Cytotoxic-antineoplastic activity of hydroquinone derivatives

Article abstract of DOI:10.1016/S0223-5234(01)01329-0

Several myrcenylhydroquinone derivatives have been evaluated for their cytotoxic activity against three neoplastic cell line cultures and compared with the activity previously observed on other neoplastic systems. Also a new series of this type of compounds has been prepared and the compounds synthesised have been evaluated by their GI₅₀ values.

Full text of DOI:10.1016/S0223-5234(01)01329-0

Eur. J. Med. Chem. 37 (2002) 177–182
Short communication
Cytotoxic–antineoplastic activity of hydroquinone derivatives
Aurora Molinari a,*, Alfonso Oliva a, Paola Reinoso a, Jose Ma. Miguel del Corral b,
Ma Angeles Castro b, Marina Gordaliza b, Mahabir P. Gupta c, Pablo Sol´ıs c,
Arturo San Feliciano b
a Instituto de Qu´ımica, Uni6ersidad Cato´lica de Valpara´ıso, Casilla 4059, Valpara´ıso, Chile
b Departamento de Qu´ımica Farmace´utica, Facultad de Farmacia, Uni6ersidad de Salamanca, E-37007 Salamanca, Spain
c Facultad de Farmacia, Uni6ersidad de Panama´, Apartado Postal 10767, Ciudad de Panama´, Panama
Received 28 May 2001; received in revised form 22 November 2001; accepted 22 November 2001
Abstract
Several myrcenylhydroquinone derivatives have been evaluated for their cytotoxic activity against three neoplastic cell line
cultures and compared with the activity previously observed on other neoplastic systems. Also a new series of this type of
compounds has been prepared and the compounds synthesised have been evaluated by their GI50 values. © 2002 Published by
´
Editions scientifiques et me´dicales Elsevier SAS.
Keywords: Prenylhydroquinones; Naphthohydroquinones; Cytotoxicity; Antineoplastic
1. Introduction
malignant melanoma [3,4]. As a continuation of these
studies, more recently we described the synthesis of new
series of prenylhydroquinones together with the evalua-
tion of their antineoplastic activity against the same
neoplastic cells. These new series were prepared
through chemical modifications, starting from the hy-
droquinonic diacetate 1 (Fig. 1), which was obtained by
Diels–Alder condensation between the monoterpene
a-myrcene and p-benzoquinone [5].
The IC50 (mM) values of selected previously reported
compounds 2–14 (Fig. 2) with high, medium and low
potency are shown in Table 3 and, as it can be ob-
served, the most potent derivatives resulted to be com-
pounds 2 and 7, which contain a saturated alkyl side
chain, and compounds 13 and 14, containing a totally
aromatized benzene ring [5]. The mentioned com-
pounds showed the strongest activity against P-388
cultured cells (IC50=0.3 mM), while those compounds
containing an additional lactone grouping, a spiroether
or a fused epoxide ring and a functionalised open side
chain, are less active. From these results, it was deduced
that a higher saturation degree of the terpenic part is
important for the activity. It was also confirmed that
It has been reported that terpenylquinones/hy-
droquinones and related compounds are active against
several types of neoplastic cells [1,2]. Some compounds
of this type were prepared through Diels–Alder con-
densation between a-myrcene and quinones and
showed fair cytotoxic activities against cultured cells of
P-388 murine leukaemia, A-549 human lung carcinoma,
HT-29 human colon carcinoma and Mel-28 human
Fig. 1. Structure of the starting hydroquinonic diacetate 1.
* Correspondence and reprints.
E-mail address: amolinar@ucv.cl (A. Molinari).
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(01)01329-0

178
A. Molinari et al. / European Journal of Medicinal Chemistry 37 (2002) 177–182
Fig. 2. Selected prenylhydroquinone derivatives 2–14.
Fig. 3. The synthesis of new hydroquinone derivatives 17–22
the aromatisation of the ring fused to the quinone
moiety improved the cytotoxic potency, as was previous-
ly reported [3,4].
In order to get more information about the cyto-
toxic–antineoplastic properties of this type of deriva-
tive, we have prepared and evaluated the activity of a
new compound 15 (Fig. 2), designed taking into ac-
count the structure of compounds with better IC50
values (2, 7, 13 and 14). Also, from epoxides 9 and 16
used as starting materials we have prepared and tested

A. Molinari et al. / European Journal of Medicinal Chemistry 37 (2002) 177–182
179
the bioactivity behaviour of a new family of derivatives
3. Biological results and discussion
17–22 (Fig. 3). Complementarily, compounds 2–14
have been submitted to further evaluation assays on the
neoplastic cell lines: MCF-7, mammary gland car-
cinoma, H-460, human lung carcinoma and SF-268
central nervous system carcinoma.
For comparison purposes, these selected derivatives
2–14 previously evaluated (through their IC50 values)
on P-388, A-549, HT-29 and Mel-28 neoplastic cells,
have now been evaluated (GI50) on cultures of MCF-7,
H-460 and SF-268 cells. The GI50 results of cytotoxicity
evaluation of these compounds and the reported IC50
values are included in Table 3. In general, the GI50
values observed with the new cell systems, are higher
than those IC50 values found in previous assays [5]. In
spite of the fact that the cell lines and the experimental
methods to evaluate the bioactivity are different, it can
be seen in Table 3 that those compounds with better
IC50 values (2, 7, 13 and 14) also display better GI50
values against the new group of cells. Furthermore, the
new GI50 values confirm the importance of the satu-
rated side chain and of the complete aromatisation of
the ring fused to the quinone moiety, for the antineo-
plastic cytotoxicity of these kind of compounds.
It is interesting to note that according to these re-
sults, it could be predicted that a hydroquinonic deriva-
tive containing a fused aromatic ring and a saturated
side chain should lead to a compound with higher
potency than 2, 7, 13 and 14. In fact, this was confir-
med by preparing and evaluating compound 15, which
simultaneously contains both mentioned structural
characteristics. The GI50 values found for this new
compound were B0.3, 0.4 and B0.3 mM, representing
a cytotoxicity enhancement by, at least, a factor of
5–17 with respect to 2, 7, 13 and 14 on the new cell
lines.
2. Chemistry
Compounds 2–14 and 16 were prepared from the
hydroquinonic diacetate 1 as starting material, by
means of reactions such as epoxidation with m-chlorop-
erbenzoic acid (MCPBA), catalytic hydrogenation by
bubbling H2 in the presence of Pd/CaCO3 or Pd/C,
reduction of aldehydes or epoxides with lithium alu-
minium hydride (LAH), oxidation of the aldehydes
with pyridinium dichromate (PDC), methylation of car-
boxylic acid by diazomethane, acid hydrolysis of epoxi-
des with HCl/H2O/t-BuOH and acetylation of alcohols
with acetic anhydride/pyridine, according to previously
reported procedures [3–6]. Compound 15 was prepared
by aromatisation of compound 7 with 2,3 dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ). The new polyfunc-
tional derivatives of the naphthohydroquinonic
diacetate 1, were prepared by oxidative cleavage of
epoxides 9 and 16, using periodic acid to afford the
ketoaldehydes 17 and 18. Compounds 19, 20, 21 and 22
were obtained by reduction/acetylation or oxidation/
methylation of 17 or 18 (Fig. 3).
Physical and analytical data of compounds 15 and
17–22 are given in Section 4, while the most significant
The new compounds 17 and 19–22 were similarly
evaluated for their cytotoxicity and the GI50 values
found for each compound are shown in Table 4.
1
data of H- and 13C-NMR spectra are listed in Tables 1
and 2, according to carbon numbering (Fig. 4).
Table 1
1H-NMR data of compounds 15, 17–22 synthesised
a
H
15
17
18
19
20
21
22
1
0.85 d (6.6)
0.85 d (6.5)
–
0.85 d (6.5)
–
0.85 d (6.6)
–
3
4
5
6
7
8
9
10
3%
–
–
–
–
–
–
4.10 t (6.6)
–
2.41 t (7.3)
–
9.50 t (2.0)
3.52 d (2.0)
3.65 s
–
7.10 s
7.10 s
2.26 s
2.27 s
2.00 s
–
–
–
–
4.12 m
–
–
5.07 m
4.12 m
–
–
–
4.03 t (6.4)
1.87 m
2.49 t (7.1)
–
2.39 t (7.4)
–
9.50 t (1.9)
3.54 d (2.0)
3.63 s
0.85 d (6.5)
7.10 s
7.10 s
2.26 s
2.27 s
–
–
–
–
2.39 t (7.2)
–
3.59 s
–
3.73 s
0.85 d (6.6)
7.08 s
7.08 s
2.27 s
2.30 s
–
5.03 m
4.14 t (7.6)
–
7.37–7.77m
7.37–7.77m
7.37–7.77m
0.85 d (6.6)
7.10 s
7.70 s
2.40 s
2.43 s
–
–
–
–
–
3.59 s
3.61 s
–
7.08 s
7.08 s
2.30 s
2.27 s
2.01 s
–
–
–
–
0.85 d (6.5)
6.99 s
6.99 s
2.35 s
2.35 s
2.04 s
1.93 s
–
6.98 s
6.98 s
2.34 s
2.34 s
2.03 s
2.01 s
1.94 s
–
4%
Ac
Ac
Ac
Ac
Ac
MeO
–
–
–
–
–
–
3.62 s
3.73 s
a According to carbon numbering given in Fig. 4.

180
A. Molinari et al. / European Journal of Medicinal Chemistry 37 (2002) 177–182
Table 2
13C-NMR data of compounds 15, 17–22 synthesised
a
C
15
22.6
17
22.4
18
19
22.5
20
21
22.5
22
1
2
–
–
–
–
–
–
29.2
27.9
36.6
38.6
142.0
128.6
121.6
121.6
22.6
127.8
144.0
117.7
116.7
144.4
126.2
169.5
21.1
169.5
21.1
–
27.8
38.3
21.6
41.5
206.4
196.0
42.7
42.6
22.4
126.0
147.0
122.1
122.5
147.4
128.2
169.0
20.8
168.8
20.8
–
27.9
26.7
21.1
31.7
73.7
63.0
34.4
38.6
22.6
130.0
147.3
121.6
121.7
147.3
130.3
169.5
21.0
169.3
21.0
171.0
23.0
170.5
23.4
–
27.9
33.1
21.6
36.3
206.7
170.3
42.0
42.1
22.5
127.5
146.9
122.2
122.2
147.0
128.1
168.8
20.8
168.9
20.9
–
3
4
5
6
7
8
9
10
1%
2%
3%
4%
5%
64.2
21.2
41.2
205.9
197.0
42.5
47.2
–
126.0
147.0
122.5
122.5
147.2
126.8
168.9
20.8
168.9
20.9
171.0
21.2
–
63.0
26.7
30.4
72.9
63.9
38.7
38.7
–
130.0
147.3
128.8
129.8
147.3
130.7
169.2
20.9
169.4
21.1
170.5
23.0
170.9
23.7
171.1
24.8
–
63.4
23.7
33.1
205.6
171.1
42.1
37.6
–
127.5
146.9
122.3
122.3
147.1
127.9
168.8
20.8
168.9
20.8
170.3
20.9
–
6%
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
MeO
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
52.3
–
–
–
–
–
–
–
52.3
–
–
a Carbon numbering given in Fig. 4.
In general, the GI50 values found for the hy-
droquinone derivatives 17 and 19–22 are higher than
those found for the naphthohydroquinones and show
that these polyfunctional monocyclic structures are less
cytotoxic. These results are in agreement with those
observed for the previously reported series [3–5]. The
presence of oxygenated functions in the non-quinonic
part of the structure decreased the cytotoxic potency.
The slightly enhanced bioactivity of ketoaldehyde 17
could probably be justified on the interaction of this
electrophilic function with free amino or other nucleo-
philic groups of the receptor biomolecules. This be-
haviour has also been observed for the neoplastic
activity of cyclolignan aldehydes [7].
a Bruker AC 200 spectrophotometer. Chemical shifts
are expressed in ppm followed by multiplicity and
coupling constants (J) in hertz. Elemental analysis of C
and H were obtained with a Perkin–Elmer 2400 Series
II CHN Elemental Analyzer within 90.4% of the
theoretical values. Column chromatographic separa-
tions were performed on Silicagel 60, 230-400 mesh
ASTM.
Using reactions according to previously reported
procedures, the following new compounds were
prepared:
4.1.1. Fully aromatized diacetate 15
Obtained by aromatisation of compound 7 with
DDQ in refluxing benzene [3,4] (86%); m.p. 52–53 °C;
IR cm−1 3067 (aromatic CH), 2954–2866 (aliphatic
CꢀH), 1763 (CꢁO ester); 1H-NMR (Table 1);
4. Experimental protocols
4.1. Chemistry
All the compounds evaluated were prepared by pre-
viously reported procedures [3–6] and characterised by
IR on a Perkin–Elmer FT IR 1600 spectrophotometer,
as film over sodium chloride discs. NMR spectra were
1
recorded at 200 MHz for H and 50 MHz for 13C in
Fig. 4. Numbering of basic carbon skeletal of compounds 15 and
17–22.
deuterochloroform using TMS as internal reference, on

A. Molinari et al. / European Journal of Medicinal Chemistry 37 (2002) 177–182
181
Table 3
Cytotoxicity of several hydroquinonic derivatives of 1 against neoplastic cultured cells
a
Compound
P-388
A-549 a
HT-29 a
Mel-28 a
MCF-7 b
H-460 b
SF-268 b
Avarol c
2
3
4
5
6
7
3.1
0.3
2.6
2.9
3.0
6.0
1.5
6.4
14.5
3.7
6.2
6.0
1.5.
6.4
14.5
14.9
6.2
6.0
1.5
6.4
14.5
3.7
5.2
nt
1.5
6.1
\25.6
\28.7
10.0
1.6
nt
3.5
nt
1.2
11.4
\25.6
\28.7
11.1
19.8
\25.6
16.8
15.8
5.6
3.1
0.3
1.5
1.5
1.5
2.2
8
1.4
2.9
2.9
2.9
2.9
8.7
2.9
9
6.9
6.9
6.9
13.8
15.1
13.8
\25.0
3.6
18.5
\30.0
\27.0
\25.0
3.2
\27.0
\30.0
\27.0
\25.0
3.3
15.5
10
11
12
13
14
15
15.1
13.8
\25.0
0.3
0.3
nt
15.1
13.8
\25.0
3.6
2.9
nt
15.1
13.8
\25.0
3.6
2.9
nt
\30.0
\27.0
\25.0
3.9
2.4
B0.3
2.9
nt
2.2
B0.3
5.9
0.4
nt: not tested.
a IC50 values, mM.
b GI50 values, mM.
c IC50 values included for comparison purposes [1,2].
13C-NMR (Table 2). Anal. Found: C, 72.90; H, 7.09.
Calc. for C20H24O4: C, 73.17; H, 7.32%.
CH), 1772 (CꢁO ester), 1731 (CꢁO ester); 1H-NMR
(Table 1); 13C-NMR (Table 2). Anal. Found: C, 59.52;
H, 6.52. Calc. for C23H30O10: C, 59.23; H, 6.44%.
4.1.2. Ketoaldehyde diacetate 17
4.1.6. Ketoester diacetate 21
Obtained by degradative H5IO6 oxidation of the
epoxide 9 in aqueous tetrahydrofuran [3,4] (72%); oil;
IR cm−1 3084 (aromatic CH), 2954–2869 (aliphatic
CH), 2725 (aldehyde CH), 1765 (CꢁO ester), 1721 (CꢁO
aldehyde); 1H-NMR (Table 1); 13C-NMR (Table 2).
Anal. Found: C, 66.23; H, 7.10. Calc. for C20H26O6: C,
66.30; H, 7.18%.
Obtained by NaClO2 oxidation of 17, followed by
CH2N2 methylation in anhydrous ethyl ether [8] (64%);
m.p. 107–109 °C; IR cm−1 3065 (aromatic CH), 2967–
2880 (aliphatic CH), 1767 (CꢁO ester); 1H-NMR (Table
1); 13C-NMR (Table 2). Anal. Found: C, 64.29; H,
7.36. Calc. for C21H28O7: C, 64.29; H, 7.14%.
4.1.7. Ketoester triacetate 22
4.1.3. Ketoaldehyde triacetate 18
Obtained by NaClO2 oxidation of 18, followed by
CH2N2 methylation in anhydrous ethyl ether. (71%);
m.p. 114–115 °C; IR cm−1 3061 (aromatic CH), 2954–
2849 (aliphatic CH), 1772 (CꢁO ester) 1742 (CꢁO ester);
1H-NMR (Table 1); 13C-NMR (Table 2). Anal. Found:
C, 58.70; H, 6.10. Calc. for C20H24O9: C, 58.82; H
5.88%.
Obtained by degradative H5IO6 oxidation of the
epoxide 16 in aqueous tetrahydrofuran (66%); oil; IR
cm−1 3060 (aromatic CH), 2960–2870 (aliphatic CH)
2731 (aldehyde CH), 1765 (CꢁO ester), 1720 (CꢁO
aldehyde); 1H-NMR (Table 1); 13C-NMR (Table 2).
Anal. Found: C, 60.26; H, 5.90. Calc. for C19H22O8: C,
60.32; H, 5.87%.
4.1.4. Tetraacetate 19
Obtained by LAH reduction of 17 in anhydrous ethyl
ether, followed by pyridine/acetic anhydride acetylation
[3–6] (53%); m.p. 98–99 °C; IR cm−1 3061 (aromatic
CH), 2952–2863 (aliphatic CH), 1769 (CꢁO ester, 1739
Table 4
Cytotoxicity of hydroquinone derivatives 17, 19–22 against neoplastic
cultured cells
1
(CꢁO ester); H-NMR (Table 1); 13C-NMR (Table 2).
a
Compound
MCF-7
H-460 a
SF-268 a
Anal. Found: C, 63.85; H, 7.50. Calc. for C24H34O8: C,
64.00; H, 7.56%.
17
19
20
21
22
4.6
\22.0
\21.0
\25.0
\25.0
7.7
\22.0
\21.0
\25.0
\25.0
4.6
\22.0
\21.0
\25.0
\25.0
4.1.5. Pentaacetate 20
Obtained by LAH reduction of 18, followed by pyri-
dine/acetic anhydride acetylation (59%); m.p. 82–83
°C; IR cm−1 3068 (aromatic CH), 2919–2860 (aliphatic
a GI50 values, mM.

182
A. Molinari et al. / European Journal of Medicinal Chemistry 37 (2002) 177–182
4.2. Bioacti6ity
the ‘Direccio´n de Investigacio´n de la Vicerrector´ıa de
Investigacio´n y Estudios Avanzados de la Universidad
Cato´lica de Valpara´ıso’, Chile (Proyecto D.I. 125.713/
99 and 125.739/01) and the ‘Junta de Castilla y Leo´n’,
Spain (Consejer´ıa de Educacio´n y Cultura. SA –71/00
B). This work has been developed under the auspices of
the ‘Programa Iberoamericano de Ciencia y Tecnolog´ıa
para el Desarrollo, CYTED, SubPrograma X’.
4.2.1. Antineoplastic assays for IC50
Cells were seeded into 16 mm wells (multidishes,
NUNC 42001) at concentrations of 1×104 (P-388),
2×104 (A-549, HT-29 and MEL-28) cells well−1, re-
spectively, in 1 mL aliquots of MEM10FCS medium,
containing the compound to be evaluated at the con-
centrations tested. In each case, a set of control wells
was incubated in the absence of sample and counted
daily to ensure the exponential growth of cells. After 3
days at 37 °C, under 10% CO2, 98% humid atmosphere,
P388 leukaemic cells were observed through an inverted
microscopy and the degree of inhibition was determined
by comparison with the controls, whereas A-549, HT-
29 and MEL-28 were stained with crystal violet before
examination [7].
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4.2.2. Antineoplastic assays for GI50
The human tumour cell line of the cancer screening
(MCF-7, H-460 and SF-268) were grown in a RPMI
1640 medium containing 5% foetal bovine serum and 1
mM
L-glutamine. Cells were inoculated into 96-well
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from 5000 to 40 000 cells well−1, depending on the
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CO2, 95% air and 100% relative humidity for 24 h prior
to addition of compound to be tested. Dilutions at
twice the intended test concentration were added at
time zero, in 100 mL aliquots, to the microtiter plates
wells. Usually, test compounds were evaluated at 5–10
fold dilution. Incubation lasted for 48 h in 5% CO2 and
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Acknowledgements
The authors acknowledge the financial support from