Mol Divers DOI 10.1007/s11030-014-9548-0

FULL-LENGTH PAPER

Novel triazole alcohol antifungals derived from fluconazole: design, synthesis, and biological activity Seyedeh Mahdieh Hashemi · Hamid Badali · Mohammad Ali Faramarzi · Nasrin Samadi · Mohammad Hosein Afsarian · Hamid Irannejad · Saeed Emami

Received: 24 April 2014 / Accepted: 13 August 2014 © Springer International Publishing Switzerland 2014

Abstract A series of new triazole alcohol antifungals were designed by replacing one of the triazolyl moiety from fluconazole with a distinct 4-amino-3-mercapto1,2,4-triazole motif, which is found in some antimicrobial agents. The antimicrobial susceptibility testing of target compounds demonstrated that the direct analogs of fluconazole (difluorophenethyl-triazoles) were less active against fungi, Electronic supplementary material The online version of this article (doi:10.1007/s11030-014-9548-0) contains supplementary material, which is available to authorized users. S. M. Hashemi Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran H. Badali · M. H. Afsarian Department of Medical Mycology & Parasitology/Invasive Fungi Research Center (IFRC), Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran M. A. Faramarzi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran N. Samadi Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Quality Assurance Research Center, Tehran University of Medical Sciences, Tehran, Iran H. Irannejad · S. Emami (B) Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran e-mail: [email protected] Present address: M. H. Afsarian Departments of Microbiology, Fasa University of Medical Sciences, Fasa, Iran

while compound 10h containing dichloro substitutions on both phenyl rings of the molecule had potent activity against yeasts including Candida albicans (four strains) and Cryptococcus neoformans (MICs = 2–8 µg/mL). Also, compound 10h was active against Candida parapsilosis, Epidermophyton floccosum, and Trichophyton mentagrophytes, while it showed no activity against Gram-positive and Gramnegative bacteria. Finally, a molecular docking study suggested that compound 10h interacts suitably with lanosterol 14α-demethylase, which is the key enzyme in ergosterol biosynthesis. Keywords Antifungal activity · Azole antifungals · Fluconazole · Triazoles · Lanosterol 14α-demethylase

Introduction Treating infections caused by drug-resistant fungi species is regarded as one of the most essential challenges for medicine to date [1]. Despite the introduction of many novel antifungal drugs in recent years, the incidence and mortality of invasive fungal infections have dramatically increased in otherwise healthy individuals, especially in patients infected with human immunodeficiency virus (HIV), intravenous drug abusers, patients using broad spectrum antibiotics, patients with severe underlying diseases or undergoing solid organ transplantation [2–4] Numerous antifungal drugs are available for the treatment of invasive fungal infections. These include polyenes (amphotericin B and nystatin), pyrimidines (5-fluorocyto sine), allylamines (naftifine and terbinafine), echinocandins (caspofungin, anidulafungin and micafungin) and azoles (fluconazole, voriconazole and itraconazole) [5]. Azole antifungals are considered as first-line therapy in current clinical

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Mol Divers Fig. 1 Structures of some marketed triazole alcohol antifungals

HO N

HO N N N F

N N

N

N N

F

N

Voriconazole

CH3

HO N

N N

CN

F S

F

123

N

N N

CH3 O N F

Cl N

F

Ravuconazole

use because of their high therapeutic index, broad spectrum activities, and superior safety profile. Although azoles have a pivotal role in the antifungal armamentarium, some of them still suffer from serious drug resistance issues. Thus, there is a real need for discovering novel antifungal azoles [6,7]. Azole antifungal agents block the synthesis of ergosterol by competitive inhibition of lanosterol 14α-demethylase (CYP51), which is the key enzyme that catalyzes the oxidative removal of the 14α-methyl group of lanosterol to give 14,15-desaturated intermediates in ergosterol biosynthesis. This selective inhibition of CYP51 would result in depletion of ergosterol and accumulation of lanosterol. Ergosterol is the main component of the fungal cell membrane and regulates membrane fluidity and solidity. Ultimately, the net effect of azoles is inhibition of fungal growth and proliferation [8,9]. The necessary pharmacophore of azole antifungals is the N -phenethyl azole structure in which the N-3(4) of the azole ring and the phenyl ring centroid play a key role [10]. Structurally, one of the important groups of azole antifungals is formed by the triazole alcohols. Fluconazole, a bistriazolylpropan-2-ol derivative, was the first triazole alcohol antifungal drug available in the market and widely used for candidiasis. But its ample use and narrow antifungal spectrum, particularly the modest activity against non-Candida species, have influenced its therapeutic efficacy in the clinic [11]. Replacing one of the triazole rings in the fluconazole structure with other heterocyclic moieties resulted in novel triazole alcohol antifungal drugs such as voriconazole, ravuconazole, and albaconazole (Fig. 1). Among various heterocyclic systems, 1,2,4-triazole-3thiols have gained importance as they mediate antibacterial

N

FN

F

Fluconazole

HO

CH3 F

Albaconazole

and antifungal properties. For example, a series of 4-amino3-mercapto-1,2,4-triazoles were described by Collin et al. as potent antifungal agents [12]. In view of above results and as an extension of our studies on the development of azole antifungal agents [13–17], we designed a new series of triazole alcohols by insertion of a 4-amino-3-mercapto-1,2,4-triazole motif instead of the 1,2,4-triazol-1-yl group in the fluconazole structure. As illustrated in Fig. 2, the pharmacophoric features of the molecule were preserved while two pharmacologically compatible molecules were combined in one frame. Herein, we report the synthesis, in vitro antifungal activity and molecular docking study of 1-[(4-amino-5-aryl-4H -1,2,4-triazol-3yl)sulfanyl]-2-(2,4-dihalophenyl)-3-(1H -1,2,4-triazol-1-yl) propan-2-ol derivatives as new fluconazole analogs.

Results and discussion Chemistry The general synthetic route to target compounds 9a–i and 10a–i is presented in Scheme 1. As depicted in Scheme 1, the target compounds were synthesized from the key intermediates, oxirans 4 and 4-amino-3-mercapto-1,2,4-triazoles 8. The synthesis of compounds 4 was started from 2,4dihalophenacyl chloride 1. The reaction of compounds 1 with 4-amino-1,2,4-triazole in refluxing 2-propanol, gave aminotriazolium chloride 2. Deamination of compound 2 occurred in diluted hydrochloric acid in the presence of sodium nitrite to afford 1-(2,4-dihalophenacyl)-1,2,4triazole 3 [13,15]. The oxirane intermediate 4 was obtained

Mol Divers Fig. 2 Design of new triazole alcohols as fluconazole analogs

R

N N S

N

Ar

NH2 Unusual triazoles with potential antifungal activity

OH N

N N N F

N N

Replacement of triazole with new motif

N N

OH N

N N

S X

N

Ar

NH2 F

X

Fluconazole

by Corey-Chaykovsky epoxidation of compound 3 in the presence of trimethylsulfoxonium iodide and NaOH [18]. To obtain 4-amino-3-mercapto-1,2,4-triazole intermediates 8a– i, first the acid hydrazide 6 was prepared from ethyl ester 5 by treating with hydrazine hydrate in absolute ethanol [19]. The acid hydrazide 6 was allowed to react with carbon disulphide in the presence of potassium hydroxide in methanol to give the corresponding potassium dithiocarbazinate 7. This salt underwent ring closure with an excess of hydrazine hydrate to give 4-amino-5-aryl-4H -1,2,4-triazole3-thiol (8a-i) [1,21,22]. In the final step, treatment of the oxirane 4 with the appropriate thiol 8 in the presence of NaHCO3 in absolute ethanol at room temperature gave final compounds 9a–i and 10a–i (Table 1). Structures of the newlysynthesized compounds were confirmed on the basis of 1 H NMR, 13 C NMR, IR and mass spectroscopy. For unambiguous assignment, Fig. 3 presents atom numbering for the general structure. The spectral data confirm the structures of synthesized compounds as interpreted in the experimental section. For example, the 1 H NMR spectrum of compound 9d showed a singlet for NH2 group at δ 5.98 ppm. The Ha and Hb protons of methylene connected to the sulfur atom displayed two doublets at 3.70 and 3.93 ppm with coupling constants of 14.0 Hz. The broad singlet signal at 6.89 ppm is related to the alcoholic hydroxyl group. The diastereotopic protons Hc and Hd appeared as two doublets at 4.66 and 4.70 ppm, with coupling constants of 14.4 Hz. The methoxy group showed a singlet at 3.82 ppm. The protons of the difluorophenyl group showed resonances at 6.96–7.47 ppm. The aromatic protons on the methoxyphenyl moiety displayed two doublets at 7.06 and 7.89 ppm, with coupling constants of 8.8 Hz. The signals related to the triazole ring were found as two singlets at 7.79 and 8.34 ppm. The target compounds 9a–i and 10a–i possess a stereogenic center and were obtained as a mixture of enantiomers.

Designed compounds

It should be noted that many of marketed azole antifungals such as miconazole, ketoconazole, itraconazole, and terconazole are racemic mixtures. Biology The minimal inhibitory concentrations (MICs) of target compounds 9a–i and 10a–i were determined by microdilution method [20]. The title compounds were evaluated against eight human pathogenic fungi: Candida albicans (four strains), Candida parapsilosis, Cryptococcus neoformans, Epidermophyton floccosum, and Trichophyton mentagrophytes. Fluconazole was used as reference antifungal drug. Compounds, in the racemic form, were dissolved in the DMSO and serially diluted in growth medium (concentration range was 0.125–64 µg/mL). The inoculum suspension was added to each well and incubated at 35 ◦ C. The microdilution plates were examined visually after 24–48 h and 72–96 h of incubation for yeast and filamentous fungi, respectively. MIC was defined as the minimum inhibitory concentration of test compound which resulted in total inhibition of the fungal growth. All susceptibility testing was performed in triplicate. The results of antifungal assays are summarized in Table 2. A survey on obtained MIC values revealed that amongst the difluoro derivatives 9a–i only 4-amino-5-(2,4-dichlorophenyl)-triazole derivative 9h showed significant antifungal activity against C. albicans (MICs = 16–32 µg/mL). In the case of dichloro series 10a–i, compounds 10a and 10h were active against all Candida strains. The ranges of observed MIC values for compounds 10a and 10h were 4–32 and 2–8 µg/mL, respectively. However, compounds 10c,d showed remarkable activity against some tested strains of C. albicans (MIC values = 2–8 µg/mL). In addition, the MIC values of tested compounds against Candida parapsilosis revealed that only compound 10h

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Mol Divers

O

O

Cl

OEt

Ar X

X 1a,b

5a-i

a

d O

N

NH2 O

N N X

X

N H

Ar

Cl

2a,b

6a-i

b

e N

O N X

O

N

N H

Ar

H N

X

SK S

7a-i

3a,b c

f N

O N

N N N N NH2 8a-i

Ar X

NH2

X 4a,b

SH

g N

OH

N N X

N N

S

N

Ar

H2N X 9a-i: X= F 10a-i: X= Cl Scheme 1 Synthesis of the title compounds 9a–i and 10a–i. Reagents and conditions: a 4-amino-1,2,4-triazole, 2-propanol, reflux, 12 h; b NaNO2 , HCl, H2 O, 0 ◦ C→ r.t., overnight; c TMSI, NaOH, toluene, 60 ◦ C, 3 h; d N2 H4 , EtOH, reflux, 5 h; e KOH, CS2 , MeOH, r.t., overnight; f N2 H4 , reflux, 3 h; then HCl; g NaHCO3 , EtOH, r.t., 24 h

had growth inhibitory activity. Furthermore, compound 10h with MIC value of 4 µg/mL exhibited good activity against Cryptococcus neoformans. Also, compounds 9e, 10b, 10d and 10h showed marginal inhibitory activity against Epidermophyton floccosum. The standard drug fluconazole was inactive against Trichophyton mentagrophytes, while compound 10h was active possessing MIC value of 32 µg/mL.

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As seen from the data, the alteration of electron-donating and electron-withdrawing substituents on the 5-phenyl-4aminotriazole part of the difluoro series (9, X = F) does not improve the antifungal activity, with the exception of the 2,4-dichlorophenyl substituent (Ar = 2,4-Cl2 -Ph) as found in compound 9h. On the other hand, when the dichlorophenylethyl-triazole scaffold was considered as the basic structure (compounds 10a-i), the intrinsic antifungal activity was preserved, and subsequently the substituent of 5-phenyl-4-aminotriazole could modulate the antifungal activity profile. It seems that in the designed compounds, the dichlorophenylethyl-azole scaffold is more favorable, as found in other azole antifungals (miconazole, ketoconazole and itraconazole, Fig. 4). The comparison of MIC values of compound 10h with those of unsubstituted compound 10a revealed that the introduction of 2,4-dichlorophenyl (Ar = 2,4-Cl2 -Ph) on the adjunct triazole increased the antifungal activity. The effect of a second chlorine added to the chlorophenyl residue was critical and provided activity to compound 9h from the difluoro- series. Since the antibacterial activity of 4-amino-3-mercapto1,2,4-triazoles has been reported previously [21–23], thus the newly synthesized compounds were screened for antibacterial activity. The MIC values of compounds 9a–i and 10a–i were determined against Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633), E. coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Klebsiella pneumoniae (ATCC 10031) based on the conventional agar dilution method [24]. All compounds showed no significant activity (MICs, >100 µg/mL) against tested Gram-positive and Gramnegative bacteria, with the exception of compound 9f, which was found to be active against B. subtilis, E. coli, and K. pneumonia with MIC values of 25–50 µg/mL. Moreover, compounds 10d and 10g exhibited weak activity against K. pneumonia (MICs = 100 µg/mL). Computational studies According to our know-how and to the literature [8–10], the synthesized compounds block the synthesis of ergosterol by competitive inhibition of the lanosterol 14α-demethylase (CYP51). Hence, in order to rationalize the antifungal activity data obtained and to provide more straightforward information into the interactions between ligand and enzyme for further rational drug design, molecular docking was performed for both enantiomeric forms of compound 10h. Since no experimental X-ray crystallographic data were available for the various fungal CYP51 enzymes and because the crystallographic structure of the complex between cytochrome P450 14α-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) and fluconazole (ID 1EA1, 2.2 Å) has proven to have high protein sequence similarity with various

Mol Divers Table 1 Chemical structures of target compounds 9a–i and 10a–i

N N

OH N

N

N NH2

S X

N

Ar

X MW

Formulaa

Yield (%)

Ph

429.45

C19 H17 F2 N7 OS

87

4-OH-Ph

445.45

C19 H17 F2 N7 O2 S

67 78

Entry

Compound

X

Ar

1

9a

F

2

9b

F

3

9c

F

4-Me-Ph

443.47

C20 H19 F2 N7 OS

4

9d

F

4-OMe-Ph

459.47

C20 H19 F2 N7 O2 S

77

5

9e

F

4-F-Ph

447.44

C19 H16 F3 N7 OS

64

6

9f

F

4-Cl-Ph

463.89

C19 H16 ClF2 N7 OS

69 61

7

9g

F

3-Br-Ph

508.34

C19 H16 BrF2 N7 OS

8

9h

F

2,4-Cl2 -Ph

498.34

C19 H15 Cl2 F2 N7 OS

52

9

9i

F

4-Pyridyl

430.43

C18 H16 F2 N8 OS

81

10

10a

Cl

Ph

462.36

C19 H17 Cl2 N7 OS

83

11

10b

Cl

4-OH-Ph

478.35

C19 H17 Cl2 N7 O2 S

63

12

10c

Cl

4-Me-Ph

476.38

C20 H19 Cl2 N7 OS

76

13

10d

Cl

4-OMe-Ph

492.38

C20 H19 Cl2 N7 O2 S

74

14

10e

Cl

4-F-Ph

480.35

C19 H16 Cl2 FN7 OS

67

15

10f

Cl

4-Cl-Ph

496.8

C19 H16 Cl3 N7 OS

72

16

10g

Cl

3-Br-Ph

541.25

C19 H16 BrCl2 N7 OS

59

17

10h

Cl

2,4-Cl2 -Ph

531.25

C19 H15 Cl4 N7 OS

68

18

10i

Cl

4-Pyridyl

463.34

C18 H16 Cl2 N8 OS

85

a

The results of elemental analyses for C, H and N were within ±0.4 % of the theoretical values

3

2 Hb N Hd HCOH H N 1' a 5 N N S 3 N 1 6' 2 X NH2 N 5 6 5

'

3' 4' 5'

3 4

X Fig. 3 Atom numbering of general structure of compounds 9 and 10 for NMR assignments

fungal CYP51 enzymes, thus it has been used extensively for rational design of azole antifungals [25–27]. Accordingly, we also used it for our docking study. Using fluconazole as a reference drug, it was docked into the enzyme active site in order to check and validate the accuracy of our docking protocol. The binding mode of fluconazole obtained by docking was very similar to native co-crystallized one, where the re-docking experiment gave a RMSD value of 1.3 Å, and a favorable free energy value (−5.04 kcal/mol). Moreover the binding interactions

between the docked fluconazole and the amino acid residues were the same as those between the co-crystallized ligand in the active site (Supplementary material). Investigating the binding mode of compound (R)-10h revealed that the triazole ring points towards the heme plane and a ring nitrogen atom coordinates to the iron ion. The distance between the triazole nitrogen atom and the heme iron is 2.5 Å and the calculated free energy of binding is −2.39 kcal/mol. The dichlorophenyl group linked to the aminotriazolyl moiety is situated in a lipophilic space constituted by Phe78, Leu321, Val434, Met433, and Ile323 and makes nonpolar and hydrophobic contacts. The aminotriazolyl moiety takes part in π –π interactions with Tyr76 and the amino group is interacting with His259, Phe78 and Met79 through van der Waals contacts. The dichlorophenyl group attached to the carbinol stereo-center is in contact with Arg96 and Gln72 (Fig. 5). The binding mode of the enantiomeric form (S)-10h was mainly the same as (R)-10h but some differences were observed (Fig. 6). For the (S)-enantiomeric form, the coordinated triazole nitrogen atom is situated far from the heme iron compared to the (R)-enantiomer at distance of 3.8 Å.

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Mol Divers Table 2 In vitro antifungal activities (MICs, µg/mL) of target compounds 9a–i and 10a–i against pathogenic fungia

a

The results were recorded after 24–48 h incubation for yeasts and 72–96 h for filamentous fungi b C.a.I Candida albicans (ATCC 18804), C.a. II Candida albicans (PTCC 6027), C.a. III Candida albicans clinical isolate (IFRC 36), C.a. IV Candida albicans clinical isolate (IFRC 37), C.n. Cryptococcus neoformans (IFRC 192), C. p. Candida parapsilosis (ATCC 22019), T.m. Trichophyton mentagrophytes (CBS 435.73), E. f., Epidermophyton floccosum (IFRC 702), FLZ fluconazole, c Not determined

Compounds

C.a. Ib

C.a. II

C.a. III

C.a. IV

C. p.

C.n.

E. f.

T.m.

9a

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

9b

>64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

9c

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

9d

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

9e

> 64

> 64

> 64

> 64

> 64

> 64

64

> 64

9f

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

9g

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

9h

32

16

NDc

ND

ND

> 64

ND

ND

9i

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

10a

32

4

4

8

> 64

> 64

> 64

> 64

10b

64

> 64

64

64

> 64

> 64

32

> 64

10c

> 64

2

> 64

8

> 64

> 64

> 64

> 64

10d

> 64

8

> 64

4

> 64

> 64

32

> 64

10e

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

10f

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

10g

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

10h

4

8

2

2

32

4

64

32

10i

> 64

> 64

> 64

> 64

> 64

> 64

> 64

> 64

FLZ

2

0.25

0.5

0.5

1

2

16

> 64

Fig. 4 Examples of azole antifungal drugs containing dichlorophenylethyl-azole scaffold

O

Cl N

O

N

N

O

N

N Cl

Cl

O N

O

Cl

Cl Cl Miconazole

Ketoconazole

N

N

O

O

N

N

N N

O

O Cl

N N Cl Itraconazole

The dichlorophenyl group attached to the chiral center is differently located and is oriented towards Phe83, Ala256 and Met99. The aminotriazolyl ring of (S)-10h was rotated by

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180◦ compared to the same group of its enantiomer so that it interacts with Met79 and Phe78. The dichlorophenyl part connected to the aminotriazolyl was overlaid on the corre-

Mol Divers

most compounds showed high potential of reproductive risk, compounds 9h,i and 10h,i, as well as fluconazole are expected to have medium risk of reproductive effect. The predicted drug-likeness of difluoro compounds 9a–i was low, while dichloro derivatives 10a–i had higher drug-likeness values. Interestingly, the most potent compound (10h) had a positive value of drug-likeness and the best value of drugscore.

Conclusion

Fig. 5 a (R)-enantiomer of 10h in the active site of cytochrome P450 14α-sterol demethylase. For clarity only amino acids within 7 Å distance from the docked ligand are shown. b 2D map of (R)-10h in the active site of enzyme

sponding part of its (R)-enantiomer and the interacting amino acids bound similarly. Finally, the free energy of binding for (S)-10h was −2.25 kcal/mol, which is in consistent with the observation that both enantiomers of 10h have almost equivalent potency against MTCYP51 14α-sterol demethylase. The physicochemical and drug-like properties of synthesized compounds 9a–i and 10a–i were predicted using OSIRIS Property Explorer program (http://www.organic-che mistry.org/prog/peo/) in comparison with the standard drug fluconazole. According to the results (Supplementary material), all compounds including fluconazole had no potential of mutagenicity, tumorigenicity and irritant effects. Although

A series of new triazole alcohol antifungals namely 1-[(4amino-5-aryl-4H -1,2,4-triazol-3-yl)sulfanyl]-2-(2,4-dihalophenyl)-3-(1H -1,2,4-triazol-1-yl)propan-2-ols were synthesized and evaluated against a panel of pathogenic fungi and bacteria. The antimicrobial susceptibility testing demonstrated that the dichlorophenethyl-triazole skeleton is more favorable than difluorophenethyl-triazole, and that the secondary dichlorophenyl moiety on the adjunctive triazole ring improves the antifungal activity. Among the tested compounds, compound 10h containing dichloro substituents on both phenyl rings of the molecule had potent activity against C. albicans (four strains) and C. neoformans (MICs = 2–8 µg/mL). Also, compound 10h was active against C. parapsilosis, E. floccosum, and T. mentagrophytes, while it showed no activity against Gram positive and Gram negative bacteria. Finally, our molecular docking study suggested that compound 10h interacts suitably with lanosterol 14αdemethylase enzyme, which is the key enzyme in ergosterol biosynthesis. Because the 4-amino-3-mercapto-1,2,4-triazole motif represents a new type of side chain in triazole alcohol antifungal agents, further structural modification and optimization is required to obtain more information about structure-activity relationships and stereoselectivity of compounds. The antifungal activity profile and pharmacological properties may be more favorable as the designed side chain becomes longer and straighter. The results of this study will be helpful for designing and developing newer antifungal agents.

Experimental section Chemistry All starting materials and reagents were procured from Sigma-Aldrich and Merck Company. The key intermediates 4 and 8 were synthesized according to the literature [18,19,21– 23]. Commercial solvents were used without any pretreatment. Melting points were determined in glass capillary tubes on a Stuart Scientific apparatus and are uncorrected. IR spectroscopy was performed on a FT-IR Perkin Elmer spectrom-

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Fig. 6 (S)-enantiomer 10h in the active site of cytochrome P450 14α-sterol demethylase. For clarity only amino acids within 7 Å distance from the docked ligand are shown

eter in solid phase KBr. All NMR spectra were recorded on a Bruker ultrashield Avance III spectrometer, working at 400 MHz for 1 H and 100 MHz for 13 C NMR. Chemical shifts (δ values) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. The mass spectra were obtained using a HP 5937 Mass Selective Detector (Agilent technologies). Thin layer chromatography (TLC) analysis was carried out on Merck precoated silica gel plates (Kieselgel 60 F254 ). Visualization was achieved by exposure to iodine vapors and/or under UV light (254 nm). As oxiran intermediate 4 was racemic, all compounds 9a–i and 10a–i were obtained as a mixture of enantiomers, having no observable optical activity.

General procedure for the synthesis of compounds 9a–i and 10a–i To a suspension of sodium hydrogen carbonate (1.17 mmol) and 4-amino-5-aryl-4H -1,2,4-triazole-3-thiol (8, 1 mmol) in absolute ethanol (10 mL) was added oxiran 4 (1 mmol), and the reaction mixture was stirred at room temperature for 1 day. After completing the reaction (monitored by TLC), the precipitated product was collected by filtration, washed with water and methanol to give pure title compounds (9a–i and 10a–i).

1-[(4-Amino-5-phenyl-4H-1,2,4-triazol-3-yl) sulfanyl]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol (9a) Yield: 87 %; m.p. 213–214 ◦ C; IR (νmax , cm−1 ): 3147, 1617, 1494, 1463, 1421, 1205, 1133, 1100, 1013, 984, 955, 848,

123

770, 691, 645, 595, 518; 1 H NMR (400 MHz, CDCl3 ) δ: 3.56 (d, 1H, J = 14.8 Hz, Ha ), 3.95 (d, 1H, J = 15.2 Hz, Hb ), 4.58 (d, 1H, J = 14.0 Hz, Hc ), 4.61 (s, 2H, NH2 ), 4.74 (d, 1H, J = 14.0 Hz, Hd ), 6.83–6.90 (m, 2H, H-3, H-5), 7.49– 7.55 (m, 3H, H-3, H-4 and H-5 ), 7.69–7.77 (m, 1H, H-6), 7.80 (s, 1H, OH), 7.86 (s, 1H, H-5 Triazole), 7.92–7.95 (m, 2H, H-2 and H-6 ), 8.29 (s, 1H, H-3 Triazole); MS (m/z, %) 430 (M+1, 2), 347 (18), 221 (100), 176 (86), 139 (30), 127 (50), 104 (72), 77 (68). 1-{[4-Amino-5-(4-hydroxyphenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (9b) Yield: 67 %; m.p.: 192–193 ◦ C; IR (νmax , cm−1 ): 3313, 3120, 1613, 1519, 1467, 1270, 1136, 984, 956, 865, 841, 757, 680, 581, 457; 1 H NMR (400 MHz, DMSO-d6 )δ: 3.69 (d, 1H, J = 13.6 Hz, Ha ), 3.91 (d, 1H, J = 14.0 Hz, Hb ), 4.66 (d, 1H, J = 14.4 Hz, Hc ), 4.70 (d, 1H, J = 14.0 Hz, Hd ), 5.94 (s, 2H, NH2 ), 6.84 (d, 2H, J = 8.4 Hz, H-3 and H-5 ), 6.96–7.01 (m, 1H, H-3), 7.16–7.23 (m, 1H, H-5), 7.41–7.47 (m, 1H, H-6), 7.75 (d, 2H, J = 8.8 Hz, H-2 and H-6 ), 7.79 (s, 1H, H-5 Triazole), 8.35 (s, 1H, H-3 Triazole), 10.09 (br s, 1H, OH Phenolic); 13 C NMR (δ, 100 MHz, DMSO-d6 ): 40.72 (d, JC,F = 4.8 Hz, CH2 -S), 57.05 (d, JC,F = 3.5 Hz, C-OH), 74.44 (d, JC,F = 4.8 Hz, CH2 -N), 104.47 (t, JC,F = 26.3 Hz, 2,4-F2 Ph C-3), 111.44 (dd, JC,F = 20.3 and 2.1 Hz, 2,4-F2 Ph C-5), 115.86 (C-3 and C-5 ), 117.27 (C-1 ), 125.44 (dd, JC,F = 12.8 and 3.1 Hz, 2,4-F2 Ph C-1), 129.77 (C-2 and C-6 ), 130.66 (dd, JC,F = 9.4 and 6.0 Hz, 2,4-F2 Ph C6), 145.56 (C-5 Triazole), 151.12 (C-3 Triazole), 153.74 (C5 Aminotriazole), 154.65 (C-3 Aminotriazole), 159.30 (dd, JC,F = 245.7 and 12.2 Hz, 2,4-F2 Ph C-2), 160.02 (C-4 ), 162.45 (dd, JC,F = 244.8 and 12.2 Hz, 2,4-F2 Ph C-4).

Mol Divers

1-{[4-Amino-5-(4-methylphenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (9c)

1-{[4-Amino-5-(4-fluorophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (9e)

Yield: 78 %; m.p.: 197–198 ◦ C; IR (νmax , cm−1 ): 3444, 3165, 1638, 1615, 1503, 1400, 1278, 1137, 1049, 957, 842, 684, 517; 1 H NMR (400 MHz, DMSO-d6 ) δ: 2.36 (s, 3H, CH3 ), 3.72 (d, 1H, J = 13.6 Hz, Ha ), 3.94 (d, 1H, J = 14.0 Hz, Hb ), 4.66 (d, 1H, J = 14.4 Hz, Hc ), 4.71 (d, 1H, J = 14.4 Hz, Hd ), 6.00 (s, 2H, NH2 ), 6.86 (br s, 1H, OH), 6.97–7.02 (m, 1H, H-3), 7.17–7.23 (m, 1H, H-5), 7.31 (d, 2H, J = 8.0 Hz, H-3 and H-5 ), 7.41–7.47 (m, 1H, H-6), 7.79 (s, 1H, H-5 Triazole), 7.83 (d, 2H, J = 8.0 Hz, H-2 and H-6 ), 8.34 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSO-d6 ): 21.41 (CH3 ), 40.72 (d, JC,F = 4.2 Hz, CH2 -S), 57.02 (d, JC,F = 3.8 Hz, C-OH), 74.39 (d, JC,F = 4.8 Hz, CH2 -N), 104.49 (t, JC,F = 26.8 Hz, 2,4-F2 Ph C-3), 111.44 (dd, JC,F = 20.6 and 2.8 Hz, 2,4-F2 Ph C-5), 124.31 (C-1 ), 125.37 (dd, JC,F = 12.8 and 3.5 Hz, 2,4-F2 Ph C-1), 128.11 (C-2 and C-6 ), 129.50 (C-3 and C-5 ), 130.65 (dd, JC,F = 9.5 and 5.7 Hz, 2,4-F2 Ph-C-6), 145.55 (C-5 Triazole), 151.15 (C-3 Triazole), 154.32 (C-5 Aminotriazole), 154.44 (C-3 Aminotriazole), 159.31 (dd, JC,F = 245.6 and 13.0 Hz, 2,4F2 Ph C-2), 162.46 (dd, JC,F = 244.9 and 12.3 Hz, 2,4-F2 Ph C-4).

Yield: 64 %; m.p.: 222–223 ◦ C; IR (νmax , cm−1 ): 3243, 3107, 1620, 1509, 1275, 1225, 1134, 1013, 957, 843, 730, 677, 516; 1 H NMR (400 MHz, DMSO-d ) δ: 3.72 (d, 1H, J = 14.0 Hz, 6 Ha ), 3.95 (d, 1H, J = 14.0 Hz, Hb ), 4.67 (d, 1H, J = 14.4 Hz, Hc ), 4.71 (d, 1H, J = 14.4 Hz, Hd ), 6.02 (s, 2H, NH2 ), 6.81 (br s, 1H, OH), 6.96–7.02 (m, 1H, H-3), 7.17–7.23 (m, 1H, H-5), 7.36 (t, 2H, J = 8.8 Hz, H-3 and H-5 ), 7.41–7.47 (m, 1H, H-6), 7.79 (s, 1H, H-5 Triazole), 8.00 (dd, 2H, J = 9.2 and 5.6 Hz, H-2 and H-6 ), 8.34 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSO-d ): 40.82 (d, J 6 C,F = 4.5 Hz, CH2 -S), 56.96 (d, JC,F = 3.7 Hz, C-OH), 74.33 (d, JC,F = 4.7 Hz, CH2 -N), 104.49 (t, JC,F = 26.9 Hz, 2,4F2 Ph C-3), 111.44 (dd, JC,F = 20.8 and 2.4 Hz, 2,4-F2 Ph C-5), 116.03 (d, JC,F = 21.7 Hz, C-3 and C-5 ), 123.68 (d, JC,F = 3.2 Hz, 2,4-F2 Ph C-1 ), 125.30 (dd, JC,F = 12.9 and 3.2 Hz, 2,4-F2 Ph C-1), 130.60 (d, JC,F = 8.4 Hz, C-2 and C-6 ), 145.55 (C-5 Triazole), 151.15 (C-3 Triazole), 153.65 (C-5 Aminotriazole), 154.50 (C-3 Aminotriazole), 159.32 (dd, JC,F = 245.7 and 12.3 Hz, 2,4-F2 Ph C-2), 162.47 (dd, JC,F = 244.9 and 12.4 Hz, 2,4-F2 Ph C-4), 163.33 (d, JC,F = 245.8 Hz, C-4 ).

1-{[4-Amino-5-(4-methoxyphenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (9d)

1-{[4-Amino-5-(4-chlorophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (9f)

Yield: 77 %; m.p.: 206–207 ◦ C; IR (νmax , cm−1 ): 3260, 3156, 2833, 1614, 1509, 1494, 1465, 1273, 1136, 1101, 1084, 955, 848, 832; 1 H NMR (400 MHz, DMSO-d6 )δ: 3.70 (d, 1H, J = 14.0 Hz, Ha ), 3.82 (s, 3H, OCH3 ), 3.93 (d, 1H, J = 14.0 Hz, Hb ), 4.66 (d, 1H, J = 14.4 Hz, Hc ), 4.70 (d, 1H, J = 14.4 Hz, Hd ), 5.98 (s, 2H, NH2 ), 6.89 (br s, 1H, OH), 6.96–7.02 (m, 1H, H-3), 7.06 (d, 2H, J = 8.8 Hz, H-3 and H-5 ), 7.17–7.23 (m, 1H, H-5), 7.41–7.47 (m, 1H, H-6), 7.79 (s, 1H, H-5 Triazole), 7.89 (d, 2H, J = 8.8 Hz, H-2 and H-6 ), 8.34 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSO-d6 ): 40.76 (d, JC,F = 4.6 Hz, CH2 -S), 55.76 (OCH3 ), 57.02 (d, JC,F = 3.9 Hz, C-OH), 74.41 (d, JC,F = 4.7 Hz, CH2 -N), 104.49 (t, JC,F = 26.3 Hz, 2,4-F2 Ph C-3), 111.43 (dd, JC,F = 20.5 and 3.6 Hz, 2,4-F2 Ph C-5), 114.38 (C-3 and C-5 ), 119.48 (C-1 ), 125.40 (dd, JC,F = 13.0 and 3.7 Hz, 2,4-F2 Ph C-1), 129.73 (C-2 and C-6 ), 130.65 (dd, JC,F = 9.8 and 5.6 Hz, 2,4-F2 Ph C-6), 145.56 (C-5 Triazole), 151.14 (C-3 Triazole), 154.04 (C-5 Aminotriazole), 154.28 (C-3 Aminotriazole), 159.31 (dd, JC,F = 245.7 and 12.1 Hz, 2,4-F2 Ph C-2), 160.78 (C-4 ), 162.46 (dd, JC,F = 245.1 and 12.4 Hz, 2,4-F2 Ph C-4).

Yield: 69 %; m.p.: 212–213 ◦ C; IR (νmax , cm−1 ): 3244, 3132, 1650, 1619, 1508, 1421, 1397, 1274, 1204, 1134, 1014, 957, 832, 724, 677, 516; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.73 (d, 1H, J = 13.6 Hz, Ha ), 3.96 (d, 1H, J = 13.6 Hz, Hb ), 4.67 (d, 1H, J = 14.4 Hz, Hc ), 4.72 (d, 1H, J = 14.4 Hz, Hd ), 6.04 (s, 2H, NH2 ), 6.78 (br s, 1H, OH), 6.96–7.02 (m, 1H, H-3), 7.16–7.23 (m, 1H, H-5), 7.41–7.47 (m, 1H, H6), 7.59 (d, 2H, J = 8.8 Hz, H-3 and H-5 ), 7.80 (s, 1H, H-5 Triazole), 7.99 (d, 2H, J = 8.4 Hz, H-2 and H-6 ), 8.34 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSOd6 ): 40.85 (d, JC,F = 4.5 Hz, CH2 -S), 56.95 (d, JC,F = 3.7 Hz, C-OH), 74.31 (d, JC,F = 4.7 Hz, CH2 -N), 104.50 (t, JC,F = 26.4 Hz, 2,4-F2 Ph C-3), 111.44 (dd, JC,F = 20.5 and 2.7 Hz, 2,4-F2 Ph C-5), 125.28 (dd, JC,F = 12.8 and 3.4 Hz, 2,4-F2 Ph C-1), 125.98 (C-1 ), 129.09 (C-2 and C-6 ), 129.87 (C-3 and C-5 ), 130.65 (dd, JC,F = 9.4 and 5.7 Hz, 2,4-F2 Ph C-6), 134.99 (C-4 ), 145.56 (C-5 Triazole), 151.15 (C-3 Triazole), 153.45 (C-5 Aminotriazole), 154.78 (C-3 Aminotriazole), 159.32 (dd, JC,F = 245.8 and 12.3 Hz, 2,4-F2 Ph C-2), 162.47 (dd, JC,F = 245.0 and 12.4 Hz, 2,4F2 Ph C-4).

123

Mol Divers

1-{[4-Amino-5-(3-bromophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (9g) Yield: 61 %; m.p.: 203–204 ◦ C; IR (νmax , cm−1 ): 3448, 3253, 3139, 1648, 1618, 1508, 1420, 1273, 1207, 1135, 1099, 955, 797, 675, 515; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.73 (d, 1H, J = 13.6 Hz, Ha ), 3.96 (d, 1H, J = 13.6 Hz, Hb ), 4.66 (d, 1H, J = 14.4 Hz, Hc ), 4.71 (d, 1H, J = 14.4 Hz, Hd ), 6.06 (s, 2H, NH2 ), 6.75 (s, 1H, OH), 6.96–7.02 (m, 1H, H-3), 7.16– 7.23 (m, 1H, H-5), 7.41–7.45 (m, 1H, H-6), 7.48 (t, 1H, J = 8.0 Hz, H-5 ), 7.70 (ddd, 1H, J = 8.4, 1.6 and 1.2 Hz, H-4 ), 7.79 (s, 1H, H-5 Triazole), 7.96 (dt, 1H, J = 8.4 and 1.2 Hz, H-6 ), 8.17 (t, 1H, J = 1.6 Hz, H-2 ), 8.34 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSO-d6 ): 40.82 (d, JC,F = 4.8 Hz, CH2 -S), 56.92 (d, JC,F = 3.7 Hz, C-OH), 74.27 (d, JC,F = 4.6 Hz, CH2 -N), 104.51 (t, JC,F = 26.7 Hz, 2,4F2 Ph C-3), 111.44 (dd, JC,F = 20.6 and 2.4 Hz, 2,4-F2 Ph C-5), 122.13 (C-3 ), 125.24 (dd, JC,F = 12.9 and 3.5 Hz, 2,4-F2 Ph C-1), 127.14 (C-6 ), 129.27 (C-5 ), 130.39 (C-4 ), 130.64 (dd, JC,F = 9.4 and 5.9 Hz, 2,4-F2 Ph C-6),131.22 (C-1 ), 132.91 (C-2 ), 145.55 (C-5 Triazole), 151.16 (C-3 Triazole), 153.00 (C-5 Aminotriazole), 154.96 (C-3 Aminotriazole), 159.32 (dd, JC,F = 246.0 and 12.3 Hz, 2,4-F2 Ph C-2), 162.47 (dd, JC,F = 244.9 and 12.4 Hz, 2,4-F2 Ph C-4). 1-{[4-Amino-5-(2,4-dichlorophenyl)-4H-1,2,4-triazol3-yl]sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (9h) Yield: 52 %; m.p.: 191–193 ◦ C; IR (νmax , cm−1 ): 3450, 1618, 1556, 1499, 1272, 1206, 1138, 1050, 965, 811, 676, 612,560; 1 H NMR (400 MHz, DMSO-d ) δ: 3.74 (d, 1H, J = 13.6 Hz, 6 Ha ), 3.98 (d, 1H, J = 13.6 Hz, Hb ), 4.68 (d, 1H, J = 14.4 Hz, Hc ), 4.73 (d, 1H, J = 14.0 Hz, Hd ), 5.79 (s, 2H, NH2 ), 6.83 (br s, 1H, OH), 6.96–7.01 (m, 1H, H-3), 7.15– 7.22 (m, 1H, H-5), 7.40–7.47 (m, 1H, H-6), 7.55–7.59 (m, 2H, H-3 and H-5 ), 7.76–7.85 (m, 2H, H-6 and H-5 Triazole), 8.36 (s, 1H, H-3 Triazole); MS (m/z, %) 498 (M + 1, 5), 259 (46), 224 (100), 171 (26), 141 (66), 113 (21), 82 (74), 58 (48). 1-{[4-Amino-5-(4-pyridyl)-4H-1,2,4-triazol-3-yl] sulfanyl}-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol (9i) Yield: 81 %; m.p.: 232–233 ◦ C; IR (νmax , cm−1 ): 3444, 3237, 3101, 1655, 1617, 1601, 1508, 1494, 1419, 1396, 1272, 1133, 1100, 1010, 955, 849, 702, 667, 518; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.76 (d, 1H, J = 13.6 Hz, Ha ), 3.98 (d, 1H, J = 13.6 Hz, Hb ), 4.67 (d, 1H, J = 14.4 Hz, Hc ), 4.72 (d, 1H, J = 14.4 Hz, Hd ), 6.13 (s, 2H, NH2 ), 6.71 (br s, 1H, OH), 6.96–7.02 (m, 1H, H-3), 7.16–7.23 (m, 1H, H-5), 7.41–7.47

123

(m, 1H, H-6), 7.80 (s, 1H, H-5 Triazole), 7.96 (d, 2H, J = 6.0 Hz, H-2 and H-6 ), 8.34 (s, 1H, H-3 Triazole), 8.72 (d, 2H, J = 6.4 Hz, H-3 and H-5 ); 13 C NMR (δ, 100 MHz, DMSOd6 ): 40.79 (d, JC,F = 4.8 Hz, CH2 -S), 56.89 (d, JC,F = 3.8 Hz, C-OH), 74.23 (d, JC,F = 4.5 Hz, CH2 -N), 104.52 (t, JC,F = 26.8 Hz, 2,4-F2 Ph C-3), 111.44 (dd, JC,F = 20.8 and 2.7 Hz, 2,4-F2 Ph C-5), 121.78 (C-2 and C-6 ), 125.19 (dd, JC,F = 12.8 and 3.4 Hz, 2,4-F2 Ph C-1), 130.64 (dd, JC,F = 9.5 and 5.8 Hz, 2,4-F2 Ph C-6), 134.25 (C-1 ), 145.55 (C-5 Triazole), 150.54 (C-3 and C-5 ), 151.17 (C-3 Triazole), 152.32 (C-5 Aminotriazole), 155.80 (C-3 Aminotriazole), 159.34 (dd, JC,F = 245.9 and 12.2 Hz, 2,4-F2 Ph C-2), 162.47 (dd, JC,F = 245.0 and 12.5 Hz, 2,4-F2 Ph C-4). 1-[(4-Amino-5-phenyl-4H-1,2,4-triazol-3-yl) sulfanyl]-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol (10a) Yield: 83 %; m.p.: 227–228 ◦ C; IR (νmax , cm−1 ): 3435, 3148, 1586, 1507, 1463, 1374, 1273, 1209, 1135, 1049, 1011, 863, 830, 793, 771, 698; 1 H NMR (500 MHz, CDCl3 ) δ: 3.55 (d, 1H, J = 15.02 Hz, Ha ), 4.18 (d, 1H, J = 14.98 Hz, Hb ), 4.70 (d, 1H, J = 14.01 Hz, Hc ), 4.80 (d, 1H, J = 14.04 Hz, Hd ), 5.61 (br s, 2H, NH2 ), 7.01 (dd, 1H, J = 8.61 and 2.13 Hz, H-5), 7.24 (d, 1H, J = 2.11 Hz, H-3), 7.32–7.37 (m, 3H, H-3, H-4 and H-5 ), 7.58 (d, 1H, J = 8.63 Hz, H-6), 7.60 (s, 1H, H-5 Triazole), 7.87 (dd, 2H, J = 7.98 and 2.1 Hz, H-2 and H-6 ), 8.25 (br s, 1H, H-3 Triazole); MS (m/z, %) 462 (M+1, 7), 379 (36), 301 (45), 252 (38), 192 (57), 181 (84), 104 (100), 77 (69). 1-{[4-Amino-5-(4-hydroxyphenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (10b) Yield: 63 %; m.p.: 221–223 ◦ C; IR (νmax , cm−1 ): 3120, 1614, 1587, 1509, 1462, 1373, 1278, 1129, 1010, 977, 864, 789, 658, 587, 522; 1 H NMR (500 MHz, DMSO-d6 ) δ: 3.71 (d, 1H, J = 14.6 Hz, Ha ), 4.32 (d, 1H, J = 13.3 Hz, Hb ), 4.78 (d, 1H, J = 14.1 Hz, Hc ), 4.96 (d, 1H, J = 15.4 Hz, Hd ), 5.96 (s, 2H, NH2 ), 6.86 (d, 2H, J = 8.8 Hz, H-3 and H-5 ), 7.20 (br s, 1H, OH), 7.35 (d, 1H, J = 9.3 Hz, H-5), 7.57–7.60 (m, 2H, H-3 and H-6), 7.76 (d, 2H, J = 8.7 Hz, H-2 and H-6 ), 7.78 (s, 1H, H-5 Triazole), 8.36 (s, 1H, H-3 Triazole), 9.92 (br s, 1H, OH Phenolic); MS (m/z, %) 477 (M+, 4), 400 (79), 372 (8), 259 (64), 224 (100), 203 (14), 181 (55), 159 (33), 127 (94), 101 (24), 82 (87), 55 (51). 1-{[4-Amino-5-(4-methylphenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (10c) Yield: 76 %; m.p.: 205–206 ◦ C; IR (νmax , cm−1 ): 3452, 3148, 1651, 1509, 1462, 1273, 1134, 1084, 1007, 819, 794, 722,

Mol Divers

677, 655, 587; 1 H NMR (400 MHz, DMSO-d6 ) δ: 2.36 (s, 3H, CH3 ), 3.74 (d, 1H, J = 14.0 Hz, Ha ), 4.33 (d, 1H, J = 14.0 Hz, Hb ), 4.79 (d, 1H, J = 14.4 Hz, Hc ), 4.98 (d, 1H, J = 14.4 Hz, Hd ), 6.02 (s, 2H, NH2 ), 7.14 (br s, 1H, OH), 7.31 (d, 2H, J = 8.0 Hz, H-3 and H-5 ), 7.35 (dd, 1H, J = 8.6 and 2.4 Hz, H-5), 7.58 (d, 1H, J = 8.4 Hz, H-6), 7.59 (d, 1H, J = 2.0 Hz, H-3), 7.78 (s, 1H, H-5 Triazole), 7.83 (d, 2H, J = 8.0 Hz, H-2 and H-6 ), 8.37 (s, 1H, H-3 Triazole). MS (m/z, %) 474 (M+1, 8), 424 (5), 378 (40), 313 (20), 256 (37), 218 (100), 159 (45), 118 (85), 55 (77).

1-{[4-Amino-5-(4-methoxyphenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (10d) Yield: 74 %; m.p.: 222–223 ◦ C; IR (νmax , cm−1 ): 3242, 3148, 2835, 1650, 1613, 1511, 1466, 1374, 1264, 1184, 1135, 1085, 916, 832, 793, 677, 587; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.72 (d, 1H, J = 14.0 Hz, Ha ), 3.81 (s, 3H, OCH3 ), 4.33 (d, 1H, J = 13.6 Hz, Hb ), 4.78 (d, 1H, J = 14.4 Hz, Hc ), 4.97 (d, 1H, J = 14.4 Hz, Hd ), 6.01 (s, 2H, NH2 ), 7.06 (d, 2H, J = 8.8 Hz, H-3 and H-5 ), 7.17 (s, 1H, OH), 7.35 (dd, 1H, J = 8.6 and 2.0 Hz, H-5), 7.59 (d, 1H, J = 8.8 Hz, H-6), 7.59 (d, 1H, J = 2.0 Hz, H-3), 7.79 (s, 1H, H-5 Triazole), 7.88 (d, 2H, J = 8.8 Hz, H-2 and H-6 ), 8.36 (s, 1H, H-3 Triazole); MS (m/z, %) 493 (M + 1, 2), 456 (14), 409 (63), 366 (23), 331 (67), 259 (17), 222 (100), 181 (56), 134 (81), 90 (31), 55 (47).

1-{[4-Amino-5-(4-fluorophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (10e) Yield: 67 %; m.p.: 236–237 ◦ C; IR (νmax , cm−1 ): 3102, 2819, 1657, 1587, 1508, 1376, 1277, 1158, 1136, 1013, 918, 840, 793, 728, 682, 652, 587; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.75 (d, 1H, J = 13.6 Hz, Ha ), 4.36 (d, 1H, J = 14.0 Hz, Hb ), 4.79 (d, 1H, J = 14.4 Hz, Hc ), 4.99 (d, 1H, J = 14.0 Hz, Hd ), 6.04 (s, 2H, NH2 ), 7.08 (s, 1H, OH), 7.32–7.39 (m, 3H, H-5, H-3 and H-5 ), 7.58 (d, 1H, J = 2.0 Hz, H-3), 7.60 (d, 1H, J = 8.4 Hz, H-6), 7.79 (s, 1H, H-5 Triazole), 8.00 (dd, 2H, J = 9 and 5.6 Hz, H-2 and H-6 ), 8.36 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSO-d ): 55.64 (C-OH), 75.74 6 (CH2 -N), 116.05 (d, JC,F = 21.7 Hz, C-3 and C-5 ), 123.61 (d, JC,F = 3.1 Hz, C-1’), 127.52 (2,4-Cl2 Ph C-5), 130.42 (2,4-Cl2 Ph C-6), 130.61 (d, JC,F = 8.6 Hz, C-2 and C6 ), 131.62 (2,4-Cl2 Ph C-2), 131.75 (2,4-Cl2 Ph C-4), 133.68 (2,4-Cl2 Ph C-3), 138.14 (2,4-Cl2 Ph C-1), 145.67 (C-5 Triazole), 151.13 (C-3 Triazole), 153.69 (C-5 Aminotriazole), 154.62 (C-3 Aminotriazole), 163.36 (d, JC,F = 246.0 Hz, C-4 ).

1-{[4-Amino-5-(4-chlorophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1yl)propan-2-ol (10f) Yield: 72 %; m.p.: 228–229 ◦ C; IR (νmax , cm−1 ): 3145, 2827, 1586, 1508, 1464, 1378, 1275, 1206, 1142, 1083, 1016, 918, 861, 831, 794, 722, 656, 582; 1 H NMR (400 MHz, DMSOd6 ) δ: 3.76 (d, 1H, J = 13.6 Hz, Ha ), 4.36 (d, 1H, J = 13.6 Hz, Hb ), 4.79 (d, 1H, J = 14.4 Hz, Hc ), 4.99 (d, 1H, J = 14.4 Hz, Hd ), 6.06 (s, 2H, NH2 ), 7.04 (s, 1H, OH), 7.35 (dd, 1H, J = 8.8 and 2.4 Hz, H-5), 7.52–7.59 (m, 2H, H-3 and H-6), 7.59 (d, 2H, J = 8.8 Hz, H-3 and H-5 ), 7.79 (s, 1H, H-5 Triazole), 7.98 (d, 2H, J = 8.8 Hz, H-2 and H-6 ), 8.36 (s, 1H, H-3 Triazole); MS (m/z, %) 495 (M+, 2), 460 (18), 415 (100), 372 (25), 335 (97), 258 (15), 226 (71), 159 (54), 111 (40), 55 (46).

1-{[4-Amino-5-(3-bromophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (10g) Yield: 59 %; m.p.: 219–220 ◦ C; IR (νmax , cm−1 ): 3241, 3137, 1649, 1508, 1459, 1427, 1374, 1273, 1134, 1083, 1007, 863, 798, 713, 676, 651; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.76 (d, 1H, J = 14.0 Hz, Ha ), 4.36 (d, 1H, J = 14.0 Hz, Hb ), 4.79 (d, 1H, J = 14.4 Hz, Hc ), 4.99 (d, 1H, J = 14.0 Hz, Hd ), 6.07 (s, 2H, NH2 ), 6.97 (s, 1H, OH), 7.35 (dd, 1H, J = 8.6 and 2.0 Hz, H-5), 7.48 (t, 1H, J = 8.0 Hz, H-5 ), 7.58 (d, 1H, J = 2.0 Hz, H-3), 7.59 (d, 1H, J = 8.8 Hz, H-6), 7.71 (ddd, 1H, J = 8.4, 1.6 and 0.8 Hz, H-4 ), 7.79 (s, 1H, H-5 Triazole), 7.96 (dt, 1H, J = 8.4 and 0.8 Hz, H-6 ), 8.17 (t, 1H, J = 1.6 Hz, H-2 ), 8.35 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSO-d6 ): 55.60 (C-OH), 75.65 (CH2 -N), 122.14 (C-3 ), 127.15 (2,4-Cl2 Ph C-5), 127.52 (C-6 ), 129.21 (2,4-Cl2 Ph C-6), 130.39 (2,4-Cl2 Ph C-2), 130.44 (2,4-Cl2 Ph C-4), 131.23 (2,4-Cl2 Ph C-3), 131.61 (C-5 ), 131.75 (C-4 ), 132.95 (C-1 ), 133.69 (C-2 ), 138.04 (2,4-Cl2 Ph C-1), 145.66 (C-5 Triazole), 151.15 (C-3 Triazole), 153.03 (C-5 Aminotriazole), 155.05 (C-3 Aminotriazole).

1-{[4-Amino-5-(2,4-dichlorophenyl)-4H-1,2,4-triazol-3yl]sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol1-yl)propan-2-ol (10h) Yield: 68 %; m.p.: 200–201 ◦ C; IR (νmax , cm−1 ): 3336, 3123, 1637, 1596, 1513, 1464, 1372, 1276, 1135, 1090, 977, 872, 813, 673; 1 H NMR (400 MHz, DMSO-d6 )δ: 3.78 (d, 1H, J = 13.6 Hz, Ha ), 4.38 (d, 1H, J = 13.6 Hz, Hb ), 4.80 (d, 1H, J = 14.4 Hz, Hc ), 5.00 (d, 1H, J = 14.4 Hz, Hd ), 5.81 (s, 2H, NH2 ), 6.98 (s, 1H, OH), 7.36 (dd, 1H, J = 8.6 and 2.4 Hz, H-5), 7.58–7.61 (m, 4H, H-3, H-6, H-5 and H-6 ),

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7.79 (s, 1H, H-5 Triazole), 7.83 (t, 1H, J = 1.2 Hz, H-3 ), 8.36 (s, 1H, H-3 Triazole); 13 C NMR (δ, 100 MHz, DMSOd6 ): 55.62 (C-OH), 75.60 (CH2 -N), 125.45 (2,4-Cl2 Ph C-5), 127.54 (C-5 ), 127.86 (2,4-Cl2 Ph C-6), 129.80 (2,4-Cl2 Ph C-3), 130.46 (C-6 ), 131.65 (C-3 ), 131.74 (2,4-Cl2 Ph C2), 133.73 (2,4-Cl2 Ph C-4), 134.08 (C-2 ), 135.01 (C-4 ), 136.23 (C-1 ), 138.04 (2,4-Cl2 Ph C-1), 145.66 (C-5 Triazole), 151.16 (C-3 Triazole), 152.86 (C-5 Aminotriazole), 153.92 (C-3 Aminotriazole). 1-{[4-Amino-5-(4-pyridyl)-4H-1,2,4-triazol-3-yl] sulfanyl}-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol (10i) Yield: 85 %; m.p.: 235– 236 ◦ C; IR (νmax , cm−1 ): 3444, 1655, 1600, 1509, 1462, 1374, 1275, 1210, 1132, 1084, 1064, 1008, 917, 863, 831, 702, 656; 1 H NMR (400 MHz, DMSO-d6 ) δ: 3.80 (d, 1H, J = 13.6 Hz, Ha ), 4.38 (d, 1H, J = 13.2 Hz, Hb ), 4.80 (d, 1H, J = 14.4 Hz, Hc ), 5.00 (d, 1H, J = 14.4 Hz, Hd ), 6.14 (s, 2H, NH2 ), 6.93 (s, 1H, OH), 7.35 (dd, 1H, J = 8.6 and 2.0 Hz, H-5), 7.58 (d, 1H, J = 2.4 Hz, H-3), 7.58 (d, 1H, J = 8.8 Hz, H-6), 7.79 (s, 1H, H-5 Triazole), 7.96 (d, 2H, J = 6.4 Hz, H-2 and H-6 ), 8.35 (s, 1H, H-3 Triazole), 8.72 (d, 2H, J = 6.0 Hz, H-3 and H-5 ); MS (m/z, %) 463 (M+1, 0.2), 365 (24), 256 (32), 218 (100), 192 (80), 162 (72), 119 (74), 105 (65), 78 (72), 51 (78). Biological activity assays Broth dilution antifungal susceptibility testing The MICs of compounds were determined for yeast and filamentous fungi by micro-dilution method according to the guidelines of CLSI documents M27-A3 and M38-A2, respectively [20]. A stock solution of compounds was prepared in DMSO and then diluted in the standard RPMI-1640 medium (Sigma Chemical Co.) buffered to pH 7.0, containing 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma) and Lglutamine, without bicarbonate. The serial dilutions of compounds were dispensed into 96-well micro-dilution trays at a final concentration of 0.125–64 µg/mL. Plates were stored at −70 ◦ C until they were used. The inoculum suspensions, including mostly nongerminated conidia, were diluted in RPMI 1640 medium. The final concentration of the stock inoculum suspensions of the tested isolates ranged from 0.5– 3×103 and 0.5–3×104 CFU/mL for yeast species and filamentous species, respectively. After inoculation, the microdilution plates were incubated at 35◦ C and examined visually after 24–48 h and 72–96 h of incubation for yeast and filamentous fungi, respectively. The endpoint MIC was the lowest concentration of the compound at which the test strain does not demonstrate visible growth.

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Agar dilution antibacterial susceptibility testing The minimum inhibitory concentrations (MICs) of compounds 9a-i and 10a–i were determined by agar dilution method [24]. The test compounds (10.0 mg) were dissolved in DMSO (1 mL) and then diluted with water (9 mL) and stored as stock solutions. Further serial dilution with molten sterile Mueller-Hinton agar was performed to obtain the required final concentrations (0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µg/mL). The medium containing the test compounds was dispensed into a sterile Petri-dish and allowed to solidify. Petri-dishes were inoculated with 1–5×104 CFU and incubated at 37 ◦ C for 18 h. The MIC was defined as the lowest concentration of the test compound, which resulted in no visible growth on the plate.

Docking study The X-ray coordinate of cytochrome P450 14α-sterol demethylase from M. tuberculosis (MTCYP51) in complex with fluconazole (PDB ID: 1EA1) was retrieved from Protein Data Bank (http://www.pdb.org). Pymol 1.6.0 (The PyMOL Molecular Graphics System, Schrödinger, LLC) was used for visualizing and handling the protein structure. Structures of compounds have been prepared using MarvinSketch 5.5.0.1 (ChemAxon: Budapest, Hungary; www.chemaxon. com). The lowest energy conformations were generated using semi-empirical AM1 method in Gaussian98. All docking studies have been performed with AutoDock 4.2 software, employing AutoDock Tools (ADT) graphical interface in MGLTools 1.5.6. For the protein and ligands, Kollman and Gasteiger charges were computed respectively and the non-polar hydrogen atoms were merged with ADT, thus preparing the appropriate PDBQT files for the molecular docking studies. The Lamarckian genetic algorithm (LGA) was used as a search engine. The active site was defined using AutoGrid 4.2. The grid size was set to 20×20×20 points with a grid spacing of 1.0 Å centered on the mass of original ligand in the crystal structure complex. A maximum number of energy evaluations were set to 2,500,000. For each of the 20 independent runs, a maximum number of 27,000 LGA operations were generated on a single population of 150 individuals. Operator weights for crossover, mutation, and elitism were set to 0.80, 0.02, and 1, respectively. 2D map of the ligand-active site complex was made by LigPlot 1.4.4. Acknowledgments This work was supported by a grant from the Research Council of Mazandaran University of Medical Sciences, Sari, Iran. This work was related to the Ph.D. thesis of SMH (Faculty of Pharmacy, Mazandaran University of Medical Sciences).

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