Identification of Novel Antifungal Skeleton of Hydroxyethyl Naphthalimides with Synergistic Potential for Chemical and Dynamic Treatments

The invasion of pathogenic fungi poses nonnegligible threats to the human health and agricultural industry. This work exploited a family of hydroxyethyl naphthalimides as novel antifungal species with synergistic potential of chemical and dynamic treatment to combat the fungal resistance. These prepared naphthalimides showed better antifungal potency than fluconazole towards some tested fungi including Aspergillus fumigatus, Candida tropicalis and Candida parapsilosis 22019. Especially, thioether benzimidazole derivative 7f with excellent anti-Candida tropicalis efficacy (MIC = 4 μg/mL) possessed low cytotoxicity, safe hemolysis level and less susceptibility to induce resistance. Biochemical interactions displayed that 7f could form a supramolecular complex with DNA to block DNA replication, and constitute a biosupermolecule with cytochrome P450 reductase (CPR) from Candida tropicalis to hinder CPR biological function. Additionally, 7f presented strong lipase affinity, which facilitated its permeation into cell membrane. Moreover, 7f with dynamic antifungal potency promoted the production and accumulation of reactive oxygen species (ROS) in cells, which destroyed the antioxidant defence system, led to oxidative stress with lipid peroxidation, loss of glutathione, membrane dysfunction and metabolic inactivation, and eventually caused cell death. The chemical and dynamic antifungal synergistic effect initiated by hydroxyethyl naphthalimides was a reasonable treatment window for prospective development.


Introduction
Pathogenic fungal diseases account for about 60% of human and animal diseases, which have the characteristics of great harmfulness, wide spread and difficult to control thoroughly [1]. Recently, the widely used chemical agents may cause drug resistance of pathogens and form ecological hidden dangers that are difficult to predict. Therefore, it is urgent to develop novel antifungal agents with high effectivity and safety to meet the needs of survival and development of mankind. For the purpose of solving this huge challenge, it is a pragmatic tactic to discover new means to heighten the fungicidal effects [2,3]. In the methods to overcome resistance, the integration of dynamic treatment dominated by reactive oxidative species (ROS) with traditional chemical treatment may express a strategy to defeat fungi [4,5]. The effectivity of chemical drug treatment is self-explanatory, and the excess expression of ROS, the dominators of dynamic treatment, directly causes the imbalance of redox system and oxidative stress, which can trigger DNA mutation, damage cell lipids and proteins and ultimately result in cell death [6,7]. Moreover, pathological causes the imbalance of redox system and oxidative stress, which can trigger DNA mut tion, damage cell lipids and proteins and ultimately result in cell death [6,7]. Moreove pathological cells are more likely to be exposed to oxidative stress, so enhancing intrace lular ROS levels and impairing antioxidant systems can disturb the balance of prooxidan antioxidant environment of compromised cells and trigger cell death [8,9]. Therefore, a tifungal agents that efficaciously trigger the generation and accumulation of ROS displa a conspicuous battery of drug candidates worthy of further evaluation for sufferers wi fungal infection in clinical trials.
Naphthalimide moiety as a unique skeleton with large tricyclic planar configuratio cycloheximide and naphthalene framework, has been supposed as a DNA-targetin chemotherapy backbone toward compromised cells [10][11][12][13]. It can intercalate into the ba pair of DNA double strands, causing the double strands to rupture, which in turn affec DNA synthesis and leads to DNA damage [14][15][16]. The amido group presented in nap thalimide moiety can bind non-covalently with a variety of functional enzymes includin lipase to exert antifungal activity. Modifications of naphthalimido moiety at the N-pos tion and 4-position have a prominent effect on the interactions with enzymes and DN [17][18][19]. Besides, numerous molecules containing naphthalimido moiety have bee proved to be expected triggers for the production and accumulation of ROS by means DNA damage channel, which would tremendously facilitate its application in medicin chemical biology [20][21][22][23]. Therefore, naphthalimido moiety was considered as a promi ing chemical and dynamic antifungal structural backbone by manipulating supramolecu lar interactions and ROS regulation. Ethanol has long been applied as disinfectants in lif and introduction of hydroxyethyl fragment as hydrogen bond donor, can affect supram lecular interaction with biomolecules and might helpfully improve antifungal activiti [24][25][26][27].
With respect to the foregoing, taking advantage of the structure and biochemic properties, hydroxyethyl fragment was merged into the N-position of naphthalimide co and the bromine atom at 4-position was replaced by amines, ethers and thioethers to a ford desirable potential antifungal molecules ( Figure 1). The structural properties, bindin effects with DNA and antifungal activities of target naphthalimide compounds were a sessed to investigate its chemicobiological behaviors. The medicinal chemical potentia of highly active compound were further elaborated, including toxicity and haemolytic a sessment, ADME study, resistance development, lipase affinity, biochemical interaction with DNA and cytochrome P450 reductase, up-regulation of ROS and ROS-mediate apoptosis pathways, to explore its application possibility.

Chemistry
Novel naphthalimido hybrids modified by hydroxyethyl fragment were derived starting from commercial 4-bromo-1,8-naphthalic anhydride. As outlined in Schemes 1 and 2, the available 4-bromo-1,8-naphthalic anhydride 1 was treated with ethanolamine in the presence of ethanol to offer hydroxyethyl naphthalimido intermediate 2 with 86.7% yield. Intermediate 2 was further reacted with amines, ethers and thioethers to give the target amine derivatives 3a-b, 4a-c and 5, hydroxyl derivatives 6a-c, mercaptoazoles 7a-f and sulfhydrypyrimidines 8a-d with moderate to good yields [28,29]. The chemical structures of all novel hydroxyethyl naphthalimides were confirmed by 1 H NMR, 13 C NMR and HRMS spectra, and the purities were determined by HPLC spectra. In the 13 C NMR spectra for hydroxyethyl naphthalimides, the chemical shifts around 160-165 ppm were primarily attributed to the carbons in carbonyl groups of naphthalimide backbone, while in the 1 H NMR spectra, the chemical shifts in the range of 8.85-7.23 ppm were deemed as the aromatic hydrogens (H-Ar) fused in naphthalimide backbone. Furthermore, the HRMS results were consistent with the structures of novel hydroxyethyl naphthalimides that displayed in the schemes, and purity analysis showed that the purities of all hydroxyethyl naphthalimides were above 95%.

Supramolecular Interaction of Thioether Benzimidazole 7f with DNA
The specific relationship between DNA and thioether benzimidazole 7f was studied. With a fixed amount of DNA, absorption spectra were measured with increasing concentrations of 7f. The DNA peak at 260 nm in Figure 3A proportionally disappeared with adding amount of 7f. A weak hypochromicity between compound 7f and DNA was demonstrated, and a slight red shift at maximum absorption wavelength was observed possibly due to the reason that the aromatic chromophore of thioether benzimidazole 7f intercalated into the helix of DNA following the increasement of the π-π conjugation [30,31].
The activities of almost all the target compounds towards A. fumigatus and C. tropicalis were stronger than that of fluconazole. In symmetric amine series 3a-b, the same antifungal values were observed, and diethylamine derivative 3b showed higher DNA binding ability. In the hybridization of multiple hydroxyethyl fragments, derivative 4c with three hydroxyethyl moiety exerted outstanding DNA affinity, indicating that multiple hydroxyethyl fragments were advantageous for non-covalent binding to DNA. Among mercaptoazoles modified hydroxyethyl naphthalimides 7a-f, thioether benzimidazole 7f gave better anti-C. tropicalis efficacy (MIC = 4 μg/mL) than fluconazole based on the antifungal activities presented, which was consistent with its excellent DNA binding ability. Similarly, sulfhydrypyrimidine 8d in sulfhydrypyrimidine series 8a-d performed remarkable DNA binding ability, and its antifungal activities shared prominent inhibitory efficacy, more potent than 8a-c. Given antifungal potential of hydroxyethyl naphthalimides, thioether benzimidazole 7f was used as model compound for farther exploration.

Supramolecular Interaction of Thioether Benzimidazole 7f with DNA
The specific relationship between DNA and thioether benzimidazole 7f was studied. With a fixed amount of DNA, absorption spectra were measured with increasing concentrations of 7f. The DNA peak at 260 nm in Figure 3A proportionally disappeared with adding amount of 7f. A weak hypochromicity between compound 7f and DNA was demonstrated, and a slight red shift at maximum absorption wavelength was observed possibly due to the reason that the aromatic chromophore of thioether benzimidazole 7f intercalated into the helix of DNA following the increasement of the π-π conjugation [30,31].  To expound the binding mode between thioether benzimidazole 7f and DNA, the existing dyes both commercial acridine orange (AO) and marketable 4 ,6-diamidino-2phenylindole (DAPI) were used as spectral probes referring the reported literature [32]. Figure 3B-D, the intensity of 7f decreased obviously at 537 nm, which suggested that 7f could embed into DNA by competing with AO. Moreover, the changes of fluorescence intensity of AO-DNA and DAPI-DNA with different concentrations of 7f was compared, and it was found that the effect of 7f on AO-DNA was stronger than that of DAPI-DNA, indicating that 7f was mainly intercalated into DNA rather than small groove binding with DNA.

Cytotoxicity, Hemolysis Assays and Resistance Development Assay
The cytotoxicity and hemolysis undergoing with thioether benzimidazole 7f were implemented to assess its underlying toxicity. Cytotoxic result showed that compound 7f had little effect on the growth of LO2 cell line (IC 50 = 163 µM) in the high concentration (100 µg/mL), and after exposure to compound 7f for 1 h, hemolytic rate was lower than 5% at anti-C. tropicalis concentration, indicating that compound 7f presented relative biosecurity ( Figure 4A,B). These compounds could selectively target fungal cell membranes due to an electrostatic distinction on the membranes between fungi and mammalian cells [33,34]. Thus, the tendency of resistant development of 7f against C. tropicalis was conducted, and fluconazole was selected as a positive control ( Figure 4C) [35][36][37][38]. The MIC values of thioether benzimidazole 7f almost remained consistent throughout the 16 passages, whereas that of reference drug fluconazole increased dramatically after the eighth passage. The result from the resistance study showed that C. tropicalis was unable to develop rapid resistance against compound 7f.
To expound the binding mode between thioether benzimidazole 7f and DNA, t existing dyes both commercial acridine orange (AO) and marketable 4′,6-diamidino phenylindole (DAPI) were used as spectral probes referring the reported literature [3 As indicated in Figure 3B-D, the intensity of 7f decreased obviously at 537 nm, wh suggested that 7f could embed into DNA by competing with AO. Moreover, the chang of fluorescence intensity of AO-DNA and DAPI-DNA with different concentrations of was compared, and it was found that the effect of 7f on AO-DNA was stronger than th of DAPI-DNA, indicating that 7f was mainly intercalated into DNA rather than sm groove binding with DNA.

Cytotoxicity, Hemolysis Assays and Resistance Development Assay
The cytotoxicity and hemolysis undergoing with thioether benzimidazole 7f w implemented to assess its underlying toxicity. Cytotoxic result showed that compound had little effect on the growth of LO2 cell line (IC50 = 163 μM) in the high concentrati (100 μg/mL), and after exposure to compound 7f for 1 h, hemolytic rate was lower th 5% at anti-C. tropicalis concentration, indicating that compound 7f presented relative osecurity ( Figure 4A,B). These compounds could selectively target fungal cell membran due to an electrostatic distinction on the membranes between fungi and mammalian ce [33,34]. Thus, the tendency of resistant development of 7f against C. tropicalis was co ducted, and fluconazole was selected as a positive control ( Figure 4C) [35][36][37][38]. The M values of thioether benzimidazole 7f almost remained consistent throughout the 16 p sages, whereas that of reference drug fluconazole increased dramatically after the eigh passage. The result from the resistance study showed that C. tropicalis was unable to d velop rapid resistance against compound 7f.

Pharmacokinetic Properties
The online softwares PreADMET and SwissADME were performed to further search the pharmacokinetic properties and druggability of thioether benzimidazole

Pharmacokinetic Properties
The online softwares PreADMET and SwissADME were performed to further research the pharmacokinetic properties and druggability of thioether benzimidazole 7f ( Table 2). The Lipinski rule, a crucial determinant in drug design and exploitation, was applied to assess theoretical pharmacological activity of thioether benzimidazole 7f [39]. Thioether benzimidazole 7f possessed the same bioavailability score with fluconazole and abided by Lipinski rule, which proved that 7f equipped good pharmacokinetic properties. Besides, thioether benzimidazole 7f displayed III category acute oral toxicity and passive response for blood-brain barrier (BBB) criteria, which indicated that compound 7f was uninjurious for oral administration. All pharmacokinetic parameters revealed that thioether benzimidazole 7f implemented considerable pharmacokinetic profile and outstanding drug-likeness.

Lipase Affinity of Thioether Benzimidazole 7f
Moreover, thioether benzimidazole 7f presented strong lipase affinity, which facilitated its permeation into cell membrane. As a crucial enzyme responsible for hydrolysis of lipids, lipase widely existed in plants, animals and microorganisms. Especially, the phospholipid layer on the surface of fungi contains a large number of lipases, and antifungal agents with strong lipase affinity can more easily combine with the cell membrane. Lipase is a single spherical polypeptide composed of more than 400 amino acid residues, including seven fixed fluorescent tryptophan [40]. Therefore, when the compound binds with lipase, the physiological environment of tryptophan residues and the enzyme structure will be significantly changed, and the corresponding fluorescence intensity will be decreased (λ ex = 290 nm, λ em = 340 nm). As shown in Figure 5, the fluorescence intensity of lipase at 340 nm decreased with the increase in the amount of compound 7f, indicating that compound 7f had strong lipase affinity.

Lipase Affinity of Thioether Benzimidazole 7f
Moreover, thioether benzimidazole 7f presented strong lipase affinit tated its permeation into cell membrane. As a crucial enzyme responsible of lipids, lipase widely existed in plants, animals and microorganisms. phospholipid layer on the surface of fungi contains a large number of lip fungal agents with strong lipase affinity can more easily combine with the c Lipase is a single spherical polypeptide composed of more than 400 amino including seven fixed fluorescent tryptophan [40]. Therefore, when the co with lipase, the physiological environment of tryptophan residues and the ture will be significantly changed, and the corresponding fluorescence in decreased (λex = 290 nm, λem = 340 nm). As shown in Figure 5, the fluorescen lipase at 340 nm decreased with the increase in the amount of compound that compound 7f had strong lipase affinity.

Membrane Damage Assay
Membrane depolarization undergoing with 7f was explored using a fluorescent probe diSC35. The diSC35 dye entering the active cell is separated by the inner and outer membranes of the fungal cell membrane, and its fluorescence gets quenched. However, the fluorescence intensity of diSC35 dye will increase following get out of the cell if the fungal membrane is depolarized by antifungal agents. As displayed in Figure 6A, compared with the dye labeled by untreated strain, a time-dependent increase was observed in the fluorescence intensity of the dye for C. tropicalis treated with thioether benzimidazole 7f, which indicated that 7f could interact with the cell membrane of C. tropicalis and cause its membrane depolarization. cause its membrane depolarization.
Moreover, the membrane permeability of C. tropicalis treated by thioether benzimidazole 7f was detected through estimating the uptake efficiency of propidium iodide (PI). As a living cell membrane impenetrable dye, PI can permeate the membranes of dead C. tropicalis strains, but cannot enter integrated living membranes [41][42][43]. The fact of a concentration-dependent growth in the PI fluorescence verified the potential of thioether benzimidazole 7f to cause physical destruction of the C. tropicalis membranes as depicted in Figure 6C. Further, the PI uptake could be visually confirmed. In Figure 6D, the red fluorescence appearance of PI dye for C. tropicalis incubated with compound 7f was distinctly observed, demonstrating that compound 7f could efficiently destroy the membrane integrity of C. tropicalis.
In addition to the transformation of membrane permeability, the leakage of proteins from C. tropicalis strains treated by thioether benzimidazole 7f was assessed employing standard Bradford assay. The result of protein leakage from C. tropicalis was presented in Figure 6B. It is proof that a dose-dependent enhancement in protein leakage was observed from C. tropicalis treated by thioether benzimidazole 7f, which indicated membrane damage and loss of cellular integrity for C. tropicalis strains.  Moreover, the membrane permeability of C. tropicalis treated by thioether benzimidazole 7f was detected through estimating the uptake efficiency of propidium iodide (PI). As a living cell membrane impenetrable dye, PI can permeate the membranes of dead C. tropicalis strains, but cannot enter integrated living membranes [41][42][43]. The fact of a concentration-dependent growth in the PI fluorescence verified the potential of thioether benzimidazole 7f to cause physical destruction of the C. tropicalis membranes as depicted in Figure 6C. Further, the PI uptake could be visually confirmed. In Figure 6D, the red fluorescence appearance of PI dye for C. tropicalis incubated with compound 7f was distinctly observed, demonstrating that compound 7f could efficiently destroy the membrane integrity of C. tropicalis.
In addition to the transformation of membrane permeability, the leakage of proteins from C. tropicalis strains treated by thioether benzimidazole 7f was assessed employing standard Bradford assay. The result of protein leakage from C. tropicalis was presented in Figure 6B. It is proof that a dose-dependent enhancement in protein leakage was observed from C. tropicalis treated by thioether benzimidazole 7f, which indicated membrane damage and loss of cellular integrity for C. tropicalis strains.

Supramolecular Interaction of Compound 7f with Cytochrome P450 Reductase
Cytochrome P450 reductase (CPR) (PDB ID: 6T1U) as an attractive target to investigate the antifungal mechanism was subjected into ligand-receptor docking to rationalize the observed antifungal activity and understand the possible mechanism. Compound 7f could form a biosupramolecular complex with CPR from C. tropicalis by multiple hydrogen bonds and other non-covalent interactions (Figure 7). The O atom of carbonyl group at 1-position in naphthalimide was bound to H atom of amino group in SER-441 with a space distance of 1.8 Å, and the H atom of hydroxyethyl segment could interact with O atom of carboxyl group in ASP-677 with a space distance of 1.9 Å. The N atom and H atom of benzimidazole fragment took part in hydrogen bonds reciprocity with TRP-679 and GLU-460 residues with a space distance of 2.3 Å and 1.9 Å, respectively. All these non-covalent interactions indicated that compound 7f could interact with cytochrome P450 reductase to disturb its biological function [44][45][46]. Cytochrome P450 reductase (CPR) (PDB ID: 6T1U) as an attractive target to gate the antifungal mechanism was subjected into ligand-receptor docking to ra the observed antifungal activity and understand the possible mechanism. Comp could form a biosupramolecular complex with CPR from C. tropicalis by multip gen bonds and other non-covalent interactions (Figure 7). The O atom of carbon at 1-position in naphthalimide was bound to H atom of amino group in SER-44 space distance of 1.8 Å, and the H atom of hydroxyethyl segment could interac atom of carboxyl group in ASP-677 with a space distance of 1.9 Å. The N atom and of benzimidazole fragment took part in hydrogen bonds reciprocity with TRP GLU-460 residues with a space distance of 2.3 Å and 1.9 Å, respectively. All th covalent interactions indicated that compound 7f could interact with cytochro reductase to disturb its biological function [44][45][46].

ROS-Mediated Dynamic Treatment
In addition to intrinsic structural advantages by supramolecular interacti DNA and CPR, thioether benzimidazole 7f could induce the up-regulation of ROS to cause inevitable impairment for cells. Additionally, thioether benzimid induced ROS production on the basis of fluorometric method by 2′,7′-dichlorof diacetate (DCFH-DA) dye was evaluated [47][48][49][50]. The fluorescence intensity of D dye at 528 nm preincubated by C. tropicalis strain and thioether benzimidazole 7f, a concentration-dependent augment, which obviously inferred that thioether b azole 7f could trigger ROS accumulation in Figure 8A. Reactive nitrogen inter (RNIs), such as NO, ONOOand S-nitrosothiols, are similar to ROS and can pathogen tissues independently or synergistically by acting on nucleic acids, pr lipids of pathogen [51]. As provided in Figure 8C, the variation trend of intracellu in C. tropicalis strains was estimated by Griess's reaction. It was proof from th quences that time and dose-dependent changes in RNIs production were noticed tropicalis treated by thioether benzimidazole 7f. The maximum generation of R tropicalis strains was acquired at 4 h with diverse contents of thioether benzimid and the generation of RNIs reduced and held constants after 4 h.

ROS-Mediated Dynamic Treatment
In addition to intrinsic structural advantages by supramolecular interactions with DNA and CPR, thioether benzimidazole 7f could induce the up-regulation of cytotoxic ROS to cause inevitable impairment for cells. Additionally, thioether benzimidazole 7finduced ROS production on the basis of fluorometric method by 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) dye was evaluated [47][48][49][50]. The fluorescence intensity of DCFH-DA dye at 528 nm preincubated by C. tropicalis strain and thioether benzimidazole 7f, occurred a concentration-dependent augment, which obviously inferred that thioether benzimidazole 7f could trigger ROS accumulation in Figure 8A. Reactive nitrogen intermediates (RNIs), such as NO, ONOOand S-nitrosothiols, are similar to ROS and can eradicate pathogen tissues independently or synergistically by acting on nucleic acids, proteins or lipids of pathogen [51]. As provided in Figure 8C, the variation trend of intracellular RNIs in C. tropicalis strains was estimated by Griess's reaction. It was proof from the consequences that time and dose-dependent changes in RNIs production were noticed from C. tropicalis treated by thioether benzimidazole 7f. The maximum generation of RNIs in C. tropicalis strains was acquired at 4 h with diverse contents of thioether benzimidazole 7f, and the generation of RNIs reduced and held constants after 4 h.
showed a continuous weakening of the GSH activity, and it was widely proved that the accumulation of ROS was advantageous to conquer the antioxidant defense system (Figure 8D). Moreover, the oxidative damage of the C. tropicalis undergoing treatment was assessed by Alamar blue (Resazurin) assay based on fluorescence spectra [55]. After cell was damaged, the Alamar blue dye turned into oxidation state (resazurin) from reduction state (resorufin) entering the cell, and the solution gradually changed from pink to blue ( Figure 8E,F).

Measurement of Metabolic Activity
Alamar blue (Resazurin) assay was applied to assess the intracellular metabolic activity of the C. tropicalis during treatment and analyze the cell activity and cell proliferation of C. tropicalis strains [56]. Alamar blue does not exhibit fluorescence in the oxidized state, but in the reduced state, it occurs a reduction product by pink or red fluorescence. The Alamar blue dye entering the viable cells was reduced by metabolic intermediates (NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD) and cytochromes, released into the outside of cells, and transformed from the non-fluorescent indigo blue to the fluorescent pink. However, inactive or damaged cells possessed lower metabolic activity and lower corresponding signals. The result displayed in Figure 9 showed that the Excessive ROS and RNIs are in an unbalanced state with the antioxidant protection mechanism, leading to occurrence of oxidative stress and dysfunction of cells. Membrane lipid peroxidation is one of the manifestations of oxidative stress. Malondialdehyde (MDA) is an extremely significant product of membrane lipid peroxidation, so the determination of MDA can help to understand the degree of membrane lipid peroxidation and further understand the degree of oxidative damage [52,53]. The production of MDA in C. tropicalis treated by 7f appeared a dose-dependent increase, which revealed the appearance of membrane lipid peroxidation and oxidative damage ( Figure 8B).
Glutathione is a marker for assessing oxidative stress, and exists in both reduced form (GSH) and oxidative form (GSSG). The production of excess ROS in the organism interferences the equilibrium of the redox system and leads to the conversion of GSH into GSSG. This degree of GSH to GSSG transformation results in a reduction in GSH activity as an indicator of oxidative stress that can be quantified through the Ellman experiment [54]. The experimental result of C. tropicalis integrated with increasing amount of 7f showed a continuous weakening of the GSH activity, and it was widely proved that the accumulation of ROS was advantageous to conquer the antioxidant defense system ( Figure 8D). Moreover, the oxidative damage of the C. tropicalis undergoing treatment was assessed by Alamar blue (Resazurin) assay based on fluorescence spectra [55]. After cell was damaged, the Alamar blue dye turned into oxidation state (resazurin) from reduction state (resorufin) entering the cell, and the solution gradually changed from pink to blue ( Figure 8E,F).

Measurement of Metabolic Activity
Alamar blue (Resazurin) assay was applied to assess the intracellular metabolic activity of the C. tropicalis during treatment and analyze the cell activity and cell proliferation of C. tropicalis strains [56]. Alamar blue does not exhibit fluorescence in the oxidized state, but in the reduced state, it occurs a reduction product by pink or red fluorescence. The Alamar blue dye entering the viable cells was reduced by metabolic intermediates (NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD) and cytochromes, released into the outside of cells, and transformed from the non-fluorescent indigo blue to the fluorescent pink. However, inactive or damaged cells possessed lower metabolic activity and lower corresponding signals. The result displayed in Figure 9 showed that the metabolic activity of C. tropicalis reduced upon treatment with thioether benzimidazole 7f. At the increased concentrations of compound 7f, metabolic activity was gradually decreased and finally metabolized inert. Thus, the decrease in metabolic activity clearly showed that the damage of cell membrane of C. tropicalis upon interacting with compound 7f observably impeded the cellular respiration of C. tropicalis, which disorganized respiration and caused metabolic arrest and loss of cell viability.
Molecules 2022, 27, x FOR PEER REVIEW 11 of 2 metabolic activity of C. tropicalis reduced upon treatment with thioether benzimidazol 7f. At the increased concentrations of compound 7f, metabolic activity was gradually de creased and finally metabolized inert. Thus, the decrease in metabolic activity clearly showed that the damage of cell membrane of C. tropicalis upon interacting with compound 7f observably impeded the cellular respiration of C. tropicalis, which disorganized respi ration and caused metabolic arrest and loss of cell viability.

Synergistic Effect of Chemical and Dynamic Antifungal Treatment for Hydroxyethyl Naphthalimide Antifungals
Based on the above, the prepared hydroxyethyl naphthalimides exhibited large in hibitory potentiality against the C. tropicalis strain through a synergistic effect of chemica and dynamic treatment, including DNA damage, membrane disruption, protein leakage metabolic deactivation and oxidative damage ( Figure 10).

Synergistic Effect of Chemical and Dynamic Antifungal Treatment for Hydroxyethyl Naphthalimide Antifungals
Based on the above, the prepared hydroxyethyl naphthalimides exhibited large inhibitory potentiality against the C. tropicalis strain through a synergistic effect of chemical and dynamic treatment, including DNA damage, membrane disruption, protein leakage, metabolic deactivation and oxidative damage ( Figure 10). metabolic activity of C. tropicalis reduced upon treatment with thioether benzimidazole 7f. At the increased concentrations of compound 7f, metabolic activity was gradually decreased and finally metabolized inert. Thus, the decrease in metabolic activity clearly showed that the damage of cell membrane of C. tropicalis upon interacting with compound 7f observably impeded the cellular respiration of C. tropicalis, which disorganized respiration and caused metabolic arrest and loss of cell viability.

Synergistic Effect of Chemical and Dynamic Antifungal Treatment for Hydroxyethyl Naphthalimide Antifungals
Based on the above, the prepared hydroxyethyl naphthalimides exhibited large inhibitory potentiality against the C. tropicalis strain through a synergistic effect of chemical and dynamic treatment, including DNA damage, membrane disruption, protein leakage, metabolic deactivation and oxidative damage ( Figure 10).

Instruments and Chemicals
Melting points were recorded on X-6 melting point apparatus and were uncorrected. TLC analysis was done using pre-coated silica gel plates. The 1 H NMR and 13 C NMR spectra were recorded on a Bruker AVANCE III 600 MHz spectrometer using TMS as an internal standard. The chemical shifts (δ) were reported in parts per million (ppm), the coupling constants (J) were expressed in hertz (Hz) and signals were described as singlet (s), doublet (d), triplet (t) as well as multiplet (m). The high resolution mass spectra (HRMS) were recorded on Bruker Impact II (Bremen, Germany). The purity was measured by HITACHI primaide (Japan). All raw materials and solvents were commercially available and were used without further purification.

Antifungal Assay
The newly synthesized compounds 2, 3a-b, 4a-c, 5, 6a-c, 7a-f and 8a-d were evaluated for their antifungal activities against Candida albicans (C. albicans), Candida albicans ATCC 90023 (C. albicans 90023), Candida tropicalis (C. tropicalis), Aspergillus fumigatus (A. fumigatus), Candida parapsilosis ATCC 22019 (C. parapsilosis 22019). A spore suspension in sterile distilled water was prepared from one day old culture of the fungi growing on Sabouraud Agar (SA) media. The final spore concentration was 1-5 × 10 3 spore mL −1 . The tested compounds and reference fluconazole were dissolved in DMSO to prepare the stock solutions, and diluted in sterile RPM1 1640 medium (Neuronbc Laboraton Technology C1., Ltd., Beijing, China) to get eleven wanted concentrations of each tested compound. These dilutions were inoculated and incubated at 37 • C for 24 h.

UV Absorption Spectra of Fluorophores with DNA
UV spectra were recorded at room temperature on a TU-2450 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells. The stock solutions of fluorophores were prepared in DMSO. Tris-HCl buffer solution (pH = 7.4) was prepared by mixing and diluting Tris (tris(hydroxymethyl)aminomethane) solution with HCl solution. Tris and HCl were analytical purity. Sample masses were weighed on a microbalance with a resolution of 0.1 mg. All other chemicals and solvents were commercially available, and were used without further purification.

Competitive Reaction of Compound 7f and AO or DAPI with DNA
The fluorescence emission spectra of compound 7f with AO-DNA and DAPI-DNA were recorded. The stock solution of compound 7f was prepared in DMSO, and acridine orange (AO) and 4 ,6-diamidino-2-phenylindole (DAPI) were prepared in distilled water. Tris-HCl buffer solution (pH = 7.4) was prepared by mixing and diluting Tris (tris(hydroxymethyl)aminomethane) solution with HCl solution. Tris and HCl were analytical purity. All other chemicals and solvents were commercially available, and were used without further purification.

Measurement of Intracellular ROS Production
Intracellular ROS was measured using standard 2,7-dichlorofluoroscein diacetate (DCFH-DA) assay [57,58]. Then, 10 6 CFU/mL of Candida tropicalis was treated with increas-ing concentrations of compound 7f for 6 h at 37 • C and 200 rpm. Following treatment, both control and treated cells were washed with PBS and incubated with 100 µM DCFH-DA probe for 30 min in dark at 37 • C. The green fluorescence originating from the oxidative cleavage of DCFH-DA to DCF was measured in a microplate reader with an excitation wavelength of 485 nm and emission wavelength of 528 nm. The increase in intracellular ROS production in cells treated with compound 7f in comparison to control cells was plotted.

Measurement of RNIs by Griess's Reaction
RNIs was measured using a spectrophotometric analysis of the total nitrite performed by using Griess's reagent [59,60]. The Candida tropicalis suspension (100 µL) were incubated with 100 µL of compound 7f (2 × MIC, 8 × MIC) at different times (1, 2, 3, 4, 5 and 6 h) at 37 • C. Then, 50 µL of 2% sulfanilamide in 5% (v/v) HCl and 50 µL of 0.1% N-(1naphthyl)ethylenediamine dihydrochloride aqueous solution were added. The formation of the azo dye was measured 15 min later by spectrophotometry at 540 nm. The OD was directly proportional to the nitrite content of the standard solution. Results were expressed respect to control without compound 7f.

Measurement of MDA
Malondialdehyde (MDA) content of cell-free extract was determined using microplate reader. Briefly, cell-free extract was mixed with TBA/TCA/HCl (15%, 0.37%) at a reagent/sample ratio of 2:1 (v/v), placed in a boiling water bath for 15 min, cooled to room temperature, and centrifuged at 1000× g for 10 min at room temperature. The absorbance of the solution was read at 535 nm against the blank using microplate reader.

Measurement of Intracellular Glutathione (GSH) Activity
The activity of intracellular GSH was determined using standard Ellman's assay [61]. Then, 10 6 CFU/mL of Candida tropicalis was treated with increasing concentrations of compound 7f for 6 h at 37 • C and 200 rpm. Following treatment, both control and treated cells were centrifuged at 5000 rpm for 5 min, washed with PBS, and lysed. The lysed cells were further centrifuged, and the clear supernatant was collected. The supernatant was mixed with 50 mM Tris-HCl and 100 mM 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and incubated for 30 min in dark at 37 • C. The absorbance of the resulting solution was measured at 412 nm using microplate reader.

Measurement of Alamar Blue Assay
Following 48 h of C. tropicalis growth, the media were replaced with fresh media containing increasing concentrations of compound 7f (MIC, 2 × MIC, 4 × MIC, 6 × MIC and 8 × MIC). The strain was treated with compound 7f for 24 h at 37 • C in a moist environment under static conditions. Following 24 h of treatment, the media were removed from the wells, and the strain was washed twice with PBS carefully to remove planktonic cells. Then, 100 µL of LB broth containing 10 µL of 5 µg/mL resazurin was added to the wells, and the plate was incubated for 45 min at 37 • C. Then, took photos for these wells, and fluorescence was measured at 571 nm excitation and 590 nm emission.

Drug Resistance Development Assay
The strain of C. tropicalis was exposed to sub-MICs of compound 7f for sustained passages, which determined every 24 h after propagation of C. tropicalis cultures and then the MIC of 7f were determined against each passage of the strain. To make comparative analysis, fluconazole was used as the control experiment. The experiment was sustained for 16 passages.

Hemolysis Assay
After washing and resuspending in PBS, 2% of human red blood cell was added to a 96-well plate with 100 µL per well. Then, the same volume of compound 7f in various concentrations was added. 0.5% Triton X-100 (v:v) and PBS were used as positive control and negative control, respectively. After co-incubation at room temperature for one hour, the plate was centrifuged at 1500 rpm for 10 min. The absorbance of 100 µL of the supernatant was measured at 450 nm. The experiments were performed in triplicate, and the hemolysis percentage was calculated as follows: Hemolysis (%) = (A 7f − A PBS )/(A Triton − A PBS ) × 100%.

In Vitro Cytotoxicity
The cytotoxicity assays were determined with LO2 cells under normal training conditions. LO2 cells were inoculated into a sterile 96-well plates with a density of 4 × 10 −4 cells·mL −1 . Compound 7f was put in DMSO and diluted with culture media. After 24 h, 7f were put in the cultured LO2 cells for 24 h. Cell viability was determined by measuring the absorbance of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide (MTT) assay at 570 nm. Each test was conducted in triplicate.

Membrane Depolarization Assay
Candida tropicalis strain in their mid log phase (OD 600 = 0.4-0.5) were washed with a buffer solution (5 mM HEPES buffer, 5 mM glucose, pH 7.2) and redispersed in the same buffer to an OD 600 of 0.1. The redispersed cells were then incubated with 0.4 µM of 3,3 -dipropylthiadicarbocyanine iodide (diSC35) dye for 1 h at 37 • C, following which 100 mM KCl was added to the suspensions. After incubation with dye, the Candida tropicalis strain was treated with compound 7f at MIC concentration, and the fluorescence of the treated cells was monitored periodically over a period of 1 h in fluorescence photometer set to an excitation wavelength of 622 nm and emission wavelength of 670 nm. Increase in fluorescence with time indicated membrane depolarization.

Protein Leakage Assay
Candida tropicalis (10 6 CFU/mL) was treated with increasing concentrations of compound 7f for 6 h at 37 • C and 200 rpm. Following treatment, the cell was pelleted down at 5000 rpm for 5 min, and the cell-free supernatant was collected. The concentration of leaked proteins in the supernatant was measured using standard Bradford assay.

Measurement of Metabolic Activity
The metabolic activity of C. tropicalis was measured using Alamar blue assay which is based on the ability of cells to convert a purple nonfluorescent dye resazurin to its pink fluorescent reduced form resofurin. Then, 10 6 CFU/mL of C. tropicalis was treated with increasing concentrations of compound 7f for 6 h at 37 • C and 200 rpm. Both control and treated cells were incubated with 25 µL of 50 µg/mL resazurin solution for 1 h at 37 • C. The metabolic conversion of resazurin to pink colored resofurin was quantified spectrophotometrically by measuring absorbance at 571 nm.

Molecular Docking
The structure of cytochrome P450 reductase (CPR) employed in the docking calculations was obtained using RCSB Protein Data Bank (PDB ID: 6T1U). The structures of compound 7f were drawn with ChemDraw 19.0. Docking analyses were performed with the Sybyl-X 2.0 and pymol program. The gird size was set to be 45 × 45 × 45 and the grid point spacing was set at default value 0.375 Å. The Lamarkian genetic algorithm (LGA) was applied for the conformational search.

Conclusions
In conclusion, a desirable family of hydroxyethyl naphthalimides with synergistic chemical and dynamic antifungal treatment were favourably discovered. These prepared compounds showed significant antifungal potency towards some tested fungi including A. fumigatus, C. tropicalis and C. parapsilosis 22019. Especially, thioether benzimidazole 7f with excellent DNA binding ability gave better anti-C. tropicalis efficacy than fluconazole.
Moreover, 7f presented low cytotoxicity, safe hemolysis level and no obvious resistance. The strong lipase affinity of 7f facilitated its permeation into cell membrane to cause membrane dysfunction. The studies of biological mechanisms directed by ROS and RNIs indicated prominent enhancement of intracellular oxidative damage with membrane lipid peroxidation and oxidization of GSH into GSSG, which destructed the antioxidant defence system of C. tropicalis and caused cell death. Under the collective participation of chemical and dynamic antifungal treatment in the killing of C. tropicalis, the fact that disruption of biological function for DNA and CPR, metabolic inactivation was displayed. By extending on this base, a battery of chemical biological studies implied that hydroxyethyl naphthalimides should be hopeful to be further exploited as specific antifungal drugs.