Narrow emission dyes, compositions containing the same, and methods of making and using the same

Abstract

The present application provides chlorin derivatives with narrowband emission. In some embodiments, the bacteriochlorin derivative is PEGylated. In some embodiments, the chlorin derivative has high water solubility (e.g., 10 mg / mL or higher). In some embodiments, the chlorin derivative is PEGylated and has high water solubility. The chlorin derivative may contain a bioconjugable group for forming a conjugate (e.g., with an antibody or nanoparticle). The chlorin derivative and its conjugate can be used for imaging and therapeutic applications. A method for synthesizing the chlorin derivative is also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 850,442, filed on May 20, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The presently disclosed subject matter generally relates to chlorin derivatives having narrow emission wavelengths (in some embodiments, water-soluble chlorin derivatives having narrow emission wavelengths), their conjugates, and methods of making and using the chlorin derivatives and their conjugates.

[0004] background

[0005] A large and growing number of applications require fluorescent dyes that are water-soluble and suitable for conjugation with other substances ranging from nanoparticles to biological targeting agents. Such applications include, for example, flow cytometry, cell and whole organism imaging, sensing, and photodynamic therapy. Among these applications, chlorin molecules are of particular interest because chlorins typically emit in the red (i.e., 600-700 nm) spectral range, making them one of the few chromophores that can be used for photochemical studies in the red spectral region.

[0006] The success of the above applications depends on many factors, including (1) significant solubility in saline solutions to avoid intermolecular aggregation (and excited-state quenching), (2) minimal nonspecific binding to cellular components, (3) incorporation of a single reactive group for conjugation to avoid cross-linking and product mixtures, and (4) robust synthesis to provide sufficient quantities for experiments. However, the large hydrophobic face of chlorins can pose a challenge to aqueous solubility.

[0007] Thus, there is a continuing need to provide additional chlorin derivatives, including but not limited to those with improved aqueous solubility (e.g., aqueous solubility above 1 mg / mL), particularly those that can also be easily conjugated to a variety of substances and / or are characterized by narrow absorption and emission bands.

[0008] Overview

[0009] This overview lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and alternatives to these embodiments. This overview is merely an example of numerous different embodiments. Mention of one or more representative features of a given embodiment is also exemplary. Such an embodiment may generally be present or absent the features mentioned; similarly, these features may be applied to other embodiments of the presently disclosed subject matter, whether or not listed in this overview. To avoid excessive repetition, this overview does not list or indicate all possible combinations of such features.

[0010] In some embodiments, the presently disclosed subject matter provides compounds of formula (I):

[0011]

[0012] Where: M is metal or -H, -H; R5, R 10 and R 15 Independently selected from H, alkoxy and a linking group having the formula: -L1-(X1-L2) p -G, wherein p is 0 or 1; L1 is an alkylene group; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) wherein q is an integer from 1 to 24 q - alkylene, alkylene or substituted alkylene, optionally wherein the substituted alkylene is alkylene substituted by one or more groups comprising a polyoxyethylene chain and / or an amide group; G is a bioconjugable group; and R2, R3, R 12 and R 13 independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a radical having the formula -L1-(X1-L2) p -G linking group and a solubilizing group, wherein the solubilizing group is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , wherein w is an integer from 0 to 5, inclusive, and Rs is a group having the formula:

[0013] -X2-(L3) z -R 17 ,

[0014] wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH- or triazolyl; L3 is -C(=O)-alkylene-C(=O)-NH-, and R 17 Selected from -(C2H4O) m -R 18 、-C(=O)C2H4-(OC2H4) m OR 18 and (C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, wherein m is an integer of 4 or greater, optionally wherein m is an integer of 8 or greater; n is an integer of 1 to 5; R 18 is lower alkyl, optionally methyl; R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) mR 18 ; The conditions are: R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0015] In some embodiments, R5, R 10 and R 15 Independently selected from H, methoxy and having the formula -L1-(X1-L2) p -G linker. In some embodiments, R 10 It has the formula -L1-(X1-L2) p -G linker. In some embodiments, R 10 It has the formula -L1-(X1-L2) p -G linking group, L1 is phenylene, p is 1; X1 is -C(=O)NH-, L2 is alkylene, and G is selected from carboxylic acid and active ester. In some embodiments, R 10 yes:

[0016]

[0017] In some embodiments, R3 and R 13 Each is independently -aryl-(R s ) w , optionally wherein R3 and R 13 Each is -phenyl-(R s )2. In some embodiments, each R s With the formula -X2-(L3) z -R 17 , wherein z is 0, X2 is -CH2NHC(=O)-, and R 17 -(C2H4O) m -R 18 , wherein m is an integer from 12 to 24. In some embodiments, R3 and R 13 Each is:

[0018]

[0019] In some embodiments, the compound is:

[0020]

[0021] In some embodiments, the presently disclosed subject matter provides a composition comprising a covalent conjugate formed between: (a) a compound of formula (I), provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15 At least one of the (a) is a linker; and (b) is one or more selected from the group consisting of small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones and growth factors.

[0022] In some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising a compound of formula (I) or a conjugate formed between: a compound of formula (I), provided that R2, R3, R5, R 10 , R 12 , R 13 and R 15 At least one of the following is a linker; and one or more of the group consisting of small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors; and a pharmaceutically acceptable carrier.

[0023] In some embodiments, the presently disclosed subject matter provides a method for detecting a target. In some embodiments, the target is a compound, a cell, or a particle, and further, wherein the method comprises labeling the target with a conjugate formed between: a compound of formula (I), provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15 At least one of is a linker; and one or more of the group consisting of a small molecule, an antigen, a microparticle, a nanoparticle, a polymer, a peptide, a protein, an antibody or antibody fragment, a nucleic acid, a hormone, and a growth factor. In some embodiments, the method comprises using flow cytometry.

[0024] In some embodiments, the presently disclosed subject matter provides methods for imaging cells, tissues, and / or organisms. In some embodiments, the methods include using a compound of formula (I) or a conjugate formed between: a compound of formula (I), provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15 At least one of the is a linker; and one or more of the group consisting of a small molecule, an antigen, a microparticle, a nanoparticle, a polymer, a peptide, a protein, an antibody or an antibody fragment, a nucleic acid, a hormone, and a growth factor.

[0025] In some embodiments, the presently disclosed subject matter provides a method of treating a disease in an individual in need of such treatment. In some embodiments, the method comprises: administering to the individual a compound of formula (I); a conjugate formed between: a compound of formula (I), provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15 at least one of which is a linker; and one or more of the group consisting of a small molecule, an antigen, a microparticle, a nanoparticle, a polymer, a peptide, a protein, an antibody or an antibody fragment, a nucleic acid, a hormone and a growth factor; or a pharmaceutical composition of the compound or conjugate; and irradiating at least a portion of the individual with light, optionally wherein the disease is a hyperproliferative disease, further optionally wherein the disease is cancer.

[0026] In some embodiments, the presently disclosed subject matter provides water-soluble chlorin dyes having a solubility in aqueous solution greater than about 1 mg / ml, optionally having a solubility in aqueous solution of about 2.5 mg / ml or more; further optionally having a solubility in aqueous solution of about 10 mg / ml or more.

[0027] In some embodiments, the presently disclosed subject matter provides methods of preparing synthetic intermediates for compounds of formula (I):

[0028]

[0029] Where: M is a metal or two hydrogens (i.e. -H, -H); R5, R 10 and R 15 Independently selected from H, alkoxy and a linking group having the formula: -L1-(X1-L2) p -G, wherein p is 0 or 1; L1 is an alkylene group; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) wherein q is an integer from 1 to 24 q -alkylene-, alkylene or substituted alkylene, optionally wherein the substituted alkylene is alkylene substituted by one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bioconjugable group; and R2, R3, R 12 and R 13 independently selected from H, halogen, cyano, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, having the formula -L1-(X1-L2) p -G linking group and a solubilizing group, wherein the solubilizing group is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w, wherein w is an integer from 0 to 5, inclusive, and Rs is a group having the formula: -X2-(L3) z -R 17 , wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH- or triazolyl; L3 is -C(=O)-alkylene-C(=O)-NH-, and R 17 Selected from -(C2H4O) m -R 18 、-C(=O)C2H4-(OC2H4) m OR 18 and -(C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, wherein m is an integer of 4 or greater, optionally wherein m is an integer of 8 or greater; n is an integer of 1 to 5; R 18 is lower alkyl, optionally methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; The conditions are: R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w ; wherein the method comprises: (a) providing a compound having formula (I'):

[0030]

[0031] Where: M is a metal or -H, -H; R5', R 10 ' and R 15 ' is independently selected from H, alkoxy,

[0032] and

[0033] R2', R3', R 12 ' and R 13 ' are each independently selected from H, ester, carboxylic acid, formyl, acetyl,

[0034]

[0035] The conditions are: R2', R3', R 12 ' and R 13 At least one of '

[0036] as well as

[0037] (b) contacting the compound provided in step (a) with a dioxane solution comprising 4 molar (M) HCl to provide a compound of formula (I"):

[0038]

[0039] Where: M is metal or -H, -H; R5", R 10 ” and R 15 " is independently selected from H, alkoxy,

[0040]

[0041] And R2”, R3”, R 12 ” and R 13 " is independently selected from H, ester, carboxylic acid, formyl, acetyl,

[0042]

[0043]

[0044] The conditions are: R2”, R3”, R 12 ” and R 13 At least one of the

[0045]

[0046] In some embodiments, the compounds of the presently disclosed subject matter are:

[0047]

[0048] Or a pharmaceutical composition comprising the same or a conjugate thereof. In some embodiments, the compound is conjugated to one or more of the group consisting of small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones and growth factors.

[0049]

[0013] Accordingly, one object of the presently disclosed subject matter is to provide chlorin derivatives, in some embodiments water-soluble chlorin derivatives, conjugates thereof, pharmaceutical compositions comprising the derivatives and / or conjugates, and methods of using and preparing the same.

[0050] These and other purposes are achieved in whole or in part by the subject matter disclosed herein. Further, the purposes of the subject matter disclosed herein, other purposes and advantages of the subject matter disclosed herein, which have been stated above, will be apparent to those skilled in the art upon examination of the following description, drawings and embodiments.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1

[0046] Scheme 1 is an exemplary route to representative WH building blocks for the synthesis of dibromo-chlorin derivatives of the presently disclosed subject matter.

[0053] Figure 2

[0066] Scheme 2 is an exemplary route to representative EH building blocks for the synthesis of dibromo-chlorin derivatives of the presently disclosed subject matter.

[0054] Figure 3 is Scheme 3, an exemplary route for synthesizing compound CP-1.

[0055] Figure 4 is Scheme 4, an exemplary route for synthesizing the western half of Compound C-1.

[0056] Figure 5 Scheme 5 is an exemplary route for synthesizing the eastern half of compound C-1 and the cyclized intermediate compound C-11.

[0057] Figure 6 is Scheme 6, an exemplary route for the final step of synthesizing Compound C-1.

[0058] Figure 7 is Scheme 7, an exemplary route for the final step in the synthesis of Compound 4. DETAILED DESCRIPTION OF THE INVENTION

[0060] The presently disclosed subject matter will now be described more fully hereinafter, in which some, but not all, embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0061] I. Definitions

[0062] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

[0063] While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0064] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those of ordinary skill in the art. References to the technology employed herein are intended to represent technology commonly understood in the art, including variations to those technologies or substitutions of equivalent technologies apparent to those skilled in the art. Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth for ease of explanation of the subject matter of the present disclosure.

[0065] In describing the presently disclosed subject matter, it should be understood that a number of techniques and steps are disclosed, each of which has separate benefits, and each may also be used in combination with one or more or in some cases all other disclosed techniques.

[0066] Therefore, for the sake of clarity, this specification will avoid repeating every possible combination of individual steps in an unnecessary manner. However, the specification and claims should be read with the understanding that such combinations are fully within the scope of the invention and claims.

[0067] In accordance with long-standing patent law conventions, the terms "a," "an," and "the" when used in this application (including the claims) mean "one or more." For example, the phrase "fluorescent particles and / or nanoparticles" means one or more fluorescent particles and / or nanoparticles, including multiple identical fluorescent particles and / or nanoparticles. Similarly, the phrase "at least one," when used herein to refer to an entity, means, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more entities, including but not limited to integer values ​​from 1 to 100 and greater than 100.

[0068] Unless otherwise indicated, all numerical values ​​used in the specification and claims expressing amounts of ingredients, reaction conditions, and the like are to be understood as being modified in all instances by the term "about". When referring to a measurable value such as mass, weight, time, volume, concentration, or percentage, the term "about" is meant to include variations from the specified amount, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1%, as such variations are suitable for practicing the disclosed methods. Therefore, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the presently disclosed subject matter.

[0069] As used herein, the term "and / or" when used in the context of a list of entities refers to the entities present individually or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0070] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term that means the specified elements and / or steps are present, but other elements and / or steps may be added and still fall within the scope of the relevant subject matter.

[0071] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specifically described. It is noteworthy that when the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the elements listed in that clause; other elements are not excluded from the claim as a whole.

[0072] As used herein, the phrase "consisting essentially of" limits the scope of the associated disclosure or claim to the specified materials and / or steps and those that do not materially affect the basic and novel characteristics disclosed and / or claimed subject matter. For example, fluorescent microparticles and / or nanoparticles may "consist essentially of a polymer matrix and at least one chlorin associated therewith," meaning that the polymer matrix is ​​the only polymer matrix in which the fluorescent microparticles and / or nanoparticles are present.

[0073] With respect to the terms "comprising," "consisting of," and "consisting essentially of," when any of these three terms are used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. For example, in some embodiments, the presently disclosed subject matter relates to fluorescent microparticles and / or nanoparticles. One of ordinary skill in the art, upon reading this disclosure, will understand that the presently disclosed subject matter thus encompasses fluorescent microparticles and / or nanoparticles consisting essentially of a polymer matrix of the presently disclosed subject matter associated therewith and at least one chlorin associated therewith, as well as fluorescent microparticles and / or nanoparticles consisting of a polymer matrix of the presently disclosed subject matter and at least one chlorin associated therewith.

[0074] As used herein, "halogen" refers to any suitable halogen, including -F, -Cl, -Br, and -I.

[0075] As used herein, "thiol" refers to a -SH group.

[0076] As used herein, "azido" refers to a -N3 group.

[0077] As used herein, "cyano" refers to a -CN group.

[0078] As used herein, "hydroxy" refers to an -OH group.

[0079] As used herein, "nitro" refers to a -NO2 group.

[0080] As used herein, "alkyl" alone or as part of another group refers to a straight or branched chain hydrocarbon containing 1 or 2 to 10, 20 or 50 carbon atoms (e.g., C1 to C4 alkyl; C4 to C 10 Alkyl; C 11 To C 50 The term "alkyl" refers to a group consisting of a alkyl radical having a carbon atom or a branched chain. ... Unless otherwise indicated, the term "alkyl" or "lower alkyl" is intended to include substituted and unsubstituted alkyl and substituted and unsubstituted lower alkyl, and these groups may be substituted by groups selected from the group consisting of halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocycloalkyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkyloxy, cycloalkylalkoxy, aryloxy, arylalkoxy, heterocyclyloxy, heterocycloalkyloxy, mercapto, alkyl-S(O) m 、Haloalkyl-S(O) m 、Alkenyl-S(O) m , Alkynyl-S(O) m 、Cycloalkyl-S(O) m 、Cycloalkylalkyl-S(O) m 、Aryl-S(O) m 、Arylalkyl-S(O) m 、Heterocyclyl-S(O) m 、Heterocycloalkyl-S(O) m, amino, carboxyl, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocyclic amino, heterocyclic alkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, wherein m=0, 1, 2 or 3.

[0081] As used herein, "alkylene" refers to a difunctionalized straight chain, branched chain or cyclic alkyl group, which may be substituted or unsubstituted, and wherein "alkyl" is as defined above.

[0082] As used herein, "alkenyl" alone or as part of another group refers to a straight or branched chain hydrocarbon containing 1 or 2 to 10, 20 or 50 carbon atoms (e.g., C1 to C4 alkenyl; C4 to C 10 Alkenyl; C H To C 50 The term "alkenyl" or "lower alkenyl" refers to alkyl, alkyl, or lower alkenyl (1-4 carbon atoms), which contains 1-4 double bonds in the positive chain. Representative examples of alkenyl include but are not limited to vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadienyl, etc. Unless otherwise indicated, the term "alkenyl" or "lower alkenyl" is intended to include substituted and unsubstituted alkenyl or lower alkenyl, and these groups can be substituted by the groups described above for alkyl and lower alkyl.

[0083] As used herein, "alkenylene" refers to a difunctionalized straight chain, branched chain or cyclic alkenyl group, which may be substituted or unsubstituted, and wherein "alkenyl" is as defined above.

[0084] As used herein, "alkynyl" as used alone or as part of another group refers to a straight or branched chain hydrocarbon (e.g., C1 to C4 alkynyl; C4 to C 10 Alkynyl; C 11 To C 50 Alkynyl, or lower alkynyl 1-4 carbon atoms), which contains 1 triple bond in the positive chain. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, etc. Unless otherwise indicated, the term "alkynyl" or "lower alkynyl" is intended to include substituted and unsubstituted alkynyl or substituted and unsubstituted lower alkynyl, and these groups may be substituted with the same groups as described above for alkyl and lower alkyl.

[0085] As used herein, "alkynylene" refers to a difunctionalized straight chain, branched or cyclic alkynyl group, which may be substituted or unsubstituted, and wherein "alkynyl" is as defined above.

[0086] As used herein, "alkylidene chain" refers to a difunctionalized straight chain, branched chain and / or cyclic organic group that may be substituted or unsubstituted, saturated or unsaturated, and may optionally contain one, two or three heteroatoms selected from the group consisting of N, O and S. Examples include, but are not limited to, alkylene, alkenylene, alkynylene, arylene, alkarylene and aralkylene. See, e.g., U.S. Pat. No. 6,946,533. The alkylidene chain may contain any suitable number of carbon atoms (e.g., C1 to C4; C4 to C 10 ; C 10 To C 20 ; C 20 To C 50 ).

[0087] "Alkoxy" as used herein alone or as part of another group refers to an alkyl or lower alkyl group as defined herein attached to the parent molecular moiety through an oxy-O- group. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0088] "Acyl" as used herein alone or as part of another group refers to a -C(O)R group, wherein R is any suitable substituent, such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl, or other suitable substituents described herein.

[0089] "Haloalkyl" as used herein alone or as part of another group refers to at least one halogen as defined herein attached to a parent molecular moiety via an alkyl as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, etc.

[0090] "Perhaloalkyl" used alone or as part of another group herein refers to an alkyl group in which each hydrogen atom in the alkyl group is replaced by halogen. In some embodiments, the perhaloalkyl is an alkyl group in which each hydrogen atom in the alkyl group is replaced by fluorine. Representative perhaloalkyl is trifluoromethyl (i.e. -CF3).

[0091] "Alkylthio" as used herein alone or as part of another group refers to an alkyl group as defined herein attached to the parent molecular moiety through a thio moiety as defined herein. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

[0092] "Aryl" as used herein alone or as part of another group refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, etc. Unless otherwise indicated, the term "aryl" is intended to include substituted and unsubstituted aryl groups, and these groups may be substituted with the same groups as described above for alkyl and lower alkyl.

[0093] As used herein, "alkylene" is a difunctionalized aryl group which may be substituted or unsubstituted, and wherein "aryl" is as defined above.

[0094] "Aralkyl" as used herein alone or as part of another group refers to an aryl group as defined herein attached to a parent molecular moiety via an alkyl group as defined herein. Representative examples of aralkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphthalen-2-ylethyl, and the like.

[0095]

[0046] "Alkarylene" and "aralkylene" as used herein alone or as part of another group refer to a difunctional group comprising at least one arylene group and at least one alkyl, alkenyl or alkynyl group as defined herein.

[0096] As used herein, "amino" refers to the group -NH2.

[0097]

[0066] "Alkylamino" as used herein alone or as part of another group refers to the group -NHR, wherein R is alkyl.

[0098]

[0066] "Aralkylamino" as used herein alone or as part of another group refers to the group -NHR, where R is arylalkyl.

[0099] As used herein, "disubstituted amino" as used alone or as part of another group refers to the group -NR a R b , where R a and R b Independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl and heterocycloalkyl.

[0100] As used herein, "amido" as used alone or as part of another group refers to the group -NR a R b , where R a is acyl as defined herein, and R b is selected from the group consisting of hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl and heterocycloalkyl.

[0101]

[0066] "Acyloxy" as used herein alone or as part of another group refers to the group -OR, wherein R is acyl as defined herein.

[0102]

[0046] "Ester" as used herein alone or as part of another group refers to a -C(O)OR group, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0103] As used herein, "formyl" refers to a -C(O)H group.

[0104] As used herein, "carboxylic acid" refers to a -C(O)OH group.

[0105] As used herein, "sulfoxyl" refers to a compound of the formula -S(O)R, wherein R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0106] As used herein, "sulfonyl" refers to a compound of the formula -S(O)(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0107] As used herein, "sulfonate" refers to a compound of the formula -S(O)(O)OR, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0108] As used herein, "sulfonic acid" refers to a compound of formula -S(O)(O)OH.

[0109] As used herein, "amide" as used alone or as part of another group refers to -C(O)NR a R b Group, where R a and R b is any suitable substituent, for example H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0110] "Sulfonamide" alone or as part of another group refers to -S(O)2NR a R b Group, where R a and R b is any suitable substituent, for example H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0111] "Urea" alone or as part of another group refers to -N(R c )C(O)NR a R b Group, where R a , Rb and R c is any suitable substituent, for example H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0112] "Alkoxyamido" alone or as part of another group refers to -N(R a )C(O)OR b Group, where R a , R b is any suitable substituent, for example H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0113] "Aminoacyloxy" by itself or as part of another group refers to -OC(O)NR a R b Group, where R a and R b is any suitable substituent, for example H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0114] "Cycloalkyl" used alone or as part of another group herein refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3, 4, 5, 6, 7 or 8 carbons (as discussed below, in heterocyclic groups, these carbons can be replaced). Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. These rings can be optionally substituted by other substituents as described herein, such as halogen or low alkyl. Unless otherwise indicated, the term "cycloalkyl" is general and is intended to include heterocyclic groups as discussed below.

[0115] As used herein, the term "polyoxyethylene chain" refers to a poly(ethylene glycol) (PEG) group (such as, but not limited to, a poly(ethylene glycol) group having the formula -(C2H4O) n -group, wherein n is an integer of 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 or more). In some embodiments, n is an integer of 4 to 5000, 4 to 1000, 4 to 100, 4 to 50, 4 to 28 or 4 to 25. As used herein, the term "polyoxyethylene chain" may refer to monodisperse or polydisperse PEG chains and linear or branched PEG chains. "Monodisperse" refers to PEG with a polydispersity index (PDI) of 1, while polydisperse refers to PEG with a PDI greater than 1, wherein PEG includes a Gaussian distribution of chain lengths and molecular weights.

[0116] As used herein, the term "bioconjugable group" refers to a reactive chemical functional group that can form a bond (e.g., a covalent bond) with a group on another entity (e.g., a protein; a peptide; a targeting agent such as an antibody or antibody fragment; a polymer; a particle such as a nanoparticle, an organic bead, a polymer bead, or an inorganic bead; another solid support surface, etc.) to form a conjugate of one of the presently disclosed chlorin or bacteriochlorin compounds and the other entity. For example, the bioconjugable group can be an aldehyde, which can form a covalent bond with an amino group on an amino-substituted biomolecule by reductive amination, or a carboxylic acid, which can be coupled to an amino-substituted biomolecule by carbodiimide activation. The bioconjugable groups include amines (including amine derivatives) such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, and the like; carboxylic acids or acid derivatives such as N-hydroxysuccinimide (NHS) esters (more generally, active esters derived from carboxylic acids; e.g., p-nitrophenyl esters), acid hydrazides, and the like; and other groups such as, but not limited to, aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyls, thiols, maleimides, aziridines, acryloyls, halogen groups, biotin, 2-iminobiotin, and the like.

[0117] The term "microparticle" refers to a structure having at least one region whose size (e.g., length, width, diameter, etc.) is less than about 1,000 μm but greater than about 1000 nm. In some embodiments, the size can be less than about 500 μm, in some embodiments less than about 250 μm, in some embodiments less than about 200 μm, in some embodiments less than about 150 μm, in some embodiments less than about 125 μm, in some embodiments less than about 100 μm, in some embodiments less than about 80 μm, in some embodiments less than about 70 μm, in some embodiments less than about 60 μm, in some embodiments less than about 50 μm, in some embodiments less than about 40 μm, in some embodiments less than about 30 μm, in some embodiments less than about 20 μm, in some embodiments less than about 10 μm, and in some embodiments less than about 5 μm. In some embodiments, the size is from about 1 μm to about 250 μm (e.g., 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm).

[0118] Similarly, the term "nanoparticle" refers to a structure having at least one region whose dimension (eg, length, width, diameter, etc.) is less than about 1,000 nm. In some embodiments, the size is smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, or even less than about 20 nm. In some embodiments, the size is from about 5 nm to about 250 nm (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm).

[0119] In some embodiments, the microparticle or nanoparticle is approximately spherical. When the microparticle or nanoparticle is approximately spherical, the characteristic size can correspond to the diameter of the sphere. In addition to spherical, the microparticle or nanoparticle can be disc-shaped, plate-shaped (e.g., hexagonal plate-shaped), elliptical, polyhedral, rod-shaped, cubic or irregular.

[0120] The microparticle or nanoparticle may comprise a core region (i.e., the space between the outer dimensions of the particle) and an outer surface (i.e., the surface defining the outer dimensions of the particle). In some embodiments, the microparticle or nanoparticle may have one or more coatings surrounding or partially surrounding the microparticle or nanoparticle core. Thus, for example, a spherical microparticle or nanoparticle may have one or more concentric coatings, with each successive layer dispersed on the outer surface of a smaller layer closer to the center of the particle.

[0121] The terms "polymer" and "polymeric" refer to a chemical structure having repeating units (i.e., multiple copies of a given chemical substructure). A polymer can be formed from a polymerizable monomer. A polymerizable monomer is a molecule comprising one or more groups that can react with groups on other polymerizable monomer molecules to form a bond (e.g., a covalent bond or a coordination bond). In some embodiments, each polymerizable monomer molecule can be bonded to two or more other molecules / groups. In some cases, a polymerizable monomer will only bond to another molecule to form the end of a polymeric material.

[0122] Polymer can be organic, or inorganic, or any combination thereof. As used herein, the term "inorganic" refers to a compound or composition containing at least some atoms other than one of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorus or halides. Thus, for example, an inorganic compound or composition can contain one or more silicon atoms and / or one or more metal atoms. In some embodiments, the polymer is polystyrene, and the microparticles and / or nanoparticles are made of polystyrene. In some embodiments, the microparticles and / or nanoparticles are polystyrene beads.

[0123] As used herein, the term "porphyrin" refers to a cyclic structure generally composed of four pyrrole rings and four nitrogen atoms and two replaceable hydrogens (various metal atoms can be easily substituted). A representative porphyrin is hemin.

[0124] As used herein, the term "chlorin" is distinguished from porphyrin by having a partially saturated pyrrole ring. The basic chromophore of chlorophyll (the green pigment of plant photosynthesis) is chlorin. The terms "chlorin" and "chlorin derivative" are used interchangeably herein.

[0125] The phrase "association" refers to any interaction between two entities, such as a polymer matrix and a chlorin. In some embodiments, the polymer matrix and the chlorin are associated with each other by non-covalent bonds, such as but not limited to one or more of hydrophobic, electrostatic and van der Waals interactions. In some embodiments, because the polymer matrix (e.g., nanoparticles, microparticles, beads, etc.) comprises chlorin, the chlorin is present in the polymer matrix, so that the polymer matrix and the chlorin are associated with each other. In such embodiments, the polymer matrix is ​​also referred to as "doped by" or "doped with" chlorin, and the chlorin can be considered to be "embedded" in the polymer matrix. In some embodiments, the polymer matrix and the chlorin are associated with each other by covalent bonds (the surface of the chlorin is connected to the polymer matrix).

[0126] As used herein, "treating" refers to any manner in which one or more symptoms of a disease or condition are ameliorated or otherwise beneficially altered. Treatment also includes any pharmaceutical use of the compositions herein, such as for the treatment of a disease or condition mediated by or involving hyperproliferative tissue or neovascularization. As used herein, amelioration of the symptoms of a particular condition by administration of a particular compound or pharmaceutical composition refers to any relief attributable to or associated with the administration of the composition, whether permanent or temporary, lasting or transient.

[0127] As used herein, a "prodrug" is a compound that, after in vivo administration, is metabolized or otherwise converted by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

[0128] As used herein, "antibody" generally refers to an immunoglobulin or fragment thereof that specifically binds to an antigen to form an immune complex.Antibodies can be complete immunoglobulins of any kind, for example, IgG, IgM, IgA, IgD, IgE, chimeric or hybrid antibodies with dual or multiple antigen or epitope specificities.It can be a polyclonal antibody, preferably an affinity-purified antibody from a human or suitable animal (e.g., primate, goat, rabbit, mouse, etc.).Monoclonal antibodies are also suitable for the subject matter disclosed at present and can be preferred due to their high specificity.They are easily prepared by the following conventional operations now considered: immunizing mammals with immunogenic antigen preparations, fusing immune lymphocytes or spleen cells with immortal myeloma cell lines, and isolating specific hybridoma clones.More unconventional methods for preparing monoclonal antibodies are not excluded, such as interspecies fusion and genetic engineering operations of hypervariable regions, because the main factor affecting their effectiveness is the antigen specificity of the antibody.Newer monoclonal production techniques can also be used, such as human monoclonal, interspecies monoclonal, chimeric (e.g., human / mouse) monoclonal, genetically engineered antibodies, etc.

[0129] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. Immunoglobulin genes typically include kappa (κ), lambda (λ), alpha (α), gamma (γ), delta (δ), epsilon (ε) and mu (μ) constant region genes, as well as countless immunoglobulin variable region genes. Light chains are classified as κ or λ. In mammals, heavy chains are classified as γ, μ, α, δ or ε, which in turn define immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Other species have other light and heavy chain genes (e.g., so-called IgY produced by some birds, which is the type of immunoglobulin left by hens in their yolks), which are similarly included in the subject matter disclosed herein.

[0130] It is known that representative immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (average molecular weight of about 25 kilodaltons (kDa)) and a "heavy" chain (average molecular weight of about 50-70 kDa). The two identical polypeptide chain pairs are held together in dimer form by disulfide bonds present in the heavy chain region. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The term variable light chain (V L) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0131] Antibodies usually exist as complete immunoglobulins or in a number of well-characterized fragments (which can be produced by digestion with various peptidases). For example, an antibody molecule is digested with papain to cut the antibody at a position near the N-terminus of a disulfide bond. This produces three fragments: two identical "Fab" fragments, which have the N-termini of the light chain and the heavy chain, and an "Fc" fragment, which includes the C-terminus of the heavy chain held together by a disulfide bond. On the other hand, pepsin digests the antibody near the C-terminus of the disulfide bond in the hinge region, producing a fragment called a "F(ab)'2" fragment, which is a dimer of Fab fragments connected by disulfide bonds. The F(ab)'2 fragment can be reduced under mild conditions to destroy the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into two "Fab'" monomers. The Fab' monomer is essentially a Fab fragment with a partial hinge region. With regard to these various fragments, Fab, F(ab')2 and Fab' fragments include at least one complete antigen binding domain (called a "paratope") and are therefore able to bind to antigens.

[0132] Although various antibody fragments are defined in terms of digestion of intact antibodies, the skilled artisan will appreciate that various of these fragments (including but not limited to Fab' fragments) can be synthesized de novo chemically or by utilizing recombinant DNA methods. Thus, the term "antibody" as used herein also includes antibody fragments produced by modifying intact antibodies, or antibody fragments synthesized de novo using recombinant DNA methods. In some embodiments, the term "antibody" includes fragments having at least one antigen binding domain.

[0133] The antibody, fragment and derivative of the current disclosed theme may also include chimeric antibodies.As used herein in the context of antibodies, the term "chimeric" and grammatical variants thereof refer to antibody derivatives with constant region and variable region, the constant region being substantially or completely derived from the antibody constant region from a species, and the variable region being substantially or completely derived from the sequence of the variable region from another species. The chimeric antibody of a particular kind is a "humanized" antibody, wherein the antibody is produced by replacing the CDR of a human antibody with the complementary determining region (CDR) of, for example, a mouse antibody (see, for example, PCT International Patent Application Publication No. WO 1992 / 22653). Therefore, in some embodiments, humanized antibodies have constant regions and variable regions substantially or completely derived from corresponding human antibody regions except CDR, and CDR substantially or completely derived from mammals except humans.

[0134] The antibodies, fragments and derivatives of the currently disclosed subject matter can also be single-chain antibodies and single-chain antibody fragments. Single-chain antibody fragments contain an amino acid sequence having at least one variable region and / or CDR of a complete antibody as described herein, but lack some or all of the constant domains of those antibodies. These constant domains are not essential for antigen binding, but constitute a major part of the complete antibody structure.

[0135] Single-chain antibody fragments can overcome some problems associated with using antibodies containing part or all of the constant domains. For example, single-chain antibody fragments tend to have no undesirable interactions between biomolecules and heavy chain constant regions, or other unwanted biological activities. In addition, single-chain antibody fragments are much smaller than complete antibodies, and therefore can have greater capillary permeability than complete antibodies, allowing single-chain antibody fragments to more effectively locate and bind to target antigen binding sites. Moreover, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thereby facilitating their production. In addition, relatively small-sized single-chain antibody fragments make them less likely to cause an immune response in receptors than complete antibodies. The single-chain antibody fragments of the current disclosed subject matter include, but are not limited to, single-chain fragment variable (scFv) antibodies and derivatives thereof, such as, but not limited to, tandem two-scFv, tandem three-scFv, double antibodies (including bispecific double antibodies), three antibodies, four antibodies, miniantibodies, microbodies, tetravalent bispecific molecules, bispecific F (ab ') 2 fragments, etc.

[0136] As used herein, "infectious agent" means an invading microorganism or parasite. As used herein, "microorganism" means viruses, bacteria, rickettsiae, mycoplasmas, protozoa, fungi and similar microorganisms, and "parasite" means an infectious, usually microscopic or very small multicellular invertebrate that is susceptible to antibody-induced clearance or lysis or phagocytic destruction, or its egg cell or juvenile form, such as malarial parasites, spirochetes, etc.

[0137] As used herein, "tumor" means a neoplasm and includes benign and malignant tumors. The term specifically includes malignant tumors that may be solid (such as breast cancer, liver cancer, or prostate cancer) or non-solid (e.g., leukemia). Tumors may also be further divided into subtypes, such as adenocarcinomas (e.g., adenocarcinomas of the breast, prostate, or lung).

[0138] As used herein, "target" refers to an object intended to be detected, diagnosed, damaged or destroyed by the methods provided herein, and includes target cells, target tissues, and target compositions.

[0139] As used herein, "target tissue" and "target cell" are those tissues that are intended to be damaged or destroyed by the treatment method. The photosensitive compound binds to or aggregates in these target tissues or target cells; then when sufficient radiation is applied, these tissues or cells are damaged or destroyed. Target cells are cells in target tissues, and target tissues include, but are not limited to, tumors, solid tumors (such as (but not limited to) tumors of the head and neck, tumors of the eyes, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, and lung tumors), non-solid tumor vascular endothelial tissues, abnormal blood vessel walls; and malignant cells of hematopoietic and lymphoid tissues, neovascular tissues; other lesions of the vascular system, bone marrow, and tissues or cells associated with autoimmune diseases. Target cells also include cells that undergo significantly faster division than non-target cells.

[0140] As used herein, "non-target tissues" are all tissues of a subject that are not intended to be damaged or destroyed by a therapeutic method. These non-target tissues include, but are not limited to, healthy blood cells and other normal tissues that are not otherwise identified as targets.

[0141] As used herein, "target compositions" are those compositions intended to be damaged or destroyed by the treatment method, and may include one or more pathogens, including but not limited to bacteria, viruses, fungi, protozoa, and toxins, and cells and tissues infected or infiltrated therewith. The term "target composition" also includes but is not limited to infectious organic particles, such as prions, toxins, peptides, polymers, and other compounds that can be selectively and specifically identified as organic targets intended to be damaged or destroyed by this treatment method.

[0142] As used herein, "hyperproliferative tissue" refers to tissue that grows out of control and includes neoplastic tissue, tumors, and unrestrained blood vessel growth, such as that found in age-related macular degeneration and that commonly occurs after glaucoma surgery.

[0143] As used herein, "hyperproliferative disorders" refers to those conditions in which excessive cell proliferation caused by unregulated or abnormal cell growth is the underlying pathology, and includes uncontrolled angiogenesis. Examples of such hyperproliferative disorders include, but are not limited to, cancer or carcinoma, acute and membranoproliferative glomerulonephritis, myeloma, psoriasis, atherosclerosis, psoriatic arthritis, rheumatoid arthritis, diabetic retinopathy, macular degeneration, corneal neovascularization, choroidal hemangioma, pterygium recurrence, and scarring caused by excimer laser surgery and glaucoma filtering surgery.

[0144] As used herein, a "therapeutically effective dose" is a dose sufficient to prevent progression of a disease or cause regression of a disease or to alleviate the symptoms caused by the disease.

[0145] As used herein, "biological material" refers to tissues (such as biopsy tissue) and cells, as well as biological fluids such as blood, urine, plasma, cerebrospinal fluid, mucus, saliva, and the like.

[0146] As used herein, "irradiating" and "irradiation" include exposing an individual to all wavelengths of light. Preferably, the irradiation wavelength is selected to match the wavelength that excites the photosensitive compound. Preferably, the irradiation wavelength matches the excitation wavelength of the photosensitive compound and has low absorption by non-target tissues of the individual, including blood proteins.

[0147] Irradiation is further defined herein by its coherence (laser) or incoherence (non-laser) as well as intensity, duration and timing associated with administration using a photosensitive compound. The intensity or fluence rate must be sufficient to allow light to reach the target tissue. The duration or total fluence dose must be sufficient to allow the photosensitive compound to be sufficiently photosensitized to act on the target tissue. The timing associated with the administration of a photosensitive compound is important because 1) the administered photosensitive compound requires some time to locate the target tissue, and 2) the blood levels of many photosensitive compounds decrease over time. The radiant energy is provided by an energy source such as a laser or a cold cathode light source, which is located outside the individual, or implanted in the individual, or introduced into the individual, such as by a catheter, an optical fiber, or by taking a light source in the form of a capsule or pill (e.g., as disclosed in U.S. Patent No. 6,273,904).

[0148] Although some embodiments of the presently disclosed subject matter involve the use of light energy to implement photodynamic therapy (PDT) to destroy tumors, other energy forms are also within the scope of the presently disclosed subject matter, as will be understood by one of ordinary skill in the art. Such energy forms include, but are not limited to, thermal energy, acoustic energy, ultrasonic energy, chemical energy, light energy, microwave energy, ionizing energy (such as x-rays and gamma rays), mechanical energy, and electrical energy. For example, sonodynamically induced or activated agents include, but are not limited to, gallium-porphyrin complexes (see Yumita et al. (1997) Cancer Letters 112: 79-86), other porphyrin complexes, such as protoporphyrin and hematoporphyrin (see Umemura et al. (1996) Ultrasonics Sonochemistry 3: S187-S191); other anticancer drugs used in the presence of ultrasound therapy, such as daunorubicin and doxorubicin (see Yumita et al. (1987) Japanese Journal of Hyperthermic Oncology 3 (2): 175-182).

[0149] As used herein, "coupling agent" refers to an agent capable of coupling a photosensitizer to a targeting agent.

[0150] A "targeting agent" is a compound that is designed to localize or preferentially associate or bind to a specific tissue, receptor, infectious agent, or other area of ​​the body of the individual to be treated, such as a target tissue or target composition. Examples of targeting agents include, but are not limited to, antibodies, ligands, a member of a ligand-receptor binding pair, nucleic acids, peptide-nucleic acids (PNAs), aptamers, proteins and peptides, and liposomal suspensions, including tissue-targeted liposomes.

[0151] As used herein, "specific binding pair" and "ligand-receptor binding pair" refer to two different molecules, one of which has an area on the surface or in a cavity that specifically attracts or binds to a specific spatial or polar organization of the other molecule, resulting in the two molecules having an affinity for each other. The members of the specific binding pair are referred to as ligand and receptor (anti-ligand). The terms ligand and receptor are intended to include the entire ligand or receptor or part thereof sufficient for binding to occur between the ligand and receptor. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor-receptor, and interferons and interferon receptors; avidin and biotin or anti-biotin; antibody and antigen pairs; enzymes and substrates, drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; nucleic acid strands and complementary oligonucleotides; interleukins and interleukin receptors; and stimulatory factors and their receptors, such as granulocyte-macrophage colony stimulating factor (GMCSF) and GMCSF receptor and macrophage colony stimulating factor (MCSF) and MCSF receptor.

[0152] A "linker" is an aromatic or aliphatic group (which may be substituted or unsubstituted and may optionally contain heteroatoms such as N, O or S) that is used to couple a bioconjugable group, a cross-coupling group, a surface-linking group, a hydrophilic group, etc. to a parent molecule. Examples include, but are not limited to, aryl, alkyl, heteroaryl, heteroalkyl (e.g., oligoglycol), peptide and polysaccharide linkers, and the like.

[0153] Subjects treated by the methods of the presently disclosed subject matter for diagnostic or therapeutic purposes include human subjects and other animal subjects used for veterinary purposes (particularly mammalian subjects such as dogs, cats, horses, monkeys, chimpanzees, etc.).

[0154] More specifically, as used herein, the terms "subject," "patient," and "recipient" may be used interchangeably and may refer to a member of any invertebrate or vertebrate species. Thus, the term "subject" is intended to encompass any member of the animal kingdom, including, but not limited to, members of the phylum Chordata (e.g., Osteichthyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all orders and families subsumed therein.

[0155] The compositions and methods of the currently disclosed subject matter are particularly suitable for warm-blooded vertebrates. Therefore, the currently disclosed subject matter relates to mammals and birds. More specifically, compositions and methods derived from and / or used for mammals such as humans and other primates, and those mammals that are important due to endangerment (such as Siberian tigers), mammals that are economically important (animals raised on farms for human consumption) and / or socially important (animals raised as pets or in zoos) to humans, for example, carnivores (such as cats and dogs), swine (piglets, pigs and wild boars), ruminants (such as cattle, bulls, sheep, giraffes, deer, goats, bison and camels), rodents (such as mice, rats, hamsters, guinea pigs and rabbits), marsupials and horses are provided. Also provided are uses of the disclosed methods and compositions on birds, including those species of birds that are endangered, kept in zoos or as pets (e.g., parrots, cockatiels, etc.), and birds, more particularly domesticated birds, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, etc., because they are also of economic importance to humans. Thus, also provided are uses of the disclosed methods and compositions on livestock, including, but not limited to, domesticated swine (piglets and pigs), ruminants, horses, poultry, etc.

[0156] II .two Chlorin compounds

[0157] In some embodiments, the currently disclosed subject provides a chlorin derivative. In some embodiments, the chlorin derivative of the currently disclosed subject is a water-soluble chlorin derivative, wherein the chlorin derivative has a solubility of about 1 mg / ml (mg / mL) or higher in an aqueous solution (e.g., water, saline, PBS, etc.). For example, water solubility is provided by adding a solubilizing group comprising a PEG chain at the β-pyrrole position. In some embodiments, the PEG chain is connected to the chlorin and / or is longer than the PEG chain connected to the previously described PEGylated chlorin compound by different groups. In some embodiments, the currently disclosed chlorin has a solubility of about 2.5 mg / mL or higher in an aqueous solution. In some embodiments, chlorin has a solubility of about 5.0 mg / mL or higher in an aqueous solution. In some embodiments, chlorin has a solubility of about 10 mg / mL or higher in an aqueous solution. In some embodiments, the chlorin has a solubility in aqueous solution of about 700, 800, or 900 μM or more. In some embodiments, the chlorin has a solubility in aqueous solution of about 1, 1.5, 2, 2.5, or 3.0 mM or more.

[0158] In some embodiments, chlorin derivatives (including but not limited to water-soluble chlorin derivatives) include a linker portion containing a bioconjugable group, which can be used to conjugate the chlorin derivative to another substance, for example, another substance that can serve as a targeting agent or a substance to be detected. In some embodiments, the substance that can be conjugated to the chlorin derivative can be a small molecule (e.g., a non-polymeric synthetic molecule with a molecular weight of about 900 Daltons (Da) or less), an antigen, a microparticle, a nanoparticle, a polymer, a peptide, a protein, an antibody or an antibody fragment, a nucleic acid, a hormone, or a growth factor. The bioconjugable group can include, for example, a carboxylic acid or an active ester, a hydroxyl, an amine, a thiol, or an aldehyde. In some embodiments, the linker portion further includes an arylene group and an alkylene group. In some embodiments, the linker portion includes an arylene group and / or an alkynylene group close to the main chlorin structure, while the alkylene group is close to the bioconjugable group.

[0159] The currently disclosed chlorin may include at least one solubilizing group. In some embodiments, the solubilizing group includes one or more polyoxyethylene chains (e.g., PEG chains). In some embodiments, the solubilizing group includes at least two PEG chains. In some embodiments, the PEG chain is monodisperse and includes at least 4-CH2CH2O-repeating units. In some embodiments, the PEG chain includes at least 6, 8, 10 or 12-CH2CH2O-repeating units. Therefore, the solubilizing group may include two PEG6, PEG8, PEG10 or PEG12 groups. In some embodiments, the PEG chain includes 12 or more-CH2CH2O-repeating units (e.g., about 12 to about 24 or about 28-CH2CH2O-repeating units). In some embodiments, the compound includes two solubilizing groups connected to two different pyrrole carbon atoms. In some embodiments, each of the two solubilizing groups includes two PEG chains.

[0160] In some embodiments, the solubilizing group is a β-pyrrole substituent comprising an arylene or alkynyl-arylene group directly attached to a β-pyrrole carbon atom, but wherein the group does not contain an oxo linker directly between the PEG chain and the aryl group. In some embodiments, the solubilizing group comprises one or more amide bonds between the aryl group and the PEG chain. In some embodiments, the amide bond further comprises one or more alkylene spacers (e.g., ethylene, propylene, etc.).

[0161] In some embodiments, the chlorin derivative is a compound of Formula (I):

[0162]

[0163] in:

[0164] M is a metal or two hydrogens (i.e. -H, -H);

[0165] R5, R 10 and R 15 Independently selected from H, alkoxy and a linking group having the formula: -L1-(X1-L2) p -G, wherein p is 0 or 1, L1 is an alkylene group, X1 is -C(=O)NH- or -NHC(=O)-, and L2 is -(CH2CH2O) wherein q is an integer from 1 to 24 q - alkylene, alkylene or substituted alkylene (e.g., alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group); and G is a bioconjugable group; and

[0166] R2, R3, R 12 and R 13 independently selected from H, halogen, cyano, perhaloalkyl (e.g., perfluoroalkyl, including but not limited to trifluoromethyl), sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, having the formula -L1-(X1-L2) p -G linking group and a solubilizing group, wherein the solubilizing group is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , wherein w is an integer from 0 to 5, inclusive, wherein when w is 0, the chlorin derivative is water-insoluble (i.e., hydrophobic), and wherein when w is 1, 2, 3, 4, or 5, the chlorin derivative is water-soluble (i.e., hydrophilic), and Rs is a group having the formula:

[0167] -X2-(L3) z -R 17 ,

[0168] wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH- or triazolyl; L3 is -C(=O)-alkylene-C(=O)-NH-, and R 17 Selected from -(C2H4O) m -R 18 、-C(=O)C2H4-(OC2H4) m OR 18 and (C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, wherein m is an integer of 4 or greater (e.g., at least 8, at least 10 or greater, or at least 12 or greater); n is an integer of 1 to 5; R 18 is lower alkyl (eg, methyl); and R19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; The conditions are: R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0169] M can be any suitable metal ion (e.g., Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, C o , Fe or Zn) or is absent (for example, in which case it is replaced by two hydrogens (-H, -H), i.e., the two pyrrole nitrogen atoms of the chlorin ring are protonated. In some embodiments, M is Zn or is replaced by -H, -H. Thus, compounds of formula (I) include metal chlorin derivatives and free base chlorin derivatives.

[0170] In some embodiments, R5, R 10 and R 15 Selected from H, methoxy and having the formula -L1-(X1-L2) p -G linker. In some embodiments, R5, R 10 and R 15 At least one of them has the formula -L1-(X1-L2) p -G linker. In some embodiments, R 10 It has the formula -L1-(X1-L2) p -G's linking group.

[0171] In some embodiments, L1 is an arylene or alkylarylene group (e.g., comprising an alkenyl or alkynyl group attached to an aryl group). In some embodiments, L1 is a phenylene group or -C≡C-phenyl-. In some embodiments, p is 1, and the linking group comprises a polyoxyethylene chain that improves solubility and / or a spacer group (e.g., an alkylene spacer group) that improves the reactivity of G (compared to the reactivity of G in a comparable chlorin in which G is directly attached to L1).

[0172] In some embodiments, the linking group (eg, R5, R 10 or R 15 The formula is -L1-(X1-L2) p-G, wherein L1 is phenylene, p is 1; X1 is -C(=O)NH-, L2 is alkylene, and G is selected from carboxylic acid or active ester (e.g., NHS-ester). In some embodiments, L2 is ethylene. Therefore, in some embodiments, the linking group includes a β-alanine spacer to improve the reactivity of G.

[0173] In some embodiments, the linker (eg, R5, R 10 or R 15 )for:

[0174]

[0175] In some embodiments, at least one of R2 and R3 and R 12 and R 13 One of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w Thus, in some embodiments, the compound of formula (I) comprises at least two solubilizing groups. For example, in some embodiments, R2 and R 12 Both are -aryl-(R s ) w or -alkynyl-aryl-(R s ) w In some embodiments, R3 and R 13 Each is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0176] In some embodiments, R3 and R 13 Each is -aryl-(R s ) w In some embodiments, R3 and R 13 Each is -phenyl-(R s )2.

[0177] In some embodiments, each R s With the formula -X2-(L3) z -R 17 , wherein: z is 0, x2 is -CH2NHC(=O)-, and R 17 -(C2H4O) m -R 18 , wherein m is an integer from 12 to 24 (i.e., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24).

[0178] In some embodiments, m is 12 and R 18 In some embodiments, R3 and R 13 Each is:

[0179]

[0180] In some embodiments, the compound is C-1, which has the following structure:

[0181]

[0182] In some embodiments, the compound is compound 4, which has the following structure:

[0183]

[0184] In some embodiments, the presently disclosed subject matter provides a covalent conjugate formed between: (a) a compound of formula (I) as defined above, and provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15 At least one of (a) is a linking group; and (b) includes one or more of the group consisting of small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones and growth factors. In some embodiments, for example, a conjugate can be formed by reacting a compound of formula (I) comprising a linking group containing a carboxylic acid or an active ester (i.e., as a bioconjugable group G) with an amino group of a small molecule, peptide, protein, antibody or polymer.

[0185] III. Synthesis Methods

[0186] Methods for synthesizing chlorins that can be suitable for preparing currently disclosed chlorins are described in, for example, U.S. Patents 8,212,055 and 8,980,565, each of which is incorporated by reference in its entirety. In some embodiments, suitable trans-β-substituted chlorins (such as chlorins in which two β-chlorin substituents are halogen (e.g., Br) substituents) can be prepared, and then the β-substituents are further reacted to replace them with suitable water-soluble groups to prepare compounds of formula (I) disclosed in the present invention. Methods for synthesizing trans-β-substituted chlorins have been previously described. See, for example, U.S. Patent 6,559,374, incorporated herein by reference in its entirety.

[0187] For example, in some embodiments, the chlorin of formula (I) can be prepared by condensing a dihydrodipyrrole building block representing the "western half" (wH) of the chlorin with a building block representing the "eastern half" (EH) of the chlorin. In some embodiments, the WH building block can have the following structure:

[0188]

[0189] Among them, S1, S2, S7, S9, S 12 and S 13 is independently selected from H, aryl, substituted aryl, phenyl, cycloalkyl, alkyl, substituted alkyl, alkenyl, alkynyl, halogen, alkoxy, alkylthio, perfluoroalkyl, perfluoroaryl, pyridyl, cyano, thiocyano, nitro, amino, alkylamino, acyl, sulfoxide, sulfonyl, imino, amide and carbamoyl; or wherein S7 and S 13 or wherein S1 and S2 together form a substituted or unsubstituted alkylene or arylene. 12 For H.

[0190] The EH building block compound may have the following structure:

[0191]

[0192] Among them, S3, S4, S5, S6, and S 11 independently selected from H, aryl, substituted aryl, phenyl, cycloalkyl, alkyl, substituted alkyl, alkenyl, alkynyl, halogen, alkoxy, alkylthio, perfluoroalkyl, perfluoroaryl, pyridyl, cyano, thiocyano, nitro, amino, alkylamino, acyl, sulfoxide, sulfonyl, imino, amide and carbamoyl; or wherein S6 and S5 together form a substituted or unsubstituted alkylene or arylene; and wherein Z is halogen (e.g., Br, Cl or I).

[0193] More specifically, methods for preparing dibromo-substituted chlorins and their corresponding bromo-substituted dihydropyrroles WH and EH building blocks have been previously described. See, for example, Krayer et al. (2009) Journal of Porphyrins and Phthalocyanines 13(10): 1098-1110; Jiang et al. (2014) Organic & Biomolecular Chemistry 12: 86-103. For example, chlorin derivatives containing two bromo substituents at positions 3 and 13 of the chlorin can be prepared from the WH building block, which is prepared, for example, from 2-formylpyrrole (e.g. Figure 1 as illustrated by route 1 shown in FIG.

[0194] like Figure 1 As illustrated in Scheme 1, 2-formylpyrrole (pyrrole-2-carboxaldehyde) is first brominated using N-bromosuccinimide (NBS) to obtain bromine-substituted pyrrole a. The nitrogen atom can then be protected by a suitable protecting group Pg to obtain a protected bromine-substituted pyrrole b. In some embodiments, the protecting group can be a tosyl group, which is introduced by deprotonation of the nitrogen atom of bromopyrrole a, followed by contacting the deprotonated compound with p-toluenesulfonyl chloride to obtain N-tosylpyrrole. The protected pyrrole b is then treated with nitromethane (e.g., in the presence of potassium acetate and a slight excess of methylamine hydrochloride) to obtain an aldol condensation product c. Reduction of the carbon-carbon double bond in c with a suitable reducing agent (e.g., LiBH4) affords compound d, which is treated with mesityl oxide in the presence of a non-nucleophilic base (e.g., 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)) to undergo a Michael addition reaction. The Michael addition product e is then treated with formamide and zinc powder to undergo a reductive cyclization to afford protected dihydropyrrole f, which is deprotected to afford chlorin WH.

[0195] Suitable EH chlorin building blocks can be obtained as Figure 2 The preparation is described in Scheme 2 shown in . See also Laha et al. (2003) Organic Process Research & Development 7(6):799-812; Yu et al. (2013) Journal of Organic Chemistry 78(21):10678-10691; and Liu et al. (2016) New Journal of Chemistry 40(9):7721-7740. The EH chlorin building block in Scheme 2 contains an aryl substituent that can be used as a linking group (e.g., after methyl ester hydrolysis) or can be further derivatized (elaborate) (e.g., by reaction with an alkyl compound containing an amino group (which further contains a second functional group that can be used as a bioconjugable group or can react to form a bioconjugable group)) to give a longer linking group in the corresponding chlorin.

[0196] like Figure 2 As described in Scheme 2 shown in FIG, pyrrole can be reacted with methyl 4-formylbenzoate to give dipyrromethane g. If desired, the use of aldehydes other than methyl 4-formylbenzoate can give dipyrromethanes substituted with groups other than methyl benzoate. Vilsmeier formylation of g gives aldehyde h. Aldehyde h can be dibrominated using NBS to give dibromodipyrromethane EH.

[0197] The WH and EH building blocks can be condensed in the presence of an acid (e.g., a Bronsted or Lewis acid such as trifluoroacetic acid or toluenesulfonic acid (TsOH)) to form a condensation product, followed by oxidative cyclization in an organic solvent in the presence of a base (e.g., 2,2,6,6-tetramethylpiperidine), an oxidant (silver trifluoromethanesulfonate), and a metal salt (zinc acetate) to provide a metal chlorin. Optionally, the metal (e.g., Zn) can be displaced by an acid (e.g., trifluoroacetic acid) to provide a chlorin free base. In some embodiments, the chlorin free base can have the following structure:

[0198]

[0199] Coupling chemistry known in the art can be used, including but not limited to Stille coupling, Hiyama coupling, Suzuki coupling, Negishi coupling, Sonogashira coupling and Kumada coupling reactions, further deriving (elaborate) the halogen substituent of the dihalogenated chlorin (such as the above-mentioned dibromochlorin free base) to provide a solubilizing group. For example, the dihalogenated chlorin can be reacted with boric acid in the presence of a Pd (0) catalyst; reacted with an organotin compound in the presence of a Pd catalyst; reacted with a class halide or organosilane in the presence of a Pd catalyst; reacted with an organozinc compound in the presence of a Ni or Pd catalyst, or reacted with a Grignard reagent in the presence of a Ni or Pd catalyst. In some embodiments, the dihalogenated chlorin (e.g., dibromochlorin free base shown above) can react with an arylboronic acid Suzuki coupling reaction partner. See Jiang et al. (2015) New Journal of Chemistry 39(7):5694-5714; and Zhang et al. (2016) New Journal of Chemistry 40(9):7750-7767. In some embodiments, the arylboronic acid may include additional chemical functional groups or protected chemical functional groups that can be further derivatized after the Suzuki coupling reaction. For example, in some embodiments, the Suzuki coupling reaction may include one or more protected amino groups, which can be reacted with a suitable PEG reagent (e.g., an activated PEG ester) after deprotection. Suitable catalysts for the Suzuki coupling reaction include, but are not limited to, Pd(PPh3)4 and PdCl2(PPh3)4. Suitable solvents include water, toluene, THF, dioxane, DMF, and combinations thereof. Suitable bases for the Suzuki coupling reaction include, but are not limited to, Cs2CO3, K3PO4, NaOH, NaOEt, NaOtBu, Na2CO3, K2CO3, alkyllithium compounds, and trialkylamines (e.g., triethylamine).

[0200] In some embodiments, the compounds disclosed herein may use a tert-butyloxycarbonyl (BOC) protecting group for amino groups present on Suzuki or other types of coupling agents, optionally in combination with tert-butyl ester protection of a carboxylic acid (e.g., in a linker). Surprisingly, it was found that deprotection of the BOC group using trifluoroacetic acid (TFA) during the preparation of the compounds disclosed herein (a common BOC deprotection method) resulted in significant decomposition, particularly, for example, in compounds containing acetylenic bonds. Therefore, in some embodiments, other conditions (e.g., 4M HCl in dioxane) are used for BOC deprotection to avoid the decomposition observed when using TFA.

[0201] IV. Drug Compounds

[0202] The compounds of the presently disclosed subject matter may be provided in the form of pharmaceutically acceptable salts. Such salts include, but are not limited to, amine salts such as, but not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts such as, but not limited to, lithium, potassium and sodium; alkaline earth metal salts such as, but not limited to, barium, calcium and magnesium; transition metal salts such as, but not limited to, zinc; and other metal salts such as, but not limited to, sodium hydrogen phosphate and disodium phosphate; and also include, but are not limited to, salts of inorganic acids such as, but not limited to, hydrochlorides and sulfates; and salts of organic acids such as, but not limited to, acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and heterocyclyl esters of acidic groups including, but not limited to, carboxylic, phosphate, phosphinic, sulfonic, sulfinic, and boronic acids.

[0203] The compounds of the currently disclosed subject matter may also include prodrugs of the compounds described herein. As described above, "prodrugs" are compounds that are metabolized or otherwise converted into biological, pharmaceutical or therapeutically active forms of the compounds by one or more steps or processes after in vivo administration. In order to prepare prodrugs, the pharmaceutically active compound is modified so that the active compound will be regenerated by metabolic processes. Prodrugs can be designed to change the metabolic stability or transport characteristics of the drug, mask side effects or toxicity, improve the flavor of the drug, or change other characteristics or properties of the drug. With the knowledge of pharmacodynamic processes and drug metabolism in vivo, those skilled in the art, once knowing the pharmaceutically active compound, can design the prodrug of the compound (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, New York, United States of America, pages 388-392).

[0204] Practicality. The methods and intermediates described herein can be used to synthesize the compounds of formula (I) described herein. Such compounds themselves or in further modified form (e.g., as salts, metallated compounds, conjugates or prodrugs) can be used for diagnostic and therapeutic purposes in a manner similar to other compounds described for photodynamic therapy, such as described in U.S. Patent Application Publication No. 2004 / 0044197 to Pandey et al. and described in further detail below.

[0205] stability. An advantage of some embodiments of the chlorin compounds of the presently disclosed subject matter is their stability and absorption characteristics. Thus, the presently disclosed subject matter provides a "neat" composition consisting of an active compound of the presently disclosed subject matter (e.g., a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, or conjugate thereof (e.g., a conjugate with a targeting agent such as a protein, peptide, or antibody)), wherein the composition has or is characterized by at least 10,000 to 300,000 nm at a wavelength of about 600 to about 800 nanometers. -1 em -1 or greater in solution (it being understood that (a) the active compound must be placed in solution to determine its peak molar absorption coefficient at a specified wavelength; and (b) the compound may exhibit other peaks outside this range, or multiple peaks within this range).

[0206] In addition, the subject matter disclosed at present provides the compound of formula (I) or its pharmaceutically acceptable salt, prodrug or conjugate (for example, with the conjugate of targeting agent such as protein, peptide or antibody) or the composition consisting of the compound of formula (I) or its pharmaceutically acceptable salt, prodrug or conjugate (for example, with the conjugate of targeting agent such as protein, peptide or antibody) in solvent. The amount of solvent is not critical and can account for 0.01 or 1 to 99 or 99.99% weight of composition. The composition has or is characterized in that at least 10,000 to 300,000 M at a wavelength of about 600 to about 800 nanometers -1 em -1 or greater in solution. It should be understood that prior to determining the molar absorption, stirring may be used as needed to break up agglomerated particles back into solution, but a certain level of agglomeration may be required for practical use of the composition. Suitable solvents depend on the specific compound and the intended use of the compound, but include organic solvents, aqueous solvents, and combinations thereof.

[0207] Whether the one or more chlorin compounds are in "pure" form or mixed with a solvent, the composition has or exhibits a loss of no more than about 10, 15, or 20 weight percent of the chlorin compound of the presently disclosed subject matter (due to degradation thereof) when stored in a sealed container (e.g., a flask ampoule or vial) at room temperature in the presence of ambient light for at least 3 or 4 months. Degradation can be determined by spectroscopy, thin layer chromatography, NMR spectroscopy, and / or mass spectrometry according to known techniques.

[0208] Solubility. An advantage of some embodiments of the compounds of the presently disclosed subject matter is their water solubility. Accordingly, the presently disclosed subject matter provides compositions, including but not limited to pharmaceutical preparations, comprising, consisting of, or consisting essentially of: (a) an aqueous solvent (e.g., distilled water, saline solution, buffer solution); and (b) about 1, 2, 5, or 10 μM to 200, 300, or 500 mM of an active compound as described herein dissolved in an aqueous solvent.

[0209] Preparation of pharmaceutical compositions.The pharmaceutical compositions provided herein contain one or more compounds provided herein in a pharmaceutically acceptable carrier in a therapeutically effective amount, which can be used to prevent, treat or improve one or more symptoms of a disease or condition associated with or involving hyperproliferative tissue or new blood vessel formation. Diseases or conditions associated with hyperproliferative tissue or new blood vessel formation include, but are not limited to, cancer, psoriasis, atherosclerosis, heart disease, and age-related macular degeneration. Drug carriers suitable for administering the compounds provided herein include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

[0210] The pharmaceutical composition preferably exhibits the above-mentioned absorption characteristics and storage or stability characteristics.

[0211] Furthermore, the compounds may be formulated as the sole pharmaceutically active ingredient in a composition or may be combined with other active ingredients.

[0212] The composition comprises one or more compounds provided herein (e.g., compounds of formula (I)). In some embodiments, the compound is formulated into a suitable pharmaceutical preparation, such as a solution, suspension, tablet, dispersible tablet, pill, capsule, powder, sustained-release preparation or elixir for oral administration, or a sterile solution or suspension for parenteral administration, as well as a transdermal patch preparation and a dry powder inhaler. In some embodiments, the above-mentioned compound is formulated into a pharmaceutical composition using techniques and operations well known in the art (see, for example, Ansel (1985) Introduction to Pharmaceutical Dosage Forms, Fourth Edition, Lea & Febiger, Philadelphia, Pennsylvania, United States of America, page 126).

[0213] In the composition, effective concentrations of one or more compounds or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier. As described above, the compound can be derived into a corresponding salt, ester, enol ether or enol ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate or prodrug before formulation. The concentration of the compound in the composition can effectively deliver the amount of one or more symptoms of the disease or condition associated with or involving hyperproliferative tissue or new blood vessel formation or in which hyperproliferative tissue or new blood vessel formation is administered.

[0214] In some embodiments, the composition is formulated for single-dose administration. To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration so that the condition being treated is alleviated, prevented, or one or more symptoms are improved.

[0215] The active compound (i.e., a compound of formula (I), or a pharmaceutically acceptable salt, prodrug or conjugate thereof) is contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without producing undesirable side effects on the patient being treated. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo systems described herein and in U.S. Pat. No. 5,952,366 to Pandey et al., and then inferring the dosage for humans therefrom.

[0216] The concentration of the active compound in the pharmaceutical composition may depend on the absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosing regimen and the amount administered, and other factors known to those skilled in the art. For example, as described herein, the amount delivered is sufficient to improve one or more symptoms of a disease or condition associated with or involving hyperproliferative tissue or neovascularization.

[0217] In some embodiments, the therapeutically effective dose should produce a serum concentration of the active ingredient of about 0.1 ng / ml to about 50-100 μg / ml. In one embodiment, the therapeutically effective dose is 0.001, 0.01 or 0.1 to 10, 100 or 1000 mg of active compound per kilogram of body weight per day. The pharmaceutical dosage unit form is prepared to provide each dosage unit form of about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in some embodiments about 10 mg to about 500 mg of active ingredient, or a combination of essential ingredients.

[0218] The active ingredient can be administered in a one-time dose, or can be divided into multiple smaller doses to be administered at intervals. It should be understood that the exact dosage and treatment duration depend on the disease being treated, and can be determined empirically using known test protocols or by inference from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​can also vary with the severity of the condition to be alleviated. It should be further understood that for any specific individual, the specific dosage regimen should be adjusted over time according to the professional judgment of the personnel administering the composition or the administration of the composition, and the concentration ranges listed herein are only examples and are not intended to limit the scope and practice of the claimed composition.

[0219] In the case where the compound exhibits insufficient solubility, methods of solubilizing the compound may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of cosolvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as polyoxyethylene sorbitan esters (e.g., those sold under the trade name The derivatives of the compounds, such as prodrugs of the compounds, can also be used to formulate effective pharmaceutical compositions.

[0220] After mixing or adding the compounds, the resulting mixture can be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to improve the symptoms of the disease, disorder or condition being treated and can be determined empirically.

[0221] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing a suitable amount of a compound or a pharmaceutically acceptable derivative thereof. In some embodiments, pharmaceutical therapeutically active compounds and derivatives thereof are formulated and administered in unit dosage forms or multiple dosage forms. As used herein, "unit dosage form" refers to physically discrete units that are suitable for human and animal individuals and packaged separately as known in the art. Each unit dose contains a predetermined amount of therapeutically active compounds sufficient to produce the desired therapeutic effect together with the required pharmaceutical carrier, vehicle or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms can be administered in divided or multiple doses. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container for administration in separate unit dosage forms. Examples of multiple dosage forms include vials, tablet or capsule bottles or pint or gallon bottles. Thus, a multiple dosage form is a plurality of dosage forms that are not separated in packaging.

[0222] Liquid pharmaceutical compositions can be prepared, for example, by dissolving, dispersing or otherwise mixing the active compound as defined above (e.g., a compound of formula (I), or a pharmaceutically acceptable salt, prodrug or conjugate thereof) and optionally present pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.) to form a solution or suspension. If necessary, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting agents, emulsifiers, solubilizers, pH buffers, etc., for example acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate and other such agents.

[0223] Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences, 15th edition, 1975, Mack Publishing Company, Easton, Pennsylvania, United States of America.

[0224] Dosage forms or compositions containing active ingredients in the range of 0.005% to 100%, with the remainder consisting of non-toxic carriers, can be prepared. Methods for preparing these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%-100% active ingredient, in one embodiment 0.1-95%, and in another embodiment 75-85%.

[0225] Compositions for oral administration. Oral pharmaceutical dosage forms are solid, gel or liquid. Solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed chewable lozenges and tablets that may be enteric coated, sugar coated or film coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

[0226] Solid compositions for oral administration.In some embodiments, the formulation is a solid dosage form, in some embodiments, a capsule or tablet. Tablets, pills, capsules, lozenges, etc. may contain one or more of the following ingredients or compounds of similar nature: binder; lubricant; diluent; glidant; disintegrant; colorant; sweetener; flavoring agent; wetting agent; emetic coating; and film coating. Examples of binders include microcrystalline cellulose, tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidone, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Flow aids include, but are not limited to, colloidal silicon dioxide. Disintegrants include cross-linked sodium carboxymethylcellulose, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, for example, any approved certified water-soluble FD and C dyes, mixtures thereof; and water-insoluble FD and C dyes suspended on alumina hydrate. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, and any number of spray-dried spices. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce pleasant sensations, such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Film coatings include hydroxyethyl cellulose, gellan gum, sodium carboxymethyl cellulose, polyethylene glycol 4000 (PEG4000), and cellulose acetate phthalate.

[0227] The compound or its pharmaceutically acceptable derivative can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated into an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredients. When the dosage unit form is a capsule, in addition to the above-mentioned type of material, it can also include a liquid carrier such as a fatty oil. In addition, the dosage unit form can include various other materials that change the physical form of the dosage unit, such as sugar coating and other enteric solvents. The compound can also be administered as a component of an elixir, a suspension, a syrup, a thin slice, a spray, a chewing gum, etc. In addition to the active compound, the syrup can also contain sucrose as a sweetener and some preservatives, dyes, coloring agents and / or flavoring agents.

[0228] The active material can also be mixed with other active materials that do not impair the desired effect, or with materials that supplement the desired effect (such as antacids, H2 blockers and diuretics). The active ingredient is a compound as described herein or a pharmaceutically acceptable derivative thereof. A higher concentration of active ingredient up to about 98% by weight can be included.

[0229] In some embodiments, tablet and capsule formulations may be coated as known to those skilled in the art to modify or maintain the dissolution of the active ingredient. Thus, for example, they may be coated with conventional enteric digestible coatings such as phenyl salicylate, waxes, and cellulose acetate phthalate.

[0230] Liquid compositions for oral administration. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are oil-in-water or water-in-oil.

[0231] Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers for elixirs include solvents. Syrups are concentrated aqueous solutions of sugars (e.g., sucrose) and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed in another liquid in the form of globules. Pharmaceutically acceptable carriers for emulsions are non-aqueous liquids, emulsifiers, and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules to be reconstituted into liquid oral dosage forms include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules to be reconstituted into liquid oral dosage forms include organic acids and carbon dioxide sources. Colorants and flavorings are used in all of the above dosage forms. Solvents include glycerol, sorbitol, ethanol, and syrups. Examples of preservatives include glycerol, methyl and propyl parabens, benzoic acid, sodium benzoate, and ethanol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, gum arabic, tragacanth, bentonite and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, magnesium aluminosilicate and gum arabic. Sweeteners include sucrose, syrup, glycerol and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Carbon dioxide sources include sodium bicarbonate and sodium carbonate. Colorants include any of any approved certified water-soluble FD and C dyes and their mixtures. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce pleasant taste. For solid dosage forms, solutions or suspensions in, for example, propylene carbonate, vegetable oils and / or triglycerides are encapsulated in gelatin capsules in some embodiments. Such solutions and their preparation and packaging are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545, each of which is incorporated herein by reference in its entirety. For liquid dosage forms, for example, solutions in polyethylene glycol can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier (e.g., water) to allow for easy measurement for administration.

[0232] Alternatively, liquid or semisolid oral formulations can be prepared by dissolving and / or dispersing the active compound or salt in a vegetable oil, glycol, triglyceride, propylene glycol ester (e.g., propylene carbonate), and / or other such carriers, and / or encapsulating these solutions or suspensions in a hard or soft gelatin capsule shell. Other useful formulations include those described in U.S. Pat. No. RE28,819 and U.S. Pat. No. 4,358,603, each of which is incorporated herein by reference in its entirety. Briefly, such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether (wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol), and one or more antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

[0233] Other preparations include, but are not limited to, aqueous alcohol solutions containing pharmaceutically acceptable acetals. The alcohol used in these preparations is any pharmaceutically acceptable water-miscible solvent with one or more hydroxyl groups, including but not limited to propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal.

[0234] Injections, solutions and emulsions. In some embodiments characterized by subcutaneous, intramuscular or intravenous injection, parenteral administration is also included in this article. Injection can be prepared in a conventional form, in a liquid solution or suspension, in a solid form suitable for dissolving or suspending in a liquid before injection, or in the form of an emulsion. Injection, solution and emulsion also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if necessary, the pharmaceutical composition to be administered may also include a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, and other such agents, for example sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrin.

[0235] Implantation of a slow-release or sustained-release system is also contemplated herein so that a constant dosage level is maintained (see, e.g., U.S. Pat. No. 3,710,795, which is incorporated herein by reference in its entirety). Briefly, the compounds provided herein are dispersed in a solid internal matrix, such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers (such as hydrogels of esters of acrylic acid and methacrylic acid), collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, the The solid inner matrix is ​​surrounded by an outer polymer film that is insoluble in body fluids, such as polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride and vinyl acetate, vinylidene chloride, copolymers of ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers and ethylene / ethyleneoxyethanol copolymers. The compound diffuses through the outer polymer film in a release rate-controlling step. The percentage of active compound included in such parenteral compositions is highly dependent on its specific properties as well as the activity of the compound and individual needs.

[0236] Parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile solutions ready for injection; sterile dry soluble products (such as lyophilized powders) ready to be mixed with solvents before use, including subcutaneous tablets; sterile suspensions ready for injection; sterile dry insoluble products ready to be mixed with vehicles immediately before use; and sterile emulsions. Solutions can be aqueous or non-aqueous.

[0237] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0238] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering or chelating agents and other pharmaceutically acceptable substances.

[0239] Examples of aqueous vehicles include sodium chloride injection, Ringers Injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers Injection. Non-aqueous parenteral vehicles include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents with antibacterial or antifungal concentrations can be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending agents and dispersants include sodium carboxymethylcellulose, xanthan gum, hydroxypropyl methylcellulose and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 (available under the trade name 80). Metal ion sequestering agents or chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0240] The concentration of the pharmaceutically active compound can be adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as known in the art.

[0241] The unit dose parenteral preparation is packaged in an ampoule, a vial or a syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration should be sterile.

[0242] Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing the active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active material, which is injected as needed to produce the desired pharmacological effect.

[0243] Injections are designed for local and systemic administration. In one embodiment, the therapeutically effective dose is formulated to contain a concentration of at least about 0.1% w / w to about 90% w / w or more of the active compound in the tissue to be treated, and in certain embodiments, a concentration of more than 1% w / w of the active compound in the tissue to be treated.

[0244] The compound may be suspended in micronized or other suitable form, or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the condition and can be determined empirically.

[0245] Freeze-dried powder. Lyophilized powders, which can be reconstituted for administration as solutions, emulsions, and other mixtures, can also be used to practice the presently disclosed subject matter. They can also be reconstituted and formulated as solids or gels.

[0246] Sterile lyophilized powder is prepared by dissolving the compound provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. The solvent may contain an excipient that improves the stability of other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable substances. The solvent may also contain a buffer such as citrate, sodium phosphate or potassium phosphate or other such buffers known to those skilled in the art, in some embodiments, a buffer of about neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to obtain the desired formulation. In some embodiments, the resulting solution is dispensed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of the compound. The freeze-dried powder may be stored under appropriate conditions, for example, at about 4 ° C to room temperature.

[0247] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or another suitable carrier. The exact amount depends on the compound selected. This amount can be determined empirically.

[0248] Topical administration Topical mixtures are prepared as described for topical and systemic administration. The resulting mixtures can be solutions, suspensions, emulsions, etc., and formulated into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, douches, sprays, suppositories, bandages, skin patches, or other preparations suitable for topical administration.

[0249] The compound or its pharmaceutically acceptable derivative can be formulated into an aerosol for topical application, such as by inhalation (see, for example, U.S. Pat. Nos. 4,044,126; 4,414,209 and 4,364,923, each of which is incorporated herein by reference in its entirety, which describes aerosols for delivering steroids for the treatment of inflammatory diseases, particularly asthma). These preparations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, alone or with an inert carrier such as lactose, or in the form of a micronized powder for insufflation. In this case, the particles of the preparation will generally have a diameter of less than 50 microns in some embodiments, and a diameter of less than 10 microns in some embodiments.

[0250] The compound can be formulated for local or topical administration, for example, in the form of gels, creams and lotions, applied topically to the skin and mucous membranes (such as eyes), and applied to the eyes or applied to the brain pool or spinal application. Topical administration is intended for transdermal delivery and is also used for eye or mucosal administration or for inhalation therapy. Nasal solutions of the active compound can also be administered alone or in combination with other pharmaceutically acceptable excipients. These solutions, especially those intended for ophthalmic use, can be formulated with appropriate salts into isotonic solutions of 0.01%-10% at a pH of about 5-7.

[0251] Compositions for other routes of administration. Other routes of administration, such as transdermal patches, including iontophoresis and electrophoresis devices, and rectal administration are also contemplated herein.

[0252] Transdermal patches, including electroosmotic and electrophoretic devices, are well known to those skilled in the art. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983; 6,261,595; 6,256,533; 6,167,301; 6,024,975; 6,010715; 5,985,317; 5,983,134; 5,948,433; and 5,860,957, each of which is incorporated herein by reference in its entirety.

[0253] For example, the pharmaceutical dosage form for rectal administration is a rectal suppository, capsule and tablet for systemic effect. Rectal suppository used in this article refers to a solid for insertion into the rectum, which melts or softens at body temperature, releasing one or more pharmaceutical or therapeutic active ingredients. The pharmaceutically acceptable substance used for rectal suppositories is a base or excipient and an agent that improves the melting point. The example of a base includes a suitable mixture of monoglycerides, diglycerides and triglycerides of the glycerol of the cocoa butter (cocoa butter), glycerol-gelatin, carbowax (polyoxyethylene glycol) and fatty acids. A combination of various bases can be used. The agent that improves the melting point of the suppository includes spermaceti and wax. Rectal suppositories can be prepared by compression methods or by molding. In some embodiments, the weight of the rectal suppository is about 2 to 3 grams.

[0254] Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substances and by the same methods as for formulations for oral administration.

[0255] Targeted agents.The compounds provided herein or their pharmaceutically acceptable derivatives can also be formulated to target specific tissues, receptors, infectious agents or other areas of the individual body to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the present compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos. 6,316,652; 6,274,552; 6,271,359; 6,253,872; 6,139,865; 6,131,570; 6,120,751; 6,071,495; 6,060,082; 6,048,736; 6,039,975; 6,004,534; 5,985,307; 5,972,366; 5,900,252; 5,840,674; 5,759,542 and 5,709,874, each of which is incorporated herein by reference in its entirety.

[0256] Liposomes. In some embodiments, liposome suspensions, including liposomes targeting tissues, such as liposomes targeting tumors, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome preparations can be prepared as described in U.S. Patent No. 4,522,811, which is incorporated herein by reference in its entirety. In short, liposomes such as multilamellar vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) inside a flask. A solution of the compound provided herein in phosphate buffered saline (PBS) without divalent cations is added and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, centrifuged, and then resuspended in PBS.

[0257] Ligand.In some embodiments, the disclosed compounds can be targeted to a specific target tissue or target composition using a ligand that is specific for the target tissue or target composition, for example, using a ligand or ligand-receptor pair such as an antibody and an antigen. Antibodies against tumor antigens and pathogens are known. For example, antibodies and antibody fragments that specifically bind to markers produced by or associated with tumors or infectious lesions, including viral, bacterial, fungal and parasitic infections, and antigens and products associated with such microorganisms, are disclosed in particular in U.S. Pat. Nos. 3,927,193; 4,331,647; 4,348,376; 4,361,544; 4,468,457; 4,444,744; 4,818,709; and 4,624,846, each of which is incorporated herein by reference in its entirety. Antibodies directed against antigens such as gastrointestinal, lung, breast, prostate, ovarian, testicular, brain or lymphoid tumors, sarcomas or melanomas may be used.

[0258] A variety of monoclonal antibodies have been developed against infectious disease agents and are summarized in the review by Polin (1984) European Journal of Clinical Microbiology 3(5):387-398, indicating their ready availability. These include monoclonal antibodies (MAbs) directed against pathogens and their antigens, for example the following: antibacterial MAbs, such as those against Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Esherichia coli, Neisseria gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease, spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, or tetanus toxin;Antiprotozoan Mabs, such as those against Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Mesocestoides corti, Emeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena), Taenia ovis, Taenia saginata; antiviral MAbs, such as those against HIV-1, -2, and -3, hepatitis A, hepatitis B, hepatitis C, hepatitis D, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I and II, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, mumps virus, Sindbis virus, mouse mammary tumor virus, feline leukemia virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, Reo virus, polio virus, dengue virus, rubella virus, mouse leukemia virus; anti-mycoplasma MAbs, such as those against Acholeplasma ramerii, laidlawii), Mycoplasma arthritidis, M.hyorhinis, M.orale, M.arginini, M.pneumonia, etc.;

[0259] Suitable MAbs have been developed against most microorganisms (bacteria, viruses, protozoa, other parasites) responsible for most human infections, and many have been used for in vitro diagnostic purposes before. These antibodies and new MAbs that can be produced by conventional methods are suitable for use as target substances with compounds provided herein.

[0260] MAbs against Plasmodium can be directed against the sporozoite, merozoite, schizont, and gametocyte stages. Monoclonal antibodies have been generated against sporozoites (circumsporozoite antigens) and have been shown to neutralize sporozoites in vitro and in rodents. See Yoshida et al. (1980) Science 207:71-73. Monoclonal antibodies have been developed against T. gondii (the protozoan parasite associated with toxoplasmosis). See Kasper et al. (1982) Journal of Immunology 129:1694-1699. MAbs against Schistosoma surface antigens have been developed and have been found to be active against Schistosoma in vivo or in vitro. See Simpson et al. (1981) Parasitology 83:163-177; Smith et al. (1982) Parasitology 84:83-91; Gryzch et al. (1982) Journal of Immunology 129:2739-2743; Zodda et al. (1982) Journal of Immunology 129:2326-2328; and Dissous et al. (1982) Journal of Immunology 129:2232-2234.

[0261] Mixtures of antibody and immunoglobulin classes can be used, as can hybrid antibodies. Multispecific (including bispecific and hybrid) antibodies and antibody fragments are particularly preferred in the methods of the presently disclosed subject matter for detecting and treating target tissues, and are composed of at least two different substantially monospecific antibodies or antibody fragments, wherein at least two of the antibodies or antibody fragments specifically bind to at least two different antigens produced by or associated with the target lesion, or at least two different epitopes or molecules of marker substances produced by or associated with the target tissue. Multispecific antibodies and antibody fragments with bispecificity can be prepared similarly to the anti-tumor marker hybrids disclosed in U.S. Pat. No. 4,361,544. Other techniques for preparing hybrid antibodies are disclosed in, for example, U.S. Pat. Nos. 4,474,893 and 4,479,895 (each of which is incorporated herein by reference in its entirety) and Milstein et al. (1984) Immunology Today 5: 299.

[0262] Antibody fragments that can be used for the subject matter of the present disclosure include F(ab')2, F(ab)2, Fab', Fab, Fv, etc., including hybrid fragments. Preferred fragments are Fab', F(ab')2, Fab and F(ab)2. Any subfragment that retains the hypervariable antigen binding region of the immunoglobulin and has a size similar to or smaller than the Fab' fragment is also useful. This can include genetic engineering and / or recombinant proteins, whether single-chain or multi-chain, which are incorporated into the antigen binding site and additionally act as a targeting carrier in vivo in substantially the same manner as natural immunoglobulin fragments. Such single-chain binding molecules are disclosed in U.S. Patent No. 4,946,778, which is incorporated herein by reference. Fab' antibody fragments can be conveniently prepared by reductive cleavage of F(ab')2 fragments, which can themselves be prepared by pepsin digestion of intact immunoglobulins. Fab antibody fragments can be prepared by papain digestion of intact immunoglobulins under reducing conditions, or by cleavage of F(ab')2 fragments produced by careful papain digestion of intact immunoglobulins.

[0263] A ligand or a member of a ligand-receptor binding pair can be conjugated to a compound provided herein to target the compound to a specific target tissue or target composition. Examples of ligand-receptor binding pairs are listed in U.S. Pat. Nos. 4,374,925 and 3,817,837, each of which is incorporated herein by reference in its entirety.

[0264] Conjugated with ligand. Many compounds that can serve as targets for ligand-receptor binding pairs (more specifically, antibodies) have been identified, and techniques for constructing conjugates of such ligands with compounds of formula (I) are well known to those of ordinary skill in the art. For example, Rakestraw et al. teach the conjugation of chlorin Sn (IV) to monoclonal antibodies via covalent bonds using modified dextran carriers. See Rakestraw et al. (1990) Proceedings of the National Academy of Science of the United States of America 87: 4217-4221. The compounds disclosed herein can also be conjugated to ligands, such as antibodies, by using coupling agents. Any bond that can connect the components so that they are stable under physiological conditions for the time required for administration and treatment is suitable, but covalent bonds are preferred. The connection between the two components can be direct, for example, where the compound of formula (I) is directly connected to the targeting agent, or can be indirect, for example, where the compound of formula (I) is connected to an intermediate and the intermediate is connected to the targeting agent.

[0265] The coupling agent should function under conditions of temperature, pH, salt, solvent system and other reactants that substantially maintain the chemical stability of the photosensitizer, backbone (if present) and targeting agent. The coupling agent should stably connect the component parts, but with minimal or no denaturation or inactivation of the compound of formula (I) or the targeting agent. Many coupling agents react with amines and carboxylates to form amides, or with alcohols and carboxylates to form esters. Coupling agents are known in the art. See, for example, Bodansky (1993) Principles of Peptide Synthesis, 2nd edition., Springer & Hermanson (1996) Bioconjugate Techniques, 1st edition, Academic Press, New York, New York, United States of America.

[0266] The conjugates of the compounds provided herein with ligands such as antibodies can be prepared by coupling the compound to the targeting moiety by coupling the carboxylic acid or ester moiety on the compound to the antibody via a peptide bond through the N-terminus, or by other methods known in the art. A variety of coupling agents, including cross-linking agents, can be used for covalent conjugation. Examples of cross-linking agents include N, N'-dicyclohexylcarbodiimide (DCC), N-succinimidyl-5-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldi-thio) propionate (SPDP), o-phenylenedimaleimide (o-PDM) and sulfosuccinimidyl-4-(N-maleimido-methyl)-cyclohexane-1-carboxylate (sulfo-SMCC). See, e.g., Karpovsky et al. (1984) Journal of Experimental Medicine 160(6):1686-1701; and Liu et al. (1985) Proceedings of the National Academy of Science of the United States of America 82(24):8648-8652. Other methods include those described by Brennan et al. (1985) Science 229:81-83 and Glennie et al. (1987) Journal of Immunology 139:2367-2375.

[0267] For example, DCC is a useful coupling agent that can be used to promote the coupling of alcohol NHS to chlorin carboxylic acid groups in DMSO to form activated esters that can be crosslinked to polylysine. DCC is a carboxyl-reactive crosslinker that is commonly used as a coupling agent in peptide synthesis and has a molecular weight of 206.32. Another useful crosslinker is SPDP, a heterobifunctional crosslinker that is used with primary amines and sulfhydryls. SPDP has a molecular weight of 312.4, a spacer arm length of 6.8 angstroms, is reactive to NHS-esters and pyridyl disulfide groups, and produces cleavable crosslinks that eliminate the agent upon further reaction, allowing the photosensitizer to be directly attached to the backbone or targeting agent. Other useful coupling agents are SATA, which is used to introduce blocked SH groups for two-step crosslinking, which is deblocked by hydroxylamine-HCl and sulfo-SMCC and is reactive to amines and sulfhydryls. Other crosslinkers and coupling agents are also available from Pierce Chemical Co. Other compounds and methods for conjugating proteins to other proteins or other compositions, such as to reporter groups or chelators for metal ion labeling of proteins, particularly those involving Schiff bases as intermediates, are disclosed in European Patent EP 0 243 929 B1.

[0268] Photosensitizers containing carboxyl groups can be attached to lysine epsilon-amino groups in target polypeptides via preformed reactive esters (such as N-hydroxysuccinimide (NHS) esters) or via esters conjugated in situ via carbodiimide-mediated reactions. The same applies to photosensitizers containing sulfonic acid groups, which can be converted into sulfonyl chlorides that react with amino groups. Chlorins with carboxyl groups can be attached to amino groups on polypeptides via in situ carbodiimide methods. Chlorins can also be attached to hydroxyl groups of serine or threonine residues or to sulfhydryl groups of cysteine ​​residues.

[0269] Methods for linking the components of a conjugate (e.g., coupling a photosensitizer with a polyamino acid chain to an antimicrobial polypeptide) can use heterobifunctional cross-linking reagents. These reagents bind to a functional group in one chain and combine it with a different functional group in a second chain. These functional groups are typically amino, carboxyl, sulfhydryl, and aldehyde. There are many permutations of suitable moieties that will react with these groups and structures represented by different formulas to conjugate them together. See Hermanson (1996) Bioconjugate Techniques, 1st ed., Academic Press, New York, New York, United States of America; and Merrifield et al. (1994) Ciba Foundation Symposium 186: 5-20.

[0270] The compound or pharmaceutically acceptable derivative thereof can be packaged as an article of manufacture containing packaging material, a compound or pharmaceutically acceptable derivative thereof provided herein (which can effectively modulate the activity of hyperproliferative tissue or neovascularization, or is used to treat, prevent or improve a disease or condition mediated by hyperproliferative tissue or neovascularization, or one or more symptoms of a disease or condition in which hyperproliferative tissue or neovascularization activity is involved) within the packaging material, and a label indicating that the compound or composition or pharmaceutically acceptable derivative thereof is used to modulate the activity of hyperproliferative tissue or neovascularization, or is used to treat, prevent or improve a disease or condition mediated by hyperproliferative tissue or neovascularization, or one or more symptoms of a disease or condition in which hyperproliferative tissue or neovascularization activity is involved.

[0271] The articles provided herein include packaging materials. Packaging materials for packaging pharmaceutical products are well known to those skilled in the art. See, for example, U.S. Patents 5,323,907; 5,052,558 and 5,033,252, each of which is incorporated by reference in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging materials suitable for the selected formulation and intended administration and treatment modality. The various formulations of the compounds and compositions provided herein are contemplated as various treatments for any disease or condition in which hyperproliferative tissue or new blood vessel formation is involved as an intermediary or contributing factor to a symptom or inducement.

[0272] V. Photodynamic therapy, diagnostic and therapeutic applications

[0273] In some embodiments, the compounds of formula (I) disclosed herein (or pharmaceutically acceptable salts or conjugates thereof) can act as photosensitizers in methods for treating diseases (e.g., hyperproliferative diseases, such as cancer) involving photodynamic therapy (PDT). In short, the photosensitive compound, its conjugate or pharmaceutical composition is generally administered to the individual before the target tissue, target composition or individual is exposed to light. The photosensitive compound is administered as described elsewhere herein.

[0274] The dose of the photosensitizing compound can be clinically determined. Depending on the photosensitizing compound used, an equivalent optimal therapeutic level must be established. A certain length of time is allowed to pass so that the circulating or locally delivered photosensitizer is absorbed by the target tissue. During this waiting period, the unbound photosensitizer is cleared from the circulation, or optionally additional time can be provided to clear the unbound compound from non-target tissues. The waiting period can be clinically determined and can vary from compound to compound.

[0275] At the end of this waiting period, the bound drug is activated using a laser light source or a non-laser light source (including but not limited to an artificial light source such as a fluorescent lamp or an incandescent lamp, or a natural light source such as ambient sunlight). The illuminated area is determined by the location and size of the pathological area to be detected, diagnosed or treated. The duration of the illumination period can depend on whether detection or treatment is being performed and can be determined empirically. A total or cumulative time period of any time from about 4 minutes to about 72 hours can be used. In some embodiments, the illumination period is about 60 minutes and 148 hours. In some embodiments, the illumination period is about 2 hours and 24 hours.

[0276] The total fluence or energy of light used for irradiation, measured in Joules, is in some embodiments from about 10 Joules to about 25,000 Joules; in some embodiments from about 100 Joules to about 20,000 Joules; and in some embodiments from about 500 Joules to about 10,000 Joules. Light of a wavelength and fluence sufficient to produce the desired effect, whether for detection by fluorescence or for therapeutic treatment to destroy or damage the target tissue or target composition, is selected. Light having a wavelength that corresponds at least in part to the characteristic light absorption wavelength of the photosensitizer is preferably used to irradiate the target tissue.

[0277] The intensity or power of the light used is measured in watts, with each joule equal to one watt-second. Thus, the intensity of the light used for irradiation in the presently disclosed methods may be substantially less than 500 mW / cm 2 Since the total fluence or amount of energy of the light in joules is divided by the duration of the total exposure time in seconds, the longer the amount of time the target is exposed to the irradiation, the greater the amount of total energy or fluence that can be used without increasing the amount of light intensity used. The presently disclosed subject matter uses an amount of total fluence of irradiation that is high enough to activate the photosensitizer.

[0278] In some embodiments of photodynamic therapy using the compounds disclosed herein, the compound is injected into the mammal (e.g., human) to be diagnosed or treated. The injection level is generally about 0.1 to about 0.5 μmol / kg body weight. In the case of treatment, the area to be treated is exposed to light of the desired wavelength and energy, for example, about 10 to 200 J / cm 2 In the case of detection, fluorescence is determined upon exposure to light at a wavelength sufficient to cause the compound to fluoresce at a wavelength different from the wavelength used to illuminate the compound. The energy used in detection is sufficient to cause fluorescence and is generally significantly lower than that required for treatment.

[0279] Any photosensitive compound disclosed herein or a pharmaceutically acceptable derivative thereof can be provided in a medicine box together with instructions for implementing any method disclosed herein. The instructions can be in any tangible form, such as printed paper, a computer disk that instructs people how to implement the method, a videotape containing instructions about how to implement the method, or a computer memory that receives data from a remote location and illustrates the instructions or otherwise provides instructions to someone (such as via the Internet). For example, any of the above instructions can be used or by receiving instructions in a classroom or during the treatment of a patient using any method disclosed herein to instruct a person how to use the medicine box.

[0280] Other examples and specific examples of methods of using the compounds and compositions of the presently disclosed subject matter include, but are not limited to, the following:

[0281] (i) Treatment of opportunistic infections. The compounds, compositions and methods of the currently disclosed subject matter can be used for PDT of opportunistic infections, particularly PDT of opportunistic infections of soft tissues. For infection, particularly antimicrobial treatment (by PDT) of wound infection, the infectious organism may include (as a non-limiting example) Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli. In hospital infections, Pseudomonas aeruginosa causes 8% of surgical wound infections and 10% of bloodstream infections. In some embodiments, the individual is an immunocompromised individual, such as those suffering from AIDS or receiving immunosuppressant therapy.

[0282] (ii) Treatment of burns. Infections with Staphylococcus aureus and Gram-positive bacteria are often particularly evident in burns. The multidrug resistance of Staphylococcus aureus presents a significant medical challenge. In this regard, the compounds, compositions and methods of the presently disclosed subject matter can be used to treat opportunistic infections in burns.

[0283] (iii) Sepsis. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT treatment of individuals suffering from opportunistic infections with Vibrio vulnificus. Vibrio vulnificus, a Gram-negative bacterium, causes primary sepsis, wound infections and gastrointestinal diseases in humans.

[0284] (iv) Ulcers. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT treatment of bacteria (Helicobacter pylori) that cause ulcers. In the clinic, treatment can be performed in any suitable manner, such as by inserting a fiber optic cable (similar to an endoscope, but with a device that delivers red or near infrared light) into the stomach or diseased area.

[0285] (v) Periodontal disease. The compounds, compositions and methods of the subject matter disclosed herein are useful in PDT for the treatment of periodontal disease, including gingivitis. Periodontal disease is caused by bacterial overgrowth, such as the Gram-negative anaerobic bacterium Porphyromonas gingivalis. As with many PDT treatments, a targeting or solubilizing entity conjugated to a photoactive substance is essential for the proper delivery of the photoactive substance to the desired cells. Target oral pathogens for targeting include Porphyromonas gingivalis, Actinobacillus actinonzycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. Polymorphum, Actinomyces viscosus and Streptococcus. For such applications, compounds or compositions of the presently disclosed subject matter can be topically applied (eg, in the form of a mouthwash or rinse) followed by photoapplication using an external device, an intraoral instrument, or a combination thereof.

[0286] (vi) Atherosclerosis. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT to treat vulnerable plaques in arteries. Without wishing to be bound by any particular theory, it is believed that invading inflammatory macrophages secrete metalloproteinases that degrade the collagen lamina in the coronary arteries, leading to thrombosis, which is often fatal. Active compounds targeting such inflammatory macrophages can be used for PDT of vulnerable plaques.

[0287] (vii) Cosmetic and Dermatological Applications. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT to treat a wide range of cosmetic dermatological problems, such as hair removal, treatment of psoriasis or removal of skin discoloration. Ruby lasers are currently used for hair removal; in many laser treatments, melanin is the photosensitive chromophore. This treatment works quite well for fair-skinned people with dark hair. The compounds, compositions and methods of the presently disclosed subject matter can be used as near-infrared sensitizers for hair removal, which can target chromophores with more specific and sharp absorption bands.

[0288] (viii) Acne. The compounds, compositions and methods of the presently disclosed subject matter may be used for PDT to treat acne. Acne vulgaris is caused by Propionibacterium acne, which infects the sebaceous glands; some 80% of young people are affected. Again, the bacteria are becoming increasingly resistant to antibiotic treatment, leading to a surge in difficult-to-treat acne. Current PDT treatments for acne typically rely on the addition of aminolevulinic acid, which is converted to free base porphyrins in the hair follicles or sebaceous glands. The compounds and compositions of the presently disclosed subject matter may be administered to an individual topically or parenterally (e.g., by subcutaneous injection), depending on the particular condition.

[0289] (ix) Infectious diseases. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT to treat infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which occur extensively in the Mediterranean and Middle East, are currently treated with arsenic-containing compounds. Recently, in at least one case, PDT has been used to produce reasonable results in human patients. The use of the compounds and compositions of the presently disclosed subject matter is also useful and potentially offers advantages such as ease of synthesis and better spectral absorption characteristics.

[0290] (x) Tissue sealants. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT as tissue sealants for individuals in need thereof. Light-activated tissue sealants are attractive for sealing wounds, gluing tissues, and closing defects in tissues. There are many applications where sutures or staples are undesirable and where the use of such mechanical sealing methods often results in infection and scarring.

[0291] (xi) Neoplastic Diseases. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT to treat neoplastic diseases or cancers, including skin cancer, lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma, leukemia, lymphoma, squamous cell carcinoma, melanoma, plaque-stage cutaneous T-cell lymphoma and Kaposi's sarcoma.

[0292] In addition to PDT, the compositions provided herein can also be used as imaging enhancers in diagnostic imaging techniques, or for marking target tissues or target compositions for diagnostic radiology. In the field of modern medicine, there are a variety of treatment methods, including magnetic resonance imaging (MRI) for disease diagnosis. Early detection of cancer should improve the ability to cure and eliminate cancerous tissues. Early diagnosis of precancerous areas and microcancers is an important theme in modern cancer treatment. MRI has become a powerful tool in clinical settings because it is non-invasive and produces accurate volumetric reproduction of individuals. The image is created by applying one or more orthogonal magnetic field gradients to an individual or sample, while exciting nuclear spins with radio frequency pulses in a typical nuclear magnetic resonance (NMR) experiment. After collecting data using various gradient fields, deconvolution generates one-dimensional, two-dimensional or three-dimensional images of samples / individuals. Typically, images are based on NMR signals from protons of water, where the signal intensity in a given volume element is a function of water concentration and relaxation time. Local changes in these parameters provide a sharp contrast observed in MR images.

[0293] MRI contrast agents act by increasing the relaxation rate, thereby increasing the contrast between water molecules in the area where the imaging agent accumulates and water molecules in other parts of the body. However, the effect of the agent is to reduce both T1 and T2, the former resulting in greater contrast and the latter resulting in lower contrast. Therefore, this phenomenon is concentration-dependent, and there is generally an optimal concentration of the paramagnetic substance for maximum efficacy. This optimal concentration may vary with the specific agent used, the imaging site, the imaging mode (i.e., spin echo imaging, saturation recovery, inversion recovery, and / or various other strongly T1-dependent or T2-dependent imaging techniques), and the composition of the medium in which the agent is dissolved or suspended. These factors and their relative importance are known in the art. See, for example, Pykett (1982) Scientific American 246:78; and Runge et al. (1983) American Journal of Radiology 141:1209. When MRI contrast agents are used diagnostically, they perfuse blood vessels, enhance vascular contrast, and report organ lesions and infiltration. However, labeling specific tissues for diagnostic radiology remains a difficult challenge for MRI. Efforts to develop cell- and tissue-specific MRI image enhancers by modifying existing immunological techniques have been the focus of much research in diagnostic radiology. For example, antibodies labeled with paramagnetic ions, usually the gadolinium chelate Gd-DTPA, have been generated and tested for their effects on MRI contrast of tumors and other tissues. See U.S. Pat. No. 5,059,415, which is incorporated herein by reference in its entirety. Unfortunately, it has been found that the relaxivity of Gd bound to the antibody is only slightly better than that of unbound Gd-DTPA. See Paajanen et al. (1990) Magnetic Resononance in Medicine 13: 38-43.

[0294] MRI is commonly used to detect 1 H nuclei. However, MRI is able to detect the NMR spectra of other nuclear species, including 13 C. 15 N. 31 P and 19 F. 19 F is not abundant in organisms. By adding isotopes that can be used for MRI to the compositions provided herein, such as 13 C. 15 N. 31 P or 19 F, especially 19 F, and administered to a subject, the compounds provided herein accumulate in the target tissue and, due to having an MRI-identifiable isotope (such as 19F), subsequent MR imaging will produce NMR data with enhanced signals from the target tissue or target composition. Therefore, the disclosed compounds can be used as image enhancement agents and provide markers for specific target tissues or target compositions for diagnostic radiology (including MRI).

[0295] In addition to PDT, the compositions provided herein can be used to detect individual target cells, target tissues or target compositions. When the compounds provided herein are used to detect target tissues or target compositions, the compounds are introduced into the individual and sufficient time is allowed for the compounds to accumulate in the target tissues or associate with the target compositions. The treatment area is then irradiated, usually using light with energy sufficient to cause fluorescence of the compound, and the energy used is usually significantly lower than the energy required for photodynamic therapy treatment. Fluorescence is determined when exposed to light of the desired wavelength, and the amount of fluorescence can be qualitatively or quantitatively associated with the presence of the compound by methods known in the art.

[0296] The compositions provided herein can also be used to diagnose the presence of an infectious agent, or to identify the composition of an infectious agent in an individual. The compounds provided herein can be conjugated to one or more ligands specific for the infectious agent, such as antibodies or antibody fragments, which selectively associate with the infectious agent, and after allowing the target compound sufficient time to associate with the infectious agent and clear from non-target tissues, the compound can be visualized, such as by exposure to light with energy sufficient to cause compound fluorescence, or by using diagnostic radiological imaging, including MRI. For example, any of the compounds provided herein can be conjugated with antibodies targeting suitable Helicobacter pylori antigens and formulated into a pharmaceutical formulation that, when introduced into an individual, releases the conjugated compound to the location of the gastric mucus / epithelial layer where the bacteria are found. After the compound selectively associates with the target infectious agent and any unbound compound is cleared from non-target tissues for a sufficient period of time, the individual can be examined to determine whether there is any Helicobacter pylori. This can be detected, for example, by MRI due to 19 F substituents, or by illuminating the suspected target area with light of sufficient energy to cause the compound to fluoresce (e.g., by using fiber optics) and detecting the fluorescence of any target compound.

[0297] In some embodiments, the compounds disclosed herein or their conjugates can be used for flow cytometry. Flow cytometry is known and described in, for example, U.S. Patent Nos. 5,167,926; 5,915,925; 6,248,590; 6,589,792 and 6,890,487, each of which is incorporated by reference in its entirety. In some embodiments, the particles (such as cells) to be detected are labeled with luminescent compounds such as phosphors or fluorophores for detection. Labeling can be performed by any suitable technique, such as coupling the luminescent compound to another compound such as an antibody, which in turn specifically binds to the particle or cell (by ingesting or internalizing the luminescent compound into the cell or particle, by non-specific adsorption of the luminescent compound to the cell or particle, etc.). The active compounds described herein can be used for flow cytometry, such as such luminescent compounds, and the flow cytometry techniques (including fluorescence activated cell sorting or FACS) can be performed based on the present disclosure according to known techniques or variants thereof that are obvious to those skilled in the art. Example

[0298] The following examples provide illustrative embodiments. In view of this disclosure and the general level of skill in the art, those skilled in the art will appreciate that the following examples are intended to be exemplary only and that numerous variations, modifications and alterations may be employed without departing from the scope of the subject matter disclosed herein.

[0299] Example 1

[0300] Synthesis of compound CP-1

[0301] like Figure 3 The di-BOC protected Suzuki coupling partner 1 (CP-1) was prepared as shown in Scheme 3.

[0302] 1-Bromo-3,5-bis(bromomethyl)benzene (CP-1a). N-Bromosuccinimide (NBS, 35.60 g, 200.0 mmol) was added to a flame-dried 3-neck 1 L round bottom flask (RBF) with a stir bar, equipped with a glass stopper, a condenser with a septum, and a rubber septum. The NBS was dried under high vacuum for 30 minutes, then the flask was flushed with argon and acetonitrile (ACN, 400 mL) was added via cannula to about half the volume (~450 mL). 1-Bromo-3,5-dimethylbenzene (15.26 g, 80.0 mmol) was added via a syringe, followed by a brief opening of the system under an argon stream and a large addition of solid azobisisobutyronitrile (AIBN, 0.670 g, 4.00 mmol). The flask was heated to a gentle reflux (oil bath set at 90° C.) under argon.

[0303] After 16 hours, the reaction mixture was transferred to a single-neck 1L RBF and concentrated to remove ACN. The solid residue was further dried under high vacuum, suspended in dichloromethane (DCM, 75mL), and heated to a slight boil. The mixture was balanced to room temperature and filtered with DCM washing. The filtrate was concentrated, dried under high vacuum, and recrystallized in ethanol (EtOH, 55mL in total) when heated in a water bath set to 65 ° C. The solid was filtered and washed with ice-cold EtOH, then dried under high vacuum. Compound CP-1a (17.40g, 51%) in the form of a white crystalline solid was isolated.

[0304] 1 H NMR (400MHz, CDCl3) δ4.41 (s, 4H), 7.34 (s, 1H), 7.47 (d, J = 2.0Hz, 2H).

[0305] 2,2'-((5-bromo-1,3-phenylene)bis(methylene))bis(isoindoline-1,3-dione). (CP-1b). Compound CP-1a (18.43 g, 53.75 mmol) was dried in a 500 mL RBF with a stir bar. The flask was flushed with argon and dimethylformamide (DMF, 215 mL, 0.25 M) was added. The clear colorless solution was stirred and potassium phthalimide (23.37 g, 123.63 mmol) was added portionwise. The flask was equipped with a condenser topped with a drying tube and heated in an oil bath set at 90°C.

[0306] After 16 hours, the mixture was cooled to room temperature, diluted with water (1 L in total), and extracted with chloroform (400, 300 and 200 mL, 1× each). The organic layers were combined and washed with 0.2N NaOH aqueous solution (500 mL) and water (500 mL). The organic layer was separated, dried over sodium sulfate, filtered and concentrated. The solid was further dried under high vacuum, then transferred to a filter and washed with room temperature ether (Et2O) (3×). Compound CP-1b (17.98 g, 70%) was isolated as a white powdery solid.

[0307] 1 H NMR (400MHz, CDCl3) δ4.78 (s, 4H), 7.42-7.47 (m, 3H), 7.78-7.70 (m, 4H), 7.87-7.82 (m, 4H).

[0308] (5-Bromo-1,3-phenylene)dimethylamine (CP-1c). Compound CP-1b (7.63 g, 16.06 mmol) was suspended in EtOH (70.0 mL) and heated in an 85°C oil bath. Hydrazine hydrate (4.88 mL, 80.29 mmol) was added in one portion and the flask was capped with a condenser. The mixture was further heated at reflux temperature for 15 minutes and then the reaction mixture was gradually cooled to room temperature.

[0309] 6N HCl aqueous solution was added until the solution was acidic by litmus test (20 mL in total). The resulting mixture was heated to reflux temperature again. The flask was flushed with argon, stirred for 1 hour, then cooled and chilled in an ice bath. The mixture was filtered to obtain a clear light amber solution. The filtrate was cooled in an ice bath and basified with 2N NaOH aqueous solution (30 mL in total). The aqueous layer was extracted with chloroform (3×75 mL). The organic layers were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to about 10 mL volume. White residue was noted on the wall of the flask. The remaining solution was filtered, and the filtrate was concentrated to obtain 2.3 g of light yellow oil. Store in a refrigerator.

[0310] The sample solidified after being stored at 4°C overnight and was further dried under high vacuum to afford 2.047 g (59%) of compound CP-1c as an amber semisolid.

[0311] Di-tert-butyl((5-bromo-1,3-phenylene)bis(methylene))dicarbamate (CP-1d). Compound CP-1c (2.00 g, 9.11 mmol) was added to a flame-dried 250 mL RBF with a stirring bar. The flask was evacuated and flushed with argon. Tetrahydrofuran (THF, 45 mL) was added and the flask was lowered into a water bath. Diisopropylethylamine (3.83 mL, 21.86 mmol) was added and the heterogeneous mixture was cooled in an ice bath. Boc anhydride (4.86 g, 21.86 mmol) was prepared as a solution in THF (10 mL) and added dropwise in 1 mL portions. The solution was stirred at 0 °C for 1 hour and then allowed to equilibrate to room temperature.

[0312] The reaction was stirred at room temperature overnight. The mixture was concentrated to give a white solid, which was redissolved in ethyl acetate (EtOAc, 70 mL). The organic phase was washed with saturated NH4Cl aqueous solution, water, saturated NaHCO3 aqueous solution and brine (1×50 mL each). The organic layer was then dried over sodium sulfate, filtered and concentrated to a light amber oil that crystallized on standing. The solid was washed with cooled 1:1 Et2O / / hexane in a glass filter. The solid was dried under high vacuum to give 3.50 g (93%) of compound CP-1d in the form of a white powdery solid.

[0313] Coupling partner 1 (CP-1). Dimethyl sulfoxide (DMSO, reagent grade, 20.0 mL) was added to a 100 mL RBF and argon was introduced under stirring for 45 minutes. Compound CP-1d (1.25 g, 3.01 mmol), bis(pinacol)diboron (0.917 g, 3.61 mmol), potassium acetate (0.886 g, 9.03 mmol) and Pd(dppf)Cl2 (0.066 g, 0.090 mmol) were added together to a dry 250 mL RBF and the flask was evacuated for 30 minutes. The flask was purged with argon and degassed DMSO was added. The solution was frozen in a dry ice / acetone bath and placed under vacuum, then thawed under argon. The reaction mixture was then heated in an oil bath at 85°C.

[0314] After 16 h, the reaction mixture was cooled to room temperature, diluted in EtOAc (100 mL), and washed with brine (3 x 100 mL).The organic layer was dried over sodium sulfate, filtered, and concentrated.

[0315] The concentrate rinsed with minimal DCM was added to a 40 g silica column and eluted with DCM containing 0.2% MeOH. The main product fractions were combined and concentrated to obtain a clear oil. After further drying under high vacuum in the presence of a stirring rod, the product solidified. Compound CP-1 (1.224 g, 88%) was isolated as a white waxy solid.

[0316] Example 2

[0317] Synthesis of compound C-1

[0318] 4-Bromopyrrole-2-carboxaldehyde (C-1a). Figure 4 As shown in Scheme 4, a stirred solution of pyrrole-2-carboxaldehyde (70.0 g, 737 mmol) in THF (737 mL) was cooled to 0° C. in a 2 L round bottom flask under argon atmosphere. NBS (133 g, 737 mmol, reagent grade, not recrystallized) was added in one portion. The reaction mixture was stirred at 0° C. under argon for 15 min, and then the solvent was removed on a rotary evaporator.

[0319] The obtained solid was dried under high vacuum for 2 hours. Water (370mL) was added to the flask and the suspension was filtered with a Buchner funnel. The filter cake was washed with another 370mL of water. The solid material after filtration was transferred to a 2L Erlenmeyer flask and dissolved in 180mL of hot ethanol (78°C) by refluxing in a hot water bath. Hot water (1400mL, 100°C) was added once. After cooling to room temperature, the product crystallized from the solution. The mixture was further cooled to -10.0°C for 2-4 hours to promote more crystallization. The mixture was filtered by vacuum filtration and dried under high vacuum for 24-48 hours to obtain light brown crystals 4-bromopyrrole-2-carboxaldehyde (C-1a, 104g, 81%).

[0320] 1 H NMR (400MHz, CDCl3) δ6.91-7.03 (m, 1H), 7.05-7.17 (m, 1H), 9.48 (s, 1H), 9.68 (br s, 1H).

[0321] 4-Bromo-2-formyl-N-tosylpyrrole (C-1b). A suspension of 90% NaH (10.3 g, 429 mmol) in anhydrous THF (352 mL) was stirred in a 1 L oven-dried round-bottom flask, evacuated, and cooled to 0°C under argon. The mixture was treated with compound C-1a (62.0 g, 356 mmol) in batches over ~15 minutes. The mixture was stirred at 0°C for 30 minutes and then treated with p-toluenesulfonyl chloride (67.9 g, 356 mmol). The reaction was stirred at room temperature for 3 hours and then water (200 mL) was slowly added to quench the reaction.

[0322] Ethyl acetate (200mL) was added, the organic layer was separated, and washed with brine (100mL), dried (~50g Na2SO4), filtered, and concentrated to an oily liquid on a rotary evaporator. The oily liquid was dried overnight under high vacuum in a 1L round-bottomed flask. The resulting crude solid was dissolved in hexane / ethyl acetate (600mL, 5: 1) by refluxing in a hot water bath. After cooling to room temperature, the product crystallized from the solution. The mixture was further cooled to -10°C for 3 hours to promote additional crystallization. The mixture was filtered by vacuum filtration and the filtered brown crystals were dried under high vacuum to obtain compound C-1b (94g, 81%).

[0323] 1H NMR (400MHz, CDCl3) δ2.44 (s, 3H), 6.77 (s, 1H), 7.31 (d, J = 13.40Hz, 1H), 7.36 ( d, J=8.25Hz, 2H), 7.60 (s, 1H), 7.76 (d, J=8.25Hz, 2H), 8.44 (d, J=13.40Hz, 1H).

[0324] 4-Bromo-2-(2-nitrovinyl)-N-tosylpyrrole (C-1c). A stirred mixture of 4-bromo-2-formyl-N-tosylpyrrole (C-1b, 84.2 g, 257 mmol), potassium acetate (20.1 g, 205 mmol), methylamine hydrochloride (13.8 g, 205 mmol) and acetic acid (1.00 mL) in about 99.5% pure ethanol (90.0 mL) in a 500 mL round bottom flask was treated with nitromethane (34.6 mL, 641 mmol). The mixture was stirred for 2 hours, then water (200 mL) was added and the resulting yellow precipitate was filtered by vacuum filtration. The solid filter material was washed with water (500 mL) and then with cold ethanol (1.2 L, 0° C.) until the eluent was clear. The filtered yellow solid was dried under high vacuum overnight to give 4-bromo-2-(2-nitrovinyl)-N-tosylpyrrole (C-1c, 79 g, 83%).

[0325] 1 H NMR (400MHz, CDCl3) δ2.44 (s, 3H), 6.77 (s, 1H), 7.31 (d, J = 13.40Hz, 1H), 7.36 ( d, J=8.25Hz, 2H), 7.60 (s, 1H), 7.76 (d, J=8.25Hz, 2H), 8.44 (d, J=13.40Hz, 1H).

[0326] 4-Bromo-2-(2-nitroethyl)-N-tosylpyrrole (C-1d). A solution of compound C-1c (74.0 g, 199 mmol) in anhydrous THF (1 L) was cooled to -10°C (ice-acetone (1:1)) under argon in a 2 L round bottom flask. The solution was treated with 95% LiBH4 (4.34 g, 199 mmol) in one portion under vigorous stirring. The reaction mixture was stirred at -10°C for 30 minutes until all the starting material disappeared. After completion, the reaction mixture was quenched by slowly adding cold saturated NH4Cl aqueous solution (340 mL, 0°C). The mixture was stirred for 5 minutes and extracted with ethyl acetate (340 mL), dried (43 g anhydrous Na2SO4), concentrated on a rotary evaporator to a dark brown solid, and dried under high vacuum in a 2 L round bottom flask for 2 hours. The crude solid material was dissolved in isopropanol (IPA, 1.2 L) by refluxing in a hot water bath. After cooling to room temperature, the product crystallized from the solution. The mixture was further cooled at -10°C for 4 hours to promote more crystallization. The mixture was filtered by vacuum filtration and the filtered light brown crystals were dried under high vacuum overnight to give compound C-1d (47 g, 62%).

[0327] 1 H NMR (400MHz, CDCl3) δ2.44 (s, 3H), 3.39 (t, J = 7.01Hz, 2H), 4.60 (t, J = 7.01Hz, 2H), 6.10 ( d, J=1.93Hz, 1H), 7.32 (d, J=1.93Hz, 1H), 7.35 (d, J=7.98Hz, 2H), 7.69 (d, J=7.98Hz, 2H).

[0328] 6-(4-bromo-1-toluenesulfonyl-1 H

[00136] 1,1-dimethoxy-4-methyl-3-penten-2-one (C-1e). A mixture of compound C-1d (13.3 g, 35.7 mmol) and 1,1-dimethoxy-4-methyl-3-penten-2-one (10.5 g, 107.0 mmol, 3.0 equiv.) in RBF was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 16 mL, 107.0 mmol, 3.0 equiv.). The resulting mixture was stirred at room temperature for 1 hour and diluted with EtOAc. The organic layer was washed with (3X water, brine), dried (Na2SO4) and concentrated. Any excess 1,1-dimethoxy-4-methyl-3-penten-2-one was removed under high vacuum for 16 hours. The resulting crude product was dissolved in a minimum amount of CH2Cl2 (8 mL) and prepared as a silica cake. The silica cake was eluted on a 120 g SiO2 column with a 25-40% EtOAc / hexanes gradient over 58 min. The major fractions were combined, concentrated and dried under high vacuum rotary evaporator to afford 11.4 g (68%) of C-1e as a brown solid.

[0329] 1 H NMR (400MHz, CDCl3) δ1.11 (s, 3H), 1.24 (s, 3H), 2.13 (s, 3H), 2.40 (AB, J = 17.8Hz, 1H), 2.43 (s, 3H), 2.55 (AB, J = 17.8Hz, 1H), 3.18 (AB, J = 16.2Hz, 1H), 3.36 (ABX, 3J=16.2Hz, 2J=11.8Hz, 1H), 5.14 (AB, J=11.8Hz, 1H), 6.00-6.02 (m, 1H), 7.22-7.24 (m, 1H), 7.34 (AB, J=8.2Hz, 2H), 7.64 (AB, J=8.2Hz, 2H).

[0330] 4-Bromo-1-tosyl-2-((3,3,5-trimethyl-3,4-dihydro-2H-pyrrol-2-yl)methyl)-1H-pyrrole (C-1f). A solution of compound C-1e (10.5 g, 22.3 mmol) in THF (105 mL) in RBF was treated with HCO2NH4 (28.1 g, 446 mmol) and zinc powder (28.3 g, 446 mmol). The resulting suspension was vigorously stirred at room temperature for 2 hours. The reaction mixture was filtered through a silica (50 g) frit pad. The filter cake was eluted with EtOAc (500 mL). The filtrate was concentrated to a fluffy light brown solid. The residue was dissolved in CH2Cl2 and prepared as a silica cake. The silica cake was eluted on a 120 g SiO2 column with 75-80% hexane in EtOAc for 60 minutes. The single major product was combined, concentrated and dried under high vacuum to afford 6.29 g (67%) of C-1f as a light brown solid.

[0331] 1 H NMR (400MHz, CDCl3) δ0.88 (s, 3H), 1.07 (s, 3H), 1.97 (s, 3H), 2.28 (AB, J=16.8Hz, 1H), 2.36 (AB, J=16.8Hz, 1H), 2.41 (s, 3H), 2.63 (ABX, 2J=16 .1Hz, 3J=10.2Hz, 1H), 2.92 (ABX, 2J=16.1Hz, 3J=3.8Hz, 1H), 3.67-3.70 (m, 1H), 6.25-6.28 (m, 1H), 7.28-7.30 (m, 3H), 7.68 (AB, J=8.2Hz, 2H).

[0332] 4-Bromo-2-((3,3,5-trimethyl-3,4-dihydro-2H-pyrrol-2-yl)methyl)-1H-pyrrole (C-1g). Compound C-1f (2.79 g, 6.59 mmol) in RBF was treated with tetra-n-butylammonium fluoride (TBAF, 19.8 mL, 1.0 M in THF, 19.8 mmol, 3 equiv.) and the reaction was stirred at reflux (64-67°C) for 1 hour. Saturated aqueous NaHCO3 solution (68 mL) was added followed by ethyl acetate (68 mL). The mixture was extracted with ethyl acetate (134 mL). The organic layer was dried (anhydrous Na2SO4), concentrated on a rotary evaporator to a dark brown oil, and dried under high vacuum for 2 hours. The residue was dissolved in DCM and prepared as a silica cake. The cake was eluted on a 40 g SiO2 column with a 25-33% EtOAc / hexane gradient over 55 minutes. The major product fractions were combined, concentrated and dried under high vacuum to afford 1.56 g (88%) of compound C-1g as a light brown solid.

[0333] 1 H NMR (400MHz, CDCl3) δ0.92 (s, 3H), 1.11 (s, 3H), 2.03 (s, 3H), 2.28 (AB, J = 16.8Hz, 1H), 2.38 (AB, J = 16.8Hz, 1H), 2.54 (ABX, 2J = 14.9 Hz, 3J=11.8Hz, 1H), 2.69 (ABX, 2J=11.8Hz, 3J=2.5Hz, 1H), 3.56-3.62 (m, 1H), 5.85-5.94 (m, 1H), 6.63-6.69 (m, 1H), 9.72-10.01 (br s, 1H).

[0334] Synthesis of the eastern half of chlorin and its cyclization with the western half Figure 5 shown.

[0335] 5-(4-(Methoxycarbonyl)phenyl)dipyrromethane (C-1h). A mixture of pyrrole (183.9 g, 2741 mmol, 190 mL, 45 equiv.) and methyl 4-formylbenzoate (10.0 g, 60.9 mmol) in a 500 mL RBF was degassed under argon for 30 minutes. Trifluoroacetic acid (TFA, 1.18 mL, 0.25 equiv.) was added and stirred for another 30 minutes under argon. Thin layer chromatography (TLC) of an aliquot of the reaction mixture using CH2Cl2 / hexane (2:1) showed complete conversion of methyl 4-formylbenzoate to C-1h. The reaction mixture was diluted with 400 mL CH2Cl2 and washed with 200 mL 0.1N NaOH. The organic layer was then washed with brine (200 mL), dried over (20 g anhydrous Na2SO4), filtered and concentrated to dryness on a rotary evaporator. The crude solid was recrystallized using 200 mL of methanol / water (10:1) and filtered by vacuum filtration to give a light brown solid. Multiple recrystallizations using methanol / water (10:1) followed by filtration in a 150 mL glass frit gave 13.5 g (79%) of 5-(4-(methoxycarbonyl)phenyl)dipyrromethane (C-1h). Alternatively, the residue was dissolved in CH2Cl2 and prepared as a silica cake. The cake was eluted on a 120 g SiO2 column with a gradient of 75-100% CH2Cl2 in hexanes for 70 min. The major product fractions were combined, concentrated and dried under high vacuum to give a light brown solid.

[0336] 1-Formyl-5-(4-(methoxycarbonyl)phenyl)dipyrromethene (C-1i). Vilsmeier reagent was prepared according to the reported procedure (see Laha et al., J. Org. Chem. 2006, 71, 4092-4102). A sample of anhydrous DMF (8.0 mL) was treated with POCl3 (1.90 mL, 1.2 equiv., 20.3 mmol) under argon and stirred at 0°C for 20 min in an oven-dried 50 mL round-bottom flask. The resulting mixture was added (via cannula) to a second oven-dried, evacuated and argon-flushed round-bottom flask (250 mL) containing a solution of 5-(4-(methoxycarbonyl)phenyl)dipyrromethene (C-1h) (5.43, 19.4 mmol) in DMF (35 mL) at 0°C. After 1.5 h, saturated NaHCO3 solution (87 mL) was gently added. The obtained mixture was stirred overnight and extracted with ethyl acetate. The organic layers were combined, washed (brine), dried at anhydrous Na2SO4, filtered and concentrated. The residue was dissolved in CH2Cl2, and prepared as a silica cake (10 g). The cake was eluted with 0-40% EtOAc gradient in hexane for 51 minutes on 80 g SiO2 posts. The main product fractions were combined, concentrated and dried under high vacuum to obtain 3.37 g (56%) C-1i, which was a light brown solid.

[0337] 8,9-Dibromo-1-formyl-5-(4-(methoxycarbonyl)phenyl)dipyrromethane (C-1j). C-1i (1.40 g, 4.54 mmol) in THF (45 mL) was treated with recrystallized NBS (1.62 g, 9.08 mmol) in an oven-dried round-bottom flask (250 mL) at -78°C. After 1 h, the cooling bath was removed, the reaction mixture was allowed to warm to -20°C, and a mixture of hexanes and water (1:1, 13 mL) was added. The resulting mixture was diluted with ethyl acetate, washed with brine, dried (Na2SO4) and concentrated. The residue was dissolved in CH2Cl2 and prepared as a silica cake or slurry (~6.0 g). The cake was eluted on a 80 g SiO2 column with a 0-20% EtOAc / hexanes gradient over 86 min. The major product fractions were combined, concentrated and dried under high vacuum to afford 2.00 g (94%) of C-1j as a yellow solid.

[0338] 3,13-Dibromo-10-(4-(methoxycarbonyl)-18,18-dimethylchlorin Zn(II) (C-1k). A suspension of 3,4,5,6-tetrahydro-1,3,3-trimethyldipyrrole (C-1g, 0.63 g, 2.35 mmol) and 8,9-dibromo-1-formyl-5-(4-(methoxycarbonyl)phenyl)-dipyrromethene (C-1j, 1.09 g, 2.35 mmol)) in CH2Cl2 (35 mL) was treated with p-toluenesulfonic acid monohydrate (2.23 g, 11.7 mmol) in methanol (12 mL) and stirred at room temperature for 40 minutes. The sample was examined by Uv-vis for a strong broad band at about 480 nm. The resulting mixture was treated with 2,2,6,6-tetramethylpiperidine for 5-10 minutes (7.92 mL, 46.9 mmol). The reaction mixture was concentrated on a rotary evaporator, the resulting brown solid was suspended in acetonitrile (235 mL) and treated with zinc acetate (6.47 g, 35.2 mmol), 2,2,6,6-tetramethylpiperidine (15.9 mL, 93.9 mmol) and silver trifluoromethanesulfonate (1.82 g, 7.04 mmol). The resulting suspension was refluxed for 18-24 hours. The reaction mixture was cooled and concentrated, and the residue was dissolved in CH2Cl2 and filtered through a silica bed on a glass powder until the eluent was clear. The residue was prepared into a silica cake and eluted on a 120 g SiO2 column with a hexane solution containing 50% CH2Cl2 for 3 minutes and kept at the gradient for another 15 minutes. The gradient was changed to hexane containing 70% CH2Cl2 for 3 minutes and kept at the gradient for another 5 minutes. The gradient was subsequently changed to hexane containing 80% CH2Cl2 for 5 minutes and kept at the gradient for 20 minutes. All green fractions were combined after TLC to give C-1k as a green solid (318 mg; 20% yield).

[0339] 3,13-Dibromo-10-(4-(methoxycarbonyl)-18,18-dimethylchlorin (C-11). C-1k (49 mg, 0.070 mmol) was placed in a round-bottom flask. To the flask was added 5.0 mL of a mixture of CH2Cl2 / TFA (4.90 / 0.10 ml), and the resulting mixture was stirred for 2 hours. The mixture was quenched with saturated aqueous NaHCO3 solution (20 mL), washed with brine, dried (Na2SO4), filtered and concentrated. The concentrate was recrystallized in hexanes / CH2Cl2 (20 mL) to give compound C-11 as a dark powder (34 mg, 75%).

[0340] like Figure 6 As shown in Scheme 6, compound C-11 is then used to prepare compound C1 via intermediate compounds C-1m and C-1n.

[0341] Suzuki coupling precursor (C-1m). A mixture of compound C-11 (63.2 mg, 100 μmol), coupling partner CP-1 (102 mg, 220 μmol), Pd(PPh 3 ) 4 (69.4 mg, 60.0 μmol) and Cs 2 CO 3 (196 mg, 60.0 μmol) was placed in a 25 mL RBF. The flask was placed under high vacuum for 1 hour and then further degassed by three evacuation-refill cycles. Toluene / DMF (2: 1, 10 mL in total) was added via a syringe and the solution was heated at 90° C. for 18 hours.

[0342] After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with aqueous NaHCO3, and dried over Na2SO4. The resulting mixture was concentrated and purified by chromatography [silica 40 g, hexane / ethyl acetate (0-75%)] to give compound C-1m (94.9 mg, 83%) as a green solid.

[0343] Suzuki coupling precursor with tert-butyl protected β-alanine linker (C-1n). Compound C-1m (38.1 mg, 33.3 μmol) in THF (13.3 mL) and MeOH (6.7 mL) was treated with 1.0 M NaOH aqueous solution (6.7 mL). The reaction mixture was stirred at room temperature in the dark under argon. After 3.5 hours, the reaction mixture was diluted with EtOAc, washed with 1.0% HCl aqueous solution, dried over Na2SO4 and concentrated.

[0344] To the crude product residue was added O-(N-succinimidyl)-N,N,N',N'-tetramethylurea Tetrafluoroborate (TSTU, 40.1 mg, 133 μmol), CH2Cl2 (8.3 mL) and TEA (18.5 μL, 133 μmol). The reaction mixture was stirred at room temperature in the dark under argon. After 1 hour, the reaction mixture was diluted with EtOAc, washed with brine, dried over Na2SO4 and concentrated.

[0345] β-Alanine tert-butyl ester hydrochloride (45.3 mg, 333 μmol) and CsCO (109 mg, 333 μmol) were added to the obtained residue. The system was evacuated, flushed with argon, and CHCl (8.3 mL) was added. The reaction mixture was stirred at room temperature in the dark under argon. After 3 hours, the reaction mixture was diluted with EtOAc, washed with brine, dried and concentrated with NaSO. Chromatographic purification [silicon dioxide 12 g, hexane / ethyl acetate (0-100%)] gave compound C-1n (40.2 mg, 97%) as a green solid.

[0346] MS: found 1257.04, calculated 1256.68 [(M+H) + , M=C 72 H 89 N9O 11 ].

[0347] C-1. Compound C-1n (12.6 mg, 10.0 μmol) in 10 mL RBF was treated with 4.0 M HCl (2.5 mL) in dioxane. The reaction mixture was stirred at room temperature in the dark under argon. After 1.5 hours, the reaction mixture was placed under high vacuum for 16 hours.

[0348] To the resulting residue was added CH3(OC2H4) 12 CONHS(mPEG 12 -NHS, 54.9 mg, 80.0 μmol) and Cs2CO3 (52.2 mg, 160 μmol), and the flask was evacuated and purged with inert gas. DMF (2.5 mL) was added, and the reaction mixture was stirred at room temperature. After 1 hour, the reaction mixture was chromatographed [C18 gold, 15.5 g, H2O / CH3CN (0-40%)] to obtain C-1 as a green solid (4.0 mg, 13%). The synthesis of C-1 was subsequently scaled up so that starting from compound C-1n (155.3 mg, 124 μmol), 252 mg of compound C-1 (66% yield) was obtained.

[0349] MS: found 1541.94, calculated 1541.86 [M+2H] 2+ ; Found 1553.24, calculated 1552.85 [(M+H+Na] 2+ ; Found 1563.89, calculated 1563.84 [(M+2Na] 2+ ; abs 417, 651nm (PBS); λ em 656nm (PBS). Quantum yield: 26% (PBS); FWHM: 20nm (PBS).

[0350] The solubility of chlorins can be assessed by measuring the absorbance of a series of diluted chlorin stock solutions. See Jiang et al. (2014) Organic & Biomolecular Chemistry 12: 86-103. The solubility of C-1 was determined to be >10 mg / mL (PBS).

[0351] Example 3

[0352] Synthesis of compound 4

[0353] Sonogashira coupled NIRvana 680 precursor (2). Figure 7 Compound 4 (also referred to herein as NIRvana 680) was synthesized as described in Scheme 7 shown in FIG.

[0354] Compound C237 was prepared as follows: A mixture of 5-ethynyl-1,3-benzenedicarboxylic acid (500 mg, 2.63 mmol), tert-butyl N-(2-aminoethyl)carbamate (2.08 mL, 13.2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 2.00 g, 10.4 mmol) and 4-dimethylaminopyridine (DMAP, 1.48 g, 13.2 mmol) was dissolved in DMF (3.3 mL). The flask was stirred at room temperature for 16 hours. The reaction mixture was directly loaded onto silica gel and chromatographed [silica, CH2Cl2 / MeOH (0-10%)] to provide a white solid. By 1 The resulting solid contained DMAP by H NMR, so it was redissolved in ethyl acetate and washed with 1.0% aqueous HCl, dried over Na2SO4 and concentrated to afford C237 (938 mg, 75%) as a white solid. A mixture of compound 1 (60.0 mg, 0.095 mmol), coupling partner C237 (225 mg, 0.474 mmol, 5.0 equiv.) and PdCl2(PPh3)2 (17 mg, 0.024 mmol, 0.25 equiv.) was placed in an oven-dried 100 mL Schlenk flask. The flask was placed under high vacuum for 0.5 h and then further degassed by three evacuate-refill cycles. Toluene / Et3N (2:1, 9 mL total) was added via syringe and the solution was heated at 85-90°C for 24 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with aqueous NaHCO3, brine, dried over Na2SO4 and concentrated. The residue was dissolved in a minimum amount of CH2Cl2 and prepared as a silica cake. The cake was eluted on a 40 g silica column with CH2Cl2 / MeOH (0-1%) to give compound 2 (132 mg, 98%) as a dark solid. MS: [M+H] + Calculated value: 1419.68; measured value: 1419.86.

[0355] Sonogashira coupled NIRvana 680 precursor with tert-butyl protected β-alanine linker. Compound 2 (45.8 mg, 32.3 μmol) in THF (6.0 mL) and MeOH (3.0 mL) was treated with 1.0 M aqueous NaOH (3.0 mL). The reaction mixture was stirred at room temperature in the dark. After 4.5 hours, the reaction mixture was quenched with 1.0 M aqueous HCl (4.5 mL), diluted with EtOAc (25 mL), washed with brine (25 mL), dried over Na2SO4 (4.0 g), filtered, concentrated and dried under high vacuum. To the crude product residue were added TSTU (19.4 mg, 64.4 μmol, 2.0 equiv.), CH2Cl2 (10.0 mL, 3.2 mM) and triethylamine (9.0 μL, 194 μmol, 6.0 equiv.). The reaction mixture was stirred at room temperature in the dark under argon for about 1 hour. To the resulting residue were added β-alanine tert-butyl ester hydrochloride (17.6 mg, 96.8 μmol, 3.0 equiv.), triethylamine (27.0 μL, 64.4 μmol, 2.0 equiv.), and the reaction was stirred at room temperature overnight. Additional TEA (54 μL, 12 equiv.) and β-alanine tert-butyl ester hydrochloride (52.8 mg, 9.0 equiv.) were added, and the reaction was stirred overnight. The reaction was then heated to reflux for 10 minutes. The reaction mixture was diluted with CH2Cl2 (15 mL), washed with brine, the organic layer was separated and dried over anhydrous Na2SO4, filtered and concentrated. Chromatographic purification [silica 12 g, wet loaded and eluted with CH2Cl2 / MeOH (0-1%)] gave compound 3 (38.2 mg, 77%) as a dark solid. MS: [M+H] + Calculated value: 1532.76; measured value: 1533.11.

[0356] NIRvana 680 (Compound 4). Compound 3 (38.2 mg, 24.9 μmol) in a scintillation vial with a sealed screw cap was evacuated and flushed with argon. HCl solution (4.0 M in dioxane, 2.5 mL) was added under argon. The reaction mixture was stirred at room temperature in the dark under argon. After 1.5 hours, the reaction mixture was placed under a high argon flow with an outlet needle to remove the solvent. After completion, the vial was dried under vacuum overnight. CH3O(OC2H4) was added to the resulting residue. 12CH2CH2COONHS ester (109.1 mg, 149.5 μmol, 6.0 equiv.) and Cs2CO3 (81.2 mg, 249.2 μmol, 10 equiv.) were added, and the flask was evacuated and purged with inert gas. DMF (3.0 mL) was added, and the reaction mixture was protected from light and stirred at room temperature under argon. After 1.5 hours, CH3O(OC2H4) was added. 12 CH2CH2COONHS ester (109.1 mg, 149.5 μmol, 6.0 equiv.) and Cs2CO3 (81.2 mg, 249.2 μmol, 10 equiv.) were added and the vial was evacuated and flushed with argon for 10 minutes, then stirring was continued under an argon atmosphere. After 3.5 hours, the reaction was concentrated. The sample was dissolved in water containing 30% ACN (2.0 mL) and then subjected to reverse phase preparative LC using a water gradient containing 30-80% ACN for 45 minutes. The main product peaks were combined, concentrated, transferred to a storage bottle of ACN, concentrated, and dried under high vacuum to give 17.5 mg (20%) of compound 4 as a green residue. MS: [M+2H] 2+ Calculated value: 1767.45; measured value: 1767.54.

[0357] Example 4

[0358] Flow cytometry

[0359] instrument : Samples were analyzed on a 19-parameter LSR-IISORP flow cytometer (BD Biosciences, San Jose, California, USA) equipped with 7 lasers (355, 405, 488, 532, 561, 594 and 633 nm) or an LSRFortessa (BD Biosciences, San Jose, California, USA) equipped with 5 lasers (355, 405, 488, 561 and 640 nm) and using FACSDiva 8.0 acquisition software. C-1 data were collected in channel A of a 100 mW 405 nm laser with a 630 nm long pass (LP) filter and a 660 / 20 nm band pass (BP) filter. Data for chlorin H2C12-PEG6-NHS used a 635 nm LP and 655 / 40 nm BP filter.

[0360] Antibody bioconjugation The NHS ester of C-1 was prepared as described above for the preparation of the intermediate NHS ester in the synthesis of C-1n using TSTU and TEA in CH2Ci2 and showed a single peak in HPLC-MS (MS: found 1591 [M+2H] 2+ ,1602[M+H+Na]2+ . A solution was prepared in a microcentrifuge tube from 106 μL of 9.4 mg / mL (1.0 mg) anti-human CD8 mouse monoclonal antibody (clone UCHT-4, Leinco Technologies, Inc., St. Louis, MO, USA), 15 μL of 1 M bicarbonate (pH 8.4), and 44 μL of PEGylated dye NHS ester in PBS (5 to 20 molar equivalents). The tube was protected from light and gently rotated at room temperature for 1-2 hours. The reaction was quenched for another hour by adding 15 μL of 200 μM Tris at room temperature. The bioconjugate was purified using a Sephadex G50M, G75M, or G100M size exclusion chromatography column eluted with PBS. Antibody bioconjugates prepared from dyes with longer PEG chains (12 units or more) are typically purified using a G75M or G100M vehicle. The column fractions are characterized by absorption at 280 nm (protein) and red or NIR dye absorption maxima. The fluorophore to protein (F / P) labeling ratio of the combined fractions was determined from these two maxima corrected for dye absorption at 280 nm.

[0361] Cell staining :Cryopreserved human peripheral blood mononuclear cells (PBMCs) were obtained from ZenBio. Inc. (Research Triangle Park, North Carolina, USA; product SER-PBMC-F), thawed and prepared for staining according to the supplier's instructions. The cells were divided into six 1.5 mL microcentrifuge tubes and centrifuged at 400xg (2000rpm) for 5 minutes. The cells were washed three times with wash buffer (PBS containing 0.5% BSA) and resuspended in 0.5 mL wash buffer. The aliquots were diluted 1:2 with trypan blue and the cell number and viability were determined by counting 4 nL squares on a hemocytometer. The viability was usually >96%. The cells were diluted to 1x10 using wash buffer. 6 / mL and aliquot 50 μL (500,000 cells) into microcentrifuge tubes. Typically, the maximum labeled antibody concentration is 4.74 μg / 5x10 5 Cells (designated as 3.16X) were added and half-log dilutions were prepared. For all antibodies except the control antibody, these dilutions were made so that 15 μL was added to the cell aliquot. The cells and antibodies were mixed and incubated for 30 minutes at room temperature. Each tube was washed twice with 1 mL of wash buffer, and the cells were then resuspended in 0.5 mL of wash buffer containing 1% formaldehyde. Before characterization by flow cytometry, the samples were filtered through nylon filter cloth into flow cell tubes.

[0362] Positive control bioconjugates were selected from CD8 (UCHT-4)-fluorescein isothiocyanate (FITC) (Leinco Technologies, Inc., St. Louis, MO, USA; Catalog No. C119), CD4 (RPA-T4)-BUV737 antibody (BD Biosciences, San Jose, CA, USA; Catalog No. 564306), and / or CD8 (UCHT-4) DY650 antibody (Leinco Technologies, Inc., St. Louis, MO, USA; Catalog No. C2064), as required, and titrated with PBMCs by the same general procedure. Staining index (SI) was calculated from mean fluorescence intensity (MFI) values ​​as follows according to Maecker et al. (Cytometry A. 2004, 62, 169-173):

[0363] SI = (mean: positive - mean: background) / (2 x SD background)

[0364] Table 1 below shows the staining index data of the titration of the anti-CD8 bioconjugate of C-1 and the anti-CD8 bioconjugate of H2C12-PEG6-NHS (previously described a chlorin with three PEG6 chains connected by 10-phenyl, see Liu et al., Molecules, 2018, 23, 130). For comparison, an anti-CD8 bioconjugate prepared by FITC (Leinco Technologies, Inc., St. Louis, Missouri, USA; cat#C119) is also provided.

[0365] Table 1

[0366] Staining Index Data for Titration of Anti-CD8 Dye Conjugates

[0367] dye F / P Ratio Maximum staining index <![CDATA[H2C12-PEG6-NHS Chlorin e6 PEGylation]]> 2.1 0 C-1 3.8 11.5 FITC Undetermined 63

[0368] These results indicate that the PEGylation design of C-1 significantly enhances performance.

[0369] References

[0370] All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entirety, to the extent they supplement, explain, provide context for, or teach the methods, techniques and / or compositions employed herein.

[0371] It will be appreciated that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

1. Compounds of formula (I): in: M is a metal or two hydrogens; R5 and R 15 is H; R 10 for R2 and R 12 is H; R3 and R 13 are independently selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , wherein w is an integer from 0 to 5, inclusive, and R S is a group having the formula: <h2 style=";text-align:left;direction:ltr">-X2-(L3)<h2 style=";text-align:left;direction:ltr"> z <h2 style=";text-align:left;direction:ltr"> -R<h2 style=";text-align:left;direction:ltr"> 17 <h2 style=";text-align:left;direction:ltr"> , wherein: z is 0; X2 is -CH2NHC(=O)- or -C(=O)NH-alkylene-NH-; L3 is -C(=O)-alkylene-C(=O)-NH-, and R 17 Selected from -(C2H4O) m -R 18 and -C(=O)C2H4-(OC2H4) m OR 18 , wherein m is an integer of 4 or more; R 18 It is a lower alkyl group.

2. The compound of claim 1, wherein m is an integer of 8 or greater.

3. The compound of claim 1, wherein R 18 It's methyl.

4. The compound of any one of claims 1 to 3, wherein R3 and R 13 Each is -aryl-(R s ) w .

5. The compound of claim 4, wherein R3 and R 13 Each is -phenyl-(R s )2.

6. The compound of claim 5, wherein each R s With the formula -X2-(L3) z -R 17 ,in: z is 0, X2 is -CH2NHC(=O)-, and R 17 -(C2H4O) m -R 18 , wherein m is an integer from 12 to 24.

7. The compound of claim 6, wherein R3 and R 13 Each is:

8. The compound of any one of claims 1 to 3, wherein the compound is:

9. A composition comprising a covalent conjugate formed between: (a) a compound according to any one of claims 1 to 8; and (b) one or more of the group consisting of small molecules, antigens, microparticles, polymers, peptides, antibodies, hormones and growth factors.

10. A composition comprising a covalent conjugate formed between: (a) a compound according to any one of claims 1 to 8; and (b) one or more of the group consisting of nanoparticles, proteins, antibody fragments and nucleic acids.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10, and a pharmaceutically acceptable carrier.

12. A method for detecting a target, wherein the target is a compound, a cell or a particle, wherein the method comprises labeling the target with the composition of claim 9 or 10.

13. The method of claim 12, wherein the method comprises using flow cytometry.

14. A method of imaging a cell, tissue or organism, wherein the method comprises using a compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10.

15. Use of a compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10 or a pharmaceutical composition according to claim 11 for the preparation of a medicament for treating a disease in a subject in need thereof.

16. The use according to claim 15, wherein the disease is a hyperproliferative disease.

17. The use according to claim 15, wherein the disease is cancer.

18. A water-soluble chlorin dye comprising a compound according to any one of claims 1 to 8 or a composition according to claim 9 or 10 or a pharmaceutical composition according to claim 11, and having a solubility in an aqueous solution of higher than 1 mg / ml.

19. The water-soluble chlorin dye of claim 18, which has a solubility of 2.5 mg / ml or more in an aqueous solution.

20. The water-soluble chlorin dye of claim 19, which has a solubility of 10 mg / ml or more in an aqueous solution.

21. A method for preparing a synthetic intermediate of a compound according to any one of claims 1 to 8: The method comprises: (a) providing a compound having formula (I'): in: M is metal or -H, -H; R5' and R 15 'For H; R 10 'for R2' and R 12 ' is H; and R3' and R 13 'Independently selected from as well as (b) contacting the compound provided in step (a) with a dioxane solution comprising 4 molar (M) HCl to provide a compound of formula (I"): in: M is metal or -H, -H; R5" and R 15 ” is H; R 10 "for R2" and R 12 ” is H; and R3" and R 13 "Independently selected from 22. A compound having one of the following formulae: or a conjugate thereof, wherein the compound is conjugated to one or more of the group consisting of a small molecule, an antigen, a microparticle, a polymer, a peptide, an antibody, a hormone and a growth factor.

23. A compound having one of the following formulae: or a conjugate thereof, wherein the compound is conjugated to one or more of the group consisting of nanoparticles, proteins, antibody fragments and nucleic acids.

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