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The synthesis and screening for anti-bacterial, -cancer, -fungicidal and -viral activities of some complexes of palladium and nickel
£ inorg, nucl Chem. Vol. 41, pp. 1245-1249 Pergamon Press Ltd., 1979. Printed in Great Britain
THE SYNTHESIS AND SCREENING FOR ANTI-BACTERIAL, -CANCER, -FUNGICIDAL AND -VIRAL ACTIVITIES OF SOME COMPLEXES OF PALLADIUM AND NICKEL ROBERT D. GRAHAM and DAVID R. WILLIAMSt Department of Chemistry, University of Wales Institute of Science and Technology, Cardiff, CFI 3NU, Wales (Received 2 December 1976; receivedfor publication 26 January 1979)
Abstract--Several complexes of palladium and nickel have been synthesized and subjected to a variety of in vitro/in vivo screens. Of the complexes submitted the most positive antibacterial was (TrC3HsPdCI)~,antrivirals were cisPd(NH3)2Cl2 and (NH4)2PdCI4, anticancers were cis and transPd(NH3)2Cl2, cisPd(NH3)2CI4, Na2PdCIr, (NH4).,PdCI4, ethylenediamineH_,.PdCl4, Pd(NH~)4-PdCI4, (zrC3H~PdCI)2, Pd(cyclopentylamine)2Cl,, Pd(cyclohexylamine)2Cl2, and some palladium asparaginate complexes, and the most toxic complexes were (~rC3HsPdCI)2 and Ni(py)4(NCO)2.There appears to be different structural criteria which are necessary for active anticancer Pd complexes as compared to active Pt complexes. The advantages of introducing Pd into drugs are discussed.
INTRODUCTION In 1963, Furst suggested the addition of group VIIIB metals to anticancer drugs in order to facilitate their action and in 1969 Rosenberg et al. reported the first platinum complex having anticancer activity in its own right (i.e. it was not the metal complex of any other anticancer drug)[1-4]. This compound, cis-dichlorodiammineplatinum(II), has been found to be successful against a wide spectrum of cancers and clinical trials have been successfully carried out in several hospitals[5]. There have been reviews and conferences concering the chemistry of complexes related to cisPt(NH3)2CI2, postulating reasons why group VIIIB catnplexes in particular ought to be carcinostatic, suggesting ways in which the design of such complexes might be improved and reasoning that amino-acids ought to be incorporated into these drugs[l,5,6]. In general, there has been a great deal of conjecture and rather less scientific facts. Thus, the mechanisms through which cisPt(NH3)2Cl2 exhibits anticancer activity have not been firmly established. The complex may either have a direct and selective action for DNA inside cells forming polymer (probably to bases such as guanine) to Pt 2+ to polymer chelate rings or there may be an indirect mechanism which functions through the immunological system of the animal. This paper reports some additional data in the form of the syntheses and screening results for a series of related palladium and nickel complexes. Quite recently Davidson et al. have reported cisPt(NH3)2(H20)2 ~+ to have antiviral activity[7] and so our complexes were screened for antibacterial, antiviral and antifungicidal activities in addition to the anticancer screening. Table 1 lists the complexes studied in this work; whenever known compounds were prepared and purified, elemental analyses agreed to within acceptable limits ?Details of syntheses and elemental analyses data for the complexes screened are available as Supplementary Publication reference UWlST DRW No. 2, 8 pages, on application to Departmental Administrator, Department of Chemistry, UWIST, Cardiff CFI 3NU enclosing U.K. £1.00 to cover postage and packing.
with those published in the literature. The compounds fall into six general types. (1) The introduction of Pd(II) is in line with our conviction that we ought to extend our attentions to metals in addition to Pt and cis and transPd(NH3)2Cl2 are analogues of Rosenberg's cisPt(NH3):CI2. In general Pd(II) complexes are more reactive than those of Pt(II) and rapid ligand exchange may occur[8]. However, our previous studies[9] found that the complexes were sufficiently stable to permit the determination of their stability constants and there are literature reports of Pd(II) complexes of 6-mercaptopurine having carcinostatic activity with respect to Adenocarcinoma 755 and Sarcoma 180110]. (2) Although Pd(IV) compounds might be expected to be rapidly reduced in tissue, carcinostatic activity for Pt has been reported for cisPt(NH3)2Ch, cisPt(ethylenediamine)Cl4 and Na2[Pt(mercaptopurine)2CL,]•2 H20[II]. (3) zr Allylpalladium chloride has a low charge density and this could assist the solubility in, or transport through, cell membranes. Further since this complex is devoid of nitrogens Pd-amine-DNA bonds are not possible (This type of carcinostatic activity involving DNA has been suggested as an alternative to the more popular halide ion loss approach). (4) Enhanced therapeutic indices were obtained when ammonias in the Pt series were replaced with cyclopentylamine and cyclohexylamine [l l] and 1-adamantanamine hydrochloride is known to be a potent antiviral agent[12, 131. ( 5 ) Amino-acid anions used as ligands have the possibilities of introducing cancer cell specificity into the complexes (lymphatic and myeloid leukaemia cells possess mechanisms f o r selectively aquiring asparagine, serine and glutamine from the extracellular fluid[14-18l) and they have the two advantages that they are non-toxi~c when released within a cell and also they chelate Pd(II) in cis positions and so prevent cis C1- from racemising. (6) The Ni(II) complexes complete the d ~ metals iin periodic group VIIIB and Gillard et al. have reported bacteriostatic activity using pyridine ligands [6, 19].
1245
1246
R.D. GRAHAM and D. R. WILLIAMS Table 1. Six general classifications of complexes synthesised and screened for biologicalactivity
1. cisPd(NH3)2CI2 transPd(NH3)2CI2 (NH4)2PdCI4 EthylenediamineH2.PdCL~ PdethylenediarnineCI2
2. cisPd(NH3)2Cl4 Na2PdCI6 Pd(NH3)4.PdCI4 Pd(ethylenediamine)2.PdCI4
3. (IrC3H~PdCI)2
5. PdasparagineCl2 Pd(asparagine)2[2 isomers] Pd(glutamine)2 PdserineCl2 Pd(serineh Pd(D-penicillamine)Cl2
4. Pd(cyclopentylamine),zCI2 Pd(cyclohexylamine)2Cl2[2 isomers] Pt(cyclohexylamine)2CI2 Pd(adamantanamine)2CI2
6. Ni(pyridine)4CI2 Ni(pyridine)4NCO Ni(adamantanamine)4(H20)2.S04
SCREENING METHODS AND RESULTS Preliminary toxicity studies. The compounds were administered to mice in doses of 50--100mgkg -~ (0.2ml) -~ either intraperitoneally or subcutaneously, once daily for 5 days. After nine days the animals were examined visually at post-mortem for signs of toxicity. In vitro antimicrobial studies. The antimicrobiai activities of the compounds were investigated by the serial dilution method using a broth media. Gram-positive and gram-negative bacteria and saprophytic and dermatophyte fungi were used at 250 #g ml-~. Antiviral studies. The compounds were added to growing cultures of cells infected with viruses (Herpes hominis, Adenovirus (SVI7), Influenza (Hong Kong and Iksha), Parainfluenza (I and III) and Rhinovirus (IB, 2, 5 and 9)) and the activities were assessed by observing the reduction in the cytopathic effect in the infected cells. Toxicity was recorded by adding the compound to uninfected cells and observing its effect on cell growth. Antitumour studies. (a) Sarcoma 180. A suspension of Sarcoma 180 tumour cells (6 mg per dose of 0.1 ml) was prepared and injected subcutaneously into mice. On the nextday, the animals were treated with the compound being screened (at 25, 50 or 100mgkg -R dose-~). This dose was repeated daily for another 8 days (dose volume = 0.2 mi). N-methyl formamide (200 mg kg-' dose -j) was used as a control treatment. Fourteen days after implantation the animals were killed and tumours were removed and weighed. For each group, the average tumour to mouse ratio was calculated and the percentage inhibitions for the treated animals compared with the untreated controls were calculated. (b) Landschutz ascites. Mice were injected intraperitoneally with Landschutz ascites cells (2× 106) and treated subcutaneously with the compounds being screened. The schedules and doses were the same as those used in the Sarcoma 180 experiments. Ten days after inoculation the ascitic fluid was drained from each .group of mice, its volume was measured and the total ~'Therapeuticindex, T.I. = = LD~ID~ where LDsois the dose that was lethal to 50% of all animals and I I ~ is the minimum dose required to produce 90~ tumour regression. SThis latter value could well be higher since the solid screened was several months old and, since the complexwas not a chelate, cis --)trans racemisationmight well have occurred.
number of cells per mouse was calculated using a haemocytometer. The percentage inhibition was determined by comparing the treated animals with the untreated control animals. (c) Landschutz ascites "in vitro/in vivo" tests were performed by tumour cells being suspended in various concentrations of the metal complex being screened (37° for 2 hr). These cells were then inoculated into mice and the viability of the complex suspension was assessed according to ability to form tumours. DISCUSSION Table 2 lists the results found in the screens listed. Of the complexes submitted the most positive antibacterial was (~rC3HsPdCI)2, antivirals were cisPd(NH3)2Cl2 and (NH4)2PdCI4, anticancers were cis and transPd(NH3)2CI2, cisPd(NH3)2CL~, (1rC3HsPdCi)2, Na2PdCl6, (NH4)2PdCI4, enH2.PdCl4, Pd(NH3)4"PdCI4, Pd(cyclopent)2Cl2, Pd(cyclohex)2Cl2 and the palladium asparaginate complexes, and the most toxic complexes were (~rC3HsPdCI)2 and Ni(pyh(NCO)2. These carcinostatic palladium compounds do not completely obey the structural criteria found for the active platinum complexes [5] in that (a) some anticancer compounds such as Na2PdCl6, (NH4)2PdCL and enH2.PdCLs do not possess nitrogen donor atoms as part of their carrier ligands, these nitrogens having at least one active hydrogen; (b) Metal-metal stacking such as occurs in cisPt(NH~)2CI2 cannot occur with the octahedral Pd complexes; (c) It has been found that, for Pt compounds, tumour selectivity arises from N substitution (for example, substituting cyclopentylamine for ammonia). We have now found that changing the central metal ion (Pd 2+ for Pt 2+) also affects the selectivity; consider the therapeutic indicest of cisPt(NH3)zCl2, T.I. = 8 against Connors ADJ/PC6A turnout, cisPd(NH3)2Cl2, T.I.=0 against Conners ADJ/PC6A tumour or T.I. = >4 against our Landschutz ascites celis;~: (d) PdCl6: - and PdCL 2- are active negative ions whereas the active Pt complexes were all neutral[l 1]. Cleare and Hoeschele have found that Pd complexes which were analogues of the most active Pt complexes had far inferior" activities in their tumour test systems[20]. Our cisPd(NH3)2Cl2 and Pd(cyclohex)2Cl2 results do not support this concept which is based upon the observation that Pd complexes exchange ligands too rapidly for the drugs to be present as the same complex
(zrC3HsPdCI)2 yellow green powder
Pden2.PdCh pale pink powder Pden2CI2 bright yellow crystals
no activity at 12.5
no activity at 250
Adeno 50% at 50, Herp and Hong nil at 50, Iksha 75% at 50, Para nil at 50, Rhino nil at 12.5
Adeno 40% at 50, Herp and Hong nil at 50, lksha 50% at 12.5, Para nil at 50, Rhino, nil at 12.5 no activity at 250
(NH4)2PdCI4 dark brown crystals
enH2PdCl4 dark purple powder Pd(NH3hPdCI~ pink powder
no activity at 250
no activity at 250
Adeno 60% at 50, Herp 70% at 50, Hong nil at 50, lksha 50% at 50, Para nil at 50, Rhino nil at 50. no activity at 250
Antiviral screening
NazPdCI6 bright crimson powder
rust brown powder
cisPd(NH3)2Ch
mustard coloured powder
transPd(NH3hCI2
cisPd(NH3hCl2
Compound and appearance
1.9
7.8 1.9 15.6
100% at and 42% at 100% at and 34% at nil at 12.5
3.9 1.9 0.04
nil at 50
91% at 12.5
34% at 25
49% at 50
36% at 50
92% at 50
48% at 50
75% at 12.5
40% at 12.5
50% at 100
27% at 50
27% at 50
11% at 50
2.5% at 50
86% at 100
43% at 50
1.6% at 25
Anticancer screening (b) (c)
62.5
100% at 15.6
100% at 76% at 100% at and 84% at
100% at 15.6 and 55% at 1.9 100% at 7.8 and 25% at 1.9 100% at 15.6 and 60% at 3.9
100% at 1.95
(a)
Table 2. Screening results for metal complexes
toxic at 12.5
nil at 50
only in 1 cell system at 50
nil at 50
nil at 50
nil at 50
nil to 50
toxic at 100 mg/kg
nil at 50
Cytotoxicity (/zg/ml)
Positive bactericidal activity for both gram positive and gram negative bacteria, this compound exhibited fungicidal activity in several screens except Aspergillus niger It is dermonecrotic when administered intradermaUy to guinea pigs
marginal activity against Mycobacteriura BCG at 250 ~g/ml
no bactericidal or fungicidal activity at 250 tzg/ml
no bactericidal or fungicidal activity at 250 tzg/ml
Other observations
~2
m
~r
t't'-
ca.
no activity at 250
Antiviral screening
100% at and 27% at 100% at and 64% at 100% at and 54% at 100% at and 12% at 100% at and 26% at 96% at and 60% at 100% at 31.2 125
62.5 250
7.8 125
1.0 62.5
1.9 62.5
1.9 31
250
50 25 50 25 50 25 50 25
37% at 50
14.7%at 70.3% at 100% at 31.25 31.2% at 6.4% at 100% at 3.9 11.1%at 63.0% at 100% at 1000 63.3% at 49.6% at
Anticancer screening (b)
100% at 3.9
(a)
nil
4.0% at 50 41.2% at 25 13.3% at 50 68.4%at 25 nil at 50 27.4 at 25 nil at 50 nil at 25
(c)
toxic at 100
Cytotoxicity (/zg/ml)
no bactericidal or fungicidal activity at 250 #g/ml All screening eliminated by anaphylaxis
marginally active against Mycobactevium BCG at 250 p~g/ml
marginally active against Mycobacterium BCG at 250/~g/ml
Other observations
Antiviral screens. Adeno = adenovirus, Herp = herpesvirus, Hong = influenza Hong Kong, Iksha = influenza Iksha, Para = para-influenza, and Rhino = rhinovirus. The percentage inhibition and the concentration (~g/ml) are noted. Anticancer screens. (a) = Landschutz ascites in vitro~in vivo test. The percentage inhibition and concentration (p.g/ml) are noted. (b) = Landschutz ascites in vioo system. The percentage inhibition and concentration (mg/kg/dose) are noted. (c) = Sarcoma 180 in vivo system. The percentage inhibition and concentration (mg/kg/dose) are noted.
Pd(D-pen)Cl2 mustard coloured powder Ni(pyhCl2 pale blue powder Ni(pyXNCO)4 pale blue powder Ni(adamh(H2Oh.S04 pale green powder
Pd(ser)CI2 red-brown soln in H20 Pd(serh yellow soln in H20
Pd(glnh pale yellow powder
Pd(asnh lilac grey isomer
Pd(asnh pale yellow isomer
Pd(cyclopenthCI2 bright yellow powder Pd(cyclohexhCl2 red isomer Pd(cyclohexhCl2 pink isomer Pt(cyclohexhCl2 grey powder Pd(adam)2CI2 straw coloured powder Pd(asn)CI2 red soin in H20
appearance
Compoundand
Table 2. (Contd)
>.
e~
g
>. >
The synthesis and screening for anti-bacterial activities when they reach their sites of anticancer acticity, unless the complexes were deactivated by chelation. This dichotomy underlines the necessity of screening compounds against more than one test system (for example, PdenCI2 had a T.I. of - 0 in the Cleare and Hoeschele screens and ca. 3 in ours). Advantages o[ using Pd complexes in chemical and biochemical research. (i) Both PtenCl2 and PdenCl2 have recently undergone structural determination by X-ray crystallography; the Pd system was considerably easier to study because of the lower atomic weight of the central metal ion[21]. (ii) Clearly, there must be a move towards screening complexes of many metals against those diseases currently attracting the most research attention. To Rosenberg's Pt series, Cleare and Connors have already added Rh(Ill) and Ir(III) (Rh complexes showing some antitumour properties--especially merRh(NH3)3Cl3--against Sarcoma 18015]) and now the list of metal complexes investigated as carcinostats is increased by the Pd and Ni results shown in the tables. Future work. The encouraging results of screening cisPt(NH3)2CI2 in animals and of clinical trials in man has initiated a search for similar, related, compounds having antitumour activity. This paper draws attention to the necessity for research into several facets of this work. (i) Although complexes of metals other than Pt may not immediately produce better drugs, it is still most desirable to correlate drug effectiveness with physical parameters (for example, solubility in water, distribution coefficients into immiscible solvents, concentrations of complexes present at cancer cells pHs [9]), (ii) One such very important property is that of mixedligand complex equilibria in solution. It is essetnial, if theories of anticancer activity are to be forthcoming, to know which complexes exist in bodily fluids at physiological pH values and chloride concentrations (we have recently concluded a mixed-ligand research project to examine this aspect[9]). (iii) The combination therapy of two antitumour metal complexes seems to be a logical extension to screening studies
1249
Acknowledgements--The authors thank Johnson-Matthey Chemicals Ltd for loans of palladium salts and Glaxo Research Ltd for performing screening tests, and Messers D. Latto and D. C. Tipney for skillful technical assistance.
REFERENCES
I. Platinum Coordination Complexes in Cancer Chemotherapy (Edited by T. A. Connors and J. J. Roberts). SpringerVerlag, Berlin (1974). 2. A. Furst, Chemistry of Chelation in Cancer. C. C. Thomas, Springfield, Illinois. American Lectures in Living Chemistry Series. Gen. Editor N. Kugelmass (1963). 3. B. Rosenberg, L. Van Camp, J. E. Trosko and V. H. Mansour, Nature 222, 385 (1969). 4. B. Rosenberg, Plat. Metals Rev. 15(2), 42 (1971). 5. T. A. Connors, Plat. Metals Rev. 17, 98 (1973). 6. S. Reslova, A. J. Thomson and R. J. P. Williams, Structure and Bonding, Vol. l I, p. I. Springer-Verlag, Berlin (1972). 7. J. P. Davidson, M. J. Cleare and B. Rosenberg, Platinum Coordination Complexes in Cancer Chemotherapy (Edited by T. A. Connors and J. J. Roberts), p. 98. Springer-Verlag, Berlin (1974). 8. F. Basolo and R. G. Pearson, Mechanisms o/ Inorganic Reactions, I lth Edn. Wiley, New York (1967). 9. R. D. Graham and D. R. Williams,JCSDalton 11230974). 10. S. Kirschner, Y.-K. Wei, D. Francis and J. G. Bergman, /. Med. Chem. 9, 369 (1966), 11. M. J. Cleare and J. D. Hoeschele, Proc. VlIth Int. Chemotherapy Cong., Prague, 13 (1971); Plat. Metals Rev. 17, 2 (1973). 12. W. L. Davies et al., Science 144, 862 (1964). 13. J. S. Wishnok, J. Chem. Ed. 50, 780 (1973). 14. E. Grundmannand H. F. Oettgen, Cancer Research. SpringerVerlag, Berlin (1970). 15. M. J. Beard, Brit. Med. J. 1, 191 (1970). 16. J. E. Kid& J. Exp. Med. 98, 565, 583 (1953). 17. J. D. Broome, Nature 191, 1114(1961). 18. F. P. Dwyer, Chelating Agents and Metal Chelates (Edited by F. P. Dwyer and D. P. Mellor). Academic Press, London (1964). 19. R. J. Bromfield,R. H. Dainty, R. D. Gillard and B. T. Heaton, Nature 223, 735 0969). 20. M. J. Cleare, Coord. Chem. Rev. 12,349(1974). 21. J. Iball, M. MacDougalland S. Scrimgeour, Acta Cryst. B31, 1672 (1975).
THE SYNTHESIS AND SCREENING FOR ANTI-BACTERIAL, -CANCER, -FUNGICIDAL AND -VIRAL ACTIVITIES OF SOME COMPLEXES OF PALLADIUM AND NICKEL ROBERT D. GRAHAM and DAVID R. WILLIAMSt Department of Chemistry, University of Wales Institute of Science and Technology, Cardiff, CFI 3NU, Wales (Received 2 December 1976; receivedfor publication 26 January 1979)
Abstract--Several complexes of palladium and nickel have been synthesized and subjected to a variety of in vitro/in vivo screens. Of the complexes submitted the most positive antibacterial was (TrC3HsPdCI)~,antrivirals were cisPd(NH3)2Cl2 and (NH4)2PdCI4, anticancers were cis and transPd(NH3)2Cl2, cisPd(NH3)2CI4, Na2PdCIr, (NH4).,PdCI4, ethylenediamineH_,.PdCl4, Pd(NH~)4-PdCI4, (zrC3H~PdCI)2, Pd(cyclopentylamine)2Cl,, Pd(cyclohexylamine)2Cl2, and some palladium asparaginate complexes, and the most toxic complexes were (~rC3HsPdCI)2 and Ni(py)4(NCO)2.There appears to be different structural criteria which are necessary for active anticancer Pd complexes as compared to active Pt complexes. The advantages of introducing Pd into drugs are discussed.
INTRODUCTION In 1963, Furst suggested the addition of group VIIIB metals to anticancer drugs in order to facilitate their action and in 1969 Rosenberg et al. reported the first platinum complex having anticancer activity in its own right (i.e. it was not the metal complex of any other anticancer drug)[1-4]. This compound, cis-dichlorodiammineplatinum(II), has been found to be successful against a wide spectrum of cancers and clinical trials have been successfully carried out in several hospitals[5]. There have been reviews and conferences concering the chemistry of complexes related to cisPt(NH3)2CI2, postulating reasons why group VIIIB catnplexes in particular ought to be carcinostatic, suggesting ways in which the design of such complexes might be improved and reasoning that amino-acids ought to be incorporated into these drugs[l,5,6]. In general, there has been a great deal of conjecture and rather less scientific facts. Thus, the mechanisms through which cisPt(NH3)2Cl2 exhibits anticancer activity have not been firmly established. The complex may either have a direct and selective action for DNA inside cells forming polymer (probably to bases such as guanine) to Pt 2+ to polymer chelate rings or there may be an indirect mechanism which functions through the immunological system of the animal. This paper reports some additional data in the form of the syntheses and screening results for a series of related palladium and nickel complexes. Quite recently Davidson et al. have reported cisPt(NH3)2(H20)2 ~+ to have antiviral activity[7] and so our complexes were screened for antibacterial, antiviral and antifungicidal activities in addition to the anticancer screening. Table 1 lists the complexes studied in this work; whenever known compounds were prepared and purified, elemental analyses agreed to within acceptable limits ?Details of syntheses and elemental analyses data for the complexes screened are available as Supplementary Publication reference UWlST DRW No. 2, 8 pages, on application to Departmental Administrator, Department of Chemistry, UWIST, Cardiff CFI 3NU enclosing U.K. £1.00 to cover postage and packing.
with those published in the literature. The compounds fall into six general types. (1) The introduction of Pd(II) is in line with our conviction that we ought to extend our attentions to metals in addition to Pt and cis and transPd(NH3)2Cl2 are analogues of Rosenberg's cisPt(NH3):CI2. In general Pd(II) complexes are more reactive than those of Pt(II) and rapid ligand exchange may occur[8]. However, our previous studies[9] found that the complexes were sufficiently stable to permit the determination of their stability constants and there are literature reports of Pd(II) complexes of 6-mercaptopurine having carcinostatic activity with respect to Adenocarcinoma 755 and Sarcoma 180110]. (2) Although Pd(IV) compounds might be expected to be rapidly reduced in tissue, carcinostatic activity for Pt has been reported for cisPt(NH3)2Ch, cisPt(ethylenediamine)Cl4 and Na2[Pt(mercaptopurine)2CL,]•2 H20[II]. (3) zr Allylpalladium chloride has a low charge density and this could assist the solubility in, or transport through, cell membranes. Further since this complex is devoid of nitrogens Pd-amine-DNA bonds are not possible (This type of carcinostatic activity involving DNA has been suggested as an alternative to the more popular halide ion loss approach). (4) Enhanced therapeutic indices were obtained when ammonias in the Pt series were replaced with cyclopentylamine and cyclohexylamine [l l] and 1-adamantanamine hydrochloride is known to be a potent antiviral agent[12, 131. ( 5 ) Amino-acid anions used as ligands have the possibilities of introducing cancer cell specificity into the complexes (lymphatic and myeloid leukaemia cells possess mechanisms f o r selectively aquiring asparagine, serine and glutamine from the extracellular fluid[14-18l) and they have the two advantages that they are non-toxi~c when released within a cell and also they chelate Pd(II) in cis positions and so prevent cis C1- from racemising. (6) The Ni(II) complexes complete the d ~ metals iin periodic group VIIIB and Gillard et al. have reported bacteriostatic activity using pyridine ligands [6, 19].
1245
1246
R.D. GRAHAM and D. R. WILLIAMS Table 1. Six general classifications of complexes synthesised and screened for biologicalactivity
1. cisPd(NH3)2CI2 transPd(NH3)2CI2 (NH4)2PdCI4 EthylenediamineH2.PdCL~ PdethylenediarnineCI2
2. cisPd(NH3)2Cl4 Na2PdCI6 Pd(NH3)4.PdCI4 Pd(ethylenediamine)2.PdCI4
3. (IrC3H~PdCI)2
5. PdasparagineCl2 Pd(asparagine)2[2 isomers] Pd(glutamine)2 PdserineCl2 Pd(serineh Pd(D-penicillamine)Cl2
4. Pd(cyclopentylamine),zCI2 Pd(cyclohexylamine)2Cl2[2 isomers] Pt(cyclohexylamine)2CI2 Pd(adamantanamine)2CI2
6. Ni(pyridine)4CI2 Ni(pyridine)4NCO Ni(adamantanamine)4(H20)2.S04
SCREENING METHODS AND RESULTS Preliminary toxicity studies. The compounds were administered to mice in doses of 50--100mgkg -~ (0.2ml) -~ either intraperitoneally or subcutaneously, once daily for 5 days. After nine days the animals were examined visually at post-mortem for signs of toxicity. In vitro antimicrobial studies. The antimicrobiai activities of the compounds were investigated by the serial dilution method using a broth media. Gram-positive and gram-negative bacteria and saprophytic and dermatophyte fungi were used at 250 #g ml-~. Antiviral studies. The compounds were added to growing cultures of cells infected with viruses (Herpes hominis, Adenovirus (SVI7), Influenza (Hong Kong and Iksha), Parainfluenza (I and III) and Rhinovirus (IB, 2, 5 and 9)) and the activities were assessed by observing the reduction in the cytopathic effect in the infected cells. Toxicity was recorded by adding the compound to uninfected cells and observing its effect on cell growth. Antitumour studies. (a) Sarcoma 180. A suspension of Sarcoma 180 tumour cells (6 mg per dose of 0.1 ml) was prepared and injected subcutaneously into mice. On the nextday, the animals were treated with the compound being screened (at 25, 50 or 100mgkg -R dose-~). This dose was repeated daily for another 8 days (dose volume = 0.2 mi). N-methyl formamide (200 mg kg-' dose -j) was used as a control treatment. Fourteen days after implantation the animals were killed and tumours were removed and weighed. For each group, the average tumour to mouse ratio was calculated and the percentage inhibitions for the treated animals compared with the untreated controls were calculated. (b) Landschutz ascites. Mice were injected intraperitoneally with Landschutz ascites cells (2× 106) and treated subcutaneously with the compounds being screened. The schedules and doses were the same as those used in the Sarcoma 180 experiments. Ten days after inoculation the ascitic fluid was drained from each .group of mice, its volume was measured and the total ~'Therapeuticindex, T.I. = = LD~ID~ where LDsois the dose that was lethal to 50% of all animals and I I ~ is the minimum dose required to produce 90~ tumour regression. SThis latter value could well be higher since the solid screened was several months old and, since the complexwas not a chelate, cis --)trans racemisationmight well have occurred.
number of cells per mouse was calculated using a haemocytometer. The percentage inhibition was determined by comparing the treated animals with the untreated control animals. (c) Landschutz ascites "in vitro/in vivo" tests were performed by tumour cells being suspended in various concentrations of the metal complex being screened (37° for 2 hr). These cells were then inoculated into mice and the viability of the complex suspension was assessed according to ability to form tumours. DISCUSSION Table 2 lists the results found in the screens listed. Of the complexes submitted the most positive antibacterial was (~rC3HsPdCI)2, antivirals were cisPd(NH3)2Cl2 and (NH4)2PdCI4, anticancers were cis and transPd(NH3)2CI2, cisPd(NH3)2CL~, (1rC3HsPdCi)2, Na2PdCl6, (NH4)2PdCI4, enH2.PdCl4, Pd(NH3)4"PdCI4, Pd(cyclopent)2Cl2, Pd(cyclohex)2Cl2 and the palladium asparaginate complexes, and the most toxic complexes were (~rC3HsPdCI)2 and Ni(pyh(NCO)2. These carcinostatic palladium compounds do not completely obey the structural criteria found for the active platinum complexes [5] in that (a) some anticancer compounds such as Na2PdCl6, (NH4)2PdCL and enH2.PdCLs do not possess nitrogen donor atoms as part of their carrier ligands, these nitrogens having at least one active hydrogen; (b) Metal-metal stacking such as occurs in cisPt(NH~)2CI2 cannot occur with the octahedral Pd complexes; (c) It has been found that, for Pt compounds, tumour selectivity arises from N substitution (for example, substituting cyclopentylamine for ammonia). We have now found that changing the central metal ion (Pd 2+ for Pt 2+) also affects the selectivity; consider the therapeutic indicest of cisPt(NH3)zCl2, T.I. = 8 against Connors ADJ/PC6A turnout, cisPd(NH3)2Cl2, T.I.=0 against Conners ADJ/PC6A tumour or T.I. = >4 against our Landschutz ascites celis;~: (d) PdCl6: - and PdCL 2- are active negative ions whereas the active Pt complexes were all neutral[l 1]. Cleare and Hoeschele have found that Pd complexes which were analogues of the most active Pt complexes had far inferior" activities in their tumour test systems[20]. Our cisPd(NH3)2Cl2 and Pd(cyclohex)2Cl2 results do not support this concept which is based upon the observation that Pd complexes exchange ligands too rapidly for the drugs to be present as the same complex
(zrC3HsPdCI)2 yellow green powder
Pden2.PdCh pale pink powder Pden2CI2 bright yellow crystals
no activity at 12.5
no activity at 250
Adeno 50% at 50, Herp and Hong nil at 50, Iksha 75% at 50, Para nil at 50, Rhino nil at 12.5
Adeno 40% at 50, Herp and Hong nil at 50, lksha 50% at 12.5, Para nil at 50, Rhino, nil at 12.5 no activity at 250
(NH4)2PdCI4 dark brown crystals
enH2PdCl4 dark purple powder Pd(NH3hPdCI~ pink powder
no activity at 250
no activity at 250
Adeno 60% at 50, Herp 70% at 50, Hong nil at 50, lksha 50% at 50, Para nil at 50, Rhino nil at 50. no activity at 250
Antiviral screening
NazPdCI6 bright crimson powder
rust brown powder
cisPd(NH3)2Ch
mustard coloured powder
transPd(NH3hCI2
cisPd(NH3hCl2
Compound and appearance
1.9
7.8 1.9 15.6
100% at and 42% at 100% at and 34% at nil at 12.5
3.9 1.9 0.04
nil at 50
91% at 12.5
34% at 25
49% at 50
36% at 50
92% at 50
48% at 50
75% at 12.5
40% at 12.5
50% at 100
27% at 50
27% at 50
11% at 50
2.5% at 50
86% at 100
43% at 50
1.6% at 25
Anticancer screening (b) (c)
62.5
100% at 15.6
100% at 76% at 100% at and 84% at
100% at 15.6 and 55% at 1.9 100% at 7.8 and 25% at 1.9 100% at 15.6 and 60% at 3.9
100% at 1.95
(a)
Table 2. Screening results for metal complexes
toxic at 12.5
nil at 50
only in 1 cell system at 50
nil at 50
nil at 50
nil at 50
nil to 50
toxic at 100 mg/kg
nil at 50
Cytotoxicity (/zg/ml)
Positive bactericidal activity for both gram positive and gram negative bacteria, this compound exhibited fungicidal activity in several screens except Aspergillus niger It is dermonecrotic when administered intradermaUy to guinea pigs
marginal activity against Mycobacteriura BCG at 250 ~g/ml
no bactericidal or fungicidal activity at 250 tzg/ml
no bactericidal or fungicidal activity at 250 tzg/ml
Other observations
~2
m
~r
t't'-
ca.
no activity at 250
Antiviral screening
100% at and 27% at 100% at and 64% at 100% at and 54% at 100% at and 12% at 100% at and 26% at 96% at and 60% at 100% at 31.2 125
62.5 250
7.8 125
1.0 62.5
1.9 62.5
1.9 31
250
50 25 50 25 50 25 50 25
37% at 50
14.7%at 70.3% at 100% at 31.25 31.2% at 6.4% at 100% at 3.9 11.1%at 63.0% at 100% at 1000 63.3% at 49.6% at
Anticancer screening (b)
100% at 3.9
(a)
nil
4.0% at 50 41.2% at 25 13.3% at 50 68.4%at 25 nil at 50 27.4 at 25 nil at 50 nil at 25
(c)
toxic at 100
Cytotoxicity (/zg/ml)
no bactericidal or fungicidal activity at 250 #g/ml All screening eliminated by anaphylaxis
marginally active against Mycobactevium BCG at 250 p~g/ml
marginally active against Mycobacterium BCG at 250/~g/ml
Other observations
Antiviral screens. Adeno = adenovirus, Herp = herpesvirus, Hong = influenza Hong Kong, Iksha = influenza Iksha, Para = para-influenza, and Rhino = rhinovirus. The percentage inhibition and the concentration (~g/ml) are noted. Anticancer screens. (a) = Landschutz ascites in vitro~in vivo test. The percentage inhibition and concentration (p.g/ml) are noted. (b) = Landschutz ascites in vioo system. The percentage inhibition and concentration (mg/kg/dose) are noted. (c) = Sarcoma 180 in vivo system. The percentage inhibition and concentration (mg/kg/dose) are noted.
Pd(D-pen)Cl2 mustard coloured powder Ni(pyhCl2 pale blue powder Ni(pyXNCO)4 pale blue powder Ni(adamh(H2Oh.S04 pale green powder
Pd(ser)CI2 red-brown soln in H20 Pd(serh yellow soln in H20
Pd(glnh pale yellow powder
Pd(asnh lilac grey isomer
Pd(asnh pale yellow isomer
Pd(cyclopenthCI2 bright yellow powder Pd(cyclohexhCl2 red isomer Pd(cyclohexhCl2 pink isomer Pt(cyclohexhCl2 grey powder Pd(adam)2CI2 straw coloured powder Pd(asn)CI2 red soin in H20
appearance
Compoundand
Table 2. (Contd)
>.
e~
g
>. >
The synthesis and screening for anti-bacterial activities when they reach their sites of anticancer acticity, unless the complexes were deactivated by chelation. This dichotomy underlines the necessity of screening compounds against more than one test system (for example, PdenCI2 had a T.I. of - 0 in the Cleare and Hoeschele screens and ca. 3 in ours). Advantages o[ using Pd complexes in chemical and biochemical research. (i) Both PtenCl2 and PdenCl2 have recently undergone structural determination by X-ray crystallography; the Pd system was considerably easier to study because of the lower atomic weight of the central metal ion[21]. (ii) Clearly, there must be a move towards screening complexes of many metals against those diseases currently attracting the most research attention. To Rosenberg's Pt series, Cleare and Connors have already added Rh(Ill) and Ir(III) (Rh complexes showing some antitumour properties--especially merRh(NH3)3Cl3--against Sarcoma 18015]) and now the list of metal complexes investigated as carcinostats is increased by the Pd and Ni results shown in the tables. Future work. The encouraging results of screening cisPt(NH3)2CI2 in animals and of clinical trials in man has initiated a search for similar, related, compounds having antitumour activity. This paper draws attention to the necessity for research into several facets of this work. (i) Although complexes of metals other than Pt may not immediately produce better drugs, it is still most desirable to correlate drug effectiveness with physical parameters (for example, solubility in water, distribution coefficients into immiscible solvents, concentrations of complexes present at cancer cells pHs [9]), (ii) One such very important property is that of mixedligand complex equilibria in solution. It is essetnial, if theories of anticancer activity are to be forthcoming, to know which complexes exist in bodily fluids at physiological pH values and chloride concentrations (we have recently concluded a mixed-ligand research project to examine this aspect[9]). (iii) The combination therapy of two antitumour metal complexes seems to be a logical extension to screening studies
1249
Acknowledgements--The authors thank Johnson-Matthey Chemicals Ltd for loans of palladium salts and Glaxo Research Ltd for performing screening tests, and Messers D. Latto and D. C. Tipney for skillful technical assistance.
REFERENCES
I. Platinum Coordination Complexes in Cancer Chemotherapy (Edited by T. A. Connors and J. J. Roberts). SpringerVerlag, Berlin (1974). 2. A. Furst, Chemistry of Chelation in Cancer. C. C. Thomas, Springfield, Illinois. American Lectures in Living Chemistry Series. Gen. Editor N. Kugelmass (1963). 3. B. Rosenberg, L. Van Camp, J. E. Trosko and V. H. Mansour, Nature 222, 385 (1969). 4. B. Rosenberg, Plat. Metals Rev. 15(2), 42 (1971). 5. T. A. Connors, Plat. Metals Rev. 17, 98 (1973). 6. S. Reslova, A. J. Thomson and R. J. P. Williams, Structure and Bonding, Vol. l I, p. I. Springer-Verlag, Berlin (1972). 7. J. P. Davidson, M. J. Cleare and B. Rosenberg, Platinum Coordination Complexes in Cancer Chemotherapy (Edited by T. A. Connors and J. J. Roberts), p. 98. Springer-Verlag, Berlin (1974). 8. F. Basolo and R. G. Pearson, Mechanisms o/ Inorganic Reactions, I lth Edn. Wiley, New York (1967). 9. R. D. Graham and D. R. Williams,JCSDalton 11230974). 10. S. Kirschner, Y.-K. Wei, D. Francis and J. G. Bergman, /. Med. Chem. 9, 369 (1966), 11. M. J. Cleare and J. D. Hoeschele, Proc. VlIth Int. Chemotherapy Cong., Prague, 13 (1971); Plat. Metals Rev. 17, 2 (1973). 12. W. L. Davies et al., Science 144, 862 (1964). 13. J. S. Wishnok, J. Chem. Ed. 50, 780 (1973). 14. E. Grundmannand H. F. Oettgen, Cancer Research. SpringerVerlag, Berlin (1970). 15. M. J. Beard, Brit. Med. J. 1, 191 (1970). 16. J. E. Kid& J. Exp. Med. 98, 565, 583 (1953). 17. J. D. Broome, Nature 191, 1114(1961). 18. F. P. Dwyer, Chelating Agents and Metal Chelates (Edited by F. P. Dwyer and D. P. Mellor). Academic Press, London (1964). 19. R. J. Bromfield,R. H. Dainty, R. D. Gillard and B. T. Heaton, Nature 223, 735 0969). 20. M. J. Cleare, Coord. Chem. Rev. 12,349(1974). 21. J. Iball, M. MacDougalland S. Scrimgeour, Acta Cryst. B31, 1672 (1975).