4862 Vol. 35 (1987)

Chem. Pharm. Bull. 35(12)4862-4867(1987)

Antifungal Properties of Solanum Alkaloids

GENJIRO KUSANO, AKIRA TAKAHASHI, KAZUHIRO SUGIYAMA and SHIGEO NOZOE*

Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan

(Received March 30, 1987)

The antifungal activities of several solanum alkaloids and their derivatives were examined against a variety of fungal strains such as Candida albicans and Trichophyton spp. Solacongestidine

(I) showed the strongest activity (minimum inhibitory concentration) against C. albicans (0.8 ƒÊg/ml), T. rubrum (0.4 ƒÊg/ml) and Cryptococcus albidus (0.78 ƒÊg/ml), and also showed lesser activities against a wide range of fungi. Solafloridine (II) and verazine (VII) showed activities against C. albicans (3.1 and 6.2 ƒÊg/ml, respectively) and T. rubrum (25 and 3.1 ƒÊg/ml, respectively), while other alkaloids such as solasodine (III), tomatidine (IV), tomatillidine (V) and solanocapsine

(VI) and related compounds (VIII-XIX) showed much lower activities. Solacongestidine also prolonged the survival time of mice infected with C. albicans.

Keywords--antifungal activity; solanum alkaloid; solacongestidine; solafloridine; verazine; Candida albicans; Trichophyton rubrum; Trichophyton mentagrophytes; Cryptococcus albidus

A number of azasteroids and steroidal alkaloids with substantial antimicrobial activity have been reported,1-11) no useful drugs have yet been found among these com- pounds. In the course of our research on antifungal compounds obtained from higher plants and mushrooms, we found that some solanum alkaloids such as solacongestidine (I) and solafloridine (II) showed strong activity against Candida albicans, Trichophyton rubrum, Cryptococcus neoformans and so on. Because the antifungal activities of these compounds showed no depression when tested in the presence of 10% serum, these alkaloids were chosen as targets of further research aimed at the development of antifungal agents for treatment of some systemic mycoses. Furthermore, solacongestidine (I) showed potent inhibition of cholesterol biosynthesis from 24,25-dihydrolanosterol. The experimental results suggested that one of the sites of its action is the step of 14ƒ¿-demethylation of dihydrolanosterol. Because the step of 14ƒ¿-demethylation is common to the biosyntheses of cholesterol and ergosterol from lanosterol, solacongestidine (I) was expected to inhibit ergosterol biosynthesis from lanosterol, resulting in disturbances of fungal growth.12) Therefore, some stock solanum alkaloids and related compounds were examined for antifungal activity. In this article we wish to report the results and to discuss their significance.

Experimental

Solacongestidine (I),13) solafloridine (II),13) solasodine (HI),14) tomatidine (IV),14) tomatillidine (V),15a,b and solanocapsine (VW') were obtained as a result of research on solanum alkaloids in the Steroids Section of the National Institute of Arthritis, Metabolic Diseases and Digestive Diseases (NIAMDD) of the National Institutes of Health (NIH), U.S.A. Verazine (VII) was given by Prof. K. Kaneko in the Faculty of Pharmaceutical Sciences of Hokkaido University (Sapporo, Japan). Dihydrosolasodine (VIII) was prepared by reduction of solasodine (III) with NaBH4 in methanol.17) The N-methyl derivative (IX) of VIII was prepared by treatment of VIII with methyl iodide in N,N-dimethylformamide. 3,16-O-Diacetylpseudosolasodine (X) was prepared by treatment of solasodine (III) with No. 12 4863 acetic anhydride containing zine chloride.18) 16-O-Acetylpseudosolasodine (XI) was obtained by stirring X in 1% methanolic Na2CO3 solution at room temperature, followed by recrystallization from methanol after Si02 chromatography. 3,16-O-Diacetylpseudotomatidine (XII) and 16-O-acetylpseudotomatidine (XIII) were obtained from tomatidine (IV) by similar treatment. A mixture (XIV) of glycosides of solacongestidine and solafloridine was obtained from the alkaloids fraction of the fruits of Solanum congestiflorum. The mixture provided solacongestidine and solafloridine on incubation with Taka-diastase.19) Dehydrosolacongestidine (XV) was prepared by oxidation of solacongestidine (I) with the Jones reagent.13) Dihydrosolacongestidine (XVI) was obtained by hydrogenation in the presence of platinum dioxide.") The quaternary salt (XVII) of solacongestidine was prepared by treatment of solacongestidine with methyl iodide in acetone containing anhydrous Na2CO3.13) These compounds (VIII—XVII) have been described in the cited references (synthetic methods and physical properties). 27-Norsolacongestidine (XIX) was synthesized with reference to the method which Schreiber and Adam applied to synthesize solaconges- tidine,20) as follows. An n-hexane solution of n-butyl lithium (15%) (0.64 ml, 1 mmol) was added to an anhydrous ether solution (4 ml) containing 2-bromopyridine 95 ill (1 mmol), and the mixture was stirred at -40 •Ž for 30 min. An anhydrous ether solution (10 ml) containing pregnenolone acetate (350 mg, 0.978 mmol) was added to the above solution over 30 min. Stirring was continued for another hour, the reaction temperature was allowed to rise to -20℃ ,and the mixture was stirred for a further 30 min. Then, the temperature was allowed to rise to 0 •Ž and ether

(20 ml) containing water (1 ml) was added. The ether layer was separated and the aqueous layer was extracted with ether twice. The combined ether solution was shaken with 3% HC1 three times. The acidic solution was alkalized with ammonia water to give a precipitate. After SiO2 chromatography of the products, 3ƒÀ-acetoxy-20-(pyridy1-2)-pregn-5- en-20-ol (26.5 mg, 6.2%) and 20-(pyridyl-2)-pregn-5-ene-3,20-diol (XVIII) (105.8 mg, 27.4%) were obtained. NMR

(CDC13) ppm of the acetate: 0.75 (3H, s, C18-3H), 1.04 (3H, s, C19-3H), 1.59 (3H, s, C21-3H), 2.02 (3H, s, OCOCH3), 4.34 (1H, m, C3-H), 5.34 (1H, m, C6-H), 7.00-7.04 (2H, m, 3'-H and 5'-H on the pyridyl ring), 7.66 (1H, m, 4'-H), 8.43 (1H, m, 6'-H). NMR (CDC13) ppm of diol: 0.94 (3H, s, C18-3H), 1.02 (3H, s, C19-3H), 1.59 (3H, s, C21-3H), 3.35

(1H, m, C3ƒ¿-H), 5.30 (1H, m, C6-H), 7.00-7.40 (2H, m, 3'-H and 5'-H on the pyridyl ring), 7.64 (1H, m, 4'-H), 8.42 (1H, m, 6'-H). After confirmation of the structure by nuclear magnetic resonance (NMR) spectroscopy, phosphorus oxychloride (0.5 ml) was added to pyridine (3.5 ml) containing the acetate (108.7 mg), with cooling on ice. The reaction temperature was increased to the reflux point for 2 h and then the solvent was evaporated off under reduced pressure. After alkalization of the residue with ammonia water, extraction with ether was carried out. The ether layer was washed with water and dried over Na2SO4. The products were subjected to Si02 chromatography and the expected dehydrate (43.5 mg, 41.7%) was eluted with n-hexane—CHC13 (2 : 1). Mass spectrum (MS) m/z: 420 (M +1), 405. IR (CHC13) cm-1: 1720 (OCOCH3), 1584, 1560, 1465, 1430 (pyridyl ring), 905 (•„C =CH2). NMR (CDC13) ppm: 0.59 (3H, s, C18-3H), 0.96 (3H, s, C19-3H), 2.00 (3H, s, OCOCH3), 4.35 (1H, m, C3ƒ¿-H), 5.2-5.6 (3H, m, C6-H, C2, -2H), 7.0-7.5 (2H, m, 3'-H and 5'-H on the pyridyl ring), 7.66 (1H, m, 4'-H), 8.63 (1H, m, 6'-H). Next, the dehydrate (104.4 mg, 0.25 mmol) was dissolved in glacial acetic acid (7 ml) and hydrogenated in the presence of Pt02

(20 mg) by stirring overnight. After alkalization of the reaction solution with ammonia water, it was extracted with ether. The ether layer was washed with saturated saline solution and dried over Na2SO4, followed by chromatog- raphy on Si02. Elution with CHC13—MeOH (10 : 1) provided 3/3-acetoxy-22,26-imino-27-nor-cholestane (53.4 mg, 50%). MS m/z: 429 (NV), 414, 84 (base peak). IR (CHC13) cm-1: 1720 (OCOCH3). NMR (CDC13) ppm: 0.64 (3H, s, C18-3H), 0.81 (3H, s, C19-3H), 0.91 (3H, d, J= 5.6 Hz, C21-3H), 2.00 (3H, s, OCOCH3), 2.55 (2H, m, C26-2H), 3.06

(1H, m, C22-H), 4.62 (1H, m, C3ƒ¿-H). Iminonorcholestanyl acetate (52.0 mg) was hydrolyzed by stirring with 2% methanolic KOH at room temperature for 2 h. After usual treatment, 22,26-imino-27-norcholestan-3ƒÀ-ol (48.9 mg) was obtained. MS m/z: 387

(M +), 84 (base peak). IR (CHC13) cm': 3600 (OH). NMR (CDC13) ppm: 0.64 (3H, s, C18-3H), 0.79 (3H, s, C19-3H), 0.92 (3H, d, J= 5.7 Hz, C21-3H), 2.56 (2H, m, C26-2H), 3.10 (1H, m, C22-H), 3.54 (1H, m, C3ƒ¿-H). The N-chloride

(47.2 mg) of the above imino alcohol was prepared by treatment of the alcohol (45.0 mg) with N-chlorosuccinimide in CH2C12 (6 ml) at -10•Ž. 27-Norsolacongestidine (XIX, 21.1 mg) was obtained by refluxing the chloride (42.2 mg) in 5% methanolic KOH for 2 h. MS m/z: 385 (M t), 370, 356, 111 (base peak). IR (CHC13) cm-1: 3625 (OH), 1650

(C =N). NMR (CDC13) cm": 0.68 (3H, s, C18-3H), 0.80 (3H, s, C19-3H), 1.06 (3H, d, J=6.7 Hz, C21-3H), 3.40 (1H, m, C3ƒ¿-H). The in vitro antifungal activities of these compounds (I—XIX) were assayed by the serial two-fold dilution method on Sabouraud glucose agar (1st screening test). The results were expressed as the values of minimum inhibitory concentration (MIC: pg/ml). The following fungi were used for the assay: Candida albicans (50157),

(55463), C. pseudotropicalis (56363), C. famata (50866), C. kefyr (59763), Cryptococcus albidus (C-3), C. neoformans (58063), Rhodotorula glutinis (59663), Trichosporon cutaneum (51271), Geotrichum candidum (30266), Aspergillus fumigatus (22167), A. candidus (21967), Fonsecaea pedrosoi (11758), Microsporum gypseum (11668), Phialophora verrucosa (24172), Sporothrix schenckii (30166), Trichophyton rubrum sc., and T. mentagrophytes sc. The mother fungi were inoculated on a Sabouraud agar slant and incubated at 27 •Ž for 2 d (yeast fungi) or for 7-14 d (thread fungi). Emulsion having 106 cells/ml of yeast fungi was prepared by adjusting the volume with saline solution containing 0.1% (w/v) Tween-80. Emulsion having 106 spores/ml was prepared as follows. The above saline solution (4 ml) was added to an incubated slant and the surface of the slant was scraped with a platinum loop. The 4864 Vol. 35 (1987)

solution containing the spores was filtered through two sheets of gauze and used to prepare the above emulsion. The emulsion (0.01 ml) was added to each well of a NUNCRON R 24-well plate (NUNC K.K.) containing 0.1 ml of two- fold dilutions of the test alkaloids and 0.9 ml of Sabouraud glucose agar. Incubation was carried out at 27 •Ž for 2 d

(C. albicans and Cryptococcus neoformans), for 5 d (Aspergillus fumigatus and Microsporum gypseum) or for 7 d (other fungi). . In the 2nd screening test to examine the effect of serum addition, MICs were assessed by using Sabouraud glucose agar containing 10% horse serum. The therapeutic effect of solacongestidine (I) was assayed in mice infected intravenously with C. albicans sc.

(mice-SPF-ddY type, 4 weeks old, male). The toxicity of I was determined on oral and intraperitoneal administration to mice.

Results

The antifungal activities of several solanum alkaloids against C. albicans and Trichophyton spp. are shown in Table I. Solacongestidine (I) showed the strongest inhibition of C. albicans and T. rubrum, followed by verazine (VII) and solafloridine (II). The MICs of solasodine (III), tomatidine (IV), tomatillidine (V) and dihydrosolasodine (VII) were more than 100 ƒÊg/m1 against four tested fungi. The inhibitory activities of solacongestidine and solafloridine were the same as in the 1st screening test in spite of the presence of 10% serum. Because many toxic compounds with substantial antifungal activity show marked loss of activity in the presence of serum, the results of the 2nd screening test with solacongestidine and solafloridine are of considerable interest. The spectrum of antifungal activities of solacongestidine against more than a dozen fungi is shown in Table II. It is interesting that solacongestidine inhibited Cryptococcus albidus, C. neoformans, C. famata and Trichosporon cutaneum, while it showed MIC values of more than 100 ƒÊg/m1 against C. tropicalis, C. collielosa, Geotrichum candidum, Aspergillus fumigatus, A. candidus, and so on. The results of the bioassay in vivo with solacongestidine are shown in Fig. 1. For

TABLE I. The Antifungal Activities (MIC,ƒÊg/ml) of Some Solanum Alkaloids against Candida albicans, Trichophyton rubrum, and T. mentagrophytes

TABLE II. The Antifungal Activities of Solacongestidine against Various Fungi No. 12 4865

Fig. 1. The Therapeutic Efficacy of Solacongestidine in Mice Infected with Candida albicans, as Compared with Ketoconazole Values are given in mg per kg body weight of mouse.

TABLE III. The Antifungal Activities (MIC, ƒÊg/m1) of Some Derivatives of Solanum Alkaloids against Candida albicans (C.a), Trichophyton rubrum (T.r),

and T. mentagrophytes (T.m)

comparison, ketoconazole was also examined. It was concluded that solacongestidine prolonged the survival time of mice infected with C. albicans, compared to the untreated mice. The toxicity of solacongestidine to mice was as follows: three mice out of 3 died when 300 mg/kg of solacongestidine was given orally, but all of three mice tested survived when given 300 mg/kg by i.p. administration. Dihydrosolasodine (VIII), its N-methyl derivative (IX), 3,16-0-diacetylpseudosolasodine (X), 16-0-acetylpseudosolasodine (XI), 3,16-0-diacetylpseudotomatidine (XII), 16-0-acetyl- 4866 Vol. 35 (1987)

pseudotomatidine (XIII), a mixture (XIV) of glycosides of solacongestidine and solafloridine, dehydrosolacongestidine (XV), dihydrosolacongestidine (XVI), its quaternary salt (XVII), 20-

(pyridy1-2)-pregn-5-ene-3ƒÀ,20-diol (XVIII) and 27-norsolacongestidine (XIX) were examined for growth inhibition of C. albicans, Trichophyton mentagrophytes, and T. rubrum. N- Methyldihydrosolasodine (VIII) showed on MIC of 25 ƒÊg/ml but the other compounds had MIC values of more than 100 ƒÊg/ml. It is interesting that 27-norsolacongestidine did not show substantial antifungal activity.

Discussion

Solacongestidine (I), a steroidal alkaloid obtained from Solanum congestiflorum, showed strong inhibition of the growth of C. albicans, T. rubrum and C. albidus. The MICs were 0.4- 1 ƒÊg/ml, and the activity was not decreased in the presence of serum. Because the in- traperitoneal administration of 300 mg/kg of solacongestidine (I) did not kill any of three mice, the toxicity of I was thought not to be so high. Thus, solacongestidine was chosen as a target of research aimed at the development of antifungal drugs. As a next step, various solanum alkaloids and related compounds were examined to look for more prospective. compounds, although no compound more active than solacongestidine

(I) has been found so far. A number of azasteroids and steroidal alkaloids with substantial antimicrobial activity have been reported. The following compounds have been reported to show MIC values of 1-50 ƒÊg/ml: 2-methy1-2-aza-5a-cholestane," 3-methy1-3-aza-5ƒ¿-cholestane," 1 ',4',5',6'- tetrahydropyridino[a-4,3]-4-aza-5-cholestene,2) 2 ',3'-dihydropyrimidazolino[a-4,3]-4-aza-5- cholestene,2) hexahydropyrimido[a-4,3]-4-aza-5-cholestene,2) tetrahydroimidazolino[a-4,3]-4- aza-5-cholestene,2) androstane-17-oxime O-dimethylaminoethyl derivatives,3) 16ƒÀ-amino-17- hydroxy-20-ketopregnene derivatives,4) 17ƒÀ-amino-3,5-androstadiene,5) 17ƒÀ-amino-5-andro- stene,5) 4-aza-22-oxa-5ƒ¿-cholestane,6) 4-methyl-4-aza-22-oxa-5ƒ¿-cholestane,6) 17ƒÀ-iso- pentyloxy-4-aza-5ƒ¿-androstane,7) 17ƒÊ-isopentyloxy-4-methy1-4-aza-5ƒ¿-androstane,7) 4,17ƒ¿- dimethy1-4-aza-5ƒ¿-androstan-17ƒÊ-ol acetate,8) and tomatidine (22 ƒÊg/ml gave 100% inhibi- tion against Polyporus versicolor, and 45-50% inhibition against Pyricularia oryzae and Rhizoctonia solani) It is noteworthy that 15-aza-24-methylene-D-homocholesta-8,14-dien-3fl- ol almost completely inhibited the growth of Ustilago maydis at 0.25 pg/ml.10) Although useful antifungal drugs have not been established in the categories of azasteroids and steroidal alkaloids, our results and the reported data seem to suggest that effective compounds may be discovered or created. It is interesting that a minor change in the side chain moiety can decrease the activity. For example, solacongestidine (a C 25R methyl group) showed strong inhibition, while verazine (a C25s methyl group) showed lower activity and 27-norsolacongestidine showed much lower activity ( •„ 100 ƒÊg/ml). It seems important that solacongestidine showed strong inhibition against C. albicans, T. rubrum and C. albidus, while it showed lower inhibition against C. pseudotropicalis (MIC 6.25 ƒÊg/ml), T. mentag- rophytes (1.5), C. neoformans (1.56), C. famata (3.13), and T. cutaneum (3.13). The MICs against several other fungi such as A. fumigatus and C. tropicalis were more than 100 ƒÊg/ml. It appears that the antifungal activity of solacongestidine (I) may be specific to certain genera of fungi. Our research will be continued with the aim of finding prospective compounds among azasteroids and steroidal alkaloids that show specific inhibition against C. albicans, taking account of the results of the report that steroidal alkaloids bearing a basic nitrogen atom in ring F, shared or unshared with ring E, with bonding capabilities a to the steroid plane may be teratogenic.11)

Acknowledgement Our thanks are due to Sankyo Co. for the biological assays. No. 12 4867

References and Notes

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