HSP90-targeting conjugates and formulations thereof

Abstract

Conjugates of active substances linked to a targeting moiety, such as at least one HSP90 binding moiety, via a linker have been designed. Such conjugates can provide improved spatio-temporal delivery of active substances; improved biodistribution and penetration in tumors; and / or reduced toxicity. Methods of making the conjugates and formulations thereof are provided. Methods of administering such formulations to a subject in need thereof, for example, to treat or prevent cancer, are provided.

Description

[0001] This application is a divisional application of application No. 2019800220712, filed on April 3, 2019, and entitled “HSP90-targeted conjugates and preparations thereof”.

[0002] Citation of Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 653,106 filed on April 5, 2018, U.S. Provisional Patent Application No. 62 / 731,543 filed on September 14, 2018, and U.S. Provisional Patent Application No. 62 / 787,799 filed on January 3, 2019, the contents of each of which are incorporated herein by reference in their entirety. Field of the Invention

[0004] The present invention relates to the use of molecules targeting heat shock proteins, including heat shock protein 90 (HSP90), for example, for the treatment of cancer. Background Art

[0005] Heat shock protein 90 (HSP90) is a molecular chaperone that is important for maintaining the stability and function of numerous client proteins. It is considered a major therapeutic target for anticancer drug development. Summary of the invention

[0006] The present invention provides a conjugate comprising an active substance coupled to an HSP90 targeting moiety via a linker and a pharmaceutical composition comprising such a conjugate, and also provides methods for preparing and using such a conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A The biodistribution of Conjugate 1 in NCI-H460 tumors is shown. Figure 1B The biodistribution of Conjugate 1 in NCI-H69 tumors is shown. Figure 1C The biodistribution of Conjugate 1 in H460 tumors is shown.

[0008] Figure 2A The biodistribution of Conjugate 2 in NCI-H69 tumors is shown. Figure 2B The biodistribution of Conjugate 2 in NCI-H460 tumors is shown.

[0009] Figure 3A The biodistribution of conjugate 3 in NCI-H69 tumors is shown. Figure 3B The biodistribution of conjugate 3 in NCI-H460 tumors is shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Applicants have designed HSP90-targeted conjugates containing active substances. Such targeting can, for example, improve the amount of active substances at the site and reduce the toxicity of the active substances to the subject. The HSP90-targeted conjugates of the present invention have deep and rapid tumor penetration. The high accumulation and long retention time of HSP90-targeted conjugates enable the use of cytotoxic and non-cytotoxic payloads, such as radionuclides, chemotherapeutic agents, kinase inhibitors or immuno-oncology modulators.

[0011] As used herein, "toxicity" refers to the ability of a substance or composition to be harmful or toxic to a cell, tissue organism, or cellular environment. Hypotoxicity refers to a reduced ability of a substance or composition to be harmful or toxic to a cell, tissue organism, or cellular environment. Such reduced toxicity or hypotoxicity can be relative to a standard measure, relative to a treatment, or relative to the absence of a treatment.

[0012] Toxicity can be further measured relative to weight loss of the subject, wherein weight loss greater than 15% of body weight, greater than 20% of body weight, or greater than 30% of body weight indicates toxicity. Other toxicity metrics can also be measured, such as patient performance metrics, including lethargy and general malaise. Neutropenia or thrombocytopenia can also be a measure of toxicity.

[0013] Pharmacological toxicity indicators include increased AST / ALT levels, neurotoxicity, renal damage, GI damage, etc.

[0014] In addition, conjugates containing an HSP90 targeting moiety linked to an active substance are predicted to have reduced toxicity to cells that do not overexpress HSP90 compared to the active substance alone. Without being bound by any particular theory, applicants believe that this feature is due to the reduced ability of the conjugated active substance to be retained in normal cells relative to tumor cells.

[0015] In some embodiments, when the active substance and the targeting moiety are connected to form a conjugate through a linker, the active substance and the targeting moiety have a synergistic effect. The efficacy of the conjugate is better than that of the active substance and / or the targeting moiety alone.

[0016] In some embodiments, when the active substance is linked to the targeting moiety via a cleavable linker, the potency of the active substance is reduced. Upon cleavage of the linker at the target site, such as a tumor site, the active substance is released and full potency is restored.

[0017] It is an object of the present invention to provide improved compounds, compositions and formulations for spatiotemporal drug delivery.

[0018] Another object of the present invention is to provide methods for preparing improved compounds, compositions and formulations for spatiotemporal drug delivery.

[0019] It is also an object of the present invention to provide methods of administering the improved compounds, compositions and formulations to individuals in need thereof.

[0020] I. Conjugates

[0021] The conjugate includes an active substance or a prodrug thereof connected to a targeting moiety (e.g., a molecule capable of binding to HSP90) via a linker. The conjugate may be a conjugate between a single active substance and a single targeting moiety, such as a conjugate having a structure XYZ, wherein X is a targeting moiety, Y is a linker, and Z is an active substance.

[0022] In some embodiments, the conjugate contains more than one targeting moiety, more than one linker, more than one active substance, or any combination thereof. The conjugate may have any number of targeting moieties, linkers, and active substances. The conjugate may have the structure XYZYX, (XY) n -Z, X-(YZ) n , X n -YZ, XYZ n 、(XYZ) n 、(XYZY) n -Z, wherein X is a targeting moiety, Y is a linker, Z is an active substance, and n is an integer between 1 and 50, 2 and 20, for example, 1 and 5. X, Y and Z may be the same or different at each occurrence, for example, the conjugate may contain more than one type of targeting moiety, more than one type of linker and / or more than one type of active substance.

[0023] The conjugate may contain more than one targeting moiety attached to a single active substance. For example, the conjugate may include an active substance and multiple targeting moieties, each attached via a different linker. The conjugate may have the structure XYZYX, where each X is a targeting moiety that may be the same or different, each Y is a linker that may be the same or different, and Z is an active substance.

[0024] The conjugate may contain more than one active substance attached to a single targeting moiety. For example, the conjugate may include a targeting moiety and multiple active substances each attached via a different linker. The conjugate may have the structure ZYXYZ, where X is a targeting moiety, each Y is a linker that may be the same or different, and each Z is an active substance that may be the same or different.

[0025] A. Active substances

[0026] The conjugate as described herein contains at least one active substance (first active substance). The conjugate may contain more than one active substance, which may be the same or different from the first active substance. The active substance may be a therapeutic agent, a prophylactic agent, a diagnostic agent or a nutrient. A variety of active substances are known in the art, and they or their analogs and derivatives can be used in the conjugates described herein. The active substance may be a protein or peptide, a small molecule, a nucleic acid or a nucleic acid molecule, a lipid, a sugar, a glycolipid, a glycoprotein, a lipoprotein or a combination thereof. In some embodiments, the active substance is an antigen, an adjuvant, a radioactive agent, an imaging agent (e.g., a fluorescent moiety) or a polynucleotide. In some embodiments, the active substance is an organometallic compound or a radioactive element. The active substance has a chemical functional group for covalently linking to a linker, or is modified to an analog or derivative for covalently linking to a linker.

[0027] In certain embodiments, the active substance of the conjugate constitutes a predetermined molar weight percentage of about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of the active substance of the conjugate can also be expressed in terms of a ratio relative to the targeting ligand. For example, the present teachings provide a ratio of active substance to ligand of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0028] Radioactive Materials

[0029] In some embodiments, the active substance Z is a radioactive substance or chemical moiety, such as a metal chelating group, that is bound to a radionuclide (such as a radioisotope). Various radionuclides have emission properties, including α, β, γ, and Auger emission, which can be used for therapeutic and / or diagnostic purposes. For example, the active material Z may contain radioactive isotopes such as Y-90, Y-86, I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Zr-89, Sr-89, Ho-166, Sm-153, Cu-67, Cu-64, Lu-177, Ac-225, Pb-203, Bi-213, Th-227, Pb-212, Ra-223, P-32, Sc-47, Br-77, Rh-105, Pd-103, Ag-111, Pr-142, Pm-149, Gd-159, Ir-194 and Pt-199.

[0030] In some embodiments, the active substance comprises an imaging probe, such as a radioactive label (e.g., a radioisotope). Non-limiting examples of radioisotopes for imaging include I-124, I-131, In-111, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-60, Cu-67, Cu-64, Lu-177, Ac-225, Bi-213, Th-227, Pb-212, Ra-223, P-32, Sc-47, Br-76, Br-77, Rh-105, Pd-103, Ag-111, Pr-142, Pm-149, Gd-159, In-111, Ir-194, Pt-199, Tc-99m , Co-57, Ga-66, Ga-67, Ga-68, Kr-81m, Rb-82, Sr-92, Tl-201, Y-86, Zr-89, C-11, N-13, O-15 and F-18.

[0031] In some embodiments, the active substance Z comprises a radioactive substance, a chelator, or a radioactive substance linked to a chelator. Conjugates comprising a radioactive substance (e.g., a radioisotope) linked to a chelator are radioactive analogs of conjugates having only a chelator or having a chelator linked to a non-radioactive isotope.

[0032] The chelating agent can be a metal chelating agent that binds to a metal (including a metal nuclide). The chelating agent can also be a portion connected to a non-metallic active substance. The chelating agent can be acyclic or macrocyclic. Non-limiting examples of chelating agents include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); DOTA derivative: DO3A; diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA); DTPA derivatives: 2-(p-SCN-Bz)-6-methyl-DTPA, CHX-A"-DTPA and cyclic anhydride of DTPA (CA-DTPA); 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA); NOTA derivatives (e.g., BCNOTA, p-NCS-Bz-NOTA, BCNO T); 6-hydrazinonicotinamide (HYNIC); ethylenediaminetetraacetic acid (EDTA); N,N′-ethylene-di-L-cysteine; N,N′-bis(2,2-dimethyl-2-mercaptoethyl)ethylenediamine-N,N′-diacetic acid (6SS); 1-(4-carboxymethoxybenzyl)-N-N′-bis[(2-mercapto-2,2-dimethyl)ethyl]-1,2-ethylenediamine-N,N′-diacetic acid (B6SS); deferoxamine (DFO); 1,1,1-tris(aminomethyl)ethane (TAME); tris(aminomethyl)ethane-N,N,N′,N′,N″,N″-hexaacetic acid (TAME Hex); O-hydroxybenzyliminodiacetic acid; 1,4,7-triazacyclononane (TACN); 1,4,7,10-tetraazacyclododecane (cyclen); 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA); 1-(1-carboxy-3-carboxypropyl)-4,7-bis-(carboxymethyl)-1,4,7-triazacyclononane (NODAGA); 1,4,7-tris(2-mercaptoethyl)-1,4,7-triazacyclononane (triazacyclononane-TM); 1,4,7-triazacyclononane-N,N′,N″ -tri(methylenephosphonic acid) acid (NOTP); 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA); 1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (PEPA); 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N",N"',N"",N""'-hexaacetic acid (HEHA); 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC); and derivatives or analogs thereof.

[0033] In some embodiments, the chelating agent is a polyaminocarboxylic acid material, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), or a derivative thereof. They can coordinate with metals such as Fe, In, Ga, Zr, Y, Bi, Pb, or Ac.

[0034]

[0035] In some embodiments, the chelating agent is a macrocyclic material: 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N",N"',N"",N""'-hexaacetic acid (HEHA), or a derivative thereof.

[0036] Non-limiting examples of DTPA and its derivatives are:

[0037]

[0038]

[0039] Non-limiting examples of DOTA and its derivatives are:

[0040]

[0041] In some embodiments, the conjugates of the present disclosure comprise DOTA, DOTAGA or any derivative / analog thereof as a chelating agent. Any chelating agent disclosed in Eisenwiener et al., Bioorg Med Chem Lett., vol. 10(18): 2133 (2000) (the contents of which are incorporated herein by reference in their entirety) can be used as a chelating agent, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-(2-carboxyethyl) (DOTAGA) or 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, 10-(1,2-dicarboxyethyl) (DOTASA).

[0042]

[0043] Other non-limiting examples of chelating agents are:

[0044]

[0045]

[0046] B. Connector

[0047] The conjugate contains one or more connectors connecting active substances and targeting moieties. Connector Y is combined with one or more active substances and one or more targeting ligands to form a conjugate. Connector Y is connected to targeting moiety X and active substance Z through a functional group, and the functional group is independently selected from ester bonds, disulfides, amides, acylhydrazones, ethers, carbamates, carbonates, sulfonamides, alkyls, aryls, heteroaryls, thioethers and ureas. Alternatively, the connector can be connected to a targeting ligand or an active drug by a group such as provided by the conjugation between thiol and maleimide, azide and alkynes. The linker is independently selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted with one or more groups, each group being independently selected from the group consisting of halogen, cyano, nitro, hydroxy, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl or heterocyclyl are each optionally substituted with one or more groups, each group being independently selected from the group consisting of halogen, cyano, nitro, hydroxy, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl.

[0048] In some embodiments, the linker comprises a cleavable cleavable functional group. The cleavable functional group can be hydrolyzed in vivo, or can be designed to be hydrolyzed enzymatically, for example, by cathepsin B. As used herein, a "cleavable" linker refers to any linker that can be physically or chemically cleaved. Examples of physical cleavage can be cleavage by light, radioactive emission, or heat, while examples of chemical cleavage include cleavage by redox reaction, hydrolysis, pH-dependent cleavage, or cleavage by enzymes. For example, the cleavable functional group can be a disulfide bond or a carbamate bond.

[0049] In some embodiments, the alkyl chain of the linker may be optionally interrupted by one or more atoms or groups selected from -O-, -C(=O)-, -NR, -OC(=O)-NR-, -S-, -SS-. The linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid. In some embodiments, the linker Y may be X'-R 1 -Y'-R 2 -Z', and the conjugate may be a compound according to Formula Ia:

[0050]

[0051] wherein X is the targeting moiety defined above; Z is the active substance; X', R 1 , Y', R 2 and Z' are as defined herein.

[0052] X' is absent or is independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or more natural or unnatural amino acids, thio or succinimidyl; R 1 and R 2 is absent or contains alkyl, substituted alkyl, aryl, substituted aryl, polyethylene glycol (2-30 units); Y' is absent or is substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol (2-30 units) or amide; Z' is absent or is independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, thio or succinimidyl. In some embodiments, the linker can connect one active substance molecule to two or more ligands, or one ligand to two or more active substance molecules.

[0053] In some embodiments, the linker Y can be A m , and the conjugate may be a compound according to formula Ib:

[0054]

[0055] wherein A is defined herein and m=0-20.

[0056] A in Formula Ia is a Spacer unit that is absent or independently selected from the following substituents. For each substituent, the dashed line indicates the site of substitution by X, Z, or another independently selected A unit, wherein X, Z, or A may be attached to either side of the substituent:

[0057]

[0058]

[0059] wherein z = 0-40, R is H or optionally substituted alkyl, and R' is any side chain present in a natural or unnatural amino acid.

[0060] In some embodiments, the conjugate may be a compound according to Formula Ic:

[0061]

[0062] wherein A is defined above, m=0-40, n=0-40, x=1-5, y=1-5, and C is a branching element defined herein.

[0063] C in Formula Ic is a branching unit containing 3 to 6 functional groups for covalently linking a spacer unit, a ligand or an active drug, wherein the functional groups are selected from amines, carboxylic acids, thiols or succinimide, including amino acids such as lysine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid and cysteine.

[0064] C. HSP90 targeting moiety

[0065] Targeting ligands (also referred to as targeting moieties) as described herein include any molecule that can bind to one or more HSP90 proteins. Such targeting ligands can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates or lipids.

[0066] The targeting moiety X can be any HSP90 binding moiety, such as, but not limited to, natural compounds (e.g., geldanamycin and radicicol) and synthetic compounds such as the geldanamycin analog 17-AAG (i.e., 17-allylaminogeldanamycin), a series of purine backbone HSP90 inhibitors, including PU24FC1 (He H. et al., J. Med. Chem., vol. 49:381 (2006), the contents of which are incorporated herein by reference in their entirety), BIIB021 (Lundgren K. et al., Mol. Cancer Ther., vol. 8(4):921 (2009), the contents of which are incorporated herein by reference in their entirety), 4,5-diarylpyrazoles (Cheung KM et al., Bioorg. Med. Chem. Lett., vol. 15:3338 (2005), the contents of which are incorporated herein by reference in their entirety), 3-aryl, 4-carboxamide pyrazoles (Brough P.A. et al., Bioorg. Med. Chem. Lett., vol. 15:5197 (2005), the contents of which are incorporated herein by reference in their entirety), 4,5-diaryl isocyanate (Brough P.A. et al., Bioorg. Med. Chem. Lett., vol. 15:5197 (2005), the contents of which are incorporated herein by reference in their entirety), azoles (Brough PA et al., J. Med. Chem., vol. 51: 196 (2008), the contents of which are incorporated herein by reference in their entirety), 3,4-diarylpyrazole resorcinol derivatives (Dymock BW et al., J. Med. Chem., vol. 48: 4212 (2005), the contents of which are incorporated herein by reference in their entirety), thieno [2,3-d] pyrimidines (WO2005034950 of VERNALIS et al., the contents of which are incorporated herein by reference in their entirety), aryl triazole derivatives of Formula I in EP2655345 of Giannini et al. (the contents of which are incorporated herein by reference in their entirety), or any other example of an HSP90 binding ligand or derivative / analog thereof.

[0067] In some embodiments, the HSP90 binding moiety may be a heterocyclic derivative containing three heteroatoms. WO2009134110 by MATULIS et al. (the contents of which are incorporated herein by reference in their entirety) discloses 4,5-diarylthiadiazoles that show good HSP90 binding affinity. Even though they have fairly modest cell growth inhibition, they can also be used as HSP90 binding moieties in the conjugates of the present invention. Another class of aza-heterocyclic adducts, i.e., triazole derivatives or analogs thereof, can be used as HSP90 binding moieties in the conjugates of the present invention. For example, 1,2,4-triazole skeletons have been documented in a large number of literatures as having HSP90 inhibitory properties. WO2009139916 by BURLISON et al. (Synta Pharmaceuticals Corp., the contents of which are incorporated herein by reference in their entirety) discloses tricyclic 1,2,4-triazole derivatives that inhibit HSP90 at high micromolar concentrations. Any tricyclic 1,2,4-triazole derivatives or derivatives / analogs thereof disclosed in WO2009139916 can be used as the HSP90 binding moiety in the conjugate of the present invention. Any trisubstituted 1,2,4-triazole derivatives or derivatives / analogs thereof disclosed in WO 2010017479 and WO 2010017545 (Synta Pharmaceuticals Corp.) (the contents of which are incorporated herein by reference in their entirety) can be used as the HSP90 binding moiety in the conjugate of the present invention. In another example, ganetespib (formerly known as STA-9090, or its highly soluble phosphate prodrug STA-1474) or derivatives / analogs thereof containing triazolone disclosed in WO2006055760 (Synta Pharmaceuticals Corp., the contents of which are incorporated herein by reference in their entirety) can be used as the HSP90 binding moiety in the conjugate of the present invention.

[0068]

[0069] In some embodiments, ganetespib or a derivative / analog thereof may be used as a targeting moiety. Non-limiting examples of ganetespib derivatives / analogs are shown below.

[0070]

[0071]

[0072] In some embodiments, Onalespib (AT13387) or its derivatives / analogs may be used as the targeting moiety in the conjugates of the present invention. Non-limiting examples of Onalespib and Onalespib derivatives / analogs are shown below.

[0073]

[0074]

[0075] Any HSP90 ligand or HSP90 inhibitor disclosed in WO2013158644, WO2015038649, WO2015066053, WO2015116774, WO2015134464, WO2015143004, WO2015184246 (the contents of which are incorporated herein by reference in their entirety) or a derivative / analog thereof can be used as the HSP90 binding portion in the conjugate of the present invention, such as:

[0076] Formula I wherein R1 can be an alkyl, aryl, halide, carboxamide or sulfonamide; R2 can be an alkyl, cycloalkyl, aryl or heteroaryl, wherein when R2 is a 6-membered aryl or heteroaryl, R2 is substituted at the 3-position and the 4-position relative to the point of attachment on the triazole ring through which the linker L is attached; and R3 can be SH, OH, -CONHR4, aryl or heteroaryl, wherein when R3 is a 6-membered aryl or heteroaryl, R3 is substituted at the 3-position or the 4-position;

[0077] Formula II Wherein R1 can be alkyl, aryl, halo, carboxamido, sulfonamido; and R2 can be optionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examples of such compounds include 5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H-1,2,4-triazole-3-carboxamide and 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide;

[0078] Formula III wherein X, Y and Z may independently be CH, N, O or S (with appropriate substitutions and satisfying the valence of the corresponding atoms and the aromaticity of the ring); R1 may be alkyl, aryl, halide, carboxamido or sulfonamido; R2 may be substituted alkyl, cycloalkyl, aryl or heteroaryl, wherein the linker L is directly attached to these rings or is attached to extended substitutions on these rings; R3 may be SH, OH, NR4R5 and -CONHR6, to which the effector moiety may be attached; R4 and R5 may independently be H, alkyl, aryl or heteroaryl; and R6 may be alkyl, aryl or heteroaryl, having at least one functional group to which the effector moiety may be attached; or

[0079] Formula IV Wherein R1 can be alkyl, aryl, halo, carboxamido or sulfonamido; R2 and R3 are independently C1-C5 hydrocarbon groups optionally substituted by one or more hydroxyl, halogen, C1-C2 alkoxy, amino, mono- or di-C1-C2 alkylamino; 5 to 12-membered aryl or heteroaryl; or R2 and R3 together with the nitrogen atom to which they are attached form a 4 to 8-membered monocyclic heterocyclic group in which up to 5 ring atoms are selected from O, N and S. Examples of such compounds include AT-13387.

[0080] The HSP90 targeting moiety can be Ganetespib, Luminespib (AUY-922, NVP-AUY922), Debio-0932, MPC-3100, Onaspib (AT-13387), SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71, or a derivative / analog thereof.

[0081]

[0082]

[0083] The HSP90 targeting moiety may be SNX5422 (PF-04929113), or any other HSP90 inhibitor disclosed in US 8080556 (Pfizer), WO2008096218 (Pfizer), WO2006117669 (Pfizer), WO2008059368 (Pfizer), WO2008053319 (Pfizer), WO2006117669 (Pfizer), EP1885701 (Novartis), EP1776110 (Novartis), EP2572709 (Novartis), WO2012131413 (Debiopharm), or WO2012131468 (Debiopharm), the contents of each of which are incorporated herein by reference in their entirety.

[0084]

[0085] The HSP90 targeting moiety may also be PU-H71 (an HSP90 inhibitor 124 I radiolabeled for PET imaging) or its derivatives / analogs.

[0086] A conjugate comprising SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71 or AT13387 may have the following structure:

[0087]

[0088]

[0089] In some embodiments, the targeting moiety comprises an imaging probe, such as a radioactive label (eg, a radioisotope). Non-limiting examples of radioactive isotopes include 1-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67, Cu-64, Lu-177, Ac-225, Bi-213, Th-227, Pb-212, Ra-223, P-32, Sc-47, Br-77, Rh-105, Pd-103, Ag-111, Pr-142, Pm-149, Gd-159, Ir-194, Pt-199, Tc-99m, Co-57, Ga-67, Kr-81m, Rb-82, Sr-92, Tl-201, C-11, N-13, O-15, and F-18.

[0090] In some embodiments, the conjugates of the invention comprise more than one targeting moiety. For example, the conjugates may comprise 2, 3, 4 or 5 HSP90 targeting moieties.

[0091] Extracellular HSP90 (eHSP90)

[0092] In normal cells, when cells are under environmental stress (such as heat, drugs, cytokines, UV and / or gamma rays), HSP90 secretion occurs. The main function of extracellular HSP90 (eHSP90) is to help tissue repair by promoting cell migration at the edge of damaged tissue to the damaged area. However, in tumors, constitutively activated oncogenes trigger the secretion of HSP90 even in the absence of any environmental stress. Hsp90 eHSP90α secreted by tumors promotes the migration of tumors and tumor stromal cells during invasion and metastasis. The extracellular promotion function of HSP90α depends on the 115-amino acid fragment (F-5) on the surface of HSP90 (Li et al., Int Rev Cell Mol Biol., vol. 303: 203-235 (2013), the contents of which are incorporated herein by reference in their entirety). It has been shown that eHSP90 is present on the surface of tumor cells and can also be internalized (Crowe et al., ACS Chem. Biol., vol. 12: 1047-1055 (2017)). Therefore, the surface expression of eHSP90 in tumor cells represents a target for selectively directing therapy to tumors rather than healthy cells. Therefore, eHSP90 (especially eHSP90α) may be a good target for treating tumors.

[0093] In some embodiments, the targeting moiety selectively binds to eHSP90. In some embodiments, the targeting moiety binds to the F-5 region of eHSP90.

[0094] In some embodiments, the targeting moiety has low cell permeability and preferentially binds to cell surface eHSP90. In some embodiments, the targeting moiety is cell impermeable and binds only to eHSP90. In some embodiments, the conjugate comprising the targeting moiety has low cell permeability or is cell impermeable.

[0095] In some embodiments, the targeting moiety comprises HS-23, HS-131 (disclosed in Crowe et al., ACS Chem. Biol., vol. 12: 1047-1055 (2017), the contents of which are incorporated herein by reference in their entirety), or DMAG-N-oxide (cell-impermeable to 17-AAG disclosed in Tsutsumi et al., Oncogene, vol. 27(17): 2478-2487 (2008) (the contents of which are incorporated herein by reference in their entirety)) or an analog / derivative thereof, the structure of which is shown below.

[0096]

[0097]

[0098] In certain embodiments, one or more targeting moieties of the conjugate are present in a predetermined molar weight percentage of about 0.1% to about 10%, or about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of the targeting moiety of the conjugate can also be expressed as a ratio to the active agent(s), for example, as a ratio of ligand to active agent of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0099] Non-limiting examples of conjugates

[0100] In some examples, the conjugate comprises an HSP90 targeting moiety, such as a ganetespib analog or derivative (such as TM1, TM2, TM3, TM4, TM5 or TM8), and a chelator for a radioactive substance. The radioactive substance may comprise any radioactive isotope, such as Lutetium 177 (Lu177 or 177 Lu). Lutetium isotope 177 ( 177 Lu) makes the conjugate radioactive. The chelating agent can be DOTA.

[0101] A non-limiting example is compound 1 (or conjugate 1), wherein the targeting moiety is TM5 and the active species comprises DOTA and lutetium atoms:

[0102]

[0103] Where Lu refers to 175 Lu, and can be 177 Lu substitution to generate a radioactive analog of the conjugate.

[0104] In some embodiments, the conjugate comprises an HSP90 targeting moiety, such as a ganetespib analog or derivative (such as TM1, TM2, TM3, TM4, TM5 or TM8), which is connected to a chelator of a radioactive substance using a linker. The linker may comprise a spacer consisting of at least one amino acid or its analog (such as 2 amino acids or their analogs, 3 amino acids or their analogs, 4 amino acids or their analogs, or 5 amino acids or their analogs). The amino acid or its analog may be a D amino acid. The amino acid or its analog may be anionic (e.g., DGlu), cationic (e.g., DLys) or uncharged (e.g., Sar, wherein Sar = N-methylglycine). The spacer may be selected from: DGlu-DGlu-DLys, DLys-DLys-DGlu, DGlu-DGlu-DGlu, DLys-DLys-DLys, Sar-DLys-Sar, Sar-Sar-Sar and Sar-DGlu-Sar. Without wishing to be bound by any theory, the spacer affects the biodistribution of the conjugate and may reduce liver uptake of the conjugate.HSP90 binding affinity is maintained regardless of the charge present on the spacer.

[0105] In some embodiments, the conjugate has the structure of Formula A:

[0106]

[0107] Wherein AA1, AA2 and AA3 are amino acids. Non-limiting examples of conjugates comprising the structure of formula A include:

[0108] Table 1: Conjugates comprising the structure of formula A

[0109]

[0110] The structure of the compound is shown below:

[0111]

[0112]

[0113]

[0114] In one embodiment, the conjugate is Compound 2 (or Conjugate 2), which comprises TM5 as a targeting moiety; DGlu-DGlu-DGlu as a spacer; DOTA as a chelator; and a lutetium atom:

[0115] Where Lu refers to 175 Lu, and can be used 177 Lu substitution to obtain the radioactive analog of the conjugate.

[0116] In one embodiment, the conjugate comprises two HSP90 targeting moieties.The conjugate may be compound 9 (or conjugate 9), which comprises two TM5 moieties and comprises a DOTAGA derivative as a chelating agent.

[0117]

[0118] Where Lu refers to 175 Lu, and can be used 177 Lu substitution to obtain the radioactive analog of the conjugate.

[0119] Lutetium (Lu) in conjugate 2, 3, 4, 5, 6, 7, 8, 9 or 10 can be replaced by Lu177 ( 177 Lu) or any other radioactive isotope to obtain a radioactive analogue of the conjugate.

[0120] D. Pharmacokinetic Regulation Unit

[0121] The conjugate of the present invention may further comprise at least one external linker connected to a reactive group that reacts with a functional group on a protein or engineered protein or its derivative / analog / mimetic, or at least one external linker connected to a pharmacokinetic modulating unit (PMU). The external linker connecting the conjugate and the reactive group or the pharmacokinetic modulating unit may be a cleavable linker that allows the release of the conjugate. Thus, the conjugate can be separated from the protein or the pharmacokinetic modulating unit as desired.

[0122] Any reactive group or PMU disclosed in WO2017 / 197241 (such as a PMU comprising a polymer) can be linked to the conjugate of the present invention, the contents of which are incorporated herein by reference in their entirety.

[0123] In some embodiments, the conjugate includes a protein binding reactive group connected to its active substance. In some embodiments, the conjugate includes a protein binding reactive group connected to its targeting portion. In some embodiments, the conjugate includes a protein binding reactive group connected to its connector. The reactive group is reversibly or irreversibly bound to the protein. The protein can be a naturally occurring protein, such as a serum or plasma protein, or a fragment thereof. Specific examples include Fc neonatal receptor (FcRn), thyroxine binding protein, transthyretin, alpha 1-acid glycoprotein (AAG), transferrin, fibrinogen, albumin, immunoglobulin, alpha-2-macroglobulin, lipoprotein, or a fragment thereof. The reactive group can be bound to such a protein by covalent bonds or non-covalent interactions (such as hydrogen bonds, ionic bonds, van der Waals interactions and hydrophobic bonds).

[0124] In some embodiments, the protein binding reactive group can bind to serum proteins through non-covalent interactions. For example, the reactive group can be a weak affinity (10 -4 Up to 10 -5 M) saturated fatty acids bound to albumin. Non-limiting examples of such fatty acids may include myristic acid (a fatty acid with 14 carbon atoms) and palmitic acid (a fatty acid with 16 carbon atoms). Other non-limiting examples of reactive groups include naphthoylsulfonamido groups, diphenylcyclohexanol phosphate groups, 6-(4-(4-iodophenyl)butyrylamino)hexanoate groups ('Albu'-tags), a series of peptides including SA21 with a DICLPRWGCLW core sequence disclosed by Dennis et al. in J. Biol. Chem., vol. 277: 35035 (2002) (the contents of which are incorporated herein by reference in their entirety) (a cyclic peptide Ac-RLIEDICLPRWGCLWEDD-NH2 with 18 amino acids).

[0125] Protein binding reactive groups can contain the following structures:

[0126]

[0127] In some embodiments, the protein binding reactive group may comprise any peptide-fatty acid albumin binding ligand disclosed in Zorzi et al., Nature Communications, vol. 8: 16092, (2017) (the contents of which are incorporated herein by reference in their entirety). These peptide-fatty acid albumin binding ligands comprise fatty acids linked to short peptides (e.g., heptapeptides) via amino acid side chains. The fatty acids can be linked to the short peptides via the carboxyl groups on their lysine side chains. The fatty acids bind to albumin with an affinity in the micromolar range, while the short peptides enhance affinity by forming additional contacts with albumin. The peptide-fatty acid ligands may have the following general structure:

[0128]

[0129] Wherein X = any amino acid (eg, Gly or Ser), K = Lys, and n = 12 (myristic acid), 14 (palmitic acid), or 16 (stearic acid).

[0130] In some embodiments, any albumin binding functional group disclosed in US 9670482 (Bicycle Therapeutics) (the contents of which are incorporated herein by reference in their entirety) can be used as a protein binding reactive group in the present application. In some embodiments, the protein binding reactive group comprises a fluorene ring and non-covalently and / or reversibly binds to albumin. As a non-limiting example, the protein binding reactive group comprises a fluorene methyloxycarbonyl group (FMOC). Optionally, the protein binding reactive group comprises at least one amino acid, such as Lys, Trp, Gly or Phe, connected to FMOC. For example, the small molecule can comprise Fmoc-Lys-, Fmoc-Gly-, Fmoc-Phe-, Fmoc-GGSGD-, Fmoc-FGGGD-, Fmoc-FGSGD-, Fmoc-WGSGD-, Fmoc-WGGGA or Fmoc-Trp-GGG.

[0131]

[0132] In one embodiment, the conjugate comprises a reactive group that non-covalently binds to albumin. The binding to albumin is reversible. The conjugate may comprise TM5 as an HSP90 targeting moiety and a DOTAGA derivative as a chelating agent. The conjugate may be compound 10 (or conjugate 10). Lutetium (Lu) in conjugate 10 may be replaced with Lu177 ( 177 Lu) or any other radioisotope to produce a radioactive analog of the conjugate.

[0133]

[0134] II. Preparation

[0135] In some embodiments, the composition is administered to a human, human patient, or subject.For the purposes of this disclosure, the phrase "active ingredient" generally refers to a conjugate as described herein.

[0136] Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be appreciated by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non-human mammals. It is fully understood that modifications to pharmaceutical compositions suitable for administration to humans are made in order to make the compositions suitable for administration to various animals, and such modifications can be designed and / or performed with routine (if any) experiments by ordinary veterinary pharmacologists in the art. Subjects to whom the pharmaceutical compositions are intended to be administered include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or poultry, including commercially relevant poultry such as poultry, chickens, ducks, geese, and / or turkeys.

[0137] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the steps of combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, dividing, shaping and / or packaging the product into the desired single or multiple dose units.

[0138] The pharmaceutical compositions of the present invention can be prepared, packaged and / or sold in batches in a single unit dosage form and / or in a plurality of single unit dosage forms. As used herein, a "unit dose" is an individual amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to the subject, and / or a suitable fraction of this dose, such as, for example, half or one-third of this dose.

[0139] The relative amounts of active ingredients, pharmaceutically acceptable excipients and / or any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size and / or condition of the subject being treated, and further depending on the route by which the composition will be administered. For example, the composition may contain 0.1% to 100%, e.g., .5 to 50%, 1-30%, 5-80%, at least 80% (w / w) of active ingredient.

[0140] The conjugates of the invention may be formulated using one or more excipients to: (1) increase stability; (2) allow for sustained or delayed release (e.g., from a depot formulation of a monomaleimide); (3) alter biodistribution (e.g., target the monomaleimide compound to a specific tissue or cell type); (4) alter the release characteristics of the monomaleimide compound in vivo. Non-limiting examples of excipients include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickeners or emulsifiers, and preservatives. Excipients of the invention may also include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, hyaluronidases, nanoparticle mimetics, and combinations thereof. Thus, the formulations of the invention may include one or more excipients, each in an amount that together increases the stability of the monomaleimide compound.

[0141] excipient

[0142] The pharmaceutical preparation may additionally contain pharmaceutically acceptable excipients such as those suitable for the particular dosage form desired, as used herein, including any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like. Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients for formulating pharmaceutical compositions and known techniques for preparing the same. Unless any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0143] In some embodiments, the purity of a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. In some embodiments, the excipient is approved for human and for veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0144] Pharmaceutically acceptable excipients for making pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Such excipients may optionally be included in the pharmaceutical compositions.

[0145] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, the like, and / or combinations thereof.

[0146] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate Sodium lauryl sulfate, quaternary ammonium compounds, and the like and / or combinations thereof.

[0147] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], monoolein, sorbitan monooleate[ 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. ), polyoxyethylene ethers (e.g. polyoxyethylene lauryl ether [ 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F 68, 188. Cetyltrimethylammonium bromide, Cetylpyridinium chloride Benzalkonium chloride, docusate sodium, the like and / or combinations thereof.

[0148] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husk, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate and larch arabinogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salt; silicic acid; polymethacrylate; wax; water; alcohol; etc.; and combinations thereof.

[0149] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetyltrimethylammonium bromide, cetylpyridinium chloride Chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidurea, phenol, phenoxyethanol, phenylethanol, phenylmercuric nitrate, propylene glycol and / or thimerosal. Exemplary antifungal preservatives include but are not limited to butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and / or sorbic acid. Exemplary alcohol preservatives include but are not limited to ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate and / or phenylethanol. Exemplary acidic preservatives include but are not limited to vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetyltrimethylammonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT Methylparaben, 115. II. NEOLONE TM , KATHON TM and / or

[0150] Exemplary buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, calcium phosphate, calcium phosphate hydroxide, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, the like, and / or combinations thereof.

[0151] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0152] Exemplary oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, canola oil, coriander oil, Brazilian palm oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, tuberose oil, fish oil, linseed oil, geraniol, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, macadamia oil, lavender oil, lavender oil, lemon oil, litsea cubeba oil, macademia oil, Oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0153] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0154] Application

[0155] The conjugates of the invention may be administered by any route that produces a therapeutically effective result. These routes include, but are not limited to, enteral, gastrointestinal, epidural, oral, transdermal, epidural / peridural, intracerebral (into the brain), intraventricular (into the ventricles of the brain), epithelial (applied on the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous injection (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin to achieve systemic distribution), transmucosal (diffusion through mucosal membranes), insufflation (nasal inhalation), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in the form of ear drops. In certain embodiments, the composition can be administered in a manner that allows the composition to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0156] The formulations described herein contain an effective amount of the conjugate in a pharmaceutical carrier suitable for administration to an individual in need thereof. The formulations can be administered parenterally (e.g., by injection or infusion). The formulations or variations thereof can be administered in any manner including enteral, topical (e.g., to the eye), or by pulmonary administration. In some embodiments, the formulations are administered topically.

[0157] A. Parenteral Preparations

[0158] The conjugates may be formulated in the form of solutions, suspensions or emulsions for parenteral delivery, such as injection or infusion. The formulations may be administered systemically, regionally or directly to the organ or tissue to be treated.

[0159] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, such as solutions or suspensions; solid forms suitable for preparing solutions or suspensions after adding a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions and microemulsions thereof, liposomes or creams.

[0160] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oil (such as vegetable oil (e.g., peanut oil, corn oil, sesame oil, etc.)), and combinations thereof. Proper fluidity can be maintained, for example, by the use of a coating agent (such as lecithin); by maintaining a desired particle size in the case of a dispersion; and / or by the use of a surfactant. In some cases, an isotonic agent is included, such as one or more sugars, sodium chloride, or other suitable agents known in the art.

[0161] Solutions and dispersions of the conjugate may be prepared in water or another solvent or dispersion medium suitable for admixture with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH adjusters, and combinations thereof.

[0162] Suitable surfactants may be anionic, cationic, amphiphilic or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium and sodium, potassium, ammonium, long-chain alkyl sulfonates, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinates, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinates, such as sodium bis-(2-ethylthioxy) sulfosuccinates; and alkyl sulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetyltrimethylammonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples of the nonionic surfactant include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearyl monoisopropanolamide and polyoxyethylene hydrogenated tallow amide. Examples of amphiphilic surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoylamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0163] The preparation may contain a preservative to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The preparation may also contain an antioxidant to prevent degradation of the active substance.

[0164] The formulation is typically buffered to pH 3-8 after reconstitution for parenteral administration. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. If 10% sucrose or 5% dextrose is used, no buffer may be required.

[0165] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinyl pyrrolidone, dextran, carboxymethyl cellulose, and polyethylene glycol.

[0166] Sterile injectable solutions can be prepared by the following steps: the conjugate is incorporated in the required amount in an appropriate solvent or dispersion medium together with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized conjugates into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above.

[0167] Pharmaceutical preparations for parenteral administration may be in the form of sterile aqueous solutions or suspensions of conjugates formed by one or more polymer-drug conjugates. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS) and isotonic sodium chloride solution. The preparation may also be a sterile solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent (such as 1,3-butanediol).

[0168] In some cases, the formulation is distributed or packaged in liquid form. Alternatively, the formulation for parenteral administration may be packaged in solid form, such as obtained by lyophilizing a suitable liquid formulation. The solid may be reconstituted with an appropriate carrier or diluent prior to administration.

[0169] Solutions, suspensions or emulsions for parenteral administration may be buffered with an effective amount of a buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known to those skilled in the art, and some examples of suitable buffers are acetate, borate, carbonate, citrate and phosphate buffers.

[0170] Solutions, suspensions or emulsions for parenteral administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art, and some examples include glycerol, sucrose, dextrose, mannitol, sorbitol, sodium chloride and other electrolytes.

[0171] Solutions, suspensions or emulsions for parenteral administration may also contain one or more preservatives to prevent bacterial contamination of ophthalmic preparations. Suitable preservatives are known in the art and include polyhexamethylene biguanide (PHMB), benzalkonium chloride (BAK), stabilized oxychloride complex (also known as ), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal and mixtures thereof.

[0172] Solutions, suspensions or emulsions for parenteral administration may also contain one or more excipients known in the art such as dispersing agents, wetting agents and suspending agents.

[0173] B. Mucosal topical preparations

[0174] The conjugate can be formulated for topical application to mucosal surfaces. Dosage forms suitable for topical application include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids and transdermal patches. The formulation can be formulated for transmucosal, transepithelial or transendothelial administration. The composition contains one or more chemical permeation enhancers, membrane permeants, membrane transporters, softeners, surfactants, stabilizers, and combinations thereof. In some embodiments, the conjugate can be administered in the form of a liquid formulation (such as a solution or suspension), a semisolid formulation (such as a lotion or ointment), or a solid formulation. In some embodiments, the conjugate is formulated into a liquid, including a solution and a suspension, such as an eye drop; or formulated into a semisolid formulation for mucosal, such as an eye, or administered vaginally or rectally.

[0175] "Surfactants" are surface-active substances that reduce surface tension and thereby increase the emulsification, foaming, dispersion, diffusion and wetting properties of a product. Suitable nonionic surfactants include emulsifying waxes, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbates, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glyceryl monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the nonionic surfactant is stearyl alcohol.

[0176] An "emulsifier" is a surface-active substance that promotes the suspension of one liquid in another and the formation of a stable mixture or emulsion of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include gum arabic, anionic emulsifying wax, calcium stearate, carbomer, cetearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glyceryl monostearate, glyceryl monooleate, hydroxypropylcellulose, hydroxypropylmethylcellulose, lanolin, hydrate, lanolin alcohol, lecithin, medium chain triglycerides, methylcellulose, mineral oil and lanolin alcohol, sodium dihydrogen phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamer, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, propylene glycol alginate, self-emulsifying monostearate glyceryl, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth gum, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glyceryl stearate.

[0177] Suitable penetration enhancer types are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithin, cholates, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses and imides), macrocyclic compounds (such as macrolides, ketones and anhydrides and cyclic ureas), surfactants, N-methylpyrrolidone and its derivatives, DMSO and related compounds, ionic compounds, azones and related compounds, and solvents (such as alcohols, ketones, amides, polyols (e.g., diols)). Examples of these types are known in the art.

[0178] Drug administration

[0179] The present invention provides methods comprising administering a conjugate as described herein to a subject in need thereof. The conjugate as described herein may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with working memory impairment). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of action, and the like.

[0180] The compositions of the present invention are generally formulated in dosage unit form to facilitate administration and uniformity of dosage. However, it should be understood that the total daily dosage of the compositions of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the administration time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or concomitantly with the specific compound used; and similar factors well known in the medical field.

[0181] In some embodiments, the compositions of the invention may be administered once or more a day in an amount sufficient to deliver about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 ...01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about The dosage level of about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg, about 25 mg / kg to about 50 mg / kg, about 50 mg / kg to about 100 mg / kg, about 100 mg / kg to about 125 mg / kg, about 125 mg / kg to about 150 mg / kg, about 150 mg / kg to about 175 mg / kg, about 175 mg / kg to about 200 mg / kg, about 200 mg / kg to about 250 mg / kg of the subject's body weight is administered to obtain the desired treatment, diagnosis, prevention or imaging effect. The desired dose can be delivered three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks or every four weeks. In some embodiments, multiple administrations (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations) can be used to deliver the desired dose. When multiple administrations are employed, split dosing regimens such as those described herein may be used.

[0182] In the pharmaceutical composition, the concentration of the conjugate can be about 0.01 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 25 mg / mL, about 0.5 mg / mL to about 10 mg / mL, or about 1 mg / mL to about 5 mg / mL.

[0183] As used herein, "fractionated dose" is a single unit dose or total daily dose divided into two or more doses, for example, a single unit dose is administered twice or more. As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / once / single route / single contact point (i.e., a single administration event). As used herein, a "total daily dose" is the amount given or prescribed within a 24-hour time period. It can be administered in a single unit dose form.

[0184] Dosage form

[0185] The pharmaceutical compositions described herein can be formulated into dosage forms described herein, such as topical, intranasal, intratracheal, or injectable (eg, intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous) dosage forms.

[0186] Liquid dosage form

[0187] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, including, but not limited to, water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran methanol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments of parenteral administration, the composition may be mixed with a solubilizer (such as ), alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof.

[0188] Injectable

[0189] Injectable preparations (e.g., sterile injectable aqueous or oily suspensions) can be prepared according to known techniques and may include suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenteral acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Among the acceptable solvents and solvents that can be used include, but are not limited to, water, USP Ringer's solution, and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspension media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injectables.

[0190] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0191] To prolong the effect of the active ingredient, it is desirable to slow down the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor water solubility. The absorption rate of the compound depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form. Alternatively, delayed absorption of the compound administered parenterally can be achieved by dissolving or suspending the compound in an oily solvent. Injectable reservoir forms are prepared by forming a microcapsule matrix of the compound in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of the compound to the polymer and the properties of the specific polymer used, the compound release rate can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Reservoir-type injectable preparations can be prepared by embedding the compound in a liposome or microemulsion compatible with body tissues.

[0192] Pulmonary preparations

[0193] The formulations described herein as being suitable for pulmonary delivery may also be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations may be administered by sniffing, i.e., by rapid inhalation through the nasal passage from a powder container held close to the nose.

[0194] Preparations suitable for nasal administration may, for example, contain as little as about 0.1% (w / w) and up to 100% (w / w) of active ingredients, and may contain one or more additional ingredients described herein. Pharmaceutical compositions may be prepared, packaged and / or sold in the form of preparations suitable for buccal administration. Such preparations may, for example, be in the form of tablets and / or lozenges prepared using conventional methods, and may, for example, contain about 0.1% to 20% (w / w) of active ingredients, wherein the remainder may contain orally soluble and / or degradable compositions and optionally one or more additional ingredients described herein. Alternatively, preparations suitable for buccal administration may include powders and / or aerosolized and / or atomized solutions and / or suspensions containing active ingredients. Such powdered, aerosolized and / or aerosolized preparations may have an average particle and / or droplet size in the range of about 0.1nm to about 200nm when dispersed, and may further include one or more of any additional ingredients described herein.

[0195] General considerations in formulating and / or manufacturing pharmaceutical preparations can be found, for example, in Remington: The Science and Practice of Pharmacy 21st Edition, Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0196] Coating or shell

[0197] Tablets, dragees, capsules, pills and granules solid dosage forms with coatings and shells (such as enteric coatings and other coatings known in the art of pharmaceutical formulation) can be prepared. They may optionally contain opacifiers and may have a composition that allows them to release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose (lactose / milk sugar) and high molecular weight polyethylene glycols.

[0198] VI. Methods of Using the Conjugates

[0199] The conjugates as described herein may be administered as appropriate to treat any hyperproliferative disease, metabolic disease, infectious disease, or cancer. The formulations may be administered by injection, orally, or topically, typically to a mucosal surface (lung, nose, mouth, buccal, sublingual, vaginal, rectal) or to the eye (intraocular or transocular).

[0200] In various embodiments, a method for treating a subject with cancer is provided, wherein the method comprises administering a therapeutically effective amount of a conjugate, a salt form thereof, as described herein to a subject with cancer, suspected of having cancer, or having a predisposition to cancer. According to the present invention, cancer includes any disease or disorder characterized by uncontrolled cell proliferation (e.g., hyperproliferation). Cancer may be characterized by a tumor (e.g., a solid tumor) or any neoplasm.

[0201] In some embodiments, the cancer is a solid tumor. Large drug molecules have limited penetration in solid tumors. The penetration rate of large drug molecules is very slow. On the other hand, small molecules such as the conjugates of the present invention can quickly and deeply penetrate solid tumors. Regarding the penetration depth of the drug, although it has a more sustained pharmacokinetics, large molecules penetrate less. Small molecules such as the conjugates of the present invention penetrate deeper. Dreher et al. (Dreher et al., JNCI, vol. 98 (5): 335 (2006), the contents of which are incorporated herein by reference in their entirety) studied the penetration of dextran with different sizes into tumor xenografts.

[0202] In one embodiment, the conjugate of the invention reaches a distance of at least about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 75 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, about 1000 μm, about 1100 μm, about 1200 μm, about 1300 μm, about 1400 μm, or about 1500 μm from the vascular surface of the tumor in a solid tumor. The zero distance is defined as the vascular surface of the tumor, and each distance greater than zero is defined as the distance to the nearest vascular surface measured in three dimensions.

[0203] In another embodiment, the conjugate of the invention penetrates into the core of the tumor. As used herein, the "core" of a tumor refers to the central region of the tumor. The distance from any part of the core region of the tumor to the vascular surface of the tumor is about 30% to about 50% of the length or width of the tumor. The distance from any part of the core region of the tumor to the center point of the tumor is less than about 20% of the length or width of the tumor. The core region of the tumor is roughly the central 1 / 3 of the tumor.

[0204] In another embodiment, the conjugate of the invention penetrates into the middle of a solid tumor. As referred to herein, the "middle" of a tumor refers to the middle region of the tumor. The distance from any part of the middle region of the tumor to the vascular surface of the tumor is about 15% to about 30% of the length or width of the tumor. The distance from any part of the middle region of the tumor to the center point of the tumor is about 20% to about 35% of the length or width of the tumor. The middle region of the tumor is roughly between the center 1 / 3 of the tumor and the outer 1 / 3 of the tumor.

[0205] In some embodiments, the subject may not otherwise have an indication for treatment with the conjugate. In some embodiments, the method comprises using a cancer cell, including but not limited to a mammalian cancer cell. In some cases, the mammalian cancer cell is a human cancer cell.

[0206] In some embodiments, the conjugates taught by the present invention have been found to inhibit cancer and / or tumor growth. They may also reduce cell proliferation, invasiveness and / or metastasis, thereby making them suitable for treating cancer.

[0207] In some embodiments, the conjugates taught by the present invention can be used to prevent the growth of tumors or cancers, and / or to prevent the metastasis of tumors or cancers. In some embodiments, the compositions taught by the present invention can be used to shrink or destroy cancers.

[0208] In some embodiments, the conjugates provided herein are suitable for inhibiting the proliferation of cancer cells. In some embodiments, the conjugates provided herein are suitable for inhibiting cell proliferation, such as inhibiting the rate of cell proliferation, preventing cell proliferation, and / or inducing cell death. In general, the conjugates as described herein can inhibit the cell proliferation of cancer cells, or inhibit the proliferation of cancer cells and / or induce cell death of cancer cells. In some embodiments, compared with untreated cells, after treatment with the conjugates of the present invention, cell proliferation is reduced by at least about 25%, about 50%, about 75% or about 90%. In some embodiments, compared with untreated cells, after treatment with the conjugates of the present invention, the cell cycle arrest marker phosphorylated histone H3 (PH3 or PHH3) increases by at least about 50%, about 75%, about 100%, about 200%, about 400% or about 600%. In some embodiments, the apoptosis marker cleaved caspase-3 (CC3) is increased by at least 50%, about 75%, about 100%, about 200%, about 400%, or about 600% after treatment with the conjugate of the invention compared to untreated cells.

[0209] Furthermore, in some embodiments, the conjugates of the invention are effective in inhibiting tumor growth in various types of tumors, whether measured as a net size (weight, surface area, or volume) or as a rate over time.

[0210] In some embodiments, the size of the tumor is reduced by about 60% or more after treatment with the conjugate of the invention. In some embodiments, the size of the tumor is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, measured by weight and / or area and / or volume.

[0211] Cancers that can be treated by the methods taught by the present invention typically occur in mammals. Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs, horses, pigs, sheep, goats and cattle. In various embodiments, cancers include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (monocytes, myeloblasts, adenocarcinomas, angiosarcomas, astrocytomas, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, bile duct cancer, bladder cancer, brain cancer, breast cancer, bronchial cancer, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, Colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt lymphoma, dysplastic changes (dysplasia and metaplasia), embryonal carcinoma, endometrial carcinoma, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, liver cancer, hepatocellular carcinoma, hormone-insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphatic endothelial cell sarcoma (lymphagioendotheliosarcoma), lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin and non-Hodgkin), malignancies and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin and uterus, lymphoid malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma, Non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors (carcinomas and sarcomas), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenstrom macroglobulinemia, testicular tumors, uterine cancer, and Wilms tumor.Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urethral cancer, kidney cancer, urothelial cancer, female genital tract cancer, uterine cancer, gestational trophoblastic disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, bone and soft tissue sarcoma, Kaposi sarcoma, neural cancer, eye cancer, meningeal cancer, cancer), glioblastoma, neuroma, neuroblastoma, schwannoma, solid tumors arising from hematopoietic malignancies such as leukemia, metastatic melanoma, recurrent or persistent epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumors, colorectal cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, malignant glioma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine cancer, refractory malignancies, triple-negative breast cancer, HER2-amplified breast cancer, nasopharyngeal carcinoma cancer), oral cancer, biliary tract cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN), non-thyroid medullary cancer, recurrent glioblastoma multiforme, neurofibromatosis type 1, CNS cancer, liposarcoma, leiomyosarcoma, salivary gland cancer, mucosal melanoma, acral / lentigo melanoma, paraganglioma, pheochromocytoma, advanced metastatic cancer, solid tumors, triple-negative breast cancer, colorectal cancer, sarcoma, melanoma, renal cancer, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancies.

[0212] In one embodiment, the conjugate described herein or a formulation containing the conjugate described herein is used to treat small cell lung cancer. About 12%-15% of lung cancer patients suffer from small cell lung cancer. The survival rate of metastatic small cell lung cancer is very low. The five-year survival rate after diagnosis is less than 5%. In the United States, the incidence of small cell lung cancer is about 26K-30K.

[0213] In some embodiments, the conjugates described herein or formulations containing the conjugates described herein are used to treat patients with tumors that express or overexpress HSP90.

[0214] The conjugates of the present invention are characterized by relatively low toxicity to organisms while maintaining the efficacy of inhibiting, for example, slowing or stopping tumor growth. As used herein, "toxicity" refers to the ability of a substance or composition to be harmful or toxic to cells, tissue organisms, or the cell environment. Low toxicity refers to a reduced ability of a substance or composition to be harmful or toxic to cells, tissue organisms, or the cell environment. Such reduced toxicity or low toxicity can be relative to a standard measure, relative to a treatment, or relative to the absence of a treatment. For example, the conjugates of the present invention may have a lower toxicity than the active substance portion Z administered alone. For a conjugate comprising DM1, its toxicity is lower than DM1 administered alone.

[0215] Toxicity can be further measured relative to the weight loss of the subject, wherein weight loss of more than 15% of body weight, more than 20% of body weight, or more than 30% of body weight indicates toxicity. Other toxicity metrics, such as patient performance metrics, including lethargy and general malaise, can also be measured. Neutropenia, thrombocytopenia, white blood cell (WBC) count, complete blood cell (CBC) count may also be a measure of toxicity. Pharmacological indicators of toxicity include elevated transaminase (AST / ALT) levels, neurotoxicity, renal damage, GI damage, etc. In one embodiment, the conjugate of the present invention does not cause a significant change in the subject's weight. After treatment with the conjugate of the present invention, the subject's weight loss is less than about 30%, about 20%, about 15%, about 10%, or about 5%. In another embodiment, the conjugate of the present invention does not cause a significant increase in the subject's AST / ALT level. After treatment with the conjugate of the present invention, the subject's AST or ALT level increases by less than about 30%, about 20%, about 15%, about 10%, or about 5%. In yet another embodiment, the conjugate of the invention does not cause a significant change in the CBC or WBC count of the subject after treatment with the conjugate of the invention. After treatment with the conjugate of the invention, the CBC or WBC level of the subject decreases by less than about 30%, about 20%, about 15%, about 10%, or about 5%.

[0216] Combination therapy

[0217] In some embodiments, the conjugate of the present invention is combined with at least one additional active substance. The active substance can be any suitable drug. The conjugate and at least one additional active substance can be administered simultaneously, sequentially or in any order. The conjugate and at least one additional active substance can be administered at different doses, at different dosing frequencies or via different routes, as long as it is appropriate.

[0218] In some embodiments, the additional active substance affects the biodistribution (i.e., tissue distribution) of the conjugate of the present invention. For example, radioactive substances can accumulate in the kidneys and may cause potential radiotoxicity problems to the kidneys and surrounding organs. Additional active substances can reduce kidney accumulation or retention time. Preferably, the renal uptake of the conjugate is reduced while not affecting the tumor uptake of the conjugate. Protect the kidneys and surrounding organs without reducing the efficacy of the conjugate. In a non-limiting example, the conjugate of the present invention can be co-administered with at least one amino acid or its analog. The amino acid or its analog can be a positively charged basic amino acid, such as lysine (L-lysine or D-lysine) or arginine, or a combination thereof. In another non-limiting example, the conjugate of the present invention can be co-administered with an active substance (such as an HSP90 inhibitor) that binds to HSP90. Any ligand discussed in the "HSP90 targeting moiety" section, such as ganetespib or its derivatives / analogs, can be used. In another non-limiting example, the conjugate of the present invention can be co-administered with monosodium glutamate (MSG) or glutamic acid. In yet another non-limiting example, the conjugate of the present invention can be administered in combination with amifostine (Ethyol, WR-2721), a bovine gelatin-containing solution succinylated gelatin (Gelofusine), or an albumin fragment. The molecular weight of the albumin fragment can be 3 to 50 kDa.

[0219] The additional active substance can be selected from any active substance described herein, such as a drug for treating cancer. It may also be a cancer symptom relief drug. Non-limiting examples of symptom relief drugs include: octreotide or lanreotide; interferon, cypoheptadine or any other antihistamine. In some embodiments, the conjugate of the present invention does not have drug-drug interference with the additional active substance. In one embodiment, the conjugate of the present invention does not inhibit cytochrome P450 (CYP) isozymes. CYP isozymes can include CYP3A4 midazolam, CYP3A4 testosterone, CYP2C9, CYP2D6, CYP1A2, CYP2C8, CYP2B6 and CYP2C19. Additional active substances can be administered concomitantly with the conjugate of the present invention.

[0220] In another example, the conjugates of the invention can be combined with moderate doses of chemotherapeutic agents such as mitomycin C, vinblastine, and cisplatin (see Ellis et al., Br J Cancer, vol. 71(2):366-370 (1995), the contents of which are incorporated herein by reference in their entirety).

[0221] In yet another example, a patient may first receive a pharmaceutically effective dose of an unconjugated active agent and then receive a pharmaceutically effective dose of a conjugate comprising the same active agent.

[0222] The conjugates described herein or formulations containing the conjugates described herein can be used to deliver therapeutic agents, prophylactic agents or diagnostic agents to selective tissues of individuals or patients in need. For example, the conjugates of the present invention are used to deliver radioactive substances to selective tissues. These tissues can be tumor tissues. The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic or preventive response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. The dosage unit form as used herein refers to a physically discrete unit suitable as a unit dose for a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired treatment.

[0223] V. Sets and Equipment

[0224] The present invention provides a variety of kits and devices for conveniently and / or effectively performing the methods of the present invention. Typically, the kit includes sufficient amounts and / or numbers of components to allow the user to perform multiple treatments on a subject and / or conduct multiple experiments.

[0225] In one embodiment, the invention provides a kit for inhibiting tumor cell growth in vitro or in vivo, comprising a conjugate of the invention or a combination of conjugates of the invention, optionally in combination with any other active substances.

[0226] The kit may also include packaging and instructions and / or a delivery agent that forms the formulation composition. The delivery agent may include saline, buffered solution, or any delivery agent disclosed herein. The amounts of the components may be varied to enable consistent, reproducible, higher concentration saline or simple buffer formulations to be obtained. The components may also be varied to increase the stability of the conjugate in buffered solution over a period of time and / or under a variety of conditions.

[0227] The present invention provides devices that can incorporate the conjugates of the present invention. These devices contain stable formulations that can be used for immediate delivery to a subject in need thereof, such as a human patient. In some embodiments, the subject suffers from cancer.

[0228] Non-limiting examples of devices include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices, multilayer microfluidic devices. The devices can be used to deliver the conjugates of the invention according to single, multiple or divided dosing regimens. The devices can be used to deliver the conjugates of the invention through biological tissues, intradermally, subcutaneously or intramuscularly.

[0229] VI. Definitions

[0230] The term "compound" as used herein is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. In the present application, compound can be used interchangeably with conjugate. Therefore, conjugate as used herein is also intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure.

[0231] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present disclosure containing asymmetrically substituted carbon atoms can be separated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by resolving racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also be present in the compounds described herein, and all such stable isomers are encompassed in the present disclosure. Cis and trans geometric isomers of the disclosed compounds are described, and can be separated in the form of a mixture of isomers or in separated isomeric forms.

[0232] The disclosed compounds also include tautomeric forms. Tautomeric forms are produced by the exchange of a single bond with an adjacent double bond and the accompanying proton migration. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactam pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions of a heterocyclic system, such as 1H-imidazoles and 3H-imidazoles, 1H-1,2,4-triazoles, 2H-1,2,4-triazoles and 4H-1,2,4-triazoles, 1H-isoindoles and 2H-isoindoles, and 1H-pyrazoles and 2H-pyrazoles. Tautomeric forms can be in equilibrium or spatially locked into a form by appropriate substitution.

[0233] The disclosed compounds also include all isotopes of atoms present in the intermediates or final compounds. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to the different number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0234] The compounds and salts of the present disclosure can be prepared by conventional methods in combination with solvents or water molecules to form solvates and hydrates.

[0235] As used herein, the term "subject" or "patient" refers to any organism to which a conjugate can be administered, for example, for experimental, therapeutic, diagnostic and / or preventive purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, guinea pigs, cows, pigs, sheep, horses, dogs, cats, hamsters, llamas, non-human primates, and humans).

[0236] As used herein, the terms "treating" or "preventing" may include preventing a disease, disorder, or condition from occurring in an animal that may be susceptible to the disease, disorder, and / or condition but has not yet been diagnosed as having the disease, disorder, or condition; inhibiting the disease, disorder, or condition, e.g., hindering its progression; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease, disorder, or condition may include ameliorating at least one symptom of a particular disease, disorder, or condition, even without affecting the underlying pathophysiology, such as treating pain in a subject by administering an analgesic, even if the agent does not treat the cause of the pain.

[0237] As used herein, "target" will mean the site to which the targeted construct binds. The target may be in vivo or in vitro. In certain embodiments, the target may be a cancer cell found in a leukemia or tumor (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast, and colon, as well as other carcinomas and sarcomas). In other embodiments, the target may refer to a molecular structure to which a targeting moiety or ligand binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate, or enzyme. The target may be a tissue type, such as neuronal tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.

[0238] "Target cells" that can serve as targets for the methods or conjugates are typically animal cells, such as mammalian cells. The methods of the invention can be used to alter the cellular function of living cells in vitro (i.e., in cell culture) or in vivo (where the cells form part of or are otherwise present in animal tissue). Thus, target cells may include, for example, blood, lymphoid tissue, cells lining the digestive tract (such as oral and pharyngeal mucosa), cells forming the villi of the small intestine, cells lining the large intestine, cells lining the respiratory system (nasal passages / lungs) of animals (which can be contacted by inhalation of the invention), dermal / epidermal cells, cells of the vagina and rectum, cells of internal organs (including cells of the placenta), and the so-called blood / brain barrier, etc. In general, target cells express at least one type of HSP90. In some embodiments, the target cell may be a cell that expresses HSP90 and is targeted by the conjugates described herein, and is close to cells affected by the release of the active substance of the conjugate. For example, HSP90-expressing blood vessels close to a tumor may be targeted, and active substances released at this site will affect the tumor.

[0239] The term "therapeutic effect" is well known in the art and refers to a local or systemic effect caused by a pharmacologically active substance in an animal, particularly a mammal, and more particularly a human. Thus, the term means any substance intended to enhance desired physical or mental development and conditions in the diagnosis, cure, alleviation, treatment or prevention of a disease, disorder or condition in an animal (e.g., a human).

[0240] The term "modulate" is art-recognized and refers to upregulation (i.e., activation or stimulation), downregulation (i.e., inhibition or suppression), or both in combination or separately, of a response. Modulation is typically compared to a baseline or reference that can be internal or external to the therapeutic entity.

[0241] As used herein, "parenteral administration" means administration by any method other than through the digestive tract (enteral) or non-invasive topical routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intraosseously, intracerebrally, intrathecally, intramuscularly, subcutaneously, subconjunctivally, by injection, and by infusion.

[0242] As used herein, "topical administration" means non-invasive administration to the skin, orifices, or mucous membranes. Topical administration can be delivered locally, i.e., the therapeutic agent can provide a local effect in the area of ​​delivery without or with minimal systemic exposure. Some topical formulations can provide a systemic effect, for example, via absorption into the bloodstream of an individual. Topical administration can include, but is not limited to, dermal and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ocular administration, and rectal administration.

[0243] As used herein, "enteral administration" means administration via absorption in the gastrointestinal tract. Enteral administration may include oral and sublingual administration, gastric administration or rectal administration.

[0244] As used herein, "pulmonary administration" means administration into the lungs via inhalation or intratracheal administration. As used herein, the term "inhalation" refers to the uptake of air into the alveoli. The uptake of air can occur through the mouth or nose.

[0245] The terms "sufficient" and "effective", as used interchangeably herein, refer to the amount (e.g., mass, volume, dosage, concentration, and / or time period) required to achieve one or more desired results. A "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of at least one symptom or a specific condition or illness, to achieve a measurable increase in life expectancy, or to generally improve the quality of life of a patient. Therefore, the therapeutically effective amount depends on the specific biologically active molecule and the specific condition or illness to be treated. The therapeutically effective amount of many active substances (such as antibodies) is known in the art. The therapeutically effective amount of the compounds and compositions described herein, for example, for treating a specific illness, can be determined by techniques that are completely within the skill of a skilled person such as a physician.

[0246] The terms "biologically active substance" and "active substance" as used interchangeably herein include, but are not limited to, physiologically or pharmacologically active substances that act locally or systemically in the body. Biologically active substances are substances used to treat (e.g., therapeutic agents), prevent (e.g., prophylactic agents), diagnose (e.g., diagnostic agents), cure, or alleviate a disease or disorder; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after they have been placed in a predetermined physiological environment.

[0247] The term "prodrug" refers to a substance that is converted into a biologically active form in vitro and / or in vivo, including small organic molecules, peptides, nucleic acids or proteins. Prodrugs may have applicability because, in some cases, they may be easier to administer than the parent compound (active compound). For example, prodrugs may have biological availability by oral administration, while the parent compound is not. Prodrugs may also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs may also have less toxicity compared to the parent. Prodrugs may be converted into parent drugs by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, NJ (1962) Drug Latentiation, edited by Jucker, Progress in Drug Research, 4:221-294; Morozowich et al. (1977) Application of Physical Organic Principles to Prodrug Design, edited by EB Roche, Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad.Pharm.Sci.; EB Roche (1977) Bioreversible Carriers in Drug Design, Theory and Application, APhA; H. Bundgaard (1985) Design of Prodrugs, Elsevier; Wang et al (1999) Prodrug approaches to the improved delivery of peptide peptide drug, Curr.Pharm.Design.5(4):265-287; Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and ProdrugStrategies, Adv.Drug.Delivery Rev.27:235-256; Mizen et al. (1998). The Use of Estersas Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm.Biotech.11:345-365; Gaignault et al. (1996) Designing Prodrugs and Bioprecursors I. Carrier Prodrugs,Pract.Med.Chem. 671 - 696; M. Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, G. L. Amidon, P. I. Lee and E. M. Topp eds., Transport Processes in Pharmaceutical Systems, Marcell Dekker, pp. 185 - 218; Balant et al. (1990) Prodrugs for the improvement of drug absorption via different routes of administration, Eur. J. Drug Metab. Pharmacokinet., 15(2):143 - 53; Balimane and Sinko (1999). Involvement of multiple transporters in the oral absorption of nucleoside analogues, Adv. Drug Delivery Rev., 39(1 - 3):183 - 209; Browne (1997). Fosphenytoin (Cerebyx), Clin. Neuropharmacol. 20(1):1 - 12; Bundgaard (1979). Bioreversible derivatization of drugs--principle and applicability to improve the therapeutic effects of drugs, Arch. Pharm. Chemi. 86(1):1 - 39; H. Bundgaard ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisher et al. (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2):115 - 130; Fleisher et al. (1985) Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting, Methods Enzymol.112:360-81; FarquharD et al. (1983) Biologically Reversible Phosphate-Protective Groups, J.Pharm.Sci., 72(3):324-325; Han, HK et al. (2000) Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1):E6; Sadzuka Y. (2000) Effective prodrug liposome and conversion to active metabolite, Curr. Drug Metab., 1(1): 31-48; DM Lambert (2000) Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm. Sci., 11 Supplement 2: S15-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87. .

[0248] As used herein, the term "biocompatible" refers to a substance and any metabolites or degradation products thereof that are substantially non-toxic to the recipient and do not cause any significant adverse effects to the recipient. In general, a biocompatible substance is one that does not induce a significant inflammatory response or immune response when administered to a patient.

[0249] As used herein, the term "biodegradable" generally refers to a substance that will degrade or corrode under physiological conditions into smaller units or chemicals that can be metabolized, eliminated, or excreted by a subject. Degradation time varies with composition and morphology. Degradation time can range from hours to weeks.

[0250] The term "pharmaceutically acceptable" as used herein refers to compounds, substances, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications within the scope of reasonable medical judgment, commensurate with a reasonable benefit / risk ratio, according to the guidelines of institutions such as the U.S. Food and Drug Administration (USFood and Drug Administration). "Pharmaceutically acceptable carrier" as used herein refers to all components of a pharmaceutical formulation that promotes the delivery of a composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, expanders, fillers, stabilizers and combinations thereof.

[0251] The term "molecular weight" as used herein generally refers to the mass or average mass of a substance. If it is a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways, including gel permeation chromatography (GPC) or capillary viscometry. In other words, the molecular weight can be expressed in terms of number average molecular weight (M). n ) weight average molecular weight (M w ) are used to report GPC molecular weights. Capillary viscometry provides estimates of molecular weight in the form of inherent viscosity measured from dilute polymer solutions using a specific set of concentration, temperature, and solvent conditions.

[0252] As used herein, the term "small molecule" generally refers to an organic molecule having a molecular weight of less than 2000 g / mol, less than 1500 g / mol, less than 1000 g / mol, less than 800 g / mol, or less than 500 g / mol. Small molecules are non-polymeric and / or non-oligomeric.

[0253] As used herein, the term "hydrophilic" refers to a substance having strong polar groups that readily interact with water.

[0254] As used herein, the term "hydrophobic" refers to a substance's lack of affinity for water; a tendency to repel and not absorb water as well as not dissolve in or mix with water.

[0255] As used herein, the term "lipophilic" refers to a compound having an affinity for lipids.

[0256] As used herein, the term "amphiphilic" refers to a molecule that combines hydrophilic and lipophilic (hydrophobic) properties. As used herein, an "amphiphilic substance" refers to a substance that contains a hydrophobic or more hydrophobic oligomer or polymer (e.g., a biodegradable oligomer or polymer) and a hydrophilic or more hydrophilic oligomer or polymer.

[0257] As used herein, the term "targeting moiety" refers to a moiety that binds or localizes to a specific location. The moiety can be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The location can be a tissue, a specific cell type, or a subcellular compartment. In some embodiments, the targeting moiety can specifically bind to a selected molecule.

[0258] As used herein, the term "reactive coupling group" refers to any chemical functional group that can react with the second functional group to form a covalent bond. The selection of reactive coupling groups is within the capabilities of those skilled in the art. Examples of reactive coupling groups may include primary amines (-NH2) and amine reactive linking groups, such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imino esters, carbodiimides, anhydrides and fluorophenyl esters. Most of these conjugates are conjugated to amines by acylation or alkylation. Examples of reactive coupling groups may include aldehydes (-COH) and aldehyde reactive linking groups, such as hydrazides, alkoxyamines and primary amines. Examples of reactive coupling groups may include thiol groups (-SH) and sulfhydryl reactive groups, such as maleimide, haloacetyl and pyridyl disulfide. Examples of reactive coupling groups may include photoreactive coupling groups such as aryl azides or diaziridines. The coupling reaction may include the use of a catalyst, heat, a pH buffer, light, or a combination thereof.

[0259] As used herein, the term "protecting group" refers to a functional group that can be added to and / or substituted to another desired functional group to protect the desired functional group from certain reaction conditions, and is selectively removed and / or replaced to deprotect or expose the desired functional group. Protecting groups are known to those skilled in the art. Suitable protecting groups may include those described in Greene and Wuts, Protective Groups in Organic Synthesis, (1991). Acid-sensitive protecting groups include dimethoxytrityl (DMT), tert-butyl carbamate (tBoc) and trifluoroacetyl (tFA). Base-sensitive protecting groups include 9-fluorenylmethoxycarbonyl (Fmoc), isobutyryl (iBu), benzoyl (Bz) and phenoxyacetyl (pac). Other protecting groups include acetylaminomethyl, acetyl, tert-amyloxycarbonyl, benzyl, benzyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl, 2-bromobenzyloxycarbonyl, tert-butyl, tert-butyloxycarbonyl, 1-benzyloxycarbonylamido-2,2,2-trifluoroethyl, 2,6-dichlorobenzyl, 2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl, 2,4-dinitrophenyl, dithiasuccinyl , formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl, 4-methylbenzyl, o-nitrophenylsulfinyl, 2-phenyl-2-propyloxycarbonyl, α-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthene, benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester, p-nitrophenyl ester, phenyl ester, p-nitrocarbonate, p-nitrobenzyl carbonate, trimethylsilyl and pentachlorophenyl ester.

[0260] As used herein, the term "activated ester" refers to an alkyl ester of a carboxylic acid, wherein the alkyl group is a good leaving group that renders the carbonyl group susceptible to nucleophilic attack by molecules carrying an amino group. Thus, the activated ester is susceptible to aminolysis and reacts with an amine to form an amide. The activated ester contains a carboxylate group -CO2R, wherein R is a leaving group.

[0261] The term "alkyl" refers to a saturated aliphatic group, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0262] In some embodiments, a straight chain or branched chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., C1-C 30 (for straight chain), C3-C 30In some embodiments, cycloalkyl groups have 3-10 carbon atoms in their ring structure, such as 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples and claims is intended to include both "unsubstituted alkyl" and "substituted alkyl", wherein the latter refers to an alkyl portion having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0263] Unless otherwise specified the number of carbons, "lower alkyl" as used herein means an alkyl group as defined above but having 1 to 10 carbons or 1 to 6 carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. In some embodiments, an alkyl group is a lower alkyl group. In some embodiments, a substituent designated as an alkyl group in this article is a lower alkyl group.

[0264] Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain may themselves be substituted where appropriate. For example, the substituents of the substituted alkyl may include halogen, hydroxyl, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate) and silyl as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester), -CF3, -CN, etc. Cycloalkyl may be substituted in the same manner.

[0265] As used herein, the term "heteroalkyl" refers to a linear or branched or cyclic carbon-containing group or a combination thereof containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein phosphorus and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized. Heteroalkyl may be substituted as defined above for alkyl.

[0266] The term "alkylthio" refers to an alkyl group as defined above having a sulfide group attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio and ethylthio. The term "alkylthio" also encompasses cycloalkyl, alkene and cycloalkenyl groups and alkynyl groups. "Arylthio" refers to an aryl or heteroaryl group. Alkylthio may be substituted as defined above for alkyl.

[0267] The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.

[0268] As used herein, the term "alkoxyl" refers to an alkyl group having an oxygen group connected thereto as defined above. Representative alkoxy groups include methoxy, ethoxy, propoxy and tert-butoxy. "Ether" is two hydrocarbons covalently linked by oxygen. Therefore, the substituent of an alkyl group that causes the alkyl group to become an ether is an alkoxy group or is similar to an alkoxy group, such as one of available-O-alkyl,-O-alkenyl and-O-alkynyl groups. Aryloxy can be represented by-O-aryl or O-heteroaryl, wherein aryl and heteroaryl are defined as follows. Alkoxy and aryloxy can be substituted as described above for alkyl.

[0269] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, such as moieties represented by the following general formula:

[0270]

[0271] Among them, R9, R 10 and R' 10 Each independently represents hydrogen, alkyl, alkenyl, -(CH2) m -R8, or R9 and R 10 Together with the N atom to which they are attached, they complete a heterocycle having 4 to 8 atoms in the ring structure; R8 represents aryl, cycloalkyl, cycloalkenyl, heterocycle or polycycle; and m is 0 or an integer ranging from 1 to 8. In some embodiments, R9 or R 10 Only one of them can be a carbonyl group, for example, R9, R 10 In other embodiments, the term "amine" does not encompass amides, for example, where R9 and R 10 In another embodiment, R9 and R 10 (and optionally R' 10) each independently represents hydrogen, alkyl or cycloalkyl, alkenyl or cycloalkenyl, or alkynyl. Thus, the term "alkylamine" as used herein means an amine group as defined above having a substituted (as described above for alkyl) or unsubstituted alkyl group attached thereto, i.e. R9 and R 10 At least one of them is an alkyl group.

[0272] The term "amido" is well known in the art as an amino-substituted carbonyl group and includes moieties that can be represented by the following general formula:

[0273]

[0274] Among them, R9 and R 10 As defined above.

[0275] As used herein, "aryl" refers to a C5-C 10 In a broad sense, "aryl" as used herein includes 5-, 6-, 7-, 8-, 9- and 10-membered monocyclic aromatic groups which may include 0 to 4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, The aromatic ring may be substituted at one or more ring positions with one or more substituents, including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic moiety, -CF3, -CN; and combinations thereof.

[0276] The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared by two adjacent rings (i.e., "fused rings"), in which at least one ring is aromatic, e.g., the other one or more cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclic rings. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzo Azolyl, benzo oxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisophthalazolyl oxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolyl, isoindolyl, isoquinolinyl, isothiazol ... oxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, Oxazolyl, 1,2,3- Oxazolyl, 1,2,4- Oxazolyl, 1,2,5- Oxazolyl, 1,3,4- Oxazolyl, Oxazolidinyl, Azolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthroline, phenazinyl, phenothiazinyl, phenanthroline Thiol, phenanthracene azine, phthalazine, piperazine, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyrido oxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienothiazolyl, The rings may be substituted as defined above for "aryl".

[0277] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (eg, an aromatic or heteroaromatic group).

[0278] As used herein, the term "carbocycle" refers to an aromatic or non-aromatic ring in which every atom of the ring is carbon.

[0279] As used herein, "heterocycle" or "heterocyclic" refers to a cyclic group attached via a monocyclic or bicyclic ring carbon or nitrogen, containing 3-10 ring atoms, such as 5-6 ring atoms, and optionally containing 1-3 double bonds and optionally substituted by one or more substituents, wherein the ring atoms consist of carbon and 1 to 4 heteroatoms, each of which is selected from: non-peroxide oxygen, sulfur and N(Y), wherein Y is absent or is H, O, (C1-C 10 )alkyl, phenyl or benzyl. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzo Azolyl, benzo oxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisophthalazolyl oxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolyl, isoindolyl, isoquinolinyl, isothiazolyl, isothiazolyl, oxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, Oxazolyl, 1,2,3- Oxazolyl, 1,2,4- Oxazolyl, 1,2,5- Oxazolyl, 1,3,4- Oxazolyl, Oxazolidinyl, oxazolyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthroline, phenazinyl, phenothiazinyl, phenanthroline Thiol, phenanthracene azine, phthalazine, piperazine, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyrido oxazole, pyridoimidazole, pyridothiazole, pyridyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienothiazolyl, The heterocyclic group may optionally be substituted at one or more positions as defined above for alkyl and aryl by one or more substituents such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3 and -CN.

[0280] The term "carbonyl" is art-recognized and includes moieties such as those represented by the following general formula:

[0281]

[0282] Where X is a bond or represents oxygen or sulfur, and R 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl, R' 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl. When X is oxygen and R 11 or R' 11 When X is oxygen and R 11 As defined above, the moiety is referred to herein as a carboxyl group, and in particular when R 11 When X is oxygen, and R' 11 When X is hydrogen, the formula represents a "formate". In general, when the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl". When X is sulfur and R 11 or R' 11 When X is sulfur and R 11 When X is sulfur, and R' 11 When X is a bond and R 11 When X is a bond and R 11 When is hydrogen, the above formula represents an "aldehyde" group.

[0283] As used herein, the term "monoester" refers to an analog of a dicarboxylic acid, wherein one carboxylic acid is functionalized to an ester and the other carboxylic acid is a free carboxylic acid or a salt of a carboxylic acid. Examples of monoesters include, but are not limited to, monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic acid, and maleic acid.

[0284] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Examples of heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur, and selenium. Other suitable heteroatoms include silicon and arsenic.

[0285] As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, -Br or -I; the term "mercapto" means -SH; the term "hydroxy" means -OH; and the term "sulfonyl" means -SO2-.

[0286] The term "substituted" as used herein refers to all permissible substituents of the compounds described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogen, hydroxyl, or any other organic group containing any number of carbon atoms, e.g., 1-14 carbon atoms, in the form of a linear, branched or cyclic structure, and optionally including one or more heteroatoms such as oxygen, sulfur or nitrogen groups. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C 20 Cyclic groups, substituted C3-C 20 Cyclic groups, heterocyclic groups, substituted heterocyclic groups, amino acids, peptides and polypeptide groups.

[0287] The heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" includes the implicit proviso that the substitution is consistent with the permissible valences of the atom substituted and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, or elimination.

[0288] In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents may be one or more and may be the same or different for appropriate organic compounds. Heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms.

[0289] In various embodiments, the substituents are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione, each of which is optionally substituted with one or more suitable substituents. In some embodiments, the substituents are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione, wherein the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione each may be further substituted with one or more suitable substituents.

[0290] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, thiol, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkyloxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkyloxy, azido, alkylthio, oxo, acylalkyl, carboxyester, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituents are selected from cyano, halogen, hydroxyl, and nitro.

[0291] As used herein, the term "copolymer" generally refers to a single polymeric substance comprising two or more different monomers. The copolymer may have any form, such as random, block or graft. The copolymer may have any end groups, including capped end groups or acid end groups.

[0292] The terms "polypeptide," "peptide," and "protein" generally refer to polymers of amino acid residues. As used herein, the terms also apply to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids, or are non-natural amino acids. The term "protein," as generally used herein, refers to polymers of amino acids joined to each other by peptide bonds to form polypeptides of sufficient chain length to produce tertiary and / or quaternary structure. By definition, the term "protein" excludes small peptides, which lack the necessary higher order structure necessary to be considered proteins.

[0293] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably to refer to deoxyribonucleotide or ribonucleotide polymers in linear or circular conformations and in single-stranded or double-stranded forms. These terms should not be interpreted as limiting the length of the polymer. The term can encompass known analogs of natural nucleotides, as well as nucleotides modified in base, sugar and / or phosphate moieties (such as thiophosphate backbones). In general and unless otherwise specified, analogs of a particular nucleotide have the same base pairing specificity; i.e., analogs of A will pair with T bases. The term "nucleic acid" is a term of art, which refers to a string of at least two base-sugar-phosphate monomer units. Nucleotides are monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of messenger RNA, antisense, plasmid DNA, portions of plasmid DNA, or genetic material derived from viruses. Antisense nucleic acids are polynucleotides that interfere with the expression of DNA and / or RNA sequences. The term nucleic acid refers to a string of at least two base-sugar-phosphate combinations. Natural nucleic acids have a phosphate backbone. Artificial nucleic acids may contain other types of backbones, but contain the same bases as natural nucleic acids. The term also includes PNA (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of natural nucleic acids.

[0294] A "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, but retains at least one function as the full-length protein, polypeptide or nucleic acid. A functional fragment may have more, less or the same number of residues as the corresponding native molecule, and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize with another nucleic acid) are well known in the art. Similarly, methods for determining protein function are well known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter paper binding assays, electrophoretic mobility shift assays, or immunoprecipitation assays. DNA cleavage can be determined by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, double hybrid assays, or complementation (e.g., genetic complementation or biochemical complementation). See, for example, Fields et al. (1989) Nature 340: 245-246; U.S. Pat. No. 5,585,245 and PCT WO 98 / 44350.

[0295] As used herein, the term "linker" refers to a carbon chain that may contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long. The linker can be substituted by various substituents, including but not limited to hydrogen atoms, alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic group, aromatic heterocyclic group, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkyl thioether, thiol and urea groups. Those skilled in the art will recognize that each of these groups can be substituted again. The example of the linker includes but is not limited to pH sensitivity linker, protease cleavable peptide linker, nuclease sensitive nucleic acid linker, lipase sensitive lipid linker, glycosidase sensitive carbohydrate linker, hypoxia sensitive linker, light cleavable linker, heat unstable linker, enzyme cleavable linker (e.g. esterase cleavable linker), ultrasound sensitive linker and x-ray cleavable linker.

[0296] The term "pharmaceutically acceptable counterion" refers to a pharmaceutically acceptable anion or cation. In various embodiments, a pharmaceutically acceptable counterion is a pharmaceutically acceptable ion. For example, a pharmaceutically acceptable counterion is selected from citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronide, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). In some embodiments, the pharmaceutically acceptable counter ion is selected from chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, citrate, malate, acetate, oxalate, acetate and lactate. In a specific embodiment, the pharmaceutically acceptable counter ion is selected from chloride, bromide, iodide, nitrate, sulfate, bisulfate and phosphate.

[0297] The term "pharmaceutically acceptable salt" refers to salts of acidic or basic groups present in the compounds useful in the compositions of the present invention. Compounds included in the compositions of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts (i.e., salts containing pharmacologically acceptable anions), including, but not limited to, sulfates, citrates, malates, acetates, oxalates, chlorides, bromides, iodides, nitrates, sulfates, bisulfates, phosphates, acid phosphates, isonicotinates, acetates, lactates, salicylates, citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sucrose salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). In addition to the acids mentioned above, the compounds that include an amino moiety included in the compositions of the present invention can also form pharmaceutically acceptable salts with various amino acids. The compounds included in the compositions of the present invention that are acidic in nature are capable of forming basic salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal salts or alkaline earth metal salts, and particularly calcium salts, magnesium salts, sodium salts, lithium salts, zinc salts, potassium salts and iron salts.

[0298] If the compounds described herein are obtained in the form of acid addition salts, the free base can be obtained by alkalizing a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, can be produced according to conventional procedures for preparing acid addition salts from base compounds by dissolving the free base in a suitable organic solvent and treating the solution with an acid. Those skilled in the art will recognize various synthetic methodologies that can be used to prepare non-toxic pharmaceutically acceptable addition salts.

[0299] The pharmaceutically acceptable salt may be derived from an acid selected from the group consisting of 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-ketoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid / decanoic acid, caproic acid / hexanoic acid, caprylic acid / octanoic acid acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isethionic acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phosphoric acid, propionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0300] The term "bioavailable" is art-recognized and refers to a form of the agent of the invention that allows a portion of the agent of the invention or the administered amount to be absorbed by, incorporated into, or otherwise physiologically available to a subject or patient to whom the agent of the invention is administered.

[0301] It should be understood that the following examples are intended to illustrate rather than limit the present invention. Various other embodiments and modifications to the previous descriptions and embodiments will be apparent to those skilled in the art upon reading this disclosure without departing from the spirit and scope of the present invention, and it is intended that all such embodiments or modifications are included within the scope of the appended claims. All publications and patents referenced herein are hereby incorporated herein by reference in their entirety.

[0302] It should be understood that in the following examples, some conjugates were prepared and characterized using non-radioactive metals such as Lu-175. It will be apparent to one skilled in the art that the corresponding radioactive Lu-177 analogs can be readily prepared using known methods, and that the distribution data for the Lu-175 conjugates will be representative of the distribution data for the Lu-177 analogs. Example

[0303] Example 1: Synthesis of conjugates

[0304] Any convenient method can be used to prepare the conjugate of the present invention. In a reasonable method, the conjugate is constructed by their respective components, targeting moieties, connectors and active substance moieties in some cases. As known in the art, components can be covalently bonded to each other by functional groups, wherein these functional groups can be present on the components or introduced into the components using one or more steps (such as oxidation reactions, reduction reactions, cleavage reactions, etc.). Functional groups that can be used to covalently bind components together to produce drug conjugates include: hydroxyl, sulfhydryl, amino, etc. The specific parts of the different components modified to provide covalent attachment will be selected so that the binding activity required for those components will not be adversely interfered with substantially, for example, for the binding activity of the active substance moiety, the region that does not affect the target binding activity will be modified to maintain a sufficient amount of the desired drug activity. When necessary and / or required, blocking groups can be used to protect certain parts on the components, as known in the art, see, for example, Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

[0305] Alternatively, the conjugates can be produced using known combinatorial methods to generate large libraries of potential conjugates, which can then be screened to identify bifunctional molecules with pharmacokinetic properties. Alternatively, the conjugates can be produced using medicinal chemistry and known structure-activity relationships of the targeting moiety and the active substance moiety. In particular, this approach will understand where to attach the two moieties to the linker.

[0306] Conjugate 1 can be synthesized according to the following scheme:

[0307]

[0308] TM5 (242 mg, 0.419 mmol, trifluoroacetic acid (TFA) salt, 1.20 equiv) and 2-[4,7,10-tris(2-tert-butoxy-2-oxo-ethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic acid (200 mg, 0.349 mmol) were added to a vial and dissolved in DMF (1 mL), HATU (198 mg, 0.524 mmol, 1.50 equiv) was added, followed by diisopropylethylamine (135 mg, 1.05 mmol, 182 μL, 3.00 equiv). After 3 h, additional diisopropylethylamine (135 mg, 1.05 mmol, 182 μL, 3.00 equiv) was added. The suspension was stirred at room temperature for 16 h. The crude product was purified by reverse phase chromatography (10-60% MeCN / water containing 0.1% TFA). The pure fractions were combined and the solvent was evaporated to dryness.

[0309] The residue was dissolved in TFA (5 mL). The solution was stirred at room temperature for 16 h. The crude product was purified on preparative HPLC (20-60% MeCN / water containing 0.1% TFA). The pure fractions were combined and the solvent was evaporated.

[0310] Lutetium (III) chloride (118 mg, 0.42 mmol, 1.20 equiv) was added to a vial and dissolved in 0.05 N HCl (10 mL). 0.2 N NaOAc was added until pH = 4.5, then the purified residue (496 mg, 0.35 mmol) was added and the solution was heated at 90 ° C for 30 min. Purification on Combiflash (5-40% MeCN / water containing 2% AcOH) gave conjugate 1 as a lyophilized powder (86 mg, 22% yield).

[0311] Conjugate 2-8 can be synthesized as follows:

[0312]

[0313] Each b-alanine-AA1-AA2-AA3-(three-tBuDOTA) construct was prepared using standard Fmoc conditions by solid phase peptide synthesis. Where necessary, for each of AA1, AA2, AA3, Fmoc-sarcosine, Fmoc-D-glutamic acid γ-tert-butyl ester or Fmoc-ε-Boc-D-lysine was coupled to the Fmoc-β-alanine on the 2-chlorotrityl resin, and then finally coupled to tri-tert-butyl DOTA. The peptide was cleaved from the resin using a 1% TFA solution in dichloromethane, and then purified by preparative HPLC.

[0314]

[0315] TM5 (1 eq.), protected tripeptide linker (1 eq.) and HATU (1.1 eq.) were added to a vial. DMF (10 vol.) and diisopropylethylamine (3 eq.) were added and the reaction was stirred at room temperature for 4 h and then purified by preparative HPLC. The product was evaporated to dryness and trifluoroacetic acid (10 vol.) was added. The reaction was stirred at room temperature until LCMS showed complete deprotection, and if deprotection was not complete after 1 h, it was heated to 50 ° C. After deprotection was complete, all TFA was removed under vacuum, and the remaining material was dissolved in a minimum amount of DMF and purified by preparative HPLC.

[0316]

[0317] A 10 mg / mL solution of lutetium (III) chloride in 0.05N HCl was prepared and 0.2M NaOAc (6 mL) was added to give a 6.25 mg / mL solution of lutetium (III) chloride at a pH of 4.5. The peptide (0.05 mmol) was dissolved in DMF (100 uL) and 4.5 mL of the above lutetium (III) chloride solution (28.1 mg, 0.10 mmol) was added. The solution was heated to 90 °C for 15 min, then cooled to room temperature and purified by preparative HPLC to give the product.

[0318] Synthesis of conjugate 9

[0319]

[0320] Fmoc-β-alanine was loaded onto 2-chlorotrityl resin (3.70 g, 0.54 mmol / g loading, 2.00 mmol), loaded onto a Liberty Blue peptide synthesizer, and coupled with Fmoc-D-glutamic acid γ-tert-butyl ester (0.4 M in DMF, 12 mL, 4.8 mmol), DIC (0.5 M in DMF, 10 mL, 5 mmol), and ethyl cyanohydroxyiminoacetate (1 M in DMF, 5 mL, 5 mmol) at 60°C for 20 min. The resin was washed with excess DMF, then with dichloromethane, and then treated with anhydrous TFA (20 mL) for 20 min. The TFA was expelled, and the resin was then washed with dichloromethane (2 x 20 mL), and the combined TFA / dichloromethane solution was concentrated under vacuum. The remaining residue was loaded onto a 50 g Isco C18 column and eluted with 25% to 80% acetonitrile in 0.1% TFA in water to give Fmoc-D-glutamate-β-alanine (692 mg, 1.57 mmol, 78% yield).

[0321]

[0322] TM5 HCl salt (505 mg, 1.01 mmol), Fmoc-D-glutamic acid-β-alanine (210 mg, 0.48 mmol) and HATU (385 mg, 1.01 mmol) were added to a vial. DMF (5 mL) and diisopropylethylamine (0.50 mL) were added and the reaction was stirred at 50 °C for 1 h. The reaction was cooled to room temperature and then DBU (0.75 mL) was added. The reaction was stirred for an additional 30 min and then acidified with acetic acid (2 mL) and purified by preparative HPLC eluting with 5%-45% acetonitrile in water containing 0.1% TFA to give TM5-DGlu-β-alanine-TM5 (596 mg, 0.410 mmol, 86% yield) as a TFA salt.

[0323]

[0324] (R)-tBu4-DOTAGA (Levy et al., Org. Process Res. Dev. 2009, 13, 535-542) (545 mg, 0.777 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (296 mg, 1.54 mmol) and N-hydroxysuccinimide (179 mg, 1.56 mmol) were added to a vial. Dichloromethane (10 mL) and diisopropylethylamine (0.81 mL, 4.67 mmol) were added and the reaction was stirred at room temperature for 24 h. The reaction was then diluted with additional dichloromethane (15 mL) and washed with saturated sodium bicarbonate (3 x 15 mL) and brine (10 mL). The organic layer was dried over MgSO4 and the solvent was removed in vacuo to give (R)-tBu4-DOTAGA NHS ester (610 mg, 0.764 mmol, 98% yield) as a yellow solid which was then used without further purification.

[0325]

[0326] TM5-DGlu-β-alanine-TM5 TFA salt (60.0 mg, 41.3 μmol) and (R)-tBu4-DOTAGA NHS ester (63.0 mg, 78.9 μmol) were added to a vial. DMF (3 mL) and diisopropylethylamine (0.50 mL) were added, and the solution was stirred at 50 ° C for 30 min. The reaction mixture was purified by preparative HPLC, eluting with 5%-55% acetonitrile in water containing 0.1% TFA to obtain TM5-DGlu ((R)-tBu4-DOTAGA)-β-alanine-TM5 (46.7 mg, 26.0 μmol) in the form of a TFA salt.

[0327]

[0328] TM5-DGlu((R)-tBu4-DOTAGA)-β-alanine-TM5 TFA salt (46.7 mg, 26.0 μmol) was added to a vial and dissolved in TFA (2 mL). The reaction was stirred at 40 °C for 1 h, and then all TFA was removed under vacuum. Acetonitrile (2 mL) and toluene (2 mL) were added, and the solvent was removed under vacuum again to ensure that excess TFA was removed. The remaining residue was dissolved in DMF (0.5 mL), and a solution of lutetium (III) chloride (9.3, 33.1 μmol) in pH 4.5 HCl / acetate buffer (1.5 mL) was added. 0.2 N sodium acetate (1 mL) was added, and the solution was stirred at 90 °C for 30 min. After cooling to room temperature, the reaction mixture was purified by preparative HPLC eluting with 5%-35% acetonitrile in 0.1% TFA in water to afford conjugate 9 (30.7 mg, 17.6 μmol, 67% yield).

[0329] Synthesis of conjugate 10

[0330]

[0331] Fmoc-β-alanine loaded onto 2-chlorotrityl resin (3.70 g, 0.54 mmol / g, 2.00 mmol) was loaded onto a Liberty Blue peptide synthesizer. It was then coupled with Fmoc-D-glutamic acid γ-tert-butyl ester x 3, [2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid and 5-azidopentanoic acid under standard conditions, followed by cleavage with 2% TFA in dichloromethane (40 mL) for 20 min, and all solvents removed under vacuum to give the crude peptide. The material was loaded onto a 100 g C18 Isco gold column and eluted with 25%-85% acetonitrile in water containing 0.1% TFA to give 5-azidopentanoyl-AEEA-[DGlu(tBu)]3-bAla-OH (717 mg, 0.783 mmol, 39% yield).

[0332]

[0333] Boc-1,3-diaminopropane (3.40 g, 19.5 mmol) and sodium carbonate (2.07 g 19.5 mmol) were added to the flask. THF (25 mL) and propargyl bromide (80% in toluene, 2.17 mL, 19.5 mmol) were added and the reaction was stirred at 60 °C for 1 h. The reaction was then cooled to room temperature, water (50 mL) was added, and the mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over MgSO4 and all solvents were removed under vacuum. The remaining residue was dissolved in dichloromethane (25 mL) and diisopropylethylamine (5 mL). Fmoc-OSu (6.58 g, 19.5 mmol) was added and the reaction was stirred at room temperature for 2 h. The reaction was then loaded onto a 120 g silica gel column and eluted with 0%-100% ethyl acetate in heptane to give 60 (2.95 g, 6.79 mmol, 35% yield).

[0334]

[0335] 4-(p-iodophenyl)butyric acid (600 mg, 2.07 mmol), DCC (427 mg, 2.07 mmol) and N-hydroxysuccinimide (238 mg, 2.07 mmol) were added to a flask. Dichloromethane (6 mL) was added and the reaction was stirred at room temperature for 4 h. The reaction mixture was filtered through a short pad of celite, the pad was rinsed with dichloromethane (5 mL), and the solvent was removed under vacuum to give crude 4-(p-iodophenyl)butyric acid NHS ester. DMF (8 mL), 4-aminobutyric acid (525 mg, 5.19 mmol) and diisopropylethylamine (2 mL) were added to it. The reaction was stirred at room temperature for 24 h and then loaded onto a 100 g C18 Isco gold column. Elution with 15%-85% acetonitrile in water containing 0.1% TFA gave 70 (560 mg, 1.49 mmol, 72% yield).

[0336]

[0337] 60 (841 mg, 1.93 mmol) was added to a flask and dissolved in acetonitrile (10 mL) and triethylamine (2.5 mL). The reaction was stirred at 60 °C for 1 h and LCMS confirmed complete Fmoc deprotection. The reaction mixture was cooled to room temperature and all solvents were removed under vacuum. To the remaining residue was added a solution of 70 (330 mg, 0.880 mmol) and HATU (470 mg, 1.25 mmol) in DMF (5 mL). Diisopropylethylamine (1 mL) was added and the reaction was stirred for 24 h. The reaction mixture was loaded onto a 50 g C18 Isco gold column and eluted with 10%-70% acetonitrile in water containing 0.1% TFA to give 80 (474 ​​mg, 0.832 mmol, 94% yield).

[0338]

[0339] TM5 HCl salt (248 mg, 0.496 mmol), 5-azidopentanoyl-AEEA-[DGlu(tBu)]3 (306 mg, 0.334 mmol) and HATU (176 mg, 0.468 mmol) were added to a vial. DMF (5 mL) and diisopropylethylamine (1 mL) were added and the reaction was stirred at room temperature for 2 h. 5% aqueous sodium carbonate solution (1 mL) was added and the reaction was warmed to 60 °C for 1 h. After 1 h, the reaction mixture was acidified with acetic acid (1 mL) and the reaction mixture was purified by preparative HPLC eluting with 35%-75% acetonitrile in water containing 0.1% TFA. The fractions containing the product were dried under vacuum and trifluoroacetic acid (5 mL) was added to the remaining residue. The reaction was stirred for 1 h and the excess TFA was removed under vacuum. Water (10 mL) was added, and the solution was frozen and lyophilized to afford 90 (430 mg, 0.328 mmol, 98% yield).

[0340]

[0341] 80 (50 mg, 88 μmol) and 90 (90 mg, 75 μmol) were added to a vial and DMF (4 mL) was added. A solution of copper (II) sulfate (15 mg, 94 μmol) in 0.2 N AcOH (0.3 mL) was added, followed by a solution of sodium ascorbate (38 mg, 192 μmol) in 0.2 N NaOAc (0.4 mL). The reaction mixture was heated to 50 °C and stirred at this temperature for 3 h. The reaction mixture was then cooled to room temperature and purified by preparative HPLC, eluting with 15%-65% acetonitrile in 0.1% TFA in water to give 100 (72 mg, 41 μmol, 54% yield).

[0342]

[0343] 10 (76 mg, 43 μmol) was added to a vial and dissolved in TFA (2 mL). The reaction was stirred at room temperature for 30 min, and then all excess TFA was removed under vacuum. To the remaining residue was added a solution of (R)-tBu4-DOTAGA NHS ester (63 mg, 79 μmol) in DMF (3 mL). Diisopropylethylamine (0.5 mL) was added, and the reaction was stirred at 50 °C for 2 h. The reaction mixture was then cooled to room temperature and purified by preparative HPLC, eluting with 15%-45% acetonitrile in water containing 0.1% TFA to give 11 (52.8 mg, 22.5 μmol, 52% yield).

[0344]

[0345] TFA (2 mL) was added to a vial containing 110 (52.8 mg, 22.5 μmol). The solution was heated to 40 °C for 1 h, and then all solvents were removed under vacuum. Acetonitrile (2 mL) and toluene (2 mL) were added, and the solvent was removed again under vacuum to ensure that excess TFA was removed. A solution of lutetium (III) chloride (9.3 mg, 33.1 μmol) in pH 4.5 HCl / acetate buffer (1.5 mL) was added. 0.2 N sodium acetate (1 mL) was added, and the solution was stirred at 90 °C for 30 min. After cooling to room temperature, the reaction mixture was purified by preparative HPLC, eluting with 5%-45% acetonitrile in 0.1% TFA in water to give conjugate 10 (35.6 mg, 15.5 μmol, 68% yield).

[0346] The analogs of Lutetium-177 of conjugates 2 and 10 were prepared as follows. After TFA deprotection of each chelator, the molecules were dissolved using ammonium acetate buffer (pH 6) and mixed with HCl solutions of Lu-177 having the desired activity. For both, the amount of starting buffer was sufficient to maintain the pH at pH 6 as Lu-177 was added. The reactions were then incubated at 37°C for 45 minutes (+ / - 15 minutes). After the incubation was complete (a small amount of the reaction sample was analyzed by C18 Sep Pak), purification was performed using a pretreated C18 Sep Pak. Unlabeled material was removed by eluting the Sep Pak with a 5% ethanol solution. The final product was eluted from the Sep Pak in a 50% ethanol solution. Ethanol was removed from the product using vacuum centrifugation. The labeled molecules were then brought to the desired volume in saline to provide the Lu-177 products of conjugates 2 and 10, respectively.

[0347] Example 2: In vitro studies using conjugates

[0348] HER2 degradation assay:

[0349] BT474 cells were plated at 12,000 cells per well and incubated at 37°C, 5% CO2 for 20-24 hours. After the cells were incubated, the compounds were reconstituted in DMSO to a stock concentration of 5mM. A compound plate was then prepared containing 10 DMSO dilution points. 2uL of these dilutions were then added to the cells to give a final working concentration of 5uM to 0.0003uM. The compounds and cells were incubated for 16 hours. The medium was then removed, the cells were washed, lysed, and human total EbB2 / Her2 levels were analyzed by ELISA.

[0350] HSP90 binding:

[0351] Binding of the conjugates to HSP90 was investigated using the HSP90α assay kit. The HSP90α assay kit is designed for the identification of HSP90α inhibitors using fluorescence polarization. The assay is based on competition of fluorescently labeled geldanamycin for binding to purified recombinant HSP90α. The key to the HSP90α assay kit is fluorescently labeled geldanamycin. Fluorescently labeled geldanamycin is incubated with samples containing the HSP90α enzyme to produce changes in fluorescence polarization, which can then be measured using a fluorescence reader.

[0352] Example 3: In vivo studies using conjugates

[0353] Tumor and plasma pharmacokinetics in H460 mice:

[0354] Conjugate accumulation at 24 and 48 h in tumor and plasma of mice bearing NCI-H460 tumors (lung cancer). Conjugate 1 was administered intravenously at 25 mg / kg. The levels of conjugate 1 in tumor and plasma were measured at 1 h, 24 h and 48 h and are shown in Table 1.

[0355] Table 2. Levels of Compound 1 in Tumors and Plasma

[0356]

[0357]

[0358] The plasma profile of Conjugate 1 at a dose of 5 mg / kg (via IV) in male SD rats was obtained. The plasma profile of Lutetium from Conjugate 1 was also measured. The results are shown in the table below.

[0359] Table 3. Plasma distribution of Conjugate 1 and Lu from Conjugate 1

[0360]

[0361] The biodistribution of the conjugate was studied. Conjugate 1 was administered to healthy rats at a dose of 5 mg / kg. The amount of conjugate 1 and lutetium from conjugate 1 in the spleen, kidney, brain, liver, and bone marrow of the rats was measured. The results are shown in the table below.

[0362] Table 4A. Levels of Lu from Conjugate 1 in rat tissues

[0363]

[0364] Table 4B. Levels of Conjugate 1 in Rat Tissues

[0365]

[0366] In other studies, the biodistribution of the conjugates was investigated using H460 and H69 tumor models.

[0367] Conjugate 1, Conjugate 2 and Conjugate 3 were administered at 5 mg / kg. Figure 1A Shown are the levels of conjugate 1 in tumor and liver in the H460 tumor model; Figure 1B Shown are the levels of conjugate 1 in tumor and liver in the H69 tumor model; Figure 1C Shown are the levels of conjugate 1 in tumor, liver and kidney in the H460 tumor model. Biodistribution studies of conjugate 1 showed a tumor:liver ratio of 0.1-1 at 24h, while the tumor:kidney ratio was approximately 2.

[0368] Figure 2A The levels of conjugate 2 in tumor and liver in the H69 tumor model are shown; Figure 2B The levels of conjugate 2 in the tumor, liver and kidney in the H460 tumor model are shown. The biodistribution study of conjugate 2 showed a tumor: liver ratio of 9-50 and a tumor: kidney ratio of 0.4 at 24h. The results are also shown in the table below.

[0369] Table 5A Concentration of conjugate 2 in H460 tumor model mouse tissue (μM)

[0370]

[0371] Table 5B Concentration of conjugate 2 in H69 tumor model mouse tissue (μM)

[0372]

[0373] Figure 3A Shown are the levels of conjugate 3 in tumor and liver in the H69 tumor model. Figure 3BThe levels of conjugate 3 in tumor, liver and kidney in the H460 tumor model are shown. The results are also shown in the table below.

[0374] Table 6A Concentration of conjugate 3 in H460 tumor model mouse tissue (μM)

[0375]

[0376] Table 6B Concentration of conjugate 3 in H69 tumor model mouse tissue (μM)

[0377]

[0378] Also summarized in the table below are the conjugate tumor levels at 24 h, tumor:liver ratio at 24 h, and tumor:kidney ratio at 24 h for Conjugate 1 and Conjugate 2.

[0379] Table 7. Conjugate tumor levels, tumor:liver ratio, and tumor:kidney ratio

[0380]

[0381] Tumor uptake was significantly higher for Conjugate 1 than for Conjugate 2, but the tumor:liver ratio was better for Conjugate 2. Tumor retention was similar for Conjugate 1 and Conjugate 2. Both compounds were rapidly cleared from plasma, but Conjugate 2 was slightly better in this regard.

[0382] In further studies, the conjugate and the amino acid were administered separately to mice. The tissue distribution of the conjugate co-administered with the amino acid was compared with the tissue distribution of the conjugate administered alone.

[0383] In another study, the conjugate and HSP90 ligand or inhibitor were administered to mice separately. The tissue distribution of the conjugate co-administered with the HSP90 ligand or inhibitor was compared with the tissue distribution of the conjugate administered alone.

[0384] In a similar study, the compound was administered to mice at a dose of 0.5 mg / kg. Lu levels in tumors, kidneys, and livers were measured. The average Lu in tumors, kidneys, and livers was calculated (shown in Table 8 below). Tumor uptake of conjugates 9 and 10 was significantly higher than that of conjugates 1 and 2.

[0385] Table 8. Lu levels in tumors, kidneys and liver

[0386]

[0387] Example 4: Determination of the permeability of the conjugate

[0388] To test the ability of the conjugates to enter cells, an artificial membrane permeability assay ("PAMPA") was used. PAMPA is a useful tool for predicting in vivo drug permeability of drugs that enter cells via passive transport mechanisms. LC / MS was used in conjunction with the PAMPA assay to determine the ability of the conjugates to permeate cells.

[0389] Pre-coated PAMPA plates were warmed to room temperature for at least 30 minutes prior to adding assay components.

[0390] Prepare stock solutions with the conjugates to be tested. To prepare working solutions, add 50 μL of 100 μM stock solution in DMSO + 950 μL PBS or 50 μL of 200 μM stock solution to a 96-deep well plate to give a final concentration of 5 μM or a final concentration of 10 μM, respectively. Add 300 μL of working solution containing each conjugate to be tested to the appropriate wells of the donor PAMPA plate. Add 200 μL PBS to the corresponding wells of the acceptor PAMPA plate.

[0391] The acceptor plate was lowered onto the donor plate and incubated for 5 hours. After 5 hours, a 50 μL aliquot was removed from each well of each plate and added to a new 96 deep well plate.

[0392] 100 μL of methanol containing a predetermined internal standard control compound was added to each aliquot and analyzed by LC / MS. The permeability of each conjugate was calculated.

[0393] Example 5: Biodistribution study of Lu-177 conjugate 2

[0394] The aim of this study was to determine the accumulation of Hsp90-DOTA-Lu177, a radioactive Lu177 analog of Conjugate 2 (with Lu-177, also known as Lu-177, in female nude mice bearing NCI-H460 NSCLC tumors) by scintigraphy using a gamma counter. 177 Lu-2 or 177 Lu-conjugate 2) and the radioactive Lu177 analog of conjugate 10 (having Lu-177, also known as 177 Lu-10 or 177 In vitro biodistribution of Lu-conjugate 10). Animals bearing tumors were injected with radioactive 177 Lu-2 or 177 After a period of time, the animals were euthanized and tumor and other tissue samples were collected for ex vivo gamma counting using a Perkin Elmer 2470 WIZARD gamma counter. For each tissue, the percentage of injected dose per gram of tissue (% ID / g) was calculated.

[0395] for 177 Lu-2 was administered intravenously at a dose of 3.7 MBq and ex vivo gamma counts were performed at the 6-hour time point (5 mice, data in Table 9). The tissues with the highest uptake were kidney and tumor. The median %ID / g were 4.29 and 1.21, respectively.

[0396] Table 9. Radioactivity in tissues 6 h after dosing, reported as %ID / g and as median of 5 mice.

[0397] Tumor kidney liver Eye brain marrow 6h 1.21 4.29 0.33 0.12 0.01 0.01

[0398] for 177 Lu-10 was administered intravenously at a dose of 3.7 MBq, and ex vivo gamma counts were performed at 24 h and 72 h (5 mice per time point, data in Table 9). The tissues with the highest uptake were kidney and tumor. The median %ID / g were 4.29 and 1.21, respectively.

[0399] Table 10. Radioactivity in tissues 24h and 72h after administration, reported as %ID / g and averaged from 5 mice Value report.

[0400] lung brain Tumor liver spleen kidney marrow 24h 1.2 0.0 9.2 1.9 1.4 21.7 0.3 72h 0.6 0.0 4.1 1.6 1.4 16.6 0.3

Claims

1. A conjugate comprising an active substance coupled to at least one HSP90 targeting moiety via a linker, wherein the active substance comprises a radioactive substance or a chelator of a radioactive substance.

2. The conjugate of claim 1, wherein the radioactive material comprises a radioisotope.

3. The conjugate of claim 2, wherein the radioisotope is 1-124, 1-131, In-111, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-60, Lu-177, Ac-225, Bi-213, Th-227, Pb-212, Ra-223, P-32, Sc-47, Br-76, Br-77, Rh-105, Pd-103, Ag-111, Tc-99m, Co-57, Ga-66, Ga-67, Ga-68, Kr-81m, Rb-82, Sr-92, Tl-201, Y-86, Zr-89, C-11, N-13, O-15, F-18, Y-86, Bi-212, At-211, Zr-89, Sr-89, Ho-166, Sm-153, Cu-67, Cu- 64. Pb-203, Bi-213, Th-227, Pb-212, Ra-223, P-32, Sc-47, Br-77, Rh-105, Pd-103, Ag-111, Pr-142, Pm-149, Gd-159, Ir-194 and Pt-199. The conjugate of claim 1 , wherein the active substance comprises a radioactive isotope bound to a chelating agent. The conjugate according to claim 4 , wherein the chelating agent is a polyaminocarboxylic acid substance.

6. The conjugate of claim 5, wherein the chelator is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), DOTAGA, or a derivative thereof. The conjugate according to claim 4 , wherein the chelating agent is a macrocyclic substance.

8. The conjugate of claim 7, wherein the chelator is 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane-N,N′,N",N"',N""-pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N",N"',N"",N""'-hexaacetic acid (HEHA), or a derivative thereof.

9. The conjugate according to any one of claims 1 to 8, wherein the conjugate comprises two HSP90 targeting moieties.

10. The conjugate according to any one of claims 1-9, wherein the HSP90 targeting moiety is an HSP90 inhibitor. The conjugate according to claim 10 , wherein the HSP90 inhibitor is a small molecule.

12. The conjugate according to claim 11, wherein the HSP90 inhibitor is selected from: Ganetespib, Luminespib (AUY-922, NVP-AUY922), Debio-0932, MPC-3100 or Onaspib (AT-13387), SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71, AT13387, and derivatives / analogs thereof. The conjugate according to claim 10 , wherein the HSP90 targeting moiety is ganetespib or a derivative thereof. The conjugate according to claim 13 , wherein the HSP90 targeting moiety is selected from TM1, TM2, TM3, TM4, TM5 or TM8. The conjugate according to claim 10 , wherein the HSP90 targeting moiety is onaspib or a derivative thereof. The conjugate according to claim 15 , wherein the HSP90 targeting moiety is selected from TM6 and TM7.

17. The conjugate according to any one of claims 1 to 16, wherein the linker comprises an ester group, a disulfide group, an acylamino group, an acylhydrazone group, an ether group, a carbamate group, a carbonate group or a urea group.

18. The conjugate according to any one of claims 1-17, wherein the linker is a cleavable linker.

19. The conjugate of claim 1, wherein the molecular weight of the conjugate is less than about 50,000 Da, less than about 40,000 Da, less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, less than about 3,000 Da, less than 2000 Da, less than 1500 Da, less than 1000 Da or less than 500 Da.

20. The conjugate according to claim 1, wherein the conjugate comprises at least one ganetespib or a derivative thereof as an HSP90 targeting moiety, and a lutetium atom.

21. The conjugate of claim 1, wherein the conjugate is selected from conjugates 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a pharmaceutically acceptable salt thereof.

22. The conjugate of claim 1, wherein the conjugate is a radioactive analogue of a conjugate selected from conjugates 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or a pharmaceutically acceptable salt thereof.

23. The conjugate of claim 22, wherein the conjugate is Lu177( 177 Lu) analogs, or pharmaceutically acceptable salts thereof.

24. The conjugate of claim 1, wherein the conjugate comprises a reactive group that binds to a serum protein.

25. The conjugate of claim 24, wherein the serum protein is albumin.

26. The conjugate of claim 24, wherein the conjugate is conjugate 10 or a radioactive analog thereof, or a pharmaceutically acceptable salt thereof.

27. The conjugate according to claim 26, wherein the conjugate is Lu177( 177 Lu) analogs.

28. A pharmaceutical composition comprising the conjugate of any one of claims 1-27 and at least one pharmaceutically acceptable excipient.

29. A method of reducing cell proliferation, comprising administering to the cell a therapeutically effective amount of at least one conjugate of any one of claims 1-27.

30. The method of claim 29, wherein the cell is a cancer cell.

31. The method of claim 30, wherein the cancer cell is a small cell lung cancer cell, a non-small cell lung cancer cell, a sarcoma cell, a pancreatic cancer cell, a breast cancer cell, or a colon cancer cell.

32. A method of treating cancer, comprising administering the pharmaceutical composition of claim 28.

33. The method of claim 32, wherein the cancer is a small cell lung cancer cell, a non-small cell lung cancer cell, a sarcoma cell, a pancreatic cancer cell, a breast cancer cell, or a colon cancer cell.

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