Studies on the synthesis of two hydrophilic hypocrellin derivatives with enhanced absorption in the red spectral region and on their photogeneration of O2*- and O2(1(delta)g)

To improve hydrophilicity and photoactivity of the new type of photosensitizer, hypocrellin, two new derivatives were synthesized through a mild reaction method between hypocrellion B (HB) and ethanolamine in tetrahydrofuran (THF) and their molecular structures were characterized by IR, NMR, MS and UV-Vis spectrometry. In the molecular structures of the two derivatives, the peri-hydroxylated perylenquinone structure of the parent HB is preserved and their photoresponses at 600-900 nm (the red spectral region) are enhanced markedly (the molar absorption coefficients at 650 nm for the two new derivatives are EAHB1 log epsilon = 3.72 and EAHB2 log epsilon = 3.91, respectively. In contrast, the parent compound HB exhibits little absorption at 650 nm). Electron paramagnetic resonance (EPR) spin trapping measurement and a 9,10-diphenylanthracene (DPA) bleaching method were employed to investigate the photodynamic action of two chemicals in the presence of oxygen. The quantum yields of O2(1(delta)g) generation of EAHB1 and EAHB2 are 0.08 and 0.45, respectively; the relative quantum yields of (O2*-) generation of EAHB1 and EAHB2 are 0.15 and 0.76, respectively, with the parent compound HB as the standard.     

Synthesis, characterization and photodynamic activity of phenmethylamino-demethoxy-hypocrellin B

Two phenmethylamino hypocrellin B derivatives are novel photodynamic agents synthesized by a mild reaction between hypocrellin B and phenmethylamine. The red absorption of the photosensitizers is enlarged distinctly and the peri-hydroxylated perylenequinone structure of the parent HB is preserved. 9,10-diphenyl-anthracene (DPA) bleaching and electron paramagnetic resonance (EPR) spin trapping techniques were used to study the photodynamic activities of the phenmethylamino hypocrellin B derivatives in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O2*-) generated in the process of illumination of the phenmethylamino hypocrellin B in aerobic solution were observed. The photodamage of PMAHBs to MGC803 cancer cells was investigated in vitro. The results in vitro reveal that the phenmethylamino hypocrellin B derivatives show a much less significant decrease in cytotoxicity than that of their parent HB. It exhibits higher selectivity of light-orientation, which can decrease the damage to normal tissues by irradiating the tumor tissues, and so increases the drug safety.     

Photodynamic action of amino substituted hypocrellins: EPR studies on the photogenerations of active oxygen and free radical species

A novel method has been employed to prepare 2-butylamino-2-demethoxy hypocrellin A (BADMHA) and 2-butylamino-2-demethoxy hypocrellin B (BADMHB). Both compounds exhibit stronger absorption at the phototherapeutic window (600-900 nm). The spin trapping and spin counteraction studies have shown that they are both efficient generators of the active oxygen (1O2, O2*-) in the aerobic condition. Under the anaerobic condition they generate non-oxygen free radical (semiquinone radical anion), and the active oxygen mechanism of photosensitization can be converted into non-oxygen free radical mechanism with the depletion of oxygen. The quantum yields of 1O2 generation of BADMHA and BADMHB are 0.46 and 0.44, respectively. Both are lower than those of their parent compounds HA and HB. But the productions of superoxide anion are enhanced significantly compared with HA and HB, indicating they are both favorable Type I phototherapeutic agents.     

Synthesis, characterization and photodynamic activity of amino-substituted hypocrellin derivatives

Three new hypocrellin derivatives, amino- or amino acid-substituted on the side ring of hypocrellin B (HB), were synthesized by the reactions of HB with 3-methoxypropylamine, 6-aminohexanoic acid and gamma-amino-n-butyric acid, respectively. The structures of these compounds were characterized with proton nuclear magnetic resonance spectra, infrared spectra and mass spectra. The UV-visible absorption spectra, singlet oxygen-generating quantum yield and amphiphilicities of hypocrellin derivatives were measured and compared with HB, the parent compound. These derivatives showed strong absorption in the domain of the phototherapeutic window (600-900 nm) and improved amphiphilicity. HB and the derivatives were preliminarily tested for their photodynamic effects on human oral cavity epithelial carcinoma KB cell line in vitro. Two amino acid-substituted hypocrellins showed phototoxicity to the KB cell line. At an inhibitory dosage of 50% killing only 0.51 mumol L-1 compound 3 (or 0.88 mumol L-1 compound 2) and 0.5 J cm-2 irradiation were required. The hypocrellins exhibited some dark toxicity to the KB cell line. HB and amino acid-substituted hypocrellins showed lower dark toxicity to the KB cell line than amino-substituted hypocrellins in the assessment of cell survival.     

Preclinical Assessment of Hypocrellin B and Hypocrellin B Derivatives as Sensitizers for Photodynamic Therapy of Cancer: Progress Update

Abstract— Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomelic form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 [2-( N,N -dimethylami-no)-propylamine-HB]. Extinction coefficients at 630 nm (φ₆₃₀) are 6230, 6190 and 4800, respectively; and ¹O₂ quantum yields, φ, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA-R1 permanently ablates EMToVEd tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhi-murium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.     

Photodynamic properties of dipeptide-modified hypocrellin B derivatives: the role of tyrosine and tryptophan groups

Three long-wavelength absorbing dipeptide-modified hypocrellin B (HB) derivatives, Gly-HB, Tyr-HB, and Trp-HB, were prepared for application in photodynamic therapy (PDT). Their abilities to produce free radicals and singlet oxygen were compared in detail with EPR technique, and their binding interactions with calf thymus DNA (CT DNA) were studied by absorption spectra and DNA melting temperature measurements. Tyr-HB and Trp-HB distinguish themselves from Gly-HB and HB remarkably by their significantly improved efficiencies to generate semiquinone anion radicals, superoxide anion radicals, and hydroxyl radicals, as well as their affinity to CT DNA, as the result of the electron-donating properties and intercalating abilities of tyrosine and tryptophan groups. Tyr-HB and Trp-HB show remarkably enhanced photodamage capabilities on CT DNA than their parent HB in aerobic conditions. Moreover, they possess moderate photodamage abilities on CT DNA even in anaerobic conditions, indicating the role of Type I mechanism in their photodynamic behaviors.     

Electric magnetic resonance and spectrophotometry evidence on the photodynamic activity of a new perylenequinonoid pigment

Di-cysteine substituted hypocrellin B (DCHB) is a new water-soluble photosensitizer with significantly enhanced red absorption at wavelengths longer than 600 nm over the parent compound hypocrellin B (HB). The photosensitizing properties (Type I and/or Type II mechanisms) of DCHB have been investigated in dimethylsulfoxide (DMSO) and aqueous solution (pH 7.4) using electron paramagnetic resonance (EPR) and spectrophotometric methods. In anaerobic DMSO solution, the semiquinone anion radical of DCHB (DCHB^•−) is predominantly photoproduced via self-electron transfer between excited- and ground-state DCHB species. The presence of an electron donor significantly promotes the formation of the reduced form of DCHB. When a deoxygenated aqueous solution of DCHB and an electron donor are irradiated with 532 nm light, the hydroquinone of DCHB (DCHBH₂) is formed via the disproportionation of the first-formed DCHB^•− and second electron transfer to DCHB^•− from the electron donor. When oxygen is present, singlet oxygen (¹O₂), superoxide anion radical (O₂^•−) and hydroxyl radical (^•OH) are produced. The quantum yield of ¹O₂ generation by DCHB photosensitization is estimated to be 0.54 using Rose Bengal as a reference, a little lower than that of HB (0.76). The superoxide anion radical is also significantly enhanced by the presence of electron donors. Moreover, (O₂^•−) upon disproportionation generated H₂O₂ and ultimately the highly reactive ^•OH via the Haber-Weiss reaction pathway. The efficiency of (O₂^•−) generation by DCHB is obviously enhanced over that of HB. These findings suggest that the photodynamic actions of DCHB may proceed via Type I and Type II mechanisms and that this new photosensitizer retains photosensitizing activity after photodynamic therapy-oriented chemical modification.     

Synthesis and characterization of three novel amphiphilic aminated hypocrellins as photodynamic therapeutic agents

To improve the amphiphilicities and red absorption of the hypocrellins, three novel 2-amino-2-demethoxy-hypocrellins were synthesized by the mild reactions of hypocrellin B with 4-(2-amino-ethyl)morpholine, N,N-dimethylethylenediamine and 1-(2-amino-ethyl)piperazine, respectively. The structures of these derivatives were characterized with proton nuclear magnetic resonance, infrared and mass spectra (MS). The ultraviolet-visible absorption and fluorescence spectra of the derivatives were measured and the new amino-substituted hypocrellins showed strong absorption in the domain of the phototherapeutic window (600-900 nm). Their amphiphilicities evaluated via the partition coefficients between n-octanol and phosphate-buffered saline buffer improved remarkably. Electron paramagnetic resonance spin-trapping measurements were used to investigate the photodynamic action of the three compounds in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O2.-) generated by illuminating the hypocrellin derivatives in aerobic solution were observed.     

竹红菌素类光动力药物*

天然竹红菌素（包括竹红菌甲素和乙素）作为一种新型的光动力抗肿瘤药物,与血卟啉衍生物(HpD)相比,具有单一和确定的化学组成、易纯化、暗毒性低、兼具Type I和Type II双重敏化机制等优点。本文综述了竹红菌素类光敏剂在光动力疗法领域中近五年来的最新研究进展,包括竹红菌素的化学修饰、物理包裹、与生物大分子的相互作用以及动物细胞的体外和体内光动力性质研究,并对竹红菌素类光敏剂未来的研究方向作了展望。     

Effect of structural modification on photodynamic activity of hypocrellins

Aluminum ion complexed 5,8-di-Br-hypocrellin B is a new water-soluble perylenequinonoid derivative with enhanced absorption over hypocrellin B (HB) in the phototherapeutic window (600-900 nm). Electron paramagnetic resonance and 9,10-diphenyl-anthracene bleaching methods were used to investigate the photosensitizing activity of [AL2(5,8-di-Br-HB)Cl4]n in the presence of oxygen. Singlet oxygen, superoxide anion radical and hydroxyl radical can be generated by [AL2(5,8-di-Br-HB)CL4]n photosensitization. Singlet oxygen (1O2) is formed via energy transfer from triplet-state [AL2(5,8-di-Br-HB)CL4]n to ground-state molecular oxygen. 1O2 participates in the generation of a portion of superoxide anion radical (O2.-). Besides superoxide anion radical (O2.-) may originate from the electron transfer between the triplet-state [AL2(5,8-di-Br-HB)CL4]n and the ground-state molecular oxygen. OH is formed through the Fenton-Haber-Weiss reaction and the decomposition of DMPO-1O2 adduct. Compared with HB [AL2(5,8-di-Br-HB)CL4]n primarily remains and enhances the generation efficiency of superoxide anion radical and hydroxyl radical but that of singlet oxygen decreases.     

Photodynamic action of a water-soluble hypocrellin derivative with enhanced absorptivity in the phototherapeutic window II. Photosensitized generation of active oxygen species

Cysteine-substituted hypocrellin B (Cys-HB) is a water-soluble perylenequinonoid derivative with significantly enhanced absorptivity in the range of wavelength longer than 600 nm. Electron paramagnetic resonance (EPR) measurements, quenching experiments and 9,10-diphenyl-anthracene bleaching studies were used to investigate the photodynamic action of Cys-HB in the presence of oxygen. Illumination of Cys-HB solution, in the presence of oxygen, generated singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide. The accumulation of active oxygen species was transformed into that of the semiquinone anion radical with the depletion of oxygen, detected by the spin counteraction of TEMPO radical formed via the reaction of TEMP with singlet oxygen produced by Cys-HB photosensitization. Oxygen content, Cys-HB concentration and reaction environment affected the transformation and the competition between the Type I and Type II reactions. Compared with hypocrellin B (HB), Cys-HB primarily remained similar and slightly lower capability of active oxygen-generation, confirmed to be a favorable phototherapeutic agent.     

Studies on the photoinduced sulfonation of hypocrellins

Hypocrellin A (HA) and hypocrellin B (HB) are efficient phototherapeutic agents. Irradiation of HB with visible light in the presence of sodium sulfite in water-pyridine or water-N,N-dimethylformamide (1:1, v/v) solution gives hypocrellin B-5-sulfonic acid and hypocrellin B-5,8-disulfonic acid. HA reacts similarly under the same conditions. Electron spin resonance and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trapping studies indicate that this photoreaction is initiated by the photoinduced electron transfer between HB and sodium sulfite to produce the semiquinone radical anion of HB (HB^.−) and the sulfur trioxide radical anion (·SO₃^-). The intermediates (HB^.− and 5-SO₃⁻-HB^.−) produced from the photolysis process have also been observed in absorption spectra. The attack of ·SO₃⁻ on HB at the 5 and/or 8 positions to form the sulfonated products has been verified by quenching experiments. The effects of air and pH on the sulfonation reaction have been investigated. On the basis of the experimental evidence, the reaction pathway for the photoreaction is proposed.     

Spectroscopic studies and photodynamic actions of hypocrellin B in liposomes

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg L-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of 1O2 generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB.-) to oxygen. The disproportionation of O2.- can generate H2O2 and ultimately the highly reactive .OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O2.- directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O2.-) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.     

PHOTOCHEMISTRY OF HYPOCRELLIN A (Ⅶ)——PHOTO-INDUCED ONE-ELECTRON REDUCTION OF HYPOCRELLIN PIGMENTS

The photo-induced one-electron reduction of hypocrellin A (abbreviated as HA) and hy-pocrellin B (abbreviated as HB) in the presence of cysteine or ascorbic acid has been studied in this paper. The semiquinone radical anions of HA and HB obtained from the photo-reduction of HA and HB respectively, and the following protonation and disproportionation of these radical anions were detected via the ESR and UV-Vis spectra. Through comparison of their UV-Vis spectra with those of the chemically synthesized compounds of similar structures, it has been suggested that the metastable products formed after the acceptance of net two electrons and two protons in the photoreduction of HA and HB were tetrahydroxyl-per-ylene derivatives.     

Synthesis of a novel hypocrellin B derivative and its photogeneration of semiquinone anion radical

In order to improve the photosensitizing activity of HB further, the complex of 5,8-di-Br-HB with Al(3+) was first designed and synthesized in high yield. 5,8-di-Br-HB forms a 2:1 type (metal-ligand ratio) complex with Al(3+) measured by molar ratio and continuous variation methods. The new photosensitizer was characterized by UV-Vis, IR, 1H NMR and elemental analysis measurements. Based on the above experimental results, we first proposed a polymer-like structural model of the complex. The water-solubility and red absorption of the [Al(2)(5,8-di-Br-HB)Cl(4)](n) complex are both enhanced over hypocrellin B. In addition, the EPR and spectrophotometric measurements demonstrate that semiquinone anion radical of [Al(2)(5,8-di-Br-HB)Cl(4)](n) can be produced by [Al(2)(5,8-di-Br-HB)Cl(4)](n) photosensitization. The generation efficiency of ([Al(2)(5,8-di-Br-HB)Cl(4)](n))(.-) is almost equal to that of HB(.-). These results obtained indicated that [Al(2)(5,8-di-Br-HB)Cl(4)](n)was at least a favorable Type I phototherapeutic agent.     

竹红菌素在DMF-H2O混合体系中的电化学研究

本文研究了竹红菌素HA,HB以及13-SO₃Na-DDHA在DMF-H₂O(体积分数φ=1)混合体系中的电化学氧化还原性质。结果表明,它们都为两步单电子还原过程,反应机理为:HA,HB:HA(HB)+e+H⁺⇔HAH^·(HBH^·)+e⇔HAH^·油溶性。水溶性13-SO₃Na-DDHA:(第一步)13-SO₃Na-DDHA+e⇔13-SO₃Na-DDHA^-·(质子化)13-SO₃Na-DDHA^-·+H⁺⇔13-SO₃Na-DDHAH^·;(第二步)13-SO₃Na-DDHAH^·+e⇔13-SO₃Na-DDHAH^-·.并且,HA,HB的单电子还原半醒自由基非常稳定,而13-SO₃Na-DDHA单电子还原半醌自由基存在歧化反应,其速率常数k_f=0.408.     

Hypericin and hypocrellin induced apoptosis in human mucosal carcinoma cells.

Potent photosensitizers hypocrellin A (HA), hypocrellin B (HB) and hypericin (HY) are lipid-soluble perylquinone derivatives of the genus Hypericum and have a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by HA, HB and HY are still unclear. Moreover, no reports have mentioned cell apoptosis induced by HA, HB and HY in human nasopharyngeal carcinoma (NPC) and other mucosal cells. In this study, we attempt to clarify the photodynamic effects of HA, HB and HY compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human NPC cells as well as human mucosal colon and bladder cells. Using these cell lines we investigated few hallmarks of apoptotic commitments in a drug dose dependent manner. Tumor cells photo-activated with HA, HB and HY showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by all tumor cell lines as evidenced by the externalization of phosphatidylserine. Under apoptotic conditions, Western blot analysis of poly(ADP-ribose) polymerase, a caspases substrate, showed the classical cleavage pattern (116 to 85 kDa) associated with apoptosis in HA, HB and HY-treated cell lysates. In addition, 85 kDa cleaved product was blocked by the tetrapepdide caspase inhibitors such as DEVD-CHO or z-VAD-fmk. Both inhibitors protect tumor cells from apoptosis. These results demonstrate that tumor cell death induced by HA, HB and HY is mediated by caspase proteases. This study also identifies HB as a more potent and promising photosensitizer for the treatment of mucosal cancer cells.     

Photoinduced charge injection from excited triplet hypocrellin B into TiO_2 colloid in ethanol

Photosensitization of TiO2 colloid by hypocrellin B (HB),a natural photodynamic pigment with extremely high photosta-bility,has been studied by surface enhanced Raman spec-troscopy (SERS),laser flash photolysis and electron paramagnetic resonance (EPR) techniques.The photoseiisitization of TiO2 occurred practically from the excited triplet dye and the electron injection rate constant is 1.3×106 s-1.The influences of donor and acceptor on the electron injection were investigated.     

Interaction of Hypocrellin B or Mono-cysteine Substituted Hypocrellin B with CT-DNA by Spectral Methods

The interaction of the anticancer drug hypocrellin B (FIB) or the mono-cysteine substituted hypocrellin B (MCHB) and calf thymus deoxyribonucleic acid (CT-DNA) has been investigated using spectral methods. The results of UV-visible spectra show that the HB and MCHB could intercalate into the base-stacking domain of the CT-DNA double helix. The studies of fluorescence spectra and circular dichroism(CD) spectra also support the interacalation mechanism.     

Efficient Degradation of Aqueous Rhodamine B Irradiation Under Indoor Light Using a SiO2–Hypocrellin B Complex

An efficient method for the degradation of the dye Rhodamine B (RhB) is reported. A SiO₂–hypocrellin B (SiO₂–HB) complex was found to work as a catalyst to degrade RhB in aqueous solution at room temperature and atmospheric pressure, over the pH range 1–10, under indoor light conditions. This method is capable of removing 82.68% of RhB within 20 min at pH 9. The SiO₂–HB complex was characterized by both scanning electron microscopy and surface area analysis. The reaction progress was examined using ultrahigh pressure liquid chromatography/mass spectrometry and UV–visible spectroscopy. This process represents an efficient means of decontaminating dye‐containing wastewaters in either highly acidic or weakly alkaline environments.