Chemistry and Chemical Technologies / 4. Chemical and Pharmaceutical Industry

 

Dr. Valery V. Belakhov1, Dr. Sc. Yury D. Shenin2, Dr. Sc.,

Prof. Aleksandr V. Garabadzhiu3a, Dr. Sc., Prof. Boris I. Ionin3b

 

1Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa, Israel;  2All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia; 3aDepartment of Technology of Microbiological Synthesis and  3bDepartment of Organic Chemistry, Saint-Petersburg State Technological Institute

 (Technical University), Saint-Petersburg, Russia

chvalery@techunix.technion.ac.il

 

CHEMICAL MODIFICATION OF POLYENE MACROLIDE ANTIBIOTICS AS A TOOL FOR THE PREPARATION OF NOVEL LOW-TOXIC DERIVATIVES WITH AN EXTENSIVE SPECTRUM OF BIOLOGICAL ACTIVITY

 

1. Introduction

      Polyene macrolide antibiotics (PMA) constitute an abundant group of natural compounds that embarrasses over 200 preparations revealing activity toward yeast, yeast-like, and filamentous fungi of both saprophytic and pathogenic species [1-4]. Perspectives of medical application of PMA were enhanced after revealing their antiviral activity [5-8], activity toward parasitic Protozoa [9-11], and potential use as antitumor agents [12-14]. They are particularly useful for treatment of fungal diseases complicating AIDS [15-18]. On the other hand, PMA used in medical practice do not satisfy completely requirements of clinical physicians due to restricted efficiency, in part, inapplicability for treatment of certain forms of deep mycoses [19-21]. Medical application of these antifungal preparations is also hindered by their high toxicity [22-25], instability on storage [26-30], poor solubility in water [1-4], and reduced sensitivity of these antibiotics to pathogenic fungal microorganisms [31-34]. Therefore, search for new derivatives of PMA with improved chemotherapeutic properties is being continued.

2. Results and Discussion      

   One of the promising methods of chemical modification of drugs and, in particular, PMA is hydrophosphorylation [35–37]. Our studies on the synthesis of semi-synthetic PMA derivatives have included the reactions of PMA with aromatic aldehydes and hypophosphorous acid, and an investigation of medical and biological properties of the obtained derivatives. Thus, it was interesting to prepare the hydrophosphoryl derivatives of PMA to examine their biomedical properties and to conduct a comparative analysis of their biological effectiveness with initial PMA.

    We showed that PMA levorin, nystatin, amphotericin B, mycoheptin, pimaricin and lucensomycin react with aromatic aldehydes and hypophosphorous acid with the formation of hydrophosphoryl derivatives of these antifungal antibiotics [38-43]. The studied reaction can be considered as a specific version of the Kabachnik-Fields reaction [44, 45] whose synthetic performance has been considered in detail in [46, 47]. In the first step of the process, the primary amino group of the pimaricin carbohydrate fragment is added to the carbonyl group of the aromatic aldehyde to form an azomethine intermediate. In the second step, hypophosphorous acid reacts at the C=N bond of the azomethine fragment to afford hydrophosphoryl derivatives of PMA. The general scheme of this reaction was performed for tetraene macrolide antibiotic pimaricin.

 

 

     As phosphorylating compound we used hypophosphorous acid, which is soft hydrophosphoryl reagent [48]. We used the following aldehydes: 4-hydroxybenzaldehyde, 4-methoxybenzaldehyde, 4-nitrobenzaldehyde, 4-(dimethylamino)benzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, and 4-bromobenzaldehyde. The hydrophosphoryl derivatives of PMA are solid crystalline compounds having no sharp melting point and decomposing on heating. These compounds are readily soluble in dimethylsulfoxide and dimethylformamide, moderately soluble in methanol, ethanol, and water, insoluble in chloroform, higher alcohols, ethers, dioxane, benzene and hexane.

     The biological tests demonstrated that acute toxicity (LD50) of hydrophosphoryl derivatives of PMA was 2–6 times lower than that of original antibiotics [38-43].

    Biological investigations showed that hydrophosphoryl derivatives of PMA possess a high antifungal activity toward 21 pathogens of different mycotic infections and, especially, to yeast-like fungi of the genus Candida (C. albicans, C. utilis, C. tropicalis, C. krusei, C. parapsilosis, C. guillermondii).

      It is known that chemical modification of biologically active compounds in some cases leads to a change in a range of biological activity of obtained derivatives and to reducing their toxicity [35, 36, 49-53]. Previously, various authors have found antiviral and antitumor activity which is nonspecific for polyene macrolide antibiotics [12–14]. On the base of a detailed study of molecular and biological mechanisms of lipido-membranotropic effect of PMA was formulated the hypothesis that these antifungal preparations possessed an antiviral activity [5-8]. According to this hypothesis, PMA cause a reorientation of the lipid matrix surface-membrane or virus-specific receptors of the cellular cytoplasmatic membrane, thereby, inactivating enveloped (lipid-containing) viruses or preventing the penetration of virus into sensitive cells. This hypothesis was later confirmed experimentally at the molecular and sub-cellular levels [54-58]. Hence, the mechanism of an antiviral action of PMA includes two points of application: 1) an interaction between PMA and lipids of cellular cytoplasmic membranes, which leads to a change in membrane permeability and to an antiviral activity; and 2) an interaction between PMA and lipids of the virion envelope, which appears as a viricidal effect of these antifungal agents. It was shown that the mechanism of antiviral action of PMA differs substantially from the virus-inhibitory effect of antibacterial non-polyene antibiotics [59]. Therefore, it was interesting to study the antiviral activity of hydrophosphoryl derivatives of PMA.

     The study of the toxicity of hydrophosphoryl derivatives of PMA for 10–11-day-old developing chick embryos showed that these compounds are less toxic than the original PMA. Antiviral activity of synthesized derivatives of PMA against model viruses was studied in experiments in ovo with doses 1/5 LD50. Biological studies showed that several hydrophosphoryl derivatives of PMA had high activity in relation to vaccinia virus and to types A and B influenza viruses [38-43].

     Data obtained in the study of hydrophosphoryl derivatives of PMA by a model of RNA-containing Rous sarcoma retrovirus are of particular interest, since this model is suggested as an adequate retroviral model for screening and studying drugs against AIDS [8, 60, 6]. It was found that a series of studied derivatives of PMA demonstrated high antiviral activity against RSV both in prophylactic and therapeutic schemes of the injection [38-43].  

  

3. Experimental part

3.1. Experimental physical and chemical part

    The structure of these derivatives was confirmed using 1H, 13C, and 31P NMR, as well as IR and UV spectroscopy. 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 or MeOH-d4, 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). Individuality of hydrophosphoryl derivatives of PMA was verified by TLC on Silica Gel 60 F254 plates (0.25 mm, Merck, Germany) in corresponding systems; spots were developed by UV illumination. Column chromatography was performed on Silica Gel 60 (63-200 mm, Merck, Germany).

 

3.2. Experimental medical and biological part

   Acute toxicity (LD50) of hydrophosphoryl derivatives of PMA was studied on white mongrel male mice weighing 18–20 g. The compounds were diluted with 0.5% aqueous solution of carboxymethylcellulose and tested in the form of suspensions by intraperitoneal injection. The LD50 values were calculated from the test results using the Kerber method.

     Antifungal activity of hydrophosphoryl derivatives of PMA against 21 pathogens of different mycotic infections was determined using serial dilutions in liquid nutrient medium. The method was based on successive two-fold dilutions of the tested compounds. The minimal fungistatic concentration (MFC) was established by a visual assessment of the growth of test cultures in experimental and control tubes using three repetitions.

      The toxicity of 10–11 day developing chick embryos was determined by introducing the compounds into the allantoic cavity or horionallantoic membrane (HAM) before examining the antiviral action of hydrophosphoryl derivatives of PMA. In each dilution we used six embryos which were incubated at 37°C until hatching with ovoscoping every two days. Antiviral activity of these compounds in experiments was examined by in ovo technique against DNA-containing variolovaccine virus using a L-IVP 01.72 strain. Working titers of variolovaccine virus were 10–6–10–8 ODE ml–1. Virus-containing material was prepared in a McIlavaine’s solution. The test compounds were administered at the HAM of 10–11-day-old chick embryos according to prophylactic (for 1 h prior to the introduction of the virus) and therapeutic schemes (1 h after infection). Incubation of embryos was carried out for 48–72 h at 37°C. Counting of the pocks was performed on HAM of embryo. Antiviral activity of hydrophosphoryl derivatives of PMA was examined against RNA containing viruses, oncogenic Rous sarcoma virus (RSV) [(strain RSV (RSV-1)], and infectious virus of type A ([a strain A2 Odessa 2882/82 (H3N2)] and type B ([a strain B/USSR (69)] in developing chick embryos. After determining the toxicity of compounds 10–11-day-old developing chick embryos were administered in the test with the RSV on the HAM according to prophylactic and therapeutic schemes. Number of foci of neoplastic transformation in the HAM of embryos was counted on 8th day after incubation at 37°C. Determination of antiinfluenzal action were carried out by the introduction of derivatives of hydrophosphoryl derivatives of PMA in the horionallantoic cavity of 10–11-day-old developing chick embryos by prophylactic scheme, i.e., 10–100 EID50 of virus for 1–1.5 h before incubation. The results were taken into account after 48 h of incubation of embryos at 37°C in the case of influenza virus of type A and after 72 h at 34°C in the case of influenza virus of type B. The presence of virus in embryos were determined in the reaction of hemagglutination with chicken red blood cells in the allantoic fluid of experimental and control embryos. An isotonic sodium chloride solution was injected into the control embryos instead of substances under study. Antiviral activity of the tested compounds in experiments in ovo was assessed by a protection index (PI), which was calculated by the following formula:

 

                 amount of virus-infected                amount of virus-infected

in control embryos (%)       ‾        in experiment embryos (%)

PI =   ––––––––––––––––––––––––––––––––––––––––––––––––––– × 100.

amount of virus-infected in control embryos (%)

 

 

 

 

 

 

4. Conclusions

1. The hydrophosphoryl derivatives of PMA were successfully prepared under the Kabachnik-Fields reaction conditions.

2. The studied reactions can be considered as a general method for chemical modification of polyene macrolide antibiotics via hydrophosphorylation.

3. This method opens promising direction for the preparation of novel low-toxic derivatives of PMA with improved medical and biological properties and extensive range of biological activity.

 

5. Acknowledgments

    This work was supported by the Ministry of Education and Science of Russian Federation under the framework of Federal Program "Scientific and Educational Specialists of the Innovational Russia" during 2009-2013 on the topic: "Development of new semi-synthetic derivatives and modification of  known polyene macrolide antibiotics for the preparation of highly effective antifungal drugs" (grant No. 2012-1.5-12-000-1013-005).

 

 

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SUMMARY

 

V.V. Belakhov1, Y.D. Shenin2, A.V.Garabadzhiu3a, B. I. Ionin3b

 

1Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa, Israel; 2All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia; 3aDepartment of Technology of Microbiological Synthesis and 3bDepartment of Organic Chemistry, Saint-Petersburg State Technological Institute (Technical University), Saint-Petersburg, Russia

 

CHEMICAL MODIFICATION OF POLYENE MACROLIDE ANTIBIOTICS AS A TOOL FOR THE PREPARATION OF NOVEL LOW-TOXIC DERIVATIVES WITH AN EXTENSIVE SPECTRUM OF BIOLOGICAL ACTIVITY

 

Reactions of the polyene macrolide antibiotics with aromatic aldehydes and hypophosphorous acid yielded their hydrophosphoryl derivatives. The physicochemical and biological properties of these semi-synthetic derivatives were studied. Biological investigations showed that hydrophosphoryl derivatives of polyene macrolide antibiotics were less toxic than the initial antibiotic and had antifungal and antiviral activity.

 

Keywords: polyene macrolide antibiotics, hydrophosphoryl derivatives, antifungal and antiviral activity, toxicity.