Synthesis of novel pyrazole derivatives using organophosphorus, stibine, and arsine reagents and their antitumor activities

2.1 Chemistry

When azidopyrazole 1 was treated with two molar equivalents of (cyanomethylene)triphenylphosphorane (2a) in dry THF at ambient temperature for 1 h, the triphenyl phosphoranylidenesuccinonitrile 8a was obtained (85% yield). Triphenylphosphane oxide (TPPO) was also isolated and identified (Scheme 1). Structure 8a is confirmed by a correct elemental analysis, IR, ¹H, ¹³C, ³¹P NMR, and mass spectra. Its IR spectrum revealed the presence of a strong band at 2182 cm⁻¹, which is ascribed to the N₃ absorption [13]. A possible explanation for the course of the formation of compound 8a is shown in Scheme 1. Azidopyrazole 1 reacted with P-ylide 2a to give the intermediate A with expulsion of TPPO. Moreover, intermediate A reacted with another molecule of the ylide 2a yielding intermediate B, which cyclized to intermediate C. Rearrangement of the latter gave the new ylide 8a (Scheme 1). Furthermore, when azidopyrazole 1 was allowed to react with (acetylmethylene)triphenylphosphorane (2b) in dry THF at room temperature, product 8b was separated as colorless crystals with 75% yield (Scheme 1). Elemental and mass spectral analysis of 8b led to an empirical formula C₁₄H₁₃N₅O. The IR spectrum displays absorption bands at ν = 1682 (C=O) and 1593 (cyclic C=N) cm⁻¹ but lacks the azido function group around at 2180 cm⁻¹ [13]. The ¹H NMR spectrum of 8b gave signals at δ = 1.96 (s, 3H, CH₃), at 2.64 ppm (s) for a methyl group attached to the triazole ring, at 7.34–7.54 ppm (m, 6H, H_arom, =CH), and a singlet at 9.66 ppm for the aldehydic proton. The ¹³C NMR spectrum of 8b added a good support for the proposed structure and revealed signals at 8.3 and 13.7 ppm for the two methyl groups, 136.3 and 136.6 ppm (2C, triazole), 123.2–133.2 ppm (aromatic CH), 117.4, 136.7, and 151.6 ppm (quaternary carbon atoms C-4, C-3, C-5 of pyrazole), and 183.4 ppm for a carbonyl group. The reaction presumably proceeds via 1,3-dipolar cycloaddition of the azide group of azidopyrazole 1 to the C=C bond of the ylide 2b followed by loss of TPPO from the cyclic intermediate D to give 1,2,3-triazole derivative 8b [14–16] (Scheme 1). The molecular structure of 8b was also confirmed by X-ray crystallographic analysis. An Ortep diagram of compound 8b is shown in Fig. 2. The crystal data and experimental parameters used for intensity data collection and the final results of the structure refinement are presented in Table 2 (cf. Experimental Section).

Scheme 1:

The behavior of azidopyrazole 1 toward stabilized phosphonium ylides 2a–c.

Fig. 2:

Ortep view of 8b with crystallographic atom numbering. Displacement parameters are drawn at the 30% probability level, H as spheres with arbitrary radii. Selected bond lengths (Å) and angles (deg): N1–N2 1.372(3), N3–N4 1.374(4), N4–N5 1.294(4); N1–N2–C6 120.2(2), N1–N2–C11 111.1(2), N3–N4–N5 106.8(3).

Similarly, the Wittig reaction of (dibromomethylene)triphenylphosphorane (2c) with azidopyrazole 1 afforded 4-(2,2-dibromovinyl)pyrazole 8c (Scheme 1). The IR spectrum of 8c revealed the presence of absorption bands at ν = 2125 (N₃) [13] and 626 (C–Br) [17] cm⁻¹. The structure of 8c was also deduced from its NMR (¹H, ¹³C) and mass spectra together with correct microanalyses (cf. Experimental Section).

Next, when azidopyrazole 1 was allowed to react with two molar equivalents of diethyl(cyanomethyl)phosphonate (3a) in an ethanolic sodium ethoxide solution at room temperature, products 9a and 9b were obtained (Scheme 2). The most important feature of structure 9a is the presence of a signal at δ = 23.03 (s) ppm in its ³¹P NMR spectrum and the presence of an IR band at 2114 cm⁻¹, which is ascribed to the N₃ absorption [13]. Moreover, the ¹H NMR spectrum of 9a revealed a triplet at δ = 1.32 ppm, which indicates CH₃ protons of ethyl group with J_HH = 13.4 Hz, singlet at 2.63 ppm (3H, CH₃), doublet at 2.72 ppm with ²J_HP = 22.89 Hz, doublet at 3.47 ppm with ³J_HP = 12.5 Hz (CHOH), and two quartet at 4.22 ppm (4H, ³J_HP = 12.5 Hz) for CH₂ protons of ethyl group.

Scheme 2:

The behavior of azidopyrazole 1 toward W-H reagents 3a,b.

A possible explanation for the course of the reaction of 1 with Wittig-Horner reagent 3a is shown in Scheme 3. The initial attack of the anion derived from phosphonic ester 3a on the most reactive center of 1 gave product 9avia intermediate E. The assigned product 9b presumably was formed via intermediate F. Under the influence of the base present in the reaction medium, elimination of N₂ was followed by hydrolysis F to give 9b and dialkyl phosphite (Scheme 2). The dialkyl phosphite was detected in the water layer by the development of a violet color on addition of 3,5-dinitrobenzoic acid [18]. The assigned cis configuration for 9b is supported by the chemical shift and coupling constant of the two protons CH=CHCN (J_HH = 7.6 Hz) (Scheme 2).

Scheme 3:

Formation mechanism of 9a,b.

The reaction of azidopyrazole 1 with 4-methoxybenzylphosphonate 3b was also investigated. We have found that the reaction of 1 with a molar equivalent of Wittig-Horner reagent 3b in the presence of sodium ethoxide in ethanol afforded the pure 9c (Scheme 2). The structural assignment for compound 9c was based upon elemental analysis and spectroscopic data (cf. Experimental Section). Product 9c can be obtained via intracyclization of azide 1. It has been reported that o-azido-carbaldehyde compounds can be intracyclized with good yield under the influence of a basic medium [19, 20].

The reaction of azidopyrazole 1 with diethyl phosphonate (diethyl hydrogen phosphite) (4) in dry toluene at room temperature gave 5-amino-pyrazole-4-carbaldehyde 10 with 75% yield (Scheme 4). The assignment of structure 10 was based on correct elemental analysis and molecular weight determination (MS), ¹H NMR, and IR comparison with an authentic specimen [21].

Scheme 4:

The behavior of azidopyrazole 1 toward diethylphosphonate 4.

A mechanism that accounts for the reaction of azidopyrazole 1 with diethyl phosphonate (4) is depicted in Scheme 4. The initial attack of the phosphorus lone pair at the azide group with expulsion of a nitrogen molecule gives intermediate G, which decomposes due to the unavoidable moisture to give the aminopyrazole 10 and diethylphosphite (Scheme 4) [21].

We have found that the reaction of trimethyl phosphite (5a) with azidopyrazole 1 in dry toluene proceeded at room temperature to give chromatographically pure adduct 11a (Scheme 5), which was separated as brown crystals (75% yield). The results of elemental analysis and molecular weight determination (MS) of this product correspond to C₁₄H₁₈N₃O₄P. The ³¹P NMR spectrum has one signal at δ = 23.2 ppm. The IR spectrum reveals the absence of an N₃ band [13] and the presence of both the (C=N) band at 1558 and C=O band at 1658 cm⁻¹. The structure of 11a has also been assigned on the basis of ¹H and ¹³C NMR spectra (cf. Experimental Section). Similarly, when 1 was reacted with 5b in dry toluene at room temperature, the phosphorane imine product 11b was obtained with 85% yield (Scheme 5). Structure elucidation of 11b was derived from its spectral data (cf. Experimental Section).

Scheme 5:

The behavior of azidopyrazole 1 toward trialkylphosphites 5a,b and trisdialkylaminophosphines 5c,d.

Meanwhile, performing the reaction of 1 with tris(dialkylamino)phosphanes 5c,d led to the formation of the [tris(dialkylamino)phosphoranylidene]triazenes 11c,d (Scheme 5). The structures of 11c,d have also been assigned on the basis of elemental analysis, MS, IR, ¹H, ¹³C, ³¹P NMR spectral data (cf. Experimental Section). The structure of 11c was also confirmed by X-ray crystallographic analysis. An Ortep diagram of the molecular structure of compound 11c in the crystal is shown in Fig. 3. Furthermore, this study has been extended to include the reaction of azidopyrazole 1 with triphenylstibane (6a) and triphenylarsane (6b) to establish whether they would behave in a similar manner as triphenylphosphane (6c) [22–25]. A different behavior is observed in the reaction of 1 with 6a and 6b, yielding 5-azido-4-(triphenylstiboranylidene)methyl-1H-pyrazole 12a in 75% yield and 4-(triphenylarsoranylidene)methyl-1H-pyrazole 12b in 45% yield with extrusion triphenylstibane oxide and triphenylarsane oxide, respectively (Scheme 6).

Fig. 3:

Ortep view of 11c with crystallographic atom numbering. Displacement parameters are drawn at the 30% probability level, H atoms as spheres with arbitrary radii. Selected bond lengths (Å) and angles (deg): P1–N5 1.579(4), P1–N6 1.709(4), P1–N7 1.714(4), P1–N8 1.704(4); N5–P1–N6 113.2(4), N5–P1–N7 110.8(4), N6–P1–N7 110.3(4), N5–P1–N8 106.1(4), N6–P1–N8 109.9(4), N7–P1–N8 106.3(4).

Scheme 6:

The behavior of azidopyrazole 1 toward TPP, TPSb, TPAs 7a–c.

When the pyrazole-4-carbaldehyde 1 was stirred at ambient temperature with LR, the 5-sulfido-1,2,3,4,5-thiatriazaphosphole 13 was obtained in good yield through cycloaddition reaction between the monomeric form of LR 7 and the azido group of 1 [26, 27] (Scheme 7). The elemental microanalysis, ¹H, ¹³C, ³¹P NMR, and MS data agreed with structure 13.

Scheme 7:

The behavior of azidopyrazole 1 toward LR 8.

The IR spectrum of structure compound 13 revealed the absence of an N₃ absorption band [13] and showed bands at 625 cm⁻¹ (P=S), 1585 cm⁻¹ (C=N), 1696 cm⁻¹ (C=O). The ¹H NMR shifts of compound 13 were at δ = 2.46 (s, 3H, CH₃), 3.76 (s, 3H, OCH₃), and 6.95–7.65 (m, 9H aromatic). The structure assigned to 13 was also based on a ¹³C NMR spectrum that shows signals of 55.7 (OCH₃), 105.0 (quaternary C-4, pyrazole), 113.9–145.5 (aromatic C-H, C-3, and C-5 of pyrazole), and 161.6, 161.7 (C=O) ppm.

2.2 Description of the crystal and molecular structures of 8b and 11c

Ortep plots of the molecular structures of compounds 8b and 11c are shown in Figs. 2 and 3, respectively. Both compounds crystallize in the monoclinic system possessing four molecules in the unit cell with space groups Cc for 8b and P2/n for 11c. The asymmetric unit in the two structures contains only one molecule. In 8b, there is a significant twist between the pyrazole ring and both attached triazole and benzene rings with the dihedral angles being 49.9(2)° and 62.3(2)°, respectively. The same is observed in compound 11c, with a significant twist between the pyrazole ring and the attached benzene ring with the dihedral angle being 38.4(2)°. An important feature to note in 8b is that the molecules are linked into chains by one weak hydrogen bond of the C–H···O type and a set of H···π and O···π interactions as listed in Table 1 and illustrated in Fig. 4. Molecules 11c are linked into chains by one hydrogen bond of the C–H···O type listed in Table 2 and illustrated in Fig. 5.

Fig. 4:

Packing of the molecules in the unit cell of compound 8b showing various types of intermolecular interactions as dashed lines.

Fig. 5:

Packing of the molecules in the unit cell of compound 11c showing weak hydrogen bonds as dashed lines.

2.3 Anticancer screening

Cancer is one of the diseases with a high mortality rate [28]. The current trend of research focuses on investigating the biological activity of newly synthesized compounds, aiming to develop new anticancer drugs. Derivatives of pyrazole have received considerable attention owing to their diverse chemotherapeutic potentials including their versatile anticancer activities [7]. Thus, the current study was tailored to screen invitro cytotoxic and growth inhibitory activities of most of the newly synthesized azidopyrazole compounds on human breast carcinoma cell line (MCF7) and human hepatocellular carcinoma cell line (HepG2). MCF7 and HepG2 cells were cultured as monolayers and treated with our compounds for 48 h. The rate of proliferation was assessed by the Alamar blue reduction assay. According to current data, compounds 8a, 8b, 9a, and 9b demonstrate a higher antitumor activity against MCF7 cells with low cytotoxic activity (IC₅₀ = 10.12, 1.012, 8.56, and 8.36 μm, respectively) than that of the reference drug (doxorubicin, IC₅₀ = 10.2 μm) (Table 3). Meanwhile, azidopyrazole derivatives showed less antitumor activity against HepG2 cells with higher IC₅₀ than doxorubicin (IC₅₀ = 5.61 μm). The study revealed that azidopyrazole derivatives have a potential antitumor activity against MCF7 cells; however, those derivatives are less potent against HepG2 cells. In addition, the current data stressed that compounds 9a, 9b, and 8b have the most effective antitumor activity. Compound 8b has a triazole ring that was previously reported to have a potentially broad range of biological and pharmacological activities [29]. Additionally, compounds 9a and 9b have a significant antitumor activity that may be associated with presence of the nitrile (CN) group, which has previously been reported to have a strong biological activity due to their strong hydrogen bond acceptor leading to strong versatile interactions with different proteins [30]; therefore, compounds 9a, 9b, and 8b have promising therapeutic effects that can be further studied invivo and invitro.

4.1 Chemistry

Melting points were determined in open glass capillaries using an Electrothermal IA 9100 series digital melting point apparatus (Electorthermal, Essex, UK), IR spectra were measured as KBr pellets with a Perkin-Elmer infracord spectrophotometer model 157. The ¹H and ¹³C NMR spectra were recorded in CDCl₃ or [D₆]dimethyl sulfoxide (DMSO) as solvents on a Jeol-500 spectrometer (¹H: 500 MHz; ¹³C: 125 MHz), and the chemical shifts are recorded in δ values relative to TMS as internal reference. The ³¹P NMR spectra were taken with a Varian CFT-20 spectrometer (vs. external 85% H₃PO₄ as standard). The mass spectra were recorded at 70 eV with a Kratos MS equipment or Varian MAT 311A spectrometer. Elemental analyses were performed using an Elementar Vario E1 instrument. The reported yields are of pure isolated materials obtained by column chromatography on silica gel 60 (Merk). 5-Azido-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (1) was prepared according to a reported method [31, 32].

4.2 Reaction of (cyanomethylene)triphenylphosphorane (2a) with 1

A mixture of 2a (0.60 g, 2 mmol) and 0.27 g (1 mmol) of 1 in dry THF (30 mL) was stirred for 1h. The volatile material was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography to give product 8a. TPPO was also isolated and identified.

4.3 Reaction of (acetylmethylene)triphenylphosphorane (2b) with 1

A mixture of 1 mmol of 2b (0.31 g) and 1 mmol of 1 (0.27 g) in dry THF (30 mL) was stirred for 2 h. The volatile material was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography to give product 8b. TPPO was also isolated and identified.

4.4 Reaction of (dibromomethylene)triphenylphosphorane (2c) with 1

Triphenylphosphane (0.5 g, 0.1 mmol) was added to a well-stirred solution of carbon tetrabromide (0.3 g, 0.05 mL) in DCM (3 mL). When the solution became orange (i.e. 2c was formed) [33], pyrazole 1 (0.27 g, 0.1 mmol) was added, and the mixture was stirred at room temperature for 8 h. After evaporation of the volatile material, the buff residual substance was chromatographed on a silica gel column. TPPO was also isolated and identified.

4.5 Reaction of diethyl cyanomethylphosphonate (3a) or diethyl 4-methoxybenzylphosphonate (3b) with 1

A solution of sodium ethoxide (0.14 g, 2 mmol) in absolute ethanol (30 mL) was treated with an equimolar amount of diethyl (cyanomethyl)phosphonate (3a) or diethyl 4-(methoxybenzyl)phosphonate (3b) (2 mmol). Azidopyrazole 1 (0.27 g, 1 mmol) was added, and the reaction mixture was stirred at room temperature for 4 h (TLC). The mixture was poured on a small amount of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the extracts were evaporated under reduced pressure. The residue was applied to silica gel column chromatography to give compounds 9a,b and/or 9c.

4.6 Reaction of diethyl hydrogenphosphite (4) with 1

A mixture of 1 mmol of 4 (0.17g) and 1 mmol of 1 (0.27 g) in dry toluene (30 mL) was stirred for 30 min. After evaporation of the volatiles under reduced pressure, the residue was subjected to silica gel column chromatography to yield structure 10.

4.7 Reaction of trialkylphosphite (5a,b) with 1

A mixture of 1 mmol of 5a or 5b and 1 mmol of 1 in dry toluene (30 mL) was stirred for 1–2 h (TLC). After evaporation of the volatile material, the residue was recrystallized from a proper solvent to yield compound 11a,b.

4.8 Reaction of tris(dialkylamino)phosphanes 5c,d with 1

A mixture of 5c or 5d (1 mmol) and 0.27 g (1 mmol) of 1 in dry benzene (30 mL) was stirred for 3 h. After evaporation of the volatile material under reduced pressure, the residue was subjected to silica gel column chromatography to yield 11c,d.

4.9 Reaction of triphenylstibane/triphenylarsane 6a,b with 1

A mixture of 6a or 6b (1 mmol) and 0.27 g (1 mmol) of 1 in dry toluene (30 mL) was warmed for 1–4 h (TLC). After evaporation of the volatile material, the buff residual substance was recrystallized from benzene to yield 12a,b and triphenylstibane oxide, triphenylarsane oxide were isolated and identified (m.p. and mixed m.p.).

4.10 Reaction of LR 7 with 1

A mixture of 0.20 g (0.5 mmol) of 7 and 0.27 g (1 mmol) of 1 was stirred for 15 min in dry benzene. The volatile material was evaporated under reduced pressure. The precipitate was filtered off and washed with diethyl ether, then recrystallized from benzene to afford product 13.

4.11 Crystal structure determinations of 8a and 11c

Single crystals of 8a and 11c were grown by crystallization from benzene. The diffraction data were collected on an Enraf-Nonius 590 diffractometer with a Kappa CCD detector using graphite-monochromatized MoK_α (λ = 0.71073 Å) radiation at National Research Center of Egypt [34, 35]. Reflection data have been recorded in the rotation mode using the ϕ and ω scan technique with θ_max = 34.98° for 8a and 30.04° for 11c. Unit cell parameters were determined from least-squares refinement with θ in the range 4 ≤ θ ≤ 34° for 8a and 3 ≤ θ ≤ 30° for 11c. The structures of 8a and 11c were solved by Direct Methods using Superflip [36] implemented in the program suit Crystals [37]. The refinement was carried out by full-matrix least-squares method on the positional and anisotropic temperature parameters of all non-hydrogen atoms based on F² using Crystals. All hydrogen atoms were positioned geometrically and were initially refined with soft restraints on the bond lengths and angles to regularize their geometry (C–H in the range 0.93–0.98 Å and N–H in the range 0.86–0.89 Å) and U_iso(H) = 1.2–1.5 × U_eq of the parent atom. Then, the positions were refined with riding constraints [38]. The general-purpose crystallographic tool Platon [39] was used for the structure analysis and presentation of the results. The molecular graphics were done using Ortep-iii for Windows [40] and Diamond [41]. Details of the data collection conditions and the parameters of the refinement process for 8a and 11c are given in Table 4.

CCDC 1415329 (8a) and 1415326 (11c) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre viawww.ccdc.cam.ac.uk/data_request/cif.

4.12 Anticancer evaluation