FBSA-based therapeutic and radioimaging conjugates targeting carbonic anhydrase-positive cancers

Abstract

The present disclosure relates to compositions and methods of carbonic anhydrase IX inhibitors. The present disclosure also relates to targeted conjugates of carbonic anhydrase IX inhibitors. The present disclosure also relates to the use of targeted conjugates of carbonic anhydrase IX inhibitors in methods of treating diseases and methods of disease imaging.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 62 / 548,670, filed on August 22, 2017, which is incorporated herein by reference in its entirety.

[0003] Field

[0004] The present disclosure relates to compositions and methods of carbonic anhydrase IX inhibitors. The present disclosure also relates to targeted conjugates of carbonic anhydrase IX inhibitors. The present disclosure also relates to the use of targeted conjugates of carbonic anhydrase IX inhibitors in methods of treating diseases and methods of disease imaging.

[0005] Background

[0006] The microenvironment can greatly influence the phenotypes of cancer cells within a tumor. One such microenvironmental influence is hypoxia, which is caused by poor formation of the vasculature present within the tumor (see, for example, Noman MZ, Hasmim M, Messai Y, Terry S, Kieda C, Janji B, Chouaib S. Hypoxia: a key player in anti-tumor immune response. A review in the Theme: Cellular Responses to Hypoxia. Am J Physiol Cell Physiol. 2015, 309(1): C569-C579). Studies have shown that 1%-1.5% of all genes are regulated by hypoxia (Harris AL. Hypoxia-a key regulatory factor in tumour growth. Nat Rev Cancer. 2002. 2(1): 38-47). Not surprisingly, hypoxic cancer cells can exhibit significantly different gene expression patterns. When in a hypoxic microenvironment, these changes can lead to differences in sensitivity to chemotherapy, which in turn can lead to increased aggressiveness and recurrence of cancer (Yamada S, Utsunomiya T, Morine Y, Imura S, Ikemoto T, Arakawa Y, Kanamoto M, Iwahashi S, Saito Y, Takasu C, Ishikawa D, Shimada M. Expressions of hypoxia-inducible factor-1 and epithelial cell adhesion molecule are linked with aggressive local recurrence of hepatocellular).

[0007] Due to the effects of hypoxia, efforts have been made to identify cancer-specific hypoxia markers for selective imaging. One such marker is carbonic anhydrase IX (also referred to herein as CAIX), which is expressed upon activation by hypoxia-inducible factor-1 (HIF-1). CA IX is a member of a group of metalloproteins and typically contains zinc that catalyzes the reversible hydration of carbon dioxide CA IX is among the most active CAs for the CO2 hydration reaction and contains four domains based on sequence similarity: an N-terminal proteoglycan-like (PG) domain, a CA catalytic domain, a transmembrane segment (TM), and an intracellular (IC) portion. CA IX is expressed in many cancers, including lung, colorectal, gastric, pancreatic, breast, cervical, bladder, ovarian, brain, head and neck, and oral cancers. Additionally, due to mutations in the VHL gene that result in constitutive activation of HIF-1, cancers such as clear cell renal carcinoma have been shown to upregulate CA IX up to 150-fold over basal levels. However, in normal cells, CA IX is expressed only in the epithelial cells of the stomach and gallbladder, where it does not appear to have catalytic activity.

[0008] Since CA IX has been touted as an excellent target for imaging agent-specific delivery, both small molecule-conjugates and antibody-conjugates have been created to image hypoxic tumors. For example, CA IX-specific ligands have been used to image murine xenograft models of colon, renal, and cervical cancers. In addition to human patients with clear cell renal carcinoma, CA IX-specific antibodies have been used to image murine xenograft models of clear cell renal carcinoma, head and neck, colon, and cervical cancers.

[0009] In addition, despite many efforts directed towards imaging agents targeting CA IX, in contrast, there has been little research on targeted therapy for cancers expressing CA IX. The few reports on CA IX-targeted therapy involve the use of anti-CA IX antibodies, which are directly labeled with therapeutic radionuclides (Muselaers CH, Oosterwijk E, Bos DL, Oyen WJ, Mulders PF, Boerman OC. Optimizaing lutetium 177-anti-carbonic anhydrase IX radioimmunotherapy in an intraperitoneal clear cell renal cell carcinoma xenograft model. Mol Imaging. 2014. 13:1-7), or conjugated with liposomes containing drugs (Wong BC, Zhang H, Qin L, Chen H, Fang C, Lu A, Yang Z. Carbonic anhydrase IX-directed immunoliposomes for targeted drug delivery to human lung cancer cells in vitro. Drug Des Devel Ther. 2014, 8:993-1001). To our knowledge, the efficacy of small molecule CA IX ligands directly conjugated with highly potent anticancer drugs has not been reported in in vivo mouse xenograft models.

[0010] Although small molecule conjugates have been mainly used for imaging hypoxic tumors using PET and fluorescence, there is still a need to develop other selective imaging agents. In addition, the need for developing CA IX-targeted therapy remains unmet.

[0011] Overview

[0012] In some embodiments, the present disclosure provides conjugates comprising a CA IX ligand covalently bound through a linker to at least one reagent selected from a therapeutic agent and an imaging agent, or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides conjugates comprising a CA IX ligand covalently bound to at least one reagent selected from a therapeutic agent and an imaging agent, or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides conjugates of formula B-L-A, wherein B is a binding ligand for carbonic anhydrase IX, L is an optional linker, and A is a therapeutic agent or an imaging agent. In some aspects of these embodiments, the CA IX ligand is an arylsulfonamide-containing compound.

[0013] In other embodiments, the present disclosure provides a method of imaging a cell population in a subject, the method comprising:

[0014] a. administering to the subject an effective amount of a conjugate comprising a CA IX ligand covalently bound to at least one imaging agent via a linker or a pharmaceutically acceptable salt thereof.

[0015] In other embodiments, the present disclosure provides a method of imaging a cell population in a subject, the method comprising:

[0016] a. administering to the subject an effective amount of a conjugate of formula B-L-A, wherein B is a binding ligand for carbonic anhydrase IX, L is an optional linker, and A is an imaging agent. In some aspects of these embodiments, the CA IX ligand is a compound containing an arylsulfonamide. In some aspects of these embodiments, the CA IX ligand has the following formula:

[0017]

[0018] wherein R A 、R B and m are as defined herein.

[0019] In other embodiments, the present disclosure provides a composition comprising a conjugate as described herein or a pharmaceutically acceptable salt thereof and optionally at least one pharmaceutically acceptable excipient.

[0020] In other embodiments, the present disclosure provides a conjugate as described herein for use in a method of imaging a cell population in a subject. In some aspects of these embodiments, the method comprises administering to the subject an amount of the conjugate effective to image the cells.

[0021] In other embodiments, the present disclosure provides the use of a conjugate as described herein in the preparation of a medicament for use in imaging a cell population in a subject. In some aspects of these embodiments, the method comprises administering to the subject an amount of the conjugate effective to image the cells.

[0022] In other embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising: a. administering to the subject an effective amount of a conjugate as described herein or a pharmaceutically acceptable salt thereof. In other embodiments, the method further comprises: b. identifying a patient for treatment by imaging. In some aspects of these embodiments, the imaging comprises: c. administering to the patient an effective amount of the conjugate, wherein the agent is an imaging agent as described herein; and d. identifying the patient as having a cancer expressing CAIX. In some aspects of these embodiments, the cancer is selected from lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0023] In other embodiments, the present disclosure provides conjugates as described herein for use in methods of treating cancer in a subject. In some aspects of these embodiments, the method comprises administering to the subject an amount of the conjugate effective to treat cancer. In some aspects of these embodiments, the cancer is selected from lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0024] In other embodiments, the present disclosure provides the use of a conjugate as described herein in the preparation of a medicament for use in treating cancer in a subject. In some aspects of these embodiments, the method comprises administering to the subject an amount of the conjugate effective to treat the cells. In some aspects of these embodiments, the cancer is selected from lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0025] In some embodiments, the present disclosure provides a composition comprising a conjugate as described herein or a pharmaceutically acceptable salt thereof and optionally at least one pharmaceutically acceptable excipient.

[0026] In some embodiments, the present disclosure provides a method of imaging a cell population in a subject, the method comprising: a. administering to the subject a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; b. irradiating the conjugate bound to the cells with light at a near-infrared wavelength, and c. detecting light emitted from the cells at an emission wavelength.

[0027] In some embodiments, the present disclosure provides a method of imaging a cell population in a subject, the method comprising: a. administering to the subject a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; and b. visualizing the conjugate bound to the cells by irradiating with light at a near-infrared wavelength.

[0028] In some embodiments, the present disclosure provides a method of imaging cancer in a subject, the method comprising: a. administering to the subject a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cancer cells expressing the CA IX protein; b. irradiating the conjugate bound to the cancer cells with light at a near-infrared wavelength, and c. detecting light emitted from the cancer cells at an emission wavelength. In some aspects of these embodiments, the cancer is selected from lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0029] In some embodiments, the present disclosure provides methods for imaging cancer in a subject, the methods comprising: a. administering to the subject a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cancer cells expressing the CA IX protein; and b. visualizing the conjugate bound to the cancer cells by irradiating with light of a near-infrared wavelength. In some aspects of these embodiments, the cancer is selected from the group consisting of lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0030] In other embodiments, the present disclosure provides the conjugate of the present disclosure or a pharmaceutically acceptable salt thereof for use in a method for imaging cancer in a patient. In some aspects of these embodiments, the method comprises: a. administering to the patient the conjugate or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; and b. visualizing the conjugate bound to the cells by irradiating with light of a near-infrared wavelength. In some aspects of these embodiments, the method comprises: a. administering to the subject the conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cancer cells expressing the CA IX protein; b. irradiating the conjugate bound to the cancer cells with light of a near-infrared wavelength, and c. detecting light emitted from the cancer cells at an emission wavelength. In some aspects of these embodiments, the cancer is selected from the group consisting of lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0031] In some embodiments, the present disclosure provides the use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in imaging cancer in a patient. In some aspects of these embodiments, the method comprises: a. administering to the patient the conjugate or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; and b. visualizing the conjugate bound to the cells by irradiating with light of a near-infrared wavelength. In some aspects of these embodiments, the method comprises: a. administering to the subject the conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cancer cells expressing the CA IX protein; b. irradiating the conjugate bound to the cancer cells with light of a near-infrared wavelength, and c. detecting light emitted from the cancer cells at an emission wavelength. In some aspects of these embodiments, the cancer is selected from the group consisting of lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain cancer, head and neck cancer, oral cancer, and kidney cancer.

[0032] In some embodiments, the present disclosure provides methods for imaging a cell population in vitro, the methods comprising: a. contacting the cells with a conjugate of the present disclosure to provide labeled cells, and b. visualizing the labeled cells with a fluorescent light source. In some embodiments, the present disclosure provides methods for imaging a cell population in vitro, the methods comprising: a. contacting the cells with a conjugate of the present disclosure to provide labeled cells, b. irradiating the conjugate bound to the cells with light at a near-infrared wavelength, and c. detecting light emitted from the cells at an emission wavelength.

[0033] In some embodiments, the present disclosure provides methods for imaging a cell population in a subject, the methods comprising: a. administering to the subject a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; b. detecting a radionuclide bound to the conjugate.

[0034] Embodiments of the invention are further described by the clauses listed below. It should be understood that any embodiment described herein can be used together with any other embodiment described herein, to the extent that the embodiments are not mutually inconsistent.

[0035] 1. A conjugate of formula B-L-A or a pharmaceutically acceptable salt thereof, wherein B is a binding ligand for carbonic anhydrase IX of the formula:

[0036]

[0037] wherein

[0038] each R A is independently selected from H, halogen, -OR 1 , -OC(O)R 1 , -OC(O)NR 1 R 2 , -OS(O)R 1 , -OS(O)2R 1 , -SR 1 , -S(O)R 1 , -S(O)2R 1 , -S(O)NR 1 R 2 , -S(O)2NR 1 R 2 , -OS(O)NR 1 R 2 , -OS(O)2NR 1 R 2 , -NR 1 R 2 , -NR 1 C(O)R 1 , -NR 1 C(O)OR2 , -NR 1 C(O)NR 1 R 2 , -NR 1 S(O)R 2 , -NR 1 S(O)2R 2 , -NR 1 S(O)NR 1 R 2 , -NR 1 S(O)2NR 1 R 2 , -C(O)R 1 , -C(O)OR 1 and -C(O)NR 1 R 2 ;

[0039] R B is -OR 3 , -SR 3 , -NR 3 R 4 , -S(O)2R 3 , -NR 4 C(O)R 3 or -NR 4 C(O)NR 3 R 4 ;

[0040] R 3 is independently C1 - C 10 alkyl, C2 - C 10 alkenyl or phenyl, each independently substituted with a substituent selected from: -N(R 5 ) - *, -N(R 5 ) - C1 - C6 alkyl - N(R 6 ) - *, -OC(O) - *, -OC(O)N(R 5 ) - *, -C(O) - *, -C(O)O - * and -C(O)N(R 5 ) - *; and each remaining hydrogen atom in C1 - C 10 alkyl, C2 - C 10 alkenyl or phenyl is independently optionally substituted with: C1 - C 10 alkyl, C2 - C 10 alkenyl, halogen, -OR 1 , -OC(O)R 1 , -OC(O)NR 1 R 2 , -OS(O)R 1 , -OS(O)2R 1 , -SR1 、-S(O)R 1 、-S(O)2R 1 、-S(O)NR 1 R 2 、-S(O)2NR 1 R 2 、-OS(O)NR 1 R 2 、-OS(O)2NR 1 R 2 、-NR 1 R 2 、-NR 1 C(O)R 1 、-NR 1 C(O)OR 2 、-NR 1 C(O)NR 1 R 2 、-NR 1 S(O)R 2 、-NR 1 S(O)2R 2 、-NR 1 S(O)NR 1 R 2 、-NR 1 S(O)2NR 1 R 2 、-C(O)R 1 、-C(O)OR 1 and -C(O)NR 1 R 2 ;

[0041] Each R 1 , R 2 , R 4 , R 5 and R 6 independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl and C3-C9 cycloalkyl;

[0042] L is an optional linker;

[0043] A is a therapeutic agent or an imaging agent;

[0044] m is an integer from 1 to 5; and

[0045] *Indicates the connection point to L or A.

[0046] 2. The conjugate of clause 1 or a pharmaceutically acceptable salt thereof, wherein the carbonic anhydrase IX ligand has the formula:

[0047]

[0048] wherein

[0049] R B is -OR 3 、-SR 3 、-NR 3 R 4 、-S(O)2R 3 、-NR 4 C(O)R 3 or -NR 4 C(O)NR 3 R 4 ;

[0050] R 3 is independently C1-C 10 alkyl, C2-C 10 alkenyl or phenyl, each independently substituted with a substituent selected from: -NR 5 -*, -N(R 5 )-C1-C6 alkyl -N(R 6 )-*, -OC(O)-*, -OC(O)N(R 5 )-*, -C(O)-*, -C(O)O-* and -C(O)N(R 5 );

[0051] each R 1 、R 2 、R 4 、R 5 and R 6 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl and C3-C9 cycloalkyl; and

[0052] * represents the point of attachment to L or A.

[0053] 3. The conjugate of any one of the preceding clauses or a pharmaceutically acceptable salt thereof, wherein the carbonic anhydrase IX ligand is:

[0054]

[0055] wherein * represents the point of attachment to the remainder of the conjugate.

[0056] 4. The conjugate of any one of the preceding clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises a moiety selected from: -C(O)(C 1- C 12 alkyl)C(O)-, -NH-C1-C 12 alkyl-NH-, -N(C1-C6 alkyl)-C1-C 12alkyl-N(C1-C6 alkyl)-, -C(O)CH2CH2(OCH2CH2) q NH-, -C(O)CH2CH2(OCH2CH2) q N(C1-C6 alkyl)-, -(CH2CH2O) q CH2CH2C(O)-, -NH(CH2CH2O) q CH2CH2C(O)- and -N(C1-C6 alkyl)(CH2CH2O) q CH2CH2C(O)-; wherein q is an integer from 1 to 40.

[0057] 5. A conjugate of any of the foregoing clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises at least one amino acid.

[0058] 6. A conjugate of any of the foregoing clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises at least one amino acid selected from the group consisting of 3-aminopropionic acid, aspartic acid, cysteine, and arginine.

[0059] 7. A conjugate of any of the foregoing clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises a cleavable linker.

[0060] 8. A conjugate of any of the foregoing clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises a disulfide moiety.

[0061] 9. A conjugate of any of the foregoing clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises a hydrazine moiety.

[0062] 10. A conjugate of any of the foregoing clauses or a pharmaceutically acceptable salt thereof, wherein the linker comprises a linker moiety of the following formula:

[0063]

[0064] wherein

[0065] each R 7 and R 8 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl is independently optionally substituted with: halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -OR 9 -, -OC(O)R 9 -, -OC(O)NR 9 R10 、 -OS(O)R 9 、 -OS(O)₂R 9 、 -SR 9 、 -S(O)R 9 、 -S(O)₂R 9 、 -S(O)NR 9 R 10 、 -S(O)₂NR 9 R 10 、 -OS(O)NR 9 R 10 、 -OS(O)₂NR 9 R 10 、 -NR 9 R 10 、 -NR 9 C(O)R 10 、 -NR 9 C(O)OR 10 、 -NR 9 C(O)NR 11 R 12 、 -NR 9 S(O)R 10 、 -NR 9 S(O)₂R 10 、 -NR 9 S(O)NR 11 R 12 、 -NR 9 S(O)₂NR 11 R 12 、 -C(O)R 9 、 -C(O)OR 9 or -C(O)NR 9 R 10 ;

[0066] Each X is independently a C₁-C₆ alkyl or an aryl-(C₁-C₆ alkyl), wherein each hydrogen atom in the C₁-C₆ alkyl and the aryl-(C₁-C₆ alkyl) is independently optionally substituted with: halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C 10 aryl, 5- to 7-membered heteroaryl, -OR 10 、 -OC(O)R 10 、 -OC(O)NR 9 、 -OC(O)NR 9 、 -OC(O)NR 9 R 10 、 -OS(O)R 9 、 -OS(O)₂R 9 、 -SR 9, -S(O)R 9 , -S(O)₂R 9 , -S(O)NR 9 R 10 , -S(O)₂NR 9 R 10 , -OS(O)NR 9 R 10 , -OS(O)₂NR 9 R 10 , -NR 9 R 10 , -NR 9 , -NRC(O)R 10 , -NR 9 , -NRC(O)OR 10 , -NR 9 , -NRC(O)NR 11 R 12 , -NR 9 , -NRS(O)R 10 , -NR 9 , -NRS(O)₂R 10 , -NR 9 , -NRS(O)NR 11 R 12 , -NR 9 , -NRS(O)₂NR 11 R 12 , -C(O)R 9 , -C(O)OR 9 or -C(O)NR 9 R 10 ;

[0067] Each R 9 , R 10 , R 11 and R 12 is independently selected from H, C₁-C₇ alkyl, C₂-C₇ alkenyl, C 2- C₇ alkynyl, C 3- C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C 10 aryl and 5- to 7-membered heteroaryl; and

[0068] Each * represents a covalent bond to the remainder of the conjugate.

[0069] 11. A conjugate of any one of the foregoing clauses or a pharmaceutically acceptable salt thereof, which comprises the following formula:

[0070]

[0071] wherein * represents the point of attachment to the remainder of the conjugate.

[0072] 12. A conjugate according to any one of clauses 1-10 or a pharmaceutically acceptable salt thereof, which comprises the following formula:

[0073]

[0074] where * represents a covalent bond to the remainder of the conjugate.

[0075] 13. A conjugate according to any one of clauses 1-10 or a pharmaceutically acceptable salt thereof, which comprises the following formula:

[0076]

[0077] where * represents a covalent bond to the remainder of the conjugate.

[0078] 14. A conjugate according to any one of the preceding clauses or a pharmaceutically acceptable salt thereof, which comprises a linker portion having the amino acid sequence 3-aminopropionic acid-Asp-Cys.

[0079] 15. A conjugate according to any one of clauses 1-13 or a pharmaceutically acceptable salt thereof, which comprises a linker portion having the amino acid sequence Asp-Arg-Asp-3-aminopropionic acid-Asp-Cys.

[0080] 16. A conjugate according to any one of clauses 4-15 or a pharmaceutically acceptable salt thereof, wherein q is 2.

[0081] 17. A conjugate according to any one of clauses 4-15 or a pharmaceutically acceptable salt thereof, wherein q is 4.

[0082] 18. A conjugate according to any one of the preceding clauses or a pharmaceutically acceptable salt thereof, wherein A is an imaging agent.

[0083] 19. A conjugate according to any one of the preceding clauses or a pharmaceutically acceptable salt thereof, wherein A is a fluorescent dye.

[0084] 20. A conjugate according to any one of the preceding clauses or a pharmaceutically acceptable salt thereof, wherein A is a fluorescein dye of the following formula:

[0085]

[0086] where * represents a covalent bond to the remainder of the conjugate.

[0087] 21. A conjugate according to clause 1 or a pharmaceutically acceptable salt thereof selected from the following:

[0088]

[0089] 22. A conjugate according to any one of clauses 1-18 or a pharmaceutically acceptable salt thereof, wherein A is a radioactive imaging agent.

[0090] 23. A conjugate according to any one of clauses 1-18 or a pharmaceutically acceptable salt thereof, wherein A comprises a radioisotope of a metal coordinated to a chelating group.

[0091] 24. A conjugate according to any one of clauses 1-18, 22 or 23 or a pharmaceutically acceptable salt thereof, which comprises a radioisotope selected from 111 In, 99m Tc, 64 Cu, 67 Cu, 67 Ga and 68 Ga.

[0092] 25. A conjugate according to any one of clauses 1-18 or 22-24 or a pharmaceutically acceptable salt thereof, which comprises a radioisotope 64 Cu.

[0093] 26. A conjugate according to any one of clauses 1-18 or 22-25 or a pharmaceutically acceptable salt thereof, which comprises a chelating group selected from the group consisting of: DOTA, NOTA, TETA, DOTAGA, NODAGA, DTPA, PCTA and NETA.

[0094] 27. A conjugate according to any one of clauses 1-18 or 22-26 or a pharmaceutically acceptable salt thereof, which comprises a chelating group being a NODAGA group.

[0095] 28. A conjugate according to any one of clauses 1-18 or 22-27 or a pharmaceutically acceptable salt thereof, which comprises a chelating group of the following formula:

[0096]

[0097] wherein * represents a covalent bond to the remainder of the conjugate.

[0098] 29. A conjugate according to any one of clauses 1-18 or 22-28 or a pharmaceutically acceptable salt thereof, which comprises the following formula:

[0099]

[0100] 30. A conjugate according to any one of clauses 1-17 or a pharmaceutically acceptable salt thereof, wherein A is a therapeutic agent.

[0101] 31. A conjugate according to any one of clauses 1-17 or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent is tubulysin.

[0102] 32. A conjugate according to clause 1 or a pharmaceutically acceptable salt thereof selected from the following:

[0103]

[0104]

[0105] 33. A conjugate according to any one of clauses 1-17 or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent is maytansine.

[0106] 34. A conjugate according to clause 1 having the following formula or a pharmaceutically acceptable salt thereof:

[0107]

[0108] 35. A method for imaging a cell population in a subject, the method comprising:

[0109] a. Administering to the subject an effective amount of a conjugate of formula B-L-A, wherein B is a carbonic anhydrase IX ligand of the following formula:

[0110]

[0111] wherein

[0112] each R A is independently selected from H, halogen, -OR 1 、-OC(O)R 1 、-OC(O)NR 1 R 2 、-OS(O)R 1 、-OS(O)2R 1 、-SR 1 、-S(O)R 1 、-S(O)2R 1 、-S(O)NR 1 R 2 、-S(O)2NR 1 R 2 、-OS(O)NR 1 R 2 、-OS(O)2NR 1 R 2 、-NR 1 R 2 、-NR 1 C(O)R 1 、-NR 1 C(O)OR 2 、-NR 1 C(O)NR 1 R 2 、-NR 1 S(O)R 2 、-NR 1 S(O)2R 2, -NR 1 S(O)NR 1 R 2 , -NR 1 S(O)2NR 1 R 2 , -C(O)R 1 , -C(O)OR 1 and -C(O)NR 1 R 2 ;

[0113] R B is -OR 3 , -SR 3 , -NR 3 R 4 , -S(O)2R 3 , -NR 4 C(O)R 3 or -NR 4 C(O)NR 3 R 4 ;

[0114] R 3 is independently C1-C 10 alkyl, C2-C 10 alkenyl or phenyl, each independently substituted with a substituent selected from: -NR 5 -*, -N(R 5 )-C1-C6 alkyl -N(R 6 )-*, -OC(O)-*, -OC(O)N(R 5 )-*, -C(O)-*, -C(O)O-* and -C(O)N(R 5 )-*;

[0115] Each R 1 , R 2 , R 4 , R 5 and R 6 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl and C3-C9 cycloalkyl;

[0116] L is an optional linker;

[0117] A is a therapeutic agent or imaging agent;

[0118] m is an integer from 1 - 5; and

[0119] * represents the point of attachment to L or A.

[0120] 36. The method of clause 35, wherein the carbonic anhydrase IX ligand has the formula:

[0121]

[0122] wherein

[0123] R B is -OR 3 、-SR 3 、-NR 3 R 4 、-S(O)2R 3 、-NR 4 C(O)R 3 or -NR 4 C(O)NR 3 R 4 ;

[0124] R 3 is independently C1-C 10 alkyl, C2-C 10 alkenyl or phenyl, each independently substituted with a substituent selected from: -NR 5 -*, -N(R 5 )-C1-C6 alkyl -N(R 6 )-*, -OC(O)-*, -OC(O)N(R 5 )-*, -C(O)-*, -C(O)O-* and -C(O)N(R 5 );

[0125] Each R 1 、R 2 、R 4 、R 5 and R 6 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl and C3-C9 cycloalkyl; and

[0126] * represents the point of attachment to L or A.

[0127] 37. The method of clause 35 or 36 or a pharmaceutically acceptable salt thereof, wherein the carbonic anhydrase IX ligand is:

[0128]

[0129] where * represents the point of attachment to the remainder of the conjugate.

[0130] 38. The method of any one of clauses 35 - 37 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a moiety selected from: -C(O)(C 1- C 12 alkyl)C(O)-, -NH-C1-C 12 alkyl-NH-, -N(C1-C6 alkyl)-C1-C12 alkyl-N(C1-C6 alkyl)-, -C(O)CH2CH2(OCH2CH2) q NH-, -C(O)CH2CH2(OCH2CH2) q N(C1-C6 alkyl)-, -(CH2CH2O) q CH2CH2C(O)-, -NH(CH2CH2O) q CH2CH2C(O)- and -N(C1-C6 alkyl)(CH2CH2O) q CH2CH2C(O)-; wherein q is an integer from 1 to 40.

[0131] 39. A method according to any one of clauses 35 - 38 or a pharmaceutically acceptable salt thereof, wherein the linker comprises at least one amino acid.

[0132] 40. A method according to any one of clauses 35 - 39 or a pharmaceutically acceptable salt thereof, wherein the linker comprises at least one amino acid selected from the group consisting of 3-aminopropionic acid, aspartic acid, cysteine, and arginine.

[0133] 41. A method according to any one of clauses 35 - 40 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a cleavable linker.

[0134] 42. A method according to any one of clauses 35 - 41 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a disulfide linker moiety.

[0135] 43. A method according to any one of clauses 35 - 42 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a hydrazine linker moiety.

[0136] 44. A method according to any one of clauses 35 - 43 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a linker moiety of the formula:

[0137]

[0138] wherein

[0139] each R 7 and R 8 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl is independently optionally substituted with: halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -OR 9 、-OC(O)R9 、 -OC(O)NR 9 R 10 、 -OS(O)R 9 、 -OS(O)₂R 9 、 -SR 9 、 -S(O)R 9 、 -S(O)₂R 9 、 -S(O)NR 9 R 10 、 -S(O)₂NR 9 R 10 、 -OS(O)NR 9 R 10 、 -OS(O)₂NR 9 R 10 、 -NR 9 R 10 、 -NR 9 C(O)R 10 、 -NR 9 C(O)OR 10 、 -NR 9 C(O)NR 11 R 12 、 -NR 9 S(O)R 10 、 -NR 9 S(O)₂R 10 、 -NR 9 S(O)NR 11 R 12 、 -NR 9 S(O)₂NR 11 R 12 、 -C(O)R 9 、 -C(O)OR 9 or -C(O)NR 9 R 10 ;

[0140] Each X is independently a C₁-C₆ alkyl or an aryl-(C₁-C₆ alkyl), wherein each hydrogen atom in the C₁-C₆ alkyl and the aryl-(C₁-C₆ alkyl) is independently optionally substituted with: halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C 10 aryl, 5- to 7-membered heteroaryl, -OR 10 aryl-(C₁-C₆ alkyl), and C₆-C 10 aryl, 5- to 7-membered heteroaryl, -OR 9 、 -OC(O)R 9 、 -OC(O)NR 9 R 10 、 -OS(O)R 9、-OS(O)2R 9 、-SR 9 、-S(O)R 9 、-S(O)2R 9 、-S(O)NR 9 R 10 、-S(O)2NR 9 R 10 、-OS(O)NR 9 R 10 、-OS(O)2NR 9 R 10 、-NR 9 R 10 、-NR 9 C(O)R 10 、-NR 9 C(O)OR 10 、-NR 9 C(O)NR 11 R 12 、-NR 9 S(O)R 10 、-NR 9 S(O)2R 10 、-NR 9 S(O)NR 11 R 12 、-NR 9 S(O)2NR 11 R 12 、-C(O)R 9 、-C(O)OR 9 or -C(O)NR 9 R 10 ;

[0141] Each R 9 , R 10 , R 11 and R 12 independently selected from H, C1-C7 alkyl, C2-C7 alkenyl, C 2- C7 alkynyl, C 3- C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 Aryl and 5- to 7-membered heteroaryl; and

[0142] Each * represents a covalent bond to the rest of the conjugate.

[0143] 45. The method of any one of clauses 35-44, or a pharmaceutically acceptable salt thereof, comprising the formula:

[0144]

[0145] where * represents the point of attachment to the remainder of the conjugate.

[0146] 46. The method of any one of clauses 35 - 44 or a pharmaceutically acceptable salt thereof, which comprises the following formula:

[0147]

[0148] where * represents a covalent bond to the remainder of the conjugate.

[0149] 47. The method of any one of clauses 35 - 44 or a pharmaceutically acceptable salt thereof, which comprises the following formula:

[0150]

[0151] where * represents a covalent bond to the remainder of the conjugate.

[0152] 48. The method of any one of clauses 35 - 47 or a pharmaceutically acceptable salt thereof, which comprises a linker portion having the amino acid sequence 3 - aminopropionic acid - Asp - Cys.

[0153] 49. The method of any one of clauses 35 - 48 or a pharmaceutically acceptable salt thereof, which comprises a linker portion having the amino acid sequence Asp - Arg - Asp - 3 - aminopropionic acid - Asp - Cys.

[0154] 50. The method of clause 38 or a pharmaceutically acceptable salt thereof, wherein q is 2.

[0155] 51. The method of clause 38 or a pharmaceutically acceptable salt thereof, wherein q is 4.

[0156] 52. The method of any one of clauses 35 - 51 or a pharmaceutically acceptable salt thereof, wherein A is an imaging agent.

[0157] 53. The method of any one of clauses 35 - 52 or a pharmaceutically acceptable salt thereof, wherein A is a fluorescent dye.

[0158] 54. The method of any one of clauses 35 - 53 or a pharmaceutically acceptable salt thereof, wherein A is a fluorescein dye of the following formula:

[0159]

[0160] where * represents a covalent bond to the remainder of the conjugate.

[0161] 55. The method of clause 35, wherein the conjugate is selected from:

[0162]

[0163] or a pharmaceutically acceptable salt thereof.

[0164] 56. A method according to any one of clauses 35 - 52 or a pharmaceutically acceptable salt thereof, wherein A is a radioimaging agent.

[0165] 57. A method according to any one of clauses 35 - 52 or 56 or a pharmaceutically acceptable salt thereof, wherein A comprises a radioisotope of a metal coordinated to a chelating group.

[0166] 58. A method according to any one of clauses 35 - 52, 56 or 57 or a pharmaceutically acceptable salt thereof, comprising a radio metal selected from 111 In, 99m Tc, 64 Cu, 67 Cu, 67 Ga and 68 Ga.

[0167] 59. A method according to any one of clauses 35 - 52 or 56 - 58 or a pharmaceutically acceptable salt thereof, comprising the radio metal 64 Cu.

[0168] 60. A method according to any one of clauses 35 - 52 or 56 - 59 or a pharmaceutically acceptable salt thereof, comprising a chelating group selected from the group consisting of: DOTA, NOTA, TETA, DOTAGA, NODAGA, DTPA, PCTA and NETA.

[0169] 61. A method according to any one of clauses 35 - 52 or 56 - 60 or a pharmaceutically acceptable salt thereof, comprising a chelating group that is the NODAGA group.

[0170] 62. A method according to any one of clauses 35 - 52 or 56 - 61 or a pharmaceutically acceptable salt thereof, comprising a chelating group of the following formula:

[0171]

[0172] wherein * represents a covalent bond to the remainder of the conjugate.

[0173] 63. A method according to clause 35 or a pharmaceutically acceptable salt thereof, comprising the following formula:

[0174]

[0175] 64. A composition comprising a conjugate according to any one of clauses 1 - 34 or a pharmaceutically acceptable salt thereof and optionally at least one pharmaceutically acceptable excipient.

[0176] 65. A conjugate according to any one of clauses 1 - 29 for use in a method of imaging a cell population in a subject.

[0177] 66. The conjugate of clause 65, wherein the method comprises administering to a subject an amount of the conjugate effective to image the cells.

[0178] 67. Use of a conjugate according to any one of clauses 1-29 in the preparation of a medicament for use in imaging a cell population in a subject.

[0179] 68. The use of clause 67, wherein the method comprises administering to a subject an amount of the conjugate effective to image the cells.

[0180] 69. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate according to any one of clauses 1-17 or 30-34.

[0181] 70. A conjugate according to any one of clauses 1-17 or 30-34 for use in a method of treating cancer in a subject.

[0182] 71. Use of a conjugate according to any one of clauses 1-17 or 30-34 in the preparation of a medicament for use in treating cancer in a subject.

[0183] 72. A method of imaging a cell population in vitro, the method comprising: a. contacting the cells with a conjugate according to any one of clauses 1-29 to provide labeled cells, and b. visualizing the labeled cells with a fluorescent light source or a suitable detector. Brief Description of the Drawings

[0185] Figure 1A -F shows an image of the cells, wherein Figure 1A and B are white light and fluorescence confocal microscopy of 25 nM FBSA-PEG2-FITC conjugate (conjugate 5), wherein Figure 1C and D show white light and fluorescence confocal microscopy of a competitive control of 25 nM FITC conjugate 5 plus 100-fold excess CAIX inhibitor (compound 3, 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid); wherein Figure 1E and F show only white light and fluorescence confocal microscopy of SKRC52 cells; and wherein the FITC conjugate 5 binds to the cells and competes in the presence of an excess of unconjugated inhibitor, indicating the presence of a specific receptor-specific binding event.

[0186] Figure 2 Shows the binding affinity of CAIX FITC conjugates 5, 7, and 9 in HEK293-CA9 cells, and shows that the PEG2 linker has a binding affinity at 17.94 nM. (●) Conjugate 5, K D= 17.94 nM; (▲) Conjugate 7, K D = 55.2 nM; (◆) Conjugate 9, K D = 215.7 nM.

[0187] Figure 3 Shows the binding affinities of CAIX FITC conjugates 5, 7, and 9 in SKRC52 cells, and shows that the PEG2 linker has a binding affinity at 1.28 nM. (●) Conjugate 5, K D = 1.28 nM; (▲) Conjugate 7, K D = 4.57 nM; (◆) Conjugate 9, K D = 40.89 nM.

[0188] Figure 4 Shows the binding affinity of CAIX FITC conjugate 5 in HT-29 cells, shows that the conjugate binds to the cells and competes in the presence of 100-fold excess of unconjugated inhibitor, indicating the presence of specific receptor-specific binding events. (▼) Competition; (●) Conjugate 5, K D = 4.553 nM.

[0189] Figure 5 Shows the binding affinity of CAIX FITC conjugate 7 in HEK293-CA9 cells, shows that the conjugate binds to the cells and competes in the presence of 100-fold excess of unconjugated inhibitor, indicating the presence of specific receptor-specific binding events. (▲) Competition; (●) Conjugate 7, K D = 55.2 nM.

[0190] Figure 6 Shows the binding affinity of CAIX FITC conjugate 5 in SKRC52 cells with or without hypoxia, shows that conjugate 5 binds to CAIX regardless of the presence of hypoxia. (●) Normoxic SKRC52, 4.57 nM; (▲) Hypoxic SKRC52, K D = 4.72 nM.

[0191] Figure 7 Shows the binding affinity of CAIX FITC conjugate 5 in A549 cells. (▼) Competition; (●) Conjugate 5, K D = 6.044 nM.

[0192] Figure 8 Shows the in vitro cytotoxicity of conjugate 14, where various concentrations of conjugate 14 are incubated with HEK or HEK-CA9 cells for 4 hours. After washing, the cells are incubated for 66 - 72 hours and their viability is determined by 3 3H-thymidine uptake. (●) HEK293-CA9; (▲) HEK293.

[0193] Figure 9 Show the in vitro binding of radiolabeled conjugate 17 in SKRC52 cells. (●) Conjugate 17, K D = 5.87 nM; (■) Competition.

[0194] Figure 10 Show the in vitro binding of radiolabeled conjugate 17 in A549 cells. (●) Conjugate 17, K D = 6.04 nM; (■) Competition.

[0195] Figure 11 Show the in vivo efficacy of conjugate 12 (TIW) in HT-29 xenografts (n = 3 for the conjugate 12 group and n = 2 for the competition group), where the competition group was treated with 100-fold excess of compound 3, and the mice treated with 2 μmol / kg of conjugate 12 showed tumor shrinkage while the untreated control grew. (●) Conjugate 12; (■) Competition.

[0196] Figure 12 Show the body mass of HT-29 xenograft mice treated with conjugate 12, showing that the body weight loss does not exceed 10% on the 6th day of treatment. The competition group was treated with 100-fold excess of compound 3, where n = 3 for the conjugate 12 group and n = 2 for the competition group.

[0197] Figure 13 Show the in vivo dose escalation efficacy of conjugate 14 in HT-29 xenografts (n = 1 for each dose), where after the tumors disappeared, the mice were treated with one more dose, and the last treatment was on the 6th, 8th, 12th, and 14th days for mice 1, 2, 3, and 4 / 5 respectively. Doses of 2, 1.5, and 1 μmol / kg all caused tumor shrinkage, but the tumor started to reappear on the 16th day at 1 μmol / kg. Mouse dose levels: (●) 2 μmol / kg; (■) 1.5 μmol / kg; (▲) 1 μmol / kg; (▼) 0.5 μmol / kg; (◆) 0.25 μmol / kg.

[0198] Figure 14 Show the body mass of HT-29 xenograft mice treated with various doses of conjugate 14 (n = 1 for each dose), where the highest dose of conjugate 14 still showed weight loss to conjugate 12 ( Figure 10 ) but the other doses showed acceptable body weight conditions. Mouse dose levels: (●) 2 μmol / kg; (■) 1.5 μmol / kg; (▲) 1 μmol / kg; (▼) 0.5 μmol / kg; (◆) 0.25 μmol / kg.

[0199] Figure 15 Show the in vivo efficacy of conjugate 14 in HT-29 xenografts (n = 5 per group), where conjugate 14 at 1.25 umole / kg and the competitive group of conjugate 14 at 1.25 umole / kg with 100-fold excess of compound 15 were given TIW for 3 weeks, where the competitive and tumor-only groups were sacrificed on day 16 according to the humane guidelines of the study, and where the treatment group showed stable tumor regression and disease stabilization before treatment cessation. (●) Conjugate 14; (■) Competitive; (▲) Tumor only.

[0200] Figure 16 Show the body weights of HT-29 xenograft mice treated with conjugate 14 at 1.25 umole / kg and the competitive group of conjugate 14 at 1.25 umole / kg with 100-fold excess of compound 15. (●) Conjugate 14; (■) Competitive; (▲) Tumor only.

[0201] Figure 17 Show the in vivo efficacy of conjugate 14 in A549 xenografts, where conjugate 14 at 1.25 umole / kg and the competitive group of conjugate 14 at 1.25 umole / kg with 100-fold excess of compound 15 were given TIW for 3 weeks, where the competitive and tumor-only groups were sacrificed on day 16 according to the humane guidelines of the study, and where the treatment group showed stable tumor regression and disease stabilization before treatment cessation. (●) Conjugate 14; (■) Competitive.

[0202] Figure 18 Show the body weights of A549 xenograft mice treated with conjugate 14 at 1.25 umole / kg and the competitive group of conjugate 14 at 1.25 umole / kg with 100-fold excess of compound 15. (●) Conjugate 14; (■) Competitive.

[0203] Figure 19 Show the biodistribution time-course study of radiolabeled conjugate 17 in mice bearing A549 xenografts. For each time point after injection, left bar = tumor; middle bar = stomach; right bar = kidney.

[0204] Figure 20 Show the biodistribution study of radiolabeled conjugate 17 in the A549 xenograft model. For each tissue type shown in the figure, left bar = conjugate 17; right bar = competitive.

[0205] Figure 21 Show the biodistribution of radiolabeled conjugate 17 in the SKRC52 xenograft model. For each tissue type shown in the figure, left bar = conjugate 17; right bar = competitive.

[0206] Figure 22 Show the biodistribution of radiolabeled conjugate 17 in the HT29 xenograft model.

[0207] Figure 23A and 23B Show the PET / CT of radiolabeled conjugate 17 in the SKRC52 xenograft model. Figure 23A , whole mouse. Figure 23B , extracted.

[0208] Figure 24 Show the PET / CT of radiolabeled conjugate 17 in the A549 xenograft model.

[0209] Define

[0210] As used herein, the term "alkyl" includes a chain of carbon atoms which is optionally branched and contains from 1 to 20 carbon atoms. It should be further understood that in certain embodiments, the alkyl may advantageously have a limited length, including C1-C 12 , C1-C 10 , C1-C9, C1-C8, C1-C7, C1-C6 and C1-C4. Illustratively, such particularly limited-length alkyls (including C1-C8, C1-C7, C1-C6 and C1-C4, etc.) may be referred to as "lower alkyls". Illustrative alkyls include (but are not limited to) methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl, octyl, etc. The alkyl may be substituted or unsubstituted. Typical substituents include cycloalkyl, aryl, heteroaryl, heterocycloaliphatic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, oxo, (=O), thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, C-carboxy, O-carboxy, nitro and amino, or as described in the various embodiments provided herein. It should be understood that "alkyl" may be combined with other groups (such as those provided above) to form a functionalized alkyl. For example, the combination of an "alkyl" group as described herein with a "carboxy" group may be referred to as a "carboxyalkyl" group. Other non-limiting examples include hydroxyalkyl, aminoalkyl, etc.

[0211] As used herein, the term "alkenyl" includes a chain of carbon atoms which is optionally branched, contains from 2 to 20 carbon atoms, and further contains at least one carbon-carbon double bond (i.e., C═C). It should be understood that in certain embodiments, the alkenyl may advantageously have a limited length, including C2-C 12, C2-C9, C2-C8, C2-C7, C2-C6, and C2-C4. Illustratively, such particularly limited-length alkenyl groups (including C2-C8, C2-C7, C2-C6, and C2-C4) may be referred to as "lower alkenyls". The alkenyl group may be unsubstituted or substituted as described for alkyl groups or as described in various embodiments provided herein. Illustrative alkenyl groups include (but are not limited to) vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc.

[0212] As used herein, the term "alkynyl" includes a chain of carbon atoms, which may optionally be branched and contains 2-20 carbon atoms and further contains at least one carbon-carbon triple bond (i.e., C≡C). It should be understood that in certain embodiments, each alkynyl group may advantageously have a limited length, including C2-C 12 , C2-C9, C2-C8, C2-C7, C2-C6, and C2-C4. Illustratively, such particularly limited-length alkynyl groups (including C2-C8, C2-C7, C2-C6, and C2-C4) may be referred to as "lower alkynyls". The alkenyl group may be unsubstituted or substituted as described for alkyl groups or as described in various embodiments provided herein. Illustrative alkynyl groups include (but are not limited to) ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, etc.

[0213] As used herein, the term "aryl" refers to a fully conjugated π-electron system of 6-12 carbon atoms of a monocyclic or polycyclic fused ring group of all-carbon. It should be understood that in certain embodiments, the aryl group may advantageously have a limited size, such as C6-C 10 aryl. Illustrative aryl groups include (but are not limited to) phenyl, naphthyl, and anthracenyl. The aryl group may be unsubstituted or substituted as described for alkyl groups or as described in various embodiments provided herein.

[0214] As used herein, the term "cycloalkyl" refers to a 3-15 membered all-carbon monocyclic, all-carbon 5 / 6 or 6 / 6 fused bicyclic or polycyclic fused ring (a "fused" ring system means that each ring in the system shares a pair of adjacent carbon atoms with each other ring in the system) group, wherein one or more rings may contain one or more double bonds, but the cycloalkyl group does not contain a fully conjugated π-electron system. It should be understood that in certain embodiments, the cycloalkyl group may advantageously have a limited size, such as C3-C 13 , C3-C6, C3-C6, and C4-C6. The cycloalkyl group may be unsubstituted or substituted as described for alkyl groups or as described in various embodiments provided herein. Illustrative cycloalkyl groups include (but are not limited to) cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl, adamantyl, norbornyl, norbornenyl, 9H-fluoren-9-yl, etc.

[0215] As used herein, the term "heterocycloalkyl" refers to a monocyclic or fused ring group having 3 to 12 ring atoms in one or more rings, wherein at least one ring atom is a heteroatom (such as nitrogen, oxygen or sulfur), and the remaining ring atoms are carbon atoms. The heterocycloalkyl may optionally contain 1, 2, 3 or 4 heteroatoms. The heterocycloalkyl may also have one of a variety of double bonds, including a double bond with nitrogen (such as C=N or N=N), but does not contain a completely conjugated π-electron system. It should be understood that in certain embodiments, the heterocycloalkyl may advantageously have a limited size, such as a 3- to 7-membered heterocycloalkyl, a 5- to 7-membered heterocycloalkyl, etc. The heterocycloalkyl may be unsubstituted or substituted as described for alkyl or as described in the various embodiments provided herein. Illustrative heterocycloalkyls include (but are not limited to) oxiranyl, thianaryl, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, piperazinyl, oxepanyl, 3,4-dihydro-2H-pyranyl, 5,6-dihydro-2H-pyranyl, 2H-pyranyl, 1,2,3,4-tetrahydropyridinyl, etc.

[0216] As used herein, the term "heteroaryl" refers to a monocyclic or fused ring group having 5 to 12 ring atoms containing 1, 2, 3 or 4 ring heteroatoms selected from nitrogen, oxygen and sulfur, the remaining ring atoms being carbon atoms, and further having a completely conjugated π-electron system. It should be understood that in certain embodiments, the heteroaryl may advantageously have a limited size, such as a 3- to 9-membered heteroaryl, a 3- to 7-membered heteroaryl, a 5- to 7-membered heteroaryl, a 5- to 9-membered heteroaryl, etc. The heteroaryl may be unsubstituted or substituted as described for alkyl or as described in the various embodiments provided herein. Illustrative heteroaryls include (but are not limited to) pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, tetrazolyl, triazinyl, pyrazinyl, tetrazinyl, quinazolinyl, quinoxalinyl, thienyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, indolyl and carbazoloyl, etc.

[0217] As used herein, "hydroxy" or "hydroxyl" refers to the -OH group.

[0218] As used herein, "alkoxy" refers to both -O-(alkyl) or -O-(unsubstituted cycloalkyl) groups. Representative examples include (but are not limited to) methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, etc.

[0219] As used herein, "aryloxy" refers to -O-aryl or -O-heteroaryl. Representative examples include (but are not limited to) phenoxy, pyridyloxy, furanyloxy, thienyloxy, pyrimidyloxy, pyrazinyloxy, etc.

[0220] As used herein, "mercapto" refers to the -SH group.

[0221] As used herein, "alkylthio" refers to -S-(alkyl) or -S-(unsubstituted cycloalkyl) groups. Representative examples include (but are not limited to) methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio, etc.

[0222] As used herein, "arylthio" refers to -S-aryl or -S-heteroaryl. Representative examples include (but are not limited to) phenylthio, pyridylthio, furanylthio, thienylthio, pyrimidylthio, etc.

[0223] As used herein, "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0224] As used herein, "trihalomethyl" refers to a methyl group having 3 halo substituents, such as trifluoromethyl.

[0225] As used herein, "cyano" refers to the -CN group.

[0226] As used herein, "sulfinyl" refers to the -S(O)R" group, where R" is any R group as described in the various embodiments provided herein, or R" may be hydroxy.

[0227] As used herein, "sulfonyl" refers to the -S(O)2R" group, where R" is any R group as described in the various embodiments provided herein, or R" may be hydroxy.

[0228] As used herein, "S-sulfonamido" refers to the -S(O)2NR"R" group, where R" is any R group as described in the various embodiments provided herein.

[0229] As used herein, "N-sulfonamido" refers to the -NR"S(O)2R" group, where R" is any R group as described in the various embodiments provided herein.

[0230] As used herein, "O-carbamoyl" refers to an -OC(O)NR''R'' group, where R'' is any R group as described in the various embodiments provided herein.

[0231] As used herein, "N-carbamoyl" refers to an R''OC(O)NR''- group, where R'' is any R group as described in the various embodiments provided herein.

[0232] As used herein, "O-thiocarbamoyl" refers to an -OC(S)NR''R'' group, where R'' is any R group as described in the various embodiments provided herein.

[0233] As used herein, "N-thiocarbamoyl" refers to an R''OC(S)NR''- group, where R'' is any R group as described in the various embodiments provided herein.

[0234] As used herein, "amino" refers to an -NR''R'' group, where R'' is any R group as described in the various embodiments provided herein.

[0235] As used herein, "C-acylamino" refers to a -C(O)NR''R'' group, where R'' is any R group as described in the various embodiments provided herein.

[0236] As used herein, "N-acylamino" refers to an R''C(O)NR''- group, where R'' is any R group as described in the various embodiments provided herein.

[0237] As used herein, "nitro" refers to a -NO2 group.

[0238] As used herein, "bond" refers to a covalent bond.

[0239] As used herein, "optional" or "optionally" means that the subsequent described event or condition may but need not occur, and the description includes both the case where the event or condition occurs and the case where it does not occur. For example, "heterocyclyl optionally substituted with alkyl" means that alkyl may but need not be present, and the description includes both the case where the heterocyclyl is substituted with alkyl and the case where the heterocyclyl is not substituted with alkyl.

[0240] As used herein, "independently" means that the subsequently described event or condition should be read on its own relative to other similar events or conditions. For example, in a situation where several equivalent hydrogen groups are optionally substituted by another group described in the situation, the use of "independently optionally" means that each instance of a hydrogen atom on the group can be substituted by another group, and the groups substituting each hydrogen atom can be the same or different. Or for example, in a situation where there are multiple groups, all of which are optionally selected from a group of possibilities, the use of "independently" means that each group can be selected from the group of possibilities separately from any other group, and the groups selected in such a situation can be the same or different.

[0241] As used herein, "amino acid" (also referred to as "AA") means any molecule containing an α-carbon atom covalently bonded to an amino group and an acid group. The acid group can include a carboxyl group. "Amino acid" can include molecules having one of the following formulas:

[0242]

[0243] wherein R’ is a side group and Φ contains at least 3 carbon atoms. "Amino acid" includes stereoisomers, such as D-amino acid and L-amino acid forms. Illustrative amino acid groups include (but are not limited to) 20 endogenous human amino acids and their derivatives, such as lysine (Lys), asparagine (Asn), threonine (Thr), serine (Ser), isoleucine (Ile), methionine (Met), proline (Pro), histidine (His), glutamine (Gln), arginine (Arg), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), alanine (Ala), valine (Val), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), cysteine (Cys), tryptophan (Trp), phosphoserine (PSER), sulfo-cysteine, argininosuccinic acid (ASA), hydroxyproline, phosphoethanolamine (PEA), sarcosine (SARC), taurine (TAU), carnosine (CARN), citrulline (CIT), anserine (ANS), 1,3-methylhistidine (ME-HIS), α-aminoadipic acid (AAA), β-alanine (BALA), ethanolamine (ETN), γ-aminobutyric acid (GABA), β-aminoisobutyric acid (BAIA), α-aminobutyric acid (BABA), L-allothionine (allothionine-A; CYSTA-A), L-thionine (thionine-B; CYSTA-B), cystine, alloisoleucine (ALLO-ILE), DL-hydroxylysine (hydroxylysine (I)), DL-allohydroxylysine (hydroxylysine (2)), ornithine (ORN), homocystine (HCY) and their derivatives. Together with the embodiments described herein, amino acids can be covalently linked to other parts of the conjugates described herein through their α-amino and carboxyl functional groups (i.e., in a peptide bond configuration) or through their side chain functional groups (such as the side chain carboxyl group in glutamic acid) and their α-amino or carboxyl functional groups. It should be understood that when amino acids are used together with the conjugates described herein, they can exist as zwitterions in the conjugates in which they are incorporated.

[0244] As used herein, "prodrug" refers to a compound that can be administered to a subject in a pharmacologically inactive form and then can be converted to a pharmacologically active form through normal metabolic processes (such as the hydrolysis of oxazolidine). It should be understood that the metabolic processes by which a prodrug is converted to an active drug include (but are not limited to) one or more spontaneous chemical reactions, enzyme-catalyzed chemical reactions, and / or other metabolic chemical reactions or combinations thereof. It should be realized that a variety of metabolic processes are known in the art, and the metabolic processes by which the prodrugs described herein are converted to active drugs are non-limiting. A prodrug can be a precursor compound of a drug that has a therapeutic effect on a subject.

[0245] As used herein, the term "therapeutically effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response (including, but not limited to, alleviation of the symptoms of the disease or disorder being treated) in a subject (i.e., a tissue system, animal, or human) that is sought by a researcher, veterinarian, physician, or other clinician. In one aspect, a therapeutically effective amount is the amount of an active substance that can be administered at a reasonable benefit / risk ratio applicable to any medical treatment to treat or alleviate a disease or the symptoms of a disease. In another aspect, a therapeutically effective amount is the amount of a prodrug that, when converted by normal metabolic processes, yields an amount of an active drug capable of eliciting the sought-after biological or pharmaceutical response in a subject.

[0246] It should also be appreciated that, whether referring to a single treatment or a combination treatment, the dosage is advantageously selected with reference to any toxicity or other undesirable side effects that may occur during administration of one or more of the conjugates described herein. Further, it should be appreciated that the co-therapies described herein may enable the administration of lower doses of conjugates that exhibit such toxicity or other undesirable side effects, where those lower doses are below the toxicity threshold or within the therapeutic window and below the doses administered in the absence of co-therapy.

[0247] "Administering" as used herein includes all manners of introducing the conjugates and compositions described herein into a host animal, including, but not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, etc. The conjugates and compositions described herein may be administered in unit dosage forms and / or formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and / or vehicles.

[0248] "Pharmaceutical composition" or "composition" as used herein refers to a mixture of one or more of the conjugates described herein or pharmaceutically acceptable salts, solvates, hydrates thereof with other chemical components, such as pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a conjugate to a subject. Pharmaceutical compositions suitable for delivering the conjugates and methods of preparing the same will be readily apparent to those skilled in the art. Such compositions and methods of preparation can be found, for example, in "Remington's Pharmaceutical Sciences", 19th Edition (Mack Publishing Company, 1995).

[0249] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0250] 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 to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0251] Detailed description

[0252] In accordance with the applicant's disclosure described herein, it is contemplated to combine the embodiments of the numbered clauses provided in the above summary or any combination thereof with any of the embodiments described in the detailed description portion of this patent application.

[0253] In each of the foregoing and each of the following embodiments, it should be understood that the chemical formula includes and represents not only all pharmaceutically acceptable salts of the conjugate, but also any and all hydrates and / or solvates of the conjugate chemical formula. It should be appreciated that certain functional groups (such as hydroxyl, amino, and similar groups) form complexes and / or coordination conjugates with water and / or various solvents in various physical forms of the conjugate. Accordingly, the above formula should be understood to include and represent those various hydrates and / or solvates. It should also be understood that the non-hydrate and / or non-solvate of the conjugate chemical formula is described by such chemical formula as well as the hydrates and / or solvates of the conjugate chemical formula.

[0254] In some embodiments, the present disclosure provides a conjugate of formula B-L-A, wherein B is a binding ligand for carbonic anhydrase IX, L is an optional linker, and A is a therapeutic agent and an imaging agent.

[0255] It should be appreciated that the CA IX ligands that can be used in conjunction with the present disclosure are not particularly limited in structure. Useful CAIX inhibitors can be any drug or compound that exhibits binding affinity for CAIX, such as CA IX inhibitors, CA IX agonists, or CA IX antagonists. In some aspects of these embodiments, the CAIX ligand is a compound containing an arylsulfonamide. In some aspects of these embodiments, the CAIX ligand has the following formula:

[0256]

[0257] wherein

[0258] each R A is independently selected from H, halogen, -OR 1 , -OC(O)R 1 , -OC(O)NR 1 R 2 , -OS(O)R 1 , -OS(O)2R 1 , -SR 1 , -S(O)R 1、 -S(O)2R 1 、 -S(O)NR 1 R 2 、 -S(O)2NR 1 R 2 、 -OS(O)NR 1 R 2 、 -OS(O)2NR 1 R 2 、 -NR 1 R 2 、 -NR 1 C(O)R 1 、 -NR 1 C(O)OR 2 、 -NR 1 C(O)NR 1 R 2 、 -NR 1 S(O)R 2 、 -NR 1 S(O)2R 2 、 -NR 1 S(O)NR 1 R 2 、 -NR 1 S(O)2NR 1 R 2 、 -C(O)R 1 、 -C(O)OR 1 and -C(O)NR 1 R 2 ;

[0259] R B is -OR 3 、 -SR 3 、 -NR 3 R 4 、 -S(O)2R 3 、 -NR 4 C(O)R 3 or -NR 4 C(O)NR 3 R 4 ;

[0260] R 3 is independently C1-C 10 alkyl, C2-C 10 alkenyl or phenyl, each independently substituted with a substituent selected from: -NR 5 -*, -NR 5 (CH2) m2 N(R 6 )-*, -OC(O)-*, -OC(O)N(R 5)-*, -C(O)-*, -C(O)O-* and -C(O)N(R 5 )-*;

[0261] Each R 1 , R 2 , R 4 , R 5 and R 6 is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl and C3-C9 cycloalkyl;

[0262] L is an optional linker;

[0263] A is a therapeutic agent or imaging agent;

[0264] m is an integer from 1 to 5; and

[0265] * represents the point of attachment to the remainder of the conjugate.

[0266] In some embodiments, each R A is selected from halogen, -S(O)2NR 1 R 2 and -NR 1 R 2 . In some embodiments, one R A is -S(O)2NR 1 R 2 . In some embodiments, one R A is -S(O)2NR 1 R 2 , where R 1 and R 2 are H. In some embodiments, one R A is -NR 1 R 2 . In some embodiments, one R A is -NR 1 R 2 , where R 1 is H, and R 2 is C3-C9 cycloalkyl. In some embodiments, one R A is -NR 1 R 2 , where R 1 is H, and R 2 is C3-C9 cyclooctyl. In some embodiments, one R A is -S(O)2NR 1 R 2 , and one R A is -NR 1 R 2 . In some embodiments, one RA is -S(O)2NR 1 R 2 wherein R 1 and R 2 are H, and one R A is -NR 1 R 2 wherein R 1 is H, and R 2 is C3-C9 cycloalkyl. In some embodiments, one R A is -S(O)2NR 1 R 2 wherein R 1 and R 2 are H, and one R A is -NR 1 R 2 wherein R 1 is H, and R 2 is cyclooctyl. In some embodiments, the CA IX ligand has the formula:

[0267]

[0268] where each R A , R B , R 1 and R 2 is as defined herein. In some embodiments, R 1 and R 2 are H.

[0269] In some embodiments, the CA IX ligand has the formula:

[0270]

[0271] where R B , R 1 and R 2 is as defined herein. In some embodiments, R 1 is H, and R 2 is cyclooctyl.

[0272] In some embodiments, R B is -S(O)2R 3 . In some embodiments, R B is -S(O)2R 3 , and one R A is -S(O)2NR 1 R 2 . In some embodiments, R B is -S(O)2R 3 , one R A-S(O)2NR 1 R 2 and an R A is -NR 1 R 2 In some embodiments, R B is -S(O)2R 3 an R A is -S(O)2NR 1 R 2 R 1 wherein R 1 and R 2 are H, and an R A is -NR 1 R 2 wherein R 1 is H, and R 2 is C3-C9 cycloalkyl. In some embodiments, the CA IX ligand has the formula:

[0273]

[0274] where each R A , R 1 , R 2 and R 3 is as defined herein.

[0275] In some embodiments, the CA IX ligand has the formula:

[0276]

[0277] where R 1 , R 2 and R 3 is as defined herein. In some embodiments, the CA IX ligand has the formula:

[0278]

[0279] where R 3 is as defined herein.

[0280] In some embodiments, R 3 is independently C1-C 10 alkyl, C2-C 10 alkenyl or phenyl, each independently substituted with a substituent selected from: -NR 5 -*, -NR 5 (CH2) m2 N(R 6 )-*, -OC(O)-*, -OC(O)N(R 5 )-*, -C(O)-*,-C(O)O-* and -C(O)N(R5 )-*, and C1-C 10 alkyl, C2-C 10 Each of the remaining hydrogen atoms in alkenyl or phenyl is independently optionally substituted with: C1-C 10 alkyl, C2-C 10 alkenyl, halogen, -OR 1 , -OC(O)R 1 , -OC(O)NR 1 R 2 , -OS(O)R 1 , -OS(O)2R 1 , -SR 1 , -S(O)R 1 , -S(O)2R 1 , -S(O)NR 1 R 2 , -S(O)2NR 1 R 2 , -OS(O)NR 1 R 2 , -OS(O)2NR 1 R 2 , -NR 1 R 2 , -NR 1 C(O)R 1 , -NR 1 C(O)OR 2 , -NR 1 C(O)NR 1 R 2 , -NR 1 S(O)R 2 , -NR 1 S(O)2R 2 , -NR 1 S(O)NR 1 R 2 , -NR 1 S(O)2NR 1 R 2 , -C(O)R 1 , -C(O)OR 1 and -C(O)NR 1 R 2 . In some embodiments, R 3 is C1-C substituted with a substituent selected from the following 10 alkyl: -C(O)N(R 5 ), and -C(O)-*, where R 5 is as defined herein, and * represents the point of attachment to the remainder of the conjugate.

[0281] It should be appreciated that the linker that can be used in conjunction with the present disclosure is not particularly limited in structure. The linker can be any linker having a length of about 2 to about 100 atoms and is composed of elements including C, N, O, and S that covalently link the CA IX ligand to the reagent. In some embodiments, the linker can be any linker having a length of 10 to 75 atoms along the length of the linker atom chain. In some embodiments, the linker can be any linker having a length of 15 to 60 atoms along the length of the linker atom chain. It should be appreciated that the ranges provided herein for linker length in terms of the number of atoms in the chain can include any boundary number between 2 and 100 (and including 2 and 100), such as 5, 10, 15, 20, 25, 35, 45, 60, 75, 80, and 100. In some embodiments, the linker can be any linker having a length of - about of any linker. In some embodiments, the linker can be any linker having a length of - about of any linker. In some embodiments, the linker can be any linker having a length of - about of any linker. In some embodiments, the linker can be any linker having a length of - about of any linker. It should be appreciated that the ranges provided herein for linker length can include any boundary number between 5 and 100 (and including 5 and 100).

[0282] In some embodiments, the linker comprises a cleavable linker, wherein the term "cleavable linker" refers to a linker that contains at least one bond that can be cleaved under physiological conditions (such as a pH-labile, acid-labile, base-labile, oxidation-labile, metabolism-labile, biochemical-labile, or enzyme-labile bond). It should be appreciated that such physiological conditions that cause bond cleavage do not necessarily include biological or metabolic processes, but can include standard chemical reactions, such as hydrolysis reactions, for example, at physiological pH or due to compartmentalization into organelles (such as endosomes with a pH lower than the cytoplasmic pH). In some embodiments, the cleavable linker contains a disulfide bond. In some embodiments, the linker contains a disulfide linker moiety. In some embodiments, the linker contains a hydrazide linker moiety.

[0283] In some embodiments, the cleavable linker contains a moiety having the following formula:

[0284]

[0285]

[0286] where each R 7 and R 8Independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, wherein each hydrogen atom in the C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl is independently optionally substituted with: halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -OR 9 、-OC(O)R 9 、-OC(O)NR 9 R 10 、-OS(O)R 9 、-OS(O)2R 9 、-SR 9 、-S(O)R 9 、-S(O)2R 9 、-S(O)NR 9 R 10 、-S(O)2NR 9 R 10 、-OS(O)NR 9 R 10 、-OS(O)2NR 9 R 10 、-NR 9 R 10 、-NR 9 C(O)R 10 、-NR 9 C(O)OR 10 、-NR 9 C(O)NR 11 R 12 、-NR 9 S(O)R 10 、-NR 9 S(O)2R 10 、-NR 9 S(O)NR 11 R 12 、-NR 9 S(O)2NR 11 R 12 、-C(O)R 9 、-C(O)OR 9 or -C(O)NR 9 R 10 ;

[0287] Each X is independently C1-C6 alkyl or C6-C 10 aryl-(C1-C6 alkyl), wherein the C1-C6 alkyl and C6-C 10Each hydrogen atom in aryl-(C1-C6 alkyl) is independently and optionally substituted with: halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -OR 9 、-OC(O)R 9 、-OC(O)NR 9 R 10 、-OS(O)R 9 、-OS(O)2R 9 、-SR 9 、-S(O)R 9 、-S(O)2R 9 、-S(O)NR 9 R 10 、-S(O)2NR 9 R 10 、-OS(O)NR 9 R 10 、-OS(O)2NR 9 R 10 、-NR 9 R 10 、-NR 9 C(O)R 10 、-NR 9 C(O)OR 10 、-NR 9 C(O)NR 11 R 12 、-NR 9 S(O)R 10 、-NR 9 S(O)2R 10 、-NR 9 S(O)NR 11 R 12 、-NR 9 S(O)2NR 11 R 12 、-C(O)R 9 、-C(O)OR 9 or -C(O)NR 9 R 10 ;

[0288] Each R 9 、R 10 、R 11 and R 12 is independently selected from H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl and 5- to 7-membered heteroaryl; and

[0289] Each * represents a covalent bond to the remainder of the conjugate. In some embodiments, each R 7 and R 8 is H. In some embodiments, X is a C1-C6 alkyl group.

[0290] In some embodiments, the linker comprises a moiety of the formula:

[0291]

[0292] wherein R 7 and R 8 are each independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl, wherein each hydrogen atom in the C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl is independently optionally substituted with: halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -OR 9 , -OC(O)R 9 , -OC(O)NR 9 R 10 , -OS(O)R 9 , -OS(O)2R 9 , -SR 9 , -S(O)R 9 , -S(O)2R 9 , -S(O)NR 9 R 10 , -S(O)2NR 9 R 10 , -OS(O)NR 9 R 10 , -OS(O)2NR 9 R 10 , -NR 9 R 10 , -NR 9 C(O)R 10 , -NR 9 C(O)OR 10 , -NR 9 C(O)NR 11 R 12 , -NR 9 S(O)R 10 , -NR 9 S(O)2R 10 , -NR 9 S(O)NR 11 R12 、 -NR 9 S(O)2NR 11 R 12 、 -C(O)R 9 、 -C(O)OR 9 or -C(O)NR 9 R 10 ;

[0293] Each X is independently a C1 - C6 alkyl or a C6 - C 10 aryl-(C1 - C6 alkyl), wherein each hydrogen atom in the C1 - C6 alkyl and C6 - C 10 aryl-(C1 - C6 alkyl) is independently optionally substituted with: halogen, C1 - C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C3 - C6 cycloalkyl, 3 - to 7 - membered heterocycloalkyl, C6 - C 10 aryl, 5 - to 7 - membered heteroaryl, -OR 9 、 -OC(O)R 9 、 -OC(O)NR 9 R 10 、 -OS(O)R 9 、 -OS(O)2R 9 、 -SR 9 、 -S(O)R 9 、 -S(O)2R 9 、 -S(O)NR 9 R 10 、 -S(O)2NR 9 R 10 、 -OS(O)NR 9 R 10 、 -OS(O)2NR 9 R 10 、 -NR 9 R 10 、 -NR 9 C(O)R 10 、 -NR 9 C(O)OR 10 、 -NR 9 C(O)NR 11 R 12 、 -NR 9 S(O)R 10 、 -NR 9 S(O)2R 10 、 -NR 9 S(O)NR 11 R 12 、 -NR 9 S(O)2NR 11 R 12 、 -C(O)R9 、 -C(O)OR 9 or -C(O)NR 9 R 10 ;

[0294] Each R 9 、R 10 、R 11 and R 12 is independently selected from H, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl and 5- to 7-membered heteroaryl; and

[0295] each * represents a covalent bond to the remainder of the conjugate). In some embodiments, each R 7 and R 8 is H. In some embodiments, X is C1-C6 alkyl.

[0296] In some embodiments, the linker may comprise one or more spacer moieties. It should be appreciated that the structure of the spacer moieties included in the conjugates of the present disclosure is not particularly limited structurally. In some embodiments, the linker comprises simple groups such as an alkyl chain moiety, an ether moiety (e.g., PEG), a long-chain amine moiety, an amino acid chain moiety, a hydrazine moiety, etc. and combinations thereof. In some embodiments, linkers that can be used in conjunction with the present disclosure comprise at least one moiety selected from the following: -C(O)(C 1- C 12 alkyl)C(O)-, -NH-C1-C 12 alkyl-NH-, -N(C1-C6 alkyl)-C1-C 12 alkyl-N(C1-C6 alkyl)-, -C(O)CH2CH2(OCH2CH2) q NH-, -C(O)CH2CH2(OCH2CH2) q N(C1-C6 alkyl)-, -(CH2CH2O) q CH2CH2C(O)-, -NH(CH2CH2O) q CH2CH2C(O)- and -N(C1-C6 alkyl)(CH2CH2O) qCH2CH2C(O)-; wherein q is an integer from 1 to 40. In some embodiments, q is 2. In some embodiments, q is 4. In some embodiments, q is 6. In some embodiments, q is 20. It should be appreciated that the value of q is not particularly limited and can be any value between 1 and about 40. In some embodiments, the linker may comprise an amino acid chain. In some embodiments, the linker may comprise a dipeptide, tripeptide, tetrapeptide, pentapeptide, or hexapeptide. In some embodiments, the linker may comprise naturally occurring amino acids. In some embodiments, the linker may comprise non-natural amino acids. In some embodiments, the linker comprises at least one amino acid selected from the following: 3-aminopropionic acid, aspartic acid, cysteine, and arginine. In some embodiments, the linker comprises a moiety having the amino acid sequence 3-aminopropionic acid-Asp-Cys. In some embodiments, the linker comprises a moiety having the amino acid sequence Asp-Arg-Asp-3-aminopropionic acid-Asp-Cys.

[0297] In some embodiments, the linker comprises a portion of the conjugate that comprises a moiety having the formula:

[0298]

[0299] where * represents a covalent bond to the remainder of the conjugate.

[0300] The reagent used in conjunction with any conjugate described herein can be any molecule capable of modulating or otherwise altering cell function (including pharmaceutically active compounds (e.g., therapeutic agents)), or any molecule capable of providing a measurable signal for imaging or visualizing cells or tissues (e.g., imaging agents).

[0301] Suitable molecules that can be used as therapeutic agents include (but are not limited to) peptides, oligopeptides, reverse oligopeptides, proteins, protein analogs in which at least one non-peptide bond replaces a peptide bond, apolipoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins; antigens and their antibodies; haptens and their antibodies; hormones, lipids, phospholipids, liposomes; cytotoxic agents such as microtubule inhibitors; antibiotics; analgesics; bronchodilators; beta blockers; antimicrobials; antihypertensives; cardiovascular drugs, including antiarrhythmics, cardiac glycosides, antianginals, and vasodilators; central nervous system drugs, including stimulants, psychotropic drugs, antimanic drugs, and depressants; antiviral drugs; antihistamines; cancer drugs, including chemotherapeutic agents; sedatives; antidepressants; H-2 antagonists; anticonvulsants; antiemetics; prostaglandins and prostaglandin analogs; muscle relaxants; anti-inflammatory substances; stimulants; decongestants; antiemetics; diuretics; antispasmodics; antiasthmatics; antiparkinson drugs; expectorants; antitussives; mucolytics; and mineral and nutritional supplements.

[0302] In some embodiments, the therapeutic agent can be tubulin. Native tubulin is typically a linear tetrapeptide composed of N-methylpipecolic acid (Mep), isoleucine (Ile), an unnatural amino acid called tubuvaline (Tuv), and an unnatural amino acid called tubutyrosine (Tut, an analogue of tyrosine) or an unnatural amino acid called tubuphenylalanine (Tup, an analogue of phenylalanine).

[0303] In some embodiments, the therapeutic agent is a tetrapeptide of the formula:

[0304]

[0305] wherein R 1a , R 3a , R 3a’ and R 3a” are each independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, and C 3- C6 cycloalkyl, wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, and C 3- C6 cycloalkyl is independently optionally substituted with: halogen, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, C 3- C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -OR 13a , -OC(O)R 13a , -OC(O)NR 13a R 13a’ , -OS(O)R 13a , -OS(O)2R 13a , -SR 13a , -SC(O)R 13a , -S(O)R 13a , -S(O)2R 13a , -S(O)2OR 13a , -S(O)NR 13a R 13a’ , -S(O)2NR 13a R 13a’ , -OS(O)NR 13a R 13a’ , -OS(O)2NR 13a R 13a’ , -NR 13a R 13a’ , -NR13a C(O)R 14a 、-NR 13a C(O)OR 14a 、-NR 13a C(O)NR 14a R 14a’ 、-NR 13a S(O)R 14a 、-NR 13a S(O)2R 14a 、-NR 13a S(O)NR 13a R 14a’ 、-NR 13a S(O)2NR 14a R 14a’ 、-P(O)(OR 13a )2. -C(O)R 13a 、-C(O)OR 13a or -C(O)NR 13a R 13a’ ;

[0306] R 2a , R 4a and R 12a Each independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl;

[0307] R 5a and R 6a Each is independently selected from H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, -OR 15a 、-SR 15a and-NR 15a R 15a’ , wherein C1-C6 alkyl, C2-C6 alkenyl and C 2- Each hydrogen atom in the C6 alkynyl group is independently optionally substituted by halogen, -OR 16a 、-SR 16a 、-NR 16a R 16a’ 、-C(O)R 16a 、-C(O)OR 16a or -C(O)NR 16a R 16a’ ; or R 5a and R 6a Together with the carbon atom to which they are attached, they form -C(O)-;

[0308] Each R 7a , R 8a , R 9a , R10a and R 11a are independently selected from H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, -CN, -NO2, -NCO, -OR 17a , -SR 17a , -S(O)2OR 17a , -NR 17a R 17a’ , -P(O)(OR 17a )2, -C(O)R 17a , -C(O)OR 17a and -C(O)NR 17a R 17a’ , wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl and C 2- C6 alkynyl is independently optionally substituted by: halogen, -OR 18a , -SR 18a , -NR 18a R 18a’ , -C(O)R 18a , -C(O)OR 18a or -C(O)NR 18a R 18a’ ;

[0309] Each R 13a , R 13a’ , R 14a , R 14a’ , R 15a , R 15a’ , R 16a , R 16a’ , R 17a and R 17a’ are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, C 3- C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl and 5- to 7-membered heteroaryl, wherein each hydrogen atom in C1-C7 alkyl, C2-C7 alkenyl, C 2- C7 alkynyl, C 3- C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C 10 aryl or 5- to 7-membered heteroaryl is independently optionally substituted by: halogen, -OH, -SH, -NH2 or -CO2H;

[0310] Each R 18a and R 18a’ are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, C 3-C6 cycloalkyl, 3- to 7-membered heteroalkyl, C6-C 10 aryl, 5- to 7-membered heteroaryl, -C(O)R 19a , -P(O)(OR 19a )2 and -S(O)2OR 19a , each R 19a independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C 2- C6 alkynyl, C 3- C6 cycloalkyl, 3- to 7-membered heteroalkyl, C6-C 10 aryl and 5- to 7-membered heteroaryl;

[0311] a is 1, 2 or 3; and

[0312] * represents a covalent bond to the remainder of the conjugate.

[0313] In some embodiments, the therapeutic agent has the following formula:

[0314]

[0315] wherein R 1a , R 2a , R 3a , R 3a’ , R 3a” , R 4a , R 5a , R 7a , R 8a , R 9a , R 10a , R 11a and R 12a are as defined herein, and * represents a covalent bond to the remainder of the conjugate.

[0316] In another embodiment, the therapeutic agent can be a naturally occurring tubulin or an analogue or derivative thereof of the following general formula:

[0317]

[0318] wherein R 9a and R 13a are as defined herein, and * represents a covalent bond to the remainder of the conjugate.

[0319] Conjugates of each of the foregoing tubulins are described herein.

[0320] In some embodiments, the therapeutic agent can be a naturally occurring tubulin of the following general formula:

[0321]

[0322] Factor <![CDATA[R 13a > <![CDATA[R 9a > A <![CDATA[(CH3)2CHCH2]]> OH B <![CDATA[CH3(CH2)2]]> OH C <![CDATA[CH3CH2]]> OH D <![CDATA[(CH3)2CHCH2]]> H E <![CDATA[CH3(CH2)2]]> H F <![CDATA[CH2CH3]]> H G <![CDATA[(CH3)2C=CH]]> OH H <![CDATA[CH3]]> H I <![CDATA[CH3]]> OH

[0323] The * and the & represent covalent bonds to the remainder of the conjugate.

[0324] In some embodiments, the therapeutic agent is a maytansinoid, including maytansinol and maytansinol analogs. Maytansinoids are compounds that inhibit microtubule formation and are highly toxic to mammalian cells. Examples of suitable maytansinol analogs include those having a modified aromatic ring and those having modifications at other positions. Such maytansinoids are described, for example, in U.S. Pat. Nos. 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,322,348, 4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,424,219, 4,371,533, 4,450,254, 5,475,092, 5,585,499, 5,846,545, and 6,333,410.

[0325] Examples of maytansinol analogs having a modified aromatic ring include (but are not limited to) C-19-dechloro (U.S. Pat. No. 4,256,746), C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016), and C-20-demethoxy, C-20-acyloxy (-OCOR), + / - dechloro (U.S. Pat. No. 4,294,757).

[0326] Examples of maytansinol analogs having modifications at positions other than the aromatic ring include (but are not limited to) C-9-SH (U.S. Pat. No. 4,424,219), C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No. 4,450,254), C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866), C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929), C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348), and 4,5-deoxy (U.S. Pat. No. 4,371,533).

[0327] In some embodiments, the conjugate comprises the maytansinoid DM1 containing a thiol, also known as N 2’ -deacetyl-N 2’ -(3-mercapto-1-oxopropyl)-maytansine as the cytotoxic agent. The structure of DM1 is represented by the following formula (I):

[0328]

[0329] In some embodiments, the conjugate comprises the thiol-containing maytansine alkaloid DM4, also known as N 2’ -deacetyl-N 2’ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine as the cytotoxic agent. The structure of DM4 is represented by the following formula (II):

[0330]

[0331] Other maytansines can be used in conjunction with the present disclosure, such as maytansine alkaloids containing thiols and disulfides with mono- or dialkyl substitutions on the carbon atom bearing the sulfur atom. Particularly preferred are maytansine alkaloids having (a) a C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl functional group at the C-3 position, and (b) an acylated amino acid side chain having an acyl group with a hindered mercapto group, wherein the acyl carbon atom bearing the thiol functional group has one or two substituents, said substituents being CH3, C2H5, a linear or branched alkyl or alkenyl having 1-10 carbon atoms, a cyclic alkyl or alkenyl having 3-10 carbon atoms, phenyl, substituted phenyl or a heteroaryl or heterocyclic alkyl, and further wherein one of the substituents may be H, and wherein the acyl group has a linear chain length of at least 3 carbon atoms between the carbonyl functional group and the sulfur atom.

[0332] Additional maytansines for use in the context of the present invention include the compounds represented by the following formula (III):

[0333]

[0334] wherein

[0335] Y’ represents (CR 1b R 2b ) p (CR 3b =CR 4b ) p1 C=C p2 A p3 (CR 5b P 6b ) p4 D p5 (CR 7b =CR 8b ) p6 (C=C) p7 B p8 (CR 9b R 10b ) p9 CR 11b R 12b SZ,

[0336] A, B, and D are each independently a C3-C9-cycloalkyl or C3-C9-cycloalkenyl, C6-C 10 aryl, 5- to 7-membered heteroaryl or 5- to 7-membered heteroalicyclic group,

[0337] wherein R 1b , R 2b , R 3b , R 4b , R 5b , R 6b , R 7b , R 8b , R 9b , R 10b , R 11b and R 12b each independently is H, C1-C6 alkyl, C1-C6 alkenyl, C6-C 10 aryl, or 5- to 7-membered heteroaryl or 5- to 7-membered heteroalicyclic group, wherein p1, p2, p3, p4, p5, p6, p7, p8, and p9 are each independently zero or an integer from 1 to 5, provided that at least two of p1, p2, p3, p4, p5, p6, p7, p8, and p9 are not zero at any one time.

[0338] Suitable molecules that can be used as imaging agents include (but are not limited to) dyes (such as fluorescein dyes, rhodamine dyes, near-infrared dyes) and SPECT imaging agents (such as any radionucleichelator known in the art). Examples of rhodamine dyes include (but are not limited to) 5-carboxytetramethylrhodamine (5-TAMRA), rhodamine B, rhodamine 6G, TRITC, Texas Red, rhodamine 123, sulforhodamine 101, etc. Examples of fluorescein dyes include (but are not limited to) fluorescein, 5-aminofluorescein, 6-aminofluorescein, fluorescein isothiocyanate (FITC), fluorescein-5-maleimide, NHS-fluorescein, Oregon Green, Tokyo Green, Singapore Green, Philadelphia Green, etc. Examples of near-infrared dyes include S-0456. Examples of radionucleichelators include (but are not limited to) those described in WO03 / 092742.

[0339] In some embodiments, A is a fluorescein dye of the following formula:

[0340]

[0341] where * represents a covalent bond to the rest of the conjugate.

[0342] In other embodiments, suitable imaging agents described herein include (but are not limited to) PET or SPECT imaging agents, such as radiopharmaceuticals comprising a radioisotope (also referred to as a radionuclide) of a metal coordinated to a chelating group. Illustrative radio-metals include isotopes of technetium, rhenium, gallium, gadolinium, indium, copper, etc. In some embodiments, suitable radionuclides include (but are not limited to) 111 In,[[]] 99m Tc,[[]] 64 Cu,[[]] 67 Cu,[[]] 67 Ga,[[]] 68 Ga, etc. Illustrative chelating groups that can be used in conjunction with the present disclosure include (but are not limited to) the following groups: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl) glutaric acid (DOTAGA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazanonan-1-yl) glutaric acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA), 2,2’,2”-(3,6,9-triaza-1-(2,6)-pyridinacyclodecaphane-3,6,9-triyl) triacetic acid (PCTA), 2,2’-((2-(4,7-bis(carboxymethyl)-1,4,7-triazanonan-1-yl)ethyl)azanediyl) diacetic acid (NETA), etc. Additional illustrative examples of radionuclide imaging agents are described in U.S. Patent No. 9,193,763, the disclosure of which is incorporated herein by reference.

[0343] In one embodiment, the methods described herein can be used as "subjects" in both human clinical medicine and veterinary applications. Thus, a "subject" can be administered a conjugate described herein and can be a human ("patient") or, in the case of veterinary applications, a laboratory, agricultural, domesticated, or wild animal. On the one hand, the subject can be a human patient, an experimental animal (such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee), a domestic animal (such as a dog, cat, and rabbit), an agricultural animal (such as a cow, horse, pig, sheep, goat), and a captive wild animal (such as a bear, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, and whale).

[0344] In various embodiments, the cancer described herein can be a tumorigenic population of cancer cells, including benign and malignant tumors, or the cancer can be non-tumorigenic. The cancer can arise spontaneously, or through processes such as mutations present in the patient's germline or somatic mutations, or the cancer can be chemically, virally, or radiation-induced. Cancers suitable for the invention described herein include (but are not limited to) carcinoma, sarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, and myeloma.

[0345] In some aspects, the cancer can be lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, endometrial cancer, leiomyosarcoma, rectal cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, spinal axis tumors, brainstem glioma, pituitary adenoma, cholangiocarcinoma, Hurthle cell thyroid cancer, or gastroesophageal junction adenocarcinoma.

[0346] In some embodiments, the present disclosure relates to targeted NIR imaging, wherein the conjugates of the present disclosure provide selective imaging of cells and tissues that express the CAIX protein. It should be appreciated that there is no particular limitation on the in vitro or in vivo imaging methods used in conjunction with the conjugates of the present disclosure, and any conventional in vitro or in vivo imaging methods known in the art can be used. In addition, any instrument or assay kit known in the art can be used to perform such imaging methods known in the art, including (but not limited to) fluorescence microscopy systems (such as Nikon 90i), in vivo fluorescence imaging systems (such as Caliper IVIS LuminaII imaging workstation (usually coupled to a camera, such as ISOON5160 Andor Nikon camera), etc.

[0347] In some embodiments, the present disclosure provides methods for imaging cell populations or tissues in vitro or in vivo. It should be appreciated that such in vitro methods can be carried out by any method known in the art. In some embodiments, the in vitro imaging methods described herein may include: a. contacting a cell population with a conjugate of the present disclosure to provide a conjugate that binds to cells expressing the CA IX protein; and b. visualizing the conjugate bound to the cells by irradiating with light of a near-infrared wavelength. It should be appreciated that visualizing the conjugate bound to the cells by irradiating with light of a near-infrared wavelength may include irradiating at an excitation wavelength and detecting at an emission wavelength. Thus, in some embodiments, the in vitro imaging methods described herein may include: a. contacting a cell population with a conjugate of the present disclosure to provide a conjugate that binds to cells expressing the CA IX protein, b. irradiating the conjugate bound to the cells expressing the CA IX protein with light of a near-infrared wavelength, and c. detecting the light emitted from the cancer cells at the emission wavelength.

[0348] In some embodiments, tissues (such as cancerous tumors) can be imaged according to the methods described herein. For example, in some embodiments, the in vivo imaging methods described herein may include: a. administering to a patient a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; and b. visualizing the conjugate bound to the cells expressing the CA IX protein by irradiating with light of a near-infrared wavelength. It should be appreciated that visualizing the conjugate bound to the cells by irradiating with light of a near-infrared wavelength may include irradiating at an excitation wavelength and detecting at an emission wavelength. Thus, in some embodiments, the in vivo imaging methods described herein may include: a. administering to a patient a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof to provide a conjugate that binds to cells expressing the CA IX protein; b. irradiating the conjugate bound to the cells expressing the CA IX protein with light of a near-infrared wavelength, and c. detecting the light emitted from the cancer cells at the emission wavelength. It should be appreciated that any known NIR imaging technique (diagnostic or otherwise) or instrument known in the art can be used to visualize the conjugate bound to the cells by irradiating with light of a near-infrared wavelength.

[0349] The wavelength of light used in conjunction with the imaging methods described herein can be in the near-infrared wavelength range, such as in the range of about 600 nm to about 2500 nm. Such wavelength can be a wavelength range or a single wavelength. In some embodiments, the excitation wavelength can be in the range of about 600 nm to about 2500 nm. In some embodiments, the excitation wavelength can be in the range of about 600 nm to about 900 nm. In some embodiments, the excitation wavelength can be in the range of about 700 nm to about 750 nm. In some embodiments, the excitation wavelength can be about 745 nm. In some embodiments, the emission wavelength can be in the range of about 600 nm to about 2500 nm. In some embodiments, the emission wavelength can be in the range of about 750 nm to about 900 nm. In some embodiments, the emission wavelength can be in the range of about 750 nm to about 790 nm. In some embodiments, the emission wavelength can be about 790 nm. In some embodiments, the emission wavelength can be the emission wavelength of ICG (also known as indocyanine green dye).

[0350] In other embodiments of the methods described herein, pharmaceutically acceptable salts of the conjugates described herein are provided. Pharmaceutically acceptable salts of the conjugates described herein include their acid addition salts and base salts.

[0351] Suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, edisylate, esylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hippurate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0352] Suitable base salts of the conjugates described herein are formed from bases that form non-toxic salts. Illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, monoethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulfates and hemicalcium salts.

[0353] Depending on the cancer type, route of administration, and / or whether the conjugate is administered locally or systemically as described herein, a wide range of tolerated doses are contemplated herein, including doses that fall within the range of about 1 μg / kg to about 1 g / kg. In other embodiments, doses that fall within the range of about 0.001 μmol / kg to about 1 μmol / kg may be used. The dose may be single or divided and may be administered according to various regimens, including four times a day, twice a day, three times a day, or even once every other day, once every two weeks (b.i.w.), once a week, four times a week, once a month, once a quarter, etc. In each of these cases, it should be understood that the therapeutically effective amount described herein corresponds to the circumstances of administration or to the total daily, weekly, monthly, or quarterly dose determined by the dosing regimen.

[0354] Any effective regimen for administering the conjugates described herein may be used. For example, the conjugates described herein may be administered as a single dose, or the dose may be divided and administered as a multi-dose daily regimen. Further, an alternating regimen such as 1 - 5 days a week may be used as an alternative to daily treatment, and for the purposes of the methods described herein, such intermittent or alternating daily regimens are considered equivalent to daily treatment and are contemplated. In an illustrative embodiment, a patient is treated with multiple injections of the conjugates described herein to treat cancer. In one embodiment, for example, the conjugates described herein are injected multiple times (preferably from about 2 to up to about 50 times) into a patient at intervals of 12 - 72 hours or at intervals of 48 - 72 hours. Additional injections of the conjugates described herein may be administered to the patient at intervals of days or months after the initial injection, and the additional injections may prevent recurrence of the cancer.

[0355] In one embodiment, the conjugates described herein may be administered as a formulation together with one or more pharmaceutically acceptable carriers. The carrier may be an excipient. The choice of carrier depends to a large extent on various factors, such as the particular route of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form. Medicinal compositions suitable for delivering conjugates such as those described herein and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington: The Science & Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005), which is incorporated herein by reference.

[0356] In an illustrative aspect, pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, and combinations thereof that are physiologically compatible. In some embodiments, the carrier is suitable for parenteral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Supplementary active compounds can also be incorporated into the compositions of the present invention.

[0357] In various embodiments, liquid formulations can include suspensions and solutions. Such formulations can contain a carrier (such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil) and one or more emulsifying agents and / or suspending agents. Liquid formulations can also be prepared by reconstitution of a solid.

[0358] In one embodiment, an aqueous suspension can contain the active material admixed with suitable excipients. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth, and acacia; dispersing or wetting agents that can be natural phospholipids such as lecithin; condensation products of alkylene oxides with fatty acids such as polyoxyethylene stearate; condensation products of ethylene oxide with long chain aliphatic alcohols (such as heptadecaethyleneoxycetanol); condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (such as polyoxyethylene sorbitan monooleate); or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (such as polyoxyethylene sorbitan monooleate). The aqueous suspension can also contain one or more preservatives (such as ascorbic acid, ethyl, n-propyl, or p-hydroxybenzoate); or one or more coloring agents.

[0359] In an illustrative embodiment, dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient admixed with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Additional excipients, such as coloring agents, can also be present.

[0360] In other embodiments, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride can be included in the composition. Prolonged absorption of an injectable composition can be achieved by including substances that delay absorption (such as monostearates and gelatin) in the composition.

[0361] Illustrative forms for oral administration include tablets, capsules, elixirs, syrups, and the like.

[0362] On the one hand, the conjugates described herein can be administered directly into the bloodstream, muscle, or visceral organs. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. Suitable modes for parenteral administration include needle (including micro-needle) syringes, needleless syringes, and infusion techniques.

[0363] In an illustrative aspect, parenteral formulations are generally aqueous solutions, which may contain carriers or excipients, such as salts, carbohydrates, and buffers (preferably at a pH of 3 - 9), but for some applications, they may be more suitably formulated as sterile non-aqueous solutions or in a dry form for use with a suitable vehicle, such as sterile pyrogen-free water. In other embodiments, any of the liquid formulations described herein may be suitable for parenterally administering the conjugates described herein. Preparation of parenteral formulations under sterile conditions, for example by lyophilization under sterile conditions, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. In one embodiment, the solubility of the conjugates described herein for use in the preparation of parenteral formulations can be enhanced by using appropriate formulation techniques, such as incorporating solubility enhancers.

[0364] In various embodiments, formulations for parenteral administration can be formulated for immediate and / or modified release. In an illustrative aspect, the active agent (i.e., the conjugates described herein) of the present invention can be administered in a time-release formulation, for example, in a composition comprising a slow-release polymer. The active agent can be prepared with a carrier that protects the conjugate from rapid release, such as a controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid - polyglycolic acid copolymer (PGLA). Methods for preparing such formulations are generally known to those skilled in the art. In another embodiment, the conjugates described herein or compositions comprising the conjugates can be administered continuously, where appropriate.

[0365] In one embodiment, a kit is provided. If combinations of the active conjugates described herein are to be administered, two or more pharmaceutical compositions can be combined in the form of a kit suitable for sequential or co-administration of the compositions. Such a kit contains two or more separate pharmaceutical compositions (where at least one contains the conjugate described herein), and means for separately holding the compositions (such as containers, separate bottles, or separate foil packages). In another embodiment, a composition comprising one or more of the conjugates described herein is provided in a container having a label that provides instructions for using the conjugate described herein for patient selection and / or treatment.

[0366] In one embodiment, a sterile injectable solution can be prepared by incorporating the required amount of the active agent, alone or in combination (as needed) with one or more of the above ingredients, into a suitable solvent and then filtering the solution under sterile conditions. Generally, dispersions are prepared by incorporating the conjugate into a sterile vehicle that contains a dispersion medium and any additional ingredients of the above type. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredients from their previously sterile-filtered solutions, or these ingredients may be filter-sterilized together.

[0367] The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In one embodiment, the appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0368] The conjugates described herein can contain one or more chiral centers or can otherwise exist as a variety of stereoisomers. Accordingly, it is to be understood that the invention includes pure stereoisomers as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and mixtures enriched in enantiomers or diastereomers. The conjugates described herein may exist as geometric isomers. Accordingly, it is to be understood that the invention includes pure geometric isomers or mixtures of geometric isomers.

[0369] It should be appreciated that the conjugates described herein can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention. The conjugates described herein can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the invention.

[0370] In another embodiment, the conjugates described herein are used to prepare compositions and / or dosage forms for administration of the conjugates described herein, wherein the conjugates have a purity of at least about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 99.5%. In another embodiment, the conjugates described herein are used to prepare compositions and / or dosage forms for administration of the conjugates described herein, wherein the conjugates have a purity of at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%. Examples

[0371] The following data may indicate the therapeutic and radioimaging applications of carbonic anhydrase targeting ligands. The ligand (FBSA, Scheme 1, Compound 3) was developed by Daumantas Matulis, et al. (J. Med. Chem. 2014, 57, 9435 - 9446).

[0372] As described herein, the data indicate that the PEG2 linker length or its variants may contribute to the apparent binding affinity. Some data may also suggest the design of small molecule therapeutic conjugates based on FBSA targeting, such as tubulin conjugates (Conjugate 12 (FBSA - PEG2 - tubulin B) and Conjugate 14 (FBSA - tubulin B conjugate with a hydrophilic linker)).

[0373] Abbreviations The materials used in the examples described herein include (but are not limited to) those described by the following abbreviations known to those skilled in the art:

[0374]

[0375] Example 1

[0376] Synthesis of Carbonic Anhydrase

[0377]

[0378] Scheme 1. Synthesis of FBSA (Compound 3, a high - affinity carbonic anhydrase small molecule targeting ligand).

[0379]

[0380] Synthesis of Pentafluorobenzenesulfonamide 1

[0381] A stirred mixture of pentafluorobenzenesulfonyl chloride (2.60 mL) in tetrahydrofuran was cooled to - 10 °C using an ice / NaCl cooling bath. Then, a 3.5 M ammonia in methanol / ethanol solution (6.00 mL) was added dropwise. Then, the reaction mixture was warmed to room temperature and stirred for 3.5 hours. Then, the solvent was removed in vacuo and the product was recrystallized from water. Yield: 2.7404 g, MP: 153.2 - 154.8 °C (Lit: 156 °C), IR (cm -1 ): 3343.00 (-NH 2, Asym), 3264 (-NH2, Sym).

[0382] Example 2

[0383]

[0384] Synthesis of 3 - ((2,3,5,6 - tetrafluoro - 4 - sulfamoylphenyl)sulfonyl)propanoic acid 2

[0385] Pentafluorobenzenesulfonamide (950.4 mg), triethylamine (1.400 mL), and 3-mercaptopropionic acid (368 μL) were added to a stirred solution of methanol (40 mL). The reaction mixture was then refluxed for 24 h and then dried in vacuo. The resulting residue was then dissolved in a 2:1 mixture of acetic acid and water. The mixture was then heated to 70 °C and 2 mL portions of 30% hydrogen peroxide were added every two hours until the total volume of peroxide added reached 10 mL. The reaction mixture was then stirred at 70 °C for a total of 24 h. The acetic acid was removed in vacuo and the resulting precipitate was filtered and washed with water. Yield: 0.5679 g, LC-MS (M+H2O): 383.0.

[0386] References: Robson, P.; Smith, T. A.; Stephens, R.; Tatlow, J. C., 691. Aromatic polyfluoro-compounds. Part XIII. Derivatives of penta- and 2,3,5,6-tetra-fluorothiophenol. Journal of the Chemical Society (Restored) 1963, (0), 3692 - 3703.

[0387] Example 3

[0388]

[0389] Synthesis of 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid 3.

[0390] Compound 2 (400 mg) was dissolved in DMSO (2 mL) and then cyclooctylamine (2 equiv) was added and the reaction mixture was stirred at room temperature for 24 h. The reaction was quenched with water and concentrated ammonium chloride. The resulting precipitate was extracted with ethyl acetate and dried over sodium sulfate. The extract was then dried over silica and purified by flash chromatography. Yield: 0.1662 g, LC-MS (M+1): 473.2.

[0391] Example 4

[0392]

[0393] Synthesis of (3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propionyl)cysteine 4 Compound 4 was synthesized by the following solid-phase method. H-Cys(Trt)-2-chlorotrityl resin (100 mg, 0.64 mmole / g) was successively swollen with 3 mL of dichloromethane (DCM) and 3 mL of dimethylformamide (DMF). After swelling the resin in DMF, a solution of 3-[2-[2-((9H-fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]propanoic acid (51.1 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (33.3 mg, 0.074 mmole), HATU (24.3 mg, 0.064 mmole), and DIPEA (0.034 ml, 0.192 mmole) in DMF was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Compound 4 was cleaved from the resin using a mixture of trifluoroacetic acid:triisopropylsilane:H2O:ethanedithiol (95:2.5:2.5:2.5) and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude conjugate was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 min] to give the product as a clear oil (54%). LRMS-LC / MS (m / z): C 27 H 41 F3N4O 10 S3 [M+H] + Calcd, 734.19; Found, 735.2.

[0394] Synthesis of 5 - 2 - ((((1 - (3 - carboxy - 4 - (6 - hydroxy - 3 - oxo - 3H - xanthen - 9 - yl)phenyl)-2,5 - dioxopyrrolidin - 3 - yl)thio)methyl)-16 - ((2 - (cyclooctylamino)-3,5,6 - trifluoro - 4 - sulfamoylphenyl)sulfonyl)-4,14 - dioxo - 7,10 - dioxo - 3,13 - diazahexadecanoic acid

[0395] To a solution of compound 4 (5 mg, 0.0068 mmol) in DMF (1 mL) was added fluorescein - 5 - maleimide (3.2 mg, 0.0075 mmol) and N,N - diisopropylethylamine (0.006 mL, 0.034 mmol), and the mixture was stirred at room temperature for 1 hour. The crude reaction mixture was purified by reverse - phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 minutes] to give the product as a yellow powder (61%). LRMS - LC / MS (m / z): C 51 H 54 F3N5O 17 S3 [M + H] + Theoretical value, 1161.26; Found value, 1162.2.

[0396] Example 5

[0397]

[0398] (2R,5S,8S)-8 - Amino - 5 - (carboxymethyl)-29 - ((2 - (cyclooctylamino)-3,5,6 - trifluoro - 4 - sulfamoylphenyl)sulfonyl)-2 - (mercaptomethyl)-4,7,11,27 - tetraoxo - 14,17,20,23 - tetraoxa - 3,6,10,26 - tetraazanonacosanic acid 6 synthesis

[0399] Compound 6 was synthesized by the following solid-phase method. H-Cys(Trt)-2-chlorotrityl resin (100 mg, 0.64 mmole / g) was successively swollen with 3 mL of dichloromethane (DCM) and 3 mL of dimethylformamide (DMF). After swelling the resin in DMF, a solution of Fmoc-Asp(tBu)-OH (52.7 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. The fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of Boc-DAP(Fmoc)-OH (54.6 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. The fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of 3-[2-[2-[2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (62.4 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. The fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (33.3 mg, 0.074 mmole), HATU (24.3 mg, 0.064 mmole), and DIPEA (0.034 ml, 0.192 mmole) in DMF was added.Argon was bubbled in for 2 h, and the resin was washed three times with 3 mL of DMF and three times with 3 mL of i-PrOH. The compound 3 was cleaved from the resin using a mixture of trifluoroacetic acid:triisopropylsilane:H2O:ethanedithiol (95:2.5:2.5:2.5) and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude compound 6 was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 min] to give the product as a clear oil (62%). LRMS-LC / MS (m / z): C. 38 H 60 F3N7O 16 S3 [M+H] + Theoretical value, 1023.32; found, 1024.3.

[0400] Synthesis of 5-(3-(((2R,5S,8S)-8-amino-2-carboxy-5-(carboxymethyl)-29-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)-4,7,11,27-tetraoxo-14,17,20,23-tetraoxa-3,6,10,26-tetraazanonacosanoyl)thio))-2,5-dioxopyrrolidin-1-yl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid 7.

[0401] To a solution of compound 6 (2 mg, 0.002 mmol) in DMF (1 mL) was added fluorescein-5-maleimide (0.85 mg, 0.002 mmol) and N,N-diisopropylethylamine (0.002 mL, 0.01 mmol), and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 min] to give the conjugate 7 as a yellow powder (76%). LRMS-LC / MS (m / z): C 62 H 73 F3N8O 23 S3 [M+H] + Theoretical value, 1450.39; found, 1451.3.

[0402] Example 6

[0403]

[0404] Synthesis of tert-butyl (2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethyl)carbamate 8.

[0405] To a solution of 3 - ((2 - (cyclooctylamino)-3,5,6 - trifluoro - 4 - sulfamoylphenyl)sulfonyl)propanoic acid (20 mg, 0.042 mmol) in DMF (2 ml) was added tert - butyl (2 - aminoethyl)carbamate (7.40 mg, 0.0462 mmol), and the mixture was stirred for 10 minutes at room temperature under argon. Then 1 - [bis(dimethylamino)methylene]-1H - 1,2,3 - triazolo[4,5 - b]pyridinium 3 - oxide hexafluorophosphate (16 mg, 0.042 mmol) and N,N - diisopropylethylamine (0.022 ml, 0.126 mmol) were added. The reaction mixture was stirred for 1 hour, then quenched with water (10 ml) and extracted with ethyl acetate (3 x 25 ml). The organic layers were combined, washed with brine and dried over anhydrous sodium sulfate. The solvent was concentrated in vacuo and the crude mixture was purified on silica gel using 10% methanol / dichloromethane to give compound 8 (21.4 mg, 83%) as a colorless oil. LRMS - LC / MS (m / z): C 24 H 37 for [M + H] 37 + of C

[0406] Example 7

[0407]

[0408] Synthesis of 5 - (3 - (2 - (3 - ((2 - (cyclooctylamino)-3,5,6 - trifluoro - 4 - sulfamoylphenyl)sulfonyl)propionamido)ethyl)thioureido)-2 - (6 - hydroxy - 3 - oxo - 3H - xanthen - 9 - yl)benzoic acid 9.

[0409] The tert - butoxycarbonyl protecting group of compound 8 (10 mg, 0.008 mmol) was removed with a mixture of TFA / DCM (20%, 2 ml) for 30 minutes. The TFA / DCM mixture was removed in vacuo and the product was carried directly into the next step without purification. To the vial containing the deprotected compound 5 was added DMF (1 ml) and N,N - diisopropylethylamine (0.007 ml, 0.04 mmol), and the mixture was stirred for 5 minutes, then fluorescein - 5 - maleimide (3.8 mg, 0.0088 mmol) was added and the mixture was stirred for an additional hour. The crude mixture was purified by preparative reverse - phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 minutes] without further work - up to give the product (76%) as a yellow powder. LRMS - LC / MS (m / z): C 40H 40 F3N5O 10 [M+H] of S3 + Theoretical value, 903.19; measured value, 904.2.

[0410] Example 8

[0411]

[0412] (2R,5S,8S)-8-Amino-5-(carboxymethyl)-23-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)-2-(mercaptomethyl)-4,7,11,21-tetraoxo-14,17-dioxa-3,6,10,20-tetraazatricosanoic acid 10 synthesis.

[0413] Compound 10 was synthesized by the following solid-phase method. H-Cys(Trt)-2-chlorotrityl resin (100 mg, 0.64 mmole / g) was successively swollen with 3 mL of dichloromethane (DCM) and 3 mL of dimethylformamide (DMF). After swelling the resin in DMF, a solution of Fmoc-Asp(tBu)-OH (52.7 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of Boc-DAP(Fmoc)-OH (54.6 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of fmoc-9-amino-4,7-dioxanonanoic acid (51.12 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Fmoc was removed with a piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. A solution of 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (Compound 3, 33.3 mg, 0.074 mmole), HATU (24.3 mg, 0.064 mmole), and DIPEA (0.034 ml, 0.192 mmole) in DMF was added.Argon was bubbled in for 2 h, and the resin was washed three times with 3 mL of DMF and three times with 3 mL of i-PrOH. Compound 10 was cleaved from the resin using a mixture of trifluoroacetic acid:triisopropylsilane:H2O:ethanedithiol (95:2.5:2.5:2.5) and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude conjugate was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: 0% B to 100% B in 30 min] to give the product as a clear oil (62%). LRMS-LC / MS (m / z): C. 34 H 52 F3N7O 14 [M+H] of S3 + Theoretical value, 935.27; found, 935.2. Note that compound 10 can be used as a precursor for conjugate 12 or can also be used as 99m a chelator for Tc and can be used as conjugate 10 for SPECT / CT imaging applications.

[0414] Example 9

[0415]

[0416] (3R,5S,16R,19S)-1-(2-((1R,3R)-1-Acetoxy-3-((2S,3S)-N-((butyryloxy)methyl)-3-methyl-2-(1-methylpiperidine-2-carboxamido)valeramido)-4-methylpentyl)thiazol-4-yl)-19-((S)-16-amino-1-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)-3,13-dioxo-7,10-dioxo-4,14-diazaheptadecane-17-carboxamido)-16-carboxy-3-(4-hydroxybenzyl)-5-methyl-1,6,9,18-tetraoxo-10-oxa-13,14-dithia-2,7,8,17-tetraazapentacosan-21-oic acid 12 synthesis.

[0417] A solution of saturated sodium bicarbonate (10 mL) in distilled water was continuously bubbled with argon for 30 minutes. Compound 9 (1.5 mg, 0.0016 mmol) was dissolved in argon-purged HPLC-grade water (2.0 mL), and the pH of the reaction mixture was increased to 7 using argon-purged sodium bicarbonate. Then a solution of disulfide-activated microtubulysin B (Compound 11, 1.86 mg, 0.018 mmol) supplied by ENDOCYTE in THF (0.5 mL) was added to the reaction mixture. The progress of the reaction was monitored using analytical LCMS, and after stirring for 30 minutes, the reaction was found to be complete. The conjugate 12 was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 minutes] to give the desired product. LRMS-LC / MS (m / z): C 79 H 119 F3N 14 O 25 [M + H] of S5 + Theoretical value, 1881.70; Measured value, 1881.5.

[0418] Example 10

[0419]

[0420] (2R,5S,8S)-8-Amino-5,12-bis(carboxymethyl)-18-(3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propionamido)-15-(3-guanidinopropyl)-2-(mercaptomethyl)-4,7,11,14,17-pentaoxo-3,6,10,13,16-pentaazadocosanedioic acid 13 synthesis.

[0421] Compound 13 was synthesized by the following solid-phase method. H-Cys(Trt)-2-chlorotrityl resin (100 mg, 0.64 mmole / g) was successively swollen with 3 mL of dichloromethane (DCM) and 3 mL of dimethylformamide (DMF). After swelling the resin in DMF, a solution of Fmoc-Asp(tBu)-OH (52.7 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. The fmoc was removed with piperidine solution (20% in DMF, 3x5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. The remaining steps followed the same protocol. Compound 13 was cleaved from the resin using a mixture of trifluoroacetic acid:triisopropylsilane:H2O:ethanedithiol (95:2.5:2.5:2.5) and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude conjugate was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 min] to give the product as a clear oil (62%). LRMS-LC / MS (m / z): C 48 H 74 F3N 13 O 21 [M+H] of S3 + Calculated, 1322.42; Found, 1322.38. Note that compound 13 is not only used as a precursor for conjugate 14, but is also designed to be 99m a chelator for Tc and can be used as conjugate 13 for SPECT / CT imaging applications.

[0422] Example 11

[0423]

[0424] Synthesis of (2R,5S,8S)-2-((11S,13R)-15-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N-((butyryloxy)methyl)-3-methyl-2-(1-methylpiperidine-2-carboxamido)valeramido)-4-methylpentyl)thiazol-4-yl)-13-(4-hydroxybenzyl)-11-methyl-7,10,15-trioxo-6-oxa-2,3-dithia-8,9,14-triazapentadecyl)-8-amino-5,12-bis(carboxymethyl)-18-(3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propionamido)-15-(3-guanidinopropyl)-4,7,11,14,17-pentoxo-3,6,10,13,16-pentaazadocosandioic acid 14

[0425] A solution of saturated sodium bicarbonate (10 mL) in distilled water was continuously bubbled with argon for 30 minutes (min). Compound 13 (1.2 mg, 0.0009 mmol) was dissolved in argon-purged HPLC-grade water (2.0 mL), and the pH of the reaction mixture was increased to 7 using argon-purged sodium bicarbonate. Then a solution of disulfide-activated tubulysin B (Compound 11, 1.27 mg, 0.0012 mmol) from ENDOCYTE in THF (0.5 mL) was added to the reaction mixture. The progress of the reaction was monitored using analytical LCMS, and after stirring for 30 minutes, the reaction was found to be complete. The conjugate 14 was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 minutes] to give the desired product. LRMS-LC / MS (m / z): C 93 H 141 F3N 20 O3S5 [M+H] + Theoretical value, 2267.86; Observed value, 1134.57 (half mass).

[0426] Example 12

[0427]

[0428] Synthesis of ((2S)-2-amino-3-(3-carboxy-2-(2-(3-carboxy-2-(3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propionamido)propionamido-5-guanidinopentanamido)propionamido)propionyl)-L-aspartic acid 15

[0429] Compound 15 was synthesized by the following solid-phase method. H-Cys(Trt)-2-chlorotrityl resin (100 mg, 0.64 mmole / g) was successively swollen with 3 mL of dichloromethane (DCM) and 3 mL of dimethylformamide (DMF). After swelling the resin in DMF, a solution of Boc-DAP(Fmoc)-OH (54.6 mg, 0.128 mmole), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (60.8 mg, 0.16 mmole), and N,N-diisopropylethylamine (0.056 ml, 0.32 mmole) in DMF (3 mL) was added. Argon was bubbled through for 2 h, and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Fmoc was removed with piperidine solution (20% in DMF, 3 x 5 ml), and the resin was washed 3 times with 3 mL of DMF and 3 times with 3 mL of i-PrOH. Other residues were added using the same stoichiometry. Compound 15 was cleaved from the resin using a mixture of trifluoroacetic acid:triisopropylsilane:H2O:ethanedithiol (95:2.5:2.5:2.5) and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude conjugate was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 100% B in 30 min] to give the product as a clear oil (62%). LRMS-LC / MS (m / z): C 45 H 69 F3N 12 O 20 [M+H] of S2 + Theoretical value, 1219.41; found, 1219.09. Note that compound 15 was used as a competitor to conjugate 14 in competitive experiments.

[0430] Example 13

[0431]

[0432] N-(2-Aminoethyl)-3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propanamide (16)

[0433] N-(2-Aminoethyl)-3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propanamide was synthesized by the following solid-phase method: After swelling 1,2-diaminoethane trityl polystyrene resin (100 mg, 1.7 mmol / g) with DMF (3 mL) and DCM (3 mL), a solution of 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecane-13-carboxylic acid (136 mg, 0.34 mmol), HATU (162 mg, 0.43 mmol) and DIPEA (0.09 mL, 0.51 mmol) in DMF (3 mL) was added. The reaction mixture was bubbled with argon for 2 h and the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL). Fmoc was removed with piperidine solution (20% in DMF, 3 x 5 ml) and the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL). A solution of 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (179 mg, 0.425 mmol), HATU (162 mg, 0.43 mmol) and DIPEA (0.09 mL, 0.51 mmol) in DMF was added. Argon was bubbled through for 2 h and the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL). N-(2-Aminoethyl)-3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionamido)ethoxy)ethoxy)propanamide was cleaved from the resin using a TFA:TIPS:H2O:EDT mixture (95:2.5:2.5:2.5) and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude conjugate was purified by reverse-phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 40% B in 30 min] to give the product as a white solid. LRMS-LC / MS (m / z): 674.3 [M+H] + .

[0434] Example 14

[0435]

[0436] 2,2'-(7-(21-carboxy-1-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)-3,13,18-trioxo-7,10-dioxa-4,14,17-triazaheneicosan-21-yl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid to N-(2-aminoethyl)-3-(2-(2-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propionylamino To a solution of (7-(1-carboxy-4-((2,4-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid (NODAGA-NHS, 1.4 mg, 0.009 mmol) and DIPEA (0.0016 mL, 0.034 mmol) in DMF (1 mL) was added and stirred at room temperature for 1 hour. The crude reaction mixture was purified by reverse phase HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = CH3CN, solvent gradient: from 0% B to 20% B in 30 minutes] to give the product (Conjugate 17, yield 83%) as a white powder. LRMS-LC / MS (m / z): 1031.5 [M+H] + .

[0437] Embodiment 15

[0438] use 64 General procedure for Cu radiolabeling of conjugate 17

[0439] Use 0.1N HCl 64 CuCl2 was radiolabeled by mixing it with a 50-fold excess of conjugate 17 in sodium acetate buffer (pH 10.5) and incubating at room temperature for a minimum of 15 minutes before use. To evaluate the radiochemical purity of the chelate, the solution was analyzed using an Agilent 1260 Infinity II equipped with a LabLogic Flow-RAM radio-HPLC detector and a C18 column with Laura Academia software using the following method: solvent A = 0.1% TFA, solvent B = ACN, solvent gradient: from 0% B to 100% B in 15 minutes]. After confirming that the radiochemical purity was >95%, the radiolabeled conjugate was used without purification after dilution into the appropriate solvent (cell culture medium for in vitro experiments) and (PBS, pH 7.4 for in vivo experiments).

[0440] Concentration <100μM Buffer Acetate buffer (0.5 mM, pH 10.5) Temperature Room temperature Time > 10 min

[0441] Table 1: Exemplary Chelation Conditions of FBSA-NODAGA with Copper-64.

[0442] In vitro experiments

[0443] Cell lines and cell culture

[0444] A549, HT29, and SKRC52 cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified 5% CO2 atmosphere.

[0445] Example 16

[0446] Fluorescence microscopy of FBSA-PEG2-FITC conjugate (conjugate 5).

[0447] Metastatic human renal cell carcinoma (SKRC52) cells (10 5 ) were seeded into chambered coverglass plates and allowed to grow to confluence within 24 hours (hr). The used medium was replaced with 0.5 mL of fresh medium, which contained 10% fetal bovine serum albumin and 25 nM concentration of either the FITC conjugate alone (conjugate 5) or the dye conjugate plus 100-fold excess of the CAIX inhibitor (compound 3, 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid). After incubation at 37 °C for 1 hour, the cells were rinsed twice with 1 mL of medium to remove unbound fluorescence, and 0.5 mL of fresh pre-warmed medium was added to the wells. Images were acquired using confocal microscopy (FV 1000, Olympus). The results are shown in Figure 1. FITC conjugate 5 binds to the cells and competes in the presence of an excess of un-conjugated inhibitor, indicating specific receptor-specific binding events.

[0448] Example 17

[0449] General procedure for CAIX FITC conjugate binding affinity assay

[0450] CAIX, SKRC52, HT29, or A549 cells (0.25x10 6Transfected HEK293 cells were placed into 1.5 ml centrifuge tubes containing 0.3 mL of fresh DMEM medium, and the medium contained increasing concentrations of the CAIX FITC conjugate. After incubation at 25 °C for 1 hour, the cells were rinsed twice with 1 mL of medium. The cells were transferred to a 96-well plate, and the mean fluorescence intensity (MFI) was read using flow cytometry (BD Accuri C6, BD Biosciences). The apparent K was calculated by plotting the MFI against the concentration of the FITC conjugate using GraphPad Prism 4. D For the hypoxia experiment, the cells were incubated in an hypoxia chamber for 24 - 48 hours in an atmosphere containing 1% oxygen, 5% carbon dioxide, and 94% nitrogen. The results of the binding affinity for CAIX FITC are shown in Table 2.

[0451]

[0452] Table 2. Binding affinity of the CAIX FITC conjugate in HEK293 or SKRC52 transfected with CAIX.

[0453] Example 18

[0454] General procedure for determining in vitro cytotoxicity by 3 H-thymidine uptake assay

[0455] HT-29 cells were seeded onto amine-coated 24-well plates and allowed to form a monolayer. The used medium in each well was replaced with fresh medium (0.5 mL) containing various concentrations of conjugate 12. After incubation at 37 °C for 2 hours, the cells were rinsed 3 times with fresh medium and then incubated for an additional 66 hours in fresh medium at 37 °C. The used medium in each well was replaced again with fresh medium (0.5 mL) containing 3 H-thymidine (1 μCi / ml), and the cells were incubated for an additional 4 hours. After the cells were washed 3 times with medium, they were lysed in 0.5 mL of 0.25 M NaOH. Then, the incorporation of thymidine was determined by counting the cell-associated radioactivity using a scintillation counter (PACKARD, PACKARD INSTRUMENT COMPANY). The IC was obtained from a plot of the percentage of 3 H-thymidine incorporation against the log concentration using Graph Pad Prism 4 and TABLECURVE 2D software. 50 value. The results are shown in Figure 8 and indicate that the IC of the conjugate is 5.045 nM. 50

[0456] Example 19

[0457] In vitro binding affinity of the radiolabeled conjugate 17

[0458] SKRC52, HT29, and A549 cells (~0.25x10 6 ) were seeded into 24-well plates and allowed to grow overnight. Fresh medium containing increasing concentrations of the radiolabeled conjugate 17 prepared as in Example 17 was added in the presence or absence of a 100-fold excess of the FBSA competitor compound. All concentrations were examined in duplicate or triplicate. After incubation at 25 °C for 1 hour, the cells were washed with PBS (3 x 0.5 mL). 0.5 M NaOH was added to each well. After 10 minutes, 0.45 mL of the NaOH was removed and the radioactivity was measured using a gamma counter (Packard, Packard Instrument Company). The apparent binding affinity was calculated by plotting the bound radioactivity against the concentration of the radiolabeled conjugate using GraphPad Prism4.

[0459] As Figure 9 and 10 visible, the apparent binding affinity of the radiolabeled 17 was established as low nM values using SKRC52 and A549 cells (K D = 5.87 nM in SKRC52, and K D = 6.04 nM in A549), and saturation was reached at approximately 75 nM. Competitive controls (cells treated with increasing concentrations of the radiolabeled conjugate 17 and a 100-fold excess of the competing ligand (unlabeled FBSA-COOH)) showed significantly lower bound radioactivity. Without being bound by theory, it appears that the binding of the radiolabeled 17 to the cells is a receptor-specific event.

[0460] In vivo experiments

[0461] Animal housing

[0462] Thymusless nu / nu mice were purchased from Harlan Laboratories (ENVIGO), housed in a sterile environment on a standard 12-hour light / dark cycle, and maintained on normal rodent chow. All animal procedures were approved by the PURDUE Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health.

[0463] Example 20

[0464] In vivo efficacy of the tubulin conjugate 12 in HT29 xenograft tumors.

[0465] Cells (HT-29 cells) (4.25×10 6 ) were subcutaneously injected into the shoulders of 5-6 week-old female nu / nu mice. Tumors were measured three times a week in two perpendicular directions using vernier calipers, and their volume was calculated as 0.5 x L x W 2 , where L is the longest axis (in millimeters), and W is the axis perpendicular to L (in millimeters). When the volume of the subcutaneous tumor reached ~100 - 275 mm 3 , administration of conjugate 12 was initiated. The administration solution was prepared in saline and filtered through a 0.22 μm filter. The solution was administered via tail vein injection or intraperitoneally. Each mouse received 2 μmol / kg of conjugate 12 per injection. Injections were given every other day, and the mice were weighed concomitantly. The results are shown as Figures 11 - 12 . As shown as Figure 11 , the treated mice stopped tumor growth, and the xenografts were in the process of shrinking at the time of this disclosure. In all cases, no severe toxicity was observed, and less than 10% weight loss was observed at this dosing level ( Figure 12 ).

[0466] As shown as Figures 13 - 16 , a similar experiment was performed using conjugate 14 instead of conjugate 12. A similar experiment was performed using conjugate 14 on A549 cells instead of HT-29 cells ( Figure 17 and 18 ).

[0467] Example 21

[0468] Whole body imaging and biodistribution of radiolabeled 17 in a murine xenograft model

[0469] SKRC52, HT29 or A549 cells (~3x10 6 cells per mouse) were subcutaneously injected into the shoulders of female athymic nu / nu mice. Tumor growth was measured in two perpendicular directions, and tumor volume was calculated as 0.5 x L x W 2 (L = longest axis, and W = axis perpendicular to L, in millimeters). Once the volume of the tumor reached at least 500 mm 3, the radiolabeled conjugate 17 (250 μCi) in PBS (100 μL, pH 7.4) was injected into the animals via the tail vein. To obtain whole-body images, the mice were anesthetized with isoflurane and images were acquired using a Kodak Imaging Station (In-Vivo FX, Eastman Kodak Company) combined with a CCD camera and Kodak molecular imaging software (version 4.0) with the kidneys shielded. The radioimages were obtained using the following parameters: radioisotope illumination source, 3-minute acquisition time, f-stop = 4, focal plane = 5, FOV = 160, binning = 8. The white light images were obtained using the following parameters: white light transmission illumination source, 0.175 s acquisition time, f-stop = 16, focal plane = 5, FOV = 160, and no binning. After whole-body imaging was acquired, the mice were euthanized by CO2 asphyxiation and the tissues were placed in pre-weighed gamma counting tubes after autopsy. The radioactivity of the tissues and a portion of the injected dose was measured with a gamma counter (Packard, Packard Instrument Company). The counts per minute (CPM) values were corrected for decay time and the results were calculated as the percentage of injected dose per gram of wet tissue (%ID / g).

[0470] 250 μCi of the radiolabeled conjugate 17 was injected into the tail vein of mice bearing xenografts derived from the A549 cell line. At each time point shown in Table 3, tissues were removed from the mice (n = 1 for each time point), weighed, and the radioactivity was quantified on a gamma counter. Since copper-64 emits gamma rays with an abundance of 36% at 511 keV, gamma detection methods can be used. The results are shown as Figure 19 follows.

[0471] Time (hr) Tumor: Stomach Tumor: Kidney 1 0.1 0.3 3 0.1 0.2 6 1.3 1.8 9 4.0 3.6 12 1.8 2.3 15 4.5 2.8 18 6.3 2.2

[0472] Table 3: Tumor: tissue ratios in the time-course study of the radiolabeled conjugate 17 in A549 xenografts.

[0473] As can be seen in Table 3, the uptake of the conjugate was initially very high in the stomach and kidneys. After 6 hours post-injection, the tumors began to show higher tissue uptake than the stomach and kidneys. The highest tumor: tissue ratio was shown at 9 hours. At all other subsequent test times, the tumors maintained this higher uptake.

[0474] At 18 hours post-injection, the complete tissue biodistribution of the radiolabeled conjugate 17 is shown as Figure 20As shown. After confirming that there was no sharp change in tissue uptake values between 6 - 18 hours, mainly for logistical convenience, the experiment was conducted at 18 hours. The high uptake in the tumor remained at 18 hours, and most importantly, compared to non - competing mice, the radiation counts in the tumor site of competing mice (mice co - injected with 250 μCi of radiolabeled conjugate 17 + 100 - fold molar excess of the competing molecule) were much lower. Without being bound by theory, this indicates that the radiolabeled conjugate 17 binds specifically to tumor cells in a receptor - mediated manner.

[0475] Figure 21 and Figure 22 Show the biodistribution data of these experiments using SKRC52 and HT29 xenografts 18 hours after injection. The SKRC52 experiment was conducted with a competition control, indicating that the radioactivity in the tumor was receptor - mediated. The HT29 xenograft was not tested with a competition control. Since HT29 cells express much fewer CAIX receptors on the cell surface, the tumor - to - background tissue ratio was not good at 18 hours.

[0476] Figure 23A and 23B Show whole - body images of mice with CAIX - positive xenografts after injection of radiolabeled 17. Specifically, it shows a competition experiment conducted with mice bearing SKRC52 xenografts after injecting 250 μCi of radiolabeled conjugate 17 and acquiring whole - body images of anesthetized mice 5 hours after injection. In the case of the experiment shown in Figure 23A , mice injected with the targeted PET - imaging conjugate showed bright uptake at the tumor site. However, the two mice injected with 100 - fold excess of the competing ligand (FBSA - COOH) binding 250 μCi of radiolabeled conjugate 17, shown on the left, did not show any tumor uptake at the threshold selected for this image. The tumors were removed from the mice and, as shown in Figure 23B , again showed higher tumor uptake in the targeted mice compared to the competing mice.

[0477] Using the above - mentioned method and acquiring whole - body images, the efficacy of the radiolabeled conjugate 17 was also shown in other tumor models. For example, as shown in Figure 24 , the targeted mice showed much higher tumor uptake than the competing mice. This data was also confirmed in mice bearing HT29 xenografts (data not shown).

Claims

1. A conjugate selected from the following or a pharmaceutically acceptable salt thereof:

2. A conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate is:

3. Use of the conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the manufacture of a medicament for imaging a cell population in a subject in a method comprising: a. Administer to the subject an effective amount of the conjugate or a pharmaceutically acceptable salt thereof, wherein the cell is a cancer cell expressing CA IX protein.

4. A composition comprising the conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and optionally at least one pharmaceutically acceptable excipient.

5. Use of the conjugate according to claim 1 or 2 in the manufacture of a medicament for a method for imaging a cell population in a subject, wherein the cells are cancer cells expressing the CA IX protein.

6. Use according to claim 5, wherein the method comprises administering to the subject an effective amount of the conjugate to image the cells, wherein the cells are cancer cells expressing the CA IX protein.

7. A non-diagnostic method for imaging a cell population in vitro, the method comprising: a. Contact the cell with the conjugate according to claim 1 or 2 to provide labeled cells, and b. Visualize the labeled cells with a fluorescent light source or a suitable detector, wherein the cell is a cancer cell expressing CA IX protein.

Images