the present invention discloses novel sulfenate derivatives and their bioconjugates for phototherapy of tumors and other lesions . wherein e is either a hydrogen atom or is selected from the group consisting of antibodies , peptides , peptidomimetics , carbohydrates , glycomimetics , drugs , hormones , or nucleic acids ; x is selected from the group consisting of —( r 5 ) noc —, —( r 5 ) nocch 2 o —, —( r 5 ) nocch 2 ch 2 o —, — ocn ( r 5 )—, — hnc (═ s ) nh —, and hnc (═ o ) nh —; q is either a single bond or an alkenyl , aromatic , or heteroaromatic radical derived from a compound selected from the group consisting of olefins , benzenes , naphthalenes , naphthoquinones , fluorenes , anthracenes , anthraquinones , phenanthrenes , tetracenes , naphthacenediones , pyridines , quinolines , isoquinolines , indoles , isoindoles , pyrroles , imidiazoles , oxazoles , thiazoles , pyrazoles , pyrazines , purines , benzimidazoles , furans , benzofurans , dibenzofurans , carbazoles , acridines , acridones , phenanthridines , thiophenes , benzothiophenes , dibenzothiophenes , xanthenes , xanthones , flavones , coumarins , and anthacylines ; r 1 to r 5 are independently selected from the group consisting of hydrogen , c1 - c10 alkyl , c5 - c10 aryl , c1 - c10 polyhydroxyalkyl , and c1 - c10 polyalkoxyalkyl ; and ar is an aromatic or heteroaromatic radical derived from a compound selected from the group consisting of benzenes , naphthalenes , naphthoquinones , diphenylmethanes , fluorenes , anthracenes , anthraquinones , phenanthrenes , tetracenes , naphthacenediones , pyridines , quinolines , isoquinolines , indoles , isoindoles , pyrroles , imidiazoles , oxazoles , thiazoles , pyrazoles , pyrazines , purines , benzimidazoles , furans , benzofurans , dibenzofurans , carbazoles , acridines , acridones , phenanthridines , thiophenes , benzothiophenes , dibenzothiophenes , xanthenes , xanthones , flavones , coumarins , and anthacylines . in one embodiment , sulfenates according to the present invention have the general formula shown above , wherein e is selected from the group consisting of somatostatin , st , neurotensin , bombesin , cholecystokinin , steroid , and carbohydrate receptor binding molecules , and dihydroxyindolecarboxylic acid ; x is selected from the group consisting of —( r 5 ) noc —, —( r 5 ) nocch 2 o —, —( r 5 ) nocch 2 ch 2 o —, and — hnc (═ s ) nh ; q is a single bond or an olefinic or aromatic radical derived from a compound selected from the group consisting of alkenes , benzenes , furans , pyrroles , imidazoles , oxazoles , thiophenes , anthraquinones , quinolines , isoquinolines , indoles , acridines , acridones , phenanthridines , xanthenes , xanthones , and anthacylines ; r 1 to r 5 are independently selected from the group consisting of hydrogen , c1 - c10 alkyl , c5 - c10 aryl , and c1 - c10 polyhydroxyalkyl ; and ar is an aromatic or heteroaromatic radical derived from a compound selected from the group consisting of benzenes , diphenylmethanes , fluorenes , anthraquinones , naphthacenediones , pyridines , quinolines , isoquinolines , indoles , acridines , acridones , phenanthridines , xanthenes , xanthones , and anthacylines . in an alternative embodiment , sulfenates according to the present invention have the general formula shown above , wherein e is selected from the group consisting of somatostatin , st , neurotensin , bombesin , cholecystokinin ( cck ), steroid , and carbohydrate receptor binding molecules ; x is —( r 5 ) noc —, and —( r 5 ) nocch 2 o —; q is a single bond or is selected from the group consisting of benzenes , furans , pyrroles , oxazoles , acridines , acridones , xanthenes , xanthones , and anthracyclines ; r 1 to r 5 are independently selected from the group consisting of hydrogen , and c1 - c10 alkyl ; and ar is an aromatic or heteroaromatic radical derived from a compound selected from the group consisting of benzenes , diphenylmethanes , fluorenes , anthraquinones , naphthacenediones , pyridines , quinolines , indoles , acridines , acridones , phenanthridines , xanthenes , xanthones , and anthracyclines . these compounds operate mainly by a type i mechanism as shown in fig1 , wherein — o — sr is the sulfenate moiety that produces free radicals upon photoactivation , and ar is an aromatic chromophore that undergoes photosensitization . aliphatic and aromatic sulfenates can be used for phototherapy , although aromatic sulfenates have better material handling properties , as is well known in the art ( j . amaudrut and o . wiest , the thermal sulfenate - sulfoxide rearrangement : a radical pair mechanism . journal of the american chemical society , 2000 , 122 , 3367 - 3374 , which is expressly incorporated by reference herein in its entirety ). l is a linker between the chromophore and the epitope . epitope ( e ) is a particular region of the molecule that is recognized by , and binds to , the target site on the cell . e is usually , but not always , associated with biomolecules which include hormones , amino acids , peptides , peptidomimetics , proeins , nucleosides , nucleotides , nucleic acids , enzymes , carbohydrates , glycomimetics , lipids , albumins , mono - and polyclonal antibodies , receptors , inclusion compounds such as cyclodextrins , and receptor binding molecules . specific examples of biomolecules include steroid hormones for the treatment of breast and prostate lesions , somatostatin , bombesin , and neurotensin receptor binding molecules for the treatment of neuroendocrine tumors , cholecystokinin receptor binding molecules for the treatment of lung cancer ; st receptor and carcinoembryonic antigen ( cea ) binding molecules for the treatment of colorectal cancer , dihydroxyindolecarboxylic acid and other melanin producing biosynthetic intermediates for melanoma , integrin receptor and atherosclerotic plaque binding molecules for the treatment of vascular diseases , and amyloid plaque binding molecules for the treatment of brain lesions . biomolecules for use in the present invention may also include synthetic polymers . examples of synthetic polymers include polyaminoacids , polyols , polyamines , polyacids , oligonucleotides , aborols , dendrimers , and aptamers . coupling of diagnostic and radiotherapeutic agents to biomolecules can be accomplished by methods well known in the art as disclosed in hnatowich et al ., radioactive labeling of antibody : a simple and efficient method . science , 1983 , 220 , 613 - 615 ; a . pelegrin et al ., photoimmunodiagnosis with antibody - fluorescein conjugates : in vitro and in vivo preclinical studies . journal of cellular pharmacology , 1992 , 3 , 141 - 145 ; and u . s . pat . no . 5 , 714 , 342 , each of which are expressly incorporated by reference herein in their entirety . successful specific targeting of fluorescent dyes to tumors using antibodies and peptides for diagnostic imaging of tumors has been demonstrated by us and others , for example , s . a . achilefu et al ., novel receptor - targeted fluorescent contrast agents for in vivo tumor imaging , investigative radiology , 2000 , 35 ( 8 ), 479 - 485 ; b . ballou et al ., tumor labeling in vivo using cyanine - conjugated monoclonal antibodies , cancer immunology and immunotherapy , 1995 , 41 , 247 - 263 ; k . licha et al ., new contrast agent for optical imaging : acid - cleavable conjugates of cyanine dyes with biomolecules , in biomedical imaging : reporters , dyes , and instrumentation , d . j . bornhop , c . contag , and e . m . sevick - muraca ( eds . ), proceedings of spie , 1999 , 3600 , 29 - 35 , each of which are expressly incorporated by reference herein in their entirety . therefore , the inventive receptor - targeted phototherapeutic agents are expected to be effective in the treatment of various lesions . in the process outlined in fig1 , the photoexcitation of the aromatic chromophore effects rapid intramolecular energy transfer to the sulfenate group , resulting in bond rupture and production of two reactive free radicals which cause cellular injury . for targeting purposes , external attachment of an epitope is used . if the aromatic sulfenate compounds themselves preferentially accumulate in the target tissue , however , an additional binding group may not be needed . for example , if ar is an anthracycline moiety , it will bind to cancer cells directly and would not require an epitope for targeting purposes . the synthesis of sulfenate derivatives is accomplished by the method disclosed in d . l . pasto and f . cottard , demonstration of the synthetic utility of the generation of alkoxy radicals by the photo - induced , homolytic dissociation of alkyl 4 - nitrobenzenesulfenates , tetrahedron letters , 1994 , 35 ( 25 ), 4303 - 4306 , which is expressly incorporated by reference herein in its entirety . this method generally involves the condensation of sulfenyl chlorides with alcohols in the presence of an organic base . the sulfenate derivatives of the present invention contain additional functionalities that can be used to attach various types of biomolecules , synthetic polymers , and organized aggregates for selective delivery to various organs or tissues of interest . a diol 1 is prepared by the reaction of methyl magnesium bromide with methyl 4 - hydroxybenzoate . referring to fig2 , alkylation of the resulting phenol with methyl bromoacetate , condensation of the tertiary alcohol with 4 - nitrobenzenesulfenyl chloride , and saponification of the ester affords an intermediate acid 2 . this acid 2 is then converted to the corresponding active ester using n - hydroxysuccimide ( nhs ) and dicyclohexylcarbodiimide ( dcc ). the active ester can be attached to any desired biomolecule of interest to form an aromatic sulfenate 3 . alternatively , the acid 2 can also be directly condensed with any biomolecule using automated peptide synthesizer . specifically , the biomolecule of the present invention pertains to those binding to colorectal , cervical , ovarian , lung , and neuroendocrine tumors . these include somatostatin , cholecystokinin , bombesin , neuroendrocrine , and st receptor binding compounds . an acridone derivative is prepared according to fig3 . the starting material 4 is prepared according to a standard method known to one of skill in the art , as disclosed in k . matsumura , 1 - aminoacridine - 4 - carboxylic acid ., journal of the american chemical society , 1938 , 32 , 591 - 592 , which is expressly incorporated by reference herein in its entirety . an aminoacridone 4 is converted to a phenol by a standard method of diazotization of the amino group followed by displacement of the diazonium group with sodium hydroxide . the phenol 5 is converted to the corresponding p - nitrobenzenesulfenate and then conjugated to the biomolecules directly using an automated peptide synthesizer , or indirectly by the active ester route , to form the inventive acridine derivative 6 . a typical preparation of acridine - sulfenate derivative is outlined in fig4 . a thiol 7 is prepared from the known starting material 9 - chloroacridone . it is converted to the corresponding sulfenyl chloride , condensed with methyl 3 - hydroxy - 3 - methylbutyrate , and saponified to acid 8 . the sulfenate - acid can be condensed with the desired biomolecules by the process previously described . the anthraquinone - sulfenate derivatives can be synthesized according to fig5 . a diacid chloride 10 is reacted with a lactone 11 under friedel - crafts conditions followed by saponification to the corresponding hydroxyanthraquinone 12 . it is then condensed with p - nitrobenzenesulfenyl chloride and conjugated to the desired biomolecule directly to form an inventive derivative 14 . alternatively , the lactone 10 could be hydrolyzed to the acid and then coupled to the biomolecule by conventional methods . the xanthene derivative can be prepared according to fig6 . a xanthone benzyl ether 15 is prepared from the known 4 - hydroxyxanthone by alkylation with benzylbromide . the compound 15 was converted to the ether 16 in three steps : bromination , grignard reaction with ethylformate , and grignard reaction with methylmagnesium bromide . deprotection of the t - butyl group with hcl followed by alkylation with methyl bromoacetate provides a tertiary alcohol 17 . the tertiary alcohol 17 is then condensed with p - nitrobenzenesulfenyl chloride , saponified , and conjugated to the desired biomolecules mentioned previously . the novel compositions of the present invention may vary widely depending on the contemplated application . for tumors , the biomolecule is selected from the class of tumor markers including , but not limited to , somatostatin , bombesin , neurotensin , cholecystokinin , st , estrogen , and progesterone receptor binding compounds . for vascular lesions , the biomolecule may be selected from the class of integrins , selectins , vascular endothelial growth factor , fibrins , tissue plasminogen activator , thrombin , ldl , hdl , sialyl lewisx and its mimics , and atherosclerotic plaque binding compounds . a typical synthetic scheme of a steroid - photosensitizer conjugate is shown in fig7 . estrone is protected as the t - butyl ether 19 and reduced with sodium borohydride to a mono protected estradiol 20 , which is then condensed with p - nitrobenzenesulfenyl chloride . deprotection of the t - butyl group yields the steroid - photosensitizer conjugate 21 . as previously described , some compounds accumulate in tumors or other lesions without the assistance of a bioactive carrier . administration of δ - aminolevulinic acid , an intermediate in porphyrin biosynthesis , results in a two - fold uptake of porphyrins in tumors compared to normal tissues . similarly , administration of dihydroxyindole - 2 - carboxylic acid , an intermediate in melanin biosynthesis , produces substantially enhanced levels of melanin in melanoma cells compared to normal cells . thus , a photosensitizer may be delivered to the site of lesion by attaching it to a biosynthetic intermediate , as shown in fig8 . the mono sulfenate 23 is prepared by the reaction of p - nitrobenzenesulfenyl chloride 22 with ethylene glycol and is condensed with an indole derivative 24 . hydrolysis of the diacetate provides the conjugate 25 . methods of performing therapeutic procedures with the inventive compounds are also disclosed . an effective amount of the inventive compounds in a pharmaceutically acceptable formulation is administered to a patient . for example , parenteral administration advantageously contains a sterile aqueous solution or suspension of the photosensitizer in a concentration ranging from about 1 nm to about 0 . 5 m . preferred parenteral formulations have a concentration of 1 μm to 10 mm . such solutions also may contain pharmaceutically acceptable buffers , emulsifiers , surfactants , and , optionally , electrolytes such as sodium chloride . formulations for enteral administration may vary widely , as is well known in the art . in general , such formulations are liquids , which include an effective amount of the complexes in aqueous solution or suspension . such enteral compositions may optionally include buffers , surfactants , emulsifiers , thixotropic agents , and the like . compounds for oral administration may also contain flavoring agents and other ingredients for enhancing their organoleptic qualities . formulations for topical delivery may also contain liquid or semisolid excipients to assist in the penetration of the photosensitizer . the compounds may also be delivered in an aerosol spray . the dose of the photosensitizer may vary from 0 . 1 to 500 mg / kg body weight , preferably from 0 . 5 to 2 mg / kg body weight . the photosensitizer is allowed to accumulate in the region of interest , followed by illumination with the light of wavelength 300 to 1200 nm , preferably 350 to 850 nm , at the site of the lesion . if the lesion is on the skin surface , the photosensitizer can be directly illuminated ; otherwise , endoscopic catheters equipped with a light source may be employed to achieve phototherapeutic effect . the intensity , power , duration of illumination , and the wavelength of the light may vary widely depending on the location and site of the lesions . the wavelength of light may vary from 300 to 1200 nm . the fluence rate is preferably , but not always , kept below 200 mw / cm2 to minimize thermal effects . appropriate power depends on the size , depth , and the pathology of the lesion . the novel inventive compounds have broad clinical utility which includes , but is not limited to , phototherapy of tumors , inflammatory processes , and impaired vasculature . the inventive compounds can be formulated into diagnostic or therapeutic compositions for enteral , parenteral , topical , or cutaneous administration . topical or cutaneous delivery of the photosensitizer may also include aerosol formulation , creams , gels , solutions , etc . the compounds are administered in doses effective to achieve the desired diagnostic or therapeutic effect . such doses may vary widely depending upon the particular complex employed , the organs or tissues to be examined , the equipment employed in the clinical procedure , the efficacy of the treatment achieved , and the like . these compositions contain an effective amount of the phototherapeutic agent along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated . these compositions may also include stabilizing agents and skin penetration enhancing agents . the following example illustrates a specific embodiment of the invention pertaining to the preparation and properties of a typical bioconjugate derived from bombesin , a bioactive peptide , and a phototherapeutic molecule , sulfenate . the peptide is prepared by fluorenylmethoxycarbonyl ( fmoc ) solid phase peptide synthesis strategy with a commercial peptide synthesizer from applied biosystems ( model 432a synergy peptide synthesizer ). the first peptide cartridge contains wang resin pre - loaded with an amide resin on 25 - μmole scale . the amino acid cartridges are placed on the peptide synthesizer and the product is synthesized from the c - to the n - terminal position . coupling of the fmoc - protected amino acids ( 75 μmol ) to the resin - bound free terminal amine ( 25 82 mol ) is carried out with 2 -( 1h - benzotriazol - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ( hbtu , 75 μmol )/ n - hydroxybenzotriazole ( hobt , 75 μmol ). each fmoc protecting group on the solid support was removed with 20 % piperidine in dimethylformamide before the subsequent amino acid was coupled to it . the last cartridge contains sulfenate acid , which is coupled to the peptide automatically , thus avoiding the need for post - synthetic manipulations . after the synthesis is completed , the product is cleaved from the solid support with a cleavage mixture containing trifluoroacetic acid ( 85 %): water ( 5 %): phenol ( 5 %): thioanisole ( 5 %) for 6 hours . the peptide - sulfenate conjugate is precipitated with t - butyl methyl ether and lyophilized in water : acetonitrile ( 2 : 3 ) mixture . the conjugate is purified by hplc and analyzed with lc / ms . the sulfenate - bombesin ( 7 - 14 ) conjugate has the following molecular structure : p - azidotetrafluorobenzoyl - gln - trp - ala - val - gly - his - leu - met - nh2 . as would be apparent to skilled artisans , various changes and modifications are possible and are contemplated within the scope of the invention described . it should be understood that the embodiments of the present invention shown and described in the specification are only specific embodiments of the inventors who are skilled in the art and are not limiting in any way . therefore , various changes , modifications or alterations to those embodiments may be made or resorted to without departing from the spirit of the invention and the scope of the following claims . the references cited are expressly incorporated by reference . for example , although the compositions of the present invention are primarily directed at therapy , most of the compounds containing polycyclic aromatic chromophores can also be used for optical diagnostic imaging purposes .
novel sulfenate derivatives and their bioconjugates for phototherapy of tumors and other lesions . the sulfenates of the present invention are designed to absorb low - energy ultraviolet , visible , or near - infrared region of the electromagnetic spectrum . the phototherapeutic effect is caused by direct interaction of free radicals , the reactive intermediate produced upon photofragmentation of the sulfenate moiety , with the tissue of interest .