Chemistry and Chemical Technologies / 4. Chemical and Pharmaceutical Industry

Dr. Valery V. Belakhov¹, Dr. Sc., Prof. Aleksandr V. Garabadzhiu^2a,

Dr. Sc., Prof. Boris I. Ionin^2b

¹Schulich Faculty of Chemistry, Technion – Israel Institute of Technology Israel; ^2aDepartment of Technology of Microbiological Synthesis and ^2bDepartment of Organic Chemistry, Saint-Petersburg State Technological Institute

(Technical University), Saint-Petersburg, Russia

chvalery@techunix.technion.ac.il

DIALKYLAMIDOPHOSPHATE DERIVATIVES OF AMPHOTERICIN B: PREPARATION AND ANTIFUNGAL ACTIVITY

1. Introduction

Amphotericin B is the main polyene macrolide antifungal antibiotic due to its high activity and broad spectrum of activity [1, 2]. Recently the range of its medical application has expanded in connection with its use in combination with synthetic antifungal drugs for therapy of fungal infections [3-5] and also the discovery of its antiviral [6, 7] and antitumor activity [8]. Furthermore, amphotericin B is promising in the treatment of leishmaniasis [9, 10] and is used as an anti-inflammatory drug [11], in gene therapy [12], and in combined therapy of serious fungal sepsis together with antibodies [13, 14]. Amphotericin B is especially important for treating fungal diseases that complicate AIDS [15-17]. Nevertheless, the high toxicity (in particular, nephrotoxicity) of amphotericin B [18, 19], the low absorption from the gastrointestinal tract and poor penetration into cerebrospinal fluid [3-5], a whole series of side reactions [20], and the reduced sensitivity to it of pathogenic fungal microorganisms [21, 22] have stimulated vigorous searches for different derivatives of this polyene antibiotic. The preparation of semisynthetic derivatives of amphotericin B with improved chemotherapeutic properties has been reviewed several times [17, 23-25]. It has also been demonstrated that liposomal amphotericin B is one of the modern highly effective drugs of this antibiotic with reduced toxicity [26-28]. We have previously prepared hydrophosphoryl [29], fluoroorganic [30] and N-benzyl derivatives of amphotericin B [31].

2. Experimental part

2.1. Experimental chemical part

We used commercial amphotericin B (Sigma, USA) with biological activity 740 ED/mg with specific absorption indices (E^1%_1cm) 782, 1373, and 1564 at wavelengths 362, 383, and 405 nm, respectively. Organic solvents were purified by the literature methods [32]. The ¹H and ¹³C NMR spectra were obtained on a Bruker Avance instrument (Germany) at 500 MHz (for protons) for 10-15% solutions in DMSO-d₆, internal reference TMS. The ³¹P NMR spectra were registered on a Bruker AC-200 instrument (Germany) with operating frequency 80 MHz (200 MHz for protons), external reference 85% H₃PO₄. The IR spectra were registered on a Bruker Vector 22 spectrophotometer (Germany) from KBr pellets. The UV spectra were recorded on an Ultrospec 2100 Pro-instrument (Biochrom, UK). The purity of the synthesized compounds was monitored using TLC on Silica Gel 60 F₂₅₄ plates (0.25 mm, Merck, Germany) and two solvent systems CHCl₃:MeOH:propan-1-ol:borate buffer (pH 3.14, 3:2:2:1, 1) and EtOH:NH₄OH (25% aqueous):H₂O (8:1:1, 2). Compounds were developed using UV light. The sorbent was Silica Gel 60 (63 – 200 mm, Merck, Germany).

2.2. Experimental biological part

Determination of antifungal activity of hydrophosphoryl derivatives of amphotericin B II and III relative to six test-cultures of yeast-like fungi of the genus Candida was performed by serial dilutions in liquid nutrient medium. The method is based on a serial two-fold dilution of the compounds under study. The minimum fungistatic concentration (MFK) was found by the results of visual evaluation of growth rate of a test culture in the experimental and control tubes on the basis of three replications. Amphotericin B was used as reference in all biomedical tests.

3. Results and discussion

In continuation of research on the preparation of semisynthetic derivatives of amphotericin B (I) with improved medical and biological properties, we studied the reactions of this heptaene macrolide antibiotic with dialkylphosphites in the presence of organic base and produced the corresponding dialkylamidophosphate derivatives II and III:

The reaction can be considered as a variation of Todd-Atherton reaction [33, 34], whose synthetic opportunities were generalized in [35].

The resulting derivatives II and III were solids without distinct melting points that decomposed on heating. Compounds II and III were very soluble in DMSO and DMF; slightly soluble in MeOH, EtOH, pyridine, and dioxane; and insoluble in water, acetone, CHCl₃, ether, benzene, and hexane.

The structure of the derivatives is confirmed by ¹H, ¹³C, and ³¹P NMR, IR and UV spectroscopy. In the ¹H NMR spectra of compounds II and III contained resonances for protons typical of amphotericin B [36, 37], protons of methoxy groups performed by doublet signals (δ 3.32–3.40 ppm) and protons of ethoxy groups consist of triplet signals of methyl groups (δ 1.12–1.25 ppm) and quartet signals of methylene groups (δ 3.81–3.94 ppm). The constant spin-spin interaction from the phosphorus atom [J_HР(CH₂O-P)] was determined as 10,0-10,2 Hz. The proton at the nitrogen atom (N-H) observed as a broad singlet (δ 5.12–5.23 ppm). In ¹³C NMR spectra there are signals of carbon atoms of amphotericin B [36, 37], and doublet signals of ethoxy groups were recorded (δ 16.3–17.4 ppm) with the constant of spin-spin interaction J_CP 6.7-7.0 Hz. The group OCH₂ resonances as doublet in more weak field (δ 61.5-62.7 ppm) with the constant spin-spin interaction J_CP 5.3-5.7 Hz. These were consistent with those reported in the literature [38]. ³¹P Chemical shifts δ of the synthesized derivatives of amphotericin B II and III are 15.42–15.84 ppm, that is typical for dialkylamidophosphates with tetra-coordinate phosphorus atom [39].

In IR spectra of compounds II and III in addition to absorption bands belonging to original amphotericin B [40] there are those in regions of 1230–1234 cm^–1 and 3957–3962 cm^–1 that proves the presence of phosphoryl group P=O and N–H bonds, respectively. The electronic absorption spectra of II and III had maxima at 362, 383, and 405 nm, which indicated that a heptaene conjugated system was present.

Biological investigations showed that dialkylamidophosphate derivatives of amphotericin B II and III had high antifungal activity against six test cultures of the genus Candida: Candida albicans, Candida utilis, Candida tropicalis, Candida krusei, Candida parapsilosis and Candida guillermondii, and minimal fungistatic concentration varied form 0.07 to 6.15 mkg/ml^-1.

4. Conclusion

Thus, further search for semi-synthetic derivatives of amphotericin B under the Todd-Atherton reaction conditions is a promising direction for synthesis of new potent antimycotic preparations with high antifungal activity against fungal pathogens.

5. Acknowledgments

This work was supported by the Ministry of Education and Science of Russian Federation under the framework of Federal Program "Scientific and Scientific Educational Specialists of the Innovational Russia" during 2009-2013 (grant No. 2012-1.5-12-000-1013-005).

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SUMMARY

V.V.Belakhov¹, A.V. Garabadzhiu^2a, B.I.Ionin^2b

¹Schulich Faculty of Chemistry, Technion – Israel Institute of Technology Israel; ^2aDepartment of Technology of Microbiological Synthesis and ^2bDepartment of Organic Chemistry, Saint-Petersburg State Technological Institute (Technical University), Saint-Petersburg, Russia

DIALKYLAMIDOPHOSPHATE DERIVATIVES OF AMPHOTERICIN B: PREPARATION AND ANTIFUNGAL ACTIVITY

The chemical modification of the heptaene macrolide antibiotic amphotericin B with dialkylphosphites was carried out in the conditions of Todd-Atherton reaction. It was shown that reactions of amphotericin B with different dialkylphosphites resulted in the formation of its corresponding dialkylamidophosphate derivatives. Physicochemical properties of dialkylamidophosphate derivatives of amphotericin B and their antifungal activity against Candida yeast-like fungal strains were studied.

Keywords: amphotericin B, dialkylphosphites, derivatives, antifungal activity.