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Article

Design, Synthesis, and Biological Evaluation of N′-Phenylhydrazides as Potential Antifungal Agents

by
Panpan Zhu
1,2,†,
Jinshuo Zheng
1,†,
Jin Yan
1,
Zhaoxia Li
1,
Xinyi Li
1 and
Huiling Geng
1,2,*
1
College of Chemistry & Pharmacy, Northwest A & F University, Yangling 712100, China
2
Key Laboratory of Botanical Pesticide R & D in Shaanxi Province, Northwest A & F University, Yangling 712100, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(20), 15120; https://doi.org/10.3390/ijms242015120
Submission received: 11 August 2023 / Revised: 19 September 2023 / Accepted: 10 October 2023 / Published: 12 October 2023
(This article belongs to the Section Biochemistry)
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Abstract

:
Fifty-two kinds of N′-phenylhydrazides were successfully designed and synthesized. Their antifungal activity in vitro against five strains of C. albicans (Candida albicans) was evaluated. All prepared compounds showed varying degrees of antifungal activity against C. albicans and their MIC80 (the concentration of tested compounds when their inhibition rate was at 80%), TAI (total activity index), and TSI (total susceptibility index) were calculated. The inhibitory activities of 27/52 compounds against fluconazole-resistant fungi C. albicans 4395 and 5272 were much better than those of fluconazole. The MIC80 values of 14/52 compounds against fluconazole-resistant fungus C. albicans 5122 were less than 4 μg/mL, so it was the most sensitive fungus (TSIB = 12.0). A11 showed the best inhibitory activity against C. albicans SC5314, 4395, and 5272 (MIC80 = 1.9, 4.0, and 3.7 μg/mL). The antifungal activities of B14 and D5 against four strains of fluconazole-resistant fungi were better than those of fluconazole. The TAI values of A11 (2.71), B14 (2.13), and D5 (2.25) are the highest. Further exploration of antifungal mechanisms revealed that the fungus treated with compound A11 produced free radicals and reactive oxygen species, and their mycelium morphology was damaged. In conclusion, the N′-phenylhydrazide scaffold showed potential in the development of antifungal lead compounds. Among them, A11, B14, and D5 demonstrated particularly promising antifungal activity and held potential as novel antifungal agents.

1. Introduction

The World Health Organization has reported the two worst threats to mankind’s health, that is, the continuous increase of antimicrobial resistance for commercial antimicrobials against fungal infections and the decreasing availability of effective therapies [1]. Fungal infections in humans are mainly caused by Candida, Aspergillus, and Cryptococcus [2]. However, Candida albicans (C. albicans), an adaptable microorganism, can develop resistance to antifungal agents after prolonged exposure to them. It is noteworthy that the emergence of drug-resistant fungi, such as Candida auris and Candida glabrata, further threatens the limited antifungal drugs [3,4]. As one of the four most common pathogens, C. albicans could cause blood infections [5,6]. For instance, the monitoring results of invasive candidiasis in China show that C. albicans accounts for 44.9% among a total of 32 Candida species [7]. C. albicans is a dimorphic fungus that can grow in both yeast and hyphal forms [8]. In infection, its yeast form is more likely to disseminate in the blood, while the hyphal form penetrates tissues through the production of a fungal biofilm, evades phagocytic attack, and adheres to organs [9]. It is estimated that half of adults are infected with C. albicans in their oral and gastrointestinal tracts [10]. However, under conditions of a compromised immune system, it can overgrow and cause candidiasis [11]. Currently, inhibiting the transition between the two morphologies with drugs is an important way to reduce the risk of C. albicans infection [12]. Candida infection has emerged as a significant contributor to mortality among patients with malignant tumors, HIV, and organ transplants, which has resulted in severe consequences for the health of individuals worldwide [13]. It is crucial to address fungal infections in the treatment of patients who have weakened immune systems [14]. According to the literature, developing new antifungal agents and thoroughly understanding the mechanisms of antifungal drug resistance and the use of combination therapy between conventional and repurposed drugs are the current strategies for combating the emerging threat of antifungal resistance in Candida [4,15].
Nowadays, various antifungal agents have been developed to cure candidiasis, which can be mainly categorized into four series, including azoles, polyenes, echinocandins, and pyrimidines [16]. Among them, fluconazole (FLC) and caspofungin are the most used clinical drugs [17,18]. However, some strains of fungi have displayed resistance to these drugs, which results in treatment failure [19,20]. Additionally, these antifungal agents may cause side effects such as headache, fever, and liver damage [21], highlighting the urgent need for a new generation of antifungal agents [22].
It is reported that hydrazides exhibit various pharmacological activities, such as inhibiting lipoxygenase, antiviral, antioxidant, and anticancer effects, as well as high inhibitory activity against phytopathogenic fungi and insects. Compounds 1–10 are hydrazides with these biological activities (Figure 1). When both substituents on the sides of hydrazine were phenyl rings, the corresponding compounds 1 and 2 exhibited inhibitory activity against lipoxygenase and adult anopheles gambiae, respectively. Compound 1 displayed a high level of inhibition against lipoxygenase, with a potency of up to 93% observed at a concentration of 10 μM [23]. The LD50 value of compound 2 was 220 ng/insect against adult anopheles gambiae [24]. Compounds 3 and 4, with the carbonyl side substituent of hydrazide being a benzene ring and the hydrazide side substituent being an alkyl or benzyl group, showed antiviral activity and reactivity, respectively. Compound 3 held potent antiviral activity against the histone deacetylase (HDAC) and its IC50 values against HDAC1, HDAC2, and HDAC3 were 0.5, 0.1, and 0.06 μM, respectively [25]. Benmoxin (compound 4) showed modulative reactivity towards Cu (II)–amyloid β and free radicals. Furthermore, its ability to mitigate free radicals was found to be similar to that of trolox, which is a vitamin E analyte [26]. When the carbonyl side substituent of hydrazide is heterocyclic and the hydrazine side substituent is benzene rings or benzyl groups, the corresponding compounds 5 and 6 present inhibitory activity against phytopathogenic fungi and cancer cells. Compound 5 could efficiently inhibit the growth of Gibberella zeae, Fusium oxporum, Colletotrichum higgensianum, and Score-tiorum, and their inhibitory rates were 98%, 91%, 100%, and 99%, respectively, at a concentration of 25 μg/mL [27]. Compound 6 could effectively block the T-47D in the G2/M phase of the cell cycle [28]. When the hydrazine side is substituted with a benzyl group and the carbonyl side is substituted with benzyl ether or adamantyl, the corresponding compounds 7, 8, and 9 all held antifungal activity against C. albicans. Compound 7 demonstrated a minimum inhibitory concentration range of 0.0156–0.125 mg/mL against three strains including C. albicans, C. glabrata, and C. tropicalis [29]. Compounds 8 and 9 also exhibited potent antifungal activity against C. albicans, displaying MIC values of 12.5 μM [30]. Isocarboxazid (compound 10) with benzyl and heterocyclic substituent on the hydrazine and carbonyl side, respectively, had irreversible inhibitory effects on monoamine oxidase [31]. To the best of our knowledge, there is no report about the inhibitory activity of N′-phenylhydrazides against C. albicans.
In this study, N′-phenylhydrazides were designed, synthesized, and structurally characterized. The antifungal activity against five strains of C. albicans was assessed via the broth microdilution (BMD) method, and initial structure–activity relationships (SARs) were established. To probe the antifungal mechanism, investigations were carried out involving free radical scavenging, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM).

2. Results and Discussion

2.1. Design and Synthesis of N′-Phenylhydrazides

The synthetic routes of target compounds are shown in Figure 2. Substituted benzoyl chloride was synthesized by the acylation of substituted benzoic acid with SOCl2. And a substitution reaction occurred between it and phenylhydrazine to gain series A compounds. At 0 °C, various substituted phenylhydrazine hydrochlorides were achieved through the diazotization reactions. Then, series B compounds were obtained via the condensation reaction between phenylhydrazine hydrochloride and benzoic acid under EDCI and HOBt. Series C and D compounds were prepared by the condensation reaction of different kinds of carboxylic acids with phenylhydrazine hydrochloride [27,32].
A total of fifty-two kinds of N′-phenylhydrazides belonging to four different series were synthesized, with yields up to 96%. The structures of target N′-phenylhydrazides were identified by HRMS, 1H NMR, and 13C NMR spectra. It was discovered that three of them (D2, D4, and D5) were unknown. The separated yields, melting points, and the data of HRMS, 1H NMR, and 13C NMR spectra of N′-phenylhydrazides are illustrated in the Supporting Information.

2.2. Evaluation of Antifungal Activity In Vitro

The in vitro antifungal activities of target candidates against five strains of C. albicans were screened at the concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 μg/mL through the BMD method [33,34]. FLC was selected as the positive control. Based on the data of antifungal activity, their MIC80 (the concentration of tested compounds when their inhibition rate was at 80%) and TAI (total activity index) values were calculated according to the formulas MIC80 = 10 log ⁡ A + log ⁡ 1 N × a − 80 a − b and TAI = ∑ 1 n 1 / M I C 80 , respectively. The TSI value (total susceptibility index) of every strain of fungus was evaluated as well [35]. By analyzing MIC80 and TAI values, the preliminary SARs were concluded. According to the standards of the Clinical Laboratory Standards Institute (CLSI), when MIC80 values are distributed in ≤4 μg/mL, 8–16 μg/mL, and ≥32 μg/mL, the corresponding compounds are classified as susceptible, intermediate, and resistant candidates, respectively [33]. To evaluate the potency of the compounds with greater inhibitory activity, time–inhibition rate curves were depicted for seven compounds (A1, A2, A8, A11, A12, B14, and C8) and their respective half-inhibitory times (IT50) were calculated.

2.2.1. Antifungal Activity of Target Compounds

A total of fifty-two kinds of compounds were synthesized, their antifungal activities in vitro were screened at a concentration in the range of 0.125–64 μg/mL against five strains of fungi, and their MIC80, MFC (minimal fungicidal concentration), and TAI (total activity index) values were calculated (Table 1).
All target compounds displayed definite inhibitory activities against the five tested fungi. In terms of series A compounds, A11 held the best inhibitory activity against C. albicans SC5314, 4395, and 5272, and the corresponding MIC80 values were 1.9, 4.0, and 3.7μg/mL, respectively. Thus, A11 had the highest TAI value (2.71). A8 (MIC80 = 0.7 μg/mL) showed the best inhibitory activity against C. albicans 5122. For the C. albicans 5172, A16 (MIC80 = 5.8 μg/mL) displayed the best inhibitory activity against it.
As far as series B compounds were concerned, B3 (MIC80 = 6.9 μg/mL) held the most effective antifungal activity against C. albicans SC5314. B14 had the most potent inhibitory activity against C. albicans 4395 and 5172 and the corresponding MIC80 values were 4.0 and 3.3 μg/mL, respectively. It also had the highest TAI value (2.13). For the C. albicans 5122, B3 (MIC80 = 0.7 μg/mL) displayed the best inhibitory activity against it. B5 (MIC80 = 6.3 μg/mL) showed the best inhibitory activity against C. albicans 5272.
As for series C compounds, C2 had the best inhibitory activity against C. albicans SC5314 and 5122 and the corresponding MIC80 values were 3.4 and 1.7 μg/mL, respectively. For the C. albicans 4395 and 5272, C4 displayed the best inhibitory activity against them and the corresponding MIC80 values were 8.9 and 3.8 μg/mL, respectively. C4 had the highest TAI value (1.85). C9 (MIC80 = 1.4 μg/mL) showed the most powerfully antifungal activity against C. albicans 5172.
With respect to series D compounds, D2 exhibited the most potent antifungal activity against C. albicans SC5314 and 4395 with MIC80 values of 12.2 and 5.5 μg/mL, respectively. As far as the C. albicans 5122, 5172, and 5272 were concerned, D5 displayed the best inhibitory activity against them and the corresponding MIC80 values were 2.2, 2.7, and 2.4 μg/mL, respectively. Hence, D5 had the highest TAI value (2.25).

2.2.2. The Inhibitory Efficiency of Compounds with Better Antifungal Activity

In order to evaluate the inhibitory potential of the compounds exhibiting better antifungal activity, time–inhibition rate curves were depicted for seven compounds (A1, A2, A8, A11, A12, B14, and C8) at a concentration of 3.2 μg/mL against C. albicans SC5314, and their half-inhibitory times (IT50) were calculated (Figure 3 and Figure 4) [33,34].
The time–inhibition rate curve of FLC showed a relatively smooth overall trend, whereas those of the seven tested compounds exhibited a steep rise in the inhibition rate during the 0–10 h period and were followed by a gradual flattening after 20 h. According to calculations, the IT50 value of FLC was 14.1 h. In contrast, the IT50 values of all seven tested compounds were less than 7.5 h. Compound A11, in particular, achieved the lowest IT50 value (1.4 h). Thus, this class of compounds held a more efficient antifungal efficacy, which might be attributed to their simple structure, making them easy to enter fungal cells.

2.2.3. The Analysis of Preliminary SARs

In this study, the compounds with TAI values ≥ 1.19 were defined as susceptible compounds. When 25% of N′-phenylhydrazides had MIC80 values ≤ 4 μg/mL against the fungi, the fungi were defined as sensitive. Sixteen susceptible compounds and one sensitive fungus, C. albicans 5122, were screened.
According to the above data, the TAI values of twenty-one compounds (A2, A4, A5, A6, A9, A10, A11, A13, A16, B1, B5, B7, B10, B11, B14, C1, C3, C4, C7, D1, and D5) with MIC80 ≤ 64 μg/mL were calculated according to the formula TAI = ∑ 1 n 1 / M I C 80 , which reflected the level of antifungal activity of the compounds, and the larger the index, the better the antifungal activity of the compound [35].
The total susceptibility index (TSI) of the fungi were calculated according to the formula TSI = ∑ 1 n 1 / M I C 80 , which reflected the sensitivity of every strain of C. albicans, and the greater the index, the more sensitive the fungus was to these compounds (Table 2) [35].
Standard C. albicans SC5314 exhibited a high degree of sensitivity to FLC (TSI = 14.8). For series A and B compounds, the most sensitive fungus was C. albicans 5122, and the TSI values were 11.6 and 12.0, respectively. C. albicans 5172 showed the highest sensitivity to series C and D compounds, and the TSI values were 7.8 and 11.2, respectively. Thus, C. albicans 5122 was a sensitive fungus.
The MIC80 and TAI values obtained allowed a preliminary evaluation of SARs, which can be contrasted with A1 to obtain the general trends.
First, the introduction of a chlorine atom to the ortho-, meta-, or para-position of the carbonyl side benzene ring was able to sharply enhance the antifungal activity against all or most of the fungi (A5, A10, and A15). A similar case was observed for ortho-/meta-F, para-Br, and meta-/para-CF3 substituted compounds (A2, A8, A6, A11, and A17). The more electron-withdrawing groups on ring A, the better the antifungal activity of the corresponding compounds (A12 > A7).
Second, the introduction of nine substituents, identical to those present in the series A compounds, onto the phenyl ring of the hydrazine side resulted in compounds with inferior antifungal activity compared to the series A compounds. Among the tested compounds, those with para-CH3, para-Cl, and para-F substituents (B7, B11, and B14) exhibited better antifungal activities compared to ortho-Cl and meta-Br substitutes (B1 and B5).
Third, the antifungal activity was also influenced by the various heterocycles and alkyl groups. C4 with cyclopropyl significantly improved the antifungal activities toward C. albicans 5172 and 5272. However, the activities were significantly cut down by the naphthyl group. A similar case was found for C3, C7, and C8 with polar heterocyclic groups (thienyl and furan), which were less effective than A1. The short-chain alkane counterparts (C12) possessed better activities against five strains of fungi than their long-chain alkane counterparts (C13). The longer the carbon chain between the benzene ring and the carbonyl group, the lower the antifungal activity of the corresponding compounds (C11 < C10 < C2).
Last, combining the pharmacophores of ring A (-F, -Cl, and -CF3) and B (-F and -Cl) sharply enhanced the antifungal activity of series D compounds. However, their antifungal activity still did not reach the level of A11.

2.3. The Investigation of the Antifungal Mechanism

In this research, C. albicans SC5314 was chosen as the tested fungi, and A11 with the highest TAI (2.71) value was used as the tested compound.

2.3.1. Assay of Free Radical Scavenging

In this assay, glutathione was selected as the free radical scavenger [36]. With an increase in the concentration of glutathione, the inhibition rate of A11 against C. albicans SC5314 decreased continuously. When the concentration of glutathione reached 1600 μg/mL, the antifungal effect of A11 essentially disappeared (Figure 5).
At concentrations of 1600, 800, and 400 μg/mL, glutathione exhibited low inhibitory effects on the growth of standard C. albicans SC5314, and the inhibition rate of A11 remained consistently stable at around 95%. When the concentrations of glutathione increased, the inhibitory effect of A11 was reduced. In the event that the concentration of glutathione was at 400 μg/mL, the inhibition rate of A11 decreased from 94.7% to 18.4%. On the occasion that the concentration of glutathione was raised to 800–1600 μg/mL, the inhibition rate of A11 dropped to below 20%, similar to the inhibition rate observed when glutathione was used alone. This indicated that A11 produced free radicals during the metabolism process within fungal cells.

2.3.2. Production of ROS

To monitor cellular redox processes, the common oxidative stress indicator DCFH-DA was utilized [35]. As shown in Figure 6, treatment with A11 resulted in a significantly higher fluorescence intensity of DCFH-DA (Figure 6A–C) as compared to the control groups (Figure 6D). These results suggested that A11 could impair mitochondrial function and increase the production of large numbers of ROS in C. albicans SC5314.

2.3.3. Effects of A11 on Hyphal Morphology

The impact of A11 on the hyphal morphology against C. albicans SC5314 was observed by SEM [37]. In Figure 7A–F, significant morphological changes in the filamentous form of C. albicans were observed after treatment with 20 μg/mL of A11 compared to the control group. The untreated hyphae exhibited dispersed, regular, plump, uniform, and smooth surfaces (Figure 7A,B). In contrast, the treated hyphae appeared shriveled, distorted, with rough surfaces and displayed folding, wrinkling, and invagination (Figure 7C–E). Figure 7F depicts an abnormal transition of the fungus from the yeast form to the filamentous form.
According to the electron microscopy observations, the integrity of mycelia was damaged by A11. The disruptive effect of A11 on hyphal morphology was the main reason for its inhibitory activity.

2.3.4. Preliminary Antifungal Mechanisms

Based on the research results in Section 2.3.1, Section 2.3.2 and Section 2.3.3, the metabolic processes of A11 within the cell could be preliminarily analyzed. Firstly, A11 was hydrolyzed by fungal amidase to generate phenylhydrazine. In the structure of hydrazine (H2N-NH2), the lone pairs of electrons on the nitrogen atoms probably repelled each other strongly and led to the reducibility of phenyl hydrazides. Then, under the action of P450, phenyl hydrazine was oxidized to diazo compounds and then released nitrogen gas and generated free radicals. Their reducibility could lead to the reduction of oxygen within cells, forming reactive oxygen species (ROS) as well such as superoxide anions and hydroxyl radicals. Excessive ROS and free radicals disrupted the normal physiological processes of the fungus and subsequently affected its hyphal morphology (Figure 8). This antifungal mechanism was similar to what has been previously reported [38].
The different degrees of inhibitory activity of target compounds against C. albicans stemmed from two factors. Firstly, their antifungal activity was affected by the rate of free radical production. The hydrolysis of amide bonds was the first step in the metabolism of N′-phenylhydrazines and was also a necessary condition for the formation of free radicals. The electron cloud density of the carbonyl group can be reduced and the hydrolysis of the amide bond can be promoted when the electron-absorbing group is introduced to the substituted benzoic acid, and thus the formation rate of the corresponding free radical is faster. For example, the ortho-fluoride, ortho-trifluoromethyl, meta-chlorine, meta-trifluoromethyl, and para-cyano substituted compounds (A2, A6, A10, A11, and A16) had better antifungal activity compared to the ortho-, meta-, or para-methyl and para-methoxy substituted ones (A2, A9, A13, and A14). Different substituents affect the affinity between the compound and hydrolase, the hydrolysis rate, and the antifungal activity of the compound.
Secondly, their antifungal activity depended on the stability of the generated free radicals, and the poorer the stability of the free radicals, the better the antifungal activity of the compound. The electron-withdrawing group reduced the electron cloud density of the benzene ring, thereby reducing the stability of substituted phenylhydrazine. For example, compounds substituted with meta- or para- halogen atoms (B5, B11, B12, and B14) exhibited better antifungal activity compared to compounds substituted with para methyl groups (B7).
The pathogenicity of C. albicans is closely associated with the formation of hyphae, which is also an important factor in the formation of biofilms [39]. Typically, the filamentous form of C. albicans exhibits greater pathogenicity compared to the yeast form [40]. Under normal growth conditions, the hyphae surface appears uniform, plump, and smooth, without any folds or wrinkles. In the pathological state, the microscopic appearance of the fungal hyphae exhibits signs of invagination and folding [41]. According to Figure 8, it can be observed that the treatment of C. albicans with compound A11 resulted in an abnormal morphology of the hyphae. This indicated that compound A11 exhibited a certain degree of inhibition on the formation and growth process of C. albicans hyphae. A11 underwent metabolism within the fungal cells and led to the generation of free radicals, and ROS disrupted the normal physiological processes of the fungus and subsequently damaged its hyphal morphology. Next, in vivo antifungal activity and cytotoxicity assay tests will be carried out.

3. Materials and Methods

3.1. Materials

Substituted benzoic acid, substituted phenyl, substituted phenyl hydrazine hydrochloride, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBt), dried N, N-Dimethylformamide (DMF), pyridine, heterocyclic carboxylic acids, alkyl carboxylic acid, phenylhydrazine, fluconazole, and dimethyl sulfoxide (DMSO) were all purchased from Adamas Reagent Co. Ltd. (Shanghai, China). Other analytical pure reagents and solvents were bought from local companies. And before use, a number of reagents were purified by standard procedures. Without other explicit direction, water was redistilled and ions were removed before use.
Standard C. albicans SC5314 was donated by Professor Dazhi Zhang of the Second Military Medical University. FLC-resistant fungi C. albicans 4935, 5122, 5172, and 5272 were donated by Professor Changzhong Wang of Anhui University of Chinese Medicine. At 30 °C, the tested fungi were cultured for 48 h on the reasoner’s 2A (R2A) agar plates. The dilution of suspension was made with RPMI medium 1640; a volume of 1 mL of these suspensions contained 1 × 104–1 × 105 colony forming unit. The tested compounds were diluted at the concentration of 8 μg/mL with DMSO.
A mixture of petroleum ether (PE) and ethyl acetate (EtOAc) was used as the mobile agent and thin-layer chromatography (TLC) used GF254 silica gel to monitor the progress of the reactions. An XT-4 micro melting point instrument (Tech Instrument, Beijing, China) was used to measure the melting point of every target compound. An advance neo spectrometer (400 MHz, Bruker, Billerica, MA, USA)) was applied to obtain the 1H NMR and 13C NMR spectrum of every synthesized chemical. Chemical shifts (δ values) and coupling constants (J values) were presented in ppm and Hz, respectively. The high-resolution mass spectra (HRMS) was taken advantage of to characterize every unknown compound. On a mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific, Billerica, MA, USA), the positive ion mass spectra of the samples were gained.

3.2. The Synthetic Procedure

3.2.1. Synthesis of Substituted Phenylhydrazine Hydrochloride

NaNO2 (1.90 g, 27.5 mmol, 1.1 equiv.) was added to a solution of substituted aniline (2.33 g, 25 mmol, 1 equiv.) in hydrochloric acid (60 mL, 20%, v/v) and stirred at 0 °C for 1 h. Then, the SnCl2 (9.48 g, 50 mmol, 2 equiv.) was added and stirred for 2 h at room temperature. The precipitate was filtered and washed with brine and Et2O and dried in vacuo at 40 °C overnight [32].

3.2.2. Synthesis of N′-Phenylhydrazides

EDCI (8.8 mmol, 1.1 equiv.) and HOBt (8.8 mmol, 1.1 equiv.) were added to a solution of carboxylic acid (8.0 mmol, 1 equiv.) in DMF (20 mL); then phenylhydrazine hydrochloride was added and the reaction mixture was stirred at ambient temperature under a nitrogen atmosphere for 3 h, with TLC detection. The reaction mixture was poured into H2O (300 mL), filtrated, and recrystallized by ethanol to give the desired light yellow or brownish-yellow product [27].

3.3. Antifungal Activity In Vitro

3.3.1. Determination of MIC80 and MFC Value

The antifungal activities in vitro of target compounds against five strains of fungi were determined via the BMD method with assays in 96-well plates as described in the CLSI guidelines (CLSI M27-A3 and M38-A2) [33,34]. FLC was selected as the positive control.
To prepare the stock solutions of the tested compounds, they were dissolved in 100% DMSO with a concentration of 0.8 mg/mL. Before the assay, the solutions were diluted to the concentration of 8.0 μg/mL with RPMI medium 1640. The final concentrations of tested compounds were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL. After inoculation, the plates were incubated at 30 °C for 24 h. The absorbance of each well was scanned at a wavelength of 625 nm via an ELISA reader (DNM 9602, Perlong, Bingjing, China), and the inhibition rate was calculated.
Inhibition rate (%) = [(AP − AD)/(AP − AN)] × 100%
where AP is the absorbance of positive control wells, AD is the absorbance of drug wells, and AN is the absorbance of negative control wells.
The MIC80 values were the concentration of tested compounds when the inhibition rate was at 80%. A curve was plotted with the logarithm of the concentration as the horizontal axis and the inhibition rate as the vertical axis, and the MIC80 was calculated.
MIC 80 = 10 log ⁡ A + log ⁡ 1 N × a − 80 a − b
Among them, A refers to the corresponding minimum concentration in the aforementioned gradient concentrations when the inhibition rate is just above 80%. a is the inhibition rate at concentration A, b is the inhibition rate just below a, and N represents the dilution factor.
The MFCs of all target compounds were defined as the lowest concentration of the subculture at which no visible growth was observed on solid media. The assays were carried out in triplicates of three independent experiments.

3.3.2. Time–Inhibition Rate Curves

A quantity of 6.4 mg of the tested compound was dissolved in 0.5 mL of DMSO, then added to RPMI medium 1640 to obtain a drug solution with a concentration of 3.2 μg/mL. The specific procedures are the same as those described in Section 3.3.1. The absorbances were recorded at 0 h, 4 h, 6 h, 10 h, 17.5 h, 22 h, 24 h, 40.5 h, and 70 h, respectively. Every test was conducted three times. The time–inhibition rate curves were plotted by origin software with the time represented on the x-axis and the corresponding inhibition rate on the y-axis [33,34].

3.4. The Investigation of the Antifungal Mechanism

3.4.1. Scavenging of Free Radicals Generated from A11

The impact of A11 on the emergence of free radicals of C. albicans SC5314 was evaluated. Glutathione with gradient concentrations (400, 800, and 1600 μg/mL) was set as a control group to exclude its effect on fungal growth [36]. At the concentration of 20 μg/mL, the inhibition rate of A11 against C. albicans SC5314 was close to 100%. μA11 was added to gradient concentrations (400, 800, and 1600 μg/mL) of glutathione and achieved a concentration of A11 of 20 μg/mL. As shown in Section 3.3.1, the assay should be followed.

3.4.2. Production of ROS

The influence of A11 on the generation of ROS of C. albicans SC5314 was ascertained as previously described [42]. DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate, 40 μM, 0.5 mL) was used to stain the hyphal tips of the fungal mycelium, followed by incubation in the dark at 25 °C for 20 min, and was cleansed with PBS twice, each time for 5 min. At excitation and emission wavelengths of 488 nm and 525 nm, respectively, the fluorescence images were observed and captured before and after stimulation by CLSM (TCS SP8, Leica, Wetzlar, Germany). Images were stored on a computer for further processing and analysis.

3.4.3. Analysis of the Mycelial Morphology

By SEM, the mycelial morphology of C. albicans SC5314 was observed according to the reported method [42]. Compound A11 (TAI = 2.71) and standard C. albicans SC5314 were chosen as the tested specimen and fungus, respectively.
The fungi were cultured in a thermostatic shaker at 37 °C and 200 rpm for a period of 8 to 24 h. Subsequently, they were soaked in a 4% glutaraldehyde solution for a duration of 4 h. Following this, the fungi were washed three times using a 0.1 mol/L PBS solution with a pH of 7.2. To initiate dehydration, the fungi were exposed to a series of ethanol concentrations including 30%, 50%, 70%, 80%, and 90% (v/v). Thereafter, the fungi were treated with 100% ethanol thrice, with each exposure lasting fifteen minutes. Finally, the hyphae were subjected to vacuum drying, coated with gold, and examined through SEM (S-4800, Hitachi, Beijing, China).

4. Conclusions

Fifty-two kinds of target compounds were synthesized efficiently and concisely up to 96% yield, and their structures were characterized by HRMS, 1H NMR, and 13C NMR. It was discovered that three of the compounds (D2, D4, and D5) were unknown.
The target compounds showed different degrees of inhibitory activity against C. albicans, among which A11 had the best antifungal activity for all tested fungi, and its TAI value was 2.71, which was higher than that of the positive control FLC (TAI = 1.19). The presence of an electron-absorbing group could significantly enhance the antifungal activity of the compounds in comparison to those having an electron-giving group. Particularly, compound series D showed considerably better antifungal activity than A1. The IT50 values of the seven compounds were about half that of FLC, and A11 was only 1.4 h, which had a more efficient antifungal mechanism.
Further exploration of mechanisms revealed that A11 promoted the generation of endogenous ROS and free radicals, disrupted the physiological processes of C. albicans SC5314, and impaired its filamentous morphology, ultimately inhibiting fungal growth.
In summary, A11 is expected to be a novel antifungal agent, and this research holds significant importance for the development of antifungal agents. Next, in vivo antifungal activity and cytotoxicity assays will be carried out.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242015120/s1. References [43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63] are mentioned in the Supplementary Material part.

Author Contributions

Conceptualization, P.Z. and H.G.; data curation, P.Z.; funding acquisition, H.G.; investigation, P.Z.; supervision, H.G.; writing—original draft, P.Z., J.Z., J.Y., Z.L. and X.L.; writing—review and editing, P.Z., J.Z., J.Y., Z.L. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 31572038) and the Key Research and Development Project of Shaanxi Province (2023-YBNY-242).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We extend our sincere appreciation to the Chemical Experiment Teaching and Research Center for their valuable support in conducting nuclear magnetic resonance analysis. Additionally, we are thankful to the Life Science Research Core Service for providing us with access to electron microscopy analysis and ESI-HRMS.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Dartois, V.A.; Rubin, E.J. Anti-tuberculosis Treatment Strategies and Drug Development: Challenges and Priorities. Nat. Rev. Microbiol. 2022, 20, 685–701. [Google Scholar] [CrossRef]
  2. Zhang, Y.; Li, Q.H.; Chao, W.; Qin, Y.L.; Chen, J.Y.; Wang, Y.W.; Liu, R.H.; Lv, Q.Z.; Wang, J.X. Design, Synthesis and Antifungal Evaluation of Novel Pyrylium Salt In Vitro and In Vivo. Molecules 2022, 27, 4450. [Google Scholar] [CrossRef] [PubMed]
  3. Costa, D.S.; Rodrigues, A.G. Candida albicans Antifungal Resistance and Tolerance in Bloodstream Infections: The Triad Yeast-Host-Antifungal. Microorganisms 2020, 8, 154. [Google Scholar] [CrossRef] [PubMed]
  4. Lee, Y.; Puumala, E.; Robbins, N.; Cowen, L.E. Antifungal Drug Resistance: Molecular Mechanisms in Candida albicans and Beyond. Chem. Rev. 2021, 121, 3390–3411. [Google Scholar] [CrossRef]
  5. Wijnants, S.; Vreys, J.; Van Dijck, P. Interesting Antifungal Drug Targets in the Central Metabolism of Candida albicans. Trends Pharmacol. Sci. 2022, 43, 69–79. [Google Scholar] [CrossRef] [PubMed]
  6. Nobile, C.J.; Johnson, A.D. Candida albicans Biofilms and Human Disease. Annu. Rev. Microbiol. 2015, 69, 71–92. [Google Scholar] [CrossRef] [PubMed]
  7. Xiao, M.; Sun, Z.Y.; Kang, M.; Guo, D.W.; Liao, K.; Chen, S.C.; Kong, F.; Fan, X.; Cheng, J.W.; Hou, X.; et al. Five-Year National Surveillance of Invasive Candidiasis: Species Distribution and Azole Susceptibility from the China Hospital Invasive Fungal Surveillance Net (ChIF-NET) Study. J. Clin. Microbiol. 2018, 56, e00577-18. [Google Scholar] [CrossRef] [PubMed]
  8. Mukaremera, L.; Lee, K.K.; Mora, M.H.M.; Gow, N.A.R. Candida albicans Yeast, Pseudohyphal, and Hyphal Morphogenesis Differentially Affects Immune Recognition. Front. Immunol. 2017, 8, 629. [Google Scholar] [CrossRef] [PubMed]
  9. Tits, J.; Cammue, B.P.A.; Thevissen, K. Combination Therapy to Treat Fungal Biofilm-Based Infections. Int. J. Mol. Sci. 2020, 21, 8873. [Google Scholar] [CrossRef] [PubMed]
  10. Mahboub, H.H.; Eltanahy, A.; Omran, A.; Mansour, A.T.; Safhi, F.A.; Alwutayd, K.M.; Khamis, T.; Husseiny, W.A.; Ismail, S.H.; Yousefi, M.; et al. Chitosan Nanogel Aqueous Treatment Improved Blood Biochemicals, Antioxidant Capacity, Immune Response, Immune-Related Gene Expression and Infection Resistance of Nile Tilapia. Comp. Biochem. Physiol. Biochem. Mol. Biol. 2024, 269, 110876. [Google Scholar] [CrossRef]
  11. Li, J.; Buchner, J. Structure, Function and Regulation of the Hsp90 Machinery. Biomed. J. 2013, 36, 106–117. [Google Scholar]
  12. Lee, K.H.; Park, S.J.; Choi, S.J.; Park, J.Y. Proteus Vulgaris and Proteus Mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form. Yonsei Med. J. 2017, 58, 1135–1143. [Google Scholar] [CrossRef]
  13. Arendrup, M.C.; Patterson, T.F. Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment. J. Infect. Dis. 2017, 216, S445–S451. [Google Scholar] [CrossRef]
  14. White, P.L.; Dhillon, R.; Hughes, H.; Wise, M.P.; Backx, M. COVID-19 and Fungal Infection: The Need for a Strategic Approach. Lancet Microbe 2020, 1, e196. [Google Scholar] [CrossRef]
  15. Murphy, S.E.; Bicanic, T. Drug Resistance and Novel Therapeutic Approaches in Invasive Candidiasis. Front. Cell. Infect. Microbiol. 2021, 11, 759408. [Google Scholar] [CrossRef]
  16. Maubon, D.; Garnaud, C.; Calandra, T.; Sanglard, D.; Cornet, M. Resistance of Candida spp. to Antifungal Drugs in the Icu: Where Are We Now? Intensive Care Med. 2014, 40, 1241–1255. [Google Scholar] [CrossRef]
  17. Hoang, A. Caspofungin Acetate: An Antifungal Agent. Am. J. Health-Syst. Pharm. 2001, 58, 1206–1214. [Google Scholar] [CrossRef]
  18. Richardson, K.; Cooper, K.; Marriott, M.S.; Tarbit, M.H.; Troke, P.F.; Whittle, P.J. Discovery of Fluconazole, a Novel Antifungal Agent. Rev. Infect. Dis. 1990, 12 (Suppl. S3), S267–S271. [Google Scholar] [CrossRef]
  19. Boken, D.J.; Swindells, S.; Rinaldi, M.G. Fluconazole-Resistant Candida albicans. Clin. Infect. Dis. 1993, 17, 1018–1021. [Google Scholar] [CrossRef]
  20. Pasquale, T.; Tomada, J.R.; Ghannoun, M.; Dipersio, J.; Bonilla, H. Emergence of Candida tropicalis Resistant to Caspofungin. J. Antimicrob. Chemother. 2008, 61, 219. [Google Scholar] [CrossRef]
  21. Sawant, B.; Khan, T. Recent Advances in Delivery of Antifungal Agents for Therapeutic Management of Candidiasis. Biomed. Pharmacother. 2017, 96, 1478–1490. [Google Scholar] [CrossRef] [PubMed]
  22. Carmo, A.; Rocha, M.; Pereirinha, P.; Tomé, R.; Costa, E. Antifungals: From Pharmacokinetics to Clinical Practice. Antibiotics 2023, 12, 884. [Google Scholar] [CrossRef]
  23. Tirapegui, C.; Acevedo, F.W.; Dahech, P.; Torrent, C.; Barrias, P.; Rojas, P.M.; Mascayano, C. Easy and Rapid Preparation of Benzoylhydrazides and Their Diazene Derivatives as Inhibitors of 15-Lipoxygenase. Bioorg. Med. Chem. Lett. 2017, 27, 1649–1653. [Google Scholar] [CrossRef]
  24. Clements, J.S.; Islam, R.; Sun, B.; Tong, F.; Gross, A.D.; Bloomquist, J.R.; Carlier, P.R. N′-Mono- and N, N′-Diacyl Derivatives of Benzyl and Arylhydrazines as Contact Insecticides against Adult Anopheles Gambiae. Pestic. Biochem. Physiol. 2017, 143, 33–38. [Google Scholar] [CrossRef]
  25. Kozlov, M.V.; Konduktorov, K.A.; Shcherbakova, A.S.; Kochetkov, S.N. Synthesis of N′-Propylhydrazide Analogs of Hydroxamic Inhibitors of Histone Deacetylases (HDACs) and Evaluation of Their Impact on Activities of Hdacs and Replication of Hepatitis C Virus (HCV). Bioorg. Med. Chem. Lett. 2019, 29, 2369–2374. [Google Scholar] [CrossRef]
  26. Nam, G.; Suh, J.M.; Yi, Y.; Lim, M.H. Drug Repurposing: Small Molecules against Cu(II)–Amyloid-β and Free Radicals. J. Inorg. Biochem. 2021, 224, 111592. [Google Scholar] [CrossRef]
  27. Wu, Y.Y.; Shao, W.B.; Zhu, J.J.; Long, Z.Q.; Liu, L.W.; Wang, P.Y.; Li, Z.; Yang, S. Novel 1,3,4-Oxadiazole-2-Carbohydrazides as Prospective Agricultural Antifungal Agents Potentially Targeting Succinate Dehydrogenase. J. Agric. Food Chem. 2019, 67, 13892–13903. [Google Scholar] [CrossRef] [PubMed]
  28. Zhang, H.Z.; Drewe, J.; Tseng, B.; Kasibhatla, S.; Cai, S.X. Discovery and Sar of Indole-2-Carboxylic Acid Benzylidene-Hydrazides as a New Series of Potent Apoptosis Inducers Using a Cell-Based Hts Assay. Bioorg. Med. Chem. 2004, 12, 3649–3655. [Google Scholar] [CrossRef]
  29. Turan, Z.G.; Altintop, M.D.; Ozdemir, A.; Demirci, F.; Abu, M.U.; Kaplancikli, Z.A. Synthesis and Antifungal Activity of New Hydrazide Derivatives. J. Enzym. Inhib. Med. Chem. 2013, 28, 1211–1216. [Google Scholar] [CrossRef] [PubMed]
  30. Pham, V.H.; Phan, T.P.D.; Phan, D.C.; Vu, B.D. Synthesis and Bioactivity of Hydrazide-Hydrazones with the 1-Adamantyl-Carbonyl Moiety. Molecules 2019, 24, 4000. [Google Scholar] [CrossRef]
  31. Ganrot, P.O.; Rosengren, E.; Gottfries, C.G. Effect of Iproniazid on Monoamines and Monamine Oxidase in Human Brain. Experientia 1962, 18, 260–261. [Google Scholar] [CrossRef] [PubMed]
  32. Jacob, N.; Guillemard, L.; Wencel, D.J. Highly Efficient Synthesis of Hindered 3-Azoindoles via Metal-Free C-H Functionalization of Indoles. Synthesis 2020, 52, 574–580. [Google Scholar] [CrossRef]
  33. Espinel-Ingroff, A.; Canton, E.; Peman, J.; Rinaldi, M.G.; Fothergill, A.W. Comparison of 24-Hour and 48-Hour Voriconazole Mics as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document) in Three Laboratories: Results Obtained with 2162 Clinical Isolates of Candida spp. and Other Yeasts. J. Clin. Microbiol. 2009, 47, 2766–2771. [Google Scholar]
  34. Iqbal, N.J.; Boey, A.; Park, B.J.; Brandt, M.E. Determination of in Vitro Susceptibility of Ocular Fusarium spp. Isolates from Keratitis Cases and Comparison of Clinical and Laboratory Standards Institute M38-A2 and E Test Methods. Diagn. Microbiol. Infect. Dis. 2008, 62, 348–350. [Google Scholar] [CrossRef] [PubMed]
  35. Yang, S.S.; Lv, Q.Y.; Fu, J.; Zhang, T.Y.; Du, Y.S.; Yang, X.J.; Zhou, L. New 7-Chloro-9-Methyl-2-Phenyl-3,4-Dihydro-Β-Carbolin-2-Iums as Promising Fungicide Candidates: Design, Synthesis, and Bioactivity. J. Agric. Food Chem. 2022, 70, 4256–4266. [Google Scholar] [CrossRef] [PubMed]
  36. Luque, C.J.C.; Rodríguez, Z.P.; López, O.J.C.; Garzón, I.L. Revisiting the Scavenging Activity of Glutathione: Free Radicals Diversity and Reaction Mechanisms. Comput. Theor. Chem. 2023, 1227, 114227. [Google Scholar] [CrossRef]
  37. Wang, D.; Zhang, J.; Jia, X.M.; Xin, L.; Zhai, H. Antifungal Effects and Potential Mechanism of Essential Oils on Collelotrichum gloeosporioides In Vitro and In Vivo. Molecules 2019, 24, 3386. [Google Scholar] [CrossRef] [PubMed]
  38. Kalgutkar, A.S.; Gardner, I.; Obach, R.S.; Shaffer, C.L.; Callegari, E.; Henne, K.R.; Mutlib, A.E.; Dalvie, D.K.; Lee, J.S.; Nakai, Y.; et al. A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups. Curr. Drug Metab. 2005, 6, 161–225. [Google Scholar] [CrossRef] [PubMed]
  39. Kakizaki, T.; Abe, H.; Kotouge, Y.; Matsubuchi, M.; Sugou, M.; Honma, C.; Tsukuta, K.; Satoh, S.; Shioya, T.; Nakamura, H.; et al. Live-Cell Imaging of Septins and Cell Polarity Proteins in the Growing Dikaryotic Vegetative Hypha of the Model Mushroom Coprinopsis Cinerea. Sci. Rep. 2023, 13, 10132. [Google Scholar] [CrossRef]
  40. Triastuti, A.; Vansteelandt, M.; Barakat, F.; Amasifuen, C.; Jargeat, P.; Haddad, M. Untargeted Metabolomics to Evaluate Antifungal Mechanism: A Study of Cophinforma mamane and Candida albicans Interaction. Nat. Prod. Bioprospect. 2023, 13, 1. [Google Scholar] [CrossRef]
  41. Zhang, Y.H.; Yang, S.S.; Zhang, Q.; Zhang, T.T.; Zhang, T.Y.; Zhou, B.H.; Zhou, L. Discovery of N-Phenylpropiolamide as a Novel Succinate Dehydrogenase Inhibitor Scaffold with Broad-Spectrum Antifungal Activity on Phytopathogenic Fungi. J. Agric. Food Chem. 2023, 71, 3681–3693. [Google Scholar] [CrossRef] [PubMed]
  42. Afri, M.; Frimer, A.A.; Cohen, Y. Active Oxygen Chemistry within the Liposomal Bilayer Part Iv: Locating 2′,7′-Dichlorofluorescein (DCF), 2′,7′-Dichlorodihydrofluorescein (DCFH) and 2′,7′-Dichlorodihydrofluorescein Diacetate (DCFH-DA) in the Lipid Bilayer. Chem. Phys. Lipids 2004, 131, 123–133. [Google Scholar] [CrossRef]
  43. Zhang, J.Q.; Huang, G.B.; Weng, J.; Lu, G.; Chan, A.S.C. Copper(II)-Catalyzed Coupling Reaction: An Efficient and Regioselective Approach to N′,N′-Diaryl Acylhydrazines. Org. Biomol. Chem. 2015, 13, 2055–2063. [Google Scholar] [CrossRef]
  44. Huang, Z.Y.; Zhang, Q.Q.; Zhao, Q.G.; Yu, W.Q.; Chang, J.B. Synthesis of 2-Imino-1,3,4-Thiadiazoles from Hydrazides and Isothiocyanates via Sequential Oxidation and P(NMe2)3-Mediated Annulation Reactions. Org. Lett. 2020, 22, 4378–4382. [Google Scholar] [CrossRef]
  45. Shuler, W.G.; Smith, E.A.; Hess, S.M.; McFadden, T.M.C.; Metz, C.R.; Van Derveer, D.G.; Pennington, W.T.; Mabe, P.J.; Knick, S.L.; Beam, C.F. Preparation and X-Ray Crystal Structure of 3-(4-(Dimethylamino)Phenyl)-2-(Phenylamino)Isoquinolin-1(2h)-One, 3-(4-Methoxyphenyl)-2-(Phenylamino)Isoquinolin-1(2h)-One, and 2-Methyl-N′-(4-Methylbenzoyl)-N′-Phenylbenzohydrazide from Polylithiated 2-Methylbenzoic Acid Phenylhydrazide and Methyl 4-Dimethylaminobenzoate, Methyl 4-Methoxybenzoate, or Methyl 4-Methylbenzoate. J. Chem. Crystallogr. 2012, 42, 952–959. [Google Scholar]
  46. Wang, T.T.; Gao, F.; Xue, M.; Song, Y.J.; Wang, W.H. Ethyl 3-(2-Chlorophenyl)-5-(Diethoxyphosphinoyl)-1-Phenyl-4,5-Dihydro-1h-Pyrazole-5-Carboxylate. Acta Crystallogr. Sect. E Struct. Rep. 2007, 63, o2549. [Google Scholar] [CrossRef]
  47. Liu, R.Y.; Li, Z.Z.; Liu, S.F.; Zheng, J.S.; Zhu, P.P.; Cheng, B.; Yu, R.J.; Geng, H.L. Synthesis, Structure–Activity Relationship, and Mechanism of a Series of Diarylhydrazide Compounds as Potential Antifungal Agents. J. Agric. Food Chem. 2023, 71, 6803–6817. [Google Scholar] [CrossRef] [PubMed]
  48. Liu, H.; Jia, H.; Wang, B.; Xiao, Y.; Guo, H. Synthesis of Spirobidihydropyrazole through Double 1,3-Dipolar Cycloaddition of Nitrilimines with Allenoates. Org. Lett. 2017, 19, 4714–4717. [Google Scholar] [CrossRef] [PubMed]
  49. Huang, Z.Y.; Zhang, Q.Q.; Yi, X.F.; Zhao, Z.X.; Yu, W.Q.; Chang, J.B. Synthesis of 2-Imino-1,3,4-Selenadiazoles via Tributylphosphine-Mediated Annulation of N-Aroyldiazenes with Isoselenocyanates. Adv. Synth. Catal. 2021, 363, 4894–4898. [Google Scholar] [CrossRef]
  50. Wang, W.J.; Zhang, T.; Duan, L.J.; Zhang, X.J.; Yan, M. KOt-Bu Promoted Homocoupling and Decomposition of N′-Aryl Acylhydrazines: Synthesis of Unsymmetric N′,N′-Diaryl Acylhydrazines. Tetrahedron 2015, 71, 9073–9080. [Google Scholar] [CrossRef]
  51. Sun, Y.; Ling, S.H.; Duan, Y.B.; Li, J.X.; Chen, Z.K.; Wu, X.F. Synthesis of 5-Trifluoromethyl-1,4-Dihydro-1,2,4-Triazines via Base-Mediated [3+3] Cycloaddition of Nitrile Imines and Cf3-Imidoyl Sulfoxonium Ylides. Adv. Synth. Catal. 2023, 365, 1521–1525. [Google Scholar] [CrossRef]
  52. Voronin, V.V.; Ledovskaya, M.S.; Gordeev, E.G.; Rodygin, K.S.; Ananikov, V.P. [3 + 2]-Cycloaddition of in Situ Generated Nitrile Imines and Acetylene for Assembling of 1,3-Disubstituted Pyrazoles with Quantitative Deuterium Labeling. J. Org. Chem. 2018, 83, 3819–3828. [Google Scholar] [CrossRef] [PubMed]
  53. Areephong, J.; Mattson, K.M.; Treat, N.J.; Poelma, S.O.; Kramer, J.W.; Sprafke, H.A.; Latimer, A.A.; Read de Alaniz, J.; Hawker, C.J. Triazine-Mediated Controlled Radical Polymerization: New Unimolecular Initiators. Polym. Chem. 2016, 7, 370–374. [Google Scholar] [CrossRef]
  54. Jimenez, A.X.; Palacios, F.; de los Santos, J.M. Sc(Otf)3-Mediated [4 + 2] Annulations of N-Carbonyl Aryldiazenes with Cyclopentadiene to Construct Cinnoline Derivatives: Azo-Povarov Reaction. J. Org. Chem. 2022, 87, 11583–11592. [Google Scholar] [CrossRef] [PubMed]
  55. Molina, C.L.; Chow, C.P.; Shea, K.J. Type 2 Intramolecular N-Acylazo Diels-Alder Reaction: Regio- and Stereoselective Synthesis of Bridgehead Bicyclic 1,2-Diazines. J. Org. Chem. 2007, 72, 6816–6823. [Google Scholar] [CrossRef]
  56. Hisler, K.; Commeureuc, A.G.J.; Zhou, S.Z.; Murphy, J.A. Synthesis of Indoles via Alkylidenation of Acyl Hydrazides. Tetrahedron Lett. 2009, 50, 3290–3293. [Google Scholar] [CrossRef]
  57. Yuan, C.; Ning, X.J.; Gao, T.; Zeng, Z.G.; Lee, K.; Xing, Y.L.; Sun, S.F.; Wang, G.Q. [3+2] Cycloaddition of Nitrile Imines with 3-Benzylidene Succinimides: A Facile Access to Functionalized Spiropyrazolines. Asian J. Org. Chem. 2022, 11, e202100699. [Google Scholar] [CrossRef]
  58. Sakamoto, T.; Kikugawa, Y. Synthesis of N-Phenylalkanehydrazonoyl Chlorides. Chem. Pharm. Bull. 1988, 36, 800–802. [Google Scholar] [CrossRef]
  59. Yamaguchi, J.I.; Aoyagi, T.; Fujikura, R.; Suyama, T. An Oxidative Transformation of N′-Phenylhydrazide to T-Butyl Ester Using a Copper (II) Halide-Lithium T-Butoxide System. Chem. Lett. 2001, 30, 466–467. [Google Scholar] [CrossRef]
  60. Lopez, R.R.; Romero, G.R.; Ortega, C.E.; Garrido, F.A. Dissipation Studies of Famoxadone in Vegetables under Greenhouse Conditions Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry: Putative Elucidation of a New Metabolite. J. Sci. Food Agric. 2019, 99, 5368–5376. [Google Scholar] [CrossRef]
  61. Kapkan, L.M.; Pekhtereva, T.M.; Chervinskii, A.Y.; Berdinskii, I.S. Conformation of Hydrazides. Ukr. Khim. Zh. (Russ. Ed.) 1989, 55, 404. [Google Scholar]
  62. Murata, T.; Hara, S.; Niizuma, S.; Hada, K.; Kawada, H.; Sakaitani, M.; Shimada, H.; Nakanishi, Y. Preparation of Quinazolinone and Isoquinolinone Derivatives as Antitumor Agents for Treating Cancer and/or Cancer Metastasis and Invasion. WO2015060373, 24 December 2015. [Google Scholar]
  63. Swindle, J.; Ajioka, J.; Hummel, H.S.; Robertson, S. Methods of Inhibiting Stearoyl Coa Desaturase. WO2009070533, 4 June 2009. [Google Scholar]
Ijms 24 15120 g001
Figure 1. Hydrazides with biological activity.
Figure 1. Hydrazides with biological activity.
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Figure 2. Synthesis of target compounds A1–D5.
Figure 2. Synthesis of target compounds A1–D5.
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Figure 3. Curves of time–inhibition rate for each compound.
Figure 3. Curves of time–inhibition rate for each compound.
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Figure 4. The IT50 values of the seven compounds (h).
Figure 4. The IT50 values of the seven compounds (h).
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Figure 5. Effect of glutathione on the inhibition rate.
Figure 5. Effect of glutathione on the inhibition rate.
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Figure 6. (A–C), effects of compound A11 on the ROS of C. albicans SC5314.; (D), control groups. (a), the yeast form of C. albicans; (b), the budding state of C. albicans; (c, d), the filamentous form of C. albicans.
Figure 6. (A–C), effects of compound A11 on the ROS of C. albicans SC5314.; (D), control groups. (a), the yeast form of C. albicans; (b), the budding state of C. albicans; (c, d), the filamentous form of C. albicans.
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Figure 7. Microstructure of C. albicans SC5314 under SEM. (A,B), untreated hyphae; (C–F), hyphae treated with A11.
Figure 7. Microstructure of C. albicans SC5314 under SEM. (A,B), untreated hyphae; (C–F), hyphae treated with A11.
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Figure 8. The metabolic process of A11 in fungal cells.
Figure 8. The metabolic process of A11 in fungal cells.
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Table 1. MIC80/MFC values (μg/mL) of target compounds against C. albicans.
Table 1. MIC80/MFC values (μg/mL) of target compounds against C. albicans.
Compd.R1R2C. albicans SC5314C. albicans 4395C. albicans 5122C. albicans 5172C. albicans 5272TAI
A1HH26.0/>64.015.0/>64.00.9/16.0>64.0/>64.0>64.0/>64.0<1.76
A22-FH13.0/64.04.1/>64.08.5/64.06.7/64.012.1/>64.01.79
A32-MeH49.3/>64.0>64.0/>64.013.3/>64.021.2/>64.053.0/>64.0N. A.a
A42-OMeH14.7/>64.031.0/>64.05.7/>64.07.8/>64.06.4/>64.01.61
A52-ClH25.3/>64.052.5/>64.010.8/>64.013.8/>64.013.0/>64.01.19
A62-CF3H11.7/64.04.5/>64.06.3/64.012.0/64.022.1/>64.01.66
A72-Me&4-ClH26.0/>64.0>64.0/>64.0>64.0/>64.023.4/>64.063.0/>64.0N. A.
A83-FH3.4/64.09.7/16.00.7/4.0>64.0/>64.027.0/64.0N. A.
A93-MeH25.3/>64.061.4/>64.010.5/>64.014.7/>64.017.8/>64.01.13
A103-ClH6.9/64.06.6/>64.015.0/>64.012.3/>64.059.0/>64.01.44
A113-CF3H1.9/32.04.0/>64.02.8/64.07.4/>64.03.7/64.02.71
A123,5-diFH3.6/>64.025.0/>64.01.6/8.0>64.0/>64.0>64.0/>64.0N. A.
A134-MeH13.9/>64.031.0/>64.05.0/64.014.6/64.027.0/64.01.35
A144-OMeH5.9/>64.032.0/>64.01.5/8.0>64.0/>64.0>64.0/>64.0N. A.
A154-ClH5.9/>64.032.0/>64.01.5/>64.0>64.0/>64.0>64.0/>64.0N. A.
A164-CNH47.5/>64.015.0/>64.06.3/>64.05.8/>64.07.2/>64.01.6
A174-CF3H>64.0/>64.015.0/>64.04.6/>64.09.6/>64.09.3/>64.0N. A.
A184-BrH24.4/>64.0>64.0/>64.07.4/>64.041.4/>64.0>64.0/>64.0N. A.
A194-iPrH>64.0/>64.061.3/>64.0>64.0/>64.0>64.0/>64.013.5/>64.0N. A.
A204-NO2H6.7/>64.016.0/>64.00.8/4.0>64.0/>64.0>64.0/>64.0N. A.
B1H2-Cl26.2/>64.052.3/>64.013.0/32.014.0/>64.012.5/>64.01.16
B2H3,5-diCl45.3/>64.0>64.0/>64.032.2/>64.045.0/>64.0>64.0/>64.0N. A.
B3H3-Cl6.9/>64.019.0/>64.00.7/4.0>64.0/>64.0>64.0/>64.0N. A.
B4H3-CF3>64.0/>64.0>64.0/>64.031.5/>64.058.6/>64.0>64.0/>64.0N. A.
B5H3-Br24.6/>64.060.0/>64.03.6/64.013.9/>64.06.3/>64.01.52
B6H4-I16.7/>64.0>64.0/>64.031.8/>64.030.0/>64.019.0/>64.0N. A.
B7H4-Me13.4/>64.032.0/>64.021.5/>64.025.0/>64.013.2/>64.01.14
B8H4-OMe9.2/>64.022.0/>64.00.8/4.0>64.0/>64.0>64.0/>64.0N. A.
B9H4-CN>64.0/>64.0>64.0/>64.0>64.0/>64.0>64.0/>64.062.6/>64.0N. A.
B10H4-CF3>64.0/>64.0>64.0/>64.0>64.0/>64.0>64.0/>64.0>64.0/>64.0N. A.
B11H4-Cl10.7/>64.012.3/>64.04.8/>64.07.1/>64.057.0/>64.01.56
B12H4-Br47.5/>64.014.7/>64.050.4/>64.030.0/>64.013.1/>64.01.00
B13H4-iPr14.3/>64.0>64.0/>64.021.0/>64.052.7/>64.0>64.0/>64.0N. A.
B14H4-F12.2/64.04.0/>64.03.5/>64.03.3/>64.014.7/>64.02.13
C1PhOH31.7/>64.014.3/>64.011.0/>64.06.7/>64.07.2/>64.01.50
C2PhMeH3.4/16.042.0/>64.01.7/>64.0>64.0/>64.0>64.0/>64.0N. A.
C32-furylH17.5/>64.045.8/>64.02.7/>64.07.3/>64.013.0/>64.01.64
C4CyclopropylH58.9/>64.08.9/>64.07.7/>64.03.9/>64.03.8/>64.01.85
C51-naphthylH>64.0/>64.0>64.0/>64.062.0/>64.0>64.0/>64.0>64.0/>64.0N. A.
C62-naphthylH14.6/>64.0>64.0/>64.051.8/>64.058.7/>64.0>64.0/>64.0N. A.
C72-thienylH14.2/>64.060.0/>64.07.0/>64.016.5/>64.042.0/>64.01.17
C83-thienylH13.9/>64.022.6/>64.011.6/>64.014.7/>64.0>64.0/>64.0N. A.
C93-indolylH>64.0/>64.0>64.0/>64.0>64.0/>64.01.4/>64.0>64.0/>64.0N. A.
C10PhEtH51.8/>64.016.1/>64.015.0/>64.0>64.0/>64.011.0/>64.0N. A.
C11PhPrH>64.0/>64.0>64.0/>64.022.8/>64.0>64.0/>64.015.2/>64.0N. A.
C12EtH>64.0/>64.0>64.0/>64.013.8/>64.0>64.0/>64.012.1/>64.0N. A.
C13amylH>64.0/>64.042.6/>64.034.4/>64.0>64.0/>64.028.5/>64.0N. A.
D14-Cl4-F12.2/32.05.5/>64.06.4/>64.03.7/>64.029.5/>64.01.81
D23-F3-Cl>64.0/>64.013.8/>64.09.6/>64.0>64.0/>64.07.6/>64.0N. A.
D33-CF34-F63.6/>64.0>64/>64.03.8/>64.0>64.0/>64.07.6/>64.0N. A.
D43,5-diF4-F30.4/>64.013.5/>64.06.3/>64.0>64.0/>64.07.6/>64.0N. A.
D53,5-diF3-Cl64.0/>64.0.29.3/>64.02.2/>64.02.7/>64.02.4/>64.02.25
FLC1.8/16.0>128.0/>128.058.0/>64.0>64.0/>64.0 >128.0/>128.0<1.19 b
a: N. A. means that their TAI values are not available; b: due to FLC’s MIC80 values being greater than 128 μg/mL for C. albicans 4395, the TAI value of FLC was recorded as <1.19.
Table 2. TSI values of the specific fungus to all target compounds.
Table 2. TSI values of the specific fungus to all target compounds.
Compd.SC53144395512251725272
A6.45.411.65.85.2
B5.04.812.04.85.0
C5.04.26.47.85.8
D3.45.89.211.27.6
FLC14.8<1.8 a2.6<2.6<1.8 a
a: the MIC80 values of FLC for C. albicans 4395 and 5272 were greater than 128 μg/mL, so their TSI values were recorded as <1.8.
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Zhu, P.; Zheng, J.; Yan, J.; Li, Z.; Li, X.; Geng, H. Design, Synthesis, and Biological Evaluation of N′-Phenylhydrazides as Potential Antifungal Agents. Int. J. Mol. Sci. 2023, 24, 15120. https://doi.org/10.3390/ijms242015120

AMA Style

Zhu P, Zheng J, Yan J, Li Z, Li X, Geng H. Design, Synthesis, and Biological Evaluation of N′-Phenylhydrazides as Potential Antifungal Agents. International Journal of Molecular Sciences. 2023; 24(20):15120. https://doi.org/10.3390/ijms242015120

Chicago/Turabian Style

Zhu, Panpan, Jinshuo Zheng, Jin Yan, Zhaoxia Li, Xinyi Li, and Huiling Geng. 2023. "Design, Synthesis, and Biological Evaluation of N′-Phenylhydrazides as Potential Antifungal Agents" International Journal of Molecular Sciences 24, no. 20: 15120. https://doi.org/10.3390/ijms242015120

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