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Article

Hybrid Bis-(Imidazole/Benzimidazole)-Pyridine Derivatives with Antifungal Activity of Potential Interest in Medicine and Agriculture via Improved Efficiency Methods

by
Tiberius Balaes
1,
Violeta Mangalagiu
2,3,
Vasilichia Antoci
4,
Dorina Amariucai-Mantu
4,
Dumitrela Diaconu
4,5,* and
Ionel I. Mangalagiu
4,*
1
Department of Biology, Faculty of Biology, Alexandru Ioan Cuza University of Iasi, 20A Carol 1st Bvd, 700505 Iasi, Romania
2
Institute of Interdisciplinary Research, CERNESIM Centre, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 11, 700506 Iasi, Romania
3
Faculty of Food Engineering, Stefan Cel Mare University of Suceava, 13 Universitatii Str., 720229 Suceava, Romania
4
Faculty of Chemistry, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 11, 700506 Iasi, Romania
5
Institute of Interdisciplinary Research, RECENT-AIR Centre, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 11, 700506 Iasi, Romania
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2025, 18(4), 495; https://doi.org/10.3390/ph18040495
Submission received: 24 February 2025 / Revised: 20 March 2025 / Accepted: 26 March 2025 / Published: 28 March 2025
(This article belongs to the Special Issue New Approaches to Fighting Infectious Diseases: Overcoming the Antimicrobial Resistance in Current Treatments)
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Abstract

:
Background/Objectives: Nowadays fungal infections are rising serious threats for the human health system and agriculture, mostly because of antifungal resistance, emergence of new fungal pathogens and adverse effects, pressing the scientific world for exploration of new antifungal compounds. Therefore, the aim of this work was to synthesize and to study antifungal activity against human and plant fungi of a new class of hybrid bis-(imidazole/benzimidazole)-pyridine salt derivatives. Methods: The synthesis of the hybrid derivatives was performed using both conventional thermal heating and ultrasound irradiation methods. Results: The use of ultrasound irradiation has the advantages of a dramatic decrease in reaction time and, consequently, a notable acceleration in reaction rate, a remarkable decrease in consumed energy and higher yields. The antifungal activity against five human fungal strains and for plant fungal strains was determined by the disk diffusion method and minimum inhibitory concentration. Conclusions: The tested hybrid derivatives manifest good antifungal activity against the tested strains. Some of the hybrid compounds have very good quasi-nonselective activity against the tested human and plant pathogenic fungi, in some cases close to the control drug fluconazole, respectively, to many antifungal agents commercially used for plant protection.

Graphical Abstract

1. Introduction

During the last decades fungal diseases have raised serious concerns for human health systems and agriculture (for crop protection) [1,2,3,4], mostly because of the antimycotic resistance phenomena caused by long-term use and misuse of the existing antifungal drugs and also because of the emergence of new fungal pathogens [5,6,7,8]. Recently [8], the World Health Organization (WHO) categorized invasive fungal infections as diseases of great risk for humans, with fungal pathogens being ranked into three categories of risk: medium (this includes Coccidioides spp., Candida krusei, Cryptococcus gattii, Lomentospora prolificans, Pneumocystis jirovecii, Paracoccidioides spp., Scedosporium spp. and Talaromyces marneffei), high (this includes Candida glabrata, Candida parapsilosis, Candida tropicalis, Fusarium spp., Histoplasma spp. and eumycetoma causative agents, Mucorales) and critical (this includes Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans). According to the literature data [1,2,9,10,11,12], the armamentarium used to combat fungal diseases includes four classes of drugs: allylamines, azoles, echinocandins, polyenes, and calcium-calcineurin derivatives (to some extent, there is a 5th one, the antimetabolites, which are pyrimidine derivatives), with each of them boasting its own mechanism of antifungal activity. Among them, the azole classis is the most used and representative from the market, with drugs containing imidazole and triazole azaheterocycles being the most important and efficient category of drugs used in current antifungal therapy [1,13,14]. In Figure 1 are listed some of the most representative clinically used drugs with imidazole skeleton, these include bifonazole, butoconazole, clotrimazole, econazole, miconazole, tioconazole, sertaconazole, ketoconazole, etc. [1,12,13,14,15,16].
Recently research data indicate that azine derivatives, especially those bearing azole units, have a great antifungal potential [1,15,16], with some of these compounds already being drugs on the market, Figure 1. Thus, fosmanogepix and ibrexafungerp (Figure 1) are antifungal drugs with a pyridine skeleton (bearing oxazole or triazole units) [15,16,17]. However, the number of existing drugs from the market (or existing in clinical trials) from this class is very low.
On the other hand, pathogenic fungi are a serious threat in agriculture for the growth of crops (especially cereals, fruits and vegetables), producing great economic losses worldwide every year. During the last period, the main approaches to assure the protection of crops against pathogenic fungi involve the use of pesticides, particularly those with azole and azine heterocycles skeletons, such as carbendazim, prochloraz, difenoconazole, boscalid, picarbutrazox, pyrimethanil, etc. [18,19], Figure 2.
Because of the excessive and unreasonable use of existing antifungal pesticides, many fungal strains gain resistance causing supplementary problems, especially economic ones [18,19]. Despite the recent advances in the class of azole and/or azine derivatives with antifungal properties, discovering new antifungal agents with better antifungal properties remains a main goal for scientists.
The literature describes many methods of synthesis for antifungal compounds with azole and azine skeletons, using conventional or nonconventional methods [20,21,22,23,24,25]. Over the last few years, ultrasound (US) irradiation has become a very useful tool in organic and medicinal chemistry, emerging as an extremely useful tool for a large variety of syntheses [26,27,28]. Compared with conventional thermal heating (TH) methods, US irradiation provides some undeniable advantages in organic synthesis. These include reduced reaction times, energy saving, use of smaller amounts of solvents, lesser or no side reactions, better yields, higher purities of products, the possibility to reach new selectivity and new reactivity in chemical reactions, many times cheaper costs, and ease of handling and processing [29,30,31,32,33,34]. As a result, reactions under US irradiation become environmentally friendly [29,30,31,32,33,34].
Motivated by the above considerations and having in view our previous expertise in the field of US-assisted reactions [35,36,37], as well as in imidazole and pyridine azaheterocycles with antifungal activity [38,39,40,41,42], this study aimed to obtain and determine the antifungal activity against human and plant fungi of a new class of hybrid bis-(imidazole/benzimidazole)-pyridine salt derivatives. In equal measure, we were interested in performing the synthesis of these hybrid compounds by using environmentally friendly methods under US irradiation.

2. Results and Discussions

2.1. Design, Mechanism of Action and Synthesis

In the first instance, by using the molecular hybridization approach [40,42], we designed the new class of hybrid bis-(imidazole/benzimidazole)-pyridine derivatives by combining the pharmacophoric potential of the imidazole/benzimidazole azole moiety and the pyridine azine moiety, Scheme 1.
In this respect, the first nitrogen atom from the imidazole/benzimidazole pharmacophore unit was connected with the pyridine pharmacophore unit via a methylene (-CH2−) linker at 2,6-position of pyridine moiety, leading to the desired bis-(imidazole/benzimidazole)-pyridine hybrids. Having in view that the existing imidazole drugs from the market usually have linked to the second nitrogen atom from an imidazole moiety a para-Y-phenyl graph, we decided to introduce into our hybrid structures such a substituent and to study the influence against antifungal activity of this graph, by using bioisosterism lead modifications as Y-substituents, from the para position of benzoyl moiety were chosen the -Cl, -Br, -NO2 and -C6H5 (phenyl, -Ph) moieties. Also, being salts, the solubility in water of our compounds will increase, and we expect a serious improvement of pharmacokinetic properties, especially absorption.
As to the mechanism of antifungal action, it is well documented that imidazole derivatives are antifungal agents that block fungal sterol/ergosterol biosynthesis by inhibiting lanosterol 14 α-demethylase enzyme CYP51 [1,2] while pyridine drugs are cell wall inhibitors [15,16,17]. Moreover, the binding site of CYP51 contains a heme cofactor that can coordinate with an imidazole or triazole ring of azole drugs. Having in view these considerations, we are expecting that our hybrid compounds will act as multitarget ligands that could inhibit the CYP51 enzyme and produce inhibition of cell wall synthesis.
To synthesize our hybrid bis-(imidazole/benzimidazole)-pyridine derivatives, we used an adaptation of a previous method described by us [40,42], which involves a direct and efficient two-step reaction pathway, Scheme 2. In the first step, 2,6-bis(chloromethyl)pyridine 1 reacts with imidazole (2a)/benzimidazole (2b) via an N1-alkylation reaction, leading to the hybrid bis-(imidazole/benzimidazole)-pyridine 3a,b. In the second step, compounds 3a,b suffer a quaternization reaction of the second N3-nitrogen atom from imidazole/benzimidazole unit with α–bromo-para-Y-substituted-acetophenones 4a–d, leading to the desired final products, the hybrid bis-(imidazole)-pyridine salts 5a–d and hybrid bis-(benzimidazole)-pyridine salts 6a–d.
Under conventional TH, the N-alkylation reactions have some major drawbacks, such as long reaction time (480–720 min), high amounts of consumed energy, moderate to good yields (around 45–85%), and large amounts of used solvents. Having in view these considerations and applying the principle of green chemistry, we decided to study these reactions by using US technology. The results obtained are presented in Table 1.
The data listed in Table 1 reveal that the use of US irradiation in the studied N-alkylation reactions has some certain advantages in terms of a dramatic decrease in reaction time (by four to seven folds) and, consequently, a notable acceleration of reaction rate, a remarkable decrease in the consumed energy, and higher yields (with about 10–15%). Unfortunately, the amount of solvent used was not possible to be decreased.
The structures of the hybrid compounds 5a–d and 6a–d were proven by elemental (C, H, N) and spectral analysis: IR, 1H-NMR and 13C-NMR. If we consider compound 5a as representative for the series, the most informative signals furnished by 1H-NMR spectrum are those of the aliphatic methylene protons (2H from CH2-α and 2H from CH2-β, respectively), and the aromatic protons H2′ (from imidazole ring), H2″ and H3″ (from para-phenyl ring). The proton signals from the two methylene groups appear as singlet, at a very high chemical shift, unusual for this type of proton: 6.16 ppm (H from CH2-β) and 5.73 ppm (H from CH2-α). This displacement to high chemical shift is due to the powerful electron withdrawing effect of the adjacent positive nitrogen atom N3′ from the imidazole moiety and carbonyl group (in the case of CH2 from β position), respectively, nitrogen atom N1′ from imidazole and α-pyridine ring (in the case of CH2 from α position). The imidazole protons H2′ are the most unshielded from the spectra, being situated at 9.25 ppm (singlet), due to the powerful unshielded effect of the positive nitrogen atom N3′ and the strong electron withdrawing effect of N1′ nitrogen from imidazole ring. The next unshielded protons are H2″ from the phenyl ring moiety (8.11–8.09 ppm, doublet, J = 8.4 Hz), this proton being strongly unshielded by the electron withdrawing effect of the adjacent carbonyl ketone group (protons are in orto position relative to ketone functionality) and phenyl moiety (Y substituent) from the para-position of benzoyl moiety. The H2″ protons are coupled with H3″ (8.05–8.03 ppm, doublet, J = 8.4 Hz), also at high chemical shifts, for the same reasons as in the case of H2″ protons. In the 13C-NMR spectrum, the most informative data are furnished by the signals corresponding to carbon atoms of the carbonyl group, C1″ and C4″ (from phenyl ring moiety) and the two aliphatic carbons from the methylene groups (C from CH2-α and C from CH2-β). The signal of the most unshielded carbon (from carbonyl ketone group) appears at 190.5 ppm, typical for a C=O carbonyl alkyl-aryl ketone group. The next unshielded carbons are C1″ and C4″ (from phenyl ring moiety) due to the powerful electron withdrawing effect of the carbonyl ketone and phenyl moiety (Y substituent) from the para-position of benzoyl moiety. Thus, C1″ carbon appears at a very high chemical shift (153.6 ppm), being in ipso position relative to ketone functionality and para position relative to phenyl moiety (Y substituent). The C4″ carbon appear also at very high chemical shifts (145.7 ppm), being in para position relative to ketone functionality and ipso position relative to phenyl moiety (Y substituent). The aliphatic carbons from methylene groups appear at a very high chemical shift, unusual for this type of carbon: 55.6 ppm (carbon from CH2-β), 52.7 ppm (carbon from CH2-α). The same explications for such a high chemical shift displacement of this aliphatic methylene carbons given in the case of protons remain valid in the case of carbons. All the remaining signals from NMR spectra are in accordance with the proposed structures. See also Supplementary Materials for the 1H- and 13C-NMR spectra of compounds and Figure 3 for atom numbering.

2.2. Antifungal Results

The in vitro antifungal activity against human and plant fungi of the hybrid bis-(imidazole/benzimidazole)-pyridine salt derivatives 5a–d and 6a–d was evaluated by the Kirby–Bauer disk diffusion method [43,44,45], using a Sabouraud nutrient agar medium for antifungal assay. The antifungal activity was determined against five fungal strains of medical importance (Rhodotorula sp., Candida albicans wild type, C. parapsilosis wild type—strains kindly offered by Dr. Simona Matiut from the Praxis Clinical Laboratory, Iasi, Romania, obtained from biological specimens; C. albicans ATCC 10231, C. parapsilosis ATCC 22019) and against four filamentous fungal strains of agriculture importance, from the culture collection of Fungal Research Laboratory, Faculty of Biology, Alexandru Ioan Cuza University of Iasi, isolated from fungal attack on cultivated plants (Aspergillus niger wild type, A. flavus wild type, Cladosporium cladosporioides wild type, Rhizopus nigricans wild type).
In Table 2 are listed the results of the antifungal assay determined by the disk diffusion method, against the five fungal strains of medical importance, for the hybrid derivatives 5a–d and 6a–d. The positive control was fluconazole and nystatin while the negative control consists of sterile filter paper disks (with no antimicrobial compounds) soaked with DMSO 3%. The results of the antifungal assay are displayed as diameters of inhibition zones (in mm), where the larger diameter is the most active compound.
The data from Table 2 reveal that some of the hybrid bis-(imidazole/benzimidazole)-pyridine derivatives 5a–d and 6a–d manifest a good antifungal activity against the tested strains, the hybrid derivatives 5a and 6a have a quasi-nonselective antifungal activity, while the hybrids 5c and 6c are active against C. albicans wild type only. Against the fungal strains C. parapsilosis ATCC 22019, C. parapsilosis wild type and Rhodotorula sp., the bis-(imidazole)-pyridine hybrid 5a (Y = -Ph) have excellent antifungal activity (with diameters of inhibition zones of 20 mm, 19 mm, 19 mm), superior or equal to the control drug fluconazole (18 mm, 19 mm, 19 mm). Against the remaining two strains (C. albicans wild type and C. albicans ATCC 10231) the hybrid 5a has good-to-moderate antifungal activity (18 mm and 23 mm), close to the control drug fluconazole (26 mm and 24 mm). The bis-(benzimidazole)-pyridine hybrid 6a (Y = -Ph) also has good antifungal activity against all five fungal strains (diameters of inhibition zones in the range of 7.5–13 mm), inferior to control drug fluconazole but still significant.
The bis-(imidazole)-pyridine hybrid 5c (Y = -Br) and bis-(benzimidazole)-pyridine hybrid 6c (Y = -Br) manifest a weak antifungal activity only against C. albicans wild type, with a diameter of inhibition zones of 7 mm and 7.5 mm, respectively.
In the next step of the antifungal assay, for the four active hybrid derivatives 5a, 5c, 6a and 6c, the minimum inhibitory concentration (MIC) was determined using the broth microdilution assay method [46,47]. The results of the MIC antifungal assay are displayed in Table 3.
The data from Table 3 reveal that the tested hybrid derivatives are active to a low concentration against the tested fungal strains, with a MIC value in the range of 3.9 to 62.5 µg/mL. The bis-(imidazole/benzimidazole)-pyridine derivatives 5a and 6a (with phenyl group (Y = -Ph) at the para position of the benzoyl moiety) are the most active (MIC in the range of 3.9 to 31.25 µg/mL), the best antifungal activity being against the fungal strains C. albicans wild type (MIC = 3.9 µg/mL in the case of hybrid 5a) and Rhodotorula sp. (MIC = 3.9 µg/mL in the case of hybrid 6a). The bis-(imidazole)-pyridine hybrid 5c (Y = -Br) and bis-(benzimidazole)-pyridine hybrid 6c (Y = -Br) manifest significant antifungal activity only against C. albicans wild type, with a MIC value of 62.5 µg/mL.
The in vitro antifungal assay against the four phytopathogenic filamentous fungal strains reveal that, from the tested hybrid bis-(imidazole/benzimidazole)-pyridine derivatives 5a–d and 6a–d, only one compound, namely the bis-(imidazole)-pyridine hybrid 5a (Y = -Ph), manifests a strong selective antifungal activity against A. niger and A. flavus, Table 4.
Thus, the hybrid 5a have a diameter of inhibition zone of 12 mm against A. niger and 9 mm against A. flavus, close to the control drug fluconazole (9 mm and 0 mm). The MIC assay (Table 5) for the hybrid 5a indicates a value of 62.5 µg/mL against A. niger and 31.25 µg/mL against A. flavus, compared to the control drug nystatin (MIC of 0.48 µg/mL against both A. niger and A. flavus).
We may notice that the MIC values for 5a against the fungal strain A. flavus are clearly higher than the MIC for nystatin; however, the values obtained are smaller than MIC obtained for many antifungal agents commercially used for plant protection [48,49].
From the SAR point of view, the comparative analysis of the data presented above denotes that the presence of a hybrid bis-(imidazole/benzimidazole)-pyridine moiety has a beneficial influence for antifungal activity against both human and plant pathogenic fungal strains. The substituent Y from the para/(4)-position of the benzoyl moiety anchored onto a N3-imidazole nitrogen atom plays also a crucial role in increasing the antifungal properties, with the presence of a phenyl ring (Y = -C6H5) being extremely beneficial for this activity; the presence of a bromine halogen moiety (Y = -Br) is also favorable to increase antifungal activity.

3. Materials and Methods

3.1. Materials and Measurements

2,6-bis(chloromethyl)pyridine (99% purity), imidazole (ACS reagent, ≥99% (titration)), benzimidazole (purity: >98.0%(GC)(T)), p-R (-Ph, -Cl, -Br, -NO2)-bromoacetophenones (99% purity) and sodium hydride (60% in mineral oil) were purchased from TCI and Sigma-Aldrich (Burlington, MA, USA). The NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bruker, Vienna, Austria) operating at 500 MHz for 1H and 125 MHz for 13C, equipped with a 5 mm PABBO detection probe. The program used for acquisition and processing of data was TopSpin 3.2 PL5 (Bruker, Vienna, Austria). The abbreviations utilized to designate chemical shift multiplicities are: s = singlet, d = doublet, dd = doublet of doublets, add = apparent doublet of doublets, t = triplet, m = multiplet. Ultrasound-assisted reactions were performed using Sonics VCX-130, USA, with a nominal power of 130 W and a frequency of 20 kHz. For this ultrasonic reactor the titanium horn (diameter: 6 mm; length: 116 mm) was fixed to the ultrasonic converter. The titanium probe tip was directly immersed in the reaction mixture. Infrared (IR) data were recorded with FTIR Cary 630 spectrophotometer (Agilent Technologies, Mulgrave, Australia) coupled to a ZnSe ATR module for measuring solid samples. Mel-Temp apparatus (Barnstead International, Dubuque, IA, USA)was used to measure the melting points of compounds and are uncorrected. Thin layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (Merck, Darmstadt, Germany). The ultrasonic bath Elma Transsonic T310 (ELMA, Stuttgart, Germany) (power 34.5 W, frequency 35 kHz) was used for solubilizing the starting materials. Elemental analyses were done using a FlashSmart CHNS/O Elemental Analyzer (Thermo Fisher, Waltham, MA, USA), with MVC. The microanalyses were in satisfactory agreement with the calculated values: C, ±0.15; H, ±0.10; N, ±0.30.

3.2. Antifungal Assay

Tested compounds have been dissolved in 20% DMSO to a final concentration of 1 mg mL−1. All the reagents used were purchased from Merck (Darmstadt, Germany).

3.2.1. Antifungal Effect Assessment

Evaluation of susceptibility to the new synthesized compounds have been determined using the Kirby–Bauer disk diffusion method, on 9 cm diameter Petri dishes with Sabouraud-agar, inoculated with suspensions of 106 CFU/mL. Tested compounds were used at 1 mg/mL final concentration per each 6 mm diameter disk. Negative controls (20% DMSO) and positive controls (nystatin 200 μg/mL and fluconazole 32 μg/mL) were also used. The cultures were incubated at 37 °C for 48 h in the case of yeasts, and at 28 °C for 5 days in the case of filamentous fungi. All experiments have been done in triplicate and repeated. The results were calculated as an average of three replicates and three diameters measured for each disk.

3.2.2. Determination of Minimum Inhibitory Concentration

Determination of minimum inhibitory concentration has been achieved only for the compounds that manifested inhibition of fungal growth in previous tests. The technique used was the broth microdilution method (MIC), following recommendation from the Clinical Standard Laboratory Institute, on 96-well round bottom plates for yeasts (with a final volume of 200 μL mixture per well) and 48-well plates for filamentous fungi (with a final volume of 400 μL mixture per well), in Sabouraud broth. Serial dilutions started at 250 μg/mL (5% DMSO) in the first well, being reduced to half subsequently. Controls with nystatin (200 μg mL−1 initial concentration) and fluconazole (32 μg/mL initial concentration) as standard antimycotics were used. Sterility controls (un-inoculated well containing media) and negative controls (inoculated wells with simple media) were also used. Inoculation has been performed with suspension of CFU as described, at a final ratio of 1:1 (inoculum versus supplemented media). Incubation was done at 37 °C for 48 h for yeasts, and at 28 °C for 3 days for filamentous fungi. A solution of 0.05% resazurin was used to read the results after incubation.
The synthesis and spectral characterization of starting materials (3a and 3b) and the synthesis under conventional TH of compounds 5b–d and 6b–d (see also Supplementary Materials), were described in a previous work published by our group [40]. The complete spectral characterization of the hybrid molecules 5b–d and 6b–d can be found there.

3.3. General Procedure for the Synthesis of Quaternary Salts 5a–d and 6a–d Under US Irradiation

The 2,6-bis(imidazolium/benzimidazolium) salts were obtained according to the following method: intermediate 3a or 3b (1 mmol) is solubilized in approximately 40 mL of acetone and p-R (-Ph, -Cl, -Br, -NO2)-bromoacetophenone (4a–d) (2.4 mmol → 3a and 4.5 mmol → 3b) is gradually added after solubilization in approximately 20 mL of acetone. The reaction mixture was then ultrasonicated for approximately 100 min (parameters for sonication: A = 100%, 5 s ON/5 s OFF pulses). TLC was used to follow the progress of the reaction. The formed precipitates were collected by filtration, washed with acetone (3 times with 10 mL) and dried in vacuum. No other purification was required.

3.4. Spectral Data of Quaternary Salts 5a–d and 6a–d (Figure 3)

In Figure 3 is presented the numbering of atoms from compounds 5a–d and 6a–d, which is also used in the description of spectral data of the hybrid derivatives.
Pharmaceuticals 18 00495 g003
Figure 3. The numbering of atoms from hybrid bis-(imidazole/benzimidazole)-pyridine derivatives 5a–d and 6a–d.
Figure 3. The numbering of atoms from hybrid bis-(imidazole/benzimidazole)-pyridine derivatives 5a–d and 6a–d.
Pharmaceuticals 18 00495 g003
1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-([1,1′-biphenyl]-4-yl)-2-oxoethyl)-1H-imidazol-3-ium) (5a)
White powder; TH: η = 56%, US: η = 61%; m.p. = 226–229 °C. 1H-NMR (500 MHz, DMSO-d6): δ = 5.73 (s, 4H, 2 × CH2-α,), 6.16 (s, 4H, 2 × CH2-β), 7.51–7.45 (m, 6H, 4 × H8″, 2 × H9″), 7.59–7.57 (d, J = 7.5 Hz, 2H, 2 × H3), 7.74–7.73 (d, J = 8.5 Hz, 4H, 4 × H7″), 7.80–7.79 (add, 4H, 2 × H4′, 2 × H5′), 7.90–7.88 (d, J = 8.5 Hz, 4H, 4 × H4″), 8.06–8.03 (t, J = 8.0 Hz, 1H, H4), 8.11–8.09 (d, J = 8.5 Hz, 4H, 4 × H3″), 9.24 (s, 2H, 2 × H2′). 13C-NMR (125 MHz, DMSO-d6): δ = 52.7, 55.6, 122.2, 123.0, 123.9, 127.0, 127.0, 128.7, 128.9, 129.1, 132.3, 137.9, 138.3, 138.9, 145.7, 153.6, 191.0. IR (ATR, ν(cm−1)): 3017, 2986, 1695, 1583, 1251. Anal. Calcd. for C41H35Br2N5O2: C, 62.37; H, 4.47; N, 8.87; Found: C, 62.47; H, 4.37; N, 8.67.
1,1′-(pyridine-2,6-diylbis(methylene))bis(3-(2-([1,1′-biphenyl]-4-yl)-2-oxoethyl)-1H-benzo[d]imidazol-3-ium) (6a)
White powder; TH: η = 46%, US: η = 64%; m.p. = 213–215 °C. 1H-NMR (500 MHz, DMSO-d6): 6.03 (s, 4H, 2 × CH2-α), 6.50 (s, 4H, 2 × CH2-β), 7.52–7.44 (m, 8H, 2 × H6′, 2 × H9″, 4 × H8″), 7.63–7.60 (t, J = 8.0 Hz, 2H, 2 × H5′), 7.71–7.69 (d, 2H, 2 × H7′, J = 8.0 Hz), 7.78–7.74 (m, 6H, 2 × H3, 4 × H7″), 7.92–7.91 (d, J = 8.5 Hz, 4H, 4 × H4″), 8.09–8.08 (m, 3H, 2 × H4′, H4), 8.18–8.16 (d, 4H, 4 × H3″), 9.90 (s, 2H, 2 × H2′). 13C-NMR (125 MHz, DMSO-d6): 50.6, 53.41, 113.48, 113.98, 122.81, 126.57, 126.74, 127.07, 128.76, 129.28, 131.65, 132.72, 133.4, 138.46, 139.06, 143.87, 145.81, 153.09, 190.91. IR (ATR, ν(cm−1)): 3020, 2973, 1673, 1591, 1545, 1467. Anal. Calcd. for C49H39Br2N5O2: C, 66.15; H, 4.42; N, 7.87; Found: C, 66.25; H, 4.32; N, 7.67.

4. Conclusions

In conclusion, we report herein the design, synthesis, structure and antifungal activity of a new class of hybrid bis-(imidazole/benzimidazole)-pyridine derivatives against human and plant pathogenic fungi. The method of synthesis for the hybrid salts is direct and efficient involving two subsequent N-alkylation reactions of the nitrogen atoms from an imidazole ring. The synthesis of the hybrid derivatives was carried out both under conventional thermal heating and ultrasound irradiation. Under ultrasound irradiation the reaction pathway has some certain advantages in terms of a dramatical decrease in reaction time (by four to seven folds), a remarkable decrease in the consumed energy, and higher yields (with about 10–15%). The structure of the hybrid bis-(imidazole/benzimidazole)-pyridine derivatives was determined by elemental and spectral (IR, NMR) analysis. The in vitro antifungal activity of the hybrid bis-(imidazole/benzimidazole)-pyridine derivatives was evaluated against human and plant pathogenic fungi and revealed that four hybrids [5a and 6a (Y = -Ph), 5c and 6c (Y = -Br)], are active to a low concentration against the tested fungal strains, with a MIC value in the range of 3.9 to 62.5 µg/mL. The best antifungal activity was manifested by the hybrid bis-(imidazole)-pyridine 5a, against both types of human and plant fungal strains, in some cases closely to the control drug fluconazole, which is superior to many antifungal agents commercially used for plant protection. The SAR correlations reveal that the presence of a hybrid bis-(imidazole/benzimidazole)-pyridine moiety has a beneficial influence on antifungal activity against both human and plant pathogenic fungal strains, and the presence of a phenyl ring (Y = -C6H5) at the para position of a benzoyl moiety from the imidazole/benzimidazole heterocycle, substantially increases antifungal properties. The results obtained make us conclude that the hybrid compound bis-(imidazole)-pyridine 5a, could be considered as a good lead candidate for future drug or pesticide development, and future research in this respect will be done.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph18040495/s1, Figure S1: 1H-NMR Spectrum of compound 5a; Figure S2: 13C-NMR Spectrum of compound 5a; Figure S3. 1H-NMR Spectrum of compound 6a; Figure S4. 13C-NMR Spectrum of compound 6a; Figure S5. 1H-NMR Spectrum of compound 5b; Figure S6. 13C-NMR Spectrum of compound 5b; Figure S7. 1H-NMR Spectrum of compound 5c; Figure S8. 13C-NMR Spectrum of compound 5c; Figure S9. 1H-NMR Spectrum of compound 5d; Figure S10. 13C-NMR Spectrum of compound 5d; Figure S11. 1H-NMR Spectrum of compound 6b; Figure S12. 13C-NMR Spectrum of compound 6b; Figure S13. 1H-NMR Spectrum of compound 6c; Figure S14. 13C-NMR Spectrum of compound 6c; Figure S15. 1H-NMR Spectrum of compound 6d; Figure S16. 13C-NMR Spectrum of compound 6d.

Author Contributions

Conceptualization, V.M., D.D. and I.I.M.; methodology, T.B., V.M., V.A. and D.A.-M.; validation, D.D., V.A. and I.I.M.; formal analysis, T.B., V.M. and D.A.-M.; investigation, T.B., V.A. and D.A.-M.; data curation, V.M., D.D. and I.I.M.; writing—original draft preparation, V.M., D.D. and I.I.M.; writing—review and editing, T.B., V.M., D.D., D.A.-M. and I.I.M.; visualization, V.M., D.D. and T.B.; supervision, I.I.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.

Acknowledgments

Authors are thankful to RECENT AIR and CERNESIM Centre within the Institute for Interdisciplinary Research at the Alexandru Ioan Cuza University of Iasi for the infrastructure used. The authors are grateful to Simona Matiut (Praxis Clinical Laboratory, Iasi, Romania) for Candida strains provided.

Conflicts of Interest

The authors declare no conflicts of interest.

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Pharmaceuticals 18 00495 g001
Figure 1. Antifungal drugs with imidazole and pyridine skeleton frequently used in current human therapy.
Figure 1. Antifungal drugs with imidazole and pyridine skeleton frequently used in current human therapy.
Pharmaceuticals 18 00495 g001
Pharmaceuticals 18 00495 g002
Figure 2. Antifungal pesticides with azole and azine skeleton frequently used in agriculture.
Figure 2. Antifungal pesticides with azole and azine skeleton frequently used in agriculture.
Pharmaceuticals 18 00495 g002
Pharmaceuticals 18 00495 sch001
Scheme 1. Design in the class of hybrid bis-(imidazole/benzimidazole)-pyridine derivatives.
Scheme 1. Design in the class of hybrid bis-(imidazole/benzimidazole)-pyridine derivatives.
Pharmaceuticals 18 00495 sch001
Pharmaceuticals 18 00495 sch002
Scheme 2. Synthesis of hybrid bis-(imidazole/benzimidazole)-pyridine salts derivatives 5a–d and 6a–d.
Scheme 2. Synthesis of hybrid bis-(imidazole/benzimidazole)-pyridine salts derivatives 5a–d and 6a–d.
Pharmaceuticals 18 00495 sch002
Table 1. Comparative study regarding the synthesis of hybrid bis-(imidazolium/benzimidazolium) salts under TH (thermal heating) versus US (ultrasound irradiation).
Table 1. Comparative study regarding the synthesis of hybrid bis-(imidazolium/benzimidazolium) salts under TH (thermal heating) versus US (ultrasound irradiation).
Compound5a5b5c5d6a6b6c6d
Yield (%) *TH5687847946715548
US6190868564735756
R.t. (min) **TH720480600720720480480720
US120100100100120120120120
* Yields were calculated in isolated product after purification; ** R.t = Reaction time.
Table 2. Antifungal assessment through Kirby–Bauer diffusion assay for fungal strains of medical importance.
Table 2. Antifungal assessment through Kirby–Bauer diffusion assay for fungal strains of medical importance.
Compound/StrainAverage Diameter of Inhibition Zone (mm) *,**
CAWCA10231CPWCP22019RH
5a18 ± 1.423 ± 1.919 ± 1.8220 ± 1.9518.5 ± 1.8
5b00000
5c7 ± 0.10000
5d00000
6a10 ± 0.87.5 ± 0.18.5 ± 0.810 ± 1.013 ± 1.1
6b00000
6c7.5 ± 0.030000
6d00000
Std-Flu26 ± 2.1524.5 ± 2.119 ± 1.8518 ± 1.8219 ± 1.75
Std-Nyst28 ± 2.1826 ± 2.1425.5 ± 2.0526.5 ± 1.9530 ± 2.6
DMSO00000
(CAW—Candida albicans wild type; CA10231—C. albicans ATCC 10231; CPW—C. parapsilosis wild type; CP22019—C. parapsilosis ATCC 22019; RH—Rhodotorula sp.; Std-Flu—reference antimycotic fluconazole; Std-Nyst—reference antimycotic nystatin; ±—standard deviation); * in bold are marked higher values of inhibition zone diameters; ** the values include the 6 mm diameter of the disks.
Table 3. Minimum inhibitory concentration (MIC) assessment for selected compounds against yeasts.
Table 3. Minimum inhibitory concentration (MIC) assessment for selected compounds against yeasts.
Compound/StrainMIC (µg/mL)
CAWCA10231CPWCP2019RH
5a3.915.6231.2531.2515.62
5c62.5NNNN
6a15.6231.2531.2531.253.9
6c62.5NNNN
Std-Flu0.120.120.977.810.48
Std-Nyst0.240.240.970.240.12
DMSO>250>250250>250125
(CAW—Candida albicans wild type; CA10231—C. albicans ATCC 10231; CPW—C. parapsilosis wild type; CP22019—C. parapsilosis ATCC 22019; RH—Rhodotorula sp.; Std-Flu—reference antimycotic fluconazole; Std-Nyst—reference antimycotic nystatin; N—not tested).
Table 4. Antifungal assessment through Kirby–Bauer diffusion assay.
Table 4. Antifungal assessment through Kirby–Bauer diffusion assay.
Compound/StrainAverage Diameter of Inhibition Zone (mm) *,**
ANAFCCRN
5a12 ± 1.09 ± 0.079 ± 0.228 ± 0.03
5b0016 ± 1.550
5c008 ± 0.050
5d0000
6a008 ± 0.057 ± 0.1
6b0000
6c0000
6d0000
Std-Flu9 ± 0.9024 ± 2.10
Std-Nyst20.5 ± 1.8513.5 ± 1.2411 ± 1.050
DMSO0000
AN—Aspergillus niger; AF—A. flavus; CC—Cladosporium cladosporioides; RN—Rhizopus nigricans; Std-Flu—reference antimycotic fluconazole; Std-Nyst—reference antimycotic nystatin; ±—standard deviation); * in bold are marked higher values of inhibition zone diameters; ** the values include the 6 mm diameter of the disks.
Table 5. Minimum inhibitory concentration assessment for selected compounds against filamentous fungi (Std-Nyst—nystatin, N-not tested).
Table 5. Minimum inhibitory concentration assessment for selected compounds against filamentous fungi (Std-Nyst—nystatin, N-not tested).
Compound/StrainMIC (µg/mL)
ANAFCCRN
5a62.531.25250250
6aNNN250
Std-Nyst0.480.48125>250
DMSO0000
AN—Aspergillus niger; AF—A. flavus; CC—Cladosporium cladosporioides; RN—Rhizopus nigricans; Std-Nyst—nystatin; N—not tested.
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MDPI and ACS Style

Balaes, T.; Mangalagiu, V.; Antoci, V.; Amariucai-Mantu, D.; Diaconu, D.; Mangalagiu, I.I. Hybrid Bis-(Imidazole/Benzimidazole)-Pyridine Derivatives with Antifungal Activity of Potential Interest in Medicine and Agriculture via Improved Efficiency Methods. Pharmaceuticals 2025, 18, 495. https://doi.org/10.3390/ph18040495

AMA Style

Balaes T, Mangalagiu V, Antoci V, Amariucai-Mantu D, Diaconu D, Mangalagiu II. Hybrid Bis-(Imidazole/Benzimidazole)-Pyridine Derivatives with Antifungal Activity of Potential Interest in Medicine and Agriculture via Improved Efficiency Methods. Pharmaceuticals. 2025; 18(4):495. https://doi.org/10.3390/ph18040495

Chicago/Turabian Style

Balaes, Tiberius, Violeta Mangalagiu, Vasilichia Antoci, Dorina Amariucai-Mantu, Dumitrela Diaconu, and Ionel I. Mangalagiu. 2025. "Hybrid Bis-(Imidazole/Benzimidazole)-Pyridine Derivatives with Antifungal Activity of Potential Interest in Medicine and Agriculture via Improved Efficiency Methods" Pharmaceuticals 18, no. 4: 495. https://doi.org/10.3390/ph18040495

APA Style

Balaes, T., Mangalagiu, V., Antoci, V., Amariucai-Mantu, D., Diaconu, D., & Mangalagiu, I. I. (2025). Hybrid Bis-(Imidazole/Benzimidazole)-Pyridine Derivatives with Antifungal Activity of Potential Interest in Medicine and Agriculture via Improved Efficiency Methods. Pharmaceuticals, 18(4), 495. https://doi.org/10.3390/ph18040495

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