Water Soluble Amide Derivatives of Polyene Antibiotic and their Antifungal Activity
Until recently the incidence of life-threatening fungal infections was considered to be too low to warrant extensive research, however over the last two decades infections caused by fungi have emerged as a growing threat to human health and there are studies indicating that the situation will become worse in the near future. Polyene antibiotics like nystatin and amphotericin B are widely employed for antifungal activities. Nystatin is used to clear candidosis and amphotericin B is useful in the treatment of deep-seated mycoses. Other polyene antibiotics like primaricin, hamycin, trichomycinare and candicidin have been used to a limited extent, for the treatment of localized mycotic infections. However, the polyene antibiotics especially aureofungin are less extensively used for the therapeutic treatment due to their toxicity and particularly less water solubility. The solubility of these antibiotics may play an important role in the enhancement of their therapeutic activity, keeping this view in mind, we have prepared water soluble amide derivatives of aureofungin, seven derivatives have been prepared and six of them were found to be active as antifungal agents when tested against Candida albicans. The structures of the derivatives were confirmed by UV and IR spectroscopy. The Minimum Inhibition Concentration (MIC) was found to be 1000 μg mL-1.
to cite this article:
P.D. Lokhande, K.R. Gawai, K.M. Kodam, B.Y. Waghmare, A.R. Chabukswar and S.C. Jagdale, 2006. Water Soluble Amide Derivatives of Polyene Antibiotic and their Antifungal Activity. Trends in Applied Sciences Research, 1: 529-533.
Over the last thirty years, the frequency of life-threatening fungal infections have increased dramatically, particularly among cancer, diabetic and immunocompromised patients (Zotchev, 2003). As many as 30% of fungal infections are leading to deaths (Ablordeppey et al., 1999). Several factors have contributed to this rise: improved recognition and diagnosis of fungal infections; prolonged survival of patients with defects in their host defense mechanisms; more invasive surgical procedures; the use of prosthetic devices and indwelling catheters; increased administration of parenteral nutrition; development of resistance fungal strains to currently available drugs; the increase in the number of patients contracting AIDS and the use of peritoneal dialysis and hemodialysis (Gallis et al., 1990).
Polyene antibiotics remains the drug of choice for life-threatening fungal
infections (Arathoon, 2001). Among the polyene antibiotics, Amphotericin B (AmB)
and Nystatin (Nys), remain the most widely employed for therapeutic purposes
for both pre-systemic and systemic fungal infections (Heimenz and Walsh, 1996).
Nystatin and amphotericin B are used to clear candidosis (Mandell and Petr,
1996). Deep seated systematic mycoses are treated with amphotericin B and successful
results have been obtained in histoplasmosis, coccidiodomycosis, blastomycosis,
cryptococcal meningitis and disseminated candidosis (Heimenz and Walsh, 1998).
The other polyenes such as pimaricin, hamycin, trichomycin and candicidin have
been used to a limited extent for the treatment of localized mycotic infections
(Georgopapadakou and Walsh, 1994).
Utilization of polyene antibiotics implies severe side effects that have restricted their use as antifungal agents, they can cause unpleasant side effects including chills, fever, lowering of blood pressure and even kidney damage (Gulati et al., 1998). The side effects of therapy can mimic the clinical appearance of serious systemic infection, complicating patient management.
The major difficulty associated with polyene antibiotics is poor water solubility (Gates and Pinney, 1993) at neutral pH. The toxic effects and poor water solubility limits the use of these class of antibiotic in the therapy. The concentrated efforts are being made to improve the water solubility. Thus, the selective toxicity and increased water solubility is the target of many research methods (Falk et al., 1999).
Aureofungin are heptane macrolide antibiotics and they are produced by Streptomyces cinnamoneus and possesses excellent antifungal activity. An aureofungin is used to control the growth of fungal infections in plants and various diseases in crops but it cannot be used for clinical purposes in animals due to its toxicity and low water solubility. The chemical modifications of amphotericin to various water soluble derivatives have resulted in low toxicity and it is useful in clinical application (Naik et al., 2001). Hence, the aim of the present study was to prepare different amide derivatives of aureofungin and evaluation for their solubility and antifungal activity which will be beneficial for exploring the clinical effectiveness of aureofungin.
Materials and Methods
All melting points are uncorrected and reported in °C. All solvents
were distilled and dried before use. IR spectra were recorded in (cm-1)
on Perkin Elmer FTIR in KBr. The UV spectra was recorded on Schimadzu UV Visible
Silica gel used for TLC was more than 200 mesh. Organic extracts were washed with brine and water successively and dried over anhydrous sodium sulphate before evaporation of solvents. Aureofungin was a generous gift from Hindustan Antibiotics Ltd., Pimpri, Pune.
Preparation of Amide Derivatives of Aureofungin
Reactions Involved in the Synthesis of Amide Derivatives
In general 500 mg (0.44 mM) of aureofungin was suspended in 20 mL of dry
DMF, stirred at room temperature and treated with 10 mM of triethyl amine, 10
mM of appropriate amide group containing compound and 10 mM of diphenyl phosphorylazide
(DPPA). The course of reaction was followed by means of TLC on silica gel using
CHCl3: MeOH: H2O, 10:6:1 (v/v/v) solvent system. After
completion of reaction the crude product was precipitated with 300 mL of dry
ether, centrifuged, dissolved in 1-butanol. After evaporation of 1-butanol,
the product was again precipitated with dry ether, centrifuged, washed three
times with ether and dried in vacuum. The purity of the prepared derivatives
(Jarzebski et al., 1981) were monitored by TLC.
The carboxyl group of aureofungin was converted to seven different amide derivatives
(R = substituted amines) with use of DPPA in presence of DMF and triethylamine
|| Structure of aureofungin
||Rf values and water solubility of amides of aureofungin
Solubility of Amides of Aureofungin in Water at Neutral pH
The solubility was checked for all the derivatives at neutral pH in distilled
water. To determine solubility a fix quantity of sample was taken and volume
of water increased till clear solution is obtained. The solubility was confirmed
by turbidemetric assay. The solubility of amides of aureofungin in mg mL-1
is given in Table 1. The successful result for all derivatives
were obtained. The enhanced water solubility is observed under same conditions.
An antifungal activity of aureofungin was tested on Candida albicans.
The antifungal activity was determined by cup and plate method (Bhat et al.,
2001) and inhibition zones shown by different amide derivatives were measured.
|| Spectroscopic data of amide derivative of aureofungin
Each experiment was repeated thrice and the mean values for zone of inhibition
is reported in Table 2. The minimum inhibition concentration
was 1000 μg mL-1. All the aureofungin derivatives except Amide-1
showed excellent antifungal activity as shown in Table 2.
Results and Discussion
The UV absorption spectra were recorded in distilled methanol. All the amide derivatives showed a sharp band at 405-406 nm as reported for parent compound (May Dean, 1976 ; Deshpande and Narshimachari, 1969). Little variation is observed in the position of other bands, but the spectra maintained the same pattern as reported except for amide-1, where the absorption peak at 380 nm is not well distinguished from other peaks. The absorption peak located at this value indicated the presence of cis double bond in the heptaene chromophore. The intensity of peaks steadily increases from lower to higher values upto 380 nm indicating the presence of cis double bond. Thus, the absorption spectra confirmed the presence of conjugated heptaene of modified aureofungin. The absorption data of amide is given in Table 3. The IR spectra are dominated by broad hydroxyl absorption centered at 3388-3405 cm-1 indicating the presence of large number of hydroxyl groups. The carbonyl stretching frequencies are not well resolved due to overlapping of bands. A strong band at 638-639 cm-1 is observed for amide derivatives except for amide-1. Several other bands in the region of 1540-1600 cm-1 are observed due to conjugated polyene chromophore. Thus, UV and IR spectrums of amide derivatives of aureofungin shows that the structure of parent molecule remain intact during modification.
Aureofungin is insoluble in water at neutral pH. The solubility in water is the main hurdle in the clinical application of these antifungal agents. The newly prepared amide derivatives of aureofungin has resulted in improved biological and physicochemical properties. The solubility of the amide derivatives has increases significantly as represented in the Table 1. The free carboxyl group and amino group of polyene macrolide have no significant activity. The carboxyl group activating agent DPPA is used for the preparation of amide derivatives of aureofungin and successful results were obtained without modifying the other functionalities in the parent molecule. The solubility in water at neutral pH is enhanced for all the amide derivatives of aureofungin. The antifungal activity is retained in all the modified derivatives of aureofungin. It has been observed that the improvement of selective toxicity is related to the absence of a free carboxyl group in the modified antibiotic molecule. Seven amide derivatives of the aureofungin, except amide-1 were found to be active as antifungal agents. The MIC was found to be 1 mg mL-1.
The complex structural and physicochemical properties of the polyene antibiotics
offers a great challenge for their clinical use. The solubility of these antibiotics
always limits their utility and results either in the toxicity and unwanted
effects. The chemical modification of the aureofungin to their amide derivatives
has resulted in the increased solubility and even the antifungal activity was
also retained, which indicate that the synthesized amide derivatives will be
useful in their actual clinical application. The further toxicity and formulation
studies on the amide derivatives will explore their exact clinical role.
Ablordeppey, S.Y., P. Fan, J.H. Ablordeppey and L. Mardenborough, 1999.
Systemic antifungal agents against AIDS-related opportunistic infections: Current status and emerging drugs in development. Curr. Med. Chem., 6: 1151-1195.
Arathoon, E.G., 2001.
Clinical efficacy of echinocandin antifungals. Curr. Opin. Infect. Dis., 14: 685-691.
Bhat, A.R., G.V. Bhat and G.G. Shenoy, 2001.
Synthesis and in vitro
antimicrobial activity of new 1, 2, 4-triazoles. J. Pharm. Pharmacol., 53: 267-272.PubMed |
Falk, R., A.J. Domb and I. Polacheck, 1999.
A novel injectable water-soluble amphotericin B-arabinogalactan conjugate. Antimicrob. Agents Chemother., 43: 1975-1981.Direct Link |
Gallis, H.A., R.H. Drew and W.W. Pickard, 1990.
Amphotericin B: 30 years of clinical experience. Rev. Infect. Dis., 12: 308-329.PubMed |
Gates, G. and R.J. Pinney, 1993.
Amphotericin B and its delivery by liposomal and lipid formulations. J. Clin. Pharmacol. Ther., 18: 147-153.PubMed |
Georgopapadakou, N.H. and T.J. Walsh, 1994.
Human mycosis: Drugs and targets for emerging pathogens. Science, 264: 371-373.CrossRef | Direct Link |
Gulati, M., S. Bajad, S. Singh and J.A. Ferdous, 1998.
Development of liposomal amphotericin B formulation. J. Microencapsulation, 15: 137-151.
Heimenz, J.W. and T.J. Walsh, 1996.
Lipid formulations of amphotericin B: Recent progress and future directions. J. Clin. Infect. Dis., 22: S133-144.PubMed |
Heimenz, J.W. and T.J. Walsh, 1998.
Lipid formulations of amphotericin B. J. Liposome Res., 8: 443-467.
Jarzebski, A., L. Falkowski and E. Borowski, 1981.
Synthesis and structure activity relationship of derivatives of amphotericin. J. Antibiotic, 35: 220-229.PubMed |
Mandell, G.L. and Jr. W.A. Petr, 1996.
Antimicrobial Agents: Penicillins, Cephalosporins and Other B-lactam Antibiotics. In: Goodman and Gilman`s. The Pharmacological Basis of therapeutics, Hardman, J.G. and L.E. Limbird (Eds.). 9th Edn., McGraw-Hill, New York, pp: 1073-1101
May Dean, L.L., 1976.
Chemistry and structure of aureofungin A and B. Ph.D. Thesis, The Graduate College, Universitiy of Illinois at Urbana-Champaign.
Naik, S.R., J. Harindran and R.K. Nanda, 2001.
Structural studies on a oxohexane polyene antibiotic (HA-1-92) produced by Streptomyces CDRIL-312. Ind. J. Chem., 40B: 1183-1186.
Zotchev, S.B., 2003.
Polyene macrolide antibiotics and their applications in human therapy. Curr. Med. Chem., 10: 211-223.