approximately 50% after 3 h; in contrast, Ac-Ala-Aib-OMe was only slightly attacked even after 6 h. The trichotoxin A-40 utilized appeared to be completely homogeneous in ten TLC systems; it also showed a largely integral amino acid ratio. According to the behavior of its cleavage products on thin layer chromatography, there were, however, a total of three N-acetylated dodecapeptides (I, 11, 111) with analogous sequences and two hexapeptides (IV and V) both beginning with proline. These five peptides were separated by silica gel column Chromatography and by multiplicative countercurrent distribution. The sequences of the peptides (Fig. 1) were determined by GLC/MS analyses of the trifluoroacetylated and esterified partial hydrolyzates. The molecular weight of the hexapeptides was obtained by field desorption mass spectrometry. The a-linkage of the valinol was demonstrated by comparison of the mass spectroscopy fragmentation patterns with those of synthetic L-Gln-valinol and L-Glu(valinol)NH,. In these experiments we observed an unusual quantitative a/y transpeptidation. The methods used provided sufficient quantities of defined partial sequences for "C-NMR investigations and activity studies. According to CD measurements, the helical part of trichotoxin is located, similarly to aIamethicin[lo1,in the N-terminal dodecapeptide. The purification, cleavage, and sequencing methods can be applied to all peptide antibiotics (alamethicins, suzukacillins, etc.) which contain Aib. Received March 12, 1979 [Z 226 IE] German version: Angew. Chem. 91. 508 (1979) CAS Registry numbers: Peptide 1. 70320-19-3; peptide 11. 70320-20-6; peptide 111, 70320-21-7; peptide IV. 70320-22-8: peptide V, 70320-23-9 trichotoxin A-40, 65216-21-9 [ I ] A . 1. Mueller, D. 0. Rudin, Nature 217, 713 (1968). [2] G. Boherm. K. Janko, D. Lerhfritz, T. Ooka. W. A. Konig, G. Jung, Biochim. Biophys. Acta 433. 182 ( 1 976). [3] G. Boherm, G. Irmscher, G. Jung, Biochim. Biophys. Acta 507, 485 (1978), and references cited therein. (41 a) 3. W Payne, R. Jakes, B. S. Harfley, Biochem. J. 117, 757 (1970); b) Yu. A. Otvhinnikov, A. A. Kiryushkin, I. V. Kozhevnikova, Zh. Obshch. Khim. 41. 2085 (1971); c) R. C. Pandey, 3. C. Cook, K. L. Rinehart. J. Am. Chem. Soc. YY. 8469 (1977); d) D. R. Martin, R. J. P. Williams, Biochem. J. 153, 181 ( 1976). [ 5 ] C. Jung, W A. Konig, D. Leibfrrtz, T. Ooka, K. Janko, G. Boheim, Biochim. Biophys. Acta 433, 164 (1975). (61 G. Irmscher, G. Bovermann, G. Boheim, C. Jung, Biochim. Biophys. Acta 507. 470 (1978). [7J G. Jung. H . Bruckner, B. Oertel, Abstr. Symp. on Chemistry of Peptides and Proteins. Grainau 1978, p. 165. [81 a) K. Kruse. W. Francke, W.A. Konig, J. Chromatogr. 170,423 (1979); b) W. A. Konrg. W. Rahn, J. Eyem, ibid. 133, 141 (1977). [9J S. Yamada, K . Achiwa, Chem. Pharm. Bull. 12, 1525 (1964). (101 G. Jung, N. Dubischar, D. Leihfrirz, Eur. J. Biochem. 54, 395 (1975).
known antitumor agentl2] cis-diamminedichloroplatinum(~1) (2). Further chemical analogies between (1) and (2) include,
for example, the ability to undergo hydrolytic dimerization via O X O - [ ~ ~or hydroxo-bridge~~~] and the selective reaction with aqueous ammonium polysulfide leading to the pentasulfide chelates (C,H,),TiS,IS1 and (NH4)2[Pt(S,),][61,respectively. To test the antitumor activity of (1) we have so far carried out a series of five trial experiments on female CF, mice. About 6 x lo6 Ehrlich ascites tumor cells['] were implanted in each mouse by intraperitoneal injection. 24 hours after transplantation (a. t.) the mice were treated (likewise intraperitoneally) with varying amounts of (1) or (2) in 0.4 ml so h tion (dimethyl sulfoxide and saline 1: 9 (v/v)). In one representative experiment 84 mice were collected into groups of six animals and treated with (1) in doses varying between 10 and 140 mg/kg. Figure 1 shows the number of deaths caused by tumors and by toxicity as well as the proportion of surviving, apparently cured animals, up to day 30 a.t. Twelve animals served as untreated controls (0.4 ml DMSO-NaCl solution; exitus resulting from tumor growth on average 14.25 days a. t.). A further six animals were used as positive controls (10 mg/kg (2); no macroscopically recognizable tumor development up to day 30 a. t.). In other series of tests, mice treated with (I) have survived for ten weeks without any sign of tumor development.
0
Titanocene Dichloride-The First Metallocene with Cancerostatic Activity'"] By Hartmut Kopf and Petra Kopf-Maier"] Dichlorobis(q5-cyclopentadienyl)titanium(~v)(titanocene dichloride) (I) was first reported some 25 years ago1'],yet its cytostatic activity has remained uninvestigated. We expected ( I ) to possibly show such an activity, since it contains a cisdichlorometal moiety in the neutral complex, like the well[*] Prof. Dr. H. Kopf Institut fur Anorganische und Analytische Chemie der Technischen Universitai Strasse des 17. Juni 135, D-1000 Berlin 12 (Germany) Dr. P. Kopf-Maier Anatomisches lnstitut der Freien Universitat Konigin-Luise-Strasse 15, D-1000 Berlin 33 (Germany)
[**I
This work was supported by the Fonds der Chemischen Industrie
Anyew Chrm. Int. Ed. Engl. 18 (1979) No. 6
0 Verlag Chemie, GmbH, 6940
121
Ill
20
-
LO 60 80 Dose [mglkgl
100
,
120
1
-
i
140
Fig. 1. Survival of mice 30 days after tumor transplantation and 29 days after treatment with ( I ) at the doses given on the abscissa. E4 Tumor deaths without signs of substance toxicity. Deaths caused by toxicity (deaths within 8 days a. I. without macroscopically visible tumor development). 0 Animals surviving on the 30th day a.t. (therapeutic range).
According to these results (1) is an effective antitumor agent with a cure rate in the therapeutic range of over 80%. (I) is the first titanium complex and the first metallocene derivative exhibiting cytostatic properties-presumably without the heavy-metal toxicity observed in the case of (2). Received: February 14. 1979 [Z 227 IE] German version: Angew. Chem. 91, 509 (1979) CAS Registry numbers: ( I ) , 1271-1 9-8; (2),15663-27-1 Weinheim, 1979
05 70-0833/ 79/0606-0477 $02.50/0
417
0
[ I ] C Wilkinron, J. M. Birmingham, J. Am. Chem. Soc. 76. 4281 (1954). [2] B. Rosenberg, L. VanCump, T. Krigu.r, Nature 205. 698 (1965): review- F. K. V. Leh. W. Wo/J J . Pharm. Sci. 65. 315 (1976) [3] Li. Thewulr. C Sch/eu.ww,Angew. Chem. 90,559 (197X): Angew. Chem. Int Ed. Engl. 17, 531 (1978). [4] R. Fuggiani, B. Lrpperr. C. J. L. Lock. B. Rosenberg, J. Am. Chem. Soc 9Y. 777 (1977). [ S ] H. KopJ B. Block. Chem. Ber. 102. 1504 (1969). 161 H KopJ I. Sasmifu- Wiramihardja. R. Goidmann, unpublished work; cf R. Goldmann, Dissertation. Technische Universitat Berlin 1978. [7] We thank Prof. Dr. E. Liss, Klinikum Steglitz der Freien Universitat Berlin. for the tumor straiii.
Thermal Generation of Pteridines and Flavines from Amino Acid Mixtures By Bettina Heinz, Walter Ried, and Klaus Dose['] Dedicated to Professor Hermann Hartmann on the occasion of his 65th birthday The thermal treatment of amino-acid mixtures['] leads to formation of high-molecular products and to some hitherto unidentified heterocyclic impurities. In addition, a vivid blue fluorescence has frequently been observed[lbl. In order to elucidate the structures of the heterocycles we heated equimolar mixtures of three of the following six amino acids: glycine, alanine, valine, lysine, aspartic acid, and glutamic acid in an oil-bath (4-6 h, 160-200 "C) or metal bath (1-2 h, 180-350°C) (cf. ["I). The UV absorption spectra of the blue to green fluorescing material (A,,,, ca. 250, 320-390, 450 nm) and the fluorescence spectra (A,,, 365 nm, A,, 420-480 nm), as well as the chromatographic analysis (cellulose, silica gel, aluminum oxide, Sephadex G 15 and Zipax ion-exchanger for HPLC) of the material isolated by dialysis and fractional extraction, indicated the presence of pteridine derivatives. Supporting evidence for the presence of such derivatives was also provided by chemical reactions (murexide reaction, coupling to diazotized sulfinic acid, color reaction with polytungstatophosphoric acid).
0
With the help of mass spectrometry we were able to detect definite pteridines; molecular ions and characteristic fragments of hydrogenated oxopteridines and aminooxopteridines (e.g. lumazine, xanthopterin, and isoxanthopterin) could be identified. In the case of sample 1, a strong molecular peak at m / e = 241 and a weaker peak at m / e = 256 suggest the fragmentation pattern of a lumichrome derivative (1) (see Table 1). For comparison, we used spectral data from the lite r a t ~ r e [ *and . ~ ] spectra of authentic samples.
M , 181
H
H
152
H
H
H
154
I
H
H 168
J
Table 1. Results of the thermolysis experiments. The starting material was fused in an oil bath (180°C. 6 h: sample 3 under N2).Compounds (I), ( 6 ) . and (7) were isolated by gel filtration (Sephadex G-15). ~~
~
Workup
Mass spectra (70 eV, 200°C)
Cornp ou n d isolated (Mol. wt. osmometric)
LYs ASP G ~ Y
Melt taken up in water. solution filtered. filtrate dialyzed, contents of tube combined and freeze dried. Crude product taken up in little water, chromatographed on cellulose column, eluate concentrated. freeze dried, dried in vacuo over PIO, at 60°C
(I)
2
Lys ASP G~Y
Melt taken up in water, solution filtered, residue hydrolyzed for 5 h with 6 N NaOH; hydrolyrate purified by chromatography on cellulose column. eluate concentrated. freeze dried, dried m i'acuo over PzO, at 60 "C
3
Lys Ala G~Y
Workup as for sample I
[a] m/e=268 (2%). 256 (4j, 242 (IS). 241 (70). 227 (5). 199 (10). 171 (52) 170 (23). 156 (32). 147 (18). 146 (18). 145 (15), 144 (14). 143 (14). 142 (23). 129 (24). 126 (18). I22 (72), 117 (23). 99 (15). 85 (8). 82 (8). 74 (8). 60 (661, 45 (52). 44 (100). 43 (100) [b] m/e= I81 (lo'%), 180 (16), 179 (15) 152 (43), 138 (22). 96 (45). 69 (27). 55 (42) [c]m / e = 168 (24%). 167 (30) 154 (20). 153 (23) 152 (42) 151 (23). 124 (13). 123 (14). 96 (45). 69 (28). 44 (100). 28 (50j, 27 (37) [d] m/e= 181 (8%). 152 (26), 138 (15). 96 (42). 69 (30). 55 (47)
Sample
Starting material (1 : 1 : I )
~~
~~
1
276
(6) 634 (?)
(7J
[a] Given in part only. [b] Isolated fragmentation series of dihydroxanthopterin (2) (or dihydroisoxanthopterin). [c] Isolated fragmentation series of the partially hydrogenated oxopteridines /3)-(S) (cf. [Z]). [d] Isolated fragmentation series of dihydroxanthopterin (2) (or dihydroisoxanthopterin): cf. sample 2. [*] Prof. Dr. W. Ried [ '1,
Dipl.-Chem. B. Heinz Institut fur Organische Chemie der Univerdtat, Lahoratorium Niederrad Theodor-Stern-Kai 7, D-6000 Frankfurt/Main 70 (Germany) Prof. Dr. K. Dose instilut fur Biochemie der Univerritkt Becherweg 30 0-6500 Main;. (Germany)
[ * ] Author to whom correspondence should be addressed
478 4791482-Anzeigen
0 Verlag Chemie, G m b H , 6940 Wtwikeini, 1979
__ -
Further characterization of the samples was provided by alkaline hydrolysis and subsequent gel filtration of the hydrolyzate on a Sephadex-G15 column with distilled water. The elution pattern at 280 nm showed essentially two peaks; the second peak consisted of substances which gave elution volumes and fluorescence colors comparable with those giv0570-083?/7'1/0600-0478 $ 02 50/0
Angeu Chem I n ! Ed Eiiyl 18 ( 1 979) V o 6
known antitumor agentl2] cis-diamminedichloroplatinum(~1) (2). Further chemical analogies between (1) and (2) include,
for example, the ability to undergo hydrolytic dimerization via O X O - [ ~ ~or hydroxo-bridge~~~] and the selective reaction with aqueous ammonium polysulfide leading to the pentasulfide chelates (C,H,),TiS,IS1 and (NH4)2[Pt(S,),][61,respectively. To test the antitumor activity of (1) we have so far carried out a series of five trial experiments on female CF, mice. About 6 x lo6 Ehrlich ascites tumor cells['] were implanted in each mouse by intraperitoneal injection. 24 hours after transplantation (a. t.) the mice were treated (likewise intraperitoneally) with varying amounts of (1) or (2) in 0.4 ml so h tion (dimethyl sulfoxide and saline 1: 9 (v/v)). In one representative experiment 84 mice were collected into groups of six animals and treated with (1) in doses varying between 10 and 140 mg/kg. Figure 1 shows the number of deaths caused by tumors and by toxicity as well as the proportion of surviving, apparently cured animals, up to day 30 a.t. Twelve animals served as untreated controls (0.4 ml DMSO-NaCl solution; exitus resulting from tumor growth on average 14.25 days a. t.). A further six animals were used as positive controls (10 mg/kg (2); no macroscopically recognizable tumor development up to day 30 a. t.). In other series of tests, mice treated with (I) have survived for ten weeks without any sign of tumor development.
0
Titanocene Dichloride-The First Metallocene with Cancerostatic Activity'"] By Hartmut Kopf and Petra Kopf-Maier"] Dichlorobis(q5-cyclopentadienyl)titanium(~v)(titanocene dichloride) (I) was first reported some 25 years ago1'],yet its cytostatic activity has remained uninvestigated. We expected ( I ) to possibly show such an activity, since it contains a cisdichlorometal moiety in the neutral complex, like the well[*] Prof. Dr. H. Kopf Institut fur Anorganische und Analytische Chemie der Technischen Universitai Strasse des 17. Juni 135, D-1000 Berlin 12 (Germany) Dr. P. Kopf-Maier Anatomisches lnstitut der Freien Universitat Konigin-Luise-Strasse 15, D-1000 Berlin 33 (Germany)
[**I
This work was supported by the Fonds der Chemischen Industrie
Anyew Chrm. Int. Ed. Engl. 18 (1979) No. 6
0 Verlag Chemie, GmbH, 6940
121
Ill
20
-
LO 60 80 Dose [mglkgl
100
,
120
1
-
i
140
Fig. 1. Survival of mice 30 days after tumor transplantation and 29 days after treatment with ( I ) at the doses given on the abscissa. E4 Tumor deaths without signs of substance toxicity. Deaths caused by toxicity (deaths within 8 days a. I. without macroscopically visible tumor development). 0 Animals surviving on the 30th day a.t. (therapeutic range).
According to these results (1) is an effective antitumor agent with a cure rate in the therapeutic range of over 80%. (I) is the first titanium complex and the first metallocene derivative exhibiting cytostatic properties-presumably without the heavy-metal toxicity observed in the case of (2). Received: February 14. 1979 [Z 227 IE] German version: Angew. Chem. 91, 509 (1979) CAS Registry numbers: ( I ) , 1271-1 9-8; (2),15663-27-1 Weinheim, 1979
05 70-0833/ 79/0606-0477 $02.50/0
417
0
[ I ] C Wilkinron, J. M. Birmingham, J. Am. Chem. Soc. 76. 4281 (1954). [2] B. Rosenberg, L. VanCump, T. Krigu.r, Nature 205. 698 (1965): review- F. K. V. Leh. W. Wo/J J . Pharm. Sci. 65. 315 (1976) [3] Li. Thewulr. C Sch/eu.ww,Angew. Chem. 90,559 (197X): Angew. Chem. Int Ed. Engl. 17, 531 (1978). [4] R. Fuggiani, B. Lrpperr. C. J. L. Lock. B. Rosenberg, J. Am. Chem. Soc 9Y. 777 (1977). [ S ] H. KopJ B. Block. Chem. Ber. 102. 1504 (1969). 161 H KopJ I. Sasmifu- Wiramihardja. R. Goidmann, unpublished work; cf R. Goldmann, Dissertation. Technische Universitat Berlin 1978. [7] We thank Prof. Dr. E. Liss, Klinikum Steglitz der Freien Universitat Berlin. for the tumor straiii.
Thermal Generation of Pteridines and Flavines from Amino Acid Mixtures By Bettina Heinz, Walter Ried, and Klaus Dose['] Dedicated to Professor Hermann Hartmann on the occasion of his 65th birthday The thermal treatment of amino-acid mixtures['] leads to formation of high-molecular products and to some hitherto unidentified heterocyclic impurities. In addition, a vivid blue fluorescence has frequently been observed[lbl. In order to elucidate the structures of the heterocycles we heated equimolar mixtures of three of the following six amino acids: glycine, alanine, valine, lysine, aspartic acid, and glutamic acid in an oil-bath (4-6 h, 160-200 "C) or metal bath (1-2 h, 180-350°C) (cf. ["I). The UV absorption spectra of the blue to green fluorescing material (A,,,, ca. 250, 320-390, 450 nm) and the fluorescence spectra (A,,, 365 nm, A,, 420-480 nm), as well as the chromatographic analysis (cellulose, silica gel, aluminum oxide, Sephadex G 15 and Zipax ion-exchanger for HPLC) of the material isolated by dialysis and fractional extraction, indicated the presence of pteridine derivatives. Supporting evidence for the presence of such derivatives was also provided by chemical reactions (murexide reaction, coupling to diazotized sulfinic acid, color reaction with polytungstatophosphoric acid).
0
With the help of mass spectrometry we were able to detect definite pteridines; molecular ions and characteristic fragments of hydrogenated oxopteridines and aminooxopteridines (e.g. lumazine, xanthopterin, and isoxanthopterin) could be identified. In the case of sample 1, a strong molecular peak at m / e = 241 and a weaker peak at m / e = 256 suggest the fragmentation pattern of a lumichrome derivative (1) (see Table 1). For comparison, we used spectral data from the lite r a t ~ r e [ *and . ~ ] spectra of authentic samples.
M , 181
H
H
152
H
H
H
154
I
H
H 168
J
Table 1. Results of the thermolysis experiments. The starting material was fused in an oil bath (180°C. 6 h: sample 3 under N2).Compounds (I), ( 6 ) . and (7) were isolated by gel filtration (Sephadex G-15). ~~
~
Workup
Mass spectra (70 eV, 200°C)
Cornp ou n d isolated (Mol. wt. osmometric)
LYs ASP G ~ Y
Melt taken up in water. solution filtered. filtrate dialyzed, contents of tube combined and freeze dried. Crude product taken up in little water, chromatographed on cellulose column, eluate concentrated. freeze dried, dried in vacuo over PIO, at 60°C
(I)
2
Lys ASP G~Y
Melt taken up in water, solution filtered, residue hydrolyzed for 5 h with 6 N NaOH; hydrolyrate purified by chromatography on cellulose column. eluate concentrated. freeze dried, dried m i'acuo over PzO, at 60 "C
3
Lys Ala G~Y
Workup as for sample I
[a] m/e=268 (2%). 256 (4j, 242 (IS). 241 (70). 227 (5). 199 (10). 171 (52) 170 (23). 156 (32). 147 (18). 146 (18). 145 (15), 144 (14). 143 (14). 142 (23). 129 (24). 126 (18). I22 (72), 117 (23). 99 (15). 85 (8). 82 (8). 74 (8). 60 (661, 45 (52). 44 (100). 43 (100) [b] m/e= I81 (lo'%), 180 (16), 179 (15) 152 (43), 138 (22). 96 (45). 69 (27). 55 (42) [c]m / e = 168 (24%). 167 (30) 154 (20). 153 (23) 152 (42) 151 (23). 124 (13). 123 (14). 96 (45). 69 (28). 44 (100). 28 (50j, 27 (37) [d] m/e= 181 (8%). 152 (26), 138 (15). 96 (42). 69 (30). 55 (47)
Sample
Starting material (1 : 1 : I )
~~
~~
1
276
(6) 634 (?)
(7J
[a] Given in part only. [b] Isolated fragmentation series of dihydroxanthopterin (2) (or dihydroisoxanthopterin). [c] Isolated fragmentation series of the partially hydrogenated oxopteridines /3)-(S) (cf. [Z]). [d] Isolated fragmentation series of dihydroxanthopterin (2) (or dihydroisoxanthopterin): cf. sample 2. [*] Prof. Dr. W. Ried [ '1,
Dipl.-Chem. B. Heinz Institut fur Organische Chemie der Univerdtat, Lahoratorium Niederrad Theodor-Stern-Kai 7, D-6000 Frankfurt/Main 70 (Germany) Prof. Dr. K. Dose instilut fur Biochemie der Univerritkt Becherweg 30 0-6500 Main;. (Germany)
[ * ] Author to whom correspondence should be addressed
478 4791482-Anzeigen
0 Verlag Chemie, G m b H , 6940 Wtwikeini, 1979
__ -
Further characterization of the samples was provided by alkaline hydrolysis and subsequent gel filtration of the hydrolyzate on a Sephadex-G15 column with distilled water. The elution pattern at 280 nm showed essentially two peaks; the second peak consisted of substances which gave elution volumes and fluorescence colors comparable with those giv0570-083?/7'1/0600-0478 $ 02 50/0
Angeu Chem I n ! Ed Eiiyl 18 ( 1 979) V o 6
