Synthesis and Characterization of New Pyrano[2,3-c]pyrazole Derivatives as 3-Hydroxyflavone Analogues

In this paper, an efficient synthetic route from pyrazole-chalcones to novel 6-aryl-5-hydroxy-2-phenylpyrano[2,3-c]pyrazol-4(2H)-ones as 3-hydroxyflavone analogues is described. The methylation of 5-hydroxy-2,6-phenylpyrano[2,3-c]pyrazol-4(2H)-one with methyl iodide in the presence of a base yielded a compound containing a 5-methoxy group, while the analogous reaction of 5-hydroxy-2-phenyl-6-(pyridin-4-yl)pyrano[2,3-c]pyrazol-4(2H)-one led to the zwitterionic 6-(N-methylpyridinium)pyrano[2,3-c]pyrazol derivative. The treatment of 5-hydroxy-2,6-phenylpyrano[2,3-c]pyrazol-4(2H)-one with triflic anhydride afforded a 5-trifloylsubstituted compound, which was further used in carbon–carbon bond forming Pd-catalyzed coupling reactions to yield 5-(hetero)aryl- and 5-carbo-functionalized pyrano[2,3-c]pyrazoles. The excited-state intramolecular proton transfer (ESIPT) reaction of 5-hydroxypyrano[2,3-c]pyrazoles from the 5-hydroxy moiety to the carbonyl group in polar protic, polar aprotic, and nonpolar solvents was observed, resulting in well-resolved two-band fluorescence. The structures of the novel heterocyclic compounds were confirmed by 1H-, 13C-, 15N-, and 19F-NMR spectroscopy, HRMS, and single-crystal X-ray diffraction data.


Introduction
Fused pyrazole derivatives represent an important class of organic compounds as they are found in a large number of biologically and chemically active compounds [1].These compounds are known for their anticancer [2], antimicrobial [3], antiviral [4], and anti-coagulant properties [5], and for their activity against CNS disorders [6].Some of the fused pyrazole moieties are present in marketed drugs, such as apixaban, sildenafil, indiplon, zaleplon, etazolate, cartazolate, allopurinol, and futibatinib, which was recently approved by the U.S. Food and Drug Administration (FDA) [7].
3-Hydroxyflavone I (Figure 1) is known as the backbone of all flavonols.Flavonols are a class of the flavonoid family, a group of naturally occurring substances with variable phenolic structures, found in fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine [16][17][18].Quercetin II and kaempferol III (Figure 1) are the most prevalent in plants and are among the flavonols that have been most investigated and reviewed for beneficial health properties, such as antioxidant, antimicrobial, hepatoprotective, and antiinflammatory properties, and other effects [19,20].Synthetic and semisynthetic flavonol derivatives have been reported in the literature in an attempt to improve the biochemical and pharmacological properties of their corresponding natural compounds.For example, synthesis and anti-Leishmania activity were reported for benzothiophene-flavonols [21].A series of spirochromone-flavonols [22] and thiophene-pyrazole-flavonols [23] was synthesized and tested as antimicrobial agents.In addition, flavonols containing an isothiazolidine ring have been found to be effective inhibitors of cyclin-dependent kinase 2 (CDK2) [24].
3-Hydroxyflavone I (Figure 1) is known as the backbone of all flavonols.Flavonols are a class of the flavonoid family, a group of naturally occurring substances with variable phenolic structures, found in fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine [16][17][18].Quercetin II and kaempferol III (Figure 1) are the most prevalent in plants and are among the flavonols that have been most investigated and reviewed for beneficial health properties, such as antioxidant, antimicrobial, hepatoprotective, and anti-inflammatory properties, and other effects [19,20].Synthetic and semisynthetic flavonol derivatives have been reported in the literature in an attempt to improve the biochemical and pharmacological properties of their corresponding natural compounds.For example, synthesis and anti-Leishmania activity were reported for benzothiophene-flavonols [21].A series of spirochromone-flavonols [22] and thiophene-pyrazole-flavonols [23] was synthesized and tested as antimicrobial agents.In addition, flavonols containing an isothiazolidine ring have been found to be effective inhibitors of cyclin-dependent kinase 2 (CDK2) [24].3-Hydroxyflavones are known as fluorescent dyes because of their typical excitedstate intramolecular photon transfer (ESIPT).ESIPT is one type of proton transfer reaction that has been the subject of considerable interest and a number of investigations in recent decades [25,26].3-Hydroxyflavones have been investigated as therapeutic imaging agents, including as fluorescence sensors and probes for the detection of the microenvironment, metal ions, and structures of proteins and DNA [27][28][29][30].For example, Jiang et al. reported the application of 3-hydroxyflavone-based ESIPT fluorescent dyes for the dynamic imaging of lipid droplets with cells and tissues [31].In a study by Kamariza et al., a 3-hydroxychromone derivative, 2-[7-(diethylamino)-9,9-dimethyl-9H-fluoren-2-yl]-3hydroxy-4H-chromen-4-one, was conjugated to trehalose and a bright solvatochromic dye was obtained that detects Mycobacterium tuberculosis in a matter of minutes [32].
The O-methylation of 3-hydroxyflavones with reagents such as diazomethane, methyl iodide, dimethyl sulfate, or dimethyl carbonate proceeded to give O-methylated flavonoids, which exhibited a variety of biological activities [33][34][35].For example, Ohtani et al. investigated the effect of 3-methoxyflavone derivatives, such as those of compound IV (Figure 1), on P-glycoprotein by measuring the potentiation of cellular accumulation and growth inhibition [36].Juvale et al. reported the inhibitory activity of 3-methoxyflavones against a breast cancer resistance protein (BVRP/ABCG2) [37].Furthermore, 3-hydroxyflavone treated with p-TsCl in the presence of a base afforded a corresponding flavone tosylate V, which was used in a Suzuki-Miyaura reaction for cross coupling with various phenyl boronic acids to give 2,3-diarylbenzopyrans [38,39].Flavone-like 2,3-diarylbenzopyrans, such as compound VI (Figure 1), have been synthesized as novel selective inhibitors of cyclooxygenase-2 [40,41].3-Hydroxyflavones are known as fluorescent dyes because of their typical excitedstate intramolecular photon transfer (ESIPT).ESIPT is one type of proton transfer reaction that has been the subject of considerable interest and a number of investigations in recent decades [25,26].3-Hydroxyflavones have been investigated as therapeutic imaging agents, including as fluorescence sensors and probes for the detection of the microenvironment, metal ions, and structures of proteins and DNA [27][28][29][30].For example, Jiang et al. reported the application of 3-hydroxyflavone-based ESIPT fluorescent dyes for the dynamic imaging of lipid droplets with cells and tissues [31].In a study by Kamariza et al., a 3-hydroxychromone derivative, 2-[7-(diethylamino)-9,9-dimethyl-9H-fluoren-2-yl]-3-hydroxy-4H-chromen-4-one, was conjugated to trehalose and a bright solvatochromic dye was obtained that detects Mycobacterium tuberculosis in a matter of minutes [32].
The O-methylation of 3-hydroxyflavones with reagents such as diazomethane, methyl iodide, dimethyl sulfate, or dimethyl carbonate proceeded to give O-methylated flavonoids, which exhibited a variety of biological activities [33][34][35].For example, Ohtani et al. investigated the effect of 3-methoxyflavone derivatives, such as those of compound IV (Figure 1), on P-glycoprotein by measuring the potentiation of cellular accumulation and growth inhibition [36].Juvale et al. reported the inhibitory activity of 3-methoxyflavones against a breast cancer resistance protein (BVRP/ABCG2) [37].Furthermore, 3-hydroxyflavone treated with p-TsCl in the presence of a base afforded a corresponding flavone tosylate V, which was used in a Suzuki-Miyaura reaction for cross coupling with various phenyl boronic acids to give 2,3-diarylbenzopyrans [38,39].Flavone-like 2,3-diarylbenzopyrans, such as compound VI (Figure 1), have been synthesized as novel selective inhibitors of cyclooxygenase-2 [40,41].
Scheme 1. Reagents and conditions: (i) appropriate carbaldehyde, NaOH, EtOH, 55 °C, 3-5 h, in accordance with ref. [44]; (ii) NaOH, EtOH, H2O2, −25 °C, 2 h, then rt, 16 h.In a subsequent step, an Algar-Flynn-Oyamada (AFO) synthetic approach was applied for the formation of novel pyrano[2,3-c]pyrazol-4(2H)-ones 3a-h.The AFO reaction is a stepwise process whereby chalcones undergo an oxidative cyclization to form flavones in the presence of alkaline hydrogen peroxide [45].The AFO reaction outcome is dependent on the choice of the base; therefore, chalcone 2a was used as a model compound for the fine-tuning of the reaction conditions.Several organic and inorganic bases (NaOH, KOH, NaOAc, TEA, and NaHCO 3 ) were screened in different mixtures of ethanol/water as a solvent and a divergent amount of hydrogen peroxide (Table S1).The best result was obtained when using NaOH in EtOH and employing 5 eq of H 2 O 2 .Stirring chalcones 2a-h with hydrogen peroxide in an alkaline ethanolic solution at −25 • C for 2 h and at room temperature overnight afforded the flavonol analogues 3a-h in poor to good yields (30-67%).The pyrano[2,3-c]pyrazol-4(2H)-ones 3e and 3g were obtained in lower yields (30-32%) when chalcones bearing naphtalen-2-yl or furan-3-yl substituents (2e and 2g, respectively) were used as starting materials in the AFO reaction.Unfortunately, the AFO reaction of (E)-3-( 4 2, path B), followed by an attack of hydrogen peroxide and subsequent oxidation to form 3a [47].
is a stepwise process whereby chalcones undergo an oxidative cyclization to form flavones in the presence of alkaline hydrogen peroxide [45].The AFO reaction outcome is dependent on the choice of the base; therefore, chalcone 2a was used as a model compound for the fine-tuning of the reaction conditions.Several organic and inorganic bases (NaOH, KOH, NaOAc, TEA, and NaHCO3) were screened in different mixtures of ethanol/water as a solvent and a divergent amount of hydrogen peroxide (Table S1).The best result was obtained when using NaOH in EtOH and employing 5 eq of H2O2.Stirring chalcones 2ah with hydrogen peroxide in an alkaline ethanolic solution at −25 °C for 2 h and at room temperature overnight afforded the flavonol analogues 3a-h in poor to good yields (30-67%).The pyrano[2,3-c]pyrazol-4(2H)-ones 3e and 3g were obtained in lower yields (30-32%) when chalcones bearing naphtalen-2-yl or furan-3-yl substituents (2e and 2g, respectively) were used as starting materials in the AFO reaction.Unfortunately, the AFO reaction of (E)-    Subsequently the methylation of compound 3h containing both the hydroxyl group and the pyridin-4-yl substituent was investigated (Scheme 3).With the alkylation reaction conditions described above (MeI, Cs2CO3, dioxane, 40 °C), a formation of zwitterionic pyrano[2,3-c]pyrazol derivative 5 as the main product was observed.The proposed mechanism for the formation of compound 5 is shown in Scheme 3. Presumably, first, as a result of the reaction of pyridinyl-containing compound 3h with methyl iodide, methylpyridinium iodide 6 was formed.This was also demonstrated when alkylating compound 3h in the absence of a base as salt 6 was obtained in a 78% yield.The subsequent treatment of methylpyridinium iodide 6 with a base led to the formation of methylpyridinium hydroxide Y, which, upon the removal of the water molecule, led to the formation of the corresponding structure 5 as a resonance hybrid with the two contributing forms A and B, zwitterionic and neutral molecular structures, respectively.Pat et al. investigated the two-photon absorption (TPA) processes in a class of 4-quinopyran chromophores.The neutral molecular structure with a quinoid geometry is the molecular ground state, while the zwitterionic configuration with a benzenoid structure contributes significantly.The bond connecting the donor and acceptor phenylene fragments is a Scheme 2. O-Methylation of compound 3a.
Subsequently the methylation of compound 3h containing both the hydroxyl group and the pyridin-4-yl substituent was investigated (Scheme 3).With the alkylation reaction conditions described above (MeI, Cs 2 CO 3 , dioxane, 40 • C), a formation of zwitterionic pyrano[2,3-c]pyrazol derivative 5 as the main product was observed.Subsequently the methylation of compound 3h containing both the hydroxyl group and the pyridin-4-yl substituent was investigated (Scheme 3).With the alkylation reaction conditions described above (MeI, Cs2CO3, dioxane, 40 °C), a formation of zwitterionic pyrano[2,3-c]pyrazol derivative 5 as the main product was observed.The proposed mechanism for the formation of compound 5 is shown in Scheme 3. Presumably, first, as a result of the reaction of pyridinyl-containing compound 3h with methyl iodide, methylpyridinium iodide 6 was formed.This was also demonstrated when alkylating compound 3h in the absence of a base as salt 6 was obtained in a 78% yield.The subsequent treatment of methylpyridinium iodide 6 with a base led to the formation of methylpyridinium hydroxide Y, which, upon the removal of the water molecule, led to the formation of the corresponding structure 5 as a resonance hybrid with the two contributing forms A and B, zwitterionic and neutral molecular structures, respectively.Pat et al. investigated the two-photon absorption (TPA) processes in a class of 4-quinopyran chromophores.The neutral molecular structure with a quinoid geometry is the molecular ground state, while the zwitterionic configuration with a benzenoid structure contributes significantly.The bond connecting the donor and acceptor phenylene fragments is a The proposed mechanism for the formation of compound 5 is shown in Scheme 3. Presumably, first, as a result of the reaction of pyridinyl-containing compound 3h with methyl iodide, methylpyridinium iodide 6 was formed.This was also demonstrated when alkylating compound 3h in the absence of a base as salt 6 was obtained in a 78% yield.The subsequent treatment of methylpyridinium iodide 6 with a base led to the formation of methylpyridinium hydroxide Y, which, upon the removal of the water molecule, led to the formation of the corresponding structure 5 as a resonance hybrid with the two contributing forms A and B, zwitterionic and neutral molecular structures, respectively.Pat et al. investigated the two-photon absorption (TPA) processes in a class of 4-quinopyran chromophores.The neutral molecular structure with a quinoid geometry is the molecular ground state, while the zwitterionic configuration with a benzenoid structure contributes significantly.The bond connecting the donor and acceptor phenylene fragments is a double bond when the molecule is neutral, while it is a single bond for the zwitterionic structure [48].

NMR Spectroscopic Investigations
The formation of 6-(hetero)aryl-5-hydroxy-2-phenylpyrano[2,3-c]pyrazol-4(2H)-ones 3a-h and their derivatives 4, 5, 6, 7, and 8a-g was confirmed through detailed analysis of their spectroscopic data.Key information for structure elucidation was obtained from NMR spectral data using a combination of standard and advanced NMR spectroscopy techniques, such as 1 H- 13 C HMBC, 1 H-13 C LR-HSQMBC, 1 H-15 N HMBC, 1 H- 13 C HSQC, 1 H-13 C H2BC, 1 H-1 H COSY, 1 H-1 H TOCSY, 1 H-1 H NOESY, and 1,1-ADEQUATE experiments.Since popular NMR prediction programs such as CSEARCH, ACD C+H predictor, as well as NMR chemical shift databases for structural dereplication depend on high-quality data with unambiguously assigned resonances [53], we carried out NMR studies with the obtained compounds to fully map all the 1 H, 13 C and 15 N NMR signals as accurately as possible.The corresponding NMR data for the selected representatives of the aforementioned new ring systems are displayed in Figures 3 and 4.
An initial comparison of the 1 H NMR spectra between chalcone 2a and compound 3a, which was isolated as the sole product, clearly indicated the disappearance of characteristic olefinic protons (δ 7.63 and 7.75 ppm) from the prop-2-en-1-one moiety.Furthermore, the 13 C NMR and DEPT, along with the 1 H- 13 C HSQC spectroscopic data of 3a, revealed the presence of two new quaternary carbons (δ 139.16 and 144.4 ppm) in the absence of two olefinic methine carbons, clearly indicating a successful oxidative cyclization to flavanol.The structure of the pyrano[2,3-c]pyrazol-4(2H)-one ring system 3a bearing phenyl substituents at sites N-2 and C-6 was further elucidated via the connectivities based on the through-space correlations from the 1 H-1 H NOESY spectrum.In this case, distinct NOEs were exhibited between the pyrazole ring proton 3-H (singlet, δ 9.38 ppm) and the neighboring phenyl group 2 (6 )-H protons (δ 8.01-8.03ppm), which confirms their proximity in space.The pyrazole 3-H proton was easily distinguished as it exhibited not only long-range HMBC correlations with neighboring N-2 "pyrrole-like" (δ −167.7 ppm) and N-1 "pyridine-like" (δ −117.0 ppm) nitrogen atoms, but also HMBC correlations with the quaternary carbons C-3a (δ 108.3 ppm) and C-7a (δ 161.2 ppm), respectively.The quaternary carbons C-5 (δ 139.16 ppm) and C-6 (δ 144.4 ppm) were assigned by comparing the long-range correlations obtained from the 1 H- 13 C HMBC and 1 H-13 C LR-HSQMBC spectra.The most downfield and significantly broadened 1 H signal resonating at δ 9.44 ppm was attributed to the hydroxyl group as it lacked correlations in the HSQC spectra.Finally, by process of elimination, the most downfield 13 C signal resonating at δ 171.8 ppm was confidently assigned to the carbonyl carbon, thus completing our assignment of the pyrano[2,3-c]pyrazol-4(2H)-one ring system.An in-depth analysis of NMR spectral data showed that the chemical shift values were highly consistent within the flavonol analogues 3a-h, thus validating the shifts for each position (Table S2).  1C HMBC, 1 H-13 C LR-HSQMBC, 1 H- 13 C H2BC, 1 H- 15 N HMBC, 1 H-1 H NOESY, and 1,1-ADEQUATE correlations, as well as 1 H NMR (italics), 13 C NMR, and 15 N NMR (bold) chemical shifts of compounds 3a (DMSO-d6), 4 (DMSO-d6), and 8f (CDCl3).
The presence of a hydroxyl group at site 5 was further confirmed by the conversion of 3a to O-methylated and O-triflated derivatives 4 and 7, respectively.While the structural elucidation of O-methylated compound 4 was straightforward and followed the same logical approach as in the case of compounds 3a-h, additional distinct NOEs were observed between the methoxy group protons (δ 3.77 ppm) and the neighboring phenyl group 2″(6″)-H protons (δ 7.96-7.98ppm).The formation of O-triflated derivative 7 was clearly distinguished from the 13 C NMR spectrum, where the CF3 group was observed as a quartet at δ 118.2 ppm (q, 1 JCF = 320.8Hz).Moreover, the 19 F NMR spectrum revealed a chemical shift of the CF3 group at δ −74.0 ppm, which is in good agreement with the data reported in the literature [54,55].The triflate intermediate 7 underwent Pd-catalyzed coupling reactions to give derivatives 8a-g, whose structures were also unambiguously elucidated.For instance, compound 8f was obtained as an E-isomer.The magnitude of the vicinal coupling between the olefinic protons Ha (δ 7.35 ppm) and Hb (δ 7.31 ppm), which exhibited an AB-spin system and appeared as two sets of doublets ( 3 JHa,Hb = 15.9Hz), unquestionably confirmed E-configuration at the C=C double bond.Lastly, the olefinic protons were easily discriminated as only the proton Ha exhibited long-range 1 H- 13 C HMBC correlations with neighboring C-4, C-5, and C-6 quaternary carbons (Figure 3).
The presence of a hydroxyl group at site 5 was further confirmed by the conversion of 3a to O-methylated and O-triflated derivatives 4 and 7, respectively.While the structural elucidation of O-methylated compound 4 was straightforward and followed the same logical approach as in the case of compounds 3a-h, additional distinct NOEs were observed between the methoxy group protons (δ 3.77 ppm) and the neighboring phenyl group 2 (6 )-H protons (δ 7.96-7.98ppm).The formation of O-triflated derivative 7 was clearly distinguished from the 13 C NMR spectrum, where the CF 3 group was observed as a quartet at δ 118.2 ppm (q, 1 J CF = 320.8Hz).Moreover, the 19 F NMR spectrum revealed a chemical shift of the CF 3 group at δ −74.0 ppm, which is in good agreement with the data reported in the literature [54,55].The triflate intermediate 7 underwent Pd-catalyzed coupling reactions to give derivatives 8a-g, whose structures were also unambiguously elucidated.For instance, compound 8f was obtained as an E-isomer.The magnitude of the vicinal coupling between the olefinic protons H a (δ 7.35 ppm) and H b (δ 7.31 ppm), which exhibited an AB-spin system and appeared as two sets of doublets ( 3 J Ha,Hb = 15.9Hz), unquestionably confirmed E-configuration at the C=C double bond.Lastly, the olefinic protons were easily discriminated as only the proton H a exhibited long-range 1 H- 13 C HMBC correlations with neighboring C-4, C-5, and C-6 quaternary carbons (Figure 3).
The NMR spectral data of compound 3h with a pyridine moiety at site 6 revealed similar chemical shifts in the pyrano[2,3-c]pyrazol-4(2H)-one ring system compared with 3a-g.The 1 H- 15 N HMBC spectrum revealed a new downfield 15 N signal resonating at δ −62.2 ppm in addition to N-2 "pyrrole-like" (δ −166.9 ppm) and N-1 "pyridine-like" (δ −117.1 ppm) nitrogen atoms from the pyrazole moiety.The formation of methylpyridinium iodide 6 via the alkylation of 3h was unambiguously confirmed from the 1 H- 15 N HMBC and 1 H-1 H NOESY spectral data.For instance, distinct NOEs were observed between the methyl group protons (δ 4.39 ppm) and the neighboring pyridinium 2 (6 )-H protons (δ 9.03 ppm).The aforementioned protons revealed strong long-range correlations with the methylpyridinium nitrogen at δ −183.3 ppm, which is in good agreement with the data reported in the literature [56].
In the case of compound 5, which can exist in two resonant forms, the 1 H- 15 N HMBC spectrum revealed a new upfield 15 N signal resonating at δ −214.4 ppm.Furthermore, distinct 1 H and 13 C signals, which appeared to be broadened, were also observed (sites 2 , 3 , 5 , and 6 ).Additionally, the key information for the structure elucidation of compounds 3h, 5, and 6 was obtained after an in-depth analysis of the long-range correlations in the 1 H- 13 C HMBC, 1 H-13 C H2BC, and 1 H- 13 C LR-HSQMBC spectra (Figure 4) [57].
Molecules 2023, 28, x 9 of 26 between the methyl group protons (δ 4.39 ppm) and the neighboring pyridinium 2″(6″)-H protons (δ 9.03 ppm).The aforementioned protons revealed strong long-range correlations with the methylpyridinium nitrogen at δ −183.3 ppm, which is in good agreement with the data reported in the literature [56].
Then, we carried out NMR studies of compounds 3h and 5 at 25 °C in TFA-d solutions (Scheme 5) to convert them to pyridinium and methylpyridinium trifluoroacetates 9 and 10, respectively.The 15 N NMR spectral data confirmed that it was easily achieved as "pyridinium-like" 15 N signals comparable to compound 6 resonating at δ −189.2 ppm and δ −183.2 ppm were observed.Moreover, in the case of compound 10, which was obtained from compound 5, the broadening of the 1 H and 13 C signals was absent.Then, we carried out NMR studies of compounds 3h and 5 at 25 • C in TFA-d solutions (Scheme 5) to convert them to pyridinium and methylpyridinium trifluoroacetates 9 and 10, respectively.The 15 N NMR spectral data confirmed that it was easily achieved as "pyridinium-like" 15 N signals comparable to compound 6 resonating at δ −189.2 ppm and δ −183.2 ppm were observed.Moreover, in the case of compound 10, which was obtained from compound 5, the broadening of the 1 H and 13 C signals was absent.Scheme 5. 15 N NMR (bold) chemical shifts of compounds 3h (DMSO-d6), 5 (DMSO-d6), 9 (TFA-d), and 10 (TFA-d).

Single-Crystal X-ray Diffraction Analysis
The asymmetric molecular structure of compound 5 is shown in Figure 5a.The single crystal is composed of compound 5 solvated with molecules of methanol.The methanol formed hydrogen bonds in the monocrystal of 5, including the hydrogen link to the O(15) (the H … O length is 1.917 Å) (Table S6).The intramolecular hydrogen bond is also observed between the O(15) enolate oxygen and the C(17)-H(17) hydrogen atom (the H … O length is 2.207 Å).The main core of compound 5 consists of the planar pyrano[2,3-c]pyrazole ring system, which possesses phenyl and the pyridin-4-yl substituents at N(2) and C(6), respectively.These substituents are slightly distorted from the pyrano[2,3-c]pyrazole plane.The phenyl ring is turned approx.10° and the pyridinyl ring for approx.6° counterclockwise when looking outward from the core.The N(19)-C( 22) bond length of the N-methylpyridinium moiety is 1.4737( 14) Å (Table S9), and the C(17)-C (18) and C(20)-C(21) bond lengths are 1.3721(15) and 1.3633(16) Å, respectively, and agree with the known bond lengths of the N-methylpyridynium salts [58].All atoms of the pyridine moiety are located in the same plane in agreement with the data reported in the literature [59].

Single-Crystal X-ray Diffraction Analysis
The asymmetric molecular structure of compound 5 is shown in Figure 5a.The single crystal is composed of compound 5 solvated with molecules of methanol.The methanol formed hydrogen bonds in the monocrystal of 5, including the hydrogen link to the O(15) (the H . . .O length is 1.917 Å) (Table S6).The intramolecular hydrogen bond is also observed between the O(15) enolate oxygen and the C(17)-H(17) hydrogen atom (the H . . .O length is 2.207 Å).The main core of compound 5 consists of the planar pyrano[2,3-c]pyrazole ring system, which possesses phenyl and the pyridin-4-yl substituents at N(2) and C(6), respectively.These substituents are slightly distorted from the pyrano[2,3-c]pyrazole plane.The phenyl ring is turned approx.10 • and the pyridinyl ring for approx.6 • counterclockwise when looking outward from the core.The N(19)-C (22) bond length of the N-methylpyridinium moiety is 1.4737( 14) Å (Table S9), and the C(17)-C (18) and C(20)-C(21) bond lengths are 1.3721(15) and 1.3633(16) Å, respectively, and agree with the known bond lengths of the N-methylpyridynium salts [58].All atoms of the pyridine moiety are located in the same plane in agreement with the data reported in the literature [59].
The selected bond lengths and angles of the pyrano[2,3-c]pyrazole ring are shown in Tables 1 and 2 12) and 1.3458( 14) Å, respectively, and agree with the known bond lengths of pyrazole compounds [63][64][65][66].The sum of the angles between the covalent bonds around the N(2) atom is 360°, which indicates that a trigonal planar geometry exists at the sp 2 -hybridized nitrogen atom.The molecules in the crystal are located in columns made up of asymmetric units held by hydrogen bonds (Figure 5b).

Optical Investigations
The optical properties of 5-hydroxy-2,6-diphenylpyrano[2,3-c]pyrazol-4(2H)-ones 3a-h in various solvents, such as polar protic (MeOH), polar aprotic (THF and DMF), and non-polar (toluene), were investigated by UV-vis spectroscopy; the compounds were also subjected to fluorimetric measurements.The UV-vis electronic absorption spectra of compounds 3a and 3b in MeOH showed the absorption maximum in the 337 and 341 nm, respectively (Figure 6a, Table 3, entries 1, 2).The presence of electron-donating substituents on the phenyl ring of compounds 3c,d resulted in a bathochromic shift of the longest wavelength absorption transition.The presence of the 4-methoxyphenyl substituent of structure 3c shifted λ max upward by 18 nm (Table 3, entry 3), and the presence of the 3,4dimethoxyphenyl substituent in structure 3d shifted λ max upward by 24 nm (Table 3, entry 4) compared to 3a, respectively.The bathochromic effect of λ max at 353 nm is also observed in the UV spectra of the naphthalene ring containing compound 3e, with a significant delocalization of 10-π electrons (Table 3, entry 5).Moreover, the replacement of the phenyl ring in the molecular structure of the products by heterocyclic rings, thiophen-2-yl, furan-3-yl, and pyridin-4-yl moieties induced a significant bathochromic shift of the near-ultraviolet band compared to that of compound 3a.Specifically, the spectra of the compounds 3f, 3g, and 3h contained intense absorption bands with λ max at 365, 360 and 355 nm, respectively (Table 3, entries 6, 7, 8).

Optical Investigations
The optical properties of 5-hydroxy-2,6-diphenylpyrano[2,3-c]pyrazol-4(2H)-ones 3a-h in various solvents, such as polar protic (MeOH), polar aprotic (THF and DMF), and non-polar (toluene), were investigated by UV-vis spectroscopy; the compounds were also subjected to fluorimetric measurements.The UV-vis electronic absorption spectra of compounds 3a and 3b in MeOH showed the absorption maximum in the 337 and 341 nm, respectively (Figure 6a, Table 3, entries 1, 2).The presence of electron-donating substituents on the phenyl ring of compounds 3c,d resulted in a bathochromic shift of the longest wavelength absorption transition.The presence of the 4-methoxyphenyl substituent of structure 3c shifted λmax upward by 18 nm (Table 3, entry 3), and the presence of the 3,4dimethoxyphenyl substituent in structure 3d shifted λmax upward by 24 nm (Table 3, entry 4) compared to 3a, respectively.The bathochromic effect of λmax at 353 nm is also observed in the UV spectra of the naphthalene ring containing compound 3e, with a significant delocalization of 10-π electrons (Table 3, entry 5).Moreover, the replacement of the phenyl ring in the molecular structure of the products by heterocyclic rings, thiophen-2-yl, furan-3-yl, and pyridin-4-yl moieties induced a significant bathochromic shift of the nearultraviolet band compared to that of compound 3a.Specifically, the spectra of the compounds 3f, 3g, and 3h contained intense absorption bands with λmax at 365, 360 and 355 nm, respectively (Table 3, entries 6, 7, 8).The fluorescence spectra of compounds 3a-h in the MeOH solution contained two well-separated fluorescence bands at around 440 and 590 nm (Figure 6b, Table 3).It is well known that the fluorescence spectra of 3-hydroxyflavone exhibit double emission due to excited-state intramolecular proton transfer (ESIPT) [67][68][69][70][71][72][73][74][75].Similarly, in compounds 3a-h, the proton transfer process (ESIPT) can occur, resulting in the formation of two forms in the excited state: the normal (N*) and tautomeric (ESIPT product, T*) forms.For example, the excitation of form N-3a leads to the excited state N*, which passes into the product T* by means of proton transfer (Figure 7).The T* form then relaxes to the ground state T form and emits fluorescence at a much longer wavelength compared to normal absorption [28,44].Therefore, the form N* of the normal emission has a Stokes shift of 8927 cm −1 , while the tautomeric product T* has a Stokes shift of 12491 cm −1 .
excited-state intramolecular proton transfer (ESIPT) [67][68][69][70][71][72][73][74][75].Similarly, in compounds 3ah, the proton transfer process (ESIPT) can occur, resulting in the formation of two forms in the excited state: the normal (N*) and tautomeric (ESIPT product, T*) forms.For example, the excitation of form N-3a leads to the excited state N*, which passes into the product T* by means of proton transfer (Figure 7).The T* form then relaxes to the ground state T form and emits fluorescence at a much longer wavelength compared to normal absorption [28,44].Therefore, the form N* of the normal emission has a Stokes shift of 8927 cm −1 , while the tautomeric product T* has a Stokes shift of 12491 cm −1 .The fluorescence quantum yield (Φ f ) of the solutions was estimated using the integrating sphere method.It appeared that the fluorescence quantum yield was sensitive to the structure of compounds 3a-h.For unsubstituted compound 3a, a high Φ f value was observed at 59.3%.The fluorescence quantum yield of 4-methoxyphenyl-group-containing compound 3c was low and did not exceed 14%.The highest Φ f value (76.1%) was measured for naphthalen-2-yl-group-containing compound 3e; the thiophen-2-yl, furan-3-yl, and pyridin-4-yl groups of compounds 3f, 3g, and 3h emitted fluorescence with the observed Φ f values of 55.8%, 42.6%, and 13.1%, respectively.It is notable that 3-hydroxyflavone had low quantum yield values in methanol (Φ f = 3%) and DMF (Φ f = 1.3%) [70].
Next, the UV-vis electronic absorption spectra of compounds 3a-h in a polar aprotic solvent, THF, showed the absorption maximum in the 339-362 nm range (Figure 8a, Table 4, entries 1-8).The fluorescence spectra (*λ ex = 380 nm) of compounds 3a-h in the THF solution showed two fluorescence bands at around 441 nm and 591 nm (Figure 8b, Table 4, entries 1-8), which were similar to the bands in MeOH.The inhibition of the ESIPT reaction by protic solvents in 3-hydroxyflavones is associated with the formation of intermolecular H-bonds, which weaken the intramolecular H-bond necessary for the ESIPT reaction [69,74].Therefore, the relative intensity of the N* band was very weak compared to that of the T* band for compounds 3a in THF as an aprotic solvent.Compounds 3c-g, especially the ones containing methoxyphenyl groups, presented dramatically decreased I N* /I T* ratios in the THF solutions compared to those in MeOH, but the 4-chlorophenyl substituent possessing compound 3b retained similar I N* /I T* ratios.However, compound 3h containing the pyridinyl substituent possessed reversed I N* /I T* ratios of 0.221 from 0.031 in MeOH.It is possible that the molecule transfered the corresponding proton to pyridine instead of to the carbonyl group.In this case, the pyridin-4-yl substituent inhibits the proton transfer process (ESIPT).
Next, the UV-vis electronic absorption spectra of compounds 3a-h in a polar aprotic solvent, THF, showed the absorption maximum in the 339-362 nm range (Figure 8a, Table 4, entries 1-8).The fluorescence spectra (*λex = 380 nm) of compounds 3a-h in the THF solution showed two fluorescence bands at around 441 nm and 591 nm (Figure 8b, Table 4, entries 1-8), which were similar to the bands in MeOH.The inhibition of the ESIPT reaction by protic solvents in 3-hydroxyflavones is associated with the formation of intermolecular Hbonds, which weaken the intramolecular H-bond necessary for the ESIPT reaction [69,74].Therefore, the relative intensity of the N* band was very weak compared to that of the T* band for compounds 3a in THF as an aprotic solvent.Compounds 3c-g, especially the ones containing methoxyphenyl groups, presented dramatically decreased IN*/IT* ratios in the THF solutions compared to those in MeOH, but the 4-chlorophenyl substituent possessing compound 3b retained similar IN*/IT* ratios.However, compound 3h containing the pyridinyl substituent possessed reversed IN*/IT* ratios of 0.221 from 0.031 in MeOH.It is possible that the molecule transfered the corresponding proton to pyridine instead of to the carbonyl group.In this case, the pyridin-4-yl substituent inhibits the proton transfer process (ESIPT).
The fluorescence spectra of compound 3a in polar aprotic solvent, DMF, contained two fluorescence bands in the regions of 428 and 589 nm and showed an IN*/IT* ratio of 0.009 (Figure 8b, Table 4, entry 9), while compound 3a in toluene contained two fluorescence bands in the region of 430 and 589 nm and showed an IN*/IT* ratio of 0.004 (Figure 8b, Table 4, entry 10).The fluorescence spectra of compound 3a in polar aprotic solvent, DMF, contained two fluorescence bands in the regions of 428 and 589 nm and showed an I N* /I T* ratio of 0.009 (Figure 8b, Table 4, entry 9), while compound 3a in toluene contained two fluorescence bands in the region of 430 and 589 nm and showed an I N* /I T* ratio of 0.004 (Figure 8b, Table 4, entry 10).Table 4. Absorption (λ abs absorption maxima and ε), fluorescence emission (λ N* em , λ T* em , ratio I N* /I T* , and quantum yield Φ f ) parameters, and Stokes shifts for 3a-h in aprotic solvents ( a THF, b DMF, and c toluene) ( * λ ex = 380 nm); sh = shoulder.

Entry
Comp. λ abs (nm) Derivative 4 with the 5-MeO substituent had an electron spectrum very close to its analog 3a (absorption maximum 306 nm and 302 nm, respectively) (Figure S1a).In the fluorescence spectrum of compound 4 in THF solution, two bands were observed at 475 and 582 nm with insignificant fluorescence (Φ f < 0.1%) (Figure S1b, Table S12, entry 1).Ormson et al. reported that the fluorescence quantum yield for 3-hydroxyflavone is much greater than for the corresponding methoxy compounds and their fluorescence lifetimes are longer [76].
Finally, we investigated the UV-vis spectra of compounds 5 and 6.In a polar aprotic solvent, THF, both compounds showed the same absorption maximum in the 528 nm (Figure 9, Table 5).The versatility of derivatives of zwitterionic chromophore, including pyran compounds, in synthetic and material applications, has been well documented [77][78][79].

General
All the chemicals and solvents were purchased from common commercial suppliers.Diffraction data were collected on a Rigaku, XtaLAB Synergy, Dualflex, HyPix diffractometer (Rigaku Corporation, Tokyo, Japan).The crystals were kept at 150.0(1) K while collecting the data.Using Olex2, the structure was solved with the ShelXT structure solution program using intrinsic phasing and refined with the olex2.refinerefinement package using Gauss-Newton minimization.The 1 H, 13 C, and 15 N NMR spectra were recorded in CDCl3 or DMSO-d6 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 GmbH, Rheinstetten, Germany) equipped with a 5 mm TCI 1 H- 13 C/ 15 N/D z-gradient cryoprobe.The chemical shifts were referenced to tetramethylsilane (TMS) and expressed in ppm.The 15 N NMR spectra were 9. UV-vis electronic absorption spectra of compounds 5 and 6 in THF.

General
All the chemicals and solvents were purchased from common commercial suppliers.Diffraction data were collected on a Rigaku, XtaLAB Synergy, Dualflex, HyPix diffractometer (Rigaku Corporation, Tokyo, Japan).The crystals were kept at 150.0(1) K while collecting the data.Using Olex2, the structure was solved with the ShelXT structure solution program using intrinsic phasing and refined with the olex2.refinerefinement package using Gauss-Newton minimization.The 1 H, 13 C, and 15 N NMR spectra were recorded in CDCl 3 or DMSO-d 6 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 GmbH, Rheinstetten, Germany) equipped with a 5 mm TCI 1 H-13 C/ 15 N/D z-gradient cryoprobe.The chemical shifts were referenced to tetramethylsilane (TMS) and expressed in ppm.The 15 N NMR spectra were referenced against neat, external nitromethane (coaxial capillary). 19F NMR spectrum (376 MHz) was obtained on a Bruker Avance III 400 instrument (Bruker BioSpin AG, Faellanden, Switzerland) with absolute referencing via δ ratio.The FT-IR spectra were recorded by ATR method on either a Bruker Vertex 70v spectrometer (Bruker Optik GmbH, Ettlingen, Germany) with an integrated Platinum ATR accessory or on a Bruker Tensor 27 spectrometer (Bruker Optik GmbH, Ettlingen, Germany) using KBr pellets.The melting points of the crystalline compounds were measured in open capillary tubes with a Buchi M 565 apparatus and are uncorrected.Mass spectra were obtained using a Shimadzu LCMS-2020 (ESI + ) spectrometer (Shimadzu Corporation, Kyoto, Japan).High-resolution mass spectra (HRMS) were measured using a Bruker MicrOTOF-Q III (ESI + ) apparatus (Bruker Daltonik GmbH, Bremen, Germany).All the reactions were performed in ovendried glassware with magnetic stirring.The reaction progress was monitored by TLC analysis on Macherey-Nagel™ ALUGRAM ® Xtra SIL G/UV254 plates (Macherey-Nagel GmbH & Co. KG, Düren, Germany) which were visualized by UV light (254 and 365 nm wavelengths).The compounds were purified by flash chromatography in glass columns (stationary phase of silica gel, high-purity grade of 9385, pore size of 60 Å, and particle size of 230-400 mesh, supplied by Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).
UV-vis spectra were recorded on a Shimadzu 2600 UV/vis spectrometer (Shimadzu Corporation, Japan).The fluorescence spectra were recorded on an FL920 fluorescence spectrometer from Edinburgh Instruments (Edinburgh Analytical Instruments Limited, Edinburgh, UK).The PL quantum yields were determined from dilute solutions by an absolute method using the Edinburgh Instruments integrating sphere excited with a Xe lamp.The optical densities of the sample solutions were ensured to be below 0.1 to avoid reabsorption effects.All the optical measurements were performed at room temperature under ambient conditions.The following abbreviations are used in reporting the NMR data: Ph, phenyl; Pyr, pyridine; Pz, pyrazole; Naph, naphtalene; and Th, thiophene.The 1 H, 13 C, and 1 H- 15

Synthetic Procedures
Compounds 2a-c and 2e-h were synthesized in accordance with the procedure described in ref. [44].

Figure 2 .
Figure 2. Mechanisms proposed for the transformation reaction of compound 2a to compound 3a.

Figure 2 .
Figure 2. Mechanisms proposed for the transformation reaction of compound 2a to compound 3a.For further modification of the obtained flavanol analogue 3a, O-alkylation reaction conditions were applied.As a result, treating 3a with methyl iodide in the presence of cesium carbonate in dioxane at 40 • C gave O-methylated compound 4 in a 79% yield (Scheme 2).

Figure 7 .
Figure 7. Depiction of the ESIPT process in 3a.Measurements of the intensity ratio of the N* and T* bands, IN*/IT*, in ESIPT compounds are used for ratiometric detection [70].A strong effect of group substitution in the compounds 3a-h was observed on the IN*/IT* fluorescence intensity ratio.4-

Figure 9 .
Figure 9. UV-vis electronic absorption spectra of compounds 5 and 6 in THF.