A dual performing vinyl-tetrazine for rapid, selective bioconjugation and functionalization of cysteine proteins

Abstract

Described herein are dual-performing bioconjugation–functionalization reagents comprising an electron-withdrawing tetrazine (Tz)-substituted vinyl (V) moiety, which serves both as a Michael acceptor for selective conjugation with cysteine (Cys) residues and as a reactive site for click chemistry with trans-cyclooctene (TCO) derivatives to introduce functional payloads.

Description

[0001] A DUAL PERFORMING VINYL-TETRAZINE FOR RAPID, SELECTIVE BIOCONJUGATION AND FUNCTIONALIZATION OF CYSTEINE PROTEINS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 711,067 filed on October 23, 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 GM130772 awarded by the National Institutes of Health. 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 in accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the USPTO Patent Center, “212443-0004-W001_sequence_listing_xml_10-OCT-2025.xml,” was created on October 10, 2025, contains 5 sequences, has a file size of 6.02 kilobytes (6,173 bytes), and is incorporated by reference in its entirety into the specification.

[0008] BACKGROUND

[0009] Chemical modification of proteins is a vital component of studies aimed at elucidating functions of proteins and developing new therapies. In this context, strategies involving site specific protein bioconjugation and functionalization have been devised and utilized for numerous purposes, such as tracking cellular events, revealing enzyme functions, imaging specific biomarkers and delivering drugs to targeted cells. Design of labeling reagents that are compatible with each of these applications faces a range of simple (e.g., introduction of fluorescent dyes) or more complicated (e.g., creation of antibody drug conjugates, ADCs) challenges.

[0010] The most common bioconjugation strategy employed to address these challenges involves direct coupling through cysteines (Cys) in native proteins. An important advantage of this approach is the relatively low abundances of these residues in proteins (2.3% in human protein). In addition, this tactic is benefitted by the unique reactivity profile of the Cys thiol moiety. Owing to this feature, many methods can be employed for Cys specific modification including those that rely on Michael addition, disulfide bond formation, a-halocarbonyl substitution, SNAr, arylation, and ring opening reactions. However, these processes generally only accomplish bioconjugation. Therefore, appropriate tags, including clickable cyclooctyne or trans-cyclooctene (TCO), photoactive, spin label, protein binding, fluorescent and biotin groups need to be preattached to the bioconjugation moiety to introduce specific functionality into proteins.

[0011] Among the currently available biorthogonal-click processes, inverse electron demand Diels-Alder (IEDDA) reactions of 1,2,4, 5-tetrazines (Tz) with strained cyclic alkenes and alkynes, which do not require catalysts, have unmatchable rates and excellent orthogonality and biocompatibility. Importantly, the high rates (up to 106M’1s-1) of IEDDA reactions minimizes both the time and amount of labeling agent required to attain high coupling yields, making them suitable for in vitro and in vivo applications. Specifically, these reactions reach completion in minutes (min) even when bioorthogonal target concentrations are very low (mM-nM). Owing to these advantageous features, IEDDA reactions have been utilized for imaging, diagnostics and therapy, as evidenced by their use for clinical anticancer drug delivery in humans.

[0012] When bulky tags utilized for bioorthogonal / click processes often disrupt the structure and / or function of the protein conjugates. To minimize these problems, pre-functionalized, small biorthogonal moieties (e.g., alkyne or azide) are incorporated into the conjugation reagents to link specific functional groups via click reactions (e.g., CuAAC). The use of two separate reagents is relevant for tuning structure and function, but this approach can potentially lead to toxicity especially when large excesses of reagents and copper catalysts are required to elevate rates of the slow click reaction. This is problematic especially in cellular protein functionalization because the click components are generally present in very low concentrations.

[0013] What is needed are compact dual functioning reagents that that carry out rapid and chemoselective Cys-selective bioconjugation and ensuing functionalization.

[0014] SUMMARY

[0015] One embodiment described herein is a compound of formula (I),

[0016]

[0017] wherein:

[0018] R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocycle, wherein R1is optionally substituted with 1-5 substituents wherein each substituent is independently selected from the group consisting of Ci-ealkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)O-i-Rx, -(X1)0-i-C(O)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -(X1)o-1-S02RX, -(X1)o-1-P02RX, -(X1)o-1-C(0)NHRX, and -(X1)0-i-C(O)NCi-4alkylRx;

[0019]

[0020] X1, at each occurrence, is -0-, -S-, -NH-, or -NCi^alkyl-;

[0021] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;

[0022] R2is Ci.6alkyl, Ci-4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2RY, -(Y1)0-i-S(O)RY, -(Y1)O-I-S02RY, -(Y1)O-I-P02RY, -(Y1)O-I-C(0)N(RY)2, -(Y1)o-i-Ci^alkylene-(Y1)O-i-RY, -(Y1)0.i-Ci.6alkylene-(Y1)o.i-C(0)RY, -(Y1)o-i-Ci-6alkylene-(Y1)0-i-C02RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S(0)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S02RY, -(Y1)o.i-Ci.6alkylene-(Y1)o-i-P02RY, or -(Y1)0-i-Ci^alkylene-(Y1)o-i-C(0)N(RY)2;

[0023] Y1, at each occurrence, is -0-, -S-, -NH-, or -NCi^alkyl-;

[0024] RY, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGY; and

[0025] Gxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, — N02, Ci-ealkyl, Ci-4haloalkyl, -CN, -OCi^alkyl, -OH, -OCi-4haloalkyl, -SH, — SCi-4alkyl, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -CO2H, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi-4alkyl, -C(O)N(Ci-4alkyl)2, -S(O)H, -S(O)Ci-4alkyl, -SO2Ci-4alkyl, -

[0026]

[0027] SO2NH2, -SO2NHCi-4alkyl, and -SO2N(Ci-4alkyl)2. In one aspect, R1is an optionally substituted 6- to 12-membered aryl.

[0028] In another aspect, the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl. In another aspect, R2is Ci-ealkyl. In another aspect, R2is methyl.

[0029] In another aspect, the compound of formula (I) is:

[0030]

[0031] Another embodiment described herein is a conjugate comprising:

[0032] a molecule comprising a cysteine residue; and

[0033] a moiety of formula (l-a),

[0034]

[0035] s

[0036] wherein a moiety of formula: ' links the cysteine residue to the moiety of formula (I- a), and wherein:

[0037] R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a C3-8cycloalkyl, or a 4- to 12-membered heterocyclyl, wherein R1is optionally substituted with 1-5 substituents, wherein each substituent is independently selected from the group consisting of Ci-ealkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)O-i-Ci-6alkylene-(X1)o-i-Rx, -(X1)0-i-C(O)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -(X1)o-1-S02RX, -(X1)o-1-P02RX, -(X1)0-I-C(O)NHRX, and -(X1)o-i-C(0)NCi-4alkylRx; X1, at each occurrence, is -0-, -S-, -NH-, or -NCi.4alkyl-;

[0038] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;

[0039] R2is Ci-6alkyl, Ci-4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2RY, -(Y1)0-i-S(O)RY, -(Y1)0-I-SO2RY, -(Y1)0-I-PO2RY, -(Y1)0-I-C(O)N(RY)2, -(Y1)o-i-Ci-ealkylene-(Y1)O-i-RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-C(0)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-C02RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S(0)RY, -(Y1)o-i-Ci-ealkylene-(Y1)o-i-S02RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-P02RY, or -(Y1)o-i-Ci-6alkylene-(Y1)0.i-C(0)N(RY)2;

[0040] Y1, at each occurrence, is -O-, -S-, -NH-, or-NCi-4alkyl-;

[0041] RY, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGY; and

[0042] Gxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -NO2, Ci-ealkyl, Ci.4haloalkyl, -CN, -OCi.4alkyl, -OH, -OCi.4haloalkyl, -SH, -SCi.4alkyl, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -CO2H, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi-4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci.4alkyl, -SO2Ci.4alkyl, -SO2NH2, -SO2NHC1.

[0043] 4alkyl, and -SO2N(Ci.4alkyl)2.

[0044] In one aspect, the molecule comprising the cysteine residue is a peptide or a protein comprising the cysteine residue. In another aspect, R1is the optionally substituted 6- to 12-membered aryl. In another aspect, the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl. In another aspect, R2is Ci-ealkyl. In another aspect, R2is methyl.

[0045] Another embodiment described herein is a method of preparing a conjugate described herein, the method comprising: providing a molecule comprising a cysteine residue; and reacting a compound described herein with the cysteine residue of the molecule, thereby providing a conjugate described herein. In one aspect, the molecule comprising the cysteine residue is a peptide or a protein. In another aspect, reacting the compound of Formula (I), with the cysteine residue occurs in the presence of a buffer solution. In another aspect, the buffer solution has a pH of 4.0 to 9.0. In another aspect, the conjugate is provided in quantitative yields.

[0046] Another embodiment described herein is a method of functionalizing a conjugate described herein, the method comprising: reacting the conjugate described herein with a functionalized payload, wherein the functionalized payload comprises a payload moiety attached to a cyclooctene, thereby providing a functionalized conjugate.

[0047] In one aspect, a linker attaches the cyclooctene to the payload.

[0048] In another aspect, the cyclooctene is trans-cyclooctene.

[0049] In another aspect, the functionalized payload is a functionalized payload of formula (II):

[0050]

[0051] wherein L1is the linker, and D1is the payload moiety.

[0052] In another aspect, the functionalized conjugate comprises a moiety formula (I l-a) or (I l-b):

[0053]

[0054] In another aspect, the functionalized payload of formula (II) is:

[0055]

[0056] In another aspect, L1is: 0 0 0

[0057]

[0058] wherein:

[0059] Z1is -0-, -S-, -NRa-, or-CRaRb-;

[0060] Raand Rb, at each occurrence, are each independently hydrogen or Ci-4alkyl; and m, at each occurrence, is 0-14,

[0061] n, at each occurrence, is 0-10, and

[0062] p, at each occurrence, is 0-10.

[0063] In another aspect, Z1is -O-. In another aspect, Raand Rb, at each occurrence, are each independently hydrogen or methyl. In another aspect, D1is a therapeutic agent, a diagnostic agent, or a targeting agent. In another aspect, the diagnostic agent is a cyanine dye. In another aspect, the therapeutic agent is an anti-cancer therapeutic agent. In another aspect, the anticancer therapeutic agent is doxorubicin or ARV- 771.

[0064] DESCRIPTION OF THE DRAWINGS

[0065] 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.

[0066] FIG. 1 illustrates the design of the dual-functional VMeTz reagent for bioconjugation and click functionalization of cysteine (Cys)-containing proteins.

[0067] FIG. 2A-C show reaction kinetics. FIG. 2A shows the reaction progression of VMeTz with L-Cys-VMeTz as determined by HPLC analysis. FIG. 2B shows the second-order rate constant for the reaction between VMeTz (1 mM) and Cys (1 mM). FIG. 2C shows the second-order rate constant for the reaction between VMeTz (10 pM) and trans-cyclooctene (TCO)-OH.

[0068] FIG. 3A-B show the chemical reaction between VTz (0.6 mM) and glutathione (GSH; 0.5 mM) (FIG. 3A) and between VMeTz (3 mM) and GSH (1 mM) (FIG. 3B).

[0069] FIG. 4A-B show the reaction of bovine serum albumin (BSA; 1.0 mM) with VTz (5.0 mM) in phosphate-buffered saline (PBS), pH 7.4 for 1 h at room temperature (RT).

[0070] FIG. 5A-E show UV-Vis spectroscopic analysis of VMeTz reacting with various concentrations of TCO-OH. FIG. 5A-E shows UV-Vis detection of the reaction of VMeTz (10 pM) with different concentrations ofTCO-OH: 20 M (FIG. 5A), 35 pM (FIG. 5B), 50 pM (FIG. 5C), 80 pM (FIG. 5D), and 100 pM (FIG. 5E) at A = 295 nm. The fobs values were calculated from nonlinear regression analysis.

[0071] FIG. 6A shows the stability of VMeTz (1 mM) in PBS buffer, pH 7.4, at room temperature as determined by high-performance liquid chromatography (HPLC). FIG. 6B shows the reaction of Pep1 (AC-GCAKFE-CONH2(SEQ ID NO: 1); 1 mM) with VMeTz (3 mM) under varying pH conditions (pH 7.4: 30 min; pH 8: 30 min; and pH 7.4: overnight). FIG. 6C shows the chemical structure of the Pep1-VMeTz conjugate. FIG. 6D-E show tandem mass spectrometry (MS / MS) spectra confirming the formation of Pep1-VMeTz.

[0072] FIG. 7A shows the reaction between Pep1 (Ac-GCAKFE-CONH2(SEQ ID NO: 1); 1 mM) and VMeTz (3 mM). FIG. 7B shows the liquid chromatography-mass spectrometry (LC-MS) spectrum of Pep1 (m / z calcd. for C41H56N12O9S: 892.4, found: 893.4 (+H+)).

[0073] FIG. 8A shows the structure of Pep 1 (Ac-GCAKFE-CONH2(SEQ ID NO: 1); 1 mM) reacted with A / -phenylmaleimide (“imide,” 1.5 mM) under different pH conditions (pH 7.4, 30 min, yield 60%; pH 8, 30 min, yield 15%). FIG. 8B shows Pep1 (SEQ ID NO: 1) reacted with N-phenylmaleimide at pH 7.4. FIG. 8C shows the LC-MS spectrum of Pep1-lmide. m / z: calcd. for C40H53N9O11S 867.35, found 898.4 (+ H+). FIG. 8D shows LC-MS spectrum for Pep1-lmide-Hydro m / z: calcd. for C40H55N9O12S 885.3, found 886.5 (+ H+).

[0074] FIG. 9A-B show the reaction scheme between Pep2 (1 mM) and VMeTz (3 mM) for 30 mins. FIG. 9C shows the LC-MS spectrum of Pep2-VMeTz (m / z: calcd. for CesHgeN-ieOisS 1408.6962, found 705.4 (+2H+)2+). FIG. 9D shows the LC-MS spectrum of Pep2-VMeTz-TCO-OH (m / z: calcd. for C76H110N14O16S 1506.7, found 754.5 (+2H+)2+). FIG. 9E-F shows MS / MS spectra confirming the modification of Pep2 by VMeTz.

[0075] FIG. 10A-B show the reaction between Pep3 (SEQ ID NO: 3) (1 mM) and VMeTz (10 mM) for 30 min. FIG. 10C shows the LC-MS spectrum of Pep3-VMeTz (m / z: calcd. for C77H93Ni9Oi5S21587.7, found 794.9 + (2H+)2+).

[0076] FIG. 11A shows the reaction of BSA (1 mM) with VMeTz (10 mM) in PBS buffer, pH 7.4, incubated overnight followed by addition of TCO-Cy5 (1 mM) and further incubation for 30 min. FIG. 11B-C show proteomic analysis of VMeTz-modified BSA protein (BSA-VMeTz). FIG. 11 D shows the circular dichroism (CD) spectra of native BSA (0.2 mg / mL) and BSA-VMeTz (200-260 nm). FIG. 11 E shows an SDS-PAGE gel analysis of the click reaction of BSA-VMeTz and TCO-Cy5, with fluorescence imaging and Coomassie staining as control.

[0077] FIG. 12A-C shows mass spectrometry data. FIG. 12A shows the protein mass spectrum of the native BSA protein. FIG. 12B shows the mass spectrum of BSA-VMeTz obtained from the reaction of BSA (1 mM) with VMeTz (10 mM) in PBS, pH 7.4, in a total volume of 50 pL (m / z: calcd. for 66626, found 66628). FIG. 12C shows MS-MS data of BSA-VMeTz.

[0078] FIG. 13A shows the reaction scheme for UbK48C (0.5 mg / mL) reacted with VMeTz (10 equiv.) at the Cys site. FIG. 13B-C show proteomic analysis of UbK48C modified by VMeTz. FIG. 13D-E show intact protein mass spectra of UbK48C (SEQ ID NO: 5) and UbK48C- VMeTz (m / z: calcd. for 8738, found 8739).

[0079] FIG. 14A shows the workflow for fluorescent imaging of HeLa cells with VMeTz. FIG. 14B shows cell images with and without VMeTz (100 pM) and tHCA (1 pM). FIG. 14C-D show immunoblot analysis of BRD4 levels in HeLa treated with ARV-771 (FIG. 14C) or TCO-ARV-771 (FIG. 14D) with VMeTz for 16 h.

[0080] FIG. 15 shows the decaging of fluorogenictHCA by protein-VMeTz to generate fluorescent HCA.

[0081] FIG. 16A shows the workflow for protein labeling with VMeTz in U87 cells. FIG. 16B-C show western blot analysis of labeled proteins in live U87 cells treated with VMeTz and TCO-biotin under various time and dose conditions. GAPDH expression was used as a loading control. FIG. 16D shows a schematic overview of the proteomics analysis workflow for generating VMeTz-labeled proteins. FIG. 16E shows the structure of Parkinson disease protein 7 (PDBid: 4RKW) and MS-MS spectrum analysis of the modified peptide.

[0082] FIG. 17 shows identification of 94 proteins modified by VMeTz, analyzed using Scaffold software (protein threshold: 99%, minimum peptides: 5, peptide threshold: 95%).

[0083] FIG. 18A shows the viability of U87 cells treated with VTz and VMeTz for 72 h. FIG. 18B shows the viability of U87 cells following treatment with Dox, TCO-Dox, and TCO-Dox + VMeTz (10 equiv, 3 h pretreatment) for 72 h. FIG. 18C shows the viability of U87 cells following treatment with ARV-771, TCO-ARV-771, and TCO-ARV-771 + VMeTz (10 equiv, 3 h pretreatment) for 72 h. FIG. 18D shows a volcano plot illustrating protein abundance (Iog2) as a function of the significance level (-Iog10). Fold change is co-treatment / control. Co-treatment group: VMeTz (1 pM, 3 h pretreatment) with TCO-ARV-771 (100 nM, 24 h). FIG. 18E shows flow cytometry apoptosis assay in U87 cells for 24 h. DMSO control, VMeTz (10 pM), TCO-ARV-771 (100 nM), VMeTz (10 equiv, 3 h pretreatment) + TCO-ARV-771 (100 nM and 1 pM), and ARV-771 (1 pM).

[0084] DETAILED DESCRIPTION

[0085] 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.

[0086] 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.

[0087] 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, “comprising,” 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.

[0088] 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.

[0089] As used herein, the term “or” can be conjunctive or disjunctive.

[0090] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0091] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0092] 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

[0093]

[0094] means “about” or “approximately.” 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.”

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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 orfemale; 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.

[0102] 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.

[0103] 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.

[0104] 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 of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0105] The term “administering” refers to any suitable route of administration to a subject, such as, but not limited to, oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, orthe implantation of a slow-release device e.g., a mini-osmotic pump, to the subject. The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0106] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, 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 Organic Chemistry, Thomas Sorrell, 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; Carruthers, Some Modern Methods of Organic Synthesis, 3rded., Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0107] The term “alkoxy,” as used herein, refers to a group -O-alkyl. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tertbutoxy.

[0108] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. The term “lower alkyl” or “Ci-ealkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “Ci.4alkyl” means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, / so-propyl, n-butyl, sec-butyl, / so-butyl, terf-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0109] The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond.

[0110] The term “alkoxyalkyl,” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0111] The term “alkylene,” as used herein, refers to a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, -CH2- -CD2-, -CH2CH2-, -C(CH3)(H)-, -C(CH3)(D)-, -CH2CH2CH2--CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2-.

[0112] The term “amide,” as used herein, means -C(O)NR- or -NRC(O)-, wherein R may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, or heteroalkyl. The term “aminoalkyl” as used herein, means at least one amino group, as defined herein, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0113] The term “amino,” as used herein, means -NRxRy, wherein Rxand Rymay be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, or heteroalkyl. In the case of an aminoalkyl group or any other moiety where amino appends together two other moieties, amino may be -NRX-, wherein Rxmay be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, or heteroalkyl.

[0114] The term “aryl,” as used herein, refers to a phenyl or a phenyl appended to the parent molecular moiety and fused to a cycloalkane group (e.g., the aryl may be indan-4-yl), fused to a 6-membered arene group (i.e., the aryl is naphthyl), or fused to a non-aromatic heterocycle (e.g., the aryl may be benzo[d][1,3]dioxol-5-yl). The term “phenyl” is used when referring to a substituent and the term 6-membered arene is used when referring to a fused ring. The 6-membered arene is monocyclic (e.g., benzene or benzo). The aryl may be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic system).

[0115] The term “cycloalkyl” or “cycloalkane,” as used herein, refers to a saturated ring system containing all carbon atoms as ring members and zero double bonds. The term “cycloalkyl” is used herein to refer to a cycloalkane when present as a substituent. A cycloalkyl may be a monocyclic cycloalkyl (e.g., cyclopropyl), a fused bicyclic cycloalkyl (e.g., decahydronaphthalenyl), or a bridged cycloalkyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptanyl). Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, and bicyclo[1.1 ,1]pentanyl.

[0116] The term “cycloalkenyl” or “cycloalkene,” as used herein, means a non-aromatic monocyclic or multicyclic ring system containing all carbon atoms as ring members and at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. The term “cycloalkenyl” is used herein to refer to a cycloalkene when present as a substituent. A cycloalkenyl may be a monocyclic cycloalkenyl (e.g., cyclopentenyl), a fused bicyclic cycloalkenyl (e.g., octahydronaphthalenyl), or a bridged cycloalkenyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptenyl). Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl. The term “carbocyclyl” means a “cycloalkyl” or a “cycloalkenyl.” The term “carbocycle” means a “cycloalkane” or a “cycloalkene.” The term “carbocyclyl” refers to a “carbocycle” when present as a substituent.

[0117] The term “1,1-carbocyclylene” means a geminal divalent group derived from a cycloalkyl.

[0118] A representative example is 1,1-C3-6cycloalkylene (i.e.,

[0119]

[0120] A further example is 1,1-

[0121] cyclopropylene (

[0122]

[0123] i.e.,

[0124] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0125] The term “haloalkyl,” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.

[0126] The term “haloalkoxy,” as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.

[0127] The term “halocycloalkyl,” as used herein, means a cycloalkyl group, as defined herein, in which one or more hydrogen atoms are replaced by a halogen.

[0128] The term “heteroalkyl,” as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, O, P and N. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides.

[0129] The term “heteroaryl,” as used herein, refers to an aromatic monocyclic heteroatomcontaining ring (monocyclic heteroaryl) or a bicyclic ring system containing at least one monocyclic heteroaromatic ring (bicyclic heteroaryl). The term “heteroaryl” is used herein to refer to a heteroarene when present as a substituent. The monocyclic heteroaryl are five or six membered rings containing at least one heteroatom independently selected from the group consisting of N, O and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). The five membered aromatic monocyclic rings have two double bonds, and the six membered aromatic monocyclic rings have three double bonds. The bicyclic heteroaryl is an 8- to 12-membered ring system and includes a fused bicyclic heteroaromatic ring system (i.e., 10TT electron system) such as a monocyclic heteroaryl ring fused to a 6-membered arene (e.g., quinolin-4-yl, indol-1-yl), a monocyclic heteroaryl ring fused to a monocyclic heteroarene (e.g., naphthyridinyl), and a phenyl fused to a monocyclic heteroarene (e.g., quinolin-5-yl, indol-4-yl). A bicyclic heteroaryl / heteroarene group includes a 9-membered fused bicyclic heteroaromatic ring system having four double bonds and at least one heteroatom contributing a lone electron pair to a fully aromatic 10TT electron system, such as ring systems with a nitrogen atom at the ring junction (e g., imidazopyridine) or a benzoxadiazolyl. A bicyclic heteroaryl also includes a fused bicyclic ring system composed of one heteroaromatic ring and one non-aromatic ring such as a monocyclic heteroaryl ring fused to a monocyclic carbocyclic ring (e.g., 6,7-dihydro-5H-cyclopenta[b]pyridinyl), or a monocyclic heteroaryl ring fused to a monocyclic heterocycle (e.g., 2,3-dihydrofuro[3,2-b]pyridi nyl) . The bicyclic heteroaryl is attached to the parent molecular moiety at an aromatic ring atom. Other representative examples of heteroaryl include, but are not limited to, indolyl (e.g., indol-1-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl (e.g., pyrazol-4-yl), pyrrolyl, benzopyrazolyl, 1 ,2,3-triazolyl (e.g., triazol-4-yl), 1 ,3,4-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl (e.g., thiazol-4-yl), isothiazolyl, thienyl, benzimidazolyl (e.g., benzimidazol-5-yl), benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl (e.g., indazol-4-yl, indazol-5-yl), quinazolinyl, 1 ,2,4-triazinyl, 1 ,3,5-triazinyl, isoquinolinyl, quinolinyl, imidazo[1,2-a]pyridinyl (e.g., imidazo[1,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.

[0130] The term “heterocycle” or “heterocyclic,” as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The term “heterocyclyl” is used herein to refer to a heterocycle when present as a substituent. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The fivemembered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocyclyls include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1 ,3-dithiolanyl, 1 ,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1 ,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a 6-membered arene, or a monocyclic heterocycle fused to a monocyclic cycloalkane, or a monocyclic heterocycle fused to a monocyclic cycloalkene, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a monocyclic heterocycle fused to a monocyclic heteroarene, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1 , 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. The bicyclic heterocyclyl is attached to the parent molecular moiety at a non-aromatic ring atom (e.g., indolin-1-yl). Representative examples of bicyclic heterocyclyls include, but are not limited to, chroman-4-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien-2-yl, 1, 2,3,4-tetrahydroisoquinolin-2-yl, 2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan-6-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), azabicyclo[3.1.0]hexanyl (including 3-azabicyclo[3.1.0]hexan-3-yl), 2,3-dihydro-1 H-indol-1-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7-oxabicyclo[2.2.1]heptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, 3-oxaspiro[5.5]undecanyl, 6-oxaspiro[2.5]octan-1-yl, and 3-oxabicyclo[3.1.0]hexan-6-yl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a 6-membered arene, ora bicyclic heterocycle fused to a monocyclic cycloalkane, or a bicyclic heterocycle fused to a monocyclic cycloalkene, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2, 5-epoxypentalene, hexahydro-2 H-2, 5-methanocyclopenta[b]furan, hexahydro-1 H-1 ,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocyclyls are connected to the parent molecular moiety at a non-aromatic ring atom.

[0131] The term “hydroxyl” or “hydroxy,” as used herein, means an -OH group.

[0132] The term “hydroxyalkyl,” as used herein, means at least one -OH group, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0133] Terms such as “alkyl,” “cycloalkyl,” “alkylene,” etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “Ci-4alkyl,” “Cs-ecycloalkyl,” “Ci-4alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C” followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, “Csalkyl” is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in “C1.4,” the members of the group that follows may have any number of carbon atoms falling within the recited range. A “Ci-4alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0134] The term “substituted” refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =0 (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0135] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0136] The term “tetrazine” refers to a substituted or unsubstituted aromatic cyclic group of 2 carbon atoms and 4 nitrogen atoms, having a single ring with three double bonds. Examples of tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine. As used herein, 1,2, 4, 5- tetrazine is referred to as a “Tz” group.

[0137] The term “cyclooctene” as used herein, refers to a substituted or unsubstituted nonaromatic cyclic alkyl group of 8 carbon atoms, having a single ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted transcyclooctene (TCO).

[0138] The term “payload” refers to an agent for delivery to a target site in a subject. Payloads include therapeutic agents, diagnostic agents, targeting agents, and the like.

[0139] The term “therapeutic agent” refers to an agent capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Therapeutic agents of the present disclosure also include prodrug forms of therapeutic agents.

[0140] The term “diagnostic agent” refers to agents that assist in diagnosing conditions or diseases. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, radionuclides, and the like. Paramagnetic agents are imaging agents that are magnetic under an externally applied field. Examples of paramagnetic agents include, but are not limited to, iron particles including iron nanoparticles and iron microparticles. Optical probes are fluorescent compounds that can be detected by excitation at one wavelength of radiation and detection at a second, different, wavelength of radiation. Optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1 ,1 -dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include, but are not limited to,3H,11C,13N,18F,19F,60Co,64Cu,67Cu,68Ga,82Rb,90Sr,90Y, "Tc, "mTc,111ln,123l,124l,125l,129l,131l,137Cs,177Lu,186Re,188Re,211At, Rn, Ra, Th, U, Pu, and241Am.

[0141] The term “targeting agent” refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and the specific target. By “stably associated” or “stable association” is meant that a moiety is bound to or otherwise associated with another moiety or structure under standard physiological conditions. Bonds may include covalent bonds and non-covalent interactions, such as, but not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. A targeting agent may be a member of a specific binding pair, such as, but are not limited to: a member of a receptor / ligand pair; a ligand-binding portion of a receptor; a member of an antibody / antigen pair; an antigen-binding fragment of an antibody; a hapten; a member of a lectin / carbohydrate pair; a member of an enzyme / substrate pair; biotin / avidin; biotin / streptavidin; digoxin / antidigoxin; a member of a DNA or RNA aptamer binding pair; a member of a peptide aptamer binding pair; and the like.

[0142] The term “linker,” “linked,” or “linking” refers to a chemical moiety that attaches two

[0143] moieties together, such as a payload moiety (-D1) and a cyclooctene moiety (e.g.,

[0144]

[0145] cyclooctane moiety (e

[0146]

[0147] .g., ). The linking can be via covalent bonds, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. The linking can be direct linkage between to the two moieties being linked, or indirectly, such as via a linker. Linkers useful in embodiments of the present disclosure include linkers having 30 carbon atoms or less in length. In some embodiments, the linkers are 1-15 carbon atoms in length, such as 1-12 carbon atoms, or 1-10 carbon atoms, or 5-10 carbon atoms in length. The types of bonds used to link the linker to the payload moiety (-D1) and to the cyclooctene or cyclooctane moiety of the present disclosure include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas. Other types of bonds may also be used in embodiments of the present disclosure. Particular linkers, such as L1, are specifically defined herein.

[0148] The term “binding agent” refers to an agent having a functional group capable of forming a covalent bond to a complementary functional group of another binding agent in a biological environment. Binding between binding agents in a biological environment may also be referred to as bioconjugation. Representative binding agents include, but are not limited to, an amine and an activated ester, an amine and an isocyanate, an amine and an isothiocyanate, thiols for formation of disulfides, an aldehyde and amine for enamine formation, an azide for formation of an amide via a Staudinger ligation. Binding agents also include bioorthogonal binding agents, which are binding agents having bioorthogonal functional groups. Bioorthogonal functional groups of bioorthogonal binding agents selectively react with a complementary bioorthogonal functional group of another bioorthogonal binding partner. Selective reaction between bioorthogonal binding partners can minimize side reactions with other binding agents, biological compounds, or other non-complementary bioorthogonal binding agents or non-complementary bioorthogonal functional groups. Bioorthogonal functional groups of bioorthogonal binding agents include, but are not limited to, an azide and alkyne for formation of a triazole via Click-chemistry reactions, trans-cyclooctene (TCO) and tetrazine (Tz) (e.g., 1,2,4,5-tetrazine), and others. The binding agents useful in the present disclosure may have a high reactivity with the corresponding binding agent so that the reaction is rapid.

[0149] The term “functionalized” refers to a moiety having a functional group attached to the moiety, such as for example a moiety having a binding agent functional group (e.g., a bioorthogonal functional group) attached thereto.

[0150] The present disclosure provides vinyl-tetrazine compounds that function as dualperforming bioconjugation-functionalization reagents. These compounds comprise a tetrazinesubstituted vinyl moiety, which exhibits strong electron-withdrawing properties and serves both as a Michael acceptor for selective conjugation with cysteine-containing biomolecules and as a reactive site for bioorthogonal functionalization. Upon conjugation to cysteine residues, the resulting tetrazine-labeled proteins are capable of undergoing inverse electron demand Diels- Alder (I EDDA) reactions with strained alkene or alkyne derivatives, thereby enabling site-specific modification with functional payloads.

[0151] As a representative example, isopropenyl-tetrazine (VMeTz) was synthesized and utilized as a 2-in-1 reagent for cysteine-selective bioconjugation and subsequent click functionalization of peptides and proteins in vitro and in live cells. VMeTz reacts with thiol-containing substrates to form mono-Michael adducts, wherein the a-methyl substituent on the vinyl group prevents multiple additions. The resulting Cys-VMeTz conjugates undergo efficient I EDDA reactions with transcyclooctene (TCO)-linked payloads, including fluorophores and biotin, for labeling and profiling applications. VMeTz further enables selective activation and intracellular delivery of TCO-caged therapeutic agents, such as doxorubicin and PROTAC ARV-771, in cancer cells, resulting in therapeutic effects comparable to the parent compounds but with reduced off-target toxicity.

[0152] Compounds

[0153] In one aspect, the present disclosure provides compounds of formula (I):

[0154]

[0155] wherein:

[0156] R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocycle, wherein R1is optionally substituted with 1-5 substituents wherein each substituent is independently selected from the group consisting of Ci^alkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)o-i-Rx, -(X1)o-i-C(0)Rx, -(X1)0.I-CO2RX, -(X1)0-i-S(O)Rx, -(X1)o-1-S02RX, -(X1)o-1-P02RX, -(X1)o-1-C(0)NHRX, and -(X1)0-i-C(O)NCi.4alkylRx; X1, at each occurrence, is -0-, -S-, -NH-, or -NCi.4alkyl-;

[0157] Rx, at each occurrence, is hydrogen, Ci.6alkyl, Ci.4haloalkyl, orGx;

[0158] R2is Ci.6alkyl, Ci.4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2RY, -(Y1)o-i-S(0)RY, -(Y1)0-I-SO2RY, -(Y1)0-I-PO2RY, -(Y1)O-I-C(0)N(RY)2, -(Y1)o-i-Ci^alkylene-(Y1)O-i-RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-C(O)RY, -(Y1)o-i-Ci^alkylene-(Y1)0-i-C02RY, -(Y1)0.i-Ci.6alkylene-(Y1)o-i-S(0)RY, -(Y1)o-i-Ci^alkylene-(Y1)o-i-S02RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-P02RY, or -

[0159]

[0160] (Y1)o-i-Ci^alkylene-(Y1)o-i-C(0)N(RY)2; Y1, at each occurrence, is -0-, -S-, -NH-, or-NCi.4alkyl-;

[0161] RY, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGY; and Gxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, -S, -NO2, Ci-ealkyl, Ci.4haloalkyl, -CN, -OCi-4alkyl, -OH, -OCi.4haloalkyl, -SH, -SCi.4alkyl, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -CO2H, -C(O)Ci.4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi.4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci.4alkyl, -SO2Ci.4alkyl, -SO2NH2, -SO2NHC1.

[0162] 4alkyl, and -SO2N(Ci-4alkyl)2.

[0163] In some instances, R1is an optionally substituted 6- to 12-membered aryl. In some instances, the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl. In some instances, R2is Ci-ealkyl. In another aspect, R2is methyl. In some instances, the compound of formula (I) is:

[0164] N

[0165] N-N

[0166]

[0167] Throughout the embodiments and description of the compounds of the invention, all instances of haloalkyl may be fluoroalkyl (e.g., any Ci-4haloalkyl may be C luoroalkyl).

[0168] Compound names and / or structures can be assigned / determined by using the Struct=Name naming algorithm as part of CH EM DRAW® ULTRA.

[0169] The compound may exist as a stereoisomer wherein asymmetric or chiral centers are present. The stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 45: 13-30 (1976). The disclosure contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this invention. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. In the compounds disclosed herein, a chiral atom depicted or described without a specific stereochemical configuration (e.g., a straight bond, not wedged or dashed bond, HC(OH)(CH3)(CH2CH3)) encompasses any stereochemical configuration at the chiral atom.

[0170] Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss et al., “Vogel’s Textbook of Practical Organic Chemistry,” 5thed. (1989), Longman Scientific & Technical, Essex CM202JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.

[0171] It should be understood that the compound may possess tautomeric forms, as well as geometric isomers, and that these also constitute embodiments of the disclosure.

[0172] In the compounds of formula (I), and any subformulas, any “hydrogen” or “H,” whether explicitly recited or implicit in the structure, encompasses hydrogen isotopes1H (protium) and2H (deuterium). Accordingly, any group comprising one or more hydrogen atoms encompasses corresponding deuterium-labeled versions of the group. For example, “-CH2-” encompasses

[0173] D

[0174]

[0175] -CD2-”; encompasses D , etc.

[0176] The present disclosure also includes isotopically-labeled compounds (e.g., deuterium labeled), where an atom in the isotopically-labeled compound is specified as a particular isotope of the atom. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36CI, respectively. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated in compounds of formula (I) are11C,13N,15O, and18F.

[0177] Isotopically-enriched forms of compounds of formula (I), or any subformulas, may generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-enriched reagent in place of a non-isotopically-enriched reagent. The extent of isotopic enrichment can be characterized as a percent incorporation of a particular isotope at an isotopically-labeled atom (e.g., % deuterium incorporation at a deuterium label).

[0178] General Synthesis

[0179] Compounds of formula (I) may be prepared by various synthetic processes. In various instances, compounds of formula (I) may be synthesized as shown below in Scheme 1. Scheme 1.

[0180] Pd-catalyst, Cu(l) source solvent, A

[0181]

[0182] i

[0183]

[0184] As shown in Scheme 1 above, compounds of formula (I) may be generated by reacting a tetrazine of formula i with a vinyl stannane of formula ii under suitable Stille coupling conditions (e.g., in the presence of Pd-catalyst (e.g., Pd(PPh3)4), Cu(l) source (e.g., CuTC), and solvent (e.g., dioxane), under reflux conditions).

[0185] Suitable reagents for reactions referenced above may be readily obtained from commercial sources or prepared by standard methods well known to those skilled in the art.

[0186] The compounds and intermediates may be isolated and purified by methods well-known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds may include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in Vogel’s Textbook of Practical Organic Chemistry, 5thed. (1989), \Longman Scientific & Technical, Essex CM202JE, England.

[0187] A disclosed compound may have at least one basic nitrogen whereby the compound may be treated with an acid to form a desired salt. For example, a compound may be reacted with an acid at or above room temperature to provide the desired salt, which is deposited, and collected by filtration after cooling. Examples of acids suitable for the reaction include, but are not limited to tartaric acid, lactic acid, succinic acid, as well as mandelic, atrolactic, methanesulfonic, ethanesulfonic, toluenesulfonic, naphthalenesulfonic, benzenesulfonic, carbonic, fumaric, maleic, gluconic, acetic, propionic, salicylic, hydrochloric, hydrobromic, phosphoric, sulfuric, citric, hydroxybutyric, camphorsulfonic, malic, phenylacetic, aspartic, or glutamic acid, and the like.

[0188] Reaction conditions and reaction times for each individual step may vary depending on the particular reactants employed and substituents present in the reactants used. Specific procedures are provided in the Examples section. Reactions may be worked up in the conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as crystallization, distillation, extraction, trituration and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or may be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commercially available, may be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above-described schemes or the procedures described in the synthetic examples section.

[0189] Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the invention. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in Wuts and Greene, in Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the invention may be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.

[0190] When an optically active form of a disclosed compound is required, it may be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization or enzymatic resolution).

[0191] Similarly, when a pure geometric isomer of a compound is required, it may be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.

[0192] It may be appreciated that the synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the invention as it is defined in the appended claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims.

[0193] Pharmaceutical Compositions

[0194] The disclosed compounds may be incorporated into pharmaceutical compositions suitable for administration to a subject. The disclosed compounds may also be provided as formulations, such as spray-dried dispersion formulations. The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the payload (e.g., therapeutic agent). A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a compound of the disclosure are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0195] The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which may serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butterand suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0196] Thus, the disclosed compounds and their pharmaceutically acceptable salts may be formulated for administration by, for example, solid dosing, eye drop, in a topical oil-based formulation, injection, parenteral, inhalation (either through the mouth or the nose), implants, or oral, buccal, topical, nasal, vaginal, or rectal administration. Techniques and formulations may generally be found in Remington’s Pharmaceutical Sciences, (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The route by which the disclosed compounds and their pharmaceutically acceptable salts are administered, and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis). The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0197] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.

[0198] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90% by weight.

[0199] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10% by weight.

[0200] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50% by weight.

[0201] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10% by weight.

[0202] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1 % by weight.

[0203] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0% by weight.

[0204] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1% by weight. Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5% by weight.

[0205] Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5% by weight.

[0206] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5% by weight.

[0207] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100% by weight.

[0208] Suitable suspending agents include AVICEL RC-591 (from EMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8% by weight.

[0209] Suitable surfactants include lecithin, Polysorbate 80, sodium lauryl sulfate, and the TWEENS (e.g., polyethylene glycol sorbitan monolaurates). Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 587-592 (1992); Remington’s Pharmaceutical Sciences, 15thed., 335-337 (1975); and McCutcheon’s Volume 1, Emulsifiers & Detergents, North American ed. 236-239 (1975). The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5% by weight.

[0210] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% by weight (or mass) of an active compound (e.g., a compound of formula (I)) and 50% to 99.99% by weight of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% by weight of active compounds and 90% to 99.9% by weight of a carrier including a diluent and a solvent.

[0211] Compositions for oral administration may have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% by weight of active compounds. The oral dosage compositions include about 50% to about 95% by weight of carriers, and more particularly, from about 50% to about 75% by weight.

[0212] Tablets may be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active compound, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which may be added for appearance. Chewable tablets may contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0213] Capsules (including implants, time release and sustained release formulations) typically include a compound (e.g., a compound of formula (I)), and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed compound, and glidants such as silicon dioxide to improve flow characteristics. Implants may be of the biodegradable or the non-biodegradable type.

[0214] The selection of ingredients in the carrier for oral compositions may depend on secondary considerations like taste, cost, and shelf stability.

[0215] Solid compositions may be coated by conventional methods, typically with pH or timedependent coatings, such that a disclosed compound is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0216] Compositions for oral administration may have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include a disclosed compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions may include one or more ingredients selected from colorants, flavors, and sweeteners.

[0217] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0218] The disclosed compounds and their pharmaceutically acceptable salts may be topically administered. Topical compositions that may be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed compound (e.g., a compound of formula (I)), and a carrier. The carrier of the topical composition may aid penetration of the compounds into the skin. The carrier may further include one or more optional components.

[0219] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods of this disclosure are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker& Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms’. Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd ed. (1976).

[0220] A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols. The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0221] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1, 2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95% by weight.

[0222] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95% by weight.

[0223] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95% by weight.

[0224] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a topical composition is typically 0% to 95% by weight.

[0225] The amount of thickener(s) in a topical composition is typically about 0% to about 95% by weight.

[0226] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95% by weight.

[0227] The amount of fragrance in a topical composition is typically about 0% to about 0.5% by weight, particularly, about 0.001% to about 0.1% by weight.

[0228] Suitable pH adjusting additives include hydrochloric acid (HCI) or sodium hydroxide (NaOH) in amounts sufficient to adjust the pH of a topical pharmaceutical composition.

[0229] Conjugates

[0230] In another aspect, the present disclosure provides conjugates derived from the compounds of formula (I). The conjugates disclosed herein comprise a molecule comprising a cysteine residue and a moiety of formula (l-a): wherein a moiety of formula:

[0231]

[0232] links the cysteine residue to the moiety of formula (I- a), and wherein:

[0233] R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocyclyl, wherein R1is optionally substituted with 1-5 substituents, wherein each substituent is independently selected from the group consisting of Ci-ealkyl, Ci.4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)O-i-Rx, -(X1)0-i-C(O)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -

[0234]

[0235] (X1)0-I-SO2RX, -(X1)0-I-PO2RX, -(X1)0-I-C(O)NHRX, and -(X1)0-i-C(O)NCi.4alkylRx; X1, at each occurrence, is -O-, -S-, -NH-, or-NCi.4alkyl-;

[0236] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;

[0237] R2is Ci.6alkyl, Ci.4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0.i-C(O)RY, -(Y1)0.I-CO2RY, -(Y1)o-i-S(0)RY, -(Y1)0-I-SO2RY, -(Y1)0-I-PO2RY, -(Y1)0-I-C(O)N(RY)2, -(Y1)O-i-Ci^alkylene-(Y1)o-i-RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-C(O)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-C02RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-S(O)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S02RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-PO2RY, or -

[0238]

[0239] (Y1)o-i-Ci-6alkylene-(Y1)o-i-C(0)N(RY)2; Y1, at each occurrence, is -O-, -S-, -NH-, or -NCi.4alkyl-;

[0240] RY, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGY; and

[0241] Gxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -N02, Ci-ealkyl, Ci-4haloalkyl, -CN, -0Ci-4alkyl, -OH, -OCi-4haloalkyl, -SH, — SCi-4alkyl, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -CO2H, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi-4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci.4alkyl, -SO2Ci-4alkyl, -SO2NH2, -SO2NHC1.

[0242] 4alkyl, and -SO2N(Ci-4alkyl)2.

[0243] In some instances, the molecule comprising the cysteine residue is a peptide or a protein. In some instances, R1is the optionally substituted 6- to 12-membered aryl. In some instances, the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl. In some instances, R2is Ci-ealkyl. In some instances, R2is methyl.

[0244] As shown in Scheme 2 below, exemplary conjugates may be prepared by reacting with a molecule comprising a cysteine residue with a compound of formula (I).

[0245] Scheme 2.

[0246]

[0247] In some instances, reacting the compound with the cysteine residue of the molecule occurs in the presence of a buffer solution. In some instances, the buffer solution has a pH of 4.0 to 9.0. In some instances, the conjugate is provided in quantitative yields.

[0248] Methods of Use

[0249] The disclosed compounds and conjugates may be used in various methods. In some instances, the disclosed compounds and conjugates may be used to selectively deliver a payload, e.g., to a subject in need thereof. The payload may be selectively delivered for therapeutic and / or diagnostic purposes. For example, the payload delivery may be targeted to specific locations within the subject, such as cysteine-containing proteins in cancer cells, while avoiding non-target tissues or organs that do not require administration of the payload.

[0250] As shown in Scheme 3, methods of selectively delivering a payload may comprise contacting cells (e.g., cancer cells) containing a cysteine-containing molecule (e.g., protein iii) with a compound of Formula (I) to form a conjugate (e.g., conjugate iv).

[0251] Scheme 3.

[0252]

[0253] Next, as shown above, the conjugate may be reacted with a functionalized payload, wherein the functionalized payload comprises a payload moiety attached to a cyclooctene, thereby providing a functionalized conjugate. As shown in Scheme 3 above, in one aspect, a linker attaches the cyclooctene to the payload. In various instances, the cyclooctene is trans-cyclooctene. In some instances, the functionalized payload is a functionalized payload of formula (II):

[0254]

[0255] wherein L1is the linker, and D1is the payload moiety. As shown above, in some instances, the functionalized conjugate comprises a moiety of formula (ll-a) or (ll-b):

[0256]

[0257] In some instances, the functionalized payload of formula (II) is:

[0258]

[0259] In some instances, L1is:

[0260] O O O

[0261]

[0262] wherein:

[0263] Z1is -0-, -S-, -NRa-, or-CRaRb-;

[0264] Raand Rb, at each occurrence, are each independently hydrogen or Ci-4alkyl; and m, at each occurrence, is 0-14,

[0265] n, at each occurrence, is 0-10, and p, at each occurrence, is 0-10.

[0266] In some instances, Z1is -O-. In some instances, Raand Rb, at each occurrence, are each independently hydrogen or methyl. In some instances, D1is a therapeutic agent, a diagnostic agent, or a targeting agent. In some instances, the diagnostic agent is a cyanine dye. In some instances, the therapeutic agent is an anti-cancer therapeutic agent. In some instances, the anticancer therapeutic agent is doxorubicin or ARV- 771.

[0267] 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 exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0268] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0269] Clause 1. A compound of formula (I),

[0270]

[0271] wherein:

[0272] R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocycle, wherein R1is optionally substituted with 1-5 substituents wherein each substituent is independently selected from the group consisting of Ci-ealkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)O-i-Rx, -(X1)0-i-C(O)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -

[0273]

[0274] (X1)0-I-SO2RX, -(X1)0-I-PO2RX, -(X1)0-I-C(O)NHRX, and -(X1)0-i-C(O)NCi-4alkylRx; X1, at each occurrence, is -0-, -S-, -NH-, or-NCi-4alkyl-;

[0275] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci.4haloalkyl, orGx;

[0276] R2is Ci.6alkyl, Ci.4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2RY, -(Y1)o-i-S(0)RY, -(Y1)0-I-SO2RY, -(Y1)O-I-P02RY, -(Y1)O-I-C(0)N(RY)2, -(Y1)o-i-Ci^alkylene-(Y1)O-i-RY, -(Y1)0-i-Ci-6alkylene-(Y1)o-i-C(0)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-C02RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-S(O)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S02RY, -(Y1)o-i-Ci-6alkylene-(Y1)0.i-P02RY, or -

[0277]

[0278] (Y1)o-i-Ci-6alkylene-(Y1)o-i-C(0)N(RY)2; Y1, at each occurrence, is -0-, -S-, -NH-, or-NCi.4alkyl-;

[0279] RY, at each occurrence, is hydrogen, Ci-salkyl, Ci.4haloalkyl, orGY; and

[0280] Gxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -N02, Ci-ealkyl, Ci.4haloalkyl, -CN, -OCi-4alkyl, -OH, -OCi.4haloalkyl, -SH, -SCi.4alkyl, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -CO2H, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi-4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci-4alkyl, -SO2Ci-4alkyl, -SO2NH2, -SO2NHC1.

[0281] 4alkyl, and -SO2N(Ci.4alkyl)2.

[0282] Clause 2. The compound of clause 1, wherein R1is the optionally substituted 6- to 12- membered aryl.

[0283] Clause s. The compound of clause 1 or 2, wherein the optionally substituted 6- to 12- membered aryl is optionally substituted phenyl.

[0284] Clause 4. The compound of any one of clauses 1-3, wherein R2is Ci-ealkyl.

[0285] Clause 5. The compound of clause 4, wherein R2is methyl.

[0286] Clause 6. The compound of any one of clauses 1-5, wherein the compound of formula (I) is:

[0287]

[0288] Clause 7. A conjugate comprising:

[0289] a molecule comprising a cysteine residue; and

[0290] a moiety of formula (l-a),

[0291]

[0292] Vs

[0293] wherein a moiety of formula ' links the cysteine residue to the moiety of formula (I- a), and wherein:

[0294] R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocyclyl, wherein R1is optionally substituted with 1-5 substituents, wherein each substituent is independently selected from the group consisting of Ci^alkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)O-i-Rx, -(X1)0-i-C(O)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -(X1)o-1-S02RX, -(X1)0-I-PO2RX, -(X1)o-1-C(0)NHRX, and -(X1)0.i-C(O)NCi.4alkylRx; X1, at each occurrence, is -0-, -S-, -NH-, or -NCi.4alkyl-;

[0295] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;

[0296] R2is Ci-6alkyl, Ci-4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2RY, -(Y1)o-i-S(0)RY, -(Y1)0-I-SO2RY, -(Y1)0-I-PO2RY, -(Y1)0-I-C(O)N(RY)2, -(Y1)o-i-Ci^alkylene-(Y1)o-i-RY, -(Y1)o.i-Ci-6alkylene-(Y1)o.i-C(0)RY, -(Y1)o-i-Ci^alkylene-(Y1)o-i-C02RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S(0)RY, -(Y1)o-i-Ci^alkylene-(Y1)o-i-S02RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-PO2RY, or -

[0297]

[0298] (Y1)o-i-Ci^alkylene-(Y1)o-i-C(0)N(RY)2;

[0299] Y1, at each occurrence, is -0-, -S-, -NH-, or-NCi.4alkyl-;

[0300] RY, at each occurrence, is hydrogen, Ci^alkyl, Ci-4haloalkyl, orGY; and

[0301] Gxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, — N02, Ci-6alkyl, Ci-4haloalkyl, -CN, — OCi_4alkyl, -OH, -OCi.4haloalkyl, -SH, -SC alkyl, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -CO2H, -C(O)Ci.4alkyl, -CO2Ci.4alkyl, -C(O)NH2, -C(O)NHCi.4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci.4alkyl, -SO2Ci.4alkyl, -SO2NH2, -SO2NHCi-4alkyl, and -SO2N(Ci-4alkyl)2.

[0302] Clause 8. The conjugate of clause 7, wherein the molecule comprising the cysteine residue is a peptide or a protein.

[0303] Clause 9. The conjugate of clause 7 or 8, wherein R1is the optionally substituted 6- to 12- membered aryl.

[0304] Clause 10. The conjugate of any one of clauses 7-9, wherein the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl.

[0305] Clause 11. The conjugate of any one of clauses 7-10, wherein R2is Ci-ealkyl.

[0306] Clause 12. The conjugate of clause 11, wherein R2is methyl.

[0307] Clause 13. A method of preparing the conjugate of any one of clauses 7-12, the method comprising:

[0308] providing a molecule comprising a cysteine residue; and

[0309] reacting the compound of any one of clauses 1-6 with the cysteine residue of the molecule, thereby providing the conjugate of any one of clauses 7-12.

[0310] Clause 14. The method of clause 13, wherein the molecule comprising the cysteine residue is a peptide or a protein.

[0311] Clause 15. The method of clause 13 or 14, wherein reacting the compound of any one of clauses 1-6, with the cysteine residue occurs in the presence of a buffer solution.

[0312] Clause 16. The bioconjugation method of clause 15, wherein the buffer solution has a pH of 4.0 to 9.0.

[0313] Clause 17. The bioconjugation method of any one of clauses 13-16, wherein the conjugate of any one of clauses 7-12 is provided in quantitative yields.

[0314] Clause 18. A method of functionalizing the conjugate of any one of clauses 7-12, the method comprising:

[0315] reacting the conjugate of any one of clauses 7-12 with a functionalized payload, wherein the functionalized payload comprises a payload moiety attached to a cyclooctene, thereby providing a functionalized conjugate.

[0316] Clause 19. The method of clause 18, wherein a linker attaches the cyclooctene to the payload. Clause 20. The method of clause 18 or 19, wherein the cyclooctene is trans-cyclooctene. Clause 21. The method of any one of clause 18-20, wherein the functionalized payload is a functionalized payload of formula (II):

[0317]

[0318] wherein L1is the linker, and D1is the payload.

[0319] Clause 22. The method of any one of clause 18-21 , wherein the functionalized conjugate comprises a moiety of formula (I l-a) or (I l-b) :

[0320]

[0321] Clause 23. The method of clause 21 or 22, wherein the functionalized payload of formula (II) is:

[0322] D1D1— 1_1

[0323] L1

[0324]

[0325] or

[0326] Clause 24. The method of any one of clauses 21-23, wherein L1is:

[0327] O O O

[0328]

[0329] wherein:

[0330] Z1is -O-, -S-, -NRa-, or-CRaRb-;

[0331] Raand Rb, at each occurrence, are each independently hydrogen or Ci-4alkyl; and m, at each occurrence, is 0-14,

[0332] n, at each occurrence, is 0-10, and

[0333] p, at each occurrence, is 0-10.

[0334] Clause 25. The method of clause 24, wherein Z1is -O-.

[0335] Clause 26. The method of clause 24 or 25, wherein Raand Rb, at each occurrence, are each independently hydrogen or methyl. Clause 27. The method of any one of clauses 21-26, wherein D1is a therapeutic agent, a diagnostic agent, or a targeting agent.

[0336] Clause 28. The method of any one of clauses 18-27, wherein the diagnostic agent is a cyanine dye.

[0337] Clause 29. The method of any one of clauses 18-27, wherein the therapeutic agent is an anticancer therapeutic agent.

[0338] Clause 30. The method of clause 29, wherein the anti-cancer therapeutic agent is doxorubicin or ARV-771.

[0339] EXAMPLES

[0340] Materials

[0341] Common materials or chemical reagents were purchased from commercial sources and used without further purification. The solvents were used by dry solvents system. All reactions were monitored by TLC or LC-MS. Purification was conducted on preparative flash column chromatography and preparative reversed-phase high performance liquid chromatography (RP-HPLC) with solvent systems specified. Nuclear magnetic resonance (NMR) spectra were recorded on automated Bruker AVIII-500 instruments. High-resolution mass spectra (HRMS) were recorded on a Bruker microTOF II instrument in positive ion mode using an Agilent G1969 API-TOF with an electrospray ionization (ESI) source. Ultraperformance liquid chromatography (UPLC) spectra for compounds were acquired using a Shimadzu LabSolutions system. All separations involved mobile phase of 0.1% TFA (v / v) in water (solvent A) and 0.1% TFA (v / v) in ACN (solvent B). Anti-BRD4 antibody (catalog no. 13440S) was purchased from Cell Signaling Technology, dilution was 1: 1000. Anti-GAPDH (catalog no. M7815) dilution was 1: 2000, and Anti-Rabbit antibodies (catalog no. MFCD00162788) dilution was 1: 1000 were purchased from Millipore Sigma. Anti-mouse IgG antibody (catalog no. A28177) was purchased from the Thermo Fisher, dilution was 1:3000. TCO-PEG3-Biotin (catalog no. CP-6014) was purchased from the Conju-Probe. HRP-Conjugated Streptavidin (catalog no. N100) was purchased from the Thermo Fisher, dilution was 1: 20000.

[0342] General Procedure for Solid-Phase Peptide Synthesis

[0343] Peptides were synthesized manually on a 0.3 mmol scale using CTC (chlorotrityl chloride) or Rink amide resins. Swelling: the resin was swelled in DMF for 20 min. Loading: the resin was treated with Fmoc-protected amino acid (6.0 equiv) and DIPEA (N,N-diisopropylethylamine,6.0 equiv) in DMF (5 mL) for 30 min, then washed with DMF three times. Fmoc removal: the resin was treated with 20% piperidine in DMF (v / v) for 20 min and washed with DMF three times. Coupling: the resin was treated with Fmoc-protected amino acid (6.0 equiv), HCTU (6.0 equiv), and DIPEA (6 equiv) in DMF (5 mL) for 1 h and then washed with DMF three times. Acetylation: the resin was treated with acetic anhydride (6 equiv) and DIPEA (6 equiv) in DMF for 35 min and washed with DMF three times. Cleavage: the resin was treated with the cocktail TFA / TIPS / H2O (95:2.5:2.5) for 3 h, precipitated from cold diethyl ether, and then purified by reverse-phase HPLC (30 x 150 mm) with a linear gradient of 3-95% (B %, v / v) at a flow rate of 17 mL / min over 40 min.

[0344]

[0345]

[0346] Cell Culture

[0347] HeLa and U87-MG cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS (GIBCO, catalog no. 10437) and 1 % penicillin / streptomycin (Thermo Fisher, catalog no.30-002-CI). The cells were grown at 37 °C with 5% CO2.

[0348] Cell Imaging

[0349] The cells were seeded in the 6-well plate and allowed to growth overnight. After the VMeTz treatments (100 pM, 1 h), the cells were washed 3 times with PBS. Then the cells were treated with tHCA (1 pM, 15 mins). After the PBS washing, the cells were fixed with 4% formaldehyde solution for 30 mins at room temperature and treated with 1* DAPI for 15 mins at room temperature. After another 3 times PBS washing, the cells were observed and imaged by using the ImageXpress Micro 4 High-Content Imaging System (Molecular Devices).

[0350] Cell Viability Assay

[0351] U87 cells were plated in 96-well plates with 6.0 x 103cells in each well and subsequently incubated for 24 h in a moist atmosphere of 5% CO2 and 37 °C. Then, different concentrations of test compounds or vehicles were added to triplicate wells. After incubation for an additional 72 h, 10 pL CCK-8 solution (Dojindo Molecular Technologies, catalog no. CK04-11) was added to each well, then the plates were incubated for 2-5 h at 37 °C. The absorbance was read at 450 nm on a Microplate Reader. The values of IC50 were calculated by the Logit method with the GraphPad software.

[0352] Immunoblot Assay

[0353] Cells were lysed in RIPA buffer supplemented with protease inhibitors. The lysates (40-60 pg protein) were then resolved by 4-12% Mini Protein Gel (Thermo Fisher) at 70 V for 10 mins and 200 V for 25 mins. Then the proteins were transferred from the gel to PVDF membrane (Bio- Rad) at 20 V for 120 mins. The membrane was incubated with primary antibody at 4 °C overnight, washed 3 times with PBS + 0.1% Tween20 (PBST), incubated secondary antibody in 5% nonfat milk for 60 mins at room temperature and then washed 3 times with PBST. The membranes were detected under the SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate (catalog no. A38555).

[0354] Apoptosis Assay

[0355] U87 cells were placed in 6-well transparent plates and then treated with test compounds and vehicle in a moist atmosphere of 5% CO2 at 37 °C for 24 h. After that, the cells were washed twice with cold Cell staining Buffer (BioLegend, catalog no. 420201 -BL) twice, and then were resuspended in Binding buffer (BioLegend, catalog no. 640914). 5 pL FITC Annexin V (BioLegend, catalog no. 640914) and 10 pL Propidium Iodide Solution (BioLegend, catalog no.

[0356] 640914) were added to the resuspended cell solution, which was then incubated for 15 min at room temperature in the dark. The analysis of stained cells was performed by a flow cytometer (BD FACS Canto II).

[0357] Proteomics Assay

[0358] In-gel digestion. Boiled clarified whole cell lysate (35 pg) was separated by 10% SDS-PAGE and stained with Bio-Safe Coomassie G-250 Stain (#1610786; Biorad, Hercules, CA). Each lane of the SDS-PAGE gel was cut into a single slice corresponding to the size BRD4 migrates on an SDS-PAGE gel. The gel slices were subjected to trypsin digestion and the resulting peptides were purified by Cis-based desalting exactly as previously described. See Kruse et al., Mol. Cell. Proteomics 16(1): 1718-1735 (2017)

[0359] Mass Spectrometry and Database Search

[0360] HPLC-ESI-MS / MS was performed in positive ion mode on a Thermo Scientific Orbitrap Fusion Lumos tribrid mass spectrometer fitted with an EASY-Spray Source (Thermo Scientific, San Jose, CA). NanoLC was performed as previously described. See Parker et al., Mol. Cell. Proteomics 18(7): 1363-1381 (2019). Tandem mass spectra were extracted from Xcalibur 'RAW files and charge states were assigned using the ProteoWizard 2.1.x msConvert script using the default parameters. The fragment mass spectra were searched against the SwissProt_2022 database (Homo sapiens, 20402 entries) using Mascot (Matrix Science, London, UK; version 2.8.0.1) using the default probability cut-off score. The search variables that were used were: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses; digestion with trypsin; a maximum of two missed tryptic cleavages; variable modifications of oxidation of methionine and phosphorylation of serine, threonine, and tyrosine. Cross-correlation of Mascot search results with X! Tandem was accomplished with Scaffold (V5.1.2; Proteome Software, Portland, OR, USA). Probability assessment of peptide assignments and protein identifications were made using Scaffold. Only peptides with > 95% probability were considered.

[0361] Label-free Peptide / Protein Quantification and Identification

[0362] Progenesis QI for proteomics software (version 2.4, Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK) was used to perform ion-intensity based label-free quantification as previously described. See Uhlorn et al., J. Am. Heart Assoc. 10(2): e018038 (2021). In brief, in an automated format, raw files were imported and converted into two-dimensional maps (y-axis = time, x-axis = m / z) followed by selection of a reference run for alignment purposes. An aggregate data set containing all peak information from all samples was created from the aligned runs, which was then further narrowed down by selecting only +2, +3, and +4 charged ions for further analysis. The samples were then grouped and a peak list of fragment ion spectra from only the top eight most intense precursors of a feature was exported in Mascot generic file (.mgf) format and searched against the SwissProt_2022 database (Homo sapiens, 20402 entries) using Mascot (Matrix Science, London, UK; version 2.8.0.1). The search variables that were used were: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses; digestion with trypsin; a maximum of two missed tryptic cleavages; variable modifications of oxidation of methionine and phosphorylation of serine, threonine, and tyrosine; 13C = 1. The resulting Mascot .xml file was then imported into Progenesis, allowing for peptide / protein assignment, while peptides with a Mascot Ion Score of < 25 were not considered for further analysis. Protein quantification was performed using only non-conflicting peptides and precursor ion-abundance values were normalized in a run to those in a reference run (not necessarily the same as the alignment reference run). Unbiased hierarchal clustering analysis (heat map) was performed in Perseus. See Tyanova et al., Nat. Methods 13: 731-740 (2016)

[0363] Synthesis and Characterization of VMeTz.

[0364] Scheme 4. Synthesis of VMeTz.

[0365] Pd(PPh3)4, CuTc

[0366] Bu3Sn dioxane, reflux

[0367] 2

[0368]

[0369]

[0370] 3-Phenyl-6-(prop-1-en-2-yl)-1,2,4,5-tetrazine (VMeTz)

[0371] A solution of 3-(methylthio)-6-phenyl-1,2,4,5-tetrazine (1) (50 mg, 0.245 mmol) and tributyl(prop-1-en-2-yl)stannane (2) (162 pL, 0.49 mmol) in 50 mL of dioxane was added Pd(PPh3)4 (43 mg, 0.037 mmol) and Copper(l) thiophene-2-carboxylate (94 mg, 0.49 mmol), then was stirred under N2atmosphere at reflux temperature for 40 min. The solvent was removed by a rotavapor, and the product was purified by flash column chromatography (hexanes / ethyl acetate = 10:1 v / v) on silica gel to give compound 4 (16 mg, 33%) as pink solid.1H NMR 58.66-8.52 (m, 2H), 7.68-7.51 (m, 3H), 6.91-6.75 (m, 1 H), 5.83 (p, J = 1.5 Hz, 1H), 2.42 (t, J = 1.3 Hz, 3H).13C NMR (101 MHz, Chloroform-d) 5 164.8, 163.5, 137.6, 132.7, 131.9, 129.4, 128.1, 124.0, 19.1. HRMS (ESI-TOF) m / z: [CHHI0N4+H]+calcd. for: 199.0973, found: 199.0978.

[0372] Results

[0373] In the initial phase of this investigation, simple vinyl-tetrazine (VTz) was synthesized and its conjugation reaction with reduced glutathione (GSH) was assessed (FIG. 2 and FIG. 3A). As expected, it was found that conjugation reaction between VTz and GSH occurs readily at RT in 10% (vol) CH3CN at pH 7.4 PBS buffer for 1 h. However, a significant amount of the byproduct GSH-( TZ)2containing 2 VTz moieties is also generated (LC-MS analysis) through conjugate addition of the mono-addition adduct GSH-VTz to a second molecule of VTz even when only 1.2 equiv. of VTz is used. Formation of a bis-adduct would be particularly problematic in protein bioconjugation since large excesses of the bioconjugate reagent are employed to enhance rate and yield, and it could interfere with characterization of the conjugate. Indeed, at least 4 conjugates (BSA-VTz, BSA-(VTz)2, BSA-(VTz)3and BSA-(VTz)4) are produced when bovine serum albumin (BSA; SEQ ID NO: 4) (containing 1 free Cys residue, 1.0 mM) is treated with 5.0 equiv. of VTz (FIG. 4). It should be noted that during our investigation, Wu and colleagues reported the use of a similar reagent, 3-vinyl-6-oxymethyl-tetrazine (VoTz), for bioconjugation and click functionalization. In these studies, only peptides were used for bioconjugation studies.

[0374] The intrinsic problem of forming multiple conjugate products with VTz stimulated the design new vinyl tetrazine derivatives. It was proposed that introducing a methyl group at the a- position of the vinyl group could sterically block the second conjugate addition reaction. To test this proposal, isopropenyl-tetrazine (VMeTz) was synthesized and its bioconjugation reaction with GSH was evaluated (FIG. 3A-B). Indeed, under the same conditions this process generates a single GSH adduct even when excess (3.0 equiv, 3.0 mM) VMeTz is used (FIG. 3B). The reaction cleanly and quantitatively to rapidly form the cysteine-modified product Cys- MeTz (FIG. 2A, FIG.

[0375] 4) with a second-order rate constant (fe) of 0.74 M’1s-1(FIG. 2B) that is comparable to the commonly used reactions of GSH with 2-chloromethyl acrylamide (1.17 M-1s’1) and ethynylphosphonamidates (0.62 M-1s-1). Moreover, “click” reaction of VMeTz with TCO-OH occurs smoothly (FIG. 5) with a rate (fe: 16.65 M’1s"1) (FIG. 2C).

[0376] In the second phase of this effort, bioconjugation reactions between VMeTz and Cys containing peptides were investigated. For this purpose, the stability of VMeTz in pH 7.4 PBS buffer was determined. The results show that no noticeable degradation of the reagent takes place within 24 h (FIG. 6A). Then, bioconjugation of peptide 1 (Pep1, AC-GC KFE-CONH?, SEQ ID NO: 1) with VMeTz was explored (FIG. 6B-C, and FIG. 7). LC-MS and HPLC analysis of the reaction mixture revealed that VMeTz readily and selectively reacts with the Cys residue in Pep1 to form the stable conjugate Pep1-VMeTz.

[0377] In contrast, the Cys adduct generated by reaction of Pep1 with the maleimide reagent / V-phenylmaleimide (NPM), which has been widely used to form antibody-drug conjugates (ADCs), 10 is labile under the same conditions and readily undergoes retro-Michael reaction (FIG.

[0378] 8). As a result, reaction of NPM with Pep1 remains incomplete (60% yield of the adduct Cys-Imide) at pH 7.4, while at pH 8.0, and only 15% of the conjugate forms (FIG. 8A). In addition, the byproduct Cys-lmide-Hydro resulting from hydrolysis of the imide moiety is also generated (FIG.

[0379] 8B, D). These undesired processes would be problematic in real-time use of NPM for biomolecule labeling, for instance in the ADCs where systemic side effects could be caused by premature release of toxic warheads. The above studies clearly show that the VMeTz modified products are sufficiently stable to minimize problem associated with NPM bioconjugation.

[0380] Next, LC-MS analysis of Pep1 -VMeTz was conducted to determine the site selectivity of the conjugation process. The results demonstrate that selective bioconjugation of the Cys residue occurs without competitive modification of nucleophilic Lys in Pep1 (FIG. 6D-E). Furthermore, similar results arise from studies of reactions of VMeTz with Pep2 (NHZ-CFSDLWKLLS-COOH (SEQ ID NO: 2), FIG. 9) and Pep3 (NH2-TFECYWCQL-COOH (SEQ ID NO: 3), FIG. 10) where Cys-selective modifications take place. Notably, both Cys residues in Pep3 are efficiently modified with VMeTz. Importantly, other common nucleophilic amino acids including Ser, Lys, Thr and Tyr do not react with VMeTz. Subsequent click reaction of the Pep2-tetrazine conjugate Pep2-VMeTz with TCO-OH (FIG. 9) proceeds efficiently and cleanly within 30 min to generate the desired conjugate Pep2-VMeTz-TCO-OH (FIG. 9). Collectively, these findings demonstrate that VMeTz is a unique dual functional reagent, which selectively and efficiently reacts with Cys residues to form conjugates that undergo rapid click reaction with TCO derivatives.

[0381] Having demonstrated that highly chemo-selective modification of peptides by VMeTz is viable, the use of this reagent for tagging cysteines in proteins was explored next. Two representative proteins were selected for this purpose including bovine serum albumin (BSA; SEQ ID NO: 4) and ubiquitin mutant UbK48C (SEQ ID NO: 5), both of which contain one Cys (natural Cys34 and engineered K48C, respectively). Bioconjugation reaction of BSA with VMeTz was carried out at physiological pH (7.4) at rt overnight. LC-MS analysis of the reaction mixture indicated that a single modification product BSA-VMeTz is formed with high efficiency (FIG. 11 and FIG. 12). This process contrasts with VTz bioconjugation of BSA, which yields at least 4 products (BSA-VTz, BSA-(VTz)2, BSA-(VTz)3 and BSA-(VTz)4) even when only 5.0 equiv. of the reagents used (FIG. 4). Moreover, proteomic MS-MS analysis indicates that labeling of BSA occurs at Cys34 (FIG. 11B-C and FIG. 12C). Also, circular dichroism (CD) spectroscopy was employed to show that no significant change takes place in the secondary structural content of the protein upon formation of BSA-VMeTz (FIG. 11 D). Finally, subsequent click reaction of BSA-VMeTz with TCO-Cy5 (1.0 mM) proceeds efficiently (30 min) to generate a fluorescent tagged protein that emits red fluorescence in the SDS-PAGE gel while no fluorescence is observed when TCO-Cy5 is not in the incubation mixture (Coomassie staining as control, FIG. 4A, 4E). The modification process is insensitive to pH and can be used for protein imaging. High reactivity and selectivity also attended bioconjugation reaction of UbK48C with VMeTz were achieved as well (FIG. 13). These protein bioconjugation studies validate that VMeTz is a viable dual functional reagent for efficient protein bioconjugation-functionalization.

[0382] Table 2. Polypeptide Sequences

[0383] SEQ ID

[0384] Name Sequence (N >C)

[0385] NO DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKT CVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLS HKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYY ANKYNGVFQECCQAEDKGACLLPKIETMREKVLTSSARQRLRCASIQKFGER ALKAWSVARLSQKFPKAE FVEVTKLVTDLTKVHKECCHGDLLECADDRADLA BSA 4 KYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDK DVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDD PHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQ VSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPV

[0386]

[0387] SEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEK QIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGP KLVVSTQTALA

[0388] Ubiquitin MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGCQLEDGRTLS

[0389]

[0390] UbK48C DYNIQKESTLHLVLRLRGG 5

[0391] Having shown that VMeTz is a useful bifunctional reagent for labeling proteins in vitro, its capacity for cellular protein labeling and functionalization was evaluated next. For this purpose, HeLa cells in pH 7.4 PBS buffer at 37 °C were incubated with VMeTz (100 pM) for 30 min (FIG.

[0392] 14A). After washing the cells with PBS buffer, non-fluorescent, fluorogenic TCO-caged hemicyanine (tHCA) was added and the mixture was incubated for 15 min. It was expected that reaction of TCO-caged hemicyanine tHCA with the Tz moiety in VMeTz would result in generation of red fluorescent HCA (FIG. 140). In contrast, the cells in the control group in which VMeTz is not added do not display red fluorescence. Observations made in the fluorescent imaging studies reveal that HeLa cells treated sequentially with VMeTz and tHCA display red emission, indicating that the bioconjugation reagent has high cell membrane permeability and click-release of HAC occurs inside cells (FIG. 14B).

[0393] Guided by these results, a TCO group caged PROATC ARV-771 prodrug TCO-ARV-771 was utilized to demonstrate the feasibility of employing VMeTz and I EDDA (“inverse electron demand Diels-Alder”) promoted bioorthogonal activation and delivery of PROTAC ARV-771 into HeLa cells for anti-cancer therapy. ARV-771 is a PROTAC that effectively degrades oncogenic BRD4 protein in tumor cells. Caging the hydroxyl group of the VHL ligand in ARV-771 abolishes its degradation ability. However, it was anticipated that ARV-771 would be liberated from the VMeTz labeled protein components of HeLa cells through reaction with TCO-ARV-771. Indeed, it was observed that significant degradation of BRD4 protein activity occurs when HeLa cells are co-treated with VMeTz and TCO-ARV-771 (FIG. 14C). However, treatment with TCO-ARV-771 alone does not degrade BRD4 activity. Furthermore, BRD4 degradation efficiency is dependent on the concentrations of TCO-ARV-771 and / or VMeTz (FIG. 14C) and the ubiquitin pathway (FIG.

[0394] 14D), which was blocked in the presence of proteasome inhibitor MG-132. Taken together, these findings clearly demonstrate that VMeTz labeled proteins promote the reaction of TCO-ARV-771 that releases ARV-771 which induces subsequent BRD4 degradation in cells.

[0395] Glioblastoma (GBM) is an aggressive phenotype associated with dismal patient survival and, as such, it is considered to be the most dangerous and fatal type of cancer.60 In the United States, approximately 13,000 new glioblastoma patients are diagnosed each year, 90% of whom die within three years. The development of efficient therapies for this cancer requires identification of new targets for treatment. Toward this end, VMeTz was used to profile Cys containing proteins in U87 cells which are widely used in therapy and mechanism of action studies of GBM.

[0396] U87 cells were incubated with VMeTz (200 pM) for 5, 30, and 60 min (FIG. 16A), and then washed with PBS and incubated with TCO-biotin for another 15 min. Ensuing cell lysis and Western Blot assays produces gels corresponding to the time-course of protein labeling given in FIG. 16B. Dose-dependent labeling was also explored using the same protocol to confirm the caging ability of VMeTz in U87 cells (FIG. 16C). Furthermore, LC-MS / MS and proteomics technology were employed to analyze the VMeTz labeled proteins in U87 cells (FIG. 16D). The scaffold reviewed data (protein threshold: 99%, min peptides: 5, peptide threshold: 95%) show that 94 proteins in U87 cells are modified by VMeTz (FIG. 17). For example, it was found that the Parkinsonism associated deglycase, known as DJ1 protein, was modified in the process. Overexpressed DJ1 protein, an oncogenic molecule, contributes to anti-apoptotic signaling and protein quality control in response to oxidative stress. Previous studies have shown that DJ1 plays an important role in self-renewal of stem cells in the U87 glioblastoma cell line. The accumulated evidence indicates that DJ1 is a promising target for cancer therapy, although the debate about its role in cancer has not been resolved to date. Therefore, labeling of DJ1 in the live U87 cells with VMeTz (FIG. 16E) may serve as a potential useful chemical tool for interrogation of the function of DJ1 and the development of potential treatments for that focus on this target.

[0397] To assess applications associated with the synergy of VMeTz and TCO caged prodrugs, studies of potential anti-cancer therapeutic ramifications were conducted utilizing U87 cells. The low toxicities (FIG. 18A) and high cell permeabilities of VMeTz (FIG. 14B) enable their use in cellular environments. A TCO caged doxorubicin (TCO-Dox) was employed as a prodrug to test the effects of VMeTz “click-release.” It was proposed that VMeTz modified Cys-containing proteins in U87 cells would trigger click-release of chemotherapeutic Dox from TCO-Dox to induce apoptosis. The results show that the VMeTz + TCO-Dox co-treatment group display toxicities (IC50 = 785 nM) that are comparable to Dox (IC50 = 632 nM), whereas the toxicity of the TCO-Dox alone is significantly lower (IC50 - 6.61 pM) (FIG. 18B). The similar toxicities observed for the VMeTz + TCO-Dox and Dox alone treatment groups suggest that VMeTz labeling U87 cells do trigger activation of the prodrug. Moreover, as demonstrated by the degradation ability of VMeTz and TCO-ARV-771 in HeLa cells (FIG. 14), the VMeTz + TCO-ARV-771 treatment strategy should be viable in U87 cells (FIG. 18C). Indeed, it was observed that co-treatment with VMeTz and TCO-ARV-771 induces a level of toxicity (IC50 = 268 nM) similar to that of ARV-771 alone (IC50 = 291 nM) while the TCO-ARV-771 prodrug has significantly reduced toxicity (IC50 = 3.64 pM). Proteomics analysis also shows the significant degradation of BRD4 protein by the co-treatment group (FIG. 18D).

[0398] To further evaluate anti-cancer effects that accompany the new approach, flow cytometry assays were utilized to measure extents of apoptosis. The results show that the VMeTz (10 pM, 3 h pretreatment) + TCO-ARV-771 (1 pM, 24 h) co-treatment group induces early apoptosis (11.9 %, FIG. 18E) in a manner that is similar to ARV-771 (1 pM, 14.8%, FIG. 18E), but a level that is higher than those of control groups including DMSO 1.31% (FIG. 18E), VMeTz (10 pM) 1.12% (FIG. 7F) and TCO-ARV-771 (100 nM) 0.82% (FIG. 18E). The finding arising from this investigation show conclusively that VMeTz bioconjugation can be used to efficiently activate TCO caged prodrugs to release anti-cancer drugs in live U87 cells.

[0399] In the study described above, a new dual functional reagent VMeTz was developed for selective labeling Cys residue and subsequent click functionalization of peptides, proteins in vitro and in live cells. VMeTz reagent was shown to be a useful conjugating reagent that efficiently and selectively produces stable single Tz-containing-Cys Michael adducts, which are applicable protein structural characterization and functionalization. The high potential of the 2-in-1 dual functional reagent has been demonstrated by its utilization in selective modification and efficient functionalization of Cys-containing peptides and proteins. Furthermore, its high cell permeability and low toxicity make VMeTz applicable to labeling and profiling Cys-containing proteins. Finally, it was also demonstrated that this dual functional probe is capable of selectively activating the delivery of the TCO-caged toxic agents Dox and PROTAC ARV-771 in cancer cells, and that this mode of delivery brings about therapeutic effects that are comparable to those of the parent drugs but with significantly reduced side effects.

Claims

CLAIMSWhat is claimed:

1. A compound of formula (I),wherein:R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocycle, wherein R1is optionally substituted with 1-5 substituents wherein each substituent is independently selected from the group consisting of Ci^alkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)O-i-Rx, -(X1)0-i-C(O)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -(X1)0-I-SO2RX, -(X1)0-I-PO2RX, -(X1)0-I-C(O)NHRX, and -(X1)0-i-C(O)NCi-4alkylRx; X1, at each occurrence, is -O-, -S-, -NH-, or-NCi.4alkyl-;Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;R2is Ci-6alkyl, Ci-4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2RY, -(Y1)0-i-S(O)RY, -(Y1)0-I-SO2RY, -(Y1)O-I-P02RY, -(Y1)0-I-C(O)N(RY)2, -(Y1)o-i-Ci^alkylene-(Y1)O-i-RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-C(0)RY, -(Y1)o-i-Ci^alkylene-(Y1)o-i-C02RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-S(O)RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-S02RY, -(Y1)o-i-Ci-6alkylene-(Y1)o-i-P02RY, or -(Y1)o-i-Ci-6alkylene-(Y1)o-i-C(0)N(RY)2; Y1, at each occurrence, is -0-, -S-, -NH-, or -NCi.4alkyl-;RY, at each occurrence, is hydrogen, Ci.6alkyl, Ci.4haloalkyl, orGY; andGxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a C3-6cycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -N02, Ci^alkyl, Ci-4haloalkyl, -CN, -OCi^alkyl, -OH, -OCi-4haloalkyl,-SH, -SCi-4alkyl, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -CO2H, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi-4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci-4alkyl, -SO2Ci-4alkyl, -SO2NH2, -SO2NHC1.4alkyl, and -SO2N(Ci-4alkyl)2.

2. The compound of claim 1 , wherein R1is the optionally substituted 6- to 12-membered aryl.

3. The compound of claim 2, wherein the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl.

4. The compound of claim 1, wherein R2is Ci-ealkyl.

5. The compound of claim 4, wherein R2is methyl.

6. The compound of claim 1, wherein the compound of formula (I) is:A conjugate comprising:a molecule comprising a cysteine residue; anda moiety of formula (l-a),NR2d-a),Swherein a moiety of formula:links the cysteine residue to the moiety of formula (I- a), and wherein:R1is a 6- to 12-membered aryl, a 5- to 12-membered heteroaryl, a Cs-scycloalkyl, or a 4- to 12-membered heterocyclyl, wherein R1is optionally substituted with 1-5 substituents, wherein each substituent is independently selected from the group consisting of Ci^alkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, -(X1)o-i-Ci^alkylene-(X1)o-i-Rx, -(X1)o-i-C(0)Rx, -(X1)0-I-CO2RX, -(X1)0-i-S(O)Rx, -(X1)0-I-SO2RX, -(X1)0-I-PO2RX, -(X1)0-I-C(O)NHRX, and -(X1)0-i-C(O)NCi-4alkylRx; X1, at each occurrence, is -O-, -S-, -NH-, or -NCi^alkyl-;Rx, at each occurrence, is hydrogen, Cvealkyl, Ci-4haloalkyl, orGx;R2is Ci.6alkyl, Ci.4haloalkyl, halogen, -CN, GY, -Y1-RY, -(Y1)0-i-C(O)RY, -(Y1)0-I-CO2Y, -(Y1)o-i-S(0)RY, -(Y1)o -I-SO2Y, -(Y1)O-I-P02RY, -(Y1)0.I-C(O)N(RY)2, -(Y1)o-i-Ci^alkylene-(Y1)O-i-RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-C(O)RY, -(Y1)o-i-Ci-6alkylene-(Y1)0-i-C02RY, -(Y1)0-i-Ci-6alkylene-(Y1)0-i-S(O)RY, -(Y1)o-i-Ci^alkylene-(Y1)o-i-S02RY, -(Y1)0-i-Ci.6alkylene-(Y1)o-i-P02RY, or -(Y1)o-i-Ci-6alkylene-(Y1)0-i-C(0)N(RY)2;Y1, at each occurrence, is -O-, -S-, -NH-, or -NCi.4alkyl-;RY, at each occurrence, is hydrogen, Ci.6alkyl, C-i.4haloalkyl, orGY; andGxand GY, at each occurrence, are independently a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gxand GY, at each occurrence, are independently optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -N02, Ci-ealkyl, Ci.4haloalkyl, -CN, — OCi-4alkyl, -OH, -OCi.4haloalkyl, -SH, — SCi-4alkyl, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -CO2H, -C(O)Ci-4alkyl, -CO2Ci-4alkyl, -C(O)NH2, -C(O)NHCi-4alkyl, -C(O)N(Ci.4alkyl)2, -S(O)H, -S(O)Ci-4alkyl, -SO2Ci-4alkyl, -SO2NH2, -SO2NHCI-4alkyl, and -SO2N(Ci-4alkyl)2.

8. The conjugate of claim 7, wherein the molecule comprising the cysteine residue is a peptide or a protein.

9. The conjugate of claim 7, wherein R1is the optionally substituted 6- to 12-membered aryl.

10. The conjugate of claim 9, wherein the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl.

11. The conjugate of claim 7, wherein R2is Ci-ealkyl.

12. The conjugate of claim 11, wherein R2is methyl.

13. A method of preparing the conjugate of claim 7, the method comprising:providing a molecule comprising a cysteine residue; andreacting the compound of claim 6 with the cysteine residue of the molecule, thereby providing the conjugate of claim 7.

14. The method of claim 13, wherein the molecule comprising the cysteine residue is a peptide or a protein.

15. The method of claim 13, wherein reacting the compound of claim 1, with the cysteine residue of the molecule occurs in the presence of a buffer solution.

16. The bioconjugation method of claim 15, wherein the buffer solution has a pH of 4.0 to 9.0.

17. The bioconjugation method of claim 13, wherein the conjugate of claim 7 is provided in quantitative yields.

18. A method of functionalizing the conjugate of claim 7, the method comprising:reacting the conjugate of claim 7 with a functionalized payload, wherein the functionalized payload comprises a payload moiety attached to a cyclooctene, thereby providing a functionalized conjugate.

19. The method of claim 18, wherein a linker attaches the cyclooctene to the payload moiety.

20. The method of claim 18, wherein the cyclooctene is frans-cyclooctene.

21. The method of claim 20, wherein the functionalized payload is a functionalized payload of formula (II):D / 1L1wherein L1is the linker, and D1is the payload moiety.

22. The method of claim 21 , wherein the functionalized conjugate comprises a moiety of formula (I l-a) or (I l-b):

23. The method of claim 21 , wherein the functionalized payload of formula (II) is:

24. The method of claim 21, wherein L1is:O O Owherein:Z1is -O-, -S-, -NRa-, or-CRaRb-;Raand Rb, at each occurrence, are each independently hydrogen or Ci^alkyl; and m, at each occurrence, is 0-14,n, at each occurrence, is 0-10, andp, at each occurrence, is 0-10.

25. The method of claim 24, wherein Z1is -O-.

26. The method of claim 24, wherein Raand Rb, at each occurrence, are each independently hydrogen or methyl.

27. The method of claim 21, wherein D1is a therapeutic agent, a diagnostic agent, or a targeting agent.

28. The method of claim 27, wherein the diagnostic agent is a cyanine dye.

29. The method of claim 27, wherein the therapeutic agent is an anti-cancer therapeutic agent.

30. The method of claim 29, wherein the anti-cancer therapeutic agent is doxorubicin or ARV-771.

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