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. 2023 Aug 29;14(10):2089–2099. doi: 10.1039/d3md00323j

Naphthylthiazoles: a class of broad-spectrum antifungals†

Mohamed Hagras a,✉, Nader S Abutaleb b,c, Hany G Ezzat a, Ehab A Salama b, Mohamed N Seleem b,d, Abdelrahman S Mayhoub a,e,✉
PMCID: PMC10583822  PMID: 37859711

Abstract

Cryptococcal infections remain a major cause of mortality worldwide due to the ability of Cryptococci to pass through the blood–brain barrier (BBB) causing lethal meningitis. The limited number of available therapeutics, which exhibit limited availability, severe toxicity and low tolerability, necessitates the development of new therapeutics. Investigating the antifungal activity of a novel series of naphthylthiazoles provided trans-diaminocyclohexyl derivative 18 with many advantageous attributes as a potential therapeutic for cryptococcal meningitis. Briefly, the antimycotic activity of 18 against cryptococcal strains was highly comparable to that of amphotericin-B and fluconazole with MIC values as low as 1 μg mL−1. Moreover, compound 18 possessed additional advantages over fluconazole; it significantly reduced the intracellular burden of Cryptococci and markedly inhibited cryptococcal biofilm formation. Initial PK assessment of 18 indicated its ability to reach the CNS after oral administration with high permeability, and it maintained therapeutic plasma concentrations for 18 h. Its antifungal activity extended to other clinically relevant strains, such as fluconazole-resistant C. auris.

Cryptococcal infections remain a major cause of mortality worldwide due to the ability of Cryptococci to pass through the blood–brain barrier (BBB) causing lethal meningitis.graphic file with name d3md00323j-ga.jpg

1. Introduction

Fungal infections, particularly those caused by Candida, Cryptococcus and Aspergillus, claim more than 1.5 million human lives annually.1 Notably, Cryptococcus has become a serious global public health threat because it afflicts millions of patients annually.1 Out of 30 known cryptococcal species that exist in the environment, two species, Cryptococcus neoformans and Cryptococcus gattii, primarily cause human infections. Cryptococcus infections range from mild pulmonary infections to severe lethal meningitis. Cryptococcal meningitis occurs via inhalation of this yeast. Most people breathe in this fungal pathogen during their life and never develop signs or symptoms of infection. One of the mechanisms of cryptococcal infections is that Cryptococcus stays hidden in the bodies of people with weakened immune systems, such as patients infected with HIV, who later may develop fatal cryptococcal meningitis.2,3 Over 220 000 new cases of cryptococcal meningitis occur annually, and around 180 000 deaths are reported annually.4 Furthermore, cryptococcal infections are the cause of approximately 15% of HIV-related deaths.5

Most of the illnesses and deaths related to cryptococcal infections are estimated to occur in resource-limited countries, particularly among people living with HIV. Therefore, the CDC is working to introduce and implement screening and treatment of cryptococcal meningitis especially in countries with a high incidence of HIV infections.6 Most worrisome is that cryptococcal infections have extended to immunocompetent healthy individuals following long-term exposure to the fungus.7 The current gold standard therapy for cryptococcal infections is the combination of amphotericin-B and 5-fluorocytosine (5-FC).8 Unfortunately, both drugs are associated with severe toxicity. For instance, a limiting factor in the use of amphotericin for the treatment of systemic fungal infections is the development of nephrotoxicity.9 Furthermore, the administration of amphotericin-B is restricted to the intravenous injection route, which is not accessible in resource-limited regions such as sub-Saharan Africa.10 Moreover, 5-FC has serious side effects including hepatotoxicity, neurotoxicity and bone marrow suppression.11 On the other hand, the oral azole monotherapy fluconazole, which serves as a maintaining and second-choice therapy for cryptococcal meningitis, is associated with severe treatment failure and relapses.12 Hence, the crucial need for development of new anticryptococcal drugs is clear.

Since the introduction of arylthiazoles as a new antibacterial class of compounds, over 500 derivatives were synthesized and tested to rigorously establish the structure–activity relationships (SARs) of this new class of antibiotics.13–27 In this regard, a nitrogenous side chain is crucial for the antibacterial activity and the hydrazine motif is one of the best cationic alternatives tested.14,26 Aromatic hydrazine-containing compounds undergo bioactivation via the formation of diazonium intermediates, which generate highly reactive aryl free radicals28,29 (Fig. 1). Therefore, these organic structures are commonly associated with idiosyncratic hepatotoxicity.30–32 For example, the well-known reported hepatotoxicity associated with the hydrazide-containing antitubercular drugs iproniazid and isoniazid is linked to the bioactivation of their hydrazine group.33–35

Fig. 1. Important observations from previous work.

Fig. 1

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One tactic to negate the possibility of diazonium formation is separating the two pharmacophoric amines (highlighted in red in Fig. 1) via a short alkyl linker. Using a short linker between the two nitrogen centers, the ethylenediamine-containing analog 7 was obtained (Fig. 1, 1st compound in Table 1). The antimicrobial activities of all newly synthesized compounds in our laboratory were routinely assessed against five microorganisms (two Gram-positive bacteria, two Gram-negative bacteria and one fungal strain). Unexpectedly, replacement of the hydrazine moiety with ethylenediamine provided compound 7 with moderate antibacterial and antifungal activities (MIC against both MRSA and Candida albicans = 32 μg mL−1). With this observation in hand, we used the ethylenediamine-containing naphthylthiazole as a lead structure to knock on the door of antifungal development. We hypothesized that the observed dual antibacterial/antifungal effects of compound 7 arose from the free-rotation of the ethylenediamine moiety, which allows the terminal amine to adjust its position and fit within target bacterial and fungal proteins in both cases. In this article, and in the absence of any data about the target protein(s), we synthesized a focused small series of conformationally-restricted isosteres for the ethylenediamine motif to retain the bioactive configuration, and consequently ameliorated the antifungal effect. The antimycotic activity of the newly prepared compounds in this article was evaluated against a large panel of clinical fungal isolates including many fluconazole-resistant species. Additionally, the pharmacokinetic profiles of the most promising derivatives were investigated to assess their potential as treatment for cryptococcal meningitis.

Initial screening (MICs, in μg mL−1) of the newly synthesized naphthylthiazoles against bacterial and fungal strains.

Tested compounds/control drugs MRSA USA300 C. difficile ATCC BAA1870 E. coli JW55031 E. coli BW25113 C. albicans SS5314 (wild-type)
7 32 64 64 >64 32
8 64 64 64 >64 >64
9 >64 >64 >64 >64 >64
10 >64 >64 >64 >64 8
11 4 16 32 >64 32
12 >64 >64 >64 >64 8
13 >64 >64 >64 >64 >64
14 >64 >64 >64 >64 >64
15 >64 >64 >64 >64 >64
16 >64 16 >64 >64 >64
17 >64 64 >64 >64 1
18 64 32 >64 >64 1
19 >64 32 >64 >64 >64
20 64 64 >64 >64 64
21 >64 64 >64 >64 32
Linezolid 1 1 8 >64 NT
Vancomycin 1 NT NT NT NT
Gentamicin NT NT ≤0.5 ≤0.5 NT
Fluconazole NT NT NT NT 0.5
Amphotericin B NT NT NT NT 1
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2. Results and discussion

2.1. Chemistry

Methylsulfone 6 was used to access nucleophilic substitution reactions on the electron-deficient carbon center at the oxadiazole position 2. The methylsulfone moiety was replaced with the amine starting materials at different rates and yields. Aliphatic open-chain derivatives such as ethylamine, propylamine and ethylenediamine provided complete conversion within one hour. However, it took up to 12 hours to consume all starting materials in the case of secondary amine-containing derivatives.

The spectroscopic data suggested two derivatives, piperidin-2-ylmethanamine and 3-aminopyrrolidine, to react in an unexpected way, in which the cyclic NH in both cases was more nucleophilic than the aliphatic NH2 and replaced the methylsulfone moiety. The 1H NMR spectra of compounds 10 and 19 revealed a broad singlet signal equivalent to two protons approximately at 1.2 ppm that were significant for aliphatic NH2. Notably, the chemical shifts of both NH, connected to an aromatic system, and NH2, connected to an alicyclic structure of compound 20, were highly distinguished from each other, in which the first one appeared as a broad singlet within the aromatic region and the second one was represented by a singlet signal, equivalent to two protons, at δ 1.29. This spectroscopic behavior for conjugated NH and NH2 connected to an aliphatic system was observed in all cases of diamino-containing compounds, such as 7, 17 and 18, in which a broad singlet of one proton was revealed between 6.7 and 8.8 ppm, and NH2 was demonstrated by a broad two-proton singlet between 1.2 and 1.8 ppm (Scheme 1).

Scheme 1. Reagents and conditions: (a) absolute EtOH, heat under reflux, 4 h, (b) absolute EtOH, NH2NH2·H2O, heat under reflux, 8 h; (c) CS2, KOH, EtOH, heat under reflux, 12 h; (d) dimethyl sulfate, KOH, H2O, stirring at 23 °C, 2 h; (e) MCPBA, dry DCM, 23 °C, 16 h; (f) appropriate amine, K2CO3, DMF, heat at 80 °C for 1–12 h.

Scheme 1

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2.2. Biology

2.2.1. Initial antimicrobial assessment and establishing SARs

All synthesized compounds were tested against two Gram-positive bacterial strains (MRSA USA300 and Clostridium difficile), two Gram-negative bacterial strains (E. coli JW55031 (tolC-mutant) and E. coli BW25113 (wild-type)), and one fungal pathogen (Candida albicans SC5314) (Table 1) (for the SAR study, see the ESI†).

2.2.2. Antifungal activity of the naphthylthiazoles

2.2.2.1. Efficacy against Candida spp. and Aspergillus spp.

After the initial screening of naphthylthiazoles against C. albicans, we investigated their efficacy against a panel of pathogenic fungal strains, starting with Candida, the fourth most common pathogen isolated from the bloodstream, which is responsible for approximately 75–88% of fungal infections.36Candida exhibits resistance to azoles, which are the predominantly prescribed drugs for Candida infections.37 Concurrently, our compounds exhibited moderate activity against C. albicans in the inhibition of growth of the tested strains at 4 μg mL−1. Notably, they maintained the same activity against fluconazole-resistant strains, such as C. albicans NR 29448. For non-albicans species, they exhibited a potent antifungal activity with MIC values ranging from 0.5 to 2 μg mL−1. Besides, these compounds were superior to fluconazole, which is the standard drug for the treatment of most fungal infections, against C. auris, C. glabrata and C. krusei strains, which exhibit resistance to fluconazole (MICs of 8 to >128 μg mL−1). Moreover, the naphthylthiazoles also exhibited moderate activity against azole-resistant Aspergillus fumigatus strains with MIC values ranging from 4 to 8 μg mL−1.

2.2.2.2. Efficacy against Cryptococcus spp.

The toxicity of amphotericin and the resistance to fluconazole limit the available treatment options for Cryptococcus spp.38 The need for anticryptococcal agents cannot be overestimated. Consequently, the anticryptococcal activity of naphthylthiazoles was explored. Our naphthylthiazoles exhibited a potent activity against C. neoformans and C. gattii strains and inhibited their growth with MIC values ranging from 1 to 2 μg mL−1. Advantageously, they were as effective as amphotericin B and fluconazole, the drugs of choice, against the tested Cryptococci (Table 3).

Antifungal activity (MICs in μg mL−1) of the naphthylthiazoles against cryptococcal clinical isolates.
Fungal strains Compounds/control antifungal drugs
17 18 Fluconazole Amphotericin-B
C. neoformans NR 41298 1 1 2 1
C. neoformans NR 41300 1 1 1 1
C. neoformans NR 48770 1 1 2 1
C. neoformans NR-41299 2 1 1 1
C. neoformans NR-48767 2 2 1 1
C. neoformans NR-48771 2 1 2 1
C. neoformans NR-48773 2 2 2 1
C. gattii NR 43210 1 1 4 1
C. gattii NR 43209 1 1 4 1
C. gattii NR-43213 2 2 2 1
C. gattii NR-43214 2 1 1 1
C. gattii NR-43216 2 2 2 1
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2.2.2.3. Toxicity profile against human eukaryotic cells

Tolerability and safety are the key factors for the development of antifungal therapeutics. The great structural similarity between fungal and human cells (both eukaryotic cells) always represents a serious obstacle during the development of new antifungal drugs. Even with currently available drugs, such as amphotericin, patients suffer from underlying nephrotoxicity and side effects on host tissues.39 Therefore, the discovery of antifungals that are safe for mammalian tissues is needed. The safety profile of compounds 17 and 18 was examined against two different mammalian cell lines, human colorectal adenocarcinoma (Caco-2) cells and fibroblast-like monkey kidney epithelial cells (Vero).

Luckily, both of the tested compounds were nontoxic to Caco-2 and Vero cells at a concentration as high as 128 μg mL−1 (Fig. 2 and 3) as shown by their 50% cytotoxic concentration (CC50), which is the concentration that leads to 50% viability of the treated cells. This concentration was 64- to 128-times higher than their corresponding MIC values against Cryptococcus. Meanwhile, amphotericin-B is toxic to Vero cells at a concentration as low as 16 μg mL−1, as reported previously. In addition, its CC50 is approximately 8 μg mL−1, at which it showed approximately 58% cell viability (Fig. 3).

Fig. 2. In vitro cytotoxicity analysis of compounds 17 and 18 (tested in triplicate at 32, 64 and 128 μg mL−1) against human colorectal cells (Caco-2) using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Data are presented as the average percentage of viable Caco-2 cells relative to DMSO (negative control). The absorbance values represent the average of three samples analyzed for the compound. Error bars represent standard deviation values.

Fig. 2

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Fig. 3. In vitro cytotoxicity analysis of A) compounds 17 and 18 and B) amphotericin-B (tested in triplicate) against fibroblast-like monkey kidney epithelial cells (Vero) using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Data are presented as the average percentage of viable Vero cells relative to DMSO (negative control). The absorbance values represent the average of three samples analyzed for the compound. Error bars represent standard deviation values.

Fig. 3

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2.2.2.4. Selectivity to fungal cells

The human body harbors a huge number of beneficial organisms (approximately 1014), which constitute the normal microbial flora. These florae play an important role in combating other pathogenic invaders, provide some essential elements to the host and help in the functionality of the host immune system. Any change or imbalance in this microflora may lead to certain infectious and noninfectious diseases, such as allergies and cancer.40 Costa et al. reported that microbiota activate the immune system and increase the levels of inflammatory mediators which was associated with increased survival of mice infected with C. gattii.41 Hence, an intact normal microbiome is essential in fighting against cryptococcal infections. Consequently, ideal antifungal drugs/compounds should exhibit a minimal effect on the normal microbiota. Therefore, we tested naphthylthiazoles against different human normal microbiota strains including Lactobacillus gasseri, L. casei and L. crispatus. Table 2 shows that both naphthylthiazoles and the control antifungal agents (fluconazole and amphotericin B) did not inhibit the normal bacterial flora. Therefore, the naphthylthiazoles showed selectivity and a broad spectrum of activities against different fungal strains without affecting the human normal microbiota (lactobacilli) (Table 4).

Antifungal activity (MICs in μg mL−1) of the naphthylthiazoles and control drugs against Candida spp. and Aspergillus spp. clinical isolates.
Fungal strains Compounds/control antifungal drugs
17 18 Fluconazole Amphotericin-B Itraconazole
C. albicans ATCC 10231 4 4 1 1 NT
C. albicans NR-29448 4 4 >128 1 NT
C. auris 381 2 2 2 0.5 NT
C. auris 382 2 2 64 1 NT
C. auris 383 1 1 64 1 NT
C. glabrata ATCC 66032 1 1 8 2 NT
C. glabrata ATCC MYA-2950 0.5 1 16 1 NT
C. parapsilosis ATCC 22019 1 1 1 0.5 NT
C. parapsilosis CAB 502638 1 1 0.5 0.5 NT
C. krusei CAB 396420 1 1 16 2 NT
C. krusei ATCC 34135 1 1 16 2 NT
Aspergillus fumigatus NR 35303 8 4 NT NT 1
Aspergillus fumigatus NR 35304 4 4 NT NT 1
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Minimum inhibitory concentrations (μg mL−1) of the tested compounds against the human normal microbiota (Lactobacillus spp.).
Bacterial strains Compounds/control antifungal drugs
17 18 Fluconazole Amphotericin-B
Lactobacillus gasseri HM-400 >128 >128 >128 >128
Lactobacillus casei HM-334 128 >128 >128 >128
Lactobacillus crispatus HM-370 >128 >128 >128 >128
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2.2.2.5. Antibiofilm activity of the naphthylthiazoles against C. neoformans

After confirming the potent anticryptococcal activity, we tested the inhibitory activities of the naphthylthiazoles against important virulence factors, such as biofilms. Cryptococcus spp. produces a polysaccharide capsule that easily forms biofilms on tissues and medical devices. Indeed, this cryptococcal biofilm initially consists of yeast cells that are embedded and attached to a polymeric polysaccharide matrix.42 The widespread use of medical devices intensifies the importance of studying these biofilms and reducing their impact on persistent and recurrent infections. For example, ventriculoperitoneal shunts are increasingly used to manage intracranial hypertension associated with cryptococcal meningoencephalitis. Cryptococcus forms highly adherent biofilms that are resistant to antimicrobial agents and host defense mechanisms over these shunts, which causes significant morbidity and mortality.43,44 Aslanyan et al. (2017)45 highlighted that cryptococcal survival inside macrophages was associated with biofilm formation, which further complicated infections. Consequently, new anticryptococcal agents with remarkable antibiofilm activities are urgently needed. To determine whether the potential therapeutic application of this class of naphthylthiazoles could be expanded beyond the mere inhibition of planktonic Cryptococci, we evaluated their ability to interfere with C. neoformans biofilm formation. As depicted in Fig. 4, the tested naphthylthiazoles showed a high potential to interfere with cryptococcal biofilm formation and surpassed fluconazole in the inhibition of cryptococcal biofilms. At 0.5 × MIC, 17 and 18 inhibited approximately 53% and 56% of the cryptococcal biofilm formation, respectively. On the other hand, fluconazole exhibited minor biofilm inhibition at the same concentration.

Fig. 4. Inhibition of Cryptococcus neoformans biofilm formation by compounds 17 and 18 and fluconazole (at 0.5× MIC). Data are presented as percentages of the Cryptococcus neoformans NR-41300 biofilm mass. The values represent the average of three samples analyzed for each test agent. Error bars represent standard deviation values. The asterisk (*) denotes statistical significance (P < 0.05) between the results for compounds 17 and 18, fluconazole and DMSO, analyzed via one-way ANOVA with post hoc Dunnett's test for multiple comparisons.

Fig. 4

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2.2.2.6. Intracellular clearance activity of the naphthylthiazoles

Macrophages are the first-line defense against microbial infections.46Cryptococci are capable of efficiently surviving and multiplying within the phagosomes of host macrophages.47 Moreover, they can escape macrophages without lysis.48 Most worrisome is that Cryptococcus spp. use macrophages as a Trojan horse, which allows their extensive invasion inside bodily tissues.49 This high capability to survive and multiply within host macrophages and escape without consequence allows the fungus to elude the host immune response, which leads to persistent infection and treatment failures.5 Consequently, we evaluated the intracellular clearance activities of compounds 17 and 18 against C. neoformans harbored inside infected macrophages. First, the safety profile of both derivatives against murine macrophage (J774) cells was tested and 17 and 18 were able to be tolerated by J774 after 24 hour exposure to a concentration as high as 64 μg mL−1 (Fig. S1†).

The intracellular clearance assay results showed that the tested compounds significantly reduced the intracellular C. neoformans. They outperformed fluconazole in reducing the intracellular C. neoformans. As shown in Fig. 5, after 24 hours, 18 and 17 generated a 0.66 and 1.04 log10 reduction of intracellular C. neoformans, which adds an advantage to this new series of antifungal agents over fluconazole, which showed limited activity in killing the intracellular Cryptococci (0.25 log10 reduction). It appears that both diaminocyclohexyl-containing naphthyl derivatives (at 4 μg mL−1) gained entry into infected macrophage cells and significantly reduced the burden of C. neoformans harbored there without affecting the host macrophages.

Fig. 5. Intracellular clearance activity of compounds 17 and 18 against C. neoformans NR-41300 present inside murine macrophage (J774) cells. Data are presented as log10 colony forming units of C. neoformans NR-41300 per mL inside infected murine macrophages after treatment with 4 μg mL−1 of 17, 18 and fluconazole (tested in triplicate) for 24 hours. Data were analyzed via one-way ANOVA, with post hoc Dunnet's test for multiple comparisons (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Asterisks (*) represent a significant difference between the treatment of J774 cells with RUK compounds in comparison to that with fluconazole.

Fig. 5

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2.2.3. Initial pharmacokinetic profiling

Cryptococci are characterized by the ability to pass through the blood–brain barrier (BBB) and cause severe lethal meningitis with hundreds of thousands of fatalities annually. The problem is further compounded by the emergence of fluconazole-resistant Cryptococci and the low tolerability and limited propensity of amphotericin B, the drug of choice in these cases, to pass through the BBB.38 Therefore, after confirming the potent anticryptococcal activity of the diaminocyclohexane derivatives 17 and 18, we assessed the pharmacokinetic behavior of both compounds. Starting with the permeability assessment, both compounds were subjected to Parallel Artificial Membrane Permeability Assay (PAMPA) tests, and the results were compared with three known FDA-approved therapeutics with variable degrees of permeability through the BBB, furosemide (low permeability), ketoprofen (moderate permeability), and propranolol (high permeability) (Table 5). Impressively, the apparent permeability coefficients of compounds 17 and 18 ranked between those of ketoprofen and propranolol. However, both of the stereoisomers possessed an almost identical microbiological profile. The trans-analog 18 exhibited an apparently enhanced permeability with a Papp value that was double the value of its corresponding cis-analog 17. This result may be due to the formation of intramolecular hydrogen bonds, in the case of 17, which increases its apparent lipophilicity above the optimum level. These results indicated that the naphthylthiazoles 17 and 18 showed increased penetration through artificial membranes, which could indicate an enhanced permeation through the BBB. In future studies, these results will be further confirmed in other assays specifically designed for testing BBB permeability.

Evaluation of apparent permeability of tested compounds and reference drugs via the PAMPA.
Compound tested Mean Pappa (×10−6 cm s−1) Remarks
Furosemide 0.0b Reference with low permeability
Ketoprofen 10.2 Reference with moderate permeability
Propranolol 76.6 Reference with high permeability
17 26.3 Moderately permeable
18 57.7 Highly permeable
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a

Mean Papp = mean apparent permeability.

b

Compound not detected in the receiver compartment (peak below the limit of detection).

With the advantageous Papp value of compound 18 near that of propranolol, we addressed its key in vivo PK parameters following a single oral dose of 25 mg kg−1 in rats. The absorption segment of the PK curve (Fig. 6) confirmed the results of the PAMPA in which compound 18 easily penetrated biological membranes and reached its effective concentration in plasma (1–2 μg mL−1) within a few minutes. Notably, compound 18 maintained its plasma concentration above its MIC value against most Cryptococci (1 μg mL−1) for almost 18 h. Additionally, a biological half-life t1/2 of 3.6 h and the obviously low clearance rate (<1 L h−1) suggest high metabolic stability and a once to twice daily therapeutic regimen.

Fig. 6. PK curve and key PK parameters in rats after oral administration at a dose of 25 mg kg−1 (average of 3 animals).

Fig. 6

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3. Conclusion

The current antifungal arsenal is missing drugs against uprising fungal attacks, which vary from candidiasis to cryptococcal meningitis, as a result of increasing resistance. This resistance invalidated most of the available tools to fight fungal infections, such as C. auris and Cryptococci, and increased the need to update the current list with new compounds to surpass resistance pathways. We introduced potential candidates for the treatment of cryptococcosis. Our compounds outperformed the first-line treatment amphotericin B in many ways. They were less toxic and possessed better penetrability through the blood brain barrier. The naphthylthiazoles inhibited cryptococcal biofilm formation and killed Cryptococci harbored inside macrophages, and were superior to other drugs in cases of nosocomial infections. Consequently, these compounds warrant further evaluation for the treatment of cryptococcal infections.

4. Experimental methods

4.1. Chemistry

General

1H NMR spectra were collected at 400 MHz and 13C spectra were determined at 100 MHz in deuterated chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6) on a Varian Mercury VX-400 NMR spectrometer. Chemical shifts are given in parts per million (ppm) on the delta (δ) scale. Chemical shifts were calibrated relative to those of the solvents. Flash chromatography was performed on 230–400 mesh silica. The progress of reactions was monitored with Merck silica gel IB2-F plates (0.25 mm thickness). Infrared spectra were recorded in potassium bromide disks on a Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometer. Mass spectra were recorded at 70 eV. High resolution mass spectra for all ionization techniques were obtained on a Finnigan MAT XL95. Melting points were determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected. All yields reported refer to isolated yields. Compounds 3–6 were prepared as reported elsewhere.17

Compounds 7–21

General procedure

To a solution of 6 (0.1 g, 0.26 mmol) in dry DMF (5 mL), an appropriate amine or aminoalcohol (0.4 mmol) was added. The reaction mixtures were then heated at 80 °C for 1–12 h. After reaction completion, as detected by TLC, the reaction mixture was cooled down to room temperature, and poured over ice water (50 mL). The formed solids were filtered and washed with 50% ethanol and recrystallized from absolute ethanol or extracted with ethyl acetate, and then dried over MgSO4 and evaporated under reduced pressure to give the desired products. Physical properties and spectral analysis of the isolated products are listed below.

N-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}ethane-1,2-diamine (7)

Following the general procedure and using ethylenediamine (24 μL, 0.4 mmol), compound 7 was obtained as a yellow solid (0.06 g, 68%); mp = 145.5 °C; 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.37 (brs, 2H), 8.10–8.0 (m, 4H), 7.59–7.53 (m, 2H), 7.31 (brs, 1H), 3.38–3.34 (m, 2H), 2.73 (s, 3H), 2.63–2.60 (m, 2H); 13C NMR (DMSO-d6) δ: 166.3, 163.5, 153.8, 152.7, 134.7, 133.3, 130.1, 129.4, 129.1, 128.1, 127.7, 127.1, 126.4, 123.6, 115.6, 57.6, 54.8, 17.3; HRMS (EI) m/z 351.1151 M+, calcd for C18H17N5OS 351.1154; anal. calc. for C18H17N5OS: C, 61.52; H, 4.88; N, 19.93%; found: C, 61.58; H, 4.93; N, 19.97%.

N-Ethyl-5-{4-methyl-2-[naphthalen-2-yl]thiazol-5-yl}-1,3,4-oxadiazol-2-amine (8)

Following the general procedure and using ethylamine (18 μL, 0.4 mmol), compound 8 was obtained as a brown solid (0.049 g, 60%); mp = 158 °C; 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.07–7.93 (m, 4H), 7.87 (brs, 1H), 7.60–7.56 (m, 2H), 3.26 (q, J = 7.2 Hz, 2H), 2.68 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ: 166.4, 163.5, 161.1, 153.8, 152.8, 134.3, 133.2, 129.9, 129.4, 129.1, 128.2, 127.5, 126.4, 123.7, 115.6, 37.9, 17.0, 15.1; HRMS (EI) m/z 336.1039 M+, calcd for C18H16N4OS 336.1045; anal. calc. for C18H16N4OS: C, 64.27; H, 4.79; N, 16.65%; found: C, 64.31; H, 4.81; N, 16.68%.

5-{4-Methyl-2-[naphthalen-2-yl]thiazol-5-yl}-N-propyl-1,3,4-oxadiazol-2-amine (9)

Following the general procedure and using propylamine (28 μL, 0.4 mmol), compound 9 was obtained as a brown solid (0.072 g, 85%); mp = 151 °C; 1H NMR (DMSO-d6) δ: 8.57 (s, 1H), 8.31 (brs, 1H), 8.09–7.94 (m, 4H), 7.61–7.58 (m, 2H), 3.23 (m, 2H), 2.71 (s,3H), 1.63 (m, 2H), 0.95 (m, 3H); 13C NMR (DMSO-d6) δ: 166.4, 163.5, 153.9, 152.5, 134.3, 133.2, 130.0, 129.4, 129.2, 128.2, 128.0, 127.6, 126.4, 123.8, 115.6, 44.9, 22.5, 17.4, 11.7; HRMS (EI) m/z 350.1191 M+, calcd for C19H18N4OS 350.1201; anal. calc. for C19H18N4OS: C, 65.12; H, 5.18; N, 15.99%; found: C, 65.19; H, 5.21; N, 16.04%.

1-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}pyrrolidin-3-amine (10)

Following the general procedure, and using 3-aminopyrrolidine dihydrochloride (63 mg, 0.4 mmol) and anhydrous potassium carbonate (0.1 g, 0.7 mmol), compound 10 was obtained as a brown solid (0.08 g, 77%); mp = 170 °C; 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.05–7.93 (m, 4H), 7.57–7.54 (m, 2H), 3.62–3.48 (m, 4H), 2.74 (s, 3H), 2.04–2.01 (m, 1H), 1.70–1.67 (m, 2H), 1.18 (brs, 2H); 13C NMR (DMSO-d6) δ: 166.4, 162.4, 153.8, 153.2, 134.3, 133.2, 129.9, 129.4, 129.2, 128.2, 128.0, 127.5, 126.5, 123.7, 115.5, 56.0, 51.3, 46.5, 34.2, 17.3; HRMS (EI) m/z 377.1323 M+, calcd for C20H19N5OS 377.1310; anal. calc. for C20H19N5OS: C, 63.64; H, 5.07; N, 18.55%; found: C, 63.69; H, 5.10; N, 18.59%.

(R)-N,N-Dimethyl-1-{5-[4-methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}pyrrolidin-3-amine (11)

Following the general procedure and using (R)-(+)-3-(dimethylamino)pyrrolidine dihydrochloride (74 mg, 0.4 mmol) and anhydrous potassium carbonate (0.1 g, 0.7 mmol), compound 11 was obtained as a yellow solid (0.07 g, 71%); mp = 150 °C; 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.07–7.93 (m, 4H), 7.57–7.56 (m, 2H), 3.80–3.63 (m, 2H), 3.42–3.40 (m, 2H), 3.20–3.15 (m, 2H), 2.74 (s, 3H), 2.18 (s, 6H), 1.84–1.74 (m, 1H); 13C NMR (DMSO-d6) δ: 166.5, 162.1, 154.0, 153.3, 134.3, 133.2, 129.9, 129.4, 129.1, 128.2, 128.0, 127.5, 126.5, 123.7, 115.4, 65.2, 51.7, 47.1, 44.2, 29.8, 17.4; HRMS (EI) m/z 405.1623 M+, calcd for C22H23N5OS 405.1623; anal. calc. for C22H23N5OS: C, 65.16; H, 5.72; N, 17.27%; found: C, 65.18; H, 5.75; N, 17.31%.

(S)-N,N-Dimethyl-1-{5-[4-methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}pyrrolidin-3-amine (12)

Following the general procedure and using (S)-(−)-3-(dimethylamino)pyrrolidine (45 μL, 0.4 mmol), compound 12 was obtained as a yellow solid (0.07 g, 68%); mp = 160 °C; 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.10–8.00 (m, 4H), 7.58–7.57 (m, 2H), 3.83–3.73 (m, 2H), 3.44–3.43 (m, 2H), 3.21–3.13 (m, 2H), 2.76 (s, 3H), 2.20 (s, 6H), 1.85–1.78 (m, 1H); 13C NMR (DMSO-d6) δ: 166.5, 162.1, 154.0, 153.3, 134.3, 133.2, 129.9, 129.4, 129.1, 128.2, 128.0, 127.5, 126.5, 123.7, 115.4, 65.2, 51.7, 47.1, 44.1, 29.7, 17.4; HRMS (EI) m/z 405.1627 M+, calcd for C22H23N5OS 405.1623; anal. calc. for C22H23N5OS: C, 65.16; H, 5.72; N, 17.27%; found: C, 65.22; H, 5.78; N, 17.31%.

(S)-1-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}pyrrolidine-2-carboxamide (13)

Following the general procedure and using l-prolinamide (45 mg, 0.4 mmol), compound 13 was obtained as a brown solid (0.06 g, 55%); mp = 193 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.06–7.95 (m, 4H), 7.59–7.57 (m, 2H), 7.36 (brs, 2H), 4.41–4.39 (m, 1H), 3.72–3.60 (m, 2H), 2.73 (s, 3H), 2.22–2.20 (m, 1H), 1.97–1.93 (m, 3H); 13C NMR (DMSO-d6) δ: 173.6, 166.7, 162.2, 154.0, 153.4, 134.3, 133.2, 129.9, 129.4, 129.2, 128.2, 128.1, 127.6, 126.5, 123.7, 115.4, 62.0, 49.0, 31.5, 24.2, 17.3; HRMS (EI) m/z 405.1260 M+, calcd for C21H19N5O2S 405.1259; anal. calc. for C21H19N5O2S: C, 62.21; H, 4.72; N, 17.27%; found: C, 62.25; H, 4.77; N, 17.31%.

(R)-1-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}pyrrolidine-2-carboxamide (14)

Following the general procedure and using d-prolinamide (45 mg, 0.4 mmol), compound 14 was obtained as a green solid (0.06 g, 59%); mp = 211 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.06–7.90 (m, 4H), 7.58–7.53 (m, 2H), 7.36 (brs, 2H), 4.41–4.40 (m, 1H), 3.71–3.60 (m, 2H), 2.75 (s, 3H), 2.22–2.20 (m, 1H), 1.95–1.93 (m, 3H); 13C NMR (DMSO-d6) δ: 173.6, 166.7, 162.2, 154.0, 153.4, 134.3, 133.2, 129.9, 129.4, 129.2, 128.2, 128.1, 127.6, 126.6, 123.7, 115.4, 62.0, 49.0, 31.5, 24.2, 17.3; HRMS (EI) m/z 405.1275 M+, calcd for C21H19N5O2S 405.1259; anal. calc. for C21H19N5O2S: C, 62.21; H, 4.72; N, 17.27%; found: C, 62.30; H, 4.77; N, 17.32%.

(S)-{1-[5-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-1,3,4-oxadiazol-2-yl]pyrrolidin-2-yl}methanol (15)

Following the general procedure and using (S)-(+)-2-pyrrolidinemethanol (40 μL, 0.4 mmol), compound 15 was obtained as a brown solid (0.09 g, 92%); mp = 152 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.10–7.93 (m, 4H), 7.59–7.57 (m, 2H), 4.77 (brs, 1H), 4.14–4.12 (m, 1H), 3.72–3.48 (m, 4H), 2.76 (s, 3H), 2.06–1.91 (m, 4H); 13C NMR (DMSO-d6) δ: 166.5, 162.4, 153.9, 153.2, 134.3, 133.2, 129.9, 129.4, 129.2, 128.2, 128.0, 127.5, 126.5, 123.7, 115.5, 61.5, 49.0, 28.3, 24.0, 23.9, 17.3; HRMS (EI) m/z 392.1319 M+, calcd for C21H20N4O2S 392.1307; anal. calc. for C21H20N4O2S: C, 64.27; H, 5.14; N, 14.28%; found: C, 64.32; H, 5.17; N, 14.32%.

(R)-{1-[5-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-1,3,4-oxadiazol-2-yl]pyrrolidin-2-yl}methanol (16)

Following the general procedure and using (R)-(−)-2-pyrrolidinemethanol (40 μL, 0.4 mmol), compound 16 was obtained as a brown solid (0.07 g, 72%); mp = 135 °C; 1H NMR (DMSO-d6) δ: 8.58 (s, 1H), 8.08–7.93 (m, 4H), 7.57–7.56 (m, 2H), 4.77 (brs, 1H), 4.12–4.10 (m, 1H), 3.68–3.40 (m, 4H), 2.74 (s, 3H), 2.04–1.88 (m, 4H); 13C NMR (DMSO-d6) δ: 166.5, 162.5, 153.9, 153.2, 134.3, 133.2, 130.0, 129.4, 129.2, 128.2, 128.1, 127.6, 126.5, 123.7, 115.5, 61.5, 49.0, 28.3, 24.0, 23.6, 17.4; HRMS (EI) m/z 392.1300 M+, calcd for C21H20N4O2S 392.1307; anal. calc. for C21H20N4O2S: C, 64.27; H, 5.14; N, 14.28%; found: C, 64.33; H, 5.19; N, 14.31%.

N-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}cyclohexane-cis-1,2-diamine (17)

Following the general procedure and using (±)-cis-1,2-diaminocyclohexane (45 μL, 0.4 mmol), compound 17 was obtained as a yellow solid (0.07 g, 75%); mp = 145 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.09–7.95 (m, 4H), 7.60–7.57 (m, 2H), 6.77 (brs, 1H), 3.89–3.87 (m, 1H), 3.04–2.99 (m, 1H), 2.74 (s, 3H), 1.76–1.55 (m, 8H), 1.30 (brs, 2H); 13C NMR (DMSO-d6) δ: 166.6, 162.4, 153.8, 153.1, 134.7, 133.8, 129.8, 129.4, 129.1, 128.4, 128.1, 127.7, 127.1, 123.8, 115.6, 53.4, 52.7, 36.0, 27.4, 27.1, 20.1, 17.3; HRMS (EI) m/z 405.1640 M+, calcd for C22H23N5OS 405.1623; anal. calc. for C22H23N5OS: C, 65.16; H, 5.72; N, 17.27%; found: C, 65.19; H, 5.76; N, 17.30%.

N-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}cyclohexane-trans-1,2-diamine (18)

Following the general procedure and using (±)-trans-1,2-diaminocyclohexane (45 μL, 0.4 mmol), compound 18 was obtained as a yellow solid (0.07 g, 65%); mp = 148 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.08–7.93 (m, 4H), 7.58–7.56 (m, 2H), 7.14 (brs, 1H), 3.60–3.47 (m, 2H), 2.73 (s, 3H), 2.63–2.56 (m, 2H), 2.00–1.99 (m, 1H), 1.87–1.85 (m, 1H), 1.64 (brs, 2H), 1.26–1.06 (m, 4H); 13C NMR (DMSO-d6) δ: 166.6, 162.4, 153.8, 153.1, 134.7, 133.6, 129.8, 129.4, 129.1, 128.8, 128.1, 127.7, 127.4, 123.2, 115.6, 54.2, 36.8, 34.7, 30.8, 25.6, 23.6, 17.3; HRMS (EI) m/z 405.1634 M+, calcd for C22H23N5OS 405.1623; anal. calc. for C22H23N5OS: C, 65.16; H, 5.72; N, 17.27%; found: C, 65.20; H, 5.76; N, 17.32%.

{1-[5-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-1,3,4-oxadiazol-2-yl]piperidin-2-yl}methanamine (19)

Following the general procedure and using 2-(aminomethyl)piperidine (45 μL, 0.4 mmol), compound 19 was obtained as a yellow solid (0.06 g, 62%); mp = 145 °C; 1H NMR (DMSO-d6) δ: 8.54 (s, 1H), 8.10–8.03 (m, 4H), 7.58–7.57 (m, 2H), 4.01–3.98 (m, 1H), 3.16–3.02 (m, 3H), 2.76 (s, 3H), 1.79–1.66 (m, 3H), 1.48 (brs, 2H), 1.29–1.20 (m, 3H), 0.87–0.085 (m, 1H); 13C NMR (DMSO-d6) δ: 168.8, 161.0, 160.1, 155.7, 134.0, 133.0, 130.9, 129.4, 128.1, 127.7, 127.4, 125.7, 125.6, 123.8, 120.5, 57.2, 47.2, 42.6, 29.1, 24.2, 19.1, 17.5; HRMS (EI) m/z 405.1622 M+, calcd for C22H23N5OS 405.1623; anal. calc. for C22H23N5OS: C, 65.16; H, 5.72; N, 17.27%; found: C, 65.22; H, 5.77; N, 17.34%.

N-{5-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]-1,3,4-oxadiazol-2-yl}cyclohexane-trans-1,4-diamine (20)

Following the general procedure and using trans-1,4-diaminocyclohexane (45 mg, 0.4 mmol), compound 20 was obtained as a green solid (0.09 g, 90%); mp = 228 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.08–7.94 (m, 4H), 7.59–7.57 (m, 2H), 7.15 (brs, 1H), 3.69–3.66 (m, 1H), 2.75 (s, 3H), 2.62–2.57 (m, 2H), 1.92 (brs, 2H), 1.83–1.80 (m, 2H), 1.35–1.17 (m, 5H); 13C NMR (DMSO-d6) δ: 166.6, 163.1, 153.8, 152.7, 134.3, 133.3, 129.8, 129.4, 128.4, 128.1, 127.6, 127.1, 126.7, 123.9, 115.6, 52.0, 49.6, 30.8, 22.9, 17.3; HRMS (EI) m/z 405.1628 M+, calcd for C22H23N5OS 405.1623; anal. calc. for C22H23N5OS: C, 65.16; H, 5.72; N, 17.27%; found: C, 65.22; H, 5.79; N, 17.31%.

2-{[5-(4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl)-1,3,4-oxadiazol-2-yl]amino}acetimidamide (21)

Following the general procedure using 2-aminoacetimidamide dihydrobromide (93 mg, 0.4 mmol) and anhydrous potassium carbonate (0.1 g, 0.7 mmol), compound 21 was obtained as a brown solid (0.05 g, 52%); mp = 208 °C; 1H NMR (DMSO-d6) δ: 8.56 (s, 1H), 8.32 (brs, 1H), 8.06–7.94 (m, 4H), 7.57–7.58 (m, 2H), 6.90 (brs, 1H), 5.68 (brs, 2H), 3.14 (s, 2H), 2.71 (s, 3H); 13C NMR (DMSO-d6) δ: 166.6, 162.4, 157.9, 153.8, 152.7, 134.0, 133.0, 129.8, 129.4, 129.1, 128.4, 128.1, 127.7, 126.4, 123.2, 115.6, 36.4, 17.3; HRMS (EI) m/z 364.1088 M+, calcd for C18H16N6OS 364.1106; anal. calc. for C18H16N6OS: C, 59.33; H, 4.43; N, 23.06%; found: C, 59.39; H, 4.49; N, 23.11%.

4.2. Microbiological assays

4.2.1. Fungal strains and culture media

The Candida, Cryptococcus, and Aspergillus strains used in this study were clinical isolates obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and BEI Resources (Manassas, VA, USA). RPMI 1640 (Thermo Fisher Scientific, Waltham, MA), 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma Aldrich, St. Louis, MO) and YPD broth and agar medium (Becton, Dickinson and Company, Franklin Lakes, NJ) were purchased from commercial vendors.

4.2.2. Determination of the minimum inhibitory concentration (MIC) of the tested compounds against a panel of Candida, Cryptococcus and Aspergillus strains

Different concentrations of the tested compounds and control drugs (fluconazole, itraconazole amphotericin) were screened against clinical isolates of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii and Aspergillus fumigatus species using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines.50,51 Minimum inhibitory concentrations (MICs) were determined as the lowest concentration of the compounds that inhibited fungal growth by 50% after incubation at 35 °C for 24 h (for Candida and Aspergillus) and 48 h (for Cryptococcus).

4.2.3. Safety profile assessment of compounds 17 and 18 against Caco-2, Vero and J774 cells

To examine the tolerability of naphthylthiazoles, their cytotoxicity profile was assessed against human colorectal adenocarcinoma (Caco-2), monkey kidney epithelial cells (Vero), and murine macrophage (J774) cells as described earlier.52–55 Briefly, Caco-2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids (1×), and penicillin–streptomycin at 37 °C with CO2 (5%). Vero cells were cultured in MEM supplemented with 10% FBS, 1 mM sodium pyruvate, and penicillin–streptomycin at 37 °C with CO2 (5%). J774 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C with CO2 (5%). The cells were incubated with different concentrations of the compounds in a 96-well plate at 37 °C with CO2 (5%) for 2 hours (for Caco-2 and Vero cells) or 24 hours (for J774 cells). Control cells were treated with DMSO at a concentration equal to that for the compound-treated samples. The assay reagent MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plates were incubated for three hours. Absorbance readings (at OD490) were taken using a kinetic microplate reader (Spectra MAX IX3, Molecular Device, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability relative to DMSO-treated control cells (average ± standard deviation).

4.2.4. Antibiofilm activity assessment

For biofilm inhibitory assessment of the tested compounds, a subinhibitory concentration (0.5 × MIC) was challenged against a cryptococcal suspension. A fungal inoculum was prepared as described previously56 with minor modifications. In brief, an overnight culture of C. neoformans was centrifuged, washed twice with PBS, adjusted to 105 cells per mL in RPMI medium and added to the wells of 96-microtiter plates containing the compounds. After incubation at 35 °C for 48 hours, the formed biofilm was rinsed with PBS, dried and stained with 0.1% crystal violet. Stained biofilms were washed with PBS and air dried for one hour. The formed biofilm was quantified via extraction of crystal violet with glacial acetic acid and measurement of the absorbance (OD595) (Spectra MAX IX3, Molecular Device, CA, USA).

4.2.5. Intracellular clearance activity of the naphthylthiazoles against Cryptococcus neoformans

The intracellular clearance activities of the tested compounds were evaluated against C. neoformans NR-41300 following a procedure reported previously.8C. neoformans NR-41300 was grown in YPD broth for 48 hours, centrifuged and washed with sterile PBS. J774 cells were exposed to C. neoformans NR-41300 cells at a multiplicity of infection of approximately 100 : 1. After 1 hour of infection, J774 cells were washed 3 times with PBS. The compounds or fluconazole (in triplicate) were subsequently added (at 4 μg mL−1). After 22 hours of incubation at 37 °C with 5% CO2, the test agents were removed, and the cells were washed with an amphotericin B (1 μg mL−1) solution for 1 hour to kill the extracellular Cryptococci. Supernatants were removed, and the cells were washed with PBS before lysing with 0.1% Triton-X 100 and plated onto YPD agar to determine viable C. neoformans inside the J774 cells. Plates were incubated at 37 °C for 48 h before counting the viable CFU mL−1. Data are presented as log10 (C. neoformans) in infected J774 cells.

4.2.6. Parallel artificial membrane permeability (PAMPA) test

The test compound was added to the donor buffer (pH 6.5) of a sandwich plate consisting of a PVDF membrane filter pretreated with lipids. The donor solution was placed in contact with the acceptor buffer at pH 7.4 with the membrane filter between, and the sandwich plate was incubated for 4 hours at ambient temperature. The amount of compound in the donor and acceptor buffer at the beginning and end of the incubation was determined by UV absorbance.

4.2.7. In vivo pharmacokinetics

Pharmacokinetic studies were performed on male naïve Sprague–Dawley (SD) rats (three animals). All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Al-Azhar University and approved by the Animal Ethics Committee of the Faculty of Medicine, Al-Azhar University, Cairo. A single oral dosing (25 mg kg−1) was administered by gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% water. Blood samples were collected over a 24 hour period post-dose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of compound 18 in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile. Two-compartmental pharmacokinetic analysis was performed on plasma concentration data in order to calculate pharmacokinetic parameters as previously reported.13–27

Abbreviations

MIC

Minimum inhibitory concentration

CFUs

Colony forming units

PAMPA

Parallel artificial membrane permeability

P app

Apparent permeability

t 1/2

Half-life

PK

Pharmacokinetics

C max

Maximum plasma concentration

t max

Time required to reach the maximum plasma concentration

C L

Clearance rate

V d

Volume of distribution

Conflicts of interest

All the authors declare that they have no conflict of interest.

Supplementary Material

MD-014-D3MD00323J-s001

Acknowledgments

This paper is based on work supported by the Science, Technology & Innovation Funding Authority (STDF) under grant number 43229 young researcher.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00323j

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