BIOINORGANIC CHEMISTRY 5, 235-239 (1976)
235
Morphological Effects of Chromium and Cobalt Complexes on Bacteria A. THEODOTOU, R.J. STRETTON, A.H. NORBURY (the late), and A .G. MASSEY Department of Chemistry, Loughborough University of Technology, Loughborough, Leicestershire LEI 13TU, Great Britain
ABSTRACT The minimum inhibitory concentration values and the effect on the morphology of Escherichia coli, of a series of chromium and cobalt (III) complexes, were determined . The cis-complexes were all active .
INTRODUCTION Filamentous forms of bacteria can be produced by a variety of agents including radiation [11, alkylating agents [21, metal complexes [2-101 and antibiotics [ 11 ] . Cis-dichlorodiamine platinum(II) and related complexes were shown by Rosenberg [31 to produce elongation in bacteria that led him to investigate their antitumor properties . The promising results obtained, for several types of tumors, led to the study of a variety of metal complexes including platinum(II) and (IV) [3,61, rhodium(l) [7,81 and (II) (101, cobalt(III) [4,91 and irridium [51 as potential antineoplastic agents. Thomson, Williams and Reslova [21 summarized the properties required in a complex for activity as (i) it must exchange only some of its ligands quickly, so the central metal must be low spin, (ii) it must be octahedral, square planar or pentagonal bi-pyramidal, (iii) two cis-leaving groups are required but not essential, and (iv) as both cis-Pt(NH 3 ) 2 CI2 , cis-Pt(en)C12 are active as antitumor agents, the biological activity is probably associated with the exchange reaction of the chloride and not due to chemical attack on other metal ligands . This work describes an investigation of the biological effects of chromium(III) and cobalt(III) complexes with a variety of bi- and tetradentate ligands on which the donor atoms are all nitrogen ; the other co-ordination positions are occupied by either halogen atoms or water molecules . Such complexes, which have octahedral coordination, are relatively substitutionally inert due to ligand field effects(D American Elsevier Publishing Company, Inc., 1976
A. THEODOTOU et al .
236
MATERIALS AND METHODS Chromium complexes were made by methods specified in Table 1 and the structures confirmed by spectral data . Escherichia colt Texas NCIB 10097 was used throughout and was grown in Roberts "C" medium [12] supplemented with glucose, 2g/ 1 ; and magnesium chloride, 0_ Ig/ l . Solutions of the required complex were made in medium "C" and sterilized by membrane filtration using Nuflow membrane filters of 0 .45 hum porosity . Graded concentrations of the appropriate complex were inoculated with an overnight culture of E . coli and incubated at 37 °C for 24 hours . The organisms were then examined microscopically, using Loeffler's methylene blue stain, for morphological variation . The minimum inhibitory concentration (MIC) was determined by a tube dilution method .
RESULTS AND DISCUSSION All the complexes tested produced filamentous forms of E . colt (Table 1), the longest filaments being produced by [Cr 1 V(dien)(O 2 ) 2 ] •H 2 0 (Fig. 1(b)) . The longest filaments were produced when E_ colt was grown in sub-inhibitory concentrations of the complex (Fig . 2) . Excluding the Cr1V complex, the other complexes follow established theory as outlined in [21, i_e ., the central atoms are of low spin [e .g_, CrIll (d 3 ), C0111(d6)1 , have the required configuration (octahedral) and in all cases the cis-form (Fig . 1) is active (although no trans complexes could be synthesized for comparison for the trien complexes, however, trans-[Cr(en) 2 C12 I Cl was shown to be inactive) . The use of different nitrogen-containing ligands en, pn, dien, trien does not seem to affect the activity to any appreciable degree, although the
TABLE 1 The Synthesis, Minimum Inhibitory Concentration Values and Morphological Effects of the Chromium and Cobalt Complexes .
Complexes K3 [Cr(ox) 3 .3H, O [Cr(trien)ox]Br_2H,O cis-[Crjtrien)CI : J CLH:O [Cr V(dien)(O, ), J .HZ O [Cr(dien)C13 J [Cr(pn), I Br, [Cr(en) 3 ] CI, cis-[Cr(en),Cl i ]Cl cis-[Co(trien)CI_IC!
Preparation [131 [131 (131 [141 [151 [161 [171 [17j [181
MIC (M x 1 (Y `) 100 50 100 6 10 100 100 100 6
Filamentation compared to cis-Pt(NH,)_C1 2 (100%) 10% 15% 10% ca. 100% 20% 20% 20% 20% 15%
aOx, oxalate ; en, ethylenediamine ; dien, diethylenetriamine ; trien, triethylenetetramine • pn, 1,2-propanediamine.
CHROMIUM AND COBALT COMPLEXES ON BACTERIA
cis-Dichiorodt(ethylene-
237
cis-o -Dichlorotriethylenetetraainechroaiua(IiI) chloride
at ine)chroeioa(lII) chloride
Dipemmiodiethylenetriaatne chroaiua(lr)
FIG. 1 . tetradentate complexes certainly seem to be less active . The bulkiness of the latter ligand may be the reason for this effect . It is interesting to note that in the Cr"'-dien complex three equivalent positions are available for exchange instead of the usual two . By far the most active complex was [Cr IV(dien)(02)21 .1120, which has a pentagonal bipyramidal structure . If the complex (Fig . 1) is actually the active medium, it is the first case of a chromium complex of this geometry to be reported as being active . However, Cr1 V compounds usually disproportionate and this particular complex might decompose when warmed 1141, more work is needed to determine what the active species is . Further research is being carried out to examine the structure-activity relationships for chromium(III) and cobalt(III) complexes with a variety of other ligands .
REFERENCES 1 H-I- Adler and A .A. Hardigree, J Bacteriol. 87, 720 (1964) . 2 . A.J . Thomson, R_J .P_ Williams, and S . Reslova, Chem. Rev. 72, 203 (1972) . 3. M .J. Cleare, Coord. Chem . Rev- 12, 349 (1974)-
238
A. THEODOTOU et aL
FIG . 2_ (a) Control: E. colt Texas grown for 24 hours at 37 °C in medium "C"_ Loeffler's methylene blue stain, X 1000 ; (b) E. coli Texas grown for 24 hours at 37 ° C for 24 hours in medium "C" in the presence of I V M cis-Pt(NH, )2C12 . Leoffler's methylene blue stain, x 1000; (c) E. coli Texas crown for 24 hours at 37 °C for 24 hours in medium "C" in the presence of 3 X I0' M [Cr (dien)(O 2 )2 ] _ Loeffler's methylene blue stain, x 1000.
CHROMIUM AND COBALT COMPLEXES ON BACTERIA
239
4. E.M. Hodnet and C .H. Moore, J. Med. Chem. 14, 1121 (1971). 5. G.M. Kolodny, Exp. Celt Res . 73, 378 (1972) . 6. G.R. Gale, L .M. Atkins, E .M. Walker, A.B . Smith, and S .J. Meishen, Proc Soc. Exp. Blot Med. 142, 1349 (1973)7. T . Giraldi, G. Zassinovich, and G . Mestroni, Chem-BioL Interact_ 9, 389 (1974). 8. C .M. Bragadin, T. Giraldi, M . Cantini, G. Zassinovich, and G . Mestroni, F E.B.S. Letters 43, 13 (1974) . 9. B.J. Crawford, D.E. Talburt, and D .A . Johnson, Bioinorg Chem. 3, 121 (1974)10. H. Simon, Diss. Abst. Inter. B 35, 1552 (1975) . 11. D.E . Hunt, R.F. Pittillo, J. Bact. 95, 712 (1968)12- R.B . Roberts, P.H. Abelson, D .B . Lownie, E.T. Bolton, and R .J. Britlen, Carnegie Inst . Wash. Publ. 607, 1955, p . 513. D.A. House and C .S. Garner, J. Am Chem. Soc. 88, 2156 (1966) . 14. D.A. House and C .S. Garner, Nature 208, 776 (1965). 15. D.A . House and C .S. Garner, Inorg. Nuc Chem. Letters 1, 137 (1965) . 16. C .L. Rollinson and J.C . Bailar, J. Am. Chem . Soc- 65, 250 (1943)17. F. Hein and S . Herzog, in Handbook of Preparative Inorganic Chemistry (G . Brauer, Ed), Vol . 2, p. 1354. 18. F . Basolo, J. Am Chem. Soc. 70, 2634 (1958) . Received 28 May 1975
235
Morphological Effects of Chromium and Cobalt Complexes on Bacteria A. THEODOTOU, R.J. STRETTON, A.H. NORBURY (the late), and A .G. MASSEY Department of Chemistry, Loughborough University of Technology, Loughborough, Leicestershire LEI 13TU, Great Britain
ABSTRACT The minimum inhibitory concentration values and the effect on the morphology of Escherichia coli, of a series of chromium and cobalt (III) complexes, were determined . The cis-complexes were all active .
INTRODUCTION Filamentous forms of bacteria can be produced by a variety of agents including radiation [11, alkylating agents [21, metal complexes [2-101 and antibiotics [ 11 ] . Cis-dichlorodiamine platinum(II) and related complexes were shown by Rosenberg [31 to produce elongation in bacteria that led him to investigate their antitumor properties . The promising results obtained, for several types of tumors, led to the study of a variety of metal complexes including platinum(II) and (IV) [3,61, rhodium(l) [7,81 and (II) (101, cobalt(III) [4,91 and irridium [51 as potential antineoplastic agents. Thomson, Williams and Reslova [21 summarized the properties required in a complex for activity as (i) it must exchange only some of its ligands quickly, so the central metal must be low spin, (ii) it must be octahedral, square planar or pentagonal bi-pyramidal, (iii) two cis-leaving groups are required but not essential, and (iv) as both cis-Pt(NH 3 ) 2 CI2 , cis-Pt(en)C12 are active as antitumor agents, the biological activity is probably associated with the exchange reaction of the chloride and not due to chemical attack on other metal ligands . This work describes an investigation of the biological effects of chromium(III) and cobalt(III) complexes with a variety of bi- and tetradentate ligands on which the donor atoms are all nitrogen ; the other co-ordination positions are occupied by either halogen atoms or water molecules . Such complexes, which have octahedral coordination, are relatively substitutionally inert due to ligand field effects(D American Elsevier Publishing Company, Inc., 1976
A. THEODOTOU et al .
236
MATERIALS AND METHODS Chromium complexes were made by methods specified in Table 1 and the structures confirmed by spectral data . Escherichia colt Texas NCIB 10097 was used throughout and was grown in Roberts "C" medium [12] supplemented with glucose, 2g/ 1 ; and magnesium chloride, 0_ Ig/ l . Solutions of the required complex were made in medium "C" and sterilized by membrane filtration using Nuflow membrane filters of 0 .45 hum porosity . Graded concentrations of the appropriate complex were inoculated with an overnight culture of E . coli and incubated at 37 °C for 24 hours . The organisms were then examined microscopically, using Loeffler's methylene blue stain, for morphological variation . The minimum inhibitory concentration (MIC) was determined by a tube dilution method .
RESULTS AND DISCUSSION All the complexes tested produced filamentous forms of E . colt (Table 1), the longest filaments being produced by [Cr 1 V(dien)(O 2 ) 2 ] •H 2 0 (Fig. 1(b)) . The longest filaments were produced when E_ colt was grown in sub-inhibitory concentrations of the complex (Fig . 2) . Excluding the Cr1V complex, the other complexes follow established theory as outlined in [21, i_e ., the central atoms are of low spin [e .g_, CrIll (d 3 ), C0111(d6)1 , have the required configuration (octahedral) and in all cases the cis-form (Fig . 1) is active (although no trans complexes could be synthesized for comparison for the trien complexes, however, trans-[Cr(en) 2 C12 I Cl was shown to be inactive) . The use of different nitrogen-containing ligands en, pn, dien, trien does not seem to affect the activity to any appreciable degree, although the
TABLE 1 The Synthesis, Minimum Inhibitory Concentration Values and Morphological Effects of the Chromium and Cobalt Complexes .
Complexes K3 [Cr(ox) 3 .3H, O [Cr(trien)ox]Br_2H,O cis-[Crjtrien)CI : J CLH:O [Cr V(dien)(O, ), J .HZ O [Cr(dien)C13 J [Cr(pn), I Br, [Cr(en) 3 ] CI, cis-[Cr(en),Cl i ]Cl cis-[Co(trien)CI_IC!
Preparation [131 [131 (131 [141 [151 [161 [171 [17j [181
MIC (M x 1 (Y `) 100 50 100 6 10 100 100 100 6
Filamentation compared to cis-Pt(NH,)_C1 2 (100%) 10% 15% 10% ca. 100% 20% 20% 20% 20% 15%
aOx, oxalate ; en, ethylenediamine ; dien, diethylenetriamine ; trien, triethylenetetramine • pn, 1,2-propanediamine.
CHROMIUM AND COBALT COMPLEXES ON BACTERIA
cis-Dichiorodt(ethylene-
237
cis-o -Dichlorotriethylenetetraainechroaiua(IiI) chloride
at ine)chroeioa(lII) chloride
Dipemmiodiethylenetriaatne chroaiua(lr)
FIG. 1 . tetradentate complexes certainly seem to be less active . The bulkiness of the latter ligand may be the reason for this effect . It is interesting to note that in the Cr"'-dien complex three equivalent positions are available for exchange instead of the usual two . By far the most active complex was [Cr IV(dien)(02)21 .1120, which has a pentagonal bipyramidal structure . If the complex (Fig . 1) is actually the active medium, it is the first case of a chromium complex of this geometry to be reported as being active . However, Cr1 V compounds usually disproportionate and this particular complex might decompose when warmed 1141, more work is needed to determine what the active species is . Further research is being carried out to examine the structure-activity relationships for chromium(III) and cobalt(III) complexes with a variety of other ligands .
REFERENCES 1 H-I- Adler and A .A. Hardigree, J Bacteriol. 87, 720 (1964) . 2 . A.J . Thomson, R_J .P_ Williams, and S . Reslova, Chem. Rev. 72, 203 (1972) . 3. M .J. Cleare, Coord. Chem . Rev- 12, 349 (1974)-
238
A. THEODOTOU et aL
FIG . 2_ (a) Control: E. colt Texas grown for 24 hours at 37 °C in medium "C"_ Loeffler's methylene blue stain, X 1000 ; (b) E. coli Texas grown for 24 hours at 37 ° C for 24 hours in medium "C" in the presence of I V M cis-Pt(NH, )2C12 . Leoffler's methylene blue stain, x 1000; (c) E. coli Texas crown for 24 hours at 37 °C for 24 hours in medium "C" in the presence of 3 X I0' M [Cr (dien)(O 2 )2 ] _ Loeffler's methylene blue stain, x 1000.
CHROMIUM AND COBALT COMPLEXES ON BACTERIA
239
4. E.M. Hodnet and C .H. Moore, J. Med. Chem. 14, 1121 (1971). 5. G.M. Kolodny, Exp. Celt Res . 73, 378 (1972) . 6. G.R. Gale, L .M. Atkins, E .M. Walker, A.B . Smith, and S .J. Meishen, Proc Soc. Exp. Blot Med. 142, 1349 (1973)7. T . Giraldi, G. Zassinovich, and G . Mestroni, Chem-BioL Interact_ 9, 389 (1974). 8. C .M. Bragadin, T. Giraldi, M . Cantini, G. Zassinovich, and G . Mestroni, F E.B.S. Letters 43, 13 (1974) . 9. B.J. Crawford, D.E. Talburt, and D .A . Johnson, Bioinorg Chem. 3, 121 (1974)10. H. Simon, Diss. Abst. Inter. B 35, 1552 (1975) . 11. D.E . Hunt, R.F. Pittillo, J. Bact. 95, 712 (1968)12- R.B . Roberts, P.H. Abelson, D .B . Lownie, E.T. Bolton, and R .J. Britlen, Carnegie Inst . Wash. Publ. 607, 1955, p . 513. D.A. House and C .S. Garner, J. Am Chem. Soc. 88, 2156 (1966) . 14. D.A. House and C .S. Garner, Nature 208, 776 (1965). 15. D.A . House and C .S. Garner, Inorg. Nuc Chem. Letters 1, 137 (1965) . 16. C .L. Rollinson and J.C . Bailar, J. Am. Chem . Soc- 65, 250 (1943)17. F. Hein and S . Herzog, in Handbook of Preparative Inorganic Chemistry (G . Brauer, Ed), Vol . 2, p. 1354. 18. F . Basolo, J. Am Chem. Soc. 70, 2634 (1958) . Received 28 May 1975
