8
10700 ( 58
6200 ( 310
7000 ( 95
23000 ( 88
40000 ( 1200
860 ( 7
b
82
182
132
164
364
20
ND
32
ND
53 ( 9
140 ( 34
110 ( 12
43 ( 8
160 ( 43
310 ( 41
8% at 1 µM
10000
ND
a
b
The IC50 values are expressed in mean ( SEM (n ) 3). 21% at 10 µM.
F igu r e 2. Changes in MAP produced by big ET-1 (1 nmol/kg
iv) at 30 and 120 min after oral administration of 70 at 30
mgequiv/kg or vehicle (PEG 400, 1 mL/kg) to conscious rats.
Values are the mean ( SEM for 4-8 animals.
F igu r e 1. Maximum increases in MAP produced by big ET-1
in anesthetized, ganglion-blocked rats. Rats were treated with
vehicle (V, 0.05 N NaOH at 1 mL/kg iv) or compound 14, 48,
41, or 49 at 10 mg/kg iv and challenged with 1 nmol/kg iv big
ET-1 15 min later. Values are the mean ( SEM of 15, 4, 4, 6,
and 2 animals for the vehicle, 14, 48, 41, and 49 groups,
respectively.
65% reduction of the pressor effect of big ET-1 at 30
and 120 min after dosing.
The effect of compound 70, the diphenyl phosphonate
prodrug of 14, on the big ET-1 pressor response in
conscious rats is shown in Figure 2. Treatment with
70 at 30 mgequiv/kg po was associated with a 63% and
Con clu sion s
Arylacetylenes were found to be excellent surrogates
for biphenyl as ECE-1 inhibitors. Within the tetrazole

Potent, Selective Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1519
New England Nuclear, Boston, MA) and 1:20000-fold diluted
rabbit antibodies that recognize specifically the carboxyl
terminal tryptophan of ET-1. Goat anit-rabbit antibodies
coupled to magnetic beads (70 µg) were then added to each
tube, and the reaction mixture was further incubated for 30
min at room temperature. The beads were pelleted using a
magnetic rack. The supernatant was decanted, and the
radioactivity in the pellet was counted in a gamma counter.
Total and nonspecific binding were measured in the absence
of nonradioactive ET-1 and anit-ET antibodies, respectively.
Under these conditions, ET-1 and big ET-1 displaced [125I]ET-
1 binding to the antibodies with IC50 values of 21 ( 2 and
260 000 ( 66 000 fmol (mean ) SEM, n ) 3-5), respectively.
series, phenylacetylene amino phosphonate 12 (Table
1) was found to be as active against ECE-1 as the
biphenyl analogue CGS 26303. Amino phosphonate
arylacetylene di- and tripeptides and amides exhibited
high potency and selectivity as ECE-1 inhibitors (Tables
2 and 4). These derivatives were shown to be the most
selective ECE-1 inhibitors reported to date. For ex-
ample, dipeptide 47 had an IC50 of 14 nM for ECE-1
and an IC50 of 2.0 µM for NEP. Amide 56 had an IC50
of 33 nM for ECE-1 and an IC50 of 6.5 µM for NEP. In
contrast, phosphoramidon, the most widely used ECE-1
inhibitor, had IC50 values of 1.2 µM and 30 nM for
ECE-1 and NEP inhibition, respectively. In addition,
2-fluoroarylacetylenes showed an increase in ECE-1
inhibitory activity compared to phenylacetylene (see
Table 1, derivatives 12 and 14). Interestingly, the
2-fluoro substituent appears to play a critical role in
NEP selectivity as seen in dipeptide derivatives 40 and
22 (Table 3). The role of the 2-fluoro substituent in
increased potency and selectivity for ECE-1 inhibition
is not clear at this time nor is the origin of the selectivity
of these inhibitors. One explanation is that the binding
of these inhibitors in the P1′ subsite alters the overall
binding mode of the inhibitor (vide supra). Further
study, including three-dimensional structural informa-
tion on the ECE-1 and NEP active sites, is needed to
elucidate these effects. Finally, these inhibitors have
shown the ability to block ET-1 production in vivo as
demostrated by the big ET-1-induced pressor response
(Figures 1 and 2). These selective ECE-1 inhibitors
could find broad application in vascular biology.
3. Da ta An a lysis: Duplicate samples were carried out in
each experiment. To determine IC50 value of an inhibitor, the
full concentration-response curve of each inhibitor was per-
formed at least three times. A nonlinear least-squares curve-
fitting program was used to fit data to a one-site model.
NEP assay was performed as previously described.12d
Big ET-1 P r essor Test. Male Sprague-Dawley rats were
anesthetized with Inactin (100 mg/kg ip) and instrumented
with catheters in the femoral artery and vein to record mean
arterial pressure (MAP) and adminster compounds, respec-
tively. A tracheostomy was performed and a cannula inserted
into the trachea to ensure airway patency. The body temper-
ature of the animals was maintained at 37 ( 1 °C by means
of a heating blanket. Following surgery, MAP was allowed to
stabilize for 30 min before interrupting autonomic neurotrans-
mission with chlorisondamine (3 mg/kg iv) and challenging
with big ET-1 (1 nmol/kg iv) 15 min later. The data are
reported as the maximum increase in MAP produced by the
big ET-1 injection.
Compound 70, a prodrug of 14, was also evaluated following
oral administration in conscious rats. To perform this study,
male Spraque-Dawley rats were anesthetized with metho-
hexital sodium (75 mg/kg ip) and instrumented with catheters
in the femoral artery and vein to measure MAP and administer
big ET-1, respectively. The catheters were then threaded
through a swivel system which enabled the animals to move
freely after regaining consciousness. On the following day,
MAP was recorded via the femoral artery catheter and
compound at 30 mg/kg or vehicle (PEG 400, 1 mL/kg) was
administered po. The animals were challenged with big ET-1
(1 nmol/kg iv) at 30 and 120 min after dosing. The changes
in MAP produced by big ET-1 were reported at 2-min intervals.
Exp er im en ta l Section
Melting points (mp) were determined on either a Thomas-
Hoover or Mel-TempII melting point apparatus and are
uncorrected. Infrared (IR) spectra were recorded on a Nicolet
5SXB FTIR spectrometer. 1H NMR spectra were recorded on
either a Bruker AC-250 or Bruker AC-300 spectrometer using
CDCl3, D2O, or DMSO-d6 as internal standard. When needed,
trifluoroacetic acid (a few drops) was used to solubilize amino
phosphonic acids in DMSO-d6. 31P NMR spectra were recorded
on a Bruker AC-300 spectrometer. 31P chemical shifts are
reported relative to 85% aqueous phosphoric acid as external
standard. 19F NMR spectra were recorded on a Bruker ARX-
500 instrument. Optical rotations were measured with a J asco
DIP-370 instrument. Mass spectra were obtained on a Mi-
croMass Platform II spectrometer. Microanalyses were per-
formed at Robertson Laboratory, Inc., Madison, NJ . All
organic solvents used were of anhydrous grade. Chromato-
graphic separations were performed on either silica gel 60, Diol
Gel, or reverse-phase MCI gel under N2 pressure. Some
chromatographic separations were performed on a Biotage
Flash40 apparatus with silica gel. Alcalase (food grade) was
aquired from Novo Nordisk (Franklinton, NC).
En zym e Assa ys. 1. ECE-1 Assa y: To assess the effect of
an inhibitor on ECE-1 activity, 10 µg of protein of membrane
preparation of CHO cells expressing human ECE-1 was
preincubated with the compound at a desired concentration
for 20 min at room temperature in 50 mM Tes, pH 7.0, and
0.005% Triton X-100 in a volume of 10 µL. Human big ET-1
(5 µL) was then added to a final concentration of 0.2 µM, and
the reaction mixture was further incubated for 2 h at 37 °C.
The reaction was stopped by adding 500 µL of radioimmu-
noassay (RIA) buffer containing 0.1% Triton X-100, 0.2%
bovine serum albumin, and 0.02% NaN3 in phosphate-buffered
saline.
Ch em istr y. Derivatives 12-15 could be prepared from
either D,L- or L-N-(tert-butoxycarbonyl)propargylglycine. De-
rivative 15 is representative.
(S)-2-[(ter t-Bu toxyca r bon yl)a m in o]p en t-4-yn oic Acid
(5). To a stirred solution of 2-[(tert-butoxycarbonyl)amino]-
pent-4-ynoic acid methyl ester (3)15 (11.1 g, 48.9 mmol) in CH3-
CN (165 mL) and 0.2 M NaHCO3 (325 mL) was added alcalase
(2.2 mL). The mixture was placed in a preheated oil bath at
40 °C and stirred for 2 h. The reaction mixture was cooled to
room temperature, Et2O was added, and the mixture stirred
for several minutes. The layers were separated, and the
aqueous layer was extracted several times with Et2O. The
combined ether extracts were dried over MgSO4, filtered, and
concentrated to give the (R)-amino ester 4 as an orange oil
(5.54 g): 1H NMR (CDCl3, 300 MHz) δ 5.38-5.35 (1H, bm),
4.48-4.42 (1H, m), 3.75 (3H, s), 2.72-2.70 (2H, m), 2.07-2.02
(1H, m), 1.43 (9H, s). The aqueous layer was acidified to pH
2-3 with 1 N HCl; then some of the water was evaporated in
vacuo. The residue was extracted several times with EtOAc.
The combined organic extracts were dried over MgSO4, filtered,
and concentrated to give 5.01 g (96%) of the (S)-amino acid 5:
1
IR (KBr) 1743, 1679, 1523, 1209 cm-1; H NMR (CDCl3, 250
MHz) δ 5.35-5.30 (1H, bd), 4.55-4.40 (1H, m), 2.8-2.7 (2H,
m), 2.05-2.00 (1H, m), 1.40 (9H, s); [R]25 +20.2 (c, 12.4 mg/
D
mL MeOH) (lit.15 [R]25D +23.5 (c, 9.1 mg/mL MeOH) (86% ee));
MS (ES-) m/z 212 (M - H). Optically pure 5 was obtained by
crystallization with (R)-(+)-R-methylbenzylamine. (R)-(+)-R-
Methylbenzylamine (4.36 g, 20.47 mmol) was added to a stirred
solution of acid 5 (86% ee) in Et2O (100 mL) which resulted in
2. Qu a n tita tion of ET-1: Diluted samples (200 µL)
obtained from the above enzyme assay were incubated at 4
°C overnight with 25 µL each of [125I]ET-1 (10 000 cpm/tube;

1520 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Wallace et al.
a white precipitate. The reaction mixture was heated to 40
°C in a water bath, and absolute EtOH (∼150 mL) was added
until a clear solution resulted. The reaction mixture was
cooled to room temperature and allowed to stand for 16 h. The
resultant crystalline needles were collected by filtration and
washed with Et2O. After drying in vacuo, the crystals were
suspended in Et2O and washed with 1 N HCl and brine. The
clear organic layer was dried (MgSO4) and concentrated to give
3.42 g (78%) of (S)-amino acid 5 as an oil: [R]25D +22.2 (c, 12.4
(1H, m), 4.81 (2H, t, J ) 7.5 Hz), 3.88 (3H, s), 3.31-3.00 (4H,
m), 0.91 (9H, s); MS (ES+) m/z 397 (M + H).
(S)-3-[5-[1-Am in o-4-(2-m eth oxyp h en yl)bu t-3-yn yl]tet-
r a zol-1-yl]p r op ion itr ile (9). A solution of tetrazole 8 (1.11
g, 2.80 mmol) in formic acid (7 mL) was heated at 40 °C for 5
h. The reaction was cooled to room temperature and concen-
trated to near dryness. The residue was taken up in EtOAc
and slowly made slightly basic (pH 8) with saturated NaHCO3.
The layers were separated, and the aqueous layer was
extracted twice with EtOAc. The combined organic extracts
were washed once with brine, dried over MgSO4, filtered, and
concentrated to give 0.80 g (97%) of the amine 9 as a near-
colorless oil which was carried forward without purification:
1H NMR (CDCl3, 300 MHz) δ 7.43 (2H, t, J ) 6.8 Hz), 6.95-
6.87 (2H, m), 4.95 (2H, ddt, J ) 36.0, 13.5, 7.5 Hz), 4.57 (1H,
t, J ) 7.5 Hz), 3.87 (3H, s), 3.22 (2H, dd, J ) 9.8, 3.0 Hz), 3.12
(2H, t, J ) 9.8 Hz), 2.00 (2H, bs).
mg/mL MeOH) (lit.15 [R]25 +23.5 (c, 9.1 mg/mL MeOH) (95%
D
ee)).
(S)-[1-[(2-Cya n oet h yl)ca r b a m oyl]b u t -3-yn yl]ca r b a m -
ic Acid ter t-Bu tyl Ester (6). To a stirred solution of acid 5
(3.21 g, 15.07 mmol) in DMF (50 mL) at room temperature
under N2 atmosphere was added HOBt (2.24 g, 16.58 mmol)
followed by triethylamine (2.73 mL, 19.59 mmol). The solution
was stirred for 10 min before 3-aminopropionitrile (1.11 mL,
15.07 mmol) was added. After the mixture stirred for 5 min,
EDCI (3.76 g, 19.59 mmol) was added. The reaction mixture
was stirred for 16 h and then partitioned between EtOAc and
H2O. The layers were separated, and the aqueous layer was
extracted with EtOAc. The combined organic extracts were
washed with H2O, 1 N HCl, saturated NaHCO3, and brine.
The organic layer was dried over MgSO4, filtered, and con-
centrated in vacuo to give 3.42 g (86%) of amide 6 as a white
solid: 1H NMR (CDCl3, 300 MHz) δ 7.00 (1H, bt, J ) 7.5 Hz),
5.30 (1H, bd, J ) 9.0 Hz), 4.32 (1H, bs), 3.57 (2H, dt, J ) 13.5,
6.8 Hz), 2.82-2.55 (4H, m), 2.14 (1H, t, J ) 3.8 Hz), 1.46 (9H,
s).
(S)-[[[1-[1-(2-Cya n oeth yl)-1H-tetr a zol-5-yl]-4-(2-m eth -
oxyp h en yl)bu t -3-yn yl]a m in o]m et h yl]p h osp h on ic Acid
Dip h en yl Ester (10). Phosphonate 10 was prepared as
described previously.5b,12d To a solution of amine 9 (0.80 g,
2.70 mmol) in EtOAc (15 mL) at 0 °C was added a 37% solution
of formaldehyde in H2O (0.26 mL, 9.49 mmol). After 1 h, the
reaction mixture was warmed to room temperature and stirred
for an additional 16 h. The reaction mixture was diluted with
EtOAc and washed with cold H2O and cold brine. The organic
layer was dried (MgSO4), filtered, and concentrated to give a
white foam. To a solution of this foam in 3:1 PhMe-THF (20
mL) was added diphenyl phosphite (0.93 mL, 4.86 mmol;
contains 10-15% phenol). The reaction mixture was heated
to 65 °C with stirring for 1.5 h. Cooling, concentration, and
purification by flash column chromatography gave 1.14 g (78%)
of amino phosphonate 10 as a white solid: IR (thin film) 2256,
(S)-[1-[(2-Cya n oet h yl)ca r b a m oyl]-4-(2-m et h oxyp h en -
yl)bu t-3-yn yl]ca r ba m ic Acid ter t-Bu tyl Ester (7). The
method of Robertson and Crisp was employed.16 To a solution
of amide 6 (1.57 g, 5.92 mmol) in DMF (30 mL) was added
PdCl2(dppf) (0.24 g, 0.30 mmol), followed by 2-iodoanisole (0.85
mL, 6.52 mmol), triethylamine (1.65 mL, 11.84 mmol), and
CuI (0.23 g, 1.18 mmol). The reaction mixture was degassed
with house vacuum, placed under N2 atmosphere, and stirred
for 0.5 h. The reaction mixture was partitioned between
EtOAc and H2O. The layers were separated, and the aqueous
layer was extracted several times with EtOAc. The combined
organic extracts were washed with H2O and brine, dried over
MgSO4, filtered, and concentrated in vacuo. Purification by
flash column chromatography (SiO2, 1:1 hexanes-EtOAc) gave
1.38 g (63%) of arylacetylene 7 as a white foam: IR (thin film)
1601, 1260, 1186, 935 cm-1 1H NMR (CDCl3, 300 MHz) δ
;
7.38-7.23 (8H, m), 7.23-7.12 (4H, m), 7.10 (2H, d, J ) 9.0
Hz), 6.92-6.81 (2H, m), 4.89-4.68 (3H, m), 3.82 (3H, s), 3.61-
3.47 (1H, m), 3.40-2.92 (5H, m), 2.62 (1H, m); 31P NMR
(CDCl3, 125 MHz) δ 17.78; MS (ES+) m/z 543 (M + H).
(S)-[[[4-(2-Met h oxyp h en yl)-1-(1H -t et r a zol-5-yl)b u t -3-
yn yl]a m in o]m et h yl]p h osp h on ic Acid Dip h en yl E st er
(11). Tetrazole 11 was prepared as described previously.5a,b
To a solution of tetrazole 10 (0.71 g, 1.31 mmol) in CH2Cl2 (10
mL) was added DBU (0.59 mL, 3.93 mmol). After 2 h, the
reaction mixture was diluted with EtOAc and washed with 1
N HCl and brine. The organic layer was dried (MgSO4),
filtered, and concentrated. Purification by flash column chro-
matography on DIOL gel (eluent: 1:1 hexanes-EtOAc) gave
0.47 g (73%) of tetrazole 11 as a foamy white solid: IR (thin
3300, 2250, 1715, 1669, 1498, 1269, 1142 cm-1 1H NMR
;
(CDCl3, 300 MHz) δ 7.40-7.31 (2H, m), 7.04 (1H, bs), 6.95-
6.89 (2H, m), 5.71 (1H, bs), 4.41 (1H, bs), 3.93 (3H, s), 3.60-
3.51 (2H, m), 3.12 (1H, dd, J ) 16.5, 6.0 Hz), 2.79 (1H, dd, J
) 16.5, 7.5 Hz), 2.62-2.56 (2H, m), 1.48 (9H, s); MS (ES+)
m/z 372 (M + H), 272 (M + H - Boc).
1
film) 1589, 1490, 1263, 1189, 943 cm-1; H NMR (CDCl3, 300
MHz) δ 7.43-7.10 (12H, m), 7.02-6.92 (2H, m), 4.61 (1H, t, J
) 3.8 Hz), 4.05 (3H, s), 3.51-3.29 (2H, m), 3.09 (2H, dq, J )
16.5, 6.8 Hz); 31P NMR (CDCl3, 125 MHz) δ 16.68; MS (ES+)
m/z 490 (M + H).
(S)-[1-[1-(2-Cya n oet h yl)-1H -t et r a zol-5-yl]-4-(2-m et h -
oxyp h en yl)bu t-3-yn yl]ca r ba m ic Acid ter t-Bu tyl Ester (8).
The method of Duncia et al. was employed with minor
modification.19 To a solution of amide 7 in MeCN (15 mL) was
added triphenylphosphine (2.26 g, 8.63 mmol). The reaction
mixture was gently heated with a heat gun until a clear
solution resulted. Upon cooling to 0 °C, a precipitate formed.
Diisopropyl azodicarboxylate (1.70 mL, 8.63 mmol) and TMSN3
(1.15 mL, 8.63 mmol) were added dropwise by syringe,
alternating 10 drops at a time, starting with diisopropyl
azodicarboxylate. During addition reaction became a clear-
yellow solution. Within minutes of completion of addition, an
off-white precipitate formed. Reaction mixture was warmed
to room temperature and stirred for 1 h at 40 °C and then at
room temperature for 16 h. The reaction mixture was diluted
with EtOAc and water, the layers were separated, and the
organic layer was washed with saturated NaHCO3 solution
and brine. The organic layer was dried (MgSO4), filtered, and
concentrated. Purification by flash column chromatography
(eluent: 60:40 hexanes-EtOAc) gave 1.18 g (86%) of tetrazole
8 as a white solid: IR (thin film) 2254, 1701, 1600, 1494, 1264,
(S)-[[[4-(2-Met h oxyp h en yl)-1-(1H -t et r a zol-5-yl)b u t -3-
yn yl]a m in o]m eth yl]p h osp h on ic Acid (15). Phosphonic
acid 15 was prepared from phosphonate 11 as previously
described.5b To a solution of phosphonate 11 (0.367 g, 0.750
mmol) in MeCN (7 mL) was added a 1.0 M solution of NaHCO3
(7 mL) followed by H2O (3 mL). The reaction mixture was
heated to 50 °C, stirred for 16 h, diluted with H2O, and
extracted with EtOAc. The aqueous layer was acidified with
1 N HCl and cooled to 0 °C. After 1 h, a white precipitate had
formed. The white solid was collected by filtration, washed
with H2O and Et2O, and dried in vacuo. Recovered 0.146 g
(58%) of phosphonic acid 15 as a white solid: mp 235 °C dec;
IR (KBr) 2800 (b), 1608, 1261, 1071, 746 cm-1; 1H NMR DMSO-
d6-TFA, 300 MHz) δ 7.28 (1H, dt, J ) 8.3, 3.0 Hz), 7.17 (1H,
dd, J ) 6.8, 3.0 Hz), 6.94 (1H, d, J ) 8.3 Hz), 6.85 (1H, t, J )
6.8 Hz), 5.19 (1H, dd, J ) 9.0, 6.0 Hz), 3.70 (3H, s), 3.52-3.17
(4H, m); 31P NMR (DMSO-d6-TFA, 125 MHz) δ 12.33; MS
(ES-) m/z 336 (M - H). Anal. (C13H16N5O4P) C, H, N.
(S,S)-5-P h en yl-2-[(3,3,3-tr iflu or o-2-m eth oxy-2-p h en yl-
p r op ion yl)a m in o]p en t -4-yn oic Acid (2-Cya n oet h yl)a -
m id e (17). Stereochemical integrity of Pd(0) cross-coupling
1
1166 cm-1; H NMR (CDCl3, 300 MHz) δ 7.32-7.23 (2H, m),
6.88 (2H, t, J ) 7.5 Hz), 5.57 (1H, d, J ) 10.5 Hz), 5.32-5.23

Potent, Selective Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1521
was shown by preparing the Mosher amides of racemic and
optically active (S)-[1-[(2-cyanoethyl)carbamoyl]-4-phenylbut-
3-ynyl]carbamic acid tert-butyl ester (7, where R ) H). The
procedures for the racemate and single enantiomer were
identical except (R)-Mosher acid chloride was used for the
racemate and (S)-Mosher acid chloride was used for the single
enantiomer. A solution of amide 7 (R ) H) (0.150 g, 0.439
mmol) in formic acid (5 mL) was heated to 40 °C for 2 h. The
reaction mixture was concentrated in vacuo to near dryness.
The residue was taken up in EtOAc and slowly made slightly
basic (pH 8) with saturated NaHCO3 solution. The layers were
separated, and the aqueous layer was extracted twice with
EtOAc. The combined organic extracts were washed once with
brine, dried over MgSO4, filtered, and concentrated to give
0.110 g (quantitative yield) of the amine as a yellow oil: 1H
NMR (CDCl3, 300 MHz) δ 7.99 (1H, bs), 7.42-7.27 (5H, m),
3.62-3.53 (3H, m), 2.97 (1H, dd, J ) 16.0, 4.0 Hz), 2.84 (1H,
dd, J ) 16.0, 7.5 Hz), 2.64 (2H, m). To a solution of the above
amine (0.060 g, 0.249 mmol) in CH2Cl2 (1 mL) was added
triethylamine (0.038 mL, 0.273 mmol) followed by (R)-(-)-R-
methoxy-R-(trifluoromethyl)phenylacetyl chloride (0.051 mL,
0.273 mmol). After stirring for 1 h, the reaction mixture was
diluted with EtOAc and washed with 1 N HCl, water,
saturated NaHCO3, water, and brine. The organic layer was
dried over MgSO4, filtered, concentrated, and passed through
a plug of silica gel (eluent: 30% EtOAc in hexanes) to give
0.100 g (91%) of the Mosher amide 16, (R,R)/(R,S)-5-phenyl-
2-[(3,3,3-trifluoro-2-methoxy-2-phenylpropionyl)amino]pent-4-
ynoic acid (2-cyanoethyl)amide: 1H NMR (CDCl3, 300 MHz) δ
7.81 (1H, d, J ) 9.0 Hz), 7.76 (1H, d, J ) 9.0 Hz), 7.58-7.00
(20H, m), 4.86-4.72 (2H, m), 3.52-3.28 (10H, m), 2.98-2.80
(4H, m), 2.51 (2H, t, J ) 7.5 Hz), 2.40 (2H, t, J ) 7.5 Hz).
Mosher amide 17 was prepared similarly, (S,S)-5-phenyl-2-
[(3,3,3-trifluoro-2-methoxy-2-phenylpropionyl)amino]pent-4-
ynoic acid (2-cyanoethyl)amide: 1H NMR (CDCl3, 300 MHz) δ
7.81 (1H, d, J ) 8.0 Hz), 7.51-7.25 (10H, m), 4.79 (1H, q, J )
7.1 Hz), 3.42 (1H, d, J ) 1.2 Hz), 3.39 (3H, s), 3.29 (2H, q, J )
6.6 Hz), 2.86 (2H, d, J ) 6.6 Hz), 2.36 (2H, t, J ) 6.5 Hz).
following literature procedures.5a,12d A solution of dimethyl-
[[[(trifluoromethyl)sulfonyl]oxy]methyl]phosphonate20 (0.263 g,
0.966 mmol) in CH2Cl2 (2 mL) was added to a solution of amine
20 (0.271 g, 0.773 mmol) and diisopropylethylamine (0.20 mL,
1.16 mmol) in CH2Cl2 at room temperature. After stirring for
16 h, the reaction mixture was diluted with EtOAc and washed
with brine. The organic layer was dried (MgSO4), filtered, and
concentrated under reduced pressure. Purification using
Flash40 chromatography system on silica gel (eluent: EtOAc)
gave 0.226 g (62%) of phosphonate 21 as a film: IR (thin film)
2246, 1741, 1675, 1243 cm-1 1H NMR (CDCl3, 300 MHz) δ
;
7.70 (1H, d, J ) 7.5 Hz), 7.48-7.38 (2H, m), 7.24-7.18 (2H,
m), 4.67-4.55 (1H, m), 3.88 (3H, d, J ) 4.5 Hz), 3.82 (3H, d,
J ) 4.5 Hz), 3.72 (3H, s), 3.52 (1H, bs), 3.28-2.89 (4H, m),
1.70-1.56 (3H, m), 0.95-0.80 (6H, m); MS (ES+) m/z 473 (M
+ H).
(S,S)-2-[[5-(2-Ch lor oph en yl)-2-[(ph osph on om eth yl)am i-
n o]p en t -4-yn oyl]a m in o]-4-m et h ylp en t a n oic Acid (24).
Phosphonic acid dipeptide 24 was prepared with slight modi-
fication of literature procedures.12d To a solution of phospho-
nate 21 (0.226 g, 0.478 mmol) in MeOH (2 mL) was added 1 N
NaOH (1.5 mL). After 1.5 h, the reaction mixture was
concentrated under reduced pressure to give the monomethyl
phosphonate (92% pure, trace of dimethyl phosphonate): 1H
NMR (D2O, 300 MHz) δ 7.45-7.38 (2H, m), 7.28-7.18 (2H,
m), 4.18 (1H, dd, J ) 9.8, 1.5 Hz), 3.50 (3H, d, J ) 9.8 Hz),
3.50-3.43 (1H, m), 2.91 (2H, d, J ) 3.0 Hz), 2.89-2.69 (2H,
m), 1.57-1.38 (3H, m), 0.59 (6H, d, J ) 4.5 Hz); 31P NMR (D2O,
121 MHz) δ 23.05. To a stirred slurry of the monomethyl
phosphonate in CH2Cl2 (7 mL) was added diisoproylethylamine
(0.42 mL, 2.39 mmol) followed by TMSBr (1.26 mL, 9.56 mmol).
After 0.5 h, the reaction mixture was concentrated under
reduced pressure without heat. To the recovered residue was
added 50 mL of H2O, and the resulting slurry was stirred for
0.5 h. The light-brown solid which formed was collected by
filtration and washed with Et2O. Recovered 0.092 g (45%) of
phosphonic acid dipeptide 24 as a light-brown solid: mp 195
°C dec; IR (KBr) 1658, 1198, 1159 cm-1; 1H NMR (DMSO-d6-
TFA, 300 MHz) δ 7.56 (1H, dd, J ) 7.4, 2.2 Hz), 7.48 (1H, d,
J ) 7.7 Hz), 7.38-7.28 (2H, m), 4.30-4.26 (2H, m), 3.32-3.12
(4H, m), 1.64-1.54 (3H, m), 0.86-0.83 (6H, m); 31P NMR
DMSO-d6-TFA, 121 MHz) δ 12.29; MS (ES-) m/z 429 (M -
H). Anal. (C18H24ClN2O6P) C, H, N.
Compounds 22-25 were prepared using standard peptide
coupling, the Pd(0) cross-coupling described above,16 and
previously reported procedures.5a,b,12d Derivative 24 is repre-
sentative.
(S,S)-2-[[2-[(ter t-Bu toxyca r bon yl)a m in o]p en t-4-yn oyl]-
am in o]-4-m eth ylpen tan oic Acid Meth yl Ester (18). Dipep-
tide 18 (0.13 g, 83%) was prepared as described for amide 6
using acid 5 (0.10 g, 0.47 mmol) and L-leucine methyl ester
hydrochloride (0.85 g, 0.47 mmol): IR (KBr) 2121, 1745, 1733,
Compounds 38-69 were prepared using standard peptide
coupling, the alcalase enzymatic resolution, and Pd(0) cross-
coupling described above16 and previously reported proce-
dures.5a,12d Derivative 41 is representative.
1
1681, 1654 cm-1; H NMR (CDCl3, 250 MHz) δ 6.72 (1H, d, J
2-[(ter t-Bu t oxyca r b on yl)a m in o]-5-(2,4-d iflu or op h en -
yl)p en t-4-yn oic Acid Meth yl Ester (26). The procedure
described for arylacetylene 7 was employed.16 Using propar-
gylglycine methyl ester 3 (10.87 g, 47.8 mmol) and 2,4-difluoro-
1-iodobenzene (6.3 mL, 52.7 mmol) gave 13.83 g (85%) of
arylacetylene 26: 1H NMR (CDCl3, 250 MHz) δ 7.33 (1H, q, J
) 7.5 Hz), 6.79 (2H, t, J ) 7.5 Hz), 5.38 (1H, d, J ) 7.5 Hz),
4.56 (1H, dt, J ) 10.7, 5.0 Hz), 3.79 (3H, s), 2.95 (2H, t, J )
5.00 Hz), 1.45 (9H, s).
) 7.4 Hz), 5.27 (1H, s), 4.64-4.56 (1H, m), 4.30-4.20 (1H, m),
3.70 (3H, s), 2.78 (1H, ddd, J ) 18.2, 5.5, 2.7 Hz), 2.57 (1H,
ddd, J ) 18.2, 5.5, 2.7 Hz), 2.05 (1H, t, J ) 2.6 Hz), 1.75-1.50
(3H, m), 1.43 (9H, s), 0.90 (6H, dd, J ) 5.8, 2.2 Hz); MS (ES+)
m/z 341 (M + H).
(S,S)-2-[[2-[(ter t-Bu t oxyca r b on yl)a m in o]-5-(2-ch lor o-
p h en yl)p en t -4-yn oyl]a m in o]-4-m et h ylp en t a n oic Acid
Meth yl Ester (19). Arylacetylene 19 (0.386 g, 69%) was
prepared as described for arylacetylene 7 using dipeptide 18
(0.420 g, 1.235 mmol) and 1-chloro-2-iodobenzene (0.17 mL,
1.420 mmol):16 IR (thin film) 2256, 1739, 1720, 1679 cm-1; 1H
NMR (CDCl3, 300 MHz) δ 7.48-7.40 (2H, m), 7.24-7.17 (2H,
m), 6.78 (1H, bs), 5.51 (1H, bs), 4.68-4.62 (1H, m), 4.42 (1H,
bs), 3.69 (3H, s), 3.15 (1H, dd, J ) 17.3, 7.5 Hz), 2.86 (1H, dd,
J ) 18.8, 7.5 Hz), 1.60-1.54 (3H, m), 1.52 (9H, s), 0.85 (6H,
dd, J ) 16.5, 7.5 Hz); MS (ES+) m/z 451 (M + H).
(S,S)-2-[[2-Am in o-5-(2-ch lor op h en yl)p en t-4-yn oyla m i-
n o]-4-m eth ylp en ta n oic Acid Meth yl Ester (20). Amine 20
(0.271 g, 90%) was prepared as previously described for amine
9 using arylacetylene 19 (0.386 g, 0.857 mmol) and carried
forward without purification: IR (thin film) 2227, 1706, 1689
cm-1; MS (ES+) m/z 351 (M + H).
(S,S)-2-[[5-(2-Ch lor oph en yl)-2-[[(dim eth oxyph osph or yl)-
m et h yl]a m in o]p en t -4-yn oyl]a m in o]-4-m et h ylp en t a n oic
Acid Met h yl E st er (21). Phosphonate 21 was prepared
(S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-5-(2,4-d iflu or op h e-
n yl)p en t-4-yn oic Acid (27). The enzymatic resolution pro-
cedure described for acid 5 was employed. Using 19 g (56.0
mmol) of methyl ester 26, alcalase resolution gave 8.72 g (96%,
based on 50% recovery) of acid 27 and ester 28. The resolved
acid was found to be of 96% enantiomeric excess: 1H NMR
(CDCl3, 250 MHz) δ 9.64 (1H, bs), 7.33 (1H, q, J ) 6.3 Hz),
6.78 (2H, t, J ) 7.5 Hz), 5.40 (1H, d, J ) 7.5 Hz), 4.65-4.53
(1H, m), 3.05-2.92 (2H, m), 1.46 (9H, s).
The enantiomeric excess of acid 27 and the 2-fluoro deriva-
tive were determined by examination of the 19F NMR spectra
of the derived Mosher amides. For comparison, the Mosher
amides of the enantiomers, isolated from the resolution, were
prepared. These were prepared as described above for amides
16 and 17 using (R)-(-)-R-methoxy-R-(trifluoromethyl)phen-
ylacetyl chloride.

1522 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Wallace et al.
(R,S)-5-(2,4-Diflu or op h en yl)-2-[(3,3,3-tr iflu or o-2-m eth -
oxy-2-p h en ylp r op ion yl)a m in o]p en t-4-yn oic a cid m eth yl
ester (29): 19F NMR (CDCl3, 470 MHz) δ -69.92 (98%), -70.08
(2%); diastereomeric excess found to be 96%.
(R,R)-5-(2,4-Diflu or op h en yl)-2-[(3,3,3-tr iflu or o-2-m eth -
oxy-2-p h en ylp r op ion yl)a m in o]p en t-4-yn oic a cid m eth yl
ester (30): 19F NMR (CDCl3, 470 MHz) δ -69.91 (9%); -70.07
(91%); diastereomeric excess estimated to be 82%.
(R,S)-5-(2,4-Flu or oph en yl)-2-[(3,3,3-tr iflu or o-2-m eth oxy-
2-p h en ylp r op ion yl)a m in o]p en t-4-yn oic a cid m eth yl es-
ter (31): 19F NMR (CDCl3, 470 MHz) δ -69.92; diastereomeric
excess found to be 98.8%.
(R,R)-5-(2,4-Flu or oph en yl)-2-[(3,3,3-tr iflu or o-2-m eth oxy-
2-p h en ylp r op ion yl)a m in o]p en t-4-yn oic a cid m eth yl es-
ter (32): 19F NMR (CDCl3, 470 MHz) δ -69.92 (8%), -70.05
(92%); diastereomeric excess estimated to be 84%.
6.79 (2H, m), 4.67-4.58 (1H, m), 3.85 (3H, d, J ) 6.8 Hz), 3.81
(3H, d, J ) 6.8 Hz), 3.74 (3H, s), 3.46 (1H, dd, J ) 8.3, 6.0
Hz), 3.25-2.79 (4H, m), 1.70-1.52 (3H, m), 0.89 (6H, t, J )
7.5 Hz); 31P NMR (CDCl3, 121 MHz) δ 27.98; MS (ES+) m/z
475 (M + H).
(S,S)-2-[[5-(2,4-Diflu or op h en yl)-2-[(p h osp h on om eth yl)-
am in o]pen t-4-yn oyl]am in o]-4-m eth ylpen tan oic Acid (41).
The method described for phosphonic acid dipeptide 24 was
employed. Using 0.337 g (0.711 mmol) of phosphonate 37 gave
0.168 g (55%) of phosphonic acid 41: mp 200 °C dec; IR (KBr)
1659, 1615, 1591, 1507, 1268, 1144, 1101, 850 cm-1; 1H NMR
DMSO-d6-TFA, 300 MHz) δ 7.56 (1H, dd, J ) 14.7, 8.5 Hz),
7.25 (1H, dt, J ) 9.6, 2.6 Hz), 7.09-7.04 (1H, m), 4.29-4.23
(2H, m), 3.27-3.11 (4H, m), 1.64-1.53 (3H, m), 0.86-0.82 (6H,
m); 31P NMR DMSO-d6-TFA, 121 MHz) δ 12.26; MS (ES-) m/z
431 (M - H). Anal. (C18H23F2N2O6P) H, N; C: calcd, 50.00;
found, 49.35.
(S )-2-[(t er t -Bu t oxyca r b on yl)a m in o]-5-(2,4-d iflu or o-
p h en yl)p en t-4-yn oic Acid Meth yl Ester (33). To a stirred
solution of acid 27 (3.36 g, 10.3 mmol) in 4:1 PhMe-MeOH
(50 mL) was added a 2.0 M solution of TMSCHN2 in hexanes
(13.0 mL, 26.0 mmol). After 1.5 h, the reaction mixture was
quenched by the addition of acetic acid and then concentrated
under reduced pressure. Ester 33 was recovered in quantati-
tive yield: 1H NMR (CDCl3, 250 MHz) δ 7.33 (1H, q, J ) 7.5
Hz), 6.79 (2H, t, J ) 7.5 Hz), 5.38 (1H, d, J ) 7.5 Hz), 4.56
(1H, dt, J ) 10.7, 5.0 Hz), 3.79 (3H, s), 2.95 (2H, t, J ) 5.00
Hz), 1.45 (9H, s).
(S)-2-Am in o-5-(2,4-d iflu or op h en yl)p en t-4-yn oic Acid
Meth yl Ester (34). The method described for amine 9 was
employed. Using 5.45 g (16.06 mmol) of 33 3.68 g (96%) of
amine 34 was recovered as a light-yellowish oil: 1H NMR
(CDCl3, 300 MHz) δ 7.38 (1H, q, J ) 7.5 Hz), 6.81 (2H, t, J )
7.5 Hz), 3.69 (3H, s), 3.72-3.62 (1H, m), 2.90 (2H, d, J ) 7.5
Hz), 1.93 (2H, bs).
(S)-5-(2,4-Diflu or op h en yl)-2-[[(d im eth oxyp h osp h or yl)-
m eth yl]a m in o]p en t-4-yn oic Acid Meth yl Ester (35). The
method described for phosphonate 21 was employed. Using
4.20 g (17.56 mmol) of amine 34 and 4.78 g (17.56 mmol) of
dim et h yl[[[(t r iflu or om et h yl)su lfon yl]oxy]m et h yl]ph os-
phonate gave 4.08 g (64%) of phosphonate 35 as an oil: 1H
NMR (CDCl3, 300 MHz) δ 7.38 (1H, q, J ) 7.5 Hz), 6.88-6.78
(2H, m), 3.83 (3H, s), 3.80 (6H, s), 3.69 (1H, t, J ) 5.3 Hz),
3.15 (2H, dt, J ) 54.8, 15.0 Hz), 2.92 (2H, d, J ) 7.5 Hz).
(S)-5-(2,4-Diflu or op h en yl)-2-[[(d im eth oxyp h osp h or yl)-
m eth yl]a m in o]p en t-4-yn oic Acid (36). To a stirred solution
of phosphonate 35 (4.08 g, 11.29 mmol) in MeOH (60 mL) was
added a 1.0 N solution of NaOH (11.3 mL). After 3.5 h, the
reaction mixture was extracted with Et2O. The remaining
aqueous layer was acidified with 1 N HCl and extracted with
EtOAc several times. The combined EtOAc extracts were
washed with H2O and brine, dried (MgSO4), filtered, and
concentrated under reduced pressure to give 3.29 g (84%) of
acid 36 as an oil: 1H NMR (DMSO-d6, 300 MHz) δ 7.52 (1H,
q, J ) 7.5 Hz), 7.46 (1H, dt, J ) 7.5, 1.5 Hz), 7.11 (1H, dt, J )
7.5, 1.5 Hz), 3.68 (6H, d, J ) 9.8 Hz), 3.54 (1H, t, J ) 5.3 Hz),
3.07 (2H, app p, J ) 15.0 Hz), 2.81 (2H, t, J ) 3.8 Hz); 31P
NMR (DMSO-d6, 121 MHz) δ 28.85; MS (ES+) m/z 348 (M +
H).
(S,S)-2-[[5-(2,4-Diflu or op h en yl)-2-[[(d im et h oxyp h os-
p h or yl)m eth yl]a m in o]p en t-4-yn oyl]a m in o]-4-m eth ylp en -
ta n oic Acid Meth yl Ester (37). To a stirred solution of acid
36 (0.310 g, 0.890 mmol) and L-leucine methyl ester hydro-
chloride (0.162 g, 0.890 mmol) in DMF (15 mL) was added
N-methylmorpholine (0.29 mL, 2.64 mmol) followed by ben-
zotriazolyl-N-oxytris(dimethylamino)phosphonium hexafluo-
rophosphate (0.592 g, 1.340 mmol). After 16 h, the reaction
mixture was partitioned between EtOAc and 1 N HCl and the
layers were separated. The organic phase was washed with
H2O, saturated NaHCO3 solution, and brine. The organic layer
was dried (MgSO4), filtered, and concentrated under reduced
pressure. Purification using Flash40 chromatography system
on silica gel (eluent: 95:5 EtOAc-EtOH) gave 0.337 g (79%)
of dipeptide phosphonate 37 as an oil: 1H NMR (CDCl3, 300
MHz) δ 7.57 (1H, q, J ) 9.0 Hz), 7.43-7.32 (1H, m), 6.88-
Ack n ow led gm en t. The authors wish to thank Drs.
Ste´phane De Lombaert, Gary Ksander, J im Stanton,
Cynthia Fink, and Paul Greenspan for helpful discus-
sions and critical reading of this manuscript. We also
wish to thank the Novartis Analytical Chemistry Staff
(Summit) for the physicochemical data on the com-
pounds presented.
Su p p or tin g In for m a tion Ava ila ble: Experimental data
for compounds 12-14, 22, 23, 25, 38-40, and 42-70 (12
pages). Ordering information is given on any current mast-
head page.
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