Narrow emission dyes, compositions comprising same, and methods of making and using same

Abstract

The present invention relates to narrow emission dyes, compositions comprising the same, and methods of making and using the same. In particular, the present application provides bacteriochlorin derivatives having narrow band emission. In some embodiments, the bacteriocin derivative is PEGylated. In some embodiments, the bacteriocin derivatives have a high water solubility (e.g., 10 mg / mL or more). In some embodiments, the bacteriocin derivatives are PEGylated and have a high water solubility. The bacteriocin derivatives may comprise a bioconjugatable group for forming a conjugate (e.g., with an antibody or nanoparticle). The bacteriocin derivatives and conjugates thereof are useful for imaging and therapeutic applications. Also provided are methods of synthesizing the bacteriocin derivatives.

Description

[0001] This application is a divisional of PCT Application No. PCT / US2020 / 033627, filed May 19, 2020, entitled “Narrow Emission Dyes, Compositions Comprising the Same, and Methods of Making and Using the Same,” which entered the Chinese national phase as Application No. 202080052180.1.

[0002] Cross Reference to Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 850,446, filed May 20, 2019, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0004] The presently disclosed subject matter relates generally to bacteriochlorophyll derivatives having narrow emission wavelengths (in some embodiments, water-soluble bacteriochlorophyll derivatives having narrow emission wavelengths), conjugates thereof, and methods of making and using the same. BACKGROUND

[0005] A large and growing number of applications require fluorescent dyes that are water-soluble and suitable for conjugation to other species ranging from nanoparticles to biologic targeting agents. Such applications include, for example, flow cytometry, cellular and whole organism imaging, sensing, and photodynamic therapy. Among these applications, bacteriochlorophyll molecules are of particular interest because bacteriochlorophylls generally absorb in the near infrared (NIR, 700-900 nm) region, making them one of the few chromophores available for photochemical studies in the NIR region.

[0006] Success in the aforementioned applications depends on a number of factors, including (1) significant solubility in saline solutions, thereby avoiding intermolecular aggregation (and excited state quenching), (2) minimal non-specific binding to cellular components, (3) addition of a single reactive group for conjugation, thereby avoiding cross-linking and mixtures of products, and (4) robust synthesis to provide sufficient quantities for experimentation. However, the large hydrophobic face of bacteriochlorophylls presents a challenge to water solubility.

[0007] Accordingly, there is a continuing need to provide additional bacteriochlorophyll derivatives, including but not limited to those having improved water solubility (e.g., water solubility higher than 1 mg / mL), particularly those that can also be readily conjugated to a variety of species. There is also a continuing need for additional bacteriochlorophylls that combine improved water solubility with narrow absorption and emission bands. SUMMARY

[0008] This summary provides an overview of several embodiments of the presently disclosed subject matter, and in many cases lists variations and alternatives of these embodiments. This summary is not exhaustive of the many different embodiments. Reference to one or more representative features of a given embodiment is likewise exemplary. Such embodiments can be present or absent, as the case can be, with the referenced features; similarly, these features can be applied to other embodiments of the presently disclosed subject matter, whether or not listed in this summary. To avoid undue repetition, this summary does not list or indicate all possible combinations of such features.

[0009] In some embodiments, the presently disclosed subject matter provides a compound of Formula (II):

[0010]

[0011] wherein: M is a metal or -H, -H; R5, R 10 and R 15 are independently selected from H, alkoxy, and a linking group of the formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1is alkylene; X1is -C(=O)NH- or -NHC(=O)-; L2is -(CH2CH2O) q alkylene, alkylene, or substituted alkylene, optionally wherein substituted alkylene is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; G is a bioconjugatable group; and R2, R3, R 12 and R 13 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linking group of the formula -L1-(X1-L2) p -G, 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 R S is a group of the formula:

[0012] -X2-(L3) z -R 17 ,

[0013] wherein: z is 0 or 1; X2is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH-, or triazolyl; L3is -C(=O)-alkylene-C(=O)-NH-, and R 17 is selected from -(C2H4O) m -R 18-C(=O)C2H4-(OC2H4) m OR 18 and - (C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, where m is an integer of 12 or greater; n is an integer from 1 to 5; R 18 is lower alkyl, optionally methyl; R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; with the proviso that at least one of R2, R3, R 12 and R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0014] In some embodiments, M is Zn. In some embodiments, R5, R 10 and R 15 are independently selected from H, methoxy, and a linking group having the formula: -L1-(X1-L2) p -G.

[0015] In some embodiments, R3and R 13 are each an ester, optionally -C(=O)OCH3. In some embodiments, R2is

[0016]

[0017] In some embodiments, each R s is a group having the formula: -X2-(L3) z -R 17 where: z is 0; X2is -C(=O)NH-alkylene-NH-; and R 17 is -C(=O)C2H4-(OC2H4) m OR 18 where m is an integer of 12 or greater, and R 18 is methyl.

[0018] In some embodiments, each R s is

[0019]

[0020] In some embodiments, each R s is a group having the formula: -X2-(L3) z -R 17wherein: z is 1; X2is -C(=0)NH-alkylene-NH-; L3is -C(=0)-propylene-C(=0)-NH-; and R 17 is -(C2H4O) n -C2H4-C(=0)NH-C(R 19 )3, wherein n is an integer from 1 to 5, optionally 4; and each R 19 is -CH2O-C2H4-C(=0)NH-(C2H4O) m R 18 wherein m is an integer of 12 or greater, optionally wherein m is 12; and R 18 is methyl.

[0021] In some embodiments, R2and R 12 are different or R3and R 13 are different. In some embodiments, one of R2and R 12 is a solubilizing group selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w and one of R2and R 12 is a linking group having the formula -L1-(X1-L2) p -G, or wherein one of R3and R 13 is a solubilizing group selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w and one of R3and R 13 is a linking group having the formula -L1-(X1-L2) p -G.

[0022] In some embodiments, R 12 is a linking group having the formula -L1-(X1-L2) p -G. In some embodiments, R 12 is a group having the formula -L1-(X1-L2) p -G;

[0023] wherein p is 0; L1is aralkynylene; and G is a bioconjugatable group. In some embodiments, R 12 is

[0024]

[0025] wherein G is selected from carboxylic acid and active ester.

[0026] In some embodiments, R 12is a group of the formula: -L1-(X1-L2) p -G;

[0027] wherein p is 1; L1is arynylene; X1is -C(=0)NH-; L2is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bioconjugatable group. In some embodiments, L1is -C≡C-(C6H4)-. In some embodiments, L2is -CH(R)-, wherein R is alkylene-NH-C(=0)-alkylene-(OC2H4) q -OR 16 wherein q is an integer from 12 to 24 and R 16 is methyl.

[0028] In some embodiments, the compound is selected from:

[0029]

[0030]

[0031]

[0032] In some embodiments, the presently disclosed subject matter provides a composition comprising a covalent conjugate formed between: (a) a compound of Formula (II), with the proviso that at least one of R2, R3, R5, R 10 , R 12 , R 13 , and R 15 is a linking group; and (b) one or more of the group consisting of small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors.

[0033] In some embodiments, the presently disclosed subject matter provides a compound of Formula (II) or a conjugate formed between: (a) a compound of Formula (II), with the proviso that at least one of R2, R3, R5, R 10 , R 12 , R 13 , and R 15 is a linking group; and (b) one or more of the group consisting of small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors; and a pharmaceutically acceptable carrier.

[0034] In some embodiments, the presently disclosed subject matter provides a method of detecting a target, wherein the target is a compound, a cell, or a particle, wherein the method comprises labeling the target with a conjugate formed between: (a) a compound of Formula (II), with the proviso that at least one of R2, R3, R5, R 10 , R 12 , R 13 , and R 15 is a linking group; and (b) one or more of the group comprising a small molecule, 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.

[0035] In some embodiments, the presently disclosed subject matter provides a method of imaging a cell, a tissue, or an organism, wherein the method comprises using a compound of Formula (II) or a conjugate formed between: (a) a compound of Formula (II), with the proviso that at least one of R2, R3, R5, R 10 , R 12 , R 13 , and R 15 is a linking group; and (b) one or more of the group comprising a small molecule, a microparticle, a nanoparticle, a polymer, a peptide, a protein, an antibody or antibody fragment, a nucleic acid, a hormone, and a growth factor.

[0036] In some embodiments, the presently disclosed subject matter provides a method of treating a disease in an individual in need of treatment thereof, the method comprising: administering to the individual a compound of Formula (II); a conjugate formed between: (a) a compound of Formula (II), with the proviso that at least one of R2, R3, R5, R 10 , R 12 , R 13 , and R 15 is a linking group; and (b) one or more of the group comprising a small molecule, a microparticle, a nanoparticle, a polymer, a peptide, a protein, an antibody or antibody fragment, a nucleic acid, a hormone, and a growth factor; or a pharmaceutical composition of the compound or the 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 a cancer.

[0037] In some embodiments, the presently disclosed subject matter provides a bacteriochlorophyll dye having a solubility in aqueous solution of higher than about 1 mg / ml, optionally having a solubility in aqueous solution of about 3.0 mg / ml or higher, further optionally having a solubility in aqueous solution of about 10 mg / ml or higher. In some embodiments, the dye has an emission wavelength higher than about 850 nanometers.

[0038] In some embodiments, the presently disclosed subject matter provides methods of making synthetic intermediates for the preparation of compounds of Formula (II):

[0039]

[0040] wherein: M is a metal or is -H, -H; R5, R 10 and R 15 are independently selected from H, alkoxy, and a linking group of the formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1is alkylene; X1is -C(=O)NH- or -NHC(=O)-; L2is -(CH2CH2O) q -alkylene, alkylene, or substituted alkylene, optionally wherein substituted alkylene is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bioconjugatable group; and R2, R3, R 12 and R 13 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linking group of the formula -L1-(X1-L2) p -G, 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 R S is a group of the formula: -X2-(L3) z -R 17 wherein: z is 0 or 1; X2is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH- or triazolyl; L3is -C(=O)-alkylene-C(=O)-NH-, and R 17 is 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 12 or greater; n is an integer from 1 to 5; R 18 is lower alkyl, optionally methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; provided that R2, R3, R 12 and R13 at least one of R2', R3', R s ) w or -alkynyl-aryl-(R s ) w ; wherein the method comprises: (a) providing a compound of Formula (II'):

[0041]

[0042] wherein: M is a metal or is -H, -H; R5', R 10 ' and R 15 ' are independently selected from H, alkoxy,

[0043] and

[0044] R2', R3', R 12 ' and R 13 ' are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl,

[0045]

[0046] with the proviso that at least one of R2', R3', R 12 ' and R 13 ' is

[0047] and

[0048] (b) contacting the compound provided in step (a) with a solution of 4 moles (M) HCl in dioxane to provide a compound of Formula (II"):

[0049]

[0050] wherein: M is a metal or is -H, -H; R5", R 10 " and R 15 " are independently selected from H, alkoxy,

[0051]

[0052] and R2", R3", R 12 " and R 13 " are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl,

[0053]

[0054] with the proviso that at least one of R2", R3", R 12 " and R 13 " isat least one of the following is present in the compound of formula (I)

[0055]

[0056] In some embodiments, the presently disclosed subject matter provides a method of preparing an asymmetric bacteriochlorophyll compound having the following formula:

[0057]

[0058] wherein: M is a metal or is -H, -H; R5, R 10 and R 15 are independently selected from H and alkoxy; and R2, R3, R 12 and R 13 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linking group, and a solubilizing group; wherein the linking group has the formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1is alkylene; X1is -C(=O)NH- or -NHC(=O)-; L2is -(CH2CH2O) q -alkylene, alkylene, or substituted alkylene, optionally wherein substituted alkylene is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; G is a bioconjugatable group; and 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 R S is a group having the formula: -X2-(L3) z -R 17 wherein: z is 0 or 1; X2is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH- or triazolyl; L3is -C(=O)-alkylene-C(=O)-NH-, and R 17 is 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 12 or greater; n is an integer from 1 to 5; R 18 is lower alkyl, optionally methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R18 ; provided that: R2and R 12 are not the same or R3and R 13 are not the same, and wherein at least one of R2, R3, R 12 and R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w ; wherein the method comprises: (a) providing a compound having the formula:

[0059]

[0060] wherein: M is a metal or is -H, -H; R5', R 10 ' and R 15 ' are independently selected from H and alkoxy; and R2', R3', R 12 ' and R 13 ' are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, and acetyl, wherein R 2’ and R 12’ are each halogen, optionally bromine, or wherein R 3’ and R 13’ are each halogen, optionally bromine; and (b) contacting the compound with a palladium catalyst, a base, and one of: (i) two different alkynes, optionally wherein the two different alkynes are each a compound having the formula:

[0061]

[0062] wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20 is an N-protected alkylamine, a protected carboxylic acid, -C(=0)-NH-alkylene-protected amine, or -C(=0)-NH-substituted alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=0)-NH-substituted alkylene-protected amine comprises a protected carboxylic acid-substituted alkylene; (ii) two different alkenes, optionally wherein the two different alkenes are each a compound having the formula:

[0063]

[0064] wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20is an N-protected alkyl amine, a protected carboxylic acid, -C(=0)-NH-alkylene-protected amine, or -C(=0)-NH-substituted alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=0)-NH-substituted alkylene-protected amine comprises a protected carboxylic acid substituted alkylene; and (iii) two different organoboronic acids; optionally wherein the two different organoboronic acids are two different aryl boronic acids or aryl boronic esters of the formula:

[0065]

[0066] wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20 is an N-protected alkyl amine, a protected carboxylic acid, -C(=0)-NH-alkylene-protected amine, or -C(=0)-NH-substituted alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=0)-NH-substituted alkylene-protected amine comprises a protected carboxylic acid substituted alkylene; and each R 21 is H or alkyl or wherein two R 21 are taken together to form an alkylene.

[0067] In some embodiments, the ratio of the two alkynes, alkenes, or organoboronic acids is adjusted to maximize the yield of the desired product based on the relative reactivity of the two alkynes, alkenes, or organoboronic acids, optionally wherein the one that is less reactive of the two is provided in a greater molar excess than the other of the two compared to the compound of step (a). In some embodiments, the yield of the desired product is greater than 50%, optionally wherein the yield of the desired product is greater than about 60%.

[0068] Accordingly, it is an object of the presently disclosed subject matter to provide water-soluble bacteriochlorins, their conjugates and pharmaceutical compositions, and methods of using and making the same.

[0069] These and other objects are achieved, in whole or in part, by the presently disclosed subject matter. Further, the above-stated objects of the presently disclosed subject matter, other objects of the presently disclosed subject matter, and advantages of the presently disclosed subject matter will become apparent to those of ordinary skill in the art, upon reading the following specification, drawings, and examples. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 is Route 1, an exemplary route for the synthesis of a representative synthetic building block for the synthesis of the presently disclosed subject matter dibromo-bacteriochlorin derivatives.

[0071] Figure 2 is Route 2, an exemplary route for the synthesis of compound CP-1.

[0072] Figure 3is Route 3, an exemplary route for synthesizing compound BC-1.

[0073] Figure 4 is Route 4, an exemplary route for synthesizing compound BC-2a.

[0074] Figure 5 is Route 5, an exemplary route for synthesizing compound BC-2.

[0075] Figure 6 is Route 6, an exemplary route for synthesizing compound BC-3.

[0076] Figure 7 is Route 7, an exemplary route for synthesizing compound BC-4.

[0077] Figure 8 is Route 8, an exemplary route for synthesizing compound BC-5.

[0078] Figure 9 is Route 9, an exemplary route for synthesizing compound BC-6.

[0079] Figure 10 is Route 10, an exemplary route for synthesizing compound BC-7a.

[0080] Figure 11 is Route 11, an exemplary route for synthesizing compound BC-7.

[0081] Figure 12 is Route 12, an exemplary route for synthesizing compound BC-8.

[0082] Figure 13 is Route 13, an exemplary route for synthesizing NIRvana 880 bis-t-butyl ester. DETAILED DESCRIPTION

[0083] The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the presently disclosed subject matter are shown. Indeed, the presently disclosed subject matter can 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. Like reference numerals can refer to like elements throughout.

[0084] I. Definitions

[0085] 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.

[0086] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. References to techniques employed herein are intended to refer to the techniques as commonly understood by one of ordinary skill in the art, including variations and alternatives to those techniques that are apparent to those skilled in the art. Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0088] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in combination with one or more or, in some cases, all of the other disclosed technologies.

[0089] Accordingly, this specification will avoid unnecessary repetition of each of various steps in an unnecessary fashion. However, the specification and claims should be read with the understanding that the specification and claims will be read in the context of being fully within the scope of the application and the claims.

[0090] In accordance with long-standing patent law precedent, the terms “a,” “an,” and “the” as used herein mean “one or more” when applied to any element that is intended to be present singularly or pluralistically (i.e., “one or more”) unless otherwise indicated. For example, the phrase “fluorescent microparticles and / or nanoparticles” means one or more fluorescent microparticles and / or nanoparticles, including a plurality of the same fluorescent microparticles and / or nanoparticles. Similarly, the phrase “at least one,” when used in the context of referring to an entity herein, 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 of 1 to 100 and greater than 100.

[0091] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” The term “about” when used in the context of a measurable value such as a mass, weight, time, volume, concentration, or percentage means that variations of ±20% are included, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% as suitable for an application of the disclosed methods. Accordingly, unless indicated to the contrary, the numerical parameters listed in the specification and attached claims are approximations. It is intended that the application claims as many embodiments of the application as are numerically possible.

[0092] As used herein, the term "and / or," when used in the context of a list of entities, means that single or combined existence of the entities is contemplated. 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.

[0093] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. "Comprising" is a term of art that is used in its broadest form and encompasses the presence of stated elements and / or steps, but also allows for the presence of additional elements and / or steps that do not materially affect the basic and novel characteristics of the subject matter.

[0094] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specifically recited. Notably, when the phrase "consisting of" appears in a clause of the body of a claim, it limits only the elements listed in that clause; other elements are not excluded from the rest of the claim.

[0095] As used herein, the phrase "consisting essentially of" limits the scope of the relevant disclosure or claim to the specified materials and / or steps, and those that do not materially affect the basic and novel characteristics of the disclosed subject matter and / or claimed subject matter. For example, a fluorescent microparticle and / or nanoparticle can "consist essentially of a polymeric matrix and at least one bacteriochlorophyll associated therewith," which means that the polymeric matrix is the only polymeric matrix in which the fluorescent microparticle and / or nanoparticle exists.

[0096] With respect to the terms "comprising," "consisting of," and "consisting essentially of," when one of the three terms is used in this document, the disclosed and claimed subject matter can include 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. Those of ordinary skill in the art, having the benefit of the present disclosure, will understand that the presently disclosed subject matter thus encompasses fluorescent microparticles and / or nanoparticles that consist essentially of a polymeric matrix of the presently disclosed subject matter and at least one bacteriochlorophyll associated therewith, as well as fluorescent microparticles and / or nanoparticles that consist of a polymeric matrix of the presently disclosed subject matter and at least one bacteriochlorophyll associated therewith.

[0097] "Halogen," as used herein, refers to any suitable halogen, including -F, -Cl, -Br, and -I.

[0098] "Mercapto," as used herein, refers to an -SH group.

[0099] "Azido," as used herein, refers to an -N3 group.

[0100] As used herein, "cyano" refers to a -CN group.

[0101] As used herein, "hydroxy" refers to an -OH group.

[0102] As used herein, "nitro" refers to a -NO2 group.

[0103] As used herein, "alkyl" refers to a straight or branched chain hydrocarbon containing from 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 Alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc. As used herein, "lower alkyl" is a subset of alkyl, and in some preferred embodiments, refers to a straight or branched hydrocarbon group containing 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. Unless otherwise indicated, the term "alkyl" or "lower alkyl" is intended to include substituted and unsubstituted alkyl or lower alkyl groups, 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, cycloalkoxy, cycloalkylalkoxy, aryloxy, arylalkoxy, heterocyclyloxy, heterocycloalkoxy, 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 、Aralkyl-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.

[0104] "Alkylene" as used herein refers to a di-functionalized straight chain, branched chain, or cyclic alkyl group, which can be substituted or unsubstituted, and wherein "alkyl" is as defined above.

[0105] "Alkenyl" as used herein 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., C1to C4alkenyl; C4to C 10 alkenyl; C 11 to C 50 alkenyl) (or lower alkenyl 1-4 carbon atoms) containing 1-4 double bonds in the normal chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2- propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4- heptadienyl, and the like. Unless otherwise specified, the term "alkenyl" or "lower alkenyl" is intended to include substituted and unsubstituted alkenyl or lower alkenyl groups, and these groups can be substituted with the same groups described above for alkyl and lower alkyl.

[0106] "Arylene" as used herein refers to a di-functionalized straight chain, branched chain, or cyclic aryl group, which can be substituted or unsubstituted, and wherein "aryl" is as defined above.

[0107] "Arylene" as used herein refers to a di-functionalized straight chain, branched chain, or cyclic aryl group, which can be substituted or unsubstituted, and wherein "aryl" is as defined above. 10 alkenyl; C 11 to C 50 alkenyl) (or lower alkenyl 1-4 carbon atoms) containing 1-4 double bonds in the normal chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2- propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4- heptadienyl, and the like. Unless otherwise specified, the term "alkenyl" or "lower alkenyl" is intended to include substituted and unsubstituted alkenyl or lower alkenyl groups, and these groups can be substituted with the same groups described above for alkyl and lower alkyl.

[0108] "Arylene" as used herein refers to a di-functionalized straight chain, branched chain, or cyclic aryl group, which can be substituted or unsubstituted, and wherein "aryl" is as defined above.

[0109] As used herein, "alkylidene chain" refers to a difunctionalized linear, branched, and / or cyclic organic group that may be substituted or unsubstituted, saturated or unsaturated, and optionally contains 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, for example, U.S. Patent No. 6,946,533. The alkylidene chain may contain any suitable number of carbon atoms (e.g., C1 to C4; C4 to C6; C7 to C8; C9 to C10; C11 to C12; C13 to C14; C15 to C16; C17 to C18; C19 to C20; C21 to C21; C22 to C23; C24 to C24; C25 to C26; C27 to C27; C28 to C29; C30 to C30; C31 to C31; C32 to C32; C33 to C33; C34 to C34; C35 to C35; C36 to C36; C37 to C37; C38 to C38; C39 to C39; C40 to C39; C41 to C41; C39 to C39; C41 to C39 ... 10 ; C 10 to C 20 ; C 20 to C 50 ).

[0110] As used herein, "alkoxy" refers to an alkyl or lower alkyl group as defined herein, attached to the parent molecular moiety through an oxy-O- radical. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentoxy, hexoxy, and the like.

[0111] As used herein, "acyl" as used alone or as part of another group refers to a -C(O)R group, where R is any suitable substituent, such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl, or other suitable substituents described herein.

[0112] "Haloalkyl," as used herein alone or as part of another group, refers to at least one halogen, as defined herein, attached to the 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, and the like.

[0113] "Perhaloalkyl", as used herein alone or as part of another group, 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., -CF ).

[0114] "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.

[0115] "Aryl" used 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 groups include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. Unless otherwise indicated, the term "aryl" is intended to encompass both substituted and unsubstituted aryl groups, and these groups can be substituted with the same groups as described above for alkyl and lower alkyl.

[0116] "Alkylene" as used herein is a bi-functionalized aryl group which can be substituted or unsubstituted, and wherein "aryl" is as defined above.

[0117] "Arylalkyl" used alone or as part of another group refers to an aryl group as defined herein attached to the parent molecular moiety through an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphthalen-2-ylethyl, and the like.

[0118] "Arylalkyl" used alone or as part of another group refers to an aryl group as defined herein attached to the parent molecular moiety through an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphthalen-2-ylethyl, and the like.

[0119] "Amino" as used herein refers to the group -NH2.

[0120] "Arylalkyl" used alone or as part of another group refers to an aryl group as defined herein attached to the parent molecular moiety through an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphthalen-2-ylethyl, and the like.

[0121] "Arylalkyl" used alone or as part of another group refers to an aryl group as defined herein attached to the parent molecular moiety through an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphthalen-2-ylethyl, and the like.

[0122] "Disubstituted amino" used alone or as part of another group refers to the group -NR a R b wherein R a and R b are independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, and heterocyclylalkyl.

[0123] "Arylalkyl" used alone or as part of another group refers to an aryl group as defined herein attached to the parent molecular moiety through an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphthalen-2-ylethyl, and the like. a R b wherein R a is acyl as defined herein, and R b is selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, and heterocyclylalkyl.

[0124] "Acyloxy" used alone or as part of another group refers to the group -OR, where R is acyl as defined herein.

[0125] "Ester" used 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.

[0126] "Formyl" as used herein refers to a -C(O)H group.

[0127] "Carboxylic acid" as used herein refers to a -C(O)OH group.

[0128] "Sulfoxyl" as used herein refers to a compound of the formula -S(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0129] "Sulfonyl" as used herein 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.

[0130] "Sulfonate" as used herein 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.

[0131] "Sulfonic acid" as used herein refers to a compound of the formula -S(O)(O)OH.

[0132] "Amide" used alone or as part of another group refers to a -C(O)NR a R b group, where R a and R b are any suitable substituents, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0133] "Sulfonamide" used alone or as part of another group refers to a -S(O)2NR a R b group, where R a and R b are any suitable substituents, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0134] "Urea" used alone or as part of another group refers to a -N(R c )C(O)NR a R b group, where Ra 、R b and R c is any suitable substituent, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0135] "Alkoxyamido" as used herein 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, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0136] As used herein, "aminoacyloxy" as used alone 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, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0137] " Cycloalkyl " used alone or as part of another group herein refers to a saturated or partially unsaturated cyclic hydrocarbon radical containing 3, 4 or 5 to 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.

[0138] As used herein, the term "polyoxyethylene chain" refers to a poly(ethylene glycol) (PEG) group (e.g., having the formula -(C2H4O) n -group, wherein n is an integer of 2 or greater (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 or greater). In some embodiments, n is an integer from 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 the PEG includes a Gaussian distribution of chain lengths and molecular weights.

[0139] As used herein, the term "bioconjugatable 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 polymeric bead, or an inorganic bead; another solid support surface, etc.) to form a conjugate of one of the presently disclosed bacteriochlorophyll compounds and the other entity. For example, the bioconjugatable group can be an aldehyde, which can form a covalent bond with an amino group on an amino-substituted biomolecule through reductive amination; or a carboxylic acid, which can be coupled to an amino-substituted biomolecule through carbodiimide activation. The bioconjugatable groups include amines (including amine derivatives) such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, etc.; 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, etc.; and other groups such as, but not limited to, aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyl groups, halogen groups, biotin, 2-imino biotin, etc.

[0140] The term "microparticle" refers to a structure having at least one region with a dimension (e.g., length, width, diameter, etc.) of less than about 1,000 μm but greater than about 1000 nm. In some embodiments, the dimension 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 dimension 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).

[0141] Similarly, the term“nanoparticle” refers to a structure having at least one region with a size (e.g., length, width, diameter, etc.) of less than about 1,000 nm. In some embodiments, the size is small (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).

[0142] In some embodiments, the microparticle or nanoparticle is approximately spherical. When the microparticle or nanoparticle is approximately spherical, the characteristic dimension can correspond to the diameter of the sphere. In addition to being spherical, the microparticle or nanoparticle can be disc-shaped, plate-shaped (e.g., hexagonal plate-like), ellipsoidal, polyhedral, rod-shaped, cubic, or irregularly shaped.

[0143] The microparticle or nanoparticle can include a core region (i.e., the space between the outer dimensions of the particle) and an outer surface (i.e., the surface that defines the outer dimensions of the particle). In some embodiments, the microparticle or nanoparticle can have one or more coating layers surrounding or partially surrounding the core of the microparticle or nanoparticle. Thus, for example, a spherical microparticle or nanoparticle can have one or more concentric coating layers, each successive layer dispersed on the outer surface of the smaller layer closer to the center of the particle.

[0144] 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 polymerizable monomers. A polymerizable monomer is a molecule that includes 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 coordinate bond). In some embodiments, each polymerizable monomer molecule can bond with two or more other molecules / groups. In some cases, a polymerizable monomer will bond with only one other molecule, forming a terminus of the polymeric material.

[0145] The polymer can be organic, or inorganic, or a combination thereof. As used herein, the term "inorganic" refers to a compound or composition that contains at least some atoms other than carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorus, or one of the 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 composed of polystyrene. In some embodiments, the microparticles and / or nanoparticles are polystyrene beads.

[0146] As used herein, the term "porphyrin" refers to a ring structure typically composed of four pyrrole rings and four nitrogen atoms, and two replaceable hydrogens (various metal atoms can readily be substituted). A representative porphyrin is hemin.

[0147] As used herein, the term "bacteriochlorin" is distinguished from porphyrin by having two partially saturated, non-adjacent (i.e., trans) pyrrole rings. The terms "bacteriochlorin" and "bacteriochlorin derivative" are used interchangeably herein.

[0148] The phrase "associated" refers to any interaction between two entities, such as between a polymer matrix and a bacteriochlorin. In some embodiments, the polymer matrix and the bacteriochlorin are associated with one another through non-covalent bonds, such as but not limited to one or more of hydrophobic, electrostatic, and van der Waals interactions. In some embodiments, the polymer matrix (e.g., nanoparticle, microparticle, bead, etc.) comprises the bacteriochlorin such that the bacteriochlorin is present within the polymer matrix, whereby the polymer matrix and the bacteriochlorin are associated with one another. In such embodiments, the polymer matrix is also referred to as "doped by" or "doped with" the bacteriochlorin, and the bacteriochlorin can be considered to be "embedded" within the polymer matrix. In some embodiments, the polymer matrix and the bacteriochlorin are associated with one another through covalent bonds that link the bacteriochlorin to the surface of the polymer matrix.

[0149] As used herein, "treatment" refers to any manner in which one or more symptoms of a disease or disorder are improved or otherwise beneficially altered. Treatment also includes any pharmaceutical use of the compositions herein, for example, for treating a disease or disorder mediated by hyperproliferative tissue or neovascularization, or a disease or disorder involving hyperproliferative tissue or neovascularization. As used herein, improvement of symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any alleviation, whether permanent or temporary, lasting or transient, that can be attributed to or associated with administration of the composition.

[0150] As used herein, a "prodrug" is a compound that is administered to the body and metabolized or otherwise converted into a biologically, pharmaceutically, or therapeutically active form of the compound after one or more steps or processes in the body.

[0151] As used herein, an "antibody" generally refers to an immunoglobulin or fragment thereof that specifically binds to an antigen to form an immunocomplex. The antibody can be an intact immunoglobulin of any class, such as IgG, IgM, IgA, IgD, IgE, chimeric or hybrid antibodies having dual or multiple antigen or epitope specificity. It can be a polyclonal antibody, preferably affinity purified from humans or suitable animals (e.g., primates, goats, rabbits, mice, etc.). Monoclonal antibodies are also suitable for the presently disclosed subject matter and can be preferred due to their high specificity. They are readily prepared by now-considered routine procedures of immunizing a mammal with an immunogenic antigen preparation, fusing immune lymphocytes or spleen cells with immortal myeloma cells, and isolating specific hybridoma clones. More unconventional methods of preparing monoclonal antibodies, such as interspecies fusions of variable regions and genetic engineering manipulations, are not excluded, as the primary concern affecting their utility is the antigen specificity of the antibody. Newer monoclonal production techniques, such as human monoclonals, interspecies monoclonals, chimeric (e.g., human / mouse) monoclonals, genetically engineered antibodies, etc., can also be used.

[0152] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The immunoglobulin genes include the kappa (K), lambda (l), alpha (a), gamma (g), delta (d), epsilon (e), and mu (m) constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. In mammals, heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Other species have other light and heavy chain genes (e.g., certain birds produce a so-called IgY, which is a type of immunoglobulin that hens lay in their egg yolks), which are similarly included in the presently disclosed subject matter.

[0153] A representative immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kilodaltons (kDa) in average molecular weight) and one "heavy" chain (about 50-70 kDa in average molecular weight). The two identical polypeptide chains of each pair are held together via disulfide bonds existing within the region of the heavy chains. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The term variable light chain (VL) is used to define the N-terminal one-third of the light chain, and the term variable heavy chain (VH) is used to define the N-terminal one-third of the heavy chain. The variable regions of each light / heavy chain pair form the antibody binding site. L) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0154] Antibodies generally exist as intact immunoglobulins or a number of well-characterized fragments produced by digestion with various peptidases. For example, papain digestion of an antibody molecule cleaves the molecule in the hinge region to produce three fragments: two identical “Fab” fragments, each with one complete light chain and one heavy chain that includes a portion of the hinge region, and a “Fc” fragment, which includes both C-terminal portions of the heavy chains linked by disulfides. On the other hand, pepsin digestion of an antibody fragment in the hinge region produces a single large “F(ab)'2” fragment that is a dimer of Fab fragments held together by two disulfides in the hinge region. A F(ab)'2 fragment can be reduced under mild conditions to break the disulfides in the hinge region, thereby converting the F(ab)'2 dimer into two “Fab'” monomers. Fab' monomers are essentially Fab fragments with part of the hinge region. With respect to these various fragments, the Fab, F(ab')2, and Fab' fragments include at least one complete antigen binding domain (termed a “paratope”), and thus are capable of binding antigen.

[0155] While various antibody fragments are defined in terms of the digestion of intact antibodies, one of skill will appreciate that various such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methods. Therefore, the term “antibody”, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methods. In some embodiments, the term “antibody” includes fragments having at least one antigen binding domain.

[0156] Antibodies, fragments, and derivatives of the presently disclosed subject matter can also include chimeric antibodies. As used herein in the context of antibodies, the term “chimeric” and grammatical variations thereof refer to antibody derivatives having a constant region that is substantially or completely derived from an antibody constant region from one species and a variable region that is substantially or completely derived from sequences of a variable region from another species. A particular kind of chimeric antibody is a “humanized” antibody, which is produced by replacing the complementarity determining regions (CDRs) of a human antibody with CDRs of, for example, a mouse antibody (see, e.g., PCT International Patent Application Publication No. WO 1992 / 22653). Thus, in some embodiments, a humanized antibody has constant and variable regions substantially or completely derived from the corresponding human antibody regions except for the CDRs, and CDRs substantially or completely derived from a mammal other than human.

[0157] The antibodies, fragments, and derivatives of the presently disclosed subject matter can also be single-chain antibodies and single-chain antibody fragments. Single-chain antibody fragments contain amino acid sequences 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 make up a substantial portion of the complete antibody structure.

[0158] Single-chain antibody fragments can overcome some of the problems associated with the use of antibodies containing some or all of the constant domains. For example, single-chain antibody fragments tend to be free of unwanted interactions between biomolecules and the heavy chain constant region, or other unwanted biological activities. In addition, single-chain antibody fragments are much smaller than complete antibodies, and thus can have greater capillary permeability than complete antibodies, allowing single-chain antibody fragments to more effectively localize and bind to target antigen binding sites. Moreover, antibody fragments can be produced in prokaryotic cells at relatively large scales, facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely to elicit an immune response in a recipient than complete antibodies. Single-chain antibody fragments of the presently 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 di-scFv, tandem tri-scFv, diabodies (including bispecific diabodies), triabodies, tetrabodies, minibodies, minibodies, tetravalent bispecific molecules, bispecific F(ab')2 fragments, and the like.

[0159] As used herein, "infectious agent" means an invading microorganism or parasite. As used herein, "microorganism" means viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi, and like microorganisms, and "parasite" means an infectious, often microscopic or very small, multicellular invertebrate animal, or an egg cell or juvenile form thereof, that is susceptible to antibody-induced clearance or lysis or phagocytic destruction, such as malaria parasites, spirochetes, and the like.

[0160] As used herein, "tumor" means a neoplasm and includes both benign and malignant tumors. The term specifically includes malignant tumors that can be solid, such as breast, liver, or prostate cancer, or non-solid, such as leukemia. Tumors can be further subdivided into subtypes, such as adenocarcinomas (e.g., adenocarcinomas of the breast, prostate, or lung).

[0161] As used herein, "target" means the object intended to be detected, diagnosed, damaged, or destroyed by the methods provided herein, and includes target cells, target tissues, and target compositions.

[0162] As used herein, "target tissue" and "target cell" are those tissues intended to be damaged or destroyed by the treatment method. The photosensitizing compound is bound to or accumulated 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 tissue, and target tissue includes, but is not limited to, tumors, solid tumors such as, but not limited to, tumors of the head and neck, tumors of the eye, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lung, vascular endothelial tissue of non-solid tumors, abnormal blood vessel walls; and malignant cells of hematopoietic and lymphoid tissues, neovascular tissue; vasculature, bone marrow, and other lesions of tissues or cells associated with autoimmune diseases. Also included among target cells are cells that undergo significantly more rapid division than non-target cells.

[0163] As used herein, "non-target tissue" is all tissue of the subject that is not intended to be damaged or destroyed by the treatment method. These non-target tissues include, but are not limited to, healthy blood cells and other normal tissues not otherwise identified as targets.

[0164] As used herein, "target composition" is those compositions intended to be damaged or destroyed by the treatment method, and can include one or more pathogens, including but not limited to bacteria, viruses, fungi, protozoa, and toxins and cells and tissues infected or infiltrated by the same. 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 such treatment methods.

[0165] As used herein, "hyperproliferative tissue" refers to tissue that grows out of control, and includes neoplastic tissue, tumors, and unregulated vascular growth such as that found in age-related macular degeneration and often occurring after glaucoma surgery.

[0166] As used herein, "hyperproliferative disorder" denotes those conditions disorders that will result from unregulated or abnormal cell growth as the underlying pathology, and includes uncontrolled angiogenesis. Examples of such hyperproliferative disorders include, but are not limited to, cancer or carcinoma, acute and membranous proliferative glomerulonephritis, myeloma, psoriasis, atherosclerosis, psoriatic arthritis, rheumatoid arthritis, diabetic retinopathy, macular degeneration, corneal neovascularization, choroidal neovascularization, pterygium recurrence, and scarring resulting from excimer laser surgery and glaucoma filtration surgery.

[0167] As used herein, "therapeutically effective dose" is a dose sufficient to prevent progression of a disease or to cause regression of a disease or to be able to alleviate symptoms caused by a disease.

[0168] 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.

[0169] As used herein, "irradiating" and "irradiation" include exposing the individual to light of all wavelengths. Preferably, the wavelength of irradiation is chosen to match the wavelength that excites the photosensitizing compound. Preferably, the wavelength of radiation matches the excitation wavelength of the photosensitizing compound and has low absorption by the non-target tissues of the individual, including blood proteins.

[0170] Irradiation is further defined herein by its coherence (laser) or incoherence (non-laser) as well as intensity, duration, and timing relative to the administration of the photosensitizing compound. The intensity or fluence must be sufficient for the light to reach the target tissue. The duration or total fluence dose must be sufficient for the photosensitizing compound to be sufficiently photosensitized to act on the target tissue. The timing relative to the administration of the photosensitizing compound is important because 1) the administered photosensitizing compound needs some time to localize to the target tissue, and 2) the blood levels of many photosensitizing compounds decrease with 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 through a catheter, optical fiber, or by ingesting a capsule or pill form of the light source (e.g., as disclosed in U.S. Patent No. 6,273,904).

[0171] While some embodiments of the presently disclosed subject matter involve the use of light energy to implement photodynamic therapy (PDT) to destroy tumors, other forms of energy are within the scope of the presently disclosed subject matter, as will be appreciated by one of ordinary skill in the art. Such forms of energy include, but are not limited to: thermal energy, sonic 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, sonodynamic-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 hematin (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).

[0172] As used herein, "coupling agent" refers to an agent capable of coupling a photosensitizing agent to a targeting agent.

[0173] A “targeting agent” is a compound that is designed to localize or preferentially associate or bind to a particular tissue, receptor, infectious agent, or other region of the body of an 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 liposome suspensions, including tissue-targeting liposomes.

[0174] As used herein, “specific binding pair” and “ligand-receptor binding pair” refer to two different molecules, one of which has a region in the surface or cavity that specifically attracts or binds to a particular spatial or polar organization of the other molecule, resulting in the two molecules having an affinity for each other. The members of a specific binding pair are referred to as a ligand and a receptor (anti-ligand). The terms ligand and receptor are intended to include the entire ligand or receptor or a portion thereof sufficient for binding to occur between the ligand and the 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-alpha and tumor necrosis factor-receptor, and interferon and interferon receptor; avidin and biotin or anti-biotin; antibody and antigen pairs; enzyme and substrate, drug and drug receptor; cell surface antigen and lectin; two complementary nucleic acid strands; a nucleic acid strand and a complementary oligonucleotide; interleukin and interleukin receptor; 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.

[0175] A “linker” is an aromatic or aliphatic group (which can be substituted or unsubstituted, and which can optionally include heteroatoms such as N, O, or S) that is used to couple a bioconjugation group, a cross-coupling group, a surface attachment group, a hydrophilic group, and the like to a parent molecule. Examples include, but are not limited to, aryl, alkyl, heteroaryl, heteroalkyl (e.g., oligo glycol), peptide, and polysaccharide linkers, among others.

[0176] An individual treated by the presently disclosed subject matter for diagnostic or therapeutic purposes includes human individuals and other animal individuals for veterinary purposes, particularly mammalian individuals such as dogs, cats, horses, monkeys, chimpanzees, and the like.

[0177] More specifically, as used herein, the terms “individual,” “patient,” and “recipient” can be used interchangeably and can refer to any invertebrate or vertebrate species member. Thus, the term “individual” is intended to encompass any member of the animal kingdom, including but not limited to members of the Chordata phylum (e.g., the Actinopterygii class (bony fishes), the Amphibia class (amphibians), the Reptilia class (reptiles), the Aves class (birds), and the Mammalia class (mammals)), and all orders and families contained therein.

[0178] The compositions and methods of the presently disclosed subject matter are particularly useful in warm-blooded vertebrates. Thus, the presently disclosed subject matter relates to mammals and birds. More particularly, compositions and methods are provided that are derived from and / or for use in mammals such as humans and other primates, and those mammals that are important due to being endangered (such as Siberian tigers), economically important to humans (animals raised on farms for human consumption), and / or socially important (animals kept as pets or in zoos), e.g., carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, hamsters, guinea pigs, and rabbits), marsupials, and horses. Use of the disclosed methods and compositions on birds, including those species of birds that are endangered, raised in zoos, or kept as pets (e.g., parrots, budgerigars, etc.), as well as fowl, more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, etc., are also provided, as they are also of economic importance to humans. Thus, use of the disclosed methods and compositions on livestock is also provided, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, etc.

[0179] II. Bacteriochlorin Compounds

[0180] In some embodiments, the presently disclosed subject matter provides water-soluble bacteriochlorins, where the bacteriochlorin has a solubility of about 1 milligram per milliliter (mg / mL) or more in aqueous solution (e.g., water, saline, PBS, etc.). For example, water-solubility is provided by the addition of a solubilizing group comprising a PEG chain at the beta-pyrrole position. In some embodiments, the PEG chain is attached to the bacteriochlorin through a different group and / or is longer than the PEG chain attached to previously described PEGylated bacteriochlorin compounds. In some embodiments, the bacteriochlorin has a solubility of about 3.0 mg / mL or more in aqueous solution. In some embodiments, the bacteriochlorin has a solubility of about 5.0 mg / mL or more in aqueous solution. In some embodiments, the bacteriochlorin has a solubility of about 10 mg / mL or more in aqueous solution. In some embodiments, the bacteriochlorin has a solubility of about 500, about 600, about 700, about 800, or about 900 mM or more in aqueous solution. In some embodiments, the bacteriochlorin has a solubility of about 1, 1.5, 2, 2.5, or 3 mM or more in aqueous solution.

[0181] In some embodiments, the bacteriochlorin has an emission wavelength higher than about 700 nm. In some embodiments, the bacteriochlorin has an emission wavelength higher than about 800 nm. In some embodiments, the bacteriochlorin has an emission wavelength of about 850 nm or more.

[0182] In some embodiments, the water-soluble bacteriochlorophyll comprises a linker moiety comprising a bioconjugatable group that can be used to conjugate the bacteriochlorophyll to another substance, e.g., another substance that can act as a targeting agent or a substance to be detected. In some embodiments, the substance that can be conjugated to the bacteriochlorophyll can be a small molecule (e.g., a non-polymeric synthetic molecule having 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 antibody fragment, a nucleic acid, a hormone, or a growth factor. The bioconjugatable group can include, for example, a carboxylic acid or active ester, a hydroxyl, an amine, a thiol, or an aldehyde. In some embodiments, the linker moiety further comprises an arylene and an alkylene. In some embodiments, the linker moiety comprises an arylene and / or an alkynylene proximal to the main bacteriochlorophyll structure, while the alkylene is proximal to the bioconjugatable group.

[0183] In some embodiments, the bacteriochlorophyll comprises at least one solubilizing group. In some embodiments, the solubilizing group comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) polyoxyethylene chains (e.g., PEG chains). In some embodiments, the solubilizing group comprises at least two PEG chains. In some embodiments, the PEG chains are monodisperse and comprise at least 4 -CH2CH2O- repeat units. In some embodiments, the PEG chains comprise at least 6, 8, 10, or 12 -CH2CH2O- repeat units. Thus, the solubilizing group can comprise two PEG6, PEG8, PEG10, or PEG12 groups. In some embodiments, the PEG chains comprise 12 or more -CH2CH2O- repeat units (e.g., about 12 to about 24 or about 28 -CH2CH2O- repeat units). In some embodiments, the compound comprises two solubilizing groups attached to two different pyrrole carbons. In some embodiments, each of the two solubilizing groups comprises two PEG chains.

[0184] In some embodiments, the solubilizing group is a beta-pyrrole substituent comprising an arylene or alkynyl-arylene group directly attached to a bacteriochlorophyll 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 bonds further comprise one or more alkylene spacers (e.g., ethylene, propylene, etc.).

[0185] In some embodiments, the bacteriochlorophyll is a compound of formula (II):

[0186]

[0187] wherein:

[0188] M is a metal or is -H, -H;

[0189] R5, R 10 and R 15 are independently selected from H, alkoxy, and a linking group of the formula: -L1-(X1-L2) p -G, wherein p is 0 or 1; L1is alkylene; X1is -C(=O)NH- or -NHC(=O)-; L2is -(CH2CH2O) 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 bioconjugatable group; and

[0190] R2, R3, R 12 and R 13 are independently selected from H, halogen, cyano, perhaloalkyl (e.g., perfluoroalkyl such as perfluoromethyl), sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linking group of the formula -L1-(X1-L2) p -G, 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 R S is a group of the formula:

[0191] -X2-(L3) z -R 17 ,

[0192] wherein: z is 0 or 1; X2is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH-, or triazolyl; L3is -C(=O)-alkylene-C(=O)-NH-, and R 17 is 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 12 or greater (e.g., 12, 14, 16, 18, 20, 22, 24, 26, or 28); n is an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5); R18 is lower alkyl (e.g., methyl); and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; provided that: R2, R3, R 12 and at least one of R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0193] M can be any suitable metal ion (e.g., Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe, or Zn) or absent (e.g., in which case it is replaced by two hydrogens (-H, -H), i.e., the two nitrogen atoms of the bacteriochlorin ring are protonated. In some embodiments, M is Zn or is replaced by -H, -H. Thus, compounds of Formula (II) include metal bacteriochlorins and free base bacteriochlorins. In some embodiments, M is Zn.

[0194] In some embodiments, R5, R 10 and R 15 are independently selected from H, methoxy, and a linking group of the formula: -L1-(X1-L2) p -G. In some embodiments, R5is methoxy. In some embodiments, R 10 and R 15 are both linking groups. In some embodiments, R 15 is a linking group. Alternatively, in some embodiments, R 12 is a linking group.

[0195] In some embodiments, the linking group -L1-(X1-L2) p -G has a linking group p of 0. In some embodiments, G is a carboxylic acid or an active ester (e.g., an NHS ester). In some embodiments, L1is alkynylene, arylene (e.g., phenylene), or a divalent group comprising both alkynylene and arylene (i.e., aralkynylene). In some embodiments, L1is -C≡C-phenyl- or -C≡C-alkylene- (e.g., -C≡C-(CH2)4-). In some embodiments, the linking group is:

[0196]

[0197] optionally, wherein G is a carboxylic acid (i.e., -C(=O)OH or an active ester.

[0198] In some embodiments, the linking group is:

[0199]

[0200] Optionally, G is a carboxylic acid or an active ester thereof.

[0201] In some embodiments, p is 1 and the linking group comprises a polyoxyethylene chain to improve solubility and / or a spacer to improve the reactivity of G compared to the reactivity of G in coparfin, where G is directly connected to L1. In some embodiments, L1 is phenylene or -C≡C-phenyl-, p is 1, X1 is -C(=O)NH-, L2 is alkylene, and G is a carboxylic acid or an active ester (e.g., an NHS ester). In some embodiments, L2 is ethylene. Thus, the linking group can comprise a beta-alanine spacer to improve the reactivity of G.

[0202] In some embodiments, p is 1; L1 is arynylene (e.g., -C≡C-(C6H4)-); X1 is -C(=O)NH-; L2 is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bioconjugatable group. In some embodiments, X1 is -C(=O)NH- and L2 is -(CH2CH2O) q alkylene-. In some embodiments, q is 12 and the alkylene is ethylene. In some embodiments, L2 is methylene substituted with a group comprising a PEG chain and / or an amide group. For example, in some embodiments, L2 is -CH(R), where R is -alkylene-NH-C(=O)-alkylene-PEG-OMe. In some embodiments, R comprises two C2-C6 alkylene groups and a PEG12-PEG25 chain. In some embodiments, R is -(CH2)4-NH-C(=O)-CH2CH2-(OC2H4) 24 OMe. In some embodiments, the linking group is:

[0203]

[0204] or an active ester thereof, optionally where PEG is PEG12. In some embodiments, the linking group is:

[0205]

[0206] or an active ester thereof, optionally where PEG is PEG24.

[0207] In some embodiments, the bacteriochlorins are asymmetric. In some embodiments, R2 and R 12 are not the same or R3 and R 13 are not the same. For example, in some embodiments, R2 and R 12One of the (eg, R2) is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w solubilizing groups, and R2 and R 12 Another one (for example, R 12 ) is of the formula -L1-(X1-L2) p -G linker or a solubilizing group having a different structure (e.g., different from the solubilizing group of R2). Alternatively, in some embodiments, R3 and R 13 One of the following is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w solubilizing groups, and R3 and R 13 The other one is of the formula -L1-(X1-L2) p -G linker or solubilizing group of different structure. In some embodiments, R2 and R 12 One of R3 and R4 is a solubilizing group and the other is a linking group, or R3 and R 13 One of the groups is a solubilizing group and the other is a linking group.

[0208] In some embodiments, R3 and R 13 In some embodiments, R3 and R 13 Each is a methyl ester, ie, -C(=O)OCH3.

[0209] In some embodiments, R2 is

[0210]

[0211] In some embodiments, each R s is a group having the formula:

[0212] -X2-(L3) z -R 17 ,

[0213] wherein: z is 0; X2 is -C(=O)NH-alkylene-NH-; and R 17 -C(=O)C2H4-(OC2H4) m OR 18 , where m is an integer of 12 or greater, and R 18 In some embodiments, m is an integer from 12 to 24 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). sis

[0214]

[0215] In some embodiments, m is 24.

[0216] In some embodiments, each R s is a group having the formula:

[0217] -X2-(L3) z -R 17 ,

[0218] wherein: z is 1; X2is -C(=O)NH-alkylene-NH-; L3is -C(=O)-propylene-C(=O)-NH-; and R 17 is -(C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, wherein n is an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5); and each R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 , wherein m is an integer of 12 or greater; and R 18 is methyl. In some embodiments, m is an integer from 12 to 24. In some embodiments, n is 4. In some embodiments, m is 12.

[0219] In some embodiments, the compound is selected from BC-1, BC-2, BC-3, BC-4, BC-5, BC-6, and BC-7, i.e., a compound having the following structure:

[0220]

[0221]

[0222]

[0223] In some embodiments, the presently disclosed subject matter provides a composition comprising a covalent conjugate formed between: (a) a compound of formula (II) as defined above, with the proviso that R2, R3, R5, R 10 , R 12 , R 13 , and R 15at least one is a linking group; and (b) includes one or more of the group of small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors. In some embodiments, for example, conjugates can be formed by reacting a compound of Formula (II) comprising a linking group containing a carboxylic acid or active ester (i.e., as a bioconjugation group G) with an amino group of a small molecule, peptide, protein, antibody, or polymer.

[0224] III. Synthetic Methods

[0225] Methods of synthesizing bacteriochlorophyll that can be adapted to make the presently disclosed bacteriochlorophylls are described, for example, in U.S. Patent Nos. 8,664,260 and 8,980,565, each of which is incorporated herein in its entirety. In some embodiments, the presently disclosed compounds of Formula (II) can be prepared by preparing a suitable trans- -substituted bacteriochlorophyll (such as a bacteriochlorophyll in which both -bacteriochlorophyll substituents are halogen (e.g., Br) substituents), and then further reacting the -substituents to replace them with suitable water-soluble groups. Methods of synthesizing trans- -substituted bacteriochlorophylls have been previously described. See, e.g., Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103.

[0226] For example, in some embodiments, bacteriochlorophylls of Formula (II) can be prepared by self-condensing a dihydropyrrole synthesis block or condensing a pair of dihydropyrrole synthesis blocks in the presence of an acid in an organic solvent. In some embodiments, the dihydropyrrole synthesis block can have the following structure:

[0227]

[0228] wherein R is an acetal or aldehyde group; and S1, S2, S3, S7, and S6 are each independently selected from the group consisting of H, aryl, substituted aryl, phenyl, cycloalkyl, alkyl, substituted alkyl, alkenyl, alkynyl, halogen, alkoxy, alkylthio, perfluoroalkyl, perfluoroaryl, pyridyl, cyano, thiocyanato, nitro, amino, alkylamino, acyl, sulfoxido, sulfonyl, imino, ester, amido, and carbamoyl, and wherein S4and S5are each H or collectively form a covalent bond. In some embodiments, at least one of S1and S2is halogen.

[0229] More specifically, methods for preparing dibromo-substituted bacteriochlorins and their corresponding dihydropyrrole synthetic building blocks have been previously described. See Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103. For example, bacteriochlorins containing two bromo substituents at the 2 and 12 positions of the bacteriochlorin can be prepared from synthetic building blocks prepared from N-protected 3,4-dibromopyrroles as shown in Scheme 1 (see Figure 1 ) and Scheme 2 (see

[0230] As shown in Scheme 1 (see Figure 1 ), N-protected 3,4-dibromopyrroles (e.g., 3,4-dibromo-(N-triisopropylsilyl)pyrrole) are treated with a base such as an alkyl lithium (e.g., tert-butyllithium) and dimethyl carbonate, followed by deprotection to provide 3-bromo-4-(methoxycarbonyl)pyrrole (a). Vilsmeier formylation of a (e.g., using POCI3-DMF) provides aldehyde b. Aldehyde b can be treated with potassium acetate and a slight excess of methylamine-hydrochloride in nitromethane to provide aldol product c. Reduction of the carbon-carbon double bond in c using a suitable reducing agent (e.g., NaBH4) provides compound d, which is treated with 1,1-dimethoxy-4-methyl-3-penten-2-one in the presence of a non-nucleophilic base (e.g., 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)) to effect a Michael addition reaction. Reductive cyclization of the Michael addition product e by first deprotonating e (e.g., by treatment with anhydrous sodium methoxide) to provide a nitronate anion intermediate, followed by cyclization of this intermediate with a deoxygenating agent (e.g., TiCl3in buffered aqueous solution) provides the dihydropyrin bacteriochlorin synthetic building block (BB). Other alternative cyclization conditions include treatment of e with a metal (e.g., zinc and acetic acid in ethanol) to generate an N-oxide intermediate, followed by cyclization of this intermediate with a deoxygenating agent (e.g., (Ti(0), Zn, NaOH / methanol; Zn, NH4Cl aqueous solution / THF; FeSO4, NH4Cl aqueous solution / CH3CN; Mg or Fe, AcONH4 / methanol; Ph3P / toluene; S / toluene; NaN3 / toluene, Zn, NaI, Me3SiCl / CH3CN; etc.).

[0231] The BBs can self-condense in the presence of an acid (e.g., a Bronsted acid or a Lewis acid, such as trifluoroacetic acid, TMSOTf, or toluenesulfonic acid (TsOH)) to form a condensation product in the presence of a proton scavenger (e.g., 2,6-di-tert-butylpyridine (DTBP)) to provide bacteriochlorins. The condensation can be performed in an organic solvent, such as acetonitrile (ACN), dichloromethane (DCM), chloroform, tetrahydrofuran (THF), chlorobenzene, ethanol, and combinations thereof. Optionally, for example, when the bacteriochlorin synthesis building blocks do not contain a carbon-carbon double bond between the heterocycles, an oxidizing agent, such as air or DDQ, can be included in the condensation reaction mixture. In some embodiments, the bacteriochlorin can have the following structure:

[0232]

[0233] The halogen substituents of a dihalogenated bacteriochlorin, such as the bacteriochlorins described above, can be further elaborated using coupling chemistry known in the art, including but not limited to Stille coupling, Hiyama coupling, Suzuki coupling, Negishi coupling, Sonogashira coupling, and Kumada coupling reactions, to provide a solubilizing group. For example, the dihalogenated bacteriochlorin can be reacted with a boronic acid in the presence of a Pd(0) catalyst; with an organotin compound in the presence of a Pd catalyst; with a halide or organosilane in the presence of a Pd catalyst; with an organozinc compound in the presence of a Ni or Pd catalyst, or with a Grignard reagent in the presence of a Ni or Pd catalyst. In some embodiments, the dihalogenated bacteriochlorin (e.g., a dibromobacteriochlorin, such as the bacteriochlorin free base shown above) can be reacted with an aryl boronic 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 aryl boronic acid can comprise an additional chemical functional group or a protected chemical functional group that can be further elaborated after the Suzuki coupling reaction. For example, in some embodiments, the Suzuki coupling reaction can include one or more protected amino groups that, after deprotection, can be reacted with a suitable PEG reagent (e.g., an activated PEG ester).

[0234] In some embodiments, the presently disclosed compounds can use a tert-butyloxycarbonyl (BOC) protecting group for an amino group present on a Suzuki or other type coupling agent, optionally in combination with the use of a 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), a common BOC deprotection method, resulted in significant decomposition during the preparation of the presently disclosed compounds, for example in compounds comprising an alkyne bond. Thus, in some embodiments, BOC deprotection is performed using other conditions (e.g., 4M HC1 in dioxane).

[0235] In some embodiments, the presently disclosed subject matter provides a method of preparing an asymmetric water-soluble bacteriochlorophyllin, wherein the method comprises performing a mixed (or hetero-) coupling reaction. For example, the method can comprise providing a dihalogenated bacteriochlorophyllin (e.g., symmetric di-bromo bacteriochlorophyllin) in which two trans beta-pyrrole carbons are substituted with halogen groups (e.g., bromo groups), and performing a mixed coupling reaction (e.g., a mixed Sonogashira, Heck, or Suzuki coupling reaction) by contacting the dihalogenated bacteriochlorophyllin with two different alkynes, two different alkenes, or two different organoboronic acids (e.g., boronic acids or esters) in the presence of a suitable catalyst (e.g., a palladium catalyst, such as a palladium(0) catalyst) and a base (e.g., a trialkyl amine, such as triethylamine, or sodium or potassium acetate).

[0236] In some embodiments, the presently disclosed subject matter provides a method of preparing an asymmetric bacteriochlorophyllin compound having the following formula:

[0237]

[0238] wherein:

[0239] M is a metal or is -H, -H;

[0240] R5, R 10 and R 15 are independently selected from H and alkoxy; and

[0241] R2, R3, R 12 and R 13 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linker group, and a solubilizing group;

[0242] wherein the linker group has the following formula:

[0243] -L1-(X1-L2) p -G,

[0244] wherein p is 0 or 1 ; L1is alkylene; X1is -C(=0)NH- or -NHC(=0)-; L2is -(CH2CH20) q -alkylene, alkylene or substituted alkylene, optionally wherein substituted alkylene is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bioconjugatable group;

[0245] and 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 R S is a group of the formula:

[0246] -X2-(L3) z -R 17 ,

[0247] wherein: z is 0 or 1 ; X2is -CH2NHC(=0)-, -C(=0)NH-alkylene-NH- or triazolyl; L3is -C(=0)-alkylene-C(=0)-NH- and R 17 is selected from -(C2H40) m -R 18 , -C(=0)C2H4-(OC2H4) m OR 18 and -(C2H40) n -C2H4-C(=0)NH-C(R 19 )3, wherein m is an integer of 12 or greater; n is an integer from 1 to 5; R 18 is lower alkyl, optionally methyl; and R 19 is -CH20-C2H4-C(=0)NH-(C2H40) m R 18 ;

[0248] with the proviso that R2and R 12 are not the same or R3and R 13 are not the same, and wherein at least one of R2, R3, R 12 and R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w ; wherein the method comprises: (a) providing a compound of the formula:

[0249]

[0250] wherein:

[0251] M is a metal or is -H, -H;

[0252] R5', R 10 and R 15 are independently selected from H and alkoxy; and

[0253] R2', R3', R 12 and R 13 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, and acetyl, wherein R 2’ and R 12’ are each halogen, optionally bromine, or wherein R 3’ and R 13’ are each halogen, optionally bromine; (b) contacting the compound with a palladium catalyst, a base, and one of: (i) two different alkynes, optionally wherein the two different alkynes are each a compound of the formula:

[0254]

[0255] wherein y is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), optionally 1 or 2; and each R 20 is an N-protected alkylamine, a protected carboxylic acid, -C(=0)-NH-alkylene-protected amine, or -C(=0)-NH-substituted-alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=0)-NH-substituted-alkylene-protected amine comprises a protected carboxylic acid-substituted alkylene; (ii) two different alkenes, optionally wherein the two different alkenes are each a compound of the formula:

[0256]

[0257] wherein y is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), optionally 1 or 2; and each R 20 is an N-protected alkylamine, a protected carboxylic acid, -C(=0)-NH-alkylene-protected amine, or -C(=0)-NH-substituted-alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=0)-NH-substituted-alkylene-protected amine comprises a protected carboxylic acid-substituted alkylene; (iii) two different organoboronic acids; optionally wherein the two different organoboronic acids are two different arylboronic acids or arylboronate esters of the formula:

[0258]

[0259] wherein y is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), optionally 1 or 2; each R 20 is an N-protected alkyl amine, a protected carboxylic acid, -C(=0)-NH-alkylene-protected amine, or -C(=0)-NH-substituted alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=0)-NH-substituted alkylene-protected amine comprises an alkylene substituted with a protected carboxylic acid; and each R 21 is H or alkyl or wherein two R 21 are taken together to form an alkylene. In the resulting product (e.g., a synthetic intermediate of a compound of Formula (II)), the halo substituents at R2' and R 12 or R3' and R 13 are each replaced with a different substituent (e.g., a different -alkynyl-aryl-(R 20 ) y , -alkenyl-aryl-(R 20 ) y or aryl-(R 20 ) y group).

[0260] In some embodiments, the ratio of two alkynes, alkenes, or organoboronic acids is adjusted to maximize the yield of the desired product. For example, in some embodiments, the ratio is adjusted based on the relative reactivity of two different alkynes, alkenes, or boronic acids. The relative reactivity of two coupling partners can be determined by monitoring the reaction by reverse phase HPLC. In some embodiments, the less reactive partner of two coupling partners is provided in a greater molar excess than the second coupling partner to maximize the yield of asymmetric bacteriochlorophyll.

[0261] In some embodiments, the yield of the desired product of the mixed coupling reaction is greater than expected. Thus, in some embodiments, the yield of the desired product is greater than 50% or greater than 55%. In some embodiments, the yield is greater than 60%. In some embodiments, the yield is about 62%.

[0262] IV. Pharmaceutical Compositions

[0263] The presently disclosed subject matter compounds can 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-pyrrolidine-l'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine, and tris(hydroxymethyl)aminomethane; alkali metal salts, for example, 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 further 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, phosphoric, phosphinic, sulfonic, sulfinic, and boric acids.

[0264] The presently disclosed subject matter compounds can also include prodrugs of the compounds described herein. As described above, a "prodrug" is a compound that is metabolized or otherwise converted into a biologically, pharmaceutically, or therapeutically active form of the compound after administration in vivo through one or more steps or processes. To prepare a prodrug, a modification is made to a pharmaceutically active compound such that the active compound will be regenerated by metabolic processes. Prodrugs can be designed to alter the metabolic stability or transport characteristics of a drug, mask side effects or toxicity, improve the flavor, or alter other characteristics or properties of the drug. With knowledge of the pharmacodynamic processes and drug metabolism in the body, one of skill in the art, once a pharmaceutically active compound is known, can design a prodrug thereof (see, e.g., see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, New York, United States of America, pages 388-392).

[0265] Utility. The methods and intermediates described herein can be used to synthesize the compounds of Formula (II) described herein. Such compounds, either as such or in further modified form (e.g., in the form of salts, metallated compounds, conjugates, or prodrugs), can be used for diagnostic and therapeutic purposes in a similar manner to other compounds described for photodynamic therapy, for example, as described in U.S. Patent Application Publication No. 2004 / 0044197 to Pandey et al. and further detailed below.

[0266] Stability. Some embodiments of the bacteriochlorophyll compounds of the presently disclosed subject matter are advantageous for their stability and absorption properties. Accordingly, 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 (II), 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 a peak molar absorption coefficient in solution of at least 10,000 to 300,000 M -1 cm -1 or more at a wavelength of about 600 to about 800 nanometers. (It will be understood that (a) the active compound must be put into solution to determine its peak molar absorption coefficient at a given wavelength; and (b) the compound can exhibit other peaks outside this range, or multiple peaks within this range).

[0267] In addition, the presently disclosed subject matter provides a composition comprising or consisting essentially of a compound of Formula (II), or a pharmaceutically acceptable salt, prodrug, or conjugate thereof (e.g., a conjugate with a targeting agent such as a protein, peptide, or antibody) in a solvent. The amount of solvent is not critical and can range from 0.01 or 1 to 99 or 99.99 percent by weight of the composition. The composition has or is characterized by a peak molar absorption coefficient in solution of at least 10,000 to 300,000 M -1 cm -1 or more at a wavelength of about 600 to about 800 nanometers. It will be understood that stirring can be used as needed to break up agglomerated particles back into solution prior to determining the molar absorption, but a certain level of agglomeration can be desirable for practical application of the composition. Suitable solvents depend on the particular compound and the intended use of the compound, but include organic solvents, aqueous solvents, and combinations thereof.

[0268] Whether in "neat" form or mixed with a solvent, the bacteriochlorophyll compound(s) of the presently disclosed subject matter have or exhibit no more than about 10, 15, or 20 percent loss (due to its degradation) of the bacteriochlorophyll compound of the presently disclosed subject matter 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.

[0269] Solubility.Some embodiments of the presently disclosed subject matter are advantageous in that they are water soluble. Accordingly, the presently disclosed subject matter provides compositions, including but not limited to pharmaceutical formulations, comprising, consisting of, or consisting essentially of: (a) an aqueous solvent (e.g., distilled water, a saline solution, a buffered solution); and (b) about 1, 2, 5, or 10 mM to 200, 300, or 500 mM of an active compound as described herein dissolved in the aqueous solvent.

[0270] Formulation of pharmaceutical compositions. The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more compounds provided herein in a pharmaceutically acceptable carrier and are useful for preventing, treating, or ameliorating one or more symptoms of a disease or disorder associated with or in which hyperproliferative tissue or neovascularization is involved. Diseases or disorders associated with hyperproliferative tissue or neovascularization include, but are not limited to, cancer, psoriasis, atherosclerosis, heart disease, and age-related macular degeneration. Suitable pharmaceutical carriers for administration of the compounds provided herein include any such carriers known to those of skill in the art to be suitable for a particular mode of administration.

[0271] The pharmaceutical compositions preferably exhibit the above-mentioned absorption properties and storage or stability properties.

[0272] In addition, the compounds can be formulated as the sole pharmaceutically active ingredient in a composition or can be combined with other active ingredients.

[0273] The compositions comprise one or more compounds provided herein (e.g., a compound of Formula (II)). In some embodiments, the compounds are formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration, or sterile solutions or suspensions for parenteral administration, as well as transdermal patch formulations and dry powder inhalers. In some embodiments, the above-mentioned compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, Fourth Edition, Lea & Febiger, Philadelphia, Pennsylvania, United States of America, p. 126).

[0274] In the composition, an effective concentration of one or more compounds or pharmaceutically acceptable derivatives thereof is mixed with a suitable pharmaceutical carrier. As described above, the compounds can be derivatized to the corresponding salt, ester, enol ether or enolate ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate, or prodrug prior to formulation. The concentration of the compound in the composition is effective to deliver an amount that, upon administration, treats, prevents, or ameliorates one or more symptoms of a disease or condition associated with or involving hyperproliferative tissue or neovascularization.

[0275] In some embodiments, the composition is formulated for single dose administration. To formulate the composition, a fraction by weight of the compound is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier in an effective concentration such that the treated condition is alleviated, prevented, or one or more symptoms are ameliorated.

[0276] The active compound (i.e., a compound of Formula (II), or a pharmaceutically acceptable salt, prodrug, or conjugate thereof) is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without causing undesirable side effects in the patient 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. Patent No. 5,952,366 to Pandey et al., from which a dosage for humans is extrapolated.

[0277] The concentration of the active compound in the pharmaceutical composition can depend on the rate of absorption, inactivation, and excretion of the active compound, the physicochemical characteristics of the compound, the dosing regimen, and the amount administered, among other factors known to those skilled in the art. For example, as described herein, the amount delivered is sufficient to ameliorate one or more symptoms of a disease or condition associated with or involving hyperproliferative tissue or neovascularization.

[0278] In some embodiments, a therapeutically effective dose should result in serum concentrations of active ingredient of from about 0.1 ng / ml to about 50-100 pg / ml. In one embodiment, a therapeutically effective dose is from 0.001, 0.01, or 0.1 to 10, 100, or 1000 mg of active compound per kilogram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg, 1000 mg, or 2000 mg, and in some embodiments from about 10 mg to about 500 mg, of active ingredient, or combinations of essential ingredients, per dosage unit form.

[0279] The active ingredients can be administered at once, or multiple smaller doses can be administered at intervals of time. It will be appreciated that the exact dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0280] In cases where the compounds exhibit insufficient solubility, methods of solubilizing the compounds can be used. Such methods are known to those of skill in the art and include, but are not limited to, the use of cosolvents, such as dimethylsulfoxide (DMSO), the use of surfactants, such as polyoxyethylensorbitol esters (e.g., Tween® sold under the trade name Derivatives of the compounds, e.g., prodrugs of the compounds, can also be used to formulate effective pharmaceutical compositions.

[0281] After mixing or adding the compound, the resulting mixture can be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the chosen carrier or vehicle. Effective concentrations are sufficient to ameliorate the symptoms of the disease, disorder, or condition being treated and can be determined empirically.

[0282] 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 containing suitable amounts of the compounds or pharmaceutically acceptable derivatives thereof, and oil-water emulsions. In some embodiments, the pharmaceutically therapeutically active compounds and derivatives thereof are formulated and administered in unit-dosage or multiple-dosage forms. As used herein, "unit-dosage form" means a physically discrete unit suitable for individual administration to a human or animal subject and packaged individually as known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association 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 on a fractionated or multiple basis. Multiple-dosage forms are multiple identical unit-dosage forms packaged in a single container for administration as separate unit-dosage forms. Examples of multiple-dosage forms include vials, bottles of tablets or capsules, or pint or gallon bottles. Thus, multiple-dosage forms are multiple dosages not separated in the package.

[0283] Liquid pharmaceutically acceptable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above (e.g., a compound of Formula (II), or a pharmaceutically acceptable salt, prodrug, or conjugate thereof) and optional pharmaceutical adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, glycols, ethanol, or the like, to form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, and the like, such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine sodium oleate, and other such agents.

[0284] 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.

[0285] Dosage forms or compositions containing active ingredients can be prepared, packaged, and / or sold in a combination with another therapeutic or prophylactic agent. Such compositions are prepared following procedures known in the art.

[0286] Compositions for oral administration. Oral pharmaceutical dosage forms are either solid, gel, or liquid. Solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which can be enteric-coated, sugar-coated or film-coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

[0287] 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, and the like can contain one or more of the following ingredients or compounds of similar nature: binders; lubricants; diluents; glidants; disintegrants; coloring agents; sweetening agents; flavoring agents; wetting agents; emetic coatings; and film coatings. Examples of binders include microcrystalline cellulose, gum tragacanth, dextrose 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. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methyl cellulose, agar, and carboxymethyl cellulose. Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, as well as any number of spray dried flavorings. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds which impart a pleasant sensation to the senses of taste and smell, such as, but not limited to, peppermint 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 (PEG 4000), and cellulose acetate phthalate.

[0288] The compounds or pharmaceutically acceptable derivatives thereof can be provided in a composition that protects it against the acidic environment of the stomach. For example, the composition can be formulated to maintain its integrity in the stomach and release the active compound in the intestine. The composition can also be formulated in combination with antacids or other such ingredients. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Additionally, the dosage unit form can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar or other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, spray, chewing gum or the like. A syrup can contain, in addition to the active compound, sucrose as a sweetening agent and certain preservatives, dyes and colorants, flavors and the like.

[0289] The active material can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action such as antacids, H2 blockers, and diuretics. The active ingredient is a compound as described herein or a pharmaceutically acceptable derivative thereof. Higher concentrations of active ingredient can be included up to about 98% by weight.

[0290] In some embodiments, tablet and capsule formulations can be coated as is known to those of skill in the art to modify or sustain dissolution of the active ingredient. Thus, for example, they can be coated with conventional enteric- soluble coatings such as phenyl salicylate, wax, and cellulose acetate phthalate.

[0291] 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 formulations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are oil-in-water or water-in-oil.

[0292] Elixirs are clear, sugar- sweetened hydroalcoholic preparations. Pharmaceutically acceptable carriers for elixirs include solvents. Syrups are concentrated aqueous solutions of sugar (e.g., sucrose), and can contain a preservative. Emulsions are two-phase systems in which one liquid is dispersed in another liquid by the use of an emulsifying agent. Pharmaceutically acceptable carriers for emulsions are non- aqueous liquids, emulsifying agents, 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 a source of carbon dioxide. Coloring agents and flavoring agents are used in all of the above dosage forms. Solvents include glycerin, sorbitol, ethanol, and syrup. Examples of preservatives include glycerin, 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 emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, veegum, and acacia. Sweeteners include sucrose, syrup, glycerin, 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 and tartaric acids. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant taste sensation. For solid dosage forms, solutions or suspensions in either aqueous or non-aqueous liquids, in one embodiment, are encapsulated in gelatin capsules. Such solutions or suspensions are disclosed, for example, 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, solutions in, for example, polyethylene glycol can be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, such as water, to enable easy measurement in the amounts desired for administration.

[0293] Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oil, glycol, triglyceride, propylene glycol ester (e.g., propylene carbonate), and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Patent No. RE 28,819 and U.S. Patent No. 4,358,603, each of which is incorporated herein 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-dimethoxy methane, diglycol dimethyl ether, triglycol dimethyl ether, tetraglycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether (where 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, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and esters thereof, and dithio- carbamates.

[0294] Other formulations include, but are not limited to, aqueous alcohol solutions comprising a pharmaceutically acceptable acetal. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having 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.

[0295] Injectables, solutions, and emulsions. In some embodiments featuring subcutaneous, intramuscular, or intravenous injection, parenteral administration is also included herein. The injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectable formulations, solutions, and emulsions further comprise one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Additionally, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, e.g., sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0296] Implantation of sustained-release or continuous-release systems is also contemplated herein, such that a constant dosage level is maintained (see, e.g., U.S. Patent No. 3,710,795, incorporated herein by reference in its entirety). Briefly, the compounds provided herein are dispersed in a solid inner 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 copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, which is surrounded by an outer polymeric membrane that is insoluble in body fluids, such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, chlorobutyl rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of chloroethylene with vinyl acetate, partial dichloroethylene, ethylene and propylene, the ionomeric polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / ethylene vinyl alcohol terpolymer, and ethylene / ethyleneoxy ethanol copolymer. The compound diffuses through the outer polymeric membrane in a rate-controlling step. The percentage of active compound included in such parenteral compositions depends on the particular nature of the compound and its activity and the needs of the individual.

[0297] Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administration. The formulations for parenteral administration include sterile solutions ready for injection; sterile dry soluble products ready to be mixed with a solvent prior to use, such as lyophilized powders, including tablets for subcutaneous injection; sterile suspensions ready for injection; sterile dry insoluble products ready to be mixed with a vehicle immediately prior to use; and sterile emulsions. The solutions can be aqueous or non-aqueous.

[0298] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0299] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, masking or chelating agents, and other pharmaceutically acceptable substances.

[0300] Examples of aqueous vehicles include sodium chloride injection, Ringers Injection, isotonic dextrose injection, sterile water injection, dextrose in water, and Lactated Ringers Injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents such as benzyl alcohol or methyl parabens, chlorobutanol, propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride can be added to parenteral preparations packaged in multi-dose containers, which include phenol or cresol, mercuric chloride, benzyl alcohol, chlorobutanol, methyl and propyl esters of p-hydroxybenzoic acid, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfite. Local anesthetics include procine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, xanthan gum, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polyoxyethylene sorbitol esters (e.g., Tween 80® 80) Metal ion sequestrants or chelating agents include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol, and propylene glycol for aqueous vehicles, and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0301] 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 is known in the art.

[0302] Unit doses of parenteral formulations are packaged in ampules, vials, or syringes with needles. All formulations for parenteral administration should be sterile, as is known and practiced in the art.

[0303] Illustratively, intravenous or intra-arterial infusion of a sterile aqueous solution containing the active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oleaginous solution or suspension containing the active material injected as needed to produce the desired pharmacological effect.

[0304] The injection is designed for local and systemic administration. In one embodiment, a therapeutically effective dose is formulated to contain at least about 0.1% w / w to about 90% w / w or more of the active compound in the concentration of the tissue to be treated, in certain embodiments, more than 1% w / w of the active compound in the concentration of the tissue to be treated.

[0305] The compound can be suspended in micronized or other suitable form, or can 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 chosen carrier or vehicle. An effective concentration is sufficient to ameliorate the symptoms of the condition and can be determined empirically.

[0306] Lyophilized powders. 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 into solids or gels.

[0307] Sterile lyophilized powders are prepared by dissolving a compound provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. The solvent can contain excipients that improve the stability of the powder or other pharmacological components of the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable substances. The solvent can also include a buffer such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art, in one embodiment, a buffer at about neutral pH. The solution is then sterile filtered and lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is aliquoted into vials for lyophilization. Each vial can contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, for example, at about 4°C to room temperature.

[0308] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. To reconstitute, the lyophilized powder is added to sterile water or another suitable carrier. The precise amount depends on the compound chosen. This amount can be determined empirically.

[0309] Topical administration Topical mixtures are prepared as described for local and systemic administration. The resulting mixture can be a solution, suspension, emulsion, or the like, and is formulated into creams, gels, pastes, lotions, solutions, elixirs, tinctures, suspensions, emulsions, gels, foams, aerosols, enemas, sprays, suppositories, bandages, skin patches, or other appropriate formulations for topical administration.

[0310] A compound or a pharmaceutically acceptable derivative thereof can be formulated into an aerosol for topical application, such as by inhalation (see, e.g., 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 describe aerosols for delivering steroids for the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of aerosol or solution in a nebulizer, or in the form of finely divided powders for insufflation, alone or in combination with inert carriers, such as lactose. In such cases, the particles of the formulation will generally have diameters of less than 50 microns, in some embodiments, less than 10 microns.

[0311] The compound can be formulated for topical or local 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 cord for 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% with a pH of about 5-7.

[0312] 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.

[0313] 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. Patents 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.

[0314] For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effects. Rectal suppositories used in this article refer to solids for insertion into the rectum that melt or soften at body temperature, releasing one or more pharmaceutically or therapeutically active ingredients. Pharmaceutically acceptable substances used for rectal suppositories are bases or excipients and agents that increase the melting point. Examples of bases include cocoa butter (cocoa butter), glycerol-gelatin, carbowax (polyoxyethylene glycol) and an appropriate mixture of monoglycerides, diglycerides and triglycerides of fatty acids. A combination of various bases can be used. Agents that increase the melting point of suppositories include spermaceti and wax. Rectal suppositories can be prepared by compression methods or by molding. In one embodiment, the weight of the rectal suppository is about 2 to 3 grams.

[0315] 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.

[0316] Targeted formulationsThe compounds provided herein, or pharmaceutically acceptable derivatives thereof, can also be formulated to target specific tissues, receptors, infectious agents, or other areas of the body of the individual to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated for use in the present compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent 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.

[0317] Liposomes. In some embodiments, liposome suspensions, including liposomes targeting tissues, such as tumor-targeted liposomes, can also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations 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 the unencapsulated compound, centrifuged, and then resuspended in PBS.

[0318] Ligands.In some embodiments, the disclosed compounds can be targeted to a specific target tissue or target composition using a ligand specific for the target tissue or target composition, e.g., using a ligand or ligand-receptor pair such as an antibody and antigen. Antibodies against tumor antigens and against 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, inter alia, in Hansen et al., U.S. Patent No. 3,927,193 and Goldenberg, U.S. Patent Nos. 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 against antigens (e.g., gastrointestinal tumors, lung tumors, breast tumors, prostate tumors, ovarian tumors, testicular tumors, brain tumors, or lymphoid tumors, sarcomas, or melanomas) can be used.

[0319] A number of monoclonal antibodies have been developed against infectious disease agents and are summarized in a review by Polin (1984) European Journal of Clinical Microbiology 3(5):387-398, which indicates the ready availability. These include monoclonal antibodies (MAb) against pathogens and their antigens, such as the following: anti-bacterial MAbs, such as those against Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Esherichia coli, Neisseria gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease, Spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, or tetanus toxin;Antiprotozoal 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; anti-viral 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 viruses 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 laidlawii, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, M. pneumonia; and the like.

[0320] Suitable MAbs have been developed against most of the microorganisms (bacteria, viruses, protozoa, other parasites) responsible for most human infections, and many have been previously used for in vitro diagnostic purposes. These antibodies and new MAbs that can be produced by routine methods are suitable for use as target substances with the compounds provided herein.

[0321] MAbs against malaria parasites can be directed against the sporozoite, schizont, merozoite, and gametocyte stages. Monoclonal antibodies against sporozoites (circumsporozoite antigen) have been produced and have been shown to neutralize sporozoites in vitro and in rodents. See Yoshida et al. (1980) Science 207:71-73. Monoclonal antibodies to T. gondii (a protozoan parasite associated with toxoplasmosis) have been developed. See Kasper et al. (1982) Journal of Immunology 129: 1694-1699. MAbs against surface antigens of blood flukes have been developed and have been found to act on blood flukes 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.

[0322] Mixtures of antibodies and immunoglobulin classes can be used, as can hybrid antibodies. Multispecific (including bispecific and hybrid) antibodies and antibody fragments are particularly preferred in 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 a target lesion, or to at least two different epitopes or marker substances produced by or associated with a target tissue. Multispecific antibodies and antibody fragments with bispecificity can be prepared similarly to the anti-tumor marker hybrid disclosed in U.S. Patent No. 4,361,544. Other techniques for preparing hybrid antibodies are disclosed in, e.g., U.S. Patent 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.

[0323] Antibody fragments that can be used in the presently disclosed subject matter include F(ab')2, F(ab)2, Fab', Fab, Fv, and the like, 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 an immunoglobulin and has a size similar to or smaller than a Fab' fragment is also useful. This can include genetically engineered and / or recombinant proteins, whether single chain or multiple chain, that incorporate an antigen binding site and additionally function in vivo as targeting vehicles in essentially the same manner as native immunoglobulin fragments. Such single chain binding molecules are disclosed in U.S. Patent No. 4,946,778, which is incorporated herein by reference in its entirety. Fab' antibody fragments can be conveniently prepared by reducing cleavage of F(ab')2 fragments, which themselves can be prepared by pepsin digestion of whole immunoglobulin. Fab antibody fragments can be prepared by papain digestion of whole immunoglobulin under reducing conditions, or by cleavage of F(ab')2 fragments, which are produced by careful papain digestion of whole immunoglobulin.

[0324] A member of a ligand or 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. Patent Nos. 4,374,925 and 3,817,837, each of which is incorporated herein by reference in its entirety.

[0325] Conjugation with ligands. A number of 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 a chlorin Sn(IV) to a monoclonal antibody through a covalent bond using a modified dextran carrier. 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 a ligand, such as an antibody, by using a coupling agent. Any linkage that is capable of linking the components so that they are stable under physiological conditions for the time desired for administration and treatment is suitable, but covalent linkages are preferred. The linkage between the two components can be direct, e.g., where the compound of Formula (I) is directly linked to the targeting agent, or it can be indirect, e.g., where the compound of Formula (I) is linked to an intermediate and the intermediate is linked to the targeting agent.

[0326] The coupling agent should function under conditions of temperature, pH, salt, solvent system, and other reactants that substantially maintain the chemical stability of the photosensitizing agent, backbone (if present), and targeting agent. The coupling agent should stably link the component moieties, but such that the compound of Formula (I) or targeting agent is only minimally or not denatured or inactivated. 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, e.g., Bodansky (1993) Principles of Peptide Synthesis, 2nd Ed., Springer & Hermanson (1996) Bioconjugate Techniques, 1st Ed., Academic Press, New York, New York, United States of America.

[0327] Conjugates of the compounds provided herein and a ligand such as an antibody can be prepared by coupling the compound through a carboxylic acid or ester moiety on the compound to the antibody via a peptide bond at 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-phenylene dimaleimide (o-PDM), and sulfosuccinimidyl-4-(N-maleimido-methyl)- cyclohexane-l-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 by Glennie et al. (1987) Journal of Immunology 139: 2367-2375.

[0328] For example, DCC is a useful coupling agent that can be used to facilitate the coupling of an alcohol NHS to a chlorin carboxylic acid group in DMSO to form an activated ester that can be cross-linked to polylysine. DCC is a carboxyl reactive cross-linking agent that is commonly used as a coupling agent in peptide synthesis and has a molecular weight of 206.32. Another useful cross-linking agent is SPDP, a heterobifunctional cross-linking agent that is used with primary amines and thiols. SPDP has a molecular weight of 312.4, a spacer arm length of 6.8 angstroms, is reactive with NHS-esters and pyridyl disulfide groups, and produces a cleavable cross-link that is eliminated upon further reaction, allowing the photosensitizer to be attached directly to the backbone or targeting agent. Other useful coupling agents are SATA for the introduction of a blocked SH group for two-step cross-linking, which is de-blocked by hydroxylamine-HCl and sulfo-SMCC, is reactive with amines and thiols. Other cross-linking agents 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 Bl.

[0329] Photosensitizers containing carboxyl groups can be attached to lysine epsilon-amino groups in the target polypeptide through preformed reactive esters such as N-hydroxysuccinimide (NHS) esters or esters conjugated in situ through carbodiimide-mediated reactions. The same applies to photosensitizers containing sulfonic acid groups, which can be converted to sulfonyl chlorides that react with amines. Bacteriochlorins with carboxyl groups can be attached to amines on polypeptides through in situ carbodiimide methods. Bacteriochlorins can also be attached to the hydroxyl group of serine or threonine residues or the thiol group of cysteine residues.

[0330] Methods of linking components of conjugates, such as coupling a photosensitizer with a polyamino acid chain to an antimicrobial polypeptide, can use heterobifunctional cross-linking reagents. These reagents bind one functional group in one chain and bind it to a different functional group in a second chain. These functional groups are usually amino, carboxyl, thiol, and aldehyde. There are many permutations of suitable moieties that will react with these groups and structures represented in different formulas to conjugate them together. See Hermanson (1996) Bioconjugate Techniques, 1st Edition, Academic Press, New York, New York, United States of America; and Merrifield et al. (1994) Ciba Foundation Symposium 186:5-20.

[0331] 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 is effective for modulating the activity of hyperproliferative tissue or neovascularization, or for treating, preventing or ameliorating 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 useful for modulating the activity of hyperproliferative tissue or neovascularization, or for treating, preventing or ameliorating 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.

[0332] 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. Patent Nos. 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 material suitable for the selected formulation and intended mode of administration and treatment. Various formulations of the compounds and compositions provided herein are contemplated for various treatments of any disease or condition in which hyperproliferative tissue or neovascularization is involved as a mediator or contributory factor to a symptom or cause.

[0333] V. Photodynamic therapy, diagnostic, and therapeutic applications

[0334] In some embodiments, the compounds of formula (II) 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 photosensitizing compound, its conjugate, or pharmaceutical composition is typically administered to an individual before the target tissue, target composition, or individual is exposed to light. The photosensitizing compound is administered as described elsewhere herein.

[0335] The dosage of the photosensitizing compound can be clinically determined. Depending on the photosensitizing compound used, an equivalent optimal therapeutic level must be established. A certain amount of time is allowed to elapse for the circulating or locally delivered photosensitizer to be absorbed by the target tissue. During this waiting period, unbound photosensitizer is cleared from the circulation, or additional time may be provided to allow unbound compound to be cleared from non-target tissues. The waiting period can be clinically determined and may vary from compound to compound.

[0336] 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 artificial light sources such as fluorescent or incandescent light, or natural light sources such as ambient sunlight. The area of illumination 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. Any total or cumulative period of time from about 4 minutes to about 72 hours can be used. In some embodiments, the illumination period is from about 60 minutes and 148 hours. In some embodiments, the illumination period is from about 2 hours and 24 hours.

[0337] Preferably, the total fluence or energy of the light used for irradiation, measured in joules, is from about 10 joules to about 25,000 joules; more preferably, from about 100 joules to about 20,000 joules; most preferably, from about 500 joules to about 10,000 joules. The light is selected for 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. Light having a wavelength at least partially corresponding to the characteristic light absorption wavelength of the photosensitizer is preferably used to irradiate the target tissue.

[0338] The intensity or power of the light used is measured in watts, with one watt-second equaling one joule per femtosecond. Thus, the intensity of the light used for irradiation in the presently disclosed methods can be substantially less than 500 mW / cm 2 . Since the total fluence or 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 site 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 sufficiently high to activate the photosensitizer.

[0339] In some embodiments of photodynamic therapy using the compounds disclosed herein, the compound is injected into a mammal (e.g., a human) to be diagnosed or treated. The injection level is typically from about 0.1 to about 0.5 umol / kg body weight. In the case of treatment, the area to be treated is exposed to light of the desired wavelength and energy, e.g., from about 10 to 200 J / cm 2 . In the case of detection, fluorescence is determined upon exposure to light of a wavelength sufficient to cause the compound to fluoresce at a wavelength different from the wavelength used to irradiate the compound. The energy used in detection is sufficient to cause fluorescence and is typically significantly lower than that required for treatment.

[0340] Any of the photosensitizing compounds disclosed herein, or a pharmaceutically acceptable derivative thereof, can be provided in a kit with instructions for carrying out any of the methods disclosed herein. The instructions can be in any tangible form, such as printed paper, a computer disk instructing a person how to carry out the method, a video tape containing instructions on how to carry out the method, or a computer memory that receives data from a remote location and sets forth the instructions or otherwise provides the instructions to a person, such as through the Internet. For example, a person can be instructed how to use the kit using any of the instructions described above or by receiving the instructions in a classroom or during the course of treating a patient using any of the methods disclosed herein.

[0341] Other examples and specific examples of methods of using the presently disclosed subject matter compounds and compositions include, but are not limited to, the following:

[0342] (i) Treatment of opportunistic infections. The presently disclosed subject matter compounds, compositions, and methods can be used for PDT of opportunistic infections, particularly PDT of opportunistic infections of soft tissue. For antimicrobial treatment (by PDT) of infections, particularly wound infections, the infecting organisms can include (as non-limiting examples) 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 with AIDS or those receiving immunosuppressive therapy.

[0343] (ii) Treatment of burns. Infections with Staphylococcus aureus and gram-positive bacteria are often particularly evident in burns. The multiple drug resistance of Staphylococcus aureus presents a major medical challenge. In this regard, the presently disclosed subject matter compounds, compositions, and methods can be used for PDT treatment of opportunistic infections of burns.

[0344] (iii) Sepsis. The presently disclosed subject matter compounds, compositions, and methods can be used for PDT treatment of individuals with opportunistic infections of Vibrio vulnificus. Vibrio vulnificus, a gram-negative bacterium, can cause primary sepsis, wound infections, and gastrointestinal illness in humans.

[0345] (iv) Ulcers. The presently disclosed subject matter compounds, compositions, and methods can be used for PDT treatment of bacteria (Helicobacter pylori) that cause ulcers. In the clinic, the treatment can be carried out in any suitable manner, such as by inserting a fiber optic cable (similar to an endoscope, but with a device to deliver red or near infrared light) into the stomach or affected area.

[0346] (v) Periodontal disease. The presently disclosed subject matter compounds, compositions, and methods are useful for PDT for the treatment of periodontal disease, including gingivitis. Periodontal disease is caused by overgrowth of bacteria, such as the gram-negative anaerobe Porphyromonas gingivalis. As with many PDT treatments, a targeting or solubilizing entity conjugated to the photoactive species is essential for proper delivery of the photoactive species 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, the presently disclosed subject matter compounds or compositions can be applied topically (e.g., in the form of a mouthwash or mouthrinse), followed by light application with an external device, intraoral instrument, or combination thereof.

[0347] (vi) Atherosclerosis. The presently disclosed subject matter compounds, compositions, and methods are useful for PDT to treat vulnerable plaque in arteries. Without wishing to be bound by any particular theory, it is believed that invading inflammatory macrophages secrete metalloproteinases that degrade the thin layer of collagen in the coronary artery, leading to thrombosis, which is often fatal. Active compounds that target such inflammatory macrophages are useful for PDT of vulnerable plaque.

[0348] (vii) Cosmetic and dermatological applications. The presently disclosed subject matter compounds, compositions, and methods are useful 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 fairly well for fair-skinned people with dark hair. The presently disclosed subject matter compounds, compositions, and methods can be used as near-infrared sensitizers for hair removal, which are able to target chromophores with more specificity and sharp absorption bands.

[0349] (viii) Acne. The presently disclosed subject matter compounds, compositions, and methods can be used in PDT to treat acne. Acne vulgaris is caused by infection of sebaceous glands by Propionibacterium acne; some 80% of young people are affected. Again, bacterial resistance to antibiotic treatment is increasing, leading to a surge in untreatable acne. Current PDT treatment of acne typically relies on the addition of aminolevulinic acid, which is converted to free-base porphyrin in the hair follicle or sebaceous gland. The presently disclosed subject matter compounds and compositions can be administered to an individual topically or parenterally (e.g., by subcutaneous injection) according to the particular condition.

[0350] (ix) Infectious diseases. The presently disclosed subject matter compounds, compositions, and methods can be used in PDT to treat infectious diseases. For example, cutaneous and subcutaneous leishmaniasis, which occurs widely in the Mediterranean and Middle East regions, is currently treated using arsenic-containing compounds. Recently, PDT has been used with reasonable effect in at least one case on a human patient. The use of the presently disclosed subject matter compounds and compositions is equally useful, and potentially offers advantages such as ease of synthesis and better spectral absorption properties.

[0351] (x) Tissue sealants. The presently disclosed subject matter compounds, compositions, and methods can be used in PDT as tissue sealants in individuals in need thereof. Photoactivated tissue sealants are attractive for sealing wounds, adhering tissues, and closing defects in tissues. There are many applications in which sutures or staples are undesirable, and applications in which the use of such mechanical sealing methods often leads to infection and scarring.

[0352] (xi) Neoplastic diseases. The presently disclosed subject matter compounds, compositions, and methods can be used in PDT for the treatment of 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, patch stage cutaneous T-cell lymphoma, and Kaposi's sarcoma.

[0353] In addition to PDT, the compositions provided herein can also be used as imaging enhancers in diagnostic imaging techniques, or for labeling target tissues or target compositions for diagnostic radiology. In the modern medical field, 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 eliminate cancerous tissue. Early diagnosis of precancerous areas and microcancers is an important theme in modern cancer treatment. MRI has become a powerful tool in the clinical environment because it is non-invasive and produces accurate volume reproductions of individuals. The image is created by applying one or more orthogonal magnetic field gradients on the 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 the sample / individual. Typically, the image is based on NMR signals from the protons of water, where the signal intensity in a given volume element is a function of water concentration and relaxation time. Local variations in these parameters provide the sharp contrast observed in MR images.

[0354] The effect of MRI contrast agents is to increase the relaxation rates, thereby increasing the contrast between water molecules in the region where the imaging agent accumulates and water molecules elsewhere in the body. However, the effect of the agent is to decrease both Tl and T2, the former leading to greater contrast and the latter leading to lower contrast. Thus, the phenomenon is concentration dependent, and there is generally an optimal concentration of paramagnetic species for maximum efficacy. The optimal concentration can vary with the particular agent used, the imaging site, the imaging modality (i.e., spin echo imaging, saturation recovery, inversion recovery, and / or various other intense Tl-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, e.g., 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 are perfused into blood vessels, enhancing the contrast of the blood vessels and reporting on organ pathology and infiltration. However, it remains a formidable challenge in MRI to label specific tissues for diagnostic radiology. 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, typically gadolinium chelates Gd-DTPA, have been generated and tested for their effect on MRI contrast of tumors and other tissues. See U.S. Patent No. 5,059,415, incorporated herein in its entirety. Unfortunately, it has been found that the relaxivity of Gd bound to antibodies is only slightly better than unbound Gd-DTPA. See Paajanen et al. (1990) Magnetic Resononance in Medicine 13:38-43.

[0355] MRI is commonly used to detect NMR spectra of H nuclei in living organisms. 1 However, MRI is capable of detecting NMR spectra of other nuclei, including 13 C, 15 N, 31 P, and 19 F. 19 F is not abundant in living organisms. By incorporating an isotope useful for MRI, such as 13 C, 15 N, 31 P, or 19 F, particularly 19 F, into the compositions provided herein, and administering to an individual, the compounds provided herein will accumulate in the target tissue, and because of the presence of the MRI-identifiable isotope (such as 19The presence of the accumulated compound of F) will, upon subsequent MR imaging, produce NMR data with enhanced signal from the target tissue or target composition. Thus, the disclosed compounds can be used as image enhancing agents and provide a marker for a specific target tissue or target composition for diagnostic radiology, including MRI.

[0356] In addition to PDT, the compositions provided herein can be used to detect target cells, target tissues, or target compositions in an individual. When a compound provided herein is used to detect a target tissue or target composition, the compound is introduced into the individual and allowed sufficient time for the compound to accumulate in the target tissue or associate with the target composition. The treatment area is then illuminated, typically with light of sufficient energy to cause fluorescence of the compound, and the energy used is typically significantly less than that required for photodynamic therapy treatment. Fluorescence is determined upon exposure to light of the desired wavelength, and the amount of fluorescence can be correlated qualitatively or quantitatively to the presence of the compound by methods known in the art.

[0357] 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. A compound provided herein can be conjugated to one or more ligands specific for the infectious agent, such as an antibody or antibody fragment, which selectively associates with the infectious agent, and after sufficient time for the compound of interest to associate with the infectious agent and clear from non-target tissues, the compound can be visualized, such as by exposure to light of sufficient energy to cause fluorescence of the compound, or by use of diagnostic radiology imaging, including MRI. For example, any one of the compounds provided herein can be conjugated to an antibody that targets a suitable H. pylori antigen, and formulated into a pharmaceutical preparation 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 sufficient time for the compound to selectively associate with the infectious agent of interest and any unbound compound to clear from non-target tissues, the individual can be examined to determine whether any H. pylori is present. This can be detected, for example, by MRI to detect the presence of the compound accumulated due to the presence of the F substituent, or by illuminating the area of interest with light of sufficient energy to cause fluorescence of the compound (for example, by use of fiber optics), and detecting any fluorescence of the compound of interest. 19 The presence of the accumulated compound of F) will, upon subsequent MR imaging, produce NMR data with enhanced signal from the target tissue or target composition. Thus, the disclosed compounds can be used as image enhancing agents and provide a marker for a specific target tissue or target composition for diagnostic radiology, including MRI.

[0358] In some embodiments, the presently disclosed compounds or conjugates thereof can be used in 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, particles to be detected, such as cells, are labeled with a luminescent compound, such as a phosphor or fluorophore, 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 uptake or internalization of 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 in flow cytometry, such as this luminescent compound, which flow cytometry technique (including fluorescence activated cell sorting or FACS) can be performed based on the present disclosure, according to known techniques or variations thereof apparent to those skilled in the art.

[0359] EMBODIMENT

[0360] The following examples provide illustrative embodiments. In light of the present 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 can be employed without departing from the scope of the presently disclosed subject matter.

[0361] EMBODIMENT 1

[0362] Synthesis of compound CP-1

[0363] A di-BOC protected Suzuki coupling partner 1 (CP-1) was prepared as shown in Scheme 2 (see Figure 2 ).

[0364] 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 stir bar fitted with a glass stopper, a condenser with 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 through a cannula to about half volume (~ 450 mL). 1-Bromo-3,5-dimethylbenzene (15.26 g, 80.0 mmol) was added via syringe, followed by a brief opening of the system under a stream of argon and a large addition of solid azobisisobutyronitrile (AIBN, 0.670 g, 4.00 mmol). The flask was heated to gentle reflux under argon (oil bath set at 90 °C).

[0365] After 16 hours, the reaction mixture was transferred to a single neck 1 L RBF and concentrated to remove the ACN. The solid residue was further dried under high vacuum, suspended in dichloromethane (DCM, 75 mL), and heated to a gentle boil. The mixture was allowed to equilibrate to room temperature and filtered with DCM washes. The filtrate was concentrated, dried under high vacuum, and recrystallized in ethanol (EtOH, total 55 mL) with 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 was isolated as a white crystalline solid (17.40 g, 51%).

[0366] 1 H NMR (400 MHz, CDC13) δ 4.41 (s, 4H), 7.34 (s, 1H), 7.47 (d, J = 2.0 Hz, 2H).

[0367] 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 in portions. The flask was fitted with a condenser topped with a drying tube and heated in an oil bath set to 90 °C.

[0368] After 16 hours, the mixture was cooled to room temperature, diluted with water (total 1 L), and extracted with chloroform (400, 300, and 200 mL, 1x each). The organic layers were combined and washed with 0.2 N aqueous NaOH (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 diethyl ether (Et20) (3x). Compound CP-1b was isolated as a white powdery solid (17.98 g, 70%).

[0369] 1 H NMR (400 MHz, CDC13) δ 4.41 (s, 4H), 7.34 (s, 1H), 7.47 (d, J = 2.0 Hz, 2H).

[0370] (5-Bromo-l,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, then the reaction mixture was allowed to cool gradually to room temperature.

[0371] 6N aqueous HC1 was added until the solution was acidic by litmus test (20 mL 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 give a clear light amber solution. The filtrate was cooled in an ice bath and basified with 2N aqueous NaOH (30 mL total). The aqueous layer was extracted with chloroform (3 x 75 mL). The organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a volume of approximately 10 mL. A white residue was noted on the walls of the flask. The remaining solution was filtered, and the filtrate was concentrated to give 2.3 g of a light yellow oil. Stored in the refrigerator.

[0372] The sample was solidified after storage overnight at 4 °C and further dried under high vacuum to give 2.047 g (59%) of compound CP-1c as an amber colored semi-solid.

[0373] Di-tert-butyl ((5-bromo-l,3-phenylene)bis(methylene))dicarbamate (CP-Id). Compound CP-1c (2.00 g, 9.11 mmol) was added to a flame-dried 250 mL RBF with a stir 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, then allowed to equilibrate to room temperature.

[0374] The reaction was allowed to stir at room temperature overnight. The mixture was concentrated to give a white solid, which was re-dissolved in ethyl acetate (EtOAc, 70 mL). The organic phase was washed with saturated aqueous NH4C1, water, saturated aqueous NaHC03, and brine (1 x 50 mL each). The organic layer was then dried over sodium sulfate, filtered, and concentrated to a light amber oil that crystallized upon standing. The solid was washed with cooled 1 : 1 Et20 / hexanes in a glass frit filter. The solid was dried under high vacuum to give 3.50 g (93%) of compound CP-Id as a white powdery solid.

[0375] Coupling partner 1 (CP-1). Dimethyl sulfoxide (DMSO, reagent grade, 20.0 mL) was added to a 100 mL RBF and purged with argon for 45 minutes with stirring. Compound CP-1d (1.25 g, 3.01 mmol), bis(pinacolato)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 with a stir bar and the flask was evacuated for 30 minutes. The flask was purged with argon and the 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.

[0376] After 16 hours, 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.

[0377] The concentrated washed clean 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 give a clear oil. After further drying under high vacuum in the presence of a stir bar, the product solidified. Compound CP-1 was isolated as a white waxy solid (1.224 g, 88%).

[0378] Example 2

[0379] Synthesis of compound BC-1

[0380] The title compound was prepared from previously described dibromothymochromin (BC-SM) (see Jiang et al. (2014) Organic & Biomolecular Chemistry 12: 86-103) as shown in Scheme 3 (see Figure 3 ).

[0381] BC-1a. BC-SM (362.8 mg, 0.538 mmol), coupling partner (i.e., CP-1, see Example 1 above; 547.3 mg, 1.184 mmol), tetrakis(triphenylphosphine)palladium(0) (373.0 mg, 0.323 mmol), and cesium carbonate (525.9 mg, 1.614 mmol) were added to an oven-dried 250 mL RBF with a stir bar and the components were dried under high vacuum for 1 hour. The flask was flushed with argon and toluene / DMF (degassed, 2:1 mixture, total 53.8 mL) was added. The flask was placed in an oil bath and heated to 90 °C under argon.

[0382] After 22 hours, the reaction mixture was cooled, toluene (4x DMF volume) was added, and the mixture was concentrated to dryness. The residue was dissolved in EtOAc (200 mL) and washed with saturated aqueous NaHC03, water, and brine (150 mL each). The organic layer was dried over sodium sulfate, filtered, and concentrated to give 1.09 g of a purple residue.

[0383] The crude residue was loaded onto 3.28 g of silica and eluted with 5-30% EtOAc in DCM over a 40 g silica column for 25 minutes. The main product fractions were combined, concentrated, and dried under high vacuum to give 0.466 g (73%) of BC-la as a purple solid.

[0384] BC-lb. BC-la (146.0 mg, 0.123 mmol) was added to an oven-dried 50 mL RBF with a stir bar. The flask was evacuated and flushed with argon and THF (20 mL) was added. NBS (23.0 mg, 0.129 mmol) was dissolved in THF (4.0 mL) and added quickly dropwise. The reaction was stirred at room temperature under argon.

[0385] After 1.5 hours, the reaction was diluted in DCM (25 mL) and quenched with saturated aqueous NaHC03(25 mL). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated.

[0386] The residue was loaded onto a 12 g silica column with minimal DCM (about 5 mL) and eluted with 0-3% MeOH in DCM over 22 minutes. The main peak fractions were combined, concentrated, and dried under high vacuum to give 0.115 g (79%) of BC-lb as a purple solid.

[0387] BC-lc. BC-lb (114.5 mg, 90.6 μmol) was added to an oven-dried 25 mL RBF with a stir bar along with Pd2(dba)3(12.5 mg, 13.6 μmol) and P(o-tol)3(32.0 mg, 105.1 μmol). The flask was capped and evacuated for 30 minutes. The flask was flushed with argon, DMF (3.63 mL) was added, followed by triethylamine (0.363 mL) and 6-heptyne acid (228.7 mg, 1812.0 μmol). The flask was heated in a 40 °C oil bath and stirred under argon.

[0388] After 16 hours, the reaction was diluted with EtOAc (10x DMF volume) and washed with equal volumes of each 0.2N aqueous HC1, water, and brine, in that order. The organic layer was separated, dried over sodium sulfate, filtered, concentrated, redissolved in toluene, concentrated, and dried under high vacuum.

[0389] The residue was loaded onto silica (1.05 g) and eluted with 0-33% EtOAc in DCM over a 24 g silica column for 15 minutes, held until any starting material was eluted. The solvent was then switched to 0-4% MeOH in DCM over 15 minutes. The product peak fractions were combined, concentrated, and dried under high vacuum to give 45.0 mg (38%) of BC-1c as a dark purple solid.

[0390] BC-1. BC-1c (17.5 mg, 13.36 pmol) was added to an oven-dried 25 mL RBF with a stir bar. The flask was placed under vacuum for 30 minutes, then flushed with argon, and the vacuum / argon cycle was repeated 2 times. Hydrogen chloride solution (4.0 M in dioxane, 2.9 mL) was added in one portion with stirring, and the reaction was stirred under argon. After 30 minutes, stirring was stopped and the precipitate was allowed to settle for 10 minutes. Most of the dioxane was removed by syringe under argon. The residue was placed under high vacuum for 2 hours.

[0391] The reaction flask was flushed with argon and tributylamine (5 drops) was added and mixed with stirring. A 2:1 hexanes / THF solution (3 mL) was then added and stirred briefly. The mixture was then sonicated for 3 minutes, transferred to a 1.5 mL sample tube, and centrifuged to pellet (9,000 g x 3 min). The clear colorless supernatant was removed and the tube was capped with parafilm, punctured with a needle, and placed in the flask to dry under high vacuum overnight.

[0392] The resulting solid intermediate was transferred to a dry 25 mL RBF with a stir bar along with cesium carbonate (48.1 mg, 147.6 pmol) and mPEG11-NHS (101.2 mg, 147.6 pmol). The flask was sealed with a septum, evacuated, flushed with argon, and DMF (2.95 mL) was added. The mixture was protected from light and stirred under argon for 2 hours.

[0393] The crude reaction mixture was subjected to reverse phase chromatography on a 50 g C18 gold column. Fractions containing product were combined, concentrated, redissolved in ACN, and re-concentrated. The residue was dried under high vacuum overnight to give 7.2 mg (16%) of BC-1 as a purple semi-solid.

[0394] MS: found 1645.1, calculated 1644.4 [M+2H] 2+ ; λ abs 378, 545, 754 nm (H20); correction factor: A280 / A754 = 0.11; λ em 760 nm (H20); quantum yield: 8.2% (PBS); extinction coefficient: 132,000 M-1cm-1-1 cm -1 (380 nm, toluene); 104,000 M -1 cm -1 (751 nm, toluene); FWHM: 26 nm.

[0395] Solubility: BC-1 was dissolved in PBS, pH 7.2 (5.8 mg / 0.58 mL) to a final concentration of 10 mg / mL. A portion of this sample (“pre- centrifugation”) was diluted in PBS to 10 mM to take absorbance readings. The initial 10 mg / mL sample was centrifuged in a microcentrifuge at 14,000 g for 10 minutes. A second portion of the centrifuged sample (“post- centrifugation”) was removed and diluted in PBS to 10 mM to take absorbance readings. The data is shown in Table 1 below. No precipitate was observed in the sample. The absorbance values remained consistent (within expected error + / - 10%) from pre- to post- centrifugation. The decrease in absorbance post- centrifugation indicates the presence of insoluble precipitate in the initial sample. Therefore, it was concluded that BC-1 was solubilized in PBS, pH 7.2 at 10 mg / mL.

[0396] Table 1 Absorbance readings for solubility of BC-1

[0397] Wavelength (nm) Corrected absorbance Before centrifugation 754 0.405 542 0.180 377 0.603 After centrifugation 754 0.423 543 0.189 377 0.626

[0398] Example 3

[0399] Synthesis of compound BC-2

[0400] BC-2a was prepared as shown in Scheme 4 (see Figure 4 ). 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 loaded directly onto silica gel and chromatographed [silica, CH2Cl2 / MeOH (0-10%)] to provide a white solid. The resulting solid was found to contain DMAP by1H NMR, so it was re-dissolved in ethyl acetate and washed with 1.0% aqueous HCl, dried over Na2SO4, and concentrated to give BC-2a as a white solid (938 mg, 75%). 1 H NMR found the resulting solid to contain DMAP, so it was re-dissolved in ethyl acetate and washed with 1.0% aqueous HCl, dried over Na2SO4, and concentrated to give BC-2a as a white solid (938 mg, 75%).

[0401] Starting from the previously described dibromobacteriochlorin (BC-SM) (see Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103), as shown in Scheme 5 (see Figure 5 )Prepare BC-2.

[0402] BC-2b. A mixture of BC-SM (135 mg, 200 μmol), tert-butyl 4-ethynylbenzoate (48.5 mg, 240 μmol), Pd(PPh3)4 (23.1 mg, 20.0 μmol), and K2CO3 (276 mg, 2.00 mmol) was placed in a RBF equipped with a 3-way tap. The flask was placed under high vacuum for 1 hour and then degassed by three evacuation-refill cycles. Anhydrous DMF (20 mL) was added via syringe, and the mixture was heated at 80°C for 16 hours. 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 chromatographed [silica gold 12 g, hexane / ethyl acetate (0-40%)] to afford BC-2b (39.4 mg, 25%) as a dark solid.

[0403] BC-2c. A mixture of BC-2b (26.6 mg, 33.4 μmol), BC-2a (79.4 mg, 167 μmol) and (PPh ) PdCl (2.3 mg, 3.34 μmol) was placed in a RBF equipped with a 3-way tap. The flask was placed under high vacuum for 1 hour and then degassed through three evacuation-refill cycles. Anhydrous DMF / TEA (2:1, 10 mL) was added via a syringe, and the reaction mixture was heated at 80° C. for 16 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with aqueous NaHCO , and dried over Na SO . The resulting mixture was concentrated and chromatographed [silica, hexane / ethyl acetate (0-40%)] to afford BC-2c (11.3 mg, 28%) as a dark solid.

[0404] BC-2. BC-2c (4.4 mg, 3.7 μmol) was treated with 4.0 M HCl in dioxane (926 μL). The reaction mixture was stirred at room temperature in the dark under argon. After 4.5 hours, the reaction mixture was placed under high vacuum for 16 hours. CH3(OC2H4) was added to the resulting mixture. 24 CONHS(mPEG 24NHS, 18.0 mg, 14.8 μmol), Cs2CO3(19.3 mg, 59.2 μmol) and DMF (926 μL). The reaction mixture was stirred at room temperature and monitored by LCMS. After 2 hours, the reaction mixture was chromatographed [C1815.5 g, H2O / CH3CN (0-40%)] to give BC-2 as a green solid (10.8 mg, 93%). MS: found 1066.64, calculated 1066.55 [M+3Na] ,3+ M = C 152 H 248 N8O 59 ; λ abs 383, 533, 792 nm (H2O / CH3CN); correction factor: A280 / A791 = 0.24 (H2O / CH3CN); λ em 800 nm (DMF, estimated); extinction coefficient estimated in DMF: 106,000 M -1 cm -1 (791 nm); 83,000 M -1 cm -1 (384 nm); 23,000 M -1 cm -1 (551 nm).

[0405] Example 4

[0406] Synthesis of compound BC-3

[0407] BC-3 was prepared from BC-2 as shown in Scheme 6 (see Figure 6 ). O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU; 0.45 mg, 1.51 μmol) solid was added to a 5 mL vial with a spinning blade. The vial was sealed, evacuated and flushed with argon. BC-2 (4.3 mg, 1.37 μmol) was dissolved in DCM (0.5 mL) and transferred to the reaction vial, followed by the addition of triethylamine (0.29 uL, 2.06 μmol). The reaction mixture was stirred at room temperature for 2 hours. The amino-PEG 12 CH2CH2COOH (4.24 mg, 6.87 μmol) was added directly to the reaction mixture, followed by additional DCM (300 uL), and the mixture was stirred at room temperature under argon in the dark.

[0408] After 16 hours, the reaction was diluted with DCM and washed with saturated aqueous NH4CI (2 mL x 2) and then with water (1 mL x 1). The aqueous layer was removed by pipette and the organic layer was concentrated in a reaction vial. The residue was dissolved in ACN / water and eluted over a 15.5 g C18 Isco gold cartridge. BC-3 was isolated as a dark red semi-solid after complete drying (3.3 g, 65%).

[0409] Example 5

[0410] Synthesis of compound BC-4

[0411] BC-4 was prepared from BC-2 as shown in Scheme 7 (see Figure 7 ). BC-2 was treated with a solution of Zn(OAc)2 2H2O (30 eq) in DMF (4 mM) and heated to 80 °C for 16 hours, after which the reaction mixture was concentrated and purified by reverse phase preparative LC.

[0412] Example 6

[0413] Synthesis of compound BC-5

[0414] BC-5 was prepared as shown in Scheme 8 (see Figure 8 ). BC-5a (i.e., 7-bromo-2,3-dihydro-8-(methoxycarbonyl)-1-(1,1- dimethoxymethyl)-3,3-dimethyl-dipyrrole) was prepared as previously described. See Jiang et al., Org. Biomol. Chem. 2014, 12, 86-103.

[0415] BC-5b. Compound BC-5a (0.771 g, 2.00 mmol) was added to a flame dried 250 mL RBF with a stir bar. The flask was evacuated and flushed with argon. Acetonitrile (111.1 mL) was added via syringe. While stirring, BF3OEt2 (2.96 mL, 24.00 mmol) was added rapidly dropwise near the surface of the solvent. A rapid color change was observed. The flask was shielded from light and stirred at room temperature under a low flow of argon.

[0416] After 16 hours, triethylamine (3.68 mL, 26.4 mmol) was added. The septum was removed and the reaction mixture was stirred until the fuming subsided. The reaction mixture was concentrated and further dried under high vacuum until the flask no longer felt cool to the touch.

[0417] The residue was dissolved in DCM and prepared into a silica cake (5 g). The cake was eluted on an 80 g Si02column with a 20-60% DCM in hexanes gradient for 17 minutes. The main product fractions were combined, concentrated, and dried under high vacuum to give 0.11 g (17%) of BC-5b as a dark red solid.

[0418] BC-5c. BC-5b (56.7 mg, 0.088 mmol) and Zn(OAc)22H2O (579 mg, 2.64 mmol, 30 equiv) were added to a flame dried, argon purged 100 mL RBF with a stir bar. The flask was sealed with a septum, evacuated, and flushed with argon. DMF (13.9 mL) was added and the flask was lowered into an oil bath preheated to 80 °C and stirred under low flow argon overnight.

[0419] After 17 hours, the reaction mixture was diluted with DCM (5x DMF volume) and the organic layer was washed with saturated aqueous NaHC03(3x DMF + DCM volume) until the aqueous layer became clear. The organic layer was dried over Na2S04, filtered, concentrated, and further dried under high vacuum. The residue was transferred to DCM in a 20 mL vial and dried under high vacuum to give 61.6 mg (99%) of a dark red solid. The material was used without further purification.

[0420] BC-5d. To an oven dried 25 mL RBF with a stir bar dried under high vacuum and flushed with argon was added BC-5c (17.7 mg, 25.0 μmol), tert-butyl 4-ethynylbenzoate (12.6 mg, 62.5 μmol, 2.5 equiv), BC-2a (prepared as described above in Example 3; 17.8 mg, 37.5 μmol, 1.5 equiv), Pd(PPh3)2Cl2(4.4 mg, 6.25 μmol, 0.25 equiv), and Cul (2.4 mg, 12.5 μmol, 0.5 equiv). The relative reactivity of the two coupling partners, tert-butyl 4-ethynylbenzoate and BC-2a, was determined by monitoring the reaction product by reverse phase HPLC. The flask was sealed with a septum (the septum was also sealed to the bottom with a parafilm) and evacuated / flushed with argon (3x). Toluene (4.17 mL) was added, stirring was initiated, and triethylamine (2.08 mL) was added. The flask was lowered into an oil bath preheated to 85 °C and stirred under low flow argon.

[0421] After 16 hours, the flask was removed from the oil bath and the residue was dissolved in EtOAc in the flask to a total approximate volume of 12 mL. The reaction mixture was concentrated to dryness, then dissolved in DCM and prepared into a silica cake (2 loads total 475 mg Si02). The cake was eluted on a 12 g Si02column with 20-50% EtOAc in hexanes for 7 minutes. After the first major product eluted, the solvent system was switched to a gradient of 0-5% MeOH in DCM over 5 minutes. The desired product eluted at this stage. The major product fractions were combined, concentrated, and dried under high vacuum to give 18.9 mg (62%) of BC-5d as a green solid.

[0422] BC-5. BC-5d (18.0 mg, 14.72 μmol) was added to an oven-dried 10 mL RBF. The flask was sealed with a septum, evacuated, and flushed with argon. HC1 solution (2.0 mL) was added. The reaction was protected from light and stirred under argon.

[0423] After 4 hours, the flask was placed in a warm water bath and flushed with rapidly flowing argon with an outlet needle vent until all solvent was removed. The vent was then removed, the flask was fitted with a glass adapter and placed under high vacuum overnight. The product was carried forward without further purification.

[0424] The residue (in a 10 mL RBF) was placed under argon. The stir bar was still present. PEG 24 NHS (55.6 mg, 44.16 μmol, 3.0 equiv) solid. The flask was evacuated and flushed with argon. DMF (3.68 mL) was added, followed by a rapid addition of triethylamine (24.6 μL, 176.64 μmol, 12.0 equiv) by pipette. When the reaction was determined to be complete by LCMS, the flask was protected from light and stirred at room temperature for 1.5 hours.

[0425] Zn(OAc)2(81.0 mg, 441.6 μmol, 30.0 equiv) was added and the reaction flask was placed in an oil bath heated to 60 °C. After 2 hours, only a slight conversion was observed. The reaction mixture was heated in the microwave to 100 °C for 10 minutes, then to 110 °C for 20 minutes, after which the reaction was observed to be complete by LCMS. The reaction mixture was concentrated to remove the DMF and the residue was dissolved in 40% ACN in water (1.3 mL) and subjected to reverse phase preparative LC with a gradient of 35-85% ACN in water over 35 minutes. The major product peak was combined, concentrated, transferred to a storage vial in ACN, concentrated, and dried under high vacuum to give 13.3 mg (28% from BC-5d) of BC-5 as a green residue.

[0426] MS: found 1627.4, calculated 626.8 [M+2H] 2+ ; λ abs 345, 595, 839 (CH3CN); λ em 855 nm (CH3CN); FWHM: 35 nm (CH3CN); λ abs 345, 609, 848 (H20); λ em 863 nm (H20); FWHM: 38 nm (H20);

[0427] Example 7

[0428] Synthesis of compound BC-6

[0429] BC-6. The title compound was prepared from BC-5 as shown in Scheme 9 (see Figure 9 ) by adding N,N,N',N'-tetramethyl-O-(N-succinimidyl)urea tetrafluoroborate (TSTU; 0.61 mg, 2.03 μmol) to a 5 mL dry conical vial with a spinning frit. The vial was capped with a septum, evacuated and flushed with argon. BC-5 (6.0 mg, 1.84 μmol) dissolved in DCM (0.75 mL) was added via syringe, followed by the direct addition of triethylamine (0.39 uL, 2.77 μmol) via a pipette. The vial was shielded from light, and the mixture was stirred at room temperature. After 1.5 hours, the completion of NHS ester intermediate formation was confirmed by LCMS. The amino PEG 12 acid was added directly to the reaction vial as a solid in one batch. The vial was resealed, flushed with argon, and stirred at room temperature for 16 hours.

[0430] The DCM was removed and the sample was loaded onto a C18 250 x 20 column using a gradient of 40-70% ACN in water for 30 minutes with 40% CAN in water. The main product fractions were combined, concentrated, and dried under high vacuum to give 5.0 mg (70%) of BC-6 as a green semi-solid. UV-Visible: 346 / 392, 602, >800 nm.

[0431] Example 8

[0432] Synthesis of compound BC-7

[0433] BC-7a. The title compound was prepared from BC-7 as shown in Scheme 10 (see Figure 10The title compound was prepared. TSTU (0.444 g, 1.48 mmol) was added to a solution of 4-ethynylbenzoic acid (0.196 g, 1.34 mmol), triethylamine (0.47 mL, 3.38 mmol), and CH2Cl2(10 mL) at room temperature. The solution was stirred at this temperature for 1 hour, and H-Lys(Boc)-OtBu HCI (0.5 g, 1.48 mmol) was added. The solution was stirred at room temperature overnight, then diluted with CH2Cl2(20 mL). The CH2Cl2solution was washed with saturated aqueous NH4Cl (2 x 20 mL), water (2 x 20 mL), and brine (20 mL). The organic layer was dried, filtered, and concentrated. The crude product was dry-loaded onto silica gel (-1.5 g) and purified on a 12 g Isco column eluting with a gradient of hexanes:EtOAc (100:0 to 1 :1). The desired product was isolated as a light yellow glassy foam that solidified upon extended drying under high vacuum (0.35 g, 61%). LCMS: 7.92 min; MS: 431.

[0434] BC-7b. As shown in Scheme 11 (see Figure 11 ), BC-5c (17.7 mg, 25.0 μmol), BC-2a (23.7 mg, 50.0 μmol), BC-7a (21.5 mg, 50.0 μmol), Pd(PPh3)2Cl2(4.4 mg, 6.25 μmol), and copper(I) iodide (2.4 mg, 12.5 μmol) were added to an oven-dried, argon-purged 20 mL pressure vial with a stir bar. The vial was sealed with a septum and evacuated / filled with argon (3x). Toluene and triethylamine were added and the septum was quickly replaced with a vial cap. The vial was heated at 100 °C in a pre-heated oil bath for 16 hours.

[0435] The vial was cooled and transferred to a 100 mL RBF rinsed with EtOAc. The solvent was removed and the residue was further dried under high vacuum for 30 minutes. The sample was prepared as a silica cake (450 mg) and the cake was eluted on a Si02column (24 g) with a gradient of 0-5% MeOH in DCM for 20 minutes. The main product fractions were combined and concentrated to give 13.9 mg of BC-7b as a dark green solid.

[0436] BC-7. As further shown in Scheme 11 above, BC-7b (13.9 mg, 9.58 μmol) was dried in a 10 mL RBF. A stir bar was added and the flask was sealed with a septum, evacuated and filled with argon (2x). HCI solution (1.9 mL of 4.0 M HCI in dioxane) was added in one portion with stirring. The mixture was stirred at room temperature for 5 hours.

[0437] The flask was then needle degassed, placed in a warm water bath, and flushed with rapidly flowing argon until all liquid had been removed. The needle vent was then removed and the reaction was placed under high vacuum for 2 hours. The flask was flushed with argon and tributylamine (50 pL) was added and the mixture was stirred. Hexanes / THF (2:1, 3 mL) was then added and the solution was sonicated. The suspension was transferred to a 1.5 mL centrifuge tube and centrifuged at 11 K g for 3 minutes. The supernatant was removed. 2:1 hexanes / THF was added to the centrifuge tube which was then sonicated and centrifuged again at 11 K g for 3 minutes. This cycle was repeated once more. The supernatant was removed, the tube was covered with parafilm, punctured with a 20 gauge needle, placed in a 100 mL RBF, and placed under high vacuum for 30 minutes before being flushed with argon. 9.3 mg of a dark red solid was isolated (94%).

[0438] The deprotected product (8.8 mg, 8.53 pmol), mPEG24-NHS (64.4 mg, 51.18 pmol), and cesium carbonate (33.4 mg, 102.36 pmol) were added to an argon purged 10 mL RBF with a stir bar. The flask was evacuated and flushed with argon and DMF (2.13 mL) was added. The reaction was shielded from light and stirred at room temperature under argon. After 1.5 hours, water (0.5 mL) was added and the solution was concentrated. The residue was diluted in water (1 mL) and subjected to reverse phase preparative LC using a gradient of 10-85% ACN in water over 35 minutes. The main product fractions were combined, concentrated, and dried to yield a dark red residue (5.1 mg, 13%).

[0439] The PEGylated intermediate (6.8 mg, 1.52 pmol) was dried in a 4 mL glass vial with a stir bar, Zn(OAc)22H2O (10.0 mg, 45.73 pmol) was added followed by DMF (0.6 mL). The vial was fitted with an adapter with an argon inlet and heated to 60 °C in an oil bath. After 16 hours, the stir bar was removed and the reaction was concentrated. The residue was dissolved in water (0.7 mL) and subjected to reverse phase preparative LC using a gradient of 10-85% ACN in water over 30 minutes. 3.9 mg (57%) of BC-7 was isolated as a dark green semi-solid.

[0440] Example 9

[0441] Synthesis of compound BC-8

[0442] BC-8. As shown in Scheme 12 (see Figure 12) prepare the title compound by BC-5d.In the 10mLRBF with stirring bar, dry BC-5d (18.9mg, 15.46 μmol, prepare as described in Example 6).The flask is sealed with septum, evacuated and flushed with argon (2x).Under stirring, add HCl solution (2.1mL, 4.0M in dioxane) in batches.The mixture is stirred at room temperature for 4.5 hours.Remove solvent (about 30 minutes) by the fast argon gas stream with needle outlet.Then the flask is placed under high vacuum for 16 hours.

[0443] The residue was dissolved in DMF (3.87 mL, 4.0 mM) and triethylamine (25.9 μL, 185.5 μmol) was added. NHSPEG4-(mPEG 12 )3 ester (89.8 mg, 37.1 μmol) was added as a solid. The mixture was stirred for 1 hour.

[0444] Zn(OAc)22H2O (101.8 mg, 463.8 μmol) was added in one portion, and the reaction flask was transferred to an oil bath at 80°C. The reaction was complete after 4 hours. The solvent was removed, and the residue was dissolved in 40% ACN in water (1.4 mL) and purified by reverse-phase preparative chromatography using a 35–85% ACN in water gradient over 35 minutes. The major product, BC-8, was isolated as 12.0 mg of a dark green semisolid after complete drying (14% from BC-5d).

[0445] Example 10

[0446] Synthesis of NIRvana 880 bis-t-butyl ester

[0447] HBC12 diol. Figure 13 As shown, NIRvana 880 di-tert-butyl esters are synthesized as described in route 13. HBC12 (115.7 mg, 179.6 μmol) is added to a flame-dried RBF with a stirring rod. The flask is evacuated and flushed with argon and DCM (18.0 mL, 10 mM) is added. The solution is cooled to -78 ° C and DIBAL-H (1.0 M in toluene, 1.437 mL) is added dropwise within 2 minutes. The stirring reaction is gradually balanced to room temperature. Continue stirring for a total of 4 hours. The reaction mixture is diluted with EtOAc and quenched with a saturated Roschel salt solution. The organic layer is washed with water and brine, dried over sodium sulfate, filtered and concentrated. Isolate the dark green solid that can be used without further purification.

[0448] HBC12 dialdehyde. HBC12 diol (108.2 mg, 183.9 umol), 4 Angstrom molecular sieves (powdered, 92.0 mg, 0.5 mg / umol diol) and N-methylmorpholine (dry, 107.7 mg, 919.5 umol, 5 equiv) were added to a dried 50 mL RBF with a stir bar. The flask was evacuated and flushed with argon and DCM / ACN (9:1, 9.3 mL total, 20 mM) was added followed by the addition of tetrapropylammonium perruthenate (TPAP, 12.9 mg, 36.8 umol, 20 mol%). The flask was covered with foil and stirred at room temperature for 3.5 hours. The reaction mixture was filtered through a pad of celite covered with sand and washed with DCM. The filtrates were combined and dried over sodium sulfate. The solution was filtered, concentrated to dryness and purified on a 40 g silica gel column with 25-65% DCM in hexanes until all desired product was eluted. 36.2 mg (34%) of a red solid was isolated.

[0449] ZnHBC12 dialdehyde. HBC12 dialdehyde (8.0 mg, 13.7 umol) was added to a 25 mL dried RBF with a stir bar and Zn(OAc)22H2O (90.2 mg, 411 umol) was added. The flask was sealed with a septum, evacuated, flushed with argon and DMF (2.74 mL) was added. The flask was lowered into a preheated oil bath. The reaction was heated at 75 °C for 4 hours. The reaction was cooled, diluted with DCM and quenched with saturated aqueous sodium bicarbonate solution. The organic layer was separated and further washed with saturated aqueous sodium bicarbonate solution. The organic layer was dried over sodium sulfate, filtered and concentrated. The product was used without further purification (near quantitative yield of a solid product).

[0450] NIRvana 880 bis-tert-butyl ester. ZnHBC12 dialdehyde (10.0 mg, 15.44 umol), ethynyl benzoic acid tert-butyl ester, Pd(PPh3)2Cl2(2.7 mg, 3.86 umol) and CuI (1.5 mg, 7.72 umol) were added together to a RBF with a stir bar. The flask was sealed with a septum, evacuated and flushed with argon. Toluene / triethylamine (2:1, 3.9 mL total) was added and the reaction was heated at 85 °C for 4 hours. The reaction mixture was concentrated and purified by column chromatography. MS: [M+H] + Calculated 889.3; Found 888.3-890.4 cluster; UV (ACN): 352, 404, 613, 763, 859 nm; em max (ACN): 876 nm.

[0451] Example 11

[0452] Flow cytometry

[0453] Instrumentation Samples were analyzed on a 19-parameter LSR-II SORP flow cytometer (BD Biosciences, San Jose, CA) equipped with 7 lasers (355, 405, 488, 532, 561, 594, and 633 nm) or a LSR Fortessa (BD Biosciences, San Jose, CA) equipped with 5 lasers (355, 405, 488, 561, and 640 nm) and using FACSDiva 8.0 acquisition software. BC-1 data used a 100 mW 355 nm laser with a 690 LP filter and a 780 / 60 BP filter in channel A. Post-experiment analysis was performed using FlowJo software (version 10.0.8, FlowJo, LLC, Ashland, OR).

[0454] Antibody bioconjugation Solutions were prepared in microfuge tubes from 106 μΐ^of 9.4 mg / mL (1.0 mg) anti-human CD8 mouse monoclonal antibody (clone UCHT-4, Leinco Technologies, Inc., St. Louis, MO), 15 μΐ^of 1 M bicarbonate (pH 8.4), and 44 μΐ^of a solution of PEGylated dye NHS ester in PBS (5 to 20 molar equivalents). The tubes were protected from light and gently rotated at room temperature for 1-2 hours. The reaction was quenched by the addition of 15 μΐ^of 200 μΜ Tris at room temperature for an additional hour. The bioconjugates were purified using Sephadex G50M, G75M, or G100M size exclusion chromatography columns eluted with PBS. Antibody bioconjugates prepared from dyes with longer PEG chains (12 units or more) were typically purified using G75M or G100M media. Column fractions were characterized by absorption at 280 nm (protein) and red or NIR dye absorption maxima. The fluorophore-to-protein (F / P) labeling ratio of pooled fractions was determined from these two maxima corrected for dye absorption at 280 nm.

[0455] Cell stainingFrozen stock of human peripheral blood mononuclear cells (PBMCs) were obtained from ZenBio, Inc. (Research Triangle Park, NC, USA; product SER-PBMC-F) and thawed and prepared for staining according to the supplier's instructions. Cells were divided into six 1.5 mL microfuge tubes and centrifuged at 400 x g (2000 rpm) for 5 minutes. Cells were washed three times with wash buffer (PBS with 0.5% BSA) and resuspended in 0.5 mL wash buffer. Aliquots were diluted 1:2 with trypan blue and cell number and viability were determined by counting 4 nL squares on a hemocytometer. Viability was typically >96%. Cells were diluted to 1 x 10 6 / mL using wash buffer and 50 μL (500,000 cells) was aliquoted into microfuge tubes. Typically, the maximum antibody concentration was 4.74 μg / 5 x 10 5 Cells (designated 3.16X) and prepared for semi-log dilutions. For all antibodies except control antibodies, these dilutions were made so that 15 μL was added to the cell aliquot. Cells and antibody were mixed and incubated for 30 minutes at room temperature. Each tube was washed twice with 1 mL wash buffer and then the cells were resuspended in 0.5 mL wash buffer containing 1% formaldehyde. Samples were filtered into flow cytometry tubes through nylon mesh before being characterized by flow cytometry.

[0456] As needed, a positive control bioconjugate was selected from CD8 (UCHT-4)-fluorescein isothiocyanate (FITC) (Leinco Technologies, Inc., St. Louis, MO, USA; cat. #C119). CD4 (RPA-T4)-BUV737 antibody (BD Biosciences, San Jose, CA, USA; catalog number 564306), and / or CD8 (UCHT-4)-DY650 antibody (Leinco Technologies, Inc., St. Louis, MO, USA; catalog number C2064), and titrated with PBMCs by the same general procedure. According to Maecker et al. (2004) Cytometry A 62: 169-173, a staining index (SI) was calculated from mean fluorescence intensity (MFI) values as follows:

[0457] SI = (mean: positive - mean: background) / (2 x S.D. background)

[0458] Table 2 below shows the titration staining index data for the anti-CD8 bioconjugate of BC-1, as well as for an anti-CD8 bioconjugate of polyethyleneglycated bacteriochlorophyll similar to BC-1 (containing only PEG4 chains instead of PEG12 chains). For comparison, an anti-CD8 bioconjugate prepared from FITC (Leinco Technologies, Inc., St. Louis, MO; Catalog No. C119) is also provided.

[0459] Table 2 Titrated staining index data for anti-CD8 dye conjugates

[0460] Dye F / P ratio Maximum staining index PEG4 polyethyleneglycolated bacteriochlorophyll 2.4 21 BC-1 2.7 41 FITC Not determined 63

[0461] These results show that the performance of the polyethyleneglycated design of BC-1 is significantly enhanced compared to the PEG4 polyethyleneglycated bacteriochlorophyll.

[0462] References

[0463] All references listed herein, including but not limited to all patents, patent applications, and publications thereof, as well as scientific journal articles, are hereby incorporated by reference in their entirety as if set forth in their entirety herein for the purpose of supplementing, explaining, providing background, or teaching the methods, techniques, and / or compositions employed herein.

[0464] It will be understood that various details of the presently disclosed subject matter can 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 (II): in: M is a metal or -H, -H; R5, R 10 and R 15 are 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 with 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, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a group 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 R S 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 12 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 .

2. The compound of claim 1, wherein M is Zn.

3. The compound of claim 1 or claim 2, wherein R5, R 10 and R 15 independently selected from H, methoxy and a linking group having the formula: -L1-(X1-L2) p -G.

4. The compound of any one of claims 1 to 3, wherein R3 and R 13 Each is an ester, optionally -C(=O)OCH3.

5. The compound of any one of claims 1 to 4, wherein R2 is 6. The compound of claim 5, wherein each R s is a group having the formula: -X2-(L3) z -R 17 , in: z is 0; X2 is -C(=O)NH-alkylene-NH-; and R 17 -C(=O)C2H4-(OC2H4) m OR 18 , where m is an integer of 12 or greater, and R 18 It's methyl.

7. The compound of claim 6, wherein each R s yes:

8. The compound of claim 5, wherein each R s is a group having the formula: -X2-(L3) z -R 17 , in: z is 1; X2 is -C(=O)NH-alkylene-NH-; L3 is -C(=O)-propylene-C(=O)-NH-; and R 17 Yes – (C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, wherein n is an integer from 1 to 5, optionally 4; and each R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 , wherein m is an integer of 12 or greater, optionally, wherein m is 12; and R 18 It's methyl.

9. The compound of claim 1 or claim 2, wherein R2 and R 12 Not the same or R3 and R 13 Not the same.

10. The compound of claim 9, wherein R2 and R 12 One of the following is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w solubilizing groups, and R2 and R 12 One of them has the formula -L1-(X1-L2) p -G linking group, or, wherein R3 and R 13 One of the following is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w solubilizing groups, and R3 and R 13 One of them has the formula -L1-(X1-L2) p -G linker.

11. The compound of claim 10, wherein R 12 Is a linking group having the formula: -L1-(X1-L2) p -G.

12. The compound of claim 11, wherein R 12 is a group having the formula: -L1-(X1-L2) p -G; wherein p is 0; L1 is an arylalkynylene group; and G is a bioconjugable group.

13. The compound of claim 12, wherein R 12 yes: wherein G is selected from carboxylic acids and active esters.

14. The compound of claim 11, wherein R 12 is a group having the formula: -L1-(X1-L2) p -G; wherein p is 1; L1 is an arylalkynylene group; X1 is -C(=O)NH-; L2 is an alkylene group substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bioconjugable group. The compound of claim 14 , wherein L 1 is —C≡C—(C 6 H 4 )—.

16. The compound of claim 14 or claim 15, wherein L2 is -CH(R)-, wherein R is alkylene-NH-C(=O)-alkylene-(OC2H4) q -OR 16 , wherein q is an integer from 12 to 24 and R 16 It's methyl.

17. The compound of claim 1, wherein the compound is selected from the group consisting of:

18. A composition comprising a covalent conjugate formed between: (a) A compound of formula (II) as defined in claim 1, provided that: R2, R3, R5, R 10 、R 12 、R 13 and R 15 At least one of is a linking group; and (b) one or more of the group consisting of small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones and growth factors.

19. A pharmaceutical composition comprising the compound of claim 1 or the conjugate of claim 18, and a pharmaceutically acceptable carrier.

20. 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 conjugate of claim 18.

21. The method of claim 20, wherein the method comprises using flow cytometry.

22. A method of imaging a cell, tissue or organism, wherein the method comprises using a compound of claim 1 or a conjugate of claim 18.

23. A method of treating a disease in a subject in need thereof, the method comprising: administering to the subject the compound of claim 1 or the conjugate of claim 18 or the pharmaceutical composition of claim 19; as well as illuminating at least a portion of the individual with light, Optionally, the disease is a hyperproliferative disease, further optionally, the disease is cancer.

24. A water-soluble bacteriochlorin dye having a solubility in aqueous solution greater than about 1 mg / ml, optionally having a solubility in aqueous solution of about 3.0 mg / ml or greater; further optionally having a solubility in aqueous solution of about 10 mg / ml or greater.

25. The water-soluble bacteriochlorin dye of claim 24, wherein the dye has an emission wavelength greater than about 850 nanometers.

26. A method for preparing a synthetic intermediate of a compound of formula (II): in: M is a metal or -H, -H; R5, R 10 and R 15 are 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 an alkylene substituted with 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, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a group 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 R S 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 12 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 (II'): in: M is a metal or -H, -H; R5'、R 10 ' and R 15 'Independently selected from H, alkoxy, and R2', R3', R 12 ' and R 13 'Independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, The conditions are: R2', R3', R 12 ' and R 13 At least one of ' 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 (II"): in: M is a metal or -H, -H; R5", R 10 ” and R 15 " is independently selected from H, alkoxy, and R2”, R3”, R 12 ” and R 13 " is independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, The conditions are: R2”, R3”, R 12 ” and R 13 At least one of the 27. A method for preparing an asymmetric bacteriochlorin compound having the formula: in: M is a metal or -H, -H; R5, R 10 and R 15 are independently selected from H and alkoxy; and R2, R3, R 12 and R 13 Independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linking group, and a solubilizing group; wherein the linking group has 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 an alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; G is a bioconjugable group; and 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 R S 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 12 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 and R 12 Not the same or R3 and R 13 are not the same, and 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 the formula: in: M is a metal or -H, -H; R5'、R 10 ' and R 15 ' is independently selected from H and alkoxy; and R2', R3', R 12 ' and R 13 ' is independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl and acetyl, wherein R 2’ and R 12’ Each is halogen, optionally bromine, or wherein R 3’ and R 13’ each is halogen, optionally bromine; and (b) contacting the compound with a palladium catalyst, a base and one of: (i) two different alkynes, optionally wherein the two different alkynes are both compounds having the formula: wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20 is an N-protected alkylamine, a protected carboxylic acid, a -C(=O)-NH-alkylene protected amine, or a -C(=O)-NH-substituted alkylene protected amine, optionally wherein the substituted alkylene of the -C(=O)-NH-substituted alkylene protected amine comprises a protected carboxylic acid substituted alkylene; (ii) two different olefins, optionally wherein the two different olefins are both compounds having the formula: wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20 is an N-protected alkylamine, a protected carboxylic acid, a -C(=O)-NH-alkylene protected amine, or a -C(=O)-NH-substituted alkylene protected amine, optionally wherein the substituted alkylene of the -C(=O)-NH-substituted alkylene protected amine comprises a protected carboxylic acid substituted alkylene; and (iii) two different organic borates; optionally wherein the two different organic borates are two different arylboronic acids or arylboronic acid esters of the formula: wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20 is an N-protected alkylamine, a protected carboxylic acid, a -C(=O)-NH-alkylene-protected amine, or a -C(=O)-NH-substituted alkylene-protected amine, optionally wherein the substituted alkylene of the -C(=O)-NH-substituted alkylene-protected amine comprises an alkylene substituted with a protected carboxylic acid; and each R 21 is H or alkyl or two of R 21 Together they form an alkylene group.

28. The method of claim 27, wherein the ratio of the two alkynes, alkenes, or organoboronates is adjusted to maximize the yield of the desired product based on their relative reactivities, optionally wherein, The less reactive of the two is provided in a greater molar excess than the other of the two compared to the compound of step (a).

29. The process of claim 27 or 28, wherein the yield of the desired product is greater than 50%, optionally wherein the yield of the desired product is greater than about 60%.

30. A compound selected from the group consisting of:

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