Narrow-Emission Dyes, Compositions Containing the Same, and Methods for Preparing and Using the Same

Abstract

The present application provides bacteriochlorophyll derivatives with narrowband emission. In some embodiments, the bacteriochlorophyll derivatives are PEGylated. In some embodiments, the bacteriochlorophyll derivatives have high water solubility (e.g., 10 mg / mL or higher). In some embodiments, the bacteriochlorophyll derivatives are PEGylated and have high water solubility. The bacteriochlorophyll derivatives may comprise a bio-conjugatable group for forming conjugates (e.g., with an antibody or nanoparticle). The bacteriochlorophyll derivatives and their conjugates can be used for imaging and therapeutic applications. Also provided are methods for synthesizing the bacteriochlorophyll derivatives.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Patent Application Serial No. 62 / 850,446, filed on May 20, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical field

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

[0004] Background

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

[0006] The success of the above - mentioned applications depends on many factors, including (1) significant solubility in saline solutions to avoid intermolecular aggregation (and excited - state quenching), (2) minimal non - specific binding to cellular components, (3) addition of a single reactive group for conjugation to avoid cross - linking and mixtures of products, and (4) robust synthesis to provide sufficient quantities for experimentation. However, the large hydrophobic face of bacteriochlorophyll poses 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 above 1 mg / mL), particularly those that can also be readily conjugated with a variety of substances. There has also been a continuing need for additional bacteriochlorophylls that combine improved water solubility with narrow absorption and emission bands.

[0008] Summary

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

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

[0011]

[0012] 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 having the formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1 is a lower alkylene; X1 is -C(=O)NH- or -NHC(=O)-; L2 is a -(CH2CH2O) 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 bio-conjugatable 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 having 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, including the end values, and R S is a group having the formula:

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

[0014] Wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, -C(=O)NH-alkylene-NH-, or triazolyl; L3 is -C(=O)-alkylene-C(=O)-NH-, and R 17 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 a lower alkyl, optionally methyl; R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) mR 18 ; provided that at least one of R2, R3, R 12 and R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0015] 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.

[0016] In some embodiments, R3 and R 13 are each an ester, optionally -C(=O)OCH3. In some embodiments, R2 is

[0017]

[0018] In some embodiments, each R s is a group having the formula: -X2-(L3) z -R 17 , where: z is 0; X2 is -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.

[0019] In some embodiments, each R s is

[0020]

[0021] In some embodiments, each R s is a group having the formula: -X2-(L3) z -R 17 , where: z is 1; X2 is -C(=O)NH-alkylene-NH-; L3 is -C(=O)-propylene-C(=O)-NH-; and R 17 is -(C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, where n is an integer from 1 to 5, optionally 4; and each R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R18 , where m is an integer of 12 or greater, optionally where m is 12; and R 18 is methyl.

[0022] In some embodiments, R2 and R 12 are different or R3 and R 13 are different. In some embodiments, one of R2 and R 12 is a solubilizing group selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , and one of R2 and R 12 is a linking group having the formula -L1-(X1-L2) p -G, or where one of R3 and R 13 is a solubilizing group selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , and one of R3 and R 13 is a linking group having the formula -L1-(X1-L2) p -G.

[0023] In some embodiments, R 12 is a linking group having the following formula: -L1-(X1-L2) p -G. In some embodiments, R 12 is a group having the following formula: -L1-(X1-L2) p -G;

[0024] where p is 0; L1 is an arylalkynylene; and G is a bio-conjugatable group. In some embodiments, R 12 is

[0025]

[0026] where G is selected from carboxylic acid and active ester.

[0027] In some embodiments, R 12 is a group having the following formula: -L1-(X1-L2) p -G;

[0028] Wherein p is 1; L1 is an arylalkynyl group; X1 is -C(=O)NH-; L2 is an alkylene group substituted by one or more groups containing polyoxyethylene chains and / or amide groups; and G is a bio-conjugable group. In some embodiments, L1 is -C≡C-(C6H4)-. In some embodiments, L2 is -CH(R)-, where R is alkylene-NH-C(=O)-alkylene-(OC2H4) q -OR 16 , where q is an integer from 12 to 24 and R 16 is methyl.

[0029] In some embodiments, the compounds are selected from:[[]]

[0030]

[0031]

[0032]

[0033] In some embodiments, the presently disclosed subject matter provides a composition comprising a covalent conjugate formed between: (a) a compound of formula (II), provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15 in which at least one 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.

[0034] 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), provided that R2, R3, R5, R 10 , R 12 , R 13 and R 15 in which at least one 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.

[0035] In some embodiments, the presently disclosed subject matter provides a method for detecting a target, wherein the target is a compound, cell, or particle, and the method comprises labeling the target with a conjugate formed between: (a) a compound of formula (II), provided that: R2, R3, R5, R 10 , R 12 , R 13 and R 15at least one of which is a linking group; and (b) one or more selected from the group consisting of small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors. In some embodiments, the method includes using flow cytometry.

[0036] In some embodiments, the presently disclosed subject matter provides methods for imaging a cell, tissue, or organism, wherein the method includes using a compound of formula (II) or a conjugate formed between: (a) a compound of formula (II), provided that: R2, R3, R5, R 10 、R 12 、R 13 and R 15 at least one of which is a linking group; and (b) one or more selected from the group consisting of small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors.

[0037] In some embodiments, the presently disclosed subject matter provides methods for treating a disease in an individual in need thereof, the method including: administering to the individual a compound of formula (II); a conjugate formed between: (a) a compound of formula (II), provided that: R2, R3, R5, R 10 、R 12 、R 13 and R 15 at least one of which is a linking group; and (b) one or more selected from the group consisting of small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors; 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 cancer.

[0038] In some embodiments, the presently disclosed subject matter provides water-soluble bacteriochlorophyll dyes having a solubility higher than about 1 mg / ml in an aqueous solution, optionally having a solubility of about 3.0 mg / ml or higher in an aqueous solution; further optionally having a solubility of about 10 mg / ml or higher in an aqueous solution. In some embodiments, the dye has an emission wavelength higher than about 850 nanometers.

[0039] In some embodiments, the presently disclosed subject matter provides methods for preparing a synthetic intermediate of a compound of formula (II):

[0040]

[0041] wherein: M is a metal or is -H, -H; R5, R 10 and R 15Independently selected from H, alkoxy, and a linking group having the formula: -L1-(X1-L2) p -G; where p is 0 or 1; L1 is a divalent hydrocarbon radical; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) q -alkylene, alkylene or substituted alkylene, optionally where the substituted alkylene is alkylene substituted with one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bio-conjugatable 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 having the formula -L1-(X1-L2) p -G and a solubilizing group, where the solubilizing group is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , where w is an integer from 0 to 5, inclusive, and R S is a group having the formula: -X2-(L3) z -R 17 , where: 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 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; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; provided that: at least one of R2, R3, R 12 and R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w ; where the method comprises: (a) providing a compound having the formula (II’):

[0042]

[0043] wherein: M is a metal or -H, -H; R5’, R 10 ’ and R 15 ’ are independently selected from H, alkoxy,

[0044] and

[0045] R2’, R3’, R 12 ’ and R 13 ’ are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl,

[0046]

[0047] provided that: at least one of R2’, R3’, R 12 ’ and R 13 ’ is

[0048] and

[0049] (b) contacting the compound provided in step (a) with a dioxane solution containing 4 moles (M) of HCl to provide a compound of formula (II”):

[0050]

[0051] wherein: M is a metal or -H, -H; R5”, R 10 ” and R 15 ” are independently selected from H, alkoxy,

[0052]

[0053] and R2”, R3”, R 12 ” and R 13 ” are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl,

[0054]

[0055] provided that: at least one of R2”, R3”, R 12 ” and R 13 ” is

[0056]

[0057] In some embodiments, the presently disclosed subject matter provides a method for preparing an asymmetric bacteriochlorophyll compound having the following formula:

[0058]

[0059] Wherein: 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 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, linking group and solubilizing group; wherein the linking group has the formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1 is a lower alkylene; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) q -alkylene, alkylene or substituted alkylene, optionally wherein the substituted alkylene is an alkylene substituted by one or more groups containing a polyoxyethylene chain and / or an amide group; G is a bio-conjugatable 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, including the end values, 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 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 a lower alkyl, optionally methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; provided that: R2 and R 12 are not the same or R3 and 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-(Rs ) w ; wherein the method comprises: (a) providing a compound having the following formula:

[0060]

[0061] wherein: 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 ’ are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl and acetyl, wherein R2’ and R 12 ’ are each halogen, optionally bromine, or wherein R3’ and R 13 ’ are each halogen, optionally bromine; and (b) contacting the compound with a palladium catalyst, a base and one of the following: (i) two different alkynes, optionally wherein both of the two different alkynes are compounds having the following formula:

[0062]

[0063] 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(=O)-NH-alkyl-protected amine or -C(=O)-NH-substituted alkyl-protected amine, optionally wherein the substituted alkyl of the -C(=O)-NH-substituted alkyl-protected amine includes a protected carboxylic acid-substituted alkyl; (ii) two different alkenes, optionally wherein both of the two different alkenes are compounds having the following formula:

[0064]

[0065] 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(=O)-NH-alkyl-protected amine or -C(=O)-NH-substituted alkyl-protected amine, optionally wherein the substituted alkyl of the -C(=O)-NH-substituted alkyl-protected amine includes a protected carboxylic acid-substituted alkyl; and (iii) two different organoborate esters; optionally wherein the two different organoborate esters are two different arylboronic acids or arylborate esters of the following formula:

[0066]

[0067] wherein y is an integer from 1 to 5, optionally 1 or 2; and each R 20is an N-protected alkylamine, a protected carboxylic acid, a -C(=O)-NH-alkylidene-protected amine or a -C(=O)-NH-substituted alkylidene-protected amine, optionally wherein the substituted alkylidene of the -C(=O)-NH-substituted alkylidene-protected amine comprises an alkylidene substituted with a protected carboxylic acid; and each R 21 is H or alkyl or wherein two Rs 21 together form an alkylidene.

[0068] In some embodiments, based on the relative reactivity of the two alkynes, alkenes or organoboronic esters, the ratio of the two alkynes, alkenes or organoboronic esters is adjusted to maximize the yield of the desired product, optionally wherein, compared to the compound of step (a), the less reactive of the two is provided in a greater molar excess than the other of the two. 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%.

[0069] Accordingly, an object of the presently disclosed subject matter is to provide water-soluble bacteriochlorophylls, their conjugates and pharmaceutical compositions, and methods of using and preparing them.

[0070] These and other objects are achieved, in whole or in part, by the presently disclosed subject matter. Further, upon study of the following description, drawings, and examples, the objects of the presently disclosed subject matter set forth above, other objects of the presently disclosed subject matter, and advantages will be apparent to those of ordinary skill in the art.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is Route 1, an exemplary route for synthesizing a representative synthetic building block of the dibromo-bacteriochlorophyll derivative of the presently disclosed subject matter.

[0073] Figure 2 is Route 2, an exemplary route for synthesizing compound CP-1.

[0074] Figure 3 is Route 3, an exemplary route for synthesizing compound BC-1.

[0075] Figure 4 is Route 4, an exemplary route for synthesizing compound BC-2a.

[0076] Figure 5 is Route 5, an exemplary route for synthesizing compound BC-2.

[0077] Figure 6 is Route 6, an exemplary route for synthesizing compound BC-3.

[0078] Figure 7It is Route 7, an exemplary route for synthesizing compound BC-4.

[0079] Figure 8 It is Route 8, an exemplary route for synthesizing compound BC-5.

[0080] Figure 9 It is Route 9, an exemplary route for synthesizing compound BC-6.

[0081] Figure 10 It is Route 10, an exemplary route for synthesizing compound BC-7a.

[0082] Figure 11 It is Route 11, an exemplary route for synthesizing compound BC-7.

[0083] Figure 12 It is Route 12, an exemplary route for synthesizing compound BC-8.

[0084] Figure 13 It is Route 13, an exemplary route for synthesizing the ditert-butyl ester of NIRvana 880. DETAILED DESCRIPTION OF THE INVENTION

[0086] The presently disclosed subject matter will now be described more fully hereinafter, in which some, but not all, embodiments of the presently disclosed subject matter are described. In fact, the presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0087] I. Definitions

[0088] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the presently disclosed subject matter.

[0089] 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.

[0090] Unless otherwise defined hereinbelow, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or substitutions of equivalent techniques that are obvious to one of ordinary skill 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.

[0091] In describing the presently disclosed subject matter, it should be understood that numerous techniques and steps are disclosed. Each of these has separate benefits, and each can also be used in combination with one or more or in some cases all of the other disclosed techniques.

[0092] Accordingly, for clarity, this specification will avoid repeating every possible combination of the individual steps in an unnecessary manner. However, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0093] In accordance with long-established patent law practice, the terms "a", "an", and "the" as used in this application (including the claims) refer to "one or more". For example, the phrase "fluorescent microparticles and / or nanoparticles" refers to one or more fluorescent microparticles and / or nanoparticles, including multiple identical fluorescent microparticles and / or nanoparticles. Similarly, the phrase "at least one", when used herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more entities, including but not limited to integer values from 1 to 100 and greater than 100.

[0094] Unless otherwise indicated, all numerical values representing amounts of ingredients, reaction conditions, etc. used in the specification and claims should be understood to be modified in all instances by the term "about". When referring to measurable values such as mass, weight, time, volume, concentration, or percentage, the term "about" means including variations of the specified amount, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1%, because such variations are appropriate for practicing the disclosed methods. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0095] As used herein, the term "and / or" when used in the context of a list of entities refers to the entities present individually or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also any and all combinations and sub-combinations of A, B, C, and D.

[0096] The term "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term that means the presence of the specified elements and / or steps, but that additional elements and / or steps may be added and still fall within the scope of the relevant subject matter.

[0097] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specifically recited. It should be noted that when the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the preamble, it only limits the elements listed in that clause; other elements are not excluded from the claim as a whole.

[0098] 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 and / or which do not materially affect the basic and novel characteristics of the disclosed and / or claimed subject matter. For example, fluorescent microparticles and / or nanoparticles may "consist essentially of a polymer matrix and at least one bacteriochlorophyll associated therewith", meaning that the polymer matrix is the only polymer matrix in which the fluorescent microparticles and / or nanoparticles are present.

[0099] Regarding the terms "comprising / including", "consisting of", and "consisting essentially of", when one of these three terms is used herein, the subject matter disclosed and claimed in the present invention may include the use of any 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 will understand, upon reading this disclosure, that the presently disclosed subject matter thus encompasses fluorescent microparticles and / or nanoparticles consisting essentially of a polymer matrix of the presently disclosed subject matter of the invention and at least one bacteriochlorophyll associated therewith, as well as fluorescent microparticles and / or nanoparticles consisting of a polymer matrix of the presently disclosed subject matter of the invention and at least one bacteriochlorophyll associated therewith.

[0100] As used herein, "halogen" refers to any suitable halogen, including -F, -Cl, -Br, and -I.

[0101] As used herein, "mercapto" refers to the -SH group.

[0102] As used herein, "azido" refers to the -N3 group.

[0103] As used herein, "cyano" refers to the -CN group.

[0104] As used herein, "hydroxy" refers to the -OH group.

[0105] As used herein, "nitro" refers to the -NO2 group.

[0106] As used herein, "alkyl", used alone or as part of another group, refers to a straight-chain or branched-chain hydrocarbon having 1 or from 2 to 10, 20 or 50 carbon atoms (e.g., C1-C4 alkyl; C4-C 10 alkyl; C 11 -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, and the like. As used herein, "lower alkyl" is a subset of alkyl and, in some preferred embodiments, refers to a straight-chain or branched-chain hydrocarbon group having 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, and the like. Unless otherwise specified, the terms "alkyl" or "lower alkyl" are intended to include substituted and unsubstituted alkyl or lower alkyl, and these groups may be substituted with a group selected from: halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic group, 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 -, heterocyclic group-S(O) m -, heterocycloalkyl-S(O) m -, amino, carboxyl, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocyclic amino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, where m = 0, 1, 2 or 3.

[0107] As used herein, "alkylene" refers to a difunctionalized straight-chain, branched-chain or cyclic alkyl, which may be substituted or unsubstituted, and wherein "alkyl" is as defined above.

[0108] As used herein, "alkenyl" used alone or as part of another group refers to a straight or branched chain hydrocarbon having 1 or 2 to 10, 20 or 50 carbon atoms (e.g., C1 to C4 alkenyl; C4 to C 10 alkenyl; C 11 to C 50 alkenyl) (or lower alkenyl having 1-4 carbon atoms), which contains 1-4 double bonds in the straight chain. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadienyl, and the like. Unless otherwise specified, the term "alkenyl" or "lower alkenyl" is intended to include substituted and unsubstituted alkenyl or lower alkenyl, and these groups may be substituted by groups as described above for alkyl and lower alkyl.

[0109] As used herein, "alkenylene" refers to a difunctionalized straight, branched or cyclic alkenyl group, which may be substituted or unsubstituted, and wherein "alkenyl" is defined as above.

[0110] As used herein, "alkynyl" used alone or as part of another group refers to a straight or branched chain hydrocarbon having 1 or 20 to 10, 20 or 50 carbon atoms (e.g., C1 to C4 alkynyl; C4 to C 10 alkynyl; C 11 to C 50 alkynyl) (or lower alkynyl having 1-4 carbon atoms), which contains 1 triple bond in the straight chain. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. Unless otherwise specified, the term "alkynyl" or "lower alkynyl" is intended to include substituted and unsubstituted alkynyl or lower alkynyl, and these groups may be substituted by the same groups as described above for alkyl and lower alkyl.

[0111] As used herein, "alkynylene" refers to a difunctionalized straight, branched or cyclic alkynyl group, which may be substituted or unsubstituted, and wherein "alkynyl" is defined as above.

[0112] As used herein, "alkylidene chain" refers to a difunctionalized straight, branched and / or cyclic organic group, which may be substituted or unsubstituted, which may be saturated or unsaturated, and which may optionally contain one, two or three heteroatoms selected from the following: N, O and S. Examples include, but are not limited to, alkylene, alkenylene, alkynylene, arylene, alkarylene and aralkyl. 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 C 10 ; C 10 to C 20 ; C20 to C 50 )。

[0113] As used herein, "alkoxy," used alone or as part of another group, refers to an alkyl or lower alkyl as defined herein attached to the parent molecular moiety through an oxygen radical -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0114] As used herein, "acyl," 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 as described herein.

[0115] As used herein, "haloalkyl," used alone or as part of another group, refers to at least one halogen as defined herein attached to the parent molecular moiety through 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.

[0116] As used herein, "perhaloalkyl," used alone or as part of another group, refers to an alkyl group in which each hydrogen atom in the alkyl group is replaced by a halogen. In some embodiments, the perhaloalkyl is an alkyl group in which each hydrogen atom in the alkyl group is replaced by fluorine. A representative perhaloalkyl is trifluoromethyl (i.e., -CF3).

[0117] As used herein, "alkylthio," used alone or as part of another group, refers to an alkyl 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.

[0118] As used herein, "aryl" refers to a monocyclic carbocyclic system or bicyclic carbocyclic fused-ring system having one or more aromatic rings. Representative examples of aryl include azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. Unless otherwise specified, the term "aryl" is intended to include substituted and unsubstituted aryl, and these groups may be substituted with the same groups as described above for alkyl and lower alkyl.

[0119] As used herein, "alkylene" is a difunctionalized aryl group, which may be substituted or unsubstituted, and wherein "aryl" is as defined above.

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

[0121] "Alkylidene aryl" and "arylidene alkyl", as used herein alone or as part of another group, refer to bifunctional groups that contain at least one arylene and at least one alkyl, alkenyl, or alkynyl as defined herein.

[0122] "Amino", as used herein, refers to the group -NH2.

[0123] "Alkylamino", as used herein alone or as part of another group, refers to the group -NHR, where R is alkyl.

[0124] "Aralkylamino", as used herein alone or as part of another group, refers to the group -NHR, where R is aralkyl.

[0125] "Disubstituted amino", as used herein alone or as part of another group, refers to the group -NR a R b , where R a and R b are independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic, and heterocycloalkyl.

[0126] "Acylamino", as used herein alone or as part of another group, refers to the group -NR a R b , where R a is acyl as defined herein, and R b is selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic, and heterocycloalkyl.

[0127] "Acyloxy", as used herein alone or as part of another group, refers to the group -OR, where R is acyl as defined herein.

[0128] "Ester", as used herein alone or as part of another group, refers to the -C(O)OR group, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0129] "Formyl", as used herein, refers to the -C(O)H group.

[0130] "Carboxylic acid", as used herein, refers to the -C(O)OH group.

[0131] As used herein, "sulfoxyl" refers to a compound of the formula -S(O)R, where R is any suitable substituent, such as an alkyl, cycloalkyl, alkenyl, alkynyl or aryl group.

[0132] As used herein, "sulfonyl" refers to a compound of the formula -S(O)(O)R, where R is any suitable substituent, such as an alkyl, cycloalkyl, alkenyl, alkynyl or aryl group.

[0133] As used herein, "sulfonate" refers to a compound of the formula -S(O)(O)OR, where R is any suitable substituent, such as an alkyl, cycloalkyl, alkenyl, alkynyl or aryl group.

[0134] As used herein, "sulfonic acid" refers to a compound of the formula -S(O)(O)OH.

[0135] "Amide" as used herein, either alone or as part of another group, refers to -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 groups.

[0136] "Sulfonamide" as used herein, either alone or as part of another group, refers to -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 groups.

[0137] "Urea" as used herein, either alone or as part of another group, refers to -N(R c )C(O)NR a R b group, where R a 、R b and R c are any suitable substituents, such as H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl groups.

[0138] "Alkoxycarbonylamino" as used herein, either alone or as part of another group, refers to -N(R a )C(O)OR b group, where R a 、R b are any suitable substituents, such as H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl groups.

[0139] "Aminoacyloxy" as used herein alone or as part of another group refers to -OC(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.

[0140] "Cycloalkyl" as used herein alone or as part of another group refers to a saturated or partially unsaturated cyclic hydrocarbon group 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 with additional substituents as described herein, such as halogen or lower alkyl. Unless otherwise specified, the term "cycloalkyl" is generic and is intended to include heterocyclic groups as discussed below.

[0141] As used herein, the term "polyoxyethylene chain" refers to a moiety comprising or consisting of poly(ethylene glycol) (PEG) groups (such as a group having the formula -(C2H4O) n -, where 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" can refer to monodisperse or polydisperse PEG chains and straight-chain or branched-chain PEG chains. "Monodisperse" refers to PEG with a polydispersity index (PDI) of 1, while polydisperse refers to PEG with a PDI greater than 1, where PEG includes a Gaussian distribution of chain lengths and molecular weights.

[0142] As used herein, the term "bio-conjugatable 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 an antibody fragment; a polymer; a particle such as a nanoparticle, an organic bead, a polymer bead, or an inorganic bead; the surface of another solid support, etc.) to form a conjugate of one of the presently disclosed bacteriochlorophyll compounds and the other entity. For example, the bio-conjugatable group can be an aldehyde, which can form a covalent bond with an amino group on an amino-substituted biomolecule by reductive amination; or a carboxylic acid, which can be coupled to an amino-substituted biomolecule by carbodiimide activation. The bio-conjugatable 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-iminobiotin, etc.

[0143] The term "microparticle" refers to a structure having at least one region with a size (e.g., length, width, diameter, etc.) less than about 1,000 μm but greater than about 1000 nm. In some embodiments, the size can be less than about 500 μm, in some embodiments less than about 250 μm, in some embodiments less than about 200 μm, in some embodiments less than about 150 μm, in some embodiments less than about 125 μm, in some embodiments less than about 100 μm, in some embodiments less than about 80 μm, in some embodiments less than about 70 μm, in some embodiments less than about 60 μm, in some embodiments less than about 50 μm, in some embodiments less than about 40 μm, in some embodiments less than about 30 μm, in some embodiments less than about 20 μm, in some embodiments less than about 10 μm, and in some embodiments less than about 5 μm. In some embodiments, the size is from about 1 μm to about 250 μm (e.g., 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm).

[0144] Similarly, the term "nanoparticle" refers to a structure having at least one region with dimensions (e.g., length, width, diameter, etc.) less than about 1,000 nm. In some embodiments, the dimensions are smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm or even less than about 20 nm). In some embodiments, the dimensions are 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).

[0145] In some embodiments, the microparticle or nanoparticle is approximately spherical. When the microparticle or nanoparticle is approximately spherical, the characteristic dimension may correspond to the diameter of the sphere. In addition to spherical, the microparticle or nanoparticle can be disc-shaped, plate-shaped (e.g., hexagonal plate), oval, polyhedral, rod-shaped, cubic or irregular in shape.

[0146] 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 coatings 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 coatings, with each successive layer dispersed on the outer surface of a smaller layer closer to the center of the particle.

[0147] The terms "polymer" and "polymeric" refer to chemical structures having repeating units (i.e., multiple copies of a given chemical substructure). Polymers can be formed from polymerizable monomers. A polymerizable monomer is a molecule that contains one or more groups that can react with groups on other polymerizable monomer molecules to form bonds (e.g., covalent or coordination bonds). In some embodiments, each polymerizable monomer molecule can bond to two or more other molecules / groups. In some cases, a polymerizable monomer will only bond to another molecule, forming the end of the polymeric material.

[0148] The polymer can be organic, inorganic, or a combination thereof. As used herein, the term "inorganic" refers to a compound or composition containing at least some atoms other than one of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorus, or a halide. 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.

[0149] As used herein, the term "porphyrin" refers to a cyclic structure typically composed of four pyrrole rings and four nitrogen atoms, as well as two replaceable hydrogens (which can be readily substituted by various metal atoms). A representative porphyrin is hemin.

[0150] As used herein, the term "bacteriochlorophyll" differs from porphyrin in having two partially saturated, non-adjacent (i.e., trans) pyrrole rings. The terms "bacteriochlorophyll" and "bacteriochlorophyll derivative" are used interchangeably herein.

[0151] The phrase "association" refers to any interaction between two entities, such as between a polymer matrix and bacteriochlorophyll. In some embodiments, the polymer matrix and bacteriochlorophyll are associated with each other by non-covalent bonds, such as one or more of hydrophobic, electrostatic, and van der Waals interactions. In some embodiments, due to the polymer matrix (e.g., nanoparticles, microparticles, beads, etc.) containing bacteriochlorophyll, the bacteriochlorophyll is present within the polymer matrix, such that the polymer matrix and bacteriochlorophyll are associated with each other. In such embodiments, the polymer matrix is also referred to as "doped by" or "doped with" bacteriochlorophyll, and the bacteriochlorophyll can be considered to be "embedded" within the polymer matrix. In some embodiments, the polymer matrix and bacteriochlorophyll are associated with each other by covalent bonds (linking the bacteriochlorophyll to the surface of the polymer matrix).

[0152] 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, such as for treating diseases or disorders mediated by hyperplastic tissue or neovascularization, or diseases or disorders involving hyperplastic tissue or neovascularization. As used herein, improving the symptoms of a particular disorder by administering a particular compound or pharmaceutical composition refers to any alleviation attributable to or associated with the administration of the composition, whether permanent or temporary, long-lasting or short-lived.

[0153] As used herein, a "prodrug" is a compound that is metabolized or otherwise converted in vivo, after administration, into a biologically, pharmaceutically, or therapeutically active form of the compound by one or more steps or processes.

[0154] As used herein, an "antibody" generally refers to an immunoglobulin or a fragment thereof that specifically binds to an antigen to form an immune complex. An antibody can be any kind of intact immunoglobulin, e.g., IgG, IgM, IgA, IgD, IgE, chimeric or hybrid antibodies with dual or multiple antigen or epitope specificities. It can be a polyclonal antibody, preferably an affinity-purified antibody from humans or a suitable animal (e.g., primate, goat, rabbit, mouse, etc.). Monoclonal antibodies are also applicable to the presently disclosed subject matter and can be preferred due to their high specificity. They can be readily prepared by the now considered conventional procedures of immunizing a mammal with an immunogenic antigen preparation, fusing immunolymph or spleen cells with an immortalized myeloma cell line, and isolating specific hybridoma clones. Unconventional methods for preparing monoclonal antibodies, such as interspecies fusions of hypervariable regions and genetic engineering manipulations, are not excluded, as it is mainly the antigen specificity of the antibody that affects its utility. More recent monoclonal production techniques can also be used, such as human monoclonal, interspecies monoclonal, chimeric (e.g., human / mouse) monoclonal, genetically engineered antibodies, etc.

[0155] As used herein, the term "antibody" refers to a protein that comprises one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. Immunoglobulin genes generally include kappa (K), lambda (λ), alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ) constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as K or λ. In mammals, heavy chains are classified as γ, μ, α, δ, or ε, 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., the so-called IgY produced by certain avians, which is the type of immunoglobulin that hens deposit in their egg yolks), which are similarly included in the presently disclosed subject matter.

[0156] A known representative immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (average molecular weight of about 25 kilodaltons (kDa)) and one "heavy" chain (average molecular weight of about 50 - 70 kDa). The two pairs of identical polypeptide chains are held together in a dimer form by disulfide bonds present in the heavy chain region. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. The term variable light chain (V Land the variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0157] Antibodies typically exist as intact immunoglobulins or as a number of well-characterized fragments that can be generated by digestion with various peptidases. For example, an antibody molecule is digested with papain to cleave the antibody at a position near the N-terminus of the disulfide bond. This produces three fragments: two identical "Fab" fragments that have the N-terminus of the light and heavy chains, and an "Fc" fragment that includes the C-terminus of the heavy chain held together by disulfide bonds. On the other hand, pepsin digests the antibody near the C-terminus of the disulfide bond in the hinge region, producing a fragment called the "F(ab)′2" fragment, which is a dimer of Fab fragments linked by disulfide bonds. The F(ab)′2 fragment can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the F(ab′)2 dimer into two "Fab′" monomers. The Fab′ monomer is essentially a Fab fragment with a portion of the hinge region. With respect to these various fragments, the Fab, F(ab’)2, and Fab′ fragments include at least one intact antigen-binding domain (referred to as a "paratope") and are thus capable of binding an antigen.

[0158] Although the various antibody fragments are defined in terms of digestion of intact antibodies, one of ordinary skill in the art will understand that the various fragments, including but not limited to Fab′ fragments, can be synthesized de novo chemically or by using recombinant DNA methods. Thus, as used herein, the term "antibody" also includes antibody fragments produced by modifying intact antibodies or antibody fragments synthesized de novo using recombinant DNA methods. In some embodiments, the term "antibody" includes fragments having at least one antigen-binding domain.

[0159] The antibodies, fragments, and derivatives of the presently disclosed subject matter may also include chimeric antibodies. As used herein in the context of antibodies, the term "chimeric" and its grammatical variants refer to antibody derivatives having a constant region and a variable region, where the constant region is substantially or entirely derived from the antibody constant region of one species and the variable region is substantially or entirely derived from the sequence of the variable region of another species. A particular type of chimeric antibody is a "humanized" antibody, where the antibody is produced by replacing the complementarity-determining regions (CDRs) of a human antibody, for example, with the CDRs of a mouse antibody (see, e.g., PCT International Patent Application Publication No. WO 1992 / 22653). Thus, in some embodiments, a humanized antibody has a constant region and a variable region that are substantially or entirely derived from the corresponding human antibody regions except for the CDRs, and CDRs that are substantially or entirely derived from a mammal other than human.

[0160] 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 the amino acid sequences having at least one variable region and / or CDR of the intact antibodies described herein, but lack some or all of the constant domains of those antibodies. These constant domains are not required for antigen binding, but constitute a major part of the intact antibody structure.

[0161] Single-chain antibody fragments can overcome some problems associated with using antibodies containing partial or all of the constant domains. For example, single-chain antibody fragments tend to have no unwanted interactions between biomolecules and the heavy-chain constant region, or other unwanted biological activities. Additionally, single-chain antibody fragments are much smaller than intact antibodies and thus can have greater capillary permeability than intact 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 on a relatively large scale, facilitating their production. In addition, the relatively small size of single-chain antibody fragments makes them less likely to elicit an immune response in recipients than intact antibodies. Single-chain antibody fragments of the presently disclosed subject matter include, but are not limited to, single-chain fragment variable (scFv) antibodies and their derivatives, such as, but not limited to, tandem di-scFv, tandem tri-scFv, diabodies (including bispecific diabodies), triabodies, tetra-bodies, miniantibodies, minibodies, tetravalent bispecific molecules, bispecific F(ab′)2 fragments, and the like.

[0162] As used herein, "infectious agent" refers to an invading microorganism or parasite. As used herein, "microorganism" refers to viruses, bacteria, rickettsiae, mycoplasmas, protozoa, fungi, and similar microorganisms, and "parasite" refers to an infectious, generally microscopic or very small multicellular invertebrate, or its egg cells or juvenile forms, such as malaria parasites, spirochetes, etc., that are sensitive to antibody-induced clearance or lysis or phagocytic destruction.

[0163] As used herein, "tumor" refers to a neoplasm and includes both benign and malignant tumors. The term specifically includes malignant tumors that can be solid (such as breast cancer, liver cancer, or prostate cancer) or non-solid (e.g., leukemia). Tumors can also be further classified into subtypes, such as adenocarcinomas (e.g., adenocarcinomas of the breast, prostate, or lung).

[0164] As used herein, "target" refers to an object that is intended to be detected, diagnosed, impaired, or destroyed by the methods provided herein, and includes target cells, target tissues, and target compositions.

[0165] As used herein, "target tissue" and "target cell" are those tissues intended to be damaged or destroyed by the treatment method. The photosensitive compound binds to or accumulates in these target tissues or target cells; then when sufficient radiation is applied, these tissues or cells are damaged or destroyed. Target cells are the cells within the target tissue, and target tissues include but are not limited to tumors, solid tumors (such as (but not limited to) tumors of the head and neck, tumors of the eye, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lung), vascular endothelial tissues of non-solid tumors, abnormal blood vessel walls; as well as malignant cells of hematopoietic and lymphoid tissues, neovascular tissues; other lesions of the vascular system, bone marrow, and tissues or cells related to autoimmune diseases. Target cells also include cells that undergo significantly faster division compared to non-target cells.

[0166] As used herein, "non-target tissue" is all tissues of the subject that are 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 that have not been otherwise identified as targets.

[0167] As used herein, "target composition" is those compositions intended to be damaged or destroyed by the treatment method, and may include one or more pathogens, including but not limited to bacteria, viruses, fungi, protozoa, and toxins, as well as cells and tissues infected or infiltrated by them. The term "target composition" also includes but is not limited to infectious organic particles, such as prions, toxins, peptides, polymers, and other compounds, which can be selectively and specifically identified as organic targets intended to be damaged or destroyed by this treatment method.

[0168] As used herein, "hyperplastic tissue" refers to tissue with uncontrolled growth, and includes neoplastic tissue, tumors, and unrestrained blood vessel growth, such as the blood vessel growth found in age-related macular degeneration and commonly occurring after glaucoma surgery.

[0169] As used herein, "hyperplastic disorder" denotes those disease conditions that have unregulated or abnormal cell growth causing excessive cell proliferation as a potential pathology, and includes uncontrolled angiogenesis. Examples of such hyperplastic disorders include but are not limited to cancer or carcinoma, acute and membranoproliferative glomerulonephritis, myeloma, psoriasis, atherosclerosis, psoriatic arthritis, rheumatoid arthritis, diabetic retinopathy, macular degeneration, corneal neovascularization, choroidal hemangioma, recurrence of pterygium, and scar formation resulting from excimer laser surgery and glaucoma filtration surgery.

[0170] As used herein, "therapeutically effective dose" is a dose sufficient to prevent disease progression or cause disease regression or capable of alleviating the symptoms caused by the disease.

[0171] As used herein, "biomaterial" refers to tissues (such as biopsy tissues) and cells, as well as biological fluids such as blood, urine, plasma, cerebrospinal fluid, mucus, saliva, and the like.

[0172] As used herein, "irradiating" and "irradiation" include exposing an individual to light of all wavelengths. Preferably, the irradiation wavelength is selected to match the wavelength that excites the photosensitive compound. Preferably, the radiation wavelength matches the excitation wavelength of the photosensitive compound and has low absorption by non-target tissues of the individual (including blood proteins).

[0173] Irradiation is further defined herein by its coherence (laser) or incoherence (non-laser), as well as intensity, duration, and timing related to the administration of the photosensitive compound. The intensity or fluence rate must be sufficient for the light to reach the target tissue. The duration or total fluence dose must be sufficient to make the photosensitive compound sufficiently photosensitized to act on the target tissue. The timing related to the administration of the photosensitive compound is important because 1) the administered photosensitive compound takes some time to localize to the target tissue, and 2) the blood levels of many photosensitive compounds decrease over time. The radiation 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, an optical fiber, or by ingestion of a capsule or pill form of the light source (e.g., as disclosed in U.S. Patent No. 6,273,904).

[0174] While some embodiments of the presently disclosed subject matter relate to using light energy to perform photodynamic therapy (PDT) to destroy tumors, other forms of energy are also within the scope of the presently disclosed subject matter, as would be understood by one of ordinary skill in the art. Such forms of energy include, but are not limited to: thermal energy, acoustic energy, ultrasonic energy, chemical energy, light energy, microwave energy, ionizing energy (such as x-rays and gamma rays), mechanical energy, and electrical energy. For example, sonodynamically induced or activated agents include, but are not limited to: gallium-porphyrin complexes (see Yumita et al. (1997) Cancer Letters 112:79-86), other porphyrin complexes such as protoporphyrin and hematoporphyrin (see Umemura et al. (1996) Ultrasonics Sonochemistry 3:S187-S191); other anti-cancer drugs used in the presence of sonotherapy, such as daunorubicin and doxorubicin (see Yumita et al. (1987) Japanese Journal of Hyperthermic Oncology 3(2):175-182).

[0175] As used herein, a "coupling agent" refers to an agent capable of coupling a photosensitizer to a targeting agent.

[0176] "Targeting agent" refers to a compound that localizes to or preferentially associates or binds to a specific 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-targeted liposomes.

[0177] As used herein, "specific binding pair" and "ligand-receptor binding pair" refer to two different molecules, where one molecule has a region on a surface or in a cavity that specifically attracts or binds to a specific spatial or polar organization of the other molecule, resulting in an affinity between the two molecules 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 that is sufficient to effect binding 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-α and tumor necrosis factor-receptor, and interferon and interferon receptor; avidin and biotin or streptavidin; antibody and antigen pairs; enzymes and substrates, drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; a nucleic acid strand and a complementary oligonucleotide; interleukins and interleukin receptors; and stimulatory factors and their receptors, such as granulocyte-macrophage colony-stimulating factor (GMCSF) and GMCSF receptor and macrophage colony-stimulating factor (MCSF) and MCSF receptor.

[0178] "Linker" is an aromatic or aliphatic group (which may be substituted or unsubstituted and may optionally contain heteroatoms such as N, O, or S) that is used to couple a bio-conjugatable group, cross-coupling group, surface-linking group, hydrophilic group, etc. to a parent molecule. Examples include, but are not limited to, aryl, alkyl, heteroaryl, heteroalkyl (e.g., oligoethylene glycol), peptide, and polysaccharide linkers, etc.

[0179] Individuals treated by the methods of the presently disclosed subject matter for diagnostic or therapeutic purposes include human individuals and other animal individuals for veterinary purposes (particularly mammalian individuals such as dogs, cats, horses, monkeys, chimpanzees, etc.).

[0180] More specifically, as used herein, the terms "individual", "patient", and "recipient" may be used interchangeably and may refer to a member of any invertebrate or vertebrate species. Thus, the term "individual" is intended to encompass any member of the animal kingdom, including but not limited to members of the phylum Chordata (e.g., class Osteichthyes (bony fish), class Amphibia (amphibians), class Reptilia (reptiles), class Aves (birds), and class Mammalia (mammals)), and all orders and families contained therein.

[0181] The compositions and methods of the presently disclosed subject matter are particularly applicable to warm-blooded vertebrates. Accordingly, the presently disclosed subject matter relates to mammals and birds. More specifically, provided are compositions and methods derived from and / or for: mammals such as humans and other primates, as well as those mammals that are important due to being endangered (such as the Siberian tiger), 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 (piglets, pigs, and wild boars), ruminants (such as cows, bulls, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, hamsters, guinea pigs, and rabbits), marsupials, and horses. Also provided are uses of the disclosed methods and compositions on birds (including those species of birds that are endangered, kept in zoos, or as pets (such as parrots, cockatoos, etc.), and flying birds, more particularly domesticated flying birds, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, etc., as they are also of economic importance to humans). Accordingly, also provided are uses of the disclosed methods and compositions on livestock, including but not limited to domesticated swine (piglets and pigs), ruminants, horses, poultry, etc.

[0182] II. Bacteriochlorophyll Compounds

[0183] In some embodiments, the presently disclosed subject matter provides water-soluble bacteriochlorophylls, wherein the bacteriochlorophyll has a solubility of about 1 milligram per milliliter (mg / mL) or higher in an aqueous solution (such as water, saline, PBS, etc.). For example, water solubility is provided by adding a solubilizing group containing a PEG chain at the β-pyrrole position. In some embodiments, the PEG chain is linked to the bacteriochlorophyll through a different group and / or is longer than the PEG chains linked to previously described polyethylene glycolated bacteriochlorophyll compounds. In some embodiments, the bacteriochlorophyll has a solubility of about 3.0 mg / mL or higher in an aqueous solution. In some embodiments, the bacteriochlorophyll has a solubility of about 5.0 mg / mL or higher in an aqueous solution. In some embodiments, the bacteriochlorophyll has a solubility of about 10 mg / mL or higher in an aqueous solution. In some embodiments, the bacteriochlorophyll has a solubility of about 500, about 600, about 700, about 800, or about 900 μM or higher in an aqueous solution. In some embodiments, the bacteriochlorophyll has a solubility of about 1, 1.5, 2, 2.5, or 3 mM or higher in an aqueous solution.

[0184] In some embodiments, the bacteriochlorophyll has an emission wavelength higher than about 700 nm. In some embodiments, the bacteriochlorophyll has an emission wavelength higher than about 800 nm. In some embodiments, the bacteriochlorophyll has an emission wavelength of about 850 nm or greater.

[0185] In some embodiments, the water-soluble bacteriochlorophyll comprises a linker moiety containing a bio-conjugatable group that can be used to conjugate the bacteriochlorophyll to another substance, such as 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 bio-conjugatable group can include, for example, a carboxylic acid or an active ester, a hydroxyl group, an amine, a thiol, or an aldehyde. In some embodiments, the linker moiety further includes an arylene and an alkylene group. In some embodiments, the linker moiety includes an arylene and / or an alkynylene group close to the main bacteriochlorophyll structure, while the alkylene group is close to the bio-conjugatable group.

[0186] In some embodiments, the bacteriochlorophyll comprises at least one solubilizing group. In some embodiments, the solubilizing group includes 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 contain at least 4 -CH2CH2O- repeating units. In some embodiments, the PEG chains contain at least 6, 8, 10, or 12 -CH2CH2O- repeating units. Thus, the solubilizing group can comprise two PEG6, PEG8, PEG10, or PEG12 groups. In some embodiments, the PEG chains contain 12 or more -CH2CH2O- repeating units (e.g., about 12 to about 24 or about 28 -CH2CH2O- repeating units). In some embodiments, the compound comprises solubilizing groups attached to two different pyrrole carbons. In some embodiments, each of the two solubilizing groups comprises two PEG chains.

[0187] In some embodiments, the solubilizing group is a β-pyrrole substituent that comprises an arylene or alkynyl-arylene group directly attached to a bacteriochlorophyll pyrrole carbon atom, provided that the group does not contain an oxo linker directly between the PEG chain and the aryl group. In some embodiments, the solubilizing group contains one or more amide bonds between the aryl group and the PEG chain. In some embodiments, the amide bond further comprises one or more alkylene spacers (e.g., ethylene, propylene, etc.).

[0188] In some embodiments, the bacteriochlorophyll is a compound of formula (II):

[0189]

[0190] Wherein:

[0191] M is a metal or -H, -H;

[0192] R5, R 10 and R 15 are independently selected from H, alkoxy, and a linking group having the formula: -L1-(X1-L2) p -G, where p is 0 or 1; L1 is a hydrocarbon group; X1 is -C(=O)NH- or -NHC(=O)-; L2 is a -(CH2CH2O) q -alkylene, alkylene or substituted alkylene (e.g., alkylene substituted by one or more groups containing a polyethylene oxide chain and / or an amide group); and G is a bio-conjugatable group; and

[0193] 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 having the formula -L1-(X1-L2) p -G and a solubilizing group, where the solubilizing group is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , where w is an integer from 0 to 5, inclusive, where when w is 0, the chlorin derivative is water-insoluble (i.e., hydrophobic), and where when w is 1, 2, 3, 4 or 5, the chlorin derivative is water-soluble (i.e., hydrophilic), and R S is a group having the formula:

[0194] -X2-(L3) z -R 17 ,

[0195] where: 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 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 (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 a lower alkyl (e.g., methyl); and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; provided that: at least one of R2, R3, R 12 and R 13 is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w .

[0196] 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 replaced by -H, -H. Thus, the compounds of formula (II) include metal bacteriochlorins and free base bacteriochlorins. In some embodiments, M is Zn.

[0197] 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. In some embodiments, R5 is methoxy. In some embodiments, one of R 10 and R 15 is a linking group. In some embodiments, R 15 is a linking group. Alternatively, in some embodiments, R 12 is a linking group.

[0198] In some embodiments, for the linking group -L1-(X1-L2) p -G, the linking group p is 0. In some embodiments, G is a carboxylic acid or an active ester (e.g., NHS ester). In some embodiments, L1 is an alkynylene, arylene (e.g., phenylene), or a divalent group containing both alkynylene and arylene (i.e., arylalkynylene). In some embodiments, L1 is -C≡C-phenyl- or -C≡C-alkylene-(e.g., -C≡C-(CH2)4-). In some embodiments, the linking group is:

[0199]

[0200] Optionally, wherein G is a carboxylic acid (i.e., -C(=O)OH or an active ester.

[0201] In some embodiments, the linking group is:

[0202]

[0203] Optionally, wherein G is a carboxylic acid or a reactive ester thereof.

[0204] In some embodiments, p is 1, and the linking group comprises a polyoxyethylene chain to improve solubility and / or comprises a spacer to improve the reactivity of G compared to the reactivity of G (where G is directly linked to L1) in a comparable bacteriochlorophyll. In some embodiments, L1 is a phenylene or -C≡C-phenyl-, p is 1, X1 is -C(=O)NH-, L2 is an alkylene, and G is a carboxylic acid or a reactive ester (e.g., an NHS ester). In some embodiments, L2 is an ethylene group. Thus, the linking group may comprise a β-alanine spacer to improve the reactivity of G.

[0205] In some embodiments, p is 1; L1 is an arylalkynyl group (e.g., -C≡C-(C6H4)-); 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 bio-conjugatable 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 an ethylene group. In some embodiments, L2 is a methylene group 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 PEG12-PEG25 chains. In some embodiments, R is -(CH2)4-NH-C(=O)-CH2CH2-(OC2H4) 24 OMe. In some embodiments, the linking group is:

[0206]

[0207] or a reactive ester thereof, optionally wherein PEG is PEG12. In some embodiments, the linking group is:

[0208]

[0209] or a reactive ester thereof, optionally wherein PEG is PEG24.

[0210] In some embodiments, the bacteriochlorophyll is 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 them (e.g., R2) is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w solubilizing groups, and the other of R2 and R 12 (e.g., R 12 ) is a linking group having the formula -L1-(X1-L2) p -G or a solubilizing group having a different structure (e.g., different from the solubilizing group of R2). Alternatively, in some embodiments, one of R3 and R 13 is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w solubilizing groups, and the other of R3 and R 13 is a linking group having the formula -L1-(X1-L2) p -G or a solubilizing group of different structure. In some embodiments, one of R2 and R 12 is a solubilizing group and the other is a linking group, or one of R3 and R 13 is a solubilizing group and the other is a linking group.

[0211] In some embodiments, R3 and R 13 are each an ester. In some embodiments, R3 and R 13 are each a methyl ester, i.e., -C(=O)OCH3.

[0212] In some embodiments, R2 is

[0213]

[0214] In some embodiments, each R s is a group having the following formula:

[0215] -X2-(L3) z -R 17 ,

[0216] where: z is 0; X2 is -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. 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). In some embodiments, each R sYes

[0217]

[0218] In some embodiments, m is 24.

[0219] In some embodiments, each R s is a group having the formula:

[0220] -X2-(L3) z -R 17 ,

[0221] wherein: z is 1; X2 is -C(=O)NH-alkylene-NH-; L3 is -C(=O)-propylene-C(=O)-NH-; and R 17 is (C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, where 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 , where 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.

[0222] 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., compounds having the following structures:

[0223]

[0224]

[0225]

[0226] 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, provided that: R2, R3, R5, R 10 , R 12 , R 13 , and R 15at least one of which is a linking group; and (b) one or more of the group consisting of small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors. In some embodiments, for example, a conjugate can be formed by reacting a compound of formula (II) containing a linking group having a carboxylic acid or an active ester (i.e., as a bio-conjugatable group G) with an amino group of a small molecule, peptide, protein, antibody, or polymer.

[0227] III. Synthesis Methods

[0228] Methods for synthetic bacteriochlorophylls that are suitable for preparing the presently disclosed bacteriochlorophylls are described, for example, in U.S. Pat. Nos. 8,664,260 and 8,980,565, each of which is incorporated herein by reference in its entirety. In some embodiments, the presently disclosed compounds of formula (II) can be prepared by preparing a suitable trans-β-substituted bacteriochlorophyll (such as a bacteriochlorophyll in which two β-bacteriochlorophyll substituents are halogen (e.g., Br) substituents), and then further reacting the β-substituents to replace them with suitable water-soluble groups. Methods for synthesizing trans-β-substituted bacteriochlorophylls have been previously described. See, e.g., Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103.

[0229] For example, in some embodiments, the bacteriochlorophylls of formula (II) can be prepared by self-condensing a dipyrromethene synthetic building block or condensing a pair of dipyrromethene synthetic building blocks in an organic solvent in the presence of an acid. In some embodiments, the dipyrromethene synthetic building block can have the following structure:

[0230]

[0231] wherein R is an acetal or an aldehyde group; and S1, S2, S3, S7, and S6 are each independently selected from H, aryl, substituted aryl, phenyl, cycloalkyl, alkyl, substituted alkyl, alkenyl, alkynyl, halogen, alkoxy, alkylthio, perfluoroalkyl, perfluoroaryl, pyridyl, cyano, thiocyanato, nitro, amino, alkylamino, acyl, sulfinyl, sulfonyl, imino, ester, amido, and carbamoyl, and wherein S4 and S5 are each H or together form a covalent bond. In some embodiments, at least one of S1 and S2 is a halogen.

[0232] More specifically, methods for preparing dibromo-substituted bacteriochlorins and their corresponding pyrrole synthesis building blocks have been previously described. See Jiang et al. (2014) Organic & Biomolecular Chemistry 12: 86-103. For example, a bacteriochlorin containing two bromine substituents at the 2 and 12 positions of the bacteriochlorin can be prepared from a synthesis building block prepared from an N-protected 3,4-dibromopyrrole such as that shown in Scheme 1 (see Figure 1 ).

[0233] As shown in Scheme 1 (see Figure 1 ), the N-protected 3,4-dibromopyrrole (e.g., 3,4-dibromo-(N-triisopropylsilyl)pyrrole) is treated with a base such as an alkyllithium (e.g., tert-butyllithium) and dimethyl carbonate and then deprotected to give 3-bromo-4(-methoxycarbonyl)pyrrole (a). Vilsmeier formylation of a (e.g., using POCl3-DMF) gives aldehyde b. Aldehyde b can be treated in nitromethane with potassium acetate and a slight excess of methylamine hydrochloride to give an aldol condensation product c. Reduction of the carbon-carbon double bond in c using a suitable reducing agent (e.g., NaBH4) gives 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. The reduction cyclization reaction of the Michael addition product e, by first deprotonating e (e.g., by treatment with anhydrous sodium methoxide) to give a nitrogenate anion intermediate and then cyclizing the intermediate with a deoxygenating agent (e.g., an aqueous TiCl3 buffer solution), provides the dihydropyrrolobacteriochlorin synthesis 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 and then cyclizing the intermediate with a deoxygenating agent (e.g., (Ti(0), Zn, NaOH / methanol; Zn, aqueous NH4Cl / THF; FeSO4, aqueous NH4Cl / CH3CN; Mg or Fe, AcONH4 / methanol; Ph3P / toluene; S / toluene; NaN3 / toluene, Zn, NaI, Me3SiCl / CH3CN; etc.).

[0234] BB can self-condense in the presence of an acid (e.g., a Brønsted 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 afford bacteriochlorophyll. The condensation can be carried out in an organic solvent such as acetonitrile (ACN), dichloromethane (DCM), chloroform, tetrahydrofuran (THF), chlorobenzene, ethanol, and combinations thereof. Optionally, for example, when the bacteriochlorophyll synthetic building block does not contain a carbon-carbon double bond between the heterocycles, an oxidant such as air or DDQ can be included in the condensation reaction mixture. In some embodiments, bacteriochlorophyll can have the following structure:

[0235]

[0236] The halogen substituents of dihalobacteriochlorophylls (such as the bacteriochlorophylls described above) can be further elaborated using coupling chemistries 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 solubilizing groups. For example, dihalobacteriochlorophyll can react with boric acid in the presence of a Pd(0) catalyst; react with an organotin compound in the presence of a Pd catalyst; react with a pseudohalide or an organosilane in the presence of a Pd catalyst; react with an organozinc compound in the presence of a Ni or Pd catalyst, or react with a Grignard reagent in the presence of a Ni or Pd catalyst. In some embodiments, dihalobacteriochlorophyll (e.g., dibromobacteriochlorophyll, e.g., the bacteriochlorophyll free base shown above) can react with an arylboronic acid Suzuki coupling reaction partner. See Jiang et al. (2015) New Journal of Chemistry 39(7):5694 - 5714; and Zhang et al. (2016) New Journal of Chemistry 40(9):7750 - 7767. In some embodiments, the arylboronic acid can contain additional chemical functional groups or protected chemical functional groups 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 can react with a suitable PEG reagent (e.g., an activated PEG ester) after deprotection.

[0237] In some embodiments, the presently disclosed compounds can use a tert-butoxycarbonyl (BOC) protecting group for the amino group present on a Suzuki or other type of coupling agent, optionally in combination with tert-butyl ester protection of a carboxylic acid (e.g., in a linking group). Surprisingly, it was found that using trifluoroacetic acid (TFA) for deprotection of the BOC group (a common BOC deprotection method) during the preparation of the presently disclosed compounds resulted in significant decomposition, for example, in compounds containing an alkyne bond. Thus, in some embodiments, other conditions (e.g., 4M HCl in dioxane) are used for BOC deprotection.

[0238] In some embodiments, the presently disclosed subject matter provides methods for preparing asymmetric water-soluble bacteriochlorophylls, wherein the methods include performing a mixed (or hetero-) coupling reaction. For example, the method can include providing a dihalobacteriochlorophyll (e.g., a symmetric dibromobacteriochlorophyll) in which two trans-β-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 dihalobacteriochlorophyll with two different alkynes, two different alkenes, or two different organoboronates (e.g., boric acid 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 trialkylamine, such as triethylamine, or sodium acetate or potassium acetate).

[0239] In some embodiments, the presently disclosed subject matter provides methods for preparing asymmetric bacteriochlorophyll compounds having the following formula:

[0240]

[0241] Wherein:

[0242] M is a metal or is -H, -H;

[0243] R5, R 10 and R 15 are independently selected from H and alkoxy; and

[0244] R2, R3, R 12 and R 13 are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, linking group, and solubilizing group;

[0245] Wherein the linking group has the following formula:

[0246] -L1-(X1-L2) p -G,

[0247] wherein p is 0 or 1; L1 is a lower alkylene group; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) q -alkylene, alkylene or substituted alkylene, optionally wherein the substituted alkylene is an alkylene substituted by one or more groups containing a polyoxyethylene chain and / or an amide group; and G is a bio-conjugatable group;

[0248] 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:

[0249] -X2-(L3) z -R 17 ,

[0250] 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 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 a lower alkyl group, optionally methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ;

[0251] provided that: R2 and R 12 are not the same or R3 and 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:

[0252]

[0253] Wherein:

[0254] M is a metal or -H, -H;

[0255] R5’, R 10 ’ and R 15 ’ are independently selected from H and alkoxy; and

[0256] R2’, R3’, R 12 ’ and R 13 ’ are independently selected from H, cyano, halogen, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl and acetyl, wherein R2’ and R 12 ’ are each halogen, optionally bromine, or wherein R3’ and R 13 ’ are each halogen, optionally bromine; (b) contacting the compound with a palladium catalyst, a base and one of the following: (i) two different alkynes, optionally wherein both of the two different alkynes are compounds having the following formula:

[0257]

[0258] 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(=O)-NH-alkyl-protected amine or -C(=O)-NH-substituted alkyl-protected amine, optionally wherein the substituted alkyl of the -C(=O)-NH-substituted alkyl-protected amine includes a protected carboxylic acid-substituted alkyl; (ii) two different alkenes, optionally wherein both of the two different alkenes are compounds having the following formula:

[0259]

[0260] 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(=O)-NH-alkyl-protected amine or -C(=O)-NH-substituted alkyl-protected amine, optionally wherein the substituted alkyl of the -C(=O)-NH-substituted alkyl-protected amine includes a protected carboxylic acid-substituted alkyl; (iii) two different organic borate esters; optionally wherein the two different organic borate esters are two different arylboronic acids or arylborate esters of the following formula:

[0261]

[0262] wherein y is an integer from 1 to 5 (e.g., 1, 2, 3, 4 or 5), optionally 1 or 2; each R 20is an N - protected alkylamine, a protected carboxylic acid, a -C(=O)-NH-alkylidene protected amine or a -C(=O)-NH-substituted alkylidene protected amine, optionally wherein the substituted alkylidene of the -C(=O)-NH-substituted alkylidene protected amine comprises an alkylidene substituted with a protected carboxylic acid; and each R 21 is H or alkyl or wherein two Rs 21 together form an alkylidene. In the resulting product (e.g., a synthetic intermediate of a compound of formula (II)), the halogen substituents at R2′ and R 12 ′ or R3′ and R 13 ′ are each replaced with a different substituent (e.g., different -alkynyl-aryl-(R 20 ), y -alkenyl-aryl-(R 20 ), y or aryl-(R 20 )) y groups).

[0263] In some embodiments, the ratio of two alkynes, alkenes or organoboronates 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 the two different alkynes, alkenes or boronic esters. The relative reactivity of the two coupling partners can be determined by monitoring the reaction by reverse phase HPLC. In some embodiments, the less reactive partner of the two coupling partners is provided in a greater molar excess compared to the second coupling partner to maximize the yield of the asymmetric bacteriochlorophyll.

[0264] In some embodiments, the yield of the desired product of the cross - 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%.

[0265] IV. Pharmaceutical Compositions

[0266] The compounds of the presently disclosed subject matter 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-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine, and tris(hydroxymethyl)aminomethane; alkali metal salts such as, but not limited to, lithium, potassium, and sodium; alkaline earth metal salts such as, but not limited to, barium, calcium, and magnesium; transition metal salts such as, but not limited to, zinc; and other metal salts such as, but not limited to, sodium hydrogen phosphate and disodium phosphate; and also include, but are not limited to, salts of inorganic acids such as, but not limited to, hydrochloride and sulfate; and salts of organic acids such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, and fumarate. Pharmaceutically acceptable esters include, but are not limited to, alkyl esters, alkenyl esters, alkynyl esters, aryl esters, heteroaryl esters, aralkyl esters, heteroaralkyl esters, cycloalkyl esters, and heterocyclic esters of acidic groups (including, but not limited to, carboxylic acid, phosphoric acid, phosphinic acid, sulfonic acid, sulfinic acid, and boric acid).

[0267] The compounds of the presently disclosed subject matter may also include prodrugs of the compounds described herein. As described above, a “prodrug” is a compound that, upon administration in vivo, is metabolized or otherwise converted by one or more steps or processes into the biologically, pharmaceutically, or therapeutically active form of the compound. To prepare a prodrug, the pharmaceutically active compound is modified 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, to mask side effects or toxicity, to improve the flavor of a drug, or to alter other characteristics or properties of a drug. With knowledge of pharmacodynamic processes and in vivo drug metabolism, one of ordinary skill in the art can design a prodrug of a compound once the pharmaceutically active compound is known (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, New York, United States of America, pages 388-392).

[0268] Utility. The methods and intermediates described herein can be used to synthesize the compounds of formula (II) described herein. Such compounds, either per se or in a further modified form (e.g., as a salt, metallated compound, conjugate, or prodrug), can be used for diagnostic and therapeutic purposes in a manner similar to other compounds described for photodynamic therapy, such as those described in U.S. Patent Application Publication No. 2004 / 0044197 to Pandey et al. and described in further detail below.

[0269] Stability. Some embodiments of the bacteriochlorophyll compounds of the presently disclosed subject matter have the advantage of their stability and absorption characteristics. Accordingly, the presently disclosed subject matter provides a "pure" composition comprising 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 em -1 or greater at a wavelength of from about 600 to about 800 nanometers (it is understood that (a) the active compound must be placed in solution to determine its peak molar absorption coefficient at the specified wavelength; and (b) the compound may exhibit other peaks outside of this range, or multiple peaks within this range).

[0270] In addition, the presently disclosed subject matter provides a composition comprising 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 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 be from 0.01 or 1 to 99 or 99.99 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 em -1 or greater. It should be understood that stirring may be used as needed to break up agglomerated particles back into solution prior to determining the molar absorption, but some level of agglomeration may be desired for the 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.

[0271] Whether in "pure" form or in admixture with a solvent, one or more bacteriochlorophyll compounds, 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, the composition has or exhibits no more than about 10, 15, or 20 weight % loss of the bacteriochlorophyll compounds of the presently disclosed subject matter (due to its degradation). Degradation can be determined by spectroscopic methods, thin layer chromatography, NMR spectroscopy, and / or mass spectrometry according to known techniques.

[0272] Solubility.Some embodiments of the compounds of the presently disclosed subject matter have the advantage of their water solubility. Accordingly, the presently disclosed subject matter provides compositions, including but not limited to pharmaceutical formulations, that comprise, consist of, or consist essentially of: (a) an aqueous solvent (e.g., distilled water, saline solution, buffered solution); and (b) from about 1, 2, 5, or 10 μM to 200, 300, or 500 mM of an active compound as described herein dissolved in the aqueous solvent.

[0273] Formulations of Pharmaceutical Compositions The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more of the compounds provided herein in a pharmaceutically acceptable carrier, and the compounds can be used to prevent, treat, or ameliorate one or more symptoms of a disease or disorder associated with or involving hyperplastic tissue or neovascularization. Diseases or disorders associated with hyperplastic tissue or neovascularization include, but are not limited to, cancer, psoriasis, atherosclerosis, heart disease, and age-related macular degeneration. Pharmaceutical carriers suitable for administering the compounds provided herein include any such carriers known to those of skill in the art that are suitable for a particular mode of administration.

[0274] The pharmaceutical compositions preferably exhibit the absorption and storage or stability characteristics described above.

[0275] In addition, the compounds can be formulated as the sole pharmaceutically active ingredient in a composition or can be combined with other active ingredients.

[0276] The compositions comprise one or more of the compounds provided herein (e.g., a compound of formula (II)). In some embodiments, the compounds are formulated into suitable pharmaceutical formulations, 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 compounds described above 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, page 126).

[0277] In a composition, one or more compounds or pharmaceutically acceptable derivatives thereof at an effective concentration are admixed with a suitable pharmaceutical carrier. As described above, the compounds may be derivatized to the corresponding salts, esters, enol ethers or enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs prior to formulation. The concentration of the compounds in the composition is an amount effective to deliver, upon administration, one or more symptoms of a disease or disorder associated with or involving hyperplastic tissue or neovascularization, or to treat, prevent or ameliorate such disease or disorder.

[0278] In some embodiments, the composition is formulated for single-dose administration. To formulate the composition, a weight fraction of the compound is dissolved, suspended, dispersed or otherwise admixed in a selected carrier at an effective concentration such that the disorder being treated is alleviated, prevented, or one or more symptoms are ameliorated.

[0279] 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 producing undesirable side effects in the patient being treated. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo systems described herein and in U.S. Patent No. 5,952,366 to Pandey et al., and the dose for humans can then be extrapolated therefrom.

[0280] The concentration of the active compound in the pharmaceutical composition can depend on the absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosing regimen and the amount administered, and other factors known to those of skill in the art. For example, as described herein, the amount delivered is sufficient to ameliorate one or more symptoms of a disease or disorder associated with or involving hyperplastic tissue or neovascularization.

[0281] In some embodiments, the therapeutically effective dose should produce a serum concentration of the active ingredient of from about 0.1 ng / ml to about 50 - 100 μg / ml. In one embodiment, the therapeutically effective dose is from 0.001, 0.01 or 0.1 to 10, 100 or 1000 mg of the 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 the active ingredient, or a combination of essential ingredients, in each dosage unit form.

[0282] The active ingredient can be administered in a single dose, or it can be divided into multiple smaller doses and administered at intervals. It should be understood that the precise dosage and duration of treatment depend on 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 should be noted that the concentration and dosage values can also vary with the severity of the condition to be alleviated. It should be further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the composition or guiding the administration of the composition, and the concentration ranges listed herein are only examples and are not intended to limit the scope and practice of the claimed composition.

[0283] In cases where the compound exhibits insufficient solubility, methods for solubilizing the compound can be used. Such methods are known to those skilled in the art and include, but are not limited to, using co-solvents such as dimethyl sulfoxide (DMSO), using surfactants such as polyoxyethylene sorbitan esters (e.g., sold under the trade name ), or dissolving in an aqueous sodium bicarbonate solution. Derivatives of the compound, such as prodrugs of the compound, can also be used to formulate effective pharmaceutical compositions.

[0284] After mixing or adding the compound, the resulting mixture can be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to improve the symptoms of the disease, disorder, or condition being treated and can be determined empirically.

[0285] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions, containing a suitable amount of the compound or its pharmaceutically acceptable derivative. In some embodiments, the pharmaceutically active compounds and their derivatives are formulated and administered in unit dosage forms or multiple dosage forms. As used herein, "unit dosage form" refers to physically discrete units suitable for individual humans and animals and individually packaged as known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound together with the required pharmaceutical carrier, vehicle, or diluent sufficient to produce the desired therapeutic effect. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms can be administered in divided or multiple doses. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container for administration in separate unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or pint or gallon bottles. Thus, a multiple dosage form is a plurality of dosage forms not separated in the package.

[0286] A liquid pharmaceutical composition can be prepared, for example, 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 optionally present pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose solution, glycerol, diols, ethanol, etc.) to form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, etc., such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate and other such reagents.

[0287] The actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington′s Pharmaceutical Sciences, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, United States of America.

[0288] Dosage forms or compositions can be prepared that contain an active ingredient in the range of 0.005% to 100%, the remainder consisting of non-toxic carriers. The methods for preparing these compositions are known to those skilled in the art. The compositions contemplated may contain 0.001%-100% of the active ingredient, in one embodiment 0.1-95%, and in another embodiment 75-85%.

[0289] Compositions for Oral Administration Oral pharmaceutical dosage forms are solids, gels or liquids. Solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed chewable lozenges and tablets that may be enteric-coated, sugar-coated or film-coated. Capsules 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.

[0290] Solid Compositions for Oral AdministrationIn some embodiments, the formulation is a solid dosage form, and in some embodiments, it is a capsule or a tablet. Tablets, pills, capsules, lozenges, etc. may contain one or more of the following ingredients or compounds of similar nature: binders; lubricants; diluents; glidants; disintegrants; colorants; sweeteners; flavorants; wetting agents; emetic coatings; and film coatings. Examples of binders include microcrystalline cellulose, tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidone, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium powder, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salts, mannitol, and dibasic calcium phosphate. Glidants include, but are not limited to, colloidal silica. Disintegrants include sodium croscarmellose, sodium carboxymethyl starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Colorants include, for example, any approved and certified water-soluble FD and C dyes, mixtures thereof; and water-insoluble FD and C dyes suspended on hydrated alumina. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, as well as any number of spray-dried flavors. Flavorants include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce a pleasant sensation, 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 (PEG4000), and cellulose acetate phthalate.

[0291] The compound or its pharmaceutically acceptable derivative may be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition may be formulated as an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredients. When the dosage unit form is a capsule, in addition to materials of the above types, it may also contain a liquid carrier such as a fatty oil. Additionally, the dosage unit form may contain various other materials that alter the physical form of the dosage unit, such as sugar coatings and other enteric solvents. The compound may also be administered as a component of an elixir, suspension, syrup, wafer, spray, chewing gum, etc. In addition to the active compound, the syrup may contain sucrose as a sweetener and certain preservatives, dyes and colorants, and flavorants.

[0292] The active material may 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 the active ingredient up to about 98% by weight may be included.

[0293] In some embodiments, tablet and capsule formulations may be coated as known to those skilled in the art to alter or maintain the dissolution of the active ingredient. Thus, for example, they may be coated with conventional enteric digestible coatings such as phenyl salicylate, waxes, and cellulose acetate phthalate.

[0294] Liquid Compositions for Oral Administration Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

[0295] An elixir is a clear, sweetened hydroalcoholic preparation. Pharmaceutically acceptable carriers for elixirs include solvents. A syrup is a concentrated aqueous solution of sugar (e.g., sucrose) and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules in another liquid. 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 a liquid oral dosage form include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Colorants 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, gum arabic, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, magnesium aluminum silicate salts, and gum arabic. 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 acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any of the approved and certified water-soluble FD and C dyes and mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce a pleasant taste. For solid dosage forms, solutions or suspensions in, for example, propylene carbonate, vegetable oil, or triglyceride are, in one embodiment, encapsulated in gelatin capsules. Such solutions and their preparation and encapsulation are disclosed in U.S. Patent 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 amount of a pharmaceutically acceptable liquid carrier (e.g., water) to be easily measured for administration.

[0296] Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (such as propylene carbonate), and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those described in U.S. Patent No. RE28,819 and U.S. Patent No. 4,358,603, each of which is incorporated herein by reference in its entirety. Briefly, such formulations include, but are not limited to, those containing the compounds provided herein, dialkylated mono- or poly-alkylene glycols, including but not limited to 1,2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether (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 its esters, and dithiocarbamates.

[0297] Other formulations include, but are not limited to, hydroalcoholic solutions containing pharmaceutically acceptable acetals. The alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents 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.

[0298] Injections, Solutions and Emulsions In some embodiments characterized by subcutaneous, intramuscular, or intravenous injection, parenteral administration is also included herein. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, in solid forms suitable for dissolving or suspending in a liquid prior to injection, or in the form of emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Additionally, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such reagents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0299] In the present text, the implantation of sustained-release or controlled-release systems is also contemplated, such that a constant dosage level is maintained (see, for example, U.S. Patent No. 3,710,795, which is incorporated herein by reference in its entirety). Briefly, the compounds provided herein are dispersed within a solid internal matrix, such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, hydrophilic polymers (such as hydrogels of esters of acrylic and methacrylic acids), collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate, and the solid internal matrix 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, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride with vinyl acetate, vinylidene chloride, ethylene, and propylene, ionomeric polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, and ethylene / ethyleneoxyethanol copolymer. The compound diffuses through the outer polymeric membrane in a rate-controlling step of release. The percentage of the active compound included in such parenteral compositions highly depends on its specific nature, as well as the activity of the compound and the needs of the individual.

[0300] Parenteral administration of the composition includes intravenous, subcutaneous, and intramuscular administration. Formulations for parenteral administration include sterile solutions ready for injection; sterile dry soluble products (such as lyophilized powders) ready to be mixed with a solvent before use, including subcutaneous injection tablets; sterile suspensions ready for injection; sterile dry insoluble products ready to be mixed with a vehicle immediately before use; and sterile emulsions. The solutions can be aqueous or non-aqueous.

[0301] 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).

[0302] 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.

[0303] Examples of aqueous vehicles include sodium chloride injection, Ringers Injection, isosmotic dextrose injection, sterile water injection, dextrose and Lactated Ringers Injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. An antimicrobial agent at a bacteriostatic or fungistatic concentration can be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffering agents include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, xanthan gum, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80( 80). Masking or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0304] The concentration of the pharmaceutically active compound can be adjusted such that the injection provides an effective amount to produce the desired pharmacological effect. The exact dosage depends on the age, weight, and condition of the patient or animal, which are known in the art.

[0305] Unit doses of parenteral preparations are packaged in ampoules, vials, or syringes with needles. As is known and practiced in the art, all preparations for parenteral administration should be sterile.

[0306] 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 oily solution or suspension containing the active material, which is injected as needed to produce the desired pharmacological effect.

[0307] Injections are 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 higher of the active compound in the tissue to be treated, and in certain embodiments, higher than 1% w / w of the active compound in the tissue to be treated.

[0308] The compound can be suspended in micronized or other suitable form, or can be derivatized to produce a more soluble active product or a prodrug. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to improve the symptoms of the condition and can be determined empirically.

[0309] Lyophilized Powders Lyophilized powders, which can be reconstituted for administration as solutions, emulsions, and other mixtures, and can also be used to practice the presently disclosed subject matter. They can also be reconstituted and formulated as solids or gels.

[0310] Sterile lyophilized powders are prepared by dissolving the compounds provided herein or their pharmaceutically acceptable derivatives 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, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable substances. The solvent can also contain buffers such as citrate, sodium phosphate, 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 subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial can contain a single dose or multiple doses of the compound. The lyophilized powders can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0311] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or another suitable vehicle. The exact amount depends on the compound selected. This amount can be determined empirically.

[0312] Topical Administration Prepare topical mixtures as described for topical and systemic administration. The resulting mixtures can be solutions, suspensions, emulsions, etc., and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, douches, sprays, suppositories, bandages, skin patches, or other formulations suitable for topical administration.

[0313] The compound or its pharmaceutically acceptable derivative can be formulated as 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 and 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 an aerosol or solution of a nebulizer or as micronized powders for insufflation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation generally have a diameter of less than 50 microns in some embodiments and less than 10 microns in some embodiments.

[0314] The compounds can be formulated for topical or local administration, for example, topically applied to the skin and mucous membranes (such as the eye) in the form of gels, creams and lotions, and applied to the eye or administered intrathecally or intraspinally. Topical administration is contemplated for transdermal delivery and also for ocular or mucosal administration or for inhalation therapy. Nasal solutions of the active compounds can also be administered alone or in combination with other pharmaceutically acceptable excipients. These solutions, especially those intended for ophthalmic use, can be formulated as isotonic solutions of 0.01%-10% at a pH of about 5-7 with appropriate salts.

[0315] Compositions for Other Routes of Administration . Also covered herein are other routes of administration, such as transdermal patches, including iontophoresis and electroosmosis devices, and rectal administration.

[0316] Transdermal patches, including electroosmosis and electroosmosis devices, are well known to those skilled in the art. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983; 6,261,595; 6,256,533; 6,167,301; 6,024,975; 6,010715; 5,985,317; 5,983,134; 5,948,433; and 5,860,957, each of which is incorporated herein by reference in its entirety.

[0317] For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic action. A rectal suppository as used herein refers to a solid for insertion into the rectum that melts or softens at body temperature to release one or more pharmaceutical or therapeutic active ingredients. Pharmaceutically acceptable substances for rectal suppositories are bases or excipients and agents that raise the melting point. Examples of bases include cocoa butter (cocoa oil), glycerol-gelatin, carbowax (polyethylene glycol), and suitable mixtures of glycerol monoesters, glycerol diesters and glycerol triesters of fatty acids. Combinations of various bases can be used. Agents that raise the melting point of the suppository include cetyl 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.

[0318] Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substances and by the same methods as those for oral administration.

[0319] Targeted Preparations. The 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 herein for use with 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.

[0320] Liposomes. In some embodiments, liposome suspensions, including liposomes that target tissues, such as liposomes that target tumors, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Patent No. 4,522,811, which is incorporated herein by reference in its entirety. Briefly, liposomes such as multilamellar vesicles (MLV) can be formed by drying egg phosphatidylcholine and phosphatidylserine (7:3 molar ratio) inside a flask. A solution of the compound provided herein in phosphate buffered saline (PBS) without divalent cations is added and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, centrifuged to pellet, and then resuspended in PBS.

[0321] Ligands.In some embodiments, the disclosed compounds can use ligands that are specific for a target tissue or target composition, e.g., using a ligand or ligand-receptor pair such as an antibody and an antigen, to target a specific target tissue or target composition. Anti-tumor antigens and anti-pathogen antibodies are known. For example, antibodies and antibody fragments that specifically bind to markers produced by or associated with a tumor or infectious lesion, including viral, bacterial, fungal, and parasitic infections, as well as antigens and products associated with such microorganisms, are particularly disclosed in Hansen et al., U.S. Patent No. 3,927,193 and Goldenberg, U.S. Patents 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 lymphomas, sarcomas or melanomas) can be used.

[0322] A variety of monoclonal antibodies against infectious disease agents have been developed and summarized in the review by Polin (1984) European Journal of Clinical Microbiology 3(5): 387-398, indicating ready availability. These include monoclonal antibodies (MAbs) 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 Mab, such as those against Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Mesocestoides corti, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata; antiviral Mab, such as those against HIV-1, -2 and -3, hepatitis A, hepatitis B, hepatitis C, hepatitis D, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I and II, parvovirus B19, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, measles virus, adenovirus, human T cell leukemia virus, Epstein-Barr virus, mumps virus, Sindbis virus, murine mammary tumor virus, feline leukemia virus, lymphocytic choriomeningitis virus, papillomavirus, bluetongue virus, Sendai virus, Reo virus, poliovirus, dengue virus, rubella virus, murine leukemia virus; anti-mycoplasma Mab, such as those against Acholeplasma laidlawii, Mycoplasma arthritidis, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini, Mycoplasma pneumoniae; and so on.

[0323] Suitable Mab against most of the microorganisms (bacteria, viruses, protozoa, other parasites) responsible for most human infections have been developed and many have been previously used for in vitro diagnostic purposes. These antibodies and new Mab that can be produced by conventional methods are suitable for use as target substances with the compounds provided herein.

[0324] MAbs against Plasmodium can be directed against the sporozoite, merozoite, schizont, 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 have been developed against Toxoplasma gondii (a protozoan parasite associated with toxoplasmosis). See Kasper et al. (1982) Journal of Immunology 129: 1694-1699. MAbs against Schistosoma surface antigens have been developed and have been found to be active against Schistosoma in vivo or in vitro. See Simpson et al. (1981) Parasitology 83: 163-177; Smith et al. (1982) Parasitology 84: 83-91; Gryzch et al. (1982) Journal of Immunology 129: 2739-2743; Zodda et al. (1982) Journal of Immunology 129: 2326-2328; and Dissous et al. (1982) Journal of Immunology 129: 2232-2234.

[0325] Mixtures of antibodies and immunoglobulin classes can be used, and hybrid antibodies can also be used. Multispecific (including bispecific and hybrid) antibodies and antibody fragments are particularly preferred in the methods of the present disclosure 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 of molecules produced by or associated with a target tissue. Multispecific antibodies and bispecific antibody fragments can be prepared similar to the anti-tumor marker hybrids disclosed in U.S. Patent No. 4,361,544. Other techniques for preparing hybrid antibodies are disclosed, for example, in 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.

[0326] Antibody fragments useful for the presently disclosed subject matter include F(ab′)2, F(ab)2, Fab′, Fab, Fv, etc., including hybrid fragments. Preferred fragments are Fab′, F(ab′)2, Fab, and F(ab)2. Any sub-fragment that retains the hypervariable antigen-binding regions of the immunoglobulin and has a size similar to or less than that of the Fab′ fragment is also useful. This can include genetically engineered and / or recombinant proteins, whether single-chain or multi-chain, that incorporate the antigen-binding site and otherwise function as targeting vectors in vivo in substantially 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 reductive cleavage of F(ab′)2 fragments, which themselves can be prepared by pepsin digestion of intact immunoglobulins. Fab antibody fragments can be prepared by papain digestion of intact immunoglobulins under reducing conditions, or by cleavage of F(ab′)2 fragments generated by careful papain digestion of intact immunoglobulins.

[0327] One 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.

[0328] Conjugated with Ligands Numerous compounds have been identified that can serve as targets for ligand-receptor binding pairs (more specifically, antibodies), and the 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. taught the conjugation of chlorin Sn(IV) to a monoclonal antibody via 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. Compounds disclosed herein can also be conjugated to ligands, such as antibodies, using a coupling agent. Any bond capable of linking the components such that they are stable under physiological conditions for the time required for administration and treatment is suitable, but covalent bonds are preferred. The linkage between the two components can be direct, for example, where the compound of formula (I) is directly linked to the targeting agent, or it can be indirect, for example, where the compound of formula (I) is linked to an intermediate and the intermediate is linked to the targeting agent.

[0329] The coupling agent should function under conditions of temperature, pH, salts, solvent systems, and other reactants that substantially maintain the chemical stability of the photosensitizer, the backbone (if present), and the targeting agent. The coupling agent should stably link the component parts, but such that there is only minimal denaturation or inactivation, or no denaturation or inactivation, of the compound of formula (I) or the targeting agent. Many coupling agents react with amines and carboxylates to form amides, or with alcohols and carboxylates to form esters. Coupling agents are known in the art. See, for example, Bodansky (1993) Principles of Peptide Synthesis, 2nd ed., and Springer & Hermanson (1996) Bioconjugate Techniques, 1st ed., Academic Press, New York, New York, United States of America.

[0330] Conjugates of the compounds provided herein with ligands such as antibodies can be prepared by coupling the compound to the targeting moiety by coupling a carboxylic acid or ester moiety on the compound via a peptide bond to the antibody 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-pyridyldithio)propionate (SPDP), o-phenylenedimaleimide (o-PDM), and sulfo-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC). See, for example, Karpovsky et al. (1984) Journal of Experimental Medicine 160(6):1686-1701; and Liu et al. (1985) Proceedings of the National Academy of Science of the United States of America 82(24):8648-8652. Other methods include those described by Brennan et al. (1985) Science 229:81-83 and Glennie et al. (1987) Journal of Immunology 139:2367-2375.

[0331] For example, DCC is a useful coupling agent that can be used to promote the coupling of an alcohol NHS with a chlorophyllin carboxylic acid group in DMSO to form an activated ester that can crosslink with polylysine. DCC is a carboxyl-reactive crosslinking agent, commonly used as a coupling agent in peptide synthesis, and has a molecular weight of 206.32. Another useful crosslinking agent is SPDP, a heterobifunctional crosslinking agent that is used with primary amines and thiols. SPDP has a molecular weight of 312.4, a spacer arm length of 6.8 Å, is reactive towards NHS-esters and pyridyldisulfide groups, and produces a cleavable crosslink such that the reagent can be removed upon further reaction, allowing the photosensitizer to be directly linked to the backbone or targeting agent. Other useful coupling agents are SATA, which is used to introduce a capping SH group for two-step crosslinking and is uncapped by hydroxylamine-HCl and sulfo-SMCC and is reactive towards amines and thiols. Other crosslinking agents and coupling agents are also available from Pierce Chemical Co. Other compounds and methods for conjugating proteins to other proteins or other compositions (e.g., conjugating to a reporter group or a chelator for metal ion labeling of proteins), particularly those involving Schiff bases as intermediates, are disclosed in European Patent EP 0 243 929 B1.

[0332] Photosensitizers containing a carboxyl group can be linked to the lysine ε-amino group in a target polypeptide via a preformed reactive ester (such as an N-hydroxysuccinimide (NHS) ester) or an ester conjugated in situ via a carbodiimide-mediated reaction. This also applies to photosensitizers containing a sulfonic acid group, which can be converted to a sulfonyl chloride that reacts with an amino group. Bacteriochlorophylls having a carboxyl group can be linked to an amino group on a polypeptide by an in situ carbodiimide method. Bacteriochlorophylls can also be linked to the hydroxyl group of a serine or threonine residue or the thiol group of a cysteine residue.

[0333] Methods of linking the components of a conjugate (e.g., coupling a photosensitizer with a polyamino acid chain to an antibacterial polypeptide) can use heterobifunctional crosslinking 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 typically amines, carboxyls, thiols, and aldehydes. There are many arrangements of suitable moieties that will react with these groups and structures represented by different formulas to conjugate them together. See Hermanson (1996) Bioconjugate Techniques, 1st ed., Academic Press, New York, New York, United States of America; and Merrifield et al. (1994) Ciba Foundation Symposium 186:5-20.

[0334] The compound or a pharmaceutically acceptable derivative thereof can be packaged as an article, which contains a packaging material, the compound or a pharmaceutically acceptable derivative thereof provided herein within the packaging material (which can effectively regulate the activity of hyperplastic tissue or neovascularization, or is used for treating, preventing or improving a disease or disorder mediated by hyperplastic tissue or neovascularization, or one or more symptoms of a disease or disorder in which hyperplastic tissue or neovascularization activity is involved), and a label, and the label indicates that the compound or composition or a pharmaceutically acceptable derivative thereof is used for regulating the activity of hyperplastic tissue or neovascularization, or is used for treating, preventing or improving a disease or disorder mediated by hyperplastic tissue or neovascularization, or one or more symptoms of a disease or disorder in which hyperplastic tissue or neovascularization is involved.

[0335] The article provided herein contains a packaging material. 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 herein 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, flasks, and any packaging material suitable for the selected formulation and the intended mode of administration and treatment. A variety of formulations of the compounds and compositions provided herein are contemplated for the treatment of a variety of diseases or disorders in which hyperplastic tissue or neovascularization is involved as a mediator or contributing factor of a symptom or cause.

[0336] V. Photodynamic Therapy, Diagnostic and Therapeutic Applications

[0337] In some embodiments, the presently disclosed compounds of formula (II) (or pharmaceutically acceptable salts or conjugates thereof) can act as photosensitizers in methods for treating diseases involving photodynamic therapy (PDT) (e.g., hyperplastic diseases such as cancer). Briefly, the photosensitive compound, its conjugate or pharmaceutical composition is generally administered to an individual prior to irradiation of the target tissue, target composition or individual with light. The photosensitive compound is administered as described elsewhere herein.

[0338] The dose of the photosensitive compound can be determined clinically. An equivalent optimal therapeutic level must be established depending on the photosensitive compound used. Allow a certain length of time for the systemically or locally delivered photosensitizer to be absorbed by the target tissue. During this waiting period, unbound photosensitizer is cleared from the circulation, or optionally additional time can be provided to clear unbound compound from non-target tissues. The waiting period can be determined clinically and can vary depending on the compound.

[0339] At the end of this waiting period, the conjugated 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 lights, or natural light sources such as ambient sunlight). The illumination area is determined by the location and size of the pathological area to be detected, diagnosed, or treated. The duration of the illumination period can depend on whether a detection or treatment is being performed and can be determined empirically. Any total or cumulative time period from about 4 minutes to about 72 hours can be used. In some embodiments, the light exposure period is from about 60 minutes to 148 hours. In some embodiments, the light exposure period is from about 2 hours to 24 hours.

[0340] Preferably, the total fluence or energy of the light used for illumination, 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. Light of a wavelength and fluence sufficient to produce the desired effect is selected, whether for detection by fluorescence or for therapeutic treatment to destroy or damage the target tissue or target composition. Light having a wavelength that at least partially corresponds to the characteristic light absorption wavelength of the photosensitizer is preferably used to illuminate the target tissue.

[0341] The intensity or power of the light used is measured in watts, where one watt is equal to one watt-second per joule. Thus, the intensity of the light used for illumination in the presently disclosed method can be substantially less than 500 mW / cm 2 . Since the total fluence or energy of the light, measured in joules, is divided by the duration of the total exposure time, measured in seconds, the longer the amount of time the target is exposed to the illumination, 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 illumination that is high enough to activate the photosensitizer.

[0342] In some embodiments of performing 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 μmol / 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., about 10 to 200 J / cm 2 . In the case of detection, fluorescence is determined when exposed to light at a wavelength sufficient to cause the compound to fluoresce at a wavelength different from the wavelength used to illuminate the compound. The energy used in detection is sufficient to cause fluorescence and is typically significantly lower than the energy required for treatment.

[0343] Any of the photosensitive compounds or pharmaceutically acceptable derivatives thereof disclosed herein can be provided in a kit together with instructions for practicing any of the methods disclosed herein. The instructions can be in any tangible form, such as printed paper, a computer disk that instructs a person how to practice the method, a videotape that contains instructions on how to practice 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 via the Internet). For example, any of the above instructions can be used or a person can be instructed how to use the kit by receiving the instructions in a classroom or in the course of treating a patient using any of the methods disclosed herein.

[0344] Other examples and specific examples of methods of using the compounds and compositions of the presently disclosed subject matter include, but are not limited to, the following:

[0345] (i) Treatment of opportunistic infections. The compounds, compositions, and methods of the presently disclosed subject matter can be used for PDT of opportunistic infections, particularly PDT of opportunistic infections of soft tissues. For the antimicrobial treatment (by PDT) of infections, particularly wound infections, the infecting organisms can include (as non-limiting examples) Staphylococcus aureus, Pseudomonas aeruginosa, and 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.

[0346] (ii) Treatment of burns. Infections with Staphylococcus aureus and Gram-positive bacteria are generally particularly evident in burns. The multi-drug resistance of Staphylococcus aureus presents a major medical challenge. In this regard, the compounds, compositions, and methods of the presently disclosed subject matter can be used for the treatment of opportunistic infections in burns.

[0347] (iii) Septicemia. The compounds, compositions, and methods of the presently disclosed subject matter can be used for PDT treatment of individuals with opportunistic infections with Vibrio vulnificus. Vibrio vulnificus, a Gram-negative bacterium, causes primary septicemia, wound infections, and gastrointestinal diseases in humans.

[0348] (iv) Ulcers. The compounds, compositions, and methods of the presently disclosed subject matter can be used for PDT treatment of bacteria (Helicobacter pylori) that cause ulcers. Clinically, the treatment can be carried out in any suitable manner, such as by inserting an optical fiber cable (similar to an endoscope but having a device for delivering red or near-infrared light) into the stomach or the affected area.

[0349] (v) Periodontal diseases. The compounds, compositions, and methods of the presently disclosed subject matter are useful in PDT for treating periodontal diseases, including gingivitis. Periodontal diseases are caused by overgrowth of bacteria, such as the Gram-negative anaerobic bacterium Porphyromonas gingivalis. As with many PDT treatments, a targeting or solubilizing entity conjugated to a photoactive agent is essential for proper delivery of the photoactive agent to the desired cells. Target oral pathogens for targeting include Porphyromonas gingivalis, Actinobacillus actinonzycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatumsubsp.Polymorphum, Actinomyces viscosus, and Streptococcus. For such applications, the compounds or compositions of the presently disclosed subject matter can be administered topically (e.g., in the form of a mouthwash or gargle), and then photo-administered with an external device, an intraoral instrument, or a combination thereof.

[0350] (vi) Atherosclerosis. The compounds, compositions, and methods of the presently disclosed subject matter can be used in PDT for treating vulnerable arterial plaques. Without wishing to be bound by any particular theory, it is believed that invading inflammatory macrophages secrete metalloproteinases that degrade the collagen thin layer in coronary arteries, leading to thrombosis, which is often fatal. Active compounds targeting such inflammatory macrophages can be used in PDT for vulnerable plaques.

[0351] (vii) Cosmetic and dermatological applications. The compounds, compositions, and methods of the presently disclosed subject matter can be used in PDT for treating a wide range of cosmetic dermatological problems, such as hair removal, treating psoriasis, or removing skin discoloration. Ruby lasers are currently used for hair removal; in many laser treatments, melanin is the photosensitive chromophore. This treatment works reasonably well for fair-skinned people with dark hair. The compounds, compositions, and methods of the presently disclosed subject matter can be used as near-infrared sensitizers for hair removal, which are capable of targeting chromophores with more specific and sharp absorption bands.

[0352] (viii) Acne. The compounds, compositions and methods of the presently disclosed subject matter may be used for PDT to treat acne. Acne vulgaris is caused by Propionibacterium acne, which infects the sebaceous glands; some 80% of young people are affected. Again, bacterial resistance to antibiotic treatment is increasing, leading to a surge in difficult-to-treat acne. Current PDT treatments for acne generally rely on the addition of aminolevulinic acid, which is converted to a free base porphyrin in the hair follicles or sebaceous glands. The compounds and compositions of the presently disclosed subject matter may be administered to an individual topically or parenterally (e.g., by subcutaneous injection), depending on the particular condition.

[0353] (ix) Infectious diseases. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT to treat infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which occur extensively in the Mediterranean and Middle East, are currently treated with arsenic-containing compounds. Recently, in at least one case, PDT has been used to produce reasonable results in human patients. The use of the compounds and compositions of the presently disclosed subject matter is also useful and potentially offers advantages such as ease of synthesis and better spectral absorption characteristics.

[0354] (x) Tissue sealants. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT as tissue sealants for individuals in need thereof. Light-activated tissue sealants are attractive for sealing wounds, gluing tissues, and closing defects in tissues. There are many applications where sutures or staples are undesirable and where the use of such mechanical sealing methods often results in infection and scarring.

[0355] (xi) Neoplastic Diseases. The compounds, compositions and methods of the presently disclosed subject matter can be used for PDT to treat neoplastic diseases or cancers, including skin cancer, lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma, leukemia, lymphoma, squamous cell carcinoma, melanoma, plaque-stage cutaneous T-cell lymphoma and Kaposi's sarcoma.

[0356] 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 field of modern medicine, there are a variety of treatment methods, including magnetic resonance imaging (MRI) for disease diagnosis. Early detection of cancer should improve the ability to cure and eliminate cancerous tissue. Early diagnosis of pre-cancerous areas and micro-cancers is an important theme in modern cancer treatment. MRI has become a powerful tool in the clinical setting because it is non-invasive and produces an accurate volumetric representation of an individual. The image is created by applying one or more orthogonal magnetic field gradients to an individual or sample while exciting nuclear spins with radiofrequency pulses in a typical nuclear magnetic resonance (NMR) experiment. After data is collected using various gradient fields, deconvolution generates one-dimensional, two-dimensional, or three-dimensional images of the sample / individual. Typically, the image is based on the NMR signal 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 distinct contrast observed in MR images.

[0357] The role of MRI contrast agents is to increase the relaxation rate, thereby increasing the contrast between the water molecules in the region where the imaging agent accumulates and the water molecules in other parts of the body. However, the agent acts to decrease T1 and T2, with the former resulting in greater contrast and the latter resulting in lower contrast. Thus, the phenomenon is concentration-dependent, and there is generally an optimal concentration of the paramagnetic substance for maximum efficacy. This optimal concentration can vary with the specific agent used, the imaging site, the imaging mode (i.e., spin echo imaging, saturation recovery, inversion recovery, and / or various other strongly T1-dependent or T2-dependent imaging techniques), and the composition of the medium in which the agent is dissolved or suspended. These factors and their relative importance are known in the art. See, for example, Pykett (1982) Scientific American 246:78; and Runge et al. (1983) American Journal of Radiology 141:1209. When MRI contrast agents are used for diagnosis, they perfuse blood vessels, enhance the contrast of blood vessels, and report organ lesions and infiltrations. However, labeling specific tissues for diagnostic radiology remains a formidable challenge for MRI. Efforts to develop cell- and tissue-specific MRI image enhancers by modifying existing immunological techniques have been the focus of many studies in diagnostic radiology. For example, antibodies labeled with paramagnetic ions, typically the gadolinium chelate Gd-DTPA, have been generated and tested for their effect on the MRI contrast of tumors and other tissues. See U.S. Patent No. 5,059,415, which is incorporated herein by reference in its entirety. Unfortunately, it has been found that the relaxivity of Gd bound to antibodies is only slightly better than that of unbound Gd-DTPA. See Paajanen et al. (1990) Magnetic Resonance in Medicine 13:38-43.

[0358] MRI is commonly used to detect 1 H nuclei in a living body. However, MRI is capable of detecting the NMR spectra of other nuclear substances, including 13 C, 15 N, 31 P, and 19 F. 19 F is not abundant in a living body. By adding isotopes that can be used for MRI, such as 13 C, 15 N, 31 P, or 19 F, especially 19 F, to the compositions provided herein and administering them to an individual, the compounds provided herein will accumulate in the target tissue and, due to having MRI-identifiable isotopes such as 19The presence of the cumulative compound of (F) allows subsequent MR imaging to generate NMR data with enhanced signals from the target tissue or target composition. Thus, the disclosed compounds can be used as image enhancers and provide labeling of specific target tissues or target compositions for diagnostic radiology, including MRI.

[0359] In addition to PDT, the compositions provided herein can be used to detect target cells, target tissues, or target compositions in an individual. When the compounds provided herein are used to detect a target tissue or target composition, the compound is introduced into the individual and allowed sufficient time to accumulate in the target tissue or associate with the target composition. The treatment area is then irradiated, typically with light of energy sufficient to cause fluorescence of the compound, and the energy used is typically significantly lower than that required for treatment with photodynamic therapy. Fluorescence is determined upon exposure to light of the desired wavelength, and the amount of fluorescence can be qualitatively or quantitatively correlated with the presence of the compound by methods known in the art.

[0360] The compositions provided herein can also be used to diagnose the presence of an infectious agent or to identify the composition of an infectious agent in an individual. The compounds provided herein can be conjugated to one or more ligands specific for the infectious agent, such as antibodies or antibody fragments, which selectively associate with the infectious agent, and after allowing sufficient time for the target compound to associate with the infectious agent and clear from non-target tissues, the compound can be visualized, such as by exposure to light of energy sufficient to cause fluorescence of the compound, or by using diagnostic radiology imaging, including MRI. By way of example, any one of the compounds provided herein can be conjugated to an antibody targeting a suitable Helicobacter pylori antigen and formulated into a pharmaceutical preparation that, when introduced into an individual, releases the conjugated compound at the location of the gastric mucus / epithelial layer where the bacteria are found. After the compound has selectively associated with the target infectious agent and any unbound compound has cleared from non-target tissues for sufficient time, the individual can be examined to determine the presence of any Helicobacter pylori. This can be detected, for example, by MRI for the compound that accumulates due to 19 the presence of the F substituent, or by irradiating the suspected target area with light of energy sufficient to cause fluorescence of the compound (e.g., by using fiber optics) and detecting the fluorescence of any target compound.

[0361] In some embodiments, the presently disclosed compounds or their conjugates can be used in flow cytometry. Flow cytometry is known and described, for example, in 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 herein by reference in its entirety. In some embodiments, the particles to be detected (such as cells) are labeled with a luminescent compound such as a phosphor or fluorophore for detection. The labeling can be carried out 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 such luminescent compounds, and the flow cytometry techniques (including fluorescence-activated cell sorting or FACS) can be based on the present disclosure and carried out according to known techniques or variants thereof that are obvious to those skilled in the art. Examples

[0362] The following examples provide illustrative embodiments. Given the present disclosure and the general level of skill in the art, those skilled in the art will understand that the following examples are intended to be exemplary only, and that many variations, modifications, and alterations can be made without departing from the scope of the subject matter disclosed by the present invention.

[0363] Example 1

[0364] Synthesis of Compound CP-1

[0365] The di-BOC protected Suzuki coupling partner 1 (CP-1) was prepared as shown in Route 2 (see Figure 2 ).

[0366] 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) equipped with a stir bar, a glass stopper, a condenser with a septum, and a rubber septum. The NBS was dried under high vacuum for 30 minutes, then the flask was flushed with argon and acetonitrile (ACN, 400 mL) was added through a cannula to approximately half volume (∼450 mL). 1-Bromo-3,5-dimethylbenzene (15.26 g, 80.0 mmol) was added via syringe, followed by briefly opening the system under an argon stream and a large amount 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).

[0367] After 16 h, the reaction mixture was transferred to a single-neck 1 L RBF and concentrated to remove ACN. The solid residue was further dried under high vacuum, suspended in dichloromethane (DCM, 75 mL), and heated to gentle reflux. The mixture was equilibrated to room temperature and washed with DCM by filtration. The filtrate was concentrated, dried under high vacuum, and recrystallized in ethanol (EtOH, total 55 mL) while heating in a water bath set at 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%).

[0368] 1 H NMR (400 MHz, CDCl3) δ 4.41 (s, 4H), 7.34 (s, 1H), 7.47 (d, J = 2.0 Hz, 2H).

[0369] 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 equipped with a magnetic 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 at 90 °C.

[0370] After 16 h, the mixture was cooled to room temperature, diluted with water (total 1 L), and extracted with chloroform (400, 300, and 200 mL, 1× each). The combined organic layers were washed with 0.2 N aqueous NaOH solution (500 mL) and water (500 mL). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The solid was further dried under high vacuum, then transferred to a filter and washed with diethyl ether (Et2O) at room temperature (3×). Compound CP-1b was isolated as a white powdery solid (17.98 g, 70%).

[0371] 1 H NMR (400 MHz, CDCl3) δ 4.78 (s, 4H), 7.42 - 7.47 (m, 3H), 7.78 - 7.70 (m, 4H), 7.87 - 7.82 (m, 4H).

[0372] (5-Bromo-1,3-phenylene)dimethanamine (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 heated further at reflux temperature for 15 minutes, then the reaction mixture was allowed to cool gradually to room temperature.

[0373] 6N aqueous HCl was added until the solution was acidic to litmus test (total 20 mL). The resulting mixture was heated again to reflux temperature. The flask was flushed with argon, stirred for 1 hour, then cooled and chilled in an ice bath. The mixture was filtered to obtain a clear pale amber solution. The filtrate was cooled in an ice bath and basified with 2N aqueous NaOH (total 30 mL). The aqueous layer was extracted with chloroform (3 × 75 mL). The organic layers were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to approximately 10 mL volume. A white residue was noted on the flask wall. The remaining solution was filtered, and the filtrate was concentrated to afford 2.3 g of a pale yellow oil. Stored in the refrigerator.

[0374] The sample solidified after storage overnight at 4 °C and was further dried under high vacuum to give 2.047 g (59%) of compound CP-1c as an amber-colored semi-solid.

[0375] Di-tert-butyl ((5-bromo-1,3-phenylene)bis(methylene)) dicarbamate (CP-1d). Compound CP-1c (2.00 g, 9.11 mmol) was added to a flame-dried 250 mL RBF equipped 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. Di-tert-butyl dicarbonate (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.

[0376] The reaction was stirred overnight at room temperature. The mixture was concentrated to give a white solid, which was redissolved in ethyl acetate (EtOAc, 70 mL). The organic phase was washed with saturated aqueous NH4Cl, water, saturated aqueous NaHCO3 and brine (each 1 × 50 mL). Then, the organic layer was dried over sodium sulfate, filtered and concentrated to a pale amber oil that crystallized upon standing. The solid was washed with cooled 1:1 Et2O / / hexane on a fritted filter. The solid was dried under high vacuum to give 3.50 g (93%) of compound CP-1d as a white powdery solid.

[0377] Coupling Partner 1 (CP-1). Dimethyl sulfoxide (DMSO, reagent grade, 20.0 mL) was added to a 100 mL RBF, and argon was bubbled through with stirring for a total of 45 minutes. 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, and the flask was evacuated for 30 minutes. The flask was purged with argon and degassed DMSO was added. The solution was frozen in a dry ice / acetone bath and placed under vacuum, then thawed under argon. The reaction mixture was then heated in an oil bath at 85 °C.

[0378] After 16 h, the reaction mixture was cooled to room temperature, diluted in EtOAc (100 mL), and washed with brine (3 × 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated.

[0379] The concentrate, rinsed with minimal DCM, was loaded onto a 40 g silica column and eluted with DCM containing 0 - 2% MeOH. The major product fractions were combined and concentrated to afford 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%).

[0380] Example 2

[0381] Synthesis of Compound BC-1

[0382] The title compound was prepared from the previously described dibromobacteriochlorin (BC-SM) (see Jiang et al. (2014) Organic & Biomolecular Chemistry 12: 86 - 103) as shown in Route 3 (see Figure 3 ).

[0383] 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 equipped with a stir bar, and the components were dried under high vacuum for 1 h. 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.

[0384] After 22 h, the reaction mixture was cooled, toluene (4× the volume of DMF) was added, and the mixture was concentrated to dryness. The residue was dissolved in EtOAc (200 mL) and washed with saturated aqueous NaHCO3, 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.

[0385] The crude residue was loaded onto silica gel (3.28 g) and eluted on a 40 g silica gel column with DCM containing 5 - 30% EtOAc for 25 min. The main product fractions were combined, concentrated, and dried under high vacuum to give 0.466 g (73%) of BC-1a as a purple solid.

[0386] BC-1b. BC-1a (146.0 mg, 0.123 mmol) was added to an oven-dried 50 mL RBF equipped 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 dropwise rapidly. The reaction was stirred under argon at room temperature.

[0387] After 1.5 h, the reaction was diluted in DCM (25 mL) and quenched with saturated aqueous NaHCO3 (25 mL). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated.

[0388] The residue was loaded onto a 12 g silica gel column with a minimum amount of DCM (ca. 5 mL) and eluted with DCM containing 0 - 3% MeOH in 22 min. The main peak fractions were combined, concentrated, and dried under high vacuum to give 0.115 g (79%) of BC-1b as a purple solid.

[0389] BC-1c. BC-1b (114.5 mg, 90.6 μmol) was added to an oven-dried 25 mL RBF equipped with a stir bar together 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 min. The flask was flushed with argon, DMF (3.63 mL) was added, followed by triethylamine (0.363 mL) and 6-heptynoic acid (228.7 mg, 1812.0 μmol). The flask was heated in an oil bath at 40 °C and stirred under argon.

[0390] After 16 h, the reaction was diluted with EtOAc (10× the volume of DMF) and washed successively with equal volumes of each of 0.2 N aqueous HCl, water, and brine. The organic layer was separated, dried over sodium sulfate, filtered, concentrated, redissolved in toluene, concentrated, and dried under high vacuum.

[0391] The residue was loaded onto silica (1.05 g) and eluted on a 24 g silica column with DCM containing 0 - 33% EtOAc for 15 min, holding until any starting material was eluted. Then the solvent was switched to DCM containing 0 - 4% MeOH within 15 min. 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.

[0392] BC-1. BC-1c (17.5 mg, 13.36 μmol) was added to an oven-dried 25 mL RBF equipped with a stir bar. The flask was placed under vacuum for 30 min, then flushed with argon, and the vacuum / argon cycle was repeated twice. A solution of hydrogen chloride (4.0 M in dioxane, 2.9 mL) was added in one portion with stirring, and the reaction was stirred under argon. After 30 min, stirring was stopped and the precipitate was allowed to settle for 10 min. Most of the dioxane was removed by syringe under argon. The residue was placed under high vacuum for 2 h.

[0393] The reaction flask was flushed with argon and tributylamine (5 drops) was added and mixed with stirring. Then a 2:1 hexane / THF solution (3 mL) was added and stirred briefly. Then the mixture was sonicated for 3 min, transferred to a 1.5 mL sample tube, and centrifuged to precipitate (9,000 g × 3 min). The clear, colorless supernatant was removed, the tube was covered with parafilm, perforated with a needle, and placed in a flask to dry overnight under high vacuum.

[0394] The resulting solid intermediate, together with cesium carbonate (48.1 mg, 147.6 μmol) and mPEG11-NHS (101.2 mg, 147.6 μmol), was transferred to a dry 25 mL RBF equipped with a stir bar. The flask was sealed with a septum, evacuated, flushed with argon and DMF (2.95 mL) was added. The mixture was stirred for 2 h under argon, protected from light.

[0395] The crude reaction mixture was subjected to reverse-phase chromatography on a 50 g C18 gold column. The fractions containing the product were combined, concentrated, redissolved in ACN and reconcentrated. The residue was dried overnight under high vacuum to give 7.2 mg (16%) of BC-1 as a purple semi-solid.

[0396] MS: Found 1645.1, Calcd 1644.4 [M+2H] 2+ ; λ abs 378, 545, 754 nm (H2O); Correction factor: A280 / A754 = 0.11; λ em 760 nm (H2O); Quantum yield: 8.2% (PBS); Extinction coefficient: 132,000 M -1cm -1 (380 nm, toluene); 104,000 M -1 cm -1 (751 nm, toluene); FWHM: 26 nm.

[0397] 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 (“before centrifugation”) was diluted to 10 μM in PBS to obtain an absorbance reading. The initial 10 mg / mL sample was centrifuged at 14,000 g for 10 minutes in a microcentrifuge. A second portion of the centrifuged sample (“after centrifugation”) was removed and diluted to 10 μM in PBS to obtain an absorbance reading. The data are shown in Table 1 below. No precipitate was observed in the sample. The absorbance values remained consistent from before to after centrifugation (within the expected error of + / - 10%). The decrease in absorbance after centrifugation indicates the presence of an insoluble precipitate in the initial sample. Therefore, it was concluded that BC-1 was dissolved at 10 mg / mL in PBS, pH 7.2.

[0398] Table 1

[0399] Absorbance Readings of the Solubility of BC-1

[0400] 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

[0401] Example 3

[0402] Synthesis of Compound BC-2

[0403] As shown in Route 4 (see Figure 4 ), BC-2a was prepared. A mixture of 5-ethynyl-1,3-benzenedicarboxylic acid (500 mg, 2.63 mmol), N-(2-aminoethyl)carbamic acid tert-butyl ester (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, CH2C12 / MeOH (0-10%)] to afford a white solid. The resulting solid was found to contain DMAP by 1 1H NMR, and thus it was redissolved 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%).

[0404] Starting from the previously described bacteriochlorophyll (BC-SM) (see Jiang et al. (2014) Organic & Biomolecular Chemistry 12: 86 - 103), as shown in Route 5 (see Figure 5 ) BC-2 was prepared.

[0405] 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 an RBF equipped with a three-way tap. The flask was placed under high vacuum for 1 hour and then degassed by three evacuation - refilling 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 gel gold 12 g, hexane / ethyl acetate (0 - 40%)] to give BC-2b as a dark solid (39.4 mg, 25%).

[0406] BC-2c. A mixture of BC-2b (26.6 mg, 33.4 μmol), BC-2a (79.4 mg, 167 μmol), and (PPh3)2PdCl2 (2.3 mg, 3.34 μmol) was placed in an RBF equipped with a three-way tap. The flask was placed under high vacuum for 1 hour and then degassed by three evacuation - refilling cycles. Anhydrous DMF / TEA (2∶1, 10 mL) was added via 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 NaHCO3, and dried over Na2SO4. The resulting mixture was concentrated and chromatographed [silica gel, hexane / ethyl acetate (0 - 40%)] to give BC-2c as a dark solid (11.3 mg, 28%).

[0407] BC-2. BC-2c (4.4 mg, 3.7 μmol) was treated with a dioxane solution of 4.0 M HCl (926 μL). The reaction mixture was stirred under argon at room temperature in the dark. After 4.5 hours, the reaction mixture was placed under high vacuum for 16 hours. CH3(OC2H4) 24 CONHS(mPEG 24-NHS, 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 h, the reaction mixture was chromatographed [C18 15.5 g, H2O / CH3CN (0 - 40%)] to give BC-2 (10.8 mg, 93%) as a green solid. MS: found 1066.64, calcd 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).

[0408] Example 4

[0409] Synthesis of Compound BC-3

[0410] As shown in Route 6 (see Figure 6 ) BC-3 was prepared from BC-2. Solid O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU; 0.45 mg, 1.51 μmol) was added to a 5 mL conical flask with a spinning vane. 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 h. Then the amino PEG 12 CH2CH2COOH (4.24 mg, 6.87 μmol) was added directly to the reaction mixture, followed by the addition of additional DCM (300 μL), and the mixture was stirred at room temperature under argon and protected from light.

[0411] After 16 h, the reaction was diluted with DCM and washed with saturated aqueous NH4Cl (2 mL × 2), then with water (1 mL × 1). The aqueous layer was removed by pipette and the organic layer was concentrated in the reaction vial. The residue was dissolved in ACN / water and eluted on a 15.5 g C18 Isco gold column. BC-3 was isolated as a dark red semi-solid (3.3 g, 65%) after complete drying.

[0412] Example 5

[0413] Synthesis of Compound BC-4

[0414] As shown in Route 7 (see Figure 7 ), BC-4 was prepared from BC-2. BC-2 was treated with a solution of Zn(OAc)2·2H2O (30 equiv) in DMF (4 mM) and heated to 80 °C for 16 h, after which the reaction mixture was concentrated and purified by reverse-phase preparative LC.

[0415] Example 6

[0416] Synthesis of Compound BC-5

[0417] As shown in Route 8 (see Figure 8 ), BC-5 was prepared. 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.

[0418] BC-5b. Compound BC-5a (0.771 g, 2.00 mmol) was added to a flame-dried 250 mL RBF equipped with a stir bar. The flask was evacuated and flushed with argon. Acetonitrile (111.1 mL) was added via syringe. While stirring, BF3·OEt2 (2.96 mL, 24.00 mmol) was rapidly added near the surface of the solvent. A rapid color change was observed. The flask was protected from light and stirred at room temperature under a low flow of argon.

[0419] After 16 h, triethylamine (3.68 mL, 26.4 mmol) was added. The septum was removed and the reaction mixture was stirred until the fumes subsided. The reaction mixture was concentrated and further dried under high vacuum until the flask no longer felt cool to the touch.

[0420] The residue was dissolved in DCM and made into a silica cake (5 g). The cake was eluted on an 80 g SiO2 column with a hexane gradient of 20 - 60% DCM for 17 minutes. The main product fractions were combined, concentrated, and dried under high vacuum to afford 0.11 g (17%) of BC-5b as a dark red solid.

[0421] BC-5c. BC-5b (56.7 mg, 0.088 mmol) and Zn(OAc)2·2H2O (579 mg, 2.64 mmol, 30 equiv) were added to a flame-dried, argon-purged 100 mL RBF equipped 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 placed in an oil bath preheated to 80 °C and stirred overnight under a low flow of argon.

[0422] After 17 h, the reaction mixture was diluted with DCM (5× the volume of DMF), and the organic layer was washed with an aqueous solution of saturated NaHCO3 (3× the volume of DMF + DCM) until the aqueous layer became clear. The organic layer was dried over Na2SO4, 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 afford 61.6 mg (99%) of a dark red solid. The material was used without further purification.

[0423] BC-5d. 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 in Example 3 above; 17.8 mg, 37.5 μmol, 1.5 equiv), Pd(PPh3)2Cl2 (4.4 mg, 6.25 μmol, 0.25 equiv), and CuI (2.4 mg, 12.5 μmol, 0.5 equiv) were added to an oven-dried 25 mL RBF equipped with a stir bar that had been dried under high vacuum and flushed with argon. 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 with Parafilm at the bottom) and evacuated / flushed with argon (3×). 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 a low flow of argon.

[0424] After 16 h, 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 and then dissolved in DCM and made into a silica cake (475 mg SiO2 in 2 loadings). The cake was eluted on a 12 g SiO2 column with hexanes containing 20 - 50% EtOAc for 7 min. After the first major product was eluted, the solvent system was switched to a DCM gradient containing 0 - 5% MeOH within 5 min. The desired product was eluted at this stage. The major product fractions were combined, concentrated, and dried under high vacuum to afford 18.9 mg (62%) of BC-5d as a green solid.

[0425] 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. HCl solution (2.0 mL) was added. The reaction was protected from light and stirred under argon.

[0426] After 4 h, the flask was placed in a warm water bath and flushed with a fast stream of argon with an outlet needle vent until all the solvent was removed. Then the vent was removed, the flask was fitted with a glass adapter and placed under high vacuum overnight. The product was carried forward without further purification.

[0427] 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 was added. The flask was evacuated and flushed with argon. DMF (3.68 mL) was added, followed by rapid addition of triethylamine (24.6 μL, 176.64 μmol, 12.0 equiv) via 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 h.

[0428] 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 h, only a slight conversion was observed. The reaction mixture was heated in a microwave to 100 °C for 10 min and then to 110 °C for 20 min, after which the reaction was observed to be complete by LCMS. The reaction mixture was concentrated to remove DMF and the residue was dissolved in an aqueous solution of 40% ACN (1.3 mL) and subjected to reverse-phase preparative LC with a water gradient containing 35 - 85% ACN over 35 min. The major product peak was combined, concentrated, transferred to ACN in a storage vial, concentrated, and dried under high vacuum to afford 13.3 mg (28% from BC-5d) of BC-5 as a green residue.

[0429] MS: Measured value 1627.4, calculated value 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 (H2O); λ em 863 nm (H2O); FWHM: 38 nm (H2O);

[0430] Example 7

[0431] Synthesis of Compound BC-6

[0432] BC-6. As shown in Route 9 (see Figure 9 ) The title compound was prepared from BC-5. N,N,N′,N′-Tetramethyl-O-(N-succinimidyloxy)uronium tetrafluoroborate (TSTU; 0.61 mg, 2.03 μmol) was added to a 5 mL dry conical flask equipped with a spinning vane. 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 direct addition of triethylamine (0.39 uL, 2.77 μmol) via pipette. The vial was protected from light and the mixture was stirred at room temperature. After 1.5 h, formation of the NHS ester intermediate was determined to be complete by LCMS. The amino-PEG 12 acid was added directly as a solid batch to the reaction vial. The vial was resealed, flushed with argon and stirred at room temperature for 16 h.

[0433] The DCM was removed and the sample was loaded onto water containing 40% CAN and purified using a water gradient containing 40 - 70% ACN on a C18 250x20 column for 30 min. The major product fractions were combined, concentrated and dried under high vacuum to afford 5.0 mg (70%) of BC-6 as a green semi-solid. UV-Vis: 346 / 392, 602, >800 nm.

[0434] Example 8

[0435] Synthesis of Compound BC-7

[0436] BC-7a. As shown in Route 10 (see Figure 10) Preparation of the title compound. 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 HCl (0.5 g, 1.48 mmol) was added. The solution was stirred overnight at room temperature and then diluted with CH2Cl2 (20 mL). The CH2Cl2 solution was washed with saturated aqueous NH4Cl (2×20 mL), water (2×20 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 hexane∶EtOAc gradient (100∶0 to 1∶1). The desired product was isolated as a pale yellow glassy foam which solidified after drying under high vacuum for an extended period (0.35 g, 61%). LCMS: 7.92 min; MS: 431.

[0437] 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 equipped with a stir bar. The vial was sealed with a septum and evacuated / backfilled with argon (3x). Toluene and triethylamine were added and the septum was quickly replaced with a vial cap. The vial was heated in a preheated oil bath at 100 °C for 16 hours.

[0438] 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 SiO2 column (24 g) with a DCM gradient containing 0 - 5% MeOH for 20 minutes. The major product fractions were combined and concentrated to afford 13.9 mg of BC-7b as a dark green solid.

[0439] 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 backfilled with argon (2x). A HCl solution (1.9 mL of a 4.0 M HCl solution in dioxane) was added in one portion with stirring. The mixture was stirred at room temperature for 5 hours.

[0440] The flask was then vented with a needle, placed in a warm water bath, and flushed with a fast-flowing argon gas until all the liquid had been removed. The needle vent was then removed, and the reaction was placed under high vacuum for 2 h. The flask was flushed with argon, tri-n-butylamine (50 μL) was added, and the mixture was stirred. Then hexane / THF (2:1, 3 mL) was added, and the solution was sonicated. The suspension was transferred to a 1.5 mL centrifuge tube and centrifuged at 11 Kg for 3 min. The supernatant was removed. 2:1 hexane / THF was added to the centrifuge tube, then sonicated and centrifuged again at 11 Kg for 3 min. The cycle was repeated one more time. The supernatant was removed, the tube was covered with parafilm, punctured with a 20-gauge needle, placed in a 100 mL RBF, placed under high vacuum for 30 min, and then flushed with argon. 9.3 mg of a dark red solid (94%) was isolated.

[0441] The deprotected product (8.8 mg, 8.53 μmol), mPEG24-NHS (64.4 mg, 51.18 μmol), and cesium carbonate (33.4 mg, 102.36 μmol) were added to an argon-purged 10 mL RBF with a stir bar. The flask was evacuated, flushed with argon, and DMF (2.13 mL) was added. The reaction was protected from light and stirred under argon at room temperature. After 1.5 h, water (0.5 mL) was added, and the solution was concentrated. The residue was diluted in water (1 mL) and subjected to reversed-phase preparative LC with a 10 - 85% ACN aqueous gradient over 35 min. The major product fractions were combined, concentrated, and dried to give a dark red residue (5.1 mg, 13%).

[0442] The PEGylated intermediate (6.8 mg, 1.52 μmol) was dried in a 4 mL glass vial with a stir bar, Zn(OAc)2·2H2O (10.0 mg, 45.73 μmol) was added, followed by DMF (0.6 mL). The vial was equipped with an adapter with an argon inlet and heated to 60 °C in an oil bath. After 16 h, the stir bar was removed, and the reaction was concentrated. The residue was dissolved in water (0.7 mL) and subjected to reversed-phase preparative LC with a water gradient containing 10 - 85% ACN over 30 min. 3.9 mg (57%) of BC-7 in the form of a dark green semi-solid was isolated.

[0443] Example 9

[0444] Synthesis of Compound BC-8

[0445] BC-8. As shown in Route 12 (see Figure 12)The title compound was prepared from BC-5d. BC-5d (18.9 mg, 15.46 μmol, prepared as described in Example 6) was dried in a 10 mL RBF equipped with a stir bar. The flask was sealed with a septum, evacuated and flushed with argon (2x). HCl solution (2.1 mL, 4.0 M in dioxane) was added in one portion with stirring. The mixture was stirred at room temperature for 4.5 h. The solvent was removed by a rapid stream of argon through a needle outlet (ca. 30 min). The flask was then placed under high vacuum for 16 h.

[0446] 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) solid was added. The mixture was stirred for 1 h.

[0447] Zn(OAc)2·2H2O (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 h. The solvent was removed, and the residue was dissolved in water containing 40% ACN (1.4 mL) and purified by reverse-phase preparative chromatography with a gradient of water containing 35–85% ACN over 35 min. The major product BC-8 was isolated as a 12.0 mg dark green semi-solid after complete drying (14% from BC-5d).

[0448] Example 10

[0449] Synthesis of NIRvana 880 Di-tert-butyl Ester

[0450] HBC12 diol. As Figure 13 shown, the NIRvana 880 ditert-butyl ester was synthesized as described in Route 13. HBC12 (115.7 mg, 179.6 μmol) was added to a flame-dried RBF equipped with a stir bar. The flask was evacuated and flushed with argon and DCM (18.0 mL, 10 mM) was added. The solution was cooled to -78 °C and DIBAL-H (1.0 M in toluene, 1.437 mL) was added dropwise over 2 min. The reaction was stirred and gradually allowed to warm to room temperature. Stirring was continued for a total of 4 h. The reaction mixture was diluted with EtOAc and quenched with saturated Rochelle salt solution. The organic layer was washed with water and brine, dried over sodium sulfate, filtered and concentrated. A dark green solid was isolated and used without further purification.

[0451] HBC12 Dialdehyde. HBC12 diol (108.2 mg, 183.9 μmol), 4 Å molecular sieve (powder, 92.0 mg, 0.5 mg / μmol diol), and N-methylmorpholine (dry, 107.7 mg, 919.5 μmol, 5 equiv) were added to a dried 50 mL RBF with a stir bar. The flask was evacuated, flushed with argon, and DCM / ACN (9:1, total 9.3 mL, 20 mM) was added, then tetrapropylammonium perruthenate (TPAP, 12.9 mg, 36.8 μmol, 20 mol%) was added in one portion. The flask was covered with foil and stirred at room temperature for 3.5 h. 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 hexane containing 25 - 65% DCM until all the desired product was eluted. 36.2 mg (34%) of a red solid was isolated.

[0452] ZnHBC12 Dialdehyde. HBC12 dialdehyde (8.0 mg, 13.7 μmol) was added to a dried 25 mL RBF with a stir bar and Zn(OAc)2·2H2O (90.2 mg, 411 μmol) 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 h. The reaction was cooled, diluted with DCM, and quenched with saturated aqueous sodium bicarbonate. The organic layer was separated and further washed with saturated aqueous sodium bicarbonate. The organic layer was dried over sodium sulfate, filtered, and concentrated. The product was used without further purification (nearly quantitative yield of a solid product).

[0453] NIRvana 880 Bis(tert-butyl ester). ZnHBC12 dialdehyde (10.0 mg, 15.44 μmol), tert-butyl ethynylbenzoate, Pd(PPh3)2Cl2 (2.7 mg, 3.86 μmol), and CuI (1.5 mg, 7.72 μmol) were added together to an RBF with a stir bar. The flask was sealed with a septum, evacuated, and flushed with argon. Toluene / triethylamine (2:1, total 3.9 mL) was added, and the reaction was heated at 85 °C for 4 h. The reaction mixture was concentrated and purified by column chromatography. MS: [M+H] + Calcd 889.3; found 888.3 - 890.4 cluster; UV (ACN): 352, 404, 613, 763, 859 nm; em max (ACN): 876 nm.

[0454] Example 11

[0455] Flow Cytometry

[0456] Instrument : Samples were analyzed on a 19-parameter LSR-II SORP flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with 7 lasers (355, 405, 488, 532, 561, 594, and 633 nm) or an LSR Fortessa (BD Biosciences, San Jose, CA, USA) equipped with 5 lasers (355, 405, 488, 561, and 640 nm) using FACSDiva 8.0 acquisition software. BC-1 data were acquired using a 100 mW 355 nm laser with a 690LP filter and a 780 / 60BP filter in channel A. Post-experimental analysis was performed using FlowJo software (version 10.0.8, FlowJo, LLC, Ashland, OR, USA).

[0457] Antibody Bioconjugation : A solution was prepared in a microcentrifuge tube from 106 μL of 9.4 mg / mL (1.0 mg) anti-human CD8 mouse monoclonal antibody (clone UCHT-4, Leinco Technologies, Inc., St. Louis, MO, USA), 15 μL of 1 M bicarbonate (pH 8.4), and 44 μL of a solution of a polyethylene glycolylated dye NHS ester in PBS (5 to 20 molar equivalents). The tube was kept in the dark and gently rotated at room temperature for 1 - 2 hours. The reaction was quenched by adding 15 μL of 200 μM Tris for an additional 1 hour at room temperature. The bioconjugate was purified using a Sephadex G50M, G75M, or G100M size-exclusion chromatography column eluted with PBS. Antibody bioconjugates prepared from dyes with longer PEG chains (12 units or more) were typically purified using a G75M or G100M medium. Column fractions were characterized by absorption at 280 nm (protein) and the absorption maximum of the red or NIR dye. The fluorophore-to-protein (F / P) labeling ratio of the pooled fractions was determined from these two maxima with dye absorption correction at 280 nm.

[0458] Cell Staining:Cryopreserved human peripheral blood mononuclear cells (PBMCs) were obtained from ZenBio, Inc. (Research Triangle Park, NC, USA; product SER-PBMC-F), thawed and prepared for staining according to the supplier's instructions. The cells were aliquoted into six 1.5 mL microcentrifuge tubes and centrifuged at 400 x g (2000 rpm) for 5 minutes. The cells were washed three times with wash buffer (PBS containing 0.5% BSA) and resuspended in 0.5 mL of wash buffer. An aliquot was diluted 1:2 with trypan blue and the cell number and viability were determined by counting the 4 nL squares on a hemocytometer. Viability was typically > 96%. The cells were diluted to 1 x 10 6 / mE with wash buffer and 50 μL (500,000 cells) were aliquoted into microcentrifuge tubes. Typically, the maximum labeled antibody concentration was 4.74 μg / 5 x 10 5 cells (designated 3.16X), and semi-log dilutions were prepared. For all antibodies except the control antibody, these dilutions were made such that 15 μL was added to the cell aliquot. The cells and antibodies were mixed and incubated for 30 minutes at room temperature. Each tube was washed twice with 1 mL of wash buffer and then the cells were resuspended in 0.5 mL of wash buffer containing 1% formaldehyde. Before characterization by flow cytometry, the samples were filtered through nylon mesh into flow cytometry tubes.

[0459] As needed, positive control bioconjugates were 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, the staining index (SI) was calculated from the mean fluorescence intensity (MFI) values as follows:

[0460] SI = (mean: positive - mean: background) / (2 x S.D. background)

[0461] Table 2 below shows staining index data for titrations of the anti-CD8 bioconjugate of BC-1 and of a PEGylated bacteriochlorin similar to BC-1 (containing only PEG4 chains rather than PEG12 chains). For comparison, an anti-CD8 bioconjugate prepared with FITC (Leinco Technologies, Inc., St. Louis, Missouri, USA; catalog number C119) is also provided.

[0462] Table 2

[0463] Staining Index Data of the Titration of Anti-CD8 Dye Conjugates

[0464] Dye F / P Ratio Maximum Staining Index PEG4 Polyethylene Glycolated Bacteriochlorophyll 2.4 21 BC-1 2.7 41 FITC Not Determined 63

[0465] These results indicate that the PEGylation design of BC-1 has significantly enhanced performance compared to PEG4-PEGylated bacteriochlorin.

[0466] References

[0467] All references listed herein, including but not limited to all patents, patent applications and their publications, as well as scientific journal articles, are hereby incorporated by reference in their entirety to the extent that they supplement, explain, provide background, or teach methods, techniques, and / or compositions employed herein.

[0468] It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Further, the foregoing description is for illustrative purposes only and not for purposes of limitation.

Claims

1. A compound of formula (II): Wherein: M is Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe or Zn or is -H, -H; R5, R 10 and R 15 are independently selected from H, alkoxy, and a linking group having the following formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1 is a lower alkylene group; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) q -alkylene, alkylene or substituted alkylene, wherein the substituted alkylene is an alkylene group substituted by one or more groups containing a polyoxyethylene chain and / or an amide group; and G is a bioconjugatable group selected from carboxylic acid or active ester, hydroxyl group, amine, thiol or aldehyde; R3 and R 13 are independently selected from esters; and R2 is the following group: R 12 selected from linking groups having the formula -L1-(X1-L2) p -G, and solubilizing groups selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , where w is 1, 2, 3, 4 or 5, and R S is a group having the following formula: -X2-(L3) z -R 17 , Wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, or -C(=O)NH-alkylene-NH-; L3 is -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 from 12 to 28; n is an integer from 1 to 5; R 18 is methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 .

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

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

4. A compound according to any one of claims 1-3, wherein R3 and R 13 are each -C(=O)OCH3.

5. A compound according to any one of claims 1-3, wherein each R s is a group having the formula: -X2-(L3) z -R 17 , Wherein: z is 0; X2 is -C(=O)NH-alkylene-NH-; and R 17 is -C(=O)C2H4-(OC2H4) m OR 18 , where m is an integer from 12 to 28, and R 18 is methyl.

6. The compound of claim 5, wherein each R s is:

7. A compound according to any one of claims 1-3, wherein each R s is a group having the formula: -X2-(L3) z -R 17 , Wherein: z is 1; X2 is -C(=O)NH-alkylene-NH-; L3 is -C(=O)-propylene-C(=O)-NH-; and R 17 is –(C2H4O) n -C2H4-C(=O)NH-C(R 19 )3, where n is an integer from 1 to 5; and each R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 , where m is an integer from 12 - 28; and R 18 is methyl.

8. A compound according to any one of claims 1-3, wherein R 12 is a linking group having the formula: -L1-(X1-L2) p -G.

9. The compound of claim 8, wherein R 12 is a group having the following formula: -L1-(X1-L2) p -G; Where p is 0; L1 is an arylalkynyl group; and G is a bio-conjugable group selected from carboxylic acid or active ester, hydroxyl group, amine, thiol or aldehyde.

10. The compound of claim 9, wherein R 12 is: Where G is selected from carboxylic acid and active ester.

11. The compound of claim 8, wherein R 12 is a group having the following formula: -L1-(X1-L2) p -G; Where p is 1; L1 is an arylalkynyl group; X1 is -C(=O)NH-; L2 is an alkylene group substituted by one or more groups containing polyoxyethylene chains and / or amide groups; and G is a bio-conjugable group selected from carboxylic acid or active ester, hydroxyl group, amine, thiol or aldehyde.

12. The compound of claim 11, wherein L1 is –C≡C-(C6H4)-.

13. A compound according to claim 11 or claim 12, wherein L2 is -CH(R)-, where R is alkylene-NH-C(=O)-alkylene-(OC2H4) q -OR 16 , where q is an integer from 12 to 24 and R 16 is methyl.

14. The compound of claim 1, wherein the compound is selected from:

15. 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 which is a linking group; and (b) One or more selected from the group consisting of small molecules, microparticles, polymers, peptides, nucleic acids, antibodies, hormones and growth factors.

16. 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 in which at least one is a linking group; and (b) One or more selected from the group consisting of nanoparticles, proteins and antibody fragments.

17. A pharmaceutical composition comprising the compound of claim 1 or the composition of any one of claims 15 - 16, and a pharmaceutically acceptable carrier.

18. A method for detecting a target, wherein the target is a compound, cell or particle, and the method comprises labeling the target with the composition of any one of claims 15 - 16.

19. The method of claim 18, wherein the method comprises using flow cytometry.

20. A method for imaging a cell, tissue or organism, wherein the method comprises using the compound of claim 1 or the composition of any one of claims 15 - 16.

21. Use of the compound of claim 1 or the composition of any one of claims 15 - 16 or the pharmaceutical composition of claim 17 in the preparation of a product for treating a disease, wherein the disease is a hyperproliferative disease.

22. The use of claim 21, wherein the disease is cancer.

23. A water-soluble bacteriochlorophyll dye having the structure of the compound of formula (II) as claimed in claim 1, and having a solubility higher than 1 mg / ml in aqueous solution, optionally having a solubility of 3.0 mg / ml or higher in aqueous solution; further optionally having a solubility of 10 mg / ml or higher in aqueous solution.

24. The water-soluble bacteriochlorophyll dye of claim 23, wherein the dye has an emission wavelength higher than about 850 nm.

25. A method for preparing a synthetic intermediate of a compound of formula (II): Wherein: M is Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe or Zn or is -H, -H; R5, R 10 and R 15 are independently selected from H, alkoxy, and a linking group having the following formula: -L1-(X1-L2) p -G; wherein p is 0 or 1; L1 is a lower alkylene; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) where q is an integer from 1 to 24 q -alkylene, alkylene or substituted alkylene, wherein the substituted alkylene is an alkylene substituted by one or more groups comprising a polyoxyethylene chain and / or an amide group; and G is a bio-conjugatable group selected from a carboxylic acid or active ester, a hydroxyl group, an amine, a thiol or an aldehyde; R3 and R 13 are independently selected from esters; and R2 is the following group: R 12 selected from linking groups having the formula -L1-(X1-L2) p -G, and solubilizing groups selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , where w is 1, 2, 3, 4 or 5, and R S is a group having the following formula: -X2-(L3) z -R 17 , Wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, or -C(=O)NH-alkylene-NH-; L3 is -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 from 12 to 28; n is an integer from 1 to 5; R 18 is methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; Wherein the method comprises: (a) Providing a compound having formula (II’): Wherein: M is Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe or Zn or is -H, -H; R5’, R 10 ’ and R 15 ’ are independently selected from H, alkoxy, and R3’ and R 13 ’ are independently selected from esters; R 12 ' is selected from R2’ is and (b) contacting the compound provided in step (a) with a dioxane solution containing 4 moles (M) of HCl to provide a compound of formula (II”): wherein: M is Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe or Zn or is -H, -H; R5”, R 10 ”, and R 15 ” are independently selected from H, alkoxy, and R3” and R 13 ” are independently selected from esters; R 12 "selected from R2” is 26. A method for preparing an asymmetric bacteriochlorophyll compound having the following formula: Wherein: M is Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe or Zn or is -H, -H; R5, R 10 and R 15 are independently selected from H and alkoxy; R3 and R 13 are independently selected from esters; and R2 is the following group: R 12 selected from 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 a lower alkylene group; X1 is -C(=O)NH- or -NHC(=O)-; L2 is -(CH2CH2O) where q is an integer from 1 to 24 q -alkylene, alkylene or substituted alkylene, wherein the substituted alkylene is an alkylene group substituted by one or more groups containing a polyoxyethylene chain and / or an amide group; G is a bio-conjugatable group selected from carboxylic acid or active ester, hydroxyl, amine, thiol or aldehyde; and wherein said solubilizing group is selected from -aryl-(R s ) w and -alkynyl-aryl-(R s ) w , where w is 1, 2, 3, 4 or 5, and R S is a group having the formula: -X2-(L3) z -R 17 , Wherein: z is 0 or 1; X2 is -CH2NHC(=O)-, or -C(=O)NH-alkylene-NH-; L3 is -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 from 12 to 28; n is an integer from 1 to 5; R 18 is methyl; and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 ; The condition is that: R2 and R 12 are not the same; wherein the method comprises: (a) providing a compound having the following formula: wherein: M is Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe or Zn or is -H, -H; R5’, R 10 ’ and R 15 ’ are independently selected from H and alkoxy; R3’ and R 13 ’ are independently selected from esters; and R2’ and R 12 ’ are each halogen, optionally bromine; and (b) contacting the compound with a palladium catalyst, a base and one of the following: (i) two different alkynes, wherein both of the two different alkynes are compounds having the following 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 alkenes, wherein both of the two different alkenes are compounds having the following 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 organoboronates; wherein the two different organoboronates are two different arylboronic acids or arylboronic esters of the following 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-alkylidene-protected amine or a -C(=O)-NH-substituted alkylidene-protected amine, optionally wherein the substituted alkylidene of the -C(=O)-NH-substituted alkylidene-protected amine comprises an alkylidene substituted with a protected carboxylic acid; and each R 21 is H or alkyl or wherein two R 21 together form an alkylidene.

27. The method of claim 26, wherein based on the relative reactivity of the two alkynes, alkenes or organoboronic esters, the ratio of the two alkynes, alkenes or organoboronic esters is adjusted to maximize the yield of the desired product, optionally wherein, The one with lower reactivity among the two is provided in a greater molar excess than the other of the two.

28. The method of claim 26 or 27, wherein the yield of the desired product is greater than 50%, optionally wherein the yield of the desired product is greater than 60%.

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