Quercetin Hybrids—Synthesis, Spectral Characterization and Radical Scavenging Potential Quercetin Hybrids—Synthesis, Spectral Characterization and Radical Scavenging Potential

+359-32-261-346 Abstract: New quercetin-based derivatives are synthesized in an easily accessible one-pot manner. The method is based on the reaction of quercetin with in situ formed electrophilic N -alkoxycarbonylazolium ions. The position of the newly formed C-C bond and structure were spectrally characterized by 1D, 2D 1 H, 13 C-NMR, IR, and MS analysis. Thus, in all cases, good regioselectivity in the C-8 position for the obtained products was demonstrated. The obtained compounds were evaluated for their DPPH and ABTS free radical scavenging activity and compared to natural compounds—quercetin and rutin. Abstract: New quercetin-based derivatives are synthesized in an easily accessible one-pot manner. The method is based on the reaction of quercetin with in situ formed electrophilic N alkoxycarbonylazolium ions. The position of the newly formed C-C bond and structure were spectrally characterized by 1D, 2D 1 H, 13 C-NMR, IR, and MS analysis. Thus, in all cases, good regioselectivity in the C-8 position for the obtained products was demonstrated. The obtained compounds were evaluated for their DPPH and ABTS free radical scavenging activity and compared to natural compounds—quercetin and rutin. hybrids— 6 – 8 a , b mol ﬁles.


Introduction
Flavonoids constitute one of the most common groups of plant phenolics. In plants, flavonoids occur as aglycones, their glycosides and methylated derivatives [1,2]. Quercetin ( Figure 1) is a flavonoid aglycone abundantly found in almost all edible vegetables and fruits.

Introduction
Flavonoids constitute one of the most common groups of plant phenolics. In plants, flavonoids occur as aglycones, their glycosides and methylated derivatives [1,2]. Quercetin ( Figure 1) is a flavonoid aglycone abundantly found in almost all edible vegetables and fruits. Quercetin is a natural compound with a variety of pharmacological properties, such as anti-inflammatory, antioxidant [3,4], and anticancer activities [5,6]. This flavonoid is also known to be a potent inhibitor for several clinically and toxicologically important enzymes, such as NADH-cytochrome b5 reductase [7], NQO1 in HepG2 cells, after activation of nuclear factor E2-related factor 2 (Nrf2) [8]. In addition, quercetin has the ability to enter cells and accumulate in mitochondria. Carrasco-Pozo et al. reported a protective effect against mitochondrial dysfunction produced by indomethacin in Caco-2 cells [9]. It is proved that the lipophilization of quercetin modulates its functional Quercetin is a natural compound with a variety of pharmacological properties, such as anti-inflammatory, antioxidant [3,4], and anticancer activities [5,6]. This flavonoid is also known to be a potent inhibitor for several clinically and toxicologically important enzymes, such as NADH-cytochrome b5 reductase [7], NQO1 in HepG2 cells, after activation of nuclear factor E2-related factor 2 (Nrf2) [8]. In addition, quercetin has the ability to enter cells and accumulate in mitochondria. Carrasco-Pozo et al. reported a protective effect against mitochondrial dysfunction produced by indomethacin in Caco-2 cells [9]. It is proved that the lipophilization of quercetin modulates its functional antioxidant properties, increasing hydrophobicity and reducing water solubility [10]. In the last two years, the remarkable benefits of quercetin in the treatment of the COVID-19 virus and the resulting problems have also been established [11][12][13]. The synthetic derivatives of quercetin have attracted interest nowadays because of their diverse bioactive profile and application as antioxidant and neuroprotective agents [14] and antiviral and cytotoxic agents [15]. The mainly employed method for the preparation of quercetin derivatives is based on the reactions of O-alkylation or acylation [14,16]. Another commonly used approach is C-aminoalkylation based on the Mannich reaction [17][18][19]. In this context, Ilkei et al. reported the C-8 regioselective synthesis of three flavonoid alkaloids by the biomimetic transformation of some 5,7-dihydroxylatedflavonoids [20]. However, methods for direct C-heteroarylation and azole scafold based alkylation of quercetin are still not found in the literature.
Certainly, 2-substituted derivatives of benzothiazole and benzimidazole are important biologically active compounds with diverse medicinal applications [21][22][23]. Furthermore, the synthesis of hybrid molecules containing two pharmacophore fragments and their evaluation as potent drug candidates have been under constant escalation for the last two decades [24,25]. Additionally, the radical scavenging assays of the new synthetic compounds have been consummated to analyze their effectiveness as antioxidant agents. Many methods have been developed to determine antioxidant activity. They are all based on the generation of various free radicals that interact with antioxidants for a period of time. Some of the most applied are DPPH and ABTS-assay [3,4].
The current direction of our scientific group is developing an accessible approach to various heterocycles [26][27][28]. The aim of this study is to apply this method for obtaining new quercetin-benzazole hybrids and evaluate their radical scavenging potential.

Results and Discussion
Here we report the investigations on the application of adducts obtained from various benzazoles (benzothiazole, benzimidazole, and 5,6-dimethylbenzimidazole) (1-3) with alkyl chloroformates (4a, b) for α-amidoalkylation of quercetin (5). The above-mentioned reactions lead to products (6-8 a, b, Scheme 1). antioxidant properties, increasing hydrophobicity and reducing water solubility [10]. In the last two years, the remarkable benefits of quercetin in the treatment of the COVID-19 virus and the resulting problems have also been established [11][12][13]. The synthetic derivatives of quercetin have attracted interest nowadays because of their diverse bioactive profile and application as antioxidant and neuroprotective agents [14] and antiviral and cytotoxic agents [15]. The mainly employed method for the preparation of quercetin derivatives is based on the reactions of O-alkylation or acylation [14,16]. Another commonly used approach is C-aminoalkylation based on the Mannich reaction [17][18][19]. In this context, Ilkei et al. reported the C-8 regioselective synthesis of three flavonoid alkaloids by the biomimetic transformation of some 5,7dihydroxylatedflavonoids [20]. However, methods for direct C-heteroarylation and azole scafold based alkylation of quercetin are still not found in the literature.
Certainly, 2-substituted derivatives of benzothiazole and benzimidazole are important biologically active compounds with diverse medicinal applications [21][22][23]. Furthermore, the synthesis of hybrid molecules containing two pharmacophore fragments and their evaluation as potent drug candidates have been under constant escalation for the last two decades [24,25]. Additionally, the radical scavenging assays of the new synthetic compounds have been consummated to analyze their effectiveness as antioxidant agents. Many methods have been developed to determine antioxidant activity. They are all based on the generation of various free radicals that interact with antioxidants for a period of time. Some of the most applied are DPPH and ABTS-assay [3,4].
The current direction of our scientific group is developing an accessible approach to various heterocycles [26][27][28]. The aim of this study is to apply this method for obtaining new quercetin-benzazole hybrids and evaluate their radical scavenging potential.

Results and Discussion
Here we report the investigations on the application of adducts obtained from various benzazoles (benzothiazole, benzimidazole, and 5,6-dimethylbenzimidazole) (1-3) with alkyl chloroformates (4a, b) for α-amidoalkylation of quercetin (5). The abovementioned reactions lead to products (6-8 a, b, Scheme 1). The reaction conditions, including the ratio of the reagents, temperature, and time, are presented in Table 1. The reaction conditions, including the ratio of the reagents, temperature, and time, are presented in Table 1. The one-pot reactions were successfully completed under heating (60-80 • C) for 1.5-24 h. It was found that acetonitrile is a suitable solvent for carrying out the reactions. The best yields of products (6-8 a, b) in the range of 37% to 61% (Table 1) were obtained with a two or threefold excess of benzazole and alkyl chloroformates. Acetonitrile was found to be the optimal solvent for the reactions, while halogenated solvents, such as dichloromethane or 1,2-dichloroethane, were not sufficient due to the low solubility of quercetin. Table 1 indicates the reaction conditions under which the C-8 monosubstituted products were obtained with the highest yields. The yield of the products depended on the stability of the in situ generated N-acyliminium adducts. In this regard, ethyl chloroformate performed better than methyl chloroformate for higher-yielding products. The mixture of regioisomers was observed (monitored by TLC) with the equimolar ratio of starting reagents according to our previously published study [28]. For the reactions with benzimidazole and 5,6-dimethylbenzimidazole, the presence of Et 3 N as a hydrochloric scavenger was required. The conditions for the amidoalkylation of quercetin with Nalkoxycarbonylbenzimidazolium adduct obtained from benzimidazole (2) with methyl chloroformate (4b) were not established and, therefore, the expected product was not successfully isolated.
Analytically pure samples of only C-8 monosubstituted products were isolated by column chromatography on silica using a mixture of petroleum/diethyl ether as eluents, and the yields are indicated in Table 1. The structure of C-8 alkylated products (6-8 a,  b) were confirmed by 1D, 2D 1 H-NMR experiments. In particular, the 1 H resonances of the quercetin and the obtained hybrids are reported in the text below. Comparing these chemical shifts with the one of quercetin reported by Kyriakou et al. [29] led to the conclusion that substitution occurs in the C-8 position. The absence of signal about 6.40 ppm in 1 H-NMR spectra and the C-8 downfield shifts in the range of δ = 97.1-99.0 ppm ( 13 C-NMR spectra) for the compounds (6-8 a, b) correctly support the above-mentioned conclusion. The 1 H-NMR spectra of the analyzed compounds indicate singlets in the range of δ = 7.50-7.68 ppm for the proton on sp 3 -C-2 atom in benzazole rings. The 1 H-, 13 C-HSQC measurements proved the exact location of these characteristic signals. The resulting products were structurally characterized by 1D, 2D 1 H, 13 C-NMR, IR, and MS spectra. Original spectra are available in the Supplementary Materials section (S1).

In Vitro DPPH and ABTS Radical Scavenging Assay
The new quercetin hybrids were screened for their in vitro antioxidant property by DPPH and ABTS radical scavenging assay using quercetin and rutin as the standards. The effectiveness of the compounds to scavenge the free radicals was calculated using Equation (1): where A 0 is the absorbance of the blank sample and A 1 is the absorbance in the presence of the analysis compounds.
The free radical scavenging potential of the synthetic quercetin hybrids varied in the range of IC 50 = 6.17 ± 0.3 µM (8b)-7.26 ± 0.3 µM (8a) measured with DPPH assay, and IC 50 = 49.8 ± 3.5 µM (6a)-62.4 ± 3.5 µM (8a) with ABTS assay. The hybrid molecules had the same high antioxidant activity as natural antioxidants quercetin and rutin investigated by DPPH-assay. The radical scavenging activity of synthetic derivatives was close to the quercetin activity and higher than rutin. The summarized results are presented in Table 2.

Materials and Methods
Quercetin Melting points were measured on a Boetius PHMKO5 hot stage apparatus (Carl Zeiss Jena, Germany) and are uncorrected. IR spectra were measured on a VERTEX 70 FT-IR spectrometer (Bruker Optics, Ettlingen, Germany). High resolution mass spectral measurements were performed on a Bruker mass spectrometer. 1 H-, 13 C-NMR spectra were measured on Bruker Avance AV600 spectrometer (Bruker, Billerica, MA, USA) at BAS-IOCCP -Sofia, and chemical shifts (δ, ppm) are downfield from TMS. To average out the rotamers observed in quercetin-N-acyl benzazole hybrids reaching adequate assignment of peaks and structure determination, the spectra were measured at 80 • C in DMSO-d 6 , as indicated in the text below. TLC was done on precoated 0.2 mm Merck silica gel 60 plates. Silica gel was used for column chromatography. The absorbance of the antioxidant assay was measured by a Spectroquant Pharo 300, UV/Vis spectrophotometer. Quercetin Hybrids (6a, 6b), General Procedure

Synthesis of
The corresponding alkyl chloroformate (ethyl chloroformate-3 mmol, 0.29 mL or methyl chloroformate-3 mmol, 0.24 mL) was added dropwise to a magnetically stirred solution of benzothiazole (3 mmol, 0.33 mL) in acetonitrile (10 mL/mmol), followed immediately by quercetin (0.302 g, 1 mmol). The reaction was continued under the conditions described in Table 1. After completion of the reaction (monitored by TLC), the solvent was evaporated under reduced pressure, and the crude mixture was dry-loaded onto silica gel. The products were isolated by column chromatography on silica gel with mixtures of petroleum/diethyl ether as the eluents and successfully crystallized. Quercetin Hybrids (7a, 8a, 8b), General Procedure

Synthesis of
The corresponding alkyl chloroformate (ethyl chloroformate-2 mmol, 0.19 mL or methyl chloroformate-2 mmol, 0.16 mL) was added dropwise to a magnetically stirred at 0 • C solution of benzimidazole (2 mmol, 0.236 g) or 5,6-dimethylbenzimidazole (2 mmol, 0.292 g) and triethylamine (2 mmol, 0.28 mL) in acetonitrile (10 mL/mmol). After 20 min. another 2 mmol alkyl chloroformate were added, followed immediately by quercetin (0.302 g, 1 mmol). The reaction was then continued under the conditions described in Table 1. After completion of the reaction (monitored by TLC), the solvent was evaporated under reduced pressure, the residue was dissolved in chloroform (20 mL) and washed twice with water (40 mL). The organic layer was dried (Na 2 SO 4 ), and the crude mixture was dry-loaded onto silica gel. The products were isolated by column chromatography on silica gel with mixtures of petroleum/diethyl ether as the eluents and successfully crystallized.

DPPH Free Radical Scavenging Assay
The DPPH free radical scavenging activities were measured as previously reported by Docheva et al. [30]: 0.12 mM DPPH was dissolved in methanol. The absorbance change was measured at 515 nm on a UV-Vis spectrophotometer within 30 min. The total DPPH radical scavenging activity within 30 min was measured in triplicate in the absence of light. The blank sample was prepared as above by replacing the test sample with equivalent methanol. The radical scavenging activity (RSA%) was calculated. IC 50 value determined the effective concentration at which 50% of DPPH radicals were scavenged, and it was obtained by interpolation from linear regression analysis. A lower IC 50 value indicates a higher antioxidant activity.

ABTS Free Radical Scavenging Assay
The ABTS free radical was prepared by the method of Re et al. [32] with some modification. ABTS radical cation (ABTS •+ ) was produced by 7 mM ABTS and 2.45 mM K 2 S 2 O 8 dissolved in deionized H 2 O (the mixture stayed in the dark at room temperature for 12-16 h before use). A mixture of the reagents as 1:1 (v/v) ABTS •+ solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 734 nm. The ABTS radical scavenging activity within 10 min was measured in triplicate in the absence of light at room temperature. The percentage inhibition (%) of radical scavenging activity was calculated according to the corresponding equation of the method.
The stock solution of all compounds was prepared in a concentration of 1 mg/mL. The working solutions were prepared by dissolving aliquot parts of the stock solution with methanol. The final concentration of IC 50 was calculated according to the dilution factor. Statistical analysis from the antioxidant activity was analyzed using SPSS. Data were mean ± S.D. The experiments were performed three times. Quercetin

Conclusions
We have successfully synthesized five new quercetin-benzazole hybrids, employing an efficient one-pot approach. The applied reaction of amidoalkylation offers a convenient synthetic route for modification of quercetin by introducing a heterocyclic moiety. The radical scavenging activity of the newly obtained quercetin derivatives gives a good lead for further structural optimization.
Supplementary Materials: S1.PDF-Processed 1 H-, 13  Funding: The present study was financially supported by the National Science Fund of the Bulgarian Ministry of Education and Science (grant number KΠ 06 M59/5-2021), and by the operational program "Science and education for smart growth" 2014-2020 (grant number BG05M2OP001-1.002-0005-c01), Personalized Innovative Medicine competence centre (PERIMED).

Data Availability Statement:
The data presented in this study are available in this article and supporting information.