A luminescent dye with a narrow range of emission wavelengths, a composition containing the same, and a method for producing and using the same.

Abstract

Bacteriochlorin derivatives having narrow emission bands are provided. In some embodiments, the bacteriochlorin derivatives are PEGylated. In some embodiments, the bacteriochlorin derivatives have high water solubility (e.g., 10 mg / mL or greater). In some embodiments, the bacteriochlorin derivatives are PEGylated to have high water solubility. The bacteriochlorin derivatives can include bioconjugable groups for forming conjugates with, for example, antibodies and nanoparticles. The bacteriochlorin derivatives and their conjugates can be used for imaging and therapeutic applications. Methods for synthesizing the bacteriochlorin derivatives are also provided.

Description

Cross-reference of related applications

[0001] This invention claims priority to U.S. Provisional Patent Application No. 62 / 850,446, filed on 20 May 2019, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] This invention generally relates to bacteriochlorin derivatives, and in some embodiments, to water-soluble bacteriochlorin derivatives having a narrow range of emission wavelengths, and further to their conjugates and methods for producing and using them. [Background technology]

[0003] There is a growing need for fluorescent dyes that are water-soluble and suitable for conjugation with other substances, from nanoparticles to biological targeting agents. Such applications include, for example, flow cytometry, cellular and whole-organism imaging, sensing, and photodynamic therapy. Bacteriochlorins are of particular interest in these applications because they typically absorb in the near-infrared (NIR, 700-900 nm) region and are one of the few chromophores available for photochemical research in the NIR region. The success of the above applications depends on several important factors, including (1) significant solubility in physiological saline solutions, thereby avoiding intermolecular aggregation (and quenching of excited states); (2) minimal nonspecific binding to cellular components; (3) the incorporation of a single reactive group for conjugation, thereby avoiding crosslinking and mixing between products; and (4) robust synthesis that provides sufficient quantities for experiments. However, the large hydrophobic surface of bacteriochlorin presents a challenge to water solubility. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Therefore, there is a continuing need to provide new bacteriochlorin derivatives. These include, but are not limited to, those with improved water solubility (e.g., greater than 1 mg / mL), and especially those that can be easily conjugated with a wide variety of substances. There is also a continuing need for novel bacteriochlorins that combine improved water solubility with narrower absorption and emission bands over a wider wavelength range.

[0005] This summary lists several embodiments of the present invention, and in many cases lists variations and substitutions of these embodiments. This summary is merely an illustration of a number of different embodiments. Even if one or more representative features are mentioned in the embodiments listed, they are equally illustrative. Such embodiments can usually exist with or without the mentioned features. Similarly, these features are applicable to other embodiments of the present invention, whether or not they are listed in this summary. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features. [Means for solving the problem]

[0006] In some embodiments, the present invention provides compounds represented by formula (II): [ka] (wherein M is a metal or two hydrogen atoms; R5, R 10 and R 15 Each of these is independently selected from a hydrogen atom, an alkoxy, and a linker group represented by the following formula; -L1-(X1-L2) p -G Here, p is 0 or 1, L1 is alkylidene, X1 is -C(=O)NH- or -NHC(=O)-, and L2 is -(CH2CH2O) q-alkylene- (wherein q represents an integer of 1 to 24), alkylene, or substituted alkylene (optionally, this substituted alkylene is alkylene substituted by one or more groups including a polyoxyethylene chain and / or an amide group); G is a bio-conjugable group; and R2, R3, R 12 and R 13 are each independently a hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linker group represented by the formula -L1-(X1-L2) p G and a solubilizing group; (wherein this solubilizing group is -aryl-(R s )) w and -alkynyl-aryl-(R s )) w selected from (wherein w is an integer of 0 to 5, and R s is a group represented by the following formula. -X2-(L3) z -R 17 , here, 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 -(C2H4O) m -R 18 , -C(=O)C2H4-(OC2H4) m OR 18 and -(C2H4O) n -C2H4-C(=O)NH-C(R 19 )3 selected from, where m is an integer of 12 or more, n is an integer of 1 to 5, R 18 is lower alkyl (optionally, methyl), and R 19 is -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 .); However, at least one of R2, R3, R 12 and R 13 is -aryl-(R s )) wor -alkynyl-aryl-(R s ) w (That is the case.)

[0007] In some embodiments, M is Zn. In some embodiments, R5, R 10 and R 15 These are, independently, a hydrogen atom, methoxy, and the formula -L1-(X1-L2) p Selected from linker groups represented by -G. In some embodiments, R3 and R 13 Each of these is an ester (arbitrarily -C(=O)OCH3). In some embodiments, R2 is expressed by the following equation. [ka] In some embodiments, each R s This is a base represented by the following equation. -X2-(L3) z -R 17 (In the formula, z is 0, X2 is -C(=O)NH-alkylene-NH-, R 17 is -C(=O)C2H4-(OC2H4) m Ure 18 (In the formula, m is an integer greater than or equal to 12, R 18 It is methyl. In some embodiments, each R s It is expressed by the following formula. [ka]

[0008] In some embodiments, each Rs is a group represented by the following formula. -X2-(L3) z -R 17 (In the formula, z is 1, X2 is -C(=O)NH-alkylene-NH-, L3 is -C(=O)-propylene-C(=O)-NH, R 17 (C2H4O) n-C2H4-C(=O)NH-C(R 19 )3(In the formula, n is an integer from 1 to 5 (arbitrarily it is 4), and each R 19 -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 (In the formula, m is an integer greater than or equal to 12 (arbitrarily, m is 12), R 18 It is methyl. ) It is. ) It is. ) On some days, R2 and R 12 They are not the same, or R3 and R 13 They are not the same. On some days, R2 and R 12 One of them is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w A solubilizing group selected from R2 and R 12 The other of these is equation -L1-(X1-L2) p It is a linker group represented by G, or R3 and R 13 One of them is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w A solubilizing group selected from R3 and R 13 The other side is equation -L1-(X1-L2) p This is a linker group represented by G. In some embodiments, R 12 R is a linker group represented by the formula -L1-(X1-L2)pG. In some embodiments, R 12 This is a base represented by the following equation. -L1-(X1-L2)pG (In the formula, p is 0, L1 is aralkylene, and G is a bioconjugable group.)

[0009] In some embodiments, R 12 It is expressed by the following formula. [ka] (In the formula, G is selected from carboxylic acids and active esters.) In some embodiments, R 12 This is a base represented by the following equation. -L1-(X1-L2) p -G (In the formula, p is 1, L1 is aralkylene, X1 is -C(=O)NH-, L2 is alkylene substituted with one or more groups from the group consisting of polyoxyethylene chains and / or amide groups, and G is a bioconjugable group.) In some embodiments, L1 is -C ≡ It is C-(C6H4)-. In some embodiments, L2 is -CH(R)- (where R is -alkylene-NH-C(=O)-alkylene-(OC2H4)). q -OR 16 (In the formula, q is an integer between 12 and 24, R 16 It is methyl. ) It is. ) It is.

[0010] In some embodiments, the compound is selected from the following: [ka] [ka] [ka]

[0011] [ka] [ka] [ka]

[0012] [ka] [ka] [ka]

[0013] In some embodiments, the present invention provides compositions comprising covalently bonded conjugates formed between the following: (a) R2, R3, R5, R 10 , R 12 , R 13 and R 15 A compound represented by formula (II), wherein at least one of the groups 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. In some embodiments, the present invention provides a compound represented by formula (II) or a conjugate formed between the following: (a) R2, R3, R5, R 10 , R 12 , R 13 and R 15 A compound represented by formula (II) wherein at least one of the groups 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 This includes a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a method for detecting a target, which is a compound, a cell, or a particle, and the method labels the target with a conjugate formed between the following: (a) R2, R3, R5, R 10 , R 12 , R 13 and R 15 A compound represented by formula (II) wherein at least one of the groups 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 In some embodiments, the method includes the use of flow cytometry.

[0014] In some embodiments, the present invention provides a method for imaging a cell, tissue or organism, which includes the use of a compound represented by formula (II) or a conjugate formed between the following. (a) R2, R3, R5, R 10 , R 12 , R 13 and R 15 where at least one of them is a linking group, a compound represented by formula (II), 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, In some embodiments, the present invention provides a method for treating a disease in a patient in need of treatment, which includes the following steps. - A compound represented by formula (II), a conjugate formed between the following, (a) R2, R3, R5, R 10 , R 12 , R 13 and R 15 where at least one of them is a linking group, a compound represented by formula (II), 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, or administering to the patient a pharmaceutical composition containing this compound or this conjugate; and - Irradiating at least a part of the patient with light (Optionally, the disease is a hyperproliferative disease, and further optionally, the disease is cancer.) In some embodiments, the present invention provides a water-soluble bacteriochlorin dye having a solubility greater than about 1 mg / ml in aqueous solution (optionally, having a solubility of about 3.0 mg / ml or more in aqueous solution, and even more optionally, having a solubility of about 10 mg / ml or more in aqueous solution). In some embodiments, the dye has an emission wavelength longer than about 850 nm.

[0015] In some embodiments, the present invention provides a method for producing a synthetic intermediate of a compound represented by the following formula (II). [ka] (wherein M is a metal or two hydrogen atoms; R5, R 10 and R 15 Each of these is independently selected from a hydrogen atom, an alkoxy, and a linker group represented by the following formula; -L1-(X1-L2) p -G Here, p is 0 or 1, L1 is alkylidene, X1 is -C(=O)NH- or -NHC(=O)-, and L2 is -(CH2CH2O) q -Alkylene-(wherein q represents an integer from 1 to 24), alkylene, or substituted alkylene (optionally, this substituted alkylene is an alkylene substituted with one or more groups including a polyoxyethylene chain and / or an amide group); G is a bioconjugable group; and R2, R3, R 12 and R 13 These are, independently, hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, formula -L1-(X1-L2) p Selected from linker groups and solubilizing groups represented by G; (Here, this solubilizing group is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w Selected from (where w is an integer from 0 to 5, Rs This is a base represented by the following equation. -X2-(L3)zR 17 , Here, 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 (C2H4O) m -R 18 -C(=O)C2H4-(OC2H4) m Ure 18 and -(C2H4O) n -C2H4-C(=O)NH-C(R 19 ) Selected from 3, where m is an integer greater than or equal to 12, and n is an integer from 1 to 5, R 18 R is a lower alkyl group (optionally methyl), 19 -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 That is. )); However, R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w (That is the case.)

[0016] This method consists of the following steps: (a) A step of providing a compound represented by formula (II'); and [ka] (wherein M is a metal or two hydrogen atoms; R5',R 10 'and R 15 'Each of these is independently a hydrogen atom, an alkoxy, [ka] Selected from; R2', R3', R 12 'and R 13' are, independently, hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, [ka] Selected from, However, R2', R3', R 12 'and R 13 At least one of ' [ka] (That is the case.) (b) The compound provided in step (a) is brought into contact with a solution containing 4 moles (M) of HCl in dioxane to provide the compound of formula (II) below: [ka] (wherein M is a metal or two hydrogen atoms; R5'',R 10 '' and R 15 Each of the '' elements is independently a hydrogen atom, an alkoxy, [ka] Selected from; R2'', R3'', R 12 '' and R 13 Each of the following is independently a hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, [ka] Selected from, However, R2'', R3'', R 12 '' and R 13 At least one of the '' [ka] (That is the case.)

[0017] In some embodiments, the present invention provides a method for producing an asymmetric bacteriochlorin compound represented by the following formula (II). [ka] (wherein M is a metal or two hydrogen atoms; R5, R 10 and R 15 Each is independently selected from a hydrogen atom and an alkoxy; and R2, R3, R 12 and R 13 Each of these is independently selected from hydrogen atoms, cyano, halo, perhaloalkyl, sulfonates, sulfonamides, esters, carboxylic acids, formyl, acetyl, linker groups, and solubilizing groups; The linker group is represented by the following formula: -L1-(X1-L2) p -G Here, p is 0 or 1, L1 is alkylidene, X1 is -C(=O)NH- or -NHC(=O)-, and L2 is -(CH2CH2O) q -Alkylene-(wherein q represents an integer from 1 to 24), alkylene, or substituted alkylene (optionally, this substituted alkylene is an alkylene substituted with one or more groups including a polyoxyethylene chain and / or an amide group); G is a bioconjugable group; The solubilizing group is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w Selected from (where w is an integer from 0 to 5, R s This is a base represented by the following equation. -X2-(L3)zR 17 , Here, 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 (C2H4O) m -R 18-C(=O)C2H4-(OC2H4) m Ure 18 and -(C2H4O) n -C2H4-C(=O)NH-C(R 19 ) Selected from 3, where m is an integer greater than or equal to 12, and n is an integer from 1 to 5, R 18 R is a lower alkyl group (optionally methyl), 19 -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 That is the case. However, R2 and R 12 If R3 and R are not the same, 13 They are not the same, R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w (That is the case.)

[0018] This method consists of the following steps: (a) A step of providing a compound represented by formula (II'); and [ka] (wherein M is a metal or two hydrogen atoms; R5',R 10 'and R 15 ' is independently selected from hydrogen atoms and alkoxy atoms, R2', R3', R 12 'and R 13 Each of the following is independently selected from hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, and acetyl, and R3' and R 13 These are each Halo (or Bromo, optionally). (b) The step of contacting the compound with a palladium catalyst, a base, and one of the following: (i) Two different alkynes (optionally, both of these alkynes are compounds represented by the following formula: [ka] (In the formula, y is an integer from 1 to 5 (arbitrarily 1 or 2), and each R 20 This 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes protected carboxylic acid-substituted alkylenes). (ii) Two different alkenes (optionally, both of these alkenes are compounds represented by the following formula: [ka] (In the formula, y is an integer from 1 to 5 (arbitrarily 1 or 2), and each R 20 This 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes protected carboxylic acid-substituted alkylenes). (iii) Two different organoboronates (optionally, the two different organoboronates are two different arylboronic acids or arylboronic acid esters of the following formulas: [ka] (In the formula, y is an integer from 1 to 5 (arbitrarily 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes a protected carboxylic acid-substituted alkylene), and each R 21 is either a hydrogen atom or an alkyl group, or two R groups 21 They together form alkylenes.

[0019] In some embodiments, the ratio of the two alkynes, alkenes, or organoboronates is adjusted to maximize the yield of the desired product based on the relative reactivity of the two alkynes, alkenes, or organoboronates. Optionally, the less reactive of the two is provided in molar excess compared to the other compound in step (a). In some embodiments, the yield of the desired product is greater than 50%. Optionally, the yield of the desired product is greater than about 60%.

[0020] Therefore, the object of the present invention is to provide water-soluble bacteriochlorin, its conjugates and pharmaceutical compositions, and methods for using and producing them. These and other objectives are achieved, in whole or in part, by the present invention. Furthermore, the above-mentioned objectives of the present invention, other objectives of the present invention, and advantages of the present invention will become apparent to those skilled in the art upon consideration of the following description, figures, and examples. [Brief explanation of the drawing]

[0021] [Figure 1] Scheme 1 is an exemplary scheme for synthesizing a typical building block of the dibromo bacteriochlorin derivative of the present invention. [Figure 2] Scheme 2 is an exemplary scheme for synthesizing compound CP-1. [Figure 3] Scheme 3 is an exemplary scheme for synthesizing compound BC-1. [Figure 4] Scheme 4 is an exemplary scheme for synthesizing compound BC-2a. [Figure 5] Scheme 5 is an exemplary scheme for synthesizing compound BC-2. [Figure 6] Scheme 6 is an exemplary scheme for synthesizing compound BC-3. [Figure 7] Scheme 7 is an exemplary scheme for synthesizing compound BC-4. [Figure 8]Scheme 8 is an exemplary scheme for synthesizing compound BC-5. [Figure 9] Scheme 9 is an exemplary scheme for synthesizing compound BC-6. [Figure 10] Scheme 10 is an exemplary scheme for synthesizing compound BC-7a. [Figure 11] Scheme 11 is an exemplary scheme for synthesizing compound BC-7. [Figure 12] Scheme 12 is an exemplary scheme for synthesizing compound BC-8. [Figure 13] Scheme 13 is an exemplary scheme for synthesizing NIRvana880 bis-t-butyl ester. [Best Mode for Carrying Out the Invention]

[0022] The present invention is described more fully below. However, while some embodiments of the invention are shown there, not all are. In fact, the present invention can be carried out in many different forms and should not be construed as being limited to the embodiments described herein. These embodiments are provided to satisfy the legal requirements to which the disclosure applies.

[0023] I. Definition The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. All technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art, unless otherwise defined below. References to technologies adopted herein refer to technologies commonly understood in the art, including variations of these technologies or substitutions of equivalent technologies that would be obvious to those skilled in the art. The following terms are expected to be well understood by those skilled in the art, but their definitions are provided below to facilitate the explanation of the present invention. It will be understood that in describing the present invention, many techniques and steps will be disclosed. Each of these has its own advantages, and each may be used in conjunction with one or more, or arbitrarily all, of the other disclosed techniques. Therefore, for clarity, this explanation will refrain from unnecessarily repeating all possible combinations of individual steps. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and claims.

[0024] In accordance with long-standing patent law practice, where used herein, including in the claims, “one (a),” “one (an),” and “it” mean “one or more.” For example, the phrase “fluorescent microparticles and / or nanoparticles” means one or more fluorescent microparticles and / or nanoparticles, including multiple identical fluorescent microparticles and / or nanoparticles. Similarly, the phrase “at least one,” where used herein to refer to an entity, means, for example, that entity including, but not limited to, integer values ​​from 1 to 100 and greater than 100, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more.

[0025] Unless otherwise indicated, all numbers used herein and in the claims to represent quantities of components, reaction conditions, etc., should be understood in all cases to be modified by the term “approximately.” When referring to measurable values ​​such as mass, weight, time, volume, concentration, or percentage (%), the term “approximately” as used herein includes variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the stated value, and these variations are appropriate for carrying out the disclosed method. Accordingly, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by the invention. As used herein, when used in a context of enumerating entities, the term "and / or" refers to entities that exist 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 partial combinations of A, B, C, and D.

[0026] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is comprehensive or unrestricted and does not exclude additional elements and / or method steps that are not cited. "Comprising" is a technical term used in the language of claims, meaning that named elements and / or steps exist, but other elements and / or steps may be added and still be within the scope of the claims. Where used herein, the phrase "consisting of only" excludes any element, step, or component not expressed in the claims. Where the phrase "consisting of only" appears in a clause of the claims rather than immediately following the preamble, it limits the elements described in that clause only, and does not exclude other elements from the claims as a whole. As used herein, the phrase “essentially from” limits the claims to the materials and / or steps specified, and to the extent that it does not substantially affect the disclosure or the basic and novel features of the claims. For example, when it is said that fluorescent microparticles and / or nanoparticles “essentially from” a polymer matrix and at least one bacteriochlorin bound thereto, it means that the polymer matrix referred to is the only polymer matrix present in the fluorescent microparticles and / or nanoparticles.

[0027] With respect to the terms “contains,” “consistes of,” and “essentially consists of,” if any of these three terms is used herein, the subject matter claimed in this disclosure may include the use of any of the other two terms. For example, in some embodiments, the present invention relates to fluorescent microparticles and / or nanoparticles. After reviewing this disclosure, those skilled in the art will understand that the present invention includes fluorescent microparticles and / or nanoparticles essentially consisting of the polymer matrix of the present invention and at least one bacteriochlorin bound thereto, and fluorescent microparticles and / or nanoparticles consisting solely of the polymer matrix of the present invention and at least one bacteriochlorin bound thereto.

[0028] As used herein, "halo" refers to any suitable halogen, including -F, -Cl, -Br, and -I. As used herein, "mercapto" refers to the -SH group. As used herein, "azide" refers to the -N3 group. As used herein, "cyano" refers to the -CN group. As used herein, "hydroxyl group" refers to the -OH group. As used herein, "nitro" refers to the -NO2 group.

[0029] As used herein, "alkyl" alone or as part of another group is a linear or branched hydrocarbon containing 1 or 2 to 10, 20 or 50 carbon atoms (e.g., C1-C4 alkyl, C4-C 10 Alkyl, C 11 ~C 50Alkyl). Representative examples of alkyl groups 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, and n-decyl. As used herein, "lower alkyl" refers to a subset of alkyl groups, which are preferred in some embodiments, and refers to a linear or branched hydrocarbon group containing 1 to 4 carbon atoms. Representative examples of lower alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. The terms “alkyl” or “lower alkyl” are intended to include both substituted and unsubstituted alkyls or lower alkyls unless otherwise indicated, and these groups may be substituted with groups selected from: halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocycloalkoxy, heterocycloalkyloxy, mercapto, alkyl-S(O)m, alkenyl-S(O)m, alkynyl Lu-S(O)m, cycloalkyl-S(O)m, haloalkyl-S(O)m, cycloalkylalkyl-S(O)m, aryl-S(O)m, arylalkyl-S(O)m, heterocyclo-S(O)m, heterocycloalkyl-S(O)m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro, or cyano (wherein m is 0, 1, 2, or 3).

[0030] As used herein, "alkylene" refers to a bifunctional linear, branched, or cyclic alkyl group, which may be substituted or unsubstituted, and "alkyl" is as defined above. In this specification, "alkenyl" as used alone or as part of another group is a linear or branched hydrocarbon containing 1 or 2 to 10, 20 or 50 carbon atoms (e.g., C1-C4 alkenyl, C4-C 10 Alkenil, C 50 Alkenyls (or lower alkenyls with 1 to 4 carbon atoms) typically contain 1 to 4 double bonds in their chain. Representative examples of alkenyls include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, and 2,4-heptadienyl. The terms “alkenyl” or “lower alkenyl” include both substituted and unsubstituted alkenyls or lower alkenyls unless otherwise indicated, and these groups may be substituted with the groups described above in relation to alkyls and lower alkyls. As used herein, "alkenylene" refers to a bifunctional linear, branched, or cyclic alkenyl group that may be substituted or unsubstituted, and "alkenyl" is as defined above.

[0031] As used herein, "alkynyl" alone or as part of another group is a linear or branched hydrocarbon containing 1 or 20 to 10, 20 or 50 carbon atoms (e.g., C1-C4 alkynyl; C4-C 10 Alkinyl; C 11 ~C 50 Alkynnyls (or lower alkynyls with 1 to 4 carbon atoms) typically contain one triple bond in the chain. Representative examples of alkynyls include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, and 3-pentynyl. Unless otherwise indicated, the terms "alkynyl" or "lower alkynyl" are intended to include both substituted and unsubstituted alkynyls or lower alkynyls, and these groups may be substituted with the same groups described above in relation to alkyls and lower alkyls. As used herein, "alkynylene" refers to a bifunctional linear, branched, or cyclic alkynyl group, which may be substituted or unsubstituted, and "alkynyl" is as defined above. As used herein, “alkylidene chain” refers to a bifunctional linear, branched, and / or cyclic organic group, which may be substituted or unsubstituted, saturated or unsaturated, and may optionally contain one, two, or three heteroatoms selected from the group consisting of N, O, and S. Examples include, but are not limited to, alkylenes, alkenylenes, alkylylenes, arylenes, alkalirenes, and aralkylenes. See, for example, U.S. Patent No. 6,946,533. This alkylidene chain may contain any suitable number of carbon atoms (e.g., C1-C1). 4; C4~C 10 ;C 10 ~C 20 ;C 20 ~C 50 ).

[0032] As used herein, "alkoxy," either alone or as part of another group, means an alkyl group or lower alkyl group as defined herein, bonded to the parent molecule via an oxy group (-O-). Representative examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, t-butoxy, pentyloxy, and hexyloxy. As used herein, "acyl" alone or as part of another group refers to a -C(O)R group (wherein R is any suitable substituent as described herein, such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl, or other suitable substituents). In this specification, "haloalkyl," used alone or as part of another group, refers to at least one halogen as defined herein bonded to the parent molecule via an alkyl group as defined herein. Representative examples of haloalkyls include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl. As used herein, "perhaloalkyl" alone or as part of another group refers to an alkyl group in which each hydrogen atom of the alkyl group is replaced by a halo. In some embodiments, this perhaloalkyl is a perfluoroalkyl group, in which each hydrogen atom of the alkyl group is replaced by a fluoro. A typical perhaloalkyl group is trifluoromethyl (i.e., -CF3). In this specification, “alkylthio,” used alone or as part of another group, refers to an alkyl group as defined herein, bonded to the parent molecule via a thio moiety as defined herein. Typical examples of alkylthio include, but are not limited to, methylthio, ethylthio, t-butylthio, and hexylthio.

[0033] In this specification, "aryl," used alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryls include azlenyl, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. Unless otherwise indicated, the term "aryl" is intended to include both substituted and unsubstituted aryls, which may be substituted with the same groups described above in relation to alkyls and lower alkyls. As used herein, "arylene" refers to a difunctional aryl group which may be substituted or unsubstituted, and "aryl" is as defined above. As used herein, "arylalkyl" alone or as part of another group refers to an aryl group as defined herein, bonded to the parent molecule via an alkyl group as defined herein. Representative examples of arylalkyls include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and 2-naphtho-2-ylethyl. As used herein, "alkalene" and "aralkylene," either alone or as part of another group, refer to a difunctional group comprising at least one arylene group and at least one alkyl, alkenyl, or alkynyl group, as defined herein. As used herein, "amino" refers to the -NH2 group. In this specification, "alkylamino" used alone or as part of another group means an -NHR group (wherein R is an alkyl group). In this specification, "arylalkylamino," used alone or as part of another group, means an -NHR group (wherein R is an arylalkyl group).

[0034] In this specification, "disubstituted amino" used alone or as part of another group is -NR a R b group (in the formula, R a and R b This means that the group is independently selected from the group consisting of alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, and heterocycloalkyl. The terms "acylamino" used herein, either alone or as part of another group, are -NR a R b group (in the formula, R a R is an acyl group as defined herein, b This refers to a group selected from hydrogen atoms, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, and heterocycloalkyl atoms. As used herein, "acyloxy" alone or as part of another group means an -OR group (wherein R is an acyl group as defined herein). As used herein, "ester" alone or as part of another group refers to a -C(O)OR group (wherein R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl). As used herein, "formyl" refers to the -C(O)H group. As used herein, "carboxylic acid" refers to the -C(O)OH group.

[0035] As used herein, "sulfoxyl" refers to a compound represented by the formula -S(O)R (wherein R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl). As used herein, "sulfonyl" refers to a compound represented by the formula -S(O)(O)R (wherein R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl). As used herein, "sulfonate" refers to a compound represented by the formula -S(O)(O)OR (wherein R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl). As used herein, "sulfonic acid" refers to a compound represented by the formula -S(O)(O)OH. In this specification, "amide" used alone or as part of another group means -C(O)NR a R b group (in the formula, R a and R b ) refers to any suitable substituent such as a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl atom. In this specification, "sulfonamide" used alone or as part of another group means -S(O)2NR a R b group (in the formula, R a and R b ) refers to any suitable substituent such as a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl atom.

[0036] As used herein, "urea" alone or as part of another group refers to -N(R c )C(O)NR a R b group (in the formula, R a , R b and R c ) refers to any suitable substituent such as a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl atom. In this specification, "alkoxyacylamino" used alone or as part of another group is -N(Ra )C(O)OR b group (in the formula, R a , R b ) refers to any suitable substituent such as a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl atom. In this specification, "aminoacyloxy" used alone or as part of another group means -OC(O)NR a R b group (in the formula, R a and R b ) refers to any suitable substituent such as a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl atom. As used herein, either alone or as part of another group, “cycloalkyl” refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3, 4 or 5-6, 7 or 8 carbon atoms (these carbon atoms may be substituted with heterocyclic groups as discussed below). Representative examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may optionally be substituted with additional substituents described herein, such as halo or lower alkyl groups. The term “cycloalkyl” is general and, unless otherwise specified, is intended to include the heterocyclic groups described below.

[0037] As used herein, the term "polyoxyethylene chain" refers to a poly(ethylene glycol) (PEG) group, e.g., formula -(C2H4O) n - (wherein n is an integer greater than or equal to 2 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or greater)) refers to a portion containing or consisting solely of a group represented by the formula -. In some embodiments, n is an integer between 4 and 5000, 4 and 1000, 4 and 100, 4 and 50, 4 and 28, or 4 and 25. As used herein, the term “polyoxyethylene chain” may refer to monodisperse or polydisperse PEG chains, and linear or branched PEG chains. “Monodisperse” refers to PEG with a polydispersity index (PDI) of 1, and “polydisperse” refers to PEG with a PDI greater than 1, where the PEG includes those with a Gaussian distribution of chain length and molecular weight.

[0038] As used herein, the term “biofunciable group” refers to a reactive chemical functional group that can form a bond (e.g., a covalent bond) with a group on another entity (e.g., a protein; a peptide; a targeting agent such as an antibody or antibody fragment; a polymer; particles such as nanoparticles, organic, polymer, or inorganic beads; another solid support surface, etc.) to form a conjugate of one bacteriochlorin compound of the present invention with another entity. This biofunciable group may be, for example, an aldehyde (which can form a covalent bond with an amino group on an amino-substituted biomolecule via reductive amination) or a carboxylic acid (which can bond to an amino-substituted biomolecule via carbodiimide activation). These bioconjugable groups include, but are not limited to, amines (including amine derivatives) such as isocyanates, isothiocyanates, iodoacetamide, azides, and diazonium salts; carboxylic acids or acid derivatives such as N-hydroxysuccinimide (NHS) esters (more commonly, active esters derived from carboxylic acids; e.g., p-nitrophenyl esters) and acid hydrazides; and other groups such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyls, halo groups, biotin, and 2-iminobiotin.

[0039] The term "microparticle" refers to a structure having dimensions (e.g., length, width, diameter, etc.) of less than approximately 1,000 μm but greater than approximately 1,000 nm, and also less than approximately 5 μm. In some embodiments, these dimensions may be less than approximately 500 μm, less than approximately 250 μm, less than approximately 200 μm, less than approximately 150 μm, less than approximately 125 μm, less than approximately 100 μm, less than approximately 80 μm, less than approximately 70 μm, less than approximately 60 μm, less than approximately 50 μm, less than approximately 40 μm, less than approximately 30 μm, less than approximately 20 μm, less than approximately 10 μm, and less than approximately 5 μm. In some embodiments, this dimension is between approximately 1 μm and approximately 250 μm (for example, approximately 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). Similarly, the term “nanoparticle” refers to a structure having at least one region whose dimensions (e.g., length, width, diameter, etc.) are less than approximately 1,000 nm. In some embodiments, this dimension is smaller (e.g., less than approximately 500 nm, less than approximately 250 nm, less than approximately 200 nm, less than approximately 150 nm, less than approximately 125 nm, less than approximately 100 nm, less than approximately 100 nm, less than approximately 80 nm, less than approximately 70 nm, less than approximately 60 nm, less than approximately 50 nm, less than approximately 40 nm, less than approximately 30 nm, or less than approximately 20 nm). In some embodiments, this dimension is between approximately 5 nm and approximately 250 nm (for example, approximately 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).

[0040] In some embodiments, the microparticles or nanoparticles are substantially spherical. When the microparticles or nanoparticles are substantially spherical, their characteristic dimensions can correspond to the diameter of the sphere. In addition to being spherical, the microparticles or nanoparticles may be disc-shaped, plate-shaped (e.g., hexagonal plate-shaped), rectangular, polyhedron-shaped, rod-shaped, cubic, or irregular in shape. The microparticle or nanoparticle may include a core region (i.e., the space between the outer dimensions of the particle) and an outer surface (i.e., the surface defining the outer dimensions of the particle). In some embodiments, the microparticle or nanoparticle may have one or more coating layers surrounding or partially surrounding the core of the microparticle or nanoparticle. Thus, for example, a spherical microparticle or nanoparticle may have one or more concentric coating layers, where each continuous layer is smaller closer to the center of the particle and extends over the outer surface.

[0041] The terms "polymer" and "polymeric" refer to a chemical structure having repeating units (i.e., multiple copies of a given chemical basis structure). Polymers can be formed from polymerizable monomers. A polymerizable monomer is a molecule containing one or more parts that can react to form bonds (e.g., covalent or coordination bonds) with parts on other molecules of the polymerizable monomer. In some embodiments, each polymerizable monomer molecule can bond to two or more other molecules / parts. In some cases, a polymerizable monomer bonds to only one other molecule, forming the ends of the polymer material. The polymer may be organic, inorganic, or a combination thereof. As used herein, the term “inorganic” refers to a compound or composition containing at least several atoms other than one of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorus, or halogens. Thus, for example, an inorganic compound or inorganic composition may contain one or more silicon atoms and / or one or more metal atoms. In some embodiments, the polymer is polystyrene, and the microparticles and / or nanoparticles are made of polystyrene. In some embodiments, the microparticles and / or nanoparticles are polystyrene beads.

[0042] As used herein, the term "porphyrin" typically refers to a cyclic structure consisting of four pyrrole rings, each having four nitrogen atoms and two replaceable hydrogen atoms that can be readily substituted for various metal atoms. A typical porphyrin is hemin. As used herein, "bacteriochlorin" differs from porphyrin in that it has two partially saturated, non-adjacent (i.e., trans)pyrrole rings. The terms "bacteriochlorin" and "bacteriochlorin derivative" are used interchangeably herein.

[0043] The phrase “to bond” refers to any interaction between two entities, for example, any interaction between a polymer matrix and bacteriochlorin. In some embodiments, the polymer matrix and bacteriochlorin are bonded to each other by non-covalent bonds (such as, but not limited to, one or more of hydrophobic, electrostatic, and van der Waals interactions). In some embodiments, the polymer matrix (e.g., nanoparticles, microparticles, beads, etc.) and bacteriochlorin are bonded to each other as a result of the polymer matrix containing the bacteriochlorin so that the bacteriochlorin is present within the polymer matrix. In such embodiments, this polymer matrix is ​​also called “doped” with bacteriochlorin, and the bacteriochlorin can be considered “embedded” within the polymer matrix. In some embodiments, the polymer matrix and bacteriochlorin are bonded to each other by covalent bonds that cause the bacteriochlorin to adhere to the surface of the polymer matrix.

[0044] As used herein, “treatment” means any method by which one or more symptoms of a disease or disorder are improved or beneficially altered. “Treatment” also encompasses any pharmaceutically appropriate use of the compositions of the present invention, such as a method of use for treating disease or disorder-mediated hyperplasia or angiogenesis, or a disease or disorder in which hyperplasia or angiogenesis is involved. In this specification, improvement of symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition means any relief, whether permanent or temporary, sustained or transient, that may be caused by or related to the administration of the composition. As used herein, "prodrug" refers to a compound that, when administered in vivo, is metabolized by one or more steps or processes, or converted to an active form of the compound biologically, pharmaceutically, or therapeutically.

[0045] As used herein, “antibody” generally refers to an immunoglobulin or a fragment thereof that specifically binds to an antigen to form an immune complex. This antibody may be any class of whole immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE), or a chimeric or hybrid antibody having bi- or multi-antigen or epitope specificity. It may also be a polyclonal antibody, preferably an affinity-purified antibody obtained from a human or suitable animal (e.g., primates, goats, rabbits, mice, etc.). Monoclonal antibodies are also suitable for use in the present invention and may be preferred due to their high specificity. These are readily prepared by what are now considered conventional procedures, such as immunization of mammals with immunogenic antigen preparations, fusion of immune lymphoid or splenic cells with immortal myeloma cell lines, and isolation of specific hybridoma clones. Newer methods for preparing monoclonal antibodies (e.g., interspecies fusion and genetic engineering of hypervariable regions) are not excluded, as it is primarily the antigen specificity of the antibody that influences their usefulness. Furthermore, newer technologies for producing monoclonals (e.g., human monoclonal antibodies, interspecies monoclonal antibodies, chimeric (e.g., human / mouse) monoclonal antibodies, genetically modified antibodies, etc.) may be used.

[0046] Accordingly, the terms “antibody” and “multiple antibodies” refer to proteins comprising one or more polypeptides substantially encoded by an immunoglobulin gene or fragment thereof. Immunoglobulin genes typically include kappa (κ), lambda (λ), alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ) invariant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. In mammals, heavy chains are classified as γ, μ, α, δ, or ε, defining the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Other species have other light and heavy chain genes (for example, certain birds produce what is called IgY, an immunoglobulin type deposited in the yolk of chicken eggs), and these are also included in the present invention.

[0047] Typical immunoglobulin (antibody) structural units are known to contain tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (average molecular weight approximately 25 kDa) and one "heavy" chain (average molecular weight approximately 50-70 kDa). The two identical pairs of polypeptide chains are held together in dimer form by a disulfide bond present within the heavy chain region. The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, which is primarily involved in antigen recognition. L ) and variable heavy chain (V H The terms ) refer to these light and heavy chains, respectively. Antibodies typically exist as intact immunoglobulins or as many well-characterized fragments that can be produced by digestion with various peptidases. For example, digestion of an antibody molecule with papain cleaves the antibody at the N-terminus of the disulfide bond. This produces three fragments: two identical "Fab" fragments with the N-terminuses of the light and heavy chains, and one "Fc" fragment containing the C-terminus of the heavy chain linked by the disulfide bond. Pepsin, on the other hand, digests the antibody at the C-terminus of the disulfide bond in the hinge region, producing a fragment known as the "F(ab)'2" fragment. This is a dimer of two Fab fragments linked by the disulfide bond. This F(ab)'2 fragment is reduced under mild conditions, breaking the disulfide bond in the hinge region and converting the F(ab')2 dimer into two Fab' monomers. These Fab' monomers are essentially Fab fragments that possess a portion of this hinge region. With respect to these various fragments, Fab, F(ab)'2, and Fab fragments contain at least one intact antigen-binding domain ("paratope") and are therefore capable of binding to an antigen.

[0048] While various antibody fragments are defined in relation to the digestion of intact antibodies, those skilled in the art will understand that these various fragments (including, but not limited to, Fab' fragments) can be newly synthesized chemically or using recombinant DNA methods. Therefore, as used herein, the term “antibody” includes both antibody fragments produced by modification of the entire antibody and antibody fragments newly synthesized using recombinant DNA methods. In some embodiments, the term “antibody” includes fragments having at least one antigen-binding domain.

[0049] Furthermore, the antibodies, fragments, and derivatives of the present invention may include chimeric antibodies. The term “chimeric” and its grammatical variations as used herein in the context of antibodies refer to antibody derivatives having an invariant region substantially or exclusively derived from a certain antibody invariant region and a variable region substantially or exclusively derived from a variable region of another species. Certain types of chimeric antibodies are “humanized” antibodies, which are produced by replacing the determination site (CDR) of their human antibody with, for example, the complementarity determination site (CDR) of a mouse antibody (see, for example, International Publication WO 1992 / 22653). Thus, in some embodiments, a humanized antibody has an invariant region and a variable region that are not substantially or exclusively derived from the corresponding human antibody region, and a determination site (CDR) substantially or exclusively derived from a non-human mammal. The antibodies, fragments, and derivatives of the present invention may be single-chain antibodies and single-chain antibody fragments. Single-chain antibody fragments contain an amino acid sequence having at least one variable region and / or determination site (CDR) of the entire antibody described herein, but lack some or all of the invariant domains of the antibody. These invariant domains are not necessary for antigen binding but constitute a large part of the structure of the entire antibody.

[0050] Single-chain antibody fragments can overcome some of the problems associated with the use of antibodies that contain some or all of the invariant domain. For example, single-chain antibody fragments tend to lack undesirable interactions between biomolecules and heavy chain invariant regions, and other undesirable biological activities. Furthermore, single-chain antibody fragments are considerably smaller than whole antibodies and have higher capillary permeability, allowing them to localize more efficiently and bind to target antigen-binding sites. Also, antibody fragments are produced relatively on a large scale in prokaryotic cells, thus facilitating their production. Moreover, because single-chain antibody fragments are relatively small in size, they are less likely to elicit an immune response in recipients than whole antibodies. The single-chain antibody fragments of the present invention include, but are not limited to, single-chain fragment variable (scFv) antibodies and their derivatives, such as tandem di-scFv, tandem tri-scFv, diabodies, and further include bispecificity diabodies, triabodies, tetrabodies, mini-antibodies, mini-bodies, tetravalent bispecificity molecules, bispecificity F(ab')2 fragments, etc.

[0051] As used herein, “infectant” means an invading microorganism or parasite. As used herein, “microorganism” means viruses, bacteria, rickettsiae, mycoplasmas, protozoa, fungi and similar microorganisms, and “parasite” means an infectious, generally microscopic or very small multicellular invertebrate or its eggs or larvae that are susceptible to antibody-induced clearance or lytic or phagocytic destruction (e.g., malaria parasites, spirochets, etc.). As used herein, “tumor” means a neoplasm and includes both benign and malignant tumors. The term particularly includes malignant tumors, which may be solid (such as breast cancer, liver cancer, or prostate cancer) or non-solid (such as leukemia). These tumors may also be further classified into subtypes, such as adenocarcinomas (of the breast, prostate, or lung).

[0052] As used herein, “target” means an object intended to be detected, diagnosed, injured or destroyed by the methods of the present invention, and includes target cells, target tissues and target compositions. As used herein, “target tissue” and “target cells” refer to tissues that are intended to be damaged or destroyed by this therapeutic method. Photosensitizing compounds bind to or accumulate in these target tissues or target cells, and then, upon sufficient radiation, these tissues or cells are damaged or destroyed. Target cells are cells within target tissues, which include, but are not limited to, vascular endothelial tissue, abnormal vascular walls of tumors, solid tumors such as head and neck tumors, tumors of the eyes, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lungs, non-solid tumors and malignant cells of hematopoietic and lymphoid tissues, angiogenic tissue, and other lesions of the vascular system, bone marrow, tissues, or cells associated with autoimmune diseases. Target cells also include cells that are undergoing substantially more rapid division compared to non-target cells.

[0053] As used herein, “non-target tissue” refers to all tissues in a subject that are not intended to be injured or destroyed by this treatment method. These non-target tissues include, but are not limited to, healthy blood cells and other normal tissues that are not otherwise identified as targets. As used herein, “Target Composition” refers to a composition intended to be injured or destroyed by this therapeutic method, and includes, but is not limited to, pathogens (bacteria, viruses, fungi, protozoa, toxins, and cells and tissues infected or infiltrated by them). The “Target Composition” also includes, but is not limited to, infectious organic particles such as prions, toxins, peptides, polymers, and other compounds that can be selectively and specifically identified as organic targets intended to be injured or destroyed by this therapeutic method. As used herein, “hyperproliferative tissue” means tissue that grows uncontrollably and includes neoplastic tissue, tumors, and unsuppressed vascular growth (such as that seen in age-related macular degeneration and often occurring after glaucoma surgery). As used herein, “hyperproliferative disorder” refers to a disorder of a condition that shares excessive cell proliferation as an underlying pathology, caused by uncontrolled or abnormal cell proliferation, and includes uncontrolled angiogenesis. Examples of this hyperproliferative disorder 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, recurrent pterygium, and scarring from excimer laser surgery and glaucoma filtration surgery.

[0054] As used herein, “therapeutic dose” is a dose sufficient to prevent progression or cause regression of the disease, or a dose that can alleviate the symptoms caused by the disease. As used herein, “biological material” refers to both tissues (such as biopsy tissue) and cells, as well as bodily fluids such as blood, urine, plasma, cerebrospinal fluid, mucus, and sputum.

[0055] As used herein, “irradiation” and “irradiation” include exposing the object to light of all wavelengths. The irradiation wavelength is preferably selected to match the wavelength that excites the photosensitive compound. The emission wavelength is preferably matched to the excitation wavelength of the photosensitive compound and has low absorption by non-target tissues of the object, including blood proteins. Irradiation is further defined by its coherence (laser) or non-coherence (non-laser), as well as the intensity, duration, and timing of the administration of photosensitizing compounds. 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 for sufficient photoactivation so that the photosensitizing compound acts effectively on the target tissue. The timing of the administration of the photosensitizing compound is also important because 1) it takes some time for the administered photosensitizing compound to return to the target tissue, and 2) the blood concentration of many photosensitizing compounds decreases over time. Irradiation energy is provided by an energy source, such as a laser or cold cathode light source, which may be external to, implanted in, or introduced into the subject (e.g., by catheter or fiber optic, or by ingesting the light source in the form of a capsule or pill (e.g., U.S. Patent No. 6,273,904)).

[0056] While some embodiments of the present invention are directed towards the use of light energy for administering photodynamic therapy (PDT) to destroy tumors, other forms of energy are also within the scope of the present invention, which will be understood by those skilled in the art. Such forms of energy include, but are not limited to, thermal, sound, ultrasonic, chemical, optical, microwave, ionization (such as X-rays and gamma rays), mechanical, and electrical energy. For example, drugs that are acoustically induced or activated include, but are not limited to, gallium-porphyrin complexes (Yumita et al. (1997) Cancer Letters 112:79-86), other porphyrin complexes such as protoporphyrin and hematoporphyrin (Umemura et al. (1996) Ultrasonics Sonochemistry 3:S187-S191), and other anticancer drugs such as daunorubicin and adriamycin used in the presence of ultrasound therapy (Yumita et al. (1987) Japanese Journal of Hyperthermic Oncology 3(2):175-182). As used herein, "coupling agent" refers to a reagent capable of coupling a photosensitizer to a target agent. A “targeting agent” refers to a compound that attributes to, preferentially binds to, or associates with a specific tissue, receptor, infectant, or other area of ​​the body being treated (such as a target tissue or target composition). Examples of targeting agents include, but are not limited to, antibodies, ligands, one member of a ligand-receptor binding pair, nucleic acids, peptide-nucleic acid (PNA), aptamers, proteins and peptides, and liposome suspensions (including tissue-targeted liposomes).

[0057] As used herein, “specific binding pair” and “ligand-receptor binding pair” refer to two distinct molecules, one of which has a surface or cavity region that specifically attracts or binds to a particular spatial or polar tissue of the other molecule, causing both molecules to have affinity for each other. The two members of this specific binding pair are called the ligand and the receptor (antiligand). The terms ligand and receptor include the entire ligand or receptor, or a part thereof, sufficient for binding to occur between the ligand and the receptor. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors (e.g., 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 antibiotin); antibody and antigen pairs; enzymes and substrates, drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid chains; nucleic acid chains and complementary oligonucleotides; interleukins and interleukin receptors; and stimulating factors and their receptors (e.g., granulocyte-macrophage colony-stimulating factor (GMCSF) and GMCSF receptor, and macrophage colony-stimulating factor (MCSF) and MCSF receptor).

[0058] A "linker" is an aromatic or aliphatic group (substituted or unsubstituted, optionally containing heteroatoms such as N, O, or S) used to bond bioconjugable groups, cross-coupling groups, surface attachment groups, hydrophilic groups, etc., to a parent molecule. Examples of linkers include, but are not limited to, aryl, alkyl, heteroaryl, heteroalkyl (e.g., oligoethylene glycol), peptides, and polysaccharides. Patients (subjects) treated by the methods of the present invention for diagnosis or treatment include both human patients and patients of other animals for veterinary purposes (in particular mammalian patients such as dogs, cats, horses, monkeys, and chimpanzees). More specifically, the terms “patient,” “subject,” and “recipient” as used herein are interchangeable and may refer to any member of any invertebrate or vertebrate species. Thus, the term “patient” is intended to encompass any member of the animal kingdom, including, but not limited to, the phylum Chordata (e.g., bony fishes, amphibians, reptiles, birds, and mammals).

[0059] The compositions and methods of the present invention are particularly useful for warm-blooded vertebrates. Therefore, the present invention relates to mammals and birds. More specifically, compositions and methods derived from and / or for use in mammals are provided. These mammals include, for example, humans and other primates, and further include mammals that are important because they are endangered (such as the Siberian tiger), mammals that are economically important to humans (animals raised on farms for human consumption), and / or mammals that are socially important (animals kept as pets or in zoos). Methods for using the compositions and methods of the present invention on birds are also provided. These birds include endangered birds, birds kept in zoos, birds kept as pets (such as parrots and cockatiels), and specifically, poultry such as turkeys, chickens, ducks, geese, and guinea fowl, as they are also economically important to humans. Therefore, methods for using the compositions and methods of the present invention on livestock are also provided. These livestock include, but are not limited to, domesticated pigs (piglets and pigs for meat), ruminants, horses, and poultry.

[0060] II. Bacteriochlorin compounds In some embodiments, the present invention provides a water-soluble bacteriochlorin having a solubility of about 1 milligram / milliliter (mg / mL) or more in an aqueous solution (e.g., water, physiological saline, PBS, etc.). Water solubility is provided, for example, by adding a solubilizing group containing a PEG chain at the position of β-pyrrole. In some embodiments, this PEG chain is bonded to the bacteriochlorin via a different group and / or is longer than the PEG chain bonded to the above-mentioned PEGylated bacteriochlorin compound. In some embodiments, the solubility of this bacteriochlorin in an aqueous solution is about 3.0 mg / mL or more. In some embodiments, this bacteriochlorin has a solubility of about 5.0 mg / mL or more in an aqueous solution. In some embodiments, this bacteriochlorin has a solubility of about 10 mg / mL or more in an aqueous solution. In some embodiments, the bacteriochlorin has a solubility of about 500, about 600, about 700, about 800, about 900 μM or higher in aqueous solution. In some embodiments, the bacteriochlorin has a solubility of about 1, 1.5, 2, 2.5, or 3 mM or higher in aqueous solution. In some embodiments, the bacteriochlorin has an emission wavelength longer than about 700 nm. In some embodiments, the bacteriochlorin has an emission wavelength longer than about 800 nm. In some embodiments, the bacteriochlorin has an emission wavelength of about 850 nm or longer.

[0061] In some embodiments, the water-soluble bacteriochlorin includes a linker moiety containing a bioconjugable group that can be used to conjugate the bacteriochlorin to another substance (e.g., one that can act as a targeting agent or a detectable substance). In some embodiments, the substance that can be conjugated to this bacteriochlorin may be a small molecule (e.g., a non-polymeric synthetic molecule having a molecular weight of about 900 daltons (Da) or less), an antigen, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, or growth factors. The bioconjugable group may include, for example, a carboxylic acid or active ester, a hydroxyl group, an amine, a thiol, or an aldehyde. In some embodiments, the linker moiety further includes both an arylene group and an alkylene group. In some embodiments, the linker moiety includes an arylene and / or alkylene group proximal to the main bacteriochlorin structure, while the alkylene group is proximal to the bioconjugable group.

[0062] In some embodiments, this bacteriochlorin comprises at least one solubilizing group. In some embodiments, this solubilizing group comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) polyoxyethylene chains (e.g., PEG chains). In some embodiments, this solubilizing group comprises at least two PEG chains. In some embodiments, this PEG chain is monodisperse and comprises at least 4 -CH2CH2O- repeating units. In some embodiments, this PEG chain comprises at least 6, 8, 10 or 12 -CH2CH2O- repeating units. Thus, this solubilizing group can comprise two PEG6, PEG8, PEG10, or PEG12 groups. In some embodiments, this PEG chain comprises 12 or more -CH2CH2O- repeating units (e.g., about 12 to about 24 or about 28 -CH2CH2O- repeating units). In some embodiments, this compound comprises two solubilizing groups attached to two different pyrrolic carbons. In some embodiments, these two solubilizing groups each comprise two PEG chains. In some embodiments, this solubilizing group is a β-pyrrole substituent, and this β-pyrrole substituent comprises an arylene or alkynyl-arylene group directly bonded to a bacteriochlorin pyrrole carbon atom, but this solubilizing group does not comprise a direct oxoline linker between the PEG chain and the aryl group. In some embodiments, this solubilizing group comprises one or more amide bonds between this aryl group and the PEG chain. In some embodiments, this amide bond further comprises one or more alkylene spacers (e.g., ethylene, propylene, etc.).

[0063] In some embodiments, this bacteriochlorin is a compound represented by the following formula (II): [Chemical formula] R5, R 10 and R 15 ​is independently selected from a hydrogen atom, an alkoxy, and a linker group represented by the following formula; -L1-(X1-L2) p -G where p is 0 or 1, L1 is an alkylidene, X1 is -C(=O)NH- or -NHC(=O)-, and L2 is -(CH2CH2O) q -alkylene- (where q represents an integer from 1 to 24), alkylene, or substituted alkylene (e.g., alkylene substituted by one or more groups including a polyoxyethylene chain and / or an amide group); G is a bio-bondable group; and R2, R3, R 12 and R 13 are each independently selected from a hydrogen atom, cyano, halo, perhaloalkyl (e.g., perfluoroalkyl such as perfluoromethyl), sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, a linker group represented by the formula -L1-(X1-L2) p G and a solubilizing group; (where this solubilizing group is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w selected from (where w is an integer from 0 to 5 (where when w is 0, this chlorin derivative is water-insoluble (i.e., hydrophobic), and when w is 1, 2, 3, 4, or 5, this chlorin derivative is water-soluble (i.e., hydrophilic)), R s is a group represented by the following 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 -(C2H4O) m -R 18 、-C(=O)C2H4-(OC2H4) m OR 18 and -(C2H4O) n-C2H4-C(=O)NH-C(R 19 ) Selected from 3, where m is an integer greater than or equal to 12 (e.g., 12, 14, 16, 18, 20, 22, 24, 26, or 28), and n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), R 18 R is a lower alkyl group (e.g., methyl), 19 -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18 That is. ); However, R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w (That is the case.)

[0064] M is any suitable metal ion (e.g., Pd, Pt, Mg, Al, Ga, In, Sn, Au, Ni, Cu, Co, Fe, or Zn) or none at all (for example, in this case it can be replaced by two hydrogen atoms (-H, -H), i.e., the two nitrogen atoms of this bacteriochlorin ring are protonated). In some embodiments, M is Zn or is replaced by two hydrogen atoms (-H, H). Thus, the compounds represented by formula (II) include metallobacteriochlorin and free base bacteriochlorin. In some embodiments, M is Zn. In some embodiments, R5, R 10 and R 15 These are, independently, a hydrogen atom, methoxy, and the formula -L1-(X1-L2) p Selected from linker groups represented by -G. In some embodiments, R5 is methoxy. In some embodiments, R 10 and R 15 One of them is a linker group. In some embodiments, R 15 is a linker group. Alternatively, in some embodiments, R 12 This is a linker group. In some embodiments, formula -L1-(X1-L2) p The p of the linker group represented by -G is 0. In some embodiments, G is a carboxylic acid or an active ester (e.g., an NHS ester). In some embodiments, L1 is an alkylylene, an arylene (e.g., phenylene), or a divalent moiety containing both an alkylylene group and an arylene group (i.e., an aralkylylene group). In some embodiments, L1 is -C≡C-phenylene or -C ≡ It is a C-alkylene (e.g., -C≡C-(CH2)4-). In some embodiments, this linker group is represented by the following formula: [ka] (Optionally, G is a carboxylic acid (e.g., -C(=O)OH) or an active ester.) In some embodiments, this linker group is represented by the following formula: [ka]

[0065] In some embodiments, p is 1, and this linker group includes a polyoxyethylene chain to improve solubility, and / or a spacer to improve the reactivity of G compared to the reactivity of G in equivalent bacteriochlorins where G is directly bonded to L1. In some embodiments, L1 is phenylene or C≡C-phenyl-, p is 1, X1 is -C(=O)NH-, L2 is alkylene, and G is a carboxylic acid or active ester (e.g., NHS ester). In some embodiments, L2 is ethylene. Thus, this linker group may include a β-alanine spacer to improve the reactivity of G. In some embodiments, p is 1, L1 is aralkylene (e.g., -C≡C-(C6H4)-), X1 is -C(=O)NH-, L2 is alkylene substituted with one or more groups including a polyoxyethylene chain and / or an amide group, and G is a bioconjugable group. In some embodiments, X1 is -C(=O)NH-, and L2 is -(CH2CH2O)q-alkylene-. In some embodiments, q is 12, and the alkylene is ethylene. In some embodiments, L2 is a methylene group substituted with a PEG chain and / or a group including an amide group. For example, in some embodiments, L2 is -CH(R) (wherein R is -alkylene-NH-C(=O)-alkylene-PEG-OMe). In some embodiments, R includes two C2-C6 alkylene groups and a PEG12-PEG25 chain. In some embodiments, R is -(CH2)4-NH-C(=O)-CH2CH2-(OC2H4). 24 It is OMe. In some embodiments, this linker group is represented by the following formula or its active ester (optionally, its PEG is PEG12). [ka] In some embodiments, this linker group is represented by the following formula or its active ester (optionally, its PEG is PEG24). [ka]

[0066] In some embodiments, this bacteriochlorin is asymmetric. In some embodiments, R2 and R 12 They are not the same, or R3 and R 13 They are not the same. For example, in some variations, R2 and R 12 On the other hand (for example, R2), there is a solubilizing group, and this solubilizing group is -aryl-(R s ) w and -alkynyl-aryl-(R s )w Selected from and R2 and R 12 The other of these is equation -L1-(X1-L2) p It is a linker group represented by G, or a solubilizing group having a different structure (for example, different from the solubilizing group of R2). In some embodiments, R3 and R 13 One of these is a solubilizing group, and this solubilizing group is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w A solubilizing group selected from R3 and R 13 The other is equation -L1-(X1-L2) p G is a linker group or a solubilizing group having a different structure. In some embodiments, R2 and R 12 One of them is a solubilizing group and the other is a linker group, or R3 and R 13 One of them is a solubilizing group, and the other is a linker group. In some embodiments, R3 and R 13 These are esters, respectively. In some embodiments, R3 and R 13 These are methyl esters (i.e., -C(=O)OCH3). In some embodiments, R2 is expressed by the following equation. [ka]

[0067] In some embodiments, each R is a base represented by the following formula: -X2-(L3) z -R 17 (In the formula, z is 0, X2 is -C(=O)NH-alkylene-NH-, R 17 is -C(=O)C2H4-(OC2H4) m Ure 18 (In the formula, m is an integer greater than or equal to 12, R 18is methyl. In some embodiments, m is an integer between 12 and 24 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24). In some embodiments, each R s is represented by the following formula:

Chemical formula

[0068] In some embodiments, this compound is selected from BC-1, BC-2, BC-3, BC-4, BC-5, BC-6 and BC-7 (that is, compounds having the following structures).

Chemical formula

Chemical formula

Chemical formula

[0069] [ka] [ka] [ka]

[0070] [ka] [ka] [ka]

[0071] In some embodiments, the present invention provides compositions comprising covalent conjugates formed between: (a) R2, R3, R5, R 10 , R 12 , R 13 and R 15 A compound represented by formula (II) according to claim 1, wherein at least one of the following is a linking group: (b) one or more of the group consisting of small molecules, antigens, fine particles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones and growth factors. In some embodiments, for example, the conjugate can be formed by reacting a compound represented by formula (II) containing a linking group comprising a carboxylic acid or an active ester (i.e., as a bioconjugable group G) with an amino group of a small molecule, peptide, protein, antibody, or polymer.

[0072] III.Synthesis method Methods for synthesizing bacteriochlorins suitable for use in the present invention are described, for example, in U.S. Patents 8,664,260 and 8,980,565, which are incorporated herein by reference in their entirety. In some embodiments, the compounds of the present invention represented by formula (II) can be produced by the steps of preparing a suitable trans-beta substituted bacteriochlorin (such as a bacteriochlorin in which two beta-bacteriochlorin substituents are halo (e.g., Br) substituents), and then reacting it so that the beta substituents are replaced with suitable water-soluble groups. Methods for synthesizing trans-beta substituted bacteriochlorins are already known and are described, for example, in Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103. For example, in some embodiments, bacteriochlorin represented by formula (II) can be prepared by self-condensing dihydrodipyrine building blocks or by condensing a pair of dihydrodipyrine building blocks in an organic solvent in the presence of an acid. In some embodiments, the dihydrodipyrine building block may have the following structure: [ka] (wherein R is an acetal or aldehyde group; S1, S2, S3, S7 and S6 are each independently a hydrogen atom, an aryl, a substituted aryl, a phenyl, a cycloalkyl, an alkyl, a substituted alkyl, an alkenyl, an alkynyl, a halo, an alkoxy, an alkylthio, a perfluoroalkyl, a perfluoroaryl, a pyridyl, a cyano, a thiocyano, a nitro, an amino, an alkylamino, an acyl, a sulfoxyl, a sulfonyl, an imide, an ester, an amide and a carbamoyl; and S4 and S5 are each a hydrogen atom or together form a covalent bond. In some embodiments, at least one of S1 and S2 is a halo.)

[0073] More specifically, methods for preparing dibromo-substituted bacteriochlorins and their corresponding dihydropyrine building blocks are already known and are described, for example, in Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103. For example, a bacteriochlorin containing two bromo substituents at the 2 and 12 positions can be prepared, for example, from a building block prepared from N-protected 3,4-dibromopyrrole, as shown in Scheme 1 (see Figure 1). As shown in Scheme 1 (Figure 1), N-protected 3,4-dibromopyrrole (e.g., 3,4-dibromo-(N-triisopropylsilyl)pyrrole) is treated with an alkyllithium (e.g., t-butyllithium) and a base such as dimethyl carbonate, and then deprotected to obtain 3-bromo-4(-methoxycarbonyl)pyrrole (a). Aldehyde b is obtained by Vilsmeyer formylation of a (e.g., using POCl3-DMF). Aldehyde b is treated with potassium acetate and a slightly excess methylamine hydrochloride in nitromethane to obtain aldol condensation product c. By reducing the carbon-carbon double bond in c using a suitable reducing agent (e.g., NaBH4), compound d is obtained, which undergoes a Michael addition reaction when 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]undeca-7-ene (DBU)). The Michael addition product e is reductively cyclized by first deprotonating e (e.g., by treatment with anhydrous sodium methoxide) to obtain a nitronate anion intermediate, and then cyclized with a deoxidizing agent (e.g., aqueous solution of buffered TiCl3) to obtain dihydrodipyrine bacteriochlorin building block (BB). Alternatively, cyclization conditions include treating e with a metal (e.g., zinc in ethanol and acetic acid) to produce an N-oxide intermediate, and then cyclizing this intermediate with a deoxidizing 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.).

[0074] BB can self-condense in the presence of an acid (e.g., a Brønsted or Lewis acid such as trifluoroacetic acid, TMSOTf, or tosylic acid (TsOH)) to form a condensation product in the presence of a proton scavenger (e.g., 2,6-di-tert-butylpyridine (DTBP)) to provide bacteriochlorin. This condensation can be carried out in organic solvents such as acetonitrile (ACN), dichloromethane (DCM), chloroform, tetrahydrofuran (THF), chlorobenzene, ethanol, and combinations thereof. Optionally, if the bacteriochlorin building block does not contain a carbon-carbon double bond between heterocycles, an oxidizing agent such as air or DDQ may be included in the condensation reaction mixture. In some embodiments, the bacteriochlorin may have a structure represented by the following formula: [ka]

[0075] The halo substituents in dihalobacteriochlorins, such as the bacteriochlorin described above, can be solubilized using chemical coupling methods known in the art, such as (but not limited to) Still coupling, Hiyama coupling, Suzuki coupling, Negishi coupling, Sonogashira coupling, and Kumada coupling reactions. For example, this dihalobacteriochlorin can be further reacted with a boronic acid in the presence of a Pd(0) catalyst; with an organotin compound in the presence of a Pd catalyst; with a pseudohalide or organosilane in the presence of a Pd catalyst; with an organozinc compound in the presence of a Ni or Pd catalyst; or with a Grignard reagent in the presence of a Ni or Pd catalyst. In some embodiments, this dihalobacteriochlorin (e.g., dibromobacteriochlorin such as the bacteriochlorin free base described above) can be reacted with an arylboronic acid Suzuki coupling reaction partner. See, for example, 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, this arylboronic acid may contain additional or protected chemical functional groups that can be further refined after the Suzuki coupling reaction. For example, in some embodiments, this Suzuki coupling reaction may contain one or more protected amino groups that can react with a suitable PEG reagent (e.g., activated PEG ester) after deprotection.

[0076] In some embodiments, the compounds of the present invention may optionally use a t-butyloxycarbonyl (BOC) protecting group on an amino group present on a Suzuki or other type of coupling agent, in combination with the use of t-butyl ester protection of a carboxylic acid (optionally in combination with the use of t-butyl ester protection on a carboxylic acid, e.g., in a linker group). Surprisingly, deprotection of the BOC group during the preparation of the compounds of the present invention using trifluoroacetic acid (TFA), a common method of BOC deprotection, has been found to result in significant decomposition, for example, in compounds containing alkyne bonds. Therefore, in some embodiments, BOC deprotection is carried out under other conditions, e.g., using 4M HCl in dioxane. In some embodiments, the present invention provides a method for preparing asymmetric water-soluble bacteriochlorines, the method comprising carrying out a mixed coupling reaction or a heterocoupling reaction. For example, the method may consist of (a) providing a dihalobacteriochlorine (e.g., symmetric dibromobacteriochlorine) in which two trans-β-pyrrole carbons are substituted with halo groups (e.g., bromo groups), and (b) carrying out a mixed coupling reaction (e.g., a mixed Sonogashira, Heck, or Suzuki coupling reaction) by contacting the dihalobacteriochlorine with two different alkynes, two different alkenes, or two different organoboronates (e.g., boronic acids or esters) in the presence of a suitable catalyst (e.g., a palladium catalyst such as a palladium(0) catalyst) and a base (e.g., a trialkylamine such as triethylamine, or sodium or potassium acetate).

[0077] In some embodiments, the present invention provides a method for producing an asymmetric bacteriochlorin compound represented by the following formula. [ka] (wherein M is a metal or two hydrogen atoms; R5, R 10 and R 15 Each is independently selected from a hydrogen atom and an alkoxy; R2, R3, R 12 and R 13 Each of these is independently selected from hydrogen atoms, cyano, halo, perhaloalkyl, sulfonates, sulfonamides, esters, carboxylic acids, formyl, acetyl, linker groups, and solubilizing groups; The linker group is represented by the following formula: Formula-L1-(X1-L2) p G (Here, p is 0 or 1, L1 is alkylidene, X1 is -C(=O)NH- or -NHC(=O)-, and L2 is -(CH2CH2O) q -Alkylene-(wherein q represents an integer from 1 to 24), alkylene, or substituted alkylene (optionally, this substituted alkylene is an alkylene substituted with one or more groups including a polyoxyethylene chain and / or an amide group); G is a biocompatible group.) The solubilizing group is -aryl-(R s ) w and -alkynyl-aryl-(R s ) w Selected from (where w is an integer from 0 to 5, R s This is a base represented by the following equation. -X2-(L3)zR 17 , Here, 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 (C2H4O) m -R 18 -C(=O)C2H4-(OC2H4) m Ure 18 and -(C2H4O) n -C2H4-C(=O)NH-C(R 19 ) Selected from 3, where m is an integer greater than or equal to 12, and n is an integer from 1 to 5, R 18 R is a lower alkyl group (optionally methyl), 19 -CH2O-C2H4-C(=O)NH-(C2H4O) m R 18That is. )); However, R2 and R 12 If R3 and R are not the same, 13 They are not the same. R2, R3, R 12 and R 13 At least one of them is -aryl-(R s ) w or -alkynyl-aryl-(R s ) w (That is the case.) A method consisting of the following steps. (a) A step of providing a compound represented by the following formula (II'); and [ka] (wherein M is a metal or two hydrogen atoms; R5',R 10 'and R 15 ' is independently selected from hydrogen atoms and alkoxy atoms, R2', R3', R 12 'and R 13 Each of the following is independently selected from hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, and acetyl, and R3' and R 13 These are each Halo (or Bromo, optionally). (b) The step of contacting the compound with a palladium catalyst, a base, and one of the following: (i) Two different alkynes (optionally, both of these alkynes are compounds represented by the following formula: [ka] (In the formula, y is an integer from 1 to 5 (arbitrarily 1 or 2), and each R 20 This 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes protected carboxylic acid-substituted alkylenes). (ii) Two different alkenes (optionally, both of these alkenes are compounds represented by the following formula: [ka] (In the formula, y is an integer from 1 to 5 (arbitrarily 1 or 2), and each R 20 This 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes protected carboxylic acid-substituted alkylenes). (iii) Two different organoboronates (optionally, the two different organoboronates are two different arylboronic acids or arylboronic acid esters of the following formulas: [ka] (In the formula, y is an integer from 1 to 5 (arbitrarily 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes a protected carboxylic acid-substituted alkylene), and each R 21 is either a hydrogen atom or an alkyl group, or two R groups 21 They together form alkylenes. In the obtained product (for example, a synthetic intermediate of the compound represented by formula (II)), R2' and R 12 'or R3' and R 13 The halo substituents of ' are each different substituents (e.g., different -alkynyl-aryl-(R 20 ) y ,-alkenyl-aryl-(R 20 ) y , or aryl-(R 20 ) y It is replaced by the base.

[0078] In some embodiments, the ratio of two alkynes, two alkenes, or two organoboronates is adjusted to maximize the yield of the desired product. For example, in some embodiments, this ratio is adjusted based on the relative reactivity of two different alkynes, two different alkenes, or two different boronates. The relative reactivity of the two coupling partners can be determined by monitoring the reaction by reverse-phase HPLC. In some embodiments, the yield of asymmetric bacteriochlorin is maximized by providing a larger molar excess to the less reactive of the two coupling partners than to the second coupling partner. In some embodiments, the yield of the desired product from the mixed coupling reaction is greater than expected. Therefore, in some embodiments, the yield of the desired product is greater than 50% or greater than 55%. In some embodiments, this yield is greater than 60%. In some embodiments, this yield is approximately 62%.

[0079] IV. Pharmaceutical Compositions The compounds of the present invention can be provided as pharmaceutically acceptable salts. Such salts include, but are not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidine-1'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts such as lithium, potassium and sodium, but not limited to these; alkaline earth metal salts such as barium, calcium and magnesium, but not limited to these; transition metal salts such as zinc; and other metal salts such as sodium hydrogen phosphate and disodium phosphate, but not limited to these; as well as mineral acid salts such as hydrochloride and sulfate, but not limited to these; and organic acid salts such as acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate and fumarate, but not limited to these. pharmaceutically acceptable esters include, but are not limited to, esters of alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and heterocyclyl groups of acidic groups (including, but not limited to, carboxylic acids, phosphoric acid, phosphinic acid, sulfonic acid, sulfinic acid, and boronic acid).

[0080] The compounds of the present invention may also include prodrugs of the compounds disclosed herein. As stated above, a “prodrug” is a compound that, when administered in vivo, is metabolized by one or more steps or processes, or otherwise converted into a biologically, pharmaceutically, or therapeutically active form of the compound. To produce a prodrug, a pharmaceutically active compound is modified so that the active compound is regenerated by a metabolic process. This prodrug may be designed to alter the metabolic stability or transport properties of the drug, to block side effects or toxicity, to improve the flavor of the drug, or to alter other properties or characteristics of the drug. Thanks to knowledge of pharmacodynamic processes and drug metabolism in vivo, those skilled in the art can design prodrugs of a pharmaceutically active compound once it is known (see, for example, Nogrady (1985) Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, USA, pages 388–392).

[0081] utility The methods and intermediates of the present invention are useful for the synthesis of compounds represented by the formula (II) described herein. Such compounds, either on their own or in further modified forms (e.g., salts, metallated compounds, conjugates, or prodrugs), are useful for diagnostic and therapeutic purposes in a manner similar to other compounds described for photodynamic therapy, such as in U.S. Patent Application Publication No. 2004 / 0044197 (Pandey et al.), which are described in further detail below.

[0082] stability The advantages of some embodiments of the bacteriochlorin compounds of the present invention are their stability and absorption properties. Accordingly, the present invention relates to the active compound of the present invention (e.g., the compound represented by formula (II) or its pharmaceutically acceptable salt, prodrug or conjugate (e.g., a conjugate with a targeting agent such as a protein, peptide or antibody)) which has a wavelength of about 600 to about 800 nm and an absorption rate of about 10,000 to 300,000 M -1 cm -1 The present invention provides a composition having, or characterized by, the above-mentioned peak molar absorption coefficient in a solution (it should be understood that (a) the active compound must be placed in the solution to determine the peak molar absorption coefficient at a specified wavelength, and (b) the compound may exhibit additional peaks outside this range or multiple peaks within this range). Furthermore, the present invention provides a composition comprising, or essentially consisting of, a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, prodrug, or conjugate (e.g., a conjugate with a targeting agent such as a protein, peptide, or antibody) in a solvent. The amount of solvent is not important and may constitute 0.01 (or 1) to 99 (or 99.99)% by weight of the composition. This composition has a wavelength of about 10,000 to 300,000 M at about 600 to about 800 nm. -1 cm -1 The solution has, or is characterized by, the above peak molar absorption coefficient. Before measuring molar absorption, it is preferable to stir as necessary to return aggregated particles to the solution, although it should be understood that some degree of aggregation may be desirable when actually using the composition. The appropriate solvent depends on the compound and its intended use, but may be either an organic solvent or an aqueous solvent, or a combination thereof. This composition, whether in the form of a bacteriochlorin compound or multiple compounds in a "neat" form, or a bacteriochlorin compound or multiple compounds mixed with a solvent, will have or exhibit a loss (due to its decomposition) of not more than about 10, 15, or 20 weight percent of the bacteriochlorin compound of the present invention when stored in a sealed container (e.g., a flask ampoule or vial) at room temperature, in the absence of ambient light, for at least 3 or 4 months. This decomposition can be determined according to the prior art by spectroscopic, thin-layer chromatography, NMR spectroscopy, and / or mass spectrometry.

[0083] solubility An advantage of some embodiments of the compounds of the present invention is their water solubility. Accordingly, the present invention provides compositions comprising, or essentially comprising, pharmaceutical formulations comprising (a) an aqueous solvent (e.g., distilled water, saline solution, buffer solution) and (b) the active compound of the present invention solubilized in the aqueous solvent, ranging from about 1, 2, 5, or 10 μM to 200, 300, or 500 mM.

[0084] Formulation of pharmaceutical compositions The pharmaceutical composition of the present invention comprises one or more compounds of the present invention in a therapeutically effective amount in a pharmaceutically acceptable carrier, and the composition is useful for the prevention, treatment, or improvement of one or more symptoms of a disease or disorder related to, or involving, hyperplasia or angiogenesis. Diseases or disorders related to hyperplasia or angiogenesis include, but are not limited to, cancer, psoriasis, atherosclerosis, heart disease, and age-related macular degeneration. Suitable pharmaceutical carriers for administering the compounds of the present invention include any carrier known to those skilled in the art to be suitable for a particular mode of administration. The pharmaceutical composition preferably exhibits the above-mentioned absorption characteristics and storage properties or stability. Furthermore, this compound can be formulated as a single pharmaceutically active ingredient in this composition, or in combination with other active ingredients. This composition comprises one or more compounds of the present invention (for example, compounds represented by formula (II)). In some embodiments, these compounds are formulated into suitable formulations such as orally administered solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release formulations, or elixirs, or into sterile solutions or suspensions for parenteral administration, as well as into transdermal patch formulations and dry powder inhalers. In some embodiments, the above compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel (1985) Introduction to Pharmaceutical Dosage Forms, Fourth Edition, Lea & Febiger, Philadelphia, Pennsylvania, USA, page 126).

[0085] In this composition, one or more compounds or pharmaceutically acceptable derivatives thereof in an effective concentration are mixed with a suitable pharmaceutical carrier. The compounds may be derivatized as corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs prior to formulation, as described above. The concentration of the compound in this composition is effective in delivering, at the time of administration, an amount that treats, prevents, or improves one or more symptoms of a disease or disorder associated with, or involving, hyperplasia or angiogenesis. 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 mixed in a selected carrier at an effective concentration that alleviates, prevents, or improves one or more symptoms of a therapeutic condition. This active compound (i.e., the compound represented by formula (II), or a pharmaceutically acceptable salt, prodrug, or conjugate thereof) is contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically beneficial effect without undesirable side effects in the patient receiving treatment. This therapeutically effective concentration can be empirically determined by testing the compound in in vitro and in vivo systems as described herein and in U.S. Patent No. 5,952,366 (Pandey et al.), from which the human dose can be estimated.

[0086] The concentration of the active compound in the pharmaceutical composition depends on the absorption rate, inactivation rate, and excretion rate of the active compound, as well as the physicochemical properties of the compound, the administration schedule and dosage, and other factors known to those skilled in the art. For example, the amount delivered is sufficient to improve one or more symptoms of a disease or disorder that is associated with, or involves, hyperplasia or angiogenesis, as described herein. In some embodiments, a therapeutically effective dose should produce a serum concentration of about 50–100 μg / ml from about 0.1 ng / ml of the active ingredient. In some embodiments, this therapeutically effective dose is 0.001 (or 0.01, 0.1) mg to 10 (or 100, 1000) mg of the active compound per kilogram of body weight per day. The pharmaceutical dosing unit forms are prepared to provide a combination of the active ingredient or essential ingredient per dosing unit form ranging from about 0.01 mg (or 0.1 mg, 1 mg) to about 500 mg (or 1000 mg, 2000 mg), and in some embodiments, about 10 mg to about 500 mg. This active ingredient may be administered as a single dose or divided into several smaller doses administered at time intervals. It should be understood that the exact dosage and duration of treatment are functions of the disease being treated and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should also be noted that the concentration and dosage values ​​may vary depending on the severity of the symptoms to be alleviated. Furthermore, a specific dosing regimen for a particular patient should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of this composition. The concentration ranges shown herein are illustrative and are not intended to limit the scope or implementation of the composition of the present invention.

[0087] If this 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 a cosolvent such as dimethyl sulfoxide (DMSO), using a surfactant such as polyoxyethylene sorbitol ester (e.g., sold under the trade name TWEEN(R)), or dissolving in aqueous sodium bicarbonate. Derivatives of this compound, such as prodrugs, can also be used in formulating effective pharmaceutical compositions. When this compound (one or more) is mixed or added, the resulting mixture may be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on many factors, such as the intended mode of administration and the solubility of this compound in the selected carrier or vehicle. The effective concentration is an amount sufficient to improve the symptoms of the disease, disorder, or condition being treated and can be determined empirically.

[0088] This pharmaceutical composition is 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, comprising an appropriate amount of the compound or a pharmaceutically acceptable derivative thereof. These pharmaceutically and therapeutically active compounds and their derivatives are, in some embodiments, formulated and administered in unit or multiple dosage forms. As used herein, unit dosage forms refer to individually packaged, physically distinct units suitable for human and animal subjects, as known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, combined with the necessary pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms may be administered in fractions or multiples thereof. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container so as to be administered in separated unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or pint or gallon bottles. Thus, multiple dosage forms are multiple unit doses that are not separated during packaging.

[0089] Liquid pharmaceutically acceptable compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing the active compound defined above (e.g., the compound represented by formula (II), or its pharmaceutically acceptable salt, prodrug, or conjugate) and any pharmaceutical adjuvant in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.) to form a solution or suspension. If necessary, the administered pharmaceutical composition may also contain small amounts of non-toxic adjuvants such as wetting agents, emulsifiers, solubilizers, and pH buffers (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, and other such agents). Practical methods for preparing such dosage forms are known to those skilled in the art, or will become apparent in the future. See, for example, Remington's Pharmaceutical Sciences, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, USA. Dosage forms or compositions can be prepared that contain an active ingredient in an amount ranging from 0.005% to 100%, with the remainder being a non-toxic carrier. Methods for preparing these compositions are well known to those skilled in the art. The compositions intended may contain the active ingredient in an amount ranging from 0.001% to 100%, 0.1% to 95% in one embodiment, and 75% to 85% in another embodiment.

[0090] Composition for oral administration Oral drug dosage forms may be solid, gel, or liquid. Solid dosage forms include tablets, capsules, granules, and bulk powders. Types of oral tablets include lozenges and tablets that can be compressed and chewed, and these may be enteric coated, sugar coated, or film coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-foaming or effervescent forms in combination with other ingredients known to those skilled in the art.

[0091] Solid composition for oral administration In some embodiments, this pharmaceutical formulation is in solid dosage form, and in some embodiments, it is in the form of capsules or tablets. These tablets, pills, capsules, lozenges, etc., may contain one or more of the following components (e.g., binders; lubricants; diluents; flow enhancers; disintegrants; colorants; sweeteners; flavorings; wetting agents; emetic coatings; film coatings) or compounds of similar properties. Examples of binders include microcrystalline cellulose, tragacanth gum, glucose solution, acacia mucus, gelatin solution, molasses, polyinylpyrrolidine, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salts, mannitol, and dicalcium phosphate. Flow enhancers include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, starch glycolate sodium, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Colorants include, for example, any or mixtures thereof of approved and certified water-soluble FD and C dyes, as well as water-insoluble FD and C dyes suspended in alumina hydrate. Sweeteners include artificial sweeteners such as sucrose, lactose, mannitol, and saccharin, as well as any number of spray-dried flavors. Flavorings include natural flavors extracted from plants such as fruits and synthetic blends of compounds that produce pleasant sensations, such as peppermint and methyl salicylate (but not limited to these). Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene laural ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammonia-modified shellac, and cellulose phthalate acetate. The film coating contains hydroxyethylcellulose, gellan gum, sodium carboxymethylcellulose, polyethylene glycol 4000 (PEG4000), and cellulose phthalate acetate.

[0092] This compound, or a pharmaceutically acceptable derivative thereof, can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestines. The composition can also be formulated in combination with an antacid or other such component. If the dosage unit form is a capsule, it may include a liquid carrier such as fatty oil in addition to the above-mentioned types of materials. Furthermore, the dosage unit form may include coatings of various other materials that modify the physical form of the dosage unit, such as sugar and other enteric drugs. This compound can also be administered as a component of elixirs, suspensions, syrups, wafers, sprinkles, chewing gum, etc. The syrup may include sucrose as a sweetener, as well as certain preservatives, dyes, colorants, and flavors, in addition to the active compound. Furthermore, this active substance can be mixed with other active substances that do not impair the desired effect, or with materials that complement the desired effect, such as antacids, H2 blockers, and diuretics. This active ingredient is the compound of the present invention or a pharmaceutically acceptable derivative thereof. This active ingredient can be included in high concentrations up to about 98% by weight. In some embodiments, tablet and capsule formulations may be coated, as is known to those skilled in the art, to modify or maintain the dissolution of the active ingredient. For example, they may be coated with conventional enteric coatings such as phenylsalicylates, waxes, and cellulose phthalate acetate.

[0093] Liquid composition 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. Elixirs are clear, sweetened, aqueous alcohol pharmaceutical formulations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars such as sucrose and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed throughout another liquid in the form of small spheres. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers, and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-foaming granules, which are reconstituted into liquid oral dosage forms, include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, which are reconstituted into liquid oral dosage forms, include organic acids and carbon dioxide sources. Colorants and flavorings are used in all of the above dosage forms. Solvents include glycerin, sorbitol, ethyl alcohol, and syrups. Examples of preservatives include glycerin, methyl and propylparabens, benzoic acid, sodium benzoate, and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include surfactants such as gelatin, acacia, tragacanth, bentonite, and polyoxyethylene sorbitan monooleate. Suspensioning agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, bee gum, and acacia. Sweeteners include sucrose, syrup, glycerin, and artificial sweeteners such as saccharin. Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Carbon dioxide sources include sodium bicarbonate and sodium carbonate. Colorants include any of the approved certified water-soluble FD and C dyes and mixtures thereof. The fragrances include natural fragrances extracted from plants such as fruits and synthetic blends of compounds that produce pleasant tastes. In the case of solid dosage forms, the solution or suspension (e.g., in propylene carbonate, vegetable oil, or triglycerides) is encapsulated in gelatin capsules in one embodiment.Such solutions, as well as their preparation and encapsulation, are disclosed in U.S. Patents 4,328,245, 4,409,239, and 4,410,545, which are incorporated herein by reference in their entirety. In the case of liquid dosage forms, for example, a solution in polyethylene glycol can be diluted with a pharmaceutically acceptable liquid carrier (e.g., water) in an amount sufficient to be administered.

[0094] Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oil, glycol, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those described in U.S. Patents RE 28,819 and 4,358,603, which are incorporated herein by reference in their entirety. Simply put, such formulations include, but are not limited to, the compounds of the present invention, dialkylated mono or polyalkylene glycols (including, but not limited to, 1,2-dimethoxymethane, diglyme, triglime, tetraglime, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, and polyethylene glycol-750-dimethyl ether (where 350, 550, and 750 refer to the approximate average molecular weight of 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). Other formulations include, but are not limited to, aqueous alcohol solutions containing pharmaceutically acceptable acetals. The alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvent having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. The acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal.

[0095] Injectable drugs, solutions and emulsions In some embodiments characterized by subcutaneous, intramuscular, or intravenous injection, parenteral administration is also expected. The injectable preparation can be prepared in conventional forms, either as a liquid solution or suspension, a solid form suitable for a solution or suspension in a liquid before injection, or an emulsion. The injectable preparation, solution, and emulsion also contain one or more excipients. Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol. Furthermore, if necessary, the administered pharmaceutical composition may also contain small amounts of non-toxic adjuncts such as wetting or emulsifying agents, pH buffers, stabilizers, dissolution accelerators, and other such agents (e.g., sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin).

[0096] The implantation of sustained-release or sustained-release systems that maintain a constant level of dosage (see, for example, U.S. Patent No. 3,710,795, which is incorporated herein by reference) is also anticipated. Simply put, the compounds of the present invention are dispersed in a solid internal matrix. This solid internal matrix is, for example, 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 acrylic acid and methacrylic acid esters, collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate, which are outer polymer films (e.g. For example, polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride copolymer with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol copolymer, and ethylene / vinyl oxyethanol copolymer, which are insoluble in body fluids. During the release rate control phase, this compound diffuses through the outer polymer membrane. The percentage of the active compound in such a parenteral composition depends largely on its specific properties, as well as the compound's activity and the patient's needs.

[0097] Parenteral administration of this composition includes intravenous, subcutaneous, and intramuscular administration. Preparations for parenteral administration include: sterile solutions ready for injection; sterile, dry, soluble products (including tablets for subcutaneous injection), such as lyophilized powders, ready to be combined with a solvent immediately before use; sterile suspensions ready for injection; and sterile, dry, insoluble products ready to be combined with a vehicle and sterile emulsion immediately before use. These solutions may be aqueous or non-aqueous. When administered intravenously, suitable carriers include solutions containing physiological saline or phosphate-buffered saline (PBS), as well as thickeners and solubilizers (such as glucose, polyethylene glycol, and polypropylene glycol) and mixtures thereof. Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspensions and dispersants, emulsifiers, sequestering or chelating agents, and other pharmaceutically acceptable substances.

[0098] Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, and dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include plant-derived fixative oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungiostatic concentrations may be added to parenteral formulations packaged in multi-dose containers. These include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethylcellulose, xanthan gum, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. The emulsifier includes polysorbate 80 (TWEEN(R) 80). The metal ion sequester or chelating agent includes EDTA. The pharmaceutical carriers include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment. The concentration of the pharmaceutically active compound can be adjusted to provide an effective amount for producing the desired pharmacological effect by injection. The exact dose depends on the age, weight, and symptoms of the patient or animal, as is known in the art.

[0099] The parenteral formulations in unit doses are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration must be sterile, as is known and practiced in the art. As an example, 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 substance, which is injected as needed to produce the desired pharmaceutical effect. The injectable formulation is designed for local and systemic administration. In some embodiments, the therapeutically effective dose is formulated so that the concentration of the active compound in the treated tissue ranges from about 0.1% w / w to about 90% w / w or more, and in certain embodiments, to greater than 1% w / w. This compound may be micronized, suspended in other suitable forms, or derivatized to produce a more soluble active product or a prodrug. The form of the resulting mixture depends on many factors, such as the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration can be determined empirically, being sufficient to improve symptoms.

[0100] freeze-dried powder Lyophilized powders, which can be reconstituted for administration as solutions, emulsions, and other mixtures, can also be used to carry out the present invention. These can also be reconstituted or formulated as solids or gels. Sterile lyophilized powders are prepared by dissolving the compound of the present invention or a pharmaceutically acceptable derivative thereof in a suitable solvent. This solvent may contain excipients to improve stability, or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Suitable excipients include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. This solvent may also contain a buffer such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, and in some embodiments, a neutral pH. The solution is then sterile filtered and subsequently freeze-dried under standard conditions known to those skilled in the art to obtain the desired pharmaceutical formulation. In one embodiment, the obtained solution is divided into multiple vials for freeze-drying. Each vial may contain a single or multiple doses of the compound. The freeze-dried powder can be stored under suitable conditions such as about 4°C to room temperature. Reconstituting this lyophilized powder with sterile water for injection yields a formulation for parenteral administration. To reconstitute, the lyophilized powder is added to sterile water or another suitable carrier. The exact amount varies depending on the selected compound. This amount can be determined empirically.

[0101] Local administration Topical mixtures are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension or emulsion, and may be prescribed as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, colorant, paste, foam, aerosol, irrigation, spray, suppository, bandage, skin patch, or other formulation suitable for topical administration. This compound or its pharmaceutically acceptable derivatives can be formulated as an aerosol for topical application, such as by inhalation (see, for example, U.S. Patents 4,044,126; 4,414,209; and 4,364,923, which describe aerosols for delivering steroids useful in the treatment of inflammatory diseases, particularly asthma, and which are incorporated herein by reference in their entirety). These formulations for administration to the airways may be in the form of an aerosol or a solution for a sprayer, or as a fine powder for blowing, and may be used alone or in combination with an inert carrier such as lactose. In such cases, the particle diameter of the formulation is less than 50 μm in some embodiments and less than 10 μm in some embodiments. This compound may be formulated in the form of a gel, cream, or lotion for topical application, such as topical application to the skin or mucous membranes (such as the eyes), application to the eyes, or application to the cisterna or spinal cord. Topical administration is expected for transdermal delivery, for administration to the eyes or mucous membranes, or for inhalation therapy. This active compound can also be administered as a nasal solution alone or in combination with other pharmaceutically acceptable excipients. These solutions, particularly ophthalmic solutions, can be formulated as 0.01% to 10% isotonic solutions with a pH of approximately 5 to 7, containing appropriate salts.

[0102] Compositions for other routes of administration Other routes of administration, such as transdermal patches and rectal administration, including ionophoresis and electrophoresis devices, are also anticipated. Transdermal patches containing ionophoresis and electrophoresis devices are well known to those skilled in the art. For example, such patches are disclosed in U.S. Patents 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, which are incorporated herein by reference in their entirety. For example, pharmaceutical dosage forms for rectal administration include rectal suppositories, capsules, and tablets to produce systemic effects. As used here, rectal suppositories refer to solids for insertion into the rectum that melt or soften at body temperature, releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or vehicles and drugs to raise the melting point. Examples of these bases include cocoa butter (theobroma oil), glycerin gelatin, carbowax (polyoxyethylene glycol), and suitable mixtures of mono-, di-, and tri-glycerides of fatty acids. Various combinations of bases may be used. Drugs used to raise the melting point of suppositories include whale wax and other waxes. Rectal suppositories can be prepared by either compression or molding. In one embodiment, the weight of the rectal suppository is approximately 2 to 3 grams. Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substances and methods as formulations for oral administration.

[0103] Targeted prescriptions The compounds of the present invention or their pharmaceutically acceptable derivatives can also be formulated to target specific tissues, receptors, infections, or other areas of the patient's body being treated. Many of these targeting methods are well known to those skilled in the art. All of these targeting methods are expected to be used in the present composition. For non-limiting examples of targeting methods, see, for example, U.S. Patents 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. These are incorporated herein in their entirety.

[0104] Liposomes In some embodiments, liposome suspensions containing tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, a liposome pharmaceutical formulation can be prepared as described in U.S. Patent No. 4,522,811, which is incorporated herein by reference. Briefly, liposomes such as multilayer vesicles (MLVs) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (molar ratio 7:3) inside a flask. A solution of the compound of the present invention in phosphate-buffered saline lacking divalent cations (PBS) is added to the flask, and the flask is shaken until the lipid membrane is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, pelletized by centrifugation, and then resuspended in phosphate-buffered saline (PBS).

[0105] Ligand In some embodiments, the compounds disclosed herein can be used to target specific target tissues or target compositions using ligands specific to those target tissues or target compositions (e.g., ligands or ligand-receptor pairs (such as antibodies and antigens)). Antibodies against tumor antigens and pathogens are known. Antibodies and antibody fragments that specifically bind to markers produced by or bound to tumors or infectious lesions (including viruses, bacteria, fungi, parasitic infections, and antigens and products that bind to such microorganisms) are disclosed, in particular, in U.S. Patent No. 3,927,193 (Hansen et al.), U.S. Patent No. 4,331,647 (Goldenberg), U.S. Patent No. 4,348,376, U.S. Patent No. 4,361,544, U.S. Patent No. 4,468,457, U.S. Patent No. 4,444,744, U.S. Patent No. 4,818,709, and U.S. Patent No. 4,624,846, which are incorporated herein by reference in whole. Antibodies against antigens such as tumors of the gastrointestinal tract, lungs, breasts, prostate, ovaries, testes, brain, or lymph nodes, as well as sarcomas or melanomas, can be used.

[0106] A broad range of monoclonal antibodies against various infectious pathogens have been developed and are readily available, as summarized in Polin (1984) European Journal of Clinical Microbiology 3(5):387-398. These include monoclonal antibodies (MAb) against pathogens and their antigens such as: antimicrobial monoclonal antibodies (MAb) against Streptococcus agalactia, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae B, Treponema pallidum, Lyme disease, spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella, Mycobacterium tuberculosis, tetanus toxin, etc.; Plasmodium falciparum, mitochondrion, and mitochondrion. Antiparasitic monoclonal antibodies (MAb) against Plasmodium falciparum, Toxoplasma gondii, Trypanosoma langerum, Trypanosoma cruzi, Trypanosoma rodeciensis, Trypanosoma bursei, Schistosoma mansoni, Schistosoma japonica, Mesocestoidoid corti, Emmeria tenella, Trichinella rotundifolia, Leishmania tropica, Trichinella, Theileria parva, Tapeworm, Taenia saginata, and Taenia saginata; HIV-1, -2, and 3, A, B Antiviral monoclonal antibodies (MAb) against influenza viruses, C and D types, rabie viruses, influenza viruses, cytomegaloviruses, simplex herpes I and II, human serum parvo-like viruses, respiratory syncytial virus, varicellazoster virus, hepatitis B virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, mumps virus, Sindbisvirus, mouse mammary tumor virus, feline leukemia virus, lymphocytic choriomeningitis virus, wart virus, blue tan virus, Sendai virus, reovirus, poliovirus, dengue virus, rubella virus, murine leukemia virus, etc.; anti-mycoplasma monoclonal antibodies (MAb) against Acholeplasma leidrowii, Mycoplasma arthritis, Mycoplasma hyolinis, Mycoplasma olare, M. arginine, M. pneumonia, etc.

[0107] Appropriate monoclonal antibodies (MAbs) have been developed against most microorganisms (bacteria, viruses, protozoa, and other parasites) that cause the majority of infections in humans, and many of these have traditionally been used for in vitro diagnostic purposes. These antibodies, as well as newer monoclonal antibodies (MAbs) that can be produced by conventional methods, are suitable for use as targeted agents in conjunction with the compounds of the present invention. Monoclonal antibodies (MAbs) against the malaria parasite can be directed at the sporozoite, merozoite, schizont, and gamete mother cell stages. Monoclonal antibodies have been produced against sporozoites (perisporozoite antigens) and have been shown to neutralize sporozoites in vitro and in rodent models. See Yoshida et al. (1980) Science 207:71-73. Monoclonal antibodies against T. gondii, a parasite of protozoa involved in toxoplasmosis, have been developed. See Kasper et al. (1982) Journal of Immunology 129:1694-1699. Monoclonal antibodies (MAbs) have been developed against the cystosomra surface antigen and have been shown to act against cystosomra in vitro or in vivo. 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.

[0108] Similar to hybrid antibodies, mixtures of antibodies and immunoglobulin classes can be used. Multispecific antibodies and antibody fragments, including bispecific and hybrid antibodies, are particularly preferred in the methods of the present invention for detecting and treating target tissues, and consist of at least two distinct substantially monospecific antibodies or antibody fragments, of which at least two specifically bind to at least two distinct antigens produced or associated with the target lesion, or to at least two distinct epitopes or molecules of marker substances produced or associated by the target tissue. Multispecific antibodies and bispecific antibody fragments can be prepared in the same manner as the antitumor marker hybrids disclosed in U.S. Patent No. 4,361,544. Other techniques for preparing hybrid antibodies are disclosed, for example, in U.S. Patents No. 4,474,893 and 4,479,895, which are incorporated herein by reference in their entirety. The same techniques are also disclosed in Milstein et al. (1984) Immunology Today 5:299.

[0109] Useful antibody fragments in the present invention 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-fragments that retain the hypervariable antigen-binding region of the immunoglobulin and have a size similar to or smaller than that of the Fab' fragment are also useful. These may include genetically engineered and / or recombinant proteins (single-chain or multi-chain) that incorporate the antigen-binding site and function as a target vehicle in vivo in substantially the same manner as natural immunoglobulin fragments. Such single-chain binding molecules are disclosed in U.S. Patent No. 4,946,778, which is incorporated herein by reference in its entirety. The Fab' antibody fragment can be readily prepared by reductive cleavage of the F(ab')2 fragment, and the F(ab')2 fragment itself can be prepared by pepsin digestion of intact immunoglobulin. Fab antibody fragments can be created by papain digestion of intact immunoglobulin under reducing conditions, or by cleavage of the F(ab)2 fragment resulting from careful papain digestion of whole immunoglobulin. A ligand or one member of a ligand-receptor binding pair can be conjugated to the compound of the present invention to target the compound to a specific target tissue or target composition. Examples of ligand-receptor binding pairs are described in U.S. Patents 4,374,925 and 3,817,837, which are incorporated herein by reference in their entirety.

[0110] Conjugation to ligand Many compounds that can serve as ligand-receptor binding pairs, more specifically as antibody targets, have been identified, and the techniques for constructing conjugates of such ligands with compounds of formula (I) are well known to the usual technicians in this field. For example, Rakestraw et al. disclose the conjugation of Sn(IV) chlorin to monoclonal antibodies via covalent linkage using a modified dextran carrier. See Rakestraw et al. (1990) Proceedings of the National Academy of Science of the USA 87:4217-4221. The compounds of the present invention can also be conjugated to ligands such as antibodies by the use of coupling agents. Any linkage that can bind the components to be stable under physiological conditions for the duration required for administration and treatment is suitable, but covalent linkage is preferred. The linkage between the two components may be direct, for example, such that the compound of formula (I) is directly linked to the target drug, or indirect, for example, such that the compound of formula (I) is linked to an intermediate, which is then linked to the target drug. Coupling agents must function under conditions such as temperature, pH, salt, solvent system, and other reactants that substantially maintain the chemical stability of the photosensitizer, backbone (if present), and target agent. The coupling agent must stably link the component parts while minimizing or completely eliminating denaturation or inactivation of the compound of formula (I) or the target agent. Many coupling agents react with amines or carboxylates to form amides, or with alcohols and carboxylates to form esters. Coupling agents are known in the art. For example, Bodansky (1993) Principles of Peptide Synthesis, 2nd ed., Springer & Hermanson (1996) Bioconjugate Techniques, 1 st See Ed., Academic Press, New York, New York, USA.

[0111] Conjugates of the compounds of the present invention with ligands such as antibodies can be prepared by coupling the compound to a target moiety by coupling the carboxylic acid or ester moiety on the compound via a peptide bond to the antibody via the N-terminus, or by other methods known in the art. Various coupling agents, including crosslinking agents, can be used for covalent bonding. Examples of such crosslinking agents include N,N'-dicyclohexylcarbodiimide (DCC), N-succinimidyl-5-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), ortho-phenylene-dimaleimide (o-PDM), and sulfosuccinimidyl 4-(N-maleimide-methyl)-cyclohexane-1-carboxylate (sulfo-SMCC). For example, see 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 USA 82(24):8648-8652. Other methods are disclosed in Brennan et al. (1985) Science 229:81-83 and Glennie et al. (1987) Journal of Immunology 139:2367-2375. For example, N,N'-dicyclohexylcarbodiimide (DCC) is a useful coupling agent that can be used to promote the coupling of alcohol NHS with a chlorincarboxylic acid group in DMSO to form an active ester that can be crosslinked to polylysine. N,N'-dicyclohexylcarbodiimide (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 N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), a heterobifunctional crosslinking agent for use with primary amines and sulfhydryl groups. N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has a molecular weight of 312.4 and a spacer arm length of 6.8 angstroms. It reacts with the NHS ester and the pyridyldithio group to form a cleavable crosslink, which, upon further reaction, removes the drug and allows the photosensitizer to be directly linked to the backbone or target agent. Other useful conjugates include N-succinimidyl-5-acetylthioacetate (SATA) for introducing a blocked SH group (which is deblocked with hydroxylamine-HCl) for two-step crosslinking, and sulfosuccinimidyl-4-(N-maleimide-methyl)-cyclohexane-1-carboxylate (sulfo-SMCC), which is reactive with amines and sulfhydryls. Other crosslinking and coupling agents are also available from Pierce Chemical Co. Additional compounds and processes for conjugating proteins to other proteins or other compositions (e.g., to reporter groups or to chelating agents for metal ion labeling of proteins), particularly those involving Schiff bases as intermediates, are disclosed in European Patent EP 0 243 929B1.

[0112] Photosensitizers containing carboxyl groups can be conjugated to the lysine ε-amino group of a target polypeptide by either a pre-formed reactive ester (such as N-hydroxysuccinimide (NHS) ester) or an ester conjugated in situ via a carbodiimide reaction. The same applies to photosensitizers containing sulfonic acid groups that can be converted to sulfonyl chloride to react with the amino group. Bacteriochlorins containing carboxyl groups can be conjugated to amino groups on polypeptides by the in-situ carbodiimide method. Bacteriochlorins can also be conjugated to hydroxyl groups of serine or threonine residues, or to sulfhydryl groups of cysteine ​​residues. In methods for conjugating components of a conjugate (for example, coupling a polyamino acid chain that produces a photosensitizer to an antimicrobial polypeptide), heterobifunctional crosslinking reagents can be used. These reagents bond to a functional group on one chain and another functional group on the second chain. These functional groups are typically amino, carboxyl, sulfhydryl, and aldehyde. There are many permutations of the appropriate parts, which react with these groups and also with differently formulated structures to conjugate them together. See Hermanson (1996) Bioconjugate Techniques, 1st Ed., Academic Press, New York, New York, USA; and Merrifield et al. (1994) Ciba Foundation Symposium 186:5-20.

[0113] The compound or a pharmaceutically acceptable derivative thereof may be packaged as a product including (i) packaging material, (ii) the compound of the present invention or a pharmaceutically acceptable derivative thereof within the packaging material (which is effective for regulating the activity of hyperproliferative tissue or angiogenesis, or for treating, preventing or improving one or more symptoms of hyperproliferative tissue or angiogenesis-mediated diseases or disorders, or diseases or disorders involving hyperproliferative tissue or angiogenesis), and (iii) a label indicating that the compound or composition, or a pharmaceutically acceptable derivative thereof, is used for regulating the activity of hyperproliferative tissue or angiogenesis, or for treating, preventing or improving one or more symptoms of hyperproliferative tissue or angiogenesis-mediated diseases or disorders, or diseases or disorders involving hyperproliferative tissue or angiogenesis. The products of the present invention include packaging materials. Packaging materials for use in packaging pharmaceuticals 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. These are incorporated herein in their entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging materials suitable for the selected formulation and the intended mode of administration and treatment. A wide variety of formulations of the compounds and compositions of the present invention are expected to serve as a broad range of therapeutics for any disease or disorder in which overgrowth or angiogenesis is involved as a mediating or contributing factor to its symptoms or causes.

[0114] V. Applications in photodynamic therapy, diagnosis, and treatment In some embodiments, compounds represented by formula (II) of the present invention (or pharmaceutically acceptable salts or conjugates thereof) can act as photosensitizers in methods of treating diseases (e.g., hyperproliferative diseases such as cancer), including photodynamic therapy (PDT). Simply put, these photosensitizing compounds, conjugates, or pharmaceutical compositions are generally administered to a patient before the target tissue, target composition, or patient is irradiated with light. These photosensitizing compounds are administered as described elsewhere herein. The dose of the photosensitizer can be determined clinically. Depending on the photosensitizer used, an equivalent optimal therapeutic level needs to be established. A certain time is permitted for the circulating or locally delivered photosensitizer to be taken up by the target tissue. Unbound photosensitizers can be removed from circulation during this waiting period, or optionally, an additional time can be provided to remove these unbound compounds from non-target tissues. This waiting period can be determined clinically and varies from compound to compound. After this waiting period, the bound drug is activated using a laser light source or a non-laser light source (including, but not limited to, artificial light sources such as fluorescence or incandescence, or natural light sources such as ambient sunlight). The range of illumination is determined by the location and size of the pathological area to be detected, diagnosed, or treated. The length of illumination time may depend on whether detection or treatment is being performed, or it may be determined empirically. Any time between approximately 4 minutes and approximately 72 hours can be used as the total or cumulative time. In some embodiments, this illumination time is between approximately 60 minutes and 148 hours. In some embodiments, this illumination period is between approximately 2 hours and 24 hours.

[0115] The total fluence or energy of the light used for irradiation, measured in joules, is preferably between about 10 joules and about 25,000 joules, more preferably between about 100 joules and about 20,000 joules, and most preferably between about 500 joules and about 10,000 joules. Light with 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. To irradiate the target tissue, it is preferable to use light having a wavelength that at least partially corresponds to the characteristic light absorption wavelength of the photosensitizer. The intensity or power of the light used is measured in watts, where 1 joule is equal to 1 watt-second. Therefore, the intensity of the light used to irradiate using the method of the present invention is substantially 500 mW / cm². 2It may be less than this. Since the total fluence or energy of this light (in joules) is divided by the total exposure time (in seconds), increasing the time the target is exposed to irradiation allows the total energy or fluence to be increased without increasing the intensity of the light used. In this invention, a total fluence of irradiation that is sufficiently high to activate the photosensitizer is used.

[0116] In some embodiments of using the compounds of the present invention for photodynamic therapy, the compounds are injected into mammals (e.g., humans) for diagnosis or treatment. The injection level is typically between about 0.1 and about 0.5 umol / kg body weight. In the case of treatment, the area being treated is at the desired wavelength and energy (e.g., about 10 to 200 J / cm²). 2 The compound is exposed to light. For detection, fluorescence is measured when the compound is exposed to light of a wavelength sufficient to cause fluorescence at a wavelength different from the wavelength used for irradiation of the compound. The energy used for detection is sufficient to cause fluorescence, and is usually significantly lower than the energy required for treatment. Any one of the photosensitizing compounds of the present invention or its pharmaceutically acceptable derivatives may be supplied in a kit along with instructions for carrying out any of the methods of the present invention. These instructions may be in any specific form, such as printed paper, a computer disk instructing a person on how to carry out the method, a video cassette containing instructions on how to carry out the method, or computer memory receiving and explaining data from a remote location, or other means of providing instructions to a person (e.g., via the Internet). Any of these instructions may be used to instruct a person on how to use the kit, either after receiving instruction in a classroom or in the course of treating a patient using any of the methods of the present invention.

[0117] Additional and specific examples of methods for using the compounds and compositions of the present invention include, but are not limited to, the following: (i) Treatment of opportunistic infections: The compounds, compositions and methods of the present invention are useful for opportunistic infections, particularly soft tissue photodynamic therapy (PDT). In the case of antimicrobial treatment (via photodynamic therapy (PDT)) of infections (particularly wound infections), the infectious organisms include (as non-limiting examples) Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. In hospital-acquired infections, Pseudomonas aeruginosa is involved in 8% of surgical wound infections and 10% of bloodstream infections. In some embodiments, the patient is an immunocompromised patient, such as a patient with AIDS or a patient receiving treatment with immunosuppressants. (ii) Treatment of burns: Infections caused by Staphylococcus aureus and Gram-positive bacteria are generally particularly prevalent in burns. Multidrug resistance of Staphylococcus aureus presents a significant medical challenge. In this regard, the compounds, compositions, and methods of the present invention are useful for treating opportunistic infections of burns. (iii) Sepsis: The compounds, compositions, and methods of the present invention are useful for photodynamic therapy (PDT) treatment of patients suffering from opportunistic infections of Vibrio vulnificus. Vibrio vulnificus is a Gram-negative bacterium that causes primary sepsis, wound infections, and gastrointestinal diseases in humans. (iv) Ulcers: The compounds, compositions, and methods of the present invention are useful for photodynamic therapy (PDT) treatment of ulcer-causing bacteria (Helicobacter pylori). In hospitals, treatment can be effectively carried out by appropriate methods, such as inserting a fiber optic cable (similar to an endoscope but equipped with equipment for emitting red or near-infrared light) into the stomach or affected area.

[0118] (v) Periodontal disease: The compounds, compositions and methods of the present invention are useful in photodynamic therapy (PDT) for the treatment of periodontal disease, including gingivitis. Periodontal disease is caused by the abnormal proliferation of bacteria such as the Gram-negative anaerobic bacterium Porphyromonas gingivalis. As with many photodynamic therapy (PDT) treatments, targeted or solubilized entities combined with photoactive species are essential for the proper delivery of the photoactive species to the target cells. Targeted oral pathogens include Porphyromonas gingivalis, Actinobacillus actinon zycetemcomitans, Bacterioid forsythias, Campylobacter rectus, Eichenella collodens, Fusobacterium nucleatum subspecies polymorphism, Actinomyces viscosus, and Streptococcus. For such applications, the compounds or compositions of the present invention can be applied topically (e.g., as a mouthwash or rinse), and then light can be irradiated with an external device, an oral instrument, or a combination thereof. (vi) Atherosclerosis: The compounds, compositions, and methods of the present invention are useful in photodynamic therapy (PDT) for treating fragile atherosclerotic plaques. While we do not wish to be bound by any particular theory, it is thought that invading inflammatory macrophages secrete metalloproteinases that break down the thin layer of collagen in the coronary arteries, leading to thrombosis, which is often fatal. Active compounds that target such inflammatory macrophages are useful in photodynamic therapy (PDT) of unstable plaques.

[0119] (vii) Cosmetic and Skin Applications: The compounds, compositions and methods of the present invention are useful in photodynamic therapy (PDT) for treating a wide range of cosmetic dermatological problems, such as hair loss, the treatment of psoriasis, or the removal of skin discoloration. Ruby lasers are currently used for hair loss, and in many laser treatments, melanin is the photosensitizing chromophore. Such treatments are suitable for people with dark hair and fair skin. The compounds, compositions and methods of the present invention can be used as near-infrared sensitizers for hair loss, thereby making it possible to target chromophores with more specific and sharp absorption bands. (viii) Acne: The compounds, compositions and methods of the present invention are useful in photodynamic therapy (PDT) for the treatment of acne. Prurigo vulgaris is caused by Propionibacterium acnes, which infects the sebaceous glands, and affects approximately 80% of young people. Here again, the increasing resistance of bacteria to antibiotic treatment has led to a surge in acne that is difficult to treat. Current photodynamic therapy (PDT) treatment for acne usually relies on the addition of aminolevulinic acid, which is converted to free base porphyrin in the hair follicles or sebaceous glands. The compounds and compositions of the present invention can be administered to the patient topically or parenterally (e.g., by subcutaneous injection), depending on the specific condition.

[0120] (ix) Infectious Diseases: The compounds, compositions and methods of the present invention are useful in photodynamic therapy (PDT) for treating infectious diseases. For example, cutaneous and subcutaneous leishmaniasis, which are widespread in the Mediterranean and Middle Eastern regions, are currently treated with arsenic-containing compounds. Recently, photodynamic therapy (PDT) has been used in at least one case to produce reasonable effects in human patients. The use of the compounds and compositions of the present invention is equally useful and potentially offers advantages such as ease of synthesis and better spectral absorption properties. (x) Tissue sealants: The compounds, compositions, and methods of the present invention are useful in photodynamic therapy (PDT) as tissue sealants for patients who require them. Photoactivated tissue sealants are attractive for wound sealing, tissue bonding, and closure of tissue defects. There are many applications where sutures and staples are undesirable and the use of such mechanical sealing methods often leads to infection and scarring. (xi) Neoplastic diseases: The compounds, compositions and methods of the present invention are useful in photodynamic therapy (PDT) for treating 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.

[0121] In addition to photodynamic therapy (PDT), the compositions of the present invention can be used as image enhancers in diagnostic imaging techniques or to label target tissues or target compositions for radiological diagnosis. In the field of modern medicine, there are various therapies for diagnosing disease, including magnetic resonance imaging (MRI). Early detection of cancer should improve the chances of curing cancerous tissue. Early diagnosis of precancerous areas and microcarcinomas is a key subject in modern cancer treatment. MRI has emerged as a powerful tool in clinical practice because it is non-invasive and provides an accurate three-dimensional representation of the target. Like a typical nuclear magnetic resonance (NMR) experiment, images are created by exciting nuclear spins with radio frequency pulses to impose one or more orthogonal magnetic field gradients on the target or specimen. After collecting data in various gradient fields, deconvolution generates one-dimensional, two-dimensional, or three-dimensional images of the specimen / target. Typically, this image is based on NMR signals from water protons, and the signal intensity of the volume element is a function of water concentration and relaxation time. Local variations in these parameters provide the vivid contrast observed in MR images.

[0122] MRI contrast agents work by increasing the relaxation rate, thereby enhancing the contrast between water molecules in the area where the contrast agent is applied and water molecules in other parts of the body. However, the effect of the contrast agent decreases both T1 and T2, with the former showing increased contrast and the latter showing decreased contrast. Therefore, this phenomenon is concentration-dependent, and there is usually an optimal concentration of paramagnetic species to obtain the greatest effect. This optimal concentration varies depending on the contrast agent used, the imaging site, the imaging mode (i.e., spin echo, 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 contrast agent is dissolved or suspended. These factors and their relative importance are known in the art. See, for example, Pyket t (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, enhancing vascular contrast and reporting organ lesions and infiltrations. However, labeling specific tissues for diagnostic radiology remains a challenge for magnetic resonance imaging (MRI). Efforts to develop cell and tissue-specific MRI enhancers by modifying existing immunological techniques have been the focus of much research in diagnostic radiology. For example, antibodies labeled with paramagnetic ions (typically gadolinium chelate Gd-DTPA) have been generated and their effects on MRI contrast agents in tumors and other tissues have been tested. See U.S. Patent No. 5,059,415, which is incorporated herein by reference in its entirety. Unfortunately, the relaxation ability of antibody-bound Gd has been found to be only slightly better than that of unbound Gd-DTPA. See Paajanen et al. (1990) Magnetic Resononance in Medicine 13:38-43.

[0123] Magnetic resonance imaging (MRI) is generally used in the body. 1 It is used to detect H nuclei. However, MRI,13 C, 15 N, 31 P, 19 NMR spectra of other nuclides, such as 14F, can also be detected. 19 F is not abundant in living organisms. 13 C, 15 N, 31 P, or 19 F etc. (especially) 19 By incorporating an isotope useful for MRI (F) into the composition of the present invention and administering it to a patient, the compound of the present invention accumulates in the target tissue, and subsequently, by NMR imaging, 19 Because of the presence of accumulated compounds containing MRI-recognizable isotopes such as 1F, enhanced NMR data is generated from the target tissue or target composition. Therefore, the compounds of the present invention can be used as image enhancers and can provide labeling for specific target tissues or target compositions for diagnostic radiology, including magnetic resonance imaging (MRI). In addition to photodynamic therapy (PDT), the compositions of the present invention can be used to detect target cells, target tissues, or target compositions in a patient. When the compounds of the present invention are used to detect target tissues or target compositions, the compounds are introduced into the patient and given sufficient time for the compounds to accumulate in the target tissues or to bind to the target compositions. The treatment area is then generally irradiated with light of sufficient energy to induce fluorescence of the compounds. The energy used here is typically significantly lower than that required for photodynamic therapy. By exposure to light of a desired wavelength, fluorescence is observed, and the amount of this fluorescence is correlated qualitatively or quantitatively with the abundance of the compounds by methods known in the art.

[0124] The compositions of the present invention can also be used to diagnose the presence of an infectious agent or to diagnose the identity of an infectious agent in a patient. The compounds of the present invention can be conjugated to one or more ligands specific to the infectious agent (such as an antibody or antibody fragment) that selectively associate with the infectious agent, and the targeted compound can be visualized (e.g., by exposing it to light with sufficient energy to produce fluorescence, or by imaging using diagnostic radiology, including MRI). For example, one of the compounds of the present invention can be conjugated to an antibody targeted against a suitable Helicobacter pylori antigen, formulated into a pharmaceutical preparation and introduced into a patient, whereupon the conjugated compound is released into the gastric mucus / cortex where the bacteria are found. After sufficient time for the compound to selectively bind to the target infectious agent and for the unbound compound to be removed from the non-target tissue, the patient can be examined to determine whether Helicobacter pylori is present. This is, for example, 19 This can be done by detecting compounds that have accumulated due to the presence of the F substituent using MRI, or by irradiating the target region with light of sufficient energy to generate fluorescence from the target compound using an optical fiber, and then detecting the fluorescence from the target compound.

[0125] In some embodiments, the compounds of the present invention or their conjugates may be useful in flow cytometry. Flow cytometry is well known and is described, for example, in U.S. Patents 5,167,926, 5,915,925, 6,248,590, 6,589,792, and 6,890,487, which are incorporated herein by reference in their entirety. In some embodiments, particles to be detected, such as cells, are labeled for detection with luminescent compounds, such as phosphorescent substances or fluorophores. This labeling can be carried out by any suitable technique, such as coupling the luminescent compound to another compound, such as an antibody (which specifically binds to particles or cells), taking up or internalizing the luminescent compound into cells or particles, or non-specifically adsorbing the luminescent compound onto cells or particles. The active compounds of the present invention are useful as luminescent compounds in flow cytometry, and flow cytometry techniques (including fluorescence-activated cell sorting or FACS) can be carried out according to techniques or variations thereof that are apparent to those skilled in the art based on the disclosure of the present invention. [Examples]

[0126] The following embodiments provide exemplary designs. In light of the present invention and the general level of those skilled in the art, those skilled in the art will understand that the following embodiments are merely illustrative and that many changes, modifications, and alterations can be made without departing from the scope of the invention.

[0127] Example 1 Synthesis of compound CP-1 As shown in Scheme 2 (Figure 2), a Di-BOC-protected Suzuki coupling partner 1 (CP-1) was prepared. 1-Bromo-3,5-bis(bromomethyl)benzene (CP-1a) N-bromosuccinimide (NBS, 35.60 g, 200.0 mmol) was added to a flame-dried 1 L round-bottom flask (RBF) with a stirring rod, and a glass stopper, diaphragm-top condenser, and rubber diaphragm were fitted to the flask. After drying the N-bromosuccinimide (NBS) under high vacuum for 30 minutes, the flask was washed with argon, and acetonitrile (ACN, 400 mL) was added via cannula until it reached approximately half its volume (~450 mL). 1-bromo-3,5-dimethylbenzene (15.26 g, 80.0 mmol) was then added by syringe, followed by a brief opening of the system under argon flow, and solid azobisisobutyronitrile (AIBN, 0.670 g, 4.00 mmol) was added. The flask was heated under argon (oil bath set to 90°C) and gently refluxed. After 16 hours, the reaction mixture was transferred to a 1 L single-neck round-bottom flask (RBF) and concentrated to remove ACN. The solid residue was further dried under high vacuum, suspended in dichloromethane (DCM, 75 mL), and heated to a gentle boil. The mixture was equilibrated at room temperature and filtered by washing with DCM. The resulting filtrate was concentrated, dried under high vacuum, and recrystallized in ethanol (EtOH, 55 mL total) while heating in a water bath set at 65°C. The resulting solid was filtered, washed with ice-cooled EtOH, and then dried under high vacuum. Compound CP-1a (17.40 g, 51%) was isolated as a white crystalline solid. 1 H NMR(400 MHz, CDCl3) δ 4.41(s, 4H), 7.34(s, 1H), 7.47(d, J = 2.0 Hz, 2H).

[0128] 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 round-bottom flask (RBF) equipped with a stirring bar. The flask was washed with argon, and dimethylformamide (DMF, 215 mL, 0.25 M) was added. This colorless, transparent solution was stirred, and potassium phthalimide (23.37 g, 123.63 mmol) was added gradually. A condenser with a drying tube was attached to the flask, and it was heated in an oil bath set to 90°C. After 16 hours, the mixture was cooled to room temperature, diluted with water (total 1 L), and extracted with chloroform (400, 300, and 200 mL, 1× each). The resulting organic layers were combined and washed with 0.2N NaOH (500 mL) and water (500 mL). The organic layers were separated, dried over sodium sulfate, filtered, and concentrated. The resulting solid was further dried under high vacuum, then transferred to a filter and washed with diethyl ether (Et2O) (3x) at room temperature. Compound CP-1b was isolated as a white powdery solid (17.98 g, 70%). 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).

[0129] (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 oil bath at 85°C. Hydrazine hydrate (4.88 mL, 80.29 mmol) was added, and a condenser was placed on the flask. The mixture was further heated under reflux for 15 minutes, and then the reaction mixture was gradually cooled to room temperature. To this, 6 NaqHCl was added until the solution became acidic according to a litmus test (total 20 mL). The resulting mixture was heated again to reflux temperature. The flask was washed with argon, stirred for 1 hour, and then cooled in an ice bath. The mixture was filtered to obtain a clear, pale amber solution. The filtrate was cooled in an ice bath and basicized with 2N NaOH (total 30 mL). The aqueous layer was extracted with chloroform (3 × 75 mL). These organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated to a volume of approximately 10 mL. A white residue was observed on the wall of this flask. The remaining solution was filtered, and the filtrate was concentrated to obtain 2.3 g of yellowish oil. This was stored in a refrigerator. This sample was stored overnight at 4°C to solidify, and then dried under high vacuum to obtain 2.047 g (59%) of compound CP-1c as an amber semi-solid.

[0130] Di-t-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 round-bottom flask (RBF) equipped with a stirring rod. The flask was evacuated and washed 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. Anhydrous Boc (4.86 g, 21.86 mmol) was prepared as a solution in THF (10 mL) and added dropwise at a rate of 1 mL. The solution was stirred at 0°C for 1 hour, and then equilibrated to room temperature. The reaction mixture was stirred overnight at room temperature. This mixture was concentrated to obtain a white solid. This was redissolved in ethyl acetate (siRNA, 70 mL). This organic layer was washed with saturated aqueous NH4Cl solution, water, saturated aqueous NaHCO3 solution, and brine (1 × 50 mL each). Next, this organic layer was dried over sodium sulfate, filtered, and concentrated to obtain a pale amber oil. This was allowed to stand and crystallized. This solid was washed with cooled Et2O / hexane (1:1) using a frit filter. This solid was dried under high vacuum to obtain 3.50 g (93%) of compound CP-1d (white powdery solid).

[0131] Coupling Partner 1 (CP-1) Dimethyl sulfoxide (DMSO, reagent grade, 20.0 mL) was added to a 100 mL round-bottom flask (RBF) and bubbling with argon for a total of 45 minutes with stirring. Compound CP-1d (1.25 g, 3.01 mmol), bis(vinacolate)diborone (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 round-bottom flask (RBF), and the flask was evacuated for 30 minutes. The flask was washed with argon, and degassed DMSO was added. This solution was frozen in a dry ice / acetone bath, placed under vacuum, and then thawed under argon. The reaction mixture was heated in an oil bath at 85°C. After 16 hours, the reaction mixture was cooled to room temperature, diluted with toluene (100 mL), and washed with brine (3 × 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. This concentrate was rinsed minimally with DCM and added neat to a 40g silica column, where it was eluted with 0-2% MeOH in the DCM. The main product fractions were totaled and concentrated to obtain a clear oil. Further drying under high vacuum with stirring caused the product to solidify. Compound CP-1 was isolated as a white waxy solid (1.224g, 88%).

[0132] Example 2 Synthesis of compound BC-1 As shown in Scheme 3 (Figure 3), the compound indicated was prepared from the previously described dibromobacteriochlorin (BC-SM). See Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103. BC-1a BC-SM (362.8 mg, 0.538 mmol), coupling partner 1 (CP-1 (Example 1); 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 round-bottom flask (RBF) equipped with a stirring rod, and these components were dried under high vacuum for 1 hour. The flask was washed 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. After 22 hours, the reaction mixture was cooled, toluene (4 × DMF volume) was added, and the mixture was concentrated to dryness. The residue was dissolved in toluene (200 mL) and washed with saturated NaHCO3 aqueous solution, water, and brine (150 mL each). The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain 1.09 g of a purple residue. The crude residue was placed on silica (3.28 g) and eluted for 25 minutes using a 40 g silica column containing 5-30% siRNA in DCM. The main product fractions were combined, concentrated, and dried under high vacuum to obtain 0.466 g (73%) of BC-1a as a purple solid.

[0133] BC-1b BC-1a (146.0 mg, 0.123 mmol) was added to an oven-dried 50 mL round-bottom flask (RBF) equipped with a stirring bar. The flask was evacuated, washed with argon, and THF (20 mL) was added. N-bromosuccinimide (NBS) (23.0 mg, 0.129 mmol) was dissolved in THF (4.0 mL) and rapidly added dropwise. The reaction mixture was stirred under argon at room temperature. After 1.5 hours, the reaction product was diluted with DCM (25 mL), and the reaction was inhibited by adding saturated NaHCO3 aqueous solution (25 mL). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The residue was placed on a 12 g silica column with a minimum amount of DCM (approximately 5 mL) and eluted with 0-3% MeOH in DCM for 22 minutes. The main peak fractions were combined, concentrated, and dried under high vacuum to obtain 0.115 g (79%) of BC-1b as a purple solid.

[0134] BC-1c BC-1b (114.5 mg, 90.6 μmol) was added to an oven-dried 25 mL round-bottom flask (RBF) equipped with a stirring bar, along with Pd2(dba)3 (12.5 mg, 13.6 μmol) and P(o-tol)3 (32.0 mg, 105.1 μmol), and stirred. The flask was capped and evacuated for 30 minutes. The flask was washed with argon, and DMF (3.63 mL) was added, followed by triethylamine (0.363 mL) and 6-heptic acid (228.7 mg, 1812.0 μmol). The flask was warmed in an oil bath at 40°C and stirred under argon. After 6 hours, the reaction product was diluted with ¼ volume of HCl (10 × DMF) and sequentially washed with equal volumes of 0.2N aq HCl, water, and brine, respectively. The organic layer was separated, dried over sodium sulfate, filtered, concentrated, redissolved in toluene, concentrated, and dried under high vacuum. The residue was placed on silica (1.05 g) and eluted for 15 minutes using a 24 g silica column containing 0-33% Â in DCM, and held until the starting material was eluted. Next, the solvent was switched to 0-4% MeOH in DCM over 15 minutes. The peak fractions of the product were combined, concentrated, and dried under high vacuum to obtain 45.0 mg (38%) of BC-1c as a dark purple solid.

[0135] BC-1 BC-1c (17.5 mg, 13.36 μmol) was added to an oven-dried 25 mL round-bottom flask (RBF) equipped with a stirring rod. The flask was placed under vacuum for 30 minutes, then washed with argon, and this vacuum / argon washing cycle was repeated twice. Hydrogen chloride solution (4.0 M in dioxane, 2.9 mL) was added in bulk with stirring, and the reaction mixture was stirred under argon. After 30 minutes, stirring was stopped, and the precipitate was allowed to settle for 10 minutes. Most of the dioxane was removed under argon using a syringe. The residue was placed under high vacuum for 2 hours. The reaction flask was washed with argon, and tributylamine (5 drops) was added and mixed with stirring. Next, hexane / THF (2:1) solution (3 mL) was added and stirred briefly. Then, the mixture was sonicated for 3 minutes, transferred to a 1.5 mL sample tube, and spun down (9,000 g × 3 mins). The colorless, clear supernatant was removed, the tube was covered with Parafilm, punctured with a needle, placed in a flask, and dried overnight under high vacuum. The resulting solid intermediate was transferred to a dry 25 mL round-bottom flask (RBF) equipped with a stirring bar, along with cesium carbonate (48.1 mg, 147.6 μmol) and mPEG11-NHS (101.2 mg, 147.6 μmol). The flask was sealed with a diaphragm, evacuated, washed with argon, and DMF (2.95 mL) was added. The mixture was kept in the dark and stirred under argon for 2 hours. The crude reaction mixture was subjected to reverse-phase chromatography using a 50 g C18 gold column. The products containing each fraction were combined, concentrated, redissolved in ACN, and concentrated again. The residue was dried overnight under high vacuum to obtain 7.2 mg (16%) of BC-1 as a purple semi-solid. MS: obsd 1645.1, calcd 1644.4 [M + 2H] 2+ λabs 378, 545,754 nm (H2O); Correction factor: A280 / A754 = 0.11; λem 760 nm (H2O); Quantum yield: 8.2% (PBS); Ext coefficient: 132,000 M-1 cm-1 (380 nm, toluene); 104,000 M -1 cm -1(751 nm, toluene); FWHM: 26 nm.

[0136] 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 (referred to as "prespin") was diluted to 10 μM in PBS and its absorbance was read. The first 10 mg / mL sample was centrifugated at 14000 g for 10 minutes. A second portion of this spun sample (referred to as "postspin") was taken, diluted to 10 μM in PBS, and its absorbance was read. These data are shown in Table 1 below. No precipitate was observed in the samples. The absorbance value remained constant from prespin to postspin (expected error within + / - 10%). The decrease in absorbance in postspin indicates the presence of an insoluble precipitate in the initial sample. Therefore, it can be concluded that BC-1 dissolves in PBS (pH 7.2) at 10 mg / mL. [Table 1]

[0137] Example 3 Synthesis of compound BC-2 As shown in Scheme 4 (Figure 4), BC-2a was prepared. A mixture of 5-ethynyl-1,3-benzenedicarboxylic acid (500 mg, 2.63 mmol), N-(2-aminoethyl)carbamate tert-butyl (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). This flask was stirred at room temperature for 16 hours. The reaction mixture was placed directly on silica gel and subjected to chromatography [silica, CH2Cl2 / MeOH (0-10%)] to obtain a white solid. The obtained solid was, 1¹H NMR revealed the presence of 4-dimethylaminopyridine (DMAP). Therefore, it was redissolved in ethyl acetate, washed with a 1.0% aqueous HCl solution, dried over Na₂SO₄, and concentrated to obtain BC-2a as a white solid (938 mg, 75%).

[0138] As shown in Scheme 5 (Figure 5), BC-2 was prepared starting from the previously described dibromobacteriochlorin (BC-SM). See Jiang et al. (2014) Organic & Biomolecular Chemistry 12:86-103. BC-2b A mixture of BC-SM (135 mg, 200 μmol), tert-butyl 4-ethynylbenzoate (48.5 mg, 240 μmol), Pd(PPh3)4 (23.1 mg, 20.0 μmol), and K2CO3 (276 mg, 2.00 mmol) was placed in a round-bottom flask (RBF) equipped with a three-way tap. This flask was placed under high vacuum for 1 hour and degassed by three evacuation-replenishment cycles. Anhydrous DMF (20 mL) was added to this mixture using a syringe, and the mixture was heated at 80°C for 16 hours. After the reaction mixture cooled to room temperature, it was diluted with ethyl acetate, washed with aqueous NaHCO3, and dried over Na2SO4. The resulting mixture was subjected to chromatography [12 g silica gold, hexane / ethyl acetate (0-40%)] to obtain BC-2b as a dark solid (39.4 mg, 25%). 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 a round-bottom flask (RBF) equipped with a three-way tap. This flask was placed under high vacuum for 1 hour and degassed by three evacuation-replenishment cycles. Anhydrous DMF / TEA (2:1, 10 mL) was added by syringe, and the reaction mixture was heated at 80°C for 16 hours. After the reaction mixture cooled to room temperature, it was diluted with ethyl acetate, washed with aqueous NaHCO3, and dried over Na2SO4. The resulting mixture was subjected to concentrated chromatography [silica, hexane / ethyl acetate (0-40%)] to obtain BC-2c as a dark solid (11.3 mg, 28%). BC-2 BC-2c (4.4 mg, 3.7 μmol) was treated with 4.0 M HCl in dioxane (926 μL). The reaction mixture was stirred at room temperature in the dark under argon. After 4.5 hours, the reaction mixture was placed under high vacuum for 16 hours. The resulting mixture was treated with CH3(OC2H4) 24 CONHS (mPEG24-NHS, 18.0 mg, 14.8 μmol), Cs2CO3 (19.3 mg, 59.2 μmol), and DMF (926 μL) were added. The reaction mixture was stirred and monitored by liquid chromatography (LCMS) at room temperature. After 2 hours, the reaction mixture was subjected to chromatography [C18 15.5 g, H2O / CH3CN (0-40%)] to obtain BC-2 as a green solid (10.8 mg, 93%). MS: obsd 1066.64, calcd 1066.55 [M + 3Na] 3+ M = C152H248N8O59; λabs 383, 533, 792 nm (H2O / CH3CN); Correction factor: A280 / A791 = 0.24 (H2O / CH3CN); λem 800 nm (DMF, estimated); Ext coefficient: 106,000 M -1 cm -1 (791 nm); 83,000 M -1 cm -1 (384 nm)' 23,000 M -1cm -1 (551 nm) in DMF, estimated

[0139] Example 4 Synthesis of compound BC-3 As shown in Scheme 6 (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 vial with spin vanes. The vial was sealed, evacuated, and washed with argon. BC-2 (4.3 mg, 1.37 μmol) was dissolved in DCM (0.5 mL) and transferred to this reaction vial, after which triethylamine (0.29 μL, 2.06 μmol) was added. The reaction mixture was stirred at room temperature for 2 hours. Next, aminoPEG was added to this reaction mixture. 12 CH2CH2COOH (4.24 mg, 6.87 μmol) was added directly, followed by the addition of DCM (300 uL), and the mixture was stirred under argon at room temperature, shielded from light. After 16 hours, the reaction mixture was diluted with DCM and washed with saturated NH4Cl aqueous solution (2 mL x 2) and then with water (1 mL x 1). The aqueous layer was removed by pipette, and the organic layer was concentrated in the reaction vial. The residue was dissolved in ACN / water and eluted using a 15.5 g C18Isco gold column. After complete drying, BC-3 was isolated as a dark red semi-solid (3.3 g, 65%).

[0140] Example 5 Synthesis of compound BC-4 As shown in Scheme 7 (Figure 7), BC-4 was prepared from BC-2. BC-2 was treated with Zn(OAc)22H2O (30 equivalents) in DMF (4 mM), heated at 80°C for 16 hours, and then the reaction mixture was concentrated and purified by reverse-phase preparative LC.

[0141] Example 6 Synthesis of compound BC-5 As shown in Scheme 8 (Figure 8), BC-5 was prepared. BC-5a (i.e., 7-bromo-2,3-dihydro-8-(methoxycarbonyl)-1-(1,1-dimethyloxymethyl)-3,3-dimethyl-dipyrine) was prepared as previously described. See Jiang et al., Org. Biomol. Chem. 2014, 12, 86-103. BC-5b BC-5a (0.771 g, 2.00 mmol) was added to a flame-dried 250 mL round-bottom flask (RBF) equipped with a stirring rod. The flask was evacuated and washed with argon. Acetonitrile (111.1 mL) was added to the flask by syringe. While stirring, BF3OEt2 (2.96 mL, 24.00 mmol) was rapidly added dropwise to the surface of the solvent. A rapid color change was observed. The flask was shielded from light and stirred at room temperature under a low flow rate of argon. After 16 hours, triethylamine (3.68 mL, 26.4 mmol) was added. The diaphragm was removed, and the reaction mixture was stirred until the fuming subsided. The reaction mixture was concentrated and further dried under high vacuum until the flask was cool to the touch. The residue was dissolved in DCM and prepared as a silica cake (5 g). This cake was eluted for 17 minutes using an 80 g SiO2 column containing 20-60% DCM in hexane. The main product fractions were combined, concentrated, and dried under high vacuum to obtain 0.11 g (17%) of BC-5b as a dark red solid. BC-5c BC-5b (56.7 mg, 0.088 mmol) and Zn(OAc)22H2O (579 mg, 2.64 mmol, 30 equivalents) were added to a flame-dried 100 mL round-bottom flask (RBF) equipped with a stirring rod. The flask was sealed with a diaphragm, evacuated, and washed 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 low-flow argon. After 17 hours, the reaction mixture was diluted with DCM (5 × DMF volume), and the organic layer was washed with saturated NaHCO3 aqueous solution (3 × DMF + DCM volume) until the aqueous layer was clear. This organic layer was dried over Na2SO4, filtered, concentrated, and further dried under high vacuum. The residue was transferred to a 20 mL vial of DCM and dried under high vacuum to obtain 61.6 mg (99%) of a dark red solid. This material was carried over to the next stage without further purification.

[0142] BC-5d BC-5c (17.7 mg, 25.0 μmol), t-butyl-4-ethynylbenzoate (12.6 mg, 62.5 μmol, 2.5 equivalents), BC-2a (Example 3) (17.8 mg, 37.5 μmol, 1.5 equivalents), Pd(PPh3)2Cl2 (4.4 mg, 6.25 μmol, 0.25 equivalents), and CuI (2.4 mg, 12.5 μmol, 0.5 equivalents) were added to an oven-dried 25 mL round-bottom flask (RBF) equipped with a stirring rod, dried under high vacuum and washed with argon. The relative reactivity of the two coupling partners, t-butyl-4-ethynylbenzoate and BC-2a, was measured by monitoring the reaction product using reverse-phase HPLC. The flask was sealed with a diaphragm (the base of which was also sealed with Parafilm) and evacuated / washed with argon (3 times). Toluene (4.17 mL) was added, stirring was started, and triethylamine (2.08 mL) was added. The flask was placed in an oil bath preheated to 85°C and stirred under low-flow argon. After 16 hours, the flask was removed from the oil bath, and the residue was dissolved in ethyl acetate in the flask to a total volume of approximately 12 mL. This reaction mixture was concentrated to dryness and then dissolved in DCM to prepare a silica cake (475 mg of SiO2 in two loads). This cake was eluted for 7 minutes using a 12 g SiO2 column containing 20–50% ethyl acetate in hexane. After the initial major product was eluted, the solvent system was switched to 0–5% MeOH in a DCM gradient for 5 minutes. At this stage, the desired product was eluted. The major product fractions were combined, concentrated, and dried under high vacuum to obtain 18.9 mg (62%) of BC-5d as a green solid.

[0143] BC-5 BC-5d (18.0 mg, 14.72 μmol) was added to an oven-dried 10 mL round-bottom flask (RBF). The flask was sealed with a diaphragm, evacuated, and washed with argon. HCl solution (2.0 mL) was added. The reaction mixture was shielded from light and stirred under argon. After 4 hours, the flask was placed in a warm water bath and washed with high-performance fluid argon through the outlet needle vent until all solvent was removed. The vent was then removed, a glass adapter was attached to the flask, and it was left under high vacuum overnight. The product was used in the next step without further purification. This residue (in a 10 mL round-bottom flask (RBF)) was placed under argon. The stirring rod was still present. PEG was then added to it. 24 Solid NHS (55.6 mg, 44.16 μmol, 3.0 equivalents) was added. The flask was evacuated and washed with argon. DMF (3.68 mL) was added, and then triethylamine (24.6 μL, 176.64 μmol, 12.0 equivalents) was immediately added by pipette. The flask was protected from light and stirred at room temperature for 1.5 hours, and the reaction was determined to be complete by liquid chromatography (LCMS). Zn(OAc)2 (81.0 mg, 441.6 μmol, 30.0 equivalents) was added to this, and the reaction flask was placed in an oil bath heated to 60°C. After 2 hours, slight reaction progress was observed. The reaction mixture was microwaved at 100°C for 10 minutes, then at 110°C for 20 minutes, and the completion of the reaction was observed by liquid chromatography (LCMS). The reaction mixture was concentrated to remove DMF, and the residue was dissolved in 40% ACN (1.3 mL) in water and subjected to reverse-phase preparative LC with a water gradient of 35-85% ACN for 35 minutes. The peaks of the main product were aligned, the mixture was concentrated, transferred to a storage vial of ACN, concentrated again, and dried under high vacuum to obtain 13.3 mg (28% from BC-5d) of BC-5 as a green residue. MS: obsd 1627.4, calcd 1626.8 [M + 2H] 2+ ; λabs 345, 595, 839(CH3CN); λem 855 nm(CH3CN); FWHM: 35 nm(CH3CN); λabs 345, 609, 848(H2O); λem 863 nm(H2O); FWHM: 38 nm(H2O);

[0144] Example 7 Synthesis of compound BC-6 As shown in Scheme 9 (Figure 9), the title compound (BC-6) was prepared from BC-5. N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 0.61 mg, 2.03 μmol) was added to a 5 mL dry conical vial with spin vanes. The vial was covered with a diaphragm, evacuated, and washed with argon. BC-5 (6.0 mg, 1.84 μmol) dissolved in DCM (0.75 mL) was added by syringe, followed by the direct addition of triethylamine (0.39 μL, 2.77 μmol) by pipette. The vial was shielded from light, and the mixture was stirred at room temperature. After 1.5 hours, the formation of the NHS ester intermediate was determined to be complete by liquid chromatography (LCMS). AminoPEG was added to this reaction vial. 12The acid was added directly in bulk as a solid. The vial was resealed, washed with argon, and stirred at room temperature for 16 hours. DCM was removed, and the sample was loaded onto a C18 250 x 20 column with 40% CAN in water, followed by a 30-minute gradient with 40-70% ACN in water. The main product fractions were combined, concentrated, and dried under high vacuum to obtain 5.0 mg (70%) of BC-6 as a green semi-solid. UV-Vis: 346 / 392, 602, >800 nm

[0145] Example 8 Synthesis of compound BC-7 BC-7a As shown in Scheme 10 (Figure 20), BC-7a was prepared. O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (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). This solution was stirred at this temperature for 1 hour, and then H-Lys(Boc)-OtBu HCl (0.5 g, 1.48 mmol) was added. This solution was stirred overnight at room temperature, and then CH2Cl2 2( The solution was diluted to 20 mL. This CH2Cl2 solution was washed with saturated NH4Cl aqueous solution (2 x 20 mL), water (2 x 20 mL), and brine (20 mL). The organic layer was dried, filtered, and concentrated. The crude product was dry-loaded onto silica gel (approximately 1.5 g) and purified by elution using a 12 g Isco column with a hexane:siRNA gradient (100:0 to 1:1). The desired product was isolated as a pale yellow glassy foam and solidified by drying under high vacuum for an extended period (0.35 g, 61%). LCMS: 7.92 min; MS: 431 BC-7b As shown in Scheme 11 (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 a 20 mL pressure vial that had been oven-dried and argon-washed, equipped with a stirring rod. The vial was sealed with a diaphragm and evacuated / argon-washed (three times). Toluene and triethylamine were added, and the diaphragm was quickly replaced with a vial screw cap. The vial was heated in a preheated oil bath at 100°C for 16 hours. The vial was cooled and transferred to a 100 mL round-bottom flask (RBF) rinsed with ethyl acetate. The solvent was removed, and the residue was dried under high vacuum for a further 30 minutes. This sample was prepared as a silica cake (450 mg), and this cake was eluted for 20 minutes using a SiO2 column (24 g) containing 0–5% MeOH in a DCM gradient. The main product fractions were combined and concentrated to obtain 13.9 mg of BC-7b as a dark green solid (38%).

[0146] BC-7 As further shown in Scheme 11 above, BC-7b (13.9 mg, 9.58 μmol) was dried in a 10 mL round-bottom flask (RBF). A stirring bar was added to the flask, the flask was sealed with a diaphragm, evacuated, and washed with argon (twice). HCl solution (1.9 mL of 4.0 M HCl in dioxane) was added in bulk with stirring. The mixture was stirred at room temperature for 5 hours. The flask was then vented with a needle, placed in a warm water bath, and washed with rapid-flow argon until all liquid was removed. The needle vent was then removed, and the reaction mixture was placed under high vacuum for 2 hours. The flask was washed with argon, tributylamine (50 μL) was added, and the mixture was stirred. Hexane / THF (2:1, 3 mL) was then added, and the solution was sonicated. The suspension was transferred to a 1.5 inch centrifuge tube and rotated at 11 kg for 3 minutes. The supernatant was removed. A 2:1 hexane / THF mixture was added to a centrifuge tube, sonicated, and rotated again at 11 kg for 3 minutes. This cycle was repeated once more. The supernatant was removed, the tube was covered with Parafilm, punctured with a 20-gauge needle, placed in a 100 mL round-bottom flask (RBF), and left under high vacuum for 30 minutes, after which it was washed with argon. 9.3 mg of dark red solid (94%) was isolated. 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 a 10 mL round-bottom flask (RBF) that had been washed with argon and equipped with a stirring bar. The flask was evacuated, washed with argon, and DMF (2.13 mL) was added. The reaction mixture was shielded from light and stirred under argon at room temperature. After 1.5 hours, water (0.5 mL) was added, and the solution was concentrated. The residue was diluted with water (1 mL) and subjected to reverse-phase preparative LC using 10-85% ACN in a water gradient for 35 minutes. The main product fractions were combined, concentrated, and dried to obtain a dark red residue (5.1 mg, 13%). The PEGylated intermediate (6.8 mg, 1.52 μmol) was dried in a 4 mL glass vial with a stirring bar, to which Zn(OAc)22H2O (10.0 mg, 45.73 μmol) was added, followed by DMF (0.6 mL). An adapter with an argon inlet was attached to this vial, and the mixture was heated to 60°C in an oil bath. After 16 hours, the stirring bar was removed, and the reaction product was concentrated. The residue was dissolved in water (0.7 mL) and subjected to reverse-phase preparative LC using 10-85% ACN in a water gradient for 30 minutes. 3.9 mg (57%) of BC-7 was separated as a dark green semi-solid.

[0147] Example 9 Synthesis of compound BC-8 As shown in Scheme 12 (Figure 12), the title compound (BC-8) was prepared from BC-5d. BC-5d (Example 6) (18.9 mg, 15.46 μmol) was dried in a 10 mL round-bottom flask (RBF) equipped with a stirring bar. The flask was sealed with a diaphragm, evacuated, and washed with argon (twice). HCl solution (2.1 mL of 4.0 M HCl in dioxane) was added in bulk with stirring. The mixture was stirred at room temperature for 4.5 hours. The solvent was removed by a rapid argon stream with a needle outlet (approximately 30 minutes). The flask was then placed under high vacuum for 16 hours. The residue was dissolved in DMF (3.87 mL, 4.0 mM), and triethylamine (25.9 μL, 185.5 μmol) was added to it. NHSPEG4-(mPEG) 12 )3 ester (89.8 mg, 37.1 μmol) solid was added, and the mixture was stirred for 1 hour. Zn(OAc)22H2O (101.8 mg, 463.8 μmol) was added to the reaction flask, and the flask was transferred to an oil bath at 80°C. The reaction was completed after 4 hours. The solvent was removed, and the residue was dissolved in 40% ACN in water (1.4 mL) and subjected to reverse-phase preparative chromatography using a gradient of 35–85% ACN in water for 35 minutes. After complete drying, the main product BC-8 was isolated as 12.0 mg of dark green semi-solid (14% from BC-5d).

[0148] Example 10 Synthesis of NIRvana880 bis-t-butyl ester HBC12 dialcohol As shown in Scheme 13 (Figure 13), NIRvana 880 bis-t-butyl ester was synthesized. HBC12 (115.7 mg, 179.6 μmol) was added to a flame-dried round-bottom flask (RBF) using a stirring bar. The flask was evacuated, washed with argon, and DCM (18.0 mL, 10 mM) was added. This solution was cooled to -78°C, and DIBAL-H (1.0 M in toluene, 1.437 mL) was added dropwise for 2 minutes. The reaction mixture was stirred and gradually equilibrated to room temperature. This stirring was continued for a total of 4 hours. The reaction mixture was diluted with HCl, and the reaction was inhibited by adding saturated Rochelle salt solution. The organic layer was washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The isolated dark green solid was used in the next step without further purification. HBC12 dialdehyde HBC12 dial alcohol (108.2 mg, 183.9 μmol), 4 angstrom molecular sieve (powder, 92.0 mg, 0.5 mg / μmol dialcohol), and N-methylmorpholine (dried, 107.7 mg, 919.5 μmol, 5 equivalents) were added to an oven-dried 50 mL round-bottom flask (RBF) equipped with a stirring rod. The flask was evacuated, washed with argon, and DCM / ACN (9:1, total 9.3 mL, 20 mM) was added, followed by bulk addition of tetrapropylammonium perruthenate (TPAP, 12.9 mg, 36.8 μmol, 20 mol%). The flask was covered with foil and stirred at room temperature for 3.5 hours. The reaction mixture was filtered through a Celite pad on sand and washed with DCM. The filtrates were combined and dried over sodium sulfate. The solution was filtered, concentrated to dryness, and purified over a 40 g silica column containing 25–65% DCM in hexane until all desired products were eluted. 36.2 mg (34%) of red solid was isolated.

[0149] ZnHBC12 dialdehyde HBC12 dialdehyde (8.0 mg, 13.7 μmol) was added to a 25 mL oven-dried round-bottom flask (RBF) with a stirring bar, and Zn(OAc)22H2O (90.2 mg, 411 μmol) was added. The flask was sealed with a diaphragm, evacuated, washed with argon, and DMF (2.74 mL) was added. The flask was placed in a preheated oil bath. The reaction mixture was heated at 75°C for 4 hours. The reaction mixture was cooled, diluted with DCM, and the reaction was inhibited by adding saturated sodium bicarbonate aqueous solution. The organic layer was separated and further washed with saturated sodium bicarbonate aqueous solution. The organic layer was dried over sodium sulfate, filtered, and concentrated. This product was used in the next step without further purification (approximately quantitative yield of solid product). NIRvana880 bis-t-butyl ester ZnHBC12 dialdehyde (10.0 mg, 15.44 μmol), t-butylethynyl benzoate, Pd(PPh3)2Cl2 (2.7 mg, 3.86 μmol), and CuI (1.5 mg, 7.72 μmol) were added together to a round-bottom flask (RBF) with a stirring bar. The flask was sealed with a diaphragm, evacuated, and washed with argon. Toluene / triethylamine (2:1, total 3.9 mL) was added, and the reaction mixture was heated at 85°C for 4 hours. The reaction mixture was concentrated and purified by column chromatography. MS: [M + H] + calc. 889.3; obs. 888.3 - 890.4 cluster; UV(ACN): 352, 404, 613, 763, 859 nm; em max(ACN): 876 nm.

[0150] Example 11 Flow cytometry deviceSamples were analyzed using a 19-parameter LSR-II SORP flow cytometer (BD Biosciences, San Jose, California, USA) equipped with seven lasers (355, 405, 488, 532, 561, 594, and 633 nm) or an LSRFortessa (BD Biosciences, San Jose, California, USA) equipped with five lasers (355, 405, 488, 561, and 640 nm), further analyzed with FACSDiva 8.0 acquisition software. For BC-1 data, a 100mW 355 nm laser with a 690LP filter and a 780 / 60BP filter was used on channel A. Post-experimental analysis was performed using FlowJo software (version 10.0.8, FlowJo, LLC, Ashland, Oregon, USA). Antibody bioconjugates The 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, Missouri, USA), 15 μL of 1 M bicarbonate (pH 8.4), and 44 μL of PEGylated dye NHS ester (5-20 molar equivalents) in PBS. The tube was shielded from light and gently rotated at room temperature for 1-2 hours. The reaction was inhibited by adding 15 μL of 200 μM Tris for a further 1 hour at room temperature. The bioconjugate was purified using one of the following Sephadex G50M, G75M, or G100M size exclusion chromatography columns that eluted with PBS. Antibody bioconjugates prepared from dyes with long PEG chains (12 units or more) were purified using G75M or G100M medium. Column fractions were characterized by absorption at 280 nm (protein) and maximum absorption of red or NIR dyes. The fluorophore-to-protein (F / P) label ratio of the pooled fractions was determined by correcting for dye absorption at 280 nm from the maximum values ​​of these two parameters.

[0151] cell staining Frozen human peripheral blood mononuclear cells (PBMCs) were obtained from ZenBio, Inc. (Research Triangle Park, North Carolina, USA; Product SER-PBMC-F), thawed, and prepared for staining according to the vendor's guidelines. The cells were divided 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 washing buffer (PBS containing 0.5% BSA) and resuspended in 0.5 mL of washing buffer. Aliquots were diluted 1:2 with trypan blue, and cell number and viability were measured by counting 4 nL squares in a hemocytometer. Viability was typically >96%. These cells were then 1 x 10⁶ of cells in washing buffer. 6 The antibody was diluted to 1 / mL and 50 μL (500,000 cells) was dispensed into microcentrifuge tubes. The maximum labeled antibody concentration was 4.74 μg / 5x10⁻¹⁰. 5 Cells (specified as 3.16X) were used, and semi-logarithmic dilutions were prepared. This dilution was performed so that 15 μL of each antibody, except for the control antibody, was added to the cell aliquots. The cells and antibodies were incubated at room temperature for 30 minutes while mixing. 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. Prior to characterization by flow cytometry, the samples were filtered through a nylon filter cloth and placed into flow cytometry tubes.

[0152] If necessary, positive control bioconjugates were selected from CD8(UCHT-4)-fluorescein isothiocyanate (FITC) (Leinco Technologies, Inc., St. Louis, Missouri, USA; catalog number C119), CD4(RPA-T4)-BUV737 antibody (BD Biosciences, San Jose, California, USA; catalog number 564306), and / or CD8(UCHT-4)-DY650 antibody (Leinco Technologies, Inc., St. Louis, Missouri, USA; catalog number C2064), and titrated using peripheral blood mononuclear cells (PBMCs) using the same general procedure. The staining index (SI) was calculated from the mean fluorescence intensity (MFI) value according to Maecker et al. (2004) Cytometry A 62:169-173 as follows. SI = (Mean: Positive - Mean: Background) / (2 × SD Background) Table 2 below shows the staining index data for titration of the BC-1 anti-CD8 bioconjugate, and the staining index data for titration of a PEGylated bacteriochlorin anti-CD8 bioconjugate similar to BC-1, but containing a PEG4 chain instead of a PEG12 chain. For comparison, data for an anti-CD8 bioconjugate prepared from FITC (Leinco Technologies, Inc., St. Louis, Missouri, USA; catalog number C119) is also provided. [Table 2] These results demonstrate a significant improvement in the performance of PEGylated designs of BC-1 compared to PEG4 PEGylated bacteriochlorin.

[0153] References All references listed herein, including but not limited to all patents, their patent applications and publications, and scientific journal articles, are incorporated herein by reference in their entirety to the extent that they supplement, explain, provide background to, or teach the methodologies, techniques and / or compositions employed herein. It should be understood that various details of the present invention disclosed herein can be modified without departing from the scope of the invention. Furthermore, the foregoing description is for illustrative purposes only and is not intended to limit the invention.

Claims

1. A compound represented by the following formula (II). 【Chemistry 1】 (wherein M is a metal or two hydrogen atoms; R ５ , R １０ and R １５ These are, independently, a hydrogen atom, an alkoxy, and are represented by the following formula: Selected from the linker groups; -L １ -(X １ -L ２ ) ｐ -G Here, p is 0 or 1, and L １ is alkylene, alkenylene, alkynylene, arylene, alkarylene, aralkylen or aralkynylene, X １ is -C(=O)NH- or -NHC(=O)-, L ２ is -(CH ２ CH ２ O) ｑ -alkylene- (wherein q represents an integer of 1 to 24), alkylene, or substituted alkylene (optionally, this substituted alkylene is alkylene substituted by one or more groups including a polyoxyethylene chain and / or an amide group); G is a carboxylic acid or an active ester, hydroxyl, amine, thiol, or aldehyde; and R ２ , R ３ , R １２ and R １３ These are, independently, hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, formula -L １ - (X １ -L ２ ) ｐ Selected from linker groups and solubilizing groups represented by G; (Here, this solubilizing group is -aryl-(R) ｓ ) ｗ and -alkynyl-aryl-(R ｓ ) ｗ Selected from (wherein w is an integer from 0 to 5, R ｓ This is a base represented by the following equation. -X ２ -(L ３ ) ｚ -R １７ 、 Here, z is either 0 or 1, X ２ ha-CH ２ NHC(=O)-, -C(=O)NH-alkylene-NH-, or triazolyl, L ３ is -C(=O)-alkylene-C(=O)-NH-, and R １７ Ha- (C ２ H ４ O) ｍ -R １８ , -C(=O)C ２ H ４ - (OC ２ H ４ ) ｍ OR １８ and -(C ２ H ４ O) ｎ -C ２ H ４ -C(=O)NH-C(R １９ ) ３ Selected from, where m is an integer greater than or equal to 12, and n is an integer from 1 to 5, R １８ R is a lower alkyl group (optionally methyl), １９ ha-CH ２ O-C ２ H ４ -C(=O)NH-(C ２ H ４ O) ｍ R １８ That is. ) ) ; However, R ２ , R ３ , R １２ and R １３ At least one of them is -aryl-(R ｓ ) ｗ or -alkynyl-aryl-(R ｓ ) ｗ And R ３ and R １３ Each of these is an ester (arbitrarily, -C(=O)OCH) ３ (It is) R ２ The following equation: 【Chemistry 2】 It is represented as R １２ However, equation -L １ - (X １ -L ２ (This is a linker group represented as p-G.)

2. The compound according to claim 1, wherein M is Zn.

3. R ５ , R １０ and R １５ However, each is independent of the hydrogen atom, methoxy, and formula -L １ - (X １ -L ２ ) ｐ A compound according to claim 1 or 2, selected from linker groups represented by -G.

4. Each R ｓ The compound according to claim 1, wherein is a group represented by the following formula. -X ２ -(L ３ ) ｚ -R １７ (where z is 0, X ２ is -C(=O)NH-alkylene-NH-, R １７ is, -C(=O)C ２ H ４ -(OC ２ H ４ ) ｍ OR １８ (where m is an integer of 12 or more, and R １８ is methyl.)).

5. Each R ｓ The compound according to claim 4, wherein the compound is represented by the following formula. 【Transformation 3】

6. The compound according to claim 1, wherein each Rs is a group represented by the following formula. -X ２ -(L ３ ) ｚ -R １７ (In the equation, z is 1, X ２ is -C(=O)NH-alkylene-NH-, and L ３ is -C(=O)-propylene-C(=O)-NH, and R １７ Ha- (C ２ H ４ O) ｎ -C ２ H ４ -C(=O)NH-C(R １９ ) ３ (In the formula, n is an integer from 1 to 5 (arbitrarily it is 4), and each R １９ is, -CH ２ O-C ２ H ４ -C(=O)NH-(C ２ H ４ O) ｍ R １８ (In the formula, m is an integer greater than or equal to 12 (arbitrarily, m is 12), R １８ It is methyl. ) It is. ) It is. )

7. R ２ and R １２ They are not the same, or R ３ and R １３ The compound according to claim 1 or 2, which is not the same.

8. R ２ and R １２ One of them is -aryl-(R ｓ ) ｗ and -alkynyl-aryl-(R ｓ ) ｗ A solubilizing group selected from and R ２ and R １２ The other of these is equation -L １ - (X １ -L ２ ) ｐ The compound according to claim 7, wherein the linker group is represented by G.

9. R １２ The compound according to claim 1, wherein the group is represented by the following formula. -L １ -(X １ -L ２ )p-G (In the formula, p is 0, L １ (where G is an aralkylene, and G is a carboxylic acid or active ester, hydroxyl, amine, thiol, or aldehyde.)

10. R １２ The compound according to claim 9, wherein the compound is represented by the following formula. 【Chemistry 4】 (In the formula, G is selected from carboxylic acids and active esters.)

11. R １２ The compound according to claim 1, wherein the group is represented by the following formula: -L １ -(X １ -L ２ ) ｐ -G (In the formula, p is 1, L １ It is aralkylene, X １ is -C(=O)NH-, and L ２ (where G is an alkylene substituted with one or more groups from the group consisting of a polyoxyethylene chain and / or an amide group, and G is a carboxylic acid or active ester, hydroxyl, amine, thiol, or aldehyde.)

12. L １ -C ≡ C - (C ６ H ４ The compound according to claim 11, wherein the compound is as follows:

13. L ２ The formula is -CH(R)- (where R is -alkylene-NH-C(=O)-alkylene-(OC) ２ H ４ ) ｑ -OR １６ (In the formula, q is an integer between 12 and 24, R １６ The compound according to claim 11 or 12, wherein is methyl.

14. A compound selected from the following: 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】

15. A covalent conjugate formed between the following: (a) R ２ , R ３ , R ５ , R １０ , R １２ , R １３ and R １５ A compound represented by formula (II) according to claim 1, wherein at least one of the groups is the linker group described in claim 1, and (b) One or more of the group that covalently bond to G of the linker group according to claim 1, comprising small molecules, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors.

16. A target detection composition comprising the covalently bonded conjugate described in claim 15.

17. A pharmaceutical composition comprising the compound described in claim 1 or the conjugate described in claim 15 and a pharmaceutically acceptable carrier.

18. A method for detecting a target in vitro, wherein the target is a compound, a cell, or a particle, and the method comprises labeling the target with the conjugate described in claim 15.

19. The method according to claim 18, wherein the method includes the use of flow cytometry.

20. A method for imaging cells, tissues, or organisms, comprising the use of the compound described in claim 1 or the conjugate described in claim 15.

21. A pharmaceutical product for treating a disease in a patient requiring treatment, and which includes the following: - The compound of claim 1, the conjugate of claim 15, or the pharmaceutical composition of claim 17 (Optionally, the disease is a hyperproliferative disease, and further optionally, the disease is cancer.)

22. A water-soluble bacteriochlorin dye comprising the compound described in claim 1, having a solubility greater than 1 mg / ml in an aqueous solution (optionally, having a solubility of 3.0 mg / ml or more in an aqueous solution, and even more optionally, having a solubility of 10 mg / ml or more in an aqueous solution).

23. A water-soluble bacteriochlorin according to claim 22, having an emission wavelength longer than 850 nm. dye.

24. The following formula (II"): [Chemistry 18] (wherein M is a metal or two hydrogen atoms; R ５ '', R １０ '' and R １５ Each of the '' elements is independently a hydrogen atom, an alkoxy, 【Chemistry 19】 Selected from; R ２ '', R ３ '', R １２ '' and R １３ Each of the following is independently a hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, 【Chemistry 20】 Selected from, However, R ２ ''but 【Chemistry 21】 A method for producing the compound of ) A method consisting of the following steps. (a) A step of providing a compound represented by formula (II'); and 【Chemistry 14】 (wherein M is a metal or two hydrogen atoms; R ５ ', R １０ 'and R １５ 'Each of these is independently a hydrogen atom, an alkoxy, 【Chemistry 15】 Selected from; R ２ ', R ３ ', R １２ 'and R １３ ' are, independently, hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, acetyl, 【Chemistry 16】 Selected from, However, R ２ 'but 【Chemistry 17】 (That is the case.) (b) A step in which the compound provided in step (a) is brought into contact with a solution containing 4 moles (M) of HCl in dioxane to provide the compound of formula (II").

25. A method for producing an asymmetric bacteriochlorin compound represented by the following formula (II), 【Chemistry 1】 (wherein M is a metal or two hydrogen atoms; R ５ , R １０ and R １５ Each is independently selected from a hydrogen atom and an alkoxy; and R ２ , R ３ , R １２ and R １３ Each of these is independently selected from hydrogen atoms, cyano, halo, perhaloalkyl, sulfonates, sulfonamides, esters, carboxylic acids, formyl, acetyl, linker groups, and solubilizing groups; The linker group is represented by the following formula: -L １ -(X １ -L ２ ) ｐ -G Here, p is 0 or 1, L １ is an alkylidene, X １ is -C(=O)NH- or -NHC(=O)-, L ２ ha- (CH ２ CH ２ O) ｑ -Alkylene- (wherein q is an integer from 1 to 24), alkylene, or substituted alkylene (optionally, the substituted alkylene is an alkylene substituted with one or more groups including a polyoxyethylene chain and / or an amide group); G is a carboxylic acid or active ester, hydroxyl, amine, thiol, or aldehyde; The solubilizing group is -aryl-(R ｓ ) ｗ and -alkynyl-aryl-(R ｓ ) ｗ Selected from (wherein w is an integer from 0 to 5, R ｓ This is a base represented by the following equation. -X ２ -(L ３ )z-R １７ 、 Here, z is either 0 or 1, X ２ ha-CH ２ NHC(=O)-, -C(=O)NH-alkylene-NH-, or triazolyl, L ３ is -C(=O)-alkylene-C(=O)-NH-, and R １７ Ha- (C ２ H ４ O) ｍ -R １８ , -C(=O)C ２ H ４ - (OC ２ H ４ ) ｍ OR １８ and -(C ２ H ４ O) ｎ -C ２ H ４ -C(=O)NH-C(R １９ ) ３ Selected from, where m is an integer greater than or equal to 12, and n is an integer from 1 to 5, R １８ R is a lower alkyl group (optionally methyl), １９ ha-CH ２ O-C ２ H ４ -C(=O)NH-(C ２ H ４ O) ｍ R １８ And, R ２ The following equation: 【Chemistry 2】 (It is represented by...) However, R ２ and R １２ They are not the same, or R ３ and R １３ They are not the same, R ２ , R ３ , R １２ and R １３ At least one of them is -aryl-(R ｓ ) ｗ or -alkynyl-aryl-(R ｓ ) ｗ (That is the case.) A method consisting of the following steps. (a) A step of providing a compound represented by formula (II'); and 【Chemistry 14】 (wherein M is a metal or two hydrogen atoms; R ５ ', R １０ 'and R １５ ' is independently selected from hydrogen atoms and alkoxy atoms, R ２ ', R ３ ', R １２ 'and R １３ Each of these is independently selected from hydrogen atom, cyano, halo, perhaloalkyl, sulfonate, sulfonamide, ester, carboxylic acid, formyl, and acetyl, R ３ 'and R １３ 'These are, respectively, Halo (or Bromo, optionally) be.) (b) The step of contacting the compound with a palladium catalyst, a base, and one of the following: (i) Two different alkynes (optionally, both of these alkynes are compounds represented by the following formula: 【Chemistry 22】 (In the formula, y is an integer from 1 to 5 (arbitrarily 1 or 2), and each R ２０ This 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes protected carboxylic acid-substituted alkylenes). (ii) Two different alkenes (optionally, both of these alkenes are compounds represented by the following formula: 【Chemistry 23】 (In the formula, y is an integer from 1 to 5 (arbitrarily 1 or 2), and each R ２０ This 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes protected carboxylic acid-substituted alkylenes). (iii) Two different organoboronates (optionally, the two different organoboronates are two different arylboronic acids or arylboronic acid esters of the following formulas): 【Chemistry 24】 (In the formula, y is an integer from 1 to 5 (arbitrarily 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, the substituted alkylene in this -C(=O)-NH-substituted alkylene-protected amine includes a protected carboxylic acid-substituted alkylene), and each R ２１ is either a hydrogen atom or an alkyl group, or two R groups ２１ (They together form alkylenes.)

26. The method according to claim 25, wherein the ratio of the two alkynes, alkenes, or organoboronates is adjusted to maximize the yield of the desired product based on the relative reactivity of the two alkynes, alkenes, or organoboronates (optionally, the less reactive of the two is provided in molar excess compared to the other compound in step (a)).

27. The method according to claim 25 or 26, wherein the yield of the desired product is greater than 50% (optionally, the yield of the desired product is greater than 60%).

28. Below formula: A compound of [unclear].

29. The carboxylic acid of the compound according to claim 28, and One or more of the following: small molecules, antigens, microparticles, nanoparticles, polymers, peptides, proteins, antibodies or antibody fragments, nucleic acids, hormones, and growth factors. A covalent conjugate formed between two bodies.

30. A composition comprising the covalent conjugate according to claim 29 for detecting a target.

31. A composition for imaging cells, tissues, or organisms, comprising the compound described in claim 28 or the conjugate described in claim 30.

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