In vitro cytotoxicity of hydrazones, pyrazoles, pyrazolo-pyrimidines, and pyrazolo-pyridine synthesized from 6-substituted 3-formylchromones

2.1 Synthesis

The experimental procedure led to the synthesis of pyrazoles 4a–f, hydrazones 5a–c and 6a–c, pyrazolo[1,5-a]pyrimidines 7a, 7b, and pyrazolo[3,4-b]pyridine 8. Derivatives 4d–f, 5b, 5c, 6a–c, and 8 are newly synthesized compounds. The products were obtained from the reaction between equimolar quantities of 3-formylchromone derivatives 1a–c, with the corresponding hydrazines 2a–c or aromatic amine 3 in anhydrous THF. The pyrazolo[3,4-b]pyridine 8 was obtained in two steps: the reaction of 1b with 3 produced a precipitate, which was then refluxed using AcOH as solvent in presence of I₂ (Scheme 1). All reactions were monitored by TLC and obtained in good yields (80–95 %). The new compounds were fully characterized using NMR, IR, and HRMS methods.

Scheme 1:

Reagents and conditions: (i) THF, reflux (1–2 h); (ii) Ac₂O/H₂SO₄, reflux (5 h); (iii) AcOH/I₂, reflux (3–4 h).

Acylated pyrazoles 4d–f showed IR bands at 3066–3068 (N=CH), 1755–1760 (O–C=O), 1622–1640 (C=O), 1583–1599 (C=C), and 1504–1505 (C=N) cm^–1. Their ¹H NMR data exhibited the same characteristic signals as already reported for pyrazoles 4a–c [34] and methyl resonances at δ=2.11–2.14 ppm.

Hydrazones 5a–c and 6a–c showed signals in the IR and NMR spectra, which were characteristic of a chromone ring. Compounds 5b and 5c had typical hydrazone IR bands at 3273–2379 (NH), 3060–3064 (N=CH), and 1516–1518 (C=N) cm^–1. ¹H NMR resonances of 5b were observed at δ=6.76 (dd, J = 2.2/8.4 Hz, 4′-H), 7.18 (d, J = 8.4 Hz, 3′-H), and 7.50 (d, J = 2.2 Hz, 6′-H) ppm corresponding to the 2,5-dichlorophenyl residue, while its 6-methyl group appeared at 2.47 ppm. The presence of the hydrazone moiety was established from signals at 8.12 (s, CH=N) and 8.60 (s, NH) ppm.

Proton signals of 5c were similar to those of 5b. The presence of fluorine at position 6 was evidenced by the multiplicity and chemical shift of the chromone protons at δ=7.89 (dd, J = 3.1/8.2 Hz, 5-H), 7.42 (ddd, J = 3.1/7.6/9.2 Hz, 7-H), and 7.52 (dd, J = 9.2/4.4 Hz, 8-H) ppm. The isomerization of 5b and 5c to pyrazole structures was discarded based on ¹³C NMR data, which showed the presence of characteristic 1,4-benzopyrone carbonyl carbons between δ=174.9–175.9 (C-4) ppm and vinylic CH=N resonances at 129.9–131.3 ppm.

Hydrazones 6a–c displayed IR bands at approximately 3265–3287 (NH), 3114–3118 (N=CH), 1515–1519 (C=N) and 1584, 1334 (N=O) cm^–1. Distinctive ¹H NMR signals for the 2,4-dinitrophenyl substituent were located at δ = 8.82–8.85 (H-3′), 8.31–8.33 (H-5′) and 8.08–8.13 (H-6′) ppm, while hydrazone-type protons were detected at 8.85–8.99 (CH=N) and 11.45–11.66 (NH) ppm. The methyl group signal of 6b was located at 2.47 ppm. Characteristic ¹³C NMR resonances for carbonyl and vinylic CH=N carbons at δ=174.6–175.1 and 125.2–125.8 ppm, excluded a pyrazole structure for 6a–c.

When chromones were substituted with either H (1a) or F (1c) at C-6, the reaction with 3 produced the expected pyrazolo[1,5-a]pyrimidines 7a and 7c. However, the reaction product between 6-methyl substituted chromone 1b with aromatic amine 3 yielded the pyrazolo[3,4-b]pyridine 8 after two steps (see general procedures). IR bands for 8 were present at 3400 (OH), 3203 (NH), 3036 (N=CH), 1634 (C=O), and 1479 (C=N) cm^–1. Its ¹H NMR spectra revealed signals of a p-hydroxy-methylphenyl residue at δ=2.53 (s, 5′-Me), 6.90 (d, J = 8.4 Hz, 3′-H), 7.22 (d, J = 2.2 Hz, 6′-H), 7.27 (dd, J = 2.2/8.4 Hz, 4′-H), and 10.09 (s, OH) ppm. The formation of the pyrazolo[1,5-a]pyrimidine structure was discarded due to the absence of the characteristic C-3 resonance between δ=93.8–96.9 ppm in the ¹³C NMR spectra. The presence of 3-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl signals at 2.26 (s, 3-Me), 8.52 (d, J = 1.8 Hz, 4-H), 8.78 (d, J = 1.8 Hz, 6-H) and 13.60 (s, NH) ppm, confirmed the formation of 8.

The formation of regioisomer 8 may arise from an imine intermediary (Scheme 2) that undergoes 1,4-addition at C-2 by attack of C-4′ from the pyrazole instead of the nitrogen atom N-2′. To the best of our knowledge, this is the first report regarding the formation of pyrazolo[3,4-b]pyridines by intramolecular attack of an sp² carbon atom.

Scheme 2:

Formation of pyrazolo[3,4-b]pyridine 8.

2.2 Brine shrimp lethality assay

The toxicity of all compounds was assayed against Artemia salina. The dose-dependent assays were carried out between 10 and 500 ppm using a negative control, Tween 80^®, at this concentration did not affect this bioassay. The IC₅₀ values of the compounds are presented in Table 1. The study showed that all tested compounds exhibited a toxic effect to the brine shrimp, with derivatives 6c, 7b, 6a and 5c [IC₅₀ = 83.0 ± 16, 94.7 ± 14, 95.3 ± 26 and 98.1 ± 14 μm respectively] among the more active ones. The observed toxicities are slightly lower than the cytotoxicity of Podophyllotoxin, a well known bioactive compound [IC₅₀ = 5.8 μm] [31].

The reasons for this toxic effect are not clear. However, the presence of fluorine increased activity, as could be observed for compounds 4c, 5c, 6c, and 7b. Such results are consistent with the general observation, which states that the presence of aromatic fluorine enhances the overall biological activity of organic compounds on a moderate scale [35, 36]. The presence of the hydroxyl group may also play a role in the toxicity, as evidenced by the increase in the IC₅₀ values for acylated derivatives 4d–f compared with 4a–c. More specific cytotoxic studies that use tumor cell lines MCF-7 (breast) and PC3 (prostate) are currently underway.

3.1 Chemistry

Melting points were determined using a Krüss-Optronic (San Diego, CA, USA) apparatus, and they were not corrected. ¹H NMR, ¹³C NMR, and DEPT-135 spectra were recorded on a JEOL Eclipse Plus 400 (400 MHz) (Peabody, MA, USA) and Bruker Avance500* (500 MHz) (Billerica, MA, USA) spectrometers, using CDCl₃ and [D₆]DMSO as solvents. IR spectra were obtained from KBr pellets with Shimadzu IR-408 (Columbia, MD, USA) equipment. The HRMS (70 eV) analyses were conducted in a JEOL JMS-AX505WA (Peabody, MA, USA) double focus mass spectrometer, using the electron impact (EI) method. Analytical thin-layer chromatography was carried out on 0.25 mm layers of silica gel PF₂₅₄ (Merck) (Kenilworth, NJ, USA).

Reagents 1a–c, 2a–c, and 3 were purchased from Aldrich (Milwaukee, WI, USA) as ‘synthetic grade’ and used without further purification. Compounds 4a–c, 5a, 7a, and 7b were prepared as described in the general procedures. Their physical constants and spectroscopic data were in agreement with those described in the literature [16, 28, 37].

3.2 General procedure for the synthesis of 4d–f

A portion of compound 4a–c (0.4 mmol) was dissolved in an excess of Ac₂O (4 mL), after which H₂SO₄ (18 m, 0.1 mL) was added and refluxed for 5 h. Once finished, 15 mL of iced water was added and extracted with CH₂Cl₂ (2 × 6 mL). The organic phase was washed with 15 % NaHCO₃ (aq.) (4 × 15 mL), and dried over Na₂SO₄, after which the solvent was evaporated under reduced pressure. The obtained solid was recrystallized from hexane-EtOAc (1:1) solvent mixture.

3.3 General procedure for the synthesis of 4a–c, 5a–c, and 6a–c

3-Formylchromone derivative 1a–c (1.0 mmol) was dissolved in hot anhydrous THF (4 mL) and a solution of hydrazine 2a–c (1.0 mmol) in THF (2 mL) was added slowly. The mixture was refluxed for 1–2 h; once cooled the solid was filtered, washed with water, and recrystallized from absolute EtOH.

3.4 General procedure for the synthesis of 7a and 7b

3-Formylchromone derivative 1a or 1c (1.0 mmol) was mixed with aminopyrazole 3 (1.0 mmol) and refluxed in 10 mL of anhydrous THF for 1 h. After cooling, the solid was filtered, washed repeatedly with hot THF, and recrystallized from EtOH to produce TLC pure compounds.

3.5 General procedure for the synthesis of 8

Equimolar quantities of 1b and 3 (1.0 mmol) were refluxed in anhydrous THF for 1–2 h, until the formation of a precipitate. Once separated from the solution, this precipitate was dissolved in AcOH (7 mL) in the presence of I₂ (1.0 mmol) and then refluxed for a period of 3–4 h. Upon completion of the TLC reaction, the mixture was poured into crushed ice and treated with NaHCO₃ and Na₂SO₃. The solid was filtered, washed with cold water, and dried.

3.6 BSLA

The assay was performed as described previously by Meyer [31] with some minor modifications. Brine shrimp eggs (Gulf Breeze^®) were hatched in artificial sea water prepared with commercial salt mixture (Instant Ocean^®), and then illuminated and oxygenated with an aquarium pump. After an incubation period of 48 h at 27 °C, 10 shrimps were transferred with a Pasteur pipette to three sample vials for each of the three doses (500, 100, 10 μg mL^–1), for a total of nine vials per sample. The compound samples (10 mg) were dissolved in CHCl₃ (5 mL). Aliquotes of testing solutions (1250, 250, or 25 μL for the 500, 100 and 10 ppm doses, respectively) were placed on vials (5 mL) and the solvent was evaporated. The residue was redissolved in 10 μL of Tween 80^®, after which artificial sea water (5 mL) was added. Survivors were counted and the percent deaths at each dose were determined. Control samples were included and assayed simultaneously. IC₅₀ values were calculated from 24 h counts using the probit analysis [38].