Microwave-assisted synthesis and antimicrobial evaluation of novel spiroisoquinoline and spiropyrido[4,3-d]pyrimidine derivatives.

Bromination of N-substituted homophthalimides and tetrahydropyrido[4,3-d]- pyrimidine-5,7-diones produces 4,4-dibromohomophthalimide and 8,8-dibromo-tetrahydropyrido[4,3-d]pyrimidine-5,7-dione derivatives, respectively, that can be used as precursors for spiro derivatives. The dibromo derivatives react with different binucleophilic reagents to produce several spiroisoquinoline and spirotetrahydropyrido[4,3-d]- pyrimidine-5,7-dione derivatives, respectively. Reaction of the dibromo derivatives with malononitrile produces dicyanomethylene derivatives which react with different binucleophiles to produce new spiro derivatives. All new compounds are prepared by using the usual chemical conditions and microwave assisted conditions. The latter conditions improved the reaction yields, reduced reaction times and ameliorated the effects on the surrounding environment as the reactions are carried out in closed systems. Structures of the newly synthesized compounds are proved using spectroscopic methods such as IR, MS, 1H-NMR and 13C-NMR and elemental analyses. Some of the newly synthesized compounds were tested for their antimicrobial activities, whereby four of them showed moderate activities and the rest showed low or no activities towards the investigated species.

1. Introduction

Spiro compounds constitute a group of generally less investigated compounds, however, recently growing efforts have been made to synthesize and characterize these compounds. Many spiro compounds possess very promising biological activities as anticancer [1,2], antibacterial [3,4], anticonvulsant [5,6,7], antituberculosis [8], anti-Alzheimer’s [9], pain-relief [10,11] and antidermatitis agents [12]. In addition to their medical uses, some spiro-compounds have found other uses in the agricultural and industrial fields. For example, they are used as antifungal agents [13], pesticides [14], laser dyes [15] and electroluminescent devices [16]. Spiro compounds have also been used as antioxidants [17,18]. Our research group is interested in using the microwave technique [3,19,20,21,22,23,24,25], as it has several advantages over conventional methods of synthesis, such as reduced reaction times, fewer effects on the environment and better reactions. In the present research, we used both the microwave technique as well as conventional methods to prepare some new spiro compounds that were then tested for their antimicrobial activities.

2. Results and Discussion

2.1. Chemistry

Homophthalic anhydride (1) was reacted with aromatic amines, namely p-toluidine and p-chloroaniline, to afford N-arylhomophthalimide derivatives 2a,b respectively, which were used as precursors for preparing new spiroisoquinolines (Scheme 1). Compounds 2a,b, having an active methylene group, reacted with two equivalents of bromine in acetic acid to produce 2-aryl-4,4-dibromoisoquinoline-1,3-(2H,4H)dione derivatives 3a,b. The mass spectrum of compound 3a displayed the expected molecular ion isomeric peaks at m/z 407 (4.8%), 409 (10.1%), 411 (5.2%). Compound 3b gave the molecular ion peaks at 427 (4.4%), 429 (9.9%), 431 (6.9%).

Scheme 1

Reactions of dibromohomophthalimides with binucleophilic reagents: synthesis of 5 and 6.

Compounds 3a,b underwent direct cyclocondensation when treated with each of o-phenylenediamine (4a) or o-aminophenol (4b) to produce 2'-aryl-1,3-dihydro-1'H-spiro-[benzo[d]imidazole-2,4'-isoquinoline]-1',3'(2'H)-diones 5a,b and 2'-aryl-1'H,3H-spiro[benzo[d]oxazole-2,4'-isoquinoline]-1',3'(2'H)-diones 5c,d, respectively (Scheme 1). The synthesis of compounds 5a–d was carried out under conventional heating conditions. Thus, when the reaction was carried out in a refluxing ethanolic piperidine solution for 5 h under TLC monitoring, the product 5a–d were obtained in 42%–51% yields.

Similarly, compounds 3a,b reacted with thiosemicarbazide under the same reaction conditions and produced 2-aryl-5'-thioxo-1H-spiro[isoquinoline-4,3'-[1,2,4]triazolidine]-1,3(2H)-diones 6a,b (Scheme 1). The analytical and spectral data of 5a–d and 6a,b were in agreement with the proposed structures (Experimental Section).

In a similar manner, when 4-(4-aryl)-6-phenyl-2-thioxo-2,3,4,4a-tetrahydropyrido-[4,3-d]-pyrimidine-5,7(6H,8H)-diones 7a,b [26] were treated with two equivalents of bromine in acetic acid, the 8,8-dibromo derivatives 8a,b were obtained. Elemental analyses as well as the spectroscopic data of 8a,b agreed with the proposed structures (Experimental Section). Heating under reflux compounds 8a,b in absolute ethanol in presence of piperidine with either of ethylenediamine or thiosemicarbazide afforded the corresponding targeted spiro compounds 4'-(4-aryl)-6'-phenyl-2'-thioxo-3',4'-dihydro-1'H-spiro- [imidazolidine-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-diones 9a,b and 4-(4-aryl)-6-phenyl-2,5'- dithioxo-3,4-dihydro-1H-spiro[pyrido[4,3-d]pyrimidine-8,3'-[1,2,4]triazolidine]-5,7(2H,6H)-diones 10a,b, respectively.

Similarly, compounds 8a,b were refluxed with o-phenylenediamine (4a) and o-aminothiophenol (4c) in absolute ethanol, in the presence of piperidine to afford the corresponding cyclized products 4'-(4-aryl)-6'-phenyl-2'-thioxo-1,3,3',4'-tetrahydro-1'H-spiro[benzo[d]-imidazole-2,8'-pyrido[4,3-d]pyrimidine]-5',7'-(2'H,6'H)-diones 11a,b and 4'-(4-aryl)-6'-phenyl-2'-thioxo-3',4'-dihydro-1'H,3H-spiro-[benzo[d]thiazole-2,8'-pyrido-[4,3-d]pyrimidine]-5',7'(2'H, 6'H)-diones 11c,d, respectively (Scheme 2). The produced compounds 9a,b, 10a,b and 11a–d gave fully consistent elemental and spectroscopic analyses data (Experimental Section).

Scheme 2

Reaction of dibromopyridopyrimidines 8a,b with binucleophilic reagents; formation of 9, 10 and 11.

On the other hand, Refluxing compounds 8a,b in ethanol/piperidine with either with malononitrile (12a) or ethyl cyanoacetate (12b) afforded the corresponding 2-(4-(4-aryl)-5,7-dioxo-6-phenyl-2-thioxo-1,2,3,4,6,7-hexahydropyrido[4,3-d]pyrimidin-8(5H)-ylidene)malononitriles (compounds 13a,b) and ethyl 2-(4-(4-aryl)-5,7-dioxo-6-phenyl-2-thioxo-1,2,3,4,6,7-hexahydropyrido-[4,3-d]pyrimidin- 8(5H)-ylidene)-2-cyanoacetates 13c,d, respectively.

Compounds 13a–d were refluxed with hydrazine hydrate in ethanol to obtain the 5-substituted-4'-(4-aryl)-5',7'-dioxo-6'-phenyl-2'-thioxo-2,2',3',4,4',5',6',7'-octahydro-1'H-spiro[pyrazole-3,8'-pyrido-[4,3-d]pyrimidine]-4-carbonitrile spiro products 14a–d, respectively. Similar treatment of compounds 13a–d with thiourea in ethanol/piperidine resulted in the formation of the 6'-substituted-4-(4-aryl)-5,7-dioxo-6-phenyl-2,2'-dithioxo-2,3,3',4,5,5',6,7-octahydro-1H,2'H-spiro-[pyrido[4,3-d]pyrimidine-8,4'-pyrimidine]-5'-carbonitriles 15a–d, respectively (Scheme 3).

Scheme 3

Reaction of 8a,b with activated methylene compounds followed by binucleophiles; formation of 14a–d and 15a–d.

Trying to take advantage of the benefits of the microwave assisted reaction conditions, compounds 5, 6, 9, 10, 11, 14 and 15 were prepared by using microwave irradiation instead of the conventional heating conditions. The results showed that much less time was needed to prepare these compounds, as well as a considerable increase in the reaction yields upon using the environmentally friendly microwave irradiation conditions. Table 1 shows a comparison in reaction times and yields between the conventional and microwave assisted methods of preparation.

Table 1

Comparison between conventional methods and microwave assisted methods of synthesis of compounds 5a–d, 6a,b, 9a,b, 10a,b, 11a–d, 14a–d and 15a–d.

Compound No.	Reaction Times		Reaction Yields (%)
	Conventional Methods *	Microwave ‡	Conventional Methods *	Microwave ‡
5a	5 h	15 min	42	91
5b	5 h	15 min	48	89
5c	5 h	15 min	47	82
5d	5 h	15 min	53	92
6a	4 h	10 min	60	94
6b	4 h	10 min	55	90
9a	2 h	5 min	65	93
9b	2 h	5 min	67	91
10a	2 h	5 min	61	91
10b	2 h	5 min	58	89
11a	2 h	5 min	47	84
11b	2 h	5 min	50	91
11c	2 h	5 min	52	88
11d	2 h	5 min	54	80
14a	3 h	7 min	37	81
14b	3 h	7 min	42	84
14c	3 h	7 min	45	89
14d	3 h	7 min	44	82
15a	3 h	7 min	47	88
15b	3 h	7 min	43	87
15c	3 h	7 min	51	90
15d	3 h	7 min	47	91

* Conventional reaction conditions: the reactants were heated under reflux in the proper solvent for 2–5 h in open systems (Experimental Section). Microwave-assisted reaction conditions: the reactants were heated in tightly closed tubes in scientific microwave oven for 5–15 min (Experimental Section).

2.2. Antimicrobial Evaluation

The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against the following microorganisms: Escherichia coli, Pseudomonas putida, Bacillus subtilis, Streptococcus lactis, Aspergillus niger, Penicillium sp. and Candida albicans. The filter paper disc diffusion method [27] was used to perform preliminary screening of the investigated compounds. The most active compounds were 5a, 6b, 9a and 9b, which showed moderate inhibition to the microorganisms. Also, compounds 5c, 6a, 11a, 11c and 15b showed slight inhibitory action. The rest of compounds showed no sensitivity at all to the tested organisms, and the results are summarized in Table 2.

Table 2

Antimicrobial activities of the newly synthesized compounds.

Comp. No.	Inhibition Zone (mm)
	Gram-Negative		Gram-Positive		Fungi		Yeast
	E. coli	P. putida	B. subtilis	S. lactis	A. niger	P. sp.	C. albicans
5a	11	13	8	10	9	8	0
5c	4	4	3	2	4	0	0
6a	8	6	7	8	6	6	0
6b	14	15	13	13	11	9	0
9a	10	10	11	10	10	8	0
9b	12	10	10	9	6	5	0
10a	0	0	0	0	0	0	0
10b	0	0	0	0	0	0	0
11a	5	5	3	3	5	4	0
11c	7	8	7	6	4	0	0
14a	0	0	0	0	0	0	0
14d	0	0	0	0	0	0	0
15b	7	5	5	4	6	3	0
15c	0	0	0	0	0	0	0
Chloram-phenicol	22	21	18	19	20	12	0
Ampicillin	24	20	19	22	24	14	14

E. coli = Escherichia coli; P. putida = Pseudomonas putida; B. subtilis = Bacillus subtilis; S. lactis = Streptococcus lactis; A. niger = Aspergillus niger; P. sp. = Penicillium sp.; C. albicans = Candida albicans. The sensitivity of microorganisms to the tested compounds is identified in the following manner: Highly sensitive = Inhibition zone 15–20 mm; Moderately sensitive = Inhibition zone: 10–15 mm; Slightly sensitive = Inhibition zone: 5–10 mm; Not sensitive = Inhibition zone: 0 mm; Each result represents the average of triplicate readings.

3. Experimental Section

3.1. General

Melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on Shimadzu FTIR-8201PC spectrophotometer (Giza, Egypt). 1H-NMR and 13C-NMR spectra were recorded on a Varian Mercury 300 MHz or Varian Gemini 200 MHz spectrometers (Giza, Egypt) using TMS as an internal standard and DMSO-d6 as solvent. Microwave reactions were performed with a Millstone Organic Synthesis Unit with touch control terminal (MicroSYNTH, Giza, Egypt) with a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The temperature of the reaction mixture was monitored using a calibrated infrared temperature control under the reaction vessel, and control of the pressure was performed with a pressure sensor connected to the septum of the vessel. Elemental analysis was carried out at the Microanalytical Center of Cairo University, Giza, Egypt.

3.1.1. 2-Aryl-4,4-dibromoisoquinoline-1,3-(2H,4H)dione Derivatives 3a,b

A solution of either of 2a (2.37 g, 0.01 mol), 2b (2.51 g, 0.01 mol) or 2c (2.72 g, 0.01 mol) in glacial acetic acid (20 mL) was heated under reflux with bromine (1.1 mL, 3.0 g, 0.02 mole) for 2 h. After cooling, the reaction mixture was poured onto ice-water and the solid that precipitated was filtered off, dried and crystallized from the proper solvent.

4,4-Dibromo-2-p-tolylisoquinoline-1,3-(2H,4H)dione (3a): white crystals after crystallization from acetic acid then washing with ethanol; 66% yield; m.p. 236–238 °C; 1H-NMR: 2.60 (s, 3H, CH3), 7.10–8.30 (m, 8H, Ar-H); 13C-NMR: 25.3 (CH3), 80.2 (sp3 C-4), 118.2, 122.7, 125.3, 126.1, 128.7, 130.0, 131.5, 134.6, 135.2, 136.4 (aromatic C), 158.4, 167.6 (2 CO); IR ν: 3066 cm−1 (aromatic CH), 2970 (aliphatic CH), 1645 (broad, 2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 407 (3.2%), 409 (6.7%), 411 (3.0%); Anal. Calcd. for C16H11Br2NO2 (407.07): C (46.98%), H (2.71%), Br (39.07%), N (3.42%); Found: C (46.7%), H (2.9%), Br (38.93%), N (3.1%).

4,4-Dibromo-2-(4-chlorophenyl)isoquinoline-1,3(2H,4H)-dione (3b): white crystals after crystallization from acetic acid then washing with ethanol; 52% yield; m.p. 216–218 °C; 1H-NMR: 7.40–8.50 (m, 8H, Ar-H); 13C-NMR: 80.2 (sp3 C-4), 121.5, 123.9, 125.8, 127.1, 128.7, 130.0, 131.5, 134.6, 135.2, 136.4 (aromatic C), 158.2, 167.5 (2 CO); IR ν: 3060 cm−1 (aromatic CH), 1645 (broad, 2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 427 (4.7%), 429 (10.3%), 431 (7.2%); Anal. Calcd.forC15H8Br2ClNO2 (429.49): C (41.95%), H (1.88%), Br (37.21%), Cl (8.25%), N (3.26%); Found: C (41.7%), H (1.7%), Br (37.1%), Cl (8.4%), N (3.1%).

3.1.2. Cyclocondensation of 3a with o-Phenylenediamine and o-Aminophenol; Formation of 5a–d

Method A: Compounds 3a,b (0.01 mol) were heated under reflux with either of o-phenylenediamine (1.08 g, 0.01 mol) or o-aminophenol (1.09 g, 0.01 mol) in absolute ethanol (25 mL) and few drops of piperidine for 5 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.

Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 15 min. The reaction mixture was treated similar to method A to obtain compounds 5a–d.

2'-(4-Tolyl)-1,3-dihydro-1'H-spiro[benzimidazole-2,4'-isoquinoline]-1',3'(2'H)-dione (5a): grey crystals after crystallization from acetic acid; 42% yield (Method A) and 91% (Method B); 229–231 °C; 1H-NMR: 2.40 (s, 3H, CH3), 3.80 (s, 2H, 2NH, D2O exchangeable), 6.40–7.70 (m, 12H, Ar-H); 13C-NMR: 25.5 (CH3), 83.4 (sp3-spiro C), 113.0, 115.9, 120.2, 122.9, 127.3, 127.8, 128.3, 128.9, 131.2, 133.7, 135.1, 135.9, 136.8 (aromatic C), 155.4, 163.6 (2 CO); IR ν: 3180 cm−1 (broad, NH), 3065 (aromatic CH), 2970 (aliphatic CH), 1655, 1640 (2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 355 (12.3%); Anal. Calcd. for C22H17N3O2 (355.39): C (74.35%), H (4.82%), N (11.82%); Found: C (74.1%), H (4.4%), N (12.1%).

2'-(4-Chlorophenyl)-1,3-dihydro-1'H-spiro[benzimidazole-2,4'-isoquinoline]-1',3'(2'H)-dione (5b): grey crystals after crystallization from acetic acid; 48% yield (Method A) and 89% (Method B); m.p. 215–217 °C; 1H-NMR: 3.90 (s, 2H, 2NH, D2O exchangeable), 6.90–8.10 (m, 12H, Ar-H); 13C-NMR: 83.4 (sp3-spiro C), 115.0, 118.1, 123.6, 125.2, 127.3, 128.8, 129.6, 130.9, 131.8, 133.7, 135.1, 135.9, 136.8 (aromatic C), 155.4, 163.6 (2 CO); IR ν: 3180 cm−1 (broad, NH), 3065 (aromatic CH), 1655, 1640 (2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 375 (11.5%), 377 (4.1%); Anal. Calcd. for C21H14ClN3O2 (375.81): C (67.12%), H (3.75%), Cl (9.43%), N (11.18%); Found: C (76.0%), H (3.5%), Cl (9.3%), N (11.3%).

2'-p-Tolyl-1,3-dihydro-1'H-spiro[benzo[d]imidazole-2,4'-isoquinoline]-1',3'(2'H)-dione (5c): grey crystals after crystallization from acetic acid; 47% yield (Method A) and 82% (Method B); m.p. 240–242 °C; 1H-NMR: 2.60 (s, 3H, CH3), 3.90 (s, 1H, NH, D2O exchangeable), 6.70–7.90 (m, 12H, Ar-H); 13C-NMR: 83.4 (sp3-spiro C), 115.4, 118.8, 122.7, 126.2, 127.3, 128.8, 129.6, 130.9, 131.8, 133.2, 136.8, 137.9, 142.8 (aromatic C), 155.4, 163.6 (2 CO); IR ν: 3190 cm−1 (broad, NH), 3065 (aromatic CH), 2975 (aliphatic CH), 1655,1645 (2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 356 (10.3%); Anal. Calcd. for C22H16N2O3 (356.37): C (74.15%), H (4.53%), N (7.86%); Found: C (74.05%), H (4.43%), N (7.75%).

2'-(4-Chlorophenyl)-1'H,3H-spiro[benzo[d]oxazole-2,4'-isoquinoline]-1',3'(2'H)-dione (5d): grey crystals after crystallization from acetic acid; 53% yield (Method A) and 92% (Method B); m.p. 220–222 °C; 1H-NMR: 3.90 (s, 1H, NH, D2O exchangeable), 6.70–8.00 (m, 12H, Ar-H); 13C-NMR: 93.8 (sp3-spiro C), 119.0, 122.1, 124.9, 125.8, 127.5, 128.9, 129.6, 131.2, 132.9, 134.7, 137.1, 139.9, 146.8 (aromatic C), 155.4, 163.6 (2 CO); IR ν: 3150 cm−1 (broad, NH), 3065 (aromatic CH), 1655,1645 (2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 376 (8.5%), 378 (2.7%); Anal. Calcd. for C21H13ClN2O3 (376.79): C (66.94%), H (3.48%), Cl (9.41%), N (7.43%); Found: C (66.6%), H (3.6%), Cl (9.1%), N (7.3%).

3.1.3. Cyclocondensation of 3a,b with Thiosemicarbazide; Formation of 6a,b

Method A: Each of compounds 3a,b (0.01 mol), was heated under reflux with thiosemicarbazide (0.91 g, 0.01 mol), absolute ethanol (25 mL) and few drops of piperidine for 4 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.

Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 10 min. The reaction mixture was treated similar to method A to obtain compounds 6a,b.

5'-Thioxo-2-p-tolyl-1H-spiro[isoquinoline-4,3'-[1,2,4]triazolidine]-1,3(2H)-dione (6a): white crystals after crystallization from acetic acid and washing with ethanol; 55% yield (Method A) and 90% (Method B); m.p. 156–158 °C; 1H-NMR: 2.40 (s, 3H, CH3), 3.10 (s, 1H, NH, D2O exchangeable), 3.60 (s, 1H, NH, D2O exchangeable), 4.00 (s, 1H, NH, D2O exchangeable), 7.20–7.90 (m, 8H, Ar-H); 13C-NMR: 23.2 (CH3), 91.7 (sp3 spiro C), 121.1, 123.4, 127.6, 128.4, 128.8, 129.8, 132.1, 134.0, 135.2, 136.8 (aromatic C), 154.9, 159.5 (2 CO), 176.9 (CS); IR ν: 3220, 3185, 3150 cm−1 (NH), 3060 (aromatic CH), 2970 (aliphatic CH), 1670,1650 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 338 (10.3%); Anal. Calcd. for C17H14N4O2S (338.36): C (60.34%), H (4.17%), N (16.56%), S(9.48%); Found: C (60.0%), H (3.9%), N (16.8%), S (9.7%).

2-(4-Chlorophenyl)-5'-thioxo-1H-spiro[isoquinoline-4,3'-[1,2,4]triazolidine]-1,3(2H)-dione (6b): white crystals after crystallization from dilute acetic acid and washing with ethanol; 60% yield (Method A) and 94% (Method B); m.p. 188–190 °C; 1H-NMR: 3.10 (s, 1H, NH, D2O exchangeable), 3.50 (s, 1H, NH, D2O exchangeable), 4.00 (s, 1H, NH, D2O exchangeable), 7.20–7.90 (m, 8H, Ar-H); 13C-NMR: 92.7 (sp3 spiro C), 127.1, 127.9, 132.6, 133.4, 134.8, 135.6, 136.1, 137.0, 137.9, 138.8 (aromatic C), 156.9, 160.7 (2CO), 180.1 (CS); IR ν: 3220, 3185, 3150 cm−1 (NH), 3060 (aromatic CH), 1670,1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 358 (12.7%) and 360 (4.3%); Anal. Calcd. for C16H11N4O2S (358.80): C (53.56%), H (3.09%), Cl (9.88%), N (15. 61%), S (8.94%); Found: C (53.4%), H (2.90%), Cl (9.6%), N (15.5%), S (8.7%).

3.1.4. Bromination of 7a,b; Formation of 8a,b

A solution of either of 7a or 7b (0.01 mol) in ethanol (20 mL) was heated under reflux with bromine (1.1 mL, 3.0 g, 0.02 mol) for 2 h. After cooling, the reaction mixture was poured onto ice-water and the solid that precipitated was filtered off, dried and crystallized from the proper solvent.

8,8-Dibromo-4-(4-chlorophenyl)-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidine-5,7(6H,8H)-dione (8a): white crystals after crystallization from absolute ethanol; 55% yield; m.p. 255–257 °C; 1H-NMR: 3.00 (s, 1H, NH, D2O exchangeable), 5.40 (s, 1H, CH), 7.20–7.70 (m, 9H, Ar-H), 11.30 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 127.1, 127.9, 132.6, 133.4, 134.8, 135.6, 136.1, 137.0, 139.9, 150.8 (aromatic C), 156.9, 160.7 (2CO), 178.0 (CS); IR ν: 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 539 (4.7%), 541 (10.7%) and 543 (7.1%); Anal. Calcd. for C19H12Br2ClN3O2S (541.64): C (42.13%), H (2.23%), Br (29.50%), Cl (6.55%), N (7.76%), S (5.92%); Found: C (42.0%), H (2.3%), Br (29.4%), Cl (6.7%), N (7.9%), S (5.8%).

8,8-Dibromo-4-(4-methoxyphenyl)-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidine-5,7(6H,8H)-dione (8b): white crystals after crystallization from absolute ethanol; 53% yield; m.p. 232–234 °C; 1H-NMR: 3.00 (s, 1H, NH, D2O exchangeable), 3.80 (s, 3H, OCH3), 5.40 (s, 1H, CH), 6.90–7.60 (m, 9H, Ar-H), 11.30 (s, 1H, NH, D2O exchangeable); 13C-NMR: 53.9 (OCH3), 55.5 (pyrimidine C-4), 83.1 (pyridine C-8), 127.4, 128.4, 132.7, 133.8, 134.9, 136.6, 137.1, 138.5, 148.3, 150.8 (aromatic C), 156.9, 160.7 (2CO), 182.0 (CS); IR ν: 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2880 (aliphatic CH), 1665, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 535 (6.5%), 537 (13.7%) and 539 (6.1%); Anal. Calcd. for C20H15Br2N3O3S (537.22): C (44.71%), H (2.81%), Br (29.75%), N (7.82%), S (5.97%); Found: C (44.8%), H (2.8%), Br (29.9%), N (7.7%), S (6.1%).

3.1.5. Cyclocondensation of 8a,b with Ethylene Diamine and Thiosemicarbazide; Formation of 9a and 10a

Method A: Each of compounds 8a,b (0.01 mol) was heated under reflux with either of ethylene diamine (0.67 mL, 0.01 mol) or thiosemicarbazide (0.91 g, 0.01 mol) in absolute ethanol (25 mL)] and few drops of piperidine for 2 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent to give 9a,b and 10a,b.

Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 5 min. The reaction mixture was treated similar to method A to obtain compounds 9a,b and 10a,b.

4'-(4-Chlorophenyl)-6'-phenyl-2'-thioxo-3',4'-dihydro-1'H-spiro[imidazolidine-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-dione (9a): white crystals after crystallization from absolute ethanol; 65% yield (Method A) and 93% (Method B); m.p. 247–249 °C; 1H-NMR: 2.60 (s, 4H, 2CH2-imidazolidine), 3.10 (s, 1H, NH, D2O exchangeable), 3.80 (s, 2H, 2NH, D2O exchangeable), 5.30 (s, 1H, CH), 7.20–7.70 (m, 9H, Ar-H), 11.30 (s, 1H, NH, D2O exchangeable); 13C-NMR: 51.2 (imidazolidine 2CH2), 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 127.1, 127.9, 132.6, 133.4, 134.8, 135.6, 136.1, 137.0, 138.5, 147.3 (aromatic C), 156.9, 160.4 (2CO), 181.0 (CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 1670, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 439 (8.7%) and 441 (3.2%). Anal. Calcd. for C21H18ClN5O2S (439.92): C (57.33%), H (4.12%), Cl (8.06%), N (15.92%), S (7.29%); Found: C (57.1%), H (4. 2%), Cl (7.9%), N (16.1%), S (7.1%).

4'-(4-Methoxyphenyl)-6'-phenyl-2'-thioxo-3',4'-dihydro-1'H-spiro[imidazolidine-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-dione (9b): white crystals after crystallization from absolute ethanol; 67% yield (Method A) and 91% (Method B); m.p. 215–217 °C; 1H-NMR: 2.60 (s, 4H, 2CH2-imidazolidine), 3.00 (s, 1H, NH, D2O exchangeable), 3.60 (s, 2H, 2NH, D2O exchangeable), 3.80 (s, 3H, OCH3), 5.40 (s, 1H, CH), 6.90–7.60 (m, 9H, Ar-H), 11.30 (s, 1H, NH, D2O exchangeable); 13C-NMR: 51.0 (imidazolidine 2CH2), 53.9 (OCH3), 55.5 (pyrimidine C-4), 83.1 (pyridine C-8), 116.4, 118.5, 125.7, 128.0, 134.9, 136.6, 137.1, 138.5, 148.3, 150.8 (aromatic C), 156.9, 160.7 (2CO), 182.0 (CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2880 (aliphatic CH), 1670, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 435 (7.1%); Anal. Calcd. for C22H21N5O3S (435.50): C (60.67%), H (4.86%), N (16.08%), S (7.36%); Found: C (60.7%), H (4.9%), N (15.9%), S (7.1%).

4-(4-Chlorophenyl)-6-phenyl-2,5'-dithioxo-3,4-dihydro-1H-spiro[pyrido[4,3-d]pyrimidine-8,3'-[1,2,4]triazolidine]-5,7(2H,6H)-dione (10a): white crystals after crystallization from absolute ethanol; 61% yield (Method A) and 91% (Method B); m.p. 225–227 °C; 1H-NMR: 3.00 (s, 1H, NH, D2O exchangeable), 3.10 (s, 1H, NH, D2O exchangeable), 3.40 (s, 1H, NH, D2O exchangeable), 5.30 (s, 1H, CH), 7.20–7.70 (m, 9H, Ar-H), 8.10 (s, 1H, NH, D2O exchangeable), 11.00 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 127.1, 127.9, 132.6, 133.4, 134.8, 135.6, 136.1, 137.0, 140.5, 152.3 (aromatic C), 156.9, 160.4 (2CO), 177.3, 181.0 (2CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 1675, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 470 (11.3%) and 441 (3.9%). Anal. Calcd. for C20H15ClN6O2S2 (470.96): C (51.01%), H (3.21%), Cl (7.53%), N (13.62%), S (7.29%); Found: C (50.9%), H (3.1%), Cl (7.3%), N (13.4%), S (7.4%).

4-(4-Methoxyphenyl)-6-phenyl-2,5'-dithioxo-3,4-dihydro-1H-spiro[pyrido[4,3-d]pyrimidine-8,3'-[1,2,4]triazolidine]-5,7(2H,6H)-dione (10b): white crystals after crystallization from absolute ethanol; 58% yield (Method A) and 89% (Method B); m.p. 206–208 °C; 1H-NMR: 3.00 (s, 1H, NH, D2O exchangeable), 3.10 (s, 1H, NH, D2O exchangeable), 3.60 (s, 1H, NH, D2O exchangeable), 3.80 (s, 3H, OCH3), 5.40 (s, 1H, CH), 6.90–7.60 (m, 9H, Ar-H), 8.00 (s, 1H, NH, D2O exchangeable), 11.30 (s, 1H, NH, D2O exchangeable); 13C-NMR: 53.3 (OCH3), 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 114.3, 115.9, 122.6, 126.4, 131.8, 135.6, 136.1, 137.0, 142.5, 150.3 (aromatic C), 160.9, 164.0 (2CO), 173.4, 181.0 (2CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 1675, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 466 (8.3%). Anal. calcd. for C21H18N6O3S2 (466.54): C (54.06%), H (3.89%), N (18.01%), S (13.75%); Found: C (53.9%), H (3.7%), N (17.9%), S (13.6%).

3.1.6. Cyclocondensation of 8a,b with o-Phenylenediamine (4a) and o-Aminothiophenol (4c); Formation of 11a–d

Method A: Each of compounds 8a,b (0.01 mol) was heated under reflux with either of o-phenylenediamine (4a; 1.08 g, 0.01 mol) or o-aminothiophenol (4c; 1.25 mL, 0.01 mol) in absolute ethanol (25 mL) and few drops of piperidine for 2 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.

Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 5 min. The reaction mixture was treated similar to method A to obtain compounds 11a–d.

4'-(4-Chlorophenyl)-6'-phenyl-2'-thioxo-1,3,3',4'-tetrahydro-1'H-spiro[benzo[d]imidazole-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-dione (11a): white crystals after crystallization from absolute ethanol; 47% yield (Method A) and 84% (Method B); m.p. 258–260 °C; 1H-NMR: 3.10 (s, 1H, NH, D2O exchangeable), 4.40 (s, 2H, 2NH, D2O exchangeable), 5.30 (s, 1H, CH), 6.60–7.50 (m, 13H, Ar-H), 12.10 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 116.8, 119.2, 127.1, 127.9, 132.6, 133.3, 134.6, 135.6, 136.1, 137.0, 138.5, 145.3, 151.4 (aromatic C), 160.9, 164.4 (2CO), 175.1 (CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3040 (aromatic CH), 2830 (aliphatic CH), 1665, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 487 (6.3%) and 489 (2.2%). Anal. Calcd. for C25H18ClN5O2S (487.96): C (61.54%), H (3.72%), Cl (7.27%), N (14.35%), S (6.57%); Found: C (61.3%), H (3.6%), Cl (7.1%), N (14.3%), S (6.4%).

4'-(4-Methoxyphenyl)-6'-phenyl-2'-thioxo-1,3,3',4'-tetrahydro-1'H-spiro[benzo[d]imidazole-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-dione (11b): white crystals after crystallization from absolute ethanol; 50% yield (Method A) and 91% (Method B); m.p. 226–228 °C; 1H-NMR: 3.10 (s, 1H, NH, D2O exchangeable), 3.80 (s, 3H, OCH3), 4.10 (s, 2H, 2NH, D2O exchangeable), 5.40 (s, 1H, CH), 6.60–7.50 (m, 13H, Ar-H), 12.20 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.3 (OCH3), 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 112.8, 116.2, 123.1, 124.9, 132.6, 133.3, 134.6, 135.6, 136.1, 137.0, 138.5, 145.4, 150.6 (aromatic C), 160.9, 164.4 (2CO), 175.1 (CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3040 (aromatic CH), 2830 (aliphatic CH), 1665, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 483 (6.8%). Anal. Calcd. for C26H21N5O3S (483.54): C (64.58%), H (4.38%), N (14.48%), S (6.63%); Found: C (64.5%), H (4.1%), N (14.6%), S (6.5%).

4'-(4-Chlorophenyl)-6'-phenyl-2'-thioxo-3',4'-dihydro-1'H,3H-spiro[benzo[d]thiazole-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-dione (11c): white crystals after crystallization from absolute ethanol; m.p. 230–232 °C, in 52% yield (Method A) and 88% (Method B); 1H-NMR: 3.10 (s, 1H, NH, D2O exchangeable), 4.50 (s, 1H, NH, D2O exchangeable), 5.30 (s, 1H, CH), 6.60–7.50 (m, 13H, Ar-H), 11.80 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.7 (pyrimidine C-4), 82.0 (pyridine C-8), 116.0, 119.6, 121.3, 125.2, 127.4, 128.5, 132.6, 133.3, 134.6, 135.6, 136.1, 137.0, 138.5, 140.3 145.3, 151.4 (aromatic C), 160.9, 164.4 (2CO), 175.1 (CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3040 (aromatic CH), 2830 (aliphatic CH), 1665, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 504 (9.8%) and 489 (3.7%). Anal. Calcd. for C25H17ClN4O2S2 (505.01): C (59.46%), H (3.39%), Cl (7.02%), N (11.09%), S (12.70%); Found: C (59.5%), H (3.2%), Cl (6.9%), N (11.2%), S (12.6%).

4'-(4-Methoxyphenyl)-6'-phenyl-2'-thioxo-3',4'-dihydro-1'H,3H-spiro[benzo[d]thiazole-2,8'-pyrido[4,3-d]pyrimidine]-5',7'(2'H,6'H)-dione (11d): white crystals after crystallization from absolute ethanol; 45% yield (Method A) and 80% (Method B); m.p. 210–212 °C; 1H-NMR: 3.10 (s, 1H, NH, D2O exchangeable), 3.80 (s, 3H, OCH3), 4.00 (s, 1H, NH, D2O exchangeable), 5.40 (s, 1H, CH), 6.60–7.50 (m, 13H, Ar-H), 12.20 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.3 (OCH3), 55.7 (pyrimidine C-4), 80.9 (pyridine C-8), 116.4, 118.2, 121.2, 125.6, 127.1, 128.5, 132.6, 133.3, 134.6, 135.6, 136.1, 137.0, 138.5, 140.3, 145.1, 151.3 (aromatic C), 160.9, 164.4 (2CO), 175.1 (CS); IR ν: 3280, 3220, 3180 cm−1 (NH), 3040 (aromatic CH), 2830 (aliphatic CH), 1665, 1645 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 500 (11.3%). Anal. Calcd. for C26H20N4O3S2 (500.59): C (62.38%), H (4.03%), N (11.19%), S (12.81%); Found: C (62.4%), H (3.9%), N (11.0%), S (12.6%).

3.1.7. Reactions of 8a,b with Malononitrile (12a) and Ethyl cyanoacetate (12b): Formation of 13a–d

To a solution of each of compounds 8a,b (0.01 mol) in absolute ethanol (30 mL) containing a catalytic amount of piperidine was added either of malononitrile (12a; 0.66 g, 0.01 mol) or ethyl cyanoacetate (12b; 1.13 mL, 0.01 mol). The reaction mixture was heated under reflux for 3 h, under TLC monitoring, then cooled and poured onto ice-cold water. The solid product that separated was filtered off, dried and crystallized from ethanol.

2-(4-(4-Chlorophenyl)-5,7-dioxo-6-phenyl-2-thioxo-1,2,3,4,6,7-hexahydropyrido[4,3-d]pyrimidin-8(5H)-ylidene)malononitrile (13a): pale yellow crystals after crystallization from absolute ethanol; 52% yield; m.p. 223–225 °C; 1H-NMR: 3.20 (s, 1H, NH, D2O exchangeable), 4.80 (s, 1H, CH), 7.20–7.50 (m, 9H, Ar-H), 12.60 (s, 1H, NH, D2O exchangeable); 13C-NMR: 55.5 (pyrimidine C-4), 81.9 (methylidine C), 107.1 (CN), 112.1, 127.1, 127.9, 132.1, 132.9, 133.8, 134.7, 135.1, 137.3, 150.8, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0 (CS); IR ν: 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2210 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 445 (12.2%) and 447 (4.7%). Anal. Calcd. for C22H12ClN5O2S (445.88): C (59.26%), H (2.71%), Cl (7.95%), N (15.71%), S (7.19%); Found: C (59.2%), H (2.5%), Cl (7.8%), N (15.6%), S (7.0%).

2-(4-(4-Methoxyphenyl)-5,7-dioxo-6-phenyl-2-thioxo-1,2,3,4,6,7-hexahydropyrido[4,3-d]pyrimidin-8(5H)-ylidene)malononitrile (13b): yellow crystals after crystallization from absolute ethanol; 50% yield; m.p. 214–216 °C; 1H-NMR: 3.10 (s, 1H, NH, D2O exchangeable), 3.80 (s, 1H, OCH3), 4.70 (s, 1H, CH), 6.90–7.50 (m, 9H, Ar-H), 12.60 (s, 1H, NH, D2O exchangeable); 13C-NMR: 54.7 (OCH3), 55.5 (pyrimidine C-4), 81.8 (methylidine C), 107.1 (CN), 112.1, 114.8, 127.3, 132.1, 132.9, 133.8, 134.6, 135.1, 137.3, 150.8, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.1 (CS); IR ν: 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2210 (CN), 1670, 1640 (2C=O), 1600, 1500 (aromatic C=C); MS (70 eV): M+ m/z 441 (15.1%); Anal. Calcd. for C23H15N5O3S (441.46): C (62.58%), H (3.42%), N (15.86%), S (7.26%); Found: C (62.6%), H (3.5%), N (15.7%), S (7.0%).

Ethyl 2-(4-(4-chlorophenyl)-5,7-dioxo-6-phenyl-2-thioxo-1,2,3,4,6,7-hexahydropyrido[4,3-d]-pyrimidin-8(5H)-ylidene)-2-cyanoacetate (13c): white crystals after crystallization from absolute ethanol; 42% yield; m.p. 188–190 °C; 1H-NMR: 1.20 (t, 3h, CH3), 3.00 (s, 1H, NH, D2O exchangeable), 4.30 (q, 2H, CH2), 5.10 (s, 1H, CH), 7.20–7.60 (m, 9H, Ar-H), 12.50 (s, 1H, NH, D2O exchangeable); 13C-NMR: 16.7 (CH3), 55.5 (pyrimidine C-4), 59.1 (CH2), 93.9 (methylidine C), 108.1 (CN), 123.4, 127.1, 127.9, 132.1, 132.9, 133.8, 134.6, 135.1, 137.3, 151.8, 157.1 (sp2 + aromatic C), 156.9, 160.7, 165.0 (3CO), 178.0 (CS); IR ν: 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2230 (CN), 1710, 1670, 1640 (3C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 492 (14.2%) and 494 (5.0%). Anal. Calcd. for C24H17ClN4O4S (492.93): C (58.48%), H (3.48%), Cl (7.19%), N (11.37%), S (6.50%); Found: C (58.3%), H (3.3%), Cl (7.1%), N (11.4%), S (6.4%).

Ethyl 2-cyano-2-(4-(4-methoxyphenyl)-5,7-dioxo-6-phenyl-2-thioxo-1,2,3,4,6,7-hexahydropyrido-[4,3-d]pyrimidin-8(5H)-ylidene)acetate (13d): white crystals after crystallization from absolute ethanol; 42% yield; m.p. 188–190 °C; 1H-NMR: 1.20 (t, 3h, CH3), 3.00 (s, 1H, NH, D2O exchangeable), 3.70 (s, 3H, CH3), 4.30 (q, 2H, CH2), 5.10 (s, 1H, CH), 6.90–7.60 (m, 9H, Ar-H), 12.30 (s, 1H, NH, D2O exchangeable); 13C-NMR: 16.7 (CH3), 53.1 (OCH3) 55.5 (pyrimidine C-4), 59.0 (CH2), 91.7 (methylidine C), 108.1 (CN), 117.7, 125.1, 126.9, 132.1, 132.9, 133.8, 133.6, 135.1, 137.3, 148.8, 156.6 (sp2 + aromatic C), 156.9, 160.7, 165.0 (3CO), 178.0 (CS); IR ν: 3220, 3180 cm−1 (NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2230 (CN), 1710, 1670, 1640 (3C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 488 (13.9%); Anal. Calcd. for C25H20N4O5S (488.52): C (61.47%), H (4.13%), N (11.47%), S (6.56%); Found: C (61.5%), H (4.0%), N (11.4%), S (6.4%).

3.1.8. Reaction of 13a–d with Hydrazine Hydrate and Thiourea: Formation of 14a–d and 15a–d

Method A: To a solution of each of compounds 13a–d (0.01 mol) in absolute ethanol (30 mL) containing a catalytic amount of piperidine was added hydrazine (0.32 mL, 0.01 mol) or thiourea (0.76 g, 0.01 mol). The reaction mixture was heated under reflux for 3 h, under TLC monitoring, then cooled and poured onto ice-cold water. The solid product that separated was filtered off, dried and crystallized from ethanol.

Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 7 min. The reaction mixture was treated similar to method A to obtain compounds 14a–d and 15a–d.

5-Amino-4'-(4-chlorophenyl)-5',7'-dioxo-6'-phenyl-2'-thioxo-2,2',3',4,4',5',6',7'-octahydro-1'H-spiro[pyrazole-3,8'-pyrido[4,3-d]pyrimidine]-4-carbonitrile (14a): white crystals after crystallization from absolute dioxane; 37% yield (Method A) and 81% (Method B); m.p. 235–237 °C; 1H-NMR: 3.20 (s, 1H, NH, D2O exchangeable), 4.10 (s, 1H, pyrazole H-4), 5.10 (s, 1H, pyrimidine H-4), 6.2 (s, 1H, NH, D2O exchangeable), 7.20–7.50 (m, 9H, Ar-H), 9.1 (s, 2H, NH2, D2O exchangeable), 12.60 (s, 1H, NH, D2O exchangeable); 13C-NMR: 39.5 (pyrazole C-4), 55.4 (pyrimidine C-4), 58.9 (spiro-C), 107.1 (CN), 110.3, 116.1, 126.9, 131.8, 132.7, 133.8, 134.6, 135.1, 137.3, 143.1, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0 (CS); IR ν: 3350, 3220, 3180 cm−1 (broad, NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C);MS (70 eV): M+ m/z 477 (9.2%) and 447 (3.5%); Anal. Calcd. for C22H16ClN7O2S (477.93): C (55.29%), H (3.37%), Cl (7.42%), N (20.52%), S (6.71%); Found: C (55.1%), H (3.1%), Cl (7.3%), N (20.4%), S (6.6%).

5-Amino-4'-(4-methoxyphenyl)-5',7'-dioxo-6'-phenyl-2'-thioxo-2,2',3',4,4',5',6',7'-octahydro-1'H-spiro[pyrazole-3,8'-pyrido[4,3-d]pyrimidine]-4-carbonitrile (14b): white crystals after crystallization from dil. dioxane; 42% yield (Method A) and 84% (Method B); m.p. 229–231 °C; 1H-NMR: 3.00 (s, 1H, NH, D2O exchangeable), 3.70 (s, 3H, OCH3), 4.10 (s, 1H, pyrazole H-4), 5.10 (s, 1H, pyrimidine H-4), 6.2 (s, 1H, NH, D2O exchangeable), 7.20–7.50 (m, 9H, Ar-H), 9.4 (s, 2H, NH2, D2O exchangeable), 12.50 (s, 1H, NH, D2O exchangeable); 13C-NMR: 39.5 (pyrazole C-4), 53.7 (OCH3), 55.4 (pyrimidine C-4), 58.9 (spiro-C), 107.1 (CN), 112.7, 114.3, 126.9, 131.8, 132.7, 133.8, 134.6, 135.1, 137.3, 143.1, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0 (CS); IR ν: 3350, 3220, 3180 cm−1 (broad, NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 473 (10.8%); Anal. Calcd. for C23H19N7O3S (477.93): C (58.43%), H, 4.04; N, 20.71; S, 6.77; Found: C (58.3%), H (4.1%), N (20.5%), S (6.6%).

4'-(4-Chlorophenyl)-5-hydroxy-5',7'-dioxo-6'-phenyl-2'-thioxo-2,2',3',4,4',5',6',7'-octahydro-1'H-spiro-[pyrazole-3,8'-pyrido[4,3-d]pyrimidine]-4-carbonitrile (14c): white crystals after crystallization from absolute ethanol; 45% yield (Method A) and 89% (Method B); m.p. 258–260 °C; 1H-NMR: 3.40 (s, 1H, NH, D2O exchangeable), 4.50 (s, 1H, pyrazole H-4), 5.40 (s, 1H, pyrimidine H-4), 6.2 (s, 1H, NH, D2O exchangeable), 7.20–7.50 (m, 9H, Ar-H), 11.1 (s, 1H, OH, D2O exchangeable), 12.60 (s, 1H, NH, D2O exchangeable); 13C-NMR: 41.1 (pyrazole C-4), 55.4 (pyrimidine C-4), 58.9 (spiro-C), 108.9 (CN), 110.3, 115.1, 126.9, 128.8, 130.1, 132.5, 134.6, 135.1, 137.3, 143.1, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0 (CS); IR ν: 3400, 3270, 3180 cm−1 (broad, NH + OH), 3060 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 478 (10.7%) and 480 (3.8%); Anal. Calcd. for C22H15ClN6O3S (478.91): C (55.17%), H (3.16%), Cl (7.40%), N (17.55%), S (6.70%); Found: C (55.0%), H (3.2%), Cl (7.3%), N (17.3%), S (6.6%).

5-Hydroxy-4'-(4-methoxyphenyl)-5',7'-dioxo-6'-phenyl-2'-thioxo-2,2',3',4,4',5',6',7'-octahydro-1'H-spiro[pyrazole-3,8'-pyrido[4,3-d]pyrimidine]-4-carbonitrile (14d): white crystals after crystallization from absolute ethanol; 44% yield (Method A) and 82% (Method B); m.p. 237–239 °C; 1H-NMR: 3.30 (s, 1H, NH, D2O exchangeable), 3.80 (s, 3H, OCH3), 4.10 (s, 1H, pyrazole H-4), 5.10 (s, 1H, pyrimidine H-4), 6.2 (s, 1H, NH, D2O exchangeable), 7.20–7.50 (m, 9H, Ar-H), 10.8 (s, 1H, OH, D2O exchangeable, 12.50 (s, 1H, NH, D2O exchangeable); 13C-NMR: 39.5 (pyrazole C-4), 53.8 (OCH3), 55.4 (pyrimidine C-4), 58.9 (spiro-C), 108.9 (CN), 112.7, 124.8, 126.9, 128.8, 132.1, 133.3, 134.6, 135.1, 137.3, 144.6, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0 (CS); IR ν: 3350, 3200, 3150 cm−1 (broad, NH + OH), 3060 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 474 (12.0%); Anal. Calcd. for C23H18N6O4S (474.49): C (58.22%), H (3.82%), N (17.71%), S (6.76%); Found: C (58.1%), H (3.6%), N (17.5%), S (6.7%).

6'-Amino-4-(4-chlorophenyl)-5,7-dioxo-6-phenyl-2,2'-dithioxo-2,3,3',4,5,5',6,7-octahydro-1H,2'H-spiro[pyrido[4,3-d]pyrimidine-8,4'-pyrimidine]-5'-carbonitrile (15a): white crystals after crystallization from dil. DMF; 47% yield (Method A) and 88% (Method B); m.p. 240–242 °C; 1H-NMR: 3.00 (s, 1H, NH, D2O exchangeable), 3.40 (s, 1H, spiro-pyrimidine H-4), 5.10 (s, 1H, pyrimidine H-4), 5.50 (s, 1H, NH, D2O exchangeable), 7.20–7.50 (m, 9H, Ar-H), 8.70 (s, 2H, NH2, D2O exchangeable), 12.00 (s, 1H, NH, D2O exchangeable); 13C-NMR: 29.0 (spiro-pyrimidine C-5), 55.4 (pyrimidine C-4), 63.9 (spiro-C), 107.8 (CN), 114.3, 119.5, 126.9, 128.8, 132.7, 133.8, 134.6, 136.1, 138.1, 143.1, 154.3 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0, 181.0 (2CS); IR ν: 3350, 3220, 3180 cm−1 (broad, NH), 3060 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C);MS (70 eV): M+ m/z 522 (10.0%) and 524 (3.8%); Anal. Calcd. for C23H16ClN7O2S2 (522.00): C (52.92%), H (3.09%), Cl (6.79%), N (18.78%), S (12.29%); Found: C (52.8%), H (3.2%), Cl (6.8%), N (18.9%), S (12.1%).

6'-Amino-4-(4-methoxyphenyl)-5,7-dioxo-6-phenyl-2,2'-dithioxo-2,3,3',4,5,5',6,7-octahydro-1H,2'H-spiro[pyrido[4,3-d]pyrimidine-8,4'-pyrimidine]-5'-carbonitrile (15b): white crystals after crystallization from dil. DMF; 43% yield (Method A) and 87% (Method B); m.p. 226–228 °C; 1H-NMR: 2.80 (s, 1H, NH, D2O exchangeable), 3.50 (s, 1H, spiro-pyrimidine H-4), 3.80 (s, 1H, OCH3), 5.00 (s, 1H, pyrimidine H-4), 5.50 (s, 1H, NH, D2O exchangeable), 6.80–7.50 (m, 9H, Ar-H), 8.50 (s, 2H, NH2, D2O exchangeable), 12.20 (s, 1H, NH, D2O exchangeable); 13C-NMR: 27.8 (spiro-pyrimidine C-5), 52.6 (OCH3), 55.7 (pyrimidine C-4), 64.2 (spiro-C), 107.8 (CN), 112.5, 116.2, 125.2, 127.0, 128.4, 129.8, 131.6, 136.1, 138.1, 139.0, 154.2 (sp2 + aromatic C), 156.9, 160.7 (2CO), 178.0, 181.0 (2CS); IR ν: 3350, 3200, 3160 cm−1 (broad, NH), 3080 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1500 (aromatic C=C); MS (70 eV): M+ m/z 517 (11.3%); Anal. Calcd. for C24H19N7O3S2 (517.58): C (55.69%), H (3.70%), N (18.94%), S (12.39%); Found: C (55.6%), H (3.5%), N (18.9%), S (12.1%).

4-(4-Chlorophenyl)-6'-hydroxy-5,7-dioxo-6-phenyl-2,2'-dithioxo-2,3,3',4,5,5',6,7-octahydro-1H,2'H-spiro[pyrido[4,3-d]pyrimidine-8,4'-pyrimidine]-5'-carbonitrile (15c): white crystals after crystallization from dioxane; 51% yield (Method A) and 90% (Method B); m.p. 255–257 °C; 1H-NMR: 3.20 (s, 1H, NH, D2O exchangeable), 3.50 (s, 1H, spiro-pyrimidine H-4), 5.10 (s, 1H, pyrimidine H-4), 5.80 (s, 1H, NH, D2O exchangeable), 7.20–7.50 (m, 9H, Ar-H), 11.10 (s, 1H, OH, D2O exchangeable), 12.40 (s, 1H, NH, D2O exchangeable); 13C-NMR: 29.0 (spiro-pyrimidine C-5), 55.4 (pyrimidine C-4), 63.9 (spiro-C), 107.8 (CN), 113.9, 119.8, 127.2, 128.8, 132.4, 133.8, 134.5, 136.5, 138.6, 152.1, 155.3 (sp2 + aromatic C), 158.9, 162.7 (2CO), 178.0, 180.5 (2CS); IR ν: 3350, 3220, 3180 cm−1 (broad, NH), 3050 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+ m/z 522 (14.0%) and 524 (4.7%); Anal. Calcd. for C23H15ClN6O3S2 (522.99): C (52.82%), H (2.89%), Cl (6.78%), N (16.07%), S (12.26%); Found: C (52.6%), H (2.9%), Cl (6.6%), N (15.9%), S (12.1%).

6'-Hydroxy-4-(4-methoxyphenyl)-5,7-dioxo-6-phenyl-2,2'-dithioxo-2,3,3',4,5,5',6,7-octahydro-1H,2'H-spiro[pyrido[4,3-d]pyrimidine-8,4'-pyrimidine]-5'-carbonitrile (15d): white crystals after crystallization from dioxane; 47% yield (Method A) and 91% (Method B); m.p. 238–240 °C; 1H-NMR: 2.90 (s, 1H, NH, D2O exchangeable), 3.60 (s, 1H, spiro-pyrimidine H-4), 3.90 (s, 1H, OCH3), 5.00 (s, 1H, pyrimidine H-4), 5.50 (s, 1H, NH, D2O exchangeable), 6.80–7.50 (m, 9H, Ar-H), 10.80 (s, 1H, OH, D2O exchangeable), 12.10 (s, 1H, NH, D2O exchangeable); 13C-NMR: 27.2 (spiro-pyrimidine C-5), 54.4 (OCH3), 57.1 (pyrimidine C-4), 64.0 (spiro-C), 108.4 (CN), 116.5, 119.6, 125.0, 127.4, 128.8, 130.3, 132.6, 136.1, 138.1, 139.0, 153.1 (sp2 + aromatic C), 158.9, 160.7 (2CO), 178.0, 181.3 (2CS); IR ν: 3350, 3200, 3160 cm−1 (broad, NH), 3080 (aromatic CH), 2850 (aliphatic CH), 2200 (CN), 1670, 1640 (2C=O), 1600, 1500 (aromatic C=C);MS (70 eV): M+ m/z 518 (14.0%), 516 (5.1%); Anal. Calcd. for C24H18N6O4S2 (518.57): C (55.59%), H (3.50%), N (16.21%), S (12.37%); Found: C (55.4%), H (3.4%), N (16.3%), S (12.3%).

3.2. Antimicrobial Screening

The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against the following microorganisms: (a) Gram-negative: Escherichia coli and Pseudomonas putide; (b) Gram-positive: Bacillus subtilis and Streptococcus lactis; (c) Fungi: Aspergillus niger and Penicillium sp.; (d) Yeast: Candida albicans. Media: Three types of specific media were used in this study:

Medium 1: Nutrient Medium for bacteria, consisting of (g/L distilled water): peptone, 5 and meat extract, 3. pH was adjusted to 7.0.

Medium 2: Potato Dextrose Medium for fungi, consisting of (g/L distilled water): Infusion from potatoes, 4 and D(+)glucose, 20. pH was adjusted to 5.5.

Medium 3: Universal Medium for yeast, consisting of (g/L distilled water): yeast extract, 3; malt extract, 3; peptone, 5 and glucose, 10. pH was adjusted to 5.5.

For solid media, 2% agar was added. All media were sterilized at 121 °C for 20 min.

3.3. Procedure (Filter Paper Diffusion Method) [27]

Proper concentrations of microbial suspensions were prepared from 1 (for bacteria) to 3 (for yeast and fungi)-day-old liquid stock cultures incubated on a rotary shaker (100 rpm). In the case of fungi, five sterile glass beads were added to each culture flask. The mycelia were then subdivided by mechanical stirring at speed No. 1 for 30 min. Turbidity of microorganisms was adjusted with a spectrophotometer at 350 nm to give an optical density of 1.0. Appropriate agar plates were aseptically surface inoculated uniformly by a standard volume (ca. 1 mL) of the microbial broth culture of the tested microorganism, namely E. coli, P. putida, B. subtilis, S. lactis, A. niger, Penicillium sp. and C. albicans. Whatman No. 3 filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at 121 °C. Test compounds were dissolved in 80% ethyl alcohol to give final concentration of 5 μg/mL. The sterile discs were impregnated with the test compounds (5 μg/disc). After the impregnated discs have been air dried, they were placed on the agar surface previously seeded with the organism to be tested. Discs were gently pressed with forceps to insure thorough contact with the media. Three discs were arranged per dish, suitably spaced apart, i.e., the discs should be separated by a distance that is equal to or slightly greater than the sum of the diameters of inhibition produced by each disc alone. Each test compound was conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to permit good diffusion before transferring them to an incubator at 37 °C for 24 h for bacteria and at 30 °C for 72 h for yeast and fungi.