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Glycoconjugated hypocrellin: synthesis of [(β-d -glucosyl)ethylthiyl]hypocrellins and photosensitized generation of singlet oxygen
Biochimica et Biophysica Acta 1472 (1999) 232^239 www.elsevier.com/locate/bba
Glycoconjugated hypocrellin: synthesis of [(L-D-glucosyl)ethylthiyl]hypocrellins and photosensitized generation of singlet oxygen Yu-Ying He, Jing-Yi An, Li-Jin Jiang * Institute of Photographic Chemistry, Academia Sinica, Beijing 100101, PR China Received 4 March 1999; received in revised form 30 June 1999; accepted 2 July 1999
Abstract In order to improve the water solubility and specific affinity for malignant tumors of hypocrellin, glycoconjugated hypocrellins have been synthesized using an improved Ko«nigs^Kno«rr reaction from mercaptoethanol substituted hypocrellin B and 2,3,4,6-tetra-O-acetyl-K-D-glucopyranosyl bromide precursors. Deprotection of glucose moieties allows the production of derivatives which had improved solubility in neutral aqueous solution and covered a range of amphiphilic character. The structures of these new protected and unprotected compounds were characterized by UV-Vis, IR, 1 H-NMR and MS data. The present strategy should prove applicable to the synthesis of other glycoconjugated perylenequinone compounds. In addition, the quantum yield of singlet oxygen generation photosensitized by these glycoconjugated hypocrellins has been determined. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Glycoconjugated hypocrellin; Photodynamic therapy; Photosensitizer; Amphiphilicity; Singlet oxygen
1. Introduction Photodynamic therapy (PDT) has received increasing attention in recent years as a new modality of selective treatment of solid tumor [1]. Although the mechanism of tumor cure is likely to require the interplay of several biological responses [2], a key point remains the ability of the photosensitizing agent to be retained to some extent by the tumor and, upon absorption of light, to generate short-lived toxic species, in particular singlet oxygen. Hypocrellins, including hypocrellin A (HA) and hypocrellin B (HB) (Chart 1), are new potential phototherapeutic agents [3^7]. Recent investigations demonstrated the
* Corresponding author. E-mail: [email protected]
photodynamic damages of hypocrellin and its derivatives to viruses, tumor cell, mixed phospholipids, pBR322 DNA and extracted cellular DNA [8^15]. These observations collectively provide a compelling rationale for the development of hypocrellin and its derivatives as PDT photosensitizers. Considerable e¡orts have been further devoted to develop photosensitizers with better tumor selectivity and higher phototherapeutic e¤ciency. Although the mechanism of photosensitizer retention by tumors is not well understood, the balance between lipophilicity and hydrophilicity is recognized as an important factor for photosensitizing e¤ciencies and tumor and cellular uptake [16^22]. However, hypocrellins are lipophilic compounds. Several amphiphilic hypocrellin derivatives have been studied so far, and most of them have ionic structure [23^27]. In the case of can-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 1 2 6 - 9
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Chart 1.
cer phototherapy, the linkages of photosensitizer with sugar moieties are of great importance because the sugar increases water solubility, membrane interaction and speci¢c a¤nity for malignant tumors [28]. Various strategies for the synthesis of glycoconjugated porphyrins, chlorines, porphines and phthalocyanines have been developed [29^39]. However, no e¡ort has been devoted to the synthesis of glycoconjugated hypocrellin derivatives until the work in our laboratory. In this paper, we report full experimental data [40] concerning the synthesis and characterization of hypocrellins with glucosyl substituents. In addition, the yield of singlet oxygen generation by these molecules is also determined. 2. Experimental section 2.1. General methods UV-visible spectra were performed on a HP 8541A spectrophotometer at room temperature. Infrared spectra were recorded on a BIO-RAD FTS165 spectrometer. 1 H-NMR spectra were obtained on a Varian XL-400 (300 MHz) and are reported in parts per million (N) relative to either tetramethylsilane (0.00 ppm) or CHCl3 (7.24 ppm). Coupling constants (J) are reported in hertz. Mass spectra were obtained on a FAB MS AEI-MS 50 Kratos spectrometer from a 3-nitrobenzyl alcohol (NBA) matrix with negative ion FAB used. Elemental analyses were carried out at the Institute of Chemistry, Academia Sinica, Beijing, China. Chemicals used were of reagent grade and used as supplied excepted where noted. Anhydrous benzene and dichloromethane (CH2 Cl2 ) were puri¢ed and then distilled under reduced pressure î molecular sieves (Fluka, activated and kept over 4 A at 500³C). Analytical thin-layer chromatography was performed on E. Merck silica gel 60 plate (2 mm). Liquid column chromatography was performed on
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E. Merck silica gel 60 (40^60 nm). Sephadex LH20 was purchased from Pharmacia LKB. The 1 O2 generation quantum yield by hypocrellins was determined by the average value from the following two methods: (1) measuring the EPR signal intensity of 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO) radical generated from the reaction of 2,2,6,6-tetramethyl-4-piperidone (TEMP) with 1 O2 generated by photosensitization of hypocrellins as a function of irradiation time; (2) measuring the absorption decrease at 374 nm of 9,10-diphenylanthracene (DPA) as a function of irradiation time. 2.2. General procedure for synthesis of protected glycoconjugated hypocrellins (6^10) Using 1 mmol monothiylated hypocrellin (2, 3) or 0.5 mmol dithiylated hypocrellin (4), 1 mmol iodine, 2 mmol freshly prepared Ag2 O, approx. 3 g powî , in 60 ml absolute bendered molecular sieve 4 A zene/dichloromethane mixture (8:1, v/v), and adding 2 mmol 2,3,4,6-tetraacetyl-K-D-glucopyranosyl bromide (5), the peracetylated monosaccharide derivatives 6^10 were obtained after 24 h at 20³C with vigorous stirring in the dark. The resulting mixture was ¢ltrated and evaporated under reduced pressure to a¡ord purple solid. This solid was subjected to thin layer chromatography (TLC) containing 1% citric acid and then eluted with acetone to obtain the condensed products. 2.2.1. 5(or 8)-Mono[(2,3,4,6-tetraacetyl-L-Dglucopyranosyl)ethylthiyl]hypocrellin (6, 7) The crude products were collected and puri¢ed on a silica gel containing 1% citric acid using a mixture of chloroform/ethyl acetate (2:1, v/v) a¡ording the title product (650 mg, 70%) after crystallization from dichloromethane/petroleum ether. FT-IR (KBr disk): 3439, 1744, 1680, 1595, 1229, 1030, 895, 800 cm31 . 1 H-NMR (300 MHz, CDCl3 ) N: 16.00 (s, 1H, exchangeable with D2 O), 15.96 (s, 1H, exchangeable with D2 O), 6.45 (s, 1H), 5.81 (d, J = 8 Hz, 1H), 5.62 (m, 1H), 4.72 (m, 1H), 4.54 (m, 1H), 4.35 (m, 1H), 4.20 (m, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 4.00 (d, J = 12 Hz, 1H), 3.97 (s, 3H), 3.83 (s, 3H), 3.30 (t, J = 7 Hz, 2H), 3.20 (d, J = 12 Hz, 1H), 2.95 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 2.12 (s, 3H), 2.08 (s, 6H), 2.02 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C46 H46 O19 S: C,
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59.09; H, 4.96. Found: C, 59.32; H, 4.89. FABMS (matrix, NBA) m/e: 933 (M 31). 2.2.2. 5(or 8)-Mono[(2,3,4,6-tetraacetyl-L-Dglucopyranosyl)ethylthiyl]-8(or 5)monohydroxyethylthiyl hypocrellins (8, 9) When 0.5 mmol compound 4, 0.5 mmol iodine, 1 mmol freshly prepared Ag2 O, approx. 1.5 g molecî and 1 mmol compound 5 in 30 ml ular sieve 4 A absolute benzene/dichloromethane mixture (8:1, v/ v) were stirred at 20³C in the dark for 24 h, the title compound was formed as a predominant product. The reaction mixture was evaporated under reduced pressure after ¢ltration, and then puri¢ed on a silica gel column containing 1% citric acid using a mixture of chloroform/ethyl acetate (2:1, v/v), a¡ording the title compound (252 mg, 50%) after crystallization from dichloromethane/petroleum ether. FT-IR (KBr disk): 3440, 1744, 1680, 1598, 1229, 1030, 895, 800 cm31 . 1 H-NMR (300 MHz, CDCl3 ) N: 15.98 (s, 1H, exchangeable with D2 O), 15.90 (s, 1H, exchangeable with D2 O), 5.80 (d, J = 8 Hz, 1H), 5.62 (m, 1H), 4.74 (m, 1H), 4.54 (m, 1H), 4.35 (m, 1H), 4.24 (m, 2H), 4.14 (s, 3H), 4.10 (s, 3H), 4.06 (s, 3H), 3.98 (s, 3H), 3.94 (d, J = 12 Hz, 1H), 3.50 (t, J = 7 Hz, 2H), 3.30 (t, J = 7 Hz, 2H), 3.16 (d, J = 12 Hz, 1H), 3.08 (t, J = 7 Hz, 2H), 2.93 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 2.14 (s, 3H), 2.08 (s, 6H), 2.04 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C48 H50 O20 S2 : C, 57.02; H, 4.99. Found: C, 57.24; H, 4.92. FABMS (matrix, NBA) m/e: 1009 (M 31). 2.2.3. 5,8-Bis[(2,3,4,6-tetraacetyl-LD-glucopyranosyl)ethylthiyl]hypocrellin (10) When 0.5 mmol compound 4, 1 mmol iodine, 2 mmol freshly prepared Ag2 O, 3 g molecular sieve î and 2 mmol compound 5 in 60 ml absolute 4 A benzene/dichloromethane mixture (8:1, v/v) were stirred at 20³C in the dark for 24 h, the title compound was produced as a predominant product. The reaction mixture was ¢ltrated, evaporated under reduced pressure, and then puri¢ed on a silica gel column containing 1% citric acid using a mixture of chloroform/ethyl acetate (2:1, v/v) a¡ording the title compound (335 mg, 50%). FT-IR (KBr disk): 3440, 1744, 1680, 1595, 1229, 1030, 895, 800 cm31 . 1 HNMR (300 MHz, CDCl3 ) N: 15.96 (s, 1H, exchangeable with D2 O), 15.90 (s, 1H, exchangeable with
D2 O), 5.82 (d, J = 8 Hz, 2H), 5.60 (m, 2H), 4.75 (m, 2H), 4.56 (m, 2H), 4.35 (m, 2H), 4.26 (m, 4H), 4.18 (s, 3H), 4.12 (s, 3H), 4.09 (s, 3H), 4.06 (s, 3H), 4.00 (d, J = 12 Hz, 1H), 3.30 (t, J = 7 HZ, 4H), 3.16 (d, J = 12 Hz, 1H), 2.92 (t, J = 7 Hz, 4H), 2.73 (s, 3H), 2.12 (s, 6H), 2.08 (s, 6H), 2.05(s, 6H), 2.01 (s, 6H), 1.96 (s, 3H). Anal. Calcd for C62 H68 O29 S2 : C, 55.51; H, 5.11. Found: C, 55.82; H, 5.05. FABMS (matrix, NBA) m/e: 1339 (M 31). 2.3. General procedure for synthesis of unprotected glycoconjugated hypocrellins (11^15) Sodium methanolate in dry methanol (100 Wl, 0.1 N) was added to a solution of protected glycoconjugated hypocrellin (50 Wmol) in dry methanol (10 ml). The mixture was stirred for 2 h at room temperature in the dark. 2.3.1. 5(or 8)-Mono[(L-D-glucosyl)ethylthiyl]hypocrellins (11, 12) After reduction under vacuum, the crude product was puri¢ed by gel ¢ltration on a Sephadex LH20 column eluted with a mixture of chloroform/methanol (5:1, v/v). The pure product was crystallized from chloroform/methanol (34 mg, 90%). FT-IR (KBr disk): 3416, 1695, 1590, 1100, 1070, 1018, 897, 800 cm31 . 1 H-NMR (300 MHz, pyridine-d5 ) N: 15.96 (s, 1H, exchangeable with D2 O), 15.93 (s, 1H, exchangeable with D2 O), 7.98 (broad, 1H, exchangeable with D2 O), 7.50 (broad, 1H, exchangeable with D2 O), 6.90 (broad, 1H, exchangeable with D2 O), 6.46 (s, 1H), 5.88 (d, J = 8 Hz, 1H), 5.67 (m, 1H), 4.77 (m, 1H), 4.60 (m, 1H), 4.37 (m, 1H), 4.25 (m, 2H), 4.10 (s, 3H), 4.07 (s, 3H), 4.02 (d, J = 12 Hz, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.30(t, J = 7 Hz, 2H), 3.22 (d, J = 12 Hz, 1H), 2.98 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C38 H38 O15 S: C, 59.51; H, 5.00. Found: C, 59.26; H, 5.04. FABMS (matrix, NBA) m/e: 765 (M 31). 2.3.2. 5(or 8)-Mono[(L-D-glucosyl)ethylthiyl]8(or 5)-hydroxyethylthiyl hypocrellins (13, 14) After solvent reduction under vacuum, the crude product was puri¢ed by gel ¢ltration on a Sephadex LH20 column eluted with a mixture of chloroform/ methanol (4:1, v/v). The pure product was crystallized from chloroform/methanol (39 mg, 92%). FT-
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IR (KBr disk): 3414, 1690, 1592, 1100, 1070, 1018, 897, 800 cm31 . 1 H-NMR (300 MHz, pyridine-d5 ) N: 15.96 (s, 1H, exchangeable with D2 O), 15.90 (s, 1H, exchangeable with D2 O), 7.96 (broad, 1H, exchangeable with D2 O), 7.50 (broad, 1H, exchangeable with D2 O), 6.90 (broad, 1H, exchangeable with D2 O), 5.89 (d, J = 8 Hz, 1H), 5.66 (m, 1H), 4.78 (m, 1H), 4.60 (m, 1H), 4.37 (m, 1H), 4.26 (m, 2H), 4.13 (s, 3H), 4.08 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.96 (d, J = 12 Hz, 1H), 3.52 (t, J = 7 Hz, 2H), 3.30 (t, J = 7 Hz, 2H), 3.20 (d, J = 12 Hz, 1H), 3.10 (t, J = 7 Hz, 2H), 2.93 (t, J = 7 Hz, 2H), 2.38 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C40 H42 O16 S2 : C, 56.99; H, 5.03. Found: C, 57.22; H, 4.98. FABMS (matrix, NBA) m/e: 841 (M 31). 2.3.3. 5,8-Bis[(L-D-glucosyl)ethylthiyl]hypocrellin (15) After solvent reduction under vacuum, the crude product was puri¢ed by gel ¢ltration on a Sephadex LH20 column eluted with a mixture of chloroform/ methanol (2:1, v/v). The pure product was crystallized from chloroform/methanol (45 mg, 90%). FTIR (KBr disk): 3410, 1685, 1590, 1100, 1070, 1018, 897, 800 cm31 . 1 H-NMR (300 MHz, pyridine-d5 ) N: 15.93 (s, 1H, exchangeable with D2 O), 15.87 (s, 1H, exchangeable with D2 O), 7.90 (broad, 2H, exchangeable with D2 O), 7.00 (broad, 4H, exchangeable with D2 O), 5.90 (d, J = 8 Hz, 2H), 5.64 (m, 2H), 4.80 (m, 2H), 4.60 (m, 2H), 4.38 (m, 2H), 4.26 (m, 4H), 4.14 (s, 3H), 4.06 (s, 3H), 4.02 (s, 3H), 4.00 (d, J = 12 Hz, 1H), 3.96 (s, 3H), 3.32 (t, J = 7 Hz, 4H), 3.18 (d, J = 12 Hz, 1H), 2.94 (t, J = 7 Hz, 4H), 2.37 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C46 H52 O21 S2 : C, 54.97; H, 5.22. Found: C, 54.76; H, 5.19. FABMS (matrix, NBA) m/e: 1003 (M 31). 3. Results and discussion 3.1. Synthesis The synthesis of glycoconjugated hypocrellins via Ko«nigs^Kno«rr reaction [41] requires the condensation of hydroxyl group(s) in hypocrellin with sugars in which hydroxyl functions are protected by suitable groups. The latter may be easily cleaved to a¡ord the water-soluble compounds without even partial de-
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struction of the sugar moieties. This was achieved using acetyl as a protecting group. There are two phenolic hydroxyl groups (positions 4 and 9) in both HA and HB (Chart 1), and one aliphatic hydroxyl group (position 14) in HA. However, these two phenolic hydroxyl groups interact with the peri-carbonyl groups (positions 3 and 10) to form a strong intramolecular hydrogen bond. The aliphatic hydroxyl group is di¤cult to react with sugar due to its steric hindrance. It can be seen that the existing hydroxyl groups in HA and HB are not reactive to sugar moieties and the incorporation of reactive hydroxyl groups into hypocrellin is essential. Therefore 5 (or 8) monomercaptoethanol substituted hypocrellin B (2, 3) and 5,8-dimercaptoethanol substituted hypocrellin B (4) were prepared according to a previous procedure [26] to provide reactive hydroxyl groups for the condensation with 2,3,4,6-tetraacetyl-K-D-glucopyranosyl bromide (5) by the improved classical Ko«nigs^Kno«rr method [41]. The acetyl groups can be easily removed by treating the products with sodium methanolate in dry methanol [42]. Such a treatment does not change the L con¢guration of the anomeric carbon of sugars [43]. Watersoluble unprotected glycoconjugated hypocrellins were puri¢ed by gel chromatography on Sephadex LH20 or by anion exchange on amberlite resin MB-3. 3.1.1. Mono(glycosylated ethylthiyl) hypocrellins (11, 12) The condensation of 2,3,4,6-tetraacetyl-K-D-glucopyranosyl bromide (5) with monomercaptoethanol substituted hypocrellin B (2, 3) under Ko«nigs^Kno«rr conditions [41] gave mono(glycosylated ethylthiyl) hypocrellins 11 and 12 (Chart 2, Scheme 1). They were then puri¢ed by chromatography on a silica gel column and identi¢ed by 1 H-NMR spectroscopy. The glycoconjugated hypocrellins were obtained in an approx. 60% yield when the ratios of compound 2 (or 3), compound 5, iodine and Ag2 O were kept at 1:2:1:2. Whereas hypocrellins 6 and 7 bearing peracetyl monosaccharides are very soluble in nonpolar solvents exclusively, the unprotected monomonosaccharide compounds 11 and 12 obtained from 6 and 7, respectively, by treatment with sodium methanolate in dry methanol [42] are soluble in alcohol and weakly soluble in neutral water.
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3.1.2. Poly(glycosylated ethylthiyl) hypocrellins (13^15) In order to obtain mono- and bis[(2,3,4,6-tetraacetyl-L-D-glucosyl)ethylthiyl]hypocrellins (8^10), 5,8-dimercaptoethanol substituted hypocrellin B (4) was condensed with compound 5 in relative proportions of 1:3 under the same conditions as those used for the synthesis of the tetraacetylglycosyl derivatives 11 and 12. The overall yield of hypocrellins was 55%. When the amount of 5 decreased, the yield of the monosugar derivative increased. When the ratio of compound 4 and compound 5 decreased up to 1:4, the monosugar derivative was detected in an approx. 10% yield whereas the diglycosylated hypocrellin (15) was obtained in a 45% yield. The unprotected monomonosaccharide compounds 13 and 14 obtained from 8 and 9 by treatment with sodium methanolate in dry methanol are soluble in alcohol and weakly soluble in neutral aqueous solution. In contrast, the dimonosaccharide compound 15 is soluble in aqueous solution. The overall glycosylation yield of hypocrellins was in£uenced signi¢cantly by the solvent and the ratio of hypocrellins (2^4), sugar, iodine and Ag2 O. Ether and ether-dichloromethane mixtures were proved to be unsuitable for the glycosylation because of insuf¢cient solubility of the sugars and too small reaction rate respectively. Benzene on the other hand was suitable with regard to reaction rate whereas the solubility of hypocrellins was poor. Therefore the optimal ratio of 8:1 for benzene and dichloromethane was used. It is known that addition of iodine enhances the rate of glycosylation under Ko«nigs^Kno«rr conditions and may suppress the formation of byproducts, especially of orthoesters [44]. Glycosylation of hypocrellins without adding iodine or with catalytical amounts of iodine resulted in the formation of
Chart 2.
peracetylated glycosides in a yield decrease by approx. 6-fold. This disadvantageous reaction was successively and drastically shifted with increasing amounts of iodine towards the exclusive formation of peracetylated glycosides (6^10) at an I2 /Ag2 O/5 ratio of 1:2:2. 3.2. 1 H-NMR characterization 1
H-NMR spectroscopy (300 MHz) was used for the characterization of protected and unprotected compounds in CDCl3 and pyridine-d5 solution, respectively. Assignments of the resonances to individual protons are based on integration and selective homonuclear decoupling experiments. The protons at 5 and 8 positions in HB (1) have similar chemical shifts (6.44 and 6.46) owing to the pseudo-symmetry of the chromophore of hypocrellin B. Therefore mono-substituted derivatives 2 and 3 cannot be separated by chromatography or distin-
Scheme 1.
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guished by 1 H-NMR spectroscopy [26,45] and thus the glycosylated compounds 11 and 12. Similarly, compounds 13 and 14 cannot be separated by chromatography or distinguished by 1 H-NMR. However, these structural isomers have consistent properties such as photophysical and photochemical properties which are su¤cient for their usage as phototherapeutic agents. The resonance of the C-1 proton of the glucosyl substituents in all protected and unprotected hypocrellins (6^15) appears as a well-de¢ned doublet (J = 8 Hz) in CDCl3 and in deuterated pyridine. This indicates a pure L con¢guration of the anomeric carbon of the sugars [43]. 3.3. Electronic spectra The electronic spectra of all glycosylated hypocrellins are very similar to those of unglycosylated hypocrellins (Table 1), but the introduction of 5 or/and 8 mercapto substituents markedly in£uences the absorption of hypocrellins and induces a red shift of approx. 30 nm for monothiylation and 50 nm for dithiylation, respectively. The red absorption of glycosylated hypocrellins has been improved signi¢cantly. This facilitates the glycoconjugated hypocrellins obtained from thiylated precursor for the clinical applications in photodynamic therapy. 3.4. Determination of the quantum yield of singlet oxygen generated by glycoconjugated hypocrellins One of the advantages of hypocrellins is the high quantum yield of singlet oxygen generation by phoTable 1 UV-visible spectra and quantum yield (P) of photosensitized 1 O2 generation of hypocrellins in (a) CHCl3 and (b) CH3 OH Compound (solvent) a
1 2,3a 4a 6,7a 8,9a 10a 11,12b 13,14b 15b
Vnm (log O) 464 490 514 490 514 514 487 512 510
(4.34), (4.32), (4.28) (4.32), (4.28) (4.28) (4.31), (4.27) (4.26)
P 554 (4.07) 580 (4.04) 580 (4.04)
580 (4.02)
0.76 0.25 0.19 0.25 0.19 0.18 0.26 0.18 0.19
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tosensitization [46]. It has been previously reported that TEMPO, a nitroxide radical detectable by electron paramagnetic resonance (EPR), was generated from the reaction of TEMP with singlet oxygen (1 O2 ) [47]. In addition, the DPA-bleaching method was con¢rmed to be an e¤cient measurement for the quantum yield of 1 O2 generation [46]. The 1 O2 quantum yield by HB (1) has been determined to be 0.76 [46]. Taking into account this value as a reference and our consistent results obtained from TEMP spin trapping and DPA-bleaching methods, the quantum yield of 1 O2 generation (P) by hypocrellin derivatives (2^4, 6^15) has been determined (Table 1). These results demonstrate that the 1 O2 quantum yield of hypocrellin derivatives (2^4, 6^15) decreases as compared with that of the parent compound (1). Moreover, the decrease of 1 O2 quantum yield results from thiylation on hypocrellin B but not from glycosylation on compounds 2^4. 4. Conclusions In this paper, we describe the synthesis and characterization of a new class of perylenequinonoid compounds on which one or two glucopyranosyl groups were linked in order to increase the solubility of the perylenequinone in aqueous solution. The present strategy may be easily used for the preparation of other neutral glycoconjugated perylenequinonoid compounds. The absorption of the resulting products shifted red and the water solubility was enhanced signi¢cantly. Furthermore, the quantum yield of photosensitized singlet oxygen generation by these new hypocrellins was determined, demonstrating that glycoconjugated hypocrellins could photosensitize the generation of singlet oxygen in a decreased quantum yield. However, the red shift of these new compounds is still not adequate for them as PDT agents on solid tumors where the irradiation light should reach as much tissue as possible. The red absorption needs to be improved in the range of phototherapeutic window (600^900 nm) for the treatment of solid tumors. The decrease of the singlet oxygen quantum yield of these compounds might somewhat a¡ect the application of these derivatives in clinical trials. However, the in vivo phototherapeutic mechanism is very complex. Additional path-
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ways, such as the generation of protons from excited states of hypocrellin, may be involved in the antiviral and antitumor activity of hypocrellin [48] by inducing intracellular pH drop, which might o¡set the e¡ect of the decrease of singlet oxygen quantum yield. It is noteworthy that these glycoconjugated hypocrellins may be potentially used as photomarkers as the parent compound HA [49,50] and antiviral agents [8^11] as well as antitumor drugs for sur¢cial tumors. Acknowledgements We are grateful to the Chinese Natural Science Foundation of China (No. 29672033). References [1] D. Kessel, Photodynamic Therapy of Neoplastic Disease, CRC Press, Boston, MA, 1994. [2] B.W. Henderson, T. Dougherty, J. Photochem. Photobiol. 55 (1992) 145^147. [3] L.J. Jiang, Chin. Sci. Bull. (1990) 1608^1616. [4] L.J. Jiang, Chin. Sci. Bull. (1990) 1681^1690. [5] Z.J. Diwu, J.W. Lown, Photochem. Photobiol. 52 (1990) 609^616. [6] Z.J. Diwu, J.W. Lown, Pharmacol. Ther. 63 (1992) 1^35. [7] Z. Diwu, J. Photochem. Photobiol. 61 (1995) 529^539. [8] J.B. Hudson, J. Zhou, L. Harris, L. Yip, G.H.N. Towers, Photochem. Photobiol. 60 (1994) 253^255. [9] M.J. Fehr, S.L. Carpenter, Y. Wannemuehler, J.W. Petrich, Biochemistry 34 (1995) 15845^15848. [10] J.B. Hudson, V. Imperial, R.P. Haugland, Z. Diwu, J. Photochem. Photobiol. 65 (1997) 352^354. [11] J. Hirayama, K. Ikebuchi, H. Abe, K.W. Kwon, Y. Ohnishi, M. Horiuchi, M. Shinagawa, K. Ikuta, N. Kamo, S. Sekiquchi, Photochem. Photobiol. 66 (1997) 697^700. [12] J. Zhang, E.H. Cao, J.F. Li, T.C. Zhang, W.J. Ma, J. Photochem. Photobiol. B Biol. 43 (1998) 106^111. [13] W.G. Zhang, M. Weng, S.Z. Pang, M.H. Zhang, H.Y. Yang, H.X. Zhao, Z.Y. Zhang, J. Photochem. Photobiol. B Biol. 44 (1998) 21^28. [14] Y.M. Xu, H.X. Zhao, Z.Y. Zhang, J. Photochem. Photobiol. B Biol. 43 (1998) 41^46. [15] W. Zou, J.Y. An, L.J. Jiang, J. Photochem. Photobiol. B Biol. 33 (1996) 73^78. [16] J. Moan, Q. Peng, J.F. Evensen, K. Berg, A. Western, C. Rimington, Photochem. Photobiol. 46 (1987) 713^721. [17] J. Moan, Photochem. Photobiol. 39 (1984) 445^449. [18] N. Brasseur, H. Ali, R. Langlois, J.E. van Lier, Photochem. Photobiol. 47 (1988) 705^711.
[19] K.R. Adams, M.C. Berenbaum, R. Bonnett, A.N. Nizhnik, A. Salgado, M.A. Valles, J. Chem. Soc. Perkin Trans. I (1992) 1465^1470. [20] P. Margaron, M.-J. Gre¨goire, V. Scasnar, H. All, J.E. van Lier, Photochem. Photobiol. 63 (1996) 217^223. [21] G. Oenbrink, P. Ju«rgenlimke, D. Gabel, Photochem. Photobiol. 48 (1988) 451^456. [22] F. Ricchelli, G. Jori, G. Moreno, F. Vinzens, C. Salet, J. Photochem. Photobiol. B Biol. 6 (1990) 69^77. [23] Y.Z. Hu, J.Y. An, L.J. Jiang, J. Photochem. Photobiol. B Biol. 17 (1993) 195^201. [24] Y.Z. Hu, J.Y. An, L.J. Jiang, J. Photochem. Photobiol. A Chem. 70 (1993) 301^308. [25] Y.Z. Hu, J.Y. An, L.J. Jiang, J. Photochem. Photobiol. B Biol. 22 (1994) 219^227. [26] Y.Y. He, J.Y. An, W. Zou, L.J. Jiang, J. Photochem. Photobiol. B Biol. 44 (1998) 45^52. [27] Y.Y. He, J.Y. An, W. Zou, L. Jiang, J. Dyes Pigments 41 (1999) 93^100. [28] C. Kieda, M. Monsigny, Invasion Metastasis 6 (1986) 347^ 366. [29] N. Ono, M. Bougauchi, K. Maruyama, Tetrahedron Lett. 33 (1992) 1629^1632. [30] Ph. Maillard, C. Huel, M. Momenteau, Tetrahedron Lett. 33 (1992) 8081^8084. [31] K. Driaf, P. Krausz, B. Verneuil, Tetrahedron Lett. 34 (1993) 1027^1030. [32] A. Bourhim, S. Czernecki, P. Krausz, J. Carbohydr. Chem. 9 (1990) 761^765. [33] A. Bourhim, O. Gaud, R. Granet, P. Krausz, M. Spiro, Synlett (1992) 563^564. [34] K. Driaf, R. Granet, P. Krausz, M. Kaouadji, F. Thomasson, A.J. Chulia, B. Verneuil, M. Spiro, J.-C. Blais, G. Bolbach, Can. J. Chem. 74 (1996) 1550^1563. [35] P. Maillard, J.-L. Guerquin-Kern, M. Momenteau, J. Am. Soc. Chem. 111 (1989) 9125^9127. [36] D. Oulmi, P. Maillard, J.-L. Guerquin-Kern, C. Huel, M. Momenteau, J. Org. Chem. 60 (1995) 1554^1564. [37] P. Maillard, J.-L. Gruerquin-Kern, C. Huel, M. Momenteau, J. Org. Chem. 58 (1993) 2774^2780. [38] P. Maillard, C. Hery, M. Momenteau, Tetrahedron Lett. 38 (1997) 3731^3734. [39] K. Kohata, Y. Yamaguchi, H. Higashio, T. Odashima, H. Ishii, Chem. Lett. (1992) 477^480. [40] For a preliminary communication, see: Y.Y. He, J.Y. An, L.J. Jiang, Tetrahedron Lett. 39 (1998) 5069^5072. [41] L. Six, K.-P. RueM, M. Lie£a«nder, Tetrahedron Lett. 24 (1983) 1229^1232. [42] G. Zemple¨n, A. Gerecs, I. Hada¨csy, Ber. 69B (1936) 1827^ 1829. [43] A.F. Casy, PMR Spectroscopy in Medicinal and Biological Chemistry, Academic Press, London, 1971, pp. 330^384. [44] B. Helferich, E. Bohn, S. Winkler, Ber. 63B (1930) 989^ 998. [45] Z.J. Diwu, J. Zimmermann, T. Meyer, J.W. Lown, Biochem. Pharmacol. 47 (1994) 373^385.
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[49] E. Koumantakis, W.Q. Liu, A. Makrigiannakis, C. Relakis, E. Unso«ld, T.G. Papazoglou, J. Photochem. Photobiol. B Biol. 37 (1997) 96^100. [50] T.G. Papazoglou, W.Q. Liu, A. Katsamouris, C. Fotakis, J. Photochem. Photobiol. B Biol. 22 (1994) 139^144.
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Glycoconjugated hypocrellin: synthesis of [(L-D-glucosyl)ethylthiyl]hypocrellins and photosensitized generation of singlet oxygen Yu-Ying He, Jing-Yi An, Li-Jin Jiang * Institute of Photographic Chemistry, Academia Sinica, Beijing 100101, PR China Received 4 March 1999; received in revised form 30 June 1999; accepted 2 July 1999
Abstract In order to improve the water solubility and specific affinity for malignant tumors of hypocrellin, glycoconjugated hypocrellins have been synthesized using an improved Ko«nigs^Kno«rr reaction from mercaptoethanol substituted hypocrellin B and 2,3,4,6-tetra-O-acetyl-K-D-glucopyranosyl bromide precursors. Deprotection of glucose moieties allows the production of derivatives which had improved solubility in neutral aqueous solution and covered a range of amphiphilic character. The structures of these new protected and unprotected compounds were characterized by UV-Vis, IR, 1 H-NMR and MS data. The present strategy should prove applicable to the synthesis of other glycoconjugated perylenequinone compounds. In addition, the quantum yield of singlet oxygen generation photosensitized by these glycoconjugated hypocrellins has been determined. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Glycoconjugated hypocrellin; Photodynamic therapy; Photosensitizer; Amphiphilicity; Singlet oxygen
1. Introduction Photodynamic therapy (PDT) has received increasing attention in recent years as a new modality of selective treatment of solid tumor [1]. Although the mechanism of tumor cure is likely to require the interplay of several biological responses [2], a key point remains the ability of the photosensitizing agent to be retained to some extent by the tumor and, upon absorption of light, to generate short-lived toxic species, in particular singlet oxygen. Hypocrellins, including hypocrellin A (HA) and hypocrellin B (HB) (Chart 1), are new potential phototherapeutic agents [3^7]. Recent investigations demonstrated the
* Corresponding author. E-mail: [email protected]
photodynamic damages of hypocrellin and its derivatives to viruses, tumor cell, mixed phospholipids, pBR322 DNA and extracted cellular DNA [8^15]. These observations collectively provide a compelling rationale for the development of hypocrellin and its derivatives as PDT photosensitizers. Considerable e¡orts have been further devoted to develop photosensitizers with better tumor selectivity and higher phototherapeutic e¤ciency. Although the mechanism of photosensitizer retention by tumors is not well understood, the balance between lipophilicity and hydrophilicity is recognized as an important factor for photosensitizing e¤ciencies and tumor and cellular uptake [16^22]. However, hypocrellins are lipophilic compounds. Several amphiphilic hypocrellin derivatives have been studied so far, and most of them have ionic structure [23^27]. In the case of can-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 1 2 6 - 9
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Chart 1.
cer phototherapy, the linkages of photosensitizer with sugar moieties are of great importance because the sugar increases water solubility, membrane interaction and speci¢c a¤nity for malignant tumors [28]. Various strategies for the synthesis of glycoconjugated porphyrins, chlorines, porphines and phthalocyanines have been developed [29^39]. However, no e¡ort has been devoted to the synthesis of glycoconjugated hypocrellin derivatives until the work in our laboratory. In this paper, we report full experimental data [40] concerning the synthesis and characterization of hypocrellins with glucosyl substituents. In addition, the yield of singlet oxygen generation by these molecules is also determined. 2. Experimental section 2.1. General methods UV-visible spectra were performed on a HP 8541A spectrophotometer at room temperature. Infrared spectra were recorded on a BIO-RAD FTS165 spectrometer. 1 H-NMR spectra were obtained on a Varian XL-400 (300 MHz) and are reported in parts per million (N) relative to either tetramethylsilane (0.00 ppm) or CHCl3 (7.24 ppm). Coupling constants (J) are reported in hertz. Mass spectra were obtained on a FAB MS AEI-MS 50 Kratos spectrometer from a 3-nitrobenzyl alcohol (NBA) matrix with negative ion FAB used. Elemental analyses were carried out at the Institute of Chemistry, Academia Sinica, Beijing, China. Chemicals used were of reagent grade and used as supplied excepted where noted. Anhydrous benzene and dichloromethane (CH2 Cl2 ) were puri¢ed and then distilled under reduced pressure î molecular sieves (Fluka, activated and kept over 4 A at 500³C). Analytical thin-layer chromatography was performed on E. Merck silica gel 60 plate (2 mm). Liquid column chromatography was performed on
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E. Merck silica gel 60 (40^60 nm). Sephadex LH20 was purchased from Pharmacia LKB. The 1 O2 generation quantum yield by hypocrellins was determined by the average value from the following two methods: (1) measuring the EPR signal intensity of 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO) radical generated from the reaction of 2,2,6,6-tetramethyl-4-piperidone (TEMP) with 1 O2 generated by photosensitization of hypocrellins as a function of irradiation time; (2) measuring the absorption decrease at 374 nm of 9,10-diphenylanthracene (DPA) as a function of irradiation time. 2.2. General procedure for synthesis of protected glycoconjugated hypocrellins (6^10) Using 1 mmol monothiylated hypocrellin (2, 3) or 0.5 mmol dithiylated hypocrellin (4), 1 mmol iodine, 2 mmol freshly prepared Ag2 O, approx. 3 g powî , in 60 ml absolute bendered molecular sieve 4 A zene/dichloromethane mixture (8:1, v/v), and adding 2 mmol 2,3,4,6-tetraacetyl-K-D-glucopyranosyl bromide (5), the peracetylated monosaccharide derivatives 6^10 were obtained after 24 h at 20³C with vigorous stirring in the dark. The resulting mixture was ¢ltrated and evaporated under reduced pressure to a¡ord purple solid. This solid was subjected to thin layer chromatography (TLC) containing 1% citric acid and then eluted with acetone to obtain the condensed products. 2.2.1. 5(or 8)-Mono[(2,3,4,6-tetraacetyl-L-Dglucopyranosyl)ethylthiyl]hypocrellin (6, 7) The crude products were collected and puri¢ed on a silica gel containing 1% citric acid using a mixture of chloroform/ethyl acetate (2:1, v/v) a¡ording the title product (650 mg, 70%) after crystallization from dichloromethane/petroleum ether. FT-IR (KBr disk): 3439, 1744, 1680, 1595, 1229, 1030, 895, 800 cm31 . 1 H-NMR (300 MHz, CDCl3 ) N: 16.00 (s, 1H, exchangeable with D2 O), 15.96 (s, 1H, exchangeable with D2 O), 6.45 (s, 1H), 5.81 (d, J = 8 Hz, 1H), 5.62 (m, 1H), 4.72 (m, 1H), 4.54 (m, 1H), 4.35 (m, 1H), 4.20 (m, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 4.00 (d, J = 12 Hz, 1H), 3.97 (s, 3H), 3.83 (s, 3H), 3.30 (t, J = 7 Hz, 2H), 3.20 (d, J = 12 Hz, 1H), 2.95 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 2.12 (s, 3H), 2.08 (s, 6H), 2.02 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C46 H46 O19 S: C,
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59.09; H, 4.96. Found: C, 59.32; H, 4.89. FABMS (matrix, NBA) m/e: 933 (M 31). 2.2.2. 5(or 8)-Mono[(2,3,4,6-tetraacetyl-L-Dglucopyranosyl)ethylthiyl]-8(or 5)monohydroxyethylthiyl hypocrellins (8, 9) When 0.5 mmol compound 4, 0.5 mmol iodine, 1 mmol freshly prepared Ag2 O, approx. 1.5 g molecî and 1 mmol compound 5 in 30 ml ular sieve 4 A absolute benzene/dichloromethane mixture (8:1, v/ v) were stirred at 20³C in the dark for 24 h, the title compound was formed as a predominant product. The reaction mixture was evaporated under reduced pressure after ¢ltration, and then puri¢ed on a silica gel column containing 1% citric acid using a mixture of chloroform/ethyl acetate (2:1, v/v), a¡ording the title compound (252 mg, 50%) after crystallization from dichloromethane/petroleum ether. FT-IR (KBr disk): 3440, 1744, 1680, 1598, 1229, 1030, 895, 800 cm31 . 1 H-NMR (300 MHz, CDCl3 ) N: 15.98 (s, 1H, exchangeable with D2 O), 15.90 (s, 1H, exchangeable with D2 O), 5.80 (d, J = 8 Hz, 1H), 5.62 (m, 1H), 4.74 (m, 1H), 4.54 (m, 1H), 4.35 (m, 1H), 4.24 (m, 2H), 4.14 (s, 3H), 4.10 (s, 3H), 4.06 (s, 3H), 3.98 (s, 3H), 3.94 (d, J = 12 Hz, 1H), 3.50 (t, J = 7 Hz, 2H), 3.30 (t, J = 7 Hz, 2H), 3.16 (d, J = 12 Hz, 1H), 3.08 (t, J = 7 Hz, 2H), 2.93 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 2.14 (s, 3H), 2.08 (s, 6H), 2.04 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C48 H50 O20 S2 : C, 57.02; H, 4.99. Found: C, 57.24; H, 4.92. FABMS (matrix, NBA) m/e: 1009 (M 31). 2.2.3. 5,8-Bis[(2,3,4,6-tetraacetyl-LD-glucopyranosyl)ethylthiyl]hypocrellin (10) When 0.5 mmol compound 4, 1 mmol iodine, 2 mmol freshly prepared Ag2 O, 3 g molecular sieve î and 2 mmol compound 5 in 60 ml absolute 4 A benzene/dichloromethane mixture (8:1, v/v) were stirred at 20³C in the dark for 24 h, the title compound was produced as a predominant product. The reaction mixture was ¢ltrated, evaporated under reduced pressure, and then puri¢ed on a silica gel column containing 1% citric acid using a mixture of chloroform/ethyl acetate (2:1, v/v) a¡ording the title compound (335 mg, 50%). FT-IR (KBr disk): 3440, 1744, 1680, 1595, 1229, 1030, 895, 800 cm31 . 1 HNMR (300 MHz, CDCl3 ) N: 15.96 (s, 1H, exchangeable with D2 O), 15.90 (s, 1H, exchangeable with
D2 O), 5.82 (d, J = 8 Hz, 2H), 5.60 (m, 2H), 4.75 (m, 2H), 4.56 (m, 2H), 4.35 (m, 2H), 4.26 (m, 4H), 4.18 (s, 3H), 4.12 (s, 3H), 4.09 (s, 3H), 4.06 (s, 3H), 4.00 (d, J = 12 Hz, 1H), 3.30 (t, J = 7 HZ, 4H), 3.16 (d, J = 12 Hz, 1H), 2.92 (t, J = 7 Hz, 4H), 2.73 (s, 3H), 2.12 (s, 6H), 2.08 (s, 6H), 2.05(s, 6H), 2.01 (s, 6H), 1.96 (s, 3H). Anal. Calcd for C62 H68 O29 S2 : C, 55.51; H, 5.11. Found: C, 55.82; H, 5.05. FABMS (matrix, NBA) m/e: 1339 (M 31). 2.3. General procedure for synthesis of unprotected glycoconjugated hypocrellins (11^15) Sodium methanolate in dry methanol (100 Wl, 0.1 N) was added to a solution of protected glycoconjugated hypocrellin (50 Wmol) in dry methanol (10 ml). The mixture was stirred for 2 h at room temperature in the dark. 2.3.1. 5(or 8)-Mono[(L-D-glucosyl)ethylthiyl]hypocrellins (11, 12) After reduction under vacuum, the crude product was puri¢ed by gel ¢ltration on a Sephadex LH20 column eluted with a mixture of chloroform/methanol (5:1, v/v). The pure product was crystallized from chloroform/methanol (34 mg, 90%). FT-IR (KBr disk): 3416, 1695, 1590, 1100, 1070, 1018, 897, 800 cm31 . 1 H-NMR (300 MHz, pyridine-d5 ) N: 15.96 (s, 1H, exchangeable with D2 O), 15.93 (s, 1H, exchangeable with D2 O), 7.98 (broad, 1H, exchangeable with D2 O), 7.50 (broad, 1H, exchangeable with D2 O), 6.90 (broad, 1H, exchangeable with D2 O), 6.46 (s, 1H), 5.88 (d, J = 8 Hz, 1H), 5.67 (m, 1H), 4.77 (m, 1H), 4.60 (m, 1H), 4.37 (m, 1H), 4.25 (m, 2H), 4.10 (s, 3H), 4.07 (s, 3H), 4.02 (d, J = 12 Hz, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.30(t, J = 7 Hz, 2H), 3.22 (d, J = 12 Hz, 1H), 2.98 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C38 H38 O15 S: C, 59.51; H, 5.00. Found: C, 59.26; H, 5.04. FABMS (matrix, NBA) m/e: 765 (M 31). 2.3.2. 5(or 8)-Mono[(L-D-glucosyl)ethylthiyl]8(or 5)-hydroxyethylthiyl hypocrellins (13, 14) After solvent reduction under vacuum, the crude product was puri¢ed by gel ¢ltration on a Sephadex LH20 column eluted with a mixture of chloroform/ methanol (4:1, v/v). The pure product was crystallized from chloroform/methanol (39 mg, 92%). FT-
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IR (KBr disk): 3414, 1690, 1592, 1100, 1070, 1018, 897, 800 cm31 . 1 H-NMR (300 MHz, pyridine-d5 ) N: 15.96 (s, 1H, exchangeable with D2 O), 15.90 (s, 1H, exchangeable with D2 O), 7.96 (broad, 1H, exchangeable with D2 O), 7.50 (broad, 1H, exchangeable with D2 O), 6.90 (broad, 1H, exchangeable with D2 O), 5.89 (d, J = 8 Hz, 1H), 5.66 (m, 1H), 4.78 (m, 1H), 4.60 (m, 1H), 4.37 (m, 1H), 4.26 (m, 2H), 4.13 (s, 3H), 4.08 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.96 (d, J = 12 Hz, 1H), 3.52 (t, J = 7 Hz, 2H), 3.30 (t, J = 7 Hz, 2H), 3.20 (d, J = 12 Hz, 1H), 3.10 (t, J = 7 Hz, 2H), 2.93 (t, J = 7 Hz, 2H), 2.38 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C40 H42 O16 S2 : C, 56.99; H, 5.03. Found: C, 57.22; H, 4.98. FABMS (matrix, NBA) m/e: 841 (M 31). 2.3.3. 5,8-Bis[(L-D-glucosyl)ethylthiyl]hypocrellin (15) After solvent reduction under vacuum, the crude product was puri¢ed by gel ¢ltration on a Sephadex LH20 column eluted with a mixture of chloroform/ methanol (2:1, v/v). The pure product was crystallized from chloroform/methanol (45 mg, 90%). FTIR (KBr disk): 3410, 1685, 1590, 1100, 1070, 1018, 897, 800 cm31 . 1 H-NMR (300 MHz, pyridine-d5 ) N: 15.93 (s, 1H, exchangeable with D2 O), 15.87 (s, 1H, exchangeable with D2 O), 7.90 (broad, 2H, exchangeable with D2 O), 7.00 (broad, 4H, exchangeable with D2 O), 5.90 (d, J = 8 Hz, 2H), 5.64 (m, 2H), 4.80 (m, 2H), 4.60 (m, 2H), 4.38 (m, 2H), 4.26 (m, 4H), 4.14 (s, 3H), 4.06 (s, 3H), 4.02 (s, 3H), 4.00 (d, J = 12 Hz, 1H), 3.96 (s, 3H), 3.32 (t, J = 7 Hz, 4H), 3.18 (d, J = 12 Hz, 1H), 2.94 (t, J = 7 Hz, 4H), 2.37 (s, 3H), 1.96 (s, 3H). Anal. Calcd for C46 H52 O21 S2 : C, 54.97; H, 5.22. Found: C, 54.76; H, 5.19. FABMS (matrix, NBA) m/e: 1003 (M 31). 3. Results and discussion 3.1. Synthesis The synthesis of glycoconjugated hypocrellins via Ko«nigs^Kno«rr reaction [41] requires the condensation of hydroxyl group(s) in hypocrellin with sugars in which hydroxyl functions are protected by suitable groups. The latter may be easily cleaved to a¡ord the water-soluble compounds without even partial de-
235
struction of the sugar moieties. This was achieved using acetyl as a protecting group. There are two phenolic hydroxyl groups (positions 4 and 9) in both HA and HB (Chart 1), and one aliphatic hydroxyl group (position 14) in HA. However, these two phenolic hydroxyl groups interact with the peri-carbonyl groups (positions 3 and 10) to form a strong intramolecular hydrogen bond. The aliphatic hydroxyl group is di¤cult to react with sugar due to its steric hindrance. It can be seen that the existing hydroxyl groups in HA and HB are not reactive to sugar moieties and the incorporation of reactive hydroxyl groups into hypocrellin is essential. Therefore 5 (or 8) monomercaptoethanol substituted hypocrellin B (2, 3) and 5,8-dimercaptoethanol substituted hypocrellin B (4) were prepared according to a previous procedure [26] to provide reactive hydroxyl groups for the condensation with 2,3,4,6-tetraacetyl-K-D-glucopyranosyl bromide (5) by the improved classical Ko«nigs^Kno«rr method [41]. The acetyl groups can be easily removed by treating the products with sodium methanolate in dry methanol [42]. Such a treatment does not change the L con¢guration of the anomeric carbon of sugars [43]. Watersoluble unprotected glycoconjugated hypocrellins were puri¢ed by gel chromatography on Sephadex LH20 or by anion exchange on amberlite resin MB-3. 3.1.1. Mono(glycosylated ethylthiyl) hypocrellins (11, 12) The condensation of 2,3,4,6-tetraacetyl-K-D-glucopyranosyl bromide (5) with monomercaptoethanol substituted hypocrellin B (2, 3) under Ko«nigs^Kno«rr conditions [41] gave mono(glycosylated ethylthiyl) hypocrellins 11 and 12 (Chart 2, Scheme 1). They were then puri¢ed by chromatography on a silica gel column and identi¢ed by 1 H-NMR spectroscopy. The glycoconjugated hypocrellins were obtained in an approx. 60% yield when the ratios of compound 2 (or 3), compound 5, iodine and Ag2 O were kept at 1:2:1:2. Whereas hypocrellins 6 and 7 bearing peracetyl monosaccharides are very soluble in nonpolar solvents exclusively, the unprotected monomonosaccharide compounds 11 and 12 obtained from 6 and 7, respectively, by treatment with sodium methanolate in dry methanol [42] are soluble in alcohol and weakly soluble in neutral water.
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3.1.2. Poly(glycosylated ethylthiyl) hypocrellins (13^15) In order to obtain mono- and bis[(2,3,4,6-tetraacetyl-L-D-glucosyl)ethylthiyl]hypocrellins (8^10), 5,8-dimercaptoethanol substituted hypocrellin B (4) was condensed with compound 5 in relative proportions of 1:3 under the same conditions as those used for the synthesis of the tetraacetylglycosyl derivatives 11 and 12. The overall yield of hypocrellins was 55%. When the amount of 5 decreased, the yield of the monosugar derivative increased. When the ratio of compound 4 and compound 5 decreased up to 1:4, the monosugar derivative was detected in an approx. 10% yield whereas the diglycosylated hypocrellin (15) was obtained in a 45% yield. The unprotected monomonosaccharide compounds 13 and 14 obtained from 8 and 9 by treatment with sodium methanolate in dry methanol are soluble in alcohol and weakly soluble in neutral aqueous solution. In contrast, the dimonosaccharide compound 15 is soluble in aqueous solution. The overall glycosylation yield of hypocrellins was in£uenced signi¢cantly by the solvent and the ratio of hypocrellins (2^4), sugar, iodine and Ag2 O. Ether and ether-dichloromethane mixtures were proved to be unsuitable for the glycosylation because of insuf¢cient solubility of the sugars and too small reaction rate respectively. Benzene on the other hand was suitable with regard to reaction rate whereas the solubility of hypocrellins was poor. Therefore the optimal ratio of 8:1 for benzene and dichloromethane was used. It is known that addition of iodine enhances the rate of glycosylation under Ko«nigs^Kno«rr conditions and may suppress the formation of byproducts, especially of orthoesters [44]. Glycosylation of hypocrellins without adding iodine or with catalytical amounts of iodine resulted in the formation of
Chart 2.
peracetylated glycosides in a yield decrease by approx. 6-fold. This disadvantageous reaction was successively and drastically shifted with increasing amounts of iodine towards the exclusive formation of peracetylated glycosides (6^10) at an I2 /Ag2 O/5 ratio of 1:2:2. 3.2. 1 H-NMR characterization 1
H-NMR spectroscopy (300 MHz) was used for the characterization of protected and unprotected compounds in CDCl3 and pyridine-d5 solution, respectively. Assignments of the resonances to individual protons are based on integration and selective homonuclear decoupling experiments. The protons at 5 and 8 positions in HB (1) have similar chemical shifts (6.44 and 6.46) owing to the pseudo-symmetry of the chromophore of hypocrellin B. Therefore mono-substituted derivatives 2 and 3 cannot be separated by chromatography or distin-
Scheme 1.
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guished by 1 H-NMR spectroscopy [26,45] and thus the glycosylated compounds 11 and 12. Similarly, compounds 13 and 14 cannot be separated by chromatography or distinguished by 1 H-NMR. However, these structural isomers have consistent properties such as photophysical and photochemical properties which are su¤cient for their usage as phototherapeutic agents. The resonance of the C-1 proton of the glucosyl substituents in all protected and unprotected hypocrellins (6^15) appears as a well-de¢ned doublet (J = 8 Hz) in CDCl3 and in deuterated pyridine. This indicates a pure L con¢guration of the anomeric carbon of the sugars [43]. 3.3. Electronic spectra The electronic spectra of all glycosylated hypocrellins are very similar to those of unglycosylated hypocrellins (Table 1), but the introduction of 5 or/and 8 mercapto substituents markedly in£uences the absorption of hypocrellins and induces a red shift of approx. 30 nm for monothiylation and 50 nm for dithiylation, respectively. The red absorption of glycosylated hypocrellins has been improved signi¢cantly. This facilitates the glycoconjugated hypocrellins obtained from thiylated precursor for the clinical applications in photodynamic therapy. 3.4. Determination of the quantum yield of singlet oxygen generated by glycoconjugated hypocrellins One of the advantages of hypocrellins is the high quantum yield of singlet oxygen generation by phoTable 1 UV-visible spectra and quantum yield (P) of photosensitized 1 O2 generation of hypocrellins in (a) CHCl3 and (b) CH3 OH Compound (solvent) a
1 2,3a 4a 6,7a 8,9a 10a 11,12b 13,14b 15b
Vnm (log O) 464 490 514 490 514 514 487 512 510
(4.34), (4.32), (4.28) (4.32), (4.28) (4.28) (4.31), (4.27) (4.26)
P 554 (4.07) 580 (4.04) 580 (4.04)
580 (4.02)
0.76 0.25 0.19 0.25 0.19 0.18 0.26 0.18 0.19
237
tosensitization [46]. It has been previously reported that TEMPO, a nitroxide radical detectable by electron paramagnetic resonance (EPR), was generated from the reaction of TEMP with singlet oxygen (1 O2 ) [47]. In addition, the DPA-bleaching method was con¢rmed to be an e¤cient measurement for the quantum yield of 1 O2 generation [46]. The 1 O2 quantum yield by HB (1) has been determined to be 0.76 [46]. Taking into account this value as a reference and our consistent results obtained from TEMP spin trapping and DPA-bleaching methods, the quantum yield of 1 O2 generation (P) by hypocrellin derivatives (2^4, 6^15) has been determined (Table 1). These results demonstrate that the 1 O2 quantum yield of hypocrellin derivatives (2^4, 6^15) decreases as compared with that of the parent compound (1). Moreover, the decrease of 1 O2 quantum yield results from thiylation on hypocrellin B but not from glycosylation on compounds 2^4. 4. Conclusions In this paper, we describe the synthesis and characterization of a new class of perylenequinonoid compounds on which one or two glucopyranosyl groups were linked in order to increase the solubility of the perylenequinone in aqueous solution. The present strategy may be easily used for the preparation of other neutral glycoconjugated perylenequinonoid compounds. The absorption of the resulting products shifted red and the water solubility was enhanced signi¢cantly. Furthermore, the quantum yield of photosensitized singlet oxygen generation by these new hypocrellins was determined, demonstrating that glycoconjugated hypocrellins could photosensitize the generation of singlet oxygen in a decreased quantum yield. However, the red shift of these new compounds is still not adequate for them as PDT agents on solid tumors where the irradiation light should reach as much tissue as possible. The red absorption needs to be improved in the range of phototherapeutic window (600^900 nm) for the treatment of solid tumors. The decrease of the singlet oxygen quantum yield of these compounds might somewhat a¡ect the application of these derivatives in clinical trials. However, the in vivo phototherapeutic mechanism is very complex. Additional path-
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