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11Porphyrins
Порфирины
макрогэтэроцмклы
http://macroheterocycles.isuct.ru
Paper
Статья
DOI: 10.6060/mhc140377b
Erythrocytes Membrane Photodestruction Sensitized by Chlorophyll a Derivatives: Some Structure-Activity Regularities
D. V. Belykh,a@ O. G. Shevchenko,b and I. S. Tarabukinaa
Dedicated to Academician Oleg Nikolaevich Chupakhin on the occasion of his 80th birthday
aInstitute of Chemistry, Komi Scientific Center, Ural Division, Russian Academy of Sciences, 167982 Syktyvkar, Russia bInstitute of Biology, Komi Scientific Center, Ural Division RAS, 167982 Syktyvkar, Russia @Corresponding author E-mail: [email protected], [email protected]
Photoinduced hemolysis, sensitized by semi-synthetic chlorins, was studied. It was shown that the presence of chlorins without exo-cycle leads to intensive photoinduced hemolysis of erythrocytes which does not depend on the nature of substituents. The presence of exocycle in macrocyclic molecule leads to decrease of hemolysis. The activity ofphorbine derivatives becomes comparable with that of compounds without exo-cycle at the presence of substituents in the molecule, promoting hydrogen bonding.
Keywords: Chlorophyll a derivatives, methylpheophorbide a, chlorin e6, photosensitizes, erythrocytes membrane, hemolysis.
Фотоповреждение эритроцитарной мембраны, сенсибилизируемое производными хлорофилла а: некоторые закономерности «структура-активность»
Д. В. Белых,а@ О. Г. Шевченко,ь И. С. Тарабукина1
Посвящается Академику РАН Олегу Николаевичу Чупахину по случаю его 80-летнего юбилея
Институт химии Коми научного центра Уральского отделения Российской академии наук, 167982 Сыктывкар, Россия
ьИнститут биологии Коми научного центра Уральского отделения Российской академии наук, 167982 Сыктывкар, Россия
@Е-таИ: [email protected], [email protected]
В настоящей работе исследован фотогемолиз эритроцитов, сенсибилизируемый полусинтетическими хлоринами на основе фитопорфиринов. Показано, что хлорины, в молекуле которых нет экзоцикла, вне зависимости от природы заместителей вызывают активный фотосенсибилизированный лизис эритроцитов. Наличие в молекуле экзоцикла в большинстве случаев приводит к снижению степени фотогемолиза. Активность форбиновых производных становится сопоставимой с активностью соединений без экзоцикла при наличии в молекуле заместителей, способствующих образованию водородной связи.
Ключевые слова: Производные хлорофилла а, метилфеофорбид а, хлорин е6, фотосенсибилизаторы, мембрана эритроцитов, гемолиз.
Introduction
At present several porphyrins are used in clinical practice as diagnostic preparates and photosensitizes (PS) for the photodynamic therapy (PDT) of cancer.[1-9] Some chlorophyll a derivatives were found to be highly active PS with low dark toxicity.[1-6,8-29] Thus, new PS searching among chlorophyll a derivatives is of a good chance and the investigation of their activity mechanisms and "structure-activity" regularities are of a great interest for new potential PS synthesis planning. [15,21,28-32] Variation of chlorins chemical structure features such as charge, hydrophobicity and steric properties leads to the significant changing of the pigments ability to insert into the cell which can define their photodynamic effectivity.[33,34] The ability of pigments to interact with membrane structures is of a great significance for photosensitizing activity appearance in cell culture and in vivo.[35] It is well known that sub-cell localization of PS defines their biological efficiency.[16] It was established that chlorins can insert to lipid membrane and chlorin e6 derivatives demonstrate high affinity to liposomes.[36] The correlation between membrane bonding and photo hemolytic activity has been shown for chlorin e6 and its derivatives.[36] From the other hand the increase in hydrophobicity of compounds does not necessarily lead to photodynamic activity increase because of their availability for cell decrease.
Cell membrane is one of the main target of PS action.[37,38] So, enucleate mammalian erythrocytes can be convenient in vitro model for potential PS activity estimation and revealing of the influence of their structure on the biomembrane photodestruction ability.[39-45] According to the literature data the principle factor of membrane photodestruction is an ability of pophyrin to interact with membrane, but spectral properties and the singlet oxygen generation ability are of a much less role in this process.[46] So the erythrocytes photodestruction
can be used to investigate features of potential PS to interact with cell membranes. Common application of Photo-RBC test is caused by the correlation of the test results with several PS photodynamic activity in vivo.[47] Here we report on the investigation of several chlorophyll a derivatives (Scheme 1) with different substituent's ability to induce photohemolysis; also some "structure-activity" regularities were found.
Experimental
'H NMR spectra were recorded in CDCl3 on spectrometer Bruker Avance II (working frequency 300 MHz). IR spectra were recorded on spectrometer Shimadzu IR Prestige 21 in KBr (diffuse reflection). UV-Vis spectra were recorded on spectrometer Shimadzu UV-1700 (PharmaSpec) in CHCl3 in 200-1100 nm range in 10 mm quartz cuvettes, using CHCl3 as comparison sample. Monitoring the reaction proceeding was performed by TLC on Silufol plates, eluent - CCl4-acetone (4:1 vol). Column chromatography was carried out using silica gel Alfa Aesar 70/230^.
Methylpyropheophorbide a (1), methylpheophorbide a (2), methyl-13(2)-hydroxypheophorbide a (5), chlorin e6 13(1)-jV-methylamide 15(2),17(3) dimethyl ester (19), chlorin e6 13(1)-iV,iV-dimethylamide-15(2),17(3)-dimethyl ester (20) were obtained according to [48]. Methylpheophorbide a 13(2)-jV,jV-dibuthylamide
(3) was obtained according to [49].
Methyl-13(2)-iV,iV-dimethylaminomethylpheophorbide a
(4), chlorin e6 13(1)-W-(«-hexyl)amide-15(2),17(3)-dimethyl ester (22), chlorin e6 13(1)-W-(«-octyl)amide-15(2),17(3)-dimethyl ester (23), chlorin e6 3(1),3(2)-bis-(jV,iV-dimethylaminomethyl)-13(1)-jV-methylamide- 15(2),17(3)-dimethyl ester (26), chlorin e6 13(1)-iV,iV-dimethylamide-15-(1-methoxycarbomoylvmyl)-17(3)-methyl ester (35) were obtained according to [50]. Methyl-mesopheophorbide a (8),[51] methylpyropheophorbide b (9),[52] methylpheophorbide b (10),[52] methylpyropheophorbide d (11),[53] methylpheophorbide b (12),[54] methyl-3(1),3(2)-bis-(iV,iV-dimethylaminomethyl)-pyropheophorbide a (15),[55] and
C02CH3 r5
C02CH3 G
Q = G = H: R1 = Vi (16); R1 = Et (17); Q = OH, G = H, R1 = Vi (18)
R1 = Vi, R4 = CH3: R2 = R3 = H (1); R2 = C02CH3, R3 = H (2); R2 = CONBU2, R3 = H (3); R2 = C02CH3, R3 = CH2N(CH3)2 (4); R2 = C02CH3, R3 = OH (5); R2, R3 = C02CH3> CH2OH (6);
R1 = Et, R4 = CH3: R2 = R3 = H (7); R2 = C02CH3, R3 = H (8);
R1 = Vi, R4 = CH=0: R2 = R3 = H (9); R2 = C02CH3, R3 = H (10);
R1 = CH=0, R4 = CH3: R2 = R3 = H (11); R2 = C02CH3, R3 = H (12);
R1 = CH(OH)CH2OH, R4 = CH3:
R2 = R3 = H (13); R2 = C02CH3, R3 = H (14);
R1 = C(CH2N(CH3)2)=CHCH2N(CH3)2, R4 = CH3: R2 = R3 = H (15);
COX
con(ch3)2 ' h2c' nco2ch3 (35)
R1 = Vi, R4 = CH3, X = Y = OCH3: Z = NHCH3 (19); N(CH3)2 (20);
NH(CH2)nCH3 (n = 1 (21), 5 (22), 7 (23)); NHCH2CH2OH (24);
tÇy> (25);
R1 = C(CH2N(CH3)2)=CHCH2N(CH3)2, R4 = CH3, X = Y = OCH3:
Z = NHCH3 (26); NHCH2CH3 (27); NHCH2CH2OH (28); l^jjP (29);
R1 = Et, R4 = CH3, X = Y = OCH3: Z = NHCH3 (30); Z = N(CH3)CH2N(CH3)2 (31)
R1 = Vi, R4 = CH3, X = Z = NHCH2CH2OH, Y = OCH3 (32); R1 = Vi, R4 = CH3, X = Y = Z = NHCH2CH2OH (33); R1 = Vi, R4 = CH=0, X = Y = OCH3, Z = N(CH3)2 (34)
Scheme 1.
methyl-13(1)-desoxopyropheophorbide a (15) [55] were obtained according to literature procedures. Methyl-13(1)-hydroxy-13(1)desoxo-pheophorbide a (18),[57] chlorin e6 13(1)-W-(2-hydroxyethyl)amide-15(3),17(3)-dimethyl ester (24),[58] chlorin e6 13(1)-morpholinylamide-15(3),17(3)-dimethyl ester (25),[58] rodin g7 13(1)-N,N-dimethylamide-15(3),17(3)-dimethyl ester (34),[59] chlorin e6 13(1),17(3)-W,W-(2-hydroxyethyl)diamide-15(3),17(3)-dimethyl ester (32),[60] and chlorin e6 13(1),15(2),17(3)-W,W,W "-(2-hydroxyethyl)triamide (33)[60] were also obtained according to the known procedures.
Methyl 13(2)-hydroxymethylpheophorbide a (6). To the solution of 2 (100 mg, 0.165 mmol) in THF (5 ml) 110 mg of paraformaldehyde and 1 ml of Na2CO3 saturated aqua solution were added and the mixture was stirred for 1 h at room temperature. The reaction was monitored by TLC. The reaction mixture was diluted with 70 ml of chloroform, washed with 10 % aqueous HCl and then with water up to neutral reaction. The chloroform solution was dried by anhydrous Na2SO4. The product was isolated by column chromatography (elution by 40:1 (v:v) CCl4-acetone mixture, TLC monitoring). The yield of compound 6 is 67 mg (70 %). The spectral properties of the compound obtained are the same as described in literature.[611
Methyl mesopheophorbide a (7) was obtained as described for 1. Compound 8 (190.0 mg) was dissolved in 5 ml of pyridine and refluxed for 40 min. The yield of compound 7 is 117 mg (68 %). UV-Vis (CH2Cl2) X nm: 409.0, 469.0, 503.0, 534.0, 600.0, 656.0. 1H NMR (CDC23, 300 MHz) 5 ppm: 9.50 s (1H, H10), 9.23 s (1H, H5), 8.48 s (1H, H20), 5.29 d (1H, H13(2)A, J 20.0 Hz), 5.12 d (1H, H13(2)B, J 20.0 Hz), 4.49 q (1H, H18, J 6.0 Hz), 4.31 br.d (1H, H17, J 7.0 Hz), 3.86 q [2H, 8-(CH2CH3), J 8.0 Hz], 3.77-3.68 m [2H, 3-(CH2CH3)], 3.69 s [3H, 17-(CH2CH2COOCH)], 3.65 s (3H, 12-CH3), 3.32 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 2.76-2.52 m [2H, 17-(CH2CH2COOCH3)], 2.41-2.26 m [2H, 17-(CH2CH2COOCH3)], 1.84 d (3H, 18-CH3, J 8.0 Hz), 1.81-1.71 m (6H, 3-CH2CH., 8-CH2CH), 0.67 br.s (1H, I-NH), -1.56 br.s (1H, III-NH).
Methyl-3(1),3(2)-dihydroxyethylpyropheophorbide a (13). 5.6 ml of 3 % aqueous KMnO4 solution was added dropwise to the solution of 1 (312 mg) in acetone (94 ml) during 1 h. The reaction mixture was diluted with 300 ml of chloroform, washed with water. The solution obtained was dried by anhydrous Na2SO4, filtrated and evaporated. The product was isolated by column chromatography (elution by 5:1 (v:v) CCl4-acetone mixture, TLC monitoring). The yield of compound 13 is 171 mg (52 %) (as 3(1)-diastereomer mixture). UV-Vis (CH2Cl2) X nm: 410.1, 472.1, 506.0, 537.0, 607.0, 664.1. 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.68 s (1H, H10), 9.52 s (1H, H5), 8.66 s (1H, H20), 6.24-6.10 m [1H, 3-(CH(OH) CH2OH)], 5.31 d (1H, H13(2)A, J 18.0 Hz), 5.15 d (1H, H13(2)B, J 18.0 Hz), 4.62-4.05 m [4H, 3-(CH(OH)CH2OH)], H18, H17), 3.79-3.56 m [2H, 8-(CH2CH3)], 3.67/3.66 s [3H, 17-(CH2CH2COOCH.)], 3.64 s (3H, 12-CH3), 3.43/3.41 s (3H, 2-CH3), 3.24 s (3H, 7-CH3), 2.792.54 m [4H, 17-(CH2CH2COOCH3)], 1.91-1.65 m (6H, 18-CH3, 8-CH2CH), 0.91 br.s (1H, I-NH), -2.03 br.s (1H, III-NH). MS (EI) m/z: (M+) 582, (M-H20)+ 564.
Methyl 3(1),3(2)-dihydroxyethylpheophorbide a (14) was obtained as described for 13. 51 mg (32 %) of 14 (as 3(1)-diastereomer mixture) was obtained from 152 mg of 2. UV-Vis (CH2Cl2) X nm: 409.0, 469.0, 504.0, 535.0, 605.5, 662.5. 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.49 s (1H, H10), 9.36 s (1H, H5), 8.56/8.52 s (1H, H20), 6.23-6.19 m [1H, 3-(CH(OH)CH2OH)], 6.22/6.21 s (1H, H13(2)), 4.52-4.05 m (4H, 3-(CH(OH)CH2OH), H18, H17), 3.93/3.92 s (3H, 13(2)-COOCH.), 3.65-3.53 m [2H, 8-(CH2CH3)], 3.64/3.63 s [3H, 17-(CH2CH2COOCH.)], 3.62 s (3H, 12-CH3), 3.34 s (3H, 2-CH3), 3.12 s (3H, 7-CH3), 2.65-2.51 m [2H, 17-(CH2CH2COOCH3)], 2.34-2.20 m [2H, 17-(CH2CH2COOCH3)], 1.83 t (3H, 8-CH2CH3, J 9.0 Hz), 1.69-1.59 m (3H, 18-CH3), 0.08 br.s (1H, I-NH), -1.98 br.s (1H, III-NH). MS (EI) m/z: (M+) 640 (M+), 564 (M-H20)+.
Methyl-13(1)-desoxomesopheophorbide a (17). To the
solution of compound 7 in CH2Cl2 TFA (10 ml) was added and the resulted solution was stirred at 0 °C in the ice bath for 10 min. Then 624.8 mg of NaBH4 was added by parts within 30 min. The resulted mixture was stirred at room temperature for 12 h. Then mixture was diluted with 200 ml CH2Cl2 and 200 ml of water and stirred for 1 h and washed by water to remove the excess of TFA and NaBH4 reaction products. The resulted solution was dried with anhydrous Na2SO4, filtrated and evaporated. The product was isolated by column chromatography (elution by 20:1 (v:v) CCl4-acetone mixture, TLC monitoring). The yield of compound 17 is 58.9 mg (52 %). UV-Vis (CH2Cl2) X nm: 394.5, 498.0, 526.5, 584.5, 638.5. 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.85 s (1H, H10), 9.67 s (1H, H5), 8.95 s (1H, H20), 5.04-4.81 m (2H, H13(2)A, H13(2)B), 4.71 br.q [(1H, H18), 6.3 Hz], 4.53 br.d [(1H, H17), 8.7 Hz], 4.17-4.07 m [4H, H13(1)A, H13(1)B, 3-(CH2CH3)], 3.93 q [2H, 8-(CH2CH3), 7.8 Hz], 3.62 s [3H, 17-(CH2CH2COOCH)], 3.57 s (3H, 12-CH3), 3.52 s (3H, 2-CH3), 3.50 s (3H, 7-CH3), 2. 90-2.19 m [4H, 17-(CH2CH2COOCH3)], 1.90 d (3H, 18-CH3, J 7.9 Hz), 1.87-1.79 m (6H, 3-CH2CH., 8-CH2CH.), -1.62 br.s (1H, I-NH), -3.47 br.s (1H, III-NH).
Chlorin e6 3(1),3(2)-bis-(N,N-dimethylaminomethyl)-13(1)-N-methylamide 15(2),17(3) dimethyl ester (27), chlorin e6 3(1),3(2)-bis-(N,N-dimethylaminomethyl)-13(1)-N-(2-hydroxyethyl)amide 15(2),17(3) dimethyl ester (28), chlorin e6 3(1),3(2)-bis-(N,N-dimethylaminomethyl)-13(1)-morpholinylamide 15(2),17(3) dimethyl ester (29) were obtained as described for 26. 50 mg (54 %) of compound 27 was obtained from 79 mg of compound 21. UV-Vis (CH2Cl2) X nm: 398.0, 498.0, 554.0, 604.0, 659.0. 1H NMR (CDCl3, Me4Si, *the signals of cis- and trans-isomers the chemical shifts of which are different, 300 MHz) 5 ppm: 9.86 s (1H, H10), 9.74 s (1H, H5), 8.82/8.83* s (1H, H20), 7.38-7.30 m [1H, 3-C(CH2N (CH3)2)=CH(CH2N(CH3)2)], 6.42 br.t (1H, 131-CONHC2H5), 5.58 d (1H, H15(1)A, J 19.2 Hz), 5.29 d (1H, H15(1)B, J 19.2 Hz), 4.459 q (1H, H18, J 6.9 Hz), 4.39 br.d (1H, H17, J 9.6 Hz), 3.96-3.85 m (2H, 131-CONHCH2CH3), 3.85-3.75 m [2H, 8-(CH2CH3)], 3.80-3.50 m [4H, 3-C(CH2N(CH3)2)=CH(CH2N(CH3)2)], 3.83 s (3H, 152-COOCH.), 3.63/3.62* s (3H, 2-CH3), 3.60 s [3H, 17-(CH2CH2COOCH.)], 3.55 s (3H, 12-CH3), 3.37 s (3H, 7-CH3), 2.98-2.70 m [2H, 17-(CH2CH2COOCH3)], 2.60-2.00 m [2H, 17-(CH2CH2COOCH3)], 3-C(CH2N(CH.)2)=CH(CH2N(CH.)2): 2.30 s (6H), 2.26 s (3H), 2.23 s (3H); 1.78-1.69 m (6H, 18-CH3, 8-CH2CH.), 1.48 t (3H, 131-CONHCH2CH., J 7.5 Hz), -1.91 br. s (1H, I-NH), -1.70 br. s (1H, III-NH). 33 mg (37 %) of compound 28 was obtained from 77 mg of compound 24. UV-Vis (CH2Cl2) X nm: 399.0, 499.0, 604.0, 660.0. 1H NMR (CDCl3, Me4Si, *the signals of cis- and trans-isomers the chemical shifts of which are different, 300 MHz) 5 ppm: 9.84 s (1H, H10), 9.73 s (1H, H5), 8.84 s (1H, H20), 7.36-7.30 m [1H, 3-C(CH2N( CH3)2)=CH(CH2N(CH3)2)], 6.92 t (1H, 13-CONHCH2CH2OH, J 4.2 Hz), 5.62 d (1H, H15(1)A, J 18.3 Hz), 5.34 d (1H, H15(1)B, J 18.3 Hz), 4.47 q (1H, H18, J 7.2 Hz), 4.43 br.d (1H, H17, J 9.6 Hz), 4.07 t (2H, 131-CONHCH2CH2OH), 4.00-3.92 m (2H, 131-CONHCH2CH2OH), 3.85-3.79 m [2H, 8-(CH2CH3)], 3.80-3.40 m [4H, 3-C(CH2N (CH3)2)=CH(CH2N(CH3)2)], 3.77 s (3H, 152-COOCH.), 3.62 s (3H,
2-CH3), 3.61 s [3 H, 17-(CH2CH2COOCH.)], 3.56 s ( 3H, 12-CH3), 3.37 s (3H, 7-CH3), 2.84-22.50 m [4H, 17-(CH2CH2COOCH3)],
3-C(CH2N(CH3)2)=CH(CH2N(CH3)2): 2.33 s (6H), 2.26/2.23* s (3H), 2.20 s (3H); 1.78-1.68 m (6H, 18-CH3, 8-CH2CH.), -188 br.s (1H, I-NH), -1.66 br.s (1H, III-NH). 30 mg (42 %) of compound 29 was obtained from 62 mg of compound 25. UV-Vis (CH2Cl2) X nm: 398.0, 498.0, 552.0, 605.0, 659.0. 1H NMR (CDCl3, Me4Si, *the signals of cis- and trans-isomers the chemical shifts of which are different, 300 MHz) 5 ppm, prevalent isomer: 9.89 s (1H, H10), 9.75 s (1H, H5), 8.87/8.86* s (1H, H20), 7.40-7.29 m [1H, 3-C(CH2N (CH3)2)=CH(CH2N(CH3)2)], 5.83 d (1H, H15(1)A, J 18.0 Hz), 5.09 d (1H,H15(1)B, J 18.0 Hz), 4.54-4.45 m (1H, H18), 4.37 br.d (1H, H17, J 9.0 Hz), 131-C0NC4H80: 4.65-4.54 m (1H), 4.17-3.80 m (7H); 3.92-3.78 m [2H, 8-(CH2CH3)], 3.89 s (3H, 152-COOCH.), 3.68/3.67* s [3H, 17-(CH2CH2COOCH)], 3.59 s (3H, 12-CH3), 3.56 s (3H, 7-CH3), 3.38/3.37* s (3H, 2-CH3), 3.18-2.67 m [4H, 3-C(CH2N(CH3)2)
=CH(CH2N(CH3)2)], 2.65-2.35 m [4H, 17-(CH2CH2COOCH3)], 3-C(CH2N(CH3)2)=CH(CH2N(CH3)2): 2.29 s (7H), 2.27/2.26* s (2H), 2.24 s (2H), 2.17 s (1H); 1.80-1.65 m (6H, 18-CH 3, 8-CH 2CH3), -1.76 br.s (1H, I-NH), -1.91 br.s (1H, III-NH). Minor isomer: 9.86 s (1H, H10), 9.72 s (1H, H5), 8.81 s (1H, H20), 7.40-7.29 m [1H, 3 -C(CH2N(CH3)2)=CH(CH2N(CH3)2)], 5.53/5.52* d (1H, H15(1)A, J 18.0 Hz), 5.23 d (1H, H1521)B, J 18.0 Hz), 4.54-4.45 m (1H, H18), 4.37 br.d (1H, H17, J 9.0 Hz), 131-CONC^HsO: 4.65-4.54 m (1 H), 4.17-3.80 m (7H); 3.92-3.78 m [2H, 8-(CH2CH3)], 3.84 c (3H, 152-COOCH3), 3.64/3.63* c [3H, 17-(CH2CH2COO CH3)], 3.57 s (3H,
12-CH3), 3.56 s (3H, 7-CH3), 3.38/3.37* s (3H, 2-CH3), 3.18-2.67 m [4H, 3-C(CH2N(CH3)2)=CH(CH2N(CH3)2)], 2.65-2.35 m [4H, 17-(C H2CH2COO CH3)], 3-C(CH2N(CH3)2)=CH(CH2N(CH3)2): 2.29 s (7H), 2.27/2.2(5* s (2H), 2.24 s (2H), 2.17 s (1H); 1.80-1.65 m (6H, 18-CH3, 8-CH2CH3), -1.76 br.s (1H, I-NH), -1.91 br.s (1H, III-NH).
Chlorin e6 13(1)-N-ethylamide-15(2),17(3)-dimethyl ester (21) was obtained as described for 19. 66 mg (61 %) of compound 21 was obtained from 100 mg of compound 2 and 2 ml 70 % aqueous EtNH2. UV-Vis (CH2Cl2) I nm: 401.5, 500.0, 528.0, 556.0, 607.5, 662.5. 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.73 s (1H, H10), 9.67 s (1H, H5), 8.83 s (1H, H30), 8.12 dd [1H, 3-(CH=CH2), J 18.0 and 12.0 Hz], 6.40 dd [1H, 3-(CH=CHHtrans), J 18.0 and 0.9 Hz], 6.17 dd [1H, 3-(CH=CHHcis), J 12.0 and "0.9 Hz], 6.40 br.t (1H,
13-CONHCH2CH3, J6.0 Hz), 5.58 d (1H, H15(1)A, J 18.0 Hz), 5.29 d (1H, H15(1)B, J 18.0 Hz), 4.49 q (1H, H18, J 9.0 Hz), 4.40 d (1H, H17, J 9.0 Hz), 3.92-3.85 m (2H, 131-CONHCH2CH3), 1.06 t (3H, 131-CONHCH2CH3 J 7.6 Hz), 3.83 s (3H, 152-COOCH3), 3.63 s [3H, 17-(CH2CH2COOCH3)], 3.59 s (3H, 12-CH3), 3.52 s (3H, 2-CH3), 3.35 s (3H, 7-CH3), 3.85-3.70 m [2H, 8-(CH2CH3)], 2.61-2.10 m [4H, 17-(CH2CH2COOCH3)], 1.73 m (6H, 18-CH3, 8-CH2CH3), -1.57 br.s (1H, I-NH), -1.79 br.s (1H, III-NH).
Mesochlorin e6 13(1)-N-methylamide-15(2),17(3)-dimethyl ester (30) was obtained as described for 19. 29 mg (87 %) of compound 30 was obtained from 32 mg of compound 8 and 1 ml 33 % aqueous MeNH2. UV-Vis (CH2Cl2) I nm: 396.5, 496.5, 547.0, 595.0, 650.0. 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.72 s (1H, H10), 9.49 s (1H, H5), 8.76 s (1H, H20), 6.40 m (1H, 13-CONHCH3), 5.55 d (1H, H15(1)A, J 20.0 Hz), 5.28 d (1H, H15(1)B, J 20.0 Hz), 4.48 q (1H, H18, J 7.0 Hz), 4.37 br.d (1H, H17, J 9.0 Hz), 3.98-3.79 m [4H, 8-(CH2CH3), 3-(CH2CH3)], 3.85 s (3H, 152-COOCH3), 3.65 s [3H, 17-(CH2CH2COOCH3)], 3.58 s (3H, 12-CH3), 3.41 c (3H,
2-CH3), 3.37 s (3H, 7-CH3), 3.28 d (3H, 13-CONHCH3, J 4.0 Hz), 2.69-2.49 m [2H, 17-(CH2CH2COOCH3)], 2.27-2.10 m [2H, 17-(CH2CH2COOCH3)], 1.89-1.70 m (9H, 18-CH3, 3-CH2CH3, 8-CH2CH3), -1.60 br.s (1H, I-NH), -1.70 br.s (1H, III-NH).
Mesochlorin e6 13(1)-dimethylaminomethyl-13(1)-N-methyl-amide-15(2),17(3)-dimethyl ester (31) was obtained as described for 19. To the solution of compound 30 (132.4 mg) in the mixture of THF (7 ml) and AcOH (7 ml) 6is(N,N-dimethylamino)methane (1.5 ml) was added. The mixture was srirred for 24 h at room temperature. Then reaction mixture was diluted with CH2Cl2 (50 ml) and washed by water. The resulted solution was dried with anhydrous Na2SO4, filtrated and evaporated. The product was isolated by column chromatography (elution by 30:1 (v:v) CCl4-acetone mixture, TLC monitoring). The yield of compound 31 is 33.1 mg (30 %). UV-Vis (CH2Cl2) I nm: 396.5, 496.5, 547.0, 596.0, 650.5. 1H NMR (CDCl3, 4 atropoisomers, 300 MHz) 5 ppm: 9.75/9.74/9.72 s (1H, H10), 9.52/9.49 s (1H, H5), 8.79/8.78/8.74 s (1H, H20), 5.82 d (1H, H15(1)A, J 21.0 Hz), 5.30-4.99 m (2H, H15(1)B, 13(2)-CH2N(CH3)2), 4.53-4.30 m (3H, 13(2)-CH2N(CH3)2,H18, H17), 3.99-3.81 m [4H, 8-(CH2CH3), 3-(CH2CH3)], 3.91/3.87/3.84 s (3H, 152-COOCH3), 3.79/3.766 s (3 H, 2-CH3), 3.67/3.65/3.64 s [3H, 17-(CH2CH2COOCH3)], 3.58/3.57/3.53 s (3H, 12-CH3), 3.44/3.42/3.41 s (3H, 7-CH3), 3.38 s (3H, 131-CONHCH3), 2.84-2.64 m [4H, 17-(CH2CH2COOCH3)], 1.91-1.67 m (15H, 18-CH3, 8-CH2CH3,
3-CH2CH3, 13(2)-CH2N(CH3)2), -1.63 br.s (1H, I-NH), -1.78 br.s (1H, III-NH).
The cytotoxic properties and photodynamic activity of compounds were estimated using their ability to induce hemolysis in the dark and in lighting conditions. Laboratory mice erythrocyte suspension (1 %) in PBS (рН 7.4) was used in the experiments. Investigated compounds were introduced in erythrocyte suspension as a solution in 96 % ethanol. The obtained suspension was incubated in a thermostatic shaker Biosan ES-20 (Latvia) at 37 °С and gently shaking. The control samples contained 0.1 % ethanol. The filament lamp (60 W) was used for illumination taking into account that photodynamic activity of porphyrins depends on irradiation dose but not on source. Luxmeter Velleman DVM 1300 (USA) was used for illumination estimation. The degree of hemolysis after 1.5 and 3 h of incubation with investigated compounds was determined spectrophotometrically as the hemoglobin transfer in the incubation medium (Genesys 20 ThemoSpectromic, USA). The hemolysis percent was calculated against the total hemolysis of the sample at I = 524 nm.[62]
Results and Discussion
Here the methylpheophorbide a (2) and its analogs (8, 12) were used us a starting material for the chlorins synthesis. The main reaction centers of these phorbine derivatives (exo-cycle vinyl group, keto group and ester group) were used for peripheral substituent's modification. Phorbine and chlorin derivatives of a-series were obtained from methylpheophorbide a (2). Exo-cycle aminomethylation and hydroxymethylation (compounds 4 and 5), 13(2)-ester group amidation (compound 3), exo-cycle decarboxylation lead to compound 1 followed by 13(1)-keto group reduction (compounds 4 and 5), or complex (Zn-1) aminomethylation (compound 15) was used for the phorbine derivatives synthesis (Scheme 2). Pd/C catalyzed methyl pheophorbide a (2) hydrogenation leads to corresponding methyl mesopheophorbide a (8), which was used as a start material for synthesis of meso-derivatives (7, 17) (Scheme 2).
Vinyl group oxidation was used for 3-formyl and 3-(1,2-dihydroxyethyl) derivatives synthesis (Scheme 3).
Synthesis of chlorin e6 13-amide derivatives (19-25) was carried out by the reaction of methyl pheophorbide a and corresponding amine (Scheme 4).[484958-60] 13-Amide derivatives 30 (Scheme 2) and 34 (Scheme 6) were obtained by the same way.[59] The insertion of additional ethanolamine fragments (compounds 32, 33) was carried out using corresponding ester groups amidation of 13-amide (24) (Scheme 5).[60] Aminomethylation of chlorin e6 13-amide derivatives (19, 21, 24, 25) with b/'s(N,A?-dimethylamino) methane (refluxing in AcOH and THF mixture) leads to twice aminomethylated derivatives 26-29 (Scheme 4).[50]
The reaction of 6/'s(^,A7-dimethylamino)methane with mesochlorin e6 derivative 30 at room temperature leads to aminomethylation of 13-amide group with formation of compound 31 (Scheme 2). Methylpheophorbide b (10) was used for synthesis of derivatives b-series (9, 34) (Scheme 6).
As can be seen from the data presented in Figure 1, in the case of erythrocytes incubation in darkening conditions the chlorophyll a derivatives investigated at concentration of 10 mM did not cause hemolysis or it was insignificant. So it indicates their potential low cytotoxicity. The estimation of the photodynamic activity shows (Figure 1) that compounds without exo-cycle (20-35) cause active photosensitized lysis
i: (CH3)2NCH2N(CH3)2, THF/AcOH, 10-12 °C, 24 h;[50] ii: paraformaldehyde, Na2CO3, H20-THF, 20 °C, 1-3 h;[61] iii: pyridine, reflux, 5 h;[48] iv: 02(air), compound 5 was isolated from polar fractions while obtaining of 2; v: Bu2NH, PhCH3, reflux, 1.5 h;[49] vi: H2, Pd/C;[51] vii: (a) Zn(OAc)2, CHCl3/CH3OH, 20 °C, (b) (CH3)2NCH2N(CH3)2, THF-AcOH, 20 °C, (c) HCl (30 % aq.)/CHCl3;[55] viii: NaBH^ CH2Cl2/CH3OH; [55] ix: NaBH4, TI)A/CH2Cl2 0 °C; x: (CH3)2NCH2N(CH3)2, THF, reflux, 15 h;[50] xi: pyridine, reflux, 5 h; xii: NaBH4, TFA/CH2Cl2 0 °C; xiii: CH3NH2 (33 % aq.)-THF; xiv: (CH3)2NCH2N(CH3)2, THF/AcOH, 20-22 °C, 24 h.
Scheme 2.
OH
i: NaIO4-OsO4 (H2O-AcOH-THF);[53 54i ii: KMnO4, H2O-acetone. Scheme 3.
of erythrocytes regardless of the substituent's nature. In all cases a part of dead cells is greater than 80 % after 1.5 hours of incubation. The presence of exo-cycle (compounds 1-19) in most cases leads to a decrease in photohemolysis power and an increase in its induction period (Figure 1). The phorbine compounds activity becomes comparable with that of compounds without exo-cycle at presence of groups promoting hydrogen bonding in the phorbine derivatives
molecules(compounds 4, 6, 13-15). As it was noted above the photo effect is determined mainly by the interaction with the membrane and much less depends on other factors action. Furthermore, the setup of present experiment allows to level factors other than interaction with the membrane.
Therefore, the differences found in photosensitizing effect of peripheral substituents of phorbine derivatives (1-19) and chlorins without exo-cycle (20-35) suggest the
c02ch3
c02ch3
(ch3)2i
ii, from (19,21, 24,25)
n(ch3)2
c02ch3 c02ch3
(2)
r1 = h, r2 = ch3 (19); r1 = r2 = ch3 (20)
Nc=o
cö2ch3 cö2ch3 n-r2
r1
r1 = h, r2 = (ch2)nch3 (n = 1 (21), n = 5 (22), n = 7 (23)); r1 = h, r2 = ch3 (26); r1 = h, r2 = ch2ch3 (27); r1 =h, r2 = ch2ch2oh (24); r1, r2 = гглэ (25) r1 =h, r2 = ch2ch2oh (28); r1, r2 = jo (29)
i: amine or its aqueous solution, THF or CHCl3;[48-49-58"601 ii: (CH3)2NCH2N(CH3)2, THF/AcOH, reflux 20 min.™ Scheme 4.
OH
C°2CH3 db2CH3°
(2)
c=o
c'02ch3 co,ch, nh
(24)
-ch3oh
Nç=0
, C02CH3 I
I 0
H0O
(32)
hn HO^J
(33)
-OH
i: ethanolamine, CHCl3, 20 °С, 3 h;[60] ii: ethanolamine, 20° С , 6-8 h;[60] iii: ethanolamine, 20 °С, 60 h.[60] Scheme 5.
c02ch3
c02ch3 cô2ch3 n(ch3)2 (34)
i: pyridine, reflux 5 h;™ ii: CH3NH2(33 % aq)-THF.[59i Scheme 6.
nature of interaction with the erythrocyte membrane to be different for these groups of compounds and the difference is caused by the macrocyclic structure. The phorbine derivatives 1-19 seem to interact with membrane by the formation of hydrogen bonds with phospholipids, glycolipids, membrane proteins polar groups, but chlorin derivatives 20-35 are known to interact with membrane in some less specific manner. We suggest that the exo-cycle absence in the PS molecule promotes its deeper infiltration into the
lipid bilayer. It causes the macrocyclic molecule photoactive region to contact directly with the most sensitive to the oxidation sites of membrane-phospholipid polyunsaturated fatty acids. This may be the reason of independence on the nature of the substituents for chlorin derivatives 2035 to sensitize erythrocytes photohemolysis. Stronger photodynamic effect of the compounds 20-35 seems to be caused by the same reason.
Figure 1. The degree of hemolysis of erythrocytes (%) after incubation with the test compounds at concentration of 10 |M. 1-illuminance 1.7 klux, 1.5 h incubation; 2 - 1.7 klux illumination, 3 h incubation; 3 - incubation conditions dimmer, 3 h.
Conclusions
Thus, the erythrocytes photohemolysis sensitized by semisynthetic chlorins based on phytoporphyrins was studied here. It was shown that chlorins without exo-cycle (20-35) cause an active photosensitized lysis of erythrocytes regardless of the substituent's nature. The presence of exo-cycle (compounds 1-19) in the most cases leads to decrease in photohemolysis power and increase in its induction period. The phorbine compounds activity becomes comparable with activity of that without exo-cycle, but with groups promoting hydrogen bonding (compounds 4, б, 13-15) in the phorbine derivative molecules.
Acknowledgements. The study is supported by Program of UD RAS, project No 12-П-34-2009.
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Received 23.03.2014 Accepted 17.04.2014
