Synthesis, Structural Investigations, and In Vitro/In Silico Bioactivities of Flavonoid Substituted Biguanide: A Novel Schiff Base and Its Diorganotin (IV) Complexes

Quercetin is one of the most powerful bioactive dietary flavonoids. The in vivo biological study of quercetin is extremely difficult due to its very low solubility. However, diorganotin complexes of quercetin are more useful when contrasted with quercetin due to increased solubility. In the present study, quercetin, substituted biguanide synthesized in the form of Schiff base and its di-alkyl/aryl tin (IV) complexes were obtained by condensing Schiff base with respective di-alkyl/aryl tin (IV) dichloride. Advanced analytical techniques were used for structural elucidation. The results of biological screening against Gram-positive/Gram-negative bacteria and fungi showed that these diorganotin (IV) derivatives act as potent antimicrobial agents. The in silico investigation with dihydropteroate (DHPS) disclosed a large ligand–receptor interaction and revealed a strong relationship between the natural exercises and computational molecular docking results.


Introduction
Flavonoids are polyphenolic compounds found in many plants, consisting of a threecarbon chain framework connected together by two phenyl rings (C 6 -C 3 -C 6 ); two C 6 groups are two benzene rings containing a pyran ring ( Figure 1A). Flavonoids are categorized into flavonols, flavanones, flavones, isoflavones, chalcones, catechins, and anthocyanidins on the basis of chemical structure. Flavonoids are associated with a wide range of promising impacts and found to be a basic component in a variety of nutraceutical, drug, and restorative applications. The dietary intake of flavonoids may result in a reduced risk of different types of cancers, such as gastric, breast, prostate, and colorectal cancer. In addition, they are known to be powerful inhibitors for a few proteins, such as xanthine oxidase (XO), cyclooxygenase (COX), lipoxygenase, and phosphoinositide 3-kinase. Currently, as cellular dysfunction is linked to cell proliferation in an oxidative environment, it is hypothesized ROS-producing enzymes, such as xanthine oxidase and lipoxygenase, are significant in the development of new drugs [1][2][3][4].
On the other hand, organotin complexes are the most diverse segment of chemical compounds with applications ranging from material science and catalytic activities to One of these flavones is quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) ( Figure 1B). Quercetin has been found to act as a very significant chemotherapy agent with some tremendous pharmacological activities, such as anti-cancer [5,6], antimicrobial, and antitumor [7,8]. Moreover, quercetin is one of the compelling metal chelators with three potential chelating positions, i.e., 3 & 4, 5 & 7, and 3 & 4 . Furthermore, it has been observed that complexing of quercetin with metal upgrades its pharmacokinetic reactions both in vitro and in vivo [9][10][11][12][13][14].
On the other hand, organotin complexes are the most diverse segment of chemical compounds with applications ranging from material science and catalytic activities to therapeutic agents for various diseases due to their wide variety of interesting structural possibilities [27]. Organotin compounds occupied an important place in cancer chemotherapy reports [28] due to their cytotoxic effects, ability to bind with DNA, anti-proliferating nature, and apoptotic-inducing nature. Organotin complexes, especially with Schiff base ligands, have been screened for their role in anti-microbial and anti-inflammatory activities [29].
The chemical structures of various reported organometallic complexes of flavonoids support the magnificent radical scavenging ability. Valentina Uivarosi et al. found that the metal-flavonoid complexes have enhanced the physiochemical and biological properties of flavonoids [30]. In another study, Andrea Kurzwernhart et al. reported that the organometallic complexes of flavanol scaffold employing O,O chelating ligand system seizes the in vitro anticancer activity [31]. Considering the biological importance of these compounds and the challenges due to the poor solubility of quercetin, the present study discloses the synthesis, spectral investigations, biological screening, and in silico study of diorganotin (IV) derivatives of novel Schiff base, where Schiff base is obtained from quercetin and biguanide. The results of the in silico study strongly support the potential of synthesized complexes as biocidal agent [32].

Infrared Spectra
The coordination of Schiff base ligand (H 2 L) with the Sn atom was verified by evaluating the IR spectra of ligand and complexes (1-4) ( Figure S2). The IR spectra of ligand displaced a broad band at 3394-3370 cm −1 confirming v(O-H) stretching vibration of coordinated water molecules and -OH groups of the ligand. The absorption band obtained in Schiff base ligand (H 2 L) at 1657 cm −1 was found to be absorbed at lower values by 20-30 cm −1 and observed at 1627-1637 cm −1 in the spectra of diorganotin (IV) complexes [33,34]. The band at 1299 cm −1 in spectra of Schiff base ligand was attributed to v(C-O-C) stretching which remains unchanged in the spectra of diorganotin complex; therefore, indicating that there is no involvement of some oxygen atom of any ring in complexation. Furthermore, the bands obtained at values 501-520 cm −1 and 460-480 cm −1 were attributed to v(Sn-O) and v(Sn-N) vibrations, respectively [35,36] and, indeed, confirm the co-ordination between tin and Schiff base ligand.

HNMR Spectra
The 1 HNMR spectra of complexes recorded in DMSO-d6 are found to exhibit peaks according to the proposed structure( Figure S1). The absence of peak for the free 3-OH indicates deprotonation of the hydroxyl groups due to bonding with Sn (IV). The chemical shifts at 12.18-12.21 and 8.17-8.21 ppm proved that 5-OH and 7-OH groups are still present in the complexes (1-4) [37]. The aromatic protons of H 2 L and derivatives seemed identical and were observed as multiplets in the range of 6-7 ppm, indicating non-participation of aromatic ring in complex formation. The spectra of complexes consisted of new shifts at 0.79, 0.90, 0.98, and 7.36 ppm, and were attributed to -CH 3 , -C 2 H 5 , -C 4 H 9 , and phenyl protons, respectively [38].

C NMR Spectra
The 13 C NMR spectra confirmed the resonance of aromatic rings in the range of 115-157 ppm for the ligand and remained undisturbed in the spectra of complexes reflecting the non-participation of aromatic ring in bond formation with the central Sn atom. The height at 148.9 ppm, which was attributed to 3-OH carbon in the spectra of ligand, received a limit in the spectra of complexes suggesting its bonding with the tin atom. The spectra of complexes consisted of new signals due to carbon-tin bonding, at 8.51 due to -CH 3 carbon, 7.5, 33.1, and 147.9-124.1 ppm for -C 2 H 5 , -C 4 H 9 , and phenylcarbon atoms, respectively [34].

Sn NMR Spectra
The chemical shift value of 119 Sn NMR describes the coordination variety of complexes. Signal for Sn was determined at δ −126.03 in the spectra of methyl complexes, whereas at δ -156.04 ppm in ethyl and at δ −290.25 and −310.04 ppm in the complexes of butyl and phenyl, respectively [39]. These values of chemical shift certainly prove that the Penta-coordinated surroundings for tin atom, and additionally all the indicators appear as

Mass Spectra
LCMS technique has been utilized for the determination of mass, and thus the molecular weight of the compounds. The spectra of the ligand contained a peak at m/z = 669. 57

UV-Visible Spectroscopic Study of the Complexes
The UV-visible spectrum of H 2 L indicated two primary absorption bands at 365 nm (band I) and 262 nm (band II). Band I is associated with ring B (cinnamoyl system) and band II with ring A (benzoyl system), Figure 1 [44][45][46]. The depth of H 2 L (band I) decreases progressively with the addition of tin (IV) derivatives, and new absorption peaks appeared at 410, 414, 420, and 430 for methyl, ethyl, butyl, and phenyl, respectively. These shifted peaks indicated the coordination of tin (IV) to the 3-hydroxyl groups of ring C [47]. Band I bathochromic shift can be defined by the interaction of tin (IV) with the 3-OH involving ring C, producing electrical redistribution between H 2 L and its derivatives, which resulted in the π-bonding. The 5-OH does not involve (i) 3-OH with higher chelation potential than the 5-OH and (ii) the delocalization of the oxygen electrons of the 3-OH involvement was greater than 5-OH [48].

Antimicrobial Assay
In vitro antimicrobial screening tests of the synthesized ligand and its diorganotin (IV) derivatives have been carried out against four bacterial strains; two Gram-positive (Staphylococcus aureus, Bacillus subtillis) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa), as well as two fungal species (Scopulariopsis canadensis, Aspergillus niger). All tests were carried out in triplicates. Measurements of the inhibition zones were recorded as a mean value of three readings, which are shown in Table 1. Based on the pharmacological data, it has been observed that complex three or four had positive antimicrobial activities against all the examined species with better efficacy than the antibiotics used as standard (ciprofloxacin and fluconazole). Due to the presence of azomethine linkage (>C=N-), complexes have been found to inhibit microbial growth. Different mechanisms have been suggested including the inhibition of cell wall synthesis and loss of the bioactivity of fundamental enzymes, such as dihydropteroate synthase [49,50]. In the current study, the examined ligand and their metal complexes possess strong anti-microbial activities. Promising antimicrobial activities of the metal complexes may be due to the chelation ability of the ligand [51]. As per the theory, chelation effects result in enhancement of permeability of the drug into the cytoplasm of the cell [52]. Bu 2 SnL and Ph 2 SnL derivatives have a promising antimicrobial action in contrast to ligand alone. The results indicated that phenyl by-product (4) acts as an enhanced anti-microbial agent with maximum zone of inhibition, i.e., 30 mm with MIC value of 79 ppm in evaluation to butyl by-product (29 mm and 84 ppm). This effect may be due to the delocalization of π-electrons and lipid soluble nature of tin atoms, which facilitate their entry into the microbial cells [53,54]. Therefore, upon evaluating the antimicrobial ability of the diorganotin (IV) complexes and conventional antibiotics (ciprofloxacin and fluconazole), it was found that complexes (3 & 4) have considerable activity against the tested microbes, in contrast to the antibiotics that demonstrated their use as effective antibacterial/antifungal agents. Table 1. Antimicrobial screening data of the synthesized ligand and its diorganotin (IV) derivatives.

Molecular Docking Studies
Staphylococcus aureus is widely known as a Gram-positive bacterium that is a frequent cause of life-threatening infections in hospitals, such as bacteremia, toxic shock syndrome, infective endocarditis, osteomyelitis, gastroenteritis, meningitis, septic arthritis, respiratory tract infections, pulmonary infections, skin and soft tissue infections, urinary tract infections, and prosthetic device infections [55,56]. Therefore, dihydropteroate synthase (DHPS) was targeted for the molecular docking studies. The enzyme DHPS performs a key function in microbial folate biosynthesis through the manufacturing of 7,8-dihydropteroate from dihydropterin pyrophosphate (DHPP) and para-aminobenzoic acid (pABA) [57]. The loss of the bioactivity of DHPS is suggested as one of the main mechanisms of the inhibition of microbial growth [46,47]. The X-ray crystal structure of the dihydropteroate synthase (DHPS) from staphylococcus aureus (1AD4) was used as a possible target for the synthetized Schiff base and its diorganotin (IV) complexes [58][59][60][61][62]. Although the synthesized ligand (H 2 L) displayed the strongest binding affinity (−9.7 kcal/mol) to the DHPS (PDB ID: 1AD4), the in vitro antimicrobial activity of H 2 L was determined as lower than the synthesized complexes. Consistent with the literature, quercetin's low solubility appears to suppress its extensive biocidal action [15,16]. Moreover, the synthesized Me2SnL (1) and Bu2SnL (3) showed the weakest binding properties with binding affinities of 8.4 kcal/mol (Figures 2 and 3 and Table 2), although Bu2SnL (3) was determined as one of the most effective antimicrobial agent on S.aureus with maximum zone of inhibition, i.e., 29 mm with MIC value of 84 ppm (Table 1). Therefore, it is hypothesized that Bu2SnL (3) triggers additional antimicrobial mechanisms to enhance its biocidal capacity, and should be further investigated. On the other hand, the synthesized Ph2SnL (4) was determined as the most effective complex among the synthesized complexes with binding affinity of −8.9 kcal/mol in correlation with in vitro experimental results (Tables 1 and 2; Figure 2).       DNA gyrase subunit B (1KZN) is considered to be a very important target for antibacterial drug design and discovery [63]. DNA gyrase is an important bacterial protein that is involved in the replication, transcription, and the catalysis of the negative supercoiling of bacterial circular DNA [64]. Therefore, the crystal structure of E. coli 24 kDa DNA gyrase fragment was used as a possible target for the synthetized Schiff base and its diorganotin (IV) complexes. Similar to the docking studies of 1AD4, the synthesized ligand (H 2 L) showed the strongest binding affinity (−9.5 kcal/mol) to the DNA gyrase subunit B, although the in vitro antimicrobial activity of H 2 L on E. coli was lower than the synthesized complexes (Tables 1 and 2). Additionally, it was determined that the data obtained in molecular docking studies performed with DNA gyrase (1KZN) were not compatible with the results of antimicrobial tests on E. coli. Although molecular docking studies indicated that Et 2 SnL (−9.0 kcal/mol) and Bu 2 SnL (−8.5 kcal/mol) are the most active ones among the synthesized complexes, respectively, the antimicrobial tests showed that Ph 2 SnL (4) and Bu 2 SnL (3) had respectively more antimicrobial activity on E. coli than the others (Tables 1 and 2; Figure 3). It seems that the antimicrobial activity of the synthesized complexes is not directly related to the effect on DNA gyrase enzyme.
Aspergillosis is known as an invasive and potentially life-threatening infection caused by Aspergillus, and frequently observed in immunocompromised patients [65]. UDP-Nacetylglucosamine pyrophosphorylase (UAP1) was reported as a potential drug target for Aspergillus fumigatus since it plays substantial roles in the biosynthesis of fungal cell wall by catalyzing the biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide [66]. Therefore, the crystal structure of UDP-N-acetylglucosamine pyrophosphorylase (6TN3) from Aspergillus fumigatus was used as a possible target for the synthetized Schiff base and its diorganotin (IV) complexes. The molecular docking studies showed that Me 2 SnL, Et 2 SnL, and Bu 2 SnL have similar binding affinities to the UAP1 by −9.2, −9.2, and −9.1 kcal/mol, respectively (Table 2 and Figure 4). Although the synthesized complexes show strong binding affinities on UAP1, their in vitro antifungal activities are not very compatible with the molecular docking results (Tables 1 and 2). For example, Ph 2 SnL was determined as the most effective antifungal complex among the synthesized diorganotin (IV) complexes, although its binding affinity was determined as the weakest one (−7.3 kcal/mol). Consequently, it is clear that there is a need to further investigate alternative drug targets in order to fully understand the antimicrobial activity mechanisms of the synthesized complexes, and it should be noted that there are complicated biological and chemical processes for the emergence of antimicrobial activities of drugs.

Materials and Measurements
Diorganotin dichloride and quercetin.2H 2 O were purchased from Sigma Aldrich (USA). Pure biguanide was prepared from biguanide sulphate. All other solvents have been purchased from Merck (INDIA) and dried before use, according to the preferred strategies [67]. The spectroscopic evaluation was performed at SAIF lab Panjab University, Chandigarh. Elemental analysis was carried out using Perkin-Elmer instrument and IR spectral investigations were achieved with KBr pellet. The NMR ( 1 H, 13

Synthesis of Diorganotin (IV) Derivatives
The Schiff base ligand H2L (3 mmol) was dissolved in methanol solvent (25 mL) and stirred for approximately 20-30 min. Next, the above solution was refluxed with a methanolic solution of methyl (0.659 g, 3 mmol), ethyl (0.743 g,3 mmol), butyl (0.911 g, 3 mmol), and phenyl (01.031 g, 3 mmol) derivatives of diorganotin (IV) dichloride. Then, a few drops of triethylamine were added to this solution and the content was refluxed for 7-8 h. After cooling, the obtained mixture was evaporated to half of its original volume

Synthesis of Diorganotin (IV) Derivatives
The Schiff base ligand H 2 L (3 mmol) was dissolved in methanol solvent (25 mL) and stirred for approximately 20-30 min. Next, the above solution was refluxed with a methanolic solution of methyl (0.659 g, 3 mmol), ethyl (0.743 g, 3 mmol), butyl (0.911 g, 3 mmol), and phenyl (01.031 g, 3 mmol) derivatives of diorganotin (IV) dichloride. Then, a few drops of triethylamine were added to this solution and the content was refluxed for 7-8 h. After cooling, the obtained mixture was evaporated to half of its original volume under low pressure. Finally, the obtained orange-colored product was washed with methanol or diethyl ether and dried in vacuum (Scheme 2). Scheme 1. Synthesis of Schiff base.

Synthesis of Diorganotin (IV) Derivatives
The Schiff base ligand H2L (3 mmol) was dissolved in methanol solvent (25 mL) and stirred for approximately 20-30 min. Next, the above solution was refluxed with a methanolic solution of methyl (0.659 g, 3 mmol), ethyl (0.743 g,3 mmol), butyl (0.911 g, 3 mmol), and phenyl (01.031 g, 3 mmol) derivatives of diorganotin (IV) dichloride. Then, a few drops of triethylamine were added to this solution and the content was refluxed for 7-8 h. After cooling, the obtained mixture was evaporated to half of its original volume under low pressure. Finally, the obtained orange-colored product was washed with methanol or diethyl ether and dried in vacuum (Scheme 2).

Antimicrobial Activity
The in vitro antimicrobial screening of the newly designed ligand and its corresponding diorganotin (IV) derivatives were evaluated against different strains of microorganisms by determining the minimum inhibitory concentration (MIC). This activity has been evaluated using serial dilution and agar well diffusion technique [68]. The antibacterial activity was measured against Gram-positive species, such as Staphylococcus aureus (MTCC 9760), Bacillus subtillis (MTCC 1133), and Gram-negative strains, i.e., Escherichia coli (MTCC 589), Pseudomonas aeruginosa (MTCC 9048). The antifungal activity was carried out against fungal species, such as Scopulariopsis canadensis (MTCC 567) and Aspergillus niger (MTCC 9933). Ciprofloxacin and fluconazole were used as standard antimicrobials to compare the activities of tested compounds while 2% DMSO was used as a blank. The solutions were prepared by dissolving 5 mg of each compound in 5 mL of 2% DMSO as solvent. Then, 1 mL of the prepared solution was diluted with 9 mL of DMSO for the concentration of the solution to reach a value of up to 100 ppm [69,70].

Molecular Docking Studies
The crystal structures of dihydropteroate synthase (PDB ID: 1AD4), E. coli 24 kDa DNA gyrase domain (PDB ID: 1KZN), and UDP-N-acetylglucosamine pyrophosphorylase (6TN3) were obtained from the protein data bank and prepared for molecular docking using AutoDock Tool 1.5.6 [71]. Chain A of dihydropteroate synthase was determined as the ligand-binding domain for the docking study, and binding coordinates were assigned as center_x = 32.74; center_y = 7.91; center_z = 41.22. For E. coli 24 kDa DNA gyrase domain, the binding coordinates were assigned as center_x = 18.84; center_y = 24.05; center_z = 36.31. For UDP-N-acetylglucosamine pyrophosphorylase, the binding coordinates were assigned as center_x = 22.81; center_y = 60.59; center_z = −4.47. The search space was set up at 30 × 30 × 30 Å for all docking studies. Twenty possible binding conformations for each ligand were generated by AutoDock Vina suite software using genetic algorithm (GA-LS) searches [72,73]. Selected conformations of ligands and proteins were combined in Pymol software and analyzed in Discovery Studio Visualizer [74].

Structure-Activities Relationship
The back bone of the chemical structure of complexes is the presence of bi-flavonoid moiety, i.e., the presence of four benzene rings with two pyranone rings. The eight flavonoid hydroxyl groups present at 11, 12, 16, and 18th and 32, 33, 37, and 39th positions of each complex are responsible for anti-oxidative properties. Four >C=N bonds present at 6, 27, 43, and 46th positions provide excellent biocidal properties, whereas the double bonds present between the 4th and 5th positions and 25th and 26th positions of bi-flavonoid moiety are responsible for effective inhibitory activities [75]. Furthermore, complexation of Schiff base with organotin (IV) increases the biocidal potential of complex, which is possibly due to the increase in lipophilic character.

Conclusions
The current research involved the synthesis of novel, Schiff base ligand using quercetin and biguanide as initial materials. In the subsequent step, diorganotin (IV) complexes of the designed Schiff base ligand were synthesized. The spectroscopic records confirmed that the oxygen atom of the 3-OH group and imine nitrogen atoms are coordination sites for central tin atom, which result in the formation of penta-coordinated complexes. Antimicrobial screening displayed that the diorganotin (IV) complexes have greater inhibition potential than the Schiff base ligand, which may be due to the increase in lipophilicity and permeability through the cell membrane following the formation of complexes. The excessive lipid solubility of organotin compounds ensures better cell penetration and interaction with intercellular sites. Furthermore, the molecular docking proved that the complex formed in the case of 4 was the most effective agent as an antimicrobial along with the substantial interactions with DHPS (1AD4) receptor protein. Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.