Enaminones as building blocks for the synthesis of substituted pyrazoles with antitumor and antimicrobial activities.

Novel N-arylpyrazole-containing enaminones 2a,b were synthesized as key intermediates. Reactions of 2a,b with active methylene compounds in acetic acid in the presence of ammonium acetate afforded substituted pyridine derivatives 5a-d. Enaminones 2a,b also reacted with aliphatic amines such as hydrazine hydrate and hydroxylamine hydrochloride to give bipyrazoles 8a,b and pyrazolylisoxazoles 9a,b, respectively. On the other hand, treatment of 2a,b with a heterocyclic amine and its diazonium salt yielded the respective [1,2,4]triazolo[4,3-a]pyrimidines 12a,b and pyrazolylcarbonyl[1,2,4]triazolo-[3,4-c][1,2,4]triazines 14a,b. Moreover, 2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (17) was prepared via reaction of enaminone 2a with aminothiouracil (15). Cyclocondensation of 17 with the appropriate hydrazonoyl chlorides 18a-c gave the corresponding pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones 21a-c. The cytotoxic effects of compounds 2b, 14a and 17 against human breast cell line (MCF-7) and liver carcinoma cell line (HEPG2) were screened and in both lines they showed inhibition effects comparable to those of 5-fluorouracil, used as a standard. The antimicrobial activity of some products chosen as representative examples was also evaluated.

1. Introduction

The growing interest in bioactive pan class="Chemical">N-arylpyrazoles has led to an increasing demand for efficient syntheses of this class of heterocyclic compounds. Several reports have found diverse applications for cal">pan class="Chemical">N-arylpyrazoles in medicine such as antitumor [1,2,3,4,5,6,7,8,9,10,11], antiviral [12], anti-inflammatory [13] agents, or kinase inhibitors for the treatment of type 2 diabetes, hyperlipidemia, and obesity [14]. Moreover, these compounds have remarkable potential in nanomedicine applications against malignant gliomas [15]. 1-(4-Chlorophenyl)-4-hydroxy-3-substituted-1H-pyrazoles (Figure 1) were reported by the U.S. National Cancer Institute (NCI) to have pronounced anticancer activity [16,17].

Figure 1

substituted pyrazoles with potential antitumor activity.

substituted n class="Chemical">pan class="Chemical">pyrazoles with potential antical">pan class="Disease">tumor activity.

Structure modifications suggested in this work focused mainly on synthesis of pan class="Chemical">polysubstituted pyrazole analogues to form A and B, having a variety of cal">pan class="Chemical">azoles and fused azoles at position 3. These substituents at position 3 are linked directly to the pyrazole ring or through the carbonyl group in order to improve the antitumor and antimicrobial activities of such compounds. This work is an extension of an ongoing research program devoted to the synthesis and characterization of different heterocyclic ring systems endowed with potential biological activities [18,19,20,21,22,23,24,25].

2. Results and Discussion

The synthetic route for preparation of the previously unreported 3-[E-3-(N,N-dimethyl-amino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pan class="Chemical">pyrazoles (2a,b), involving condencal">pan class="Species">sation of 3-acetyl-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles (1a,b) [26] with dimethylformamide dimethylacetal (DMF-DMA) under reflux for 10 hours in the absence of solvent, is depicted in Scheme 1.

Scheme 1

Synthesis of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles (2a,b).

Synthesis of pan class="Chemical">3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles (2a,b).

The structures of 2a,b were confirmed by their spectral data (IR, MS and 1H-NMR) and elemental analyses. For example, the 1H-NMR spectrum revealed two doublet signals at δ 5.88, 7.67 ppm with coupling constant J = 13 Hz assignable to olefinic protons (CH=CH) in a trans configuration [26,27] besides two singlet signals of the pan class="Chemical">dimethylamino group at δ 2.8, 3.1 ppm.

Reactions of pan class="Chemical">enaminones 2a,b with C-nucleophiles such as n class="Species">cal">pan class="Chemical">2,4-pentanedione and ethyl 3-oxo-butanoate were carried out in glacial acetic acid in the presence of ammonium acetate and led to formation of 6-(pyrazol-3-yl)-pyridine derivatives 5a-d via nucleophilic displacement of active methylene to the dimethylamino group followed by concurrent elimination of water molecule from non-isolable intermediates 4a-d (Scheme 2). The other possible isomeric structures 4-(pyrazol-3-yl)-pyridines 7a-d were discarded based on 1H-NMR data that revealed pyridyl hydrogens at C-4, C-5 as a pair of doublets at δ 7.5, 7.7 ppm, respectively, with J = 8 Hz assignable to 6-substituted-pyridines 5a-d. The isomeric structures 7a-d should display pair of doublets corresponding to C-5, C-6 with a lower coupling constant (J = 2–3 Hz) [28].

Scheme 2

Reactions of enaminones 2a,b with active methylene compounds.

Reactions of pan class="Chemical">enaminones 2a,b with active methylene compounds.

Treatment of pan class="Chemical">enaminones 2a,b with a n class="Species">cal">pan class="Chemical">N-nucleophile such as hydrazine hydrate in absolute ethanol under reflux afforded 1H,1'H-3,3'-bipyrazoles 8a,b. The structures of the products were substantiated by the 1H-NMR spectra which displayed new pair of doublets at δ 7.53 and 7.58 ppm with (J = 7.5 Hz) corresponding to pyrazole protons at positions 4 and 5, respectively and another D2O exchangeable proton at δ 13 ppm assignable to the NH group. The products were formed via initial addition of the amino group in hydrazine to the enaminone double bond, followed by elimination of dimethylamine and water molecules to give the final isolable products 8a,b as previously mentioned [29] (Scheme 3). Similarly, enaminones 2a,b reacted with hydroxylamine hydrochloride in refluxing absolute ethanol in the presence of anhydrous potassium carbonate to yield products that may be formulated as pyrazolylisoxazoles 9a,b or its isomeric forms 10a,b. Structure 9 was assigned for the reaction products on the basis of the 1H-NMR spectral data in which a resonance for H-4 and H-5 of isoxazole appeared typically at δ 6.78 and 8.50 ppm, respectively (see Experimental). The other isomeric structures 10a,b were ruled out as the isoxazole H-3 would be expected to resonate at a higher field of δ 8.0 ppm [30]. It is thus assumed that, the products 9a,b were formed via initial condensation of amino group of hydroxylamine with carbonyl group of enaminones 2a,b followed by elimination of dimethylamine (cf. Scheme 3).

Scheme 3

Reactions of enaminones 2a,b with hydrazine hydrate and hydroxylamine.

Reactions of pan class="Chemical">enaminones 2a,b with n class="Species">cal">pan class="Chemical">hydrazine hydrate and hydroxylamine.

Next, the reactions of pan class="Chemical">enaminones 2a,b with cal">pan class="Chemical">heterocyclic amines were investigated. Refluxing of enaminones 2a,b with 3-amino-1H-[1,2,4]triazole in glacial acetic acid gave the corresponding [1,2,4]triazolo[4,3-a]pyrimidines 12a,b via non-isolable intermediates 11a,b (Scheme 4).

Scheme 4

Reactions of enaminones 2a,b with heterocyclic amines.

The structures of the products were confirmed by spectral (IR, MS and 1H-NMR) and elemental analyses (see Experimental). On the other hand, coupling of pan class="Chemical">enaminones 2a,b with diazotized cal">pan class="Chemical">3-amino-1H-[1,2,4]triazole in pyridine at low temperature afforded the respective pyrazolylcarbonyl- [1,2,4]triazolo[3,4-c][1,2,4]triazines 14a,b. The reactions proceeded by initial formation of non-isolable hydrazonals [31,32,33] 13a,b followed by elimination of water molecules to give the desired products 14a,b.

The utility of pan class="Chemical">enaminone 2a in the synthesis of annelated heterocycles was further explored via its reaction with n class="Species">cal">pan class="Chemical">6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (15) in glacial acetic acid under reflux for 6 hours. This reaction afforded the 2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one 17 via intermediate 16. Spectral (IR, MS, 1H-NMR) data and elemental analysis were in consistent with the isolated product 17.

Reactions of pan class="Chemical">enaminones 2a,b with cal">pan class="Chemical">heterocyclic amines.

For example, IR revealed three absorption bands at 3261, 3245, 1677 cm−1 assignable for 2 NH, and a C=O, respectively. The 1H-NMR spectrum also displayed a characteristic pair of doublet signals at δ 8.29, 8.48 ppm assigned to the pan class="Chemical">pyridine H-2, H-3 protons, respectively [34]. Treatment of cal">pan class="Chemical">2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (17) with the appropriate hydrazonoyl chlorides 18a-c in dioxane in the presence of triethylamine under reflux conditions furnished the corresponding pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidinones 21a-c as the end products (Scheme 5).

Scheme 5

Reactions of enaminone 2a with pyrimidinethione.

Reactions of pan class="Chemical">enaminone 2a with cal">pan class="Chemical">pyrimidinethione.

The reactions proceeded through S-alkylation [35] to give S-alkylated products 19apan class="Chemical">-c followed by Smiles rearrangement [36], cal">pan class="Disease">affording intermediates 20a-c which cyclized in situ under the employed reaction conditions via elimination of hydrogen sulfide gas to give the desired products 21a-c (cf. Scheme 5). The other isomeric structures, pyrido[2,3-d][1,2,4]triazolo[3,4-a]pyrimidinones 22a-c, were ruled out based on the 13C-NMR which revealed a signal for a carbonyl group at δ = 161.9–164 ppm which is similar to that of I (δ = 161–164 ppm) and different from its isomeric structure II (δ = 170–175) [37] (Figure 2).

Figure 2

13C NMR for azolopyrimidinones.

pan class="Chemical">13C NMR for n class="Species">cal">pan class="Chemical">azolopyrimidinones.

2.1. Antitumor Screening Test

The pan class="Disease">cytotoxicn> effects of compounds 2b, cal">pan>n class="Species">14a and 17 against human breast cell line (MCF-7) and liver carcinoma cell line (HEPG2) were evaluated using 5-fluorouracil as a standard sample in both lines. These compounds were selected by the National Cancer Institute (NCI), Cairo, Egypt. The analysis of the data obtained indicated that the values of IC50 for such compounds against human breast cell MCF-7 line are 0.863 μg/well (Figure 3), 2.33 μg/well (Figure 4), and 2.33 μg/well (Figure 5), respectively [IC50 of 5-fluorouracil as a standard sample = 0.67 μg] (Figure 6). The results indicated that biologically active compound 2b has almost the same activity as the reference drug (5-fluorouracil).

Figure 3

Effect of conc. of 2b on MCF-7 line.

Figure 4

Effect of conc. of 14a on MCF-7 line.

Figure 5

Effect of conc. of 17 on MCF-7 line.

Figure 6

Effect of conc. of 5-fluorouracil (standard) on MCF-7 line.

Effect of conc. of 2b on n class="Chemical">pan class="CellLine">MCF-7 line.

Effect of conc. of n class="Chemical">pan class="Species">14a on cal">pan class="CellLine">MCF-7 line.

Effect of conc. of 17 on n class="Chemical">pan class="CellLine">MCF-7 line.

Effect of conc. of n class="Chemical">pan class="Chemical">5-fluorouracil (standard) on cal">pan class="CellLine">MCF-7 line.

On the other hand, IC50 of compounds 2b, pan class="Species">14a and 17 against cal">pan class="Disease">liver carcinoma cell line (HEPG2) are 0.884 μg/well (Figure 7), 0.806 μg/well (Figure 8), and 4.07 μg/well (Figure 9), respectively. [IC50 of 5-fluorouracil as standard sample = 5 μg] (Figure 10). The values of IC50 indicated that the tested compounds 2b, 14a and 17 have higher cytotoxic activities against liver carcinoma cell line (HEPG2) than standard drug (5-fluorouracil). The cytotoxic activity was measured by the Skehan et al. method (see Experimental).

Figure 7

Effect of conc. of 2b on HEPG2 line.

Figure 8

Effect of conc. of 14a on HEPG2 line.

Figure 9

Effect of conc. of 17 on HEPG2 line.

Figure 10

Effect of conc. of 5-fluorouracil (standard) on HEPG2 line.

Effect of conc. of 2b on n class="Chemical">pan class="CellLine">HEPG2 line.

Effect of conc. of n class="Chemical">pan class="Species">14a on cal">pan class="CellLine">HEPG2 line.

Effect of conc. of 17 on n class="Chemical">pan class="CellLine">HEPG2 line.

Effect of conc. of n class="Chemical">pan class="Chemical">5-fluorouracil (standard) on cal">pan class="CellLine">HEPG2 line.

The results of biological screening allow the following assumptions about the structure activity relationships (n class="Chemical">pan class="Species">SAR) of these compounds:

The presence of nitrogenous fused heterocycles at position 3 of the main pan class="Chemical">pyrazole moiety, linked directly or through carbonyl group, with multn class="Species">cal">pan class="Chemical">icenters for hydrogen accepting properties are essential for activity where it can intercalate within the DNA strands.

The pan class="Chemical">3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b are essential for antical">pan class="Disease">tumor activity.

2.2. Antimicrobial Activity

The newly synthesized products 2a, 2b, 5b, 5c, 8b, 9b, 12b, pan class="Species">14a, 21a and 21b were tested for their antimicrobial activities using four species of fungi, namely cal">pan class="Species">Aspergillus fumigatus AF, Penicillium italicum PI, Syncephalastrum racemosum SR and Candida albicans CA, in addition to four bacterial species, namely Staphylococcus aureus SA, Pesudomonas aeruginosa PA, Bacillus subtilis BS and Escherichia coli EC. The organisms were tested against the activity of solutions of three different concentrations [5 mg/mL, 2.5 mg/mL, 1.25 mg/mL] of each compound and using inhibition zone diameter (IZD) in mm as criterion for the antimicrobial activity. The fungicide terbinafine and the bactericide chloramphenicol were used as references to evaluate the potency of the tested compounds under the same conditions. The results, depicted in Table 1, Table 2, Table 3, revealed that compounds 2a and 5c exhibited high degree of inhibition against SA, and BS. Compounds 9b, 12b, 14a, 21a and 21b have high inhibition effects against AF, PI, SR and SA. These compounds also exhibited moderate inhibition effect against CA and BS. All the tested compounds were reflecting no inhibition of growth against PA and EC.

Table 1

Antimicrobial activity of products 2a, 2b, 5b, and 5c.

(Sample)	2a (mg/mL)			2b (mg/mL)			5b (mg/mL)			5c (mg/mL)			Standard*
Tested Microorganism	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25
Aspergillus fumigatus (AF)	9	5	0	8	0	0	7	0	0	6	4	0	24	18	11
Penicillium italicum (PI)	7	3	0	6	0	0	5	0	0	5	3	0	19	9	4
Syncephalastrum racemosum (SR)	12	7	3	12	9	5	14	9	0	11	9	0	21	13	9
Candida albicans (CA)	9	6	3	7	4	0	9	4	0	12	9	5	19	10	6
Staphylococcus aureus (SA)	11	7	4	11	8	5	14	8	5	11	8	5	15	6	4
Pesudomonas aeruginosa (PA)	0	0	0	0	0	0	0	0	0	0	0	0	11	5	0
Bacillus subtilis (BS)	15	8	6	12	7	4	14	9	4	18	13	9	22	18	11
Escherichia coli (EC)	0	0	0	0	0	0	0	0	0	0	0	0	27	20	13

*Chloramphenicol was used as a standard antibacterial agent while, Terbinafine was used as a standard antifungal agent.

Table 2

Antimicrobial activity of products 8b, 9b, 12b, and 14a.

(Sample)	8b (mg/mL)			9b (mg/mL)			12b (mg/mL)			14a (mg/mL)			Standard*
Tested Microorganism	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25
Aspergillus fumigatus (AF)	9	7	3	22	14	9	18	11	6	16	7	3	24	18	11
Penicillium italicum (PI)	10	6	3	14	6	3	13	6	4	0	0	0	19	9	4
Syncephalastrum racemosum (SR)	9	7	4	19	12	8	16	9	6	18	12	7	21	13	9
Candida albicans (CA)	10	8	4	9	6	2	10	7	3	9	6	2	19	10	6
Staphylococcus aureus (SA)	12	7	4	12	8	5	13	8	5	11	8	5	15	6	4
Pesudomonas aeruginosa (PA)	0	0	0	0	0	0	0	0	0	0	0	0	11	5	0
Bacillus subtilis (BS)	16	9	5	15	8	5	14	10	7	10	8	4	22	18	11
Escherichia coli (EC)	0	0	0	0	0	0	0	0	0	0	0	0	27	20	13

*Chloramphenicol was used as a standard antibacterial agent while, Terbinafine was used as a standard antifungal agent.

Table 3

Antimicrobial activity of products 21a and 21b.

(Sample)	21a (mg/mL)			21b (mg/mL)			Standard*
Tested Microorganism	5	2.5	1.25	5	2.5	1.25	5	2.5	1.25
Aspergillus fumigatus (AF)	20	11	6	19	14	9	24	18	11
Penicillium italicum (PI)	17	6	4	13	6	3	19	9	4
Syncephalastrum racemosum (SR)	15	8	6	17	12	8	21	13	9
Candida albicans (CA)	10	7	3	9	6	2	19	10	6
Staphylococcus aureus (SA)	13	7	5	10	8	5	15	6	4
Pesudomonas aeruginosa (PA)	0	0	0	0	0	0	11	5	0
Bacillus subtilis (BS)	12	9	7	13	10	8	22	18	11
Escherichia coli (EC)	0	0	0	0	0	0	27	20	13

*Chloramphenicol was used as a standard antibacterial agent while, Terbinafine was used as a standard antifungal agent.

*pan class="Chemical">Chloramphenicol was used as a standard antibacterial agent while, n class="Species">cal">pan class="Chemical">Terbinafine was used as a standard antifungal agent.

Antimicrobial activity of products 8b, 9b, 12b, and pan class="Species">14a.

*pan class="Chemical">Chloramphenicol was used as a standard antibacterial agent while, n class="Species">cal">pan class="Chemical">Terbinafine was used as a standard antifungal agent.

*pan class="Chemical">Chloramphenicol was used as a standard antibacterial agent while, n class="Species">cal">pan class="Chemical">Terbinafine was used as a standard antifungal agent.

3. Experimental

3.1. General

All melting points were determined on an electrothermal Gallenkamp apparatus and are uncorrected. Solvents were generally distilled and dried by standard literature procedures prior to use. The IR spectra were measured on a Pye-Unicam SP300 instrument in pan class="Chemical">potassium bromide discs. The 1H NMR spectra were recorded on a Varian Mercury VXR-300 spectrometer (300 MHz) and the chemical shifts were related to that of the solvent cal">pan class="Chemical">DMSO-d6. The mass spectra were recorded on a GCMS-Q1000-EX Shimadzu and GCMS 5988-A HP spectrometers, the ionizing voltage was 70 eV. Elemental analyses were carried out by the Microanalytical Center of Cairo University, Giza, Egypt. Antitumor activity was evaluated by the National Institute of Cancer, Biology Department, Cairo University, Egypt. Antimicrobial activity was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. 3-Acetyl-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 1a,b [26] and hydrazonoyl halides [38,39,40,41,42,43] 18a-c were prepared following literature methods.

3.2. Synthesis of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b

A mixture of pan class="Chemical">3-acetyl-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles (1a or 1b) (0.01 mol) and cal">pan class="Chemical">dimethyl-formamide dimethylacetal (DMF-DMA) (5 mL) was refluxed for 10 hours. After cooling, methanol was added and the solid product was collected by filtration and crystallized from ethanol. The physical constants and the spectral data are shown below.

pan class="Chemin class="Species">cal">3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-phenyl-1H-pyrazole (2a). Yellow crystals, (2.89 g, 80%), m.p. 132–134 °C; IR (KBr) υ = 1,642 (CO) cm−1; 1H-NMR (CDCl3) δ = 2.82 (s, 3H, CH3), 3.00 (s, 3H, CH3), 5.88 (d, 1H, J = 13 Hz, CH=), 7.27 (d, 2H, J = 8 Hz, Ar-H), 7.67 (d, 1H, J = 13 Hz, CH=), 7.35–7.68 (m, 5H, Ar-H), 8.05 (d, 2H, J = 8 Hz, Ar-H), 8.90 (s, 1H, cal">pan class="Chemical">pyrazole-H-5) ppm; MS, m/z (%) 362 (M+, 25), 292 (30), 264 (20), 122 (15), 98 (40), 77 (100), 70 (40). Anal. Calcd. for C20H18N4O3 (362.14): C, 66.29; H, 5.01; N, 15.46. Found: C, 66.18; H, 4.93; N, 15.58%.

pan class="Chemin class="Species">cal">3-[E-3-(N,N-dimethylamino)acryloyl]-1-(4-methylphenyl)-4-(4-nitrophenyl)-1H-pyrazole (2b). Yellow crystals, (3.31 g, 88%), m.p. 148–150 °C; IR (KBr) υ = 1647 (CO) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.37 (s, 3H, Ar-CH3), 2.89 (s, 3H, CH3), 3.13 (s, 3H, CH3), 5.88 (d, 1H, J = 13 Hz, CH=), 7.35 (d, 2H, J = 8 Hz, Ar-H), 7.66 (d, 1H, J = 13 Hz, CH=), 7.82 (d, 2H, J = 8 Hz, Ar-H), 7.91 (d, 2H, J = 8 Hz, Ar-H), 8.21 (d, 2H, J = 8 Hz, Ar-H), 8.92 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 376 (M+, 25), 306 (40), 278 (20), 98 (40), 92 (85), 77 (100), 70 (40). Anal. Calcd. for C21H20N4O3 (376.15): C, 67.01; H, 5.36; N, 14.88. Found: C, 67.12; H, 5.23; N, 14.71%.

3.3. Reactions of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b with Active Methylene Compounds

To a solution of 2a or 2b (1 mmol) and pan class="Chemical">2,4-pentanedione (3a) or cal">pan class="Chemical">ethyl 3-oxobutanoate (3b) (1 mmol) in acetic acid (20 mL) was added ammonium acetate (0.156 g, 2 mmol). The reaction mixture was heated under reflux for 5 hours. After cooling, the reaction mixture was poured onto ice and the solid product was collected by filtration and crystallized from an ethanol/dioxane mixture (1:1). The physical constants, together with the spectral data for products 5a-d, are shown below.

pan class="Chemical">3-Acetyl-2-methyl-6-[4-(4-nitrophenyl)-1-phenyl-1H-pyrazol-3-yl]pyridine (5a). Yellow crystals, (0.34 g, 85%), m.p. 318–320 °C; IR (KBr) υ = 1,691 (CO) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.32 (s, 3H, CH3), 2.42 (s, 3H, COCH3), 7.35 (d, 2H, J = 8 Hz, Ar-H), 7.55 (d, 1H, J = 8 Hz, pyridyl H-4), 7.67 (d, 1H, J = 8 Hz, pyridyl H-5), 7.71–8.20 (m, 5H, Ar-H), 8.26 (d, 2H, J = 8 Hz, Ar-H), 9.01 (s, 1H, pyrazole-H-5) ppm; MS, m/z (%) 398 (M+, 60), 355 (30), 122 (25), 77 (100). Anal. Calcd. for C23H18N4O3 (398.14): C, 69.34; H, 4.55; N, 14.06. Found: C, 69.27; H, 4.68; N, 14.11%.

pan class="Chemical">3-Acetyl-2-methyl-6-[4-(4-nitrophenyl)-1-(4-methylphenyl)-1H-pyrazol-3-yl]pyridine (5b). Yellow crystals, (0.35 g, 85%), m.p. 322–325 °C; IR (KBr) υ = 1,692 (CO) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.32 (s, 3H, CH3), 2.38 (s, 3H, Ar-CH3), 2.44 (s, 3H, COCH3), 7.37 (d, 2H, J = 8 Hz, Ar-H), 7.53 (d, 1H, J = 8 Hz, pyridyl H-4), 7.61 (d, 1H, J = 8 Hz, pyridyl H-5), 7.76–8.26 (m, 4H, Ar-H), 8.29 (d, 2H, J = 8 Hz, Ar-H), 9.11 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 412 (M+, 75), 369 (30), 122 (25), 91 (50), 77 (100). Anal. Calcd. for C24H20N4O3 (412.15): C, 69.89; H, 4.89; N, 13.58. Found: C, 69.77; H, 4.78; N, 13.41%.

pan class="Chemical">Ethyl 2-methyl-6-[4-(4-nitrophenyl)-1-phenyl-1H-pyrazol-3-yl]nicotinate (5c). Pale yellow crystals, (0.35 g, 82%), m.p. 180–182 °C; IR (KBr) υ = 1,706 (CO) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 1.31 (t, 3H, J = 7 Hz, CH3), 2.38 (s, 3H, CH3), 4.35 (q, 2H, J = 7 Hz, CH2), 7.36 (d, 2H, J = 8 Hz, Ar-H), 7.52 (d, 1H, J = 8 Hz, pyridyl H-4), 7.62 (d, 1H, J = 8 Hz, pyridyl H-5), 7.81-8.20 (m, 5H, Ar-H), 8.28 (d, 2H, J = 8 Hz, Ar-H), 8.97 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 428 (M+, 60), 355 (30), 122 (25), 77 (100). Anal. Calcd. for C24H20N4O4 (428.15): C, 67.28; H, 4.71; N, 13.08. Found: C, 67.19; H, 4.62; N, 13.16%.

pan class="Chemical">Ethyl 2-methyl-6-[4-(4-nitrophenyl)-1-(4-methylphenyl)-1H-pyrazol-3-yl]nicotinate (5d). Pale yellow crystals, (0.37 g, 85%), m.p. 186–188 °C; IR (KBr) υ = 1,709 (CO) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 1.33 (t, 3H, J = 7 Hz, CH3), 2.38 (s, 3H, CH3), 2.41 (s, 3H, Ar-CH3), 4.39 (q, 2H, J = 7 Hz, CH2), 7.38 (d, 2H, J = 8 Hz, Ar-H), 7.51 (d, 1H, J = 8 Hz, pyridyl H-4), 7.59 (d, 1H, J = 8 Hz, pyridyl H-5), 7.72–8.10 (m, 4H, Ar-H), 8.29 (d, 2H, J = 8 Hz, Ar-H), 8.99 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 442 (M+, 50), 369 (30), 122 (25), 91 (100), 77 (60). Anal. Calcd. for C25H22N4O4 (442.16): C, 67.86; H, 5.01; N, 12.66. Found: C, 67.74; H, 4.92; N, 12.56%.

3.4. Reactions of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b with Hydrazine Hydrate

To a solution of the pan class="Chemical">enaminone (2a or 2b) (1 mmol) in cal">pan class="Chemical">ethanol (10 mL) was added hydrazine hydrate (1 mL) and the mixture was heated under reflux for 5 hours. The reaction mixture was acidified by HCl/ice mixture and the formed product was filtered and crystallized from ethanol.

pan class="Chemical">4-(4-Nitrophenyl)-1-phenyl-1H,1'H-3,3'-bipyrazole (8a). Yellow crystals, (0.30 g, 90%), m.p. 200–202 °C; IR (KBr) υ = 3,246 (NH) cm–1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 7.25 (d, 2H, J = 8 Hz, Ar-H), 7.35–7.97 (m, 5H, Ar-H), 7.53 (d, 1H, J = 7.5 Hz, pyrazole H-4), 7.58 (d, 1H, J = 7.5 Hz, pyrazole H-5), 8.23 (d, 2H, J = 8 Hz, Ar-H), 9.01 (s, 1H, pyrazole H-5), 13.00 (D2O-exchangeable) (s, 1H, NH) ppm; MS, m/z (%) 331 (M+, 60), 284 (20), 122 (25), 77 (100). Anal. Calcd. for C18H13N5O2 (331.11): C, 65.25; H, 3.95; N, 21.14. Found: C, 65.37; H, 3.88; N, 21.21%.

pan class="Chemical">4-(4-Nitrophenyl)-1-(4-methylphenyl)-1H,1'H-3,3'-bipyrazole (8b). Yellow crystals, (0.31 g, 90%), m.p. 172–174 °C; IR (KBr) υ = 3,226 (NH) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.36 (s, 3H, Ar-CH3), 7.23 (d, 2H, J = 8 Hz, Ar-H), 7.25–7.91 (m, 4H, Ar-H), 7.51 (d, 1H, J = 7.5 Hz, pyrazole H-4), 7.56 (d, 1H, J = 7.5 Hz, pyrazole H-5), 8.20 (d, 2H, J = 8 Hz, Ar-H), 9.00 (s, 1H, pyrazole H-5), 12.97 (D2O-exchangeable) (s, 1H, NH) ppm; MS, m/z (%) 345 (M+, 70), 299 (20), 122 (25), 91 (100), 77 (80). Anal. Calcd. for C19H15N5O2 (345.12): C, 66.08; H, 4.38; N, 20.28. Found: C, 66.12; H, 4.46; N, 20.18%.

3.5. Reactions of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b with Hydroxylamine Hydrochloride

pan class="Chemical">Hydroxylamine hydrochloride (0.07 g, 1 mmol) was added to a mixture of n class="Species">cal">pan class="Chemical">enaminone 2a or 2b (1 mmol) and anhydrous potassium carbonate (0.5 g) in absolute ethanol (20 mL). The mixture was heated under reflux for 5 hours and poured onto water. The solid product was filtered and crystallized from ethanol.

pan class="Chemical">3-[4-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-3-yl]isoxazole (9a). Yellow crystals, (0.25 g, 75%), m.p. 160–162 °C; IR (KBr) υ = 1,600 (C=N), cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 6.78 (d, 1H, J = 5 Hz, isoxazole H-4), 7.29 (d, 2H, J = 8 Hz, Ar-H), 7.36–8.21 (m, 5H, Ar-H), 8.48 (d, 2H, J = 8 Hz, Ar-H), 8.72 (d, 1H, J = 5 Hz, isoxazole H-5), 9.05 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 332 (M+, 60), 286 (20), 122 (25), 77 (100). Anal. Calcd. for C18H12N4O3 (332.09): C, 65.06; H, 3.64; N, 16.86. Found: C, 65.17; H, 3.58; N, 16.71%.

pan class="Chemical">3-[4-(4-Nitrophenyl)-1-(4-methylphenyl)-1H-pyrazol-3-yl]isoxazole (9b). Yellow crystals, (0.26 g, 75%), m.p. 170–172 °C; IR (KBr) υ = 1,601 (C=N), cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.37 (s, 3H, Ar-CH3), 6.78 (d, 1H, J = 5 Hz, isoxazole H-4), 7.35 (d, 2H, J = 8 Hz, Ar-H), 7.39–8.32 (m, 4H, Ar-H), 8.68 (d, 2H, J = 8 Hz, Ar-H), 8.79 (d, 1H, J = 5 Hz, isoxazole H-5), 9.04 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 346 (M+, 50), 300 (20), 122 (25), 91 (100), 77 (60). Anal. Calcd. for C19H14N4O3 (346.11): C, 65.89; H, 4.07; N, 16.18. Found: C, 65.77; H, 3.98; N, 16.11%.

3.6. Reactions of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b with 3-amino-1H-[1,2,4]triazole

A mixture of pan class="Chemical">enaminone 2a or 2b (1 mmol) and n class="Species">cal">pan class="Chemical">3-amino-1H-[1,2,4]triazole (0.085 g, 1 mmol), in glacial acetic acid (20 mL), was refluxed for 5 hours. The solid that formed was filtered off, and crystallized from dioxane to afford compounds 12a,b.

pan class="Chemical">5-[4-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-3-yl][1,2,4]triazolo[4,3-a]pyrimidine (12a). Yellow crystals, (0.32 g, 85%), m.p. 290–292 °C; IR (KBr) υ = 1,596 (C=N) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 7.45 (d, 2H, J = 8 Hz, Ar-H), 7.59–8.01 (m, 5H, Ar-H), 7.71 (d, 1H, J = 5 Hz, pyrimidine H-5), 8.11 (d, 2H, J = 8 Hz, Ar-H), 8.47 (s, 1H, triazole H-5), 9.01 (s, 1H, pyrazole H-5), 9.34 (d, 1H, J = 5 Hz, pyrimidine H-4) ppm; MS, m/z (%) 383 (M+, 50), 337 (40), 122 (25), 77 (100). Anal. Calcd. for C20H13N7O2 (383.11): C, 62.66; H, 3.42; N, 25.58. Found: C, 62.77; H, 3.58; N, 25.71%.

pan class="Chemin class="Species">cal">5-[4-(4-Nitrophenyl)-1-(4-methylphenyl)-1H-pyrazol-3-yl][1,2,4]triazolo[4,3-a]pyrimidine (12b). Yellow crystals, (0.34 g, 85%), m.p. 310–312 °C; IR (KBr) υ = 1,598 (C=N) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.41 (s, 3H, Ar-CH3), 7.37 (d, 2H, J = 8 Hz, Ar-H), 7.49–8.01 (m, 4H, Ar-H), 7.74 (d, 1H, J = 5 Hz, pyrimidine H-5), 8.16 (d, 2H, J = 8 Hz, Ar-H), 8.49 (s, 1H, triazole H-5), 9.03 (s, 1H, pyrazole H-5), 9.36 (d, 1H, J = 5 Hz, pyrimidine H-4) ppm; MS, m/z (%) 397 (M+, 50), 351 (40), 122 (25), 91 (70), 77 (100). Anal. Calcd. for C21H15N7O2 (397.13): C, 63.47; H, 3.80; N, 24.67. Found: C, 63.58; H, 3.62; N, 24.77%.

3.7. Coupling of 3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 2a,b with diazonium salt of 3-amino-1H-[1,2,4]triazole

To a cold solution of pan class="Chemical">enaminone 2a or 2b (1 mmol) in n class="Species">cal">pan class="Chemical">pyridine (25 mL) was added the heterocyclic diazonium salt [prepared by diazotizing 3-amino-1H-[1,2,4]triazole (0.085 g, 1 mmol) dissolved in concentrated nitric acid (2 mL) with a solution of sodium nitrite (0.07 g, 1 mmol) in water (2 mL)]. After complete addition of the diazonium salt, the reaction mixture was stirred for a further 30 min in an ice bath, and then poured onto ice/HCl mixture. The solid precipitated was filtered off, washed with water, dried and crystallized from ethanol/dioxane mixture to give the respective products 14a and 14b.

pan class="Chemical">[4-(4-Nitrophenyl)-1-phenyl-1H-3-pyrazolyl]carbonyl[1,2,4]triazolo[3,4-c][1,2,4]triazine (cal">pan class="Species">14a). Pale yellow crystals, (0.32 g, 80%), m.p. 290–292 °C; IR (KBr) υ = 1,662 (CO) cm−1; 1H-NMR (DMSO-d6) δ = 7.43 (d, 2H, J = 8 Hz, Ar-H), 7.56–7.96 (m, 5H, Ar-H), 7.77 (s, 1H, triazine H-5), 8.22 (d, 2H, J = 8 Hz, Ar-H), 8.48 (s, 1H, triazole H-5), 9.17 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 412 (M+, 50), 292 (20), 122 (50), 77 (100). Anal. Calcd. for C20H12N8O3 (412.10): C, 58.25; H, 2.93; N, 27.17. Found: C, 58.37; H, 3.02; N, 27.31%.

[4-(4-Nitrophenyl)-1-(4-methylphenyl)-n class="Chemical">1H-3-pyrazolyl]{[1,2,4]triazolo[3,4pan class="Chemical">-c][1,2,4]triazin-6-yl}-methanone (14b). Pale yellow crystals, (0.34 g, 80%), m.p. 198–200 °C; IR (KBr) υ = 1,664 (CO) cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 2.39 (s, 3H, Ar-CH3), 7.46 (d, 2H, J = 8 Hz, Ar-H), 7.51–7.99 (m, 4H, Ar-H), 7.78 (s, 1H, triazine H-5), 8.26 (d, 2H, J = 8 Hz, Ar-H), 8.49 (s, 1H, triazole H-5), 9.13 (s, 1H, pyrazole H-5) ppm; MS, m/z (%) 426 (M+, 50), 306 (20), 148 (60), 122 (50), 77 (100). Anal. Calcd. for C21H14N8O3 (426.12): C, 59.15; H, 3.31; N, 26.28. Found: C, 59.39; H, 3.22; N, 26.38%.

3.8. Synthesis of 5-[4-(4-nitrophenyl)-1-phenyl-1H-pyrazol-3-yl]-2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (17)

A mixture of pan class="Chemin class="Species">cal">3-[E-3-(N,N-dimethylamino)acryloyl]-4-(4-nitrophenyl)-1-phenyl-1H-pyrazole (2a) (1.81 g, 5 mmol) and cal">pan class="Chemical">6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (15, 0.715 g, 5 mmol) in acetic acid (20 mL) was refluxed for 6 hours. The reaction mixture was cooled and diluted with methanol and the solid product was collected by filtration and recrystallized from dioxane to give 17. Yellow crystals (0.35 g, 80%), m.p. 310–313 °C; IR (KBr) υ = 3,261, 3,245 (2 NH), 1,677 (CO), cm−1; 1H-NMR (DMSO-d6) δ = 7.42 (d, 2H, J = 8 Hz, Ar-H), 7.49–8.20 (m, 5H, Ar-H), 8.24 (d, 2H, J = 8 Hz, Ar-H), 8.29 (d, 1H, J = 7 Hz, pyridine-H), 8.48 (d, 1H, J = 7 Hz, pyridine-H), 9.05 (s, 1H, pyrazole H-5), 12.62 (s, 1H, NH), 13.14 (s, 1H, NH) ppm; MS, m/z (%) 442 (M+, 40), 396 (20), 122 (40), 77 (100). Anal. Calcd. for C22H14N6O3S (442.08): C, 59.72; H, 3.19; N, 18.99; S, 7.25. Found: C, 59.81; H, 3.14; N, 19.04; S, 7.20%.

3.9. Synthesis of pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one derivatives 21a-c

To a mixture of equimolar amounts of 17 and the appropriate pan class="Chemical">hydrazonoyl chlorides 18an class="Species">cal">pan class="Chemical">-c (1 mmol) in dioxane (15 mL) was added triethylamine (0.14 mL, 1 mmol). The reaction mixture was refluxed until all of the starting materials have disappeared and hydrogen sulfide gas ceased to evolve (6 hours, monitored by TLC). The solvent was evaporated and the residue was triturated with methanol. The solid that formed was filtered and crystallized from methanol/dioxane mixture to give compounds 21a-c.

3-Acetyl-6-[4-(4-nitrophenyl)-1-phenyl-1H-pyrazol-3-yl]-1-phenyl-1,5-dihydropyrido[2,3-d][1,2,4] triazolo[4,3-a]pyrimidin-5-one (21a). Yellow crystals, (0.45 g, 80%), m.p. 280–282 °C; IR (KBr) υ = 1,707, 1,650 (2 CO), cm−1; 1H-NMR (pan class="Chemical">DMSO-d6) δ = 2.84 (s, 3H, COCH3), 7.26 (d, 2H, J = 8 Hz, Ar-H), 7.39–7.85 (m, 10H, Ar-H), 8.03 (d, 1H, J = 7 Hz, cal">pan class="Chemical">pyridine-H), 8.27 (d, 2H, J = 8 Hz, Ar-H), 8.69 (d, 1H, J = 7 Hz, pyridine-H), 9.05 (s, 1H, pyrazole H-5) ppm; 13C-NMR (DMSO-d6) δ = 31.3, 119.8, 121.7, 122.4, 123.3, 124.5, 125.3, 127.4, 128.5, 129.1, 129.8, 131.2, 139.4, 142.5, 143.9, 146.8, 147.8, 148.1, 148.8, 152.1, 153.8, 155.3, 159.5, 164.0, 176 ppm; MS, m/z (%) 568 (M+, 25), 525 (20), 497 (40), 122 (30), 77 (100). Anal. Calcd. for C31H20N8O4 (568.16): C, 65.49; H, 3.55; N, 19.71. Found: C, 65.34; H, 3.42; N, 19.64%.

Ethyl 5-oxo-6-[4-(4-nitrophenyl)-1-phenyl-1H-pyrazol-3-yl]-1-phenyl-1,5-dihydropyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidine-3pan class="Chemical">-carboxylate (21b). Yellow crystals, (0.47 g, 80%), m.p. 250–253 °C; IR (KBr) υ = 1,719, 1,645 (2 CO), cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 1.45 (t, J = 7 Hz, 3H, CH3,), 4.57 (q, J = 7 Hz, 2H, CH2,), 7.26 (d, 2H, J = 8 Hz, Ar-H), 7.27–7.81 (m, 10H, Ar-H), 8.16 (d, 1H, J = 7 Hz, pyridine-H), 8.21 (d, 2H, J = 8 Hz, Ar-H), 8.62 (d, 1H, J = 7 Hz, pyridine-H), 9.07 (s, 1H, pyrazole H-5) ppm; 13C-NMR (DMSO-d6) δ = 31.6, 35.8, 118.9, 120.7, 122.4, 123.3, 124.6, 125.7, 127.2, 128.5, 129.3, 129.9, 131.2, 139.4, 142.5, 143.7, 146.8, 147.9, 148.2, 148.8, 152.1, 153.8, 155.3, 159.5, 163.4, 177 ppm; MS, m/z (%) 598 (M+, 25), 525 (40), 479 (40), 122 (30), 77 (100). Anal. Calcd. for C32H22N8O5 (598.17): C, 64.21; H, 3.70; N, 18.72. Found: C, 64.34; H, 3.62; N, 18.62%.

N3,1-Diphenyl-5-oxo-6-[4-(4-nitrophenyl)-1-phenyl-n class="Chemical">1H-pyrazol-3-yl]-1,5-dihydropyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidine-3pan class="Chemical">-carboxamide (21c). Yellow crystals, (0.48 g, 75%), m.p. 325−327 °C; IR (KBr) υ = 3,388 (NH), 1,697, 1,651 (2 CO), cm−1; 1H-NMR (cal">pan class="Chemical">DMSO-d6) δ = 7.23 (d, 2H, J = 8 Hz, Ar-H), 7.39–8.15 (m, 15H, Ar-H), 8.01 (d, 1H, J = 7 Hz, pyridine-H), 8.24 (d, 2H, J = 8 Hz, Ar-H), 8.62 (d, 1H, J = 7 Hz, pyridine-H), 9.01 (s, 1H, pyrazole H-5), 10.92 (s, 1H, NH) ppm; 13C-NMR (DMSO-d6) δ = 111.8, 119.8, 120.7, 121.4, 121.7, 122.4, 123.3, 124.5, 125.3, 125.9, 127.4, 128.5, 129.1, 129.8, 131.2, 139.4, 142.5, 143.9, 146.8, 147.8, 148.1, 148.8, 152.1, 153.8, 155.3, 159.5, 161.9, 168 ppm; MS, m/z (%) 645 (M+, 25), 525 (40), 122 (20), 77 (100). Anal. Calcd. for C36H23N9O4 (645.19): C, 66.97; H, 3.59; N, 19.53. Found: C, 66.84; H, 3.46; N, 19.61%.

3.10. Agar diffusion well method to determine the antimicrobial activity

The microorganism inoculums were uniformly spread using sterile cotton swab on a sterile Petri dish Malt extract pan class="Chemical">agar (for fungi) and nutrient n class="Species">cal">pan class="Chemical">agar (for bacteria). One hundred μL of each sample was added to each well (10 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24–48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, the microorganism's growth was observed. Inhibition of the bacterial and fungal growth were measured in mm. Tests were performed in triplicate [44].

3.11. Cytotoxic activity

The method applied is similar to that reported by Skehan et al. [45] using pan class="Chemical">Sulfo-Rhodamine-B stain (n class="Species">cal">pan class="Chemical">SRB). Cells were plated in 96-multiwill plate (104 cells/well) for 24 h before treatment with the tested compounds to allow attachment of cell to the wall of the plate. Different concentrations of the compound under test (0, 2.5, 5, and 10 µg/mL) were added to the cell monolayer in triplicate wells individual dose, monolayer cells were incubated with the compounds for 48 h at 37 °C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with SRB stain, excess stain was washed with acetic acid and attached stain was recovered with tris-EDTA buffer, color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified compound. The response parameter calculated was the IC50 value, which corresponds to the compound concentration causing 50% mortality in net cells (Figures 3-10).

4. Conclusions

In this study, synthetic routes to a wide variety of pan class="Chemical">azoles, fused n class="Species">cal">pan class="Chemical">azoles, and azines at the 3-position of N-arylpyrazole ring were developed using novel enaminones as building blocks. Moreover, some of the newly synthesized products were tested as antitumor and antimicrobial agents and the results obtained were promising.