WO2017051435A1 - Chlorin based compounds, a process for preparation thereof and use as photodynamic therapeutic agents and fluorescent probes - Google Patents

Chlorin based compounds, a process for preparation thereof and use as photodynamic therapeutic agents and fluorescent probes Download PDF

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WO2017051435A1
WO2017051435A1 PCT/IN2016/050318 IN2016050318W WO2017051435A1 WO 2017051435 A1 WO2017051435 A1 WO 2017051435A1 IN 2016050318 W IN2016050318 W IN 2016050318W WO 2017051435 A1 WO2017051435 A1 WO 2017051435A1
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chlorin
compounds
cells
solvent
formula
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Marydasan BETSY
Bollapalli MADHURI
Danaboyina Ramaiah
Kunchala Sridhar Rao
Chintalagiri Mohan Rao
Tavarekere Kalliah CHANDRASHEKAR
Suneesh Chandramathi KARUNAKARAN
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Council Of Scientific And Industrial Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to novel chlorin based compounds.
  • the invention also relates to process for preparation of chlorin based compounds of general Formula 1.
  • the compounds are useful as photodynamic therapy (PDT) agents (also referred to as sensitizers) in biological, biochemical and industrial applications such as in photodynamic therapeutics, diagnostics and as near infrared (NIR) fluorescence probes for cell imaging applications.
  • PDT photodynamic therapy
  • NIR near infrared
  • the present invention provides a process for the preparation of chlorin based compounds of the general Formula 1 and use of such sensitizers as NIR fluorescence probes in photodynamic therapeutic, diagnostic and biological, biochemical and industrial applications.
  • Photodynamic therapy is an emerging technique for the treatment of malignant or benign cancerous tissues.
  • This technique involves a two-step photochemical reaction resulting in the production of singlet oxygen (a reactive oxygen species) which can destroy the invasive cancerous or tumor tissues by oxidative damage.
  • This process involves a combination of photosensitizing agents, light of suitable wavelength and molecular oxygen. Initially the photosensitizer is injected to the patient and it is selectively accumulated in the target cancerous cells. In the second step the photosensitizer is irradiated with light of suitable wave length, which, in presence of molecular oxygen produces highly reactive oxygen, which is a key factor in photodynamic therapy.
  • the advantage in the present strategy over many other conventional therapies is its selectivity towards target cancerous cells and tissues. Irradiation of the photosensitizer loaded cancer cells with near infrared light using fiber optic technology minimizes damage to the adjacent healthy tissues.
  • References may be made to Kelly, J. F.; Snell, M. E. /. Urol, 115, 1976, 150-151. Kelly, J. F.; Snell, M. E.; Berenbaum, M. C. Br. J. Cancer,31, 1975, 237-244.Pariser, D. M.; Lowe, N. J.; Stewart, D. M,; Jarratt, M. T.; Lucky, A. W.; Pariser, R. J. /. Am. Acad.
  • NIR dyes have currently attracted much interest for the detection, diagnosis and treatment of cancerous cells in photodynamic therapy due to the transparency of tissues towards NIR light. Moreover, NIR dyes can be used as probes for near infrared fluorescence (NIRF) imaging.
  • NIRF near infrared fluorescence
  • a number of different photosensitizing compounds like methylene blue, rosebengal, and acridine are known to be efficient photodynamic therapy agents.
  • photosensitizers in clinical trials are cyclic tetrapyrroles or structural compounds of cyclic tetrapyrrole based chromophore, particularly porphyrin, chlorin, bacteriochlorin, expanded porphyrin and phthalocyanine compounds etc.
  • the reason behind is that the cyclic tetrapyrrolic compounds have an inherent similarity to the naturally occurring porphyrins present in living matter as a result they have little or no toxicity in the absence of light.
  • Chlorins are one of the main classes in the second generation photosensitizers which have a reduced macrocyclic aromatic ring-shaped tetrapyrrolic core.
  • porphyrins two of the exocyclic double bonds in opposite pyrrolic rings are cross-conjugated and are not required to maintain aromaticity. The reduction of one or both of these cross-conjugated bonds maintains the aromaticity and known as chlorin and bacteriochlorin respectively.
  • porphyrins, chlorins and bacterio-chlorins having 22, 20 and 18 ⁇ -electrons exhibits long-wavelength absorption and are capable of treating large and deeply seated tumors depending on wavelength region where the photosensitizers absorb light (Bownei al, 2002).
  • Vrouenraets M. B.; Visser, G. W.; Snow, G. B.; van Dongen, G. A. Anticancer Res., 23, 2003, 505-522.Nathan, T. R.; Whitelaw, D. E.; Chang, S.
  • Chlorins play a key role in many biological properties since the tetrapyrrolic core and metal coordinated cores play a vital role in many life processes.
  • Magnesium-containing chlorins are called chlorophylls, the central photosensitive pigment in chloroplasts.
  • Chlorins can form metal chelates with a large variety of metal ions, including: zinc, cobalt, copper and iron. References may be made to Mammana, A.; Asakawa, T.; Bitsch-Jensen, K.; Wolfe, A.; Chaturantabut, S.; Otani, Y.; Li, X.; Li, Z.; Nakanishi, K.; Balaz, M.; Ellestad, G.
  • Hematoporphyrin the first generation photosensitizers are mainly based on porphyrin macrocycle.
  • the hematoporphyrin compound (HpD) and its commercial variant Photofrin are facing some major drawbacks which include (a) they are a mixture of at least nine components, (b) preparation is highly sensitive to reaction conditions and (c) causes extended cutaneous photosensitivity. Prolonged patient photosensitivity (poor clearance) and lack of long wavelength absorption are the main drawbacks in the case of Photofrin.
  • photosensitizers include modified tetrapyrrolic porphyrins compounds, such as benzoporphyrin (Visudynes), chlorin (Temoporfins) and porphycene (ATMPn) and their metallated compounds having more intense long wavelength absorption. More recently another porphyrin based photosensitizer is 5, 10, 15, 20- tetrakis (meta-hydroxyphenyl) -chlorin which is commercially known as Foscan has been used clinically for the treatment of various cancers. The poor solubility in aqueous medium and the requirement of high concentrations made these dyes inconvenient for effective therapeutic applications.
  • the main objective of the present invention is to provide efficient chlorin based compounds and pharmaceutically acceptable compounds and thereof their use as PDT sensitizers in photodynamic therapeutic applications.
  • Yet another objective of the present invention is to provide efficient chlorin based compounds and or pharmaceutical acceptable compounds thereof, for use as NIR fluorescence probes in photodynamic diagnostic applications for the detection of tumors.
  • Yet another objective of the present invention is to provide efficient chlorin based compounds and or pharmaceutical acceptable compounds thereof, for use as near-infrared fluorescence sensors for biological, biochemical and industrial applications.
  • Yet another objective of the present invention is to provide chlorin based compounds of the general Formula 1 that can be used as NIR fluorescent labels in immunoassays.
  • step (iii) reacting chlorin compound obtained from step (i) or (ii) with a metal salt in a mixture of solvent under reflux at a temperature ranging between 60 to 70 °C for a period of time ranging between 10-15 h followed by washing to get a metal complex.
  • the base is selected from the group consisting of potassium carbonate, cesium carbonate, sodium-ter-butoxide and potassium-ter-butoxide.
  • the solvent is selected from the group consisting of dry methylene chloride, dry chloroform.
  • the neutralizing agent is selected from the group consisting of triethylamine, diisopropylethylamine, diethylamine.
  • the mixture of solvent used is methanol and chloroform.
  • FIG. 1 Represents structures of chlorin based dyes and their metallocompounds.
  • H&E staining of non-target spleen tissue showing no systemic-cytotoxicity of TDHPC.
  • chlorin compounds of the general Formula 1 have been synthesized.
  • Phenol groups were to render amphiphilicity to these dyes and hence increase the cell permeability and to bring about target specificity.
  • the first step of the synthetic procedure of the present invention we carried out the condensation reaction between 3,4-dimethoxybenzaldehyde and pyrrole in presence of trifluoroacetic acid gave the dipyrromethane compoundin 78% yield. Further, the corresponding porphyrin was obtained by the reaction of the intermediate dipyrromethane with 3,4-dimethoxybenzaldehyde, followed by oxidation using DDQ to yield 32 % of 3,4 dimethoxy tetraphenyl porphyrin.
  • the second step we adopted a general method for the synthesis of chlorin based photo sensitizers and are as follows.
  • the synthesis of all chlorin compounds has been achieved by using 3, 4 dimethoxytetraphenyl porphyrin as the starting material.
  • the porphyrin compounds were reduced to the corresponding methoxychlorin compound using tosyl hydrazine in pyridine, which was further hydrolyzed by reacting with boron tribromide in methylene chloride at -78 0 C.
  • the reaction was cooled to 0 °C and methanol was added. Further, the reaction mixture was neutralized with triethylamine and concentrated under reduced pressure to give an amorphous purple solid.
  • reaction time period used is preferably in the range of 15 to 24 h.
  • the yield of the chlorin compound of Formula 1 obtained is in the range of 85% to 90%.
  • the chlorin compound of Formula 1 exhibits an intense purple color having strong NIR absorption in the region 400 nm - 700 nm.
  • the chlorin compound of Formula 1 exhibits NIR emission in methanol at about 600 nm - 800 nm.
  • Yet another embodiment of the present invention is to provide efficient dyes based on chlorin and/or pharmaceutical acceptable compounds thereof, for use as near-infrared fluorescence sensors for biological and industrial applications.
  • the compounds of the Formula 1 are used in photodynamic therapy as NIR fluorescent sensors for the diagnosis of cancer.
  • Another embodiment of the present invention is to provide chlorin compounds of the general
  • Formula 1 that can be used as NIR fluorescent labels in immunoassays.
  • the present invention provides novel chlorin compounds represented by the general Formula 1 and /or compounds thereof as NIR sensitizers for photodynamic therapeutic and diagnostic, biological and industrial applications.
  • These chlorin compounds possess absorption (400-700 nm) and emission (600-750 nm) in the regions where biological chromophores do not absorb.
  • These compounds exhibited good solubility both in organic as well as aqueous medium since the substituents like hydroxyl units on these dyes render them amphiphilicity thereby improving their solubility in the aqueous media and cellular uptake and localization.
  • These dyes are having fluorescence quantum yields in the range 0.03-0.08 ⁇ 0.01. Nanosecond laser flash photolysis studies of these systems showed that they exhibit good triplet excited state quantum yield values of ca.
  • the present compound 5,10,15,20 Tetrakis (3,5-dihydroxyphenyl)chlorin(TDHPC) is novel and synthetically pure compound. It is superior to the other reported chlorin compounds, in terms of its photo physical properties, water solubility and exhibiting least cytotoxicity.
  • the procedure for its synthesis is very simple, economical with typical reaction yields of about 85%.
  • the dye showed excellent singlet oxygen generation, quantum yields of ⁇ 0.8 ⁇ 0.02 which is better than the second generation photosensitizer, Foscan (FDA approved for clinical use).
  • TDHPC hydroxyl-chlorin compound
  • TDHPC is its preferential retention in cancer cells.
  • Studies using mouse models has clearly demonstrated that TDHPC do not cause any damage to normal cells and tissues like liver, spleen etc, even when administered through intra-peritoneal or intravenous routes. We did not observe any extended photosensitivity in skin of the animals treated with TDHPC is not observed.
  • TDHPC The efficacy of TDHPC is predominantly due to its ability to generate good singlet oxygen in aqueous media. TDHPC did not affect normal tissues such as Liver, Spleen, and Kidney which makes it superior molecule compared to the existing / reported photosensitizers.
  • TDHPC is readily and preferentially taken up and retained in tumor cells in culture or in animal models. This dye does not have cytotoxic effect on its own, a property that is superior to the known compounds.
  • the tissues such as Liver, Kidney, Spleen etc of animals of treated with TDHPC did not show cytopathic effects even after irradiation.
  • the most common photo-toxic effect in skin after systemic administration of the sensitizers, as reported for many chlorin compounds is not seen with TDHPC.
  • TDHPC is a novel water soluble compound. It exhibits least cytotoxicity and has high phototoxic efficiency. This compound appears to better than the FDA approved Foscan in its photo physical and biological properties. While Foscan has a Molar extinction coefficient of 13000 at 651 nm, TDHPC has a molar coefficient of 15000 at 649 nm and a high triplet and singlet oxygen yields. Thus TDHPC is a better photosensitizer than Foscan. TDHPC has low cytotoxicity and does not affect non-target (normal) tissues or display extended photo-sensitivity of skin after systemic administration. All the cell lines used in the present study are commercially available.
  • the present invention also relates to chlorin derivatives of the general Formula 1 or pharmaceutically acceptable derivatives thereof, for use as NIR fluorescence probes in biochemical applications such as photodynamic therapy for the detection of cancer and other diseases.
  • the present invention also relates to chlorin derivatives of the general Formula 1 and/or their derivatives for both in vitro and in vivo photodynamic therapeutic treatment.
  • the present invention also relates to chlorin compounds of the general Formula 1 that can be used as NIR fluorescent labels in immunoassays.
  • Our present invention aims at the development of efficient NIR absorbing fluorescent probes based on chlorins for photodynamic and biological applications.
  • the hallmark property that we targeted in designing chlorin based photo sensitizer is its selectivity towards tumor cells compared to normal tissues.
  • the dye possesses significant fluorescent quantum yields with strong absorption in the long wavelength region, non toxic to normal tissues and in dark conditions but display better light induced toxicity (phototoxicity) and soluble in buffer at physiological pH or in aqueous medium.
  • phototoxicity light induced toxicity
  • these dye molecules are targeted specifically to cancerous cells, these molecules have immense application potential in biomedical and bio chemical applications.
  • Step2 Boron tribromide (7.4 mmol) was added to dry distilled methylene chloride (10 mL) and the mixture was cooled to -78 C.
  • the apparatus was fitted with a calcium chloride drying tube.
  • 5,10,15, 20-tetrakis (3,4-dimethoxyphenyl)chlorin(0.3 mmol) was dissolved in minimum volume of dry methylene chloride (10 ml), placed in a dropping funnel and slowly added over a period of 20 min. The mixture was stirred for 2 h at -78 C and then for 12 h at 25 C.
  • TDHPC 5,10,15,20-tetrakis(3,4- dihydroxyphenyl)chlorin)
  • SKOV3 human Ovarian Carcinoma cell line
  • the light source used for irradiation is PDT 1200 L (Waldmann, Germany)
  • All the culture media, fine biochemicals and kits used for biological experiments are from standard commercial suppliers like Sigma-Aldrich, USA., Molecular Probes, USA, Invitrogen, USA.
  • Cytotoxicity and phototoxicity of the chlorin compound of the general Formula 1 on SKOV-3 cells was investigated by using standard MTT (3, (4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay.
  • SKOV-3 cells (5 x 10 3 cells) were added to in each well of a 96-well micro titer plate in DMEM containing 10% fetal calf serum and incubated for 18 h in a humidified C0 2 -incubator at 37 °C. Increasing concentrations of chlorin compound (0, 2.5, 5, 10, 15 and 20 ⁇ in complete medium) was added.
  • the cells were subjected to light irradiation using Waldmann PDT 1200 L at 200 J/cm 2 and 50 mW/cm 2 .
  • Corresponding dark controls (cells treated with chlorin but not irradiated) were also set.
  • the MTT assay was performed after 24 h of incubation following exposure to light.
  • Half the inhibitory concentration (IC 50 ) value was determined from cell viability plot as a function of the concentration of chlorin compound. The IC 50 value was observed to be 13 ⁇ .
  • Fluorescence activated cell sorting for studying membrane damage: Propidium Iodide is non-vital fluorescent stain for nucleic acids in a cell. Cells whose membranes are damaged and dead will take up the dye and exhibit fluorescence, while live cells do not take up the dye and remain non-fluorescent.
  • SKOV-3 cells were treated with the chlorin photosensitizer for 1 h followed by irradiation at 200 J/cm 2 , 50 mW/cm 2 . Cells were trypsinized and treated with 30 ⁇ g/ml Propidium iodide and 200 ⁇ g/ml RNase A for 10 min.
  • Fluorescence activated cell sorting for cell cycle analysis: Human ovarian carcinoma cells-SKOV-3 were seeded in a 25 cm 2 flask at an appropriate density. After 24 h of incubation at 37° C, chlorin was added to the respective flasks and incubated for 1 h followed by irradiation (200 J/cm 2 , 50 mW/cm 2 ). The cells were further incubated for 12 h and 24 h post PDT following which they were harvested and fixed in 0.5 ml of 70% cold ethanol and incubated over night at 4 °C.
  • FACS Fluorescence activated cell sorting
  • Mitochondrial damage Human ovarian carcinoma cells SKOV-3 plated on cover slips were treated with chlorin photosensitizer for lh at 37 °C. The cells were subsequently irradiated at 200 J/cm 2 at a fluence of 50 mW/cm 2 . Mitotracker Red CMX Ros, a dye that localizes to mitochondria was added to the cells for 15 min. The cells were fixed in 4 % formaldehyde for 10 min, counterstained with DAPI and visualized by confocal microscopy. The mitochondria of control cells showed a fibrillar appearance while those of PDT treated cells showed a collapsed appearance, indicating mitochondrial damage of SKOV-3 cells after PDT with chlorin. Figure no. 12
  • SKOV-3xenograft-bearing mice Human ovarian carcinoma cells SKOV-3xenograft-bearing mice: Subcutaneous tumors were established by implanting 3-5 million SKOV-3 ovarian carcinoma cells into the flank of fox Nl athymic nude mice. Tumor size was measured using digital calipers and the tumor volume was calculated (mm 3 ) as (width) 2 x length x 0.5. Treatment was initiated when tumors reached a volume of 100-150 mm 3 . TDHPC was administered intravenously at 20 mg/kg body weight. The nude mice were irradiated (100 J/cm 2 and 100 mW/cm 2 ) 24 h after administration of the chlorin.
  • mice Prior to the light treatment mice were anesthetized with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg). The in vivo antitumor activity of the chlorin was evaluated in SKOV-3 xenografts in nude mice by measuring reduction in tumor volume.
  • Figure no. 13 Example 8
  • Chlorin based compounds represented by Formula 1 are novel and pure single substances.
  • Chlorin based compounds represented by Formula 1 possess absorption in the visible to near- infrared region (400-700 nm).
  • Chlorin based compounds represented by Formula 1 possess fluorescence emission in the near-infrared region (600-800 nm). Chlorin based compounds represented by Formula 1 possess emission quantum yields in the range 0.03-0.08 in aqueous media. Chlorin based compounds represented by Formula 1 possess triplet quantum yields in the range 0.5-0.85 in aqueous media. Chlorin based compounds represented by Formula 1 possess singlet oxygen quantum yields in the range 0.4-0.8 in aqueous media. Chlorin based compounds of the general Formula 1 can be used as NIR fluorescent labels in immunoassays. Chlorin based compounds of the general Formula 1 can be used for the detection and treatment of cancerous and non-cancerous diseases under physiological conditions. These novel dyes can be used as near-infrared fluorescence probes in biological and industrial applications.

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Abstract

The present invention deals with the synthesis of chlorin compounds of general Formula 1 as shown below and their compounds thereof, that can be used as photodynamic therapy (PDT) agents (also referred to as sensitizers) in biological, biochemical and industrial applications such as in photodynamic therapeutic, diagnostic and as near infrared (NIR) fluorescence probes for cell imaging applications. Formula (1) wherein, R3, R4 = OCH3or OH; R1, R2, R5 = H; M = 2H or metal selected from a group consisting of Zn, Cu, F. The present invention also provides a process for the preparation of chlorin compounds of the general Formula (1) and use of such sensitizers as NIR fluorescence probes in photodynamic therapeutic, diagnostic and biological, biochemical and industrial applications.

Description

CHLORIN BASED COMPOUNDS, A PROCESS FOR PREPARATION THEREOF AND
USE AS PHOTODYNAMIC THERAPEUTIC AGENTS AND FLUORESCENT PROBES
FIELD OF THE INVENTION
The present invention relates to novel chlorin based compounds. The invention also relates to process for preparation of chlorin based compounds of general Formula 1. The compounds are useful as photodynamic therapy (PDT) agents (also referred to as sensitizers) in biological, biochemical and industrial applications such as in photodynamic therapeutics, diagnostics and as near infrared (NIR) fluorescence probes for cell imaging applications.
Figure imgf000003_0001
Formula 1
wherein, R3, R4 = OCH3or OH; Rl5 R2, R5 = H; M = 2H or metal selected from a group consisting of Zn, Cu, Fe etc.
Particularly, the present invention provides a process for the preparation of chlorin based compounds of the general Formula 1 and use of such sensitizers as NIR fluorescence probes in photodynamic therapeutic, diagnostic and biological, biochemical and industrial applications. BACKGROUND AND PRIOR ART OF THE INVENTION
Cancer is one of the most challenging diseases in which cells become abnormal and divide without control. Photodynamic therapy (PDT) is an emerging technique for the treatment of malignant or benign cancerous tissues. This technique involves a two-step photochemical reaction resulting in the production of singlet oxygen (a reactive oxygen species) which can destroy the invasive cancerous or tumor tissues by oxidative damage. This process involves a combination of photosensitizing agents, light of suitable wavelength and molecular oxygen. Initially the photosensitizer is injected to the patient and it is selectively accumulated in the target cancerous cells. In the second step the photosensitizer is irradiated with light of suitable wave length, which, in presence of molecular oxygen produces highly reactive oxygen, which is a key factor in photodynamic therapy. References may be made to Sharman, W. M.; Allen, C. M.; Van Lier, J. E. Drug Discovery Today,4, l999, 507- 517. Juarranz, A.; Jaen, P.; Sanz-Rodriguez, F.; Cuevas, J.; Gonzalez, S. Clin. Transl. Oncol., 70,2008, 148-154. Dougherty, T. J. Photochem. Photobiol. \9 Ί , 45, 879; Diwu, S.; Zhang, C; Lown, J. W.. /. Photochem. Photobiol. A,66, \992, 99-112.; Sessler, J. L.; Nicolai, A. T.; Julian, D.; Pavel, A.; KarolinaJ.; Wataru, S.; Daniel, S.; Vincent, L.; Chris, B. B.; Andrew, T.; Bruno, A.; Greg, H.; Tarak, D. M.; Darren, J. M.; Vladimoar, K. Pure Appl. Chem, 71, 1999, 2009-2018.
The advantage in the present strategy over many other conventional therapies is its selectivity towards target cancerous cells and tissues. Irradiation of the photosensitizer loaded cancer cells with near infrared light using fiber optic technology minimizes damage to the adjacent healthy tissues. References may be made to Kelly, J. F.; Snell, M. E. /. Urol, 115, 1976, 150-151. Kelly, J. F.; Snell, M. E.; Berenbaum, M. C. Br. J. Cancer,31, 1975, 237-244.Pariser, D. M.; Lowe, N. J.; Stewart, D. M,; Jarratt, M. T.; Lucky, A. W.; Pariser, R. J. /. Am. Acad. Dermatol, 2003, 48, 227-32. Bonnett, R. Chem. Soc. Rev. ,24, 1995, 19-33. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. /. Natl. Cancer lnst.,90, \99 , 889-905.
Near-infrared (NIR) dyes have currently attracted much interest for the detection, diagnosis and treatment of cancerous cells in photodynamic therapy due to the transparency of tissues towards NIR light. Moreover, NIR dyes can be used as probes for near infrared fluorescence (NIRF) imaging. References may be made to Tsuda, A.; Nagamine, Y.; Watanabe, R.; Nagatani, Y.; Ishii, N. Nat. Chem., 2, 2010, 977-983; Ribeiro, A. O.; Tome, J. P. C; Neves, M. S.; Cavaleiro, J. A. S.; Iamamoto, Y.; Torres, T; Tetrahedron Lett., 2006, 47, 9177-9180. Pushpan, S. K.; Venkatraman, S.; Anand, V. G.; Sankar, J.; Parmeswaran, D.; Ganesan, S.; Chandrashekar, T. K. Curr. Med. Chem. - Anti -Cancer Agents, 2, 2002, 187-207; Achilefu, S.; Jimenez, H. N.; Dorshow, R. B.; Bugaj, J. E.; Webb, E. G.; Wilhelm, R. R.; Rajagopalan, R.;Johler, J.; Erion, J. L. /. Med. Chem. 2002 45, 2003-2015; Adarsh, N.; Shanmughasundaram, M.; Avirah, R.R.; Ramaiah, D. Chem. Eur. J., 18, 2012, 12655-12662.
In this context developing efficient NIR light absorbing photosensitizers or fluorescent probes based on porphyrins, chlorins, bacteriochlorins and phthalocyanines have attracted much attention in current research interests in photochemical and biological aspects. There are other squaraine as well as porphyrin based dyes developed by the inventors and showed their use for in PDT. These dyes possess favorable absorption in the near infrared region and exhibit significant triplet quantum yields. Among the various dyes developed, bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine and 2,4,6 Tri hydroxyl Phenyl Porphyrin (THPP) are found to be a promising compound in PDT as an effective NIR photo sensitizer in clinical applications through in vitro and in- vivo experiments. References may be made to Ramaiah, D.; Eckert, I.; Arun, K. T.; Weidenfeller, L.; Epe, B. Photochem. Photobiol, 2002, 76, 672-677; Suneesh, C. K.; SaneeshBabu, P. S.; Bollapalli, M.; Betsy, M.; Albish, K. P.; Asha S. N.; Sridhar Rao, K.; Srinivasan, A.; Chandrashekar, T. K.; Mohan Rao, Ch.; Radhakrishna, P.; Ramaiah, D.; ACS Chem. Biol., 2013, 8, 127-132.U.S. Pat. 6, 770, 787 B2; Ramaiah, D.; Joy, A.; Chandrasekar, N.; Eldho, N.V.; Das, S.; George, M.V; Photochem. Photobio, 1997, 65, 783-790. A number of different photosensitizing compounds like methylene blue, rosebengal, and acridine are known to be efficient photodynamic therapy agents. But a large number of photosensitizers in clinical trials are cyclic tetrapyrroles or structural compounds of cyclic tetrapyrrole based chromophore, particularly porphyrin, chlorin, bacteriochlorin, expanded porphyrin and phthalocyanine compounds etc. The reason behind is that the cyclic tetrapyrrolic compounds have an inherent similarity to the naturally occurring porphyrins present in living matter as a result they have little or no toxicity in the absence of light. These chlorin systems form a class of dyes possessing sharp and intense absorption bands in the visible to near infrared region. These reduced pyrrolic systems have good photo physical and photochemical properties compared to porphyrins and thus studied extensively since it would be highly suitable for a number of biological and industrial applications. References may be made to Nyst, H. J.; Wildeman, M. A.; Indrasari, S. R.; Karakullukcu, B.; van Veen, R. L.; Adham, M.; Stewart, F. A.; Levendag, P. C; Sterenborg, H. J.; Tan, I. B. Photodiagn. Photodyn., 9, 2012, 274-281. Moore, C. M.; Nathan, T. R.; Lees, W. R.; Mosse, C. A.; Freeman, A.; Emberton, M.; Bown, S. G. Lasers Surg. Med., 38, 2006, 356-363. Chlorins are one of the main classes in the second generation photosensitizers which have a reduced macrocyclic aromatic ring-shaped tetrapyrrolic core. In porphyrins, two of the exocyclic double bonds in opposite pyrrolic rings are cross-conjugated and are not required to maintain aromaticity. The reduction of one or both of these cross-conjugated bonds maintains the aromaticity and known as chlorin and bacteriochlorin respectively. As a result, there occurs a change in symmetry which shifts the Q-bands to red region, 640-800 nm, with high extinction coefficient. Thus porphyrins, chlorins and bacterio-chlorins having 22, 20 and 18 π-electrons exhibits long-wavelength absorption and are capable of treating large and deeply seated tumors depending on wavelength region where the photosensitizers absorb light (Bownei al, 2002). References may be made to Vrouenraets, M. B.; Visser, G. W.; Snow, G. B.; van Dongen, G. A. Anticancer Res., 23, 2003, 505-522.Nathan, T. R.; Whitelaw, D. E.; Chang, S. C; Lees, W. R.; Ripley, P. M.; Payne, H.; Jones, L.; Parkinson, M. C; Emberton, M.; Gillams, A. R.; Mundy, A. R.; Bown, S. G. /. Urol, 168, 2002, 1427-1432. Gayathri Devi, D.; Cibin, T. R.; Ramaiah, D.; Abraham, A. /. Photochem. Photobiol. B, 92,2008, 153-159.
Chlorins play a key role in many biological properties since the tetrapyrrolic core and metal coordinated cores play a vital role in many life processes. For e.g., Magnesium-containing chlorins are called chlorophylls, the central photosensitive pigment in chloroplasts. Chlorins can form metal chelates with a large variety of metal ions, including: zinc, cobalt, copper and iron. References may be made to Mammana, A.; Asakawa, T.; Bitsch-Jensen, K.; Wolfe, A.; Chaturantabut, S.; Otani, Y.; Li, X.; Li, Z.; Nakanishi, K.; Balaz, M.; Ellestad, G. A.; Berova, N. Bioorg. Med. Client., 16,2008, 6544- 6551. Zhang, P.; Steelant, W.; Kumar, M.; Scholfield, M. /. Am. Chem. Soc, 129,2001, 4526-4527. Gouterman, M. New York, Part A, Physical Chemistry, Vol 3, 1978.
Hematoporphyrin, the first generation photosensitizers are mainly based on porphyrin macrocycle. The hematoporphyrin compound (HpD) and its commercial variant Photofrin are facing some major drawbacks which include (a) they are a mixture of at least nine components, (b) preparation is highly sensitive to reaction conditions and (c) causes extended cutaneous photosensitivity. Prolonged patient photosensitivity (poor clearance) and lack of long wavelength absorption are the main drawbacks in the case of Photofrin.
To overcome these drawbacks second generation photosensitizersare developed. These include modified tetrapyrrolic porphyrins compounds, such as benzoporphyrin (Visudynes), chlorin (Temoporfins) and porphycene (ATMPn) and their metallated compounds having more intense long wavelength absorption. More recently another porphyrin based photosensitizer is 5, 10, 15, 20- tetrakis (meta-hydroxyphenyl) -chlorin which is commercially known as Foscan has been used clinically for the treatment of various cancers. The poor solubility in aqueous medium and the requirement of high concentrations made these dyes inconvenient for effective therapeutic applications. These factors forced the researchers to develop novel photosensitizing agents for medical or other biochemical applications. It is well established that the groups which increase the lipophilicity of a photosensitizer, for e.g. Hydroxyl groups, strongly affects the binding affinity of a photosensitizer toward the tumor or cancerous tissues and hence its cytotoxic activity. References may be made to Ben Dror, S.; Bronshtein, I.; Weitman, H.; Smith, K. M.; O'Neal, W. G.; Jacobi, P. A.; Ehrenberg, B. Eur. Biophys. J. ,38,2009, 847-855. Merchat et al., /. Photochem. Photobiol. B.Biol., 35: 149-157 (1996).
Our interest in this area is to design, synthesize and establish novel chlorin based photo sensitizers which can replace the existing clinically used photosensitizers like Foscan. In recent years a great variety of non-porphyrinic sensitizers like Methylene blue, phenothiaxinium dye, Rhodamine and Rosebengal, are being developed for use in PDT. However, its use as an in vivo photosensitizer is limited by its reduction by ubiquitous cellular enzymes to the colorless form, which is photodynamically inactive and in the case of Rhodamine 123, its poor phototoxicity since it possesses high fluorescence quantum yield with low triplet quantum yield. References may be made to O'Connor, A. E.; Gallagher, W. M.; Byrne, A. T. Photochem. Photobiol., 85, 2009, 1053-1074; Bonnett, R. Chem. Soc. Rev, 1995, 24, 19. Richmond, R. C; O'Hara, J. A. Photochem.Photobiol., 57, 1993, 291-297; Lin, C. W.; Shulok, J. R.; Kirley, S. D.; Cincotta, L.; Foley, J. W. Cancer Res., 51, 1991, 2710-2719.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to provide efficient chlorin based compounds and pharmaceutically acceptable compounds and thereof their use as PDT sensitizers in photodynamic therapeutic applications.
Yet another objective of the present invention is to provide efficient chlorin based compounds and or pharmaceutical acceptable compounds thereof, for use as NIR fluorescence probes in photodynamic diagnostic applications for the detection of tumors.
Yet another objective of the present invention is to provide efficient chlorin based compounds and or pharmaceutical acceptable compounds thereof, for use as near-infrared fluorescence sensors for biological, biochemical and industrial applications.
Yet another objective of the present invention is to provide chlorin based compounds of the general Formula 1 that can be used as NIR fluorescent labels in immunoassays. SUMMARY OF THE INVENTION
Accordingly, the pre la 1
Figure imgf000007_0001
Formula 1
wherein, R3, R4 = OCH3 or OH; Rl5 R2, R5 = H; M = 2H or metal selected from a group consisting of Zn, Cu, Fe.
In an embodiment of the invention, wherein the compounds are useful as NIR fluorescence probes in photodynamic applications for the detection and treatment of cancer and other diseases in human beings or animals. In another embodiment of the invention wherein a process for preparation of compounds of formula 1 wherein the process steps comprising:
(i) reducing the 3,4-dimethoxyphenylporphyrin by reacting with reducing agent in the presence of solvent and base under refluxing at a temperature ranging between 100 to 140 °C for a period ranging between 12 to 36 h to get 5,10,15,20-tetrakis(3,4- dimethoxyphenyl) chlorin;
(ii) reacting the chlorin compound obtained in step (i) with demethylating agent in a solvent at -78 °C for 2-3 h, and then for 10-12 h at 25-30 C, followed by cooling at a temperature ranging between 0-5 °C, quenching and neutralizing to get demethylated chlorin compound (TDHPC) of Formula 1 wherein R3, R4 = OH; R R2, R5 = H; M = 2H;
(iii) reacting chlorin compound obtained from step (i) or (ii) with a metal salt in a mixture of solvent under reflux at a temperature ranging between 60 to 70 °C for a period of time ranging between 10-15 h followed by washing to get a metal complex.
In yet another embodiment of the invention, wherein the reducing agent used for the reduction of porphyrin to chlorin is p-tosyl hydrazine.
In still another embodiment of the invention, wherein the solvent used is pyridine.
In an embodiment of the invention wherein the base is selected from the group consisting of potassium carbonate, cesium carbonate, sodium-ter-butoxide and potassium-ter-butoxide.
In an embodiment of the invention, wherein the dealkylating agent used for the demethylation is Boron tribromide.
In an embodiment of the invention wherein the solvent is selected from the group consisting of dry methylene chloride, dry chloroform.
In an embodiment of the invention, wherein the neutralizing agent is selected from the group consisting of triethylamine, diisopropylethylamine, diethylamine. In an embodiment of the invention, wherein the mixture of solvent used is methanol and chloroform.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Represents structures of chlorin based dyes and their metallocompounds.
FIG. 2 Absorption and fluorescence spectra of chlorin compounds of the general Formula 1 wherein, R3, R4 = OCH3; R2, Ri = H; M = 2H in chloroform.
R3, R4 = OCH3; R2, Ri = H; M = Zn in chloroform.
FIG. 3 Absorption and fluorescence spectra of chlorin compounds of the general Formula 1 inmethanol wherein, R3, R4 = OH; R2, Ri = H; M = 2H in methanol.
R3, R4 = OH; R2, Ri = H; M = Zn in methanol.
FIG. 4 Triplet absorption spectra of chlorin compounds of the general Formula 1 in DMSO wherein, R3, R4 = OCH3; R2, Ri = H; M = 2H in DMSO.
Laser Excitation wavelength, 355 nm. FIG. 5 Triplet absorption spectra of chlorin compounds of the general Formula 1 in methanol wherein, R3, R4 = OH; R2, Rj = H; M = 2H in methanol.
R3, R4 = OH; R2, Rj = H; M = Zn in methanol.
Laser Excitation wavelength, 355 nm. FIG. 6 Singlet oxygen generation efficiency of chlorin compounds of the general Formula 1 in DMSO wherein, R3, R4 = CH3; R2, Rj = H; M = 2H,
R3, R4 = CH3; R2, Rj = H; M = Zn in DMSO.
Excitation wavelength, 515 nm long pass filter. FIG. 7 Singlet oxygen generation efficiency of chlorin compounds of the general Formula 1 in methanol wherein, R3, R4 = OH; R2, Rj = H; M = 2H,
R3, R4 = OH; R2, Rj = H; M = Zn in methanol.
Excitation wavelength, 515 nm long pass filter. FIG. 8 Photototoxicity of the investigated chlorin compound of general Formula 1, wherein, R3, R4 = OH; Rj, R2, R5 = H; M = 2H in human ovarian cancer cells -SKOV-3 in dark and exposure to light (after PDT). The viability of cells is represented as a percentage over the controls. FIG. 9 Morphological images of the human ovarian cancer cells - SKOV-3, after incubation with the investigated chlorin compound of the general Formula 1 wherein, R3, R4 = OH; Rj, R2, R5 = H; M = 2H showing intake of chlorin compounds by the cancer cells within a short time period, here within one hour.
FIG. 10 Live Cell staining of human ovarian cancer cells - SKOV-3 cells using Propidium Iodide, after PDT with the investigated chlorin compound of the general Formula 1 wherein, R3, R4 = OH; Rl 5 R2, Rs = H; M = 2H.
FIG.ll Analysis of cell cycle progression in SKOV-3 cells after PDT with the investigated chlorin compound of the general Formula 1 wherein, R3, R4 = OH; Rl5 R2, R5 = H; M = 2H.
FIG.12 SKOV-3 cells showed an evident loss mitochondria (red stain) after PDT with the investigated chlorin compound of the general Formula 1 wherein, R3, R4 = OH; Rl5 R2, R5 = H; M = 2H.
FIG.13 In vivo photobiological studies with the investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl 5 R2, R4 = H; M = 2H with control and PDT subjected nude mice bearing SKOV-3 tumors (wherein chlorin intravenously administered). The tumor volumes (TV) are also indicated (NM-non-measurable).
FIG.14 Hematoxylin and Eosin staining of tumor sections after photodynamic therapy with the Investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl 5 R2, R4 = H; M = 2H.
FIG.15 Hematoxylin and Eosin staining of liver sections after photodynamic therapy with the investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl5 R2, R4 = H; M = 2H. H&E staining of non-target liver tissue showing no systemic-cytotoxicity of TDHPC.
FIG.16 Hematoxylin and Eosin staining of spleen sections after photodynamic therapy with the Investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl 5 R2, R4 = H; M = 2H. H&E staining of non-target spleen tissue showing no systemic-cytotoxicity of TDHPC. However, there is a small increase in the number of megakaryocytes after TDHPC treatment. FIG.17 Hematoxylin and Eosin staining of kidney sections after photodynamic therapy with the Investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl 5 R2, R4 = H; M = 2H. H&E staining of non-target kidney tissue showing no systemic -cytotoxicity of TDHPC. FIG.18 Hematoxylin and Eosin staining of heart sections after photodynamic therapy with the Investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl 5 R2, R4 = H; M = 2H.H&E staining of non-target Heart tissue showing no systemic-cytotoxicity of TDHPC.
FIG.19 Hematoxylin and Eosin staining of lung sections after photodynamic therapy with the investigated chlorin compounds of the general Formula 1 wherein, R3, R4 = OH; Rl5 R2, R4 = H; M = 2H. H&E staining of non-target lung tissue showing no systemic-cytotoxicity of TDHPC.
DETAILED DESCRIPTION OF THE INVENTION In the present invention, chlorin compounds of the general Formula 1 have been synthesized.
Phenol groups were to render amphiphilicity to these dyes and hence increase the cell permeability and to bring about target specificity.
As the first step of the synthetic procedure of the present invention, we carried out the condensation reaction between 3,4-dimethoxybenzaldehyde and pyrrole in presence of trifluoroacetic acid gave the dipyrromethane compoundin 78% yield. Further, the corresponding porphyrin was obtained by the reaction of the intermediate dipyrromethane with 3,4-dimethoxybenzaldehyde, followed by oxidation using DDQ to yield 32 % of 3,4 dimethoxy tetraphenyl porphyrin. As the second step, we adopted a general method for the synthesis of chlorin based photo sensitizers and are as follows. The synthesis of all chlorin compounds has been achieved by using 3, 4 dimethoxytetraphenyl porphyrin as the starting material. The porphyrin compounds were reduced to the corresponding methoxychlorin compound using tosyl hydrazine in pyridine, which was further hydrolyzed by reacting with boron tribromide in methylene chloride at -78 0 C. The reaction was cooled to 0 °C and methanol was added. Further, the reaction mixture was neutralized with triethylamine and concentrated under reduced pressure to give an amorphous purple solid. The compound was again separated using ethyl acetate-water and was re -crystallized from a mixture of methanol and chloroform to give the hydroxy substituted chlorin compounds of the general Formula 1. In an embodiment the reaction time period used is preferably in the range of 15 to 24 h.
In another embodiment the yield of the chlorin compound of Formula 1 obtained is in the range of 85% to 90%. In yet another embodiment, the chlorin compound of Formula 1 exhibits an intense purple color having strong NIR absorption in the region 400 nm - 700 nm. In yet another embodiment the chlorin compound of Formula 1 exhibits NIR emission in methanol at about 600 nm - 800 nm.
Yet another embodiment of the present invention is to provide efficient dyes based on chlorin and/or pharmaceutical acceptable compounds thereof, for use as near-infrared fluorescence sensors for biological and industrial applications.
In yet another embodiment of the invention, the compounds of the Formula 1 are used in photodynamic therapy as NIR fluorescent sensors for the diagnosis of cancer. Another embodiment of the present invention is to provide chlorin compounds of the general
Formula 1 that can be used as NIR fluorescent labels in immunoassays.
The present invention provides novel chlorin compounds represented by the general Formula 1 and /or compounds thereof as NIR sensitizers for photodynamic therapeutic and diagnostic, biological and industrial applications. These chlorin compounds possess absorption (400-700 nm) and emission (600-750 nm) in the regions where biological chromophores do not absorb. These compounds exhibited good solubility both in organic as well as aqueous medium since the substituents like hydroxyl units on these dyes render them amphiphilicity thereby improving their solubility in the aqueous media and cellular uptake and localization. These dyes are having fluorescence quantum yields in the range 0.03-0.08 ± 0.01. Nanosecond laser flash photolysis studies of these systems showed that they exhibit good triplet excited state quantum yield values of ca. 0.5- 0.85± 0.03. These chlorin systems showed quantum yields of singlet oxygen generation, φ O2) of ca. 0.46-0.8 ± 0.02, depending on the nature of the substituent present on the mesophenyl ring and the inserted metal. Hence these compounds are ideal candidates for application as NIR PDT agents and fluorescence sensors for medicinal applications in biology. The inventor also investigated the in vitro photodynamic activity of these compounds using MTT assay in an ovarian carcinoma SKOV-3 and the cytotoxicity efficacy was determined in terms of IC50 value. The chlorin dye shows no toxicity in the dark and selectively localizes to the tumor tissues. The mechanism of the biological activity of these systems has been evaluated through fluorescence activated cell sorting (FACS) analysis, cell morphological changes and MitoTracker Red staining experiments. Moreover, we have also investigated preliminary in vivo anti-tumor activity in nude mice models and which showed the considerable visual reduction of tumor within two to three weeks, when compared to the control experiments. Accordingly, these chlorin compounds are extremely useful as NIR PDT fluorescence sensors in photodynamic, therapeutic and diagnostic, biological and industrial applications.
The present compound, 5,10,15,20 Tetrakis (3,5-dihydroxyphenyl)chlorin(TDHPC) is novel and synthetically pure compound. It is superior to the other reported chlorin compounds, in terms of its photo physical properties, water solubility and exhibiting least cytotoxicity. The procedure for its synthesis is very simple, economical with typical reaction yields of about 85%. The dye showed excellent singlet oxygen generation, quantum yields of ~ 0.8 ± 0.02 which is better than the second generation photosensitizer, Foscan (FDA approved for clinical use).
The molar extinction of the hydroxyl-chlorin compound (TDHPC) is 15000 at 649 nm, compared to commercially available Foscan which is 13000 at 651 nm. Furthermore, TDHPC showed a good triplet excited state quantum yield of ca.0.84 ± 0.02 and singlet oxygen generation efficiency of cfl.0.80±0.03. TDHPC showed negligible dark toxicity and high phototoxic effect in vitro activity when compared to the clinically used sensitizers, Photofrin. The studies indicated that the biological activity of the TDHPC can be attributed predominantly to the enhanced singlet oxygen yield, which kills cancer cells with high efficiency.
One of the most promising features of TDHPC is its preferential retention in cancer cells. The preliminary results indicated TDHPC is localized to cytoplasm in the cells. The studies in vivo using nude mice models, demonstrate that the TDHPC when injected intra-tumorally, completely reduced the xenografted SKOV 3 ovarian tumour of -150 mm3 to a non-measurable level within 2 weeks post irradiation, without formation of any scars. Studies using mouse models has clearly demonstrated that TDHPC do not cause any damage to normal cells and tissues like liver, spleen etc, even when administered through intra-peritoneal or intravenous routes. We did not observe any extended photosensitivity in skin of the animals treated with TDHPC is not observed.
The efficacy of TDHPC is predominantly due to its ability to generate good singlet oxygen in aqueous media. TDHPC did not affect normal tissues such as Liver, Spleen, and Kidney which makes it superior molecule compared to the existing / reported photosensitizers.
TDHPC is readily and preferentially taken up and retained in tumor cells in culture or in animal models. This dye does not have cytotoxic effect on its own, a property that is superior to the known compounds. In addition, the tissues such as Liver, Kidney, Spleen etc of animals of treated with TDHPC did not show cytopathic effects even after irradiation. The histopathological evaluations reported in here stand testimony to this effect. The most common photo-toxic effect in skin after systemic administration of the sensitizers, as reported for many chlorin compounds is not seen with TDHPC.
TDHPC is a novel water soluble compound. It exhibits least cytotoxicity and has high phototoxic efficiency. This compound appears to better than the FDA approved Foscan in its photo physical and biological properties. While Foscan has a Molar extinction coefficient of 13000 at 651 nm, TDHPC has a molar coefficient of 15000 at 649 nm and a high triplet and singlet oxygen yields. Thus TDHPC is a better photosensitizer than Foscan. TDHPC has low cytotoxicity and does not affect non-target (normal) tissues or display extended photo-sensitivity of skin after systemic administration. All the cell lines used in the present study are commercially available. They are obtained either from American Type Culture Collection (ATCC), USA or National Center for Cell Science, Pune, India and maintained as per the company's specifications at Tissue Culture facility at CCMB, Hyderabad, India. The light source used for irradiation is PDT 1200 L (Waldmann, Germany) .All the culture media, fine biochemicals and kits used for biological experiments are from standard commercial suppliers like Sigma-Aldrich, USA, Molecular Probes, USA, Invitrogen, USA.
The present invention also relates to chlorin derivatives of the general Formula 1 or pharmaceutically acceptable derivatives thereof, for use as NIR fluorescence probes in biochemical applications such as photodynamic therapy for the detection of cancer and other diseases.
The present invention also relates to chlorin derivatives of the general Formula 1 and/or their derivatives for both in vitro and in vivo photodynamic therapeutic treatment.
The present invention also relates to chlorin compounds of the general Formula 1 that can be used as NIR fluorescent labels in immunoassays.
Our inventions on photo physical properties of tetradihydroxychlorin showed absorption in the range from 400-700 nm and fluorescence emission in the range 600-800 nm. These compounds exhibited good solubility both in organic as well as aqueous medium. These dyes are having fluorescence quantum yields in the range 0.03-0.08 ± 0.01. Nanosecond laser flash photolysis studies of these systems showed that they exhibit good triplet excited state quantum yield values of ca. 0.5- 0.85+ 0.03. These chlorin systems showed quantum yields of singlet oxygen generation, φ O2) of ca. 0.46-0.8 ± 0.02, depending on the nature of the substituent present on the mesophenyl ring and the inserted metal.
We investigated the in vitro cytotoxic and phototoxic activity of these compounds by standard MTT assay using various cancer cell lines such as IMR- 32, MCF-7, MDA MB-231 and SKOV-3. The chlorin compounds are less cytotoxic and showed better phototoxic activity in all the tested cell lines showed when compared to the clinically used sensitizer Photofrin. Data on a highly tumorigenic human ovarian cell line -SKOV-3, is presented herewith. The phototoxic effects of these systems has been evaluated through fluorescence activated cell sorting (FACS) analysis, cell morphological changes and Mitotracker red staining experiments. Moreover, we have also investigated preliminary in vivo anti-tumor activity in nude mice models and which showed the considerable visual reduction of tumor, when compared to the control animals.
In the present invention we have synthesized novel chlorin compounds and demonstrated their potential as photodynamic agents for biological and biochemical applications. Hence these compounds can be used as tumor targeting NIR fluorescent probes.
Our present invention aims at the development of efficient NIR absorbing fluorescent probes based on chlorins for photodynamic and biological applications. We have successfully synthesized chlorin based molecules having hydroxyl groups in its periphery positions which exhibit absorption and emission in the NIR region. These compounds showed good solubility in aqueous medium and favorable fluorescence intensity hence can accelerate their cellular uptake.
The hallmark property that we targeted in designing chlorin based photo sensitizer is its selectivity towards tumor cells compared to normal tissues. In addition to that the dye possesses significant fluorescent quantum yields with strong absorption in the long wavelength region, non toxic to normal tissues and in dark conditions but display better light induced toxicity (phototoxicity) and soluble in buffer at physiological pH or in aqueous medium. Moreover, since these dye molecules are targeted specifically to cancerous cells, these molecules have immense application potential in biomedical and bio chemical applications.
EXAMPLES
The following examples are given by way of illustration and therefore should not be construed to limit the scope of present invention.
Example 1
Preparation of the chlorin compound of general Formula 1
Stepl. 3,4-Dimethoxyphenylporphyrin (3.2 mmol) and p-tosyl hydrazine(32 mmol) were dissolved in dry pyridine (500 mL) in two neck round bottom flask , and activated potassium carbonate (1.3 mmol) was added. The reaction mixture was allowed to reflux at 120° C under argon atmosphere for 24 h. After 2 h, the reaction was monitored by UV-visible spectroscopy until the peak at 650nm which corresponds to chlorin was completely formed. After completion of the reaction, the reaction mixture was cooled and extracted using dichloromethane. The organic layer was concentrated, dried and was poured onto a pad of alumina (50 mm maximum diameter 150 mm length) and eluted with methylene chloride (1L). The solvent was removed under reduced pressure to give a black solid, which was column chromatographed over silica gel. Elution of the column with chloroform gave the corresponding chlorin. Yield; 83% mp>300° C; *H NMR (300 MHz, CDC13, 30° C, TMS): δ (ppm) - 1.44 (s, 2H), 3.95 (s, 12H), 4.11 (s, 6H), 4.14 (s, 6H), 4.2 (s, 4H), 7.16 (t, 4H, J= 15Hz), 7.39 (d, 4H, J= 5Hz ) ,7.67(d, 4H, J=5Hz), 8.24 (d, 2H, J= 15Hz), 8.48 (s, 2H), 8.64 (d, 2H, J= 5Hz). 13C NMR (125 MHz, CDC13, TMS) δ 206.9, 167.7, 152.6, 148.7, 147.1, 140.97, 135.6, 134.7, 131.8, 128.0, 126.6, 124.6, 123.3, 122.3, 117.6, 109.4, 77.0, 56.1. IR (KBr) :vmax 3324, 2821, 1680, 1516, 1261, 1246, 1230, 1138, 1028 cm 1. FAB-MS: m/z= Calculated 857.4; Found 857.35(M) +
Step2. Boron tribromide (7.4 mmol) was added to dry distilled methylene chloride (10 mL) and the mixture was cooled to -78 C. The apparatus was fitted with a calcium chloride drying tube. 5,10,15, 20-tetrakis (3,4-dimethoxyphenyl)chlorin(0.3 mmol) was dissolved in minimum volume of dry methylene chloride (10 ml), placed in a dropping funnel and slowly added over a period of 20 min. The mixture was stirred for 2 h at -78 C and then for 12 h at 25 C. After cooling to 0 C in an ice bath, excess of methanol was added to quench any excess of boron tribromide and to breakdown the chlorin-boron tribromide complex. Triethylamine was added to neutralize the reaction mixture and was concentrated under reduced pressure to give an amorphous purple solid, which was recrystallized from a mixture of methanol and chloroform to give of the hydroxyl chlorin TDHPC. Yield: 85% mp> 300 °C. *H NMR (300 MHz, DMSO-d6,30 °C, TMS): δ (ppm) -1.43 (s, 2H), 4.2 (s, 4H), 7.12 (t, 4H, J= 20Hz), 7.17 (d, 2H, J= 10Hz), 7.28 (s, 2H), 7.39 (d, 2H, J= 10Hz), 7. 53 (s, 2H), 8.29 (d, 2H, J=5Hz), 8.48 (s, 2H), 8.68 (d, 2H, J= 5Hz). 13C NMR (125 MHz, DMSO-d6, TMS): δ (ppm) 167.8, 152.6, 145.0, 144.6, 140.7, 137.8, 135.3, 134.4, 131.2, 127.5, 125.8, 123.7, 122.3, 121.4, 119.3, 112.1, 99.8, 48.13. IR (KBr): vmax 3187, 2694, 1577, 1525, 1512, 1500, 1431, 1354, 1255, 1112, 935cm 1.
FAB-MS: m/z= Calculated 745.2; Found 745.31 (M) + Step3. A solution of the chlorin compound5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin(2.2 m mol) in a mixture (1 :2) of methanol and chloroform 25 mL was refluxed with zinc acetate (3.1 m mol) for 6 h. The solvent was distilled off under vacuum and the residue obtained was washed with several portions of distilled water to remove the excess zinc acetate. The obtained zinc compound of the corresponding chlorin was recrystallized from a mixture (1:3) of methanol and ethyl acetate. Yield: (80%). mp>300°C; 1H NMR (500 MHz, DMSO-d6, TMS): δ (ppm)3.12 (s, 4H),7.10 (t, 4H, J= 15Hz), 7.26 (d, 4H, J=10Hz), 7.55 (d, 2H, J= 10Hz), 7.71 (s, 2H), 8.20 (d, 4H, J= 5Hz), 8.64 (d, 2H, J= 5Hz). 1JC NMR (125 MHz, DMSO-d6, TMS): δ (ppm) 167.8, 152.5, 148.0, 147.6, 141.7, 135.5, 134.7, 131.8, 128.7, 126.6, 124.7, 123.4, 117.6, 111.9, 109.5, 77.5, 56.0.IR (KBr): vmax3327, 1708, 1585, 1512, 1433, 1263, 1207 1114, 943cm 1; MALDI-TOF-MS: m/z Calculated: 808.14; Found 809.56 (M+l).
Example 2
All the cell lines used in this study are commercially available. They are obtained either from American Type Culture Collection (ATCC), USA or National Center for Cell Science, Pune, India and maintained as per the company's specifications at Tissue Culture facility at CCMB, Hyderabad, India.
We have tested the photodynamic efficacy of 5,10,15,20-tetrakis(3,4- dihydroxyphenyl)chlorin) (TDHPC) on various tumor cell lines, but we chose to work with SKOV3 (human Ovarian Carcinoma) cell line as a model (because of its better ability to form tumors in nude mice) for complete evaluation in culture and in animal experimentation. i) The light source used for irradiation is PDT 1200 L (Waldmann, Germany) ii) All the culture media, fine biochemicals and kits used for biological experiments are from standard commercial suppliers like Sigma-Aldrich, USA., Molecular Probes, USA, Invitrogen, USA.
Determination of the cytotoxic / phototoxic effect of the chlorin compound on SKOV-3 cells:
Cytotoxicity and phototoxicity of the chlorin compound of the general Formula 1 on SKOV-3 cells was investigated by using standard MTT (3, (4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. SKOV-3 cells(5 x 103 cells) were added to in each well of a 96-well micro titer plate in DMEM containing 10% fetal calf serum and incubated for 18 h in a humidified C02-incubator at 37 °C. Increasing concentrations of chlorin compound (0, 2.5, 5, 10, 15 and 20 μΜ in complete medium) was added. The cells were subjected to light irradiation using Waldmann PDT 1200 L at 200 J/cm2 and 50 mW/cm2. Corresponding dark controls (cells treated with chlorin but not irradiated) were also set. The MTT assay was performed after 24 h of incubation following exposure to light. Half the inhibitory concentration (IC50) value was determined from cell viability plot as a function of the concentration of chlorin compound. The IC50 value was observed to be 13 μΜ. Figure no. 8
Example 3
Determination of the morphological changes in SKOV-3 upon exposure to chlorin compound: Human ovarian carcinoma cells SKOV-3 were grown on cover slips and exposed to IC50 concentration of the chlorin compound of the general Formula 1 for 1 hour. These studies indicated SKOV-3 cells showed significant changes in cell morphology after treatment with chlorin followed by irradiation. Cell shape changed from a well attached and spread polygonal appearance to a rounded and loosely attached appearance, suggesting cytotoxicity. The light and dark controls showed no significant changes in cell morphology. Figure no. 9
Example 4
Fluorescence activated cell sorting (FACS) for studying membrane damage: Propidium Iodide is non-vital fluorescent stain for nucleic acids in a cell. Cells whose membranes are damaged and dead will take up the dye and exhibit fluorescence, while live cells do not take up the dye and remain non-fluorescent. For studying the Propidium Iodide uptake by cells, SKOV-3 cells were treated with the chlorin photosensitizer for 1 h followed by irradiation at 200 J/cm2, 50 mW/cm2. Cells were trypsinized and treated with 30 μg/ml Propidium iodide and 200 μg/ml RNase A for 10 min. The samples were run using FACS Calibur and analyzed using the CELL Quest software. PDT treated cells showed ca. 79 % Propidium Iodide uptake when compared to the dark and light controls, demonstrating membrane damage in the PDT treated cells. Figure no. 10
Example 5
Fluorescence activated cell sorting (FACS) for cell cycle analysis: Human ovarian carcinoma cells-SKOV-3 were seeded in a 25 cm2 flask at an appropriate density. After 24 h of incubation at 37° C, chlorin was added to the respective flasks and incubated for 1 h followed by irradiation (200 J/cm2, 50 mW/cm2). The cells were further incubated for 12 h and 24 h post PDT following which they were harvested and fixed in 0.5 ml of 70% cold ethanol and incubated over night at 4 °C. The cells were pelleted and suspended in buffer containing Propidium Iodide (60 μg/mL) and 200 μg/mL RNase and incubated for 30 min. Cell cycle analysis was done using FACS calibur (Becton Dickinson, CA, USA). All of the data generated in these experiments were recorded using CELL Quest research software (Becton-Dickinson Immunocy tome try Systems). We studied the analysis of cell cycle progression with chlorin in SKOV-3 where we observed.- 59 % sub-Gl population 12 h post-PDT which increased to ca. 76 % after 24 h post-PDT. Figure no. 11
Example 6
Mitochondrial damage: Human ovarian carcinoma cells SKOV-3 plated on cover slips were treated with chlorin photosensitizer for lh at 37 °C. The cells were subsequently irradiated at 200 J/cm2 at a fluence of 50 mW/cm2. Mitotracker Red CMX Ros, a dye that localizes to mitochondria was added to the cells for 15 min. The cells were fixed in 4 % formaldehyde for 10 min, counterstained with DAPI and visualized by confocal microscopy. The mitochondria of control cells showed a fibrillar appearance while those of PDT treated cells showed a collapsed appearance, indicating mitochondrial damage of SKOV-3 cells after PDT with chlorin. Figure no. 12
Example 7
Human ovarian carcinoma cells SKOV-3xenograft-bearing mice: Subcutaneous tumors were established by implanting 3-5 million SKOV-3 ovarian carcinoma cells into the flank of fox Nl athymic nude mice. Tumor size was measured using digital calipers and the tumor volume was calculated (mm3) as (width)2 x length x 0.5. Treatment was initiated when tumors reached a volume of 100-150 mm3. TDHPC was administered intravenously at 20 mg/kg body weight. The nude mice were irradiated (100 J/cm2 and 100 mW/cm2) 24 h after administration of the chlorin. Prior to the light treatment mice were anesthetized with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg). The in vivo antitumor activity of the chlorin was evaluated in SKOV-3 xenografts in nude mice by measuring reduction in tumor volume. Figure no. 13 Example 8
Histopathology of tumors and non-target organs: The untreated and dark control tumors showed the neoplastic cells invading from the subcutaneous to the dermal region. Stromal cells can be seen surrounding the neoplastic cells. Angiogenesis, which is critical for tumor growth and neoplastic progression, is also observed. Whereas the tumors from PDT treated animals showed cell death and tumor cells replaced by connective tissue (Figure no. 14), H&E staining of the non-target organs like liver (Figure no. 15), spleen (Figure no. 16), kidney (Figure no. 17) , heart (Figure no. 18), lung (Figure no. 19) did not show any histo- or cyto-pathic effects indicating PDT with TDHPC does not cause any systemic toxicity. ADVANTAGES OF THE PRESENT INVENTION
The chlorin dyes used for the present invention possess satisfactory properties of a NIR PDT agent and can be used as fluorescent probe in photodynamic therapeutic and diagnostic, biological, biochemical and industrial applications. The main advantages of these systems include: 1. Chlorin based compounds represented by Formula 1 are novel and pure single substances.
2. Their synthetic methodology is very economical.
3. Chlorin based compounds represented by Formula 1 possess absorption in the visible to near- infrared region (400-700 nm).
4. Chlorin based compounds represented by Formula 1 possess fluorescence emission in the near-infrared region (600-800 nm). Chlorin based compounds represented by Formula 1 possess emission quantum yields in the range 0.03-0.08 in aqueous media. Chlorin based compounds represented by Formula 1 possess triplet quantum yields in the range 0.5-0.85 in aqueous media. Chlorin based compounds represented by Formula 1 possess singlet oxygen quantum yields in the range 0.4-0.8 in aqueous media. Chlorin based compounds of the general Formula 1 can be used as NIR fluorescent labels in immunoassays. Chlorin based compounds of the general Formula 1 can be used for the detection and treatment of cancerous and non-cancerous diseases under physiological conditions. These novel dyes can be used as near-infrared fluorescence probes in biological and industrial applications.

Claims

WE CLAIM:
1. A compound of
Figure imgf000021_0001
Formula 1 wherein, R3, R4 = OCH3 or OH; Rl5 R2, R5 = H; M = 2H or metal selected from a group consisting of Zn, Cu, Fe.
The compounds as claimed in claim 1 , wherein the compounds are useful as NIR fluorescence probes in photodynamic applications for the detection and treatment of cancer and other diseases in human beings or animals.
A process for preparation of compounds of formula 1 wherein the process steps comprising;
(i) reducing the 3,4-dimethoxyphenylporphyrin by reacting with reducing agent in the presence of solvent and base under refluxing at a temperature ranging between 100 to 140 °C for a period ranging between 12 to 36 h to get 5,10,15,20-tetrakis(3,4- dimethoxyphenyl) chlorin;
(ii) reacting the chlorin compound obtained in step (i) with demethylating agent in a solvent at -78 °C for 2-3 h, and then for 10-12 h at 25-30 °C, followed by cooling at a temperature ranging between 0 - 5 °C, quenching and neutralizing to get demethylated chlorin compound (TDHPC) of Formula 1 wherein R3, R4 = OH; Rl 5 R2, Rs = H; M = 2H, (iii) reacting chlorin compound obtained from step (i) or (ii) with a metal salt in a mixture of solvent under reflux at a temperature ranging between 60 to 70 °C for a period of time ranging between 10-15 h followed by washing to get a metal complex.
The process as claimed in claim 3, wherein the reducing agent used for the reduction of porphyrin to chlorin is p-tosyl hydrazine.
The process as claimed in claim 3, wherein the solvent used is pyridine.
The process as claimed in claim 3, wherein the base is selected from the group consisting of potassium carbonate, cesium carbonate, sodium-ter-butoxide and potassium-ter-butoxide.
The process as claimed in claim 3, wherein the dealkylating agent used for the demethylation is Boron tribromide.
The process as claimed in claim 3, wherein the solvent is selected from the group consisting of dry Methylene chloride, dry chloroform.
The process as claimed in claim 3, wherein the neutralizing agent is selected from the group consisting of triethylamine, diisopropylethylamine, diethylamine.
The process as claimed in claim 3, wherein the mixture of solvent used is methanol and chloroform.
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