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. 2026 Sep 16;20:636189. doi: 10.2147/DDDT.S636189

Discovery of a Caffeic Acid Thioamide with Potent Activity against Pathogenic Fungi and Fluconazole Tolerance-Eliminating Effects in Candida albicans

Jiayin Wang 1,*, Qinli Guo 1,*, Nan Chen 2,*, Yuanying Jiang 2, Dazhi Zhang 1,3,✉, Tingjunhong Ni 2,✉
PMCID: PMC13590262  PMID: 42769125

Abstract

Background

The increasing incidence of invasive fungal infections, coupled with the rising threat of antifungal resistance and tolerance, has created an urgent demand for novel antifungal agents with distinct mechanisms of action.

Methods

In this study, a series of caffeic acid amide derivatives (59 compounds) from our in‑house library was systematically screened for in vitro antifungal activity against clinically relevant pathogenic fungi. Structure-activity relationship (SAR) analysis was performed to identify key pharmacophoric features. The lead compound F4 was further characterized via checkerboard microdilution assays, biofilm formation inhibition assays, and spot assays to evaluate its combination effects, antibiofilm potential, and ability to eliminate fluconazole (FLC) tolerance.

Results

SAR studies revealed that the ortho‑dihydroxy moiety and thioamide substitution are critical determinants of antifungal activity. F4 demonstrated potent and broad‑spectrum activity against Cryptococcus neoformans and Candida glabrata (MIC50 = 0.5–16 μg/mL), and moderate activity against drug‑resistant Candida auris (MIC50 = 1–8 μg/mL), outperforming FLC against certain strains. Furthermore, F4 significantly inhibited biofilm formation in a dose‑dependent manner across multiple species, with strain‑dependent potency relative to FLC. Checkerboard assays indicated additive effects when F4 was combined with FLC against C. auris, C. glabrata, and C. neoformans. Importantly, although F4 showed no synergy and lacked direct growth inhibition in YPD medium (MIC > 64 μg/mL) against Candida albicans SC5314, spot assays demonstrated that F4 at concentrations ≥16 μg/mL effectively eliminated FLC tolerance at 37 °C, a temperature‑dependent effect not observed at 30 °C.

Conclusion

Compound F4 emerges as a promising lead for antifungal drug development, distinguished by its broad‑spectrum activity, antibiofilm properties, and unique capacity to eliminate FLC tolerance. These findings warrant further mechanistic and in vivo investigations.

Keywords: caffeic acid amides, structure-activity relationship, antifungal, biofilm inhibition, fluconazole tolerance

Graphical Abstract

Chemical structure of antifungal lead F4 with effects and MIC values for various fungi.

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Introduction

Fungal infections represent a growing global health threat, particularly in immunocompromised populations, where invasive fungal infections (IFIs) are associated with high mortality rates, exceeding 40%.1,2 Despite the availability of antifungal agents such as azoles, polyenes, echinocandins, and flucytosine, their clinical use remains limited by toxicity, drug–drug interactions, suboptimal efficacy, and the emergence of resistance.3–5 Therefore, novel antifungal agents are urgently required.

Fluconazole (FLC) is a synthetic triazole antifungal agent that selectively inhibits fungal cytochrome P-450-dependent 14α-demethylase (Erg11), a key enzyme in the conversion of lanosterol to ergosterol, an essential component of the fungal cell membrane.6 However, the widespread clinical use of FLC has been accompanied by the increasing challenge of drug resistance and tolerance.7,8 Resistance typically arises from point mutations in the target enzyme, overexpression of efflux transporters (eg, Mdr1 and Cdr1), or the formation of protective biofilms. Tolerance, on the other hand, is defined as the ability of some fungal strains to survive and grow slowly at drug concentrations above the minimal inhibitory concentration (MIC), often mediated by adaptive stress responses or metabolic plasticity.9 Notably, tolerance is most frequently observed in Candida albicans at physiological temperature (37 °C), the most prevalent opportunistic fungal pathogen, and has also been documented in the emerging multidrug-resistant species Candida auris.10 These phenomena seriously compromise therapeutic efficacy, particularly in immunocompromised patients, and underscore the pressing need for novel antifungal strategies or combination therapies.

Caffeic acid amides have diverse therapeutic activities. They function as potent antioxidants and cytoprotective agents.11,12 In diabetes management, they act as multi-targeting inhibitors of α-glucosidase and α-amylase while improving insulin signaling.13,14 They also showed neuroprotective effects in diabetic retinopathy models by modulating extracellular signal-regulated kinase (ERK)/ protein kinase B (AKT) signaling.15 Importantly, caffeic acid amides possess significant antifungal properties.16,17 Their enhanced metabolic stability and multitargeting capabilities make them promising candidates for treating complex diseases.18,19

In our previous work, based on the structural modification of berberine, we designed and synthesized a series of caffeic acid amides. Among them, 46 derivatives were evaluated for their synergistic effects in combination with FLC against FLC‑resistant C. albicans, and most of these markedly enhanced the susceptibility of this pathogen to FLC.20 However, previous studies have focused exclusively on FLC-resistant C. albicans, leaving the broad-spectrum antifungal activity unexplored. Therefore, in this study, we screened 59 caffeic acid amide derivatives from our in-house library (24 of which had been included in the 46 compounds reported previously) and systematically evaluated their in vitro antifungal activities against clinically pathogenic fungi to identify lead compounds.

Materials and Methods

Chemicals

Caffeic acid amide derivatives were obtained from our research group.20 All compounds were synthesized starting from commercially supplied caffeic acid, cinnamic acid, (E)-3-(3,4-dimethoxyphenyl)acrylic acid, and a range of substituted amines. Subsequent chemical transformations, such as amide condensation, thionation and demethylation, were performed to afford the final derivatives. The complete synthetic routes and procedures are provided in Scheme S1 and the experimental section of the Supporting Information. Their structures were confirmed by 1H nuclear magnetic resonance (1H NMR, Table S1, Figure S1–S15) and liquid chromatography–mass spectrometry (LC-MS, Figure S16–S53). FLC and other reagents were purchased from Adamas. All compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 6.4 mg/mL. RPMI 1640 medium (Life Technologies Corporation) was used as the experimental culture medium. For the 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT)-menadione solution, XTT was dissolved in phosphate-buffered saline (PBS) at 0.5 g/L, and menadione was prepared in acetone as a 100 mM stock solution. The working solution was freshly prepared by adding 10 μL of menadione stock solution to 100 mL of XTT solution.

Strains and Culture Conditions

C. albicans SC5314, Candida glabrata strain 537, C. glabrata clinical isolates (306582, 200864, 200863, 106667, 202313, 203116, 203531, 202978), Candida krusei 4946, Candida parapsilosis ATCC 22019, Candida tropicalis 2718, Candida auris 922, C. auris clinical isolates (789, 791, 887, 888, and D12), Cryptococcus neoformans H99, C. neoformans clinical isolates (56992, HN17, D2-12, D2-13, 3-5, 47-68, 38-34, 126-5), Cryptococcus gattii clinical strains (①, ②, R265, 292) were obtained from our laboratory’s fungal culture collection (Standard strains were purchased from the Institute of Dermatology, Chinese Academy of Medical Sciences. Clinical isolates were obtained from Shanghai Changzheng Hospital and Shanghai East Hospital). Strains were cryopreserved in PBS containing 25% (v/v) glycerol and stored at −80 °C. Prior to experiments, thawed strains were revived by two serial passages on yeast extract-peptonedextrose (YPD) agar plates at 30 °C. All strains were then cultured overnight in YPD liquid medium at 30 °C in a shaking incubator at 220 rpm until they reached the exponential growth phase before each experiment.

In vitro Susceptibility Test

The minimum inhibitory concentration (MIC) values of the target compounds were determined according to the broth microdilution protocol of the Clinical and Laboratory Standards Institute (M27-A3 and M60).21,22 The final concentration of the fungal suspension in the RPMI 1640 medium was 0.5 × 103-2.5 × 103 CFU/mL. A drug-free growth control (negative control) containing fungal cells and RPMI 1640 medium, and a blank control containing only RPMI 1640 medium, were included in each assay. FLC was used as the positive control. The lowest concentration of a compound that inhibited 50% of the visible fungal growth was designated as the MIC50 value. Compounds exhibiting optimal activities were selected for subsequent in-depth investigations.

Synergistic Antifungal Assay

The synergistic antifungal activity of compound F4 in combination with FLC was evaluated using the checkerboard microbroth dilution method according to CLSI guidelines. Twofold serial dilutions of F4 and FLC were prepared in RPMI 1640 medium in 96-well plates. C. auris 922, C. glabrata 537, and C. neoformans H99 were adjusted to 1 × 103 CFU/mL and added to each well. Plates were incubated at 30 °C for 48–72 h, and optical density at 600 nm (OD600) was measured to determine fungal growth. The fractional inhibitory concentration index (FICI) was calculated as follows: FICI = (MIC50 of F4 in combination) / (MIC50 of F4 alone) + (MIC50 of FLC in combination) / (MIC50 of FLC alone). The interaction was defined by the FICI: synergy, FICI ≤ 0.5; additive, 0.5 < FICI ≤ 1; indifference, 1 < FICI ≤ 4; and antagonism, FICI > 4.

Biofilm Formation Inhibition Assay

C. auris 922, C. glabrata 537, C. neoformans H99 cells (1.0×106 CFU/mL) were suspended in RPMI 1640 medium, and 100 μL of the suspension was added to each well of a 96-well plate. The plate was incubated at 37 °C for 1 h to allow cell adhesion. After incubation, the supernatant was discarded and the wells were washed once with PBS to remove non-adherent cells. Subsequently, 100 μL of RPMI 1640 medium containing various concentrations of FLC or F4 (64, 32, 16, 8, and 0 μg/mL) was added to each well and the plate was further incubated at 37 °C for 24 h. At the end of the incubation, the medium was removed, and the biofilms were washed once with PBS. To assess metabolic activity, 100 μL of XTT-menadione solution was added to each well and incubated at 37 °C in the dark for 3 h. Finally, 70 μL of the supernatant from each well was transferred to a new 96-well plate and absorbance was measured at 490 nm (OD490).

Spot Assay

C. albicans SC5314 suspension was adjusted to a final concentration of 1 × 106 CFU/mL, and 100 μL was uniformly spread onto YPD agar plates containing gradient concentrations of compound F4 (64, 32, 16, 8, and 0 μg/mL). The plates were left undisturbed for 10–15 min until the surface was completely dry. A sterile filter paper disk was placed at the center of each plate, onto which 5 μL of FLC solution at a concentration of 40 mg/mL was added. All plates were incubated upside down at 30 °C or 37 °C for 24–48 h, and fungal growth was observed and recorded accordingly.

Results and Discussion

In vitro Antifungal Activity

Seven series of caffeic acid amide derivatives with structures provided in Figure 1 were evaluated for in vitro antifungal activity against seven common pathogenic fungi, and the results are summarized in Table 1. Structure–activity relationship analysis revealed that the ortho-dihydroxy moiety is essential for antifungal activity. Within the A series, activity was influenced by the amino acid linker. Valine (A6) showed the highest activity against C. glabrata (MIC50 = 2 μg/mL), whereas phenylalanine (A4) and isoleucine (A9) exhibited the highest activity against C. neoformans (MIC50 = 8 μg/mL). The B series exhibited moderate activity, with p-fluoromethyl phenethylamine (B4) being the most potent against C. glabrata (MIC50 = 1 μg/mL), comparable to that of FLC. In contrast, methylation (C series) or truncation (D series) of the ortho-dihydroxy moiety nearly abolished the activity, confirming its critical role. Replacing the amide bond of the C series with a thioamide to yield the E series resulted in weak antifungal activity against C. glabrata, with MIC50 values ranging from 16 μg/mL to >64 μg/mL. Notably, the F series, which bears both ortho-dihydroxy and thioamide moieties, displays broad-spectrum antifungal potential. It showed activity against all tested strains, except C. tropicalis 2718, with MIC50 values ranging from 2 to 32 μg/mL. Among these compounds, F4 was identified as the most potent, exhibiting MIC50 values of 4 μg/mL against C. krusei 4946 and C. neoformans, outperforming FLC (MIC50 = 16 μg/mL against C. krusei and 8 μg/mL against C. neoformans). F4 also exhibited moderate activity against C. albicans SC5314 with an MIC50 of 16 μg/mL. F6 and F7 likewise showed strong activity against C. neoformans, with MIC50 values of 4 μg/mL. The activity within the F series was influenced by the carbon chain length of the amino substituent, with optimal activity at n = 1, as well as by the electronic and hydrophobic properties of para-substituents on the benzylamine moiety, following the trend Br > Cl > F. Finally, in the G series, replacement of the double bond with a cyclopropyl group led to a decrease in antifungal activity, indicating that the double-bond system contributes to antifungal potency.

Figure 1.

Chemical structures of caffeic acid amide derivatives in seven series. The image shows seven series of caffeic acid amide derivatives, designated A-G. Series A (A1 to A9) comprises a caffeic acid core linked to homopiperonylamine through amino acid linkers via amide bonds. Series B (B1 to B7) comprises a caffeic acid core connected to variable R substituents via an amide bond. Series C (C1 to C11) comprises a 3,4-dimethoxycinnamic acid core connected via an amide bond to variously substituted amines. Series D (D1 to D9) comprises a cinnamic acid core connected via an amide bond to variously substituted amines. Series E (E1 to E9) comprises a 3,4-dimethoxycinnamic acid core connected via a thioamide bond to variously substituted amines. Series F (F1 to F8) comprises a caffeic acid core connected via a thioamide bond to variously substituted amines. Series G (G1 to G6) comprises a cyclopropanated caffeic acid core connected via an amide bond to variously substituted amines.

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Chemical structures of caffeic acid amides (series A–G).

Table 1.

In vitro Antifungal Activity of the Target Compounds

Comp. Structures MIC50 (μg/mL)
C. kru
4946		 C. par
22019		 C. tro
2718		 C. gla
537		 C. aur
922		 C. neo
H99		 C. alb
SC5314
A1 Line angle formula of a two benzene ring compound with two HO groups and a N carbonyl N carbonyl linker. >64 >64 >64 4 64 16 >64
A2 2D skeletal formula of a catechol linked to piperazine and an aryl dioxole via carbonyl groups. 64 >64 >64 4 >64 32 >64
A3 Skeletal formula of a dihydroxy benzene linked by amide and alkene to N containing and O containing rings. >64 >64 >64 4 >64 32 >64
A4 Skeletal line angle structure with catechol, two amide carbonyls, pyridine and benzodioxole phenyl. 32 >64 >64 4 64 8 >64
A5 Skeletal formula of a two benzene ring molecule with two amide links and two HO labels. >64 >64 >64 4 >64 32 >64
A6 Two dimensional skeletal formula with two benzene rings, two HO groups, amide linker and one wedge bond. >64 >64 >64 2 >64 32 >64
A7 Two-dimensional skeletal formula of a dihydroxy benzene linked by amides to a fused O O ring. >64 >64 >64 4 >64 16 >64
A8 2D skeletal structure with two benzene rings, ester, two amides and a sulfur substituent. >64 >64 >64 8 >64 16 >64
A9 Line angle structure with dihydroxy benzene, two amide nitrogens and a fused O ring on the right. >64 >64 >64 4 >64 8 >64
B1 Line angle formula of two hydroxylated benzene rings linked by a C double bond O and N H group. >64 >64 >64 32 >64 >64 >64
B2 Skeletal formula of a diaryl amide with two benzene rings, two HO groups and one F substituent. 64 64 >64 4 >64 32 >64
B3 Skeletal formula of a dihydroxybenzene to amide to ethylbenzene chain. 64 >64 >64 4 >64 64 >64
B4 A compound with two benzene rings, hydroxyl groups and an amide linkage. 32 64 >64 1 >64 32 >64
B5 A compound with two benzene rings, one with two hydroxyl groups. 64 64 >64 2 >64 32 64
B6 A compound with a benzene ring, amide linkage and thiophene ring. >64 >64 >64 8 >64 64 >64
B7 A compound with a benzene ring linked to a five-membered ring via an amide bond. 64 >64 >64 8 64 32 >64
C1 Line bond formula of a dimethoxyphenyl enone amide linked to a chloro substituted benzene ring. >64 >64 >64 >64 >64 >64 >64
C2 Skeletal formula of a two benzene ring amide with alkene linker, two methoxy groups and one fluorine. >64 >64 >64 >64 >64 >64 >64
C3 2D skeletal formula of a bis benzene amide with methoxy groups, an alkene and a methyl group. >64 >64 >64 >64 >64 >64 >64
C4 Line angle formula of a benzene benzene amide with methylenedioxy and methoxy substituents. >64 >64 >64 >64 >64 >64 >64
C5 Skeletal formula of a dimethoxy benzene linked by CH2 CH2 C double bond O NH to benzene. >64 >64 >64 >64 >64 >64 >64
C6 2D skeletal formula of dimethoxybenzoyl amide linked to a bromophenyl ring. >64 >64 >64 >64 >64 >64 >64
C7 A compound with two benzene rings, one with methoxy groups, linked by an amide bond. >64 >64 >64 >64 >64 >64 >64
C8 2D skeletal diaryl amide with a dimethoxy benzene ring linked to a chloro benzene ring. >64 >64 >64 >64 >64 >64 >64
C9 Line angle formula of a dimethoxy benzene amide linked to a bromo substituted benzene. >64 >64 64 >64 >64 >64 >64
C10 Line angle formula of a two benzene ring amide with methoxy and fluorine substituents. >64 >64 >64 >64 >64 >64 >64
C11 2D skeletal formula of a dimethoxyphenyl to amide to ethylphenyl compound. >64 >64 >64 >64 >64 >64 >64
D1 Skeletal formula of phenyl CH double bond CH C double bond O NH tolyl amide. >64 >64 >64 >64 >64 >64 >64
D2 A compound with two benzene rings connected by a carbonyl and amine group. >64 >64 >64 >64 >64 >64 >64
D3 A compound with two benzene rings linked by a C O N chain. >64 >64 >64 >64 >64 >64 >64
D4 Line angle structure of an amide linking an alkene phenyl chain to an ethyl phenyl chain. >64 32 >64 >64 >64 >64 >64
D5 Line angle formula of an amide linking two benzene rings, with an alkene and a chlorine substituent. >64 >64 >64 >64 >64 >64 >64
D6 Skeletal formula of phenyl alkene amide linked to bromobenzene, name not provided. >64 >64 >64 >64 >64 >64 >64
D7 Skeletal formula of phenyl C H double bond C H C double bond O N H C H subscript 2 C H subscript 2 phenyl. >64 32 >64 >64 >64 >64 >64
D8 A compound with two benzene rings connected by an amide group and a bromine substituent. >64 >64 >64 >64 >64 >64 >64
D9 Line angle formula of an alkene amide linking two benzene rings, with two methoxy groups on one ring. >64 >64 >64 >64 >64 >64 >64
E1 2D formula: two benzene rings, C=C bond, thioamide, chloro group. >64 >64 >64 >64 >64 32 >64
E2 Line angle structure of a dimethoxyphenyl alkenyl thioether NH fluorophenyl compound. >64 >64 >64 >64 >64 >64 >64
E3 Two dimensional skeletal structure with two benzene rings, enone linker, C double bond S, NH, methoxy, methyl. >64 >64 >64 16 >64 32 >64
E4 Line angle formula of a dimethoxy benzene linked by an alkene to C double bond S and N H to benzene. 64 >64 >64 32 >64 64 >64
E5 Skeletal formula of a methoxy benzene to bromo benzene S double bond N linked alkene chain. 64 >64 >64 32 >64 >64 >64
E6 Two dimensional skeletal structure with dimethoxy benzene, alkene chain, C double bond S, N H and fluorobenzene. >64 >64 >64 64 >64 >64 >64
E7 A compound with two benzene rings linked by a sulfur and nitrogen chain. >64 >64 >64 64 64 >64 >64
E8 Line angle structure of a diaryl chain with two methoxy groups, a C double bond S, NH and fluoro. >64 >64 >64 >64 >64 64 >64
E9 Skeletal structure of benzodioxole linked by C double bond C and C double bond S to NH and a phenethyl benzene. 32 >64 >64 32 >64 64 >64
F1 Skeletal formula of a two benzene ring molecule with HO groups, C double bond C chain, S and N and Cl. 8 16 64 4 4 8 8
F2 Two benzene rings joined through an S to N connecting chain. 8 16 64 4 16 16 16
F3 Line angle formula of a para dihydroxy benzene linked by carbon carbon double bond to S and N H to benzene. 8 16 64 2 16 8 16
F4 A pair of benzene rings connected by an S to N linking chain. 4 8 >64 2 2 4 16
F5 A compound with two benzene rings linked by a sulfur-nitrogen chain. 4 16 64 8 8 8 32
F6 2D skeletal formula of dihydroxybenzene linked via S and ethyl NH to chlorobenzene. 4 8 >64 2 4 4 16
F7 A compound with two benzene rings, one with hydroxyls, linked by a sulfur and nitrogen chain. 4 8 >64 4 8 4 16
F8 Two phenyl rings joined through a sulfur to nitrogen linker. 8 16 >64 2 8 8 16
G1 Line angle formula of catechol linked via epoxide chain to amide and phenyl ring. 64 >64 >64 4 >64 16 >64
G2 Line angle formula of a dihydroxyphenethyl amide linking two benzene rings. 32 >64 >64 2 32 8 32
G3 A compound with two benzene rings, one with hydroxyl groups, linked by an amide bond. 32 >64 >64 4 >64 16 64
G4 Line angle formula of a para dihydroxy benzene linked by C double bond O and N H to benzene. 32 32 >64 4 64 16 64
G5 A compound with two benzene rings linked by a carbonyl and nitrogen group. 16 64 >64 2 64 8 64
G6 A compound with two benzene rings, carbonyl and amine linkers. 32 >64 >64 2 >64 16 64
FLC – 16 1 ≤0.125 1 0.25 8 0.25
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Abbreviations: Comp., compound; MIC, minimum inhibitory concentration; FLC, fluconazole; C. kru, Candida krusei; C. par, Candida parasilosis; C. tro, Candida tropicalis; C. gla, Candida glabrata; C. aur, Candida auris; C. neo, Cryptococcus neoformans; C. alb, Candida albicans.

Collectively, these results highlight the ortho-dihydroxy moiety and sulfur substitution as key structural features that contribute to the enhanced antifungal activity of caffeic acid amides.

Lead Compounds against Clinical Isolates of Fungi

Based on the above SAR analysis and MIC50 results, the optimal compounds, F4, F6, and F8, were selected for further evaluation of their in vitro antifungal activities against clinical fungal pathogens, including eight isolates of C. glabrata, five isolates of C. auris, eight isolates of C. neoformans, and four isolates of C. gattii. MIC50 data are shown in Table 2. These three compounds generally exhibited comparable antifungal activity. However, F4 showed slightly higher potency, with MIC50 values of 0.5 μg/mL against C. glabrata 202313 and 4 μg/mL against C. neoformans 3-5, superior to those of FLC. Additionally, they showed moderate activity against drug-resistant C. auris (MIC50 range: 1–32 μg/mL), whereas FLC exhibited MIC50 values of >64 μg/mL. Collectively, F4 emerged as a potent antifungal lead, exhibiting high activity against Candida and Cryptococcus species, with notable potency against drug-resistant C. auris.

Table 2.

In vitro Antifungal Activity of the Lead Compounds

Strains MIC50 (µg/mL)
Two phenyl rings bridged by a sulfur to nitrogen linkage.F4 A compound with two benzene rings connected by a sulfur and nitrogen linker.F6 A compound with two benzene rings connected by a sulfur-nitrogen chain.F8 FLC
C. gla 306582 2 2 4 2
C. gla 200864 2 2 4 >64
C. gla 200863 2 2 4 4
C. gla 106667 2 2 4 8
C. gla 202313 0.5 1 2 1
C. gla 203116 2 2 4 8
C. gla 203531 2 2 4 4
C. gla 202978 4 4 8 16
C. aur 789 8 16 16 >64
C. aur 791 4 32 16 >64
C. aur 887 1 8 8 >64
C. aur 888 4 16 16 >64
C. aur D12 8 16 16 >64
C. neo 56992 8 8 16 1
C. neo HN17 8 8 8 4
C. neo D2-12 8 4 8 8
C. neo D2-13 8 8 8 4
C. neo 3-5 4 8 8 8
C. neo 47-68 16 8 16 2
C. neo 38-34 8 8 8 2
C. neo 126-5 16 16 16 2
C. gat ① 16 16 16 2
C. gat ② 8 8 16 16
C. gat R265 16 8 16 4
C. gat 292 16 16 16 8
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Abbreviations: MIC, minimum inhibitory concentration; FLC, fluconazole; C. gla, Candida glabrata; C. aur, Candida auris; C. neo, Cryptococcus neoformans; C. gat, Cryptococcus gattii.

Combination Antifungal Activity of Lead Compound F4

Having established the potent antifungal activity of F4, we evaluated its effects in combination with FLC against C. albicans SC5314, C. auris 922, C. glabrata 537, and C. neoformans H99 using a checkerboard microdilution assay (Figure 2A–D). Specifically, for C. albicans SC5314 (Figure 2A), the checkerboard microdilution assay did not reveal any significant synergistic effect, which is different from our previous work on FLC-resistant C. albicans 103. For C. auris 922 (Figure 2B), the combination of F4 and FLC resulted in MIC50 values of 0.5 μg/mL for F4 and 0.25 μg/mL for FLC, with a FICI value of 0.625. For C. glabrata 537 (Figure 2C), the MIC50 values were 0.25 μg/mL for both F4 and FLC, with a FICI of 0.75. For C. neoformans H99 (Figure 2D), the MIC50 values were 1 μg/mL for F4 and 2 μg/mL for FLC, yielding an FICI value of 0.625. These results indicate that F4 exhibits additive effects against C. auris 922, C. glabrata 537, and C. neoformans H99, whereas no synergy was observed against C. albicans SC5314.

Figure 2.

Four heatmaps of optical density 600 for F4 and fluconazole checkerboards labeled A, B, C, D. Image A: Heatmap with optical density 600 gradient. Horizontal axis: F4 (microg/mL) with ticks 64 to 0. Vertical axis: fluconazole (microg/mL) with ticks 2 to 0. Darkest cells at low fluconazole, especially F4 4 to 0.25. Image B: Similar heatmap. Horizontal axis: F4 (microg/mL) with ticks 16 to 0. Vertical axis: fluconazole (microg/mL) with ticks 4 to 0. Broad high density at low fluconazole, especially 0.0625 and 0.125, spanning F4 0.5 to 0.0625. Image C: Similar heatmap. Horizontal axis: F4 (microg/mL) with ticks 64 to 0. Vertical axis: fluconazole (microg/mL) with ticks 4 to 0. Maximum colour density is observed at low F4 concentrations of 0.5 to 0.25 and fluconazole concentrations of 0.0625 to 0.25. Image D: Similar heatmap. Horizontal axis: F4 (microg/mL) with ticks 16 to 0. Vertical axis: fluconazole (microg/mL) with ticks 32 to 0. High density block at F4 1 to 0.0625 across fluconazole 0.5 to 8. No numeric values printed.

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Checkerboard microdilution assay of compound F4 combined with FLC against indicated fungal strains. (A) Candida albicans SC5314. (B) Candida auris 922. (C) Candida glabrata 537. (D) Cryptococcus neoformans H99. FLC, fluconazole.

Inhibitory Effects of F4 against Biofilm Formation

In addition to evaluating the combination activity, we also investigated the ability of F4 to inhibit biofilm formation by C. albicans SC5314, C. auris 922, C. glabrata 537, and C. neoformans H99. The XTT reduction assay (Figure 3A–D) showed that F4 significantly reduced biofilm formation in the tested strains in a dose-dependent manner. For C. albicans SC5314 (Figure 3A), compound F4 was less potent than FLC at 8 μg/mL, but showed comparable activity at 64 μg/mL. For C. auris 922 (Figure 3B), the antibiofilm activity of F4 was comparable to that of FLC. For C. glabrata 537 (Figure 3C), F4 exhibited significantly superior activity compared to FLC for C. glabrata 537. However, for C. neoformans H99 (Figure 3D), FLC showed significantly higher activity than F4. These results indicate that the antibiofilm activity of F4 is strain-dependent.

Figure 3.

Grouped bar charts showing biofilm formation for F4 and fluconazole across four strains and concentrations. Image A: Bar chart with x-axis labeled Concentration (microg/mL) and y-axis labeled Biofilm formation (percent), ranging 0-120. Legend: F4 and FLC. At 0 microg/mL, both treatments are near 100 percent. From 8 to 64 microg/mL, both decrease, F4 generally higher than FLC except at 64 microg/mL where they are similar. Image B: Similar setup. F4 decreases from just over 100 percent at 0 to 40 percent at 64 microg/mL. FLC drops from mid-90 percent at 0 to 60 percent at 64 microg/mL. F4 is higher at 8 and 16 microg/mL, lower at 32 and 64 microg/mL compared to FLC. Image C: F4 decreases from 100 percent at 0 to 50 percent at 64 microg/mL. FLC remains high, 90 to 100 percent across all concentrations. F4 is consistently lower than FLC at all concentrations. Image D: F4 is 95-100 percent at 0 and 8 microg/mL, 85-90 percent at 16 microg/mL, 95 percent at 32 microg/mL, dropping to 45 percent at 64 microg/mL. FLC starts at 105 percent at 0 microg/mL, then drops to 30 percent at 8 and 16 microg/mL, 20 percent at 32 microg/mL and 10 percent at 64 microg/mL. From 8 to 64 microg/mL, F4 is higher than FLC.

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Inhibitory effects of F4 on biofilm formation. (A) Candida albicans SC5314. (B) Candida auris 922. (C) Candida glabrata 537. (D) Cryptococcus neoformans H99. FLC, fluconazole.

F4 Eliminates Fluconazole Tolerance in C. albicans SC5314

Given that compound F4 exhibited no synergistic effect against C. albicans SC5314 and was inactive in YPD medium (Table S2, MIC > 64 μg/mL), we sought to further explore its potential via a spot assay to determine whether F4 could affect FLC tolerance in this strain. Antifungal tolerance is a distinct phenomenon from resistance. Notably, C. albicans SC5314 displays temperature‑enhanced tolerance at 37 °C but not at 30 °C. To assess this, we prepared YPD agar plates containing serial dilutions of F4, while FLC was applied as disks (40 mg/mL, 5 μL per disk) at a fixed concentration on all plates. The plates were photographed after 24 and 48 h, and the inhibition zone diameters are presented in Figure 4. At low F4 concentrations (8 and 16 μg/mL), the inhibition zones at 37 °C were consistently smaller than those at 30 °C. However, at higher concentrations (32 and 64 μg/mL), the zone sizes did not differ significantly between the two temperatures. At 30 °C, no clear dose‑dependent trend was observed at either time point, with the exception that F4 at 64 μg/mL produced slightly larger zones than the other concentrations. In striking contrast, at 37 °C, a robust dose‑dependent increase in inhibition zones was evident. Moreover, at F4 concentrations ≥16 μg/mL, the zones in F4‑treated groups were significantly larger than those in the control group (without F4). Notably, at the 48 h time point, the control group exhibited pronounced FLC tolerance, whereas the inclusion of F4 at 16 μg/mL or higher effectively eliminated this tolerance. Collectively, although F4 lacked synergy with FLC against C. albicans SC5314 and showed no direct growth inhibition in YPD medium, it potently eliminated FLC tolerance at concentrations ≥16 μg/mL.

Figure 4.

Microbiology figure: Parts A & B, each with 10 agar plate photos, labeled by temperature, time, concentration.

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Spot assay of F4 eliminating FLC tolerance in C. albicans SC5314. (A) Spot assay results after 24 h of incubation. (B) Spot assay results after 48 h of incubation.

Conclusion

In conclusion, this study systematically evaluated the antifungal activity of caffeic acid amide derivatives and elucidated their key structure-activity relationships. The ortho-dihydroxy moiety on the caffeic acid amide scaffold was identified as the core pharmacophore, with additional contributions from the thioamide substitution. Among the series, F series exhibited the most potent and broad-spectrum antifungal activity, with F4 emerging as the lead compound. F4 demonstrated potent activity against C. neoformans, C. glabrata, and drug-resistant C. auris; it significantly inhibited biofilm formation in a dose-dependent manner; and it showed additive effects when combined with FLC. Importantly, F4 at concentrations ≥ 16 μg/mL eliminated FLC tolerance in C. albicans SC5314 at body temperature (37 °C), a property not observed at 30 °C, suggesting temperature-dependent activity. This finding distinguishes F4 from previously reported caffeic acid amide derivatives and highlights its potential for treating infections in which FLC tolerance poses a clinical challenge. Future studies should focus on the mechanistic basis of tolerance elimination and in vivo efficacy.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 22377092, 82473973, and 82173786) and the National Key Research and Development Program of China (No. 2022YFC2303000).

Disclosure

The author(s) report no conflicts of interest in this work.

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