Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 µg/mL against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

From Synergy to Monotherapy: Discovery of Novel 2,4,6-Trisubstituted Triazine Hydrazone Derivatives with Potent Antifungal Potency In Vitro and In Vivo.

Invasive fungal infections pose a serious threat to public health and are associated with high mortality and incidence rates. The development of novel antifungal agents is urgently needed. Based on hit-to-lead optimization, a series of 2,4,6-trisubstituted triazine hydrazone compounds were designed, synthesized, and biological evaluation was performed, leading to the identification of compound 28 with excellent in vitro synergy (FICI range: 0.094-0.38) and improved monotherapy potency against fluconazole-resistant Candida albicans and Candida auris (MIC range: 1.0-16.0 µg/mL). Moreover, 28 exhibited broad-spectrum antifungal activity against multiple pathogenic strains. Furthermore, 28 could inhibit hyphal and biofilm formation, which may be related to its ability to disrupt the fungal cell wall. Additionally, 28 significantly reduced the CFU in a mouse model of disseminated infection with candidiasis at a dose of 10 mg/kg. Overall, the triazine-based hydrazone compound 28 with low cytotoxicity, hemolysis, and favorable ADME/T characteristics represents a promising lead to further investigation.

Design, synthesis and evaluation of novel deferasirox derivatives with high antifungal potency in vitro and in vivo.

Here we designed and synthesized 58 deferasirox derivatives with the aim of discovering novel antifungal agents. Most compounds exhibited moderate to excellent in vitro antifungal activities against Cryptococcus neoformans H99 with MIC values ranging from 0.25 µg/mL to 16 µg/mL, including ten compounds with MIC values less than 1 µg/mL that were further screened against an additional six pathogenic fungi. This class of compounds showed high potency against Candida glabrata with MIC values ranging from <0.125 µg/mL to 1 µg/mL. We identified that compound 54 has high potency against 14 strains of Candida glabrata spp. and Cryptococcus spp. with MIC values ranging from <0.125 µg/mL to 1 µg/mL. In addition, compound 54 significantly reduced the CFU in a mouse model of disseminated infection with Cryptococcus neoformans H99 at a dose of 10 mg/kg, which is comparable to FLC. Further investigations on compound 54 are currently in progress.

Discovery of Novel 8-Hydroxyquinoline Derivatives with Potent In Vitro and In Vivo Antifungal Activity.

Fungal pathogens can cause life-threatening infections, yet current antifungals are inadequate at treating many of these, highlighting the importance of novel drug discovery. Here, we report hit compound L14, a novel 8-hydroxyquinoline derivative with potent and broad-spectrum antifungal activity. In vitro experiments exhibited that L14 had better activity and lower cytotoxicity than that of clioquinol and showed synergy in combination with fluconazole (FLC). In a Candida albicans-infected murine model, L14 at 2 mg/kg showed better in vivo efficacy than clioquinol at reducing fungal burden and extending the survival of C. albicans-infected mice. In addition, L14 alone or in combination with FLC had significant inhibitory activity against hypha and biofilm formation. Overall, our data indicated that 8-hydroxyquinoline derivative L14 has favorable pharmacokinetics and acceptable safety profiles and could be further investigated as a promising antifungal hit compound.

Design, synthesis and in vitro biological studies of novel triazoles with potent and broad-spectrum antifungal activity.

A series of novel triazole derivatives containing aryl-propanamide side chains was designed and synthesised. In vitro antifungal activity studies demonstrated that most of the compounds inhibited the growth of six human pathogenic fungi. In particular, parts of phenyl-propionamide-containing compounds had excellent, broad-spectrum antifungal activity against Candida albicans SC5314, Cryptococcus neoformans 22-21, Candida glabrata 537 and Candida parapsilosis 22-20 with MIC values in the range of ≤0.125 µg/mL-4.0 µg/mL. In addition, compounds A1, A2, A6, A12 and A15 showed inhibitory activities against fluconazole-resistant Candida albicans and Candida auris. Preliminary structure-activity relationships (SARs) are also summarised. Moreover, GC-MS analysis demonstrated that A1, A3, and A9 interfered with the C. albicans ergosterol biosynthesis pathway by inhibiting Cyp51. Molecular docking studies elucidated the binding modes of A3 and A9 with Cyp51. These compounds with low haemolytic activity and favourable ADME/T properties are promising for the development of novel antifungal agents.

Discovery of novel triazoles containing benzyloxy phenyl isoxazole side chain with potent and broad-spectrum antifungal activity.

As a continuation study, 29 novel triazoles containing benzyloxy phenyl isoxazole side chain were designed and synthesized based on our previous work. The majority of the compounds exhibited high potency in vitro antifungal activities against eight pathogenic fungi. The most active compounds 13, 20 and 27 displayed outstanding antifungal activity with MIC values ranging from <0.008 µg/mL to 1 µg/mL, and showed potent activity against six drug-resistant Candida auris isolates. Growth curve assays further confirmed the high potency of these compounds. Moreover, compounds 13, 20 and 27 showed a potent inhibitory activity on biofilm formation of C. albicans SC5314 and C. neoformans H99. Notably, compound 13 showed no inhibition of human CYP1A2 and low inhibitory activity against CYP2D6 and CYP3A4, suggesting a low risk of drug-drug interactions. With high potency in vitro and in vivo and good safety profiles, compound 13 will be further investigated as a promising candidate.

Discovery of Novel Tetrazoles Featuring a Pyrazole Moiety as Potent and Highly Selective Antifungal Agents.

In pursuit of developing novel azole antifungals with potent activity and high selectivity, a series of (2R,3S)-3-(substituted-1H-pyrazol-3-yl)-2-(2,4-difluorophenyl)-1-(1H-tetrazol-1-yl)butan-2-ol derivatives were designed and synthesized based on our previous work. All compounds exhibited excellent in vitro antifungal activities against Candida spp. and Cryptococcus neoformans H99 with minimum inhibitory concentration values ranging from <0.008 to 4 µg/mL, with some even showing moderate activity against Aspergillus fumigatus 7544. The most active compounds (8, 11, 15, 24, and 25) displayed outstanding antifungal activity against six fluconazole-resistant C. auris clinical isolates and showed a potent inhibitory effect on biofilm formation of C. albicans SC5314 and C. neoformans H99. In addition, compounds 11 and 15 showed no inhibition of human CYP1A2 and low inhibitory activity against CYP3A4, indicating minimal risk of drug-drug interactions. Taken together, these promising tetrazoles with high in vitro potency and good safety profiles warrant further investigation.

Novel antifungal triazoles with alkynyl-methoxyl side chains: Design, synthesis, and biological activity evaluation.

Previous work led to the rational design, synthesis and testing of novel antifungal triazole analogues bearing alkynyl-methoxyl side chains. Tests of in vitro antifungal activity showed Candida albicans SC5314 and Candida glabrata 537 gave MIC values of ≤0.125 µg/mL for most of the compounds. Among these, compounds 16, 18, and 29 displayed broad-spectrum antifungal activity against seven human pathogenic fungal species, two fluconazole-resistant C. albicans isolates and two multi-drug resistant Candida auris isolates. Moreover, 0.5 µg/mL of 16, 18, and 29 was more effective than 2 µg/mL of fluconazole at inhibiting fungal growth of the strains tested. The most active compound (16) completely inhibited the growth of C. albicans SC5314 at 16 µg/mL for 24 h, affected biofilm formation and destroyed the mature biofilm at 64 µg/mL. Several Saccharomyces cerevisiae strains, overexpressing recombinant Cyp51s or drug efflux pumps, indicated 16, 18, and 29 targeted Cyp51 without being significantly affected by a common active site mutation, but were susceptible to target overexpression and efflux by both MFS and ABC transporters. GC-MS analysis demonstrated that 16, 18, and 29 interfered with the C. albicans ergosterol biosynthesis pathway by inhibition at Cyp51. Molecular docking studies elucidated the binding modes of 18 with Cyp51. The compounds showed low cytotoxicity, low hemolytic activity and favorable ADMT properties. Importantly, compound 16 showed potent in vivo antifungal efficacy in the G. mellonella infection model. Taken together, this study presents more effective, broad-spectrum, low toxicity triazole analogues that can contribute to the development of novel antifungal agents and help overcome antifungal resistance.

Discovery of novel thiosemicarbazone derivatives with potent and selective anti-Candida glabrata activity.

A series of 21 novel compounds containing a thiosemicarbazone moiety were designed and synthesised based on hit compound 1 from our in-house compound library screening. Most compounds showed potent antifungal activity in vitro against seven common pathogenic fungi. Notably, all compounds showed high potency against Candida glabrata 537 (MIC = ≤0.0156-2 µg/mL). Of note, compounds 5j and 5r displayed excellent antifungal activity against Candida krusei 4946 and Candida auris 922. Additionally, compounds 5j and 5r also showed high potency against 15 C. glabrata isolates with MIC values ranging from 0.0625 µg/mL to 4 µg/mL, with compound 5r being slightly superior to 5j. Moreover, compound 5r has certain effect against biofilm formation of C. glabrata. Furthermore, compound 5r has minimal cytotoxicity against HUVECs with an IC50 value of 15.89 µg/mL and no haemolysis at 64 µg/mL. Taken together, these results suggest that promising lead compound 5r deserves further investigation.

Design, Synthesis and Structure-Activity Relationship Studies of Nicotinamide Derivatives as Potent Antifungal Agents by Disrupting Cell Wall.

Fungal infections pose a serious challenge to human health due to the limited paucity of antifungal treatments. Starting as a hit compound screened from our compound library, a series of nicotinamide derivatives have been successfully synthesized via a facile one-step coupling reaction of aromatic carboxylic acid and amine. The synthesized compounds were evaluated for their antifungal activity against Candida albicans SC5314. Among the 37 nicotinamide derivatives screened, compound 16g was found to be the most active against C. albicans SC5314, with an MIC value of 0.25 µg/mL and without significant cytotoxicity. The rudimentary structure-activity relationships study revealed that the position of the amino and isopropyl groups of 16g was critical for its antifungal activity. In particular, compound 16g showed potent activity against six fluconazole-resistant C. albicans strains with MIC values ranging from 0.125-1 µg/mL and showed moderate activity against the other seven species of Candida, three strains of Cryptococcus neoformans, and three strains of Trichophyton. Furthermore, compound 16g showed fungicidal, anti-hyphal, and anti-biofilm activities in vitro, which were related to its ability to disrupt the cell wall of C. albicans. Taken together, 16g is a promising compound that is fungal-specific by targeting the cell wall and could be used as a lead compound for further investigation.

Design, synthesis, and evaluation of novel tetrazoles featuring isoxazole moiety as highly selective antifungal agents.

In an effort to develop novel azole antifungals with potent activity and high selectivity, a series of (2R,3R)-3-((3-substitutied-phenyl-isoxazol-5-yl)methoxy)-2-(2,4-difluorophenyl)-1-(1H-tetrazol-1-yl)butan-2-ol derivatives were designed and synthesized based on our previously work. All compounds exhibited moderate to excellent in vitro antifungal activities against Candida albicans SC5314 and Cryptococcus neoformans H99, but inactive against Aspergillus fumigatus 7544. Among them, the most active compound 10h displayed outstanding antifungal activity against fluconazole-resistant C. albicans 103, C. glabrata 537 and C. auris 922 with MIC values of ≤0.008 µg/mL. In addition, compound 10h was superior to FLC in inhibiting the filamentation of FLC-resistant C. albicans 103. Notably, compound 10h showed no inhibition of human CYP3A4 with IC50 values of >100 µM, low cytotoxicity at 32 µg/mL and low hERG inhibition with IC50 values of 6.22 µM, suggesting a low risk of drug-drug interactions and good safety profiles. Furthermore, compound 10h exhibited excellent PK profiles and showed remarkable in vivo efficacy in a mouse model of C. albicans and C. neoformans infection. Taken together, compound 10h will be further investigated as a promising lead antifungal agent.

A Small Molecule Inhibitor of Erg251 Makes Fluconazole Fungicidal by Inhibiting the Synthesis of the 14α-Methylsterols.

Fluconazole (FLC) is widely used to prevent and treat invasive fungal infections. However, FLC is a fungistatic agent, allowing clinical FLC-susceptible isolates to tolerate FLC. Making FLC fungicidal in combination with adjuvants is a promising strategy to avoid FLC resistance and eliminate the persistence and recurrence of fungal infections. Here, we identify a new small molecule compound, CZ66, that can make FLC fungicidal. The mechanism of action of CZ66 is targeting the C-4 sterol methyl oxidase, encoded by the ERG251 gene, resulting in decreased content of sterols with the 14α-methyl group and ultimately eliminating FLC tolerance of Candida albicans. CZ66 most likely interacts with Erg251 through residues Glu195, Gly206, and Arg241. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance. IMPORTANCE Fluconazole (FLC) tolerance increases the frequency of acquired FLC resistance, and a high FLC tolerance level is associated with persistent candidemia. Multiple functional proteins, such as calcineurin, heat shock protein 90 (Hsp90), and ADP ribosylation factor, are essential for the survival of C. albicans exposed to FLC, but how these factors increase the fungicidal activity of FLC remains to be determined. In this study, we found that 14α-methylsterols replace ergosterol to allow C. albicans to survive FLC, but Erg251 inactivated by CZ66 results in loss of 14α-methylsterol synthesis and cell death of C. albicans treated with FLC. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance.

Discovery of Novel Orally Bioavailable Triazoles with Potent and Broad-Spectrum Antifungal Activity In Vitro and In Vivo.

In our continuing efforts to discover novel triazoles with improved antifungal activity in vitro and in vivo, a series of 41 novel compounds containing 1,2,3-triazole side chains were designed and synthesized via a click reaction based on our previous work. Most of the compounds showed moderate to excellent broad-spectrum antifungal activity in vitro. Among them, the most promising compound 9A16 displayed excellent antifungal and anti-drug-resistant fungal ability (MIC80 = 0.0156-8 µg/mL). In addition, compound 9A16 showed powerful in vivo efficacy on mice systematically infected with Candida albicans SC5314, Cryptococcus neoformans H99, fluconazole-resistant C. albicans 100, and Aspergillus fumigatus 7544. Moreover, compared to fluconazole, compound 9A16 showed better in vitro anti-biofilm activity and was more difficult to induce drug resistance in a 1 month induction of resistance assay in C. albicans. With favorable pharmacokinetics, an acceptable safety profile, and high potency in vitro and in vivo, compound 9A16 is currently under preclinical investigation.

Discovery of Novel Thiosemicarbazides Containing 1,3,5-Triazines Derivatives as Potential Synergists against Fluconazole-Resistant Candida albicans.

The clinical prevalence of antifungal drug resistance has been increasing over recent years, resulting in the failure of treatments. In an attempt to overcome this critical problem, we sought novel synergistic enhancers to restore the effectiveness of fluconazole against resistant Candida albicans. Based on the structural optimization of hit compound 8 from our in-house library, a series of novel 1,3,5-triazines derivatives was designed, synthesized, and biologically evaluated for synergistic activity in combination with fluconazole. Among them, compounds 10a-o, which contain thiosemicarbazides side chains, exhibited excellent in vitro synergistic antifungal potency (MIC80 = 0.125-2.0 µg/mL, FICI range from 0.127 to 0.25). Interestingly, compound 10l exhibited moderate C. albicans activity as monotherapy with an MIC80 value of 4.0 µg/mL, and also on several Cryptococcus strains (MIC80 ranging from ≤ 0.125-0.5 µg/mL) and C. glabrata (MIC80 ≤ 0.125 µg/mL). These effects were fungal-selective, with much lower levels of cytotoxicity towards human umbilical vein endothelial cells. Here, we report a series of thiosemicarbazides containing 1,3,5-triazines derivatives as potent synergists with fluconazole, and have preliminarily validated compound 10l as a promising antifungal lead for further investigation.

Design, synthesis, and in vitro evaluation of novel antifungal triazoles containing substituted 1,2,3-triazole-methoxyl side chains.

In order to develop new triazole derivatives, we optimized the lead compound a6 by structural modifications to obtain a series of (2R,3R)-3-((1-substituted-1H-1,2,3-triazol-4-yl) methoxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl) butan-2-ol, compounds 5-36. Most of the target compounds exhibited excellent in vitro antifungal activity against Candida albicans 10231 and Candida glabrata 537 with MIC ≤ 0.125 µg/mL. Of particular note, compounds 6, 22, 28, 30 and 36 were highly active against Candida neoformans 32609 with MIC ≤ 0.125 µg/mL and showed broad-spectrum antifungal activity including against fluconazole-resistant Candida auris 891. In addition, compounds 6 and 22 demonstrated inhibitory effects on filamentation in the azole-resistant C. albicans isolate. Moreover, compounds 6 and 22 were minimally toxic to HUVECs and possessed weak inhibitory effects on the human CYP3A4 and CYP2D6. SARs and docking study further indicated that ortho-substituted groups in the terminal phenyl ring can promote the compounds to improve their antifungal activity.

Discovery of Novel 7-Hydroxy-5-oxo-4,5-dihydrothieno[3,2-b]pyridine-6-carboxamide Derivatives with Potent and Selective Antifungal Activity against Cryptococcus Species.

Cryptococcus neoformans and Cryptococcus gattii can cause fatal invasive infections, especially in immunocompromised patients. However, few antifungal drugs are available to help treat cryptococcosis. In this study, by compound library screening, we presented the first report of hit compound P163-0892, which had potent in vitro and in vivo antifungal activity against Cryptococcus spp. In vitro tests showed that P163-0892 was not cytotoxic and had highly selective and strong antifungal activities against Cryptococcus spp. with MIC values less than 1 µg/mL. Synergism of P163-0892 and fluconazole was also observed in vitro. The in vivo antifungal efficacy of P163-0892 was assessed in a wax moth larval fungal infection model, and treatment with 10 mg/kg P163-0892 caused a significant reduction in fungal burden and significant extension of the survival time. Taken together, our data indicate that the hit compound P163-0892 warrants further investigation as a novel anti-Cryptococcus agent.

Design, Synthesis, and In Vitro and In Vivo Antifungal Activity of Novel Triazoles Containing Phenylethynyl Pyrazole Side Chains.

A series of triazole derivatives containing phenylethynyl pyrazole moiety as side chain were designed, synthesized, and most of them exhibited good in vitro antifungal activities. Especially, compounds 5k and 6c showed excellent in vitro activities against C. albicans (MIC = 0.125, 0.0625 µg/mL), C. neoformans (MIC = 0.125, 0.0625 µg/mL), and A. fumigatus (MIC = 8.0, 4.0 µg/mL). Compound 6c also exerted superior activity to compound 5k and fluconazole in inhibiting hyphae growth of C. albicans and inhibiting drug-resistant strains of C. albicans, and it could reduce fungal burdens in mice kidney at a dosage of 1.0 mg/kg. An in vivo efficacy evaluation indicated that 6c could effectively protect mice models from C. albicans infection at doses of 0.5, 1.0, and 2.0 mg/kg. These results suggested that compound 6c deserves further investigation.

A New Antifungal Agent (4-phenyl-1, 3-thiazol-2-yl) Hydrazine Induces Oxidative Damage in Candida albicans.

A gradual rise in immunocompromised patients over past years has led to the increasing incidence of invasive fungal infections. Development of effective fungicides can not only provide new means for clinical treatment, but also reduce the occurrence of fungal resistance. We identified a new antifungal agent (4-phenyl-1, 3-thiazol-2-yl), hydrazine (numbered as 31C) which showed high-efficiency, broad-spectrum and specific activities. The minimum inhibitory concentration of 31C against pathogenic fungi was between 0.0625-4 µg/ml in vitro, while 31C had no obvious cytotoxicity to human umbilical vein endothelial cells with the concentration of 4 µg/ml. In addition, 31C of 0.5 µg/ml could exhibit significant fungicidal activity and inhibit the biofilm formation of C. albicans. In vivo fungal infection model showed that 31C of 10 mg/kg significantly increased the survival rate of Galleria mellonella. Further study revealed that 31C-treatment increased the reactive oxygen species (ROS) in C. albicans and elevated the expression of some genes related to anti-oxidative stress response, including CAP1, CTA1, TRR1, and SODs. Consistently, 31C-induced high levels of intracellular ROS resulted in considerable DNA damage, which played a critical role in antifungal-induced cellular death. The addition of ROS scavengers, such as glutathione (GSH), N-Acetyl-L-cysteine (NAC) or oligomeric proanthocyanidins (OPC), dramatically reduced the antifungal activities of 31C and rescued the 31C-induced filamentation defect. Collectively, these results showed that 31C exhibited strong antifungal activity and induced obvious oxidative damage, which indicated that compounds with a structure similar to 31C may provide new sight for antifungal drug development.

11g, a Potent Antifungal Candidate, Enhances Candida albicans Immunogenicity by Unmasking ß-Glucan in Fungal Cell Wall.

In the course of optimizing GPI biosynthesis inhibitors, we designed and synthetized a 2-aminonicotinamide derivative named 11g. After evaluating the antifungal activity of compound 11g in vitro, we investigated the influences of 11g on fungi immunogenicity. In addition, we also took advantage of murine systemic candidiasis model to investigate the protective effects of 11g in vivo. Results show that 11g exhibited potent antifungal activity both in vitro and in vivo. Further study shows that 11g caused the unmasking of fungi ß-glucan layer, leading to stronger immune responses in macrophages through Dectin-1. These results suggest that 11g is a very promising antifungal candidate, which assists in eliciting stronger immune responses to help host immune system disposing pathogens. The discovery of 11g might expand the toolbox of fungal infection treatment.

Design, synthesis, and in vitro evaluation of novel triazole analogues featuring isoxazole moieties as antifungal agents.

In order to develop novel antifungal agents, based on our previous work, a series of (2R,3R)-3-((3-substitutied-isoxazol-5-yl)methoxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl) butan-2-ol (a1-a26) were designed and synthesized. All of the compounds exhibited good in vitro antifungal activities against eight human pathogenic fungi. Among them, compound a6 showed excellent inhibitory activity against Candida albicans and Candida parasilosis with MIC80 values of 0.0313 µg/mL. In addition, compounds a6, a9, a12, a13 and a14 exhibited moderate inhibitory activities against fluconazole-resistant isolates with MIC80 values ranging from 8 µg/mL to 16 µg/mL. Furthermore, compounds a6, a12 and a23 exhibited low inhibition profiles for CYP3A4. Clear SARs were analyzed, and the molecular docking experiment was carried out to further investigate the relationship between a6 and the target enzyme CYP51.