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Organometallics 1995, 14,1632-1636
Ionic Titanocene(IV) a-AminoAcid Complexes of DL-Phenylalanine and DL-4-Fluorsphenylalanine: Synthesis, Characterization, and Investigation of the Antimicrobial Behavior toward Escherichia coli Inis C. Tornieporth-Oetting* Institut fur Anorganische und Analytische Chemie, Technische Universitat Berlin, Strasse des 17 Juni 135, Sekr. C2, 0-10623 Berlin, Germany
Peter S . White Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599 Received October 24, 1994@ Antitumor-active titanocene dichloride (CpsTiCl2; Cp = q5-C5H5)reacts with a-amino acids (aa) in methanol to give the titanium(IV) amino acid complexes [Cp2Ti(aa)2l2+[Cl12-(aa = DL-phenylalanine, 1; ~~-4-fluorophenylalanine, 2). Metathetical reactions of 1 and 2 with 2 (3) and [ C ~ ~ T ~ ( D L equiv of AgAsF6 yielded the ionic complexes [CpzTi(~~-Phe)2l~+[AsFs124-F-Phe)2I2+[AsF6]2-(4). All complexes have been characterized by chemical analyses (C/ WN) and NMR (lH, 14N, 19F),infrared, and Raman spectroscopy. Preliminary structural data of complex 4 are also presented: monoclinic, P21/c; a = 11.751(3), b = 20.257(3), c = 16.080(4) A; ,f3 = 110.01(0)";2 = 4. I n addition, the antimicrobial behavior (against E. coli) of all complexes was determined and is discussed in comparison with the free amino acids. It was established that not only DL-4-fluorophenylalanine but also the titanocene complex 2 and, to some minor extent, also 3 and 4 cause elongation of E. coli.
Introduction Recently we became interested in the coordination behavior of the cytostatic active titanocene dichloride toward a-amino acids in order to synthesize titanium model complexes containing biologically important ligands. All reactions of Cp2TiCl2 with glycine, Lalanine, and 2-methylalanine were carried out in aqueous methanol.lS2 The synthesized complexes, which are stable toward air and moisture, were fully characterized, and it was shown by vibrational spectroscopy and X-ray structure determination that the amino acids are bound to titanium exclusivelyvia the oxygen of the carboxylato group.lz2 Continuing these studies, we became interested in complexes containing essential amino acids such as phenylalanine. Moreover, especially fluorinesubstituted analogues of the naturally occurring amino acids and nucleic acids have become established as antiviral, antitumor, and antifungal agents, and a number of potential drugs in which fluorine substitution is a key to biological activity are under intensive s t ~ d y . ~ In these instances, the electronic or polar effects of the fluorine substituent must play a significant role in the ~ we also expression of biological a ~ t i v i t y . Therefore, included one (artificial) fluorine analogue of DL-phenylalanine, p-fluorophenylalanine, in the present study. In previous studies by other investigators it was established that peptides accessible to phenylalanine during normal biosynthesis are also accessible to p-fluorophenylalanine and that there is some suppression of protein @Abstractpublished in Advance ACS Abstracts, March 1, 1995. (1)Klapotke, T. M.; Kopf, H.; Tornieporth-Oetting, I. C.; White, P. S.Angew. Chem. 1994,106,1587-1589;Angew. Chem., Int. Ed. Engl. 1994, 33, 1518-1519. (2) Klapotke, T. M.; Kopf, H.; Tornieporth-Oetting, I. C.: White, P. S. Organometallics 1994, 13, 3628-3633. (3) Bergstrom, D. E.; Swartling, D. J. In Fluorine-Containing Molecules; Liebman, J. F., Greenberg, A,, Dolbier, W. R., Jr., Eds.); VCH Publishers: New York, 1988; pp 259-308, and references therein.
0276-733319512314-1632$09.0010
synthesis by the F a n a l ~ g u e . ~ Moreover, ,~ it has been pointed out that labile proteins are of general importance in controlling cell division and that the fundamental chemical events which regulate cell division are similar in higher organisms and in b a ~ t e r i a . ~ There,~ fore, we became interested not only in the syntheses of these complexes but also in the investigation of the biological properties toward Escherichia coli.
Experimental Section General Techniques. All reactions were carried out using Schlenk technique under argon atmosphere or were carried out in SO2 atmosphere in a two-bulb glass vessel equipped with PTFE valve^.^ Titanocene dichloride (CpzTiClz)was prepared by literature methods8 The amino acids (aa) DL-phenylalanine e ) used (DL-Phe)and DL-4-fluorophenylalanine( ~ ~ - 4 - F - p hwere as commercially available without further purification (Aldrich). Methanol (Merck) and Freon-11 (CFC13, Merck) were used as supplied. Infrared spectra were recorded using a Perkin-Elmer 580 B instrument, and Raman spectra were measured with a Jobin Yvon Ramanor U 1000 spectrometer, equipped with a Spectra Physics Kr laser (A = 647.09 nm) or a Bruker F T - I m a m a n spectrometer (IFS 66" 1061, equipped with a Nd-YAG laser (1060 nm). 'H NMR and 19F NMR spectra were obtained from a Bruker -00 instrument (200 MHz, lH; 188 MHz, 19F)and were referred to TMS or CFC13, respectively. 14NNMR spectra were recorded using a Bruker ARX400 instrument operating at 28.901 MHz and were referred to external CH3N02. Elemental analyses were performed by the TU Berlin service. (4)Wheatley, D. N.; Henderson, J. Y. Nature 1974,247, 281-283, and references therein. (5) Richmond, M. H. J . Mol. Biol. 1963, 6, 284-294. (6) Smith, H. S.; Pardee, A. B. J . Bacteriol. 1970, 101, 901-909. ( 7 ) Woollins, J. D. Inorganic Experiments; VCH: Weinheim, New York, 1994; pp 217-219. ( 8 )Wilkinson, G.; Birmingham, J. M. J . Am. Chem. Soc. 1954, 76, 4281-4284.
0 1995 American Chemical Society
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Antimicrobial Action of CpzTiClz Amino Acid Complexes Synthesis of 1. CpzTiClz (0.75 g, 3.0 mmol) and w P h e (1.00 g, 6.0 mmol) were stirred in 10 mL of CH30H at room temperature. After 20 min the precipitated light orange solid was filtered off, washed several times with CFCl3, and dried in vacuo (94%,T d W >140 "C). Anal. Calcd for CzsH3zClzNz04Ti (579.36): C, 58.1; H, 5.6; N, 4.8. Found: C, 57.2; H, 5.5; N, 4.8. 'H NMR (DzO, relative to DzO int): b 7.25-7.12 (m, C&5, lOH), 6.4U6.31 (Cp, lOH), 3.93-4.03 (m, CH, 2H), 3.20-2.90 (m, CH2,4H). I4NNMR (DzO): 6 -340.4 (s, NH3+). IR (KBr, cm-1): 3450 (ms, br), 3030 (SI, 2925 (s, br, sh), 2860 (s, br, sh), 2716 (ms, sh), 2640 (ms), 2495 (m), 1665 (SI, 1605 (m, sh), 1594 (ms), 1530 (ms), 1515 (ms), 1496 (ms), 1455 (m), 1438 (ms), 1378 (s), 1352 (ms), 1312 (s), 1272 (s), 1210 (ms), 1132 (ms), 1080 (m), 1028 (ms), 1015 (m),827 (s), 810 (ms), 760 (m), 745 (ms), 702 (s), 615 (ms), 590 (m), 541 (ms), 482 (m), 442 (m), 423 (ms). Raman (647 nm, 22 "C, 20 mW, 8 scans, cm-l): 3120 (140 "C). Anal. Calcd for C Z E H ~ O C ~ Z F Z N (615.34): ZO~T~ C, 54.7; H, 4.9; N, 4.6. Found: C, 54.1; H, 4.9; N, 4.3. 'H NMR (Dz0, re1 to DzO int): 6 7.18-6.93 (m, C&F, 8H), 6.43/ 6.34 (Cp, 10H), 4.03-3.96 (m, CH, 2H), 3.18-2.94 (m, CHZ, 4H). 14N NMR (DzO): 6 -340.2 (s,NH3+). "F NMR (DzO): b -117.3 to -117.5 (m, C6H4F). 19F(1H}NMR (DzO): b -117.4 (s, C6H4F). IR (KBr, cm-I): 3436 (ms, br), 3130 ( 6 , br), 3092 (s, br), 3025 (s, br), 2926 (s, br), 2867 (s, br), 2716 (ms), 2643 (ms), 2626 (m), 2560 (m), 2492 (m), 1660 (vs), 1605 (ms), 1593 (ms), 1583 (ms), 1530 (ms), 1510 (vs), 1447 (ms), 1438 (ms), 1420 (m), 1377 (vs), 1352 (s), 1325 (vs), 1313 (vs), 1294 (s), 1275 (vs), 1224 (s), 1215 (vs), 1198 (m), 1168 (m), 1165 (m), 1160 (m), 1131 (m), 1101 (m), 1096 (m), 1045 (m), 1028 (m), 1016 (m), 880 (m), 856 (ms), 840 (ms), 830 (vs), 790 (ms), 725 (ms), 708 (m), 638 (m), 625 (ms), 614 (s), 595 (m), 570 (m), 541 (ms), 500 (m), 436 (ms). Raman (647 nm, 22 "C, 50 mW, 8 scans, cm-I): 3193 ( 2.5a(In,t) used for calculations; absorption correction was made using DIFABS.loa~bStructure analysis and refinement: The structure was solved and refined with 82 atoms, 208 parameters, and 1525 of the 4676 independent reflections with use of NRCVAXIOasbto residuals of RF = 0.151 and Rw = 0.180. All nonhydrogen atoms were anisotropically refined. (a) Gabe, E. J.;Le Page, Y.; Charland, J.-P.; Lee, F. L.; White, P. S. J.Appl. Crystallogr. 1989, 22, 384-387. (b) Le Page, Y. J.Appl. Crystallogr. 1988,21, 983-984. (13) Bond lengths: Til-01 1.89(3),Til-03 1.96(3),01-C1 1.32(7), C1-02 1.18(7),Cl-C2 1.61(8), C2-Nl 1.40(7), C2-C3 1.35(9), C3-C4 1.45(7),C5-Fl 1.33(4),and C10-F2 1.33(3)A. Bond angles: 01-Til-03 88.0(13),01-C1-02 128(5),02-Cl-C2 119(5),C1-C2N1 105(4), Cl-C2-C3 (117(5), Nl-C2-C3 121(5), and C2-C3-C4 118(5)". (14) Motherwell, S. University Chemical Laboratory, Cambridge, U.K., 1978.
Antimicrobial Action of CpzTiClz Amino Acid Complexes
Figure 4. Structure of the dication in 4 (PLUTOplot).14 Table 1. Microbiological Activity of Ionic Titanocene Amino Acid Complexes ~
1
~~
2
10-2 lo-'
4 104
3
Io-'
m
4
10-5
m
DL-Phe DL-4-F-Phe CpZTiClz USF6 "
10-2 10-4
4 104 1.2 1.3
IO-? > 10-2
4 4 1,s 4 4 IO 1.2 1.3
10-3
IO-' 10-6 10-6
10-3
10-6 10-3
4 30 4 10 2 6
" K , = bacterial count (c = O)/bacterial count (c = i); c, bactericide contentlgg-' b, The unit gg-' means grams of solid per grams of solution. Elongation of E. coli relative to the blind sample after 72 h incubation time and c(bactericide) = 0.1 MIC. [
ration of 4 starting with enantiomerically pure amino acid led only to the formation of oily products. We are still trying to get better crystals of either 3 or 4; however, with these compounds slowly decomposing in SO2 a better solvent is still yet t o be found and is likely not possible as 3 and 4 are insoluble in methanol and most of the organic solvents (including hydrocarbons, halocarbons, alcohols, ether, tetrahydrofuran, and benzene). Microbiological Aspects. Table 1 summarizes the results from the microbiological studies and gives the minimum inhibitory concentrations (MIC)as well as the K values for bactericide contents of (0.1%,K10-3) and the K values for the lowest bactericide content i with significant inhibition (~i). For comparison, the literature data obtained for CpzTiClz and KAsF6 have also been included. Whereas neither CpzTiCl2 nor KAsF6 shows strong bactericide activity compounds 1 and DL-Phe are somewhat active, but high concentrations of the bactericide are needed to get significant inhibition of the bacterial growth. Compounds 2 and DL-4-F-Phe on the other hand have strong biological activity that was again substantially exceeded by complexes 3 and 4. It follows from these experiments, therefore, that the combination of the well-known bactericide D L - Q - F - Pwith ~ ~ the antitumor drug CpnTiClz, which does not show strong bactericide behavior, results in a complex (2) that is as active as the free fluoro amino acid is. Moreover, the metathetical exchange of the chloride counterions in 1 and 2 by the very large and weakly basic octahedral AsF6- ions afforded the ionic titanocene complexes 3 and 4, which are highly active bactericides. (N.B. Neither KAsF6 nor free DL-phenylalanine shows strong inhibition of bacterial growth.)
Organometallics, Vol. 14,No. 4,1995 1635
In previous studies (protein synthesis) by other investigators it has been established that peptides accessible to phenylalanine during normal biosynthesis are also accessible to p-fluorophenylalanine and that there is some suppression of protein synthesis by the F a n a l ~ g u e .Moreover, ~~~ it has been pointed out that labile proteins are of general importance in controlling cell division and that the fundamental chemical events that regulate cell division are similar in higher organisms and in b a ~ t e r i a . ~In, ~this context, it is very interesting that the coordination of DL-6fluorophenylalanine to the titanocene fragment CpzTi, which is supposed to be the antiproliferative active moiety in antitumor titanocene complexes,15results in a complex (2) that also displays strong bactericide properties. Moreover, the replacement of the chloride ions in 2 by weakly basic AsF6- anions in 4 greatly enhanced the bacteriocidal activity. Whereas in the solid state of titanocene amino acid complexes of the type [CpzTi(aa)zl[C& there are strong cation-anion interactions with contacts of all hydrogen atoms of the NH3+ groups to chloride the related h F 6 - salts [CpzTi(aa)zl[AsF& do not show significant cation-anion interactions. This is probably due to the extremely low basicity of the AsF6- ions (which is only exceeded by "superanions" like [(OTeF5)6El-, E = As,Sb, Bill6 and may very well explain the different biological behavior of 1 vs 3 and 2 vs 4, respectively. The finding of elongation of E. coli by Rosenberg et a1.l' was the first hint to the biological activity of inorganic platinum complexes (cisplatin) and led t o the detection of pronounced antitumor effectivity against animal and human tumors.lg It was also noticed that under treatment of E. coli with organobismuth compounds the bacteria became elongated, and eventually, the antiproliferative activity of this class of compounds P ~ ~added , to cultures was e ~ t a b l i s h e d .D ~ ~L - ~ - F -when of E. coli in the exponential phase, was also reported to cause a transformation of exponential into linear With the present study we confirm these observations. Moreover, we have been able to show that not only D L - C F - Pbut ~ ~also the titanocene complex 2 and, to some minor extent, 3 and 4 cause elongation of E. coli (Table 1). Elongation is generally interpretated as evidence that the agents are effecting cell division and DNA replication. Therefore, further investigations of the coordination behavior of these complexes to DNA is desirable and should be done in future studies.
Summary This study allows the following conclusions to be drawn: (i)the reaction of CpzTiCl2 with DL-Phe and DL(15)Kopf-Maier, P.In Metal Complexes in Cancer Chemotherapy; Keppler, B. K., Ed.; VCH: Weinheim, New York, 1993;pp 259-296, and references therein. (16)Mercier, H. P. A.; Sanders, J. S. P.; Schrobilgen, G. J. J . Am. Chem. Soc. 1994, 116,2921-2937. (17)Rosenberg, B.;Camp, L.; Krigas, T. Nature 1965, 205, 698699.Rosenberg, B.;Camp, L.; Grimley, E. B.; Thomson, A. J. J . Biol. Chem. 1967, 242, 1347. Rosenberg, B.; Renshaw, E.; Camp, L.; Hartwick, J.; Drobuik, J. J. Bacteriol. 1967, 93,716. (18)Rosenberg, B.;Camp, L.; Trosko, J. E.; Mansour, V. H. Nature 1969,222, 385-386. Rosenberg, B.Cancer 1985,55, 2303. (19)Klapotke, T. M. Biol. Met. 1988, 1 , 69-76, and references therein. (20)Cohen, G. N.;Munier, R. Biochim. Biophys. Acta 1959,31,347356. (21)Gowik, P.;Klapotke, T. M. Monatsh. Chem. 1989, 120, 711714.
Tornieporth-Oetting and White
1636 Organometallics, Vol. 14, No. 4,1995
4-F-Phe led to the preparation of the first titanocene complexes (1, 2) containing essential amino acids; (ii) the metathetical reaction of 1and 2 with AgAsFs yielded the corresponding titanocene amino acid hexafluoroarsenate salts 3 and 4, which unlike all known chloride complexes contain both in solution and in the solid state “free”[Cp2Ti(aa)zl2+cations with no significant cationanion interactions; (iii) for all new complexes 1-4 the bactericide activity toward E. coli was established and increases in the order 1 < 2
