WO2000044742A1

WO2000044742A1 - Improved sensitizers for use in photodynamic therapy

Info

Publication number: WO2000044742A1
Application number: PCT/US1998/013879
Authority: WO
Inventors: Alan R. Morgan; Michael E. Menes; Ruizheng Wang
Original assignee: Lambda Pharmaceuticals, Inc.
Priority date: 1997-07-02
Filing date: 1998-07-03
Publication date: 2000-08-03

Abstract

Families of brominated, iodated or brominated and iodated squaraines and squarylium derivatives are disclosed. The derivatives have one of general formulas (IA, IB, IC, IIA, III, IV) where X is R12NR13, for example, where R12 and R13 can be hydrogen or alkyl groups, and Y and Z can be the same or different, and each is one of several moieties, examples of which are shown, designated moieties 1, 2, 3.

Description

IMPROVED SENSITIZERS FOR USE IN PHOTODYNAMIC THERAPY

REFERENCE TO RELATED APPLICATION

This is a continuation in part of a Provisional application entitled SENSITIZERS FOR USE IN PHOTODYNAMIC THERAPY, Serial No. 60/051,610, filed July 2, 1997. TECHNICAL FIELD

The present invention relates to a series of novel squaraine and squarylium derivatives that can be used as photosensitizers in photodynamic therapy ("PDT"). More specifically, the invention relates to a series of carboxylated and halogenated squaraine and squarylium derivatives. In addition, the present invention relates to pharmaceutically acceptable formulations of the new squaraine and squarylium derivatives and to the use of these new squaraine and squarylium compositions for the selective destruction of cells and tissues in PDT.

BACKGROUND OF THE INVENTION

PDT is an emerging technology for the selective destruction of cells and/or tissues. The process requires the presence of a photosensitizing agent which is capable of being taken up by target cells and tissues and which, on irradiation by light of a particular wavelength, generates species which are toxic to those cells and tissues.

It has been discovered that certain compounds are photosensitizing agents. Thus, for example, a porphyrin-derived preparation known as Photofrin®, when applied to a human or animal body, has been shown to be taken up by certain cells and tissues. The cells and tissues containing the photosensitizing agent can then be exposed to light of a certain wavelength. The light is generated and directed to the target cells and/or tissue by the use of lasers, photodiode arrays or lamps. If the target cells and tissues are located deeper into the body, light is delivered by the use of fiber-optic systems, endoscopic devices and catheters. It has also been shown that Photofrin, a certain amount of time after it is administered, is retained in greater concentrations in certain types of cells. The time period for such "selective" accumulation is 24-48 hours. Cells that show such selectivity have been characterized as hyperproliferating cells, i.e. cells which are growing at a faster rate that normal. Such cells are representative of many diseased tissues and states, including but not limited to cancer, dermatologic disease such as psoriasis, cardiovascular disease such as atherosclerosis and restenosis and diseases which are characterized by a rapid growth of blood vessels, for example, as is the case in ophthalmologic conditions such as age-related macular degeneration. Additionally, certain cells associated with immunological functions have been shown to retain photosensitizing agents selectively. Thus, photodynamic therapy to reduce the potential for graft rejection and for autoimmune disease such as rheumatoid arthritis has been reported.

Thus it can be seen that, following the deliverance of light, its absorption by the photosensitizer and the subsequent generation of species toxic to cells and tissues, cells and tissues such as those associated with cancer, dermatologic disease, cardiovascular disease, ophthalmologic diseases and immune disorders can be effectively treated.

Photodynamic therapy has advantages over many other conventional therapies due to the selectivity of the photodynamic process. For example, in the case of cancer, therapies such as chemotherapy and radiation therapy are known to have significant side-effects and are toxic to normal as well as abnormal cells. Consequently these treatments are associated with the destruction of a significant amount of normal cells and tissues. In the case of photodynamic therapy, the increased affinity of the photosensitizing agent for hyperproliferating cells such as those found in tumors reduces the potential for destruction of normal cells and tissues while increasing the potential for destruction of the lesion. In addition, the ability to direct the light specifically onto the target cells and tissues by the use of fiber-optic technology or the ability to protect adjacent normal cells or tissues by the use of filters further increases the selectivity of the photodynamic process. Furthermore, the use of photosensitizing agents which elicit no response until irradiated with light reduces significantly the potential for side-effects which may complicate the process. Thus for example, it has been shown that the only significant side-effect associated with the use of Photofrin as a photodynamic agent is a general skin response which has been characterized as a sunburn and which is associated with activation of Photofrin retained in the patient's skin following exposure to sunlight. This side-effect however, is only significant for the first 4-12 weeks following treatment during which time the patient is asked to avoid exposure of skin to strong sunlight.

It has also been shown that cells that are not characterized as hyperproliferating can also be destroyed using photodynamic therapy. In such a case, light activation of the photosensitizing agent is effected shortly after administration of the photosensitizer. At this time, the photosensitizing agent is present in many cell and tissue types and the selective retention of the photosensitizer in hyperproliferating cells has not occurred. Selectivity of treatment in this case is controlled by the delivery of light to the target tissues. For example, the photodynamic response can be controlled by the wavelength of light used. Since, as is known, light of longer wavelengths penetrates deeper into tissues, then the use of longer wavelengths of light will result in a greater depth of treatment. The biological response can also be controlled by the amount of light given, thus, the greater the amount of light, the greater the biological response. More light can be given by increasing the time of irradiation, by increasing the intensity of the light or by both. Such a regimen is useful for the treatment of diseases including but not limited to benign prostatic hypertrophy and endometriosis.

The mechanisms by which photosensitizers generate cytotoxic species following irradiation with light have not yet been fully identified in the human or animal patient. However, it is thought that the initial mechanism involves photoactivation of the photosensitizer. The next step of the reaction is dependent on the photosensitizer used. In most cases, including when Photofrin is the sensitizer used, it is thought that the photoactivated photosensitizer transfers energy to molecular oxygen, which is present in cells and tissues. This process generates singlet oxygen, a species that is known to be toxic to cells and tissues, leading eventually to cell death. The process of cell death is not well understood but is thought to involve interaction of the singlet oxygen with cellular components such as lipids and proteins, particularly those found in cell membranes. Loss of membrane integrity can eventually result in loss of cell viability. It has also recently been reported that photodynamic therapy may also result in activation of a process known as apoptosis, which is defined as programmed cell death and is the mechanism by which cells die naturally. Thus, photodynamic therapy may also be advantageous in that the mechanism of cell death may involve processes that biological systems use in their natural state to remove cells that are no longer useful.

A second mechanism by which the photodynamic effect may be generated involves not the generation of singlet oxygen, but the generation of radical species. Such species can be generated by the interaction of the photoactivated photosensitizer with oxygen to produce radical species such as superoxide and/or hydroxyl radical that are known to be toxic to cells. Alternatively, the photoactivated photosensitizer can itself react directly with cellular components to generate a free-radical type reaction which again results in loss of cell integrity and thus, of cell viability.

Although the exact mechanisms by which photodynamic therapy exerts its effects are not yet well understood and depend to some extent on the specific photosensitizer used, it is clear that photodynamic therapy has advantages over many current day procedures. The process appears to have minimum side-effects and a greater degree of selectivity for target cells and tissues. In addition, photodynamic therapy can be used in conjunction with other treatment modalities as well as in a stand-alone treatment.

Consequently, photodynamic therapy has received approval for certain indications in certain countries. Thus, in the USA, the FDA has approved the use of Photofrin as a photosensitizer in photodynamic therapy for the treatment of certain esophageal tumors and lung cancer. In Japan, the use of Photofrin has been approved for photodynamic treatment of a variety of cancers including lung, stomach and cervical; in Canada the same procedure has been approved for use in treatment of bladder cancer and in the Netherlands, France and Germany, approval has been granted for use in esophageal cancer.

Although Photofrin is the only photosensitizer to have gained approval anywhere in the world to-date, a number of photosensitizers have been proposed for use in photodynamic therapy and are in pre-clinical or clinical trials for the treatment of various indications including cancer, dermatological, cardiovascular, immunologic and ophthalmologic and urologic. Examples of photosensitizers which have received attention include Purpurins, Benzoporphyrin Derivatives, Porphycenes, Texaphyrins; Pheophorbides and Phthalocyanines Application of the use of photodynamic therapy in the treatment of cancer, cardiovascular disease, blood components, viral lesions, ophthalmologic disease, urological conditions, dermatological disease and in the treatment of immunological related disease have all been demonstrated in pre-clinical and/or clinical studies.

Notwithstanding the above, the use of these potential photodynamic agents is associated with various disadvantages which limit their usefulness in photodynamic therapy. Such disadvantages range from inefficient or costly synthetic procedures, the necessity of complex delivery vehicles in order to effect administration to patients, absorbance of light in regions of the visible spectrum which limit light production to costly and complex light devices and clinical symptoms, more specifically the presence of an unwanted skin reaction due to poor photosensitizer clearance from skin, or as has been reported with some experimental photosensitizers, pain at the treatment site during light activation.

For some time, the applicants have been searching for superior photosensitizing agents for use in photodynamic therapy. The applicants have concentrated in particular on carboxylated and halogenated squaraine and squarylium derivatives. Squaraine and squarylium derivatives have been extensively studied, mostly with respect to their use in nonlinear optics, optical recorders or as red absorbing fluorescent dyes. However, the properties which make them useful in these fields - i.e. generally water-insoluble, high fluorescent quantum yields, short triplet lifetimes - do not make them useful in PDT. We have accordingly sought new squaraine and squarylium derivatives that may be useful in PDT. The present invention is the result of this study.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a series of squaraine and squarylium derivatives having a molecular weight not greater than 10,000 Daltons and with the proviso that the final molecule contains 2-4 halogen atoms (specifically Br and/or I) and at least two carboxylic acids or acid derivatives, or that the final molecule contains 6-8 halogen atoms (specifically Br and/or I) and is of the general structure IA-IB-IC shown below, in Formula 1 , in which Y and Z can be the same or different:

Examples of the cationic forms of Y and Z are shown below as Moieties 1 through 15, where points of attachment of Y and Z to the squaraine ring are indicated by an arrow. The several R groups can be the same or different and, in general, each is hydrogen, halogen, or a monovalent moiety consisting of any combination of C,H,N,O,P, S and halogen.

In another aspect, the derivative is of the general structure HA shown below in structural formula 2

ΠA

Formula 2

Where X= NRjR₂, CRιR₂R₃ or SR and R, Y and Z have the same meanings as in Formula 1; preferably, at least one of R, R_, R₂ and R₃ is a carboxylic acid, an ester or an amino acid residue.

In still another aspect, the derivative is of the general structure IHA shown below in structural formula 3

πi

Formula 3

Where R, Y and Z are as identified above.

In still another aspect, the derivative is of the general structure INA shown below in structural formula 4

IN

Formula 4

Where X= ΝRιR₂, CRιR₂R₃ or SR and R, Y and Z are as identified above; preferably, at least one of R_\, R₂ and R₃ in CR)R₂R is a carboxylic acid, an ester or an amino acid residue.

In yet another aspect, the present invention is a pharmaceutical composition wherein the active ingredient is a composition having the structure of one of formulas 1-4, above.

In a further aspect, the present invention is one of several methods for destroying cells and tissues through a photodynamic process comprising the steps of administering an effective amount of one of the compositions of formulas 1-4 and supplying sufficient light to generate a cytotoxic effect.

DETAILED DESCRIPTION OF THE INVENTION

The essence of this invention is a family of novel photosensitizing agents and pharmaceutical compositions including such photosensitizing agents. More specifically, the novel photosensitizing agents are squaraine and squarylium derivatives which have one of the foregoing general structural formulae 1-4, wherein X, Y and R have the meanings set forth above, with the proviso that the molecular weight of the compound is not greater than 10,000 Daltons. More specifically, in one aspect, the invention provides families of brominated, iodated or brominated and iodated squaraine and squarylium derivatives having one of the general formulas:

where Y and Z can be the same or different, and each is a moiety having the structure of one of Chemical moieties 1 through 15, below, which is attached in one of the foregoing general formulas at the position indicated by an arrow:

Moiety 5 Moiety 6

Moiety 7 Moiety 8 Moiety 9

In Moieties 1 through 15, RI, R2, R3, R4, R5, R6, R7 and R8 can be the same or different, and each is Br, I, a carboxylic acid or a carboxylic acid derivative,

H, OH, OR₅, CHO, R₆NR₇ or NO₂, where R₅, R₆ and R₇ are subsequently defined, an alkyl group having from 1 to 6 carbon atoms, an alkylene group having from 2 to 4 carbon atoms, a group having the formula R₂N(R₃)₂ where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R₃ groups can be the same or different, a group having the formula R₂N(R₄)₃A where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and R₄ is an alkyl group having from 1 to 2 carbon atoms and the three R^ groups can be the same or different, a group having the formula R₂OH were R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,

CO₂R', CH₂CO₂R' or CH₂CH₂CO₂R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl, an amino acid moiety which is attached to the squaraine or squarylium derivative through a carbonyl which is a part of an amide produced by reaction between an amine function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a CO₂R', CH₂CO₂R' or CH₂CH₂CO2R' group of the squaraine or squarylium derivative, a group having the formula

O O

II CR II

| or CR15

R₂NR₃ NR₃

where R₂ is a bivalent aliphatic hydrocarbon ladical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond, R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atoms, and R15 is a lesidue of an amino acid formed by reaction between reaction between an acid function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a NHR₃ group of the squaraine oi squarylium derivative,

R9 and RIO can be the same or different, and each is

O H

C R12 or C ^■ R13

R14 where R12, R13 and R14 can be the same or different, and each is

H, OR| or CHO where Ri is an alkyl group having from 1 to 6 carbon atoms, an alkyl group having from 1 to 6 carbon atoms, an alkylene gioup having from 2 to 4 carbon atoms, a group having the formula R₂N(R )₂ where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 caibon atoms, wherein any carbon to carbon bond is either a single oi a double bond, and not moie than one is a double bond, R₃ is hydrogen or an alkyl group having fiom 1 to 2 caibon atoms and the two R groups can be the same or different, a group having the formula R₂N(R )₃A where R₂ is a bivalent aliphatic hydrocarbon radical having fiom 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single oi a double bond, and not moie than one is a double bond, A is a physiologically acceptable anion, and R_j is an alkyl group havmg from 1 to 2 carbon atoms and the three R₄ groups can be the same 01 different, a group having the formula R₂OH were R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,

CO₂R', CH₂CO₂R' or CH₂CH₂CO₂R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms,

Rl l is:

H

R12

R13

Where R12 and R13 can be the same or different, and each has the meaning set forth above, Where R_, R₆ and R₇ can be the same or different, and each is an alkyl group having from 1 to 6 carbon atoms, or R9 or RIO, as previously defined, And where X is:

R12

R12NR13 or C R13 or SRI 2, and

R14

R12, R13 and R14 can be the same or different, and have the meanings set forth above,

W is O, S, Se, Te or NR12, where R12 has the meaning set forth above, with the proviso that the squaraine or squaryilium derivative has from 2 to 4 substituents which are bromine, iodine or bromine and iodine and at least two substituents which are carboxylic acids or carboxylic acid derivatives, or has from 6 to 8 substituents which are bromine, iodine, or bromine and iodine. Preferred families of derivatives according to the invention have the following formulas:

Where R1-R6, R9 and RIO can be the same or different, and each has the meaning set forth above. Preferably, the derivatives of this family have the structure:

wherein R2\ R3\ R4' and R5', R2 and R4 H, Br or I, R5 is t-butyl, R2, and RI and R3 are alkoxy groups having from 1 to 6 carbon atoms. Another preferred family of compounds has the structure:

Where RI through R6 are Br, I, or alkyl groups having from 1 to 6 carbon atoms.

Still another preferred family has the structure:

Where R5 on the heterocyclic ring is an alkyl group having from 1 to 6 carbons, RI is an alkoxy group having from 1 to 6 carbon atoms, and R2 and R4 on the heterocyclic ring and R2 through R5 on the carbocyclic ring are H, Br or I. Another preferred family has the structure:

Where X, R2 through R5, R9 and RIO have the meanings set forth above. Most desirably, X has the structure:

R12

C ^■ R13

R14

Where R12 is H and R13 and R14 are alkyl groups having from 1 to 6 carbon atoms.

Still another preferred family of derivatives has the general formula:

Where R2\ R3\ R4\ R5', R2 and R4 are H, Br or I, R5 is tertiary butyl, and RI and R3 are alkoxy groups having from 1 to 6 carbon atoms.

DETAILED DISCLOSURE OF THE INVENTION

The synthesis of squaraines is well described in the literature, for example, in "the Chemistry of Squaraines", Schmidt, Arthur H., Oxocarbon (1980) pp 185-231, Edited by Robert West, Academic: New York Press, in Angew. Chem. Int. Ed. (1966), 5, pp 894, in Dyes and Pigments 1988, 9, 85-107, ibid 1989, 11, 21-35 and in numerous U.S. patents (5077160; 5210301; 5500156; 5256620; 4942141; 5543086; 5329019; 5416214; 5627014; 5492795; 5227499 and references cited therein).

In general, squaric acid (Aldrich Chemical Company) is condensed with the corresponding reactant (1-16 in figure 5) using a Dean Stark apparatus to remove water during reaction. The solvent can be an alcohol/benzene solvent mixture, n-butanol, toluene, ethanol etc. A base such as pyridine, triethylamine or quinoline can also be added to aid reaction. The resulting product can be collected and purified, preferably by recrystalization, distillation or chromatography. This procedure leads to the synthesis of squaraine derivatives in which both Z and Y are the same. EXAMPLE 1-1 Synthesis of a symmetrical squaraine of general formula 1 in which Y and Z are the Moiety 6, above, where R5=R6=Me, R2=isopropyl

For the synthesis of the squaraine in which Y and Z are Moiety 7, above, with R5=R6=Me, R2=isopropyl, guaiazulene (R1=R3=R4=H; Aldrich Chemical Company) is used as reactant in the above general procedure. Guaiazulene (2 equivalents) is taken up in toluene containing a small amount of triethylamine and squaric acid (one equivalent) added. The solution is refluxed, with removal of water, until the reaction is complete by thin layer chromatography (TLC). The product was isolated and purified by column chromatography.

EXAMPLE 1-2 Bromination of the product formed in Example 1-1 So that R1=R3=R4 of the guaiazulene units = Br

Bromination of the product of Example 1-1 was effected at positions RI, R3 and R4 by dissolving the product in glacial acetic acid and adding six equivalents of bromine in acetic acid. After stirring for several hours, the tribromo derivative was isolated and purified using a standard work up. The reaction sequence described in Examples 1-1 and 1-2 is shown below.

The synthesis of unsymmetrical squaraines (where Y and Z are not the same) is also well documented in the literature. The use of two different reactants in the above described procedure for the synthesis of symmetric squaraines will lead to a statistical mixture of three squaraine products - the two symmetric squaraines derived from each of the two reactants used and the unsymmetrical squaraine from reaction of squaric acid with each reactant. To avoid lower yields and timely and costly separation, the controlled sequential addition of both reactants is in many cases advantageous. Such chemistry is described in detail in J. Org. Chem. 1992, 57, 3278-3286, in Dyes and Pigments 1988, 9, 85-107, ibid 1989, 1 1, 21-35 and in U.S. patents 5500156, 5106713 and 5977160.

A typical procedure for the synthesis of unsymmetrical squaraines is described in U.S. patent 5627014 and outlined below.

EXAMPLE 2-1

Preparation of an unsymmetrical squaraine derivative of general formula 1

Where R = t-butyl; R₂, R₄ = H; R₃, R₅ = OMe; R₇ = H.

Tetrachlorocyclobutenone was prepared as described in Angew. Chem. Int.Ed. 1966, 5, 893. 5,7-Dimethoxy-2-[l,l-dimethylethyl]benz-4H-pyran-4-one was prepared from methyl 4,4-dimethyl-3-oxopentanoate as described in US Patent 5627014 (Example 53). A suspension of the above generated pyrone and tetrachlorocyclobutane in N,N- dimethylformamide was heated (90 C) with stirring, in the presence of the base triethylamine, for three hours. The reaction was then cooled, rotoevaporated and the residue purified by column chromatography to give the monosubstituted squaraine. Hydrolysis of the chloro groups by treatment with triflic acid followed by water generated the corresponding oxo- species.

Reaction with the second component of the desired squaraine, in this case dimethylanilene following the same methods described generated the desired squaraine. The reaction sequence is shown below.

EXAMPLE 2-2

Bromination of the Unsymmetrical Squaraine Prepared

In Example 2- 1

The squaraine prepared as described in Example 2-1 was brominated by dissolving the product in glacial acetic acid and adding six equivalents of bromine in acetic acid. After stirring for several hours, the hexabromo derivative where R₂, R _ι R₇ = Br was isolated and purified using a standard work up. EXAMPLE 3

Iodination of the Unsymmetrical Squaraine Prepared

In Example 2- 1

The squaraine prepared in Example 2-1 was iodinated by reaction with an excess of iodine monochloride, in glacial acetic acid. The mixture was stirred at 50-60° for 2 hrs, water added and the product filtered off and air-dried. The product was purified by recrystalization from methanohdichloromethane.

EXAMPLE 4-1 Preparation of a squaraine of general formula 2 where X = NHMe

Where R = t-butyl; R₂, R₄ = H; R₃, R₅ = OMe; R₇ = H.

Dimethyl sulfate is added to the product from Experiment 2-1 in dichloromethane and the solution refluxed for twenty-four hours. The solution was washed with water and the solvent removed by rotoevaporation. The residue was taken up in dichloromethane and methylamine added. Twelve hours later, the reaction mixture was quenched with aqueous, sodium bicarbonate and the organic layer collected, reduced to small volume and passed through a silica gel column. The product was collected and recrystalized from dichloromethane.

EXAMPLE 4-2 Bromination of the product of Experiment 4- 1

The above product from Example 4-1 was taken up in glacial acetic acid and six equivalents of bromine added. After twenty-four hours, the solution was washed with water, the organic layer collected and the product collected and purified by solvent evaporation and column chromatography. The desired product, where R₂, R , R₇ = Br. was collected in modest yield.

EXAMPLE 5-1 Preparation of a Squaraine of general formula 2 where X = CHRjR₂

Where R, = CH₃CO-, R₂ = CO₂Et, R₇ = H.

The reaction sequence is shown below.

dimethylanilene

CH₃COCH₂C0₂Et

dimethylanilene

The dibutyl ester of squaric acid (available from Aldrich Co) was treated with one equivalent of dimethylanilene and triethylamine in dichloromethane at reflux for twelve hours. The organic layer is removed and the mono substituted squaric acid derivative is purified by column chromatography. The mono substituted derivative was then taken up in THF and ethyl acetoacetate and DBU added in slight excess. The mixture was stirred for six hours, and the solvent then removed by rotoevaporation. The residue was washed with ether, and then taken up in dichloromethane. One equivalent each of dimethylanilene and triethylamine was added and the solution refluxed for six hours. The solvent was removed and the product purified by column chromatography.

EXAMPLE 5-2 Bromination of Product Obtained in Example 5-1

The product obtained in Example 5-1 was brominated following the method described above in Example 1 -2. The octabromosquaraine (R₇ = Br) was isolated in good yield. To form derivatives of general formulae 3 and 4, squaric acid is replaced in the above examples and reaction sequences by croconic acid or the disodium salt thereof.

Example 1 describes the synthesis of a symmetrical squaraine of general formula 1 where both Y and Z are the same and are Moiety 6, above. Example 2 describes the synthesis of an unsymmetrical squaraine of general formula 1 in which Z is derived from N,N- dimethylanilene (Moiety 3) and Y is derived from a methylbenzpyrrylium salt (Moiety 1). These procedures can also be used to make any symmetrical squaraine derivatives from any of Moieties 1 through 15, above, or to make any unsymmetrical squaraine derivative from combining any two different ones of the Moieties. Thus, for example, the use of ortho-N,N-

dimethylaminoisophthalic acid (Moiety 3) would result in generation of a symmetrical squaraine bearing four carboxylic acid residues as shown below, while combining ortho-N,N- dimethylaminoisophthalic acid with N,2,3-trimethylpyrrole (Moiety 9) would generate the unsymmetrical squaraine bearing two carboxylic acid residues. As also described in Examples 1-3, such squaraines can easily be substituted with bromide or iodide to prepare halogenated squaraine derivatives.

Examples 4 and 5 describe the synthesis of squaraine and sqaurylium derivatives of general formulae 2. Those skilled in the art will appreciate that a variety of amines can be used to prepare additional compounds of general formula 2 where

and that compounds which contain an activated methylene group (e.g. malonic acid, ethyl cyanoacetate, malononitrile) can be used to prepare additional compounds in which X = CR)R₂R₃. In addition, those skilled in the art will appreciate what additional changes in starting materials are necessary, and how to synthesize the changed materials that are required to produce squaraine and squarylium derivatives having the structures of formulae 1-4 where Y and Z are the foregoing moieties, where X= NRιR₂, CR|R₂R₃ or SR with the proviso that at least one of R, R₂ and R₃ in CR₁R₂R₃ is a carboxylic acid, ester or an amino acid residue and where any R group may be the same or different and each is hydrogen, halogen, or a monovalent moiety consisting of any combination of C,H,N,O,P, S and halogen, with the proviso that the molecular weight of the compound is not greater than 10,000 Dal tons and with the further proviso that the final molecule contains 2-4 halogen atoms (specifically Br and/or I) and at least two carboxylic acids or acid derivatives, or that the final molecule contains 6-8 halogen atoms (specifically Br and/or I).

Administration and Use

The compounds of the invention are useful as photosensitizers and can be used to destroy cells and tissues following activation by light. In general, the squaraine and sqaurylium derivatives are administered in a therapeutic amount to a human or animal patient in whom it is desired to destroy certain cells or tissues. The administration may be intravenous, intramuscular or topical. Following accumulation of the photosensitizer in the target cells and tissues, the target cells and tissues are exposed to light of a wavelength which causes the photosensitizer to become cytotoxic. Thus, the target cells and tissues are destroyed. The process may also be applied to an in vitro situation where, for example, blood or blood products collected from a human or animal patient can be treated photodynamically and then readministered to the same or to another human or animal patient.

The compositions are formulated into pharmaceutical compositions for administration using techniques which are well known to those skilled in the art and which are described in general in texts such as Remington's Pharmaceutical Sciences. More specifically, the preferred formulations are those prepared in conventional forms in which suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like or emulsions based on medium chain triglycerides, non-ionic solubilizers such as Cremophor or Tween 80 or phospholipids such as EYP. For applications such as in the treatment of dermatologic diseases such as psoriasis, the compositions may be topically administered using standard topical compositions involving penetrating solvents, or in the form of lotions, creams, gels or ointments. It is necessary only for the solution or emulsion to be one which is physiologically acceptable and of a suitable concentration or dilutable to a suitable concentration for administration. An indefinitely large number of such solutions and emulsions will be apparent to those skilled in the relevant art from thr foregoing disclosures.

Typical indications for which these compositions have utility include, but are not limited to, cancer, dermatologic applications, cardiovascular applications, urologic applications, ophthalmologic applications, immunologic applications, treatment of viral and fungal conditions and treatment of blood or blood products.

The following example is illustrative of a method used to assess the photodynamic potential of the compositions of this invention.

EXAMPLE 11

In Nivo Biological Response - Tumor Treatment

The squaraine

synthesized as indicated above, is dissolved in saline containing 1 % ethanol to give a solution with a final photosensitizer concentration of 0.5mg/ml. The solution is filtered through a 0.22 micron Millipore filter. Ten C3H/HeJ mice with 0.5mm diameter subcutaneous RIF tumors in the flank are injected IN with 0.75 mg/kg body weight of the above solution. After 24 hours, the tumor area is exposed to light at 650-670nm for 30 minutes at a power density of lOOmW/cm". The light source may be a Xenon arc lamp or an argon-pumped dye laser. Beginning twenty-four hours following light treatment, the treatment site appears cyanotic in nature and tumors are seen to become flat and non-palpable. An eschar forms over the treatment site. At 7 days post treatment, 100% of animals are shown to have responded to the photodynamic therapy. At thirty days post treatment, 35% of animals remain tumor free. Normal tissue surrounding the tumor and included in the treatment site is shown to be minimally affected by the photodynamic treatment, indicating a selectivity for response in the tumorous tissue.

The data clearly demonstrates these compounds to be effective to cause a cytotoxic response, at low doses when compared to Photofrin, which is significant in the target tissues and minimal in surrounding healthy tissue. These are desirable features since lowering the amount of photosensitizer needed to generate the desired effect reduces further the possibility of unwanted side-effects while the reduced response to normal tissue indicates that patients are not subjected to the prolonged skin sensitivity reported with Photofrin.

It is to be understood that this invention is not limited to the particular compounds, compositions, methods of use or synthesis as described herein. It is also understood that the terminology used is for the purpose of describing particular embodiments only and is not intended to be limiting. It is also to be understood that various changes may be made and equivalents substituted without departure from the scope and spirit of the invention. The scope of the present invention will be limited only by the appended claims.

Claims

We claim:

1. Brominated, iodated or brominated and iodated squaraines and squarylium derivatives having one of the general formulas:

Moiety 4

Moiety 6

Moiety 10 Moiety 12

Moiety 13 Moiety 14 Moiety 15

where Rl, R2, R3, R4, R5, R6, R7 and R8 can be the same or different, and each is Br, I, a carboxylic acid or a carboxylic acid derivative,

H, OH, OR_s, CHO, RaNR₇ or N0₂, where R₅, R₆ and R₇ are subsequently defined, an alkyl group having from 1 to 6 carbon atoms, an alkylene group having from 2 to 4 carbon atoms, a group having the formula R₂N(R₃)₂ where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R₃ groups can be the same or different, a group having the formula R N(R_t)₃A where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and * is an alkyl group having from 1 to 2 carbon atoms and the three R_j groups can be the same or different, a group having the formula R₂OH were R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,

CO₂R', CH₂CO₂R' or CH₂CH₂CO₂R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl, an amino acid moiety which is attached to the squaraine or squarylium derivative through a carbonyl which is a part of an amide produced by reaction between an amine function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a CO₂R', CH₂CO R' or CH₂CH₂CO₂R' group of the squaraine or squarylium derivative, a group having the formula

where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atoms, and R15 is a residue of an amino acid formed by reaction between reaction between an acid function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a NHR₃ group of the squaraine or squarylium derivative,

R9 and RIO can be the same or different, and each is O H

C R12 or C R13

R14 where R12, R13 and R14 can be the same or different, and each is:

H, OR] or CHO where Ri is an alkyl group having from 1 to 6 carbon atoms, an alkyl group having from 1 to 6 carbon atoms, an alkylene group having from 2 to 4 carbon atoms, a group having the formula R₂N(R₃)₂ where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R₃ groups can be the same or different, a group having the formula R₂N(R )₃A where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and R_j is an alkyl group having from 1 to 2 carbon atoms and the three R groups can be the same or different, a group having the formula R₂OH were R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,

CO₂R', CH₂CO₂R' or CH₂CH₂CO2R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms,

Rl l is:

H

R12

R13

Where R12 and R13 can be the same or different, and each has the meaning set forth above,

Where R₅, R₆ and R₇ can be the same or different, and each is an alkyl group having from

1 to 6 carbon atoms, or R9 or RIO, as previously defined,

And where X is:

R12

R12NR13 or C R13 or SRI 2, and

R14 R12, R13 and R14 can be the same or different, and have the meanings set forth above,

W is O, S, Se, Te or NR12, where R12 has the meaning set forth above, with the proviso that the squaraine or squaryilium derivative has from 2 to 4 substituents which are bromine, iodine or bromine and iodine and at least two substituents which are carboxylic acids or carboxylic acid derivatives, or has from 6 to 8 substituents which are bromine, iodine, or bromine and iodine.

2. A derivative as claimed in claim 1 which has the general formula:

3. A derivative as claimed in claim 1 which has the general formula:

4. A derivative as claimed in claim 1 which has the general formula:

5. A derivative as claimed in claim 1 which has the general formula:

6. A derivative as claimed in claim 4 wherein each of Rl through R6 is Br, I, a carboxylic acid, a carboxylic acid derivative, or an alkyl group having from 1 to 6 carbons.

7. A derivative as claimed in claim 5 wherein each of Rl through R6 is Br, I, a carboxylic acid, a carboxylic acid derivative, R₃, or an alkyl group having from 1 to 6 carbons.

8. A derivative as claimed in claim 3 which has the formula:

wherein each of R2 through R5 is Br or I, and each of R9 and RIO is an alkyl group having from 1 to 6 carbons.

9. A derivative as claimed in claim 3 which has the following formula:

wherein R2\ R3\ R4' and R5' are Br or I, R5 is t-butyl, R2 and R4 are H, and Rl and R3 are alkoxy groups having from 1 to 6 carbon atoms.