WO2017027721A1 - Near-ir light-cleavable conjugates and conjugate precursors - Google Patents

Abstract

Embodiments of near-infrared light-cleavable heptamethine cyanine-based conjugates, particularly targeting agent-drug conjugates, according to Formula (I) or Formula (II) and conjugate precursors are disclosed. The disclosed targeting agent-drug conjugates are useful for targeted delivery and release of a drug. Methods of making and using the conjugates and precursors also disclosed.

Description

NEAR-IR LIGHT-CLEAVABLE CONJUGATES AND CONJUGATE PRECURSORS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 62/204,381, filed August 12, 2015, which is herein incorporated by reference in its entirety.

FIELD

This disclosure concerns conjugates, particularly targeting agent-drug conjugates comprising heptamethine cyanine fluorophores, conjugate precursors, and methods of making and using the conjugates.

BACKGROUND

The recent clinical success of antibody-drug conjugates has validated the benefits of combining macromolecule and small molecule therapeutics. Within this exciting area, a remaining challenge is to identify linker strategies that provide improved cleavage selectivity with site- specific drug delivery. An appealing solution would be to develop antibody-drug cleavage chemistry that relies on an external stimulus which can be applied in a site-specific fashion.

Light in the near-IR range (e.g., 650-900 nm) has unique potential in this context. These wavelengths exhibit significant tissue penetration, minimal toxicity, and, moreover, are clinically validated for both diagnostic and therapeutic applications. Near-IR fluorescence imaging is routine in certain clinical contexts and innovative applications, such as methods to optically define tumor margins during surgery, are being developed. Light-based therapeutic modalities using phototoxic small molecules have an extensive history in the treatment of cancer and skin disorders.

Existing approaches, however, rely on intracellular processes that use endogenous, often enzymatic, reactions with little inherent tumor selectivity. As a consequence, benign tissue uptake of the antibody through either antigen-specific or antigen-independent mechanisms can lead to off- target drug release with resulting dose-limiting toxicities, especially in organs responsible for catabolizing antibody-drug conjugates. Moreover, premature release in circulation can be a significant issue.

SUMMARY

This disclosure concerns embodiments of targeting agent-drug conjugates comprising heptamethine cyanine fluorophores, precursors of the conjugates, and methods of making and using the conjugates and precursors. The targeting agent promotes preferential or targeted delivery of the drug to a target site. Embodiments of the disclosed conjugates undergo photodegradation when irradiated with near-infrared light, which produces intramolecular cleavage and release of the drug. Advantageously, some embodiments of the conjugates are fluorophores, and fluorescence is lost upon photodegradation and drug release. Embodiments of the disclosed targeting agent-drug conjugates are useful for site-specific delivery and selective activation with concomitant drug release. Fluorescence levels of the administered conjugate may be monitored to visualize the location of the conjugate within a subject and/or as an indicator of drug release.

Targeting agent-drug conjugates and intermediate conjugates comprising a drug and a reactive group have a chemical structure according to Formula I or Formula II, or a

pharmaceuticall acceptable salt thereof:

wherein R³ is a drug-containing moiety and one of R¹ and R⁴ includes a targeting agent or a reactive group suitable for further conjugation to a targeting agent. With reference to Formulas I and II, m isl, 2, 3, 4, or 5; n is 1, 2 or 3;one of R¹ and R⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R⁴ is -(CH2)_X-L2-R^a, where x is an integer

> 1, L₂ is a linker moiety or is absent, and R^a is 0)N(H)R^b, -N(H)C(0)R^b,

-N(H)R^b, or -SR^b where R^b is a targeting agent,

R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl; R³ is -Li-C(0)-X-drug, where Li is a linker moiety or is absent and X is O, N(H), or N(CH₃); R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate; R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate; each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d

independently is H or alkyl; and each ring A independently is a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring. In some embodiments, each Y is C(CH3)2 or each Y is S.

The two heterocycle moieties may be substantially the same or different from one another. In some embodiments, each Y is the same and R⁵-R⁹ are identical to R¹⁰-R¹⁴, respectively. In certain embodiments, R⁶-R⁹ and Rⁿ-R¹⁴ are H. In any or all of the above embodiments, R⁵ and R¹⁰ may be -(Ct^SOs^". In any or all of the above embodiments, R³ may be

where X is O, N(H), or N(CH₃), and R¹⁵-R²² independently are H, alkyl, -N0₂, -NR¾, -NR^e ₃, alkoxy, or sulfonate, wherein each R^e independently is H, halo, or alkyl. In some embodiments,

>^^X"Drug

R¹⁵-R¹⁹ are H. In certain embodiments, R³ is ⁰ . In any or all of the above embodiments, the drug may be an anti-cancer drug. In one embodiment, the drug is combretastatin A4.

where q and r independently are 1, 2, 3, 4, or 5. In some embodiments, R^a is -N(H)C(0)R^b or -C(0)N(H)R^b and R^b is an antibody. In an independent embodiment, one of R¹ and R⁴ is lower alkyl and the other of R¹ and R⁴ is

-(CH₂)_X-L₂-R^a wherein R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is a targeting agent. In one embodiment, R^b is panitumumab. In an independent embodiment, R^b is

panitumumab and R³ is -C(0)-0-combretastatin A4.

In any or all of the above embodiments, the conjugate may have a structure according to Formula II. In one embodiment, ring A is a fused heteroaryl ring including one nitrogen atom. In an independent embodiment, ring A is substituted with optionally substituted sulfonate. In another independent embodiment, ring A is a fused phenyl ring.

A pharmaceutical composition comprises (i) a conjugate according to Formula I or Formula II wherein R^b is a targeting agent, and (ii) a pharmaceutically acceptable carrier.

Embodiments of precursor compounds for preparing the disclosed targeting agent-drug con ugates have a chemical structure according to Formula III or IV, or a salt thereof:

wherein R²³ is a protecting group and one of R¹ and R²⁴ is an alkynyl group. With respect to Formulas III and IV, m, n, R², R⁵-R¹⁴, Y, and ring A are as previously defined; one of R¹ and R²⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R²⁴ is -(CH2)u-C≡CH where u is 1, 2, 3, 4, or 5; and R²³ is a protecting group. In some embodiments, the protecting group is i<?ri-butyloxycarbonyl (BOC) or 9-fluorenylmethyloxycarbonyl (FMOC). In certain embodiments, one of R¹ and R²⁴ is lower alkyl and the other of R¹ and R²⁴ is

A method of using a conjugate as disclosed herein includes providing a conjugate according to Formula I or II, or a pharmaceutically acceptable salt thereof, wherein R^b is a targeting agent and wherein if Y is C(R^d)2, at least one R^d is other than H, and subsequently irradiating the conjugate with targeted application of an effective quantity of light having a selected wavelength in the near- infrared range and a selected intensity to induce a cleavage reaction and release the drug from the conjugate. In some embodiments, irradiating the conjugate with targeted application of light comprises irradiating the conjugate with a laser that produces light having a wavelength of 680- 700 nm. In any or all of the above embodiments, the method may further include monitoring a level of fluorescence of the conjugate, and ceasing irradiation when the level of fluorescence falls below a target level.

In any or all of the above embodiments, the method may include (i) providing a biological sample including, or suspected of including, a target molecule; (ii) contacting the biological sample with the conjugate, wherein the targeting agent of the conjugate is capable of recognizing and binding to the target molecule; and (iii) subsequently irradiating the biological sample with the targeted application of light.

In any or all of the above embodiments, the method may further include (i) identifying a subject as having a condition that may be treated with the drug; (ii) administering a therapeutically effective amount of the conjugate or a pharmaceutical composition comprising the conjugate to the subject; and (iii) subsequently irradiating the conjugate by targeted application of an effective quantity of light having a wavelength in the near-infrared range and a selected intensity to a targeted portion of the subject, thereby releasing the drug from at least some molecules of the conjugate. In some embodiments, the subject has a tumor and the targeted portion of the subject includes an area proximate a location of the tumor. In any or all of the above embodiments, the effective quantity of light applied to the targeted portion may be from 10-250 J/cm².

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a reaction scheme showing photo-induced cleavage of a drug from an exemplary targeting agent-drug conjugate as disclosed.

FIG. 2 is a schematic diagram illustrating one embodiment of a method for using the disclosed targeting agent-drug conjugates to treat a subject having a tumor by injection of the conjugate followed by targeted delivery of light of a desired wavelength to the external surface of the skin.

FIG. 3 shows the formation of two constitutional isomers that occurred upon quenching trifluoroacetic acid-mediated Boc deprotection of a precursor compound and acylation with combretastatin A4-chloroformate.

FIG. 4 is absorption spectra of a panitumumab-combretastatin A4 conjugate (CY-Pan-CA4) and a panitumumab-phenol conjugate (CY-Pan-Phenol).

FIGS. 5A and 5B show SDS-PAGE analysis of CY-Pan-CA4 and CY-Pan-Phenol with Coomassie blue staining (FIG. 4A) or fluorescence (FIG. 4B).

FIG. 6 is a diagram showing the formation of CY-Pan-CA4 and the yield of free CA4 in the presence and absence of 690 nm irradiation (50 J).

FIG. 7 shows initial absorption and emission curves (690 nm excitation) for CY-Pan-CA4.

FIG. 8 shows fluorescence emission over time of CY-Pan-CA4 with 690 nm irradiation.

FIG. 9 is an HPLC calibration curve for CA4.

FIG. 10 shows HPLC chromatograms at 300 nm of CA4 (upper panel), photolysis of CY- Pan-CA4 at t = 18h (middle panel), and a dark control of Cy-Pan-CA4 at t = 18 h (lower panel).

FIG. 11 shows confocal microscopy images of live MDA-MG-468 (panels 1-3) and MCF-7 (panels 4-6) cells treated with Hoechst 3342 (1 μΜ) and CY-Pan-CA4 (100 nm). Panels 1 and 4 are fluorescence emission from Hoechst 3342; panels 2 and 5 are fluorescence emission from CY- Pan-CA4; panels 3 and 6 are differential interference contrast images.

FIG. 12 shows flow cytometric analysis of live MDA-MB-468 and MCF-7 cells treated with 100 nM CY-Pan-CA4.

FIG. 13 is a graph showing light-dependent (690 nm, 30 J) cytotoxicity of CY-Pan-CA4, CA4, and Pan against MDA-MB-468 cells (continuous dose). FIG. 14 is a bar graph showing light-dependent (690 nm, 30 J) cytotoxicity of internalized CY-Pan-CA4, CA4, and Pan against MDA-MB-468 and MCF-7 cells (media exchange).

FIGS. 15A and 15B are in vivo serial fluorescence images of CY-Pan-CA4 in A432-tumor- bearing mice (right dorsum) taking from the ventral (15A) and dorsal (16B) sides.

FIGS. 16A and 16B are graphs showing raw fluorescence intensity (16A) and tumor-to- background ratio (16B) of CY-Pan-CA4 in the tumor and liver of A432-tumor-bearing mice (right dorsum) as a function of time post-injection. Error bars represent the standard deviation of n = 5 mice.

FIG. 17 shows in vivo serial fluorescence images of A431 tumors (implanted in both sides of the dorsum) pre- and post-irradiation with various doses of near-IR light at 2 days post-injection of CY-Pan-CA4, and a graph of tumor-to-background ratio versus near-IR light dose (first bar in each pair = irradiated; second bar in each pair = not irradiated). Only the right tumor was irradiated; the left tumor was covered with aluminum foil. DETAILED DESCRIPTION

This disclosure concerns embodiments of targeting agent-drug conjugates comprising heptamethine cyanine fluorophores, precursors of the conjugates, and methods of making and using the conjugates and precursors. A near-IR uncaging strategy uses the heptamethine cyanine fluorophore scaffold as the caging component. Irradiation with an effective quantity of near- infrared light induces cleavage of the drug from the targeting agent-drug conjugate.

This approach allows target- specific delivery of bioactive small molecules using near-IR optical tools already employed in various clinical settings. An advantageous feature is that the fluorescent properties of the conjugate can be used to evaluate targeting agent-target engagement. Moreover, following administration of therapeutic light doses, the loss of that signal allows real- time assessment of drug release.

Certain embodiments of the disclosed conjugates comprise an antibody and an anti-cancer agent, and are useful for site-specific delivery of the anti-cancer agent to a tumor. By separating the drug payload release process from cellular internalization, the extracellular targeting agent-drug conjugate fraction will release the drug cargo into the local tumor environment. Advantageously, this localized release should effectively transfer molecules from antigen-positive cells to adjacent antigen-negative cells achieving bystander effects, which can be critical for therapeutic efficacy.

I. Definitions

The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term "about." Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word "about" is recited.

Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.),

Hawley's Condensed Chemical Dictionary, published by John Wiley & Sons, Inc., 1997 (ISBN 0- 471-29205-2). Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a

Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Aliphatic: A substantially hydrocarbon-based compound, or a radical thereof (e.g., C₆Hi3, for a hexane radical), including alkanes, alkenes, alkynes, including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Unless expressly stated otherwise, an aliphatic group contains from one to twenty-five carbon atoms; for example, from one to fifteen, from one to ten, from one to six, or from one to four carbon atoms. The term "lower aliphatic" refers to an aliphatic group containing from one to ten carbon atoms. An aliphatic chain may be substituted or unsubstituted. Unless expressly referred to as an "unsubstituted aliphatic," an aliphatic group can either be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in an aliphatic chain, or up to one substituent for each carbon of a -C=C- double bond in an aliphatic chain, or up to one substituent for a carbon of a terminal methine group).

Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or other functionality.

Alkoxy: A group having the structure -OR, where R is a substituted or unsubstituted alkyl. Methoxy (-OCH3) is an exemplary alkoxy group. In a substituted alkoxy, R is alkyl substituted with a non-interfering substituent.

Alkoxy carbonyl: A group having the structure -(O)C-O-R, where R is a substituted or unsubstituted alkyl.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chain may be branched, unbranched, or cyclic (cycloalkyl). The term lower alkyl means the chain includes 1-10 carbon atoms. Unless otherwise specified, the term alkyl encompasses substituted and unsubstituted alkyl.

Alkyl carbonyl: A group having the structure -(O)C-R, where R is a substituted or unsubstituted alkyl.

Alkyl sulfonate: A group having the structure -R-SO3^", where R is a substituted or unsubstituted alkyl.

Amino: A group having the structure -N(R)R' where R and R' are independently hydrogen, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, or other functionality. A "primary amino" group is -NH2.

"Mono-substituted amino" means a radical -N(H)R substituted as above and includes, e.g., methylamino, (l-methylethyl)amino, phenylamino, and the like. "Di-substituted amino" means a radical -N(R)R' substituted as above and includes, e.g., dimethylamino, methylethylamino, di(l-methylethyl)amino, and the like. The term amino also encompasses charged tri-substituted amino groups, e.g. , -N(R)(R')R"⁺ where R, R', and R" are independently hydrogen, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfano, or other functionality.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. In avian and reptilian species, IgY antibodies are equivalent to mammalian IgG.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer, respectively, to these light and heavy chains.

The structure of IgY antibodies is similar to the structure of mammalian IgG, with two heavy ("nu" chains; approximately 67-70 kDa) and two light chains (22-30 kDa). The molecular weight of an IgY molecule is about 180 kDa, but it often runs as a smear on gels due to the presence of about 3% carbohydrate. Heavy chains (H) of IgY antibodies are composed of four constant domains and one variable domain, which contains the antigen-binding site.

As used herein, the term "antibodies" includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999). As used herein, the term "antibodies" includes antibodies comprising one or more unnatural (i.e., non-naturally occurring) amino acids (e.g., / acetyl-phenylalanine, p-azidomethyl phenylalanine (pAMF)) to facilitate site-specific conjugation.

Antibodies for use in the methods of this disclosure can be monoclonal or polyclonal, and for example specifically bind a target such as the target antigen. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using

Antibodies: A Laboratory Manual, CSHL, New York, 1999.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. As used herein, a "target antigen" is an antigen (including an epitope of the antigen) that is recognized and bound by a targeting agent. "Specific binding" does not require exclusive binding. In some embodiments, the antigen is obtained from a cell or tissue extract. In some embodiments, the target antigen is an antigen on a tumor cell. An antigen need not be a full-length protein. Antigens contemplated for use include any immunogenic fragments of a protein, such as any antigens having at least one epitope that can be specifically bound by an antibody.

Aryl: A monovalent aromatic carbocyclic group of, unless specified otherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., quinoline, indole, benzodioxole, and the like), provided that the point of attachment is through an atom of an aromatic portion of the aryl group and the aromatic portion at the point of attachment contains only carbons in the aromatic ring. If any aromatic ring portion contains a heteroatom, the group is a heteroaryl and not an aryl. Aryl groups are monocyclic, bicyclic, tricyclic or tetracyclic. Unless otherwise specified, the term aryl encompasses substituted and unsubstituted aryl.

Biological sample: As used herein, a "biological sample" refers to a sample obtained from a subject (such as a human or veterinary subject) or other type of organism, such as a plant, bacteria or insect. Biological samples from a subject include, but are not limited to, cells, tissue, serum, blood, plasma, urine, saliva, cerebral spinal fluid (CSF) or other bodily fluid. In particular examples of the method disclosed herein, the biological sample is a tissue sample.

Conjugate: Two or more moieties directly or indirectly coupled together. For example, a first moiety may be covalently coupled to a second moiety. Indirect attachment is possible, such as by using a "linker" (a molecule or group of atoms positioned between two moieties). DMP: Dess-Martin periodinane

Drug: As used herein, the term "drug" refers to a substance which has a physiological effect when administered to a subject, and is intended for use in the treatment, mitigation, cure, prevention, or diagnosis of disease or used to otherwise enhance physical or mental well-being. The term "small molecule drug" refers to a drug having a molecular weight < 1,000 Daltons.

An anti-cancer drug is a drug that is used to treat malignancies. Exemplary anti-cancer drugs include, but are not limited to, abiraterone, actinomycin D, altretamine, amifostine, anastrozole, asparaginase, bexarotene, bicalutamide, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil cisplatin, cladribine, clodronate, combretastatin A4, cyclophosphamide, cyproterone, cytarabine, dacarbazine, daunorubicin, degarelix, diethylstilbestrol, docetaxel, doxorubicin, duocarmycin DM, epirubicin, ethinyl estradiol, etoposide, exemestane, 5-fluorouracil, fludarabine, flutamide, folinic acid, fulvestrant, gemcitabine, goserelin, ibandronic acid, idarubicin, ifosfamide, irinotecan, lanreotide, lenalidomide, letrozole, leuprorelin, medroxyprogesterone, megestrol, melphalan, mesna, methotrexate, octreotide, pamidronate, pemetrexed, mitocmycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pentastatin, pipbroman, plicamycin, procarbazine, raltitrexed, stilbestrol, streptozocin, tamoxifen, temozolomide, teniposide, topotecan, triptorelin, vinblastine, vincristine, vinorelbine, and zolendronic acid.

Effective amount or therapeutically effective amount: An amount sufficient to provide a beneficial, or therapeutic, effect to a subject or a given percentage of subjects.

Epitope: An antigenic determinant. Epitopes are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope based on the three dimensional structure of the antibody and the matching (or cognate) epitope.

Halogen: The terms halogen and halo refer to fluorine, chlorine, bromine, iodine, and radicals thereof.

Heteroaliphatic: An aliphatic compound or group having at least one heteroatom, i.e. , one or more carbon atoms has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. Heteroaliphatic compounds or groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" groups. Heteroalkyl: An alkyl group as defined above containing at least one heteroatom, such as N, O, S, or S(0)n (where n is 1 or 2). Unless otherwise specified, the term heteroalkyl encompasses substituted and unsubstituted heteroalkyl.

Heteroaryl: An aromatic compound or group having at least one heteroatom, i.e. , one or more carbon atoms in the ring has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwise specified, the term heteroaryl encompasses substituted and unsubstituted heteroaryl.

Ligand: A molecule that binds to a receptor, having a biological effect.

Linker: A molecule or group of atoms positioned between two moieties. As used herein, the term "linker" refers to a group of atoms positioned between the cyanine fluorophore and a targeting agent or reactive group, or to a group of atoms positioned between the cyanine fluorophore and a drug.

Near-infrared (near-IR, NIR): Wavelengths within the range of 650-2500 nm. Unless otherwise specified, the terms "near-infrared" and "NIR" as used herein refer to wavelengths within the range of 650-900 nm.

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more targeting agent-drug conjugates as disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pharmaceutically acceptable salt: A biologically compatible salt of a disclosed conjugate, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically acceptable acid addition salts are those salts that retain the biological effectiveness of the free bases while formed by acid partners that are not biologically or otherwise undesirable, e.g., inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (See, for example, S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66:1-19, which is incorporated herein by reference.)

Protecting group: When synthesizing organic compounds, often a specific functional group cannot survive the required reagents or chemical environments. These groups must be protected. A protecting group, or protective group, is introduced into a molecule by chemical modification of a functional group in order to obtain chemoselectivity in a subsequent chemical reaction. Various exemplary protecting or protective groups are disclosed in Greene's Protective Groups in Organic Synthesis, by Peter G. M. Wuts and Theodora W. Greene (October 30, 2006), which is incorporated herein by reference.

Specific binding partner: A member of a pair of molecules that interact by means of specific, non-covalent interactions that depend on the three-dimensional structures of the molecules involved. Exemplary pairs of specific binding partners include antigen/antibody, hapten/antibody, receptor/ligand, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, carbohydrate/lectin, biotin/avidin (such as biotin/streptavidin), and virus/cellular receptor. Substituent: An atom or group of atoms that replaces another atom in a molecule as the result of a reaction. The term "substituent" typically refers to an atom or group of atoms that replaces a hydrogen atom, or two hydrogen atoms if the substituent is attached via a double bond, on a parent hydrocarbon chain or ring. The term "substituent" may also cover groups of atoms having multiple points of attachment to the molecule, e.g. , the substituent replaces two or more hydrogen atoms on a parent hydrocarbon chain or ring. In such instances, the substituent, unless otherwise specified, may be attached in any spatial orientation to the parent hydrocarbon chain or ring. Exemplary substituents include, for instance, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic (e.g. , haloalkyl), haloalkoxy, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, isocyano, isothiocyano, oxo, sulfonamide, sulfhydryl, thio, and thioalkoxy groups.

Substituted: A fundamental compound, such as an aryl or aliphatic compound, or a radical thereof, having coupled thereto one or more substituents, each substituent typically replacing a hydrogen atom on the fundamental compound. Solely by way of example and without limitation, a substituted aryl compound may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene. Again solely by way of example and without limitation, a long-chain hydrocarbon may have a hydroxyl group bonded thereto.

Sulfonate-containing group: A group including SO3^". The term sulfonate-containing group includes -SO3^" and -RSO3^" groups, where R is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Target: An intended molecule to which a disclosed targeting agent-drug conjugate is capable of specifically binding. Examples of targets include proteins and nucleic acid sequences present in tissue samples. A target area is an area in which a target molecule is located or potentially located.

Targeting agent: An agent that promotes preferential or targeted delivery to a target site, for example, a targeted location in a subject's body, such as a specific organ, organelle, physiologic system, tissue, or site of pathology such as a tumor, area of infection, or area of tissue injury.

Targeting agents function by a variety of mechanisms, such as selective concentration in a target site or by binding to a specific binding partner. Suitable targeting agents include, but are not limited to, proteins, polypeptides, peptides, glycoproteins and other glycoslyated molecules, oligonucleotides, phospholipids, lipoproteins, alkaloids, and steroids. Exemplary targeting agents include antibodies, antibody fragments, affibodies, aptamers, albumin, cytokines, lymphokines, growth factors, hormones, enzymes, immune modulators, receptor proteins, antisense oligonucleotides, avidin, nano particles, and the like. Particularly useful of targeting agents are antibodies, nucleic acid sequences, and receptor ligands, although any pair of specific binding partners can be readily employed for this purpose.

II. Conjugates

Conjugates comprising a heptamethine cyanine fluorophore, a drug, and a targeting agent or a reactive group suitable for further conjugation have a chemical structure according to Formula I or Formula II, or a pharmaceutically acceptable salt thereof.

With respect to Formulas I and II, m isl, 2, 3, 4, or 5; n is 1, 2 or 3; one of R¹ and R⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R⁴

integer > 1, -C(0)N( ^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b where R^b is a targeting agent or R^b is

R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl; R³ is

-Li-C(0)-X-drug, where Li is a linker moiety or is absent and X is O, N(H), or Ν(0¾); R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate; R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate; each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d independently is H or alkyl; and each ring A independently is a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring.

In some embodiments, R² is -CH₂- and m is 1, 2 or 3. In an independent embodiment, (R²)m collectively is phenyl. The two heterocycle moieties,

and , may be identical or different from one another. In certain embodiments, the two heterocycle moieties are identical. R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate. In some embodiments, R⁵ and R¹⁰ are alkyl sulfonate, such as -(CH2)4S03^". R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate. In certain embodiments, R⁶-R⁹ and Rⁿ-R¹⁴ are H. Each Y independently is

C(R^d)2, S, O, Se, or N(R^d) wherein each R^d independently is H or alkyl. In some examples, each Y independently is C(CH3)2 or S. In one embodiment, when R^b is a targeting agent and Y is C(R^d)2, at least one R^d is other than H.

In some embodiments, R⁸ and R⁹, together with the carbon atoms to which they are bound, collectively form a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring. Similarly, R¹³ and R¹⁴, together with the carbon atoms to which they are bound, collectively may form a 6- membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring. These 6-membered fused rings are shown as Ring A in Formula II. In one embodiment, ring A is a 6-membered fused heteroaryl ring with one nitrogen heteroatom. In another embodiment, ring A is a fused phenyl ring. Ring A may be substituted with one or more optionally substituted sulfonate groups.

R³ is -Li-C(0)-X-drug, where Li is a linker moiety or is absent and X is O, N(H), or N(CH3). In one embodiment, Li is absent. In some embodiments, Li is aryl or heteroaryl substituted with at least one substituent comprising a substituted or unsubstituted aliphatic or heteroaliphatic moiety, wherein the aryl or heteroaryl ring is the site of attachment to the nitrogen atom and the substituent is bonded to the -C(0)-X-drug moiety. In certain embodiments, R³ is:

-NR^e2, -NR^E3, alkoxy, or sulfonate, wherein each R^e independently is H, halo, or alkyl. In some examples, R¹⁵-R¹⁹ are H. In certain embodiments, Li is absent and R³ is -C(0)-0-drug.

The drug can be any drug capable of conjugation to the remainder of the R³ moiety. In some embodiments, the drug is a small-molecule drug, e.g., a drug having a molecular weight < 1,000 Daltons. In certain embodiments, the drug moiety is an anti-cancer drug. In one embodiment, the drug is an anti-breast cancer drug. In one embodiment, the drug is a combretastatin, such as combretastatin A4 (CA4). CA4 displays potent growth inhibitory activity via inhibition of microtubule polymerization and has been the subject of numerous clinical trials despite significant vascular system-associated toxicity. A targeted strategy to deliver CA4 could alleviate these undesirable side effects. In an independent embodiment, the drug is a duocarmycin, such as duocarmycin DM. The duocarmycins are cytotoxic antibiotics that are DNA minor groove- binding alkylating agents, and are suitable for use against solid tumors. Another exemplary drug is hemiasterlin, a natural product that disrupts microtubule dynamics and, in some doses,

depolymerizes microtubules.

Exemplary -X-Drug moieties include, but are not limited to:

One of R¹ and R⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl. In some embodiments, either R¹ or R⁴ is alkyl, such as lower alkyl. For example, R¹ or R⁴ may be methyl. ¹ and R⁴ is -(CH2)_X-L2-R^a, where x is an integer > 1, L2 is absent or a linker

-N(H)C(0)R^b, -N(H)R^b, or -SR^b where R^b is a targeting agent or R^b is

, or

In some embodiments, L2 is aliphatic, heteroaliphatic, or heteroaryl-aliphatic. In one embodiment, R^a is -C(0)N(H)R^b or -N(H)C(0)R^b. In an independent embodiment, R^b is a targeting agent. Exemplary targeting agents include, but are not limited to, antibodies, ligands, nucleic acid strands, and the like. In certain examples, the targeting agent is an antibody. In one embodiment, R^a is -C(0)N(H)R^b or -N(H)C(0)R^b and R^b is an antibody. In an independent embodiment, R^b is a ligand, e.g., a ligand capable of binding to a receptor on a cell surface.

Exemplary antibodies include antibodies capable of recognizing and binding to a target molecule, such as a biomarker associated with a disease, infection, or environmental exposure. The antibodies may be modified antibodies that include one or more unnatural amino acids to facilitate site-specific conjugation. Biomarkers include, but are not limited to, proteins, peptides, lipids, metabolites, and nucleic acids. In some embodiments, the antibody is capable of recognizing and binding to a tumor biomarker, such as a protein only found in or on tumor cells or to a cell-surface receptor associated with one or more cancers. For example, panitumumab is a human monoclonal antibody that recognizes and binds to human epidermal growth factor receptor 1 (HER1); HER1 is overexpressed in numerous tumor types and is also associated with some inflammatory diseases. Brentuximab is a monoclonal antibody that targets a cell-membrane protein CD30, which is expressed in classical Hodgkin lymphoma and systemic anaplastic large cell lymphoma.

Trastuzumab and pertuzumab are monoclonal antibodies that bind to the HER2/neu receptor, which is over-expressed in some breast cancers.

In one embodiment, R^b is panitumumab. In another embodiment, R^b is trastuzumab. In one embodiment, R^b is panitumumab and R³ is -C(0)-0-combretastatin A4. In an independent embodiment, R^b is trastuzumab and R³ is -C(0)-0-duocarmycin DM. In another independent embodiment, R^b is trastuzumab and R³ is -C(0)-X-hemiasterlin, where X is O or N(H). In an independent embodiment, R^a or R^b i is

and the conjugate according to Formula I or Formula II is an intermediate that may be used for further conjugation reactions, such as conjugation to a targeting agent.

Exemplary RVR⁴ groups include:

where q and r independently are 1, 2, 3, 4, or 5. In certain examples, one of R¹ and R⁴ is

In one embodiment, R¹ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, and R⁴ is -(CH2)x-L2-R^a. In an independent embodiment, R¹ is -(CH2)_X-L2-R^a and R⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R. In another independent embodiment, R¹ is lower alkyl and R⁴ is -(CH₂)_X-L₂-R^a where R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is an antibody. In yet another independent embodiment, R⁴ is lower alkyl and R¹ is

-(CH₂)_X-L₂-R^a where R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is an antibody.

In one embodiment, the conjugate has a structure according to Formula II, and ring A is a heteroaryl ring including one nitrogen atom. In an independent embodiment, the conjugate has a structure according to Formula II, and ring A is substituted with optionally substituted sulfonate. In another independent embodiment, conjugate has a structure according to Formula II, and ring A is a fused phenyl ring.

As shown in FIG. 1 with an exemplary antibody-drug conjugate, embodiments of the disclosed conjugates undergo photodegradation when irradiated with near-IR light, which uncages the C4'-nitrogen and renders C4'-N bond hydrolytically labile. Subsequently, the irradiation induces cyclization of the uncaged amine onto a pendant carbamate group and hydrolysis to release the drug payload. The photodegradation involves a singlet oxygen-mediated regioselective cyanine polyene cleavage process that proceeds through dioxetane intermediates. III. Precursor Compounds

Embodiments of a precursor compound useful for making the conjugates according to Formulas I and II have a structure accordin to Formula III or Formula IV, or a salt thereof.

(IV)

With respect to Formulas III and IV, m, n, R², R⁵-R¹⁴, Y, and ring A are as previously defined; one of R¹ and R²⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R²⁴ is -(CH2)_U-C≡CH where u is 1, 2, 3, 4, or 5; and R²³ is a protecting group. In some embodiments, the protecting group is an amine protecting group. Exemplary amine protecting groups include, but are not limited to, Boc (i<?ri-butyloxycarbonyl) and Fmoc (9-fluorenylmethyloxycarbonyl).

R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl. In certain embodiments, R² is -CH2- and m is 1, 2 or 3. In an independent embodiment, (R²)m collectively is phenyl.

R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate. In some embodiments, R⁵ and R¹⁰ are alkyl sulfonate, such as -(CH2)4S03^". R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate. In certain embodiments, R⁶-R⁹ and Rⁿ-R¹⁴ are H. Each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d independently is H or alkyl. In some examples, each Y independently is C(CH3)2 or S. The two heterocycle moieties may be substantially the same or different from one another. In certain embodiments, each Y is the same and R⁵-R⁹ are identical to R¹⁰-R¹⁴, respectively.

In some embodiments, R⁸ and R⁹, together with the carbon atoms to which they are bound, collectively form a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring. Similarly, R¹³ and R¹⁴, together with the carbon atoms to which they are bound, collectively may form a 6- membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring. These 6-membered fused rings are shown as Ring A in Formula IV. In one embodiment, ring A is a 6-membered fused heteroaryl ring with one nitrogen heteroatom. In another embodiment, ring A is a fused phenyl ring. Ring A may be substituted with one or more optionally substituted sulfonate groups.

In one embodiment, R¹ is lower alkyl and R²⁴ is -(CH2)_U-C≡CH. In an independent embodiment, R¹ is -(CH2)_U-C≡CH and R²⁴ is lower alkyl. In certain embodiments, R¹ and R²⁴ may undergo a spontaneous rearrangement (i.e., the substituents switch positions) when R²³ is removed during subsequent conjugation of a drug to the precursor compound.

Exemplary precursor molecules include, but are not limited to:

IV. Synthesis

Embodiments of the disclosed conjugates and precursor compounds are synthesized from cyanine fluorophores. An exemplary synthesis of a precursor compound according to Formula III or Formula IV is shown in Scheme 1.

Scheme 1

Exemplary compound 1 is commercially available (IR-783, Sigma- Aldrich). A Boc- protected amine 2 is synthesized by reaction of tosyl-pentyne with ethanolamine, followed by reaction with triethylamine and di-i-butyl dicarbonate to provide a protected nitrogen. .i¾- ·' ;._··!·,

2 Steps

The hydroxy group is replaced with a substituted amine by reaction with Dess-Martin periodinane (DMP) followed by reaction with a desired amine, e.g., methylamine.

Compound 1 undergoes C4'-substitution with 2 in high yield (81%) to afford 3 as shown Scheme 1. Removal of Boc and addition of drug to 3 produces 4 (Scheme 2, where R = drug, e. combretastatin A4 as shown).

Scheme 2

Under many conditions, a 1:1 mixture of two distinct N- linked cyanine products is obtained. These products are constitutional isomers comprising the configuration of 4 and an isomer in which the alkynyl and N-linked methyl groups are reversed (i.e., with reference to Formulas III and IV, the alkynyl group is at R¹ and the methyl group is at R²⁴). The isomers arise from the intermediate di te formation.

Compound 4 is produced in high yield by deprotection of the Boc group in neat

trifluoroacetic acid, followed by careful aqueous bicarbonate quench at -10 °C, and addition of the drug to the cooled mixture. In contrast, quenching at room temperature and allowing for equilibration prior to drug addition produces a mixture of the isomers.

A conjugate according to Formula I or Formula II where R^b is a reactive group is prepared by addition of a linker and a reactive group to the drug-functionalized precursor. In one example, an NHS ester 7 is formed by copper-catalyzed cycloaddition of a linker (e.g., azido-PEG₄-acid 5), and subsequent transformation of the acid to the NHS ester with TSTU 6 (Ν,Ν,Ν', N'-tetramethyl- 0-(N-succinimidyl)uronium tetrafluoroborate) (Scheme 3).

Scheme 3

A targeting agent is then reacted with 7 to produce a targeting agent-drug conjugate according to Formula I or Formula II. The conjugate is purified, e.g., by size-exclusion chromatography. In some embodiments, the targeting agent is an antibody. In one embodiment, the anti-HERl antibody panitumumab was reacted with 7 to provide a panitumumab- combretastatin A4 conjugate. The NHS ester reacts readily with targeting agents including one or more primary amine groups. A comparable maleimidyl-functionalized compound is useful when the targeting agent includes one or more sulfhydryl groups.

In some embodiments, a dibenzocylooctyne is reacted with the alkynyl group of 4. The targeting agent, e.g., an antibody, is activated with an azide-containing molecule. The

dibenzocylooctyne-functionalized heptamethine cyanine-drug conjugate readily reacts with the azide-functionalized targeting agent to form a stable triazole and produce the targeting agent-drug conjugate according to Formula I or Formula II. Advantageously, the reaction does not require a Cu(I) catalyst. In one embodiment, the targeting agent is trastuzumab and the drug is duocarmycin DM. In another embodiment, the targeting agent is trastuzumab and the drug is hemiasterlin. V. Pharmaceutical Compositions

This disclosure also includes pharmaceutical compositions comprising at least one conjugate as disclosed herein. Some embodiments of the pharmaceutical compositions include a pharmaceutically acceptable carrier and at least one conjugate. Useful pharmaceutically acceptable carriers and excipients are known in the art. The pharmaceutical compositions comprising one or more conjugates may be formulated in a variety of ways depending, for example, on the mode of administration and/or on the location to be imaged. Parenteral formulations may comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients may include, for example, nonionic solubilizers, such as cremophor^®, or proteins, such as human serum albumin or plasma

preparations. If desired, the pharmaceutical composition to be administered may also contain nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

The form of the pharmaceutical composition will be determined by the mode of administration chosen. Embodiments of the disclosed pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation. Generally, embodiments of the disclosed

pharmaceutical compositions will be administered by injection, systemically, or orally.

Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. The composition may take such forms as suspension, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. For example, parenteral administration may be done by bolus injection or continuous infusion. Alternatively, the conjugate may be in powder form for reconstitution with a suitable vehicle, e.g. sterile water, before use.

Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

Oral formulations may be liquid (e.g. , syrups, solutions or suspensions), or solid (e.g. , powder, tablets, or capsules). Oral formulations may be coupled with targeting ligands for crossing the endothelial barrier. Some conjugate formulations may be dried, e.g. , by spray-drying with a disaccharide, to form conjugate powders. Solid compositions prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, mannitol, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g. , potato starch or sodium starch glycolate); or wetting agents (e.g. , sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophor^® or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the fluorophore, as is well known.

For rectal and vaginal routes of administration, the conjugate(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation or insufflation, the conjugate(s) can be conveniently delivered in the form of an aerosol spray or mist from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane,

trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Certain embodiments of the pharmaceutical compositions comprising conjugates as described herein may be formulated in unit dosage form suitable for individual administration of precise dosages. The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the conjugate. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The amount of conjugate administered will depend at least in part on the subject being treated, the target (e.g. , the size, location, and characteristics of a tumor), and the manner of administration, and is known to those skilled in the art. Within these bounds, the formulation to be administered will contain a quantity of the conjugate disclosed herein in an amount effective to provide a therapeutically effective dose of the drug to the subject being treated when the conjugate is irradiated with NIR light to release the drug from the conjugate. In some embodiments, the pharmaceutical composition includes a second therapeutic agent other than the conjugate. The second agent may be, for example, an anti-tumor agent or an angiogenesis inhibitor. VI. Photo-induced Cleavage

Embodiments of the disclosed conjugates according to Formula I or Formula II are photoactivated by application of light having a desired wavelength, intensity, and/or surface area to a pre-selected target area for an effective period of time. Photoactivation results in cleavage of the drug from the conjugate. The wavelength is selected within the near-infrared range, e.g. , from 650 nm to 2500 nm, such as from 650-900 nm. In some embodiments, the light source is a laser that produces light having a wavelength of 680-700 nm. Suitable light intensities may range from 1 mW to 750 mW depending on the target site and method of application. Near-infrared light sources can be obtained from commercial sources, including Thorlabs (Newton, NJ), Laser Components, USA (Hudson, NH), ProPhotonix (Salem, NH) and others. In some embodiments, the effective quantity of NIR light is 10-250 J, such as 10-200 J, 10-150 J, or 10-100 J. When irradiating a target area (e.g., an area proximate a tumor), the effective quantity of NIR light may be 10-250 J/cm², such as 10-200 J/cm², 10-150 J/cm², or 10-100 J/cm².

In some in vivo embodiments, irradiation is performed by external application of light to a targeted area of a subject. NIR light is capable of penetrating transcutaneously into tissue to a depth of several centimeters. In other embodiments, irradiation may be performed by internal application of light, such as by using an endoscope, a fiber optic catheter, or an implantable fluorescence device. Internal application may be used when the target tissue, such as a tumor, is located at a depth that is unsuitable for external light application. For example, an endoscope may be used for light delivery into the lungs, stomach, or bladder.

The surface area for light application is generally selected to include target tissue, e.g. , a tumor or portion of a tumor, or an area of skin external to the target tissue. When targeted application of external light is desired for an in vivo biological sample, the surface area can be controlled by use of an appropriate light applicator, such as a micro-lens, a Fresnel lens, or a diffuser arrangement. For targeted internal light application, a desired endoscope or fiber optic catheter diameter can be selected. In some applications, an indwelling catheter filled with a light scattering solution may be internally placed proximate the target tissue, and an optical fiber light source may be inserted into the catheter (see, e.g. , Madsen et al , Lasers in Surgery and Medicine 2001, 29, 406-412). Irradiation is performed for a period of time sufficient to deliver an amount of irradiation effective to induce cleavage of the drug from at least some molecules of the conjugate. In some embodiments, the effective amount of irradiation is at least 10 J/cm², such as at least 30 J/cm², at least 50 J/cm², or at least 100 J/cm². Effective amounts of irradiation may range from 10- 250 J/cm², such as from 30-100 J/cm².

VII. Methods of Use

Conjugates according to Formula I or Formula II are suitable for in vivo, ex vivo, or in vitro use. In some embodiments, when R^b is a targeting agent and Y is C(R^d)2, then at least one R^d is other than H. The conjugate is irradiated with targeted application of an effective quantity of light having a selected wavelength in the near-infrared range and a selected intensity to induce a cleavage reaction and release the drug from at least some molecules of the conjugate. For example, drug may be released from at least 10%, at least 20% at least 40%, at least 60%, or at least 80% of the conjugate molecules when the conjugate is irradiated with an effective quantity of light. In some embodiments, from 10-100% of the drug is released, such as from 20-100%, from 40-100%, from 60-100%, or from 80-100%. In one embodiment, the conjugate is evaluated in the absence of a biological sample to confirm that the particular conjugate will undergo photodegradation when irradiated with near-IR light.

A fluorescence level of the conjugate may be monitored during irradiation, and irradiation may be ceased when the fluorescence level falls below a target level. Fluorescence decreases as drug is released from the conjugate. Thus, the fluorescence level might be monitored to determine when a desired or sufficient proportion of the conjugate has undergone cleavage and drug release.

A biological sample may be contacted in vivo, ex vivo, or in vitro with the conjugate according to Formula I or Formula II. Following contact with the conjugate, the biological sample is irradiated with near-IR radiation to induce a cleavage reaction and release the drug from the conjugate. In some embodiments, a period of time is allowed to lapse between administration of the conjugate and application of near-IR radiation, thereby providing time for the conjugate to accumulate at and bind to the target site. The period of time may be several hours to several days, such as from 1-7 days or from 12 hours-2 days.

In some embodiments, the conjugate according to Formula I or II comprises a targeting agent capable of recognizing and binding directly or indirectly, in vitro, in vivo, or ex vivo, to a target (e.g. , an antigen or a receptor) present or suspected of being present in the biological sample. In one embodiment, the biological sample is visualized under conditions suitable to produce near- IR fluorescence if the conjugate is present in the biological sample. Fluorescence also confirms presence of the target in the biological sample. Excess unbound conjugate may be removed from the biological sample (e.g. , by washing a tissue sample) prior to visualizing the sample to detect fluorescence.

In one non-limiting example, a biological sample (e.g. , a tissue sample) that may comprise a target is contacted with a conjugate according to Formula I or II comprising an antibody capable of recognizing and binding to the target. In another non-limiting example, a biological sample that may comprise a target is combined with a first antibody capable of recognizing and binding to the target; subsequently, the biological sample is contacted with a conjugate comprising an anti- antibody antibody. In another non-limiting example, the biological sample is contacted with a conjugate comprising a ligand capable of binding to a receptor. For instance, substituent R^b may be a receptor ligand capable of binding to a receptor on a cell surface.

In some embodiments, a subject is identified as having a condition that may be treated with a drug. A therapeutically effective amount of a conjugate according to Formula I or II comprising the drug or a pharmaceutical composition comprising the conjugate is administered to the subject. A therapeutically effective amount of the conjugate is an amount sufficient to release a

therapeutically effective dose of the drug when irradiated by targeted application of an effective quantity of light having a wavelength in the near-infrared range and a selected intensity to a targeted portion of the subject. In certain embodiments, the light source provides light with a wavelength of 680-700 nm and an intensity of 300-700 mW/cm². In one embodiment, the light has a wavelength of 690 nm and an intensity of 500 mW/cm². The effective amount may range from 10-250 J/cm², such as from 10-100 J/cm², or 30-100 J/cm².

In one embodiment, the subject has a tumor and a conjugate according to Formula I or II comprises a targeting agent capable of recognizing and binding to an antigen or ligand-binding receptor of the tumor. Suitable tumors include, but are not limited to, solid tumor masses, such as intraperitoneal tumors (e.g., ovarian, prostate, colorectal), breast tumors, or head/neck tumors. The targeting agent may be, for example, an antibody that recognizes and binds to the tumor antigen. A therapeutically effective amount of the conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the conjugate, is administered to the subject by any suitable means including, but not limited to, parenteral, intravenous, subcutaneous, oral, rectal, vaginal, or topical administration. The administered conjugate is irradiated by targeted application of NIR light to an area proximate a location of the tumor. For example, with reference to FIG. 2, a subject 100 with a tumor 110 may be treated with a conjugate comprising an anti-tumor drug and an antibody or ligand capable of recognizing and binding to an antigen or receptor on a tumor cell surface. Administration of the conjugate to the subject may impair growth of the tumor and/or cause tumor regression.

In certain embodiments, prior to targeted application of NIR light, the administered conjugate is visualized by exposing the tumor to light having a wavelength suitable for exciting the cyanine fluorophore, thereby inducing fluorescence and visualizing the tumor. In some examples, the tumor site is exposed by surgical incision prior to exposing the tumor to light. The tumor is excised using the area of fluorescence as guidance. Remaining conjugate and/or tumor tissue is then be irradiated by targeted application of NIR light as described above to release the drug and treat any non-excised cancerous tissue.

In the example shown in FIG. 2, the conjugate 120 is administered via intravenous injection. A period of time is allowed to elapse during which the conjugate preferentially accumulates at the tumor site as the antibody or ligand moiety binds to the tumor. A target portion of the subject subsequently is selectively irradiated with an effective amount of NIR light energy of a desired wavelength using an external light applicator 130. The light applicator 130 applies the photoactivation energy to a target area limited to the region of the tumor 110, thereby selectively inducing cleavage of the conjugate molecules in and around the tumor 110 and targeting delivery of the anti-tumor drug released from the conjugate.

A therapeutically effective amount of a second agent may be co-administered with the conjugate or salt thereof. The conjugate (or salt thereof) and the second agent may be administered either separately or together in a single composition. The second agent may be administered by the same route or a different route. If administered concurrently, the conjugate (or salt thereof) and the second agent may be combined in a single pharmaceutical composition or may be administered concurrently as two pharmaceutical compositions. The second agent may be, for example, an antitumor agent or an angiogenesis inhibitor.

In another embodiment, an in vitro or ex vivo evaluation may be performed to determine whether a targeting agent-drug conjugate according to Formula I or II will effectively bind to a tissue sample obtained from a subject having, or suspected of having, a condition that may be treated or ameliorated by the drug and/or to determine whether the drug may be effective for the subject's condition. The conjugate comprises a drug and a moiety at R^b thought to be capable of binding to or associating with the target molecule. In one non-limiting example, R^b is a receptor ligand or antibody capable of binding to a target receptor. The conjugate is combined with the tissue sample, and the sample is subsequently irradiated with an effective amount of near-IR light. In one embodiment, the tissue sample is washed to remove excess, unbound conjugate, and fluorescence of the tissue sample is assessed. Fluorescence indicates that the conjugate has bound to the tissue sample. Following irradiation with near-IR light, fluorescence may again be assessed. A decrease in (or cessation of) fluorescence indicates release of the drug. The drug's efficacy also may be assessed, e.g., by assessing cytotoxicity.

Embodiments of conjugates according to Formula I or Formula II wherein R^b comprises a succinimidyl or maleimidyl group are suitable for customized conjugation to a targeting agent of choice. In one non-limiting example, a tumor sample is obtained from a subject, and a conjugate comprising a drug that may be effective against the tumor is selected. An antibody that specifically recognizes and binds to an antigen on the tumor is prepared by methods known to one of ordinary skill in the art. The prepared antibody is then reacted with R^b of the selected conjugate to provide a customized conjugate suitable for administration to the subject.

Precursor compounds according to Formula III or Formula IV are suitable for customized conjugation to a selected drug and a selected targeting agent. In one embodiment, the precursor compound is used by a pharmaceutical company to develop a conjugate having a desired combination of drug and targeting agent. In another embodiment, the precursor compound is used by a researcher or clinician to develop conjugates having desired combinations of drugs and targeting agents useful for research purposes or for developing a customized conjugate for treating a subject.

VIII. Kits

Kits are also a feature of this disclosure. Embodiments of the kits include at least one conjugate according to general Formula I or II or a precursor compound according to general Formula III or IV. In one embodiment, the kit includes a conjugate according to Formula I or II wherein R^b is a targeting agent, e.g. , an antibody. In another embodiment, the kit includes an intermediate compound wherein R^a or R^b is

and the kit may be used to prepare a further conjugate comprising a desired targeting agent, wherein the targeting agent is capable of reacting with the intermediate compound to provide a conjugate comprising the targeting agent. In yet another embodiment, the kit includes a precursor compound according to Formula III or Formula IV, and the kit is used to prepare a conjugate having a desired targeting agent and a desired drug.

In some embodiments, the kits also include at least one solution in which the conjugate or precursor compound may be dissolved or suspended. The kits also may include one or more containers, such as a disposable test tube or cuvette. The kits may further include instructions for using the conjugate according to Formula I or II, for forming a further conjugate with a desired targeting agent if R^b is a reactive group, and/or for preparing a conjugate comprising a desired targeting agent and a desired drug from the precursor compound according to Formula III or IV. In some embodiments, the kits further include reagents suitable for conjugating the compound according to Formula I, II, III, or IV to a targeting agent and/or for conjugate the compound according to Formula III or IV to a drug.

In some embodiments of the kits, the conjugate or precursor compound is provided as a solid, and the solution is provided in liquid form. In one embodiment, the solution is suitable for dissolving a conjugate according to Formula I or II so that the dissolved conjugate may be administered to a subject or so that a dissolved conjugate wherein R^b is a reactive group (an intermediate compound comprising a drug and a reactive group) may be conjugated to a targeting agent. In an independent embodiment, the solution is suitable for dissolving a precursor compound according to Formula III or IV for subsequent conjugation to a drug and/or targeting agent. The solution may be provided at a concentration suitable for the intended use.

Alternatively, the solution may be provided as a concentrated solution, which is subsequently diluted prior to use. In certain embodiments, the conjugate or precursor compound is premeasured into one or more containers (e.g. , test tubes or cuvettes). IX. Examples

General Materials and Methods. Unless stated otherwise, reactions were conducted in oven- dried glassware under an atmosphere of nitrogen or argon using anhydrous solvents (passed through activated alumina columns). All commercially obtained reagents were used as received. Azido-PEG₄-acid 5 was purchased from Aurum Pharmatech, Inc. (Howell, New Jersey). (∑)- Combretastatin A4 and IR-783 were purchased from Sigma- Aldrich. Flash column

chromatography was performed using normal phase silica gel (60 A, 230-400 mesh, RediSep® Normal-phase Silica Flash Columns) or reversed phase (100 A, 20-40 micron particle size, RediSep® Rf Gold® Reversed-phase C18) on a CombiFlash® Rf 200i (Teledyne Isco, Inc.).

Analytical LC/MS was performed using a Shimadzu LCMS-2020 Single Quadrupole utilizing a Kinetex 2.6 μηι C18 100 A (2.1 x 50 mm) column obtained from Phenomenex Inc. Runs employed a gradient of 0→90% MeOH/0.1 % aqueous formic acid over 4.5 minutes at a flow rate of

0.2 niL/min. ¾ NMR and ¹³C NMR spectra were recorded on Varian and Bruker spectrometers (at 400 or 500 MHz or at 100 or 125 MHz) and are reported relative to deuterated solvent signals. Data for ¾ NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. Data for ¹³C NMR spectra are reported in terms of chemical shift. IR spectra were recorded on a Varian 640-IR spectrometer and are reported in terms of frequency of absorption (cm ¹). Absorption curves for quantum yield measurements were performed on a Shimadzu UV-2550 spectrophotometer operated by UVProbe 2.32 software. Fluorescence traces and quantum yield measurements were recorded on a PTI QuantaMaster steady-state

spectrofluorimeter operated by FelixGX 4.2.2 software, with 5 nm excitation and emission slit widths, 0.1 s integration rate, and enabled emission correction. Data analysis and curve fitting were performed using MS Excel 2011 and GraphPad Prism 6. Light intensity measurements were performed with a Thorlabs PM200 optical power and energy meter fitted with an S120VC standard Si photodiode power sensor (200 - 1100 nm, 50 nW - 50 mW). Flow cytometry was performed at the CCR Flow Cytometry Core (NCI- Frederick) and microscopy was performed at the Optical Microscopy and Analysis Laboratory (NCI-Frederick). See JOC Standard Abbreviations and Acronyms for abbreviations (http://pubs.acs.org/userimages/ContentEditor/1218717864819/ j oceah_abbreviations .pdf) .

Example 1

Synthesis and Characterization of Panitumumab-Combretastatin A4 Conjugate (CY-Pan-CA4)

(S3): A round bottom flask was charged with tosyl-pentyne SI (10 g, 42 mmol) and immersed in a room temperature water bath (Hugenberg et al , J. Med. Chem. 2013, 56:6858). Ethanolamine (15 mL, 248 mmol) was added slowly in one portion and the reaction was stirred neat for 3 hours at room temperature. The reaction was then diluted with water (100 mL) and brine (100 mL) and extracted with diethyl ether (10 x 75 mL). The combined organic layers were dried over MgS0₄, filtered and concentrated to afford 5 grams of crude S2 (-80% by *H NMR analysis), which was used directly in the next step. To a solution of crude S2 (5 g, -35 mmol) in THF (10 mL) under argon was added triethylamine (9.7 mL, 70 mmol) and di-i-butyl dicarbonate (8.4 g, 39 mmol) in succession. Vigorous gas evolution occurred for 10 seconds immediately after B0C2O addition, and the resulting clear yellow solution was stirred for 1 hour at room temperature thereafter. After concentration of the solvent in vacuo the crude oil was purified by silica gel chromatography (0→10 % MeOH/DCM) to afford S3 (3.0 g, 32 % from SI) as a colorless oil. ¾ NMR (CDCb, 400 MHz) J3.74 (q, / = 5.0 Hz, 2H), 3.46 - 3.36 (m, 2H), 3.33 (t, / = 7.2 Hz, 2H), 3.17 (br s, 1H), 2.20 (td, J = 7.0, 2.7 Hz, 2H), 1.96 (t, J = 2.7 Hz, 1H), 1.83 - 1.69 (m, 2H), 1.46 (s, 9H); ¹³C NMR (CDCI3, 100 MHz) δ\5ΊΛ, 83.5, 80.3, 68.8, 62.6, 50.4, 47.7, 28.4, 27.4, 15.9; IR (thin film) 3431, 3306, 2932, 2118, 1667, 1478, 1366, 1162 cm ¹; MS (ESI) calculated for

C12H22NO3 (MH⁺) 228.2, observed 228.2.

(S4): To a solution of S3 (400 mg, 1.76 mmol) in DCM (9 mL) was added Dess-Martin periodinane (780 mg, 1.85 mmol). The hazy light yellow solution was stirred at room temperature for 45 minutes. The reaction was diluted with ethyl acetate (50 mL), and the organic layer was washed with saturated aqueous Na2S2(¾ (2 5 mL), saturated aqueous NaHCCb, and brine. The organic layer was dried over MgS0₄, filtered and concentrated in vacuo to afford S4 (370 mg, 93 % yield) as a colorless oil. ¾ NMR (CDCI3, 400 MHz) 59.59 (s, 1H), 4.04 - 3.82 (m, 2H), 3.46 - 3.31 (m, 2H), 2.30 - 2.16 (m, 2H), 1.97 (t, J = 2.1 Hz, 1H), 1.82 - 1.68 (m, 2H), 1.52 - 1.39 (m, 9H); ¹³C NMR (CDCI3, 100 MHz) J198.8, 155.4, 83.3, 80.8, 69.0, 57.8, 47.8, 28.3, 27.4, 15.8; IR (thin film) 2932, 2118, 1736, 1667, 1418, 1366, 1162 cm ¹; MS (ESI) calculated for C12H20NO3 (MH⁺) 226.1, observed 226.2.

(2): To a solution of S4 (190 mg, 0.84 mmol) in DCM (10 mL) was added methylamine (420 \L, 3.38 mmol, 33 wt % in ethanol) in one portion at room temperature. The orange/brown solution was stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (267 mg, 1.27 mmol) solid was charged in one portion, and the hazy brown mixture was stirred for 2 hours. The mixture was diluted with DCM (100 mL) and washed with 1M NaOH (30 mL). The organic layer was washed with brine, and then dried over MgS0₄ and filtered. The volatiles were concentrated in vacuo, and the brown residue was purified by silica gel chromatography (0→20 % MeOH/DCM with 1 % Et₃N) to afford 2 (101 mg, 50 % yield) as a colorless oil. ¾ NMR (CDCb, 500 MHz) J3.43 - 3.19 (m, 4H), 2.83 - 2.65 (m, 2H), 2.45 (s, 3H), 2.18 (td, / = 7.0, 2.7 Hz, 2H), 1.95 (t, / = 2.7 Hz, 1H), 1.79 - 1.66 (m, 2H), 1.44 (s, 9H); ¹³C NMR (CDCb, 125 MHz) J155.7, 83.6, 79.7, 68.8, 50.2, 46.9, 46.8 (observed by HSQC), 36.1, 28.4, 27.4, 15.9; IR (thin film) 3254, 2932, 2792, 2118, 1686, 1413, 1365, 1163 cm ¹; MS (ESI) calculated for C13H25N2O2 (MH⁺) 241.2, observed 241.3.

81 % yield

(3): To a solution of IR-783 (1, 81 mg, 0.097 mmol) in DMF (0.9 mL) was added Boc- diamine 2 (70 mg, 0.29 mmol) and NN-diisopropylethylamine (67 uL, 0.39 mmol). The green solution was sparged with argon for 5 minutes, then heated to 105 °C in a sealed vial for

50 minutes. LC/MS analysis of the dark blue reaction showed complete consumption of 1. The reaction was cooled to 30 °C and charged with 4-(trifluoromethyl)-benzyl bromide (45 μί, 0.29 mmol) and NN-diisopropylethylamine (33 μί, 0.19 mmol). The remaining amount of amine 2 co-eluted with the product 3. The 4-(trifluoromethyl)-benzyl bromide derivatized the residual 2, generating a non-polar adduct, which facilitated isolation of 3. After 2 hours, LC/MS analysis showed complete consumption of the remaining portion of 2. The reaction was diluted with saturated aqueous NaHCCb (10 mL), water (5 mL), and acetonitrile (0.5 mL), and stirred for 20 minutes at room temperature. The entire mixture was loaded directly onto a pre-packed 50 g C18 column and purified by reversed-phase chromatography (5→45 % MeCN/ water). The solvent was removed in vacuo to afford 3 (76 mg, 81 % yield) as a dark blue solid. *H NMR (CD3OD, 400 MHz, 70 °C) £7.71 (d, / = 13.4 Hz, 2H), 7.44 - 7.28 (m, 4H), 7.23 - 7.09 (m, 4H), 6.00 (d, / = 13.4 Hz, 2H), 4.15 - 3.96 (m, 4H), 3.92 - 3.81 (m, 2H), 3.60 - 3.51 (m, 2H), 3.45 (s, 3H), 3.37 - 3.32 (m, 2H), 2.93 - 2.83 (m, 4H), 2.65 - 2.50 (m, 4H), 2.26 - 2.14 (m, 3H), 2.04 - 1.90 (m, 8H), 1.91 - 1.83 (m, 2H), 1.80 - 1.71 (m, 2H), 1.68 (s, 12H), 1.43 (s, 9H); IR (thin film) 2924, 2866, 2114, 1686, 1509, 1365, 1255 cm ¹; MS (ESI) calculated for C51H69N4O8S2 (Mr) 929.5, observed 929.7. With N-alkyl carbamates 3, 4, and S6-S8, high temperature NMR (70 °C) was required to resolve the carbamate rotamers. However, these sulfonates proved unstable at elevated temperatures for prolonged time in various deuterated solvents, which precluded obtaining ¹³C NMR spectra.

(4): To a solution of combretastatin A4 (31 mg, 0.10 mmol) in THF (3 mL) was added triethylamine (19 μί, 0.13 mmol). After stirring for 5 minutes phosgene (62 μί, 0.090 mmol, 15 wt % toluene) was added, resulting in a white precipitate. This reaction mixture of combretastatin A4 chloroformate (S5) was stirred for 1 hour at room temperature. In a separate 25 mL round bottom flask, 3 (35 mg, 0.037 mmol) was dissolved in neat trifluoroacetic acid (400 μί, 5.23 mmol) under argon at room temperature. The dark red solution was stirred for 20 minutes at room temperature, then subsequently cooled to -10 °C and diluted with THF (3 mL). A vent needle was inserted in the septum, and a sodium bicarbonate (588 mg, 7.00 mmol) solution in 7 mL of water was added slowly with vigorous stirring. To this dark blue mixture was added the THF suspension of S5 generated above in one portion. The -10 °C bath was removed and the reaction was allowed to warm to room temperature with vigorous stirring. After 15 minutes the THF was removed in vacuo, and MeCN (1 mL) and water (5 mL) were added to the crude reaction mixture. The entire mixture was loaded directly onto a pre-packed 30 g C18 column and purified by reversed-phase

chromatography (5→45 % MeCN/water). The solvent was removed in vacuo to afford 4 (40 mg, 93 % yield) as a dark blue solid. ¾ NMR (CD₃OD, 400 MHz, 70 °C) Si.69 (d, / = 13.3 Hz, 2H), 7.40 - 7.27 (m, 4H), 7.21 - 7.03 (m, 5H), 6.99 - 6.89 (m, 2H), 6.61 - 6.33 (m, 4H), 5.96 (d, J = 13.3 Hz, 2H), 4.12 - 3.92 (m, 6H), 3.77 (s, 3H), 3.75 - 3.66 (m, 5H), 3.63 (s, 6H), 3.53 - 3.38 (m, 5H), 2.91 - 2.84 (m, 4H), 2.57 - 2.46 (m, 4H), 2.29 - 2.14 (m, 3H), 2.01 - 1.89 (m, 10H), 1.90 - 1.75 (m, 4H), 1.62 (s, 12H); IR (thin film) 2967, 2931, 2117, 1716, 1507, 1365 cm ¹; MS (ESI) calculated for C65H79N4O12S2 (Mr) 1171.5, observed 1171.8.

(S6): To a 1 dram vial was added 4 (11.5 mg, 0.01 mmol), cupric sulfate (0.3 mg,

0.002 mmol) and sodium ascorbate (1.0 mg, 0.005 mmol). The headspace was flushed with argon, and a water/i-butanol (1.0 mL, 1: 1 v/v) solution of 5 (3.6 mg, 0.013 mmol) was added to the solid admixture at room temperature. The deep blue reaction was stirred for 4 hours, at which time LC/MS indicated consumption of 4. The reaction was diluted with water (3 mL) and purified directly by reversed phase chromatography (5→95 % MeCN/water). The solvent was removed in vacuo to afford S6 (10 mg, 70 % yield) as a dark blue solid. ¾ NMR (CD₃OD, 400 MHz, 70 °C) δ 7.74 (s, 1H), 7.68 (d, J = 13.4 Hz, 2H), 7.43 - 7.25 (m, 4H), 7.23 - 7.02 (m, 5H), 7.00 - 6.90 (m, 2H), 6.61 - 6.38 (m, 4H), 5.95 (d, / = 13.4 Hz, 2H), 4.46 (t, / = 5.1 Hz, 2H), 4.07 - 3.93 (m, 6H), 3.86 - 3.80 (m, 2H), 3.78 - 3.52 (m, 29H), 3.50 - 3.40 (m, 4H), 2.92 - 2.83 (m, 4H), 2.74 (t, / = 7.5 Hz, 2H), 2.56 - 2.43 (m, 6H), 2.11 - 1.88 (m, 10H), 1.87 - 1.74 (m, 2H), 1.60 (s, 12H); IR (thin film) 2930, 2868, 1720, 1509, 1371 cm ¹; MS (ESI) calculated for C76H101N7O18S2 (Mr) 1462.7, observed 1463.2.

(7): To a 1 dram vial was added S6 (5.5 mg, 0.0037 mmol) and N,N,N',N'-tetramethyl-0-

(N-succinimidyl)uronium tetrafluoroborate (6, 1.1 mg, 0.0041 mmol). The headspace was flushed with argon, and DCM (0.8 mL) and MeCN (0.2 mL) were charged to the vial, followed by N,N-diisopropylethylamine (0.7 μί, 0.0041 mmol). The deep blue solution was stirred for 1 hour, at which time LC/MS indicated consumption of S6. The solvent was removed in vacuo, and the blue residue was triturated in ethyl acetate (1 mL) by repeated vortex mixing and sonication. The fine suspension was centrifuged, the supernatant decanted, and the pellet resuspended in diethyl ether (1 mL). The reslurry procedure was repeated twice with diethyl ether, and the pellet was placed under vacuum (< 0.1 Torr) for 1 hour to afford 7 (5.0 mg, 85 % yield) as a dark blue solid. MS (ESI) calculated for C80H103N8O20S2 (Mr) 1559.7, observed 1560.2.

(S7): In a 25 mL round bottom flask 3 (40 mg, 0.041 mmol) was dissolved in neat trifluoroacetic acid (440 μί, 5.74 mmol) under argon at room temperature. The dark red solution was stirred for 20 minutes at room temperature, then subsequently cooled to -10 °C and diluted with THF (6 mL). A vent needle was inserted in the septum, and a sodium bicarbonate (645 mg, 7.68 mmol) solution in 8 mL of water was added slowly with vigorous stirring. To this dark blue mixture was added neat phenyl chloroformate (15 μL·, 0.023 mmol). The -10 °C bath was removed and the reaction was allowed to warm to room temperature with vigorous stirring. After 15 minutes the THF was removed in vacuo, and MeCN (1 mL) and water (6 mL) were added to the crude reaction mixture. The entire mixture was loaded directly onto a pre-packed 30 g CI 8 column and purified by reversed-phase chromatography (5→45 % MeCN/water). The solvent was removed in vacuo to afford S7 (33 mg, 83 % yield) as a dark blue solid. ¾ NMR (CD₃OD, 400 MHz, 70 °C) δ 7.72 (d, / = 13.4 Hz, 2H), 7.43 - 7.25 (m, 6H), 7.21 - 7.08 (m, 5H), 7.09 - 7.00 (m, 2H), 5.98 (d, / = 13.4 Hz, 2H), 4.12 - 3.93 (m, 6H), 3.85 - 3.68 (m, 2H), 3.56 - 3.43 (m, 5H), 2.94 - 2.80 (m, 4H), 2.56 (t, / = 6.5 Hz, 4H), 2.37 - 2.17 (m, 3H), 2.08 - 1.80 (m, 12H), 1.63 (s, 12H); IR (thin film) 2967, 2931, 2118, 1714, 1505, 1365 cm ¹; MS (ESI) calculated for C53H65N4O8S2 (M^") 949.4,

(S8): To a 1 dram vial was added S7 (3.5 mg, 0.0036 mmol), cupric sulfate (0.6 mg, 0.004 mmol) and sodium ascorbate (1.4 mg, 0.007 mmol). The headspace was flushed with argon, and a water/i-butanol (1.0 mL, 1: 1 v/v) solution of 5 (4.0 mg, 0.014 mmol) was added to the solid admixture at room temperature. The deep blue reaction was stirred for 2 hours, at which time LC/MS indicated consumption of S7. The reaction was diluted with water (1 mL) and purified directly by reversed phase chromatography (5→40 % MeCN/water). The solvent was removed in vacuo to afford S8 (3.5 mg, 78 % yield) as a dark blue solid. ^lH NMR (CD3OD, 400 MHz, 70 °C) δ 7.76 (s, 1H), 7.70 (d, 7 = 13.5 Hz, 2H), 7.41 - 7.28 (m, 6H), 7.20 - 7.09 (m, 5H), 7.06 - 7.01 (m, 2H), 5.97 (d, J = 13.5 Hz, 2H), 4.47 - 4.43 (m, 2H), 4.10 - 3.94 (m, 6H), 3.85 - 3.78 (m, 2H), 3.78 - 3.73 (m, 2H), 3.70 (t, / = 6.5 Hz, 2H), 3.62 - 3.52 (m, 12H), 3.52 - 3.45 (m, 5H), 2.92 - 2.83 (m, 4H), 2.79 - 2.72 (m, 2H), 2.58 - 2.51 (m, 4H), 2.48 (t, / = 6.5 Hz, 2H), 2.11 - 1.99 (m, 2H), 2.01 - 1.90 (m, 8H), 1.91 - 1.82 (m, 2H), 1.61 (s, 12H); IR (thin film) 2930, 2868, 1721, 1506, 1371 cm ¹; MS (ESI) calculated for C64H86N7O14S2 (Mr) 1240.6, observed 1240.9.

(S9): To a 1 dram vial was added S8 (2.5 mg, 0.0020 mmol) and N,N,N,N-tetramethyl-0- (N-succinimidyl)uronium tetrafluoroborate (6, 0.6 mg, 0.002 mmol). DCM (0.8 mL) and MeCN (0.2 mL) were charged to the vial, followed by NN-diisopropylethylamine (0.5 μί, 0.002 mmol). The deep blue solution was stirred for 1 hour, at which time LC/MS indicated consumption of S8. The solvent was removed in vacuo, and the blue residue was triturated in ethyl acetate (0.5 mL) by repeated vortex mixing and sonication. The fine suspension was centrifuged, the supernatant decanted, and the pellet resuspended in diethyl ether (0.5 mL). The procedure was repeated twice with diethyl ether, and the pellet was placed under vacuum (< 0.1 Torr) for 1 hour to afford S9 (1.8 mg, 67 % yield) as a dark blue solid. MS (ESI) calculated for C68H89N₈Oi6S₂ (Mr) 1337.6, observed 1337.9. S9 was prepared as a negative control for 7.

Assignment of Isomeric C4'-N-Alkyl Cyanines 4 and S10

Initial studies on the Boc removal, carbamate formation sequence revealed that many conditions provided a -1: 1 mixture of two distinct N- linked cyanine products. Further analysis revealed these to be constitutional isomers comprising the desired C4'-N-linkage, 4, and the isomer arising from the intermediate diamine equilibrating through C4' addition/elimination sequences prior to carbamate formation (FIG. 3). The observation of the two constitutional isomers, 4 and S10, occurred upon quenching the TFA-mediated Boc deprotection of 3 at room temperature and allowing for equilibration prior to acylation with chloroformate S5. It was ultimately determined that deprotection of the Boc group in neat TFA, careful aqueous bicarbonate quench at -10 °C, and addition of the chloroformate of CA4 to the cooled mixture provided only 4 in excellent yield (93%). After separation of 4 and S10 by preparative HPLC, NMR analysis revealed 4 possessed C4'-N-methyl ¾ and ¹³C (assigned by HSQC) chemical shifts consistent with other known dialkylamine cyanines (Gorka et al. , JACS 2014, 136: 14153; Nani et al. J. Org. Lett. 2015, 17:302). Alternatively, the upfield shifts of the N-methyl resonance of S10 in the ¾ and ¹³C spectra are indicative of an N-methyl tertiary carbamate (Gopin et al , Bioconj. Chem. 2006, 17: 1432). Determination of Quantum Yields and Molar Absorption Coefficients

Quantum yields (Of) were determined in MeOH relative to IR783 ( ί = 0.084 in MeOH), from plots of integrated fluorescence intensity vs. absorbance, according to the following relationship:

where subscripts st and x denote standard and test sample, respectively, Φ is the fluorescence quantum yield, Grad is the gradient of the integrated fluorescence intensity vs. absorbance plot, and η is the refractive index of the solvent. Measurements were performed in 10 mm path length quartz cuvettes (Hellma 111-QS), maintained at 25 °C, with the absorbance of all dye solutions < 0.08 in order to maximize illumination homogeneity and optical transparency and minimize reabsorption effects. IR783 standard and test dye solutions were excited at 10 nm below their absorption maxima. The quantum yield of S6 in MeOH relative to IR783 was <Df = 0.16.

Molar absorption coefficients (ε) were determined in 1 : 1 (v/v) MeOH/PBS (pH 7.4) using Beer's law, from plots of absorbance vs. concentration. Measurements were performed in 10 mm path length quartz cuvettes (Hellma 111-QS), maintained at 25 °C, with absorbance at the highest concentration < 0.08 (see above).

Procedure for Panitumumab Conjugation (Bhattacharyya et al., Medchemcomm 2014, 5: 1337) Panitumumab was incubated with 1.3 equiv. of 7 to provide the CY-Pan-CA4 conjugate.

All steps were performed under reduced lighting. To 250 of 1 M PBS (pH 8.5) in a 1.5 mL microcentrifuge tube was added 500 of panitumumab (from a 20 mg/mL commercial stock acquired from Amgen). In a separate tube, 23 uL of a 5 mM DMSO solution of 7 or S9 was quickly premixed with 250 of 1 M PBS (pH 8.5), and then immediately transferred to the panitumumab solution. The resulting mixture was gently pipetted and inverted and incubated at room

temperature for 1 h on a rocking platform. Three identical G10 Sephadex^® columns (GE

Healthcare) were primed with 6 column volumes each of 0.9% (w/v) saline. The reaction was partitioned equally between the three columns (-300 μί) and eluted with 0.9% saline. The first eluting blue band was collected, affording 2.5 mL of a saline solution. HPLC analyses of the conjugates revealed no detectable amounts of free combretastatin A4 or phenol. An aliquot of each fraction was diluted tenfold into 1: 1 (v/v) MeOH/PBS (50 mM, pH 7.4) and analyzed by UV-vis in a plastic UV cuvette (FIG. 4). The absorption values at 280 and 700 nm were obtained, and the dye and antibody concentrations were determined from Beer's law (C = Α/εΙ), with Sdy_e = 55,000 M ¹ cm and Santit>ody = 200,000 M ¹ cm ¹, corresponding to the molar extinction coefficients of S6/S8 and panitumumab in 1: 1 (v/v) MeOH/PBS, respectively. A correction factor of 25% and 5% for S6 and S8, respectively, was applied to account for the absorption contribution of the free dye at 280 nm. The degree of labeling (DOL), the average number of dye molecules per antibody, was determined from the quotient of dye concentration to antibody concentration. CY-Pan-CA4 possessed a DOL of 1.2 and protein concentration of 18 μΜ, and CY-Pan-Phenol a DOL of 1.1 and protein concentration of 21 μΜ. The antibody conjugate solutions were filtered through a 0.22 μιη sterile filter (Acrodisc) and stored at 4 °C. SDS-PAGE

Sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to assess the purity of the antibody conjugates. NuPAGE 4-12% Bis-Tris gels (Life Technologies #IM-8042) were loaded with 15 μg of CY-Pan-CA4 and CY-Pan-Phenol (1 : 1:2 conjugate/NuPAGE LDS sample buffer/lX PBS) and run under non-reducing conditions in IX MES SDS running buffer at 180 V for 40 min. A BenchMark Pre-Stained Protein Standard (Life Technologies #10748010) was used for molecular weight comparison. Gels were stained with SimplyBlue SafeStain (Life Technologies #LC6060) for 1 h and imaged with white transillumination (60 ¹ s exposure time) (FIG. 5A). Fluorescence images were obtained using an ImageQuant LAS 4000 (GE Healthcare) with red epi light (630 nm) excitation and a 670 nm emission filter (FIG. 5B). Exposure time was 60 s.

To a round bottom flask Boc-Cy7 3 (11.3 mg, 0.012 mmol) was dissolved in neat trifluoroacetic acid (250 μί) under argon at room temperature. The dark red solution was stirred for 20 minutes at room temperature, then subsequently cooled to 0 °C and diluted with THF (4 mL). A sodium bicarbonate (500 mg) solution in 10 mL of water was added slowly with vigorous stirring. In a separate vessel duocarmycin DM (5.6 mg, 0.010 mmol) was dissolved in MeCN (0.5 mL) at 0 °C under argon. Diisopropylethylamine (4 μί, 0.022 mmol) and 4-nitrophenylchloroformate (40 mg, 0.20 mmol) in MeCN (0.5 mL) were added in succession. The clear, light yellow solution was stirred for 20 minutes, after which time the reaction was combined with the cyanine/aqueous THF solution. The reaction was heated to 37 °C for 45 minutes, and the THF was subsequently removed in vacuo. The entire mixture was loaded directly onto a pre-packed 30 g CI 8 column and purified by reversed-phase chromatography (5→55 % MeCN/water). The solvent was removed in vacuo to afford 8 (5.5 mg, 41 % yield) as a dark blue solid. MS (ESI) calculated for

C73H₈5ClN70ioS₂ (M-) 1318.5, observed 1318.9.

To a 1 dram vial was added 8 (0.6 mg, 0.0005 mmol), cupric sulfate (0.1 mg, 0.0009 mmol) and sodium ascorbate (0.4 mg, 0.002 mmol). The headspace was flushed with argon, and a water/i-butanol (0.5 mL, 1 : 1 v/v) solution of azido-PEG4-acid 5 (0.2 mg, 0.0005 mmol) was added to the solid admixture at room temperature. The deep blue reaction was stirred for 30 minutes, at which time LC/MS indicated consumption of 5. The reaction was diluted with water (2 mL) and purified directly by reversed phase chromatography (5→95 % MeCN/ 0.1% (NH^CCb/water). The solvent was removed in vacuo to afford 9 (0.4 mg, 75 % yield) as a dark blue solid. MS (ESI) calculated for C84H107CIN10O16S2 (M^~) 1610.7, observed 1610.4. Compound 9 can further be reacted with N,N,N\N'-tetramethyl-0-(N-succinimidyl)uronium tetrafluoroborate (6) to afford a succinimidyl ester-functionalized compound suitable for conjugation to a targeting agent. Example 3

Photochemical Cleavage and Stability of CY-Pan-CA4

To assess the photochemical cleavage and dark stability, a solution of CY-Pan-CA4 in DMEM/FBS was irradiated with 50 J of 690 nm light administered from a convenient LED source (20 mW/cm²), or left in the dark, and then incubated for 18 h at 37 °C. The irradiated sample provided an excellent yield of free CA4, 61%, as measured by HPLC, whereas the unirradiated sample provided only negligible quantities (1%) of the free drug (FIG. 6).

The absorption and fluorescence emission of the conjugate were measured, and the consequence of irradiation on the fluorescence emission was also evaluated. The CY-Pan-CA4 conjugate maintained the near-IR optical properties of unconjugated S6 ( abs = 700 nm, em = 795 nm, FIG. 7).

The decay of the fluorescent signal during the photobleaching of CY-Pan-CA4 was determined as follows. A 500 nM solution of CY-Pan-CA4 was prepared by dilution of a saline stock solution into DMEM/FBS (9: 1 v/v) and 150 of this solution was transferred to a 3 mm path length quartz cuvette (Hellma 101-QS). The sample was irradiated (open to air) with 10 J doses of 690 nm light between fluorimeter readings. The sample was excited at 690 nm and the integrated emission intensity from 740-800 nm was extracted and plotted against light dose.

Experiments were run in triplicate and plotted with error bars derived from the standard deviation. Irradiation using 690 nm light reduced the emission of CY-Pan-CA4 (FIG. 8), with little remaining fluorescence after the 50 J of light fluence used for drug release. The latter result indicates that the irradiation-induced disappearance of cyanine emission will serve as a useful marker for drug release.

The HPLC yields of combretastatin A4 (CA4) during the photolysis experiment were determined by an external calibration method (Pigini et al. , Rapid Commun. Mass Spectrom. 2006, 20: 1031). A calibration curve was constructed with varying concentrations of CA4 plotted against the integrated area of the compound peak (FIG. 9). The solutions for calibration were generated from a 1 mM DMSO stock solution of CA4 via dilution in 1 : 1 (v/v) saline/MeCN to afford 1, 5, 10, and 20 μΜ solutions. The calibration samples were analyzed on an Agilent 1260 Infinity HPLC utilizing an Eclipse Plus 5 μιη C18 110 A (4.6 x 250 mm) column with a gradient of 5→95% (8 min) to 95→5% (2 min) MeCN/0.1% aqueous formic acid at a flow rate of 2.0 niL/min. This method of sample preparation and analysis was identical to what is used in the photolysis experiment (see below) to ensure consistency between the calibration curves and the experimental runs. A CY-Pan-CA4 saline solution (effective dye concentration of 22.4 μΜ) was diluted into DMEM/FBS (9: 1 v/v) to afford 100 μΐ. of a 10 μΜ solution. The sample was irradiated in a microcentrifuge tube with 50 J of 690 nm light at room temperature. The sample was subsequently heated for 18 hours at 37 °C, concurrently with a non-irradiated dark control. The reaction was diluted with 100 of acetonitrile to precipitate proteins, vortexed, and centrifuged for 1 min at 6,000 rpm. The CA4 concentration was divided by the original dye concentration to determine the yield of the released CA4. The photolysis was performed in experimental triplicate. FIG. 10 shows chromatograms of CA4 at 300 nm (top panel), photolysis of Cy-Pan-CA4 at t = 18 h at 300 nm (middle panel), and a dark control of CY-Pan-CA4 at t = 18 h at 300 nm (lower panel).

Example 4

In Vitro Characterization of CY-Pan-CA4

MDA-MB-468 (HER1 overexpression) and MCF-7 (normal HER1 expression) human breast cancer cell lines were obtained from the NCI DTP, DCTD Tumor Repository. MDA-MB- 468 was cultured in RPMI supplemented with 2 mM L-glutamine, 11 mM D-glucose, 24 mM sodium bicarbonate, 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. MCF-7 was cultured in DMEM supplemented with 4 mM L-glutamine, 25 mM D-glucose, 44 mM sodium bicarbonate, 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Both cell lines were grown at 37 °C in an atmosphere of 20% O2 and 5% CO2. Stock cultures were maintained in continuously exponential growth by weekly passage of the appropriate number of cells following trypsinization with 0.25% Trypsin-EDTA (0.9 mM) in PBS.

Fluorescence Confocal Microscopy

MDA-MB-468 or MCF-7 cells (5 x 10⁴ cells/well) were plated on Nunc Lab-Tek^® II chambered #1.5 German borosilicate coverglass (Thermo Fisher Scientific, Inc.) and allowed to adhere overnight. Cells were incubated with 100 nM CY-Pan-CA4 for 3 h, washed twice with PBS, incubated with 1 μΜ Hoechst 33342 for 0.5 h, washed twice with PBS, and imaged.

Fluorescence microscopy was performed using a Zeiss LSM 780 confocal microscope at 63x magnification using a plan-apochromat oil immersion objective. Near-IR fluorescence was imaged using a HeNe633 laser (633 nm excitation, 650 nm longpass emission) and Hoechst 33342 using an Argon/2 laser (488 nm excitation, 505-550 bandpass emission). Differential interference contrast (DIC) was collected using the Argon/2 laser. Image processing was conducted with Fiji.

FIG. 11 shows fluorescence confocal microscopy images of live MDA-MG-468 (panels 1- 3) and MCF-7 (panels 4-6) cells treated with Hoechst 3342 (1 μΜ) and CY-Pan-CA4 (100 nm). Panels 1 and 4 are fluorescence emission from Hoechst 3342; panels 2 and 5 are fluorescence emission from CY-Pan-CA4; panels 3 and 6 are differential interference contrast images. The results show that only HER1+ (MDA-MB-468) cells exhibited characteristic antibody labeling.

Specific cellular labeling was also confirmed using fluorescence activated cell sorting (FACS). MDA-MB-468 or MCF-7 cells were seeded into 6-well plates (1 xlO⁶ cells/well) and allowed to adhere overnight. Cells were incubated with 100 nM CY-Pan-CA4 for 3 h, washed twice with PBS, trypsinized, and suspended in PBS supplemented with 2% fetal bovine serum. Flow cytometric analysis for near-IR fluorescence signal was performed at the CCR Flow

Cytometry Core (NCI-Frederick) using a BD LSRII Fortessa analyzer operating a laser line at 647 nm (FIG. 12). Data processing was conducted with FlowJo vX.0.7.

The results of the fluorescence confocal microscopy and FACS indicate that the binding specificity of PAN was preserved in the immunoconjugate.

CY-Pan-CA4 was evaluated to determine whether it elicits a cytotoxic effect in the same cell lines in a light- and antigen-dependent fashion. Cell viability was determined with continuous exposure to a wide concentration range of CY-Pan-CA4 to examine the full biological effect of cleaved vs. uncleaved conjugate. MDA-MB-468 or MCF-7 cells were seeded into 96-well plates (5 x 10⁴ cells/well) and allowed to adhere overnight. Initial seeding densities were such to ensure cells remained in exponential growth for the duration of the assay. For the continuous dose assay, media was replaced with that containing CY-Pan-CA4, CY-Pan-Phenol, CA4, Pan, or DMSO. Cells were exposed to 30 J of irradiation from a 690 nm LED (10 mW/cm²) or kept dark. For the pre-incubation assay, media was replaced with that containing test compounds, incubated for 24 h at 37 °C in the dark, media replaced with fresh, inhibitor free media, and irradiated as above.

Following a 72 h incubation period at 37 °C, 20 uL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) from a 5 mg/mL stock in PBS was added to each well and incubated for 4 h at 37 °C. Media was removed, 100 of DMSO added to each well to solubilize MTT formazan, and absorbance at 550 nm was recorded using a microplate reader. Drug effects were expressed as % cell viability relative to the DMSO (no inhibitor) control. Half-maximal inhibitory concentrations (IC50) were obtained from sigmoidal curve fits of % viability vs. concentration data using GraphPad Prism 6. All experiments were conducted in quadruplicate, with error bars representing the standard deviation.

Irradiation of cells in the presence of CY-Pan-CA4 with 30 J of 690 nm light led to a growth inhibitory activity (IC50 = 16 nM) that nearly matched that of CA4 alone (11 nM) (FIG. 13). By contrast, the absence of irradiation significantly diminished this growth inhibitory effect (IC50 = 1.1 μΜ), providing additional evidence for the high dark stability of the conjugated form. Finally, as expected, the antibody alone had no effect on cell viability over the concentration range examined (IC50 > 2 μΜ). The internalized and cell-surface bound antibody fraction were evaluated by preincubation and then media exchange. MDA-MB-468 (HER1+) and MCF-7 (HER1-) cells were incubated with CY-Pan-CA4 (100 nM) for 24 h, the media was replaced, irradiation was carried as above, and cell viability was evaluated. A significant reduction in cell viability was observed only upon 690 nm irradiation in the HER1+ cell line, with little effect in either the HER1- cell line or in the absence of irradiation (FIG. 14). No effect on viability was apparent using a version of the antibody conjugate that releases only biologically inactive phenol, indicating that the observed cytotoxicity is solely a consequence of drug release.

Example 5

In Vivo Characterization of CY-Pan-CA4

The in vivo properties of CY-Pan-CA4 were examined. Specifically, conjugate stability, tumor localization, and modulation of fluorescence signal by external irradiation were evaluated. Fluorescence signal modulation was used to provide an initial, albeit surrogate, measure for drug release.

To examine the biodistribution of the CY-Pan-CA4 conjugate, a xenograft tumor model with dorsal A431 (HER1+) tumors was used. All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the National Cancer Institute/NIH Animal Care and Use

Committee. Six-week-old to 8-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick). During treatment, mice were anesthetized with isoflurane. A431 cells (2 x 10⁶) were injected subcutaneously in the right dorsum of each mouse. Experiments were performed at 8-9 days after cell injection. Tumors reaching approximately 5-7 mm were selected for the study.

In order to evaluate the biodistribution and tumor accumulation of CY-Pan-CA4, serial ventral and dorsal fluorescence images of mice were obtained before and 0, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 24, 48, 72, 96, 120, 144, and 168 h after i.v. injection of CY-Pan-CA4 via the tail vein with a Pearl Imager (LI-COR Biosciences, Lincoln, NE) using an 800 nm fluorescence channel

(FIGS. 15A and 15B, ventral side and dorsal side, respectively). Regions of interest (ROIs) were placed on the fluorescent images with a white light reference to measure fluorescence intensities of the tumor, the liver, and the left dorsum (i.e. background tissue on the opposite side of the tumor) (FIG. 16A). Pearl Cam Software (LI-COR Biosciences) was used for calculating the average fluorescence intensity within each ROI. Tumor-to-background ratio (TBR, FIG. 16B) was calculated using the following formula (n = 5):

mean tumor intensity - mean background intensity

mean non-tumor intensity - mean background intensity

Significant selective tumor accumulation was observed, with high tumor-to-background ratios between 1-7 days. Tumor accumulation was maximal between 2-3 days (FIGS. 15A-15B, 16A-16B).

To evaluate the effect of irradiation on the fluorescent signal, mice were implanted with two tumors, one in each side of the dorsum, administered CY-Pan-CA4, and, after 2 days, one of the tumors was irradiated with increasing light doses from a 690 nm laser. CY-Pan-CA4 (100 μg) was administered intravenously via the tail vein. Two days post-injection, the right dorsum tumor was exposed to 0, 10, 30, 50, and 100 J/cm² doses of 690 nm light (500 mW/cm²) using a laser system (BWF5-690-8-600-0.37; B&W Tek Inc., Newark, DE). The left dorsum tumor was covered with aluminum foil. Immediately after exposing tumors to the indicated dose, serial dorsal fluorescence images of mice were obtained with a Pearl Imager (LI-COR Biosciences) using an 800 nm fluorescence channel. Regions of interest (ROIs) were placed on the fluorescent images with a white light reference to measure fluorescence intensities of both tumors and normal dorsum adjacent to the tumors (i.e. background tissue on the opposite side of the tumor). Pearl Cam Software (LI-COR Biosciences) was used for calculating the average fluorescence intensity within each ROI. TBR was calculated as above (n = 6).

The fluorescence signal of only the irradiated tumor decreased in a light-dependent manner (FIG. 17). Notably, the light doses applied were similar to or compared favorably to those used for conventional photodynamic therapy-type applications.

Example 6

Treatment of Solid Tumors with the Disclosed Conjugates A subject having a solid tumor is identified and selected for treatment. The subject may be selected based on a clinical presentation and/or by performing tests to demonstrate presence of a solid tumor.

The subject is treated by administering a conjugate according to Formula I or Formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof at a dose determined by a clinician to be therapeutically effective. The conjugate is administered by any suitable means, such as parenteral, intravenous, or subcutaneous injection. In some instances, the conjugate is injected directly into the tumor. In some examples, the location of the conjugate is monitored by exposure to light having a wavelength suitable for inducing fluorescence of the cyanine fluorophore, thereby exciting the cyanine fluorophore, and detecting fluorescence of the conjugate. Monitoring may be performed after a period of time sufficient to allow binding of the conjugate to the tumor.

The administered conjugate subsequently is irradiated by targeted application of an effective quantity of light having a wavelength in the near-infrared range and a selected intensity to a targeted portion of the subject, thereby releasing the drug from at least some molecules of the conjugate. Advantageously, the targeted portion of the subject is proximate the tumor. Irradiation may be performed after a period of time sufficient to allow binding of the conjugate to the tumor. For example, irradiation may be performed several hours to several days after administration of the conjugate, such as from 1-7 days after administration of the conjugate. In some instances, drug release is assessed by monitoring a decrease in fluorescence emission of the conjugate in vivo.

In some instances, at least a portion of the tumor is surgically excised prior to targeted application of near-infrared light with subsequent release of the drug from at least some molecules of the conjugate. Fluorescence-guided surgery is used to determine the location and extent of tissue excision.

A therapeutically effective amount of a second agent may be co-administered with the conjugate or salt thereof. The conjugate (or salt thereof) and the second agent may be administered either separately or together in a single composition. The second agent may be administered by the same route or a different route. If administered concurrently, the conjugate (or salt thereof) and the second agent may be combined in a single pharmaceutical composition or may be administered concurrently as two pharmaceutical compositions. The second agent may be, for example, an antitumor agent or an angiogenesis inhibitor. Representative embodiments are described in the numbered paragraphs below.

1. A conjugate having a chemical structure according to Formula I or Formula II, or a pharmaceuticall acceptable salt thereof:

wherein m isl, 2, 3, 4, or 5; n is 1, 2 or 3; one of R¹ and R⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R⁴ is -(CH2)_X-L2-R^a, where x is an integer > 1, L₂ is a linker moiety or is absent, and R^a is 0)N(H)R^b, -N(H)C(0)R^b,

-N(H)R^b, or -SR^b where R^b is a targeting agent

; R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl; R³ is -Li-C(0)-X-drug, where Li is a linker moiety or is absent and X is O, N(H), or N(CH₃); R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate; R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate; each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d

independently is H or alkyl; and each ring A independently is a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring.

2. The conjugate according to paragraph 1, wherein each Y is the same and R⁵-R⁹ are identical to R¹⁰-R¹⁴, respectively.

3. The conjugate according to paragraph 1 or paragraph 2, wherein R⁶-R⁹ and Rⁿ-R¹⁴ are H.

4 The conjugate according to any one of paragraphs 1-3, wherein R³ is

, where X is O, N(H), or N(CH₃), and R¹⁵-R²² independently are H, alkyl, -N0₂, -NR^e2, -NR^e3, alkoxy, or sulfonate, wherein each R^e independently is H, halo, or alkyl.

5. The conjugate according to paragraph 4, wherein R¹⁵-R¹⁹ are H.

e according to any one of paragraphs 1-5, wherein one of R¹ and R⁴

, or where q and r independently are 1, 2, 3, 4, or 5.

7. The conjugate according to any one of paragraphs 1-6, wherein each Y is C(CH3)2.

8. The conjugate according to any one of paragraphs 1-6, wherein each Y is S. 9. The conjugate according to any one of paragraphs 1-8, wherein R^a is -C(0)N(H)R^b or -N(H)C(0)R^b and R^b is an antibody.

10. The conjugate according to any one of paragraphs 1-8, wherein R⁵ and R¹⁰ are

11. The conjugate according to any one of paragraphs 1-10, wherein R³ is ⁰

e according to any one of paragraphs 1-11, wherein one of R¹ and R⁴ is

where q and r independently are 1, 2, 3, 4, or 5.

13. The conjugate according to any one of paragraphs 1-12, wherein R¹ is lower alkyl and R⁴ is -(CH₂)_X-L₂-R^a wherein R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is a targeting agent.

14. The conjugate according to any one of paragraphs 1-12, R⁴ is lower alkyl and R¹ is -(CH₂)_X-L₂-R^a wherein R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is a targeting agent.

15. The conjugate according to any one of paragraphs 1-14, wherein the conjugate has a chemical structure according to Formula II and ring A is a fused heteroaryl ring including one nitrogen atom.

16. The conjugate according to any one of paragraphs 1-14, wherein the conjugate has a chemical structure according to Formula II and ring A is a fused phenyl ring.

17. The conjugate according to any one of paragraphs 1-16, wherein the conjugate has a chemical structure according to Formula II and ring A is substituted with optionally substituted sulfonate.

18. The conjugate according to any one of paragraphs 1-17, wherein the drug is an anticancer drug.

19. The conjugate according to paragraph 18, wherein the drug is an anti-breast cancer drug. The conjugate according to any one of paragraphs 1-17, wherein -X-Drug

21. The conjugate according to any one of paragraphs 1-17, wherein R^b is panitumumab and the R³ is -C(0)-0-combretastatin A4.

22. A pharmaceutical composition comprising a conjugate according to any one of paragraphs 1-21 wherein R^b is a targeting agent and a pharmaceutically acceptable carrier.

23. A precursor compound having a chemical structure according to Formula III or IV, or a salt thereof:

wherein m is 1, 2, 3, 4, or 5; n is 1, 2 or 3; one of R¹ and R²⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R²⁴ is -(CH₂)_U-C≡CH where u is 1, 2, 3, 4, or 5; R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl; R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate; R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate; each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d independently is H or alkyl; each ring A independently is a 6- membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring; and R²³ is a protecting group.

24. The precursor compound of paragraph 23, wherein the protecting group is i<?ri-butyloxycarbonyl (BOC) or 9-fluorenylmethyloxycarbonyl (FMOC).

25. The precursor compound of paragraph 23 or paragraph 24, wherein R¹ is lower alkyl and R²⁴ is -(CH₂)_U-C≡CH.

26. The precursor compound of paragraph 23 or paragraph 24, wherein R¹ is

-(CH₂)u-C≡CH and R²⁴ is lower alkyl.

27. A method, comprising: providing a conjugate according to any one of paragraphs 1- 21, wherein R^b is a targeting agent and wherein if Y is C(R^d)2, at least one R^d is other than H; and subsequently irradiating the conjugate with targeted application of an effective quantity of light having a selected wavelength in the near-infrared range and a selected intensity to induce a cleavage reaction and release the drug from the conjugate.

28. The method of paragraph 27, wherein irradiating the conjugate with targeted application of light comprises irradiating the conjugate with a laser that produces light having a wavelength of 680-700 nm.

29. The method of paragraph 27 or paragraph 28, further comprising: monitoring a level of fluorescence of the conjugate; and ceasing irradiation when the level of fluorescence falls below a target level.

30. The method of any one of paragraphs 27-29, further comprising: providing a biological sample including, or suspected of including, a target molecule; contacting the biological sample with the conjugate, wherein the targeting agent of the conjugate is capable of recognizing and binding to the target molecule; and subsequently irradiating the biological sample with the targeted application of light.

31. The method of any one of paragraphs 27-29, further comprising: identifying a subject as having a condition that may be treated with the drug; administering a therapeutically effective amount of the conjugate or a pharmaceutical composition comprising the conjugate to the subject; and subsequently irradiating the conjugate by targeted application of an effective quantity of light having a wavelength in the near-infrared range and a selected intensity to a targeted portion of the subject, thereby releasing the drug from at least some molecules of the conjugate.

32. The method of paragraph 31, wherein the subject has a tumor and the targeted portion of the subject includes an area proximate a location of the tumor. 33. The method of paragraph 31 or paragraph 32, wherein the effective quantity of light applied to the targeted portion is from 10-250 J/cm².

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

What is claimed is:

1. A conjugate having a chemical structure according to Formula I or Formula II, or a pharmaceuticall acceptable salt thereof:

wherein m isl, 2, 3, 4, or 5;

n is 1, 2 or 3;

one of R¹ and R⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of ¹ and R⁴ is -(CH2)_X-L2-R^a, where x is an integer > 1, L2 is a linker moiety or is

absent, and R^a is (0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b where R^b is a

targeting agent,

R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl;

R³ is -Li-C(0)-X-drug, where Li is a linker moiety or is absent and X is O, N(H), or N(CH₃);

R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate;

R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate;

each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d independently is H or alkyl; and

each ring A independently is a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring.

2. The conjugate according to claim 1, wherein each Y is the same and R⁵-R⁹ are identical to R¹⁰-R¹⁴, respectively..

3. The conjugate according to claim 1 or claim 2, wherein R⁶-R⁹ and Rⁿ-R¹⁴ are H. 4 The conjugate according to any one of claims 1-3, wherein R^J is

, where X is O, N(H), or N(CH₃), and R¹⁵-R²² independently are H, alkyl, -N0₂, -NR^E2, -NR^E3, alkoxy, or sulfonate, wherein each R^e independently is H, halo, or alkyl.

5. The conjugate according to claim 4, wherein R¹⁵-R¹⁹ are H.

6. The conjugate according to any one of claims 1-5, wherein one of R¹ and R⁴ is

, or ' where q and r independently are 1, 2, 3, 4, or 5.

7. The conjugate according to any one of claims 1-6, wherein each Y is C(C]¾)2.

8. The conjugate according to any one of claims 1-6, wherein each Y is S.

9. The conjugate according to any one of claims 1-8, wherein R^a is -C(0)N(H)R^b or -N(H)C(0)R^b and R^b is an antibody.

10. The conjugate according to any one of claims 1-8, wherein R⁵ and R¹⁰ are -(CH₂)₄S0₃-.

11. The conjugate according to any one of claims 1-10, wherein R³ is

'Drug

O e according to any one of claims 1-11, wherein one of R¹ and R⁴ is

where q and r independently are 1, 2, 3, 4, or 5.

13. The conjugate according to any one of claims 1-12, wherein R¹ is lower alkyl and R⁴ is -(CH₂)_X-L₂-R^a wherein R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is a targeting agent.

14. The conjugate according to any one of claims 1-12, R⁴ is lower alkyl and R¹ is -(CH₂)_X-L₂-R^a wherein R^a is -C(0)N(H)R^b, -N(H)C(0)R^b, -N(H)R^b, or -SR^b and R^b is a targeting agent.

15. The conjugate according to any one of claims 1-14, wherein the conjugate has a chemical structure according to Formula II and ring A is a fused heteroaryl ring including one nitrogen atom.

16. The conjugate according to any one of claims 1-14, wherein the conjugate has a chemical structure according to Formula II and ring A is a fused phenyl ring.

17. The conjugate according to any one of claims 1-16, wherein the conjugate has a chemical structure according to Formula II and ring A is substituted with optionally substituted sulfonate.

18. The conjugate according to any one of claims 1-17, wherein the drug is an anticancer drug.

19. The conjugate according to claim 18, wherein the drug is an anti-breast cancer drug The conjugate according to any one of claims 1-17, wherein -X-Drug

21. The conjugate according to any one of claims 1-17, wherein R^b is panitumumab and the R³ is -C(0)-0-combretastatin A4.

22. A pharmaceutical composition comprising a conjugate according to any one of claims 1-21 wherein R^b is a targeting agent and a pharmaceutically acceptable carrier.

23. A precursor compound having a chemical structure according to Formula III or IV, or a salt thereof:

wherein m is 1, 2, 3, 4, or 5;

n is 1, 2 or 3;

one of R¹ and R²⁴ is alkyl, cycloalkyl, alkoxy, -(O)C-R, or -(O)C-O-R, wherein R is alkyl, and the other of R¹ and R²⁴ is -(CH₂)_U-C≡CH where u is 1, 2, 3, 4, or 5;

R² is C(R^C)2 wherein each R^c independently is H, halo, alkyl, or aryl, or (R²)_m collectively is phenyl;

R⁵ and R¹⁰ independently are H, alkyl, alkoxy, or alkyl sulfonate;

R⁶-R⁹ and Rⁿ-R¹⁴ independently are H, alkyl, amino, alkoxy, or alkyl sulfonate;

each Y independently is C(R^d)2, S, O, Se, or N(R^d) wherein each R^d independently is H or alkyl;

each ring A independently is a 6-membered fused aliphatic, heteroaliphatic, aryl, or heteroaryl ring; and

R²³ is a protecting group.

24. The precursor compound of claim 23, wherein the protecting group is

i<?ri-butyloxycarbonyl (BOC) or 9-fluorenylmethyloxycarbonyl (FMOC).

25. The precursor compound of claim 23 or claim 24, wherein R¹ is lower alkyl and R²⁴

26. The precursor compound of claim 23 or claim 24, wherein R¹ is -(CH2)_U-C≡CH and R²⁴ is lower alkyl. 27. A method, comprising:

providing a conjugate according to any one of claims 1-21, wherein R^b is a targeting agent and wherein if Y is C(R^d)2, at least one R^d is other than H; and

subsequently irradiating the conjugate with targeted application of an effective quantity of light having a selected wavelength in the near-infrared range and a selected intensity to induce a cleavage reaction and release the drug from the conjugate.

28. The method of claim 27, wherein irradiating the conjugate with targeted application of light comprises irradiating the conjugate with a laser that produces light having a wavelength of 680-700 nm.

29. The method of claim 27 or claim 28, further comprising:

monitoring a level of fluorescence of the conjugate; and

ceasing irradiation when the level of fluorescence falls below a target level. 30. The method of any one of claims 27-29, further comprising:

providing a biological sample including, or suspected of including, a target molecule; contacting the biological sample with the conjugate, wherein the targeting agent of the conjugate is capable of recognizing and binding to the target molecule; and

subsequently irradiating the biological sample with the targeted application of light.

31. The method of any one of claims 27-29, further comprising:

identifying a subject as having a condition that may be treated with the drug;

administering a therapeutically effective amount of the conjugate or a pharmaceutical composition comprising the conjugate to the subject; and

subsequently irradiating the conjugate by targeted application of an effective quantity of light having a wavelength in the near-infrared range and a selected intensity to a targeted portion of the subject, thereby releasing the drug from at least some molecules of the conjugate.

32. The method of claim 31, wherein the subject has a tumor and the targeted portion of the subject includes an area proximate a location of the tumor.

33. The method of claim 31 or claim 32, wherein the effective quantity of light applied to the targeted portion is from 10-250 J/cm².