Hydrazino-aza and N-azapeptoids with therapeutic potential as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2003.09.018

The ubiquitin-proteasome-mediated degradation pathway plays an important role in regulating protein turnover in eucaryotic cells and, consequently, regulates both cell proliferation and cell death. The proteasome influences many cellular regulatory signal

Full text of DOI:10.1016/j.bmc.2003.09.018

Bioorganic & Medicinal Chemistry 11 (2003) 4881–4889
Hydrazino-Aza and N-Azapeptoids with Therapeutic Potential as
Anticancer Agents
Karine Bouget,a Sandrine Aubin,a Jean-Guy Delcros,b Yannick Arlot-Bonnemainsc,* and
Michele Baudy-Floc’ha,*
a
` ` ´
Laboratoire de Synthese et Electrosynthese Organiques (SESO), CNRS UMR 6510, Universite de Rennes I,
´ ´
´
Avenue du General Leclerc, F-35042 Rennes Cedex, France
´
b
´
´
´
´
Groupe de Recherche en Therapeutique Anticancereuse (GRETAC), Faculte de Medecine, Universite de Rennes I,
2 Avenue du Pr Leon Bernard, CS 34317, 35043 Rennes Cedex, France
´ ´
´
Universite Rennes I, 2 Avenue du Pr Leon Bernard, CS 34317, 35043 Rennes Cedex, France
c
´
´
´
´ ´
GroupeCycleCellulaire, CNRSUMR 6061Genetique etDeveloppement, IFR97Genomique Fonctionnelle etSante, Faculte deMedecine,
Received 28 March 2003; accepted 15 September 2003
Abstract—The ubiquitin-proteasome-mediated degradation pathway plays an important role in regulating protein turnover in
eucaryotic cells and, consequently, regulates both cell proliferation and cell death. The proteasome influences many cellular reg-
ulatory signals and is thus a potential target for pharmacological agents. The study of proteasome function has led to the identifi-
cation of several natural and synthetic compounds that can act as tumor cell growth inhibitors. In this study, we have developed a
series of hydrazino-aza and N-azapeptoids, analogues of Ac-Leucyl-Leucyl-Norleucinal (ALLN) a non-specific peptidyl aldehyde
inhibitor of the proteasome. These peptide analogues share a common backbone and bear different C- and N-terminal functions.
Their antiproliferative activity on murine leukemia L1210 cells is reported here.
# 2003 Elsevier Ltd. All rights reserved.
In cells, protein degradation is a key pathway for the
destruction of abnormal or damaged proteins as well as
for the elimination of proteins whose presence is no
longer required.1 Among the various cell proteases, the
proteasome, a multicatalytic macromolecular complex,
is specifically required for the specific degradation of
ubiquitinated proteins. In particular, the proteasome
ensures the elimination of numerous proteins that play
critical roles in cell functions all along cell cycle
progression.2ꢀ4 The proteasome is relevant in human
cancer because the cell cycle, tumor growth and survival
are governed by a large repertoire of intracellular pro-
teins that are regulated by the ubiquitin-mediated pro-
teasome pathway. For this reason, the target
degradation of key regulatory proteins is an essential
element of cell cycle control.5 Malignant cells are more
sensitive to the loss of proteasome activities and studies
comparing normal and malignant cells have shown that
proteasome inhibition sensitizes malignant cells to
apoptosis.6 Consequently, proteasome inhibition has
become a new and potentially significant strategy in
cancer therapy.7,8 In the development of new antitumor
agents, one proteasome inhibitor, the dipeptide boronic
acid analogue PS-341, that leads to an early stage of
apoptosis and overcomes drug resistance in human
myeloma cells in vitro may offer a promising new
approach for treating cancer.9
Peptidyl aldehyde inhibitors, such as ALLN (Ac-Leu-
cyl-Leucyl-Norleucinal) or MG 132 (Z-Leucyl-Leucyl-
Leucinal) (Scheme 1), were first synthesized to improve
the different enzymatic activities of the proteasome.10
But these inhibitors are not specific for the proteasome
and they also inhibit thiol proteases such as cathepsin B
and calpains.11 Furthermore, the substituent adjacent to
the aldehyde is not configurationally stable, due to the
acidity of the a-proton. Thus far, peptide inhibitors
modified at the C-terminal position have been descri-
bed. These include peptide trifluoromethyl ketones,
chloromethyl ketones,12 (often exploited as serine pro-
teases), vinyl sulfones,13 a, b-epoxy ketones,14 a-keto
aldehydes,15 a-keto amides16 and boronates.17 Most of
*Corresponding authors. Tel.: +33-2-2323-6933; fax: +33-2-2323-6738;
tel.: +33-2-2323-4526; fax: +33-2-2323-4478;e-mail: michele.baudy-flo-
ch@univ-rennnes1.fr (M. Baudy-Floc’h);. e-mail: yannick.arlot@ univ-
rennnes1.fr (Y. Arlot-Bonnemains).
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.018

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K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
Scheme 1. Peptide aldehyde inhibitors of proteasome.
these inhibitors are too reactive, unstable, nonspecific or
membrane impermeable. However, PS-341, a boronate
peptide inhibitor has emerged and is actually in clinical
trials for cancer treatment.12,18
Scheme 2. Adopted symbolism for amino acid analogues.
The development of oligomeric peptidomimetics is cur-
rently the focus of increasing attention. It represents an
interesting strategy to design analogues of structurally
and physiologically important targets, as it avoids ste-
reochemical constraints and contribute to enhanced
proteolytic resistance. Some significant results have
already been obtained following this route in particular
with the most widely studied azapeptides,19 peptoids,20
retropeptoids,21 ureapeptoids,22 amino oxypeptoids,23
b-peptoids,24 in which side chains are linked to nitrogen
atoms.
hydrazino-aza 5 or N-azapeptoids 6 with correct chains
to mimic known proteasome inhibitors.
Hybrid pseudopeptoids 5 or 6 were then deprotected
under standard conditions using piperidine in ether
(PG=Fmoc) or hydrochloric acid in ether (PG=Boc).
Different electrophilic groups, to give reversible or irre-
versible interaction with the active site of the protea-
some, were then introduced in the C-terminal position.
By nucleophilic substitution on the nitrogen atom of 7 or
8 target inhibitors 9 or 10 were obtained: respectively, a
trifluoroketone by reaction of trifluoroacetic anhydride,
a ketoester by reaction of ethyl oxalyl chloride.
Formylation of 5 occurred using pentafluorophenyl-
formate generated in situ by coupling formic acid with
pentafluorophenol in the presence of DCC.26
Our main focus is the synthesis of what we call hydra-
zino-azapeptoids, a new class of peptoid analogues.25
These modified peptides, which have no chiral center,
are built with Na-substituted hydrazino acetic acid
monomers (Nah aa) and a C-terminal aza amino acid
unit (aza aa) or an N-substituted azaglycine (N-aza aa)
monomer (Scheme 2). These ‘hybrid’ pseudopeptides
have nitrogen-enriched peptidic backbones, with nitro-
gen atoms carrying various side chains mimicking both
proteinogenic or nonproteinogenic amino acids (Scheme
3). Here we investigated the in vitro anti-proliferative
activity of different synthetic hydrazino-azapeptoids
and N-azapeptoids, on murine leukemia L1210 cells.
As dipeptidyl boronic acids are new potent proteasome
inhibitors,9,12,17,18 we synthesized analogues 11 with a
boronic acid in C-terminal position by condensation of
substituted hydrazine 8 with ortho-, meta- or para-for-
myl benzene boronic acid.
Biological study
Effect of the different hydrazino-azapeptoids on the
growth of the L1210. We examined the effect of the dif-
ferent compounds on the growth of L1210 leukemia
cells. IC50s after 24 or 48 h culture are reported in Table
1.
Results and Discussion
Synthesis of hydrazino-azapeptoids 5 and 6
We set out to prepare a series of hybrid pseudopeptides
as new analogues of Ac-Leu-Leu-Norleucinal (ALLN)
or Z-Leu-Leu-Leucinal (MG 132): the hydrazino-aza-
peptoids and the hydrazino N-aza peptoids with mod-
ified C-terminal position (Scheme 4).
Among the synthesized hydrazino-N-azapeptoids, Boc-
NahLeu-N-aza-Leu-CF3 10a, PhCO-NahLeu-N-aza-
Leu-CO2Et 10e and Boc-NahLeu-N-azaLeu-H 10g had
no effect on L1210 cell growth after 24 h culture. The
compounds Boc-NahLeu-N-azaLeu-o-C6H4B(OH)2 11a
and Boc-NahLeu-N-azaLeu-p-C6H4B(OH)2 11c bearing
a C-terminus function ortho or para benzene boronic
acid had a moderate anti-proliferative activity with IC50
of 18.5 and 10.5 mM, respectively, after 48 h.
The synthesis of hydrazino-aza and N-azapeptoids can
be accomplished using a two-step procedure to form the
hydrazinopeptoids backbones (Scheme 3), which were
then deprotected and reacted with suitable nucleophiles
(Scheme 4). In a first step, N,N-substituted carbazates 1
or N,N0-substituted carbazates 2,19a were treated with
bromoacetyl bromide and pyridine in dichloromethane
at 0 ꢁC to afford bromo acetylated hydrazines 3 or 4 in
35–75% yield. Nucleophilic substitution of compounds
3 or 4 by N,N0-substituted carbazates 2 affords the
Boc-NahLeu-N-azaLeu-CH2Br 1 0 h,Boc-NahLeu-N-
azaPhe-CH2Br 10i, Z-NahLeu-N-azaPhe-CH2Br 10k
and Boc-NahLeu-N-azaLeu-CH2Cl 10l were the more
potent inhibitors of L1210 cell growth with IC50s lower
than 2 mM. It should be noted that the most potent

K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
4883
Scheme 3. Reagents and conditions: (i) BrCH2COBr, C5H5N/CH2Cl2, 0 ꢁC; (ii) PG1NHNHCH2CH(CH3)2, CHCl3, Á, 24 h.
Scheme 4. Reagent and conditions: (i) PG1=Fmoc/piperidine, Et2O, 15 h (62–84%), PG1=Boc/HCl/ Et2O, NaHCO3 (62%), PG1=Z: H2 (1 atm),
5% Pd/C, 2-propanol, 18 h (84%); (ii) (CF3CO)2O, TEA, Et2O, rt, 6 h (65–82%); (iii) ClCOCO2Et, TEA, Et2O, rt, 6 h (48–56%); (iv) HCO2H,
C6F5OH, DCC, CHCl3, rt, 4 h (85%); (v) BrCOCH2Br or ClCOCH2Cl, pyridine, Et2O, 0 ꢁC, 5 h (57–60%); (vi) HCOC6H4B(OH)2, Et2O, rt, 1 h (92–
96%).
compounds bear a bromoacetyl functionality at the C
terminus position. Those bromoacetyl analogues are
slightly more potent than ALLN.
Although they have been synthesized to mimic ALLN
(Ac-Leu-Leu-Norleu-H) or MG132 (Z-Leu-Leu-Leu-
H), the ability of these compounds to inhibit the pro-
teolytic activity of the proteasome has not been eval-
uated yet. Whether the observed anti-proliferative effect
in L1210 cells is related or not to proteasome inhibition
remains to be analyzed. The potency of the present
analogues yet remains low compared to that of Borte-
zomib, a potent proteasome inhibitor which have
Some of our new pseudopeptides are potent inhibitors
of cell proliferation. These ‘hybrid’ peptidomimetics are
C-modified hydrazino N-azapeptoid based compounds
bearing diverse electrophilic functional groups such as
boronic acid 11a, 11c, chloromethylcarbonyl 10l.

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K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
167 ꢁC. H NMR (CDCl3, d): 0.89 (d, J=6.7 Hz, 6H),
1.81 (m, 1H), 3.59 (br s, 2H), 3.93 (br s, 2H), 4.26 (t,
J=7 Hz, 1H), 4.74 (br d, 2H), 6.84 (s, 1H), 7.32–7.89
(m, 8H). 13C NMR (CDCl3, d): 19.9, 26.1, 26.2, 47.4,
54.9, 66.9, 120.2, 125.1, 127.1, 128.2, 141.4, 143.1, 154.5,
169.3. Anal. calcd for C21H23BrN2O3: C, 58.47; H, 5.34;
Br, 18.56; N, 6.50. Found: C, 56.13; H, 4.93; Br, 19.44;
N, 6.89.
1
Table 1. IC50 of some of the synthesized inhibitors
Analogues
IC50 (mM)
24 h
48 h
ALLN
10a
10e
10g
10h
10i
10k
10l
11a
11c
3.04ꢃ0.01
>80
>80
3.33ꢃ0.10
>80
1.21ꢃ0.10
2.18ꢃ0.76
0.70ꢃ0.15
8.10ꢃ0.80
52.0ꢃ18.0
17.6ꢃ0.6
0.73ꢃ0.15
1.33ꢃ0.58
—
3.81ꢃ0.38
18.5ꢃ1.84
10.5ꢃ2.4
BrCH2CO-N-azaNorleu-OFm [4b, R=(CH2)3CH3,
PG=Fmoc]. The title compound was synthesized from
N-n-butyl-N0-Fmoc hydrazine 2b (3.1 g, 10 mmol) fol-
lowing the general procedure for 3a. 4b (2.3 g, 52%).
Mp 155 ꢁC. H NMR (CDCl3, d): 0.87 (t, J=7.0 Hz,
1
3H), 1.24 (br, 2H), 1.36 (br, 2H), 1.79 (s, 2H), 3.57 (s,
2H), 3.60 (br s, 2H), 4.20 (t, J=6.5 Hz, 1H), 4.67 (br s,
2H), 7.25–7.40 (m, 8H). 13C NMR (CDCl3, d): 13.7,
19.7, 26.3, 28.2, 47.3, 47.7, 66.9, 120.1, 124.6, 127.1,
127.9, 141.5, 143, 154.7, 168.8. Anal. for C21H23BrN2O3:
C, 58.47; H, 5.34; Br, 18.56; N, 6.50. Found: C, 58.34;
H, 5.44; Br, 18.20; N, 6.64.
entered clinical trials.5 Nevertheless, the present study
allowed the identification of appealing leads. Pharmaco-
modulations of those analogues are in progress in order
to enhance their antiproliferative activity.
Experimental
BrCH2CO-N-azaLeu-Ot-Bu [4d, R=CH2CH(CH3)2,
PG=Boc]. The title compound was synthesized from
N-i-butyl-N0-Boc hydrazine 2d (1.9 g, 10 mmol) follow-
ing the general procedure for 3a: (2.4 g, 78%). Mp
General methods
NMR spectra were run at 200, 300 (1H) or 75.5 MHz
(13C). HR-MS were obtained from the ‘Centre Regional
de Mesures Physiques de l’Ouest’, using MS/MS Mass
spectrometer ZAB Spec TOF. Elemental analyses were
performed by the analytical laboratory (CNRS, Lyon).
Thin-layer chromatography was performed on silica gel
60 F254 plates from Merck. Melting points were taken
with a LEICA system Kofler.
96 ꢁC. H NMR (CDCl3, d): 0.98 (d, J=7.0 Hz, 6H),
1
1.55 (s, 9H), 1.99 (m, 1H), 2.86–4.22 (br, 2ꢂ2H), 6.80 (s,
1H). 13C NMR (CDCl3, d): 20.4, 26.6.1, 26.9, 28.6, 55.7,
82.9, 154.3, 169.9. Anal. calcd for C11H21BrN2O3:
C,.42.73; H, 6.85; Br, 25.84; N, 9.08. Found: C, 42.53;
H, 6.86; Br, 25,92; N, 9.06.
BrCH2CO-N-azaPhe-Ot-Bu [4e, R=CH2C6H5, PG=
Boc]. The title compound was synthesized from N-ben-
zyl-N0-Boc hydrazine 2e (2.2 g, 10 mmol) following the
Boc, Fmoc or Z-protected alkyl or aralkyl hydrazines 1
and 2 were prepared according to literature procedures
by reduction of Boc or Z-protected hydrazones, derived
from the condensation of Boc or Z-carbazate with
either aldehyde or ketone.19c,25
general procedure for 3a (2.4 g, 68%). Mp 76 ꢁC. H
1
NMR (CDCl3, d): 1.35 (s, 9H), 3.87 (s, 2H), 4.14–5.20
(br s, 2H), 6.73 (s, 1H), 7.17–7.27 (m, 5H). 13C NMR
(CDCl3, d): 27.1, 28.5, 51.3, 83.1, 128.7, 129.5, 129.7,
135.1, 154.3, 169.5. Anal. calcd for C14H19BrN2O3:
C,.49.11; H, 5.60; Br, 23.07; N, 8.19. Found: C, 48.98;
H, 5.62; Br, 22.93; N, 8.12.
BrCH2CO-azaLeu-OFm
[3a,
R=CH2CH(CH3)2,
PG=Fmoc]. To a stirred and cooled (0 ꢁC) solution of
N-i-butyl-N-Fmoc hydrazine 1a (3.1 g, 10 mmol) in
CH2Cl2 (10 mL) and pyridine (0.95 g, 12 mmol), was
added a solution of bromoacetyl bromide 2 (2.5 g,
12 mmol) in CH2Cl2 (10 mL). The resulting mixture was
stirred over a period of 5 h, and washed three times with
water (50 mL). The organic layer was dried (Na2SO4),
filtered and evaporated in vacuo to give the crude pro-
duct 3a (1.94 g, 45%) which was considered sufficiently
BrCH2CO-N-azaPhe-OBz (4f, R=CH2C6H5, PG=Z).
The title compound was synthesized from N-benzyl-N0-
Z hydrazine 2f (2.1 g, 10 mmol) following the general
procedure for 3a (2.5 g, 66%). Mp 76 ꢁC. 1H NMR
(CDCl3, d): 3.98 (s, 2H), 4.15–5.40 (br s, 2H), 5.19 (s,
2H), 6.79 (s, 1H), 7.34–7.40 (m, 10H). 13C NMR
(CDCl3, d): 26.7, 51.2, 68.7, 128.6, 128.7, 128.8, 129.1,
129.3, 129.4, 134.8, 135.5, 155.2, 169.4. Anal. calcd for
C17H17BrN2O3: C,.54.11; H, 3.56; Br, 21.22; N, 7.43.
Found: C, 54.56; H, 4.67; Br, 20.45; N, 7.54.
pure for the following reactions. Mp 133 ꢁC. H NMR
1
(CDCl3, d): 0.83 (br s, 6H), 1.73 (br s, 1H), 2.81 (br s,
2H), 3.26 (br s, 2H), 4.22 (br t, 1H), 4.55 (br d, 2H),
7.25–7.77 (m, 8H), 8.28 (s, 1H). 13C NMR (CDCl3, d):
19.8, 26.2, 26.7, 47.1, 56.8, 67.5, 119.9, 124.7, 127.1,
127.7, 141.3, 143.5, 155.9, 164.8. Anal. calcd for
C21H23BrN2O3 : C, 58.47; H,5.34; Br, 18.56; N, 6.50.
Found: C, 58.52; H, 5.50; Br, 17.98; N, 6.64.
Boc-NꢀhLeu-azaLeu-OFm [5a, R=CH2CH(CH3)2,
PG=Fmoc, PG1=Boc]. To a stirred solution of N-i-
butyl-N0-Boc hydrazine 2 (4.7 g, 25 mmol) in chloroform
(10 mL) was added slowly the a-bromohydrazide 3a
(4.3 g, 10 mmol) in chloroform (5 mL). The reaction
mixture was refluxed over a period of 24 h, and after
cooling washed three times with water (50 mL), once
with HCl 2 N (50 mL), once with NaHCO3 1 N (50 mL)
BrCH2CO-N-azaLeu-OFm [4a, R=CH2CH(CH3)2,
PG=Fmoc]. The title compound was synthesized from
N-i-butyl-N0-Fmoc hydrazine 2a (3.1 g, 10 mmol) fol-
lowing the general procedure for 3a (4.1 g, 95%). Mp

K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
4885
and once with water (50 mL). The organic phase was
dried over Na2SO4. The solvent was removed under
reduced pressure, and the crude product was purified by
silica gel chromatography (hexane–EtOAc as eluent 1:1)
to afford 5a (3.9 g, 73%); oil; 1H NMR (CDCl3, d): 0.87
(d, J=6.5 Hz, 12H), 1.33 (s, 9H), 1.57 (m, 1H), 1.76 (m,
1H), 2.44 (d, J=6.5 Hz, 2H), 3.30 (br, 2H), 3.34 (s, 2H),
4.15 (t, J=6.7 Hz, 1H), 4.32 (d, J=6.7 Hz, 2H), 5.59 (s,
1H), 7.20–7.75 (m, 8H), 10.02 (s, 1H) two rotameric
forms are observed in proportion 90/10, only data of the
major compound are reported. 13C NMR (CDCl3, d):
18.3, 19.6, 25.3, 26.0, 28.6, 47.2, 59.2, 61.5, 62.1, 66.8,
82.1, 124.9, 127.1, 127.6, 141.4, 142.4, 154.8, 156.8,
171.3. Anal. calcd for C30H42N4O5: C,.66.88; H, 7.86;
N, 10.41. Found: C, 66.89; H, 7.78; N, 10.38.
sized from a-bromohydrazide 4d (3.1 g, 10 mmol) and
N-i-butyl-N0-Z hydrazine (2.2 g, 10 mmol) followꢁing the
1
general procedure for 5a. (2.9 g, 65%). Mp 90 C. H
NMR (CDCl3, d): 0.82 (d, J=6.5 Hz, 6H), 0.89 (d,
J=6.5 Hz, 6H), 1.44 (s, 9H), 1.62 (m, 1H), 1.77 (m, 1H),
2.53 (d, J=6 Hz, 2H), 3.18–3.51 (br s, 2H), 3.38 (s, 2H),
5.04 (s, 2H), 7.35 (m, 5H), 8.44 (s, 1H), 9.37 (s, 1H). 13C
NMR (CDCl3, d): 20.3, 20.9, 26.2, 26.4, 28.2, 54.9, 60.1,
65.2, 65.8, 80.6, 128.0, 128.2, 128.7, 137.1, 154.6, 156.3,
171.2. Anal. for C23H38N4O5 : C, 61.31; H, 8.50; N,
12.43. Found: C, 61.14; H, 8.56; N, 12.48.
Z-NꢀhLeu-N-azaPhe-Ot-Bu
(6e,
R=CH2C6H5,
PG=Boc, PG1=Z). The title compound was synthe-
sized from a-bromohydrazide 4e (3.5 g, 10 mmol) and
N-i-butyl-N0-Z hydrazine (2.2 g, 10 mmol) following the
Boc-NꢀhLeu-N-azaLeu-OFm [6a, R=CH2CH(CH3)2,
PG=Fmoc, PG1=Boc]. The title compound was syn-
thesized from a-bromohydrazide 4a (4.3 g, 10 mmol)
following the general procedure for 5a. (3.7 g, 68%); oil;
1H NMR (CDCl3, d): 0.83 (d, J=6.7 Hz, 6H), 0.87 (d,
J=6.7 Hz, 6H), 1.34 (s, 9H), 1.59 (m, 1H), 1.78 (m, 1H),
2.37 (d, J=6.7 Hz, 2H), 3.32 (s, 2H), 3.43 (br, 2H), 4.14
(t, J=6.5 Hz, 1H), 4.43 (d, J=6.5 Hz, 2H), 6.01 (s, 1H),
7.15–7.73 (m, 8H), 8.87 (s, 1H). 13C NMR (CDCl3, d):
19.6, 20.7, 25.4, 28.6, 47.2, 60.9, 62.2, 64.4, 82.1, 119.9,
124.9, 127.2, 127.6, 141.4, 143.9, 156.6, 156.8, 169.9.
Anal. calcd for C30H42N4O5: C,.66.88; H, 7.86; N,
10.41. Found: C, 66.79; H, 7.69; N, 10.31.
general procedure for 5a. (3.5 g, 72%). Mp 98 ꢁC. H
1
NMR (CDCl3, d): 1.05 (d, J=6 Hz, 6H), 1.52 (s, 9H),
1.82 (m, 1H), 2.72 (d, J=5.75 Hz, 2H), 3.78 (s, 2H),
4.25–5.55 (br s, 2H), 5.09 (s, 2H), 6.94 (s, 1H), 7.43 (m,
10H), 7.64 (s, 1H). 13C NMR (CDCl3, d): 21.1, 26.7,
28.5, 51.4, 60.5, 66.6, 67.2, 82.3, 128.3, 128.5, 128.9,
129.1, 129.7, 135.8, 136.6, 154.4, 156.6, 172.1. Anal. for
C26H36N4O5: C, 64.46; H, 7.44; N, 11.57. Found: C,
64.20; H, 7.45; N, 11.63.
Boc-NꢀhLeu-N-azaPhe-OBn (6f, R=CH2C6H5, PG=Z,
PG1=Boc). The title compound was synthesized from
a-bromo hydrazide 4f (3.8 g, 10 mmol) and N-i-butyl-
N0-Boc hydrazine (1.9 g, 10 mmol) following the general
Boc-NꢀhLeu-N-azaNorleu-OFm [6b, R=(CH2)3CH3,
PG=Fmoc, PG1=Boc]. The title compound was syn-
thesized from a-bromohydrazide 4b (4.3 g, 10 mmol)
following the general procedure for 5a. (4.2 g, 78%). oil.
1H NMR (CDCl3, d): 0.83 (d, J=6.3 Hz, 6H), 0.88 (t,
J=6.8 Hz, 3H), 1.34 (s, 9H), 1.10–1.49 (m, 4H), 1.62 (m,
1H), 2.33 (d, J=6.3 Hz, 2H), 3.37 (br, 2H), 3.44 (s, 2H),
4.14 (t, J=6.5 Hz, 1H), 4.43 (d, J=6.5 Hz, 2H), 5.85 (s,
1H), 7.20–7.74 (m, 8H), 8.96 (s, 1H). 13C NMR (CDCl3,
d): 13.7, 19.6, 21.3, 25.3, 28.5, 30.5, 47.2, 49.4, 55.4,
62.1, 64.3, 82.1, 119.9, 124.9, 127.2, 127.6, 141.4, 143.9,
156.3, 156.8, 169.2. Anal. calcd for C30H42N4O5: C,
66.88; H, 7.86; N, 10.41. Found: C, 66.76; H, 7.82; N,
10.39.
procedure for 5a. (4.2 g, 69%). oil. H RMN (CDCl3,
1
d): 0.87 (d, J=6.5 Hz, 6H), 1.40 (s, 9H), 1.65 (m, 1H),
2.47 (d, J=6.75 Hz, 2H), 3.58 (s, 2H), 4.77 (br s, 2H),
5.15 (s, 2H), 6.08 (s, 1H), 7.36 (m, 10H), 8.39 (s, 1H).
13C NMR (CDCl3, d): 19.6, 25.3, 28.6, 49.9, 55.3, 62.1,
65.3, 82.1, 127.3, 124.9, 128.5, 128.6, 128.9, 134.6,
137.3, 155.8, 156.8, 169.7. Anal. for C26H36N4O5: C,
64.46; H, 7.44; N, 11.57. Found: C, 64.50; H, 7.35; N,
11.53.
Boc-NꢀhLeu-NH-NHi-Bu (7a, R=CH2CH(CH3)2, PG1=
Boc). The hydrazino-azapeptoid 5a (5.4 g, 10 mmol)
was dissolved in ether (5 mL). A solution of piperidine
(1.7 g, 20 mmol) in ether (3 mL) was added dropwise
and the mixture was allowed to stir for 15 h. Evapora-
tion of the solvent and trituration of the crude
product with ethanol afforded a white precipitate of
dibenzofulvene–piperidine adduct. After filtration of
this secondary product, evaporation of the solvent and
addition of ether gave the desired product 7a (1.95 g,
PhCO-NꢀhLeu-N-azaLeu-OFm [6c, R=CH2CH(CH3)2,
PG=Fmoc, PG1=PhCO]. The title compound was
synthesized from a-bromohydrazide 4c (4.3 g, 10 mmol)
and N-i-butyl-N0-COPh hydrazine 1f (1.9 g, 10 mmol)
following the general procedure for 5a. (3.4 g, 62%); oil.
1H NMR (CDCl3, d): 0.77 (d, J=6.7 Hz, 6H), 0.86 (d,
J=6.7 Hz, 6H), 1.65 (m, 1H), 1.81 (m, 1H), 2.57 (d,
J=6.7 Hz, 2H), 3.28 (d, J=6.2 Hz, 2H), 3.61 (br s, 2H),
4.11 (t, J=7 Hz, 1H), 4.42 (d, J=7 Hz, 2H), 7.19–7.75
(m, 13H), 8.43 (s, 1H), 9.10 (s, 1H). 13C NMR (CDCl3,
d): 17.7, 20.8, 24.7, 26.4, 47.2, 54.7, 60.9, 61.6, 64.3,
119.9, 124.9, 127.2, 127.6, 128.3, 128.6, 133.4, 141.4,
144.0, 156.5, 168.9, 170.1. Anal. calcd for C32H38N4O4:
C, 70.81; H, 7.06; N, 10.33. Found: C, 71.01; H, 7.04; N,
10.32.
1
62%). H NMR (CDCl3, d): 0.86 (d, J=6.7 Hz, 6H),
0.89 (d, J=7 Hz, 6H), 1.36 (s, 9H), 1.67 (m, 2ꢂ1H), 2.43
(d, J=6.7 Hz, 2H), 2.56 (d, J=7 Hz, 2H), 3.32 (s, 2H),
5.54 (s, 1H), 9.31 (s, 1H). Anal. calcd for C16H34N4O3:
C,.58.14; H, 10.38; N, 16.96. Found: C, 58.35; H, 10.32;
N, 16.85.
Boc-NꢀhLeu-Ni-Bu-NH2
[8a,
R=CH2CH(CH3)2,
PG1=Boc]. The title compound 8a was synthesized
from 6a (5.4 g, 10 mmol) following the general proce-
dure for 7a. (2.7 g, 84%); mp 104 ꢁC. 1H NMR (CDCl3,
d): 0.93 (d, J=6.7 Hz, 6H), 0.97 (d, J=6.7 Hz, 6H), 1.45
(s, 9H), 1.76 (m, 1H), 2.04 (m, 1H), 2.64 (d, J=6.7 Hz,
Z-NꢀhLeu-N-azaLeu-Ot-Bu [6d, R=CH2CH(CH3)2,
PG=Boc, PG1=Z]. The title compound was synthe-

4886
K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
2H), 3.35 (d, J=6.7 Hz, 2H), 3.93 (d, J=6.2 Hz, 2H),
4.03 (s, 2H), 6.69 (s, 1H). 13C NMR (CDCl3, d): 20.3,
21.1, 26.1, 27.1, 28.7, 57.1, 58.1, 65.8, 79.9, 155.8, 173.2
two rotameric forms are observed in proportion 85/15,
only data of the major compound are reported. Anal.
calcd for C15H32N4O3: C, 56.96; H, 10.13; N, 17.72.
Found: C, 56.73; H, 10.19; N, 17.73.
Boc-NꢀhLeu-NBz-NH2 (8f, R=CH2C6H5, PG1=Boc).
To a solution of 6f (5.4 g, 10 mmol) in 2-propanol
(50 mL) was added Pd/C 10% (60 mg per mmol, 6 mg).
After 18 h of stirring under H2 atmosphere, the solution
was filtered on Celite and the solvent was evaporated to
afford 8f (2.9 g, 84%). Mp 90 C. H NMR (CDCl3, d):
1.01 (d, J=6.5 Hz, 6H), 1.52 (s, 9H), 1.82 (m, 1H), 2.76
(d, J=6.5 Hz, 2H), 3.86 (s, 2H), 4.05 (s, 2H), 4.77 (s,
2H), 6.98 (s, 1H), 7.41 (m, 5H). 13C NMR (CDCl3, d):
21.1, 27.1, 28.8, 53.2, 59.0, 65.8, 79.8, 127.3, 128.4,
129.0, 129.3, 135.9, 155.7, 173.1. Anal. calcd for
C18H30N4O3 : C, 61.69; H, 8.63; N, 15.99. Found: C,
61.16; H, 8.66; N, 15.71.
ꢁ
1
Boc-NꢀhLeu-Nn-Bu-NH2 [8b, R=(CH2)3CH3, PG1=
Boc]. The title compound was synthesized from 6d
(5.4 g, 10 mmol) following the general procedure for 7a
(2.5 g, 78%); mp 105 ꢁC. H NMR (CDCl3, d): 0.93 (d,
1
J=6.7 Hz, 6H), 0.94 (t, J=7 Hz, 3H), 1.34 (m, 2H), 1.43
(s, 9H), 1.57 (m, 2H), 1.73 (m, 1H), 2.61 (d, J=6.7 Hz,
2H), 3.50 (t, J=7 Hz, 2H), 3.88 (br s, 2H), 4.31 (br s,
2H), 6.79 (br s, 1H). 13C NMR (CDCl3, d): 14.1, 20.1,
21.1, 26.9, 28.6, 30.2, 49.3, 57.9, 59.6, 65.9, 79.1, 155.8,
173.2, two rotameric forms are observed in proportion
90/10, only data of the major compound are reported.
Anal. calcd for C15H32N4O3: C, 56.96; H, 10.13; N,
17.72. Found: C, 56.77; H, 9.99; N, 17.57.
Boc-NꢀhLeu-azaLeu-CF3 [9a, R=CH2CH(CH3)2, PG1=
Boc, R1=CF3]. To a stirred and cooled solution (0 ꢁC)
of 7a (1.6 g, 5 mmol) in ether (5 mL) and triethylamine
(0.6 g, 5.5 mmol) was added dropwise trifluoroacetic
anhydride (1.2 g, 5.5 mmol) in 5 mL of ether. The mix-
ture was allowed to stir for 6 h. The reaction mixture
was washed three times with water (30 mL). The organic
layer was dried (Na2SO4), filtered and evaporated in
vacuo to give the crude product 9a which precipitate
slowly in ether: (0.7 g, 65%). Mp 105 ꢁC. 1H NMR
(CDCl3, d): 0.87 (d, J=7 Hz, 12H), 1.37 (s, 9H), 1.57
(m, 1H), 1.88 (m, 1H), 2.50 (br, 2H), 3.07–3.86 (br, 2H),
3.42 (s, 2H), 5.81 (s, 1H), 10.70 (s, 1H). 13C NMR
(CDCl3, d): 20.1, 20.7, 26.1, 26.9, 28.4, 56.2, 62.3, 69.1,
81.8, 119.3, 157.3, 158.5, 170.1. Anal. calcd for
C17H31F3N4O4 : C, 49.52; H, 7.52; F, 13.84; N, 13.59.
Found: C, 49.77; H, 7.80; F, 13.42; N, 13.88.
PhCO-NꢀhLeu- Ni-Bu-NH2 [8c, R=CH2CH (CH3)2,
PG1=PhCO]. The title compound 8c was synthesized
from 6e (5.4 g, 10 mmol) following the general proce-
dure for 7a. (3.5 g, 84%); oil. 1H NMR (CDCl3, d): 0.91
(d, J=6.7 Hz, 6H), 0.98 (d, J=6.5 Hz, 6H), 1.81 (m,
1H), 2.02 (m, 1H), 2.81 (d, J=6.7 Hz, 2H), 3.34 (d,
J=6.5 Hz, 2H), 3.91 (s, 2H), 4.12 (s, 2H), 7.345–7.83
(m, 5H), 8.79 (s, 1H) two rotameric forms are observed
in proportion 85/15, only data of the major compound
are reported. Anal. calcd for C18H30N4O2: C, 64.64; H,
9.04; N, 16.75. Found: C, 64.54; H, 8.89; N, 16.89.
Boc-NꢀhLeu-N-azaLeu-CF3 [10a, R=CH2CH(CH3)2,
PG1=Boc, R1=CF3]. The title compound was synthe-
sized from 8a (1.6 g, 5 mmol) following the general pro-
cedure for 9a. (2.7 g, 65%). Mp 110 ꢁC. 1H NMR
(CDCl3, d): 0.84 (d, J=6.7 Hz, 6H), 0.88 (d, J=6.7 Hz,
6H), 1.35 (s, 9H), 1.67 (m, 1H), 1.77 (m, 1H), 2.31 (d,
J=6.7 Hz, 2H), 3.33 (d, J=6.7 Hz, 2H), 3.35 (s, 2H),
5.39 (s, 1H), 11.22 (s, 1H). 13C NMR (CDCl3, d): 20.8,
21, 26.3, 27.4, 28.5, 55.8, 64.3, 67.9, 81.9, 119.8, 156.1,
157.4, 169.2. Anal. calcd for C17H31F3N4O3: C, 49.51;
H, 7.52; F, 13.83; N, 13.59. Found: C, 49.30; H, 7.83; F,
13.51; N, 13.86.
Z-NꢀhLeu-Ni-Bu-NH2
(8d,
R=CH2CH(CH3)2,
PG1=Z]. A solution of hydrazino-azapeptoid 6d (4.5 g,
10 mmol) in ether (10 mL) was cooled to 0 ꢁC. A satu-
rated solution of HCl in ether (20 mL) was added drop-
wise, and the solution was allowed to warm to room
temperature. A white precipitate was formed, which was
collected by filtration. The residue was dissolved in
NaHCO3 1 N (20 mL) and extracted with ether
(3ꢂ20 mL). The organic layers was dried under
Na2S2O4 and evaporated to give product 8d as an oil
1
(2.2 g, 62%). H NMR (CDCl3, d): 0.93 (d, J=7.5 Hz,
6H), 0.97 (d, J=6.75 Hz, 6H), 1.77 (m, 1H), 2.02 (m,
1H), 2.70 (d, J=6.75 Hz, 2H), 3.33 (d, J=7.5 Hz, 2H),
3.85–4.13 (br s, 2H), 4.01 (s, 2H), 5.13 (s, 2H), 7.26 (s,
1H), 7.36 (s, 5H). 13C NMR (CDCl3, d): 20.6, 21.2, 26.7,
28.6, 56.1, 63.2, 67.2, 128.3, 128.6, 128.9, 136.5, 154.7,
157.1. Two rotameric forms are observed in proportion
95/5; only data of the major compound are reported.
Anal. calcd for C18H29N4O3: C, 61.87; H, 8.36; N,
16.03. Found: C, 61.75; H, 8.32; N, 15.95.
Boc-NꢀhLeu-N-azaNorleu-CF3 [10b, R=(CH2)3CH3,
PG1=Boc, R1=CF3]. The title compound was synthe-
sized from 8b (1.6 g, 5 mmol) following the general pro-
cedure for 9a (3.3 g, 81%). Mp 90–95 ꢁC. 1H NMR
(CDCl3, d): 0.80 (d, J=6.7 Hz, 6H), 0.86 (t, J=7 Hz,
3H), 1.24 (m, 2H), 1.35 (s, 9H), 1.45 (m, 2H), 1.65 (m,
1H), 2.32 (d, J=7 Hz, 2H), 3.34 (s, 2H), 3.49 (br, 2H),
5.73 (s, 1H), 11.32 (s, 1H). 13C NMR (CDCl3, d): 14.1,
20.3, 20.9, 26.3, 28.5, 29.3, 48.2, 64.1, 67.7, 81.6, 113.2,
156.2, 157.3, 168.5. Anal. calcd for C17H31F3N4O4 : C,
49.52; H, 7.52; F, 13.84; N, 13.59. Found: C, 49.68; H,
7.68; F, 13.77; N, 13.55.
Z-NꢀhLeu-NBz- NH2 (8e, R=CH2C6H5, PG1=Z).
The title compound 8e was synthesized from 6e (4.9 g,
10 mmol) following the general procedure for 8d. (2.5 g,
64%); oil. 1H NMR (CDCl3, d): 0.96 (d, J=6.5 Hz, 6H),
1.77 (m, 1H), 2.50 (s, 1H), 2.75 (d, J=7 Hz, 2H), 3.73 (s,
2H), 4.02 (s, 2H), 4.66 (s, 2H), 5.11 (s, 2H), 7.26 (s, 1H),
7.35 (m, 2ꢂ5H). Anal. calcd for C21H27N4O3: C,.65.78;
H, 7.10; N, 14.61. Found: C, 65.56; H, 7.05; N, 14.52.
PhCO-NꢀhLeu-N-azaLeu-CF3 [10c, R=CH2CH(CH3)2,
PG1=PhCO, R1=CF3]. The title compound was syn-
thesized from 8c (2.1 g, 5 mmol) following the general
ꢁ
1
procedure for 9a (4.2 g, 82%). Mp 144 C. H NMR
(CDCl3, d): 0.87 (d, J=6.5 Hz, 6H), 0.99 (d, J=6.5 Hz,

K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
4887
6H), 1.68 (m, 1H), 1.86 (m, 1H), 2.54 (d, J=6.5 Hz,
2H), 3.44 (s large, 2H), 3.51 (s, 2H), 7.46–7.76 (m, 5H),
7.89 (s, 1H), 11.90 (s, 1H). 13C NMR (CDCl3, d): 20.8,
21.1, 26.5, 27.3, 55.9, 63.7, 67.5, 127.8, 129.1, 132.1,
132.9, 155.9, 156.7, 168.8, 169.8. Anal. calcd for
C19H27F3N4O3: C, 54.81; H, 6.49; F, 13.46; N, 13.70.
Found: C, 55.15; H, 6.53; F, 13.28; N, 13.61.
1H), 11.01 (s, 1H). 13C NMR (CDCl3, d): 14.1, 14.2,
20.3, 21.0, 26.4, 28.6, 29.3, 48.1, 63.1, 63.6, 67.4, 80.9,
155.7, 156.6, 159.5, 169.1. Anal. calcd for C19H36N4O6:
C, 54.81; H, 8.65; N, 13.46. Found: C, 54.62; H, 8.81; N,
13.48.
Boc-NꢀhLeu-N-azaLeu-H [10g, R=CH2CH (CH3)2,
PG1=Boc, R1=H]. To a stirred and cooled solution
(0 ꢁC) of pentafluorophenol (0.92 g, 5 mmol) in ether
(5 mL) was added formic acid (0.28 g, 6 mmol) and
DCC (1.03 g, 5 mmol). After 10 min, 8a (0.8 g, 2.5 mmol)
in solution of chloroform (5 mL) was added to the mix-
ture and stirred at room temperature. After 4 h, the
reaction mixture was diluted by chloroform (20 mL) and
DIEA (0.58 g, 5 mmol) was added. The reaction mixture
was washed with HCl 1 N (10 mL), NaHCO3 5%
(10 mL) and water (10 mL). The organic layer was dried
(Na2SO4), filtered and evaporated in vacuo to give 10g
as a precipitate (2.92 g, 85%). Mp 124 ꢁC. 1H NMR
(CDCl3, d): 0.94 (d, J=6.5 Hz, 6H), 0.98 (d, J=6.5 Hz,
6H), 1.46 (s, 9H), 1.75 (m, 1H), 1.94 (m, 1H), 2.47 (d,
J=6.5 Hz, 2H), 3.48 (s, 2H), 3.50 (d, J=6.5 Hz, 2H),
5.69 (s, 1H), 8.12 (s, 1H), 10.34 (s, 1H). 13C NMR
(CDCl3, d): 20.7, 21.0, 26.5, 27.1, 28.7, 55.3, 63.8, 67.0,
81.4, 156.8, 159.8, 169.7. Anal. calcd for C16H32N4O4:
C, 55.76; H, 9.36; N, 16.27. Found: C, 55.74; H, 9.47; N,
16.18.
Boc-NꢀhLeu-azaLeu-CO2Et [9d, R CH2CH (CH3)2,
PG1=Boc, R1=CO2Et]. To a stirred and cooled solu-
tion (0 ꢁC) of 7a (1.6 g, 5 mmol) in ether (5 mL) and
triethylamine (0.6 g, 5.5 mmol) was added dropwise
ethyl oxalyl chloride (0.75 g, 5.5 mmol) in 5 mL of ether.
The mixture was allowed to stir for 6 h. The reaction
mixture was washed three times with water (30 mL). The
organic layer was dried (Na2SO4), filtered and evapo-
rated in vacuo to give the crude product 9d, which
slowly precipitates in ether (2.3 g, 56%). Mp 147 ꢁC. H
1
NMR (CDCl3, d): 0.86 (d, J=6.5 Hz, 6H), 0.90 (d,
J=6.5 Hz, 6H), 1.24 (t, J=7.2 Hz, 3H), 1.38 (s, 9H),
1.57 (m, 1H), 1.85 (m, 1H), 2.47 (d, J=7 Hz, 2H), 3.38
(s, 2H), 3.40 (d, J=6.7 Hz, 2H), 4.18 (q, J=7.2 Hz, 2H),
5.75 (s, 1H), 10.40 (s, 1H). 13C NMR (CDCl3, d): 14.3,
20.2, 20.8, 26.3, 26.8, 28.5, 54.6, 62.2, 62.7, 68.8, 81.7,
157.1, 162.5, 163.6, 169.8. Anal. calcd for C19H36N4O6 :
C, 54.81; H, 8.65; N, 13.46. Found: C, 54.26; H, 8.49; N,
13.04.
Boc-NꢀhLeu-N-azaLeu-CO2Et [10d, R=CH2CH(CH3)2,
PG1=Boc, R1=CO2Et]. The title compound was syn-
thesized from 8a (1.6 g, 10 mmol) following the general
procedure for 9d (2.24 g, 54%). Mp 100–105 ꢁC. 1H
NMR (CDCl3, d): 0.99 (d, J=6.5 Hz, 12H), 1.49 (t,
J=7 Hz, 3H), 1.51 (s, 9H), 1.78 (m, 1H), 1.94 (m, 1H),
2.49 (d, J=7 Hz, 2H), 3.48 (s, 2H), 3.54 (d, J=7 Hz,
2H), 4.45 (q, J=7 Hz, 2H), 5.91 (s, 1H), 11.06 (s, 1H).
13C NMR (CDCl3, d): 13.7, 18.8, 19.5, 25.7, 28.5, 54.1,
60.3, 62.1, 63.2, 82.1, 149.1, 156.8, 162.4. Anal. calcd for
C19H36N4O6: C, 54.81; H, 8.65; N, 13.46. Found: C,
54.85; H, 8.58; N, 13.31.
Boc-NꢀhLeu-N-azaLeu-CH2Br [10h, R=CH2CH(CH3)2,
PG1=Boc, R1=CH2Br]. To a stirred and cooled solu-
tion (0 ꢁC) of 8a (1.6 g, 5 mmol) in dichloromethane
(10 mL) and pyridine (0.6 g, 6 mmol) was added drop-
wise bromo acetyl bromide (1.2 g, 6 mmol) in 5 mL of
dichloromethane. The mixture was allowed to stir for
5 h. The reaction mixture was washed three times with
water (50 mL). The organic layer was dried (Na2SO4),
filtered and evaporated in vacuo to give the crude pro-
duct 10hꢁwhich precipitate slowly in ether (1.27 g, 58%).
1
Mp 105 C. H NMR (CDCl3, d): 0.95 (d, J=6.5 Hz,
6H), 0.98 (d J=6.5 Hz, 6H), 1.48 (s, 9H), 1.75 (m, 1H),
1.91 (m, 1H), 2.50 (d, J=7 Hz, 2H), 3.46 (d, J=7 Hz,
2H), 3.49 (s, 2H), 3.89 (s, 2H), 5.79 (s, 1H), 10.39 (s,
1H). 13C NMR (CDCl3, d): 18.9, 19.5, 19.7, 25.2, 26.1,
27.5, 55.0, 55.4, 60.8, 64.4, 78.8, 154.8, 171.1, 174.1.
Anal. calcd for C17H33N4O4Br: C, 46.68; H, 7.60; N,
12.81; Br, 18.27. Found: C, 46.56; H, 7.52; N, 12.75; Br,
18.14.
PhCO-NꢀhLeu-N-azaLeu-CO2Et [10e, R=CH2CH(CH3)2,
PG1=PhCO, R1=CO2Et]. The title compound was
synthesized from 8c (2.1 g, 10 mmol) following the
general procedure for 9d. (2.85 g, 55%). Mp 105 ꢁC. H
1
NMR (CDCl3, d): 0.77 (d, J=6.7 Hz, 6H), 0.86 (d,
J=6.7 Hz, 6H), 1.34 (t, J=7.2 Hz, 3H), 1.70 (m, 1H),
1.76 (m, 1H), 2.47 (d, J=7 Hz, 2H), 3.35 (d, J=7 Hz,
2H), 3.49 (s, 2H), 4.31 (q, J=7.2 Hz, 2H), 7.33–7.67 (m,
5H), 7.83 (s, 1H), 11.16 (s, 1H). 13C NMR (CDCl3, d):
13.7, 18.7, 19.5, 25.6, 28.5, 54.1, 60.3, 62.1, 63.2, 82.1,
149.1, 156.9, 162.5, 168.4. Anal. calcd for C21H32N4O5:
C, 60.00; H, 7.62; N, 13.33. Found: C, 59.69; H, 7.69; N,
12.96.
Boc-NꢀhLeu-N-azaPhe-CH2Br (10i, R=CH2C6H5, PG1=
Boc, R1=CH2Br). The title compound was synthesized
from 8f (3.5 g, 10 mmol) following the general procedure
ꢁ
1
for 10h (2.9 g, 62%); mp 135 C; H NMR (CDCl3, d):
0.81 (d, J=6.5 Hz, 6H), 1.44 (s, 9H), 1.65 (m, 1H), 2.50
(d, J=6 Hz, 2H), 3.52 (s, 2H), 3.85 (s, 2H), 4.85 (br s,
2H), 5.69 (s, 1H), 7.33 (m, 5H), 10.15 (s,1H). 13C NMR
(CDCl3, d): 19.5, 25.3, 28.4, 36.5, 54.1, 54.5, 62.1, 82.1,
125.7, 125.4, 127.3, 138.5, 156.6, 168.2, 170.5. Anal.
calcd for C20H31N4O4Br: C, 50.96; H, 6.63; N, 11.89;
Br, 16.95. Found: C, 50.69; H, 6.60; N, 11.82; Br, 16.79.
Boc-NꢀhLeu-N-azaNorleu-CO2Et [10f, R=(CH2)3CH3,
PG1=Boc, R1=CO2Et]. The title compound was syn-
thesized from 8a (1.6 g, 10 mmol) following the general
ꢁ
1
procedure for 9d (2.28 g, 55%). Mp 105 C; H NMR
(CDCl3, d): 0.83 (d, J=6.7 Hz, 6H), 0.85 (t, J=7.2 Hz,
3H), 1.17–1.41 (m, 4H), 1.30 (t, J=7.2 Hz, 3H), 1.32 (s,
9H), 1.61 (m, 1H), 2.30 (d, J=6.7 Hz, 2H), 3.34 (s, 2H),
3.46 (t, J=7.2 Hz, 2H), 4.24 (q, J=7.2 Hz, 2H), 5.93 (s,
Z-NꢀhLeu-N-azaLeu-CH2Br [10j, R=CH2CH(CH3)2,
PG1=Z, R1=CH2Br]. The title compound was synthe-
sized from 8d (3.5 g, 10 mmol) following the general

4888
K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
1
procedure for 10h (3.3 g, 69%); oil; H NMR (CDCl3,
d): 0.85 (d, J=7 Hz, 2ꢂ6H), 1.65 (m, 1H), 1.79 (m, 1H),
2.52 (d, J=7 Hz, 2H), 2.63 (d, J=6.75 Hz, 2H), 3.64 (s,
2H), 4.59 (s, 2H), 5.12 (s, 2H), 6.58 (s, 1H), 7.45 (m,
5H), 10.24 (s, 1H). 13C NMR (CDCl3, d): 18.7, 19.5,
25.3, 25.6, 36.5, 53.6, 60.3, 61.6, 120.3, 125.7, 128.8,
147.2, 151.2, 168.5, 171.5. Anal. calcd for
C20H32N4O4Br: C, 50.85; H, 6.83; N, 11.86; Br, 16.91.
Found: C, 50.75; H, 6.78; N, 11.78; Br, 16.82.
58.6, 64.8, 78.4, 128.6, 129.6, 129.9, 134.2, 135.9, 138.2,
141.5, 156.8, 171.8. 11B NMR (DMSO-d6/Et2OBF3, d):
30 (br s). Anal. calcd for C22H37N4O5 B: C, 58.93; H,
8.32; N, 12.50; B, 2.41. Found: C, 58.69; H, 8.32; N,
12.50; B, 2.45.
Boc-NꢀhLeu-N-azaLeu-CHPh-p-B(OH)2 (11c). The title
compound was synthesized from 8a (1.8 g, 5 mmol) and
4-formylbenzene boronic acid (0.8 g, 5.5 mmol) follow-
ing the general procedure for 11a: (2.5 g, 96%). Mp
186 ꢁC. H NMR (DMSO-d6, d): 0.83 (d, J=6.75 Hz,
1
Z-NꢀhLeu-N-azaPhe-CH2Br
(10k,
R=CH2C6H5,
PG1=Z, R1=CH2Br). The title compound was synthe-
sized from 8e (3.8 g, 10 mmol) following the general
12H), 1.31 (s, 9H), 1.59 (m, 1H), 1.95 (m, 1H), 2.58 (d,
J=6.75 Hz, 2H), 3.76 (d, J=6.75 Hz, 2H), 4.07 (s, 2H),
7.46 (s, 1H), 7.64–7.81 (m, 4H), 7.93 (s, 1H), 8.10 (s,
2H). 13C NMR (DMSO-d6, d): 20.3, 20.9, 25.1, 26.5,
28.4, 46.5, 58.7, 64.8, 78.5, 126.3, 134.7, 136.1, 136.5,
140.5, 154.8, 171.6. 11B NMR (DMSO-d6/Et2OBF3, d):
30 (br s). Anal. calcd for C22H37N4O5 B: C, 58.93; H,
8.32; N, 12.50; B, 2.41. Found: C, 58.69; H, 8.32; N,
12.50; B, 2.45.
1
procedure for 10h oil (3 g, 60%). H NMR (CDCl3, d):
0.83 (d, J=6.75 Hz, 6H), 1.68 (m, 1H), 2.50 (d, J=7 Hz,
2H), 3.54 (s, 2H), 3.75 (s, 2H), 4.77 (s, 2H), 5.01 (s, 2H),
6.67 (s, 1H), 7.35 (m, 5H), 10.04 (s, 1H). 13C NMR
(CDCl3, d): 19.5, 25.3, 36.5, 53.7, 54.8, 61.6, 120.3,
125.4, 127.3, 128.8, 138.4, 147.1, 151.2, 168.2, 170.4.
Anal. calcd for C23H29N4O4Br: C, 54.66; H, 5.78; N,
11.09; Br, 15.81. Found: C, 54.32; H, 5.65; N, 10.98; Br,
15.78.
Biological Assay
Boc-NꢀhLeu-N-azaLeu-CH2Cl [10l, R=CH2CH (CH3)2,
PG1=Boc, R1=CH2Cl]. The title compound was syn-
thesized from 8a (3.2 g, 10 mmol) following the general
All reagents used in the biological assay were purchased
from Merck, (Darmstadt, Germany) or Sigma (Chemi-
cal CO. (St Louis, MO). DFMO was obtained from
ILEX Oncology (San Antonio, TX, USA). Compounds:
The compounds were all dissolved in DMSO and stored
at minus 80 ꢁC as a stock solution 100 mM. For experi-
mental procedure, the compounds were diluted from
stock solution in filtered growth medium and used
immediately. Data are given as means values of three
experiments. Comparisons between means are made
using the Student’s t-test assuming significance at
p<0.01.
procedure for 10h (2.2 g, 57%); mp 135 ꢁC; H NMR
1
(CDCl3, d): 0.95 (d, J=6.5 Hz, 6H), 0.98 (d, J=6.5 Hz,
6H), 1.47 (s, 9H), 1.75 (m, 1H), 1.88 (m, 1H), 2.48 (d,
J=7 Hz, 2H), 3.48 (br, 2ꢂ2H), 4.10 (s, 2H), 5.63 (s,
1H), 10.34 (s, 1H). 13C NMR (CDCl3, d): 18.8, 19.6,
25.3, 25.7, 28.5, 38.5, 54.2, 60.3, 62.1, 82.1, 156.8, 165.2,
168.5. Anal. calcd for C17H33N4O4Cl: C, 52.04; H, 8.41;
N, 14.29; Cl, 8.92. Found: C, 51.95; H, 8.48; N, 14.12;
Cl, 8.95.
Boc-NꢀhLeu-N-azaLeu-CHPh-o-B(OH)2 (11a). To a
stirred solution of 8a (1.8 g, 5 mmol) in ether (5 mL) was
added dropwise 2-formylbenzene boronic acid (0.8 g,
5.5 mmol) in solution of ether (5 mL), a white pre-
cipitate appeared. After 1 h stirring, the precipitate was
filtered and washed with ether. (2.4 g, 94%). Mp 159 ꢁC.
1H NMR (DMSO-d6, d): 0.79 (d, J=6.75 Hz, 6H), 0.82
(d, J=6.75 Hz, 6H), 1.29 (s, 9H), 1.56 (m, 1H), 1.97 (m,
1H), 2.55 (d, J=6.75 Hz, 2H), 3.68 (d, J=6.75 Hz, 2H),
4.06 (s, 2H), 7.24–7.51(m, 4H), 7.76 (d, J=7.25 Hz, 1H),
8.16 (s, 2H), 8.26 (s, 1H). 13C NMR (DMSO-d6, d):
20.3, 20.9, 24.9, 26.5, 28.5, 46.9, 58.6, 64.7, 78.5, 126.1,
128.8, 129.3, 134.1, 136.7, 138.1, 142.5, 154.8, 171.7. 11B
NMR (DMSO-d6/Et2OBF3, d): 30 (br s). Anal. calcd for
C22H37N4O5 B: C, 58.93; H, 8.32; N, 12.50; B, 2.41.
Found: C, 58.69; H, 8.32; N, 12.50; B, 2.45.
Analysis of the cytotoxicity
Cell culture line. L1210 murine leukemia cells were
grown in RPMI 1640 medium (Eurobio, Les Ulis,
France) supplemented with 10% fetal calf serum, 2 mM
glutamine penicillin (100 Units/mL), and streptomycin
(50 mg/mL) (BioMerieux Marcy l’Etoile, France) (full
medium). Cells were cultivated at 37 ꢁC under a humi-
dified 5% CO2 atmosphere and maintained in exponen-
tial growth as described by Delcros et al.27
In vitro evaluation of cytotoxicity. The effect of the dif-
ferent hydrazino-aza and N-azapeptoides on cell growth
was assayed in sterile 96 wells microtiter tissue plates
(Becton Dickinson, Oxnard, CA, USA). The cells were
seeded at 5ꢂ105 cells/mL of medium (100 mL per well).
The different hydrazino-aza and N-azapeptoides (5 mL/
per well) at the appropriate concentration (10 nM–
1 mM) were added at the time of seeding. The incu-
bations were performed at 37 ꢁC during 24 and 48 h.
After exposure to the compounds, cell growth was
determined by measuring the formazan formation from
3-(4,5 - dimethylthiazol - 2yl) - 2,5 - diphenyltetrazolium
(MTT) as previously described.28 A Titertek Multiskan
MCC/340 microplate reader (Labsystems, Cergy Pontoise,
France) was used for absorbance measurements (540 nm).
Boc-NꢀhLeu-N-azaLeu-CHPh-m-B(OH)2 (11b). The
title compound was synthesized from 8a (1.8 g, 5 mmol)
and 3-formylbenzene boronic acid (0.8 g, 5.5 mmol) fol-
lowing the general procedure for 11a. (2.3 g, 92%). Mp
110 ꢁC. H NMR (DMSO-d6, d): 0.92 (d, J=6.75 Hz,
1
6H), 0.95 (d, J=6.75 Hz, 6H), 1.41 (s, 9H), 1.54 (m,
1H), 1.68 (m, 1H), 2.68 (d, J=6.75 Hz, 2H), 4.01 (s
large, 2H), 4.13 (s, 2H), 7.45 (s large, 1H), 7.84 (s, 1H),
7.88 (s, 1H), 8.05 (s, 1H), 8.17 (s, 1H), 8.23 (s, 2H). 13C
NMR (DMSO-d6, d): 20.3, 20.9, 24.9, 26.5, 28.5, 46.8,

K. Bouget et al. / Bioorg. Med. Chem. 11 (2003) 4881–4889
4889
Acknowledgements
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This work was supported by the ‘Ligue Nationale Con-
tre le Cancer (Bretagne)’. The ‘Conseil Regional de
Bretagne’ and the ‘CNRS’ (Centre Nationale de la
Recherche Scientifique).
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