Steroids xxx (2016) xxx–xxx

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Steroids journal homepage: www.elsevier.com/locate/steroids

Synthesis and antifungal activity of bile acid-derived oxazoles Lucía R. Fernández a, Laura Svetaz b, Estefanía Butassi b, Susana A. Zacchino b, Jorge A. Palermo a, Marianela Sánchez a,⇑ a UMYMFOR – Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 3° Piso, (1428) Buenos Aires, Argentina b Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, CP2000, Rosario, Argentina

a r t i c l e

i n f o

Article history: Received 25 November 2015 Received in revised form 11 January 2016 Accepted 26 January 2016 Available online xxxx Keywords: Bile acids Candida albicans Oxazoles Dakin–West reaction

a b s t r a c t Peracetylated bile acids (1a–g) were used as starting materials for the preparation of fourteen new derivatives bearing an oxazole moiety in their side chain (6a–g, 8a–g). The key step for the synthetic path was a Dakin–West reaction followed by a Robinson–Gabriel cyclodehydration. A simpler model oxazole (12) was also synthesized. The antifungal activity of the new compounds (6a–g) as well as their starting bile acids (1a–g) was tested against Candida albicans. Compounds 6e and 6g showed the highest percentages of inhibition (63.84% and 61.40% at 250 lg/mL respectively). Deacetylation of compounds 6a–g, led to compounds 8a–g which showed lower activities than the acetylated derivatives. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Bile acids are enantiomerically pure, abundant and inexpensive commercial compounds with a wide range of biological activities [1]. The pharmacological interest in these acids is directly related to the fact that liver cells can specifically recognize such natural ligands. This fact makes them ideal building blocks for the synthesis of novel molecules that can be recognized at the molecular level [2]. The particular conformation, reactivity and amphiphilic properties of bile acids were also exploited in supramolecular chemistry and biotechnological applications [3–4]. Small heterocycles such as imidazole, thiazole and oxazole are present in both marine and terrestrial natural products. In a biosynthetical sense, thiazole and oxazole are masked aminoacids, usually formed by cyclization of serine and cysteine, and are frequent structural fragments in cyclic peptides. Secondary metabolites with these structural fragments have shown diverse biological properties, which include antitumoral, antibacterial and antiviral activities [5]. In medicinal chemistry, an emergent practice is to bind two or more bioactive molecules as a way to improve the biological properties of the starting components [6,7]. In this context, it was expected that a compound combining a bile acid core and an oxazole moiety, may retain intrinsic biological properties belonging to both fragments. For example, synthetic ⇑ Corresponding author. E-mail address: [email protected] (M. Sánchez).

steroidal isoxazoles have already shown a great variety of biological activities [8] and more recently, 6,5 fused cholestane oxazoles have been tested for antifungal activity [9]. In previous work, our group has used the strategy of bioconjugation to prepare compounds which combined bile acids with quinones and Cinchona alkaloids [10–12]. In this work, 14 new compounds were synthesized using acetylated bile acids and the 4-methyl ester of aspartic acid as starting materials. The synthetic strategy had as key steps a Dakin-West reaction followed by a Robinson–Gabriel cyclodehydration [13]. The parent acetylated bile acids and their resulting derivatives together with a low-molecular weight compound representing the oxazole fragment itself were evaluated for antifungal activity against Candida albicans.

2. Experimental 2.1. General The bile acids chenodeoxycholic, lithocholic, deoxycholic, hyodeoxycholic, cholic, ursodeoxycholic and hyocholic and all other reagents were obtained from Sigma–Aldrich Co. (St Louis, MO, USA). All chemicals and solvents were of analytical grade. Silicagel 60 H (Merck) was used for dry column flash chromatography. TLC was carried out on Merck Silicagel 60 F254 plates. TLC plates were analyzed by visualization under UV light (254 nm), exposition to iodine vapors or by spraying with either 2% vanillin

http://dx.doi.org/10.1016/j.steroids.2016.01.014 0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

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L.R. Fernández et al. / Steroids xxx (2016) xxx–xxx

in H2SO4 (cc) or 0.2% ninhydrin in ethanol. Among the solvents, CH2Cl2 was distilled from phosphorous pentoxide and Me2CO was distilled from potassium permanganate prior to their use in the syntheses. UV spectra were obtained on a Hewlett Packard 8453 spectrophotometer and IR spectra were obtained on an FT-IR Nicolet Magna 550 instrument. Optical rotations were measured on a Perkin-Elmer 343 polarimeter, whereas 1H and 13C NMR data were acquired using Bruker Avance-2 (500 MHz) and AC-200 (200 MHz) spectrometers, in CDCl3 or CD3OD. Proton chemical shifts were referenced to the residual signal of protonated CDCl3 at d 7.26 or d 3.31 when CD3OD was used, and 13C NMR were referenced to the central peak of CDCl3 at 77.0 ppm or CD3OD at 49.0 ppm. Homonuclear 1H connectivities were determined by COSY experiments. The edited reverse-detected single quantum heteronuclear correlation (DEPT–HSQC) experiment allowed the determination of carbon multiplicities, as well as one-bond proton–carbon connectivities, and the heteronuclear multiple bond correlation (HMBC) experiments allowed the determination of long-range proton–carbon correlations. All 2D NMR experiments were performed using standard pulse sequences. HRESI mass spectra were recorded using a Bruker MicrOTOF QII mass spectrometer. HPLC separations were performed using HPLC-grade solvents, a Thermo Separations Spectra Series P100 pump, a Thermo Separations Refractomonitor IV RI detector, a Thermo Separations SpectraSeries UV 100 UV detector, and a Phenomenex Bondclone 10 C18 (10 lm, 8 mm
300 mm) column. UV detection was performed at 220 nm. Peracetylated compounds 1a–g were obtained by standard methods, using Ac2O/DMAP/Pyr. Compounds 3 and 9 were prepared according to previously reported procedures [14,15]. 2.2. Chemistry 2.2.1. Methyl (5-methyl-2-(3a,7a-diacetoxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (6a) To a solution of the peracetylated bile acid 1a (119 mg, 0.25 mmol) in dry CH2Cl2 (5 mL), under inert atmosphere and stirring at 0 °C, a solution of 1.25 mmoles (5 equiv, 0.10 mL) of oxalyl chloride in 2 mL of dry CH2Cl2 was added dropwise. After 3 h, the solvent was evaporated at reduced pressure and the acid chloride 2a was redissolved in dry acetone and used as such in the following step. Sodium carbonate (83.8 mg, 0.88 mmol, 3.5 equiv) was suspended in 2 mL of a 1:1 mixture of pyridine and acetone at 0 °C. Then, the 4-methyl ester of aspartic acid (3) (153.5 mg, 0.5 mmol, 2 equiv) was added, followed by a slow addition of the corresponding bile acid chloride (2a) in acetone (2 mL), the resulting mixture was stirred for 30 min. After this time, the solution was adjusted to pH = 3 by adding HCl and extracted with CH2Cl2 (3 x 15 mL). The combined organic phases were concentrated to yield a yellowish oil (4a) that was utilized without further purification. Compound 4a was dissolved in 0.6 mL of pyridine under stirring with the addition of a catalytic amount of 4-dimethylaminopyridine (DMAP). Slowly, 0.6 mL of acetic anhydride was added and the reaction mixture was kept at 90 °C for one hour. Then, the mixture was acidified to pH = 3 with HCl and extracted with 10 mL of CH2Cl2 (3 times). The combined organic layers were successively washed with 2 N HCl and water. A yellow oil (5a) was obtained after concentration and used in the following step. Ketoamide 5a was dissolved in 3 mL of DMF with stirring under inert atmosphere, and then 6.25 lL of phosphoryl chloride (POCl3) were added dropwise. The reaction mixture was heated up to 90 °C for twenty minutes, then diluted with 20 mL of water and finally extracted with diethyl ether (3
15 mL). The combined organic phases were washed with water, dried over magnesium sulfate and evaporated under reduced pressure. The crude product was chromatographed on

silicagel with cyclohexane:EtOAc (9:1) as mobile phase to give 6a (7.4 mg, 5.1% from 1a) as a yellowish oil. 1H NMR (500 MHz, CDCl3): 4.59 (1H, tt, J = 11.4, 4.4 Hz, H-3), 4.87 (1H, q, J = 2.9 Hz, H-7), 0.63 (3H, s, H-18), 0.93 (3H, s, H-19), 0.96 (3H, d, J = 5.8 Hz, H-21), 2.73 (1H, ddd, J = 15.2, 11.2, 4.3 Hz, H-23a), 2.56 (1H, ddd, J = 14.7, 10.0, 6.0 Hz, H-23b), 2.23 (3H, s, H-60 ), 3.45 (2H, s, H-70 ), 3.71 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 608.3581 (calc. for C34H51NNaO+7, 608.3558). IR (film, cm1): 2937, 2871, 1734. aD (CHCl3, c = 0.39) = +15.8°. UV (CHCl3, 1/Mcm): e241 = 388, e269 = 300. 2.2.2. Methyl (5-methyl-2-(3a-acetoxy-24-nor-5b-cholan-23-yl)-1,3oxazol-4-yl)acetate (6b) The title compound was prepared in 6.2% yield (8 mg) as an oil from 1b (105 mg, 0.25 mmol) via the procedure used to prepare 6a. 1 H NMR (500 MHz, CDCl3): 4.71 (1H, tt, J = 11.4, 4.7 Hz, H-3), 0.63 (3H, s, H-18), 0.92 (3H, s, H-19), 0.95 (3H, d, J = 6.2 Hz, H-21), 2.73 (1H, ddd, J = 15.3, 11.3, 4.4 Hz, H-23a), 2.56 (1H, ddd, J = 14.8, 10.1, 5.9 Hz, H-23b), 2.23 (3H, s, H-60 ), 3.45 (2H, s, H-70 ), 3.71 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 550.3522 (calc. for C32H49NNaO+5, 550.3503). IR (film, cm1): 2939, 2867, 1735. aD (CHCl3, c = 0.75) = +23.3°. UV (CHCl3, 1/Mcm): e242 = 995, e268 = 828. 2.2.3. Methyl (5-methyl-2-(3a,12a-diacetoxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (6c) The compound was prepared in 24.2% yield (35.4 mg) as an oil from 1c (119 mg, 0.25 mmol) via the procedure used to prepare 6a. 1 H NMR (500 MHz, CDCl3): 4.69 (1H, m, H-3), 5.07 (1H, brs, H-12), 0.71 (3H, s, H-18), 0.90 (3H, s, H-19), 0.84 (3H, d, J = 6.1 Hz, H-21), 2.71 (1H, ddd, J = 15.3, 10.8, 3.8 Hz, H-23a), 2.54 (1H, ddd, J = 15.3, 10.2, 5.8 Hz, H-23b), 2.22 (3H, s, H-60 ), 3.44 (2H, s, H-70 ), 3.70 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 608.3611 (calc. for C34H51NNaO+7, 608.3563). IR (film, cm1): 2925, 2869, 1733, 1623. aD (CHCl3, c = 0.56) = +79.4°. UV (CHCl3, 1/Mcm): e241 = 304, e268 = 178. 2.2.4. Methyl (5-methyl-2-(3a,6a-diacetoxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (6d) The compound was prepared in 9.5% yield (13.9 mg) as an oil from 1d (119 mg, 0.25 mmol) applying the procedure used to prepare 6a. 1H NMR (500 MHz, CDCl3): 4.69 (1H, m, H-3), 5.14 (1H, dt, J = 12.3, 4.8 Hz, H-6), 0.63 (3H, s, H-18), 0.96 (3H, s, H-19), 0.95 (3H, d, J = 6.1 Hz, H-21), 2.72 (1H, m, H-23a), 2.57 (1H, ddd, J = 15.3, 10.2, 5.9 Hz, H-23b), 2.22 (3H, s, H-60 ), 3.42 (2H, s, H-70 ), 3.70 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 608.3589 (calc. for C34H51NNaO+7, 608.3563). IR (film, cm1): 2925, 2869, 1736. aD (CHCl3, c = 0.23) = +22.2°. UV (CHCl3, 1/Mcm): e242 = 726, e270 = 562. 2.2.5. Methyl (5-methyl-2-(3a,7a,12a-triacetoxy-24-nor-5b-cholan23-yl)-1,3-oxazol-4-yl)acetate (6e) The compound was obtained in 16% yield (25.7 mg) as an oil from 1e (133 mg, 0.25 mmol) by the same procedure used for 6a. 1 H NMR (500 MHz, CDCl3): 4.57 (1H, tt, J = 11.3, 4.3 Hz, H-3), 4.90 (1H, q, J = 3.2 Hz, H-7), 5.08 (1H, t, J = 2.9 Hz, H-12), 0.72 (3H, s, H-18), 0.91 (3H, s, H-19), 0.85 (3H, d, J = 5.8 Hz, H-21), 2.72 (1H, m, H-23a), 2.55 (1H, ddd, J = 15.4, 10.0, 6.0 Hz, H-23b), 2.22 (3H, s, H-60 ), 3.44 (2H, s, H-70 ), 3.70 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 666.3662 (calc. for C36H53NNaO+9, 666.3613). IR (film, cm1) 3448, 2950, 2872, 1735, 1654, 1648. aD (CHCl3, c = 0.36) = +66.4°. UV (CHCl3, 1/Mcm): e242 = 631, e272 = 183.

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L.R. Fernández et al. / Steroids xxx (2016) xxx–xxx Table 1 C NMR (125 MHz, CDCl3) data for compounds 6a–g and 12.

13

C

6a

6b

6c

6d

6e

6f

6g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 3-OCOCH3

35.1 27.0 74.4 34.8 41.1 31.5 71.4 38.1 34.3 35.0 20.8 39.7 42.9 50.6 23.7 28.2 55.8 11.9 22.9 35.6 18.6 33.3 25.2 170.8

35.2 26.8 74.6 32.4 42.1 26.5a 28.4a 36.0 40.6 34.8 21.0 40.3 42.9 56.7 24.4 27.2 56.1 12.2 23.5 35.7 18.5 33.4 25.3 170.8

34.9 26.8 74.4 32.4 42.0 26.0a 27.1a 35.9 34.6 34.2 25.8 76.1 45.2 49.7 23.6 27.6 47.7 12.6 23.2 35.1 17.8 33.2 25.2 170.7

35.2 26.6 73.8 26.4 45.5 71.1 31.5 34.8 40.0 36.2 20.9 40.0 43.1 56.3 24.3 28.3 56.0 12.2 23.4 35.6 18.5 33.4 25.2 170.7a

34.8 27.1 74.3 34.9 41.1 31.4 70.9 37.9 29.1 34.5 25.8 75.6 45.2 43.6 23.0a 27.4a 47.3 12.4 22.8 34.9 17.8 33.2 25.0 170.7

34.7 26.6 73.7 33.0 42.2 33.0 73.8 40.2 39.6 34.2 21.4 40.1 43.8 55.4 25.8 28.6 55.1 12.2 23.4 35.6 18.7 33.4 25.3 170.8

35.2 26.9 73.9 28.9 45.6 70.1 70.9 37.4 34.2 36.3 20.8 39.4 43.0 50.4 23.6 28.1 55.7 11.9 23.2 35.5 18.5 33.3 25.1 170.7

3-OCOCH3

21.7

21.6

21.5

21.6

21.7

170.6

7-OCOCH3

21.8

21.8

12-OCOCH3

a

21.6 21.1

170.6 170.6

14.0

170.4

21.6

6-OCOCH3 7-OCOCH3

12-OCOCH3 20 40 50 60 70

22.0a

170.6a

6-OCOCH3

12

170.8 21.6a

170.3 21.3

170.7

21.6

21.6

70 -COOCH3

163.3 127.5 145.0 10.2 32.0 171.1

163.4 127.5 144.8 10.2 32.0 171.2

163.2 127.6 145.0 10.2 32.0 171.1

163.3 127.6 145.0 10.2 32.0 171.1

163.2 127.6 144.9 10.2 32.0 171.1

163.3 127.6 144.9 10.2 32.0 171.1

163.2 127.5 144.9 10.2 32.0 171.1

159.5 127.8 145.1 10.2 32.0 171.1

70 -COOCH3

52.3

52.3

52.3

52.3

52.3

52.3

52.3

52.3

Values may be interchanged.

2.2.6. Methyl (5-methyl-2-(3a,7b-diacetoxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (6f) The compound was prepared in 38% yield (55.8 mg) as an oil from 1f (119 mg, 0.25 mmol) via the procedure used to prepare 6a. 1H NMR (500 MHz, CDCl3): 4.66 (1H, tt, J = 10.7, 5.1 Hz, H-3), 4.76 (1H, td, J = 10.9, 5.2 Hz, H-7), 0.67 (3H, s, H-18), 0.98 (3H, s, H-19), 0.96 (3H, d, J = 5.9, H-21), 2.71 (1H, m, H-23a), 2.56 (1H, ddd, J = 15.3, 9.9, 5.9 Hz, H-23b), 2.23 (3H, s, H-60 ), 3.45 (2H, s, H-70 ), 3.70 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 608.3647 (calc. for C34H51NNaO+7, 608.3558). IR (film, cm1): 2923, 2872, 2851, 1733. aD (CHCl3, c = 0.60) = +40.1°. UV (CHCl3, 1/Mcm): e241 = 1357, e268 = 1707. 2.2.7. Methyl (5-methyl-2-(3a,6a,7a-triacetoxy-24-nor-5b-cholan23-yl)-1,3-oxazol-4-yl)acetate (6g) The compound was obtained in 36% yield (57.9 mg) as an oil from 1g (133 mg, 0.25 mmol) by the same procedure used for 6a. 1 H NMR (500 MHz, CDCl3): 4.56 (1H, m, H-3), 5.10 (1H, dd, J = 5.4, 3.5 Hz, H-6), 5.20 (1H, t, J = 3.1 Hz, H-7), 0.63 (3H, s, H-18), 0.98 (3H, s, H-19), 0.95 (3H, d, J = 5.8 Hz, H-21), 2.71 (1H, ddd, J = 15.0, 11.0, 4.2 Hz, H-23a), 2.56 (1H, ddd, J = 14.5, 9.9, 6.1 Hz, H-23b), 2.22 (3H, s, H-60 ), 3.43 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+Na]+ 666.3655 (calc. for C36H53NNaO+9, 666.3613). IR (film, cm1): 2944, 2871, 1740, 1623. aD (CHCl3, c = 0.86) = +21.7°. UV (CHCl3, 1/Mcm): e241 = 1048, e269 = 890.

2.2.8. Methyl (5-methyl-2-(3a,7a-dihydroxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (8a) A solution of NaOH in MeOH (20%) was added to compound 6a (21.8 mg) up to a final concentration of 1 mg/mL, and the mixture was kept under reflux for 24 h. The solvent was removed under reduced pressure and the residue was dissolved in H2O adjusting the pH to 5 with HCl before extraction with EtOAc. Evaporation of the organic layer led to the intermediate 7a which was treated with an excess of CH2N2 in ethyl ether and stirred for 4 h at room temperature. The solvent was evaporated under a N2 stream and the residue was dissolved in MeOH for further preparative HPLC purification with MeOH:H2O 85:15 as elution solvent. This procedure led to 7.4 mg of compound 8a as a transparent oil (tR = 68.5 min, 39% yield). 1H NMR (500 MHz, CD3OD): 3.37 (1H, m, H-3), 3.79 (1H, brs, H-7), 0.68 (3H, s, H-18), 0.93 (3H, s, H-19), 1.00 (3H, d, J = 6.2 Hz, H-21), 2.75 (1H, m, H-23a), 2.62 (1H, m, H-23b), 2.25 (3H, s, H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/ z [M+H]+ 502.3542 (calc. for C30H48NO+5, 502.3532). IR (film, cm1): 3383, 2927, 2865, 1741. UV (CHCl3, 1/Mcm): e218 = 3620, e288 = 1380. 2.2.9. Methyl (5-methyl-2-(3a-hydroxy-24-nor-5b-cholan-23-yl)-1,3oxazol-4-yl)acetate (8b) A solution of NaOH in MeOH (20%) was added to compound 6b (14.2 mg) up to a final concentration of 1 mg/mL, and the mixture

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L.R. Fernández et al. / Steroids xxx (2016) xxx–xxx

Table 2 C NMR (125 MHz, CD3OD) data for compounds 8a–g.

13

a

C

8a

8b

8c

8d

8e

8f

8g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 20 40 50 60 70 70 -COOCH3

36.6 31.4 72.9 40.5 43.2 35.9 69.1 40.8 34.1 36.7 21.8 41.0 43.7 51.5 24.6 29.2 57.1 12.1 23.4 36.7 18.8 34.4 25.7 165.1 128.5 146.9 9.7 31.9 172.5

36.5 31.2 72.4 37.2 43.6 27.7 29.2 37.2 41.9 35.7 21.9 41.5 43.9 57.9 25.3 28.4 57.2 12.4 23.9 36.7 18.8 34.3 25.7 165.1 128.5 146.9 9.7 31.9 172.5

36.5 31.1 72.6 37.2 43.6 28.4 27.5 37.5 34.8 35.3 29.9 74.0 47.6 49.3 24.9 28.6 48.0 13.2 23.7 36.7 17.6 34.4 25.7 165.2 128.5 146.9 9.8 31.9 172.5

36.8 31.1 72.4 30.0 49.9 68.7 35.6 36.2 41.3 36.9 21.9 41.3 44.0 57.2 25.3 29.2 57.6 12.4 24.1 36.7 18.8 34.3 25.7 165.1 128.5 146.9 9.4 31.9 172.5

36.5 31.2 72.9 40.5 43.2 35.9 69.1 41.0 27.9 35.8 29.6 74.0 47.5 43.0 28.7 24.2 47.9 13.0 23.2 36.7 17.7 34.4 25.7 165.2 128.5 146.9 9.7 31.9 172.5

36.1 31.0 72.1 38.6a 44.1 38.0a 72.0 44.5 40.7 35.2 22.4 41.5 44.8 57.5 27.9 29.6 56.3 12.6 23.9 36.6 19.0 34.4 25.7 165.1 128.5 146.9 9.7 31.9 172.5

36.8 31.4 72.8 33.3 49.5 70.7 72.9 33.9 40.0 37.0 21.8 40.9 43.8 51.4 24.5 29.2 57.1 12.1 23.7 36.7 18.8 34.4 25.7 165.1 128.5 146.9 9.7 31.9 172.5

70 -COOCH3

52.6

52.6

52.6

52.6

52.6

52.6

52.6

Values may be interchanged.

was kept under reflux overnight. Applying the same procedure as above, the intermediate 7b was obtained and further methylated. Purification by preparative HPLC purification with MeOH:H2O 90:10 as elution solvent led to 3.6 mg of compound 8b as a transparent oil (tR = 49 min) with a 27% yield. 1H NMR (500 MHz, CD3OD): 3.54 (1H, m, H-3), 0.68 (3H, s, H-18), 0.95 (3H, s, H-19), 0.99 (3H, d, J = 6.2 Hz, H-21), 2.75 (1H, m, H-23a), 2.61 (1H, m, H-23b), 2.25 (3H, s, H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/z [M+H]+ 496.3602 (calc. for C30H48NO+4, 486.3583). IR (film, cm1): 3390, 2927, 2862, 1745. UV (CHCl3, 1/Mcm): e218 = 4914. 2.2.10. Methyl (5-methyl-2-(3a,12a-dihydroxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (8c) Using the same procedure mentioned for 8b, 10.4 mg of compound 8c (45% yield) was obtained as an oil from 6c (26.7 mg) (HPLC conditions: MeOH:H2O 85:15, tR = 75 min). 1H NMR (500 MHz, CD3OD): 3.52 (1H, m, H-3), 3.96 (1H, t, J = 2.8 Hz, H-12), 0.70 (3H, s, H-18), 0.93 (3H, s, H-19), 1.05 (3H, d, J = 6.4 Hz, H-21), 2.76 (1H, m, H-23a), 2.62 (1H, m, H-23b), 2.25 (3H, s, H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/z [M+H]+ 502.3511 (calc. for C30H48NO+5, 502.3532). IR (film, cm1): 3378, 2934, 1743, 1577. UV (CHCl3, 1/Mcm): e219 = 4301. 2.2.11. Methyl (5-methyl-2-(3a,6a-dihydroxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (8d) The compound was prepared in 15% yield (3.9 mg) as an oil from 6d (29.3 mg) via the procedure used to obtaine 8b (HPLC conditions: MeOH:H2O 85:15, tR = 37.5 min). 1H NMR (500 MHz, CD3OD): 3.51 (1H, m, H-3), 4.01 (1H, dt, J = 12.1, 4.7 Hz, H-6), 0.68 (3H, s, H-18), 0.93 (3H, s, H-19), 0.99 (3H, d, J = 6.2 Hz, H-21), 2.75 (1H, m, H-23a), 2.62 (1H, m, H-23b), 2.25 (3H, s,

H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/z [M+H]+ 502.3491 (calc. for C30H48NO+5, 502.3532). IR (film, cm1): 3357, 2932, 2865, 1743. UV (CHCl3, 1/Mcm): e219 = 54827. 2.2.12. Methyl (5-methyl-2-(3a,7a,12a-trihydroxy-24-nor-5b-cholan23-yl)-1,3-oxazol-4-yl)acetate (8e) The compound was prepared in 29% yield (6.5 mg) as an oil from 6e (27.4 mg) via the procedure used to prepare 8b (HPLC conditions: MeOH:H2O 80:20, tR = 66.5 min). 1H NMR (500 MHz, CD3OD): 3.38 (1H, m, H-3), 3.80 (1H, m, H-7), 3.95 (1H, t, J = 2.7 Hz, H-12), 0.71 (3H, s, H-18), 0.92 (3H, s, H-19), 1.06 (3H, d, J = 6.4 Hz, H-21), 2.77 (1H, ddd, J = 15.1, 10.2, 4.7 Hz, H-23a), 2.66 (1H, m, H-23b), 2.25 (3H, s, H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/z [M+H]+ 518.3458 (calc. for C30H48NO+6, 518.3432). IR (film, cm1) 3392, 2927, 2864, 1740. UV (CHCl3, 1/Mcm): e218 = 6461, e271 = 1441. 2.2.13. Methyl (5-methyl-2-(3a,7b-dihydroxy-24-nor-5b-cholan-23yl)-1,3-oxazol-4-yl)acetate (8f) The compound was prepared in 51% yield (8.4 mg) as an oil from 6f (19.4 mg) via the procedure used to prepare 8b (HPLC conditions: MeOH:H2O 80:20, tR = 45 min). 1H NMR (500 MHz, CD3OD): 3.49 (1H, m, H-3), 3.49 (1H, m, H-7), 0.70 (3H, s, H-18), 0.97 (3H, s, H-19), 1.00 (3H, d, J = 6.1 Hz, H-21), 2.75 (1H, ddd, J = 14.7, 10.1, 4.6 Hz, H-23a), 2.61 (1H, m, H-23b), 2.25 (3H, s, H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/z [M+H]+ 502.3536 (calc. for C30H48NO+5, 502.3532). IR (film, cm1): 3362, 2929, 2863, 1742. UV (CHCl3, 1/Mcm): e219 = 2020. 2.2.14. Methyl (5-methyl-2-(3a,6a,7a-trihydroxy-24-nor-5b-cholan23-yl)-1,3-oxazol-4-yl)acetate (8g) The compound was prepared in 50% yield (6.0 mg) as an oil from 6g (14.9 mg) via the procedure used to prepare 8b (HPLC conditions: MeOH:H2O 80:20, tR = 55 min). 1H NMR (500 MHz, CD3OD: 3.34 (1H, m, H-3), 3.78 (1H, m, H-6), 3.77 (1H, m, H-7), 0.68 (3H, s, H-18), 0.93 (3H, s, H-19), 1.01 (3H, d, J = 6.2 Hz, H-21), 2.76 (1H, ddd, J = 14.6, 10.1, 4.5 Hz, H-23a), 2.62 (1H, m, H-23b), 2.25 (3H, s, H-60 ), 3.49 (2H, s, H-70 ), 3.69 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CD3OD): see Table 2. ESI-MS m/z [M+H]+ 518.3461 (calc. for C30H48NO+6, 518.3432). IR (film, cm1): 3285, 2926, 2854, 1735. UV (CHCl3, 1/Mcm): e219 = 771, e281 = 826. 2.2.15. Methyl 2-(2,5-dimethyloxazol-4-yl)acetate (12) To a solution of 0.7 mmoles of compound 9 in 1 mL of pyridine, 1 mL of Ac2O and a catalytic amount of DMAP were added. The reaction mixture was left at room temperature under stirring for 30 min. During the next half hour the temperature was set at 90 °C. Then, the mixture was acidified to pH = 3 with HCl and extracted with 10 ml of CH2Cl2 (3 times). The combined organic layers were successively washed with 2 N HCl and water. After evaporation of the solvent under reduced pressure, the resulting oil (10) was used in the next step without further purification. A solution of 197 mg of compound 10 in 1 mL of DMF was treated with POCl3 using the procedure previously described for compounds 6a–g. The crude product (11) was used as such in the next step. To a solution of 47.1 mg of compound 11 in 6 mL of MeOH, 0.1 equiv of K2HPO4 were added, and the reaction mixture was heated under reflux for 3 h with stirring. The solvent was then evaporated under reduced pressure, and the residue was partitioned between H2O and cyclohexane. The aqueous layer was liophilized and purified by dry column flash chromatography on silicagel (with

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a gradient of cyclohexane:EtOAc) which afforded 8 mg of compound 12 as a colorless oil (yield 6.7%). 1H NMR (500 MHz, CDCl3): 2.37 (3H, s, H-23), 2.23 (3H, s, H-60 ), 3.44 (2H, s, H-70 ), 3.71 (3H, s, 70 -COOCH3). 13C NMR (125 MHz, CDCl3): see Table 1. ESI-MS m/z [M+H]+ 170.0825 (calc. for C8H12NO+3, 170.0812). IR (film, cm1): 3366, 2954, 2927, 2853, 1743. UV (CHCl3, 1/Mcm): e261 = 15.9. 2.3. Antifungal activity evaluation 2.3.1. Microorganisms and media For the evaluation of antifungal activity, the standardized strain C. albicans ATCC 10231 from the American Type Culture Collection (ATCC, Rockville, MD, USA), was used. The strain was grown on a Sabouraud-chloramphenicol agar slant for 48 h at 30 °C, maintained on slopes of Sabouraud-Dextrose agar (SDA, Oxoid) and sub-cultured every 15 days to prevent pleomorphic transformations. The inoculum suspension was obtained according to reported procedures [16] and adjusted to 1–5
103 cells with colony forming units (CFU)/mL. 2.3.2. Fungal growth inhibition percentage determination Broth microdilution technique was performed in 96-well microplates according to the guidelines of the Clinical and Laboratory Standards Institute for yeasts (M27-A3) [16]. For the assay, compound test wells (CTWs) were prepared with stock solutions of

each compound in DMSO (maximum concentration 6 1%), diluted with RPMI-1640, to final concentrations of 250–0.98 lg/mL. An inoculum suspension (100 lL) was added to each well (final volume in the well = 200 lL). A growth control well (GCW) (medium, inoculum, the same amount of DMSO used in a CTW, but compound-free) and a sterility control well (SCW) (sample, medium and sterile water instead of inoculum) were included in each plate. Microtiter trays were incubated in a moist, dark chamber at 30 °C, 48 h. Microplates were read in a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA, USA). Amphotericin B was used as positive control. Tests were performed in triplicate. Reduction of growth for each compound concentration was calculated as follows: % of inhibition = 100  (OD405 CTW  OD405 SCW)/(OD405 GCW  OD405 SCW). Inhibition percentages ± SEM vs. concentrations of each compound were used for constructing doses–response curves by using SigmaPlotÒ 11.0 software. 2.3.3. Statistical analysis The unpaired Student’s test was used to analyze the data (p < 0.05, calculated using a two-tailed test, was considered significant). 3. Results In this work, fourteen new bile acid derivatives bearing an oxazole moiety in the side chain (6a–g and 8a–g) were synthesized

O HO

COOCH 3

O

NH 2 3 +

HO

(ii)

6´

O COOCH3 (iii)

O

NH

5´ 4´

COOCH 3 (iv) O

COOCH 3

O

NH

7´

N 2´

RCOCl

R 4a-g

2a-g

R

R 5a-g

6a-g (v)

(i) 6´

RCOOH 1a-g

5´ 4´

7´

(vi)

COOCH 3

O

7a-g

N 2´

R' 8a-g 21

R3

1 3

AcO

10 5

H

H

20 23

19 11

R=

21

R3

H 14 8 7

19 11

13 16

R '= H

R2 R1

1 3

HO

10 5

H

20 23

H 14

H7

8

13 16

H R2

R1

a R 1: H 2, R 2: α-OAc, R 3 : H 2

a R1 : H 2, R2 : α-OH, R 3: H2

b R1 : H 2, R2 : H 2, R3 : H 2

b R 1: H2, R 2: H2, R 3 : H2

c R 1: H 2, R 2: H 2, R 3: OAc

c R1 : H 2, R2 : H 2, R3 : OH

d R1 : OAc, R 2 : H2, R 3 : H2

d R 1: OH, R 2: H2, R 3: H 2

e R 1: H 2, R 2: α-OAc, R 3 : OAc

e R1 : H 2, R2 : α-OH, R 3: OH

f R 1 : H 2, R 2 : β-OAc, R3 : H 2

f R 1: H2, R 2: β-OH, R 3 : H2

g R1 : OAc, R 2 : α-OAc, R3 : H 2

g R 1: OH, R 2: α-OH, R3 : H2

Fig. 1. Synthesis of compounds 6a–g and 8a–g were R and R0 represent the corresponding steroid core. (i) oxalyl chloride, CH2Cl2, 0 °C, 3 h (ii) Na2CO3, Py:Me2CO, 0 °C, 300 ; (iii) Ac2O, DMAP, Py, 90 °C, 1 h; (iv) POCl3, DMF; 90 °C, 200 ; (v) NaOH, MeOH, reflux, 16–24 h; (vi) CH2N2, ethyl ether, RT, 4 h.

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L.R. Fernández et al. / Steroids xxx (2016) xxx–xxx

O H2N

N

(ii)

(i) HN

O

5´

2´

O O

23

O

O

OH O

N

(iii) O

O

COOCH3

7´

O

9

10

OCH2CH2Ph

11

6´

4´

12

Fig. 2. Synthesis of compound 12 (i) Ac2O, Py, DMAP, RT to 90 °C, 1 h; (ii) POCl3, DMF; 90 °C, 200 ; (iii) K2HPO4, MeOH, reflux, 3 h.

was prepared from b-phenylethyl aspartate (9) by a Dakin–West reaction. The antifungal activity of the peracetylated bile acids 1a–g, the oxazole derivatives (6a–g, 8a–g) and compound 12 against C. albicans ATCC 10231 was evaluated by the standardized microbroth dilution method M-27A3 [16]. The results are expressed as percentages of inhibition of the fungus in the range of 250–3.9 lg/ mL and are displayed in Table 3. The results showed that compound 12 is almost devoid of activity with only 2.1% of inhibition at 250 lg/mL while peracetylated bile acids 1a–g possess low to moderate antifungal activities (11.7–40.1% at 250 lg/mL). Regarding the antifungal behavior of oxazoles 6a–g, it is interesting to note that 6a, 6d–g, which have an -OAc group either at C-6 (R1) or C-7 (R2) or at both positions, showed a significant enhancement (p < 0.05) of the fungal inhibitory capacity compared to their parent compounds 1a, 1d–g. For example, compound 6a displayed a 46.2% inhibition at 250 lg/mL while 1a and 12 produced 21.1% and 2.1% of inhibition respectively. Fig. 3 depicts

using the peracetylated bile acids (1a–g) as starting materials (Fig. 1). The acyl chlorides of the peracetylated acids (2a–g) were prepared by a standard technique with oxalyl chloride under inert atmosphere. Amides (4a–g) were produced by reaction of the corresponding acyl chlorides with the b-methyl protected form of aspartic acid [13]. The protection of the b-carboxyl group of aspartic acid was necessary to achieve good yields in the following step of the synthetic route in which compounds 4a–g were converted to ketoamides 5a–g by a Dakin–West reaction. This key step of the synthesis introduced the methyl group necessary for the Robinson–Gabriel cyclodehydration, which provided oxazoles 6a–g. The final products 8a–g were obtained by refluxing 6a–g with a methanolic solution of NaOH followed by a standard methylation with CH2N2. In order to evaluate the role of the covalent bond between the steroids and the oxazole in the enhancement of the antifungal activity, a simplified oxazole (12) was synthesized (Fig. 2), bearing a methyl group instead of a norcholane one at C-20 . This compound

Table 3 Comparative antifungal activities (inhibition percentages) of the bile acids 1a–g, oxazoles 6a–g, their deacetylated derivatives 8a–g and 12 against Candida albicans ATCC 10231.

21

R3

1 3

AcO

23

H 14

10

H

5

H

19

13 16 1

8

H

7

3

R2

AcO

R1

1a-g

10 5

H

4´

20 23

11

H 14 H7

8

13 16

2´

N

7´

19 1

H

3

HO

R1

O

R3

COOCH 3

R2

6´

21

5´

O

R3

COOH

20

11

19

6´

21

4´

20 23

11

H 14

5´

2´

13 16

N

COOCH 3 7´

6´ 5´

10 5

H

6a-g

H

8 7

O

H R2

23

2´

4´

N

COOCH 3 7´

R1 12

8a-g

Inhibition percentages (%) of series 1, 6 and 8 and compound 12 at different concentrations (in lg/mL) N°

R1

R2

R3

250

125

62.5

31.2

15.6

7.8

3.9

LogP

1a 6a 8a 1b 6b 8b 1c 6c 8c 1d 6d 8d 1e 6e 8e 1f 6f 8f 1g 6g 8g 12 Amph

H2 H2 H2 H2 H2 H2 H2 H2 H2 OAc OAc OH H2 H2 H2 H2 H2 H2 OAc OAc OH -

a-OAc a-OAc a-OH

H2 H2 H2 H2 H2 H2 OAc OAc OH H2 H2 H2 OAc OAc OH H2 H2 H2 H2 H2 H2 –

21.1 ± 1.1 46.2 ± 0.2 38.3 ± 1.8 29.7 ± 3.2 19.9 ± 0.5 29.71 ± 1.47 40.1 ± 2.1 35.8 ± 4.5 22.62 ± 1.83 16.1 ± 1.8 29.6 ± 0.6 25.4 ± 2.2 28.3 ± 1.3 63.8 ± 1.2 4.3 ± 2.4 16.3 ± 0.1 33.1 ± 0.7 22.5 ± 3.2 11.7 ± 1.9 61.4 ± 0.8 14.0 ± 0.1 2.1 ± 0.0 100

11.1 ± 1.0 24.2 ± 0.7 16.3 ± 2.2 14.8 ± 0.7 7.6 ± 0.2 15.58 ± 0.15 19.7 ± 1.1 18.2 ± 2.4 12.94 ± 0.55 11.2 ± 2.1 14.5 ± 1.1 14.8 ± 0.6 0.2 ± 0.0 10.0 ± 2.7 3.2 ± 0.7 6.4 ± 0.1 15.0 ± 0.7 6.2 ± 3.1 1.5 ± 0.1 39.4 ± 3.0 4.7 ± 1.1 1.7 ± 0.1 100

4.5 ± 0.6 13.8 ± 0.4 9.8 ± 0.1 8.7 ± 0.0 2.2 ± 0.5 8.06 ± 0.21 9.9 ± 2.3 10.3 ± 2.7 5.93 ± 0.26 5.6 ± 0.2 6.1 ± 1.0 8.4 ± 0.4 0.1 ± 0.0 9.2 ± 0.6 0 2.9 ± 0.3 13.1 ± 1.3 0 0 13.2 ± 0.3 0 0.9 ± 0.1 100

2.5 ± 0.3 8.0 ± 0.0 4.4 ± 0.2 6.2 ± 1.0 3.1 ± 0.4 2.94 ± 0.47 5.5 ± 1.7 7.6 ± 1.1 4.66 ± 0.01 4.9 ± 0.1 3.6 ± 1.0 3.0 ± 1.0 0.1 ± 0.0 5.1 ± 1.0 0 0.7 ± 0.1 9.2 ± 2.1 0 0 2.3 ± 0.5 0 0.9 ± 0.3 100

1.0 ± 0.2 4.2 ± 0.1 3.3 ± 0.8 4.4 ± 0.4 0.3 ± 0.0 2.93 ± 0.60 3.0 ± 0.1 2.3 ± 0.3 2.42 ± 0.41 3.3 ± 0.1 2.4 ± 0.9 0.5 ± 0.7 0.1 ± 0.0 4.3 ± 0.2 0 0 2.7 ± 0.0 0 0 0.6 ± 0.4 0 0.6 ± 0.2 100

0.6 ± 0.1 3.4 ± 0.0 1.8 ± 0.6 3.8 ± 0.2 0 1.97 ± 1.09 3.0 ± 0.0 4.4 ± 0.5 1.24 ± 0.08 2.6 ± 0.2 1.3 ± 0.3 0.4 ± 0.2 0 3.3 ± 0.2 0 0 1.8 ± 0.0 0 0 0.4 ± 0.1 0 0.4 ± 0.1 100

0.5 ± 0.1 3.1 ± 0.6 1.2 ± 0.7 2.2 ± 0.6 0 0 2.2 ± 0.8 2.6 ± 0.9 1.23 ± 0.35 1.2 ± 0.1 0.8 ± 0.2 1.0 ± 0.1 0 2.3 ± 0.1 0 0 0.9 ± 0.1 0 0 0.2 ± 0.1 0 0 100

4.59 5.42 4.96 5.52 6.35 6.12 4.66 5.49 5.03 4.59 5.42 4.96 3.73 4.56 3.87 4.59 5.42 4.96 3.94 4.77 4.08 0.6

H2 H2 H2 H2 H2 H2 H2 H2 H2 a-OAc a-OAc a-OH b-OAc b-OAc b-OH a-OAc a-OAc a-OH –

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1a 6a 12

1d 6d 12

60

Inhibition (%)

Inhibition (%)

60

40

20

40

20

0

0 0

50

100

150

200

250

300

0

50

100

Concentration (μg/mL)

150

200

250

300

250

300

Concentration (μg/mL)

70 1e 6e 12

60

1f 6f 12

60

Inhibition (%)

Inhibition (%)

50

40

30

40

20

20

10

0

0 0

50

100

150

200

250

0

300

50

100

150

200

Concentration (μg/mL)

Concentration (μg/mL) 70 1g 6g 12

60

Inhibition (%)

50

40

30

20

10

0 0

50

100

150

200

250

300

Concentration (μg/mL) Fig. 3. Comparative doses–response curves of the antifungal activity of the hybrid compounds 6a, 6d–g and of the moieties 12 and bile acids 1a, 1d–g.

the comparative inhibition curves of 1a, 1d–g as well as of 6a, 6d–g and 12, at all concentrations tested, and clearly shows that 6a and 6d–g display significantly higher activities than their constitutive moieties (1a, 1d–g and 12) on their own. It is also interesting to note that 6e, 6g and also 6a, which are the three most active compounds all share a 7a-acetate which seems to be important for the biological activity [17]. At the same time, com-

pounds 6b and 6c, which lack an -OAc on C-6 or C-7, showed a slight activity decrease. The deacetylated compounds 8a, 8d–g (Table 3) all showed lower activities than 6a, 6d–g. This trend of increased activity of acetylated (6a, 6d–g) vs. non acetylated compounds (8a, 8d–g), which can be clearly seen in Fig. 4, is obviously related to an increased lipophilicity, since acetylation alters the facial

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L.R. Fernández et al. / Steroids xxx (2016) xxx–xxx

6a 8a

6d 8d

60

Inhibition (%)

Inhibition (%)

60

40

40

20

20

0

0 0

50

100

150

200

250

300

0

50

100

150

200

250

300

250

300

Concentration (μg/mL)

Concentration (μg/mL) 70 6e 8e

60

6f 8f

60

Inhibition (%)

Inhibition (%)

50

40

30

40

20

20

10 0

0 0

50

100

150

200

250

0

300

50

100

150

200

Concentration (μg/mL)

Concentration (μg/mL) 70 6g 8g

60

Inhibition (%)

50

40

30

20

10

0 0

50

100

150

200

250

300

Concentration (μ g/mL) Fig. 4. Comparative doses–response curves of the antifungal activity of the hybrid compounds 6a, 6d–g and their deacetylated derivatives 8a, 8d–g.

amphiphilic conformation of these compounds, especially of the concave hydrophilic a-face [17]. This is also confirmed by comparison of the calculated LogP values of compounds of the 1, 6 and 8 series. It is evident that compounds of the 6 series have higher LogP, which correlates with an increase in antifungal activity [18].

4. Discussion In the present work, fifteen new oxazoles were synthesized, fourteen of which are bile acid derivatives. Overall yields were variable, and the formation of the amides was the critical step

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L.R. Fernández et al. / Steroids xxx (2016) xxx–xxx

due to the difficulties in handling the unstable acid chlorides. The acetylated bile acids used as starting materials as well as the synthetic compounds were tested for their antifungal activity against C. albicans. The results showed an increase of the activity for the acetylated oxazolic derivatives, which was generally lost upon deacetylation. On the other hand, the simple oxazole 12 was found to be almost inactive. The activity of a structurally related oxazoline derivate from chenodeoxycholic acid was previously evaluated against Saccharomyces cerevisiae resulting in a MIC of 178 lg/mL [19], which is in the same order of magnitude of our results. Shamsuzzaman et al. [9] reported that steroidal 6,5 fused oxazoles were active against C. albicans by the disk diffusion method. As, in both cases, the parent steroids were not evaluated, it is not possible to assess if the activity of the synthetic derivatives was improved. Our results showed that the modification of the antifungal capacity could be ascribed to the introduction of the oxazole moiety into the bile acid molecule. Although the synthesized compounds were not very active, the present results support the hypothesis that a covalent bond between a steroidal core and an oxazole fragment can lead to a compound with more potent biological activity than the separated structures. Further work must be done to improve the antifungal capacity of these compounds, but compounds 6e and 6g are good starting points for additional development. In this respect, the oxazole fragment incorporated to the side chain can be further functionalized in the search for a more active derivative. Acknowledgements This research was supported by grants from CONICET (PIP 2010-2012 N° 516), UBA (Program 2014-2017 N° 704) and ANPCyT (PICT-2010 N° 1808 and N° 0608). We thank Dra. Gabriela Cabrera, Ing. José Gallardo and Lic. Gernot Eskuche (UMYMFOR-CONICET) for recording mass and NMR spectra. L.R.F. thanks CONICET for a doctoral fellowship. J.A.P. and M.S. are researchers of CONICET. Associate Professor S.Z., doctoral fellow E.B. and researcher assistant of CONICET L.S. thank to CONICET, National University of Rosario and ANPCyT (PICT 2014-1170). Appendix A. Supplementary data

9

References [1] A. Enhsen, W. Kramer, G. Wess, Bile acids in drug discovery, Drug Discovery Today 3 (1998) 409–418. [2] O. Bortolini, A. Medici, S. Poli, Biotransformations on steroid nucleus of bile acids, Steroids 62 (1997) 564–577. [3] E. Virtanen, E. Kolehmainen, Use of bile acids in pharmacological and supramolecular applications, Eur. J. Med. Chem. 2004 (2004) 3385–3399. [4] H. Svobodá, V. Noponen, E. Kolehmainen, E. Virtanen, Recent advances in steroidal supramolecular gels, RSC Adv. 2 (2012) 4985–5007. [5] J. Zhong, Muscarine, imidazole, oxazole, and thiazole alkaloids, Nat. Prod. Rep. 28 (2011) 1143–1191. [6] S. Raghavan, P. Manogaran, K. Kumari, K.K. Gadepalli Narasimha, B. Kalpattu Kuppusami, P. Mariyappan, et al., Synthesis and anticancer activity of novel curcumin–quinolone hybrids, Bioorg. Med. Chem. Lett. 25 (2015) 3601–3605. [7] J.C. Coa, W. Castrill, W. Cardona, M. Carda, V. Ospina, J.A. Muñoz, et al., Synthesis, leishmanicidal, trypanocidal and cytotoxic activity of quinoline– hydrazone hybrids, Eur. J. Med. Chem. 101 (2015) 746–753. [8] S.V. Drach, R.P. Litvinovsaya, V.A. Khripach, Steroidal 1,2-oxazoles. Synthesis and biological activity, Chem. Heterocycl. Compd. 36 (2000) 233–255. [9] Shamsuzzaman, M.S. Khan, M. Alam, Z. Tabassum, A. Ahmad, A.U. Khan, Synthesis, antibacterial and antifungal activities of 6,5 fused steroidal oxazoles in cholestane series, Eur. J. Med. Chem. 45 (2010) 1094–1097. [10] G.E. Siless, M.E. Knott, M.G. Derita, S.A. Zacchino, L. Puricelli, J.A. Palermo, Synthesis of steroidal quinones and hydroquinones from bile acids by Barton radical decarboxylation and benzoquinone addition. Studies on their cytotoxic and antifungal activities, Steroids 77 (2012) 45–51. [11] A. Leverrier, J. Bero, M. Frédérich, J. Quetin-Leclercq, J.A. Palermo, Antiparasitic hybrids of Cinchona alkaloids and bile acids, Eur. J. Med. Chem. 66 (2013) 355– 363. [12] A. Leverrier, J. Bero, J. Cabrera, M. Frédérich, J. Quetin-Leclercq, J.A. Palermo, Structure–activity relationship of hybrids of Cinchona alkaloids and bile acids with in vitro antiplasmodial and antitrypanosomal activities, Eur. J. Med. Chem. 100 (2015) 10–17. [13] A.G. Godfrey, D.A. Brooks, L.A. Hay, M. Peters, J.R. McCarthy, D. Mitchell, Application of the Dakin–West reaction for the synthesis of oxazolecontaining dual PPARra/c agonists, J. Org. Chem. 68 (2003) 2623–2632. [14] C.R. Reddy, B. Latha, Synthesis of ()-dihydropinidine, (2S,6R)-isosolenopsin and (+)-monomorine via a chiral synthon from L-aspartic acid, Tetrahedron: Asymmetry 22 (2011) 1849–1854. [15] J. Luijten, E.J. Vorenkamp, A.J. Schouten, Reversible helix sense inversion in surface-grafted poly(b-phenethyl-L-aspartate) films, Langmuir 23 (2007) 10772–10778. [16] Clinical and Laboratory Standards Institute, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard (CLSI document M27-A3), 3rd ed., Clinical and Laboratory Standards Institute; NCCLS, Wayne, PA, 2008. 28, pp. 1–25. [17] D.B. Salunke, B.G. Hazra, V.S. Pore, M.K. Bhat, P.B. Nahar, M.V. Deshpande, New steroidal dimers with antifungal and antiproliferative activity, J. Med. Chem. 47 (2004) 1591–1594. [18] P.C. Leal, A. Mascarello, M. Derita, F. Zuljan, R.J. Nunes, S. Zacchino, et al., Relation between lipophilicity of alkyl gallates and antifungal activity against yeasts and filamentous fungi, Bioorg. Med. Chem. Lett. 19 (2009) 1793–1796. [19] P.B. Hylemon, R.J. Fricke, E.H. Mosbach, Antibacterial properties of bile acid oxazoline compounds, Steroids 40 (1982) 347–357.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2016.01. 014.

Please cite this article in press as: L.R. Fernández et al., Synthesis and antifungal activity of bile acid-derived oxazoles, Steroids (2016), http://dx.doi.org/ 10.1016/j.steroids.2016.01.014