Synthesis, Hemolytic Activity, and In Silico Studies of New Bile Acid Dimers Connected with a 1,2,3-Triazole Ring

The synthesis of bile acid conjugates plays a significant role in pharmacology and organic chemistry. These complex compounds are widely studied due to their potential therapeutic applications (e.g., drug carriers or antibacterial agents) and their impact on interactions with biological target systems. It is important to determine the biological activity of the obtained conjugates with potential pharmacological applications. The research aimed to synthesize acyl conjugates of bile acids, determine the influence of acyl groups on potential antibacterial activity and evaluate the impact of conjugation on hemolytic activity. New acetyl bile acid acetyl dimers were synthesized using the “Click Chemistry” reaction, aiming to investigate their hemolytic and antibacterial activity. The structures of all compounds were confirmed through spectral analysis techniques, including 1H and 13C nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), and electrospray ionization-mass spectrometry (ESI-MS). The PM5 semiempirical method was also used to estimate the heat of formation of individual conjugates, and the prediction of activity spectra for substances (PASS) technique was used to determine the pharmacokinetic potential of compounds. Docking studies indicate that obtained conjugates have the potential ability to inhibit the biosynthesis of Lipid II and block DNA gyrase. These compounds can therefore be treated as potential candidates for antibacterial compounds. Research findings suggest that conjugating bile acids and their derivatives through 1,2,3-triazole ring, results in final products with reduced hemolytic activity.


INTRODUCTION
Steroids, as a subgroup of natural products, have long been a subject of interest in scientific research due to their potential applications across various fields, including organic chemistry and biomedicine. 1,2Steroids, crucial compounds found in all living organisms, play pivotal roles in many biological processes such as regulation of metabolism, immune response, and reproductive function. 4ile acids�derivatives of cholesterol synthesized predominantly in the liver, form a unique subset within the steroid group. 5Traditionally associated with digestion and fat solubilization, bile acids have recently been identified as signaling molecules involved in glucose and energy metabolism regulation.Their amphipathic properties facilitate the solubilization and emulsification of fats during digestion. 6Bile acids act as signaling molecules by binding to receptors like the nuclear receptor farnesol-X-receptor (FXR) and the G-protein-coupled receptor TGR5.The distinctive structural characteristics of bile acids, including their fused four-ring core and specific hydroxyl group positions, contribute to their diverse biological activities. 7,8These compounds' stereochemistry and amphiphilic nature make them suitable for various biomedical applications e.g., as antimicrobial agents. 9−18 Their ability to form stable complexes with guest molecules highlights their utility in molecular recognition and host−guest interactions. 19,20he concept of "Click Chemistry" has revolutionized the synthesis of complex molecules, offering effective and selective reactions for chemical modification. 21,22The Huisgen 1,3dipolar cycloaddition is a highly efficient technique for synthesizing disubstituted 1,2,3-triazole rings, which are resistant to oxidation, hydrolysis, and reduction.−30 These conjugates can address the limitations of traditional steroids, such as restricted bioavailability and dose-related toxicity.The initial high hemolytic activity of bile acids can be reduced through chemical modifications.These modifications often introduce linkers like the 1,2,3-triazole ring, which is not recognized by bacteria or fungi.
Before experimental research, computational investigations are crucial to predict the chemical activity and physicochemical properties of synthesized compounds. 31,32Computational modeling provides valuable insights into structure−activity relationships and facilitates the rational design of bile acid derivatives with optimized pharmacological profiles.Molecular docking is an effective tool for predicting interactions between potential drugs and target bacterial proteins, accelerating the development of new antibacterial therapies.
This research aims to synthesize steroid conjugates by combining acetate derivatives of bile acids with reduced hemolytic activity and to evaluate their potential antibacterial properties.The hemolytic activity of the synthesized compounds was assessed, and molecular docking studies were conducted to predict their antibacterial efficacy.These investigations were carried out to determine the impact of conjugation on the hemolytic activity and to explore the potential of these compounds as candidates for antibacterial drug
The basic building block of all steroids is the cyclopentanoperhydrophenanthrene ring system is not a source of many useful infrared (IR) signals.The vibrational bands corresponding to C−C bonds were notably weak and were obscured by overlapping signals in the fingerprint region.Stretching vibrations of C−H bonds merged into one broad band, for conjugate structure, between 2952 and 2867 cm −1 .These signals are present for all compounds (13−21).The symmetric carbonyl group ν(C�O) stretching vibration induces characteristic bands at 1736−1741 cm −1 in the Fourier-transform-IR (FT-IR) spectrum, serving as essential signals for all products.Moreover, strong characteristic signals in the region 1247−1215 cm −1 are present, which are assigned to the ν(C−O).Compound (13), which is a derivative of lithocholic acids does not contain an acetate group in its molecule.Consequently, its FT-IR spectrum exhibits an All compounds (13−21) showed signals originating from 3β-H and 3β′-H protons.In the case of compounds derived from deoxycholic or cholic acids, signals originating from the following protons 12β-H/12′β-H (14−21) and 7β-H/7′β-H (15, 18, 19−21) were also present.
Compounds ( 13) and ( 14), a series of products from azidoacetate derivatives of lithocholic acid, had a 3′β-H signal in the form of a multiplet at 4.89−4.81ppm.3β-H signals occurred as multiplets with values ranging from 5.10 to 5.03 (13), 5.06 and 4.99 (14).Compound ( 14) also showed a signal from the 7β-H proton as a singlet at 5.08 ppm.In the case of compound (15), the 12′β-H proton was visible in the form of a distinct singlet at 5.09 ppm, however, the signals from 7′β-H, 3β-H, 3′β-H overlapped, creating a multiplet at 4.96−4.80ppm.
Compounds ( 16) and ( 17), a series of products from azidoacetate derivatives of deoxycholic acid, had a 3′β-H signal in the form of a multiplet at 4.87−4.79ppm.Compound (17) also showed signals from the 3β-H proton at 5.06−4.99ppm in the form of a multiplet.Moreover, the signals from 12β′-H and 12β-H were visible as singlets at 5.09 and 5.08 ppm.In the case
In the case of compounds (19−21), which are a series of products from azidoacetate cholate derivatives, all signals from the 3′β-H group were present in the form of a multiplet at 4.74−4.66ppm.In compound (19)  2.
As evident, the end products (13−21) exhibit lower HOF values compared to their substrates.The highest HOF values are observed for the compound (13), while the lowest is for (21).The presence of hydroxyl groups in the bile acid structure, as well as their substitution by acetate groups, affects the determinant value of the HOF.Moreover, the incorporation of OAc groups facilitates the formation of stable host− guest complexes through intramolecular hydrogen bonds.Consequently, an increase in the number of OAc groups in the bile acid skeleton results in a decrease in the HOF value.These complexes can be stabilized via hydrogen bonding or electrostatic interactions stemming from the OAc groups within the bile acid molecule.

Prediction of Activity
Spectra for Substances.−40 Biological activity predicted for a potential compound with the highest probability (focal activities) has also been selected and presented in Table 2.The most frequently predicted types of biological activity are cryoprotectant and hypolipemic.On the other hand, conjugates (18,  20−21) have an antiviral activity and (13−15, 19, 21)  Adenomatous polyposis inhibitor activity.
2.4.Hemolytic Activity.Bile acids exhibit different effects on the molecular structure of cell membranes dependent on their chemical structure and concentration. 41,42Hydrophobic bile acids like lithocholic and deoxycholic acid provoke hemolysis in red blood cells (RBC) in a dose-dependent manner by enhancing the cell membrane permeability to ions. 43Conversely, at sublytic concentrations, bile acid molecules serve to stabilize the lipid bilayer of cell membranes and modulate the activity of membrane proteins such as MRP1. 44,45Considering the amphiphilic properties of bile acids and their derivatives, it is essential to evaluate their hemolytic activity before any biomedical uses Therefore, all compounds underwent in vitro evaluation for cytotoxicity using a hemolytic assay with human RBC.Based on literature data and the results obtained, it can be concluded that the dimerization of acetyl derivatives of bile acids results in a reduction in hemolytic activity. 46In the case of lithocholic (LA) and cholic acid (CA), it can be seen that the result of acylates to derivatives LA-Ac and CA-Ac is a slight increase in hemolytic activity.It is worth noting, however, that LA's activity is much higher than the tested deoxycholic acid (DA) and CA.Acylation of DA increases its hemolytic activity to a value of 90%.For all compounds obtained, the hemolytic activity value was 13−28%, as shown in Figure 3. LA-Ac and DA-Ac conjugation with other acetyl bile acid derivatives results in obtaining dimers with lower hemolytic activity than the initial one.Only in the case of CA-Ac derivatives, an increase in hemolytic activity by 10−20% can be observed when conjugates with other acetyl derivatives.The above conclusions are particularly visible for compounds ( 13), (18), and ( 21) which are dimers of acetyl derivatives of lithocholic, deoxycholic and cholic acids (marked with the same color as the substrates in Figure 3).

Molecular Docking.
In the modern world, the growing threat from antibiotic-resistant bacteria poses an urgent need for scientists and researchers to discover new chemical compounds with antibacterial properties.Drugresistant bacteria are becoming more common, which makes existing treatments less effective.Therefore, the search for new compounds with antibacterial activity is becoming a key goal of scientists around the world.,It is known that steroid derivatives may have antibacterial activities. 47hree representative compounds were selected for virtual screening: (13), (17), and (21).These are dimers of lithocholic, deoxycholic and cholic acids respectively, which additionally differ in the number of acetyl groups present.The presence or absence of these groups may therefore influence the ability to dock to selected active sites.Numerous mechanisms of antibacterial action exist.For example, antibacterial glycopeptides show activity by inhibiting Lipid II of bacteria. 47Lipid II plays a crucial role in bacterial cell wall synthesis as it serves as a precursor molecule for the formation of peptidoglycan, an essential component of bacterial cell walls.Peptidoglycan provides structural integrity and protection to bacterial cells, making the biosynthesis of Lipid II a vital process for bacterial survival and proliferation.In turn, fluoroquinolones, by blocking DNA gyrase, effectively destroy Gram-positive bacteria. 47,48DNA gyrase, which belongs to topoisomerases, is a key enzyme involved in many life processes of bacteria, including replication, transcription and recombination of bacterial DNA.It is composed of two   subunits�A (responsible for cell division processes) and B (responsible for ATP hydrolysis). 49ue to literature reports confirming the antibacterial activity of steroids, molecular docking was performed. 47,48Docking involved activity to inhibit Lipid II and DNA gyrase of compounds ( 13), (17), and (21).
To assess the antibacterial activity of selected compounds, the first step is To assess the arrangement and type of interactions of a typical representative of fluoroquinolones− ciprofloxacin in the GyrB binding pocket (PBD: 3U2D). 50hen compare them with the arrangement and type of interactions created by the tested compounds.Docked ciprofloxacin (docking score −7.169 kJ/mol) forms stable hydrogen bond interactions with Ser129 (2.68 Å), while protonated piperazine forms a salt bridge with Glu58 (3.86 Å) and an additional hydrogen bond also with Glu58 (2.95 Å) and Asn54 (2.80 Å).
All three molecules ( 13), (17), and (21).show a similar skeletal conformation, when binding to the binding pocket of DNA gyrase.These compounds take the shape of the letter "U", with the inflection region at the level of the triazole ring, and occupy the same space as ciprofloxacin.The results are presented in Figure 4.
For compound (13), the triazole forms a hydrogen interaction with Ser129 (2.91 Å) similar to that in the case of ciprofloxacin or other sterol derivatives described by Ansari et al. 49 Both carbonyl groups of the bile acid side chain form a total of three hydrogen bonds−one double with Arg144 (2.37 and 2.34 Å) and one with Gln91 (2.17 Å).The results are presented in Figure 4A.Compound (17) (Figure 4B) similar to compound (13), it forms a hydrogen interaction with Ser129 (1.96 Å), but not through the triazole ring but through the carbonyl group at C-26 (derived from the propioloacetate derivative).Moreover, the carbonyl group present in the steroid side chain also forms hydrogen bonds with Arg144 (2.39 Å) while the carbonyl oxygen forms a hydrogen bond with Lys93 (2.12 Å).Compound (21) (Figure 4D) adopts a similar conformation as ( 13) and ( 17), forming hydrogen interactions with Ser129 (2.07 Å) and only one hydrogen bond between Tyr63 (2.28) with the oxygen of the bile acid side chain carbonyl group.None of the four introduced acetate groups interacts with the DNA gyrase binding pocket.
As shown in Table 3, compound ( 13) may have the best potential to inhibit DNA gyrase.It has a similar docking score and creates the largest number of hydrogen bonds.Compound (21), in turn, has the highest docking score and creates only two interactions with Ser129 (2.07 Å) and Tyr63 (2.28 Å).
The tested compounds were also docked to the crystal structure of Staphylococcus aureus membrane receptor transglycosylase (PDB: 3VMT) 51 with parent Lipid II as a reference.The tested compounds ( 13) and ( 17), similarly to  DNA gyrase, adopt a bent U-shaped conformation where the triazole ring (which is the inflection point) squeezes into the binding pocket, occupying the same space as the parent crystallized compound−LHI301 which is shown in Figure 5.
In the case of compound ( 13) (docking score −5.423 kcal/ mol), the triazole forms an interaction with Glu102 (2.70 Å), while the carbonyl oxygen directly attached to the triazole through C-26 interacts with a hydrogen bond with Lys113 (2.09 Å).The carbonyl oxygen located on the other side of the triazole ring at C-30 also forms a hydrogen bond with Lys248 (2.36 Å).Furthermore, the carbonyl oxygen of the bile acid side chain forms a hydrogen bond with Gln240 (2.04 Å).For compound (17) (docking score −5.144 kcal/mol), the hydrogen bond with Glu102 (1.87 Å) is not formed with the triazole ring but with the carbonyl group at C-30 (from the azidoacetate derivative part).The ester group attached directly to the triazole (from the propionate derivative part), in turn, forms two hydrogen interactions with Ser132 (2.52 Å) and Gly130 (2.46 Å).The second ester group attached to the bile acid side chain (from the azide derivative part) also forms two hydrogen interactions with Arg117 (2.55 Å) and Lys248 (2.43 Å).Compound (21) did not dock to the crystal structure of the molecular target.Taking into account the above and analyzing the data from Table 4, both compounds ( 13) and (17) have the potential to exhibit antibacterial activity.

CONCLUSIONS
This study presents the synthesis and chemical characterization of conjugates of bile acid derivatives connected via a 1,2,3triazole ring (13−21).The results obtained in this study confirm that the conjugation of acetyl derivatives of lithocholic and deoxycholic acid esters causes a significant reduction in hemolytic activity of the obtained products ( 13) and ( 17).In the case of a dimer conjugate of cholic acid derivatives (21), an increase in hemolytic activity can be observed compared with cholic acid and its acyl derivative.The results indicate that for bile acid derivatives with high initial hemolytic activity, conjugation is a convenient method of obtaining new compounds with reduced hemolytic activity.This approach circumvents the challenge posed by the high hemolytic activity of these compounds.
Molecular docking studies prove that the compounds ( 13) and ( 17) exhibit possible binding affinity toward the protein targets of interest (3U2D and 3VMT).Inhibition of both protein's activity could indicate the tested compound's antibacterial properties.Docking results suggest that the antibacterial activity primarily stems from interactions associated with triazole rings or carbonyl groups, rather than acetate groups.It is therefore worth considering the possibility of other modifications of the hydroxyl groups.
Bacteria can develop resistance to commonly used antibiotics, making these drugs ineffective.This poses a serious threat to humanity.Therefore, there is an urgent need to search for new antibacterial drug candidates.The compounds obtained in this study appear valuable based on the results.This prompts further in vitro tests on pathogenic bacterial strains.
absence of signals attributed to the ν(C�O) and ν(C−O) functional groups.The most characteristic signals of compounds (13−21) in the range of 3.90−8.50ppm in the 1 H NMR spectra are shown in Figure 1.The diagnostic proton signals of the triazole ring 28-CH in all bioconjugates (13−21), featuring a 1,2,3-triazole ring are manifested as a singlet approximately at 8.28−8.25 ppm.The protons from the methylene groups 29-CH 2 directly bonded to the triazole ring exhibit signals at approximately 5.21−5.18ppm.However, in the case of compounds (19−21) (methyl cholate azidoacetate derivatives), these signals appear as a double singlet at 5.21 ppm.

Figure 3 .
Figure 3. Hemolytic activity of compounds tested (0.1 mg/mL) after 1 h incubation at 37 C. A hemolysis degree higher than 5% indicates the cell membrane-perturbing activity of compounds.PBS�negative control, no hemolysis.* Adapted from data in ref 46.

Figure 4 .
Figure 4. (A−C) Binding modes with hydrogen bonds of key amino acids of docked compounds (13)-magenta, (17)-red, (21)-black.(D) The surface of the gyrase DNA with docked compounds on.The yellow dotted line represents the hydrogen bond.

Figure 5 .
Figure 5. Binding modes with hydrogen bonds of key amino acids of docked compounds: (A) compound (13) magenta, (B) compound (17) red.The yellow dotted line represents the hydrogen bond.

Table 2 .
Probability "To Be Active" (PA) Values for the Predicted Biological Activity of Dimmers(13−21)

Table 3 .
Docking Scores and Hydrogen Bonds Formed with Representative Compounds (13),(17), and (21) a Ciprofloxacin is used as a reference with good antibacterial activity. a

Table 4 .
Docking Scores and Hydrogen Bonds Formed with Representative Compounds(13)and(17)