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9Porphyrins
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Макрогэтэроцмклы
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http://macroheterocycles.isuct.ru
DOI: 10.6060/mhc160857p
Conjugates of Pyropheophorbide a with Androgen Receptor Ligands
Vladimir A. Zolottsev,a Olga V. Zazulina,a Galina E. Morozevich,a Maria G. Zavialova,a Alexander Y. Misharin,a Roman A. Novikov,b Vladimir P. Timofeev,b Oskar I. Koifman,c and Gelii V. Ponomareva@
aInstitute of Biomedical Chemistry, 119121 Moscow, Russia
hEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia c Research Institute ofMacroheterocycles, Ivanovo State University of Chemistry and Technology, 153000 Ivanovo, Russia @Corresponding author E-mail: gelii@yandex.ru
Two new conjugates of pyropheophorbide a with testosterone anddihydrotestosterone: 173[2-(17fi-hydroxy-3-oxopregn-4-en-21-oylamido)ethylamido]pyropheophorbide a (10) and 173[2-(17fi-hydroxy-3-oxopregnan-21-oylamido)ethyl-amido]pyropheophorbide a (11) were synthesized. IC50 for conjugates 10 and 11 at 96 h incubation in LNCaP and PC-3 prostate carcinoma cells were 1.4 ¡M and 3.3 ¡M for compound 10, and 4.5 ¡M and 6.1 ¡M for compound 11, respectively. Irradiation with light at wavelength of660 nm increased toxicity of the conjugates.
Keywords: Conjugates, pyropheophorbide a, testosterone, dihydrotestosterone, prostate carcinoma cells, cytotoxicity.
Конъюгаты пирофеофорбида а с лигандами андрогенового рецептора
В. А. Золотцев^ О. В. Зазулина^ Г. Е. Морозевич^ М. Г. Завьялова^ А. Ю. Мишарин^ Р. А. Новиков* В. П. Тимофеев* О. И. Койфман^ Г. В. Пономарев^
аИнститут биомедицинской химии, 119121 Москва, Россия
ъИнститут молекулярной биологии им. В.А. Энгельгардта РАН, 119991 Москва, Россия
СНИИ химии макрогетероциклических соединений, Ивановский государственный химико-технологический
университет, 153000 Иваново, Россия
®Е-шаИ: gelii@yandex.ru
Синтезированы два новых конъюгата пирофеофорбида а с тестостероном и дигидротестостероном: 173[2-(17^-гидрокси-3-оксопрегн-4-ен-21-оиламидо)этиламидо]пирофеофорбид а (10) и 173[2-(17@-гидрокси-3-оксопрегнан-21-оиламидо)этиламидо]пирофеофорбид а (11). 1С50 для конъюгатов 10 и 11 при 96-часовой инкубации в клетках карциномы простаты LNCaP и РС-3 составляет 1.4 мкМ и 3.3 мкМ для соединения 10 и 4.5 мкМ и 6.1 мкМ для соединения 11, соответственно. Облучение светом длиной волны 660 нм приводило к многократному повышению токсичности конъюгатов.
Ключевые слова: Конъюгаты, пирофеофорбид а, тестостерон, дигидротестостерон, клетки карциномы простаты, цитотоксичность.
Макрогетероциклы /Macroheterocycles 2017 10(1) 77-80
© ISUCT Publishing
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Tetrapyrrolic macrocycles, porphyrins and chlorins, owing to their unique photochemical and photophysical properties have wide range of biomedical applications such as optical imaging, fluorescent labeling, photodynamic inactivation of microbial infections, and photodynamic therapy of solid tumors. Coupling of macrocycles with fragments of biological active molecules improves delivery and distribution of macrocycle-based compounds to a specific location within the cells, facilitates its transport through receptor or drug mediated endocytosis, and affects its biological activity.11-31 Synthesized earlier conjugates of macrocycles with polyamines, amino acids, peptides, peptidomimetics, antibiotics, nucleotides, carbohydrates, bile acids, lipids, steroids, etc, revealed prospective implications in biomedical studies and photodynamic therapy.[4-19]
In this study we have synthesized conjugates of pyropheophorbide a with androgen receptor ligands -testosterone and dihydrotestosterone. Androgen receptor is known to be an important drug target for treatment of prostate cancer. Modern trends in preparation and application of various steroid conjugates targeting androgen receptor have been reviewed.[20, and the re£ therein] Until now conjugates of testosterone and dihydrotestosterone with tetrapyrrolic macrocycles have not been reported. Synthesis of new conjugates 10 and 11 is presented in the Scheme 1.
Testosterone 1 and dihydrotestosterone 4 were transformed to steroid blocks 2 and 5 by three steps including consecutive protection of carbonyl functions with formation of 1,3-dioxolanes, oxidation of 170-hydroxyl groups, and Reformatsky reaction of obtained 17-ketones with Zn and ethyl bromoacetate.[21,22] The aforementioned reaction is known to pass stereoselectively and give
appropriate 170-OH isomer. Removal of ethylene ketal and ethyl ester protective groups in compounds 2 and 5 led to 21-carboxylic acids 3 and 6 in 49 % and 58 % overall yields (based on compounds 1 and 4, respectively). Compounds 3 and 6 were transformed to related ^-hydroxysuccinimide esters 3a and 6a by treatment with ^-hydroxysuccinimide in the presence of ^.^'-dicyclohexylcarbodiimide (DCC). HRMS, 1H NMR, 13C NMR data for compounds 2, 3, 3a, 5,
6 and 6a are given in Supplementary section.
Pyropheophorbide a derivative comprising primary amino group (compound 8[23]) was prepared from pyropheophorbide a 7 through formation of pentafluorophenyl ester 7a, followed by its treatment with excess of ethylene diamine. Compound 9[24] comprising Boc-protected amino group was prepared from pentafluorophenyl ester 7a by same reaction with mono-Boc ethylene diamine.[25]
Condensation of ^-hydroxysuccinimide esters of steroid acids 3a and 6a with 173[(2-aminoethyl)amido] pyropheophorbide a (8) led to the target conjugates 10 and 11,[26 27] respectively. These conjugates were isolated as individual compounds. Their structures were completely characterized by HRMS, 1H NMR, 13C NMR and electron absorption spectra.
Absorption spectra of conjugates 9, 10 and 11 in CH2Cl2 were very close to those for pyropheophorbide a
7 and 173[(2-aminoethyl)amido]pyropheophorbide a (8). 1H NMR spectra of conjugates 10 and 11 displayed strong high field shifts for H-18' and H-19' methyl protons in comparison with those in spectra of non conjugated steroids (s, 0.53 ppm and s, 0.91 ppm for compound 10 instead of s, 0.95 ppm and s, 1.19 ppm for compound 3; s, 0.56 ppm and s, 0.78 ppm for compound 11 instead of s, 0.92 and s, 1.01 ppm for compound 6). The H-4' resonance for compound 10 (s,
Scheme 1. (a) N-OSu, DCC/CH2Cl2; (b) CF3COOC6F5/CH2Cl2; (c) H2N(CH2)2NH2/CH2Cl2; (d) BocNH(CH2)2NH2/CH2Cl2. 78 Макрогетер0циmbl /Macroheterocycles 2017 70(1) 77-80
V. A. Zolottsev et al.
5.46 ppm) was also shifted in high field compared with those for compound 3(s, 5.73 ppm). These spectral peculiarities apparently were caused by influence of macrocycle on steroid moiety; close effects were reported earlier for conjugates of pyropheophorbide a with cholesterol.[28] Resonance of tert-butyl protons in conjugate 9 (s, 1.21 ppm) was also shifted in high field compared to those usually observed for Boc-amides (s, 1.4 ppm).
Speculating that steroid fragments may affect affinity conjugates 10 and 11 to prostate carcinoma cells, we investigated viability of androgen-sensitive LNCaP and androgen-insensitive PC-3 cells in the presence of these conjugates and 173[(2''-tert-butyloxycarbonylamidoethyl)-amido]pyropheophorbide a (9) (as reference compound). Two experiments were carried out: in the Experiment 1 we have measured LNCaP and PC-3 cells viability at 96 h incubation with compounds 9, 10 and 11; in the Experiment 2 we have compared dark toxicity and photo toxicity of conjugates in the same cells at short time incubation (labeling - 18 h; irradiation - 10 min; incubation without compounds -24 h). Cell viability was measured with MTT method.[29] The protocol used is given in supplementary section. Student's t-test was used to estimate average values for all cases. All Student's t-tests were calculated by an online calculator (http://www.graphpad. com/quickcalcs/ttestl. cfm), confidence interval for each case did not exceed 6 % of the mean.
The results demonstrated that coupling of pyropheo-phorbide a with testosterone and dihydrotestosterone led to conjugates toxic in LNCaP and PC-3 cells. Figures 1a and 1b (see Supplementary section) showed that conjugates 10 and 11 were highly toxic in both prostate carcinoma cells at 96 h incubation; conjugate 10 being significantly more potent cytotoxic agent than conjugate 11, steroid-free conjugate 9 exhibited rather low effect on cells viability.
Figures 1c and 1d showed that both conjugates 10 and 11 decreased LNCaP and PC-3 cells viability at short time incubation (dark toxicity), though less potently than at 96 h incubation; conjugate 9 at short time incubation stimulated proliferation of LNCaP, rather than PC-3 cells. Irradiation (LED AFS "Spectrum", Laser medical centrum Ltd, Moscow, Russia; wavelength of 660 nm, 10 min) potently increased toxicity of conjugates in all cases. However, at short time incubation (either with irradiation, or without irradiation) cells viability remained rather high (-20 % for PC-3 cells, -40 % for LNCaP cells) even at 50 ^M and 100 ^M of conjugates 10 and 11. IC50 for conjugates 10 and 11 at 96 h incubation in LNCaP and PC-3 prostate carcinoma cells were 1.4 ^M and 3.3 ^M for compound 10, and 4.5 ^M and 6.1 ^M for compound 11, respectively (Table 1, Supplementary section).
In cocnclusion, conjugates of pyropheophorbide a with androgen receptor ligands - testosterone and dihydrotesto-sterone - were synthesized. These conjugates were found to exhibit potent dark and photo toxicity in prostate carcinoma cells. We speculate that further investigation of uptake, distribution, subcellular localization, and possible participation in signaling and regulatory pathways of these compounds and related steroid conjugates may be helpful for development of new photo sensitizers possessing high specificity and activity.
Acknowledgments. Authors acknowledge Mr. Mikhail Muraviev, the head of "Laser medical centrum Ltd", kindly providing LED AFS "Spectrum" for photo toxicity experiments. This work was supported by Russian Foundation for Basic Research (project No 15-04-02426), Russian Science Foundation (project No. 14-23-00204) and Programs for Basic Research of Russian State Academy of Sciences for 2013-2020 and "Molecular and cell biology" of Presidium of Russian Academy of Science.
References and Notes
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11. Nikolaeva I.A., Misharin A.Y., Ponomarev G.V., Timofeev V.P., Tkachev Y.V. Bioorg. Med. Chem. Lett. 2010, 20, 2872.
12. Nikolaeva I.A., Morozova J.V., Zavialova M.G., Novikov R.A., Tkachev Y.V., Timofeev V.P., Misharin A.Y., Ponomarev G.V. Macroheterocycles 2010, 3, 150.
13. Battogtokh G., Liu H-B., Bae S-M., Chaturvedi P.K., Kim Y.W., Kim I-W., Ahn W.S. J. Photochem. Photobiol., B 2012, 110, 50.
14. Hahn F., Schmitz K., Balaban T.S., Brase S., Schepers U. ChemMedChem. 2008, 3, 1185.
15. Kascakova S., Hofland L.J., De Bruijn H.S., Ye Y., Achilefu S., van der Wansem K., van der Ploeg-van den Heuvel A., van Koetsveld P.M., Brugts M.P., van der Lelij A-J., Sterenborg H.J. C.M., ten Hagen T.L.M., Robinson D.J., van Hagen M.P. PLoS One 2014, 9(8), e104448.
16. Khan E.H., Ali H., Tian H., Rousseau J., Tessier G., Shafiullaha, van Lier J.E. Bioorg. Med. Chem. Lett. 2003, 13, 1287.
17. Koivukorpi J., Sievänen E., Kolehmainen E., Kral V.Molecules 2007, 12, 13.
18. Mammana A., Asakawa T., Bitsch-Jensen K-B., Wolfe A., Chaturantabut S., Otani Y., Li X., Li Z., Nakanishi K., Balaz M., Ellestad G. A., Berova N. Bioorg. Med. Chem. 2008, 16, 6544.
19. Zhylitskaya H.A., Zhabinskii V.N., Litvinovskaya R.P., Lettieri R., Monti D., Venanzi M., Khripach V.A., Drasar P. Steroids 2012, 77, 1169.
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21. Bittler D., Laurent H., Rach P.,Topert, M. US Patent 5 010 071, Apr. 23, 1991.
22. Oliveto E.P. In: Organic Reactions in Steroid Chemistry, Vol. II (Fried J., Edwards J.A., Eds.). Van Nostrand Reinhold Co.; 1972. p. 139.
Макрогетер0циmbl /Macroheterocycles 2017 10(1) 77-80
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23. 173[(2''-Aminoethyl)amido]pyropheophorbide a (8). The mixture of pentafluorophenylpyropheophorbide a 7a (202 mg, 0.29 mmol), ethylene diamine (580 |xL, 520 mg, 8.65 mmol) and abs. CH2Cl2 (10 mL) was stirred for 2 h, then the mixture was poured into 0.1 M CH3COONa buffer (pH 5.20 mL), extracted with CH2Cl2 (2x20 mL), the combined extract was washed with brine (20 mL), dried over Na2SO4, and evaporated. Then the residue was dissolved in THF (30 mL), the solution was dried over granulated KOH, followed by evaporation to dryness. The obtained black powder (139 mg, 0.24 mmol, 83 %) was used without further purification; the analytical sample was purified by TLC in CHCl3:MeOH:NH4OH (90:9:1) mixture. HRMS, calculated for [C, NOJ+: 577.3291, found: 577.3292. 1H
L 35 41 6 2J 7
NMR 5 ppm: -1.70, 0.33 (each 1H, br.s, N-H); 1.62 (3H, t, J=7.6 Hz, 82-H); 1.75 (3H, d, J=7.3 Hz, 18-CH3); 3.18, 3.37, 3.41 (each 3H, s, 2-, 7-, 12-CH3); 4.23, 4.45 (each 1H, m, 171-H and 81-H); 4.98, 5.19 (each 1H, d, J=19.7 Hz, 172-H); 6.13 (1H, dd, J=11.5 Hz and J=1.4 Hz, 32-H, cis); 6.24 (1H, dd, J=17.9 Hz and J=1.4 Hz, 32-H, trans); 7.95 (1H, dd, J=11.5 Hz and J=17.9 Hz, 31-H); 8.50, 9.24, 9.30 (each 1H, s, 5-, 10-, 20-H); 13C NMR 5 ppm: 11.18; 11.81; 12.05; 17.36; 19.37; 23.01; 28.30; 30.17; 30.88; 32.80; 40.92; 41.65; 48.01; 49.97; 51.70; 92.92; 97.08; 103.88; 106.03; 122.65; 128.10; 129.18; 131.50; 135.78; 135.96; 136.13; 137.68; 144.92; 148.86; 150.65; 155.11; 160.37; 171.68; 172.36; 196.14. UV-Vis (CH2Cl2) Xmax nm (e): 413 (85,000); 507 (8,900); 538 (8,000); 609 (7,000X 665 (35,200).
24. 173[(2''-tert-Butyloxycarbonylamidoethyl)amido]-pyropheophorbide a (9). Compound 9 was synthesized from pentafluorophenylpyropheophorbide a 7a (88 mg, 0.13 mmol) and mono-Boc-ethylene diamine (42 mg, 0.26 mmol) according the procedure described in ref.[29] and isolated by silica gel flash chromatography in CHCl3:MeOH:NH4OH (90:9:1) mixture. After evaporation compound 9 (43 mg, 0.06 mmol, 43 %) was obtained as black powder. HRMS, calculated for [C40H49N6O4]+: 677.3815, found: 677.3818. 1H NMR 5 ppm: -1.74, 0.36 (each 1H, br.s, N-H); 1.21 (9H, s, t-Bu); 1.59 (3H, t, J=7.6 Hz, 83-H); 1.76 (3H, d, J=7.3 Hz,18-CH3); 3.17, 3.27, 3.37 (each 3H, s, 2-, 7-, 12-CH3); 4.25, 4.47 (each 1H, m, 171-H and 81-H); 5.01, 5.21 (each 1H, d, J=19.7 Hz,172-H); 6.12 (1H, dd, J=11.5 Hz and J=1.4 Hz, 32-H, cis); 6.23 (1H, dd, J=17.9 Hz and J=1.4 Hz, 32-H, trans); 7.92 (each 1H, dd, J=11.5 Hz and J=17.9 Hz, 31-H); 8.51, 9.12, 9.28 (each 1H, s, 5-, 10-, 20-H). 13C NMR 5 ppm: 11.25; 11.76; 12.14; 17.41; 19.04; 23.15; 28.24; 28.46; 30.44; 33.02; 40.36; 40.63; 48.09; 50.08; 51.85; 79.56; 93.07; 97.14; 103.89; 106.08; 122.55; 128.06; 129.25; 130.28; 131.63; 135.86; 135.99; 136.23; 137.89; 141.61; 145.01; 148.95; 150.69; 155.19; 160.50;
171.80; 173.01; 196.26. UV-Vis (CHCL) I nm (e): 413
2 2 max
(85,000); 507 (8,900); 538 (8,000); 609 (7,000); 665 (35,200).
25. Krapcho A.P., Kuell C.S. Synth. Commun. 1990, 20, 2559.
26. 173[2"-(17'^-Hydroxy-3'-oxopregn-4'-en-21'-oylamidoethyl) amido]pyropheophorbide a (10): The mixture of compounds 3a (30 mg, 69 ^mol), 8 (33 mg, 57 ^mol), dry Py (3 mL), and dry THF (5 mL) was stirred at r. t. for 16 h, then evaporated to dryness with toluene, and the residue was applied on the top a silica gel column. The column initially was washed with CHCl3:(CH3)2CO:AcOH (75:24:1) to remove byproducts, then washed with 5 mL CHCl3, and finally the target product was eluted with CHCl3:MeOH:7M NH3 solution in MeOH (93:5:2,
by vol). After evaporation the compound 10 (38 mg, 42 ^mol, 73 %) was obtained as black powder. HRMS, calculated for [C56H69N6O5]+: 905.5329, found: 905.5327. Щ NMR 5 ppm: -1.86 (1H, br.s, N-H); 0.53, 0.91 (each 3H, s, H-18' and H-19' in steroid moiety); 1.61 (3H, t, J=7.6 Hz, 82-H in pyropheophorbide moiety), 1.74 (3H, d, J=7.3 Hz, 18-CH3 in pyropheophorbide moiety), 3.19, 3.36, 3.39 (each 3H, s, 2-, 7-, 12-CH3 in pyropheophorbide moiety), 4.23, 4.46 (each 1H, m, 171-H and 81-H in pyropheophorbide moiety), 4.98, 5.17 (each 1H, d, J=19.7 Hz, 172-H in pyropheophorbide moiety), 5.46 (1H, s, H-4' in steroid moiety), 6.15 (1H, dd, J=11.5 Hz and J=1.4 Hz, 32-H, cis in pyropheophorbide moiety), 6.17 (1H, br. t, J=5.2 Hz, NH-CO); 6.25 (1H, dd, J=17.9 Hz and J=1.4 Hz, 32-H, trans in pyropheophorbide moiety), 6.71 (1H, br.t, J=5.2 Hz, NH-CO); 7.90 (1H, dd, J=11.5 Hz and J=17.9 Hz, 31-H in pyropheophorbide moiety), 8.58, 9.30 9.35 (each 1H, s, 5-, 10-, 20-H in pyropheophorbide moiety) 13C NMR 5 ppm: 11.29; 12.00; 12.15; 13.60; 17.16; 17.32 19.51; 20.36; 20.51; 23.17; 23.31; 30.35, 31.32; 32.59; 32.78 33.83; 35.46; 36.00; 38.42; 39.59; 42.43; 45.97; 48.10; 49.62 50.10; 51.92; 53.33; 81.77; 93.88; 97.10; 103.93; 106.51 123.09; 123.67; 128.30; 129.04; 130.53; 132.19; 135.81 136.35; 136.50; 137.83; 141.97; 144.94; 146.94; 149.36 153.65; 155.65, 161.35; 171.17; 172.46; 173.59; 173.84: 174.03; 196.12; 199.41. UV-Vis (CH2Cl2) Xmax nm (e): 413 (86,400); 507 (8,700); 538 (7,800); 609 (6,900)7 667 (36,000).
27. 173[2''-(17 'ß-Hydroxy-3 '-oxopregnan-21 '-oylamidoethyl) amido]pyropheophorbide a (11). The synthesis of compound 11 was carried out from compounds 6a (26 mg, 60 ^mol) and 8 (30 mg, 52 ^mol) using the procedure described in ref.[25] Compound 11 (33 mg, 37 ^mol, 69 %) was obtained as black powder. HRMS, calculated for [C56H71N6O5]+: 907.5486, found: 907.5490. 1H NMR 5 ppm: -1.67, (1H, br.s, N-H); 0.56, 0.78 (each 3H, s, H-18' and H-19' in steroid moiety); 1.65 (3H, t, J=7.6 Hz, 82-H in pyropheophorbide moiety); 1.77 (3H, d, J=7.3 Hz, 18-CH3 in pyropheophorbide moiety); 3.21, 3.37, 3.45 (each 3H, s, 2-, 7-, 12-CH3 in pyropheophorbide moiety); 4.27, 4.46 (each 1H, m, 171-H, 81-H in pyropheophorbide moiety); 5.02, 5.21 (each 1H, d, J=19.7 172-H in pyropheophorbide moiety); 5.86 (1H, br.t, J=5.2 Hz, NH-CO); 6.14 (1H, dd, J=11.5 Hz and J=1.4 Hz, 32-H, cis in pyropheophorbide moiety), 6.20 (1H, dd, J=17.9 Hz and J=1.4 Hz, 32-H, trans in pyropheophorbide moiety), 6.58 (1H, br. t, J=5.2 Hz, NH-CO); 7.93 (1H, dd, J=11.5 Hz and J=17.9 Hz, 31-H in pyropheophorbide moiety), 8.52, 9.30 9.33 (each 1H, s, 5-, 10-, 20-H in pyropheophorbide moiety) 13C NMR 5 ppm: 11.39; 12.03; 13.62; 14.02; 17.34; 19.31 20.61; 23.05; 23.36; 28.42; 28.74; 29.69; 31.46; 31.87; 32.98 33.90; 35.25; 35.66; 36.16; 38.48; 39.72; 42.49; 46.00; 46.63 48.04; 49.95; 50.03; 51.72; 53.67; 81.85; 93.11; 97.11; 103.85 105.86; 122.65; 124.15; 125.29; 128.22; 129.02; 130.01 131.73; 135.97; 136.32; 137.58; 137.86; 141.70; 144.98 148.66; 148.93; 160.45; 171.97; 173.58; 174.20; 174.48 196.31; 211.49. UV-Vis (CH2Cl2) Xmax nm (e): 413 (85,900) 507 (8,500); 538 (8,000); 609 (6,500")Г667 (35,800).
29. Ponomarev G.V., Solovieva M.N., Dugin N.O., Zavialova M.G. Mehtiev A.R., Misharin A.Yu., Novikov R.A., Tkachev Y.V. Popenko V.I., Timofeev V.P. Bioorg. Med. Chem. 2013, 21 5420.
30. Mosmann T. J. Immunol. Methods 1983, 65, 55.
Received 28.08.2016 Accepted 05.02.2017
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