Molecular modeling, synthesis and biological evaluation of caffeic acid based Dihydrofolate reductase inhibitors

Abstract

Dihydrofolate reductase (DHFR) is an enzyme that plays a crucial role in folate metabolism, which is essential for cell growth and division. DHFR has been identified as a molecular target for numerous diseases due to its significance in various biological processes. DHFR inhibitors can disrupt folate metabolism by inhibiting DHFR, leading to the inhibition of cell growth. So, a series of caffeic acid derivatives were designed, synthesized, characterized and evaluated for their in vitro ability to inhibit DHFR, as well as their antimicrobial and anticancer properties. Among all synthesized compounds, compound CE11 exhibited the highest DHFR inhibitory activity, with an IC₅₀ value of 0.048 µM, which is approximately four times more potent than methotrexate. Compound CE11 exhibited similar docking performance to methotrexate, binding to the same site and engaging key residues such as Glh30, Phe31, Phe34, and Ser59. It also fit snugly in the hydrophobic pocket of modeled protein. Moreover, substantial hydrophobic interactions were noted between the ligand and the hydrophobic amino acid residues of DHFR. This effectively secured the derivative within the restricted substrate cavity. Furthermore, compound CE11 demonstrated a significant anticancer activity against MCF-7 breast cancer cell line, with an IC₅₀ value of 5.37 ± 0.16 µM. Compounds CE3 and CE15 displayed better antibacterial activity compared to trimethoprim and comparable to ampicillin against the gram-positive bacteria S. aureus. Moreover, compounds CE3 and CE15 have shown better antibacterial activity than standard trimethoprim, ampicillin and tetracycline against the gram-negative bacteria, particularly P. aeruginosa and E. coli. Molecular docking analysis of CE3 revealed that it firmly entrapped into the active site of enzyme through hydrophobic interaction with hydrophobic residues. Additionally, it forms hydrogen bonds with important amino acid residues Ala7, Asn18, and Thr121 with excellent docking score and binding energy (-9.9, -71.77 kcal/mol). These interactions might be contributed to the significant DHFR inhibition and antimicrobial activity. The generated model holds potential value in facilitating the development of a novel category of DHFR inhibitors as anticancer and antimicrobial agents.

Introduction

DHFR (E.C. 1.5.1.3) is a ubiquitous enzyme found in almost all eukaryotic and prokaryotic cells. It plays pivotal role in the biosynthesis of tetrahydrofolate (THF) by catalyzing the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate using NADPH as a coenzyme. THF is the active form of folic acid which serves as a vital cofactor in one-carbon transfer reactions such as DNA synthesis, amino acid metabolism and purines biosynthesis. Inhibiting this enzyme results in a deficiency of the active form of folic acid, this disrupts nucleotide biosynthesis and ultimately causes cell death. Hence, it is a significant target for therapeutic purposes in biochemistry and medicinal chemistry and it has been validated as an effective therapeutic target for treating bacterial infections and cancers [1,2,3,4,5,6,7,8]. This is evidenced by the use of standard DHFR inhibitors as anticancer drugs such as methotrexate (MTX), raltitrexed, pemetrexed, pralatrexate, and trimetrexate, as well as the use of trimethoprim (TMP) as an antibacterial agent. However, the absence of novel DHFR inhibitors in the pipeline for decades, coupled with persistent resistance to existing DHFR inhibitors, has driven concentrated research in this area [9]. MTX faces resistance through various mechanisms, such as elevated expression of the DHFR protein due to DHFR gene amplification, increased methotrexate efflux facilitated by the over expression of ATP-binding cassette transporters, reduced methotrexate uptake via the reduced folate carrier, decreased methotrexate polyglutamation by folypolyglutamate synthetase, reduced methotrexate affinity due to DHFR mutation, or combinations of these mechanisms [10,11,12,13,14]. The standard antimicrobial DHFR inhibitor TMP also encounters resistance through mechanisms such as the overproduction of resistant chromosomal DHFR enzymes and the activation of alternative metabolic pathways [15,16,17]. Hence, there remains a significant potential in this area to pursue the discovery of novel and effective DHFR inhibitors, offering new opportunities to overcome resistance. To address the limitations of current DHFR inhibitors, researchers have shifted their focus toward developing natural compounds as DHFR inhibitors. It is essential to acknowledge that many plant-derived compounds remained largely untapped in drug discovery. Phytochemicals may serve as alternatives to synthetic DHFR inhibitors due to their inherent anticancer and antimicrobial properties and their ability to protect essential cellular components, such as proteins, DNA and lipids from oxidation [18].

In the search of a novel structural class for DHFR inhibition, caffeic acid was chosen as a lead to design derivatives. In our previous investigation, which aimed to identify natural-source compounds capable of inhibiting DHFR, caffeic acid showed notable activity and favorable binding scores [19]. Caffeic acid is a secondary plant metabolite found in foods like coffee, wine, tea and widely used remedies such as propolis. It has a range of biological applications such as antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic activities and shows potential in treating skin-related diseases. Despite numerous studies have reported on the antimicrobial and anticancer effects of caffeic acid but its mechanisms of action remain largely unknown [20, 21]. Furthermore, caffeic acid and its derivatives have received little attention or underexplored as DHFR inhibitors, highlighting an opportunity to explore these compounds in developing a novel class of DHFR inhibitors to target this crucial therapeutic pathway. Thus, Caffeic acid stands out as a promising lead for chemical modification in the development of novel and effective DHFR inhibitors. To our knowledge, no comprehensive computational study has yet been conducted to identify safe caffeic acid based derivatives as DHFR inhibitors. With this goal in mind, our study was undertaken to determine the pharmacophore necessary for interaction with key amino acid residues of DHFR.

Developing a novel drug is a complex, time consuming and costly process; however, advancements in computer aided drug design, particularly structure based drug design (SBDD) provide solutions to help streamline this process. SBDD allows for the screening of thousands of ligands, predicting their affinity for specific disease targets. Therefore, virtual screening has become a widely accepted technique for identifying potential hits and excluding non-complementary compounds [22]. In this study, we concentrated on developing novel caffeic acid based class of compounds as DHFR inhibitors. Ligands with high binding scores were selected, and their absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties were evaluated before synthesis to validate in silico results. So, this study focused on the design, synthesis and biological evaluation of novel category of DHFR inhibitors. The synthesized derivatives were evaluated for their in vitro DHFR inhibition potential as well as antimicrobial and anticancer activities. This study offers valuable insights and a foundation for developing a new class of DHFR inhibitors with enhanced efficacy and reduced side effects.

Result and discussion

Molecular modeling

The molecular docking technique was utilized to screen out the potential compounds from in house library of caffeic acid derivatives, as it is a commonly used method to exclude compounds that do not exhibit strong interactions with the target protein. Structure-based virtual screening (SBVS) of caffeic acid derivatives was carried out by using Schrodinger's Maestro Glide software to identify novel category of derivatives as DHFR inhibitors. To design antimicrobial agents, the three-dimensional coordinates of the S. aureus trimethoprim-resistant variant, S1DHFR (PDB ID: 2W9S), co-crystallized with trimethoprim was used [23]. To target cancer, the homo sapiens DHFR with PDB ID 1U72, co-crystallized with methotrexate, was chosen.

Grid generation

The receptor grid generation tool of Maestro was utilized to define the regions where ligand docking calculations would occur for the protein receptors. This involved picking the molecules of modeled protein as these structures contained co-crystallized ligands: trimethoprim for 2W9S and methotrexate for PDB ID 1U72. The receptor grid generation process established a grid-based representation of the binding site for subsequent ligand docking simulations.

Docking site validation

Docking site validation was carried out as it is crucial to validate the docking protocol before proceeding to experimental validation in the wet lab. Docking site validation was performed by separating the co-crystallized ligand from the energy-minimized DHFR protein and subsequently re-docking it back into the active site. When the calculated RMSD (Root mean square deviation) value found below 2 Å, it validates the computational docking protocol. Hence, RMSD between the co-crystallized ligand and the docked pose was calculated using Maestro's structure alignment task and superposition tool. The obtained RMSD values were 0.5619 Å for PDB ID 2W9S and 1.1945 Å for PDB ID 1U72, indicating satisfactory docking protocol.

Molecular docking interaction with PDB ID 2W9S (microbe DHFR)

It was observed that caffeic acid exhibited promising interactions with DHFR, establishing it as a prime candidate for the development of novel DHFR inhibitors [19]. Therefore, using caffeic acid as a lead candidate, alterations were introduced to the carboxylic acid functional group of the lead compound to design a diverse library of caffeic acid derivatives. The designed ligands having superior and comparable docking scores and binding energies than the standard drugs TMP and MTX were reserved for further investigation to elucidate their mechanistic interactions with DHFR. Selected ligands were subsequently synthesized in the laboratory and their in vitro DHFR inhibitory potential was assessed to validate docking studies results.

DHFR PDB ID 2W9S was used as a molecular target to design caffeic acid derivatives as antimicrobial agents. Among all the designed derivatives the docking pose of the most active antimicrobial compound CE3 revealed the formation of hydrogen bonds with amino acid residues Ala 7, Asn 18, and Thr 121. Furthermore, this compound exhibited an outstanding docking score and binding energy (− 9.9; − 71.77 kcal/mol) better than the standard drug TMP. It was observed that ligand was firmly entrapped into the binding site of the enzyme through hyodrophobic interaction with non-polar amino acid residues Ile 5, Val 6, Ala 7, Ile 14, Leu 20, Ile 31, Ile50, Phe 92, Tyr 98 and Tyr 109 as shown in Figs. 1 and 2. Saturated ring system (2-isopropyl-5-methylcyclohexanol) in compound CE3 might help the compound to enter into the hydrophobic pocket of the DHFR enzyme by increasing its affinity for the non-polar amino acids in the pocket. This might prevent substrate (dihydrofolate) binding to the enzyme and ultimately reduce the enzyme activity. Ligand CE3 occupied same space of active site of DHFR as TMP, as shown in superimposed image of CE3 and TMP (Fig. 3). Docking pose of second most active antibacterial compound CE15 formed hydrogen bond with essential amino acids residues of active site such as Ala 7 and Phe 92 and pi-pi stacking with Phe 92 which might be responsible for potency of compound (Figs. 4 and 5) with excellent docking score and binding energy (− 9.7, − 68.84 kcal/mol). Anilides of caffeic acid (CAm7) also shown good interactions with protein by forming three hydrogen bonds with residues Ala 7, Asn 18, and, Thr 121 additionally, it is worth noting that the compound also demonstrated impressive results in terms of docking score of −9.5 and a binding energy of − 64.5 kcal/mol. It is observed that compound CAm7 was firmly entrapped into the active site of enzyme through hydrophobic interaction with Ile 14, Leu 20, Trp 22, Ile 31, Phe 92, and Tyr 98 residues. The formation of hydrogen bonds was identified as a crucial factor for the effective binding of the ligands within the enzyme. It is noteworthy that nearly every selected molecule displayed better and comparable docking scores in comparison to the standard trimethoprim, along with excellent binding energies, as tabulated in Table 1. Molecular docking analysis revealed that nearly all the synthesized ligands occupied the common core of enzymes having surroundings residues are Ile5, Val6, Ala7, Ile14, Asn 18, Gln 19, Leu20, Trp22, Asp 27, Leu 28, Ile 31, Thr 46, Ile50, Leu 54, Phe92, Gly 93, Gly 94, Tyr 98, Tyr 109, and Thr 121 as shown in Fig. 6. This type of findings also supported by Ikram, M. et al., 2023 and Adnane Aouidate, 2017 [24, 25].

Fig. 1

2D pose of compound CE3 encircled by non-polar and polar amino acid residues, demonstrating interactions with DHFR (PDB ID 2W9S)

Fig. 2

3D view of compound CE3 in the binding pocket of DHFR (PDB ID 2W9S) with surrounded amino acid residues

Fig. 3

Superimposed image of compound CE3 (green) and trimethoprim (white) in the binding pocket of DHFR (PDB ID 2W9S) receptor with amino acids residues present in proximity of active site

Fig. 4

2D pose of compound CE15 encircled by non-polar and polar amino acid residues, demonstrating diverse interactions with DHFR (PDB ID 2W9S)

Fig. 5

3D view of compound CE15 in the binding pocket of DHFR (PDB ID 2W9S) with surrounded amino acid residues

Table 1 Molecular docking interaction with DHFR PDB ID 2W9S and DHFR inhibition assay of potential synthesized compounds and standard drug

Fig. 6

Protein–ligand interaction fingerprintings of designed hits showcasing interaction with amino acids residues of DHFR active site

Molecular docking interaction with PDB ID 1U72 (human DHFR)

To design caffeic acid derivatives as anticancer agents, molecular docking was performed by using the homo sapien DHFR (PDB ID 1U72) as molecular target. Among all the designed derivatives compound CE11 emerged out as most potent anticancer compound exhibited similar docking interactions to MTX, binding to the same site and engaged with key amino acid residues residues such as Glh30, Phe31, Phe34, and Ser59 as shown in Fig. 7 and tabulated in Table 2. It also fit snugly into the hydrophobic pocket of modeled protein as depicted in Figs. 8 and 9. This type of interactions results aligned well with Wang et al., 2017 and El-Gazzar Y.I. et al., 2017 results [26, 27].

Fig. 7

Superimposed image of compound CE11 (pink) and methotrexate (green) in the binding pocket of DHFR (PDB ID 1U72) receptor with amino acids residues present in proximity of active site

Table 2 Molecular docking interaction analysis of potential anticancer compounds and standard drugs with PDB ID 1U72

Fig. 8

2D pose of compound CE11 within DHFR (PDB ID: 1U72) binding pocket, showing interactions with surrounding non-polar and polar residues

Fig. 9

3D view of compound CE11 in the active site pocket of DHFR (PDB ID 1U72) with surrounded important amino acid residues

Binding pocket of microbe DHFR and human DHFR

The objective of this research was to identify specific structural characteristics necessary for inhibiting microbe-DHFR and to design new compounds that selectively target it, utilizing both computational analysis and laboratory techniques. While DHFR is found in both humans and bacteria, the ability to selectively inhibit the enzyme is crucial for designing effective ligands. To develop selective inhibitors for microbe-DHFR, a virtual screening protocol was utilized, using two protein targets: microbe DHFR and human DHFR. A library of caffeic acid derivatives was constructed and screened against microbe DHFR and the screened compounds were further tested for selectivity against human DHFR. Understanding the composition of the active sites in these two target proteins is essential for exploiting their differences in the development of specific and selective ligands. A comparative analysis of the two target proteins has unveiled discrepancies in the amino acids constituting the catalytic triad, as well as other crucial amino acids responsible for ligand interactions. The in silico studies provided additional insights that were not possible to infer from the experimental data alone. Based on in silico data, the amino acids found in proximity to the active site of microbe DHFR were Ile 5, Val 6, Ala7, Asn18, Leu20, Asp27, Leu28, Ile 31, Ile 50, Leu 54, Phe 92, and Tyr 98. It is postulated that a designed ligand interacting with these residues would be an effective inhibitor of DHFR. This result aligned well with Azzam R.A. et al., 2020 findings [28]. On the other hand, for human DHFR inhibition, crucial interactions involved amino acid residues Ile7, Val8, Ala9, Leu22, Glh30, Phe31, Tyr33, Phe34, Gln35, Val115, Ile60, Pro61, Leu67, Val115, and Tyr121. These observations were further supported by Canh Pham, E. et al., 2022, Sharma K et al., 2018 [29, 30]. The most effective anticancer compound CE11 with MTX and the most effective antibacterial compound CE3 with TMP may be seen in superimposed pictures (Figs. 7 and 3) respectively, to showcase comparable pharmacophores and occupy the same active site regions within the binding pocket of DHFR. In summary, these findings offer valuable insights for the development of selective inhibitors, illuminating the precise interactions needed to effectively target microbe DHFR and human DHFR.

Systematic ADMET evaluation of caffeic acid derivatives

ADMET analysis plays a pivotal role in drug development, facilitating rapid and preliminary testing of ADMET qualities before subjecting compounds to in vitro evaluation. In this study, the ADMETlab 2.0 online tool was utilized to predict comprehensive pharmacokinetic properties of selected compounds [31,32,33]. The ADMET prediction of the synthesized compounds indicated that they adhere to Lipinski's rules of five, ensuring their drug-like characteristics. Intestinal absorption (Caco-2), membrane and skin permeability, P-glycoprotein substrate or inhibitor, and human intestinal absorption (HIA), were considered as these factors influence drug absorption. Predictions for drug distribution encompassed plasma protein binding (PPB), volume of distribution (VD) and blood–brain barrier permeability (BBB) values. Metabolism predictions were based on CYP (cytochrome P450) models for substrate or inhibition, while excretion was assessed using clearance (CL) and drug half-life (T1/2). Toxicity predictions encompassed parameters like AMES toxicity, hERG inhibition, rat oral acute toxicity, respiratory toxicity, skin sensitization, and carcinogenicity.

The evaluation of ADMET properties indicated that synthesized compounds demonstrated excellent human intestinal absorption, as predicted by Caco-2 permeability, MDCK permeability, P-glycoprotein inhibition (Pgp), and HIA values (Table 3). This advantage might be attributed to the lipophilicity of compounds, facilitating their passage through biological membranes. Human oral bioavailability predictions (F20%) suggested that most synthesized compounds lacked oral bioavailability, except for CE3, CE11, and CAm7 compounds, which exhibited good oral bioavailability. Distribution assessment through PPB, VD, and BBB permeability revealed high plasma protein binding for all compounds, impacting distribution and indicating a need for customization in this area. However, distribution assessed by VD and BBB for the synthesized compounds was found to be excellent. Analysis revealed that the compounds could moderately inhibit most cytochrome P450 family isoenzymes involved in drug metabolism. Excretion, evaluated in terms of clearance, highlighted that the novel derivatives exhibited excellent total clearance. Regarding toxicity, the last parameter studied in the ADMET profile, AMES toxicity predictions for the derivatives indicated that most synthesized compounds were free from potential mutagenicity, except for CE15, CE17 and CAm7. Additionally, compounds CE8, CE15, and CAm7 showed positive results for respiratory toxicity, while all others were free from this toxicity. However, all the synthesized compounds were devoid of the probability of rat oral acute toxicity and carcinogenicity, except for CE15, CE17, and CAm7, which showed positive carcinogenicity results. Importantly, all derivatives demonstrated no likelihood of cardiotoxicity, indicating no inhibitory effect on hERG except CE11.

Table 3 ADMET data of synthesized caffeic acid derivatives

Chemistry

Previous literature research has discussed specific features that serve as pharmacophoric characteristics for active DHFR inhibitors. These features include the placement of the basic nitrogen atom, the carbonyl function, hydrophobic regions, the formation of ester bonds and their relative diameters [15, 27]. The synthetic approach used to synthesize the designed compounds is outlined in Scheme 1 [34, 35]. The structural elucidation of the synthesized derivatives was carried out using FTIR, ¹H NMR, ¹³C NMR and elemental analysis. Briefly, 3-(3,4-dihydroxyphenyl)acryloyl chloride was synthesized by adding thionyl chloride to caffeic acid using pyridine as a catalyst. In the second step, esters were synthesized by adding different aromatic alcohols to 3-(3,4-dihydroxyphenyl)acryloyl chloride in ether. Completion of reaction was checked by cessation of hydrogen chloride evolution and single spot TLC. Anilides of caffeic acid were synthesized by adding substituted aniline to 3-(3,4-dihydroxyphenyl)acryloyl chloride in ether. Subsequently, anilide precipitates were subjected to treatment with 5% hydrochloric acid solution, followed by a 4% sodium carbonate solution and water to eliminate any remaining aniline. The confirmation of ester formation was established by observing the formation of the ester's carbonyl (C=O) linkage between caffeic acid and the alcohol. This confirmation was further supported by the disappearance of the O–H stretching vibration (bonded of carboxylic acid group) peak, which typically occurs in the 3000–2500 cm⁻¹ range for caffeic acid. In addition, the appearance of a distinct C=O ester peak in the range of 1750–1648 cm⁻¹ was observed in the synthesized compounds [36]. The FTIR spectrum of the synthesized derivatives displayed distinct peaks corresponding to the stretching vibrations of aromatic and aliphatic CH bonds. These peaks were observed at approximately 3000–3084 cm⁻¹ for aromatic C-H stretching and 2834–2872 cm⁻¹ for aliphatic CH stretching, respectively. Missing of broad band of carboxylic acid between 3200–2500 cm⁻¹ also indicates conversion of caffeic acid into product. The formation of ester and anilide compounds is further confirmed by the ¹H NMR signals, as indicated by the disappearance of the carboxylic acid peak at approximately δ 12 in all the synthesized compounds.

Scheme 1

Synthetic procedure to synthesize caffeic acid derivatives

Biological evaluation

Dihydrofolate reductase inhibition

To assess the effectiveness and validate the findings from molecular docking, the in vitro DHFR inhibitory effect of the synthesized derivatives was evaluated. This evaluation was conducted using the DHFR assay kit (Sigma-Aldrich, catalog number CS0340) by employing a spectrophotometric method. The enzymatic degradation of dihydrofolic acid to tetrahydrofolic acid was monitored at 340 nm for 180 s, following the procedure recommended by the enzyme assay kit manufacturer. The IC₅₀ (concentration required to inhibit 50% of enzymatic activity) was determined and is presented in Table 1. The in vitro activity results were consistent with the dry lab findings, with only a few minor deviations observed. Methotrexate, which is a known standard for DHFR inhibition, was included in the screening under identical conditions for comparison purposes. Additionally, caffeic acid, identified as a potential hit, and trimethoprim were also screened using the same conditions. To better understand the way compounds work, the promising synthesized derivatives were tested against DHFR. Methotrexate was used as positive control. The outcomes demonstrated the great potency of these compounds at micromolar concentrations to inhibit the target enzyme. Compared to the parent molecule caffeic acid, all synthesized compounds have greater activity. Compound CE11 stands out as the most potent among all the synthesized compounds with an IC₅₀ value of 0.048 µM, which is lower than the IC₅₀ of the methotrexate (IC₅₀: 0.088 µM) as compared in Fig. 10. This might be due to the good lipophilic character of this compound, a character required for DHFR inhibition [37]. Compound CE3 also exhibited strong inhibition against DHFR, with an IC₅₀ 0.182 µM. Similarly, compound CAm7 demonstrated good inhibition against DHFR with an IC₅₀ value of 1.10 µM. Because the IC₅₀ range for ester and anilides derivatives of caffeic acid was between 0.048 and 16.92 µM, our findings stipulate that DHFR may be the molecular target of these analogues and that they may have an antimetabolite effect.

Fig. 10

Comparison of DHFR inhibition (IC₅₀ values) between standard methotrexate and synthesized compounds

Antimicrobial activity

The lowest concentration of an antimicrobial agent necessary to stop the development of a microbe after 18 to 24 h is known as Minimum Inhibitory Concentration (MIC) [38]. In vitro antimicrobial screening of the synthesized compounds in μg/ml was determined by using the serial dilution method against bacteria (gram-negative and gram-positive) and fungal strains using trimethoprim (DHFR Inhibitor), ampicillin (antibacterial), tetracycline (antibacterial), fluconazole (antifungal) and voriconazole (antifungal) as standard drugs. DMSO was used as a solvent for dissolving both the reference and sample derivatives. The synthesized compounds were subjected to testing to assess their antibacterial effectiveness against S. aureus (ATCC FDA209-P), E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853). Additionally, their antifungal activity was also assessed against C. albicans (ATCC SC5314) and A. niger (MTCC 1688). Each synthesized compound exhibited antibacterial efficacy, as illustrated in Table 4 and Fig. 11. Notably, compound CE3 displayed the most potent antibacterial activity against S. aureus, surpassing trimethoprim (MIC 16 µg/ml) and demonstrating a comparable efficacy to ampicillin (MIC 4 µg/ml) with a MIC value of 4 µg/ml. Compound CE3 displayed substantial efficacy against gram-negative bacteria, namely P. aeruginosa and E. coli, with MIC values of 2 µg/ml for each of these microorganisms. These MIC values are superior to those of trimethoprim, which recorded 8 µg/ml for both P. aeruginosa and E. coli, as well as ampicillin, which had MIC values of 8 µg/ml for P. aeruginosa and 4 µg/ml for E. coli. Compound CE15 demonstrated significant antibacterial activity against S. aureus, P. aeruginosa and E. coli, with MIC 8 µg/ml, 4 µg/ml and 4 µg/ml, respectively. These values surpass those of TMP and are comparable to the antibacterial efficacy of ampicillin. Similarly, CAm7 demonstrated significant effectiveness compared to the standard, with MIC values of 8 µg/ml, 8 µg/ml, and 4 µg/ml against S. aureus, P. aeruginosa, and E. coli, respectively. The compound's inhibitory activity against fungal strain C. albicans was not as strong as their antibacterial activity. The only compounds with moderate action were CE3, CE12 and CE17. In general, the antibacterial activity was influenced by both the scaffold type and the substituents on the aromatic moiety. It is concluded from this research that compounds having more hydrophobic character and containing electron-withdrawing groups on their aromatic ring exhibit significant antimicrobial activity.

Table 4 Antimicrobial activity screening results of synthesized derivatives and standard drugs

Fig. 11

Antimicrobial screening results of synthesized compounds and standard drugs

Anticancer screening

The synthesized compounds were evaluated for their potential as anticancer compounds using the MCF-7 breast cancer cell line. The results, presented in Table 2, indicate that compound CE11 exhibited remarkable anticancer activity with IC₅₀ values of 5.37 ± 0.16 as shown in Fig. 12. This finding aligned well with the outcomes of docking study results and DHFR inhibition tests. This suggests that CE11 could acts as a potential DHFR inhibitor. It might be due to that it formed hydrogen bonds with crucial amino acids residue of binding site such as Glh30 and Ser59, along with the formation of pi-pi stacking interactions with Phe31 and Phe34. These results support the notion that caffeic derivatives can be effectively designed as DHFR inhibitors. Furthermore, compounds CE3 and CE17 demonstrated moderate anticancer activity against the MCF-7 breast cancer cell line. While their activity might not be as potent as CE11, but it still indicates a potential for further investigation and optimization. Overall, these findings highlight the promising anticancer activity of the synthesized derivatives; particularly compound CE11, in targeting MCF-7 breast cancer cells.

Fig. 12

Comparison of anticancer activity (IC₅₀ values) between standard methotrexate and potential synthesized compounds (MCF7, human breast cancer cell line)

Structure activity relationship

Aliphatic esters, aromatic esters, and anilides, of caffeic acid were designed, synthesized and subjected for their DHFR inhibitory properties, as well as their antimicrobial and anticancer properties. To explore the correlation between the structural characteristics of the newly synthesized caffeic derivatives and their biological effectiveness, the following observations were observed: synthesized caffeic acid derivatives indicatethat esters, amides and anilides having bulky aromatic groups were more active as evidenced by low MIC value of CE3, CE11 and CAm7 (Fig. 13). Results of biological screening and in vitro DHFR inhibition indicatethat substitution of aromatic ring at o- and m- position by electron-donating substituent and presence of N atom led to a more active compound as evidenced by the good antibacterial activity of CE3 and CAm7. Aromatic esters are more active compounds than aliphatic esters. The superiority of aromatic esters over anilides and amides, is also supported by the findings of El-Gazzar et al., (2017) and Sarova et al., (2011) [27, 39]. After a comprehensive analysis of biological activities and molecular docking interactions, it can be inferred that the presence of a phenolic free hydroxyl group in the caffeic acid moiety is essential for binding to crucial amino acid residues within the DHFR binding site.

Fig. 13

Explicate of some structure–activity relationship

Experimental

In silico studies protocol

Molecular docking

In silico docking analysis was done with Glide module of Maestro software. X-ray protein structures of DHFR co-crystallized with ligands was retrieved from RCSB site having PDB ID: 2W9S and 1U72 for antimicrobial and anticancer activity, respectively [40, 41]. Selection of PDB IDs was done on the basis of resolution and species. Protein preparation tool of Schrödinger was utilized to prepare proteins Co-crystallized enzyme (DHFR) structure directly downloaded from Protein Data Bank on maestro workspace interface followed by pre-process steps, water molecules were removed and Epik tool were used to ionize heteroatoms at biological pH to maintain biosimilar environment. After pre-process, finally energy minimized structure was obtained using OPLS3e as force field [42,43,44,45]. Docking site validation was done by splitting co-crystallized ligands from minimized prepared protein complex and then re-docked to the active site. For the 2W9S and 1U72 PDB ID, the RMSD values were found to be 0.56 and 1.19, respectively as shown in Fig. 14. This is more than satisfactory to approve the docking protocol. To analyze interaction of protein and ligands docking with extra precision was used.

Fig. 14

RMSD calculation between the co-crystallized ligand and docked pose using the structure alignment superposition tool. a RMSD for PDB ID 2W9S; b RMSD for PDB ID 1U72

Ligand design

Library of caffeic acid derivatives was built using the chemdraw ultra software and LigPrep tool of Maestro was used to prepare ligands for energy minimization and to correct the coordinates, stereochemistry, generate tautomers to obtain appropriate conformation. 32 stereo isomers per ligand were allowed; at target pH 7 ± 2 were set as a default option and force field was OPLS3e and Epik was used for ionization. The primary goal of this research was to identify specific structural characteristics necessary for inhibition of DHFR and to design novel category of compounds that could inhibit DHFR. Caffeic acid was found to form favorable interaction with DHFR, hence, an in-house library was created, encompassing various classes of caffeic acid derivatives such as esters, amides, anilides, hydrazides, Schiff bases, hybrid derivatives with heterocyclic rings, and previously reported derivatives of caffeic acid. Additionally, a library of analogous compounds with different substituent of same class was constructed, totaling approximately seven hundred derivatives. This approach was designed to examine how different class and substituent’s influence the effectiveness of the compounds. Molecular interaction analysis suggests that the presence free phenolic hydroxyl functional groups of lead are crucial for establishing hydrogen bonds with key amino acid residues of DHFR. So, alterations were made to the carboxylic acid functional group of the lead compound to make a diverse library of compounds. The designed ligands demonstrated superior or comparable docking scores and binding energies relative to standard drugs, TMP and MTX, were thus selected for further investigation to elucidate their mechanistic interactions with DHFR. Thus, molecular docking studies helped identify the most promising compounds for subsequent synthesis and evaluation.

ADMET and drug likeness study

ADMET and drug-likeness studies are important in drug designing and development as they allow for a quick and provisional testing of pharmacokinetics and toxicity parameters before the compounds are completely inspected in vitro. ADMETlab 2.0 online tool was utilized to predict comprehensive pharmacokinetic properties and to provide an overall understanding of ADMET properties for selected compounds [46]. Drug absorption of were predicted by considering various factors, such as intestinal absorption (Caco-2), membrane and skin permeability, P-glycoprotein substrate or inhibitor, and human intestinal absorption (HIA), were considered. Predictions for drug distribution encompassed plasma protein binding (PPB), blood–brain barrier permeability (BBB) and volume of distribution (VD), values. Metabolism predictions were based on CYP (cytochrome P 450) models for substrate or inhibition, while excretion was assessed using drug half-life (T_1/2) and clearance (CL). Toxicity predictions encompassed parameters like hERG inhibition, AMES toxicity, respiratory toxicity, rat oral acute toxicity, skin sensitization and carcinogenicity. Lipinski's rules of five also predicted to ensure drug-like characteristics of drugs [47,48,49,50,51].

Binding free energies and MM/GBSA

The Prime module of Maestro was utilized for MM/GBSA energy calculations to determine the free binding energy of designed derivatives. The VSGB solvation model was utilized, and default settings were applied to calculate the binding energy. It is a well accepted approach to find out the free binding energy of the ligands to the target protein to rationalize the experimental results and virtual screening findings. This approach serves to differentiate between substances that act as drugs and those that merely bind to the target [52, 53]. The energy difference was calculated using the equation:

$$\Delta E\hspace{0.17em}=\hspace{0.17em}Ecomplex\hspace{0.17em}-\hspace{0.17em}Eligand- Eprotein.$$

Synthesis of caffeic acid derivatives

Based on the results from in silico molecular modelling, derivatives of the lead molecule caffeic acid were chosen and they were synthesized by using the scheme 1 [34]. The derivatives were synthesized and the progress and completion of the reaction were monitored through the use of TLC. Characterization of synthetic derivatives was done by using physicochemical and spectral determination.

General synthetic procedure to synthesize caffeic acid esters

To synthesize the caffeic acid ester, firstly 3-(3,4-dihydroxyphenyl) acryloyl chloride was synthesized by progressively adding thionyl chloride (0.3 mol) to the 0.25 mol of caffeic acid. Pyridine (1–2 drops) was added as a catalyst, and it was stirred and refluxed at 80 °C for 3–4 h. TLC was used to track the reaction progression and then extra thionyl chloride was distilled off. The product obtained was used for further synthetic step. The desired ester derivatives were synthesized by adding a solution of various alcohols (0.05 mol) in 50 ml diethyl ether to a solution of 3-(3,4-dihydroxyphenyl)acryloyl chloride (0.05 mol) in diethyl ether (50 ml). The mixture was heated using a water bath until the emission of hydrogen chloride ceased. The confirmation of the reaction's completion was additionally verified through a single-spot TLC. The resulting precipitates were then separated by filtration using a suction pump, followed by multiple washes with water. Finally, the purified product was obtained by recrystallization with alcohol.

General synthetic procedure to synthesize amides/anilides of caffeic acid

A solution of 0.1 mol of aniline was prepared by dissolving it in 50 ml of diethyl ether. Simultaneously, a separate solution of 0.1 mol of 3-(3,4-dihydroxyphenyl) acryloyl chloride, which had been synthesized in a prior step, was also dissolved in 50 ml of diethyl ether and kept at a temperature range of 0–10 °C. The amine/aniline solution was added drop wise to the solution of 3-(3,4-dihydroxyphenyl)acryloyl chloride while maintaining the temperature at 0–10 °C. The resulting solution was stirred for 30 min, during which the anilide precipitated out. The precipitate was then separated from the solution using filtration. Anilide product so obtained was treated with water, 4% sodium carbonate and 5% hydrochloric acid to eliminate any remaining aniline residues and then alcohol was used for further purification of final product [35].

Characterization of caffeic acid derivatives

Compound 2-isopropyl-5-methylcyclohexyl 3-(3,4-dihydroxyphenyl)acrylate (CE3): Mol. Wt.: 318.41; Yield 72.2%; R_f 0.58; m.p 188–189 °C; Light brown crystals; FTIR in cm⁻¹ 3614 (O–H, phenol), 2952 (C-H, aromatic), 1261 (C-O, alcohol), 1695 (C=O, ester), 1601 (C=C, phenyl), 1175 (C-O str., ester), 1H NMR (400 MHz, DMSO-d6) in δ: 7.44 (s, 1H; OH), 7.40 (s, 1H; OH), 7.03 (d, J = 2.1 Hz, 1H; aliphatic-H), 6.97 (d, J = 2.1 Hz, 1H; ethylene-H), 6.95 (d, J = 2.1 Hz, 1H, ethylene-H), 6.77 (s, 1H; Ar–H), 6.75 (s, 1H; Ar–H), 3.21—3.07 (m, 9H; 3CH3), 1.82 (ddt, J = 12.2, 3.8, 2.0 Hz, 1H; aliphatic-H); 13C NMR (DMSO-d6) in δ: 168.24, 148.38, 127.74, 116.17, 115.84, 114.65, 69.95, 50.00, 48.20, 47.05, 45.63, 34.81, 31.62, 25.50, 24.15, 23.59, 22.78,, 21.49, 20.95, 16.82, 16.51; Anal. Calculated for C₁₉H₂₆O₄: C, 71.67; H, 8.23; Found: C, 71.69; H, 8.25.

Compound Phenyl 3-(3,4-dihydroxyphenyl)acrylate (CE8): Mol. Wt.: 256.257; Yield 69.5%; R_f 0.73; m.p 201–202 °C; Brown powder; FTIR in cm − 1: 3328 (O–H, phenol), 2952 (C–H, aromatic), 750 (C-H), 1708 (C=O, ester), 1595 (C=C, phenyl), 1217 (C-O, ester); 1H NMR (400 MHz, DMSO-d6) in δ: 8.88 (m, 2H; Ar–H), 8.47 (m, 1H; ethylene-H), 7.97(m, 1H; Ar–H), 7.49 (d, 2H; Ar–H), 7.25 (d, 1H; ethylene-H), 6.55 (s, 2H; OH); 13C NMR (DMSO-d6) in δ: 150.18, 144.22, 129.82, 126.99, 116.10, 56.58, 32.23; Anal. Calculated for C15H12O4: C, 70.32; H, 4.72; Found: C, 70.34; H, 4.72.

Compound 1-(isopropylamino)−3-(naphthalen-1-yloxy)propan-2-yl 3-(3,4-dihydroxyphenyl)acrylate (CE11): Mol. Wt.: 421.492; Yield 75.5%; R_f 0.78; m.p 198–199 °C; Light brown powder; FTIR in cm⁻¹: 3638 (O–H, phenol), 3114 (C-H, aromatic), 1700 (C = O, ester), 1525 (C = C, phenyl), 1271 (C-O, ester), 1H NMR (400 MHz, DMSO-d6) in δ: 9.79 (s, 1H; OH), 8.79 (dd, 1H; Ar–H), 8.65 (d, J = 8.6 Hz, 1H; ethylene-H), 8.36 (d, 1H; ethylene-H), 8.31—8.25 (m, 2H; Ar–H), 7.90 (d, 1H; Ar–H), 7.88 (dd, J = 3.1, 1.8 Hz, 1H; Ar–H), 7.62 (d, J = 2.1 Hz, 1H; Ar–H), 7.53 (dd, J = 7.1, 4.4, 2.1 Hz, 3H; Ar–H), 7.49 (s, 1H; OH), 7.44 (q, J = 7.9 Hz, 2H; methine-H), 6.98 (d; 2H; Ar–H), 6.77 (dd; 2H; Ar–H), 6.18 (d, J = 15.9 Hz, 1H; Ar–H), 4.49 – 4.32 (m, 3H; CH3), 4.16 (dd, J = 10.0, 5.1 Hz, 6H; 2 CH3); 13C NMR (DMSO-d6) in δ: 158.20, 157.58, 156.67, 155.41, 155.22, 155.03, 154.96, 151.65, 150.39, 135.37, 100.03, 95.43, 80.00, 76.91, 76.84, 48.84; Anal. calculated for C₁₆H₁₅NO₃: C, 71.24; H, 6.46; N, 3.32 Found: C, 71.11; H, 6.41; N, 3.38.

Compound Phenethyl 3-(3,4-dihydroxyphenyl)acrylate (CE12): Mol. Wt.: 284.311; yield 67.3%; R_f 0.67; m.p 195–196 °C; Dark brown powder; FTIR in cm⁻¹ 3675 (O–H, phenol), 3061 (C-H, aromatic), 1041 (C–O,), 1648 (C=O, ester), 1495 (C=C, phenyl), 1169 (C–O, ester); ¹H NMR (400 MHz, DMSO-d₆) δ 7.30 (m, 2H; Ar- H), 7.27 (t, 1H; methylene-H), 7.26 (d, 1H; Ar- H), 7.24 (d, 1H; Ar- H), 7.22 (t, 1H; Ar- H), 7.21–7.16 (m, 1H; Ar- H), 7.07 (s, 1H; Ar- H), 4.39 (t, J = 6.7 Hz, 1H; Ar- H), 3.66 (t, J = 7.1 Hz,2H; methylene-H), 2.77 (t, J = 7.1 Hz, 2H; methylene-H); ¹³C NMR (DMSO-d₆) δ 146.08, 145.96, 139.98, 129.40, 129.35, 129.31, 128.84, 128.78, 128.58, 127.04, 126.81, 126.28, 119.98, 109.11, 65.18, 62.77, 35.03. Anal. calculated for C₁₇H₁₆O₄: C, 71.83; H, 5.67; Found: C, 71.85; H, 5.69.

Compound Benzyl 3-(3,4-dihydroxyphenyl)acrylate (CE13): Mol. Wt.: 270.284; Yield 61.7%; R_f 0.6; m.p 190–191 °C; Light yellow crystal; FTIR in cm⁻¹ 3675 (O–H, phenol), 3061 (C-H, aromatic), 1041 (C-O), 1648 (C=O, ester), 1495 (C=C, phenyl), 1169 (C–O, ester); ¹H NMR (400 MHz, DMSO-d₆) δ 10.40 (s, 2H; OH), 9.15 (s, 2H; methylene-H), 7.46 (d, 1H; Ar–H), 7.18 (dd, 2H; Ar–H), 7.07 (d, 3H; Ar–H), 6.96 (d, 2H; Ar–H), 6.82 (t, 1H; Ar–H). ¹³C NMR (DMSO-d₆) δ 161.96, 139.39, 136.75, 134.24, 131.84, 129.38, 128.95, 128.84, 127.94, 125.67; Anal. calculated for C₁₆H₁₄O₄: C, 71.10; H, 5.22; Found: C, 71.15; H, 5.23.

Compound Quinolin-8-yl 3-(3,4-dihydroxyphenyl)acrylate (CE15): Mol. Wt.: 307.305; Yield 75.34%; R_f 0.6; m.p 122–123 °C; Light brown powder; FTIR in cm⁻¹: 3677 (O–H, phenol), 1747 (C = O, ester), 3216, (C-H, aromatic), 1542 (C=C, phenyl), 1699 (C=C, alkene), 1411 (ring, quinoline), 1653 (C = N, quinoline); ¹H NMR (400 MHz, DMSO-d₆) δ 9.31 (dd, 1H; naphthalene-H), 9.18 (dd, 1.4 Hz, 3H; naphthalene-H), 9.11 (dd, 2H; naphthalene-H), 8.09 (d, 3H; Ar–H), 7.78 (d, J = 2.0 Hz, 2H; ethylene-H), 7.77 (s, 1H; OH), 6.94 (s, 1H; OH). ¹³C NMR (DMSO-d₆) δ 148.85, 147.00, 146.14, 144.66, 130.98, 130.24, 129.07, 122.82, 118.89, 116.60, 114.29, 109.76, 109.22; Anal. calculated for C₁₈H₁₃NO₄: C, 70.35; N, 4.57; H, 4.26; Found: C, 70.85; N, 4.62; H, 4.20.

Compound 2-aminophenyl 3-(3,4-dihydroxyphenyl)acrylate (CE17): Mol. Wt.: 271.272; Yield 52.8%; R_f 0.72; m.p 151–152 °C; Cream colored crystal; FTIR in cm⁻¹ 3739 (N–H, amino), 3612 (O–H str., phenol), 3113 (C-H, aromatic), 1693 (C=O, ester), 1521 (C=C, phenyl), 1095 (C-O, ester), ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (d, 1H; ethylene-H), 8.51–8.45 (m, 1H; Ar–H), 7.97 (dd, 2H; Ar–H), 7.46 (d, J = 7.5 Hz, 2H; Ar–H), 7.26 (d, 1H; Ar–H), 7.22 (d, J = 1.7 Hz, 1H; ethylene-H), 7.20 (s, 1H; OH), 7.18 (s, 1H; OH), 6.55 (s, 1H; Ar–H), 3.85 (s, 2H; NH₂); ¹³C NMR (DMSO-d₆) δ 144.05, 139.96, 129.34, 128.59, 127.09, 126.29, 116.09; Anal. calculated for C₁₅H₁₃NO₄: C, 66.41; N, 5.16; H, 4.83; Found: C, 66.93; N, 5.23; H, 4.63.

Compound 3-(3,4-dihydroxyphenyl)-N–o-tolylacrylamide (CAm7): Mol. Wt.: 180.16; Yield 72.2%; R_f 0.77; m.p 202–203 °C; Black colored powder; FTIR in cm⁻¹ FTIR 3609 (O–H, phenol), 1450 (C=O, anilides), 2928 (C-H, aromatic), 2859 (C–H, aliphatic), 1689 (C=C, Ar), 3385 (N–H, anilide); ¹H NMR (400 MHz, DMSO-d₆) δ 10.48 (s, 1H, NH), 8.83 (s, 1H; OH), 8.13 – 8.08 (m, 1H; Ar–H), 7.42 (s, 1H; OH), 7.35—7.11 (m, 4H, menthol-H), 7.07–7.01 (m, 2H; Ar–H), 6.85—6.79 (d, 1H; ethylene-H), 6.68 (d, 1H; ethylene-H), 2.88 (d, 3H, CH3). ¹³C NMR (DMSO-d₆) δ 146.06, 145.51, 137.90, 130.47, 129.67, 123.54, 116.32, 115.29, 114.37, 21.00; Anal. calculated for C₁₆H₁₅NO₃: C, 71.36; H, 5.61; N, 5.20; O, 17.82 Found: C, 71.35; H, 5.62; N, 5.19 Anal. calculated for C₁₆H₁₅NO₃: C, 71.36; N, 5.21; H, 5.61; Found: C, 71.86; N, 5.25; H, 6.13.

Biological evaluation

DHFR inhibition assay

Spectrophotometric assay method was used to measure the enzyme-catalyzed conversion of dihydrofolic acid to tetrahydrofolic acid at 340 nm for 180 s in order to test the ability of the synthesized derivatives to inhibit DHFR activity. This assay utilized the Dihydrofolate reductase assay kit (Sigma-Aldrich, catalogue number CS0340). The kit was tested on recombinant DHFR, NIH 3T3, A431 and CHO cell lines and kidney, brain, liver and muscle tissue extract from rat. Stock solutions of different concentrations of the synthesized compounds were prepared. Dihydrofolic acid, NADPH, standard drug solution, assay buffer and DHFR stock solutions were prepared in accordance with the guidelines mentioned in the technical bulletin that was provided with the assay kit. All stock solutions were kept on ice except the assay buffer. A calculated amount of assay buffer (190 μL) was placed into each well of a 96-well transparent microplate, followed by 6 μL of diluted DHFR, which was thoroughly mixed. For inhibition assay, different concentrations of serially diluted synthesized compounds (0.01–10 μM) and diluted NADPH (1.2 μL) was added into the wells. After that, the microplate was covered with parafilm and the contents mixed by inversion. Dihydrofolic acid substrate (1 μL) was added and then the absorbance was measured at 340 nm in kinetic mode using an ELISA reader (Gen BioTek Microplate Reader) for 180 s at room temperature. The slope for each test inhibitor sample was determined by plotting the relationship between absorbance and time. Equation 1 was used to get the relative inhibition percentages [28, 54, 55].

$$\% relative\, inhibition = (slope \,of [EC] - slope \,of [S]/slope\, of [EC]) \times 100$$

(1)

where, EC: enzyme control.

S: sample of interest.

Antimicrobial activity

Using the broth microdilution method, the MIC of all the synthesised compounds were assessed for their antibacterial and antifungal activity. The newly synthesized compounds underwent testing against various bacterial strains, including S. aureus (ATCC FDA209-P), P. aeruginosa (TCC 27853) and E. coli (ATCC 25922), as well as fungi strains C. albicans (ATCC SC5314) and A. niger (MTCC 1688). Individual MIC (µg/mL) value was determined by serial dilution technique using Mueller Hinton Broth (MHB) media for bacterial growth and Rosswell Park Memorial Institute (RPMI) media for fungal growth. ampicillin, trimethoprim, tetracycline, voriconazole and fluconazole were used as standard drugs for MIC calculations in same condition as benchmarks. The strains were cultured on MHA for bacterial species (incubated for 24 h at 37 ± 1 °C) and RPMI medium was utilized for cultivating fungal species, specifically for 48 h at 37 ± 1 °C for C. albicans and for 7 days at 25 ± 1 °C for A. niger. The turbidity of the spore suspension was corrected to 0.5 McFarland (for bacteria) by adding distilled water. Stock solutions of synthesized compounds were prepared in DMSO solvent and serially diluted to the concentrations of 64.0, 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5, 0.25 and 0.12 µg/mL to determine MIC value. Each spore (0.1 ml) was put to each well holding the finally serially diluted targeted drug concentration, standards and controls. Optically clear (MIC-0) endpoint criteria, defining MIC as the concentration resulting in 99.9% suppression of visible bacterial and fungal growth following incubation, were used. CFU (Colony forming unit) was also determined by taking 100 µl culture from twelfth well and was dispensed to the tubes containing 900µL saline and were serially diluted. Spore spotting was conducted on agar plates for each spore, followed by incubation for 24 h at 37 ± 1 °C (bacterial spores), for 48 h at 37 ± 1 °C (C. albicans) and for 7 days at 25 ± 1 °C (A. niger) to assess the CFU count. To precise the results duplicate testing was performed on three different days [56,57,58,59].

Anticancer activity

The MTT Assay was employed to evaluate the cytotoxic impact of the selected synthesized compounds on the MCF-7 cell line (sourced from NCCS, Pune). Cultured at a density of 10,000 cells per well in 96-well plates, the cells were incubated for 24 h in DMEM medium supplemented with 10% FBS and 1% antibiotic solution at 37 °C in a 5% CO₂ environment. Subsequently, the cells were exposed to varying concentrations of the derivatives (ranging from 1000 to µM), prepared in an incomplete media. After 24-h incubation period, MTT solution at a final concentration of 250 µg/ml was introduced to the cell cultures and incubated for an additional 2 h. Upon completion of the experiment, the culture supernatant was aspirated and the cellular matrix was dissolved in 100 µl of DMSO. The absorbance of the solution was measured using an Elisa plate reader (iMark, Biorad, USA) at wavelengths of 540 nm. This process allowed the evaluation of the cytotoxicity levels of the synthesized compounds on the MCF-7 cell line. Conducting measurements in triplicates enhances the reliability and reproducibility of the acquired results [60,61,62,63].

Conclusion

Novel category of DHFR inhibitors were designed, synthesized, characterized and assessed for their capability to inhibit DHFR, as well as their antimicrobial and anticancer properties. To prevent late-stage failures, it is crucial to examine the initial pharmacokinetic parameters. The pharmacokinetic data indicates that all the synthesized compounds could potentially be considered as drugs. The compounds exhibited significant inhibitory potency against bacterial strains and moderate activity against fungi. Specifically, compounds CE3, CE15 and CAm7 demonstrated excellent antibacterial effects, particularly against gram-negative bacteria, possibly attributed to their hydrophobic nature. Additionally, compound CE11 exhibited remarkable anticancer potential against the breast cancer cell line MCF7, with an IC₅₀ 5.37 ± 0.16 µM. All the synthesized compounds displayed superior DHFR inhibition activity compared to the parent compound caffeic acid. Among them, CE11 exhibited the highest DHFR inhibitory activity, with an IC₅₀ value of 0.048 µM, making it approximately four times more effective than methotrexate. CE11, the most active DHFR inhibitor and anticancer compound, demonstrated excellent docking scores and binding energy (−9.6, −72.12 kcal/mol) which is comparable to methotrexate. It bound to the same site and interacted with key residues such as Glh30, Phe31, Phe34, and Ser59. CE11 also snugly fit into the hydrophobic pocket of the modeled protein due to its lengthened structure, targeting the hydrophobic back pocket. Furthermore, the bulkiness of CE11 allowed favorable interactions within the hydrophobic pocket, promoting contacts within the DHFR enzyme's active site. The anticancer efficacy was further confirmed through in vitro DHFR enzyme inhibition studies. Docking analysis of the most active antimicrobial agent, CE3, revealed its strong entrapment in the active site of enzyme, with hydrophobic interactions involving hydrophobic residues and the formation of three hydrogen bonds with residues Ala 7, Asn 18, and Thr 121. It exhibited excellent docking scores and binding energy (−9.9, −71.77 kcal/mol). The enzyme inhibition assays verified that the novel compounds operate through a DHFR-mediated mechanism of action. This study identified novel structural types of antimicrobial and anticancer agents that could serve as lead molecules for further research and development in these areas.