Design, synthesis, and pharmacological evaluation of the aqueous prodrugs of desmethyl anethole trithione with hepatoprotective activity

Article abstract of DOI:10.1016/j.ejmech.2010.03.029

A metabolite-based prodrug strategy to increase the solubility of anethole trithione was reported to facilitate the clinical application of this hepatoprotective agent. Water-soluble analogs of anethole trithione were synthesized via substituting the meth

Full text of DOI:10.1016/j.ejmech.2010.03.029

European Journal of Medicinal Chemistry 45 (2010) 3005e3010
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and pharmacological evaluation of the aqueous prodrugs
of desmethyl anethole trithione with hepatoprotective activity
*
Pei Chen, Yu Luo, Li Hai, Shan Qian, Yong Wu
West China School of Pharmacy, Sichuan University, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A metabolite-based prodrug strategy to increase the solubility of anethole trithione was reported to
facilitate the clinical application of this hepatoprotective agent. Water-soluble analogs of anethole
trithione were synthesized via substituting the methyl group of anethole trithione with the simple
hydrophilic alkylamino group, and subjected to physiochemical, pharmacological and metabolic studies.
The prodrugs displayed increased solubility as well as other physiochemical properties favorable for
parenteral use. Among the analogs synthesized, the compound 5a exhibited best hepatoprotective
activity at the dose of 2.0 mg/kg in mice equal to that of anethole trithione. The in vivo metabolic
investigation demonstrated that the straight-side chain prodrug 5a could convert to desmethyl anethole
trithione in vivo, while the ring-side chain prodrug 5d could not. The hepatoprotective activity of the
prodrugs might result from the active metabolite desmethyl anethole trithione.
Received 18 October 2009
Received in revised form
25 February 2010
Accepted 19 March 2010
Available online 25 March 2010
Keywords:
Design
Synthesis
Prodrugs
Ó 2010 Elsevier Masson SAS. All rights reserved.
Metabolite
Desmethyl anethole trithione
Hepatoprotective activity
1. Introduction
a novel prodrug strategy based on this hypothesis. Expected to
undergo the similar metabolism pathways as ATT in vivo, a series
The dithiolthione compound [1] anethol trithione (ATT),
[5-(p-methoxy-phenyl)-1,2-dithiol-3-thione] (Fig. 1) is consid-
ered to protect the liver against various hepatotoxic substances
via increasing the activity of phase II enzymatic detoxification
systems such as glutathione S-transferase, glutathione reductase,
UDP-glucuronosyltransferase, and quinone reductase [2e4]. It
has been widely used in clinic to treat the symptoms associated
with hepatobiliary dysfunctions without any major adverse
reactions noted [5,6]. Despite the high potency and low toxicity,
the poor water solubility of ATT has long been a concern as it
results in low dissolution and limited dosage forms, and has
hindered the clinical use of ATT. Most studies [7e10] seeking ATT
derivatives with improved water solubility employed the
strategy of introducing hydrophilic groups to either 1,2-dithiole-
3-thione or the benzene ring of ATT, however few candidates
were found to possess hepatoprotective activity superior or equal
to that of ATT. Von R. Gmelin and F. Lagler [11] reported that ATT
was rapidly broken down in animal and human with the
formation of desmethyl ATT (ATX). We hypothesize that the
metabolite ATX or the course of conversion to it is attributable to
the pharmacodynamic activity of ATT, and seek to develop of
of prodrugs was synthesized via coupling a simple hydrophilic
alkylamine hydrochloride salt group or quaternary ammonium
group with metabolite ATX, and was screened in physiochemical
studies in vitro and the pharmacological evaluations in mice
model.
2. Chemistry
In order to efficiently improve the hydrophilicity of 1, the methyl
group was substituted with either ethylamine hydrochloride or
quaternary ammonium groups. These prodrugs were prepared
using the procedures illustrated in Schemes 1 and 2. According to
the variation in the amino groups, two methods (A and B) were
developed to obtain the final water-soluble products starting from
2, which were obtained by demethylation of 1. The attempt to
demethylate 1, however, failed due to the high stability of phenol
ether to concentrated sulfuric acid, concentrated Hydrochloride, or
Lewis acid. The attempt finally succeeded by treatment of anthol-
trithione with pyridine hydrochloride under N2 atmosphere at
215ꢀC through which a satisfactory yield was obtained [11]. Method
A (Scheme 1) was found suitable for the efficient preparation of the
amine salt derivatives of anethol trithione. Compound 3, which is
commercially available, coupled directly with 2 in the presence of
potassium carbonate to yield the amine derivatives, which were
* Corresponding author. Tel./fax: þ86 028 85503666.
E-mail address: wyong@scu.edu.cn (Y. Wu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.029

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P. Chen et al. / European Journal of Medicinal Chemistry 45 (2010) 3005e3010
Fig. 1. ATT, ATX and target compounds 5(aeg).
then quickly converted to the corresponding hydrochlorides 5
(aed) with chloride hydrogeneTHF solution.
the activities of the serum marker enzymes AST and ALT in experi-
mental mice plasma.
However, Method A was not suitable in the preparation of
quaternary ammonium salt prodrugs. Therefore, Method
B
4. Results and discussion
(Scheme 2) was alternatively employed. O-alklation of 2 with
1,2-dibromoethane was first afforded 5-(bromoethoxyl phenyl)-
1,2-dithiol-3-thione 4, then 4 was quaterisated with triethylamine,
pyridine, or N,N-dimethylpyridine to yield the desired compounds.
4.1. Aqueous solubility and stability study in vitro
4.1.1. Aqueous solubility
The solubility of these compounds in water is shown in Table 1.
As expected, the aqueous solubility of the synthetic prodrugs was
significantly higher than that of 1 and 2. Moreover, amine salt
prodrugs obviously had more solubility than quaternary ammo-
nium salt prodrugs.
3. Pharmacology
To characterize the prodrugs, studies in vitro were carried out in
two aspects: aqueous solubility and stability.
The aqueous solubility studies were designed to determine
whether the prodrugs dissolved in aqueous medium and if so, to
what extent.
4.1.2. Stability in mice plasma and phosphate buffer solution
At 37ꢀC, the investigated compounds were stable, and few
decomposed under the experimental pH except at 12.02. At 60ꢀC,
the compounds were stable under the following pH values: 1.10,
4.02, and 6.84. Moreover, the investigated compounds were stable
in mice plasma.
The above studies in vitro demonstrated that these prodrugs
possessed preliminary and favorable physicochemical properties
for parenteral use.
The stability of the prodrugs was studied using the HPLC
method in mice blood plasma and phosphate buffer at pH 1.10, 4.02,
6.84, 9.14, and 12.02, respectively, under 37ꢀC and 60ꢀC.
In addition to 5e as a representative of quaternary ammonium
salt prodrugs, 5a, 5b, 5c, and 5d were also selected for pharmaco-
logical evaluation owing to their good aqueous solubility. The eval-
uation was carried out by intraperitoneally injecting mice with the
synthesized prodrugs at three dose levels separately (1, 2, 4 mg/kg
body weight) and 0.5 h later with CCl4 (5 mL/kg as a 0.2% peanut oil
solution, body weight), a model hepatotoxin to induce experimental
liver injury [12e14]. ATT was employed as the reference drug. The
hepatoprotective effect of these prodrugs was studied by assaying
4.2. Pharmacology
All prodrugs used in the study decreased the alanine trans-
aminase (ALT) and aspartate transaminase (AST) levels in serum
Scheme 1. Method A condition and reagent (a) pyridine hydrochloride 215 ꢀC 3 h (b) compound 5a: 40% NaOH tetrabutylammonium bromide r.t 48 h, compound: 5b 5c 5d K2CO3
DMSO 60 ꢀC 3e6 h (c) dry hydrochloride THF 0e5 ꢀC 5 h.

P. Chen et al. / European Journal of Medicinal Chemistry 45 (2010) 3005e3010
3007
Scheme 2. Method B condition and reagent (d) 1,2-dibromoethane K2CO3 DMSO 60 ꢀC 6 h (e) pyridine 110 ꢀC 10 min.
compared with the CCl4 model group, but the values of ALT and AST
varied in a large range. Compound 5a was the most active, and its
effect on decreasing the level of ALT and AST was equal to that of 1
at the dose of 2.0 mg/kg and 4.0 mg/kg, while 5b, 5c, 5d, and 5e
were much less effective than 1 in all dose levels (Table 2).
We found an interested finding that 5a and 1, which both
possessed a straight chain at the O position, were more effective
than the other prodrugs possessing a ring chain at the same posi-
tion. This seemed to indicate that the structure of the side chains
had some potential relationship with the efficacy of the prodrugs.
However, if the hepatoprotective activity of 1 was associated with
its metabolism process or metabolite 2 itself as we postulated, the
structure of the side chain would be of little concern to the activity
of the prodrugs. Thus the structure-activity relationships above
mentioned seem not to support our postulate when these prodrugs
undergo the similar metabolism pathways as 1.
administered with 5d, which had low hepatoprotective activity,
indicated indirectly the rationality of our strategy.
Moreover, the relationship between the structure of the side
chain and the metabolite gave us a valuable implication for future
research on more potential prodrugs of 2. Extensive side chain
structureemetabolite relationship studies and the further phar-
macokinetics of 5a are currently in progress.
6. Experimental protocols
6.1. Chemistry
TLC was performed using precoated silica gel GF254 (0.2 mm),
while column chromatography was performed using silica gel
(100e200 mesh). The melting point was measured on a YRT-3
melting point apparatus (Shantou Keyi instrument & Equipment
Co. Ltd, Shantou, China). IR spectra were obtained on a Per-
kineElmer 983 (Perkin Elmer, Norwalk, CT, USA). Elemental anal-
yses were performed by Atlantic Microlab, Atlanta, GA, USA. NMR
spectra were taken on a Varian INOVA400 (Varian, Palo Alto, CA,
USA) using CDCl3, d6-DMSO and D2O as solvent. Chemical shifts are
In order to find out whether the major metabolites of these
prodrugs were in accordance with that of 1 in mice, 5a and 5d, as
representatives of straight-chain prodrugs and ring-chain prodrugs
respectively, were selected for metabolism investigation. Mean-
while, an HPLC determination method for 5a, 5d, and 2 in mice
plasma was established.
The results, as illustrated in Figs. 2 and 3, demonstrated that 2
could be detected in mice plasma after injection of 5a, but not in
mice injected with 5d. This finding proved that different activities
between the straight-side chain prodrug 5a and the ring-side chain
prodrug 5d were due to the fact that the former could convert to 2
in vivo, while the latter could not. In summary, the hep-
atoprotective activity of the prodrugs might result from their
capability of converting to 2 in vivo.
Table 2
Effect of 1, 5a, 5b, 5c, 5d, 5e on the Levels of ALT, AST in Serum of Mice (mean ꢁ S.D).
Compound Group
Quantity Doses
(mg/kg)
ALT(IU/L)
AST(IU/L)
Normala 10
0
0
65.3 ꢁ 13.5*
89.8 ꢁ 22.1*
964.3 ꢁ 419.8
269.6 ꢁ 90.4*
265.9 ꢁ 159.4*
407.1 ꢁ 234.0*
Modelb
Lowc
10
10
788.6 ꢁ 336.6
1
1 mg/kg 150.7 ꢁ 69.0
2 mg/kg 112.4 ꢁ 64.2*
4 mg/kg 177.2 ꢁ 94.2*
1 mg/kg 216.5 ꢁ 170.3* 310.9 ꢁ 150.4*
2 mg/kg 109.7 ꢁ 79.8* 215.7 ꢁ 38.4*
4 mg/kg 171.6 ꢁ 118.3* 284.1 ꢁ 167.7*
Middled 10
Highe
10
5a
5b
5c
5d
5e
Low
Middle
High
10
10
10
5. Conclusions
In the present work, a metabolite-based prodrug approach for
water-soluble hepatoprotective analogs of 1 was described on the
hypothesis that 2 played an essential role in hepatoprotective
activity during or after 1 was metabolized into it in vivo according
to Von R. Gmelin and F. Lagler’s study.
The trials in vitro showed that these prodrugs were more
soluble than 1 or 2 in water, and were stable in mice plasma and
acidic buffer solution. These properties render the prodrugs serv-
able by injection.
In vivo, prodrug 5a exhibited good hepatoprotective activity
equal to that of 1 in the screening dose of 2 mg/kg and 4 mg/kg.
It was a potential prodrug candidate for further pharmacokinetics
study.
The metabolism study of 5a, indicating that the good hep-
atoprotective activity of the prodrugs might result from its capa-
bility of converting to 2, supported directly our prodrug strategy.
Furthermore, the fact that no metabolite 2 was found in mice
Low
Middle
High
10
10
10
1 mg/kg 595.4 ꢁ 342.1
2 mg/kg 468.0 ꢁ 183.1
4 mg/kg 457.7 ꢁ 219.8
851.7 ꢁ 534.5
552.5 ꢁ 187.2
824.9 ꢁ 585.5
501.2 ꢁ 308.8
582.9 ꢁ 169.2
607.8 ꢁ 191.7
Low
Middle
High
10
10
10
1 mg/kg 416.6 ꢁ 124.4
2 mg/kg 535.9 ꢁ 155.9
4 mg/kg 561.5 ꢁ 252.5
Low
Middle
High
10
10
10
1 mg/kg 169.3 ꢁ 70.9*
920.6 ꢁ 519.6
2 mg/kg 259.6 ꢁ 86.7*
416.8 ꢁ 242.9*
4 mg/kg 382.9 ꢁ 174.6* 423.9 ꢁ 103.8*
Low
Middle
High
10
10
10
1 mg/kg 426.1 ꢁ 265.4
2 mg/kg 630.1 ꢁ 506.1
4 mg/kg 685.0 ꢁ 248.6
559.2 ꢁ 328.5
617.8 ꢁ 448.9
784.3 ꢁ 323.8
*Compared with model group P < 0.01.
a
The mice of this group were intraperitoneally injected with saline solution
alone.
b
The mice of this group were intraperitoneally injected with CCl4 at a dose of
5 mL/kg as a 0.2% peanut oil solution.
c
The mice were intraperitoneally injected with the investigated compounds at
a dose of 1 mg/kg, and 0.5 h later with CCl4 at a dose of 5 mL/kg as a 0.2% peanut oil
solution.
d
The mice were intraperitoneally injected with the investigated compounds at
Table 1
a dose of 2 mg/kg, and 0.5 h later with CCl4 at a dose of 5 mL/kg as a 0.2% peanut oil
solution.
The Aqueous Solubility of the Compounds at 25 ꢁ 1 ꢀC.
e
The mice were intraperitoneally injected with the investigated compounds at
Drug
1
2
5a
5b
5c
5d
5e
5f
5g
a dose of 4 mg/kg, and 0.5 h later with CCl4 at a dose of 5 mL/kg as a 0.2% peanut oil
solution.
mg/mL
<0.01
<0.13
>20
>20
>20
>20
2.3
2.8
3.5

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P. Chen et al. / European Journal of Medicinal Chemistry 45 (2010) 3005e3010
15 mmol), and dichloromethane (20 mL) were then added and
stirred for 48 h at room temperature. The reaction mixture was
poured into water (100 mL), and the aqueous layer was extracted
with dichloromethane (3 ꢂ 50 mL). The organic layers were
combined and washed with water (50 mL) and brine (50 mL), and
then dried (Na2SO4). After filtration, the filtrate was evaporated,
and the remaining residue was subjected to column chromatog-
raphy using petroleumeacetone (10:1) as eluent. The base of 5a
was obtained as brown oil (2.0 g, 66.2%).
Dry hydrochloride gas was continually added to the brown oil
in anhydrous THF (20 mL) under constant stirring until the
precipitation stopped increasing. The reaction mixture was
filtered, and the cake was dried in a vacuum desiccator to yield 5a
(2.1 g, 93.6%), m.p. 190e192ꢀC (found: C, 46.79; H, 4.83; Cl, 10.63;
N, 4.19; O, 4.79; S, 28.83. Calcd for C13H16ClNOS3: C, 46.76; H, 4.83;
Cl, 10.62; N, 4.19; O, 4.79; S, 28.81%) 1H NMR D2O: 7.63 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.36 (s, 1H, ¼ CH), 7.07 (d, 2H, J ¼ 8.4 Hz, AreH),
4.48 (t, 2H, J ¼ 4.4 Hz, OCH2CH2), 3.69 (t, 2H, J ¼ 4.4 Hz, OCH2CH2),
Fig. 2. The Concentration of 5a, 2 in Mice Plasma after the Injection of 5a.
expressed in d(ppm), with tetramethylsilane (TMS) functioning as
3.06 (s, 6H, -CH3ꢂ2). IR (KBr pellets cmꢃ1
) y 3039, 3009, 2952,
the internal reference. The following abbreviations were used:
s ¼ singlet, d ¼ doublet, t ¼ triplet, and m ¼ multiplet; coupling
constants (J) were expressed in Hz. Mass spectra were recorded on
an Agilent 1946B ESI-MS instrument (Agilent, Palo Alto, CA, USA).
Anethol trithione (ATT) was purchased from ChengDu GuoJia
United Pharmaceutical Ltd. (Chengdu, P. R. China), while 3(aed)
was purchased from J&K Scientific Ltd. (Beijing China).
2690, 2518, 2481, 1604, 1575, 1481, 1424, 1294, 1259, 1202, 1060,
1024; ESIeMS: 298 (Mþþ1).
6.1.3. General procedure for the synthesis of 5b, 5c, and 5d
To the mixture of 2 (0.1 g, 0.44 mmol), 3 (0.66 mmol), and K2CO3
(0.304 g, 2.2 mmol), DMSO (3 mL) was added under stirring and
argon protection, which was heated to 60ꢀC and reacted for 2 h.
After completion of the reaction (monitored by TLC), the reaction
mixture was poured into cold water (25 mL) and extracted with
dichloromethane (3 ꢂ 15 mL). The combined organic layers were
washed with water (1 ꢂ 5 mL) and brine (1 ꢂ 5 mL), and then dried
(Na2SO4). After filtration, the filtrate was evaporated under reduced
pressure. The residue was subjected to column chromatography
using petroleumeacetone (10:1) as eluent. The bases of 5b, 5c, or
5d were obtained as brown oil. Compound 5b: (0.096 g, 64%), 5c:
(0.122 g, 85.4%), 5d: (0.102 g, 68.4%).
6.1.1. 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (2,ATX)
Pyridine hydrochloride (68.0 g, 590 mmol) was added to 1
(25.0 g, 100 mmol) in a dry flask, mixed and then heated to melt at
215ꢀC under argon protection for 20 min. After being cooled to
100ꢀC, warmed water (200 mL) was added and hot-filtered. The
cake was placed in a beaker, and 10%NaOH (300 mL) was added.
This was stirred for 4 h, filtered, the cake dissolved in water (3 L),
then adjusted to pH 2 with concentrated hydrochloride. The red
precipitation was filtered and washed to neutral using water, then
dried in a vacuum desiccator to yield a yellow solid (18.0 g, 76.5%),
m.p. 172e174 (found: C, 47.78; H, 2.67; O, 7.08; S, 42.53. Calcd for
C9H6OS3: C, 47.76; H, 2.67; O, 7.07; S, 42.50%); 1H NMR d6-DMSO:
10.47 (br, 1H, -OH), 7.84 (d, 2H, J ¼ 8.8 Hz, AreH), 7.70 (s, 1H, ¼ CH),
6.88 (d, 2H, J ¼ 8.8 Hz, AreH).
Dry hydrochloride gas was added to brown oil in anhydrous THF
(20 mL) and constantly stirred until the precipitation stopped
increasing. The reaction mixture was filtered, and the cake was dried
in a vacuum desiccator to yield the corresponding hydrochloride salt.
6.1.3.1. 5-(4-(2-morpholinoethoxy)phenyl)-3H-1,2-dithiole-3-thione
hydrochloride(5b). Obtained from 2 (0.1 g, 0.44 mmol) and 3b
(0.122 g, 0.66 mmol) as yellow solid. (0.090 g, 57.8%), m.
p.210e212ꢀC (found: C, 47.93; H, 4.83; Cl, 9.44; N, 3.73; O, 8.52; S,
25.62. Calcd for C15H18ClNO2S3: C, 47.92; H, 4.83; Cl, 9.43; N, 3.73; O,
8.51; S, 25.59%) 5b in base form: 1H NMR CDCl3: 7.61 (d, 2H,
J ¼ 8.8 Hz, AreH), 7.40 (s, 1H, ¼ CH), 6.98 (d, 2H, J ¼ 8.8 Hz, AreH),
4.18 (t, 2H, J ¼ 5.6 Hz, ArOCH2CH2), 2.85 (t, 2H, J ¼ 5.6 Hz,
ArOCH2CH2), 3.75 (t, 4H, J ¼ 4.8 Hz, CH2OeCH2), 2.59e2.62 (m, 4H,
6.1.2. 5-(4-(2-(dimethylamino)ethoxy)phenyl)-3H-1,2-dithiole-3-
thione hydrochloride (5a)
40%NaOH (20 mL) was added to 2 (2.3 g, 10 mmol) in a flask and
mixed for 30 min; tetrabutylammonium bromide (0.65 g, 2 mmol),
2-chloro- N,N-dimethyl eethanamine hydrochloride (2.16 g,
NCH2CH2OCH2CH2); IR (KBr pellets cmꢃ1
) y 3033, 2976, 2930, 2861,
2654, 2578, 2459, 1603, 1479, 1422, 1288, 1250, 1195, 1080, 1027;
ESIeMS: 340 (Mþþ1).
6.1.3.2. 5-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-3H-1,2-dithiole-3-
thione hydrochloride (5c). Obtained from 2 (0.1 g, 0.44 mmol) and
3c (0.112 g, 0.66 mmol) as yellow solid. (0.118 g, 74.1%), m.
p.216e218ꢀC (found: C, 50.09; H, 5.04; Cl, 9.86; N, 3.89; O, 4.44; S,
26.73. Calcd for C15H18ClNOS3: C, 50.05; H, 5.04; Cl, 9.85; N, 3.89; O,
4.44; S, 26.72%); 1H NMR D2O: 7.76(d, 2H, J ¼ 8.4 Hz, AreH), 7.53
(s, 1H, ¼ CH), 7.14(d, 2H, J ¼ 8.4 Hz, AreH), 4.50(t, 2H, J ¼ 4.4 Hz,
OCH2CH2), 3.76(t, 2H, J ¼ 4.4 Hz, OCH2CH2), 3.84e3.80(m, 2H,
CH2NCH2), 3.27e3.33(m, 2H, CH2NCH2), 2.10e2.30(m, 4H,
NCH2CH2CH2); IR (KBr pellets cmꢃ1
) y 3063, 2946, 2877, 2668,
2598, 2478, 1598, 1489, 1420, 1282, 1261, 1187, 1056, 1023. ESIeMS:
Fig. 3. The Concentration of 5d, 2 in Mice Plasma after the Injection of 5d.
324 (Mþþ1).

P. Chen et al. / European Journal of Medicinal Chemistry 45 (2010) 3005e3010
3009
6.1.3.3. 5-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-3H-1,2-dithiole-3-
thione hydrochloride (5d). Obtained from (0.1g, 0.44 mmol) and 3d
(0.121g, 0.66 mmol) as yellow solid. (0.105 g, 63.4%), m.p.215e217ꢀC
(found: C, 51.41; H, 5.39; Cl, 9.49; N, 3.75; O, 4.28; S, 25.74. Calcd for
C16H20ClNOS3: C, 51.38; H, 5.39; Cl, 9.48; N, 3.75; O, 4.28; S, 25.72%);
1H NMR D2O: 7.66 (d, 2H, J ¼ 8.8 Hz, AreH), 7.39 (s, 1H, ¼ CH), 7.08
(d, 2H, J ¼ 8.0 Hz, AreH), 4.48 (s, 2H, OCH2CH2), 3.14 (t, 2H,
J ¼ 12.4 Hz, OCH2CH2), 3.63e3.70 (m, 4H, CH2NCH2), 1.80e2.05 (m,
6.1.5.3. N,N,N-triethyl-2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
ethanaminium bromide(5g). Obtained from 4 (0.333 g, 1.0 mmol)
and Et3N (20 mL) as yellow solid. (0.305 g, 70.1%), m.p. 226e228ꢀC
(found: C, 47.01; H, 5.58; Br, 18.41; N, 3.22; O, 3.68; S, 22.16. Calcd
for C17H24BrNOS3: C, 46.99; H, 5.57; Br, 18.39; N, 3.22; O, 3.68; S,
22.14%); 1H NMR d6-DMSO: 7.94 (d, 2H, J ¼ 8.8 Hz, AreH), 7.81
(s, 1H, ¼ CH), 7.14 (d, 2H, J ¼ 8.8 Hz, AreH), 4.50 (t, 2H, J ¼ 4.4 Hz,
ArOCH2CH2), 3.71 (t, 2H, J ¼ 4.4 Hz, ArOCH2CH2), 3.30e3.40 (m,
6H, NCH2CH2CH2CH2); IR (KBr pellets cmꢃ1
)
y
3063, 2952, 2875,
6H, CH2CH3ꢂ3), 1.23 (m, 9H, CH2CH3ꢂ3); IR (KBr pellets cmꢃ1
) y
2730, 2489, 1602,1489, 1420, 1288, 1260, 1191, 1063, 1021; ESI-MS:
3043, 2986, 1603, 1484, 1421, 1291, 1264, 1202, 1024; ESI-MS: 354
338 (Mþþ1)
(Mþ).
6.1.4. 5-(4-(2-bromoethoxy)phenyl)-3H-1,2-dithiole-3-thione (4)
To the mixture of 2 (0.226 g, 1.0 mmol), 1,2-dibromoethane
(0.544 g, 3.0 mmol), and K2CO3 (0.690 g, 5.0 mmol), DMSO (3 mL)
was added under constant stirring and argon protection. It was
then heated to 60ꢀC and reacted for 2 h. After completion of the
reaction, (monitored by TLC), the reaction mixture was poured into
cold water (25 mL) and extracted with dicholoroethane
(3 ꢂ 20 mL). The combined organic layers were washed with water
(1 ꢂ 15 mL) and brine (1 ꢂ 15 mL), then dried (Na2SO4). After
filtration, the filtrate was concentrated under reduced pressure,
and the residue was subjected to column chromatography using
petroleumeacetone (10:1) as eluent. Compound 4 was obtained
as brown solid. (0.256 g, 76.9%), m.p.134e136 ꢀC (found: C, 39.65;
H, 2.72; Br, 23.98; O, 4.80; S, 28.87. Calcd for C11H9BrOS3: C, 39.64;
H, 2.72; Br, 23.97; O, 4.80; S, 28.86%); 1H NMR CDCl3: 7.63(d, 2H,
J ¼ 8.8 Hz, AreH), 7.40(s, 1H, ¼ CH), 7.00(d, 2H, J ¼ 8.8 Hz, AreH),
4.36(t, 2H, J ¼ 6.0 Hz, ArOCH2CH2), 3.68(t, 2H, J ¼ 6.0 Hz, BrCH2CH2).
6.2. Aqueous solubility and stability
6.2.1. Aqueous solubility
Each prodrug measuring 200.0 mg in accurate weight was
placed in a 50 mL beaker. Then 10 mL water was added and
exposed to ultrasonic oscillation for 5min and stirred for 24 h in
a 25ꢀC constant temperature water bath. It was centrifuged at 10
000 rpm for 10 min, and the supernatant was filtered through
a microporous membrane and injected to the HPLC system for
determination.
6.2.2. Stability
The stability in phosphate buffer solution was investigated at
five pH values: 1.10, 4.02, 6.84, 9.14, and 12.02 with two
temperature degrees: 37ꢀC and 60ꢀC. The concentration was
determined by HPLC in the following intervals: 0, 1, 2, 4, 8, 16,
and 24 h.
The stability in mice plasma was investigated using the
6.1.5. General procedure for the synthesis of 5e, 5f, and 5g
The mixture of 4 (0.333 g, 1.0 mmol) and excess pyridine, DMAP,
or Et3N was heated to 110ꢀC then stirred for 10 min or 6 h. After
being cooled to room temperature, the precipitation was filtered
and dried in the vacuum desiccator to yield yellow 5e, 5f, and 5g,
respectively.
following steps: precisely 1.0 mL water solution (50 mg/mL) of the
target compound was added into 5 mL mice plasma at 37ꢀC. After
mixing, it was kept in a 37 ꢁ 1ꢀC constant water bath, and the
200 mL sample was withdrawn at the following time points: 0, 1, 2,
4, 8, and 12 h. These were added to 400
mL acetonitrile, mixed for
5min, then centrifuged at 10 000 rpm for 5 min. The 20
m
L
supernatant was then injected into the HPLC system for
determination.
6.1.5.1. 1-(2-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl)pyr-
idinium bromide(5e). Obtained from 4 (0.333 g, 1.0 mmol) and
pyridine (20 mL) as yellow solid. (0.312 g, 75.5%), m.p. 160ꢀC
(found: C, 46.63; H, 3.42; Br, 19.39; N, 3.40; O, 3.88; S, 23.34 Calcd
for C16H14BrNOS3: C, 46.60; H, 3.42; Br, 19.38; N, 3.40; O, 3.88; S,
23.33%) 1H NMR d6-DMSO: 9.15 (d, 2H, J ¼ 6.8 Hz, N¼CHCH), 8.66
(t, 1H, J ¼ 8.0 Hz, N¼CHCHC-H), 8.20 (dd, 2H, J1 ¼ 6.8 Hz, J2 ¼ 7.6 Hz,
N¼CHCH), 7.89 (d, 2H, J ¼ 8.8 Hz, AreH), 7.78 (s, 1H, ¼ CH), 7.08
(d, 2H, J ¼ 8.8 Hz, AreH), 4.63 (t, 2H, J ¼ 5.2 Hz, ArOCH2CH2), 5.08
6.3. Pharmacology
6.3.1. Drug and chemical agents
Compounds 5a, 5b, 5c, 5d, and 5e were synthesized and
identified in our laboratory. ATT was purchased from ChengDu
GuoJia United Pharmaceutical Ltd. (Chengdu, P. R. China). CCl4
and peanut oil were purchased from J&K Scientific Ltd. (Beijing
China).
ALT and AST were investigated by the West China Medical
Centre of Sichuan University. Data were expressed as
mean ꢁ standard deviation and were analyzed using SPSS version
11.5 software.
(t, 2H, J ¼ 4.8 Hz, ArOCH2CH2); IR (KBr pellets cmꢃ1
) y 3055,
2926,2880, 1633, 1603, 1486, 1421, 1289, 1263, 1201, 1025. ESIeMS:
332 (Mþ).
6.1.5.2. 4-(dimethylamino)-1-(2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)
phenoxy)ethyl) pyri -dinium bromide(5f). The mixture of 4 (0.333 g,
1.0 mmol) DMAP (0.244 g, 2.0 mmol) and DMF (10 mL) was heated
to 100ꢀC and stirred for 6h. DMF was evaporated under reduced
pressure, then 10 mL petroleum was added to the residue and
stirred overnight in order to precipitate. The mixture was filtered,
and the cake was dried in a vacuum desiccator to yield yellow
solid. (0.320 g, 70%), m.p. 152e154ꢀC (found: C, 47.50; H, 4.20; Br,
17.55; N, 6.16; O, 3.51; S, 21.13 Calcd for C18H19BrN2OS3 C, 47.47; H,
4.20; Br, 17.54; N, 6.15; O, 3.51; S, 21.12%); 1H NMR d6-DMSO: 8.34
(d, 2H, J ¼ 7.6 Hz, CH-N¼CH), 7.89 (d, 2H, J ¼ 8.8 Hz, CH ¼ CH-
N¼CHeCH), 7.76 (s, 1H, ¼ CH), 7.05e7.07 (m, 4H, AreH), 4.61
(t, 2H, J ¼ 4.4 Hz, ArOCH2), 4.47 (t, 2H, J ¼ 4.4 Hz, ArOCH2CH2); IR
6.3.2. Test animals
Adult male Kunming mice weighing 20e22 g were obtained
from the animal center of Sichuan University. The animals were left
for two days to acclimatize to animal room conditions and were
maintained on standard pellet diet and water ad libitum. Food was
withdrawn on the day before the experiment, but free access to
water was allowed. Since the experiment could be completed
within 48 h, there was no significant change in the mices’ body
weight during the experiment. All animals received human care,
and the study protocols complied with the guidelines of the animal
center of Sichuan University. Throughout the experiments, the
animals were handled according to the international ethical
guidelines for the care of laboratory animals.
(KBr pellets cmꢃ1
1025. MS: 375 (Mþ).
) y 3045, 1651, 1602, 1482, 1423, 1284, 1250, 1180,

3010
P. Chen et al. / European Journal of Medicinal Chemistry 45 (2010) 3005e3010
6.3.3. CCl4-induced acute liver damage model in mice and
investigated compounds treatment
acetonitrile (200:200:150 (v/v)) was used as the mobile phase at
a flow rate of 1.0 mL/min. The detection was performed at a wave-
The mice were randomly divided into five groups of ten. In the
normal control group, the mice were intraperitoneally injected
with saline solution alone. In the model group, the mice were
intraperitoneally injected with CCl4 at a dose of 5 mL/kg as a 0.2%
peanut oil solution. The low dose group of mice was intraperito-
neally injected with the investigated compounds at a dose of
1 mg/kg, while the middle dose group was intraperitoneally
injected with the investigated compounds at a dose of 2 mg/kg.
The high dose group was intraperitoneally injected with the
investigated compounds at a dose of 4 mg/kg.
length of 354 nm after an injection volume of 20 mL.
6.4.4. Preparation of calibration standards and quality control
samples
The calibration standards of 5a, 5d, and 2 consisting of six points
ranging from 0.05 to 31.11 mg/mL were prepared by adding appro-
priate amounts of the standard solution into blank plasma. Quality
control (QC) samples were prepared by adding the proper 5a, 5d,
and 2 into blank plasma in order to create concentrations in three
levels.
The mice in the low, middle, and high dose groups were
subsequently injected intraperitoneally with CCl4 at a dose of
5 mL/kg as a 0.2% peanut oil solution 0.5 h after pretreatment with
the investigated compounds.
Sixteen hours after the introduction of CCl4, all the mice were
sacrificed, and blood samples withdrawn from the orbital sinus
were collected and allowed to clot; they were then centrifuged at
3 500 rpm for 10 min. The serum was separated and used for the
assay of ALT and AST.
6.4.5. Sample preparation
Precisely 200
nitrile. After being laid down in the ultrasonic instrument for 5 min
and being centrifuged at 10 000 rpm for 5 min, 20 L aliquots of the
mL of mice plasma was added into 400 mL aceto-
m
supernatant were injected into HPLC for the assay.
6.4.6. Method validation
The selectivity of the method was checked for interference from
plasma. The standard curve consisting of six points ranging from
6.4. Metabolism study
0.05 to 31.11 mg/mL was developed. Quality control samples as LQC,
MQC, and HQC were used to determine the intra- and inter-day
precision and accuracy of the assay. Drug concentrations in the
plasma samples along with the same day-standard curve samples
were calculated using a linear equation.
6.4.1. Chemical agents and animal
Compounds 5a, 5d, and 2 were synthesized and identified in our
laboratory.
The animals were handled in accordance with the procedures
detailed in Section 6.3.2.
References
6.4.2. Animal test
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The mice were randomly divided into ten groups of four and were
intraperitoneally injected with the investigated compounds
respectivelyatadoseof10.00mg/kgbodyweight. Theblood samples
(0.5 mL) withdrawn from the orbital sinus were collected into
heparinized tubes at the following post-dose intervals: 0.02, 0.08,
0.25, 0.5, 0.75,1,1.5, 3, 5, and 8 h. Plasma was separated immediately
through centrifugation (3 000 rpm, 5 min) and then processed in
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6.4.3. Instrumentation and chromatography
The Waters liquid chromatographic system employed was an
LC-10A liquid chromatographic system (Shimadzu Japan). The
analysis was carried out on
(200 mm 4.6 mm,
a
m
SinoChrom ODS-C18 column
ꢂ
5
m), thermostated at 25ꢀC,
[14] Chun-Kwan Wong, Vincent E.C. Ooi, Put O. Ang Jr., Hydrobiologia 512 (2004)
267e270.
0.05 mol Lꢃ1KH2PO4 (adjust pH to 3 with H3PO4): methanol: