COMMUNICATIONS density in a photometer. The concentrations of ligand I . complex 2, a 1:cisplatin mixture, cisplatin, and photofrin were lo-' in01 L- I . Photofrin, an oligomer fraction of hematoporphyrin derivative enriched to 80-90%. is at present the only phorphyrin used in clinical tests."] The data of Table 1 correspond to the vitality of the cells with respect t o untreated control groups, which define 100%. Ligand Free Acid Porphyrin and Its Conjugated Monoanion"" Jurgen Braun, Christian Hasenfratz, Reinhard Schwesinger, and Hans-Heinrich Limbach" Porphyrin (PorH,) and its derivatives are amphoteric, and the two inner protons are highly mobile between the four nitrogen atom sites (Scheme 1). This tautomerism takes place both in the Table 1. Cunqi;irison of the antitumor activity of hgand I . complex 2, 1,cisplatin m o l L - ' i n vitro mixture. cisplatin. and Photofrin in a concentration of touards the MDA-MB-231 minnmdtumor cell line i n water. The value? are the accragc 01 I 6 me,iwreinents. which \:tried by about 10-20%. I 2 I 'cisplatin ci\platin Phoiofi-in 110pgiiiL-') without 11-radiation \\ith irradi'ition > 100 81 63 38 14 53 49 44 100 20 1 was not active without irradiation; it became tumor-destructive only on irradiation, reducing the cell proliferation to about 81 Yo. By complexation with platinum the activity was improved. Complex 2 had cytostatic potential even without irradiation and reduced the vitality of the cells to values of approximately 63%. On irradiation the activity of the complex increased. m d t h e cell proliferation was reduced to 38 %. This demonstrates the additivity of the phototoxic effect of the porphyrin and the cytostatic effect of the platinum in the complex. Cisplatin alone and together with 1 reduced the cell proliferation to about 50%. On irradiation of the combination l/cisplatin the additional photodynamic effect of ligand 1 operated (reduction to 14%). Photofrin attained vitality values of20 % in a concentration of 10 pgmL-'. If a molecular weight of 1000 is assumed for Photofrin, the concentration is 10-TmolL-'. Rcccivcd: June 30. 1994 [Z7085 IE] German version: Aiigrii. Clicvit 1994. 106, 2305 R. Pctttiel-. P I i ~ ~ r ~ i ~ ~ ~ i i ~ Molecirlur, ; r i . \ ~ ~ t i ~Cdlulur ~~i. ntrd Mrilir 01 Aspects. S p r i n y r . Bei-lin. 1988. p. 2.  T J Dougherr). .4ih P h o t o d i e f i r . 1992. 17. 275. [:] M. Landthalcr. A. Ruck. R.-M. Szeimics. H(tttr~irt1993. 44, 69.  T, J. L>ougheriy. Plioriidicn7. P1iotoh;ol. 1987. 46. 569. [51 R. Pottier. J. c' Kennedy. J. Pho~ocliriii.Pkotohiol. B 1990. 8. I . 161 A. J. Bonnell. J. C. Kennedy. R. A Jones. P. Nadeau. R . Pottier. J. Pliorodx~in. Ph~iiohii~l B 1990. 6 . 30'1.  B. ti. Kepplrr. M~/id ('onip/c.\-r.s iii Cancw T I t ~ m i p rVCH. Weinheim. 1993. [XI B. ti. Keppler. : V m l i r . Ciicrii. 7?di. Luh. 1987, 35. 1029.  H . Ohei-meier. P1i.D. Thesis. Universitlt Regensburg. 1994. [IO] h.S C',iiighe). .I D. Alben. W. Y. Fujiinoto, J. L. York. J. Orx. (%em. 1966. 31. 163 I [ I I1 K M S m i t h . R. ti. Pmdey. F.-Y. Shim. N . W. Smith. P. lakovides. T. J Dougher11. P I O , S P l E /It!. Soc. Opt. E I I , ~1992. . 1654, 274. [I?] 1 : Virilct Ixiuder. in p. >250 C.yield 89%. I R (KBr): i = 1 7 2 0 c m - ' ( C O ) : ' H N\qI< ( 2 5 0 M H / . [DJDMSO): 6 =11.20 (s, ? H : COOH). 10.41. 10.36. 102.7. 10 I 6 14s. 4 H : = r H ) . 5.86. 5.81 (2s. 4 H ; Cff,NMe,j. 4.33 (m.4H: =CC'H,). 3.Y2. .?.XI. 3.71. 3.63 (4s. 1 2 H ; = C r H 3 ) . 3 16 ( s . 1ZH; N M e ? ) , ca. - 4 [I>IL. =iiH). [I31 H . Hiunnci-. F Maiterth, B. Treittinger, Clieni. Bcr.. i n press. 1141 2: Broun pouder, m.p. >250 C, yield 50%. I R (KBrj: i. =1560cm-i ( C O )c i n I : PI LISIMS: (glycero1:DMSO): d z : 625 [ I + HI': ' H NMR (250 Rlllz. [DJDMSO). B =10.46. 10.41, 10.32. 10.29 (4s, 4 H ; = CH). 5.30 (m.3 H . C'H,NMe,). 4.36 (m. 4 H : =CCH,). 3.94 (m. 6 H : NH,). 3 74. 3.65 ( 2 s . 1211. =CCH,). 3 IX(m.4H:O,CCH,),2.81 ( 2 s . 12H:NMe,). -3.88(s. 2 H . = N H ) . Correct CHN analysis. [I51 H Bi-unncr. F. Maitcrth. €3. Treittinger. %. .Vuiur/or.\ch. B 1992. 47. 942. [l6] Waldm;inn Lichttechnik. Type PDT 1200, distance of lamp 1.0 ni; intensity 5 0 niU'ciii-" pmrer 60 Jcm-': R.-M. Szeimies. R. Hein. W Biumler. A . Heinc. bl L.indth.iler. Acrrr Drwii. L+no.eol. 1988. 74. 31 5. [l ] It 11 It tl Scheme 1. The tautomerism of porphyrin PorH, according to ref. [ic.i.j] liquid['.21and in the solid stater3.'] and has been studied from a theoretical standpoint as a model reaction for double proton transfer in polyatomic molecule^.^^^ The basicity of PorH,, which led to the frequently used name "metal-free base porphyrin", enables a protonation by acids generating the stable dication PorH:+,[i'."l probably via a monoprotonated intermediate PorH:. The acidity of porphyrin is manifested in the release of two protons during the formation of metalloporphyrins. However, so far the term "free acid porphyrin" has not been justified because the conjugated metal-free monoanion PorH (Scheme 2)-judged as unstable['] has never been observed experimentally. This paper shows that PorH- is a stable species which can easily be generated from PorH, by reaction with very strong organic bases in aprotic medium, and can be chardcterized by NMR and UVjVIS spectroscopy. Moreover. it is shown that PorH- is also subject to a degenerate intramolecular tautomerisin of the remaining single proton (Scheme 2 a). The dynamics observed for this process shed new light on the mechanism of the porphyrin tautomerism. [*I Prof. Dr. H.-H. Limbach. Dr. J. [**I Brdun Institut fur Organische Chemie der Freien Universitit Tdkusstrassc 3. D-14195 Berlin (FRG) Tclefax: Int. code +(30) 838-5320 Prix .-Do/. Dr. R. Schuesinger. DiplLChem. C . HasenfratL Institut f i r Organische Chemie der Universitit Freiburg Thls work way supported by the Deutsche Forschtingsgemein~chaftand the Fonds der Chemischen Industrie. COMMUNICATIONS a) 3 5 7 1300 254 200 1300 t-hept-P4Hi Scheme 2. a ) The tautomei-ism o f t h c conjugated porphyrin monoanion PorH-. h) Structure of the protonated phospharene basc t-hept-P4H + . A - < 10 To avoid complications in the N M R experiments arising from I4N quadrupole relaxation. "N-labeled porphyrin['] was employed as a precursor of PorH-. The choice of the base was critical. Conventional inorganic bases immediately form metalloporphyrins such as PorM - M or PorM;['I neutral organic bases Bare required which. on one hand. must be strong enough to remove an inner proton of PorH,. On the other hand, the structure of the base should be such that fast intermolecular proton transfer between BH+ and PorH- is slow on the N M R time scale. Higher phosphazene bases, for example. "t-heptP4"= 1.I .I ,5,5.5-hexakis(dimethylamino)-3-(1 ,I .2,2-tetramethylpropyl)imino - 3 - [tris(dimethylamino)phosphoranylidene]amino1 1.',3i5.51',-1 .4-triphosphazadiene (shown in its protonated form i n Scheme 2 b) were suitable. The syntheses and properties of phosphazene bases have been described recentIy.['l Figure 1 a depicts the superposed experimental and calculated N M R signals of the inner protons of "N-labeled porphyrin in [D,]THF.[3j. l o ] Because of the low solubility of PorH,, the concentration was only 1 mM. As established previ~usly,[~j] the signal of the inner protons of PorH, appears at 6 = -3.5 and exhibits a doublet at low temperature, arising from scalar coupling with the attached 'N nucleus and indicating localized inner protons. The coupling constant is 'JjH, 1 5 N z 98 Hz and decreases slightly with increasing temperature. A differential line broadening of the 'H-"N doublet, observed at low temperature, arises from reduced molecular mobility and interference between the 'H-'H and the 'H-"N dipolar interaction, as well as the chemical shift anisotropy." As the temperature is increased. rotational diffusion becomes faster. and the asymmetry disappears. At the same time, a doublet-pentet transition is observed at about 250 K, arising from a fast intramolecular proton transfer between all four nitrogen atoms. The line-shape changes were calculated['". 3h1 by varying the rate constant 2kHH, which corresponds to the inverse average lifetime of a given tautomer in Scheme 1. In the presence of phosphazene bases, the solubility of porphyrin is drastically increased. The superposed experimental and calculated high field signals of a 15 mM solution of PorH, in [DJTHF in the presence of a twofold excess of t-hept-P4 are depicted in Figure 1 b. Signs of PorH, can no longer be detected, but a new species is observed. At 152 K again a 'H I 'N doublet is observed at Ci = - 2.7 typical for- a n inner porphyrin proton are similar to (Fig. 1 b). The scalar coupling constants 'J,H,15N those of the parent compound. The differential line broadening is more pronounced as the spectra were taken at lower temper+ - -3.5 & -4 6/PPm 7 -2.5 -3 6/PPm Fig. 1. Superposed experimental and calculated variable-temperature 500 M H r ' H NMR signals of the inner protons of "N-labeled PorH, dissolved in [DJTHF (a) (adapted from ref. [3j]) and PorH- (15 mki) generated by dissolving the parent compound together with a twofold excess of t-hept-P4 ( b ) . The sample contained approxnnarively 34% PorD- which doea not contribuie 10 the signals. 6.5 ps 60 pulses, 9 kHr spectral width. 3.5 f repetition time. up to 3000 scans for (a) ;ind 600 for (b) [lo]. atures. As the temperature is raised, the doublet-pentet transition proving a fast intramolecular tautomerism of a central porphyrin proton could be observed as in the parent compound; however, the transition occurs already around 200 K instead of 250 K, which indicates that the tautomerism of the new porphyrin species is much faster than in the parent compound. This observation can only be explained by the formation of a stable conjugated monoanion PorH -. Its structure is confirmed by analyzing the signals of protons attached to the peripheral carbon atoms. At 152 K, two exchange-broadened lines arising from the rnethine protons 5H!20H and 1 0 H / l 5 H appear at 6 = 9.79 and 9.73 (PorH,: 6 =10.5['h1). Furthermore, as expected, four exchange-broadened singlets appear at 6 = 9.04. 8.90, 9.1 5, and 8.93 for the fl-pyrrole protons, which are tentatively assigned to the 7H/18H, 8H/17H, 2 H / 3 H , and 12H/ 13 H positions. These signals broaden and coalesce into a single line at about 200 K. In contrast, at low temperatures PorH, contributes only two j7-pyrrole signals to the ' H spectra.[lhlThe N M R signals of t-hept-P4 and t-hept-P4H+ were not analyzed. Subsequent N M R experiments showed that the formation of conjugated porphyrin monoanion is not restricted to the parent compound but is a common feature of substituted porphyrins. for example, tetraphenylporphyrin (TPP) and octaethylporphyrin (OEP). The changes in the UVjVIS and fluorescence spectra upon deprotonation of PorH, are illustrated in Figure 2. The spectra of PorH, (Figs. 2a and 2b) were previously recorded and discussed.['. The transition of lowest energy (Q, band, So + S;) appearing for PorH, at 615 nm is shifted in PorH- to 600 nm (Fig. 2c). The assignment to the 0-0 transition is confirmed by the fluorescence data (Fig. 2 d ) . No evidence of a band calculated to appear at 700 nrn["'could be found, but the Soret band of PorH, at 390 nm is shifted to 420 nm in the monoanion and is more intense. In addition, major spectral changes are observed in the region between 480 and 580 nm. COMMUNICATIONS Gerritren. H. Rumpel, Furudur Disc.um C h i r . S w . 1982. 74, X22. I) P. Stilbs. M. E. Moseley. J. Chein. So< Furudui. Ti-uns. 2 1980. 76, 729: m ) P. Stilbs, J. Mugn. R e o n . 1984, 58, 152: n) I. Hennig. H.-H. Limbach. J. A i n Chem Soc. 1984. 106. 869; o ) H.-H. Limbach. Di.ironm Y M R Spairrouipi'In rhc Pre\cn(e of Kinrfic Isuropp~Effccr.s ( N M R B[I\ICPrint.. Pi-og 1990. 26, Chap. 2). a) M. Schlabach, B. Wehrle. H -H. Limbach. E. Bunnenberg. A. Knierzinger. A Y. L. Shu. B. R. Tolf, C. Djerassi. J Ani. Chrm Suc. 1986. /OX. 3x56: b) M . Schlabach. H. Rumpel. H.-H. Limbach. AnReii C/iiwi. 1989. 101. X4; Anxcii.. C'hcrn. b 7 r . Ed. EngL 1989.28.78: c ) M. Schlobach. H.-H I.imbach, E. Bunnenberg. A. Y. L. Shu. B. R. Tolf. C. Djer2lssi. J. An?. Chmii. SJC. 1993. (15. 4554: d ) M J. Crosswell. M. M. Hal-ding. S. Sternhell, /hid 1986. I O X , 3608; c ) M . J. Crosswell, L. D. Field, M. M. Harding, S Sternhell, ;hid 1987. IOY. 2335. a) H.-H. Limbach. J. Hennig, R. D Kendrick. C. S. Ymnoni. J. A m C/JNII. 600 700 Sor. 1984. 106. 4059: b) H.-H. Limbach. B. Wehrlc. H . Zimmrrinann. R. D. Kendrick. C. S. Yannoni, jhrd. 1987. 109, 929: c) A!igiw. Chrrii 1987. 99. 241 : Arigcii. Chm7. I n r . Ed. E q / . 1987. 26. 247: d ) B. Wehrle. H.-H. Liinbach. M . Kocher. 0 . Ermer. E. Vogel. ibid 1987.99.914 and 1987.26.934. c ) B. Wehrle. H. Zimmermann. H.-H. Limbach. Ber. B u n v c n g ~ Plijs. . Chrw~ 1987, 91. 941 : f') H.-H. Limbach. B. Wehrle. M. Schlabach. R D. Kendrick. C' S Yannoui. J. Mugn. Rcson. 1988. 77. 84: g ) L Frydman. A. C . Olibieri. L. E. Diaz. B. Frydman. F. G. Morin. C. L. Mayne. D M. Grant. A. D. Adler. J. A m . Chw!. S o ( , 1988. 110, 336: h) L. Frydman. A. C . Olivieri. L. E. Diad. B. A. Valasinas. B. Frydman. i h d 1988. 110, 5651 ; I ) M. Schlabach. B. Wehrlc. J. Bratin, G I 0-0 Scherer. H.-H. Limbach. BPi.. 8itfiwng<e\ P / i u Chiwi. 1992. 96, 821: j ) J Braun. M. Schlabach. B. Wehrle. M. Kocher. E Vogel. H.-H. Limbach. J. Am. C'hen7. Sue. 1994, 116. 6593. a ) S. Volker. J. H. van der Waals, ;Mu/. P h w 1976. 32. 1703, b, S Viilkcr. R. 400 500 600 700 600 700 Macfarlane, IBM J RE.T.Dei'. 1979. 33, 547. h/nm + A/nm a ) J. Almlof. Inr. J. Quunrur17 Chetn. 1974. 8. 915; b) H.-H. Limbach. J. Hennig. Fig. 2. 'I) Room temperature UV,'VIS and b) fluorescence spectra of PorH, disJ. C ' h t w P h j s 1979. 71. 3120: c) H.-H Limbach. J. Hennig. J. Stulr. i h i d . 1983. solved in T H E excitation wave length 570 nm. c) and d) Corresponding spectra of 78. 5432: d ) H.-H. Limbach. h i d 1984. 81). 5343: e) V. A. Kusmitsky. K. N . PorH- 't-tIt.pt-P4H- in T H F D optical density: I : Intensity. Solovyov. J. Mid. Slruc.1. 1980. 65. 219: f ) A. Sarai. J. Cheiw. P/jJ.\. 1982. 76. 5554; g)ihid. 1984. KO, 5431: h ) G I Bersuker. V. Z. Polinger. Chon. P h n . 1984. 86. 57: i) K. M . M e n . C. H. Reynolds, J Chrm. So(,. Chcni. Coniiwurr. 1988. 90: J ) Z . Smedarchina. W. Siebrand. T. A Wildman. Chcni. l'/ij\ Lctr. To estimate the p K , of free acid porphyrin, a titration 1988. 143, 395: k) D. C. Raalings. E. R. Davidson. M . Gouterman. Theor. Chnn. Actu 1982. 61. 227. experiment of the more soluble OEP with the base t-buJ. V. Knop. A Knop. Z. .Yulirr~orwh A 1970. 25. 1720. 1726. P4=3-r~~r.r-butylimino-l, 1,1,5.5,5-hexakis(diniethylamino)-3'%labeled porphyrin was synthesized froin "NH,CI (95",b enrichment. [tris(di1nethylamino)phosphoranylidene]a1ni~io)-1L~,3~~,5~.~-Chemotrade. Leipzig) with slightly modified versions of the procedures dc1.4-triphosphazadiene1' 31 was performed. The deprotonation of scribed for the unlabeled compounds. See also R. M. Silverstein, E. E Ryskiewicr. S. W. Chaikin. J. Am. Chmi. SCJC.. 1954. 76.4485: F. R. Longo. E. J. OEP was followed by monitoring the Soret band shift. A t conThorne. A. D. Adler, S. Dym. J. I l e w u c J d . C/iuii. 1975, 12. 1305. centrations of 0.01 M of t-bu-P4 in dry T H E O E P was deprotoJ. Arnold, D. Y. Dawson. C. G. Hoffman. J, Am. Chcm. .TM. 1993. 115. nated to approximately 85% at room temperature. From this 2707. result we extrapolate using a procedure described previously" 31 R. Schwesinger, C. Hasenfratz. H. Schlemper. L Walr, E.-M. Peters, K. Peters. H. G. von Schnering. A n g n i . C'/icrii 1993. 105. 1420; Angeii,. ('hcni. Inr. Ed. an absolute pK, value for OEP of 37 to 38 in acetonitrile. For the B i g / . 1993, 32. 1361 parent compound PorH, we expect a slightly lower value of The N M R samples were prepared on a vacuum line as described in Ref. 11 01 about 35 to 36. [ D J T H F was dried over potassium-s0dium:anthracene. In conclusion. we have shown that the conjugated monoanion a ) H. Shimidzu. J. Clieni. Pk s. 1964.40,3357: b) H. Riiterjans, E. Kaun. W. E Hull. H.-H. Limbach. Nuc/c ' A i i i b Res. 1982.10.7027:~)R. H . Griffey.C. D. PorH of porphyrin is a stable species in aprotic solvents and Poulter, Z. Yamaizumi. S. Nishimura. R. E. Hurd, J. Am. Chem. Sor. 1982. can easily be generated by dissolving the parent compound with f04. 5811 : d) M. Gueron, J. L. Leroy. R. H. Griffey. ihid. 1983, 105, 7262. an appropriate strong metal-free base. The labels "free acid a) A. Gamgee, Z. Biol. ( M u n i c h ) 1897. 34. 505: b) M. Gouterinan. J Cheni. porphyrin" and "free base porphyrin" are, therefore, equally Phvs. 1959, 30, 1 1 3 9 ; ~ibid. ) 1960. 33. 1523.d) J. Mol. S p C t r o . x . 1961, 6. 138: e) R. J. Abraham. Mu/. Phjs. 1961.4. 145; f ) H. Kuhn, W. Huber, I l r l ~ 'Chin?. . justified. The tautomerism of PorH- is much faster than that of Actu 1959. 42, 363; g) H. C. Longuet-Higgins, C. W. Rector. J. R . Platt. J PorH,. This observation can be explained by the fact that the Chem.Pl1p.s. 1950, 18, 1174: h) G . R Seely, ihid. 1957. 27. 125: i) J. E. Falk. single proton transfer in PorH- is degenerate (Scheme 2a) but Purp/iprin.c unit Metulli)purp/ijriris, Elsevier. Amsterdam. 1964: J) M. Gouterleads to c i s intermediates of higher energies in the case of PorH, man, G. E. Khalil. J. M u / . SppPcrrusc 1974. 53, 88. R. Schwesinger. H. Schlemper, A n g m . Chrm. 1987, 99. 1212, Angcw. C/wn?. (Scheme I ) .l3j1The tunneling probabilities at low temperatures hi.Ed. EngI. 1987. 26, 1167. are, therefore. much higher for PorH- than for PorH,, at com- 'M - ~ parable barrier heights. Studies of the kinetic H/D and solid state isotope effects of PorH - tautomerism. which could confirm this interpretation, are under way. Received: May 11. 1994 [Z 6928 IE] German \'ersion: Angew Cherri. 1994. 106. 2302 [l] a ) C B Storm. Y. Teklu. J. Ani. Chm?.Soc. 1972, 94, 1745; b) C. B. Storm, Y. Teklu. E A. Sokoloski. Ann. N. Y. A wd . Scr. 1973,206,631 :c) R. J. Abraham, G. E. Hawkes. K. M. Smith. Tein&i/ron Lerr. 1974. 1483: d) J. Clieni. Sue. Pcrkni Truns 2 1974. 1483, e) H. J. C. Yeh, M. Sato, I . Morishima. J Mugn. Re.soii. 1977. 20. 365; f) D Gust. J. D. Roberts. J Am. Chiun. SOC.1977. 99, 3637: 9 ) C S. Irving. A. Lapidot. J Cheni. SOC.Cheni. Corntnun. 1977. 184; h) H:H. Limbach. I. Hennig, J Cliani. Sue. F a r a d q Trrms. 2 1979, 75.752: i) J. Hennig. H.-H. Limbach. J Mrign Rerun. 1982, 49, 322: j) S. S . Eaton, G. R. Eaton. J. A m C / ~ a n ?Soc. . 1977, 99, 1601: k) H.-H. Limbach. J. Hennig. D .