- Email: [email protected]
Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging
T
Marina V. Mal'shakovaa, , Yana I. Pylinab, Dmitry V. Belykha ⁎
a b
Institute of Chemistry of the Komi Science Centre, Ural Division of Russian Academy of Sciences, 167000, 48 Pervomayskaya st., Syktyvkar, Russia Institute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, 167000, 28 Kommunisticheskaya st., Syktyvkar, Russia
ARTICLE INFO
ABSTRACT
Keywords: Chlorin e6 D-Galactopyranose Cytotoxicity Amide
We synthesized new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via carbon atom in position 6 of the galactose fragment. Galactose fragments were inserted by alkylation of the amino groups of chlorin e6 amides with one and two ethylene diamine fragments on the macrocycle periphery with triflate of diacetone galactose, followed by removal of diisopropylidene protection by 70% aqueous trifluoroacetic acid. The synthesized compounds were shown to be capable of penetrating the membrane of HeLa cells; they have intense red fluorescence inside the cell and have phototoxic properties towards HeLa cells (upon LED irradiation at 660 nm and light exposure value of 12 J/cm2). These properties, along with water solubility, allow us to consider the synthesized compounds to be promising as potential antitumor PSs and diagnostic compounds for visualizing malignant tumors and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases.
Porphyrins and their analogues are intensively studied as preparations for diagnostics and photodynamic therapy of oncological diseases1,2,3–5. Chlorins obtained by chemical modification of peripheral substituents of chlorophyll a and its derivatives (such as methylpheophorbide a (1), Scheme 1) have several advantages in comparison with porphyrins when used in the diagnostics and therapy of oncological diseases, such as their relatively low toxicity and good photophysical characteristics.1,2,3,5 Therefore, the development of new anticancer photosensitizers (PSs) and diagnostic preparations for oncology based on chlorophyll a derivatives attracts much attention, and some of these compounds are already used in clinical practice.1,2,3,5 The most important areas of PS improvement are increasing bioavailability and tropism for malignant neoplasms, as well as enabling the PS molecule to actively penetrate the cell membrane. The insertion of fragments of carbohydrate molecules onto the periphery of the macrocycle of PS based on porphyrins and their analogues, as a rule, helps to improve the properties of these PSs in this respect.6–8,9,10–13,14,15–22,1 In particular, the properties of PSs based on chlorophyll a derivatives can thus be improved.11,16,21,1 To further improve PSs and diagnostic preparations based on chlorophyll a derivatives, the conjugation of the latter with galactose appears to be the most promising. Galactose contains a sufficiently large number of hydroxyl groups to significantly increase the bioavailability of the PS molecule. In addition, a fragment of galactose molecule as part of various conjugates is capable of non-covalent ⁎
interactions with cellular structures, due to which tropism of the conjugates towards malignant neoplasms increases and the conjugates acquire the ability to actively penetrate the cell membrane.6,10,11,14,15–17,19,21,1 The data from the literature suggest that the interaction with cellular structures is significantly influenced by the method of binding of the galactose molecule fragment to macrocycle fragments. For example, in terms of absorption by cancer cells, conjugation of the galactose fragment via the hydroxyl group in position 1 is less advantageous than conjugation via the hydroxyl group in position 3,6 which can be explained by the participation of the hydroxyl group in position 1 in binding to the galectin-1 receptor as a hydrogen bond acceptor. Most of the conjugates with tetrapyrrole macrocycles described in the literature were synthesized on the basis of tetramezoarylporphyrins and phthallocyanines.6–10,12–15,17–22,1 Relatively few conjugates with chlorophyll a derivatives were described, and in most cases conjugation was implemented via the hydroxyl group in position 1.11,16,1 Therefore, the synthesis of chlorophyll a derivatives with galactose fragments linked to the chlorin macrocycle via carbon atoms in different positions of the galactose molecule would be of interest. In the present work, chlorine e6 derivatives with two and four galactose fragments on the periphery of the macrocycle linked via the carbon atom in position 6 of the galactose molecule were synthesized (Scheme 1), and dark and photoinduced cytotoxicities and ability of new compounds to penetrate the HeLa cell membrane were assessed.
Corresponding author. E-mail addresses: [email protected] (M.V. Mal'shakova), [email protected] (Y.I. Pylina), [email protected] (D.V. Belykh).
https://doi.org/10.1016/j.bmcl.2019.07.019 Received 22 April 2019; Received in revised form 5 July 2019; Accepted 9 July 2019 Available online 10 July 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
OTf
O H2N
NH N N HN
O
NH2
NH N
OCH3 OCH3
O
O
O
N
O OCH3
H
(2) [(2)]
O
OCH3
(1) H2N
O
NH2
N
O OCH3 O
i, ii (one flask)
H2N
N
O
(6)
N
H
O
NH2
O OCH3
NH N N
H O
9, iii
OH
N OH
N HN
OH
OCH3
(4)
O
v
N HN O
O
O
O
N HN O OCH3
O
O
O
NH N
O
O O
NH N
H
H
N
(3)
O
O
(9)
OCH3
iv
NH2
O
O
N HN
N HN
i
O
NH N
9, iii
O O
HO
O
HO
OH
HO
N
O OCH3
OH O N
(5)
OH
H
OH
OH
O
HO
HO
OH
HO
OH OH
NH N
NH N
iv
N HN
N HN H
O
O O
O
N
O O
N
O
O N
O OCH3 O
O
O
O
O (7)
H
O O
N O
O
O
H
O
O
O
O HO
O
N
O
O OH OCH3 OH
HO OH
N
OH
H O N
O
N
OH
HO
O
HO
O
HO
HO (8)
HO
OH
HO
OH OH
Scheme 1. i: CHCl3, r. t., 2 h [25]; ii: r. t., 20 h25; iii: THF, reflux, 3 h (yield: 3 (48%), 7 (22%)); iv: TFA(90%)-H2O, 3–4 h (yield: 4 (81%), 8 (76%)); v: TFA(90%)H2O, 48 h (yield of 5 (35%)).
The insertion of fragments of diacetone galactose onto the periphery of the chlorin macrocycle was carried out by alkylation of amino groups of chlorin e6 derivatives 2, 6 by the action of triflate diacetone galactose 9 (boiling in THF in the presence of sodium acetate as base and two- or four-fold molar excess of 9 in the case of chlorins 2 and 6, respectively), followed by the removal of diisopropylidene protection (hydrolysis in 90% aqueous TFA) (Scheme 1). The mono- and diamino derivatives of chlorin e6 2 and 6 were synthesized on the basis of methylpheophorbide a 1 (obtained from the spirulina biomass as in Kustov et al.23) by the action of ethylene diamine according to the methods developed by us earlier.24 The spectral characteristics of the compounds 1, 2, and 6 that were described earlier correspond to the literature data.23,24 Alkylation of amino chlorins 2 and 6 with a twofold (relative to the number of amino groups in the molecule) molar excess of triflate diacetone galactose allows to insert two diacetone galactose fragments into each amino group and thus to obtain chlorins with two and four carbohydrate fragments. Removal of protection from conjugate 7 takes place without the formation of byproducts, since the ester group in position 15 does not undergo hydrolysis under the reaction conditions. In the case of conjugate 3, in addition to removal of the diisopropylidene protection, hydrolysis of the ester group in position 17 is possible, and the formation of chlorin 5 with the carboxyl group in position 17 is
also possible, aside from the target product 4. Chlorin 5 is of interest not only as a possible byproduct, but also as a more hydrophilic derivative, the presence of both carboxyl and alkylated amino groups in the molecule of which determines the possibility of its existence in solution in the zwitterionic form (Fig. 1). Therefore, we purposefully prepared chlorin 5 by means of a more prolonged exposure to 90% aqueous TFA on chlorin 3 (Scheme 1). Using chlorin 5 as an internal standard in the analysis of the reaction mixture for preparation of compound 4, we showed that the hydrolysis of the ester group in position 17 after 3 h exposure of compound 3 to 90% aqueous TFA does not occur, therefore no chlorin 5 was detected in the reaction mixture. The structure of the de novo synthesized compounds was established on the basis of data from NMR, IR and UV–vis spectroscopy, as well as mass spectrometry. Mass spectra (ESI) of conjugates with fragments of diacetone galactose 3, 7 and galactose 5, 8 show peaks, the m/z values of which correspond to protonated molecular ions with structures of 3, 5, 7, and 8. In the case of compounds 3 and 4, peaks corresponding to adducts with the sodium cation are observed. The mass spectrum of compound 4, in addition to the peak of the adduct with the sodium cation, has a peak the m/z value of which corresponds to the hydrogenated molecule ([M+2H]). In the UV–vis spectra of all de novo synthesized compounds, absorption bands characteristic of the chlorin 2065
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
Fig. 1. Light micrographs (upper row) and fluorescent micrographs (lower row) of HeLa cells after 40 min exposure to compounds (10 μM) and without them. IC50 ± SE, for Photolon a drug applied in clinical practice is 31.09 ± 4.92 μM (dark toxicity) and 4.01 ± 0.91 μM (phototoxicity).
chromophore are observed. In the 1H and 13C NMR spectra of conjugates 3–5, 7, and 8, the signals of the chlorin macrocycle and fragments of diacetone galactose (compounds 3, 7) or galactose (compounds 4, 5, and 8) are observed, with the ratio of the intensities of the signals of protons of the chlorin macrocycle and carbohydrate fragments in the spectra 1H NMR corresponding to the presence of two (for compounds 3–5) and four (compounds 7 and 8) carbohydrate fragments in the molecule, which correlates with the data of mass spectrometry. The presence in the spectra of 1H NMR of chlorins with fragments of diacetone galactose 3 and 7 singlet signals that correspond, judging by the integral intensity, to the methyl groups of the diisopropylidene protection of two (in compound 3) and four (in compound 7) fragments indicates that the reaction is selective and the isopropylidene protection at the conditions of its carrying out is persistent. The presence of isopropylidene protection is also manifested in the 13C NMR spectra (signals of carbon atoms of methyl groups and quaternary carbon atoms associated with them). In the 1H and 13C NMR spectra of compounds 4, 5 and 8, there are no signals corresponding to protons and carbon atoms of the diisopropylidene protection, whereas the signals of protons of hydroxyl groups formed after removal of diisopropylidene fragments are observed, which confirms the complete removal of the protection. The removal of the diisopropylidene protection results in the opportunity for isomerization of compounds 4, 5, and 8 in solution, which is reflected in the broadening or splitting of the signals of some carbon atoms of the galactose fragments in the 13C NMR spectra of these compounds. However, for most signals, diastereomeric differences are not visible. In the 1H NMR spectrum of compound 5, in contrast to compound 4, there is no signal of the methyl group in position 17, which confirms the hydrolysis at this position. In addition, at 12 m.d. there is a broadened signal of the proton of the carboxyl group which is formed during hydrolysis. The presence of this signal indicates the absence of a zwitterionic form 5 in DMSO. As already noted, the presence of galactose fragments increases the solubility of porphyrin compounds in water. We obtained preliminary data on the solubility of compounds 4, 5, and 8 in water. When dissolved in water, compounds 4, 5, and 8 generate fluorescent in UV light solutions, which indicates the presence of single molecules in the aqueous phase, that is, the formation of a true solution. For all compounds studied, it is possible to obtain a solution containing at least 0.1 mg/ml without the use of auxiliary substances, which indicates the
possibility of obtaining water-soluble forms. To assess the ability of the compounds to penetrate the cell membrane and the possibility of their use for visualization of tumor cells, we used fluorescence microscopy. The cells were incubated with the compounds under study and then the fluorescence of the cells was examined. Intense red fluorescence of cells indicates the ability of compounds 4, 5 and 8 to penetrate the cell membrane (Fig. 1). In addition, the intense fluorescence of the studied compounds inside the cells indicates that these compounds interact with cellular structures as single molecules, which can be used to visualize tumors for diagnostic purposes. Thus, we can conclude that all the synthesized compounds are of interest as potential diagnostic drugs. To evaluate the dark and photoinduced (LED-irradiated at a wavelength of 660 nm and light exposure value of 12 J/cm2) cytotoxicities of compounds 4, 5 and 8 towards HeLa cells, IC50 values were determined as in our previous studies25 (Fig. 1). The obtained data indicate that conjugates 4, 5 and 8 exert a photoinduced cytotoxic action on HeLa cells, and the maximum photoinduced effect is observed for conjugates 5 and 8: IC50 values under dark exposure to these compounds are 31 and 38 times correspondingly higher than that under photosensitized exposure. Collation of IC50 values (calculated as a reactant – chlorine e6) for the Photolon a drug applied in clinical practice26–28 allows to make a conclusion on comparable or better ratio of dark and photoinduced cytotoxicity for all conjugates with galactose studied. In the present work, we describe the synthesis of new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via the carbon atom in position 6 of the galactose fragment. The synthesized compounds were shown to be able to penetrate the cell membrane, to have intense red fluorescence, and to be characterized by a high photoinduced cytotoxicity towards HeLa cells. The most promising from this point of view were conjugates 5 and 8, the IC50 values of which for dark cytotoxicity are 31 and 38 times correspondingly greater than for photoinduced one. Considerable photodynamic activity of conjugates 5 and 8, in combination with water solubility, allow to consider these compounds to be promising for further research as antitumor PSs and diagnostic preparations for visualizing malignant neoplasms and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases (that can be utilized as theranostic agents). 1 H and 13C NMR spectra of the synthesized compounds were 2066
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
[M+Na]+ (calcd. for C50H66N6NaO15+, 1013.4). 1H NMR (300 MHz, DMSO-D7): 9.78 (1H, s, H-10); 9.74 (1H, s, H-5); 9.13 (1H, s, H-20); 9.08 (1H, t, NH-13(1) (amide), J = 4.5 Hz); 8.30 (1H, dd, H-3(1), J = 11.7, 17.7 Hz); 6.43 (1H, d, H-3(2) (trans), J = 17.7 Hz); 6.16 (1H, d, H-3(2) (cis), J = 11.7 Hz); CH2-15(1): 5.55 (1H, d, J = 19.2 Hz), 5.34 (1H, d, J = 19.2 Hz); H-1a, H-1b: 6.55 (1H, m), 6.16 (1H, m); H-2a, H2b, H-3a, H-3b, H-4a, H-4b, H-5a, H-5b, OH-1a, OH-1b, OH-2a, OH-2b, OH3a, OH-3b, OH-4a, OH-4b, OH-5a, OH-5b: 4.99 (2H, m), 4.75 (2H, m), 4.56 (2H, m), 4.35 (3H, m), 4.13 (1H, m), 3.83 (4H, m), 3.63 (4H, m); 4.44 (1H, d, H-17, J = 9.0 Hz); 4.64 (1H, q, H-18, J = 7.2 Hz); 3.86 (2H, m, CH2-8(1)); 3.74 (3H, s, CH3-15(3)); 3.60 (3H, s, CH3-17(4)); 3.54 (3H, s, CH3-12(1)); 3.52 (3H, s, CH3-2(1)); 3.32 (3H, s, CH3-7(1)); CH2-6a, CH2-6b: 3.84 (2H, m), 3.66 (2H, m); CH2-13(2), CH2-13(3): 3.83 (2H, m), 3.58 (2H, m); CH2-17(1), CH2-17(2): 2.36 (2H, m), 2.17 (2H, m); 1.70 (3H, t, CH3-8(2), J = 7.8 Hz); 1.66 (3H, d, CH3-18(1), J = 7.2 Hz); −1.80 (1H, s, I-NH); −2.08 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.83 (C-3(1)); 122.40 (C-3(2)); 101.26 (C-10); 98.99 (C-5); 103.21 (C-1a, C-1b); 94.58 (C-20); 93.22, 98.04, 99.00, 67.99, 69.31, 70.13, 72.66, 74.17 (C-4a, C-4b, C-3a, C-3b, C-2a, C-2b, C5a, C-5b); 55.76, 55.17 (C-6a, C-6b); 53.14 (C-17); 52.39 (C-15(3)); 51.83 (C-17(4)); 48.55 (C-18); 38.57, 38.41 (C-13(2), C-13(3)); 37.21 (C-15(1)); 31.07, 29.92 (C-17(1), C-17(2)); 23.43 (C-18(1)); 19.44 (C8(1)); 18.28 (C-8(2)); 12.51 (C-12(1)); 12.23 (C-2(1)); 11.47 (C-7(1)). Conjugate 5. To the 100 mg (0.087 mmol) of compound 3 10 ml 70% trifluoroacetic acid was added. The reaction mixture was shaken at room temperature for 48 h. Next, the reaction mixture was evaporated at low pressure. Evaporated precipitate was chromatographed on silochrom C-120 (eluent: trichloromethane – methanol, 2:1), (TLC, 95% ethanol). 30 mg (35% yield) was obtained as black crystalline powder. UV (C2H5OH (80%)) λmax/nm (Irelative (%)): 661 (27), 606 (3), 527 (3), 499 (9), 399 (94). UV (H2O) λmax/nm (Irelative (%)): 663 (25), 503 (1), 399 (100). IR (KBr), ν/cm−1: 3338 (OeH), 1728 (C]O, ester), 1680 (“amide-I”), 1601 (“chlorine band”), 1543 (“amide-II”). Mass spectrum (ESI), m/z: 977.7 [M+H]+ (calcd. for C49H65N6O15+, 977.5). 1H NMR (300 MHz, DMSO-D7): 12.00 (1H, br s, COOH-17(3)); 9.80 (1H, s, H10); 9.74 (1H, s, H-5); 9.14 (1H, s, H-20); 8.29 (1H, dd, H-3(1), J = 12.0, 17.4 Hz); 6.67 (1H, t, NH-13(1) (amide), J = 4.5 Hz); 6.43 (1H, d, H-3(2) (trans), J = 18.3 Hz); 6.16 (1H, d, H-3(2) (cis), J = 11.7 Hz); CH2-15(1): 5.53 (1H, d, J = 18.3 Hz), 5.42 (1H, d, J = 19.2 Hz); H-1a, H-1b: 6.67 (1H, m), 6.39 (1H, m); H-2a, H-2b, H-3a, H-3b, H-4a, H-4b, H-5a, H-5b, OH-1a, OH-1b, OH-2a, OH-2b, OH-3a, OH3b, OH-4a, OH-4b, OH-5a, OH-5b: 5.40 (2H, m), 4.85 (4H, m), 4.44 (4H, m), 3.91 (4H, m), 3.58 (4H m); 4.47 (1H, d, H-17, J = 10.2 Hz); 4.65 (1H, q, H-18, J = 7.0 Hz); 3.84 (2H, m, CH2-8(1)); 3.74 (3H, s, CH315(3)); 3.56 (3H, s, CH3-12(1)); 3.52 (3H, s, CH3-2(1)); 3.31 (3H, s, CH3-7(1)); CH2-6a, CH2-6b: 3.36 (2H, m), 2.69 (2H, m); 3.84 (4H, m, CH2-13(2), CH2-13(3)); CH2-17(1), CH2-17(2): 2.31 (2H, m), 2.15 (2H, m); 1.65 (6H, m, CH3-8(2), CH3-18(1)); −1.77 (1H, s, I-NH); −2.03 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.81 (C-3(1)); 122.44 (C-3(2)); 101.39 (C-10); 98.97 (C-5); 103.20 (C-1a, C-1b); 94.63 (C-20); 102.32, 97.94, 93.36, 83.07, 73.85, 72.34, 69.73, 68.95 (C-2a, C-2b, C3a, C-3b, C-4a, C-4b, C-5a, C-5b); 60.44, 56.60 (C-6a, C-6b); 53.20 (C-17); 52.49 (C-15(3)); 48.60 (C-18); 38.57, 38.41 (C-13(2), C-13(3)); 37.38 (C-15(1)); 31.22, 29.99 (C-17(1), C-17(2)); 23.50 (C-18(1)); 19.42 (C8(1)); 18.31 (C-8(2)); 12.54 (C-12(1)); 12.30 (C-2(1)); 11.48 (C-7(1)). Conjugate 7. To the solution 340 mg (0.49 mmol) of diaminochlorine 6 in 30 ml of the dry tetrahydrofuran 850 mg (2.17 mmol) of 1,2:3,4-Di-Oisopropylidene-D-galactopyranose trifluoromethanesulfonic and 178 mg (2.17 mmol) sodium acetate was added. The mixture was refluxed for 4 h (TLC, tetrachloromethane – acetone, 4:2). Next, the reaction mixture was diluted with 70 ml of trichloromethane and sodium acetate was washed with water. After that, the mixture was dried over dehydrated sodium sulfate and evaporated at low pressure. Evaporated precipitate was chromatographed on silica gel (Alfa Aesar 70–230 mesh) (eluent: tetrachloromethane – acetone, 5:1). 180 mg (22% yield) was obtained as deep blue crystalline powder. UV (CHCl3) λmax/nm (Irelative (%)): 664 (31), 609
recorded on Bruker AVANCE-II-300 (working frequency 300 MHz and 75 MHz for NMR 1H and 13C respectively) using standard impulse Bruker software for one and two-dimensional experiments. Infrared spectra were measured in KBr tablets on the “IR Prestige 21” device (Shimadzu). Mass spectra were recorded on the “ThermoFinnigan LCQ Fleet” device. UV–Vis spectra were recorded on a spectrometer UV1700 (Shimadzu) with the wavelength range of 200–1100 nm. The samples were analyzed in quartz cuvettes (10 mm thick). The reaction was controlled using TLC method on Sorbfil slides. Extraction of the reaction products was done using column chromatography on silica gel Alfa Aesar 70–230 mesh and silochrom C-120 (SiO2*nH2O). Monoaminochlorin 2 and diaminochlorin 6 were obtained according to Belykh et al.24. Diacetone-D-galactose was obtained by the method,29 and Triflate Diacetone-D-galactose was obtained according to literature method.30 Conjugate 3. To the solution 300 mg (0.45 mmol) of aminochlorine 2 in 30 ml of the dry tetrahydrofuran 400 mg (1.02 mmol) of 1,2:3,4-DiO-isopropylidene-D-galactopyranose trifluoromethanesulfonic and 84 mg (1.02 mmol) sodium acetate was added. The mixture was refluxed for 3 h (TLC, tetrachloromethane – acetone, 4:1). Next, the reaction mixture was diluted with 70 ml of trichloromethane and sodium acetate was washed with water. After that, the mixture was dried over dehydrated sodium sulfate and evaporated at low pressure. Evaporated precipitate was chromatographed on silica gel (Alfa Aesar 70–230 mesh) (eluent: tetrachloromethane – acetone, 10:1). 250 mg (48% yield) was obtained as deep blue crystalline powder. UV (CHCl3) λmax/nm (Irelative (%)): 663 (30), 607 (2), 529 (2), 501 (8), 403 (98). IR (KBr), ν/cm−1: 3086 (CeH, vinyl group), 1738 (C]O, ester), 1657 (“amide-I”), 1601 (“chlorine band”), 1514 (“amide-II”). Mass spectrum (ESI), m/z: 1151.8 [M+H]+ (calcd. for C62H83N6O15+, 1151.6), 1173.7 [M+Na]+ (calcd. for C62H82N6NaO15+, 1173.6). 1H NMR (300 MHz, DMSO-D7): 9.79 (1H, s, H-10); 9.75 (1H, s, H-5); 9.12 (1H, s, H-20); 8.78 (1H, t, J = 12.3 Hz, NH-13(1) (amide)); 8.30 (1H, dd, H-3(1), J = 11.6, 17.9 Hz); 6.44 (1H, d, H-3(2) (trans), J = 17.7 Hz); 6.17 (1H, d, H-3(2) (cis), J = 11.7 Hz); CH2-15(1): 5.56 (1H, d, J = 18.9 Hz) and 5.33 (1H, d, J = 18.6 Hz); 5.46 (2H, d, H-1a, H-1b, J = 4.5 Hz); 4.65 (1H, m, H-18); 4.60 (2H, d, H-3a, H-3b, J = 8.1 Hz); 4.44 (1H, m, H-17); 4.40 (2H, d, H-4a, H-4b, J = 7.8 Hz); 4.31 (2H, H-2a, H-2b, J = 3.3 Hz); 4.03 (2H, m, H-5a, H-5b); 3.83 (2H, q, CH2-8(1), J = 8.0 Hz); 3.73 (3H, s, CH3-15(3)); 3.59 (3H, s, CH3-17(4)); 3.53 (6H, s, CH3-12(1), CH3-2(1)); 3.32 (3H, s, CH3-7(1)); CH2-6a, CH26b: 2.95 (2H, m), 2.79 (2H, m); CH2-13(2), CH2-13(3): 3.83 (2H, m), 3.58 (2H, m); CH2-17(1), CH2-17(2): 2.71 (2H, m), 2.35 (2H, m); 1.69 (3H, t, CH3-8(2), J = 7.5 Hz); 1.65 (3H, d, CH3-18(1), J = 6.9 Hz); CH38a, CH3-9a, CH3-11a, CH3-12a, CH3-8b, CH3-9b, CH3-11b, CH3-12b: 1.51 (6H, s), 1.36 (6H, s), 1.22 (6H, s), 1.18 (6H, s); −1.79 (1H, s, I-NH); −2.07 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.84 (C-3(1)); 122.40 (C-3(2)); 101.32 (C-10); 98.97 (C-5); 96.31 (C-1a, C-1b); 94.55 (C-20); 71.42 (C-4a, C-4b); 70.66 (C-3a, C-3b); 70.54 (C-2a, C-2b); 65.37 (C-5a, C-5b); 54.77, 54.03 (C-6a, C-6b); 53.12 (C-17); 52.27 (C-15(3)); 51.77 (C-17(4)); 48.52 (C-18); 38.41 (C-13(2), C-13(3)); 37.21 (C15(1)); 32.54, 31.07 (C-17(1), C-17(2)); 26.46, 26.37, 25.24, 24.45 (C8a, C-9a, C-11a, C-12a, C-8b, C-9b, C-11b, C-12b); 23.41 (C-18(1)); 19.40 (C-8(1)); 18.27 (C-8(2)); 12.51 (C-12(1)); 12.07 (C-2(1)); 11.47 (C7(1)). Conjugate 4. To the 150 mg (0.13 mmol) of compound 3 10 ml 90% trifluoroacetic acid was added. The reaction mixture was shaken at room temperature for 3 h. Next, the reaction mixture was evaporated at low pressure. Evaporated precipitate was chromatographed on silochrom C-120 (eluent: trichloromethane – methanol, 3:1), (TLC, 95% ethanol). 105 mg (81% yield) was obtained as black crystalline powder. UV (CHCl3-C2H5OH (1:1)) λmax/nm (Irelative (%)): 663 (31), 609 (4), 530 (4), 501 (1), 402 (99). UV (H2O), λmax/nm (Irelative)): 664 (27), 502 (1), 400 (99). IR (KBr), ν/cm−1: 3338 (OeH), 1728 (C]O, ester), 1680 (“amide-I”), 1601 (“chlorine band”), 1543 (“amide-II”). Mass spectrum (ESI), m/z: 992.3 [M+2H]+ (calcd. for C50H68N6O15+, 992.5), 1013.7 2067
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
(3), 529 (3), 501 (9), 403 (99). IR (KBr), ν/cm−1: 1738 (C]O, ester), 1662 (“amide-I”), 1601 (“chlorine band”), 1516 (“amide-II”). Mass spectrum (ESI), m/z: 1663.6 [M+H]+ (calcd. for C87H123N8O24+, 1663.9). 1H NMR (300 MHz, CDCl3): 9.70 (1H, s, H-10); 9.68 (1H, s, H-5); 8.83 (1H, s, H20); 8.15 (1H, dd, H-3(1), J = 12.0, 17.4 Hz); 7.66 (1H, t, NH-13(1) (amide), J = 4.5 Hz); 6.40 (1H, d, H-3(2) (trans), J = 18.3 Hz); 6.29 (1H, m, 17(3)-NH (amide)); 6.18 (1H, d, H-3(2) (cis), J = 11.1 Hz); CH2-15(1): 5.72 (1H, d, J = 18.9 Hz), 5.23 (1H, d, J = 18.3 Hz); H-1a, H-1b, H-1c, H1d: 5.39 (2H, d, 5.4 Hz), 5.22 (2H, d, 4.5 Hz); H-2a, H-2b, H-2c, H-2d, H-3a, H-3b, H-3c, H-3d, H-4a, H-4b, H-4c, H-4d, H-5a, H-5b, H-5c, H-5d: 4.43 (2H, d, J = 8.1 Hz), 4.26 (2H, d, J = 9.0 Hz), 4.14 (6H, m), 3.97 (4H, m), 3.71 (2H, m); 4.50 (1H, q, H-18, J = 7.0 Hz); 4.57 (1H, d, H-17, J = 7.5 Hz); 3.84 (2H, m, CH2-8(1)); 3.85 (3H, s, CH3-15(3)); 3.61 (3H, s, CH3-12(1)); 3.53 (3H, s, CH3-2(1)); 3.37 (3H, s, CH3-7(1)); CH2-6a, CH2-6b, CH2-6c, CH2-6d: 2.97 (4H, m), 2.60 (4H, m); CH2-13(2), CH2-13(3): 3.97 (4H, m); 3.20 (4H, m, CH2-17(1), CH2-17(2)); CH2-17(4), CH2-17(5): 2.38 (2H, m), 1.97 (2H, m); 1.76 (6H, m, CH3-18(1), CH3-8(2)); CH3-8a, CH3-9a, CH311a, CH3-12a, CH3-8b, CH3-9b, CH3-11b3, CH3-12b, CH3-8c, CH3-9c, CH311c, CH3-12c, CH3-8d, CH3-9d, CH3-11d, CH3-12d: 1.50 (6H, s), 1.33 (6H, s), 1.28 (6H, s), 1.12 (6H, s), 1.11 (6H, s), 0.96 (6H, s), 0.88 (6H, s), 0.58 (6H, s); −1.59 (1H, m, I-NH); −1.84 (1H, m, III-NH). 13C NMR (75 MHz, CDCl3): 129.13 (C-3(1)); 121.49 (C-3(2)); 100.98 (C-10); 98.76 (C-5); 96.44, 96.23 (C-1a, C-1b, C-1c, C-1d); 93.43 (C-20); 71.75, 71.55, 70.65, 70.52, 70.43, 70.33, 65.42, 65.14 (C-2a, C-2b, C-2c, C-2d, C-3a, C-3b, C-3c, C-3d, C-4a, C-4b, C-4c, C-4d, C-5a, C-5b, C-5c, C-5d); 54.03, 54.77 (C-6a, C6b); 53.12 (C-17); 52.88 (C-18); 52.05 (C-15(3)); 38.16 (C-13(2), C-13(3)); 37.21 (C-15(1)); 37.47, 37.00 (C-17(1), C-17(2)); 32.71, 30.50 (C-17(4), C-17(5)); 26.07, 26.00, 25.85, 25.60, 24.70, 24.60, 24.00, 23.47 (C-8a, C9a, C-11a, C-12a, C-8b, C-9b, C-11b, C-12b, C-8c, C-9c, C-11c, C-12c, C-8d, C9d, C-11d, C-12d); 23.16 (C-18(1)); 19.75 (C-8(1)); 17.77 (C-8(2)); 12.08 (C-12(1)); 12.17 (C-2(1)); 11.37 (C-7(1)). Conjugate 8. To the 130 mg (0.078 mmol) of compound 7 10 ml 90% trifluoroacetic acid was added. The reaction mixture was shaken at room temperature for 4 h. Next, the reaction mixture was evaporated at low pressure. Evaporated precipitate was chromatographed on silochrom C-120 (eluent: trichloromethane – methanol, 1:1), (TLC, 95% ethanol). 80 mg (76% yield) was obtained as black crystalline powder. UV (C2H5OH (70%)) λmax/nm (Irelative (%)):661 (25), 605 (3), 526 (3), 499 (8), 399 (87). UV (H2O), λmax/nm (Irelative)): 658 (22), 603 (3), 501 (8), 400 (93). IR (KBr), ν/cm−1: 3341 (OeH), 1676 (“amide-I”), 1603 (“chlorine band”), 1543 (“amide-II”). Mass spectrum (ESI), m/z: 1343.5 [M+H]+ (calcd. for C63H91N8O24+, 1343.6). 1H NMR (300 MHz, DMSO-D7): 9.81 (1H, s, H-10); 9.78 (1H, s, H-5); 9.16 (1H, s, H-20); 9.38 (1H, m, NH-13(1) (amide)); 8.08 (1H, m, NH-17(3) (amide)); 8.33 (1H, dd, H-3(1), J = 12.0, 17.4 Hz); 6.49 (1H, d, H-3(2) (trans), J = 18.3 Hz); 6.22 (1H, d, H-3(2) (cis), J = 12.0 Hz); CH2-15(1): 5.52 (1H, d, J = 18.6 Hz), 5.30 (1H, d, J = 18.3 Hz); H-1a, H-1b, H-1c, H-1d, H-2a, H-2b, H-2c, H-2d, H-3a, H-3b, H-3c, H-3d, H-4a, H-4b, H-4c, H-4d, H5a, H-5b, H-5c, H-5d, CH2-6a, CH2-6b, CH2-6c, CH2-6d, OH-1a, OH-1b, OH-2a, OH-2b, OH-3a, OH-3b, OH-4a, OH-4b, OH-1c, OH-1d, OH-2c, OH2d, OH-3c, OH-3d, OH-4c, OH-4d: 5.42 (6H, m), 4.96 (6H, m), 4.34 (6H, m), 3.84 (4H, m), 3.63 (9H, m), 3.33 (9H, m), 3.16 (4H, m); 4.63 (1H, q, H-18, J = 6.7 Hz); 4.42 (1H, d, H-17, J = 7.2 Hz); 3.87 (2H, m, CH28(1)); 3.74 (3H, s, CH3-15(3)); 3.56 (3H, s, CH3-12(1)); 3.55 (3H, s, CH3-2(1)); 3.34 (3H, s, CH3-7(1)); 3.37 (4H, m, CH2-13(2), CH2-13(3)); 2.53 (4H, m, CH2-17(1), CH2-17(2)); 2.13 (4H, m, CH2-17(4), CH217(5)); 1.70 (6H, m, CH3-8(2), CH3-18(1)); −1.72 (1H, s, I-NH); −2.00 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.82 (C-3(1)); 122.51 (C-3(2)); 101.36 (C-10); 98.97 (C-5); 68.75, 69.35, 69.69, 70.53, 72.10, 73.51, 76.47, 77.70, 93.26, 93.39, 97.81, 97.94, 98.97, 102.22, 102.32, 103.12 (C-1a, C-1b, C-1c, C-1d, C-2a, C-2b, C-2c, C-2d, C-3a, C-3b, C-3c, C3d, C-4a, C-4b, C-4c, C-4d, C-5a, C-5b, C-5c, C-5d, C-6a, C-6b, C-6c, C-6d); 94.65 (C-20); 53.29 (C-17); 52.55 (C-15(3)); 48.81 (C-18); 37.47 (C15(1)); 32.67, 32.75 (C-13(2), C-13(3)); 29.47, 30.07 (C-17(1), C17(2)); 30.73, 30.79 (C-17(4), C-17(5)); 23.55 (C-18(1)); 19.44 (C8(1)); 18.31 (C-8(2)); 12.57 (C-12(1)); 12.27 (C-2(1)); 11.50 (C-7(1)).
Investigation of cytotoxicity of the compounds and estimation of the ability to penetrate the cell membrane. In this work, we used human cervical cancer cell line (HeLa) (BioloT, Russia). The cells were cultured in a growth medium DMEM/F12 (PAA Laboratories GmbH, Austria) containing 10% of fetal bovine serum (FBS) (HyClone, USA), without antibiotics at 37 °C and 5% CO2. Investigation of dark and phototoxic activity was carried out according to the procedure described in Pylina et al.25. The number of live cells was assessed by the FMCA method as described in Lindhagen et al.31 The experiments were performed in 10 independent microcultures for each concentration. The data was checked for artifacts using Grabbs criterion before used for IC50 calculation. The IC50 was calculated from the compound concentration in relation to the survival index, SE – standard error. The ability of compound to enter the cell was evaluated by fluorescent microscopy according to the procedure described in Pylina et al.25. References 1. Singh S, Aggarwal A, Bhupathiraju NVSDK, Arianna G, Tiwari K, Michael C. Glycosylated porphyrins, phthalocyanines, and other porphyrinoids for diagnostics and therapeutics. Chem Rev. 2015;115:10261–10306. https://doi.org/10.1021/acs. chemrev.5b00244. 2. Ethirajan M, Chen Y, Joshi P, Pandey RK. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem Soc Rev. 2011;40:340–362. https://doi. org/10.1039/b915149b. 3. Ormond AB, Freeman HS. Dye sensitizers for photodynamic therapy. Materials. 2013;6:817–840. https://doi.org/10.3390/ma6030817. 4. Abrahamse H, Hamblin MR. New photosensitizers for photodynamic therapy. Biochem J. 2016;473:347–364. https://doi.org/10.1042/BJ20150942. 5. Brandis AS, Salomon Y, Scherz A. Chlorophyll sensitizers in photodynamic therapy. Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications. Netherlands. 2006:461–483. https://doi.org/10.1007/1-4020-45166_32. 6. Pereira PMR, Silva S, Ramalho JS, et al. The role of galectin-1 in in vitro and in vivo photodynamic therapy with a galactodendritic porphyrin. Eur J Cancer. 2016;68:60–69. https://doi.org/10.1016/j.ejca.2016.08.018. 7. Feng L, Xujun H, Li L, Li W, Tianjun L. A novel water-soluble near-infrared glucoseconjugated porphyrin: synthesis, properties and its optical imaging effect. J Porphyrins Phthalocyanines. 2011;15:217–222. https://doi.org/10.1142/ S1088424611003252. 8. Arja K, Elgland M, Appelqvist H, Konradsson P, Lindgren M, Nilsson KPR. Synthesis and characterization of novel fluoro-glycosylated porphyrins that can be utilized as theranostic agents. Chem Open. 2018;7:495–503. https://doi.org/10.1002/open. 201800020. 9. Fadlan A, Tanimoto H, Ito T, et al. Synthesis, photophysical properties, and photodynamic activity of positional isomers of TFPP-glucose conjugates. Bioorg Med Chem. 2018;26:1848–1858. https://doi.org/10.1016/j.bmc.2018.02.031. 10. Pereira PMR, Rizvi W, Bhupathiraju NVSDK, et al. Carbon-1 versus carbon-3 linkage of D-galactose to porphyrins: Synthesis, uptake, and photodynamic efficiency. Bioconjugate Chem. 2018;29:306–315. https://doi.org/10.1021/acs.bioconjchem. 7b00636. 11. Park Young K, Bayarmaa Bold, Cun Cui Bing, Quan Bai Jin, Woo-kyoung Lee, Key Shim Young. Binding affinities of carbohydrate-conjugated chlorins for galectin-3. Bull Korean Chem Soc. 2008;29:130–134. https://doi.org/10.5012/bkcs.2008.29.1. 130. 12. Ming W, Zuo-Wei Y, Yang L, et al. Glycosyl-modified diporphyrins for in vitro and in vivo fluorescence imaging. ChemBioChem. 2013;14:979–986. https://doi.org/10. 1002/cbic.201300065. 13. Vicente MGH, Bhupathiraju NVSDK, Hu X, et al. Synthesis and in vitro evaluation of BBB permeability, tumor cell uptake and cytotoxicity of a series of carboranylporphyrin conjugates. J Med Chem. 2014;57:6718–6728. https://doi.org/10. 1021/jm500786c. 14. Iqbal Z, Masilela N, Nyokong T, Lyubimtsev A, Hanack M, Zieglerb T. Spectral, photophysical and photochemical properties of tetra- and octaglycosylated zinc phthalocyanines. Photochem Photobiol Sci. 2012;11:679–686. https://doi.org/10. 1039/c2 pp05348a. 15. Iqbal Z, Lyubimtsev A, Herrmann T, Hanack M, Ziegler T. Synthesis of octaglycosylated zinc(II) phthalocyanines synthesis of octaglycosylated zinc(I). Synthesis. 2010;18:3097–3104. https://doi.org/10.1055/s-0030-1258178. 16. Grin MA, Lonin IS, Lakhina AA, et al. 1,3-Dipolar cycloaddition in the synthesis of glycoconjugates of natural chlorins and bacteriochlorins. J Porphyrins Phthalocyanines. 2009;13:336–345. https://doi.org/10.1142/S1088424609000425. 17. Silva S, Pereira PMR, Silva P, et al. Porphyrin and phthalocyanine glycodendritic conjugates: synthesis, photophysical and photochemical properties. Chem Commun. 2012;48:3608–3610. https://doi.org/10.1039/c2cc17561d. 18. Iqbal Z, Lyubimtsev A, Hanack M, Ziegler T. Anomerically glycosylated zinc(II) naphthalocyanines. Tetrahedron Lett. 2009;50:5681–5685. https://doi.org/10.1016/ j.tetlet.2009.07.127. 19. Iqbal Z, Lyubimtsev A, Hanack M, Ziegler T. Synthesis and characterization of 1,8(11),15(18),22(25)-tetraglycosylated zinc(II) phthalocyanines. J Porphyrins
2068
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al. Phthalocyanines. 2010;14:494–498. https://doi.org/10.1142/S1088424610002343. 20. Koifman OI, Hanack M, Syrbu SA, Lyubimtsev AV. Phthalocyanine conjugates with carbohydrates: synthesis and aggregation in aqueous solutions. Russ Chem Bull. 2013;62:896–917. https://doi.org/10.1007/s11172-013-0121-2. 21. Gang Zheng, Graham A, Shibata M, et al. Synthesis of a-galactose-conjugated chlorins derived by enyne. Metathesis as galectin-specific photosensitizers for photodynamic therapy. J Org Chem. 2001;66:8709–8716. https://doi.org/10.1021/ jo0105080. 22. Lyubimtsev A, Iqbal Z, Crucius G, Syrbu S, Ziegler T, Hanack M. Synthesis of glycosylated metal phthalocyanines and naphthalocyanines. J Porphyrins Phthalocyanines. 2012;16:434–463. https://doi.org/10.1142/S1088424612300030. 23. Kustov AV, Belykh DV, Smirnova NL, Khudyaeva IS, Berezin DB. Partition of methylpheophorbide a, dioxidine and their conjugate in the 1-octanol/phosphate saline buffer biphasic system. J Chem Thermodyn. 2017;115:302–306. https://doi.org/10. 1016/j.jct.2017.07.031. 24. Belykh DV, Mal’shakova MV. Interaction of methylpheophorbide (a) with ethylenediamine. Butlerov Commun. 2014;39:35–42. 25. Pylina YI, Shadrin DM, Shevchenko OG, et al. Dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives with oligoethylene glycol substituents on the periphery of their macrocycles. Int J Mol Sci. 2017;18. https://doi.org/10.
3390/ijms18010103 103/1-103/14. 26. Samtsov MP, Tarasau DS, Kaplevsky KN, Voropay ES, Petrov PT, Istomin Yu.P. Fluorescence diagnosis of damage to tumor tissues during photodynamic therapy with the photosensitizer photolon. J Appl Spectrosc. 2016;83:79–84. https://doi.org/ 10.1007/s10812-016-0246-9. 27. Copley L, Watt P, Wirtz KW, Parker MI, Leaner VD. Photolon, a chlorin e6 derivative, triggers ROS production and light-dependent cell death via necrosis. Int J Biochem Cell Biol. 2008;40:227–235. https://doi.org/10.1016/j.biocel.2007.07.014. 28. Istomina YP, Lapzevicha TP, Chalaua VN, Shliakhtsin SV, Trukhachova TV. Photodynamic therapy of cervical intraepithelial neoplasia grades II and III with Photolon. Photodiagn Photodyn Ther. 2010;7:144–151. https://doi.org/10.1016/j. pdpdt.2010.06.005. 29. Berichte der Deutschen, Chemischen Gesellschaft. Acetone compounds of the sugars and their derivatives. IV. Constitution of diacetonegalactose. [Abteilung] Abhandlungen. 1925; 58: 2585–2589. 30. Szarek WA, Hay GW, Doboszewsk B. Utility of tris(dimethy1amino)sulphonium difluorotrimethylsilicate (TASF) for the rapid synthesis of deoxyfluoro sugars. J Chem Soc, Chem Commun. 1985;10:663–664. https://doi.org/10.1039/c39850000663. 31. Lindhagen L, Nygren P, Larsson R. The fluorometric microculture cytotoxicity assay. Nat Protoc. 2008;3(8):1364–1369. https://doi.org/10.1038/nprot.2008.114.
2069
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging
T
Marina V. Mal'shakovaa, , Yana I. Pylinab, Dmitry V. Belykha ⁎
a b
Institute of Chemistry of the Komi Science Centre, Ural Division of Russian Academy of Sciences, 167000, 48 Pervomayskaya st., Syktyvkar, Russia Institute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, 167000, 28 Kommunisticheskaya st., Syktyvkar, Russia
ARTICLE INFO
ABSTRACT
Keywords: Chlorin e6 D-Galactopyranose Cytotoxicity Amide
We synthesized new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via carbon atom in position 6 of the galactose fragment. Galactose fragments were inserted by alkylation of the amino groups of chlorin e6 amides with one and two ethylene diamine fragments on the macrocycle periphery with triflate of diacetone galactose, followed by removal of diisopropylidene protection by 70% aqueous trifluoroacetic acid. The synthesized compounds were shown to be capable of penetrating the membrane of HeLa cells; they have intense red fluorescence inside the cell and have phototoxic properties towards HeLa cells (upon LED irradiation at 660 nm and light exposure value of 12 J/cm2). These properties, along with water solubility, allow us to consider the synthesized compounds to be promising as potential antitumor PSs and diagnostic compounds for visualizing malignant tumors and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases.
Porphyrins and their analogues are intensively studied as preparations for diagnostics and photodynamic therapy of oncological diseases1,2,3–5. Chlorins obtained by chemical modification of peripheral substituents of chlorophyll a and its derivatives (such as methylpheophorbide a (1), Scheme 1) have several advantages in comparison with porphyrins when used in the diagnostics and therapy of oncological diseases, such as their relatively low toxicity and good photophysical characteristics.1,2,3,5 Therefore, the development of new anticancer photosensitizers (PSs) and diagnostic preparations for oncology based on chlorophyll a derivatives attracts much attention, and some of these compounds are already used in clinical practice.1,2,3,5 The most important areas of PS improvement are increasing bioavailability and tropism for malignant neoplasms, as well as enabling the PS molecule to actively penetrate the cell membrane. The insertion of fragments of carbohydrate molecules onto the periphery of the macrocycle of PS based on porphyrins and their analogues, as a rule, helps to improve the properties of these PSs in this respect.6–8,9,10–13,14,15–22,1 In particular, the properties of PSs based on chlorophyll a derivatives can thus be improved.11,16,21,1 To further improve PSs and diagnostic preparations based on chlorophyll a derivatives, the conjugation of the latter with galactose appears to be the most promising. Galactose contains a sufficiently large number of hydroxyl groups to significantly increase the bioavailability of the PS molecule. In addition, a fragment of galactose molecule as part of various conjugates is capable of non-covalent ⁎
interactions with cellular structures, due to which tropism of the conjugates towards malignant neoplasms increases and the conjugates acquire the ability to actively penetrate the cell membrane.6,10,11,14,15–17,19,21,1 The data from the literature suggest that the interaction with cellular structures is significantly influenced by the method of binding of the galactose molecule fragment to macrocycle fragments. For example, in terms of absorption by cancer cells, conjugation of the galactose fragment via the hydroxyl group in position 1 is less advantageous than conjugation via the hydroxyl group in position 3,6 which can be explained by the participation of the hydroxyl group in position 1 in binding to the galectin-1 receptor as a hydrogen bond acceptor. Most of the conjugates with tetrapyrrole macrocycles described in the literature were synthesized on the basis of tetramezoarylporphyrins and phthallocyanines.6–10,12–15,17–22,1 Relatively few conjugates with chlorophyll a derivatives were described, and in most cases conjugation was implemented via the hydroxyl group in position 1.11,16,1 Therefore, the synthesis of chlorophyll a derivatives with galactose fragments linked to the chlorin macrocycle via carbon atoms in different positions of the galactose molecule would be of interest. In the present work, chlorine e6 derivatives with two and four galactose fragments on the periphery of the macrocycle linked via the carbon atom in position 6 of the galactose molecule were synthesized (Scheme 1), and dark and photoinduced cytotoxicities and ability of new compounds to penetrate the HeLa cell membrane were assessed.
Corresponding author. E-mail addresses: [email protected] (M.V. Mal'shakova), [email protected] (Y.I. Pylina), [email protected] (D.V. Belykh).
https://doi.org/10.1016/j.bmcl.2019.07.019 Received 22 April 2019; Received in revised form 5 July 2019; Accepted 9 July 2019 Available online 10 July 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
OTf
O H2N
NH N N HN
O
NH2
NH N
OCH3 OCH3
O
O
O
N
O OCH3
H
(2) [(2)]
O
OCH3
(1) H2N
O
NH2
N
O OCH3 O
i, ii (one flask)
H2N
N
O
(6)
N
H
O
NH2
O OCH3
NH N N
H O
9, iii
OH
N OH
N HN
OH
OCH3
(4)
O
v
N HN O
O
O
O
N HN O OCH3
O
O
O
NH N
O
O O
NH N
H
H
N
(3)
O
O
(9)
OCH3
iv
NH2
O
O
N HN
N HN
i
O
NH N
9, iii
O O
HO
O
HO
OH
HO
N
O OCH3
OH O N
(5)
OH
H
OH
OH
O
HO
HO
OH
HO
OH OH
NH N
NH N
iv
N HN
N HN H
O
O O
O
N
O O
N
O
O N
O OCH3 O
O
O
O
O (7)
H
O O
N O
O
O
H
O
O
O
O HO
O
N
O
O OH OCH3 OH
HO OH
N
OH
H O N
O
N
OH
HO
O
HO
O
HO
HO (8)
HO
OH
HO
OH OH
Scheme 1. i: CHCl3, r. t., 2 h [25]; ii: r. t., 20 h25; iii: THF, reflux, 3 h (yield: 3 (48%), 7 (22%)); iv: TFA(90%)-H2O, 3–4 h (yield: 4 (81%), 8 (76%)); v: TFA(90%)H2O, 48 h (yield of 5 (35%)).
The insertion of fragments of diacetone galactose onto the periphery of the chlorin macrocycle was carried out by alkylation of amino groups of chlorin e6 derivatives 2, 6 by the action of triflate diacetone galactose 9 (boiling in THF in the presence of sodium acetate as base and two- or four-fold molar excess of 9 in the case of chlorins 2 and 6, respectively), followed by the removal of diisopropylidene protection (hydrolysis in 90% aqueous TFA) (Scheme 1). The mono- and diamino derivatives of chlorin e6 2 and 6 were synthesized on the basis of methylpheophorbide a 1 (obtained from the spirulina biomass as in Kustov et al.23) by the action of ethylene diamine according to the methods developed by us earlier.24 The spectral characteristics of the compounds 1, 2, and 6 that were described earlier correspond to the literature data.23,24 Alkylation of amino chlorins 2 and 6 with a twofold (relative to the number of amino groups in the molecule) molar excess of triflate diacetone galactose allows to insert two diacetone galactose fragments into each amino group and thus to obtain chlorins with two and four carbohydrate fragments. Removal of protection from conjugate 7 takes place without the formation of byproducts, since the ester group in position 15 does not undergo hydrolysis under the reaction conditions. In the case of conjugate 3, in addition to removal of the diisopropylidene protection, hydrolysis of the ester group in position 17 is possible, and the formation of chlorin 5 with the carboxyl group in position 17 is
also possible, aside from the target product 4. Chlorin 5 is of interest not only as a possible byproduct, but also as a more hydrophilic derivative, the presence of both carboxyl and alkylated amino groups in the molecule of which determines the possibility of its existence in solution in the zwitterionic form (Fig. 1). Therefore, we purposefully prepared chlorin 5 by means of a more prolonged exposure to 90% aqueous TFA on chlorin 3 (Scheme 1). Using chlorin 5 as an internal standard in the analysis of the reaction mixture for preparation of compound 4, we showed that the hydrolysis of the ester group in position 17 after 3 h exposure of compound 3 to 90% aqueous TFA does not occur, therefore no chlorin 5 was detected in the reaction mixture. The structure of the de novo synthesized compounds was established on the basis of data from NMR, IR and UV–vis spectroscopy, as well as mass spectrometry. Mass spectra (ESI) of conjugates with fragments of diacetone galactose 3, 7 and galactose 5, 8 show peaks, the m/z values of which correspond to protonated molecular ions with structures of 3, 5, 7, and 8. In the case of compounds 3 and 4, peaks corresponding to adducts with the sodium cation are observed. The mass spectrum of compound 4, in addition to the peak of the adduct with the sodium cation, has a peak the m/z value of which corresponds to the hydrogenated molecule ([M+2H]). In the UV–vis spectra of all de novo synthesized compounds, absorption bands characteristic of the chlorin 2065
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
Fig. 1. Light micrographs (upper row) and fluorescent micrographs (lower row) of HeLa cells after 40 min exposure to compounds (10 μM) and without them. IC50 ± SE, for Photolon a drug applied in clinical practice is 31.09 ± 4.92 μM (dark toxicity) and 4.01 ± 0.91 μM (phototoxicity).
chromophore are observed. In the 1H and 13C NMR spectra of conjugates 3–5, 7, and 8, the signals of the chlorin macrocycle and fragments of diacetone galactose (compounds 3, 7) or galactose (compounds 4, 5, and 8) are observed, with the ratio of the intensities of the signals of protons of the chlorin macrocycle and carbohydrate fragments in the spectra 1H NMR corresponding to the presence of two (for compounds 3–5) and four (compounds 7 and 8) carbohydrate fragments in the molecule, which correlates with the data of mass spectrometry. The presence in the spectra of 1H NMR of chlorins with fragments of diacetone galactose 3 and 7 singlet signals that correspond, judging by the integral intensity, to the methyl groups of the diisopropylidene protection of two (in compound 3) and four (in compound 7) fragments indicates that the reaction is selective and the isopropylidene protection at the conditions of its carrying out is persistent. The presence of isopropylidene protection is also manifested in the 13C NMR spectra (signals of carbon atoms of methyl groups and quaternary carbon atoms associated with them). In the 1H and 13C NMR spectra of compounds 4, 5 and 8, there are no signals corresponding to protons and carbon atoms of the diisopropylidene protection, whereas the signals of protons of hydroxyl groups formed after removal of diisopropylidene fragments are observed, which confirms the complete removal of the protection. The removal of the diisopropylidene protection results in the opportunity for isomerization of compounds 4, 5, and 8 in solution, which is reflected in the broadening or splitting of the signals of some carbon atoms of the galactose fragments in the 13C NMR spectra of these compounds. However, for most signals, diastereomeric differences are not visible. In the 1H NMR spectrum of compound 5, in contrast to compound 4, there is no signal of the methyl group in position 17, which confirms the hydrolysis at this position. In addition, at 12 m.d. there is a broadened signal of the proton of the carboxyl group which is formed during hydrolysis. The presence of this signal indicates the absence of a zwitterionic form 5 in DMSO. As already noted, the presence of galactose fragments increases the solubility of porphyrin compounds in water. We obtained preliminary data on the solubility of compounds 4, 5, and 8 in water. When dissolved in water, compounds 4, 5, and 8 generate fluorescent in UV light solutions, which indicates the presence of single molecules in the aqueous phase, that is, the formation of a true solution. For all compounds studied, it is possible to obtain a solution containing at least 0.1 mg/ml without the use of auxiliary substances, which indicates the
possibility of obtaining water-soluble forms. To assess the ability of the compounds to penetrate the cell membrane and the possibility of their use for visualization of tumor cells, we used fluorescence microscopy. The cells were incubated with the compounds under study and then the fluorescence of the cells was examined. Intense red fluorescence of cells indicates the ability of compounds 4, 5 and 8 to penetrate the cell membrane (Fig. 1). In addition, the intense fluorescence of the studied compounds inside the cells indicates that these compounds interact with cellular structures as single molecules, which can be used to visualize tumors for diagnostic purposes. Thus, we can conclude that all the synthesized compounds are of interest as potential diagnostic drugs. To evaluate the dark and photoinduced (LED-irradiated at a wavelength of 660 nm and light exposure value of 12 J/cm2) cytotoxicities of compounds 4, 5 and 8 towards HeLa cells, IC50 values were determined as in our previous studies25 (Fig. 1). The obtained data indicate that conjugates 4, 5 and 8 exert a photoinduced cytotoxic action on HeLa cells, and the maximum photoinduced effect is observed for conjugates 5 and 8: IC50 values under dark exposure to these compounds are 31 and 38 times correspondingly higher than that under photosensitized exposure. Collation of IC50 values (calculated as a reactant – chlorine e6) for the Photolon a drug applied in clinical practice26–28 allows to make a conclusion on comparable or better ratio of dark and photoinduced cytotoxicity for all conjugates with galactose studied. In the present work, we describe the synthesis of new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via the carbon atom in position 6 of the galactose fragment. The synthesized compounds were shown to be able to penetrate the cell membrane, to have intense red fluorescence, and to be characterized by a high photoinduced cytotoxicity towards HeLa cells. The most promising from this point of view were conjugates 5 and 8, the IC50 values of which for dark cytotoxicity are 31 and 38 times correspondingly greater than for photoinduced one. Considerable photodynamic activity of conjugates 5 and 8, in combination with water solubility, allow to consider these compounds to be promising for further research as antitumor PSs and diagnostic preparations for visualizing malignant neoplasms and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases (that can be utilized as theranostic agents). 1 H and 13C NMR spectra of the synthesized compounds were 2066
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
[M+Na]+ (calcd. for C50H66N6NaO15+, 1013.4). 1H NMR (300 MHz, DMSO-D7): 9.78 (1H, s, H-10); 9.74 (1H, s, H-5); 9.13 (1H, s, H-20); 9.08 (1H, t, NH-13(1) (amide), J = 4.5 Hz); 8.30 (1H, dd, H-3(1), J = 11.7, 17.7 Hz); 6.43 (1H, d, H-3(2) (trans), J = 17.7 Hz); 6.16 (1H, d, H-3(2) (cis), J = 11.7 Hz); CH2-15(1): 5.55 (1H, d, J = 19.2 Hz), 5.34 (1H, d, J = 19.2 Hz); H-1a, H-1b: 6.55 (1H, m), 6.16 (1H, m); H-2a, H2b, H-3a, H-3b, H-4a, H-4b, H-5a, H-5b, OH-1a, OH-1b, OH-2a, OH-2b, OH3a, OH-3b, OH-4a, OH-4b, OH-5a, OH-5b: 4.99 (2H, m), 4.75 (2H, m), 4.56 (2H, m), 4.35 (3H, m), 4.13 (1H, m), 3.83 (4H, m), 3.63 (4H, m); 4.44 (1H, d, H-17, J = 9.0 Hz); 4.64 (1H, q, H-18, J = 7.2 Hz); 3.86 (2H, m, CH2-8(1)); 3.74 (3H, s, CH3-15(3)); 3.60 (3H, s, CH3-17(4)); 3.54 (3H, s, CH3-12(1)); 3.52 (3H, s, CH3-2(1)); 3.32 (3H, s, CH3-7(1)); CH2-6a, CH2-6b: 3.84 (2H, m), 3.66 (2H, m); CH2-13(2), CH2-13(3): 3.83 (2H, m), 3.58 (2H, m); CH2-17(1), CH2-17(2): 2.36 (2H, m), 2.17 (2H, m); 1.70 (3H, t, CH3-8(2), J = 7.8 Hz); 1.66 (3H, d, CH3-18(1), J = 7.2 Hz); −1.80 (1H, s, I-NH); −2.08 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.83 (C-3(1)); 122.40 (C-3(2)); 101.26 (C-10); 98.99 (C-5); 103.21 (C-1a, C-1b); 94.58 (C-20); 93.22, 98.04, 99.00, 67.99, 69.31, 70.13, 72.66, 74.17 (C-4a, C-4b, C-3a, C-3b, C-2a, C-2b, C5a, C-5b); 55.76, 55.17 (C-6a, C-6b); 53.14 (C-17); 52.39 (C-15(3)); 51.83 (C-17(4)); 48.55 (C-18); 38.57, 38.41 (C-13(2), C-13(3)); 37.21 (C-15(1)); 31.07, 29.92 (C-17(1), C-17(2)); 23.43 (C-18(1)); 19.44 (C8(1)); 18.28 (C-8(2)); 12.51 (C-12(1)); 12.23 (C-2(1)); 11.47 (C-7(1)). Conjugate 5. To the 100 mg (0.087 mmol) of compound 3 10 ml 70% trifluoroacetic acid was added. The reaction mixture was shaken at room temperature for 48 h. Next, the reaction mixture was evaporated at low pressure. Evaporated precipitate was chromatographed on silochrom C-120 (eluent: trichloromethane – methanol, 2:1), (TLC, 95% ethanol). 30 mg (35% yield) was obtained as black crystalline powder. UV (C2H5OH (80%)) λmax/nm (Irelative (%)): 661 (27), 606 (3), 527 (3), 499 (9), 399 (94). UV (H2O) λmax/nm (Irelative (%)): 663 (25), 503 (1), 399 (100). IR (KBr), ν/cm−1: 3338 (OeH), 1728 (C]O, ester), 1680 (“amide-I”), 1601 (“chlorine band”), 1543 (“amide-II”). Mass spectrum (ESI), m/z: 977.7 [M+H]+ (calcd. for C49H65N6O15+, 977.5). 1H NMR (300 MHz, DMSO-D7): 12.00 (1H, br s, COOH-17(3)); 9.80 (1H, s, H10); 9.74 (1H, s, H-5); 9.14 (1H, s, H-20); 8.29 (1H, dd, H-3(1), J = 12.0, 17.4 Hz); 6.67 (1H, t, NH-13(1) (amide), J = 4.5 Hz); 6.43 (1H, d, H-3(2) (trans), J = 18.3 Hz); 6.16 (1H, d, H-3(2) (cis), J = 11.7 Hz); CH2-15(1): 5.53 (1H, d, J = 18.3 Hz), 5.42 (1H, d, J = 19.2 Hz); H-1a, H-1b: 6.67 (1H, m), 6.39 (1H, m); H-2a, H-2b, H-3a, H-3b, H-4a, H-4b, H-5a, H-5b, OH-1a, OH-1b, OH-2a, OH-2b, OH-3a, OH3b, OH-4a, OH-4b, OH-5a, OH-5b: 5.40 (2H, m), 4.85 (4H, m), 4.44 (4H, m), 3.91 (4H, m), 3.58 (4H m); 4.47 (1H, d, H-17, J = 10.2 Hz); 4.65 (1H, q, H-18, J = 7.0 Hz); 3.84 (2H, m, CH2-8(1)); 3.74 (3H, s, CH315(3)); 3.56 (3H, s, CH3-12(1)); 3.52 (3H, s, CH3-2(1)); 3.31 (3H, s, CH3-7(1)); CH2-6a, CH2-6b: 3.36 (2H, m), 2.69 (2H, m); 3.84 (4H, m, CH2-13(2), CH2-13(3)); CH2-17(1), CH2-17(2): 2.31 (2H, m), 2.15 (2H, m); 1.65 (6H, m, CH3-8(2), CH3-18(1)); −1.77 (1H, s, I-NH); −2.03 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.81 (C-3(1)); 122.44 (C-3(2)); 101.39 (C-10); 98.97 (C-5); 103.20 (C-1a, C-1b); 94.63 (C-20); 102.32, 97.94, 93.36, 83.07, 73.85, 72.34, 69.73, 68.95 (C-2a, C-2b, C3a, C-3b, C-4a, C-4b, C-5a, C-5b); 60.44, 56.60 (C-6a, C-6b); 53.20 (C-17); 52.49 (C-15(3)); 48.60 (C-18); 38.57, 38.41 (C-13(2), C-13(3)); 37.38 (C-15(1)); 31.22, 29.99 (C-17(1), C-17(2)); 23.50 (C-18(1)); 19.42 (C8(1)); 18.31 (C-8(2)); 12.54 (C-12(1)); 12.30 (C-2(1)); 11.48 (C-7(1)). Conjugate 7. To the solution 340 mg (0.49 mmol) of diaminochlorine 6 in 30 ml of the dry tetrahydrofuran 850 mg (2.17 mmol) of 1,2:3,4-Di-Oisopropylidene-D-galactopyranose trifluoromethanesulfonic and 178 mg (2.17 mmol) sodium acetate was added. The mixture was refluxed for 4 h (TLC, tetrachloromethane – acetone, 4:2). Next, the reaction mixture was diluted with 70 ml of trichloromethane and sodium acetate was washed with water. After that, the mixture was dried over dehydrated sodium sulfate and evaporated at low pressure. Evaporated precipitate was chromatographed on silica gel (Alfa Aesar 70–230 mesh) (eluent: tetrachloromethane – acetone, 5:1). 180 mg (22% yield) was obtained as deep blue crystalline powder. UV (CHCl3) λmax/nm (Irelative (%)): 664 (31), 609
recorded on Bruker AVANCE-II-300 (working frequency 300 MHz and 75 MHz for NMR 1H and 13C respectively) using standard impulse Bruker software for one and two-dimensional experiments. Infrared spectra were measured in KBr tablets on the “IR Prestige 21” device (Shimadzu). Mass spectra were recorded on the “ThermoFinnigan LCQ Fleet” device. UV–Vis spectra were recorded on a spectrometer UV1700 (Shimadzu) with the wavelength range of 200–1100 nm. The samples were analyzed in quartz cuvettes (10 mm thick). The reaction was controlled using TLC method on Sorbfil slides. Extraction of the reaction products was done using column chromatography on silica gel Alfa Aesar 70–230 mesh and silochrom C-120 (SiO2*nH2O). Monoaminochlorin 2 and diaminochlorin 6 were obtained according to Belykh et al.24. Diacetone-D-galactose was obtained by the method,29 and Triflate Diacetone-D-galactose was obtained according to literature method.30 Conjugate 3. To the solution 300 mg (0.45 mmol) of aminochlorine 2 in 30 ml of the dry tetrahydrofuran 400 mg (1.02 mmol) of 1,2:3,4-DiO-isopropylidene-D-galactopyranose trifluoromethanesulfonic and 84 mg (1.02 mmol) sodium acetate was added. The mixture was refluxed for 3 h (TLC, tetrachloromethane – acetone, 4:1). Next, the reaction mixture was diluted with 70 ml of trichloromethane and sodium acetate was washed with water. After that, the mixture was dried over dehydrated sodium sulfate and evaporated at low pressure. Evaporated precipitate was chromatographed on silica gel (Alfa Aesar 70–230 mesh) (eluent: tetrachloromethane – acetone, 10:1). 250 mg (48% yield) was obtained as deep blue crystalline powder. UV (CHCl3) λmax/nm (Irelative (%)): 663 (30), 607 (2), 529 (2), 501 (8), 403 (98). IR (KBr), ν/cm−1: 3086 (CeH, vinyl group), 1738 (C]O, ester), 1657 (“amide-I”), 1601 (“chlorine band”), 1514 (“amide-II”). Mass spectrum (ESI), m/z: 1151.8 [M+H]+ (calcd. for C62H83N6O15+, 1151.6), 1173.7 [M+Na]+ (calcd. for C62H82N6NaO15+, 1173.6). 1H NMR (300 MHz, DMSO-D7): 9.79 (1H, s, H-10); 9.75 (1H, s, H-5); 9.12 (1H, s, H-20); 8.78 (1H, t, J = 12.3 Hz, NH-13(1) (amide)); 8.30 (1H, dd, H-3(1), J = 11.6, 17.9 Hz); 6.44 (1H, d, H-3(2) (trans), J = 17.7 Hz); 6.17 (1H, d, H-3(2) (cis), J = 11.7 Hz); CH2-15(1): 5.56 (1H, d, J = 18.9 Hz) and 5.33 (1H, d, J = 18.6 Hz); 5.46 (2H, d, H-1a, H-1b, J = 4.5 Hz); 4.65 (1H, m, H-18); 4.60 (2H, d, H-3a, H-3b, J = 8.1 Hz); 4.44 (1H, m, H-17); 4.40 (2H, d, H-4a, H-4b, J = 7.8 Hz); 4.31 (2H, H-2a, H-2b, J = 3.3 Hz); 4.03 (2H, m, H-5a, H-5b); 3.83 (2H, q, CH2-8(1), J = 8.0 Hz); 3.73 (3H, s, CH3-15(3)); 3.59 (3H, s, CH3-17(4)); 3.53 (6H, s, CH3-12(1), CH3-2(1)); 3.32 (3H, s, CH3-7(1)); CH2-6a, CH26b: 2.95 (2H, m), 2.79 (2H, m); CH2-13(2), CH2-13(3): 3.83 (2H, m), 3.58 (2H, m); CH2-17(1), CH2-17(2): 2.71 (2H, m), 2.35 (2H, m); 1.69 (3H, t, CH3-8(2), J = 7.5 Hz); 1.65 (3H, d, CH3-18(1), J = 6.9 Hz); CH38a, CH3-9a, CH3-11a, CH3-12a, CH3-8b, CH3-9b, CH3-11b, CH3-12b: 1.51 (6H, s), 1.36 (6H, s), 1.22 (6H, s), 1.18 (6H, s); −1.79 (1H, s, I-NH); −2.07 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.84 (C-3(1)); 122.40 (C-3(2)); 101.32 (C-10); 98.97 (C-5); 96.31 (C-1a, C-1b); 94.55 (C-20); 71.42 (C-4a, C-4b); 70.66 (C-3a, C-3b); 70.54 (C-2a, C-2b); 65.37 (C-5a, C-5b); 54.77, 54.03 (C-6a, C-6b); 53.12 (C-17); 52.27 (C-15(3)); 51.77 (C-17(4)); 48.52 (C-18); 38.41 (C-13(2), C-13(3)); 37.21 (C15(1)); 32.54, 31.07 (C-17(1), C-17(2)); 26.46, 26.37, 25.24, 24.45 (C8a, C-9a, C-11a, C-12a, C-8b, C-9b, C-11b, C-12b); 23.41 (C-18(1)); 19.40 (C-8(1)); 18.27 (C-8(2)); 12.51 (C-12(1)); 12.07 (C-2(1)); 11.47 (C7(1)). Conjugate 4. To the 150 mg (0.13 mmol) of compound 3 10 ml 90% trifluoroacetic acid was added. The reaction mixture was shaken at room temperature for 3 h. Next, the reaction mixture was evaporated at low pressure. Evaporated precipitate was chromatographed on silochrom C-120 (eluent: trichloromethane – methanol, 3:1), (TLC, 95% ethanol). 105 mg (81% yield) was obtained as black crystalline powder. UV (CHCl3-C2H5OH (1:1)) λmax/nm (Irelative (%)): 663 (31), 609 (4), 530 (4), 501 (1), 402 (99). UV (H2O), λmax/nm (Irelative)): 664 (27), 502 (1), 400 (99). IR (KBr), ν/cm−1: 3338 (OeH), 1728 (C]O, ester), 1680 (“amide-I”), 1601 (“chlorine band”), 1543 (“amide-II”). Mass spectrum (ESI), m/z: 992.3 [M+2H]+ (calcd. for C50H68N6O15+, 992.5), 1013.7 2067
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al.
(3), 529 (3), 501 (9), 403 (99). IR (KBr), ν/cm−1: 1738 (C]O, ester), 1662 (“amide-I”), 1601 (“chlorine band”), 1516 (“amide-II”). Mass spectrum (ESI), m/z: 1663.6 [M+H]+ (calcd. for C87H123N8O24+, 1663.9). 1H NMR (300 MHz, CDCl3): 9.70 (1H, s, H-10); 9.68 (1H, s, H-5); 8.83 (1H, s, H20); 8.15 (1H, dd, H-3(1), J = 12.0, 17.4 Hz); 7.66 (1H, t, NH-13(1) (amide), J = 4.5 Hz); 6.40 (1H, d, H-3(2) (trans), J = 18.3 Hz); 6.29 (1H, m, 17(3)-NH (amide)); 6.18 (1H, d, H-3(2) (cis), J = 11.1 Hz); CH2-15(1): 5.72 (1H, d, J = 18.9 Hz), 5.23 (1H, d, J = 18.3 Hz); H-1a, H-1b, H-1c, H1d: 5.39 (2H, d, 5.4 Hz), 5.22 (2H, d, 4.5 Hz); H-2a, H-2b, H-2c, H-2d, H-3a, H-3b, H-3c, H-3d, H-4a, H-4b, H-4c, H-4d, H-5a, H-5b, H-5c, H-5d: 4.43 (2H, d, J = 8.1 Hz), 4.26 (2H, d, J = 9.0 Hz), 4.14 (6H, m), 3.97 (4H, m), 3.71 (2H, m); 4.50 (1H, q, H-18, J = 7.0 Hz); 4.57 (1H, d, H-17, J = 7.5 Hz); 3.84 (2H, m, CH2-8(1)); 3.85 (3H, s, CH3-15(3)); 3.61 (3H, s, CH3-12(1)); 3.53 (3H, s, CH3-2(1)); 3.37 (3H, s, CH3-7(1)); CH2-6a, CH2-6b, CH2-6c, CH2-6d: 2.97 (4H, m), 2.60 (4H, m); CH2-13(2), CH2-13(3): 3.97 (4H, m); 3.20 (4H, m, CH2-17(1), CH2-17(2)); CH2-17(4), CH2-17(5): 2.38 (2H, m), 1.97 (2H, m); 1.76 (6H, m, CH3-18(1), CH3-8(2)); CH3-8a, CH3-9a, CH311a, CH3-12a, CH3-8b, CH3-9b, CH3-11b3, CH3-12b, CH3-8c, CH3-9c, CH311c, CH3-12c, CH3-8d, CH3-9d, CH3-11d, CH3-12d: 1.50 (6H, s), 1.33 (6H, s), 1.28 (6H, s), 1.12 (6H, s), 1.11 (6H, s), 0.96 (6H, s), 0.88 (6H, s), 0.58 (6H, s); −1.59 (1H, m, I-NH); −1.84 (1H, m, III-NH). 13C NMR (75 MHz, CDCl3): 129.13 (C-3(1)); 121.49 (C-3(2)); 100.98 (C-10); 98.76 (C-5); 96.44, 96.23 (C-1a, C-1b, C-1c, C-1d); 93.43 (C-20); 71.75, 71.55, 70.65, 70.52, 70.43, 70.33, 65.42, 65.14 (C-2a, C-2b, C-2c, C-2d, C-3a, C-3b, C-3c, C-3d, C-4a, C-4b, C-4c, C-4d, C-5a, C-5b, C-5c, C-5d); 54.03, 54.77 (C-6a, C6b); 53.12 (C-17); 52.88 (C-18); 52.05 (C-15(3)); 38.16 (C-13(2), C-13(3)); 37.21 (C-15(1)); 37.47, 37.00 (C-17(1), C-17(2)); 32.71, 30.50 (C-17(4), C-17(5)); 26.07, 26.00, 25.85, 25.60, 24.70, 24.60, 24.00, 23.47 (C-8a, C9a, C-11a, C-12a, C-8b, C-9b, C-11b, C-12b, C-8c, C-9c, C-11c, C-12c, C-8d, C9d, C-11d, C-12d); 23.16 (C-18(1)); 19.75 (C-8(1)); 17.77 (C-8(2)); 12.08 (C-12(1)); 12.17 (C-2(1)); 11.37 (C-7(1)). Conjugate 8. To the 130 mg (0.078 mmol) of compound 7 10 ml 90% trifluoroacetic acid was added. The reaction mixture was shaken at room temperature for 4 h. Next, the reaction mixture was evaporated at low pressure. Evaporated precipitate was chromatographed on silochrom C-120 (eluent: trichloromethane – methanol, 1:1), (TLC, 95% ethanol). 80 mg (76% yield) was obtained as black crystalline powder. UV (C2H5OH (70%)) λmax/nm (Irelative (%)):661 (25), 605 (3), 526 (3), 499 (8), 399 (87). UV (H2O), λmax/nm (Irelative)): 658 (22), 603 (3), 501 (8), 400 (93). IR (KBr), ν/cm−1: 3341 (OeH), 1676 (“amide-I”), 1603 (“chlorine band”), 1543 (“amide-II”). Mass spectrum (ESI), m/z: 1343.5 [M+H]+ (calcd. for C63H91N8O24+, 1343.6). 1H NMR (300 MHz, DMSO-D7): 9.81 (1H, s, H-10); 9.78 (1H, s, H-5); 9.16 (1H, s, H-20); 9.38 (1H, m, NH-13(1) (amide)); 8.08 (1H, m, NH-17(3) (amide)); 8.33 (1H, dd, H-3(1), J = 12.0, 17.4 Hz); 6.49 (1H, d, H-3(2) (trans), J = 18.3 Hz); 6.22 (1H, d, H-3(2) (cis), J = 12.0 Hz); CH2-15(1): 5.52 (1H, d, J = 18.6 Hz), 5.30 (1H, d, J = 18.3 Hz); H-1a, H-1b, H-1c, H-1d, H-2a, H-2b, H-2c, H-2d, H-3a, H-3b, H-3c, H-3d, H-4a, H-4b, H-4c, H-4d, H5a, H-5b, H-5c, H-5d, CH2-6a, CH2-6b, CH2-6c, CH2-6d, OH-1a, OH-1b, OH-2a, OH-2b, OH-3a, OH-3b, OH-4a, OH-4b, OH-1c, OH-1d, OH-2c, OH2d, OH-3c, OH-3d, OH-4c, OH-4d: 5.42 (6H, m), 4.96 (6H, m), 4.34 (6H, m), 3.84 (4H, m), 3.63 (9H, m), 3.33 (9H, m), 3.16 (4H, m); 4.63 (1H, q, H-18, J = 6.7 Hz); 4.42 (1H, d, H-17, J = 7.2 Hz); 3.87 (2H, m, CH28(1)); 3.74 (3H, s, CH3-15(3)); 3.56 (3H, s, CH3-12(1)); 3.55 (3H, s, CH3-2(1)); 3.34 (3H, s, CH3-7(1)); 3.37 (4H, m, CH2-13(2), CH2-13(3)); 2.53 (4H, m, CH2-17(1), CH2-17(2)); 2.13 (4H, m, CH2-17(4), CH217(5)); 1.70 (6H, m, CH3-8(2), CH3-18(1)); −1.72 (1H, s, I-NH); −2.00 (1H, s, III-NH). 13C NMR (75 MHz, DMSO-D7): 129.82 (C-3(1)); 122.51 (C-3(2)); 101.36 (C-10); 98.97 (C-5); 68.75, 69.35, 69.69, 70.53, 72.10, 73.51, 76.47, 77.70, 93.26, 93.39, 97.81, 97.94, 98.97, 102.22, 102.32, 103.12 (C-1a, C-1b, C-1c, C-1d, C-2a, C-2b, C-2c, C-2d, C-3a, C-3b, C-3c, C3d, C-4a, C-4b, C-4c, C-4d, C-5a, C-5b, C-5c, C-5d, C-6a, C-6b, C-6c, C-6d); 94.65 (C-20); 53.29 (C-17); 52.55 (C-15(3)); 48.81 (C-18); 37.47 (C15(1)); 32.67, 32.75 (C-13(2), C-13(3)); 29.47, 30.07 (C-17(1), C17(2)); 30.73, 30.79 (C-17(4), C-17(5)); 23.55 (C-18(1)); 19.44 (C8(1)); 18.31 (C-8(2)); 12.57 (C-12(1)); 12.27 (C-2(1)); 11.50 (C-7(1)).
Investigation of cytotoxicity of the compounds and estimation of the ability to penetrate the cell membrane. In this work, we used human cervical cancer cell line (HeLa) (BioloT, Russia). The cells were cultured in a growth medium DMEM/F12 (PAA Laboratories GmbH, Austria) containing 10% of fetal bovine serum (FBS) (HyClone, USA), without antibiotics at 37 °C and 5% CO2. Investigation of dark and phototoxic activity was carried out according to the procedure described in Pylina et al.25. The number of live cells was assessed by the FMCA method as described in Lindhagen et al.31 The experiments were performed in 10 independent microcultures for each concentration. The data was checked for artifacts using Grabbs criterion before used for IC50 calculation. The IC50 was calculated from the compound concentration in relation to the survival index, SE – standard error. The ability of compound to enter the cell was evaluated by fluorescent microscopy according to the procedure described in Pylina et al.25. References 1. Singh S, Aggarwal A, Bhupathiraju NVSDK, Arianna G, Tiwari K, Michael C. Glycosylated porphyrins, phthalocyanines, and other porphyrinoids for diagnostics and therapeutics. Chem Rev. 2015;115:10261–10306. https://doi.org/10.1021/acs. chemrev.5b00244. 2. Ethirajan M, Chen Y, Joshi P, Pandey RK. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem Soc Rev. 2011;40:340–362. https://doi. org/10.1039/b915149b. 3. Ormond AB, Freeman HS. Dye sensitizers for photodynamic therapy. Materials. 2013;6:817–840. https://doi.org/10.3390/ma6030817. 4. Abrahamse H, Hamblin MR. New photosensitizers for photodynamic therapy. Biochem J. 2016;473:347–364. https://doi.org/10.1042/BJ20150942. 5. Brandis AS, Salomon Y, Scherz A. Chlorophyll sensitizers in photodynamic therapy. Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications. Netherlands. 2006:461–483. https://doi.org/10.1007/1-4020-45166_32. 6. Pereira PMR, Silva S, Ramalho JS, et al. The role of galectin-1 in in vitro and in vivo photodynamic therapy with a galactodendritic porphyrin. Eur J Cancer. 2016;68:60–69. https://doi.org/10.1016/j.ejca.2016.08.018. 7. Feng L, Xujun H, Li L, Li W, Tianjun L. A novel water-soluble near-infrared glucoseconjugated porphyrin: synthesis, properties and its optical imaging effect. J Porphyrins Phthalocyanines. 2011;15:217–222. https://doi.org/10.1142/ S1088424611003252. 8. Arja K, Elgland M, Appelqvist H, Konradsson P, Lindgren M, Nilsson KPR. Synthesis and characterization of novel fluoro-glycosylated porphyrins that can be utilized as theranostic agents. Chem Open. 2018;7:495–503. https://doi.org/10.1002/open. 201800020. 9. Fadlan A, Tanimoto H, Ito T, et al. Synthesis, photophysical properties, and photodynamic activity of positional isomers of TFPP-glucose conjugates. Bioorg Med Chem. 2018;26:1848–1858. https://doi.org/10.1016/j.bmc.2018.02.031. 10. Pereira PMR, Rizvi W, Bhupathiraju NVSDK, et al. Carbon-1 versus carbon-3 linkage of D-galactose to porphyrins: Synthesis, uptake, and photodynamic efficiency. Bioconjugate Chem. 2018;29:306–315. https://doi.org/10.1021/acs.bioconjchem. 7b00636. 11. Park Young K, Bayarmaa Bold, Cun Cui Bing, Quan Bai Jin, Woo-kyoung Lee, Key Shim Young. Binding affinities of carbohydrate-conjugated chlorins for galectin-3. Bull Korean Chem Soc. 2008;29:130–134. https://doi.org/10.5012/bkcs.2008.29.1. 130. 12. Ming W, Zuo-Wei Y, Yang L, et al. Glycosyl-modified diporphyrins for in vitro and in vivo fluorescence imaging. ChemBioChem. 2013;14:979–986. https://doi.org/10. 1002/cbic.201300065. 13. Vicente MGH, Bhupathiraju NVSDK, Hu X, et al. Synthesis and in vitro evaluation of BBB permeability, tumor cell uptake and cytotoxicity of a series of carboranylporphyrin conjugates. J Med Chem. 2014;57:6718–6728. https://doi.org/10. 1021/jm500786c. 14. Iqbal Z, Masilela N, Nyokong T, Lyubimtsev A, Hanack M, Zieglerb T. Spectral, photophysical and photochemical properties of tetra- and octaglycosylated zinc phthalocyanines. Photochem Photobiol Sci. 2012;11:679–686. https://doi.org/10. 1039/c2 pp05348a. 15. Iqbal Z, Lyubimtsev A, Herrmann T, Hanack M, Ziegler T. Synthesis of octaglycosylated zinc(II) phthalocyanines synthesis of octaglycosylated zinc(I). Synthesis. 2010;18:3097–3104. https://doi.org/10.1055/s-0030-1258178. 16. Grin MA, Lonin IS, Lakhina AA, et al. 1,3-Dipolar cycloaddition in the synthesis of glycoconjugates of natural chlorins and bacteriochlorins. J Porphyrins Phthalocyanines. 2009;13:336–345. https://doi.org/10.1142/S1088424609000425. 17. Silva S, Pereira PMR, Silva P, et al. Porphyrin and phthalocyanine glycodendritic conjugates: synthesis, photophysical and photochemical properties. Chem Commun. 2012;48:3608–3610. https://doi.org/10.1039/c2cc17561d. 18. Iqbal Z, Lyubimtsev A, Hanack M, Ziegler T. Anomerically glycosylated zinc(II) naphthalocyanines. Tetrahedron Lett. 2009;50:5681–5685. https://doi.org/10.1016/ j.tetlet.2009.07.127. 19. Iqbal Z, Lyubimtsev A, Hanack M, Ziegler T. Synthesis and characterization of 1,8(11),15(18),22(25)-tetraglycosylated zinc(II) phthalocyanines. J Porphyrins
2068
Bioorganic & Medicinal Chemistry Letters 29 (2019) 2064–2069
M.V. Mal'shakova, et al. Phthalocyanines. 2010;14:494–498. https://doi.org/10.1142/S1088424610002343. 20. Koifman OI, Hanack M, Syrbu SA, Lyubimtsev AV. Phthalocyanine conjugates with carbohydrates: synthesis and aggregation in aqueous solutions. Russ Chem Bull. 2013;62:896–917. https://doi.org/10.1007/s11172-013-0121-2. 21. Gang Zheng, Graham A, Shibata M, et al. Synthesis of a-galactose-conjugated chlorins derived by enyne. Metathesis as galectin-specific photosensitizers for photodynamic therapy. J Org Chem. 2001;66:8709–8716. https://doi.org/10.1021/ jo0105080. 22. Lyubimtsev A, Iqbal Z, Crucius G, Syrbu S, Ziegler T, Hanack M. Synthesis of glycosylated metal phthalocyanines and naphthalocyanines. J Porphyrins Phthalocyanines. 2012;16:434–463. https://doi.org/10.1142/S1088424612300030. 23. Kustov AV, Belykh DV, Smirnova NL, Khudyaeva IS, Berezin DB. Partition of methylpheophorbide a, dioxidine and their conjugate in the 1-octanol/phosphate saline buffer biphasic system. J Chem Thermodyn. 2017;115:302–306. https://doi.org/10. 1016/j.jct.2017.07.031. 24. Belykh DV, Mal’shakova MV. Interaction of methylpheophorbide (a) with ethylenediamine. Butlerov Commun. 2014;39:35–42. 25. Pylina YI, Shadrin DM, Shevchenko OG, et al. Dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives with oligoethylene glycol substituents on the periphery of their macrocycles. Int J Mol Sci. 2017;18. https://doi.org/10.
3390/ijms18010103 103/1-103/14. 26. Samtsov MP, Tarasau DS, Kaplevsky KN, Voropay ES, Petrov PT, Istomin Yu.P. Fluorescence diagnosis of damage to tumor tissues during photodynamic therapy with the photosensitizer photolon. J Appl Spectrosc. 2016;83:79–84. https://doi.org/ 10.1007/s10812-016-0246-9. 27. Copley L, Watt P, Wirtz KW, Parker MI, Leaner VD. Photolon, a chlorin e6 derivative, triggers ROS production and light-dependent cell death via necrosis. Int J Biochem Cell Biol. 2008;40:227–235. https://doi.org/10.1016/j.biocel.2007.07.014. 28. Istomina YP, Lapzevicha TP, Chalaua VN, Shliakhtsin SV, Trukhachova TV. Photodynamic therapy of cervical intraepithelial neoplasia grades II and III with Photolon. Photodiagn Photodyn Ther. 2010;7:144–151. https://doi.org/10.1016/j. pdpdt.2010.06.005. 29. Berichte der Deutschen, Chemischen Gesellschaft. Acetone compounds of the sugars and their derivatives. IV. Constitution of diacetonegalactose. [Abteilung] Abhandlungen. 1925; 58: 2585–2589. 30. Szarek WA, Hay GW, Doboszewsk B. Utility of tris(dimethy1amino)sulphonium difluorotrimethylsilicate (TASF) for the rapid synthesis of deoxyfluoro sugars. J Chem Soc, Chem Commun. 1985;10:663–664. https://doi.org/10.1039/c39850000663. 31. Lindhagen L, Nygren P, Larsson R. The fluorometric microculture cytotoxicity assay. Nat Protoc. 2008;3(8):1364–1369. https://doi.org/10.1038/nprot.2008.114.
2069