Synthesis and cytotoxic evaluation of 6-(3-pyrazolylpropyl) derivatives of 1,4-naphthohydroquinone-1,4-diacetate

Article abstract of DOI:10.1002/ardp.200900041

Several new 6-(3-pyrazolylpropyl) derivatives of 1,4-naphthohydroquinone-1, 4-diacetate (NHQ-DA) have been prepared by chemical modifications of the Diels-Alder adduct of a-myrcene and 1,4-benzoquinone. All these new compounds and precursors have been eva

Full text of DOI:10.1002/ardp.200900041

Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
A. Molinari et al.
591
Full Paper
Synthesis and Cytotoxic Evaluation of 6-(3-Pyrazolylpropyl)
Derivatives of 1,4-Naphthohydroquinone-1,4-diacetate
Aurora Molinari1, Alfonso Oliva1, Claudia Ojeda1, Josꢀ M. Miguel del Corral2, M. Angeles Castro2,
Carmen Cuevas3, and Arturo San Feliciano2
1 Instituto de Quꢀmica, Pontificia Universidad Catꢁlica de Valparaꢀso, Valparaiso, Chile
2 Depto de Quꢀmica Farmacꢂutica, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Espaꢃa
3 Pharma Mar S. A., Madrid, Espaꢃa
Several new 6-(3-pyrazolylpropyl) derivatives of 1,4-naphthohydroquinone-1,4-diacetate (NHQ-
DA) have been prepared by chemical modifications of the Diels–Alder adduct of a-myrcene and
1,4-benzoquinone. All these new compounds and precursors have been evaluated in vitro for
their cytotoxicity against cultured human cancer cells of MB-231 breast-adeno carcinoma, A-549
lung carcinoma, and HT-29 colon carcinoma. GI50 values ranged in and below the micromolar
concentration level.
Keywords: Antineoplastic cytotoxicity / a-Myrcene / 1,4-Naphthohydroquinone / Pyrazole /
Received: February 27, 2009; accepted: June 7, 2009
DOI 10.1002/ardp.200900041
[15–21]. Functionalized 1,4-benzoquinones are of cur-
rent interest in medicinal chemistry and their use as
potential synthetic building block is now a powerful
tool in the design and synthesis of nitrogen-, oxygen-,
and sulfur-containing heterocyclic drug candidates [22,
23].
Introduction
Natural and synthetic substituted quinones and hydro-
quinones are a class of compounds with important bio-
logical functions in bioenergetic transport [1], oxidative
phosphorylation decoupling [2–4], or electron transfer
process [5]. Also, they have diverse pharmacological
activities such as anticancer [6–9], antimicrobial [10,
11], antiviral [12], and anti-inflammatory activities [13].
Their antitumor properties and mechanism of action
are attributed to an inhibition of mitochondrial elec-
tron transport and decoupling of oxidative phosphoryla-
tion by the one-electron redox process of the quinone/
hydroquinone couple, involving semi-quinonic radicals
which induce an increase in reactive oxygen species for-
mation [2–4, 14]. Moreover, they can act as topoisomer-
ase inhibitors, via DNA intercalation and reduction of
the quinone moiety by oxido-reductases (DT-diaphorase)
Related to the cytotoxic-antineoplastic activities of ter-
penylhydroquinones, we have described the synthesis
and studies on the structure-activity relationships of
prenyl-1,2 or 1,4-naphthohydroquinone diacetates 1 and
2 (NHQ-DA) (Fig. 1) prepared via Diels–Alder cyclocon-
densation of a-myrcene and 1,2-, 1,4-, or 2-substituted-
1,4-benzoquinones, followed by further chemical modifi-
cations of the terpenyl side chain, leading to several fam-
ilies of substituted NHQ-DA, taking in account that ace-
tate groups are hydrolysable either in vitro at neutral pH
or in vivo by the action of lipases and esterases, to liber-
ate the active NHQ derivative [24–27]. These families dis-
play cytotoxicity IC50 or GI50 values in the lM- and nM-
level against several neoplastic cell lines, some of the
compounds tested showed a moderate selectivity against
P-388, Mel-28, SKBR-3, K-562, and PANC-1 cell lines, and
from structure-activity studies it was deduced that full
aromatization is an important structural factor to
enhance the cytoxicity [28–33]. Some heterocyclic-sub-
stituted terpenylhydroquinones are also cytotoxic, and,
Correspondence: Aurora Molinari, Instituto de Quꢀmica, Facultad de
Ciencias Bꢄsicas y Matemꢄticas, Pontificia Universidad Catꢁlica de Val-
paraꢀso, Av. Brasil 2950, Valparaꢀso, Chile.
E-mail: amolinar@ucv.cl
Fax: +56 32 227-3422
Abbreviation: 1,4-Naphthohydroquinone-1,4-diacetate (NHQ-DA)
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592
A. Molinari et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
Table 1. Cytotoxicity (GI50a), lM) for compounds 10a–c to 15a–c.
Compound
MB-231b)
A-549c)
HT-29d)
3e)
1.01
0.872
1.65
1.41
5.65
3.06
0.860
4.93
1.28
0.219
2.40
1.28
0.208
0.490
1.28
0.371
1.45
4.11
1.22
1.04
1.83
0.368
6.52
1.00
0.09
1.42
3.44
3.55
4.46
14.1
2.62
14.6
12.8
6.30
1.71
7.20
6.30
1.51
7.08
6.30
1.59
3.04
18.0
3.71
>30
1.79
1.64
3.04
2.73
9.24
3.28
4.63
5.26
8.25
5.91
6.32
8.25
0.63
4.36
8.25
1.35
4.56
6.30
1.74
9.99
>30
4e)
5e)
6e)
7e)
8e)
10 a
10 b
10 c
11 a
11 b
11 c
12 a
12b
12 c
13 a
13 b
13 c
14 a
14 b
14 c
15 a
15 b
15 c
Doxorubicinf)
Figure 1. General structure of prenyl-1,2 and 1,4-naphthohy-
droquinone derivatives 1 and 2.
recently, we have reported the synthesis and evaluation
of some pyridyl, imidazolyl, and benzimidazolyl NHQ-
DA 3–8 (Fig. 2). They were evaluated with A-549, HT-29,
and MB-231 cell lines giving GI50 values between 0.9 to
14.1 lM as it is shown in Table 1 for comparison pur-
poses [34].
Over the past few years, much interest has been given
to the remarkable pharmacological activities of pyrazole
derivatives as antibacterial-antifungal [35], anti-inflam-
matory [36], and potential anticancer agents [37–40].
Modified structures of the (+)-CC-1065 antibiotic isolated
from Streptomices zelensis fungus [41], 1-(4-chlorophenyl)-
4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide ana-
logs [42], 1-(2-chloro-2-phenylethyl)-6-methylthio-1H-pyra-
zolo[3,4-d]pyrimidines-4-amino substituted [43], 1,3-di-
aryl-5-(2,5-dimethoxyphenyl)pyrazoles [44], pyrazofurin
[45, 46], 1-arylmethyl-3-aryl-1H-pyrazole-5-carbo-hydra-
zides [47], ethyl 1-(2k-hydroxy-39-aroxypropyl)-3-aryl-1H-
pyrazole-5-carboxylates [48], indeno-[1,2-c] substituted
pyrazoles [49], and pyrazole emodin compounds [50] are
examples of pyrazolyl derivatives screened against a wide
variety of leukemia, lung, colon, ovarian, renal, prostate,
and melanoma human tumor cell lines. Emodin is an
anthraquinone structurally related to daunorubicin and
mitoxantrone used clinically for the treatment of various
tumors [51]. By itself, emodin has a low or insignificant
cytotoxicity against several cancer cell lines; however the
incorporation of a pyrazole ring into its anthraquinone
core enhances significatively the antitumor activity
30.0
1.57
14.3
>30
0.834
8.01
18.4
0.10
0.07
a)
The drug concentration that causes 50% of growth inhibi-
tion.
Breast-adenoma carcinoma.
Lung carcinoma.
Colon carcinoma.
b)
c)
d)
e)
f)
Included for comparison purposes.
Reference drug.
against the same cell lines [50]. Keeping in view all these
above facts, we have designed and synthesized a new fam-
ily of pyrazolyl-substituted NHQ-DA derivatives by com-
bining the pyrazol group with the NHQ core in order to
analyze the effect of the heterocyclic substituent on the
cytotoxic-antineoplastic properties of this kind of NHQ.
All the synthesized compounds have been screened in
vitro for anticancer activity against A-549, HT-29, and MB-
231 neoplastic cell lines.
Figure 2. Structure of heterocy-
clic-substituted 1,4-terpenylnaph-
thohydroquinones 3–8.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
Derivatives of 1,4-Naphthohydroquinone-1,4-diacetate
593
Reagents and conditions: (a) NaBH4, MeOH, 15 s, r.t.; (b) TsCl, Pyr, 08C, 4 h; (c) LiBr, acetone, r.t., 23 h; (d) 3-Methyl-1-phenyl-2-pyrazolin-5-one, K2CO3, DMF, 458C, 45 h;
(e) 3-Methyl-2-pyrazolin-5-one, NaOAc, DMF, CS2, 458C, r.t., 20 h; (f) 3-Methyl-1-phenyl-2-pyrazolin-5-one, NaOAc, DMF, CS2, 458C, r.t., 20 h.
Scheme 1. Synthetic pathway for the new 1,4-naphthohydroquinone derivatives 10a–c to 15a–c.
pared from 3-methyl-2-pyrazolin-5-one and 3-methyl-1-
phenyl-2-pyrazolin-5-one by a similar deprotonation proc-
Results and discussion
Chemistry
ess with sodium acetate in DMF, but the reaction with the
bromides was achieved after the addition of carbon disul-
fide [52, 53]. Physical and analytical data of compounds
The new pyrazolyl-substituted terpenyl-1,4-hydroqui-
none diacetates 13a–c, 14a–c, and 15a–c were prepared
using the aldehydes 9a–c as starting material (Scheme 1),
which were obtained from the Diels–Alder cycloadduct
of a-myrcene and 1,4-benzoquinone [28–32]. These alde-
hydes were reduced to the corresponding alcohols 10a–c,
converted to the p-toluensulfonates 11a–c, and then to
the bromides 12a–c by previously reported procedures
[34]. The pyrazolyl derivatives 13a–c, 14a–c, and 15a–c
were synthesized using the carbanion chemistry of 5-pyr-
azolones. 3-Methyl-2-pyrazolin-5-one was deprotonated at
the a-carbon with potassium carbonate in DMF, reacting
through its enolate form with the bromides 12a–c to
afford the pyrazolyloxy derivatives 13a–c. The pyrazolyl-
dithiocarbonyloxy derivatives 14a–c and 15a–c were pre-
are given in the experimental part while the 1H and 13
C
chemical shifts are shown in Tables 2 and 3, according to
carbon numbering of compounds given in Scheme 1.
Bioactivity
A conventional colorimetric assay was set up to estimate
the GI50 values, i.e. the drug concentration that causes
50% cell-growth inhibition after 72 h of continuing expo-
sure to the test compounds [54, 55]. Ten serial dilutions
(from 10 to 0.026 lg/mL) for each sample were evaluated
in triplicate. All 30 values found were used to define a
nonlinear regression line for each compound, with auto-
matic detection and discarding of outlier points and
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594
A. Molinari et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
Table 2. 1H-NMR data for compounds 10a–c to 15a–c.
H
10a
10b
10c
11a
11b
11c
12a
12b
12c
2
3
5
6
7
7.17 s
7.21 s
7.62 s
–
7.39 dd
(8.6;1.5)
7.78 d (8.6)
2.84 t (8.0)
1.91 m
3.64 t (6.4)
–
6.90 s
6.90 s
3.09 s
–
6.87 s
6.87 s
2.71 m
1.60 m
1.57 m
7.19 s
7.22 s
7.60 s
–
7.37 dd
(8.6;1.4)
7.80 d (8.6) 3.01 m
2.85 t (7.4)
2.03 m
6.92 s
6.92 s
3.13 s
–
6.87 s
6.87 s
2.45 m
1.80 m
1.80 m
7.19 s
7.23 s
7.66 s
–
7.40 dd
(8.6;1.6)
7.81 d (8.1) 3.09 m
2.96 d (7.3) 2.02 m
2.21 m
3.41 t (6.4)
–
–
–
6.93 s
6.93 s
3.20 s
–
6.88 s
6.88 s
2.80 m
1.61 m
1.61 m
5.58 m
5.45 s
5.61 s
8
9
3.22 m
2.16 t (7.4) 1.20 m
1.73 m 1.55 m
3.66 t (6.4) 3.66 t (6.6) 4.03 t (6.0)
–
2.75 m
2.68 m
1.23 m
1.50 m
4.04 t (6.4)
2.80 m
1.30 m
1.877 m
2.11 t (7.4)
1.82 m
4.03 t (6.2)
10
11
13
14
16
OAc
OAc
2.02 m
3.42 t (6.7) 3.43 t (6.8)
–
–
–
–
–
7.79 d (8.2) 7.78 d (8.2) 7.79 d (8.2)
7.79 d (8.2) 7.78 d (8.2) 7.79 d (8.2)
2.46 s
2.41 s
2.45 s
–
–
–
–
–
–
–
2.43 s
2.32 s
2.30 s
2.43 s
2.31 s
2.30 s
–
2.43 s
2.43 s
2.33 s
2.31 s
2.31 s
2.30 s
2.46 s
2.46 s
2.31 s
2.30 s
2.30 s
2.30 s
H
13a
13b
13c
14a
14b
14c
15a
15b
15c
2
3
5
6
7
7.19 s
7.22 s
7.60 s
–
7.37 dd
(8.6;1.4)
7.80 d (8.6)
2.96 t (7.4)
2.18 m
4.08 t (8.9)
5.45 s
2.27 s
–
7.74 m
7.42 m
7.24 m
–
2.46 s
2.39 s
–
6.93 s
6.93 s
3.20 s
–
6.79 s
6.79 s
2.70 m
1.60 m
1.60 m
7.19 s
7.23 s
7.65 s
–
7.40 dd
(8.6;1.4)
7.81 d (8.6) 3.12 m
2.93 t (7.4)
2.14 m
6.93 s
6.93 s
3.21 m
–
6.89 s
6.89 s
2.70 m
1.60 m
1.60 m
7.26 s
7.26 s
7.71 s
–
6.94 s
6.94 s
3.24 m
–
6.81 s
6.81 s
2.80 m
1.63 m
1.58 m
5.57 m
5.62 bs
7.74 m
5.63 bs
8
9
3.09 m
2.26 t (7.7) 1.23 m
1.98 m 1.50 m
2.80 m
2.70 m
1.32 m
1.89 m
3.40 t (7.2)
–
–
2.81 s
–
–
–
7.86m
3.03 t (7.5)
2.24 m
3.49 t (7.4)
–
3.1 m
2.84 m
2.26 t (7.1)
1.96 m
3.37 t (7.3)
–
2.28 t (6.5) 1.36 m
1.97 m 2.00 m
3.41 t (7.3) 2.90 t (6.5)
–
–
10
11
13
15
16
17
18
19
20
OAc
OAc
NH
OH
4.08 t (6.3) 4.08 t (7.2) 3.40 t (7.2)
5.47 s
2.27 s
–
7.70 m
7.40 m
7.23 m
–
2.31 s
2.31 s
–
5.50 s
2.28 s
–
7.70 m
7.48 m
7.24 m
–
2.45 s
2.42 s
–
–
–
–
–
–
–
2.72 s
–
–
–
2.75 s
–
–
–
2.74 s
–
2.67 s
–
2.71s
–
7.86 m
7.51 m
7.37m
2.52 s
2.50 s
–
7.81 m
7.46 m
7.32m
2.34 s
2.32 s
–
7.80 m
7.42 m
7.30m
2.33 s
2.31 s
–
–
–
–
2.47 s
2.45 s
11.74 bs
11.74 bs
2.34 s
2.31 s
11.70 bs
11.70 bs
2.33 s
2.33 s
11.05 bs
11.05 bs
–
–
–
13.77 s
13.75 s
13.73 s
direct calculation of GI50 values. The antineoplastic cyto- activity decrease must be also related to the lost of the
toxicity of all the compounds synthesized was evaluated naphthalenic character of compounds 10b–15b and
in vitro against the tumoral cell lines A-549 lung carci- 10c–15c. Furthermore, the presence of a pyrazolyl sub-
noma, HT-29 colon adenocarcinoma, MB-231 breast carci- stituent in the side chain of aromatized terpenyl-1,4-ben-
noma and the mean of the results obtained from tripli- zohydroquinones has a slightly enhancing effect against
cate assays are shown in Table 1.
MB-231 when the cytotoxic activity of them is compared
From the evaluation of the data reported in Table 1, with those observed in heterocyclic derivatives 3–8
the following general observations can be made. All the (Table 1) reported by us [34] recently. However, in spite of
compounds are cytotoxic against the three cell lines this slight enhancement, the anticancer activities in gen-
assayed, most of them show GI50 values ranging between eral found for compounds 10a–c–15a–c against the
0.2 and 10.0 lM and these values are better against the three cell lines assayed are better than most of those
MB-231 cell line. Except for 14a, the GI50 of all the fully reported in 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole-3-
aromatized compounds 10a–15a are under the lM-level, carboxylic acid hydrazide analogs (A-549: a0.01–33.2 lM;
probably due to the naphthalenic character of the hydro- HT-29: 0.25–40.1 lM) and related indeno-pyrazolyl deriv-
quinone moiety [14]. Besides, the cytotoxic activity is atives (A-549: 10.8–37.8 lM; HT-29: 15.4–44.0 lM; MB-
diminished as the saturation of the ring A increases; this 231: 2.89–87.1 lM) [42, 49]. Finally, alcohols 10a–b, p-tol-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
Derivatives of 1,4-Naphthohydroquinone-1,4-diacetate
595
Table 3. 13C-NMR data for compounds 10a–c to 15a–c.
C
10a
10b
10c
11a
11b
11c
12a
12b
12c
1
2
3
4
4a
5
6
7
8
8a
9
10
11
12
13
14
15
16
144.3
116.8
117.7
143.9
127.2
120.0
140.9
128,4
121.7
126.1
32.3
33.8
61.9
–
146.1
119.9
119.2
146.1
133.1
27.3
141.7
117.2
25.0
133.3
33.3
30.3
62.5
–
146.0
119.6
119.6
146.6
131.0
30.1
53.5
40.5
23.6
130.9
32.3
32.8
63.1
–
144.8
117.1
117.9
144.2
127.7
120.5
139.3
128.1
121.9
126.3
31.7
146.0
120.0
120.0
146.0
128.1
26.5
133.0
118.1
25.1
146.6
119.7
119.7
146.6
133.4
27.6
54.5
32.6
23.5
133.0
30.0
144.3
117.0
117.9
143.9
127.7
120.5
139.4
128.3
122.0
126.3
33.7
34.1
32.9
–
146.1
120.0
120.0
146.1
128.2
27.4
132.1
118.2
25.2
128.3
35.2
30.3
33.3
–
146.6
119.6
119.6
146.6
130.9
27.8
32.4
34.8
23.5
130.9
30.3
30.1
34.0
–
128.1
32.5
27.3
30.2
69.3
27.4
70.6
69.7
132.8
127.8
129.8
143.9
21.5
128.1
127.9
129.8
144.7
21.6
130.8
127.9
129.9
144.9
21.7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
OAc
OAc
OAc
OAc
20.9
21.0
169.5
169.4
20.8
20.8
169.3
169.3
20.9
20.8
169.4
169.3
21.0
20.9
169.4
169.3
20.9
20.8
169.2
169.1
20.9
20.8
169.3
169.3
21.0
21.0
169.4
169.3
20.9
20.8
169.2
169.2
20.9
20.8
169.4
169.3
C
13a
13b
13c
14a
14b
14c
15a
15b
15c
1
2
3
4
4a
5
6
7
8
8 a
9
10
11
12
13
14
15
16
17
18
19
20
OAc
OAc
OAc
OAc
144.3
117.1
117.9
143.9
127.7
120.4
138.8
129.0
120.6
126.3
30.3
32.3
70.6
129.0
86.4
154.7
14.5
139.7
121.6
128.8
125.8
–
20.9
21.0
169.4
169.4
146.1
120.0
120.0
146.0
128.2
26.6
148.7
117.9
25.1
128.3
33.1
27.4
71.2
132.4
86.3
145.9
120.0
120.0
145.5
130.9
31.0
54.5
40.5
22.4
130.9
30.3
28.1
71.2
131.5
86.4
154.7
14.5
140.0
121.6
128.7
125.0
–
144.3
117.0
117.9
143.9
127.7
120.2
139.8
128.2
121.9
126.3
35.3
29.1
33.2
215.0
11.1
139.8
141.3
15.8
–
146.0
120.0
119.9
146.0
128.4
25.3
141.4
118.0
25.2
128.2
36.2
27.4
35.4
215.3
11.81
132.4
163.2
15.8
–
146.7
119.8
119.7
146.7
130.8
26.1
32.6
35.6
20.4
130.8
30.1
29.4
34.2
215.6
112.0
131.0
163.4
16.0
–
144.2
118.0
118.0
144.0
127.2
120.1
139.0
129.1
120.5
126.0
32.5
146.8
119.9
118.1
146.1
132.9
25.4
139.6
119.4
25.1
132.4
36.3
27.4
33.1
213.4
11.3
128.2
157.6
18.1
137.1
121.7
129.0
127.7
20.9
146.6
120.0
120.0
146.5
131.0
26.3
32.8
34.6
20.3
130.8
30.0
33.6
34.7
29.4
34.1
214.3
113.1
135.4
155.2
14.1
138.8
119.3
130.3
125.9
20.8
213.9
11.5
131.4
157.9
13.9
140.0
119.7
130.6
126.0
20.7
154.8
14.5
138.8
121.6
128.7
125.7
–
20.8
20.8
169.1
169.1
–
–
–
–
–
–
–
–
–
21.0
21.0
169.2
169.2
21.0
21.0
169.4
169.5
20.9
20.8
169.3
169.2
21.0
20.9
169.5
169.4
20.8
169.3
169.3
20.8
169.2
169.2
20.7
169.0
169.0
uensulfonates 11a–b, and bromides 12a–b are more HT-29 tumor cell lines to be considered as useful tem-
cytotoxic than those previously reported, related deriv- plates and obtain more potent antitumor agents via deri-
atives from 1,2-benzoquinone [33].
vation or structural variation. Due to the structural che-
In conclusion, novel pyrazolyl-substituted-1,4-NHQ-DA lating metal-complex properties of the 4-dithiocarbonyl-
were synthesized and tested for their anticancer activ- oxy-5-hydroxy-pyrazolyl moiety [56, 57] in compounds
ities. The results show that several of these compounds 14a–c and 15a–c, the antitumor activity of metal-com-
exhibit remarkable activities against MB-231, A-549, and plexes of them could be also explored.
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596
A. Molinari et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
The authors acknowledge financial support from the Comisiꢀn 6-(3-Hydroxypropyl)-5,6,7,8-tetrahydro-1,4-
Nacional de Investigaciꢀn Cientꢁfica y Tecnolꢀgica, CONICYT naphthoquinone diacetate 10c
Purified by CC with hexane/ethyl acetate 1:2 as eluent (53%); oil;
(Project Fondecyt 1060447), Direcciꢀn de Investigaciꢀn de la
Vicerrectorꢁa de Investigaciꢀn y Estudios Avanzados de la Pon-
tificia Universidad Catꢀlica de Valparaꢁso (Project DI 125.796/
06), and Junta de Castilla y Leꢀn, Spain (Project SA114A06).
IR cm– 1: 3418 (alcoholic OH), 30587 (aromatic CH), 2933 (ali-
phatic CH), 1760 (C=O ester); HMRS calc. for C17H22O5: 306.14676;
found: 306.14515.
This research was performed under the auspices of the ”Pro-
grama Iberoamericano CYTED-Subprograma X”.
General procedure for the synthesis of p-toluensulfonates
11a–c
0.66 mmol of the corresponding alcohol 2a–c dissolved in pyri-
The authors have declared no conflict of interest.
dine (0.2 mL) and p-toluensulfonyl chloride (0.14 g, 0.73 mmol)
were stirred for 4 h at 08C. The reaction mixture was poured in
ice/water and then extracted with CH2Cl2 (465 mL). The organic
layer was washed with 2 N HCl (366 mL), H2O until neutral pH
and dried with anhydrous Na2SO4. After filtration, the solution
was evaporated to dryness to afford the crude corresponding
alcohol which was purified by CC.
Experimental
6-[3-(p-Tosyloxy)propyl]-1,4-naphthohydroquinone
diacetate 11a
Chemistry
Solvents and reagents were purchased from commercial sources
and purified by standard procedures as necessary. Aldehydes
1a–c were prepared by previously reported procedures [29]. The
IR spectra were recorded on a Perkin Elmer FT IR 1600 spectro-
photometer (Perkin Elmer, USA) as a film over NaCl discs. 1H and
13C-NMR spectra were recorded on an Avance 400 Digital NMR
Bruker spectrophotometer (Bruker Bioscience, USA) at 440.132
MHz for 1H and 100.623 MHz for 13C by using solutions in CDCl3
and TMS as internal reference. Chemical shifts were expressed
in ppm, followed by multiplicity and coupling constant (J) in Hz.
High resolution mass spectra (HRMS) were run on a VGTS 250
spectrometer (TriTech Microelectronics, USA) working at 70 eV.
The progress of synthetic procedures were monitored by TLC
with silicagel 60 F254 (0.25 mm thick; Merck, Germany) alumi-
num sheets and column chromatographies were performed on
silicagel 60 (Merck, 230–400 mesh ASTM), using mixtures of sol-
vents with variable proportions as eluent.
Purified by CC with hexane/ethyl acetate 2:1 as eluent (83%);
solid; m.p.: 86–908C; IR cm– 1: 3058 (aromatic CH), 2958 (ali-
phatic CH), 1766 (C=O ester); HMRS calc. for C24H24O7S:
456.12432; found: 456.12421.
6-[3-(p-Tosyloxy)propyl]-5,8-dihydro-1,4-
naphthohydroquinone diacetate 11b
Purified by CC with hexane/ethyl acetate 3:2 as eluent (56%); oil;
IR cm– 1: 3022 (aromatic CH), 2913 (aliphatic CH), 1761 (C=O
ester); HMRS calc. for C24H26O7S: 458.13998; found: 458.13988.
6-[3(p-Tosyloxy)propyl]-5,6,7,8-tetrahydro-1,4-
naphthohydroquinone diacetate 11c
Purified by CC with hexane/ethyl acetate 3:2 as eluent (83%); oil;
IR cm– 1: 3051 (aromatic CH), 2930 (aliphatic CH), 1762 (C=O
ester); HMRS calc. for C24H28O7S: 460.155648; found: 460.15292.
General procedure for the synthesis of alcohols 10a–c
2.2 mmol of the aldehydes 9a–c dissolved in methanol (60 mL)
were stirred at r.t. for 15 s with NaBH4 (0.041 g, 1.07 mmol). After
dilution with H2O (60 mL), the product was extracted with Et2O
(3610 mL) and the organic layer was dried over anhydrous
Na2SO4. After filtration, the solution was evaporated to dryness
to afford the crude corresponding alcohol 10a–c purified then
by CC.
General procedure for the synthesis of bromides 12a–c
2.19 mmol of the corresponding p-tosyloxy diacetate dissolved
in dry acetone (25 mL) was stirred with LiBr (1.90 g, 21.9 mmol)
for 23 h at r.t. The reaction mixture was evaporated to dryness
and then was treated with water (50 mL) and extracted with Et2O
(2660 mL). The organic layer was washed with H2O (3650 mL)
and dried with anhydrous Na2SO4. After filtration, the solution
was concentrated to dryness to afford the corresponding bro-
mide compound which was purified by CC.
6-(3-Hydroxypropyl)-1,4-naphthohydroquinone diacetate
10a
6-(3-Bromopropyl)-1,4-naphthohydroquinone diacetate
12a
Purified by CC with hexane/ethyl acetate 3:1 as eluent (70%); oil;
IR cm– 1: 3058 (aromatic CH), 2930 (aliphatic CH), 1760 (C=O
ester); HMRS calc. for C17H17BrO4: 364.03101; found: 364.03184.
Purified by CC with hexane/ethyl acetate 1:2 as eluent (57%); oil;
IR cm– 1: 3413 (alcoholic OH), 3051 (aromatic CH), 2940 (aliphatic
CH), 1762 (C=O ester); HMRS calc. for C17H18O5: 302.11544; found:
302.11221.
6-(3-Hydroxypropyl)-5,8-dihydro-1,4-naphthoquinone
diacetate 10b
Purified by CC with hexane/ethyl acetate 1:2 as eluent (65%); oil;
IR cm– 1: 3413 (alcoholic OH), 3014 (aromatic CH), 2927 (aliphatic
CH), 1760 (C=O ester); HMRS calc. for C17H20O5: 304.13110; found:
304.13177.
6-(3-Bromopropyl)-5,8-dihydro-1,4-naphthohydroquinone
diacetate 12b
Purified by CC with hexane/ethyl acetate 3:1 as eluent (69%); oil;
IR cm– 1: 3022 (aromatic CH), 2936 (aliphatic CH), 1761 (C=O
ester); HMRS calc. for C17H19BrO4: 366.04667; found: 366.03372.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
Derivatives of 1,4-Naphthohydroquinone-1,4-diacetate
597
phatic CH), 1761 (C=O ester); HMRS calc. for C22H22N2O5S2:
458.09716; found: 458.09745.
6-(3-Bromopropyl)-5,6,7,8-tetrahydro-1,4-
naphthohydroquinone diacetate 12c
Purified by CC with hexane/ethyl acetate 3:1 as eluent (70%); oil;
IR cm– 1: 3058 (aromatic CH), 2930 (aliphatic CH), 1760 (C=O
ester); HMRS calc. for C17H21BrO4: 368.06233; found 368.06094.
6-[3-(5-Hydroxy-3-methylpyrazolyl-4-
dithiocarbonyloxy)propyl]-5,8-dihydro-1,4-
naphthohydroquinone diacetate 14b
General procedure for the synthesis of the pyrazolyloxy
derivatives 13a–c
Purified by CC with hexane/ethyl acetate 1:1 as eluent (28%); oil;
IR cm– 1: 3440–2540 (enolic OH), 3029 (aromatic CH), 2931 (ali-
phatic CH), 1760 (C=O ester); HMRS calc. for C22H24N2O5S2:
460.11282; found: 460.11098.
K2CO3 (0.042 g, 0.304 mmol), 3-methyl-1-phenyl-2-pyrazolin-5-
one (0.030 g, 0.30 mmol) in DMF (5 mL) were stirred 3 h at 458C
under nitrogen atmosphere, followed by the addition of the cor-
responding bromide (0.280 mmol). The mixture was stirred 42 h
at 458C, then poured in water / ice and extracted with CH2Cl2
(4625 mL). The organic layer was washed with 2 N HCl (3620
mL), water (4620 mL) and dried with anhydrous Na2SO4. After
filtration, the solution was concentrated to dryness and the
crude product was purified by CC.
6-[3-(5-Hydroxy-3-methylpyrazolyl-4-
dithiocarbonyloxy)propyl]-5,6,7,8-tetrahydroquinone
diacetate 14c
Purified by CC with hexane/ethyl acetate 1:1 as eluent (25%); oil;
IR cm– 1: 3390–2580 (enolic OH), 3050 (aromatic CH), 2960 (ali-
phatic CH), 1760 (C=O ester); HMRS calc. for C22H26N2O5S2:
462.12848; found: 462.12890.
6-[3-(3-Methyl-1-phenylpyrazolyl-5-oxy)propyl]-1,4-
naphthohydroquinone diacetate 13a
6-[3-(5-Hydroxy-3-methyl-1-phenylpyrazolyl-4-
dithiocarbonyloxy)propyl]-1,4-naphthohydroquinone
diacetate 15a
Purified by CC with hexane/dichloromethane/ethyl acetate 3:6:1
as eluent (31%); oil; IR cm– 1: 3700–2850 (enolic OH), 3049 (aro-
matic CH), 2931 (aliphatic CH), 1762 (C=O ester); HMRS calc. for
C28H26N2O5S2: 534.12848; found: 534.12948.
Purified by CC with hexane/dichloromethane/ethyl acetate 1:8:1
as eluent (33%); oil; IR cm– 1: 3058 (aromatic CH), 2935 (aliphatic
CH), 1764 (C=O ester); HMRS calc. for C27H26N2O5: 458.18428;
found: 458.18367.
6-[3-(3-Methyl-1-phenylpyrazolyl-5-oxy)propyl]-5,8-
dihydro-1,4-naphthohydroquinone diacetate 13b
Purified by CC with hexane/dichloromethane/ethyl acetate 1:8:1
as eluent (37%); oil; IR cm– 1: 3043 (aromatic CH), 2935 (aliphatic
CH), 1761 (C=O ester); HMRS calc. for C27H28N2O5: 460.19994;
found: 460.19993.
6-[3-(5-Hydroxy-3-methyl-1-phenylpyrazolyl-4-
dithiocarbonyloxy)propyl]-5,8-dihydroquinone-1,4-
naphthohydroquinone diacetate 15b
Purified by CC with hexane/dichloromethane/ethyl acetate 3:6:1
as eluent (28%); oil; IR cm– 1: 3700–2820 (enolic OH), 3049 (aro-
matic CH), 2912 (aliphatic CH), 1760 (C=O ester); HMRS calc. for
C28H28N2O5S2: 536.14414; found: 536.14828.
6-[3-(3-Methyl-1-phenylpyrazolyl-5-oxy)propyl]-5,6,7,8-
tetrahydro-1,4-naphthohydroqui-none diacetate 13c
Purified by CC with hexane/dichloromethane/ethyl acetate 1:8:1
as eluent (28%); oil; IR cm– 1: 3065 (aromatic CH), 2938 (aliphatic
CH), 1766 (C=O ester); HMRS calc. for C27H30N2O5: 462.21560;
found: 462.21548.
6-[3-(5-Hydroxy-3-methyl-1-phenylpyrazolyl-4-
dithiocarbonyloxy)propyl]-5,6,7,8-tetrahydro-1,4-
naphthohydroquinone diacetate 15c
Purified by CC with hexane/dichloromethane/ethyl acetate 3:6:1
as eluent (22%); oil; IR cm– 1: 3601–2740 (enolic OH), 3070 (aro-
matic CH), 2983 (aliphatic CH), 1766 (C=O ester); HMRS calc. for
C28H30N2O5S2: 538.15980; found: 538.15942.
General procedure for the synthesis of the
pyrazolyldithiocarbonyloxy derivatives 14a–c and 15a–c
NaOAc (0.134 g, 1.63 mmol), 3-methyl-2-pyrazolin-5-one or 3-
methyl-1-phenyl-2-pyrazolin-5-one (1.60 mmol) in DMF (4 mL)
were stirred 3 h at 458C under nitrogen atmosphere, followed by
the addition of CS2 (0.20 mmol). The mixture was stirred 3 h at
458C and after addition of the corresponding bromide, it was
stirred 14 h at the same temperature. The reaction mixture was
poured into water/ice and extracted with CH2Cl2 (4625 mL). The
organic layer was washed with 2 N HCl (3620 mL), water (4620
mL) and dried with anhydrous Na2SO4. After filtration, the solu-
tion was concentrated to dryness and the crude product was
purified by CC.
Bioactivity
A colorimetric assay using sulforhodamine B (SRB) was adapted
for a quantitative measurement of cell growth and viability, fol-
lowing a previously described method [31, 40]. Cells were seeded
in a 96-well microtiter plates, at 56103 cells/well in aliquots of
195 lL RPMI medium and were allowed to attach to the plate sur-
face by growing in a drug-free medium for 18 h. Afterwards, sam-
ples were added in aliquots of 5 lL (dissolved in DMSO/H2O, 3:7).
After 72 h exposure, the antitumor effect was measured by the
SRB method: cells were fixed by adding 50 mL cold 50% (wt/vol)
trichloroacetic acid (TCA) and incubating for 60 min at 48C.
Plates were washed with deionized water and dried; 100 lL of
SRB solutions (0.4% w/v in 1% acetic acid) was added to each
microtiter well and incubated for 10 min at room temperature.
Unbound SRB was removed by washing with 1% acetic acid.
6-[3-(5-Hydroxy-3-methylpyrazolyl-4-
dithiocarbonyloxy)propyl]-1,4-naphthohydroquinone
diacetate 14a
Purified by CC with hexane/ethyl acetate 1:1 as eluent (28%); oil;
IR cm– 1: 3420–2510 (enolic OH), 3049 (aromatic CH), 2945 (ali-
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598
A. Molinari et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 591–599
Plates were air-dried and bound stain was solubilized with Tris
buffer. Optical densities were read on an automated spectropho-
tometer plate reader at a single wavelength of 490 nm. Data
analyses were generated automatically by LIMS implementation.
Using control OD values (C), test OD values (T), and time zero OD
values (To), the drug concentration that causes 50% growth
inhibition (GI50) was calculated from the equation:
[23] S. Anke, F. Christine, L. Peter, A. Zafar, Tetrahedron 2006,
62, 4800–4806.
[24] B. Testa, in Drug Metabolism, Burger's Medicinal Chemistry
(Ed.: M. E. Wolff), 5th Ed., Wiley, New York 1995, Vol. I, pp.
129–180.
[25] D. A. Williams, in Drug Metabolism Foye's Principles of Medic-
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Lippinkott Williams & Wilkins, Baltimore 2002, pp. 174–
233.
1006[(T–To)/C–To] = 50.
[26] J. Cadwell, S. C. Mitchell, in Metabolic Patways, Comprehen-
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Each value in Table 1 represents the mean from triplicate
determinations. The therapeutic agent and quinine derivative,
doxorubicin, was used as reference drug.
[27] C. P. Balant, E. Doelker, in Metabolic Considerations, Burger's
Medicinal Chemistry (Ed.: M. E. Wolff), 5th Ed., Wiley, New
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