Design synthesis and biological evaluation of lipophilic analogs of anethol trithione

Article abstract of DOI:10.2174/1570180811007010747

16 ADT carboxylate esters were prepared and tested for their chemical characteristics, stability, bioavailability, potency and toxicity. Considering the good bioavailability, 3a was selected for the pharmacology test, and the result showed that the potency of 3a was remarkably higher than that of ATT. Additionally, 3a was also chosen for the acute toxicity test. The result indicated that the prodrug 3a was more safer than ATT. The study suggested the feasibility to improve the bioavailability of ATT by using prodrug strategy.

Full text of DOI:10.2174/1570180811007010747

Letters in Drug Design & Discovery, 2010, 7, 747-753
747
Design, Synthesis and Biological Evaluation of Lipophilic Analogs of
Anethol Trithione
Xiao-Cen Li1, Wei Fan1, Li Hai1, Shan Qian1, Qui-Qi Xiao2 and Mei Guan*,2
1Key Laboratory of Drug Targeting, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041,
P.R. China
2West China Hospital, Sichuan University, No. 37, GuoXue road, Chengdu, Sichuan 610041, P.R. China
Received May 12, 2010: Revised July 30, 2010: Accepted August 11, 2010
Abstract: 16 ADT carboxylate esters were prepared and tested for their chemical characteristics, stability, bioavailability,
potency and toxicity. Considering the good bioavailability, 3a was selected for the pharmacology test, and the result
showed that the potency of 3a was remarkably higher than that of ATT. Additionally, 3a was also chosen for the acute
toxicity test. The result indicated that the prodrug 3a was more safer than ATT. The study suggested the feasibility to im-
prove the bioavailability of ATT by using prodrug strategy.
Keywords: Synthesis, Anethol trithione, Lipophilicity, Bioavailability, Prodrugs.
INTRODUCTION
results and considering the structural characteristics of the
active constituent ADT, 16 ADT carboxylate esters prodrugs
(Fig. (1)) were prepared by means of esterification with ADT
in order to improve the biological availability of this active
drug. The purpose of this study was to investigate the stabil-
ity, bioavailability and toxicity of the designed and synthe-
sized ADT analogs.
Anethol trithione (ATT), a sulfur heterocyclic compound
first discovered in cruciferous vegetables, is a relatively new
chologogue. ATT and its analogues have been studied as
sialagogue, antischistosomial, choleretic and hepatic protec-
tant [1, 2], which are widely used in clinic for treating symp-
toms associated with hepatobiliary dysfunctions without any
major adverse reactions being noted [3]. ATT can increase
human cholinergic, adrenergic responsiveness and prevent
the up-regulation in muscarinic acethylcholine receptor den-
sity. Also it is a potent inducer of phase II enzymatic detoxi-
fication systems and can increase the activity of glutathione
reductase and glutathione peroxidase, improve the level of
glutamyl cysteine synthetase and glutathione (GSH) which
can deactivate many chemicals by conjugation, resulting in
the enhancement of hepatic cell activity and bilifaction [4,
5]. Besides the above effects, Zhang and co-works found that
ATT analogues could prevent cellular superoxidant damages
and carcinogenesis produced [6].
RESULT AND DISCUSSION
Chemistry
As prodrugs of ADT, the designed compounds should
simultaneously achieve the following features: 1. great lipo-
philicity, 2. high conversion efficiency of the prodrug to the
active molecule ADT, 3. good biological availability of the
active drug. In order to improve the fat-solubility of ADT,
the methyl group of ATT was removed to afford active form
ADT and then coupled with various aromatic or aliphatic
acid via ester linkage at -O position of ADT. We hoped that
the lipophilic group of these acids were able to enhance the
lipophilicity of the prodrug and caused high uptake rate in
intestinal tract. The ester linkage of these lipophilic groups
would make sure that these compounds could be swiftly hy-
drolyzed by the blood esterase and release active drug ADT.
These analogs were prepared by the procedures illustrated in
Scheme 1. We performed the first step by treatment the ATT
with pyridine hydrochloride under N2 atmosphere at 215 oC
with satisfied yield (84.5%) to afford ADT (2). Then com-
pound 2 was coupled with different acid groups in the pres-
ence of DCC and DMAP to afford 3a–3p with yields
43.8%-96.0%. In total, 16 new compounds 3a–3p were pre-
Previous research described that ATT was firstly metabo-
lized into 4-hydroxy-anethole trithione (ADT) in plasma and
then ADT was conjugated with sulfuric acid or glycuronic
acid to excrete. The primary pharmacodynamic active con-
stituent was ADT, the demethyl metabolite of ATT, since
ATT lose methyl group swiftly in liver after administration
[7]. However, the marketed dose form of ATT was tabella
which caused the bad absorption of the drug in intestinal
tract. Besides this, it must be noted that the rate of metabo-
lism of ATT was comparatively low which limited the
pharmacodynamic effect of ADT. Lots of efforts had been
done to improve the bioavailability of ATT such as changing
the dosage form [8], but no report has been published report-
ing the structural modification of ATT to increase the
bioavailbiity in vivo. Inspired by those previous research
1
pared, their structures were confirmed by IR, HNMR, ESI-
MS. In general, IR spectra showed the ArC=C peak at 1448–
1608 cm–1, the C=S stretching vibrations at 1164–1189 cm–1,
and the C-O stretching vibrations at 1012–1049 cm–1. In the
nuclear magnetic resonance spectra (1H-NMR), the signals
of the respective protons of the synthesized compounds were
verified on the basis of their chemical shifts, mutiplicities,
and coupling constants. The spectra showed the alkene
(C=CHCS) proton as a singlet at7.25–7.44 ppm.
*Address correspondence to this author at the West China Hospital, Sichuan
University, No. 37, GuoXue road, Chengdu, Sichuan 610041, P.R. China;
Tel: 86-28-85503235; Fax: 86-28-85503235; E-mail: tgxx903@163.com
1570-1808/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

748 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 10
Li et al.
S
S
S
S
O
O
O
S
S
R
1
ATT
ADT
3a
3gR=Heptadecyl
3mR=2,2-Diphenylethyl
R=Methyl
3bR=Ethyl
S
3n
R=3,5-Dimethoxyphenyl
3hR=Tertiary butyl
S
3o R=1,2,3,4-Tetrahydronaphthyl
3p R= 5,6,7,8-Tetrahydronaphthyl
3cR=Propenyl 3i R=2-Phenylvinyl
HO
3j R=2-Fluoro-phenyl
3kR=4-Chloro-phenyl
R=Hydrocinnamyl
3d
R=Isopropyl
3e
R=Phenyl
S
3l
2
3f R=Benzyl
Fig. (1). ATT, ADT and ADT carboxylate esters 3a-3p.
O
S
S
S
S
S
a
b,
R-COOH
S
HO
O
R
O
S
S
S
3a-3p
3g R=Heptadecyl
3l R=Hydrocinnamyl
2
1
3a R=Methyl 3b R=Ethyl 3c R=Propenyl 3d R=Isopropyl 3e R=Phenyl 3f R=Benzyl
3h R=Tertiary butyl
3m R=2,2-Diphenylethyl
3i R=2-Phenylvinyl
3j R=2-Fluoro-phenyl
3k R=4-Chloro-phenyl
3n R=3,5-Dimethoxyphenyl 3o R=1,2,3,4-Tetrahydronaphthyl
3p R= 5,6,7,8-Tetrahydronaphthyl
o
Scheme 1. The synthetic route of 3a-p. Regents and condition: (a) pyridine hydrochloride, 215 C, 3h; (b) DCC, DMAP, CH2Cl2, 25 oC,
overnight.
pH Stability in Aqueous Buffers
9.5 buffer. Generally, the stability of these compounds was
determined by the pH deviation from neutral value, the more
deviation from neutral value the pH had, the more unstable
these compounds were.
In order to achieve hepatic protectant effects, the ADT
moiety of these analogs should be released from those forms
in plasma by hydrolysis of esterase. Therefore, 3a, 3d, 3e, 3f
and 3i were selected to the stability test in solutions under
different pH and plasma at 37 oC in vitro.
Plasma Stability In Vitro
The results of the test in aqueous buffer solutions was
listed in Fig. (2), which showed that these compounds are
moderately stable in physiological conditions. These com-
pounds were partially hydrolyzed to parent drug at pH 7.4,
indicating that the compounds will undergo hydrolysis in the
physiological environment. The compounds appeared to be
unstable in pH 1.0, and comparatively stable in pH 6.0 and
To determine the possibility of esterase hydrolysis, the
analogs synthesized were incubated with mice plasma at
37 C (Table 1). The result showed that all the test com-
pounds were unstable in plasma, as was expected, so as to
swift release the active drug ADT after absorbed in intestinal
tract. From the five compounds, 3a and 3e could be totally
hydrolyzed by the esterase in plasma in 180 minutes, how-
o
Fig. (2). Stability of ADT analog 3a, 3d, 3e, 3f, 3i under different pH at 37 oC (remained concentration%).

Design, Synthesis and Biological Evaluation of Lipophilic Analogs
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 10 749
Table 1. Stability of ADT Analog 3a, 3d, 3e, 3f, 3i in Mice Plasma at 37 oC (Remained Concentration%)
Compound
Time (min)
60
0
30
120
180
3a
3d
3e
3f
100.0
100.0
100.0
100.0
100.0
13.2
17.4
14.2
14.5
16.1
7.5
9.6
1.8
4.2
2.7
4.8
5.4
nda
1.8
nd
8.7
10.1
9.1
1.23
2.3
3i
and: not detected.
ever, we found that the hydrolytic process of 3d,3f and 3i
were slower compared with 3a and 3e, and these analogs
could not get complete hydrolysis in test time. This might
contribute to the steric hinderance of coupled acid in the ana-
logs that the esterase could not reach the substrate and hy-
drolyzed the compounds effectively.
ach (pH 1-3), of which the structure possessed large polarity
and could not be effectively absorbed. Additionally, the
ADT concentrations of the compounds 3a, 3d, 3f, 3i in
plasma were higher than that of ADT during 180 as expect.
The Cmax of 3a, 3d, 3f and 3i were 660.6μg/ml, 236.1μg/ml,
212.1mg/ml and 453.3μg/ml, which were much higher than
that of ATT (48.4μg/ml). Pharmacokinetic parameters of
ADT in mice after i.g. administration of the prodrugs and
ATT were reported in Table 3. The results in Table 3 showed
that in the case of orally administrating 3a, 3d, 3f, 3i, AUC0–t
in plasma were significantly higher than that after the ad-
ministration of ATT. The REs were enhanced to 4.65, 1.82,
3.22, and 1.57 times that of ATT for compounds 3a, 3d, 3f,
and 3i, and the CEs were increased to 13.65, 4.88, 4.38, and
9.77 times that of ATT, respectively. It was notable that the
AUC0–t and Cmax of 3a was much higher than that of other
test compounds. These data indicated that more prodrug 3a
were transferred to plasma and could be easily hydrolysed in
plasma to the active ingredient ADT and render better
bioavailability.
In Vivo Evaluation
To understand the in vivo behavior of the designed ana-
logs, we assessed the plasma pharmacokinetics of the syn-
thesized compounds and the control (ATT) in mice. Com-
pound 3a, 3d, 3e, 3f, 3i and ATT were subjected to bioavail-
ability evaluation as these compounds were expected to pos-
sess better lipophilicity than ATT. Freshly prepared solutions
of these compounds in peanut oil were administrated to mice
by intragastric injection in a single dose equivalent to
250mg/kg of ATT. At specific time intervals the animals
were sacrificed, and then blood samples were collected for
analysis by HPLC method which was established and ap-
plied to investigate the major metabolite (ADT) of these
compounds and the results was outlined in Table 2.
Pharmacology Investigation
In this assay, we found that all the compounds could not
be detected at the first time interval (5 minute), which may
be due to the high metabolic rate of them in plasma. The
results showed that all compounds except 3f could reach the
peak values of blood drug concentration of ADT more swift
than ATT (120 min). This could be caused by the better
lipid-solubility of the compounds than ATT, which could
enhance the uptake rate in mice intestine. From the in vitro
stability test, decomposition of 3f was quit fast in pH 1.0,
and this could explain the low bioavailability of 3f in vivo.
Compound 3f might be quickly hydrolyzed to ADT in stom-
3a was selected for pharmacology evaluation in view of
the good bioavailability. Acute liver lesion induced by CCl4
was the most classic experimental model of liver lesion [9].
The hepatoprotective effects of these prodrugs were investi-
gated by the treatment with mice at three dose levels respec-
tively, and the mice were sequentially induced liver damage
by CCl4. The results showed that 3a could decreased the
alanine transaminases (ALT) and aspartate transaminases
(AST) level in serum compared with the CCl4 control group,
and its effect on decreasing the level of ALT and AST was
Table 2. The ADT Concentration after Intragastric Administration of the Compounds (ng.ml-1)
Compound
Time (min)
5
10
15
30
45
60
120
180
3a
65.3
64.5
nd
101.2
120.5
2.7
178.2
236.1
10.0
145.8
145.7
17.2
243.2
156.7
17.0
660.6
63.4
22.5
156.2
49.7
46.7
41.0
57.6
21.1
212.1
9.1
nd a
35.2
19.6
120.6
4.3
3d
3e
3f
3i
5.6
9.9
21.8
14.1
77.6
14.9
16.0
159.7
21.4
453.3
29.1
245.1
33.4
156.3
37.2
ATT
48.4
25.0
and: not detected.

750 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 10
Li et al.
Table 3. Pharmacokinetic Parameters of ADT after Intragastric Administration of the Compounds and ATT
Parameters
Compounds and ATT
3a
3d
3e
3f
3i
ATT
CL/F(L/min/kg)a
AUC0-t(ng.min/mL)b
MRT0-t(min)c
t1/2(min)d
5.49
34452.00
81.24
83.76
60
14.47
13447.87
67.77
72.67
15
12.09
3263.16
99.01
69.32
60
58.80
23854.51
108.54
143.61
120
21.14
11617.08
35.35
25.84
15
12.96
7401.13
98.45
174.37
120
Tmax(min)e
Cmax(ng/mL)f
REg
660.60
4.65
236.1
1.82
22.5
212.1
3.22
453.3
1.57
48.4
-
0.46
CEh
13.65
4.88
0.46
4.38
9.37
-
aCL/F: clearance.
bAUC0-t: area under the concentration time curve.
cMRT0-t: the mean residence time.
dt1/2: biological half life.
eTmax: time of maximum concentration.
fCmax: maximum concentration.
grelative uptake efficiency.
hconcentration efficiency.ꢀ
silica gel (100-200 mesh). Melting point was measured on an
YRT-3 melting point apparatus. IR spectra were obtained on
a Perkin-Elmer983. NMR spectra were taken on a Varian
INOVA400 and on a Bruker AC-E200. Chemical shifts are
expressed in ꢀ (ppm) with tetramethylsilane (TMS) as inter-
nal reference and coupling constants (J) are expressed in Hz.
Mass spectra were recorded on an Agilent 1946B ESI-MS
instrument.
superior to ATT at all the three doses as expected (Table 4).
Similar to ATT, 3a exhibited the highest hepatoprotective
ability at the middle dose (2mg/kg) that decreased the level
of ALT and AST to 98.2±15.4 IU/L and 132.7±34.8 IU/L
respectively. All mentioned above indicated that 3a could be
used as a promising prodrug of ADT, which possessed better
bioavailability and potency than ATT.
Acute Toxicity Test
The synthesis route of the ATT analogs (3a-p), starting
from a commercially available ATT, is outlined in Scheme
1. As a representative example, the general synthetic proce-
dure of 3a is provided below. The synthetic procedure of
compounds 3b-3p is similar to that of compound 3a.
Because of the good bioavailability and potency, the
safety of this compound 3a is a non-negligible factor. To
study the acute toxicity of 3a, we used the method of maxi-
mum dose. ICR mice were administered saturated solution of
3a in peanut oil at a constant volume of 40ml/kg. As a result,
no mouse died within the whole 14 days, which indicated
that the maximum tolerated dose (MTD) of ADT was no less
than 3.25 g/kg (saturated concentration of 3a: 0. 0813 g/ml).
It is known that the LD50 of ATT is 1.480 g/kg [10], so the
result suggested that compound 3a was more safe than ATT.
Chemistry
Synthesis of 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione
(2)
A mixture of ATT 1 (10.05g, 40mmol) and anhydrous
pyridine hydrochloride (34.07g, 290 mmol) was heated to
220°C for 20 min under argon atmosphere. The system was
cooled to room temperature, and then 100ml water was
poured in and stirred for 5 min. The suspension was filtered,
and the filter cake was collected. The solid was dissolved in
100ml of 10% NaOH aqueous solution and stirred for10 min,
EXPERIMENTAL SECTION
Materials and Apparatus
TLC was performed using precoated silica gel GF254
(0.2mm), and column chromatography was performed using
Table 4. Effect of ATT and Compounds 3a on the Levels of ALT, AST in Serum of Model Mice
Compound
Group
n
Doses (mg/kg)
ALT( X±SD, IU/L)
AST( X±SD, IU/L)
Normal
Model
Low
10
10
10
10
10
10
10
10
0
0
1
2
4
1
2
4
65.3±14
789±37
151±29
112±14
177±94
111±12
98.2±15
87.7±26
89.8±18
964±90
270±90
266±59
407±34
174±31
133±35
288±52
ATT
3a
Middle
High
Low
Middle
High

Design, Synthesis and Biological Evaluation of Lipophilic Analogs
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 10 751
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl stearate (3g)
then filtered, the filtrate was acidified to pH 1 by conc HCl
to form massive solid, then filtered and the filtter cake was
dried in vacuo to afford 2 as orange solid (7.65g, 84.5%).
MP: 172-174°C. 1HNMR (CDCl3): ꢀ 7.67 (dt, 3 H, J1=8.8Hz,
J2=2.0Hz), 7.38 (s, 1 H), 7.24 (dt, 2 H, J1 = 8.8 Hz, J2 = 2.0
Hz). IR (KBr): 3179, 3050, 1608, 1474, 1197, 1023cm-1. MS
m/z: 227 (M+H+).
Yield: 79.3%. MP: 85–86°C. 1HNMR (CDCl3, 400
MHz): ꢀ 7.68 (dt, 2H, J1=8.4Hz, J2=2.1H), 7.41 (s,1H), 7.23
(dt, 2H, J1=8.8Hz, J2=2.0Hz), 2.59 (t, 2H, J=7.2Hz), 1.76 (q,
2H, J=7.2Hz), 1.40 (br,28H). IR (KBr): 3031, 2953, 2920,
1747, 1599, 1516, 1460, 1379, 1221, 1188, 1018cm-1. MS
m/z: 493 (M+H +)
Synthesis of 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl acetate
(3a)
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl pivalate (3h)
1
Yield: 43.8%. MP: 112–113°C. HNMR (CDCl3, 400
To a solution of DCC (271mg, 1.20mmol) and DMAP
(15mg, 0.12mmol) in CH2Cl2, acetic acid (66mg, 1.10mmol)
was added and the reaction mixture was stirred for 30 min,
and then compound 2 (226mg, 1.00mmol) was added to this
slurry. The mixture was stirred at room temperature over
night, then the white precipitate was filtered off and CH2Cl2
was removed in vacuo, the residue was purified on a silica-
gel chromatography column to get compound 3a (239mg,
MHz): ꢀ 77.79 (d, 2H, J=8.8Hz), 7.43 (s, 1H), 7.21 (d, 2H,
J=8.4Hz), 1.39 (s, 9H). IR (KBr): 2961, 2930, 1741, 1598,
1516, 1395, 1368,1231, 1168, 1020cm-1. MS m/z: 311 (M+H
+
)
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl cinnamate (3i)
1
Yield: 91.2%. MP: 161–162°C. HNMR (CDCl3, 400
MHz): ꢀ 7.90 (d, 1H, J=16Hz), 7.71 (dt, 2H, J1=8.8Hz,
J2=2.0Hz), 7.60 (m,2H), 7.44 (m,3H), 7.42 (s,1H), 7.33 (dt,
2H, J1=8.8Hz, J2=2.0Hz), 6.62 (d, 1H, J=16Hz). IR (KBr):
3059, 1729, 1630, 1597, 1448, 1309, 1189, 1025cm-1. MS
m/z: 357 (M+H +)
1
89%) as a red solid. MP: 140-142°C. HNMR (CDCl3): ꢀ
7.69 (dt, 3 H, J1=8.8Hz, J2=2.0Hz), 7.40 (s, 1 H), 7.24 (dt, 2
H, J1 = 8.6 Hz, J2 = 2.4 Hz), 2.34 (s, 3 H). IR (KBr): 3028,
2922, 1763, 1594, 1516, 1363, 1185, 1023 cm-1. MS m/z:
269 (M+H+).
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 2-fluorobenzoate
(3j)
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl propionate (3b)
1
1
Yield: 78.9%. MP: 170–171°C. HNMR (CDCl3, 400
Yield: 80.4%. MP: 110-111°C. HNMR (CDCl3): ꢀ 7.68
MHz): ꢀ 8.12 (td, 1H, J1=7.6Hz, J2=2.0Hz), 7.75 (d, 2H,
J=8.4Hz), 7.66 (m, 1H), 7.45 (s, 1H), 7.40 (dt, 2H, J=8.8Hz),
7.32 (m, 1H), 7.24 (m, 1H). IR (KBr): 3058, 1745, 1721,
1601, 1455, 1292, 1249, 1171, 1037cm-1. MS m/z: 349
(M+H +).
(dt, 2H, J1=8.4Hz, J2=2.0H), 7.41 (s, 1H), 7.23 (dt, 2H,
J1=8.8Hz, J2=2.0Hz), 2.63 (q, 2H, J=7.6Hz), 1.29 (t, 3H,
J=7.6Hz). IR (KBr): 3107, 2959, 2935, 1759, 1597, 1452,
1228, 1174, 1025cm-1. MS m/z: 283 (M+H+).
(E)-4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl but-2-enoate
(3c)
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 4-chlorobenzoate
(3k)
1
Yield: 94.4%. MP: 126-127°C. HNMR (CDCl3): ꢀ 7.62
1
Yield: 91.1%. MP: 188–189°C. HNMR (CDCl3, 400
(dt, 2H, , 7.36 (s, 1H), 7.20 (dt, 2H, J1=8.8Hz, J2=2.0Hz),
7.15 (dq, 1H, J1=15.6Hz, J2=1.6Hz), 5.99 (dq, 1H,
J1=15.6Hz, J2=1.6Hz), 1.93 (dd, 3H, J1=6.8Hz, J2=1.6Hz).
IR (KBr): 3063, 1753, 2935, 1654, 1596, 1520, 1314, 1174,
1026, 978 cm-1. MS m/z: 295 (M+H+).
MHz): ꢀ 8.14 (dt, 2H, J1=8.8Hz, J2=2.0Hz), 7.74 (dt, 2H,
J1=8.8Hz, J2=2.0Hz), 7.53 (dt, 2H, J1=9.2Hz, J2=2.4Hz),
7.44 (s, 1H), 7.37 (dt, 2H, J1=8.8Hz, J2=2.0Hz). IR (KBr):
2924, 1736, 1593, 1520, 1229, 1173, 1091, 1029cm-1. MS
m/z: 365 (M+H +).
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl isobutyrate (3d)
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 3-phenylpropanoate
(3l)
Yield: 51.9%. MP: 99–100°C. 1HNMR (CDCl3, 400
MHz): ꢀ 7.67 (dt, 2H, J1=8.8Hz, J2=2.0Hz), 7.41 (s, 1H),
7.22 (dt, 2H, J1=8.8Hz, J2=2.0Hz ), 2.84 (m, 1H), 1.33 (d,
6H, J=7.2Hz). IR (KBr): 3046, 2977, 2932, 1748, 1386,
1164, 1027 cm-1. MS m/z: 297 (M+H +)
1
Yield: 76.8%. MP: 192–194°C. HNMR (CDCl3, 400
MHz): ꢀ 7.67 (dt, 2H, J1=8.4Hz, J2=2.0Hz), 7.45 (m, 1H),
7.40 (s, 1H), 7.32-7.36 (m, 4H), 7.15 (dt, 2H, J1=8.8Hz,
J2=2.0Hz), 3.10 (t, 2H, J=7.6Hz), 2.94 (t, 2H, J=8.0Hz). IR
(KBr): 3062, 1736, 1598, 1524, 1480, 1228, 1188, 1028cm-1.
MS m/z: 359 (M+H +).
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl benzoate (3e)
1
Yield: 97.5%. MP: 161–162°C. HNMR (CDCl3, 400
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 3,3-diphenylpropan-
oate (3m)
MHz): ꢀ 8.22 (dt, 2H, J1=8.4Hz, J2=2.0Hz), 7.75 (dt, 2H,
J1=8.8Hz, J2=2.0Hz), 7.68(tt, 1H, J1=7.6Hz, J2=1.2Hz), 7.54
(t, 2H, J=8.4Hz), 7.44(s, 1H), 7.38(dt, 2H, J1=8.8Hz,
J2=2.0Hz). IR (KBr): 3061, 1736, 1598, 1451, 1267, 1180,
1012 cm-1. MS m/z: 331 (M+H +)
1
Yield: 89.4%. MP: 156–158°C. HNMR (CDCl3, 400
MHz): ꢀ 7.58 (d, 2H, J=8.8Hz), 7.35 (s, 1H), 7.30 (s, 10H),
6.88 (d, 2H, J=8.8Hz), 4.64 (t, 1H, J=8.0Hz), 3.33 (d, 2H,
J=8.4Hz). IR (KBr): 2924, 1759, 1597, 1523, 1489, 1221,
1171, 1020cm-1. MS m/z: 435 (M+H +).
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 2-phenylacetate (3f)
1
Yield: 85.9%. MP: 134–136°C. HNMR (CDCl3, 400
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 3,5-dimethoxybenz-
oate (3n)
MHz): ꢀ7.65 (d, 2H, J=8.4Hz), 7.38 (d, 5H, J=4Hz), 7.25 (s,
1H), 7.20 (d, 2H, J=8.4Hz), 3.88 (s, 2H). IR (KBr): 3028,
2914, 1755, 1601, 1453 1353, 1231, 1169, 1026 cm-1. MS
m/z: 345 (M+H +)
1
Yield: 86.1%. MP: 122–123°C. HNMR (CDCl3, 400
MHz): ꢀ 7.75 (d, 2H, J=8.4Hz), 7.45 (s, 1H), 7.37 (d, 2H,

752 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 10
Li et al.
J=8.0Hz), 7.34 (d, 2H, J=2.4Hz), 6.76 (t, 1H, J=2.4Hz),
3.88(s,6H). IR (KBr): 2836, 1733, 1591, 1519, 1230, 1166,
1042cm-1. MS m/z: 391 (M+H +).
Bioavailability Evaluation in Mice In Vivo
According to the requirements of the National Act on the
usage of experimental animals (P. R. China), the Sichuan
University Animal Ethical Experimentation Committee, ap-
proved all procedures of our in vivo studies. The ICR mice
were separated into 6 groups (8 in each) and intragastric ad-
ministration of a solution of ATT and five analogs in peanut
oil at a dose of compound equivalent to 250mg per kg body
weight respectively was done. Mice from each group were
sequentially sacrificed at 5, 10, 15, 30, 45, 60, 120 and
180min after administration and blood samples were col-
lected from the ocular artery directly after removing eyeball,
and were treated following the same procedure described for
in vitro test to obtain plasma samples. These samples were
collected to analysis the concentration of ADT in each time
by HPLC method.
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 1,2,3,4-tetrahydro-
naphthalene-1-carboxylate (3o):
Yield: 96.0%. MP: 25–26°C. 1HNMR (CDCl3, 400
MHz): ꢀ 7.67 (d, 2H, J=8.4Hz), 7.41 (s, 1H), 7.23 ( d, 2H,
J=8.8Hz), 7.33-7.16 (m, 4H), 4.10 (t, 1H, J=6.0Hz), 2.87( m,
2H, J=6.4Hz), 2.4-1.8 (m, 4H). IR (KBr): 3026, 2935, 1753,
1741, 1593, 1452, 1221, 1176, 1029cm-1. MS m/z: 385
(M+H +).
4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 5,6,7,8-tetrahydro-
naphthalene-1-carboxylate (3p):
Yield: 96.0%. MP: 25–26°C. 1HNMR (CDCl3, 400
MHz): ꢀ 7.96 (d, 1H, J=8.0Hz), 7.74 (dt, 2H, J1=8.8Hz,
J2=2.0Hz), 7.44(s, 1H), 7.36 (dt, 2H, J1=8.8Hz, J2=2.0Hz),
7.34 (br, 1H), 7.23 (d, 1H, J=7.6Hz), 3.15 (t, 2H, J=4.8Hz),
2.87 (t, 2H, J=6.0Hz), 1.8-1.9 (br, 4H). IR (KBr): 3026,
2930, 1757, 1741, 1598, 1450, 1220, 1174, 1028 cm-1. MS
m/z: 385 (M+H +).
CCl4-Induced Acute Liver Damage Model in Mice and
Treatment of Investigated Compounds
The ICR mice (20-22g) were randomly divided into five
groups, and each contained 10. In normal group and model
group, the mice were injected intraperitoneally, 5ml/kg sa-
line solution and CCl4 at a dose of 5ml/kg as a 0.2% peanut
oil solution, respectively. In low, middle and high dose
group, the mice were intraperitoneally injected with the in-
vestigated compound at a dose of 1mg/kg, 2mg/kg and
4mg/kg, respectively. The low, middle and high dose groups
were followed by intraperitoneal injection with CCl4 at a
dose of 5ml/kg as a 0.2% peanut oil solution 0.5 hour after
pretreatment with tested compound. According to the previ-
ously reported methods with some modification[9], 16 hours
after introduction of CCl4, all mice were sacrificed and blood
sample withdrawn from orbital sinus was collected and al-
lowed to clot, then centrifuged at 3500rpm for 10min. The
serum was separated and used for assay of alanine transa-
minases (ALT) and aspartate transaminases.
Pharmacological Evaluation
General Method
All chemicals and reagents were analytical grade, and ob-
tained commercially. All liquid reagents were distilled be-
fore use. ICR mice weighing 18-22g were received from
Sichuan Industrial Institute of Antibiotics (P.R. China) and
were maintained empty stomach for 24h. Since the experi-
ment could be finished within 48 h, there was no significant
change in mice’s body weights during the experiment.
The HPLC system consisted of an SPD-10A variable
UV-VIS detector and a set of Model LC-10AT liquid chro-
matography including a manometric module as well as a
dynamic mixer from Shimadzu. The mobile phase consists
of pure water and methanol, which was filtered through
0.45mm membrane filter before use. A phenomenex ODS
column (4.6ꢁ200mm, 5μm) was eluted with the mobile
phase (methanol/water: 10/3, v/v) at a flow rate of
1.0ml/min. The eluate was monitored by measuring the ab-
sorption at 355nm with a sensitivity of AUFS 0.01 at 25°C.
Acute Toxicity Test
Sixty ICR mice were divided into three groups: test
group, control group and vehicle group. Every group con-
sisted of 10 males and 10 females, and all drugs were intra-
gastricly administered in a constant volume of 40ml/kg. The
three groups were treated once with following drugs respec-
tively: 3a saturated solution of peanut oil, normal saline and
peanut oil. Animals were observed closely during the period
30 min to 1 hour after being administered. The number of
dead animals was counted at two weeks, body weights were
noted once per week. The body weight of ADT group had no
statistics difference compared with control group and vehicle
group (On Day 0, ADT group: 20.1 ± 1.2 g vs. control
group: 20.0 ± 1.2 g and vehicle group: 20.2 ± 1.3 g; On Day
7, ADT group: 27.3 ± 0.9 g vs. control group: 27.7 ± 0.9 g
and vehicle group: 27.6 ± 0.9 g; On Day 14, ADT group:
34.8 ± 1.3 g vs. control group: 35.1 ± 1.1 g and vehicle
group: 34.8 ± 1.1 g; all of the P > 0.05).
pH Stability Test In Vitro
An aqueous solution of the compounds (50 ug/mL) at a
volume of 1.0 ml was incubated in 10ml buffer under differ-
ent pH (1, 6, 7.4, 9.5) at 37°C under the protection of nitro-
gen. At a predetermined time interval, a 20 μL portion of the
solution was removed, and the concentration of the com-
pounds was measured by HPLC as described.
Plasma Stability Test In Vitro
Aliquots of the compounds aqueous solution at a volume
of 0.1 ml was incubated in 0.4 ml mice plasma at 37°C under
the protection of nitrogen. 100μl of the samples were with-
drawn at predetermined time points and mixed with 200 μL
acetonitrile. After centrifugation at 8000 rmp/min for 7 min,
20 μl of the supernatant was collected to monitor the disap-
pearance of the compounds by HPLC analysis.
Statistical Analysis
The clearance(CL/F), area under the concentration time
curve(AUC0-t), the mean residence time(MRT0-t), biological
half life(t1/2) and maximal concentration(Tmax) were calcu-
lated by Data and max Statistics (DAS, Shanghai, China).
The RE and CE were calculated to evaluate the delivery

Design, Synthesis and Biological Evaluation of Lipophilic Analogs
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 10 753
property of the prodrugs. The value of RE and CE were de-
fined as following:
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In summary, we have designed and developed a method
of synthesis for different ATT analogs. Five of the synthe-
sized compounds were subjected to in vitro stability test and
in vivo biological evaluation. The experimental data high-
lighted that after oral administration of ATT analogs 3a, 3d,
3e, 3f and 3i, concentrations and AUC0–t of ADT in plasma
were significantly higher than that of ATT at the same dose.
This verified that the prodrugs has the ability to enhance the
bioavailability of ADT. Besides this, toxicity test results in-
dicated compound 3a was safer than ATT. In conclusion,
these compounds could be used as potential candidates for
the prodrug of ADT in clinical trial for the treatment of he-
patobiliary dysfunctions.
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