A novel aryl-hydrazide from the marine lichen Lichina pygmaea: Isolation, synthesis of derivatives, and cytotoxicity assays

Article abstract of DOI:10.1016/j.bmcl.2010.06.013

A new aryl-hydrazide l-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of l-glutamic acid with phenylhydrazine moieties were useful

Full text of DOI:10.1016/j.bmcl.2010.06.013

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4582–4586
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel aryl-hydrazide from the marine lichen Lichina pygmaea: Isolation,
synthesis of derivatives, and cytotoxicity assays
*
Catherine Roullier, Marylène Chollet-Krugler , Pierre van de Weghe, Françoise Lohézic-Le Devehat,
Joël Boustie
EA 4090 ‘Substances licheniques et photoprotection’, Faculté des Sciences pharmaceutiques et Biologiques, Université Européenne de Bretagne, Université de Rennes 1,
2 Av. du Pr. Léon Bernard, 35043 Rennes Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new aryl-hydrazide
L-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of
Received 26 April 2010
Revised 1 June 2010
Accepted 2 June 2010
Available online 8 June 2010
Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of
L
-glutamic
acid with phenylhydrazine moieties were useful to elucidate the structure of 3 and to carry out biological
assays. Thus, the cytotoxicity of the ortho-, meta-, and para-hydroxyl isomers along with their respective
benzyl intermediates, and a natural methoxylated analog, were evaluated on murine and human mela-
noma cells (B16, A375). The para-hydroxyl isomer 6 was found to be the most active (IC50 = 1.6 lM)
on B16 cells.
Keywords:
Lichen
Pygmeine
Ó 2010 Elsevier Ltd. All rights reserved.
Aryl-hydrazide
Cytotoxic activity
Lichina pygmaea
As a continuation of our ongoing work1 on bioactive secondary
metabolites isolated from lichens, we focused on Lichina pygmaea,
which is a shrubby black marine lichen, found on the rocky shore
at the seaside, in the intertidal region. This lichen results from
the symbiosis of a fungus and a Calothrix cyanobacterium. Forming
dark cushions of some millimeters thick, it suffers from harmful UV
radiation, exposure to sun and reverberation, a salty environment,
immersion phases, and the violence of waves. So, it is assumed that
this lichen may possess protective secondary metabolites, and
antioxidant properties are claimed for a mycosporine compound.2,3
Two amino acid derivatives had previously been isolated, namely
mycosporine-serinol (1), which has a good UVB filter profile, and
temperature for 2 h, then methanol (1.5 L ꢀ 7) at 45 °C for 2 h,
and methanol 50% in water (1.5 L ꢀ 3) at 45 °C for 2 h. A first frac-
tionation was conducted on the filtrate of the methanolic extract of
L. pygmaea by a multiple dual-mode centrifugal partition chroma-
tography method (MDM CPC) previously described,4 using BuOH/
AcOH/H2O (4:1:5) as the biphasic solvent system. Fraction Lp6 re-
sulted in the isolation of compound 2 (121 mg) and fraction Lp8 in
the isolation of compound 1 (25 mg). Fraction Lp7 was evaporated
to dryness (1.065 g), and the residue was further partitioned
between CH2Cl2 and H2O. The aqueous layer was concentrated
and subjected to chromatography over a reversed-phase silica gel
the L
-glutamic acid 5-[(2,4-dimethoxyphenyl)-hydrazide] (2).4 This
O
last compound was found to have antioxidant activities,5 and is
claimed to be a myeloperoxidase inhibitor as isolated from Penicil-
lium cultures.6 A third compound (3) has additionally been isolated
from a crude methanolic extract of L. pygmaea, by Centrifugal Par-
tition Chromatography (CPC) (Fig. 1). Low quantities obtained and
ambiguous spectroscopic data for structure identification led us to
synthesize analogs which were also tested for cytotoxicity in a pre-
liminary assay on melanoma cell lines.
OCH3
3'
H3CO
4'
5'
2' OCH3
(S)
HO
NH2
NH
H
4
2
HOH2C
N
5
1
OH
N
1'
(S)
6'
H
3
O
O
OH OH
Mycosporine serinol 1
L-glutamic acid 5-[(2,4-dimethoxy
phenyl)-hydrazide] 2
HO
NH2
H
HO
NH2
H
N
N
OH
Extraction by three successive solvents was performed on 270 g
OH
N
of the dried lichen,7 namely ethyl acetate (1.5 L ꢀ 3) at room
N
(S)
O
H
H
O
O
O
Agaritine
Unknown 3
* Corresponding author. Tel.: +33 2 232 348 96; fax: +33 2 232 347 04.
E-mail address: marylene.chollet@univ-rennes1.fr (M. Chollet-Krugler).
Figure 1. Structures of compounds isolated from L. pygmaea, and the similar
agaritine.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.013

C. Roullier et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4582–4586
4583
Table 1
NMR spectroscopic data (270 MHz, D2O) of isolated compounds 2 and 3, compared with synthetic o-, m- and p-isomers 4–6
Position
2
3
4
5
6
a
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
2
3
4
176.3
56.6
28.7
174.4*
54.7
26.7
174.4*
54.7
26.7
175.2*
55.6
27.6
175.7*
56.0
28.0
3.70, m
2.08, m
2.41, m
3.77, t (6.1)
2.18, m
2.50, m
3.77, t (6.2)
2.17, m
2.50, m
3.78, t (6.2)
2.17, m
2.50, m
3.77, t (6.2)
2.17, m
32.1
30.1
30.1
30.9
31.4
2.49, m
5
176.9
132.8
157.0
102.1
151.2
107.6
116.8
58.4
175.2*
136.1
144.4
116.0
121.6
122.3
114.4
175.2*
136.1
144.4
116.0
121.6
122.3
114.4
176.1*
150.5
101.7
158.1
109.5
132.2
107.1
176.4*
142.4
117.4*
118.0*
151.9
118.0*
117.4*
10
20
6.36, d (2.2)
6.81, s
6.81, s
30
6.55, d (2.5)
6.85, s
6.85, s
6.85, s
6.85, s
6.86, s
6.86, s
6.86, s
6.86, s
40
6.44, m
7.15, t (8.1)
6.44, m
50
6.43, dd (8.7, 2.5)
6.71, d (8.7)
3.68, s
6.81, s
6.81, s
60
OCH3 (20)
OCH3 (40)
58.5
3.76, s
a
dC described by Hilbig et al.9
Not attributed signals.
*
(C-18 Hydro ChromabondÒ) using H2O/AcOH (0.1%) and increasing
gradients of MeOH (10%, 50% to 100% MeOH). The first fraction that
eluted with H2O/AcOH (0.1%) was collected in eight subfractions.
The seventh subfraction was evaporated to dryness (40 mg) and
the residue was made to precipitate in ACN/MeOH (90:10) to
obtain 20 mg of a yellow powder. It was finally subjected to a
semi-preparative reversed-phase HPLC (HypersilÒ BDS, 250 ꢀ
R2
R1
NH2 HOOC
HN COOBn
TBTU 1.5 eq.
DIPEA 3 eq.
R3
NH
.HCl
+
(S)
11-13
COOBn
1 eq.
DMF, rt
60-75%
1.2 eq.
R1
R2
R3
7
8
9
10
OBn
H
H
OBn
H
H
H
OBn
10 mm, 5 lm, 1.0 mL/min; UV detection at 254 nm) using H2O
H
(0.1% AcOH) for 45 min, and a MeOH/H2O non-linear gradient
(10% for 17 min, 50% for 22 min, and then 100% for 15 min), yield-
ing compound 3, tR 51.2 min (3 mg).
R2
R2
R1
R1
R3
R3
COOBn H2,Pd/C
HN
NH2
H
N
H
N
A first attempt for structure determination of this newly
described compound 3 was performed using NMR and MS.8 The
HRESIMS analysis of 3 displayed a molecular ion peak at m/z
254.1144 [M+H]+, confirmed by the presence of two other peaks
at m/z 276.0966 and m/z 292.0693 standing for [M+Na]+ and
[M+K]+, respectively, suggesting the molecular formula
MeOH, rt
COOBn
N
H
COOH
(S)
N
(S)
H
O
O
75-95%
R1
R2
R3
R1
R2
R3
11
12
13
OBn
H
H
H
OBn
H
H
H
4
5
6
OH
H
H
H
OH
H
H
H
OH
OBn
C
11H15N3O4. 1H NMR spectrum showed two signals at 2.18 and
Scheme 1. Synthesis of compounds 4–6.
2.50 ppm, respectively, each integrating for two protons and an-
other signal for one proton at 3.77 ppm, indicating the neighboring
of an oxygen or a nitrogen atom. Four protons were also observed
in the aromatic zone, as a broad singlet at 6.85 ppm. Close struc-
tural relationships between compound 3 and the previously iso-
lated compound 2 appeared, as a similar aliphatic proton pattern
was observed in 1H NMR (Table 1). Both compounds also presented
a yellow coloration with sulfuric anisaldehyde and a positive pink
reaction to ninhydrin reagent on TLC plates, confirming the pres-
ence of an amino residue in the molecule. These data along with
distinct Rf values (0.45 for 2 and 0.26 for 3 in CHCl3/MeOH/H2O
6:4:1), suggested a similar skeleton differing from the aromatic
ring substitution. Therefore, three possible structures 4–6 have
emerged as an option and the hydroxyl group position had to be
OBn
NaNO2 1.6 eq.
OBn
N
OBn
SnCl2 3.3 eq.
H2O
NH2
NH
.HCl
NH2
N
HCl 6 M, 0 °C
HCl 6 M, 0 °C
69%
8
1 eq.
Scheme 2. Preparation of (2-benzyloxy-phenyl)-hydrazine, hydrochloride 8.
6.73
H
6.58
114.2
H
119.1
OH
2.36
2.36
H
H
N
144.3
NH2
H
137.2
119.1
H
3.28
171.2
27.0
53.5
29.9
H
N
112.2
6.64
COOH
169.9
H
H
6.64
H
O
H
1.92
6.71
1.92
OH
OH
NH2
NH2
H
H
Figure 3. Structure of pygmeine and its 1H and 13C NMR chemical shift assignments
in DMSO-d6, with selected HMBC correlations.
N
OH
N
OH
N
N
(S)
O
Pygmeine 4
(S)
O
H
H
O
O
5
HO
ascertained (Fig. 2). The para-hydroxyl compound 6 was the only
one described in literature, as xanthodermine, isolated from the
Basidiomycete mushroom Agaricus xanthoderma.9 NMR proton
profiles were similar, but not conclusive and 13C NMR data sug-
gested the presence of six non equivalent aromatic carbons, which
was not consistent with the para-hydroxyl compound. Due to the
NH2
H
N
OH
N
H
(S)
O
Xanthodermine 6
O
Figure 2. Possible structures of compound 3.

4584
C. Roullier et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4582–4586
Table 2
Cytotoxicity results on melanoma cells (A375 and B16) after a 24 h or a 48 h exposure
Compound
A375
B16
24 h
% Inh at 50
48 h
% Inh at 50
24 h
% Inh at 50
48 h
% Inh at 50 lM
IC50
(
l
M)a
l
M
IC50
(
lM)
lM
IC50
>50
20 2.5
12
1.6 0.2
21
8
5
(
l
M)
l
M
IC50
27 1.5
14
12 0.2
0.7 0.15
(
l
M)
1
4
5
6
>50
>50
>50
>50
29
28
>50
19
11
11
8
12
77
80
4
>50
>50
>50
>50
30
29
>50
12
20
19
14
31
69
73
19
100
34
100
100
100
96
100
100
100
85
100
100
100
100
100
100
100
2
1
11
12
13
Etoposide
2
1
6
1
1
2
1.5
4.5
15
14.5
3
2
0.6 0.1
0.13 0.07
5
100
6
9
a
Each result is the mean SD of at least two experiments in triplicate.
limited quantities of the purified compound 3, only the 1H NMR
data were unambiguously informative and the synthesis of the
three optional derivatives appeared to be appropriate to determine
the structure and to facilitate biological assays.
Therefore synthesis of the three isomers was performed
through amide coupling of L-glutamic acid (with protecting groups
group. Due to the limited quantities of the isolated compound 3,
the optical rotation could not be recorded but it was demonstrated
on a chiral HPLC column that this compound presented exactly the
same retention time than the synthetic compound 4.19 It could
then be assumed that the 2-position stereochemistry may be ‘S’
as the biosynthetic pathway to this molecule probably implies
on the amino acid functions) and benzyloxy phenylhydrazines, fol-
lowed by cleavage of the protecting groups (Scheme 1). Different
approaches were given in previous studies for the coupling of
the natural L-glutamic acid.
This compound, which is reported here for the first time as a
natural product, and from a lichen, was named pygmeine. An anal-
ogous compound, the 1-(5-glutamyl)-2-methylhydrazine, where
the aryl group is replaced by a methyl group, was shown to be a
product of sheep brain glutamine synthetase by Meister and
co-workers in 1972.20 They demonstrated that methylhydrazine
could act as a substrate of this enzyme, in place of ammonia. It
could be assumed that pygmeine may also be synthesized by a glu-
tamine synthetase-like enzyme.
L-glutamic acid and a phenylhydrazine. Xanthodermine was
synthesized by Hilbig9 using HOTDO (2,5-diphenyl-4-hydroxy-3-
oxo-2,3-dihydrothiophene-1,1-dioxide) as a coupling reagent.10
Agaritine, a similar structure, was obtained using (1) the amino
acid azide derivative,11,12 (2) a mixed anhydride formed from the
amino acid and ethyl chloroformate,13 or (3) DCC (dicyclohexylcar-
bodiimide) coupling the Z-Glu-OBn and the corresponding phen-
ylhydrazine (in low yields: 1%, 25%, and 33%, respectively).14
Each isomer here was promptly synthesized in a two-step-reac-
tion procedure from the commercially available Z-Glu-OBn 7 and
the corresponding benzyloxyphenylhydrazine 8–10 (Scheme 1).
The 2-benzyloxyphenylhydrazine 8 was prepared from the 2-ben-
zyloxyaniline via the formation of a diazonium intermediate
(Scheme 2).15,16 The first step consisted in forming the C–N bond
between the protected glutamic acid 7 and the phenylhydrazine
derivative 8–10 using TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate) as coupling reagent in the
presence of the N,N-diisopropylethylamine Hünig’s base (DIPEA)
in DMF at room temperature.17 Compounds 4–6 were then ob-
tained after cleavage of the benzyl protecting groups of the isolated
11–13, with an overall yield of 48–66%.18
Compounds 2, 4–6, and 11–13 were tested for their cytotoxic
activity on human (A375) and murine (B16) melanoma cell lines,
and compared to the positive control etoposide (Table 2).21 As
shown by the toxicity at the highest 50 lM concentration tested,
the A375 cell line was found less sensitive than the B16 to these
compounds. The cytotoxicity observed for protected intermediates
(11–13) was higher than the corresponding final compounds (4–6)
on A375 but comparable on B16. It could be assumed that the pro-
tected compounds are less polar and therefore penetrate cell mem-
branes more readily. Once inside the cells they may be acted upon
by non-specific esterases. After a 24 and 48 h-exposure on B16, the
most active compound was the natural xanthodermine 6, which
strongly inhibited the cell growth with an IC50 at 1.6 lM (9 lM
for etoposide). Therefore xanthodermine, which was previously
described as being active against Bacillus strains,9 was found to
be tenfold more cytotoxic than compound 4, suggesting the p-hy-
droxyl position was enhancing the activity.
The spectroscopic data of each synthetic isomer 4–6 were com-
pared to those of the isolated compound 3 (Table 1). The proton
pattern in the aromatic zone was not consistent with the meta-hy-
droxyl derivative 5, as the four proton signals were split into three
peaks, corresponding to H-20, H-40/H-60 and H-50 chemical shifts.
Xanthodermine 6 and compound 4 should typically have the
AA0BB0 and the ABCD splitting pattern respectively, but they both
exhibited a broad singlet signal in the aromatic zone as well as
the isolated compound 3. A 0.05 ppm shift can be noticed for this
1H NMR signal in D2O and in DMSO-d6, the aromatic zone was ex-
tended from 6.56 to 6.76 ppm for 3 and 4, while the aromatic pro-
tons of compound 6 appeared between 6.51 and 6.63. The
comparison of the 13C NMR spectra clearly suggested the natural
compound 3 was corresponding to the synthetic structure 4,
namely the o-hydroxyl isomer. Such a similarity was confirmed
as the same correlations could be observed through the 2D COSY
and HMBC spectra in DMSO-d6 (Fig. 3), for the semi-purified com-
pound 3 and the synthetic compound 4. A correlation between the
hydrazide proton (dH 6.71, s) and carbon signals at dC = 112.2,
144.3, and 171.2 ascertained the ortho position of the hydroxyl
As a conclusion, a new aryl-hydrazide L-glutamic acid derivative
was isolated from a marine lichen L. pygmaea, and was shown to
have some activity on melanoma cancer cells. Synthesis of analogs
gave a hit compound which is xanthodermine, with a promising
cytotoxicity on B16. As most melanoma cells are rich in tyrosinase,
it can be expected that this kind of compound would be metabo-
lized by this enzyme, like the
c-glutaminyl-p-hydroxybenzene
(GHB) whose structure is very close, and therefore have specificity
against melanoma cells.22 This is in accordance with recent works
on aryl hydrazines and hydrazides, presenting them as promising
anticancer compounds.23
Acknowledgments
The authors wish to thank the ARC foundation (Association
pour la Recherche sur le Cancer) for a Ph.D. Grant to C.R., Dr. Kris-
tina Articus for the lichen determination, Sourisak Sinbandhit and

C. Roullier et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4582–4586
4585
120.6, 120.6, 126.9, 127.2, 127.2, 127.3, 127.5, 127.8, 127.9, 135.4, 136.4, 136.7,
138.1, 145.0, 170.3, 171.4; HRESIMS m/z 590.2270 [M+Na]+ (calcd for C33
H33N3O6Na, 590.2267).
Philippe Jehan (CRMPO) for valuable comments in structure deter-
mination and Isabelle Rouaud for contributing to biological assays.
N2-(Benzyloxycarbonyl)-
L
-glutamic acid 1-(benzyl ester) 5-[(3-benzyloxy-phenyl)-
hydrazide] (12): The peptide-bond-forming reaction described above (see 11)
was employed using (3-benzyloxy-phenyl)-hydrazine hydrochloride
(242 mg, 0.97 mmol), Z-Glu-OBn (299 mg, 0.81 mmol), TBTU (390 mg,
Supplementary data
9
7
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.013.
1.22 mmol) and DIPEA (0.420 mL, 2.5 mmol), in anhydrous DMF (5 mL).
Reaction went to completion in 2 h. The reaction mixture was then poured
into 1 M NaHSO4 (30 mL) and extracted with EtOAc (30 mL). The organic layer
was washed consecutively with 1 M NaHSO4 (30 mL), aqueous NaHCO3
(3 ꢀ 20 mL), and aqueous NaCl (20 mL). After drying (Na2SO4), the solvent
was evaporated under reduced pressure. The residue was filtrated on silica,
using CH2Cl2/MeOH (95:5) as eluent, evaporated again, and finally triturated in
Et2O, to provide hydrazide 12 as a white precipitate (343 mg, 76%): mp 109–
References and notes
1. Boustie, J.; Lohézic-Le Dévéhat, F. In Botanical Medicine in Clinical Practice;
Preedy, V. R., Ed.; CAB International: Wallingford, 2008; pp 356–365.
2. De La Coba Luque, F.; Aguilera Arjona, J.; Lopez Figueroa, F. Spain patent
2007026036, 2007.
3. De la Coba Luque, F.; Aguilera Arjona, J.; Figueroa, F. L.; de Galvez, M. V.;
Herrera, E. J. Appl. Phycol. 2009, 21, 161.
4. Roullier, C.; Chollet-Krugler, M.; Bernard, A.; Boustie, J. J. Chromatogr., B 2009,
877, 2067.
5. Roullier, C.; Chollet-Krugler, M.; Lohézic-Le Dévéhat, F.; Rouaud, I.; Boustie, J.
Abstract of Papers, the 50th Annual Meeting of the American Society of
Pharmacognosy, Honolulu, HI; American Society of Pharmacognosy: Honolulu,
HI, 2009; Abstract P-187.
11 °C; ½a 2D0
ꢁ4 (c 1, CHCl3); UV (CH2Cl2) kmax (log e) 232 (3.95), 283 (3.43) nm;
ꢂ
IR (KBr) mmax 3312, 3032, 1745, 1691, 1645, 1600, 1538, 1496, 1452, 1341,
1269, 1186, 1153, 1056, 1003, 826, 736, 695 cmꢁ1 1H NMR (DMSO-d6,
;
500 MHz, 60 °C) d 1.86–2.31 (4H, m), 4.17 (1H, m), 5.02 (2H, s), 5.04 (2H, s),
5.14 (2H, s), 6.31–6.36 (3H, m), 7.00 (1H, m), 7.31–7.42 (15H, m), 7.50 (1H, s),
9.48 (1H, s); 13C NMR (DMSO-d6, 500 MHz, 60 °C) d 26.1, 29.1, 53.2, 65.1, 65.5,
68.7, 98.6, 104.4, 104.8, 126.9, 127.1, 127.1, 127.2, 127.4, 127.8, 127.8, 127.8,
128.0, 128.9, 135.4, 136.4, 136.9, 150.3, 155.6, 158.9, 170.5, 171.4; HRESIMS m/
z 590.2269 [M+Na]+ (calcd for C33 H33N3O6Na, 590.2267).
N2-(Benzyloxycarbonyl)-
L-glutamic acid 1-(benzyl ester) 5-[(4-benzyloxy-phenyl)-
hydrazide] (13): The peptide-bond-forming reaction described above (see 11)
was employed using (4-benzyloxy-phenyl)-hydrazine hydrochloride 10
6. Tanimoto, T.; Tanaka, I.; Nakajima, M. Japan patent JP 2001131133, 2001.
7. Lichen material was collected on the west coast of France, on the rocky shore of
Dinard (48°38.090 N, 02°08.150 W), in December 2007. It was sorted out,
washed and dried under ambient atmosphere. After macroscopic and
(318 mg, 1.29 mmol), Z-Glu-OBn
7 (407 mg, 1.09 mmol), TBTU (528 mg,
1.64 mmol) and DIPEA (0.570 mL, 3.4 mmol), in anhydrous DMF (8 mL).
Reaction went to completion in 2 h and was treated as described above (see
11), to provide hydrazide 13 as a white precipitate (436 mg, 71%): mp 163–
microscopic observations, comparing to
a reference sample of the ‘Des
Abbayes’ herbarium, it was identified as Lichina pygmaea (Lightf.) C. Agardh.
and a voucher specimen is kept in the laboratory with the reference JB/07/98.
8. Spectral data for the isolated compound pygmeine (3). 1H NMR (D2O, 270 MHz) d
2.18 (2H, m), 2.50 (2H, m), 3.77 (1H, t, J = 6.1 Hz), 6.85 (4H, s); 13C NMR (D2O,
62.5 MHz) d 26.7, 30.1, 54.7, 114.4, 116.0, 121.6, 122.3, 136.1, 144.4, 174.4,
175.2; 1H NMR (DMSO-d6, 500 MHz) d 1.91 (2H, m), 2.31 (2H, m), 3.31 (1H, t,
J = 6.3 Hz), 6.58 (1H, m), 6.64 (2H, m), 6.71 (1H, s), 6.73 (1H, ps d, J = 7.7 Hz);
13C NMR (DMSO-d6, 125 MHz) d 26.9, 29.8, 53.4, 112.1, 114.2, 119.1, 119.1,
137.2, 144.3, 170.1, 171.1; HRESIMS m/z 254.1144 [M+H]+ (calcd for
165 °C; ½a 2D0
ꢁ5 (c 1, CHCl3); UV (CH2Cl2) kmax (log e) 238 (4.12), 298 (3.35) nm;
ꢂ
IR (KBr) mmax 3295, 3033, 1728, 1688, 1629, 1543, 1507, 1453, 1380, 1276,
1236, 1171, 1063, 824, 747, 695 cmꢁ1 1H NMR (DMSO-d6, 500 MHz, 60 °C) d
;
1.90 (1H, m), 2.08 (1H, m), 2.31 (2H, m), 4.17 (1H, m), 5.06 (2H, s), 5.15 (4H, s),
6.71–6.81 (4H, m), 6.95 (1H, d, J = 7.3 Hz), 7.32–7.38 (13H, m), 7.50 (2H, d,
J = 6.6 Hz), 7.66 (1H, s), 9.67 (1H, s); 13C NMR (DMSO-d6, 500 MHz, 60 °C) d
26.1, 29.1, 53.2, 65.1, 65.5, 69.5, 113.2, 115.0, 126.9, 127.0, 127.1, 127.2, 127.2,
127.5, 127.8, 127.9, 127.9, 128.0, 135.5, 136.4, 137.2, 143.0, 151.3, 170.5,
172.4; HRESIMS m/z 590.2262 [M+Na]+ (calcd for C33 H33N3O6Na, 590.2267).
C11H16N3O4, 254.1141).
18.
L-Glutamic acid 5-[(2-hydroxyphenyl)-hydrazide] (4): A catalytic quantity of 10%
9. Hilbig, S.; Andries, T.; Steglich, W.; Anke, T. Angew. Chem. 1985, 97, 1063.
10. Hollitzer, O.; Seewald, A.; Steglich, W. Angew. Chem. 1976, 88, 480.
11. Kelly, R. B.; Daniels, E. G.; Hinman, J. W. J. Chem. Soc. 1962, 27, 3229.
12. Hinman, J. W.; Kelly, R. B. U.S. patent 3,288,848, 1966.
13. Wallcave, L.; Nagel, D. L.; Raha, C. R.; Jae, H.-S.; Bronczyk, S.; Kupper, R.; Toth, B.
J. Org. Chem. 1979, 44, 3752.
Pd/C (about 10 mg) were added to a suspension of hydrazide 11 (482 mg,
0.85 mmol) in 40 mL of MeOH. H2 was applied from a balloon on the top of the
flask with stirring for 5 h. Then, the mixture was filtered, the filtrate
evaporated to yield hydrazide 4 as a pale yellow powder (225 mg, 89%): mp
(degradation) 144–146 °C; ½a D20
ꢁ2 (c 1, MeOH); UV (MeOH) kmax (log e) 207
ꢂ
(4.31), 233 (3.82), 282 (3.43) nm; IR (KBr) mmax 3039, 2609, 1659, 1650, 1644,
14. Datta, S.; Hoesch, L. Helv. Chim. Acta 1987, 70, 1261.
15. Witte, J.; Boekelheide, V. J. Org. Chem. 1972, 37, 2849.
1633, 1613, 1557, 1537, 1504, 1455, 1392, 1247, 1201, 748, 666, 542,
469 cmꢁ1 1H NMR (D2O, 270 MHz) d 2.17 (2H, m), 2.50 (2H, m), 3.77 (1H, t,
;
16. (2-Benzyloxy-phenyl)-hydrazine,
hydrochloride
(8):
2-benzyloxy-aniline
J = 6.2 Hz), 6.86 (4H, s); 13C NMR (D2O, 67.5 MHz) d 26.7, 30.1, 54.7, 114.4,
116.0, 121.6, 122.3, 136.1, 144.4, 174.4, 175.2; 1H NMR (DMSO-d6, 500 MHz) d
1.92 (2H, m), 2.36 (2H, m), 3.28 (1H, t, J = 6.3 Hz), 6.58 (1H, m), 6.64 (2H, m),
6.71 (1H, s), 6.73 (1H, ps d, J = 7.1 Hz), 10.02 (2H, br s); 13C NMR (DMSO-d6,
125 MHz) d 27.0, 29.9, 53.5, 112.2, 114.2, 119.1, 119.1, 137.2, 144.3, 169.9,
171.2; HRESIMS m/z 252.0987 [MꢁH]ꢁ (calcd for C11H14N3O4, 252.0984).
(535 mg, 2.6 mmol) was added dropwise to an ice-cooled solution of HCl 6 M
(10 mL). The mixture was stirred at 0 °C, and treated dropwise with sodium
nitrite (291 mg, 4.2 mmol), dissolved in water (5 mL). The mixture was stirred
vigorously during 60 min at 0 °C. Then tin (II) chloride (1.963 g, 8.5 mmol)
dissolved in HCl 6 M (15 mL) was added dropwise and the solution was stirred
at 0 °C for an additional 2 h. The reaction mixture was alkalized at pH >11 with
NaOH (12.5 M), and extracted with diethyl ether (3 ꢀ 100 mL). The organic
layer was dried on Na2SO4 and hydrochloric acid in methanol (2 mL) was
added to the solution to provide hydrazine 8 as a white precipitate (450 mg,
L-Glutamic acid 5-[(3-hydroxyphenyl)-hydrazide] (5): A catalytic quantity of 10%
Pd/C (about 10 mg) were added to a suspension of hydrazide 12 (210 mg,
0.37 mmol) in 15 mL of MeOH. H2 was applied from a balloon on the top of the
flask with stirring for 4 h. Then, the mixture was filtered, the filtrate
evaporated to yield hydrazide 5 as a pale yellow powder (59 mg, 63%): mp
(degradation) 173–174 °C; IR (KBr) mmax 3242, 3053, 2601, 2125, 1681, 1659,
1651, 1643, 1633, 1605, 1567, 1556, 1537, 1519, 1504, 1454, 1415, 1153, 993,
69%): mp 136–138 °C; UV (MeOH) kmax (log
nm; IR (KBr) max 3252, 2863, 2688, 1600, 1538, 1504, 1472, 1449, 1382, 1338,
1256, 1220, 1121, 1020, 916, 876, 737, 696 cmꢁ1 1H NMR (CD3OD, 270 MHz) d
e) 208 (4.43), 232 (3.85), 276 (3.52)
m
;
5.20 (2H, s), 6.93–7.09 (4H, m), 7.31–7.49 (5H, m); 1H NMR ((CD3)2CO,
270 MHz) d 5.18 (2H, s), 6.85–6.97 (2H, m), 7.08 (1H, dd, J = 7.8, 1.6 Hz), 7.20
(1H, dd, J = 7.6, 1.9 Hz), 7.33–7.43 (3H, m), 7.55 (2H, d, J = 6.5 Hz); 13C NMR
(CD3OD, 67.5 MHz) d 71.7, 114.0, 116.9, 122.4, 125.2, 128.7, 129.2, 129.6,
135.1, 138.1, 149.5; HRESIMS m/z 237.1003 [M+Na]+ (calcd for C13H14N2ONa,
237.1004).
766, 689, 542, 483, 457 cmꢁ1 1H NMR (D2O, 270 MHz) d 2.17 (2H, m), 2.50 (2H,
;
m), 3.78 (1H, t, J = 6.2 Hz), 6.36 (1H, d, J = 2.2 Hz), 6.44 (2H, m), 7.15 (1H, t,
J = 8.1 Hz); 13C NMR (D2O, 67.5 MHz) d 27.6, 30.9, 55.6, 101.7, 107.1, 109.5,
132.2, 150.5, 158.1, 175.2, 176.1; HRESIMS m/z 252.0986 [MꢁH]ꢁ (calcd for
C11H14N3O4, 252.0984).
17. N2-(Benzyloxycarbonyl)-
L-glutamic acid 1-(benzyl ester) 5-[(2-benzyloxy-phenyl)-
L-Glutamic acid 5-[(4-hydroxyphenyl)-hydrazide] (6): A catalytic quantity of 10%
Pd/C (about 10 mg) were added to a suspension of hydrazide 13 (384 mg,
0.68 mmol) in 40 mL of MeOH. H2 was applied from a balloon on the top of the
flask with stirring for 4 h. Then, the mixture was filtered, the filtrate
evaporated to yield hydrazide 6 as a pale yellow powder (160 mg, 93%): mp
(degradation) 190–192 °C; IR (KBr) mmax 3266, 3024, 2601, 2101, 1651, 1583,
hydrazide] (11): Z-Glu-OBn 7 (407 mg, 1.1 mmol), hydrazine hydrochloride 8
(318 mg, 1.3 mmol, 1.2 equiv) and TBTU (528 mg, 1.6 mmol, 1.5 equiv) were
dissolved in DMF (8 mL), and the solution was cooled at ꢁ15 °C. DIPEA
(0.570 mL, 3.3 mmol, 3 equiv) was added and the mixture stirred for 2 h at
room temperature. The reaction mixture was then poured into 1 M NaHSO4
(60 mL) and extracted with EtOAc (150 mL). The organic layer was washed
consecutively with 1 M NaHSO4 (60 mL), aqueous NaHCO3 (3 ꢀ 40 mL), and
aqueous NaCl (40 mL). After drying (Na2SO4), the solvent was evaporated
under reduced pressure. The residue was filtrated on silica, using CH2Cl2/MeOH
(95:5) as eluent, evaporated again, and finally triturated in Et2O, to provide
1557, 1513, 1505, 1408, 1233, 978, 824, 668, 514 cmꢁ1 1H NMR (D2O,
;
270 MHz) d 2.17 (2H, m), 2.49 (2H, m), 3.77 (1H, t, J = 6.2 Hz), 6.81 (4H, s); 1H
NMR (DMSO-d6, 270 MHz) d 1.90 (2H, m), 2.30 (2H, m), 3.23 (1H, t, J = 6.2 Hz),
6.51–6.63 (4H, ps s), 7.12 (1H, s), 9.81 (1H, s); 13C NMR (DMSO-d6, 67.5 MHz) d
27.1, 30.0, 53.6, 113.9, 115.3, 141.7, 150.4, 169.8, 171.6; HRESIMS m/z
252.0991 [MꢁH]ꢁ (calcd for C11H14N3O4, 252.0984).
hydrazide 11 as a white precipitate (436 mg, 71%): mp 151–153 °C; ½a D20
ꢁ1 (c
ꢂ
19. HPLC experiments were performed on a chiral column (ChiralpakÒ AD-H,
Chiral technologies Europe, Illkirch, France). Each sample was diluted in
1, CHCl3); UV (CH2Cl2) kmax (log e) 237 (3.81), 284 (3.33) nm; IR (KBr) mmax
3293, 3032, 1731, 1688, 1651, 1600, 1538, 1503, 1447, 1378, 1345, 1305, 1262,
1238, 1208, 1130, 1064, 1018, 977, 907, 862, 737, 697, 477 cmꢁ1 1H NMR
;
ethanol at a concentration of 0.1 mg/mL, and after passing through a 0.45-
membrane filter, 20 were injected into the column, using heptane/
isopropanol (60:40) as an eluent, with flow rate of 0.7 mL/min. Peak
lm
lL
(DMSO-d6, 500 MHz, 60 °C) d 1.87 (1H, m), 2.06 (1H, m), 2.27 (2H, m), 4.15 (1H,
m), 4.98 (2H, s), 5.05 (2H, s), 5.14 (2H, s), 6.65 (2H, d, J = 8.8 Hz), 6.80 (2H, d,
J = 8.8 Hz), 7.29–7.41 (15H, m), 7.65 (1H, d, J = 4.5 Hz), 9.47 (1H, s); 13C NMR
(DMSO-d6, 500 MHz, 60 °C) d 26.0, 29.2, 53.2, 65.1, 65.5, 69.3, 111.5, 111.8,
a
detection was carried out online using a UV detector at 254 nm. The retention
time of 3 and 4 was 6.13 min.

4586
C. Roullier et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4582–4586
20. Rueppel, M. L.; Lord Lundt, S.; Gass, J. D.; Meister, A. Biochemistry 1972, 11,
2839.
RPMI) followed by extraction with a mixture (150 lL) ethanol/acetic acid/
water (50:1:49). The absorbance was measured at 540 nm using a microplate
spectrophotometer system. Results are presented as percentage of control
values and IC50 values were determined graphically from concentration-effect
curves of at least three experiments.
21. Cytotoxic activity of compounds was determined for B16 (murine melanoma
cells), and A375 (human melanoma cells), by the neutral red uptake (NRU)
assay based on the initial protocol described by Borefreund and Puerner
(1985).24 Briefly, 96-well tissue culture plates were seeded with B16 and A375
at, respectively, 10 ꢀ 103 and 12 ꢀ 103 cells/well. The plates were then
incubated at 37 °C in a humidified 5% CO2 incubator for 24 h. Cells were then
exposed to dilutions of the test compounds in RPMI medium for 24 and 48 h.
Etoposide was used as a positive control and cell viability was assessed using
22. Boekelheide, K.; Graham, D. G.; Mize, P. D.; Koo, E. H. J. Invest. Dermatol. 1980,
75, 322.
23. Gohil, V. M.; Agrawal, S. K.; Saxena, A. K.; Garg, D.; Gopimohan, C.; Bhutani, K.
K. Indian J. Exp. Biol. 2010, 48, 265.
24. Borenfreund, E.; Puerner, J. A. Toxicol. Lett. 1985, 24, 119.
the NRU assay. It consisted of a 3 h-incubation with neutral red (50 l
g mLꢁ1 in