# Synthesis, Antifungal Evaluation, and Structure-activity Relationship of Chlorinated Imidazopyridinylphenylpropenone Derivatives against Aspergillus fumigatus

Deto Ursul Jean-Paul N'guessan, Songuigama Coulibaly, Avi Tanguy Kouao, Alain A Kacou, Kpongbo Etienne Angora, Fabrice Koffi, Ismaila Diallo, Mahama Ouattara

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Deto Ursul Jean-Paul N'guessan, Songuigama Coulibaly, Avi Tanguy Kouao, Alain A Kacou, Kpongbo Etienne Angora, et al.. Synthesis, Antifungal Evaluation, and Structure-activity Relationship of Chlorinated Imidazopyridinylphenylpropenone Derivatives against Aspergillus fumigatus. Chemical Science International Journal, 2025, 34 (6), pp.138-148. 10.9734/CSJI/2025/v34i61011. hal-05439497

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# Synthesis, Antifungal Evaluation, and Structure–activity Relationship of Chlorinated Imidazopyridinyl-phenylpropenone Derivatives against *Aspergillus fumigatus*

Deto Ursul Jean-Paul N'guessan <sup>a*</sup>, Songuigama Coulibaly <sup>a</sup>, Avi Tanguy Kouao <sup>a</sup>, Alain A. Kacou <sup>a</sup>, Kpongbo Etienne Angora <sup>b,c</sup>, Fabrice Koffi <sup>a</sup>, Ismaila Diallo <sup>a</sup> and Mahama Ouattara <sup>a</sup>

## Authors' contributions

This work was carried out in collaboration among all authors. All authors read and approved the final manuscript.

## Article Information

DOI: https://doi.org/10.9734/CSJI/2025/v34i61011

**Open Peer Review History:**

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/150113

**Original Research Article**

Received: 28/10/2025

Published: 30/12/2025

<footnote><sup>a</sup> Department of Therapeutic Chemistry and Organic Chemistry, UFR Pharmaceutical and Biological, Côte d'Ivoire.</footnote>

<footnote><sup>b</sup> Department of Parasitology and Mycology, UFR Pharmaceutical and Biological Sciences, Felix Houphouët-Boigny University, 01 BP V34 Abidjan, Côte d'Ivoire.</footnote>

<footnote><sup>c</sup> Laboratory of Parasitology of the Angré University Hospital, Abidjan, Côte d'Ivoire.</footnote>

<footnote>*Corresponding author: E-mail: nguessanursul@gmail.com;</footnote>

<footnote>**Cite as:** Deto Ursul Jean-Paul N'guessan, Songuigama Coulibaly, Avi Tanguy Kouao, Alain A. Kacou, Kpongbo Etienne Angora, Fabrice Koffi, Ismaila Diallo, and Mahama Ouattara. 2025. "Synthesis, Antifungal Evaluation, and Structure–activity Relationship of Chlorinated Imidazopyridinyl- Phenylpropenone Derivatives Against Aspergillus Fumigatus". Chemical Science International Journal 34 (6):138–148. https://doi.org/10.9734/CSJI/2025/v34i61011.</footnote>
ABSTRACT

**Background:** *Aspergillus fumigatus* is the leading causative agent of invasive aspergillosis, a deadly opportunistic fungal infection in immunocompromised patients. The increasing emergence of azole-resistant strains necessitates the urgent development of new antifungal molecules.

**Objective:** This study reports the synthesis, structural characterization, and evaluation of the antifungal activity of ten novel chlorinated chalcone-imidazo[1,2-a]pyridine hybrid derivatives against a clinical strain of *A. fumigatus*.

**Methods:** The compounds were synthesized by Claisen-Schmidt condensation between acetylated imidazo[1,2-a]pyridine derivatives and various substituted aromatic aldehydes. Structural characterization was performed by ¹H and ¹³C NMR. Antifungal activity was determined by microplate scattering with calculation of minimum inhibitory concentrations (MICs).

**Results:** Ten compounds were obtained with varying yields (45–83%). Spectral analysis confirmed the formation of α,β-unsaturated structures with *E* geometry. The MICs ranged from 73.16 to 319.74 µM, with optimal activity for the 3-nitro derivative (MIC = 73.16 µM) and the Chlorinated derivatives (MIC = 75.48 µM). The 2-methoxyphenyl derivative (MIC = 76.50 µM) and the unsubstituted compound (MIC = 84.27 µM) show promising activity.

**Conclusion:** Structure-activity relationship studies reveal the importance of lipophilicity and steric hindrance. These chalcone-imidazopyridine hybrids represent promising structures for the development of new antifungal agents against *A. fumigatus*.

**Keywords:** *Aspergillus fumigatus*; chalcone; imidazo[1,2-a]pyridine; antifungal; structure-activity relationship; azole resistance.

1. INTRODUCTION

*Aspergillus fumigatus* is the most formidable fungal pathogen among the species of the genus Aspergillus, responsible for more than 90% of cases of invasive aspergillosis (Latgé, 1999). This opportunistic fungal infection primarily affects immunocompromised patients, particularly those with prolonged neutropenia, organ transplantation, or immunosuppressive therapy (Patterson et al., 2016). With a mortality rate that can reach 50% despite available treatments, invasive aspergillosis represents a major therapeutic challenge (Brown et al., 2012).

The standard treatment relies on azole antifungals, primarily voriconazole and itraconazole, supplemented by amphotericin B and echinocandins (Ullmann et al., 2018). However, the alarming emergence of *A. fumigatus* strains. Azole-resistant *Fumigatus*, linked to the intensive agricultural use of azole fungicides and mutations in the cyp51A gene, severely compromises therapeutic efficacy (Rybak et al., 2019, Burks et al., 2021). This cross-resistance between medical and agricultural antifungals represents an emerging public health problem requiring the urgent development of new classes of antifungals (Fisher et al., 2018).

Chalcones, biosynthetic precursors of flavonoids, constitute a class of natural compounds with a

broad spectrum of biological activities (Zhuang et al., 2017). These α,β-unsaturated ketones have demonstrated promising antifungal properties against various pathogenic fungal species, including *Aspergillus* spp. (Batovska and Todorova, 2010). Their mechanism of action involves the inhibition of ergosterol biosynthesis, disruption of the fungal cell wall, and induction of oxidative stress (López et al., 2001, Song et al., 2025).

Imidazo[1,2-a]pyridine, a fused bicyclic heterocyclic system, is a preferred pharmacophore in medicinal chemistry (Bagdi et al., 2015). This structure is present in many commercially available drugs, including zolpidem (a hypnotic) and alpidem (an anxiolytic), demonstrating its pharmacological versatility (Narayan et al., 2024). Molecular hybridization between chalcones or isoster acrylonitrile and imidazo [1,2-a]pyridine represents a rational strategy for optimizing antifungal activity (Mellado et al., 2020, N'Guessan et al., 2021, N'guessan et al., 2025).

This study presents the synthesis of ten new chalcone-imidazo [1,2-a]pyridine hybrid derivatives, their complete structural characterization by NMR, and the systematic evaluation of their antifungal activity against *A. fumigatus*. A detailed structure-activity relationship analysis is provided to guide the future optimization of these compounds (Li et al., 2025).
## 2. METHODOLOGY

### 2.1 General Chemistry

#### 2.1.1 Materials and reagents

All commercial reagents were used without further purification (Sigma-Aldrich, Merck). Melting points were determined on a Kofler apparatus and are not corrected. Yields reported are for purified products.

#### 2.1.2 Instrumentation

¹H and ¹³C NMR spectra were recorded on a Bruker Avance 300 MHz spectrometer, using CDCl₃ or DMSO-d₆ as deuterated solvents. Chemical shifts (δ) are expressed in ppm relative to tetramethylsilane (TMS) as an internal reference. Coupling constants (J) are expressed in Hz.

## 2.2 Chemical Synthesis

### 2.2.1 Preparation of acetylated precursors

3-Acetyl-6-chloro-2-methylimidazo[1,2-a]pyridine were prepared according to published methods (N'guessan et al., 2025).

To obtain the imidazopyridinyl-arylpropenones (3a-3j), 3-acetyl-2-methyl-6-Chloro-imidazo[1,2-a]pyridine (1) was first synthesized by condensation of 5-Chloro-2-aminopyridine (a)

with 3-chloropentane-2,4-dione (b) under reflux in ethanol, and the solution was then neutralized with ammonium hydroxide (NH₄OH). The compound (1) thus obtained was then subjected to a Claisen–Schmidt condensation, in the presence of base, with a series of substituted aromatic aldehydes (2a–2j), leading to the desired imidazopyridinyl-arylpropenones (Claisen and Claparède, 1881). After neutralization with a 20% hydrochloric acid solution, the products were isolated by precipitation and then purified by recrystallization (Fig. 1).

### 2.2.2 General synthesis of chalcone-imidazopyridine hybrids

To a solution of the acetylated imidazo[1,2-a]pyridine derivative (1 mmol) in anhydrous ethanol (15 mL), the appropriate aromatic aldehyde (1.2 mmol) and a solution of potassium hydroxide (2 mmol) in distilled water (3 mL) are successively added. The reaction mixture is kept under magnetic stirring at room temperature for 24 hours. The reaction progress is monitored by thin-layer chromatography (TLC) using a dichloromethane/methanol eluent mixture (9:1).

After neutralization by the dropwise addition of dilute hydrochloric acid (20%) until the pH is neutral, the resulting precipitate is filtered through sintered glass, washed thoroughly with cold water, and then dried under vacuum. Purification is carried out by recrystallization in hot ethanol (N'guessan et al., 2025, Claisen and Claparède, 1881).

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Synthetic route to 6-Chloroimidazopyridinyl-phenylpropenones (3a–3j). Reagents and conditions: (i) EtOH / NH₄OH; (ii) NaOH, EtOH</figcaption></figure>
## 2.3 Biological Evaluation

### 2.3.1 Chemical and biological materials

#### 2.3.1.1 Chemical materials

All test compounds, synthesized and characterized as pure powders by the Research Unit of Organic Chemistry and Therapeutic Chemistry, were provided for antifungal evaluation. Itraconazole (Sigma Aldrich) was used as the reference antifungal agent. Solvents included analytical grade methanol, dimethyl sulfoxide (DMSO), and distilled water. DMSO served as the primary solvent for stock solutions of poorly water-soluble compounds (Gonzalez-Jimenez et al., 2023, Kathiravan et al., 2012).

#### 2.3.1.2 Fungal strain and culture conditions

Clinical isolates of *Aspergillus fumigatus* 487957, characterized by the Parasitology-Mycology Unit (CHU d'Angré), were used. Culture media included Sabouraud-chloramphenicol agar for strain maintenance and Tryptone Soya Broth (TSB, Oxoid) for susceptibility testing. Methylthiazolyltetrazolium (MTT) was used for viability assessment.

The antifungal susceptibility profile of the clinical *A. fumigatus* 487957 isolate was initially confirmed using a disk diffusion assay on Casitone agar, following standard protocols. This ensured the strain's responsiveness to conventional agents (Clinical and Laboratory Standards Institute, 2017).

**Inoculum Preparation:** The strain was cultured on Sabouraud-chloramphenicol agar at 37°C for 48h. Spores were suspended in sterile water and adjusted to a 0.5 McFarland standard turbidity (~0.4-5 x 10⁶ spores/mL).

**Procedure:** The adjusted inoculum was spread evenly on Casitone agar plates. Commercial antifungal disks (fluconazole, nystatin, 5-fluorocytosine, amphotericin B, econazole) were applied. Plates were incubated at 30°C for 24h, after which inhibition zone diameters were measured.

### 2.3.2 Determination of minimum inhibitory concentrations (MICs)

The minimum inhibitory concentration (MIC) of test compounds and itraconazole was

determined using a broth microdilution method in 96-well plates, adapted from standard guidelines (Clinical and Laboratory Standards Institute, 2017).

**Compound Dilution Range:** Twofold serial dilutions were prepared from 100 µg/mL stock solutions (in DMSO) using TSB as diluent.

**Fungal Inoculum Preparation:** Spores from a 7-day-old *A. fumigatus* culture on Sabouraud-chloramphenicol agar were harvested into TSB. The suspension was vortexed, spore density quantified with a Malassez chamber, and adjusted to 0.5 McFarland standard. A final 1:50 dilution in TSB yielded a working inoculum of ~0.08-1 x 10⁵ CFU/mL.

**Microplate Preparation:** In a sterile 96-well plate, 100 µL of each test compound or itraconazole (initial concentration 10 µg/mL) was added to the first row (A1-A10). Then, 50 µL of TSB was added to wells in rows B-H. A twofold serial dilution was performed by transferring 50 µL from row A to row B, mixing, and repeating down to row H, leaving 50 µL in each well after dilution. Finally, 50 µL of the standardized fungal inoculum was added to all test and growth control wells.

**Evaluation Protocol:** In each well, 100 µL of medium containing the test compound and 100 µL of conidin suspension (10⁴ CFU/mL) were added. Itraconazole was used as the reference antifungal. The microplates were incubated at 37°C for 48 hours. The MIC was defined as the lowest concentration that completely inhibited visible fungal growth.

**Wells included:**

- Test wells: Inoculum + compound (serial concentrations).

- Growth control: Inoculum + TSB (no compound).

- Sterility control: TSB only (no inoculum, no compound).

- Plates were incubated at 37°C for 48 hours.

After incubation, fungal viability was assessed using MTT.

**Procedure:** 40 µL of aqueous MTT solution was added to each well. Plates were incubated at room temperature for 30 minutes.
Visual Reading: Metabolic reduction of MTT by viable fungi produces a purple formazan precipitate. Wells were examined for color and turbidity:

Purple/turbid wells: Indicated fungal growth.
Clear/yellow wells: Indicated growth inhibition.

The MIC was defined as the lowest compound concentration that produced a well visually comparable to the sterility control (clear, no purple color), indicating complete inhibition of metabolic activity.

### 2.3.3 Quality controls

Growth controls (medium + inoculum without antifungal), sterility controls (medium alone), and solvent controls (DMSO at the maximum tested concentration) were included in each series of experiments. Each test was performed in triplicate independently.

## 3. RESULTS AND DISCUSSION

### 3.1 Chemical Synthesis and Yields

A series of ten chalcone-imidazo[1,2-a]pyridine hybrid derivatives was successfully synthesized by Claisen-Schmidt condensation under mild basic conditions (Fig. 1). This method offers the advantage of operational simplicity and compatibility with various aromatic substituents. The yields obtained (45 - 83%) are satisfactory and consistent with those reported in the literature for this type of condensation (Claisen and Claparède, 1881). The observed yield variations can be attributed to the electronic influence of the aromatic substituents on the reactivity of the corresponding aldehydes. Electron-withdrawing groups (nitro) tend to activate the aldehyde, while electron-donating groups (methoxy, hydroxyl) exert a moderate deactivating effect.

Ten compounds were obtained with varying yields (45 – 83%) (Table 1).

### 3.2 Structural Characterization

#### 3.2.1 ¹H NMR spectroscopy analysis

The ¹H NMR spectra unambiguously confirm the formation of the expected chalcone structures. Diagnostic signals include:

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-phenylprop-2-en-1-one (3a)

Pale Green solid, Yield : 68%. Recrystallization in water/ethanol (1:1).

¹H NMR (300 MHz, CDCl₃) δ 9.91 – 9.84 (m, 1H, Ph-4), 7.80 (d, *J* = 15.5 Hz, 1H, CH=CH), 7.63 – 7.54 (m, 3H, Ph-2,6 and CH=CH), 7.41 (dd, *J* = 9.4, 2.0 Hz, 1H, H7), 7.26 (s, 1H, H5), 7.00 – 6.89 (m, 2H, Ph-3,5), 2.89 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 161.7, 151.9, 145.3, 142.9, 130.6 (CH=CH), 130.1, 129.9, 128.8, 127.6, 127.1, 126.6, 122.5, 121.9 (CH=CH), 116.7, 116.5, 114.5, 113.7, 55.4, 52.8, 40.3, 18.5.

Mp: 155-159°C.

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(3-chlorophenyl)prop-2-en-1-one (3b)

Yellow Solid, Yield: 62 %. Recrystallization in water

¹H NMR (300MHz, DMSO-d6) δ: 9.66 (d, 1H, *J* = 4.5 Hz, H5);

7.75 (d, 1H, *J* = 16 Hz, CH=CH); 7.55 - 7.68 (m, 3H, CH₃-Ph-6, H7 and H8); 7.48 (d, 1H, *J* = 16 Hz, CH=CH); 7.15 - 7.44 (m, 4H, CH₃-Ph-3,4,5 and H6); 2.89 (s, 3H, CH₃), 2.33 (s, 3H, CH₃-Ph).

¹³C NMR (75MHz, DMSO-d6) δ: 179.4 (C=O), 152.0, 146.5, 141.5 (CH=CH), 140.9, 132.0, 128.4, 125.4, 124.9, 124.5, 122.5, 121.7 (CH=CH), 120.3, 120.2, 117.1, 114.7, 21.1, 18.1. Mp: 148-152°C.

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(2-hydroxyphenyl)prop-2-en-1-one (3c)

Yellow solid, Yield: 49 %. Recrystallization in water

¹H NMR (300 MHz, CDCl₃) δ 13.21 (s, 1H, OH), 9.42 (s, 1H, H-5), 8.47 (dd, *J* = 2.6, 0.7 Hz, 1H, H-7), 7.75 (dd, *J* = 8.4, 2.6 Hz, 2H, H8, CH=CH), 7.51 (dd, *J* = 7.7, 1.7 Hz, 1H, CH=CH), 7.31 (d, *J* = 0.7 Hz, 1H, Ph-6), 7.08 – 6.94 (m, 3H, PH-3,4,5), 2.82 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 165.3 (C=O), 161.9 (C-OH), 155.9, 147.8, 138.1, 134.2, 133.6 (CH=CH), 130.4, 121.1 (CH=CH), 119.3, 118.9, 117.3.

Mp: 128 - 132 °C;

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(2-methoxyphenyl)prop-2-en-1-one (3d)
Brown solid, Yield: 58%. Recrystallization in water/ethanol (1:1)

¹H NMR (300 MHz, DMSO) δ 9.77 (dd, *J* = 2.0, 0.9 Hz, 1H), 7.94 (d, *J* = 15.7 Hz, 1H), 7.81 – 7.63 (m, 5H), 7.49 – 7.40 (m, 1H), 7.14 (d, *J* = 8.3 Hz, 1H), 7.04 (t, *J* = 7.4 Hz, 1H), 3.93 (d, *J* = 3.2 Hz, 3H), 2.82 (s, 3H).

¹³C NMR (75 MHz, DMSO) δ 179.86 (OCH₃), 137.44 (C=O), 132.0 (CH=CH), 129.9, 129.7, 125.9, 125.08, 121.4 (CH=CH), 120.8, 117.2, 111.8, 55.7, 18.0 (OCH₃).

Mp: 164 -168°C.

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(3-methoxyphenyl)prop-2-en-1-one (3e)

Brown solid, Yield: 64%. Recrystallization from ethanol

¹H NMR (300MHz, DMSO-d6) δ: 9.66 (d, 1H, *J* = 4.5 Hz, H5); 7.75 (d, 1H, *J* = 16 Hz, CH=CH); 7.55 - 7.68 (m, 3H, CH₃-Ph-6, H7 and H8); 7.48 (d, 1H, *J* = 16 Hz, CH=CH); 7.15 - 7.44 (m, 4H, CH₃-Ph-3,4,5 and H6); 2.89 (s, 3H, CH₃), 2.33 (s, 3H, CH₃-Ph).

¹³C NMR (75MHz, DMSO-d6) δ: 179.4 (C=O), 152.0, 146.5, 141.5, 140.9, 132.0, 128.4, 125.4, 124.9, 124.5, 122.5, 121.7 (CH=CH), 120.3, 120.2, 117.1, 114.7, 21.1, 18.1 CH₃.

Mp: 159-163°C.

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (3f)

Green solid, Yield: 58%. recrystallization in Ethyl Acetate/ethanol (1:1)

¹H NMR (300 MHz, DMSO) δ 9.77 (dd, *J* = 2.1, 0.9 Hz, 1H, H7), 7.88 (d, *J* = 15.6 Hz, 1H, CH=CH), 7.80 – 7.54 (m, 4H, H-5, H-8, Ph-3), 7.51 (d, 1H, *J* = 15.8 Hz, CH=CH), 6.72 – 6.58 (m, 2H, Ph-5,6), 3.96 – 3.81 (m, 6H, 2,4-0CH₃), 2.80 (s, 3H, (s, 3H, CH₃).

¹³C NMR (75 MHz, DMSO) δ 179.9 (C=O), 137.8, 131.5, 129.6 (CH=CH), 125.9, 122.4, 117.1 (CH=CH), 115.92, 106.3, 98.5, 55.8, 55.5, 18.0 (CH₃).

Mp: 168 - 172 °C;

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(m-tolyl)prop-2-en-1-one (3g)

Brown solid, yield: 45 %, Recrystallization in water/ethanol (1:1).

¹H NMR (300 MHz, CDCl₃) δ 9.91 - 9.84 (m, 1H, H8), 7.80 (d, *J* = 15.5 Hz, 1H, CH=CH), 7.63 – 7.54 (m, 3H, H7, PH-2,4), 7.41 (dd, *J* = 9.4, 2.0 Hz, 1H, H7), 7.26 (s, 1H, H-5), 7.00 – 6.89 (m, 2H, PH-5,6), 2.90 (s, 3H, CH₃); 2.35 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 180.28 (C=O), 161.7, 151.92, 145.3, 142.9, 130.6, 130.1, 129.9, 128.8 (CH=CH), 127.6, 127.1, 126.6, 122.5, 121.9, 116.7 (CH=CH), 116.5, 114.5, 113.7, 55.4, 52.8, 40.3, 18.5 (CH₃).

Mp: 160 - 164 °C;

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(p-tolyl)prop-2-en-1-one (3h)

Pale yellow solid, yield: 80%. Recrystallization from ethanol

1H NMR (300 MHz, CDCl₃) δ 9.90 (dd, *J* = 2.1, 0.8 Hz, 1H, H-7), 7.83 (d, *J* = 15.5 Hz, 1H, CH=CH), 7.63 – 7.53 (m, 3H, Ph-2,6, H-8), 7.49 – 7.41 (m, 2H, Ph-3,5), 7.26 (d, *J* = 7.9 Hz, 2H, H-5, CH=CH), 2.90 (s, 3H, CH₃), 2.42 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 143.2, 141.1, 132.1, 130.1, 129.8, 128.4, 127.1, 123.2, 122.6, 116.7, 121.5 (CH₃), 18.4 (CH₃).

Mp: 160 - 164 °C;

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(2-nitrophenyl)prop-2-en-1-one (3i)

Brown solid, yield: 53%. Recrystallization in water/ethanol (1:1)

¹H NMR (300 MHz, CDCl₃) δ 9.84 (dd, *J* = 2.1, 0.9 Hz, 1H, H-5), 8.22 – 8.13 (m, 2H, H-8, H-7), 7.92 – 7.70 (d, *J* = 15.5 Hz, 1H, CH=CH), 7.64 (m, 2H, ), 7.69 – 7.62 (m, 2H), 7.58 (s, 1H), 7.51 (dd, *J* = 9.4, 2.1 Hz, 1H), 2.93 (s, 3H), 1.27 (d, *J* = 4.4 Hz, 1H), 0.87 (s, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 179.9 (C=O), 130.8, 128.8 (CH=CH), 124.9, 124.3, 116.9 (CH=CH), MP: >260°C

(E)-1-(6-chloro-2-methylimidazo[1,2-a]pyridin-3-yl)-3-(3-nitrophenyl)prop-2-en-1-one (3j)

Brown solid, 70%. Solid, Recrystallization in water/ethanol (1:1)
¹H NMR (300 MHz, CDCl₃) δ 9.92 (dd, *J* = 2.1, 0.9 Hz, 1H, H-7), 8.37 – 8.27 (m, 2H, H-8, Ph-5), 7.92 – 7.74 (m, 3H, Ph-2,4,6), 7.69 – 7.62 (m, 2H, CH=CH), 7.58 (s, 1H), 7.51 (dd, *J* = 9.4, 2.1 Hz, 1H, CH=CH), 2.93 (s, 3H), 1.27 (d, *J* = 4.4 Hz, 1H), 0.87 (s, 1H).

¹³C NMR (75 MHz, CDCl₃) δ: 180.0 (C=O), 139.9, 130.8, 128.8, 127.2 (CH=CH), 124.9, 124.3, 116.9 (CH=CH), 18.0 (CH₃). MP: 168 -172°C

H-imidazo proton (δ 9.42–10.05 ppm): This signal, usually observed as a broad singlet or a doublet of doublets, corresponds to the proton at position 5 of the imidazopyridine. The significant deshielding results from the electron-withdrawing effect of the adjacent nitrogen and the partial dehydration of the aromatic system.

β-vinyl proton (δ 7.75–7.94 ppm): This signal consistently appears as a doublet with a coupling constant *J* ≈ 15.5–15.7 Hz, characteristic of the E geometry of the C=C double bond. This trans stereochemistry is thermodynamically favored and results from the basic conditions of condensation.

Aromatic protons (δ 6.58–8.60 ppm): The complex signals in this region correspond to the protons of the substituted phenyl and imidazopyridine rings. Analysis of coupling patterns allows for structural assignment.

Methyl and methoxy groups (δ 2.05–3.93 ppm): The signals from the CH₃ groups of imidazopyridine (δ 2.05–2.93 ppm) and the methoxy groups (δ 3.85–3.93 ppm) appear as singlets integrating for 3H.

### 3.2.2 ¹³C NMR spectroscopy analysis

¹³C spectra provide complementary structural information:

Carbonyl (δ 165.24–180.31 ppm): The signal from the carbonyl carbon of the chalcone is a reliable structural marker. Variations in chemical shift reflect the local electronic environment.

Aromatic Quaternary Carbons (δ 130–180 ppm): These signals correspond to bonding carbons and carbons bearing substituents. Their reduced multiplicity in proton decoupling facilitates identification.

Aromatic Carbons CH (δ 106–138 ppm): The signals from carbons bearing a hydrogen atom have normal intensity and confirm the degree of aromatic substitution.

Aliphatic Carbons (δ 17.97–55.84 ppm): Methyl and methoxy groups exhibit characteristic signals in this region.

## 3.3 Evaluation of Antifungal Activity

The results of the antifungal susceptibility testing revealed that the clinical strain of *A. fumigatus* 487957 is resistant to fluconazole, amphotericin B, and 5-fluorocytosine, but susceptible to nystatin and econazole.

### 3.3.1 Minimum inhibitory concentrations

The results of antifungal activity against the clinical strain *A. fumigatus* are presented in Table 2.

### 3.3.2 Structure-activity relationship analysis (SAR)

Systematic analysis of the results reveals several important trends for optimizing antifungal activity:

#### 1. Influence of lipophilicity

The 2-methoxyphenyl derivative exhibits the best activity (MIC = 76.50 µM), suggesting that the introduction of a methoxy group in the ortho position significantly improves activity. This improvement can be attributed to:

- Increased lipophilicity promoting membrane permeation

- Optimization of hydrophobic interactions with the fungal target

- Modulation of the electron density of the conjugated system

#### 2. Effect of polarity

The 2-hydroxyphenyl derivative shows the lowest activity (MIC = 319.74 µM), confirming the negative impact of polar groups. The hydroxyl group can:

- Decrease the lipophilicity necessary for cell penetration

- Alter interactions with lipid membrane components
### 3. Impact of steric hindrance

The 2,4-dimethoxyphenyl compound exhibits intermediate activity (MIC = 140.13 µM), lower than the monomethoxy derivative. This suggests that excessive steric hindrance can compromise interaction with the biological target.

### 4. Influence of substituent position

The derivatives methylated at the meta (160.89 µM) and para (160.89 µM) positions show comparable but lower activities than the orthomethoxy derivative, indicating the importance of substituent position.

### 5. Role of Chlorinated Derivatives

The compounds in the 6-chloro-2-methylimidazopyridine series show interesting potential:

- (Non substituted, 3-chloro derivative, 3-Nitro derivative, 2-methoxyle derivative): promising activity

- (meta derivative compounds): favorable activity profile (MIC = 73.16 – 160.89 µM)

- (2-electro withdraw substituent derivative): low activity

The introduction of chlorine at position 6 of the imidazopyridine appears to favorably modulate the activity, probably by increasing lipophilicity and modifying electronic properties.

### 3.4 Comparison with Reference Antifungals

Itraconazole, the reference azole antifungal, has a significantly higher MIC (<1.11 µM) compared to the tested compounds. This difference in activity can be explained by:

- The extensive pharmacological optimization of itraconazole

- Its high specificity for the fungal target (CYP51A)

- Its optimized bioavailability and pharmacokinetic properties

<table><caption>Table 1. Structures and yields of synthesized compounds</caption><thead><tr><th>Structure</th><th>R</th><th>Yield (%)</th></tr></thead><tbody><tr><td rowspan="10">[Chemical Structure]</td><td>H</td><td>68</td></tr><tr><td>3-Cl</td><td>62</td></tr><tr><td>2-OH</td><td>48</td></tr><tr><td>2-OCH<sub>3</sub></td><td>58</td></tr><tr><td>3-OCH<sub>3</sub></td><td>64</td></tr><tr><td>2,4-OCH<sub>3</sub></td><td>45</td></tr><tr><td>4-CH<sub>3</sub></td><td>74</td></tr><tr><td>3-CH<sub>3</sub></td><td>53</td></tr><tr><td>2-NO<sub>2</sub></td><td>83</td></tr><tr><td>3-NO<sub>2</sub></td><td>71</td></tr></tbody></table>

<table><caption>Table 2. Antifungal Activity of 6-chloro imidazo[1,2-a]pyridinyl-phenylpropenone derivatives against Clinical isolates of Aspergillus fumigatus 487957</caption><thead><tr><th>Structure</th><th>R</th><th>MIC (µM)</th></tr></thead><tbody><tr><td rowspan="10">[Chemical Structure]</td><td>H</td><td>84.27</td></tr><tr><td>3-Cl</td><td>75.48</td></tr><tr><td>2-OH</td><td>319.74</td></tr><tr><td>2-OCH<sub>3</sub></td><td>76.50</td></tr><tr><td>3-OCH<sub>3</sub></td><td>153.00</td></tr><tr><td>2,4-OCH<sub>3</sub></td><td>140.13</td></tr><tr><td>4-CH<sub>3</sub></td><td>160.89</td></tr><tr><td>3-CH<sub>3</sub></td><td>160.89</td></tr><tr><td>2-NO<sub>2</sub></td><td>292.63</td></tr><tr><td>3-NO<sub>2</sub></td><td>73.16</td></tr><tr><td>Itraconazole</td><td></td><td>&lt;1.11</td></tr></tbody></table>
Nevertheless, chalcone-imidazopyridine hybrids
offer several potential advantages:

- A different mechanism of action that can overcome azole resistance

- A customizable chemical structure for optimization

- Potential for the development of new therapeutic classes

4. CONCLUSION

This study demonstrates the antifungal potential
of 6- chloroimidazo[1,2-a]pyridinyl-
phenylpropenone derivatives against Clinical
isolates of Aspergillus fumigatus. The ten
synthesized compounds exhibit varying activities
(MIC: 73.16–319.74 µM), with optimal efficacy for
derivatives bearing lipophilic substituents in the
ortho position, particularly the 2-methoxyphenyl
derivative.

Detailed structure-activity relationship analysis
reveals the critical importance of lipophilicity and
substituent position for antifungal activity. Polar
groups such as hydroxyl exert a detrimental
effect, while methoxy substituents significantly
enhance activity.

Chlorinated derivatives of the series also show
promising potential, suggesting that the
introduction of halogens onto imidazopyridine is
a viable optimization strategy.

Although the MICs obtained remain higher than
those of the reference itraconazole, these results
open up interesting perspectives for the
development of new classes of antifungals. The
increasing emergence of resistance to azoles
makes the exploration of alternative mechanisms
of action, which these chalcone-imidazopyridine
hybrids could offer, crucial.

These compounds thus constitute promising
starting structures for the rational optimization of
antifungal agents active against clinical strain of
A. fumigatus, potentially contributing to the
therapeutic arsenal against resistant invasive
fungal infections.

DISCLAIMER (ARTIFICIAL INTELLIGENCE)

Author(s) hereby declare that NO generative AI
technologies such as Large Language Models
(ChatGPT, COPILOT, etc) and text-to-image
generators have been used during writing or
editing of this manuscript.

ACKNOWLEDGEMENTS

The authors thank Félix Houphouët-Boigny
University for its institutional support and access
to research.

COMPETING INTERESTS

Authors have declared that no competing
interests exist.

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