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11Порфирины
Porphyrins
Макрогэтэроцмклы
Статья
Paper
http://macroheterocycles.isuct.ru
DOI: 10.6060/mhc140500b
Novel pH-Independent Amphophilic Chlorophyll a Derivatives with Oligoethylene Glycol Substituents as a Hydrophilic Part: Synthesis and Hydrophilicity Estimation
D. V. Belykh,@ O. M. Startseva, and S. A. Patov
Institute of Chemistry, Komi Scientific Center, Ural Division, Russian Academy of Sciences, 167982 Syktyvkar, Russia @Corresponding author E-mail: [email protected], [email protected]
Several novel pH-independent amphiphilic chlorophyll a derivatives with oligoethylene glycol substituents as a hydrophilic part were synthesized, and hydrophilicity estimation was carried out using their mobility on reverse phase HPLC data. It was shown, that the oligoethylene glycol substituent insertion significantly increases the hydrophilicity of the whole molecule. The most important structural factors affecting hydrophylicity are the presence or absence of the exocycle (exocycle opening results in hydrophobicity decrease in case of the same length oligoethylene glycol chain), and the position of the oligoethylene glycol substituent (increase in length of the spacer between the macrocycle and oligoethylene glycol substituent leads to increase in hydrophilicity). Oligoetylene glycol chain elongation does not lead to appreciable increase in hydrophilicity. So more available di-, tri- and tetraethylene glycols may be used for chlorophyll a derivatives hydrophilization instead of the less available penta- and hexamers.
Keywords: Chlorophyll a derivatives, methylpheophorbide a, chlorin e6, photosensitizes, hydrophilicity, oligoethylene glycol.
Новые рН-независимые амфифильные производные хлорофилла а с фрагментами олигоэтиленгликолей в качестве гидрофильной части: синтез и оценка гидрофильности
Д. В. Белых® О. М. Старцева, С. А. Патов
Институт химии Коми научного центра Уральского отделения Российской академии наук, 167982 Сыктывкар, Россия
@E-mail: [email protected], [email protected]
Синтезирован ряд новых рН-независимых амфифильных производных хлорофилла а с фрагментами олигоэтиленгликолей в качестве гидрофильной части и выполнена оценка гидрофильности полученных соединений на основе данных об их хроматографической подвижности на обращенной фазе. Показано, что внедрение олигоэтиленгликольного фрагмента значительно увеличивает гидрофильность молекулы в целом. Из структурных факторов наиболее сильно влияет наличие/отсутствие экзоцикла (размыкание экзоцикла приводит при одинаковой длине олигоэтиленгликольной цепочки к уменьшению гидрофобности), а так же положение олигоэтиленгликольного фрагмента (увеличение длины спейсера между фрагментом олигоэтиленгликоля и макроциклом приводит к повышению гидрофильности). Удлинение олигоэтиленгликольной цепочки не приводит к заметному увеличению гидрофильности. В связи с этим для гидрофилизации производных хлорофилла а можно использовать более доступные ди-, три- и тетраэтиленгликоли вместо менее доступных пента- и гексамеров.
Ключевые слова: Производные хлорофилла а, метилфеофорбид а, хлорин в6, гидрофильность, фотосенсибилизаторы, олигоэтиленгликоль.
Introduction
It is known that chlorophyll a derivatives are intensively investigated as photosensitizers (PS) for photodynamic therapy (PDT) in various fields of medicine (oncology,[1-6] otolaryngology,17-81 ophthalmology,[910] surgery,[11] treatment of fungal diseases[12]). Some of them have been already used in clinical practice.[1-3] The formation of amphiphilic properties of chlorin PS molecule enhances photodynamic action through more effective interaction of PS with cell membranes, that increases the efficiency of PDT in many cases.[1314] Taking into account that the porphyrin macrocycle is hydrophobic the hydrophilic moieties introduction to the macrocycle periphery is of great interest. The oligoethylene glycol substituents (where the number of ethylene glycol units varies from 2 to 6) were used as hydrophobic moiety here. There is only fragmentary information about such derivatives with di- or triethylene glycol substituents.[15-18] And the influence of the structure of these compounds on the hydrophilicity was not studied systematically. Here we report the synthesis of several phorbines (6-21) and chlorins (22-32) with di-, tri-, tetra-, penta- and hexaethylene glycol substituents using methyl pheophorbide a (1) and its derivatives (2-5) as an initial material (Scheme 1). The influence
of the oligoethylene glycol chain length, its position at the macrocycle periphery and the structure of macrocycle on the hydrophilicity of compounds obtained was estimated using their mobility on the reverse phase HPLC data.
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). HPLC was carried out by Thermo finnigan surveyor (PDA) instrument, pump with auto assembler, Hypersil C18 column 100x2/1 mm, temperature 22 °C, gradient elution (from a mixture of 1 % aqueous trifluoroacetic acid-methanol, 40:60 by volume, to pure methanol for 50 min, flow rate 0.4 ml/min). UV-Vis detection was realized at 400 nm. Mass spectra were obtained by Thermo finnigan LCQ Flut (ESI) instrument. 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^. Methyl pheophorbide a (1) was obtained according to[19]. Methyl pyropheophorbide a (2) and chlorin e6 13(1)-jV-methylamide-15,17-dimethyl ester (4) was obtained according to[20].
n = 1-3
n = 1-3
(19-21)
n = 1-3
n = 1 (6, 11, 14, 19, 22, 27, 30); n = 2 (7, 12, 15, 20, 23, 28, 31); n = 3 (8, 13, 16, 21, 24, 29, 32); n = 4 (9, 17, 25); n = 5 (10, 18, 26)
i: collidine, reflux, 30-40 min; ii: CH3NH2/H2O, THF, r.t., 1-2 h; iii: H2O-HCl/acetone; iv: HOCH2(CH2OCH2)nOCH2OH (n = 1-3), H2SO4(conc), r. t. 12-16 h; v: HOCH2(CH2OCH2)nOCH2OH (n = 1-5), 2-chloro-iV-methylpyridmium iodide, DMAP, toluene, reflux 1-3 h; vi: HOCH2(CH2OCH2)nOCH2OH (n =2 4, 5), 2-chlöro-iV-methylpyridmium iodide, DMAP, THF, reflux 1-2 h.
Scheme 1.
Pyropheophorbide a (3). Methyl pyropheophorbide a (2) (61 mg, 0.11 mmol) was dissolved in acetone (4 ml), concentrated hydrochloric acid (0.5 mL, HCl 30-33 %) was added to the resulting solution and left for 20 hours in the darkness at room temperature. The reaction mixture was diluted with chloroform (50 ml), and hydrochloric acid was removed by water until neutral reaction of washing waters. The resulted chloroform solution was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The evaporation residue was chromatographed on silica gel (eluent: CCl4-acetone from 70:1 to 2:1). The eluate containing the major product was evaporated. 25.2 mg (42 %) of compound 3 was obtained as dark-blue crystals. Spectral data of the compound obtained are identical to those described in[21]. 1Н NMR (CDCl3, 300 MHz) 5 ppm: 9.55 s (1H, Н10), 9.44 s (1H, H5), 8.61 s (1H, H20), 8.03 dd [1H, 3-(C#=CH2), J 18.0 and 11.4 Hz], 6.32 d [1Н, 3-(CH=CH#tnJ, J 17.6 Hz], 6.21 d [1Н, 3-(CH=CH#is), J 11.4 Hz], 5.31 d (1H, Н13(2)А, J 19.8 Hz), 5.16 d (1H, Н13(2)В, J 20.1 Hz), 4.54 q (1H, H18, J 7.3 Hz), 4.36 br.d (1H, H17, J 7.3 Hz), 3.70 s (3H, 15-(СН2СООС#Д 3.73 q (2H, 8-CH2CH3, J 7.7 Hz), 3.44 s (3H, 2-СН3), 3.27 s (3H, 7-СН3), 2.86-2.24 m [4H, 17-(СН2СН2СООСН3)], 1.86 d (3H, 18-СН^, J 7.3 Hz), 1.72 t (8-СН2СН, J 77 Hz), -1.65 br.s (1H, III-NH).
Chlorin e613(1)-N-methylamide 15-methyl ester (5). Chlorin e6 13(1)-V-methylamide-15,17-dimethyl ester (4) (70 mg, 0.11 mmol) was dissolved in acetone (3 ml), concentrated hydrochloric acid (0.5 mL, HCl 30-33 %o) was added to the resulting solution and left for 22 hours in the darkness at room temperature. The reaction mixture was diluted with chloroform (50 ml), and hydrochloric acid was washed with water until neutral reaction of washing waters. The resulted chloroform solution was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue after evaporation was chromato-graphed on silica gel (eluent: CCl4-acetone from 70:1 to 2:1) The eluate containing the major product was evaporated. 41.1 mg (60 %o) of compound 5 was obtained as dark-green crystals. Spectral data of the compound obtained are identical to those described in™. 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.73 s (1H, H10), 9.67 s (1H, H5), 8.83 s (1H, H20), 8.12 dd [1H, 3-(C#=CH2), J 18.0 and 11.4 Hz], 6.50-6.42 br.m (1H, 13-CONHCH3), 6.39 d [1H, 3-(CH=CH#tnJ, J 18.0 Hz], 6.18 d [1H, 3-(CH=CH#cis), J 11.4 Hz], 5.52 d (1H,s H15(1)A, J 19.1 Hz), 5.33 d (1H, H15(1)B, JC19.1 Hz), 4.47 q (1H, H18, J 7.0 Hz), 4.40 br.d (1H, H17, J 9.2 Hz), 3.82 s (3H, 15-(CH2COOCH), 3.88-3.78 m (2H, 8-CH2CH3), 3.58 s (3H, 12-CH3), 3.52 s (3H, 2-CH3), 3.35 s (3H, 7-CH3), 3.28 d (3H, 13-CONHCH, J 4.8 Hz), 2.73-22.21 m (4H, 17-(CH2CH2COOH)), 1.74 t (3H, 8-Ш2СЯ,, J 7.0 Hz), 1.73 d ((3H, 18-CH3, J 6.8 Hz), -1.82 br.s (1H, III-NH).
Trans-esterification of the ester group at position 13(2) of methyl pheophorbide a exocycle (general procedure, preparation of derivatives 6-10). To a solution of 50-200 mg of methyl pheophorbide a (1) (0.083-0.330 mmol) in 10.5 mL of toluene 3-4 times molar excess of dimethylaminopyridine (DMAP), two-times molar excess of 2-chloro-jV-methylpyridinium iodide and corresponding oligoethylene glycol (5-20 mmol) was added. The resulted mixture was heated under reflux for 1-3 hours (TLC monitoring, eluent: CCl4-acetone 2:1). The reaction mixture was diluted with 50 ml chloroform, transferred to a separatory funnel and washed with 5-10 % hydrochloric acid for removing of DMAP and 2-chlo-ro-jV-methylpyridinium iodide excess; then hydrochloric acid was removed by washing with distilled water until neutral reaction of washing waters. The resulting solution was dried over anhydrous sodium sulfate and evaporated under reduced pressure at 40-50 °C. The residue after evaporation was chromatographed on silica gel (eluted with CCl4:acetone in ratios ranging from 40:1 to 5:1). The eluate, containing the main substance, was evaporated under reduced pressure. The residue after evaporation was crystallized first from a mixture of chloroform with hexane, and then from a mixture of chloroform with methanol.
Methyl pheophorbide a 13(2) diethylene glycol ester (6). 75.3 mg (67 %) of compound 6 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 7:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained in reaction of 100 mg (0.165 mmol) of 1, 0.5 ml (5.3 mmol) of diethylene glycol, 80.0 mg (o.656 mmol) of DMAP and 83.4 mg (0.328 mmol) of 2-chloro-iV-methylpyridinium iodide in 5 ml of toluene for 3 hours at full conversion of the starting compound 1. Mass spectrum (ESI), m/z: for MH+ (C39H45N4O7) calcd. 681.3, found 681.4. UV-Vis (CHCl3) I nm (relative intensity, %): 670 (34.9%), 613 (5.5%), 531 (7.2%), 501 (9.2%), 404 (100.0%). IR (KBr) cm-1: 3493 (v OH); 3395 (v NH of chlorin cycle); 2957 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2739 (vCH CH2-O-, glycol); 1736 (v C=O, ester); 1695 (v C=O, exo cycle); 1616 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.55 s (1H, H10), 9.42 s (1H, H5), 8.59 s (1H, H20), 8.03 dd [1H, 3-(CH=CH2), J 18.5 and 11.6 Hz], 6.33 dd [1H, 3-(CH=CHHtrans), J 17.4 and
1.4 Hz], 6.32 s (1H, H13(2)), 6.22 dd [1H, 3-(CH=CHHcis), J 11.4 and 1.5 Hz], 4.61-4.45 m (3H, 13(2)-COOCH2CH2OCH2CH2OH, H18), 4.28 br.d (1H, H17, J 8.5 Hz), 3.85-3.50 m [6H, 8-(CH2CH3), 13(2)-COOCH2CH2OCH2CH2OH], 3.72 s (3H, 12-CH3), 3.(51 s [3H, 17-(CH2CH2COOCH)], 3.44 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 2.77-2.15 m [4H, 17-(CH2CH2COOCH3)], 1.86 d (3H, 18-CH3, J 7.3 Hz), 1.74 t (3H, 8-CH£H J 7.4 Hz), 0.62 br.s (1H,
1-NH), -1.55 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.53 s (1H, H10), 9.39 s (1H, H5), 8.53 s (1H, H20), 8.07-7.97 m [1H, 3-(CH=CH2)], 6.33 dd [1H, 3-(CH=CHHtnJ, J 17.4 and 1.4 Hz], 6.21 s (1H, H13(2)), 6.22 dd [1H, 3-(CH=CHHJ, J 11.4 and
1.5 Hz], 4.61-4.45 m (3H, 13(2)-COOCH2CH2OCH2CH2OH, H18), 4.28 br.d (1H, H17, J 8.5 Hz), 3.85-3.50 m [6H, 8-(CH2CH3), 13(2)-COOCH2CH2OCH2CH2OH], 3.69 s (3H, 12-CH3), 3.48 s [3H, 17-(CH2CH2COOCH)], 3.42 s (3H, 2-CH3), 3.27 s (3H, 7-CH3), 2.77-2.15 m [4H, 17-(CH2CH2COOCH3)], 1.86 d (3H, 18-CH3, J 7.3 Hz), 1.74 t (3H, 8-CH2CH3, J 7.4 Hz), 0.72 br.s (1H, I-NH), -1.37 br.s (1H, III-NH).
Methyl pheophorbide a 13(2) Methylene glycol ester (7). 73.7 mg (67 %) of compound 7 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 9:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained in reaction of 100 mg (0.165 mmol) of 1, 0.5 ml (3.6 mmol) of triethylene glycol, 80.0 mg (0.656 mmol) DMAP and 80.0 mg (0.325 mmol) of 2-chloro-V-methylpyri-dinium iodide in 5 ml of toluene for 3 hours at full conversion of the starting compound 1. Mass spectrum (ESI) m/z: for MH+ (C41H49N4O8) calcd. 725.3, found 725.3; for MNa+ (C41H48N4O8Na) calcd. 747.3 found 747.5. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (46.6%), 611 (9.1°%), 538 (10.5%), 507 (11.2%), 414 (100%). IR (KBr) cm-1: 3460 (v OH); 3392 (v NH of chlorin cycle); 2957
(vCHas CH3); 2926 (vCHas CH2); 2870 (Vchs ch3); 2741 (VCH
glycol); 1735 (v C=O, ester); 1695 (v C=O, exocycle); 1616 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm: Signals of 13(2) R-diastereomer: 9.55 s (1H, H10), 9.41 s (1H, H5), 8.60 s (1H, H20), 8.03 dd [1H, 3-(CH=CH2), J 17.6 and 11.4 Hz], 6.33 dd [1H, 3-(CH=CHHtrans), J 17.9 and 1.1 Hz], 6.31 s (1H, H13(2)), 6.22 dd [1H, 3-(CH=CHHcis), J 11.4 and 1.4 Hz], 4.58-4.45 m (3H, 13(2)-COOCH2CH2OCHi,CH2OCH2CH2OH, H18), 4.28 m (1H, H17), 3.72 s (3H, 12-CH3), 3.59 s [3H, 17-(CH2CH2COOCH3)], 3.44 s (3H,
2-CH3), 3.27 s (3H, 7-CH3), 3.82-3.(54 m 5H and 3.57-3.41 m 4H [8-(CH2CH3), 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH], 3.36 t [2H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH, J 4.4 Hz], 3.33-3.24 m [13(2)-COOCH2CH2OCH2CH2OCH2CH2OH], 2.752.18 m [4H, 17-(CH2CH2COOCH3)], 1.86 d (3H, 18-CH3, J 7.0 Hz), 1.73 t (3H, 8-CH2CH3, J 7.7 Hz), 0.58 br.s (1H, I-NH), -1.59 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.51 s (1H, H10), 9.37 s (1H, H5), 8.53 s (1H, H20), 8.01 dd [1H, 3-(CH=CH2), J 17.6 and 11.0 Hz], 6.35-6.28 m [1H, 3-(CH=CHHtrans)], 6.23-6.19 m [1H, 3-(CH=CHHcis)], 6.21 s (1H, H13(2)), 4.58-4n45 m (3H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH, H18), 4.28 m (1H, H17), 3.69 s (3H, 12-CH3), 3.61 s [3H, 17-(CH2CH2COOCH3)], 3.42 s
(3H, 2-CH3), 3.25 s (3H, 7-CH3), 3.82-3.64 m 5H and 3.57-3.41 m 4H [8-(CH;CH3), 13(2)-COO CH2CH2OCH2CH2OCH2CH;OH], 3.36 t [2H, 13(2)-COOCH2CH2OCH2CH2OCH2CH;OH, J 4.4 Hz], 3.82-3.64 m 5H and 3.57-3.41 m 4H [8-(CH;CH3), 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH], 3.36 t [2H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH, J 4.4 Hz], 3.33-3.24 m [4H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH], 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH], 2.75-2.18 m [4H, 17-(CH;CH;COOCH3)], 1.86 d (3H, 18-CH3, J 7.0 Hz), 1.7- t (3H, 8-CH2CH3, J 7.0 Hz), 0.69 br.s (1H, I-NH), -1.41 br.s (1H, III-NH).
Methyl pheophorbide a 13(2) tetraethylene glycol ester (8). 63.5 mg (50 %) of compound 8 (13(-)-diastereomers mixture, 13(2)-R/13(2)-S 7:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained in reaction of 100 mg (0.165 mmol) of 1, 0.5 ml (-.9 mmol) of tetraethylene glycol, 60.0 mg (0.491 mmol) of DMAP and 60.0 mg (0.317 mmol) of --chloro-iV-methylpyridinium iodide in 5 ml of toluene for 3 hours at full conversion of the starting compound 1. Mass-spectrum (ESI) m/z: for MH+ (C43H53N4O9) calcd. 769.4, found 769.3; for MNa+ (C43H52N4O9Na) calcd. "791.3, found 791.3. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (46.8%), 610 (9.1%), 538 (13.6°%), 507 (11.2%), 414 (100%). IR (KBr) cm-1: 3489 (v OH); 3391 (v NH of chlorin cycle); 2955 (vCHas CH3); 2927 (vCHas CH2); 2870 (vCHs CH3); 2739 (vCH CH2-O-, glycol); 1736 (v C=O, ester); 1697 (v C=O, exo cycle); 1616 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.57 s (1H, H10), 9.44 s (1H, H5), 8.60 s (1H, H20), 8.04 dd (1H, 3(1)-H, J 17.2 and 11.4 Hz), 6.34 d (1H, 3(2)-Htrans J 17.2 Hz), 6.30 s (1H, H13(2)), 6.23 d (1H, 3(2)-Hcis 11.4 Hz), 4.67-4.40 m (3H, 13(2)-COOCH2CH2OCH2CH2OCH2cH2OH, H18), 4.35-4.23 m (1H, H17), 3.72 s (3H, 12-CH3), 3.58 s [3H, 17-(CH;CH;COOCH3)], 3.44 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.82-3.65 m 4H, 3.65-3.53 m 2H and 3.52-3.39 m 2H [8-(CH;CH3), 13(2)-COOCH2CH;OCH2CH2OCH2CH2OH], 3.36 m [2H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OH], 3.32-3.20 m [2H 13(2)-COOCH2CH2OCH-CH2OCH2CH2OH], 2.61-2.24 m (4H, 17-(CH2CH2COOCH3), 1.85 d (3H, 18-CH3, J 6.9 Hz),
I.74 t (3H, 8-CH;CH3, J 7.3 Hz), 0.58 br.s (1H, I-NH), -1.58 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.53 s (1H, H10), 9.40 s (1H, H5), 8.54 s (1H, H20), 8.04 dd (1H, 3(1)-H, J 17.2 and 11.4 Hz), 6.34 d (1H, 3(2)-^ J 17.2 Hz), 6.20 s (1H, H13(2)), 6.23 d (1H, 3(2)-Hcis 11.4 Hz),™4.61-4.40 m (3H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, H18), 4.35-4.-3 m (1H, H17), 3.72 s (3H, 12-CH3), 3.5 8 s [3H, 17-(CH;CH;COOCH3)], 3.44 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.82-3.65 m (4H), 3.65-3.53 m (2H), 3.52-3.39 m (4H), 3.39-3.33 m (2H) and 3.32-3.20 m (4H) [8-(CH;CH3), 13(2)-COOCH2CH2OCH2CH2OCH2CH2OCH2CH2 OH], 2.61-2.24 m (4H, 17-(CH2CH;COOCH3), 1.85 d -3H, - 8-CH3, J 6.9 Hz), 1.74 t (3H, 8-CH;CH3, J 7.3 Hz), 0.68 br.s (1H, I-NH), -1.41 br.s (1H, III-NH).
Methyl pheophorbide a 13(2) pentaethylene glycol ester (9). 78.1 mg (58 %) of compound 9 (13(-)-diastereomers mixture, 13(2)-R/13(2)-S 7:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained in reaction of 100 mg (0.165 mmol) of 1, 0.5 ml (-.4 mmol) of pentaethylene glycol, 60.0 mg (0.491 mmol) of DMAP and 60.0 mg (0.317 mmol) of --chloro-V-methylpyridinium iodide in 5 ml of toluene for 3 hours at full conversion of the starting compound 1. Mass-spectrum (ESI) m/z: for MH+ (C45H57N4O10) calcd. 813.4, found 813.3; for MNa+ (C43H52N4O9Na) (C45H57N4O10Na) calcd. 835.4, found 835.3. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (59.6%), 610 (12.2%), 538 (14.3%), 508 (15.3%), 414 (100%). IR (KBr) cm-1: 3485 (v OH); 3393 (v NH of chlorin cycle); 2955 (vCHas CH3); 2924 (vCHas CH2); 2870 (vCHs CH3); 2742 (vCH CH;-O-, glycol); 1736 (v C=O, ester); 1695 (v C=O, exo cycle); 1616 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.57 s (1H, H10., 9.44 s (1H, H5), 8.60 s (1H, H20), 8.04 dd (1H, 3(1)-H, J 17.2 and
II.4 Hz), 6.34 d (1H, 3(2)-H J 18.3 Hz), 6.30 s (1H, H13(2)), 6.-3
d (1H, 3(2)-Hcis 11.7 Hz), 4.54-4.45 m (3H, 13(2)-COOCH2CH2O CH2CH2OCH2CH2OCH2CH2OCH2CH2OH, H18), 4.32-4.25 m (1H, H17), 3.73 s (3H, 12-CH3), 3.58 s [3H, 17-(CH2CH2COOCH3)], 3.45 s (3H, 2-CH3), 3.29 s (3H, 7-CH3), 3.82-3.36 m 16H, 3.32-3.20 m 2H [8-(CH2CH3), 13(2)-COOCH2CH2OCH2CH2OCH2CH2OCH2CH2 OCH2CH2OH], 2.65-2.20 m (4H, 17-(CH2CH2COOCH3), 1.85 d (3H, 18-CH3, J 7.3 Hz), 1.74 t (3H, 8-CH2CH3, J 7.3 Hz), 0.56 br.s (1H, I-NH), -1.59 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.54 s (1H, H10), 9.40 s (1H, H5), 8.54 s (1H, H20), 8.12-7.97 m (1H, 3(1)-H), 6.34 d (1H, 3(2)-Htrans J 18.3 Hz), 6.23 d (1H, 3(2)-Hcis 11.7 Hz), 6.20 s (1H, H13(2)), 4.54-4.45 m (3H, 13(2)-COOCH2cH2OC H2CH2OCH2CH2OCH2CH2OCH2CH2OH, H18), 4.32-4.25 m (1H, H17), 3.70 s (3H, 12-CH3), 3.61 s [3H, 17-(CH2CH2COOCH3)], 3.43 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.82-3.36 m 16H, 3.32-3.20 m 2H [8-(CH,CH3), 13(2)-COOCH2CH2OCH2CH2OCH2CH2OCH2C H2OCH2CH2OH], 2.65-2.20 m (4H, 17--(CH£H^,COOCH3), 1.85 d (3H, 18-CH3, J7.3 Hz), 1.74 t (3H, 8-CH,CH3, J 7.3 Hz), 0.68 br.s (1H, I-NH), -1.33 br.s (1H, III-NH).
Methyl pheophorbide a 13(2) hexaethylene glycol ester (10). 66.7 mg (47 %) of compound 10 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 7:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained in reaction of 100 mg (0.165 mmol) of 1, 0.5 ml (1.99 mmol) of hexaethylene glycol, 60.0 mg (0.491 mmol) of DMAP and 60.0 mg (0.317 mmol) of 2-chloro-iV-methylpyridinium iodide in 5 ml of toluene for 3 hours at full conversion of the starting compound 1. Mass-spectrum (ESI) m/z: for MH+ (C47H61N4On) calcd. 857.4, found 857.0. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (46.9%), 611 (8.8%), 538 (10.3%), 508 (11.1%), 414 (100%). IR (KBr) cm4: 3485 (v OH); 3391 (v NH of chlorin cycle); 2953 (vCHas CH3); 2922 (vCHas CH,); 2870 (vCHs CH3); 2743 (vCH CH,-O-, glycol); 1736 (v C=O, ester); 1695 (v C=O, exo cycle); 1(518 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.56 s (1H, H10), 9.43 s (1H, H5), 8.60 s (1H, H20), 8.04 dd (1H, 3(1)-H, J 17.6 and 11.4 Hz), 6.33 dd (1H, 3(2)-Htrans J 17.9 and 1.1 Hz), 6.30 s (1H, H13(2)), 6.22 dd (1H, 3(2)-Hcis, 11™4 and 1.1 Hz), 4.56-4.44 m (3H, 13(2)-CO OCH2CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, H18), 4.28 br.dt (1H, H17, 8.4 and 2.9 Hz), 3.72 s (3H, 12-CH3), 3.58 s [3H, 17-(CH2CH2COOCH3)], 3.44 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.80-3.67 m 6H, 3.66-3.41 m 13H, 3.38-3.29 m 5H [8-(CH2CH3), 13(2)-COOCH2CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OCH2 CH2OH], 2.74-2.47 m (2H, 17-(CH2CH2COOCH3), 2.45-2.16 m (2H, 17-(CH2CH2COOCH3), 1.85 d (3H, 18-CH3, J 7.0 Hz), 1.74 t (3H, 8-CH2CH3, J 7.3 Hz), 0.56 br.s (1H, I-NH), -1.60 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.53 s (1H, H10), 9.39 s (1H, H5), 8.54 s (1H, H20), 8.04 m (1H, 3(1)-H), 6.33 dd (1H, 3(2)-Htrans J 17.9 and 1.1 Hz), 6.19 s (1H, H13(2)), 6.22 dd (1H, 3(2)-Hcis, 11.4 and 1.1 Hz), 4.56-4.44 m (3H, 13(2)-COOCH2CH2OCH2CH20 CH2CH2OCH2CH2OCH2CH2OH, H18), 4.28 br.dt (1H, H17, 8.4 and 2.9 Hz)2 3.71 s (3H, 12-CH3), 3.59 s [3H, 17-(CH2CH2COOCH3)], 3.43 s (3H, 2-CH3), 3.26 s (3H, 7-CH3), 3.80-3.67 m 6H, 3.66-3.41 m 13H, 3.38-3.29 m 5H [8-(CH2CH3), 13(2)-COOCH2CH2OCH2C H2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH], 2.74-2.47 m (2H, 17-(CH2CH2COOCH3), 2.45-2.16 m (2H, 17-(CH2CH2COOCH3), 1.85 d (3H, 18-CH3, J 7.0 Hz), 1.74 t (3H, 8-^^ J 7.3 Hz), 0.64 br.s (1H, I-NH), -1.42 br.s (1H, III-NH).
Trans-esterification of propionate substituent in position 17 by the action of di-, tri- and tetraethylene glycol (generalprocedure, preparation of derivatives 11-16, 19-21). 30-100 mg of the starting chlorophyll a derivative (about 0.049-0.165 mmol) was dissolved in a mixture of 10.5 ml of corresponding oligoethylene glycol with 0.1-0.3 ml of concentrated sulfuric acid. In case of poor solubility of the starting chlorin, 1-2 ml of chloroform was added to a mixture (until complete dissolution of the starting compound). The resulting solution was allowed to stand for 17-H h in a dark place at 18-H °C. The reaction mixture was diluted with 100-300 ml of water, and the precipitate was filtered through a paper filter, washed with 200-400 ml of water and dried in air. The resulting chlorins
mixture was chromatographed on silica gel (eluent: CCl4-acetone from 70:1 to 2:1). The eluate containing the major product was evaporated and residue after evaporation was re-precipitated from chloroform-hexane mixture.
Pheophorbide a 17-diethylene glycol ester (11). 57.1 mg (51 %) of compound 11 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 6:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained with transesterification of 100 mg (0.165 mmol) of 1 in a mixture of 10 ml of diethylene glycol, 0.3 ml of concentrated sulfuric acid and 2 ml of chloroform for 19 hours at complete conversion of starting compound 1. Mass-spectrum (ESI) m/z: for MH+ (C39H45N4O7) calcd. 681.3, found 681.3. UV-Vis (CHCl3) I nm (relative intensity, %): 663 (45.1%), 611 (9.5%), 538 (11.2°%), 508 (12.1%), 414 (100%). IR (KBr) cm-1: 3466 (v ОН); 3395 (v NH of chlorin cycle); 2957 (vCHas CH3); 2926 (vCHas CH2); 2870 (vCHs CH3); 2739 (vCH CH2-O-, glycol); 1738 (v C=O, ester); 1697 (v C=O, exo cycle); 1616 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.54 s (1H, H10), 9.40 s (1H, H5),
8.60 s (1H, H20), 8.01 dd (1H, 3(1)-H, J 17.6 and 11.4 Hz), 6.32 d (1H, 3(2)-Htrans J 17.6 Hz), 6.32 s (1H, H13(2)), 6.21 d (1H, 3(2)-Hcis 11.4 Hz), 4.50 qd ™1H, H18, J 7.3 and 1.5 Hz), 4.27 br.dt (1H, H177j 7.9 and 2.2 Hz), 4.21-4.05 m [2H, 17-CH2CH2COOC#2CH2OCH2CH2OH], 3.91 s (3H, 13(2)-COOCH3), 3.72 s (3H, 12-CH3), 3.44 s (3H, 2-CH3), 3.25 s (3H, 7-CH3), 3.82-3.36 m [8H, 8-(C#2CH3), 17-(CH2CH2COOCH2CH2OCH2CH2OH)], 2.75-2.49 m (2H, 17-CH2CH2C OOCH2CH2OCH2CH2OH), 2.48-2.34 and 2.32-2.19 (both m 1H, 17-(CH2CH2COOCH2CH2OCH2CH2OH), 1.86 d (3H, 18-CH3, J 7.3 Hz), 1.72 t (3H, 8-CH2CH3, J 7.3 Hz), 0.58 br.s (1H, I-NH), -1.59 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.51 s (1H, H10), 9.36 s (1H, H5), 8.54 s (1H, H20), 8.05-7.94 m (1H, 3(1)-H), 6.34 d (1H, 3(2)-Htrans J 18.3 Hz), 6.23 d (1H, 3(2)-Hcis 11.7 Hz), 6.20 s (1H, H13(2)), 4.53 br.q (1H, H18, J 7.2 Hz), 4.34 br.t(1H, H17, J 7.7 Hz),4.21-4.05m[2H,17-(CH2CH2C00CH2CH20CH2CH20H)], 3.87 s (3H, 13(2)-COOCH3), 3.71 s (3H, 12-CH3), 3.42 s (3H, 2-CH3), 3.24 s (3H, 7-CH3), 3.82-3.36 m [8H [8-(CH2CH3), 17-(CH2CH2COOCH2CH2OCH2CH2OH)], 2.75-2.49 m (2H, 17-СН2СH2COOCH2CH20CH2CH2OH), 2.48-2.34 and 2.32-2.19 (both m 1H, 17-С H2CH2CO OCH2CH2OCH2CH2OH), 1.86 d (3H, 18-CH3, J 7.3 Hz), 1.72 t (3H, 8-CH2CH3, J 7.3 Hz), 0.67 br.s (1H, I-NH), -1.42 br.s (1H, III-NH).
Pheophorbide a 17-triethylene glycol ester (12). 21.7 mg (36 %) of compound 12 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 5:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained with transesterification of 50 mg (0.082 mmol) of 1 in a mixture of 3 ml of triethylene glycol, 0.2 ml of concentrated sulfuric acid for 20 hours at complete conversion of starting compound 1. Mass-spectrum (ESI) m/z: for MH+ (C41H49N4O8) calcd. 725.4, found 725.3. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (45.8%), 610 (8.9%), 538 (10.4%), 508 (11.1%), 414 (100%). IR (KBr) cm-1: 3464 (v OH); 3393 (v NH of chlorin cycle); 2957 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2743 (vCH CH2-O-, glycol); 1736 (v C=O, ester)2 1695 (v C=O, exo cycle); Ш6 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.58 s (1H, H10), 9.44 s (1H, H5),
8.61 s (1H, H20), 8.04 dd (1H, 3(1)-H, J 17.6 and 11.4 Hz), 6.34 dd (1H, 3(2)-Htrans J 17.6 and 1.5 Hz), 6.31 s (1H, H13(2)), 6.23 dd (1H, 3(2)-Hcis 117 and 1.5 Hz), 4.51 br.q (1H, H18, J 7.3 Hz), 4.26 br.d (1H, H17 J 7.7 Hz), 4.22-4.06 m [2H, 17-(CH2CH2COOCH2C H2OCH2CH2OCH2CH2OH)], 3.91 s (3H, 13(2)-COOCH3), 3.73 s (3H, 12-CH3), 3.45 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.80-3.39 m [12H [8-(CH2CH3), 17-(CH2CH2COOCH2CH2OCH2CH2OCH 2CH2OH)], 2.73-2.48 m (2H, 17-CH2CH2COOCH2CH2OCH2CH2 OCH2CH2OH), 2.47-2.32 and 2.31-2.18 (both m 1H, 17-CH2CH2 COOCH2CH2OCH2CH2OCH2CH2OH), 1.85 d (3H, 18-CH3, J 7.0 Hz), 1.742 t (3H, 8-CH2CH3, J 6.9 Hz), 0.58 br.s (1H, I-NH3, -1.58 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.54 s (1H, H10), 9.40 s (1H, H5), 8.55 s (1H, H20), 8.08-7.97 m (1H, 3(1)-H), 6.34 dd (1H, 3(2)-H J 17.6 and 1.5 Hz), 6.23 dd (1H, 3(2)-H . 11.7 and
I.5 Hz), 6.19 s (1H, H13(2)), 4.51 br.q (1H, H18, J 7.3 Hz), 4.26 br.d (1H, H17, J 7.7 Hz), 4.22-4.06 m [2H, 17-(CH2CH2COOCH2CH2O CH2CH2OCH2CH2OH)], 3.86 s (3H, 13(2)-COOCH3), 3.70 s (3H, 12-CH3), 3.43 s (3H, 2-CH3), 3.27 s (3H, 7-CH3), 3.80-3.39 m [12H [8-(CH2CH3), 17-(CH2CH2COOCH2CH2OCH2CH2OCH2CH2OH)], 2.73-2.48 m (2H, 17-CH2CH2COOCH2CH2OCH2CH2OCH2CH2O H), 2.47-2.32 and 2.31-2.18 (both m 1H, 17-(CH2CH2COOCH2CH2 OCH2CH2OCH2CH2OH), 1.85 d (3H, 18-CH3, J 7.0 Hz), 1.74 t (3H, 8-CH2CH3, J 6.9 Hz), 0.62 br.s (1H, I-NH), -1.40 br.s (1H, III-NH).
Pheophorbide a 17-tetraethylene glycol ester (13). 57 mg (45 %) of compound 13 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 7:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained with transesterification of 100 mg (0.165 mmol) of 1 in a mixture of 5 ml of tetraethylene glycol, 0.25 ml of concentrated sulfuric acid for 18 hours at complete conversion of starting compound 1. Mass-spectrum (ESI) m/z: for MH+ (C43H53N4O9) calcd. 769.4, found 769.3, for MNa+ (C43H52N4O9Na) calcd. 791.4, found 791.3, for MK+ (C43H52N4O9K) calcd. 807.3, found 807.2. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (46.9%), 611 (9.3%), 558 (4.4%), 538 (10.7%), 414 (100%). IR (KBr) cm-1: 3464 (v OH); 3393 (v NH of chlorin cycle); 2955 (vŒ- CH3); 2926 (vŒas CH2); 2870 (vŒs CH3); 2741 (vŒ CH2-O-, glycol); 1738 (v C=O, ester); 1697 (v C=O, exo cycle); 1618 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.62 s (1H, H10), 9.48 s (1H, H5), 8.65 s (1H, H20), 8.05 dd (1H, 3(1)-H, J 17.6 and 11.4 Hz), 6.34 d (1H, 3(2)-Htrans J 17.6 Hz), 6.31 s (1H, H13(2)), 6.24 d (1H, 3(2)-Hcis J
II.7 Hz), 4.52 br.q (1H, H18, J 7.7 Hz), 4.27 br.d (1H, H17, J'88.1 Hz), 4.23-4.07 m [2H, 17-(CH2CH2COOCH2CH2OCH2CH2OCH2 CH2OCH2CH2OH)], 3.91 s (3H 13(2)-COOC^H3)2 3.74 s (3H, 12-CH^, 3.46 s (3H, 2-CH3), 3.29 s (3H, 7-CH3), 3.82-3.38 m [16H [8-(CH2CH3), 17-(CH2CH2COOCH2CH2OCH2CH2OCH2CH2OCH2 СН2ОЩ, 2.77-2.47 m (2H, 17-CH2CH2COOCH2CH2OCH2CH2O CH2CH2OCH2CH2OH), 2.46-2.34 and 2.33-2.17 (both m 1H, 17-CH2CH2COOCH2CH2OCH2CH2OCH2 CH2OCH2CH2OH), 1.86 d (3H, 18-CH3, J 7Ю Hz), 1.74 t (3H, 8-CH2CH3, J 7.7Hz), 0.48 br.s (1H, I-NH), -1.61 br.s (1H, III-NH). Signals of 13(2)R-diastereomer: 9.59 s (1H, H10), 9.44 s (1H, H5), 8.60 s (1H, H20), 8.08-7.96 m (1H, 3(1)-H), 6.34 d (1H, 3(2)-Htrans J 17.6 Hz), 6.24 d (1H, 3(2)-Hcis J 11.7 Hz), 6.20 s (1H, H13(2)), 4l 2 br.q (1H, H18, J 7.7 Hz), 4.37-431 m (1H, H17), 4.23-4.07 m [2H, 17-(CH2CH2COOCH2CH2OCH2C H2OCH2CH2OCH2CH2OH)], 3.87 s (3H, 13(2)-COOCH3), 3.71 s (3H, 12-CH3), 3.44 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.82-3.38 m [16H [8-(CH2CH3), 17-(CH2CH2COOCH2CH2OCH2CH2OCH2CH2 OCH2CH2OH)], 2.77-2.47 m (2H, 17-CH2CH2COOCH2CH2OCH2 CH2OCH2CH2OCH2CH2OH), 2.46-2.34 and 2.33-2.17 (both m 1H, 17-C H2CH2CO OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH), 1.86 d (3H, 18-CH3, J 7.0 Hz), 1.74 t (3H, 8-CH2CH3, J 7.7 Hz), 0.58 br.s (1H, I-NH), -1.44 br.s (1H, III-NH).
Pyropheophorbide a 17-diethylene glycol ester (14). 15.1 mg (24 %) of compound 14 as a dark blue-black crystalline powder was obtained with transesterification of 50 mg (0.091 mmol) of 2 in a mixture of 3 ml of diethylene glycol, 0.15 ml of concentrated sulfuric acid for 18 hours at complete conversion of starting compound 2. Mass-spectrum (ESI) m/z: for MH+ (C37H43N4O5) calcd. 623.3, found 623.4. UV-Vis (CHCl3) I nm (relative intensity, %): 668 (44.7%), 611 (10.0%), 540 (11.6%), 509 (12.5%), 414 (100 %). IR (KBr) cm-1: 3429 (v OH); 3393 (v NH of chlorin cycle); 2960 (vŒ- CH3); 2926 (vŒas CH2); 2868 (vŒs CH3); 2731 (vŒ CH2-O-, glycol); 1732 (v C=O, ester); 1692 (v C=O, exo cycle); 1622 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.56 s (1H, H10), 9.44 s (1H, H5), 8.61 s (1H, H20), 8.04 dd (1H, 3(1)-H, J 17.8 and 11.7 Hz), 6.33 d (1H, 3(2)-Htrans J 17.6 Hz), 6.22 d (1H, 3(2)-Hcis 11.4 Hz), 5.33 d (1H, H13(2)A, J 2(12 Hz), 5.16 d (1H, H13(2) В, J 20.1 Hz), 4.54 br.q (1H, H18, J 7.0 Hz), 4.39-4.30 m (1H, H17), 4.26-4.16 m [2H, 17-CH2CH2COOCH2CH2OCH2CH2OH], 3.74 q (2H, 8-(CH2CH3), J 7.7 Hz)2 3.71 s (3H, 12-CH3), 3.45 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 3.73-3.66 m 1H and 3.66-3.57 m
3Н (17-СН2СН2СООСН2СЯ2ОСН2СЯ2ОН), 3Н 3.53 t (2Н, 17-СН2СН2СООСН2СН2ОСЯ2СН2ОН, J 4.4 Hz), 2.85-2.55 m (2Н, 17-СЯ2СН2СООСН2СН2ОСН2СН2ОН), 2.45-2.29 m (2Н, 17-(СН2СЯ2СООСН2СН2ОСН2СН2ОН), 1.86 d (3Н, 18-СН3, J 7.3 Hz), 1.73 t (3Н, 8-СН2СЯ3, J 7.3 Hz), 0.48 br.s (1Н, I-NH), -1.63 br.s (1Н, III-NH).
Pyropheophorbide a 17-triethylene glycol ester (15). 35.5 mg (29 %) of compound 15 as a dark blue-black crystalline powder was obtained with transesterification of 100 mg (0.182 mmol) of 2 in a mixture of 5 ml of triethylene glycol, 0.25 ml of concentrated sulfuric acid for 17 hours at complete conversion of starting compound 2. Mass-spectrum (ESI) m/z: for MH+ (C39H47N4O6) calcd. 667.3, found 667.4. UV-Vis (СНС13) X nm (relative; intensity, %): 668 (45.0%), 610 (9.2%), 539 (10.8%), 509 (11.7%), 414 (100%). IR (KBr) cm-1: 3442 (v ОН); 3393 (v NH of chlorin cycle); 2959 (vCHas CH3); 2924 (vŒ- CH2); 2868 (vCHs CH3); 2737 (vCH CH2-O-, glycol); 1732 (v C=O, ester); 1692 (v C=O, exo cycle); 1618 («chlorin band»). 1Н NMR (CDCl3, 300 MHz) 5 ppm: 9.61 s (1Н, Н10), 9.49 s (1Н, Н5), 8.65 s (1Н, H20), 8.06 dd (1Н, 3(1)-Н, J 18.0 and 11.4 Hz), 6.34 dd (1Н, 3(2)-^ J 18.0 and 1.3 Hz), 6.23 dd (1Н, 3(2)-Hcis 11.4 and 1.3 Hz), 5.34s d (1Н, Н13(2)А, J 19.8 Hz), 5.17 d (1Н, Н13(2)В, J 19.8 Hz), 4.56 qd (1Н, Н18, J 7.3 and 2.2 Hz), 4.37 br.d (1Н, Н17, J 7.3 Hz), 4.20 dd (2Н, 17-СН2СН2СООСЯ2СН2ОСН2СН2ОН J 5.5 and 4.0 Hz), 3.76 q (2Н, 8-(СЯ2СН3), J 7.7 Hz), 3.73 s (3Н, 12-СН3), 3.46 s (3Н, 2-СН3), 3.30 s (3Н, 7-СН3), 3.67-3.48 m (11Н, 17-СН2СН2СООСН2СЯ2ОСЯ2СЯ2ОСЯ2СЯ2ОЯ), 2.86-2.55 m (2Н, 17-СЯ2СН2СООСН2СН2ОСН2СН2ОСН2СН2ОН), 2.45-2.28 m (2Н, 17-(С Н2СЯ2СООС Н2С Н2ОС Н2С Н2ОС Н2С Н2ОН), 1.86 d (3Н, 18-СН3, J7.3 Hz), 1.74 t (3Н, 8-СН2СЯ3, J7.3 Hz), 0.36 br.s (1Н, I-NH), -1.66 br.s (1Н, III-NH).
Pyropheophorbide a 17-tetraethyleneglycolester(16). 37.7 mg (28 %) of compound 16 as a dark blue-black crystalline powder was obtained with transesterification of 100 mg (0.182 mmol) of 2 in a mixture of 5 ml of triethylene glycol, 0.25 ml of concentrated sulfuric acid for 17 hours at complete conversion of starting compound 2. Mass-spectrum (ESI) m/z: for MH+ (C41H50N4O7) calcd. 711.4, found 711.4. UV-Vis (СНа3) X nm (relative intensity, %): 668 (44.4%), 610 (8.5%), 539 (9.9%), 509 (11.0%), 414 (100%). IR (KBr) cm-1: 3443 (v ОН); 3393 (v NH of chlorin cycle); 2959 (vŒ- CH3); 2924 (vŒ- CH2); 2870 (vŒs CH3); 2734 (Vch CH^O-, glycol); 1732 (v С=О, ester); 1692 (v С=О, exo cycle); 1618 («chlorin band»). 1Н NMR (CDCl3, 300 MHz) 5 ppm: 9.54 s (1Н, Н10), 9.43 s (1Н, Н5), 8.61 s (1Н, H20), 8.05 dd (1Н, 3(1)-Н, J 18.0 and 11.7 Hz), 6.33 d (1Н, 3(2)-^^, J 18.0 Hz), 6.21 d (1Н, 3(2)-H.s, 11.3 Hz), 5.32 d (1Н, Н13(2)А, J 19.8 Hz), 5.16 d (1Н, Н13(2)В, J 19.8 Hz), 4.54 br.q (1Н, Н18, J 7.0 Hz), 4.36 br.d (1Н, Н17, J 7.7 Hz), 4.20 m (2Н, 17-СН2СН2СООСЯ2СН2ОСН2СН2ОСН2СН2ОН), 3.73 q (2Н, 8-(СЯ2СН3), J 7.9 Hz), 3.71 s (3Н, 12-СН3), 3.45 s (3Н, 2-СН3), 3.28 s (3Н, 7-СН3), 3.67-3.48 m (11Н, 17-СН2СН2СООСН2СЯ2ОСЯ2СЯ2ОСЯ2СЯ2ОЯ), 2.83-2.54 m (2Н, 17-СЯ2С Н2СООС Н2С Н2ОС Н2С Н2ОС Н2С Н2ОСН2СН2ОН), 2.452.26 m (2Н, 17-(СН2СЯ2СООС Н2С Н2ОС Н2С Н2ОС Н2С Н2ОСН2С Н2ОН), 1.85 d (3Н, 18-СН3, J 7.0 Hz), 1.74 t (3Н, 8-СН2СЯ3, J 7.7 Hz), 0.52 br.s (1Н, I-NH), -1.65 br.s (1Н, III-NH).
Pheophorbide a 13(2).17-bisdiethylene glycol ester (19). 14.1 mg (42 %) of compound 19 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 8:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained with transesterification of 30 mg (0.040 mmol) of 6 in a mixture of 3 ml of diethylene glycol, 0.15 ml of concentrated sulfuric acid for 22 hours at complete conversion of starting compound 6. Mass-spectrum (ESI) m/z: for MH+ (C42H51N4O9) calcd. 755.4, found 755.3. UV-Vis (СНа3) X nm (relative intensity, %): 668 (47.3%), 611 (9.9%), 539 (11.3%), 508 (11.8%), 414 (100%). IR (KBr) cm-1: 3458 (v ОН); 3393 (v NH of chlorin cycle); 2959 (vCHas CH3); 2926 (vCHas CH2); 2870 (vCHs CH3); 2740 (vCH CH2-O-, glycol); 1734 (v С=О, ester); 1695 (v С=О, exo cycle); 1616 («chlorin band»). 1Н NMR (CDCl 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer:
9.61 s (1Н, Н10), 9.47 s (1Н, Н5), 8.65 s (1Н, Н20), 8.04 dd (1Н, 3(1)-Н, J 18.0 and 11.7 Hz), 6.34 d (1Н, 3(2)-^^ J 17.9 Hz), 6.36 s (1Н, Н13(2)), 6.24 d (1Н, 3(2)-H.s 11.4 Hzf4.68-4.47 m (3Н, 13(2)-СООСЯ2СН2ОСН2СН2ОН, Ъ18), 4.36-4.27 m (1Н, Н17), 4.25-4.13 m [2Н, 17-СН2СН2СООСЯ2СН2ОСН2СН2ОН], 3.88-3.48 m [16Н, 8-СЯ2СН3, 13(2)-СООСН2СЯ2ОСЯ2СЯ2ОЯ, 17-СН2СН2СООСН2СЯ2ОСЯ2СЯ2ОЯ], 3.73 s (3Н, 12-ССН3), 3.45 s (3Н, 2-СНД 3.29 s (3Н, 7-СН3), 2.78-2.55 m (2Н, 17-СЯ2СН2СООСН2ССН2ОСН2СН2ОН), 2.53-2.23 m (2Н, 17-(СН2С//2СООСН2СН2ОСН2СН2ОН), 1.88 d (3Н, 18-СН3, J 7.3 Hz), 1.74 t (3Н, 8-СН2СЯ3, J 7.3 Hz), 0.52 br.s (1Н, I-NH), -1.57 br.s (1Н, III-NH). Signals of 13(2)S-diastereomer: 9.59 s (1Н, Н10), 9.44 s (1Н, Н5), 8.59 s (1Н, Н20), 8.04 dd (1Н, 3(1)-Н, J 18.0 and 11.7 Hz), 6.34 d (1Н, 3(2)-^^ J 17.9 Hz), 6.24 d (1Н, 3(2)-H.s 11.4 Hz), 6.19 s (1Н, H13®), 4.68-4.47 m (3Н, 13(2)-СООСЯ2СН2ОСН2СН2ОН, Н18), 4.36-4.27 m (1Н, Н17), 4.25-4.13 m [2Н, 17-СН2СН2СООСЯ2СН2ОСН2СН2ОН], 3.883.48 m [16Н, 8-СЯ2СН3, 13(2)-СООСН2СЯ2ОСЯ2СЯ2ОЯ, 17-СН2СН2СООСН2СЯ^ОСЯ2СЯ2ОН], 3.76 s (3Н, 12-ССН3), 3.44 s (3H, 2-СН3)2 3.30 s (3Н, 7-СН3), 2.78-2.55 m (2Н, 17-СЯ2СН2СООСН2ССН2ОСН2СН2ОН), 2.53-2.23 m (2Н, 17-(СН2СЯ2СООСН2СН2ОСН2СН2ОН), 1.88 d (3Н, 18-СН3, J 7.3 Hz), 1.744 t (3Н, 8-СН2СЯ3, J 7.3 Hz), 0.62 br.s (1Н, I-NH), -1.42 br.s (1Н, III-NH).
Pheophorbide a 13(2).17-bistriethylene glycol ester (20). 40.0 mg (42 %) of compound 20 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 7:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained with transesterification of 80 mg (0.095 mmol) of 7 in a mixture of 5 ml of triethylene glycol, 0.25 ml of concentrated sulfuric acid for 18 hours at complete conversion of starting compound 7. Mass-spectrum (ESI) m/z: for MH+ (C46H59N4O11) calcd. 843.4, found 843.3. UV-Vis (СНа3) X nm (relative intensity, %): 668 (44.9%), 610 (9.7%), 538 (11.4%), 507 (12.9%), 414 (100%). IR (KBr) cm-1: 3460 (v ОН); 3388 (v NH of chlorin cycle); 2959 (vCHas CH3); 2922 (vCHas CH2); 2872 (vCHs CH3); 2736 (vCH Ш2-О-, glycol); 1736 (v С=О, ester); 1697 (v С=О, exo cycle); 1611 («chlorin band»). 1Н NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.57 s (1Н, Н10), 9.43 s (1Н, Н5), 8.61 s (1Н, Н20), 8.03 dd (1Н, 3-СЯ=СН2, J 17.6 and 11.4 Hz), 6.33 d (1Н, 3-СН^НЯ.^ J 17.6 Hz), 6.34 s (1Н, Н13(2)), 6.22 d (1Н, 3-СН^СНН^ J 11.7 Hz), 4.57-4.48 m (3Н, 13(2)-СООСЯ2СН2ОСН2СН2ОСН2СН2ОН, Н18), 4.30 br.d (1Н, Н17, J 7.7 Hz), 4.22-4.08 m [2Н, 17-СН2СН2СООСЯ2СН2ОСН2СН2ОСН2СН2ОН], 3.86-3.30 m [22Н, 8-СЯ2СН3, 13(2)-СООСН2СЯ2ОСЯ2СЯ2ОСЯ2СЯ2ОЯ, 17-СH2СH2С00СH2СЯ20СЯ2СЯ20СЯ2СЯ20Н], 3.72 s (3Н, 12-СН3), 33.45 s (3Н, 2-СН3), 3.27 s (3Н, 7-СН3), 2.78-2.55 m (2Н, 17-СЯ2СH2C00СH2СH20СH2СH20СH2СH20H), 2.53-2.23 m (2Н,
17-(СН2СЯ2СООСН2СН2ОСН2СН2ОСН2СН2ОН), 1.86 d (3Н,
18-СН32 J 7.0 Hz), 1.74 t (3Н, 8-СН2СЯ3, J 7.3 Hz), 0.57 br.s (1H, I-NH), -1.59 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.53 s (1H, H10), 9.39 s (1H, H5), 8.55 s (1H, H20), 8.03 dd (1H, 3-СЯ=СН2, J 17.6 and 11.4 Hz), 6.33 d (1H, 3-СН^НЯ.^ J 17.6 Hz), (5.21 s (1H, H13(2)), 6.22 d (1H, 3-СН^НН^ J 11.7 Hz), 4.57-4.48 m (3H, 13(2)-СООСЯ2СН2ОСН2СН2ОС Н2СН2ОН, H18), 4.30 br.d (1H, H17, J 7.7 Hz), 4.22-4.08 m [2H, 17-СН2СН2СООСЯ2СН2ОСН2СН2ОСН2СН2ОН], 3.86-3.30 m [22H, 8-СЯ2СН3, 13(2)-СООС Н2СЯ20СЯ2СЯ20СЯ2СЯ20Я, 17-СН2СН2С00СН2СЯ20СЯ2СЯ20СЯ2СЯ20Щ, 372 s (3Н, 12-СН3), 3.44 s (3Н, 2-СН3), 3.266 s (3Н, 72СН3), 2.78-2.55 m (2Н, 17-СЯ2СН2СООСН2СН2ОСН2СН2ОСН2СН2ОН), 2.53-2.23 m (2Н, 17-(ССН2СЯ2СООССН2СН2ОССН2СН2ОССН2СН2ОН), 1.86 d (3Н, 18-СН3, J 7.0 Hz), 1.74 t (3Н, 8-СН2СЯ3, J 7.3 Hz), 0.68 br.s (1H, I-NH), -1.41 br.s (1H, III-NH).
Pheophorbide a 13(2).17-bistetraethylene glycol ester (21). 25.0 mg (27 %) of compound 21 (13(2)-diastereomers mixture, 13(2)-R/13(2)-S 5.5:1 according to 1H NMR) as a dark blue-black crystalline powder was obtained with transesterification of 76 mg
(0.099 mmol) of 8 in a mixture of 5 ml of tetraethylene glycol, 0.25 ml of concentrated sulfuric acid for 19 hours at complete conversion of starting compound 8. Mass-spectrum (ESI) m/z: for MH+ (C^HNO,,) calcd. 931.5, found 931.5; for MNa+ (C HN О Na)
v 50 67 4 13y 7 ' v 50 66 4 13 7
calcd. 953.5, found 953.4; for MK+ (C50H66N4O13K) calcd. 969.4, found
969.3. UV-Vis (CHCl3) X nm (relative intensity, %): 669 (41.5%), 612 (8.3%), 535 (9.5%), 504 (11.6%), 414 (100%). IR (KBr) cm-1: 3485 (v OH); 3393 (v NH of chlorin cycle); 2959 (vCHas CH3); 29— (vCHas CH2); 2872 (vCHs CH3); 2736 (vCH CH2-O-, glycol); 1736 (v C=O, ester); 16295 (v C=O, exo cycle); 1616 («chlorin band»). 1H NMR (CDCl3, 300 MHz) 5 ppm. Signals of 13(2)R-diastereomer: 9.59 s (1H, H10), 9.45 s (1H, H5), 8.64 s (1H, H20), 8.04 dd (1H, 3-CH=CH2, J 17.6 and 11.7 Hz), 6.33 d (1H, 3-CH=CHHtrans J 17.6 Hz), 6.32 s (1H, H13(2)), 6.23 d (1H, 3-CH=CHHcis J 117 Hz), 4.57-4.46 m (3H, 13(2)-COOCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, H18), 4.30 br.d (1H, H17, J 7.7 Hz), 4.21-4.05 m [2H, 17-CH2CH2COO CH2CH2OCH2CH2OCH2CH2OCH2CH2OH], 3.86-3.30 m [30H, 8-ch2ch3, 13 (2)-cooc h2ch2och2ch2och2ch2och2ch2oh, 17-СН2СН2С00СН2СН20СН2СН20СН2СН20СН2СН20Н], 3.72 s (3H, 12-CH3), 3.45 s (3H, 2-CH3), 3.28 s (3H, 7-CH3), 2.782.55 m (2H, 17-CH2CH2COOCH2CH2OCH2CH2OCH2CH2OCH2 CH2OH), 2.53-2.23 m (2H, 17-(CH2CH2COOCH2CH2OCH2CH2 OCH2CH2OCH2CH2OH), 1.86 d (3H 18-CH3, J 1.3 Hz), 1.73 t (3H, 8-СН2СН3, J 7.7 Hz), 0.48 br.s (1H, I-NH), -1.63 br.s (1H, III-NH). Signals of 13(2)S-diastereomer: 9.56 s (1H, H10), 9.41 s (1H, H5), 8.58 s (1H, H20), 8.02 dd (1H, 3-СН=СН2, J 17.9 and 11.4 Hz), 6.33 d (1H, 3-CH=CHHtrans J 17.6 Hz), 6.21 s (1H, H13(2)), 6.23 d (1H, 3-CH=CHHcis J 11.7"hz), 4.57-4.46 m (3H, 13(2)-С00СН2СН20СН2СН20СН2СН20СН2СН20Н, H18), 4.30 br.d (1H, H1^ J 7.7 Hz), 4.21-4.05 m [2H, 17-СН2СН2СООСН2СН2О CH2CH2OCH2CH2OCH2CH2OH], 3.86-3.30 m [30H, 8-СН2СН3, 13(2 )-СООСН2СН2ОСН2СН2ОСН2СН2ОСН2СН2ОН, 17-C H2(C Н2СООСН2СН2ОСН2СН2ОСН2СН2ОСН2СН2ОН], 3.72 s (3H, 12-CH3), 3.44 s (3H, 2-CH3), 3.27 s (3H, 7-CH3), 2.78-2.55 m (2H, 17-CН)CH2COOCH2CH2OCH2CH2OCH2CH2OCH2CH2OH), 2.532.23 m (2H, 17-(CH2CН2COOCH2CH2OCH2CH2OCH2CH2OCH2C H2OH), 1.86 d (3H, 18-CH3, J 7.3 Hz), 1.73 t (3H, 8-СН2СН3, J 7.7 Hz), 0.56 br.s (1H, I-NH), -1.44 br.s (1H, III-NH).
Recovering of exocycle of chlorophyll a phorbin derivatives with oligotetraethylene glycol fragments (general procedure, synthesis of compounds 22-29, 30-32). 33 % Aqueous methylamine solution (1.0-1.5 ml) was added to solution of start phorbin derivative (50-100 mg, approximately 0.081-0.165 mmol) in THF (5-7 ml) and stirred for 20-60 minutes (until complete conversion of the starting compound, control TLC). The reaction mixture was diluted with 50 ml of chloroform and washed with 1 % hydrochloric acid (200 ml) to remove an excess of methylamine and then with distilled water until neutral reaction of wash waters. The resulting solution was dried over anhydrous sodium sulfate then the solvent was distilled off. The residue after evaporation was chromatographed on silica gel (eluent - mixture of CCl4:acetone = 30:1 ^ 5:1).
Chlorin e6 13(1)-N-methylamide-17-methyl-15-diethylene glycol ester (22). 31.3 mg (60 %) of compound 22 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on 50 mg (0.073 mmol) of compound 6 in 5 ml of THF for 20 min at complete conversion of starting compound 6. Mass-spectrum (ESI) m/z: for MH+ (C^H^N^) calcd.
712.4, found 712.3. UV-Vis (CHCl3) X nm (relative intensity, %): 663 (31.2%), 607 (3.9%), 501 (9.9%), 403 (100%). IR (KBr) cm-1: 3377 (v OH); 3305 (v NH of chlorin cycle); 2959 2957 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2733 (vCH CH2-O-, glycol); 1734 (v C=O, ester); 1653 (v C=O, «amide I»); 1616 («chlorin band»); 1547 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm. 9.74 s (1H, H10), 9.69 s (1H, H5), 8.86 s (1H, H20), 8.13 dd (1H, 3-СН=СН2, J 18.3 and 11.7 Hz), 7.31-7.22 m (1H, 13-CON#CH3), 6.40 d (1H, 3-СН=СНН , J 18.3 Hz), 6.18 d (1H, 3-CH=CHH . J 11.7 Hz),
trans' 7' v ' cis 7'
5.59 d (1H, 15-CНAHBCOOCH2CH2OCH2CH2OH, J 19.4 Hz), 5.40 br.d (1H, 15-CHA//BCOOCH2CH2OCH2CH2OH, J 19.4 Hz),
4.52 q (1H, H18, J 7.0 Hz), 4.45 br.d (1H, H17, J 8.8 Hz), 4.37-4.11 m (2H, 15-CH2COOСН2CH2OCH2CH2OH), 3.84 q (2H, 8-СН2СН3, J 7.7 Hz), 3.63 s (3H, 17-CH2CH2COOСН3), 3.60 s (3H, 12-СНД 3.54 s (3H, 2-CH3), 3.36 s (3H, 7-CH3), 3.28 d (3H, 13-CONHC#3, J 4.4 Hz), 3.19-22.79 m (6H, 15-CH2COOCH2СН2OСН2СН2OH), 2.60-2.46 and 2.35-2.22 (both m 1H, 17-СН2CH2COOCH3), 2.162.01 and 1.98-1.83 (both m 1H, 17-СН2СН2С OOCH3), 1.76 t (3H, 8-СН2СН3, J 7.3 Hz), 1.72 d (3H, 18-CH3, J7.0 Hz), -1.63 br.s (1H, I-NH), -1.83 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide-17-methyl-15-triethylene glycol ester (23). 38.7 mg (74 %) of compound 23 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on 50 mg (0.069 mmol) of compound 7 in 5 ml of THF for 20 min at complete conversion of starting compound 7. Mass-spectrum (ESI) m/z: for MH+ (C42H54N5O8) calcd. 756.4, found 756.3. UV-Vis (CHCl3) X nm (relative intensity, %): 662 (30.8%), 607 (3.8%), 501 (9.9%), 403 (100%). IR (KBr) cm-1: 3380 (v OH); 3309 (v NH of chlorin cycle); 2957 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2735 (vCH CH2-O-, glycol); 1734 (v C=O, ester)2 1651 (v C=O, «amide I»); 1600 («chlorin band»); 1549 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.74 s (1H, H10), 9.69 s (1H, H5), 8.85 s (1H, H20), 8.13 dd (1H, 3-СН=СН2, J 18.0 and 11.4 Hz), 7.16-7.04 m (1H, 13-CON#CH3), 6.40 d (1H, 3-СН=СНН ,J 18.0 Hz), 6.18 d (1H, 3-CH=CHH . J 11.4 Hz), 5.57
trans' 7' v ' cis 7'
br.d (1H, 15-CНAHBCOOCH2CH2OCH2CH2OCH2CH2OH, J 18.7 Hz), 5.40 br.d (1H, 15-CHAНBCOOCH2CH2OCH2CH2OCH2CH2OH, J 18.7 Hz), 4.51 q (1H, H18, J7.3 Hz), 4.44 br.d (1H, H17, J8.8 Hz), 4.38-4.16 m (2H, 15-CH2COOСН2CH2OCH2CH2OCH2CH2OH), 3.83 q (2H, 8-СН2СН3, J 7.3 Hz), 3.64 s (3H, 17-СН2СН2С00СН3), 3.58 s (3H, 12-CH3), 3.53 s (3H, 2-CH3), 3.3(5 s (3H, 7-CH3), 3.28 br.d (3H, 13-CONHC#3, J 4.0 Hz), 3.52-3.09 m (6H, 15-CH2COOCH2СН2OСН2СН2OCH2CH2OH), 2.65-2.02 m (4H, 17-СН2СН2COOCH3), 2.00-1.65 m (4H, 15-cн2œ0cн2cн20cн2cн20cн2cн20н ), 1.75 t (3H, 8-сн2сн3, J 73 Hz), 1.72 d (3H, 18-CH3, J 7.3 Hz), -1.61 br.s (1H, I-NH3, -1.82 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide-17-methyl-15-tetraethylene glycol ester (24). 33.5 mg (62 %) of compound 24 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 50 mg (0.065 mmol) of compound 8 in 5 ml of THF for 20 min at complete conversion of starting compound 8. Mass-spectrum (ESI) m/z: for MH+ (C44H58N5O9) calcd. 800.4, found 800.3, for MNa+ (C^N^Na) calcd. 822.4- found 822.4. UV-Vis (CHCl3) X nm (relative5 intensity, %): 663 (31.1%), 606 (4.5%), 501 (10.8%), 403 (100%). IR (KBr) cm-1: 3380 (v OH); 3309 (v NH of chlorin cycle); 2955 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2737 (vCH CH2-O-, glycol); 1734 (v C=O, ester); 1651 (v C=O, «amide I»); 1601 («chlorin band»); 1551 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm. 9.75 s (1H, H10), 9.70 s (1H, H5), 8.85 s (1H, H20), 8.14 dd (1H, 3-СН=СН2, J 18.3 and 11.7 Hz), 7.43-7.34 m (1H, 13-CON#CH3), 6.40 d (1H, 3-СН=СНН , J 18.0 Hz), 6.19 d (1H, 3-CH=CHH . J 11.4 Hz),
trans cis
5.59 br.d (1H, 15-CНAHBC00CH2CH20CH2CH20CH2CH20CH2C H2OH, J 18.0 Hz), 5.41 br.d (1H, 15-CHA^BCOOCH2CH2OCH2C H2OCH2CH2OCH2CH2OH, J 18.0 Hz), 4.51 q (1H, H18, J 7.3 Hz), 4.45 br.d (1H, H17, J 8.8 Hz), 4.36-4.14 m (2H, ^-C^COO^ CH2OCH2CH2OCH2CH2OCH2CH2OH), 3.85 q (2H, 8-СН2СН3, J 7.3 Hz), 3.63 s (3H, 17-CH2CH2C 00СН3), 3.61 s (3H, 12-CH3), 3.54 s (3H, 2-CH3), 3.37 s (3H, 7-CH3), 3.29 d (3H, 13-CONHC#3, J 4.0 Hz), 3.19-2.79 m (12H, 15-CH2œ0CH2СН20СН2СН20СН2 СН20СН2СН20Н), 2.62-2.17 m (4H, 17-СН2СН2С OOCH3), 1.76 2 (3H, 8-СН2СН3, J 7.3 Hz), 1.72 d (3H, 18-CH3, J 7.0 Hz), -1.63 br.s (1H, I-NH), -1.84 br.s (1H, III-NH).
Chlorin e613(1)-N-methylamide 17-methyl-15-pentaethylene glycol ester (25). 36.8 mg (71 %) of compound 25 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 50 mg (0.062 mmol) of compound 9 in 5 ml of THF for 20 min at complete conversion
of starting compound 9. Mass-spectrum (ESI) m/z: for MH+ (C46H62N5O10) calcd. 844.4, found 844.3, for MNa+ (C46H61N5O10Na) calcd. 866.44found 866.3, for MK+ (C46H61N5O10K) calcd. 8812.4 found 882.3. UV-Vis (СНа3) X nm (relative intensity, %): 656 (32.3%), 602 (4.9%), 498 (11.0%), 398 (100%). IR (KBr) cm-1: 3380 (v ОН); 3309 (v NH of chlorin cycle); 2955 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2737 (vCH CH2-0-, glycol); 1732 (v C=0, ester); 1651 (v С=О, «amide I»); 1601 («chlorin band»); 1551 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.75 s (1H, H10), 9.70 s (1H, H5), 8.85 s (1H, H20), 8.14 dd (1H, 3-СЯ=СН2, J 18.0 and 11.4 Hz), 7.47-7.35 m (1H, 13-C0NHCH3), 6.40 d (1H, 3-CH=CHHtrans, J 18.0 Hz), 6.19 d (1H, 3-CH=CHHcis J 11.4 Hz), 5.59 br.d (1H,
15-сяанвсоосн2сн2осн2сн2оСн2сн2осн2сн2осн2сн2
ОН, J 19.4 Hz), 5.41 brd (1H, 15-CHAHBC00СН2СН20СН2СН2 0СН2СН20СН2СН20СН2СН20Н, J 19.41 Hz), 4.51 q (1H, H18, J 7.0 Hz), 4.45 brd (1H, H17, J 8.8 Hz), 4.36-4.13 m (2H, 15-CH2C 00СЯ2CH20CH2CH20CH2CH20CH2CH20CH2CH20H), 3.85 q (2H, 8-СЯ2СН3, J7.6 Hz), 3^4 s (3H, 17-СН2СН2С00СЯ3), 3.62 s (3H, 12-CH3), 3.54 s (3H, 2-CH3), 3.37 s (3H, 7-CH3), 3.30 d (3H, 13-CONHCH3, J 4.3 Hz), 3.19-2.79 m (18H, 15-СН2С00СН2СЯ2 0СЯ2СЯ20СЯ2СЯ20СЯ2СЯ20СЯ2СЯ20Н), 2.62-2.00 m (4H, 172 СЯ2СЯ2С00СН3), 1.76 t (3H, 8-СН2СЯ3, J 7.6 Hz), 1.72 d (3H, 18-C H32 J 7.0 Hz), -1.63 br.s (1H, I-NH ), -1.85 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide-17-methyl-15-hexaethylene glycol ester (26). 29.5 mg (57 %) of compound 26 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 50 mg (0.058 mmol) of compound 10 in 5 ml of THF for 20 min at complete conversion of starting compound 10. Mass-spectrum (ESI) m/z: for MH+ (C48H66N5O11) calcd. 888.5, found 888.4, for MNa+ (C48H65N5O11Na) calcd. 910.5 found 910.4, for MK+ (C48H65N5O11K) calcd. 926.4 found 926.3. UV-Vis (CHCl3) X nm (relative intensity, %): 663 (31.6 %), 607 (4.4 %), 501 (10.3 %), 403 (100 %). IR (KBr) cm-1: 3380 (v ОН); 3308 (v NH of chlorin cycle); 2953 (vCHas CH3); (vCHas CH2); 2868 (vCHs CH3); 2741 (vCH CH2-0-, glycol); 1734 (v C=0, ester); 1651 (v C=0, «amide I»); 1601 («chlorin band»); 1549 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.75 s (1H, H10), 9.69 s (1H, H5), 8.85 s (1H, H20), 8.14 dd (1H, 3-СЯ=СН2, J 16.9 and 10.7 Hz), 7.54-7.33 m (1H, 13-C0NHCH3), 6.40 d (1H, 3-СН=СНН , J 16.9 Hz), 6.19 d (1H, 3-CH=CHH ., J 10.3 Hz),
trans' 77 v ' cis' 77
5.59 br.d (1H, 15-CHAHBC00CH2CH20CH2CH20CH2CH20C H2CH20CH2CH20CH2CH20H, J 19.4 Hz), 5.41 brd (1H, 15-СЯ AHBC0 0CH2CH20CH2CH20CH2CH20CH2CH20CH2CH20C H2CBH20H, J 19.4 Hz), 4.63-4.39 m (2H, H18 and H17), 4.37-4.10 m (2H, 15-CH2C00СЯ2CH20CH2CH20CH2CH20CH2CH20C H2CH20CH2CH20H), 3.93-3.79 m (2H, 8-СЯ2СН3), 3.64 s (3H, 17-СН2СН2С00СЯ3), 3.62 s (3H, 12-CH3), 3.54 s (3H, 2-CH3), 3.36 s (3H 7-CH3), 3.30 d (3H, 13-C0NHC#3, J 4.3 Hz), 3.192.79 m (22H, 15-СН2С00СН2СЯ20СЯ2СЯ20СЯ2СЯ20СЯ2СЯ2 0СЯ2СЯ20СЯ2СЯ20Н), 2.62-2.00 m (4H, 17-СЯ2СЯ2СООССH3), 1.76 t(3H, 8-СН2СЯЯ3, J 7.6 Hz), 1.72 d (3H, 18-CH3, J 7.0 Hz), -1.(53 br.s (1H, I-NH), -1.84 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide-15-methyl-17-diethylene glycol ester (27). 30.4 mg (58 %) of compound 27 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 50 mg (0.073 mmol) of compound 11 in 5 ml of THF for 20 min at complete conversion of starting compound 11. Mass-spectrum (ESI) m/z: for MH+ (C40H50N5O7) calcd. 712.4, found 712.3. UV-Vis (CHCl3) X nm (relative intensity, %): 663 (29.6%), 607 (3.6%), 501 (9.2%), 403 (100%). IR (KBr) cm-1: 3382 (v ОН); 3308 (v NH of chlorin cycle); 2957 (vŒas CH3); 2926 (vŒas CH2); 2868 (vŒs CH3); 2733 (vŒ CH2-0-, glycol); 1734 (v C=0, ester); 1647 (v C=0, «amide I»); 1601 («chlorin band»); 1549 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.75 s (1H, H10), 9.69 s (1H, H5), 8.85 s (1H, H20), 8.13 dd (1H, 3-СЯ=СН2, J 17.6 and 11.7 Hz), 6.58-6.46 m (1H, 13-CON#CH3), 6.40 dd (1H, 3-CH=CHHtrans, J 17.9 and 1.1 Hz), 6.19 dd (1H, 3-CH=CHH., J 11.7 and 1.1™Hz), 5.55 d (1H, 15-
CHAHBCO2CH3, J 19.0 Hz), 5.32 d (1H, 15-CHAHBCO2CH3, J 19.0 Hz), 4.51 q (1H, H18, J 7.3 Hz), 4.44 br.d (1H, H17, J 8.4 Hz), 4.16 dd (2H, 17-CH2CH2C00СЯ2CH20CH2CH20H, J 5.9 and 3.7 Hz), 3.84 q (2H, 8-СЯ2СН3, J 7.3 Hz), 3.86 s (3H, 15-CH2C02C0CH3), 3.60 s (3H, 12-CH3), 3.53 s (3H, 2-CH3), 3.673.49 m (5H, 17-СН2СН2С00СН2СЯ20СЯ2СН20Я), 3.48-3.43 m (2H, 17-СН2СН2С00СН2СН20СН2СЯ20Н), 3^6 s (3H, 7-CH3), 3.31 d (3H, 13-C0NHC#3, J 4.8 Hz2, 2.60-2.45 m 1H, 2.35-2.16 m 2H and 2.14-2.00 m 1H (17-СЯ2СЯ2С00СН2СН20СН2СН20Н), 1.77 d (3H, 18-CH3, J 7.0 Hz), 1.75 t (3H, 8-СН2СЯ3, J 7.3 Hz), -1.60 br.s (1H, I-NH ), -1.78 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide 15-methyl-17-triethylene glycol ester (28). 5.0 mg (16 %) of compound 28 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 30 mg (0.041 mmol) of compound 12 in 3 ml of THF for 1 h at complete conversion of starting compound 12. Mass-spectrum (ESI) m/z: for MH+ (C42H54N5O8) calcd. 756.4, found 756.4, for MNa+ (C42H53N5O8Na) calcd. 778.4 found 778.4, for МК+ (C42H53N5O8K) calcd. 5794.3 found 794.3. UV-Vis (CHCl3) X nm (relative 5ntensity, %): 663 (31.6%), 608 (5.4%), 500 (11.7%), 402 (100%). IR (KBr) cm-1: 3382 (v ОН); 3307 (v NH of chlorin cycle); 2957 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2733 (vCH CH2-0-, glycol); 1734 (v C=0, ester); 1647 (v C=0, «amide-I»); 1601 («chlorin band»); 1551 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.76 s (1H, H10), 9.70 s (1H, H5), 8.87 s (1H, H20), 8.13 dd (1H, 3-СЯ=СН2, J 17.6 and 11.4 Hz), 6.66-6.52 m (1H, 13-C0NHCH3), 6.40 d (1H, 3-CH=CHHtrans, J 17.6 Hz), 6.20 d (1H, 3-CH=CHHcis, J 11.7 Hz), 5.56 d (1H, t15S-CHAHBCO2CH3, J 19.1 Hz), 5.33 dcis(1H, 15-CHAHBCO2CH3, J 19.1 Hz), 4.52 q (1H, H18, J 7.3 Hz), 4.45 br.d (1H, H17, J 8.8 Hz), 4.15 dd (2H, 17-СН2СН2С00СН2СН20СН2СН20СН2СН20Н, J 5.1 and 4.0 Hz), 3.844 q (2H, 8-СЯ2СН3, J 7.7 Hz), 3.86 s (3H, 15-CH2C02C0CH3), 3.59 s (3H, 12-CH3), 3.54 s (3H, 2-CH3), 3.58-3.33 m (10H, 17-СН2СН2С00СН2СЯ20СЯ2СЯ20СЯ2СН20Я), 3.36 s (3H, 7-CH3), 3.30 d (3 H, 13-CONHCH3, J 4.4 Hz), 2.61-2.47 m 1H and 2.34-2.0 4 m 3H (17-СЯ2СЯ2С00СН2СН20СН2СН20СН2СН20Н), 1.77 d (3H, 18-CH3, J7.3 Hz), 1.75 t (3H, 8-СН2СЯ3, J7.7Hz), -1.65 br.s (1H, I-NH), -1.81 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide 15-methyl-17-tetraethylene glycol ester (29). 10.3 mg (21 %) of compound 29 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 20 mg (0.026 mmol) of compound 13 in 3 ml of THF for 1 h at complete conversion of starting compound 13. Mass-spectrum (ESI) m/z: for MH+ (C44H58N5O9) calcd. 800.4, found 800.3, for MNa+ (C44H57N5O9Na) calcd. 822.4 found 822.4, for МК+ (C44H57N5O9K) calcd. 838.4 found 838.4. UV-Vis (CHCl3) X nm (relative intensity, %): 663 (31.5%), 607 (4.2%), 500 (10.0%), 403 (100%). IR (KBr) cm-1: 3382 (v ОН); 3306 (v NH of chlorin cycle); 2955 (vCHas CH3); 2924 (vCHas CH2); 2868 (vCHs CH3); 2735 (vCH CH2-0-, glycol); 1734 (v C=0, ester); 1651 (v C=0, «amide-I»C 1601 («chlorin band»); 1549 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.74 s (1H, H10), 9.69 s (1H, H5), 8.86 s (1H, H20), 8.13 dd (1H, 3-СЯ=СН2, J 17.6 and 11.4 Hz), 6.57-6.46 m (1H, 13-C0NHCH3), 6.40 d (1H, 3-CH=CHHtrans, J 17.6 Hz), 6.19 d (1H, 3-CH=CHHis, J 11.7 Hz), 5.55 d (1H, 15-CHAHBCO2CH3, J 18.7 Hz), 5.31 d (1н, 15-CHAHBCO2CH3, J 18.7 Hz), 4.52 q (1H, H18, J 7.0 Hz), 4.42 br.d (1H, H17, J 8.8 Hz), 4.244.12 m (2H, 17-СН2СН2С00СЯ2СН20СН2СН20СН2СН20СН2С H20H), 3.86 s (3H, 15-CH2C02C0CH3), 3.84 q (2H 8-СЯ2СН3, J 7.3 Hz), 3.59 s (3H, 12-CH3X 3.51 s (3H, 2-CH3), 3.64-3.34 m (14H, 17-СН2СН2С00СН2СЯ20СЯ2СЯ20СЯ2СЯ20СЯ2СЯ20Н), 3.36 s (3H, 7-CH3), 3.30 d (3H, 13-C0NHCH3, J 4.8 Hz), 2.642.46 m 1H and 2.36-2.05 m 3H (17-СЯ2СЯ2С00СН2СН20СН2С H20CH2CH20CH2CH20H), 1.76 d (3H, 18-CH3, J 7.0 Hz), 1.75 t (3H, 8-СН2СЯ3, J 7.3 Hz), -1.61 br.s (1H, I-NH), -1.78 br.s (1H, III-NH).
Chlorin e613(1)-N-methylamide 15,17-bis(diethylene glycol) ester (30). 14.1 mg (42 %) of compound 30 as a dark blue-black
crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 30 mg (0.040 mmol) of compound 19 in 3 ml of THF for 1 h at complete conversion of starting compound 19. Mass-spectrum (ESI) m/z: for MH+ (C43H5(.N5O9) calcd. 786.4, found 786.3. UV-Vis (CHCl3) X nm (relative intensity, %): 663 (28.8%), 607 (3.2%), 501 (8.5%), 403 (100%). IR (KBr) cm-1: 3382 (v OH); 3309 (v NH of chlorin cycle); 2957 (vCHas CH3); 2926 (vCHas CH2); 2868 (vCHs CH3); 2733 (vCH CH2-O-, glycol); 1730 (v C=O, ester); 1637 (v C=O, «amide-I»); 1601 («chlorin band»); 1552 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.78 s (1H, H10), 9.73 s (1H, H5), 8.88 s (1H, H20), 8.14 dd (1H, 3-СН=СН2, J 17.8 and 11.7 Hz), 7.23-7.04 m (1H, 13-CON#CH3), 6.40 d (1H, 3-СН=СНН , J 17.6 Hz), 6.20 d (1H, 3-CH=CHH ., J 11.7 Hz),
trans cis
5.59 d (1H, 15-CНAHBC00CH2CH20CH2CH20H, J 19.1 Hz), 5.42 d (1H, 15-CHAНBC00CH2CH20CH2CH20H, J 19.1 Hz), 4.52 q (1H, H18, J 7.7 Hz), 4.49 br.d (1H, H17, J 8.0 Hz), 4.404.25 m (2H, 15-CH2C00СН2CH20CH2CH20H), 4.21 br.t (2H, 17-CH2CH2C00СН2CH[20CH2CH20H, J 4.42 Hz), 3.85 q (2H, 8-СН2СН3, J 8.1 Hz), 3.71-341 m (12H, 17-СН2СН2С00СН2СН20СН2СН20Н, 15-СН2С00СН2СН20СН2СН20Н), 3260 s (3H, 12-CH3), 3.54 s (3H, 2-CH3), 3.37 s (3H, 7-CH3), 3.30 d (3H, 13-CONHCH3, J 4.4 Hz), 2.60-2.00 m (4H, 17-СН2СН2С00СН2СН20СН2СН20Н),
1.74 d (3H, 18-CH3, J 8.0 Hz), 1.75 t (3H, 8-СН2СН3, J 8.4 Hz), -1.66 br.s (1H, I-NH), -1.85 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide 15,17-bis(triethylene glycol) ester (31). 14 mg (43 %) of compound 31 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 30 mg (0.040 mmol) of compound 20 in 3 ml of THF for 1 h at complete conversion of starting compound 20. Mass-spectrum (ESI) m/z: for MH+ (C^HNO,, ) calcd. 875.5, found 874.4, for MNa+ (tHNONa)
47 64 5 11 47 63 5 11
calcd. 896.4 found 896.4, МК+ (C H NO,K) calcd. 912.4, found
47 63 5 11
912.3. UV-Vis (CHCl3) X nm (relative intensity, %): 663 (28.8%), 607 (3.2%), 501 (8.5%), 403 (100%). IR (KBr) cm-1: 3382 (v OH); 3317 (v NH of chlorin cycle); 2955 (vCHas CH3); 2924 (vCHas CH2); 2870 (vCHs CH3); 2737 (vCH CH2-O-, glyc;ol); 1730 (v C=0, ester); 1645 (v C=O, «amide-I»); 1601 («chlorin band»); 1551 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.77 s (1H, H10), 9.72 s (1H, H5), 8.88 s (1H, H20), 8.14 dd (1H, 3-СН=СН2, J 17.6 and 11.7 Hz), 7.42-7.32 m (1H, 13-CON#CH3), 6.41 d (1H, 3-СН^НН^, J 17.6 Hz), 6.20 d (1H, 3-CH=CHHis, J 11.7 Hz), 5.59 d (1H, 15-CНAHвœ0CH2CH20CH2CH20CH-Cн20H, J 18.7 Hz), 5.44 d (1H, 15-CHAНвœ0CH2CH20CH2CH2CH20CH2CH20H, J 18.7 Hz), 4.53 q (1H, H18, J7.3 Hz), 4.50 br.d (1H, H17, J 8.0 Hz), 4.354.20 m (2H, 15-CH2C00СН2CH20CH2CH20CH2CH20H), 4.16 dd (2H, 17-cн2cн2c00cн2cн20cн2cн20cн2cн20н, J 4.7 and 3.1 Hz), 3.85 q (2H, 8-СН2СН3, J "7.7 Hz), 3.65-3.33 m 12H and 3.22-2.80 m 8H (17-СН2СН2С00СН2СН20СН2СН20СН2СН20Н, 15-СН2С00СН2СН20СН2СН20СН2СН20Н), 3.61 2 (3H, 12-C H3), 3.51 s (3H, 2-CH3), 3. 37 s (3 H, "7-CH3), 3.22 9 d (3H, 13-CONHC#3, J 4.8 Hz), 2.59-2.22 m (4H, 17-СН2СН2С00СН2СН20СН2СН20Н),
1.75 t (3H, 8-СН2СН3, J 7.0 Hz), 1.74 d (3H, 18-CH3, J 6.2 Hz), -1.70 br.s (1H, I-NH), -1.87 br.s (1H, III-NH).
Chlorin e6 13(1)-N-methylamide 15,17-bis(tetraethylene glycol) ester (32). 15.0 mg (46 %) of compound 31 as a dark blue-black crystalline powder was obtained by the action of 33 % aqueous methylamine solution (1 ml) on the 30 mg (0.040 mmol) of compound 21 in 3 ml of THF for 1 h at complete conversion of starting compound 21. Mass-spectrum (ESI) m/z: for MH+ (C^H^O^) calcd. 962.5, found 962.8, for MNa+ (C^H^O^Na) calcd. 9854.5, found 984.7, МК+ (C^H^O^K) calcd. 1000.5, found 1000.3. UV-Vis (CHCl3) X nm (relative; intensity, %): 663 (29.7%), 607 (4.3%), 501 (10.2%), 403 (100%). IR (KBr) cm-1: 3382 (v OH); 3312 (v NH of chlorin cycle); 2955 (vCHas CH3); M— (vCHas CH2); 2870 (vCHs CH3); 2743 (vCH CH2-O-, glycol); 1732 (v C=O, ester); 1645 (v C=O, «amide-I»); 1601 («chlorin band»); 1551 («amide-II»). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.74 s (1H, H10), 9.69 s (1H, H5), 8.85 s (1H, H20), 8.10 dd (1H, 3-СН=СН J
18.0 and 11.7 Hz), 7.47-7.36 m (1H, 13-CON#CH3), 6.37 d (1H, 3-СН=СНН , J 18.0 Hz), 6.17 d (1H, 3-CH=CHH ., J 11.7 Hz),
trans cis
5.56 d (1H, 15-CНAHBC00CH2CH20CH2CH20CH2CH20CH2CH2 OH, J 18.3 Hz), 5.38 d (1H, 15-0^^000^0^00^0^0 H2OCH2CH2OCH2CH2OH, J 18.3 Hz), 4.50 q (1H, H18, J 7.7 Hz), 4.49-4.41 m(1H, H17), 4.33-4.10 m (2H, 15-CH2C00СН2CH20CH 2CH2OCH2CH2OCH2CH2OH), 4.12 dd (2H, 17-СН2СН2С00СН2С H2OCH2CH2OCH2CH2OCH2CH2OH, J 5.1 and 3.7 Hz), 3.82 q (2H, 8-СН2СН3, J 7.7 Hz), 3.64-3.30 m 22H and 3.11-2.82 m 8H (17-C Н2СН2С00СН2СН20СН2СН20СН2СН20СН2СН20Н, 15-CH2CO 0СН2СН20СН2СН20СН2СН20СН2СН20Н), 3.58 s (3H, 12-CH3), 3.49 s (3H, 2-CH3), 3.34 s (3H, 7-CH3), 3.26 d (3H, 13-CONHC#3, J 4.4 Hz), 2.57-2.17 m (4H, 17-СН2СН2С00СН2СН20СН2СН20С H2CH2OH), 1.72 t (3H, 8-СН2СН3, J 7.7 Hz), 1.70 d (3H, 18-CH3, J 7.7 Hz), -1.76 br.s (1H, I-NH), -L90 br.s (1H, III-NH).
Pyropheophorbide a 17-pentaethylene glycol ester (17). To a solution of 30.0 mg (0.055 mmol) of pyropheophorbide a (3) in 5 ml of chloroform 15.0 mg of DMAP, 18.1 mg 2-chloro-jV-methylpyridinium iodide and 0.1 ml of pentaethylene glycol was added. The mixture was boiled under reflux for 1 hour. The reaction was monitored by TLC (eluent: CCl4 -acetone = 1:1). The reaction mixture was diluted with 50 ml chloroform, transferred to a separatory funnel and washed with 5-10 % hydrochloric acid for removing of DMAP and 2-chloro-V-methylpyridinium iodide excess. And then hydrochloric acid was washed by distilled water until neutral reaction of wash waters. The resulting solution was dried over anhydrous sodium sulfate and evaporated under reduced pressure at 40-50 °C. The residue after evaporation was chromatographed on silica gel (eluted with CCl4-acetone in ratios ranging from 30:1 to 1:1). The fraction containing the basic substance was evaporated and precipitated with hexane. 15 mg (36 %) of compound 17 was obtained. Mass-spectrum (ESI) m/z: for MH+ (C43H55N4O8) calcd. 755.4, found 755.4, for MNa+ (C43H52N4O9Na) calcd. 777.4, found 777.4, MK+ (C43H52N4O9K) calc d. 793.4, found 793.3. UV-Vis (CHCl3) X nm (relative intensity, %): 668 (46.9%), 611 (9.3%), 558 (4.4%), 538 (10.7%), 414 (100%). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.54 s (1H, H10), 9.43 s (1H, H5), 8.61 s (1H, H20), 8.04 dd (1H, 3-C#=CH2, J 18.0 and 11.7 Hz), 6.32 d (1H, 3-CH=CHH , J 18.0 Hz), 6.21 d (1H, 3-CH=CHH.,
trans cis
J 11.7 Hz), 5.31 d (1H, H13(2)A , J 20.2 Hz), 5.16 d (1H, Н13(2)В, J 20.2 Hz), 4.54 q (1H, H18, J 7.3 Hz), 4.36 br.d (1H, H17, J 7.3 Hz), 4.25-4.16 m (2H, 17-СН2СН2С00СН2СН20(СН2СН20)3СН2С H2OH), 3.73 q (2H, 8-(СН2СН3), J 7.3 Hz), 3.71 s ^H, 12-CH3), 3.45 s (3H, 2-CH3), 3.78-3.46 m (18H, СН2СН2С00СН2СН20( СН2СН20)3СН2СН20Н), 3.27 s (3H, 7-CH3), 2.83-2.57 m [22H, 17-^0^0000^(0^00^)^^), 2.44-2.29 m [2H, 17-СН2СН2С00СН2(СН20СН2)4СН20Н), 1.86 d (3H, 18-CH3, J 7.0 Hz), 1.73 t (8-СН2СН3, J 7.3 Hz), 0.43 br.s (1H, I-NH), -3.67 br.s (1H, III-NH).
Pyropheophorbide a 17-pentaethylene glycol ester (18). To a solution of 33.0 mg (0.060 mmol) of pyropheophorbide a (3) in 5 ml of chloroform 15.9 mg of DMAP, 15.3 mg of 2-chloro-V-methylpyridinium iodide and 0.1 ml of hexaethylene glycol was added. The mixture was boiled under reflux for 1.5 hour. Hereinafter the synthesis and isolation of the reaction product was carried out analogously to the procedure of synthesis of pentaethylene glycol derivative 17. The yield of compound 18 was 15 mg (30 %). Massspectrum (ESI) m/z: for MH+ (C45H59N4O9) calcd. 799.4, found 799.6, for MNa+ (C45H58N4O9Na) calcd. 821.4, found 821.5, MK+ (C43H52N4O9K) calcd. 837.4, found 837.4. UV-Vis (CHCl3) X nm (relative intensity, %): 668 (46.9%), 611 (9.3%), 558 (4.4%), 538 (10.7%), 414 (100%). 1H NMR (CDCl3, 300 MHz) 5 ppm: 9.48 s (1H, H10), 9.38 s (1H, H5), 8.60 s (1H, H20), 8.01 dd (1H, 3-CH=CH2, J 17.5 and 11.5 Hz], 6.30 d [1H, 3-CH=CHHtrans, J 17.6 Hz), 6.19 d (1H, 3-CH=CHHcis, J 12.1 Hz), 5.30 d (1H, H13(2)A , J 20.0 Hz), 5.15 d (1H, H13(2)B,ciJ 20.1 Hz), 4.53 q (1H, H18, J 7.1 Hz), 4.34 br.d (1H, H17, J 7.1 Hz), 4.25-4.16 m (2H, 17-СН2СН2С00СН2 CH2O(CH2CH2O)4CH2CH2OH), 3.71 s (3H, 12-CH3), 344 s (3H,
2-СН3), 3.78-3.46 m (24Н, 8-СЯ2СН3, СН2СН2С00СН2СЯ2 0(СЯ2СЯ20)4СЯ2СЯ20Н), 3.24 s (3Н, 7-СН3), 2.83-2.57 m [2Н, 17-СЯ2СН2С00СН2(СН20СН2)4СН20Н), 2.444-2.29 m (2Н, 17-СН2СЯ2С00СН2(СН20СН2)4СН20Н), 1.86 d (3Н, 18-СН3, J 7.0 Hz), 1.73 t (8-СН2СЯ3, J 7.3 Hz), 0.43 br.s (1H, I-NH), -1.67 br.s (1H, III-NH).
Results and Discussion
The transesterification reaction of the ester groups at positions 17 and 13(2) (under acid catalysis and with the activation of 2-chloro-^-methylpyridinium iodide, respectively) and the esterification of the carboxyl group at position 17 were used for insertion of oligoethylene glycol substituent's to the macrocycle periphery (compounds 6-32, Scheme 1). The transformation of phorbins to chlorins was carried out by exocycle opening under the action of methylamine. The structure of the compounds obtained was confirmed using IR, UV-Vis and NMR spectroscopy and mass spectrometry data. The insertion of oligoethylene glycol substituent leads to a decrease in the chromatographic mobility on normal phase (TLC Silufol) compared with the both initial ester and carboxylic acid derivatives. The peaks of protonated molecular ions and, in many cases, the peaks of sodium and potassium adduct cations of compounds 6-32 are observed in mass spectra (ESI). The oligoethylene glycol fragment is manifested in the IR spectrum as a weak band at 2730-2750 cm-1 region, this band corresponds to the stretching vibrations of the methylene group linked to the etheric oxygen atom C-H bond. In the 'H NMR spectra the oligoethylene glycol fragments appear as multiplets at the regions of 4.60-4.00 and 3.90-3.00 ppm, typical for the methylene protons near the ester, alcohol and ether oxygen atoms. The trans-esterification of the ester group leads to absence of the corresponding methyl singlet in the Щ NMR spectrum of the product that allows to distinguish methoxyl substitution at the positions 13(2) and 17. Chemical shifts of these proton signals in the methyl pheophorbide a *H NMR spectra differ significantly from each other and, at the same time, slightly change during the transition from one to another derivative. So the ester methyl group signals can provide a reliable source of information about the direction of the reaction (see Figure 1, which presents the Щ NMR spectra of methylpheophorbide a (1) and its diethylen glycol esters (6 and 11) with different positions of glycol substituent as an example). The investigation of NMR spectra of all 13(2)-carbomethoxy derivatives shows that all of them are 13(2) diastereomer mixture with a significant prevalence of 13(2)-R diastereomer.
Opening of phorbin derivatives exocycle is manifested in the IR and NMR spectra by the same way as more simple derivatives we have observed previously.122-261 In the IR spectra of chlorin e6 derivatives 22-32 the 13(1)-keto group absorption band about 1700 cm-1 is absent and absorption bands of amide groups ("amide-I» in the region of 16401650 cm-1 and «amide-II» in the region 1530-1550 cm-1) are present. The singlet of the proton in position 13(2) at 6.256.35 ppm is absent and the signals of protons of the methylene group which is formed by opening exocycle (AB multiplet system at 5-6 ppm region) as well as the methylamide group proton signals (broad quartet or unresolved multiplet of NH
proton and doublet of the methyl moiety) are observed in the Щ NMR spectra of compounds 22-32. Preparation of di-, tri- and tetraethylene glycol 17-esters was carried out by the action of the corresponding diol excess on the corresponding 17-methyl ester at presence of sulfuric acid. Di-, tri- and tetraethylene glycol acted simultaneously as a reactant and a solvent. In the case of derivatives 1-3, the solubility of which in a mixture of sulfuric acid and oligothylene glycol is low, a small amount of chloroform was added to reaction mixture up to the complete dissolution of the starting chlorin precipitate. As a result, the trans-esterification of the ester group at the 17-position substituent to form the target products takes place. Exocycle ester group trans-esterification does not occur under these conditions. Esterification of chlorins 3 and 5 carboxy group by penta- and hexaethyleneglycol, stimulated by 2-chloro-^-methylpyridinium iodide as activating agent, was used for corresponding esters synthesis because of low availability of penta- and hexaethyleneglycols.[27-32] The corresponding derivatives were obtained in the case of pyropheophorbide a. Under the action of penta- and hexaethyleneglycol on chlorin 5 at the same conditions, there was a formation of a complex mixture of unidentified compounds, but the target products were not obtained. The same activating agent was also used by us to synthesize 13(2) ethers and methylpheophorbides 6-10 with the di-, tri-, tetra-, penta- and hexaethylene glycol fragments: it is known that the ester group exocycle methylpheophorbide has a relatively high chemical activity and, therefore, can undergo a trans-esterification reaction.[33-36] Synthesis of chlorin e6 derivatives with oligoethylene glycol substituents at position 15 (22-26) was carried out by the action of methylamine on phorbin derivatives 6-10. Chlorin e6 derivatives with oligoethylene glycol substituents at position 17 (27-29) were obtained by the same way (the phorbin derivatives 11-13 exo ring recovering by the action of methylamine).
Trans-esterification of phorbin derivatives (6-10) ester groups at position 17 was used for synthesis of phorbin derivatives with two fragments of oligoethylene glycol (1921). Chlorin e6 derivatives with two oligoethylene glycol fragments (30-32) can also be synthesized by the action of methylamine on phorbin derivatives 19-21. Efforts to obtain the same derivatives directly from methylamide 4 by trans-esterification of both ester groups of this compound were unsuccessful.
Separation of even a slight excess of the diol from the reaction product is the most time-consuming step of the process. The more polar is the derivative obtained, the more difficult is its separation. In this regard, the synthesis of oligoethylene glycol derivatives should be designed so, that the step involving interaction with oligoethylene glycol would lead to the production of the most possible hydrophobic compound and subsequent conversion to form more hydrophilic derivatives held without oligoethylene glycols. Thus, when two substituents are inserted (compounds 30-32), it is advisable to obtain phorbin derivatives 19-21 first, and thereafter exocycle opening. Similarly, chlorin e6 derivatives with oligoethylene glycol fragments at position 17 (27-29) are more convenient to receive via appropriate 17-ester pheophorbide a 11-13.
For the hydrophilicity estimation of biologically active substances the characteristics of their distribution between
13(2)-c02ch3 i2-CH3
17-c02ch3
7-ch,
2-ch,
-1-1-1-1-1-j-I-r-1-1-1-1-1-1-1-1-1-1-1-1-1-1-r-
4.00 3.80 3.60 3.40 3.20
I2-CH3
17-c02ch3
7-ch, 2-ch,
3.00
B
-1-r-1-1-1-1-1-1-1-1-1-r
4.00 3.80 3.60
—I-1-1-1—I-1-1-1-1-1-1-1—
3.40
3.20
3.00
13(2)-c02ch3 I2-CH3
7-ch,
2-ch,
. 13(2)i—
CO2CH3 i o
do2ch3
-1-1-1-1-1-1-r
4.00 3.80
—1-1-j-1-1-1-1-1-1-1-1-1-1-1-1-1-1-[-r
3.60 3.40 3.20 3.00
ho o a'
Figure 1. 1H NMR spectra of methylpheophorbide a (1) and its diethylene glycol esters 6 (B) and 11 (C) (CDCl 300 MHz, 3-4 ppm).
the aqueous and «fat phase» are used and currently octanol is commonly used as the «fat phase».[37] Furthermore, it was shown that the ratio of distribution correlates with retention times of compounds by reverse phase chromatography.[38] Thus the chromatographic mobility of the compounds by reverse phase chromatography may serve as a quantitative criterion for the hydrophilic properties estimation along with the distribution between octanol and water. The higher the mobility, the greater the hydrophilicity of the compounds investigated, and the greater the difference in retention times, the more different hydrophilicity. The insertion of any oligoethylene glycol fragment in any position of the macrocycle significantly increases chromatographic mobility on reversed phase (retention time is reduced by 3-5 minutes). A similar effect is achieved by the introduction of the second oligoethylene glycol fragment. Comparison of the oligoethylene glycol derivatives 6-32 chromatographic characteristics (Table 1) reveals the following structure features influencing on the hydrophilicity of the compounds obtained. The exocycle presence/absence and oligoethylene glycol fragment position in the macrocycle are the structural factors of a most significant influence. Transition from phorbin derivatives to chlorin derivatives leads to increase in the hydrophilicity of the compounds when other structural characteristics (oligoethylene glycol chain length and number of identical oligoethylene glycol alternates) are equal. For example, the transition of 13(2)-glycol derivatives 6-10 to the corresponding chlorins 22-26 leads to retention
time decreasing of approximately 0.5-1.5 min. Similarly, retention time of 2.3 min decreased in case of transition from phorbin derivatives with glycol fragment in position 17 (11-13) to chlorin derivatives with the same fragment at the same position (27-29). When other structural characteristics are equal the isomeric derivatives with oligoethylene glycol moiety at position 17 have higher chromatographic mobility than derivatives with oligoethylene glycol moiety at position 13(2) (in the case of phorbin derivatives) or at position 15 (in the case of chlorins). For example, the retention time of chlorin e6 derivatives with oligoethylene glycol moiety at position 17 (27-29) is approximately 2 min lower than the retention time of analogous derivatives with oligoethylene glycol moiety at position 15 (6-8). In the case of phorbin derivatives the difference in the retention time for analogous pairs is about 1 min. The oligoethylene glycol chain length influencing on the chromatographic mobility of derivatives is significantly lower than the effect of other structural factors. The retention time of these derivatives is similar and the difference of retention time values for structural analogs is lower than 0.5 min in most cases. It is interesting that the monotonous increasing of chromatographic mobility with oligoethylene glycol chain length growing was not observed in many cases because of complex interactions of oligoethylene glycol moiety with stationary phase. Thus, results of hydrophylicity estimation of the oligoethylene glycol chlorophyll a derivatives reported above allowed to conclude that more available di-, tri- and tetraethylene glycol
Table 1. Retention time of chlorins with oligoethylene glycol fragments (Thermo finnigan surveyor (PDA, column Hypersil C18 100x2/1 mm, gradient elution (from a mixture of 1% aqueous trifluoroacetic acid - methanol (40:60 by volume) to pure methanol for 50 min, flow rate 0.4 ml/min) (*) - chlorophyll a derivatives without oligoethylene glycol fragments for comparison.
Gl = OCH2(CH2OCH2)nCH2OH X = Gl X = OCH3 X = Y = Y = OCH3 Y = Gl X Y Gl X = Gl Y = OCH3
1 37.76 (6) 36.68 (11) 29.13 (19) 41.64 (14) 36.14 (22)
2 37.54 (7) 36.56 (12) 33.28 (20) 41.48 (15) 36.46 (23)
3 36.81 (8) 36.39 (13) 31.90 (21) 41.22 (16) 36.22 (24)
4 36.46 (9) 43.09 (17) 35.93 (25)
5 36.58 (10) 40.74 (18) 36.12 (26)
(*) 41.16 (1) 45.92 (2)
Y O' X
Gl = OCH2(CH2OCH2)nCH2OH X = OCH,
Y = Gl
X = Y = Gl
34.01 (27) 34.43 (28) 33.98 (29)
27.03 (30) 31.19 (31) 30.09 (32)
40.96 (4)
n
can be used for the synthesis of hydrophilic derivatives instead of the less available penta- and hexamers.
Conclusion
Thus, chlorophyll a phorbin and chlorin derivatives with oligoethylene glycol fragments at the macrocycle periphery were synthesized in this study and the hydrophi-licity estimation of the compounds obtained based on their chromatographic mobility on reverse phase was carried out. The introduction of oligoethylene glycol moiety has been shown to increases significantly the hydrophilicity of the whole molecule. Among structural factors the presence/ absence of exocycle (exocycle opening leads to the hydro-phobicity decrease), as well as position of oligoethylene glycol fragment (the moving of oligoethylene glycol fragment from the macrocycle leads to the increase in hydro-philicity due to more effective solvation of this fragment) are the most important. The monotonous increase of chro-matographic mobility with oligoethylene glycol chain length growing was not observed in many cases, the most likely, due to the complex nature of oligoethylene glycol moiety interaction with the stationary phase. Oligoethylene glycol chain lengthening does not lead to any appreciable increase in hydrophilicity, so for hydrophilizing chlorophyll derivatives may be used more available di-, tri- and tetraethylene glycol instead of less available penta- and hexamers.
Acknowledgements. This work was supported by RFBR (grant № 14-03-01061 a)
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Received 08.05.2014 Accepted 24.05.2014
