Hydrogen peroxide responsive theranostics

Abstract

Described herein are hydrogen peroxide responsive theranostics for cancer-selective activation of DNA alkylators and real-time fluorescence monitoring in living cells.

Description

[0001] HYDROGEN PEROXIDE RESPONSIVE THERANOSTICS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 627,441 , filed on January 31 , 2024, which is incorporated by reference herein in its entirety.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number CA277656-01 awarded by the National Cancer Institute. The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831 and PCT Rule 13fer. The Sequence Listing XML file submitted in the USPTO Patent Center, “020871-0017-W001_sequenceJisting_.xmL14-JAN-2025.xml,” was created on January 14, 2025, contains 2 sequences, has a file size of 4.0 kilobytes (4,096 bytes), and is incorporated by reference in its entirety into the specification.

[0008] BACKGROUND

[0009] Cancer is still an ongoing major health concern worldwide as the current first or second leading cause of premature death in most developed countries. Although recent advancements in chemotherapeutics and early diagnosis have generally reduced cancer mortality rate, there are still unmet needs to improve the treatment efficacy, reduce undesirable side effects, and enable simultaneous diagnosis of cancer. The theranostic strategies combine the modalities of both cancer diagnosis and therapeutics into one, which concurrently monitor and treat cancer. Theranostic agents have the potential for simultaneous diagnosis of early-stage cancer, selective delivery of therapeutic drug to tumors, monitoring drug activity in real-time within biological setting, and minimizing the unwanted side effects, which will allow the development of effective and personalized medicine with improved therapeutic indexes for cancer patients. However, there are many challenges to rationally design targeted theranostics. Continued research into advancing theranostic approaches for targeting tumor cells is of importance to cancer treatment.

[0010] The unique characteristics of cancer cells offer promising strategies for targeting cancer. It has been well-established that cancer cells are under higher degree of basal level oxidative stress than normal cells, reflected by increased presence of reactive oxygen species (ROS), such as H2O2, superoxide free radicals, etc. Deregulated ROS generation has been attributed to numerous endogenous factors, such as mitochondrial dysfunction caused by mutations of the mitochondrial DNA, malfunctioning antioxidant system, etc. As a result, cancer cells proliferate and operate with higher H2O2concentrations relative to normal, healthy cells. This intrinsic hallmark of cancer cells present opportunities for developing tumor-selective diagnostic and therapeutic strategies. Since boron moieties, such as boronic acids and esters, comprise of an electron-deficient trivalent boron element with an empty p-orbital, they are susceptible to nucleophilic attack, specifically by H2O2. This deboronation event induces a change from an electron withdrawing boron group to an electron donating hydroxyl group, which restores the reactivity of the masked functional groups. Taking advantage of the chemoselective reaction between boronic acids and their esters with H2O2, the first boron-based H2O2-responsive profluorescent probe was developed for detecting H2O2. This work was extended to cellular systems. Thereafter, a plethora of studies have identified a wide variety of H2O2-activated fluorescent probes and numerous non-toxic prodrugs capable of H2O2-induced activation to release fluorescent reporters or cytotoxic species specifically in oxidatively stressed cancer cells. Of the numerous fluorescent scaffolds, coumarins and fluoresceins are widely used in biological systems. The boronate ester analogues of coumarin and fluorescein have been developed as H2O2-responsive fluorogenic probes for detecting cellular H2O2.

[0011] What are needed are hydrogen peroxide responsive theranostics for cancer-selective activation of DNA alkylators and real-time fluorescence monitoring in living cells.

[0012] SUMMARY

[0013] In one embodiment, described herein is a theranostic compound or pharmaceutically acceptable salt thereof, comprising: a compound of formula (I), wherein:

[0014] X is halo;

[0015] R1, at each occurrence, is independently a linker; and

[0016] R2, at each occurrence, is independently a boronic acid, a boronic ester, a boronate, a boronamine, hydrogen, or C1-6alkyl; and a fluorophore. in one aspect, the compound of claim 1 , wherein the linker has a formula (II): wherein:

[0017] R3is independently hydrogen or C1-3alkyl;

[0018] L1is C1-15alkylene;

[0019] L2is C1-10alkylene or -(CH2)0-5-Y--(CH2)0-5--;

[0020] Y is C(O), N(RX), or C(O)-N(RX);

[0021] Rxis hydrogen or C1-3alkyl; and

[0022] G1is a 6- to 12-membered aryl, C3-10carbocyclyl, or a 4- to 12-membered heterocyclyl, optionally substituted with halogen, cyano, C1-6alkyl, C1-6haloalkyl, -OR1a, -SR13, CO2R1a, -C(O)R1a, -SO2R1a, -N(R1a)2, -CO2N(R1a)2, or -NO2;

[0023] R1a, at each occurrence, are each independently hydrogen or C1-3alkyl; m = 0—1 ; and n = 0-1 .

[0024] In another aspect, the compound of claim 1 , wherein the linker has a formula (III): wherein

[0025] L3is C1-6alkylene, C(O), -O-C(O)-O-, -N(RX)-, or-O-; and w = 0-1 .

[0026] In another aspect, the compound of claim 1 , wherein the fluorophore is fluorescein, rhodamine, cyanine, coumarin, BODIPY, napthalamide, xanthenone, (benzopyranylidene) malononitrile, a derivative thereof, or a pharmaceutically acceptable salt thereof in another aspect, the compound of claim 1 , wherein X is chloro, in another aspect, the compound of claim 1 , wherein the compound has a formula l-a: in another aspect, the compound of claim 1, wherein the theranostic has a formula l-b:

[0027] wherein Q is a fluorophore.

[0028] In another aspect, R3is hydrogen. In another aspect, the compound of claim 7, wherein m ~ 0 and n = 0. In another aspect, Q is coumarin, fluorescein, a derivative thereof, or a salt thereof. In another aspect, the theranostic compound or a pharmaceutically acceptable salt thereof, is selected from a group consisting of:

[0029]

[0030] In another aspect, the theranostic compound or a pharmaceutically acceptable salt thereof, is selected from a group consisting of:

[0031] In another aspect, the compound has a formula of l-c: wherein Q is a fluorophore.

[0032] In another aspect, R1is hydrogen or C1-6alkyl. In another aspect, w= 1 . In another aspect, L3is - O--C(O)-O-. In another aspect, the fluorophore is a is coumarin, fluorescein, a derivative thereof, or a pharmaceutically acceptable salt thereof. In another aspect, the theranostic, or pharmaceutically acceptable salt thereof, is selected from a group consisting of:

[0033] Another embodiment described herein is a method for treating and diagnosing a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of a theranostic compound, or pharmaceutically acceptable salt thereof. In one aspect, the disease or disorder is associated with oxidative stress. In another aspect, the disease or disorder is cancer. In another aspect, the cancer is selected from breast cancer, brain cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, leukemia, or other cancers. Another embodiment described herein is the use of a theranostic compound, or a sait thereof, for the preparation of a medicament for the treatment of a disease or disorder associated with oxidative stress in a subject.

[0034] DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] FIG. 1 shows a phosphorimager radiogram of denaturing PAGE analysis from DNA-ICL reaction of a 49-mer DNA duplex 28 in the presence of 1a, 1c or 2 (1 mM) with and without H2O2. The ICL reaction was performed in a mixture of DMSO and pH 8 phosphate buffer with 3:7 ratio by incubation at RT for 16 h.

[0037] FIG. 2 shows a comparison of DNA alkylation induced by 1a, 1c, or 2. Phosphorimager images of 20% denaturing PAGE analysis of DNA duplex 28 treated under different conditions: lanes 1- 3, duplex 28 was incubated with 1a, 1c, or 2 at RT for 16 h in the presence of H2O2(without heating in 1.0 M piperidine); lane 4, control sample with 28 incubated in phosphate buffer only (without H2O2) followed by heating in 1.0 M piperidine at 90 °C for 30 min; lane 5, control sample with 28 incubated with H2O2(6.0 mM) at RT for 16 h followed by heating in 1 .0 M piperidine at 90 °C for 30 min; lanes 6-8, duplex 28 was incubated with 1a, 1c, or 2 at RT for 16 h in the absence of H2O2followed by heating in 1.0 M piperidine at 90 °C for 30 min; lanes 9-11 : duplex 28 was incubated with 1a, 1c, or 2 at RT for 16 h in the presence of H2O2followed by heating in 1.0 M piperidine at 90 °C for 30 min; lane 12: G+A sequencing.

[0038] FIG. 3A-C show dose-dependent apoptosis of MDA-MB-468 cancer cells or MCF-10A human mammary epithelial cells with 1a, 1c, and 2. FIG. 3A shows MDA-MB-468 cancer cells with 1a, 1c, and 2 in the absence or presence of a safe dose of PQ (6.0 μM) after 48 h incubation at 37 °C (n = 4). FIG. 3B shows MCF-10A cells with 1a, 1c, 2 after 48 h incubation at 37 °C (n = 4). FIG. 3C shows MDA-MB-468 cancer cells with paraquat (PQ) after 48 h incubation at 37 °C (n = 2).

[0039] FIG. 4A-B show H2O2levels and a standard curve. FIG. 4A shows the H2O2levels produced in MDA-MB-468 cells and MCF-10A cells as determined using the Amplex Red Assay (Invitrogen, A22188). The data are presented as the mean ± SD from three independent experiments (n = 3). The samples were combined with an equal amount of Amplex Red reagent before the assay, resulting in a two-fold adjustment to the final H2O2concentration. FIG. 4B shows a H2O2standard curve. Data are represented as mean ± SD from three independent experiments (n = 3).

[0040] FIG. 5A-E show comparisons of cellular DNA damage in MDA-MB-468 cells and MCF- 10A cells treated with 1a and 1c. FIG. 5A shews an alkaline comet assay of MDA-MB-468 cells and MCF10A ceils treated with 1a and 1c for 48 h at 37 °C. The OxiSelect Comet Assay Kit (abeam: ab238544) was employed for the analysis of DNA damage. The comet images were captured using an EVOS Digital Inverted Microscope at 10* magnification. FIG. 5B--E show quantification in head DNA (FIG. 5B), tail DNA (FIG. 5C), tail moment (FIG. 5D). and tail olive moment (FIG. 5E) between MDA-MB-468 cells and MCF-10A cells treated with 1a or 1c using Tritek CometScore Software to analyze the images obtained from the alkaline comet assay. Each data point is the result of three independent replicate experiments (n = 3), and the data are expressed as the mean ± SD.

[0041] FIG. 6A-B show fluorescence and UV spectroscopy of 1a. 1c and 2 (2 μM) in the presence or absence or of H2O2. FIG. 6A shows the fluorescence spectra of 1a, 1c and 2 (2 μM) in the presence or absence of H2O2measured in PBS: DMSO (10:1). FIG. 6B show the UV / VIS absorbance spectra of 2 (2 μM) with or without H2O2. The activation of 1c and 2 (2 μM) with 10 mM H2O2was carried out in PBS: DMSO (10: 1) by incubation at RT for 1 h with agitation prior to analysis.

[0042] FIG. 7A-B show fluorescence responses of theranostic 1c and 2 (2 μM, 10% DMSO, 90% pH 7.4 PBS buffer) towards H2O2. FIG. 7A shows a time-course kinetic measurement of fluorescence response of 1c and 2 under pseudo-first order conditions with 1 mM H2O2measured at RT (n = 2) (FIG. S23-S24). FIG. 7B shows the change in fluorescence intensity at 400-570 nm for 1c and 505-620 nm for 2 with increasing concentrations of H2O2(0-10 mM) (1c and 2 were incubated with H2O2at RT for 1 h (n = 2).

[0043] FIG. 8. Fluorescence responses of theranostic 1c (a) and 2 (b) (2 μM, 10% DMSO, 90% PBS buffer) in various pH conditions with and without H2O2(100 μM) [1c and 2 were incubated at RT for 8 h (n = 2) (FIG. S27-S28)].

[0044] FIG. 9 shows the conversion of the neutral form 2" to a di-anionic form 2" under basic medium.

[0045] FIG. 10 shows the fluorescence responses of 1c and 2 (2 μM) towards various ROS (100 μM) after incubation at RT for 12 h (n = 2) (the fluorescence intensity was determined in a mixture of 10% DMSO and 90% PBS buffer pH 7.4).

[0046] FIG. 11A-B show the detection of 1a, 1c, or 2-alkylated DNA via fluorescence measurement. FIG. 11A shows a 49-mer DNA duplex 28 (SEQ ID NO: 1-2) (2 μM) incubated with 1a, 1c, or 2 (1 mM) in the presence or absence of H2O2or tracker dyes (controls) SYTO 9, MitoRed CMXRos, Hoechst 33342 (1 mM) at RT for 24 h (n = 2) (The non-covalently bound compounds were removed by precipitation and size-exclusion chromatography). FIG. 11 B shows cellular DNA isolated from MDA-MB-468 after incubation with 50 μM of either theranostic (1a, 1c, 2) or tracker dye (SYTO 9, MitoTracker Red CMXRos, Hoechst 33342) for 20 h at 37 °C (n = 3).

[0047] FIG. 12 shows confocal images of MDA-MB-468 ceils treated with 1a, 1c, or 2 (5 μM) and co-labeled with MitoTracker Red CMXRos (200 nM) in the presence or absence of H2O2(100 μM) or pre-treatment of PQ (500 μM). Cells were incubated with the theranostics for 0.5 h followed by addition of H2O2and incubated for another 1 .5 h, or cells were pre-treated with 500 μM PQ for 10 h at 37 °C followed by treatment of theranostics and incubation for another 1.5 h.

[0048] FIG. 13 shows confocal images of MDA-MB-468 cells treated with 1a, 1c, or 2 (5 μM) and co-labeled with SYTO 9 (500 nM) in the presence or absence of H2O2(100 μM) or pre-treatment of PQ (500 μM). Cells were incubated with the theranostics at 37 °C for 0.5 h followed by addition of 100 μM H2O2and incubation for another 1.5 h, or cells were pre-treated with 500 μM PQ for 10 h at 37 °C followed by treatment of theranostics and incubation for another 1 .5 h.

[0049] FIG. 14A-H show in vivo efficacy and selectivity. FIG. 14A shows changes of mice body weight after a 5-day treatment with 1c at doses of 5, 10, or 20 mg / kg. FIG. 14B-H show in vivo efficacy evaluation of 1c in xenograft athymic nude. Mice were administered IP with vehicle or 1c at a dose of 5 mg / kg for 8-weeks, 5-day per week. Data are expressed as an average ± SD (n = 3 for variable group and n = 2 for vehicle group. FIG. 14B shows time-dependent body weight changes; FIG. 14C shows time-dependent tumor growth measured by caliper. FIG. 14D shows tumor weight of mice after treatment with vehicle or 1c. FIG. 14E shows the appearance of mice organs after treatment. FIG. 14F shows tumors dissected from mice after treatment. FIG. 14G shows the average weight of xenograft nude mice organs. The significance was determined by two-way ANOVA; n = 3 for treated group and n = 2 for control group, n.s. P > 0.05, (**) P < 0.01 , and (***) p < 0.001 vs. control group. FIG. 14H shows H&E staining of liver and kidney of 1c or vehicle-treated mice. Liver: arrow (→ ) as the central vein; Kidney: arrow (→ ) as the glomerulus and arrowhead (») as the renal tubules).

[0050] FIG. 15 shows the fluorescence spectra of blood plasma in methanol obtained after IP administration of 1 c or vehicle and blood obtained at specified time points (n = 1 , slit width λexand λem= 12 nm). DETAILED DESCRIPTION

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0052] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0053] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “camprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0054] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0055] As used herein, the term “or” can be conjunctive or disjunctive.

[0056] As used herein, the term “and / or” refers to both the conjuctive and disjunctive.

[0057] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0058] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term "about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0059] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0060] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15- 30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C + 10%; about 20 °C ± 10%; about 22 °C + 10%; about 25 °C ± 10%; about 27 °C + 10%; ~20 °C, -22 °C, ~25 °C, or -27 °C, at standard atmospheric pressure.

[0061] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0062] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0063] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein. As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art

[0064] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0065] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate, in one embodiment, the subject is a human.

[0066] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0067] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0068] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction cf the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0069] Definitions of specific functional groups and chemical terms are described in more detail herein. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5thed, John Wiley & Sons, Inc., New York, 2001 ; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modem Methods of Organic Synthesis, 3rded, Cambridge University Press, Cambridge, 1987.

[0070] As used herein, the term “alkyl” refers to a straight or branched hydrocarbon having from 1 to 12 (e.g., C1--C12) carbon atoms and includes, for example, methyl, ethyl, n-propyl. isopropyl, n-butyl, sec-butyl, isobutyi, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, and the like.

[0071] As used herein, the term “aikyene” refers to a -CH2.

[0072] As used herein, the term “alkenyl” refers to straight and branched hydrocarbon having from 2 to 12 carbon atoms (e.g., C2-C12) and at least one double bond and includes, but is not limited to, ethenyl, 3-buten-1-yl, 2-ethenylbutyl, 3-hexen-1-yl, and the like. The term “alkenyl” includes cycloalkenyl, and heteroaikenyi in which 1 to 3 heteroatoms selected from O, S, N, or substituted nitrogen may replace carbon atoms.

[0073] As used herein, the term “alkynyl” refers to straight and branched hydrocarbon having from 2 to 12 carbon atoms (e.g., C2-C12) and at least one triple bond and includes, but is not limited to, ethynyl, 3-butyn-1-yl, propynyi, 2-butyn-1-yl, 3-pentyn-1-yl, and the like.

[0074] As used herein, the term “cycioalkyl” refers to a monocyclic or polycyclic hydrocarbyl group having from 3 to 8 carbon atoms (e.g., C3-C8), for instance, cyclopropyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclobutyl, adamantyl, norpinanyl. decalinyl, norbornyl, cyclohexyl, and cyclopentyl. Such groups can be substituted with groups such as hydroxy, keto, amino, alkyl , and dialkylamino, and the like. Also included are rings in which 1 to 3 heteroatoms replace carbons. Such groups are termed “heterocyclyl,” which means a cycioalkyl group also bearing at least one heteroatom selected from O, S, N. or substituted nitrogen. Examples of such groups include, but are not limited to, oxiranyl, pyrrolidinyl, piperidyl, tetrahydropyran, and morpholine.

[0075] As used herein, the term “alkoxy” refers to a straight or branched chain alkyl groups having 1—10 carbon atoms (e.g., C2-C10) and linked through oxygen. Examples of such groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyloxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy. In addition, alkoxy refers to polyethers such as -O- (CH2)2-O-CH3, and the like.

[0076] The alkyl, alkenyl, alkoxy, and alkynyl groups described herein are optionally substituted (i.e. , may be substituted, but are not necessarily substituted), preferably by 1 to 3 groups selected from NR4R5, phenyl, substituted phenyl, thio C1-C6alkyl, C1-C6alkoxy, hydroxy, carboxy, C1-C6alkoxycarbonyl, halo, nitrile, cycloalkyl, and a 5- or 6-membered carbocyclic ring or heterocyclic ring having 1 or 2 heteroatoms selected from nitrogen, substituted nitrogen, oxygen, and sulfur. “Substituted nitrogen” means nitrogen bearing C1-C6alkyl or (CH2)pPh where p is 1 , 2, or 3. Perhalo and polyhalo substitution is also included.

[0077] Examples of substituted alkyl groups include, but are not limited to, 2-aminoethyl, 2- hydroxyethyl, pentachloroethyl, trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, ethoxycarbonylmethyl, 3-phenylbutyl, methanylsulfanylmethyl, methoxymethyl, 3-hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl, 3-cyclopropylpropyi, pentafluoroethyl, 3-morpholinopropyl, piperazinylmethyl, and 2-(4-methylpiperazinyl)ethyl.

[0078] Examples of substituted alkynyl groups include, but are not limited to, 2-methoxyethynyl, 2-ethylsulfanylethynyl, 4-(1-piperazinyl)-3-(butynyl). 3-phenyl-5-hexynyl, 3-diethylamino-3- butynyl, 4-chloro-3-butynyl, 4-cyclobutyl-4-hexenyl, and the like.

[0079] Typical substituted alkoxy groups include aminomethoxy, trifluoromethoxy, 2- diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy, 6-carboxhexyloxy, and the like.

[0080] Further, examples of substituted alkyl, alkenyl, and alkynyl groups include, but are not limited to, dimethylaminomethyl, carboxymethyl, 4-dimethylamino-3-buten-1-yl, 5- ethylmethylamino-3-pentyn-1-yl, 4-morpholinobutyl, 4-tetrahydropyrinidylbutyl, 3-imidazolidin-1- ylpropyl, 4-tetrahydrothiazol-3-yl-butyl, phenylmethyl, 3-chlorophenylmethyl, and the like.

[0081] As used herein, the term “anion” means a negatively charged species such as chloride, bromide, trifluoroacetate, or triethylammonium. The term “cation” refers to a positively charged species, such as sodium, potassium, or ammonium.

[0082] As used herein, the term “acyl” refers to alkyl or aryl (Ar) group having from 1 -10 carbon atoms bonded through a carbonyl group, i.e., R-C(O)-. For example, acyl includes, but is not limited to, a C1-C6alkanoyl, including substituted alkanoyl, wherein the alkyl portion can be substituted by an amine, amide, carboxylic, or heterocyclic group. Typical acyl groups include acetyl, benzoyl, and the like.

[0083] As used herein, the term “aryl” refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system where at least one of the rings in the ring system is an aromatic hydrocarbon ring and any other aromatic rings in the ring system include only hydrocarbons. In some embodiments, a monocyclic aryl group can have from 6 to 14 carbon atoms and a polycyclic aryl group can have from 8 to 14 carbon atoms (e.g., C8-C14). The aryl group can be covalently attached to the defined chemical structure at any carbon atom(s) that result in a stable structure. In some embodiments, an aryl group can have only aromatic carbocyclic rings, e.g., phenyl, 1- naphthyl, 2-naphthyl, anthracenyl, phenanthrenyl groups, and the like. In other embodiments, an aryl group can be a polycyclic ring system in which at least one aromatic carbocyclic ring is fused (i.e., having a bond in common with) to one or more cycloalkyl orcycioheteroalkyl rings. Examples of such aryl groups include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5,6-bicyciic cycloalkyl / aromatic ring system), cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl / aromatic ring system), imidazoline (i.e., a benzimidazolinyl group, which is a 5,6-bicyclic cycloheteroalkyl / aromatic ring system), and pyran (i.e., a chromenyl group, which is a 6,6-bicyclic cycloheteroalkyl / aromatic ring system). Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like.

[0084] As used herein, the terms “halogen" or “halo” refer to fluorine, bromine, chlorine, or iodine. As used herein, the term “haloalkyl” refers to an alkyl group having one or more halogen substituents. In some embodiments, a haloalkyl group can have 1 to 10 carbon atoms (e.g., C1- C8). Examples of haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCI3, CHCI2, CH2CI, C2CI5, and the like. Perhaloalkyl groups, i.e., alkyl groups wherein all the hydrogen atoms are replaced with halogen atoms (e.g., CF3and C2F5), are included within the definition of “haloalkyl.” For example, a C1-10haloalkyl group can have the formula Jherein X is F, Cl, Br, or I, i is an integer in the range of 1 to 10, and j is an integer in the range of 0 to 21 , provided that j is less than or equal to 21+1 .

[0085] As used herein, the term “heteroaryl” refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from O, N, and S or a polycyclic ring system where at least one of the rings in the ring system is aromatic and contains at least one ring heteroatom. A heteroaryl group can have from 5 to 14 ring atoms (e.g., C5-C14), and contains 1~ 6 ring heteroatoms (e.g., N, O, S, P, or the like), in some embodiments, heteroaryl groups can include monocyclic heteroaryl rings fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, or non-aromatic cycloheteroalkyl rings. The heteroaryl group can be covalently attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O-O, S-S, or S-O bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S,S-dioxide). Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazoiyi, benzothiazolyl, benzisothiazoiyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, 1 / T-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyrdazinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl groups, and the like. Further examples of heteroaryl groups include 4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like.

[0086] As used herein, the term “lower alkenyl” refers to alkenyl groups which contains 2 to 6 carbon atoms (e.g., C2-C6). An alkenyl group is a hydrocarbyl group containing at least one carbon-carbon double bond. As defined herein, it may be unsubstituted or substituted with the substituents described herein. The carbon-carbon double bonds may be between any two carbon atoms of the alkenyl group. It is preferred that it contains 1 or 2 carbon-carbon double bonds and more preferably one carbon-carbon double bond. The alkenyl group may be straight chained or branched. Examples include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2- butenyl, 2-methyl- 1-propenyl, 1 ,3-butadienyl, and the like.

[0087] As used herein, the term “lower alkynyl” refers to an alkynyl group containing 2-6 carbon atoms (e.g., C2-C6). An alkynyl group is a hydrocarbyl group containing at least one carbon- carbon triple bond. The carbon-carbon triple bond may be between any two-carbon atom of the alkynyl group, in an embodiment, the alkynyl group contains 1 or 2 carbon-carbon triple bonds and more preferably one carbon-carbon triple bond. The alkynyl group may be straight chained or branched. Examples include but are not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl and the like.

[0088] As used herein, the term “carbalkoxy” refers to an alkoxycarbonyl group, where the attachment to the main chain is through the carbonyl group, e.g., -C(O)--. Examples include but are not limited to methoxy carbonyl, ethoxy carbonyl, and the like.

[0089] As used herein, the term “oxo” refers to a double-bonded oxygen (i.e., =O). It is also to be understood that the terminology C(O) refers to a -C=O group, whether it be ketone, aldehyde or acid or acid derivative. Similarly, S(O) refers to a -S-O group.

[0090] As used herein, the term “cycloalkyl” refers to a non-aromatic carbocyclic group including cyclized alkyl, alkenyl, and alkynyl groups. A cycloalkyl group can be monocyclic (e.g., cyclohexyl) or polycyclic (e.g., containing fused, bridged, and / or spiro ring systems), wherein the carbon atoms are located inside or outside of the ring system. A cycloalkyl group can have from 3 to 14 ring atoms (e.g., from 3 to 8 carbon atoms for a monocyclic cycloalkyl group and from 7 to 14 carbon atoms for a polycyclic cycloalkyl group). Any suitable ring position of the cycloalkyl group can be covalently linked to the defined chemical structure. Examples of cycloalkyl groups include cyclopropyl, cyclobutyi, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, noracryl, adamantyl, and spiro[4.5]decanyl groups, as well as their homologs, isomers, and the like.

[0091] As used herein, the term “heteroatom” refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, sulfur, phosphorus, and selenium.

[0092] As used herein, the term “cycloheteroalkyl” refers to a non-aromatic cycloalkyl group that contains at least one (e.g., one, two, three, four, or five) ring heteroatom selected from O, N, and S, and optionally contains one or more (e.g., one, two, or three) double or triple bonds. A cycloheteroalkyl group can have from 3 to 14 ring atoms and contains from 1 to 5 ring heteroatoms (e.g., from 3-6 ring atoms for a monocyclic cycloheteroalkyl group and from 7 to 14 ring atoms for a polycyclic cycloheteroalkyl group). The cycloheteroalkyl group can be covalently attached to the defined chemical structure at any heteroatcm(s) or carbon atom(s) that results in a suitable structure. One or more N or S atoms in a cycloheteroalkyl ring may be oxidized (e.g., morpholine Af-oxide, thiomorpholine S-oxide, thiomorpholine S,S-dioxide). Cycloheteroalkyl groups can also contain one or more oxo groups, such as phthalimidyl, piperidinyl, oxazolidinoxyl, dioxo-pyrimidinyl, pyridin-2(1H)-onyl, and the like. Examples of cycloheteroalkyl groups include, among others, morpholinyl, thiomorpholinyl, pyranyl, imidazolidinyl, imidazolinyl, oxazolidinyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, piperazinyl, azetidine, and the like.

[0093] As used herein, the term “theranostic” refers to a molecule that is able to both detect or diagnose a medical condition and also treat.

[0094] A series of arylboronate prodrugs bearing an inactive DNA alkylating functional group masked with an arylboronate that can be activated selectively by H2O2to allow DNA alkylations have been developed. These compounds are harmless until they enter a cancer cell, where H2O2transforms them into an active alkylating agent. The in vivo study suggested that these prodrugs are particularly effective against triple negative breast cancer (TNBC) cells that maintain high levels of H2O2; yet the prodrugs present minimal toxicity to normal tissues. In addition, they (e.g., FAN-NM-CH3) showed reduced side effects and improved selectivity in comparison to clinically used DNA alkylating agents (e.g., chlorambucil). However, the existing H2O2-activated DNA alkylating agents do not have diagnosis potential, which prevents further investigation of their mechanism of function in complex biological settings, such as in cultured cells and in vivo, and hinders the further development of these agents as tumor-selective therapeutics for a wide variety of cancer. In this study, FAN-NM-CH3 (Table 1) was used as the lead compound to develop H2O2- responsive theranostic agents containing both H2O2-activatable DNA alkylator with therapeutic effect and fluorogenic reporters capable of monitoring molecular activity.

[0095] Bifunctional alkylating agents produce DNA interstrand cross-links (ICLs) or covalent adducts with DNA nucleobases, which prevent strand separation and block DNA replication and transcription resulting in cell death. A variety of DNA alkylating agents have been approved by FDA and widely used for cancer treatment, such as nitrogen mustard analogues chlorambucil, cyclophosphamide, melphalan, bendamustine, etc. They are effective anticancer drugs against fast dividing malignant cells yet suffer severe limitations due to nonspecific activity resulting in toxicity to normal, healthy cells. Nevertheless, in the absence of improved agents, the current alkylating chemotherapeutics are still used for cancer treatment. Therefore, more selective DNA- targeting agents are needed to reduce unwanted side effects and allow simultaneous monitoring and treatment of cancer. In this study, several H2O2-responsive theranostic agents (1a, 1c and 2) were developed comprising of H2O2-activatable therapeutic and diagnostic effectors, such as nitrogen mustard functional groups capable of cross-linking DNA or other nucleophilic biological targets, along with a hydroxycoumarin or fluorescein-derived moiety as the fluorogenic reporters to monitor molecular activity. These newly developed nitrogen mustard analogues can thereby allow selective activation of the molecules to deliver drug and imaging unit at H2O2rich areas such as cancerous cells to achieve therapeutic treatment with concomitant florescence detection. Described herein is the design, synthesis, and biological investigation of theranostic agents derived from both nitrogen mustard and fluorophore as anticancer and bioimaging agents.

[0096] Table 1. Theranostic Agents

[0097]

[0098] A theranostic agent can also include compounds:

[0099]

[0100]

[0101]

[0102]

[0103] A theranostic agent can further include compounds:

[0104]

[0105]

[0106] A theranostic can include compounds:

[0107] Triple negative breast cancer (TNBC) is a notoriously difficult disease to treat, and many of the existing TNBC chemotherapeutics lack tumor selectivity and the capability for simultaneously visualizing and monitoring their own activity in the biological context. However, TNBC cells have been known to generate high levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). To this end, three novel small molecule theranostics 1a, 1c, and 2 consisting of both H2O2~responsive nitrogen mustard prodrug and profluorophores have been designed, synthesized, and evaluated as the targeted cancer therapeutics and bioimaging agents. The three theranostics comprise boronate esters that deactivate nitrogen mustard functional groups and fiuorophores but allow their selective activation through H2O2-specific oxidative deboronation for the release of the active drug and fluorophore. The three theranostics demonstarted H2O2-inducible DNA-alkylating capability and fluorescence turn-on properties in addition to selective anticancer activity. They are particularly effective in killing TNBC MDA-MB- 468 ceils with high H2O2level while safe to normal epithelial MCF-1 OA cell. The conjugated boron- masked fiuorophores in 1c and 2 are highly responsive towards H2O2, which enabled tracking of the theranostics in live cells’ mitochondria and nucleus. The theranostics 1a, 1c, and 2 are capable of both selective release of the active drug to take effect in H2O2-rich cancer sites and simultaneously monitoring its activity. This single molecule system is of utmost importance to understand the function, efficacy, and mechanism of the H2O2-activated prodrugs within the living recipient.

[0108] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplasy embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0109] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0110] Clause 1 . A theranostic compound or pharmaceutically acceptable salt thereof, comprising: a compound of formula (I), wherein:

[0111] X is halo;

[0112] R1, at each occurrence, is independently a linker; and

[0113] R2, at each occurrence, is independently a boronic acid, a boronic ester, a boronate, a boronamine, hydrogen, or C1-6alkyl; and a fluorophore.

[0114] Clause 2. The compound of clause 1 , wherein the linker has a formula (II): wherein:

[0115] R3is independently hydrogen or C-1-3alkyl;

[0116] L1is C1-15alkylene;

[0117] L2is C1-10alkylene or -(CH2)0-5-Y-(CH2)0-5-;

[0118] Y is C(O), N(RX), or C(O)-N(RX);

[0119] Rxis hydrogen or C1-3alkyl; and

[0120] G1is a 6- to 12-membered aryl, C3-10carbocyclyl, or a 4- to 12-membered heterocyclyl, optionally substituted with halogen, cyano, C1-6alkyl, C1-6haloalkyl, -OR13, -SR13, CO2R1a, -C(O)R1a, -SO2R13, -N(R13)2, -CO2N(R13)2, or -NO2;

[0121] R1a, at each occurrence, are each independently hydrogen or C1-3alkyl; m = 0-1 ; and n - 0—1 .

[0122] Clause 3. The compound of clause 1 or 2, wherein the linker has a formula (III): wherein

[0123] L3is C1-6alkylene, C(O), -O-C(O)--O-, -N(RX)-, or -O-; and w = 0—1 .

[0124] Clause 4. The compound of any one of clause 1-3, wherein the fluorophore is fluorescein, rhodamine, cyanine, coumarin, BODIPY, napthaiamide, xanthenone, (benzopyranylidene) malononitrile, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0125] Clause 5. The compound of any one of clause 1-4, wherein X is chloro.

[0126] Clause 6. The compound of any one of clause 1-5, wherein the compound has a formula I- a:

[0127] Clause 7. The compound of any one of clause 1-6, wherein the theranostic has a formula I- b: wherein Q is a fluorophore.

[0128] Clause 8. The compound of any one of clause 1-7, wherein R3is hydrogen.

[0129] Clause 9. The compound of any one of clause 1—8, wherein m = 0 and n = 0.

[0130] Clause 10. The compound of any one of clause 1-9, wherein Q is coumarin, fluorescein, a derivative thereof, or a salt thereof.

[0131] Clause 11. The compound of any one of clause 1-10, wherein the theranostic compound or a pharmaceutically acceptable salt thereof, is selected from a group consisting of:

[0132]

[0133] Clause 12. The compound of any one of clause 1-11 , wherein the theranostic compound or a pharmaceutically acceptable salt thereof, is selected from a group consisting of:

[0134] Clause 13. The compound of any one of clause 1—12, wherein the compound has a formula of l-c: wherein Q is a fluerophore.

[0135] Clause 14. The compound of any one of clause 1—13, wherein R:is hydrogen or C1-6alkyl.

[0136] Clause 15. The compound of any one of clause 1—14, wherein w = 1 .

[0137] Clause 16. The compound of clause any one of clause 1—15, wherein L3is -O--C(O)--O-.

[0138] Clause 17. The compound of any one of clause 1—16, wherein the fluorophore is a is coumarin, fluorescein, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0139] Clause 18. The compound of any one of clause 1—17. wherein the theranostic, or pharmaceutically acceptable salt thereof, is selected from a group consisting of:

[0140] Clause 19. A method for treating and diagnosing a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of the compound of any one of clause 1—18, or pharmaceutically acceptable salt thereof.

[0141] Clause 20. The method of clause 19, wherein the disease or disorder is associated with oxidative stress.

[0142] Clause 21 . The method of clause 19 or 20, wherein the disease or disorder is cancer.

[0143] Clause 22. The method of clause 19—21 , wherein the cancer is selected from breast cancer, brain cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, leukemia, or other cancers

[0144] Clause 23. Use of the theranostic compound of clause 1—18, or a salt thereof, for the preparation of a medicament for the treatment of a disease or disorder associated with oxidative stress in a subject.

[0145] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disciosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0146] EXAMPLES

[0147] Example 1

[0148] General Methods

[0149] Synthesis of 1a

[0150] Scheme 1. Synthetic Route for Theranostic 1a

[0151]

[0152] 5-Amino~2~bromobenzyl alcohol (3b) An oven-dried 100 mL round bottom flask was charged with 5-amino-2-bromobenzoic add

[0153] 3a (2.84 g, 4.6 mmol) and purged under nitrogen. Dry THF (5 mL) was added to the flask and the mixture was cooled to 0 °C under ice-water bath. A solution of borane (1.0 M in THF) was added dropwise with stirring. The reaction was kept under ice-water bath until the evolution of gas subsided, and the mixture was slowly warmed to RT and stirred overnight. Then, the reaction was cooled under ice-water bath and the reaction was quenched with water (1 mL). The mixture was extracted with ethyl acetate (3* 30 mL) and the organic layers were collected and dried over Na2SO4. The solvent was evaporated in vacuo and the crude product was purified by column chromatography (silica gel; 2:1 , hexanes: EtOAc) to obtain 3b as a white solid (3.1 g, 93%).1H NMR (500 MHz, MeOD) 6 7.10-7.08 (1 H, m), 6.80 (1 H, d, J ~ 2.7 Hz), 6.43-6.41 (1 H, m), 4.44 (2H, s).13C NMR (125 MHz, MeOD) δ 148.77, 141.39, 132.33, 114.32, 114.11 , 105.86, 63.14 (the NMR spectra were in agreement with those reported by Barbasiewicz et al., Chem. - Eur. J. 18(45): 14237-14241 (2012).

[0154] A 100 mL round bottom flask was charged with 3b (3.1 g, 15.34 mmol) and methanol (30 mL). DMF-DMA was added, and the reaction mixture was heated to 50 °C with stirring for 12 h. After the completion of the reaction as monitored by TLC, the solvent was evaporated in vacuo and the mixture was extracted with ethyl acetate (3× 30 mL), then washed with brine (3×). The organic layers were combined, dried over Na2SO4, and concentrated. The resulting residue was purified by column chromatography over silica gel (1 :1 , hexanes / EtOAc) to afford 3c as a yellow oil (3.95 g, quantitative).1H NMR (500 MHz, MeOD) 6 7.57 (1 H, s), 7.28 (1 H, d, 8.2 Hz), 7.05 (1 H, d, J = 2.7 Hz), 6.69 (1 H, dd, J = 2.8, 8.5 Hz), 4.51 (1 H, s), 3.01-2.86 (6H, m).13C NMR (125 MHz, MeOD) δ 155.10, 151.08, 140.66, 132.37, 121.26, 120.83, 114.49, 63.34, 33.55. HRMS (ESI) calculated [M+H] for C10H14BrN2O4257.02840; found: 257.02976. A 100 mL oven-dried flask was charged with 3c (3.4 g, 13.2 mmol) and purged with argon. Dry THF (20 mL) was added, and the mixture was cooled to 0 °C in ice-water bath. The septum was opened and NaH (60% in oil, 1.27 g, 30.5 mmol) was added in portions with stirring. The apparatus was kept in the dark and the solution was slowly warmed to RT and stirred until the evolution of gas subsided. A solution of propargyl bromide (80% in toluene) was slowly added dropwise. The reaction mixture was stirred for an additional 24 h, cooled in ice-water bath, and slowly quenched with water (5 mL). The resulting mixture was extracted with ethyl acetate (3* 30 mL). The organic layers were combined, dried over NaSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 1 :1 , hexanes / EtOAc) to afford 4 as a yellow oil (3.4 g, 88%).1H NMR (500 MHz, MeOD) δ 7.65 (1 H, s), 7.43-7.39 (1 H, m), 7.10 (1 H, s), 6.83 (1 H, dd, 2.5, 8.5 Hz), 4.62 (2H, s), 4.27 (2H, d, J = 1.8 Hz), 3.03 (6H, d, 31.2 Hz), 2.93 (1 H, s).13C NMR (125 MHz, MeOD) 5 155.10, 151.08, 140.66, 132.37, 121.26, 120.83, 114.49, 63.34, 13.06. HRMS (ESI) calculated [M+H]+for C13H16BrN2O+295.04405; found: 295.04568.

[0155] A 100 mL round bottom flask was charged with 4 (3.4 g, 11.5 mmol) and ethanol (35 mL). Ethylenediamine (3.6 mL, 54.3 mmol) was slowly added dropwise with stirring. The mixture was heated to 80 °C and stirred overnight. Upon completion as judged by TLC, the resulting mixture was cooled to 0 °C, neutralized with 1 M HCI, and extracted with ethyl acetate (3× 30 mL). The organic layers were combined, washed with brine (3×), dried over NaSO4, then filtered. The solvent was evaporated in vacuo. The crude product was isolated by column chromatography (silica gel; 3:1 , hexanes / EtOAc) to obtain 5 as a yellow oil (2.1 g, 91%).1H NMR (500 MHz, MeOD) δ 7.12-7.10 (1 H, m), 6.74-6.73 (1 H, m), 6.44 (1 H, dd, J = 2.8, 8.5 Hz), 4.78 (2H, d, J = 4.1 Hz), 4.74 (2H, s), 4.44-4.43 (2H, m), 4.13-4.12 (2H, m), 2.79-2.77 (1 H, m).13C NMR (500 MHz, MeOD) δ 144.05, 136.38, 132.01 , 115.21 , 115.11 , 109.96, 78.48, 73.90, 69.96, 56.81. HRMS (ESI) calculated [M+H]+for C10H11NOBr 240.00185; found: 240.00451.

[0156] 2,2'-((4-Bromo-3-((prop-2-yn-1-yloxy)methyl)phenyl)azanediyl)bis(ethan-1-ol) (6)

[0157] A 100 mL round bottom flask was charged with 5 (2.1 g, 8.7 mmol), calcium carbonate (2.0 g, 19.7 mmoi), Nai (0.4 g, 2.6 mmol) and distilled H2O (22 mL). The reaction mixture was stirred at RT for 0.5 h. 2-Chloroethanol (1.8 mL, 26.9 mmol) was slowly added to the reaction mixture dropwise and the mixture was heated to reflux for 12 h. The reaction mixture was slowly cooled to RT and the second portion of 2-chloroethanol (1.8 mL, 26.9 mmol) was added. The reaction mixture was heated to reflux with stirring for 12 h (monitored by TLC), slowly cooled to RT and filtered. The solution was extracted with ethyl acetate (3 x 30 mL) and washed with brine (3*). The organic layers were collected, dried over NaSO4, and evaporated in vacuo. The crude product was isolated by column chromatography (silica gel; 1 :2, hexanes / EtOAc) to afford 6 as a yellow oil (1.49 g, 52%).1H NMR (500 MHz, MeOD) δ 7.19 (1 H, d, 7.5 Hz), 6.76 (1 H, d, J = 3.2 Hz), 6.51 (1 H, dd, J = 3.2, 8.9 Hz), 4.48 (2H, s), 4.13 (2H, d, J = 2.8 Hz), 3.61 (4H, t, J = 6.0 Hz), 3.43 (OH, t, J = 6.2 Hz), 3.29 (OH, s), 2.81-2.79 (1 H, m).13C NMR (125 MHz, MeOD) δ 147.50, 136.66, 132.54, 112.88, 112.83, 107.91 , 79.07, 74.89, 70.96, 58.84, 56.99, 53.62. HRMS (ESI) calculated [M+H]+for 328.05428; found 328.05663.

[0158] A 100 mL round bottom flask was charged with 6 (1.49 g, 4.1 mmol) and dry DCM (25 mL). The reaction mixture was cooled to 0 °C with stirring. Triethylamine and methanesulfonyl chloride were slowly added dropwise. The reaction mixture was stirred at 0 °C for 0.5 h, slowly warmed to RT, and quenched with water (5 mL) after completion (3 h) as observed by TLC. The resulting mixture was extracted with ethyl acetate (3× 20 mL) and washed with brine (3×). The organic layers were collected, dried over NaSO4, filtered and the solvent was evaporated in vacuo to obtain a crude product as a yellow oil. The resulting crude product without further purification was dissolved in DMF (25 mL) and anhydrous LiCI (0.48 g, 11.3 mmol) was added. The reaction mixture was heated to 70 °C overnight with stirring and monitored by TLC. The solvent was evaporated in vacuo. The crude product was diluted with water and extracted with ethyl acetate (3* 20 mL). The organic layers were combined, washed with brine (3x). and dried over NaSO4. The solvent was evaporated in vacuo and the crude product was isolated by column chromatography (silica gel; 25:1 , hexanes / EtOAc) to obtain 7 as a yellow oil (96%).1H NMR (500 MHz, CDCI3) δ 7.31 (3H, d, J = 8.9 Hz), 6.77 (1 H, d, 4.4 Hz), 6.47 (1 H, dd, J ~ 3.2, 8.9 Hz), 4.56 (2H, s), 4.20 (2H, d, 2.4 Hz), 3.67 (4H, t, 6.7 Hz), 3.56 (4H, t, 6.7 Hz), 2.43 (1 H, t, 2.4 Hz).13C NMR (125 MHz, CDCI3) δ 144.30, 136.78, 132.36, 112.11 , 111.96, 109.36, 78.39, 73.99, 70.12, 56.85, 52.63, 39.19. HRMS (ESI) calculated [M+H]+calcd. for 364.99433; found: 365.32072.

[0159] 2,4-Dihydroxybenzaldehyde (S2)

[0160] A 250 mL round bottom flask was charged with acetonitrile (80 mL) and DMF (20.4 mL). The reaction mixture was cooled to 0 °C under an ice-water bath for 15 mins with stirring. POCI3(14.64 mL, 156.48 mmol) was slowly added dropwise, and the reaction mixture was stirred for an additional 30 minutes. Then, m-dihydroxybenzene (14.64 g, 133.2 mmol) was added to the mixture in one portion and the mixture was stirred for an additional 1 h. The resulting crystals were filtered and washed with a small amount of cold acetonitrile. The crude product was further purified by recrystallization from water to obtain S2 as a solid white powder (40%).!H NMR (500 MHz, DMSO) δ 10.85 (1 H, s), 10.63 (1 H, s), 9.93 (1 H, s), 7.54 (1 H, d, 8.7 Hz), 6.40 (1 H, dd, 2.1 , 8.5 Hz), 6.32 (1 H, d, 2.3 Hz).13C NMR (125 MHz, DMSO) δ 191.34, 165.62, 163.70, 133.21 , 115.70, 109.09, 102.64 (the NMR spectra were in agreement with those reported by Zhang et al., J. Asian Nat. Prod. Res. 19(9): 903-909 (2017). N-Acetyl glycine (S3)

[0161] Glycine (37.5 g, 0.5 mol) was dissolved in water (150 mL) in a 500 mL round bottom flask with stirring. Then, acetic anhydride (95 mL, 1 moi) was added in one portion to the reaction mixture. The resuiting reaction mixture was vigorously stirred at RT for 40 mins, then kept in icewater bath for 12 h. The resulting precipitate was filtered, washed with ice cold water, and recrystallized from boiling water to obtain A / -acetyl glycine S3 as a solid white crystal (86%).1H NMR (500 MHz, DMSO) δ 8.16 (t, J = 5.2 Hz, 1 H), 3.72 (d, J = 5.8 Hz, 2H), 1.85 ppm (s, 3H).13C NMR (125 MHz, DMSO) δ 171.90, 170.04, 41.07, 22.76 (the NMR spectra were in agreement with those reported by Rajbongshi et al., Cryst. Growth Des. 12(4); 1823-1829 (2012).

[0162] 3-Azido-7-hydroxycoumarin (8)

[0163] A mixture of 2,4-dihydroxybenzaldehyde (2.76 g), / V-acetylglycine (2.34 g), and anhydrous sodium acetate (4.46 g, 60 mmol) in acetic anhydride (100 mL) was refluxed under stirring for 4 h. The reaction mixture was poured onto ice to give a yellow precipitate, which was filtered and washed with ice water. The precipitate was then refluxed in a solution of concentrated HCI and ethanol (2:1 30 mL) for 1 h and cooled in an ice water bath. NaNO2(2.76 g, 40 mmol) was added to the solution which was stirred for 5-10 minutes. Then, NaN3(3.96 g, 60 mmol) was added in portions to the reaction mixture while stirring for another 15 mins. The resulting brown precipitate was filtered and washed with water. The product was dried in vacuo to obtain 8 as a brown solid powder (37%).1H NMR (500 MHz, DMSO): δ 8.16 (1 H, t, J ~ 5.2 Hz), 3.72 (2H, d, J = 5.8 Hz), 1.85 (3H, s).13C NMR (125 MHz, DMSO): δ 171 .90, 170.04, 41.07, 22.76 (the NMR spectra were in agreement with those reported by Sivakumar et al., Org. Lett. 6(24): 4603-4606 (2004).

[0164] 3-(4-(((5-(Bis(2-chloroethyl)amino)-2-bromobenzyl)oxy)methyl)-1 H-1,2,3-triazol-1-yl)-7-hydroxy- 2H-chromen-2-one (9)

[0165] A 50 mL round bottom flask was charged with 8 (0.68 g, 3.3 mmol), 7 (1.1 g, 3.0 mmol), sodium ascorbate (0.12 g, 0.06 mmol), and copper(ll) sulfate pentahydrate (0.08 g, 0.03 mmol) in waterethanol (1 :1 , 30 mL). The reaction mixture was stirred vigorously overnight in the dark at RT. The solvent was removed, and the crude product was isolated by column chromatography (silica gel; 2:1 , hexanes / EtOAc) to afford 9 as a yellow-white powder (1.24 g, 73%).1H NMR (500 MHz, DMSO) δ 10.90 (s, 1 H), 8.60 (d, 16.0 Hz, 2H), 7.76 (d, 8.6 Hz, 1 H), 7.38 (d, 7 = 8.9

[0166] Hz, 1 H), 6.94-6.91 (m, 2H), 6.87 (d, 7 = 2.1 Hz, 1 H), 6.68 (q, 7 = 4.0 Hz, 1 H), 4.74 (s, 2H), 4.57 (s, 2H), 3.74 ppm (s, 8H).13C NMR (125 MHz, DMSO) δ 162.92, 156.79, 155.12, 146.47, 144.49, 137.96, 136.79, 133.41 , 131.45, 125.39, 119.81 , 114.76, 113.63, 113.53, 110.82, 109.14, 102.64, 71.61 , 63.45, 52.45, 41.45. HRMS (ESI) calculated [M+H]+for 567.01960; found: 567.02401.

[0167] A 50 mL round bottom flask was charged with bispinacolatodiboron (0.54 g, 2.13 mmol), anhydrous potassium acetate (0.42 g, 4.28 mmol), Pd(dppf)CI2(0.052 g, 0.074 mmol) and 9 (0.807 g, 1.42 mmol), which was purged with argon, dissolved in 1 ,4-dioxane (15 mL) and heated to 85 °C for 12 h. Upon completion as judged by TLC, the reaction mixture was diluted with ethyl acetate (5 mL), filtered, and extracted with water and ethyl acetate. The organic layers were collected and washed with brine (3×), dried over NaSO4, and the solvent was evaporated in vacuo. The resulting crude product was isolated by column chromatography (silica gel; 2:1 , hexanes / EtOAc) to afford 1a as a yellow powder. The resulting product was further purified by trituration in diethyl ether to afford 1a as a yellow powder (0.06 g, 11 %).1H NMR (500 MHz, MeOD) 5 8.48 (s, 1 H), 8.44 (s, 1 H), 7.59 (dd, J = 1 .5, 8.7 Hz, 1 H), 6.84 (q, J = 3.6 Hz, 1 H), 6.80 (d, J = 2.4 Hz, 1 H), 6.76 (d, 7 = 2.6 Hz, 1 H), 6.59 (dd, 7 = 2.7, 8.5 Hz, 1 H), 4.71 (s, 2H), 3.74 (t, J = 6.9 Hz, 4H), 3.62 (t, J = 6.8 Hz, 4H), 1.25 (s, 12H).13C NMR (125 MHz, MeOD) δ 162.95, 156.68, 155.16, 148.70, 145.99, 144.91 , 137.69, 135.67, 130.52, 124.36, 119.44, 114.17, 110.91 , 110.59, 109.77, 102.01 , 83.07, 71 .80, 62.93, 52.66, 40.19, 23.81 . HRMS (ESI) calculated [M+H]’ for . 19430; found: 615.20240.

[0168] Synthesis of 1c Scheme 2. Synthetic Route for Theranostic 1o 11-Bromoundec-1-yne (16)

[0169] A round bottom flask was charged with 10-undecyn-1-ol and CBr4in DCM (100 mL). The reaction mixture was stirred in ice bath for 0.5 h. Triphenylphosphine in DCM was slowly added dropwise to the mixture. Then, the reaction mixture was stirred at RT for an additional 2 h, then concentrated and poured into n-hexanes, and filtered. The filtrate was concentrated and purified by column chromatography (50:1 , hexanes / EtOAc).1H NMR (500 MHz, CDCI3) δ 3.42 (t, J ~ 6.9 Hz, 2H), 2.21-2.17 (m, 2H), 1.95 (t, J= 2.7 Hz, 1 H), 1.90-1.83 (m, 2H), 1.57-1.50 (m, 2H), 1.46- 1.38 (m, 4H), 1.32 ppm (m, 1.8 Hz, 7H).13C NMR (125 MHz, CDCh) δ 84.68, 68.11 , 33.97,

[0170] 32.82, 29.28, 28.98, 28.70, 28.68, 28.45, 28.14, 18.39 (the NMR spectra were in agreement with those reported by Kim et al., Macromolecules 50(17):6489-6500 (2017). A 250 mL round bottom flask was charged with 3c (5.22 g, 20.30 mmol) and NaH (1 .46 g, 60.90 mmol) in anhydrous THF (20 mL) at 0 °C. The resulting mixture was stirred at RT until the evolution of hydrogen gas subsided. 11 -Bromoundec- 1-yne (6) (3.59 mL, 24.36 mmol) was subsequently added at 0 °C. The reaction mixture was stirred at RT for 48 hrs. After completion as judged by TLC, the reaction mixture was quenched with 10 mL of water. The aqueous layer was extracted with EtOAc (3* 20 mL) and washed with brine (3*). The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by column chromatography (1 :1 , hexane / ethyl acetate) over silica gel to afford 17 as a yellow oil (6.86 g, 83%).1H NMR (500 MHz, CDCI3) δ 7.32 (d, J = 8.4 Hz, 1 H), 7.06 (d, 2.6 Hz, 1 H),

[0171] 6.81 (dd, 2.5, 8.4 Hz, 1 H), 3.46 (t, J ~ 6.7 Hz, 1 H), 2.10 (dt, J = 2.6, 7.1 Hz, 1 H), 1.87 (t, J = 2.6 Hz, 1 H), 1.56 (quint, J = 7.9 Hz, 1 H), 1.44 (quint, J 7.6 Hz, 1 H), 1.33 (m, J 3.0 Hz, 1 H), 1.23 (m, 3.9 Hz, 1 H).13C NMR (125 MHz, CDCI3) δ 152.29, 137.40, 131.87, 120.8 120.18,

[0172] 115.16, 83.77, 71.12, 70.01 , 67.06, 28.70, 28.43, 28.40, 27.71 , 27.45, 25.15, 17.37. HRMS (ESI) calculated [M+H] for 407.16925; found: 407.15880.

[0173] A 250 mL round bottom flask was charged with 17 (6.86 g, 16.84 mmol) and ethanol (20 mL). Ethylenediamine (2.25 mL, 2.02 mol) was added dropwise at RT. The reaction mixture was heated to 80 °C and stirred at 80 °C overnight. After completion of the reaction as judged by TLC, the crude mixture was neutralized to pH 7, extracted with EtOAc (3× 30 mL) and washed with brine (3*). The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (3:1 , hexane / EtOAc) over silica gel to afford 18 as a yellow oil (5.16 g, 87%). NMR (500 MHz, CDCI3) δ 7.27 (d, J = 8.4 Hz, 1 H), 6.84 (d, J = 2.9 Hz, 1 H), 6.49 (d, J ~ 5.5 Hz, 1 H), 6.48 (d, J = 11.3 Hz, 1 H), 3.55 (t, 6.6 Hz,

[0174] 2H), 2.20 (dt, 2.6, 7.1 Hz, 2H), 1.96 (t, 2.6 Hz, 1 H), 1.66 (d, 8.0 Hz, 2H), 1.57 (quint, J

[0175] = 9.6 Hz, 2H), 1.54 (quint, 7.5 Hz, 2H), 1.41 (m, J = 11.6 Hz, 4H), 1.33 ppm (m, 4.3 Hz,

[0176] 7H).13C NMR (125 MHz, CDCI3) δ 145.92, 138.61 , 132.85, 115.40, 115.35, 110.13, 84.83, 71.99, 71.05, 68.12, 29.75, 29.45, 29.06, 28.74, 28.59, 26.19, 18.41. HRMS (ESI) calculated [M+H]+for = 352.12705; found: 352.12960.

[0177]

[0178] A 250 mL round bottom flask was charged with 13 (5.16 g, 14.65 mmol), 2-chloroethanol (2.95 mL, 43.94 mmol), CaCO3(4.40 g, 43.94 mmol) and Nal (0.44 g, 2.93 mmol) in 50 mL of water. The reaction mixture was heated to 100 °C for 24 h, then extracted with EtOAc (3x 20 mL) and washed with brine (3×). The organic layers were collected, dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (2:1 , hexane / EtOAc) over silica gel to afford 19 as a yellow oil (3.86 g, 52%).1H NMR (500 MHz, CDCI3) δ 7.33 (d, J = 8.6 Hz, 1 H), 6.85 (d, J= 3.9 Hz, 1 H), 6.51 (dd, J= 2.9, 9.1 Hz, 1 H), 4.52 (s, 2H), 3.87 (t, 5.1

[0179] Hz, 4H), 3.60 (t, J = 4.9 Hz, 4H), 3.56 (t, J = 6.2 Hz, 2H), 3.09 (s, 2H), 2.22-2.18 (2H, m), 1.96 (t, 0= 2.5 Hz, 1 H), 1.70-1.63 (2H, m), 1.57-1.51 (2H, m), 1.46-1.39 (4H, m), 1.37-1.30 (8H, m).13C NMR (125 MHz, CDCI3) δ 147.60, 138.48, 132.84, 113.56, 113.40, 109.50, 84.84, 72.10, 71.04, 68.10, 61.13, 46.06, 29.76, 29.46, 29.43, 29.06, 28.74, 28.49, 26.22, 18.41. HRMS (ESI) calculated [M+K]+for 440.08333; found: 440.17999.

[0180] A 250 mL round bottom flask was charged with 19 (3.86 g, 9.76 mmol) and Et3N (3.06 mL, 21.91 mmol) in 50 mL DCM. Then, MsCI (1.70 mL, 21.91 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at RT for 4 h. After completion of the reaction as judged by TLC, the reaction mixture was diluted with water extracted with DCM (3* 20 mL) and washed with brine (3*). The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The resulting residue was dissolved in 50 mL DMF and LICI (0.93 g, 21.91 mmol) was added. After being stirred at 70 °C for 9 h, the mixture was diluted with water, extracted with DCM (3× 20 mL), washed with brine. The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (25:1 , hexane / EtOAc) to afford 20 as a yellow foam (96%, 4.02 g).1H NMR (500 MHz, CDCI3) δ 3.74 (d, J ~ 6.9 Hz, 5H), 3.65 (d, J = 6.8 Hz, 5H), 3.63 (d, J = 0.5 Hz, 5H), 3.57 (t, J = 6.5 Hz, 2H), 2.20 (dt, J = 2.6, 7.1 Hz, 2H), 1 .96 (t, J = 2.6 Hz, 1 H), 1.67 (quint, J = 8.0 Hz, 2H), 1 .57 (quint, J = 9.5 Hz, 2H), 1 .54 (m, 7.5 Hz, 2H), 1.42 ppm (m, J= 7.6 Hz, 5H).13C NMR (125 MHz, CDCI3) δ 145.56, 139.01 ,

[0181] 133.18, 112.35, 112.18, 109.63, 84.81 , 72.06, 71.13, 68.08, 53.56, 40.30, 29.83, 29.46, 29.07, 28.75, 28.50, 26.31 , 18.41 . HRMS (ESI) calculated [M+H]+for 476.11171 ; found: 476.11428.

[0182] A 100 mL round bottom flask was charged with 20 (1 g, 2.10 mmol), CuSO4·5 H2O (0.42 mmol), sodium ascorbate (0.84 mmol) and 8 (0.25 mmol) in 40 mL of H2O:EtOH (1 :1). The reaction mixture was stirred at RT in the dark for 24 h. After completion of the reaction as judged by TLC, the mixture was extracted with EtOAc and washed with brine (3×). The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by column chromatography over silica (2:1 hexane / EtOAc) to obtain yellow oil 21 (1.20 g, 84%). 'H NMR (500 MHz, CDCI3) δ 9.10 (s, 1 H), 8.51 (s, 1 H), 8.39 (s, 1 H), 7.55 (d, J = 9.2 Hz, 1 H), 7.35 (d, 8.8 Hz, 1 H), 7.03-7.00 (m, 2H), 6.84 (d, 3.2 Hz, 1 H), 6.48 (q, 4.0 Hz, 1 H), 4.52 (s,

[0183] 2H), 3.73 (t, 7.0 Hz, 5H), 3.63 (t, 6.9 Hz, 5H), 3.58 (t, 6.6 Hz, 2H), 2.83 (t, 7.7 Hz,

[0184] 2H), 1.80-1.71 (m, 3H), 1.71-1.63 (m, 3H), 1.41 (m, J = 7.1 Hz, 12H), 1.34 ppm (m, J = 8.7 Hz, 12H).13C NMR (125 MHz, CDCI3) δ 161.87, 156.50, 154.78, 148.47, 145.58, 138.82, 134.42, 133.19 ,130.33, 122.19, 119., 79, 114.98, 112.41 , 112.26, 111.08, 109.69, 103.28, 72.10, 71.17, 53.51 , 40.30, 29.77, 29.45, 29.26, 29.23, 29.17, 26.28, 25.46. HRMS (ESI) calculated [M+H]+for 679.14480; found: 679.15367.

[0185]

[0186] A 100 mL round bottom flask was charged with 21 (1.20 g, 1.76 mmol) and 50 mL DCM. DI PEA was subsequently added to the reaction mixture at RT. Tritiate anhydride (0.36 mL, 2.12 mmol) was added dropwise at 0 °C. The resulting mixture was stirred for 6 h at RT. After completion of the reaction as judged by TLC, the mixture was diluted with water and extracted with DCM. The organic layer was washed with brine. The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography over silica gel (3:1 , Hexane / EtOAc) to obtain 22 as a yellow oil (0.54 g, 38%).1H NMR (500 MHz, CDCI3) δ 8.63 (s, 1 H), 8.42 (s, 1 H), 7.80 (d, 8.7 Hz, 1 H), 7.42 (d, 2.4 Hz, 1 H), 7.37 (q, J =

[0187] 3.6 Hz, 1 H), 6.86 (d, J = 3.2 Hz, 1 H), 6.49 (q, J = 4.0 Hz, 1 H), 4.52 (s, 2H), 3.75 (t, J = 7.0 Hz, 4H), 3.64 (t, 6.9 Hz, 4H), 3.57 (t, J = 6.5 Hz, 2H), 2.83 (t, 7.7 Hz, 2H), 1.76 (q, 7.5 Hz, 2H), 1.72-1.64 (m, 2H), 1.46-1.33 ppm (m, 11 H).13C NMR (125 MHz, CDCI3) δ 154.83, 152.77, 151.08, 149.14, 145.56, 139.01 , 133.16, 130.71 , 130.36, 124.02, 121.72, 120.00, 118.94, 118.31 , 116.71 , 112.33, 112.16, 110.48, 109.61 , 72.05, 71.13, 53.54, 40.30, 29.82, 29.47, 29.30, 29.22, 2918, 26.31 , 25.61.19F NMR (470 MHz, CDCI3) δ -72.45. HRMS (ESI) calculated [M+H]+for 811.09408; found: 811.10760. A 50 mL round bottom flask was charged with 22 (0.54 g, 0.66 mmol), bis(pinacolato)diboron (0.68 g, 2.66 mol), KOAc (0.39 g, 3.99 mmol), and Pd(dppf)CI2(0.047 g, 0.066 mmol) in 1,4-dioxane (20 mL). The reaction mixture was stirred under argon at 85 °C for 12 h. After completion of the reaction as judged by TLC, the reaction mixture was diluted with EtOAc and filtered. The filtrate was diluted with water, extracted with EtOAc (3× x10 mL), and washed with brine (3*). The organic layers were collected, dried over Na2SO4and concentrated. The resulting residue was purified by column chromatography (2:1 , hexane / EtOAc) to afford 1c as a yellow foam (0.10 g, 17%).1H NMR (500 MHz, CDCI3) δ 8.51 (s, 2H), 8.36 (s, 2H), 7.77 (s, 2H), 7.72 (d, J = 7.6 Hz, 2H), 7.65 (d, 8.3 Hz, 1 H), 7.58 (d, J = 7.7 Hz, 2H), 6.86 (d, 7 = 2.5

[0188] Hz, 1 H), 6.58 (q, J = 3.6 Hz, 1 H), 4.68 (s, 2H), 3.70 (t, 7.1 Hz, 6H), 3.57 (t, 7 = 6.9 Hz, 6H),

[0189] 3.43 (q, 7.0 Hz, 3H), 2.74 (t, 7.7 Hz, 3H), 1.67 (t, J = 7.3 Hz, 3H), 1.57 (t, 7.4 Hz, 3H),

[0190] 1.31 (s, 19H), 1.24 (s, 16H), 1.17 ppm (s, 18H).13C NMR (125 MHz, CDCI3) δ 155.96, 151.95, 148.80, 148.27, 147.79, 138.07, 135.17, 132.30, 131.20, 127.98, 127.88, 123.95, 122.50, 121.85, 120.25, 110.08, 109.42, 84.63, 83.11 , 75.04, 71.94, 70.71 , 53.25, 40.40, 30.00, 29.58, 29.35, 29.29, 29.23, 26.40, 25.63, 24.89, 24.87. HRMS (ESI) calculated [M+H]+for 837.40979; found: 837.41761.

[0191] Synthesis of 2

[0192] Scheme 3. Synthetic route for Theranostic 2

[0193] A 250 mL round bottom flask was charged with fluoresceinamine that was dissolved in methanol. NaNO2(1.1 g, 16.41 mmol) in water (20 mL) and HCI (20 mL, 5 M) was added to the solution. Then, NaN3(1.37 g, 21.10 mmol) in water (30 mL) was added dropwise to the reaction mixture through the dropping funnel. The reaction mixture was stirred at RT for 2 h. After completion as judged by TLC, the solution was concentrated, filtered, and washed with cold water. The resulting solid was lyophilized to yield 23a as an orange powder (1 .07 g, 82%).1H NMR (500 MHz, DMSO) δ 10.17 (s, 2H), 7.65 (d, 1.7 Hz, 1 H), 7.52 (dd, 2.4, 8.3 Hz, 1 H), 7.30 (d, J =

[0194] 8.5 Hz, 1 H), 6.69 (d, J = 2.4 Hz, 2H), 6.62-6.55 (4H, m) (the NMR spectra were in agreement with those reported by Yang et al., ACS Sens. 4(5): 1190-1196 (2019).

[0195]

[0196] A 100 mL round bottom flask was charged with 23a (0.535 g, 1.43 mmol) and DCM (20 mL). DIPEA (1 mL, 5.73 mmol) was subsequently added to the reaction mixture at RT. Inflate anhydride (0.58 mL, 3.44 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at RT for 8 h. After completion of the reaction as judged by TLC, the mixture was diluted with water and extracted with DCM. The organic layer was washed with brine. The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography over silica gel (10:1 , Hexane / EtOAc) to afford 23b as a white solid powder (0.26 g, 28%).1H NMR (500 MHz, CDCI3) δ 7.63 (d, 1.9 Hz, 1 H), 7.27 (dd, J = 2.1 , 8.3 Hz, 1 H),

[0197] 7.23 (d, J = 2.4 Hz, 2H), 7.09 (d, J = 8.2 Hz, 1 H), 6.99 (d, J = 2.4 Hz, 2H), 6.97 (d, J = 2.5 Hz, 2H), 6.91 (s, 2H), 6.90 ppm (s, 1 H).19F NMR (470 MHz, CDCI3) δ -72.66. HRMS (ESI) calculated [M+H]+for 637.97572; found: 637.97742.

[0198] A 100 mL round bottom flask was charged with 20 (0.72 g, 1.51 mmol), CuSO4-5 H2O (0.08 g, 0.30 mmol), NaAsc (0.12 g, 0.60 mmol) and 23a (0.68 g, 1.81 mmol) in 40 mL of H2O:EtOH (1 :1). The reaction mixture was stirred at RT for 24 h. After completion of the reaction as judged by TLC, the mixture was extracted with EtOAc and washed with brine (3×). The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by column chromatography over silica (1 :1 hexane / EtOAc) to obtain 24 as a yellow oil (1.21 g, 94%).1H NMR (500 MHz, CDCI3) δ 8.25 (1 H, d, J = 1.7 Hz), 8.19 (1 H, dd, 2.0, 8.3 Hz), 7.80 (1 H, s), 7.27 (4H, d, J = 9.0 Hz), 7.26 (4H, d, J = 2.3 Hz), 7.00 (2H, d, 2.4 Hz), 6.99 (2H, d, J = 2.4 Hz), 6.95 (2H, s), 6.93 (2H, s), 6.78 (1 H, d, 3.2 Hz), 6.42 (1 H, dd, 3.2, 8.8 Hz), 4.42 (2H, s), 3.65 (4H, t, 7.0 Hz), 3.55 (4H, t, 6.9 Hz), 3.48 (2H, t, 6.5 Hz), 2.76 (2H, t, J = 7.6 Hz), 1.73-1.64 (2H, m), 1.63-1.55 (2H, m), 1.38-1.23 (12H, m). HRMS (ESI) calculated [M+H]+for 849.18158; found: 849.19558

[0199] A 100 mL round bottom flask was charged with 24 (1.21 g, 1.42 mmol) and DCM (20 mL). DIPEA (1 mL, 5.7 mmol) was subsequently added to the reaction mixture at RT. Triflate anhydride (0.57 mL, 3.44 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at RT for 8 h. After completion of the reaction as judged by TLC, the mixture was diluted with water and extracted with DCM. The organic layer was washed with brine. The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography over silica gel (10:1 , Hexane / EtOAc) to afford 25 as a white solid powder (0.25 g, 16%).1H NMR (500 MHz, CDCI3) δ 8.25 (d, 1.6 Hz, 2H), 8.19 (q, J - 3.4 Hz, 2H), 7.80 (s,

[0200] 2H), 7.28 (t, 5.8 Hz, 6H), 7.26 (d, 2.4 Hz, 6H), 7.00 (d, 2.4 Hz, 1 H), 6.98 (d, 2.4 Hz, 2H), 6.95 (s, 2H), 6.93 (s, 1 H), 6.78 (d, J = 3.0 Hz, 2H), 6.42 (d, J = 11.9 Hz, 1 H), 6.41 (d, J = 5.7 Hz, 1 H), 3.65 (t, J = 7.0 Hz, 6H), 3.55 (t, J = 6.9 Hz, 6H), 3.48 (t, J = 6.5 Hz, 3H), 2.75 (t, J = 7.6 Hz, 3H), 1.68 (t, 7.3 Hz, 3H), 1.58 (t, 7.4 Hz, 3H), 1.37-1.24 ppm (m, 16H).13C NMR (125

[0201] MHz, CDCh) 6 166.10, 150.31 , 150.25, 149.44, 149.05, 138.39 138.24, 132.33, 128.82, 126.88, 126.35, 124.52, 118.92, 117.82, 117.49, 116.91 , 116.37, 115.22, 112.64, 112.28, 109.95, 79.39, 70.93, 70.17, 53.16, 52.41 , 38.75, 28.77, 28.68, 28.44, 28.42, 28.25, 28.16, 28.13, 25.29, 24.51.1SF NMR (470 MHz, CDCI3) δ -72.64. HRMS (ESI) calculated [M+H]+for

[0202] 1113.08015; found: 1113.09918.

[0203]

[0204] A 50 mL round bottom flask was charged with 25 (0.25 g, 0.22 mmol), bis(pinacolato)diboron (0.34 g, 1.35 mmol), KOAc (0.20 g, 2.02 mmol), and Pd(dppf)CI2(0.016 g, 0.023 mmol) in 1 ,4-dioxane (20 mL). The reaction mixture was stirred under argon at 85 °C for 16 h. After completion of the reaction as judged by TLC, the mixture was diluted with EtOAc and filtered. The filtrate was extracted with water and EtOAc and washed with brine (3*)- The organic layers were collected, dried over Na2SO4and concentrated. The resulting residue was purified by column chromatography (3:1. hexane / EtOAc) to afford 2 as a yellow oil (0.078 g, 31 %).1H NMR (500 MHz, CDCI3) δ 8.08 (q, 3.4 Hz, 1 H), 7.79 (s, 1 H), 7.69 (d, J = 0.8 Hz, 2H), 7.63 (d,

[0205] 8.4 Hz, 1 H), 7.39 (q, 3.0 Hz, 3H), 7.17 (t, J = 8.1 Hz, 1 H), 6.81 (s, 4H), 6.80 (s, 4H), 6.50

[0206] (q, 3.7 Hz, 1 H), 4.68 (s, 2H), 3.70 (t, J = 7.1 Hz, 5H), 3.56 (t, 7.1 Hz, 5H), 3.44 (t, 6.6

[0207] Hz, 2H), 2.74 (t, J = 7.6 Hz, 2H), 1 .71-1.63 (m, 3H), 1.61-1 .53 (m, 3H), 1 .36-1 .30 (m 6H), 1 .28 (s, 30H), 1.23 (s, 15H), 1.16 ppm (s, 8H).13C NMR (125 MHz, CDCI3) δ 167.20, 152.05, 149.53, 148.84, 146.92, 146.82, 139.81 , 137.59, 137.05, 132.16, 131.39, 130.99, 130.91 , 128.85, 128.51 , 126.33, 125.82, 124.38, 122.78, 119.41 , 117.82, 114.91 , 109.33, 108.79, 83.27, 82.12, 81.38, 75.78, 73.96, 70.88, 69.68, 52.39, 39.23, 28.98, 28.55, 28.48, 28.44, 28.31 , 28.24, 28.18, 25.39, 24.57, 23.84. HRMS (ESI) calculated [M+H]+for 1117.53687; found: 1117.55805.

[0208] Synthesis of Other Molecules

[0209]

[0210] A solution of 7 (1.0 g, 3.20 mmol) in dry tetrahydrofuran (THF) (20 mL) was cooled to -78 °C under argon. Then, n-BuLi (2.05 mL, 2.5 M in hexane) was added slowly at -78 °C within 10 min. After 30 min, trimethylsilyl chloride (0.81 mL, 6.41 mmol) was added to the solution. The reaction mixture was allowed to warm to RT, stirred for another 6 h, quenched with saturated NH4CI solution at 0 °C, then extracted with DCM (3* 75 mL), washed with water, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (45:1 hexane / EtOAc) to afford 11 as an oil (0.54 g, 62%).1H NMR (500 MHz, CDCI3) δ 7.38 (d, J = 8.5 Hz, 1 H), 6.84 (d, J = 3.3 Hz, 1 H), 6.51 (dd, 3.3, 8.1 Hz, 1 H), 4.64 (s, 2H), 4.29 (s, 2H), 3.75 (t, J = 6.7 Hz, 4H), 3.65 (t, J = 6.3 Hz, 4H), 0.23 (s, 9H).13C NMR (125 MHz, CDCI3) δ 145.99, 137.91 , 133.32, 112.77, 112.72, 109.99, 101.12, 92.11 , 71.02, 58.63, 53.52, 40.39, -0.14. HRMS (ESI) calculated [M+H]+for 436.02604; found: 436.02657

[0211] A solution of 9 (0.33 g, 0.58 mmol) in DCM (10 mL) was added with DI PEA (0.15 mL, 0.87 mmol) at RT. Then, triflate anhydride (0.12 mL, 0.70 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at RT for 8 h. After completion of the reaction as judged by TLC, the mixture was diluted with water and extracted with DCM. The organic layer was washed with brine. The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography over silica gel (7:1 , hexane / EtOAc) to afford 13 as a yellow solid powder (0.25 g, 61 %).1H NMR (500 MHz, CDCI3): 5 8.65 (s, 1 H), 8.57 (s, 1 H), 7.74 (s, 1 H), 7.72 (s, 1 H), 7.34 (d, J ~ 2.2 Hz, 1 H), 7.31-7.28 (2H, m), 4.78 (s, 2H), 4.59 (s, 2H), 3.68 (t, J = 6.6 Hz, 4H), 3.57 (t, 6.6 Hz, 4H).13C NMR (125 MHz, CDCI3) 5 153.65, 151.87, 150.25,

[0212] 144.53, 136.90, 132.38, 130.31 , 129.50, 122.82, 122.74, 118.93, 118.01 , 117.10, 116.38, 111.93, 111.74, 109.52, 109.06, 70.98, 62.79, 52.58, 39.37.19F NMR (470 MHz, CDCI3) δ -72.36. HRMS (ESI) calculated [M+H]+for 698.96888; found: 698.97879.

[0213] Other Synthetic Routes

[0214] The following schemes provide synthetic routes or propose synthetic routes for additional theranostic molecules. Scheme 4

[0215] Proposed Synthetic Route for Theranostics 10a-f Scheme 5

[0216] Synthetic Route for Theranostic 11a-c

[0217]

[0218] Scheme 6

[0219] Synthetic Route for Theranostics 12b

[0220]

[0221] Scheme 7

[0222] Proposed Synthetic Route for Theranostic 13 instruments and Reagents for Assays

[0223] Confocal imaging was performed on a Zeiss LSM 710 confocal laser scanning fluorescence microscope with 63× oil-immersion objective lens or EVOS-FL on 40x coverslip corrected objective lens. CellTiter-Glo® (cell viability) assay was performed with Tecan Freedom EVO liquid handling system equipped with a 100 nL pin tool (V&P Scientific) and the resulting luminescence was measured with Infinite M1000 (Tecan) plate reader. In-solution fluorescence assay was performed on Perkin Elmer LS 55. UV / VIS absorbance assay was performed on Shimadzu UV2401. Quantification of radioactive (32P) DNA was performed on a Storm 860 Molecular Dynamic Phosphorimager equipped with ImageQuantVersion 5.2 software.1H-NMR and13C-NMR was performed on 500 MHz Bruker and analyzed with TopSpin 3.6.5. HRMS was performed on Shimadzu 9030 triple quad TOP. The molecule’s purity was determined by an ACQUITY CSH C18 column (2.1 mm x 50 mm, 1.7 pm particle size) using gradient: 0-10 min 5%-100% MeCN in A, 10.0-12.0 min 100% MeCN in A, 12.0-13.0 min 100%-5% MeCN in A, 13.0-15.0 min 5% MeCN in A at a flow rate of 0.5 mL / min (solution A: water).

[0224] Concentration Dependent DNA Cross-Linking Assay with Duplex DNA

[0225] ICL formation and cross-linking yields were analyzed using denaturing polyacrylamide gel electrophoresis (PAGE) followed by phosphorimager analysis. The DNA cross-linking abilities of the theranostic molecules were investigated using a32P-labeled 49-mer oligonucleotide (28; Table 2) then subjected to 20% denaturing PAGE analysis. The32P-labelled oligonucleotide (1.0 μM) was annealed with 1 .5 equiv. of the complementary strand by heating to 90 °C for 5 min in PBS (10 mM, pH 8) and 100 mM NaCI, followed by slow cooling to RT overnight. The32P-labeled duplex DNA (2 μL, 1.0 μM) was mixed with 1.0 M NaCI (2 μL), 100 mM potassium phosphate (2 μL, pH 8), H2O2(2, 4 or 6 equivalence in 2 μL) and 333 μM to 3.33 mM 1a, 1c, or 2 in DMSO (6 μL, resulted in a concentration range of 100 μM to 10 mM) and H2O to give a final volume of 20 μL. The reaction mixture was incubated at RT for 16 h and quenched by an equal volume of 90% formamide loading buffer, then subjected to 20% denaturing polyacrylamide gel analysis.

[0226] Interstrand Cross-Link Formation and Time-Dependent Kinetic Study with Duplex DNA

[0227] Aliquots containing 0.1 μM32P-labeled oligonucleotide duplex 28, 100 mM NaCI, 10 mM potassium phosphate buffer (pH 8), and 0.5 mM or 1 mM of 1a, 1c, or 2 with H2O2(2, 4 or 6 equivalence) were incubated at RT or 37 °C for various time periods and quenched by 90% formamide loading buffer and stored at -20 °C until 20% denaturing PAGE analysis. Determination of DNA Monoalkylations

[0228] A 60 μL solution containing 0.1 μM32P-labeled oligonucleotide duplex 28, 100 mM NaCI, 10 mM potassium phosphate buffer (pH 8), and H2O2(2, 4, or 6 mM) and 1 mM of 1a, 1c, or 2 were incubated at RT for 16 h. Then, DNA 28 was precipitated out of the solution by addition of 20 μL calf-thymus DNA (25 pg / mL), 10 μL NaOAc (3 M) and 270 μL of cold ethanol. The resulting mixture was inverted several times, incubated at -80 °C for 30 mins, then centrifuged for 5 mins at 12,500 rpm. The resulting supernatant was discarded, and the DNA pellet was again subjected to second precipitation. The purified DNA was dissolved in 45 μL of autoclaved distilled H2O and separated into three aliquots. One aliquot (5 μL) was used as a control without further treatment, the second aliquot (20 μL) was heated in PBS (pH 7.0) at 90 °C for 30 mins, and the third was heated in 1.0 M piperidine (10 μL) at 90 °C for 30 mins. The mixtures were dried in vacuo and quenched by an equal volume of 90% formamide loading buffer and autoclaved water, then subjected to 20% denaturing polyacrylamide gel analysis.

[0229] Cytotoxicity Evaluation in Cells

[0230] Cell Culture

[0231] The triple-negative breast cancer cell line MDA-MB-468 and the normal cell line MCF10A were purchased from the American Type Culture Collection. MDA-MB-468 cells were cultured in L-15 Leibovitz media (Thermo Scientific: 41300070), complemented with non-essential amino acids (100× solution, Hyclone: SH30238.01 , 5.0 mL), fetal bovine serum (Biowest: SI 620, 50 mL), penicillin, and streptomycin (100* solution, Hyclone: SV30010, 5.0 mL), and maintained at 37 °C in an environment of 100% air at 100% relative humidity. MCF10A cells were sustained in Lonza media kit MEGM (CC-3150), supplemented with 100 ng / mL Cholera toxin (Sigma Aldrich: C8052), within a 5% CO2incubator at 37 °C.

[0232] Cell Viability (Cytotoxicity) Assay

[0233] The cells were inoculated into 96-well microtiter plates in 40 μL plating densities ranging from 3,500 to 10,000 cells / well. After cell inoculation, the microtiter plates were incubated at 37 °C for 1 h prior to the addition of the theranostics. Theranostic molecules were dissolved in DMSO at 20 mM and serially diluted 10—12 times by 50%. 400 nanoliters of the serially diluted solutions was added to the cell plate (1 :100 dilution) using Tecan Freedom EVO liquid handling system equipped with a 100 nL pin tool (V&P Scientific) resulting in final concentrations of 0.195 μM to 200 μM or 0 μM as the control. Following addition, the plates were incubated at 37 °C for 48 h. Then, aliquots of 30 μL CellTiter-Glo® (Promega) were added to the wells and the resulting plate was incubated for 15 minutes. The plate was then measured for luminescence by Infnite M1000 (Tecan) plate reader.

[0234] H2O2Measurement

[0235] The H2O2levels were determined using an Amplex Red Hydrogen Peroxide Assay (Invitrogen, A22188) as per the manufacturer's protocol. Cellular measurements: 25 x 103-50 × 103cells were plated into 384 Well Black, Optically Clear Polymer Bottom Plate (Thermo Scientific Catalog: 142761) in 40 μL(Final volume). After 48 h of incubation, the cells were washed twice with 1 x KRPG buffer (Krebs-Ringer phosphate consists of 145 mM NaCI, 5.7 mM sodium phosphate, 4.86 mM KCI, 0.54 mM CaCI2, 1.22 mM MgSO4, 5.5 mM glucose, pH 7.35) and incubated for 5 h in 40 μL KRPG buffer. Then, 20 μL of this KRPG buffer was transferred into another 384 Well Black, Optically Clear Polymer Bottom Plate (Thermo Scientific) in triplicate and mixed with an equal amount of Amplex Red reagent (50 μM Amplex Red and 0.1 U / mL HRP final concentrations). After 5 h incubation, fluorescence was measured (Ex / Em: 560 / 590 nm) on an infinite M1000 (Tecan) microplate reader. Standard curve measurements: 10 μM stock of freshly prepared H2O2in KRPG buffer was serially diluted 2-fold into 4 different concentrations and a blank with KRPG buffer. Then, 20 μL of this KRPG buffer was transferred into another 384 Well Black, Optically Clear Polymer Bottom Plate (Thermo Scientific™ Catalog: 142761) in triplicate and mixed with an equal amount of Amplex Red reagent (50 μM Amplex Red and 0.1 U / mL HRP final concentrations). After 30 min incubation, fluorescence was measured (Ex / Em: 560 / 590 nm) on an infinite M1000 (Tecan) microplate reader. The final concentration of H2O2was adjusted by two-fold to account for the dilution by the same volume of amplex red reagent.

[0236] Alkaline Comet Assays

[0237] The comet assay was performed according to manufacturer’s protocol (Abeam: ab238544). Cells were seeded in 6-well Tissue culture, surface treated, sterile, clear plates (VWR: 10062-892) at a cell density of 105 ceils per well. Upon 90% confluency, cells were treated at varied conditions for 48 h. Cells were gently removed from the 6-well plate by scraping with a rubber policeman in 1 mL ice-cold PBS (without Mg2 hand Ca2f). Cell suspension was centrifuged at 700 x g for 5 mins. Supernatant was discarded. Finally, cells were resuspended in PBS and further diluted to obtain 1 x 105cells / mL. Cell samples (20 μL) were mixed gently with the warm agarose (180 μL, 37 ° C). 150 μL / well of this mix was transferred onto a pre-warmed glass slide and maintained at 4 ° C in the dark for 30 minutes to let the agarose solidify. The slides were carefully immersed into a small basin containing pre-chilled lysis buffer at 4 °C for 3 h in the dark to lyse the cell membrane. The Lysis buffer was then replaced with pre-chilled alkaline unwinding solution (300 mM NaOH, 1 mM EDTA) at 4 °C for 30 minutes in the dark to denature DNA. The slides were then gently transferred into a horizontal electrophoresis chamber filled with pre-chilled alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH > 13). A voltage of 35 V was applied for 30 minutes. The slides were then removed and immersed slowly to rinse twice with pre-chilled DI water for 2 minutes followed by cold 70% ethanol for 5 minutes. The slides were allowed to air dry for 1 h in the dark. 100 μL / well of diluted Vista Green DNA dye was added onto the agarose. The slides were then incubated at room temperature for 15 minutes. Comets were analyzed under an EVOS FL Digital Inverted Microscope at 10× magnification and DNA damage was qualified using TriTek CometScore Software.

[0238] Fluorescence Measurements of DNA Alkylations Induced by 1a, 1c, and 2 in Living Cells

[0239] Approximately 1.2 million MDA-MB-468 cells were plated on 6-well plates containing L-15 media and incubated overnight. The media was replaced with fresh L-15 (1 mL). Then, the cells were treated with either 1a, 1c, 2, MitoTracker Red CMXRos, SYTO 9 or Hoechst 33342 (50 μM) or DMSO (1 % v / v) by incubation at 37 °C for 20 h. The resulting cell cultures were trypsinized, transferred into 5 mL centrifuge tubes and the media was replaced with 100 μL PBS (pH 7.4), 100 μL ceil lysis buffer and 5 μL of proteinase K (20 mg / mL). The resulting solutions were heated at 56 °C and periodically vortexed for 1 h. The DNA in solution was extracted with phenol / chloroform / isoamyl alcohol (25:24:1). The aqueous phase was collected and dried overnight in vacuo to produce a solid mixture. The mixture was resuspended by addition of 45 μL H2O, 15 μL NaCI (5 M) and 300 μL cold ethanol. DNA was subsequently precipitated from the solution by incubation at -80 °C for 30 minutes. The mixture was centrifuged at 12,500 rpm for 5 mins and the supernatant was discarded. The resulting DNA was precipitated from the mixture three times, then further purified by size-exclusion chromatography using Sephadex G-25. The final DNA product was dried in vacuo overnight. The isolated DNA was dissolved in 80 μL DMSO and 720 μL PBS (pH 7.4) for spectroscopic measurements. DNA absorbance and fluorescence were investigated via Shimadzu 2401 and Perkin Elmer LS 55 using Aex and Aem 12.0 nm slit width.

[0240] Colocalization Imaging

[0241] General methods for CLSM (Confocal Laser Scanning Microscope) imaging. MDA-MB- 468 cells were plated on 22 mm glass coverslips and incubated overnight. Then, the L-15 media was replaced, and the cells were incubated with 5 μM of 1a, 1c, or 2 in DMSO / L-15 (1 % v / v) at 37 °C for 2 h. After washing with PBS (pH 7.4) (3×), the cells were subjected to CLSM imaging on Zeiss LSM 710 for 1a and 2, and EVOS-FL for 1c. Theranostic 1a was measured at excitation wavelength of 405 nm and an emission detection range of 435-542nm, 1o was measured at excitation wavelength of 360 nm and an emission detection of 447 nm, and 2 was measured at excitation wavelength of 488 nm and an emission detection range of 513-590 nm. For the colocalization experiments, the cells were loaded with MitoTracker Red CMXRos (200 nM) or SYTO 9 (500 nM) or Hoechst 33342 (500 nM) for another 30 mins. MitoTracker Red CMXRos was excited at 561 nm and emission was collected at 595-697 nm; SYTO 9 was excited at 488 nm and emission was collected at 495-542 nm; Hoechst 33342 was excited at 405 nm and emission was collected at 416-590 nm on Zeiss LSM 710. SYTO 9 was excited at 470 nm and emission was collected at 525 nm; MitoTracker Red was excited at 530 nm and emission was collected at 593 nm on EVOS-FL. The resulting images were analyzed using Imaged and Pearson’s correlation coefficient was calculated using JaCoP.

[0242] Theranostic Responsiveness Towards Exogenous H2O2in Living Cells

[0243] MDA-MB-468 cells were incubated with 1c or 2 (5 μM) at 37 °C for 0.5 h. Then, 100 μM H2O2was added to the samples that were further incubated for an additional 1.5 h. For colocalization analysis, cells were co-labeled with either MitoTracker Red, SYTO 9 or Hoechst 33342 at the last 0.5 h of incubation time. The resulting live cells were imaged on EVOS-FL or Zeiss LSM 710.

[0244] Permeability Assay

[0245] The permeability was determined by parallel-artificial-membrane permeation assay that was performed with the Millipore MultiScreen filter plates (MAIPNTR10) and Millipore transport receiver plates (MATRNPS50) using a 5% by volume n-hexadecane in n-hexane solution to create the artificial layer. The absorbance readings were completed with a Corning Costar 96 well UV plate. 1 x phosphate buffered saline (PBS) was prepared in 500 mL batches using 18 MQ water and adjusted to pH 7.4 or pH 6.4 with HCI and NaOH. All the absorbance readings were performed on an Infinite M1000 plate reader.

[0246] The artificial membrane was prepared by carefully pipetting 15 μL of the 5% (v / v) hexadecane in hexane solution to each of the wells of the donor plate. The plate was placed in a fume hood for 3 h to ensure complete evaporation of the hexane. Then, 300 μL of PBS with 5% (v / v) DMSO was added to each of the wells of the acceptor plate. The hexadecane treated donor plate was then placed on top of the acceptor plate to ensure that the underside of the membrane is completely in contact with the solution in each of the acceptor wells. Each compound’s solution was prepared in triplicate in a separate 96-weil plate to 300 μM (9 μL of 10 mM compound solution in DMSO, 6 μL DMSO, and 285 μL buffer). Then, 150 μL of the compound solution was added to the donor wells. For each plate, carbamazepine (medium-high permeability), verapamil (high permeability), and ranitidine (low permeability) were used as standard molecules for reference.

[0247] The lid was placed on the plates and the entire plate sandwich was placed into a closed container with a wet paper towel along the bottom to circumvent evaporation during the incubation process. The container was then placed on a reciprocal shaker for agitation at about 100 rpm. The time at the beginning of the incubation was recorded, as this is a thermodynamic-based assay. The incubation was then allowed to continue for approximately eighteen hours.

[0248] The next day, the plates were removed from the incubation container and the time of the end of the incubation period was noted. The donor plate was removed and 50 μL of the acceptor solution was transferred to the UV plate. Drug solutions at the theoretical equilibrium concentration (300 μM) was also prepared and transferred to the UV plate. The absorbance of the solutions in the UV plate was then scanned from 250-600 nm with 1 nm steps and a 5 nm bandwidth.

[0249] The relative permeability (cm / s) of the small molecules was calculated with Equation 2: where VDis the volume of the donor well in cm3(150 μL), VAis the volume in the acceptor well in cm3(300 μL), A is the active surface area of the membrane in cm2(0.283 cm2), T is the incubation time of the assay in seconds, is the absorbance of the compound in the acceptor well after the incubation period, and is the absorbance of the compound at the concentration of the theoretical equilibrium (as if the donor and acceptor solutions were simply combined). The equation is derived from equation 2 shown above, described previously, in which the change in concentration of the solute is time dependent.

[0250] Solubility Assay

[0251] About 2 mg of each compound was added to 500 μL of PBS buffer at pH 7.4, vortexed for 10 s, sonicated for 2 min and agitated with a reciprocal shaker in a closed vial for 24 h. The mixtures were transferred to an Eppendorf tube and centrifuged for 5 min at 16000 x g followed by filtration through 0.22 pm cellulose acetate spin X centrifuge filter (Costar). To 200 μL of filtrate transferred to a new Eppendorf tube, 200 μL of acetonitrile was added. Then, the sample was diluted subsequently in 50:50 acetonitrile / PBS buffer if necessary. After mixing, 50 μL of the sample was transferred into a 384 well plate (Coring UV star, 781801) for UV detection at 250- 600 nm (Tecan M1000). The assay was carried out with three independent samples of each compound. The concentration of each solution was determined with a calibration curve in 50:50 acetonitrile / PBS buffer. The absorbance of the corresponding acetonitrile / PBS blank were recorded and subtracted from the absorbance of calibration curve solutions and from the samples.

[0252] Example 2

[0253] Design and Synthesis of H2O2-Activated Theranostic Agents

[0254] Previous studies have shown that FAN-NM-CH3was the most promising H2O2-activated anticancer prodrug that showed in vivo efficacy and selectivity with drug-like property. FAN-NM- CH3was used as the parent compound for designing H2O2-responsive theranostics. The four designed theranostic agents 1a-c and 2 contain an aromatic nitrogen mustard moiety conjugated with a fluorescent reporter, either a hydroxycoumarin- (1a-c) or fluorescein-derived moiety (2), through a copper-catalyzed azide alkyne click (CuAAC) reaction. Nitrogen mustard moiety acts as an effector that can cross-link DNA via alkylation while the fluorescence group can monitor H2O2-induced alkylation via fluorescence detection. Compound 1a was synthesized starting from 3a (Scheme 8). First, the carboxylic acid analogue 3a was converted to benzyiic alcohol 3b by reduction with borane in THF. Then, the amine group of 3b was protected by dimethyl-formamide dimethyiacetal (DMF-DMA) to form an amidine intermediate 3c, which underwent selective alkylation at the benzyiic alcohol position with propargyl bromide to afford propargyl ether 4. A subsequent deprotection of 4 by ethylenediamine (en) restored the free amine 5 that was converted to N,N-bisalkylation product 6 in 52% yield in the presence of 2-chloroethanol under basic conditions. Formation of the mono-alkylated intermediate was also observed from the reaction, which can be further converted to 6 by repeat treatment with 2-chloroethanol. The diol 6 was transformed to bischloride 7 via mesylation followed by chlorination. 7-Hydroxycoumarin azide 3 was conjugated with 7 via CuAAC cycloaddition reaction with copper(ll) sulfate pentahydrate and ascorbic acid yielding the coumarin mustard conjugate 9. Compound 8 was prepared as described in Scheme 1 . Finally, Miyaura borylation of 9 afforded the target molecule 1a.

[0255] Scheme 8. Synthesis of 1a.

[0256]

[0257] The goal was to synthesize diboronate theranostic 1b consisting of both inactivated fluorophore and masked nitrogen mustard. However, significant challenges were faced due to the incompatibility of CuAAC and palladium catalyzed borylation reaction. For example, borylation of 7 resulted in dimerization between two alkyne functionalities forming the dimeric product 10 (Scheme 9). Attempts to prevent formation of 10, such as protection at the terminal alkyne with TMS group (→ 11) followed by borylation through either lithiation or Pd-catalyzed reactions failed to produce the desired product 1b. The conversion of 9 to the diboronate ester 1b via a triflate intermediate 13 was attempted. However, deboronation product 14 was observed after column chromatographic isolation on silica, which was caused by protodeboronation reaction. In another attempt to purify the final product by recrystallization, only the cyclization product 15, was isolated and identified.

[0258] Scheme 9. Failed attempts to convert 7 or 9 to 1b.

[0259] To prevent cyclization and stabilize the resulting diboronate product, 1c was designed with an extended hydrocarbon linker between phenyl bromide and alkyne functional group. Unlike the monoboronate 1a with a persistent active fluorescence, the diboronate ester 1c consists of both an inactive mustard and coumarin fluorophore, giving 1c bath profluorogenic and prodrug properties. Simiiar to the synthesis of 9, compound 21 was prepared starting from 3c via ether formation (→ 17) by treatment with 11 -bromoundecyne 16 (Scheme 10). Preparation of 16 was performed as described (Scheme 2). The following reactions of 17 involved deprotection (-->18), N,N-bisalkylation (→ 19), chlorination (→ 20), followed by CuAAC reaction to generate the hydroxy coumarin analogue 21. Compound 21 was converted to trifiate derivative 22 that underwent boryiation yielding 1o. Due to the modularity of click reaction, other fluorescent molecules can be appended to the nitrogen mustard scaffold and readily access other theranostic molecules. For example, fluorescein analogue 2 was synthesized via CuAAC reaction between fluorescein azide 23a and the alkynyiated nitrogen mustard 20 (→ 24), followed by triflation (→ 25) and Miyaura borylation (→ 2). Alternatively, 23a can be first triflated to furnish 23b and subsequently conjugated with 20 via CuAAC reaction to form 25, which affords a higher total yield towards the synthesis of 2.

[0260] Scheme 10. Synthesis of Profluorogenic Theranostic 1o and 2.

[0261] The Chemical Reactivity and Selectivity of 1a, 1c, and 2 towards DNA

[0262] It has been well established that DNA alkylation was the source of the cytotoxicity of nitrogen mustard analogues. Thus, the reactivity and selectivity of 1a, 1c, and 2 were studied by determining their capability to form DNA interstrand cross-link (ICLs) or mono-alkylation using a 49-mer DNA duplex 28 in the absence or presence of H2O2(FIG. 1). The reaction of 1a, 1c and 2 with duplex 28 was carried out at 3:7 ratio of DMSO to a pH 8 phosphate buffer. The organic solvent DMSO helps to dissolve the monomers while phosphate buffer is similar to physiological condition with a more aqueous phase (i.e., 60-70% water in cells). An optimal ratio of 2:1 for H2O2to compound was used for DNA alkylation assay. No ICL formation was observed with 1a without H2O2(FIG. 1 lane 4) while a combination of 1a (1.0 mM) and H2O2(2.0 mM) led to 32% ICL yield (FIG. 1 lane 3). These data suggested that 1a is an effective H2O2-activated DNA interstrand cross-linking agent with the boronate group capable of masking the reactivity of nitrogen mustard while allowing its activation by H2O2. Time-dependent study suggested that the ICL reaction of 1a was completed within 16 h at RT while a slightly quicker ICL reaction (12 h) was observed at physiological temperature 37 °C. A separate study of increasing H2O2and 1a concentrations showed that the maximum ICL yield (31.6 ± 0.6%) was achieved with 1 mM 1a and 2 mM of H2O2after 16 h incubation at RT while less than 1 .2 ± 0.8% ICL yield was observed without H2O2(n = 2, FIG. 1).

[0263] In contrast, no ICL products were detected for 1c and 2 even with extended reaction time (24 h) and / or elevated temperature (37 °C) (FIG. 1 lanes 5 and 7). Efficient DNA ICL formation observed for 1a but not for 1c and 2 suggested that the extended hydrocarbon linker and large side chains may prevent alkylation from occurring due to the increased steric hindrance preventing efficient interaction between these molecules and DNA. However, the DNA ICL assay cannot tell whether mono-alkylation occurred with 1c and 2 as the mono-alkylated products cannot be detected by this cross-linking assay. It is well established that nitrogen mustard analogues mainly alkylate DNA at the nucleophilic N7-position of purines producing N7-alkylated dG or dAs that easily undergo deglycosylation upon heating under basic conditions leading to the cleavage of DNA strands. Thus, the heat stability of DNA duplex 28 was studied after treatment with 1a, 1c, and 2. Duplex 28 was first incubated with these compounds in the presence of H2O2for 16 h, then precipitated to obtain pure DNA that were heated to 90 °C in 1.0 M piperidine for 30 min (FIG. 2). The DNA cleavage bands were observed at dG and dA sites for all three compounds but not for controls (FIG. 2), which suggested that alkylation reaction of 1a, 1c, and 2 occurred with DNA at G-A sites. However, DNA cleavage induced by 1c (41%) and 2 (53%) were in a lesser extent than those induced by 1a (71 %). Collectively, these data suggested that 1c and 2 can be activated by H2O2to induce DNA mono-alkylation but not DNA interstrand crosslinking, which is likely due to enhanced steric hindrance introduced by the extended side chain.

[0264] Efficient Oxidative Deboronation is crucial for Activation of Nitrogen Mustard To determine the effect of the side chains on the release of the active theranostic, the chemical reactivity of these compounds with H2O2was determined. Compounds 1a, 1c, and 2 was incubated in the presence of 1 .5 equivalence of H2O2in a mixture of DMSO-d6 (450 μL) and a pH 8 phosphate buffer (50 μL) made with D2O. The reaction was monitored by1H NMR analysis for 3-8 hours. The shift of CT-H (1a) from δ 1.239 ppm to 1.149 ppm (27) suggested that oxidative deboronation efficiently occurred with 1a to yield the active theranostic 1a". Hydroxyl pinacolborane 26 spontaneously underwent hydrolysis to generate dihydroxyl pinacol 27 (5 1.064 ppm) and boric acid. After 1 hour, 89% of 1a boronate ester was consumed and converted to the phenol product 1a". The pseudo first-order reaction rate of deboronation was calculated based on the C1 '-H (1a) at 5 1.2396 ppm of the phenyl boronate ester 1a, determined as of 3.7 x 10-4s-1. A similar phenomenon was observed with 1c and 2, which showed efficient oxidative deboronation reactions with a reaction rate of 1.85x 10-4s-1for 1c and 5.47 x 10-4s-1for 2 determined at 1.2314 ppm (1c) and at 1.2223 ppm (2), respectively.

[0265] Scheme 12. H2O2-lnduced deboronation reaction of 1c and 2 [the reaction of 1c and 2 (4.47μM) with 3 eq. of H2O2or 4.5 eq. H2O2, respectively was monitored by1H-NMR spectroscopy.

[0266] The kinetics of H2O2-induced deboronation of the profluorophore functionalities was undetermined for 1c to 1c” and 2 to 2” based on the boronate ester peaks due to the overlapping of C14 -H (1c and 2) and the methyl group of the dihydroxy pinacol 27 at around 1.06 ppm (Scheme 12). As an alternative, the shift of C12 -H (1c) and C13 -H (1c) (ortho to the boronate ester) at about 7.80 ppm was observed to have moved upfield at 6.60 ppm. This suggested efficient activation of 1c’s coumarin fluorophore by H2O21which was observed to disappear over time with a rate of 3.12 x 10-4s-1. Similarly, the neighboring ortho protons C16 -H (2) at approximately 6.91 ppm transitioning to C16”-H (2) at 6.67-6.73 ppm were used to evaluate the reaction rate of fluorescein activation, which was calculated at 2.57 x 10-4s-1. The data collectively suggested fast rates of activation or deboronation of all three theranostics by H2O2with an order of 1c > 1a > 2 for the activation of the boronate aromatic mustard. However, it took 16-hour incubation time to reach the maximum DNA ICL yields at RT, which is much slower than the oxidative deboronation reaction. Both data imply that the rate limiting step in ICL formation are alkylation events.

[0267] In vitro Cytotoxicity and Selectivity

[0268] After having demonstrated that 1a, 1c, and 2 can be activated by H2O2to release the reactive alkylating nitrogen mustard species, the anticancer effect and selectivity was assessedwith triple negative breast cancer (TNBC) cell line MDA-MB-468 and normal human mammary epithelial MCF-10A cells (incubated in 5% CO2) (FIG. 3). Cells were treated with 1a, 1c, and 2 at various concentrations (0-200 μM) and incubated for 48 h at 37 °C. Then, CeliTiter- Glo® assay was performed to determine cell viability. Compounds 1a and 1c showed concentration-dependent antiproliferative effects against MDA-MB-468 cells with an IC50of 3.9 ± 0.6 μM and 4.2 ± 1.5 μM for 1a and 1c, respectively (n ~ 4) (FIG. 3A). However, the IC50of 2 was not achieved with MDA-MB-468 cell. The cell viability did not appreciably change when the MDA- MB-468 cancer cells were treated with 2 at concentrations higher than 3.125 μM. Compound 2 afforded slightly higher alkylation yields than 1c, which was expected to cause higher in vitro cytotoxicity; however, 2 displayed despairingly lower cell inhibition compared to 10. Other factors, such as water solubility, cell membrane permeability, etc. might affect the cellular toxicity in the biological setting besides DNA-alkylation. The poor water solubility of 2 is likely one of the causes leading to its low cytotoxicity (Table 3). The aqueous solubility decreases in the order of 1a, 1c and 2. Permeability is also an important factor in determining whether the molecules can enter in the intracellular matrix. To this end, parallel-artificial-membrane permeation assays (PAMPA) were performed at physiological pH which showed that most to least permeable were in the order of 1c, 2 and 1a (1c > 2 > 1a). A lower cell permeability of 2 than 1c may be one of the reasons for its lower cytotoxicity. In contrast, the enhanced permeability of 1c likely increased its anticancer effect. Although 1c showed a lower alkylating capability than 1a, similar cytotoxicity was observed with both 1c and 1a. Due to the ionizable capability of the A / ,A / ,A / -triazole moiety, and the fact that existing data suggests some malignant cancer cells may lower the pH of their microenvironment in contrast to normal cells, permeability was also measured at pH 6.4. Slightly reduced permeability far all compounds were observed at pH 6.4 in comparison to pH 7.4.

[0269] Nevertheless, the permeability order is the same under both conditions (1c > 2 > 1a).

[0270] To evaluate the selectivity of these compounds to cancer cells, human mammary epithelial MCF-10A cells were tested for toxicity with 1a and 1c through cell viability assay. Both compounds showed significantly reduced cytotoxicity towards MCF-10A cells (FIG. 3B) in comparison to MDA-MB-468 cancer cells (FIG. 3A). In particular, 1c is 12 times more cytotoxic toward cancer cells (IC50of 4.2 ± 1 .5 pM) than normal cells (IC50of 49.7 ± 2.1 pM) (FIG. 3A-B). Having established that these compounds showed selective cytotoxicity toward MDA-MB-468 cells but spare normal cells, the correlation between the efficacy and H2O2level was investigated. Thus, the H2O2level in MDA-MB-468 and MCF-10A cells were determined using the Amplex Red hydrogen peroxide assay kit (Invitrogen, A22188). After incubation of ~ (25-50) x 103cells at 37 °C for 5 h, MDA-MB-468 cells showed ~2 pM H2O2level that was approximately 2-fold higher than that observed in the normal MCF-10A cells (1 pM) (FIG. 4A). To quantify the H2O2released by both cell lines, a H2O2standard curve was prepared (FIG. 4B). The respective H2O2concentrations released by cells were calculated using the linear fit equation of the line, it is important to note that the prepared samples were combined with an equal amount of Amplex Red reagent before the assay, resulting in a two-fold adjustment to the final H2O2concentration.

[0271] Collectively, these data support that the premise of higher H2O2release in cancer cells relative to normal cells is essential for the H2O2-induced cytotoxicity and selectivity of these prodrugs (1a and 1c) towards cancer cells. One possible scenario is that H2O2accumulation in cancer cells activates prodrugs to release alkylating intermediates that induce DNA damages to drive extensive cell death. DNA damage, such as DNA ICL formation, DNA alkylation, and strand breaks, pose a significant hinderance to both replication and transcription processes that are essential for proper functioning of cells. Consequently, the cytotoxicity and selectivity of these compounds can potentially be associated with their capacity to induce DNA damage in cancer cells. An alkaline comet assay was performed with both MDA-MB-468 cells and normal MCF- 10A cells using OxiSelect Comet Assay Kit (abeam: ab238544). Cells were treated with 5 μM and 10 μM concentrations of 1a and 1c at 37 °C for 48 h and analyzed with an EVOS Digital Inverted Microscope at 10× magnification and TriTek CometScore Software (FIG. 5A-B). These data suggested that DNA damage was observed with MDA-MB-468 cancer cells treated with 1a and 1c but not with normal MCF-10A cells treated under the same conditions (FIG. 5A). Both 1a and 1c induced substantial amount of tail DNA % and olive moment in MDA-MB-468 cancer cells in a concentration dependent manner, whereas such observations were minimal or not with normal MCF-10A cells (FIG. 5B). These findings provide compelling evidence that 1a and 1c induce cellular DNA damage in cancer cells as a result of H2O2activation, which underscores a strong correlation between H2O2level, DNA damage, and their respective cytotoxicity.

[0272] Furthermore, combination of a H2O2-amplifying agent, such as paraquat (PQ), greatly enhanced the anticancer effect of 1a and 1c. For example, the IC50of 1a in MDA-MB-468 cell line decreased from 3.9 μM in the absence of PQ to 1 .7 ± 0.4 μM in combination with a safe dose of PQ (6 μM) (FIG. 3A, C). The same phenomenon was observed with 1c that showed approximately 50% decrease of the IC50in the presence of 6.0 μM PQ (FIG. 3A). The later study suggested that PQ greatly promoted the fluorescence response of 1a and 1c in MDA-MB-468 cells suggesting the accumulation of H2O2induced by PQ (FIG. 12 and 13). In sum, these data provide clear evidence that H2O2accumulated in cancer cells actively contributed to the treatment efficacy of these H2O2-activated prodrugs.

[0273] Spectroscopic Properties of 1a, 1c, and 2

[0274] To investigate the diagnostic potential of prodrugs 1a, 1c, and 2, the spectral properties, including UV / Vis and fluorescence spectroscopy were determined. Both 1a and 1c containing coumarin fluorophore showed a maximum absorption in the UV region (Table 4). Compound 1a in its original form displayed fluorescence with an emission wavelength of 480 nm, a quantum yield of 0.17, and fluorescence brightness of 22 without addition of H2O2(Table 4 and FIG. 6). In contrast to 1a, the fluorescence emission of coumarin in 1c is quenched due to the presence of an electron-withdrawing boronate ester group, which allows its activation by H2O2to turn on the fluorescence. The quantum yield and fluorescence brightness of 1c increase approximately by a factor of 2 after the addition of H2O2. The boron masked fluorescein analogue 2 showed no absorption in the visible region of 400-600 nm as well as no fluorescence. However, H2O2- induced deboronation of 2 resulted in marked increase in absorbance at 498 nm with a high molar absorptivity. Furthermore, the activated fluorescein 2 has significantly enhanced fluorescence emission intensity at 574 nm with a quantum yield of 5.31 and a high brightness.

[0275] Having confirmed that H2O2can activate the fluorescence of 1c and 2, the kinetics of H2O2mediated deboronation were analyzed using fluorescence intensity under pseudo-first order conditions with 1c and 2 (2 μM) and H2O2(1.0 mM). Compound 1c showed an observed rate constant of 1.7 x 10-4s-1which is slightly faster than that of 2 (1.2 x 10-4s-1) (FIG. 7A). FIG. 7B illustrates the fluorescence response of 1c and 2 in increasing concentrations of H2O2(0-10 mM).

[0276] The marked fluorescence increase was observed with 1c and 2 with increasing H2O2concentration.

[0277] As the pH values vary in different cell conditions, the pH dependence of fluorescence activation of 1c and 2 (FIG. 8) was investigated. In the absence of H2O2, 1c and 2 showed weak fluorescence at various pH conditions (pH 4—11) after incubation at RT for 8 h. On the other hand, the presence of H2O2(100 μM) resulted in fluorescence emission increase with both 1c and 2 at basic conditions. Coumarin analogue 1c showed a maximum fluorescence intensity at about pH 9.5 but dropped at pH lower or higher than 9.5. The fluorescence activity of the fluorescein derivative 2 continuously increased with increasing pH values as the basic conditions favor the formation of di-anion form 2"' that shows higher fluorescence brightness than the neutral form 2" (FIG. 9). This observation is consistent with previous reports of fluorescein’s higher fluorescence brightness under basic medium.

[0278] Since there are many other biologically relevant ROS species influencing the function of these prodrugs, the selectivity of 1c and 2 towards H2O2was tested among a wide variety of ROS species, such as superoxide (O2), tert- butoxy radical (fBuO ), hypochlorite (OCI-, tertbutylhydroperoxide (TBHP), nitric oxide (NO·), and hydroxyl radical (HO ) (FIG. 10). The highest fluorescence enhancement was observed with 1c and 2 in the presence of H2O2while the fluorescence intensity of 1c and 2 maintained at the background level with other ROS species. These data suggested that both 1c and 2 were preferentially responsive toward H2O2among various biologically relevant ROS species.

[0279] Fluorescence Detection of 1a, 1c, or 2-alkylated DNA in Living Cells

[0280] Having established that 1a, 1c, or 2 can be activated by H2O2to induce DNA alkylation and turn on fluorescence, the diagnostic utility of these compounds was further evaluated for diagnosing DNA modification in H2O2rich sites using a 49-mer DNA duplex as well as MDA-MB- 468 cancer cells. Previously,32P-labeled DNA was to detect DNA ICL formation and alkaline- labile lesions formed by 1a, 1c, or 2 via gel electrophoresis assay, which is impractical for cellular DNA detection. Therefore, the possibility of measuring fluorescence of 1a, 1c, or 2 alkylated to DNA was tried to distinguish the covalently bound theranostic from commercially available tracker dyes with non-covalent or indirect interactions with DNA. The DNA duplex 28 was treated with 1a, 1c, or 2 (1.0 mM) with or without H2O2(2 equivalence per boronate ester functional group) for 24 h at RT. The alkylated DNA was isolated through precipitation and size exclusion chromatography which can quickly remove the unreacted molecules. The DNA samples treated with 1a, 1c, or 2 (1.0 mM) without H2O2(control) showed background fluorescence signal suggesting that the non-covalently bound theranostic agents were efficiently removed by precipitation. On the other hand, DNA duplex 28 treated with 1a, 1c, or 2 in the presence of H2O2showed approximately eight-fold or greater increase in fluorescence relative to the control samples without H2O2(FIG. 11 A). To further test the reliability of this method, duplex 23 was incubated with three tracker dyes, SYTO 9, MitoT racker Red CMXRos and Hoechst 33342, which produced low fluorescence indistinguishable from the background. The results supported that the DNA tracker dyes with only non-covalent interactions (i.e., SYTO 9, MitoTracker Red CMXRos and Hoechst 33342) can be efficiently removed without causing “background” fluorescence. Collectively, these data suggested that the novel nitrogen mustard derived theranostics 1a, 1c and 2 can form strong covalent bonds with DNA to permanently mark H2O2rich sites.

[0281] Finally, the diagnostic utility of 1a, 1c, and 2 was investigated in cell culture using MDA- MB-468 cells. The cells were incubated with 1a, 1c, or 2 (50 μM) at 37 °C for 20 h (FIG. 11B). Then, the DNA was isolated from the cells by chemical lysis, DNA extraction, and followed by DNA precipitation and size-exclusion chromatography. The resulting purified cellular DNA showed greatly enhanced fluorescence intensity, which suggested that 1a, 1c, and 2 covalently attached to the cellular DNA via alkylation. Fluorescence emissions increase in the order of 2, 1a, and 1c. An unexpected weak fluorescence response was observed with the DNA isolated from the cells incubated with 2, which was likely due to its low aqueous solubility as demonstrated in Table 3. To provide evidence that the fluorescence detected in cellular DNA samples is unlikely caused by non-covalently bound compounds, three tracker dyes with only non-covalent interaction, SYTO 9, MitoRed and Hoechst 33342 with MDA-MB-468 cells was tested. No fluorescence or the background level fluorescence was observed with all cellular DNA samples isolated from MDA-MB-468 cells incubated with SYTO 9, MitoRed or Hoechst 33342 (n = 3). Collectively, these data suggested that all three theranostics 1a, 1c, and 2 can be activated in cells to form covalent bonds with DNA and turn on fluorescence, which allows their use as a permanent fluorescent label for determination of H2O2-rich sites in cell specific compartments via bioimaging techniques.

[0282] Theranostic Molecules 1a, 1c, and 2 Accumulate in the Mitochondria

[0283] After having demonstrated the capability of detecting 1a, 1c, and 2-alkylated DNA in cancer cells via fluorescence measurement, a colocalization study was performed to track the location of these molecules in living cells. Both nucleus and mitochondria are possible targets of 1a, 1c, and 2 as they contain abundant DNAs. Since boron-based theranostics 1a, 1c, and 2 are initially inert and inactive molecules which require H2O2for activation, the mitochondria is more likely targeted by these compounds due to the production of high level H2O2in the organelle. In supporting this hypothesis, the colocalization assay was performed with live MDA-MB-468 cells at both nucleus and mitochondria organelles using MitoTracker Red CMXRos for mitochondria visualization and SYTO 9 or Hoechst 33342 for nuclear DNA stain. Mitochondria colocalization assay was performed by colabeling MitoTracker Red CMXRos with 1a, 1c, or 2 (5 μM) in MDA- MB-468 cells (FIG. 12). Initially, live MDA-MB-468 cells were treated with 1a, 1c, or 2 (5 μM) for 2 h without addition of exogenous H2O2. A significant blue fluorescence signal was observed with the cells treated with 1a (FIG. 12), which suggested efficient cellular uptake of 1a. In contrast, samples treated with 1c or 2 displayed significantly lower fluorescence signal because of low H2O2concentrations in this short time frame (FIG. 12). However, the fluorescence signal slightly increased with longer incubation time. In a separate experiment, treatment of MDA-MB-468 cells with 1c or 2 for 0.5 h followed by addition of H2O2(100 μM) and incubation for an additional 1.5 h, greatly enhanced the intracellular fluorescence signal (FIG. 12). As shown in FIG. 12, the confocal images of the cells treated with 1a moderately overlapped with MitoTracker Red in the mitochondria (Pearson’s correlation coefficient, r= 0.56). Similarly, cells simultaneously loaded with exogenous H2O2(100 μM), 1c or 2, and MitoTracker Red also produced modest correlation (r= 0.54 and 0.74; respectively) (FIG. 12). The data suggested that these compounds are likely to accumulate in mitochondria. Having shown that both 1c and 2 fluorescence are responsive towards exogenous H2O2, the effect of endogenously generated H2O2was tested using PQ, a toxin known to induce mitochondrial dysfunction at the electron transport chain and ultimately cause cellular oxidative stress and marked uptake in ROS production. Cells pre-treated with PQ (500 μM) followed by subsequent incubation of either 1c or 2 (5 μM) for 1.5 h demonstrated significant fluorescence enhancement as opposed to samples without PQ (FIG. 12). The resulting Co-labeled samples with MitoTracker Red showed higher accumulation in mitochondria for both 1c and 2 in PQ treated cells (r= 0.79 and 0.93; respectively) (FIG. 12).

[0284] In contrast to the mitochondria, preferential accumulation in the nucleus of 1a, 1c, and 2 was not observed (FIG. 13). Cells co-labeled with 1a and nucleus tracking dye, SYTO 9, led to r of -0.28 (FIG. 13). Cells treated with 1c and 2 in the presence of exogenous H2O2(100 μM) were observed to have r of 0.07 or “0.08 (FIG. 13), respectively. Similarly, colocalization assays with PQ pre-treated cells showed no significant accumulation in the nucleus (1c: r = 0.03 and 2: r ~ “0.14), rather the results suggest random disordered appearance of these theranostics inside and outside of the nucleus relative to SYTO 9 or Hoechst 33342 (FIG. 13).

[0285] In sum, both 1c and 2 are weakly fluorescent theranostics that are highly responsive towards H2O2activation as de-boronation induces rapid and significant increase in fluorescence turn-on. The responsiveness of 1c and 2 extend towards both exogenously and endogenously generated H2O2at physiologically relevant concentrations in living cells.

[0286] Described herein is the design and synthesis of three novel H2O2-responsive theranostic agents (1a, 1c, and 2) comprising of a bisalkylating functional group (nitrogen mustard) masked by a boronate group and a fluorescence reporter, such as a coumarin (1a) or a boronate- deactivated coumarin (1c) or a boronate-masked fluorescein moiety (2). The monoboronate 1a with a shorter linker attached to coumarin reporter can be activated by H2O2to produce efficient DNA ICL formation with the 49-mer DNA duplex 23. However, only mono-alkylation products but no DNA ICL formation were observed with 1c or 2 comprising of an 11 -carbon linker attached to the fluorescence reporter upon H2O2-activation. The extra-long carbon linker plus bulky fluorophore may increase the steric hindrance preventing the efficient interaction of 1c or 2 with DNA therefore inhibiting bis-alkylation from occurring with 28. Compounds 1a and 1c showed selective cytotoxicity toward cancer cells but spared normal cells. The in vitro cytotoxicity assay demonstrated that 1a and 1c greatly inhibited MDA-MB-468 cancer ceil growth with IC50of 3.9 ± 0.6 μM for 1a and 4.2 + 1 .5 μM for 1c, while very low cytotoxicity was observed for 2. In contrast, 1a and 1c showed significantly lower cytotoxicity toward normal cell line MCF-10A (IC50= 8.3 + 0.7 μM and 49.7 ± 2.1 μM, respectively).

[0287] NMR kinetics displayed that H2O2induced fast and efficient oxidative deboronation reactions with all three theranostics 1a, 1c, and 2 to form the active phenol products therefore restoring their alkylating capability and fluorescence. In terms of the activation of the nitrogen mustard moiety, 1c was slightly faster than either 1a or 2. Among 1a, 1c, and 2, compound 1a showed the best water solubility and 1c displayed the best cell permeability with medium aqueous solubility, which may contribute to their cytotoxicity. However, the poorest water solubility was observed with 2, which may account for its low cytotoxicity. Collectively, these data suggested that H2O2-induced DNA alkylating capability, the biochemical and biophysical properties, such as water solubility, cell membrane permeability, etc. combine to affect the cytotoxicity of 1a, 1c, and 2.

[0288] Further investigation suggested that MDA-MB-468 cancer cells showed higher H2O2level than normal MCF-10A cells, which may contribute to the selective cytotoxicity of 1a and 1c towards cancer cells. The comet assay demonstrated that 1a and 1c induced cellular DNA damage in MDA-MB-468 cells but not in MCF-10A cells. This data provided evidence that 1a and 1c can be activated by high H2O2level in cancer cells to release DNA damaging species but not in normal ceils that contain lower H2O2concentration.

[0289] In addition to the therapeutic effect, 1a, 1c, and 2 demonstrated diagnosis potential due to the presence of fluorescence reporters. Compound 1a comprises of a persistent active coumarin fluorophore that can be directly visualized in live cells by fluorescence measurement. On the other hand, 1c and 2’s fluorescence is highly responsive toward H2O2over time and at various concentrations due to the presence of fluorophores masked by a H2O2-responsive boronate group, which allows their use for detecting H2O2-rich sites in cells. In addition, no visible wavelength absorption was observed for 2 in its original form until H2O2activation removes the boronate esters to enable the fluorescein functionality’s isomerization between closed and open form. Activated 2 demonstrates promising safe bioimaging capability with an absorption in the visible region (498 nm), whereas 1a and activated 1c have maximal absorption in the UV region (395 nm and 350 nm; respectively). Because 1c and 2 showed negligible fluorescence signal at different pH values (from pH 4 to pH 11), the pH stability further supports structural integrity and selectivity under various conditions.

[0290] Furthermore, 1c and 2 showed selectivity for H2O2among various ROS with significant fluorescence intensity enhancement with various concentrations of H2O2. These promising properties allow the convenient detection of DNA alkylation induced by 1a, 1c, and 2 by fluorescence measurement in replacement of radioactive 32P-labeling. Strong fluorescence was detected with a 49-mer DNA duplex (28) treated with 1a, 1c or 2 loaded with H2O2but not for samples without H2O2. The diagnostic utility of 1a, 1c, or 2 was further demonstrated with live cells. Fluorescence signal was detected far the cellular DNAs isolated from MDA-MB-468 cells treated with 1a, 1c or 2. The intensity of fluorescence emission was in the order of 1c > 1a > 2. Compound 1c showed higher fluorescence signal than 1a, which is likely due to the higher cell permeability of 1c than 1a. On the other hand, the poorest water solubility of 2 may lead to its poor cellular uptake therefore leading to the lowest fluorescence intensity. Considering 2 showed excellent fluorescence properties, further modification will be needed to enhance its water solubility.

[0291] Finally, all three compounds 1a, 1c, and 2 promoted fluorescence response to both exogenous and endogenous H2O2in live cancer cells. Although 1a has persistent fluorescence independent of H2O2level in cancer cells, H2O2can activate 1a to form the active nitrogen mustard species that greatly enhances its DNA alkylating capability leading to accumulation of 1a in cancer cells. On the other hand, H2O2not only increases the alkylating capability of 1c and 2 but also turns on their fluorescence, therefore leading to significantly enhanced fluorescence signal. The confocal colocalization analysis demonstrated the abundant presence of these H2O2-activated theranostic agents 1a, 1c, and 2 in the mitochondria, the organelle with high level of H2O2. Whereas random occurrences in the nucleus were observed from colocalization measurements.

[0292] These compounds appear to be the first H2O2-responsive DNA-alkylating nitrogen mustard small-molecule theranostics capable of simultaneously providing both therapeutic effector and imaging reporter in one integrated molecule. These molecules allow tracking H2O2- activated DNA alkylating agents and aid the understanding of mechanistic function in live cells. Future efforts are aimed towards using the work herein as a foundation of the design and synthesis for improved H2O2-responsive theranostics capable of greater DNA-alkylating selectivity, balanced biophysical and enhanced photophysical features for biological applications against cancer diseases. Therefore, this work provides important guidance for further development of clinically useful H2O2-activated therapeutic agents.

[0293] Example 3

[0294] / n vivo Efficacy and Selectivity

[0295] In vitro studies have demonstrated that theranostic 1c showed the greatest selectivity toward cancer cells. See Saxon et al., Eur. J. Med. Chem. 27Q: 116695 (2024). Therefore, the anticancer efficacy and selectivity of 1c was further assessed in vivo with Xenograft mice model. First, CD1 mice were used to identify safe doses. Mice were administered with 1c at doses of 5, 10 and 20 mg / kg per day for 5 days and observed for an additionai 5 days for latent side effects. No obvious toxicity and an absence of weight loss were observed with all mice, indicating that 1c is safe to mice without apparent undesirable side effects (FIG. 14A). As a result, a dose of 5 mg / kg was used to establish the in vivo efficacy of 1c with athymic nude mice xenografted with breast cancer cells (FIG. 14B-H). The mice were subcutaneously implanted with MDA-MB-468 cancer cells. After one week, tumors were developed in mice that were treated with 5 mg / kg 1c for 8-weeks. The body weights of the mice were measured every other day (FIG. 14B), which displayed continuous increase throughout the treatment without weight loss or treatment-related mortality. The vehicle mice showed uncontrollable tumor volume increase with a final 2037 ± 422% growth from the initial starting size (FIG. 14C). In contrast, 1c greatly inhibited tumor growth with a final size increase of 235 ± 18% from the initial size. The average tumor volume (346 ± 122 mm3) of 1c-treated mice was only 14% of mean volume in control group (2491 ± 1125 mm3). The calculated tumor growth inhibition rate of 1c [IR (%) = [1 - (mean volume of treated tumors) / (mean volume of control tumors) x 100] was 86%. At the end of 8-week treatment, the mice were sacrificed, and tumors were excised (FIG. 14D-H). The average tumor weight for 1c- treated mice was 228 ± 157 mg in comparison to 924 ± 190 mg for vehicle (FIG. 14D). No obvious color or weight changes were observed with the internal organs of 1c-treated mice compared to the control group, including liver, spleen, kidney, lung, and heart (FIG. 14E). These data suggest that 1c at a dosage of 5 mg / kg greatly inhibited tumor growth for MDA-MB-468 xenografts without adverse effects.

[0296] Since the liver functions to filter blood for the removal of foreign toxins and is susceptible to drug-induced fatty liver disease (DIFLD),1 the harvested liver tissue was stained with hematoxylin and eosin (H&E) to assess drug-related organ injuries. As shown in FIG. 14G, no vacuoles from microvesicular and macrovesicular drug-induced lipid droplets (LDs)2 were observed in 1c-treated group. The other common organ susceptible to drug injury is the kidney, which also functions to filter blood of unwanted toxins at the glomerulus and renal tubule. Assessing the kidneys by H&E staining presented unremarkable results. The tissue appeared no tubular vacuoles or dilation from necrosis nor the presence of immune (eosinophils, neutrophils and mononuclear) cells that is indicative of inflammation (FIG. 14G). In sum, H&E histological staining found no drug-induced damage, which further supports the safety profile of theranostic 1c towards normal tissue.

[0297] Detection of Theranostic 1c by Fluorescence Spectroscopy Drug detection in vivo often requires expensive, labor intensive methods and instrumentation. However, theranostic 1c consists of boronated coumarin moiety with profluorogenic properties, which allows facile and simple fluorescence visualization of the molecule’s activation in complex matrices such as blood. In support of this hypothesis, nude xenograft mice were administered a single treatment of 1c in 10 mg / kg dose and consequently sacrificed after 0.5 h, 1 h and 4 h (n = 1). The blood collected from cardiopuncture was diluted in MeOH, centrifuged for plasma isolation, and measured for fluorescence (FIG. 15). Subsequent excitation at λexof 350 nm and collection of the emission wavelengths resulted in spectra with marked fluorescence enhancement in blood plasma at 0.5 h (λmsx= 423 nm) and 1 h (λmax= 526 nm). No observable fluorescence could be detected at 4 h period distinct from the vehicle. The data implies that theranostic 1c undergoes rapid activation in vivo. The fluorescence discrepancy in 0.5 h and 1 h blood plasma also suggests different activation or other reactions may have taken place at the coumarin functionality. In contrast, the untreated control blood plasma produced negligible fluorescence similar to the 4 h sample.

Claims

CLAIMSWhat is claimed:

1. A therancstic compound or pharmaceutically acceptable salt thereof, comprising: a compound of formula (I),wherein: X is halo;R1, at each occurrence, is independently a linker; and R2, at each occurrence, is independently a boronic acid, a boronic ester, a boronate, a boronamine, hydrogen, or C1-6alkyl; and a fluorophore.

2. The compound of claim 1 , wherein the linker has a formula (II):wherein:R3is independently hydrogen or Ci-3alkyl;L' is C1 -15alkylene;L2is C1-10alkylene or -(CH2)0-5-Y--(CH2)0-5--;Y is C(O), N(RX), or C(O)-N(RX);Rxis hydrogen or C1 -3alkyl; andG1is a 6- to 12-membered aryl, C3-10carbocyclyl, or a 4- to 12-membered heterocyclyl, optionally substituted with halogen, cyano, C1-6alkyl, C1-6haloalkyl, -OR1a, -SR13, CO2R1a, -C(O)R1a, -SO2R1a, -N(R1a)2, -CO2N(R1s)2, or -NO2;R1a, at each occurrence, are each independently hydrogen or C1-3alkyl; m ~ 0-1 ; and n= 0-1.

3. The compound of claim 1 , wherein the linker has a formula (III):whereinL3is C1-6alkylene, C(O), -O-C(O)-O~, ~N(RX)-, or-O-; and w = 0-1 .

4. The compound of claim 1 , wherein the fluorophore is fluorescein, rhodamine, cyanine, coumarin, BODIPY, napthaiamide, xanthenone, (benzopyranylidene) maiononitriie, a derivative thereof, or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1 , wherein X is chloro.

6. The compound of claim 1 , wherein the compound has a formula l-a:

7. The compound of claim 1 , wherein the theranostic has a formula l-b:wherein Q is a fluorophore.

8. The compound of claim 7, wherein R3is hydrogen.

9. The compound of claim 7, wherein m ~ 0 and n = 0.

10. The compound of claim 7, wherein Q is coumarin, fluorescein, a derivative thereof, or a salt thereof.

11. The compound of claim 7, wherein the theranostic compound or a pharmaceutically acceptable salt thereof, is selected from a group consisting of:2.

13. The compound of claim 1 , wherein the compound has a formula of l-c:wherein Q is a fluorophore.

14. The compound of claim 13, wherein R1is hydrogen or C1-6alkyl.

15. The compound of claim 13, wherein w ~ 1.

16. The compound of claim 13, wherein L3is -O-C(O)-O-.

17. The compound of claim 13, wherein the fluorophore is a is coumarin, fluorescein, a derivative thereof, or a pharmaceutically acceptable salt thereof.

18. The compound of claim 13, wherein the theranostic, or pharmaceutically acceptable salt thereof, is selected from a group consisting of:

19. A method for treating and diagnosing a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of the compound of claim 11 or 18, or pharmaceutically acceptable salt thereof.

20. The method of claim 19, wherein the disease or disorder is associated with oxidative stress.21 . The method of claim 20, wherein the disease or disorder is cancer.

22. The method of claim 21 , wherein the cancer is selected from breast cancer, brain cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, leukemia, or other cancers.

23. Use of the theranostic compound of claim 11 or 18, or a salt thereof, for the preparation of a medicament for the treatment of a disease or disorder associated with oxidative stress in a subject.

Images