Design and synthesis of hydrazinopeptides and their evaluation as human leukocyte elastase inhibitors

Article abstract of DOI:10.1021/jm980419o

The name hydrazinopeptide designates peptidic structures in which one of the native CONH links is replaced by a CONHNH (hydrazido) fragment. In this paper, we report the synthesis of such hydrazinohexapeptides (3-5) analogous to Z-Ala-Ala-Pro-Val-Ala-Ala-

Full text of DOI:10.1021/jm980419o

J . Med. Chem. 1998, 41, 4833-4843
4833
Design a n d Syn th esis of Hyd r a zin op ep tid es a n d Th eir Eva lu a tion a s Hu m a n
Leu k ocyte Ela sta se In h ibitor s
Laure Guy, J oe¨lle Vidal,* and Andre´ Collet*
Ste´re´ochimie et Interactions Mole´culaires (UMR CNRS 117), EÄ cole Normale Supe´rieure de Lyon, 46 alle´e d’Italie,
69364 Lyon Cedex 07, France
Augustin Amour and Miche`le Reboud-Ravaux*
De´partement de Biologie Supramole´culaire et Cellulaire, Laboratoire d’Enzymologie Mole´culaire Fonctionnelle, Institut J acques
Monod (UMR CNRS 7592/ Universite´s Paris VI and VII), 2 place J ussieu, Tour 43, 75251 Paris Cedex 05, France
Received J uly 15, 1998
The name hydrazinopeptide designates peptidic structures in which one of the native CONH
links is replaced by a CONHNH (hydrazido) fragment. In this paper, we report the synthesis
of such hydrazinohexapeptides (3-5) analogous to Z-Ala-Ala-Pro-Val-Ala-Ala-NHiPr (6), a
substrate of human leukocyte elastase (HLE; EC 3.4.21.37), cleaved by this serine protease
between the Val4 and Ala5 residues. In hydrazinopeptides 3-5, the Ala5, Val4, or Pro3 residue,
respectively, of the model peptide, has been replaced by the corresponding R-L-hydrazino acid.
In 3, the bond likely to be cleaved by HLE is the one involving the CONHNH link, while in 4
and 5, this link is normally shifted away by one or two amino acid units from the catalytic
serine. On incubation with HLE, hydrazinopeptide 3 proved to be a substrate and was cleaved
between Val4 and NHAla5, like peptide 6. In contrast, 4 and 5 proved to bind to HLE without
being cleaved, featuring properties consistent with reversible competitive inhibition. General
guidelines for the synthesis of hydrazinopeptides are also reported in this paper. These
guidelines take into account the chemical specificity of hydrazino acids, while being fully
compatible with the conventional peptide coupling techniques. The utilization of orthogonally
bisprotected hydrazino acids 1 where the Nâ and NR atoms bear a Boc and a Bzl group,
respectively, is recommended for the easy construction of such hydrazinopeptides.
Ch a r t 1. Hydrazinopeptides and Related
Pseudopeptides
In tr od u ction
Among the variety of pseudopeptide structures cur-
rently investigated in organic and medicinal chemistry,1
little attention has been devoted to hydrazinopeptides,
in which one of the native amino acids has been replaced
by the corresponding R-hydrazino acid, to form a -CO-
NH-NH-C* (hydrazide) link (Chart 1). This modifica-
tion of the peptide bond was first introduced by Niedrich2
in the mid-1960s for a series of eledoisin analogues, to
increase the resistance toward proteolysis while keeping
the physiological activity of the natural peptide unaf-
fected. This goal has been met, at least partially, in
these eledoisin mimics.3 The subsequent lack of interest
for this class of pseudopeptides is probably due to the
difficulty of the synthesis of L- or D-R-hydrazino acids
and to the absence of efficient methodologies for their
insertion in peptide chains.
During the past decade, several methods allowing the
preparation of L- or D-R-hydrazino acids 1 either free,4
Nâ-protected,5,6 or Nâ,NR-orthogonally bisprotected7 have
been reported. This circumstance has renewed the
interest for hydrazinopeptides in making these com-
pounds more easily accessible. Short hydrazinopeptide
models incorporating one hydrazino acid coupled with
one or two amino acids have been synthesized. Struc-
tural studies (X-rays, NMR and IR spectroscopies,
molecular modeling)8-10 have revealed in these com-
pounds a common conformational bias, called a hy-
drazino turn,11 which folds the peptide backbone locally
by way of a well-defined intramolecular bifurcated
H-bonding pattern (see Chart 1).
We address here the problem of the development of
hydrazinopeptidic inhibitors for serine proteases. Hu-
man leukocyte elastase (HLE; EC 3.4.21.37) which is
involved in the pathogenesis of chronic inflammatory
diseases such as pulmonary emphysema was chosen as
10.1021/jm980419o CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/30/1998

4834 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Guy et al.
Ch a r t 2. Hydrazinopeptides 2-5 and the HLE Peptidic
Substrate 6a
Sch em e 1. Preparation of Nâ-Boc- and
Nâ-Boc,NR-Bzl-L-R-hydrazino Acids
a
The cleavage site of 2, 3, and 6 in the presence of HLE is
indicated by -//-.
the target enzyme.12-14 These diseases are associated
with a functional or genetic deficiency of the major
plasmatic inhibitor of HLE, R1-PI, which normally
regulates the activity of this enzyme. The replacement
of the scissile bond of a peptidic substrate by a
pseudopeptidic linkage that may resist proteolytic action
has been a successful approach to the design of potent
synthetic inhibitors for non-serine proteases such as
HIV protease.15 In contrast, little attention has been
directed to the development of pseudopeptidic inhibitors
for serine proteases such as HLE. The best-character-
ized pseudopeptidic inhibitors of HLE are the azapep-
tides16 (Chart 1), in which a nitrogen atom replaces the
R-carbon of an amino acid residue. Excellent titrants
of the elastase active site have been obtained with
azapeptides displaying a good leaving group.17 Re-
cently, a peptidomimetic aminimide (Chart 1) has been
shown to inhibit porcine pancreatic elastase (PPE; EC
3.4.21.36), another serine protease closely related to
HLE.18 All these pseudopeptides contain a CO-N-N
fragment like the hydrazinopeptides. In preliminary
work we have shown that the hydrazinohexapeptide 2
(Chart 2) was a substrate of HLE and PPE and was
cleaved by these enzymes in the same way as the
corresponding hexapeptide.19 This result prompted us
to explore more systematically the interaction of HLE
with tailored hydrazinopeptides. We report here the
discovery of the first compounds of this class (4 and 5)
which bind to HLE without being cleaved and fea-
ture properties consistent with reversible competitive
inhibition.
Resu lts a n d Discu ssion
Design P r in cip les. Peptide 6 was chosen as the
model substrate because its Ala1-Ala2-Pro3-Val4 frag-
ment appears to be one of the best known sequences
for binding to the S1-S4 sites of HLE.22 This sequence
places the substrate in the active site in a way that
promotes a selective cleavage of the peptide bond
involving the Val4 carbonyl23 (Chart 2). The Ala5-Ala6
fragment on the C-terminus side was preferred to the
Ile5-Leu6 one used in our preliminary study (see 2); the
purpose of this change was to reduce the hydrophobicity,
responsible for a poor solubility of the peptide in water,
and to decrease the steric hindrance around the ex-
pected cleavage site. This last requirement is important
if one wants to probe the sensitivity of the hydrazide
link toward HLE hydrolysis. The choice of a CONHiPr
end group instead of a CO2Me group was made princi-
pally to avoid possible complications due to ester hy-
drolysis. Finally, a benzyloxycarbonyl group (Z) was
fixed on the N-terminus side to serve as a chromophoric
group for monitoring the HLE interaction experiments
by HPLC. The nature of the N-terminus group seems
to have little influence on the enzymatic hydrolysis
parameters in the case of HLE and related elastases.22
The model peptide 6 was prepared by conventional
liquid-phase peptide coupling techniques.24
Hydrazinopeptides 3-5 were derived from structure
6 by replacing the Ala5, Val4, or Pro3 unit, respectively,
by the corresponding hydrazino acid. In 3, the bond
likely to be cleaved by the protease is precisely the one
involving the hydrazide link, while in 4 and 5 the
hydrazide group is normally shifted away by one or two
amino acid units from the catalytic serine, if the
positioning of the three molecules in the enzyme is the
same.
Hyd r a zin o Acid s. The L-hydrazino acid building
blocks 1 required for the syntheses of 2-5 were pre-
pared by reaction of the corresponding L-amino acids 7
(Et4N+ salts) with oxaziridine 8, following our recently
established methodology (Scheme 1).7 The transfer of
the N-Boc group from the oxaziridine to the nucleophilic
amino acid nitrogen (Ala, Val, Ile, Pro) was generally
Another general aspect of this work is the develop-
ment of guidelines for the synthesis of hydrazinopep-
tides, which take into account the chemical specificity
of hydrazino acids while being fully compatible with the
conventional peptide coupling techniques. In particular,
we wished to examine how the presence of a potentially
nucleophilic NR site may affect the C-coupling and Nâ-
coupling reactions of hydrazinoalanine (NHAla), hy-
drazinovaline (NHVal), hydrazinoisoleucine (NHIle),
and hydrazinoproline (NHPro) with normal amino acids.
Our conclusion is that the incorporation of R-hydrazino
acids into pseudopeptidic chains is generally best ac-
complished by means of orthogonally bisprotected hy-
drazino acids 1, where the Nâ and NR atoms bear a Boc
and a Bzl protecting group, respectively. These hy-
drazino acid building blocks are themselves accessible
in one step from the corresponding N-benzyl amino
acids.20 Thus further developments of this class of
pseudopeptides, and more generally of bioactive struc-
tures containing hydrazino acids,21 should no longer be
restricted by synthetic issues.

Design and Synthesis of Hydrazinopeptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4835
Sch em e 2. Hydrazinopeptide Synthesis
leading to undesired products such as the diketopip-
erazine 13a (FAB M + H ) 373) and the mixture of
related oligomers 13b (n ) 2-5, FAB M + Na ) 599.2,
785.2, 971.4, 1157.9, respectively). This is why this
coupling, in the presence of DCC, can only be successful
when the activated ester is formed in situ; under these
conditions, hindered hydrazino acids such as 1c may
give acceptable yields probably because the NR is
partially masked. The DCC activation worked reason-
ably well for the coupling of Boc-NHPro 1d , where the
NR is tertiary. This is illustrated by the synthesis of
9d (entry 4).
i
Mixed anhydrides prepared from ClCO2 Bu (iBocCl)
cannot be used for the activation of NR-unprotected
hydrazino acids in hydrazinopeptide synthesis (e.g., see
entry 2). Once the anhydride has been formed, its acyl
group is immediately transferred to the NR to give 14
(Scheme 3). The coupling can nevertheless be effected
if 2 equiv of iBocCl is employed. This is of little practical
i
interest, however, because the Boc group fixed on the
NR is not as easy to remove as a conventional peptide
protecting group.
Most of the above problems can be circumvented by
using Nâ-Boc,NR-Bzl-bisprotected hydrazino acids. Sev-
eral activation methods were tested for the coupling of
Boc-NH(Bzl)Ala (1e) to Ala-Ala-NHiPr to give 9e (en-
tries 5-8). The PyBop activation27 was found to be the
most efficient method here. The mixed anhydride
activation also worked well, but a significant amount
of unreacted starting material was always recovered.
This is possibly due to a too short activation time (2 min
at -15 °C); this question was not investigated further.
fast (1 h at -15 °C in CH2Cl2). The reaction worked
quite well with proline (1d : 95%) and with N-benzyl
amino acids25 (7: PGR ) Bzl) bearing a secondary amino
group (1e,f,g: 88%, 69%, 75%). With unprotected amino
acids (7: PGR ) H), the results were not as good. In
fact, BocNHAla (1a ) and BocNHVal (1b) could only be
isolated in modest yield by this method, while no
BocNHIle (1c) could be recovered. This is likely due to
the fact that the 4-cyanobenzaldehyde released when
oxaziridine 8 has transferred its N-Boc group can in
turn react either with the starting material (7) to give
the corresponding Schiff base or with the product (1) to
give oxazolidinone heterocycles. These Nâ-Boc-mono-
protected derivatives 1a -c could be easily obtained by
catalytic hydrogenolysis of the NR-benzyl group of 1e-g
(H2, 1 atm, EtOH, Pd/C). The reaction was quantitative,
and no detectable cleavage of the hydrazide N-N bond
occurred under these conditions.26
In the following sections we describe the construction
of the hydrazinopeptidic chain of 2-5. The strategy
outlined in Scheme 2 is similar to that used in peptide
synthesis (Boc methodology, liquid-phase synthesis),
and we only focus here on the coupling steps involving
the hydrazino acid units.
Cou p lin g of N-P r otected Hyd r a zin o Acid s to
N-F r ee P ep tid e Der iva tives. We have summarized
in Table 1 some representative results on the activation
of mono- or bis-N-protected hydrazino acids 1a -g and
on their coupling with appropriate precursors of 2-5
on the C-terminus side. The coupling of NR-unprotected
derivatives 1a ,c to Ala-NHiPr and Leu-OMe was real-
ized in modest yield in the presence of DCC and suitable
additives (HOSu and HOBt) to give 9a ,c (entries 1 and
3). Despite its weak basicity (pKb ) 10.2 in H2O for
the related MocNHVal, Moc ) methoxycarbonyl), the
unprotected NR is sufficiently nucleophilic to make the
intermediate activated ester unstable (12 in Scheme 3),
No detectable racemization of the Nâ-Boc, NR-Bzl hy-
drazino acid units occurred under these coupling con-
ditions. This was demonstrated by the fact that the
diastereomer of 9e, Boc-NH(Bzl)(D)Ala-(L)Ala-NHiPr,
which was prepared separately to serve as a reference,
1
was not detected by 500-MHz H NMR in any of the
coupling reactions listed in Table 1, leading to 9e.
It was important to make sure that the NR-Bzl group
in these hydrazinopeptide precursors could be removed
by catalytic hydrogenolysis, without cleavage of the
N-N bond. This was checked with 9f which, on reaction
with H2 in EtOH (1 atm) in the presence of 10% Pd/C,
at room temperature, was quantitatively converted to
Boc-NHVal-Ala-Ala-NHiPr (9b).
F or m a tion of th e Hyd r a zid e Lin k . Experiments
on the coupling of N-protected amino acids to the Nâ-
terminus of substrates 10 to give hydrazinopeptide
precursors 11 are assembled in Table 2. Compounds
10a -e were prepared from the corresponding 9a -e by
cleavage of the Nâ-Boc under usual conditions (3 M HCl
in ethyl acetate). It had been observed in previous
works2,3,8-11,19 that the coupling of completely depro-
tected hydrazines such as 10a -c took place regioselec-
tively on the Nâ when both the incoming amino acid and
the hydrazino acid bear bulky side chains. The present
results support this trend. In the cases we have
investigated, fair coupling yields were obtained by using
the mixed anhydride activation method (entries 3 and
4). This method allowed a short access to hydrazi-
nopeptide 4, which was obtained in 55% yield by
coupling 10b with the tripeptidic fragment Z-Ala-Ala-
Pro. In contrast, the coupling of valine to 10a , bearing

4836 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Guy et al.
Ta ble 1. Activation of the Carboxylic Group and Coupling of N-Protected Hydrazino Acids to Amino Derivatives
entry
hydrazino acid
1
activation
product
no.
yield (%)
1
2
3
4
5
6
7
8
9
Boc-NHAla
Boc-NHVal
Boc-NHIle
Boc-NHPro
Boc-NH(Bzl)Ala
Boc-NH(Bzl)Ala
Boc-NH(Bzl)Ala
Boc-NH(Bzl)Ala
Boc-NH(Bzl)Val
Boc-NH(Bzl)Ile
Boc-NH(Bzl)Ile
1a
1b
1c
1d
1e
1e
1e
1e
1f
DCC/HOSu
ClCO2 Bu
Boc-NHAla-Ala-NHiPr
9a
14
9c
9d
9e
9e
9e
9e
9f
44
a
i
Boc-NH(iBoc)Val
DCC/HOBt
DCC/HOSu
DCC/HOSu
DCC/HOPfp
Boc-NHIle-Leu-OMe
67
75
46
67
64b
82
60c
57d
72
Boc-NHPro-Val-Ala-Ala-NHiPr
Boc-NH(Bzl)Ala-Ala-NHiPr
Boc-NH(Bzl)Ala-Ala-NHiPr
Boc-NH(Bzl)Ala-Ala-NHiPr
Boc-NH(Bzl)Ala-Ala-NHiPr
Boc-NH(Bzl)Val-Ala-Ala-NHiPr
Boc-NH(Bzl)Ile-Leu-OMe
Boc-NH(Bzl)Ile-Leu-OMe
i
ClCO2 Bu
PyBop
DCC/HOSu
i
10
11
1g
1g
ClCO2 Bu
9g
9g
PyBop
a
b
iBoc, Isobutyloxycarbonyl; Viret, J . The`se de Doctorat, Universite´ Paris VI, 1988. 26% of the starting material 1e was recovered;
the corrected yield is therefore 90%. c The diastereomeric Boc-NH(Bzl)(D)Ala-(L)AlaNHiPr was not detected by 1H NMR (500 MHz). 24%
d
of the starting material 1g was recovered; the corrected yield is 81%.
Sch em e 3. Side Reactions in the Coupling of
NR-Unprotected Hydrazino Acids
Ch a r a cter iza tion . Hydrazinopeptides 2-5 were
purified by chromatography on silica gel and isolated
as amorphous or microcrystalline solids. They were
homogeneous by HPLC. The hydrazinopeptides proved
to be much more soluble in water and organic solvents
than the corresponding hexapeptides. They were fully
characterized by high-field NMR (1D, COSY, and ROE-
SY) and by mass spectroscopy (FAB and electrospray
1
techniques). The H NMR 500-MHz spectra of 2-5 all
showed the presence of a minor species (<15%), in slow
exchange with the major one. This was demonstrated
by 2D experiments showing among other things the
existence of correlations between the NH of the two
forms. The most likely interpretation of this equilibri-
um is the presence of two conformational isomers
1
associated with the Pro3 amide bond.31 The H NMR
spectra of hydrazinopeptides 2-5 are in almost identical
with those of the normal peptide, except for the hy-
drazino acid residues which exhibit the following specific
features in DMSO-d6 (Table 3): (i) the NâH of the
hydrazide link is shifted downfield (+1.1 to +1.5 ppm)
with respect to the NH of a normal amide link; (ii) the
NRH is observed between 4 and 5.2 ppm; (iii) the CRH
is shifted upfield (-0.8 to -1.1 ppm) with respect to the
corresponding amino acid. The FAB mass spectra of
hydrazinopeptides 2-5 and 11a showed prominent (M
+ H)+ ions with little fragmentation. Among the weak
fragment ions, the ion bearing the hydrazide link
showed in general a remarkable intensity (10-20% of
the molecular base peak in 2, 3, and 11a to 100% in 5),
certainly due to its stabilization by the NR nitrogen of
the hydrazide link (Chart 3).
a NHAla terminus, proved to be less satisfactory (entry
1). To improve on this, we examined the utilization of
the corresponding NR-Bzl derivative 10e, using Boc-Val
as the acylating substrate (entries 6-9). Although the
bulkiness of the benzyl group seems to make the Nâ-
acylation more difficult, the Boc-N-carboxyanhydride
(UNCA) activation method28 proved quite satisfactory
(entry 9).
Hyd r a zin op ep tid es 2-5. The final coupling steps
are summarized in Scheme 4. For 5, a linear synthesis
was followed throughout. At the last step, the coupling
of Z-Ala-OSu with 15b required the use of 2 equiv of
Et3N, because the acidolysis of the Boc group in 9b left
15b with its hydrazide NR protonated. For 2-4, as well
as for the model hexapeptide 6, a 3+3 segment synthesis
was preferred. This choice, which significantly shortens
the synthesis, was made because there is little racem-
ization risk in a segment synthesis when the activated
amino acid is a proline, such as in 16.29 Moreover, the
bulkiness of the acylating segment 16 allowed the
utilization of NR-unprotected hydrazines (10b, 15a ,c) on
the C-terminus side.30 It is noteworthy that the hy-
drazinopeptides 2-4 were eventually isolated in better
yields than the peptide 6. This is not due to the
chemistry but rather to the fact that 6 is difficult to
purify because of its poor solubility and its tendency to
form gels in organic solvents.
Ch a r t 3. Possible Stabilization Mechanism of the Ion
Bearing the Hydrazide Link (left) in Mass Spectroscopy
In ter a ction w ith HLE. To determine the potential
of peptide 6 and hydrazinopeptides 3-5 to behave as
substrates, compounds 3-6 were spectrophotometrically
scanned between 220 and 400 nm in the presence of
HLE or PPE at pH 8.0 and 25 °C. A significant time-
dependent variation of the absorption spectrum was
observed only in the case of hydrazinopeptide 3 and
peptide 6. This experiment suggested that 3 was
enzymatically cleaved. The reaction mixtures were
analyzed by HPLC with UV detection at 220 nm. The
retention times (tR) of 3, 4, 5, and 6 were 14.6, 15.0,

Design and Synthesis of Hydrazinopeptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4837
Ta ble 2. Formation of the Hydrazide Link
entry
activated substrate
Nâ-free hydrazine (a)
10
hydrazinopeptide product
no.
yield (%)
1
2
3
4
5
6
7
8
9
Z-Val-OSu
Boc-Ala-Ala-Pro-OSu
Z-Ala-Ala-Pro-OCO2 Bu
Z-Val-OCO2 Bu
Boc-Ala-OSu
Boc-Val-OSu
Boc-Val-OCO2 Bu
Boc-Val/PyBop
Boc-Val-NCA
NHAla-Ala-NHiPr
10a
10b
10b
10c
10d
10e
10e
10e
10e
Z-Val-NHAla-Ala-NHiPr
11a
11b
4
11c
11d
11e
11e
11e
11e
40b
NHVal-Ala-Ala-NHiPrc
NHVal-Ala-Ala-NHiPr
NHIle-Leu-OMe
Boc-Ala-Ala-Pro-NHVal-Ala-Ala-NHiPr
Z-Ala-Ala-Pro-NHVal-Ala-Ala-NHiPr
Z-Val-NHIle-Leu-OMe
25d
i
55
60
42
i
NHPro-Val-Ala-Ala-NHiPr
NH(Bzl)Ala-Ala-NHiPr
NH(Bzl)Ala-Ala-NHiPr
NH(Bzl)Ala-Ala-NHiPr
NH(Bzl)Ala-Ala-NHiPr
Boc-Ala-NHPro-Val-Ala-Ala-NHiPr
Boc-Val-NH(Bzl)Ala-Ala-NHiPr
Boc-Val-NH(Bzl)Ala-Ala-NHiPr
Boc-Val-NH(Bzl)Ala-Ala-NHiPr
Boc-Val-NH(Bzl)Ala-Ala-NHiPr
no reaction
i
22
28
60
a
b
Obtained after cleavage of the Boc group in 9 (Table 1) by anhydrous HCl in AcOEt. The coupling between Z-Val-OSu and Ala-
Ala-NHiPr gave the corresponding tripeptide in 32% yield. c Hydrogenolysis of 9f (H2, EtOH, 1 atm, Pd/C) led to Boc-NHVal-Ala-Ala-
NHiPr (9b) quantitatively. The coupling between Boc-Ala-Ala-Pro-OSu and Val-Ala-Ala-NHiPr gave the corresponding hexapeptide in
d
44% yield.
Sch em e 4. Final Steps for the Synthesis of
Hydrazinopeptides 2-5 and Peptide 6
evaluating the competitive effect of 6 on the HLE
amidolysis of the chromogenic substrate MeOSuc-Ala-
Ala-Pro-Val-pNA;33 kcat was deduced from the values of
k
cat/Km and Km (Table 4).
Although the replacement of the amide bond of 6 by
a hydrazide bond in 3 does not prevent HLE hydrolysis,
the catalytic efficiency is decreased by a factor of 2.2,
as a result of a 5.4 times lower affinity of 3 compared
to 6. Both 6 and 3 behaved as normal substrates with
no accumulation of acyl-enzyme, as confirmed experi-
mentally, indicating that neither compound behaved as
an acylating agent of HLE or PPE. This distinguishes
hydrazinopeptides from azapeptides, which are efficient
acylating agents of serine proteases.
Unlike 6 and 3, hydrazinopeptides 4 and 5 did not
behave as substrates of HLE and PPE. Their potential
inhibitory properties toward HLE and PPE were studied
using suitable chromogenic substrates (MeOSuc-Ala-
Ala-Pro-Val-pNA and Suc-Ala-Ala-Ala-pNA, respec-
tively). Both 4 and 5 acted as competitive inhibitors of
HLE and PPE (Table 5). The total recovery of the
enzyme activities after separation of the enzyme from
the inhibitor by filtration indicated that the inhibitions
were reversible. Hydrazinopeptide 5 was a better
inhibitor than 4 toward both elastases and was more
potent toward HLE.
14.5, and 15.4 min, respectively (20-min 0-100% ac-
etonitrile gradient). In the presence of HLE or PPE,
the peak areas corresponding to 3 and 6 decreased with
the incubation time while a new peak (tR ) 13.7 min)
appeared. The retention time of this peak was the same
as that of peptide Z-Ala-Ala-Pro-Val. Amino acid analy-
sis of the isolated fractions corresponding to the 13.7-
min peak observed in the incubation of 3 and 6 yielded
two alanyl, one prolyl, and one valyl residue. This
means that hydrazinopeptide 3 and peptide 6 behave
as substrates for both elastases and are cleaved by these
enzymes after the valyl residue (i.e., between Val4 and
Ala5, see Chart 2). In contrast, HPLC analyses did not
reveal any significant change in the chromatograms of
hydrazinopeptides 4 and 5 after a 4-h incubation with
HLE or PPE, which indicated that neither 4 nor 5 was
hydrolyzed by these enzymes under these conditions.
The kinetic parameters of the HLE-catalyzed hy-
drolysis of hydrazinopeptide 3 were derived from ab-
sorption data at 250 nm (∆ꢀ ) 629 M-1 cm-1 between 3
and the end products; 25 °C and pH 8.0). The kinetic
parameters kcat and Km were calculated by iterative
least-squares fits to the Michaelis equation (Table 4).32
The catalytic efficiency (kcat/Km) of the HLE-catalyzed
hydrolysis of peptide 6 was determined by HPLC at
concentrations of 6 below Km to allow linearization of
the Michaelis equation: v ≈ kcat/Km × ([HLE] × [6]).
The Michaelis constant Km was then established by
It is noteworthy that the affinity of HLE for hydrazi-
nopeptide 5 is 2-fold higher than its affinity for peptide
6 (Km(6)/Ki(5) ) 2.0). The presence of the hydrazide
bond in 5 therefore slightly enhances the binding to the
enzyme active site with respect to the normal peptide
(this is not true for 4, however). The reason the Val4-
Ala5 CONH bond is not cleaved in these compounds is
not yet clear. Earlier X-ray, NMR, and IR studies on
short models indicate that the hydrazinopeptide bond
of 4 and 5 could form a hydrazino turn8-11 (see Chart
1). This feature may modify the position of the Val4-
Ala5 peptide bond within the enzyme active site in such
a way as to make its hydrolysis more difficult. In short
hydrazinopeptidic fragments, the existence of a hy-
drazino turn has been inferred from the observation that
1
the H NMR chemical shift of the CONH hydrogen of
the i+1 amino acid linked to the (i)-hydrazino acid unit
was almost insensitive to the solvent polarity (on going
from CDCl3 to DMSO-d6).11 This feature has been
ascribed to the existence of the bifurcated H-bond
sketched in Chart 1. Things are a little bit more
complicated in the present hydrazinohexapeptides, which
must be much more flexible than the previously inves-
1
tigated short models. Nevertheless, careful H NMR

4838 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Guy et al.
Ta ble 3. Selected 1H NMR Chemical Shifts for Hydrazinopeptides and Related Peptides (DMSO-d6, 30 °C)a
δ (ppm)
entry
hydrazinopeptide or peptide
no.
NRH
4.83
NâH or NH
CRH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Boc-NHAla -Ala-NHiPr
9a
9e
8.16
7.96
7.00
9.36
8.98
7.90
9.30
7.93
8.12
7.70
6.71
9.16
7.70
8.01
7.70
6.63
9.12
8.30
9.16
7.75
9.33
9.12
3.30
3.46
3.91
3.13
3.50
4.26
3. 34
4.21
3.07
2.97
3.77
2.99
4.07
3.18
3.03
3.71
3.22
4.24
3.23
4.18
3.44
3.41
4.50
Boc-NH(Bzl)Ala -Ala-NHiPr
Boc-Ala -Ala-NHiPr
Z-Val-NHAla -Ala-NHiPr
11a
11e
5.21
Boc-Val-NH(Bzl)Ala -Ala-NHiPr
Boc-Val-Ala -Ala-NHiPr
Z-Ala-Ala-Pro-Val-NHAla -Ala-NHiPr
Z-Ala-Ala-Pro-Val-Ala -Ala-NHiPr
Boc-NHVa l-Ala-Ala-NHiPr
Boc-NH(Bzl)Va l-Ala-Ala-NHiPr
Boc-Va l-Ala-Ala-NHiPr
3
6
9b
9f
5.17
4.73
Z-Ala-Ala-Pro-NHVa l-Ala-Ala-NHiPr
Z-Ala-Ala-Pro-Va l-Ala-Ala-NHiPr
Boc-NHIle-Leu-OMe
4
6
9c
9g
4.03
4.70
Boc-NH(Bzl)Ile-Leu-OMe
Boc-Ile-Leu-OMe
Z-Val-NHIle-Leu-OMe
11c
2
5.00
5.00
Boc-Val-Ile-Leu-OMe
Z-Ala-Ala-Pro-Val-NHIle-Leu-OMe
Z-Ala-Ala-Pro-Val-Ile-Leu-OMe
Boc-Ala-NHP r o-Val-Ala-Ala-NHiPr
Z-Ala-Ala-NHP r o-Val-Ala-Ala-NHiPr
Z-Ala-Ala-P r o-Val-Ala-Ala-NHiPr
11d
5
6
a
The data correspond to the residues shown in bold.
Ta ble 4. Kinetic Parameters for the Hydrolysis of Peptide 6
and Hydrazinopeptide 3 Catalyzed by HLE at 25 °C and pH
8.0a
Oehme, hydrazinopeptides may find applications in
many other aspects of medicinal chemistry. Future
works in this area will be facilitated by the availability
of building units such as NR-benzyl-Nâ-Boc-protected L-
and D-hydrazino acids and by the development of
synthetic guidelines such as those described here,
allowing their incorporation in peptide chains or in other
potentially interesting structures.
compd
kcat (s-1
)
Km (µM)
kcat/Km (M-1 s-1
)
6
3
0.88 ( 0.14
2.24 ( 0.28
131 ( 7
712 ( 171
6776 ( 1016
3146 ( 865
a
For chromatographic reasons, sodium chloride was omitted
from the buffer used (0.05 M Hepes, 10% (v/v) DMSO).
Ta ble 5. Inhibition Constants by Hydrazinopeptides 4 and 5
toward HLE and PPE at pH 8.0 and 25 °C (0.05 M Hepes, 10%
(v/v) DMSO)
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. Melting points were
measured on a Perkin-Elmer DSC7 microcalorimeter, with
simultaneous check of purity. Rotations were measured on a
Perkin-Elmer 241 polarimeter at 25 °C using a 1-dm quartz
cell. 1H NMR spectra were recorded at 30 °C on a Bruker AC
200 (200 MHz) or a Varian Unity+ (500 MHz) spectrometer
using the solvent as internal standard (δ ) 0 ppm for TMS);
s ) singlet, d ) doublet, t ) triplet, q ) quartet, quint )
quintet, m ) multiplet, dd ) double doublet, br ) broad peak.
1H NMR peaks were assigned from 2D experiments at 500
MHz (COSY and ROESY sequences). FAB and electrospray
mass spectra were obtained respectively on Fison ZAB VG and
Hewlet Packard 5989A MS Engine mass spectrometers at the
Service Central d'Analyses du CNRS (Vernaison, France),
where the elemental analyses were also performed. Flash
chromatography was performed on columns of silica gel 60
(Merck; 0.040-0.063 mm). Dimethoxyethane (DME) and N,N-
dimethylformamide (DMF) were distilled over CaH2 under
atmospheric and reduced pressure, respectively. Tetrahydro-
furan (THF) was dried and distilled over Na/benzophenone.
Dry CH2Cl2 was obtained after filtration over basic alumina
(activity I). Peptides (6, 16, dipeptides or tripeptides described
in Table 1) were prepared by conventional liquid-phase peptide
coupling techniques (see details and characterization in Sup-
porting Information) using N-Boc or N-Z-L-R-amino N ′-hy-
droxysuccinimide esters (Boc-Ala-OSu, Z-Ala-OSu, Boc-Val-
OSu, or Z-Val-OSu), purchased from Bachem (Switzerland).
Z or Bzl protective groups were removed by atmospheric
hydrogenolysis in methanol or 95% ethanol using 5% pal-
ladium on wet activated carbon (Degussa type E101). Boc
protective group was removed by acidolysis with dry hydrogen
chloride (3 M) in AcOEt (1 mL/mmol). Nâ-Boc-NR-Bzl-L-R-
hydrazino acids 1a -f were prepared according to ref 7.
L-N-Ben zyl-N-(ter t-b u t oxyca r b on yla m in o)isoleu cin e
(1g). The reaction7 of N-benzyl-L-isoleucine (0.884 g, 4 mmol)
Ki (µM)
compd
HLE
PPE
4
5
595 ( 85
65 ( 4
397 ( 44
146 ( 5
studies on 3-6 as a function of the solvent polarity
(Supporting Information) suggest that a hydrazino turn
structuration may exist in 3 (cleaved by HLE) and 5
(not cleaved), but not in 4 (not cleaved). This suggests
that the resistance of 4 and 5 toward enzymatic cleavage
might have different origins.
Con clu sion
In this study, we have described the synthesis of a
series of hydrazinohexapeptides analogous to a HLE
substrate. Two of these compounds behave as normal
substrates, while the other two proved to be reversible
inhibitors of this enzyme, at the micromolar level. Even
though the mechanistic details of the enzyme-hydrazi-
nopeptide interactions are still lacking, such a simple
transformation of a HLE peptidic substrate into a
protease-resisting inhibitor with increased affinity (at
least for 5) opens interesting prospects in the search for
new classes of bioactive principles. Other families of
proteases such as HIV proteases, for which potent
azadipeptide inhibitors have recently been discovered,34
are obvious targets for such studies. Moreover, as
suggested by the pioneering works of Niedrich and

Design and Synthesis of Hydrazinopeptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4839
with oxaziridine 8 (1.03 g, 4.2 mmol) afforded 1.01 g (75%) of
1g as a white solid: mp ) 104 °C; [R]25D ) +34.3 (c 1, MeOH);
1H NMR (200 MHz, CDCl3) (two conformers in a 55:45 ratio)
δ 0.83 (m, 3H, CH3δ), 0.92 (d, J ) 7 Hz, 3H, CH3γ), 1.25 (m,
1H, CHâ), 1.35 (s, 9H, Boc), 1.87 (m, 2H, CH2γ), 3.21 (d, J )
6 Hz, 1H, CHR), 3.91 (m, 2H, CH2N), 6.57 (0.55 H, NH), 7.04
(0.45 H, NH), 7.30 (m, 5H, Ph), 11.08 (br s, 1H, COOH). Anal.
(C14H28N4O4) C, H, N.
amorphous solid: [R]25D ) -21.1 (c 1.0, CH2Cl2); 1H NMR (500
MHz, DMSO-d6) δ 1.04 (d, J ) 6.5 Hz, 3H, CH3 iPr), 1.05 (d,
J ) 6.5 Hz, 3H, CH3 iPr), 1.15 (d, J ) 6.5 Hz, 3H, CH3 Ala),
1.19 (d, J ) 6.5 Hz, 3H, CH3 NHAla), 1.24 (s, 9H, Boc), 3.46
i
(m, 1H, CHR NHAla), 3.80 (m, 3H, CH Pr, CH2Ph), 4.23 (m,
1H, CHR Ala), 7.24-7.43 (m, 5H, Ph), 7.64 (d, J ) 6.5 Hz, 1H,
i
NH Pr), 7.96 (s, 1H, NHâ NHAla), 8.36 (br s, 1H, NH Ala).
Anal. (C21H34N4O4‚0.75H2O) C, H, N.
Boc-NH(Bzl)Ala -Ala -NHiP r (9e). Gen er a l P r oced u r e
B, Usin g th e DCC-HOP fp Cou p lin g Meth od . Compound
9e was prepared by a procedure similar to A, using pentafluo-
rophenol (HOPfp) (0.092 g, 0.5 mmol), DCC (0.103 g, 0.5
mmol), Boc-NH(Bzl)Ala (1e) (0.147 g, 0.5 mmol), and then Ala-
NHiPr (0.052 g, 0.5 mmol) in DMF (0.6 mL). After removal
of DMF and dissolution in CH2Cl2, the organic layer was
washed by water, dried over Na2SO4, and concentrated in
vacuo. Flash chromatography [CH2Cl2/Et2O (1/1)] afforded
0.136 g (67%) of 9e.
Boc-NHAla -Ala -NHiP r (9a ). Gen er a l P r oced u r e A,
Usin g th e DCC-HOSu Cou p lin g Meth od . N-Hydroxysuc-
cinimide (0.299 g, 2.6 mmol) and DCC (0.536 g, 2.6 mmol) were
added to a cooled (5 °C) solution of BocNHAla (1a ) (0.530 g,
2.6 mmol) in dry DME (5 mL). After stirring for 22 h at 5 °C
and filtration, the filtrate was reacted with Ala-NHiPr (pre-
pared by hydrogenolysis of 0.687 g of Z-Ala-NHiPr, 0.270 g,
2.6 mmol) for 18 h at room temperature. Filtration and
washing of the solid by CH2Cl2 afforded 0.510 g of crude 9a .
A second crop of 9a (0.749 g) was obtained after concentra-
tion of the filtrate under reduced pressure and washing of the
solid by water. After dissolution in boiling AcOEt, filtration
of insoluble impurities, and purification by flash chromatog-
raphy [CH2Cl2/MeOH (98/2)], 0.365 g of pure 9a was obtained
(44%) as a white amorphous solid: [R]25D ) -76.2 (c 1.0, CH2-
Cl2); 1H NMR (500 MHz, DMSO-d6) δ 1.04 (d, J ) 6.5 Hz, 3H,
CH3 iPr), 1.06 (d, J ) 6.5 Hz, 3H, CH3 iPr), 1.10 (d, J ) 7.5
Hz, 3H, CH3 NHAla), 1.19 (d, J ) 5.5 Hz, 3H, CH3 Ala), 3.35
Boc-NH(Bzl)Ala -Ala NHiP r (9e). Gen er a l P r oced u r e C,
Usin g th e Mixed An h yd r id e Cou p lin g Meth od . To Boc-
NH(Bzl)Ala (1e) (0.151 g, 0.5 mmol) in dry THF (2.5 mL) under
Ar at -20 °C were added Et3N (0.069 mL, 0.5 mmol) and
isobutyl chloroformate (0.065 mL, 0.5 mmol). After the
mixture stirred for 5 min, a solution of Ala-NHiPr (0.052 g,
0.5 mmol) in dry THF (3 mL) was added. The mixture was
allowed to warm and was stirred for 2 h at room temperature.
After removal of THF and dissolution in CH2Cl2, the organic
layer was washed twice with water, dried over Na2SO4,
concentrated in vacuo, and then flash chromatographed [CH2-
Cl2/MeOH (95/5)], affording 0.133 g (64%) of pure 9e and 0.039
g (26%) of starting material 1e (corrected yield: 90%).
i
(m, 1H, CHR NHAla), 3.81 (quint, J ) 7.5 Hz, 1H, CH Pr),
4.14 (m, 1H, CH Ala), 4.83 (br s, 1H, NHR NHAla), 7.54 (br s,
1H, NH iPr), 7.85 (br s, 1H, NH Ala), 8.17 (br s, 1H, NHâ
NHAla). Anal. (C14H28N4O4‚0.75H2O) C, H; N: calcd, 16.98;
found, 16.40.
Boc-NHVa l-Ala -Ala -NHiP r (9b). Compound 9b was ob-
tained by catalytic hydrogenation of 9f (100%): 1H NMR (500
MHz, DMSO-d6) δ 0.89 (d, J ) 6.5 Hz, 3H, CH3 NHVal), 0.91
(d, J ) 6.5 Hz, 3H, CH3 NHVal), 1.03 (d, J ) 6.0 Hz, 3H, CH3
iPr), 1.04 (d, J ) 6.0 Hz, 3H, CH3 iPr), 1.18 (d, J ) 7.5 Hz, 3H,
CH3 Ala3), 1.23 (d, J ) 7.5 Hz, 3H, CH3 Ala1), 1.83 (m, 1H,
CHâ NHVal), 3.08 (d, J ) 5.5 Hz, 1H, CHR NHVal), 3.80 (o, J
Boc-NH(Bzl)Ala -Ala -NHiP r (9e). Gen er a l P r oced u r e
D, Usin g th e P yBop Cou p lin g Meth od . To a cooled (0 °C)
solution of Ala-NHiPr (0.0965 g, 0.74 mmol) and Boc-NH(Bzl)-
Ala (1e) (0.212 g, 0.72 mmol) in dry CH2Cl2 (1 mL) were added
PyBop (0.388 g, 0.74 mmol) and Et3N (0.205 mL, 1.48 mmol).
The mixture was allowed to warm and was stirred for 18 h at
room temperature. After evaporation of the solvent, the crude
material was purified by flash chromatography [CH2Cl2/MeOH
(97/3)] to yield 0.239 g (82%) of 9e.
i
) 7.5 Hz, 1H, CH Pr), 4.19 (m, 2H, CHR Ala2, CHR Ala3),
i
4.74 (br s, 1H, NHR NHVal), 7.58 (d, J ) 7.5 Hz, 1H, NH Pr),
Boc-NH(Bzl)(D)Ala -(L)Ala -NHiP r . This compound was
prepared by procedure D from Boc-NH(Bzl)(D)Ala (0.0227 g,
0.081 mmol) and Ala-NHiPr (0.0105 g) in 64% (0.020 g) yield:
1H NMR (500 MHz, DMSO-d6) δ 1.04 (d, J ) 6.5 Hz, 3H, CH3
iPr), 1.05 (d, J ) 6.5 Hz, 3H, CH3 iPr), 1.18 (d, J ) 6.5 Hz, 3H,
CH3 Ala), 1.20 (d, J ) 6.5 Hz, 3H, CH3 NHAla), 1.24 (s, 9H,
7.71 (br s, 1H, NH Ala3), 7.94 (br s, 1H, NH Ala2), 8.12 (br s,
1H, NHâ NHVal).
Boc-NHP r o-Va l-Ala -Ala -NHiP r (9d ). This compound was
prepared by procedure A using HOSu (0.184 g, 1.8 mmol), DCC
(0.371 g, 1.8 mmol), BocNHPro (0.414 g, 1.8 mmol) in DMF
(1.8 mL) and then HCl Val-Ala-Ala-NHiPr (0.606 g, 1.8 mmol)
and Et3N (0.250 mL, 1.8 mmol). The mixture was stirred for
2 days at room temperature. The solvent was removed, and
the crude material was precipitated in water and then filtered
and recrystallized (DME) to afford 0.490 g of 9d . Purification
of the mother liquors by flash chromatography [CH2Cl2/MeOH
(95/5)] afforded 0.210 g of 9d as a white solid (75%): mp >
i
Boc), 3.45 (m, 1H, CHR NHAla), 3.81 (m, 1H, CH Pr), 3.74
and 3.88 (AB system, J AB ) 13 Hz, 2H, CH2Ph), 4.18 (m, 1H,
CHR Ala), 7.24-7.39 (m, 5H, Ph), 7.57 (d, J ) 6.5 Hz, 1H, NH
iPr), 7.96 (s, 1H, NHâ NHAla), 8.20 (br s, 1H, NH Ala).
Boc-NH(Bzl)Va l-Ala -Ala -NHiP r (9f). This compound was
prepared by procedure A using HOSu (0.230 g, 2 mmol), DCC
(0.412 g, 2 mmol), Boc-NH(Bzl)Ala (1e) (0.588 g, 2 mmol), and
then HCl Ala-Ala-NHiPr (0.475 g, 2 mmol) and Et3N (0.273
mL, 2 mmol). The mixture was stirred for 3 days at room
temperature. The solvent was removed and the crude material
was precipitated in water and then filtered and washed with
Et2O and pentane. Recrystallization (acetone) afforded 0.605
200 °C; [R]25 ) -55.8 (c 0.55, MeOH); 1H NMR (500 MHz,
D
DMSO-d6) δ 0.86 (d, J ) 6.5 Hz, 6H, CH3 Val), 1.01 (d, J )
6.0 Hz, 3H, CH3 iPr), 1.02 (d, J ) 6.0 Hz, 6H, CH3 iPr), 1.14
(d, J ) 7.0 Hz, H, CH3 Ala3), 1.17 (d, J ) 7.0 Hz, 3H, CH3
Ala4), 1.37 (s, 9H, Boc), 1.58 (m, 1H, CH2γ NHPro), 1.73 (m,
1H, CH2γ NHPro), 2.09 (m, 2H, CH2â NHPro), 2.76 (m, 1H,
CH2δ NHPro), 3.14 (m, 1H, CH2δ NHPro), 3.36 (dd, J ) 10.0
Hz, J ) 5.5 Hz, CH2R NHPro), 3.77 (o, J ) 6.5 Hz, 1H, CH
iPr), 4.03 (br s, 1H, CHR Val), 4.13 (quint, J ) 7.0 Hz, 1H,
CHR Ala3), 4.23 (quint, J ) 7.0 Hz, 1H, CHR Ala4), 7.57 (d, J
g of 9f (60%) as a hygroscopic solid: [R]25 ) -18.3 (c 1.0,
D
MeOH); 1H NMR (500 MHz, DMSO-d6) δ 0.83 (d, J ) 6.5 Hz,
3H, CH3 NHVal), 1.04 (m, 3H, CH3 Val, CH3 iPr), 1.18 (d, J )
10.0 Hz, 3H, CH3 Ala3), 1.26 (m, 12H, CH3 Ala2, Boc), 1.90
(m, 1H, CHâ NHVal), 2.97 (d, J ) 9.5 Hz, 1H, CHR NHVal),
i
) 7.5 Hz, 1H, NH Pr), 7.72 (d, J ) 7.0 Hz, 1H, NH Ala3),
i
7.76 (br s, 1H, NH Ala4), 8.27 (br s, 1H, NH Val), 8.33 (br s,
3.81 (m, 3H, CH Pr, CH2Ph), 4.18 (m, 1H, CHR Ala3), 4.36
1H, NHâ NHPro). Anal. (C24H44N6O6) C, H, N.
(m, 1H, CHR Ala2), 7.20-7.42 (m, 5H, Ph), 7.65 (d, J ) 6.5
i
Boc-NH(Bzl)Ala -Ala -NHiP r (9e). This compound was
prepared by procedure A using HOSu (0.391 g, 3.4 mmol), DCC
(0.700 g, 3.4 mmol), Boc-NH(Bzl)Ala (1e) (0.999 g, 3.4 mmol),
and then Ala-NHiPr (0.353 g, 3.4 mmol) in DME (2 mL). After
stirring for 4 days at room temperature and removal of the
solvent, the crude product was dissolved in CH2Cl2. The
organic phase was washed three times with water, dried over
Na2SO4, and concentrated in vacuo. Flash chromatography
[CH2Cl2/Et2O (8/2)] afforded 0.500 g (36%) of 9e as a white
Hz, 1H, CH Pr), 7.70 (br s, 1H, NHâ NHVal), 7.81 (br s, 1H,
NH Ala3), 8.29 (br s, 1H, NH Ala2). Anal. (C26H43N5O5‚
0.5H2O) C, H, N.
Boc-NH(Bzl)Ile-Leu -OMe (9g). This compound was pre-
pared by procedure C starting from Boc-NH(Bzl)Ile (1g) (0.291
g, 0.86 mmol), N-methylmorpholine instead of Et3N (0.095 mL,
0.86 mmol), and HCl LeuOMe (0.157 mg, 0.86 mmol) dissolved
in dry DMF (2 mL). After removal of THF and dissolution in
CH2Cl2, the organic layer was washed with water, aqueous

4840 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Guy et al.
i
5% KHSO4, and aqueous 5% KHCO3. Purification by flash
chromatography [CH2Cl2/MeOH (98/2)] afforded 0.230 g of 9g
(57%) followed by 0.070 g (24%) of the starting material 1g
(corrected yield: 81%).
Compound 9g was also prepared by procedure D using Boc-
NH(Bzl)Ile (1g) (0.084 g, 0.25 mmol), HCl LeuOMe (0.545 g,
0.30 mmol), PyBop (0.130 g, 0.25 mmol), and Et3N (0.105 mL,
0.75 mmol) in CH2Cl2 (0.5 mL). Flash chromatography [CH2-
6H, Ph, NH Ala1), 7.61 (d, J ) 7.5 Hz, 1H, NH Pr), 7.77 (d, J
) 7.5 Hz, 1H, NH Ala6), 7.94 (d, J ) 7.5 Hz, 2H, NH Ala2,
NH Ala5), 9.16 (d, J ) 5.5 Hz, 1H, NHâ NHVal); MW
C
33H52N8O8 calcd 689.3986 (M + H), found 689.3998 (M + H,
HRFABMS). Anal. (C33H52N8O8‚H2O) C, H, N.
Boc-Ala -NHP r o-Va l-Ala -Ala -NHiP r (11d ). This com-
pound was prepared by procedure E using Boc-Ala-OSu (0.360
g, 1.6 mmol), HCl NHPro-Val-Ala-Ala-NHiPr (10e) (0.564 g,
1.26 mmol), and Et3N (0.351 mL, 2.52 mmol) in DMF (2 mL).
After the mixture stirred for 2 days, the solvent was concen-
trated in vacuo and 11d was precipitated by addition of water.
Recrystallization (iPrOH) gave 0.313 g (42%) of 11d as a white
solid: dec > 240 °C; 1H NMR (500 MHz, DMSO-d6) (two
conformers) δ 0.84 (m, 6H, CH3 Val), 1.01 (d, J ) 5.5 Hz, 3H,
CH3 iPr), 1.03 (d, J ) 5.5 Hz, 3H, CH3 iPr), 1.14-1.21 (m, 9H,
CH3 Ala1, CH3 Ala4, CH3 Ala5), 1.61 (m, 1H, CH2γ Pro), 1.77
(m, 2H, CH2γ Pro, CH2â Pro), 2.14 (m, 2H, CH2â Pro, CHâ
Val), 2.87 (m, 1H, CH2δ Pro), 3.14 (m, 1H, CH2δ Pro), 3.44
Cl2/MeOH (98/2)] afforded 0.083 g of 9g (72%): mp ) 74 °C;
1
[R]25 +13.9 (c 1.0, MeOH); H NMR (200 MHz, DMSO-d6) δ
D
0.76-0.93 (m, 12H), 1.19-1.28 (m, 10H), 1.45-1.91 (m, 5H),
3.03 (d, J ) 8.7 Hz, 1H, CHR NHIle), 3.61 (s, 3H), 3.77 (br s,
2H), 4.35 (br s, 1H, CHR Leu), 7.06-7.32 (m, 5H, Ph), 7.70
(br s, 1H, NHBoc), 8.57 (d, J ) 6.5 Hz, 1H, NH). Anal.
(C25H41N3O5) C, H, N.
Z-Va l-NHAla -Ala -NHiP r (11a ). Gen er a l P r oced u r e E,
for Acyla tion w ith Activa ted Ester s. A mixture of Z-Val-
OSu (0.392 g, 1.02 mmol), HCl NHAla-Ala-NHiPr (10a ) (0.257
g, 1.02 mmol), and Et3N (0.286 mL, 2.04 mmol) in dry DMF
(2 mL) was stirred for 4 days at room temperature. After
evaporation of the solvent, the crude product was precipitated
by addition of water, filtered, and washed several times with
pentane. Extraction of the aqueous phase with CH2Cl2 gave
another crop of crude 11a . Recrystallization (iPrOH) yielded
0.183 g (40%) of 11a as a white solid: dec at 190 °C; 1H NMR
(500 MHz, DMSO-d6) δ 0.81 (d, J ) 12.5 Hz, 3H, CH3 Val),
0.82 (d, J ) 12.5 Hz, 3H, CH3 Val), 1.03 (d, J ) 11.0 Hz, 3H,
CH3 iPr), 1.05 (d, J ) 11.0 Hz, 3H, CH3 iPr), 1.12 (d, J ) 7.0
Hz, 3H, CH3 NHAla), 1.18 (d, J ) 7.0 Hz, 3H, CH3 Ala), 1.88
(m, 1H, CHâ Val), 3.37 (m, 1H, CHR NHAla), 3.75 (m, 1H,
i
(m, 1H, CHR Pro), 3.78 (m, 1H, CH Pr), 3.89 (m, 1H, CHR
Ala1), 4.08 (m, 1H, CHR Val), 4.14 (m, 1H, CHR Ala5), 4.23
(m, 1H, CHR Ala4), 6.80 (br s, 1H, NH Ala1), 7.50 (d, J ) 7.5
i
Hz, 1H, NH Pr), 7.72 (m, 2H, NH Ala4, NH Ala5), 8.60 (d, J
) 8.5 Hz, 1H, NH Val), 9.33 (s, NHâ Pro). Anal. (C33H52N8O8‚
H2O) C, H, N.
Boc-Va l-NH(Bzl)Ala -Ala -NHiP r (11e). To a solution of
Boc-Val-NCA (0.100 g, 0.46 mmol) in dry THF (1 mL) under
Ar and at room temperature were added a solution of HCl NH-
(Bzl)Ala-Ala-NHiPr (10e) (0.134 g, 0.46 mmol) in dry THF (5
mL) and then Et3N (0.064 mL, 0.46 mmol). The mixture was
stirred for 2 h at room temperature. After evaporation of the
solvent and dilution with CH2Cl2, the organic layer was
washed with aqueous 5% KHCO3. Purification by flash
chromatography [CH2Cl2/MeOH (95/5)] afforded 0.140 g (60%)
of pure 11e.
This compound was also prepared by procedure C using Boc-
Val (0.115 g, 0.53 mmol) and HCl NH(Bzl)Ala-Ala-NHiPr (10e)
(0.151 g, 0.44 mmol). After stirring for 19 h at 5 °C,
concentration in vacuo, and dissolution in CH2Cl2, the organic
layer was washed twice with water, dried over Na2SO4, and
concentrated in vacuo. Flash chromatography [CH2Cl2/MeOH
(95/5)] afforded 0.050 g (22%) of pure 11e.
i
CHR Val), 3.81 (m, 1H, CH Pr), 4.16 (m, 1H, CHR Ala), 5.01
(s, 2H, CH2Ph), 5.21 (m, 1H, NHR NHAla), 7.29 (m, 1H, NH
i
Val), 7.35 (br s, 5H, Ph), 7.65 (d, J ) 7.5 Hz, 1H, NH Pr), 7.92
(m, 1H, NH Ala), 9.37 (d, J ) 5.0 Hz, 1H, NHâ NHAla). Anal.
(C22H35N5O5) C, H, N.
Boc-Ala -Ala -P r o-NHVa l-Ala -Ala -NHiP r (11b). This com-
pound was prepared by procedure E using Boc-Ala-Ala-Pro-
OSu (0.227 g, 0.5 mmol), HCl NHVal-Ala-Ala-NHiPr (10b)
(1.756 g, 0.5 mmol), and Et3N (0.069 mL, 0.5 mmol) in DMF
(1.5 mL). The mixture was stirred for 6 days. Purification
by flash chromatography [CH2Cl2/MeOH (95/5)] afforded 0.083
g of 11b (25%): 1H NMR (500 MHz, DMSO-d6) (two conformers
in a 86:14 ratio) δ 0.87 (d, J ) 7.0 Hz, 3H, CH3 Val), 0.90 (d,
J ) 7.0 Hz, 3H, CH3 Val), 1.01 (d, J ) 6.5 Hz, 3H, CH3 iPr),
1.02 (d, J ) 6.5 Hz, 3H, CH3 iPr), 1.15 (m, 9H, CH3 Ala1, CH3
Ala2, CH3 Ala6), 1.22 (d, J ) 7.0 Hz, 3H, CH3 Ala5), 1.36 (m,
9H, CH3 Boc), 1.70-2.00 (m, 5H, CH2â Pro, CH2γ Pro, CHâ
NHVal), 2.91 (br s, 1H, CHR NHVal), 3.52 (m, 2H, CH2δ Pro),
This compound was also prepared by procedure D using Boc-
Val (0.0212 g, 0.097 mmol) and HCl NH(Bzl)Ala-Ala-NHiPr
(10e) (from 0.0378 g 9e, 0.093 mmol). After stirring for 36 h
at room temperature and diluting with CH2Cl2, the organic
layer was washed by aqueous 5% citric acid, aqueous 5%
KHCO3, and water. Purification by flash chromatography
[CH2Cl2/MeOH (98/2)] afforded 0.013 g (28%) of pure 11e: dec
i
at 110 °C; [R]25 +1.3 (c 0.75, CH2Cl2); 1H NMR (500 MHz,
3.78 (m, 1H, CH Pr), 3.94 (m, 1H, CHR Ala1), 4.27-4.34 (m,
D
3H, CHR Pro, CHR NHVal, CHR Ala6), 4.51 (m, 1H, CHR
Ala2), 5.02 (m, 1H, NHR NHVal), 6.86 (d, J ) 7.0 Hz, 1H, NH
DMSO-d6) δ 0.61 (d, J ) 7.0 Hz, 3H, CH3 Val), 0.70 (d, J )
7.0 Hz, 3H, CH3 Val), 1.05 (d, J ) 7.5 Hz, 3H, CH3 iPr), 1.06
(d, J ) 7.5 Hz, 3H, CH3 iPr), 1.18 (d, J ) 6.0 Hz, 3H, CH3
Ala), 1.21 (d, J ) 5.5 Hz, 3H, CH3 NHAla), 1.37 (s, 9H, Boc),
1.70 (o, J ) 6.0 Hz, 1H, CHâ Val), 3.50 (q, J ) 7.5 Hz, 1H,
CHR NHAla), 3.59 (t, J ) 9.0 Hz, 1H, CHR Val), 3.82 (o, J )
i
Ala1), 7.62 (d, J ) 7.5 Hz, 1H, NH Pr), 7.78 (d, J ) 7.5 Hz,
1H, NH Ala6), 7.83 (d, J ) 7.5 Hz, 1H, NH Ala2), 7.96 (d, J )
7.0 Hz, NH Ala5), 9.17 (d, J ) 5.5 Hz, NHâ NHVal).
Z-Ala -Ala -P r o-NHVa l-Ala -Ala -NHiP r (4). This compound
was prepared by procedure C using Z-Ala-Ala-Pro-OH (0.128
g, 0.3 mmol, obtained after treatment of Z-Ala-Ala-Pro-OtBu
by TFA in CH2Cl2) in THF (4.5 mL) and a solution of HCl
NHVal-Ala-Ala-NHiPr (10b) (0.105 g, 0.3 mmol) and Et3N
(0.041 mL, 0.3 mmol) in DMF (1.3 mL). The mixture was
stirred for 18 h at 5 °C. After concentration in vacuo and
dissolution in CH2Cl2, the organic layer was washed twice by
water, dried over Na2SO4, and concentrated in vacuo. Flash
i
7.5 Hz, 1H, CH Pr), 3.89 (s, 2H, CH2Ph), 4.21 (quint, J ) 7.0
Hz, 1H, CHR Ala), 6.57 (d, J ) 9.0 Hz, 1H, NH Val), 7.21-
7.32 (m, 4H, Ph), 7.42 (d, J ) 9.0 Hz, 1H, Ph), 7.66 (d, J ) 7.0
i
Hz, 1H, NH Pr), 8.45 (br s, 1H, NH Ala), 8.98 (s, 1H, NHâ
NHAla). Anal. (C26H43N5O5‚H2O) C, H, N.
Z-Ala -Ala -NHP r o-Va l-Ala -Ala -NHiP r (5). This compound
was prepared by procedure E using Z-Ala-OSu (0.170 g, 0.45
mmol) and HCl Ala-NHPro-Val-Ala-Ala-NHiPr (0.275 g, 0.45
mmol). After the mixture stirred for 60 h at room temperature,
the solvent was removed and the precipitate washed with hot
acetone. Flash chromatography [CH2Cl2/MeOH (95/5)] gave
0.120 g (30%) of 5: 1H NMR (500 MHz, DMSO-d6) (two
conformers in a 91:9 ratio) δ 0.83 (m, 6H, CH3 Val), 0.99 (m,
6H, CH3 iPr), 1.20-1.08 (m, 12H, CH3 Ala1, CH3 Ala2, CH3
Ala5, CH3 Ala6), 1.60 (m, 1H, CHR NHPro), 1.74 (m, 1H, CH2â
NHPro), 1.79 (m, 2H, CH2γ NHPro), 2.08 (m, 1H, CHâ Val),
2.15 (m, 1H, CH2â NHPro), 2.81 (d, J ) 8.0 Hz, 1H, CH2δ
NHPro), 3.13 (m, 1H, CH2δ NHPro), 3.41 (m, 1H, CHR NHPro),
chromatography [CH2Cl2/MeOH (95/5)] afforded 0.115 g of 4
1
(55%): dec at 130 °C; [R]25 -124.9 (c 1.0, MeOH); H NMR
D
(500 MHz, DMSO-d6) (two conformers in a 86:14 ratio) δ 0.86
(d, J ) 6.5 Hz, 3H, CH3 NHVal), 0.89 (d, J ) 6.5 Hz, 3H, CH3
NHVal), 1.00 (d, J ) 7.0 Hz, 3H, CH3 iPr), 1.01 (d, J ) 7.0 Hz,
3H, CH3 iPr), 1.15 (m, 9H, CH3 Ala1, CH3 Ala2, CH3 Ala6),
1.20 (d, J ) 7.0 Hz, 3H, CH3 Ala5), 1.65-2.00 (m, 5H, CH2â
Pro, CH2γ Pro, CHâ NHVal), 2.99 (m, 1H, CHR NHVal), 3.52
i
(m, 2H, CHδ Pro), 3.76 (m, 1H, CH Pr), 4.03 (m, 1H, CHR
Ala1), 4.20 (m, 3H, CHR Pro, CHR Ala5, CHR Ala6), 4.47 (m,
1H, CHR Ala2), 4.91 (m, 3H, CH2Ph, NHR NHVal), 7.32 (m,
i
3.75 (o, J ) 6.5 Hz, 1H, CH Pr), 4.03 (m, 2H, CHR Ala1, CHR

Design and Synthesis of Hydrazinopeptides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4841
3.48 (m, 1H, CH2δ Pro), 3.57 (m, 1H, CH2δ Pro), 3.79 (m, 1H,
Val), 4.11 (quint, J ) 6.5 Hz, 1H, CHR Ala6), 4.18 (quint, J )
7.0 Hz, 1H, CHR Ala2), 4.27 (m, 1H, CHR Ala5), 4.93 (d, J AB
) 12.5 Hz, 1H, NH Ala1), 5.03 (d, J AB ) 12.5 Hz, 2H, CH2Ph),
7.32 (m, 5H, Ph), 7.45 (d, J ) 7.0 Hz, 1H, NH Ala1), 7.51 (d,
i
CH Pr), 4.02 (m, 2H, CHR Ala1, CHR Val), 4.13 (m, 1H, CHR
Ala6), 4.13 (m, 1H, CHR Pro), 4.50 (m, 1H, CH2R Ala2), 4.98
(s, 1H, CH2Ph), 5.17 (br s, 1H, NHR NHAla), 7.32 (m, 6H, NH
Ala1, Ph), 7.65 (d, J ) 7.5 Hz, 1H, NHiPr), 7.73 (d, J ) 8.5
Hz, 1H, NH Val), 7.88 (d, J ) 7.5 Hz, 1H, NH Ala6), 7.95 (d,
J ) 7.5 Hz, 1H, NH Ala2), 9.30 (d, J ) 5.0 Hz, 1H, NHâ
NHAla); MW C33H52N8O8 calcd 689.3986 (M + H), found
689.3994 (M + H, HRFABMS). Anal. (C33H52N8O8‚H2O) C,
H.
i
J ) 7.0 Hz, 1H, NH Pr), 7.72 (d, J ) 7.5 Hz, 1H, NH Ala5),
7.84 (d, J ) 7.5 Hz, 1H, NH Ala6), 7.98 (d, J ) 7.5 Hz, 1H,
NH Ala2), 8.49 (d, J ) 8.0 Hz, 1H, NH Val), 9.12 (s, 1H, NHâ
NHPro); MW C33H52N8O8 calcd 689.3986 (M + H), found
689.3985 (M + H, HRFABMS). Anal. (C33H52N8O8‚0.5H2O)
C, H, N.
Boc-Ala -Ala -P r o-Va l-Ala -Ala -NHiP r . This compound was
prepared by procedure E using Boc-Ala-Ala-Pro-Osu (0.122 g,
0.27 mmol) and HCl Val-Ala-Ala-NHiPr (0.090 g, 0.27 mmol).
After stirring for 3 days and solvent concentration in vacuo,
the product was precipitated by addition of water. Extraction
of the aqueous phase with CH2Cl2 gave a further crop of Boc-
Ala-Ala-Pro-Val-Ala-Ala-NHiPr. A solution of the crude prod-
uct in CH2Cl2/MeOH was decolorized over activated charcoal.
Chromatography on a column of silica gel [CH2Cl2/MeOH (1/
1)] afforded 0.055 g of pure Boc-Ala-Ala-Pro-Val-Ala-Ala-NHi-
Z-Ala -Ala -P r o-Va l-NHIle-Leu -OMe (2). This compound
was prepared by procedure C using Z-Ala-Ala-Pro-OH (0.074
g, 0.19 mmol, obtained after treatment of Z-Ala-Ala-Pro-OtBu
by TFA in CH2Cl2), HCl Val-NHIle-Leu-OMe (0.074 g, 0.19
mmol), and N-methylmorpholine (0.022 mL, 0.2 mmol). After
the mixture stirred for 18 h, the precipitated N-methylmor-
pholine hydrochloride was filtered off. THF was concentrated
in vacuo, and the residue was dissolved in CH2Cl2. This
organic layer was washed with aqueous 5% KHSO4, aqueous
5% KHCO3, and water, dried over Na2SO4, and concentrated.
Flash chromatography [CH2Cl2/MeOH (98/2)] afforded 0.081
g (60%) of pure 2 as a white solid: [R]25D -136.3 (c 0.65, MeOH);
1H NMR (500 MHz, DMSO-d6) (two conformers in a 84:16
ratio) δ 0.79 (d, J ) 6.5 Hz, 3H, CH3 Val), 0.80 (d, J ) 6.5 Hz,
3H, CH3 Val), 0.81-0.90 (m, 12H, CH3 NHIle, CH3 Leu), 1.16
(m, 8H, CH3 Ala1, CH3 Ala2, CH2γ NHIle), 1.47 (m, 2H, CHγ
Leu, CH2â Leu), 1.61 (m, 2H, CH2â Leu, CHâ NHIle), 1.89
(m, 5H, CH2â Val, CH2â Pro, CH2γ Pro), 3.21 (m, 1H, CHR
NHIle), 3.48 (m, 2H, CH2δ Pro), 3.57 (s, 3H, OMe), 4.04 (m,
2H, CHR Ala1, CHR Val), 4.23 (m, 1H, CHR Leu), 4.69 (m,
2H, CHR Ala2, CHR Pro), 4.98 (br s, 1H, NHR NHIle), 7.29-
7.39 (m, 6H, NH Ala1, Ph), 7.69 (d, J ) 9.5 Hz, 1H, NHVal),
7.97 (d, J ) 7.0 Hz, 1H, NH Ala2), 8.12 (d, J ) 7.0 Hz, 1H,
NH Leu), 9.16 (d, J ) 4.5 Hz, 1H, NHâ NHIle). MW
i
Pr as a white solid which was found to gelify MeOH and PrOH
(45%): 1H NMR (500 MHz, DMSO-d6) (two conformers in a
90:10 ratio) δ 081 (d, J ) 7.0 Hz, 3H, CH3 Val), 0.84 (d, J )
7.0 Hz, 3H, CH3 Val), 1.01 (d, J ) 6.5 Hz, 6H, CH3 iPr), 1.03
(d, J ) 6.5 Hz, 6H, CH3 iPr), 1.10-1.20 (m, 12H, CH3 Ala1,
CH3 Ala2, CH3 Ala5, CH3 Ala6), 1.86 (m, 4H, CH2â Pro, CH2γ
Pro), 1.96 (m, 1H, CHâ Val), 3.52 (m, 1H, CH2δ Pro), 3.57 (m,
i
1H, CH2δ Pro), 3.77 (m, 1H, CH Pr), 3.95 (m, 1H, CHR Ala1),
4.08 (m, 1H, CHR Val), 4.13 (m, 1H, CHR Ala6), 4.22 (m, 1H,
CHR Ala5), 4.41 (m, 1H, CHR Pro), 4.49 (m, 1H, CHR Ala2),
6.69 (d, J ) 7.5 Hz, 1H, NH Ala1), 7.58 (d, J ) 7.5 Hz, 1H,
i
NH Pr), 7.73 (m, 2H, NH Val, NH Ala6), 7.90 (d, J ) 7.0 Hz,
1H, NH Ala2), 7.96 (d, J ) 7.0 Hz, 1H, NH Ala5); MW
C
30H53N7O8 calcd 640.4034 (M + H), found 640.4028 (M + H,
HRFABMS). Anal. (C30H53N7O8‚H2O) C, H.
C
37H60N7O9 calcd 746.4452 (M + H), found 746.4453 (M + H,
Z-Ala -Ala -P r o-Va l-Ala -Ala -NHiP r (6). The replacement
of the protective group was done following general procedure
E starting from Z-OSu (0.016 g, 0.06 mmol) and HCl Ala-Ala-
Pro-Val-Ala-Ala-NHiPr (0.053 g, 0.05 mmol) in the presence
of Et3N (0.007 mL, 0.05 mmol). After 72 h of stirring and
solvent concentration in vacuo, the product was precipitated
by addition of water. The solid was washed with Et2O to give
0.021 g of 6 (58%): 1H NMR (500 MHz, DMSO-d6) (two
conformers in a 88:12 ratio) δ 0.81 (d, J ) 7.0 Hz, 3H, CH3
Val), 0.83 (d, J ) 7.0 Hz, 3H, CH3 Val), 1.00 (d, J ) 6.0 Hz,
6H, CH3 iPr), 1.02 (d, J ) 6.0 Hz, 6H, CH3 iPr), 1.18 (m, 12H,
CH3 Ala1, CH3 Ala2, CH3 Ala5, CH3 Ala6), 1.85 (m, 4H, CH2â
Pro, CH2γ Pro), 1.96 (m, 1H, CHâ Val), 3.52 (m, 1H, CH2δ Pro),
HRFABMS). Anal. (C37H60N7O9‚0.5H2O) C, H, N.
Boc-Ala -Ala -P r o-Va l-NHAla -Ala -NHiP r . This compound
was prepared by procedure E using Boc-Ala-Ala-Pro-OSu
(0.181 g, 0.4 mmol) and Val-NHAla-Ala-NHiPr (0.126 g, 0.4
mmol). After stirring for 5 days and concentration of the
solvent in vacuo, the crude material was precipitated by
addition of water and filtered. Extraction of the aqueous layer
with CH2Cl2 gave a further crop. Flash chromatography [CH2-
Cl2/MeOH (80/20)] afforded 0.110 g of pure Boc-Ala-Ala-Pro-
1
Val-NHAla-Ala-NHiPr (40%): [R]25 -56.4 (c 1.0, CH2Cl2); H
D
NMR (500 MHz, DMSO-d6) (two conformers in a 88:12 ratio)
δ 0.76 (d, J ) 7.0 Hz, 3H, CH3 Val), 0.79 (d, J ) 7.0 Hz, 3H,
CH3 Val), 1.01 (d, J ) 6.0 Hz, 6H, CH3 iPr), 1.08 (d, J ) 7.0
Hz, 3H, CH3 NHAla), 1.11 (d, J ) 7.0 Hz, 3H, CH3 Ala1), 1.15
(d, J ) 7.0 Hz, 6H, CH3 Ala2, CH3 Ala6), 1.34 (s, 9H, Boc),
1.70-2.00 (m, 5H, CH2â Pro, CH2γ Pro, CHâ Val), 3.34 (m,
1H, CHR Val), 3.48 (m, 1H, CH2δ Pro), 3.56 (m, 1H, CH2δ Pro),
i
3.57 (m, 1H, CH2δ Pro), 3.77 (m, 1H, CH Pr), 4.04 (m, 1H,
CHR Ala1), 4.07 (m, 1H, CHR Val), 4.13 (m, 1H, CHR Ala6),
4.21 (m, 1H, CHR Ala5), 4.50 (m, 2H, CHR Pro, CHR Ala2),
4.99 (s, 2H, CH2Ph), 7.37 (m, 6H, NH Ala1, Ph), 7.55 (d, J )
i
i
7.5 Hz, 1H, NH Pr), 7.70 (m, 2H, NH Ala6, NH Val), 7.93 (d,
3.76 (m, 1H, CH Pr), 3.93 (m, 1H, CHR Ala1), 3.99 (m, 1H,
J ) 7.0 Hz, 1H, NH Ala5), 7.96 (d, J ) 7.0 Hz, 1H, NH Ala2);
MW C33H51N7O8 calcd 674.3877 (M + H), found 674.3885 (M
+ H, HRFABMS). Anal. (C33H51N7O8‚1.5H2O) C, H, N.
CHR NHAla), 4.13 (m, 1H, CHR Ala6), 4.40 (m, 1H, CH2R Pro),
4.47 (m, 1H, CH2R Ala2), 5.17 (br s, 1H, NHR NHAla), 6.86
(d, J ) 7.0 Hz, 1H, NH Ala1), 7.64 (d, J ) 7.5 Hz, 1H, NHiPr),
7.71 (d, J ) 8.5 Hz, 1H, NH Val), 7.84 (d, J ) 7.0 Hz, 1H, NH
Ala2), 7.88 (d, J ) 7.5 Hz, 1H, NH Ala6), 9.29 (d, J ) 5.0 Hz,
1H, NHâ NHAla); MW C30H54N8O8 calcd 655.4143 (M + H),
found 655.4161 (M + H, HRFABMS). Anal. (C30H54N8O8‚H2O)
C, H, N.
Bioch em ica l P r oced u r es. Gen er a l. HLE and PPE were
purchased from Elastin Products Co. and Serva, respectively.
The chromogenic substrates, methoxysuccinyl-alanyl-alanyl-
prolyl-valyl-p-nitroanilide (MeO-Suc-Ala-Ala-Pro-Val-pNA) for
HLE and N-succinyl-alanyl-alanyl-alanyl-p-nitroanilide (Suc-
Ala3-pNA) for PPE, were obtained from Sigma. HPLC quality
acetonitrile was purchased from Touzart et Matignon (Vitry-
sur-Seine, France). Active site titrations of both elastases17,35
were carried out with N-benzyloxycarbonyl-alanyl-alanyl-
prolyl-azaalanyl p-nitrophenyl ester. Spectrophotometric mea-
surements were made with a lambda 5 Perkin-Elmer UV-vis
spectrophotometer. Reverse-phase HPLC was performed us-
ing a Waters model 510 system on a Lichrosorb C8, 5-µm
column (Interchim; 250 × 4.6 mm). The enzymatic activities
of HLE and PPE were measured at 25 °C in 0.05 M Hepes
(pH 8.0, 10% v/v DMSO).
Z-Ala -Ala -P r o-Va l-NHAla -Ala -NHiP r (3). The replace-
ment of the protective group was done following procedure E
starting from Z-OSu (0.021 g, 0.08 mmol) and 2HCl Ala-Ala-
Pro-Val-NHAla-Ala-NHiPr (0.053 g, 0.08 mmol) in the presence
of Et3N (0.022 mL, 0.16 mmol). After stirring for 72 h and
solvent concentration in vacuo, the product was precipitated
by addition of water. The solid was washed with Et2O to give
0.038 g of 3 (72%): 1H NMR (500 MHz, DMSO-d6) (two
conformers in a 87:13 ratio) δ 0.77 (d, J ) 7.0 Hz, 3H, CH3
Val) 0.79 (d, J ) 7.0 Hz, 3H, CH3 Val), 1.01 (d, J ) 6.5 Hz,
6H, CH3 iPr), 1.09 (d, J ) 7.0 Hz, 3H, CH3 NHAla), 1.15 (d, J
) 7.0 Hz, 9H, CH3 Ala1, CH3 Ala2, CH3, Ala6), 1.70-2.02 (m,
5H, CH2â Pro, CH2γ Pro, CHâ Val), 3.34 (m, 1H, CHR NHAla),
En zym a tic Hyd r olysis. Reaction mixtures containing 100
µM of compounds 3-6 in the presence or absence of enzyme

4842 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Guy et al.
Heterooctapeptides mit L-R-Hydrazino-â-phenyl-propionsau¨re
(NHPhe) statt Phenylalanin. Chem. Ber. 1967, 100, 3283-3288.
(c) Niedrich, H.; Ko¨ller, G. Synthese von Eledoisinpeptiden, die
R-Hydrazinopropionsau¨re enthalten. J . Prakt. Chem. 1974, 316,
729-740.
(200 nM HLE) were initially scanned by spectrophotometry
(from 230 to 400 nm) at different times of incubation to look
for an evolution of absorption spectrum resulting from enzy-
matic activity. The kinetic parameters of the enzymatic
hydrolyses of 3 (5-50 µM) were determined by spectropho-
tometry at 250 nm ([E]0 ) 200 nM, pH 8.0 and 25 °C).
Samples of the above reaction mixtures were taken at
different incubation times ranging from 0 to 4 h. To quench
enzyme activity, their pH was dropped to 3.5 by adding 10%
(v/v) TFA. The reaction products were then separated from
the enzyme by centrifugation/filtration on a Centricon 10
instrument (Amicon). The filtrates were concentrated by
Speed-vac and fractionated by reverse-phase HPLC on a
Lichrosorb C8, 5-µm column in Milli-Q water (0.1% TFA;
solvent A) using a 20-min (0-100%) linear gradient of aceto-
nitrile (0.07% TFA; solvent B). The flow rate was 1 mL/min.
The absorbance of the effluent was monitored at 220 nm. The
absence of spontaneous hydrolysis was also checked by HPLC.
This chromatographic technique was used to determine the
kinetic parameters for the HLE-catalyzed hydrolysis of 6 (5-
20 µM). The concentrations of the end products after 2-5 min
of incubation were quantified from their peak areas precali-
brated with peptide Z-Ala-Ala-Pro-Val.
Sites of En zym e Clea va ge. The end products obtained
after elastase-catalyzed hydrolysis of 3 and 6 were isolated
by HPLC as described above. Their amino acid composition
was determined after hydrolysis in vacuo in 6 M HCl for 24 h
at 110 °C and determination of phenylthiocarbamyl amino
acids by HPLC techniques using a Waters model 510 system
apparatus (Pico-Tag method).
En zym e In h ibition P r oced u r e. The inhibitory effects of
4 (50-250 µM), 5 (25-300 µM), and 6 (40-200 µM) toward
HLE (20 nM) or PPE (22 nM) were determined spectrophoto-
metrically at 405 nm by continuous monitoring of the release
of p-nitroaniline from the respective chromogenic substrate.
The double-reciprocal plots of v vs substrate concentration
(3) (a) Oehme, P.; Bergmann, J .; Niedrich, H.; J ung, F.; Menzel,
G.; Eichsta¨dt, M.; Rudel, M. Zur Pharmakologie von Hydrazi-
nokarbonsau¨ren, Hydrazinopeptiden und anderen Hydrazin-
derivaten. Acta Biol. Med. Ger. 1970, 25, 613-625. (b) Oehme,
P.; Katzwinkel, S.; Vogt, W. E.; Niedrich, H. Retarded Enzymatic
Degradation of Heterologous Eledoisin Sequence. Experientia
1973, 29, 947-948.
(4) Viret, J .; Gabard, J .; Collet, A. Practical Synthesis of Optically
Active R-Hydrazino Acids from R-Amino Acids. Tetrahedron
1987, 43, 891-894.
(5) Vidal, J .; Drouin, J .; Collet, A. N-Amination using N-Methoxy-
carbonyl-3-phenyloxaziridine. Direct Access to Chiral N-â-
Protected R-Hydrazinoacids and Carbazates. J . Chem. Soc.,
Chem. Commun. 1991, 435-437.
(6) Vidal, J .; Guy, L.; Ste´rin, S.; Collet, A. Electrophilic Amination:
Preparation and Use of N-Boc-3-(4-cyanophenyl)oxaziridine, a
New Reagent that Transfers a N-Boc Group to N- and C-
Nucleophiles. J . Org. Chem. 1993, 58, 4791-4793.
(7) (a) Vidal, J .; Damestoy, S.; Guy, L.; Hannachi, J .-C.; Aubry, A.;
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gave straight lines with x intercepts of -1/Km(app)
.
The
apparent Michaelis constants Km(app) {equal to Km(1 + [I]o/Ki)}
and the maximum velocities Vm were calculated with the
software package Kaleidagraph 3.0 (Abelbeck, Reading, PA)
by iterative least-squares fits to the equation for competitive
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m(app)/Vm vs inhibitor concentration (least-squares analysis).
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Su p p or tin g In for m a tion Ava ila ble: Preparation of pep-
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Lineweaver-Burk representation of the HLE-catalyzed hy-
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1H NMR spectra (500 MHz) of hydrazinohexapeptides 2-5 and
hexapeptide 6; 1H NMR chemical shift of NH in hydrazinopep-
tides 3-5 and peptide 6 as a function of DMSO-d6 content in
the solvent (11 pages). Ordering information is given on any
current masthead page.
(20) Some of these orthogonally bisprotected hydrazino acids are
commercially available (Acros Chimica).
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