Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 3 pp. 357ñ373, 2011

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 3 pp. 357ñ373, 2011 ISSN 0001-6837Polish Pharmaceutical Society

The synthesis of dihydropyrimidine derivativeshave been attracting extensive attention as a widerange of such compounds played an important role inthe field of medicinal chemistry as antiviral (1), anti-bacterial (2), antimalarial (3), antihypertensive (4)and anti-inflammatory (5) agents. In addition, pyrim-idine derivatives form the basis of a large number ofpharmacological products with anticancer and pro-tein kinase inhibitory activity (6).One of the mostfamous pyrimidine derivatives is 5-florouracil (5-Fu), considered as one of the most important drugsfor the treatment of colorectal, head and neck, pan-creatic and breast carcinomas (7). Furthermore,recent studies showed that benzofuran ring systemfused with heterocyclic moieties exhibit a wide spec-tra of pharmacological activities and especially anti-tumor activity (8ñ10). Moreover, pyrazole deriva-tives have been reported to act as antitumor agentsagainst various types of carcinomas such as lung,breast cancer and leukemia (11, 12).

Folate metabolism is considered as an impor-tant target for the development of new anticanceragents due to its role in the biosynthesis of nucleicacid precursors (13, 14). The inhibition of folate-

dependent enzymes such as thymidylate synthase,which catalyzes the reductive methylation ofdeoxyuridylate (dUMP) to thymidylate (dTMP) hasalso been recognized as an interesting target for drugdiscovery (15, 16). Classical antitumor agents thatprevent this pathway have a disadvantage that theyneed an active transport mechanism to enter thecells, which can cause tumor resistance if impaired(17, 18). Also, recent preclinical experiments onhuman liver carcinoma cell lines (HEPG2) revealedthat at higher doses of antifolate a loss of thymidy-late synthase inhibition occurs and cytotoxic effectsare preserved (19).

Based on all these findings, this work aims todesign a new series of compounds structurally con-taining benzofuranyl-pyrazole ring system conjugat-ed with pyrimidine derivatives as a trial to develop anew thymidylate synthase inhibitor that could beselective and may be a good target for drug discov-ery. For this target, some docking of the newly syn-thesized compounds was done using Autodock Vina(20). Moreover the cytotoxic activities of new fiveselected compounds were screened against HEPG2(human liver carcinoma cell line).

SYNTHESIS, ANTITUMOR ACTIVITY AND MOLECULAR DOCKING STUDYOF NOVEL BENZOFURAN-2-YL PYRAZOLE PYRIMIDINE DERIVATIVES

MAGDY I. EL-ZAHAR1,*, SOMAIA S. ABD EL-KARIM1, MOGEDDA E. HAIBA1

and MOHAMMED A. KHEDR2

1Therapeutical Chemistry Department, National Research Center, Dokki, Cairo, Egypt2Welsh School of Pharmacy, Cardiff University, UK

Abstract: A new series of (benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl) pyrimidine derivatives were synthe-sized from 3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-carbaldehyde (1) through different routes of cyclocon-densation reactions. Condensation of 1 with active methylene compounds afforded compounds 2-8. Thecyclization of 2 with chloroacetic acid, ortho substituted benzoic acid and/or ethanolamine gave compounds 9-

12. Also condensation of 2 with hydrazine hydrate followed by cyclocondensation afforded corresponding tri-azines and pyrazole derivatives 18-27. Some docking studies of the newly prepared compounds as thymidylatesynthase inhibitors have been done. Also the cytotoxic activity of some of the prepared compounds as a repre-sentative examples was evaluated against HEPG2 (human liver carcinoma cell line) in comparison with 5-fluoro-uracil (5-Fu).

Keywords: pyrimidin-2-thione, benzofuranoyl-pyrazoles, thiazolidinone, triazole, thymidylate synthaseinhibitor, Autodock Vina, antitumor activity

357

* Corresponding author: e-mail: [email protected]; fax: +20 33370931

358 MAGDY I. EL-ZAHAR et al.

EXPERIMENTAL

Chemistry

All melting points are uncorrected and weretaken in open capillary tubes using anElectrothermal IA 9100 digital melting point appa-ratus. Elemental microanalyses were carried out atMicroanalytical Laboratory, Central ServicesLaboratory, National Research Centre, Dokki,Cairo, Egypt, on Vario Elementar analyzer and werefound within ± 0.5% of the theoretical values.Infrared spectra were recorded on a FT/IR-6100,Fourier transform, infrared spectrometer (Japan)using KBr disc technique. 1H-NMR spectra weredetermined using a JEOl EX-270 NMR spectrome-ter and measured in δ scale using TMS as an inter-nal standard. The mass spectra were measured witha Finnigan MAT SSQ-7000 mass spectrometer.Follow up of the reactions and checking the purityof the compounds were made by TLC on silica gel-precoated aluminium sheets (Type 60, F 254,Merck, Darmstadt, Germany) and the spots weredetected using UV lamp at λ254 nm. The chemicalnames given for the prepared compounds areaccording to the IUPAC system.

3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazole-4-car-baldehyde (1)

This compound was prepared according toreported method in our previously published paper(21).

6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydro-4-oxo-2-thioxopyrimidine-5-car-bonitrile (2)

A mixture of compound 1 (1.44 g, 0.005 mole),ethyl cyanoacetate (0.57 mL, 0.005 mole) andthiourea (0.38 g, 0.005 mole) in sodium ethoxide(20 mL) was stirred for one hour at room tempera-ture. The reaction mixture was poured into ice coldwater and acidified by hydrochloric acid; the solidseparated was filtered and recrystallized to givecompound 2.

1-(4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydro-6-methyl-2-thioxopyrimidin-5-yl)ethanone (3) and ethyl 4-(3-benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydro-6-methyl-2-thioxopyrmidine-5-carboxylate (4)

General procedure: A mixture of compound 1(0.58 g, 0.002 mole), thiourea (0.16 g, 0.002 mole),acetylacetone and/or ethyl acetoacetate (0.002 mole)in absolute ethanol (15 mL) containing few drops ofconcentrated hydrochloric acid was refluxed for 4 h.

The formed precipitate was filtered, washed withwater several time, dried and recrystallized to givecompounds 3 and 4, respectively.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3,4,5,6,7,8- hexahydroquinazoline-2(1H)-thione (5)and 4-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3,4-dihydro-1H-indeno[1,2-d]pyrimidine-(5H)-thione (6)

General procedure: A mixture of compound 1(0.58 g, 0.002 mole), thiourea (0.16 g, 0.002 mole),cyclohexanone and/or 2-indanone (0.0025 mole) inabsolute ethanol (15 mL) containing few drops ofconcentrated hydrochloric acid was refluxed for 3 h.After cooling the formed precipitate was filtered,washed several times with water, dried and recrys-tallized to give compounds 5 and 6, respectively.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,2-dihydro-6-(1,2,3,4-tetrahydronaphthalen-7-yl)-2-oxopyridine-3-carbonitrile (7)

A mixture of compound 1 (0.58 g, 0.002 mole),ethyl cyanoacetate (0.23 mL, 0.002 mole), anhy-drous ammonium acetate (1.24 g, 0.016 mole) and6-acetyl-1,2,3,4-tetrahydronaphthalene (0.5 mL,0.002 mole) in n-butanol (10 mL) was heated underreflux for 3 h. The formed precipitate was filtered,dried and recrystallized to give compound 7.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,2-dihydro-6-(1,2,3,4-tetrahydronaphthalen-7-yl)-2-iminopyridine-3-carbonitrile (8)

A mixture of compound 1 (0.58 g, 0.002 mole),malononitrile (0.14 g, 0.002 mole), anhydrousammonium acetate (1.24 g, 0.016 mole) and 6-acetyl-1,2,3,4-tetrahydronaphthalene (0.5 mL, 0.002mole) in absolute ethanol (10 mL) was refluxed for3 h. The formed precipitate on cooling was filtered,dried and recrystallized to give compound 8.

7-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3,5-dihydro-3,5- dioxo-2H-thiazolo[3,2-a]pyrimi-dine-6-carbonitrile (9)

A mixture of compound 2 (0.42 g, 0.001 mole),chloroacetic acid (0.1 g, 0.001 mole) and anhydroussodium acetate (0.33 g, 0.004 mole) in glacial aceticacid (10 mL) and acetic anhydride (2 mL) wasrefluxed for 3 h. The reaction mixture was cooled,poured into ice cold water, the formed precipitatewas filtered and recyrstallized to give compound 9.

2-(4-Methoxybenzylidene)-7-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3,5-dihydro-3,5-dioxo-2H-thiazolo[3,2-a] pyrimidine-6-carbonitrile (10a),

Synthesis, antitumor activity and molecular docking study of novel benzofuran-2-yl... 359

7-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3,5-dihydro-3,5-dioxo-2-((thiophen-2-yl) methyl-ene)-2H-thiazolo[3,2 a]pyrimidine-6-carbonitrile(10b) and 2-((1H-indol-3-yl) methylene)-7-(3-(ben-zofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3,5-dihy-dro-3,5-dioxo-2H-thiazolo[3,2-a]pyrimidine-6-car-bonitrile (10c)

General procedure: A mixture of compound 9(0.46 g, 0.001 mole) and an appropriate aldehydenamely; p-anisaldehyde, 2-thiophenecarboxalde-hyde or indole-3-carboxaldehyde (0.001 mole) inacetic acid (10 mL) was refluxed for 8 h. The reac-tion mixture was cooled, poured into ice cold water.The formed precipitate was filtered, washed severaltimes with water, dried and recrystallized to givecompounds 10a-c, respectively.

2-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6,11-dihydro -4,6 -dioxo-4H-pyrimido[2,1-b]quina-zoline-3-carbonitrile (11a), 2-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-hydro-4,6-dioxo-4H -pyrimido[2,1-b]benzo[1,3]oxazine-3-carbonitrile(11b) and 2-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-hydro-4,6-dioxo-4H -pyrimido[2,1-b]benzo[1,3]thiazine-3-carbonitrile (11c)

General procedure: A mixture of compound 2(0.42 g, 0.001 mole) and ortho substituted benzoicacid namely: anthranilic acid, salicylic acid or thio-salicylic acid (0.001 mole) in 2% sodium ethoxide(20 mL) was refluxed for 10 h. Then cooled, pouredinto ice cold water and acidified by dilutedhydrochloric acid. The formed precipitate was fil-tered, washed several times with water, dried andrecrystallized from to give compounds 11a-c,respectively.

7-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)1,2,3,5-tetrahydro-5-oxoimidazo[1,2-a]pyrimi-dine-6-carbonitrile (12)

A mixture of compound 2 (0.42 g, 0.001 mole)and ethanolamine (0.07 mL, 0.001 mole) in iso-propyl alcohol (10 mL) was refluxed for 6 h. Thenpoured into ice cold water and acidified by dilutedhydrochloric acid. The formed precipitate was fil-tered and recrystallized to give compound 12.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,6-dihydro-2- (methylthio)-6-oxopyrimidine-5-car-bonitrile (13)

A mixture of compound 2 (2.0 g, 0.005 mole)and iodomethane (0.72 mL, 0.005 mole) in 2%sodium ethoxide (20 mL) was refluxed for 3 h. Thereaction mixture was cooled, poured into ice coldwater and acidified by diluted hydrochloric acid.

The formed precipitate was filtered and recrystal-lized to give compound 13.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-hydrazinyl-1,6-dihydro-6-oxopyrimidine-5-car-bonitrile (14)

Method A: A mixture of compound 13 (1.28 g,0.003 mole) and hydrazine hydrate 98% (0.15 mL,0.003 mole) was refluxed for 3 h. The formed pre-cipitate was filtered and washed with ethanol, driedand recrystallized to give compound 14. Method B: A mixture of compound 2 (2.0 g, 0.005mol) and hydrazine hydrate 98% (0.3 mL, 0.005mole) was refluxed for 2 h. The formed precipitatewas filtered and washed with ethanol, dried andrecrystallized to give compound 14.

6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4-chloro-1,2-dihydro-2-thioxopyrimidine-5-car-bonitrile (15)

A mixture of compound 2 (2.1 g, 0.005 mole)and phosphorus pentachloride (1.04 g, 0.025 mole)in phosphorus oxychloride (15 mL) was heated onwater bath for 6 h, then cooled and poured into icecold water. The formed precipitate was filtered,washed several times with water, dried and recrys-tallized to give compound 15.

6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4-hydrazinyl-1,2-dihydro-2-thioxopyrimidine-5-carbonitrile (16)

Hydrazine hydrate (99%) (0.1 mL, 0.002 mole)was added to compound 15 (0.86 g, 0.002 mole) inethanol (15 mL) and stirred at room temperature.After complete addition, stirring was continuousovernight. The formed precipitate was filtered andrecrystallized to give compound 16.

7-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-5,6-dihydro-5-thioxotetrazolo[1,5-f]pyrimidine-8-carbonitrile (17)

A mixture of compound 15 (0.86 g, 0.002mole) and sodium azide (0.14 g, 0.002 mole) inacetic acid (15 mL) was refluxed for 6 h. The reac-tion mixture was cooled and poured into ice coldwater. The formed precipitate was filtered, dried andrecrystallized to give compound 17.

7-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,2,3,5-tetrahydro-3,5-dioxo[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (18)

A mixture of compound 14 (0.41 g, 0.001mole) and ethyl chloroformate (0.10 mL, 0.001mole) in pyridine (10 mL) was refluxed for 6 h.


Then it was cooled and poured into ice cold watercontaining few drops of hydrochloric acid. Theformed precipitate was filtered, washed severaltimes with water, dried and recrystallized to givecompound 18.

7-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,5-dihydro-5-oxo [1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (19)

A mixture of compound 14 (0.41 g, 0.001mole) and triethylorthoformate (1 mL, 0.001 mole)in absolute ethanol (10 mL) was refluxed for 8 h.The reaction mixture was concentrated; the formedprecipitate was filtered, dried and recrystallized togive compound 19.

7-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,5-dihydro-3-methyl-5-oxo[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (20)

A mixture of compound 14 (0.41 g, 0.001mole) and acetic anhydride (0.1 mL, 0.001 mole) in

acetic acid (10 mL) was refluxed for 6 h. The reac-tion mixture was cooled and poured into ice coldwater. The formed precipitate was filtered, dried andrecrystallized to give compound 20.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,6-dihydro-6-oxo-2-(3,5-dioxopyrazolidin-1-yl)pyr-imidine-5-carbonitrile (21)

A mixture of compound 14 (0.41 g, 0.001mole) and diethyl malonate (0.16 mL, 0.001 mole)in acetic acid (10 mL) was refluxed for 8 h. Then itwas cooled and poured into ice cold water. Theformed precipitate was filtered, dried and recrystal-lized to give the compound 21.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,6-dihydro-2-(4,5-dihydro-3-methyl-5-oxopyrazol-1-yl)-6-oxopyrimidine-5-carbonitrile (22)

A mixture of compound 14 (0.41 g, 0.001mole) and ethyl acetoacetate (0.13 mL, 0.001 mole)in acetic acid (10 mL) was refluxed for 6 h. The

Scheme 1.


reaction mixture was cooled and poured into icecold water. The formed precipitate was filtered,dried and recrystallized to give compound 22.

4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,6-dihydro-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-oxopyrimidine-5-carbonitrile (23)

A mixture of compound 14 (0.41 g, 0.00 mole)and acetylacetone (0.1 mL, 0.001 mole) in aceticacid (15 mL) was refluxed for 6 h. The reaction mix-ture was cooled and poured into ice cold water. The

formed precipitate was filtered, dried and recrystal-lized to give compound 23.

8-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2,3,4,6-tetrahydro-3,4,6-trioxo-1H-primido[2,1-c][1,2,4]triazine-7-carbonitrile (24)

A mixture of compound 14 (0.41 g, 0.001mole) and diethyl oxalate (0.15 mL, 0.001 mole) inabsolute ethanol (15 mL) was refluxed for 8 h. Theprecipitate formed on cooling was filtered, dried andrecrystallized to give compound 24.

Scheme 2.


Comp.no.

2

3

4

5

6

7

8

9

10a

10b

10c

11a

11b

11c

12

13

14

15

16

17

18

19

20

21

M.p. (OC)(Cryst. solvent)

124ñ125(Benzene)

235ñ236(Ethanol)

191ñ192(Ethanol)

219ñ220(Ethanol)

258ñ259(Ethanol)

> 300(Acetic acid)

224ñ225(Ethanol)

132ñ133(Benzene)

281-283(Ethanol)

160ñ161(Isopropanol)


115ñ116(Ethanol)

121ñ122(Ethanol)

142ñ143(Ethanol)

128ñ129(Benzene)

200ñ201(Ethanol)

Method A 256ñ258

Method B258ñ260

(Acetic acid)

223ñ225(Ethanol)

280ñ282(Ethanol)

199ñ200(Ethanol)

186ñ187(Ethanol)


159ñ160(Ethanol)

182-184(Ethanol)

Yield%

83

53

51

52

89

80

65

71

59

92

71

80

83

94

61

97

31

75

91

44

78

81

69

95

76

Mol. formula(Mol. Wt.)

C22H13N5O2S(411.44)

C24H20N4O2S(428.51)

C25H22N4O3S(458.53)

C25H22N4OS(426.53)

C28H20N4OS(460.55)

C33H24N4O2

(508.57)

C33H25N5O(507.58)

C24H13N5O3S(451.46)

C32H19N5O4S(569.59)

C29H15N5O3S2

(545.59)

C33H18N6O3S(578.60)

C29H16N6O3

(496.48)

C29H15N5O4

(497.46)

C29H15N5O3S(513.53)

C24H16N6O2

(420.42)

C23H15N5O2S(425.46)

C22H15N7O2

(409.40)

C22H12ClN5OS(429.88)

C22H15N7OS(425.11)

C22H12N8OS(436.45)

C23H13N7O3

(435.39)

C23H13N7O2

(419.40)

C24H15N7O2

(433.42)

C25H15N7O4

(477.43)

Analysis (%)Calcd./Found

C

64.2264.53

66.2766.43

65.4865.72

70.4070.69

73.0273.33

77.9377.58

78.0977.88

63.8563.72

67.4867.32

63.8463.62

68.5068.83

70.1670.32

70.0270.32

67.8367.44

68.5668.67

64.9364.71

64.5464.72

61.4761.23

62.1062.41

60.5460.84

63.4563.72

65.8765.61

66.5166.83

62.8962.52

H

3.183.41

4.704.48

4.844.67

5.205.37

4.384.61

4.764.42

4.964.72

2.903.11

3.363.41

2.772.54

3.143.23

3.253.52

3.043.21

2.942.61

3.843.62

3.553.91

3.693.55

2.812.96

3.553.99

2.772.95

3.013.22

3.123.45

3.493.56

3.173.33

N

17.0216.99

13.0713.39

12.2212.53

13.1413.28

12.1712.28

11.0211.32

13.8013.51

15.5115.31

12.3012.51

12.8412.59

14.5214.34

16.9316.67

14.0814.36

13.6413.82

19.9919.73

16.6416.22

3.9523.71

16.2916.53

23.0423.32

25.6725.32

22.5222.24

23.3823.58

22.6222.87

20.5420.41

S

7.797.99

7.487.24

6.997.31

7.527.81

6.967.23

7.107.23

5.635.72

11.7511.48

5.545.31

6.245.99

7.547.31

7.467.31

7.547.78

7.357.53

Table 1. Physical and analytical data of all new compounds 2-26b


1-(4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-5-cyano-1,6-oxopyrimidin-2-yl)-4-substitutedthiosemicarbazides (25añd)

General procedure: A mixture of compound 14

(0.41 g, 0.001 mole) and an appropriate substitutedthiosemicarbazide namely: ethylthiosemicarbazide,cyclohexylthiosemicarbazide, phenylthiosemicar-bazide or o-tolylthiosemicarbazide (0.001 mole) indry benzene (20 mL) was refluxed for 6 h. Aftercooling, the formed precipitate was filtered andrecrystallized to give compounds 25a-d, respective-ly.

2-(2,5-Dioxopyrrolidin-1-ylamino)-4-(3-(benzofu-ran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (26a) and 2-(5,7-dioxo-5H-pyrrolo[3,4-b]pyridine-6(7H)-ylamino)-4-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (26b) General procedure: A mixture of compound 14

(0.41 g, 0.001 mole), and an appropriate acid anhy-dride namely: succinic anhydride or 2,3-pyridinedi-carboxylic anhydride (0.001 mole) in acetic acid (10mL) was refluxed for 6 h. The formed precipitatewas filtered, dried and recrystallized to give com-pounds 26a-b, respectively.

Antitumor activity

Materials and Methods

The aim of the present study was to illustratethe effect of some newly synthesized compounds onthe human liver carcinoma cell line (HEPG2) incomparison with 5-fluorouracil (5-FU) in a trial toget more effective and less toxic agents. Humantumor cell lines were obtained frozen in liquid nitro-gen (ñ180OC) from the American Type CultureCollection. RPMI-1640 medium (Sigma ChemicalCo., St. Louis, MO, USA). The medium was pre-pared and used for culturing and maintenance of thehuman tumor cell lines. The prepared medium waskept in a refrigerator (4OC) and checked at regularintervals for contamination. Before the use, themedium was warmed at 37OC in a water bath andsupplemented with penicillin/streptomycin andFBS. Experiments were set up using theSulforhodamine-B (SRB) assay according toSkehan et al. (22). Different concentrations of thecompounds tested (0, 5, 12.5, 25 and 50 µg mLñ1)were added to the cell monolayer. Each concentra-tion was evaluated three times (each dose was incu-bated with the cells in three different wells).Monolayer cells were incubated with the com-pounds for 48 h at 37OC.

Comp.no.

22

23

24

25a

25b

25c

25d

26a

26b

M.p. (OC)(Cryst. solvent)

218-219(Ethanol)

200-202(Ethanol)

215-216(Ethanol)

199-200(Ethanol)

225-226(Ethanol)

203-205(Ethanol)

199-200(Ethanol)

201-202(Acetic acid)

223-225(Acetic acid)

Yield%

67

78

52

54

88

80

76

51

48

Mol. formula(Mol. Wt.)

C26H17N7O3

(475.46)

C27H19N7O2

(473.49)

C24H13N7O4

(463.40)

C25H20N8O2S(496.54)

C29H26N8O2S(550.63)

C29H20N8O2S(544.59)

C30H22N8O2S(558.61)

C26H17N7O4

(491.46)

C29H16N8O4

(540.49)

Analysis (%)Calcd./Found

C

65.6865.87

68.4968.79

62.2062.54

60.4760.52

63.2663.57

63.9663.59

64.5064.79

63.5463.91

64.4464.21

H

3.603.24

4.043.68

2.832.51

4.064.28

4.764.33

3.704.11

3.973.61

3.493.11

2.982.62

N

20.6220.17

20.7120.96

21.1621.48

22.5722.81

20.3520.21

20.5820.86

20.0620.32

19.9520.18

20.7320.38

S

6.466.81

5.825.61

5.896.21

5.745.96

Table 1. cont.

364 MAGDY I. EL-ZAHAR et al.T

able

2. S

pect

ral d

ata

of th

e ne

wly

syn

thes

ized

com

poun

ds

Com

pd.n

o.

IR (

KB

r, c

m-1)

1 H-N

MR

(so

lven

t, δ,

ppm

) M

S [m

/z (

%)]

232

20, 3

127

(NH

, NH

), 2

226

(CD

Cl 3)

: 7.2

6ñ7.

98 (

m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

, 8.6

2 (s

, 1H

, pyr

azol

e ri

ng),

41

1 [M

+ ] (

21),

378

[M

+ ñS

H]

(C≡N

), 1

729

(C=O

), 1

148

(C=S

) 9.

15, 1

0.25

(s,

s, 1

H, 1

H, N

H, N

H e

xcha

ngea

ble

by D

2O(9

), 7

7 [C

6H5]

(10

0)

333

20, 3

188

(NH

, NH

), 1

720

(DM

SO-d

6): 2

.14

(s, 3

H, C

H3)

, 2.3

3 (s

, 3H

, CH

3CO

), 5

.80

(s, 1

H, t

hiox

opyr

imid

ine

428

[M+ ]

(2)

, 386

[M

+ ñC

H2C

O]

(C=O

), 1

181

(C=S

)

ring

), 7

.28ñ

7.97

(m

, 10H

, Ar-

H, b

enzo

fura

n pr

oton

s), 8

.33

(s, 1

H, p

yraz

ole

ring

),(2

), 6

2 [C

5H2]

(10

0)9.

24, 1

0.20

(s,

s, 1

H, 1

H, N

H, N

H e

xcha

ngea

ble

by D

2O)

434

02, 3

282

(NH

, NH

), 1

745

(C=O

,(D

MSO

-d6)

: 0.8

4 (t

, 3H

, CH

3C

H2)

, 2.3

0 (s

, 3H

, CH

3), 3

.83

(q, 2

H, C

H3C

H2),

45

9 [M

+ +1

] (2

5), 4

57 [

M+ ñ

1] (

19),

este

r), 1

173

(C=S

)5.

74 (

s, 1

H, t

hiox

opyr

imid

ine

ring

), 7

.26ñ

7.89

(m

, 10H

, Ar-

H, b

enzo

fura

n pr

oton

s), 8

.43

77 [

C6H

5] (

38),

44

[C3H

8] (

100)

(s, 1

H, p

yraz

ole

ring

), 9

.40,

10.

19 (

s, s

, 1H

, 1H

, NH

, NH

exc

hang

eabl

e by

D2O

)

533

99, 3

192

(NH

, NH

), 2

929

(CH

2, (D

MSO

-d6)

: 1.6

0, 1

.72

(m, m

, 4H

, 4H

, cyc

lohe

xane

rin

g), 5

.50

(s, 1

H, t

hiox

opyr

imid

ine

428

[M+ +2

] (4

), 6

3 [C

5H3]

(10

0)

cycl

ohex

ane)

, 106

3 (C

=S)

ring

), 7

.25ñ

7.98

(m

, 10H

, Ar-

H, b

enzo

fura

n pr

oton

s), 8

.49

(s, 1

H, p

yraz

ole

ring

), 9

.40,

10

.19

(s, s

, 1H

, 1H

, NH

, NH

exc

hang

eabl

e by

D2O

)

633

97, 3

140

(NH

, NH

), 2

989

(CH

2),

(DM

SO-d

6): 3

.61

(m, 2

H, C

H2

inda

none

rin

g), 6

.16

(s, 1

H, t

hiox

opyr

imid

ine

ring

),46

0 [M

+ ] (

2), 4

15 [

M+ ñH

CS]

(3)

, 11

93 (

C=S

)

6.89

ñ7.8

9 (m

, 14H

, Ar-

H, b

enzo

fura

n pr

oton

s, in

dano

ne r

ing)

, 8.7

0 (s

, 1H

, 63

[C

5H3]

(10

0)py

razo

le r

ing)

, 9.0

5, 1

0.74

(1

s, s

, H, 1

H, N

H, N

H e

xcha

ngea

ble

by D

2O)

731

38 (

NH

), 2

930

(CH

2of

(DM

SO-d

6): 1

.70,

2.7

2 (m

, m, 4

H, 4

H, t

etra

hydr

onap

htha

lene

), 7

.14ñ

7.95

(m

, 14H

, 50

8 [M

+ ] (

12),

77

[C6H

5] (

100)

tetr

ahyd

rona

phth

alen

e),

2217

(C≡N

), 1

646

(C=O

) A

r-H

, ben

zofu

ran

prot

ons,

tetr

ahyd

rona

phth

alen

e ri

ng,

pyri

dine

rin

g), 8

.00

(s, 1

H, p

yraz

ole

ring

), 9

.08

(s, 1

H, N

H, e

xcha

ngea

ble

by D

2O)

834

36, 3

202

(NH

, NH

), 2

929

(CH

2(D

MSO

-d6)

: 1.7

9, 2

.74

(m, m

, 4H

, 4H

, tet

rahy

dron

apht

hale

ne),

7.1

9ñ7.

95 (

m, 1

4H,

507

[M+ ]

(15

), 7

7 [C

6H5]

(10

0)

of te

trah

ydro

naph

thal

ene)

, A

r-H

, ben

zofu

ran

prot

ons,

tetr

ahyd

rona

phth

alen

e ri

ng, p

yrid

ine

ring

), 8

.02

(s, 1

H,

2217

(C≡N

), 1

599

(C=N

) py

razo

le r

ing)

, 9.6

0 (s

, 1H

, NH

, exc

hang

eabl

e by

D2O

)

922

22 (

C≡N

), 1

757(

C=O

), 1

692

(CD

Cl 3)

: 3.6

9 (s

, 2H

, thi

azol

e ri

ng),

7.2

5ñ7.

94 (

m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

, 45

1 [M

+ ] (

3), 7

7 [C

6H5]

(10

0)

(C=O

, thi

azol

e ri

ng)

8.28

(s,

1H

, pyr

azol

e ri

ng)

10a

(CD

Cl 3

): 3

.88

(s, 3

H, O

CH

3), 7

.25ñ

7.90

(m

, 15H

, Ar-

H, b

enzo

fura

n pr

oton

s, m

ethy

lene

569

[M+ ]

(2)

, 77

[C6H

5] (

100)

pr

oton

s), 8

.69

(s, 1

H, p

yraz

ole

ring

)

10b

(CD

Cl 3

): 7

.25ñ

7.85

(m

, 14

H, A

r-H

, ben

zofu

ran

prot

ons,

met

hyle

ne p

roto

n), 8

.70

545

[M+ ]

(2)

, 313

[C

20H

15N

3O]

(s

, 1H

, pyr

azol

e ri

ng)

(39)

, 273

[C

18H

13N

2O]

(100

)

10c

(CD

Cl 3

): 7

.25ñ

8.30

(m

, 16H

, Ar-

H, b

enzo

fura

n pr

oton

s, in

dole

pro

tons

), 8

.69

(s,

578

[M+ ]

(10

), 7

7 [C

6H5]

(10

0)1H

, pyr

azol

e ri

ng),

10.

05 (

s, 1

H, N

H-i

ndol

e ex

chan

geab

le b

y D

2O)

11a

3210

(N

H),

172

0, 1

695

(2 C

=O),

(DM

SO-d

6): 7

.26ñ

7.98

(m

, 14H

, Ar-

H, b

enzo

fura

n pr

oton

s), 8

.45

(s, 1

H, p

yraz

ole

ring

),

497

[M+ +1

] (4

), 7

7 [C

6H5]

(10

0)22

22 (

C≡N

) 9.

20 (

s, 1

H, N

H e

xcha

ngea

ble

by D

2O)

11b

2228

(C

≡N),

172

4, 1

698

(2 C

=O)

49

9 [M

+ +2

] (3

), 4

23 [

M+ ñ

C6H

2]

(58)

, 395

[42

3ñC

O]

(100

)

11c

2221

(C

≡N),

172

0, 1

692

(2 C

=O)

51

3 [M

+ ] (

5), 4

39 [

M+ ñC

6H2]

(23

), 4

11

[439

ñCO

] (5

7), 3

95 [

411ñ

NH

2] (

100)

Synthesis, antitumor activity and molecular docking study of novel benzofuran-2-yl... 365T

able

2. c

ont.

Com

pd.n

o.

IR (

KB

r, c

m-1)

1 H-N

MR

(so

lven

t, δ,

ppm

) M

S [m

/z (

%)]

12

(CD

Cl 3

): 3

.42

(m, 2

H, C

H2-

imid

azol

e ri

ng)

3.65

(m

, 2H

, CH

2im

idaz

ole

ring

), 7

.17ñ

7.95

42

3 [M

+ +3

] (2

6), 3

94 [

M+ ñC

2H2]

(m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

, 8.5

2 (s

, 1H

, pyr

azol

e ri

ng),

10.

12 (

s, 1

H, N

H(3

), 3

95 [

394+

1] (

21),

273

[C

18H

13N

2O]

exch

ange

able

by

D2O

) (6

6), 7

7 [C

6H5]

(10

0)

13

3128

(N

H),

222

3 (C

≡N),

166

7(D

MSO

-d6)

: 2.2

5 (s

, 3H

, S-C

H3)

, 7.2

5ñ7.

98 (

m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

, 25

[M

+ ] (

13),

423

[M

+ñ2

] (1

4), 4

378

(C

=O),

130

9 (S

-CH

3)

8.63

(s,

1H

, pyr

azol

e ri

ng),

9.2

1 (s

, 1H

, NH

exc

hang

eabl

e by

D2O

) [M

+ ñS

CH

3] (

14),

77

[C6H

5] (

100)

14

Bro

ad b

and

cent

ered

at 3

282

(DM

SO-d

6): 7

.24ñ

7.64

(m

, 10H

, Ar-

H, b

enzo

fura

n pr

oton

s), 7

.93

(s, 1

H, p

yraz

ole

ring

),

409

[M+ ]

(6)

, 410

[M

+ +1

] (3

1), 3

94

(NH

NH

2), 3

115

(NH

), 2

196

9.00

(s,

1H

, NH

exc

hang

eabl

e by

D2O

), 1

0.18

(s,

3H

, NH

NH

2ex

chan

geab

le b

y D

2O)

[M+ ñN

H]

(25)

, 379

[M

+ñN

2H2]

(C

≡N),

166

7 (C

=O)

(68)

, 77

[C6H

5] (

100)

15

NH

), 2

221

(C≡N

), 1

214

(C=S

) (D

MSO

-d6)

: 7.2

5ñ7.

92 (

m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

, 8.6

1 (s

, 1H

, pyr

azol

e ri

ng),

42

9, 4

31[M

+ ] (

7, 2

5), 4

30, 4

32 [

M+ +1

] 9.

20 (

s, 1

H, N

H e

xcha

ngea

ble

by D

2O)

(9, 3

0), 7

7 [C

6H5]

(10

0)

16

Bro

ad b

and

cent

ered

at 3

319

425

[M+ ]

(5)

, 263

[C

17H

15N

2O]

(100

) (N

HN

H2)

, 317

1 (N

H),

221

3 (C

≡N),

106

9 (C

=S)

17

3241

(NH

), 22

22 (C

≡N),

1148

(C=S

)45

5 [M

+.ñ1

] (1

0), 7

7 [C

6H5]

(10

0)

18

Bro

ad b

and

cent

ered

at 3

223

(DM

SO-d

6): 7

.31ñ

7.94

(m

, 11H

, Ar-

H, b

enzo

fura

n pr

oton

s, p

yraz

ole

ring

), 9

.16

434

[M+

ñ1]

(54)

, 433

[M

+ ñ2

] (4

6),

(NH

, NH

), 2

215

(C≡N

), 1

661,

(s

, 1H

, NH

exc

hang

eabl

e by

D2O

)77

[C

6H5]

(77

), 5

1 [C

4H3]

(10

0)

1596

(2C

=O)

19

3251

(NH

), 21

98 (C

≡N),

1673

(C=O

)(D

MSO

-d6)

: 7.2

6ñ7.

98 (

m, 1

2H, A

r-H

, ben

zofu

ran

prot

ons,

pyr

azol

e ri

ng, t

riaz

ole

ring

),

419

[M+ ]

(11

), 7

7 [C

6H5]

(10

0)9.

02 (

s, 1

H, N

H e

xcha

ngea

ble

by D

2O)

20

3262

(NH

), 22

16 (C

≡N),

1673

(C=O

)(D

MSO

-d6)

: 2.7

8 (s

, 3H

, CH

3), 7

.30ñ

7.96

(m

, 11H

, Ar-

H, b

enzo

fura

n pr

oton

s, p

yraz

ole

433

[M+ ]

(29

), 4

38 [

M+1

] (1

3), 7

7 ri

ng),

9.1

0 (s

, 1H

, NH

exc

hang

eabl

e by

D2O

) [C

6H5]

(10

0)

21

3225

(NH

), 22

16 (C

≡N),

1671

(C=O

) (C

DC

l 3):

2.7

5 (s

, 2H

, CH

2), 7

.04ñ

7.70

(m

, 11H

, Ar-

H, b

enzo

fura

n pr

oton

s, p

yraz

ole

477

[M+ ]

(10

), 7

7 [C

6H5]

(10

0)

ring

), 8

.77

(s, 1

H, N

H e

xcha

ngea

ble

by D

2O)

22

3122

(N

H),

221

0 (C

≡N),

at 1

667

(DM

SO-d

6): 1

.90

(s, 3

H, C

H3)

, 2.2

5 (s

, 2H

, CH

2), 7

.26ñ

7.99

(m

, 10H

, Ar-

H, b

enzo

fura

n 47

5 [M

+ ] (

13),

77

[C6H

5] (

100)

(C

=O)

prot

ons)

, 8.3

9 (s

, 1H

, pyr

azol

e ri

ng),

9.1

7 (s

, 1H

, NH

exc

hang

eabl

e by

D2O

)

23

3119

(N

H),

2212

(C

≡N),

1705

(C

=O)

(DM

SO-d

6): 2

.25

(s, 6

H, 2

CH

3), 5

.36

(s, 1

H, C

H),

7.2

9ñ7.

90 (

m, 1

0H, A

r-H

, ben

zofu

ran

473

[M+ ]

(14

), 7

7 [C

6H5]

(10

0)

prot

ons)

, 8.7

6 (s

, 1H

, pyr

azol

e ri

ng),

9.1

5 (s

, 1H

, NH

exc

hang

eabl

e by

D2O

)

24

Bro

ad b

and

cent

ered

at 3

239

(NH

),

(DM

SO-d

6): 7

.31ñ

7.57

(m

, 10H

, Ar-

H, b

enzo

fura

n pr

oton

s), 7

.92

(s, 1

H, p

yraz

ole

ring

),46

5 [M

+ +2

] (3

9), 7

7 [C

6H5]

(77

), 5

3 22

07 (

C≡N

), 1

664

(C=O

)

9.01

(s,

1 H

, NH

exc

hang

eabl

e by

D2O

) [C

4H5]

(10

0)

25a

3274

, 315

2 (N

H),

220

7 (C

≡N),

167

1 (D

MSO

-d6)

: 1.0

3 (t

, 3H

, CH

2CH

3),

3.60

(q,

2H

, CH

2C

H3)

, 7.3

0ñ7.

92 (

m, 1

0H, A

r-H

,49

6 [M

+ ] (

25),

51

[C4H

3] (

100)

(C

=O),

121

4 (C

=S)

benz

ofur

an p

roto

ns),

8.3

3 (s

, 1H

, pyr

azol

e ri

ng),

8.5

9, 9

.01,

9.1

1, 1

0.50

(s,

4H

, 4N

H

exch

ange

able

by

D2O

)


Molecular docking

Preparation of protein and ligands for docking

The crystal structure of thymidylate synthasecomplexed with RaltitrexedÆ was downloaded fromprotein data bank (http://www.pdb.org/pdb/home/home.do) with pdb code = 1HVY and the site inwhich the inhibitor was complexed was identifiedand the surrounding important residues such as Tyr258, Asp 218, Leu 221, Gly 222, Asn 226, PHE 225,PHE 80, Glu 87, IIe 108, TRP 109, Ala 312, Asn112 and Leu 192 were recognized. All compoundswere drawn using Chem3D ultra 8.0 software. Boththe protein and ligands were saved in pdbqt format.Docking was performed as in the Autodock Vinamanual and the results were visualized usingAutodock tools. Binding affinities were calculatedand the highly ranked compounds were selected anddocking was repeated for these selected compoundsto confirm their affinity.

Building of Grid box

The grid maps representing the native ligand inthe actual docking target site were calculated. Thegrid box dimensions were chosen to be sufficientlylarge to include all the previously mentionedresidues that are surrounding the complexesinhibitor.

RESULTS AND DISCUSSION

Chemistry

During our drug discovery program (23, 24)and in continuation of the recent studies concerningsynthesis and cytotoxic screening of 3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-carbaldehyde (1) (21),it was of interest to introduce compound 1 as usefulstarting material for future synthesis. Condensation

Tab

le 2

. con

t.

Com

pd.n

o.

IR (

KB

r, c

m-1)

1 H-N

MR

(so

lven

t, δ,

ppm

) M

S [m

/z (

%)]

25b

(D

MSO

-d6)

: 1.2

4ñ1.

90 (

m, 1

1H, c

yclo

hexy

l),7

.35ñ

7.93

(m

, 11H

, Ar-

H, b

enzo

fura

n,

548

[M+ ñ

2] (

4), 4

3 [C

3H7]

(10

0)pr

oton

s, p

yraz

ole

ring

), 8

.59,

8.9

7, 9

.13,

11.

43 (

s, 4

H, 4

(NH

) ex

chan

geab

le b

y D

2O)

25c

3300

, 321

0, 3

116

(4N

H),

220

8 (D

MSO

-d6)

: 7.3

7ñ7.

91 (

m, 1

6H, A

r-H

, ben

zofu

ran

prot

ons,

pyr

azol

e ri

ng),

8.7

2, 9

.29,

546

[M+ +2

] (3

3), 5

47 [

M+ +3

] (6

7),

(C≡N

), 1

666

(C=O

), 1

196

(C=S

) 9.

93, 1

1.88

(s,

4H

, 4N

H e

xcha

ngea

ble

by D

2O)

77

[C

6H5]

(10

0)

25d

33

12, 3

139

(NH

), 2

199

(C≡N

),

(DM

SO-d

6): 2

.28

(s, 3

H, C

H3)

, 7.2

6ñ7.

87 (

m, 1

5H, A

r-H

, ben

zofu

ran

prot

ons,

pyr

azol

e 55

8 [M

+ ] (

20),

106

[C

7H8N

] (9

4), 5

1 16

66(C

=O)

ring

), 8

.70,

9.2

5, 9

.80,

11.

83 (

s, 4

H, 4

NH

exc

hang

eabl

e by

D2O

)[C

4H3]

(10

0)

26a

Bro

ad b

and

cent

ered

at 3

116

(DM

SO-d

6): 3

.10

(s, 4

H, -

CH

2CH

2-),

7.3

5ñ7.

96 (

m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

,49

1 [M

+ ] (

14),

77

[C6H

5] (

100)

(2 N

H),

221

0 (C

≡N),

170

8 8.

75 (

s, 1

H, p

yraz

ole

ring

), 9

.12,

9.4

4 (s

, 2H

, 2N

H e

xcha

ngea

ble

by D

2O)

(C=O

), 1

615

(C=O

)

26b

D

MSO

-d6)

: 7.3

4ñ7.

95 (

m, 1

0H, A

r-H

, ben

zofu

ran

prot

ons)

, 8.7

3, 9

.12,

9.4

2 (m

, 4H

,54

0 [M

+ ] (

11),

77

[C6H

5] (

100)

pyri

dine

rin

g, p

yraz

ole

ring

), 1

1.93

, 12.

63 (

s, 1

H, 1

H, 2

NH

exc

hang

eabl

e by

D2O

)

Table 3. Cytotoxic activity of the selected compounds on human livercarcinoma cell line (HEPG2) compared to 5-fluorouracil (5-Fu).

Cell Line

Compd. no. HEPG2

IC50 ( µg/mL)

9 6.48

14 11.8

18 15.6

21 9.53

25b 11.5

5-Fluorouracil 27.7

IC50 = dose of the compound which reduces survival to 50%


of 1 with active methylene compounds namely:ethyl cyanoacetate, acetylacetone and/or ethyl ace-toacetate in the presence of thiourea gave the corre-sponding 2-thioxopyrimidine derivatives 2-6. Alsoone pot reaction (25) of 1 with 6-acetyl-1,2,3,4-tetrahydronaphthalene, ethyl cyanoacetate and/ormalononitrile in excess anhydrous ammoniumacetate afforded compounds 7 and 8, respectively.(Scheme 1)

Furthermore, refluxing of compound 2 withchloroacetic acid in the presence of acetic acid anhy-dride and glacial acetic acid afforded compound 9.The presence of an active methylene group in com-pound 9 was established by condensation with dif-

ferent aromatic aldehydes namely: p-anisaldehyde,2-thiophene carboxaldehyde and/ or indole-3-car-boxaldehyde in acetic acid to give compounds 10a-

c, respectively. Compound 2 was condensed withortho substituted benzoic acids namely: anthranilicacid, salicylic acid and/or thiosalicylic acid in sodi-um ethoxide yielding compounds 11a-c, respective-ly. The displacement reaction of the mercapto groupof 2 was achieved by refluxing with ethanolamine inisopropyl alcohol to give compound 12, whereasalkylation of 2 with iodomethane in sodium ethox-ide gave 13, which condensed with hydrazinehydrate (99%) in absolute ethanol afforded 14.Another route for synthesis of compound 14 was

Scheme 3.

Table 4. Docking results of the synthesized compounds

Interactions of the best mode

Compound Affinity

Interacted Main Distance AngleKcal/mol

moiety Residue Å

2 ñ9.0 CN group NH2 of Lys 308 2.07 21.4

3 ñ8.9 C=O NH2 of Lys 308 2.48 114.4

4 ñ9.6 -NH C=O of 2.64 82.0lle 108

5 ñ8.9 No action

6 ñ9.0 -NH C=O of 2.96 110Leu 221

7 ñ9.9 C=O NH2 of 2.82 121.5PHE 225


9 ñ10.4 C=N -NH2 of 2.03 13.8Asp 218

10a ñ9.1 No action

10b ñ9.5 C=O NH2 of Leu 221 2.17 43.5

10c ñ9.0 C=N NH2 of PHE 80 2.40 134.4

11a ñ9.4 No action

11b ñ9.2 No action

11c ñ9.0 No action

12 ñ9.1 C=N NH2 of Glu 87 2.91 134.9

13 ñ9.5 CN group NH2 of PHE 225 2.57 29.6

14 ñ10.7 NH2 of ñNH-NH2 -C=O of Asp 218 2.58 39.2


16 ñ9.6 -NH2 -C=O of Gly 222 2.72 93.1

17 ñ9.0 No action



20 ñ9.6 No action

21 ñ10.4 CN group NH2 of Lys 308 2.66 118.2-C=O NH2 of Leu 221 3.16 102.7

22 ñ8.9 No action

23 ñ9.7 No action

24 ñ9.5 -C=O NH2 of PHE 80 2.44 121.6

25a ñ9.3 No action

25b ñ10.3 -C=O of pyrimidinone -NH2 of Asp 218 2.49 91.3

25c ñ8.9 No action

25d ñ8.7 No action

26a ñ8.7 No action

26b ñ8.2 No action



Figure 2. The crystal structure of thymidylate synthase showing the site of action of the complexed inhibitor which was used for docking.

Figure 1. Cytotoxicity screening of compounds 9, 14, 18, 21, 25b against HEPG2


hydrate (99%) in absolute ethanol at room tempera-ture or sodium azide in refluxing acetic acid giving16 and 17, respectively. (Scheme 2)

In addition, the reported anticancer activity oftriazole (26, 27), pyrazole (28) and triazine (29, 30)derivatives against different types of carcinoma cellline prompted us to introduce these ring systems inour parent compound. Accordingly, cyclocondensa-tion of compound 14 with ethyl chloroformate, tri-ethylorthoformate or acetic anhydride afforded car-bonitriles 18, 19 and 20, respectively. The reflux of14 with active methylene compounds namely:diethyl malonate, ethyl acetoacetate and/or acetyl-acetone in acetic acid afforded compounds 21ñ23,respectively. Further, cyclocondensation of 14 withdiethyl oxalate in refluxing ethanol yielded carboni-trile 24. Conjugated N-N-S- tridentates ligand sys-tem of thiosemicarbazide (RNH-CS-NH-NH2)seems essential for anticancer activity and displayantiproliferative activity on different tumors cell line(31). Thus, we aimed to introduce such thiosemicarb-azide side chain into our parent compound via reac-tion of 14 with thiosemicarbazide derivatives name-ly: ethyl, cyclohexyl, phenyl or p-tolyl thiosemicarb-azides in dry benzene to give 25a-d, respectively.For incorporation of pyrrolidinedione moiety, whichexihibt antiproliferative activity toward murineleukemia and human colon carcinoma (32, 33), intothe key structure, compound 14 was allowed to reactwith different acid anhydrides namely: succinic acidanhydride and/or 2,3-pyridinedicarboxylic acidanhydride yielding compounds 26a and 26b, respec-tively (Scheme 3).

Antitumor activity

Five of the newly synthesized compounds (9,14, 18, 21, 25b) were chosen for screening of theirbiological cytotoxicity against human liver carcino-ma cell line (HEPG2). According to Table 3 andFigure 1, it is obvious that all of the mentioned com-pounds are highly active against human liver carci-noma cell line (HEPG2) as compared to 5-fluo-rouracil (5-Fu). In case of derivative 9 (IC50 6.48µg/mL) which contain thiazolidinone ring, it showedabout 4-fold higher activity than of the reference 5-Fu. The cytotoxic activity decreased slightly to 3-fold higher than 5-Fu in case of derivative 21 (IC50

9.53 µg/mL) which contain the pyrazolidinedionenucleus. Furthermore, a decrease was observed incase of compounds 14, 18 and 25b (IC50 11.8, 15.6,11.5 µg/mL, respectively) where the benzofuranpyrazole moiety was attached to 2-hydrazinyloxopy-rimidine, triazolopyrimidine and thiosemicarbazidepyrimidine moieties. From the above discussion, we

Figure 5. Compound 18 interaction and formation of hydrogenbond with Lys 308

Figure 3. Hydrogen bond formation of compound 9 with Asp 218

Figure 4. Hydrogen bond formation of compound 14 with Asp218.

succeeded by refluxing of 2 with hydrazine hydrate(99%) in absolute ethanol. Heating a mixture ofcompound 2 with phosphorus pentachloride/phos-phorus oxychloride in a water bath yielded carboni-trile 15, which was condensed with hydrazine


can conclude that the newly synthesized moietiesattached to benzofuran pyrazolo pyrimidine moietyare important for antitumor activity and especiallythiazolidinone ring of compound 9.

Molecular modeling of TS

Autodock Vina (20) is a new program for drugdiscovery. It provides a fast and highly accuratemolecular docking. By obtaining the calculatedbinding affinities we can rank the compounds andselect those that are expected to be of good activity.

In the present work we used the crystal struc-ture of thymidylate synthase complexed withRaltitrexed inhibitor in which the glutamate moietywas not found to have any role in the interactions ofthe complexed ligand as supposed for most ofantifolate or well known thymidylate synthaseinhibitors. Instead of that we found that the nitrogenatom in position 3 of 2-methyl-3,4-dihydro-4-quina-zolinone moiety was the main key for hydrogenbond formation with ñC=O group of Asp 218residue with distance = 2.63 Å.

This can show the important role of the pyrim-idine ring in the proposed activity. In this work wesynthesized a number of compounds that containsubstituted pyrimidine ring, thiopyrimidine or fusedpyrimidine ring system with thiazolone ring systemas in compound 9. All these compounds were dockedinto the same site of the complexed ligand that wasdetermined using pdbsum (http://www.ebi.ac.uk/thornton-srv/ databases/pdbsum/). According to thedocking results, there are five compounds with highaffinity, namely: 9, 14, 18, 21 and 25b with affinitiesof: ñ10.4, ñ10.7, ñ10.5, ñ10.4 and ñ10.3 kcal/mole,respectively. We found that the shared functionalgroups in the compounds can have common interac-tion pattern. For example; the N atom of ñC=N bondin the pyrazole ring was found interacting with -NH2

group of Asp 218 as in 9. It has been shown alsoanother hydrogen bonds formation with ñNH2 ofPHE 80 as found for compound 10c, -NH2 of PHE225 as for compound 13, and ñNH2 of Glu 87 asfound in compound 12.

On the other hand, the common cyano group inmost of the compounds has shown a fixed figure,which was responsible for the formation of hydro-gen bond with -NH2 of Lys 308. Benzofuran ringsystem did not show any observed interactions. TheñC=O groups in the 1,6-dihdro-2-pyrimidine ring ofcompounds 7, 10b and 21 were found interactingwith -NH2 of PHE 225, -NH2 of Leu 221 and -NH2

of Leu 221, respectively. The results of the dockingtogether with the measured affinities, distances, andbond angles are shown in Table 4.

An explanation for the activity of the testedcompounds could be referred to their interactions.For example; compound 9 has shown a hydrogenbond formed between the N atom of -C=N bond inpyrazole ring with the -NH2 group of Asp 218residue (2.03 Å), which is the main residue found inthe interaction profile of Raltitrexed inhibitor (Fig.3).

Also, compound 14 interacted by its -NH2

group of the hydrazine side chain with Asp 218, butwith its -C=O of the carboxylate moiety (2.58 Å)(Fig. 4).

Compound 18 has two interactions with twodifferent conformations: the first is the formation ofhydrogen bond between the cyano group and -NH2

of Lys 308 (2.52 Å) and the second between -NH oftriazole ring with ñC=O of Gly 222 (2.84 Å). Also,compound 21 has two interactions but, within oneconformation that was found forming hydrogenbond by its -CN group with -NH2 of Lys 308 (2.66

Figure 6. Compound 21 shows a single conformation that interactswith two residues (Lys 308 and Leu 221).

Figure 7. Compound 25b hydrogen bond between ñC=O groupand ñNH2 of Asp 218.


Å) and by its -C=O (found in the dioxotetrahydro-1H-pyrazol ring with -NH2 of Leu 221 (3.16 Å)(Figs. 5, 6).

Finally, compound 25b has a conformationthat poses a hydrogen bond formed between the -C=O group of pyrimidinone ring and -NH2 group ofAsp 218 (2.49 Å). That could confirm that the inter-action with such residue may be the key reason forthe activity of the tested compounds as 25b is thethird compound found forming hydrogen bond withAsp 218 (Fig. 7).

CONCLUSION

This study includes the preparation of 32derivatives, which contain benzofuran pyrazolopyrimidine as backbone skeleton attached to variousaromatic and/or heterocyclic ring systems such asthiazolidinone, quinazoline, triazole, pyrazole, tri-azine, pyrrolidine and side chain as thiosemicarb-azide derivatives. All of the compounds wereautodocked for the inhibition of the enzymethymidylate synthase via Autodock Vina. The studyshowed that most of the derivatives exhibited highaffinities towards the enzyme thymidylate synthaseexcept compounds 5, 10a, 11a-c, 17, 22, 23, 25a,25c,d and 26a,b. In order to specify the autodockstudy, five of the compounds: 9, 14, 18, 21 and 25b

were studied for cytotoxicity against human livercarcinoma cell line (HEPG2) and all showed higheractivity than that of 5-fluorouracil. This indicatesthat the heterocyclic rings thiazolidinone, pyrazo-lidinedione, triazole and side chain as hydrazinyl, inthiosemicarbazide derivatives are essential for theantitumor activity.

Acknowledgment

The authors express deep thanks to Prof. Dr.Samia Shoman, the head of Pharmacology Unit,National Cancer Institute, Cairo University for car-rying out the anticancer screening, and the staff ofCentral Services Laboratory, National ResearchCentre, Dokki, Cairo, Egypt.

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Received: 10. 05. 2010

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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 3 pp. 357ñ373, 2011