Synthesis and antiproliferative activity of aryl- and heteroaryl-hydrazones derived from xanthone carbaldehydes

Article abstract of DOI:10.1016/j.ejmech.2007.09.003

In order to explore the antiproliferative effect associated with the xanthone framework, several arylhydrazonomethyl derivatives were synthesized from various isomeric 1,3-dihydroxyxanthone carbaldehydes. Variation in the position of the aldehydic functio

Full text of DOI:10.1016/j.ejmech.2007.09.003

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1336e1343
http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and antiproliferative activity of aryl- and
heteroaryl-hydrazones derived from xanthone carbaldehydes
a
a
Martine Varache-Lembege , Stephane Moreau , Stephane Larrouture
a,!
`
Daniele Montaudon , Jacques Robert , Alain Nuhrich
´
´
,
b
b
a,
*
`
a Laboratoire de Chimie The´rapeutique, EA2962, Faculte´ de Pharmacie, Universite´ Victor Segalen Bordeaux 2, 146 Rue Le´o-Saignat,
33076 Bordeaux Cedex, France
b Laboratoire de Pharmacologie des Me´dicaments Anticance´reux, Universite´ Victor Segalen Bordeaux 2, Institut Bergonie´,
229 cours de l’Argonne, 33076 Bordeaux Cedex, France
Received 29 January 2007; received in revised form 27 July 2007; accepted 6 September 2007
Available online 15 September 2007
Abstract
In order to explore the antiproliferative effect associated with the xanthone framework, several arylhydrazonomethyl derivatives were syn-
thesized from various isomeric 1,3-dihydroxyxanthone carbaldehydes. Variation in the position of the aldehydic function led to three sets of
compounds, bearing the hydrazonomethyl chain at positions 5, 6 or 7 on the xanthone nucleus, respectively.
The antiproliferative effect of the compounds was evaluated in vitro using the MTT colorimetric method against two human cancer cell lines
(MCF-7, breast adenocarcinoma, and KB 3.1, squamous cell oral carcinoma) for two time periods (24 h and 72 h).
Among the series, four compounds exhibited interesting growth inhibitory effects against both the cell lines, with IC50 values in the micro-
molar concentration range. When compared with doxorubicin, the xanthone derivatives showed moderate cytotoxic effects. Surprisingly, unlike
doxorubicin, these compounds displayed no significant time-dependent change in the concentration causing 50% inhibitory effect in prolifera-
tion.
This unusual cytotoxicity profile led to the hypothesis that these molecules could be endowed with a mechanism of action distinct to that of
doxorubicin.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Xanthones; Hydrazones; Antiproliferative activity
1. Introduction
Auckland Cancer Society Research Centre in New Zealand
[10] emerged as an interesting compound exhibiting both ex-
cellent experimental antitumor activity and antivascular ef-
fects. Several biochemical pathways explaining the activity
of DMXAA have been established. This drug induces apopto-
sis of tumoral vascular endothelial cells via an up-regulation of
the nuclear transcription factor, NFkB, leading to the produc-
tion of TNFa and other cytokines [11,12].
Due to this particular therapeutic profile, DMXAA is con-
sidered as a very promising molecule among the novel classes
of anti-cancer drugs named vascular disrupting agents (VDAs)
[13]. Antitumor activity of DMXAA appears to be due to in-
direct effects more than direct action on DNA. In combination
with standard chemotherapy this substance is currently
During the past several years, the xanthone template has
generated a growing interest in the search for new antitumor
agents [1e6]. Thus, noticeable antiproliferative effects were
discovered in various naturally occurring xanthones, as exem-
plified by psorospermin and mangostin derivatives [7e9]. On
the other hand, within synthetic molecules, 2-(5,6-dimethyl-9-
oxo-9H-xanthen-4-yl)acetic acid (DMXAA) developed at the
* Corresponding author. Tel.: þ33 05 57 57 46 98; fax: þ33 05 57 57 47 05.
E-mail address: alain.nuhrich@chimthera.u-bordeaux2.fr (A. Nuhrich).
!
Deceased on 7 July 2006.
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.003

M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
1337
O-acetyl derivatives 4, when treated with N-bromosuccini-
mide/benzoyl peroxide in CCl4 afforded in all cases the desired
bromomethyl compounds 5 as major products.
The xanthone carbaldehydes 6 were obtained from the
bromomethyl derivatives by smooth oxidation with bis-
tetrabutylammonium dichromate (prepared by reacting tetra-
butylammoniumhydrogensulfate with potassium dichromate)
[21].
Condensation of 6 with appropriate aryl- or heteroaryl-
hydrazine salts gave the corresponding hydrazones 7aei. De-
protection by alkaline hydrolysis in mild conditions then
afforded the final 1,3-diphenolic compounds (8aei).
Fig. 1. General structure of the investigated xanthones.
investigated in the clinic (phase III trials) under the code
AS1404 and seems of particular interest for the treatment of
solid tumors [14].
3. Results and discussion
These findings allow us to think that the xanthone chromo-
phore is an attractive lead for the design of novel antitumor
drugs. On the other hand, hydrazino system has revealed good
inhibitory effects on cancer cells’ proliferation [15e17]. With
the aim of further exploring the therapeutic potential of this tri-
cyclic framework, we describe in this paper the synthesis and
a preliminary investigation of the cytotoxic profile of a series
of new compounds whose general structure is outlined in Fig. 1.
In the first experiment, the selected arylhydrazones 7 and 8
were tested for their effect on cellular viability against two hu-
man tumor cell lines (MCF-7, breast carcinoma, and KB 3.1,
oral squamous cell carcinoma). Assays were performed in vi-
tro on exponentially growing cells. The activity was evaluated
by measuring the levels of surviving cells after incubation for
24 h with the test samples, using the MTT colorimetric assay
[22], based on the ability of metabolically active cells to con-
vert the pale yellow MTT to a blue formazan product, which is
quantifiable spectrophotometrically.
2. Chemistry
The results of this primary screening are reported in Table 1.
Most of the hydrazone derivatives exhibited a rather
moderate cytotoxicity or were inactive.
However, within the series studied, four xanthones revealed
a significant activity (7g, 8e, 7h, and 8f). In terms of cell line
sensitivity, comparable responses were observed against both
the MCF-7 and the KB 3.1 cells.
For these active compounds, results appeared to be indicative
of a doseeresponse relationship, with a clear inhibition of the
percentage of living cells after exposure to 10 mM of substance.
The synthetic approach (Fig. 2) for the construction of the
xanthone nucleus is based on the method of Grover et al. [18]
and involves the condensation between phloroglucinol 1 and
3-, 4- or 5-methylsalicylic acid 2. The use of Eaton’s reagent
(phosphorus pentoxide and methanesulfonic acid: P2O5/
CH3SO3H) [19] as the coupling agent afforded the xanthones
3 in high yield (90%). The strategy used to protect the phenolic
groups is of primordial importance for governing the regiospe-
cificity of the following bromination step [20]. Thus, the
Fig. 2. Synthetic pathway for arylhydrazonomethylxanthones. Reagents: (i) P2O5/CH3SO3H; (ii) Ac2O/pyridine; (iii) NBS/DBP/CCl4; (iv) (Bu4N)2Cr2O7/CHCl3;
(v) Ar(Het))eNHeNH2/EtOH; (vi) NaHCO3/H2O.

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M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
Table 1
Effects of studied xanthones on the viability of MCF-7 and KB 3.1 cells
Comp.
R1
R2
R3
R4
MCF-7
KB 3.1
10 mM
1 mM
10 mM
1 mM
7a
8a
7b
8b
7c
8c
7d
8d
7e
8e
7f
OAc
OH
X
X
H
H
H
H
Y
Y
H
H
H
H
Z
Z
H
H
H
H
H
H
X
X
H
H
H
H
Y
Y
H
H
H
H
Z
Z
H
H
H
H
H
H
X
X
H
H
H
H
Y
Y
H
H
H
H
Z
Z
100
72
100
100
100
100
100
100
85
100
82
100
61
82
96
60
90
87
2
100
100
100
100
100
100
98
OAc
OH
100
100
100
100
70
OAc
OH
OAc
OH
41
58
95
OAc
OH
100
3
100
100
80
100
77
OAc
OH
79
12
90
3
95
96
8f
86
64
7g
8g
7h
8h
7i
OAc
OH
14
64
14
92
13
80
66
75
100
99
90
35
OAc
OH
13
70
85
93
73
80
OAc
OH
48
82
95
90
8i
97
Viability was estimated as the percentage of living cells after incubation of the cells for 24 h with two concentrations (1 mM and 10 mM) of the molecules tested
(100 indicates no activity; 0 indicates complete cell death) with final DMSO concentration of 0.1%.
From the data obtained, it is obvious that the nature of the ter-
minal ring on the hydrazono chain exerts a striking effect on the
antiproliferative activity. Within the few variations considered
in this study, the presence of a nitrogen heterocycle afforded
a clear beneficial effect with regard to antiproliferative proper-
ties. With the exception of 7g, the dihydroimidazolyl derivatives
were slightly less active than their pyridinyl counterpart (com-
pare 8e to 8h, and 8f to 8i). Introduction of a non-nitrogen
ring resulted in compounds completely devoid of activity (in
the case of phenyl derivatives 7aec and 8aec).
Intriguingly, position of the hydrazonomethyl functionality
on the third ring of the xanthone nucleus did not allow to de-
lineate clear structureeactivity relationships. In this context, it
is worth noting that similar activities are observed for 7g
(5-substituted), 8e (6-substituted) and 8f (7-substituted).
The role of the oxygenated groups in the 1- and 3-position on
thexanthone system seems rather difficult to clarify since signif-
icant activities were shown both among acetylated derivatives
and in free phenolic compounds. A plausible explanation is
that tumoral drug-metabolizing enzymes (DMEs), which are ca-
pable of biotransforming a variety of xenobiotics [23], could be
involved in the hydrolysis of diacetylated compounds, thus
releasing dihydroxyxanthone in the cellular medium. The
extended incubation periods used in our assays are consistent
with this hypothesis. Without further investigation, it was not
possible to decide if the biological response was due to the ester
itself or attributable to its hydrolyzed product.
Since the inhibitory effects of xanthones on the cell viability
were established, we then examined the impact of exposure time
on antiproliferative activity. In a further experiment, we first ex-
posed KB 3.1 and MCF-7 cells to the test compound for 24 h or
72 h and we then explored the growing capacity of the treated
cells when reincubated on fresh culture medium for a further
96-h period. Doxorubicin was used as reference substance.
Results of these assays are presented in Table 2.
In our experiments, data for antiproliferative effect of doxo-
rubicin on MCF-7 cells showed a striking evolution of the
drug concentration required to decrease cell survival by 50%
after 24-h or 72-h treatment. This proceeds from the corre-
sponding IC50 ratio values, R(24/72) ¼ 25. This observation is
in accordance with the time-dependent cytotoxic response usu-
ally reported for doxorubicin [24,25].
In contrast, evaluation of the cytotoxicity induced by the
tested hydrazonoxanthones yielded an apparently different pat-
tern. The doses causing 50% inhibitory effect did not change sig-
nificantly after 24 h and 72 h, as indicated by the R(24/72) value

M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
1339
Table 2
Effect of time of incubation on antiproliferative activity against KB 3.1 and MCF-7 cells (IC50, mM)
KB 3.1
24 h
MCF-7
24 h
72 h
R(24/72)
72 h
R(24/72)
7g
7h
11.3 ꢀ 1.1
1.6 ꢀ 0.3
4.7 ꢀ 0.9
2.4
0.9
9.3 ꢀ 1.3
7.0 ꢀ 1.0
1.3
0.8
1.8 ꢀ 0.4
6.5 ꢀ 0.3
1.9 ꢀ 0.4
5.1 ꢀ 0.6
2.4 ꢀ 0.2
5.6 ꢀ 0.5
7i
Doxorubicin
6.8 ꢀ 0.6
1.05
25.0
0.9
25.7
0.010 ꢀ 0.003
0.0004 ꢀ 0.0001
0.018 ꢀ 0.004
0.0007 ꢀ 0.0001
Cell viability after a further 96-h period in fresh culture medium was estimated as the compound concentration required for 50% growth inhibition. For each drug,
a R(24/72) value was calculated and represents the ratio of the IC50s (mM) obtained after 24-h and 72-h treatment, respectively. Data represent mean values (ꢀS.D.)
for three independent experiments.
which is always near to 1.This would indicate that the cytotoxi-
city of these compounds is not cell cycle phase-dependent.
salicylic acid 2 (9.13 g, 60 mmol) was added slowly 100 ml of
Eaton’s reagent (P2O5/CH3SO3H, Aldrich). The mixture was
warmed up to 80 ꢁC for 20 min, under stirring. After cooling to
room temperature, the reaction mixture was poured onto ice
(300 g) and stirred for 2 h. The resulting solid was collected by fil-
tration, washed with water until pH 6, and dried at 60 ꢁC to give
a reddish brown solid used in the following step without further
purification.
4. Conclusion
According to these preliminary results, it appears that the
introduction of a heteroarylhydrazonomethyl moiety on the
xanthone system is potentially of interest to obtain antiproli-
ferative compounds.
This work allowed us to identify a few active molecules able
to inhibit in vitro the growth of two human cancer lines. Com-
pared to doxorubicin, these compounds displayed both a rela-
tively weak cytotoxicity and a phase-independent cytotoxic
mechanism. Our results suggest that the antiproliferative prop-
erties induced by the xanthone framework might involve distinct
mechanisms to that of classical topoisomerase inhibitors.
All these findings support the need for further investiga-
tions to clarify the features underlying the antitumor potential
of these new xanthone derivatives.
5.1.1.1. 1,3-Dihydroxy-5-methyl-9H-xanthen-9-one (3a). Mp >
260 ꢁC; yield: 96% [20].
5.1.1.2. 1,3-Dihydroxy-6-methyl-9H-xanthen-9-one (3b). Mp >
260 ꢁC; yield: 95% [20].
5.1.1.3. 1,3-Dihydroxy-7-methyl-9H-xanthen-9-one (3c). Mp >
260 ꢁC; yield: 91% [20].
5.1.2. General procedure for the
5. Experimental
methyl-9-oxo-9H-xanthene-1,3-diyl diacetates (4)
Asuspensionofdihydroxylatedderivative3(14 g, 57.8 mmol)
in a mixture of acetic anhydride (130 ml) and pyridine (10 ml)
was stirred at reflux for 3 h. After cooling to room temperature,
the reaction mixture was poured into ice-water (400 g) and then
acidified with hydrochloric acid. The resulting precipitate was
separated and washed with water until pH 7. The expected acety-
lated compound 4 was recrystallized from ethanol.
5.1. Materials and methods
Melting points (mp) were determined using an electrothermal
capillary melting point apparatus (Digital Mel-Temp 3.0, Model
1402) and are uncorrected. IR spectra were recorded as potassium
bromide discs on a Shimadzu IR 470 spectrometer. NMR spectra
were recorded, in DMSO-d6 solution, on a Bruker AMX 500 in-
strument (1H: 500 MHz, 13C: 125 MHz). Chemical shifts are re-
ported as d (ppm) relative to TMS as internal standard.
Multiplicities in 13C NMR spectra were derived from JMOD ex-
periments. The 2D experiments (Homonuclear Correlation
{1He1H COSY} and Heteronuclear Correlations {HMQC and
HMBC}) were used to assign the signals. Thin-layer chromatog-
raphy (TLC) was performed using aluminium precoated plates
(silica gel SDS 60F 254 Whatman, 0.2 mm thickness). Column
chromatography was carried out on silica gel 60e200 mm
(Merck). Elemental analyses performed on the terminal products
were within ꢀ0.4% of the calculated values (for element indicated
in brackets).
5.1.2.1. 5-Methyl-9-oxo-9H-xanthene-1,3-diyl diacetate (4a). Mp:
173 ꢁC; yield: 62% [20].
5.1.2.2.
6-Methyl-9-oxo-9H-xanthene-1,3-diyl
diacetate
(4b). Mp: 156 ꢁC; yield: 56%; IR (cmꢂ1) 1770 (CO ester),
1650 (CO ketone); 1H NMR (d) 2.33 and 2.38 {2(s, 3H,
COeCH3)}, 2.48 (s, 3H, CH3), 7.05 (d, 1H, H2,
4J ¼ 2.2 Hz), 7.29 (dd, 1H, H7, 3J ¼ 8.1 Hz and
4J ¼ 1.3 Hz), 7.44 (d, 1H, H4, 4J ¼ 2.2 Hz), 7.45 (s, 1H,
H5), 7.99 (d, 1H, H8, 3J ¼ 8.1 Hz); 13C NMR (d) 20.8
2 ꢃ CH3 (Ac), 21.2 CH3, 109.1 C4, 112.3 C1a, 113.1 C2,
117.3 C5, 119.11 C8a, 125.5 C7, 126.0 C8, 146.8 C6, 150.4
C1, 154.7 C3, 154.8 C5a, 156.9 C4a, 168.1 and 168.6 C]O
(Ac), 173.0 C]O (xanthone). Anal. C18H14O6 (C, H).
5.1.1. General procedure for the preparation
of hydroxylated xanthones (3)
To a mixture containing phloroglucinol dihydrate 1 (9.73 g,
60 mmol, dried at 120 ꢁC overnight) and appropriate methylated
5.1.2.3. 7-Methyl-9-oxo-9H-xanthene-1,3-diyldiacetate(4c). Mp:
163 ꢁC; yield: 42%; IR (cmꢂ1) 1770 (CO ester), 1660

1340
M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
1
(CO ketone); H NMR (d) 2.34 and 2.38 {2(s, 3H, COeCH3)},
2.42 (s, 3H, CH3, 7.05 (d, 1H, H2, 4J ¼ 2.3 Hz), 7.44 (d, 1H, H4,
as eluent. The desired product 6, obtained as a yellowish white
solid, was further recrystallized from ethanol.
4J ¼ 2.3 Hz), 7.53 (dd, 1H, H6, J ¼ 8.5 Hz and J ¼ 2.7 Hz),
3
4
7.67 (d, 1H, H5, J ¼ 8.5 Hz), 7.90 (s, 1H, H8); 13C NMR (d)
5.1.4.1.
5-Formyl-9-oxo-9H-xanthene-1,3-diyl
diacetate
diacetate
3
20.1 CH3, 20.7 CH3 (Ac), 109.0 C2, 112.1 C1a, 112.9 C4,
117.4 C5, 120.9 C8a, 125.0 C8, 134.1 C7, 136.4 C6, 150.3 C1,
152.9 C5a, 154.7 C3, 156.9 C4a, 168.0 and 168.6 C]O (Ac),
173.9 C]O (xanthone). Anal. C18H14O6 (C, H).
(6a). Mp: 172 ꢁC (ethanol); yield: 29% [20].
5.1.4.2.
6-Formyl-9-oxo-9H-xanthene-1,3-diyl
(6b). Mp: 196 ꢁC (ethanol); yield: 43%; IR (cmꢂ1) 1780 (CO es-
ter), 1700 (CO aldehyde), 1660 (CO ketone); 1H NMR (d) 2.36
4
and 2.40 {2(s, 3H, COeCH3)}, 7.11 (d, 1H, H7, J ¼ 2.2 Hz),
4
3
5.1.3. General procedure for the bromomethyl-9-oxo-9H-
xanthene-1,3-diyl diacetates (5)
7.52 (d, 1H, H5, J ¼ 2.2 Hz), 7.90 (dd, 1H, H2, J ¼ 8.1 Hz
4
4
and J ¼ 1.35 Hz), 8.14 (d, 1H, H4, J ¼ 1.3 Hz), 8.27 (d, 1H,
3
A mixture of diacetate derivative 4 (5.55 g, 17 mmol), N-bro-
mosuccinimide (3.03 g, 17 mmol) and dibenzoylperoxide
(412 mg, 1.7 mmol) in carbon tetrachloride (110 ml) was re-
fluxed for 3e5 h under light (2 ꢃ 60 W). After cooling at 0 ꢁC
and stirring for 2 h, the precipitate was filtered, washed with wa-
ter (3 ꢃ 15 ml), acetone (2 ꢃ 15 ml) and diethyl ether (20 ml) to
give a solid which was recrystallized from butan-2-one.
H1, J ¼ 8.3 Hz), 10.17 (s, 1H, CHO); 13C NMR (d) 20.6 and
20.7 CH3 (Ac), 109.2 C5, 112.4 C8a, 113.5 C7, 119.8 C4,
123.2 C2, 124.6 C1a, 126.9 C1, 140.6 C3, 151.4 C8, 154.8
C4a, 155.1 C6, 157.1 C5a, 167.9 and 168.5 C]O (Ac), 173.7
C]O (xanthone), 192.0 CHO. Anal. C18H12O7 (C, H).
5.1.4.3.
7-Formyl-9-oxo-9H-xanthene-1,3-diyl
diacetate
(6c). Mp: 175 ꢁC (ethanol); yield: 51%; IR (cmꢂ1) 1780 (CO
1
ester), 1690 (CO aldehyde), 1670 (CO ketone); H NMR (d)
5.1.3.1. 5-Bromomethyl-9-oxo-9H-xanthene-1,3-diyl diacetate
(5a). Mp: 198 ꢁC (butan-2-one); yield: 50% [20].
2.34 and 2.40 {2(s, 3H, COeCH3)}, 7.13 (d, 1H, H7,
4
4J ¼ 2.2 Hz), 7.51 (d, 1H, H5, J ¼ 2.2 Hz), 7.77 (d, 1H, H4,
3
4
5.1.3.2. 6-Bromomethyl-9-oxo-9H-xanthene-1,3-diyl diacetate
3J ¼ 8.6 Hz), 8.27 (dd, 1H, H3, J ¼ 8.7 Hz and J ¼ 2.0 Hz),
4
(5b). Mp: 198 ꢁC (butan-2-one); yield: 40%; IR (cmꢂ1) 1780
8.66 (d, 1H, H1, J ¼ 1.9 Hz), 10.11 (s, 1H, CHO); 13C NMR
1
(CO ester), 1660 (CO ketone); H NMR (d) 2.34 and 2.38
(d) 20.7 CH3 (Ac), 109.3 C7, 112.3 C8a, 113.8 C5, 119.0 C4,
121.4 C1a, 129.6 C1, 132.4 C2, 133.7 C3, 150.3 C8, 155.2
C6, 156.8 C5a, 158.0 C4a, 167.9 and 168.5 C]O (Ac), 173.6
C]O (xanthone), 191.2 CHO. Anal. C18H12O7 (C, H).
{2(s, 3H, COeCH3)}, 4.84 (s, 2H, CH2Br), 7.07 (d, 1H, H2,
4J ¼ 2.1 Hz), 7.47 (d, 1H, H4, 4J ¼ 2.2 Hz), 7.52 (dd, 1H, H7,
3J ¼ 7.6 Hz and J ¼ 2.2 Hz), 7.72 (d, 1H, H5, J ¼ 1.3 Hz),
4
4
8.09 (d, 1H, H8, J ¼ 8.2 Hz); 13C NMR (d) 20.7 and 20.7
3
CH3 (Ac), 32.2 CH2Br, 109.1 C4, 112.3 C1a, 113.3 C2, 117.9
C5, 120.8 C8a, 125.6 C7, 126.3 C8, 145.8 C6, 150.3 C1,
154.5 C3, 154.9 C5a, 156.9 C4a, 168.0 and 168.7 C]O (Ac),
173.6 C]O (xanthone). Anal. C18H13BrO6 (C, H).
5.1.5. General procedure for the 9-oxo-((2-
arylhydrazono)methyl)-9H-xanthene-1,3-diyl diacetates (7)
A mixture of the appropriate carboxaldehyde 6 (340 mg,
1 mmol) and the convenient aryl or heteroaryl-hydrazine
(1.2 mol) was stirred at room temperature in 15 ml of 1-prop-
anol for 15 min and then acetic acid (about 0.5 ml) was added
dropwise to the reaction mixture. The suspension was stirred
for an additional period of 15 min and heated under reflux
for 2 h. The mixture was cooled to room temperature and fil-
tered to give hydrazone 7. The different ways of purification
are given for each compound.
5.1.3.3. 7-Bromomethyl-9-oxo-9H-xanthene-1,3-diyl diacetꢂat1e
(5c). Mp: 156 ꢁC (butan-2-one); yield: 73%; IR (cm
)
1
1770 (CO ester), 1660 (CO ketone); H NMR (d) 2.35 and
2.45 {2(s, 3H, COeCH3)}, 4.52 (s, 2H, CH2Br), 6.93
4
4
(d, 1H, H2, J ¼ 2.2 Hz), 7.32 (d, 1H, H4, J ¼ 2.3 Hz), 7.47
3
3
(d, 1H, H6, J ¼ 8.6 Hz), 7.68 (d, 1H, H5, J ¼ 8.6 Hz), 8.26
(s, 1H, H8). Anal. C18H13BrO6 (C, H).
5.1.5.1. 9-Oxo-7-((2-phenylhydrazono)methyl)-9H-xanthene-
1,3-diyl diacetate (7a). Mp: 214 ꢁC (ethanol); yield: 63% [20].
5.1.4. General procedure for the formyl-9-oxo-9H-
xanthene-1,3-diyl diacetates (6)
5.1.5.2. 9-Oxo-6-((2-phenylhydrazono)methyl)-9H-xanthene-
1,3-diyl diacetate (7b). Mp: 210 ꢁC (acetoneewater, 1:2);
yield: 42% [20].
The convenient bromomethyl derivatives 5aec (2.03 g,
5 mmol) were added to the solution of bis-tetrabutylammo-
nium dichromate [21] in 100 ml of chloroform. The reaction
mixture was heated under reflux for 5 h. After cooling at
room temperature, the crude product was filtered through sil-
ica gel (40e63 mm, 20 g) to eliminate inorganic and tetrabuty-
lammonium salts. Silica was washed with ethyl acetate
(2 ꢃ 50 ml). Evaporation of the combined organic solvents
gave a greenish brown oil. The residue was further purified
on a silica gel (63e200 mm, 30 g) column (1.6 cm in diameter,
47 cm in length) with methylene chlorideeethyl acetate (9:1)
5.1.5.3. 9-Oxo-5-((2-phenylhydrazono)methyl)-9H-xanthene-
1,3-diyl diacetate (7c). Mp: 196 ꢁC (ethanoleacetone, 9:1);
yield: 61% [20].
5.1.5.4. 9-Oxo-7-((2-(pyridin-2-yl)hydrazono)methyl)-9H-xan-
thene-1,3-diyl diacetate (7d). Mp: 211 ꢁC; yield: 83%; IR
(cmꢂ1) 3400 (NH), 1770 (CO ester), 1660 (CO ketone),
1620 (CN), 1570 (NeH); 1H NMR (d) 2.36 and 2.40

M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
1341
3
{2(s, 3H, COeCH3)}, 6.82 (t, 1H, H50, J ¼ 5.8 Hz), 7.10 (s,
4J ¼ 2.1 Hz), 7.93 (d, 1H, H2, 3J ¼ 8.3 Hz), 8.12 (s, 1H,
3
3
1H, H7), 7.34 (d, 1H, H60, J ¼ 7.9 Hz), 7.44 (s, 1H, H5),
H4), 8.15 (d, 1H, H1, J ¼ 8.3 Hz), 8.33 (s, 1H, CH]), 8.85
3
3
7.51 (t, 1H, H2, J ¼ 7.6 Hz), 7.68 (t, 1H, H40, J ¼ 7.6 Hz),
8.08 (d, 1H, H1, 3J ¼ 7.9 Hz), 8.16 (d, 1H, H30,
3J ¼ 7.3 Hz), 8.39 (d, 1H, H3, 3J ¼ 7.3 Hz), 8.66 (s, 1H,
CH]), 11.16 (s, 1H, NH). Anal. C23H17N3O6 (C, H, N).
(m, 1H, NH) 12.57 (s, 1H, NH); 13C NMR (d) 20.7 and 20.8
CH3 (Ac), 42.7 CH2eCH2, 109.0 C5, 112.4 C8a, 113.4 C7,
116.4 C4, 122.1 C1a, 123.1 C2, 126.2 C1, 139.8 C3, 147.2
CH], 150.4 Cipso, 154.8 C8, 154.9 C4a, 157.0 C6, 157.8
C5a, 168.0 and 168.6 C]O (Ac), 173.6 C]O (xanthone).
Anal. C22H19N3O6 (C, H, N).
5.1.5.5. 9-Oxo-6-((2-(pyridin-2-yl)hydrazono)methyl)-9H-xan-
thene-1,3-diyl diacetate (7e). Mp: 254 ꢁC; yield: 77%; IR
(cmꢂ1) 3200 (NH), 1780 (CO ester), 1650 (CO ketone),
5.1.5.9.
5-((2-(4,5-Dihydro-1H-imidazol-2-yl)hydrazono)-
1
1620 (CN), 1540 (NeH); H NMR (d) 2.34 and 2.39 {2(s,
methyl)-9-oxo-9H-xanthene-1,3-diyl
diacetate
(7i). Mp:
3H, COeCH3)}, 6.84 (td, 1H, H50, 3J ¼ 7.1 Hz and
4J ¼ 0.9 Hz), 7.07 (d, 1H, H7, 3J ¼ 2.2 Hz), 7.37 (d, 1H,
H60, 3J ¼ 8.4 Hz), 7.45 (d, 1H, H5, 4J ¼ 2.2 Hz), 7.69 (td,
1H, H40, 3J ¼ 6.9 Hz and 4J ¼ 1.8 Hz), 7.79 (s, 1H, H4),
259 ꢁC dec. (acetoneewater, 1:2); yield: 52%; IR (cmꢂ1) 3200
1
(NH), 1770 (CO ester), 1660 (CO ketone), 1620 (CN); H
NMR (d) 2.34 and 2.39 {2(s, 3H, COeCH3)}, 3.76 (s, 4H,
4
CH2eCH2), 7.11 (s, 1H, H7), 7.49 (s, 1H, H5, J ¼ 2.1 Hz),
3
4
7.80 (dd, 1H, H2, J ¼ 9.9 Hz and J ¼ 1.6 Hz), 8.10 (d, 1H,
7.74 (d, 1H, H4, 3J ¼ 8.8 Hz), 8.33 (s, 1H, CH]), 8.37 (d, 1H,
H3, 3J ¼ 9.2 Hz), 8.50 (s, 1H, H1), 8.72 (m, 1H, NH) 11.32 (s,
1H, NH); 13C NMR (d) 20.7 and 20.7 CH3 (Ac), 42.6 CH2e
CH2, 109.2 C5, 112.3 C8a, 113.5 C7, 118.5 C4, 121.4 C1a,
125.6 C1, 129.8 C2, 133.4 C3, 146.6 CH], 150.3 C8, 154.9
C6, 155.8 C4a, 156.8 C5a, 157.7 Cipso, 168.0 and 168.5 C]O
(Ac), 173.7 C]O (xanthone). Anal. C22H19N3O6 (C, H, N).
3
H1, J ¼ 8.2 Hz), 8.15 (s, 1H, CH]), 8.16 (dd, 1H, H30,
3J ¼ 4.9 Hz and J ¼ 0.9 Hz), 11.27 (s, 1H, NH); 13C NMR
4
(d) 20.7 and 20.7 CH3 (Ac), 106.8 C60, 109.0 C5, 112.4
C8a, 113.2 C7, 114.2 C4, 115.7 C50, 120.6 C1a, 121.6 C2,
126.2 C1, 136.2 CH], 137.9 C40, 142.4 C6, 147.5 C30,
150.3 C8, 154.7 C6, 155.1 C4a, 156.4 Cipso, 157.0 C5a,
168.1 and 168.6 C]O (Ac), 173.5 C]O (xanthone). Anal.
C23H17N3O6 (C, H, N).
5.1.6. General procedure for the 1,3-dihydroxy-
((2-arylhydrazono)methyl)-9H-xanthen-9-ones (8)
The diacetate derivative 7 (0.3 mmol) was treated with so-
dium bicarbonate (252 mg, 3 mmol) in 20 ml of 1-propanole
water (1:1). The alkaline hydrolysis was processed by heating
under reflux for 3 h and then cooling to 0 ꢁC overnight. The
phenolic hydrazone derivative 8 was collected and purified
as described below.
5.1.5.6. 9-Oxo-5-((2-(pyridin-2-yl)hydrazono)methyl)-9H-xan-
thene-1,3-diyl diacetate (7f). Mp: 228 ꢁC; yield: 88%; IR
(cmꢂ1) 3300 (NH), 1780 (CO ester), 1660 (CO ketone),
1
1620 (CN), 1540 (NeH); H NMR (d) 2.35 and 2.41 {2(s,
3
3H, COeCH3)}, 6.79 (t, 1H, H50, J ¼ 5.7 Hz), 7.10 (s, 1H,
3
H7), 7.29 (d, 1H, H60, J ¼ 8.2 Hz), 7.50 (s, 1H, H5), 7.67
(t, 1H, H40, 3J ¼ 8.7 Hz), 7.69 (d, 1H, H4, 3J ¼ 8.2 Hz),
8.11e8.15 (m, 2H, H30 and CH]), 8.22 (d, 1H, H3,
3J ¼ 9.3 Hz), 8.25 (s, 1H, H1), 10.97 (s, 1H, NH). Anal.
C23H17N3O6 (C, H, N).
5.1.6.1.
1,3-Dihydroxy-7-((2-phenylhydrazono)methyl)-9H-
xanthen-9-one (8a). Mp: 256 ꢁC dec. (acetoneewater, 1:2);
yield: 58% [20].
5.1.5.7.
7-((2-(4,5-Dihydro-1H-imidazol-2-yl)hydrazono)-
5.1.6.2.
1,3-Dihydroxy-6-((2-phenylhydrazono)methyl)-9H-
methyl)-9-oxo-9H-xanthene-1,3-diyl
diacetate
(7g). Mp:
xanthen-9-one (8b). Mp: 260 ꢁC dec. (ethanolewater, 1:1);
yield: 81% [20].
260 ꢁC (ethanol); yield: 20%; IR (cmꢂ1) 3400 (NH), 1770
1
(CO ester), 1660 (CO ketone), 1620 (CN), 1560 (NeH); H
NMR (d) 2.36 and 2.39 {2(s, 3H, COeCH3)}, 3.78 (s, 4H,
CH2eCH2), 7.09 (s, 1H, H7), 7.41 (s,1H, H5), 7.54 (t, 1H,
5.1.6.3.
1,3-Dihydroxy-5-((2-phenylhydrazono)methyl)-9H-
xanthen-9-one (8c). Mp: 272 ꢁC dec. (acetoneewater, 1:2);
yield: 48% [20].
3
3
H2, J ¼ 7.7 Hz), 8.19 (d, 1H, H1, J ¼ 7.7 Hz), 8.56 (d, 1H,
3
H3, J ¼ 7.2 Hz), 8.86 (m, 2H, CH] and NH), 11.32 (m,
1H, NH); 13C NMR (d) 20.7 and 20.9 CH3 (Ac), 42.7 CH2e
CH2, 108.9 C5, 112.1 C8a, 113.4 C7, 119.3 Cpara, 121.8
C1a, 124.2 C2, 128.0 C1, 122.2 C4, 131.6 C3, 140.8 CH],
150.4 Cipso, 152.7 C8, 154.9 C4a, 156.4 C6, 157.7 C5a,
168.1 and 168.6 C]O (Ac), 173.6 C]O (xanthone). Anal.
C22H19N3O6 (C, H, N).
5.1.6.4. 1,3-Dihydroxy-7-((2-(pyridin-2-yl)hydrazono)methyl)-
9H-xanthen-9-one (8d). Mp: 286 ꢁC dec.; yield: 92%; IR
(cmꢂ1) 3500 (OH), 3200 (NH), 1650 (CO ketone), 1610
1
(CN), 1560 (NeH); H NMR (d) 5.62 (s, 1H, H7), 5.80 (s,
3
1H, H5), 6.79 (t, 1H, H50, J ¼ 5.8 Hz), 7.31 (d, 1H, H60,
3J ¼ 7.5 Hz), 7.34 (t, 1H, H2, J ¼ 7.7 Hz), 7.66 (t, 1H, H40,
3
3J ¼ 7.7 Hz), 7.95 (dd, 1H, H1, 3J ¼ 7.5 Hz and
4J ¼ 1.5 Hz), 8.13 (d, 1H, H30, 3J ¼ 4.5 Hz), 8.20 (d, 1H,
H3, 3J ¼ 8.0 Hz), 8.60 (s, 1H, CH]), 11.08 (s, 1H, NH),
12.91 (s, 1H, OH). Anal. C19H13N3O4 (C, H, N).
5.1.5.8.
6-((2-(4,5-Dihydro-1H-imidazol-2-yl)hydrazono)-
methyl)-9-oxo-9H-xanthene-1,3-diyl
diacetate
(7h). Mp:
241 ꢁC (ethanol); yield: 86%; IR (cmꢂ1) 3200 (NH), 1770
1
(CO ester), 1660 (CO ketone), 1560 (NeH); H NMR (d)
2.34 and 2.39 {2(s, 3H, COeCH3)}, 3.78 (s, 4H, CH2e
CH2), 7.09 (d, 1H, H7, 4J ¼ 2.1 Hz), 7.45 (d, 1H, H5,
5.1.6.5. 1,3-Dihydroxy-6-((2-(pyridin-2-yl)hydrazono)methyl)-
9H-xanthen-9-one (8e). Mp: 319 ꢁC dec.; yield: 98%; IR

1342
M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
(cmꢂ1) 3450 (OH), 3200 (NH), 1650 (CO ketone), 1540 (Ne
The precipitate was collected by filtration, washed with water
and dried at 60 ꢁC.
1
H); H NMR (d) 5.88 (s, 1H, H7), 6.06 (s, 1H, H5), 6.83 (m,
3
1H, H50), 7.36 (d, 1H, H60, J ¼ 8.7 Hz), 7.67e7.70 (m, 3H,
Mp: 262 ꢁC dec.; yield: 52%; IR (cmꢂ1) 3400 (OH), 3200
(NH), 1660 (CO ketone), 1540 (NH); 1H NMR (d) 3.54 (s, 4H,
H40, H4 and H2), 8.04 (d, 1H, H1, 3J ¼ 8.1 Hz), 8.10 (s,
1H, CH]), 8.15 (m, 1H, H30), 11.19 (s, 1H, NH), 12.92
(s, 1H, OH). Anal. C19H13N3O4 (C, H, N).
4
CH2eCH2), 6.20 (d, 1H, H7, J ¼ 1.9 Hz), 6.38 (d, 1H, H5,
4J ¼ 1.9 Hz), 7.57 (d, 1H, H4, 3J ¼ 8.7 Hz), 7.68 (m, 1H,
NH), 8.18 (s, 1H, CH]), 8.26 (s, 1H, H1), 8.29 (dd, 1H,
3
4
5.1.6.6. 1,3-Dihydroxy-5-((2-(pyridin-2-yl)hydrazono)methyl)-
9H-xanthen-9-one (8f). Mp: 307 ꢁC dec.; yield: 96%; IR
(cmꢂ1) 3400 (OH), 3300 (NH), 1660 (CO ketone), 1530
(NeH); 1H NMR (d) 6.21 (s, 1H, H7), 6.39 (s, 1H, H5),
H3, J ¼ 8.8 Hz and J ¼ 1.7 Hz), 8.32 (s, 1H, NH), 12.75
(s, 1H, OH); 13C NMR (d) 47.5 CH2eCH2, 99.5 C5, 103.6
C7, 107.4 C8a, 123.2 C4, 125.1 C1a, 128.6 C1, 137.2 C2,
138.2 C3, 149.4 CH], 154.2 Cipso, 160.7 C4a 162.5 C5a,
168.1 C8, 171.4 C6, 184.7 C]O (xanthone). Anal.
C17H14N4O4 (C, H, N).
3
6.79 (m, 1H, H50), 7.28 (d, 1H, H60, J ¼ 8.4 Hz), 7.62 (d,
3
1H, H4 J ¼ 8.4 Hz), 7.67 (m, 1H, H40), 8.13e8.15 (m, 2H,
3
H30 and CH]), 8.19 (d, 1H, H3, J ¼ 7.37 Hz), 8.25 (s, 1H,
H1), 10.96 (s, 1H, NH), 12.80 (s, 1H, OH). Anal.
C19H13N3O4 (C, H, N).
5.2. Biological assays
Stock solutions (20 mM) of test compounds were first pre-
pared in dimethylsulfoxide (DMSO) and stored at ꢂ20 ꢁC. On
the day of experiment, these concentrated solutions were di-
luted to the desired final concentrations immediately prior ad-
dition to cell culture wells. The final DMSO concentration was
0.1% in each well and showed no interference with the biolog-
ical activities tested.
5.1.6.7.
7-((2-(4,5-Dihydro-1H-imidazol-2-yl)hydrazono)-
methyl)-1,3-dihydroxy-9H-xanthen-9-one (8g). After heating
under reflux for 20 h, the solution was cooled to 0 ꢁC, concen-
trated to half volume under vacuum and extracted with three
portions of ethyl acetate. The organic layer was dried
(Na2SO4), filtered and evaporated under reduced pressure.
The residual solid was triturated with hexane, filtered, recrys-
tallized from methylethylcetone and dried at 60 ꢁC.
5.2.1. Cell culture
Mp: 327 ꢁC dec.; yield: 89%; IR (cmꢂ1) 3450 (OH), 3300
1
(NH), 1640 (CO ketone), 1560 (NeH); H NMR (d) 3.77 (s,
The human tumor cell lines KB 3.1 (oral squamous cell car-
cinoma) and MCF-7 (breast adenocarcinoma) were obtained
from the American Type Culture Collection (ATCC, Mana-
ssas, VA). Cells were routinely grown with either DMEM
(KB 3.1) or RPMI 1640 medium (MCF-7), both supplemented
with 10% fetal calf serum, and a mixture of antibiotics, all ob-
tained from Biochrom AG (Berlin, Germany). They were
grown on Petri dishes (Nunc, Denmark) at 37 ꢁC in a humidi-
fied atmosphere containing 5% CO2. Cells were replicated
every 4e5 days and the medium changed once in-between.
4H, CH2eCH2), 6.22 (s, 1H, H7), 6.38 (s, 1H, H5), 7.48 (t,
3
3
1H, H2, J ¼ 7.5 Hz), 8.14 (d, 1H, H1, J ¼ 7.5 Hz), 7.51 (d,
3
1H, H3, J ¼ 7.6 Hz), 8.77 (m, 2H, CH] and NH), 11.19
(m, 1H, NH), 12.63 (s, 1H, OH); 13C NMR (d) 42.7 CH2e
CH2, 94.1 C5, 98.4 C7, 101.9 C8a, 120.3 C1a, 122.0 C4,
123.8 C2, 127.3 C1, 131.5 C3, 140.7 CH], 153.0 C4a,
156.7 C5a, 157.8 Cipso, 162.8 C8, 166.0 C6, 179.1 C]O (xan-
thone). Anal. C17H14N4O4 (C, H, N).
5.2.2. Viability assay
5.1.6.8.
6-((2-(4,5-Dihydro-1H-imidazol-2-yl)hydrazono)-
The potential effects on cell viability were investigated ac-
cording to our previously reported conditions [26], using the
MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide, SigmaeAldrich Chimie, Saint-Quentin-Fallav-
ier, France] as an indicator of metabolically active cells.
Known number of MCF-7 or KB 3.1 cells (103) were trans-
ferred into 96-well plates in a volume of 200 ml of culture me-
dium and incubated for 48 h before addition of test
compounds. Cells were then exposed for 24 h at 37 ꢁC to known
concentrations of the compound to be tested (1 or 10 mM, ex-
pressed as final concentration). After drug exposure, the culture
medium was removed and 200 ml of MTT reagent (diluted in
culture medium, 1 mg/ml) was added. Following incubation
for 4 h, the MTT/medium was removed and DMSO (200 ml)
was added to dissolve the formazan crystals. Absorbance of
the colored solution was measured on a microplate photometer
(Bio-Tek Instruments) using a test wavelength of 570 nm and
a reference wavelength of 630 nm. Results were evaluated by
comparing the absorbance of the wells containing compound-
treated cells with the absorbance of wells containing 0.1%
methyl)-1,3-dihydroxy-9H-xanthen-9-one (8h). The mixture
was refluxed for 3 h, cooled to 0 ꢁC, and filtered. The solid
was washed with water and dried at 60 ꢁC.
Mp: 273 ꢁC dec.; yield: 95% IR (cmꢂ1) 3450 (OH), 3200
1
(NH), 1650 (CO ketone), 1540 (NeH); H NMR (d) 3.47 (s,
4
4H, CH2eCH2), 6.17 (d, 1H, H7, J ¼ 2.1 Hz), 6.34 (d, 1H,
4
H5, J ¼ 2.1 Hz), 6.80 (m, 1H, NH), 7.22 (m, 1H, NH), 7.75
3
4
(dd, 1H, H2, J ¼ 8.3 Hz and J ¼ 1.2 Hz), 7.83 (d, 1H, H4,
4J ¼ 1.0 Hz), 8.01 (d, 1H, H1, 3J ¼ 8.2 Hz), 8.06 (s, 1H,
CH]), 12.92 (s, 1H, OH); 13C NMR (d) 42.5 CH2eCH2,
93.9 C5, 98.1 C, 101.9 C8a, 113.6 C4, 118.5 C1a, 122.0 C2,
124.9 C1, 141.9 CH], 144.2 C3, 148.6 Cipso, 155.6 C4a,
157.4 C5a, 162.8 C8, 166.2 C6, 179.1 C]O (xanthone).
Anal. C17H14N4O4 (C, H, N).
5.1.6.9.
5-((2-(4,5-Dihydro-1H-imidazol-2-yl)hydrazono)-
methyl)-1,3-dihydroxy-9H-xanthen-9-one (8i). The mixture
was heated under reflux for 5 h. The resulting solution was
cooled to 0 ꢁC and then allowed to stand at 0 ꢁC for 1 h.

M. Varache-Lembe`ge et al. / European Journal of Medicinal Chemistry 43 (2008) 1336e1343
1343
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DMSO alone (solvent control). Conventionally, cell viability
was estimated to be 100% in the solvent control. All experiments
were performed at least twice in triplicate.
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5.2.3. Effect of exposure time on antiproliferative activity
Tumor cells in exponential growth phase were exposed to the
test compounds for 24 or 72 h, respectively, using a series of drug
concentration between 1 mM and 100 mM. After drug exposure,
the cells were washed twice with phosphate-buffered saline and
then reincubated in fresh culture medium for a further 96 h.
The reduction in cell proliferation was measured using the
MTT assay according to the same protocol as indicated in Sec-
tion 5.2.2. Doxorubicin hydrochloride (purchased from Sigma)
was used as positive control. The concentration of substance
required for 50% growth inhibition (IC50) was estimated as
that giving a 50% decrease in absorbance as compared to con-
trols incubated simultaneously without substance.
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`
The authors wish to thank Mrs. Annie Lagardere and Mrs.
Christiane Chapey for their collaboration. This work was sup-
ported by grants from the University Victor Segalen Bordeaux
2 and the Ligue Nationale Contre le Cancer.
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