Synthesis of new oxazole hybridized with pyrazole and/or thiazole compounds: Molecular modeling and cytotoxic activity

2.1. Instruments

The melting temperature (°C) was measured using the open capillary method. Infrared (IR) spectral data (KBr discs, cm^-1) were analyzed using a ThermoNicolet IS10-FTIR spectrophotometer. Nuclear magnetic resonance (NMR) spectral data were recorded in DMSO-d₆ on a Jeol-500 MHz spectrometer. Mass spectra (MS) were recorded by a ThermoScientific GC-MS DSQII spectrometer at 70 eV. Microanalyses of the elements (C, H, and N) were recorded using a Perkin Elmer analyzer.

2.2. 2-Oxopropyl 4-(2-cyanoacetamido)benzoate (3)

A sodium carbonate solution (1.59 g, 15 mmol) in water (5 mL) was added dropwise to a solution of 4-(2-cyanoacetamido)benzoic acid (1) (3.00 g, 15 mmol) in 20 mL of DMSO in a 250 mL conical flask. The solution was stirred for 15 min, and then chloroacetone (1.38, 15 mmol) was added. The mixture was heated for 4 h on a water bath at 90°C. After cooling, the mixture was poured into ice-cold water, and the obtained solid was collected and crystallized from EtOH.

Yield = 85.8%, m.p. = 163-164°C. IR (ν/cm^-1): 3348 (N-H), 2252 (C≡N), 1730, 1687 (C=O). ¹H NMR (δ/ppm): 2.13 (s, 3H, -CH₃ of acetyl), 3.88 (s, 2H, -CO-CH₂-CN), 5.56 (s, 2H, -CO-CH₂-O), 7.68 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 10.71 (s, 1H, N-H). ¹³C NMR (δ/ppm): 24.18, 30.89, 70.43, 116.76, 119.03 (2C), 124.37, 130.51 (2C), 142.60, 164.15, 167.21, 201.88. MS for C₁₃H₁₂N₂O₄ [M]⁺: m/z = 260 (37.25%). Analysis for C₁₃H₁₂N₂O₄ (260.08): Calcd.: C, 60.00; H, 4.65; N, 10.76%. Found: C, 60.13; H, 4.71; N, 10.67%.

2.3. 2-Cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4)

To a suspension of 2-oxopropyl 4-(2-cyanoacetamido)benzoate (3) (1.30 g, 5 mmol) and acetamide (1.47 g, 25 mol) in 50 mL xylene, 47% BF₃/Et₂O (0.35 mL) was added dropwise. The solution was refluxed for 8 h, and then the solution was quenched with cold water (50 mL), and the aqueous layer was extracted with diethyl ether. The combined organic layer was dried over anhydrous MgSO₄ and evaporated under vacuum. The yellow solid was crystallized in 95% EtOH.

Yield = 71.4%, m.p. = 211-212°C. IR (ν/cm^-1): 3284 (N-H), 2254 (C≡N), 1690 (C=O). ¹H NMR (δ/ppm): 2.21 (s, 3H, oxazole-CH₃), 3.96 (s, 2H, -CO-CH₂-CN), 7.57 (s, 1H, oxazole-H5), 7.70 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 8.5 Hz, 2H), 10.64 (s, 1H, N-H). ¹³C NMR (δ/ppm): 13.82, 31.16, 116.47, 120.33 (2C), 126.06, 127.74 (2C), 134.20, 138.63, 141.56, 162.38, 163.91. MS for C₁₃H₁₁N₃O₂ [M]⁺: m/z = 241 (30.08%). Analysis for C₁₃H₁₁N₃O₂ (241.09): Calcd.: C, 64.72; H, 4.60; N, 17.42%. Found: C, 64.55; H, 4.68; N, 17.54%.

2.4. N-(4-Chlorophenyl)-2-((4-(4-methyloxazol-2-yl)phenyl)amino)-2-oxoacetohydrazonoyl cyanide (5)

A diazonium solution derived from 4-chloroaniline (1.01 g, 8 mmol) was obtained by adding sodium nitrite solution (0.56 g in 10 mL H₂O) drop by drop (for 10 min) to a suspension of 4-chloroaniline (1.01 g, 8 mmol) in conc. HCl (2.40 mL) at 0-5°C. The diazonium solution was added dropwise to a well-stirred suspension of 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4) (1.92 g, 8 mmol) in 30 mL of pyridine at 0-5°C. The mixture was stirred at 0-5°C for 2 h and then diluted with ice water. The solid was filtered, dried, and then crystallized from an EtOH-DMF mixture (5:1).

Yield = 67.5%, m.p. = 242-243°C. IR (ν/cm^-1): 3303, 3241 (N-H), 2221 (C≡N), 1667 (C=O). ¹H NMR (δ/ppm): 2.23 (s, 3H, oxazole-CH₃), 7.13 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.55 (s, 1H, oxazole-H5), 7.71 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.5 Hz, 2H), 10.47 (s, 1H, N-H), 11.85 (s, 1H, C=N-N-H). ¹³C NMR (δ/ppm): 13.77, 110.54, 111.30, 117.35 (2C), 121.22 (2C), 126.01, 127.63 (2C), 128.28, 129.41 (2C), 133.87, 138.51, 140.46, 142.16, 162.14, 162.93. MS for C₁₉H₁₄ClN₅O₂ [M]⁺: m/z = 379 (13.77%). Analysis for C₁₉H₁₄ClN₅O₂ (379.08): Calcd.: C, 60.09; H, 3.72; N, 18.44%. Found: C, 60.25; H, 3.80; N, 18.55%.

2.5. 2-Oxo-2-(thiophen-2-yl)ethyl 4-(4-amino-1-(4-chlorophenyl)-1H-pyrazole-3-carboxamido)benzoate compounds 6a-c

A 150 mL RB-flask was charged with a solution of N-(4-chlorophenyl)-2-((4-(4-methyloxazol-2-yl)phenyl)amino)-2-oxoacetohydrazonoyl cyanide (5) (1.06 g, 2.8 mmol) in 40 mL tetrahydrofuran (THF) and 0.2 mL of triethylamine. The appropriate halogenated reagent, either chloroacetone, ethyl chloroacetate, or chloroacetonitrile (2.8 mmol), was added to the previous solution, and the mixture was refluxed for 6 h. The solid obtained after cooling was filtered and crystallized from EtOH to furnish the target oxazole-pyrazole hybrid compounds 6a-c.

2.6. 2-Cyano-N-(4-(4-methyloxazol-2-yl)phenyl)-2-(4-oxo-3-phenylthiazolidin-2-ylidene)acetamide (7)

A mixture of 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4) (0.96 g, 4 mmol) in DMF (20 mL) was stirred in a 250 mL conical flask with solid potassium hydroxide (0.22 g, 4 mmol) for 10 min, and phenyl isothiocyanate (0.48 g, 4 mmol) was then added. The reaction components were stirred for 6 h at 25-30°C. In situ, ethyl bromoacetate (0.67 mL, 4 mmol) was added, and the mixture was stirred for 6 h. The solid that formed upon dilution with 30 mL ice-water was collected and purified by crystallization from EtOH.

Yield = 64.8%, m.p. = 230-231°C. IR (ν/cm^-1): 3388 (N-H), 2195 (C≡N), 1740, 1668 (C=O). ¹H NMR (δ/ppm): 2.21 (s, 3H, oxazole-CH₃), 4.01 (s, 2H, cyclic-CH₂), 7.41 (d, J = 7.5 Hz, 2H), 7.48-7.53 (m, 3H), 7.58 (s, 1H, oxazole-H5), 7.71 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.5 Hz, 2H), 9.54 (s, 1H, N-H). ¹³C NMR (δ/ppm): 13.76, 32.18, 77.97, 113.54, 120.85 (2C), 126.17, 127.73 (2C), 128.13, 129.40 (2C), 130.17 (2C), 133.82, 136.77, 138.39, 139.68, 162.05, 163.30, 170.91, 173.41. MS for C₂₂H₁₆N₄O₃S [M]⁺: m/z = 416 (25.93%). Analysis for C₂₂H₁₆N₄O₃S (416.09): Calcd.: C, 63.45; H, 3.87; N, 13.45%. Found: C, 63.34; H, 3.82; N, 13.51%.

2.7. 2-Cyano-2-(5-arylidene-4-oxo-3-phenylthiazolidin-2-ylidene)-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide compounds 8a and 8b

To a suspension of 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)-2-(4-oxo-3-phenylthiazolidin-2-ylidene)acetamide (7) (0.83 g, 2 mmol) in 30 mL EtOH and 0.1 mL of piperidine, either 4-aniladehyde or 4-chlorobenzaldehyde (2 mmol) was added. The mixture was refluxed for 4 h, and the solid obtained after cooling was filtered. The solid was subjected to purification by crystallization from an EtOH-DMF mixture (4:1) to furnish target oxazole-thiazole hybrids 8a and 8b.

2.8. DFT Computations

The Gaussian 09W built-in DFT/B3LYP/6-311⁺⁺G(d,p) routine [30-33] was employed in the investigation of the designed derivatives’ optimum spatial configurations, frontier molecular orbitals (FMOs), and electronic properties, where the GaussView [34] was utilized for analyzing the outcomes.

2.9. Cytotoxic assay

The cytotoxic effects of the synthesized oxazole hybridized with pyrazole and/or thiazole analogs were assessed on three distinct human sarcoma cell lines: MCF-7 (breast cancer), Panc-1 (epithelioid carcinoma), and HT-29 (colorectal adenocarcinoma), besides a normal cell line (WI-38). The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) test was used to conduct the assessment, as described in the supporting information file. Dasatinib was used as the reference medication for the comparative analysis [35].

2.10. Carbonic anhydrase assay

The carbonic anhydrase inhibition activity against CA IX and CA XII of the synthesized oxazole derivatives hybridized with pyrazole and/or thiazole analogs was evaluated according to the protocol of the carbonic anhydrase technique [36] (see the supplementary file).

2.11. Molecular docking

The docked analogs of all the hybrids 3-8 against carbonic anhydrase CA XII were analyzed separately and evaluated using M.O.E. 2019. The co-crystal structure of carbonic anhydrase CA XII was obtained from (PDB having PDBID 1V9E (www.rcsb.org) [37], and compared with the reference drug (acetazolamide). For protein preparation, hydrogen atoms were incorporated to correct protonation states, while water molecules and heteroatoms were removed to simplify the system. Furthermore, the structure underwent energy minimization applying the AMBER10: EHT force field to resolve steric clashes. Simultaneously, during ligand generation, energy was minimized using the MMFF94x force field, and partial charges were assigned. Additionally, the docking procedure was validated by re-docking the co-crystallized ligand into the binding site of the generated protein. The root-mean-square deviation (RMSD) between the docked and crystallographic conformations was 1.2 Å, demonstrating the reliability of the docking approach.

3.1. Synthesis of oxazole-pyrazole and oxazole-thiazole hybrids

The process of synthesizing oxazole-pyrazole and oxazole-thiazole hybrids was started by producing the versatile precursors, 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4), and its corresponding N-(4-chlorophenyl)-hydrazone compound 5, according to the following sequence of reactions. The first step involved the esterification reaction of 4-(2-cyanoacetamido)benzoic acid (1) [38] with chloroacetone by heating in DMSO and sodium carbonate at 90°C to furnish the corresponding ester, 2-oxopropyl 4-(2-cyanoacetamido) benzoate (3) (Scheme 1). In the second step, the introduction of an oxazole ring into ester compound 3 was achieved by treatment with acetamide in boiling xylene and boron trifluoride. The product was identified as 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4). The final step takes advantage of the methylene group’s reactivity in the cyanoacetamide part of compound 4, facilitating an electrophilic diazo-coupling reaction with a diazonium salt that originates from 4-chloroaniline. The reaction proceeds when 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4) was treated with 4-chlorophenyl diazonium chloride at 0-5°C in pyridine to provide the conforming arylhydrazone compound (5), N-(4-chlorophenyl)-2-((4-(4-methyloxazol-2-yl)phenyl)amino)-2-oxoacetohydrazonoyl cyanide. The structures of compounds 4 and 5 were verified based on compatible spectral data. The IR spectrum showed the absorption frequency for the carbonyl group in cyanoacetamide compound 4 at 1690 cm^-1, and at a lower wavenumber at 1667 cm^-1 in its arylhydrazone compound 5 due to the extended conjugation with the hydrazone skeleton. The ¹H NMR spectrum of compound 4 clearly indicated a singlet signal for the methylene protons at δ 3.96 ppm, while the ¹H NMR spectrum of compound 5 exhibited no signal assignable to the methylene protons and displayed the hydrazone proton as a singlet at δ 11.85 ppm.

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Scheme 1.

Synthesis of 2-cyano-N-(4-(oxazolyl)phenyl)acetamide derivatives 4 and 5.

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The first series of oxazole-pyrazole hybrids 6a-c was obtained by heating arylhydrazone compound 5 with either chloroacetone, ethyl chloroacetate, or chloroacetonitrile in THF and triethylamine (Scheme 2). The proposed mechanism begins with the nucleophilic displacement of the chlorine atom from the halogenated reagent to form the N-alkylated intermediate (A). This intermediate undergoes intramolecular cyclization via the addition of the methylene group to the nitrile function to form the pyrazole intermediate (B), which rapidly tautomerizes to produce the final aminopyrazole skeleton in hybrids 6a-c. The IR spectrum of 6a showed no absorption band assignable to the cyano function, while it displayed the absorptions of the –NH₂ and N-H groups at stretching frequencies at 3323, 3281, and 3205 cm^-1 for the NH groups, in addition to two absorptions for the carbonyl groups at 1660 (conjugate amidic carbonyl) and 1627 cm^-1 (intramolecular hydrogen bonding carbonyl of acetyl group). The ¹H NMR spectrum of compound 6a displayed two singlet signals at δ 2.21 and 2.30 ppm, which correspond to the protons of the methyl-linked oxazole and acetyl groups, respectively. The protons of the pyrazole amino group were detected as a singlet at δ 6.64 ppm. The aromatic protons appeared as four doublet signals at δ 7.34, 7.51, 7.70, and 7.82 ppm. Additionally, the protons of the oxazole-C5 and N-H groups were detected as singlet signals at δ 7.58 and 10.11 ppm, respectively.

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Scheme 2.

Synthesis of oxazole-pyrazole hybrids 6a-c.

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The synthesis of the second targeting series of oxazole-thiazole hybrids 7 and 8 was initiated by the base-catalyzed addition of 2-cyano-N-(4-(4-methyloxazol-2-yl)phenyl)acetamide (4) to phenyl isothiocyanate in stirred DMF and potassium hydroxide to produce the corresponding thiocarbamoyl intermediate (C), which underwent in situ treatment with ethyl bromoacetate and cyclization into the thiazolidine-4-one ring in hybrid compound 7 by losing water and HBr molecules (Scheme 3). The reactivity of the methylene group of the thiazolidinone ring (hybrid 7) supports the condensation reaction with aromatic aldehydes (Knoevenagel reaction). Thus, oxazole-thiazolidinone hybrid 7 was heated with either 4-anisaldehyde or 4-chlorobenzaldehyde in EtOH and piperidine to furnish the corresponding 5-benzylidene-thiazolidin-4-one compounds 8a and 8b, respectively. Spectral studies were performed to elucidate the chemical structures of 7, 8a, and 8b. The introduction of a benzylidene group at position-5 of the thiazolidinone ring lowered the absorption frequency of the carbonyl group from 1740 cm^-1 (IR spectrum of hybrid 7) to 1708 cm^-1 (hybrid 8a) and 1713 cm^-1 (hybrid 8b). The ¹H NMR spectrum of hybrid 8b indicated the absence of any signal related to the methylene group and exhibited a singlet signal at δ 7.88 ppm assignable to the olefinic proton (benzylidene system).

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Scheme 3.

Synthesis of oxazole-thiazole hybrids 7 and 8.

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3.2. Molecular modeling

The spital configuration for the cyanoacetamide conjugates 3 and 4, as well as the oxoacetohydrazonoyl cyanide 5, was planar (Figure 1). However, the resulting pyrazolyl carboxamide analogs 6a-b departed from planarity, whereas the cyanopyrazole derivative 6c conserved flatness. In 6a-b, a distortion from planarity was observed for the carboxamide group, which moved away from the plane of both phenyl and pyrazolyl rings, C³_(Ph)-C⁴_(Ph)-NH_(Car)-CO_(Car) = 2.9-3.6° and NH_(Car)-CO_(Car)-C3_(Prz)-C4_(Prz) = 24.1-24.9°, respectively. Further planarity deformation has been perceived for the chlorophenyl group, which strongly leans on the pyrazolyl’s plane, N²_(Prz)-N¹_(Prz)-C¹_(PhPrz)-C²_(PhPrz) = 48.9-52.1°. Moreover, the acetyl (6a) and carboxylate (6b) presented additional divergence from pyrazolyl’s level, e.g., N²_(Prz)-N¹_(Prz)-C⁵_(Prz)-CO_(AcPrz) = 166.1° and N²_(Prz)-N¹_(Prz)-C⁵_(Prz)-CO_(EstPrz) = -169.0°, respectively (Figure 1).

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Figure 1.

The DFT/B3LYP/6-311⁺⁺G(d,p) optimized structures of derivatives 3-6.

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Otherwise, the thiazolidinylidene derivatives 7-8 presented comparable non-planar configurations (Figure 2), where the cyanoacetamide group was tilted differentially on the phenyl and thiazolidinylidene rings, C²_(Ph)-C¹_(Ph)-NH_(Acm)-CO_(Acm) = -37.4 - -41.3° and CO_(Acm)-C_(Acm)-C²_(Tz)-S¹_(Tz) = 5.0-5.5°. Also, the cyano functional group has been moved away from the carbonyl of the cyanoacetamide joint, CO_(Acm)-C_(Acm)-CN_(Acm)-NC_(Acm) = -23.6 - -25.6°. Moreover, the N-substituent phenyl was approximately perpendicular to the thiazolidinylidene ring, C²_(Tz)-N³_(Tz)-C¹_(PhTz)-C²_(PhTz) = 88.3-90.1 (Table S1).

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Figure 2.

The DFT/B3LYP/6-311⁺⁺G(d,p) optimized structures of derivatives 7-8.

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Then, the studied hybrids’ DFT exhibited bond lengths and angles, in contrast with those belonging to analogous compounds, single crystal X-ray [39,40], divulged an appreciated congruence, since the observed inconsistencies were <0.13 Å and <14.0° (RMSD = 0.05-0.07 and 4.8-5.2), respectively. The discrepancy might have resulted from that calculations were performed on a single gaseous particle with no columbic interactions [41] (Tables S2-S3).

Furthermore, the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO), FMOs, shapes, and energies have outstanding weight owing to their substantial influence on the molecule’s performance to donate or acquire electrons [42-45]. The analogs 3 and 4 displayed comparable FMOs, where their HOMO and LUMO had been overlaid mainly on the phenylacetamide fragment with minor contribution of the oxopropyl and methyloxazolyl groups (π- and π*-orbitals, respectively). The chlorophenyl hydrazonyl 5 had FMOs spread over the entire skeleton, while the pyrazolyl derivatives 6a-c displayed analogous composition, wherein HOMO was centered on methyloxazolylphenyl carboxamide with minor involvement of pyrazole ring, and LUMO has been confined on the chlorophenylpyrazole portion. Along with, the phenyl thiazolidinylidene 7 presented equivalent HOMO configuration, on methyloxazolylphenyl carboxamide, however its LUMO was span over the whole molecule. Contrarywise, the benzylidene conjugates 8a-b have demonstrated unlike configurations, wherein the HOMO and LUMO were localized on the phenyl thiazolidinylidene cyanoacetamide segment (π- and π*-orbitals, respectively) (Figure 3 and Figure S1).

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Figure 3.

The DFT/B3LYP/6-311⁺⁺G(d,p) FMO’s 3D-plots for hybrids 3, 4, 5, 6a, 7, and 8a.

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Successively, the explored hybrids’ HOMO energies (E_H) have been presented ranged from -5.81 (for 6b) to -7.15 eV (for 3), while these of LUMO (E_L) were ranged from -1.75 (for 4) to -2.90 eV(for 8b). Also, the energy gap (ΔE_H-L) was in 3.27-5.00 eV zone, obeying the array: 5 < 8a < 8b < 6a < 6c < 6b < 7 < 4 < 3, which designated that the thiazolidinylidene derivatives 8a-b have lower gap than the pyrazolyl conjugates 6a-c (Figure 4). Furthermore, the energies of FMOs were manipulated in the estimation of certain chemical reactivity factors [43]. As the electronegativity and hardness disclosed that compound 3 had the uppermost standards (χ = 4.65 and η = 2.50 eV), while derivatives 6b and 5 presented the lowest merits (3.93 and 1.63 eV), respectively. However, the electrophilicity (ω) ranged from 3.55 (for 4) to 6.24 eV (for 8b), endorsing the undoubted electrophilicity character of these conjugates, ω > 1.5 eV, with superior tendency to give than receiving electrons, as electron donating (ω⁺) power < accepting (ω^-) [46,47] (Table 1).

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Figure 4.

Representation of explored hybrids’ FMO energies obtained from DFT/B3LYP/6-311⁺⁺G(d,p) calculations.

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Besides, the intramolecular charge-transfer and electronegativity might be empathized further from the Mulliken’s atomic charges [48]. The data showed that the cyanoacetamide nitrogen NC_(CNM) has acquired a close negative charge in 3-5 and 7-8 derivatives, -0.133 - -0.179. As well, the corresponding nitrogen of cyanoacetamide NH_(CNM) and carboxamide NH_(Car) in 6a-c have caught comparable negative charge, but slightly higher in the latter, -0.505 - -0.532 and -0.548 - -0.567, respectively. Whereas, the carbonyl oxygen of these functionals OC_(CNM) and OC_(Car) were ranged from -0.337 to -0.389. Moreover, in compound 3, the oxopropyl oxygen atoms O²C¹_(Prp), O¹C¹_(Prp) and OC²_(Prp) displayed close negative charge values, -0.366, -0.380 and -0.308, respectively, whereas the conversion of this group into oxazolyl ring led to reduction of the oxazolyl oxygen O¹_(Oxz) to be -0.280 - -0.284. Also, the oxazolyl nitrogen N³_(Oxz) were negatively charged with relative merits, -0.283 -0.292. Alternatively, in conjugate 5, the nitrogen atoms of hydrazonyl group, N_(HZ) and NH_(HZ), have diverse negative charge, -0.094 and -0.385, respectively. When they participated in formation of pyrazolyl ring in 6a-c, N²_(Prz) and N¹_(Prz), their charges were -0.162 - -0.169 and -0.031 -0.085, respectively. However, the aminopyrazolyl nitrogen NH_2(Prz) has higher negative charge, -0.802 -0.814. Further, the thiazolidinylidene sulfur S¹_(Tz) and nitrogen N³_(Tz) atoms presented opposite charges, the former was positively charged (0.173-0.194), while the latter has negative charge (-0.298 - -0.313) (Table S4).

Else, the molecular polarizability (α_total), hyperpolarizabilities (β_total), and dipole moment (μ) were evaluated [49-51] to demonstrate the distribution of electronic density and softness, which principally influence the intermolecular interactions [52]. The analogs exposed rampant dipole moment (μ) swung from 3.85 D (5) to 7.78 D (8a), implying 2.80-5.66 times the standard substance urea [53] (Table 2). Also, the conjugates 4 and 8b unveiled the least and greatest values of polarizability (α_total = 1.61-3.44×10^-23 esu), while the analog 7 showed the lowest first-order hyperpolarizability, however both of 8a and 6c displayed the highest value (β_total = 2.07-5.03×10^-30 esu). In contrast to urea [53], the considered hybrids offered larger hyperpolarizability, 5.53-13.45 times (Table 2).

3.3. Cytotoxic assay

The cytotoxicity of the synthesized analogs demonstrated varying degrees of effectiveness across the three human cancer cell lines (Panc-1, HT-29, MCF-7) and a normal lung fibroblast line (WI-38) (Table 3). Hybrids 3, 4, and 5 showed weak effectiveness, IC₅₀ valued between 18.69±0.04 - 40.61±0.19 μM (HT-29), 23.57±0.36 - 37.65±0.14 μM (MCF-7), and 38.05±0.28 - 46.32±0.21 μM (Panc-1), and less selectivity towards normal WI-38 cells. Meanwhile, the analogs of fused thiazole/pyrazole moieties 6a-c enhanced anticancer potency, mainly against MCF-7 (IC₅₀ = 7.74±0.33 - 14.09±0.02 μM), Panc-1 (IC₅₀ = 18.12±0.44 - 23.36±0.15 μM), and keeping good selectivity toward WI-38 (IC₅₀ = 70.64±0.54 - 86.07±0.48 μM). Hybrid 7 exhibited sustained results with sensible effectiveness toward all cancer lines (IC₅₀ = 11.61±0.11, 20.48±0.30 and 23.82±0.31 μM against MCF-7, HT-29 and Panc-1, respectively). Moreover, analogs 8a and 8b, which feature both oxazole and thiazole motifs, emerged as the most potent derivatives, e.g., analog 8a achieved IC₅₀ values (μM) 23.52±0.04 (Panc-1), 13.22±0.16 (HT-29), and 6.41±0.47 (MCF-7), while showing high selectivity to normal cells (82.76±0.39), demonstrating an auspicious therapeutic index. In addition, analog 8b displayed comparable results. In comparison, the drug reference (dasatinib) showed more potency as an expected (IC₅₀: 2.35-4.63 μM) over higher selectivity towards WI-38 cells (IC₅₀ = 87.28 μM), close to those of analogs 6b, 8a, and 8b. These results underline the effective role of the heterocyclic amendments in enhancing cytotoxic effectiveness and selectivity.

3.4. Structural activity relationship

Among the structures of the newly produced oxazole hybridized with pyrazole and/or thiazole analogs and their cytotoxic effectiveness, hybrid 3 has the cyano-acetamide and benzoate moiety; the ester-linked cyano-acetamide shows moderate activity, suggesting that the flexible 2-oxopropyl group may reduce its binding efficiency likened to more rigid analog. However, for hybrid 4, which possess the oxazole-ring, the direct binding between the cyano-acetamide group and the oxazole-phenyl core may improve its potency, likely due to stronger H-bonding or enhanced planarity. Meanwhile, for hybrid 5, with the hydrazonyl moiety, the appearance of both hydrazonoyl-cyanide and p-chlorophenyl groups improvements may enhanced its cytotoxic effectiveness, probably through the increase of electron-withdrawal or further hydrophobic contacts. For hybrids 6a-6c with the pyrazole ring, conjugate 6a contains the pyrazole ring and p-chlorophenyl group grows its rigidity and lipophilicity, leading to improved its cytotoxic effectiveness. Conjugate 6b has the ethyl carboxylate group likely improves its solubility or its metabolic stability, contributing to higher effectiveness, specifically against MCF-7 cells. Moreover, conjugate 6c with the nitrile group is expected to produce growth in solubility or metabolic stability, leading to its cytotoxic effectiveness. Hybrid 7 possess the thiazolidinone core, introducing a potential Michael acceptor, which may covalently modify cellular targets, increasing activity. Together with the hybrids 8a and 8b, which have the 4-oxo-3-phenylthiazolidin-2-ylidene moiety, conjugate 8a has the p-methoxy-benzylidene group, which enhances potency, possibly by improving the membrane permeability or the electronic stabilization. Furthermore, conjugate 8b with the p-chloro-benzylidene substitution maintains strong cytotoxic effectiveness, with the p-chloro group contributing to hydrophobic and electron-withdrawing effects.

3.5. Carbonic anhydrase assay

The carbonic anhydrase inhibition activity of the synthesized oxazole derivatives hybridized with pyrazole and/or thiazole analogs revealed significant variations in inhibitory potency against CA IX and CA XII, depending on the specific heterocyclic substitutions (Table S5 and Figure 5). Among the tested analogs, analog 6c exhibited the strongest inhibition towards CA IX (IC₅₀ = 0.011±0.015 μM), comparable to the reference inhibitor acetazolamide (AZA, IC₅₀ = 0.054±0.019 μM), followed by analog 6a (IC₅₀ = 0.018±0.001 μM). However, analog 8b (IC₅₀ = 0.079±0.019 μM) and analog 8a (IC₅₀ = 0.095±0.043 μM) displayed good CA IX inhibition. Analogs 3, 4, and 5, had IC₅₀ values ranging from 0.102±0.052 to 0.331±0.047 μM. Meanwhile, according to CA XII inhibition, analog 7 demonstrated the highest activity (IC₅₀ = 0.119±0.043 μM), followed by analog 8a (IC₅₀ = 0.102±0.009 μM), analog 8b (IC₅₀ = 0.114±0.009 μM), and analog 6c (0.127 μM), comparable to or approaching AZA (IC₅₀ = 0.080±0.033 μM). Excitingly, analog 6b exhibited moderate inhibition against both CA IX and CA XII isoforms, possibly due to the existence of a amino-pyrazole moiety, which affects its selectivity and potency. Moreover, analogs 3-5 still demonstrated low inhibition, indicating baseline activity. The results showed that hybridization with specific thiazole/pyrazole skeletons obviously enhances carbonic anhydrase inhibitory profiles, mainly in analogs 6a, 6c, and 8 hybrids.

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Figure 5.

Carbonic anhydrase inhibition of the of synthesized analogs.

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3.6. Molecular docking

As the docking data show, unique binding modes are associated with structural properties of the newly synthesized thiophene hybridized with pyrazole, pyridine, and/or thiazole analogs (Table 4). Analog 3 exhibited a moderate binding energy S = -5.4630 kcal/mol (RMSD 1.2173), forming H-acceptor bonds with LYS168 (2.91 Å) and ALA241 (3.18 Å), through its acetyl and nitrile groups, respectively, stabilizing the ligand within the binding pocket of PDB ID: 1V9E (Figure S2). Meanwhile, analog 4 displayed a slightly enhancement in the docking score (S = -5.5212 kcal/mol, RMSD = 1.9559), indicating a more energetic fit, with five different bindings: i) H-donors between both nitrogen (N7) and acetamide carbon (C13) with GLU234; ii) H-acceptor from oxygen (O14) with Ser1, iii) π-H interaction of the oxazole-ring with ASN10, and iv) π-cation stacking of the phenyl-ring with LYS168. These various bindings endorse a more intricate and stable binding mode (Figure S2). While analog 5 presented a lower binding score (S = -5.1156 kcal/mol, RMSD = 1.0878), based on only π-H interaction between the oxazole-ring and Asn10 (3.63 Å). The minimal binding score may cause the relatively weaker bindings compared to the other analogs (Figure S2). In the interim, analog 6a demonstrated an improved score (S = -5.7659 kcal/mol, RMSD = 1.7577), forming a single H-acceptor binding amongst the acetyl group oxygen (O29) and GLN91 (3.00 Å). This contact, while singular, might contribute to its adequate binding energy (Figure 6). However, analog 6b showed a modest energy (S = -5.3282 kcal/mol, RMSD = 1.4752), motivated by lone π-H stacking amid the pyrazole ring and GLY7 (4.15 Å), with limited binding but potentially suitable binding stability (Figure S2). Also, analog 6c showed a considerable docking energy (S = -5.6831 kcal/mol, RMSD = 1.6365), featuring a H-bond from the amino-group to THR226 (3.09 Å) and a π-H stacking amongst the oxazole-ring and SER98 (3.92 Å). The sequence of the polar and π-type bindings likely contributes to its moderate binding (Figure S2). Similarly, derivative 7 disclosed a score S = -5.6063 kcal/mol (RMSD = 1.0789), forming a single H-acceptor bond between the nitrile nitrogen (N29) and LEU48 at 3.45 Å. This binding, though explicit, appears to bargain moderate stabilization (Figure S2). Moreover, analog 8a offered a significant increasing in binding affinity (S = -6.5463 kcal/mol, RMSD 1.7158), over three interactions: an H-donor with GLU212 via the thiazolidinone sulfur (3.6 Å), an H-acceptor interaction with ASN185 via the nitrile nitrogen (3.57 Å), and a π-H stacking with PRO153 (3.89 Å). The presence of both heteroatom-based and aromatic bindings enhances its binding strength (Figure 6). Furthermore, analog 8b yielded a strong docking (S = -7.0940 kcal/mol, RMSD =1.5035), forming a π-π stacking between the oxazole ring and the aromatic system of HIS93 (3.82 Å). This specific aromatic stacking recommends high binding affinity through effective π-delocalization (Figure 6). Finally, acetazolamide (reference) established an expected good binding energy (S = -8.6276 kcal/mol, RMSD = 1.3597. It formed five bindings: H-donor bond with GLY62 (3.16 Å), two H-acceptor bonds via the sulphonyl-group with LYS168 and ASN230 (3.53 and 2.98 Å), an H–π stacking between the amino-group and HIS2 (3.31 Å), and a π-H stacking of the thiadiazole-ring with PHE229 (4.26 Å). The diversity and number of interactions recommend that (Aza) is offering a stable and energetically good fit (Figure S2).

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Figure 6.

Docking images of analogs 6a, 8a and 8b with PDB: 1V9E obtained from MOE

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3.7. Pharmacokinetic properties

In addition to the pharmacokinetic properties of newly oxazole hybridized with pyrazole and/or thiazole analogs (Table S6 and Figure S3) using the Swiss ADME program. A detailed summary of the ADME parameters, including molecular weight, lipophilicity (iLOGP), solubility, topological polar surface area (TPSA), hydrogen bond donors/acceptors, gastrointestinal (GI) absorption, blood–brain barrier (BBB) permeability, P-glycoprotein (Pgp) substrate status, and Lipinski’s rule compliance, has been provided in the Supporting Information (Table S6 and Figure S3).

Overall, the compounds exhibited favorable in silico pharmacokinetic properties and satisfactory drug-likeness profiles. All analogs showed high GI absorption, suggesting good oral bioavailability potential, and none were predicted to permeate the BBB or behave as Pgp substrates, traits that reduce CNS-related side effects and multidrug resistance risk. Most analogs complied fully with Lipinski’s rule of five, with acceptable values for TPSA and hydrogen bonding, supporting their potential as orally active agents. Notably, analogs 3 and 4, being highly soluble with low molecular weight, possessed optimal oral drug-likeness characteristics. Analogs of higher molecular weight, such as 6a–c, 7, and 8a–b, were within acceptable physicochemical parameters and possessed uniform bioavailability scores (0.55), moderate solubility, and balanced lipophilicity. These findings suggest that their pharmacokinetic potential was not compromised by structural class. Together, these in silico results maintain the efficacy of these synthesized hybrids as lead compounds for development. Comparative figures and extensive ADME data are presented in Supporting Information for reference (Table S6 and Figure S3).