Oxadiazolylthiazoles as novel and selective antifungal agents

Journal Pre-proof Oxadiazolylthiazoles as novel and selective antifungal agents Mohamed Hagras, Ehab A. Salama, Ahmed M. Sayed, Nader S. Abutaleb, Ahmed Kotb, Mohamed N. Seleem, Abdelrahman S. Mayhoub PII:

S0223-5234(20)30013-1

DOI:

https://doi.org/10.1016/j.ejmech.2020.112046

Reference:

EJMECH 112046

To appear in:

European Journal of Medicinal Chemistry

Received Date: 13 October 2019 Revised Date:

20 December 2019

Accepted Date: 6 January 2020

Please cite this article as: M. Hagras, E.A. Salama, A.M. Sayed, N.S. Abutaleb, A. Kotb, M.N. Seleem, A.S. Mayhoub, Oxadiazolylthiazoles as novel and selective antifungal agents, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112046. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Graphical abstract

Oxadiazolylthiazoles as Novel and Selective Antifungal Agents

Mohamed Hagrasǁa, Ehab A. Salamaǁb, Ahmed M. Sayeda, Nader S. Abutalebb, Ahmed Kotba, Mohamed N. Seleemb,c** and Abdelrahman S. Mayhouba,d*

a.

Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar

University, Cairo 11884, Egypt. b.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue

University, West Lafayette, IN 47907, USA. c.

Purdue Institute of Inflammation, Immunology, and Infectious Disease, West

Lafayette, IN 47907, USA. d.

University of Science and Technology, Nanoscience Program, Zewail City of

Science and Technology, October Gardens, 6th of October, Giza 12578, Egypt.

Corresponding Authors ** MNS; email, [email protected] * ASM; e-mail, [email protected] ǁ

These two authors contributed equally

Abstract. Studying the structure-activity relationships (SAR) of oxadiazolylthiazole antibiotics unexpectedly led us to identify ethylenediamine- and propylenediamine-analogs as potential antimycotic novel lead structures. Replacement of the ethylenediamine moiety for the lead compound 7 with cis-diaminocyclohexyl group (compound 18) significantly enhanced the antifungal activity. In addition to the high safety margin of 18 against mammalian cells, it showed highly selective broad-spectrum activity against fungal cells without inhibiting the human normal microbiota. The antifungal activity of 18 was investigated against 20 drugresistant clinically important fungi, including Candida species, Cryptococcus, and Aspergillus fumigatus strains. In addition to the low MIC values that mostly ranged between 0.125 – 2.0 µg/mL, compound 18 outperformed fluconazole in disrupting mature Candida biofilm.

Keywords: antifungal; Fluconazole-resistant Candida spp; cryptococcal infections; Candida auris

1.

Introduction. Fungal infections claim more than 1.5 million human lives each year,

exceeding those killed by either malaria or tuberculosis [1]. Infections caused by fungi vary from mild superficial infection to life-threatening diseases (like meningitis and pneumonia), severe chronic conditions (as chronic pulmonary aspergillosis), and recurrent infections such as candidiasis [2, 3]. Three major pathogens (Cryptococcus, Candida and Aspergillus) are the most common causes of fungal infections [1]. Candida is the main cause of invasive fungal infections (invasive candidiasis), particularly in intensive care patients, which is considered as the fourth most common bloodstream infection in the United States [4, 5]. The mortality rate of candidemia can reach 50%, while invasive aspergillosis mortality rate reaches about 30-50% [3]. The available treatment for fungal infections is restricted to polyenes (e.g. nystatin and amphotericin B), azoles (e.g. fluconazole and voriconazole), echinocandins (e,g, micafungin and caspofungin), and antimetabolites (e.g. 5-fluorocytosine) [6]. Clinical management of fungal infections is highly compromised by the emergence of fungal isolates that exhibit resistance to current antifungal agents. Further compounding the problem is the emergence of multidrug and pandrug resistant Candida auris strains [7, 8]. Azoles, particularly fluconazole, are the most commonly used antifungals because they are highly advantageous over other antifungal drugs in terms of cost, safety, oral bioavailability, and ability to cross the blood-brain barrier [9]. Unfortunately, the repeated use of fluconazole for the treatment of fungal infections has resulted in the emergence of multidrug resistance fungal isolates exhibiting resistance to other azoles, such as itraconazole and voriconazole [10]. Recently, C. krusei showed resistance against fluconazole without prior exposure (innate resistance) [11]. Moreover, several isolates of Candida and Aspergillus species, which are responsible for 1.4 million causalities annually, became highly resistant to fluconazole [12]. In

addition, amphotericin B, which is used for invasive fungal infections, is known to cause serious nephrotoxicity limiting its use for treatment in certain patients [13]. Moreover, the cost of amphotericin B liposomes formulation, with reduced toxicity, is prohibitively expensive [14]. Additionally, 5-fluorocytosine has serious side effects including hepatotoxicity, neurotoxicity and bone marrow suppression [15]. Furthermore, even though echinocandins are generally well tolerated and safer than amphotericin B, they are associated with hepatic dysfunction and adverse reactions including rash, pruritus, facial swelling, bronchospasm, and vasodilatation. They also, have poor oral bioavailability and are highly expensive [16]. Such uprising resistance to antifungals with the underlining toxicity and adverse reactions of those agents, in addition to difficulties in registering new compounds for fighting resistant strains of fungi have left medical community in an urgent and unmet need to develop new potent antifungals.

Figure 1. Overview of the previous efforts. Since 2014, our group was involved in the development of polysubstituted thiazoles as novel antibacterial agents [17-31]. Regularly, the antimicrobial activities of all newly synthesized compounds in our laboratory are assessed against five microorganisms (two Gram-positive bacteria, two Gram-negative bacteria and one fungal strain). In this regard, the hydrazinly structure I (Figure 1) was one of the best cationic replacements with potent activity against multidrug-resistant staphylococci [30]. Because pendant hydrazine motif is bioactivated to

putative aryl free radicals (via a diazonium intermediate) [32, 33], which leads to idiosyncratic hepatotoxicity [34-36], we decided to further derivatize the cationic part. Therefore, additional methylene units were added between, and ethylenediamine II and propylenediamine III derivatives were obtained. Interestingly, by increasing the length of carbon chain between the two amino groups, the antimicrobial activity shifted towards fungal cells (Table 1). Therefore, it was believed that the free rotation of the open-chain carbon linker between the two amino groups allowed the terminal nitrogen to adjust its position to hit either the bacterial or the fungal target. With complete absence of any knowledge about the possible fungal target, the present work adopts a lead-based discovery approach to explore the structure-activity relationships (SAR) of this promising class of antimycotics. In this avenue, we tried to build a library of conformationally-restricted diamine head with a special focus on the stereochemistry of the nitrogenous centers. In order to extend the understanding to the SAR of this novel class of antimicrobials, hydroxyl group was also used as a possible bio-isostere to the terminal nitrogen. 2.

Results and discussion

2.1. Chemistry. Connecting the desired amine-containing side chains by the electron-deficient carbon C2 of oxadiazole ring was achieved by nucleophilic substitution reaction on the methylsulfone moiety, which is a good leaving group. Amine synthons are vastly varying in terms of their ability to substitute the methylsulfone group, in which small open-chain primary amines such as ethylenediamine or its N,N-dimethylamino derivative were efficient in this reaction (high yield in short time). On the other hand, secondary cyclic amines took longer time for reaction completion. Among the cyclic secondary amine structures, the trans-diaminocyclohexane reacted faster than its cis-isomer.

The important methylsulfone-containing intermediate 6 was obtained by allowing the acid hydrazide 4 to react with carbon disulfide and the obtained thiol-containing intermediate was then converted into the methylsulfone 6 via methylation followed by peracid-mediated oxidation (Scheme 1). It is important to mention that, all stereocenters were assigned based on that of starting materials as the one-step nucleophilic substitution reaction is not expected to affect the stereochemistry of the reactants.

2.2. Biological results and discussion

2.2.1. Initial antimicrobial screening. At the outset of this study, all synthesized compounds were initially 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 SS5314) (Table 1). As mentioned earlier, ethylenediamine-containing derivative 7 showed moderate antibacterial and weak antifungal activities with MIC values against MRSA and C. albicans of 8 and 32 µg/mL, respectively. Converting the terminal primary amine to tertiary one (compound 9) or replacement with imidine motif (compound 10) had negative impact on the overall antimicrobial effect. On the other hand, increasing carbon chain length between the two amines provided the propylenediamine-containing analog 11 that revealed one-fold improvement in the antimycotic activity (MIC = 16 µg/mL) without any antibacterial effect (MICs = 64 µg/mL or higher against the tested bacterial strains). Of note, replacement of the terminal amino group with hydroxyl one nullified the antimicrobial effect as observed with compounds 8 and 12-16 regardless of the linker length or the stereochemistry of the substituents. Next, nitrogenous cyclic side chains have been tried to explore the right configuration for the two amino groups. This selected small set of nitrogenous cycles contains 1,2-, 1,3- and 1,4diamino substituents. The 1,2-diaminocyclohexane side chains (compounds 18 and 19) gave varied results as the cis analog 19 was with negligible antimicrobial activity. On the other hand, the trans-diamine derivative 18 showed notable potent selectivity against Candida with MIC value of 1 µg/mL, without inhibiting any of the bacterial strains tested. Finally, other tested cyclic derivatives 20-27 were all void from any antifungal activity as shown in Table 1.

Table 1. Initial antimicrobial screening MICs (µg/mL) Tested compounds/ controlled drugs

C. difficile MRSA

E. coli

E. coli

C. albicans ATCC

USA300 JW55031 BW25113

SS5314 (wild-type) BAA1870

7

8

>64

>64

>64

32

8

>64

>64

>64

32

>64

9

64

32

>64

16

32

10

>64

>64

>64

64

>64

11

>64

>64

>64

64

16

12

>64

>64

>64

32

>64

13

>64

>64

>64

32

>64

14

>64

>64

>64

32

>64

15

>64

32

>64

16

>64

16

>64

>64

>64

>64

>64

17

32

32

>64

8

8

18

>64

>64

>64

64

1

19

64

>64

>64

16

64

20

32

32

>64

16

>64

21

32

16

>64

16

>64

22

>64

>64

>64

16

>64

23

>64

>64

>64

32

>64

24

>64

>64

>64

>64

>64

25

>64

>64

>64

32

>64

26

>64

64

>64

32

>64

27

>64

>64

>64

32

>64

MICs (µg/mL) Tested compounds/ controlled drugs

C. difficile MRSA

E. coli

E. coli

C. albicans ATCC SS5314 (wild-type)

USA300 JW55031 BW25113 BAA1870

Linezolid

1

8

>64

1

NT

Vancomycin

1

NT

NT

NT

NT

Gentamicin

NT

≤0.5

≤0.5

NT

NT

Fluconazole

NT

NT

NT

NT

>64

Amphotericin B

NT

NT

NT

NT

1

2.2.2. Profiling of Antifungal activity. Compound 18 was then evaluated against a panel of clinical fungal isolates including Candida albicans, non-albicans Candida species (C. auris, C. glabrata, C. parapsilosis, and C. krusei), in addition to Aspergillus and Cryptococcus strains. Our results showed that 18 exhibited a broad-spectrum activity against pathogenic yeasts and molds (Table 2). C. albicans is the most prevalent fungal species causing various fungal infection in mouth, throat, vagina along with the deadly candidemia [3, 37]. Control of C. albicans infections is more compromised due to the development of high resistance to the azoles; including fluconazole [38]. Hence, new compounds capable of maintaining their potency against fluconazole-resistant strains are highly desirable. Compound 18 inhibited the growth of tested strains of C. albicans at concentrations ranging from 2 to 8 µg/mL (Table 2). Interestingly, it was capable of inhibiting the fluconazole-resistant C. albicans NR 29448. Moreover, the efficacy of 18 extended to include other non-albicans Candida species such as C. auris, C. glabrata, C. parapsilosis, and C. krusei. Candida auris is an emerging multidrugresistant human fungal pathogen causing invasive infections, several nosocomial outbreaks, and

death [39]. Importantly, compound 18 exhibited potent activity against C. auris with MIC values ranging from 2 to 4 µg/mL. In addition, compound 18 maintained its superiority over fluconazole where it efficiently inhibited the fluconazole-resistant C. auris strains. Bloodstream infections due to C. auris resulted in a 60% mortality rate, which exceeded severe bacterial and viral infections [40, 41]. Unlike other Candida species, C. auris can live on the skin and on healthcare-surfaces for a long time, allowing rapid spread and dissemination of fatal infections. Most worrisome in C. auris associate infection is the high level of antifungal-resistance which has never been seen in other Candida infections. About one-third of the detected strains were resistant to two classes of antifungal drugs and some strains were resistant to all three drug classes used to treat fungal infections [41, 42]. Additionally, compound 18 was found to be superior to both fluconazole and amphotericin B against C. glabrata. C. glabrata is a common causative agent for candidemia, vulvovaginal candidiasis and candiduria [43]. Moreover, 18 was as effective as fluconazole and amphotericin B against C. parapsilosis and outperformed fluconazole against C. krusei strains. Both candida strains are known for their resistance to several antifungal drugs [44]. Furthermore, compound 18 was as potent as fluconazole and amphotericin B against Cryptococcus neoformans inhibiting their growth at a concentration ranging from 1 to 2 µg/mL. It was also, superior to fluconazole and equipotent to amphotericin B against the tested C. gattii. Both strains are causing cryptococcosis which is responsible for more than 650,000 annual deaths [45-47]. Compound 18 exhibited moderate activity against molds (Aspergillus fumigatus) strains, that are resistant to fluconazole, with MIC values ranging from 4 to 8 µg/mL. These results

collectively, indicated that 18 exhibited a broad-spectrum antifungal activity, a character that is highly needed for a new antifungal compound. Table 2. Antifungal activity (MIC in µg/mL) of compound 18 against Candida spp, Cryptococcus spp and Aspergillus fumigatus clinical isolates Fungal strains

Compound/ Control antifungal drugs

18

Fluconazole

Amphotericin B

2

1

1

8 8 4 2 4 0.5

1 >128 2 128 >128 8

0.125

16

1 1 0.5 1 2 2 1

NT NT NT NT NT NT

C. parapsilosis ATCC 22019

1

1

0.5

NT

C. parapsilosis CAB 502638

1

0.5

0.5

NT

C. krusei CAB 396420

1

16

2

NT

C. krusei ATCC 34135

2

16

2

NT

C. neoformans NR 41298

2

2

1

NT

C. neoformans NR 41300

2

1

1

NT

C. neoformans NR 48770

1

2

1

NT

C. gattii NR 43210

1

4

1

NT

C. gattii NR 43209

1

4

1

NT

8

>64

NT

1

4

>64

NT

1

C. albicans SS5314 (wildtype) C. albicans ATCC 10231 C. albicans NR 29448 C. auris 381 C. auris 383 C. auris 384 C. glabrata ATCC 66032 C. glabrata ATCC MYA2950

Aspergillus fumigatus NR 35303 Aspergillus fumigatus NR 35304 NT1; Not tested

Itraconazole NT1 NT

2.2.3. Effect on human microbiota. Under normal circumstances, Candida is a non‐ pathogenic, commensal microorganism inhabiting the gut, the oral cavity as well as the female reproductive tract [48]. The human microbiota prevents Candida species shifting from a harmless commensal microbe to a disease‐causing pathogen in the oral cavity, gut and vaginal

tract [49-51]. Normal microbiota, particularly lactobacilli, interferes with Candida infections through immunomodulation of the host epithelial barrier and competition for Candida binding sites reducing its adhesion to the host epithelial cells [50, 52]. Furthermore, lactobacilli interfere with Candida biofilm formation and retard its hyphal formation, which is an essential step during infection [53, 54]. Ideally, antifungal drugs/compounds should exhibit a minimal effect on the normal microbiota, particularly lactobacilli. Therefore, we tested 18 against different lactobacilli including Lactobacillus gasseri, L. casei and L. crispatus. 18, similar to the standard antifungal agents (fluconazole and amphotericin B), did not inhibit lactobacilli. Thus, 18 possessed a selective broad-spectrum activity against different fungal strains without harming the human normal microbiota (lactobacilli). Table 3. The minimum inhibitory concentrations (MIC in µg/mL) of compound 18 against human normal microbiota strains Compound/ Control antifungal drugs

Tested Strains 18

Fluconazole

Amphotericin B

Lactobacillus gasseri HM-400

128

>128

>128

Lactobacillus casei HM-334

>128

>128

>128

Lactobacillus crispatus HM370

>128

>128

>128

2.2.4. Toxicity profile. A major limitation of the current antifungal therapy is its potential toxicity to host tissues [55]. For instance, azoles such as fluconazole and itraconazole are associated with mild hepatotoxicity that required discontinuation of treatment in some patients. Amphotericin B causes serious nephrotoxicity [13, 56]. Thus, new antifungals, that are safe to mammalian tissues, are urgently needed. Consequently, the safety profile of 18 was evaluated against human colorectal adenocarcinoma (Caco-2) cells. After 24-hours of exposure time, the compound was found to be nontoxic to Caco-2 up to a concentration of 64 µg/mL where 100%

of the cells were viable (Figure 2A). This represents up to 64-fold higher than its corresponding antifungal activity. In addition, we tested the toxicity of 18 against fibroblast-like monkey kidney epithelial cells (Vero). In accordance with its high tolerability to Caco-2 cells, the compound was found to be non-toxic to Vero cells CC50% higher than 64 µg/mL (Figure 2B. This represents up to 64-fold higher than its MICs against the tested fungal strains. On the other hand, amphotericin B was toxic to Vero cells at the concentration of 16 µg/mL and its CC50 was about 8 µg/mL (Figure 3). Consequently, 18 possessed another advantage that is highly needed in the antifungal drug discovery.

Figure 2. In vitro cytotoxicity analysis of 18 (tested in triplicates at 32, 64 and 128 µg/mL for 24 hours) against A) human colorectal cells (Caco-2), and B) fibroblast-like monkey kidney epithelial cells (Vero), using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Data are presented as the average percentage of viable cells relative to DMSO (negative control). The absorbance values represent an average of three samples analyzed for the compound. Error bars represent standard deviation values. CC50 (the compound’s concentration that resulted in 50% cell viability) of compound 18 was determined.

Figure 3. In vitro cytotoxicity analysis of Amphotericin B (tested in triplicates for 24 hours) 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 an average of three samples analyzed for the compound. Error bars represent standard deviation values. CC50 (the compound’s concentration that resulted in 50% cell viability) of amphotericin B was determined. 2.2.5. Antibiofilm activity. Biofilms are complex structures capable of forming on the surface of medical devices such as indwelling catheters, shunts, dentures, and implanted prosthetic devices. Microbes in biofilms can cause a wide range of persistent infections [57]. Notably, C. albicans is the most frequently isolated pathogen from fungal biofilms, which are highly resistant to currently available antifungals [58]. For instance, C. albicans cells embedded in biofilms are nearly 1,000-fold more resistant than planktonic cells against fluconazole [59]. Consequently, the development of new antifungal agents with improved antibiofilm activities are urgently needed. To determine whether the potential therapeutic application of 18 could be expanded beyond merely inhibiting planktonic Candida, 18 ability to interfere with C. albicans biofilm formation and disrupt the preformed mature C. albicans biofilm were investigated. 2.2.5.1. Efficacy against Candida albicans biofilm formation. As shown in Figure 4, compound 18 was nearly as effective as fluconazole in inhibiting biofilm formation. At 0.5 × MIC, it inhibited 40% of C. albicans NR-29448 biofilm formation while fluconazole (at 0.5 × MIC) inhibited about 50% of the biofilm formation at the same concentration.

Figure 4. Inhibition of Candida albicans biofilm formation by 18 and fluconazole. Data are presented as a percent of Candida albicans NR-29448 biofilm mass. The values represent an average of three samples analyzed for each test agent. Error bars represent standard deviation values. An asterisk (*) denotes statistical significance (P < 0.05) between results for compound 18 or fluconazole and the DMSO analyzed via one-way ANOVA with post-hoc Dunnet’s test for multiple comparisons. 2.2.5.2. Efficacy against Candida albicans mature biofilm. In addition to interference with biofilm formation, we investigated 18’s ability to disrupt the pre-formed mature biofilms. At 64 µg/mL, 18 significantly disrupted the mature biofilm by about 16%. Conversely, fluconazole, at the same concentration, did not significantly disrupt the pre-formed biofilm. Thus, in addition to the ability of compound 18 to inhibit C. albicans planktonic cells, it suppressed biofilm formation of Candida and disrupted the intact Candida biofilm.

Figure 5. Biofilm eradication activity of 18 and fluconazole (tested at the concentration of 64 µg/mL). Data are presented as a percent of Candida albicans NR-29448 biofilm mass. The values represent an average of three samples analyzed for each test agent. Error bars represent standard deviation values. Data were analyzed via one-way ANOVA with post-hoc Dunnet’s test for multiple comparisons. An asterisk (*) denotes statistical significance (P < 0.05) between results for compound 18 or fluconazole and the DMSO. (#) denotes statistical significance (P < 0.05) between results for compound 18 and fluconazole. 2.2.6. Time kill kinetics assay. In order to investigate the killing kinetics of the promising compound 18, a time-kill assay was conducted at 5 × MIC and 10 × MIC against Candida albicans SS5314 (wild-type) (Figure 6). Compound 18 exhibited a fungistatic activity against C. albicans at both tested concentrations. Similarly, fluconazole exhibited a fungistatic activity

against the tested strain at both 5 × MIC and 10 × MIC, as reported earlier [60]. On the other hand, amphotericin B, as reported earlier

[61] exhibited a rapid fungicidal activity, by

completely eradicating the initial C. albicans count within two hours .

Figure 6. Killing kinetics of compound 18, fluconazole (tested in triplicates at 5 × MIC and 10 × MIC), and amphotericin B (tested in triplicates at 5 × MIC), against Candida albicans SS5314 (wild-type) over a 36-h incubation period at 37°C. DMSO (solvent for the compound) served as a negative control, and fluconazole and amphotericin B served as control drugs. The error bars represent standard deviation values obtained from triplicate samples used for each test agent. 2.2.7. Multi-step resistance study against MRSA. A multi-step resistance study Figure 7 was conducted to investigate the resistance development towards oxadiazolylthiazoles. As shown in Figure 7, The MIC values of compound 18 and fluconazole remained stable along the 10 passages without any increase. This result indicated that C. albicans was unable to develop a rapid resistance to compound 18 and fluconazole.

Figure 7. Multi-step resistance study of compound 18 and fluconazole against Candida albicans SS5314 (wild-type). Fungi were serially passaged over a 10-days period, and the broth microdilution assay was used to determine the MIC of each test agent against Candida albicans SS5314 after each successive passage. A four-fold increase in MIC would be indicative of development of fungal resistance to the test agent.

3.

Conclusion. We are proposing a fungistatic antifungal compound with a broad spectrum

of activity against the most important pathogenic fungi. Compound 18 inhibited pathogenic strains of C.

albicans and non-albicans species including fluconazole-resistant strains. In

addition, compound 18 was capable of inhibiting clinically-important Cryptococcus and Aspergillus strains. Moreover, compound 18 did not affect human microbiota and exhibited excellent tolerability to the mammalian cells. Additionally, it surpassed the fluconazole in disrupting C. albicans biofilm formation and not rapid resistance was developed against it even after 10-passeges. Accordingly, this compound warrants further investigation as a novel antifungal agent. Experimental 4.1. Chemistry 4.1.1. General. 1H NMR spectra were done 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). The infrared spectra were recorded in potassium bromide disks on 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 from 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 [62] 4.1.2. Compounds 7-27. General procedure. To a solution of methylsulfone 6 (0.1 g, 0.26 mmol) in dry DMF (5 mL), a proper amine (0.4 mmol) was added. The reaction mixtures were heated at 80 °C for a period between 0.5 and 12 h, and then poured over ice water (50 mL), the formed solid was filtered and washed with 50% ethanol and recrystallized from absolute ethanol give the desired products. Physical properties and spectral analysis of isolated products are listed below: 4.1.2.1.

N-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-yl}ethane-

1,2-diamine (7). Following the general procedure, and using ethylenediamine (21 µL, 0.4 mmol), compound 7 was obtained as yellow solid (0.07 g, 82%) mp = 175 °C; 1H NMR (DMSOd6) δ: 7.99 (d, J = 8.4 Hz, 2H), 7.84 (brs, 1H), 7.50 (d, J = 8.4 Hz, 2H), 3.50-3.48 (m, 2H), 2.67 (s, 3H), 2.59-2.55 (m, 2H), 1.78 (brs, 2H), 1.28 (s, 9H); 13C NMR (DMSO-d6) δ: 166.4, 163.8, 154.8, 153.8, 153.1, 130.1, 126.7, 126.4, 114.9, 52.4, 37.5, 35.0, 31.3, 17.3; HRMS (EI) m/z 357.1630 M+, calcd for C18H23N5OS 357.1623; Anal. Calc. for: (C18H23N5OS): C, 60.48; H, 6.49; N, 19.59%; Found: C, 60.53; H, 6.54; N, 19.62%.

4.1.2.2.

2-{[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]amino}ethan-1-ol (8). Following the general procedure, and using 2-aminoethan-1-ol (21 µL, 0.4 mmol), compound 8 was obtained as yellow solid (0.08 g, 85%) mp = 245 °C; 1H NMR (DMSO-d6) δ: 7.87 (d, J = 8.4 Hz, 2H), 7.81 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 4.76 (brs, 1H), 3.55-3.54 (m, 2H), 3.40-3.38 (m, 2H), 2.65 (s, 3H), 1.31 (s, 9H); 13C NMR (DMSO-d6) δ: 166.6, 163.5, 154.2, 153.8, 153.5, 130.1, 126.7, 126.4, 114.9, 59.3, 45.8, 35.0, 31.5, 17.3; HRMS (EI) m/z 358.1461 M+, calcd for C18H22N4O2S 358.1463; Anal. Calc. for: (C18H22N4O2S): C, 60.31; H, 6.19; N, 15.63%; Found: C, 60.36; H, 6.24; N, 15.67%. 4.1.2.3.

N-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-yl}-N,N-

dimethylethane-1,2-diamine (9). Following the general procedure, and using N,Ndimethylenediamine (35 µL, 0.4 mmol), compound 9 was obtained as yellow solid (0.06 g, 63%) mp = 190 °C; 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.75 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 3.83-3.80 (m, 2H), 2.96-2.93 (m, 2H), 2.66 (s, 3H), 2.16 (s, 6H), 1.28 (s, 9H); 13C NMR (DMSO-d6) δ: 166.6, 163.6, 154.3, 153.7, 152.8, 130.0, 126.7, 126.6, 115.0, 58.2, 45.6, 41.1, 35.1, 31.3, 17.3; HRMS (EI) m/z 385.1932 M+, calcd for C20H27N5OS 385.1936; Anal. Calc. for: (C20H27N5OS): C, 62.31; H, 7.06; N, 18.17%; Found: C, 62.33; H, 7.09; N, 18.17%. 4.1.2.4.

2-{[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-yl]amino}

acetimidamide (10). Following the general procedure, using 2-aminoacetimidamide dihydrobromide (93 mg, 0.4 mmol) and potassium carbonate anhydrous (0.1 g, 0.7 mmol), compound 10 was obtained as brown solid (0.06 g, 59%) mp = 245 °C; 1H NMR (DMSO-d6) δ: 1

H NMR (DMSO-d6) δ: 7.90 (d, J = 8.4 Hz, 2H), 7.76 (brs, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.33

(brs, 2H), 3.80 (s, 2H), 2.63 (s, 3H), 1.30 (s, 9H); 13C NMR (DMSO-d6) δ: 166.6, 163.5, 160.1, 154.5, 153.8, 153.5, 129.8, 126.4, 126.0, 114.9, 56.5, 35.3, 31.2, 17.3; HRMS (EI) m/z 375.1593

M+, calcd for C18H22N6OS 375.1576; Anal. Calc. for: (C18H22N6OS): C, 58.36; H, 5.99; N, 22.68%; Found: C, 58.42; H, 6.02; N, 22.73%. 4.1.2.5. N-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-yl}propane1,3-diamine (11). Following the general procedure, and using propane-1,3-diamine (29 µL, 0.4 mmol), compound 11 was obtained as yellow solid (0.07 g, 79%) mp = 240 °C; 1H NMR (DMSO-d6) δ: 7.93 (d, J = 8.4 Hz, 2H), 7.81 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 3.45-3.42 (m, 2H), 2.68 (s, 3H), 2.58-2.56 (m, 2H), 1.67 (brs, 2H), 1.29 (s, 9H), 1.25-1.21 (m, 2H); 13C NMR (DMSO-d6) δ: 166.6, 163.5, 154.5, 153.4, 153.1, 130.1, 126.7, 126.4, 114.9, 42.7, 37.5, 35.0, 32.3, 31.3, 17.3; HRMS (EI) m/z 371.1785 M+, calcd for C19H25N5OS 371.1780; Anal. Calc. for: (C19H25N5OS): C, 61.43; H, 6.78; N, 18.85%; Found: C, 61.45; H, 6.82; N, 18.86%. 4.1.2.6.

3-{[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]amino}propan-1-ol (12). Following the general procedure, and using 3-aminopropan-1-ol (30 µL, 0.4 mmol), compound 12 was obtained as yellow solid (0.07 g, 77%) mp = 270 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.85 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 4.48 (brs, 2H), 3.50-3.47 (m, 2H), 3.27-3.23 (m, 2H), 2.66 (s, 3H), 1.73-1.71 (m, 2H), 1.30 (s, 9H);

13

C NMR

(DMSO-d6) δ: 166.5, 163.6, 154.3, 153.7, 152.8, 130.0, 126.7, 126.6, 114.9, 58.5, 41.9, 35.1, 32.4, 31.3, 17.3; HRMS (EI) m/z 372.1626 M+, calcd for C19H24N4O2S 372.1620; Anal. Calc. for: (C19H24N4O2S): C, 61.27; H, 6.49; N, 15.04%; Found: C, 61.27; H, 6.53; N, 15.07%. 4.1.2.7,

3-{[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]amino}propane-1,2-diol (13). Following the general procedure, and using 3-aminopropane1,2-diol (36 µL, 0.4 mmol), compound 13 was obtained as yellow solid (0.05 g, 74%) mp = 220 °C; 1H NMR (DMSO-d6) δ: 7.96 (d, J = 8.4 Hz, 2H), 7.80 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 4.83 (brs, 1H), 4.58 (brs, 1H), 3.65-3.63 (m, 1H), 3.36-3.32 (m, 2H), 3.18-3.15 (m, 2H), 2.65 (s,

3H), 1.30 (s, 9H);

13

C NMR (DMSO-d6) δ: 166.6, 163.9, 154.2, 153.8, 153.1, 130.0, 126.6,

126.4, 114.9, 70.2, 64.0, 46.1, 35.1, 31.3, 17.3; HRMS (EI) m/z 388.1574 M+, calcd for C19H24N4O3S 388.1569; Anal. Calc. for: (C19H24N4O3S): C, 58.74; H, 6.23; N, 14.42%; Found: C, 58.76; H, 6.27; N, 14.45%. 4.1.2.8.

(R)-3-{(5-[2-(4-(tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]amino}propane-1,2-diol (14). Following the general procedure, and using (R)-3aminopropane-1,2-diol (36 µL, 0.4 mmol), compound 14 was obtained as yellow solid (0.06 g, 61%) mp = 215 °C; 1H NMR (DMSO-d6) δ: 7.93 (d, J = 8.4 Hz, 2H), 7.84 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 4.83 (brs, 1H), 4.61 (brs, 1H), 3.65-3.62 (m, 1H), 3.50-3.47 (m, 2H), 3.15-3.12 (m, 2H), 2.71 (s, 3H), 1.27 (s, 9H); 13C NMR (DMSO-d6) δ: 166.5, 163.9, 154.2, 153.7, 152.8, 129.8, 126.6, 126.4, 115.0, 70.0, 64.0, 46.5, 35.0, 31.3, 17.3; HRMS (EI) m/z 388.1580 M+, calcd for C19H24N4O3S 388.1569; Anal. Calc. for: (C19H24N4O3S): C, 58.74; H, 6.23; N, 14.42%; Found: C, 58.78; H, 6.25; N, 14.42%. 4.1.2.9.

(S)-3-{(5-[2-(4-(tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]amino}propane-1,2-diol (15). Following the general procedure, and using (S)-3aminopropane-1,2-diol (36 µL, 0.4 mmol), compound 15 was obtained as yellow solid (0.06 g, 66%) mp = 265 °C; 1H NMR (DMSO-d6) δ: 7.90 (d, J = 8.4 Hz, 2H), 7.83 (brs, 1H), 7.53 (d, J = 8.4 Hz, 2H), 4.85 (brs, 1H), 4.59 (brs, 1H), 3.81-3.80 (m, 1H), 3.66-3.63 (m, 2H), 3.16-3.10 (m, 2H), 2.65 (s, 3H), 1.28 (s, 9H); 13C NMR (DMSO-d6) δ: 166.5, 163.9, 154.3, 153.7, 152.8, 130.0, 126.6, 126.4, 115.0, 70.2, 64.0, 46.5, 35.0, 31.3, 17.3; HRMS (EI) m/z 388.1586 M+, calcd for C19H24N4O3S 388.1569; Anal. Calc. for: (C19H24N4O3S): C, 58.74; H, 6.23; N, 14.42%; Found: C, 58.75; H, 6.23; N, 14.44%.

4.1.2.10.

2-{[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]amino}propane-1,3-diol (16). Following the general procedure, and using 2-aminopropane1,3-diol (36 mg, 0.4 mmol), compound 16 was obtained as orange solid (0.06 g, 59%) mp = 190 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.69 (brs, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.74 (brs, 2H), 3.80-3.78 (m, 1H), 3.53-3.50 (m, 4H), 2.65 (s, 3H), 1.29 (s, 9H);

13

C NMR

(DMSO-d6) δ: 166.3, 163.5, 154.2, 153.8, 152.7, 129.8, 126.6, 126.4, 114.9, 60.4, 57.9, 35.0, 31.2, 17.3; MS (m/z) 388; HRMS (EI) m/z 388.1565 M+, calcd for C19H24N4O3S 388.1569; Anal. Calc. for: (C19H24N4O3S): C, 58.74; H, 6.23; N, 14.42%; Found: C, 58.77; H, 6.28; N, 14.46%. 4.1.2.11.

N-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-

yl}cyclohexane-trans-1,4-diamine (17). Following the general procedure, and using trans-1,4diaminocyclohexane (45 mg, 0.4 mmol), compound 17 was obtained as yellow solid (0.09 g, 83%) mp = 227 °C; 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 6.61 (brs, 1H), 3.80-3.70 (m, 2H), 2.63 (s, 3H), 2.0-1.92 (m, 4H), 1.79-1.76 (m, 4H), 1.45 (brs, 2H), 1.28 (s, 9H);

13

C NMR (DMSO-d6) δ: 166.5, 162.9, 154.3, 153.7, 152.7, 130.0, 126.6,

126.4, 115.0, 52.2, 49.6, 35.1, 34.2, 31.5, 31.3, 17.3; HRMS (EI) m/z 411.2099 M+, calcd for C22H29N5OS 411.2093; Anal. Calc. for: (C22H29N5OS): C, 64.20; H, 7.10; N, 17.02%; Found: C, 64.21; H, 7.16; N, 17.06%. 4.1.2.12.

N-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-

yl}cyclohexane-cis-1,2-diamine (18). Following the general procedure, and using (±)-cis-1,2diaminocyclohexane (45 µL, 0.4 mmol), compound 18 was obtained as brown solid (0.08 g, 73%) mp = 229 °C; 1H NMR (DMSO-d6) δ: 7.82 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.96 (brs, 1H), 3.73-3.68 (m, 2H), 2.67 (s, 3H), 1.92-1.89 (m, 1H), 1.68-1.63 (m, 3H), 1.41 (brs, 2H), 1.29 (s, 9H), 1.25-1.21 (m, 4H); 13C NMR (DMSO-d6) δ: 166.6, 163.5, 154.2, 153.8, 152.7,

130.5, 126.7, 126.3, 114.9, 53.8, 51.3, 35.3, 34.7, 31.2, 28.1, 20.4, 20.1, 17.3; HRMS (EI) m/z 411.2100 M+, calcd for C22H29N5OS 411.2093; Anal. Calc. for: (C22H29N5OS): C, 64.20; H, 7.10; N, 17.02%; Found: C, 64.25; H, 7.12; N, 17.06%. 4.1.2.13.

N-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-

yl}cyclohexane-trans-1,2-diamine (19). Following the general procedure, and using (±)-trans1,2-diaminocyclohexane (45 µL, 0.4 mmol), compound 19 was obtained as green solid (0.09 g, 86%) mp = 165 °C; 1H NMR (DMSO-d6) δ: 7.90 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.68 (brs, 1H), 3.82-3.80 (m, 2H), 2.67 (s, 3H), 2.10-2.0 (m, 2H), 1.85 (brs, 2H), 1.70-1.67 (m, 2H), 1.30 (s, 9H), 1.18-1.13 (m, 4H); 13C NMR (DMSO-d6) δ: 166.6, 162.8, 154.5, 153.8, 153.1, 130.1, 126.7, 126.4, 114.9, 54.5, 37.5, 35.0, 31.3, 28.4, 25.3, 23.6, 23.2, 17.3; HRMS (EI) m/z 411.2103 M+, calcd for C22H29N5OS 411.2093; Anal. Calc. for: (C22H29N5OS): C, 64.20; H, 7.10; N, 17.02%; Found: C, 64.22; H, 7.14; N, 17.05%. 4.1.2.14.

1-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-

yl}pyrrolidin-3-amine (20). Following the general procedure, using 3-amino-pyrrolidine dihydrochloride (60 mg, 0.4 mmol) and potassium carbonate anhydrous (0.1 g, 0.7 mmol), compound 20 was obtained as yellow solid (0.07 g, 77%) mp = 178 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 3.60-3.58 (m, 3H), 3.19-3.15 (m, 2H), 2.65 (s, 3H), 1.87-1.85 (m, 2H), 1.73 (brs, 2H), 1.26 (s, 9H);

13

C NMR (DMSO-d6) δ: 166.5, 162.4,

154.3, 153.7, 153.2, 129.9, 126.7, 126.6, 114.9, 55.9, 51.0, 46.5, 35.1, 34.0, 31.3, 17.3; HRMS (EI) m/z 383.1786 M+, calcd for C20H25N5OS 383.1780; Anal. Calc. for: (C20H25N5OS): C, 62.64; H, 6.57; N, 18.26%; Found: C, 65.64; H, 6.63; N, 18.31%. 4.1.2.15.

{1-[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]piperidin-2-yl}methanamine (21). Following the general procedure, and using 2-

(aminomethyl)piperidine (45 µL, 0.4 mmol), compound 21 was obtained as canary yellow solid (0.06 g, 54%) mp = 246 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 3.85-3.80 (m, 2H), 2.90-2.89 (m, 2H), 2.71 (s, 3H), 1.80-1.60 (m, 7H), 1.42 (brs, 2H), 1.29 (s, 9H);

13

C NMR (DMSO-d6) δ: 166.6, 163.1, 154.5, 153.8, 153.1, 130.1, 126.7, 126.4,

114.9, 58.6, 49.6, 42.3, 35.0, 31.3, 29.1, 24.6, 19.4, 17.3; HRMS (EI) m/z 411.2089 M+, calcd for C22H29N5OS 411.2093; Anal. Calc. for: (C22H29N5OS): C, 64.20; H, 7.10; N, 17.02%; Found: C, 64.20; H, 7.14; N, 17.07%. 4.1.2.16.

(S)-{1-[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]pyrrolidin-2-yl}methanol (22). Following the general procedure, and using (S)-(+)-2pyrrolidinemethanol (40 µL, 0.4 mmol), compound 22 was obtained as yellow solid (0.06 g, 62%) mp = 184 °C; 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 4.85 (brs, 1H), 3.93-3.90 (m, 1H), 3.53-3.50 (m, 4H), 2.67 (s, 3H), 2.03-1.93 (m, 4H), 1.28 (s, 9H); 13C NMR (DMSO-d6) δ: 166.6, 162.4, 154.2, 153.5, 153.1, 130.1, 126.4, 126.0, 114.9, 61.8, 61.4, 48.9, 35.0, 31.2, 28.1, 23.9, 17.3; HRMS (EI) m/z 398.1775 M+, calcd for C21H26N4O2S 398.1776; Anal. Calc. for: (C21H26N4O2S): C, 63.29; H, 6.58; N, 14.06%; Found: C, 63.35; H, 6.61; N, 14.10%. 4.1.2.17.

(R)-{1-[5-(2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-

yl]pyrrolidin-2-yl}methanol (23). Following the general procedure, and using (R)-(-)-2pyrrolidinemethanol (40 µL, 0.4 mmol), compound 23 was obtained as yellow solid (0.07 g, 69%) mp = 158 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 4.84 (brs, 1H), 3.90-3.89 (m, 1H), 3.53-3.48 (m, 4H), 2.67 (s, 3H), 1.98-1.92 (m, 4H), 1.28 (s, 9H);

13

C NMR (DMSO-d6) δ: 166.5, 162.4, 154.3, 153.7, 153.2, 130.0, 126.6, 126.4, 114.9,

61.58, 61.54, 49.0, 35.1, 31.3, 28.1, 23.9, 17.3; HRMS (EI) m/z 398.1774 M+, calcd for

C21H26N4O2S 398.1776; Anal. Calc. for: (C21H26N4O2S): C, 63.29; H, 6.58; N, 14.06%; Found: C, 63.30; H, 6.62; N, 14.09%. 4.1.2.18. (R)-1-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-yl}-N,Ndimethylpyrrolidin-3-amine (24). Following the general procedure, and using ((R)-(+)-3(dimethylamino)pyrrolidine dihydrochloride (74 mg, 0.4 mmol) and potassium carbonate anhydrous (0.1 g, 0.7 mmol), compound 24 was obtained as brown solid (0.05 g, 51%) mp = 137 °C; 1H NMR (DMSO-d6) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 3.69-3.58 (m, 5H), 3.23-3.20 (m, 1H), 2.65 (s, 3H), 2.17 (s, 6H), 1.88-1.82 (m, 1H), 1.29 (s, 9H);

13

C NMR

(DMSO-d6) δ: 166.6, 162.2, 154.4, 153.8, 153.7, 129.9, 126.6, 126.4, 114.9, 65.2, 51.3, 47.2, 44.1, 35.1, 31.2, 29.8, 17.3; HRMS (EI) m/z 411.2091 M+, calcd for C22H29N5OS 411.2093; Anal. Calc. for: (C22H29N5OS): C, 64.20; H, 7.10; N, 17.02%; Found: C, 64.25; H, 7.16; N, 17.05%. 4.1.2.19. (S)-1-{5-[2-(4-(Tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-yl}-N,Ndimethylpyrrolidin-3-amine (25). Following the general procedure, and using (S)-(-)-3(dimethylamino)pyrrolidine (45 µL, 0.4 mmol), compound 25 was obtained as brown solid (0.06 g, 55%) mp = 158 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 3.71-3.68 (m, 3H), 3.50-3.45 (m, 2H), 3.24-3.21 (m, 1H), 2.66 (s, 3H), 2.17 (s, 6H), 1.85-1.79 (m, 1H), 1.29 (s, 9H); 13C NMR (DMSO-d6) δ: 166.6, 162.2, 154.4, 153.9, 153.4, 129.9, 126.63, 126.60, 114.8, 65.2, 51.7, 47.7, 44.1, 35.1, 31.3, 29.7, 17.3; HRMS (EI) m/z 411.2093 M+, calcd for C22H29N5OS 411.2077; Anal. Calc. for: (C22H29N5OS): C, 64.20; H, 7.10; N, 17.02%; Found: C, 64.24; H, 7.14; N, 17.05%. 4.1.2.20.

(S)-1-{5-[2-(4-(tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-

yl}pyrrolidine-2-carboxamide (26). Following the general procedure, and using L-prolinamide

(45 mg, 0.4 mmol), compound 26 was obtained as yellow solid (0.08 g, 73%) mp = 192 °C; 1H NMR (DMSO-d6) δ: 7.90 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.15 (brs, 2H), 4.28-4.26 (m, 1H), 3.63-3.53 (m, 2H), 2.65 (s, 3H), 2.26-2.23 (m, 1H), 1.95-1.93 (m, 3H), 1.28 (s, 9H); 13C NMR (DMSO-d6) δ: 173.6, 166.7, 162.2, 154.4, 153.8, 153.4, 129.9, 126.6, 126.4, 114.8, 61.9, 49.0, 35.1, 31.5, 31.3, 24.2, 17.2; HRMS (EI) m/z 411.1729 M+, calcd for C21H25N5O2S 411.1740; Anal. Calc. for: (C21H25N5O2S): C, 61.29; H, 6.12; N, 17.02%; Found: C, 61.32; H, 6.17; N, 17.04%. 4.1.2.21.

(R)-1-{5-[2-(4-(tert-butyl)phenyl)-4-methylthiazol-5-yl]-1,3,4-oxadiazol-2-

yl}pyrrolidine-2-carboxamide (27). Following the general procedure, and using D-prolinamide (45 mg, 0.4 mmol), compound 27 was obtained as canary yellow solid (0.06 g, 56%) mp = 202 °C; 1H NMR (DMSO-d6) δ: 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.15 (brs, 2H), 4.28-4.26 (m, 1H), 3.62-3.53 (m, 2H), 2.63 (s, 3H), 2.26-2.23 (m, 1H), 1.97-1.92 (m, 3H), 1.28 (s, 9H);

13

C NMR (DMSO-d6) δ: 173.6, 166.7, 162.2, 154.4, 153.8, 153.4, 129.9, 126.7, 126.6,

114.8, 61.9, 49.0, 35.1, 31.5, 31.3, 24.2, 17.3; HRMS (EI) m/z 411.1738 M+, calcd for C21H25N5O2S 411.1729; Anal. Calc. for: (C21H25N5O2S): C, 61.29; H, 6.12; N, 17.02%; Found: C, 66.29; H, 6.16; N, 17.042%. 4.2. Microbiological assay 4.2.1. Microbial Strains and culture media. Bacterial and fungal strains used in this study were clinical isolates obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), Centers for Disease Control and prevention (CDC) and Biodefense and Emerging Infections Research Resources Repository (BEI Resources) (Manassas, VA, USA). RPMI 1640 (Thermo Fisher Scientific, Waltham, MA), yeast peptone dextrose (YPD) broth, YPD agar, cation adjusted Mueller-Hinton broth, brain heart infusion broth, and lactobacilli MRS broth

(Becton, Dickinson and Company, Franklin Lakes, NJ) were purchased from commercial vendors. Phosphate buffered saline (PBS) was purchased from Fisher Scientific (Waltham, MA). Yeast extract, L-cysteine, vitamin K, 3-(N-Morpholino)propanesulfonic acid (MOPS) and hemin were obtained from Sigma-Aldrich (St. Louis, MO). 4.2.2. Initial screening against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentrations (MICs) of the tested compounds and control drugs were determined using the broth microdilution method, according to guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI) [63] against clinically-relevant bacterial strains. 4.2.3. Antifungal activity of compound 18 against Candida, Cryptococcus and Aspergillus clinical isolates. The broth microdilution assay was used to determine the MIC of compound 18 and control antifungal drugs (fluconazole, itraconazole and amphotericin B) against Candida, Cryptococcus and Aspergillus clinical isolates following the guidelines provided by the Clinical and Laboratory Standards Institute for yeasts [64] and molds [65]. Plates containing fungal isolates with 18 and control drugs were incubated aerobically at 37 °C for 24 hours (for Candida and Aspergillus strains) and 48 hours (for Cryptococcus strains) before determining the MIC values by visual inspection. MICs reported are the minimum concentrations of the compound and

control

drugs

that

inhibited

the

visual

fungal

growth

by

50%.

4.2.4. Antimicrobial evaluation of 18 against the human normal bacterial flora. Broth microdilution was utilized in order to determine the MICs of 18 against the human microflora as described elsewhere [66]. 4.2.5. In vitro cytotoxicity evaluation of 18 against human colorectal adenocarcinoma (Caco-2) cells and monkey kidney epithelial cells (Vero). Compound 18 was assayed for its toxicity profile against a human colorectal adenocarcinoma (Caco-2) cell line and fibroblast-like

monkey kidney epithelial cells (Vero) at concentrations 16, 32, 64 and 128 µg/mL as described in previous reports [24, 25]. 4.2.6. Antibiofilm assessment 4.2.6.1. Evaluation of compound 18’s ability to inhibit Candida albicans biofilm formation. Compound 18’s ability to inhibit C. albicans biofilm formation was investigated using the crystal violet assay as described elsewhere [67, 68]. 4.2.6.2. Evaluation of compound 18’s activity against pre-formed mature C. albicans biofilm. The biofilm disrupting activity of 18 was assessed using crystal violet assay as reported earlier [25, 62]. 4.2.6.3. Time-kill kinetics assay against C. albicans. A time kill assay was performed for compound 18 against Candida albicans SS5314 (wild-type), as described previously [69], and detailed in the supporting information. 4.2.6.4. Multi-step resistance study against C. albicans. The capability of Candida albicans SS5314 (wild-type) to develop resistance against oxadiazolylthiazole 18 was investigated via a multistep resistance study as described previously. [70] Resistance was defined as a greater than 4-fold increase in the initial MIC .[71] Conflict of interests There are no conflicts to declare. Supporting information. The supporting information is available free of charge on the journal website. Detailed microbiological procedures, 1H and compounds.

13

C NMR spectra of all new described

Acknowledgments. This work was funded by Academy of Scientific Research and Technology, JESOUR-D program (Cycle 11; Project ID: 3092). The authors would like to thank CDC and BEI Resources for providing the clinical isolates used in this study.

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Highlights: •

Replacement ethylenediamine motif with cis-diaminocyclohexane remarkably enhanced the antimycotic effect

•

Stereochemistry of the two amines is critical as the trans-isomer is 64-times less active

•

Compound 18 is effective vs. fluconazole-resistant albicans and non-albicans candida

•

Compound 18 is more efficient in clearing cryptococcal infections than fluconazole

•

Compound 18 is selective to fungal cells with antibiofilm activity

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: