Design, Synthesis and Antifungal Evaluation of Novel Pyrylium Salt In Vitro and In Vivo.

Nowadays, discovering new skeleton antifungal drugs is the direct way to address clinical fungal infections. Pyrylium salt SM21 was screened from a library containing 50,240 small molecules. Several studies about the antifungal activity and mechanism of SM21 have been reported, but the structure-activity relationship of pyrylium salts was not clear. To explore the chemical space of antifungal pyrylium salt SM21, a series of pyrylium salt derivatives were designed and synthesized. Their antifungal activity and structure-activity relationships (SAR) were investigated. Compared with SM21, most of the synthesized compounds exhibited equivalent or improved antifungal activities against Candida albicans in vitro. The synthesized compounds, such as XY10, XY13, XY14, XY16 and XY17 exhibited comparable antifungal activities against C. albicans with MIC values ranging from 0.47 to 1.0 μM. Fortunately, a compound numbered XY12 showed stronger antifungal activities and lower cytotoxicity was obtained. The MIC of compound XY12 against C. albicans was 0.24 μM, and the cytotoxicity decreased 20-fold as compared to SM21. In addition, XY12 was effective against fluconazole-resistant C. albicans and other pathogenic Candida species. More importantly, XY12 could significantly increase the survival rate of mice with a systemic C. albicans infection, which suggested the good antifungal activities of XY12 in vitro and in vivo. Our results indicated that structural modification of pyrylium salts could lead to the discovery of new antifungal drugs.

1. Introduction

Candida, Aspergillus and Cryptococcus are the main opportunistic pathogens leading to human fungal infections [1]. During the last three decades, invasive fungal infections (IFIs) have killed about 1.6 million people each year and pose a serious threat to human health, especially among people with HIV infection, cancer, organ transplants and autoimmune diseases [2]. In recent years, systemic Candida infection has risen to fourth place among the nosocomial blood-borne infections [3,4]. At the same time, the mortality rate of candidemia is high. According to the statistics, the 90-day mortality rate of solid-organ transplant recipients with candidiasis is 22–44% [5]. These studies indicate that pathogenic fungi are a serious and increasing threat to human health. Solving the problem of fungal infections is an important means to improve the recovery rate of patients with immunodeficiencies [6].

Despite advances in antifungal therapy, invasive fungal infection remains the major cause of morbidity and death among immunocompromised patients [7]. Clinically, three major classes of drugs are available, which include azoles, echinocandins and polyenes, but the mortality of patients with invasive fungal infection is still as high as 40% [8,9]. In addition, the long-term use of antifungal drugs causes the problem of drug resistance. Multi-center investigations of candidiasis in China showed that about 0.5–2.0% of C. albicans was resistant to fluconazole. Moreover, the survival time of patients infected with drug-resistant C. albicans was significantly lower than that of patients infected with sensitive strains [10]. With continuous emergence of drug resistance and the undesired side effects of available antifungal drugs, effective agents with new structures and targets are urgently needed.

In 2014, Wong et al screened 50,240 compounds for their antifungal activities using the yeast-to-hyphae inhibitory phenotype. Among them, lipophilic pyrylium salt SM21 (Figure 1 XY2 in this study) showed good antifungal activities in vitro and in vivo [11]. Related mechanistic studies demonstrate that SM21 could inhibit the function and fusion of mitochondria in C. albicans [12,13]. However, the structure–activity relationship of SM21 is not clear. In our studies, a series of pyrylium salts were rationally designed and synthesized base on the chemical space investigation of XY2 (Figure 1). The structure–activity relationship of pyrylium salts was preliminarily discussed and a compound numbered XY12 was obtained, which showed stronger antifungal activity and lower cytotoxicity. Our research provided a new lead compound for the development of antifungal drugs.

Figure 1

Design and evaluation of new antifungal agents based on lead compound SM21 (XY2).

2. Results and Discussion

2.1. Chemistry

Except for commercially available pyrylium salt derivatives, new pyrylium salt derivatives XY3-20 were synthesized from XY1A or XY1B (Figure 1). The synthesis of the target compounds XY3-17, XY19 and XY20 was shown in Scheme 1, Scheme 2 and Scheme 3, respectively. Pyrylium salts were used as cationic dyes [14] and the methods to prepare these types of compounds were reported [15]. The target compounds XY3-17 and XY19 can be obtained efficiently by a dehydration condensation reaction of methyl pyrylium salt XY1A or XY1B and a different substituted aldehyde A1-16 under the heating condition in acetic anhydride, with single trans-olefin selectivity (Scheme 1 and Scheme 2). The structures of new pyrylium salts were determined by the high resolution mass spectra and 1H, 19F and 13C-NMR spectra shown in the Supplementary Materials. All the synthesized compounds contained triflate (CF3SO3−) as the counter anions were established from the HRESIMS data in negative ion mode and the peak of −79 ppm in the 19F-NMR spectra. The 1H-NMR signals at δH 1.40 (s, 9 H) and δH 7.64 (s, 2 H) could be attributed to the pyrylium motif. The trans-olefin was confirmed by the doublet olefinic proton signals with a coupling constant of 15.2 Hz.

Scheme 1

The synthetic route for the target compounds XY2-17.

Scheme 2

The synthetic route for the target compound XY19.

Scheme 3

The synthetic route for the target compound XY20.

Moreover, the neutral analogue XY20 was prepared by the Horner-Wadsworth-Emmons reaction. The treatment of aldehyde A11 with dibenzyl phosphate S1 under alkaline condition afforded E-type olefin XY20 in a 94% yield (Scheme 3).

2.2. Structure-Activity Relationship (SAR) Studies

The main purpose of this study is to explore the chemical space and analyze the structure-activity relationship of pyrylium salt derivatives against fungi to find novel skeleton antimicrobial agents with high efficiency and low toxicity. A series of pyrylium salt derivatives were designed and synthesized and their antifungal activity was evaluated in vitro by standard pathogenic fungi C. albicans SC5314. These results are summarized in Table 1.

Table 1

In vitro antifungal activities of the target compounds against pathogenic C. albicans SC5314.

Compounds	R	MIC (μM)	Compounds	R	MIC (μM)
XY1A	-	>179.7	XY11		>104.7
XY2	N(CH3)2	0.51	XY12		0.24
XY3	OCH3	67.5	XY13		0.95
XY4	OH	34.8	XY14		1.0
XY5	H	144.1	XY15		3.8
XY6	F	138.5	XY16		0.47
XY7	CN	>136.4	XY17		0.94
XY8	SO2CH3	>122.6	XY18		8.7
XY9	COOCH3	>127.4	XY19		3.6
XY10		0.49	XY20		>177.1

Firstly, the structure of XY2 (SM21) was analyzed. Pyrylium salt (XY1A) and 4-(Dimethylamino)benzaldehyde (A1) showed no significant activity (MIC > 179.7 μM), which revealed that both fragments of the motif were required for antifungal activity. Pyrylium salt is a kind of cationic dye [14], in which the terminal groups have a dramatic effect on their properties, such as the properties of electron transition and spectral properties. Based on these properties, a series of pyrylium salt compounds (XY2-17) with different substituents were synthesized. Interestingly, a formal positive correlation between antifungal activity and spectral properties was observed. It is to be noted that the electron-donating levels, generated predominantly by terminal groups, certainly determined the antifungal activity. Compounds XY7, XY8 and XY9, bearing a strong electron-withdrawing group, had no antifungal activity. At the same time, pyrylium salt derivatives without a substituent (XY5) or with a fluorine substituent (XY6) showed low activity, and the MIC was higher as 138.5 μM. As expected, the antifungal activity of methoxyl XY3 (MIC = 67.5 μM) and phenolic hydroxyl XY4 (MIC = 34.8 μM) were also much weaker than XY2. The above results prompted us to further study the substituent groups on nitrogen. By replacing dimethylamine (XY2) with diethylamine (XY10) and diphenylamine (XY11), the results showed the activity of diphenylamine decreased significantly and diethylamine (MIC = 0.49 μM) was equal to dimethylamine (MIC = 0.51 μM). Taken together, these results indicate that N,N-dialkylaniline as the terminal group is a necessary, but not sufficient, prerequisite for antifungal activity.

Next, a series of cyclic alkylamine derivatives were designed and synthesized for the antifungal evaluation (XY12-15). To our surprise, ring size was directly associated with the antifungal activity, with the five-membered ring being the most potent antifungal agent with an MIC value of 0.24 μM (XY12), whereas the activity of both four-membered and six-membered rings decreased (XY13-14, MIC = 0.95–1.0 μM). In addition, the introduction of a further heteroatoms such as morpholine, resulted in the decline in the activity (XY15, MIC = 3.8 μM). On this basis, the introduction of the fluoro-substituted and hydroxyl-substituted derivatives (XY16-17) exhibited decreased activity (0.47 μM, 0.95 μM). It is possible that steric hindrance of substituents affects the activity of compounds. Thus far, we obtained the best compound, XY12.

Finally, to evaluate the effect of substitutions on the pyrylium ring, the dicyclopentyl-substituted derivative XY18 (commercially available) and diphenyl-substituted derivative XY19 were synthesized, starting with the methyl pyrylium salt (XY1B) and aldehyde A11 using the standard synthetic route described above (Scheme 2); they exhibited a decline in antifungal potency (8.7 and 3.6 μM, respectively) compared to tert-butyl-substituted counterparts (XY2, XY12). XY20 was obtained by the Horner–Wadsworth–Emmons reaction (Scheme 3), and its pyrylium ring was replaced by a benzene ring. The inactivation of XY20 (>177.1 μM) indicated the necessity of the cation (Table 1). This result further implied that this lipophilic pyrylium dye shows antifungal activity, probably via targeting mitochondria [12,13,16,17].

2.3. Pharmacological Activity of

To further investigate the antifungal activity of XY12, a variety of Candida strains were used to test the MIC of XY12 and XY2. Compared with the antifungal activity of XY2, the MIC of XY12 decreased about one-fold. XY12 showed enhanced antifungal bioactivity against most of the tested Candida species with the MIC ranging from 0.12 μM to 0.97 μM, including C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis (Table 2). The compound XY12 also showed excellent activity against fluconazole-resistant C. albicans 100 and 901 with the MIC value of 0.24 μM and 0.12 μM (Table 2). By comparing the radar charts, we clearly found that the MICs of XY12 against the pathogenic Candida were lower than those of XY2 (Figure 2), which indicated the stronger anti-Candida activities of XY12.

Table 2

Antifungal activity of XY14 against Candida.

Candida Isolates	MIC (μM)
	XY12	XY2	Fluconazole
C. albicans SC5314	0.24	0.51	0.82
C. albicans Y0109	0.24	0.51	0.40
C. albicans 100	0.24	1.0	>209.1
C. albicans 901	0.12	2.1	>209.1
C. glabrata 537	0.49	4.1	0.82
C. glabrata 896	0.97	2.1	1.6
C. glabrata 8535	0.24	2.1	6.5
C. tropicalis 753	0.24	2.1	1.6
C. tropicalis 112936	0.49	2.1	6.5
C. tropicalis 750	0.49	2.1	3.3
C. parapsilosis 90018	0.49	4.1	1.6
C. parapsilosis 22019	0.24	4.1	3.3
C. parapsilosis 700	0.24	2.1	6.5
C. krusei 463	0.97	1.0	13.1
C. krusei 629	0.97	2.1	6.5

Figure 2

MICs of XY12 and XY2 against Candida species showed by Radar chart.

To investigate the cytotoxicity of XY12 and XY2, the viability of human umbilical vein endothelial cells (HUVECs) was assayed by CCK-8 agents. As shown in Figure 3, 7.8 μM of XY12 showed almost no toxicity to HUVECs and the IC50 of XY12 was about 34.6 μM. However, a significant inhibitory effect of XY2 was observed at the concentration of 0.51 μM. Meanwhile, the IC50 of XY2 was about 1.8 μM, which indicated that the cytotoxicity of XY12 was reduced by about 20-fold. Therefore, structural modification of XY2 at the terminal groups not only improves the antifungal activity, but also reduces the cytotoxicity.

Figure 3

Cytotoxicity of compounds XY2 and XY12 detected by human umbilical vein endothelial cells (HUVECs). HUVECs were treated with different concentrations of compounds XY2 (A) and XY12 (B) for 24 h. Cell viability was detected by CCK-8 assay. Unpaired t test, * p < 0.05, *** p < 0.001.

Finally, we compared the in vivo antifungal activity of XY12 and XY2 using the systemic candidiasis model. Treated with 10 mg/kg of XY12, the average survival time and survival rate of mice increased significantly. However, XY2 did not show any protective efficacy against C. albicans infection at the dose of 10 mg/kg. When the dose reduced to 5 mg/kg, the protective effect of XY12 was lost. However, increasing the dose of XY12 not only failed to improve the protective effect, but also showed toxicity in vivo. The survival time of mice injected with 20 mg/kg of XY12 was shorter than that of mice injected with the solvent (Figure 4). Although XY12 showed moderate protective effect in vivo, it also caused abdominal irritation in mice, which was reflected by the writhing responses. Therefore, further structural modification of XY12 was required to enhance its antifungal effect and reduce its irritation to mammals.

Figure 4

Survival of mice infected with C. albicans systemically. Female ICR mice were infected with C. albicans 1.5 × 106 CFU through tail vein; 10 mg/kg of XY2 or 5, 10, 20 mg/kg of XY12 was injected intraperitoneally once a day for 5 days and the survival of mice was observed for 30 days. Log-rank (Mantel–Cox) test, *** p < 0.001, ns—no significance.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

All the starting materials were commercially available reagents and used without further purification. 1H, 19F and 13C-NMR spectra were recorded at 500 MHz for 1H, at 126 MHz for 13C and at 282 MHz for 19F in CDCl3 with the BRUKER DRX-500 or 300M NMR spectrometer (Bruker, Billerica, MA, USA). Coupling constants were given in hertz (Hz). GC-MS was conducted using the Agilent MSD Trap XCT (for ESI) and Q-Tof (for HR-ESI-MS) (Agilent Technologies, Santa Clara, CA, USA) and were recorded on the Shimadzu/QP2010 Plus (Kyoto, Japan). All reactions were monitored by thin-layer chromatography (TLC) using silica-gel plates (silica gel 60 F254 0.25 mm).

3.1.2. Synthesis

This was the general procedure for the synthesis of XY3-17, 19: To a mixture of XY1A or XY1B (0.168 mmol) and A1-16 (0.202 mmol), acetic anhydride (1.0 mL) was added under an argon atmosphere. The mixture was allowed to stir at 130 °C for 3 h before it was cooled to room temperature and a precipitate formed. The precipitate was washed with diethyl ether (3 × 20 mL), which can be directly used or purified by flash column chromatography for purification to provide XY3-17 (yield: 55–75%) and XY19 (yield: 83%) as yellow or dark blue powder. The purity was identified by 1H-NMR.

This was the general procedure for the synthesis of XY20: To a stirred solution of S1 (0.176 mmol) in DMF, NaH (0.264 mmol) at 0 °C was added under an argon atmosphere. The mixture was allowed to stir at that temperature for 30 min before a solution of A11 (0.211 mmol) in DMF was added. The mixture was allowed to stir at room temperature for 24 h before it was quenched with saturated aq. NaHCO3 was extracted with EtOAc (3 times). The combined organic phases were washed with brine, dried over anhydrous Na2SO4 and filtered. The solvent was evaporated under vacuum, and the residue was subjected to flash column chromatography for purification using petroleum ether/EtOAc (10%) as eluent to give XY20 (59.8 mg, yield: 94%) as a white powder.

XY3: An orange powder. Yield 71% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.42 (d, J = 16.1 Hz, 1 H), 7.92 (d, J = 8.8 Hz, 2 H), 7.85 (s, 2 H), 7.37 (d, J = 15.7 Hz, 1 H), 6.90 (d, J = 8.7, 2 H), 3.83 (d, J = 2.7 Hz, 3 H), 1.48 (d, J = 1.9 Hz, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 183.0, 165.2, 164.4, 152.8, 133.7, 127.8, 120.8, 115.2, 113.2, 55.8, 38.6, 28.2 ppm. HRMS (m/z): [M]+ calcd for C22H29O2+ 325.2162, found 325.2166; [M]− calcd for CF3SO3−,148.9526, found 148.9529.

XY4: An orange powder. Yield 59% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 9.36 (s, 1 H) 8.05 (d, J = 15.6 Hz, 1 H), 7.62–7.52 (m, 4 H), 7.05 (d, J = 15.7 Hz, 1 H), 6.88 (d, J = 8.3 Hz, 2 H), 1.48 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 183.5, 164.7, 162.3, 152.3, 133.5, 126.8, 119.5, 117.2, 112.4, 38.6, 28.0 ppm. HRMS (m/z): [M]+ calcd for C21H27O2+ 311.2006, found 311.2012; [M]− calcd for CF3SO3−,148.9526, found 148.9528.

XY6: A brown powder. Yield 68% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.40 (d, J = 16.1 Hz, 1 H), 7.98 (dd, J = 8.9, 5.4 Hz, 2 H), 7.95 (s, 2 H), 7.44 (d, J = 16.0 Hz, 1 H), 7.08–7.02 (m, 2 H), 1.52 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7, −113.8 ppm; 13C-NMR (126 MHz, CDCl3): δ = 184.4, 165.7, 165.5 (d, J = 256.5 Hz), 151.0, 133.5 (d, J = 9.1 Hz), 131.10 (d, J = 2.9 Hz), 123.13 (d, J = 2.0 Hz), 116.70 (d, J = 21.9 Hz), 114.3, 38.9, 28.3 ppm. HRMS (m/z): [M]+ calcd for C21H26FO+ 313.1962, found 313.1972; [M]− calcd for CF3SO3−,148.9526, found 148.9527.

XY7: A brown powder. Yield 64% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.32 (d, J = 16.1 Hz, 1 H), 8.10 (s, 2 H), 8.00 (d, J = 8.0 Hz, 2 H), 7.61 (d, J = 8.2 Hz, 2 H), 7.58 (d, J = 16.2 Hz, 1 H), 1.54 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 186.0, 165.2, 148.2, 138.5, 132.9, 130.8, 126.2, 118.3, 115.4, 114.7, 39.2, 28.4. HRMS (m/z): [M]+ calcd for C22H26NO+ 320.2009, found 320.2015; [M]− calcd for CF3SO3−,148.9526, found 148.9530.

XY8: A brown powder. Yield 67% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.15 (d, J = 16.2 Hz, 1 H), 8.05 (d, J = 8.3 Hz, 2 H), 8.00 (s, 2 H), 7.78 (d, J = 8.1 Hz, 2 H), 7.37 (d, J = 16.2 Hz, 1 H), 2.96 (s, 3 H), 1.56 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 186.0, 164.7, 147.1, 142.1, 139.3, 131.3, 128.0, 126.0, 115.3, 44.3, 39.2, 28.3 ppm. HRMS (m/z): [M]+ calcd for C22H29O3S+ 373.1832, found 373.1839; [M]− calcd for CF3SO3−,148.9526, found 148.9527.

XY9: A yellow powder. Yield 72% (purity > 90%). 1H-NMR (500 MHz, CDCl3): δ = 8.41 (d, J = 16.1 Hz, 1 H), 8.05 (s, 2 H), 7.99 (d, J = 8.2 Hz, 2 H), 7.96 (d, J = 8.4 Hz, 2 H), 7.63 (d, J = 16.0 Hz, 1 H), 3.91 (s, 3 H), 1.51 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 185.3, 166.3, 165.5, 149.9, 138.5, 133.0, 130.4, 130.3, 125.6, 115.0, 52.5, 39.1, 28.3 ppm. HRMS (m/z): [M]+ calcd for C23H29O3+ 353.2111, found 353.2118; [M]− calcd for CF3SO3−,148.9526, found 148.9528.

XY10: A dark blue powder. Yield 59% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.23 (d, J = 15.0 Hz, 1 H), 7.85 (d, J = 8.7 Hz, 2 H), 7.37 (s, 2 H), 7.06 (d, J = 15.1 Hz, 1 H), 6.74 (d, J = 9.0 Hz, 2 H), 3.50 (q, J = 7.1 Hz, 4 H), 1.42 (s, 18 H), 1.24 (t, J = 7.1 Hz, 6 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 178.5, 160.7, 153.6, 153.2, 123.4, 116.1, 112.8, 45.5, 37.8, 28.2, 12.9 ppm. HRMS (m/z): [M]+ calcd for C25H31NO+ 366.2791, found 366.2793; [M]− calcd for CF3SO3−,148.9526, found 148.9530.

XY11: A dark blue powder. Yield 57% (purity > 92%). 1H-NMR (500 MHz, CDCl3): δ = 8.35 (d, J = 15.4 Hz, 1 H), 7.78 (d, J = 8.7 Hz, 2 H), 7.68 (s, 2 H), 7.37 (m, 4 H), 7.24–7.17 (m, 7 H), 6.96 (d, J = 8.5 Hz, 2 H), 1.48 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 181.3, 163.5, 153.7, 152.9, 145.4, 133.8, 130.0, 127.0, 126.9, 126.2, 122.4, 119.8, 119.4, 119.1, 112.1, 38.4, 28.3 ppm. HRMS (m/z): [M]+ calcd for C33H36NO+ 462.2791, found 462.2795; [M]− calcd for CF3SO3−,148.9526, found 148.9526.

XY12: A dark blue powder. Yield 75% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.23 (d, J = 15.0 Hz, 1 H), 7.84 (d, J = 8.5 Hz, 2 H), 7.35 (s, 2 H), 7.06 (d, J = 15.1 Hz, 1 H), 6.64 (d, J = 8.7 Hz, 2 H), 3.50–3.47 (m, 4 H), 2.10–2.04 (m, 4 H), 1.42 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.6 ppm; 13C-NMR (126 MHz, CDCl3): δ = 178.5, 160.6, 153.6, 153.1, 123.7, 122.2, 119.7, 116.1, 113.7, 48.5, 37.9, 28.2, 25.4 ppm. HRMS (m/z): [M]+ calcd for C25H34NO+ 364.2635, found 364.2644; [M]− calcd for CF3SO3−,148.9526, found 148.9529.

XY 13: A dark blue powder. Yield 61% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.27 (d, J = 15.1 Hz, 1 H), 7.86 (d, J = 8.8 Hz, 2 H), 7.45 (s, 2 H), 7.13 (d, J = 15.1 Hz, 1 H), 6.90 (d, J = 8.7 Hz, 2 H), 3.56 (m, 4 H), 1.73–1.69 (m, 6 H), 1.44 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 183.7, 164.0, 149.4, 133.0, 122.7, 121.7, 119.5, 118.9, 113.6, 54.8, 38.8, 28.2, 24.1, 22.1 ppm. HRMS (m/z): [M]+ calcd for C26H36NO+ 378.2791, found 378.2792; [M]− calcd for CF3SO3−,148.9526, found 148.9529.

XY14: A dark blue powder. Yield 63% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.22 (d, J = 15.2 Hz, 1 H), 7.81 (d, J = 8.4 Hz, 2 H), 7.37 (s, 2 H), 7.05 (d, J = 15.1 Hz, 1 H), 6.37 (d, J = 8.6 Hz, 2 H), 4.14 (t, J = 7.5 Hz, 4 H), 2.48 (m, 2 H), 1.42 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 178.7, 160.8, 155.0, 153.6, 123.9, 116.2, 111.2, 51.5, 37.9, 28.2, 16.2 ppm. HRMS (m/z): [M]+ calcd for C24H32NO+ 350.2478, found 350.2484; [M]− calcd for CF3SO3−,148.9526, found 148.9530.

XY15: A dark blue powder. Yield 67% (purity > 90%). 1H-NMR (500 MHz, CDCl3): δ = 8.32 (d, J = 15.2 Hz, 1 H), 7.89 (d, J = 8.5 Hz, 2 H), 7.60 (s, 2 H), 7.24 (d, J = 16.3 Hz, 1 H), 6.92–6.86 (m, 1 H), 3.88–3.75 (m, 1 H), 3.5–3.39 (m, 1 H), 1.45 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7 ppm; 13C-NMR (126 MHz, CDCl3): δ = 180.7, 163.0, 154.9, 153.2, 134.7, 125.2, 118.3, 114.0, 111.5, 66.5, 46.9, 38.2, 28.2 ppm. HRMS (m/z): [M]+ calcd for C25H34NO2+ 380.2584, found 380.2589; [M]− calcd for CF3SO3−,148.9526, found 148.9523.

XY16: A dark blue powder. Yield 63% (purity > 92%). 1H-NMR (500 MHz, CDCl3): δ = 8.30 (d, J = 15.0 Hz, 1 H), 7.87 (d, J = 8.5 Hz, 2 H), 7.47 (s, 2 H), 7.14 (d, J = 15.0 Hz, 1 H), 6.59 (d, J = 8.3 Hz, 2 H), 5.42 (d, J = 52.0 Hz, 1 H), 3.72–3.54 (m, 4 H), 2.49–2.35 (m, 1 H), 2.29–2.11 (m, 1 H), 1.43 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.7,−173.5–−179.0 (m) ppm; 13C-NMR (126 MHz, CDCl3): δ = 179.3, 161.7, 153.7, 152.5, 124.1, 122.3, 119.8, 116.8, 113.5, 92.39 (d, J = 176.4 Hz), 54.80 (d, J = 23.1 Hz), 46.0, 38.0, 32.05 (d, J = 21.8 Hz), 28.2 ppm. HRMS (m/z): [M]+ calcd for C25H33FNO+ 382.2541, found 382.2544; [M]− calcd for CF3SO3−,148.9526, found 148.9523.

XY17: A dark blue powder. Yield 55% (purity > 92%). 1H-NMR (500 MHz, CDCl3): δ = 8.33 (d, J = 15.0 Hz, 1 H), 7.90 (d, J = 8.5 Hz, 2 H), 7.49 (s, 2 H), 7.16 (d, J = 15.0 Hz, 1 H), 6.65 (d, J = 8.4 Hz, 2 H), 5.46 (d, J = 4.0 Hz, 1 H), 3.75 (dd, J = 12.5, 4.7 Hz, 1 H), 3.67–3.59 (m, 2 H), 3.56 (d, J = 12.5 Hz, 1 H), 2.30–2.25 (m, 2 H), 1.45 (s, 18 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.6 ppm; 13C-NMR (126 MHz, CDCl3): δ = 179.4, 161.7, 153.8, 152.5, 124.1, 122.3, 119.7, 116.9, 113.5, 73.2, 54.0, 46.3, 38.0, 31.1, 28.3 ppm. HRMS (m/z): [M]+ calcd for C25H34NO2+ 380.2584, found 380.2590; [M]− calcd for CF3SO3−,148.9526, found 148.9528.

XY19: A dark blue powder. Yield 83% (purity > 95%). 1H-NMR (500 MHz, CDCl3): δ = 8.31 (s, 2 H), 8.25 (d, J = 15.8 Hz, 1 H), 8.20 (d, J = 7.7 Hz, 4H), 7.94 (d, J = 8.2 Hz, 2 H), 7.82–7.78 (m, 2 H), 7.75–7.66 (m, 4 H), 7.53 (d, J = 8.1 Hz, 2 H), 7.47 (d, J = 15.8 Hz, 1 H), 3.87–3.76 (m, 4 H), 2.37–2.29 (m, 4 H) ppm; 19F-NMR (282 MHz, CDCl3) δ = −78.6 ppm; 13C-NMR (126 MHz, CDCl3): δ = 169.6, 160.4, 147.9, 144.6, 135.7, 132.6, 130.5, 128.7, 128.0, 123.6, 121.0, 118.6, 113.8, 57.9, 24.3 ppm. HRMS (m/z): [M]+ calcd for C29H26NO+ 404.2009, found 404.2014; [M]− calcd for CF3SO3−,148.9526, found 148.9528.

XY20: A white powder. Yield 94% (purity > 95%). 1H-NMR (300 MHz, CDCl3): δ = 7.44 (d, J = 8.6 Hz, 2 H), 7.36–7.33 (m, 2 H), 7.31 (m, 1 H), 7.06 (d, J = 16.2 Hz, 1 H), 6.95 (d, J = 16.2 Hz, 1 H), 6.59 (d, J = 8.2 Hz, 2 H), 3.45–3.27 (m, 4 H), 2.18–1.91 (m, 4 H), 1.38 (s, 18 H) ppm; 13C-NMR (75 MHz, CDCl3): δ = 151.0, 147.5, 137.5, 128.5, 127.8, 125.0, 121.2, 120.5, 112.0, 47.8, 35.0, 31.6, 25.6 ppm. HRMS (m/z): [M + H]+ calcd for C25H35N+ 362.2842, found 362.2841.

3.2. In Vitro Antifungal Activity

The standard C. albicans SC5314 was donated by Professor William A. Fonzi of Georgetown University in the United States. Clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis were isolated from patients of Changhai Hospital and identified by biochemistry and morphology. The antifungal minimum inhibitory concentration (MIC) was measured by serial dilution in 96-well plates with RPMI 1640 medium as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI M27-A3). The MIC is defined as the concentration that reduced fungal growth by more than 95%. All compounds were dissolved in DMSO. The OD600 was determined after Candida was cultured at 30 °C for 48 h. The MIC of each compound was calculated by OD600. All the antifungal activity was detected at least two times. The in vitro antifungal activities of XY1A-20 were evaluated by the C. albicans standard isolate SC5314.

3.3. Cytotoxicity against Human Umbilical Vein Endothelial Cells (HUVECs)

HUVECs were cultured to detect the cytotoxicity of XY12 and XY2 as described. HUVECs were cultured in DMEM containing 10% fetal bovine serum (FBS). Then, a 100 μL suspension of HUVECs (1 × 105 cells/mL) was added to 96-well tissue culture plates and incubated at 37 °C for 3 h. After incubation, the supernatant was replaced by 100 μL of fresh DMEM complete mediums containing different concentrations of XY12 and XY2. After incubation for 24 h, the supernatant was added with 10 μL of CCK-8 agents and cultured at 37 °C for 2 h. Finally, cell viability was assessed by measuring OD450 [18].

3.4. In Vivo Antifungal Activity

8-week-old female ICR mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). The mice were injected with a 200 μL PBS suspension of C. albicans SC5314 (7.5 × 106 CFU/mL) through the tail vein. XY12 and XY2 compounds were solved in the DPH buffer (5% DMSO, 30% PEG 400 and 65% ddH2O) and injected intraperitoneally 2 h after infection. XY12 and XY2 were injected in the mice once a day for 5 days. After that, the mice were observed daily for survival.

4. Conclusions

In summary, based on the lead compound SM21 (XY2), we synthesized a series of pyrylium salt derivatives and evaluated their antifungal activities. Most of the designed compounds exhibited moderate in vitro antifungal activities against C. albicans. Among them, the most promising antifungal agent was coded as XY12. Compared with the lead compound XY2, XY12 showed lower cytotoxicity and higher activity against Candida species, including the fluconazole-resistant C. albicans. Additionally, the in vivo antifungal activity of XY12 was also enhanced. The SARs study demonstrated that the electron-donating level of terminal groups on the benzene ring determined antifungal activity. At the same time, further activity and selectivity strongly depended on the ring size and steric hindrance of terminal cyclic alkylamine. The character of lipophilic cations responsible for activity implies that this type of pyrylium dye exhibited the antifungal activity, probably via targeting mitochondria. Researches on the antifungal mechanisms of pyrylium salts will provide more information for further structural modifications.