2300 Vol. 35 (1987)

Chem. Pharm. Bull. 35( 6 )2300-2304(1987),

Occurrence of Marinobufotoxin and Telocinobufotoxin Homologs in the Skin of bankorensis BORBOuR1)

KAZUTAKESHIMADA, YOSHIHIRO SATO, and TOSHIONAMBARA*

PharmaceuticalInstitute, Tohoku University, Aobayama, Sendai 980, Japan

(ReceivedNovember 13, 1986) The occurrenceof marinobufagin3-succinyl-L-arginine and 3-glutaryl-L-arginineesters, and telocinobufagin3-glutaryl-L-arginine ester, together with seven known bufogenins, in the skinof Bufo bankorensisBORBOUR, is reported. The structureswere elucidatedby degradativemeans and/ordirect comparisonwith authenticsamples. These compounds were assayed for inhibitory activitytowards guinea pig Na+, K+-adenosine triphosphatase. Keywords-Bufo bankorensis;toad ; bufogenin;marinobufotoxin homolog; telo- cinobufotoxinhomolog; sodium-potassium-activated ATPase inhibition

In a series of studies on toad venom,2) we characterized the cardiac in the skin of Formosan toad, Bufo bankorensis BORBOUR, and assayed them for Na +-adenosine triphosphatase (Na+, K +-ATPase; EC 3.6.1.3) inhibitory activity.3) Five toads were sacrificed by freezing in dry ice, and the skins were immediately flayed off and extracted with ethanol. The ethanolic extract was concentrated in vacuo and the residue was subjected to column chromatography on silica gel. Subsequent separation of the bufogenin and mixture was repeatedly done by high-performance liquid chromat- ography (HPLC) on a reversed-phase column. Three new (5a, 5b, 10) were isolated as colorless amorphous substances, which gave negative ninhydrin and positive Sakaguchi tests. Upon hydrolysis with 6 N hydrochloric acid, arginine was produced and identified by thin-layer chromatography (TLC). When subjected to enzymic hydrolysis with a hog pancreas lipase preparation followed by methylation with diazomethane, these bufotoxins afforded bufogenin 3-hemi- carboxylate methyl esters (3a, 3b, 8). Compounds 3a, 3b gave mass spectral (MS) data (m/z 382, 364, 213) ascribable to the marinobufagin residue. Pairs of fragment ions (3a: m/z 133, 115; 3b: m/z 147, 129) suggested the dicarboxylic acid moieties to be succinic acid and glutaric acid monomethyl esters, respectively.4) The MS of 8 also showed characteristic fragment ions (m/z 384, 366, 323, 147, 129) due to telocinobufagin and a glutaric acid monomethyl ester residue.4,5) The absolute configuration of the residue was elucidated to be L as judged from the substrate specificity of the enzyme used.2) These data prompted us to synthesize marinobufagin 3-succinyl-L-arginine and 3- glutaryl-L-arginine esters (5a, 5b), and telocinobufagin 3-glutaryl-L-arginine ester (10) as authentic samples. Marinobufagin (1) and telocinobufagin (6) were treated with the approp- riate dicarboxylic acid anhydride in the presence of 4-(dimethylamino)pyridine6) to give marinobufagin 3-hemisuccinate (2a) and 3-hemiglutarate (2b), and telocinobufagin 3-hemi- glutarate (7) in satisfactory yields, respectively. Methylation with diazomethane afforded the methyl esters, which showed the same chromatographic behaviors (HPLC and TLC) as the compounds derived from the natural source (3a, 3b, 8). Treatment of bufogenin 3- hemicarboxylates (2a, 2b, 7) with p-nitrophenol and N,N'-dicyclohexylcarbodiimide provided the p-nitrophenyl esters (4a, 4b, 9), which in turn were condensed with L-arginine to give the No. 6 2301

1: R=H 6: R= H 2: R=CO(CH2),,COOH 7: R = CO(CH2)3COOH 3: R CO(CH2),,COOCH3 8: R=CO(CH2)3COOCH3

4: R=CO(CH2)C00•¬NO2 9: R=CO(CH2)3C00•¬NO2

5: R=CO(CH2)CONFIFI(CH2)3NHNH2 10: R=CO(CH2)3CONHH(CH2)3NHNH2

COOH NH COOH NH

a: n=2, b: n=3, c: n=4

Chart 1

TABLE I. Inhibition of Na+,K+-ATPase by Marinobnfotoxin and Telocinobufotoxin Homologs

desired bufogenin 3-hemicarboxylate-L-arginine esters. The chromatographic behaviors of these synthetic specimens were identical with those of the natural products in HPLC on various reversed-phase columns with different solvent systems. These results support the structures marinobufagin 3-succinyl-L-arginine and 3-glutaryl-L-arginine esters, and telocino- 3-glutaryl-L-arginine ester for 5a, 5b and 10, respectively (Chart 1). Bufalin, resibufogenin, , , telocinobufagin, marinobufagin, and gamabufotalin were also isolated and characterized by direct comparison with authentic samples. Lin et al.7) identified resibufogenin, bufotalin, hellebrigenol, and dehydrobufotonin from Formosan "Ch'an Su ," a mixture of from Bufo bufo asiaticus STEINDACHNERand Bufo bankorensis BORBOUR.No further investigation on the venom of the latter toad has been carried out. Recently, we disclosed the existence of 3-glutaryl-L-arginine ester together with other bufotoxin homologs in Korean toad venom.8 It should be noted that bufotoxin homologs having glutaric acid as a dicarboxylic acid moiety commonly exist in toad venom. 2302 Vol. 35 (1987)

In order to clarify the structure—activity relationship, the naturally occurring bufotoxin homologs and synthetic marinobufagin 3-adipoyl-L-arginine ester (5c) were tested for inhibitory activity towards Na±,K+-ATPase. The molar concentrations of these compounds giving half-maximal inhibition (I50) of Na+,1( +-ATPase from guinea pig heart are listed in Table I. Among the marinobufotoxin homologs, the suberoyl-L-arginine ester91 and succinyl-

L-arginine ester exhibited the highest and lowest potencies, respectively. This result is consistent with that obtained for gamabufotalitoxin and cinobufotoxin homologs.3)

Further studies on cardiac steroids in toad venoms are being conducted in these laboratories, and the details will be reported elsewhere.

Experimental

All melting points were taken on a micro hot-stage apparatus and are uncorrected. Optical rotations were measured with a JASCO DIP-4 automatic polarimeter. MS measurements were run on a Hitachi M-52 spectrometer. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded using tetramethylsilane as an internal standard on a JEOL FX-90A spectrometer at 90 MHz. Abbreviations: s= singlet, d = doublet, dd = doublet of doublets, and m = multiplet. Silica gel HF254 and Silica gel 60 (70-230 mesh) (E. Merck AG, Darmstadt) were used for preparative TLC and column chromatography, respectively. A hog pancreas lipase preparation and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) and Nakarai Chemicals Ltd. (Kyoto), respectively. HPLC was carried out on a Toyo Soda 803A chromatograph equipped with an ultraviolet detector (280 nm) at a flow rate of 1 ml/min, unless otherwise stated. Extraction of Steroidal Components Five toads (Bufo bankorensis BORBOUR) obtained from Vivarium Co.

(Tokyo) were sacrificed by freezing in dry ice. The skins were immediately flayed off and extracted with EtOH (200 ml) for 6 months. After removal of insoluble materials by filtration through a Celite layer, the filtrate was concentrated in vacuo below 50 •Ž to give a brown oily residue (116 mg). Isolation of Bufogenins and Bufotoxins The residue obtained above was chromatographed on Silica gel 60

(15 cm x 2 cm i.d.) employing hexane-AcOEt (1 : 1) and AcOEt as eluents. Further purification of the dried eluate by HPLC on a Develosil ODS-5 column (Nomura Chem. Co., Seto; 5 ,um, 15 cm x 0.4 cm i.d.) with CH3CN-F120 (5 : 7, 1 : 2, and 4 : 9) as eluents gave the following bufogenins: bufalin (1 mg), resibufogenin (4 mg), arenobufagin (1 mg), bufotalin (1 mg), telocinobufagin (2 mg), marinobufagin (2 mg), and gamabufotalin (1 mg). These bufogenins were identified by direct comparison with authentic samples (MS, 1H-NMR spectra and chromatographic behavior4.5'")). Further chromatography on silica gel with AcOEt-MeOH (1 : 1) as described above gave a crude bufotoxin mixture, which in turn was subjected to further separation by HPLC on a LiChrosorb RP-18 column (E. Merck AG; 5pm, 15 cm x 0.4 cm i.d.) with CH3CN-0.5% (NH4)2CO3 (2 : 7) to give two new bufotoxins (tR 5a, 7.0 min, 1 mg; tR 5b, 10.0 min, 1 mg). Separation on a Develosil ODS-5 column (CH3CN-H20 (4 : 11)) also gave a new bufotoxin (tR 10, 5.8 min, 1 mg). Structure Elucidation of New Bufotoxins All new bufotoxins showed negative ninhydrin and positive Sakaguchi tests. Each new bufotoxin (ca. 100 jig) was heated with 6 N HCI (0.2 ml) for 8 h. A portion of the resulting solution was subjected to two-dimensional TLC on Silica gel G (E. Merck AG) using CHC13-Me0H-17% NH4OH

(2 : 2 : 1) and phenol-H2O (3 : 1) as developing solvents, and arginine was detected by means of the ninhydrin test. Each new bufotoxin (5a, 5b, 10, ca. 100 jig) obtained from the natural source was dissolved in 1% NaCI-MeOH (9 : 1)

(0.5 ml) and incubated with a hog pancreas lipase preparation (ca. 500 fig) at 37C for 2 h. The incubation mixture was extracted with AcOEt, and the extract was washed with H20 and then evaporated down in vacua. The residue was dissolved in Me0H (0.05 ml) and treated with ethereal CH2N2. After usual work-up, the crude product obtained was subjected to preparative TLC using benzene-AcOEt (1 : 1) as a developing solvent. The adsorbents correspond- ing to the spots of Rf 0.52 (3a), 0.61 (3b) and 0.22 (8) were scraped off and extracted with AcOEt. The products were identical with the methyl esters of synthetic marinobufagin 3-hemisuccinate (3a) and 3-hemiglutarate (3b), and telocinobufagin 3-hemiglutarate (8) with respect to MS and chromatographic behavior. HPLC: LiChrosorb RP-18, CH3CN-H20 (1 : 1, tR 3a, 4.7 min; 3b, 5.9 min; 8, 4.1 min), Me0H-H20 (2 : 1; tR 3a, 4.7 min; 3b, 5.6 min; 8, 4.4 min). Develosil ODS-5, CH3CN-H20 (1 : 1, tR 8, 5.0 min), Me0H-H20 (2 : 1; tR 8, 6.3 min). TLC: benzene-AcOEt (1 : 1: RI 3a, 0.52; 3b, 0.61; 8, 0.22). New bufotoxins (5a, 5b, 10) obtained from the natural source showed the same chromatographic behavior as the synthetic samples. HPLC: LiChrosorb RP-18, CH3CN-0.5% (NH4)2CO3 (2: 7; 1R 5a, 7.8 min; 5b, 10.7 min) (4 : 15; tR 10, 14.5 min). Me0H-0.5% (NH4)2CO3 (1 : 1; tR 5a, 4.5 min; 5b, 5.5 min) (4 : 5; tR 10, 7.7 min). Develosil ODS-5, CH3CN-0.5% (NH4)2CO3 (2 : 5; 1, 5a, 4.0 min; 5b, 5.1 min) (4 : 15; 1, 10, 17.5 min). Me0H-0.5% (NH4)2CO3 (5 : 4; tR 5a, 4.2 min; 5b, 5.0 min) (4 : 5, tR 10, 14.2 min). TSKgel ODS-80TM (Toyo Soda

Co., 5 pm, 15 cm x 0.4 cm i.d.), CH3CN-0.5% (NH4)2CO3 (1 : 3; tR 5a, 13.5 min; 5b, 18.0 min). Me0H-0 .5% (NH4)2CO3 (1 : 1; 0.8 ml/min; tR 5a, 10.0 min; 5b, 12.2 min). Procedure for the Preparation of Bufotoxin Homologs Each bufogenin (1, 6; 30 mg) was refluxed with succinic No. 6 2303

anhydride, glutaric anhydride, or adipic anhydride (17 mg) and 4-(dimethylamino)pyridine (56 mg) in pyridine (2 ml) for 5 h. An additional amount of dicarboxylic acid anhydride (17 mg) was added to the mixture every 1 h. After extraction with AcOEt, the organic layer was washed with 5% HC1, H20 and dried over anhydrous Na2SO4. After evaporation of the solvent, a portion (1 mg) of the residue (30 mg) was redissolved in Me0H (0.1 ml) and treated with CH2N2 in the usual manner. The crude product was purified by preparative TLC using benzene-AcOEt (1 : 1) as a developing solvent. The adsorbents corresponding to the spots of Rf 0.52 (3a), 0.61 (3b), 0.63 (3c), and 0.22 (8) were extracted with AcOEt. The dried extracts showed the following MS data. 3a: m/z 514, 382, 364, 213, 133, 115. 3b: m/z 528, 382, 364, 213, 147, 129. 3c: m/z 542, 382, 364, 213, 161, 143.8: m/z 530,. 384, 366, 323, 147, 129. A solution of each bufogenin 3-hemicarboxylate (30 mg) in AcOEt (2 ml) was treated with p-nitrophenol (30 mg) and N,N'- dicyclohexylcarbodiimide (50 mg), and the reaction mixture was allowed to stand at room temperature for 6 h. After removal of the precipitate by filtration, the filtrate was evaporated down in vacuo and the residue was subjected to preparative TLC. The adsorbents corresponding to the spots of Rf 0.51 (4a: benzene-AcOEt (1 : 1)), 0.61 (4b: benzene-AcOEt (1 : 1)), 0.41 (4c: benzene-AcOEt (2 : 1)) and 0.55 (8: benzene-AcOEt (1 : 4)) were scraped off and extracted with AcOEt. The dried extracts showed the following 1H-NMR spectral data in CDC13. 4a: 6 0.78 (3H, s, 18-CH3), 1.00 (3H, s, 19-CH3), 2.85 (4H, m, CO(CH2)2C0), 3.51 (1H, s, 15a-H), 5.30 (1H, br s, W1,2=7 Hz, 3a-H),

6.25 (1H, d, J=9.8 Hz, 23-H), 7.22 (1H, d, J=2.5 Hz, 21-H), 7.30 (2H, d, J=9.6 Hz, •¬NO2), 7.78 (1H, dd,

J=9.8, 2.5 Hz, 22-H), 8.28 (2H, d, J=9.6 Hz, •¬NO2). 4b: 6 0.78 (3H, s, 18-CH3), 1.00 (3H, s, 19-CH3), 3.53 (1H,

s, 15a-H), 5.29 (1H, br s, W112= 7 Hz, 3a-H), 6.25 (1H, d, J=9.8 Hz, 23-H), 7.22 (1H, d, J=2.5 Hz, 21-H), 7.30 (2H, d,

J=9.6 Hz, •¬NO2), 7.78 (1H, dd, J=9.8, 2.5 Hz, 22-H), 8.28 (2H, d, J=9.6 Hz, •¬NO2).0 4c: 6 0.78 (3H, s,

18-CH3), 0.99 (3H, s, 19-CH3), 3.51 (1H, s, 15a-H), 5.28 (1H, brs, W112= 7 Hz, 3a-H), 6.24 (1H, d, J=9.8 Hz, 23-H),

7.21 (1H, d, J=2.5 Hz, 21-H), 7.29 (2H, d, J=9.6 Hz, •¬NO2), 7.78 (1H, dd, J=9.8, 2.5 Hz, 22-H), 8.27 (2H, d,

J=9.6 Hz, •¬NO2). 8: 6 0.74 (3H, s, 18-CH3), 0.98 (3H, s, 19-CH3), 5.26 (1H, br s, W112 = 8 Hz, 3a-H), 6.22 (1H,

d, J=9.8 Hz, 23-H), 7.18 (1H, d, J=2.6 Hz, 21-H), 7.27 (2H, d, J=9.5 Hz, •¬NO2),-0 7.76 (1H, dd, J=9.8, 2.6 Hz,

22-H), 8.20 (2H, d, J=9.5 Hz, •¬NO2). L-Arginine (20 mg) in H2O (1 ml) was added to a solution of each active

ester (20 mg) in pyridine (1 ml), and the reaction mixture was allowed to stand at room temperature for 12 h. The resulting solution was diluted with H20 (100 ml) and percolated through a column packed with Amberlite XAD-4 resin (18 cm x 1.1 cm i.d.). After washing of the column with water (400 ml), the desired compound was eluted with

Me0H (100 ml) and dried. The residue obtained was subjected to column chromatography (15cm x 0.6 cm i.d.) on silica gel with CHC13-Me0H-H20 (80 : 20 : 2.5). The dried eluate was recrystallized from Me0H-ether to give the corresponding bufotoxin homolog as a white amorphous substance: 5a (24 mg). mp 195-200 •Ž (dec.). [a]i; + 24.0•‹

(c= 0.13, Me0H). Anal. Calcd for C34H48N409. 1/2H20: C, 59.72; H, 7.52; N, 8.19. Found: C, 59.55; H, 7.70; N, 7.73. 4-1-NMR (CDC13-CD3OD) 6: 0.80 (3H, s, 18-CH3), 1.00 (3H, s, 19-CH3), 2.60 (4H, br s, W112= 7.2 Hz, CO(CH2)2C0), 3.55 (1H, s, 15a-H), 5.19 (1H, br s, /2 = 8 Hz, 3a-H), 6.25 (1H, d, J=9.8 Hz, 23-H), 7.28 (1H, d, J=2.5 Hz, 21-H), 7.82 (1H, dd, J = 9 .8, 2.5 Hz, 22-H). 5b (20 mg). mp 183-190 •Ž (dec.). [c]2 + 15.1•‹ (c=0.13, Me0H). Anal. Calcd for C35HSON409. H20: C, 61.03; H, 7.61; N, 8.13. Found: C, 60.75; H, 7.69; N, 7.86. 1H-NMR

(CDC13-CD3OD) 6: 0.76 (3H, s, 18-CH3), 1.00 (3H, s, l9-CH3), 3.54 (1H, s, 15a-H), 5.19 (1H, br s, W112 = 8 Hz, 3a- H), 6.26 (1.H, d, J=9.8 Hz, 23-H), 7.27 (1H, d, J=2.5 Hz, 21-H), 7.78 (1H, dd, J=9.8, 2.5 Hz, 22-H). Sc (20 mg). mp 183-186 •Ž (dec.). [G13.3 + 36.0•‹ (c = 0.13, Me0H). Anal. Calcd for C361152N409. 3/2H20: C, 60.74; H, 7.79; N, 7.87. Found: C, 60.97; H, 7.73; N, 7.59. 1H-NMR (CDC13-CD30D) 6: 0.79 (3H, s, 18-CH3), 1.00 (3H, s, 19-CH3), 3.57

(1H, s, 15a-H), 5.23 (1H, br s, /2 = 8 Hz, 3a-H), 6.27 (1H, d, J=9.8 Hz, 23-H), 7.30 (1H, d, J=2.5 Hz, 21-H), 7.87. (1H, dd, J =9 .8, 2.5 Hz, 22-H). 10 (21 mg). mp 180-185 °C (dec.). [a]l; + 15.4° (c = 0.13, Me0H). Anal. Calcd for C35H52N409.2H20: C, 59.30; H, 7.96; N, 7.90. Found: C, 59.19; H, 7.56; N, 7.65. 'H-NMR (CDC13-CD3OD) 6: 0.75 (3H, s, 18-CH3), 0.97 (3H, s, 19-CH3), 5.22 (1H, br s, W112 = 8 Hz, 3a-H), 6.30 (1H, d, J=9.8 Hz, 23-H), 7.34 (1H, d, J=2.6 Hz, 21-H), 7.97 (1H, dd, J=9.8, 2.6 Hz, 22-H). Assay for the Inhibition of Na+,K +-ATPase The samples were tested for inhibitory activity towards Na + ,K +-

ATPase (10 iumol Pi/mg ) from guinea pig heart by the method described in the previous paper.3) The concentration of a compound required for 50% inhibition was defined as the I50 value.

Acknowledgement The authors thank the staff of the central analytical laboratory of this institute for elemental analyses and spectral measurements. This work was supported in part by a grant from the Ministry of Education, Science and Culture. 2304 Vol. 35 (1987)

References and Notes

1) Part CCXXX of "Studies on Steroids," by T. Nambara; Part CCXXIX: K. Shimada, F. Xie, T. Niwa, T. Wakasawa, and T. Nambara, J. Chromatogr., in press. In this paper marinobufagin-3-O-ylsuccinyl-L-arginine was named marinobufagin 3-succinyl-L-arginine ester as previously reported. Other related compounds were also similarly designated. 2) K. Shimada, N. Ishii, and T. Nambara, Chem. Pharm. Bull., 34, 3454 (1986) and references cited therein. 3) K. Shimada, K. Ohishi, H. Fukunaga, J. S. Ro, and T. Nambara, J. Pharmacobio-Dyn., 8, 1054 (1985). 4) P. Brown, Y. Kamano, and G. R. Pettit, Org. Mass Spectrom., 6, 613 (1972). 5) P. Brown, Y. Kamano, and G. R. Pettit, Org. Mass Spectrom., 6, 47 (1972). 6) G. Hofle and W. Steglich, Synthesis, 1972, 619. 7) C.-N. Lin, M.-I. Chung, E.-C. Wang, I.-J. Chen, S.-C. Cheng, M. Arisawa, M. Shimizu, and N. Morita, Shoyakugaku Zasshi, 38, 175 (1984). 8) K. Shimada, J. S. Ro, K. Ohishi, and T. Nambara, Chem. Pharm. Bull., 33, 2767 (1985). 9) K. Shimada and T. Nambara, Chem. Pharm. Bull., 27, 1881 (1979). 10) K. Shimada, M. Hasegawa, K. Hasebe, Y. Fujii, and T. Nambara, Chem. Pharm. Bull., 24, 2995 (1976). 11) The Li, value of this compound reported in the previous paper2) should be corrected.