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FT IR Studies on the active component of hematoporphyrin derivative
Volume125, number 4
CHEMICAL PHYSICS
LETTERS
11 April 1986
FT IR STUDIES ON THE ACTIVE COMPONENT OF HEMATOPORPHVRIN DERIVATIVE S.H. MODIANO and B.T. LIM Department
of Chemistry,
Wayne State University, Detroit, MI 48202, USA
Received 20 December 1985; in final form 14 January 1986
It is shown by FT IR that neither a dihematoporphyrin ether nor a dihematoporphyrin structure for the major component of photofrin II which is used in phokradiation therapy.
1, Introduction Since the early 1960’s, the hematoporphyrin derivative (HpD) has provided clinical investigators a sensitive means of diagnosis and a selective method for eradication of tumors [ 1,2]. Its emerging importance in photoradiation therapy is evidenced by research efforts directed at the fundamental problems related to the mechanism of photosensitization and the increasing clinical applications for cancer therapy. One of the most important problems involving HpD is the chemical identity of the tumor-localizing component(s). HpD, prepared for clinical use, is derived from hematoporphyrin by its acetylation using a mixture of acetic and sulfuric acid followed by alkaline hydrolysis of the acetylated product. The procedure produces a complex mixture of porphyrins which can be separated into an active and inactive fraction by either gel filtration or HPLC. The inactive fraction has been shown to contain hemotoporphyrin (HP), hydroxyethylvinyldeuteroporphyrin (l-IV), and protoporphyrin (PP) [3-g]. The identity of the active component is not totally resolved. Dougherty et al. [lo] identified the active component of HpD as a dihematoporphyrin ether (DHE) which is an hydroxyethylvinyldeuteroporphyrin molecule and a hematoporphyrin molecule joined by an ether linkage at the 2 and 4 positions on the porphyrin ring. ln addition to the dimeric ether, the active component has been characterized as an aggregated fraction which can be hydrolyzed to known porphyrins [ 1I] and as a metal 0 009.2614/86/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
ester can be supported as the
complex involving zinc and Hp [ 121. A suggestion has also been made that the dimer linkage is an ester linkage rather than an ether bond [ 131. In this report we wish to reexamine the identity of the active component of HpD utilizing FT IR which allows the structural features of molecules to be determined directly. It will be shown that no spectral support was obtained for the dimeric ester as a major component of HpD, nor was the presence of an ether linkage clearly demonstrated.
2. Experimental HpD was obtained from Photofrin Medical Inc., Raritan NJ, under the trade name of Photofrin II in the form of an injectable solution in isotonic saline at the concentration of 2.5 mg/cc. Removal of the solvent from Photofrin II was performed with lyophilization. Hematoporphyrin dihydrochloride was purchased from Sigma Chemical Co., St. Louis MO. Protoporphyrin was kindly provided by D. Kessel of Wayne State University, Department of Medicine and Pharmacology. Deuteroporphyrin dimethylester was purchased from Aldrich Chemical, Milwaukee, WI. All infrared spectra were taken using a Nicolet FT IR 20 DX spectrophotometer. The compounds were examined as solids in KBr disks.
333
CHEMICAL PHYSICS LETTERS
Vohme 125, number 4
3. Results and discussion Figs. 1 and 2 show the FT IR spectra of the monomers (presented for comparison) and Photofrin II (PH II) over the range of 4600-400 cm-l. The spectra for protoporphyrin, deuteroporphyrin dimethyl ester, and hematoporphyrin are consistent with the data which has already been reported [ 141. HEMATOPORPHYRIN
‘ROTOPORPHYR
IN
IEUTEROPORPHYRIN ~IMETHYL ESTER
Fig. 1. The FT IR spectra of hematoporphyrin, ptotoporphyrin, and deuteroporphyrin dimethylester dispersed in KBr pellets.
334
11 April 1986
When the spectrum of PH II is compared with those of the other porphyrins, the characteristic carbonyl group frequency which is readily seen in the monomers is absent in PH II. What is seen instead are two strong bands at 1563 and 1403 cm-l. It is apparent that these bands result from the formation of carboxyl salts made from the carboxylic acid functional groups in PH II. The band at 1563 cm-l is due to the strong asymmetric OCO stretching vibration and the band at 1403 cm-l is due to the weaker symmetric stretch [ 151. Also, the absence of a carbonyl group frequency at 1730 cm-1 attributable to an ester linkage leads to the conclusion that the ester-linked dimeric structure is incorrect. Assignments for some of the other bands in PH II include the NH stretch at 33 12 cm-l [ 161, the asymmetric and symmetric methyl C-H stretch, 2967 and 2922 cm-l respectively, and the out-of-plane deformation of the methane, CH, at 832 cm-l [ 141. One additional feature noted for the spectrum of PH II is the absence of the strong, broad band due to the C-O-C asymmetric stretch in the range of 11401085 cm-l usually found for ethers [15]. Although it can be argued that this band may not be observable in symmetrical ether compounds, this band was found in ar-methylbenzyl ether, a model compound used in comparison with PH II (spectrum not shown). The most significant result of the PH II spectrum is the absence of two strong bands; the one near 1730 cm-l due to the carbonyl of an ester and the other in the range of 1140-1085 cm-1 indicating the presence of an ether. Since neither band was present, we can conclude that no evidence was found from the infrared spectroscopic data to support either the ester or the ether-linked dimeric structure as the major active component of HpD. The infrared spectroscopic data aside, the evidence upon which the chemical structures of the active component are based is neither overwhelming nor unambiguous. It is possible that the active component has not been sufficiently resolved to allow the determination of its detailed chemical structure. Since FT IR does not lend added support to the proposed structures, other approaches are necessary to further characterize the chemical identity of the active component.
Volume 125, number 4
CHEMICAL PHYSICS LETTERS
PHOTOFRIN
moo.
0
POOO.
11
0
POOO.
0
ZPOO.
1000.0
0
YAVPNIJWBERS
Fig.
1400.0
IOOO.0
800.00
600.00
4oo.oa
us-l>
2. The FT IR spectrum of Photofrin II dispersed in KBr pellets.
Acknowledgement This work was supported
11 April 1986
by N.I.H. Grant 1 R01
CA 34075.
References [l] R. Lipson, E. Baldes and A. Olsen, J. Natl. Cancer Inst. 26 (1961) 1. [2] T. Dougherty, I. Kaufman, A. Goldfarb, K. Weishaupt, D. Boyle and A. Mittleman, Cancer Res. 38 (1978) 2628. [ 31 R. Bonnet, R.J. Ridge and P.A. Scourides, J. Chem. Sot. Perkin I(1981) 3135. [4] M.C. Berenbaum, R. Bonnet and P.A. Scourides, Brit. J. Cancer 45 (1982) 571. [5] J. Moan, T. Christensen and S. Sommer, Cancer Letters 15 (1982) 161. [6] D. Kessel and T.C. Chou, Cancer Res. 43 (1983) 1994. [7] A.G. Swincer, V.C. Trenerry and A.D. Ward, in: Porphyrin localization and treatment of tumors, eds. D.R. Doiron and C.J. Comer (Liss, New York, 1984) pp. 285-300.
[8] P.A. Cowled, I.J. Forbes, A.G. Swincer, V.C. Trenerry and A.D. Ward, Photochem. Photobiol. 41 (1985) 445. [ 91 D. Kessel and ML. Cheng, Photochem. Photobiol. 41 (1985) 277. [lo] T. Dougherty, W. Potter and K. Weishaupt, in: Porphyrin localization and treatment of tumors, eds. D.R. Doiron and C.J. Comer (Liss, New York, 1984) pp. 301-304. [ll] A. Andreoni and R. Cubeddu, Chem. Phys. Letters 100 (1983) 503. [ 121 A.A. Lamola and M. Sassaroll, Chem. Phys. Letters 112 (1983) 539. 1131 D. Kessel, private communication, Department of Medicine and Pharmacology, Wayne State University. [ 141 W.S. Caughey, J.O. Alben, W.Y. Fujimoto and J.L. York, J. Org. Chem. 31 (1966) 2631. [ 151 N.B. Colthup, L.H. Daly and S.E. Wiberley, Introduction to infrared and Raman spectroscopy (Academic Press, New York, 1964). (161 S.F. Mason, J. Chem. Sot. (1958) 976.
335
CHEMICAL PHYSICS
LETTERS
11 April 1986
FT IR STUDIES ON THE ACTIVE COMPONENT OF HEMATOPORPHVRIN DERIVATIVE S.H. MODIANO and B.T. LIM Department
of Chemistry,
Wayne State University, Detroit, MI 48202, USA
Received 20 December 1985; in final form 14 January 1986
It is shown by FT IR that neither a dihematoporphyrin ether nor a dihematoporphyrin structure for the major component of photofrin II which is used in phokradiation therapy.
1, Introduction Since the early 1960’s, the hematoporphyrin derivative (HpD) has provided clinical investigators a sensitive means of diagnosis and a selective method for eradication of tumors [ 1,2]. Its emerging importance in photoradiation therapy is evidenced by research efforts directed at the fundamental problems related to the mechanism of photosensitization and the increasing clinical applications for cancer therapy. One of the most important problems involving HpD is the chemical identity of the tumor-localizing component(s). HpD, prepared for clinical use, is derived from hematoporphyrin by its acetylation using a mixture of acetic and sulfuric acid followed by alkaline hydrolysis of the acetylated product. The procedure produces a complex mixture of porphyrins which can be separated into an active and inactive fraction by either gel filtration or HPLC. The inactive fraction has been shown to contain hemotoporphyrin (HP), hydroxyethylvinyldeuteroporphyrin (l-IV), and protoporphyrin (PP) [3-g]. The identity of the active component is not totally resolved. Dougherty et al. [lo] identified the active component of HpD as a dihematoporphyrin ether (DHE) which is an hydroxyethylvinyldeuteroporphyrin molecule and a hematoporphyrin molecule joined by an ether linkage at the 2 and 4 positions on the porphyrin ring. ln addition to the dimeric ether, the active component has been characterized as an aggregated fraction which can be hydrolyzed to known porphyrins [ 1I] and as a metal 0 009.2614/86/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
ester can be supported as the
complex involving zinc and Hp [ 121. A suggestion has also been made that the dimer linkage is an ester linkage rather than an ether bond [ 131. In this report we wish to reexamine the identity of the active component of HpD utilizing FT IR which allows the structural features of molecules to be determined directly. It will be shown that no spectral support was obtained for the dimeric ester as a major component of HpD, nor was the presence of an ether linkage clearly demonstrated.
2. Experimental HpD was obtained from Photofrin Medical Inc., Raritan NJ, under the trade name of Photofrin II in the form of an injectable solution in isotonic saline at the concentration of 2.5 mg/cc. Removal of the solvent from Photofrin II was performed with lyophilization. Hematoporphyrin dihydrochloride was purchased from Sigma Chemical Co., St. Louis MO. Protoporphyrin was kindly provided by D. Kessel of Wayne State University, Department of Medicine and Pharmacology. Deuteroporphyrin dimethylester was purchased from Aldrich Chemical, Milwaukee, WI. All infrared spectra were taken using a Nicolet FT IR 20 DX spectrophotometer. The compounds were examined as solids in KBr disks.
333
CHEMICAL PHYSICS LETTERS
Vohme 125, number 4
3. Results and discussion Figs. 1 and 2 show the FT IR spectra of the monomers (presented for comparison) and Photofrin II (PH II) over the range of 4600-400 cm-l. The spectra for protoporphyrin, deuteroporphyrin dimethyl ester, and hematoporphyrin are consistent with the data which has already been reported [ 141. HEMATOPORPHYRIN
‘ROTOPORPHYR
IN
IEUTEROPORPHYRIN ~IMETHYL ESTER
Fig. 1. The FT IR spectra of hematoporphyrin, ptotoporphyrin, and deuteroporphyrin dimethylester dispersed in KBr pellets.
334
11 April 1986
When the spectrum of PH II is compared with those of the other porphyrins, the characteristic carbonyl group frequency which is readily seen in the monomers is absent in PH II. What is seen instead are two strong bands at 1563 and 1403 cm-l. It is apparent that these bands result from the formation of carboxyl salts made from the carboxylic acid functional groups in PH II. The band at 1563 cm-l is due to the strong asymmetric OCO stretching vibration and the band at 1403 cm-l is due to the weaker symmetric stretch [ 151. Also, the absence of a carbonyl group frequency at 1730 cm-1 attributable to an ester linkage leads to the conclusion that the ester-linked dimeric structure is incorrect. Assignments for some of the other bands in PH II include the NH stretch at 33 12 cm-l [ 161, the asymmetric and symmetric methyl C-H stretch, 2967 and 2922 cm-l respectively, and the out-of-plane deformation of the methane, CH, at 832 cm-l [ 141. One additional feature noted for the spectrum of PH II is the absence of the strong, broad band due to the C-O-C asymmetric stretch in the range of 11401085 cm-l usually found for ethers [15]. Although it can be argued that this band may not be observable in symmetrical ether compounds, this band was found in ar-methylbenzyl ether, a model compound used in comparison with PH II (spectrum not shown). The most significant result of the PH II spectrum is the absence of two strong bands; the one near 1730 cm-l due to the carbonyl of an ester and the other in the range of 1140-1085 cm-1 indicating the presence of an ether. Since neither band was present, we can conclude that no evidence was found from the infrared spectroscopic data to support either the ester or the ether-linked dimeric structure as the major active component of HpD. The infrared spectroscopic data aside, the evidence upon which the chemical structures of the active component are based is neither overwhelming nor unambiguous. It is possible that the active component has not been sufficiently resolved to allow the determination of its detailed chemical structure. Since FT IR does not lend added support to the proposed structures, other approaches are necessary to further characterize the chemical identity of the active component.
Volume 125, number 4
CHEMICAL PHYSICS LETTERS
PHOTOFRIN
moo.
0
POOO.
11
0
POOO.
0
ZPOO.
1000.0
0
YAVPNIJWBERS
Fig.
1400.0
IOOO.0
800.00
600.00
4oo.oa
us-l>
2. The FT IR spectrum of Photofrin II dispersed in KBr pellets.
Acknowledgement This work was supported
11 April 1986
by N.I.H. Grant 1 R01
CA 34075.
References [l] R. Lipson, E. Baldes and A. Olsen, J. Natl. Cancer Inst. 26 (1961) 1. [2] T. Dougherty, I. Kaufman, A. Goldfarb, K. Weishaupt, D. Boyle and A. Mittleman, Cancer Res. 38 (1978) 2628. [ 31 R. Bonnet, R.J. Ridge and P.A. Scourides, J. Chem. Sot. Perkin I(1981) 3135. [4] M.C. Berenbaum, R. Bonnet and P.A. Scourides, Brit. J. Cancer 45 (1982) 571. [5] J. Moan, T. Christensen and S. Sommer, Cancer Letters 15 (1982) 161. [6] D. Kessel and T.C. Chou, Cancer Res. 43 (1983) 1994. [7] A.G. Swincer, V.C. Trenerry and A.D. Ward, in: Porphyrin localization and treatment of tumors, eds. D.R. Doiron and C.J. Comer (Liss, New York, 1984) pp. 285-300.
[8] P.A. Cowled, I.J. Forbes, A.G. Swincer, V.C. Trenerry and A.D. Ward, Photochem. Photobiol. 41 (1985) 445. [ 91 D. Kessel and ML. Cheng, Photochem. Photobiol. 41 (1985) 277. [lo] T. Dougherty, W. Potter and K. Weishaupt, in: Porphyrin localization and treatment of tumors, eds. D.R. Doiron and C.J. Comer (Liss, New York, 1984) pp. 301-304. [ll] A. Andreoni and R. Cubeddu, Chem. Phys. Letters 100 (1983) 503. [ 121 A.A. Lamola and M. Sassaroll, Chem. Phys. Letters 112 (1983) 539. 1131 D. Kessel, private communication, Department of Medicine and Pharmacology, Wayne State University. [ 141 W.S. Caughey, J.O. Alben, W.Y. Fujimoto and J.L. York, J. Org. Chem. 31 (1966) 2631. [ 151 N.B. Colthup, L.H. Daly and S.E. Wiberley, Introduction to infrared and Raman spectroscopy (Academic Press, New York, 1964). (161 S.F. Mason, J. Chem. Sot. (1958) 976.
335