New Generation of Hybrid Pyrazole–Tetrazole Tetrapodal Compounds: Synthesis and Biological Activities

Abstract

The elaboration of a new family of tetrapodal molecules L1–L3 bearing two pyrazole–tetrazole units is presented. The structure assigned to such molecules was verified by various techniques, including FTIR, NMR, HRMS and elemental analysis. The ability of these tetrapods to inhibit the growth of four fungal strains was examined, and the obtained results showed that they have some antifungal potency in the range of 12–16 mm. The alpha-amylase inhibition activity of these molecules was also evaluated. The obtained IC₅₀ values (1.34 × 10⁻¹–1.2 × 10⁻² mg/mL) demonstrated that all compounds are potent enough to inhibit this enzyme much better than the positive control acarbose (2.6 × 10⁻¹ mg/mL). A docking study on the porcine alpha-amylase was performed, and the results were in good correlation with the experimental results.

1. Introduction

The biological properties of pyrazole and its derivatives have been the subject of several studies. This is due to their pharmacological properties, including antimicrobial [1,2,3,4,5], antiviral [6,7,8], anti-inflammatory and [9,10,11] antitumoral [12,13,14,15] activities.

Tetrazole and its derivatives have also garnered much interest in the pharmacological fields [16]. The significance attributed to this five-membered ring containing four nitrogen atoms is due to the tetrazole–carboxylic acid bioisosterism [17]. Indeed, both groups exhibit nearly the same pKa values as well as planar electronic delocalization [18,19,20].

Therefore, the assembling of these two N-heterocycles into one structure could lead to hybrid compounds with potent activity. Faria et al. [21] demonstrated the synthesis of a series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives with good activity against the Leishmania braziliensis. In addition, the development of a family of (2R,3S)-3-(substituted-1H-pyrazol-3-yl)-2-(2,4-difluorophenyl)-1-(1H-tetrazol-1-yl)butan-2-ol compounds was described by Chi and coworkers [22]. These molecules showed high antifungal activity against six fluconazole-resistant C. auris clinical isolates. In a recent study, Metre et al. [23] reported the anticancer activity of two series: 4-((5-(1-aryl-1H-pyrazol-3-yl)-2H-tetrazol-2yl)-6-substituted-2H-chromen-2-one and 4-((5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazol-2yl)-6-substituted-2H-chromen-2-one. Our team has also elaborated pyrazole–tetrazole hybrid compounds in different skeletons [24,25,26,27,28]. These compounds showed good biological activity as antimicrobial [24], antidiabetic [27] and vasorelaxant [28] agents.

As a continuation of these works, we present in this paper a new family of tetrapodal compounds bearing two pyrazole–tetrazole subunits and differing by the nature of the lariat side. The antifungal activities of these compounds against four fungal strains were examined.

Their potencies in inhibiting the alpha-amylase enzyme were also evaluated. In fact, this enzyme is responsible for the degradation of longer chains of polysaccharides, such starch and glycogen, to shorter chains such maltose. This makes it a good target to treat diabetes-related diseases [29]. A docking study was also carried out to correlate the experimental and computational data.

2. Materials and Methods

2.1. Reagents and Instruments

Chemicals (reagent grade) and solvents were procured from Aldrich Chemical Co. and used as received. NMR analysis was carried out using a Bruker AC 500 spectrometer (Bruker France SAS, Wissembourg, France). The spin resonances are displayed as chemical shifts (δ) in parts per million (ppm) and referenced to the residual peak of CDCl3 (7.26 and 77 ppm for ¹H NMR and ¹³C NMR, respectively). The spin multiplicities are presented as s, d, t, q and m for singlet, doublet, triplet, quartet and multiplet, respectively. Mass spectrometry was carried out using a Platform II Micromass instrument (ESI+, CH₃CN/H₂O: 50/50) and a Micromass Q-TOF micro MS spectrometer (Bruker France SAS, Wissembourg, France). Melting points were measured using IA9100 (Electrothermal) apparatus (Reagecon Diagnostics Ltd., Shannon, Irland). Elemental analysis was carried out via an EA 3000 Elemental Analyzer (EuroVector S.p.A., Milan, Italy). A Shimadzu FT-IR 8400s spectrophotometer (SpectraLab Scientific Inc., Markham, Canada) was used for the FT-IR analysis. Compound 1 was obtained following procedures described in our recent work [25].

2.2. Synthesis

2.2.1. (1-((2-Ethyl-2H-tetrazol-5-yl)methyl)-5-methyl-1H-pyrazol-3-yl)methanol 2

Compound 1 (18.9 mmol) in THF (70 mL) was slowly added to a THF solution (80 mL) of LiAlH₄ (26.3 mmol). Thereafter, the reaction was agitated for 4 h at 65 °C. After cooling, we added the following at 0 °C: (i) water (1 mL), (ii) NaOH (0.125 g) in water (1 mL) and (iii) water (3 mL). Then, the mixture was heated for 1/2 h and the formed solid was eliminated by filtration. The filtrate was concentrated, and then, the obtained residue was purified by column chromatography on silica gel (diethyl ether/methanol (97/3), R_f = 0.37) to produce 2 as a colorless oil (78%). ¹H NMR (CDCl3) δ: 1.63 (t, 3H, J = 7.4 Hz, CH₃CH₂); 2.37 (s, 3H, CH₃Pz); 4.61 (q, 2H, J = 7.4 Hz CH₂-NTz); 4.62 (s, 2H, HO-CH₂-); 5.48 (s, 2H, Tz-CH₂-Pz); 6.06 (s, 1H, HPz). ¹³C NMR (CDCl3) δ: 11.41 (CH₃Pz); 14.58 (CH₃-CH₂); 44.17 (CH₂-Tz); 48.72 (Tz-CH₂-Pz); 59.26 (-CH₂-OH); 104.69 (HCPz); 140.38 (CPzCH₃); 152.09 (CPz-CH₂); 162.22 (CTz). HRMS m/z: calculed for C₉H₁₅N₆O [M + H]⁺, 223.1307; found: 223.1310. Anal. Calcd for C₉H₁₅N₆O: C, 48.64; H, 6.35; N, 37.81. Found: C, 48.75; H, 6.49; N, 37.95. IR (KBr) cm⁻¹: 3203, 1452.

2.2.2. 5-((3-(Chloromethyl)-5-methyl-1H-pyrazol-1-yl)methyl)-2-ethyl-2H-tetrazole 3

A solution of SOCl₂ (16.4 mmol) in CH₂Cl₂ (25 mL) was added to compound 2 (10.6 mmol) in CH₂Cl₂ (20 mL). The resulting mixture was maintained under stirring at room temperature for 3 h. Thereafter, it was neutralized using sodium bicarbonate solution, and the CH₂Cl₂ solution was dried using anhydrous magnesium sulfate. After evaporation, the chloride derivative, compound 3, was obtained in its pure state in a 75% yield. ¹H NMR (CDCl3) δ: 1.63 (t, 3H, J = 7.3 Hz, CH₃CH₂); 2.31 (s, 3H, CH₃Pz); 4.43 (s, 2H, Cl-CH₂-); 4.59 (q, J = 7.3 Hz, 2H, CH₂-NTz); 5.51 (s, 2H, Tz-CH₂-Pz); 6.10 (s, 1H, HPz). ¹³C NMR (CDCl3) δ: 11.34 (CH₃Pz); 14.56 (CH₃-CH₂); 38.98 (-CH₂-Cl); 44.13 (CH₂-Tz); 51.75 (Tz-CH₂-Pz); 105.54 (HCPz); 140.41 (CPzCH₃); 147.19 (CPz-CH₂); 162.08 (CTz). HRMS m/z: calculed for C₉H₁₄N₆Cl [M + H]⁺, 241.0890; found: 241.0897. Anal. Calcd for C₉H₁₃N₆Cl: C, 44.91; H, 5.44; N, 34.92. Found: C, 44.98; H, 5.54; N, 35.07. IR (KBr) cm⁻¹: 2984.

2.2.3. Synthesis of Tetrapodal Structures L1–L3

A DMF solution (20 mL) of compound 3 (6 mmol) was added to a DMF solution (25 mL) of the primary amine (3 mmol) and sodium carbonate (20 mmol). Subsequently, the mixture was agitated at 45 °C for 48 h. Thereafter, the solid material was eliminated by filtration and the filtrate was concentrated under reduced pressure. All tetrapodal ligands were purified by column chromatography on silica gel with diethyl ether/EtOH (95/5).

3-(bis((1-((2-ethyl-2H-tetrazol-5-yl)methyl)-5-methyl-1H-pyrazol-3-yl)methyl)amino)propan-1-ol L1

Yellow oil. Yield = 41%. ¹H NMR (CDCl3) δ: 1.57 (t, 6H, J = 7.3 Hz,, CH₃-CH₂); 1.71 (m, 2H, CH₂-CH₂-CH₂); 2.31 (s, 6H, CH₃-Pz); 2.67 (t, 2H, J = 7 Hz, N-CH₂-(CH₂)₂-OH); 3.61 (s, 4H, Pz-CH₂-N); 3.66 (s, 2H, J = 6.3Hz, CH₂-OH); 4.58 (m, 4H, CH₂-NTz); 5.44 (s, 4H, Tz-CH₂-Pz); 6.08 (s, 2H, HPz). ¹³C NMR (CDCl3) δ: 11.33 (CH₃Pz); 14.49 (CH₃-CH₂-Tz); 27.83 (CH₂-CH₂-CH₂-); 44.08 (CH₂-Tz); 48.62 (CH₂-CH₂-N-CH₂); 51.00 (Pz-CH₂-N); 52.41 (Tz-CH₂-Pz); 63.33 (CH₂-OH); 106.30 (CHPz); 140.27 (PzC(CH₃)); 148.70 (PzC-CH2); 162.27 (CTz). HRMS m/z: calculed for C₂₁H₃₄N₁₃O [M + H]⁺, 484.3009; found: 484.3006. Anal. Calcd for C₂₁H₃₃N₁₃O: C, 52.16; H, 6.88; N, 37.65; Found: C, 52.21; H, 6.97; N, 37.77. IR (KBr) cm⁻¹: 3263, 1561, 1162.

2-(4-(bis((1-((2-ethyl-2H-tetrazol-5-yl)methyl)-5-methyl-1H-pyrazol-3-yl)methyl)amino)phenyl)ethan-1-ol L2

Brown oil. Yield = 41%. ¹H NMR (CDCl3) δ: 1.62 (t, 6H, J = 7.4 Hz, 6H, CH₃-CH₂-N); 2.30 (s, 6H, CH₃); 2.73 (t, 2H, J = 6.5 Hz, CH₂-CH₂-OH); 3.78 (t, 2H, J = 6.5 Hz, CH₂-CH₂-OH); 4.49 (s, 4H, Pz-CH₂-N); 4.63 (q, 4H, J = 7.4 Hz, CH₂-NTz); 5.46 (s, 4H, Ph-CH₂-Pz); 5.91 (s, 2H, HPz); 6.82 (d, 2H, J = 5 Hz, HPh (m)); 7.01 (d, 2H, J = 5 Hz HPh (o)). ¹³C NMR (CDCl3): δ= 11.31 (CH₃-Pz); 14.47 (CH₃-CH₂-NTz); 38.14 (CH₂-CH₂-OH); 44.01 (CH₂-NTz); 48.53 (Pz-CH₂-N); 48.93 (Tz-CH₂-Pz); 63.88 (CH₂-OH); 105.07 (CHPz); 113.17 (CPh (o)); 125.70 (CPh (m)); 129.57 (CPh-CH₂-); 139.90 (CPz-CH₃); 147.65 (CPh-N); 150.46 (N-CPz-CH₂); 162.26 (CTz). HRMS m/z: calculed for C₂₆H₃₆N₁₃O [M + H]⁺, 546.3166; found: 546.3165. Anal. Calcd for C₂₆H₃₅N₁₃O: C, 57.23; H, 6.47; N, 33.37. Found: C, 57.37; H, 6.59; N, 33.45. IR (KBr) cm⁻¹: 3275, 1567, 1167.

N,N-bis((1-((2-ethyl-2H-tetrazol-5-yl)methyl)-5-methyl-1H-pyrazol-3-yl)methyl)propan-1-amine L3

Yellow oil. Yields: 42%. ¹H NMR (CDCl3) δ: 0.78 (t, 3H, J = 7.4 Hz, CH₃-CH₂-CH₂); 1.53–1.61 (m, 6H, J = 7.3 Hz, CH₃-CH₂-N; 5H, CH₃-CH₂-CH₂); 2.32 (s, 6H, CH₃-Pz); 2.43 (m, 2H, CH₃-CH₂-CH₂-N); 3.69 (s, 4H, Pz-CH₂-N); 4.58 (m, 4H, CH₂-NTz); 5.43 (s, 4H, Tz-CH₂-Pz); 6.20 (s, 2H, HPz). ¹³C NMR (CDCl3) δ: 11.08 (CH₃Pz); 11.77 (CH₃-CH₂-CH₂-N); 14.82 (CH₃-CH₂-Tz); 24.39 (CH₂-CH₂-CH₂); 44.13 (CH₂-Tz); 48.52 ((CH₂)₂N-CH₂); 50.71 (Pz-CH₂-N); 54.07 (Tz-CH₂-Pz); 106.73 (CHPz); 140.17 (PzC(CH₃)); 147.94 (PzC-CH2); 162.63 (CTz). HRMS m/z: calculed for C₂₁H₃₄N₁₃ [M + H]⁺, 468.3060; found: 468.3062. Anal. Calcd for C₂₁H₃₃N₁₃: C, 53.94; H, 7.11; N, 38.94. Found: C, 54.13; H, 7.27; N, 39.09. IR (KBr) cm⁻¹: 2983.

2.3. Antifungal Activity Determination

The antifungal potency of tetrapodal compounds L1–L3 was tested against four fungal strains: Geotrichum candidum sp., Aspergillus niger HO32, Penicillium digitatum P22 and Rhodotorula glutinis ON209167.1. These fungi were isolated in our Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohammed Premier university, Oujda, Morocco. Their effectiveness was evaluated by determining the inhibition zone diameters (see Supplementary Materials) [24]. Cycloheximide served as the positive control.

2.4. Alpha-Amylase Inhibition Activity Determination

The inhibition potency of ligands L1–L3 on the pancreatic α-amylase enzyme was assessed following the procedure reported in our previous paper (see Supplementary Materials) [21]. The inhibition percentage (%) was determined by Formula (1):

$$\begin{gathered} \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)= \\ {\displaystyle \frac{\left(\mathrm{O}\mathrm{D}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}540\mathrm{n}\mathrm{m}-\mathrm{O}\mathrm{D}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}540\mathrm{n}\mathrm{m}\right)-(\mathrm{O}\mathrm{D}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}540\mathrm{n}\mathrm{m}-\mathrm{O}\mathrm{D}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k})}{\mathrm{O}\mathrm{D}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}540\mathrm{n}\mathrm{m}-\mathrm{O}\mathrm{D}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}540\mathrm{n}\mathrm{m}}}\times 100 \end{gathered}$$

(1)

while the Half-maximal inhibitory concentration (IC₅₀) was calculated graphically by using function (2).

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\left(\%\right)=\mathrm{f}(\log \left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right))$$

(2)

2.5. Molecular Docking Study

The docking study was carried out using the “iGemdock program” [30]. The 3D structure of porcine pancreatic alpha-amylase complexed with acarbose (1OSE) was extracted from the Protein Data Bank (https://www.rcsb.org/, accessed on 15 June 2024). The 3D structures of compounds L1–L3 were optimized using Chem3D Ultra 8.0. The binding energy was determined using the following equation: Binding Energy = Van der Waal’s (V) energy + hydrogen-bonding (H) energy + electrostatic (E) energy. The parameters used during the calculations were as follows: population size: 200; generations: 70; solutions: 3 (drug screening). The 3D and 2D docked poses were displayed via RasMol 2.7.3 (www.rasmol.org, accessed on 15 June 2024) and BIOVIA Discovery Studio Visualizer 2021 v21.1.0.20298 (https://discover.3ds.com/discovery-studio-visualizer-download, accessed on 15 June 2024), respectively.

3. Results and Discussion

3.1. Synthesis

Tetrapodal hybrid molecules L1–L3 were synthesized in three steps, as displayed in Scheme 1 molecules.

The first step was the reduction of ester compound 1 to the compound 2 using LiAlH₄. Thereafter, alcohol 2 was converted to its corresponding chlorinated compound 3. The success of this step was confirmed by carbon 13 NMR. In fact, the ¹³C NMR spectrum of chlorinated derivative 3 shows the appearance of a peak at 39 ppm. This signal is assigned to the carbon attached to the chlorine atom. The final step was the elaboration of tetrapodal ligands L1–L3. This was conducted by reacting two equivalents of compound 3 and one equivalent of the primary amine in the presence of Na₂CO₃ as a base. The structure of target compounds L1–L3 was ensured by spectroscopic and spectrometric analysis.

3.2. Antifungal Screening

The widespread issue of fungal resistance to various anti-infective medications emphasizes the urgent necessity of finding new and powerful antimicrobial agents. In this context, the antifungal potency of L1–L3 compounds was evaluated against some fungal strains. In addition, cycloheximide was used as a positive control, while DMSO was used as a negative one.

The data displayed in

Table 1. Antifungal potency in inhibition diameter (mm) of L1–L3 compounds (NA: no activity).

Compound	Geotrichum candidum	Aspergillus niger	Penicillium digitatum	Rhodotorula glutinis
L1	13 ± 01	14 ± 01	12 ± 01	15 ± 01
L2	14 ± 02	15 ± 01	14 ± 01	14 ± 01
L3	16 ± 00	13 ± 01	14 ± 00	14 ± 02
Cycloheximide	30 ± 02	31 ± 01	30 ± 03	30 ± 02
DMSO	NA	NA	NA	NA

prove that compounds L1–L3 bear some antifungal potencies against the examined strains. Indeed, the diameter of inhibition was found to be in the range of 14–16 mm. On the other hand, no clear correlation between the nature of the lateral arm and antifungal activity was observed. Furthermore, the antifungal potencies of L1–L3 were less than that of the positive control used.

3.3. Alpha-Amylase Inhibition Activity

Tetrapodal compounds L1–L3 were evaluated for their in vitro alpha-amylase inhibition activity. Acarbose and DMSO were employed as positive and negative controls, respectively, in this study. All of the results are regrouped in

Table 2. The IC₅₀ of compounds L1–L3 and their binding energies to the active site of alpha-amylase (NA: no activity).

Compound	Structure	α-Amylase IC50 (mg/mL)	Binding Energy (Kcal/mol)
L1		1.34 × 10−1 ± 1.7 × 10−2	−115.89
L2		6.5 × 10−2 ± 7 × 10−3	−122.30
L3		1.2 × 10−2 ± 8 × 10−3	−123.01
Acarbose		2.6 × 10−1 ± 8 × 10−2
DMSO		NA

.

The nature of the side arm is the only differentiating factor for compounds L1–L3. For tetrapodal compound L3, the presence of a propyl chain as the lateral arm demonstrates potent alpha-amylase activity compared to acarbose. The presence of a hydroxyl group at the extremity of the propyl lateral arm (L1) unexpectedly decreases the alpha-amylase inhibition. Indeed, the presence of this group on a flexible chain may distribute the stability of the enzyme–ligand complex. Finally, adding a phenyl group into the lateral arm of ligand L1 leads to the compound L2 with a less flexible one. This structural modification enhances twice the alpha-amylase inhibition potency of L2 compared to L1. Nevertheless, its alpha-amylase activity still less than that of L3.

3.4. Docking of Compounds L1–L3

A molecular docking study was undertaken to examine the interactions of compounds L1–L3 with porcine pancreatic alpha-amylase. Research indicates that the active site of α-amylase is a V-shaped cavity near its center, defined by three key residues: ASP197, ASP300 and GLU233 [31]. These residues are vital for the enzyme’s function, where GLU233 plays a crucial role as a general acid in the initial phase of the catalytic process [31].

Initially, the docking protocol was validated by redocking acarbose, as described in our recent publication [31]. Then, the same settings were used to dock the tetrapodal compounds. The results are displayed in Table 2 and Figure 1.

The obtained results indicate that all tetrapodal compounds are engaged in multiple interactions with the residues of the active site, albeit in varying ways. Nevertheless, all of the molecules form carbon–hydrogen bonds with the critical residue GLU233. Furthermore, we observed that the alpha-amylase compound complexes are stabilized through numerous interactions with ASP300. This could potentially explain the strong alpha-amylase inhibition activity demonstrated by compounds L1–L3. Additionally, the calculated binding energies were −115.89, −122.30 and −122.97 Kcal/mol for L1, L2 and L3, respectively. These findings align well with the obtained experimental data.

4. Conclusions

During this work, we presented the synthesis of a new generation of tetrapodal compounds L1–L3 bearing two pyrazole–tetrazole units. Their structures, as well as those of the intermediates, were determined by NMR and FTIR spectroscopic methods and spectrometric analyses, such as HRMS and elemental analysis. The evaluation of the antifungal activity of L1–L3 evidenced that they possess enough potency to inhibit the fungal growth of Geotrichum candidum, Aspergillus niger, Penicillium digitatum and Rhodotorula glutinis. Furthermore, the alpha-amylase potencies of these compounds were also examined. The obtained results showed that the L1–L3 compounds have high activity (2–4 times more than that of acarbose used as a positive control). Finally, a docking study was also conducted on the alpha-amylase enzyme. The resulting data were found to be in good agreement with those experimentally obtained.