INTRODUCTION:

Although candida species are frequently found as benign organisms in the digestive systems of healthy people, they can cause a wide range of harmful disorders in impaired hosts^1-3. Due to the lack of antifungal medicines, treating serious candida infections has been difficult. Although Amphotericin B was the essential treatment for a long time, it was highly toxic and must be administrated intravenously.

On the other hand, flucytosine is limited in use due to its possibility to cause myelosuppression and the marked increase in random resistance^4-5. However, the discovery of oral bioavailable azole antifungal medications began to change the approach to treating serious candida infections⁶. Ketoconazole, the earliest of these medicines to be used, was effective in treating chronic fungal infection of the mucosa and skin. Soon after these medications were introduced, reports of clinical problems associated with increased resistance to ketoconazole developed following extended treatment⁷. However, this issue hasn’t been overcome until the following introduction of fluconazole. Fluconazole is a triazole derivative (soluble in water) with very good bioavailability after oral intake, has been extensively utilized to cure a variety of candida-related infections^8-12. The mechanism by which it acts is by inhibiting the enzyme Sterol 14-α demethylase (The crucial enzyme involved in the biochemical synthesis of ergosterol), where the ergosterol is an essential member required for fungal cell membrane formation^13-18.

As we mentioned above, fluconazole is widely used to provide treatment for various infections caused by fungi ¹⁹. However, indiscriminate use of fluconazole has developed resistance to it^20-23. The ERG11 gene in Candida species encodes Sterol 14-α demethylase (the target of azole antifungals). One of the resistance mechanisms is a conformational shift of the target sterol 14-α demethylase (ERG11) caused by ERG11 gene mutation^24,25.

Finally, the synthesis of new fluconazole derivatives represents a good solution to overcome the resistance among it. The Structural modifications of the new derivatives can improve antifungal activity and help overcome the resistance mechanisms.

This research aims to design, develop and synthesize a series of novel fluconazole derivatives using analytical techniques such as TLC, IR Spectroscopy, Mass Spectrometry, and NMR to identify the structure of the synthesized compound.

Moreover, molecular modeling studies were undertaken to determine the interaction of these compounds with the target enzyme (Sterol 14-α demethylase), and determine the ability of these compounds to inhibit ergosterol biochemical synthesis pathway, which is the aim of azole antifungal medications.

MATERIALS AND METHODS:

Fluconazole was supplied by Mediotic Labs Pharmaceuticals Industries, Homs, Syria. The enzyme code (pdb id:1EA1) was sourced from the Protein data bank (pdb). The Molegro Virtual Docker (MVD) version 2022.5.5 was used to dock the compounds to test their activity, calculate binding energy, and determine the bonds with the enzyme's amino acids. Marvin sketch program version 21.2 was used to design the compounds and then applied to the Molegro program. The Melting point was found out using a BÜCHI Melting point apparatus. (BÜCHI Labor technik, Switzerland). aluminum sheets coated with silica gel 60 F254(Macherey-Nagel Germany) were used for analytical Thin Layer Chromatography (TLC, RF Values), the mobile phase used was 9\1 ethyl acetate and ethanol. IR Spectra were determined using an ATR-FTIR Spectrometer from Bruker (Bruker, Billerica, Massachusetts). The Mass spectrum (MS) was determined using a Mass Spectrometer. ¹H and ¹³C NMR Spectroscopy were determined by JOEL-ECA NMR Spectrophotometer (JOEL, Tokyo, Japan) using deuterated dimethyl sulfoxide as a dissolving agent.

Procedure of synthesis:

Synthesis of compound b1

2-((1-(2,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)propan-2-yl)oxy)acetic acid

In a suitable round flask, (0.5g, 8.9mmol) of potassium hydroxide (KOH) was dissolved using 8ml of water, then a reflux distillation column was set up.

(0.61g, 2mmol) of Fluconazole was added to the mixture by stirring and hitting at (60-70℃) for 10 minutes.

Then, 6ml of 50% chloroacetic acid aqueous solution was gradually added with stirring and heating at the same temperature under reflux for 1hour.

The mixture was cooled to 25℃ and then (HCL) was used to acidify the mixture to a PH≈3.

After that, the mixture was placed in an ice cold bath to help to form a white precipitate. Then, the formed precipitate was separated by filtering and dried in an oven at 60℃ for 2hours.

Sterol 14-α demethylase structure:

Sterol 14-α demethylase (Cyp51) is a crucial enzyme in the sterol biochemical synthesis pathway in fungi; It carries out the 14α demethylase of lanosterol. The critical reaction in the biochemical synthesis of ergosterol, a main component forming cell membranes of fungi²⁶.

The Protein data bank provided the Three-dimensional crystallized structure of Sterol 14-α demethylase (pdb id:1EA1), which is presented in Figure 1.

It has a resolution of 2.1 Å, and It is a monopeptide chain of 455 amino acid residues.

The crystallized structure includes two ligands: a hem cofactor (HEM), which is important for enzyme activity, and Fluconazole (TPF) an azole inhibitor.

Figure 1. Co-Crystallized structure of the Sterol 14-α demethylase (pdb id: 1EA1).

Docking Studies:

Protein Preparation:

The Sterol 14-α demethylase protein is prepared by entering it into the MVD program. A list of its amino acid residues is displayed, and any amino acid residues with possible faults are marked in the list and identified and colored in red or yellow in the three-dimensional structure of the protein.

There are two types of residue errors: the presence of deleted atoms or bond faults, and they are corrected if any errors occur. In addition, water molecules removed from the protein's crystal formula.

Specify the binding cavity:

The binding cavity is specified by the presence of the native ligand (Fluconazole) within it, which has a volume of 142.33Å and is suitable for the psa (polar surface area) of the studied compounds, as shown in Figure 2.

Figure 2. Sterol 14-α demethylase (pdb id: 1EA1) binding cavity.

Docking Procedure:

The Molegro program (MVD) was used for molecular docking. The parameters listed below in Table 1 were used for docking with the Sterol 14-α demethylase enzyme.

Table 1. Docking parameters used for molecular docking studies with Sterol 14-α demethylase.

Parameters	Value
Scoring Function	PLANTS Score
Binding site radius	15
Searching algorithm	Iterated Simplex
Max Iteration	100
Max population size	20
Simplex Evaluation(max steps)	2000
Tolerance	0.01
Max number of poses	5

Validation of Docking Protocol:

Molecular docking was performed to evaluate the ability of compounds a1-a7/b1-b7/c1-c6 to inhibit the enzyme Sterol 14-α demethylase in addition to calculating the binding energies.

But first, a suitable process has to be determined and validated before starting the molecular docking.

A method was developed in which the ligand (Fluconazole) bonded to the crystallized structure (pdb id:1EA1) was re-docked and the standard deviation (Rmsd) was calculated. The value for the used protocol was (0.97Å), which is acceptable because, in computer-aided drug design, standard deviation value below 2Å is considered a threshold. After verifying the validity of the protocol used, this procedure performed molecular docking for the compounds a1-a7/b1-b7/c1-c6.

RESULTS AND DISCUSSIONS:

-Chemistry

Compound b1

2-((1-(2,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)propan-2-yl)oxy)acetic acid was synthesized by reacting Fluconazole with chloroacetic acid in an alkaline aqueous medium. The reaction yielded 55.5%.

Figure 3 summarizes the synthetic pathways and the compound’s b1 chemical structure.

The compound was identified using Infrared absorption spectroscopy, Mass spectra, ¹H and ¹³C NMR spectra, as described in the analytical data part.

Figure 3. Synthesis of compound 1b.

Docking:

The compounds were designed using the Marvin sketch program, stored in molecular 2 format, and then applied to the Molegro Virtual Docker (MVD).

All applied compounds were bound to the enzyme and given binding energies. Also, all applied compounds formed H-bonds and Van Der Waals bonds with the enzyme, with varied bond distances and binding energies. All the studied compounds had higher binding energies than the native ligand (fluconazole), and some of them were even higher than the reference compound (the reference was chosen based on previous research an ox-diazole fluconazole derivative)⁶. The structure of the Fluconazole and the reference compound are mentioned in (Figure 4), wherein structure (a) represents fluconazole, while structure (b) represents the reference compound. Forming H-bonds with both Arg96 and Thr260 is essential for good binding energy with sterol 14-α demethylase (pdb id: 1EA1). Forming more H-bonds with other amino acids results in increased binding energies.

By looking at a1-a7 group compounds and interpreting the results of the binding energies mentioned in Table 2, we notice that they gave higher binding energies than both b1-b7 and c1-c6 groups compounds. This is due to the ability of compounds a1-a7 to form a H-bond with His259, because they contain an NH group in their chemical structure.

Compound a6 achieved the highest binding energy among the compounds in group a1-a7 (ΔG = -206.84Kcal\mol), as we notice due to its capability to form a H-bond with His259 via its NH group, in addition to forming H-bonds with Thr260 and Arg96 via the Triazole groups (The predicted binding positions of compound a6 and its interactions with amino acid residues in the pocket of Sterol 14-α demethylase are shown in Figure 5 and 6). Compound a7 also showed a binding energy close to compound a6 (ΔG = -197.17Kcal\mol), as this compound was able to form H-bonds with His259 in addition to Val43, because of the hydroxyl and carboxyl group in its structure.

We conclude from above that compounds which are able to form a H-bond with His259 and Val435 have clearly high binding energies.

The compounds of the b1-b7 group gave slightly lower binding energies than the compounds of the a1-a7 group but still formed important H-bonds with Thr260 and Arg96.

We can see that compound b1 formed two H-bonds with Arg96 through the carbonyl group presented in its structure, which gave a good binding energy (ΔG = -165.10Kcal\mol).

Compound b2 formed a H-bond with Ala256, and thus giving a relatively good binding energy (ΔG = -162.97Kcal\mol). Compounds b6 and b7 gave the highest binding energy compared to the other compounds in group b1-b7 (ΔG=-170.20Kcal\mol, ΔG= -172.72kcal\mol) respectively. This is due to the fact that compound b6 formed an additional H-bond with Gln72 and compound b7 formed a H-bond with Arg96 via the carbonyl group in its structure.

Moving on to the compounds c1-c6 group, we can see that their binding energies are lower compared to the groups a1-a7 and b1-b7, except compound 6c, which gave a good binding energy (ΔG = -193.84Kcal\mol).

Looking at the results of the association of this group c1-c6 with amino acids in Table 3 we can see the importance of the carbonyl group forming a H-bond, as most of the compounds in this group formed a H-bond with either Arg96 or Thr260. Compound c3 formed an additional hydrogen bond with Ala256 via the carbonyl group.

Finally, regarding Van Der Waals bonds, all compounds formed Van Der Waals bonds with several amino acids, which helped in the stabilization of the studied compounds with the enzyme.

Table 3: lists all compound's associations with the amino acids.

Figure 4. Structure of Fluconazole and Reference compound.

Figure 5.6. Predicted binding positions of compound a6 and its interactions with amino acids residues in the pocket of sterol 14-α demethylase.

Table 2. Binding energies (Kcal\mol) of the designed compounds compared to Fluconazole (-134.09Kcal\mol) and the Reference (-192.49Kcal\mol).

Compound	Moldock Score Kcal\mol	Compound	Moldock Score Kcal\mol	Compound	Moldock Score Kcal\mol
Compound	Moldock Score Kcal\mol	Compound	Moldock Score Kcal\mol	Compound	Moldock Score Kcal\mol
a 1	-164.11	b 1	-165.1	c 1	-158.47
a 2	-194.2	b 2	-162.97	c 2	-154.63
a 3	-190.85	b 3	-155.67	c 3	-160.38
a 4	-192.82	b 4	-150.27	c 4	-160.7
a 5	-199.29	b 5	-168.08	c 5	-162.42
a 6	-206.84	b 6	-170.2	c 6	-193.84
a 7	-197.17	b 7	-172.72

Table 3. The interactions of the designed compounds with amino acids of Sterol 14-α demethylase enzyme.

Ligand	Residue	H-Bond Associations	Bond Lengths	Bond energies	Van Der Waals Association
Ligand	Residue	H-Bond Associations	(A⁰)	(Kcal\mol)	Van Der Waals Association
a 1	Thr260	OH…. N triazole (Ligand)	3.07	-2.5	Thr260
	Arg96	NH2….N triazole (Ligand)	3.37	-1.16	Leu100
					Phe255
a 2	Thr260	OH…. N triazole (Ligand)	3.4	-1	Leu100
	Tyr76	OH…..N triazole (Ligand)	3.12	-2.42	Met433
					Met79
					Phe255
a 3	Thr260	OH…. N triazole (Ligand)	3.08	-2.5	Thr260, Leu100
	Arg96	NH2…..N triazole (Ligand)	3.07	-1.67	Phe255, Met433
					Val435, Phe78
					Arg96,His259
a 4	Thr260	OH…. N triazole (Ligand)	2.85	-2.5	Thr260, Ala256
	Arg96	NH2…. N triazole (Ligand)	2.88	-2.5	Leu100, Met79
	His259	N…..NH (Ligand)	3.45	-0.07	Met433, Phe255
					His259
a 5	Thr260	OH... N triazole (Ligand)	3.07	-2.5	Ala256, Thr260
	Gln72	NH2…. N triazole (Ligand)	3.19	-0.17	Leu100, Phe255
	Arg96	NH2... N triazole (Ligand)	3.37	-1.14	Met433, His259
	His259	N…..NH (Ligand)	3.45	-0.74	Pro230, Arg96
					Gln72
a 6	Thr260	H…. N triazole (Ligand)	3.06	-2.5	Thr260, Ala256
	Arg96	NH2...N triazole (Ligand)	3.33	-1.33	Leu100, Phe255
	His259	N…..NH (Ligand)	3.4	-1.01	Met433, Ile323,
					Gln72
a 7	Thr260	OH…. N triazole Ligand)	3.01	-2.5	Thr260, Phe255
	Arg96	NH2... N triazole (Ligand)	3.37	-0.7	Leu100, Arg96
	Val435	NH2….CO (Ligand)	2.99	-2.5	Pro320, Val435
	His259	N…..OH(Ligand)	2.61	-1.97	Met433, Phe78
					His259
b 1	Thr260	OH…. N triazole (Ligand)	3.57	-0.16	Thr260, Arg96
	Arg96	NH2….CO (Ligand)	3.1	-2.49	Leu100,Phe255
	Arg96	NH…..CO(Ligand)	3.53	-3.53
b 2	Thr260	OH…. N triazole (Ligand)	2.64	-2.5	Thr260.Ala256
b 2	Ala256	NH…..N triazole (Ligand)	3.12	-0.6	Leu100,Phe255
b 3	Thr260	OH…...N triazole (Ligand)	3.45	-0.76	Ala256, Thr260
	Arg96	NH2…..O (Ligand)	3.4	-0.93	Leu100, Phe255
					Arg96
b 4	Thr260	OH…..N triazole (Ligand)	3.07	-2.5	Ala256, Leu100
b 4	Thr260	OH…..N triazole (Ligand)	3.07	-2.5	Phe255.Met79
b 5	Thr260	OH…...N triazole (Ligand)	3.29	-1.55	Thr260, Leu100
b 5	Gln72	NH2…..OH (Ligand)	3.15	-2.25	Phe255,Gln72
b 6	Thr260	OH…...N triazole (Ligand)	3.04	-2.5	Thr260.Leu100
	Arg96	NH2... N triazole (Ligand)	3.25	-1.73	Phe255.Arg96
	Gln72	NH2…..N triazole (Ligand)	3.59	-0.06
b 7	Thr260	OH…. N triazole (Ligand)	2.68	-2.5	Ala256, Thr260
	Arg96	NH2…..CO (Ligand)	3.26	-1.72	Phe255.Leu100
					Met79
c 1	Thr260	OH…. N triazole (Ligand)	3.12	-2.38	Thr260.Leu100
c 1	Arg96	NH2…..CO (Ligand)	3.41	-0.56	Phe255
c 2	Thr260	OH…...CO (Ligand)	3.12	-2.38	Thr260, Leu100
c 2	Arg96	NH2…..N triazole (Ligand)	3.41	-0.56	Phe255
c 3	Thr260	OH…. N triazole (Ligand)	3.58	-0.11	Arg96, Phe255
	Arg96	NH2…. N triazole (Ligand)	2.94	-1.71	Ala256.Leu100
	Ala256	NH….CO (Ligand)	3.01	-3.01	Thr260
c 4	Thr260	OH…. N triazole (Ligand)	3.19	-2.05	Thr260, Leu100
	Arg96	NH2…..CO (Ligand)	2.86	-0.67	Phe255, Leu321
					Arg96
c 5	Thr260	OH…. N triazole (Ligand)	3.11	-2.44	Thr260, Leu100
c 5	Arg96	NH2…..CO(Ligand)	2.92	-0.63	Arg96,Phe255
c 6	Thr260	OH…. N triazole (Ligand)	3.34	-1.3	Leu100, Met433
c 6	Arg96	NH2…..N triazole (Ligand)	3.16	-1.48	Phe255,Met79
Fluconazole	Arg96	NH2....OH(Ligand)	3.55	-0.25	Leu100,Thr260
Fluconazole	Thr260	OH…..N triazole (Ligand)	2.9	-0.88	Leu100,Thr260
Reference	Thr260	OH…. N triazole (Ligand)	3.07	-2.5	Thr260, Leu100
	Arg96	NH2... N triazole (Ligand)	3.34	-1.29	Phe255, Met433
	Met433	O…. NH2 (Ligand)	3.1	-0.76	Arg96,Gln72
	Ile323	O…..NH2 (Ligand)	3.59	-0.03

Analytical Data:

Compound b1:

2-((1-(2,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)propan-2-yl)oxy)acetic acid

White powder, Yield: 55.5%, melting point: 112-115℃, TLC: the mobile phase was ethyl acetate: ethyl alcohol:9:1, in which the retention factor for compound 1b is 0.10. IR spectrum (νmax, cm^-1): 1727(C=O), 3117(OH Carboxylic),1245(C-O, Ether), the IR spectrum of the compound b1 is shown in Figure 7. ¹H-NMR spectrum (DMSO-d6, δ, ppm): 2.47(M, 1H, CH), 4.54(D, 2H, CH2), 4.72(D, 1H, CH), 6.36(S, 2H, CH2), 6.84(S, 1H, Ar-H), 7.15(D, 2H, Ar-H), 7.74(S, 2H, NH-Triazole), 8.28(S, 2H, NH-Triazole).¹³C-NMR (DMSO-d6, δ, ppm): 55.40, 74.20, 104.39, 111.38, 123.69, 129.01, 145.86, 151.18, 158.57, 160.63, 161.55, 163.55. Mass spectrum (m/z, ESI): showed molecular ion [M + H]⁺ peak at 365.26 representing C15H14F2N6O3, and its fragments are 127.06, 169.14, 220.17.

Figure 7. IR Spectrum for compound b1.

CONCLUSION:

The above article details the characterize and synthesis of novel fluconazole derivatives as antifungal medicines targeting the sterol 14-α demethylase enzyme. A novel fluconazole derivative was synthesized with a good yield by reacting fluconazole with an alkyl halide in an aqueous alkaline medium. The newly synthesized compounds’ structure was identified using ¹H and ¹³C NMR, Mass, and IR spectroscopies. The efficiency of the synthesized and designed compounds was determined by docking them into the sterol 14-α demethylase (pdb id:1EA1) binding cavity using the Molegro program (MVD), the binding energies of these compounds were calculated and compared to the interaction energies of EA1's native ligand (fluconazole) and the reference compound (chosen based on earlier research, ox diazole fluconazole derivative).

Based on the previous results, we conclude that all of the characterized compounds achieved greater binding energy than fluconazole, and some of them even gave greater binding energy than the reference compound. This was the result of the structural modifications and the addition of functional groups capable of forming additional hydrogen bonds with amino acids within the sterol 14-α demethylase enzyme.

Finally, more research is needed to evaluate the efficacy of this compounds as antifungal medicines. We hope that these compounds show promise and provide the desired efficacy.

ACKNOWLEDGMENTS:

The authors thank the Marburg University, Institute of Pharmacy, Department of Pharmaceutical Chemistry, Germany, for the analysis of our synthesized compound using Mass spectra, ¹H-NMR and ¹³C-NMR.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest regarding the publication of this paper.

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Received on 26.02.2025 Revised on 19.06.2025

Accepted on 07.08.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5935-5941.

DOI: 10.52711/0974-360X.2025.00858

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