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 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 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.

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 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.

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.

New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo.

In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 µg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 µg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

Design, synthesis, and structure-activity relationship studies of novel triazole agents with strong antifungal activity against Aspergillus fumigatus.

The incidence of invasive fungal infections has dramatically increased for several decades. In order to discover novel antifungal agents with broad spectrum and anti-Aspergillus efficacy, a series of novel triazole derivatives containing 1,2,3-benzotriazin-4-one was designed and synthesized. Most of the compounds exhibited stronger in vitro antifungal activities against tested fungi than fluconazole. Moreover, 6m showed comparable antifungal activity against seven pathogenic strains as voriconazole and albaconazole, especially against Aspergillus fumigatus (MIC = 0.25 µg/ml), and displayed moderate antifungal activity against fluconazole-resistant strains of Candida albicans. A clear SAR study indicated that compounds with groups at the 7-position resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.

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.

Chemogenomic Profiling of the Fungal Pathogen Candida albicans.

There is currently a small number of classes of antifungal drugs, and these drugs are known to target a very limited set of cellular functions. We derived a set of approximately 900 nonessential, transactivator-defective disruption strains from the tetracycline-regulated GRACE collection of strains of the fungal pathogen Candida albicans This strain set was screened against classic antifungal drugs to identify gene inactivations that conferred either enhanced sensitivity or increased resistance to the compounds. We examined two azoles, fluconazole and posaconazole; two echinocandins, caspofungin and anidulafungin; and a polyene, amphotericin B. Overall, the chemogenomic profiles within drug classes were highly similar, but there was little overlap between classes, suggesting that the different drug classes interacted with discrete networks of genes in C. albicans We also tested two pyridine amides, designated GPI-LY7 and GPI-C107; these drugs gave very similar profiles that were distinct from those of the echinocandins, azoles, or polyenes, supporting the idea that they target a distinct cellular function. Intriguingly, in cases where these gene sets can be compared to genetic disruptions conferring drug sensitivity in other fungi, we find very little correspondence in genes. Thus, even though the drug targets are the same in the different species, the specific genetic profiles that can lead to drug sensitivity are distinct. This implies that chemogenomic screens of one organism may be poorly predictive of the profiles found in other organisms and that drug sensitivity and resistance profiles can differ significantly among organisms even when the apparent target of the drug is the same.

Design, synthesis, and in vitro evaluation of novel antifungal triazoles.

Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and synthesized. Most of the compounds exhibited potent in vitro antifungal activities against 8 fungal isolates. Especially, compounds a10, a13, and a14 exhibited superior or comparable antifungal activity to ravuconazole against all the tested fungi. Structure-activity relationship study indicated that replacing 4-cyanophenylthioazole moiety of ravuconazole with fluorophenylisoxazole resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.

Design, synthesis, and SAR study of 3-(benzo[d][1,3]dioxol-5-yl)-N-benzylpropanamide as novel potent synergists against fluconazole-resistant Candida albicans.

Based on our previous discovery and SAR study on the lead compounds 7d, 5 and berberine which can significantly enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans, a series of 3-(benzo[d][1,3]dioxol-5-yl)-N-(substituted benzyl)propanamides were designed, synthesized, and evaluated for their in vitro synergistic activity in combination with fluconazole. The series 2a-f were designed by replacing the amide moiety of the lead compound 7d with retro-amide moiety, and compounds 2a and 2b showed more activity than the lead 7d. Furthermore, introducing biphenyl moiety into series 2d-f afforded series 3a-r, most of which exhibited significantly superior activity to the series 2d-f. Especially, compound 3e, at a concentration of 1.0µg/ml, can enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans from 128.0µg/ml to 0.125-0.25µg/ml. A clear SAR of the compounds is discussed.

Design, synthesis, and evaluation of caffeic acid amides as synergists to sensitize fluconazole-resistant Candida albicans to fluconazole.

A series of caffeic acid amides were designed, synthesized, and their synergistic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The title caffeic acid amides 3-30 except 26 exhibited potent activity, and the subsequent SAR study was conducted. Compound 3, 5, 21, and 34c, at a concentration of 1.0 µg/ml, decreased the MIC80 of fluconazole from 128.0 µg/ml to 1.0-0.5 µg/ml against the fluconazole-resistant C. albicans. This result suggests that the caffeic acid amides, as synergists, can sensitize drug-resistant fungi to fluconazole. The SAR study indicated that the dihydroxyl groups and the amido groups linking to phenyl or heterocyclic rings are the important pharmacophores of the caffeic acid amides.

Berberine inhibits fluphenazine-induced up-regulation of CDR1 in Candida albicans.

Over-expression of the Candida drug resistance gene CDR1 is a common mechanism generating azole-resistant Candida albicans in clinical isolates. CDR1 is transcriptionally activated through the binding of the transcription factor Tac1p to the cis-acting drug-responsive element (DRE) in its promoter. We previously demonstrated that the combination of fluconazole (FLC) and berberine (BBR) produced significant synergy when used against FLC-resistant C. albicans in vitro. In this study, we found that BBR inhibited both the up-regulation of CDR1 mRNA and the transport function of Cdr1p induced by fluphenazine (FNZ). Further, electrophoretic mobility shift assays suggested that the transcription activation complex of protein-DRE was disrupted by BBR, and electrospray ionization mass spectrometry analysis showed that BBR bound to the DRE of CDR1. Thus we propose that BBR inhibits the FNZ-induced transcriptional activation of CDR1 in C. albicans by blocking transcription factor binding to the DRE of CDR1. These results contribute to our understanding of the mechanism of synergistic effect of BBR and FLC.

[Derivatization of berberine based on its synergistic antifungal activity with fluconazole against fluconazole-resistant Candida albicans].

Abstract: Our previous work revealed berberine can significantly enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans, which suggested that berberine has synergistic antifungal activity with fluconazole. Preliminary SAR of berberine needs to be studied for the possibility of investigating its target and SAR, improving its drug-likeness, and exploring new scaffold. In this work, 13-substitutited benzyl berberine derivatives and N-benzyl isoquinoline analogues were synthesized and characterized by 1H NMR and MS. Their synergetic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The 13-substitutited benzyl berberine derivatives 1a-1e exhibited comparable activity to berberine, which suggested that the introduction of functional groups to C-13 can maintain its activity. The N-benzyl isoquinolines, which were designed as analogues of berberine with its D ring opened, exhibited lower activity than berberine. However, compound 2b, 2c, and 4b showed moderate activity, which indicated that berberine may be deconstructed to new scaffold with synergistic antifungal activity with fluconazole. The results of our research may be helpful to the SAR studies on its other biological activities.

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.

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.

Synthesis and antifungal evaluation of novel triazole derivatives bearing a pyrazole-methoxyl moiety.

Life-threatening invasive fungal infections pose a serious threat to human health. A series of novel triazole derivatives bearing a pyrazole-methoxyl moiety were designed and synthesized in an effort to obtain antifungals with potent, broad-spectrum activity that are less susceptible to resistance. Most of these compounds exhibited moderate to excellent in vitro antifungal activities against Candida albicans SC5314 and 10,231, Cryptococcus neoformans 32,609, Candida glabrata 537 and Candida parapsilosis 22,019 with minimum inhibitory concentration (MIC) values of ≤0.125 µg/mL to 0.5 µg/mL. Use of recombinant Saccharomyces cerevisiae strains showed compounds 7 and 10 overcame the overexpression and resistant-related mutations in ERG11 of S. cerevisae and several pathogenic Candida spp. Despite being substrates of the C. albicans and Candida auris Cdr1 drug efflux pumps, compounds 7 and 10 showed moderate potency against five fluconazole (FCZ)-resistant fungi with MIC values from 2.0 µg/mL to 16.0 µg/mL. Growth kinetics confirmed compounds 7 and 10 had much stronger fungistatic activity than FCZ. For C. albicans, compounds 7 and 10 inhibited the yeast-to-hyphae transition, biofilm formation and destroyed mature biofilm more effectively than FCZ. Preliminary mechanism of action studies showed compounds 7 and 10 blocked the ergosterol biosynthesis pathway at Erg11, ultimately leading to cell membrane disruption. Further investigation of these novel triazole derivatives is also warranted by their predicted ADMET properties and low cytotoxicity.

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.