Synthesis and Antimicrobial, Anticancer and Anti-Oxidant Activities of Novel 2,3-Dihydropyrido[2,3-d]pyrimidine-4-one and Pyrrolo[2,1-b][1,3]benzothiazole Derivatives via Microwave-Assisted Synthesis

In our attempt towards the synthesis and development of effective antimicrobial, anticancer and antioxidant agents, a novel series of 2,3-dihydropyrido[2,3-d]pyrimidin-4-one 7a–e and pyrrolo[2,1-b][1,3]benzothiazoles 9a–e were synthesized. The synthesis of 2-(1,3-benzo thiazol-2-yl)-3-(aryl)prop-2-enenitrile (5a–e) as the key intermediate was accomplished by a microwave efficient method. Via a new variety oriented synthetic microwave pathway, these highly functionalized building blocks allowed access to numerous fused heteroaromatic such as 7-amino-6-(1,3-benzo thiazol-2-yl)-5-(aryl)-2-thioxo-2,3dihydropyrido [2,3-d]pyrimidin-4(1H)-one 7a–e and 1-amino-2-(aryl)pyrrolo[2,1-b][1,3]benzothiazole-3-carbonitrile derivatives 9a–e in order to study their antimicrobial and anticancer activity. The present investigation offers effective and rapid new procedures for the synthesis of the newly polycondensed heterocyclic ring systems. All the newly synthesized compounds were evaluated for antimicrobial, anticancer and antioxidant activity. Compounds 7a,d, and 9a,d showed higher antimicrobial activity than cefotaxime and fluconazole while the remaining compounds exhibited good to moderate activity against bacteria and fungi. An anticancer evaluation of the newly synthesized compounds against the three tumor cell lines (lung cell NCI-H460, liver cancer HepG2 and colon cancer HCT-116) exhibited that compounds 7a, d, and 9a,d have higher cytotoxicity against the three human cell lines compared to doxorubicin as a reference drug. These compounds also exhibited higher antioxidant activity and a great ability to protect DNA from damage induced by bleomycin.

We have also reported here the synthesis of pyrrolo[2,1-b] [1,3]benzothiazole bearing aryl and heteroaryl derivatives under microwave irradiation in the hope of obtaining novel antimicrobial and anticancer agents with excellent yield. Nowadays, the microwave irradiation method is a steady and attractive method for the synthesis of polycondensed heterocyclic compounds due to rapid, simple, and high yields [38,39].

Chemistry
The synthetic strategy of 2-(1,3-benzothiazol-2-yl)-3-(aryl)prop-2-enenitrile derivatives 5a-e includes two steps outlined in Scheme 1. Synthesis of 2-cyano -methyl-1,3-benzothiazol 3 by a newly highly efficient method with high yield (92%) under microwave irradiation at 40 °C for 10 min by the reaction of 2-amino thiophenol 1 with malononitrile 2 in ethanol (10 mL) and acetic acid as a catalyst is compared to the old method with low yield and long reaction time [40]. Knoevenagel condensation of active methylene 3 with appropriate aromatic aldehyde using absolute ethanol in the presence of a catalytic amount of triethylamine under MWI at 60 °C afforded compounds 5a-e as shown in Table 1. The progress of the reaction was followed by TLC (petroleum ether (80-90): ethylacetate, ratio: 3:1). The structure of the newly synthesized compounds was established by spectral data and elemental analysis. Accordingly, with all of the previous observations of the biological importance and in continuation of our program in the synthesis of pyrido [2,3-d]pyrimidine [37], this study aims to design and develop highly selective and efficacious antimicrobial and anticancer agents of a novel series of pyrido [2,3-d]pyrimidine derivatives bearing different heterocyclic and aryl moieties such as benzothiazole, thiophene, furan, piperonal, naphthalene, and fluorophenyl as a side chain and various aryl derivatives by the microwave irradiation technique.
We have also reported here the synthesis of pyrrolo[2,1-b] [1,3]benzothiazole bearing aryl and heteroaryl derivatives under microwave irradiation in the hope of obtaining novel antimicrobial and anticancer agents with excellent yield. Nowadays, the microwave irradiation method is a steady and attractive method for the synthesis of polycondensed heterocyclic compounds due to rapid, simple, and high yields [38,39].

Chemistry
The synthetic strategy of 2-(1,3-benzothiazol-2-yl)-3-(aryl)prop-2-enenitrile derivatives 5a-e includes two steps outlined in Scheme 1. Synthesis of 2-cyano -methyl-1,3benzothiazol 3 by a newly highly efficient method with high yield (92%) under microwave irradiation at 40 • C for 10 min by the reaction of 2-amino thiophenol 1 with malononitrile 2 in ethanol (10 mL) and acetic acid as a catalyst is compared to the old method with low yield and long reaction time [40]. Knoevenagel condensation of active methylene 3 with appropriate aromatic aldehyde using absolute ethanol in the presence of a catalytic amount of triethylamine under MWI at 60 • C afforded compounds 5a-e as shown in Table 1. The progress of the reaction was followed by TLC (petroleum ether (80-90): ethylacetate, ratio: 3:1). The structure of the newly synthesized compounds was established by spectral data and elemental analysis.  Under microwave irradiation conditions, the treatment of the reactive intermed 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol a °C generated the desired product 7a-e with (49-53%) yield within 45 min. Interesti the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 1 afforded the most satisfactory results of the correspon 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4-one rivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DM ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2.  Under microwave irradiation conditions, the treatment of the reactive intermediates 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol at 100 °C generated the desired product 7a-e with (49-53%) yield within 45 min. Interestingly, the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 150 °C afforded the most satisfactory results of the corresponding 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4-one derivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DMF or ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2.  Under microwave irradiation conditions, the treatment of the reactive intermediates 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol at 100 °C generated the desired product 7a-e with (49-53%) yield within 45 min. Interestingly, the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 150 °C afforded the most satisfactory results of the corresponding 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4-one derivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DMF or ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2.  Under microwave irradiation conditions, the treatment of the reactive intermediates 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol at 100 °C generated the desired product 7a-e with (49-53%) yield within 45 min. Interestingly, the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 150 °C afforded the most satisfactory results of the corresponding 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4-one derivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DMF or ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2.  Under microwave irradiation conditions, the treatment of the reactive intermediates 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol at 100 °C generated the desired product 7a-e with (49-53%) yield within 45 min. Interestingly, the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 150 °C afforded the most satisfactory results of the corresponding 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4-one derivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DMF or ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2.  Under microwave irradiation conditions, the treatment of the reactive intermediates 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol at 100 °C generated the desired product 7a-e with (49-53%) yield within 45 min. Interestingly, the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 150 °C afforded the most satisfactory results of the corresponding 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4-one derivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DMF or ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2. Under microwave irradiation conditions, the treatment of the reactive intermediates 5a-e with 6-aminothiouracil 6 using triethylamine as a catalyst in absolute ethanol at 100 • C generated the desired product 7a-e with (49-53%) yield within 45 min. Interestingly, the use of dimethylformamide instead of ethanol as a solvent without a catalyst at 150 • C afforded the most satisfactory results of the corresponding 7-amino-(1,3-benzothiazol-2-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4-one derivatives 7a-e in high yield within 25 min as shown in (Table 2).
Nevertheless, heating under reflux of compounds 5a-e with 6 whether in DMF or ethanol\TEA for a long time afforded (7a-e) as shown in Scheme 2.  In this research, we have also studied the possibility of the multicomponent reaction of 2-cyanomethyl-1,3-benzothiazol 3, 6-aminothiouracil 6, and appropriate aldehydes 4a-e under microwave irradiation conditions at 150 °C and TLC control. Pyrido[2,3-d]pyrimidine derivatives 7a-e were accumulated, the reaction condition has been established in Table 3. On the other hand, the same reaction was carried out by conventional heating under reflux in DMF as shown in Scheme 2. Furthermore, in our progression in the development of the highly efficient method in the synthesis of newly fused heterocyclic compounds with expected biological activity. we report the synthesis of novel pyrrolo [  In this research, we have also studied the possibility of the multicomponent reaction of 2-cyanomethyl-1,3-benzothiazol 3, 6-aminothiouracil 6, and appropriate aldehydes 4a-e under microwave irradiation conditions at 150 °C and TLC control. Pyrido[2,3-d]pyrimidine derivatives 7a-e were accumulated, the reaction condition has been established in Table 3. On the other hand, the same reaction was carried out by conventional heating under reflux in DMF as shown in Scheme 2. Furthermore, in our progression in the development of the highly efficient method in the synthesis of newly fused heterocyclic compounds with expected biological activity. we report the synthesis of novel pyrrolo [  In this research, we have also studied the possibility of the multicomponent reaction of 2-cyanomethyl-1,3-benzothiazol 3, 6-aminothiouracil 6, and appropriate aldehydes 4a-e under microwave irradiation conditions at 150 °C and TLC control. Pyrido[2,3-d]pyrimidine derivatives 7a-e were accumulated, the reaction condition has been established in Table 3. On the other hand, the same reaction was carried out by conventional heating under reflux in DMF as shown in Scheme 2. Furthermore, in our progression in the development of the highly efficient method in the synthesis of newly fused heterocyclic compounds with expected biological activity. we report the synthesis of novel pyrrolo [  In this research, we have also studied the possibility of the multicomponent reaction of 2-cyanomethyl-1,3-benzothiazol 3, 6-aminothiouracil 6, and appropriate aldehydes 4a-e under microwave irradiation conditions at 150 °C and TLC control. Pyrido[2,3-d]pyrimidine derivatives 7a-e were accumulated, the reaction condition has been established in Table 3. On the other hand, the same reaction was carried out by conventional heating under reflux in DMF as shown in Scheme 2. Furthermore, in our progression in the development of the highly efficient method in the synthesis of newly fused heterocyclic compounds with expected biological activity. we report the synthesis of novel pyrrolo [  In this research, we have also studied the possibility of the multicomponent reaction of 2-cyanomethyl-1,3-benzothiazol 3, 6-aminothiouracil 6, and appropriate aldehydes 4a-e under microwave irradiation conditions at 150 °C and TLC control. Pyrido[2,3-d]pyrimidine derivatives 7a-e were accumulated, the reaction condition has been established in Table 3. On the other hand, the same reaction was carried out by conventional heating under reflux in DMF as shown in Scheme 2. Furthermore, in our progression in the development of the highly efficient method in the synthesis of newly fused heterocyclic compounds with expected biological activity. we report the synthesis of novel pyrrolo[2,1-b] [1,3]benzothiazole derivatives 9a-e,   In this research, we have also studied the possibility of the multicomponent reaction of 2-cyanomethyl-1,3-benzothiazol 3, 6-aminothiouracil 6, and appropriate aldehydes 4a-e under microwave irradiation conditions at 150 • C and TLC control. Pyrido[2,3-d]pyrimidine derivatives 7a-e were accumulated, the reaction condition has been established in Table 3. On the other hand, the same reaction was carried out by conventional heating under reflux in DMF as shown in Scheme 2. Furthermore, in our progression in the development of the highly efficient method in the synthesis of newly fused heterocyclic compounds with expected biological activity. we report the synthesis of novel pyrrolo[2,1-b] [1,3]benzothiazole derivatives 9a-e, Scheme 4, via microwave-assisted three-component reactions and the evaluation of their cytotoxicity, leading to the discovery of some new heterocycles with potent cytotoxic activity higher than or similar to doxorubicin as standard drug.
Initially, under microwave irradiation of three-component of 2-cyanomethyl -1,3benzothiazol 3, benzoyl cyanide 8 and, appropriate aldehyde such as 4-fluorobenzaldhyde 4d was employed to optimize the reaction conditions as shown in (Table 4). Firstly, (MWI, 100 • C, 25 min) solvent screening showed that the usage of methanol or ethanol as polar solvents was beneficial while in dichloroethane no reaction occurred (entry 2). The presence of bases also increases the yield percentage as shown in Table 4. DBU was shown to be an effective base catalyst more than TEA. So, EtOH/DBU was preferred as the optimal solvent/catalyst system and 120 • C was selected as the most convenient reaction temperature (Table 4, entry 7) in view of the highest yield of 9d (87%). Under the optimized reaction conditions, various aldehydes 4a-e were treated with 2-cyanomethyl-1,3-benzothiazol 3 and benzoyl cyanide 8, as illustrated in Table 4, while treatment of the three components under conventional heating took a long time and afforded a moderate yield. Scheme 4, via microwave-assisted three-component reactions and the evaluation cytotoxicity, leading to the discovery of some new heterocycles with potent cytot tivity higher than or similar to doxorubicin as standard drug. Initially, under microwave irradiation of three-component of 2-cyano -1,3-benzothiazol 3, benzoyl cyanide 8 and, appropriate aldehyde s 4-fluorobenzaldhyde 4d was employed to optimize the reaction conditions as sh (Table 4). Firstly, (MWI, 100 °C , 25 min) solvent screening showed that the u methanol or ethanol as polar solvents was beneficial while in dichloroethane no occurred (entry 2). The presence of bases also increases the yield percentage as sh Table 4. DBU was shown to be an effective base catalyst more than TEA. So, EtO was preferred as the optimal solvent/catalyst system and 120 °C was selected as t convenient reaction temperature (Table 4,    A reasonable reaction mechanism for the synthesis of 1-amino pyrrolobenzothiazole-3carbonitrile derivatives 9a-e is shown in Scheme 5. The reaction involves a base-catalyzed two-step formation of 2-arylidene cyanomethyl 1,3-benzothiazoles via a Knoevenagel condensation between 2-cyanomethyl-1,3-benzothiazol and aldehydes, followed by [4+1] cycloaddition of benzoyl cyanide.
Finally, as illustrated in Table 5, the corresponding products 9a-e were successfully achieved in high yield compared with the three-component reaction through the treatment of the reactive intermediates 5a-e with benzoyl cyanide 8 under microwave irradiation at 120 • C for 20-25 min in EtOH/DBU, while conventional heating in ethanol/TEA took a long time. As is evident, the yield afforded from the treatment of 5a-e with benzoyl cyanide 8 was the best, compared to the yield from multicomponent reactions under microwave irradiation. So, it is now settled that microwaves can greatly speed up reactions and improve overall yield.
A reasonable reaction mechanism for the synthesis of 1-amino pyrrolobenzothiazole-3-carbonitrile derivatives 9a-e is shown in Scheme 5. The reaction involves a base-catalyzed two-step formation of 2-arylidene cyanomethyl 1,3-benzothiazoles via a Knoevenagel condensation between 2-cyanomethyl-1,3-benzothiazol and aldehydes, followed by [4+1] cycloaddition of benzoyl cyanide. Finally, as illustrated in Table 5, the corresponding products 9a-e were successfully achieved in high yield compared with the three-component reaction through the treatment of the reactive intermediates 5a-e with benzoyl cyanide 8 under microwave irradiation at 120 °C for 20-25 min in EtOH/DBU, while conventional heating in ethanol/TEA took a long time. As is evident, the yield afforded from the treatment of 5a-e with benzoyl cyanide 8 was the best, compared to the yield from multicomponent reactions under microwave irradiation. So, it is now settled that microwaves can greatly speed up reactions and improve overall yield.

Antimicrobial Activity and Structure Activity Relationship
The values of MIC of the synthesized compounds against the tested microorganisms are displayed in Tables 6 and 7. Benzothiazole arylidine derivatives 5a-e exhibited good to moderate antimicrobial activities. These derivatives increased the antimicrobial activity when treated with 6-aminothiouracil to afford 2,3-dihydropyrido [2,3-d] pyrimidine-4-one derivatives. The investigations showed significant inhibitory effects against bacteria with the majority of the compounds with MIC values of (4-20 μmol L -1 ) ( Table  6). It was found that compounds 7a,d were found to be more potent against bacteria with MIC value ranging from (4-12 μmol L -1 ) than cefotaxime with MIC value (6-12 μmol L -1 ), while compounds 7b, 7c and 7e exhibited good activity against all the bacterial strains. Scheme 5. Synthetic mechanism of pyrrolobenzothiazole-3-carbonitrile derivatives 9a-e. The values of MIC of the synthesized compounds against the tested microorganisms are displayed in Tables 6 and 7. Benzothiazole arylidine derivatives 5a-e exhibited good to moderate antimicrobial activities. These derivatives increased the antimicrobial activity when treated with 6-aminothiouracil to afford 2,3-dihydropyrido [2,3-d] pyrimidine-4-one derivatives. The investigations showed significant inhibitory effects against bacteria with the majority of the compounds with MIC values of (4-20 µmol L -1 ) ( Table 6). It was found that compounds 7a,d were found to be more potent against bacteria with MIC value ranging from (4-12 µmol L -1 ) than cefotaxime with MIC value (6-12 µmol L -1 ), while compounds 7b, 7c and 7e exhibited good activity against all the bacterial strains. This high efficacy may be attributed to the presence of a benzothiazol and thiophene moiety as compound 7a and the presence of an electron withdrawing group (fluoro) in the para-position of the phenyl ring attached to the pyridopyrimidine backbone 7d. Compound 7a had equipotent activity with MIC (6, 12 µmol L -1 ) as cefotaxime against Bacillus subtilis and Chlamydia pneumonia, respectively. Compound 7e was also equipotent with MIC (8 µmol L -1 ) as cefotaxime against Salmonella typhi. Pyrrolo[2,1-b] [1,3]benzothiazole derivatives 9a-e could effectively inhibit the growth of the tested bacteria strains. Compound 9a was more potent against Staphylococcus aureus while it had equipotent efficacy against Bacillus subtilis and Chlamydia pneumonia to cefotaxime; this was attributed to the presence of pyrrolo [2,1-b] [1,3]benzothiazole with thiophene moiety as a side chain. Compound 9d with MIC (4-10 µmol L -1 ) had exceptional activity toward all bacteria due to the presence of pyrrolobenzothiazole with p-fluorophenyl substituent which increased the antibacterial potency compared to the reference drug. On the other hand, compounds 9b, 9c and 9e exhibited good inhibition activity against all bacterial strains (Table 6).  The newly synthesized compounds were evaluated for their antifungal activity against three fungal strains displayed in (Table 7). Most of the synthesized compounds exhibited good to moderate inhibition activities against all the fungal strains. Compound 9d exhibited more potent activity than fluconazole against Candida albicans and Ganoderma lucidum while it had equipotent activity against Aspergillus flavus. Compound 9a exhibited stronger activity than fluconazole against Candida albicans and excellent activity against Aspergillus flavus and Ganoderma lucidum. Compound 9b had good inhibition activity against Aspergillus flavus and Candida albicans but moderate activity toward Ganoderma lucidum. Compound 9e also had promising activity against all fungal strains.
Compounds 7a,d had good inhibition activity against all fungus, while compounds 5a-e, 7b, 7c and 7e exhibited good to moderate activities. The introduction of benzothiazole, thiophene, and p-fluorophenyl moieties to the pyridopyrimidine derivatives and the presence of pyrrolobenzothiazole derivatives with thiophene and p-fluorophenyl side chains might be responsible for the antifungal activity enhancement of these compounds.

Cytotoxicity Screening and Structure Activity Relationship (SAR)
The cytotoxic activity of all the newly synthesized compounds benzothiazole arylidine 5a-e, pyrido[2,3-d]pyrimidine 7a-e and pyrrolo [2,1-b] [1,3] benzothiazole derivatives 9a-e were evaluated against three tumor cell lines (human lung cell NCI-H460, liver cancer HepG2 and colon cancer HCT-116) by MTT assay. The three human cancer cell lines were provided by the National Cancer Institute (NCI, Cairo, Egypt). Doxorubicin was used as the positive control. The cytotoxic activities are expressed as the median growth inhibitory concentration (IC 50 ) and are provided in (Table 8). From the results, it is evident that most of the newly synthesized compounds showed potent to moderate cytotoxic activity against the three tumor cell lines. The results of the series of compound 5 indicated that the type of the side chain on the benzothiazole arylidine derivatives occupied significant roles in the cytotoxic activity. Compounds 5a with IC 50 values of (3.61, 3.14 and 4.20 µmol L −1 ) and 5d with IC 50 values of (3.04, 3.20 and 3.38 µmol L −1 ) showed good antitumor activity toward all tumor cell lines, while the other compounds 5b, 5c and 5e exhibited moderate activity in the range of (IC 50 = 5.82-10.20 µmol L −1 ) compared to doxorubicin. The series of 2,3-dihydropyrido[2,3-d]pyrimidine-4-one derivatives 7a-e exhibited higher cytotoxic activity than the series of pyrrolo [2,1-b] [1,3] benzothiazole 9a-e. Table 8 shows 2,3-dihydropyrido[2,3-d]pyrimidine-4-one 7a containing benzothiazol and thiophene as a biologically active side chain with IC 50 values of (0.66, 0.45 and 0.70 µmol L −1 ), benzothiazol and p-fluorophenyl moieties 7d with IC 50 values of (0.28, 0.39 and 0.14 µmol L −1 ), pyrrolo [2,1-b] [1,3]benzothiazole 9a with benzothiazol and thiophene with IC 50 values of (1.10, 0.49 and 0.89 µmol L −1 ), benzothiazol and p-fluorophenyl 9d side chain moieties with IC 50 values of (0.45, 0.47 and 0.59 µmol L −1 ) were found to be more potent and efficacious than the reference drug doxorubicin with IC 50 values of (1.98, 0.52 and 1.12 µmol L −1 ). It was noticeable that the presence of side chain p-fluorophenyl improved the antitumor activity more than thiophene side chain in addition to the basic skeleton.
Generally, the type of substituent plays an important role in antitumor activity and the presence of the basic skeleton of fused heterocyclic compounds improves the cytotoxic activity in cancer cells.

Bleomycin-Dependent DNA Damage
The pro-oxidant activities of compounds 7a-e and 9a-e were assessed by their effects on bleomycin-induced DNA damage. Analysis of the data in Table 9 showed that, compounds 7a (0.053), 7d (0.045), and 9d (0.064) were found to be more potent than the reference drug Trolox (0.067). Moreover, compound 9a (0.069) had almost equipotent activity, that means these compounds have great ability to protect DNA from the damage induced by bleomycin. The rest of compounds 7b, 7c, 7e, 9b, 9c and 9e exhibited excellent protection against DNA damage.

Reagents and Materials
Melting points were recorded on an advanced melting point apparatus, SMP30, Stuart (Bibby Scientific, London, UK). Microanalytical data were gathered with a Vario Elementar apparatus (Shimadzu-Japan). Elemental analyses of all compounds were within ± 0.4% of the theoretical values. The IR spectra (KBr) were recorded on a Perkin Elmer 1650 spectrometer (USA). 1 H and 13 C NMR spectra (500 MHz for 1 H and 125 MHz for 13 C NMR) were recorded on JEOL ECA-500 (Shimadzu) instruments.
Chemical shifts were expressed in ppm relative to SiMe 4 as an internal standard in DMSO-d 6 or (CDCl 3 ) as a solvent. Mass spectra were recorded on a 70 eV Finnigan SSQ 7000 spectrometer (Thermo-Instrument System Incorporation, USA). Follow up of the reaction and the purity of the compounds was checked on aluminum plates coated with silica gel (Merck, Germany) on Microwave Advanced Flexible Synthesis Platform 1900 W (flexiWAVE-Milestone, Italy), producing continuous irradiation and equipped with a simultaneous external air-cooling system. Chemicals and solvents (Analar ≥ 99%) were purchased from Sigma-Aldrich (USA).

Syntheses
General procedure. A mixture of compound 3 (0.87 g, 5 mmol), (5 mmol) of the appropriate aromatic aldehyde (thiophene-2-carboxaldehyde, 5-methylfuran-carbox-aldhyde, 1-naphthaldhyde, 4-fluorobenzaldhyde, and piperonal) 4a-e and TEA in ethanol was subjected to MWI at 60 • C for 4-8 min. The progress of the reaction was followed by TLC (petroleum ether (80-90): ethyl acetate, ratio:3:1). After cooling, solids were obtained, filtered, and recrystallized from a proper solvent to give 5a-e, respectively. General procedure: (Method 1) under microwave irradiation. (Method 1a): A mixture of compounds 5a-e (5 mmol) and 6-aminothiouracil 6 (0.7 g, 5 mmol) in DMF was placed in a microwave process vial. The vial was closed and subjected to microwave irradiation for 25 min at 150 • C/or in EtOH/TEA for 45 min at 100 • C. The progress of the reaction was followed by TLC. After cooling to room temperature, the solid formed was filtered off, washed with ethanol, dried, and crystallized from DMF.
(Method 1b): A mixture of 2-cyanomethyl-1,3-benzothiazol 3 (0.87 g, 5 mmol), 6-amino thiouracil 6 (0.7 g, 5 mmol), and appropriate aldehydes 4a-e (5 mmol) in DMF was placed in a microwave process vial. The vial was closed and subjected to microwave irradiation for 50 min at 150 • C. The progress of the reaction was followed by TLC. After cooling to room temperature, the solid formed was filtered off, washed with ethanol, dried, and crystallized from DMF.
(Method 2a): A mixture of compounds 5a-e (10 mmol) and 6-aminothiouracil 6 (1.43 g, 10 mmol) was heated under reflux in DMF/or EtOH/TEA for several hours. The reaction mixture was allowed to cool to room temperature and the solid formed was filtered off, dried, and crystallized from DMF.
(Method 1b): A mixture of compounds 5a-e and benzoyl cyanide 8 in EtOH/DBU was placed in a microwave process vial. The vial was closed and subjected to microwave irradiation for (20-25) min at 120 • C. Cooling the mixture to room temperature, the solid precipitate was collected by filtration, dried and crystallized from dioxane.
(Method 2a): A mixture of 2-cyanomethyl-1,3-benzothiazol 3 (5 mmol), benzoyl cyanide 8 (5 mmol) and appropriate aldehyde 4a-e was heated under reflux in ethanol and trimethylamine for an appropriate time. The reaction mixture was allowed to cool to room temperature and the solid formed was filtered off, dried and crystallized from dioxane.
(Method 2b): A mixture of compounds 5a-e (5 mmol) and benzoyl cyanide 8 (5 mmol) was heated under reflux in ethanol and trimethylamine for an appropriate time. The reaction mixture was allowed to cool to room temperature and the solid formed was filtered off, dried and crystallized from dioxane.  [42]. Serial dilutions were prepared from the stock solution by dissolving 5 mg of each test compound in 1 mL of DMSO. The plates were incubated at 37 • C for 24 h. DMSO was used as a solvent control which had no effect on bacterial growth. Results of antimicrobial activities are summarized in Tables 6 and 7. 3.3.2. Antitumor Activity Human carcinoma cell lines (lung cell NCI-H460, liver cancer HepG2 and colon cancer HCT-116) were provided by the National Cancer Institute (NCI, Cairo, Egypt). (FBS) fetal bovine serum from Gibco Invitrogen Co. (UK). Dimethyl sulfoxide (DMSO) and doxorubicin were from Sigma Chemical Co. (USA).

In Vitro Anticancer Activity by MTT Assay
Cell culture: All the synthesized compounds were tested in vitro for anticancer activity against three tumor cell lines (lung cell NCI-H460, liver cancer HepG2 and colon cancer HCT-116). Cells were cultured in humidified atmosphere at 37 • C with 5% CO 2 . The cell density was 2 × 10 3 seeded in a 96-multiwell plate in 0.1mL DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% (FBS) fetal bovine serum for 24 h. The tested compounds were dissolved in DMSO as a stock solution (0.1 mol L -1 ). After 48 h of incubation, cells were treated with different concentrations of the tested compounds (5,12,25, and 50 µmol L −1 ). The medium cells of the control group contained 0.2% DMSO. For each individual dose triplicate wells were performed. To each well, MTT in phosphate buffered saline (PBS, 5 mg/mL) was added and incubated for 4 h at 37 • C. After that, MTT reagent was removed and added (DMSO, 100 µL) to each well to dissolve formazan crystals. A measure of 100µM of Doxorubicin was used as a standard drug and the optical density was determined at 570 nm with a DTX 880 multimode detector. The calculation of the IC50 was done by performing 4 concentrations of the compounds on the cells in triplicate and the results were analyzed using SPSS software (Version 20.0).

Bleomycin-Dependent DNA Damage
Assay mixtures [44] contained DNA (0.5 mg/mL), bleomycin sulfate (0.05 mg/mL), FeCl 3 (50 mM), MgCl 2 (5 mM), and selected synthesized compounds to be examined at different concentrations. To start the reaction, Trolox was added which was used as positive control and was incubated at 37 • C for 1 h. After the incubation period, 0.05 mL of EDTA (0.1 M) was added to terminate the reaction. the addition of 0.5 mL of (TBA) thiobarbituric acid (1%, w/v) and 0.5 mL HCI (25%, v/v) then heated in the water bath for 10 min at 80 • C to assay DNA damage. the extent of DNA damage was measured after centrifugation, by the increase in absorbance at 532 nm.

Conclusions
The present investigation offers effective and rapid new procedures for the synthesis of the newly polycondensed heterocyclic ring systems. All the newly synthesized com-pounds showed antimicrobial activity against bacteria and fungi. Among the synthesized compounds, compounds 7a,d, and 9a,d showed more potent inhibitory activities than cefotaxime and fluconazole while the remaining compounds exhibited good to moderate activity against bacteria and fungi. Compounds 7a,d, and 9a,d also exhibited higher cytotoxicity for all tested cell lines compared to doxorubicin. These derivatives exhibited higher antioxidant activity compared to Trolox, also manifested the best protective effect against DNA damage induced by Bleomycin. Based on the anticancer and antioxidant activity, we can consider 2,3-dihydropyrido[2,3-d]pyrimidine-4-one and pyrrolo [2,1-b] [1,3]benzothiazole as highly potential molecules for the development of novel anticancer drugs.
Supplementary Materials: The following are available online. Figure S1. 1 H-NMR and 13 C-NMR spectra for all compounds.