Phosphinic tripeptides as dual angiotensin-converting enzyme C-domain and endothelin-converting enzyme-1 inhibitors

Article abstract of DOI:10.1021/jm9010803

A new series of phosphinic inhibitors able to interact with both angiotensin-converting enzyme (ACE) C-domain and endothelin-converting enzyme-1 (ECE-1), while sparing neprilysin (NEP), has been developed. The most potent and selective inhibitor in this series (compound 8_F2) displays K _i values of 0.65 nM, 150 nM, 14 nM and 6.7 μM toward somatic ACE C-domain, ACE N-domain, ECE-1, and NEP, respectively. Remarkably, in this series, the inhibitor's ability to discriminate between ECE-1 and NEP was observed to depend on the stereochemistry of the residue present in the inhibitor's P₁′ position. After iv administration, compound 8_F2 (10 mg/kg) lowered mean arterial blood pressure by 24 ± 2 mmHg in spontaneously hypertensive rats, as compared with controls. Mixed ACE/ECE-1 inhibitor may lead to a new generation of vasopeptide inhibitors that should reduce the levels of angiotensin-II and endothelin-1, without interfering with bradykinin cleavage.

Full text of DOI:10.1021/jm9010803

208 J. Med. Chem. 2010, 53, 208–220
DOI: 10.1021/jm9010803
Phosphinic Tripeptides as Dual Angiotensin-Converting Enzyme C-Domain and Endothelin-Converting
Enzyme-1 Inhibitors
Nicolas Jullien,†,§ Anastasios Makritis,‡ Dimitris Georgiadis,‡ Fabrice Beau,† Athanasios Yiotakis,‡ and Vincent Dive*,†
†
ꢀ
CEA, DSV, Service d’Ingenierie Moleculaire des Proteines (SIMOPRO), Bat 152, CE-Saclay, Gif/Yvette 91191 Cedex, France and
ꢀ
ꢀ
‡Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, Panepistimiopolis Zografou 15771, Athens,
§
Greece. Present address: Institut de Radioprotection et de Surete Nucleaire (IRSN), DRPH/SRBE/LRPAT, Bat 05, BP17,
Fontenay-aux-roses 92262, France.
ꢀ
ꢀ
Received July 21, 2009
A new series of phosphinic inhibitors able to interact with both angiotensin-converting enzyme (ACE)
C-domain and endothelin-converting enzyme-1 (ECE-1), while sparing neprilysin (NEP), has been
developed. The most potent and selective inhibitor in this series (compound 8F2) displays Ki values of
0.65 nM, 150 nM, 14 nM and 6.7 μM toward somatic ACE C-domain, ACE N-domain, ECE-1, and
NEP, respectively. Remarkably, in this series, the inhibitor’s ability to discriminate between ECE-1 and
0
NEP was observed to depend on the stereochemistry of the residue present in the inhibitor’s P1 position.
After iv administration, compound 8F2 (10 mg/kg) lowered mean arterial blood pressure by 24 ( 2
mmHg in spontaneously hypertensive rats, as compared with controls. Mixed ACE/ECE-1 inhibitor
may lead to a new generation of vasopeptide inhibitors that should reduce the levels of angiotensin-II
and endothelin-1, without interfering with bradykinin cleavage.
Introduction
ECE-1 inhibitors and ultimately triple inhibitors blocking
ACE-NEP-ECE-1 simultaneously.7 The dual ACE-NEP in-
hibitor omapatrilat (Chart 1) was reported in a clinical trial to
be more effective than the use of a single ACE inhibitor.
However, the higher incidence of angioedema observed in
patients treated with omapatrilat versus patients treated with
ACE inhibitors has halted the development of omapatrilat
and raised concerns about the risk/benefit ratio of dual ACE-
NEP inhibitors for therapeutic applications.8 While a risk of
angioedema associated with triple ACE-NEP-ECE-1 inhibi-
tor treatment remains to be evaluated carefully, it has been
suggested that NEP inhibition in a context of either dual or
triple inhibitor treatment might be responsible for the occur-
rence of unwanted side effects.7 Inhibiting NEP results in
increased concentrations of BK and ET-1, two peptides
associated with life-threatening side effects. The above con-
cerns led us to consider the development of dual ACE/ECE-1
inhibitors, able to spare NEP. Targeting both ACE and ECE-
1 should lower plasma concentrations of the two most potent
vasoconstrictive peptides, Ang-II and ET-1. However, suc-
cessful development of dual ACE/ECE-1 inhibitors relies on
the possibility of identifying compounds able to differentiate
ECE-1 from NEP while maintaining good potency toward
ACE. Comparison between the crystallographic structures of
NEP and ECE-1 reveals only slight differences in the topology
oftheiractivesites.9,10 The most significant differencebetween
these two peptidases, which could be exploited in the inhibitor
design, is the replacement of Met579 in NEP by Val603 in ECE-
The major involvement of membrane-bound zinc metallo-
peptidases like angiotensin-converting enzyme (ACE,a EC
3.4.15.1), neutral endopeptidase (neprilysin, NEP, EC
3.4.24.11), and endothelin-converting enzyme-1 (ECE-1, EC
3.4.24.71) in the control of peptide hormones exerting vaso-
constrictive (angiotensin-II (Ang-II), endothelin-1 (ET-1))
and vasodilatory (natriuretic peptides, bradykinin (BK)) ac-
tivities has made these enzymes the key targets for developing
antihypertensive drugs.1-4 Despite the success of ACE inhi-
bitor therapy in treatment of hypertensive patients, blood
pressure control remains suboptimal in a significant propor-
tion of patients on this therapy or on other antihypertensive
drug treatment.5 This has justified the development of new
strategies for improving treatment of patients with high blood
pressure, a major risk factor for cardiovascularcomplications.
To address this concern, dual inhibitors able to target potently
ACE and NEP were first developed,6 followed by dual NEP/
*To whom correspondence should be addressed. Phone:
330169082603. Fax: 330169089071. E-mail: vincent.dive@cea.fr.
a Abbreviations: ACE, angiotensin-converting enzyme; ECE-1, en-
dothelin-converting enzyme 1; NEP, neprilysin; MMPs, matrix metal-
loproteinases; MCA, (7-methoxycoumarin-4-yl)acetyl; Dnp, 2,4-
dinitrophenyl; DpaOH, N3-(2,4-dinitrophenyl)-L-diaminopropionyl;
Ac2O, acetic anhydride; AcOH, acetic acid; AcOEt, ethyl acetate; Boc,
tert-butoxycarbonyl; DCR, dipolar cycloaddition; EDC, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide; HOBt, N-hydroxy benzo-
triazole; DIPEA, N,N-diisopropylethylamine; TIS, triisopropylsilane;
But, tert-butyl; Cbz, benzyloxycarbonyl; DCM, dichloromethane;
0
1, which leads in the in ECE-1 crystal structure to a larger S1
pocket, as compared with NEP. This consideration, together
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPM H3PO2,
3
aminodiphenylmethane hypophosphite; Et2O, diethyl ether; EtOH,
ethanol; HMDS, 1,1,1,3,3,3-hexamethyldisilazane; MeOH, methanol;
NCS, N-chlorosuccinimide; Np, para-nitrophenyl; PE 40-60 °C, pet-
roleum ether 40-60 °C; TMSCl, chlorotrimethylsilane; TFA, trifluoro-
acetic acid.
0
with the presence in ACE of an S1 subsite forming a very large
cavity,11 led us to envisage the development of0phosphinic
derivatives containing bulky side chains in their P1 position as
r
pubs.acs.org/jmc
Published on Web 11/09/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 209
Chart 1. Chemical Structure of ACE, NEP, and ECE-1 Inhibitors
potential dual ACE/ECE-1 inhibitors. Among the phosphinic
peptides we have developed in our laboratory as potent ACE
inhibitors, compound 21 (RXPA380 in Chart 1) possesses
both interesting structural features and selectivity profile.12,13
Like phosphoramidon (Chart 1), one of the first phosphorus-
co0ntaining peptide inhibitor of ECE-1, 21 also contains in its
P2 position a free Trp residue. Moreover, on the functional
side, 21 is a potent and selective inhibitor of the ACE C-
domain, the ACE active site responsible for the conversion of
Ang-I into Ang-II and thus for the blood pressure control.14
Somatic ACE contains two functional zinc-active sites,
termed the N and C-domain, which displayed different phy-
siological selectivity profiles and which can be differentiated
by specific N- and C-domain selective inhibitors.12,15 Com-
parison of the crystal structures of the N- and C-domain in
interaction with their selective inhibitors has shown that only
few mutations in the N- and C-domain active site were
responsible for their different profiles of selectivity toward
substrates and inhibitors.13,16
The above considerations have led us to start from the
0
structure of 21 and see whether its modification at the P1
position by bulky side chains may provide new inhibitors
possessing the desired dual ACE C-domain and ECE-1
selectivity.
Results
Chemistry. A series of phosphinic tripeptides were synthe-
sized, bearing a Cbz-protected pseudophenylalanine in the
0
P1 position, natural amino acid residues in the P2 position,
and isoxazoline- (1-4), biphenyl- (6), or isoxazole- (5, 7, and
8) containing side chains in the P1 position.
0
The synthesis of isoxazoline-containing tripeptides 1-4
started from the phospha-Michael addition of the bis-
(trimethylsilyl) phosphonite derived from the Cbz-protected
aminophosphinic analogue of L-phenylalanine 9 to ethyl
R-allyl acrylate 10 (Scheme 1).17,18 From this reaction,
phosphinic pseudodipeptide 11 was obtained as a mixture
of two diastereoisomers in a 3:1 ratio. The isomer in excess
0
possesses the S-stereochemistry in the P1 position, as has
been discussed in a previous report by our group.18 Building
block 11 was extended to tripeptides 12 and 13 after alkaline
removal of the ethylester and carbodiimide-mediated cou-
pling of H-Ala-OBut (for 12) or H-Trp-OMe (for 13) with the
resulting diacid.
At the next synthetic step, compounds 12 and 13 served as
ideal dipolarophilic substrates for the formation of isoxazo-
line rings in their P10 position via a 1,3-dipolar cycloaddition
(DCR) between the lateral terminal alkene and suitable
nitrile oxides (Scheme 1).19 These nitrile oxides are formed
in situ by the corresponding aryl aldoximes after oxidative
chlorination followed by base-induced dehydrochlorination
of the intermediate hydroximinoyl chlorides. Correct choice
of the chlorination conditions is essential in order to avoid
possible oxidative decomposition of the substrate.18 In par-
ticular, common bleach (aqueous NaOCl solution)20 is
compatible with tripeptide 12 but not with tripeptide 13
because the indole ring is prone to oxidation by this reagent.
Thus, compound 2 was produced (as a mixture of 4 dia-
stereoisomers) from the alkaline deprotection of the inter-
mediate cycloaddition product between 12 and benzonitrile
oxide. In the case of 13, the terminal methyl ester was cleaved
and the one-pot Huisgen protocol was applied to the product
involving N-chlorosuccinimide as an oxidant.21 Under these

210 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Scheme 1. Synthesis of Isoxazoline Derivatives 1-4a
Jullien et al.
Scheme 2. Synthesis of Biphenyl Derivative 6a
a Reagents and conditions: (a) HCl H-Trp-OMe, EDC HCl, HOBt,
DIPEA, CH2Cl2, rt, 2 h; (b) NaOH, MeOH, H2O, rt, 48 h, 81%, two steps.
3
3
stereochemically pure tripeptides 5F1 (R,S,S) and 5F2 (R,R,S)
on a preparative scale, a sequence of crystallization resolving
steps in carefully selected synthetic intermediates was devised
(Scheme 3). Initially, diacid 15 was coupled with H-Trp-OMe
to afford an 1:1 diastereoisomeric mixture of tripeptides 16 (R,
S,S) and 17 (R,R,S). Attempts to separate the mixture by
crystallization with CH2Cl2 furnished a white crystalline pre-
cipitate corresponding to 16 (R,S,S), while a 1:3 diastereoiso-
meric mixture of 16 (R,S,S) and 17 (R,R,S) was isolated from
the filtrates [enriched to the more soluble 17 (R,R,S)]. Applica-
tion of the one-pot Huisgen protocol to isomer 16 (R,S,S), for
the introduction of the isoxazole ring, and alkaline deprotection
of the resulting tripeptide 18 (R,S,S) efficiently led to pure
diastereoisomer 5F1 (R,S,S).18 Transformation of the 1:3 dia-
stereoisomeric mixture of 16 (R,S,S) and 17 (R,R,S) to the
corresponding isoxazole derivatives by the one-pot Huisgen
protocol afforded an 1:3 diastereoisomeric mixture of 18 (R,S,S)
and 19 (R,R,S). Gratifyingly, from the latter mixture, isomer 19
(R,R,S) proved to be less soluble in CHCl3 than 18 (R,S,S). On
this basis, 19 (R,R,S) was isolated as a crystalline solid after
crystallization by CHCl3, while a 3:1 mixture of 18/19 was
recovered from the filtrates. Compound 5F2 was finally ob-
tained after alkaline hydrolysis of methylester 19 (R,S,S). The
stereochemical identity of 5F1 (R,S,S) was confirmed by the
conversion of the previously described pure R,S-diastereo-
isomer of 15 (noted as 150 in Scheme 3) to tripeptide 18.18 This
result unequivocally proves0 that derivative 5F1 possesses
the S-stereochemistry in the P1 position and, consequently, deri-
vative 5F2 possesses the R-stereochemistry in the same position.
Finally, by following the one-pot Huisgen protocol, com-
pound 15 was converted to isoxazole derivative 20 after 1,3-
dipolar cycloaddition reaction with benzonitrile oxide
(Scheme 3). Tripeptides 7 and 8 were obtained as mixtures
of 2 diastereoisomers (1:1 ratio) after coupling of 20 with the
appropriate amino acid and final deprotection. Isolation of
fractions I (first eluting isomer, R,S,S) and II (second eluting
isomer, R,R,S) from mixtures 7 and 8 was achieved by means
of semipreparative RP-HPLC. [In each case, the stereochem-
ical identity of the first eluting fraction was confirmed by a
separate small-scale synthesis of 7F1 and 8F1 starting from the
stereochemically pure diacid 150.]
a Reagents and conditions: (a) HMDS, 110 °C, 1 h, then 10, 100 °C,
3 h, then EtOH, 70 °C, 20 min, 84%; (b) NaOH, EtOH, H2O, rt, 12 h;
(c) HCl H-Ala-OtBu, EDC HCl, HOBt, DIPEA, CH2Cl2, rt, 1.5 h,
3
3
93%, two steps; (d) HCl H-Trp-OMe, EDC HCl, HOBt, DIPEA,
3
3
CH2Cl2, rt, 1.5 h, 80%, two steps; (e) PhCHdNOH, common bleach,
Et3N, CH2Cl2, rt, 16 h; (f) NaOH, EtOH, H2O, rt, 24 h, 88%, two steps;
(g) NaOH, MeOH, H2O, rt, 24 h; (h) p-R-PhCHdNOH, NCS, pyridine
(cat.), CHCl3, 45 °C, 3-4 h, then phosphinic substrate, Et3N, 45 °C, 3 d,
92% (for 1), 93% (for 3), 90% (for 4), two steps.
conditions, the indole ring remained intact and target
compounds 1, 3, and 4 were obtained as mixtures of four
diastereoisomers in high yields.
For the synthesis of biphenyl phosphinic tripeptide 6,
compound 14 was prepared according to a published proce-
dure22 and was subjected to carbodiimide-mediated coupling
with H-Trp-OMe and subsequent alkaline deprotection.
Compound 6 was obtained as an 1:1 mixture of (R,R,S)
and (R,S,S) diastereoisomers (Scheme 2).
In Vitro Potency and Selectivity Profile. Introduction of a
0
The synthesis of isoxazole-containing tripeptides 5, 7, and
8 started from the previously described propargylic diacid
15 (1:1 diastereoisomeric mixture).18 For the synthesis of
phenyl substituted isoxazoline side chain in the P1 position
of 21 resulted in new compounds (1-4) displaying high
potency toward ACE, a result in agreement with the presence

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 211
Scheme 3. Synthesis of Isoxazole Derivatives 5F1, 5F2, 7, and 8a
a Reagents and conditions: (a) PhCHdNOH, NCS, pyridine (cat.), CHCl3, 45 °C, 3-4 h, then 15, Et3N, 45 °C, 3 d, 4 repetitions, 83%; (b) HCl H-Phe-
3
OtBu, EDC HCl, HOBt, DIPEA, CH2Cl2, rt, 2 h; (c) NaOH, MeOH, H2O, rt, 24 h, 87%, two steps; (d) HCl H-Tyr(OtBu)-OtBu, EDC HCl, HOBt,
DIPEA, CH2Cl2, rt,2h;(e) HCO2H, TIS, rt,24h, 90%, threesteps;(f) HCl H-Trp-OMe,EDC HCl, HOBt,DIPEA,CH2Cl2, rt, 1.5h;(g) crystallizationby
3
3
3
3
3
CH2Cl2, 33% of 16 þ 43% of 16/17 (1:3), two steps; (h) PhCHdNOH, NCS, pyridine (cat.), CHCl3, 45 °C, 3-4 h, then phosphinic substrate, Et3N, 45 °C,
3 d, 70% (for 18); (j) crystallization by CHCl3, 59% for 19, two steps; (i) NaOH, MeOH, H2O, rt, 24 h, 86% (for 5F1), 94% (for 5F2).
0
of a very large and deep cavity S1 cavity in this enzyme
(Table 1). In contrast, the size of these side chains has more
pronounced effects on the potency of these 21 analogues
when tested with NEP and ECE-1. Interestingly, in this series
only 1 and 2 displayed potency toward NEP and ECE-1, a
result showing that the S10 subsite in these two enzymes is of
much reduced size compared with ACE. With an alanine
probed with these two additional compounds. As shown in
0
Table 1, ACE can accommodate such P1 substituents, but
their effects on potency differ considerably between NEP
and ECE-1. While a biphenyl provided a potent NEP
inhibitor (Ki 52 nM), this side chain was not tolerated by
ECE-1 (Ki > 10 μM). In contrast, ECE-1 displayed a marked
preference for the isoxazole side chain, the replacement of the
isoxazoline with the isoxazole side chain resulting in a 65-
fold increase in affinity (Ki 910 nM versus Ki 14 nM). The
same substitution only provided a 10-fold increase in the
inhibitor affinity for NEP. The above phosphinic peptides
were evaluated as a mixture of four diastereoisomers, ex-
cepting 5 and 6, which were synthesized as a mixture of two
0
instead of a tryptophan in its P2 position, 2 exhibited a
higher potency toward NEP, as compared with ECE-1, but
the inverse situation0was observed with 1, which contains a
Trp residue in its P2 position. This result concurs with the
ability of ECE-1 to accept a bulky residue in the inhibitor’s
0
P2 position. It also suggests that there is a relationship
0
be0tween t0he size of the side chain present in the inhibitor’s
P1 and P2 positions, which differs inNEP andECE-1. ECE-1
can tolerate the simultaneous presence of two bulky side
chains in these positions, unlike NEP.
diastereoisomers (a single asymmetric center in their P1
position). To assess the influence of the side chain stereo-
0
chemistry in the inhibitor’s P1 position, the two diastereoi-
somers of compound 5 (5F1 and 5F2) were prepared through a
sequence of crystallization resolving steps and their respec-
tive Ki values for ACE, NEP, and ECE-1 were determined.
As expected from previous studies, a better potency toward
0
Further changes in the P1 side chain structure were
invest0igated in order to exploit subtle differences between
the S1 subsites of NEP and ECE-1. Instead of an isoxazoline
side chain, two other “isosteric side chains” were assessed.
The isoxazole side chain in 5 incorporates an additional
double bond in the heterocycle, as compared with the
isoxazoline ring. This difference results in a different orienta-
tion of these two side chains with respect to the inhibitor
scaffold and thus may influence the potency of the inhibitor
toward NEP and ECE-1. The same0 objective applied to 6,
containing a biphenyl side in the P1 position. In this com-
pound, the two aromatic rings are perpendicular to each
other in their lower energy conformation, while in 5, the two
cycles are almost coplanar. Thus, here again subtle differ-
0
ACE was observed when the CR carbon in the P1 position
had an S configuration. While the same trend held true for
NEP, the most potent ECE-1 inhibitor displayed the R
configuration. This difference in stereoselectivity provided
a compound (5F2), which behaved as a dual ACE C-domain/
ECE-1, with Ki values of 36 and 8 nM, respectively, for these
two peptidases, but which displayed much lower potency
with NEP (1850 nM). Toward the ACE N-domain, this
compound exhibited a Ki value of 1250 nM, so 5F2 is the
first example of a dual ACE C-domain/ECE-1 inhibitor.
However, preliminary in vivo evaluation of this inhibitor in
lowering blood pressure in spontaneously hypertensive rats
0
ences between the S1 cavity of NEP and ECE-1 can be

212 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Jullien et al.
Table 1. Potency of Phosphinic Peptide Inhibitors toward ACE C-Domain, NEP, and ECE-1
(SHR) indicated a short duration of action (few minutes,
see below). Previous studies have suggested that high bind-
ing to plasma proteins might be responsible for such a
short-lived effect23 by limiting the inhibitor’s access to the
enzyme active. This led us to evaluate the extent of bind-
ing of compound 5F2 to plasma proteins from this animal
model.
Inhibitor Potency in the Presence of SHR Rat Plasma
Proteins. To test the potential binding of compound 5F2 to
SHR rat plasma proteins, the potency of 5F2 in blocking
ACE activity in diluted rat plasma (1/50 and 1/100), com-
plemented or not with serum albumin (5 μM final con-
centration) [in plasma 1/100, the concentration of serum
albumin is still around 5 μM], was evaluated using an assay

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 213
various substituted isozazoline side chains (Table 2).18 But
due to the sharp binding preference of MMPs for phosphinic
0
peptide inhibitors containing in their P1 position side chains
with an S configuration, 5F2, 7F2, and 8F2 were observed to be
weak inhibitors of MMP-13 (Table 3). For the same reason,
5F2, 7F2, and 8F2 are expected to display weak potency toward
other MMPs.25
Effect on Blood Pressure. The in vivo efficacy of com-
pounds 5F2 and 8F2 was evaluated by studying their ability to
lower the blood pressure of SHR rats (n = 6). In the control
group, SHR rats had elevated mean arterial pressure (MAP)
of 156 ( 4 mmHg. Compound 5F2 at a iv bolus dose of 10
and 30 mg/kg reduced the MAP to 148 ( 4 mm and 96 (
7 mmHg, respectively. However, this potent effect was very
short-lived, as after 5 min the blood pressure increased to
baseline. In contrast, compound 8F2 at 3 mg/kg and 10 mg/kg
doses reduced the MAP to 147 ( 6 and 132 ( 5 mmHg,
respectively, an effect that lasted 40 min.
Discussion and Conclusions
Several phosphorus-containing pseudopeptides have been
reported previously as moderate or potent inhibitors of ECE-
1. Phosphoramidon was probably the first example of such
compounds (Chart 1). Lloyd et al. prepared more stable
analogues of phosphoramidon by replacing the phosphona-
mide moiety (PO2-NH) by a phosphinic function (PO2CH2),
but these derivatives displayed similar potencies toward NEP
and ECE-1 (compound 22, Chart 1).26 Better potency toward
ECE-1 was observed with compound 23 (SCH-54,470,
Chart 1),27 but the major breakthrough in potency and
selectivity was observed with compound compound 24
Figure 1. Apparent Ki values (numbers on the top of bars) dis-
played by compounds 5F2 (dark gray), 7F2 (dotted line), and 8F2
(black) in different serum media.
based on the cleavage of a fluorogenic substrate.24 The
apparent Ki values of 5F2, determined under these different
conditions, are reported in Figure 1. The huge variation in
the apparent Ki values, from 36 nM to 1520 in plasma 1/50,
demonstrates that compound 5F2 binds strongly to proteins
present in rat plasma, probably to albumin as shown by the
higher Ki value observed for this compound when the plasma
1/100 is complemented with this protein (500 versus 1510
nM). To alleviate this drawback, two additional compounds
were synthesized by replacing the Trp residue by Phe and Tyr
residues, as Trp is known to favor binding to albumin. Such
0
(CGS 35066, Chart 1), which has within the P1 side chain a
dibenzofuranyl.23 Recently, highly selective ECE-1 inhibitors
have been reported, but their chemical structures do not
incorporate phosphoryl group.28,29 Considering all these struc-
tures led us to suggest that further optimization of selective
ECE-1 inhibitors can be achieved with a series of phosphinic
compounds by keeping respectively in their structure a Z-Phe
0
0
moiety in the P1 position, a Trp-OH in the P2 position, and
0
substitution of the P2 position by Phe and Tyr residues led to
four additional compounds (resolved by reverse-HPLC),
which confirm the above finding of the key role of the
various long side chains in their P1 positions. Starting from the
chemical structure of 21, these simple ideas led us to rapidly
identify a series of potent triple ACE/NEP/ECE-1 inhibitors
(5F1, 7F1, and 8F1) or dual ACE/ECE-1 inhibitors (5F2, 7F2, and
0
configuration of the P1 position in this series of compounds,
in affording dual ACE C-domain/ECE-1 inhibitors. Com-
0
0
8F2). These data demonstrate the key role played by the P1 side
chain configuration in discriminating between ECE-1 and
NEP. It has been previously argued that NEP, because i0t has
pounds 5F1, 7F1, and 8F1 with the S configuration in the P1
position behaved as triple ACE/NEP/ECE-1 inhibitors.
Interestingly, compounds 7F2 and 8F2 exhibited higher po-
tency toward the ACE C-domain, but more reduced potency
toward NEP, while maintaining similar potency toward
ECE-1. Toward the N-domain, 7F2 and 8F2 exhibited
Ki values of 185 and 150 nM, respectively. Remarkably,
the substitution of the Trp residue by Phe and Tyr resulted
in a marked reduction in inhibitor binding to serum proteins.
In diluted rat plasma (1/100), complemented with 5 μM of
serum albumin, 8F2 displayed an apparent Ki value of 20 nM
toward ACE, while in the same media, 5F2 was much less
potent (Ki 1510 nM).
Potency toward Matrix-Metalloproteinases (MMPs). Fi-
nally, compounds 5, 7, and 8 were tested against MMP-13, a
zinc metalloproteinas0e which is also characterized by the
presence of a deep S1 cavity in its active site. Compounds
5F1, 7F1, and 8F1 behaved as rather potent inhibitors of
MMP-13, confirming our previous findings which were
based on phosphinic peptide containing in their P10 position
0
a larger S1 cavity than ECE-1, can accommodate large P1 side
chains without stereochemical preference.30 Here, the stereo-
chemistry of compounds 5, 7, and 8 has more pronounced
effect on their affinity toward NEP than ECE-1. A second
factor contributing to ECE-1 selectivity was that the R con-
figuration reduces the affinity of compounds 5, 7, and8 toward
NEP, as compared with the S configuration, but the reverse
situation was observed for ECE-1. Recent efforts to develop
inhibitors exhibiting similar selectivity profiles, based on a
series of substituted urea congeners, failed to identify com-
pounds with ACE-ECE-1 selectivity.31
Compounds 5F2, 7F2, and 8F2 retain quite good selectivity
for the C-domain of ACE, as compared with 21. Determina-
tion of the 3D-structure crystal of 21 complexed with the ACE
C-domain has not unveiled the molecular determinants of 21
responsible for its unique selectivity toward the ACE C-
0
domain. The presence of a Trp in the P2 position of the

214 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Jullien et al.
Table 2. Potency of Phosphinic Peptide Inhibitors toward ACE C-Domain, NEP, and ECE-1
Table 3. Inhibition of MMP-13
should be mentioned that assays to dock 8F2 in the crystal
structure of ACE C-domain in a “standard binding mode”,
the one adopted by 21 in this crystal structure, resulted in a
5F1
7F1
8F1
5F2
7F2, μM
8F2
Ki nM 19 ( 2 30 ( 3 24 ( 2 1200 ( 20
>10
4000 ( 35
0
0
model in which both the P1 and P2 inhibitor side chain atoms
0
0
inhibitors has been proposed to contribute to this selectivity, a
suggestion that fits with the selectivity displayed by com-
pounds 5F2. The good potencyexhibited0bythiscompoundfits
also with the observation of a large S1 cavity in the crystal
structures of the ACE C-domain/21 complex. However, it
exhibit steric clashes with atoms of the S1 and S2 subsites of
ACE C-domain. Thus, the exact mode of 8F2 binding in the
active sites of these enzymes remains to be experimentally
determined. Such structures would certainly be of great value
in revealing unexpected binding modes and thus be the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 215
starting point for the development of nonpeptide inhibitors
with a similar selectivity profile.
ESI mass spectral analysis was performed on a mass spectro-
meter MSQ Surveyor, Finnigan, using direct sample injection.
Negative or positive ion ESI spectra were acquired by adjusting
the needle and cone voltages accordingly. High-resolution mass
spectroscopy (HRMS) data for compounds 5F1-8F1 and
5F2-8F2 were recorded using a 4800 MALDI-TOF mass spec-
trometer (Applied Biosystems, Foster City, CA). Detection and
resolution were optimized (delay time extraction and detector
voltage multiplier) in positive reflectron mode in the m/z range
of 200-1000 to obtain an accuracy of less than 5 ppm. All
compounds were diluted in 50% acetonitrile/water with 0.1%
TFA at a concentration of 100 μM or 0.1 mg/mL. Then 0.5 μL of
each sample was spotted on a MALDI standard plate using the
dried-droplet method with 0.5 μL of R-cyano-4-hydroxycin-
namic acid matrix solution at 10 mg/mL in 50% acetonitrile/
water with 0.1% TFA. Each spectrum was the result of
1000-2000 shots (20 different positions into each spot and 50
shots per subspectrum) and internal calibration was applied by
using 4-HCCA matrix m/z.
RP-HPLC analyses were performed on a Hewlett-Packard
1100 model (C18-Cromasil-RP, 5 μm, UV/vis detector, flow:
0.5 mL/min, 254 and/or 280 nm detection). The following
gradients of buffers A and B (A buffer: 90% H2O with 0.1%
TFA, 10% CH3CN; B buffer: 10% H2O with 0.09% TFA, 90%
CH3CN) were used: method 1 (0 min, 30% B; 20 min, 60% B;
50 min, 100% B; 60 min, 40% B); method 2 (0 min, 0% B;
10 min, 25% B; 45 min, 75% B; 60 min, 100% B); method 3
(0 min, 0% B; 10 min, 35% B; 35 min, 100% B; 40 min, 35% B).
Purity of Compounds. Purity of compounds after preparative
reverse-phase HPLC was assessed by analytical HPLC using
both isochratic and gradient elution. On the basis of this criteria,
all compounds possess purity at >95%.
Blocking in vivo the C-domain of ACE by 21 was observed
to prevent the conversion of Ang-I into Ang-II but not to
abrogate the cleavage of BK by the N-domain, free of
inhibitor.12 On the basis of these observations, one may
speculate that compounds 5F2, 7F2, and 8F2 should behave in
the same manner, blocking Ang-I conversion but not inter-
fering in vivo with the BK degradation. Also, based on the
weak potency displayed by these compounds toward NEP,
the cleavage of BK by this peptidase should also occur in
presence of these inhibitors. Thus, the increase in BK levels,
which has been observed with the simultaneous blockade of
ACE and NEP and seems to be responsible for angioedema,
should not be observed with this novel series of vasopeptidase
inhibitors.7 Increase in ET-1 concentration has been also
implicated in the development of side effects associated with
dual ACE/NEP inhibitors through the blockade of NEP by
these compounds. By targeting ECE-1, while sparing NEP,
8F2 should result in vivo in a reduction of ET-1 levels.
Compound 8F2 was observed to reduce the MAP of SHR rats
by 6 and 15% at iv doses of 3 and 10 mg/kg. These preliminary
experiments are quite promising, but further work is required
to determine the full dose-effect of compound 8F2 on blood
pressure. It will be mandatory also to examine the effect of
compound 8F2 on key vasoactive peptide mediators to explain
its effect on blood pressure. This could be of fundamental
importance, as to the best of our knowledge compound 8F2
possesses a unique selectiveprofile andsomightbe agood tool
to improve understanding of the complex interplay between
the different vasoactive peptide mediators. Finally, 8F2 may
alsofind important applications in diseases where bothAng-II
and ET-1 overproduction is a main concern, like cardiovas-
cular end-organ damage32 or particular cancers.33,34
Chemistry. (1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenyl-
ethyl[2-(ethoxycarbonyl)-pent-4-enyl] Phosphinic Acid (11). A
mixture of aminophosphinic acid 9 (4.0 g, 12.5 mmol) and
HMDS (13.1 mL, 62.5 mmol) was heated at 110 °C for 1 h
under Ar, then ethyl R-allyl acrylate (10) (2.63 g, 18.8 mmol)
was added dropwise. The resulting mixture was stirred at
100 °C for additional 3 h and slowly cooled to 70 °C. Then,
dry EtOH (13 mL) was added portionwise, still under Ar. The
cooled mixture was stirred at this temperature for 20 min. The
solvent was removed under vacuum and the residue was
purified by column chromatography (CHCl3/MeOH/AcOH
7:0.3:0.3) to afford 4.8 g (84%) of compound 11 as a white
solid; mp 120-122 °C; Rf = 0.65 (CHCl3/MeOH/AcOH
Experimental Section
General. Solvents were purchased as anhydrous grade and
˚
stored over 4 A activated molecular sieves before use. Reagents
were purchased from Aldrich, Acros, Fluka, Novabiochem, and
Bachem and were used without further purification. Column
chromatography was performed on silica gel (E. Merck, 70-230
mesh). TLC analyses were performed on silica gel plates (E.
Merck silica gel 60F254), and components were visualized by the
following methods: UV light absorbance and/or charring after
spraying with a solution of NH4HSO4. A CHCl3/MeOH/AcOH
7:2:1 solvent system was used for all Rf values reported in the
Experimental Section unless otherwise noted. Melting points
(measured on an electrothermal apparatus) are uncorrected.
Optical rotation data were acquired on a Perkin-Elmer 343
polarimeter at 25 °C.
1
3
7:0.5:0.5). H NMR (200 MHz, CD3OD) δ 1.22 (t, JHH
=
6.8 Hz, 3H, CH2CH3), 1.63-1.89 (m, 1H, PCHH), 1.96-2.20
(m, 1H, PCHH), 2.24-2.44 (m, 2H, CH2CHdCH2), 2.58-3.02
(m, 2H, PCH2CH, PCHCHHPh), 3.18-3.34 (m, 1H,
PCHCHHPh), 3.94-4.19 (m, 3H, CH2CH3, PCH), 4.93-
5.16 (m, 4H, CHdCH2, PhCH2O), 5.48-5.81 (m, 1H, CHd
CH2), 7.12-7.29 (m, 10H, Ar). 13C NMR (50 MHz, CD3OD) δ
14.4, 28.8 (d, JPC = 91.4 Hz), 34.6, 38.9 (d, JPC = 9.5 Hz), 40.1,
53.2 (d, JPC = 106.8 Hz), 61.9, 66.4, 119.1, 127.2, 127.8, 128.4,
128.7, 129.5, 129.7, 130.2, 130.5, 135.8, 139.3, 139.5, 157.5 (d,
JPC = 5.1 Hz), 175.9 (d, JPC = 7.8 Hz). 31P NMR (81 MHz,
CD3OD) δ 49.2, 49.7. ES-MS m/z: calcd for [C24H30NO6P -
H]- 458.2; found 458.1.
NMR spectra were recorded on either a Varian 200 MHz
Mercury or a Bruker 500 MHz Avance III spectrometer at 26
°C. For NMR characterization, DMSO-d6 containing 1%
1
CF3COOD was used as a solvent unless otherwise noted. H
and 13C spectra are referenced according to the residual peak of
the solvent based on literature data.35 31P NMR chemical shifts
are reported in ppm downfield from 85% H3PO4 (external
standard). 13C and 31P NMR spectra are fully proton decoupled.
For compounds 1-4, 6, 11-13, 16, and 18-20, 1H NMR peak
assignments are based on 2D COSY experiments. For com-
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl[2-({[(1S)-
2-(tert-butyloxy)-1-methyl-2-oxoethyl]amino}carbonyl)-pent-4-
enyl] Phosphinic Acid (12). Phosphinic pseudodipeptide 11 (750
mg, 1.63 mmol) were dissolved in EtOH (14 mL), and the
solution was cooled to 0 °C. NaOH (1 M, 13 mL) was added
portionwise, and the mixture was stirred at rt for 12 h. After
acidification with 2 M HCl, ethanol was evaporated, and the
residue was diluted with water and extracted with AcOEt
(40 mL). The organic phase was washed with H2O and brine,
dried over Na2SO4, and evaporated under vacuum. The residue
was dried over P2O5 for 24 h, and addition of CH2Cl2 (10 mL)
followed. To the resulting suspension, DIPEA (0.81 mL,
pounds 5F1-8F1 and 5F2-8F2, a full assignment of 1H and 13
C
NMR spectra, acquired on a 500 MHz instrument, is provided
in Supporting Information. This assignment was performed on
the basis of 1D DEPTQ and 2D COSY, TOCSY, HSQC, and
HMBC spectra that were processed and analyzed with the
Bruker TOPSPIN 2.0 program.

216 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Jullien et al.
4.9 mmol), hydrochloric L-alanine tert-butyl ester (311 mg, 1.71
mmol), HOBt (220 mg, 1.63 mmol), and EDC HCl (1.88 g, 9.78
evaporated under vacuum. Compound 1 (319 mg, 92%) was
obtained as a white solid after purification by column chroma-
tography (CHCl3/MeOH/AcOH 7:0.35:0.3) and precipitation
by a Et2O/light petroleum (1:9) mixture; mp 121-124 °C (dec);
Rf = 0.49. 1H NMR (200 MHz) δ 1.35-2.18 (m, 4H, CH2CHO,
PCH2), 2.54-3.52 (m, 7H, CHCH2Ph, CHCONH, CH2indolyl,
CH2CdN), 3.65-4.22 (m, 1.75H, PCH, CHO of first dia-
stereoisomer), 4.22-4.57 (m, 1.25H, CHO of second diastereo-
isomer, NHCHCO), 4.78-5.04 (m, 2H, PhCH2O), 6.83-7.80
(m, 22H, Ar, indole-NH, OCONH), 7.98-8.19 and 8.36-8.52
(m, 1H, NHCHCO). 13C NMR (50 MHz) δ 27.6 (d, JPC = 88.2
Hz), 28.6 (d, JPC = 88.2 Hz), 32.7 (br), 33.2, 36.9, 37.1, 42.3, 52.6
(d, JPC = 97.8 Hz), 53.8, 65.1, 78.8/79.1, 110.6, 111.2, 112.1,
118.4, 118.6, 118.7, 120.7, 123.7, 126.2, 127.1, 127.2, 127.5,
128.2, 128.3, 128.9, 129.0, 131.3, 136.8, 137.3, 138.2, 138.6,
155.8 (d, JPC = 3.2 Hz), 156.2, 172.1, 173.5, 173.9. 31P NMR
(81 MHz) δ 45.8, 45.9, 46.6, 47.1. HPLC tR(2) = 38.9, 40.1(d)
min, (4 isomers). HRMS m/z: calcd for [C40H41N4O8P þ H]þ
737.2740; found 737.2750.
3
mmol) were added. The mixture was stirred for 1.5 h at rt. Then
it was diluted with CH2Cl2 (40 mL) and the solution was washed
with 1 M HCl (3 ꢀ 10 mL), H2O (10 mL), 10% NH4HCO3 (2 ꢀ
1.5 mL), 1 M HCl (2 ꢀ 10 mL), and brine (10 mL). The organic
phase was dried over Na2SO4 and evaporated under vacuum.
Compound 12 (850 mg, 93%) was obtained as a white solid after
addition of a Et2O/light petroleum (1:3) mixture to the residue
and filtration of the precipitate; mp 111-113 °C; Rf = 0.44/0.38
1
(CHCl3/MeOH/AcOH 7:0.5:0.5). H NMR (200 MHz) δ 1.23
3
(d, JHH = 6.7 Hz, 3H, CHCH3), 1.34 [s, 9H, C(CH3)3],
1.51-2.05 (m, 2H, PCH2), 2.11-3.17 (m, 5H, CH2CHdCH2,
PCH2CH, PCHCH2Ph), 3.79-4.09 (m, 1H, PCH), 4.19-4.37
(m, 1H, NHCHCO), 4.64-5.17 (m, 4H, CHdCH2, PhCH2O),
5.46-5.82 (m, 1H, CH = CH2), 7.04-7.44 (m, 10H, Ar), 7.63 (d,
3JHH = 9.6 Hz, OCONH), 8.39 (d, 3JHH = 9.6 Hz, NHCHCO).
13C NMR (50 MHz) δ 17.1, 27.5 (d, JPC = 88.6 Hz), 27.8, 33.1,
38.1, 47.9, 52.6 (d, JPC = 95.9 Hz), 65.1, 81.1, 126.2, 127.0, 127.5,
128.1, 128.4, 128.9, 137.2, 138.5 (d, 1H, JPC = 13.6 Hz), 156.1,
172.6 (d, 1H, JPC = 7.9 Hz). 31P NMR (81 MHz) δ 46.2-46.9.
HPLC tR(1) = 25.5, 26.6 min (2 isomers). ES-MS m/z: calcd for
[C29H40N2O7P þ H]þ 559.3; found 559.1.
(2S)-2-{[3-(40,50-Dihydro-30-phenyl-50-isoxazolyl)-2-{[hydroxyl-
(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]amino}ethyl)phosphinyl]-
methyl}-1-oxopropyl]amino} Propanoic Acid (2). Benzaldoxime
(0.12 g, 1.0 mmol) was dissolved in CH2Cl2 (6 mL), and addition
of phosphinic pseudodipeptide 12 (140 mg, 0.25 mmol) and
Et3N (35 μL, 0.25 mmol) followed. To this solution, common
bleach (4.5% aqueous solution of NaOCl, 1.65 mL, 1.0 mmol)
was slowly added, and the resulting biphasic mixture was
vigorously stirred at rt for 16 h. Then, the mixture was diluted
with CH2Cl2 (15 mL) and 1 M HCl was added for acidification
of the aqueous layer. The aqueous phase was separated and the
organic phase was washed with 1 M HCl (2 ꢀ 10 mL), H2O (10
mL) and brine (10 mL). Then, the organic phase was dried over
Na2SO4 and evaporated under vacuum. The residue was purified
by column chromatography (eluent: CHCl3/MeOH/AcOH =
70:3.5:3) and the pure cycloaddition product was dissolved in
ethanol (2 mL). The resulting solution was cooled to 0 °C and
1 M NaOH (2 mL) was added portionwise. After stirring at rt for
24 h, EtOH was evaporated, the residue was diluted with H2O,
and the resulting solution was washed with Et2O (2 ꢀ 5 mL). The
aqueous phase was acidified with 2 M HCl, and extractions with
AcOEt (3 ꢀ 10 mL) followed. The organic phase was washed
with H2O and brine, dried over Na2SO4, and evaporated under
vacuum. Compound 2 (137 mg, 88%) was obtained as a white
solid after addition of a Et2O/light petroleum (2:1) mixture to
the residue and filtration of the precipitate; mp 165-171 °C
(dec); Rf = 0.39. 1H NMR (200 MHz) δ 1.27 (appt, 3JHH = 6.9
Hz, 3H, CHCH3), 1.64-2.40 (m, 4H, CH2CHO, PCH2),
2.59-3.23 (m, 4H, CHCH2Ph, CHCONH, CHHCdN),
3.34-3.63 (m, 1H, CHHCdN), 3.76-4.08 (m, 1H, PCH),
4.10-4.33 (m, 1H, CHO), 4.49-4.77 (m, 1H, CHCH3),
4.78-5.10 (m, 2H, PhCH2O), 6.92-7.77 (m, 16H, Ar,
OCONH), 8.15-8.55 (m, 1H, NHCHCO). 13C NMR (50 MHz)
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl[2-({[(1S)-
1-(1H-indol-3-ylmethyl)-2-methoxy-2-oxoethyl]amino}carbonyl)-
pent-4-enyl] Phosphinic Acid (13). For the synthesis of 13,
phosphinic pseudodipeptide 11 (2.0 g, 4.4 mmol) was saponified
and coupled to L-tryptophan methyl ester according to the
synthetic protocol described for the preparation of compound
12. Treatment of the residue obtained after the final workup
with a mixture of Et2O/light petroleum (2:1) and filtration of the
precipitate furnished compound 13 (2.2 g, 80%) as a white solid;
mp 184-187 °C; Rf = 0.44 (CHCl3/MeOH/AcOH 7:0.5:0.5).
1H NMR (200 MHz) δ 1.42-2.12 (m, 2H, PCH2), 2.13-2.30 (m,
2H, CH2CHdCH2), 2.56-2.87 (m, 2H, PCH2CH, CHHPh),
2.90-3.42 (m, 3H, CH2indolyl, CHHPh), 3.59 (s, 3H, OCH3),
3.83-4.15 (m, 1H, PCH), 4.27-4.58 (m, 1H, NHCHCO),
4.62-5.02 (m, 4H, CHdCH2, PhCH2O), 5.05-5.28 (m,
0.25H, CHdCH2, first diastereoisomer), 5.41-5.63 (m, 0.75H,
CHdCH2, second diastereoisomer), 6.83-8.41 (m, 18H, Ar,
NH). 13C NMR (50 MHz) δ 26.7 (d, JPC = 89.7 Hz), 27.7 (d,
JPC = 89.7 Hz), 33.1, 33.6, 37.7 (d, JPC = 10.1 Hz), 47.6, 51.5 (d,
JPC = 101.3 Hz), 51.8, 53.6, 65.4, 110.6, 111.1, 111.3, 116.8,
118.2, 120.8, 123.5, 126.2, 126.7, 127.1, 127.3, 127.4, 127.6,
128.2, 128.3, 129.0, 135.3, 135.7, 136.1, 136.2, 137.2, 138.3,
138.6, 156.3 (d, JPC = 4.7 Hz), 173.2 (d, JPC = 7.5 Hz). 31P
NMR (81 MHz) δ 46.7, 47.4. HPLC tR(2) = 43.9 min (2
isomers). ES-MS m/z: calcd for [C34H38N3O7P þ H]þ 632.3;
found 632.5.
(2S)-2-{[3-(40,50-Dihydro-30-phenyl-50-isoxazolyl)-2-{[hydroxyl-
(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]amino}ethyl)phosphinyl]-
methyl}-1-oxopropyl]amino} 1H-Indole-3-propanoic Acid (1).
Phosphinic pseudotripeptide 13 (300 mg, 0.47 mmol) was dis-
solved in MeOH (5 mL), and the solution was cooled to 0 °C.
NaOH (1 M, 5.0 mL) was added portionwise, and the mixture
was stirred at rt for 24 h. Then, MeOH was evaporated and the
residue was diluted with H2O (10 mL) and acidified with 2 M
HCl. The white precipitate was filtered off and dried over P2O5
for 24 h. In a separate flask, benzaldoxime (340 mg, 2.8 mmol)
was dissolved in CHCl3 (15 mL), and 3-4 drops of pyridine were
added. Then NCS (374 mg, 2.8 mmol) was added at rt and after
10 min the resulting mixture was stirred at 45 °C for 3-4 h. In
this solution, the dry tripeptide obtained from the saponifica-
tion step was added, followed by slow addition of Et3N (254 μL,
3.8 mmol) at the same temperature. The reaction mixture was
stirred for 3 days at 45 °C. Then it was concentrated under
vacuum and the residue was diluted with AcOEt (30 mL) and
washed with 1 M HCl (2 ꢀ 10 mL), H2O (10 mL), 10%
NH4HCO3 (2 ꢀ 1 mL), 1 M HCl (2 ꢀ 10 mL), and brine
(10 mL). The organic phase was dried over Na2SO4 and
δ 16.9, 17.0, 17.1, 17.3, 27.5 (d, JPC = 88.4 Hz), 28.7 (d, JPC
=
88.4 Hz), 29.3 (d, JPC = 88.4 Hz), 33.0 (br), 36.0, 36.4, 37.6, 47.8/
47.9, 51.8 (d, JPC = 103.7 Hz), 52.0 (d, JPC = 103.7 Hz), 52.7 (d,
JPC = 103.7 Hz), 65.2, 79.1/79.3, 126.3, 126.6, 127.1, 127.6,
128.2, 128.4, 128.9, 129.1, 129.7, 130.1, 135.8, 137.3, 138.5 (d,
J
PC = 14.1 Hz), 156.2 (d, JPC = 3.4 Hz), 156.7 (br), 173.3, 173.5,
173.7, 174.1, 174.3. 31P NMR (81 MHz) δ 46.2, 46.9, 47.0, 47.5.
HPLC tR(3) = 22.6-26.8 min, (4 isomers). HRMS m/z: calcd
for [C32H36N3O8P þ H]þ 622.2318; found 622.2303.
(2S)-2-{[3-(40,50-Dihydro-30-[400-trifluoromethyl]phenyl-50-iso-
xazolyl)-2-{[hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]methyl}-1-oxopropyl]amino} 1H-Indole-
3-propanoic Acid (3). For the synthesis of 3, phosphinic pseu-
dodipeptide 13 (80 mg, 0.13 mmol) and 4-trifluoromethyl-
benzaldoxime were subjected to the same synthetic procedure
as described for the preparation of compound 1. Compound 3
(97 mg, 93%) was obtained as a white solid; mp 147-151 °C
1
(dec); Rf = 0.60. H NMR (200 MHz) δ 1.35-2.12 (m, 4H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 217
CH2CHO, PCH2), 2.58-3.49 (m, 7H, CHCH2Ph, CHCONH,
CH2indolyl, CH2CdN), 3.65-4.19 (m, 1.75H, PCH, CHO of
first diastereoisomer), 4.23-4.52 (m, 1.25H, CHO of second
diastereoisomer, NHCHCO), 4.77-5.02 (m, 2H, PhCH2O),
6.96-7.85, 7.97-8.25 and 8.48-8.85 (m, 22H, Ar, NH). 13C
NMR (50 MHz) δ 27.6 (d, JPC = 86.2 Hz), 28.3 (d, JPC = 84.9
Hz), 32.6 (br), 33.6, 36.9, 37.1, 42.3, 52.6 (d, JPC = 98.6 Hz),
53.5, 65.1, 78.7/79.1, 108.0, 110.6, 111.2, 112.1, 118.4, 118.6, 118.7,
120.7, 122.3, 123.7, 125.2, 126.2, 127.1, 127.2, 127.5, 128.2, 128.3,
136.3, 137.2, 137.3, 137.9, 138.2 (d, JPC = 10.1 Hz), 138.6,
140.1, 140.2, 156.1 (appt, JPC = 3.8 Hz), 173.2, 173.4, 173.5,
173.6. 31P NMR (81 MHz) δ 46.4, 47.2. HPLC tR(3) = 32.9, 34.2
min (2 isomers). HRMS m/z: calcd for [C43H42N3O7P þ H]þ
744.2760; found 744.2757.
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl[(2S)-2-
({[(1S)-1-(1H-indol-3-ylmethyl)-2-methoxy-2-oxoethyl]amino}-
carbonyl)-pent-4-ynyl] Phosphinic Acid (16). For the synthesis of
16, phosphinic pseudodipeptide 15 (5.0 g, 11.6 mmol) was
coupled to L-tryptophan methyl ester according to the synthetic
protocol described for the preparation of compound 6. After the
end of the coupling reaction, the mixture was diluted with CH2Cl2
(200 mL) and the solution was washed with 1 M HCl (4 ꢀ 30 mL),
H2O (30 mL), and brine (30 mL). The organic phase was dried over
Na2SO4, evaporated under vacuum, and a Et2O/light petroleum
(2:8) mixture was added to the residue. The precipitate was filtered,
washed with Et2O, and recrystallized by CH2Cl2 (∼10 mL/g).
After filtration, compound 16 (2.44 g, 33%) was isolated as a white
solid; mp 161-171 °C; Rf = 0.59; [R]D20 = -6.2 (c = 1.0, AcOH).
1H NMR (200 MHz) δ 1.71-2.17 (m, 2H, PCH2), 2.37-3.29 (m,
8H, CHCH2Ph, CH2indolyl, CHCH2CtCH), 3.54(s, 3H, OCH3),
3.82-4.09 (m, 1H, PCH), 4.45-4.67 (m, 1H, NHCHCO),
4.73-5.06 (m, 2H, PhCH2O), 6.82-7.60 (m, 16H, Ar, indole-
NH), 7.67 (d, 3JHH = 9.4 Hz, 1H, OCONH), 8.42 (d, 3JHH = 7.2
Hz, 1H, NHCHCO). 13C NMR (50 MHz) δ 22.1 (d, JPC = 7.5
Hz), 27.2, 27.4 (d, JPC = 88.7 Hz), 32.9, 38.1, 52.3 (d, JPC = 103.9
Hz), 51.6, 53.1, 65.2, 71.9, 81.7, 109.2, 111.3, 117.9, 118.3, 120.8,
123.7, 126.0, 126.9, 127.1, 127.4, 128.0, 128.1, 128.9, 136.1, 137.6,
138.2 (d, JPC = 14.1 Hz), 156.0 (d, JPC = 4.0 Hz), 172.0, 172.5 (d,
128.9, 129.2, 129.4, 131.3, 136.8, 137.3, 138.2, 138.6, 155.7 (d, JPC
=
2.4 Hz), 156.2, 172.3, 173.9 (d, JPC = 7.1 Hz). 31P NMR (81 MHz)
δ 45.7, 45.9, 46.6, 47.1. HPLC tR(2) = 44.4, 46.0 min (4 isomers).
HRMS m/z: calcd for [C41H40F3N4O8P þ H]þ 805.2614; found
805.2604.
(2S)-2-{[3-(30-[1,10-Biphenyl]-400-yl-40,50-dihydro-50-isoxazolyl)-
2-{[hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]amino}ethyl)-
phosphinyl]methyl}-1-oxopropyl]amino} 1H-Indole-3-propanoic
Acid (4). For the synthesis of 4, phosphinic pseudodipeptide
13 (80 mg, 0.13 mmol) and (1,10-biphenyl)-4-carbaldehyde
oxime were subjected to the same synthetic procedure as de-
scribed for the preparation of compound 1. Compound 4
(95 mg, 90%) was obtained as a white solid; mp 174-177 °C
1
(dec); Rf = 0.71. H NMR (200 MHz) δ 1.34-2.17 (m, 4H,
CH2CHO, PCH2), 2.55-3.52 (m, 7H, CHCH2Ph, CHCONH,
CH2indolyl, CH2CdN), 3.65-4.22 (m, 1.75H, PCH, CHO of
first diastereoisomer), 4.26-4.57 (m, 1.25H, CHO of second
diastereoisomer, NHCHCO), 4.78-5.02 (m, 2H, PhCH2O),
6.83-8.19 (m, 26H, Ar, indole-NH, OCONH), 8.28-8.52 (m,
1H, NHCHCO). 13C NMR (50 MHz) δ 27.4 (d, JPC = 84.9 Hz),
28.5 (d, JPC = 84.9 Hz), 32.7 (br), 33.4, 36.9, 37.3, 42.3, 52.3 (d,
J
PC = 9.5 Hz). 31P NMR (81 MHz) δ 46.6. HPLC tR(3) = 27.1
JPC = 98.3 Hz), 53.6, 65.4, 78.8/79.1, 110.7, 111.2, 112.1, 118.4,
min. ES-MS m/z: calcd for [C34H36N3O7P þ H]þ 630.2; found
630.2. The filtrates were concentrated and recrystallized by CH2Cl2
(∼10 mL/g). After filtration, the new filtrates were concentrated
and triturated with a Et2O/light petroleum (3:7) mixture. The
mixture was filtrated and a white solid was obtained corresponding
to a 1:3 mixture of 16/17 (3.21 g, 43%).
118.6, 118.7, 120.6, 123.7, 126.2, 127.1, 127.2, 127.5, 128.3,
128.9, 129.0, 131.4, 136.8, 137.3, 138.2, 138.6, 139.6, 155.8 (d,
J
PC = 2.4 Hz), 156.1, 172.1, 173.8 (d, JPC = 7.1 Hz). 31P NMR
(81 MHz) δ 45.9, 46.1, 46.8, 47.1. HPLC tR(2) = 46.0, 47.4, 48.0
min, (4 isomers). HRMS m/z: calcd for [C46H45N4O8P þ H]þ
813.3053; found 813.3057.
(2S)-2-({3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]-(2S)-2-[(3-phenylisoxazol-5-yl)methyl]-
1-oxopropyl}amino) 1H-Indole-3-propanoic Acid, Methyl Ester
(18). Benzaldoxime (1.35 g, 11.1 mmol) was dissolved in CHCl3
(30 mL), and 5 drops of pyridine were added. Then, NCS (1.48 g,
11.1 mmol) was added at rt and after 10 min the resulting
mixture was stirred at 45 °C for 3 h. In this solution, phosphinic
tripeptide 16 (700 mg, 1.11 mmol) was added, followed by slow
addition of Et3N (1.8 mL, 12.9 mmol) at the same temperature.
The reaction mixture was stirred for 4 days at 45 °C. Then, it was
diluted with CHCl3 (70 mL) and the resulting solution was
washed with 1 M HCl (2 ꢀ 30 mL). The organic phase was
concentrated (without prior drying) and the residue was recrys-
tallized twice by AcOEt (∼25 mL/g). Compound 18 (580 mg,
70%) was isolated as a white solid after filtration of the
precipitate and washings with Et2O; mp 193-195 °C; Rf =
0.61; [R]D20 = -4.4 (c = 0.25, AcOH). 1H NMR (200 MHz) δ
1.73-2.13 (m, 2H, PCH2), 2.62-2.89 (m, 1H, CHCHHPh),
2.94-3.55 (m, 6H, CHCHHPh, CH2indolyl, CHCH2isoxazolyl),
3.40 (s, 3H, OCH3), 3.83-4.10 (m, 1H, PCH), 4.40-4.60 (m,
1H, NHCHCO), 4.72-5.06 (m, 2H, PhCH2O), 6.51 (s, 1H, H of
isoxazole ring), 6.85-7.87 (m, 22H, Ar, OCONH, indole-NH),
8.73 (d, 3JHH = 7.2 Hz, 1H, NHCHCO). 13C NMR (50 MHz) δ
27.2, 28.5 (d, JPC = 93.0 Hz), 29.4, 32.8, 37.4, 51.7, 52.5 (d,
JPC = 103.8 Hz), 53.4, 65.2, 100.2, 109.3, 111.5, 118.0, 118.4,
121.0, 123.9, 126.3, 126.5, 127.0, 127.6, 128.2, 128.3, 128.9,
129.1, 130.1, 136.1, 137.2, 138.4 (d, JPC = 14.1 Hz), 156.1 (d,
(2S)-2-{[3-(1,10-Biphenyl)-2-{[hydroxyl(2-phenyl-(1R)-1-{[(ben-
zyloxy)carbonyl]amino}ethyl)phosphinyl]methyl}-1-oxopropyl]-
amino} 1H-Indole-3-propanoic Acid (6). Phosphinic pseudodi-
peptide 14 (65 mg, 0.12 mmol) was suspended in CH2Cl2
(1.0 mL), and DIPEA (42 μL, 0.24 mmol), L-tryptophan methyl
ester (28 mg, 0.13 mmol), HOBt (16 mg, 0.12 mmol), and
EDC HCl (138 mg, 0.72 mmol) were added to the resulting
3
suspension. The mixture was stirred for 2 h at rt. Then the
solvent was evaporated, AcOEt (15 mL) and 1 M HCl (5 mL)
were added, and the organic layer was separated and washed
with 1 M HCl (2 ꢀ 5 mL), H2O (5 mL) and brine (5 mL). The
organic phase was dried over Na2SO4, evaporated under va-
cuum, and the residue was purified by column chromatography
(CHCl3/MeOH/AcOH 70:1:1). The resulting tripeptide was
suspended in MeOH (5 mL), and 1 M NaOH (2 mL) was added
portionwise at 0 °C. After stirring at rt for 48 h, MeOH was
evaporated, the residue was diluted with water and acidification
followed with the addition of 2 M HCl. The mixture was
extracted with AcOEt (3 ꢀ 15 mL) and the combined organic
layers were washed with H2O and brine, dried over Na2SO4, and
evaporated under vacuum. Compound 6 (70 mg, 81%) was
obtained as a white solid after addition of Et2O to the residue
and filtration of the precipitate; mp 196-204 °C (dec); Rf =
0.61. 1H NMR (200 MHz) δ 1.57-2.09 (m, 2H, PCH2),
2.56-3.24 (m, 7H, CHCH2Ph, ArCH2CHCONH, CH2indolyl),
3.82-4.08 (m, 1H, PCH), 4.44-4.64 (m, 1H, NHCHCO),
4.74-5.02 (m, 2H, PhCH2O), 6.86-7.77 (m, 26H, Ar,
J
PC = 3.4 Hz), 161.7, 171.6, 172.3, 172.8 (d, JPC = 10.9 Hz). 31
P
3
indole-NH, OCONH), 8.31 and 8.41 (d, JHH = 7.4 Hz, 1H,
NMR (81 MHz) δ 45.4. HPLC tR(3) = 31.1 min. ES-MS m/z:
NHCHCO). 13C NMR (50 MHz) δ 27.1 (d, JPC = 88.6 Hz), 27.6
(d, JPC = 88.6 Hz), 27.3, 27.4, 32.9 (br), 52.1 (d, JPC = 103.2
Hz), 52.6 (d, JPC = 103.2 Hz), 52.9, 53.1, 65.1/65.2, 109.9, 111.5,
118.3, 118.4, 120.1, 123.8, 126.2, 126.4, 126.6, 127.0, 127.1,
127.2, 127.3, 127.6, 128.2, 128.3, 128.9, 129.1, 129.8, 136.2,
calcd for [C41H41N4O8P þ H]þ 749.3; found 749.2.
(2S)-2-({3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]-(2R)-2-[(3-phenylisoxazol-5-yl)methyl]-
1-oxopropyl}amino) 1H-Indole-3-propanoic Acid, Methyl Ester
(19). A 1:3 mixture of 16/17 (500 mg, 0.79 mmol), isolated during

218 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Jullien et al.
the synthesis of 16, was subjected to 1,3-DCR according to the
synthetic protocol followed for the synthesis of 18. After the end
of the reaction, the mixture was diluted with CHCl3 (100 mL)
and the resulting solution was washed with 1 M HCl (3 ꢀ 20
mL), H2O (20 mL), and brine (10 mL). The organic phase was
dried over Na2SO4, evaporated under vacuum, and the residue
was refluxed with CHCl3 (50 mL). The hot mixture was filtrated,
and the filtrates were concentrated and dissolved to a minimum
quantity of CHCl3. A white solid (corresponding to 19) was
precipitated after 24 h at room temperature which was isolated
after filtration. A second crop of 19 was obtained after repeating
the same crystallization procedure to the filtrates. The combined
J= 2.2 Hz, indole-NH). 13C NMR (125.7 MHz, DMSO-d6) δ27.3,
29.8, 29.8, 30.7, 33.1, 38.1, 52.5 (d, JPC = 106 Hz), 54.3, 64.7, 99.9,
110.3, 111.3, 118.1, 118.3, 120.8, 123.5, 125.7, 126.4, 126.8, 127.1,
127.3, 127.9, 128.1, 128.7, 128.9, 129.0, 129.9, 136.1, 137.5, 139.6,
155.8, 161.5, 171.9, 173.0, 173.5. HPLC tR(3) = 30.7 min. HRMS
m/z: calcd for [C40H39N4O8P þ H]þ 735.2583; found 735.2587.
3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]amino}-
ethyl)phosphinyl]-2-[(3-phenylisoxazol-5-yl)methyl] Propanoic
Acid (20). Benzaldoxime (340 mg, 2.8 mmol) was dissolved in
CHCl3 (15 mL), and 3 drops of pyridine were added. Then NCS
(374 mg, 2.8 mmol) was added at rt and after 10 min the resulting
mixture was stirred at 45 °C for 3-4 h. In this solution,
phosphinic dipeptide 15 (300 mg, 0.7 mmol) was added, fol-
lowed by slow addition of Et3N (0.58 mL, 4.2 mmol) at the same
temperature. The reaction mixture was stirred for 3 days at
45 °C. Then, it was concentrated under vacuum, and the residue
was dissolved in 5% NaHCO3 (10 mL). The resulting solution
was washed with Et2O (4 ꢀ 10 mL), and the aqueous phase was
acidified with 1 M HCl and extracted with AcOEt (5 ꢀ 15 mL).
The combined organic layers were washed with H2O (10 mL)
and brine (10 mL), dried over Na2SO4, and evaporated under
vacuum. The reaction was repeated three more times in order to
completely consume the unreacted starting material. Com-
pound 20 (320 mg, 83%, 1:1 diastereoisomeric mixture) was
obtained as a yellowish solid after addition of Et2O to the
final residue, boiling of the suspension, filtration of the hot
mixture, and washings with Et2O; mp 168-170 °C (dec); Rf =
0.42. 1H NMR δ 1.67-1.93 (m, 1H, PCHH), 1.98-2.22 (m, 1H,
PCHH), 2.59-2.86 (m, 1H, CHCHHPh), 2.92-3.29 (m, 4H,
CHCHHPh, CHCH2isoxazolyl), 3.76-4.01 (m, 1H, PCH),
4.70-4.95 (m, 2H, PhCH2O), 6.72 (s, 1H, H of isoxazole ring),
6.97-8.07 (m, 16H, Ar, NH). 13C NMR δ 27.6 (d, JPC = 88.5
Hz), 27.8 (d, JPC = 88.5 Hz), 29.2, 29.3, 32.7 (br), 37.5, 37.8, 52.3
(d, JPC = 105.0 Hz), 52.6 (d, JPC = 105.0 Hz), 65.2, 100.6, 126.3,
126.6, 127.0, 127.1, 127.6, 128.2, 128.3, 128.8, 129.2, 130.2,
137.3, 138.4, (d, JPC = 13.5 Hz), 156.1 (d, JPC = 3.2 Hz),
161.8, 171.3, 174.5 (d, JPC = 9.8 Hz). 179.5. 31P NMR δ 45.4,
45.6. HPLC tR(3) = 27.6 min (2 isomers). ES-MS m/z: calcd for
[C29H29N2O7P - H]þ 547.2; found 547.3.
solids were washed with hot Et2O to afford compound 19 (350
20
mg, 59%) as a white solid; mp 218-220 °C; Rf = 0.66; [R]D
=
-28.6 (c = 0.50, DMSO). 1H NMR (200 MHz) δ 1.64-1.89 (m,
1H, PCHH), 1.93-2.18 (m, 1H, PCHH), 2.61-2.82 (m, 1H,
CHCHHPh), 2.89-3.34 (m, 6H, CHCHHPh, CH2indolyl,
CHCH2isoxazolyl), 3.51 (s, 3H, OCH3), 3.80-4.08 (m, 1H,
PCH), 4.40-4.60 (m, 1H, NHCHCO), 4.76-5.03 (m, 2H,
PhCH2O), 6.39 (s, 1H, H of isoxazole ring), 6.88-7.76 (m,
3
22H, Ar, OCONH, indole-NH), 8.71 (d, JHH = 7.0 Hz, 1H,
NHCHCO). 13C NMR (50 MHz) δ 27.3, 27.5 (d, JPC = 88.5
Hz), 29.7, 32.9, 37.5, 51.3, 51.8, 53.3, 65.1, 100.2, 109.4, 111.5,
118.0, 118.5, 121.1, 123.8, 126.3, 126.4, 127.0, 127.6, 128.2,
128.3, 128.7, 129.1, 130.1, 136.1, 137.2, 138.4 (d, JPC = 14.2
Hz), 156.0 (d, JPC = 3.9 Hz), 161.6, 171.4, 172.2, 172.4 (d, JPC
=
9.7 Hz). 31P NMR (81 MHz) δ 46.1. HPLC tR(3) = 30.7 min.
ES-MS m/z: calcd for [C41H41N4O8P þ H]þ 749.3; found 749.3.
(2S)-2-({3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]-(2S)-2-[(3-phenylisoxazol-5-yl)methyl]-
1-oxopropyl}amino) 1H-Indole-3-propanoic Acid (5F1). Phosphi-
nic pseudotripeptide 18 (211 mg, 0.28 mmol) was dissolved in
MeOH (3 mL), and the solution was cooled to 0 °C. NaOH (1 M,
1.25 mL) was added portionwise, and the mixture was stirred at
rt for 24 h. Then MeOH was evaporated, the aqueous residue
was acidified with 2 M HCl, and the precipitate was filtrated.
Compound 5F1 (177 mg, 86%) was obtained as a white solid
after washings of the solid product with hot Et2O; mp 186-
189 °C; Rf = 0.49; [R]D20 = -7.8 (c = 0.25, AcOH). 1H NMR
(500.1 MHz, DMSO-d6) δ 1.63-1.79 (m, 2H, PCH2), 2.69 (dt,
J = 6.0, 13.7 Hz, 1H, CHCHHPh), 3.02-3.13 (m, 5H, CHCH2i-
soxazolyl, CHHindolyl, CHCHHPh), 3.24 (dd, J = 4.8, 14.6
Hz, 1H, CHHindolyl), 3.69-3.77 (m, 1H, PCH), 4.21-4.33 (m,
1H, NHCHCO), 4.72 (d, J = 13.0 Hz, 1H, PhCHHO), 4.91 (d,
J = 13.0 Hz, 1H, PhCHHO), 6.53 (s, 1H, H of isoxazole ring),
6.94-7.72 (m, 21H, Ar, OCONH), 8.86 (br s, 1H, NHCHCO),
10.83 (d, J = 1.8 Hz, 1H, indole-NH). 13C NMR (125.7 MHz,
DMSO-d6) δ 26.8, 29.5, 30.7, 33.2, 38.7, 53.1 (d, JPC = 106 Hz),
53.5, 53.9, 64.7, 99.7, 110.3, 111.3, 118.1, 118.2, 120.8, 125.7,
126.4, 126.8, 127.2, 127.3, 127.9, 128.1, 128.8, 128.9, 129.0,
129.8, 136.0, 137.4, 139.6, 155.9, 161.5, 172.2, 173.1, 173.3.
HPLC tR(3) = 29.4 min. HRMS m/z: calcd for [C40H39N4O8P þ
H]þ 735.2583; found 735.2599.
(2S)-2-({3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]-(2S)-2-[(3-phenylisoxazol-5-yl)methyl]-
1-oxopropyl}amino)-3-phenyl Propanoic Acid (7F1) and (2S)-2-
({3-[Hydroxyl(2-phenyl-(1R)-1{[(benzyloxy)carbonyl]amino}ethyl)
Phosphinyl]-(2S)-2-[(3-phenylisoxazol-5-yl)methyl]-1-oxopro-
pyl}amino)-3-phenyl Propanoic Acid (7F2). Phosphinic pseudo-
dipeptide 20 (150 mg, 0.27 mmol) was suspended in CH2Cl2 (2.3
mL), and DIPEA (141 μL, 0.81 mmol), hydrochloric L-pheny-
lalanine tert-butyl ester (75 mg, 0.29 mmol), HOBt (37 mg, 0.27
mmol), and EDC HCl (310 mg, 1.62 mmol) were added to the
3
resulting suspension. The mixture was stirred for 2 h at rt. Then
the solvent was evaporated, AcOEt (30 mL) and 1 M HCl (15
mL) were added, and the organic layer was separated and
washed with 1 M HCl (2 ꢀ 10 mL), H2O (10 mL), and brine
(10 mL). The organic phase was dried over Na2SO4, evaporated
under vacuum, and the residue was purified by column chro-
matography (CHCl3/MeOH/AcOH 70:1:1). The resulting tri-
peptide was suspended in MeOH (10 mL) and 1 M NaOH (4
mL) was added portionwise at 0 °C. After stirring at rt for 24 h,
MeOH was evaporated, the residue was diluted with water, and
acidification followed with the addition of 2 M HCl. The
mixture was extracted with AcOEt (3 ꢀ 15 mL), and the
combined organic layers were washed with H2O and brine,
dried over Na2SO4, and evaporated under vacuum. Compound
7 (165 mg, 87%) was obtained as a white solid after addition of
Et2O to the residue and filtration of the precipitate; mp 172-177
°C (dec); Rf = 0.41. HPLC tR(3) = 30.1, 31.5 min (2 isomers).
ES-MS m/z: calcd for [C38H38N3O8P þ H]þ 696.2; found 696.2.
Diastereoisomers 7F1 and 7F2 were isolated after separation by
preparative reverse-phase HPLC (AIT column 250 mm ꢀ
10 mm, kromasil C18, 10 μm, 3 mL/min) under isocratic elution
(2S)-2-({3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]-(2R)-2-[(3-phenylisoxazol-5-yl)methyl]-
1-oxopropyl}amino) 1H-Indole-3-propanoic Acid (5F2). For the
synthesis of 5F2, phosphinic pseudotripeptide 19 (112 mg, 0.15
mmol) was saponified and treated according to the synthetic
protocol described for the preparation of compound 5F1. Com-
pound 5F2 (103 mg, 94%) was obtained as a white solid; mp 183-
185 °C; Rf = 0.52; [R]D20 = -21.5 (c = 1.0, DMSO). 1H NMR
(500.1 MHz, DMSO-d6) δ 1.62-1.76 (m, 2H, PCH2), 2.62-2.70
(m, 1H, CHCHHPh), 2.72-2.80 (m, 1H, CHHisoxazolyl), 2.95
(dd, J = 7.9, 15.3 Hz, 1H, CHHisoxazolyl), 2.98 (dd, J = 9.1, 14.8
Hz, 1H, CHHindolyl), 3.06-3.12 (m, 2H, CHCH2isoxazolyl,
CHCHHPh), 3.18 (dd, J = 4.7, 14.8 Hz, 1H, CHHindolyl),
3.66-3.81 (m, 1H, PCH), 4.34-4.42 (m, 1H, NHCHCO), 4.81
(d, J = 13.0 Hz, 1H, PhCHHO), 4.89 (d, J = 13.0 Hz, 1H,
PhCHHO), 6.47 (s, 1H, H of isoxazole ring), 6.93-7.67 (m, 21H,
Ar, OCONH), 8.67 (d, J = 7.4 Hz, 1H, NHCHCO), 10.77 (d, 1H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 219
(43% CH3CN in 0. 1% TFA) tR (7F1) = 30.7 min; tR (7F2) =
34.7 min.
(dd, J = 9.0, 13.7 Hz, 1H, CHHCHCOOH), 2.90-2.95 (m, 2H,
CHHCHCOOH, CHHisoxazolyl), 2.99-3.07 (m, 1H,
CHCH2isoxazolyl), 3.09 (d, J = 14 Hz, 1H, PCHCHHPh),
3.63-3.69 (m, 1H, PCH), 4.16 (dt, J = 4.7, 8.4 Hz, 1H,
NHCHCO), 4.80 (d, J = 13.0 Hz, 1H, PhCHHO), 4.89 (d,
J = 13.0 Hz, 1H, PhCHHO), 6.53-6.58 (m. 2H, Hε Tyr), 6.61
(s, 1H, H of isoxazole ring), 6.93-7.80 (m, 18H, Ar, OCONH),
8.52 (br s, 1H, NHCHCO). 13C NMR (125.7 MHz, DMSO-d6) δ
7F1: 1H NMR (500.1 MHz, DMSO-d6) δ 1.56-1.60 (m, 2H,
PCH2), 2.59-2.66 (m, 1H, PCHCHHPh), 2.83 (dd, J = 5.6, 15.4
Hz, 1H, CHHisoxazolyl), 2.89 (dd, J = 10.5, 13.5 Hz, 1H,
CHHCHCOOH), 2.90-2.98 (m, 1H, CHCH2isoxazolyl), 3.02
(dd, J = 8.5, 15.3 Hz, 1H, CHHisoxazolyl), 3.08 (d, J = 13.0,
Hz, 1H, PCHCHHPh), 3.14 (dd, J = 3.9, 13.5 Hz, 1H,
CHHCHCOOH), 3.56-3.69 (m, 1H, PCH), 3.98-4.10 (m,
1H, NHCHCO), 4.74 (d, J = 13.1 Hz, 1H, PhCHHO), 4.91
(d, J = 13.1 Hz, 1H, PhCHHO), 6.62 (s, 1H, H of isoxazole
ring), 7.06-7.81 (m, 21H, Ar, OCONH), 8.84 (d, J = 7.0 Hz,
1H, NHCHCO). 13C NMR (125.7 MHz, DMSO-d6) δ 29.9,
30.0, 31.9 (d, JPC = 83.0 Hz), 33.6, 36.5, 39.0, 53.4 (d, JPC = 105
Hz), 55.4, 64.6, 99.8, 125.6, 125.9, 126.5, 126.8, 126.9, 127.3,
127.8, 127.9, 128.1, 128.9, 129.0, 129.0, 129.2, 129.9, 137.5,
139.0, 140.0, 140.2, 155.8, 161.6, 172.4, 172.7, 173.1. HRMS,
m/z: calcd for [C38H38N3O8P þ H]þ 696.2474; found 696.2507.
30.2, 30.3, 31.5 (d, JPC = 87 Hz), 33.2, 36.4, 38.3, 52.5 (d, JPC
=
105 Hz), 55.8, 64.6, 99.9, 114.7, 125.5, 126.4, 126.8, 127.3, 127.8,
128.1, 128.7, 128.9, 129.0, 129.9, 137.6, 140.1, 155.5, 155.8,
161.5, 172.3, 173.1. 173.6. HRMS, m/z: calcd for [C38H38-
N3O9P þ H]þ 712.2424; found 712.2443
Inhibitor Potency. Enzymes and inhibitors were incubated for
45 min before the initiation of the reaction by substrate addition.
Assays were carried out at 25 °C in 50 mM Hepes (pH 6.8), 200
mM NaCl, 10 μM ZnCl2, and 0.02% Brij-35. Continuous assays
were performed by recording the fluorescence increase at 405 nm
(εex = 320 nm) induced by the cleavage of fluorogenic sub-
strates, using black, flat-bottomed, 96-well nonbinding surface
plates (Corning-Costar, Schiphol-RijK, The Netherlands).
Fluorescence signals were monitored using a Fluoroscan
Ascent photon counter spectrophotometer (Thermo-Labsys-
tems, Courtaboeuf, France) equipped with a temperature con-
trol device and a plate shaker. The substrate and enzyme
concentrations for the experiments were chosen so as to remain
well below 10% of substrate utilization and to observe initial
rates. For each inhibitor, percentage inhibition was determined
in triplicate experiments at five inhibitor concentrations, chosen
to observe a 20-80% range of inhibition. Ki values were
determined using the method proposed by Horovitz and
Leviski.36
7
F2: 1H NMR (500.1 MHz, DMSO-d6) δ 1.59-1.63 (m, 2H,
PCH2), 2.59-2.68 (m, 2H, CHHisoxazolyl, PCHCHHPh), 2.81
(dd, J = 10.0, 13.8 Hz, 1H, CHHCHCOOH), 2.90 (dd, J = 8.5,
15.5 Hz, 1H, CHHisoxazolyl), 3.00-3.10 (m, 3H, CHCH2isox-
azolyl, PCHCHHPh, CHHCHCOOH), 3.68-3.78 (m, 1H,
PCH), 4.28-4.36 (m, 1H, NHCHCO), 4.82 (d, J = 13.0 Hz,
1H, PhCHHO), 4.89 (d, J = 13.0 Hz, 1H, PhCHHO), 6.57 (s,
1H, H of isoxazole ring), 7.00-7.78 (m, 21H, Ar, OCONH),
8.64 (d, J = 7.9 Hz, 1H, NHCHCO). 13C NMR (125.7 MHz,
DMSO-d6) δ 30.1, 30.2, 31.7 (d, JPC = 88.0 Hz), 33.1, 37.0, 38.4,
52.4 (d, JPC = 106 Hz), 54.9, 64.6, 99.9, 125.6, 126.0, 126.4,
126.8, 127.3, 127.8, 127.9, 128.1, 128.8, 128.9, 129.0, 129.0,
129.9, 137.6, 138.3, 139.9, 155.8, 161.5, 172.0, 173.2, 173.4.
HRMS, m/z: calcd for [C38H38N3O8P þ H]þ 696.2474; found
696.2483.
ACE. Inhibition assays were performed using Mca-Ala-Ser-
Asp-Lys-Dpa-OH from Enzo Life Sciences, as substrate (8 μM)
and human somatic ACE (0.5 nM) from R&D systems (929-ZN-
010). The Ki values for the N- and C-domains were obtained as
previously described.37
ECE-1. Inhibition assays were performed using Mca-Arg-
Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)OH from Enzo Life
Sciences, as substrate (8 μM, Km = 4.6 μM) and human ECE-
1 (0.1 nM) from R&D Systems (1784-ZN-010). The Km value
was estimated according to the direct linear plot.38
NEP. Inhibition assays were performed using Mca-Arg-Pro-
Pro-Gly-Phe-Ser-Pro-Dpa-OH from Enzo Life Sciences, as
substrate (5 μM, Km = 2 μM) and human NEP (0.5 nM) from
R&D Systems (1182-ZN-010). The Km value was estimated
according to the direct linear plot.38
(2S)-2-({3-[Hydroxyl(2-phenyl-(1R)-1-{[(benzyloxy)carbonyl]-
amino}ethyl)phosphinyl]-2-[(3-phenylisoxazol-5-yl)methyl]-1-oxo-
propyl}amino)-3-(4-hydroxy-phenyl) Propanoic Acid (8). For the
synthesis of 8, phosphinic pseudodipeptide 20 (175 mg, 0.32
mmol) was coupled to L-tyrosine(tert-butyl ether) tert-butyl
ester and the product was subjected to saponification according
to the synthetic protocol described for the preparation of
compound 7. The saponified product was dissolved in HCO2H
(5 mL), TIS (0.1 mL) was added, and the resulting solution was
stirred for 24 h at rt. Then, HCO2H was evaporated and Et2O
was added to the residue. Compound 8 (205 mg, 90%) was
obtained as a white solid after filtration of the precipitate and
washing with Et2O; mp 189-192 °C (dec); Rf = 0.40. HPLC
tR(3) = 26.3, 26.8 min (2 isomers). ES-MS m/z: calcd for
[C38H38N3O9P þ H]þ 712.2; found 712.2. Diastereoisomers
8F1 and 8F2 were isolated after separation with preparative
reverse-phase HPLC (AIT column 250 mm ꢀ 10 mm, kromasil
C18, 10 μm, 3 mL/min) under isocratic elution (52% CH3CN in
0. 1% TFA) tR (8F1) = 21.8 min; tR (8F2) = 27.8 min.
MMP-13. Inhibition assays were performed using Mca-Pro-
Leu-Gly-Leu-Dpa-Ala-Arg-NH2, as substrate (13 μM, Km
=
8.5 μM) and human MMP-13 (3 nM) from R&D Systems (511-
MM-010), as previously described.39
Ki Values in the Presence of Serum Proteins. Rat plasma was
diluted (1/50 or 1/100) in 50 mM Hepes (pH 6.8), 200 mM NaCl,
10 μM ZnCl2 and 0.02% Brij-35 at 25 °C. ACE (final concen-
tration 1 nM) and inhibitors at various concentrations were
incubated in the diluted plasma for 45 min. The inhibition assays
were initiated by adding to these solutions Mca-Ala-Ser-Asp-
Lys-Dpa-OH, as substrate (15 μM).24
Measurement of MAP. Male SHR rats (250 g, 12 weeks old,
from Charles River, France) were anesthetized with Nesdonal
(50 mg/kg, ip) and instrumented with an electronic transducer
(SPR 407, Millar Instruments, Houston, TX) inserted into the
femoral artery to measure mean arterial blood pressure (MAP)
and with catheters in veins to administer inhibitors. A tracheot-
omy was performed and a cannula inserted in the trachea to
ensure airway patency. The body temperature of the animals
was maintained at 37 °C by means of a heating blanket.
Following surgery, MAP was allowed to stabilize for 20 min
before injection of inhibitors or vehicle (0.09% NaCl) and
measured for 60 min (n = 6/dose).
8
F1: 1H NMR (500.1 MHz, DMSO-d6) δ 1.53-1.64 (m, 2H,
PCH2), 2.64 (dt, J = 5.5, 13.2 Hz, 1H, PCHCHHPh), 2.75 (dd,
J = 10.0, 14.0 Hz, 1H, CHHCHCOOH), 2.88 (dd, J = 5.2, 15.0
Hz, 1H, CHHisoxazolyl), 2.91-3.01 (m, 1H, CHCH2isoxazolyl),
2.97 (dd, J = 5.5, 15.0 Hz, 1H, CHHCHCOOH), 3.04 (t, J = 7.7
Hz, 1H, CHHisoxazolyl), 3.10 (dt, J = 3.5, 14.5 Hz, 1H,
PCHCHHPh), 3.59-3.67 (m, 1H, PCH), 3.92-4.01 (m, 1H,
NHCHCO), 4.73 (d, J = 13.0 Hz, 1H, PhCHHO), 4.92 (d, J =
13.0 Hz, 1H, PhCHHO), 6.58-6.62 (m. 2H, Hε Tyr), 6.64 (s, 1H,
H of isoxazole ring), 6.95-7.81 (m, 18H, Ar, OCONH), 8.86 (d,
J = 7.4 Hz, 1H, NHCHCO). 13C NMR (125.7 MHz, DMSO-d6)
δ 29.8, 31.9 (d, JPC = 83.0 Hz), 33.6, 36.2, 38.9, 53.4 (d, JPC
=
105 Hz), 55.9, 64.6, 99.7, 114.8, 125.5, 126.5, 126.8, 127.3, 127.8,
128.1, 128.9, 129.0, 129.1, 129.9, 130.1, 137.5, 140.1, 140.2,
155.5, 155.8, 161.6, 172.7, 172.8, 173.2. HRMS, m/z: calcd for
[C38H38N3O9P þ H]þ 712.2424; found 712.2457.
8F2: 1H NMR (500.1 MHz, DMSO-d6) δ 1.51-1.66 (m, 2H,
PCH2), 2.60-2.67 (m, 2H, PCHCHHPh, CHHisoxazolyl), 2.71

220 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Jullien et al.
Supporting Information Available: NMR characterization
of compounds 5F1, 5F2, 7F1, 7F2, 8F1, and 8F2. This material
is available free of charge via the Internet at http://pubs.
acs.org.
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