NAcSDKP analogues resistant to angiotensin-converting enzyme

Article abstract of DOI:10.1021/jm9701132

Two series of analogues of the tetrapeptide NAcSDKP, an inhibitor of hematopoietic stem cell proliferation, were prepared, and their enzymatic stability toward rabbit lung angiotensin-converting enzyme (ACE) was evaluated as well as their capacity to inhibit NAcSDKP hydrolysis by this enzyme. In the first series, each of the peptide bonds has been successively replaced by an aminomethylene bond. In the second one, the C-terminus of the peptide has been modified by decarboxylation or amidation. The results reported here indicate that all of these molecules but one have good stability toward the enzyme but none of the compounds is able to inhibit NAcSDKP hydrolysis by ACE.

Full text of DOI:10.1021/jm9701132

J . Med. Chem. 1997, 40, 3963-3968
3963
Notes
NAcSDKP An a logu es Resista n t to An gioten sin -Con ver tin g En zym e
Sandrine Gaudron, Marie-The´re`se Adeline, Pierre Potier, and J osiane Thierry*
Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette, France
Received February 25, 1997X
Two series of analogues of the tetrapeptide NAcSDKP, an inhibitor of hematopoietic stem cell
proliferation, were prepared, and their enzymatic stability toward rabbit lung angiotensin-
converting enzyme (ACE) was evaluated as well as their capacity to inhibit NAcSDKP hydrolysis
by this enzyme. In the first series, each of the peptide bonds has been successively replaced
by an aminomethylene bond. In the second one, the C-terminus of the peptide has been modified
by decarboxylation or amidation. The results reported here indicate that all of these molecules
but one have good stability toward the enzyme but none of the compounds is able to inhibit
NAcSDKP hydrolysis by ACE.
The peptide NAcSDKP (1), first isolated from foetal
calf bone marrow, has been demonstrated to be a
negative regulator of stem cell proliferation.1 Thus its
use during chemotherapy has been proposed to prevent
the hematopoietic stem cells from proliferating and
being destroyed by the cytotoxic phase-dependent drugs.
Clinical trials using cytosine arabinoside and cyclophos-
phamide as anti-cancer drugs and NAcSDKP as a
protecting drug have been initiated.2
In vitro stability studies of NAcSDKP (1) in human
plasma have demonstrated the role of angiotensin-
converting enzyme (ACE) in the catabolism of the
peptide3 resulting in cleavage of the Asp-Lys bond.
Further studies have shown 1 to be a natural and
specific substrate for the N-terminal active site of
human ACE.4
In vivo, peptides are subject to proteolysis and rapid
elimination. Many strategies have been devised during
the past decade to design biologically active peptides
resistant to proteolysis.6 Among these, cyclization or
replacement of the scissile bond by diverse amide bond
surrogates have been most often used.5,7,8 In order to
get more stable molecules for therapeutic use and to
gain structural information about the peptide backbone,
each of the amide bonds of the tetrapeptide 1 has been
successively replaced by the so-called reduced bond
giving the analogues 2, 3, and 4.
The proposed mechanism for the hydrolysis of angio-
tensin I by ACE involves an ionic interaction between
the C-terminal carboxylate group and a protonated
arginine side chain in the active site of ACE. This
model has been successfully exploited to design inhibi-
tors of ACE.9 We hypothesized that the suppression of
this ionic bond would reduce the affinity of the substrate
for the enzyme and therefore increase the half-life of
the molecule in physiological medium. Thus, two
analogues lacking the carboxylate moiety have been
synthesized: one with a carboxamide C-terminus 5 and
the other lacking the C-terminal carboxylate group 6.
The present paper deals with the synthesis and the
stability studies toward ACE of the five following
peptides and pseudopeptides analogues of 1 (Figure 1).
F igu r e 1. NAcSDKP analogues. The arrows indicate the site
of modification.
As such manipulations of the peptide bond have
sometimes yielded inhibitors, and also in view of the
resemblance of NAcSDKP with lisinopril, a well-known
inhibitor of ACE, the inhibitory potency of these mol-
ecules toward the hydrolysis of NAcSDKP by ACE has
also been assessed.
Ch em istr y
The different syntheses were realized step by step
using mainly the mixed anhydride methodology.10 R-ami-
no groups were protected with Boc, the side chain
groups and the C-terminus with benzyl alcohol-derived
protecting groups in the case of 2 and 4; R-amino groups
were protected with Z and the side chain groups and
C-terminus with tert-butyl alcohol-derived protecting
groups in the case of 3.
Several methods are available for the replacement of
amide bond with the aminomethylene bond Ψ(CH2NH).
X Abstract published in Advance ACS Abstracts, November 1, 1997.
S0022-2623(97)00113-1 CCC: $14.00 © 1997 American Chemical Society

3964 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Ta ble 1. Analytical Properties of the Analogues
Notes
Ta ble 2. Stability toward Enzymatic Degradation by Rabbit
Lung ACE of 1 and Analogues
HPLCa
FAB-HRMSb
calculated
% residual peptidea
t ) 30 min t ) 24 h
compd
k′1
k′2
formula
found
peptide
2
3
4
5
6
9.28
8.17
4.11
8.4
8.3
7.6
3.92
7.1
8.9
C20H36N5O8
474.2564
474.2564
474.2564
487.2516
444.2458
474.2554
474.2562
474.2562
487.2518
444.2461
C
C
C
20H36N5O8
20H36N5O8
20H35N6O8
AcSDKP
43.6 ( 1.6
99.7 ( 0.3
100 ( 0
0
2
3
4
5
6
97.6 ( 2.4
100 ( 0
99.4 ( 0.6
96.7 ( 3.3
0
10.1
Cl9H34N5O7
99.7 ( 0.3
99.9 ( 0.1
36.2 ( 0.5
a
k′l and k′2 refer to the HPLC gradient described in the
b
Experimental Section. High-resolution fast atom bombardment
a
mass spectroscopy.
Amount of intact peptide after 30 min or 24 h incubation time
was estimated from the area of the corresponding peak observed
at 215 nm in HPLC and compared to the value given at t ) 0.
However, the method in general use involves reductive
alkylation of the R-amine of an amino acid (or peptide)
ester with an aldehyde derived from a Weinreb amide.12
The latter compound was obtained by coupling the acid
group of suitably protected amino acids with N,O-
dimethylhydroxylamine. The hydroxamate derivatives
of Boc-Ser(Bzl)-OH, Z-Asp(O-But)-OH, and Boc-Lys(Z)-
OH were prepared and converted into aldehydes as
described in refs l3 and 14. Reduction conditions
(temperature, amount of LiAlH4) should be carefully
controlled to prevent overreduction or epimerization of
the aldehyde.11
The amide analogue 5 was prepared via amidation
of the tripeptide Z-Asp(O-But)-Lys(Boc)-Pro-OH by re-
acting the tripeptide, activated through its mixed
anhydride, with aqueous ammonia. Hydrogenolysis of
Z-Ser-Asp(O-But)-Lys(Boc)-Pro-NH2 and subsequent
acetylation were performed very efficiently in a single
step by adding acetylimidazole to the catalyst in ethyl
acetate.
The decarboxylated analogue 6 was prepared from the
pyrrolidide derivative of Boc-Lys(Z)-OH which was
obtained through the mixed anhydride of Boc-Lys(Z)-
OH treated with pyrrolidine. The following reactions
consisted of a classical peptide synthesis sequence.
Acetylation of the N-terminus serine proceeded very
smoothly with acetylimidazole in DMF, in presence of
triethylamine in the case of trifluoroacetate salt. Final
deprotection of benzyl alcohol-derived protecting groups
was performed by hydrogenolysis with 10% Pd/C, and
tert-butyl alcohol-derived protecting groups were cleaved
off by acidolysis with trifluoroacetic acid. Crude pep-
tides were purified by preparative reverse phase HPLC
using mixtures of acetonitrile and water containing 0.1%
trifluoroacetic acid as an eluent. The purified peptides
were characterized by their FAB or LSI mass spectrum,
and their purity was checked by HPLC analysis in two
systems of solvents (Table 1).
or 50 µM) with rabbit lung angiotensine-converting
enzyme before adding the radiolabeled NAcSD[3H]KP
diluted with some cold peptide (5 µM). The tritium label
was located in the lysine side chain.15 The conditions
used for incubation are those used in the stability study.
Two control experiments were carried out: the first one
was a simple hydrolysis of NAcSD[3H]KP and the
second one was an hydrolysis of NAcSD[3H]KP in the
presence of captopril (1 µM). After 30 min at 37 °C, the
incubation medium was analyzed by high-voltage elec-
trophoresis.4 Comparison of the radioactivity associated
with residual NAcSD[3H]KP in the different sets of
experiments was used to quantify the effect of the
analogue on the hydrolysis of NAcSDKP.
Resu lts a n d Discu ssion
HPLC analysis of the incubation media of 1 (or each
one of the pseudopeptides) with ACE showed, after 30
min, 44% of residual peptide 1 and 100% of residual
pseudopeptides (Table 2). After a 24 h incubation, there
was still 98% of residual 2 whereas 100% and 99% of
pseudopeptides 3 and 4 were remaining (Table 2).
These results illustrate the fact that when a peptide
bond is modified even at a distance from the hydrolyzed
bond, improved stability of the resulting analogue
toward proteolysis can be observed.
The same stability can be seen for the amido analogue
(100% after 30 min 98% residual analogue after 24 h)
(Table 2). Conversely, the decarboxylated peptide is
hydrolyzed with a time course similar to that observed
for the parent peptide 1 (36% after 30 min, 0% of
residual 6 after 24 h) (Table 2). This result led us to
conclude that our hypothesis relying upon an ionic
interaction between the C-terminal carboxylate group
of angiotensin I and a protonated arginine side chain
in the active site of ACE was not valid in the case of
our compounds and that the interaction of 1 or the
analogues with the enzyme is probably different from
the one proposed for angiotensin I.
As shown in Table 3, the addition of the analogues 2,
3, 4, or 5 did not change the extent of hydrolysis of
NAcSDKP, suggesting that these molecules have weaker
affinities than the parent peptide 1 for the active site
of ACE or are not recognized by the enzyme. The
peculiar properties of pseudopeptides, especially the
ability of the peptide amide bond surrogate to be
protonated at physiological pH, might explain the weak
binding to the active site of the enzyme.
Nonetheless, all of the analogues described here
retain full biological activity as inhibitors of the entry
into cycle of two different primitive hematopoietic
cells: HPP-CFC and CFU-GM.16
ACE Degr a d a tion Stu d y
The stability of each of the analogues was checked
by incubating them with rabbit lung ACE for 30 min or
24 h in comparison to NAcSDKP (1). The conditions
used for the hydrolysis are those described by Rousseau
and al.4 HPLC analysis of the reaction mixture was
performed. The amount of residual peptide was as-
sessed by measuring the area of the peak corresponding
to the residual tetrapeptide 1 or analogues recorded at
215 nm.
ACE In h ibition in Vitr o Assa ys
The ability of ACE resistant analogues to inhibit the
hydrolysis of NAcSDKP (1) was studied by preincubat-
ing each one of them at different concentrations (0.5, 5,

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3965
Ta ble 3. Inhibitory Potency of Captopril and Analogues in
NAcSDKP Hydrolysis by Rabbit Lung ACE
To this solution was added 10% Pd/C (about 20% by weight).
The reaction was stirred under an atmosphere of hydrogen at
atmospheric pressure and room temperature. The catalyst
was removed by filtration through a filter paper and the
filtrate concentrated under reduced pressure.
compound
% residual peptide 1a
1
54.4 ( 2.3
99.2 ( 0.4
46.7 ( 6.2
54.0 ( 0.7
46.9 ( 3.8
41.7 ( 3.7
49.3 ( 9.4
46.0 ( 1.8
50.3 ( 6.5
48.8 ( 2.0
47.3 ( 7.5
49.7 ( 5.1
50.1 ( 8.4
46.0 ( 7.5
captopril
2, 50 µM
2, 51 µM
2, 0.5 µM
3, 50 µM
3, 5 µM
3, 0.5 µM
4, 50 µM
4, 5 µM
4, 0.5 µM
5, 50 µM
5, 5 µM
5, 0.5 µM
Cou p lin g Rea ction s. Mixed An h yd r id e Meth od . The
N-protected amino acid was dissolved in tetrahydrofuran (5
mL/mmol). To this solution cooled to -15 °C was added
N-methylmorpholine (1 equiv) and isobutyl chloroformate (1
equiv). After 5 min, the temperature was lowered to -20 °C
and the cooled solution of C-protected amino acid or peptide
(1.1 equiv) dissolved in a minimum amount of dimethyl-
formamide or dichloromethane was added along with N-
methylmorpholine (1.1 equiv) in the case of the trifluoracetate
salt. After 1 h at -10 °C, the temperature was allowed to
warm to room temperature. Completion of the reaction was
monitored by TLC. The reaction mixture was then concen-
trated under reduced pressure, and the residue was taken up
with ethyl acetate and 5% citric acid. The organic layer was
washed with water, 5% sodium bicarbonate, water, and brine
and dried over sodium sulfate. Evaporation of the solvent
yielded the crude product which was either purified through
column chromatography or used in the next step when
homogeneous by TLC.
Acetyla tion Rea ction . The N-deprotected peptide or its
trifluoroacetate salt was dissolved in DMF (2 mL/mmol).
Acetylimidazole (1.1 equiv) was added to the solution, cooled
on a ice bath. Triethylamine (1.1 equiv) was added in the case
of the salt. The reaction mixture was stirred at room tem-
perature. Completion of the reaction was monitored by TLC.
The reaction mixture was evaporated down and the residue
was taken up with 0.5 N HCl and ethyl acetate. The organic
layer was washed with water until the pH was neutral and
then with brine and then was dried over sodium sulfate.
Evaporation of the solvent yielded the crude product which
was purified through column chromatography before the last
deprotection step.
a
Amount of remaining NAcSDKP after a 30 min incubation
time following a 30 min preincubation with analogues or captopril
at 37 °C.
The good stability of the three pseudopeptides and of
the analogue 5 along with their full biological activity
turns these compounds into attractive candidates for
clinical trials and useful tools for further studies aimed
at elucidating the still elusive mechanism of action of
NAcSDKP.
Exp er im en ta l Section
Gen er a l Meth od s. All protected amino acids were pur-
chased from Bachem AG or Novabiochem and are of L-
configuration. Thin-layer chromatography (TLC) were run on
silica gel precoated plates (60 F-254, Merck). Solvent systems
were (A) dichloromethane/methanol, 98/2; (B) dichloromethane/
methanol, 95/5; (C) dichloromethane/methanol, 9/1; (D) ethyl
acetate/heptane, 1/1; (E) 1-butanol/acetic acid/eau, 4/1/1. UV
light, ninhydrin, and/or Pataki reagent were used for detection.
Protected peptides were purified by column chromatography
on Merck silica gel 60 (40-63 µm).
Nr-Acet yl-L-ser yl-Ψ(CH 2NH )-L-a sp a r t yl-L-lysyl-L-p r o-
lin e (2). The dipeptide Boc-Lys(Z)-Pro-Bzl was obtained by
coupling of Boc-Lys(Z)-OH with the HCl,Pro-OBzl via the
mixed anhydride method. The crude product obtained as a
syrup was purified on silica gel using CH2Cl2/MeOH (99/1) as
an eluent. Yield: 68%. Rf(B) ) 0.65, Rf(D) ) 0.23. FAB-MS:
All reagents and solvents were of analytical grade and used
as supplied except for THF, which was either distilled from
sodium/benzophenone or filtered through a column of basic
alumina immediately prior to use, and DMF, which was
distilled from ninhydrin under reduced pressure and stored
over 4 Å molecular sieves. Protected peptides were character-
ized by their FAB or LSI mass spectrum. Mass spectra were
obtained using a Kratos MS 80 mass spectrometer using a
xenon FAB gun with glycerol, thioglycerol, or nitrobenzyl
alcohol as a matrix. HPLC purifications were performed on a
C-18 Beckmann Ultrasphere column (5 µm, 10 × 250 mm)
using either a gradient or an isocratic elution with a mixture
of acetonitrile and water containing 0.1% trifluoracetic acid
m/z 590 (MNa)+, 568 (MH)+, 468 (MH)+ - Boc, 434 (MH)+
Z.
-
Boc-Lys(Z)-Pro-Bzl was deprotected by treatment with TFA
for 2 h and 30 min at room temperature. Boc-Asp(OBzl)-OH
was coupled with the trifluoracetate salt for 5 h via the mixed
anhydride method and purified on column (AcOEt/heptane,
1/1). Yield: 81%. Rf(B) ) 0.4, Rf(D) ) 0.10. FAB-MS: m/z
795 (M + Na)+, 773 (M + H)+, 673 (M + H)+ - Boc, 639 (M +
H)+ - Z.
Boc-Ser(OBzl)-N(CH3)OCH3 was prepared as described by
Martinez5 and purified on column (CH2Cl2/MeOH, 99/1).
Yield: 47%. Rf(D) ) 0.43, Rf (A) ) 0.4. CI-MS: m/z 338
(MH)+, 282 (MH)+ - But, 238 (MH)+ - Boc.
The tripeptide Boc-Asp(OBzl)-Lys(Z)-Pro-Bzl (0.372 g, 0.5
mmol) was deprotected by treatment with TFA for 2 h at room
temperature. The salt was dried under reduced pressure. The
aldehyde prepared by the reduction of Boc-Ser(Bzl)-N(CH3)-
OCH3 (0.75 mmol) according to the method of Martinez5 was
dissolved in a mixture of MeOH/AcOH (99/1) containing the
trifluoroacetate salt obtained from the deprotection of Boc-Asp-
(OBzl)-Lys(Z)-Pro-Bzl (0.50 mmol). Sodium cyanoborohydride
(0.036 g) was added portionwise. After 1 h, the reaction
mixture was worked up as described in ref 5. The crude
product (0.490 g) was purified on column (CH2Cl2/MeOH, 98.5/
1.5). Yield: 70%. Rf(B) ) 0.20, Rf(C) ) 0.50. FAB-MS: m/z
936 (MH)+, 836 (MH)+ - Boc, 882 (MH)+ - Z.
Boc-Ser(Bzl)-Asp(OBzl)-Lys(Z)-Pro-Bzl (0.290 g, 0.31 mmol)
was deprotected by treatment with TFA for 1 h and 30 min at
room temperature. The trifluoroacetate salt (0.110 g, 0.31
mmol) dissolved in DMF (0.6 mL) containing Et3N (0.047 mL,
0.031 mmol) was reacted with acetylimidazole (0.034 g, 0.031
mmol). After 6 h and 30 min of stirring, the reaction mixture
was concentrated under reduced pressure. Workup afforded
at a flow rate of 3 mL min-1
. Elution was monitored by
recording absorbance at 215 nm. The fractions were pooled,
concentrated, and lyophilized. Pure peptides were character-
ized by their high-resolution LSI mass spectrum recorded on
a Fisons (VG ZabSpec-T) mass spectrometer. HPLC analysis
for purity control was performed on a Waters Nova-Pak
column C-18 (4 µm particle size, 3.9 × 150 mm) with a solvent
system consisting of a binary system of water and acetonitrile
containing 0.1% TFA at a 1 mL min-1 flow rate with monitor-
ing at 215 nm. The solvent programs involved the following
linear gradients: (1) 0-50% acetonitrile over 50 min, (2)
0-80% acetonitrile over 40 min. k′ values are reported in
these two solvent systems.
Ch em istr y: Typ ica l P r oced u r es. Boc Acid olysis. The
protected peptide was dissolved in a 1/1 mixture of dichlo-
romethane and trifluoroacetic acid (10-30 equiv). After the
mixture was stirred for 1-2 h at room temperature, the
solvents were removed in vacuum after dilution with dichlo-
romethane. The trifluoracetate salt was generally triturated
with a mixture of dry ether and petroleum ether (1/3, v/v) and
dried under vacuum for several hours.
Hyd r ogen olysis. The protected peptide was dissolved in
either ethanol or methanol/water (9/1) for final deprotection.

3966 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Notes
the crude product (0.235 g) purified on column (CH2Cl2/MeOH
(98/2)). Yield: 62%. Rf(B) ) 0.36, Rf(C) ) 0.68. FAB-MS: m/z
878 (MH)+, 778 (MH)+ - Boc, 744 (MH)+ - Z.
(MH)+, 324 (MH)+ - Boc, 290 (MH)+ - Z. IR (film): 3332,
2975, 2937, 2869, 1708-1702, 1655-1650, 1523-1519, 1500,
1456, 1391, 1367, 1250, 1170 cm-1. Anal. (C21H33O6N3) C, H,
N, O. [R]D ) -13.1 (c 1.0, MeOH).
The peptide Ac-Ser(Bzl)-Ψ(CH2NH)-Asp(OBzl)-Lys(Z)-Pro-
Bzl was dissolved in 10% aqueous EtOH with 10% Pd/C
catalyst (0.030 g). The suspension was stirred for 20 h under
an atmosphere of hydrogen and purified by RP-HPLC; isocratic
The hydroxamate derivative (l.44 g, 3.96 mmol) dissolved
in dry THF (7 mL) was reduced with AlLiH4 (0.325 g, 8.8
mmol) at 0 °C under stirring as described in ref 5. Boc-Lys-
(Z)-H was obtained as an oil (1.224 g, 85%), which was
immediately used without purification. Rf(A) ) 0.18, Rf(D) )
0.42. 1H-NMR (200 MHz, CDCl3): δ 9.55 (s, 1H), 7.35 (s, 5H),
5.35 (s, 1H), 5.1 (s, 2H), 4.9 (broad s, 1H), 3.2 (m, 2H), 2-1.4
elution with CH3CN/H2O/TFA (95/5/0.1), flow rate 3 mL min-1
.
N r-Ace t yl-L-se r yl-L-a sp a r t yl-Ψ(CH 2N)-L-lysyl-L-p r o-
lin e (3). Z-Lys(Boc)-Pro-OBut was obtained by coupling Z-Lys-
(Boc)-OH (2.66 g, 7 mmol) with H-Pro-OBut (1.32 g, 7.7 mmol)
via the mixed anhydride method. After 6 h of stirring, the
reaction mixture was worked up as usual to afford an oil (3.6
g). Yield: 96%. Rf(B) ) 0.26, Rf(D) ) 0.21. The crude product
(1.08 g, 2 mmol) was dissolved in EtOH (40 mL). Then 10%
Pd/C (0.120 g) was added, and the suspension was stirred for
4 h and 30 min under an atmosphere of hydrogen. Yield:
0.672 g (84%). Rf(C) ) 0.34. FAB-MS: m/z 422 (MNa)+, 400
(MH)+, 344 (MH)+ - But, 300 (MH)+ - Boc, 288 (MH)+ - 2But,
244 (MH)+ - But - Boc.
(m, 6H), 1.4 (s, 9H). MS (CI): m/z 365 (MH)+, 309 (MH)+
-
But, 265 (MH)+ - Boc. IR (film): 3342, 3006, 2976, 2935, 2867,
1718-1686, 1533-1509, 1456, 1392, 1367, 1251, 1166 cm-1
.
HCl,H-Pro-OBzl (1.136 g, 4.7 mmol) and the aldehyde Boc-
Lys(Z)-H (1.2 g, 3.36 mmol) were dissolved in a solution of
MeOH-AcOH (99:1). NaBH3CN (0.252 g, 4 mmol) was added
portionwise over 45 min. The reaction was monitored by TLC.
After 1 h, the reaction mixture was worked up. Chromatog-
raphy on column (solvent A) gave Boc-Lys(Z)-Ψ(CH2N)-Pro-
OBzl. Yield: 0.985 g (53%). Rf(A) ) 0.3, Rf(D) ) 0.29. FAB-
MS: m/z 576 (MNa)+, 554 (MH)+, 454 (M+H)+ - Boc, 418
(MH)+ - Z.
Boc-Lys(Z)-Ψ(CH2N)-Pro-OBzl (0.744 g, 1.3 mmol) was
deprotected by treatment by TFA (20 equiv). The trifluoro-
acetate salt was triturated with petroleum ether and dried
under vacuum. Boc-Asp(OBzl)-OH (0.394 g, 1.2 mmol) was
coupled with the dipeptide salt via the mixed anhydride
method. After 6 h, the usual workup gave Boc-Asp(OBzl)-Lys-
(Z)-Ψ(CH2N)-Pro-OBzl as an oil homogeneous in TLC. Yield:
0.577 g (57%). Rf (CH2Cl2/MeOH, 97/3) ) 0.23, Rf(D) ) 0.22.
FAB-MS: m/z 781 (MNa)+, 759 (MH)+, 623 (MH)+ - Z.
Boc-Asp(OBzl)-Lys(Z)-Ψ(CH2N)-Pro-OBzl (0.577 g, 0.76 mmol)
was treated by a 1/1 mixture of CH2Cl2/TFA. Boc-Ser(Bzl)-
OH (0.204 g, 0.69 mmol) was coupled with the tripeptide salt
via the mixed anhydride method. After 5 h of stirring, the
usual workup gave Boc-Ser(Bzl)-Asp(OBzl)-Lys(z)-Ψ(CH2N)-
Pro-OBzl as an oil. Yield: 0.495 g (70%). Rf(CH2Cl2/MeOH,
97/3) ) 0.23, Rf(D) ) 0.1. FAB-MS: m/z 958 (MNa)+, 936
(MH)+, 800 (MH)+ - Z.
Boc-Ser(Bzl)-Asp(OBzl)-Lys(Z)-Ψ(CH2N)-Pro-OBzl (0.480 g,
0.5 mmol) was treated by a 1/1 mixture of TFA/CH2Cl2. The
trifluoracetate salt was dissolved in DMF (3 mL). Acetylimi-
dazole (0.062 g, 0.56 mmol) followed by triethylamine (0.078
mL, 0.56 mmol) was added at room temperature. After 4 h
and 30 min of stirring, the reaction mixture was worked up.
Purification on column (AcOEt/MeOH, 99/1) gave Ac-Ser(Bzl)-
Asp(OBzl)-Lys(Z)-Ψ(CH2N)-Pro-OBzl. Yield: 0.187 g (42%).
Rf(CH2Cl2/MeOH, 97/3) ) 0.2, Rf(AcOEt) ) 0.25. FAB-MS:
m/z 900 (MNa)+, 878 (M + H)+, 788 (MH)+ - Bzl, 742 (MH)+
- Z.
NR-(Benzyloxycarbonyl)-O-tert-butyl-L-aspartyl N,O-dimeth-
ylhydroxamate was prepared and reduced into aldehyde as
described by Martinez.5 The aldehyde (2 mmol) was dissolved
in MeOH/AcOH (99/1) (7 mL) containing H-Lys(Boc)-Pro-OBut
(1 mmol). Sodium cyanoborohydride (0.094 g) was added
portionwise. After 2 h and 30 min, the reaction mixture was
worked up. Chromatography on column (CH2Cl2/MeOH, 99/
1, then CH2Cl2/MeOH, 98/2) gave Z-Asp(OBut)-Ψ(CH2NH)-Lys-
(Boc)-Pro-OBut. Yield: 56%; Rf(B) ) 0.28, Rf(C) ) 0.61,
Rf(AcOEt/heptane, 2/1) ) 0.11. FAB-MS: m/z 691 (MH)+, 635
(MH)+ - But, 557 (MH)+ - Z.
Z-Asp(OBut)-Ψ(CH2NH)-Lys(Boc)-Pro-OBut (0.34 mmol) was
dissolved in 10% aqueous EtOH (9.9 mL) with 10% Pd/C (0.044
g). The suspension was stirred under an atmosphere of
hydrogen for 24 h. Yield: 0.277 g (100%). Rf(C) ) 0.42. FAB-
MS: m/z 557 (MH)+, 501 (MH)+ - But.
Z-Ser(But)-OH (0.132 g, 0.45 mmol) was coupled with the
tripeptide so obtained via the mixed anhydride method. After
6 h, the usual workup gave Z-Ser(But)-Asp(OBut)-Ψ(CH2NH)-
Lys(Boc)-Pro-OBut as a white foam (0.351 g). Purification on
column (AcOEt/hexane, 1/1). Yield: 0.233 g (70%). Rf(B) )
0.37, Rf(C) ) 0.63. FAB-MS: m/z 834 (M + H)+, 778 (M +
H)+ - But.
Z-Ser(But)-Asp(OBut)-Ψ(CH2NH)-Lys(Boc)-Pro-OBut (0.2
mmol) was dissolved in 10% aqueous EtOH (4.4 mL) with 10%
Pd/C (0.035 g). The suspension was stirred under an atmo-
sphere of hydrogen overnight. Yield: 0.110 g (66%). Rf(B) )
0.32, Rf(C) ) 0.48. The resulting peptide (0.140 g, 0.2 mmol)
was dissolved in DMF (0.5 mL) and reacted with acetylimi-
dazole (0.033 g, 0.3 mmol). After 4 h of stirring, the usual
workup gave Ac-Ser(But)-Asp(OBut)-Ψ(CH2NH)-Lys(Boc)-Pro-
OBut. Purification was on column (AcOEt/MeOH, 99/1).
Yield: 0.100 g (74%). Rf(B) ) 0.37, Rf(C) ) 0.55. FAB-MS:
m/z 742 (MH)+, 686 (MH)+ - But.
Ac-Ser(Bzl)-Asp(OBzl)-Lys(Z)-Ψ(CH2N)-Pro-OBzl (0.180 g,
0.2 mmol) was dissolved in 10% aqueous MeOH (10 mL). 10%
Pd/C (0.036 g) was added, and the suspension was stirred
overnight under an atmosphere of hydrogen. The crude
peptide was purified by RP-HPLC using the following gradient
solvent system: 0% to 5% CH3CN over 20 min, flow rate 3
Ac-Ser(But)-Asp(OBut)-Ψ(CH2NH)-Lys(Boc)-Pro-OBut (0.056
g, 0.067 mmol) was dissolved in TFA/H2O (300 µL/30 µL). The
solution was stirred for 5 h at room temperature. The reaction
mixture was concentrated under reduced pressure after ad-
dition of H2O (1 mL) and then lyophylized. Purification by
RP-HPLC, gradient solvent system: 0% to 3% CH3CN over
mL min-1
.
Nr-Acetyl-L-ser yl-L-a sp a r tyl-L-lysyl-L-p r olin a m id e (5).
Z-Lys(Boc)-Pro-OBzl was obtained from Z-Lys(Boc)-OH (1.54
g, 4 mmol), and HCl,Pro-OBzl (1.06 g, 4.4 mmol) was coupled
via the mixed anhydride method. The crude product was
purified on silica gel using CH2Cl2/MeOH (99/1) as an eluent
to give Z-Lys(Boc)-Pro-OBzl as a white foam. Yield: 1.76 g
(77%). Rf(A) ) 0.22; Rf(D) ) 0.24. FAB-MS: m/z 590 (MNa)+,
10 min, 3% CH3CN over l5 min, flow rate 3 mL min-1
.
Nr-Acet yl-L-ser yl-L-a sp a r t yl-L-lysyl-Ψ(CH 2-N)-L-p r o-
lin e (4). NR-(tert-Butoxycarbonyl)-Nꢀ-(benzyloxycarbonyl)-L-
lysyl N,O-dimethylhydroxamate: Boc-Lys(Z)-OH (2.28 g, 6
mmol) was dissolved in CH2Cl2 (30 mL) containing N,O-
dimethylhydroxylamine hydrochloride (0.702 g, 7.2 mmol).
DCC (1.238 g, 6 mmol) and DMAP (0.366 g, 3 mmol) were
added at 0 °C followed by DIPEA (1.24 mL, 7.2 mmol). The
reaction mixture was stirred overnight, and then the solvent
was evaporated under reduced pressure. The same workup
as for the coupling reaction yielded Boc-Lys(Z)-N(CH3)OCH3.
Purification was on column (solvent A). Yield: 1.7 g (66%).
Rf(CH2Cl2/MeOH, 97/3) ) 0.25, Rf(D) ) 0.2. 1H-NMR (300
MHz, CDCl3): δ 7.4 (s, 5H), 5.25 (broad s, 1H), 5.1 (s, 2H), 4.9
(broad s, 1H), 4.65 (broad s, 1H), 3.75 (s, 3H), 3.2 (m, 5H),
1.8-1.4 (m, 6H), 1.4 (s, 9H). FAB-MS: m/z 446 (MNa)+, 424
568 (MH)+, 512 (MH)+ - But, 468 (MH)+ - Boc, 434 (MH)+
Z, 378 (MH)+ - Boc - Bzl, 334 (MH)+ - Boc - Z.
-
Z-Lys(Boc)-Pro-OBzl (1 g, 1.75 mmol) was dissolved in 10%
aqueous MeOH (33 mL); 10% Pd/C (0.2 g) was added, and the
suspension was stirred under an atmosphere of hydrogen
overnight. Yield: 0.565 g (94%). Rf(E) ) 0.55. FAB-MS: m/z
366 (MNa)+, 344 (MH)+, 288 (MH)+ - But, 244 (MH)+ - Boc.
Z-Asp(OBut)-OH (0.323 g, 1 mmol) was coupled with the
dipeptide H-Lys(Boc)-Pro-OH via the mixed anhydride method.
After 5 h of stirring, the reaction mixture was worked up as
usual. The crude product was purified on silica gel using (CH2-

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3967
Cl2/MeOH/AcOH, 97/3/0.5) as an eluent. Yield: 0.450 g (60%).
0.39. FAB-MS: m/z 838 (MNa)+, 816 (MH)+, 716 (MH)+ - Boc,
682 (MH)+ - Z, 582 (MH)+ - Boc - Z.
Rf(CH2Cl2/MeOH/AcOH, 97/3/0.5) ) 0.11. FAB-MS: m/z 693
(M2Na)+ - H, 671 (MNa)+, 615 (MNa)+ - But, 549 (MH)+
Boc, 537 (MNa)+ - Z, 515 (MNa)+ - But - Boc, 493 (MH)+
But - Boc, 437 (MNa)+ - Boc - Z.
-
-
The tripeptide (0.181 g, 0.22 mmol) was deprotected with
TFA. The trifluoracetate salt dissolved in DMF (0.5 mL) was
treated with acetylimidazole (0.026 g, 0.24 mmol) and trieth-
ylamine (0.034 mL, 0.23 mmol). After 4 h at room tempera-
ture, the usual workup and purification of the crude product
on column (CH2Cl2/MeOH, 96/4) gave Ac-Ser(Bzl)-Asp(OBzl)-
Lys(Z)-pyrrolidide as an oil. Yield: 0.121 g (74%). Rf(B) )
0.31, Rf(AcOEt/MeOH, 95/5) ) 0.16. FAB-MS: m/z 780
(MNa)+, 758 (MH)+.
Ac-Ser(Bzl)-Asp(OBzl)-Lys(Z)-pyrrolidide (0.100 g, 0.13 mmol)
was dissolved in 10% aqueous MeOH (7.7 mL). Pd/C (10%,
0.020 g) was added, and the suspension was stirred overnight
under an atmosphere of hydrogen. Purification by RP-HPLC
gradient elution: 0-10% CH3CN over 35 min. tR ) 25 min.
FAB-MS: m/z 466 (MNa)+, 444 (MH)+.
To a stirred solution of the tripeptide Z-Asp(OBut)-Lys(Boc)-
Pro-OH (0.129 g, 0.2 mmol) in THF (5 mL) cooled to -15 °C
was added N-methylmorpholine (0.022 mL, 0.2 mmol) followed
by isobutyl chloroformate (0.028 mL, 0.2 mmol). After 5 min
of stirring at -15 °C, a cold 34% ammonia solution (0.2 mL)
was added at -20 °C. After 1 h of stirring at a temperature
below -10 °C and an additional hour at a temperature below
0 °C, the reaction mixture was concentrated under reduced
pressure. The residue was dissolved in ethyl acetate and 5%
citric acid. The aqueous phase was extracted with ethyl
acetate. The combined organic layers were washed with H2O
and brine, then dried over Na2SO4, and concentrated under
reduced pressure to afford Z-Asp(OBut)-Lys(Boc)-Pro-NH2 as
a white foam. Purification was on column with CH2Cl2/MeOH
(95/5) as an eluent. Yield: 0.105 g (81%). Rf(B) ) 0.24, Rf-
(AcOEt/MeOH, 99/1) ) 0.45. FAB-MS: m/z 670 (MNa)+, 648
ACE Degr a d a tion Stu d y. NAcSDKP was a gift from
IPSEN-BIOTECH (Paris, France). ACE from lung rabbit was
a commercial enzyme purchased from Sigma. A 150 µL of a
solution of ACE (0.12 unit/mL) in Tris-maleate buffer (100 mM
Tris, 50 mM NaCl, 10-5 M ZnSO4) at pH 7 was preincubated
at 37 °C for 30 min, and 30 µL of a solution of the tetrapeptide
NAcSDKP (1) or the analogues (300 µM) in buffer were added.
Incubation of the medium containing the enzyme (0.1 unit/
mL) and 1 or one of the analogues (50 µM) was carried out at
37 °C for either 30 min or 24 h. At the end of incubation time,
aliquots (50 µL) were withdrawn and enzymatic hydrolysis was
stopped by addition of 50 µL of a 1% TFA solution. RP-HPLC
analysis of these aliquots was performed twice using a Nova-
Pak C-18 column (Waters) (3.9 × 150 mm, 4 µM, 80 Å).
Elution was monitored by recording UV absorbance at 215 nm.
The solvents system used for elution consisted of H2O/0.1%
TFA and CH3CN/0.1% TFA. Different conditions of elution
were used: for 2, 3, and 6, a linear gradient of 0-5% CH3CN
over 25 min, for 5 a linear gradient of 0-5% CH3CN over 15
min, and for 4 which is the most polar, an isocratic elution
with water/CH3CN/TFA (90/10/0.1, v/v/v) containing octane
sulfonic acid (5 mM). A calibration curve has been set up for
the six compounds in the 5-50 µm range to check the linearity
of the response at 215 nm and deduce the concentration of
the residual compound from the area of the corresponding
peak. The results shown in Table 2 are the mean values of
two experiments run in triplicate.
ACE In h ibition Assa y. A 30 µL portion of the solutions
of 2, 3, 4, or 5 (at the following concentrations: 1.5, 15, or 150
µM) or 30 µL of a 3 µM solution of captopril was preincubated
with ACE (45 µL, 0.12 unit/mL) in Tris-maleate buffer (100
mM Tris, 50 mM NaCl, 10-5 M ZnSO4) at pH 7 at 37 °C for 30
min. At this point, the hydrolysis was initiated by adding
NAcSD[3H]KP (3.6 µCi) and 15 µL of a 30 µM solution of 1.
Incubation of the medium containing the enzyme (0.1 unit/
mL) with 1 (5 µM) and one of the analogue (0.5, 5, or 50 µM)
or captopril (1 µM) as a control, was carried out for 30 min at
37 °C. Enzymatic hydrolysis was stopped by freezing the
incubation medium at -80 °C. It was analyzed by high-voltage
paper electrophoresis as described by Rousseau et al.4 The
residual radioactivity associated with NAcSD[3H]KP at t ) 30
min was compared with the one at t ) 0. Each experiment
was run in duplicate. The results shown in Table 3 are the
mean values of two experiments.
(MH)+, 614 (MNa)+ - But, 548 (MH)+ - Boc, 534 (MH)+
ProNH2, 514 (MH)+ - Z, 492 (MH)+ - But - Boc.
-
Z-Asp(OBut)-Lys(Boc)-Pro-NH2 (0.152 g, 0.23 mmol) was
dissolved in MeOH (6 mL); 10% Pd/C(0.030 g) was added, and
the suspension was stirred under an atmosphere of hydrogen
for 2 h. Yield: 0.111 g (94%). Rf(B) ) 0.08, Rf(C) ) 0.35. FAB-
MS: m/z 536 (MNa)+, 514 (MH)+, 480 (MNa)+ - But, 458
(MH)+ - But, 414 (MH+) - Boc, 400 (MH)+ - ProNH2.
Z-Ser-OH (0.045 g, 0.19 mmol) was coupled with the
tripeptide H-Asp(OBut)-Lys(Boc)-Pro-NH2 (0.105 g, 0.2 mmol)
via the mixed anhydride method. After 5 h of stirring, the
reaction mixture was concentrated under reduced pressure.
The usual workup gave Z-Ser-Asp(OBut)-Lys(Boc)-Pro-NH2.
Purification was on column (CH2Cl2/MeOH, 94/6). Yield:
0.140 g (80%). Rf(B) ) 0.18, Rf(C) ) 0.43. FAB-MS: m/z 757
(MNa)+, 735 (MH)+, 701 (MNa)+ - But, 635 (MH)+ - Boc, 601
(MH)+ - Z, 579 (MH)+ - But - Boc, 501 (MH)+ - Boc - Z,
465 (MH)+ - Boc - But - ProNH2.
Z-Ser-Asp(OBut)-Lys(Boc)-Pro-NH2 (0.080 g, 0.011 mmol)
was dissolved in AcOEt (2 mL); 10% Pd/C (0.016 g) and
acetylimidazole (0.014 g, 0.013 mmol) was added and the
suspension was stirred under an atmosphere of hydrogen
overnight. The catalyst was removed by filtration on a Celite
pad, and the filtrate was concentrated under reduced pressure.
Purification on column (CH2Cl2/MeOH, 9/1). Yield: 0.050 g
(71%); Rf(C) ) 0.29. FAB-MS: m/z 665 (MNa)+, 643 (MH)+,
609 (MNa)+ - But, 543 (MH)+ - Boc, 487 (MH)+ - But - Boc,
373 (MH)+ - Boc - But - ProNH2.
Ac-Ser-Asp(OBut)-Lys(Boc)-Pro-NH2 (0.039 g, 0.06 mmol) in
solution in 200 µL of TFA containing 20 µL of H2O was stirred
at room temperature for 90 min. The reaction mixture was
concentrated under reduced pressure and the residue was
triturated twice with dry ether. After removal of ether, the
solid white residue was taken up in 1.5 mL of H2O and
lyophilized. Purification by RP-HPLC, elution gradient con-
sisting of two solvents (A, H2O/0.1% TFA; B, CH3CN/0.1% TFA;
100% to 80% A over 20 min; tR ) 13 min), flow rate 3 mL min-1
.
Nr-Acetyl-L-ser yl-L-aspar tyl-L-lysylpyr r olidide (6). Boc-
Lys(Z)-OH (0.76 g, 2 mmol) in THF (10 mL) was coupled with
pyrrolidine (0.18 mL, 2.2 mmol) via the mixed anhydride
method. The usual workup gave Boc-Lys(Z)-pyrrolidide as a
white foam. Yield: 0.786 g (91%). Rf(B) ) 0.23. IC-MS: m/z
434 (MH)+, 343 (MH)+ - Boc, 300 (MH)+ - Z.
Ack n ow led gm en t. This work was supported by
CNRS and Ipsen-Biotech. One of us (S.G.) was a student
from IFSBM (Villejuif) and received support from it. We
are indebted to L. Serani and O. Laprevote for recording
high-resolution mass spectra.
Boc-Lys(Z)-pyrrolidide (0.775 g, 1.34 mmol) was dissolved
in CH2Cl2/TFA (20 equiv) for deprotection. Boc-Asp(OBzl)-OH
(0.499 g, l.55 mmol) was coupled with the dipeptide salt (1.7
mmol) via the mixed anhydride method. The usual workup
provided Boc-Asp(OBzl)-Lys(Z)-pyrrolidide as an oil. Yield:
0.771 g (78%). Rf(A) ) 0.22.
Refer en ces
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(2) Carde, P.; Chastang, C.; Goncalve`s, E.; Mathieu-Tubiana, N.;
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The dipeptide (0.303 g, 0.47 mmol) was deprotected with
trifluoroacetic acid. The trifluoracetate salt was coupled with
Boc-Ser(Bzl)-OH via the mixed anhydride method. Purifica-
tion of the crude product on silica gel with solvent A as an
eluent yielded Boc-Ser(Bzl)-Asp(OBzl)-Lys(Z)-pyrrolidide as a
white foam. Yield: 0.181 g (71%). Rf(A) ) 0.19, Rf(AcOEt) )

3968 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Notes
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J M9701132