JBCS

INTRODUCTION

Fungal infections represent a serious health problem. Since the late 1960s, when antibiotic therapy was already developed, accounting for a reduction in deaths by bacterial infections, a drastic rise in fungal diseases was noticed, which today accounts for a large number of deaths worldwide.^1,2 The increasing incidence of opportunistic fungal infections has some factors such as the growing number of immunodeficient patients (patients on anti-tumor therapy, transplanted, AIDS carriers or diabetics) or people experiencing some kind of stress.³ There is a need for new antifungal therapeutic agents, since treatment failure is very common in immunocompromised patients, with high incidence of mortality.

The papulacandins are a group of naturally occurring glycolipids, isolated from the fermentation broths of Papularia sphaerosperma.⁴ The papulacandins A, B and C are spiroglycosides derived from the disaccharide lactose, acylated at positions 3 and 6' with residues of unsaturated fatty acids containing some chiral centers. The papulacandin D is a monosaccharide spiroglucoside derived from glucose acylated at position 3 with the same fatty acid residue present in this position of papulacandins A, B and C (Figure 1).

 

 

The papulacandins show potent fungicidal activity against different strains of Candida sp. (MIC range of 0.1-12.5 µg mL^-1).⁴ The success of these substances in the treatment of fungal infections caused by Pneumocystis carinii, a species that reaches immunocompromised patients with high mortality, has been reported.⁵ However, these compounds are not active against other pathogenic fungi like Aspergillus sp., causing lung infections of difficult treatment.⁴ Another limitation of papulacandins is their lower activity in vivo than in vitro, due to strong binding to plasma proteins.⁶

Literature reports the enzyme (1,3)-β-D-glucan synthase as the molecular target of papulacandins.^5,7 Since these compounds exert their action by inhibiting the (1,3)-β-D-glucans synthesis, major structural components of the fungal cell wall absent in mammalian cells, the papulacandins have become an important chemical class to be explored for the design and discovery of analogues or derivatives which may be useful against fungal infections in vitro and in vivo.

Recently, Kaaden and coworkers synthesized four papulacandin D analogues (Figure 2) modified in side chain, and evaluated their antifungal activity against Candida albicans. The compounds 1 and 4 were inactive while compounds 2 and 3 showed some activity. The papulacandin D derivative 2, containing a palmitoyl acyl residue as side chain, showed MIC of 88 µg mL^-1, representing the most active compound of the series.⁸

 

 

Considering these findings, we describe herein the synthesis of simplified papulacandin D analogues, bearing a palmitoyl residue as side chain, since it is present in the most active compound synthesized and evaluated by Kaaden and coworkers.⁸ The spiroketal ring of papulacandin D was replaced by aromatic glycosides derived from 4-hydroxy-3-methoxybenzaldehyde or 3-hydroxybenzaldehyde, which are very cheap and more accessible synthetically (Figure 3). Since SAR studies on papulacandin B showed that modifications on the aglycone had, in general, little influence on activity⁹ and that no study has been carried out on Papulacandin D, we though these modifications could shed some light about the influence of the aglycone in this particular series.

 

 

 

RESULTS AND DISCUSSION

Chemistry

The synthetic route (Scheme 1) involved the reaction of 4-hydroxy-3-methoxybenzaldehyde or 3-hydroxybenzaldehyde with peracetylated glucosyl bromide 5 in the presence of lithium hydroxide, according to published procedure.¹⁰ This is one of the most used methods for this purpose and was employed in the preparation of peracetylated glycosides 6 and 7 in 51% and 61% yields, respectively. Then, the peracetylated glucosides 6 and 7 were deacetylated using potassium hydroxide/methanol solution,¹¹ affording 8 and 9 in quantitative yields.

 

 

Interestingly, the compound 9 was obtained as a possible oligomer as observed at ¹H and ¹³C NMR spectra, which did not prevent the next planned derivative (11) was obtained accordingly. The next step consisted on the selective protection of C4 and C6 hydroxyl groups of the saccharide unit as benzylidene acetal. This protecting group was chosen due to its stability and easy removal in mild conditions.¹² Thus, the reaction of glucosides 8 or 9 with benzaldehyde and zinc chloride afforded the acetals 10 and 11 in 72% and 60% yields, respectively. When 10 was reacted with 1.2 equivalent of palmitic acid and N,N'-dicyclohexylcarbodiimide in dichloromethane, in the presence of catalytic amount of 4-dimethylaminopyridine, a mixture of three compounds was obtained. This result is not unexpected, in view of the difficulties for the regioselectivity acylation of β-glucosides.¹³ Besides, for structure-activity relationship studies it is interesting to evaluate the antifungal activity of each component of the mixture. Thus, the mixture was submitted to flash column chromatography (hexane/ethyl acetate, 9:1) which allowed for the isolation of 3-O-palmitoyl derivative 12 (31% yield), 2-O-palmitoyl derivative 13 (27% yield) and 2,3-di-O-palmitoyl derivative 14 (13% yield). Each isolated compound was characterized by 1D and 2D ¹H and ¹³C NMR experiments. The most important information from these experiments is the downfield shift of H-2 and/or H-3 of the saccharide moiety upon esterification, as it is well stablished in the literature.¹⁴ Finally, the removal of the acetal protecting group was carried out in the presence of HCl in acetone, affording the papulacandin D analogues 18-20 in 92%, 88% and 83% yields, respectively.¹⁵

Under the same conditions as described for 10, acylation of 11 also afforded a three-component mixture which was purified by flash chromatography to furnish 3-O-palmitoyl derivative 15 (24% yield), 2-O-palmitoyl derivative 16 (40% yield) and 2,3-di-O-palmitoyl derivative 17 (18% yield). Removal of 4,6-O-benzylidene protecting group afforded the corresponding deprotected neoglycolipids 21-23 in 86%, 89% and 72% yields, respectively.

Besides the 1D and 2D NMR experiments involving the protected acylated glycosides, as described above, the unprotected neoglycolipids 18-23 were also submitted to HMBC experiments. The results for compounds 18-20 are shown below (Figure 4). Thus, through correlation observed between H-3, H-2 or H-3 and H-2 protons (carbohydrate) with carbonyl carbon of acetyl group (palmitoyl side chain) we could unequivocally identify each isomer (acylated at positions 2 or 3) and the 2,3-di-acylated analogue.

 

 

In vitro assays

Compounds 6-23 were evaluated against Candida albicans, C. krusei, C. parapsilosis and C. glabrata according to published protocol.¹⁶ Most compounds were inactive and three displayed low antifungal activity, two against Candida albicans (compounds 7 and 23) and one against Candida tropicalis (compound 17) (Table 1).

 

 

Compounds 18 and 21, close analogues of the papulacandin D were inactive, while papulacandin D derivative 2 showed MIC of 88 µg mL^-1, as related by Kaaden and coworkers, indicating that classical aryl glycosides can't replace the spiro glucoside.⁸ Nevertheless, it remains to verify if a classical glucoside with a 2,4-dihydroxy-substituted aglycone which resembles the natural product more closely, could be active. The palmitoyl side chain was not essential to the active compound 7, devoid of such a chain, and this may suggest this compound may have had its antifungal activity by another mechanism of action, as this chain is essential for activity of the papulacandins. All the active compounds bear a 3-formylphenyl aglycone while no compound bearing the 4-formyl-2-methoxyphenyl aglycone displayed activity, indicating that a 3-formyl group, which is more reactive than that at position 4, possibly have a contribution on activity. A hydrophilic/lipophilic balance also seems to be important, since not all compounds bearing the 3-formylphenyl aglycone were active.

 

CONCLUSION

Six new neoglycolipids related to papulacandin D were synthesized along with isomeric 2-acylated and 2,3-diacylated compounds. These compounds as well as synthetic intermediates were evaluated against four Candida species. The closely related papaulacandin D analogues 18 and 21 were inactive, but two 2,3-diacylated (compounds 17 and 23) and one peracetylated (compound 7) showed low activity against Candida albicans (7 and 23) or Candida tropicalis (17), with a MIC of 200 µg mL^-1. All the active compounds are derived from 3-hydroxybenzaldehyde and represent a starting point for the design of new antifungal candidates.

 

SUPPLEMENTARY MATERIAL

¹H and ¹³C NMR spectra of all synthesized compounds are found in the supplementary material and have free access at http://quimicanova.sbq.org.br.

 

ACKNOWLEDGEMENTS

The authors thank CNPq, CAPES and FAPEMIG for fellowships and financial support.

 

EXPERIMENTAL

Physical measurements

Melting points were determined on Microquímica MOAs 301 apparatus and were uncorrected. IR spectroscopy was performed on Spectrum One Infrared Spectrometer, Perkin-Elmer. ¹H-NMR and ¹³C-NMR spectra were obtained on Bruker Avance DRX-200 (200 MHz FT NMR) and DRX-400 (400 MHz FT NMR) in deuterated chloroform or dimethylsulfoxide. The specific optical rotation [α]_D were measured on Perkin Elmer 341 polarimeter, at 20 ºC. Reaction courses and product mixtures were monitored by thin-layer chromatography (TLC) on silica gel-G TLC plates (Merck). For chromatograph, column grade silica gel (0.040-0.063 mm mesh size) was employed.

Synthesis of the compounds

Synthesis of peracetylated glucosides 6 and 7: general procedure

A solution of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (5, 6.15 mmol) in acetone (20 mL) was added to a solution of 4-hydroxy-3-methoxybenzaldehyde or 3-hydroxybenzaldehyde (18 mmol) em 1.0 mol L^-1 lithium hydroxide (10 mL) and the solution stirred for 2 hours at room temperature and monitored by TLC. After removal of acetone, the resulting suspension was extracted with dichloromethane (3 x 50 mL). The crude product was washed with 10% sodium hydroxide (3 x 30 mL), water and dried on anhydrous sodium sulphate. After filtration and removal of the solvent under reduced pressure, the crude product was recrystallized from isopropyl alcohol, affording compounds 6 and 7.

4-formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (6)

This compound was obtained as white crystals; yield: 51%; m.p. 139.1-139.9 ºC; [α]_D -41.0º (c 0.63, CHCl₃); IR (υ/cm^-1): 2944 (sp³ C-H), 1752 (ester C=O), 1694 (aldehyde C=O), 1591, 1510, 1475 (Ar. C=C). ¹H-NMR (200 MHz, CDCl₃) δ: 9.8 (1H, s, CHO), 7.4 (1H, dd, J₄= 1.7 Hz e J₃ = 8.6 Hz, H-9), 7.2 (1H, s, H-11), 7.2 (1H, d, J₃ = 8,6 Hz, H-12), 5.3-5.1 (4H, m, H-1, H-2, H-3 and H-4), 4.2 (1H, dd, J₃ = 5.0 Hz e J₂ = 12.2 Hz, H-6), 4,1 (1H, dd, J₃ = 2.5 Hz e J₂ = 12.2 Hz, H-6'), 3.8 (3H, s, OCH₃), 3.9-3.8 (1H, m, H-5), 2.0-2.0 (12H, s, OCOCH₃). ¹³C-NMR (50 MHz, CDCl₃) δ: 191.4 (1C, CHO), 170.6-169.3 (4C, ester C=O), 151.2 (1C, C-7), 151,1 (1C, C-8), 132.9 (1C, C-10), 125.4 (1C, C-11), 118.2 (1C, C-12), 110.9 (1C, C-9), 99.8 (1C, C-1), 72.5 (1C, C-5), 72.3 (1C, C-3), 71.1 (1C, C-2), 68.7 (1C, C-4), 62.0 (1C, C-6), 56.2 (1C, OCH₃), 20.7 (4C, OCOCH₃).

3-formylphenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (7)

This compound was obtained as white crystals; yield: 61%; m.p. 103.6-105.9 ºC; [α]_D -37.5º (c 0.58, CHCl₃); IR (υ/cm^-1): 3065 (Ar. C-H), 2975, 2941 (sp³ C-H), 1747 (ester C=O), 1697 (aldehyde C=O), 1592, 1483 (Ar. C=C). ¹H-NMR (200 MHz, CDCl₃) δ: 9.9 (1H, s, CHO), 7.5-7.2 (4H, m, H-8, H-10, H-11 and H-12), 5.3-5.1 (4H, m, H-1, H-2, H-3 and H-4), 4.2-4.1 (2H, m, H-5 and H-6), 3.9 (1H, m, H-6'), 2.1-2.0 (12H, s, OCOCH₃). ¹³C-NMR (50 MHz, CDCl₃) δ: 191.1 (1C, CHO), 170.3-168.9 (4C, ester C=O), 157.0 (1C, C-7), 137,6 (1C, C-9), 130.5 (1C, C-11), 125.6 (1C, C-10), 123.4 (1C, C-12), 114.9 (1C, C-8), 98.3 (1C, C-1), 72.3-67.9 (4C, C-2, C-3, C-4 and C-5), 61.3 (1C, C-6), 20.36-20.32 (4C, OCOCH₃).

Synthesis of deacetylated glucosides 8 and 9: general procedure

The peracetylated glucosides (1 mmol) were solubilized in a solution of KOH in MeOH (20 mL, 1.0 mol L^-1) and stirred at room temperature for 30 min. After the completion of the reaction, as observed by TLC, the mixture was neutralized with IRA-120 resin. The resin was filtered off and washed with methanol. The collected filtrate was concentrated in vacuum to afford glucosylated derivatives 8 and 9.

4-formyl-2-methoxyphenyl β-D-glucopyranoside (8)

This compound was obtained as white crystals; yield: 99%; m.p. 183.0-184.6 ºC; [α]_D -52.4º (c 0.61, DMSO); IR (υ/cm^-1): 3365 (OH), 2891 (sp³ C-H), 1688 (aldehyde C=O), 1589, 1508, 1465 (Ar. C=C). ¹H-NMR (200 MHz, DMSO-d₆) δ: 9.8 (1H, s, CHO), 7.5 (1H, d, J₃ = 8.2 Hz, H-9), 7.4 (1H, s, H-11), 7.2 (1H, d, J₃ = 8.2 Hz, H-8), 5.3 (1H, s, OH), 5.1-5.0 (2H, sl, OH), 5.1 (1H, d, J₃ = 7.4 Hz, H-1), 4.5 (1H, s, OH), 3.8 (3H, s, OCH₃), 3.6 (1H, m, H-6), 3.4-3.2 (5H, m, H-2, H-3, H-4, H-5 and H-6'). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 191.6 (1C, CHO), 151.7 (1C, C-7), 149,3 (1C, C-8), 130.5 (1C, C-10), 125.4 (1C, C-11), 114.5 (1C, C-12), 110.5 (1C, C-9), 99.4 (1C, C-1), 77.1 (1C, C-5), 76.8 (1C, C-3), 73.1 (1C, C-2), 69.5 (1C, C-4), 60.6 (1C, C-6), 55.6 (1C, OCH₃).

3-formylphenyl β-D-glucopyranoside (oligomer) (9)

This compound was obtained as white crystals; yield: 100%; m.p. 103.6-105.9 ºC; [α]_D -37.5º (c 0.19 DMSO). ¹H-NMR (200 MHz, DMSO-d₆) δ: 10.0-9.8 (1H, s, CHO), 7.59-7.54 (3H, m, Ar-H), 7.53-7.29 (2H, m, Ar-H), 7.14-7.06 (4H, m, Ar-H), 5.06-4.22 (12H, m, OH, sugar), 3.71-3.18 (10H, m, OH, sugar). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 192.8 (1C, CHO), 157.8-157.2 (3C), 156.9 (1C), 139.2 (1C), 137.6 (1C), 137.4 (1C), 130.4 (1C), 130.3 (1C), 129.2 (1C), 123.4 (1C), 123.1 (1C), 122.6 (1C), 122.5 (1C), 116.6 (1C), 116.1 (1C), 116.3 (1C), 114.9 (1C), 100.8-100.2 (4C), 94.1 (1C), 80.3-80.2 (2C), 77.0-76.4 (4C), 74.1-60.5 (1C).

Synthesis of benzylidene acetals 10 and 11: general procedure

To a solution of 7.95 mmol of ZnCl₂ in 16 mL (0.137 mol) of benzaldehyde was added 3.18 mmol of 8 or 9, and stirred for 1 hour at room temperature, when noticed the completion of the reaction by TLC. The mixture was poured into a beaker containing ice water and ethyl ether, and upon stirring a solid formed, which was collected by filtration and washed alternately with ice water and hexanes several times.

4-formyl-2-methoxyphenyl 4,6-O-benzylidene-β-D-glucopyranoside (10)

This compound was obtained as white crystals; yield: 72%; m.p. 191.4-193.0 ºC; [α]_D -62.5º (c 0.60, DMSO); IR (υ/cm^-1): 3364 (OH), 2880 (sp³ C-H), 1683 (aldehyde C=O), 1590, 1506, 1465 (Ar. C=C). ¹H-NMR (200 MHz, DMSO-d₆) δ: 9.9 (1H, s, CHO), 7.5-7.3 (8H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14 and H-16), 5.7 (1H, d, J₃ = 5.4 Hz, OH), 5.6 (1H, s, H-7), 5.5 (1H, d, J₃ = 4.8 Hz, OH), 5.2 (1H, d, J₃ = 7.4 Hz, H-1), 4.2 (1H, m, H-6 eq.), 3.7-3.5 (5H, m, H-2, H-3, H-4, H-5 and H-6 ax.), 3.3 (3H, s, OCH₃). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 192.9 (1C, CHO), 157.6 (1C, C-12), 137.6 (1C, C-13), 130.5 (1C, C-8), 128.9 (1C, C-15), 128.0-116.4 (5C, C-9, C-9', C-10, C-10' and C-11), 100.7 (1C, C-1), 100.5 (1C, C-7), 80.3-67.8 (4C, C-2, C-3, C-4 and C-5), 65.9 (1C, C-6).

3-formylphenyl-4,6-O-benzylidene-β-D-glucopyranoside (11)

This compound was obtained as white crystals; yield: 60%; m.p. 158.5-160.3 ºC; [α]_D -10.3º (c 0.19, DMSO); IR (υ/cm^-1): 3379 (OH), 2884 (sp³ C-H), 1686 (aldehyde C=O), 1585, 1487, 1465 (Ar. C=C). ¹H-NMR (200 MHz, DMSO-d₆) δ: 9.9 (1H, s, CHO), 7.5-7.1 (9H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14, H-15 and H-17), 5.7 (1H, d, J₃ = 4.4 Hz, OH), 5.6 (1H, s, H-7), 5.5 (1H, d, J₃ = 3,4 Hz, OH), 5.2 (1H, d, J₃ = 7.2 Hz, H-1), 4.2 (1H, m, H-6 eq.), 3.7-3.3 (5H, m, H-2, H-3, H-4, H-5 and H-6 ax.). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 192.9 (1C, CHO), 157.6 (1C, C-12), 137.6 (1C, C-14), 130.5-126.3 (6C, C-9, C-9', C-10, C-10', C-11 and C-16), 123.5 (1C, C-15), 122.6 (1C, C-17), 116.4 (1C, C-13), 100.7 (1C, C-1), 100.5 (1C, C-7), 80.3-67.8 (4C, C-2, C-3, C-4 and C-5), 65.9 (1C, C-6).

Synthesis of acylated benzylidene acetals 12-17: general procedure

To a solution of 0.745 mmol of palmitic acid, 0.745 mmol of N,N-dicyclohexylcarbodiimide (DCC) in 40 mL of dichloromethane was added, at -5 ºC, 0.621 mmol of 10 or 11 and 15 mg of dimethylaminopyridine (DMAP). The mixture was stirred at -5 ºC for six hours when the solvent was concentrated and the solid (dicyclohexylurea) was collected by filtration. The filtrate was concentrated to dryness affording a mixture of three products, as observed by TLC. The mixture was submitted to column chromatography (hexane/ethyl acetate 9:1) which afforded the pure products 12 (31%), 13 (27%), 14 (13%), 15 (24%), 16 (40%) and 17 (18%).

4-formyl-2-methoxyphenyl 3-O-palmitoyl-4,6-O-benzylidene-β-D-glucopyranoside (12)

This compound was obtained as white crystals; yield: 31%; m.p. 149.3-1151.3ºC; [α]_D -33.7º (c 0.29 CH₂Cl₂); IR (υ/cm^-1): 3323 (OH), 3036 (ar. C-H), 2926, 2850 (sp³ C-H), 1731 (ester C=O), 1691 (aldehyde C=O), 1571, 1535, 1507, 1465 (Ar. C=C). ¹H-NMR (400 MHz, CDCl₃) δ: 9.9 (1H, s, CHO), 7.4-7.2 (8H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14 and H-16), 5.5 (1H, s, H-7), 5.3 (1H, t, J₃ = 9.6 Hz, H-3), 5.0 (1H, d, J₃ = 8.0 Hz, H-1), 4.3 (1H, dd, J_eq = 4.0 Hz e J_ax = 12.0 Hz, H-6 eq.), 3.9-3.8 (4H, m, OCH₃ and H-6 eq.), 3.8 (1H, t, J₃ = 10 2 Hz, H-2), 3.7 (1H, t, J₃ = 9.4 Hz, H-4), 3.6 (1H, m, H-5), 2.4 (2H, t, J₃ = 7.6 Hz, methylene), 1.3-1.2 (28H, m, methylene), 0.8 (3H, t, J₃ = 6.8 Hz, methyl). ¹³C-NMR (100 MHz, CDCl₃) δ: 190.8 (1C, CHO), 173.6 (1C, ester C=O), 151.0 (1C, C-12), 150.8 (1C, C-13), 136,7 (1C, C-8), 132.9 (1C, C-15), 128.2 (2C, C-10 and C-10'), 126.0 (2C, C-9 and C-9'), 129.8-118.4 (4C, C-11, C-14, C-16 and C-17), 102.9 (1C, C-1), 101.5 (1C, C-7), 78.1 (1C, C-4), 73.1 (1C, C-2), 72.9 (1C, C-3), 68.4 (1C, C-6), 66.9 (1C, C-5), 56.1 (1C, OCH₃), 34.3-22.6 (14C, methylene), 14.0 (1C, methyl).

4-formyl-2-methoxyphenyl 2-O-palmitoyl-4,6-O-benzylidene-β-D-glucopyranoside (13)

This compound was obtained as white crystals; yield: 27%; m.p. 83.6-85.2 ºC; [α]_D +12.0º (c 0.16, CH₂Cl₂); IR (υ/cm^-1): 3326 (OH), 2918, 2850 (sp³ C-H), 1741 (ester C=O), 1685 (aldehyde C=O), 1590, 1508, 1466 (Ar. C=C). ¹H-NMR (400 MHz, CDCl₃) δ: 9.9 (1H, s, CHO), 7.5-7.1 (8H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14 and H-16), 5.5 (1H, s, H-7), 5.2 (1H, t, J₃ = 8.0 Hz, H-2), 5.2 (1H, d, J₃ = 7.6 Hz, H-1), 4.4 (1H, dd, J_eq = 4.8 Hz e J_ax = 10.4 Hz , H-6 eq.), 4.0 (1H, t, J₃ = 8.8 Hz, H-3), 3.9 (3H, s, OCH₃), 3.8 (1H, t, J₃ = 10.4 Hz, H-6 ax.), 3.7 (1H, t, J₃ = 9.4 Hz, H-4), 3.6 (1H, m, H-5), 2.4-2.3 (2H, m, methylene), 1.6 (2H, t, J₃ = 7.6 Hz, methylene) 1.3-1.2 (24H, m, methylene), 0.8 (3H, t, J₃ = 6.8 Hz, H-methyl). ¹³C-NMR (100 MHz, CDCl₃) δ: 190.8 (1C, CHO), 172.9 (1C, ester C=O), 151.1 (1C, C-12), 150.7 (1C, C-13), 136,7 (1C, C-8), 132.5 (1C, C-15), 128.3 (2C, C-10 and C-10'), 126.5 (2C, C-9 and C-9'), 129.3-117.1 (4C, C-11, C-14, C-16 and C-17), 102.2 (1C, C-7), 99.7 (1C, C-1), 80.7 (1C, C-4), 73.3 (1C, C-2), 72.2 (1C, C-3), 68.5 (1C, C-6), 66.5 (1C, C-5), 56.0 (1C, OCH₃), 34.2-22.6 (14C, methylene), 14.1 (1C, methyl).

4-formyl-2-methoxyphenyl 2,3-di-O-palmitoyl-4,6-O-benzylidene-β-D-glucopyranoside (14)

This compound was obtained as white crystals; yield: 13%; m.p. 81.5-83.1 ºC; [α]_D -81.6º (c 0.19, CH₂Cl₂); IR (υ/cm^-1): 2953, 2916, 2849 (sp³ C-H), 1746 (ester C=O), 1696 (aldehyde C=O), 1593, 1507, 1467 (ar. C=C). ¹H-NMR (400 MHz, CDCl₃) δ: 9.8 (1H, s, CHO), 7.4-7.1 (7H, m, H-9, H-9', H-10, H-10', H-11, H-14 and H-16), 7.1 (1H, d, J₃ = 8.8 Hz, H-13), 5.5 (1H, s, H-7), 5.4 (1H, t, J₃ = 9.2 Hz, H-3), 5.3 (1H, t, J₃ = 9.2 Hz, H-2), 5.2 (1H, d, J₃ = 7.6 Hz, H-1), 4.3 (1H, dd, J_eq = 4.8 Hz e J_ax = 10.4 Hz, H-6 eq.), 3.8 (3H, s, OCH₃), 3.8-3.7 (3H, m, H-4, H-5 and H-6 ax.), 2.3-2.2 (4H, m, methylene), 1.6-1.2 (52H, m, methylene), 0.8 (6H, t, J₃ = 6.8 Hz, methyl). ¹³C-NMR (100 MHz, CDCl₃) δ: 190.8 (1C, aldehyde C=O), 172.8 (1C, ester C=O), 172.1 (1C, ester C=O), 151.0 (1C, C-12), 150.8 (1C, C-13), 136,6 (1C, C-8), 132.7 (1C, C-15), 128.2 (2C, C-10 and C-10'), 126.0 (2C, C-9 and C-9'), 129.1-110.6 (4C, C-11, C-14, C-16 and C-17), 101.5 (1C, H-7), 110.1 (1C, C-1), 78.1 (1C, C-4), 71.6 (1C, C-2), 71.1 (1C, C-3), 68.4 (1C, C-6), 66.8 (1C, C-5), 56.0 (1C, OCH₃), 34.1-24.9 (28C, methylene), 14.5 (2C, methyl).

3-formylphenyl 3-O-palmitoyl-4,6-O-benzylidene-β-D-glucopyranoside (15)

This compound was obtained as white crystals; yield: 24%; m.p. 91.4-92.6 ºC; [α]_D +16.0º (c 0.10 CH₂Cl₂); IR (υ/cm^-1): 3554 (OH), 2921, 2852 (sp³ C-H), 1732 (ester C=O), 1701 (aldehyde C=O), 1589, 1488 (Ar. C=C). ¹H-NMR (400 MHz, CDCl₃) δ: 9.9 (1H, s, CHO), 7.5-7.2 (9H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14, H-15 and H-17), 5.5 (1H, s, H-7), 5.3 (1H, t, J₃ = 9.0 Hz, H-3), 5.1 (1H, d, J₃ = 7.6 Hz, H-1), 4.4-4.3 (1H, m, H-6 eq.), 3.9-3.6 (4H, m, H-2, H-4, H-5 and H-6 ax.), 3.4 (1H, s, OH), 2.4 (2H, t, J₃ = 7.4 Hz, methylene), 1.6 (2H, m, methylene), 1.2 (24H, m, methylene), 0.8 (3H, t, methyl). ¹³C-NMR (100 MHz, CDCl₃) δ: 191.8 (1C, CHO), 174.4 (1C, ester C=O), 157.5 (1C, C-12), 137.9 (1C, C-8), 136,9 (1C, C-14), 136.9 (1C, ar.), 128.4 (2C, C-10 and C-10'), 126.8 (2C, C-9 and C-9'), 130.5-116.6 (5C, C-11, C-13, C-15, C-16 and C-17), 101.6 (2C, C-7 and C-1), 78.2 (1C, C-4), 73.8 (1C, C-5), 73.4 (1C, C-3), 68.6 (1C, C-6), 66.7 (1C, C-2), 34.5-22.8 (14C, methylene), 14.2 (1C, methyl).

3-formylphenyl 2-O-palmitoyl-4,6-O-benzylidene-β-D-glucopyranoside (16)

This compound was obtained as white crystals; yield: 40%; m.p. 97.2-98.6 ºC; [α]_D +23.8º (c 0.23, CH₂Cl₂); IR (υ/cm^-1): 3439 (OH), 3071 (ar. C-H), 2917, 2850 (sp³ C-H), 1732 (ester C=O), 1700 (aldehyde C=O), 1597, 1584, 1483 (Ar. C=C). ¹H-NMR (400 MHz, CDCl₃) δ: 9.96 (1H, s, CHO), 7.5-7.2 (9H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14, H-15 and H-17), 5.5 (1H, s, H-7), 5.2-5.1 (2H, m, H-2 and H-1), 4.4 (1H, dd, J_eq = 4.2 Hz e J_3ax = 10.0 Hz, H-6 eq.), 3.9 (1H, t, J₃ = 8.4 Hz, H-3), 3.8-3.6 (3H, m, H-4, H-5 and H-6 ax.), 2.3 (2H, t, J₃ = 7.4 Hz, methylene), 1.6 (2H, q, J₃ = 7.0 Hz, methylene), 1.2 (24H, s, methylene), 0.8 (3H, t, methyl). ¹³C-NMR (100 MHz, CDCl₃) δ: 191.7 (1C, CHO), 173.2 (1C, ester C=O), 157.5 (1C, C-12), 138,0 (1C, C-8), 136.9 (1C, C-14), 128.5 (2C, C-10 and C-10'), 126.5 (2C, C-9 and C-9'), 130.5-115.7 (5C, C-11, C-13, C-15, C-16 and C-17), 102.1 (1C, C-7), 99.3 (1C, C-1), 80.7 (1C, C-4), 73.6 (1C, C-5), 72.3 (1C, C-2), 68.6 (1C, C-6), 66.6 (1C, C-3), 34.4-22.8 (14C, methylene), 14.3 (1C, methyl).

3-formylphenyl 2,3-di-O-palmitoyl-4,6-O-benzylidene-β-D-glucopyranoside (17)

This compound was obtained as white crystals; yield: 18%; m.p. 60.3-63.1 ºC; [α]_D +8.9º (c 0.22, CH₂Cl₂); IR (υ/cm^-1): 2910, 2850 (sp³ C-H), 1747 (ester C=O), 1698 (aldehyde C=O), 1596, 1536, 1484, 1467 (Ar. C=C). ¹H-NMR (400 MHz, CDCl₃) δ: 9.9 (1H, s, CHO), 7.6-7.2 (9H, m, H-9, H-9', H-10, H-10', H-11, H-13, H-14, H-15 and H-17), 5.5 (1H, s, H-7), 5.4 (1H, t, J₃ = 8.8 Hz, H-2), 5.3 (1H, t, J₃ = 8.8 Hz, H-3), 5.2 (1H, d, J₃ = 7.6 Hz, H-1), 4.4 (1H, dd, J_eq = 4.8 Hz e J_ax = 10.4 Hz, H-6 eq.), 3.8 (2H, m, H-4 and H-6 ax.), 3.7-3.6 (1H, m, H-5), 2.3-2.2 (4H, m, methylene), 1.5 (4H, m, methylene), 1.2 (48H, sl, methylene), 0.8 (6H, t, J₃ = 6.6 Hz, methyl). ¹³C-NMR (100 MHz, CDCl₃) δ: 191.3 (1C, CHO), 172.7 (1C, ester C=O), 172.2 (1C, ester C=O), 157.2 (1C, C-12), 137,9 (1C, C-8), 136.6 (1C, C-14), 128.2 (2C, C-10 and C-10'), 126.1 (2C, C-9 and C-9'), 130.3-116.1 (5C, C-11, C-13, C-15, C-16 and C-17), 101.5 (1C, C-7), 99.4 (1C, C-1), 78.1 (1C, C-4), 71.6 (1C, C-2), 71.2 (1C, C-3), 68.4 (1C, C-6), 66.7 (1C, C-5), 34.1-22.6 (28C, methylene), 14.1 (2C, methyl).

Synthesis of neoglycolipids 18-23: general procedure

To a solution of 0.389 mmol of acylated benzylidene acetals in 20 mL of acetone, was added 15 drops of HCl, at 0 ºC. The reaction mixture was stirred at room temperature and the reaction monitored by TLC. After the completion of the reaction, the mixture was neutralized with IRA 400 resin. The resin was filtered off and the filtrate was concentrated in vaccum to afford neoglycolipids 18-23.

4-formyl-2-methoxyphenyl 3-O-palmitoyl-β-D-glucopyranoside (18)

This compound was obtained as white crystals; yield: 92%; m.p. 134.4-135.8 ºC; [α]_D +83.3º (c 0.14 DMSO); IR (υ/cm^-1): 3323 (OH), 2919, 2850 (sp³ C-H), 1733 (ester C=O), 1702 (aldehyde C=O), 1625, 1592, 1575, 1535, 1508 (Ar. C=C). ¹H-NMR (400 MHz, DMSO-d₆) δ: 9.8 (1H, s, CHO), 7.5 (1H, dd, J₄ = 1.8 Hz e J₃ = 8.4 Hz, H-9), 7.4 (1H, d, J₄ = 1.8 Hz, H-11), 7.3 (1H, d, J₃ = 8.4 Hz, H-8), 5.5 (1H, d, J₃ = 6.4 Hz, OH), 5.2 (2H, m, H-1 e OH), 4.9 (1H, t, J₃ = 9.4 Hz, H-3), 4.6 (1H, t, J₃ = 5.6 Hz, OH), 3.8 (3H, s, OCH₃), 3.6 (1H, dd, J₃ = 5.2 Hz e J₂ = 10.4 Hz, H-6), 3.5-3.3 (4H, m, H-2, H-4, H-5 and H-6), 2.3 (2H, t, J₃ = 7.4 Hz, methylene), 1.7-1.2 (26H, m, methylene), 0.8 (3H, t, J₃ = 6.8 Hz, methyl). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 191.4 (1C, CHO), 172.3 (1C, ester C=O), 151.4 (1C, C-7), 149.2 (1C, C-8), 130.6 (1C, C-10), 125.2 (1C, C-11), 114.5 (1C, C-12), 110.5 (1C, C-9), 98.9 (1C, C-1), 77.4 (1C, C-5), 76.7 (1C, C-3), 71.0 (1C, C-2), 67.2 (1C, C-4), 60.1 (1C, C-6), 55.6 (1C, OCH₃), 33.7-22.0 (14C, methylene), 13.8 (1C, methyl).

4-formyl-2-methoxyphenyl 2-O-palmitoyl-β-D-glucopyranoside (19)

This compound was obtained as white crystals; yield: 88%; m.p. 135.1-137.0 ºC; [α]_D +27.7º (c 0.14 DMSO); IR (υ/cm^-1): 3322 (OH), 2920, 2849 (sp³ C-H), 1718 (ester C=O), 1688 (aldehyde C=O), 1624, 1591, 1536, 1510, 1462 (Ar. C=C). ¹H-NMR (400 MHz, DMSO-d₆) δ: 9.8 (1H, s, CHO), 7.5 (1H, d, J₃ = 8.0 Hz, H-9), 7.4 (1H, s, H-11), 7.3 (1H, d, J₃ = 8.4 Hz, H-8), 5.4 (1H, d, J₃ = 5.2 Hz, OH), 5.3-5.2 (2H, m, H-1 and OH), 4.8 (1H, t, J₃ = 8.6 Hz, H-2), 4.6 (1H, s, OH), 3.7 (3H, s, OCH₃), 3.7-3.4 (5H, m, H-3, H-4, H-5, H-6 and H-6'), 2.2 (2H, m, methylene), 1.4-1.0 (26H, m, methylene), 0.8-0.8 (3H, m, methyl). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 191.4 (1C, CHO), 171.6 (1C, ester C=O), 151.3 (1C, C-7), 149.5 (1C, C-8), 131.1 (1C, C-10), 124.9 (1C, C-11), 115.5 (1C, C-12), 110.8 (1C, C-9), 97.9 (1C, C-1), 77.3 (1C, C-5), 73.7 (1C, C-2), 73.0 (1C, C-3), 69.6 (1C, C-4), 60.4 (1C, C-6), 55.7 (1C, OCH₃), 33.6-22.0 (14C, methylene), 13.8 (1C, methyl).

4-formyl-2-methoxyphenyl 2,3-di-O-palmitoyl-β-D-glucopyranoside (20)

This compound was obtained as white crystals; yield: 83%; m.p. 93.5-95.0 ºC; [α]_D +90.0º (c 0.66 DMSO); IR (υ/cm^-1): 3433 (OH), 2955, 2849 (sp³ C-H), 1743 (ester C=O), 1689 (aldehyde C=O), 1589, 1508, 1471 (Ar. C=C). ¹H-NMR (400 MHz, DMSO-d₆) δ: 9.9 (1H, s, CHO), 7.5 (1H, dd, J₄ = 1.8 Hz e J₃ = 8.0 Hz, H-9), 7.4 (1H, d, J₄ = 1.8 Hz, H-11), 7.4 (1H, d, J₃ = 8.0 Hz, H-8), 5.4 (1H, d, J₃ = 7.6 Hz, H-1), 5.2 (1H, t, J₃ = 9.0 Hz, H-3), 5.1 (1H, t, J₃ = 9.0 Hz, H-2), 4.9 (1H, s, OH), 4.0 (1H, s, OH), 3.8 (3H, s, OCH₃), 3.9-3.7 (4H, m, H-4, H-5, H-9 and H-6'), 2.3-2.2 (2H, m, methylene), 1.6-1.5 (2H, m, methylene), 1.3-1.2 (52H, m, methylene), 0.8 (6H, t, J₃ = 6.6 Hz, methyl). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 191.1 (1C, CHO), 172.8 (1C, ester C=O), 172.0 (1C, ester C=O), 152.2 (1C, C-7), 150,9 (1C, C-8), 132.8 (1C, C-10), 125.4 (1C, C-11), 117.1 (1C, C-12), 111.5 (1C, C-9), 99.4 (1C, C-1), 77.7 (1C, C-5), 75.5 (1C, C-3), 71.8 (1C, C-2), 68.7 (1C, C-4), 61.5 (1C, C-6), 56.2 (1C, OCH₃), 34.3-22.9 (28C, methylene), 14.1 (2C, methyl).

3-formylphenyl 3-O-palmitoyl-β-D-glucopyranoside (21)

This compound was obtained as white crystals; yield: 86%; m.p. 74.5-76.0 ºC; [α]_D +19.8º (c 0.70 DMSO); IR (υ/cm^-1): 3404 (OH), 2919, 2850 (sp³ C-H), 1721 (ester C=O), 1681 (aldehyde C=O), 1608, 1589, 1486, 1469 (Ar. C=C). ¹H-NMR (400 MHz, DMSO-d₆) δ: 9.9 (1H, s, CHO), 7.6-7.5 (3H, m, H-9, H-10 and H-12), 7.3 (1H, d, J₃ = 7.6 Hz, H-8), 5.5 (1H, d, J₃ = 5.6 Hz, OH), 5.2 (1H, d, J₃ = 5.6 Hz, OH), 5.1 (1H, d, J₃ = 7.6 Hz, H-1), 4.9 (1H, t, J₃ = 9.4 Hz, H-3), 4.6 (1H, s, OH), 3.7-3.6 (1H, m, H-6), 3.5-3.3 (4H, m, H-2, H-4, H-5 and H-6'), 2.3 (2H, t, J₃ = 7.2 Hz, methylene), 1.5 (2H, q, J₃ = 7.0 Hz, methylene), 1.2 (24H, m, methylene), 0.8 (3H, t, J₃ = 6.8 Hz, methyl). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 192.7 (1C, CHO), 172.2 (1C, ester C=O), 157.6 (1C, C-7), 137,5 (1C, C-9), 130.3-116.5 (3C, C-8, C-10 and C-11), 122.5 (1C, C-12), 100.0 (1C, C-1), 77.2 (1C, C-3), 76.7 (1C, C-5), 71.1 (1C, C-2), 67.4 (1C, C-4), 60.2 (1C, C-6), 33.7-22.0 (14C, methylene), 13.8 (1C, methyl).

3-formylphenyl 2-O-palmitoyl-β-D-glucopyranoside (22)

This compound was obtained as white crystals; yield: 89%; m.p. 136.0-138.0 ºC; [α]_D +103.4º (c 0.11 DMSO); IR (υ/cm^-1): 3509 (OH), 2920, 2849 (sp³ C-H), 1721 (ester C=O), 1703 (aldehyde C=O), 1596, 1529, 1489, 1466 (Ar. C=C). ¹H-NMR (400 MHz, DMSO-d₆) δ: 9.9 (1H, s, CHO), 7.5-7.2 (4H, m, H-8, H-9, H-10 and H-12), 5.3 (1H, s, OH), 5.2 (1H, d, J₃ = 8.0 Hz, H-1), 5.2 (1H, sl, OH), 4.8 (1H, t, J₃ = 8.8 Hz, H-2), 4.6 (1H, sl, OH), 3.7 (1H, m, H-6), 3.5-3.3 (4H, m, H-3, H-4, H-5 and H-6'), 2.2 (2H, t, J₃ = 7.2 Hz, methylene), 1.5 (2H, q, J₃ = 7.2 Hz, methylene), 1.2-1.1 (24H, m, methylene), 0.8 (3H, t, J₃ = 6.8 Hz, methyl). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 192.5 (1C, CHO), 171.8 (1C, ester C=O), 157.2 (1C, C-7), 137.5 (1C, C-9), 130.4(1C, C-10), 123.4 (1C, C-11), 122.5 (1C, C-12), 116.3 (1C, C-8), 97.8 (1C, C-1), 77.2 (1C, C-5), 73.7 (1C, C-3), 73.2 (1C, C-2), 69.6 (1C, C-4), 60.3 (1C, C-6), 33.7-22.0 (14C, methylene), 13.9 (1C, methyl).

3-formylphenyl 2,3-di-O-palmitoyl-β-D-glucopyranoside (23)

This compound was obtained as white crystals; yield: 72%; m.p. 107.0-108.0 ºC; [α]_D +20.4º (c 0.58 DMSO); IR (υ/cm^-1): 3397 (OH), 2955, 2849 (sp³ C-H), 1731 (ester C=O), 1687 (aldehyde C=O), 1593, 1491, 1472 (Ar. C=C). ¹H-NMR (400 MHz, DMSO-d₆) δ: 9.9 (1H, s, CHO), 7.6 (1H, d, J₃ = 7.6 Hz, H-10), 7.5-7.4 (2H, m, H-9 and H-12) 7.3 (1H, d, J₃ = 7.6 Hz, H-8), 5.5-5.2 (2H, m, H-1 and OH), 5.1 (1H, t, J₃ = 9.1 Hz, H-3), 4.9 (1H, t, J₃ = 9.1 Hz, H-2), 4.7 (1H, d, J₃ = 5.6 Hz, OH), 3.7-3.3 (4H, m, H-4, H-5, H-6 and H-6'), 2.3-0.8 (56H, m, methylene), 0.8 (6H, t, J₃ = 6.4 Hz, methyl). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 191.3 (1C, CHO), 172.6 (1C, ester C=O), 172.0 (1C, ester C=O), 152.4 (1C, C-7), 150,5 (1C, C-8), 132.4 (1C, C-10), 125.0 (1C, C-11), 117.0 (1C, C-12), 111.9 (1C, C-9), 100.2 (1C, C-1), 77.5 (1C, C-5), 75.0 (1C, C-3), 71.3 (1C, C-2), 68.2 (1C, C-4), 61.0 (1C, C-6), 34.4-22.6 (28C, methylene), 14.0 (2C, methyl).

In vitro antifungal susceptibility testing

The reference Candida albicans (ATCC 18804), C. krusei (ATCC 20298), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750) were obtained from the American Type Culture Collection. Susceptibility assays were performed according to the CLSI (Clinical and Laboratory for Standards Institute), broth microdilution reference method M27-A2.¹⁶Candida species were cultured on SDA (Difco Laboratories, Detroit, MI, USA) for 48 h at 35 ºC. The inoculum concentration was determined by measuring the transmittance of fungal suspensions at 530 nm. The compounds 6-23 were primarily dissolved in dimethylsulfoxide (DMSO) and diluted in sodium bicarbonate-free RPMI 1640 medium (Sigma, St Louis, MO, USA) buffered with 165 mmol L^-1 morpholine propanesulphonic acid (MOPS; Sigma), pH 7.0, and supplemented with 4 mmol L^-1 L-glutamine. Fungal inoculum concentrations were adjusted to 1 x 10³ to 5 x 10³ cells mL^-1. One hundred microlitres of each test solution was distributed in sterile flat-bottom 96-well microplates (Difco) followed by the addition of 100 µL of inoculum. All the compounds were tested at a concentration range of 0.25-250 µg mL^-1. Serial twofold dilutions were prepared as described in CLSI document M27-A2. Microplates were incubated at 35 ºC for 48 h. RPMI medium without the compounds and the solvents to be used as a control for the growth and sterility. Fluconazole (Pfizer, São Paulo, Brazil) was included as positive antifungal control. The endpoints were determined visually by comparing with the growth in the drug-free control well. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that did not allow for the detection of any visual fungal growth.

 

REFERENCES

1. Souza, T. B.; Orlandi, M.; Coelho, L. F. L.; Malaquias, L. C. C.; Dias, A. L. T.; Carvalho, R. R.; Silva, N. C.; Carvalho, D. T.; Med. Chem. Res. 2013, 23, 496. DOI: http://dx.doi.org/10.1007/s00044-013-0669-2

2. Vandeputte, P.; Ferrari, S.; Coste, A. T.; Int. J. Microbiol. 2012, 1.

3. Bierman, B.; Bubalo, J.; U. S. Pharm. 2006, 1, 3.

4. Traxler, P.; Gruner, J.; Auden, J. A. L.; J. Antibiot. 1977, 30, 289. DOI: http://dx.doi.org/10.7164/antibiotics.30.289 PMID: 324958

5. Schmatz, D. M.; Romanchek, M. A.; Pittarelli, L. A.; Schwartz, R. E.; Fromtling, R. A.; Nollstadt, K. H.; Vanmiddelesworth, F. L.; Wilson, K. E.; Turner, M. J.; Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 5950. DOI: http://dx.doi.org/10.1073/pnas.87.15.5950 PMID: 2198575

6. Yeung, C. M.; Klein, L. L.; Lartey, P. A.; Bioorg. Med. Chem. Lett. 1996, 6, 819. DOI: http://dx.doi.org/10.1016/0960-894X(96)00113-8

7. Bartizal, K.; Abruzzo, G.; Trainor, C.; Krupa. D.; Nollstadt, K.; Schwartz, R.; Hammond, M.; Balkovec, J.; Vanmiddlesworth, F.; Antimicrob. Agents Chemother. 1992, 36, 1648. DOI: http://dx.doi.org/10.1128/AAC.36.8.1648 PMID: 1416847

8. Kaaden, M.; Breukink, E.; Pieters, J.; Beilstein J. Org. Chem. 2012, 8, 732. DOI: http://dx.doi.org/10.3762/bjoc.8.82 PMID: 23015820

9. Traxler, P.; Tosch, W.; Zak, O.; J. Antibiot. 1987, 40, 1146. DOI: http://dx.doi.org/10.7164/antibiotics.40.1146 PMID: 3680026

10. Jacobsson, M.; Malmberg, J.; Ellervik, U.; Carbohydr. Res. 2006, 341, 1266. DOI: http://dx.doi.org/10.1016/j.carres.2006.04.004 PMID: 16650391

11. Souza, T. B.; Raimundo, P. O.; Andrade, S. F.; Hipólito, T. M.; Silva, N. C.; Dias, A. L. T.; Ikegaki, M.; Rocha, R.; Coelho, L. F. L.; Veloso, M. P.; Carvalho, D. T.; Dias, D. F.; Carbohydr. Res. 2015, 410, 1. DOI: http://dx.doi.org/10.1016/j.carres.2015.04.002 PMID: 25933362

12. Hall, D. M.; Carbohydr. Res. 1980, 86, 158. DOI: http://dx.doi.org/10.1016/S0008-6215(00)84593-0

13. Barrett, A. G. M.; Pena, M.; Willardsen, J. A.; J. Org. Chem. 1996, 61, 1082. DOI: http://dx.doi.org/10.1021/jo951895e

14. Furihata, K.; Seto, H.; Tetrahedron Lett. 1999, 40, 6271. DOI: http://dx.doi.org/10.1016/S0040-4039(99)01233-2

15. Bell, D. J.; Lorber, J.; J. Chem. Soc. 1940, 453. DOI: http://dx.doi.org/10.1039/jr9400000453

16. Wayne, P. A.; National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A2. National Committee for Clinical Laboratory Standards, 2002.