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Article

New Derivatives of Chalcones, Chromenes, and Stilbenoids, Complexed with Methyl-β-Cyclodextrin with Antioxidant Properties and Antibacterial Synergism with Antibiotics

by
Igor D. Zlotnikov
1,
Sergey S. Krylov
2,
Natalya G. Belogurova
1,
Alexander N. Blinnikov
2,
Victor E. Kalugin
2 and
Elena V. Kudryashova
1,*
1
Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1/3, 119991 Moscow, Russia
2
N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
Biophysica 2024, 4(4), 667-694; https://doi.org/10.3390/biophysica4040044
Submission received: 8 November 2024 / Revised: 5 December 2024 / Accepted: 8 December 2024 / Published: 13 December 2024
(This article belongs to the Topic Designing New Antimicrobials Based on Known Valuable Heterocycles as Building Blocks)
Browse Figures
Figure 1
<p>UV/vis absorption spectra of the investigated dyes and chalcones in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (<b>a</b>) Congo Red 8 µM; (<b>b</b>) Methylene blue 15 µM; (<b>c</b>) Malachite green 10 µM; (<b>d</b>) R351-ClO<sub>4</sub><sup>−</sup> 2.5 µM; (<b>e</b>) sample 9–50 µM; (<b>f</b>) Brilliant green 35 µM, toluidine blue 5 µM, gentian violet 3 µM, sudan III 7 µM. PBS (0.01M, pH 7.4). DMSO could be added to enhance the solubility of free samples. T = 37 °C.</p> "> Figure 1 Cont.
<p>UV/vis absorption spectra of the investigated dyes and chalcones in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (<b>a</b>) Congo Red 8 µM; (<b>b</b>) Methylene blue 15 µM; (<b>c</b>) Malachite green 10 µM; (<b>d</b>) R351-ClO<sub>4</sub><sup>−</sup> 2.5 µM; (<b>e</b>) sample 9–50 µM; (<b>f</b>) Brilliant green 35 µM, toluidine blue 5 µM, gentian violet 3 µM, sudan III 7 µM. PBS (0.01M, pH 7.4). DMSO could be added to enhance the solubility of free samples. T = 37 °C.</p> "> Figure 2
<p>FTIR spectra of the investigated «drug candidates» in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (<b>a</b>) chalcone (9), (<b>b</b>) 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate (17). PBS (0.01M, pH 7.4). T = 37 °C.</p> "> Figure 3
<p><sup>1</sup>H NMR spectra of the «drug candidate» sample 9: (<b>a</b>) in free form in d<sub>6</sub>-DMSO; (<b>b</b>) complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol) in D<sub>2</sub>O with predicted peak correlations. T = 25 °C. (<b>c</b>) The proposed structure of the chalcone 9—MCD complex obtained during computer modeling. Carbon atoms are indicated in green (MCD) and blue (the guest molecule, compound <b>9</b>); oxygen atoms are indicated in red. hydrogen—white, sulfur—yellow, nitrogen—blue. The purple sphere is Na<sup>+</sup>. The simulation was performed using the PyMOL program. (<b>d</b>) Schematic cyclodextrin torus representation.</p> "> Figure 3 Cont.
<p><sup>1</sup>H NMR spectra of the «drug candidate» sample 9: (<b>a</b>) in free form in d<sub>6</sub>-DMSO; (<b>b</b>) complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol) in D<sub>2</sub>O with predicted peak correlations. T = 25 °C. (<b>c</b>) The proposed structure of the chalcone 9—MCD complex obtained during computer modeling. Carbon atoms are indicated in green (MCD) and blue (the guest molecule, compound <b>9</b>); oxygen atoms are indicated in red. hydrogen—white, sulfur—yellow, nitrogen—blue. The purple sphere is Na<sup>+</sup>. The simulation was performed using the PyMOL program. (<b>d</b>) Schematic cyclodextrin torus representation.</p> "> Figure 4
<p>ABTS antioxidant test of the “drug candidates” complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol): (<b>a</b>) Dyes and fluorophores; (<b>b</b>) Chalcone and stilbene derivatives. PBS (0.01M, pH 7.4). T = 37 °C.</p> "> Figure 4 Cont.
<p>ABTS antioxidant test of the “drug candidates” complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol): (<b>a</b>) Dyes and fluorophores; (<b>b</b>) Chalcone and stilbene derivatives. PBS (0.01M, pH 7.4). T = 37 °C.</p> "> Figure 5
<p>The structures of the drug candidates in the relationships with antioxidant and antibacterial properties. The circles highlight significant fragments of molecules.</p> "> Figure 6
<p>Confocal laser scanning microscopy images of (<b>a</b>,<b>b</b>) <span class="html-italic">E. coli</span> cells and (<b>c</b>,<b>d</b>) <span class="html-italic">Lactobacilli</span> cells, stained with R6G (10 µg/mL) in free form or complexed with MCD (100 µg/mL). λexci, max = 488 nm, λemi = 530–580 nm. The scale bar is 20 µm.</p> "> Scheme 1
<p>Synthesis scheme of compound <b>17</b>.</p> "> Scheme 2
<p>Synthesis scheme of Xanthylium derivatives R351 salts compounds.</p> "> Scheme 3
<p>Synthesis scheme of the chalcone and stilbenoid derivatives.</p> ">
Versions Notes

Abstract

:
Cyclodextrins (CDs) are natural cyclic oligosaccharides with the ability to form inclusion complexes with various organic substances. In this paper, we investigate the potential of CD complex formation to enhance the antibacterial activity and antioxidant properties of poorly soluble bioactive agents, such as chalcones, chromenes, stilbenoids and xanthylium derivatives, serving as potential adjuvants, in comparison with standard antiseptics. The interaction of these bioactive agents with the hydrophobic pocket of methyl-β-cyclodextrin (MCD) was confirmed using spectroscopic methods such as UV-vis, FTIR, 1H and 13C NMR, mass-spectrometry. CD-based delivery system allows for combining multiple active agents, improving solubility, antibacterial efficacy by enhancing penetration into target bacterial cells (E. coli selectivity demonstrated via confocal microscopy). Novel compounds of chalcones and stilbenoids derivatives additionally enhance efficacy by inhibiting bacterial efflux pumps, increasing membrane permeability, and inhibiting bacterial enzymes, and showed a synergy when used in combination with metronidazole. The intricate relationship between the structural characteristics and functional properties of chalcones and stilbenoids in terms of their antibacterial and antioxidative capabilities is revealed. The substituents within aromatic rings significantly influence this activity, where position of electron-donating methoxy groups playing a crucial role. Among chalcones, stilbenoids, ana xanthyliums, the compounds caring a benzodioxol ring, analogous to natural bioactive compounds like apiol, dillapiol, and myristicin, emerge as prominent antibacterial activity. To explore the possibility to create theranostic formulations, we used fluorescent markers to visualize target cells, antiseptics to provide antibacterial activity, and bioactive agents as chalcones acting as adjuvants. Additionally, new antioxidant compounds were found such as Xanthylium derivative (R351) and chromene derivative: 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate: the pronounced antioxidant properties of these substances are observed comparable to quercetin in the efficiency. Rhodamine 6G, gentian violet, and Congo Red exhibit good antioxidant properties, although their activity is an order of magnitude lower than that of quercetin. However, they have remarkable potential due to their multifaceted nature, including the ability to visualize target cells. The most effective theranostic formulation is the combination of the antibiotic (metronidazole) + dye/fluorophore (methylene blue/rhodamine 6G) for visualization of target cells + adjuvant (chalcones or xanthylium derivatives) for antiinflammation effect. This synergistic combination, results in a promising theranostic formulation for treating bacterial infections, with enhanced efficiency, selectivity and minimizing side effects.

1. Introduction

Infectious diseases caused by pathogenic bacteria represent a significant threat to society, constituting one of the leading causes of mortality in developing countries and posing a critical challenge for developed nations [1,2,3,4,5,6,7]. The urgent need for the development of novel, more efficient, and safe therapeutic approaches is paramount.
The complex and protracted treatment involving high-dose medications often leads to the emergence of a multitude of undesirable side effects, while bacterial strains simultaneously cultivate resistance and multidrug resistance (MDR) mechanisms [8,9,10,11,12,13]. It is well-established that modern antibacterial agents of the third and fourth generations effectively eliminate pathogenic bacteria under in vitro conditions, often requiring relatively low dosages. However, their efficacy in vivo is significantly compromised, as they exhibit limited penetration into the targeted sites of bacterial localization, including within alveolar macrophages. The low bioavailability of certain drugs necessitates the administration of high doses, particularly in cases such as atypical intracellular pneumonia and pulmonary tuberculosis, which can persist for extended periods, posing a significant risk to the overall health of the patient.
Theranostic system that combines diagnostics and therapy into one molecular container, which allows early detection and effective treatment of diseases with minimal impact on healthy tissues of the body [14,15,16,17,18,19,20,21,22,23,24,25]. Examples of theranostic include radionuclide, photodynamic, and thermotherapy. Herein, we suggest employing dyes and fluorophores as components of a synergistic theranostic formulation to enable the simultaneous diagnosis and treatment of diseases, including photodynamic therapy. This technology is embodied in therapeutics, which represents a unique fusion of fluorescent dye and an antimicrobial agent in a single formulation. This approach allows for simultaneous visualization of the infection and targeted elimination of pathogens, resulting in more effective treatment while minimizing the adverse effects associated with traditional therapies.
Here, we propose to a combination of fluorophores and drugs, specifically antibiotics. The fluorophores used in the diagnostic system must satisfy the requirements of safety, biocompatibility, and effectiveness of their antimicrobial action. We have selected a set of promising fluorophores based on their biological activity, taking into account the literature indications on their anti-inflammatory properties. Some examples of bioactive fluorophores are listed below (Table 1).
Methylene blue exhibits remarkable antibacterial properties, particularly effective against fungal infections [26,27,28,29,30,31,32]. The mode of action is related to its capacity to induce oxidative stress within microorganisms, leading to damage to their cellular membranes. Crystal violet (gentian violet), not only serves as a stain for staining bacteria, but also demonstrates activity against a wide range of Gram-positive and Gram-negative bacteria, operating by disrupting the integrity of their cell walls [33,34]. The rhodamines 123, B, 6G, and their derivatives are employed as fluorophores, exhibiting pronounced antibacterial effects [35,36,37,38]. The mechanism of action relies on the photodynamic process, which involves the generation of reactive oxygen species (ROS) under the influence of light (550–700 nm). Curcumin has been highlighted in numerous studies for its potent antibacterial and anti-inflammatory attributes. Curcumin interacts with bacterial cell membranes, permeating into cells and disrupting metabolic processes [39,40,41,42,43,44,45].
The employment of dyes and fluorophores possessing antibacterial activity presents a promising avenue in the battle against infections, particularly in the context of increasing antibiotic resistance. However, further exploration is required to elucidate their mechanisms of action, optimize their application, and assess their clinical efficacy. Combining these substances with conventional antibiotics may prove to be a strategic approach to enhance their antimicrobial potency and overcome antibiotic resistance.
The fluorophores exert antibacterial or cytotoxic effects through various mechanisms (Table 1).
Table 1. Mechanisms of antibacterial or cytotoxic effects of dyes and fluorophores.
Table 1. Mechanisms of antibacterial or cytotoxic effects of dyes and fluorophores.
MechanismDescriptionExamples
Intercalation in DNAA wide range of dyes are capable of binding to DNA, interfering with the processes of replication and transcription processes. Dues establish hydrogen bonds with the bases of DNA, thereby hindering their functional activity.Acridine orange [28,46,47],
Methylene blue [28],
Ethidium bromide [28,48,49],
Proflavine, doxorubicin [50]
Damage to the Cell MembraneCertain dyes can interact with the phospholipids of a bacterial cell membrane, resulting in its destruction and ultimately leading to cell lysis. Lipophilic compounds tend to accumulate within the membrane, disrupting the functionality of ion pumps and enhancing proton permeability. The toxic effects of these compounds on membranes can manifest themselves through mechanisms such as membrane blockage and expansion of membranes.Sudan dyes [51,52],
Indocarbocyanine derivatives [53],
Alexa and Atto [54]
Oxidative StressFluorophores like rhodamine contribute to oxidative stress within cells by inducing the production of reactive oxygen species (ROS). These ROS cause damage to cellular structures, including lipids, proteins, and DNADoxorubicin [55],
Fluorescein and rhodamine derivatives [56]
Ionization and Metabolic DisordersDisruption metabolic processes, hindering the synthesis of ATP or the biosynthesis of cellular components, inhibition of mitochondrial oxidative phosphorylationRhodamine 6G [57], Fluorescein Analogues [58]
Use in photodynamic therapyPhotodynamic therapy (PDT) is a minimally invasive treatment that involves the use of a photosensitizing agent, usually a dye, and a specific wavelength of light. When a photosensitizing agent absorbs light, it generates ROS that damage target cells. The dyes used in PDT are carefully selected to ensure that they can penetrate the tissues and affect certain cells or structures of the body. They are usually water-soluble or fat-soluble, depending on the location of the desired exposure.Phthalocyanines, Porphyrins, Chlorins [59,60,61,62], Methylene blue [31], Rhodamine 123 [63]
As candidates to the antibacterial agents here we are exploring a set of bioactive substances of different class such as chalcones, chromenes, stilbenoids and, xanthylium derivatives, in comparison with the well-known antiseptic agents, with the aim of uncovering their novel properties and expanding their applications. The investigation of the antibacterial potential of “drug candidates” assumes particular significance in light of the escalating issue of multidrug resistance. They can serve as alternative antimicrobial agents or be combined with conventional antibiotics or cytostatic drugs to enhance their efficacy.
The use of adjuvants derived from natural extracts, chalcones, cyano-methoxychromene, stilbenoids and their derivatives in a single delivery system with antibiotics is expected to enhance the efficacy of drugs while reducing the risk of side reactions. These compounds may exhibit synergistic effects with antibiotics, potentially mitigating the development of bacterial resistance.
A notable group of promising compounds in this category are stilbenoids and chalcones [64,65,66,67,68], that have been gaining attention among researchers due to their promising bioactive properties. Stilbenoids are chemical compounds based on the structure of so-called E-stilbene, a condensed ring system with the formula R-C6H4-CH=CH-C6H4-R1, where R and R1 represent various substituents. Naturally compounds stilbenoids exhibit a wide range of biological activities, including antitumor and antimalarial effects. Moreover, many stilbene derivatives demonstrate antibacterial activity, as exemplified by a compound extracted from the leaves of Combretum woodii. These substances have anti-inflammatory, antibacterial, antiviral, antioxidant, antitumor, cardiovascular and neuroprotective properties. They are low-molecular-weight compounds, such as resveratrol, pterostilbene and many others. Tamoxifen and raloxifene are synthetic stilbene derivatives approved by the FDA and are prescribed for various skin conditions [69]. Due to their poor water solubility, stilbenoid derivatives face significant challenges in medical applications. Chalcones and stilbenoids are being considered as potential novel medications for the treatment of inflammatory conditions, cancer, and infections.
Chalcone, stilbenoid and chromene derivatives hold significant promise in biomedicine due to their diverse and potent biological activities. These classes of bioactive compounds often exhibit antioxidant, anti-inflammatory, antimicrobial, and anticancer properties, stemming from their characteristic conjugated π-electron systems and the ability to readily modify their structures to fine-tune their activity. Their relatively simple structures allow for facile synthesis and modification, enabling the development of targeted drug candidates with improved efficacy and reduced side effects. The diverse biological activities of these scaffolds make them attractive lead to a broad range of therapeutic applications, including the treatment of infectious diseases, and various types of inflammation conditions.
In this paper, we also discuss a class of chromenes (Benzopyran derivatives)—substances with various biological properties that are widely found in nature [70,71]. Compounds with a chromene framework exhibit many properties, including antibacterial and fungicidal action, anti-inflammatory and antioxidant activity. However, chromenes are practically insoluble in water, which makes their use difficult. To address this challenge, it became necessary to synthesize water-soluble chromene derivatives. These derivatives would solve the problem of low solubility in water, making it easier to use chromenes in various applications.
For the delivery of a combine theranostic composition, we propose using cyclodextrin as the basis for creating a joint delivery system. Cyclodextrins (CDs) are natural cyclic oligosaccharide with a unique ability to form inclusion complexes with various molecules (as well as the possibility of forming double complexes), including medicinal substances and radioactive isotopes, which makes it an ideal candidate for the creation of theranostic delivery systems. Cyclodextrins (CDs) are proposed as a delivery system for combined theranostic compositions due to their ability to form inclusion complexes. Many promising bioactive compounds, including chalcones and chromenes, suffer from poor solubility. CD inclusion complexes significantly enhance the solubility and bioavailability of these compounds, leading to improved antibacterial and antioxidant activities. The hydrophobic CD cavity protects the encapsulated molecules from degradation, while the hydrophilic exterior facilitates delivery. This targeted delivery, combined with increased stability and concentration at the site of action, results in significantly enhanced therapeutic efficacy, overcoming limitations inherent in the free compounds [72,73,74,75,76]. The advantages of using CD to enhance the therapeutic potential of “drug candidates”:
  • Enhanced aqueous solubility: This attribute is particularly crucial for substances with limited solubility in water, as enhanced bioavailability translates into more potent antibacterial efficacy.
  • Resistance to degradation: Incorporation into the CD shields dyes from the deleterious effects of light, heat, and oxygen, thereby enhancing their stability and prolonging their shelf life.
  • Optimized targeted delivery: The use of cyclodextrins enhances the precision of dye delivery to the site of infection, mitigating toxicity to healthy cells. This feature is particularly advantageous in bacterial infection treatment, as it minimizes adverse reactions.
  • Enhanced antibacterial and antioxidant properties of the formed inclusion complexes were observed for several drugs: the encapsulation has enhanced the antimicrobial activity of carvacrol against both E. coli and Salmonella bacteria [77]. The antibacterial nanofibers made of perillaldehyde and hydroxypropyl-γ-cyclodextrin were developed. The researchers showed that these nanofibers had improved water solubility, thermostability, and antioxidant activity [78]. Baicalein-hydroxypropyl-β-cyclodextrin inclusion complex prepared using supercritical antisolvent technology was designed to enhance the solubility, antioxidant activity, and antibacterial activity of baicalein [79].
In our study, we investigated a range of biologically active substances of natural origin as «drug candidates» in comparison with known antibiotics, to assess their potential for use in the development of a highly effective theragnostic system that incorporates a fluorophore and an antibacterial agent.

2. Materials and Methods

2.1. Reagents

2.1.1. Chemicals

Rhodamine 6G (R6G), methyl-β-cyclodextrin (MCD) were obtained from Sigma-Aldrich (St. Louis, MI, USA). Dyes, salts for the preparation of buffer solutions, NaOH, and HCl were produced by Reachim (Helicon, Moscow, Russia). Components for LB medium were bactotrypton, agarose and yeast extract (Helicon, Russia), NaCl (Sigma Aldrich, USA). Properties of methyl-β-cyclodextrin (MCD): Mn = 1310 g/mol, –OH groups (2, 3, 6) are replaced by –OCH3 in random order. On average, there are 7–9 methyl groups per β-CD molecule, distributed between the primary and secondary rims.

2.1.2. Preparation of–1-Methyl-3-(2-amino-7-methoxy-3-cyanochromene-4-yl) and Pyridinium Methanesulfate (Sample 17, Scheme 1)

Stage 1. Preparation of 2-Amino-7-methoxy-4-(pyridine-3-yl)-3-cyanochromene—sample 17x.
Biophysica 04 00044 sch001
Scheme 1. Synthesis scheme of compound 17.
Scheme 1. Synthesis scheme of compound 17.
Biophysica 04 00044 sch001
A measure of 0.3 g of triethylamine was added to a solution of 1.07 g (10 mmol) nicotinic aldehyde, 0.66 g (10 mmol) malonic nitrile and 1.24 g (10 mmol) 3-methoxyphenol in 12 mL of methanol. The reaction mixture was boiled for 4 h, then cooled to 0–5 °C and left overnight. The precipitate was filtered, washed with cold methanol and dried. A sample of 1.7 g of the substance (sample 17x, 60%) was obtained. Tmelt = 224–227 °C. 1H NMR spectrum (DMSO-d6): 3.76 s(3H, OCH3), 4.82 s(1H, Hch(4)), 6.62 s (1H, Hch(8)), 6.72 d (1H, Hch(6)), 6.94d (1H, Hch(5)), 7.04 s ((2H, NH2), 7.34 t(1H, Hpd(5)), 7.55 d (1H,Hpd(4)), 8,46 d (1H, Hpd(6)), 8,50 s (1H, Hpd(2)).
Stage 2. Preparation of Sample 17 (1-methyl-3-(2-amino-7-methoxy-3-cyanochromene-4-yl) pyridinium methanesulfate).
A mixture of 0.56 g (2 mmol) of Sample 17x and 0.32 g (2.6 mmol) of dimethyl sulfate in 5 mL of methanol was boiled for 4 h. Then, the solvent was evaporated at reduced pressure and the residue was ground in a mixture of ethyl acetate-petroleum ether = 1:2. After the start of crystallization, the mixture was left overnight. The fallen crystals were filtered, washed with a mixture of ethyl acetate-petroleum ether = 1:2 and dried. The yield is 0.6 g (80%). Tmelt = 186–192 °C. 1H NMR spectrum (DMSO d6): 3.40 s (3H, N-CH3), 3.76 s (3H, N-CH3), 3.76 s (3H, CH3O), 4.36 s (3H, CH3OSO3−), 5.12 s(1H, Hch(4)), 6.63 s (1H, Hch(8)), 6.76 d (1H, Hch(6)), 8.90 d (1H, Hpd(6)), 9,04 s (1H, Hpd(2)).

2.1.3. Synthesis of R351 Compounds of Xanthylium Derivatives

The synthesis of R351 compounds of xanthylium derivatives was carried out by the Claisen-Schmidt condensation reaction from apiol-tetralone and 2,4-dihydroxybenzaldehyde in methanol with the addition of HCl or HClO4 (Scheme 2).

2.1.4. Synthesis of the Chalcone and Stilbenoid Derivatives

Samples, from 7 to 15 inclusive, are obtained by the Claisen-Schmidt reaction in an aqueous alcohol medium in the presence of alkali (Scheme 3). The reaction between ketones and sulfo-anisic aldehyde proceeds at 20 °C for 6–8 h. The precipitate that has fallen out of the solution is filtered, washed with alcohol, and dried. The yield of the condensation product is 95–98%. If the corresponding nitrile derivatives are used instead of ketones, the reaction proceeds at 0–5 °C [80,81].

2.1.5. Spectral Data of the Chromene, Chalcone, and Stilbenoid Derivatives

The complete NMR and mass spectra are presented in the Supplement—Figure S1.
Sample 7: 1H NMR (300 MHz, DMSO-d6) δ 8.34 (d, J = 2.5 Hz, 1H), 8.09–7.96 (m, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.5 Hz, 2H), 7.47–7.37 (m, 1H), 7.19 (d, J = 8.7 Hz, 1H), 3.91–3.85 (m, 6H). 13C NMR (126 MHz, DMSO-d6) δ 158.04, 142.36, 135.65, 134.01, 131.18, 130.31, 129.36, 129.05, 128.68, 125.41, 124.72, 118.12, 112.11, 107.08, 55.70, 39.28.
Sample 8: 1H NMR (300 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.00 (dd, J = 8.7, 2.5 Hz, 1H), 7.86 (s, 1H), 7.79–7.66 (m, 2H), 7.16 (d, J = 8.7 Hz, 1H), 7.12–6.99 (m, 2H), 3.85 (d, J = 11.6 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ 159.59, 157.72, 140.21, 135.87, 130.54, 130.25, 126.81, 126.44, 124.96, 118.25, 114.42, 112.03, 106.80, 55.60, 55.24.
Sample 9: 1H NMR (300 MHz, DMSO-d6) δ 8.27 (d, J = 2.4 Hz, 1H), 8.02 (dd, J = 8.6, 2.5 Hz, 1H), 7.92 (s, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.03 (dd, J = 11.1, 1.7 Hz, 2H), 6.08 (s, 2H), 3.92 (s, 3H), 3.86 (s, 3H), 2.10 (s, 0H). 13C NMR (126 MHz, DMSO-d6) δ 157.89, 149.02, 143.27, 141.38, 135.81, 135.35, 130.61, 130.52, 128.82, 124.74, 118.21, 112.06, 106.73, 105.70, 101.78, 99.48, 56.35, 55.62.
Sample 10: 1H NMR (300 MHz, DMSO-d6) δ 8.17–8.09 (m, 3H), 7.92 (dd, J = 8.6, 2.4 Hz, 1H), 7.72 (s, 2H), 7.67 (t, J = 7.3 Hz, 1H), 7.58 (t, J = 7.4 Hz, 2H), 7.11 (d, J = 8.5 Hz, 1H), 3.87 (s, 3H). 1H NMR (300 MHz, DMSO-d6) δ 8.14, 8.13, 8.12, 7.94, 7.93, 7.91, 7.90, 7.85, 7.72, 7.70, 7.67, 7.65, 7.60, 7.58, 7.55, 7.13, 7.10, 3.87, 2.09.
Sample 11: 1H NMR (300 MHz, DMSO-d6) δ 8.19–8.06 (m, 3H), 7.88 (dd, J = 8.6, 2.4 Hz, 1H), 7.79–7.60 (m, 2H), 7.10 (d, J = 8.6 Hz, 3H), 3.87 (d, J = 6.3 Hz, 6H), 3.39 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 187.16, 162.95, 158.14, 143.03, 135.69, 131.37, 130.67, 130.52, 128.72, 125.72, 119.41, 113.90, 112.14, 55.69, 55.44.
Sample 12: 1H NMR (300 MHz, DMSO-d6) δ 8.07 (d, J = 2.3 Hz, 1H), 7.90 (dd, J = 8.6, 2.4 Hz, 1H), 7.69 (ddd, J = 14.4, 7.3, 2.2 Hz, 4H), 7.08 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 4.34 (q, J = 5.0 Hz, 4H), 3.85 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 186.97, 158.20, 147.70, 143.26, 143.22, 135.80, 131.28, 131.21, 129.00, 125.66, 122.42, 119.24, 117.23, 117.06, 112.12, 64.45, 63.83, 55.64.
Sample 14: 1H NMR (300 MHz, DMSO-d6) δ 8.09 (d, J = 2.4 Hz, 1H), 7.89 (td, J = 7.9, 6.8, 2.2 Hz, 2H), 7.75 (d, J = 15.5 Hz, 1H), 7.67 (d, J = 15.5 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.11 (dd, J = 8.5, 4.6 Hz, 2H), 3.93–3.79 (m, 9H). 13C NMR (126 MHz, DMSO-d6) δ 187.16, 158.10, 152.92, 148.65, 142.93, 135.56, 131.39, 130.57, 128.80, 125.77, 123.09, 119.40, 112.15, 110.75, 110.46, 55.72, 55.64, 55.44.
Sample 15: 1H NMR (300 MHz, DMSO-d6) δ 8.15–8.03 (m, 3H), 7.90 (dd, J = 8.7, 2.3 Hz, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.74–7.68 (m, 2H), 7.10 (d, J = 8.6 Hz, 1H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 188.07, 158.43, 144.43, 136.66, 135.60, 131.77, 131.72, 130.35, 128.91, 126.92, 125.49, 119.12, 112.19, 55.73.
Sample 17: 1H NMR (300 MHz, DMSO-d6) δ 9.02 (s, 2H), 8.91 (d, J = 6.0 Hz, 2H), 8.45 (d, J = 8.1 Hz, 2H), 8.11 (t, J = 7.2 Hz, 2H), 7.26 (s, 4H), 7.13 (d, J = 11.4 Hz, 0H), 7.03 (d, J = 8.6 Hz, 2H), 6.79–6.68 (m, 2H), 6.63 (d, J = 2.3 Hz, 2H), 5.12 (s, 2H), 4.38 (s, 6H), 3.77 (d, J = 1.8 Hz, 5H), 3.69 (d, J = 7.0 Hz, 1H).
R351-Cl−: 1H NMR (300 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.16 (d, J = 8.9 Hz, 1H), 7.45 (dd, J = 8.9, 2.2 Hz, 1H), 7.36 (d, J = 2.3 Hz, 1H), 6.33 (s, 2H), 4.15 (s, 3H), 3.93 (s, 3H), 3.02 (dd, J = 16.0, 6.7 Hz, 5H).

2.1.6. High-Resolution Mass Spectrometry

High-resolution mass spectra (HRMS) were measured on a Bruker micrOTOF II instrument using electrospray ionization (ESI). The mass spectrometry data of the studied compounds are presented in Supplement (Figure S2).

2.2. Methyl-β-Cyclodextrin (MCD) Inclusion Complexes Obtaining

2.2.1. Complexation in an Aqueous Buffer Solution

To 2–3 mg of the substance, 1 mL of MCD solution (10–30 mg) in PBS (10 mM, pH 7.4) was added. The molar ratio of cyclodextrin to the guest was varied from 0.25 to 10. The mixture was incubated at 37 °C for 1 h. After that, it was centrifuged at 14,000 rpm for 5 min to separate insoluble fractions. The obtained complexes were analyzed spectroscopically (UV and FTIR) to determine the solubility of the compound and the parameters of the complex formation.

2.2.2. Complexation in Organic Solvent Suspension

In this procedure, 2–3 mg of aromatic substance was mixed with 10–30 mg of MCD, and then 100–200 µL of acetonitrile was added. The molar ratio of the cyclodextrin to the guest was varied from 0.25 to 10. The mixture was thoroughly mixed using the rubbing method. The suspension was incubated at 37 °C for 1 h, after this the complexes were dried in a stream of dry air or by freeze drying.

2.2.3. Calculation of Solubility of Compounds and Dissociation Constants of Complexes with MCD

The calculation of the solubility of compounds and the dissociation constants of complexes with MCD was carried out as described earlier [73,82].

2.3. Molecular Absorption Spectroscopy and CD Spectroscopy in the UV/Visible Range

UV/visible absorption spectra of solutions of the studied substances and their MCD-complexes were recorded on the AmerSham Biosciences UltraSpec 2100 pro device (Chicago, IL, USA). The final solutions were obtained from concentrated (10 mg/mL) solutions of samples of drug+MCD complexes in 10% DMSO by diluting with a PBS buffer to the desired concentration (0.0005–0.05 mg/mL). Circular dichroism spectra of samples (1–5 mg/mL in PBS) were recorded on Jasco J-815 CD Spectrometer (JASCO, Tokyo, Japan).

2.4. FTIR Spectroscopy

FTIR spectra of samples in the dispersed state (5–10 mg/mL per drug substance in PBS without DMSO) were obtained using a Bruker Tensor 27 spectrometer (Bruker, Ettlingen, Germany), FTIR spectra of samples in the solid state were obtained using a MICRAN-3 IR microscope (Simex, Novosibirsk, Russia) equipped with a cooled MCT detector. The spectra were obtained in the ATR mode.

2.5. NMR Spectroscopy

The 1H NMR and 13C NMR spectra of free substances in d6-DMSO and complex substances with MCD in D2O were registered on a Bruker DRX-500 device [working frequencies of 500.13 MHz (1H) and 125.76 MHz (13C)]. Chemical shifts were expressed in parts per million (ppm) and assigned to the appropriate NMR solvent peaks.

2.6. Microbiology Experiments

2.6.1. Bacterial Strains

In this study, we utilized two bacterial strains: Escherichia coli (ATCC 25922) obtained from the National Resource Center of the Russian Collection of Industrial Microorganisms at SIC “Kurchatov Institute”, and Lactobacillus, which was a commercially available lactobacillus liquid concentrate. The cultures were cultivated for 18–20 h at 37 °C to achieve an approximate concentration of 2 × 107 CFU/mL. This concentration was determined through measurement of optical density (A600) and plating onto Petri dishes. The nutrient medium employed, Luria–Bertani, possessed a pH of 7.2 and was agitated at 120 rpm.

2.6.2. Study of the Antibacterial Activity of Compounds

Suspensions of cells containing 107 CFUs were subjected to incubation with solutions of the studied substances and the MCD-complexes. The final solutions were obtained from concentrated stock solutions 10% DMSO of samples (3–5 mg/mL) in the complex with MCD in it by diluting with the cell liquid medium to the desired drug concentration (25 µM–5 mM). The resulting DMSO concentration did not exceed 0.1%. In an independent experiment, it was shown that DMSO in concentrations below 1% does not affect the survival of E. coli cells. The incubation period was 24 h at 37 °C. Following incubation, cell viability was assessed through optical density measurement (A700) and confirmed via plating of the cell suspensions onto plates. These plates were subsequently incubated at 37 °C for a further 24 h to allow for colony formation. Colony-forming units (CFUs) were subsequently determined.
MIC values are indicated where possible (not all of the substances show sufficient activity). Additionally, the IC25 concentration parameters are given, at which a 25% inhibition of bacterial growth is achieved.

2.6.3. Visualization of Plates Using Fluorescent Images

Fluorescent images of plates containing E. coli (107 CFUs/0.5 mL) placed on 20 mL agar were acquired using the UVP BioSpectrum Imaging System (BioSpectrum, UVP, Upland, CA, USA). The fluorophore wells had a diameter of 0.9 cm, with fluorophore (R6G or FITC, or R351) concentrations of 0.1–1 mg/mL. The excitation wavelength (λexci, max) was set at 480 nm, and emission wavelengths ranged between 515 and 570 nm.

2.7. CLSM of Staining of Bacterial Cells Using a Theranostic Fluorescent Preparation

The study involved visualizing the staining of bacterial cells using a theranostic fluorescent preparation. Bacterial cells, with a concentration of 5 × 106 cells per mL, were incubated with different concentrations of R6G and complexes with MCD. After incubation, the cells were subjected to double washing for 5 min at 8000× g and then transferred to 96-well plates. The cells were subsequently fixed with paraformaldehyde and filled with 70 μL of a solution containing 50% glycerol and PBS. Confocal laser scanning microscopy (CLSM) images were acquired using an Olympus FluoView FV1000 confocal microscope equipped with a spectral scanning unit and a transmitted light detector. The measurements were performed using an Olympus UPLSAPO 40X NA 0.90 dry objective lens. Fluorescence emission was collected through emission windows set at 510–560 nm (green channel) and 560–610 nm (red channel).

2.8. Antioxidant Activity Using ABTS Assay

The capacity of the drug formulations to neutralize free radicals was assessed using the ABTS assay, as outlined in papers [83,84]. This method involved measuring the absorbance at a wavelength of 734 nm of an ABTS cation radical in the presence of the drugs. The results are presented in comparison with the reference antioxidant quercetin.

3. Results and Discussion

3.1. Drug Candidates

Table 2 present the compounds under investigation encompasses a variety of antimicrobial dyes, including “drug candidates” chalcone, stilbenoid, cyano-7-methoxychromene and xanthylium derivatives, R351, as well as antiseptic agents and antibiotics (Table 2).
To create a combined theranostic antibacterial system, we have selected derivatives of natural compounds such as chalcones and stilbenoids and dyes/fluorophores as study objects, as they exhibit a broad spectrum of pharmacological activities, including antibacterial and antioxidant properties. These substances can act in synergy, amplifying the efficacy of antibiotic therapy, for example, metronidazole main component enhancement. To optimize the synergistic effect, we have developed a common delivery system to create effective drugs system for combating infections.
Such dyes as crystal violet, Congo red, Sudan III, malachite green, rhodamine 6G, methylene blue, ponceau 4R and toluidine blue have antibacterial activity against various microorganisms. Xanthylium derivatives such as R351-ClO4− and R351-Cl- are considered as perspective antibacterial agents. Some chalcones and stilbenoids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria [85,86]. Among other applications, they can serve to inhibit the efflux of antibiotics in bacteria that are resistant or have developed multiple resistances [67]. Previous studies of chalcons and stilbenoids that do not contain sulfogroups revealed their antibacterial and cytostatic properties, but their low solubility in water limited their use. Here, the sulfo-derivatives of chalcons and stilbenoids are investigated as perspective antibacterials. The introduction of sulfogroups, increasing solubility, creates new opportunities that require studying the effect on the activity of water. Benzalkonium chloride and chlorhexidine are well-known antiseptics with high antibacterial activity. Metronidazole is an antibiotic that is effective against anaerobic bacteria, while clotrimazole is an antifungal drug with antibacterial properties.
The issue of suboptimal drug efficacy and the development of bacterial resistance necessitates the search for innovative solutions. We suggest a theranostic agent by combining the primary drug with adjuvants derived from the class of chalcones or dyes/fluorophores. Theranostics encompasses the diagnosis of infections and inflammatory conditions, as well as their concurrent treatment. Of particular interest is the study of the effect of CD as a molecular container on the effectiveness of antibacterial and anti-inflammatory agents. We expected that the use of CD for the inclusion of drugs and adjuvants could increase their effectiveness and bioavailability, which opens new therapeutic options. This approach can also be used to create antioxidant formulations, expanding the range of applications of cyclodextrins in medicine.

3.2. Molecular Absorption Spectroscopy of Dyes and Their Complexes with MCD

Figure 1a–f presents the absorption spectra of the investigated dyes in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD). Upon the formation of these complexes, there is a discernible decrease in absorption intensity, which can be attributed to the formation of a complex that affects the physicochemical properties of the dyes. The incorporation of dyes such as Congo red, Methylene Blue, and others into the hydrophobic interior of MCD results in several critical changes.
Firstly, shifts in the absorption maximum were detected. For example, the inclusion of Congo red in the MCD is accompanied by a shift in the absorption maximum in the long-wavelength region from 490 to 495 nm (Figure 1a), and in the case of Xanthylium derivatives R351, a shift is observed in the short-wavelength region from 520 to 512 nm, which correlates with alterations in their microenvironment upon interaction with MCD.
Secondly, there is a decline in the potency of absorption for most of the dyes due to the influence of radiation shielding. The development of these complexes results in a partial protection of dye molecules from interaction with the solvent, potentially diminishing their capacity for absorbing light.
Moreover, modifications in the spectral pattern occur when dye–MCD complexes of the “guest–host” type are formed. In the case of Malachite Green (Figure 1c), there is an augmentation in the proportion of the short-wavelength component by 425 nm, which corresponds to an increase in the fraction of monomeric species (dissociation of microcrystals). Analogous alterations are observed for xanthylium derivatives such as R351 (Figure 1d).
Table 3 presents the physico-chemical characteristics of the dyes, including their molar weights and molar absorptivity coefficients. These data provide valuable insights into prediction the behavior of these dyes in various chemical environments and optimize their use in applications ranging from staining biological tissues to developing colored materials.
MCD, a cyclic oligosaccharide, enhances the solubility of various compounds, including dyes in aqueous solution. In the presence of MCD at a concentration of 5 mM, the solubility typically increases by several orders of magnitude compared to that in a PBS solution (Table 3). The mechanism underlying this increase in solubility involves the formation of inclusion complexes between MCD molecules and dye molecules. The cyclic structure of MCD allows it to form a cavity that accommodates dye molecules, reducing their hydration energy and consequently increasing the solubility in PBS.
The investigation of the spectral patterns of chromophores in their complexes with MCD allowed us to determine ehe dissociation constants of dye or chalcone complexes with MCD to describe the stability of these guest–host inclusion complexes with the studied compounds; the values are of the order of 10−3–10−4 M (for highly soluble compounds, this parameter reaches about 10−2 M), indicating to the relative strength of the complex and sufficient to increase the solubility of the bioactive substance and improve bioavailability, as well as to create combined unified delivery systems several components of drug + dye + adjuvant at once.

3.3. FTIR Spectroscopy of Dyes and Their Complexes with MCD

Fourier Transform Infrared (FTIR) spectroscopy is a powerful analytical technique used to identify functional groups and molecular interactions in a variety of compounds, including dyes and the complexes with MCD. This technique provides information about the oscillations of molecular bonds, allowing for the analysis of molecular structures and the assessment of complex formation. Figure 2 presents the FTIR spectra of the investigated dyes in free form in a buffer solution and complexed with MCD.
FTIR spectra of the dyes and compounds under investigation, such as chalcones and others, typically exhibit distinctive absorption peaks associated with specific functional groups, such as –CH2–, –NH–, –C=O, and aromatic C=C stretches. For example, peaks at 3000–2800 cm−1 correspond to ν(C-H), while peaks in the region of 1500–1600 cm−1 often relate to aromatic C=C oscillation, 1150–1050 cm−1 C-O stretching oscillation. MCD displays distinct peaks associated with its molecular structure, the most important 1150–1050 cm−1 C-O-C stretching oscillation.
In the FTIR spectrum of a chalcone derivative 9 (Figure 2a), prominent peaks can be found at 1650–1580 cm−1 ν(C=C), 2210 cm−1 ν(C-N nitrile), 1300–1250 cm−1 ν(SO3−). When chalcone 9 or sample 17 (Figure 2a,b) form complexes with MCD, the ν(C=C) around 1600 cm−1 peak as well as other analytically significant peaks (for example, corresponding to the sulfogroup, ether bonds) exhibit decreased intensity, suggesting interaction with the cyclodextrin cavity. Additionally, a slight shift in the characteristic maxima towards the low-frequency range (from 1 to 5 cm−1), for instance, in oscillations with aromatic C=C, can be employed as an additional marker of the formation of MCD complex with chalcones.
In the FTIR spectrum of a chromene derivative (17) (Figure 2b), prominent peaks can be found at 1740–1720 ν(C=C in pyridinium), 1680–1580 cm−1 ν(C=C in methoxychromene system), 2190 cm−1 ν(C-N nitrile), 1060 cm−1 ν(C–C) and 1280–1190 cm−1 ν(CH3SO4−). Upon complexation of chromene derivative with MCD, the spectral features corresponding to the functional groups within the drug molecule undergo alterations due to the establishment of hydrogen bonding interactions or Van der Waals forces. In particular, a displacement of the O-H stretch band may suggest the formation of hydrogen bonds between the drug molecule and the MCD component. This is manifested in a reduction in the intensity of characteristic peaks of the compound upon its incorporation into the hydrophobic pocket of the MCD. Simultaneously, charged groups, such as C=N+ or the counterion CH3SO4−, being highly polar, remain exposed to the aqueous environment rather than fully immersed within the MCD pocket. These groups engage in hydrogen bonding interactions due to their accessibility to water molecules. Consequently, there is a more pronounced decrease in the absorption peak at 1280–1190 cm−1 associated with CH3SO4− compared to the rest of the molecular complex involving MCD.
FTIR spectra of other chalcones and their complexes with MCD are presented in Supplement (Figure S3). The inclusion of chalcones into the hydrophobic cavity of the MCD occurs mainly in the more hydrophobic part (ring), which is reflected in a decrease in the intensity of the corresponding peaks in the FTIR spectrum. For example, for chalcone 12, there is a decrease in the peak intensity of 1600–1550 cm−1, and an increase in the peak intensity at 1640 cm−1, which indicates the inclusion of a ring A with a benzodioxol “nose” into the hydrophobic cavity, and conversely, the orientation of the ring B with a methoxy and sulfogroup into the solvent.
It is known that the main force of the guest–host complexation is the hydrophobic interactions of aromatic systems of bioactive substances and the hydrophobic structure of cyclodextrin (enthalpy factor), with the simultaneous displacement of water (entropy factor) [87,88]. Dyes or fluorophores often contain functional oxygen- and/or nitrogen-containing (N) groups, which can participate in the formation of hydrogen bonds with hydroxyl groups of MCD, which further stabilizes the complex. This information is crucial for optimizing the use of these compounds in various applications, such as in drug delivery and in the encapsulation of dyes. The use of FTIR in combination with other techniques, such as nuclear magnetic resonance (NMR) and UV-Vis spectroscopy, can provide a more comprehensive understanding of the complex phenomenon under study.

3.4. 1H NMR Spectroscopy of Chalcones and Their Complexes with Methyl-β-Cyclodextrin

1H nuclear magnetic resonance spectroscopy (NMR) is a highly effective method for elucidating the structural features of organic molecules, as well as shedding light on the intricate mechanisms underlying drug interactions with molecular targets, such as chalcones complexed with MCD. The NMR spectra of chalcones are presented in Figure 3, with a particular focus on compound 9.
Analysis of the chalcone spectrum (sample 9, Figure 3a) reveals a number of characteristic features in its structure. Protons in the benzodioxol ring can resonate in the range from 6.7 to 7.4 ppm for the aromatic fragment and from 3 to 4 ppm for the oxol-fragment. Protons attached to the carbon atoms of the double bond give a signal at about 8.3 ppm. The methoxy groups give a signal in the range from 3.0 to 4.0 ppm. The spectrum of the MCD-chalcone complex (Figure 3b) also contains signals from the CD. H1, H1′ protons give the lowest-field signals, observed at 5.25, 5.04 ppm. It is sensitive to the environment of the MCD molecule and may vary depending on the presence of a guest in the cavity. Protons H-2, H-3, H-4, H-5, H-6 give signals in the range of 3.2–4.1 ppm.
The proposed structure of the complex obtained during computer modeling is shown in Figure 3c; it confirms the immersion of aromatic fragments of chalcone (compound 9) into the hydrophobic layer of cyclodextrin. The hydrophobic aromatic core is submerged within the MCD, with the benzodioxol «nose» protruding from the MCD’s surface, and is capable of engaging with a second MCD molecule akin to a lid. The sulfogroup, owing to its high polarity and charge, is directed into a solution. During the complexation of chalcone with MCD, drug signals are shifted due to changes in the microenvironment of the aromatic molecule from the DMSO to the environment of the MCD: for example, for protons of the aromatic ring, the shift occurs from 7.1–7.0 ppm to 7.3–7.2 ppm (due to a more hydrophobic microenvironment inside the MCD), and for methoxy groups, on the contrary, there is a shift into a strong field (due to a more hydrophilic microenvironment, since these groups “stick out” from the aromatic systems and contact with the OH groups of MCD). This indicates the inclusion of the aromatic core of the drug molecule into the cyclodextrin cavity, and the successful formation of the guest–host complex.

3.5. Investigation of the Antibacterial Potency of Novel Compounds

In our research, we explore the antibacterial capabilities of novel promising chemical compounds as «drug candidate» chalcones, chromene and stilbenoids, xanthylium derivatives, conducting comparative analysis with standard antibiotics. Of particular interest is the synergistic interaction between these novel compounds with metronidazole.
Table 4 provides information on the viability of E. coli cells (%) and the synergistic coefficient with metronidazole for various compounds. High viability: Samples such as Chalcone derivative (8–14) (90% at 1 mM) and Congo Red (94% at 1 mM) demonstrate low efficacy as the antibacterial agent. However, chalcones 9, 12, and 15 exhibit moderate antibacterial activity, with a survival rate of 75–80% of cells (Table 2), owing to the presence of a benzodioxole moiety or a bromine atom functioning as a substituent for ring A. This benzodioxole fragment serves as a crucial component providing the antibacterial properties inherent in natural compounds like apiol as recently shown [73]. Indeed, among the novel compounds tested containing benzodioxole fragment and methoxy-groups, xanthylium R351-Cl− derivatives demonstrated the pronounced antioxidant activity, comparable to Clotrimazole. Low viability of bacterial cells is achieved by the action of Benzalkonium chloride (5% at 1 mM) and Malachite Green (12% at 1 mM) on them, which indicates their high antibacterial activity. These substances can serve both for topical use and as an adjuvant to the main component—metronidazole.
MIC values are indicated where possible, since not all substances are sufficiently active. Additionally, the IC25 concentration parameters are given, at which a 25% inhibition of bacterial growth is achieved. The presented data correlates with similar data described in the literature.
The synergistic coefficient shows how many times the combo formulation is more active than single substances. The coefficient with metronidazole is an important indicator for creating a combined formulation for potentially overcoming drug resistance. The values of the synergistic coefficient (k ≥ 1) indicate that the combination of the substance with metronidazole affects their effectiveness. For example, Chalcone (9) (benzodioxole apiol-like moiety) and xanthylium R351 derivatives have a coefficient of 1.36 and 1.2, which indicates a synergistic effect. We have recently demonstrated that apiol shows a synergistic effect with antibiotics such as levofloxacin due to efflux inhibition [73]. Rhodamine 6G, Benzalkonium chloride, and Chlorhexidine also demonstrate high synergy with metronidazole (up to 1.95). Values below 1, such as those obtained for Chalcone derivative 10 (0.9) and Ponceau 4R (0.95), indicate a lack of synergy or antagonistic behavior.
Metronidazole showed the viability of E. coli cells at the level of 15% at 1 mM, indicating its pronounced antibacterial activity compared with most of the listed chalcone derivatives. Some other tested compounds, such as Congo Red and Sudan III, show high cell viability.
Thus, we have demonstrated a variety of antibacterial properties of the tested substances. Chalcone derivatives showed low to moderate efficacy, while agents such as Benzalkonium chloride, Chlorhexidine, dyes methylene blue, rhodamine 6G, malachite green, gentian violet had a strong suppressive effect on E. coli. The results of the synergistic analysis with metronidazole also confirm the effectiveness of these dyes, including for the further creation of complex formulations to improve antibacterial activity.
The most effective formulation involves the combination of a primary drug, such as metronidazole, with a dye or fluorophore, such as methylene blue or rhodamine 6G, for target cell visualization. Additionally, an adjuvant, such as chalcones, is used to inhibit efflux. These formulations demonstrate significant therapeutic potential, enabling simultaneous visualization of target cells and therapeutic action. In the creation of this combined formulation, CDs play a crucial role. MCD enhances the solubility and bioavailability of drugs, as well as augmenting their antibacterial effects. Furthermore, MCD enables the development of a unified system for delivering multiple components to target cells for both visualization and elimination purposes.

3.6. Comparative Analysis of Antioxidant Activity

Antioxidants play a crucial role in preventing oxidative stress, a condition that can lead to cellular damage and the onset of various diseases. Their significance lies in their capacity to neutralize free radicals, which are unstable molecules responsible for oxidative stress.
Several methods exist for evaluating the antioxidant capacity of compounds:
  • The DPPH assay measures the ability of a substance to scavenge free radicals generated by DPPH (2,2-diphenyl-1-picrylhydrazyl).
  • The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay assesses the capacity of antioxidants to quench ABTS radicals.
  • The oxygen radical absorbance capacity (ORAC) assay determines the protective effect against oxidative stress.
In our study, we employed the ABTS method to evaluate the antioxidant potential of the tested substances of different classes (Figure 4).
Quercetin has been chosen as the benchmark, exhibiting remarkable antioxidant activity in a variety of tests. This activity is attributed to the presence of multiple hydroxyl groups, which enhances its capacity to neutralize free radicals. The antioxidant activity of various substances was evaluated by measuring the rate of ABTS radical neutralization, comparing it with the activity of quercetin, a well-known antioxidant.
It was found that dyes, such as rhodamine 6G, gentian violet and Congo red, exhibit moderate antioxidant activity, approximately 50% and 25%, respectively, of quercetin activity at concentrations of 1 and 0.01 mg/mL. These substances are effective at concentrations, exceeding 50–100 µM. Among the compounds tested, xanthylium R351 derivatives demonstrated the highest antioxidant activity, comparable to quercetin. The most potent antioxidant was 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium me-thanesulfate (17), exhibiting high activity even at concentrations as low as 0.1 mg/mL due to its ability to readily interact with radicals. For chalcones 7–15, antioxidant activity is virtually nonexistent (Figure 4b), with even an inverse correlation between activity and concentration observed, suggesting a non-specific mode of action.
The impact of cyclodextrin on the antioxidative potential of dyes is a critical aspect that is contingent upon a multitude of factors. MCD enhances the solubility of the dye in aqueous solutions, rendering it more susceptible to interaction with reactive species, thereby augmenting its antioxidative capacity. The formation of a complex with cyclodextrin alters the electronic distribution within the dye molecule, potentially affecting its microenvironment and influencing its capacity to scavenge free radicals. Furthermore, the spatial arrangement of the dye complexed with MCD, could enhance its antioxidative properties. For instance, we observed that complexes of methylene blue with MCD exhibited a 15% improvement in antioxidative activity compared to free methylene blue, and there was up to a remarkable 20–25% increase for Xanthylium—R351 (Figure 4). So, here we identified several new compounds with moderate antioxidant activity. The pronounced antioxidant properties of these substances are observed for Xanthylium derivative—R351 and 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate (sample 17). The charged groups conjugated to the condensed aromatic structures play an important role in enhancing antioxidant activity. These substances hold promise as potential antioxidants.

3.7. The Structure and Activity Relationship (SAR) Studies

The intricate relationship between the structural characteristics and functional properties of chalcones and stilbenoids in terms of their antibacterial and antioxidative capabilities is a complex and multifaceted phenomenon, contingent upon several critical structural elements. The presence of conjugated π-electron systems within the chalcone and stilbenoid frameworks is pivotal for both activities, enabling the delocalization of electrons for reactive oxygen species (ROS) uptake and interaction with bacterial targets. The substituents within aromatic rings significantly influence this activity, with electron-donating groups such as hydroxyl and methoxy groups playing a crucial role.
Among the chalcones and stilbenoids, compounds 9, 12, and 15 stand out as the most potent in terms of antibacterial activity (Table 4, Figure 5). Compounds 9 and 12 feature a benzodioxol ring, analogous to natural compounds like apiol, dillapiol, and myristicin, while compound 15 incorporates a benzene ring with a bromine substitution. So, the compounds (9 and 12) as well as the Xanthylium derivatives R351-Cl, featuring a benzodioxole moiety, emerge as prominent antioxidants among the array of chalcones and stilbenoids. It is noteworthy that compounds with either an unsubstituted or a monosubstituted A ring exhibit the lowest levels of antioxidant and antibacterial activity. Moreover, the cyano group in compound 9 assumes a critical role in facilitating both antioxidant and antibacterial properties.
While the B ring remains consistent across compounds 7–15, precluding definitive conclusions regarding its specific contribution, but the presence of polar groups, such as the sulfogroup in the meta position and methoxy in the para position, significantly enhances solubility. To optimize the structures of chalcones and stilbenoids for enhanced therapeutic efficacy, further systematic modifications of substituents and comprehensive analysis of biological activities are essential.
Figure 5 further elucidates crucial components for the manifestation of biological activity and pharmacophoric moieties. Analogous investigations into pharmacophores have been conducted, for instance, for licarin A derivatives, yielding two distinct pharmacophoric classes: vanillin- and isoeugenol-like [89]. The significant fragments comprising methyl, methoxy, acetyl, and hydroxy functional groups are noteworthy. This implies that the arguments we have presented truly reflect the potential mechanisms underlying the activity exhibited by chalcones and stilbenoids, which can be attributed to the presence of specific chemical groups.

3.8. CLSM Visualization of Bacterial Cells Stained with Theranostic Formulation

CLSM (confocal laser scanning microscopy) used to visualize the penetration of a theranostic formulation containing rhodamine 6G (R6G) into E. coli bacteria and lactobacilli. Selective penetration into E. coli cells is observed, which is confirmed by a more intense fluorescent signal in these cells compared to lactobacilli. Again, the rhodamine complex with cyclodextrin demonstrates significantly more effective staining of bacterial cells compared to free R6G (Figure 6). These observations open up prospects for the use of this formulation as a theranostic agent for the visualization and therapy of bacterial infections. The results obtained can be used for the (1) detection and localization of bacterial infection; selective penetration into E. coli allows you to visualize infected areas without affecting other cells (the ratio of the fluorescence intensity between the signal and background in the case of E. coli case is approximately four times greater than that observed in the lactobacillus case); (2) develop the targeted drug delivery system directly to bacterial cells.
The substances under consideration for biomedical applications will be employed in conjunction with an antibiotic such as metronidazole to facilitate the visualization of specific cells or tissues and to address infections. The theranostic formulation antibiotics with rhodamine 6G and MCD can be applied for the development of new tools for the diagnosis and treatment of bacterial infections. Such formulations could find applications, for instance, in the field of dentistry for diagnosing and treating bacterial infections, such as stomatitis, gingivitis, and the treatment of cysts.

3.9. Discussion of the Results and Their Biomedical Significance

The study of cyclodextrin (MCD) as a molecular depot for bioactive compounds in theranostic applications is of great importance.
MCD offers several key benefits:
  • Improved solubility for compounds such as chalcones, stilbenoids, dyes, and xanthylium derivatives, making them more bioavailable.
  • Enhanced bioavailability due to protection from degradation, which prolongs circulation and allows for higher concentrations in target tissues.
  • Unified delivery system for combining multiple active agents in a single system, opening up new possibilities for developing theranostic formulations.
  • Efficacy of antibacterial compounds by increasing their concentration at the site of infection.
Confocal microscopy has shown that MCD improves the penetration of compounds into target bacterial cells, with high selectivity for E. coli compared to Lactobacilli. Fluorescent markers such as rhodamine allow visualization of the target cells.
Our research explored several classes of compounds, revealing novel properties and potential therapeutic applications.
(1) Chalcones are naturally occurring compounds with diverse biological activities, including anti-tubulin properties. Chalcone derivatives demonstrated significant anti-proliferative activity against different cancer cell lines (SK-OV-3, PC-3, HCT-116, A375, A549) [81]. This suggests potential as novel anticancer agents.
(2) Chromenes are heterocyclic compounds with established roles in various biological activities [70,89]. Our newly synthesized chromene derivative 17 showed potent antioxidant activity. This suggests potential applications in treating oxidative stress-related diseases and bacterial infections.
(3) Stilbenoids, such as resveratrol, possess significant antioxidant and anti-inflammatory properties. Stilbenoids (on the example of combretastatin derivatives) displayed enhanced cytotoxicity against human cancer cell lines (HeLa, SK-OV-3, A549, and HT-29) [71,90].
(4) Dyes and Xanthylium derivatives. We investigated well-known dyes, discovering photophysical properties with significant potential for application in fluorescence imaging, photosensitizers and photodynamic therapy.
Natural compounds like chalcones and stilbenoids may have additional effects:
  • Inhibition of efflux in bacterial cells, increasing the intracellular concentration of the drug. Previously, we have demonstrated the phenomenon of efflux inhibition in bacteria by the components of natural extracts, such as apiol, dillapiol, and similar molecules [73], which are «building blocks» of chalcones (samples 12 and 14), and xanthylium derivatives R351.
  • Increased permeability of cell membranes due to local defects.
  • Bacterial enzyme inhibition that provides selective antibacterial activity.
Potential applications include treating bacterial infections such as skin infections and wounds, delivering antibiotics to bacterial colonies, and combining with anti-inflammatory drugs for anti-inflammatory therapy. Compared to other systems, MCD systems allow the targeted delivery of drugs to specific areas. Fluorescent markers make it easy to detect infection, chalcones and stilbenoids inhibit efflux, and the main antibiotic has antibacterial effect. Additionally, new antioxidant compounds were found such as Xanthylium derivative—R351 and chromene derivative 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate: the pronounced antioxidant properties of these substances are observed comparable to quercetin in the efficiency. The charged groups conjugated to the condensed aromatic structures play an important role in enhancing antioxidant activity. These substances hold promise as potential antioxidants. and the activity of CD helps minimize the harmful effects of bacterial toxins and reactive oxygen species on the human body. We have presented new and well-known compounds with new properties that combine the drug with fluorophore/dyes and an efflux inhibitor, creating a promising theranostic formulation.

4. Conclusions

The development of theranostic agents that combine antibiotics with cyclodextrin-based auxiliaries is a groundbreaking approach to revolutionizing drug delivery systems. These innovative compounds have the potential to improve treatment outcomes, reduce side effects, offer a more targeted approach, and help fight antibiotic-resistant pathogens.
Our goal was to develop a combination drug that can both diagnose and treat infections and inflammatory processes. In search of antibacterial agents, we explore a wide range of biologically active substances, including chalcones, chromenes, stilbenoids and xanthyl derivatives. We compare these compounds with well-established antiseptics to identify new properties and expand their application.
To create combined products, we strive to optimize the composition by adding dyes/fluorophores+ drugs+ excipients (antioxidants/adjuvants/efflux inhibitors). This study explores the use of cyclodextrin inclusion complexes as a universal platform for improving the delivery and biological activity of various antimicrobial and antioxidant substances.
The study thoroughly examines the interaction between the selected compounds and methyl-β-cyclodextrin (MCD) using spectroscopic techniques such as ultraviolet-visible (UV-Vis), infrared (IR), and nuclear magnetic resonance (NMR). The observed spectral shifts and variations in peak intensity are strong evidence for the formation of inclusion complexes, indicating that the compounds are effectively encapsulated in the hydrophobic MCD cavity. For many compounds, this encapsulation exceeds 80%, which can increase solubility up to ten times. This process can lead to improved bioavailability, stability, and individual release profiles, as has been shown for other antibacterial drugs.
The study also examines the antioxidant potential of compounds included in MCD delivery systems, comparing them to well-known reference compounds such as quercetin. This is important to minimize the toxic effects of pathogens on the body and neutralize dangerous forms of oxygen. The study identified several new compounds with high antioxidant activity, such as xanthyl derivatives and the chromene derivative 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)—pyridinium methanesulfate. For these compounds, the concentration of the radical half-life is an order of magnitude higher than that of quercetin, which is known as a good antioxidant. The presence of condensed aromatic frameworks also seems to increase the antioxidant capacity.
Dyes and/or fluorophores such as rhodamine 6G, R351, and gentian violet are an important component of the combined theranostic preparation—they have remarkable potential due to their multi-faceted nature, including the ability to visualize target cells.
An important component of the combined theranostic drug is the adjuvants of the main drug (metronidazole, levofloxacin and other antibiotics): chalcones and certain dyes can inhibit the process of outflow in bacterial cells and bacterial dehydrogenase, increasing the effectiveness of the main antibacterial compound, such as metronidazole. The observed synergistic interactions between certain compounds and traditional antibiotics, especially when combined with metronidazole, highlight the potential for synergistic therapy and the ability to overcome antibiotic resistance. For example, chalcon derivatives (7 and 9, which have methoxy-and sulfo-groups in the-B ring) and dyes such as rhodamine 6G and methylene blue have shown strong synergistic effects with metronidazole, indicating their potential to significantly increase the effectiveness of antibiotics.
This study presents formulations of promising” drug candidates “ with a molecular container (MCD) that, when combined, exhibit improved biopharmaceutical and theranostic properties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biophysica4040044/s1, Figure S1. 1H and 13C NMR spectra of the investigated «drug candidates» in free form in d6-DMSO: (a,b) sample 7; (c,d) sample 8; (e,f) sample 9; (g,h) sample 10; (i,j) sample 11; (k,l) sample 12; (m,n) sample 14; (o,p) sample 15; (q) sample 17; (r) theoretical 13C NMR spectral data for sample 17; (s) sample R351-Cl−; (t) theoretical 13C NMR spectral data for sample R351-Cl−. Figure S2. Mass-spectra of the investigated «drug candidates» in free form: (a) sample 7; (b) sample 8; (c) sample 9; (d) sample 14; (e) sample 15. Figure S3: FTIR spectra of the investigated «drug candidates» in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (a) chalcone 10; (b) chalcone 12; (c) chalcone 15. PBS (0.01M, pH 7.4). T = 37 °C.

Author Contributions

Conceptualization, E.V.K. and I.D.Z.; methodology, I.D.Z., N.G.B., S.S.K. and E.V.K.; formal analysis, I.D.Z., A.N.B., V.E.K. and S.S.K.; investigation, I.D.Z., S.S.K., N.G.B. and E.V.K.; data curation, I.D.Z. and E.V.K.; writing—original draft preparation, I.D.Z.; writing—review and editing, E.V.K.; project supervision, E.V.K.; funding acquisition, E.V.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Science Foundation, grant number 24-25-00104.

Institutional Review Board Statement

Cell lines were obtained from the Lomonosov Moscow State University Depository of Live Systems Collection (Moscow, Russia).

Data Availability Statement

The data presented in this study are available in the main text and in the Supplementary Materials.

Acknowledgments

This work was performed using the following equipment from the program for the development of Moscow State University: FTIR microscope MICRAN-3, Jasco J-815 CD Spectrometer, and AFM microscope NTEGRA II.

Conflicts of Interest

The authors declare no conflicts of interest.

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Biophysica 04 00044 sch002
Scheme 2. Synthesis scheme of Xanthylium derivatives R351 salts compounds.
Scheme 2. Synthesis scheme of Xanthylium derivatives R351 salts compounds.
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Scheme 3. Synthesis scheme of the chalcone and stilbenoid derivatives.
Scheme 3. Synthesis scheme of the chalcone and stilbenoid derivatives.
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Figure 1. UV/vis absorption spectra of the investigated dyes and chalcones in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (a) Congo Red 8 µM; (b) Methylene blue 15 µM; (c) Malachite green 10 µM; (d) R351-ClO4− 2.5 µM; (e) sample 9–50 µM; (f) Brilliant green 35 µM, toluidine blue 5 µM, gentian violet 3 µM, sudan III 7 µM. PBS (0.01M, pH 7.4). DMSO could be added to enhance the solubility of free samples. T = 37 °C.
Figure 1. UV/vis absorption spectra of the investigated dyes and chalcones in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (a) Congo Red 8 µM; (b) Methylene blue 15 µM; (c) Malachite green 10 µM; (d) R351-ClO4− 2.5 µM; (e) sample 9–50 µM; (f) Brilliant green 35 µM, toluidine blue 5 µM, gentian violet 3 µM, sudan III 7 µM. PBS (0.01M, pH 7.4). DMSO could be added to enhance the solubility of free samples. T = 37 °C.
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Figure 2. FTIR spectra of the investigated «drug candidates» in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (a) chalcone (9), (b) 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate (17). PBS (0.01M, pH 7.4). T = 37 °C.
Figure 2. FTIR spectra of the investigated «drug candidates» in free form in a buffer solution and complexed with methyl-cyclodextrin (MCD): (a) chalcone (9), (b) 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate (17). PBS (0.01M, pH 7.4). T = 37 °C.
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Figure 3. 1H NMR spectra of the «drug candidate» sample 9: (a) in free form in d6-DMSO; (b) complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol) in D2O with predicted peak correlations. T = 25 °C. (c) The proposed structure of the chalcone 9—MCD complex obtained during computer modeling. Carbon atoms are indicated in green (MCD) and blue (the guest molecule, compound 9); oxygen atoms are indicated in red. hydrogen—white, sulfur—yellow, nitrogen—blue. The purple sphere is Na+. The simulation was performed using the PyMOL program. (d) Schematic cyclodextrin torus representation.
Figure 3. 1H NMR spectra of the «drug candidate» sample 9: (a) in free form in d6-DMSO; (b) complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol) in D2O with predicted peak correlations. T = 25 °C. (c) The proposed structure of the chalcone 9—MCD complex obtained during computer modeling. Carbon atoms are indicated in green (MCD) and blue (the guest molecule, compound 9); oxygen atoms are indicated in red. hydrogen—white, sulfur—yellow, nitrogen—blue. The purple sphere is Na+. The simulation was performed using the PyMOL program. (d) Schematic cyclodextrin torus representation.
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Figure 4. ABTS antioxidant test of the “drug candidates” complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol): (a) Dyes and fluorophores; (b) Chalcone and stilbene derivatives. PBS (0.01M, pH 7.4). T = 37 °C.
Figure 4. ABTS antioxidant test of the “drug candidates” complexed with methyl-cyclodextrin (MCD) (1:5 mol/mol): (a) Dyes and fluorophores; (b) Chalcone and stilbene derivatives. PBS (0.01M, pH 7.4). T = 37 °C.
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Figure 5. The structures of the drug candidates in the relationships with antioxidant and antibacterial properties. The circles highlight significant fragments of molecules.
Figure 5. The structures of the drug candidates in the relationships with antioxidant and antibacterial properties. The circles highlight significant fragments of molecules.
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Figure 6. Confocal laser scanning microscopy images of (a,b) E. coli cells and (c,d) Lactobacilli cells, stained with R6G (10 µg/mL) in free form or complexed with MCD (100 µg/mL). λexci, max = 488 nm, λemi = 530–580 nm. The scale bar is 20 µm.
Figure 6. Confocal laser scanning microscopy images of (a,b) E. coli cells and (c,d) Lactobacilli cells, stained with R6G (10 µg/mL) in free form or complexed with MCD (100 µg/mL). λexci, max = 488 nm, λemi = 530–580 nm. The scale bar is 20 µm.
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Table 2. The list of compounds studied.
Table 2. The list of compounds studied.
Dyes and Fluorophores
Gentian (Crystal) Violet
Biophysica 04 00044 i001		Congo red
Biophysica 04 00044 i002		Sudan III
Biophysica 04 00044 i003		Malachite Green
Biophysica 04 00044 i004
Rhodamine 6G
Biophysica 04 00044 i005		Methylene blue
Biophysica 04 00044 i006		Ponceau 4R
Biophysica 04 00044 i007		Toluidine blue
Biophysica 04 00044 i008
AntisepticsDrugs
Benzalkonium chloride
Biophysica 04 00044 i009		Chlorhexidine
Biophysica 04 00044 i010		Metronidazole
Biophysica 04 00044 i011		Clotrimazole
Biophysica 04 00044 i012
Xanthylium derivativesChromenes
R351-ClO4−
Biophysica 04 00044 i013		R351-Cl−
Biophysica 04 00044 i014		Sample 17x
Biophysica 04 00044 i015		Sample 17
Biophysica 04 00044 i016
Chalcones and stilbenoids
Sample 7
Biophysica 04 00044 i017		Sample 8
Biophysica 04 00044 i018		Sample 9
Biophysica 04 00044 i019		Sample 10
Biophysica 04 00044 i020
Sample 11
Biophysica 04 00044 i021		Sample 12
Biophysica 04 00044 i022		Sample 14
Biophysica 04 00044 i023		Sample 15
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Table 3. Spectral parameters of dyes and physico-chemical parameters of their inclusion complexes with MCD. PBS (0.01M, pH 7.4). T = 37 °C.
Table 3. Spectral parameters of dyes and physico-chemical parameters of their inclusion complexes with MCD. PBS (0.01M, pH 7.4). T = 37 °C.
CompoundMolar Mass, g/molλmax,abs, nmε, M−1·cm−1Solubility in PBS, mMSolubility in PBS in Presence of 5 mM MCD, mMKdis (Compound-MCD), M
Gentian Violet408 (chloride salt)59580,0000.0250.4(3.1 ± 0.2) × 10−4
Congo Red697 (sodium salt)335, 490121,0001013(6.7 ± 0.4) × 10−3
Sudan III248360, 50035,0000.0050.03(1.0 ± 0.15) × 10−3
Brilliant Green483 (hydrosulfate salt)425, 63092,0002124(1.4 ± 0.3) × 10−2
Malachite Green365 (chloride salt)425, 62593,0002224(3.3 ± 0.5) × 10−2
Rhodamine 6G479 (chloride salt)500, 526116,00036.5(1.3 ± 0.2) × 10−3
Methylene Blue320 (chloride salt)610, 66085,0000.10.4(1.6 ± 0.1) × 10−3
Ponceau 4R376 (sodium salt)340, 51056,000812(2.0 ± 0.3) × 10−3
Toluidine Blue304 (chloride salt)63040,0001519(3.8 ± 0.4) × 10−3
Xanthylium Derivatives R351389 (R351-Cl−)
453 (R351-ClO4−)		370, 52069,0000.070.15(4.3 ± 0.5) × 10−3
Table 4. Antibacterial activity of the studied substances against E. coli: MIC and IC25 values as well as the viability of E. coli bacteria depending on the substance’s concentrations. Synergism with metronidazole. Complex formulations of MCD compounds (1:5 mol/mol) were tested. The chemical structures of the compounds are shown in Table 2.
Table 4. Antibacterial activity of the studied substances against E. coli: MIC and IC25 values as well as the viability of E. coli bacteria depending on the substance’s concentrations. Synergism with metronidazole. Complex formulations of MCD compounds (1:5 mol/mol) were tested. The chemical structures of the compounds are shown in Table 2.
SampleCell Viability of E. coli, %MIC Value, mMIC25 *, mMSynergy Coefficient with Metronidazole
1 mM0.1 mM25 µM
Chalcone derivative 787 ± 598 ± 2100 ± 1>55 ± 11.08
Chalcone derivative 892 ± 396 ± 2100 ± 2>5>51.01
Chalcone derivative 979 ± 494 ± 3100 ± 2>51.5 ± 0.31.36
Chalcone derivative 1098 ± 199 ± 1100 ± 1>5>50.89
Chalcone derivative 1197 ± 2100 ± 1100 ± 1>5>51.06
Chalcone derivative 1275 ± 791 ± 497 ± 2>51.0 ± 0.11.03
Chalcone derivative 1493 ± 297 ± 2100 ± 1>5>50.97
Chalcone derivative 1577 ± 887 ± 393 ± 4>51.1 ± 0.11.02
Chromene derivative 1799 ± 1100 ± 1100 ± 1>5>50.97
Xanthylium derivative R351-ClO4−86 ± 290 ± 499 ± 1>55 ± 11.01
Xanthylium derivative R351-Cl−68 ± 479 ± 391 ± 27 ± 10.4 ± 0.051.16
Clotrimazole48 ± 665 ± 880 ± 32.5 ± 0.30.06 ± 0.011.24
Benzalkonium chloride5 ± 229 ± 394 ± 20.8 ± 0.20.040 ± 0.0051.55
Chlorhexidine20 ± 139 ± 387 ± 51.3 ± 0.40.044 ± 0.0071.95
Gentian Violet24 ± 351 ± 580 ± 41.5 ± 0.30.06 ± 0.011.23
Congo red94 ± 298 ± 2100 ± 1>5>51.05
Sudan III84 ± 990 ± 598 ± 1>5>51.04
Toluidine blue74 ± 587 ± 396 ± 3>51.0 ± 0.21.12
Methylene blue38 ± 455 ± 782 ± 42.1 ± 0.20.035 ± 0.0041.53
Ponceau 4R86 ± 290 ± 497 ± 1>5>50.95
Malachite Green12 ± 634 ± 552 ± 81.1 ± 0.20.015 ± 0.0031.28
Rhodamine 6G27 ± 543 ± 288 ± 31.7 ± 0.40.07 ± 0.011.70
Metronidazole15 ± 337 ± 475 ± 80.25 ± 0.050.025 ± 0.003-
* IC25 concentration parameters are given, at which a 25% inhibition of bacterial growth is achieved. The final concentration of DMSO did not exceed 0.1%, and was also taken into account as a control when calculating the antibacterial effect.
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Zlotnikov, I.D.; Krylov, S.S.; Belogurova, N.G.; Blinnikov, A.N.; Kalugin, V.E.; Kudryashova, E.V. New Derivatives of Chalcones, Chromenes, and Stilbenoids, Complexed with Methyl-β-Cyclodextrin with Antioxidant Properties and Antibacterial Synergism with Antibiotics. Biophysica 2024, 4, 667-694. https://doi.org/10.3390/biophysica4040044

AMA Style

Zlotnikov ID, Krylov SS, Belogurova NG, Blinnikov AN, Kalugin VE, Kudryashova EV. New Derivatives of Chalcones, Chromenes, and Stilbenoids, Complexed with Methyl-β-Cyclodextrin with Antioxidant Properties and Antibacterial Synergism with Antibiotics. Biophysica. 2024; 4(4):667-694. https://doi.org/10.3390/biophysica4040044

Chicago/Turabian Style

Zlotnikov, Igor D., Sergey S. Krylov, Natalya G. Belogurova, Alexander N. Blinnikov, Victor E. Kalugin, and Elena V. Kudryashova. 2024. "New Derivatives of Chalcones, Chromenes, and Stilbenoids, Complexed with Methyl-β-Cyclodextrin with Antioxidant Properties and Antibacterial Synergism with Antibiotics" Biophysica 4, no. 4: 667-694. https://doi.org/10.3390/biophysica4040044

APA Style

Zlotnikov, I. D., Krylov, S. S., Belogurova, N. G., Blinnikov, A. N., Kalugin, V. E., & Kudryashova, E. V. (2024). New Derivatives of Chalcones, Chromenes, and Stilbenoids, Complexed with Methyl-β-Cyclodextrin with Antioxidant Properties and Antibacterial Synergism with Antibiotics. Biophysica, 4(4), 667-694. https://doi.org/10.3390/biophysica4040044

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