Utility of Pyrazolylchalcone Synthon to Synthesize Azolopyrimidines under Grindstone Technology

Abstract

A series of pyrazolyl-triazolo[1,5-a]pyrimidines, pyrazolyl-tetrazolo[1,5-a]pyrimidines, pyrazolyl-benzo[4,5]imidazo[1,2-a]pyrimidines and bis-azolopyrimidines were prepared by reaction of pyrazolyl-chalcones or its bis-pyrazolyl-chalcones with the appropriate heterocyclic amines as aminotriazole, aminotetrazole, 2-aminobenzimidazole and 4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-amine by grinding method. The newly synthesized compounds have been characterized on the basis of elemental analysis and spectral data (IR, ¹H- and ¹³C-NMR, Mass). Moreover, the newly synthesized products were screened for their in vitro antibacterial activities and the results showed that compounds 5f and 11d exhibited excellent activities compared with penicillin G and streptomycin as reference drugs.

α,β-Unsaturated ketones display a wide range of pharmacological properties, including antimicrobial, anticancer, anxiolytic, antiviral, and antitubercular activities.^1–6) On the other hand, pyrazolines constitute an interesting class of heterocycles due to their synthetic versatility and effective biological activities such as antiviral, analgesic, anti-inflammatory, antidepressant, anticonvulsant, antibacterial, antifungal, antitubercular, antiamoebic and anticancer activities.^7–14) Moreover, grinding method contributes to the development of a green strategy for the preparation of organic compounds in high yields with fewer waste products, simple, efficient, economical and environmentally benign compared to classical procedures.^15–20)

In continuation of our previous work to discover new bioactive heterocyclic compounds,^21–31) we aimed here to synthesize new pyrazolyl-azolopyrimidines via ecofriendly method to evaluate their antibacterial activity.

Results and Discussion

Chemistry

This article deals with the behavior of some heterocyclic amines toward α, β-unsaturated carbonyl compounds under ecofriendly conditions thus, when pyrazolyl chalcones 1a–f³²⁾ were allowed to react with 5-amino-1,2,4-triazole via grinding in a mortar at room temperature, in the presence of few drops of glacial acetic acid as catalyst for 10–20 min, it yielded triazolopyrimidines 5a–f (Chart 1).

Chart 1. Synthesis of Compounds 5a–h

Similarly, when pyrazolyl chalcones 1a–c were submitted to react with 5-amino-tetrazole under the same conditions afforded the tetrazolopyrimidines 5g and h, respectively (Chart 1).

To account for the formation of products 5, it was suggested that intermediate 3 is initially formed via aza-Michael 1,4-addition of the amino group of compound 2 to the electron-deficient carbon of the chalcones 1 which undergoes dehydrative cyclization to give the intermediate 4 which undergoes dehydrogenation leading to the more thermodynamically stable product 5 as shown in Chart 1.

Theoretically there are three paths for the reaction: Path (a) is a kinetically favor because the amino group at position 5 is sp³ hybridized and more nucleophilic than other nitrogen atoms (nucleophilicity is kinetically controlled), path (b) is not recommended because endocyclic amino group is sp²-hybridized and less nucleophilic and ruled out. Also path (c) is not acceptable because the carbonyl group of α,β-unsaturated compounds has weak electrophilic properties due to its conjugation with double bond, thus also can be ruled out. Thus compounds 8a–h are not obtained.

The elemental analysis together with the data derived from IR, ¹H-NMR and mass spectra (MS) are in agreement with the proposed structure 5. The ¹H-NMR spectra of compound 5a, taken as example, revealed the presence of three singlet signals at δ 2.60, 8.30 and 8.63 ppm, assigned for the methyl, triazole and pyrazole protons, in addition to the expected signals for the aryl and pyrimidine protons. The MS of each of products 5 revealed the presence of a molecular ion peak (m/z) which is consistent with the structure of the respective compound (see Experimental).

Also when heterocyclic chalcones 1a, c, d and f were allowed to react with 2-aminobenzimidazole (10) under the same conditions, afforded 4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-2-aryl/or heteroaryl benzo[4,5]imidazo[1,2-a]pyrimidine derivatives 11a–d, respectively, and not compounds 12a and b (Chart 2) as elucidated from spectroscopic tools (cf. Experimental).

Chart 2. Synthesis of Compounds 11a–d

In a similar manner, interaction of the heterocyclic chalcones 1a and c with 4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-amine (13) under ecofriendly conditions, yielded pyrido[2′,3′:3,4] pyrazolo[1,5-a]pyrimidines 14a and b, respectively, and not compounds 15a and b (Chart 3). The structure 15 was substantiated by elemental analysis and spectral data. For example, the ¹H-NMR spectra of products 15 revealed the presence of a characteristic singlet signal at δ 7.32 ppm, assigned for the pyridine proton, in addition to the expected signals of the methyl and aryl protons. The MS revealed a molecular ion peak at the correct values.

Chart 3. Synthesis of Compounds 14a and b

On the other hand, when the bis-chalcone 16³²⁾ reacted with the appropriate 3-amino-1,2,4-triazole (2a), 5-aminotetrazole (2b), 2-aminobenzimidazole (10) and 4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-amine (13), yielded the bis-aza Micheal adduct 17a, b, 19 and 21 (Chart 4). The other products 18a, b, 20 and 22 were discarded because the nucleophilicity of the exo-amino group is higher than the endo-imino group. The structure of the products 7a–g was deduced from their spectral data (IR, ¹H-NMR, and electrospray ionization (ESI)-MS) and elemental analyses, which are listed in Experimental.

Chart 4. Synthesis of Compounds 17a, b, 19 and 21

Antimicrobial Evaluation

The synthesized compounds were evaluated for their in vitro antibacterial activity at 5 mg/mL using agar well diffusion method against Staphylococcus aureus and Bacillis subtilis as examples of Gram-positive bacteria as well as Pseudomonas aeruginosa and Escherichia coli as examples of Gram-negative bacteria. The results of testing for antibacterial effects summarized in Table 1 showed that the new derivatives tested displayed variable in vitro antibacterial actions. In general, the chemical structure of the whole molecule, comprising the nature of the heterocyclic system as well as the type of the substituted function present in the heterocyclic ring structure, has a pronounced effect on activity (Fig. 1).

Table 1. In Vitro Antibacterial Activity of the Tested Compounds by Well Diffusion Agar Assay Expressed as Inhibition Zone Diameter (mm) in the Form of the Mean±Standard Deviation (S.D.)

Tested compounds	Gram-positive bacteria		Gram-negative bacteria
	Bacillus subtilis	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa
5a	16.1±0.3	12.1±0.3	8.1±0.3	11.1±0.3
5b	16.5±0.4	7.8±0.5	11.8±0.9	9.4±0.7
5c	17.7±0.2	11.4±0.4	9.2±0.3	7.3±0.6
5d	21.4±0.6	20.1±0.4	14.9±0.8	19.2±0.7
5e	19.1±0.4	18.5±0.3	16.3± 0.6	15.8±0.3
5f	25.3±0.2	23.6±0.6	20.1±0.5	20.7±0.4
5g	18.6±0.4	13.7±0.6	15.1±0.4	16.6±0.5
11a	16.7±0.3	16.4±0.3	10.4±0.3	8.1±0.3
11b	18.5±0.3	13.7±0.3	9.4±0.3	6.8±0.3
11c	18.5±0.5	20.4±0.6	15.5±0.3	18.4±0.2
11d	24.1±0.3	20.2±0.4	19.0±0.3	20.3±0.3
14a	18.3±0.5	12.7±0.3	9.3±0.5	9.0±0.8
14b	16.3±0.4	14.1±0.6	10.8±0.3	12.1±0.4
17a	14.6±0.3	13.2±0.4	6.1±0.4	6.7±0.6
17b	9.9±0.6	9.4±0.8	8.9±0.9	8.1±0.7
19	8.4±0.3	8.5±0.5	5.1±0.7	9.3±0.2
21	15.3±0.7	14.9±0.5	15.7±0.6	13.4±0.5
Penicillin G	26.2±0.3	24.6±0.3	—	—
Streptomycin	—	—	26.7±0.5	20.6±0.8

Fig. 1. The Most Active Two Heterocycles towards Bacteria

• From the screening results, it can be seen that:
  • • Compounds 5h and 11d showed excellent activity against Gram-positive bacteria and Gram-negative bacteria comparable to the reference drugs.
  • • All the tested compounds have more activities towards Gram-positive bacteria than Gram-negative bacteria.
  • • For Gram-positive bacteria, most of the tested compounds have more activities towards Bacillis subtilis than Staphylococcus aureus as Gram-positive bacteria than Gram-negative bacteria.
  • • For Gram-negative bacteria, most of the tested compounds have more activities towards Pseudomonas aeruginosa than Escherichia coli.
  • • Bis-derivatives 17a, b and 19 showed no activities against bacteria.
  • • Most of the tested compounds have moderate activities towards bacteria.

Experimental

Chemistry

All melting points (mp) 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 potassium bromide discs. ¹H-NMR (300 MHz), ¹³C-NMR (75 MHz) were run in deuterated dimethyl sulfoxide (DMSO-d₆). Chemical shifts were related to that of the solvent. The MS 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 at the Microanalytical Centre of Cairo University, Giza, Egypt. Antibacterial activity of the products was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.

General Method

A mixture of pyrazolyl-chalcone 1 (1 mmol) and the appropriate heterocyclic amine (2a, b or 10 or 13) (1 mmol) was grinded with catalytic drops of acetic acid, in a mortar at room temperature for 10–20 min (monitored through TLC). The reaction mixture was poured into water and the solid product was collected by filtration followed by washing with ethanol. The crude product was then recrystallized from the appropriate solvent to give pure products 5a–h, 11a–d, and 14a, b, respectively. Compounds 5a–h, 11a–d, and 14a, b with their physical constants and spectral data are depicted as shown below:

7-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (5a)

Yellow solid (78%); mp 260–262°C (AcOH); IR: v 1650 (C=N), 2963, 2999, 3060 (C–H) cm⁻¹; ¹H-NMR δ: 2.60 (s, 3H, CH₃), 7.44–7.88 (m, 11H, Ar-H and pyrimidine-H), 8.30 (1H, s, triazole-H), 8.63 (s, 1H, pyrazole-H); MS m/z (%): 352 (M⁺, 14), 288 (39), 185 (100), 77 (60). Anal. Calcd for C₂₁H₁₆N₆ (352.14): C, 71.58; H, 4.58; N, 23.85. Found: C, 71.46; H, 4.49; N, 23.76%.

7-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-5-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5b)

Yellow solid (79%); mp 272–274°C (dioxane); IR: v 1651 (C=N), 2933, 3000, 3061 (C–H) cm⁻¹; ¹H-NMR δ: 2.32 (s, 3H, CH₃), 2.61 (s, 3H, CH₃), 7.44–7.89 (m, 10H, Ar-H and pyrimidine-H), 8.27 (1H, s, triazole-H), 8.62 (s, 1H, pyrazole-H); ¹³C-NMR (DMSO-d₆) δ: 12.0, 18.4 (CH₃), 121.2, 122.1, 124.4, 125.3, 128.6, 128.7, 129.2, 130.2, 132.1, 134.6, 138.2, 141.6, 141.8, 143.3, 147.5, 154.0 (Ar-C); MS m/z (%): 368 (M⁺, 27), 288 (62), 185 (100), 77 (86). Anal. Calcd for C₂₂H₁₈N₆ (368.17): C, 72.11; H, 4.95; N, 22.94. Found: C, 72.04; H, 4.83; N, 22.71%.

5-(4-Chlorophenyl)-7-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (5c)

Yellow solid (82%); mp 286–288°C (N,N-dimethylformamide (DMF)); IR: v 1653 (C=N), 2929, 3060 (C–H) cm⁻¹; ¹H-NMR δ: 2.59 (s, 3H, CH₃), 7.51–7.97 (m, 10H, Ar-H and pyrimidine-H), 8.32 (1H, s, triazole-H), 8.64 (s, 1H, pyrazole-H); MS m/z (%): 388 (M⁺+2, 19), 386 (M⁺, 53), 185 (23), 188 (19), 77 (100). Anal. Calcd for C₂₁H₁₅ClN₆ (386.10): C, 65.20; H, 3.91; N, 21.72. Found: C, 65.15; H, 3.75; N, 21.57%.

7-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-5-(thiophen-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (5d)

Yellow solid (78%); mp 259–261°C (DMF); IR: v 1650 (C=N), 2961, 3093 (C–H) cm⁻¹; ¹H-NMR δ: 2.60 (s, 3H, CH₃), 7.21–7.84 (m, 9H, Ar-H and pyrimidine-H), 8.19 (1H, s, triazole-H), 8.64 (s, 1H, pyrazole-H); MS m/z (%): 358 (M⁺, 22), 185 (31), 118 (26), 77 (100). Anal. Calcd for C₁₉H₁₄N₆S (358.10): C, 63.67; H, 3.94; N, 23.45. Found: C, 63.52; H, 3.88; N, 23.32%.

5-(Furan-2-yl)-7-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (5e)

Yellow solid (70%); mp 272–274°C (DMF); IR: v 1652 (C=N), 2927, 3064 (C–H) cm⁻¹; ¹H-NMR δ: 2.58 (s, 3H, CH₃), 6.67–7.56 (m, 9H, Ar-H and pyrimidine-H), 7.89 (1H, s, triazole-H), 8.47 (s, 1H, pyrazole-H); ¹³C-NMR (DMSO-d₆) δ: 12.1 (CH₃), 112.9, 116.2, 121.0, 121.3, 123.3, 125.3, 128.4, 128.6, 129.2, 128.2, 141.4, 143.2, 145.7, 148.1, 151.0, 157.2 (Ar-C); MS m/z (%): 342 (M⁺, 47), 185 (29), 118 (33), 77 (100). Anal. Calcd for C₁₉H₁₄N₆O (342.12): C, 66.66; H, 4.12; N, 24.55. Found: C, 66.51; H, 4.04; N, 24.38%.

5-(1,3-Diphenyl-1H-pyrazol-4-yl)-7-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-4,7-dihydro[1,2,4]triazolo[1,5-a]pyrimidine (5f)

Yellow solid (76%); mp 250–252°C (DMF); IR: v 1652 (C=N), 2924, 2963, 3056 (C–H) cm⁻¹; ¹H-NMR δ: 2.58 (s, 3H, CH₃), 7.41–7.98 (m, 15H, Ar-H and pyrimidine-H), 8.42 (1H, s, triazole-H), 8.62, 9.32 (2s, 2H, 2pyrazole-H); MS m/z (%): 358 (M⁺, 22), 185 (31), 118 (26), 77 (100). Anal. Calcd for C₃₀H₂₂N₈ (494.20): C, 72.86; H, 4.48; N, 22.66. Found: C, 72.72; H, 4.39; N, 22.53%.

7-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine (5g)

Yellow solid (78%); mp 260–262°C (AcOH); IR: v 1651 (C=N), 2933, 2999, 3061 (C–H), 3433 (NH) cm⁻¹; ¹H-NMR δ: 2.60 (s, 3H, CH₃), 7.45–7.88 (m, 11H, Ar-H and pyrimidine-H), 8.82 (s, 1H, pyrazole-H); MS m/z (%): 353 (M⁺, 36), 326 (74), 288 (33), 185 (69), 77 (94), 58 (100). Anal. Calcd for C₂₀H₁₅N₇ (353.14): C, 67.98; H, 4.28; N, 27.75. Found: C, 67.85; H, 4.20; N, 27.66%.

5-(4-Chlorophenyl)-7-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine (5h)

Yellow solid (74%); mp 271–273°C (DMF); IR: v 1654 (C=N), 2971, 3061 (C–H), 3439 (NH) cm⁻¹; ¹H-NMR δ: 2.59 (s, 3H, CH₃), 7.51–7.92 (m, 10H, Ar-H and pyrimidine-H), 8.82 (s, 1H, pyrazole-H); MS m/z (%): 389 (M⁺+2, 4), 387 (M⁺, 15), 322 (31), 185 (44), 118 (21), 77 (100). Anal. Calcd for C₂₀H₁₄ClN₇ (387.10): C, 61.94; H, 3.64; N, 25.28. Found: C, 61.78; H, 3.53; N, 25.21%.

4-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylbenzo[4,5]imidazo[1,2-a]pyrimidine (11a)

Yellow solid (75%); mp 247–249°C (DMF); IR: v 1652 (C=N), 2925, 3065 (C–H) cm⁻¹; ¹H-NMR δ: 2.59 (s, 3H, CH₃), 7.51–7.93 (m, 15H, Ar-H and pyrimidine-H), 8.62 (s, 1H, pyrazole-H); MS m/z (%): 401 (M⁺, 33), 371 (64), 185 (89), 133 (25), 77 (100). Anal. Calcd for C₂₆H₁₉N₅ (401.16): C, 77.79; H, 4.77; N, 17.44. Found: C, 77.64; H, 4.69; N, 17.35%.

2-(4-Chlorophenyl)-4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine (11b)

Yellow solid (77%); mp 255–257°C (DMF); IR: v 1655 (C=N), 2924, 3062 (C–H) cm⁻¹; ¹H-NMR δ: 2.59 (s, 3H, CH₃), 7.51–7.92 (m, 14H, Ar-H and pyrimidine-H), 8.63 (s, 1H, pyrazole-H); MS m/z (%): 437 (M⁺+2, 5), 435 (M⁺, 16), 322 (64), 185 (97), 118 (22), 77 (100). Anal. Calcd for C₂₆H₁₈ClN₅ (435.13): C, 71.64; H, 4.16; N, 16.07. Found: C, 71.51; H, 4.08; N, 16.02%.

4-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-2-(thiophen-2-yl)-benzo[4,5]imidazo[1,2-a]pyrimidine (11c)

Yellow solid (73%); mp 241–243°C (AcOH); IR: v 1655 (C=N), 2925, 3092 (C–H) cm⁻¹; ¹H-NMR δ: 2.58 (s, 3H, CH₃), 7.17–7.80 (m, 13H, Ar-H and pyrimidine-H), 8.53 (s, 1H, pyrazole-H); MS m/z (%): 407 (M⁺, 14), 294 (31), 185 (100), 118 (31), 77 (81), 58 (92). Anal. Calcd for C₂₄H₁₇N₅S (407.12): C, 70.74; H, 4.21; N, 17.19. Found: C, 70.72; H, 4.09; N, 17.10%.

2-(1,3-Diphenyl-1H-pyrazol-4-yl)-4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (11d)

Yellow solid (79%); mp 283–285°C (dioxane); IR: v 1652 (C=N), 2924, 3059 (C–H) cm⁻¹; ¹H-NMR δ: 2.58 (s, 3H, CH₃), 7.41–7.96 (m, 20H, Ar-H and pyrimidine-H), 8.42 (s, 1H, pyrazole-H), 9.33 (s, 1H, pyrazole-H); MS m/z (%): 543 (M⁺, 19), 430 (24), 245 (50), 185 (72), 115 (18), 77 (100). Anal. Calcd for C₃₅H₂₅N₇ (543.22): C, 77.33; H, 4.64; N, 18.04. Found: C, 77.26; H, 4.52; N, 17.92%.

8,10-Dimethyl-4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-2-phenylpyrido[2′,3′:3,4]pyrazolo[1,5-a]pyrimidine (14a)

Yellow solid (82%); mp 260–262°C (DMF); IR: v 1650 (C=N), 2935, 2998, 3061 (C–H) cm⁻¹; ¹H-NMR δ: 2.59 (s, 3H, CH₃), 2.67 (s, 3H, CH₃), 2.85 (s, 3H, CH₃), 7.32 (s, 1H, pyridine-H), 7.43–7.92 (m, 11H, Ar-H and pyrimidine-H), 8.62 (s, 1H, pyrazole-H); MS m/z (%): 430 (M⁺, 13), 288 (62), 185 (100), 118 (26), 77 (61). Anal. Calcd for C₂₇H₂₂N₆ (430.19): C, 75.33; H, 5.15; N, 19.52. Found: C, 75.19; H, 5.11; N, 19.48%.

2-(4-Chlorophenyl)-8,10-dimethyl-4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)pyrido[2′,3′:3,4]pyrazolo[1,5-a]pyrimidine (14b)

Yellow solid (80%); mp 277–279°C (DMF); IR: v 1650 (C=N), 2919, 2998, 3064 (C–H) cm⁻¹; ¹H-NMR δ: 2.59 (s, 3H, CH₃), 2.73 (s, 3H, CH₃), 2.89 (s, 3H, CH₃), 7.32 (s, 1H, pyridine-H), 7.50–7.92 (m, 10H, Ar-H and pyrimidine-H), 8.62 (s, 1H, pyrazole-H); MS m/z (%): 466 (M⁺+2, 12), 464 (M⁺, 43), 232 (27), 185 (31), 118 (25), 77 (100). Anal. Calcd for C₂₇H₂₁ClN₆(464.15): C, 69.75; H, 4.55; N, 18.08. Found: C, 69.63; H, 4.47; N, 18.01%.

Reactions of Bis-Chalcone 16 with Heterocyclic Amines 2a, b, 10 and 13

A catalytic drops of acetic acid was added to a mixture of bis-chalcone 16 (0.498 g, 1 mmol) and the appropriate heterocyclic amine (2a, b or 10 or 13) (1 mmol) in a mortar. The reaction mixture was grinded at room temperature for 15–25 min, poured into water and the solid products were collected by filtration then washed with ethanol. The crude products were recrystallized from appropriate solvent to give pure products 17a, b, 19, 21, respectively.

1,4-Bis(7-(5-methyl-1-phenyl-1H-pyrazol-4-yl)[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)benzene (17a)

Yellow solid (70%); mp 241–243°C (DMF); IR: v 1648 (C=N), 2921, 3052 (C–H), cm⁻¹; ¹H-NMR δ: 2.72 (s, 6H, 2CH₃), 7.51–7.62 (m, 10H, Ar-H), 7.90 (s, 2H, 2 pyrimidine-H), 8.07 (s, 4H, Ar-H), 8.45 (s, 2H, 2 triazole-H), 8.66 (s, 2H, 2 pyrazole-H); MS m/z (%): 626 (M⁺, 14), 539 (27), 379 (63), 185 (100), 118 (30), 77 (82). Anal. Calcd for C₃₆H₂₆N₁₂ (626.24): C, 69.00; H, 4.18; N, 26.82. Found: C, 68.94; H, 4.11; N, 26.70%.

1,4-Bis(7-(5-methyl-1-phenyl-1H-pyrazol-4-yl)tetrazolo[1,5-a]pyrimidin-5-yl)benzene (17b)

Yellow solid (68%); mp 293–296°C (DMF); IR: v 1654 (C=N), 2926, 3056 (C–H), cm⁻¹; ¹H-NMR δ: 2.61 (s, 6H, 2CH₃), 7.51–7.68 (m, 10H, Ar-H), 7.73 (s, 2H, 2 pyrimidine-H), 7.98 (s, 4H, Ar-H), 8.08 (s, 2H, 2 triazole-H), 8.66 (s, 2H, 2 pyrazole-H); MS m/z (%): 628 (M⁺, 8), 539 (23), 498 (71), 185 (80), 118 (44), 77 (100). Anal. Calcd for C₃₄H₂₄N₁₄ (628.23): C, 64.96; H, 3.85; N, 31.19. Found: C, 64.85; H, 3.68; N, 31.08%.

1,4-Bis(4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)benzene (19)

Yellow solid (67%); mp 277–279°C (AcOH); IR: v 1650 (C=N), 2925, 2964, 3063 (C–H), cm⁻¹; ¹H-NMR δ: 2.61 (s, 6H, 2CH₃), 7.51–7.64 (m, 18H, Ar-H and 2 pyrimidine-H), 7.96 (s, 4H, Ar-H), 8.17 (s, 2H, 2 triazole-H), 8.65 (s, 2H, 2 pyrazole-H); MS m/z (%): 724 (M⁺, 12), 498 (28), 315 (47), 185 (71), 77 (83), 58 (100). Anal. Calcd for C₄₆H₃₂N₁₀ (724.28): C, 76.23; H, 4.45; N, 19.32. Found: C, 76.19; H, 4.37; N, 19.24%.

1,4-Bis(8,10-dimethyl-4-(5-methyl-1-phenyl-1H-pyrazol-4-yl)pyrido[2′,3′:3,4] Pyrazolo[1,5-a]pyrimidin-2-yl)benzene (21)

Yellow solid (74%); mp 294–296°C (DMF); IR: v 1642 (C=N), 2921, 3056 (C–H), cm⁻¹; ¹H-NMR δ: 2.60 (s, 6H, 2CH₃), 2.82 (s, 6H, 2CH₃), 2.88 (s, 6H, 2CH₃), 7.15 (s, 2H, 2 pyridine-H), 7.50–7.60 (m, 10H, Ar-H), 7.81 (s, 2H, 2 pyrimidine-H 8.19 (s, 4H, Ar-H), 8.69 (s, 2H, 2 pyrazole-H); MS m/z (%): 782 (M⁺, 6), 444 (25), 317 (52), 185 (100), 77 (78), 58 (52). Anal. Calcd for C₄₈H₃₈N₁₂ (782.33): C, 73.64; H, 4.89; N, 21.47. Found: C, 73.42; H, 4.83; N, 21.33%.

Antibacterial Activity Assay

The preliminary antimicrobial activity was investigated on a dozen of newly synthesized compounds in order to increase the selectivity of these derivatives towards test microorganisms. The antimicrobial profile was tested using a modified well diffusion method.^33,34) Briefly, 100 µL of the test bacteria was grown in 10 mL of fresh media Mueller–Hinton and Sabaroud agar (Oxoid, U.K.) until they reached a count of approximately 10⁸ cells/mL. A hundred microliter of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained and tested for susceptibility by well diffusion method. A hundred microliter of each sample (at 5 mg/mL) was added to each well (10 mm diameter holes cut in the agar gel). The plates were incubated for 24–48 h at 37°C. After incubation, the microorganism’s growth was observed. The plates were done in triplicate and the resulting inhibition zone diameters were measured in millimeters and used as criterion for the antimicrobial activity. The size of the clear zone is proportional to the inhibitory action of the compound under investigation. Penicillin G and streptomycin (Sigma-Aldrich, U.S.A.) were used as a positive control against Gram-positive and Gram-negative bacteria, respectively. Solvent control (DMSO) was included in every experiment as negative control.

Conclusion

In conclusion, we have reported a simple and efficient solvent-free grinding method for synthesis of new azolopyrimidine derivatives and its bis-derivatives in good yields. The synthesized compounds were evaluated for their in vitro antibacterial activity at 5 mg/mL using agar well diffusion method against a representative panel of pathogenic strains and the results indicated that compounds 5f and 11d showed excellent activity against bacteria.

Conflict of Interest

The authors declare no conflict of interest.

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