Tetrazine allyl acetate as a dual functional BIS-thiol bioconjugation-functionalization agent

Abstract

Described herein is a dual-functional tetrazine–allyl reagent that enables site-selective disulfide modification in peptides and proteins, serving both as a Michael acceptor for bis-thiols and as a reactive handle for Inverse Electron-Demand Diels–Alder (IEDDA) click chemistry to introduce functional payloads.

Description

[0001] 212443-0005-W001

[0002] TETRAZINE ALLYL ACETATE AS A DUAL FUNCTIONAL BIS-THIOL BIOCONJUGATIONFUNCTIONALIZATION AGENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 711,085 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, “212443-0005-W001_sequence_listing_xml_10-OCT-2025.xml,” was created on October 10, 2025, contains 10 sequences, has a file size of 9.75 kilobytes (9,986 bytes), and is incorporated by reference in its entirety into the specification.

[0008] BACKGROUND

[0009] Owing to their high target affinity and specificity, proteins have rapidly become a new family of therapeutics, as evidenced by data showing that they comprise 12 out of 20 top selling drugs in 2023. Site-selective conjugation reactions for protein modifications with small chemical groups, such as fluorophores or bioactive compounds, have facilitated biomedical research and therapeutic development. In this context, bioorthogonal click reactions have been broadly employed as chemical tools for protein modifications. Among these processes, the inverse electron demand Diels-Alder (IEDDA) reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooctene (TCO) is exceptional because of its rapid rate (up to 106M-1s’1), excellent orthogonality, catalyst-free nature, biocompatibility and unrivaled click-release capacity. As a result, the IEDDA reaction of Tz with TCO has become a versatile bioorthogonal tool for use in chemical biology and drug delivery. The rapid rate of this process is particularly appealing in the context of modification of proteins in their native environment where they exist in very low concentrations.

[0010] Currently, two common strategies are utilized in lEDDA-based site-selective protein modification including the genetic code expansion method in which Tz or a TCO moiety is introduced via incorporation of noncanonical amino acids, and the tag-based posttranslational 212443-0005-W001

[0011] attachment approach utilizing enzymes like lipoic acid protein ligase A. However, the genetic code expansion method is often impeded by low yields and intricate synthesis processes that limit scalability and accessibility, while the tag-based posttranslational attachment strategy require initial genetic fusion of a specific peptide sequence (e.g., lipoate acceptor peptide) to the protein of interest.

[0012] In contrast, attachment of desired functionalities to natural amino acid residues in proteins is a straightforward, cost-effective approach for protein bioconjugation Owing to its relatively low abundance and the unique nucleophilicity of its sulfhydryl group, the cysteine (Cys) moiety is an ideal site for highly chemoselective protein conjugation. However, few proteins in their natural state contain free cysteine residues although they can be introduced by using genetic engineering. Disulfide rebridging strategy, involving reduction of a disulfide bond to form two free Cys sulfhydryl groups is an alternative approach, particularly in forming antibody-drug conjugates (ACDs). An assortment of reagents have been designed to react highly selectively rebridge the liberated bis-thiols such as dibromomaleimides, divinylpyrimidines, b / s-vinylsulfones, 2-chloromethyl acrylamide, double thiol-ene reagent, a-chlorothioesters, bispentafluorophenyl sulfonamide, heteroaryl nitriles, 3-bromo-5-methylene pyrrolones, and vinylphosphonothiolate. However, these reagents can only accomplish protein and antibody bioconjugation. Therefore, an additional clickable moiety needs to be pre-attached to the bioconjugation group for subsequent introduction of specific functionality such as drug payloads into proteins in ADCs.

[0013] What is needed are dual functional bis-thiol bioconjugation-functionalization agents.

[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 heterocyclyl, wherein R1is optionally substituted with 1-5 substituents, wherein each substituent is independently selected from the group consisting of Ci-salkyl, 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, -

[0019]

[0020] (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-; 212443-0005-W001

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

[0022] Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -NO2, Ci-ealkyl, Ci.

[0023] 4haloalkyl, -CN, -OCi-4alkyl, -OCi-4haloalkyl, -OH, - SCi.4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci.4alkyl, -CO2H, -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; and

[0024] R2is a leaving group.

[0025] In one 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.

[0026] In another aspect, the leaving group at R2is -Y1-C(O)RY, -Y1-S(O)RY, -Y1-CO2RY, -Y1-SO2RY, -Y1-PO2RY, -Y1RY, -Br, -Cl, -I, -CN, -Y1-Ci-6alkylene-(Y1)0-i-RY,

[0027] -Y1-Ci-6alkylene-(Y1)0-i-C(O)RY, -Y1-Ci-6alkylene-(Y1)o-i-S(0)RY,

[0028] -Y1-Ci-6alkylene-(Y1)0-i-CO2RY, -Y1-Ci-6alkylene-(Y1)0-i-SO2RY, or

[0029] -Y1-Ci-6alkylene-(Y1)0-i-PO2RY;

[0030] RY, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGY;

[0031] Y1, at each occurrence, is -O-, -NH-, -NCi-4alkyl-, or-S-; and

[0032] GY, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein GY, at each occurrence, is optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -NO2, Ci-ealkyl, C1-4haloalkyl, -CN, -OCi-4alkyl, -OCi-4haloalkyl, -OH, - SCi-4alkyl, -SH, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -C(O)Ci.4alkyl, -CO2H, -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.

[0033] In another aspect, the leaving group at R2is -Y1-C(0)RYor-Y1-SO2RY. In another aspect, Y1is -O-. In another aspect, RYis Ci-ealkyl or Ci-4haloalkyl. In another aspect, RYis -CH3 or -CF3. In another aspect, the leaving group is -O-C(O)CH3.

[0034] In another aspect, the compound of formula (I) is: 212443-0005-W001

[0035]

[0036] Another embodiment described herein is a conjugate comprising:

[0037] a molecule comprising two cysteine residues; and

[0038] a moiety of formula (l-a),

[0039]

[0040] \S\

[0041] V

[0042] wherein a moiety of formu

[0043]

[0044] SX

[0045] la ' links the two cysteine residues to the moiety of formula (l-a), and wherein:

[0046] 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, -

[0047]

[0048] (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-;

[0049] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;

[0050] X1, at each occurrence, is -O-, -S-, -NH-, or -NCi.4alkyl-; and

[0051] Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a C3-6cycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is 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, -0Ci-4alkyl, -OCi-4haloalkyl, -OH, — SCi-4alkyl, -SH, -NH2, -NHCi.4alkyl, -N(Ci.4alkyl)2, -C(O)H, -C(O)Ci.4alkyl, -CO2H, -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. 212443-0005-W001

[0052] In one aspect, the molecule comprising at least two cysteine residues is a peptide or a protein. 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.

[0053] Another embodiment described herein is a method of preparing a conjugate described herein, the method comprising:

[0054] providing a molecule comprising at least two cysteine residues; and

[0055] reacting the compound of formula (I) with two cysteine residues of the molecule, thereby providing the conjugate.

[0056] In one aspect, the molecule comprising at least two cysteine residues is a peptide or a protein. In another aspect, reacting the compound of formula (I), with the two cysteine residues occurs in the presence of a buffer solution.

[0057] In another aspect, the buffer solution has a pH of 4.0 to 9.0.

[0058] In another aspect, the conjugate is provided in quantitative yields.

[0059] Another embodiment described herein is a method of functionalizing a conjugate, the method comprising: reacting a conjugate described herein with a functionalized payload, wherein the functionalized payload comprises a payload attached to a cyclooctene, thereby providing a functionalized conjugate. In another aspect, a linker attaches the cyclooctene to the payload. In another aspect, the cyclooctene is trans-cyclooctene. In another aspect, the functionalized payload is a functionalized payload of formula (II):

[0060]

[0061] wherein L1is the linker, and D1is the payload.

[0062] In another aspect, the functionalized conjugate comprises a moiety of formula (I l-a) or (I l-b) :

[0063]

[0064] In another aspect, the functionalized payload of formula (II) is: 212443-0005-W001

[0065]

[0066] In another aspect, L1is:

[0067] O O

[0068] RaRb

[0069]

[0070] wherein:

[0071] Z1is -O-, -S-, -NRa-, or-CRaRb-;

[0072] Raand Rb, at each occurrence, are each independently hydrogen or Ci-4alkyl; and m, at each occurrence, is 0-14

[0073] n, at each occurrence, is 0-10, and

[0074] p, at each occurrence, is 0-10.

[0075] 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.

[0076] DESCRIPTION OF THE DRAWINGS

[0077] 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.

[0078] FIG. 1 shows the dual functionality of the 2-in-1 reagent TzAA, enabling site-selective incorporation of a tetrazine (Tz) tag into bi-cysteine / disulfide motifs within peptides or proteins, followed by inverse electron-demand Diels-Alder (I EDDA) based conjugation with transcyclooctene (TCO)-linked substances..

[0079] FIG. 2 shows HPLC analysis of TzAA in phosphate-buffered saline (PBS, pH 7.4) before and after 24 hours of incubation, demonstrating its stability under physiological conditions. 212443-0005-W001

[0080] FIG. 3A-B the reaction between TzAAand cysteine to form TzAA-2Cys (FIG. 3A), and the corresponding HPLC trace comparing TzAA alone and the reaction mixture post-incubation with cysteine (FIG. 3B).

[0081] FIG. 4 shows the chemical structure of Somatostatin-Tz (SST-Tz), a tetrazine-modified peptide.

[0082] FIG. 5 shows HPLC analysis of the reaction of Somatostatin (SEQ ID NO: 5) and TzAA. FIG. 6A-C show analytical characterization of Somatostatin-Tz. FIG. 6A shows the Electrospray Ionization (ESI) mass spectrum of Somatostatin-Tz with calculated and observed m / z values for mono-, di-, and tri-protonated species. HPLC-MS (ESI) mlz calcd. for C87H115N22O19S2 (M+H)+1835.8, found 1836.2; calcd. for C87HII6N22OI9S2 (M+2H)2+918.4, found 918.5. FIG. 6B shows circular dichroism (CD) spectra comparing native Somatostatin (SEQ ID. NO: 5) and Somatostatin-Tz (0.2 mg / mL in water, 195-260 nm). FIG. 6C shows click functionalization of Somatostatin-Tz with TCO-Cy5, TCO-biotin, and TCO-PEG12.

[0083] FIG. 7 shows high-resolution mass spectra of Somatostatin-Tz, confirming the expected molecular weights for various protonation states. HPLC-MS (ESI) mlz calcd. for C87H115N22O19S2 (M+H)+1835.8, found 1836.2. Calcd. for C87H116N22O19S2 (M+2H)2+918.4, found 918.5. Calcd. for C87H117N22O19S2 (M+3H)3+612.6, found 612.8.

[0084] FIG. 8 shows HPLC analysis of Somatostatin-Tz under different stability conditions. FIG. 9 shows the HPLC trace of the crude reaction mixture resulting from the conjugation of Somatostatin-Tz with TCO-functionalized reagents.

[0085] FIG. 10 shows mass spectrometric characterization of Somatostatin-Cy5. H PLC-MS (ESI) m / z calcd. for C133H178N24O31S5 (M+2H)2+1383.6, found 1383.9. Calcd. for C133H179N24O31S5 (M+3H)3+922.7, found 922.8.

[0086] FIG. 11 shows mass spectrometric characterization of Somatostatin-Biotin. HPLC-MS (ESI) m / z calcd. forCn4Hi62N24O26S3 (M+2H)2+1189.6, found 1189.7. Calcd. for C114H163N24O26S3 (M+3H)3+793.4, found 793.4.

[0087] FIG. 12 shows mass spectrometric characterization of Somatostatin-PEG12. HPLC-MS (ESI) m / z calcd. for C123H183N21O35S2 (M+2H)2+1289.1, found 1289.4. Calcd. for C123H184N21O35S2 (M+3H)3+859.8, found 859.8.

[0088] FIG. 13A-D show TzAA modification of cyclic bioactive peptides. FIG. 13A show structures of oxytocin (SEQ ID NO: 6), vasopressin (SEQ ID NO: 7), and octreotide (SEQ ID NO: 8) post-TzAA modification. FIG. 13B shows CD spectra of native and Tz-modified oxytocin (0.2 mg / mL in water) (195-260 nm). FIG. 13C shows comparative reduction stability of oxytocin-Tz (200 pM) versus native oxytocin (200 pM) in the presence of glutathione (GSH, 5 mM) in PBS 212443-0005-W001

[0089] (pH 7.4). FIG. 13D shows flow cytometry of apoptosis assays. Effects of OT (3.0 pM), OT-Tz (3.0 pM), and oxytocin-Tz (3.0 pM) + TCO-OH (3.0 pM) on the induction of apoptosis in MCF-7 cells. MCF-7 were incubated in the presence and absence of the indicated compounds for 48 h, and then Annexin and 7-AAD staining for flow cytometry experiments were performed. Representative dot-plot graphs of each group.

[0090] FIG. 14 shows a HPLC trace of the crude reaction mixture between oxytocin (SEQ ID NO: 6) and TzAA.

[0091] FIG. 15 shows a HPLC trace of the crude reaction mixture between vasopressin and TzAA.

[0092] FIG. 16 shows a HPLC trace of the crude reaction mixture between octreotide and TzAA. FIG. 17 shows mass spectrometric data for Oxytocin-Tz. HPLC-MS (ESI) mlz calcd. for C54H77N16O12S2 (M+H)+1205.5, found 1205.6. Calcd. for C54H78N16O12S2 (M+2H)2+603.2, found 603.4.

[0093] FIG. 18 shows mass spectrometric data for Vasopressin-Tz. HPLC-MS (ESI) mlz calcd. for C57H76N19O12S2 (M+H)+1282.5, found 1282.7. Calcd. for C57H77N19O12S2 (M+2H)2+641.8, found 641.8.

[0094] FIG. 19 shows mass spectrometric data for Octreotide-Tz. HPLC-MS (ESI) m / z calcd. for C60H77N14O10S2 (M+H)+ 1217.5, found 1217.4. Calcd. for C6oH76Ni4OioS2Na (M+Na)+1239.5, found 1239.2. Calcd. for C60H78N14O10S2 (M+2H)2+609.3, found 609.3.

[0095] FIG. 20 shows CD spectra comparing native oxytocin (SEQ ID NO: 6) and Oxytocin-Tz (0.2 mg / mL in water).

[0096] FIG. 21 shows CD spectra comparing native vasopressin (SEQ ID NO: 7) and Vasopressin-Tz (0.2 mg / mL in water).

[0097] FIG. 22 shows CD spectra comparing native octreotide (SEQ ID NO: 8) and Octreotide-Tz (0.2 mg / mL in water).

[0098] FIG. 23 shows HPLC analysis of Oxytocin-Tz stability under various conditions.

[0099] FIG. 24 shows HPLC analysis of Vasopressin-Tz stability under various conditions.

[0100] FIG. 25 shows HPLC analysis of Octreotide-Tz stability under various conditions.

[0101] FIG. 26 shows HPLC analysis for the stability of oxytocin (200 pM in PBS, pH 7.4) in the presence of glutathione (GSH, 5 mM), using L-Phe-OH (400 pM) as an internal standard.

[0102] FIG. 27 shows HPLC analysis for stability of Oxytocin-Tz (200 pM in PBS, pH 7.4) in the presence of glutathione (GSH, 5 mM), using L-Phe-OH (400 pM) as an internal standard.

[0103] FIG. 28A schematically shows site-selective modification of lysozyme (SEQ ID NO: 9) with TzAA. FIG. 28B shows the mass spectrum of the native lysozyme. Calcd. for 14691.14, found 212443-0005-W001

[0104] 14693.16. FIG. 28C shows the mass spectrum of the Tz-modified lysozyme-Tz. Calcd. for 14887.22, found 14890.48. FIG. 5D shows the mass spectrum of the C6-C128 fragment generated by trypsin digestion of lysozyme-Tz. HRMS (ESI) m / z calcd. for C70H109N22O20S2 (M+3H)3+547.2538, found 547.2552. FIG. 28E shows the CD spectra of native lysozyme and Lysozyme-Tz (0.2 mg / mLin pH 7.4 PBS) (190-260 nm). FIG. 28F shows an SDS-PAGE gel analysis of Cy5 functionalized lysozyme-Tz, with fluorescence imaging (top gels) and Coomassie staining (bottom gels).

[0105] FIG. 29 shows mass spectra of native lysozyme (SEQ ID NO: 9). Calcd. for 14691.14, found 14693.16.

[0106] FIG. 30 shows mass spectra of Tz-modified lysozyme-Tz. Calcd. for 14887.22, found 14890.48.

[0107] FIG. 31 shows the sequence profile of lysozyme and the expected site of TzAA modification.

[0108] FIG. 32 shows the chemical structure and mass spectrum of the lysozyme C6-C128 fragment. HRMS (ESI) m / z calcd. for C70H109N22O20S2 (M+3H)3+547.2538, found 547.2552.

[0109] FIG. 33A-C show one-pot disulfide reduction and rebridging of salmon calcitonin (sCT) using TzAA. FIG. 33A shows site-selective modification of sCT with TzAA. FIG. 33B shows HPLC traces showing reaction progress (black) vs. purified sCT reference (red). FIG. 33C shows the mass spectrum of Tz-modified sCT-Tz. m / z: calcd. for 3627.8, found 908.5 (+4H+)4+, 1211 (+3H+)3+and 1816.3 (+2H+)2+.

[0110] FIG. 34A-D show structural and analytical data for sCT and sCT-Tz. FIG. 34A shows structures of salmon calcitonin (sCT) (SEQ ID NO: 10), Re-sCT (reduced form), and Tz modified sCT-Tz. FIG. 34B shows the mass spectrum of native sCT. m / z calcd. for 3429.7, found 859.0 (+4H+)4+, 1144.9 (+3H+)3+and 1717.2 (+2H+)2+. FIG. 34C shows the mass spectrum of sCT-Tz. m / z: calcd. for 3431.7, found 859.4 (+4H+)4+, 1145.5 (+3H+)3+and 1718.2 (+2H+)2+. FIG. 34D shows CD spectra of native sCT and Tz modified sCT-Tz (0.25 mg / mL in water).

[0111] FIG. 35A-D show bioconjugation of TzAA with unprotected peptides. FIG. 35A shows the reaction scheme for cyclization / bioconjugation of TzAA with protecting group free peptides. FIG.

[0112] 35B shows a HPLC trace of RGD peptide conjugation with TzAA. FIG. 35C shows the HPLC stability analysis of RGD-Tz under various conditions. FIG 35D shows the mass spectrum (ESI) of RGD-Tz. m / z calcd. for C40H53N14O9S2 (M+H)+937.4, found 937.0. Calcd. for C40H54N14O9S2 (M+2H)2+469.2, found 469.0.

[0113] FIG. 36 shows the HPLC trace of the crude reaction mixture between AcNH-CMYIEALDRYAC-CONH2 (EBP, SEQ ID NO: 2) and TzAA. 212443-0005-W001

[0114] FIG. 37 shows the HPLC trace of the crude reaction mixture between H2N-CPIEDRPMC-CONH2 (RPM; SEQ ID NO: 3) and TzAA.

[0115] FIG. 38 shows the HPLC trace of the crude reaction mixture between AcNH-CTPSPFSHC-CONH2 (TCP-1; SEQ ID NO: 4) and TzAA.

[0116] FIG. 39 shows the mass spectrum of EBP-Tz. HPLC-MS (ESI) m / z calcd. for C75H107N20O19S3 (M+H)+1687.7, found 1687.7. Calcd. for C75Hio8N2oOi9S3 (M+2H)2+844.4, found 844.5.

[0117] FIG. 40 shows the mass spectrum of RPM-Tz. HPLC-MS (ESI) m / z calcd. for C53H80N17O13S3 (M+H)+1258.5, found 1258.3. Calcd. for C53H81N17O13S3 (M+2H)2+629.8, found 629.9.

[0118] FIG. 41 shows the mass spectrum of TCP-1-Tz. HPLC-MS (ESI) m / z calcd. for C54H71N16O13S2 (M+H)+1215.5, found 1215.6. Calcd. for C54H72N16O13S2 (M+2H)2+608.3, found 608.4.

[0119] FIG. 42 shows HPLC analysis of the stability of EBP-Tz under various conditions.

[0120] FIG. 43 shows HPLC analysis of the stability of RPM-Tz under various conditions.

[0121] FIG. 44 shows HPLC analysis of the stability of TCP-1-Tz under various conditions. FIG. 45A-D demonstrate cellular applications of RGD-Tz. FIG. 45A shows fluorescent microscope images (60 x camera) of HeLa cells incubated with DMSO and RGD-Tz (500 nM) in pH 7.4 PBS for 3 h, washed three times with PBS and then incubated with TCO-Cy5 (5.0 pM) for 15 min. DMSO as blank control, the Hoechst stain was used to locate the nuclei. FIG. 45B shows decaging of TCO-Dox prodrug via an RGD-Tz mediated click-release reaction. FIG. 45C shows HPLC monitoring of the click-release process promoted by treatment of RGD-Tz (20 pM) with TCO-Dox (10.0 pM). FIG. 45D shows cell viabilities of HeLa cells treated with different concentrations (10.00, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020 pM) of Dox, RGD-Tz, TCO-Dox, and RGD-Tz (10 pM) + TCO-Dox group for 72 h.

[0122] FIG. 46 shows fluorescence microscopy of HS-27 cells treated with DMSO or RGD-Tz (500 nM), followed by incubation with TCO-Cy5 (5.0 pM) and imaging. After the 3 h incubation of HS-27 cells with DMSO or RGD-Tz (500 nM), the cells were washed three times with PBS and then treated with TCO-Cy5 (5.0 pM) for another 15 min. After the cells were washed by PBS three times, the images were taken under the 60 x camera.

[0123] FIG. 47 shows cell viability data for HS-27 cells treated with doxorubicin, RGD-Tz, TCO-Dox, or the combination of RGD-Tz + TCO-Dox over 72 hours. 212443-0005-W001

[0124] DETAILED DESCRIPTION

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] As used herein, the term “or” can be conjunctive or disjunctive.

[0130] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0131] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0132] 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 212443-0005-W001

[0133] system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.” 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.”

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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. 212443-0005-W001

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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 212443-0005-W001

[0144] compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0145] 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.

[0146] The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0147] 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.

[0148] 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.

[0149] 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, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0150] The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond. 212443-0005-W001

[0151] 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.

[0152] 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-.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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).

[0157] 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.

[0158] 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 212443-0005-W001

[0159] 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), ora 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.

[0160] 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.

[0161] The term “1,1-carbocyclylene” means a geminal divalent group derived from a cycloalkyl.

[0162] A representative example is 1,1-C3-6cycloalkylene (i.e.,

[0163]

[0164] A further example is 1,1-

[0165] cyclopropylene (

[0166]

[0167] i.e.,

[0168] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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- 212443-0005-W001

[0174] 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.

[0175] 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, 212443-0005-W001

[0176] 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, ora 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.

[0177] The term “hydroxyl” or “hydroxy,” as used herein, means an -OH group. 212443-0005-W001

[0178] 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.

[0179] 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,” “C3-6cycloalkyl,” “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 “Ci.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).

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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).

[0184] 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. 212443-0005-W001

[0185] 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.

[0186] 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.

[0187] 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.

[0188] The term “linker,” “linked,” or “linking” refers to a chemical moiety that attaches two

[0189] moieties together, such as a payload moiety (-D1) and a cyclooctene moiety (e.g.,

[0190]

[0191] 212443-0005-W001

[0192] cyclooctane moiety (e

[0193]

[0194] .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.

[0195] 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. 212443-0005-W001

[0196] 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.

[0197] Described herein is a dual-functional tetrazine allyl acetate reagent (TzAA), which serves as a versatile chemical tool for the direct, site-selective incorporation of a tetrazine (Tz) moiety into native peptides and proteins via disulfide rebridging (FIG. 1). The electron-withdrawing nature of the Tz group activates the allyl acetate functionality within TzAA, enabling selective substitution reactions with disulfide-derived bis-thiols. The resulting bisthioether conjugates contain a Tz moiety that functions as a reactive center for inverse electron-demand Diels-Alder (I EDDA) reactions with trans-cyclooctene (TCO)-linked functional tags.

[0198] Experimental results demonstrate the broad applicability and efficiency of TzAA-mediated conjugation, yielding well-defined peptide and protein conjugates under mild conditions without compromising biological activity. The bisthioether linkages formed exhibit superior stability compared to conventional thiol-maleimide conjugates and native disulfide bonds.

[0199] Additionally, TzAA enables efficient cyclization of unprotected peptides, generating cyclic peptides with enhanced metabolic stability and distinct biological functions. The Tz group incorporated via TzAA serves as a modular handle for subsequent I EDDA-based functionalization with TCO-linked fluorophores, biotin, and polyethylene glycol (PEG) derivatives. These cyclic conjugates can be further adapted into probes for selective protein targeting and high-affinity ligands for biomolecular imaging.

[0200] Moreover, the I EDDA reaction facilitates activation of TCO-caged prodrugs, enabling targeted drug delivery to tumor cells with reduced systemic toxicity. Collectively, these findings underscore the utility of TzAA as a robust platform for site-selective peptide and protein labeling and for expanding the scope of I EDDA chemistry in the functionalization of therapeutically relevant biomolecules.

[0201] Compounds

[0202] In one aspect, the present disclosure provides compounds of formula (I):

[0203]

[0204] wherein:

[0205] 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 212443-0005-W001

[0206] 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, -

[0207]

[0208] (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-;

[0209] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx; and

[0210] Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is 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, -OCi-4haloalkyl, -OH, — SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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; and

[0211] R2is a leaving group.

[0212] 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.

[0213] In some instances, the leaving group at R2is -Y1-C(O)RY, -Y1-S(O)RY, -Y1-CO2RY, -Y1-SO2RY, -Y1-PO2RY, -Y1RY, -Br, -Cl, -I, -CN, -Y1-Ci.6alkylene-(Y1)0-i-RY,

[0214] -Y1-Ci-6alkylene-(Y1)0-i-C(O)RY, -Y1-Ci-6alkylene-(Y1)0-i-S(O)RY,

[0215] -Y1-Ci-6alkylene-(Y1)o-i-C02RY, -Y1-Ci-6alkylene-(Y1)0-i-SO2RY, or

[0216] -Y1-Ci-6alkylene-(Y1)0-i-PO2RY;

[0217] RY, at each occurrence, is hydrogen, Ci^alkyl, Ci-4haloalkyl, orGY;

[0218] Y1, at each occurrence, is -O-, -NH-, -NCi^alkyl-, or-S-; and

[0219] GY, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a C3-6cycloalkyl, or a 4- to 6-membered heterocyclyl, wherein GY, at each occurrence, is optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -NO2, Ci-ealkyl, C1- 4haloalkyl, -CN, -0Ci-4alkyl, -OCi-4haloalkyl, -OH, - SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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.

[0220] In some instances, the leaving group at R2is -Y1-C(0)RYor -Y1-SO2RY. In some instances, Y1is -O-. In some instances, RYis Ci-6alkyl or Ci-4haloalkyl. In some instances, RY 212443-0005-W001

[0221] is -CH3 or -CF3. In some instances, the leaving group is -0-C(0)CH3. In some instances, the compound of formula (I) is:

[0222]

[0223] 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 Ci-4fluoroalkyl).

[0224] Compound names and / or structures can be assigned / determined by using the Struct=Name naming algorithm as part of CH EM DRAW® ULTRA.

[0225] 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 Appt. 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.

[0226] 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.

[0227] 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.

[0228] 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 212443-0005-W001

[0229] (deuterium). Accordingly, any group comprising one or more hydrogen atoms encompasses corresponding deuterium-labeled versions of the group. For example, “-CH2-” encompasses D

[0230] “

[0231]

[0232] -CD2-”; encompasses D , etc.

[0233] 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.

[0234] 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).

[0235] General Synthesis

[0236] 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.

[0237] Pd-catalyst, Cu(l) source solvent, A

[0238]

[0239]

[0240] 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(PPhs)4), Cu(l) source (e.g., CuTC), and solvent (e.g., dioxane), under reflux conditions).

[0241] 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. 212443-0005-W001

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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 212443-0005-W001

[0246] 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.

[0247] 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).

[0248] 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.

[0249] 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.

[0250] Pharmaceutical Compositions

[0251] 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.

[0252] 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. 212443-0005-W001

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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. 212443-0005-W001

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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. 212443-0005-W001

[0268] Suitable suspending agents include AVICEL RC-591 (from FMC 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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 212443-0005-W001

[0274] disclosed compound, and glidants such as silicon dioxide to improve flow characteristics. Implants may be of the biodegradable or the non-biodegradable type.

[0275] The selection of ingredients in the carrier for oral compositions may depend on secondary considerations like taste, cost, and shelf stability.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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 212443-0005-W001

[0281] 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, Bankers Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms’. Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd ed. (1976).

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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. 212443-0005-W001

[0286] 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.

[0287] The amount of thickener(s) in a topical composition is typically about 0% to about 95% by weight.

[0288] 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.

[0289] 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.

[0290] Suitable pH adjusting additives include hydrochloric acid (HCI) or sodium hydroxide (NaOH) in amounts sufficient to adjust the pH of a topical pharmaceutical composition.

[0291] Conjugates

[0292] 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):

[0293] •JA 'xN

[0294] R

[0295]

[0296] 1N (|-a),

[0297] Ys\

[0298] S\

[0299] wherein a moiety of formula ' links the two cysteine residues to the moiety of formula (l-a), and wherein:

[0300] 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-6alkyl, Ci-4haloalkyl, halogen, -CN, =0, =S, -NO2, Gx, -X1-Rx, 212443-0005-W001

[0301] -(X1)o.i-Ci-6alkylene-(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)o-1-C(0)NHRX, and -(X1)0.i-C(O)NCi.4alkylRx; X1, at each occurrence, is -0-, -S-, -NH-, or -NCi^alkyl-;

[0302] Rx, at each occurrence, is hydrogen, Ci.6alkyl, Ci.4haloalkyl, orGx;

[0303] X1, at each occurrence, is -O-, -S-, -NH-, or-NCi.4alkyl-; and

[0304] Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -N02, Ci-salkyl, Ci-4haloalkyl, -CN, -OCi-4alkyl, -OCi-4haloalkyl, -OH, — SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, — N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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.

[0305] In some instances, the molecule comprising at least two cysteine residues 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.

[0306] As shown in Scheme 2 below, exemplary conjugates may be prepared by reacting with a molecule comprising at least two cysteine residues with a compound of formula (I).

[0307] Scheme 2.

[0308]

[0309] In some instances reacting the compound with the two cysteine residues 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.

[0310] Methods of Use

[0311] 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 212443-0005-W001

[0312] 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.

[0313] As shown in Scheme 3, methods of selectively delivering a payload may comprise contacting cells (e.g., cancer cells) containing a molecule comprising at least two residues (e.g., protein iii) with a compound of Formula (I) to form a conjugate (e.g., conjugate iv).

[0314] Scheme 3.

[0315]

[0316] 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 (H):

[0317]

[0318] wherein L1is the linker, and D1is the payload moiety. In some instances, the functionalized conjugate comprises a moiety of formula (I l-a) or (ll-b):

[0319]

[0320] In some instances, the functionalized payload of formula (II) is: 212443-0005-W001

[0321]

[0322] L1is:

[0323] O 0 0

[0324]

[0325] wherein:

[0326] Z1is -0-, -S-, -NRa-, or-CRaRb-;

[0327] Raand Rb, at each occurrence, are each independently hydrogen or C^alkyl; and m, at each occurrence, is 0-14

[0328] n, at each occurrence, is 0-10, and

[0329] p, at each occurrence, is 0-10.

[0330] In some instances, Z1is -0-. 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

[0331] 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 212443-0005-W001

[0332] 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.

[0333] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0334] Clause 1. A compound of formula (I),

[0335]

[0336] wherein:

[0337] 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.6alkyl, Ci.4haloalkyl, halogen, -CN, =0, =S, -N02, 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, -

[0338]

[0339] (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-;

[0340] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx; and

[0341] Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Ca-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is 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, -OCi-4haloalkyl, -OH, — SCi-4alkyl, -SH, -NH2, -NHCi.4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci.4alkyl, -CO2H, -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; and

[0342] R2is a leaving group.

[0343] Clause 2. The compound of clause 1, wherein R1is the optionally substituted 6- to 12- membered aryl. 212443-0005-W001

[0344] Clause s. The compound of clause 1 or 2, wherein the optionally substituted 6- to 12- membered aryl is optionally substituted phenyl.

[0345] Clause 4. The compound of any one of clauses 1-3, wherein:

[0346] the leaving group at R2is -Y1-C(O)RY, -Y1-S(O)RY, -Y1-CO2RY, -Y1-SO2RY, -Y1-PO2RY, -Y1RY, -Br, -Cl, -I, -CN, -Y1-Ci.6alkylene-(Y1)0-i-RY, -Y1-Ci-6alkylene-(Y1)0-i-C(O)RY, -Y1-Ci.6alkylene-(Y1)0-i-S(O)RY, -Y1-Ci-Salkylene-(Y1)0-i-CO2RY, -Y1-Ci-6alkylene-(Y1)0-i-SO2RY, or -Y1-Ci-6alkylene-(Y1)0-i-PO2RY;

[0347] RY, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGY;

[0348] Y1, at each occurrence, is -O-, -NH-, -NCi-4alkyl-, or-S-; and

[0349] GY, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a C3-6cycloalkyl, or a 4- to 6-membered heterocyclyl, wherein GY, at each occurrence, is optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -NO2, Ci-ealkyl, C1-4haloalkyl, -CN, -0Ci-4alkyl, -OCi.4haloalkyl, -OH, -SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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.

[0350] Clause 5. The compound of any one of clauses 1-4, wherein the leaving group at R2is -Y1- C(O)RYor-Y1-SO2RY.

[0351] Clause 6. The compound of clause 4 or 5, wherein Y1is -0-.

[0352] Clause 7. The compound of any one of clauses 4-6, wherein RYis Ci-ealkyl or Ci-4haloalkyl. Clause 8. The compound of clause 7, wherein RYis -CH3or -CF3.

[0353] Clause 9. The compound of any one of clauses 4-8, wherein the leaving group is -O- C(O)CH3.

[0354] Clause 10. The compound of clause 1 , wherein the compound of formula (I) is:

[0355]

[0356] Clause 11. A conjugate comprising:

[0357] a molecule comprising two cysteine residues; and

[0358] a moiety of formula (l-a), 212443-0005-W001

[0359]

[0360] Ys\

[0361]

[0362] V

[0363] hSX

[0364] w erein a moiety of formula ' links the two cysteine residues to the moiety of formula (l-a), and wherein:

[0365] 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)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^alkyl-;

[0366] Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;

[0367] X1, at each occurrence, is -0-, -S-, -NH-, or -NCi^alkyl-; and

[0368] Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is 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, -OCi-4haloalkyl, -OH, -SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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.

[0369] Clause 12. The conjugate of clause 11, wherein the molecule comprising at least two cysteine residues is a peptide or a protein.

[0370] Clause 13. The conjugate of clause 11 or 12, wherein R1is the optionally substituted 6- to 12- membered aryl.

[0371] Clause 14. The conjugate of clause 13, wherein the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl.

[0372] Clause 15. A method of preparing the conjugate of any one of clauses 11-14, the method comprising: 212443-0005-W001

[0373] providing a molecule comprising at least two cysteine residues; and

[0374] reacting the compound of any one of clauses 1-10 with two cysteine residues of the molecule, thereby providing the conjugate of any one of clauses 11-14.

[0375] Clause 16. The method of clause 15, wherein the molecule comprising at least two cysteine residues is a peptide or a protein.

[0376] Clause 17. The method of clause 15 or 16, wherein reacting the compound of any one of clauses 1-10, with the two cysteine residues occurs in the presence of a buffer solution. Clause 18. The method of clause 17, wherein the buffer solution has a pH of 4.0 to 9.0. Clause 19. The method of any one of clauses 15-17, wherein the conjugate is provided in quantitative yields.

[0377] Clause 20. A method of functionalizing a conjugate, the method comprising:

[0378] reacting the conjugate of any one of clauses 11-14 with a functionalized payload, wherein the functionalized payload comprises a payload attached to a cyclooctene, thereby providing a functionalized conjugate.

[0379] Clause 21. The method of clause 20, wherein a linker attaches the cyclooctene to the payload. Clause 22. The method of clause 20 or 21 , wherein the cyclooctene is frans-cyclooctene. Clause 23. The method of clause 21, wherein the functionalized payload is a functionalized payload of formula (II):

[0380]

[0381] wherein L1is the linker, and D1is the payload.

[0382] Clause 24. The functionalization method of clause 23, wherein the functionalized conjugate comprises a moiety of formula (I l-a) or (ll-b):

[0383]

[0384] Clause 25. The method of clause 23 or 24, wherein the functionalized payload of formula (II) is: 212443-0005-W001

[0385]

[0386] or

[0387] Clause 26. The method of any one of clauses 23-25, wherein L1is:

[0388] O 0 0

[0389] O O O Rb

[0390]

[0391] wherein:

[0392] Z1is -O-, -S-, -NRa-, or-CRaRb-;

[0393] Raand Rb, at each occurrence, are each independently hydrogen or Ci-4alkyl; and m, at each occurrence, is 0-14

[0394] n, at each occurrence, is 0-10, and

[0395] p, at each occurrence, is 0-10.

[0396] Clause 27. The method of clause 26, wherein Z1is -O-.

[0397] Clause 28. The method of clause 26 or 27, wherein Raand Rb, at each occurrence, are each independently hydrogen or methyl.

[0398] Clause 29. The method of any one of clauses 23-28, wherein D1is a therapeutic agent, a diagnostic agent, or a targeting agent.

[0399] Clause 30. The method of clause 29, wherein the diagnostic agent is a cyanine dye.

[0400] Clause 31. The method of clause 29, wherein the therapeutic agent is an anti-cancer therapeutic agent.

[0401] Clause 32. The method of clause 31 , wherein the anti-cancer therapeutic agent is doxorubicin or ARV-771.

[0402] EXAMPLES

[0403] Materials

[0404] General 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- 212443-0005-W001

[0405] 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. High performance liquid chromatography (HPLC) spectra for compounds were acquired using a Shimadzu LabSolutions system.

[0406] Synthesis and Characterization of TzAA

[0407] Scheme 4. Synthesis of TzAA

[0408]

[0409] Synthesis

[0410]

[0411] 2-(6-Phenyl-1 ,2,4,5-tetrazin-3-yl)prop-2-en-1-ol (4).

[0412] A solution of 3-(methylthio)-6-phenyl-1,2,4,5-tetrazine (1) (645 mg, 3.16 mmol) and tert-butyldimethyl((2-(tributylstannyl)allyl)oxy)silane (2) (2.19 g, 4.75 mmol) in 150 mL of dioxane was added Pd(PPh3)4 (548 mg, 0.47 mmol) and copper(l) thiophene-2-carboxylate (1.20 g, 6.29 mmol), then was stirred under N2 atmosphere at reflux temperature for 40 min. See Xie et al., Angew. Chem. Int. Ed. 59: 16967-16973 (2020); Darwish et al., Org. Lett. 10: 861-864 (2008). 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 crude 3. To crude 3 in 50 212443-0005-W001

[0413] mL of MeOH, AcCI (674 pL, 9.48 mmol) was added, then stirred for 20 min at room temperature. Then saturated NaHCCh solution was added to adjust pH to 7-8. The product was extracted into ethyl acetate (3 * 20 mL) and dried with Na2SO4, and was purified by flash column chromatography (hexanes / ethyl acetate = 4:1 v / v) on silica gel to give 4 (280 mg, 41%) as pink solid.1H NMR (500 MHz, CDCI3): 58.62-8.60 (m, 2H), 7.66-7.59 (m, 3H), 6.99 (s, 1H), 6.10 (m, 1H), 4.85 (s, 2H).13C NMR (125 MHz, CDCI3): 6 164.0, 163.7, 140.5, 133.1, 129.5, 128.2, 124.2, 63.3. HRMS (ESI) m / z calcd. for CnHnN4O (M+H)+215.0933, found 215.0927.

[0414]

[0415] 2-(6-Phenyl-1,2,4,5-tetrazin-3-yl)allyl acetate (TzAA)

[0416] To a solution of 4 (110 mg, 0.51 mmol) in 10 mL of DCM, AcCI (73 pL, 1.03 mmol) was added, then triethylamine (167 pL, 1.20 mmol) was added, the resulting mixture was stirred for 1 h at room temperature. The solvent was removed by a rotavapor, and the product was purified by flash column chromatography (hexanes / ethyl acetate = 4:1 v / v) on silica gel to give Tz2 (98 mg, 75%) as pink solid.1H NMR (500 MHz, CDCh): 6 8.63-8.61 (m, 2H), 7.66-7.59 (m, 3H), 7.06 (m, 1H), 6.11 (m, 1H), 5.34 (s, 2H), 2.14 (s, 3H).13C NMR (125 MHz, CDCI3): 5170.6, 164.0, 163.1, 136.6, 133.1, 131.7, 129.5, 128.3, 125.4, 63.0, 21.1. HRMS (ESI) m / z calcd. for Ci3Hi2N4NaO2 (M+Na)+279.0858, found 279.0851.

[0417] Cell Culture

[0418] Human fibroblast HS-27 cells, HeLa cells, and MCF-7 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.

[0419] Cell Imaging

[0420] The cells were seeded in the 6-well plate and allowed to growth overnight. After the treatments, the cells were fixed with 4% formaldehyde solution for 30 min at room temperature and washed 3 times with PBS. Then the cells were treated with 1x Hoechst and 5 pM TCO-Cy5 (Click chemistry tools, catalog no. 1089-5) for 15 min at room temperature. After another 3 times 212443-0005-W001

[0421] PBS washing, the cells were observed and imaged by using the ImageXpress Micro 4 High-Content Imaging System (Molecular Devices).

[0422] Cell Viability Assay

[0423] Cells were plated in 96-well plates with 6.0 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 1-4 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.

[0424] Apoptosis Assay

[0425] MCF-7 cells were placed in 6-well transparent plates and then treated with test compounds and vehicles in a moist atmosphere of 5% CO2 at 37 °C for 48 h. After that, the cells were washed with cold Cell staining Buffer (BioLegend, catalog no. 420201 -BL) twice, and then were resuspended in Binding buffer (BioLegend, catalog no. 640914). FITC Annexin V (5 pL, BioLegend, catalog no. 640914) and propidium iodide Solution (10 pL, BioLegend, catalog no.

[0426] 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).

[0427] Preparation of RGD, EBP, RPM, and TCP-1 Liner Peptides

[0428] RGD (ACNH-CRGDFC-CONH2, SEQ ID NO: 1), EBP (AcNH-CMYIEALDRYAC-CONH2, SEQ ID NO: 2), RPM (H2N-CPIEDRPMC-CONH2, SEQ ID NO: 2) and TCP-1 (AcNH-CTPSPFSHC-CONH2, SEQ ID NO: 4) peptides were synthesized manually using a modified 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol using the commercially available A / -o-Fmoc-protected amino acids as the substrates and the rink amide resin as the solid support. A solution of 20% piperidine in DMF was used to remove the Fmoc protecting group, and O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) was used as the carboxyl group activating agent. An excess of the Fmoc-protected amino acid (4 equiv.), HCTU (4 equiv.), and DIPEA (8 equiv.) in DMF were used for each coupling at room temperature. For the A / -terminal acetylation, acetic anhydride (10 equiv.) and DIPEA (10 equiv.) in DMF were added and the mixture was stirred at room temperature for 30 min. After washing 212443-0005-W001

[0429] with DMF and CH2CI2, the resin was treated with a solution of TFA / TIPS / H2O (95:2.5:2.5) for 2 h to cleave the peptide from the resin and remove the protecting groups. The resin was removed by filtration and the filtrate was precipitated by the addition of cold diethyl ether, and then purified by reverse-phase HPLC (30 * 150 mm) with a linear gradient of 3-95% (B %, v / v) at a flow rate of 17 mL / min over 40 min.

[0430] Sequences of Peptides and Proteins

[0431] The following peptides and proteins were used in the experiments described herein.

[0432] Table 1. Sequences 0 Peptides and Proteins

[0433] SEQ ID

[0434] Name Sequence

[0435] NO:

[0436] RGD ACNH-CRGDFC-CONH21 EBP ACNH-CMYIEALDRYAC-CONH22 RPM H2N-CPI EDRPMC-CON H23 TCP-1 ACNH-CTPSPFSHC-CONH24 Somatostatin AGCKNFFWKTPTSC* 5 Oxytocin CYIQNCPLG-NH2* 6 Vasopressin CTFQNCPRG-NH2* 7 OctreotidedFCFdTKTCTt* 8 KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGY NTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNAC

[0437] Lysozyme 9 HLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRC QNRDVRQYVQGCGV

[0438] Salmon Calcitonin

[0439] CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP-NH210 (sCT) _

[0440] Somatostatin, vasopressin, oxytocin, and octreotide are cyclic peptides formed by disulfide linkages between the two cysteines (bolded) forming cystines.

[0441] For Oxtreotide,dF anddT indicates D-amino acids; Tfindicates the non-standard amino acid \>OH

[0442] threoninol,H2N H

[0443]

[0444] Results

[0445] Synthesis, Stability and Reactivity of TzAA

[0446] The dual functioning TzAA reagent was synthesized using a 3-step sequence and fully characterized (Scheme 4). A stability test performed by incubation in a pH 7.4 PBS buffer and HPLC monitoring indicated that TzAA is stable formore than 24 h (FIG. 2). The reactivity of TzAA was evaluated by reacting it (200 pM) with Cys (2 mM) in a pH 7.4 PBS / MeCN 9 / 1 (v / v) for 2 h 212443-0005-W001

[0447] (FIG. 3A). This process takes place with complete conversion to form the bis-cysteine adduct TzAA-2Cys and minimal amounts of side products (FIG. 3B).

[0448] TzAAfor Disulfide Modification of Peptides and Proteins

[0449] The cyclic peptide hormone somatostatin, which plays a key role in regulating the endocrine system and contains an accessible disulfide bond, was employed to assess the capability of TzAA as a reagent for modifying disulfide moieties in peptides and proteins (Table 2). The disulfide bond in the peptide was first reduced by using tris(2-carboxyethyl)phosphine (TCEP, 30 min, pH 7.4 PBS / MeCN (9:1)) to generate two free thiol groups, and then the reaction mixture was treated with TzAA and incubated for 30 min. Different amounts of TCEP and TzAA were screened for optimization purposes. HPLC analysis of the crude reaction mixtures showed that modification occurs with high efficiency (77% based on HPLC quantification and 75% isolated yield) when 1.2 equiv of TCEP and 1.1 equiv of TzAA are used (Table 2, entry 5, FIG. 4-5). The Somatostatin-Tz conjugate (FIG. 4) was isolated and characterized using mass spectrometry (FIG. 6A and FIG. 7). 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 Somatostatin-Tz (FIG. 6B). It is noteworthy that one primary objective of using this rebridging technology is to enhance the stability of modified peptides under various adverse storage and metabolic conditions. A stability test to explore this issue showed that Somatostatin-Tz remains stable in PBS at different pHs (FIG. 8). The native disulfide bridge is particularly vulnerable in reductive environments and prone to be reduced in the presence of free thiols. To assess its reductive stability, Somatostatin-Tz was incubated in pH 7.4 PBS containing GSH (2 mM). No noticeable degradation occurs (HPLC) over a 24 h period (FIG. 8). 212443-0005-W001

[0450] Table 2. Optimization of Modification of Somatostatin with TzAA

[0451]

[0452] Somatostatin-Tz

[0453] Entry TCEP (equiv.) TzAA (equiv.) HPLC yield (%) 1 2.0 1.1 42 2 2.0 1.5 44 3 2.0 2.0 36 4 1.5 1.1 57 5 1.2 1.1 77 (75)* Reaction conditions: To a solution of somatostatin (1.0 equiv. 200 pM) in PBS (pH 7.4) : MeCN = 9 : 1 was added TCEP. The mixture was incubated at rt for 30 min. Then TzAA was added, and the resulting mixture was incubated at rt for 30 min. *lsolated yield by PREP HPLC.

[0454] As shown above, TzAA was designed not only to carry out disulfide rebridging, but also for the Tz moiety to participate in ensuing click functionalization to introduce new tags. The latter feature was explored by employing tetrazine-modified peptides, prepared using TzAA, for creation of well-defined conjugates containing an array of functional groups including the fluorophore Cy5, biotin and a PEG chain (FIG. 6C). For these purposes, TCO-Cy5, TCO-biotin and TCO-PEG12 were individually incubated with Somatostatin-Tz in pH 7.4 PBS. The IEDDA reactions proceed smoothly within 30 min to cleanly (HPLC) form the corresponding adducts Somatostatin-Cy5, Somatostatin-biotin and Somatostatin-PEG12 (FIG. 9). The three modified peptides were also isolated in high yields (84-92%) and their structures were validated by using mass spectrometry (FIG. 10-13).

[0455] To rigorously test its generality, the operationally simple and effective TzAA based protocol was applied to sequential conjugation and functionalization of other bioactive peptides including the neuro-hypophysial nonapeptide hormones oxytocin (OT) and related arginine vasopressin (FIG. 13). Both peptides were observed to undergo smooth re-bridging reactions with TzAA (FIG. 212443-0005-W001

[0456] 14-15) to yield Oxytocin-Tz (OT-Tz) and Vasopressin-Tz in high respective yields of 85% and 70% after purification. The cyclic octapeptide octreotide, which is pharmacologically related to somatostatin and a potent inhibitor of growth hormone, was also tested (FIG. 16). Performance of the one-pot reduction and bioconjugation operation led to formation of Octreotide-Tz in a 62% yield. Production of all three modified cyclic peptides was confirmed by using HPLC-MS (FIG.

[0457] 17-19), and they were shown to possess secondary structures that are similar to those of the native forms using CD analysis (FIG. 13B, FIG. 20-22) and to have high stabilities under different conditions including different pHs and the presence of GSH (FIG. 23-225).

[0458] A study was conducted to prepare and evaluate the stability of OT-Tz relative to that of the parent peptide in pH 7.4 PBS containing a physiological level of GSH (5 mM). The results reveal that OT-Tz has significantly greater stability than OT itself in that it does not undergo observable degradation over a 2 h period (FIG. 13C and FIG. 26-27). Because OT has been utilized in the several anti-cancer treatment protocols, including those targeted at breast, ovarian and prostate cancers, the cancer cell apoptotic activity of OT-Tz was assessed. OT induces apoptosis in oxytocin receptor (OTR)-expressing tumors resulting in inhibition of cell proliferation. Thus, an overexpressed OTR MCF-7 cancer cell line was used to probe the apoptotic activity of OT-Tz with OT as a positive control. Flow cytometry analysis showed that OT-Tz induces a level of early apoptosis (11.7%) that is similar to that promoted by OT (10.1%) (FIG. 13D). Furthermore, the apoptotic activity of the IEDDA functionalized counterpart OT-Tz-TCO formed by click reaction of OT-Tz with TCO-OH was evaluated (FIG. 14). Indeed, OT-Tz-TCO promotes a comparable level of early apoptosis (11.2%). These results suggest that the bioconjugation and click modification has minimal effects on OT activity. Taken together, the studies demonstrate that TzAA is a valuable reagent for carrying out peptide modification under mild conditions, and without significant interruption of peptide structure and function.

[0459] Having shown that TzAA is a useful bifunctional reagent for labeling peptides, its capacity to functionalize more complex proteins was assessed next. Lysozyme (PDB id: 1JUG), which possesses four disulfide bonds with only the one between C6-C128 being solvent-accessible, was selected for investigation (FIG. 28A). This protein was subjected to reduction using 2.5 equiv of TCEP in pH 7.4 PBS buffer (2 h) and then treated with TzAA (2.5 equiv) and incubated overnight. Mass spectrometric analysis confirmed that only a single Tz moiety was present in the modified lysozyme (FIG. 28B-C and FIG. 29-30). To pinpoint the modification site, Tz-lysozyme was analyzed utilizing LC-MS / MS. Specifically, the fragment containing the C6-C128 disulfide bond generated by trypsin digestion, contains an additional TzAA derived disulfide bridge (FIG.

[0460] 28A, D and FIG. 30-32). In addition, CD spectra of both native and the modified lysozyme 212443-0005-W001

[0461] indicate that these proteins have similar secondary and tertiary structures (FIG. 28E). Finally, subsequent IEDDA reaction of Tz-lysozyme with TCO-Cy5 yielded a tagged protein whose SDS-PAGE profile contains a fluorescent band (FIG. 28F), demonstrating the potential of the protocol for protein imaging.

[0462] The protocol is also demonstrated for the conjugation of salmon calcitonin (sCT), a 32 amino acid peptide hormone secreted by parafollicular cells of the thyroid in humans and other chordates in the ultimopharyngeal body. This peptide is used for the treatment of a range of bone conditions including postmenopausal osteoporosis, Paget’s disease, and hypercalcaemia. It contains a disulfide bridge at Cys1-Cys7 that can be reduced to give two sulfhydryl groups for rebridging conjugation with TzAA. The one-pot operation via TCEP (1.2 equiv) reduction (30 min) of the disulfide bridge followed by direct treatment with TzAA (1.2 equiv, 30 min) delivered the sCT-Tz in 73% isolate yield (FIG. 33A-B and FIG. 34A). It is noted that in the rebridging reaction with 2,3-dibromomaleimide, pH had to be adjusted to 6.2 to achieve high reaction efficiency. The Tz modified sCT-Tz was characterized by MS (FIG. 33C and FIG. 34B-C). The modification did not alter the secondary structure significantly based on CD analysis (FIG. 34D). Collectively, these studies validate that TzAA is a viable reagent for bioconjugation and functionalization of proteins without only minimally disrupting their native structures and functions.

[0463] TzAA for bis-Cysteine Modification of Peptides

[0464] Having demonstrated the capability of TzAA as a reagent for efficient disulfide bond targeting bioconjugation of peptides and proteins, its utility in a new cyclization method for generating cyclic peptides was explored. Cyclic peptides are a fascinating class of molecules in modern drug discovery because they have unique features associated with high binding affinity and specificity, proteolytic stability, low toxicity, and improved membrane permeability.53,54 In addition to their clinical potential, cyclic peptides have the unique properties that make them useful research tools, such as probes that selectively modulate target proteins and high-affinity ligands for biomolecular imaging. Having demonstrated the capability of TzAA as a reagent for highly site-selective, efficient disulfide bond targeting bioconjugation of peptides and proteins, its role in a new cyclization method for generating cyclic peptides was explored. Toward this end, 4 peptides were selected, which contain 2 Cys residues and have distinct structures and biological functions, as model substrates for TzAA mediated cyclization and click functionalization studies (FIG. 35A). The specific peptides employed in the effort are AcNH-CRGDfC-CONH2(labeled as RGD; SEQ ID NO: 1) that is a tumor-targeting peptide against the av[33 integrin, AcNH-CMYIEALDRYAC-CONH2 (labeled as EBP; SEQ ID NO: 2) that targets EGFR-overexpressing tumor cells, H2N- 212443-0005-W001

[0465] CPIEDRPMC-CONH2(labeled as RPM; SEQ ID NO: 3) that has high affinity for poorly differentiated colon carcinoma cells, and AcNH-CTPSPFSHC-CONH2(labeled as TCP-1; SEQ ID NO: 4) that localizes in the metabolically active part of colon tumors and neighboring blood vessels. These target peptides were prepared using solid-phase peptide synthesis and then purified. Cyclization / bioconjugation reactions of the peptides with TzAA processes take place rapidly (30 min) (FIG. 35A-B and FIG. 36-38), efficiently (only 1.1 equiv of TzAA needed) and chemo-selectively under mild reaction conditions to produce RGD-Tz (92%), EBP-Tz (83%), RPM-Tz (81%) and TCP-1-Tz (92%). All Tz bridged peptides were characterized by using HPLC-MS (FIG. 35D and FIG. 39-41), and they were shown to have high stability at various pHs and in the presence of GSH (FIG. 35C and FIG. 42-44).

[0466] The Tz-conjugated peptide RGD-Tz was utilized for subsequent click functionalization and biological investigations. RGD-Tz contains an RGD motif specific for binding to integrin and targets cancer cells through binding to surface integrins. To determine if the conjugated peptide possess this binding specificity, fluorescence imaging studies were conducted with HeLa cells that overexpress avp3integrin. The cells were treated with RGD-Tz (500 nM) (blank DMSO treated one as control) for 3 h, and then incubated with the fluorescent labelling agent TCO-Cy5 for 15 min. The washed RGD-Tz and TCO-Cy5 treated cells were found to display red emission that corresponds to Cy5 fluorescence (FIG. 45A). In contrast, cells treated only with DMSO do not emit fluorescence. Moreover, RGD-Tz treated HS-27 cells (normal cells with low avp3 integrin expression) display only very weak fluorescence (FIG. 46), suggesting that RGD-Tz selectively targets tumor cells that overexpress avp3integrin.

[0467] TCO-caged doxorubicin (Dox) TCO-Dox was employed as the model prodrug to demonstrate that IEDDA reaction of RGD-Tz can be employed in a sequence to selectively deliver cytotoxic agents to cancer cells (FIG. 45B). It was expected that the carbamate-linked TCO group at the allylic position in TCO-Dox would suppress Dox activity and that IEDDA reaction of TCO-Dox with RGD-Tz would trigger release of Dox. Indeed, HPLC monitoring of the reaction of RGD-Tz (20 pM) with TCO-Cy5 (10 pM) in pH 7.4 PBS at room temperature showed that free Dox is indeed generated to the extent of 90% within 4 h (FIG. 45C).

[0468] Having established that RGD-Tz enters HeLa cells via an avp3integrin promoted pathway and that it effectively undergoes click-release reaction with TCO-Dox, the next step was to evaluate if this sequence can be utilized to bring about cancer cell death. For this purpose, HeLa cells were incubated with RGD-Tz (10 pM) for 3 h, washed with pH 7.4 PBS buffer, and then treated with TCO-Dox (10.00, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02 pM). The results of cytotoxicity assays reveal that treatment with TCO-Dox and RGD-Tz individually has 212443-0005-W001

[0469] only a small effect on the viability of HeLa cells (ICso > 10 pM each). However, when treatment with a combination of these substances causes a high level of cytotoxicity (ICso = 0.98 pM) that is comparable to that of free Dox (ICso = 0.66 pM) (FIG. 45D). Furthermore, applying the same protocol using low av[33 integrin-expressing normal fibroblast HS-27 cells causes a significantly lower cytotoxicity (ICso > 1,000 nM) compared to that of free Dox (ICso - 109 nM) (FIG. 47). These results demonstrate that the TCO-tetrazine prodrug strategy can be employed to selectively target and activate prodrugs in av|33 integrin-positive cancer cells.

[0470] Its exceptional rate, excellent orthogonality and biocompatibility and unique release capacity gives the click reaction between Tz and TCO a high propensity for biological applications even in humans. However, the lack of practical methods for direct incorporation of the Tz moiety into peptides and proteins for bioconjugation hinders the use of this protocol for biomedically related purposes. As a result of this issue, the simple and widely applicable 2-in- 1 dual functional TzAA reagent was developed for site-specific installation of a single Tz group into peptides or proteins via selective reaction with bis-cysteine / disulfide moieties. The facility of this process is a consequence of the electron-withdrawing capacity of the Tz group and its promotion of a double Michael cascade reaction with disulfide derived bis-thiols to generate stable bisthioether conjugates. As the results of the effort described above demonstrate, structurally diverse peptides and proteins which contain disulfide bonds, or two cysteines effectively and selectively participate in this unique bioconjugation reaction. The resulting Tz functionalized cyclic peptides and proteins display higher stabilities than their corresponding disulfide counterparts. Furthermore, the tetrazine-functionalized peptides or proteins undergo rapid IEDDA click reactions with various TCO-modified substances, such as dyes, biotin and polymers to form stable bioconjugates. In addition, TzAA serves as a new peptide stapling reagent, which can be utilized to create cyclic structures from protecting group free peptide precursors under physiological conditions and in high yields. Also, cyclic peptides generated in this manner display distinct biological functions and their Tz moiety enables tagging functional groups for imaging and drug activation purposes. These features make the strategy particularly appealing in biomedical and translational research. Overall, in the above effort a simple dual functional bioorthogonal reagent was developed for site-selective protein labeling and manipulation and showed that the straightforward bioconjugation / functionalization strategy has relevance to biomedicine.

Claims

1. 212443-0005-W0012.CLAIMS3.What is claimed:

1. A compound of formula (I),6. 8.wherein:9.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, -10.

11. (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-;12.Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx; and13.Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is 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, -OCi-4haloalkyl, -OH, — SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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; and14.R2is a leaving group.

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:18.the leaving group at R2is -Y1-C(0)RY, -Y1-S(O)RY, -Y1-CO2RY, -Y1-S02RY, -Y1-P02RY, -Y1RY, -Br, -Cl, -I, -CN, -Y1-Ci.6alkylene-(Y1)0-i-RY, 212443-0005-W00119.-Y1-Ci.6alkylene-(Y1)o-i-C(0)RY, -Y1-Ci-6alkylene-(Y1)o.i-S(0)RY, -Y1-Ci-6alkylene-(Y1)0-i-CO2RY, -Y1-Ci-6alkylene-(Y1)0-i-SO2RY, or -Y1-Ci-5alkylene-(Y1)0-i-PO2RY;20.RY, at each occurrence, is hydrogen, Ci.6alkyl, Ci.4haloalkyl, orGY;21.Y1, at each occurrence, is -O-, -NH-, -NCi^alkyl-, or-S-; and22.GY, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a C3.ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein GY, at each occurrence, is optionally substituted with 1-4 substituents, wherein each substituent is independently selected from the group consisting of halogen, =0, =S, -NO2, Ci-salkyl, Ci-4haloalkyl, -CN, -OCi-4alkyl, -OCi-4haloalkyl, -OH, — SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, — N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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.

5. The compound of claim 4, wherein the leaving group at R2is -Y1-C(0)RYor -Y1-SO2RY.

6. The compound of claim 4, wherein Y1is -O-.

7. The compound of claim 4, wherein RYis Ci .ealkyl or Ci.4haloalkyl.

8. The compound of claim 7, wherein RYis -CH3or -CF3.

9. The compound of claim 4, wherein the leaving group is -O-C(O)CH3.

10. The compound of claim 1, wherein the compound of formula (I) is:

30.

11. A conjugate comprising:33.a molecule comprising two cysteine residues; and34.a moiety of formula (l-a), 212443-0005-W00135.•A -jN37.

38. R1N (|-a),39.Ys\40.w e e n m i t o f41.

42. V43.h i o ySX44.r a e f ormula ' links the two cysteine residues to the moiety of formula (l-a), and wherein:45.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)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-;46.Rx, at each occurrence, is hydrogen, Ci-ealkyl, Ci-4haloalkyl, orGx;47.X1, at each occurrence, is -O-, -S-, -NH-, or -NCi^alkyl-; and48.Gx, at each occurrence, is a phenyl, a 5- to 6-membered heteroaryl, a Cs-ecycloalkyl, or a 4- to 6-membered heterocyclyl, wherein Gx, at each occurrence, is 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, -OCi-4haloalkyl, -OH, -SCi-4alkyl, -SH, -NH2, -NHCi-4alkyl, -N(Ci-4alkyl)2, -C(O)H, -C(O)Ci-4alkyl, -CO2H, -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.

12. The conjugate of claim 11, wherein the molecule comprising at least two cysteine residues is a peptide or a protein.

13. The conjugate of claim 11, wherein R1is the optionally substituted 6- to 12-membered aryl.

14. The conjugate of claim 13, wherein the optionally substituted 6- to 12-membered aryl is optionally substituted phenyl.212443-0005-W00115. A method of preparing the conjugate of claim 11 , the method comprising:53.providing a molecule comprising at least two cysteine residues; and54.reacting the compound of any one of claims 1-10 with two cysteine residues of the molecule, thereby providing the conjugate of any one of claims 11-14.

16. The method of claim 15, wherein the molecule comprising at least two cysteine residues is a peptide or a protein.

17. The method of claim 15, wherein reacting the compound of any one of claims 1-10, with the two cysteine residues occurs in the presence of a buffer solution.

18. The method of claim 17, wherein the buffer solution has a pH of 4.0 to 9.0.

19. The method of claim 15, wherein the conjugate is provided in quantitative yields.

20. A method of functionalizing a conjugate, the method comprising:60.reacting the conjugate of claim 11 with a functionalized payload, wherein the functionalized payload comprises a payload attached to a cyclooctene, thereby providing a functionalized conjugate.

21. The method of claim 20, wherein a linker attaches the cyclooctene to the payload.

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

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

65. 67.wherein L1is the linker, and D1is the payload.

24. The functionalization method of claim 23, wherein the functionalized conjugate comprises a moiety of formula (I l-a) or (I l-b):212443-0005-W00170.

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

74.

26. The method of any one of claims 23, wherein L1is:

78.

79. wherein:80.Z1is -O-, -S-, -NRa-, or-CRaRb-;81.Raand Rb, at each occurrence, are each independently hydrogen or Ci-4alkyl; and m, at each occurrence, is 0-1482.n, at each occurrence, is 0-10, and83.p, at each occurrence, is 0-10.

27. The method of claim 26, wherein Z1is -O-.

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

29. The method of any one of claims 23, wherein D1is a therapeutic agent, a diagnostic agent, or a targeting agent.212443-0005-W00130. The method of claim 29, wherein the diagnostic agent is a cyanine dye.

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

32. The method of claim 31, wherein the anti-cancer therapeutic agent is doxorubicin or ARV- 771.

Images