SITE-SPECIFIC RUTHENIUM(II) AND COBALT(III) ANTITUMOR AGENTS

Abstract: A coordination complex of the formula (R)2---M---(Y)2 wherein M comprises a suitable transition metal, e.g. cobalt or ruthenium, R comprises 1,10-phenanthroline or a substituted derivative thereof, Y comprises a labile ligand, e.g. chloride, tartrate, malonate or ascorbate ion and R and Y are bonded to M by coordination bonds. A complex of this invention may be used for covalently labeling DNA with a complex of the formula (R)2---M, wherein R and M are as previously defined. A complex of this invention which contains cobalt may also be used in a method for nicking DNA by effecting single-stranded scission of at least one phosphodiester bond of the DNA with ultraviolet radiation. A complex of this invention is further useful in a method for killing tumor cells. A pharmaceutical conposition for the treatment of tumor cells in a subject may be prepared containing an effective anti-tumor amount of a complex of this invention and a pharmaceutically acceptable carrier. Such a composition may be used for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells.

Description: SITE-SPECIFIC RUTHENIUM (II) AND CQBAT.Tfτm ANTTTΠMOR AGENTS The invention described herein was made with government support under grant number GM 33309 from the National Institutes of Health, United States Department of Health and Human Services. The government has certain rights in this invention. ga ggQtnd of the Invention. Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Recently there has been increased attention focused on the binding of metal complexes to nucleic acids and nucleic acid constituents (1) . This interest stems in large part from the successful application of cis-di- chlorodiammineplatinum(II) (cis-DDP or cisplatin) as an antitumor drug (2) . See also, U.S. Patent Nos. 4,273,755 (1981); 4,302,446 (1981); 4,310,515 (1982); 4,339,437 (1982) and 4,451,447 (1984). More recently chiral transition metal complexes have been utilized in designing specific probes for nucleic acid struc¬ ture. The tris(phenanthroline) complexes of zinc(II) (3) and ruthenium(ll) (4) display enantiomeric selec¬ tivity in binding to DNA by intercalation. Because of their high specificity in intercalative binding to right- or left-handed DNAs, enantiomers of tris(4,7- diphenylphenanthroline) ruthenium(II) and cobalt(III) provide respectively spectroscopic probes *(5) and cleaving agents (6) that are DNA conformation-specific. Such complexes bind to DNA only under suitable intercalating conditions, and do not bind to DNA in a covalent fashion. It has now been discovered that certain bis-substituted metal complexes of phenanthrolines are capable of bind- ing covalently and stereospecifically to DNAs. Such complexes are useful in stereospecific labeling and cleavage of DNAs and are further useful as antitumor agents. Summary of the Invention This invention involves a coordination complex of the formula (R) 2 M (Y) wherein R comprises 1,10- phenanthroline or a substituted derivative thereof; M comprises a suitable transition metal, e.g. ruthenium or cobalt; Y comprises a labile ligand, e.g. chloride, tartrate, malonate or ascorbate ion; and R and Y are bonded to M by coordination bonds. 10 This invention also concerns a method for covalently labeling DNA with a complex of the formula (R) 2 M, where R and M are as " defined above. This method in¬ volves contacting the DNA with a complex of this inven- 15 tion under suitable binding conditions such that com¬ plex covalently binds to 'the DNA. This invention further concerns a labeled DNA molecule comprising DNA to which a complex of the formula 20 (R) 2 M is covalently bound wherein R comprises 1,10- phenanthroline or a substituted derivative thereof, M comprises a transition metal, e..g. ruthenium or cobalt, and R is bonded to M by a coordination bond. _? _ This invention further concerns a method for nicking DNA by effecting single-stranded scission, i.e. break¬ age of at least one of the phosphodiester bonds along the DNA. This method involves contacting the DNA with a cobalt-containing complex of this invention under 30 suitable binding conditions such that the complex covalently binds to the DNA to form an adduct and irra¬ diating the adduct so formed with a sufficient dose of ultraviolet radiation of an appropriate wavelength to nick the DNA. An appropriate wavelength for the ultra¬ 35 violet radiation of this method is a wavelength of ultraviolet radiation absorbed by the ligand bands of the complex used. Another embodiment of this invention is a method for killing a portion of a population of appropriate tumor cells. This method involves contacting the tumor cells under suitable conditions with an effective amount of a coordination complex of this invention to kill the tumor cells. Where the tumor cells are present in a subject, e.g. a human or animal, the contacting may suitably be effected by administering the coordination complex to the subject. Where the complex used in this embodiment is a cobalt-containing complex the method may further involve irradiating the tumor cells with a suitable dose of ultraviolet radiation of an appropri¬ ate wavelength at a suitable time after the tumor cells have been contacted with the complex, permitting the complex to nick DNA. This invention further involves a pharmaceutical compo¬ sition for the treatment of tumor cells in a subject which comprises an effective antitumor dose of a com¬ plex of this invention and a pharmaceutically accepta¬ ble carrier. This invention additionally concerns a method for treating a subject, e.g. a human or animal, afflicted with tumor cells so as to cause regression of the tumor cells. This method involves administering to the sub¬ ject by a suitable ' route a pharmaceutical composition of this, invention in an amount sufficient to cause regression of the tumor cells. Administration may be parenteral or may be topical. Furthermore as in previ¬ ous embodiments, where the complex is a cobalt-con¬ taining complex the method may further involve irradi- ating the tumor cells with a suitable dose of ultra¬ violet radiation of an appropriate wavelength. In this method the tumor cells may be irradiated at a suitable time after administration of the pharmaceuti¬ cal composition to the subject permitting the complex to nick DNA. Brief Description of the Figures Figure 1. Plot of (1,10-phenanthroline) 2 RuCl 2 binding to calf thymus DNA as a function of time; r is the ratio of bound ruthenium to nucleotide concentrations. Figure 2. Circular dichroism of the supernatant after ethanol precipitation of the ruthenium complex bound to B-DNA. Binding to B-DNA is stereoselective and leads to enrichment of the supernatant in the unbound delta isomer (inset) . Detailed Description of the Invention The present invention involves a coordination complex of the formula (R) 2 Co(III) (Y) 2 wherein R compris¬ es 1,10-phenanthroline or a substituted derivative thereof; Y comprises a labile ligand, e.g. chloride, tartrate, malonate or ascorbate ion, and R and Y are bonded to the Co(III) by coordination bonds. A "substituted derivative" as the phrase is used herein is a compound obtained by replacing one or more hydrogen atoms present in 1,10-phenanthroline with one or more moieties having the characteristic that the complex containing the resulting compound binds to DNA. Merely by way of example, the substituted derivative of 1,10-phenanthroline may be 4,7-diamino-l,10- phenanthroline; 3,8-diamino-l,10-phenanthroline; 4,7- diethylenediamine-1,10-phenanthroline; 3 ,8-diethylene- diamine-l,10-phenanthroline; 4,7-dihydroxyl-l,10-phen- anthroline; 3,8-dihydroxyl-l,10-phenanthroline; 4,7-di- nitro-l,10-phenanthroline; 3,8-dinitro-l,10-phenanthro- line; 4,7-diphenyl-l,10-phenanthroline; 3 ,8-diphenyl- 1,10-phenanthroline; 4,7-dispermine-l,10-phenanthro- line or 3,8-dispermine-l,10-phenanthroline. Unless otherwise specified, the complex of this invention is a racemic mixture of enantiomers. Several such complexes including bis(l,10-phenanthro- line)ruthenium(II) dichloride, bis(4,7-diphenyl-l,10- phenanthroline)-ruthenium(II) dichloride, and bis(4,7- diphenyl-l,10-phenanthroline)cobalt(III) tartrate have been prepared and their DNA-binding properties studied. Each of these neutral species is chiral, octahedral, and contains two inert ligands (the diamines) and two labile ligands in a _£is.-orientation. One embodiment involves a method for covalently label¬ ing DNA with a complex of the formula (R) 2 M where R is as defined above, M in this and other embodiments of the invention is a suitable transition metal, i.e. a transition metal capable of forming an octahedral com¬ plex with 1,10-phenanthroline or a substituted deriva¬ tive thereof and R is bound to M by a coordination bond. Presently preferred transition metals are ruthe- nium(II) and cobalt(III). According to this method the DNA is contacted with a complex of the formula (R) 2 M (Y) 2 ' where R, M and Y are as defined above, the contacting being under suitable conditions such that the (R) 2 M complex covalently bonds to the DNA. " The invention also concerns a labeled DNA molecule comprising DNA to which a complex of the formula (R) M as defined previously, is covalently bound. Preferably, the labeled DNA is produced by the method described above. A further embodiment of this invention concerns a meth¬ od for nicking DNA by effecting single-stranded scis¬ sion, i.e. breakage, of at least one phosphodiester bond along the DNA. This method involves contacting the DNA with a cobalt(III)-containing complex of the formula (R) 2 Co(III) (Y) 2 . as previously defined, the contacted being under suitable conditions such that the (R) 2 Co(III) complex covalently binds to the DNA to form an adduct. The adduct so formed is then irradiated with a sufficient dose of ultraviolet radia¬ tion of an appropriate wavelength to nick the DNA. In this and other embodiments an appropriate wavelength is a wavelength of ultraviolet radiation which is absorbed by the ligand bands of the complex used. Still another embodiment of this invention is a method for killing a portion of a population of appropriate tumor cells. The method involves contacting the tumor cells under suitable conditions with an effective a- mount of a coordination complex of the formula (R) 2 (Y) , as previously defined, to kill the tumor cells. In a presently preferred embodiment, the suitable transition metal, M, is cobalt(lll). if the tumor cells are present in a subject, e.g. a human or animal, the tumor cells may be contacted with the coor¬ dination complex by administering the complex to the subject. When a cobalt(III)-containing complex is used, the method may further involve irradiating the tumor cells with a suitable dose of ultraviolet radia¬ tion, as previously defined, . at a suitable time after the tumor cells have been contacted with the complex, permitting the complex to nick DNA. This invention further concerns a pharmaceutical compo¬ sition for the treatment of tumor cells in a subject. The composition comprises an effective anti-tumor a- mount of a complex of the formula (R) 2 M (Y) ' as defined above, and a pharmaceutically acceptable carri¬ er. Preferably, the suitable transition metal, M, is ruthenium(II) or cobalt(III), with cobalt(III) being especially preferred. Suitable carriers include ster¬ ile saline or buffer-containing solutions or other carriers known in the art such as those used with cis- platin. Still another embodiment of this invention is a method for treating a subject, e.g. a human or animal, af¬ flicted with tumor cells so as to cause regression of the tumor cells. The method involves administering to the subject by a suitable route a pharmaceutical compo- sition as described above in an amount sufficient to cause regression of the tumor cells. Suitable routes of administration include parenteral administration and topical administration, e.g. in cases such as skin cancers where the tumor cells are located on or near an exposed surface of the subject. Furthermore, if the complex used is a cobalt(III) -containing complex, the method may additionally involve irradiating the tumor cells with a suitable dose of ultraviolet radiation of an " appropriate wavelength permitting the complex to nick DNA. In this method the irradiation should be conducted at a suitable time after administration of the composition to the subject, i.e. to permit the complex to interact with the DNA. Experimental Details Unlike the corresponding tris-substituted complexes, the bis-analogues of this invention are not coordi- natively saturated. The cis-oriented chlorides are good leaving groups, permitting DNA base substitution at those positions. Indeed the aqueous chemistry of ruthenium(II) , and bis(l,10-phenanthroline)dichlororu- thenium(II) [also referred to as (phen) 2 RuCl 2 ] in par- ticular, resembles reactions of platinu (II) . The complex (phen) 2 R U ci 2 binds covalently to DNA. The neutral ruthenium complex moreover shows similarities to the anticancer drug cis-dichlorodiammineplatinum(II) (cis-DDP) in its binding characteristics, with respect to rates of reaction, DNA conformational changes, and the preferential binding to guanine sites. The ruthe¬ nium complex offers two potentially interesting advan¬ tages. First, the enantiomers show chiral selectivity. The complexes represent covalent-binding analogues to the chiral tris-phenanthroline cations. One enantio- mer binds preferentially to right-handed B-DNA. The other enantiomer binds to left-handed DNA preferential¬ ly and even converts sequences from the B- to Z-form. Secondly the organic ligand framework for these octahe- dral complexes permits additional specificity to be built in. Thus the stereochemistry in these ruthenium complexes provides a basis for the design of site-spe¬ cific covalent binding drugs. One illustrative embodiment, namely (phen) 2 Ruci , binds covalently to the DNA duplex and exhibits striking enantiomeric selectivity, different from that observed in the intercalation of corresponding M(phen) 3 complex¬ es with DNA. In one experiment, race ic (phen) 2 RuCl 2 ( 7 ) ( 50 ^M) was incubated in " buffer containing 10% ethanol, 50 mM NaN03- 5 mM Tris at pH 7.1, either at ambient tempera¬ tures or 37°C for variable amounts of time with calf thymus DNA (500 μM nucleotide) (8). Immediately after the incubation period, NaCl and 95% ethanol were added to quench the reaction and precipitate the DNA. Un¬ bound ruthenium, more soluble in the ethanol supernatant, remained in solution. After centrifuga- tion, the supernatant was assayed spectrophotometri- cally, compared to controls lacking ruthenium or DNA, and levels of bound and free metal complex were deter¬ mined. This experiment measured only covalent binding to the DNA. The procedure was repeated using the coordinatively saturated tris(phenanthroline) t ruthenium cation, (phen)3 u^ + , which binds to DNA by intercalation (4) . Under these assay conditions no binding to DNA was observed. A plot of the extent of coordination to DNA by the (phen) Ru ^ + cation as a function of time is shown in Figure 1. A maximum binding ratio of Θ.045, or one (phen) 2 Ru ^ + moiety for every 11 base pairs, is obtained at about 3 1/2 hours. This dependence on time likely reflects both the kinetics of hydrolysis of (phen) 2 RuCl 2 and ligand substitution (9), e.g. the association of the metal complex with the DNA. Significant enantiomeric discrimination accompanies this covalent binding. The circular dichrois of the supernatant, the unbound fraction, is shown in Figure 2. The solution is appreciably enriched in the less favored isomer. Optically enriched (phen) 2 RuCl 2 solu ¬ tions have not been obtained previously using more conventional methods. While the rate of racemization of the complexes of this invention is slow in the buff¬ er system used, significant solvent dependence in the racemization rate has been observed. These observa¬ tions support similar observations previously reported (10) . The magnitude of the rotation in the ultravio¬ let region is approximately 5 times larger than that seen earlier for (phen^Ru 2 * solutions at comparable levels of intercalative binding. Hence, the degree of chiral selectivity for this covalent adduct appears substantially greater than for (phen) 3 Ru 2+ . Based upon exciton theory (11), it was expected that the rotational " strength of pure enantiomers of (phen) 2 RuCl 2 n the vicinity of the ligand absorption would be one half that of (phen) 3 R U C1 2 . Since pure enantiomers of (phen) 2 RuCl 2 have not been isolated, the relative ratio of affinities of the two. enantiomers has not yet been determined. The absolute configuration of the isomer preferred has, however, been assigned. Based upon simple exciton theory (11) and the identical circular dichroism (CD) in the ultraviolet region to that for (phen) 3 Ru 2+ (12), the CD given in Figure 2 has been assigned to the delta isomer. In contrast to the binding specificity seen with (phen) β Ru * - * " , it is lamb- da-(phen) 2 RU 2+ that binds preferentially to B-DNA. The enantiomeric discrimination of the bis (phenanthro¬ line)ruthenium complex in binding to B-DNA must there¬ fore differ from tris (phenanthroline) cation not only in degree but also in the structural basis for the stereoselectivity. Ruthenium(II) complexes have a high affinity for the heterocyclic bases of DNA (14) . A likely site of metallation would be the N-7 atom of guanine, which is readily accessible in the major groove of the DNA .duplex. Initial intercalation is probable; immediate hypochromic changes in the rutheni- um charge transfer band are evident upon the addition of DNA. However further spectroscopic changes become evident on a time scale comparable to the binding given in Figure 1 and these changes must reflect covalent binding to the helix. From an initially intercalated position, the lambda isomer is well oriented for cova¬ lent binding to base positions above and below. Model building shows that the delta isomer cannot be similar¬ ly aligned for covalent binding, since the other non- stacked phenanthroline ligand is considerably crowded by the right-handed helical column (base and sugar- phosphate groups) . This bifunctional coordination oriented by initial intercalation could account for the high stereoselectivity observed. It is interesting that in the case of intercalation by (phen^Ru , the delta isomer, which has the same helical screw sense as the right-handed B-DNA, is preferred, while here metal- lation of base positions seems to require the lambda configuration, that is a structure complementary to the B-DNA helix. Recently the photoactivated stereospecific cleavage of DNA by chiral tris-substituted phenanthroline complexes of Cobalt(III), e.g. the tris (4,7-diphenyl-l,10- phenanthroline) or "DIP" cobalt complexes, has been reported (6) . The corresponding chiral bis enantiomers, e.g. bis(DIP)Co(III) tartrate, which have DNA binding properties analogous to those of the cor¬ responding bis-ruthenium complexes have now been found to cleave DNA photochemically and at sites different from (DIP) Co +* ^ . Bis (DIP)Co(III) for example may cleave DNA specifically at homopurine sites upon ultra¬ violet irradiation. The stereoselective covalent binding to DNA of (phen) 2 RUC1 2 , substituted analogs thereof, e.g. the bis-DIP complex, and the corresponding cobalt ana¬ logues likely has significant biological consequences. The neutral (phen) 2 RuCl 2 , for example, may be consid¬ ered an octahedral analogue for cis-Pt(NH 3 ) 2 C1 2 (14) . Results from laboratories in Australia more than ten years ago which indicated antibacterial, virostatic, and antileukemic activity __. vitro of tris unsubstitut- ed phenanthroline complexes of ruthenium(ll) (15) ; recent reports of antitumor activities and toxicities of various ruthenium complexes (1,15) , the possible similarities between Ru(phen) 2 Cl 2 and cis-DDP in inter¬ actions with DNA; and the striking stereoselectivity observed with the complexes of this invention all sup¬ port .potential chemotherapeutic application of these chiral complexes. Complexes of this invention have in fact been screened with respect to cytotoxicity, and the results presented in Table I show the complexes to be highly potent in yitro. Additionally, the cobalt complexes of this invention may exhibit photochemical activation with ultraviolet irradiation. These compounds are potentially very effective anti¬ tumor drugs. The advantages such compounds provide over cis-platin include lower heavy-metal toxicity, greater selectivity owing to stereochemistry, greater site specificity given the organic ligands (not present in cis-platin) and the possibility of .linkage to monoclonal antibodies, and easier and less expensive preparation. Furthermore the cobalt (III) nicking ac¬ tivity may permit localization by photolysis in vivo. Table I Cytotoxicity Results of Cobalt and Ruthenium Complexes Compound Cell Line a ID 50 (μg/ml) b Ru(phen) 2 ci 2 L1210 4.7 P815 7.0 Ru(DIP) 2 Cl 2 LI210 3.2 P815 3.5 Co(DIP) 2 Cl 2 LI210 0.44 P815 0.48 a LI210 and P815 are mouse leukemia cell lines D determined by the method of Burchenal, J.H. et al., CANCER RESEARCH £2.:2598-2600 (1982) Experimental Method and Materials Phenanthroline CQn.pls_.e_ Racemic (phen) 2 Ru(ll)Cl 2 was prepared as follows: To a solution of 3mmoles RuCl 3 * 3H 2 0 in 30 ml dimethylfor- mamide was added 6 mmoles phenanthroline monohydrate. The solution was allowed to reflux for 3h during which time the solution turned a deep violet in color. After being reduced in volume to about 20 ml, the solution was cooled at 0°C and a deep black solid as crude prod¬ uct was obtained. The product was recrystallized twice from 100 ml 50% ethanol saturated with lithium chlo¬ ride. Racemic mixtures of other complexes of this invention were prepared by analogous methods substitut¬ ing the appropriate 1,10-phenanthroline compound for 1,10-phenanthroline. See also (7); 1,10- phenanthroline, .4,7-diphenyl-l,10-phenanthroline, and other chemical reagents were obtained from Aldrich Chemical Co., St. Louis, Missouri. Spectroscopic data for Ru(phen) 2 ci 2 in ETOH was as follows: ε = 1.08xl0 4 M "1 cm "1 at 496 nm; e = 7.25xl0 4 M~ 1 cm~ 1 at 267 nm. In aqueous solution the complex may be consid¬ ered a mixture of hydrolyzed species. Bis (4,7-diphenyl-l,10-phenanthroline)cobalt(III) chlo¬ ride, ( DIP) 2 co(lll)Cl 2 , was prepared as follows: 4,7- diphenyl-l,10-phenanthroline (Aldrich) was dissolved in a minimum volume of ethanol to which one half stoichio- metric CoCl 2 *6H 2 0 was added. The green-brown solution was oxidized by using Br 2 /H 2 o, and a heavy orange pre¬ cipitate formed immediately. The solution was refluxed for lh, and concentrated hydrochloride was added. The bromine oxidation was then repeated. The crude complex was recrystallized in aqueous ethanol. Other cobalt (III) complexes of this invention may be prepared by this method by substituting the appropriate ligand for 4,7-diphenyl-l,10-ρhenanthroline. Buffers and DNA Calf thymus DNA was obtained from Sigma Chemical Compa- ny, St. Louis, Missouri and purified by phenol extrac¬ tion using previously described methods (8)-. DNA con¬ centrations per nucleotide were determined spectropho- tometrically by assuming ε 2 g 0 6000M~ 1 cm "1 (18) . Buffers were also obtained from Sigma. DNA cleavage The cobalt complex is added to the DNA sample in a solution buffered to about 7.1 e.g., in buffer contain¬ ing 10% ethanol, 50 mM NaN0 3 , 5 mM Tris at pH 7.1, either at ambient temperature or 37°C. The solution is then irradiated' at 315 nm with a 1000 W Hg/xenon lamp (narrowed to 315+5 nm with a monochrometer) for about 90 seconds to about 1 hour and the precipitate washed with ethanol. In vitro screening For cell culture studies, a modification of the tech¬ nique of Fischer (4) was used. The cells were, incubat¬ ed in McCoy's Medium 5A with 15% fetal calf serum. The initial inoculum was 40,000 to 60,000 leukemic cells/ml. For studies of the inhibition of cell growth, 0.1 ml of a 20-fold concentration of the drug in question was added to 2 ml of media containing 4 X 10 4 cells/ml in Linbro tissue culture multiwell plates and allowed to incubate at 37° in 5% CO, for 96 hours. By this time, growth to approximately 10° cells/ml occurred in the control wells. The contents of each well were agitated to resuspend the cells and counted on a Coulter Counter. The percentage of inhibition of growth and the concentrations inhibiting cell growth by 50% were calculated. Cell culture experiments were done with mouse leukemia cell lines L1210 and P815 which may be obtained from the American Type Culture Collection (ATCC) , Rockville, Maryland. References 1. (a) Lippard, S.J., Ed. "Platinum, Gold, and Other Metal Chemotherapeutic Agents," A.C.S. Syrapos. Ser. No. 209; American Chemical Society, Washington 1983; (b) Barton, J.K. and Lippard, S.J., Met. Ions Biol. , 1980, 1:31; (c) Marzilli, L.G. ; Kistenmacher, T.J.; Eichhorn, G.L., Met. Ions Biol., 1980, 1:179; (d) Mar¬ tin, R.B. and Mariam, Y.H. , Met. Ions. Biol. Syst., 1979, J£:57; (e) Marzilli, L.G., Prog. Inorg. Chem. , 1977, 11:255; (f) Hodgson, D.J. , Prog. Inorg. Chem., 1977, 21:211. 2. Rosenberg, B.; Van Camp, L.; Trosko, J.E.; Mansour, V.H.; Nature (London), 1969, 222:385; (b) Rosenberg, B.; Van Camp, L., Cancer Res., 1970, 1Q.:1979; (c) for a review, see Hill, J.M.; Loeb, E.; MacLellan, A.; Hill, N.V. ; Khan, A.; King, J.J.; Cancer Chemother. Rep., 1975, 5_S.:647; (d) Lippard, S.J., Acct. Chem. Res., 1978, 11:211; (e) Hacker, M.P.; Doyle, E.B.; Krakoff, I.H., Eds. "Platinum Coordination Com¬ plexes in Cancer Chemotherapy"; Martinus Nijhoff: Boston 1984. 3. Barton, J.K.; Dannenberg, J.J.; Raphael, A.L., J. Amer. Chem. Soc. , 1982, 10i.:4967. 4. Barton, J.K.; Danishefsky, A.T.; Goldberg, J.M. , J. Airier. Chem. Soc, 1984, 106:2172. 5. Barton, J.K.; Basile, L.A.; Danishefsky, A.; Alex- andrescu. A.; Proc. Natl. Acad. Sci. USA, 1984, 8.1:1961. 6. Barton, J.K. and Raphael, A.L., J. Amer. Chem. Soc, 1984, 106:2466. 7. See Sullivan, B.P.; Salmon, D.J.; Meyer, T.J.; Inorg. Chem., 1978, 17:3334. 8. Barton, J.K. and Lippard, S.J., Biochemistry, 1979 12:2661. 9. Isied, S. S. and .Taube, H. , Inorg. Chem., 1976, 15:3070. 10. Arce Segues, J.A.; Gillard, R.D.; Smalley, D.H.; Williams, P.A.; Inorg. Chim. Acta, 1980, 41:211. 11. Bosnich, B., Ace. Chem. Res., 1968, 2:266. 12. McCaffrey, A.J.; Mason, S.F.; Norman, B.J.; J. Chem. Soc A, 1969: 1428. 13. (a) Clarke, M.J.; Taube, H. ; J. Amer. Chem. Soc, 1974, 9_£:5413; (b) Clarke, M.J., Inorg. Chem., 1977; 1≤.:738; (c) Clarke, M.J.; Buchbunder, M. , Inorg. Chim. Acta, 1978, 21:L87; (d) Clarke, M.J., Inorg. Chem., 1980, 1≤.:1103; (d) Graves, B.J.; Hodgson, D.J., J. Amer. Chem. Soc, 1974, 101:5608. 14. The bite size for cis-DDP is 3.35 A and for bis(diamine)-dichlororuthenium(II) complexes is 3.49 A. See respectively Milburn, G.H.W.; Truter, M.R., J. Chem. Soc A, 1966: 1609; and Pank, V.; Klaus, J.; von Deuton K.; Feigel, M.; Bruder, H. ; torn Dieck, H. , Tran¬ sition Met. Chem., 1981, £:185. 15. (a) F.P. Dwyer, I.K. Reid, Shulman, G.M. Laycock and S. Dixson Aust. ___ Exp. Biol. Med. Sci. 41, 203 (1969) ; " (b) ' F.P. Dwyer, E. Mayhew, E.M.F. Roe, and A. Shulman Brit. __. Cancer 1£, 195 (1965); (c) A. Shulman and D.O. White Chem. Biol. Inter. 6_, 407 (1973); (d) A. Shulman and G.M. Laycock Chem. fii2_L_. Inter. 1£, 89 (1977). 16. (a) Clarke, M.J., Met. Ions Biol. Syst., 1980, 11:231; (b) Giraldi, Sawa, G.; Berloli, G. ; Mestroni, G.; Zassinovich, G.; Cancer Res.. 1977, H:26 (c) Yasbin, R.E..; Matthews, C.R.; Clarke, M.J.; Chem. Biol. Interactions, 1980, 1983, 45.:2; Tsuruo, T.; Iida, H.;. Tsukagoshi, S. Sakurai, Y.; Jap. J. Can. Res., 1980, H:151. 17. Wells, " R.D. et al., Mol. Biol., 1970, 54:465.

Claims: What is Claimed is: 1. A coordination complex of the formula (R) 2 Co(III) (Y) 2 ' wherein R comprises 1,10- phenanthroline or a substituted derivative thereof; Y comprises a labile ligand and Y and R are bonded to the Co(III) by coordination bonds. 2. A complex of claim 1, wherein Y is a chloride, tartrate, malonate or ascorbate ion. 3. A complex of claim 1, wherein the substituted derivative of 1,10-phenanthroline comprises 4,7-di- amino-1,10-phenanthroline; 3,8-diamino-l,10-phenan- throline; 4,7-diethylenediamine-l,10-phenanthroline; 3,8-diethylenediamine-l,10-phenanthroline; 4,7-dihy- droxy1-1,10-phenanthroline; 3,8-dihydroxy1-1,10-phenan- throline; 4,7-dinitro-l,10-phenanthroline; 3,8-dinitro- 1,10-phenanthroline; 4,7-dipheny1-1,10-phenanthroline; 3,8-diphenyl-l,10-phenanthroline; 4,7-dispermine-l,10- phenanthroline, or 3,8-dispermine-l,l0-phenanthroline. 4. A method for covalently labeling DNA with a com¬ plex of the formula (R) 2 M wherein R comprises 1,10- phenanthroline or a substituted derivative thereof, M comprises a suitable transition metal, and R is bonded to M by a coordination bond, which comprises contacting the DNA with a complex of the formula (R) 2 _ (Y) 2 , wherein Y is a labile ligand and is also bonded to M by a coordination bond, the contacting being under suita¬ ble conditions such that the complex covalently binds to the DNA. A method of claim 4, wherein M is ruthenium(II) 6. A method of claim 4, wherein M is cobalt(III). 7. A method of claim 4, wherein the substituted de¬ rivative of 1,10-phenanthroline comprises 4,7-diamino- 1,10-phenanthroline; 3,8-diamino-l,10-phenanthroline; 4,7-diethylenediamine-l,10-phenanthroline; 3,8-diethy- lenediamine-1,10-phenanthroline; 4,7-dihydroxyl-l,10- phenanthroline; 3,8-dihydroxyl-l,10-phenanthroline; 4,7-dinitro-l,l0-phenanthroline; 3,8-dinitro-l,10-phen- anthroline; 4,7-diphenyl-l,10-phenanthroline; 3,8-di- pheny1-1,10-phenanthroline; 4,7-dispermine-l,10-phen- anthroline, or 3,8-dispermine-l,10-phenanthroline. 8. "A labeled DNA molecule comprising DNA to which a complex of the formula (R) 2 M is covalently bound, wherein R comprises 1,10-phenanthroline or a substitut¬ ed derivative thereof,- M comprises a suitable transi¬ tion metal and R is bonded to M by a coordination bond. 9. A labeled DNA molecule of claim 8, wherein M is ruthenium(II) . 10. A labeled DNA molecule of claim 8, wherein M is cobalt (III) . 11. A labeled DNA molecule of claim 8, wherein the substituted derivative of 1,10-phenanthroline comprises 4,7-diamino-l,10-phenanthroline; 3,8-diamino-l,10-phen- anthroline; 4,7-diethylenediamine-l,10-phenanthroline; 3,8-diethylenediamine-l,10-phenanthroline; 4,7-dihy- droxyl-l,10-phenanthroline; 3,8-dihydroxy1-1,10-phenan- throline; 4,7-dinitro-l,l0-phenanthroline; 3,8-dinitro- 1,10-phenanthroline; 4,7-diphenyl-l,10-phenanthroline; 3,8-dipheny1-1,10-phenanthroline; 4,7-dispermine-l,10-. phenanthroline, or 3,8-dispermine-l,l0-phenanthroline. 12. A DNA molecule labeled with a complex of the for¬ mula (R) 2 -—M wherein R is 1,10-phenanthroline or a substituted derivative thereof, M is a suitable transi¬ tion metal and R is bonded to M by a coordination bond, the labeled DNA molecule being produced by the method of claim 4. 13. A method for nicking DNA by effecting breakage of at least one phosphodiester bond along the DNA which comprises contacting the DNA with a complex of claim 1 under suitable conditions such that the complex covalently binds to the DNA to form an adduct and irra¬ diating the adduct so formed with a sufficient dose of ultraviolet radiation of an appropriate wavelength to nick the DNA. 14. A method for killing a portion of a population of appropriate tumor cells which comprises contacting the tumor cells under suitable conditions with an effective amount of a coordination complex of the formula (R) 2 M (γ) 2 to km the tumor cells, wherein R comprises 1,10-phenanthroline or a substituted deriva¬ tive thereof, M comprises a suitable transition metal; Y comprises a labile ligand and Y and R are bonded to M by coordination bonds. 15. A method of claim 14, wherein M is ruthenium(II) . 16. A method of claim 14, wherein M is cobalt(III). 17. A method of claim 14, wherein the substituted derivative of 1,10-phenanthroline comprises 4,7-di- amino-1,10-phenanthroline; 3,8-diamino-1,10-phenanthro- line; 4,7-diethylenediamine-l,10-phenanthroline; 3,8- diethylenediamine-l,10-phenanthroline; 4,7-dihydroxyl- 1,10-phenanthroline; 3,8-dihydroxy1-1,10-phenanthro- line; 4,7-dinitro-l,10-phenanthroline; 3,8-dinitro- 1,10-phenanthroline; 4,7-dipheny1-1,10-phenanthroline; 3,8-dipheny1-1,10-phenanthroline; 4,7-dispermine-l,10- phenanthroline, or 3,8-dispermine-l,l0-phenanthroline. 18. A method of claim 14, wherein the tumor cells are present in a subject and the contacting is effected by administering the coordination complex to the subject. 19. A method of claim 16 which further comprises irra- - diating the tumor cells with a suitable dose of ultra¬ violet radiation of an appropriate wavelength at a suitable time after the tumor cells have been contacted with the complex, permitting the complex to nick DNA. 20. A pharmaceutical composition for the treatment of tumor cells in a subject which comprises an effective anti-tumor amount of a complex of the formula (R) 2 tø (γ) 2 an< * ι a pharmaceutically acceptable car¬ rier, wherein R comprises 1,10-phenanthroline or a substituted derivative thereof, M comprises a suitable transition metal; Y comprises a labile ligand and Y and R are bonded to M by coordination bonds. 21. A pharmaceutical composition of claim 20, wherein M is ruthenium(II) . 22. A pharmaceutical composition of claim 20, wherein M is cobalt(III) . 23. A pharmaceutical composition of claim 20, wherein the substituted derivative of 1,10-phenanthroline com¬ prises 4,7-diamino-l,10-phenanthroline; 3,8-diamino- 1,10-phenanthroline; 4,7-diethylenediamine-l,10-phenan- throline; 3,8-diethylenediamine-l,10-phenanthroline; 4,7-dihydr.oxyl-l,10-phenanthroline; 3,8-dihydroxyl- 1,10-phenanthroline; 4,7-dinitro-l,10-phenanthroline; 3,8-dinitro-l,10-phenanthroline; 4,7-diphenyl-l,10- phenanthroline; 3,8-dipheny1-1,10-phenanthroline; 4,7- dispermine-l,10-phenanthroline, or 3,8-dispermine-l,10- phenanthroline. 24. A method for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells which comprises administering to the subject by a suitable route a composition of claim 20 in an amount sufficient to cause regression of the tumor cells. 25. A method for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells which comprises administering to the subject by a suitable route a composition of claim 21 in an amount sufficient to cause regression of the tumor cells. 26. A method for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells which comprises administering to the subject by a suitable route a composition of claim 22 in an amount sufficient to cause regression of the tumor cells. 27. A method for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells which comprises administering to the subject by a suitable route a composition of claim 23 in an amount sufficient to cause regression of the tumor cells. 28. A method of claim 24, wherein the route of admini¬ stration is parenteral. 29. A method of claim 24, wherein the route of admini¬ stration is topical. 30. A method of claim 26 which further comprises irra¬ diating the tumor cells with a suitable dose of ultra¬ violet radiation of an appropriate wavelength permit¬ ting the complex to nick DNA at a suitable time after the administration of the composition to the subject.

Inventors: BARTON JACQUELINE K, BARTON, JACQUELINE, K.
Applicants: UNIV COLUMBIA, THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF
Application year: 1986
Main classification: C12Q1
IPC codes: C07F15, C12Q1