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New Water-soluble pyropheophorbide a derivatives as possible agents for photodynamic therapy of cancer
Tetrahedron Letters, Vol.32, No.38, pp 5107-5110, 1991 Printed in Great Britain
0040-4039/91 $3.00 + .00 Pergamon Press pie
New Water-soluble Pyropheophorbide a Derivatives as Possible Agents for Photodynamic Therapy of Cancer Tomoyuki Ando, Yoko Suzuki, Rieko Geka, Kazuhiro Irie,* Koichi Koshimizu, Takeshi Takemura,a Susumu Nakajimab and Isao Sakatac Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan aRe.search Institute of Applied Electricity, HokkaidoUniversity,Sapporo 060, Japan bDivision of Surgical Operation, Asahikawa Medical College, Asahikawa 078, Japan CToyo-Hakka Co. Ltd., Okayama 719-03, Japan
Key Words: Photodynamic therapy (PDT); photosensitizer; pyropheophorbide a (pyroPPBa); tumor retention; rapid clearance. Abstract: Five new water-soluble pyroPPBa derivatives, whose vinyl groups at position 2 were converted into hydrophilic groups,
were synthesized, and their in vivo tumor retention was measured by the N2-pulsed laser spectrofluorometry method. The results suggested that these derivatives were advantageous to reduce hyperphotosensitivity in skin, a major side effect of photodynamic therapy.
Photodynamic therapy (PDT) has been proved useful in the treatment of cancer.1 Hematoporphyfin derivative (HPD), which is obtained from hematoporphyrin by acetylation and subsequent alkaline hydrolysis, is a photosensitizer used universally for PDT.1 However, HPD has several disadvantages: relatively weak absorption (e 5,000) at 630 nm and complexity as a mixture of undefined porphyrins and their dimers and/or oligomers. 1 A great number of dyes such as phthalocyanine, purpurin, bactefiochlorin, chlorin e6 and its aspartyl derivative (MACE) have been reported as new potential photosensitizers in the last several years.1 Among these dyes, pheophorbide a (PPBa) (1), which is a degradation product of chlorophyll a, has received increasing attention because of its strong absorption (e 50,000) at 670 nm, high production of 102 and abundance in green plants.2 Since the proton at position 10 of PPBa (1) is activated as an cx-proton of [~-keto ester to cause epimerization, cleavage offing V and oxidative degradation in the presence of a base,3 we selected a more stable pyropheophorbide a (pyroPPBa) (2a) as a lead compound to generate new agents for PDT.
In this
communication we describe the syntheses of new water-soluble pyroPPBa derivatives, their physicochemical properties (absorption, fluorescence and triplet lifetime) and in vivo tumor retention. Since pyroPPBa (2a) is hardly soluble in water, introduction of hydrophilic groups into 2a was carried out to increase its water-solubility. First, dicyclohexylamine (DCHA) salt of 2a was condensed with L-aspartic acid dimethyl ester hydrochloride using water soluble carbodiimide (WSC) to give 3a, whose alkaline hydrolysis resulted in the corresponding 7-aspartyl derivative (3b). However, the water-solubility of 3b was not enough for in vivo bioassay, indicating that the vinyl group at position 2 of 2a must be additionally converted into some
hydrophilic groups. Compound 4b with a 2'-hydroxyethyl group at position 2 was therefore synthesized from
5107
5108
,--OR R
|t!
COR'
1 2a 2b 3a 3b
COR'
R CO2Me H H H H
R' OH OH OMe Asp(OMe)2 Asp
CO2R' R H H Ac Ac H A~ H
4a 4b 5 7a 7b 8a 8b
R' OMe OH OH Gly(OEt) Gly Asp(OMe)2 Asp
R CHO CO2H CO2H COAsp(OMe)2 COAsp COGIy(OEt) COGly
6 9a 9b 10 a 10 b 1 la 11 b
R t
1Vie H Me H Me H
Abbreviations; Asp(OMe)2: NHCH(CO2Me)CH2CO2Me, Asp: NHCH(CO2FI)CH2CO2H, Gly(OEt): NHCH2CO2Et, Gly: NHCH2CO2H, Ac: OCOMe.
Table 1. Photochemical Properties of Water-soluble pyroPPBa Derivatives in the Aquatic Media.a
Compound
Visible absorption Fluorecence (emission) 0% HSAb 3%HSAc 0% lISAb 3%HSAc (Soret band) (Qband) (Soretband) (Q band) )maax (nm) (nm) (nm) (nm) 2~max(nm) Of (nm) Of
Triplet lifetimec,d (ms)
7b
380
660
413
662
653
0.02
666
0.7
1.4
8b
380
660
415
662
660
0.05
667
1.0
1.5
9b
380
660
413
666
660
0.05
675
1.0
1.5
10b
380
663
413
668
662
0.1
674
1.0
1.3
lib
375
664
414
668
661
0.05
676
1.0
1.7
aAbbreviations; HSA: human serum albumin, Of: relative fluorescence yield, bMeasured in water containing 0.9% sodium chloride. CMeasured in water containing 0.9 % sodium chloride and 3% lISA. dDetermined by measurement of the delayed fluorescence.
'
5109
methyl pyroPPBa (2b) by the method of Smith et aL4 Since some degree of improvement was observed in the water-solubility of 4b, we planned to synthesize two types of pyroPPBa derivatives: 7b and 8b with some amino acid residues at position 7, and 9b, 10b and l i b with more hydrophilic groups (e.g. a carboxymethyl or an amino acid residue) at position 2. After the hydroxyl group at position 2 of 4b was protected by acetylation, condensation of a DCHA salt of this acetylated derivative (5) with glycine ethyl ester hydrochloride and L-aspartic acid dimethyl ester hydrochloride gave, respectively, 7a and 8a, whose alkaline treatment afforded the corresponding 7-glycyl and 7-aspartyl derivatives (Tb and 8b). Oxidation of the 2-aldehyde derivative (6)5 by use of chromic acid and sulfric acid in water (Jones reagent) gave the 2-carboxymethyl compound (9a), whose hydrolysis afforded 9b. The use of other oxidizing agents as bromine or metachloroperbenzoic acid gave only disappointing results: treatment of 6 with bromine gave the ~-bromo derivative. Condensation of the DCHA salt of 9a with glycine ethyl ester hydrochloride and Laspartic acid dimethyl ester hydrochloride using WSC gave, respectively, 10a and l l a , whose alkaline treatment afforded the corresponding 2-aspartyl and 2-glycyl derivatives (10b and l i b ) . Compound 7b, 8b, 9b, 10b and l i b showed sufficient water-solubility (> 5 mg/ml) for in vivo bioassay.6 The structure of these derivatives was confirmed by UV, IH NMR, FAB-MS and/or elemental analysis.7 Table 1 shows absorption, fluorescence, relative fluorescence intensity and triplet lifetime of 7b, 8b, 9b, 10b and l i b in two aquatic media. Compounds 7b, 8b, 9b, 10b and l i b demonstrated similar photochemical properties. In a 0.9 % aqueous sodium chloride solution, the wavelength of Q band of each derivative was shorter than that of the fluorescence, and the fluorescent intensity was fairly weak. However, these abnormal phenomena were cancelled by the addition of human serum albumin (HSA). These results suggest that each derivative exists mainly as an aggregated form in the 0.9 % aqueous sodium chloride solution. This type of aggregation is possibly due to the flatness of these pyroPPBa derivatives, and is not observed in the chlorin e6 derivatives. 8 The spectral change by the addition of HSA suggests that these pyroPPBa derivatives exist as monomer and/or protein-bound forms after intravenous injection. Tissue transportation and biodistribution of photosensitizing drugs are thought to be affected by the nature of drugs such as water-solubility, aggregation, protein binding and so on.1 These findings might give a clue clarifying the tissue transportation of drugs. Triplet lifetime of all derivatives in Table 1 was ca. 1 ms. Our previous study8,9 suggests that these values would promise a potent photocytotoxicity. Tumor retention of these derivatives was measured by the N2-pulsed laser spectrofluorometry method.10 Syrian golden hamsters with a nitrosoamine-induced pancreatic cancer were subjected to injection of a test compound (4mg/kg). After 2, 6 or 24 hr, the animals were exsanguinated and dissected, and the organs (tumor, liver, kidney and tung) were excised. The test compound-derived surface fluorescence of each organ (tumor, liver, kidney and lung) and serum was detected using a N2-pulsed laser equipped with observation systems described in ref. 6. The fluorescence derived from all derivatives in Table 1 decreased to undetectable levels on the normal organs and serum within 24 hr. Compound l l a showed significant fluorescence on tumor at 24 hr after injection. These results suggest that excretion of these compounds from normal organs and serum occurred within 24 hr. This rapid clearance property is comparable to that reported for mono-aspartyl chlorin e6 (MACE), which showed low skin hyperphotosensitivity. 11 The prolonged skin hyperphotosensitivity after PDT treatment is a serious problem in PDT. In this regard, further studies on compounds of this type would clear the way for obtaining practical new agents for PDT.
5110
Ackowledgements The authors thank Professor J. Oda of the Institute for Chemical Research at Kyoto University and Mr. R. Imamura of Faculty of Science at Kyoto University for 1H NMR measurements. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
1.
2.
3.
4.
5. 6. 7.
8.
9. 10.
11.
References and Footnotes For review see: Dougherty, T. J. Photochem. Photobiol. 1987, 45, 879-889. Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use; Bock, G.; Harnett, S. Eds., Ciba Foundation Symposium, John Willey and Sons, 1989, 146. Roeder, B.; Kricheldorff, K. Stud. Biopys. 1986, 114, 171-182. Roeder, B. Stud. Biopys. 1986, 114, 183-186. Kenner, G. W.; McCombie, S. W.; Smith, K. M. J. Chem. Soc. Perkin ! 1973, 2517-2523. Seely, G. R.: The Structure and Chemistry of Functional Groups. In Chlorophylls; Vernon, L. P.; Seely, G. R. Eds.; Academic Press: New York, 1966; pp. 67-143. Smith, K. M.; Goff, D. A.; Simpson, D. J. J. Am. Chem. Soc. 1985, 107, 4946-4954. Compound 6 was obtained from methyl pyroPPBa (2b) by the method of Smith et al.4 with some modifications. Preparation method of a stock solution: a test compound dissolved in an equivalent of 0.1 N NaOH was diluted with 1/15 M phosphate buffer (pH 7.4) to 5 mg/ml. Structure of la, 2a, 2b, 3a, 4a, 7a, 8a, 10a and lla, was conf'mned by UV, 1H NMR, FAB-MS and elemental analysis. For 3b, 4b, 5, 6, 7b, 8b, 9a, 9b, 10b and lib, the structural confirmation was achieved by UV, 1H NMR and FAB-MS. FAB-MS was measured using 3-nitrobenzylalcohol or glycerol as a matrix. Ando, T.; Irie, K.; Koshimizu, K.; Takemura, T.; Nishino, H.; Iwashima, A.; Nakajima, S.; Sakata, I. Tetrahedron 1990, 46, 5921-5930. Takemura, T.; Nakajima, S.; Sakata, I. Photochem. Photobiol. 1989, 50, 339-344. Nakajima, S.; Hayashi, H.; Omote, Y.; Yamazaki, Y., Hirata, S.; Maeda, T.; Kubo, Y.; Takemura, T.; Kakiuchi, Y.; Shindo, Y.; Koshimizu, K.; Sakata, I. J. Photochem. Photobiol. B: biology 1990, 7, 189-198. Gommer, C. J.; A. Ferrario, A. Cancer Res. 1990, 50, 3985-3990. Nelson, J. S,; Roberts, W. G.; Berns, M. W. Cancer Res. 1987, 47, 4681-4685.
(Received in Japan 16 May 1991)
0040-4039/91 $3.00 + .00 Pergamon Press pie
New Water-soluble Pyropheophorbide a Derivatives as Possible Agents for Photodynamic Therapy of Cancer Tomoyuki Ando, Yoko Suzuki, Rieko Geka, Kazuhiro Irie,* Koichi Koshimizu, Takeshi Takemura,a Susumu Nakajimab and Isao Sakatac Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan aRe.search Institute of Applied Electricity, HokkaidoUniversity,Sapporo 060, Japan bDivision of Surgical Operation, Asahikawa Medical College, Asahikawa 078, Japan CToyo-Hakka Co. Ltd., Okayama 719-03, Japan
Key Words: Photodynamic therapy (PDT); photosensitizer; pyropheophorbide a (pyroPPBa); tumor retention; rapid clearance. Abstract: Five new water-soluble pyroPPBa derivatives, whose vinyl groups at position 2 were converted into hydrophilic groups,
were synthesized, and their in vivo tumor retention was measured by the N2-pulsed laser spectrofluorometry method. The results suggested that these derivatives were advantageous to reduce hyperphotosensitivity in skin, a major side effect of photodynamic therapy.
Photodynamic therapy (PDT) has been proved useful in the treatment of cancer.1 Hematoporphyfin derivative (HPD), which is obtained from hematoporphyrin by acetylation and subsequent alkaline hydrolysis, is a photosensitizer used universally for PDT.1 However, HPD has several disadvantages: relatively weak absorption (e 5,000) at 630 nm and complexity as a mixture of undefined porphyrins and their dimers and/or oligomers. 1 A great number of dyes such as phthalocyanine, purpurin, bactefiochlorin, chlorin e6 and its aspartyl derivative (MACE) have been reported as new potential photosensitizers in the last several years.1 Among these dyes, pheophorbide a (PPBa) (1), which is a degradation product of chlorophyll a, has received increasing attention because of its strong absorption (e 50,000) at 670 nm, high production of 102 and abundance in green plants.2 Since the proton at position 10 of PPBa (1) is activated as an cx-proton of [~-keto ester to cause epimerization, cleavage offing V and oxidative degradation in the presence of a base,3 we selected a more stable pyropheophorbide a (pyroPPBa) (2a) as a lead compound to generate new agents for PDT.
In this
communication we describe the syntheses of new water-soluble pyroPPBa derivatives, their physicochemical properties (absorption, fluorescence and triplet lifetime) and in vivo tumor retention. Since pyroPPBa (2a) is hardly soluble in water, introduction of hydrophilic groups into 2a was carried out to increase its water-solubility. First, dicyclohexylamine (DCHA) salt of 2a was condensed with L-aspartic acid dimethyl ester hydrochloride using water soluble carbodiimide (WSC) to give 3a, whose alkaline hydrolysis resulted in the corresponding 7-aspartyl derivative (3b). However, the water-solubility of 3b was not enough for in vivo bioassay, indicating that the vinyl group at position 2 of 2a must be additionally converted into some
hydrophilic groups. Compound 4b with a 2'-hydroxyethyl group at position 2 was therefore synthesized from
5107
5108
,--OR R
|t!
COR'
1 2a 2b 3a 3b
COR'
R CO2Me H H H H
R' OH OH OMe Asp(OMe)2 Asp
CO2R' R H H Ac Ac H A~ H
4a 4b 5 7a 7b 8a 8b
R' OMe OH OH Gly(OEt) Gly Asp(OMe)2 Asp
R CHO CO2H CO2H COAsp(OMe)2 COAsp COGIy(OEt) COGly
6 9a 9b 10 a 10 b 1 la 11 b
R t
1Vie H Me H Me H
Abbreviations; Asp(OMe)2: NHCH(CO2Me)CH2CO2Me, Asp: NHCH(CO2FI)CH2CO2H, Gly(OEt): NHCH2CO2Et, Gly: NHCH2CO2H, Ac: OCOMe.
Table 1. Photochemical Properties of Water-soluble pyroPPBa Derivatives in the Aquatic Media.a
Compound
Visible absorption Fluorecence (emission) 0% HSAb 3%HSAc 0% lISAb 3%HSAc (Soret band) (Qband) (Soretband) (Q band) )maax (nm) (nm) (nm) (nm) 2~max(nm) Of (nm) Of
Triplet lifetimec,d (ms)
7b
380
660
413
662
653
0.02
666
0.7
1.4
8b
380
660
415
662
660
0.05
667
1.0
1.5
9b
380
660
413
666
660
0.05
675
1.0
1.5
10b
380
663
413
668
662
0.1
674
1.0
1.3
lib
375
664
414
668
661
0.05
676
1.0
1.7
aAbbreviations; HSA: human serum albumin, Of: relative fluorescence yield, bMeasured in water containing 0.9% sodium chloride. CMeasured in water containing 0.9 % sodium chloride and 3% lISA. dDetermined by measurement of the delayed fluorescence.
'
5109
methyl pyroPPBa (2b) by the method of Smith et aL4 Since some degree of improvement was observed in the water-solubility of 4b, we planned to synthesize two types of pyroPPBa derivatives: 7b and 8b with some amino acid residues at position 7, and 9b, 10b and l i b with more hydrophilic groups (e.g. a carboxymethyl or an amino acid residue) at position 2. After the hydroxyl group at position 2 of 4b was protected by acetylation, condensation of a DCHA salt of this acetylated derivative (5) with glycine ethyl ester hydrochloride and L-aspartic acid dimethyl ester hydrochloride gave, respectively, 7a and 8a, whose alkaline treatment afforded the corresponding 7-glycyl and 7-aspartyl derivatives (Tb and 8b). Oxidation of the 2-aldehyde derivative (6)5 by use of chromic acid and sulfric acid in water (Jones reagent) gave the 2-carboxymethyl compound (9a), whose hydrolysis afforded 9b. The use of other oxidizing agents as bromine or metachloroperbenzoic acid gave only disappointing results: treatment of 6 with bromine gave the ~-bromo derivative. Condensation of the DCHA salt of 9a with glycine ethyl ester hydrochloride and Laspartic acid dimethyl ester hydrochloride using WSC gave, respectively, 10a and l l a , whose alkaline treatment afforded the corresponding 2-aspartyl and 2-glycyl derivatives (10b and l i b ) . Compound 7b, 8b, 9b, 10b and l i b showed sufficient water-solubility (> 5 mg/ml) for in vivo bioassay.6 The structure of these derivatives was confirmed by UV, IH NMR, FAB-MS and/or elemental analysis.7 Table 1 shows absorption, fluorescence, relative fluorescence intensity and triplet lifetime of 7b, 8b, 9b, 10b and l i b in two aquatic media. Compounds 7b, 8b, 9b, 10b and l i b demonstrated similar photochemical properties. In a 0.9 % aqueous sodium chloride solution, the wavelength of Q band of each derivative was shorter than that of the fluorescence, and the fluorescent intensity was fairly weak. However, these abnormal phenomena were cancelled by the addition of human serum albumin (HSA). These results suggest that each derivative exists mainly as an aggregated form in the 0.9 % aqueous sodium chloride solution. This type of aggregation is possibly due to the flatness of these pyroPPBa derivatives, and is not observed in the chlorin e6 derivatives. 8 The spectral change by the addition of HSA suggests that these pyroPPBa derivatives exist as monomer and/or protein-bound forms after intravenous injection. Tissue transportation and biodistribution of photosensitizing drugs are thought to be affected by the nature of drugs such as water-solubility, aggregation, protein binding and so on.1 These findings might give a clue clarifying the tissue transportation of drugs. Triplet lifetime of all derivatives in Table 1 was ca. 1 ms. Our previous study8,9 suggests that these values would promise a potent photocytotoxicity. Tumor retention of these derivatives was measured by the N2-pulsed laser spectrofluorometry method.10 Syrian golden hamsters with a nitrosoamine-induced pancreatic cancer were subjected to injection of a test compound (4mg/kg). After 2, 6 or 24 hr, the animals were exsanguinated and dissected, and the organs (tumor, liver, kidney and tung) were excised. The test compound-derived surface fluorescence of each organ (tumor, liver, kidney and lung) and serum was detected using a N2-pulsed laser equipped with observation systems described in ref. 6. The fluorescence derived from all derivatives in Table 1 decreased to undetectable levels on the normal organs and serum within 24 hr. Compound l l a showed significant fluorescence on tumor at 24 hr after injection. These results suggest that excretion of these compounds from normal organs and serum occurred within 24 hr. This rapid clearance property is comparable to that reported for mono-aspartyl chlorin e6 (MACE), which showed low skin hyperphotosensitivity. 11 The prolonged skin hyperphotosensitivity after PDT treatment is a serious problem in PDT. In this regard, further studies on compounds of this type would clear the way for obtaining practical new agents for PDT.
5110
Ackowledgements The authors thank Professor J. Oda of the Institute for Chemical Research at Kyoto University and Mr. R. Imamura of Faculty of Science at Kyoto University for 1H NMR measurements. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
1.
2.
3.
4.
5. 6. 7.
8.
9. 10.
11.
References and Footnotes For review see: Dougherty, T. J. Photochem. Photobiol. 1987, 45, 879-889. Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use; Bock, G.; Harnett, S. Eds., Ciba Foundation Symposium, John Willey and Sons, 1989, 146. Roeder, B.; Kricheldorff, K. Stud. Biopys. 1986, 114, 171-182. Roeder, B. Stud. Biopys. 1986, 114, 183-186. Kenner, G. W.; McCombie, S. W.; Smith, K. M. J. Chem. Soc. Perkin ! 1973, 2517-2523. Seely, G. R.: The Structure and Chemistry of Functional Groups. In Chlorophylls; Vernon, L. P.; Seely, G. R. Eds.; Academic Press: New York, 1966; pp. 67-143. Smith, K. M.; Goff, D. A.; Simpson, D. J. J. Am. Chem. Soc. 1985, 107, 4946-4954. Compound 6 was obtained from methyl pyroPPBa (2b) by the method of Smith et al.4 with some modifications. Preparation method of a stock solution: a test compound dissolved in an equivalent of 0.1 N NaOH was diluted with 1/15 M phosphate buffer (pH 7.4) to 5 mg/ml. Structure of la, 2a, 2b, 3a, 4a, 7a, 8a, 10a and lla, was conf'mned by UV, 1H NMR, FAB-MS and elemental analysis. For 3b, 4b, 5, 6, 7b, 8b, 9a, 9b, 10b and lib, the structural confirmation was achieved by UV, 1H NMR and FAB-MS. FAB-MS was measured using 3-nitrobenzylalcohol or glycerol as a matrix. Ando, T.; Irie, K.; Koshimizu, K.; Takemura, T.; Nishino, H.; Iwashima, A.; Nakajima, S.; Sakata, I. Tetrahedron 1990, 46, 5921-5930. Takemura, T.; Nakajima, S.; Sakata, I. Photochem. Photobiol. 1989, 50, 339-344. Nakajima, S.; Hayashi, H.; Omote, Y.; Yamazaki, Y., Hirata, S.; Maeda, T.; Kubo, Y.; Takemura, T.; Kakiuchi, Y.; Shindo, Y.; Koshimizu, K.; Sakata, I. J. Photochem. Photobiol. B: biology 1990, 7, 189-198. Gommer, C. J.; A. Ferrario, A. Cancer Res. 1990, 50, 3985-3990. Nelson, J. S,; Roberts, W. G.; Berns, M. W. Cancer Res. 1987, 47, 4681-4685.
(Received in Japan 16 May 1991)