The present invention relates to new compounds useful in photodynamic therapy (PDT). More particularly, the invention relates to new porphyrin derivatives and methods of using them in PDT.

Presently, PDT constitutes one of the more promising new modalities being explored for use in a variety of medical applications. For example, PDT is a well- recognized treatment for the destruction of tumors.

PDT utilizes the ability of a selectively retained photosensitizer to elicit an efficient photodynamic reaction upon activation with tissue penetrating light. The advantages of PDT are its cost effectiveness, simplicity of use, and lack of severe toxic side effects or disfigurement exhibited by the standard modes of cancer treatment.

Protoporphyrin IX (Photofrin TR) became the first porphyrin-type agent approved by the U. S. Food and Drug Administration (FDA) for a variety of cancer treatments. It has been used for the treatment of esophageal cancer and early and late stages of lung cancer in the U. S., Canada, Japan,'France, and the Netherlands.

Although the exact mechanism is unknown, it is believed that Photofrin TR is selectively taken up into tumor cells because of their higher metabolism and the slightly acidic nature of their cell walls. The tumor is then selectively irradiated with white light using fiber optic technology capable of accurately delivering the light to the target cells.

It is generally accepted that the excited state of porphyrin sensitizes the formation of singlet oxygen. The highly reactive activated oxygen then reacts with various components in the cell such as proteins, nucleic acids, and lipids. Cell death is caused by damage to vital molecular components that prevents metabolism or cell division. Cells can also become apoxic, and die due to lack of oxygen.

There are, however, disadvantages in prior compounds that have been used in photodynamic therapy. First, Photofrin TR and its generic forms are an inhomogeneous mixture of multimers of (protoporphyrin IX) x wherein x = 1 to 3. It is not a pure compound, and to date the active species is not known. This uncertainty can cornplicate dosageing.

Second, the dimeric (x = 2) and trimeric (x = 3) forms of Photofrin TR are not soluble enough to be used under physiological conditions. The insolubility leads to difficulty in administering the drug.

Third. Photofrin TR has lipophilic properties, which prevents it from readily entering cell walls, and an overall negative charge, which hinders its delivery into nuclear DNA.

Thus, there is a need of new compounds for photodynamic therapy in which the active species is known. There is an additional need of new compounds for photodynamic therapy that can more easily cross cell membranes and bind to nuclear DNA.

SUMMARY OF INVENTION It has now been discovered that these and other objectives can be achieved by providing a meso-tetraarylporphyrin compound having the following formula: Formula 1. or derivatives thereof, wherein two or three of R, R', R", or R"'represent cationic groups and the remaining R, R', R", or R"'groups represent one neutral polar group and one non-polar group or two neutral polar groups.

Another aspect of the invention provides a meso-tetraarylporphyrin compound suitable for photodynamic therapy having Formula 1. or derivatives thereof, wherein at least one of R, R', R"and R'represents X-SA wherein SA represents a saccharide and X = CH2 or S. The remaining R, R', R"or R"'groups are one or more cationic groups, neutral polar groups or non-polar groups.

Another aspect of this invention is related to a method of killing cells by first adding to the cells a meso-tetraarylporphyrin compound having Formula 1 or derivatives thereof wherein two or three of R, R', R", or R"'represent cationic groups and the remaining R, R', R", or R"'groups represent one neutral polar group and one non-polar group or two neutral polar groups. Alternatively, the invention is related to a method of killing cells by first adding to the cells'a meso-tetraarylporphyrin compound having Formula 1 or derivatives thereof, wherein at least one of R, R', R"and R"' represents X-SA wherein SA represents a saccharide and X = CH2 or S. The remaining R, R', R"or R"'groups are one or more cationic groups, neutral polar groups or non-polar groups. The next step is to irradiate the cells at a wavelength sufficient to cause cell death.

DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides a novel meso- tetraarylporphyrin compound suitable for photodynamic therapy having Formula 1 or derivatives thereof, wherein two or three of R, R', R", or R"'represent cationic groups and the remaining R, R', R", or R"'groups represent one neutral polar group and one non-polar group or two neutral polar groups. For example, the meso- tetraarylporphyrin compounds include compounds described above having two or three cationic groups and two neutral polar groups; two cationic groups, one neutral polar group and one non-polar group; and three cationic groups and one neutral polar group.

It is preferred that the cationic groups be located adjacent to each other on the meso-tetraarylporphyrin compound. For example, if the meso-tetraarylporphyrin compound represented by Formula 1 has two cationic groups, it is preferred the cationic groups be represented by R and R', R'and R", R"and R"', or R"'and R.

In this specification, meso-tetraarylporphyrin derivatives refer to meso- tetraarylporphyrin compounds wherein one or two pyrrole double bonds are oxidatively or reductively converted to single bonds. Some examples of derivatives wherein one double bond is converted to a single bond are the 3-4 dihydroporphyrins (chlorins) of the meso-tetraarylporphyrin compounds described above (see Formula 2A) and the corresponding dihydroxy and dioxo derivatives. Some examples of derivatives wherein two double bonds are converted to two single bonds are the a- tetrahydroporphyrins shown in Formula 2B, the b-tetrahydroporphyrins shown in Formula 2C, and the corresponding tetrahydroxy and tetraoxo derivatives.

Formula 2 In Formula 2, the circle in the porphyrin ring represents 2H+ or M.

Any cationic group can be used for purposes of this invention. Preferred cationic groups include, but are not limited to, mono (lower N-alkyl) pyridinyl, tri (lower alkyl) ammonium, di (lower alkyl) sulfonium, or tri (lower alkyl) phosphonium.

The mono (lower N-alkyl) pyridinyl ring may be bonded at any position to the meso-tetraarylporphyrin ring, i. e. 2-, 3-, or 4-mono (lower N-alkyl) pyridinyl.

In this specification, lower alkyl groups are preferably 1 to 3 carbons in length (CI-C3). Some examples of lower alkyl groups include methyl, ethyl, n-propyl and isopropyl.

Any neutral polar group can be used in this invention. Preferred neutral polar groups include, but are not limited to, hydroxy groups, amino groups, and saccharides.

The saccharides can be linked to the porphyrin compound by oxygen (O- glycoside linkage), sulfur (S-glycoside linkage) or carbon (C-glycoside linkage). The preferred glycoside linkages are S-glycoside linkages and C-glycoside linkages, more preferably the C-glycoside linkages.

Saccharides which can be used in this invention can be monosaccharides, disaccharides or polysaccharides. The saccharides can be in either the D or L configuration. Monosaccharides can be either aldoses or ketoses. The number of carbons of the saccharide can be from three carbons to about six carbons. An example of a three carbon sugar is glyceraldehyde. Examples of four carbon sugars include erythrose and threose. Examples of five carbon sugars include ribose, arabinose, xylose and lyxose. Examples of six carbon sugars include allose, altrose, glucose, mannose, gulose, idose, galactose and talose. In a preferred embodiment, the saccharide is galactose.

Non-polar groups preferably include lower alkyl, as defined above.

Preferred embodiments of these new meso-tetraarylporphyrin compounds are listed in Table 1 below.

Table 1<BR> Table 1. Characterization of Selected Porphyrin Compounds Table 1 Table 1. Characterization of Selected Porphyrin Compounds Porphyrin R R' R" R'" KB1(x10-6M) KB2(x10-6M) KOct/Water @X174 photocleavage 1 OH Me Me PyMe+ 0.3 179 >2500 poor 2 OH Me PyMe+ PyMe+ 1.5 46 7.23 excellent 3 OH PyMe+ Me PyMe+ 2.3 55 16.5 good 4 OH OH PyMe+ PyMe+ 0.8 86 4.02 excellent 5 OH PyMe+ OH PyMe+ 0.8 75 6.25 poor 6 OH OH OH PyMe+ 1.7 245 >2500 good 7 OH PyMe+ PyMe+ PyMe+ good 8 OH OH Me N(Me)3+ Each row in Table 1 represents a new meso-tetraarylporphyrin compound.

KB I (X I0-6M) stands for the first DNA binding constant and represents increased binding g of the meso-tetraarylporphyrin compound to DNA as the constant decreases; KB2x 10-6M stands for the second DNA binding constant; Xl7 stands for a commercially available plasmid DNA and represents the ability of compounds to bind and cleave DNA as the number increases; and Koct/water is the octanol/water partition coefficient, and represents the ability of compounds to dissolve in organic liquid and pass through cell membranes as the number increases.

In another emodiment, the present invention also provides a meso- tetraarylporphyrin compound suitable for photodynamic therapy having Formula 1 or derivatives thereof. At least one of R, R', R"and R"'represents X-SA wherein SA represents a saccharide and X = CH2 or S. The remaining R, R', R"or R"'groups represent one or more cationic groups, neutral polar groups or non-polar groups. The cationic groups, neutral polar groups, non-polar groups and saccharides are the same as those described above.

Compounds in this embodiment include those having one, two, three or four saccharides. Any remaining R, R'. R"and R"'groups can be cationic groups, neutral polar groups, or non-polar groups as described above. Where there are two cationic groups, the cationic groups are preferably adjacent to each other, also as described above.

In another embodiment, themeso-tetraarylporphyrin compounds of Formula 1 and Formula 2 further contain a metal ion. The metal ion is preferably located within the porphyrin ring. An example of this structure is as follows.

Formula 3 The metal ion can be any metal ion that forms a stable complex with the porphyrin ring. Metal ions include, but are not limited to, ions of Sn, Fe, Mn, Co, Zn, Cr and Cu. It is preferred that the oxidation states for each of the metals ions described above are Sn (IV), Fe (III), Mn (III), Co (III), Zn (II), Cr (IV) or Cu (II).

In a preferred embodiment, the minimum octanol-water partition coefficient (Kocuwater) of the meso-tetraarylporphyrin compound is at least about 2, preferably at least about 5, and more preferably at least about 10. The maximum octanol-water partition coefficient (Koctzwater) of the meso-tetraarylporphyrin compound is at least about 25, preferably at least about 30, and more preferably at least about 40.

The present invention also provides a method of killing cells by first adding a meso-tetraarylporphyrin compound of the present invention, or derivatives thereof, to cells. Preferably, the meso-tetraarylporphyrin compound enters the cells. The cells are then irradiated at a wavelength sufficient to cause cell death.

Cells which can be targeted can be either in vivo or ex vivo, and in both cases they can be either on the surface of the cell or in the interior. In vivo cells are cells inside the body of an organism, such as a mammal, e. g. a human. Ex vivo cells are cells outside the body of an organism. The ex vivo cells can be prokaryotic or eukaryotic, such as cells in bodily fluids, e. g. blood. Some examples of cells that can be targeted include, but are not limited to, tumor cells, microorganisms including bacterial cells, viral infected cells such as cells in a supply of blood, and diseased or defective cells, such as in the case of certain ocular cells.

The meso-tetraarylporphyrin compounds exhibit their cytotoxic effect at any wavelength of light that kills targeted cells. It is preferred that this wavelength of light be from about 400nm to about 750nm, more preferably from about 600nm to about 700nm.

The derivatives of the present generally absorb light at wavelengths higher than the corresponding porphyrins. For example, the wavelength of light absorbed by the chlorins are preferably from about 550nm to about 750nm, more preferably from about 700nm to about 750nm.

Light-induced killing of solid tumors according to the invention can be carried out on any solid tumors which are accessible to light from conventional sources (e. g. a xenon arc lamp, an incandescent white light) or from a laser. If a tumor is on the body surface, any light source can be employed that provides light at the appropriate wavelengths to activate the meso-tetraarylporphyrins. Preferably, the light source can deliver 50 to 200 mW per square centimeter of treated area. The light can be delivered for example by means of a tunable argon-laser (e. g. a 5 watt argon ion pumped tunable dye laser, Coherent, model Innova 100, Palo Alto, Calif.) using DCM (Exiton Chemical Co., Dayton, Ohio). Similar lasers are also commercially available from, for example, Spectra Physics, Mountain View, Calif. However, a projector light source may also be employed.

For cells, including tumor cells within the body, which are inaccessible to direct light sources, light is administered via optical fibers and the light source is a laser.

The meso-tetraarylporphyrin derivatives of the invention are made by methods known in the art. See, for example, Adler, et al., J. Org. Chem. 32,476-484 (1967) and references cited therein. Generally, a 2-, 3-, or 4-substituted benzaldehyde, or a substituted benzaldehyde derivative, such as a 2-, 3-, or 4-substituted tetrafluorobenzaldehyde, is condensed with pyrrole.

The glycosidic linkages described above are typically made under standard Adler conditions whereby the C-or S-sugar substituted benzaldedhyde and the pyrrole (0.2 to 0.05 M in acetic or propionic acid) are stirred at reflux for 1-3 hours. The concentration, acid, and duration of reaction are varied to optimize the yields. An alternative method for the S-sugars is to substitute the 4'F oftetraperfluorophenylporphyrin in DMF using the salt of the thiosugar. The synthetic steps are diagramed below in Formula 4. R' OH H N H H y N 1. BF, OEt. CH, C6. NaCI 1 2. DDQ H tu Nô N ruz go 2 bon OU H2,P0 5% an C MeOH.EtOAC R N R/ R H 5 R =H N R oh "OH 8 R'= R=N OU Formula 4 The derivatives of the meso-tetraarylporphyrin compounds can also be made by methods known in the art. For example, reduced derivatives, such as chlorins, can be made by methods of photoreduction or chemical reduction of porphyrin as described in Mauzaroll, The Photoreduction of the Porphyrins: Structure of the Products, JACS 1962, 24-45.

Alternatively, porphyrins can be reduced to the dihydro level using sodium dithionate at pH 6-7 in the dark. Similarly, porphyrin can be irradiated with monochromatic light, or with white light, in 0.1 M to 0.5M EDTA at a pH of about 5.8 to produce first stage reduction (dihydroporphyrin) and second stage reduction (tetrahydroporphyrin) products.

EXAMPLES The following example serve to provide further appreciation of the invention but are not meant in any way to restrict the effectice scope of the invention.

EXAMPLE 1 Svnthesis of meso-tetraphenvlporphvrin (TPP) Freshly distilled pyrrole (56 ml, 0.8 mole) and 80 ml (0.8 mole) of reagent grade benzaldehyde were added to 3.1 of refluxing reagent grade propionic acid. (Note: crystalline material was not directly obtained if acetic was used. After refluxing for 30 min, the solution was cooled to room temperature and filtered, and the filter cake was washed thoroughly with methanol. After a hot water wash, the resulting purple crystals were air dried, and finally dried in vacuo to remove absorbed acid to yield 25 g (20% yield) of TPP. Spectrophotometric analysis showed that only 1% of the TPP yield remained in the filtrate and also that the filtered material was about 3% tetraphenylchlorin (TPC) by weight.

Crude TPP produced by the above procedure was easily purified by a simple batchwise technique. TPP (l. Og) and 100 g of fuller's earth (Florex) were stirred with enough, 1,1,1-trichloroethane to make about 1 1. of mixture. This immediately passed through a 0.25-in. bed of solvent-washed fuller's earth on a 6-in. sintered-glass vacuum funnel. There was a loss of approximately 80% of the starting material in this purification procedure.

EXAMPLE 2 Purification of meso-tetraarylporphvrin Crude TPP produced from Example 1 was purified by entrainement sublimation using nitrogen gas as the carrier. A horizontal furnace with one or two hot zones was used with the temperature of the hotter zone adjusted to 325°, while the cooler zone was maintained at 250°. During the sublimitation process, nitrogen gas was allowed to flow at a rate of 1 ft3/hr. against atmospheric pressure. In this method, large, single-crystal needles of TPP were obtained.

EXAMPLE 3 Purification of meso-tetraarvlporphyrin Crude TPP produced from Example 1 was purified by vacuum sublimation in a horizontal furnace using vacuum ion pumping and a thermal gradient. The material was sublimed through a 12-in. diffusion path (325-350°F) at position of initial material to 150-200°F at the collection region) at a pressure which must be maintained at less than 1 X 10-7 torr. This method yielded octahedral and rhombohedral crystals.

EXAMPLE, 4 Bioactivitv As an indicator of bioactivity, the eight novel porphyrins thus found were incubated with a supercoiled plasmid DNA (-X 174) for 2-12 hours in the dark at room temperature, and then irradiated with continuous 50 foot-candles white light with a 450 nm cut-off filter that eliminates blue light for up to 90 minutes at 35 °C. To test for sequence specificity of the resultant single strand nicks (and amplify the signal), some samples were treated with S I nuclease. Gel electrophoresis shows that the plasmid DNA treated with the selected porphyrins derivatives was degraded.