Synthesis, antitrichinnellosis and antiprotozoal activity of some novel thieno[2,3-d]pyrimidin-4(3H)-ones containing benzimidazole ring

Article abstract of DOI:10.1016/j.ejmech.2010.09.050

Some novel thieno[2,3-d]pyrimidin-4(3H)-ones containing benzimidazol-2-yl-thioethyl- and benzimidazol-2-yl-methanethioethyl moiety in second position of the pyrimidine ring were synthesized in order to determine their antitrichinellosis and antiprotozoal effects. The structures of the compounds were confirmed by IR, ¹H NMR and elemental analysis. The antiparasitic screening showed that the benzimidazole derivatives of thieno[2,3-d]pyrimidin-4(3H)-ones exhibited higher activity against Trichinella spiralis in vitro in comparison albendazole. The most active compound, 2-[2-(5-nitro-1H-benzimidazol-1-yl)ethyl]-5,6,7,8-tetrahydro[1]benzothieno[2, 3-d]pyrimidin-4(3H)-one 22 revealed 95% activity at a dosage of 5 mg/kg mw after 24 h, while compounds 8 and 10 applied at the same dose showed efficacy of 90% after 48 h. The compound 2-{2-[(5(6)-nitro-1H-benzimidazol-2-yl)thio]ethyl}-5,6, 7,8-tetrahydro[1]-benzothieno[2,3-d]pyrimidin-4(3H)-one 11 exhibited 90% efficacy after 24 h. The pharmaco-therapeutic study in vivo on invaded with Lamblia muris white mice showed 100% effectiveness of the compounds 8, 10, 11, 13-15 and 22, 23 after five-days-treatment course.

Full text of DOI:10.1016/j.ejmech.2010.09.050

European Journal of Medicinal Chemistry 45 (2010) 5856e5861
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitrichinnellosis and antiprotozoal activity of some novel
thieno[2,3-d]pyrimidin-4(3H)-ones containing benzimidazole ring
,
b
a
c
Anelia Ts. Mavrova a , Dimitar Vuchev , Kameliya Anichina , Nikolay Vassilev
*
a Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
b Medicinal University, Section of Parasitology and Tropical deseases, Plovdiv, Bulgaria
c Institute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2010
Received in revised form
20 September 2010
Accepted 21 September 2010
Available online 25 September 2010
Some novel thieno[2,3-d]pyrimidin-4(3H)-ones containing benzimidazol-2-yl-thioethyl- and benzimi-
dazol-2-yl-methanethioethyl moiety in second position of the pyrimidine ring were synthesized in order
to determine their antitrichinellosis and antiprotozoal effects. The structures of the compounds were
confirmed by IR, 1H NMR and elemental analysis.
The antiparasitic screening showed that the benzimidazole derivatives of thieno[2,3-d]pyrimidin-4
(3H)-ones exhibited higher activity against Trichinella spiralis in vitro in comparison albendazole. The
most active compound, 2-[2-(5-nitro-1H-benzimidazol-1-yl)ethyl]-5,6,7,8-tetrahydro[1]benzothieno
[2,3-d]pyrimidin-4(3H)-one 22 revealed 95% activity at a dosage of 5 mg/kg mw after 24 h, while
compounds 8 and 10 applied at the same dose showed efficacy of 90% after 48 h. The compound 2-{2-[(5
(6)-nitro-1H-benzimidazol-2-yl)thio]ethyl}-5,6,7,8-tetrahydro[1]-benzothieno[2,3-d]pyrimidin-4(3H)-
one 11 exhibited 90% efficacy after 24 h.
Keywords:
Benzimidazoles
Thieno[2,3-d]pyrimidin-4(3H)-ones
Antitrichinellosis activity
Antiprotozoal activity
The pharmaco-therapeutic study in vivo on invaded with Lamblia muris white mice showed 100%
effectiveness of the compounds 8, 10, 11, 13e15 and 22, 23 after five-days-treatment course.
Ó 2010 Published by Elsevier Masson SAS.
1. Introduction
derivatives were shown to be active in vitro against protozoan
parasites as Trichomonas vaginalis, Giardia lamblia, Entamoeba
Thieno[2,3-d]pyrimidines are an important group of biolog-
ical active compounds due to their diverse pharmacological
properties as antitumour [1,2], antiviral [3], antibacterial [4] and
antituberculosis agents [5] and their synthesis has been in focus
of considerable attention in the synthetic organic chemistry as
well as in the medicinal chemistry. Many thieno[2,3-d]pyrrimi-
din-4-ones were synthesized as potential inhibitors of Pneu-
mocystis carinii and Toxoplasma gondii dihydrofolate reductase
[6] as well as high inhibitory effect against FP-2 of Plasmodium
falciparum [7].
hystolica Leishmania major and Acanthamoeba polyphaga, others
were active in vitro against Trypanosoma brucei rhodesience and
Plasmodium Falciparum [12e15]. Following, mebendazole cam-
bendazole, oxfendazole, oxibendazole and flubendazole many
benzimidazole derivatives have been studied for their antiparasitic
properties [16,17]. Regardless of the high efficacy of the above
mentioned drugs the definitive treatment of trichinellosis, one of
the most disseminated tissue helmintosis and as well as the
problem with the medication of protozoa diseases, remains
pending.
Benzimidazole is another fused heteroaromatic system of great
interest. Benzimidazole derivatives occupied unique place in
medicinal chemistry. The demonstration of a potent biological
activity from the different substituted benzimidazoles as antiviral
[8,9], anticancer agents [10e11] support further the importance of
benzimidazol-2-yl-thiol moiety as pharmacophore for generating
new chemotherapeutical agents. Selected benzimidazole
On the base of the above mentioned facts it is of a pharma-
cological interest to synthesized new derivatives of benzimid-
azole-2-thiols, containing thieno[2,3-d]pyrimidin-4-one moiety
and to perform preliminary study of their antiparasitic proper-
ties. Probably the introduction of benzimidazole ring in the
structure of a thienopyrimidinone molecule can contribute to
its interaction with the biological target. In this paper we
describe the synthesis of new thienopyrimidinone derivatives of
1H-benzimidazol-2-yl-thiole and their activity in vitro against
Trichinella spiralis as well as their effect against Paramaecium
caudatum and Lamblia muris.
* Corresponding author. Tel.: þ35928163207; fax: þ3592685488.
E-mail address: anmav@abv.bg (A.Ts. Mavrova).
0223-5234/$ e see front matter Ó 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.09.050

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 45 (2010) 5856e5861
5857
2. Chemistry
potassium carbonate in acetonitrile as well as in the presence of
sodium hydroxide and resulted to the formation of compounds
22e24.
The synthesis of thieno[2,3-d]pyrimidin-4(3H)-one derivatives,
containing benzimidazole ring is illustrated and outlined in Fig. 1.
The starting 3-ethoxycarbonyl-2-amino-thiophenes 3e4 were
synthesized according to the method, described by Gewaldt et al.
[18]. 2-(2-Chloroethyl)-thieno[2,3-d]pyrimidin-4(3H)-ones 5e6
were obtained by passing a stream of dry hydrogen chloride gas
through a solution of the appropriate 2-amino-3-ethoxycarbonyl-
thiophene and 3-chloro-propanonitrile.
The structures of all new compounds were established by IR, 1H
NMR as well as elemental analysis. Detailed assignment of the 1H
NMR and some of the 13C NMR spectra of the synthesized
compounds is given in the Experimental. The elemental analyses
indicated by the symbols of the element were within ꢀ0.4% of the
theoretical values.
The nucleophilic substitution between the thieno[2,3-d]
pyrimidinones 5e6 and the appropriated benzimidazol-2-ylth-
iols in the presence of sodium hydroxide resulted in the
formation of 7e17 and the reaction of 5e6 with benzimidazol-
2-yl-methanethiols yielded in compounds 18e21. The interac-
tion between the starting thienopyrimidin-4-ones and 4-(un)
substituted-1H-benzimidazoles was performed out in the
presence of tetrabutylammonium bromide (TBAB) and dry
3. Pharmacology
3.1. Antihelmintic activity
The parasitological study in vitro showed that most of the tested
compounds possess higher activity than the activity of albendazole
against T. spiralis. The results are given in Table 1.
O
R
COOC2H5
NH2
R
OC2H5
R
a
b
R1
R1
R1
O
S
S
NH
C
3 - 4
1 - 2
HCl .HN
Cl
3: R = R1 = (CH2)4;
1: R + R1 = (CH2)4
2: R= CH3; R1 = COOC2H5
4: R = CH3, R1 = COOC2H5
O
R
O
R
b
e (f)
NH
NH
R1
R1
S
N
S
N
Cl
N
N
5 - 6
5: R = R1 = (CH2)4;
6: R = CH3, R1 = COOC2H5
R3
22 - 24
22: R = R1 = (CH2)4, R3 = H;
23: R =CH3, R1 = COOC2H5, R3 =H
24: R =CH3, R1 = COOC2H5, R3 = NO2
d
c
R3
O
R
O
N
R
NH
R1
NH
N
R1
N
S
N
S
S
N
S
R2
R3
N
7 -17
R2
18 - 21
7: R + R1 = (CH2)4, R2 = R3 = H;
8: R + R1 = (CH2)4, R2 = CH3, R3 = H;
18: R = R1 = (CH2)4, R2 = R3 = H;
9: R + R1 = (CH2)4, R2 = C2H5, R3 = H;
19: R = R1 = (CH2)4, R2 = H, R3 = CH3;
10: R + R1 = (CH2)4, R2 = C3H7, R3 = H;
11: R + R1 = (CH2)4, R2 = NO2, R3 = H;
12: R = CH3, R1 = COOC2H5, R2 = R3 = H;
13:R = CH3, R1 = COOC2H5, R2 =CH3; R3 = H;
14: R = CH3, R1 = COOC2H5, R2 = C2H5, R3 = H;
15: R = CH3, R1 = COOC2H5, R2 = H, R3 = Cl;
16: R = CH3, R1 = COOC2H5, R2 = H, R3 = NO2;
17: R = CH3, R1 = COOC2H5, R2 = H, R3 = CH3
20:R = CH3, R1 = COOC2H5, R2 = R3 = H;
21: R = CH3, R1 = COOC2H5, R3 = CH3;
Fig. 1. Synthesis of benzimidazole derivatives of thieno[2,3-d]pyrimidin-4(3H)-ones. Regents and conditions: (a) ethyl cyanoacetate, sulfur, diethyl amine, 20 or 60 ꢁC; (b) dioxan, 2-
chloro-propionitrile, dry hydrogen chloride gas; 20 ꢁC; (c) NaOH, benzimidazol-1-ylthiol; reflux; (d) NaOH, benzimidazol-2-ylmethanethiol, reflux; (e) ethanol, NaOH, benz-
imidazoles, reflux; (f) acetonitrile, benzimidazole, K2CO3, TBAB.

5858
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 45 (2010) 5856e5861
Table 1
activity in comparison to the compounds 14e16 and 23. The
compound 22 comprising unsubstituted benzimidazole ring, but
not benzimidazolthiols moiety was distinguished for the highest
activity e 95.1%, while compound 23 exhibited 85.3% larvocide effect.
From the thienopyrimidinones, containing benzimidazolthioethyl
group the highest activity after 24 h revealed compound 11 e 90.1%,
while compounds 8 and 10 possessed the same effect after 48 h.
Due to the obtained results for the antirichinellosis activity,
further research is currently in progress for the antirichinellosis
activity in intestinal and muscle phase.
Antihelminthicactivity of compounds 8, 10, 11, 14e16 and 22, 23 against Trichinella
spiralis.
b
Comp.
Efficacy (%) a after 24 h
Efficacy (%) after 48 h
g/ml
(20 g/ml)
5
m
g/ml
5 m
(20
m
g/ml) c
m
8
10
11
14
40.05
84.80
90.10
39.07
0.00
89.90
90.05
90.10
59.75
5.09
15
16
22
23
50.00
95.05
79.80
10.6
80.05
95.05
85.30
14.8
4.2. Antiprotozoal activity in vitro
Six of the new synthesized compounds, 8, 10e11, 14e16 and
22e23 were subjected to a preliminary test for antiprotozoal
activity in vitro on Paramaecium caudatum. Immediately after the
treatment, the motions of the Paramaecia became more intensively
and chaotically, but after 2 min they began to grow weaker and
slower. After 15e20 min the paramaecia cells were motionless and
the cell content becomes turbid. A multitude of drop formations of
the cell content, permeated through the cell membrane appeared
on membrane outside, probably due to cell membrane lysis. That
phenomenon was an indicator for the antiprotozoal activity of the
tested compounds.
Albendazole
a
Control e 96 parasites.
p < 0.05.
concentration of albendazole.
b
c
3.2. Antiprotozoal activity
An indicator test for antiprotozoal activity against Paramaecium
caudatum was carried out. The response of Paramaecium caudatum
was observed by means of electronic microscope.
3.3. Effect against Lamblia muris
4.3. Antiprotozoal activity in vivo
The study was performed with white mice, invaded with Lam-
blia muris. The invaded mice were treated with the tested
compounds and the mice faeces were studied for availability of
Lamblia muris cysts.
The compounds mentioned above demonstrated high efficacy in
the implemented study for antiprotozoal activity.
The screening in vivo was performed using immature white
mice, infected at equal conditions with Lamblia muris. The mice
faeces were examined stained with Lugol’s solution and observed
under microscope for the availability of Lamblia cysts. The invaded
animals were separated from the other mice in a 40-member
group.
Thirty mice were divided in six groups, each of them of five
mice. The six groups of mice were treated p. o. with each one of the
compounds 8, 10e11, 14e16 and 22e23 at a dose of 0.5
mg/ml pro
die for 5 days cure course. Five mice were not treated and were used
as control samples. One group of five mice was treated only with
DMSO. In the control study, accomplished five-days after treatment
no Lamblia muris cysts were observed in each group of the treated
with the compounds mice. That indicated a significant anti-
protozoal activity. In the control group and in the group treated
with DMSO an increasing of Lamblia muris was determined.
4. Results and discussion
To synthesize thieno[2,3-d]pyrimidin-4(3H)-one containing
benzimidazole moiety we use the nucleophillic substitution of
different substituted benzimidazol-2-ylthiols as well as benzimi-
dazol-2-yl-methanethiols with suitable substituted thienopyr-
imidinones. The reaction between compounds 5e6 and the
respective benzimidazol-2-ylthiols was carried out in ethanol
medium under reflux for 3e6 h to afford compounds 7e21.
The molar ratio thienopyrimidinone/benzimidazolthiols/sodium
hydroxide was 1.0:1.0:1.8 and in the reaction of 5e6 and the ben-
zimidazol-2-yl-methanethiols the molar ratio was 1.14:1.0:2.4. The
yields of compounds 7e21 were in the range 49e80%, while that of
compounds 22e24 were between 49 and 90%.
5. Conclusion
4.1. Antihelminthic activity
New benzimidazole derivatives of thieno[2,3-d]pyrimidin-4-
ones were synthesized and studied for their antiparasitic activity.
The in vitro screening for antinematode effect showed that the
compounds exhibit significant antiparasitic activity at concentra-
The parasitological experiment in vitro ascertained that the
tested compounds possessed a different antihelminthic effect
expressed in suppressing the motor activity of Trichinella larvae
and in losing of the spiral form, which is a mark for not viability. In
control samples both in physiological solution and in DMSO prac-
tically all Trichinella larvae were in spiral form namely vital. The
observed difference between the antitrichinellosis efficacy of the
compounds and that of albendazole was statistically significant
(p < 0.05). The results obtained by the in vitro test for the activity of
the compounds 8, 10e11, 14e16 and 22e23 proved the efficacy of
the tested compounds (excluding compound 15). It deserves to be
underlined that the studied compounds revealed higher activity
tion of 5 mg/ml. All tested benzimidazole derivatives of 4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine excluding compound 15 exhibited
higher activity against Trichinella spiralis larvae in comparison to
albendazol, furthermore some of the compounds demonstrated
antitrichinellosis activity, which surpassed many times that of
albendazole.
The indicator test on Paramaecium caudatum revealed anti-
protozoal activity of all tested compounds. The pharmaco-thera-
peutic study in vivo on invaded with Lamblia muris white mice,
showed 100% effectiveness of compounds after five-days-treat-
ment course. During the control microscopic study of mice faeces
no Lamblia muris cysts were found. That fact indicated that all
studied compounds possess remarkable antiprotozoal efficacy.
than albendazole at concentration of 5
was used at concentration of 20 g/ml.
mg/ml, while albendazole
m
The compounds 8, 10e11 and 22 containing tetrahy-
drobenzothienopyrimidin-4-one cycle showed higher antihelminthic

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 45 (2010) 5856e5861
5859
These results confirmed also the hypothesis that the introduc-
6.2. General procedure for preparation of compounds 7e17
tion of a1H-benzimidazolyl-2-thiol as well as 1-H-benzimidazole
moiety in the structure of thieno[2,3-d]pyrimidine ring is favour-
able to the interaction of these molecules with the biological
targets.
To a solution of 0.13 g (3.3 mmol) sodium hydroxide in ethanol
(5 ml), (1.8 mmol) of 1,5-(un)substituted-2-mercapto-benzimid-
azole 3aeg was added. The solution was refluxed 1 h and 1.8 mmol
of thieno[2,3-d]pyrimidin-4(3H)-one (2aeb) was added and the
mixture was heated by reflux for two-three hours. The reaction
solution was concentrated under reduced pressure. To the dry
residue was added diethyl ether and the obtained precipitate was
filtered.
6. Experimental part
Melting points (mp) were determined on an Electrothermal AZ
9000 3MK4 apparatus and were uncorrected. The thin layer chro-
matography (TLC, Rf values) was performed on F254 or silica gel
plates F254 (Merck, 0.2 mm thick) and visualization was effected
with ultraviolet light. IR spectra were recorded on a Bruker Equinox
55 spectrophotometer as potassium bromide discs. All NMR spectra
were recorded on a Bruker Avance DRX 250 spectrometer (Bruker,
Faelanden, Switzerland) operating at 250.13 MHz for 1H and
62.89 MHz for 13C, using a dual 5 mm 1H/13C probehead. Chemical
shifts were expressed relative to tetramethylsilane (TMS) and were
6.2.1. 2-[2-(1H-benzimidazol-2-ylthio)ethyl]-5,6,7,8-tetrahydro[1]
benzothieno[2,3-d]pyrimidin-4(3H)-one (7)
Yield: 66.4%; Mp: 213e215 ꢁC; re-crystallized with ethanol;
Rf ¼ 0.55, mobile phase: chloroform/heptane/ethanol e 3:2:0.5; 1H
NMR (DMSO): 1.74 (m, 4H, (CH2)2eBzth); 2.7 (bt, 2H, CH2); 2.85 (bt,
2H, CH2); 3.08 (t, 2H, CH2ePyr); 4.25(t, 2H, CH2eS); 7.23e7.48(m,
4H, Ar); 12.29 (s, 1H, NH, exchangeable with D2O); Analysis: C,
59.66; H, 4.74; N, 14.65; S, 16.77; Found: C, 59.69; H, 4.69; N, 14.55;
S, 16.81.
reported as
d (ppm). The measurements were carried out at
ambient temperature (300 K). The microanalyses for C, H, N and S
were performed on PerkineElmer elemental analyzer.
The 2-amino-thiophenes (3e4) were synthesized by conden-
sation of cyclohexanone or ethyl acetoacetate with ethyl cyanoa-
cetate and sulfur in the presence of diethyl amine according to [18].
The 1-(un)substituted-2-mercapto-benzimidazoles were synthe-
sized in good yields through two methods, namely by fusing 1-alkyl-
benzimidazoles with sulfur at 260 ꢁC and by the reaction of 4-(un)
substituted-1,2-diamino-benzene with potassium ethyl-dithiocar-
bonate in the presence of sodium hydroxide in ethanol medium [19].
Benzimidazole, 4-nitro-benzimidazole and 4-chloro-benzimidazole
are commercially available.
6.2.2. 2-{2-[(1-methyl-1H-benzimidazol-2-yl)thio]ethyl}-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (8)
Yield: 68.6%; Mp: 228e230 ꢁC, re-crystallized with ethanol;
Rf ¼ 0. 53, mobile phase: chloroform/heptane/ethanol e 3:2:0.5; 1H
NMR (DMSO): 1.75 (m. 4H. 2(CH2)2eBzth); 2.63 (bt, 2H, CH2eBzth);
2 (dt, 4H, 2(CH2)2eBzth, CH2ePyr); 3.69 (s, 3H, CH3); 4.26 (t, 2H,
CH2eS); 7.02 (m, 2H, Ar); 7.45 (m, 2H, Ar); 12.30 (s, 1H, NH,
exchangeable with D2O); Analysis: C, 60.58; H, 5.08; N, 14.13; S,
16.17; Found: C, 60.55; H, 5.11; N, 14.08; S, 16.21.
The synthesis of 5(6)-(un)substituted-benzimidazol-2-yl-meth-
anethiols was performed by refluxing the respective 1,2-diamino-
benzene hydrochloride with mercaptoacetic acid according to the
procedure given in [20].
6.2.3. 2-{2-[(1-ethyl-1H-benzimidazol-2-yl)thio]ethyl}-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (9)
Yield: 62.50%; Mp: 247e249 ꢁC; Rf ¼ 0.55, mobile phase: chlo-
roform/heptane/ethanol e 3:2:0.5; 1H NMR (DMSO): 1. 25 (t,
J ¼ 7.1 Hz, 3H, CH3); 1.75(m, 4H, 4H, (CH2)2eBzth); 2.65 (bt, 2H,
CH2eBzth); 2.86(t, 2H, CH2eBzth); 2.95(t, 2H, CH2ePyr); 4.31 (q,
J ¼ 7.1 Hz, 2H, CH2eN); 4.64 (t, 2H, CH2eS); 7.22 (m, 2H, Ar); 7.51
(m, 2H, Ar); 12.31 (s, 1H, NH, exchangeable with D2O). 13C NMR
(DMSO): 12.7(CH3), 22.2(CH2), 22.6(CH2), 24.4(CH2), 25.5(CH2),
34.0(CH2ePyr), 38.8(CH2eN), 42.1(CH2eS), 109.2(Ar-CH), 109.8(Ar-
CH), 119.8(CeBzth), 122.5(Ar-CH), 122.6(Ar-CH), 127.7(CeBzth),
130.3(Ar-C), 130.8(Ar-C), 131.2(SeC]N), 158.1(SeCeNeBzth), 163.5
(NeC]NeBzth), 167.7(C]O); Analysis: C, 61.43; H, 5.40; N, 13.65;
S, 15.62; Found: C, 61.48; H, 5.36; N, 13.60; S, 15.58.
6.1. General procedure for the preparation of 5e6
A stream of dry hydrogen chloride gas was passed through
a solution of 0.002 mol of compound 3 or 4 and 2.15 g (0.002 mol) of
3-chloro-propanonitrile in dry dioxane for
5 h at ambient
temperature. At the beginning of the reaction a formation of ami-
dine hydrocloride has been observed, which is turned later in the
reaction solution, forming the pyrimidinone ring. The reaction
solution was poured in ice-water (40 ml) and basified with 10%
ammonium hydroxide. The obtained precipitate was filtered and
crystallized from ethanol.
6.2.4. 2-{2-[(1-propyl-1H-benzimidazol-2-yl)thio]ethyl}-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (10)
Yield: 64.2% Mp: 254e256 ꢁC (decomp); Rf ¼ 0.59, mobile
phase: chloroform/heptane/ethanol e 3:2:0.5; 1H NMR (DMSO):
0.89 (t, 3H, CH3); 1.68e1.79 (m, 6H, (CH2)2eBzth, CH2eCH3); 2.66
(bt, 2H, CH2eBzth); 2.86 (bt, 2H, CH2eBzth); 2.96 (t, 2H, CH2ePyr);
4.23 (t, 2H, CH2eN); 4.65 (t, 2H, CH2eS); 7.17e72 (m, 2H, Ar);
7.47e7.53(m, 2H, Ar); 12.34 (s, 1H, NH, exchangeable with D2O);
Analysis: C, 62.23; H, 5.70; N, 13.20; S, 15.10; Found: C, 62.18; H,
5.768; N, 13.25; S, 15.08.
6.1.1. 2-(2-chloroethyl)-5,6,7,8-tetrahydrobenzothieno[2,3-d]
pyrimidin-4(3H)-one (5)
Yield: 85%; mp: 300e302 ꢁC (decomp); re-crystallized with
ethanol; Rf ¼ 0.62; mobile phase: chloroform/heptane/ethanol e
3:2:1; 1H NMR (DMSO): 1.77 (m, 4H, (CH2)2eBzth); 2.72 (bt, 2H,
CH2); 2.85 (bt, 2H, CH2); 3.08 (t, 2H, CH2ePyr); 4.01 (t, 2H, CH2eCl);
12.33 (s, 1H, NH, exchangeable with D2O); Analysis: Calc.: C, 53.63;
H, 4.88; N,10.42; S, 11.93; Found: C, 53.59; H, 4.92; N,10.39; S,11.90.
6.1.2. 2-(2-chloroethyl)-6-ethoxycarbonyl-5-methyl-thieno[2,3-d]
pyrimidin-4(3H)-one (6)
6.2.5. 2-{2-[(5(6)-nitro-1H-benzimidazol-2-yl)thio]ethyl}-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (11)
Yield: 74.2%; Mp: 271e272 ꢁC; re-crystallized with ethanol;
Rf ¼ 0.65, mobile phase: chloroform/heptane/ethanol e 3:2:1; 1H
NMR (DMSO): 1277 (t, 3H, CH3); 2.78 (s, 3H, CH3); 3.08 (t, 2H,
CH2ePyr); 4.232 (q, 2H, eCH2eO); 4.41 (t, 2H, CH2eCl); 12.33 (s,1H,
NH, exchangeable with D2O). Analysis: Calc.: C, 47.92; H, 4.36; N,
9.31; S, 10.66; Found: C, 47.97; H, 4.31; N, 9.38; S, 10.69.
Yield:38.47%;Mp:212e214 ꢁC;re-crystallizedwithetherechloro-
form; Rf ¼ 0.53, mobile phase: ethyl acetate/heptane e 3:2; 1H NMR
(DMSO): 1.74 (m, 4H, 2CH2); 2.72 (m, 4H, 2CH2); 2.84(t. 2H, CH2); 3.01
(t. 2H, CH2); 7.68 (d, 1H, Ar, J3 ¼ 11.37 Hz); 7.79 (m, 2H, Ar); 11.55
(bs, 2H, 2NH, exchangeable with D2O). 13C NMR (DMSO): 21.7(CH2),
22.4(CH2), 24.3(CH2), 25.2(CH2), 32.8(CH2ePyr), 40.5(CH2eS), 103.4

5860
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 45 (2010) 5856e5861
(Ar-CH), 109.5(Ar-CH), 116.4(Ar-CH), 131.1(Ar-C), 139.2(Ar-C), 140.8
(Ar-C), 157.0(SeC]N), 158.5(SeCeNeBzth), 162.6(NeC]NeBzth),
176.4(C]O); Analysis: Calc.: C, 53.38; H, 4.01; N, 16.38; S, 15.00;
Found: C, 53.31; H, 4.06; N, 16.33; S, 15.06.
(CH3), 32.5(CH2ePyr), 42.2(CH2eS), 61.5(CH2O), 106.4(Ar-CH), 110.3
(Ar-CH), 119.4(Ar-CH), 121.4(CeBzth), 122.4(CeBzth), 132.0
(CeBzth), 136.6(Ar-C), 143.5(Ar-C), 143.6(Ar-C), 158.2(SeC]N),
158.4(SeCeNeBzth), 159.4(OeC]O), 162.4(NeC]NeBzth), 166.3
(C]O); Analysis: Calc.: C, 49.66; H, 3.73; N, 15.24; S, 13.96; Found:
C, 49.68; H, 3.70; N, 15.26; S, 13.96.
6.2.6. Ethyl 2-[2-(1H-benzimidazol-2-ylthio)ethyl]-5-methyl-4-
oxo-3,4-dihydrothieno-[2,3-d]pyrimidine-6-carboxylate (12)
Yield: 68.0%; Mp: 251e253 ꢁC; Rf ¼ 0.50, mobile phase: chlo-
roform/heptane/ethanol e 3:2:0.5; 1H NMR: 1.27 (t, 3H, CH3); 2.79
(s, 3H, CH3); 2.88 (t, 2H, CH2ePyr); 4.26 (q, 2H, eCH2eO); 4.6 (t, 2H,
eCH2eS); 6.99e7.48 (m. 3H, Ar); 12.23 (s, 1H, NH, exchangeable
with D2O); Analysis: Calc.: C, 55.05; H, 4.38; N, 13.52; S, 15.47;
Found: C, 55.09; H, 4.40; N, 13.50; S, 15.43.
6.2.11. Ethyl 5-methyl-2-{2-[(5(6)-methyl-1H-benzimidazol-2-yl)
thio]ethyl}-4-oxo-3,4-dihydrothieno [2,3-d]pyrimidine-6-
carboxylate (17)
Yield: 92%; Mp 237e239 ꢁC; Rf ¼ 0.58, mobile phase: chloro-
form/heptane/ethanol e 3:2:1; 1H NMR (DMSO): 1.29 (t, 3H, CH3);
2.49 (s, 3H, CH3); 2.78 (s, 3H, CH3); 2.89 (t, 2H, CH2); 4.24 (q, 2H,
CH2); 4.58 (t, 2H, CH2); 6.97 (m, 2H, Ar); 7.32 (m, 1H, Ar); Analysis:
Calc.: C, 56.06; H, 4.70; N, 13.07; S, 14.97; Found: C, 56.04; H, 4.73;
N, 13.11; S, 14.99.
6.2.7. Ethyl 5-methyl-2-{2-[(1-methyl-1H-benzimidazol-2-yl)thio]
ethyl}-4-oxo-3,4-dihydro-thieno[2,3-d]pyrimidine-6-carboxylate
(13)
Yield: 64.5%; Mp: 244e248 ꢁC (decomp), re-crystallized with
6.3. General procedure for preparation of compounds 18e21
ethanol; Rf
¼
0.68, mobile phase: chloroform/heptane/
ethanol ¼ 3:2:1; 1H NMR (DMSO): 1.27 (t, 3H, CH3); 2.81 (s, 3H,
CH3); 2.87 (t, 2H, CH2); 3.71 (s, 3H, CH3); 4.21 (q, 2H, CH2); 4.64 (t,
2H, 2H, CH2); 7.22 (m, 2H, Ar); 7.43 (m, 2H, Ar); Analysis: Calc. C,
56.06; H, 4.70; N, 13.07; S, 14.97; Found: C, 56.09; H, 4.68; N, 13.05;
S, 14.94.
A solution of 0.46 g (0.012 mol) sodium hydroxide in absolute
ethanol and 0.8 g (4.9 mmol) benzimidazol-2-yl-methylthiol was
refluxed for 1 h. After that 5.6 mmol of the thieno[2,3-d]pyrimidin-
4(3H)-one 5 or 6 was added and the solution was heated by reflux
for 2 h. After cooling the obtained precipitate was filtered and
crystallized from ethanol.
6.2.8. Ethyl 5-methyl-2-{2-[(1-ethyl-1H-benzimidazol-2-yl)thio]
ethyl}-4-oxo-3,4-dihydrothieno-[2,3-d]pyrimidine-6-carboxylate
(14)
6.3.1. 2-(benzimidazol-2-yl)-methylthioethyl-5,6,7,8-
tetrahydrobenzothieno[2,3-d]pyrimidin-4(3H)-one (18)
Yield: 72.4%; Mp-196e198 ꢁC; Rf ¼ 0.48, mobile phase chloro-
Yield: 52.5%; Mp: 236e239 ꢁC, re-crystallization with diethyl
ether/chloroform; Rf ¼ 0.48, mobile phase: ethyl acetate/heptane
3:2; 1H NMR (DMSO): 1.274 (dt, 3H, 2CH3); 2.781(s, 3H, CH3); 2.830
(t, 2H, CH2ePyr); 4.237 (q, 2H, eCH2eN); 4.347 (q, 2H, eCH2eO);
4.634 (t, 2H, eCH2eS); 7.115e7.248 (m. 2H, Ar); 7.434e7.483(m. 2H,
Ar); 12.312 (s, 1H, NH, exchangeable with D2O). 13C NMR (DMSO):
12.7(CH3), 14.2(CH3), 15.4(CH3), 36.0(CH2ePyr), 38.8(CH2eN), 42.6
(CH2eS), 60.0(CH2O), 109.2(Ar-CH), 109.7(Ar-CH), 122.0(Ar-CH),
122.0(Ar-CH), 120.4(CeBzth), 130.8(CeBzth), 131.4(Ar-C), 141.2(Ar-
C), 143.8(Ar-C), 158.0(SeC]N), 158.6(SeCeNeBzth), 159.4(OeC]
O), 163.0(NeC]NeBzth), 167.7(C]O); Analysis: Calc.: C, 56.99; H,
5.01; N, 12.66; S, 14.49; Found: C, 56.96; H, 5.05; N, 12.63; S, 14.51.
form/heptane/ethanol
e
3:2:1; 1H NMR: 1.76 (m, 6H, 4H,
(CH2)2eBzth, CH2ePyr); 2.52 (bt, 2H, CH2eBzth); 2.69(bt, 2H,
CH2eBzth); 2.90 (t, 2H, CH2eS); 3.94 (s, 2h, SeCH2eBzi); 7.31e7.43
(m, 4H, Ar); 12.33 (s, 1H, NH, exchangeable with D2O.) Analysis:
Calc.: C, 60.27; H, 5.56; N, 14.06; S, 16.09; Found: C, 60.21; H, 5.58;
N, 14.09; S, 16.04.
6.3.2. 2-{2-[(5(6)-methyl1-1H-benzimidazol-2-ylmethyl)thio]
ethyl}-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one
(19)
Yield 83%; Mp-168e170 ꢁC; Rf ¼ 0.52, mobile phase chloroform/
heptane/ethanol e 3:2:1; 1H NMR: 1.77(m, 6H, 4H, (CH2)2eBzth,
CH2ePyr); 2.51 (bt, 2H, CH2eBzth); 2.69 (bt, 2H, CH2eBzth); 2.88 (t,
5H, CH2eS, CH3); 3.93 (s, 2H, SeCH2eBzi); 7.31e7.43 (m, 3H, Ar);
12.31 (s, 1H, NH, exchangeable with D2O); Analysis: Calc.; C, 61.43;
H, 5.40; N, 13.65; S, 15.62; Found: C, 61.41; H, 5.43; N, 13.62; 3; S,
15.64.
6.2.9. Ethyl 5-methyl-2-{2-[(5(6)-chloro-1H-benzimidazol-2-yl)
thio]ethyl}-4-oxo-3,4-dihydro-thieno [2,3-d]pyrimidine-6-
carboxylate (15)
Yield: 54.0%; Mp. 167e169 ꢁC re-crystallized with diethyl ether/
chloroform; Rf ¼ 0.56, mobile phase ethyl acetate/heptane e 3:2;
1H NMR(DMSO): 1.26 (t, 6H, 2CH3); 2.80 (s, 3H, CH3); 2.88(t, 2H,
CH2ePyr); 4.24 (q, 2H, eCH2eO); 4.60 (t, 2H, eCH2eS); 7.00e7.50
(m. 3H, Ar); 12.23 (s, 1H, NH, exchangeable with D2O); 13C NMR
(DMSO): 14.2(CH3), 15.1(CH3), 28.2(CH2ePyr), 42.4(CH2eS), 60.5
(CH2O), 109.0(Ar-CH), 110.7(Ar-CH), 119.8(Ar-CH), 121.8(CeBzth),
122.2(CeBzth), 130.2(CeBzth), 132.2(Ar-C), 143.9(Ar-C), 144.1(Ar-
C), 162.2(SeC]N), 162.4(SeCeNeBzth), 162.5(OeC]O), 166.9
(NeC]NeBzth), 169.1(C]O). Analysis: Calc.: C, 50.83; H, 3.82; Cl,
7.90; N, 12.48; S, 14.28; Found: C, 50.85; H, 3.80; Cl, 7.92; N, 12.47; S,
14.29.
6.3.3. Ethyl 2-{2-[(1H-benzimidazol-2-ylmethyl)thio]ethyl}-5-
methyl-4-oxo-3,4-dihydro-thieno[2,3-d]pyrimidine-6-carboxylate
(20)
Yield 49%; Mp-178e180 ꢁC; Rf ¼ 0.50, mobile phase chloroform/
heptane/ethanol e 3:2:1; 1H NMR: 1.30 (t, 3H, CH3); 2.78 (s, 3H,
CH3); 2.88 (t, 2H, CH2ePyr); 2.98 (s, 3H, CH3); 3.33 (t, 2H, eCH2eS);
4.1 (s, 2H, SeCH2); 4.32 (q, 2H, eCH2eO); 7.31e7.44 9(m, 3H, Ar);
12.31 (s, 1H, NH, exchangeable with D2O); Analysis: Calc: C, 55.79;
H, 5.15; N, 13.01; S, 14.90; Found: C, 55.81; H, 5.18; N, 13.04; S, 14.88.
6.2.10. Ethyl 5-methyl-2-{2-[(5(6)-nitro-1H-benzimidazol-2-yl)
thio]ethyl}-4-oxo-3,4-dihydro thieno-[2,3-d]pyrimidine-6-
carboxylate (16)
6.3.4. Ethyl 2-{2-[(5(6)-methyl-1H-benzimidazol-2-ylmethyl)thio]
ethyl}-5-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxylate (21)
Yield: 55.8%; Mp. 241e243 ꢁC; Rf ¼ 0.49, mobile phase ethyl
acetate/heptane e 3:2; 1H NMR(DMSO): 1.27 (t, J ¼ 7.0 Hz, 3H, CH3);
2.76 (s, 3H, CH3); 2.86 (t, 2H, CH2ePyr); 4.26 (q, J ¼ 7.0 Hz,
2H, eCH2eO); 4.65 (t, 2H, eCH2eS); 7.73e8.10 (m. 3H, Ar); 12.29 (s,
1H, NH, exchangeable with D2O); 13C NMR (DMSO): 14.6(CH3), 15.2
Yield: 49%; Mp: 163e165 ꢁC; Rf ¼ 0.55, mobile phase chloro-
form/heptane/ethanol e 3:2:1; 1H NMR: 1.30 (t, 3H, CH3); 2.79
(s, 3H, CH3); 2.88 (t, 2H, CH2ePyr); 2.98 (s, 3H, CH3); 3.33 (t,
2H, eCH2eS); 4.08 (s, 2H, SeCH2); 4.32 (q, 2H, eCH2eO); 7.31e7.44
9(m, 3H, Ar); 12.32 (s, 1H, NH, exchangeable with D2O); Analysis:

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 45 (2010) 5856e5861
5861
Calc.: C, 56.73; H, 5.44; N, 12.60; S, 14.43; Found: C, 56.70; H, 5.41;
N, 12.64; S, 14.41.
in DMSO. The used concentration was 5
m
g/ml. The samples were
incubated in “humid” chamber with thermostat at 37 ꢁC. The
microscopy control for vitality of the Trichinella larvae was carried
out 24 as well as 48 h after treatment using stereomicroscope
MBC-9.
6.4. General procedure for preparation of compounds 22e24
Method A: To a solution of 0.1 g (2.6 mmol) sodium hydroxide in
40 ml ethanol, 1.7 mmol of the appropriated 1H-benzimidazole was
added and the mixture was refluxed for 1 h. After that 3.4 mmol of
thieno[2,3-d]pyrimidinone 2a or 2b was added and the solution
was refluxed for 5 h more. The ethanol was removed under reduced
pressure. The remaining product was dissolved in chloroform and
the organic layer was washed with water and dried. The chloroform
was removed under reduced pressure and the obtained residue
crystallized.
6.5.2. Indicator test for antiprotozoal activity on Paramaecium
caudatum
Culture of Paramaecium caudatum, obtained through macer-
ating of dry hay was used. The tested compounds were dissolved
in DMSO at concentration 1
mg/ml. To a drop from the solution
of each compound in a Petri dish, 2e3 drops of the Paramecia
culture were added. The reaction of Paramecia was observed
stereomicroscopically.
Method B: To a solution of 0.01 mol of the respective benz-
imidazole in acetonitrile (50 ml), 2.7 g (0.02 mol) anhydrous K2CO3,
0.9 g (0.03 mol) thieno[2,3-d]pyrimidinone 2a or 2b was added. The
mixture was stirred at ambient temperature and monitored by TLC
over the reaction period. After completion of the reaction, the
mixture was filtered to separate the solid K2CO3, the organic
solvent was evaporated under reduced pressure and the residue
crystallized.
6.5.3. Antiprotozoal activity in vivo against Lamblia muris
Thirty white mice, divided in six groups were treated p. o. with
each one of the compounds 8, 10e11, 14e16 and 22e23. The tested
compounds were introduced in the oesophagus of each mouse by
means of thin metallic probe in a single time dose of 0.5 mg/ml pro
die as a five-days-treatment course. The compounds were used as
DMSO solutions in volume of 0.5 ml. The first control group was
without treatment and the mice in the second control group were
given only the solvent (DMSO) during five days. After treatment
during five days the mice faeces were studied microscopic for the
availability of Lamblia muris cysts.
6.4.1. 2-[2-(5(6)-nitro-1H-benzimidazol-1-yl)ethyl]-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one, 22
Yield: 53% (method A), 74% (method B); Mp: 196e198 ꢁC;
Rf ¼ 0.50, mobile phase: benzene/methanol e 2:1. 1H NMR (DMSO):
1.77 (t, 4H, 2CH2); 2.73 (t, 2H, CH2); 2.87 (t, 2H, CH2); 3.06 (t, 2H,
CH2); 3.99 (t, 2H, CH2); 7.54 (m, 4H, Bz); 12.35 (s, 1H, NH,
exchangeable with D2O); Analysis: Calc.: C, 57.71; H, 4.33; N, 17.71;
O, 12.14; S, 8.11; Found: C, 57.73; H, 4.30; N, 17.69; S, 8.15.
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3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxylate, 23
Yield 75% (method A), 85% (method B); Mp-231e233 ꢁC,
benzene/methanol 2:1, Rf
¼
0,68, mobile phase: benzene/
methanol ¼ 2:1 1H NMR (DMSO): 1.37 (t, 3H, CH3); 2.85 (s, 3H,
CH3); 3.28 (t, 2H, CH2); 4.35 (q, 2H, OeCH2); 4.75 (t, 2H, NeCH2);
7.49e8.36 (m, 5H, CHeBzi), 12.33 (s, 1H, NH, exchangeable with
D2O); Analysis: Calc.: C, 59.67; H, 4.74; N, 14.65; S, 8.38; Found: C,
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6.4.3. Ethyl 5-methyl-2-[2-(5(6)-nitro-1H-benzimidazol-1-yl)
ethyl]-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxylate, 24
Yield: 51% (method A), 71.5% (method B); Mp e 248e250 ꢁC;
Rf ¼ 0.52, mobile phase: benzene/methanol ¼ 2:1; 1H NMR
(DMSO): 1.31 (t, 3H, CH3); 2.88 (s, 3H, CH3); 3.27 (2H, CH2); 4.25 (q,
2H, CH2); 4.83 (t, 2H, CH2); 7.86 (m 2H, CHeBzi); 8.22 (m, 1H,
CHeBzi); 8.66 (m,1H, CHeBzi); 12.48 (s, 1H, NH, exchangeable with
D2O); Analysis: Calc.: C, 53.39; H, 4.01; N, 16.38; S, 7.50; Found: C,
53.36; H, 4.05; N, 16.36; S, 7.53.
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6.5. Biological screening
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6.5.1. Antitrichinellosis activity in vitro
The parasitological pharmaco-therapeutic experiments in vitro
for antitrichinellosis activity of the tested compounds were carried
out according to Campbell’s method [21,22].
Encapsulated Trichinella spiralis larvae were used in the para-
sitological experiment in vitro, 100 specimens for 1 ml physiolog-
ical solution. The tested benzimidazole derivatives were dissolved