Antioxidant Activity of Novel Fused Heterocyclic Compounds Derived ...

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quinazolinone 8, respectively. The IR spectrum of the dihy- dropyrimidine 6 showed a broad peak centered at 3206 due to νOH and νNH and the carbonyl band ...
Chem. Pharm. Bull. 63, 866–872 (2015)

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Regular Article

Antioxidant Activity of Novel Fused Heterocyclic Compounds Derived from Tetrahydropyrimidine Derivative Marwa Sayed Salem,*,a Mahmoud Farhat,b Asma Omar Errayes,b and Hassan Mohamed Fawzy Madkoura a

 Synthetic Organic Chemistry Laboratory, Chemistry Department, Faculty of Science, Ain Shams University; Abbasiya, Cairo 11566, Egypt: and b Chemistry Department, Faculty of Science, Tripoli University; Tripoli 13494, Libya. Received June 1, 2015; accepted June 27, 2015 6-(Benzo[d][1,3]dioxol-5-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile has been utilized for synthesis of the fused heterocyclic compounds namely thiazolopyrimidines, tetrazolopyrimidine, pyrimidoquinazoline, pyrimidothiazolopyrimidine, pyrimidothiazolotriazine and pyrrolothiazolopyrimidine derivatives. The newly synthesized compounds were characterized by IR, 1H-NMR, 13C-NMR, and mass spectral data. Antioxidant activities of all synthesized compounds were investigated. Key words

arylidene; tetrahydropyrimidine; thiazolo[3,2-a]pyrimidine; antioxidant activity

Pyrimidines are of great importance in fundamental metabolism, being an integral part of DNA and RNA, found in the three bases uracil, thymine and cytosine of the six present in the nucleotides.1) They are found to possess diverse biological properties as bactericides, fungicides, viricides, insecticide, and meticides2) and antioxidants.3) Many derivatives of pyrimidine have been used as therapeutic agents.4) Several triazolo and pyrazolopyrimidine derivatives are found to possess antifungal and antilieishmanial activity.5) Certain pyrimidine derivatives are known to display antimalarial6) antifilarial activities and also potent inhibitors of cancer cell proliferation.7–9) In the recent years, a lot of attention has been drawn by the pyrimidine derivatives due to their diverse range of activities, especially calcium channel blocker property.10) The most general and widely used route to synthesize pyrimidines involves the combination of a reagent containing the N–C–N skeleton namely (urea, thiourea and guanidine) with C–C–C unit such as 1,3-diketones and diesters. In continuation of our previous work,11) thiourea is employed as the N–C–N unit and is condensed with arylidene ethyl cyanoacetate to complete the pyrimidine ring namely 6-(benzo[d][1,3]dioxol-5-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 1. The present work aimed at utilization of thioxotetrahydropyrimidine 1 as a scaffold for synthesis of different heterocyclic compounds and studies their biological activities as antioxidant agent.

Results and Discussion

Chemistry When tetrahydropyrimidine 1 was submitted to react with bromomalononitrile in aqueous alcoholic potassium carbonate solution enaminonitrile 2 was obtained. The structural features of enaminonitrile 2 were identified on the basis of coupling band exhibited at ν 3391 and 3291 cm−1 due to the amino NH2 functionality and disappearance of νC=S. 1 H-NMR spectrum revealed D2O-exchangeable singlet at δ 8.48 ppm due to amino group. Thiazolopyrimidine derivative 3 can be obtained via reaction of tetrahydropyrimidine 1 with chloroacetamide. The structural features of thiazolopyrimidine derivative 3 were

established by elemental analysis as well as spectral data. The structure of compound 3 also confirmed chemically via condensation reaction with substituted aromatic aldehydes namely, 4-chlorobenzaldehyde and/or 4-methoxybenzaldehyde to afford the corresponding benzylidene derivatives 4a and b, respectively. Chlorination of tetrahydropyrimidine 1 with a mixture of phosphorus pentachloride and phosphorus oxychloride as a chlorinating reagent gave the chloropyrimidine derivative 5. The structure of compound 5 was assigned from its spectroscopic data, a qualitative and quantitative elemental analysis which indicates the presence of chlorine. A chemical evidence for the structure assignment of compound 5 is the reaction with glycine, sodium azide and/or anthranilic acid to afford dihydropyrimidine 6, tetrazolopyrimidine 7 and pyrimidoquinazolinone 8, respectively. The IR spectrum of the dihydropyrimidine 6 showed a broad peak centered at 3206 due to νOH and νNH and the carbonyl band of a carboxylic acid at 1700 cm−1. This spectrum pattern reveals the possibility of two interconvertable forms for the product 6 via 1,3-proton shift. Alkylation of tetrahydropyrimidine 1 with ethyl iodide in the presence of sodium ethoxide furnished S-alkylated product 9, which has been chlorinated via reaction with phosphorus pentachloride in the presence of phosphorus oxychloride to afford the chlorinated product 10. Pyrimidine derivative 10 underwent thiation under the effect of thiourea to give thioxodihydropyrimidine 11. The IR spectrum of thioxodihydropyrimidine 11 displayed the appearance of νNH and νC=S at 3194 and 1240, respectively. 1H-NMR of compound 11 showed D2O-exchangeable signal at 13.02 ppm due to NH proton. Alkaline hydrolysis of tetrahydropyrimidine 1 using 10% alcoholic sodium hydroxide solution gave dioxotetrahydropyrimidine derivative 12 (cf. Chart 1). Enaminonitrile 2 is the key starting material for design and synthesis of fused novel heterocyclic systems such as pyrimidothiazolopyrimidine derivatives 13 and 14 and pyrimidothiazolotriazine 15. Thus, when enaminonitrile 2 was allowed to react with formamide, formic acid and/or sodium nitrite, it afforded pyrimidothiazolopyrimidine derivatives 13 and 14 and

 To whom correspondence should be addressed.  e-mail: [email protected] *  © 2015 The Pharmaceutical Society of Japan

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(i) BrCH(CN)2, K 2CO3, EtOH; (ii) ClCH 2CONH 2, fused AcONa, EtOH; (iii) Ar1CHO, fused AcONa, EtOH; (iv) POCl3, PCl5; (v) NH 2CH 2COOH, Ac2O; (vi) NaN3, glacial AcOH; (vii) anthranilic acid, EtOH; (viii) EtI, EtONa; (ix) NH 2CSNH 2, EtOH; (x) NaOH 10%.

Chart 1

pyrimidothiazolotriazine 15 respectively. Treatment of enaminonitrile 2 with ethyl chloroacetate and /or carbon disulfide in pyridine furnished ethyl-2-(7-(benzo[d][1,3]dioxol-5-yl)-2,6dicyano-5-oxo-5H-thiazolo[3,2-a]pyrimidin-3-ylamino)acetate (16) and/or (7-(benzo[d][1,3]dioxol-5-yl)-2,6-dicyano-5-oxo5H-thiazolo[3,2-a]pyrimidin-3-yl)carbamodithioic acid (17), respectively (cf. Chart 2). In one of our previous publications,12) it has been reported that, the effect of boiling triethyl orthoformate on enaminonitrile resulted in ethyl formamidate derivative such as 18. In the present work, reaction of neat triethyl orthoformate with enaminonitrile 2 under reflux gave pyrrolothiazolopyrimidine derivative 19 indicating that cyclization on the cyano functionality takes place after the nucleophilic attack of the amino group on the electronically deficient carbon atom of triethyl orthoformate. Treatment of enaminonitrile 2 with diethyl malonate in ethanolic solution of sodium ethoxide afforded N-(7(benzo[d][1,3]dioxol-5-yl)-2,6-dicyano-5-oxo-5H-thiazolo[3,2̈ H is more acidic than a]pyrimidin-3-yl)acetamide (20). As –N C–H bond, the basic ethoxide ion abstracts a proton from NH2 ̈ ⊖ which attacks the carbonyl carbon of groups to generate N

the ester group through tetrahedral mechanism leading to departure of the good leaving ethoxide group (cf. Chart 3). Pharmacology Antioxidant Evaluation The antioxidant activities of the synthesized compounds were determined and listed in Table 1 and Fig. 1. The results revealed that all compounds were found to be potent. Moreover, the results showed that nearly three compounds 1, 6 and 9 were found to be the most potent levels of activity. Additionally, compounds 2, 4b, 8, 11, 14, 17, 19 and 20 were found to have moderate activity. The following points were noticed. On comparison between the compounds 1, 6 and 9, it was noticed that compound 6 indicating that the presence of COOH group was more effective than the tetrahydropyrimidine 1, while conversion of the C=S group in tertrahydropyrimidine 1 to -C-S-Et group in compound 9, resulted in low activity of 9. On the other hand when C=O in compound 9 convert into C=S in compound 11 antioxidant activity decrease. Chloropyrimidine 5 is less active than tetrahydropyrimidine 1 but more active than pyrimidioquinazoline 8. Compound 19 is more potent antioxidant than compounds 14, 17 and 20 that is due

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to the presence of =NH group.

Experimental

All melting points were measured on a Gallenkamp electric melting point apparatus and are uncorrected. The infrared spectra were recorded using potassium bromide disks on a Pye Unicam SP-3–300 infrared spectrophotometer. 1H- and

C-NMR experiments were run at 300 MHz on a Varian Mercury VX-300 NMR spectrometer using tetramethylsilane (TMS) as internal standard in deuterated chloroform or dimethylsulphoxide. Chemical shifts are quoted as δ. The mass spectra were recorded on Shimadzu GCMS-QP-1000EX mass spectrometers at 70 eV. All the spectral measurements as well as the elemental analyses were carried out at the Micro analytical Center of Cairo University. All the newly synthesized compounds gave satisfactory elemental analyses. The reactions and the purity of all new compounds were monitored by TLC. Synthesis 3-Amino-7-(benzo[d][1,3]dioxol-5-yl)-5-oxo-5H-thiazolo[3,2-a]pyrimidine-2,6-dicarbonitrile (2) A mixture of tetrahydropyrimidine 111) (2.73 g, 0.01 mol), bromomalononitrile (1.44 g, 0.01 mol) and potassium carbonate (1.38 g, 0.01 mol/25 mL H2O) in ethanol (25 mL) was heated under reflux for 2 h, cooled and then poured onto ice with stirring. The resulted solid product was filtered, dried and recrystallized from ethanol to give enaminonitrile 2 as orange crystals. mp 260–262°C, yield 60%. FT-IR (KBr, cm−1): 3391, 3291 νNH2, 3081 νCH aromatic, 2909 νCH aliphatic, Table 1.

(i) HCONH 2; (ii) HCOOH; (iii) NaNO2, AcOH, HCl; (iv) ClCH 2COOEt, pyridine; (v) CS2, pyridine; (vi) CH(OEt)3; (vii) CH 2(COOEt)2, EtONa.

Total Antioxidant Capacity of the Synthesized Compounds

Compound

Total antioxidant capacity (mg AAE/g compound)

1 2 3 4a 4b 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20

308.33±1.25 132.54±1.65 69.01±2.55 20.38±1.85 102.74±1.35 112.15±2.40 436.85±2.25 97.25±2.75 109.01±1.15 225.87±1.50 82.34±2.45 176.46±1.60 56.46±1.30 76.07±1.95 163.91±2.80 95.68±1.20 81.56±2.45 123.91±2.45 176.46±1.75 145.88±3.30

Results are (mean±S.D.) (n=3) and AAE.

Chart 2

Chart 3

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Fig. 1.

Total Antioxidant Capacity of the Synthesized Compounds

2201 νC≡N, 1687 νC=O. 1H-NMR (300 MHz, DMSO-d6): 8.48 (s, 2H, NH2, D2O-exchangeable), 7.63–7.11 (m, 3H, Ar-H) and 6.17 (s, 2H, O–CH2–O). 13C-NMR (75 MHz, DMSO-d6): 102.2 (O–CH2–O), 107.88, 108.38, 112.89, 115.65, 124.60, 127.99, 129.26, 147.67, 150.83, 151.28, 161.35, 165.19 (C=O). MS m/z: 339 (M+2]+·), 338 (M+1]+·), 337 (M+·). Anal. Calcd for C15H7 N5O3S (337.31): C, 53.41; H, 2.09; N, 20.76; S, 9.51. Found: C, 53.26; H, 1.98; N, 20.81; S, 9.42. 7-(Benzo[d][1,3]dioxol-5-yl)-3,5-dioxo-3,5-dihydro-2Hthiazolo[3,2-a]pyrimidine-6-carbonitrile (3) To a solution of tetrahydropyrimidine 1 (2.73 g, 0.01 mol) in ethanol (20 mL), chloroacetamide (0.93 g, 0.01 mol) and sodium acetate (1.23 g, 0.015 mol), was added. The reaction mixture was refluxed for 3 h, then left to cool; the precipitated solid was filtered, dried and recrystallized from ethanol to give thiazolopyrimidine 3 as yellow crystals, mp 195–197°C, yield 77%. FT-IR (KBr, cm−1): 3033 νCH aromatic, 2919 νCH aliphatic, 2224 νC≡N, 1724, 1695 νC=O, 1654 νC=N. 1H-NMR (300 MHz, DMSO-d6): 7.61–7.10 (m, 3H, Ar-H), 6.16 (s, 2H, O–CH2–O) and 4.13 (s, 2H, S–CH2–C=O). MS m/z: 313 (M+·). Anal. Calcd for C14H7 N3O4S (313.29): C, 53.67; H, 2.25; N, 13.41; S, 10.23. Found: C, 53.45; H, 2.12; N, 13.31; S, 10.15. Reaction of 7-(Benzo[d][1,3]dioxol-5-yl)-3,5-dioxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carbonitrile (3) with Aromatic Aldehydes A mixture of thiazolopyrimidine 3 (1.5 g, 0.005 mol), 4-chlorobenzaldehyde and/or 4-methoxybenzaldehyde (0.005 mol) and sodium acetate (0.66 g, 0.0055 mol), in ethanol (20 mL) was refluxed for 3 h. After cooling the reaction mixture the precipitated solid was collected by filtration, dried and recrystallized from ethanol to give benzylidene derivatives 4a, b, respectively. 2-(4-Chlorobenzylidene)-7-(benzo[d][1,3]dioxol-5-yl)-3,5dioxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carbonitrile (4a) Yellow crystals, mp 270–272°C, yield 68%, FT-IR (KBr, cm−1): 3127 νCH aromatic, 2828 νCH aliphatic, 2227 νC≡N, 1721, 1665 νC=O. 1H-NMR (300 MHz, DMSO-d6): 7.26–7.08 (m, 7H, Ar-H), and 6.19–6.11 (m, 3H, O–CH2–O, –CH=C–S). MS m/z: 435 (M+·). Anal. Calcd for C21H10ClN3O4S (435.84): C, 57.87; H, 2.31; Cl, 8.13; N, 9.64; S, 7.36. Found: C, 57.75; H, 2.42; Cl, 8.24; N, 9.73; S, 7.27.

2-(4-Methoxybenzylidene)-7-(benzo[d][1,3]dioxol-5-yl)-3,5dioxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carbonitrile (4b) White crystals, mp >300°C, yield 72%, FT-IR (KBr, cm−1): 3141 νCH aromatic, 2980 νCH aliphatic, 2205 νC≡N, 1713 and 1662 νC=O. 1H-NMR (300 MHz, DMSO-d6): 9.87 (s, 1H, –CH=C-S), 7.31–6.94 (m, 7H, Ar-H), 6.07 (s, 2H, O–CH2–O) and 3.87 (s, 3H, –OCH3). 13C-NMR (75 MHz, DMSO-d6): 77.89 (OCH3), 101.26 (O–CH2–O), 107.42, 107.49, 108.33, 108.42, 120.15, 122.49, 122.51, 132.64, 146.72, 148.37, 158.55, 165.86 (2C=O, C=N), 170.90. MS m/z: 431 (M+·). Anal. Calcd for C22H13N3O5S (431.42): C, 61.25; H, 3.04; N, 9.74; S, 7.43. Found: C, 61.12; H, 3.00; N, 9.65; S, 7.34. 6 - ( Ben zo[d ][1,3]dioxol-5-yl) - 4 - chloro -2-thioxo -1,2dihydropyrimidine-5-carbonitrile (5) A mixture of tetrahydropyrimidine 1 (2.73 g, 0.01 mol), and phosphorus pentachloride (2.5 g, 0.01 mol) in phosphorus oxychloride (7 mL, 0.01 mol) was heated on a water bath for 8 h. The reaction mixture was cooled then poured onto crushed ice, collect the product by filtration, dried and recrystallized from ethanol to afford thioxodihydropyrimidine derivative 5 as beige crystals, mp >300°C, yield 86%. FT-IR (KBr, cm−1): 3202 νNH, 3151 νCH aromatic, 2987 νCH aliphatic, 2225 νC≡N, 1652 νC=N, 1220 νC=S. 1H-NMR (300 MHz, DMSO-d6): 13.07 (br s, 1H, -NH, D2O-exchangeable), 7.22–7.07 (m, 3H, Ar-H) and 6.14 (s, 2H, O–CH2–O). MS m/z: 291 (M+·). Anal. Calcd for C12H6ClN3O2S (291.71): C, 49.41; H, 2.07; Cl, 12.15; N, 14.40; S, 10.99. Found: C, 49.25; H, 2.12; Cl, 12.00; N, 14.52; S, 11.10. 2-(6-(Benzo[d ][1,3]dioxol-5-yl)-5-cyano-2-thioxo-1,2dihydropyrimidin-4-ylamino)acetic Acid (6) A mixture of thioxodihydropyrimidine derivative 5 (2.92 g, 0.01 mol) and glycine (0.75 g, 0.01 mol) in acetic anhydride (10 mL) was refluxed for 6 h, cooled and The solid formed was collected by filtration, dried and recrystallized from ethanol to yield compound 6 as black crystals, mp 240–242°C, yield 89%. FT-IR (KBr, cm−1): br centered at 3206 νOH & νNH, 2219 νC≡N, 1700 νC=O acid, 1606 νC=N. MS m/z: 330 (M+·). Anal. Calcd for C14H10N4O4S (330.32): C, 50.91; H, 3.05; N, 16.96; S, 9.71. Found: C, 50.85; H, 2.99; N, 16.79; S, 9.67. These data are satisfied and convincing for both forms of compound 6.

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7-(Benzo[d][1,3]dioxol-5-yl)-5-thioxo-5,6-dihydrotetrazolo[1,5-f ]pyrimidine-8-carbonitrile (7) To a solution of thioxodihydropyrimidine derivative 5 (2.92 g, 0.01 mol) in glacial acetic acid (30 mL), sodium azide (0.65 g, 0.01 mol), was added. The reaction mixture was refluxed for 5 h and allowed to cool. The solid formed was collected, dried and recrystallized from ethanol to give compound 7 as brown crystals, mp >300°C, yield 63%. FT-IR (KBr, cm−1): 3217 νNH, 2216 νC≡N, 1612 νC=N. 1H-NMR (300 MHz, DMSO-d6): 7.77–7.07 (m, 3H, Ar-H), 6.13 (s, 2H, O–CH2–O) and 3.40 (br s, 1H, –NH, D2O-exchangeable). MS m/z: 298 (M+·). Anal. Calcd for C12H6N6O2S (298.28): C, 48.32; H, 2.03; N, 28.17; S, 10.75. Found: C, 48.23; H, 1.98; N, 28.23; S, 10.89. 3 - ( Ben zo[d ][1,3]d ioxol-5 -yl) -10 - oxo -1-t h ioxo -2 ,10 dihydro-1H-pyrimido[6,1-b]quinazoline-4-carbonitrile (8) A mixture of thioxodihydropyrimidine derivative 5 (2.92 g, 0.01 mol), and anthranilic acid (1.37 g, 0.01 mol) in ethanol (50 mL) was refluxed for 6 h, cooled, filtered, dried and recrystallized from ethanol to afford compound 8 as pale brown crystals, mp 270–272°C, yield 68%. FT-IR (KBr, cm−1): 3346, 3209 νNH, 2216 νC≡N, 1645 νC=O pyrimidinone, 1605 νC=N, 1247 νC=S. 1H-NMR (300 MHz, DMSO-d6): 8.10 (br s, 1H, –NH, D2O-exchangeable), 7.50–7.13 (m, 7H, Ar-H), 6.17 (s, 2H, O–CH2–O). MS m/z: 374 (M+·). Anal. Calcd for C19H10N4O3S (374.37): C, 60.96; H, 2.69; N, 14.97; S, 8.56. Found: C, 60.82; H, 2.58; N, 14.89; S, 8.47. 4-(Benzo[d ][1,3]dioxol-5-yl)-2-(ethylthio)-6 -oxo-1,6 dihydropyrimidine-5-carbonitrile (9) A solution of tetrahydropyrimidine 1 (2.73 g, 0.01 mol) in ethanolic solution of sodium ethoxide (20 mL), ethyl iodide (1.82 g, 0.01 mol), was added then refluxed for 6 h. Left to cool, the solid was filtered, dried and recrystallized from ethanol to give compound 9 as yellow crystals, mp 274–275°C, yield 55%. FT-IR (KBr, cm−1): 3167 νNH, 3082 νCH aromatic, 2969 νCH aliphatic, 2234 νC≡N, 1705 νC=O pyrimidinone, 1672 νC=N. 1 H-NMR (300 MHz, DMSO-d6): 13.10 (s, 1H, –NH, D2O-exchangeable), 7.24–7.10 (m, 3H, Ar-H), 6.17 (s, 2H, O–CH2–O), 3.45 (q, 2H, –CH2CH3, J=6.0 Hz) and 1.55 (t, 3H, –CH2CH3, J=6.0 Hz). 13C-NMR (75 MHz, DMSO-d6): 14.30, 14.57, 62.78, 100.08, 102.54, 108.99, 109.34, 126.21, 129.97, 154.42, 154.80, 178.65. MS m/z: 301 (M+·). Anal. Calcd for C14H11N3O3S (301.32): C, 55.80; H, 3.68; N, 13.95; S, 10.64. Found: C, 55.68; H, 3.75; N, 13.78; S, 10.60. 4 -(Ben zo[d ][1,3]dioxol-5-yl)- 6 - chloro-2-(ethylthio)pyrimidine-5-carbonitrile (10) A mixture of S-alkylated pyrimidine 9 (3.01 g, 0.01 mol), was heated under reflux in phosphorus oxychloride (7 mL, 0.01 mol) and phosphorus pentchloride (2.5 g, 0.01 mol) for 8 h, cooled and poured onto ice. The precipitated solid was filtered off, dried and recrystallized from ethanol to give compound 10 as brown crystals, mp 220–222°C, yield 73%. FT-IR (KBr, cm−1): 3205 νCH aromatic, 2914 νCH aliphatic, 2222 νC≡N, 1655 νC=N. 1H-NMR (300 MHz, DMSO-d6): 7.30–6.95 (m, 3H, Ar-H), 6.17 (s, 2H, O–CH2–O), 3.43 (q, 2H, –CH2CH3, J=7.5 Hz), 1.06 (t, 3H, –CH2CH3, J=7.5 Hz). MS m/z: 319 (M+·). Anal. Calcd for C14H10ClN3O2S (319.77): C, 52.59; H, 3.15; Cl, 11.09; N, 13.14; S, 10.03. Found: C, 52.45; H, 3.03; Cl, 11.00; N, 13.02; S, 10.14.

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4-(Benzo[d][1,3]dioxol-5-yl)-2-(ethylthio)-6-thioxo-1,6dihydropyrimidine-5-carbonitrile (11) A mixture of chloropyrimidine derivative 10 (3.19 g, 0.01 mol), thiourea (0.76 g, 0.01 mol), in ethanol (20 mL) was refluxed for 6 h, cooled. The precipitated solid was filtered, dried and recrystallized from ethanol to afford compound 11 as orange crystals, mp 212–214°C, yield 88%. FT-IR (KBr, cm−1): 3194 νNH, 2218 νC≡N, 1667 νC=N, 1240 νC=S. 1H-NMR (300 MHz, DMSO-d6): 13.02 (br s, 1H, –NH, D2O-exchangeable), 7.16–6.95 (m, 3H, Ar-H), 6.16 (s, 2H, O–CH2–O), 3.50 (q, 2H, –CH2CH3, J=6.9 Hz) and 1.06 (t, 3H, –CH2CH3, J=6.9 Hz). MS m/z: 317 (M+·). Anal. Calcd for C14H11N3O2S2 (317.39): C, 52.98; H, 3.49; N, 13.24; S, 20.21. Found: C, 52.89; H, 3.36; N, 13.11; S, 20.03. 6 - ( B e n z o [d ] [1, 3] d i o x o l -5 -y l ) -2 , 4 - d i o x o -1, 2 , 3 , 4 tetrahydropyrimidine-5-carbonitrile (12) A solution of tetrahydropyrimidine 1 (2.73 g, 0.01 mol) in sodium hydroxide (20 mL, 10%), was refluxed for 8 h, cooled, acidified with dil. HCl to give precipitates, which were filtered, dried and recrystallized from ethanol to give compound 12 as yellow crystals, mp 268–270°C, yield 70%. FT-IR (KBr, cm−1): 3440 νNH, 2232 νC≡N, 1702 νC=O, 1660 νC=N,. 1H-NMR (300 MHz, DMSO-d6): 13.09 (s, 2H, –2NH, D2O-exchangeable), 7.25–7.09 (m, 3H, Ar-H), 6.17 (s, 2H, O– CH2–O). 13C-NMR (75 MHz, DMSO-d6): 90.17, 102.10, 108.39, 109.02, 114.81, 122.51, 123.70, 147.08, 150.47, 158.50, 160.21, 176.04. MS m/z: 257 (M+·). Anal. Calcd for C12H7 N3O4 (257.2): C, 56.04; H, 2.74; N, 16.34. Found: C, 55.98; H, 2.63; N, 16.22. 4 - A m i n o -7- (1, 3 - b e n z o d i o x o l -5 -y l ) - 9 - o x o - 9H pyrimido[4′,5′:4,5][1,3]thiazolo[3,2-a]pyrimidine-8-carbonitrile (13) A solution of enaminonitrile 2 (1.69 g, 0.005 mol) in formamide (10 mL) was heated under reflux for 4 h, cooled and poured onto ice. The product was collected by filtration, dried and recrystallized from ethanol to give pyrimidothiazolopyrimidine 13 as brown crystals, mp 154–156°C, yield 42%. FT-IR (KBr, cm−1): 3313, 3190 νNH2, 2207 νC≡N, 1668 νC=O. 1 H-NMR (300 MHz, DMSO-d6): 9.44 (br s, 2H, –NH2, D2Oexchangeable), 8.40 (s, 1H, pyrimidine-H2), 7.42–6.84 (m, 3H, Ar-H), 6.13 (s, 2H, O–CH2–O). MS m/z: 364 (M+·). Anal. Calcd for C16H8N6O3S (364.34): C, 52.75; H, 2.21; N, 23.07; S, 8.80. Found: C, 52.61; H, 2.12; N, 23.18; S, 8.74. 7-(1,3-Ben zodioxol-5-yl)- 4,9-dioxo-1,4 -dihyd ro -9Hpyrimido[4′,5′:4,5][1,3]thiazolo[3,2-a]pyrimidine-8-carbonitrile (14) A solution of enaminonitrile 2 (1.69 g, 0.005 mol) in formic acid (10 mL) was heated under reflux for 10 h, cooled and then poured onto ice. The precipitate solid was collected by filtration, dried and recrystallized from ethanol to give compound 14 as brown crystals, mp 234–236°C, yield 45%. FT-IR (KBr, cm−1): 3392 νNH, 2219 νC≡N, 1683 νC=O. 1H-NMR (300 MHz, DMSO-d6): 12.67 (s, 1H, –NH, D2O-exchangeable), 8.44 (s, 1H, pyrimidine-H2), 8.23–7.14 (m, 3H, Ar-H) and 6.19 (s, 2H, O–CH2–O). MS m/z: 365 (M+·). Anal. Calcd for C16H7 N5O4S (365.32): C, 52.60; H, 1.93; N, 19.17; S, 8.78. Found: C, 52.54; H, 1.79; N, 19.02; S, 8.78. 7- (1, 3 - B e n z o d i o xo l -5 -y l ) - 4 - c h l o r o - 9 - o xo - 9H pyrimido[2′,1′:2,3][1,3]thiazolo[4,5-d ][1,2,3]triazine-8carbonitrile (15) A solution of sodium nitrite in 10 mL H2O was added to a cold solution of enaminonitrile 2 (1.69 g, 0.005 mol), in acetic

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acid (30 mL) and conc. HCl (15 mL), After complication of the addition the ice bath was removed and stirring continued for an additional 2 h. The crude product was filtered, dried then recrystallized from ethanol to afford 15 as brown crystals, mp 92–94°C, yield 78%. FT-IR (KBr, cm−1): 2210 νC≡N, 1670 νC=O pyrimidinone, 1621 νC=N. 1H-NMR (300 MHz, DMSO-d6): 8.38–6.91 (m, 3H, Ar-H), 6.21 (s, 2H, O–CH2–O). MS m/z: 385 (M+1]+·), 384 (M+·). Anal. Calcd for C15H5ClN6O3S (384.76): C, 46.82; H, 1.31; Cl, 9.21; N, 21.84; S, 8.33. Found: C, 46.77; H, 1.27; Cl, 9.00; N, 21.69; S, 8.21. Ethyl-2-(7-(benzo[d][1,3]dioxol-5-yl)-2,6-dicyano-5-oxo-5Hthiazolo[3,2-a]pyrimidin-3-ylamino)acetate (16) A mixture of enaminonitrile 2 (1.69 g, 0.005 mol), ethyl choloroacetate (0.61g, 0.01 mol) in 20 mL dry pyridine was heated under reflux for 6 h, left to cool, acidified with cold dilute hydrochloric acid, filtered, dried and recrystallized from ethanol to give compound 16 as black crystals, mp 255–257°C, yield 80%. FT-IR (KBr, cm−1): 3338 νNH, 2197 νC≡N, 1730 νC=O ester, 1668 νC=O pyrimidinone, 1635 νC=N. 1H-NMR (300 MHz, DMSO-d6): 8.27–7.28 (m, 4H, Ar-H, –NH, D2Oexchangeable), 6.13 (s, 2H, O–CH2–O), 4.63 (q, 2H, –CH2CH3, J=7.2 Hz), 4.27 (m, 2H, HN–CH2–CO), 1.55 (t, 3H, –CH2CH3, J=7.2 Hz). MS m/z: 423 (M+·). Anal. Calcd for C19H13N5O5S (423.4): C, 53.90; H, 3.09; N, 16.54; S, 7.57. Found: C, 53.78; H, 2.99; N, 16.45; S, 7.48. (7-(Benzo[d ][1,3]dioxol-5-yl)-2,6 -dicyano-5-oxo-5Hthiazolo[3,2-a]pyrimidin-3-yl)carbamodithioic Acid (17) To a solution of enaminonitrile 2 (1.69 g, 0.005 mol) in pyridine (7 mL), carbon disulfide (10 mL) was added. The reaction mixture was refluxed for 8 h, left to cool, acidified with cold dilute hydrochloric acid to give precipitate, which was filtered, dried and recrystallized from ethanol to give compound 17 as pale brown crystals, mp 220–222°C, yield 68%. FT-IR (KBr, cm−1): 3301 νNH, 2210 νC≡N, 1710 νC=O pyrimidinone, 1640 νC=N, 1247 νC=S. 1H-NMR (300 MHz, DMSO-d6): 14.11 (s, 1H, –SH, D2O-exchangeable), 13.59 (s, 1H, –NH, D2O-exchangeable), 7.65–7.07 (m, 3H, Ar-H), 6.20 (d, 2H, O–CH2–O, J=12.0 Hz). MS m/z: 414 (M+1]+·), 413 (M+·). Anal. Calcd for C16H7 N5O3S3 (413.45): C, 46.48; H, 1.71; N, 16.94; S, 23.27. Found: C, 46.37; H, 1.66; N, 16.78; S, 23.14. 6-(1,3-Benzodioxol-5-yl)-2-ethoxy-3-imino-8-oxo-3H,8Hpyrrolo[2′,3′:4,5][1,3]thiazolo[3,2-a]pyrimidine-7-carbonitrile (19) A solution of enaminonitrile 2 (1.69 g, 0.005 mol) in triethyl orthoformate (10 mL), was heated under reflux for 8 h, excess of triethyl orthoformate was removed by distillation under reduced pressure. The solid formed was filtered, dried and recrystallized from ethanol to give compound 19 as brown crystals, mp 170–174°C, yield 69%. FT-IR (KBr, cm−1): 3344 νNH, 2199 νC≡N, 1710 νC=O pyrimidinone, 1624 νC=N. 1H-NMR (300 MHz, DMSO-d6): 7.69–7.12 (m, 3H, Ar-H), 6.11 (s, 2H, O–CH2–O), 4.52 (s, 1H, –NH, D2O-exchangeable), 4.30 (q, 2H, –CH2CH3, J=6.9 Hz), 1.27 (t, 3H, –CH2CH3, J=6.9 Hz). MS m/z: 393 (M+·). Anal. Calcd for C18H11N5O4S (393.38): C, 54.96; H, 2.82; N, 17.80; S, 8.15. Found: C, 54.85; H, 2.74; N, 17.68; S, 8.00. N-(7-(Benzo[d][1,3]dioxol-5-yl)-2,6-dicyano-5-oxo-5Hthiazolo[3,2-a]pyrimidin-3-yl)acetamide (20) A mixture of enaminonitrile 2 (1.69 g, 0.005 mol), diethyl malonate (0.01 mol) in solution of sodium ethoxide (0.05 g sodium in 30 mL absolute ethanol) was heated under reflux

for 6 h, cooled and acidified with cold dilute hydrochloric acid, filtered, dried and recrystallized from ethanol to give compound 15 as brown crystals, mp 280–282°C, yield 81%. FT-IR (KBr, cm−1): 3389, 3221 νNH, 2225, 2192 (2 νC≡N), 1673 νC=O pyrimidinone, 1628 νC=O amide. 1H-NMR (300 MHz, DMSOd6): 8.48 (br s, 1H, –NH, D2O-exchangeable), 7.62–7.11 (m, 3H, Ar-H), 6.17 (s, 2H, O–CH2–O) and 1.26 (s, 3H, –CH3). MS m/z: 381 (M+2]+·), 380 (M+1]+·), 379 (M+·). Anal. Calcd for C17H9N5O4S (379.35): C, 53.82; H, 2.39; N, 18.46; S, 8.45. Found: C, 53.69; H, 2.24; N, 18.38; S, 8.31. Determination of Total Antioxidant Capacity (TAC) The antioxidant activity (AOA) of a compound was determined according to phosphomolybdenum method13) using ascorbic acid as standard. This assay is based on the reduction of MoVI to MoV by the sample analyte and subsequent formation of a green colored [phosphate=MoV] complex at acidic pH. In this method, 0.5 mL of the compound (100 µg/mL) in methanol was combined in dried vial with 5 mL of reagent solution (0.6 M sulfuric acid, 28 m M sodium phosphate and 4 m M ammonium molybdate solutions). The vials containing the reaction mixture were capped and incubated in a thermal block at 95°C for 90 min. After the samples had cooled at room temperature, the absorbance was measured at 695 nm against a blank. The blank consisted of all reagents and solvents without the sample and it was incubated under the same conditions. All experiments were carried out in triplicate. The antioxidant activity of the sample was expressed as the number of ascorbic acid equivalent (AAE). The phosphomolybdenum assay is based on the reduction of MoVI to MoV by antioxidant compounds and the formation of a green phosphate/MoV complex with a maximal absorption at 695 nm.13–18) Statistical Analysis All data were presented as mean±standard deviation (S.D.) using SPSS 13.0 program.19)

Conclusion

A variety of fused and non fused heterocyclic systems containing pyrimidine nucleus have been synthesized from the reaction of tetrahydropyrimidine with different reagents. All the synthesized pyrimidines are potent antioxidants. In particular the tetrahydropyrimidine 1, dihydropyrimidine 6 and S-alkylated product 9 showed the most antioxidant activity (AOA) expressed in 308.33±1.25, 436.85±2.25 and 225.87±1.50 mg AAE/g compound (AAE) using ascorbic acid as standard. Acknowledgment The authors gratefully thank Dr. Mosad Ahmed Ghareeb and his colleagues in Biochemistry and Medicinal Chemistry Department, Theodor Bilharz Research Institute (TBRI) for performing antioxidant screening of the synthesized compounds. Conflict of Interest interest.

References

The authors declare no conflict of

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