Antihelminthic activity of some newly synthesized 5(6)-(un)substituted-1H-benzimidazol-2-ylthioacetylpiperazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2006.07.005

Piperazine derivatives of 5(6)-substituted-(1H-benzimidazol-2-ylthio)acetic acids were synthesized by using two methods and studied for antihelminthic activity. The antiparasitic screening showed that compounds 18-24 exhibited higher activity against Trichinella spiralis in vitro in comparison to methyl 5-(propylthio)-1H-benzimidazol-2-yl-carbamate (albendazole). Most active were compounds 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-1H-benzimidazo le 21 and 2-{[2-oxo-2-(4-benzhydrylpiperazin-1-yl)ethyl]thio}-5(6)-methyl-1(H)-ben zimidazole 19 as well as 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-5(6)-methyl-1( H)-benzimidazole 23 with efficacy of 96.0%, 98.4% and 100%, respectively. The tested derivatives 15-19 and 20-23 were less active against Syphacia obvelata in vivo than albendazole and exhibited the same efficacy as piperazine, but in twice lower concentration.Compounds 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-1H-benzimidazo le 21, 1,4-bis[(5(6)-methyl-1(H)-benzimidazol-2-ylthio)acetyl]piperazine 17 and 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-5(6)-methyl-1( H)-benzimidazole 23 had higher efficacies of 73%, 76%, and 77%, respectively.

Full text of DOI:10.1016/j.ejmech.2006.07.005

European Journal of Medicinal Chemistry 41 (2006) 1412e1420
http://www.elsevier.com/locate/ejmech
Original article
Antihelminthic activity of some newly synthesized
5(6)-(un)substituted-1H-benzimidazol-2-ylthioacetylpiperazine derivatives
a
b
Anelia Ts. Mavrova a, , Kamelya K. Anichina , Dimitar I. Vuchev ,
*
Jordan A. Tsenov c, Pavletta S. Denkova c, Magdalena S. Kondeva d, Mitka K. Micheva d
a Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
b National Center of Infectious and Parasitic Diseases, 26 Yanko Sakazov Blvd., 1504 Sofia, Bulgaria
c Institute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
d Department of Pharmacology and Toxicology, Medicinal University of Sofia, 2 Dunav street, 1504 Sofia, Bulgaria
Received 25 May 2006; received in revised form 4 July 2006; accepted 4 July 2006
Available online 22 September 2006
Abstract
Piperazine derivatives of 5(6)-substituted-(1H-benzimidazol-2-ylthio)acetic acids were synthesized by using two methods and studied for
antihelminthic activity.
The antiparasitic screening showed that compounds 18e24 exhibited higher activity against Trichinella spiralis in vitro in comparison to
methyl 5-(propylthio)-1H-benzimidazol-2-yl-carbamate (albendazole). Most active were compounds 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-
2-oxoethyl}thio)-1H-benzimidazole 21 and 2-{[2-oxo-2-(4-benzhydrylpiperazin-1-yl)ethyl]thio}-5(6)-methyl-1(H )-benzimidazole 19 as well
as 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-5(6)-methyl-1(H )-benzimidazole 23 with efficacy of 96.0%, 98.4% and 100%,
respectively.
The tested derivatives 15e19 and 20e23 were less active against Syphacia obvelata in vivo than albendazole and exhibited the same efficacy
as piperazine, but in twice lower concentration.Compounds 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-1H-benzimidazole 21,
1,4-bis[(5(6)-methyl-1(H )-benzimidazol-2-ylthio)acetyl]piperazine 17 and 2-({2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)-5(6)-
methyl-1(H )-benzimidazole 23 had higher efficacies of 73%, 76%, and 77%, respectively.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Benzimidazoles; 1H-Benzimidazol-2-ylthioacetylpiperazines; Antitrichinellosis activity; Antinematode activity; Hepatotoxicity
1. Introduction
action, which is expressed in the interruption and consequent
blockade of microtubule assembly in nematode, cestode and
trematode cells as well as in the violation of the glycose trans-
port and fumarase inhibition [11e13]. Some of the benzimid-
azole drugs used in the parasitological praxis belong to the
group of 2-amino-1H-benzimidazole analogues as benzimid-
azole carbamates, while the others contain 2-mercapto-1-
H-benzimidazole moiety. It appeared that the benzimidazoles,
which have been used in the chemotherapy of trichinellosis
were unable to kill Trichinella spiralis larvae at an advanced
stage of development. This fact indicates that the search for
better drugs is still necessary.
The benzimidazole compounds possess broad spectrum of
biological properties and were investigated for their antiviral
(anti-HIV), anticancer and antibacterial activities [1e10].
Some of them found application in the above-mentioned fields
of the medicine praxis. Many benzimidazole derivatives are
widely used for the treatment of parasitic diseases. Their effi-
cacy against helminthes and fungi resulted from their mode of
* Corresponding author. Tel.: þ359 2 8163207; fax: þ359 2 9238401.
E-mail addresses: anmav@abv.bg (A.Ts. Mavrova), dvutchev@yahoo.com
(D.I. Vuchev), magdalenakondeva@hotmail.com (M.S. Kondeva).
The pathway that has been exploited successfully in the
search for antiparasitic agents is the one for the synthesis of
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.07.005

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
1413
polyamines playing essential roles in cell proliferation and cell
differentiation. Many diamine analogues, which interfere with
polyamines’ metabolism have been synthesized and their anti-
parasitic effects against protozoa have been proved [14e17].
Among the diamines we choose piperazine because it is one
of the earliest drugs used against ascaridosis and enterobiosis
and which is in the structure of many antihelminthic pharma-
ceutical drugsadministered today. The piperazine compounds
applied in the parasitological praxis exert hyperpolarization
of muscle membrane and act as chloride channel opener and
GABA agonist. From the other side the piperazine cycle par-
ticipates as pharmacophore in the structure of many drugs, be-
longing to different pharmacological groups.
On that reason the synthesis of new benzimidazoles contain-
ing piperazine cycle and the investigation of their antihelminthic
activity in order to compare it to the efficacy of albendazole and
to the activity of the most important commercial antiparasitic
drug ivermectin are of pharmacological interest.
In this paper we describe the synthesis of new piperazine
derivatives of (1H-benzimidazol-2-ylthio)acetic acid and their
activity in vivo against Syphacia obvelata and T. spiralis as
well as their hepatotoxicity. In our previous paper we
reported the synthesis and antitrichinellosis activity of
some 2-substituted-[1,3]-thiazolo[3,2-a]benzimidazol-3(2H)-
ones and consecutively this paper is a continuation of our
work [18].
Compounds 16, 17, 19, 22, 24 and 25 were prepared also
through direct condensation of (benzimidazol-2-ylthio)acetic
acid with the corresponding piperazine at 5e10 ꢀC in the pres-
ence of DCC for 20e25 h (Method B).
The chemical structures of compounds 15e26 were estab-
lished by elemental analyses, IR, 1H NMR and 13C NMR spec-
tra as well as mass spectral data for compounds 15, 16, 20 and
21 and the results are presented in Section 7. The elemental
analyses indicated by the symbols of the element were within
ꢁ0.4% of theoretical values. The IR spectra of piperazine de-
rivatives 15e26 were quite similar. The characteristic absorp-
tion bands at 1640e1660 were observed in the IR spectra.
The analysis of the NMR spectra was done taking into
consideration the electronic effect of the respective substitu-
ents and the structure numbering used for the description of
1
the H NMR spectra is given in Fig. 1. For compounds 21
and 22 the 2D heteronuclear 1He13C correlation spectra
(HMQC technique e heteronuclear multiple quantum correla-
tion) were recorded to confirm the assignment of the signals in
1H and 13C spectra.
3. Pharmacology
3.1. Antihelminthic activity
The 1H-benzimidazol-2-ylthioacetylpiperazine derivatives
18e24 were evaluated for their activity against T. spiralis in
vitro as well as their antinematode activity against S. obvelata
in vivo. The hepatotoxicity effect of compounds 15, 17, 18, and
20e23 on the viability of isolated rat hepatocytes in vitro was
studied.
2. Chemistry
The synthesis of the piperazine derivatives of (1H-benzimi-
dazol-2-ylthio)acetic acids is carried out as depicted in Figs. 1
and 2.
1H-Benzimidazole-2-thiols 1e3 were commercially avail-
able and compound 4 was prepared by refluxing ethanole
water solution of sodium hydroxide, carbon disulphide and
4-chloro-1,2-diamino-benzenes [19,20].
4. Results
4.1. Antitrichinellosis activity
The 5(6)-(un)substituted-(1H-benzimidazol-2-ylthio)acetic
acids (5e8) were obtained through William’s reaction of
5(6)-(un)substituted-1H-benzimidazole-2-thiols (1aed) with
chloroacetic acid in the presence of sodium hydroxide by re-
fluxing [20]. The yields were in the range of 59e75%.
1,3-Thiazolo[3,2-a]benzimidazol-3(2H )ones 9e10 and 12
were synthesized by heating 5(6)-(un)substituted-(1H-benzi-
midazol-2-ylthio)acetic acids 5, 6, 8 with acetic anhydride in
pyridine medium on steam bath [20]. The cyclization of (ben-
zimidazol-2-ylthio)acetic acids 7 in the presence of dicyclo-
hexyl carbodiimide (DCC) at room temperature resulted in
compound 11.
The in vitro parasitological study showed that most of the
tested compounds exhibited higher activity than albendazole’s
effect against T. spiralis and comparable to that of ivermectin
(Table 1). Compounds 21, 23 and 19 demonstrated 96.0%,
98.2% and 100% activities, respectively, at a dose of
200 mg/mL after 48 h, expressed in suppressing motor activity
of larvae and opening of their spiral form. In the control samples
with physiological solution and the control samples with
DMSO all T. spiralis larvae had spiral form i.e. are vital [11,12].
4.2. Activity against S. obvelata
The hydrolysis of [1,3]-thiazolo[3,2-a]benzimidazol-3(2H )-
ones with an appropriated N-monosubstituted piperazine in
ethanol medium under refluxing conditions for 3 h resulted in
the piperazine derivatives of (benzimidazol-2-ylthio)acetic
acids 15e26 (Method A). Compounds 13 and 14 were obtained
from 2-(2-hydroxy-ethylamino)-ethanol and the corresponding
4-substituted-anilines through the procedure given in Ref. [21];
piperazine and benzhydrylpiperazine were commercially
available.
The results for antinematode activity determined three days
after finishing the three-day-treatment course of invasion with
S. obvelata white mice are systematized and the average num-
ber of parasites of each experimental group is given in Table 2.
The data indicated that the tested compounds exhibit different
antinematode effect which is less than the effect of the referent
albendazole. The most active compounds were 17, 21 and 23
with 76%, 73% and 77% activities, respectively [22].

1414
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
O
R
R
R
5 H
N
N
a
6 CH3
7 NO2
8 Cl
SH
S
OH
N
H
N
H
(59-75 %)
5-8
1-4
(50-71%)
b (or c)
2
NH
R
3
7
6
R
9
H
N
5
N
19
9
14
R
10 CH3
11 NO2
12 Cl
N
S
N
d
18
11 S
O
17
N
H
(45%)
13
16
15 R = H
O
15
9-12
e
R1
R1
13 CH3
14 Cl
HN
N
f
g
(48%, 62%)
(57-58%)
(60-70%)
13,14
17'
R1
R
16'
13'
10
18'
19'
11
12
11
10
9
8
14'
9
NH
12
18
17
11'
N
8
7
6
13
2
2
9'
N
16
S
7
19
3
7'
O
N
21 20
2
N
O
5
26
13
N
3
6
16
21
R
N
3
6
N
O
5
25
33
18 S
32
29
14
22
R
7 N
O
27
24
5
N
19
16
N
H
20
14
13
31
30
9
11 S
R
23
24 S
N
18
17
N
H
26
N
H
20-26
R
R1
CH3
Cl
18,19
16,17
20 H
21 H
R
18 H
R
19 CH3
22 CH3 CH3
23 CH3 Cl
24 NO2 CH3
25 NO2 Cl
26 Cl Cl
16 H
17 CH3
Fig. 1. Synthesis of 1H-benzimidazol-2-ylthioacetylpiperazine derivatives 15e26 (Method A) and structure numbering used in NMR spectra description. Reagents
and conditions: (a) ClCH2COOH, NaOH, ethanol; (b) Ac2O/Py, reflux; (c) DDC/Py, reflux; (d) piperazine hexahydrate, C2H5OH, reflux; (e) 9, 10, piperazine hexa-
hydrate; (f) benzhydrylpiperazine ethanol, reflux; (g) C2H5OH, reflux.
4.3. Hepatotoxicity
ylthio)acetamides (Method A). This non-traditional reaction of-
fers the possibility for preparing large number of compounds by
means of a simple procedure through introduction of various
substituents both in the 5(6)-position of thiazolobenzimidazole
ring and in the second place of thiazole cycle. Some 2-[(2-oxo-
2-piperazin-1-ylethyl)thio]-1H-benzimidazoles were obtained
by direct condensation between 5(6)-(un)substituted-(1H-ben-
zimidazol-2-ylthio)acetic acids and N-substituted piperazines
in the presence of DCC in pyridine medium (Method B). Irre-
spective of the less number of reaction stages of Method B,
Method A was preferred because of the simple work-up proce-
dure and the higher purity of the synthesized substances.
The hepatotoxicity of compounds 15, 17, 18, and 20e23 on
the viability of isolated rat hepatocytes was determined to-
wards the control group (untreated cells) as well as compared
to the hepatotoxicity of albendazole and the results are given
in Table 3 [23,24].
5. Discussion
5.1. Chemistry
In a basic medium the condensed thiazole cycle underwent
a cleavage [25]. Because of these properties of the above-men-
tioned compounds we choose the reaction between [1,3]-
thiazolo[3,2-a]benzimidazol-3(2H )-one and amines among
the different methods for synthesis of (benzimidazol-2-
5.2. Antitrichinellosis activity
The most piperazine derivatives, excluding 18 and 24, ex-
hibited significant larvocide activity against T. spiralis larvae.

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
1415
R
NH
NH
S
O
N
O
N
R
N
a
b
R
N
S
OH
S
(53%)
O
N
H
(62%)
N
H
N
R
5 - 7
15 R = H
5 H
N
O
6 CH3
7 NO2
R
N
S
N
H
R1
13 CH3
14 Cl
c
d
HN
N
R1
(64%)
(68-71%)
17
R
17 CH3
13, 14
R1
N
N
N
N
R
N
R
N
S
O
S
O
N
H
N
H
22, 24, 25
19
R
R1
22 CH3 CH3
24 NO2 CH3
25 NO2 Cl
R
19 CH3
Fig. 2. Synthesis of compounds 15, 17, 19, 22, 24 and 25 (Method B). Reagents and conditions: (a) piperazine hexahydrate, DCC/Py, 5e10 ꢀC; (b) compound 6,
piperazine hexahydrate, DCC/Py, 5e10 ꢀC; (c) compound 6, benzhydrylpiperazine, DCC/Py, 5e10 ꢀC; (d) DCC/Py, 5e10 ꢀC.
In parasitological experiment in vitro for antitrichinellosis ac-
tivity it was ascertained that the tested compounds 18, 19, and
20e24 exert antihelminthic effect, expressed in suppressing
motor activity of larvae and opening of their spiral form. In
the first control group with physiological solution and in the
second with DMSO practically all larvae were in spiral form.
All piperazine derivatives of (benzimidazol-2-ylthio)acetic
acid exhibited higher activity against T. spiralis larvae in re-
gards to the activity of albendazole, moreover compounds 19,
21 and 23 manifested antitrichinellosis activity, which
surpassed five times the activity of albendazole. Compounds
19e23 demonstrated higher effect against T. spiralis larvae at
a concentration of 200 mg/mL than ivermectin after 24 h. At
the same concentration the efficacy of compounds 19, 21, and
23 was also higher than the efficacy of ivermectin after 48 h.
Compounds 22e24 containing methyl substituent at 5(6)-
position of benzimidazole ring showed higher antihelminthic
activity in comparison to the unsubstituted compounds 18,
20 and 21. Compound 19 having methyl group in the 5(6)-
position of benzimidazole ring was distinguished for 92.7%
efficacy at a concentration of 200 mg/mL, while compound
18 exhibited 13.6% larvocide effect at the same concentration
after 24 h. The products possessing chlorine atom as substituent
at fourth position of benzene cycle (21 and 23) were more
active than those containing methyl group at the same place
(20 and 22).
Due to the obtained good results antitrichinellosis effect in
vitro, further research is currently in progress for the antitrichi-
nellosis activity in intestinal and muscle phase.
5.3. Antihelminthic activity against S. obvelata
The analysis of the results obtained by the study of the tested
and the control groups ascertained statistically significant differ-
ence in the level of parasites’ invasion ( p < 0.05). Albendazole
and piperazin hexahydrate were used as referent compounds.
The control test carried out with DMSO as well as the control
group of white mice (without treatment) did not indicate real re-
duction of the infection and the number of parasites was equal.
All tested products excluding 15 and 18 showed a significant
antihelminthic effect. The efficacyof each compound was estab-
lished according to Ref. [22] (Eq. (1)).
E ¼ ½ðIc ꢂ ItÞ=Icꢃ ꢄ 100
ð1Þ

1416
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
Table 1
Antihelminthic activity of compounds 18e24 against Trichinella spiralis
Compound no.
Efficacy (%) after 24 h
Somp. no.
Efficacy (%) after 48 h
50 mg/mL
100 mg/mL
200 mg/mL
50 mg/mL
100 mg/mL
200 mg/mL
18
19
11.2
37.4
19.5
29.6
26.5
35.8
11.4
10.6
45.4
13.6
75.8
48.0
64.7
56.8
72.0
15.6
10.7
48.7
13.6
92.7
78.8
82.6
80.6
86.5
31.2
13.3
54.2
18
19
19.5
44.0
21.0
33.5
34.7
41.0
14.3
14.8
62.1
19.7
88.0
63.0
83.0
63.0
92.7
16.0
15.1
78.4
53.9
100
20
21
20
21
80.0
96.0
82.1
98.4
37.8
17.4
88.3
22
23
22
23
24
Albendazole
Ivermectin
24
Albendazole
Ivermectin
E e efficacy, Ic e intensity of invasion in the control group
e.g. the number of parasites; It e intensity of invasion in the
treatment group.
Effective compound was accepted compound, which pos-
sesses an efficacy higher than 50%.
microscopic study of the intestinal content both in the experi-
mental animals and in the control groups. On the grounds of
this observation we accepted that the tested compounds ex-
hibited antihelminthic activity, expressed only against imagi-
nal forms of nematode but not against larvae.
The highest activity against S. obvelata manifested com-
pounds 17, 21 and 23 (E ¼ 73%, 70% and 71% in a dose of
20 mg/kg mw). The antinematode activity of compounds 16,
20 and 19 in a dose of 20 mg/kg mw was similar to the activity
of piperazine hexahydrate in a dose of 100 mg/kg mw, but in
comparison to albendazole all tested compounds were less ac-
tive. The five times increase of the dose exerts insignificant in-
fluence on the therapeutic effect.
The obtaining of an acceptable doseeresponse curve depends
on the biological characteristic of the parasites and on how much
is previously known about the biological activity and the safety
of the newly tested compounds, therefore additional experiments
are required using lower or higher concentrations.
5.4. Hepatotoxicity
The purpose of our study using isolated rat hepatocytes was
to compare whether the new compounds showed more toxic
effect than albendazole. As it is well known that albendazole
exerts hepatotoxic effect, we used it as a control, versus which,
to trace the effect of the new compounds (more toxic/less
toxic) [26,27]. The effect of the compounds on cell viability
was investigated by using a trypan blue test [23,24].
The results obtained by the study showed that albendazole
reduced the cells vitality by 56%. The piperazine containing
benzimidazole 20 was less hepatotoxic than albendazole and
preserved with 15% the cell viability regarding to albendazole,
while the most active compounds 23 and 19 showed hepato-
toxicity comparable to that of albendazole (7%, respectively,
2%). Compounds 20 and 19 comprising methyl group at
5(6)-position of benzimidazole cycle were less hepatotoxic
At a concentration of 0.200 mg/kg mw ivermectin showed
an efficacy of 64% that is lower than the efficacy of com-
pounds 17, 21 and 23 at a concentration of 20 mg/kg mw.
By the establishment of the number of parasites after treat-
ment, a large number of nematode larvae were observed by the
Table 2
Efficacy (E ) of compounds 15e23 against Syphacia obvelata
Compound no.
20 mg/kg mw
100 mg/kg mw
Average number of parasites in groupsa
E (%)
Average number of parasites in groupsa
E (%)
15
16
84.4 ꢁ 20.58
37.0 ꢁ 10.5
25.2 ꢁ 26.7
83.4 ꢁ 11.9
36.6 ꢁ 42.2
32.0 ꢁ 7.87
34.8 ꢁ 25.6
28.2 ꢁ 14.1
29.4 ꢁ 27.4
14.2 ꢁ 7.36
e
11
61
73
12
63
66
70
68
71
85
81.4 ꢁ 9.2
34.2 ꢁ 11.6
22.8 ꢁ 21.2
79.0 ꢁ 15.8
29.9 ꢁ 30.1
30.2 ꢁ 14.7
25.6 ꢁ 13.9
28.4 ꢁ 15.1
21.8 ꢁ 8.9
0.8 ꢁ 1.3
14
64
76
17
69
68
73
70
77
99
61
17
18
19
20
21
22
23
Albendazole
Piperazine hexahydratf
Ivermectinb
37.0 ꢁ 22.1
33.87 ꢁ 24.7
94.0 ꢁ 44.9
p < 0.05.
At concentration of 0.200 mg/kg mw.
64
Control group
94.0 ꢁ 44.9
a
b

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
1417
Table 3
Effect of benzimidazole derivatives on the cell viability
7. Experimental part
Compounds
no.
Cell viability
Effect versus
control (%)
Effect versus
albendazole (%)
Melting points (mp) were determined on an Electrothermal
AZ 9000 3MK4 apparatus and were uncorrected. The thin layer
chromatography (TLC, Rf values) was performed on silica gel
60 plates F254 (Merck, 0.2 mm thick) using mobile phase benze-
ne:methanol e 3:1, and visualization was effected with ultravi-
olet light. IR spectra were recorded on a Specord 71 IR
spectrophotometer as potassium bromide discs. All NMR spec-
tra were recorded on a Bruker Avance DRX 250 spectrometer
(Bruker, Faelanden, Switzerland) operating at 250.13 MHz for
1H and at 62.89 MHz for 13C, using a dual 5 mm 1H/13C probe-
head. The studied compounds were dissolved in CDCl3 or
DMSO-d6 (see Section 5.1 for details). Chemical shifts were ex-
pressed relative to tetramethylsilane (TMS) and were reported
as d (ppm). The measurements were carried out at ambient tem-
perature (300 K). The atom numbering used for description of
the spectra is shown in Fig. 1.
Mass spectrometric measurements were performed using
an LCQ DECA instrument (Thermo Finnigan, Palo Alto,
CA, USA). Compounds were solubilized in CH3OH. Solu-
tions (10ꢂ6 M) of compounds were prepared and directly in-
fused into the ESI source. The ions were produced using
spray voltage, capillary voltage and capillary temperature of
4 kV, 8 kV and 220 ꢀC, respectively. The microanalyses for
C, H, N and S were performed on PerkineElmer elemental
analyzer.
Control
Albendazole
93 ꢁ 3.5a
41 ꢁ 2.0a
27 ꢁ 4.3a,b
34 ꢁ 2.9a,b
20 ꢁ 1.6a,b
42 ꢁ 2.9a
47 ꢁ 4.5a
29 ꢁ 2.5a,b
28 ꢁ 4.0a,b
44 ꢁ 2.9a
100
56Y
71Y
63Y
78Y
55Y
49Y
69Y
70Y
53Y
15
17
18
19
20
21
22
23
a
34Y
17Y
51Y
15[
29Y
32Y
7[
p < 0.05 versus control.
p < 0.05 versus albendazole.
b
than their unsubstituted analogues 21 and 18 (R ¼ H), but this
relation was not affirmed by the derivatives 20 and 22. Com-
pound 18, which showed the lowest activity both against
T. spiralis larvae and S. obvelata was most hepatotoxic
compared to albendazole.
6. Conclusion
New piperazine derivatives of 5(6)-substituted-(1H-benzi-
midazol-2-ylthio)acetic acids were synthesized by using two
methods and studied for antihelminthic activity.
The in vitro and in vivo screening for antinematode effect
showed that the compounds exhibit significant antiparasitic ac-
tivity. All tested piperazine derivatives of (1H-benzimidazol-2-
ylthio)acetic acids exhibited higher activity against T. spiralis
larvae in comparison to albendazole, furthermore some of the
compounds (21, 23 and 19) demonstrated antitrichinellosis ac-
tivity, which surpassed five times the activity of albendazole.
With respect to ivermectin compounds 20, 21 and 19, 22e
23 demonstrated higher effect against T. spiralis larvae at
a concentration of 200 mg/mL after 24 h. At the same concen-
tration the efficacy of compounds 19, 21 and 23 was also
higher than the efficacy of ivermectin after 48 h.
The pharmaco-therapeutic study in vivo showed that the
tested compounds were less active than albendazole against
S. obvelata and have equal efficacy in a twice lower concentra-
tion related to piperazine. Moreover it was ascertained that the
5(6)-substituted-1H-benzimidazol-2-ylthioacetylpiperazine
derivatives were active against the mature nematodes but not
against the larvae. Nevertheless all tested compounds ex-
hibited 60e77% efficacy and as it is known effective com-
pounds were accepted compounds which demonstrate
efficacy higher than 50%.
7.1. Chemistry
7.1.1. General procedure for compound 4
4-Chloro-1,2-diamino-benzene (0.019 mol) and water
(3 mL) were added to a solution of sodium hydroxide
(0.022 mol) in ethanol (20 mL) and carbon disulphide
(0.022 mol). The mixture was heated under reflux for 3 h. Char-
coal is then added cautiously, and after the mixture has been re-
fluxed for 10 min the charcoal was removed by filtration. The
filtrate is heated to 60e70 ꢀC and quenched with warm water
(70ꢀ, 20 mL), and then 50% acetic acid (9 mL) was added by
good stirring. The product was separated and after cooling in re-
frigerator for 3 h the crystallization was completed. Yield: 69%.
7.1.2. General procedure for compounds 5e8
Solution of sodium hydroxide (0.012 mol) in ethanol
(14 mL) and 5(6)-(un)substituted-1H-benzimidazole-2-thiol
(0.0067 mol) was refluxed for 1 h. After cooling, chloroacetic
acid (0.0067 mol) was added and the refluxing was continued
for 1e5 h more. Then the reaction mixture was cooled, poured
into water and acidified with diluted acetic acid. The crystal-
lized product was filtrated, carefully washed with water and
re-crystallized. Yield: 59e75%.
The most active 5(6)-(un)substituted-1H-benzimidazol-2-
ylthioacetylpiperazine derivatives possess hepatotoxicity com-
parable or less than that of albendazole.
These results also confirmed the hypothesis that the intro-
duction of a piperazine ring into the structure of 5(6)-(1H-ben-
zimidazol-2-ylthio)acetic acid is favourable for the interaction
of these molecules with the biological targets.
7.1.3. General procedures for compounds 9e12
Method A: solution of 5(6)-(un)substituted-(1H-benzimida-
zol-2ylthio)acetic acid (0.104 mol) and acetic anhydride
(19.5 mL) in dry pyridine (65 mL) was heated by refluxing

1418
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
for 10 min. Products were crystallized as pale orange sediment
after cooling and water (100 mL) was added in small portions
by stirring. Yield: 50e71%.
C22H22N6O2S2: C e 56.63, H e 4.75, N e 18.01; found:
C e 56.75, H e 4.98, N e 18.11.
Method B: DCC (0.0144 mol) in pyridine (10 mL) was
added dropwise to compounds 5e8 (0.0144 mol) diluted in
dry pyridine (60 mL). The solution was stirred for 10e12 h
at 5e10 ꢀC. The precipitate of dicyclohexylurea was removed
and the filtrate was evaporated under reduced pressure to dry-
ness. The residue was dissolved in chloroform and filtrated af-
ter heating with charcoal. After cooling and filtering the
products were re-crystallized.
7.1.4.3. 1,4-Bis[(5(6)-methyl-1(H )-benzimidazol-2-ylthio)acetyl]
piperazine 17 Yield e 62% (Method A), 70% (Method B); mp.
137e139 ꢀC; re-crystallized with ethanol; Rf ¼ 0.48; mobile
1
phase, benzene:methanol ¼ 3:1; H NMR (DMSO-d6): 2.38 (s,
6H, 2CH3); 3.58 (m, 8H, 2CH2eNeCH2); 4.43 (s, 4H,
2SCH2CO); 6.93 (dd, 2H, Bzi, J ¼ 8.16 Hz); 7.21e7.29 (m,
4H, Bzi); 12.46 (br s, 2H, 2NH). Analysis: calculated for
C24H26N6O2S2: C e 58.28, H e 5.30, N e 16.99, S e 12.97;
found: C e 58.15, H e 5.42, N e 16.81, S e 13.09.
7.1.4. General procedures for compounds 15e26
7.1.4.4. 2-{[2-Oxo-2-(4-benzhydrylpiperazin-1-yl)ethyl]thio}-
1H-benzimidazole 18 Yield e 58% (Method A); mp. 194e
196 ꢀC; re-crystallized with ethanol; Rf ¼ 0.62; 1H NMR
Method A: a mixture of 3.16 mmol [1,3]thiazolo[3,2-a]ben-
zimidazol-3(2H )-one and 3.79 mmol N-substituted piperazine
in 12 mL ethyl alcohol was refluxed for 3 h. The solvent was
distillated under reduced pressure and the obtained oil product
was washed with water several times in order to remove the
excess of piperazine. By addition of ethanol to the oil the com-
pounds crystallized out.
Method B: to 3.9 mmol N-substituted piperazine, dissolved
in 5 mL dry pyridine solution of 3.9 mmol DCC in 5 mL pyr-
idine was added. The solution was cooled down to 5e10 ꢀC by
stirring and 3.9 mmol of (1H-benzimidazol-2ylthio)acetic acid
was added. The stirring went on for 20e25 h at the same tem-
perature. The obtained precipitate of dicyclohexylurea was re-
moved through filtration and the native solution was
evaporated to dryness in vacuum. The obtained oil product
was washed with water several times in order to remove the
excess of piperazine, after that ethanol was added and the
compounds crystallized out.
6
(CDCl3): 2.38 (s, 4H, CH2eNe2CH2); 3.50e3.66 (m, 4H,
7
3CH2eNe5CH2); 4.00 (s, 2H, SCH2CO); 4.20 (s, 1H, CH-
(Ph)2); 7.18e7.55 (m, 14H, 2Ph; Bzi); 13C NMR: 167.7
(C]O), 149.2 (N]CeS), 141.8 (2Ce8C, 16C), 139.0
(2Ce26C, 27C), 128.6 (4CHe9C, 13C, 17C, 21C), 127.7
(4CHe10C, 12C, 18C, 20C), 127.2 (2CHe11C, 19C), 122.2
(2CHe30C, 31C), 114.5 (2CHe29C, 32C), 75.7 (7CH), 51.6
(2CH2), 51.2 (6CH2), 46.6 (3CH2), 42.6 (5CH2), 33.5 (22CH2);
ESI mass spectral data: NOþ e m/z 443 (100); NNaþ e m/z
465 (12); MS, m/z (%): 443 (100), 357, 167. Analysis: calcu-
lated for C26H26N4OS2: C e 70.56, H e 5.92, N e 12.66; found:
C e 70.71, H e 6.10, N e 12.83.
7.1.4.5. 2-{[2-Oxo-2-(4-benzhydrylpiperazin-1-yl)ethyl]thio}-
5(6)-methyl-1H-benzimidazole 19 Yield e 57% (Method A),
64% (Method B); mp. 149e151 ꢀC; re-crystallized with ethanol;
Rf ¼ 0.60; mobile phase, benzene:methanol ¼ 3:1; 1H NMR
2
7.1.4.1. 2-[(2-Oxo-2-piperazin-1-ylethyl)thio]-1H-benzimidazole
15 Piperazine was used in twofold excess; yield e 45% (Method
A), 53%(MethodB);mp.93e96 ꢀC;re-crystallizedwithethanol;
Rf ¼ 0.81; mobile phase, benzene:methanol ¼ 3:1; 1H NMR
(CDCl3): 2.44 (m, 7HeCH3 and CH2eNe6CH2); 3.59e3.72
3
(m, 4H, CH2eNe5CH2); 3.95 (s, 2H, SCH2CO); 4.25 (s 1H,
7CHe(Ph)2); 7.01e7.42 (m, 13H, Ph and Bzi); 13C NMR: 167.6
(C]O), 148.6 (N]CeS), 141.8 (2Ce8C, 16C), 139.0 (2Ce26C,
27C), 132.0 (30C), 128.6 (4CHe9C, 13C, 17C, 21C), 127.7
(4CHe10C, 12C, 18C, 20C), 127.2 (2CHe11C, 19C), 123.6
(31CH), 113.9 (2CHe29C, 32C), 75.7 (7CH), 51.6 (2CH2), 51.2
(6CH2), 46.6(3CH2), 42.5(5CH2), 33.7(22CH2), 21.5(CH3). Anal-
ysis: calculated for C27H28N4OS: C e71.02, H e6.18, N e12.27,
S e 7.02; found: C e 70.15, H e 6.09, N e 12.19, S e 7.21.
2
(DMSO-d6): 2.61e2.74 (m, 4H, CH2eNHe6CH2); 3.37e3.60
3
(m, 4H, CH2eNHe5CH2); 4.39 (s, 2H, SCH2CO); 7.08e7.15
(m, 2He17CH, 18CH, Arebz); 7.43 (m, 2He16CH, 19CH, Are
bz); 7.45 (br s, NH); 13C NMR: 165.5 (C]O), 149.9 (C]Ne
S), 140.8 (2Ce13C, 14C, Ar), 121.4 (2CHe17CH, 18CH, Ar),
113.7 (2CHe16CH, 19CH, Ar), 46.93 (2CH2eNH), 45.73 (6CH2e
NH), 45.32 (3CH2eN), 42.82 (5CH2eN), 35.18 (9CH2eS).
Analysis: calculated for C13H16N4OS: C e 56.50, H e 5.84, N
e 20.27, S e 11.60; found: C e 56.68, H e 5.61, N e 20.39, S
e 11.48.
7.1.4.6. 2-({2-[4-(4-Methylphenyl)piperazin-1-yl]-2-oxoethyl}thio)
-1H-benzimidazole 20 Yield e 68% (Method A); mp. 183e
185 ꢀC; re-crystallized with ethanol; Rf ¼ 0.38; mobile phase,
1
benzene:methanol ¼ 3:1; H NMR (CDCl3): 2.28 (s, 3H, CH3);
7.1.4.2. 1,4-Bis[(1H-benzimidazol-2-ylthio)acetyl]piperazine
16 The ratio between 9 and piperazine was 2:1; yield e 48%
(Method A); mp. 275e277 ꢀC; re-crystallized with ethanol;
Rf ¼ 0.61; mobile phase, benzene:methanol ¼ 3:1; 1H NMR
(CDCl3): 2.88e2.97 (m, 4H, 2CH2eNHe6CH2); 3.54e3.71
(m, 4H, 3CH2eNHe5CH2); 3.95 (s, 4H, 2SCH2CO); 7.18e
7.23 (m, 8H, 2Arebz); 7.56 (br s, 2H, 2NH); ESI, m/z (%):
3.12e3.19 (m, 4H 2CH2eNHe6CH2); 3.72e3.87 (m, 4H,
3CH2eNe5CH2); 4.10 (s, 2H, SCH2CO); 6.82e6.85 (m,
2He8CH, 12CH); 7.08e7.11 (m, 2He9CH, 11CH); 7.18e7.24
(m, 2He24CH, 25CH); 7.52e7.57 (m, 2He23CH, 26CH); 13C
NMR: 167.7 (C]O), 149.2 (N]CeS), 148.4 (7C), 138.9 (20C,
9
21C), 130.4 (10C), 129.8 (2CHe11CH, CH), 122.4 (2CHe24CH,
25CH), 117.1 (2CHe8CH, 12CH), 114.3 (2CHe23CH, 26CH),
50.1 (2CH2), 49.8 (6CH2), 46.5 (3CH2), 42.4 (5CH2), 33.8
(16CH2), 20.4 (CH3); ESI, m/z (%): NOþ e 367 (100); NNaþ e
NOþ e 467 (100); NNaþ e 489 (10); MS, m/z (%): NOþ
467 (100), 277, 191, 163, 118. Analysis: calculated for
e

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
1419
389 (28); MS, m/z(%): NOþ e367 (100), 177, 134, 119. Analysis:
calculated for C20H22N4OS: C e 65.55, H e 6.05, N e 15.29;
found: C e 65.39, H e 6.20, N e 15.37.
(Method B); mp. 210e213 ꢀC (decomp.); Rf ¼ 0.66; mobile
phase, benzene:methanol ¼ 3:1; 1H NMR (DMSO-d6): 2.19 (s,
3H, CH3); 3.09 (m, 4H, 2CH2eNe6CH2); 3.65 (m, 4H, 3CH2e
Ne5CH2); 4.53 (s, 2H, SCH2CO); 6.86 (m, 2He8CH, 12CH,
Ar); 7.03 (m, 2He9CH, 11CH, Ar); 7.57 (d, 1H, 26CH,
J ¼ 8.16 Hz); 8.04 (br d, 1H, 25CH, J ¼ 8.16 Hz); 8.28 (br s,
1H, 23CH); 13.2 (br s, 1H, NH); 13C NMR: 165.3 (C]O),
156.2 (N]CeS), 148.7 (7C), 143.7 (21C), 142.1 (24C), 139.6
(20C), 129.5 (2CHe9C, 11C), 128.3 (10C), 117.5 (25CH), 116.2
(2CHe8C, 12C), 113.4 (26CH), 110.3 (23CH), 49.08 (2CH2),
48.68 (6CH2), 45.37 (3CH2), 41.57 (5CH2), 35.29 (16CH2), 20.1
(CH3). Analysis: calculated for C20H21N5O3S: C e 58.38, H e
5.14, N e 17.02.
7.1.4.7. 2-({2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)
-1H-benzimidazole 21 Yield e 70% (Method A); mp. 187e
188 ꢀC; re-crystallized with ethanol; Rf ¼ 0.50; mobile phase,
1
benzene:methanol ¼ 3:1; H NMR (CDCl3): 3.09e3.15 (m, 4H,
3
2CH2eNe6CH2); 3.69e3.85 (m, 4H, CH2eNe5CH2); 4.16 (s,
2H, SCH2CO); 6.78e6.83 (m, 2He8CH, 12CH); 7.18e7.26 (m,
4He9CH, 11CH, 24CH and 25CH); 7.49e7.54 (m, 2He23CH,
26CH); 13C NMR: 167.9 (C]O), 149.2 (N]CeS), 149.1 (7C),
9
139.2 (2Ce20C, 21C), 129.1 (2CHe11C, C), 125.8 (10C), 122.4
(2CHe24C, 25C), 117.9 (2CHe8C, 12C), 114.3 (2CHe23C, 26C),
49.5 (2CH2), 49.2 (6CH2), 46.3 (3CH2), 42.2 (5CH2), 33.8
(16CH2); ESI, m/z (%): NOþ e 387 (100); NNaþ e 409 (12);
MS, m/z (%): NOþ e 387 (100), 197, 154, 119. Analysis: calcu-
lated for C19H19ClN4OS: C e 58.98, H e 4.95, N e 14.48; found:
C e 58.81, H e 5.07, N e 14.59.
7.1.4.11. 2-({2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)
-5(6)-nitro-1(H )-benzimidazole 25 Yield e 65% (MethodA), 71%
(Method B); mp. 240e242 ꢀC (decomp.); Rf ¼ 0.67; mobile
1
phase, benzene:methanol ¼ 3:1; H NMR (DMSO-d6): 3.09e
3.21 (m, 4H, 2CH2eNe6CH2); 3.59e3.71 (m, 4H, 3CH2e
8
Ne5CH2); 4.54 (s, 2H, SCH2CO); 6.98 (m, 2H, CH j 12CH);
9
7.1.4.8. 2-({2-[4-(4-Methylphenyl)piperazin-1-yl]-2-oxoethyl}thio)
-5(6)-methyl-1(H )-benzimidazole 22 The ratio between 10 and pi-
perazine was 1:2; yield e 68% (Method A), 74% (Method B); mp.
180e182 ꢀC; re-crystallized with ethanol; Rf ¼ 0.68; mobile phase,
7.26 (m, 2H, CH j 11CH); 7.59 (d, 1H, 26CH, J ¼ 8.23 Hz);
8.05 (dd, 1H, 25CH, J ¼ 1.9, 8.23 Hz); 8.29 (d, 1H, 23CH,
J ¼ 1.9 Hz); 13C NMR: 166.4 (C]O), 157.2 (N]CeS), 150.5
(7C), 146.5 (21C), 143.1 (24C), 142.6 (20C), 129.7 (2CHe9C,
11C), 123.8 (10C), 118.4 (25CH), 118.3 (2CHe8C, 12C), 114.5
(26CH), 111.0 (23CH), 49.20 (2CH2), 48.85 (6CH2), 46.12
(3CH2), 42.35 (5CH2), 36.18 (16CH2). Analysis: calculated for
C19H18ClN5O3S: C e 52.84, H e 4.20, N e 16.22, S e 7.42;
found: C e 52.99, H e 4.20, N e 16.41, S e 7.31.
1
benzene:methanol ¼ 3:1; H NMR (CDCl3): 2.28 (s, 3H, CH3e
Ar); 2.44 (s, 3H, CH3eBzi); 3.09e3.15 (m, 4H, 2CH2e
3
Ne6CH2); 3.60e3.79 (m, 2H, CH2eNe5CH2); 4.08 (s, 2H,
8
SCH2CO); 6.79e6.85 (m, 2H, CH j 12CH); 7.00 (dd, 1H, 25H,
J ¼ 1.37, 8.22 Hz); 7.05e7.09 (m, 2H, 9H and 11H); 7.27 (d, 1H,
23H, J ¼ 1.37 Hz); 7.39 (d, 1H, 26H, J ¼ 8.22 Hz); 13C NMR:
167.5 (C]O), 148.6 (N]CeS), 148.4 (7C), 139.0 (20C), 137.7
(21C), 132.1 (24C), 130.3 (10C), 129.7 (2CHe9C, 11C), 123.7
(25CH), 117.0 (2CHe8C, 12C), 114.1 (26CH), 113.9 (23CH), 49.9
(2CH2), 49.7 (6CH2), 46.3 (3CH2), 42.3 (5CH2), 34.0 (16CH2),
21.5 (27CH3), 20.4 (15CH3); ESI, m/z (%): NOþ e 381 (100),
NNaþ e403 (31); MS, m/z: 205, 177, 133, 177, 134, 119. Analysis:
calculated for C21H24N4OS: C e 66.29, H e 6.36, N e 14.72;
found: C e 66.19, H e 6.51, N e 14.91.
7.1.4.12. 2-({2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)
-5(6)-chloro-1(H )-benzimidazole 26 Yield e 63% (Method A);
mp. 176e179 ꢀC, Rf ¼ 0.78; mobile phase, benzene:methanol ¼
1
2
3:1; H NMR (DMSO-d6): 3.13e3.24 (m, 4H, CH2eNe6CH2);
3.61e3.71 (m, 4H, 3CH2eNe5CH2); 4.47 (s, 2H, SCH2CO);
6.98 (m, 2H, CH and 12CH); 7.14 (dd, 1H, 25CH, J ¼ 1.96,
8
8.51 Hz); 7.26 (m, 2H, CH and 11CH); 7.41e7.49 (2H, 23CH j
9
26CH, overlapped); 12.74 (br s, 1H, NH). Analysis: calculated
for C19H18Cl2N4OS: C e 54.16, H e 4.31, N e 13.30, S e
7.61; found: C e 54.39, H e 4.45, N e 13.12, S e 7.49.
7.1.4.9. 2-({2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-oxoethyl}thio)
-5(6)-methyl-1(H )-benzimidazole 23 Yield e 69% (Method A);
mp. 172e174 ꢀC; re-crystallized with ethanol; Rf ¼ 0.73; mobile
1
phase, benzene:methanol ¼ 3:1; H NMR (CDCl3): 2.39 (s, 3H,
7.2. Biological screening
CH3); 3.04 (m, 4H, 2CH2eNe6CH2); 3.59e3.740 (m, 4H,
8
3CH2eNe5CH2); 4.13 (s, 2H, SCH2CO); 6.74 (m, 2H, CH and
7.2.1. Antitrichinellosis activity
12CH); 6.96e6.99 (d, 1H, 25CH, J ¼ 7.99 Hz); 7.18 (m, 2H, 9CH
and 11CH); 7.26 (br s, 1H, 23CH); 7.38 (d, 1H, 26CH,
J ¼ 7.99 Hz); 10.78 (br s, 1H, NH); 13C NMR: 167.4 (C]O),
149.1 (N]CeS), 148.5 (7C), 139.0 (20C), 137.5 (21C), 132.2
(24C), 129.0 (2CHe9C, 11C), 125.5 (10C), 123.8 (25CH), 117.8
(2CHe8C, 12C), 114.1 (26CH), 113.8 (23CH), 49.3 (2CH2), 49.0
(6CH2), 46.0 (3CH2), 42.1 (5CH2), 34.1 (16CH2), 21.6 (CH3). Anal-
ysis: calculated for C20H21ClN4OS: C e 59.91, H e 5.28, N e
13.97; found: C e 60.07, H e 5.18, N e 13.71.
The parasitological pharmaco-therapeutical experiments in
vitro for antitrichinellosis activity of investigated compounds
were carried out according to Campbell’s method [11,12].
Encapsulated T. spiralis larvae were used in the parasito-
logical pharmaco-therapeutic experiment in vitro, 100 speci-
mens for 1 mL physiological solution. The tested
benzimidazole derivatives were dissolved in DMSOewater.
The used concentrations are given in Table 3. The samples
were incubated in ‘‘humid’’ chamber with thermostat at
37 ꢀC. The control for vitality of the T. spiralis larvae was car-
ried out 24 h and 48 h after the treatment using stereomicro-
scope MBC-9.
7.1.4.10. 2-({2-[4-(4-Methylphenyl)piperazin-1-yl]-2-oxoethyl}thio)
-5(6)-nitro-1(H )-benzimidazole 24 Yield e 60% (Method A), 68%

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A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 41 (2006) 1412e1420
7.2.2. Antihelminthic activity against S. obvelata
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mice for every one of the tested compounds. Each of the con-
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7.2.3. Hepatotoxicity test
Rathepatocyteswereusedtoexaminethe hepatotoxicityofthe
synthesized compounds. The rat was anesthetized with sodium
pentobarbital. In situ liver perfusion and cell isolation were per-
formed (Fau etal. [24]). Thecells were counted under microscope
and the viability was estimated by trypan blue (0.05%) exclusion.
Theinitialviabilityaveraged90%. Incubationswerecarriedoutin
25-mL Erlenmeyer flasks. The cells were diluted with Krebse
Ringer-carbonate buffer, pH ¼ 7.4. Each flask contained 3 mL
of the cell suspension (i.e., 9 ꢄ 106 hepatocytes) and the corre-
sponding compound in concentration of 250 mM. The incubation
was performed under a carbogen atmosphere.
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Acknowledgement
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Thanks are due to National Science Fund/Ministry of Edu-
cation and Science grant X1408 and Science Fund/Chemical
Technology and Metallurgy grant 10135 for financial support
of this work.
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