Synthesis and Characterization of Novel Heterocyclic Chalcones from 1-Phenyl-1H-pyrazol-3-ol

An efficient synthetic route to construct diverse pyrazole-based chalcones from 1-phenyl-1H-pyrazol-3-ols bearing a formyl or acetyl group on the C4 position of pyrazole ring, employing a base-catalysed Claisen–Schmidt condensation reaction, is described. Isomeric chalcones were further reacted with N-hydroxy-4-toluenesulfonamide and regioselective formation of 3,5-disubstituted 1,2-oxazoles was established. The novel pyrazole-chalcones and 1,2-oxazoles were characterized by an in-depth analysis of NMR spectral data, which were obtained through a combination of standard and advanced NMR spectroscopy techniques.

The structural simplicity and therapeutic potential have motivated the design and development of synthetic chalcones with enhanced activity and potency [18].
Chalcones are usually synthesized from aromatic aldehydes and aliphatic aldehydes or ketones via the Claisen-Schmidt condensation reaction in the presence of base or acid catalysts [19][20][21][22]. Other procedures were efficiently employed for the synthesis of chalcones, including the Pd-catalysed Suzuki cross-coupling reaction between the appropriate Molecules 2022, 27, 3752 2 of 32 cinnamoyl chloride and phenylboronic acid or benzoyl chloride and phenylvinylboronic acid or Heck coupling reaction between aryl iodide and an unsaturated ketone [23][24][25]. The Wittig olefination reaction of triphenylbenzoylmethylene phosphoranes and benzaldehydes and the Julia-Kocienski olefination technique of heteroaryl-sulfonyl phenylethanones and benzaldehydes were also applied to give chalcones in efficient yields [26].
Among the synthetic chalcone derivatives, heterocyclic chalcones are important for medicinal chemistry, as most biologically active chemical entities contain a heterocyclic scaffold [31,32]. For example, Pd(II) or Pt(II) complexes containing chalcone I displayed good anticancer and antimicrobial activities [33], while the structure of pyridine-chalcone derivative II was developed as a potential anti-tubulin agent, with antiproliferative activity against a panel of cancer cell lines ( Figure 1) [34]. The anticancer activity was also reported for indolizinyl compound III, with the potential to induce the caspase-dependent apoptosis of human lymphoma cells [35] or quinoxalinyl derivative IV, which was active against MCF-7-cell lines [36], and thiophen-2-yl derivative V, which was active against colorectal carcinoma cells by causing apoptosis [37]. Chalcone VI showed remarkable inhibition potency against AChE and MAO-B enzymes and, therefore, can be further developed as a novel, phenothiazine-based, dual-targeting inhibitor for neurogenerative diseases [38], while compound VII acts as a tissue transglutaminase inhibitor [39]. Compound VIII and analogues were designed as promising anti-tubercular agents by combining in silico design, QSAR-driven virtual screening, synthesis, and experimental evaluation. The synthesized nitroaromatic chalcone derivatives were also active against Mycobacterium tuberculosis strains resistant to isoniazid or rifampicin [40]. In addition, indole-based chalcone IX was reported to act as a nonselective COX-1 and COX-2 inhibitor and showed anti-inflammatory and antioxidant activities in vivo [41]. Moreover, pyrazole-based chalcone derivatives X-XII were also synthesized and investigated. Compound X was evaluated for its anti-inflammatory activity, and compounds XI and XII showed potential activity as chemotherapeutic agents for the treatment of hepatocellular carcinoma (HCC), as they caused cell cycle arrest at the G2/M phase and induced apoptotic cell death [42,43].
An in-depth analysis of NMR spectral data, which were obtained through a combination of standard and advanced NMR spectroscopy techniques, such as 1 H- 13 C HMBC, 1 H- 13 C HSQC, 1 H- 13 C H2BC, 1 H- 15 N HMBC, 1 H- 15 N LR-HSQMBC, 1 H-1 H TOCSY, 1 H-1 H COSY, 1 H-1 H NOESY and 1,1-ADEQUATE experiments, provided the key information in the establishment of structural assignments and predominant configuration, due to conformations in a solvent of novel pyrazole-chalcones. The synthesis and biological activity of the related compounds has been reported in previous works, but no data on conformational analysis supported by NMR experiments were given [58][59][60][61].

as a catalyst and
Molecules 2022, 27, 3752 8 of 32 Cs 2 CO 3 as a base and by performing the reaction at 80 • C with a microwave irradiation potency of 150 W in EtOH. 1-[1-Phenyl-3-(pyridinyl)-1H-pyrazol-4-yl]ethan-1-ones 11a,b were obtained in 63-64% yields. The reaction was carried out in the presence of KBr, which is known to suppress triflate reduction by stabilizing the cationic (σ-aryl)-palladium transition state [62]. 4-Acetylpyrazoles 11a,b were further employed in the Claisen-Schmidt condensation reaction with different carbaldehydes. The reaction was performed under the above-described conditions in the presence of ethanolic NaOH at 55 • C. As a result, when 1-[1-phenyl-3-pyridinyl-1H-pyrazol-4-yl]ethan-1-ones 11a,b were reacted with benzaldehyde, 4-methyl-or 4-(trifluoromethoxy)benzaldehyde Eand Z-chalcones (12a-f and 13a-f, respectively) were obtained in fair to good total yields (51-70%). The NMR spectra of inseparable mixtures showed the presence of both isomers in different ratios, with a predominance of the E-isomer. In contrast, compounds 12g-j were obtained only as pure E-isomers. This latter observation can be explained by the strong electron-donor capacity of the 4-methyloxy group based on the resonance structure, which results in increased electron density of the enone moiety, while the electronegativity of the 4-trifluoromethyl group has a significance for decreased electron density of the enone moiety. It is known in most cases that the E-isomer is more stable from the perspective of thermodynamics, which makes it the predominant configuration among the chalcones [27]. starting materials decomposed. Refluxing the reaction mixture of coupling partners in EtOH in the presence of Pd(OAc)2 and Cs2CO3 led to the formation of product 11a with only a 20% yield. The best result of Suzuki cross-coupling was accomplished using Pd(PPh3)4 as a catalyst and Cs2CO3 as a base and by performing the reaction at 80 °C with a microwave irradiation potency of 150 W in EtOH. 1-[1-Phenyl-3-(pyridinyl)-1H-pyrazol-4-yl]ethan-1-ones 11a,b were obtained in 63-64% yields. The reaction was carried out in the presence of KBr, which is known to suppress triflate reduction by stabilizing the cationic (σ-aryl)-palladium transition state [62]. 4-Acetylpyrazoles 11a,b were further employed in the Claisen-Schmidt condensation reaction with different carbaldehydes. The reaction was performed under the above-described conditions in the presence of ethanolic NaOH at 55 °C. As a result, when 1-[1-phenyl-3-pyridinyl-1H-pyrazol-4yl]ethan-1-ones 11a,b were reacted with benzaldehyde, 4-methyl-or 4-(trifluoromethoxy)benzaldehyde E-and Z-chalcones (12a-f and 13a-f, respectively) were obtained in fair to good total yields (51-70%). The NMR spectra of inseparable mixtures showed the presence of both isomers in different ratios, with a predominance of the Eisomer. In contrast, compounds 12g-j were obtained only as pure E-isomers. This latter observation can be explained by the strong electron-donor capacity of the 4-methyloxy group based on the resonance structure, which results in increased electron density of the enone moiety, while the electronegativity of the 4-trifluoromethyl group has a significance for decreased electron density of the enone moiety. It is known in most cases that the Eisomer is more stable from the perspective of thermodynamics, which makes it the predominant configuration among the chalcones [27]. It is widely accepted that a large and constant difference in the magnitudes of the 3 JHH coupling constants of the olefinic protons in E-Z isomers can be used for structural elucidation, which in our case were larger by approximately 3 Hz for the predominant E-isomer (15.6-15.7 Hz), while the minor Z-isomer provided significantly lower coupling constant values (12.7-12. 8 Hz). Moreover, the 1D selective NOESY experimental data clearly It is widely accepted that a large and constant difference in the magnitudes of the 3 J HH coupling constants of the olefinic protons in E-Z isomers can be used for structural elucidation, which in our case were larger by approximately 3 Hz for the predominant E-isomer (15.6-15.7 Hz), while the minor Z-isomer provided significantly lower coupling constant values (12.7-12. 8 Hz). Moreover, the 1D selective NOESY experimental data clearly showed that upon irradiation of the olefinic protons of the minor Z-isomer, the expected NOEs between them were observed. In the case of the major E-isomer, the olefinic protons exhibited only appropriate correlations with neighboring aromatic protons, therefore, unambiguously confirming the correct configuration (Figures S1-S3).
As expected, the NMR spectral data of compound 12g revealed a distinct difference in chemical shifts in the 1H-pyrazol-4-yl moiety compared with the other series of pyrazolochalcones, due to the pyridin-3-yl substituent on the third position of the pyrazole ring ( Figure 4). The key information for structure elucidation of the pyridin-3-yl moiety was obtained from the 1 H-1 H TOCSY spectrum. The results clearly showed a spin system of four protons, which were mostly downfield. Moreover, a comparison between the 1 H-1 H COSY spectra and the 1 H-1 H TOCSY spectra showed a complete absence of COSY cross-peaks between one of the protons, with the remainder from the aforementioned spin system. This finding strongly hinted at a neighboring quaternary carbon at site 3 , which was unambiguously assigned from 1,1-ADEQUATE spectral data, where the protonated pyridine carbons C-2 (δ 149.0 ppm) and C-4 (δ 135.9 ppm) showed a sole correlation with C-3 at δ 127.5 ppm. The remainder of the protonated pyridine carbons were easily assigned from the appropriate correlations in the 1 H- 13 C H2BC spectrum. The 3-(pyridinyl)-1H-pyrazol-4-yl heterocyclic system contains three nitrogen atoms. The chemical shifts of the N-1 and N-2 atoms of compound 12g were δ −161.8 and δ −78.2 ppm, respectively. The pyridin-3-yl substituent nitrogen resonated at δ −70.6 ppm.
showed that upon irradiation of the olefinic protons of the minor Z-isomer, the expected NOEs between them were observed. In the case of the major E-isomer, the olefinic protons exhibited only appropriate correlations with neighboring aromatic protons, therefore, unambiguously confirming the correct configuration (Figures S1-S3).
literature [67,68], i.e., treating compound 9a with hydroxylamine in the presence of NaOH in MeOH/H2O (95/5 v/v), led to a mixture of 1,2-oxazoles 14 and 15 with a poor total yield of 23%. The formation of intermediate reaction products, such as isoxazolines or oximes, could be also identified by HPLC/MS data (HPLC data of crude reaction mixture are provided in Figure S194, followed by MS data in Figures S195-S198). The regioselective formation of pyrazole-isoxazoles 14 and 15 was confirmed by NMR studies ( Figure 5). As expected, the 1 H, 13 C and 15 N NMR chemical shifts and the relevant correlations in the two-dimensional NMR spectra of these two isomeric 1,2-oxazoles were highly similar. The unambiguous formation of 1,2-oxazole (isoxazole) moiety was easily deduced from 1 H- 15 N HMBC spectral data, as it clearly showed a distinct longrange correlation between the isoxazole methine 4′-H proton and nitrogen N-2′, which resonated at δ −18.6 and −19.6 ppm for compounds 15 and 14, respectively, and this is in good agreement with the data reported in the literature [69]. The 2 Hz optimized 1 H- 15 N HMBC spectra hinted in favor of these structures. For instance, the conversion of chalcone 4a provided an 1,2-oxazole derivative, in which the pyrazole 5-H proton (singlet, δ 8.33 ppm) exhibited not only long-range HMBC correlations throughout the 1H-pyrazol-4-yl moiety, but also a weak correlation with the oxazole N-2′ nitrogen at δ −19.6 ppm was observed. Meanwhile, the 1,2-oxazole derivative obtained from chalcone 9a was assigned to structure 15, due to the correlation with the neighboring protons 2″(6″)-H (δ 7.88 ppm) from the phenyl moiety. The aforementioned protons from the pyrazole and phenyl moieties in the 1 H- 13 C HMBC spectrum showed three-bond connectivities with the appropriate isoxazole quaternary carbons C-3′ and C-5′, which allowed us to confirm the correct structure assignments afterwards via the analysis of JCN couplings. The regioselective formation of pyrazole-isoxazoles 14 and 15 was confirmed by NMR studies ( Figure 5). As expected, the 1 H, 13 C and 15 N NMR chemical shifts and the relevant correlations in the two-dimensional NMR spectra of these two isomeric 1,2oxazoles were highly similar. The unambiguous formation of 1,2-oxazole (isoxazole) moiety was easily deduced from 1 H- 15 N HMBC spectral data, as it clearly showed a distinct longrange correlation between the isoxazole methine 4 -H proton and nitrogen N-2 , which resonated at δ −18.6 and −19.6 ppm for compounds 15 and 14, respectively, and this is in good agreement with the data reported in the literature [69]. The 2 Hz optimized 1 H- 15 N HMBC spectra hinted in favor of these structures. For instance, the conversion of chalcone 4a provided an 1,2-oxazole derivative, in which the pyrazole 5-H proton (singlet, δ 8.33 ppm) exhibited not only long-range HMBC correlations throughout the 1H-pyrazol-4-yl moiety, but also a weak correlation with the oxazole N-2 nitrogen at δ −19.6 ppm was observed. Meanwhile, the 1,2-oxazole derivative obtained from chalcone 9a was assigned to structure 15, due to the correlation with the neighboring protons 2 (6 )-H (δ 7.88 ppm) from the phenyl moiety. The aforementioned protons from the pyrazole and phenyl moieties in the 1 H- 13 C HMBC spectrum showed three-bond connectivities with the appropriate isoxazole quaternary carbons C-3 and C-5 , which allowed us to confirm the correct structure assignments afterwards via the analysis of J CN couplings.
literature [67,68], i.e., treating compound 9a with hydroxylamine in the presence of NaOH in MeOH/H2O (95/5 v/v), led to a mixture of 1,2-oxazoles 14 and 15 with a poor total yield of 23%. The formation of intermediate reaction products, such as isoxazolines or oximes, could be also identified by HPLC/MS data (HPLC data of crude reaction mixture are provided in Figure S194, followed by MS data in Figures S195-S198). The regioselective formation of pyrazole-isoxazoles 14 and 15 was confirmed by NMR studies ( Figure 5). As expected, the 1 H, 13 C and 15 N NMR chemical shifts and the relevant correlations in the two-dimensional NMR spectra of these two isomeric 1,2-oxazoles were highly similar. The unambiguous formation of 1,2-oxazole (isoxazole) moiety was easily deduced from 1 H- 15 N HMBC spectral data, as it clearly showed a distinct longrange correlation between the isoxazole methine 4′-H proton and nitrogen N-2′, which resonated at δ −18.6 and −19.6 ppm for compounds 15 and 14, respectively, and this is in good agreement with the data reported in the literature [69]. The 2 Hz optimized 1 H- 15 N HMBC spectra hinted in favor of these structures. For instance, the conversion of chalcone 4a provided an 1,2-oxazole derivative, in which the pyrazole 5-H proton (singlet, δ 8.33 ppm) exhibited not only long-range HMBC correlations throughout the 1H-pyrazol-4-yl moiety, but also a weak correlation with the oxazole N-2′ nitrogen at δ −19.6 ppm was observed. Meanwhile, the 1,2-oxazole derivative obtained from chalcone 9a was assigned to structure 15, due to the correlation with the neighboring protons 2″(6″)-H (δ 7.88 ppm) from the phenyl moiety. The aforementioned protons from the pyrazole and phenyl moieties in the 1 H- 13 C HMBC spectrum showed three-bond connectivities with the appropriate isoxazole quaternary carbons C-3′ and C-5′, which allowed us to confirm the correct structure assignments afterwards via the analysis of JCN couplings.  Then, in order to avoid any ambiguity in the structure assignment of regioisomeric 1,2-oxazoles, the 15 15 N-labeling in azaheterocycles is an important method for studying molecular structures, which significantly expands the possibilities of using standard NMR meth-ods [70]. The 15 N-labeled aromatic heterocyclic structures typically have well-resolved 1 H-15 N (J HN ) and 13 C-15 N (J CN ) coupling constants, including additional splitting of the corresponding signals in the standard proton decoupled 1D 13 C NMR and 1D 1 H NMR spectra [71,72].
Then, in order to avoid any ambiguity in the structure assignment of regioisomeric 1,2-oxazoles, the 15 N-labeled pyrazole-isoxazoles 16 and 17 were synthesized by analogy to 14 and 15. The treatment of chalcone 9a with 15 N-hydroxylamine hydrochloride produced an inseparable mixture of regioisomers 16 and 17 in a ratio of about 8:1 (Scheme 6). The selective 15 N-labeling in azaheterocycles is an important method for studying molecular structures, which significantly expands the possibilities of using standard NMR methods [70]. The 15 N-labeled aromatic heterocyclic structures typically have well-resolved 1 H-15 N (JHN) and 13 C-15 N (JCN) coupling constants, including additional splitting of the corresponding signals in the standard proton decoupled 1D 13 C NMR and 1D 1 H NMR spectra [71,72]. In the case of 15 N-labeled pyrazole-isoxazoles 16 and 17, the analysis of 1 H-15 N (JHN) coupling constants 3 JH4′-N2′ did not provide significant information regarding the correct structure confirmation, and were 1.23 Hz and 1. 31 Hz for major and minor regioisomers, respectively. As expected, the unambiguous structure assignment of regioisomeric 1,2oxazoles was achieved after a careful analysis of the 13 Hz), and C-5′ ( 2 JC5′-N2′ = 1.52 Hz) in the 1,2-oxazole moiety, which is in good agreement with the data reported in the literature [73]. Moreover, the 2 JCN and 3 JCN couplings were observed for the signals from the pyrazole fragment. These 13 C-15 N spin-spin interactions with adjacent phenyl and pyrazole moieties were an additional criterion to confirm the final structures of the pyrazole-isoxazoles 16 and 17.

General Information
All starting materials were purchased from commercial suppliers and were used as received. Microwave reactions were conducted using a CEM Discover synthesis unit (CEM Corp., Matthews, NC, USA) and performed in glass vessels (capacity: 10 mL), sealed with a septum. The pressure was controlled by a load cell connected to the vessel. The temperature of the contents of the vessel was monitored using a calibrated infrared temperature controller, mounted under the reaction vessel. All experiments were performed with stirring. Flash column chromatography was performed on silica gel, 60 Å In the case of 15 N-labeled pyrazole-isoxazoles 16 and 17, the analysis of 1 H-15 N (J HN ) coupling constants 3 J H4 -N2 did not provide significant information regarding the correct structure confirmation, and were 1.23 Hz and 1. 31 Hz for major and minor regioisomers, respectively. As expected, the unambiguous structure assignment of regioisomeric 1,2-oxazoles was achieved after a careful analysis of the 13 C-15 N (J CN ) coupling constants, which were obtained from a 13 C NMR spectrum. The 13 C-15 N spin-spin interaction was observed for the signals of the major regioisomer C-3 ( 1 J C3 -N2 = 2.89 Hz), C-4 ( 2 J C4 -N2 = 1. 23 Hz) and C-5 ( 2 J C5 -N2 = 1.39 Hz) from the 1,2-oxazole moiety, as well as the 2 J CN and 3 J CN couplings from the adjacent phenyl ring. The minor regioisomer provided similar data, where the 1 J CN coupling constants were higher than 2 J CN coupling constants, C-3 ( 1 J C3 -N2 = 2.25 Hz), C-4 ( 2 J C4 -N2 = 1.11 Hz), and C-5 ( 2 J C5 -N2 = 1.52 Hz) in the 1,2-oxazole moiety, which is in good agreement with the data reported in the literature [73]. Moreover, the 2 J CN and 3 J CN couplings were observed for the signals from the pyrazole fragment. These 13 C-15 N spin-spin interactions with adjacent phenyl and pyrazole moieties were an additional criterion to confirm the final structures of the pyrazole-isoxazoles 16 and 17.

General Information
All starting materials were purchased from commercial suppliers and were used as received. Microwave reactions were conducted using a CEM Discover synthesis unit (CEM Corp., Matthews, NC, USA) and performed in glass vessels (capacity: 10 mL), sealed with a septum. The pressure was controlled by a load cell connected to the vessel. The temperature of the contents of the vessel was monitored using a calibrated infrared temperature controller, mounted under the reaction vessel. All experiments were performed with stirring. Flash column chromatography was performed on silica gel, 60 Å (230-400 µm, Merck). Thin-layer chromatography was carried out on silica gel plates (Merck Kieselgel 60 F254) and visualized by UV light (254 nm). The melting points were determined on a Büchi M-565 melting point apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and were uncorrected. The IR spectra were recorded on a Bruker Vertex 70v FT-IR spectrometer (Bruker Optik GmbH, Ettlingen, Germany) using neat samples or on a Bruker Tensor 27 (Bruker Optik GmbH, Ettlingen, Germany) spectrometer using KBr pellets and were reported in frequency of absorption (cm −1 ). Mass spectra were obtained on a Shimadzu LCMS-2020 (ESI + ) spectrometer (Shimadzu Corporation, Kyoto, Japan). High-resolution mass spectra were measured on a Bruker MicrOTOF-Q III (ESI + ) apparatus (Bruker Daltonik GmbH, Bremen, Germany). The 1 H, 13 C and 15 N NMR spectra were recorded in CDCl 3 or DMSOd 6 solutions at 25 • C on a Bruker Avance III 700 (700 MHz for 1 H, 176 MHz for 13 C, and 71 MHz for 15 N) spectrometer (Bruker BioSpin AG, Fallanden, Switzerland), equipped with a 5 mm TCI 1 H- 13 C/ 15 N/D z-gradient cryoprobe and a Bruker Avance III 400 (400 MHz for 1 H, 101 MHz for 13 C, and 40 MHz for 15 N) spectrometer (Bruker BioSpin AG), using a 5 mm directly detecting BBO probe. The chemical shifts (δ), expressed in ppm, were relative to tetramethylsilane (TMS). The 15 N NMR spectra were referenced to neat, external nitromethane (coaxial capillary). Full and unambiguous assignment of the 1 H, 13 C and 15 N NMR resonances was achieved using a combination of standard NMR spectroscopic techniques [74], such as DEPT, COSY, TOCSY, NOESY, ROESY, gs-HSQC, gs-HMBC, H2BC, LR-HSQMBC and 1,1-ADEQUATE experiments [75]. The following abbreviations are used in reporting NMR data: Ph, phenyl; Pz, pyrazole; Pyr, pyridine; Naph, naphtalene; Quin, quinoline; Th, thiophene; Ox, 1,2-oxazole. 1 H-, 13 C-, and 1 H- 15

General Procedure for the Synthesis of 3b,c
Phosphoryl chloride (0.37 mL, 4 mmol) was added dropwise to DMF (0.31 mL, 4 mmol) at −10 • C. Then, 2b,c (1 mmol) was added to the Vilsmeier-Haack complex, and the reaction mixture was heated at 70 • C for 1 h. After the neutralization with 10% aq NaHCO 3 , the precipitate was filtered off and recrystallized from DCM to produce pure 3b,c.

General Procedure for the Synthesis of 5a-c
To a solution of 4g-j (1 mmol) in toluene (3 mL), TFA (3mL) was added. The reaction mixture was stirred at room temperature for 24 h. Toluene and trifluoroacetic acid were evaporated. The residue was recrystalized from ACN to produce pure 5a-c.