Conjugates for targeted therapy

Abstract

The present disclosure provides novel conjugates or pharma- ceutically acceptable salts thereof, comprising a conjugate active pharmaceutical moiety or a prodrug thereof, a targeting module and a linker therebetween, the conjugates or pharma- ceutically acceptable salts thereof being useful for treating disease, recurrence or progression in a subject, or for increasing the chances of survival over a relevant period in subjects diagnosed with a disease.

Description

[Technical field]

[0001] The present disclosure relates to novel conjugates, in particular conjugates for use in targeted therapy. [Background technology]

[0002] Selective delivery of drugs to defined disease sites is one of the most important goals of clinical therapy. For example, in the case of the treatment of certain tumors, there is still a need for delivery systems that can target anticancer drugs to tumor sites while minimizing off-target effects. Although chemotherapy has already made great progress, current treatments are still unsatisfactory and the prognosis of most patients remains poor. One of the main challenges is the occurrence of undesirable side effects.

[0003] Drugs are usually formulated as medicaments for topical, oral, intravenous or intramuscular administration. Drugs administered along these routes usually require doses much larger than the actual drug amount used at the lesion site. Also, toxic drugs are administered in amounts that may limit long-term use or cumulative application.

[0004] For example, in colorectal cancer (CRC), the fourth most common and second most deadly cancer in the world, 5-fluorouracil (5-FU) has been used as the basic treatment for metastatic colorectal cancer (mCRC) for over 40 years, usually in combination with leucovorin, with or without oxaliplatin, as standard first-line therapy (Cunningham et al., Ann Oncol 2009, 20, 244-50). However, it has been reported that the average response rate of patients receiving 5-FU-based chemotherapy was only about 23% (Sotos et al., Cancer Treat Rev 1994, 20, 11-49). Irinotecan, a prodrug of SN-38, is an important chemotherapy agent in the second-line treatment of fluorouracil-refractory stage IV CRC patients, but stage II studies have shown that the response rate of irinotecan alone in fluorouracil-resistant patients is only about 27% (Shimada et al., J Clin Oncol 1993, 11, 909-13). Furthermore, the low selectivity of 5-FU-based regimens and irinotecan may cause side effects in mCRC therapy, such as neutropenia, diarrhea, nausea, vomiting, and stomatitis (Falcone et al., J Clin Oncol 2001, 19, 3456-62).

[0005] Therefore, there is a need for improved methods of targeting the delivery of drugs to specific organs, tissues, cells and / or subcellular organelles. Summary of the Invention

[0006] The present disclosure provides novel conjugates or pharma- ceutically acceptable salts thereof, having an active pharmaceutical moiety or a prodrug thereof, a targeting module and a linker therebetween, the conjugates or pharma- ceutically acceptable salts thereof being applied to treat disease, recurrence or progression in a subject, or to increase the chances of survival over a relevant period in a subject diagnosed with a disease.

[0007] In one embodiment of the first aspect of the present disclosure, a compound of formula I: ALB (I) or a pharma- ceutically acceptable salt thereof. During the ceremony, A is an active pharmaceutical moiety or a prodrug thereof; B is the target module, L is of formula II: A linker represented by TIFF2025513000000002.tif19170, During the ceremony, R 1 Ha-SR 2 or TIFF2025513000000003.tif16170, R 2 is R 3 , -SR 3 or TIFF2025513000000004.tif22170, R 3 Ha-(CH 2 )nR 4 and R 4 is R 5 , -OC(O)-R 5 , -C(O)-R 5 , -C(O)NH-R 5 , -C(O)-, -NHC(O)-R 5 , -OC(O)OR 5 or -C(O)-Z-NH-phenylene-R 5 and R 5 Ha-(CH 2 ) m - and Z is -Val-Cit-, -Phe-Lys-, -Val-Ala- or -Gly-Phe-Leu-Gly-, p is an integer from 2 to 9; n is an integer from 2 to 9; m is 0 or 1, and The dotted lines are covalent bonds. However, the conjugate is not a compound selected from the group consisting of: TIFF2025513000000005.tif78170TIFF2025513000000006.tif41170and TIFF2025513000000007.tif75170

[0008] In one embodiment of the conjugate or pharma- ceutically acceptable salt thereof, A is an anticancer therapeutic moiety. In some embodiments, A is selected from 5-fluorouracil (5-FU), SN-38, irinotecan, lonidamine, bleomycin, mitomycin, cisplatinum, doxorubicin, DM-1, taxol, cabazitaxel, floxuridine, or FdUMP.

[0009] In one embodiment of the conjugate or pharma- ceutically acceptable salt thereof, B is a glucose transporter specific binding agent. In some embodiments, B can be selected from phloretin optionally substituted with glucose, mannose, or 2-fluoro-D-glucose.

[0010] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, R 1 Ha-SR 2 and R 2 teeth TIFF2025513000000008.tif21170, R 3 Ha-(CH 2 )n -R 4 and R 4 -C(O)-R 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 5, and m is 0.

[0011] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, R 1 Ha-SR 2 and R 2 Ha-SR 3 and R 3 Ha-(CH 2 ) n -R 4 and R 4 HA-OC(O)-R 5 OR-OC(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 2, and m is 0.

[0012] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, R 1 is SR 2 and R 2 teeth TIFF2025513000000009.tif20170, R 3 Ha-(CH 2 ) n -R 4 and R 4 is -C(O)-Z-NH-phenylene-R 5 and R 5 Ha-(CH 2 ) m -, Z is -Val-Cit-, p is 2, n is 4 and m is 1.

[0013] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, R 1 teeth TIFF2025513000000010.tif16170, R3 Ha-(CH 2 ) n -R 4 and R 4 -C(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 3, and m is 1.

[0014] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, R 1 Ha-SR 2 and R 2 teeth TIFF2025513000000011.tif22170, R 3 Ha-(CH 2 ) n -R 4 and R 4 -C(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 4, and m is 1.

[0015] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, R 1 Ha-SR 2 and R 2 Ha-SR 3 and R 3 (CH 2 ) n -R 4 and R 4 -C(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 3, and m is 1.

[0016] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, the conjugate has formula III: Represented as TIFF2025513000000012.tif22170, During the ceremony, R is OH or The file is TIFF2025513000000013.tif22170.

[0017] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, the conjugate is selected from the group consisting of: TIFF2025513000000014.tif50170TIFF2025513000000015.tif19170TIFF2025513000000016.tif25170TIFF2025513000000017.tif28170 and TIFF2025513000000018.tif58170In the formula, R is OH or The file is TIFF2025513000000019.tif22170.

[0018] One example of the second aspect of the present disclosure is a method of treating disease, recurrence or progression in a subject, or increasing the chances of survival over a relevant period in a subject diagnosed with a disease, comprising administering to the subject a conjugate as described herein, or a pharma- ceutically acceptable salt thereof.

[0019] In one embodiment of the method, the disease is a cancer selected from solid or liquid cancers or their metastases, including, but not limited to, squamous cell carcinoma, lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, head and neck cancer, lymphoma, leukemia, myeloma, and myeloproliferative neoplasms.

[0020] In one embodiment of the method, the subject is difficult to treat, refractory to the drug, or resistant to the drug.

[0021] In one embodiment of the method, A is an active pharmaceutical moiety or a prodrug thereof, and the active pharmaceutical moiety is an anticancer therapeutic moiety. In some embodiments, A is selected from 5-fluorouracil (5-FU), SN-38, irinotecan, lonidamine, bleomycin, mitomycin, cisplatin, doxorubicin, DM-1, taxol, cabazitaxel, floxuridine, or FdUMP.

[0022] In one embodiment of the method, B is a glucose transporter specific binding agent. In one embodiment, B is phloretin, optionally substituted with glucose, mannose, or 2-fluoro-D-glucose.

[0023] In one embodiment of the method, the conjugate has formula III: Represented as TIFF2025513000000020.tif23170, During the ceremony, R is OH or The file is TIFF2025513000000021.tif22170.

[0024] In one embodiment of the method, the conjugate is selected from the group consisting of: TIFF2025513000000022.tif63170TIFF2025513000000023.tif24170TIFF2025513000000024.tif35170TIFF2025513000000025.tif34170 and TIFF2025513000000026.tif59170In the formula, R is OH or The file is TIFF2025513000000027.tif21170.

[0025] One embodiment of the third aspect of the present disclosure is a composition comprising a therapeutically effective amount of the aforementioned conjugate or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, excipient or vehicle.

[0026] These and other aspects will become apparent from the following description of the preferred embodiments in combination with the following drawings, although changes and modifications can be made without departing from the spirit and scope of the novel concepts of the present disclosure. [Brief description of the drawings]

[0027] Figure 1 shows the structures of 5-FU (1), phlorizin (2), phloretin (3), SN-38 (28), irinotecan (39), and the designs of compounds 4–9, 30–31, 37–38, and 42–45.

[0028] Figures 2a)-2d) show the in vitro plasma stability and release profiles of compounds 5, 7, and 9 under different conditions, where Figure 2a) is the analysis of compound 7 in human plasma using RP-HPLC, Figure 2b) is the stability of compounds 5, 7, and 9 in human plasma at 37°C, Figure 2c) is the analysis of compound 7 with GSH (5mM) using RP-HPLC, and Figure 2d) is the in vitro release profile of compound 7 at GSH concentrations (5μM, 1mM, and 5mM) and the formation of 5-FU at 1mM and 5mM GSH, and the experimental data are expressed as mean ± standard error of the mean (SEM) (n=3).

[0029] Figures 3a)-3d) show the in vitro plasma stability and release profiles of compounds 30 and 31 under different conditions, where Figure 3a) is the stability in human plasma at 37°C, Figure 3b) is the stability of SN-38 at 5 μM or 5 mM and its release rate, Figure 3c) is the analysis of compound 30 with GSH (5 mM) using RP-HPLC, and Figure 3d) is the analysis of compound 31 with GSH (5 mM) using RP-HPLC. The experimental data are expressed as mean ± standard error of the mean (SEM) (n=3).

[0030] Figure 4 shows the inhibitory effects of compounds 16 and 17 on GLUT-1 activity by analysis of 2-NBDG absorbance, and the experimental data are expressed as mean ± standard error of the mean (SEM) (n = 4, ns, not significant, ****P < 0.0001 (two-way ANOVA)).

[0031] Figures 5a)-5d) show immunohistochemical staining photographs of cathepsin B in paraffin-embedded colon sections: Figure 5a) healthy mouse, scale bar = 200 μm, magnification x400; Figure 5b) healthy mouse, scale bar = 50 μm, magnification x100; Figure 5c) CRC mouse, scale bar = 200 μm, magnification x400; and Figure 5d) CRC mouse, scale bar = 50 μm, magnification x100. The immunohistochemical marker of cathepsin B was detected using DAB substrate (brown). Cell nuclei were counterstained using hematoxylin (blue / purple).

[0032] Figure 6a)-6d) show the schematic diagram of 5-FU-based compounds in CRC mouse model; Figure 6b) macroscopic photograph of mouse colon tumor; Figure 6c) tumor area and Figure 6d) body weight change in mice treated with control group (PBS only), 5-FU (50 mg / kg), compound 7 (50 mg / kg), and 5-FU + compound 17 (10 mg / kg 5-FU and 30 mg / kg compound 17, dose based on compound 7). Experimental data are expressed as mean ± standard error of the mean (SEM) (n=5). ****P<0.0001, or *P<0.05 (one-way ANOVA).

[0033] Figures 7a)-7d) show: Figure 7a) schematic diagram of SN-38-based compounds in CRC mouse model, Figure 7b) macroscopic photograph of mouse colon tumor, Figure 7c) comparative diagram of tumor area in intraperitoneal (ip) injection group, Figure 7d) tumor area in intravenous (iv) injection group, Figure 7e) weight change diagram of mice treated with control group (5% DMA+2% Tween80+93% PBS), irinotecan (20 mg / kg), compound 31 (20 mg / kg), and irinotecan + compound 17 (20 mg / kg irinotecan and 14 mg / kg compound 17) via ip injection, Figure 7f) weight change diagram of mice treated with control group (10% DMSO+20% Cremophor+10% Na 2 CO 3 The weight change diagrams of mice treated with SN-38 (5% in double distilled water + 60% D5W), irinotecan (40 mg / kg), compound 38 (40 mg / kg), compound 38 (20 mg / kg), compound 37 (40 mg / kg), compound 37 (20 mg / kg), SN-38 + compound 17 (12 mg / kg SN-38 and 13 mg / kg compound 17, based on compound 38 at a dose of 40 mg / kg), SN-38 + compound 16 (10 mg / kg SN-38 and 15 mg / kg compound 16, based on compound 37 at a dose of 40 mg / kg), compound 17 (13 mg / kg), and compound 16 (15 mg / kg) are shown. Experimental data are expressed as mean ± standard error of the mean (SEM) (n = 3-5). ****P<0.0001, ***P<0.001, **P<0.01 or *P<0.05 (one-way analysis of variance).

[0034] Figures 8a)-8c) show the pharmacokinetic profiles of compound 7 and 5-FU in mice after iv and ip injections. Figures 8a) and 8b) show plots of plasma concentrations as a function of time after iv injection of compound 7 (50 mg / kg) and ip injection of compound 7 and 5-FU (50 mg / kg). Data points showing concentrations below the detection limit of the LC-MS / MS method used were arbitrarily placed just below 0.003 μg / mL. Figure 8c) shows the distribution of 5-FU 1 hour after ip injection of free 5-FU (9.5 mg / kg) and compound 7 (50 mg / kg, 5-FU equivalent dose) in BALB / c mice. Experimental data are expressed as mean ± standard error of the mean (SEM) (n=3). *P<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] definition

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present document, including definitions, shall prevail. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

[0037] Throughout this specification, unless otherwise specified, the word "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of other elements or integers, or group of elements or integers.

[0038] As used herein, the term "conjugate" refers to two or more molecules covalently linked into a larger construct. In some embodiments, the conjugate comprises one or more specific binding molecules covalently linked to one or more therapeutic molecules.

[0039] As used herein, the terms "Formula I" and "Formula III" may be referred to as "conjugates of the invention" in the following text. Such terms are also defined to include all forms of the conjugates of Formula I or Formula III, including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs, and metabolites thereof. For example, the conjugates of the invention or pharma- ceutically acceptable salts thereof can exist in unsolvated and solvated forms. When tightly bound to a solvent or water, the conjugate has a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, such as in channel solvates and hygroscopic compounds, the water / solvent content depends on humidity and drying conditions. In such cases, nonstoichiometry is the norm.

[0040] Stereoisomers of formula I or formula III include optical isomers, such as cis and trans isomers, R and S enantiomers, diastereomers, geometric isomers, rotamers, configurational isomers, and tautomers of the compounds of the invention, including compounds exhibiting more than one type of isomerism, and mixtures thereof, such as racemates and diastereomeric pairs.Also included are acid addition or base addition salts in which the counterion is optically active (e.g., D-lactate or L-lysine) or racemic (e.g., DL-tartrate or DL-arginine).

[0041] When any racemate crystallizes, two different types of crystals are possible. The first type is the aforementioned racemate (true racemate), which produces one homogeneous form of crystal containing both enantiomers in equimolar amounts. The second type is a racemic mixture or aggregate, which produces two forms of crystals, each containing a single enantiomer, in equimolar amounts.

[0042] The conjugate of formula I or formula III may show tautomerism, and such tautomers are also considered as compounds of the present invention.All such tautomeric forms and their mixtures are included in the scope of the conjugate of formula I or formula III.Tautomers exist as a mixture of tautomeric sets in solution.In solid form, one tautomer usually predominates.Although one tautomer may be described, the present invention also includes all tautomers of the conjugate of formula I or formula III and its salts.

[0043] The phrase "pharmaceutically acceptable salts" as used herein includes salts of acidic or basic groups that may be present in the compounds described herein, unless otherwise defined. The compounds that are essentially basic and are used in the methods of the present invention can form 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 include, for example, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, Ethyl succinate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, Non-toxic acid addition salts, i.e., salts which contain pharmacologically acceptable anions, include nitrates, oleates, oxalates, pamoates (embonates), palmitates, pantothenates, phosphates / diphosphates, polygalacturonates, salicylates, stearates, subacetates, succinates, tannates, tartrates, theoclates, tosylates, triethiodides, and valerates.

[0044] As used herein, the term "active pharmaceutical moiety" refers to the portion of an active pharmaceutical ingredient (API) that has a physiological, pharmaceutical, pharmacological or therapeutic effect. The term is intended to include the API in any suitable form, such as, for example, its pharmaceutically acceptable salt, complex, solvate or prodrug, or, where relevant, in any stereoisomeric form, including any enantiomeric or racemic form, or any combination thereof. Illustrative examples of APIs include, but are not limited to, hormones, peptides, small molecules or prodrugs thereof. In another embodiment, the active pharmaceutical moiety can be a biomolecule that includes one or more chemical groups that are normally synthesized in vivo, including, but not limited to, amino acids, nucleotides, polysaccharides, monosaccharides, lipids or combinations thereof.

[0045] As used herein, the term "prodrug" refers to any substance, molecule or entity that is in a form based on that substance, molecule or entity that acts as a therapeutic agent after chemical or physical changes. Prodrugs are covalently linked or sequestered in a particular manner and can be released or converted to an active pharmaceutical moiety before, during, or after administration to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compound such that the modifications are cleaved to the parent compound, either by routine manipulation or in vivo. Prodrugs include compounds that are bonded to any group that, when administered to a mammalian subject, hydroxy, amino, thiol, or carboxyl groups are cleaved to form hydroxy, amino, thiol, or free carboxyl groups, respectively.

[0046] As used herein, the term "targeting module" refers to a portion of a molecular structure that can directly or indirectly bind to a target of interest (e.g., a receptor on a cell surface or a protein in a tissue) and thus facilitate delivery of the molecular structure of the present invention to the target of interest. In certain instances, the targeting module can direct the molecular structure to a location adjacent to a target cell. In other instances, the targeting module can specifically bind to a molecule on the surface of a target cell, or the targeting module can specifically bind to a second molecule that can specifically bind to a molecule on the surface of a target cell. In certain instances, once the targeting module binds to the target of interest, the targeting module can internalize the molecular structure of the present invention, allowing it to move into the cytoplasm of the target cell. The targeting module can be an antibody or ligand for a cell surface receptor, or a molecule that binds to said antibody or ligand, thereby indirectly targeting the molecular structure of the present invention to a target site (e.g., the surface of a selected cell).

[0047] As used herein, the term "glucose transporter" refers to a sodium-dependent glucose transporter (SGLT) or facilitative glucose transporter (GLUT). SGLTs transport glucose into cells against a concentration gradient by coupling active transport of sugar with passive transport of sodium across the cell membrane. The inward sodium gradient in the apical domain of epithelial cells is maintained by ATP-driven active extrusion of sodium across the basolateral domain on the abluminal surface of the cell. Phlorizin specifically and competitively inhibits both SGLT-1 and SGLT-2 and has no effect on GLUT1-12.

[0048] Among GLUT1-12, the insulin-independent glucose transporter GLUT-1 is widely overexpressed in human cancers, and high expression of these proteins in tumor biopsy samples correlates with the prognosis of malignant cancers, making it an attractive therapeutic target. Phloretin is a known, naturally abundant GLUT-1 inhibitor. Also, many glucose transporter 1-4 inhibitors have been identified, including cytochalasin B, WZB117, GLUT-1, GLUT-2, BAY-876, Chromopynone-1, Glutor, Glupin, and NV-5440, as disclosed in ChemBioChem 2020, 21, 45-52.

[0049] As used herein, the term "anti-cancer therapeutic moiety" refers to the portion of an anti-cancer drug, including all chemical or physical interventions used in cancer treatment. Anti-cancer drugs include chemotherapeutic drugs such as cytotoxic or immunotoxic drugs, as well as radiolabeled antibodies, peptides, and chemicals that can emit alpha, beta, and gamma rays, as well as electrons. In addition, radiation therapy includes charged particles such as photons, electrons, positrons, muons, protons, alpha particles, and heavy atomic nuclei from accelerators of sufficiently high energy, as well as neutrons and gamma rays. In some embodiments, the anti-cancer drug can be selected from 5-fluorouracil (5-FU), SN-38, irinotecan, lonidamine, bleomycin, mitomycin, cisplatin, doxorubicin, DM-1, taxol, cabazitaxel, floxuridine, or FdUMP.

[0050] As used herein, the term "substituted" means that one or more hydrogens on a specified atom are replaced with a selection from a specified group, provided that the replacement does not exceed the normal valence of the specified atom in its existing state, and provided that the replacement results in a stable compound.

[0051] As used herein, the term "optionally substituted" refers to a group that is unsubstituted or substituted with one or more substituents.

[0052] As used herein, "treatment" and "treating" include both preventive (i.e., prophylactic) or therapeutic (i.e., curative and / or palliative) treatment. Thus, the terms "treatment" and "treating" particularly include, in a defined form, therapeutic treatment of patients who have already developed said condition. Therapeutic treatment may be symptomatic treatment to alleviate symptoms of a particular indication, or causal treatment to reverse or partially reverse the indication condition, or to stop or slow the progression of the disease. Thus, the conjugates, compositions and methods of the present disclosure can be used, for example, as therapeutic treatment over a period of time, and for chronic therapy. Furthermore, the terms "treatment" and "treating" include preventive therapeutic treatment, i.e., treatment of patients at risk of developing the conditions mentioned herein above, thus reducing said risk.

[0053] As used herein, the term "therapeutically effective amount" refers to an amount of a conjugate of the present disclosure that (i) treats or prevents a particular disease or condition, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease or condition, or (iii) prevents or delays the onset of one or more symptoms of a particular disease or condition described herein.

[0054] "Cancer," "tumor," and similar terms include precancerous, neoplastic, transformed, and cancerous cells, and can refer to solid or non-solid cancers (see, e.g., Edge et al., AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009)). Cancer includes both benign and malignant neoplasms (abnormal growths). "Transformation" is a spontaneous or induced phenotypic change, e.g., cellular immortalization, morphological changes, abnormal cell proliferation, loss of contact inhibition, fixation, and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Transformation can result from infection with a transforming virus and uptake of new genomic DNA or uptake of exogenous DNA, but can also occur spontaneously or following exposure to carcinogens.

[0055] Conjugates and their general preparation procedures

[0056] In one embodiment of the first aspect of the disclosure, the conjugate has formula I: ALB (I) or a pharma- ceutically acceptable salt thereof.

[0057] A is an active pharmaceutical moiety or a prodrug thereof, in particular an anti-cancer therapeutic moiety, which in some embodiments of the disclosure is 5-fluorouracil (5-FU), SN-38, irinotecan, lonidamine, bleomycin, mitomycin, cisplatin, doxorubicin, DM-1, taxol, cabazitaxel, floxuridine, or FdUMP.

[0058] B is a targeting module. In some embodiments of the present disclosure, B is a targeting module that targets tumor cells, such as a transporter-specific binding agent. In some embodiments, the glucose transporter is a sodium-dependent glucose transporter (SGLT) or a facilitative glucose transporter (GLUT). In some other embodiments, B can be selected from phloretin, optionally substituted with glucose, mannose, or 2-fluoro-D-glucose.

[0059] Cancer cells, compared to non-cancerous cells, rely on the anaerobic glycolytic pathway to a greater extent than mitochondrial oxidative phosphorylation, the so-called Warburg effect. There are two categories of glucose transporters that allow cancer cells to take up glucose: (i) facilitative GLUTs (1-4) and (ii) secondary active sodium-glucose cotransporters (SGLT-1-2). Without wishing to be limited by theory, it is believed that tumor cells overexpress glycolytic enzymes and glucose transporters, both of which correlate with the invasiveness and metastatic potential of cancer. Specimens from CRC patients treated with 5-FU were found to have higher transcript levels of glucose transporters in those refractory to therapy, and overexpression of glucose transporters is known to correlate with 5-FU resistance in colon cancer cells via free radical scavenging by pyruvate.

[0060] Phloretin, a known naturally abundant GLUT-1 inhibitor, has been investigated for its anticancer activity, and preclinical studies have demonstrated its ability to overcome anticancer drug treatment resistance when administered in combination with anticancer drugs. Phlorizin is a distinct SGLT-1 inhibitor from phloretin in that it has an additional glucose conjugate at the orthohydroxy group of the phenyl moiety. Because these glucose transporters are physiologically expressed in a wide range of cells and tissues, phloretin or phlorizin has the ability to selectively target SGLT-1 or GLUT-1 on cancer cells.

[0061] L is of formula II: A linker represented by TIFF2025513000000028.tif20170, During the ceremony, R 1 Ha-SR 2 or TIFF2025513000000029.tif16170, R 2 is R 3 , -SR 3 or TIFF2025513000000030.tif21170, R 3 Ha-(CH 2 ) n -R 4 and R 4 is R 5 , -OC(O)-R 5 , -C(O)-R 5 , -C(O)NH-R 5 , -C(O)-, -NHC(O)-R 5 , -OC(O)OR 5 or -C(O)-Z-NH-phenylene-R 5 and R 5 Ha-(CH 2 ) m - and Z is -Val-Cit-, -Phe-Lys-, -Val-Ala- or -Gly-Phe-Leu-Gly-, p is an integer from 2 to 9; n is an integer from 2 to 9; m is 0 or 1, and The dotted lines are covalent bonds.

[0062] L is redox sensitive and / or biodegradable in the tumor environment. When the conjugate is in a normal environment, L is intact and connects A and B. When the conjugate is in a GSH-rich or cathepsin B-rich environment, L is degraded and A is released to exhibit pharmaceutical activity.

[0063] Examples of L include, but are not limited to, redox-sensitive and / or biodegradable linkers, succinimidyl thioethers, disulfide linkers, triazoles, valine-citrulline dipeptides, p-aminobenzyl spacers, and ether bonds.

[0064] In one embodiment, R 1 Ha-SR 2 and R 2 teeth TIFF2025513000000031.tif22170, R 3 Ha-(CH 2 ) n -R 4 and R 4 -C(O)-R 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 5, and m is 0.

[0065] In one embodiment, R 1 Ha-SR 2 and R 2 Ha-SR 3 and R 3 Ha-(CH 2 ) n -R 4 and R 4 HA-OC(O)-R 5 OR-OC(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 2, and m is 0.

[0066] In one embodiment, R 1 is SR 2 and R 2 teeth TIFF2025513000000032.tif22170, R 3 Ha-(CH 2 ) n -R 4 and R 4is -C(O)-Z-NH-phenylene-R 5 and R 5 Ha-(CH 2 ) m -, Z is -Val-Cit-, p is 2, n is 4 and m is 1.

[0067] In one embodiment, R 1 teeth TIFF2025513000000033.tif18170, R 3 Ha-(CH 2 ) n -R 4 and R 4 -C(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 3, and m is 1.

[0068] In one embodiment, R 1 Ha-SR 2 and R 2 teeth TIFF2025513000000034.tif22170, R 3 Ha-(CH 2 ) n -R 4 and R 4 -C(O)OR 5 and R 5 Ha-(CH 2 ) m -, p is 2, n is 4, and m is 1.

[0069] In one embodiment, R 1 Ha-SR 2 and R 2 Ha-SR 3 and R 3 (CH 2 ) n -R 4 and R 4 -C(O)OR 5 and R 5 Ha-(CH 2 )m -, p is 2, n is 3, and m is 1.

[0070] In some embodiments of the present disclosure, the conjugate is not a compound selected from the group consisting of: TIFF2025513000000035.tif72170TIFF2025513000000036.tif38170and TIFF2025513000000037.tif74170

[0071] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, the conjugate has formula III: Represented as TIFF2025513000000038.tif23170, During the ceremony, R is OH or The file is TIFF2025513000000039.tif22170.

[0072] In one embodiment of the conjugate or a pharma- ceutically acceptable salt thereof, the conjugate is selected from the group consisting of: TIFF2025513000000040.tif58170TIFF2025513000000041.tif22170TIFF2025513000000042.tif30170TIFF2025513000000043.tif32170 and TIFF2025513000000044.tif57170, where R is OH or The file is TIFF2025513000000045.tif22170.

[0073] In some embodiments of the present disclosure, in compounds 4-7, 30-31, and 44-45, phloretin 3 (or phlorizin 2) is linked to 5-FU, SN-38, or irinotecan via a redox-sensitive linker. Compounds 4-5 used succinimide thioether (moderately sensitive linker), whereas compounds 6-7 and 30-31 instead incorporate a disulfide linker (highly sensitive linker). Compounds 8-9 and 42-43 are also provided, which incorporate a triazole phosphorus conjugate as a stable linker-bearing group. Due to the overexpression of cathepsin B in the tumor microenvironment, compounds 37 and 38 are constructed with a valine-citrulline dipeptide, a p-aminobenzyl spacer, and an ether bond, and released SN-38 by the activity of cathepsin B and 1,6-elimination.

[0074] The compounds of formula I of the present disclosure are prepared according to general chemical synthesis procedures. The preparation of embodiments of the compounds of the present disclosure is described below.

[0075] Pharmaceutical Compositions and Methods of Treatment of the Present Disclosure

[0076] One embodiment of the second aspect of the present disclosure is a method for treating disease, recurrence or progression in a subject, or for increasing the chances of survival over the relevant period in a subject diagnosed with a disease, comprising administering to the subject a conjugate as described herein, or a pharma- ceutically acceptable salt thereof.

[0077] In some embodiments of the present disclosure, a series of drug conjugates including 5-FU, SN-38, irinotecan, and phlorizin or phloretin linked with succinimide thioether, disulfide, triazole, or cathepsin B sensitive linkers have been designed, synthesized, and biologically evaluated. For 5-FU based prodrugs, most of these compounds were less potent than 5-FU against CRC cell lines, but compound 7 incorporating 5-FU, phloretin, and disulfide bonds showed good stability (t) in human plasma. 1 / 2= 13 h) and instability in the presence of 5 mM GSH (t 1 / 2 =10.3h) and its cytotoxicity against HCT-116 and HT-29 cell lines was similar to that of 5-FU. In an orthotopic mouse CRC model, compound 7 showed excellent antitumor efficacy and low toxicity, reducing tumor volume by 67% (5-FU only reduced tumor volume by 33%) without reducing mouse body weight. The colon targeting effect was the result of high tissue penetration and GLUT-1 targeting efficacy, resulting in significant tumor inhibition compared to 5-FU. In SN-38-based prodrugs, compound 31 showed similar cytotoxicity to SN-38 in cell assays, but showed superior antitumor efficacy to irinotecan by reducing tumor volume by 70% (irinotecan only reduced tumor volume by 27%) in the absence of 65% SN-38 via the ip route. Also, compound 38 (40 or 20 mg / kg) showed a superior therapeutic effect to irinotecan (40 mg / kg) in a dose-dependent manner by iv injection when only 43% or 22% of SN-38 was administered. Furthermore, both compounds 31 and 38 did not reduce body weight during treatment. Thus, targeting GLUTs with cytotoxic small molecule drugs conjugated with glutathione-sensitive and / or cathepsin B linkers is proposed as a novel strategy for CRC treatment.

[0078] In one embodiment, the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of the disclosed conjugate. Such a method comprises administering to the subject a conjugate of the present disclosure in an amount sufficient to treat the condition. For example, the cancer includes, but is not limited to, the group consisting of lung cancer, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, head and neck cancer, lymphoma, leukemia, myeloma, and myeloproliferative neoplasm.

[0079] The conjugates may be formulated into pharmaceutical compositions that can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Pharmaceutically acceptable carriers and diluents are familiar to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers and / or diluents include saline and sterile water, and may include antioxidants, buffers, bacteriostatic agents, and other common additives as required. The compositions may also be formulated as pills, capsules, granules, or tablets that contain diluents, dispersants and surfactants, adhesives, and lubricants in addition to the compounds of the present disclosure.

[0080] The pharmaceutical compositions within the scope of the present invention include all compositions that combine the compound of the present invention with a pharma-ceutically acceptable carrier.In one embodiment, the compound is present in the composition in an amount effective to achieve its intended therapeutic purpose.Although individual needs may vary, determining the optimal range of effective amount of each compound is within the skill of the art.

[0081] The conjugates of the present disclosure may be useful in combination with one or more second therapeutic agents, particularly those therapeutic agents applied in the treatment and / or prevention of the conditions and diseases set out above.

[0082] In one embodiment, the conjugate is present in the composition in an amount effective to achieve its intended therapeutic purpose. Although individual needs may vary, determination of optimal ranges of effective amounts of each compound is within the skill of the art.

[0083] The conjugates of the present disclosure may be useful in combination with one or more second therapeutic agents, particularly therapeutic agents applied in the treatment and / or prevention of the conditions and diseases described herein.

[0084] For oral administration, suitable pharmaceutical compositions of the present invention include powders, granules, pills, tablets, lozenges, chewable tablets, gels, and capsules, as well as liquids, syrups, suspensions, elixirs, and emulsions.These compositions may also include antioxidants, flavorings, preservatives, suspending agents, thickening and emulsifying agents, coloring agents, flavorings, and other pharma-ceutically acceptable additives.Formulations for oral administration can be formulated to be immediate or sustained release, which sustained release includes delayed release, sustained release, pulsed release, controlled release, targeted release, and program controlled release.

[0085] For parenteral administration, the compound of the present invention is directly administered into blood, muscle or internal organs via intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous or other injection or infusion.Parenteral preparations can be prepared in aqueous injection solutions that may contain, in addition to the compounds of the present disclosure, buffers, antioxidants, bacteriostatic agents, salts, carbohydrates and other additives that are commonly used in such solutions.Parenteral administration can be immediate release or sustained release (such as injection or implanted depot).

[0086] The conjugates of the present disclosure can also be administered topically to the skin or mucosa, into the dermis (intradermally), or transdermally. Typical formulations include gels, hydrogels, lotions, solutions, creams, ointments, dressings, foams, skin patches, wafers, implants, and microemulsions. Compounds of the present invention can also be administered via inhalation or intranasal administration, for example, using dry powders, aerosol sprays, or drops. Additional routes of administration of compounds of the present invention include vaginal administration, rectal administration (by suppository, pessary, or enema), ocular administration, and aural administration. Working Example

[0087] chemical method

[0088] Synthetic Route

[0089] Compounds 4–9, 30–31, 37–38, and 42–45 were constructed by sequential derivatization of the central linker (Scheme 1). Triethylene glycol and TsCl were dissolved in ethanol / H 2 O to produce a tosylated intermediate, which was then added with a solution of potassium thioacetate in acetone. After heating the mixture under reflux for 1 h, compound 11 was obtained in 85% yield. Hydrolysis of compound 11 was achieved using potassium carbonate (0.05 M) to give compound 12 in 80% yield, the free thiol of which was protected by 2-methyl-2-propanethiol to give compound 13 in 71% yield. After mesylation and bromide displacement, the brominated compound 14 was obtained in 57% yield. Alkylation of phlorizin (3) with compound 14 using potassium carbonate as base gave compound 15 (54%) as the major product. The regioselective nature of this alkylation is probably due to the electroattractive effect of the carbonyl group at the para position of the benzyl group, which reduces the steric hindrance at this position. Deprotection of the tert-butylthiol with 4.0 N NaOH and TCEP gave compound 16 in 86% yield, which was refluxed with 1.0 N HCl at 90 °C for 3 h and then deprotected with NaHCO 3 (aqueous solution) to give compound 17 (73% yield).

[0090] Phlorizin and phlorizin azide-containing derivatives 20 and 21 were prepared similarly. Methanesulfonyl chloride (MsCl) was added slowly with stirring to a solution of triethylene glycol and triethylamine in DCM at 0° C. After 20 min, the reaction mixture was concentrated and the residue was dissolved in EtOH and added NaN 3 After refluxing for 24 h, the azido linker 18 was obtained. This was mesylated and the mesyl group was subsequently exchanged for bromine using lithium bromide to give compound 19, which was used to derivatize phlorizin to give the desired intermediate 20. After hydrolysis, the phloretin derivative 21 was obtained. TIFF2025513000000046.tif129170 Scheme 1. Synthetic route to phloridzin and phloretin conjugates

[0091] N-1 alkyl group-substituted 5-FU derivatives have low cytotoxicity and are not converted to 5-FU in vivo. Anticipating this problem, an ester functional group was designed into the linker, and it was hypothesized that this ester functional group would be cleavable by endogenous esterases in the tumor microenvironment. Reaction of 5-FU with formaldehyde, followed by 6-maleimidocaproic acid, 4-(pyridine-2-disulfide)-butanoic acid or 4-pentynoic acid, and coupling reagents dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) gave compounds 22-24, respectively (Scheme 2). Compounds 16 and 17 were conjugated with the maleimide of 5-FU (22) to give compounds 4 and 5, respectively, and the carboxylic acid of 23 was reacted to give compounds 6 and 7, respectively. Compounds 20 and 21 were conjugated with the alkyne-containing 5-FU (24) under click conditions (Cu(I)-catalyzed azide-alkyne cycloaddition) to give compounds 8 and 9, respectively. TIFF2025513000000047.tif114170 Scheme 2. Synthetic route for conjugates 4-9

[0092] Using mercaptoethanol 25) as a starting material, compounds 30 and 31 were synthesized through four steps (Scheme 3). First, compound 25 was coupled with aldrithiol-2 in the presence of acetic acid to give compound 26. Second, the hydroxy group of compound 26 was reacted with p-nitrophenyl chloroformate to give compound 27. Compound 27 was then reacted with compound 28 (SN-38) to give compound 29, which was then added with compound 16 or compound 17 in THF under nitrogen conditions, respectively, to give compound 30 or 31.

[0093] The synthesis of compounds 37 and 38 was shown in Scheme 4. Fmoc-val-osu (32) was reacted with L-citrulline in THF / water / dimethoxymethane (DMM) by using sodium bicarbonate as a base to give compound 33 (Fmoc-val-cit). Compound 33 was then reacted with p-aminobenzyl alcohol in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to give compound 34 (Fmoc-val-cit-pab-OH). To synthesize compound 36 containing an ether bond, the benzyl alcohol of compound 34 was replaced with a bromine leaving group by adding HBr at a concentration of 33% in glacial acetic acid to give intermediate 35. Intermediate 35 was added to a mixture of compound 28 and cesium carbonate in DMF. After stirring for 20 min, piperidine was added to the reaction mixture for Fmoc deprotection to give val-cit-pab-O-SN-38 in a one-pot reaction. Subsequently, val-cit-pab-O-SN-38 was reacted with TEA and 5-maleimidovaleric acid-NHS in DMF to give compound 36. Finally, compound 36 was coupled with compound 16 or 17 in MeOH to give compound 37 or 38, respectively.

[0094] The synthesis of compounds 42-45 is disclosed in Scheme 5. Irinotecan was subjected to a coupling reaction with 4-pentynoic acid or 6-maleimidocaproic acid in the presence of DCC and DMAP to give compounds 40 or 41, respectively. Subsequently, the alkyne-containing 40 was conjugated with compounds 20 or 21 under click conditions to give compounds 42 and 43. Meanwhile, maleimide 41 was conjugated with compounds 16 or 17 to give compounds 44 and 45, respectively. TIFF2025513000000048.tif74170 Scheme 3. Synthetic route for conjugates 30 and 31 TIFF2025513000000049.tif149170 Scheme 4. Synthetic route for conjugates 37 and 38 TIFF2025513000000050.tif207170 Scheme 5. Synthetic route for conjugates 42-45

[0095] General chemicals.

[0096] All reagents and solvents used in the synthesis were of reagent grade and were used without further purification. HPLC analysis was performed on a HITACHID-2000Elite system equipped with a BDS HYPERSIL C18 (5 μm, 4.6 × 250 mm) column. The mobile phase was ACN and ddH 2 O or NaH 2 PO 4 The mixture of buffers was filtered through a 0.45 μm membrane filter before use. The column was eluted with the mobile phase at a flow rate of 1.0 mL / min. The eluate was monitored by measuring the absorbance at 254 nm or 265 nm at 25 °C. The purity of all final products was confirmed to be greater than 95% by HPLC before in vitro and in vivo use. Thin layer chromatography (0.25 mm, E. Merck Silica Gel 60F) was used to determine the purity of the final products. 254 ) was used to monitor the progress of the reaction. Plates were visualized by UV (state wavelength) or by staining with cerium or ninhydrin and heating. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were acquired on a Bruker AV-400 (400 MHz) and a Bruker AV-600 (600 MHz) (see Supplementary Data). Chemical shifts are referenced to the residual solvent peak in parts per million (δ): 6 -DMSO, 1 Hδ=2.50, 13 Cδ=39.52; CD 3 In the case of O.D. 1 Hδ=3.31, 13 Cδ=49.00;d 6 - In the case of acetone, 1 Hδ=2.05, 13 Cδ = 29.84, 206.26; CDCl 3 in the case of, 1 Hδ=7.26, 13Cδ=77.16. Coupling constants (J) are given in Hertz (Hz). Splitting patterns are indicated as s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), and m (multiplet). Mass spectra were acquired using a Burker bioTOF III and are reported in m / z.

[0097] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl 6-(3-((2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propionyl)-5-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanoate (4) To a solution of compound 16 (35 mg, 0.06 mmol) in dry methanol (2 mL) was added compound 22 (21 mg, 0.06 mmol). The reaction mixture was stirred for 10 min and then concentrated in vacuo. The residue was purified by column chromatography (silica gel, DCM / MeOH=12 / 1) to give compound 4 (53 mg, 0.06 mmol, >99%) as a foam. 1 H NMR (600MHz, CD 3OD) δ 7.88 (d, J = 6 Hz, 1H, H-29), 7.07 (d, J = 8.2 Hz, 2H, H-2, H-6), 6.67 (d, J = 8.2 Hz, 2H, H-3, H-5), 6.31 - 6.29 (m, 1H, H-3’), 6.11 - 6.09 (m, 1H, H-5’), 5.61 (s, 2H, H-23), 5.11 (d, J = 7.2 Hz, 1H, H-1’’), 4.15 (t, J = 4.8 Hz, 2H), 4.03 - 4.00 (m, 1H), 3.90 (d, J = 4.2 Hz, 1H), 3.81 (t, J = 4.2 Hz, 2H), 3.77 - 3.70 (m, 3H), 3.67 (t, J = 4.2 Hz, 2H), 3.63 (t, J = 5.4 Hz, 2H), 3.50 - 3.45 (m, 5H), 3.42 - 3.30 (m, 3H), 3.17 - 3.11 (m, 1H), 3.09 - 3.05 (m, 1H), 2.89 - 2.81 (m, 3H), 2.50 (dd, J 1 = 15.6, 3 Hz, 1H), 2.33 (t, J = 7.2 Hz, 2H, H-21), 1.60 - 1.58 (m, 2H, H-20), 1.52 - 1.50 (m, 2H, H-18), 1.28 - 1.26 (m, 2H, H-19) ppm; 13 C NMR (150 MHz, CD 3 OD) 206.8, 178.9, 177.2, 174.6, 167.2, 166.2, 161.9, 159.6 (d, J CCF = 26.1 Hz), 156.3, 151.0, 141.4 (d, J CF = 232.5 Hz), 133.7, 130.5 (d, J CCF = 33.1 Hz), 130.4 (2C), 116.0 (2C), 107.6, 102.1, 97.1, 95.2, 78.4, 74.7, 72.1, 71.7, 71.6, 71.2, 71.1, 70.4, 69.0, 62.5, 49.8, 47.0, 40.8, 39.4, 37.3, 34.3, 32.0, 30.6, 28.0, 26.9, 25.0 ppm; HRMS (ESI TOF-MS) C 42 H 52 FN 3 O 18 SNa + [M + Na] + Calculated value: 960.2843, Experimental value: 960.2847.

[0098] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl 6-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanoate (5) To a solution of compound 17 (22 mg, 0.05 mmol) in dry MeOH (1 mL) was added compound 22 (19 mg, 0.05 mmol). The reaction mixture was stirred for 10 min and then concentrated in vacuo. The residue was purified by column chromatography (silica gel, DCM / MeOH=12 / 1) to give compound 5 (39 mg, 0.05 mmol, >99%) as a foam. 1 H NMR (400 MHz, CD 3 OD) δ7.89(d, J=6Hz, 1H, H-29), 7.05(d, J=8.4Hz, 2H, H-2, H-6), 6.68(d, J=8.4Hz, 2H, H-3, H-5), 5.92( s, 2H, H-3', H-5'), 5.61(s, 2H), 4.09(t, J=4.4Hz, 2H), 4.09-3.99(m, 1H), 3.81(t, J=4.4Hz, 2H), 3.76- 3.72(m, 2H), 3.69-3.64(m, 4H), 3.42(t, J=7Hz, 2H), 3.21-3.20(m, 1H), 3.11-3.07(m, 2H), 2.87-2.83 (m, 3H), 2.47(dd, J=14.8, 3.6Hz, 1H), 2.34(t, J=7.3Hz, 2H), 1.62-1.50(m, 4H), 1.29-1.27(m, 2H)ppm; 13 C NMR (100 MHz, CD 3 OD) δ206.7, 178.9, 177.1, 174.5, 166.5, 165.5(2C), 159.7(d, J CCF =27.4Hz), 156.4, 151.3, 141.6(d, J CF =227Hz), 133.8, 130.5(d, J CCF=32.2Hz), 130.3(2C), 116.1(2C), 106.1, 94.8(2C), 72.1, 71.7, 71.6, 71.2, 70. 4,68.7,47.4,40.7,39.4,37.2,34.3,32.0,31.2,28.0,26.9,25.0ppm;HRMS(ESI TOF-MS)C 36 H 42 FN 3 O 13 SNa + [M+Na] + Calculated value: 798.2315, Experimental value: 798.2319.

[0099] 5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl 4-((2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propionyl)-5-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)butanoate((6) To a solution of compound 16 (76 mg, 0.13 mmol) in THF was added compound 23 (50 mg, 0.13 mmol) and phosphate buffer (pH=8.0). The mixture was stirred at ambient temperature for 2 h. The solvent was removed by vacuum. The residue was purified by column chromatography (silica gel, DCM / MeOH=15 / 1 to 10 / 1) to give compound 6 (107 mg, 0.13 mmol, >99%) as an oil. 1 H NMR (400 MHz, CD 3OD) δ7.89(t, J=6.1Hz, 1H), 7.07(t, J=8.4Hz, 2H), 6.68(t, J=8.4Hz, 2H), 6.32(d, J=2.2Hz, 1H), 6.13(d, J=2.2Hz, 1H), 5.62(s, 2H), 5.10-5.03(m, 1H), 4.64(t, J=7.2Hz, 1H), 4.17-4.05(m, 2H) , 3.90(dd, J=12.4Hz, 2.0Hz, 1H), 3.83-3.95(m, 2H), 3.59-3.73(m, 7H), 3.44-3.54(m, 6H), 3.34 -3.39(m, 1H), 2.87(m, 4H), 2.72(t, J=7.0Hz, 2H), 2.49(t, J=7.1Hz, 2H), 1.98(t, J=7.0Hz, 2H); 13 C NMR (100 MHz, CD 3 OD) δ205.5, 172.9, 166.0, 165.0, 160.6, 159.8(d, J CCF =27Hz), 155.0, 149.8, 140.0(d, J CF =233Hz), 132.4, 129.3(d, J CCF =35Hz), 129.1(2C), 114.7(2C), 106.3, 100.8, 95.8, 93.8, 77.1, 77.1, 73.4, 70.0(2C ), 69.8, 69.5(2C), 67.6, 66.3, 61.1, 45.8, 38.0, 37.3, 31.7, 29.3, 23.6ppm;HRMS(ESI TOF-MS)C 36 H 45 FN 2 O 16 S 2 + [M+H] + Calculated value: 845.2267, Experimental value: 845.2279.

[0100] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl 4-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)butanoate (7) To a solution of compound 17 (55 mg, 0.13 mmol) in THF was added compound 23 (50 mg, 0.13 mmol) and phosphate buffer (pH 8.0). The mixture was stirred at ambient temperature for 2 h. The solvent was removed by vacuum. The residue was purified by column chromatography (silica gel, DCM / MeOH=20 / 1) to give compound 7 (85 mg, 0.12 mmol, 92%) as a solid. 1 H NMR (600 MHz, d 6 -acetone) δ7.93(d, J=6.4Hz, 1H), 7.09(d, J=8.4Hz, 2H), 6.75(d, J=9.4Hz, 2H ), 5.69(s, 2H), 6.01(s, 2H), 4.15(t, J=4.6Hz, 2H), 3.81(t, J=4.3Hz, 2H), 3.70(t, J=6.5Hz, 2H), 3.66-3.60(m, 4H), 3.35(t, J=8.0Hz, 2H), 2.89-2.7 7(m, 4H), 2.78(t, J=7.2Hz, 2H), 2.52(t, J=7.2Hz, 2H), 2.07-2.04(m, 2H); 13 C NMR (150 MHz, d 6 -acetone) δ 205.8, 173.3, 166.1, 165.3, 157.9 (d, J CCF =27Hz), 156.4, 150.1, 140.9(d, J CF =231Hz), 133.4, 130.2(2C), 129.0(d, J CCF =34.5Hz), 115.2(2C), 105.7, 94.0(2C), 71.4, 71.3, 71.0, 70.1, 70.0, 68.5, 47.0, 39.3, 38.1, 32.7, 30.5, 30.3, 24.7ppm. HRMS(ESI TOF-MS)C 30 H 35 FN 2 O 11 S 2 + [M+H] + Calculated value: 683.1739, Experimental value: 683.1763.

[0101] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl 3-(1-(2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propionyl)-5-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoate (8) To a solution of compound 20 (95 mg, 0.16 mmol) in EtOH / water (3 / 1, 2 mL) was added tris(benzyltriazolemethyl)amine (TBTA, 18 mg, 0.03 mmol), sodium ascorbate (NaASC, 20.8 mg, 0.10 mmol), copper(II) sulfate pentahydrate (3.3 mg, 0.01 mmol) and compound 24 (38 mg, 0.16 mmol). The reaction mixture was stirred for 12 h and then concentrated in vacuo. The mixture was dissolved in EtOAc and extracted with water. The organic layer was concentrated in vacuo and purified by column chromatography (silica gel, DCM / MeOH=10 / 1) to give compound 8 (117 mg, 0.14 mmol, 88%) as a foam. 1 H-NMR (600MHz, CD 3 OD) δ7.81(d, J=6Hz, 1H, H-24), 7.77(s, 1H, H-13), 7.06(d, J=8.4Hz, 2H, H-2, H-6), 6.67(d, J=8.4Hz, 2H, H-3, H-5), 6.30(d, J=2 .4Hz, 1H, H-3'), 6.12 (d, J=2.4Hz, 1H, H-5'), 5.59 (s, 2H, H-18), 5.07 (d, J=7.2Hz, 1H, H-1''), 4.50 (t, J=4.8Hz, 2H, H-12), 4.1 5(t, J=4.2Hz, 2H, H-7), 3.88(dd, J=10, 2Hz, 1H), 3.80(t, J=4.8Hz, 2H), 3.79-3.77(m, 2H), 3.68(q, J=6Hz, 1H), 3.66-3.61(m, 4 H), 3.5-3.4(m, 5H), 3.38-3.34(m, 1H), 2.94(t, J=7.8Hz, 2H, H-16), 2.88(t, J=7.2Hz, 2H, H-β), 2.72(t, J=7.2Hz, 2H, H-17)ppm;13 C NMR (150 MHz, CD 3 OD) δ206.8, 173.5, 167.2, 166.2, 161.8, 159.5(d, J CCF =26Hz), 156.4, 150.9, 146.9, 141.3(d, J CF =232Hz), 133.7, 130.5(d, J CCF =34Hz), 130.3(2C), 124.4, 116.0(2C), 107.6, 102.1, 97.1, 95.1, 78.5, 78.4, 74.7, 71. 6,71.5,71.4,71.1,70.4,70.3,68.9,62.4,51.3,47.0,34.0,30.6,21.4ppm;HRMS(ESI TOF-MS)C 37 H 45 FN 5 O 16 + [M+H] + Calculated value: 834.2840, Experimental value: 834.2855.

[0102] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl 3-(1-(2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propionyl)-5-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoate (9) To a solution of compound 21 (62.3 mg, 0.14 mmol) in EtOH / water (v / v=3:1, 2 mL) was added TBTA (7.9 mg, 0.01 mmol), sodium ascorbate (NaASC, 45 mg, 0.22 mmol), copper(II) sulfate pentahydrate (5.6 mg, 0.02 mmol) and compound 24 (35.7 mg, 0.15 mmol). The reaction mixture was stirred for 12 h and then concentrated in vacuo. The mixture was then dissolved in EtOAc and extracted with water. The organic layer was concentrated in vacuo and purified by column chromatography (silica gel, DCM / MeOH=14 / 1) to give compound 9 (62.9 mg, 0.09 mmol, 64%) as a foam. 1 H NMR (400 MHz, d 6 -DMSO)δ8.11(d, J=6.4Hz, 1H, H-24), 7.85(s, 1H, H-13), 7.05(d, J=8.4Hz, 2H, H-2, H-6), 6.69(d , J=8.4Hz, 2H, H-3, H-5), 5.96-5.96(m, 2H, H-3', H-5'), 5.59(s, 2H, H-18), 4.48(t, J=5Hz, 2H, H- 12), 4.12-4.05(m, 2H), 3.82(t, J=5Hz, 2H), 3.75-3.65(m, 2H), 3.59-3.51(m, 4H), 3.27(t, J=7.6 Hz, 2H), 2.89(t, J=7.2Hz, 2H, H-16), 2.80(t, J=7.4Hz, 2H, H-β)), 2.7(t, J=7.2Hz, 2H, H-17)ppm; 13 C NMR (100 MHz, CD 3 OD) δ206.7, 173.6, 166.5, 165.6(2C), 159.7(d, J CCF =26Hz), 156.5, 151.1, 147.0, 141.3(d, J CF =232Hz), 133.9, 130.4(d, J CCF =34Hz), 130.3(2C), 124.4, 116.1(2C), 106.1, 94.8(2C), 71.6, 71.6, 71.4, 70.5, 70.3, 68.7, 51.3, 47.4, 34.0, 31.2, 21.5ppm;HRMS(ESI TOF-MS)C 31 H 35 FN5 O 11 + [M+H] + Calculated value: 672.2312, Experimental value: 672.2318.

[0103] S-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)ethanethioate (11) Triethylene glycol (compound 10, 50.0 g, 333 mmol) in THF (300 mL) was cooled to 0° C. in an ice bath. NaOH (4.40 g, 110 mmol) in water (200 mL) was then added and stirred for 10 min. Tosyl chloride (TsCl, 6.34 g, 33.3 mmol) in dry THF (50 mL) was added dropwise (approximately 30 min). After stirring for an additional 2 h at ambient temperature, the reaction was poured into water and extracted with ether. The aqueous layer was re-extracted with DCM. The combined organic layers were washed with MgSO 4 The mixture was dried at 40° C. and concentrated in vacuo to give a colorless oil. To the intermediate in dry acetone (200 mL) was then added potassium thioacetate (3.8 g, 33.3 mmol). The reaction mixture was heated to reflux for 1 h. The undissolved solids were filtered off and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, Hex / EtOAc=1 / 1) to give compound 11 (5.89 g, 28.3 mmol, 85%) as a yellow oil. 1 H NMR (400 MHz, CDCl 3 )δ3.70-3.54(m, 10H), 3.06(t, J=6.4Hz, 2H), 2.30(s, 3H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ195.5, 72.4, 70.2, 69.6(2C), 61.6, 30.4, 28.6ppm;HRMS(ESI TOF-MS)C 8 H 17 O 4 S+Na + [M+Na] + Calculated value: 231.0662, Experimental value: 231.0672.

[0104] 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (12) To a stirred solution of compound 11 (5.7 g, 27.40 mmol) in dry MeOH (50 mL) was added potassium carbonate (345 mg, 2.50 mmol). The reaction mixture was diluted with NH 4 It was quenched with Cl solution and filtered to remove the precipitate. The filtrate was concentrated in vacuo. The residue was dissolved in EtOAc and washed with water. The organic layers were combined and washed with MgSO 4 The residue was concentrated in vacuo and purified by column chromatography (silica gel, Hex / EtOAc=1 / 1) to give compound 12 (3.63 g, 21.86 mmol, 80%) as a yellow oil. 1 H NMR (400 MHz, CDCl 3 )δ3.74-3.71(m, 4H), 3.67-3.65(m, 4H), 3.60-3.57(m, 2H), 2.90(t, J=6.4Hz, 2H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ72.5, 70.3, 70.2, 69.5, 61.6, 38.3ppm;HRMS(ESI TOF-MS)C 6 H 15 O 3 S + [M+H] + Calculated value: 167.0736, Experimental value: 167.0735.

[0105] 2-(2-(2-(tert-butyldihydrothio)ethoxy)ethoxy)ethanol (13) To a stirred solution of compound 12 (1.43 g, 8.56 mmol) in EtOH (5 mL), 2-methyl-2-propanethiol (9.7 mL, 7.76 g, 86.04 mmol) was added. The reaction mixture was cooled to 0° C. and a solution of iodine (3.0 g) in EtOH (20 mL) was added dropwise until the color of the reaction changed from colorless to dark brown. After 12 h, saturated NaHCO 3 The solution was added until the pH was > 7. The reaction solution was concentrated in vacuo. The residue was then dissolved in EtOAc and saturated NaHCO 3 The combined organic layers were extracted with MgSO 4The mixture was dried at 40° C., filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, Hex / EtOAc=2 / 1) to give compound 13 (1.54 g, 6.06 mmol, 71%) as a yellow oil. 1 H NMR (400 MHz, CDCl 3 )δ3.71-3.68(m, 4H), 3.66-3.61(m, 4H), 3.61-3.57(m, 2H), 2.86(t, J=6.8Hz, 2H), 1.30(s, 9H)ppm; 13 C NMR (100 MHz, CDCl 3 ) δ72.4, 70.2, 69.7(2C), 61.6, 47.7, 36.6, 29.7(3C)ppm. HRMS(ESI TOF-MS)C 10 H 22 O 3 S 2 + [M+H] + Calculated value: 255.1083, Experimental value: 255.1089.

[0106] 1-(2-(2-(2-bromoethoxy)ethoxy)ethyl)-2-(tert-butyl)disulfane (14) To a stirred solution of compound 13 (70.0 mg, 0.28 mmol) in dry DCM (2 mL) was added TEA (33.5 mg, 0.33 mmol) at 0° C. and the reaction mixture was stirred for 10 min. To the reaction mixture was added methanesulfonyl chloride (32.0 mg, 0.28 mmol) over 10 min. The reaction mixture was extracted with water followed by MgSO 4 The mixture was dried over 500° C. to give the mesylated intermediate in DCM. To the above solution was then added LiBr (60.2 mg, 0.69 mmol) in THF (1 mL). The residue was stirred at 55° C. for 18 h. The reaction mixture was concentrated and extracted with EtOAc and water, followed by MgSO. 4 The residue was concentrated and purified by column chromatography (silica gel, Hex / EtOAc=8 / 1) to give compound 14 (50 mg, 0.16 mmol, 57% yield, 2 steps) as a colorless oil. 1 H NMR (400 MHz, CDCl 3)δ3.77(t, J=6Hz, 2H), 3.68-3.65(m, 2H), 3.63-3.58(m, 4H), 3.44(t, J=6.8Hz, 2H), 2.83(t, J=6.8Hz, 2H), 1.28(s, 9H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ71.1, 70.3, 70.2, 69.8, 47.7, 39.7, 29.7(3C)ppm;HRMS(ESI TOF-MS)C 10 H 21 Bro 2 S 2 Na + [M+Na] + Calculated value: 339.0059, Experimental value: 339.0066.

[0107] 1-(4-(2-(2-(2-(tert-butyldisulfanyl)ethoxy)ethoxy)ethoxy)-2-hydroxy-6-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3-(4-hydroxyphenyl)propan-1-one (15) Phlorizin (3, 1.0 g, 2.29 mmol) in dry DMF (10 mL) was added with K 2 CO 3 (436 mg, 3.15 mmol) was added at ambient temperature and stirred for 10 min. Compound 14 (1.0 g, 3.16 mmol) was added and the reaction mixture was heated at 55° C. for 12 h. The reaction mixture was concentrated in vacuo. The residue was extracted three times with water / EtOAc. The organic layers were combined and concentrated in vacuo. The residue was purified by column chromatography (silica gel, (DCM / MeOH=10 / 1) to give compound 15 (837 mg, 1.25 mmol, 54%) as a foam. 1 H NMR (400 MHz, CD 3OD) δ7.06(d, J=8.4Hz, 2H, H-2, H-6), 6.68(d, J=8.4Hz, 2H, H-3, H-5), 6.33(d, J=2.4Hz, 1 H, H-3'), 6.14(d, J=2.4Hz, 1H, H-5'), 5.08(d, J=7.6Hz, 1H, H-1''), 4.15(t, J=4.4Hz, 2H ), 3.91-3.88(m, 1H), 3.82(t, J=4.4Hz, 2H), 3.71-3.62(m, 5H), 3.61-3.60(m, 2H), 3.50- 3.44(m, 5H), 3.34(d, J=9.2Hz, 1H), 2.90-2.84(m, 4H), 1.30(s, 9H, H-14, H-15, H-16)ppm; 13 C NMR (150 MHz, CD 3 OD) δ206.8, 167.2, 166.2, 161.8, 156.3, 133.7, 130.3(2C), 116.0(2C), 107.6, 102.1, 97.1, 95.1, 7 8.4,74.7,71.6,71.3,71.1,70.7,70.5,68.9,62.4,48.4,47.0,40.9,30.6,30.2(3C)ppm;HRMS(ESI TOF-MS)C 31 H 44 NaO 12 S 2 Na + [M+Na] + Calculated value: 695.2166, experimental value: 695.2174.

[0108] 1-(2-hydroxy-4-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)-6-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3-(4-hydroxyphenyl)propan-1-one (16) To a stirred solution of compound 15 (156 mg, 0.23 mmol) in dry THF (5 mL) was added TCEP (133 mg, 0.46 mmol) and 4.0 N NaOH solution (1 mL). The reaction mixture was stirred for 5 h and then concentrated in vacuum. The residue was dissolved in EtOAc and the precipitate was filtered. The filtrate was concentrated in vacuum and purified by column (silica gel, DCM / MeOH=10 / 1) to give compound 16 (115 mg, 0.20 mmol, 86%) as a foam. 1 H NMR (400 MHz, CD 3 OD) δ7.06(d, J=8.4Hz, 2H, H-2, H-6), 6.68(d, J=8.4Hz, 2H, H-3, H-5), 6.33(d, J=2.4Hz, 1H, H- 3'), 6.14 (d, J=2.4Hz, 1H, H-5'), 5.08 (d, J=7.2Hz, 1H, H-1''), 4.15 (t, J=4.4Hz, 1H, H-7), 3.9 1(dd, J=10, 2Hz, 1H), 3.84-3.81(m, 2H), 3.71-3.68(m, 3H), 3.64-3.62(m, 2H), 3.59(t, J=6.4H z, 2H), 3.50-3.43(m, 5H), 3.39-3.37(m, 1H), 2.88(t, J=7.5Hz, 2H), 2.64(t, J=6.4Hz, 2H)ppm; 13 C NMR (100 MHz, CD 3 OD) δ206.8, 167.3, 166.3, 161.8, 156.3, 133.7, 130.3(2C), 116.0(2C), 107.6, 102.1, 97.1, 9 5.1,78.5,78.4,74.7,74.1,71.6,71.1(2C),70.5,68.9,62.4,47.0,30.7,24.6ppm;HRMS(ESI TOF-MS)C 27 H 36 O 12 SNa + [M+Na] + Calculated value: 607.1820, experimental value: 607.1821.

[0109] 1-(2,6-dihydroxy-4-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)phenyl)-3-(4-hydroxyphenyl)propan-1-one (17) To a solution of compound 16 (210 mg, 0.36 mmol) in EtOH (2 mL) was added 1.0 N NCl (1 mL). The mixture was heated to reflux for 3 h. The reaction mixture was cooled and the solvent was evaporated in vacuo. The residue was extracted with water and EtOAc and washed with MgSO 4 The organic layers were combined and concentrated in vacuo. The residue was purified by column chromatography (silica gel, Hex / EtOAc=1 / 1) to give compound 17 (111 mg, 0.26 mmol, 73%) as a foam. 1 H NMR (400 MHz, CDCl 3 )δ7.00(d, J=8.4Hz, 2H, H-2, H-6), 6.70(d, J=8.4Hz, 2H, H-3, H-5), 5.87(s, 2H, H-3', H-5'), 4.00(t, J=3.2Hz, 2H), 3.79-3.77(m, 2H), 3.71-3.69(m, 2H), 3.63-3.61(m, 2H), 3.54(t, J=6.4Hz, 2H), 3.27(t, J=7.6Hz, 2H), 2.83(t, J=7.6Hz, 2H), 2.62-2.57(m, 2H); 13 C NMR (100 MHz, CDCl 3 )δ205.2, 164.5(2C), 153.7, 133.4, 129.5(2C), 115.3(2C), 94.5(2C), 77.2, 72.8, 70.4, 70.4, 69.9, 69.4, 67.0, 45.8, 29.8, 23.9;HRMS(ESI TOF-MS)C 21 H 26 O 7 SNa + [M+Na] + Calculated value: 445.1291, Experimental value: 445.1328.

[0110] 2-(2-(2-azidoethoxy)ethoxy)ethanol (18) To triethylene glycol (compound 10, 50.0 g, 333 mmol) in dry DCM (250 mL) was added TEA (37.0 g, 366 mmol) at 0° C. and the reaction mixture was stirred for 10 min. Subsequently, methanesulfonyl chloride (30.5 g, 266 mmol) was added to the reaction mixture over 20 min. The reaction mixture was extracted with water and diluted with MgSO 4 The mixture was dried over 1000 ml and concentrated to give 2-(2-(2-hydroxyacetate)ethoxy)ethyl methanesulfonate. Sodium azide (21.6 g, 332 mmol) was added to the mesylated intermediate in EtOH (250 ml) and heated to reflux overnight. Water was added and the mixture was extracted with DCM. The organic layer was washed with MgSO 4 The mixture was dried at 40° C., concentrated and purified by flash column chromatography (silica gel, EtOAc / Hex=3 / 1 to 1 / 5) to give compound 18 (12.1 g, 69.1 mmol, 26%, 2 steps) as a colorless oil. 1 H-NMR (200 MHz, CDCl 3 )δ3.68-3.64(m, 2H), 3.63-360(m, 6H), 3.57-3.53(m, 2H), 3.34(t, J=5.2Hz, 2H)ppm; 13 C-NMR (50 MHz, CDCl 3 )δ72.4, 70.5, 70.2, 69.8, 61.5, 50.5ppm;HRMS(ESI TOF-MS)C 6 H 14 N 3 O 3 + [M+H] + Calculated value: 176.1030, Experimental value: 176.1029.

[0111] 1-Azido-2-(2-(2-bromoethoxy)ethoxy)ethane (19) To a stirred solution of compound 18 (8.0 g, 45.7 mmol) in dry DCM (100 mL) was added TEA (9.25 g, 91.4 mmol) at 0° C. and the reaction mixture was stirred for 10 min. Subsequently, methanesulfonyl chloride (6.28 g, 54.8 mmol) was added to the reaction mixture over 20 min. The reaction mixture was extracted with water and diluted with MgSO 4The mesylated intermediate was dried over 500 ml of THF to give the mesylated intermediate in DCM. Lithium bromide (11.9 g, 137 mmol) was dissolved in THF (150 mL) and added to the mesylated intermediate and reaction mixture. The residue was stirred at 45° C. for 18 h. The reaction mixture was concentrated and extracted with EtOAc and water, and the EtOAc layer was collected and washed with MgSO 4 The mixture was dried at 40° C., followed by filtration. The filtrate was concentrated and purified by flash column chromatography (silica gel, Hex / EtOAc=4 / 1) to give compound 19 (6.9 g, 29.1 mmol, 64%, 2 steps) as a colorless oil. 1 H NMR (200 MHz, CDCl 3 )δ3.73(t, J=6Hz, 2H), 3.6-3.5(m, 6H), 3.4(t, J=6Hz, 2H), 3.3(t, J=5Hz)ppm; 13 C NMR (50 MHz, CDCl 3 )δ70.9, 70.3, 70.2, 69.7, 50.3, 30.1ppm.

[0112] 1-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-2-hydroxy-6-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3-(4-hydroxyphenyl)propan-1-one (20) Phlorizin (compound 3, 2.31 g, 5.30 mmol) in dry DMF (15 mL) was added at ambient temperature 2 CO 3 (1.11 g, 8.03 mmol) was added and stirred for 10 min. Compound 19 (1.66 g, 7.00 mmol) was added and the reaction mixture was heated at 55° C. for 12 h. The reaction mixture was concentrated in vacuo. The residue was extracted three times with water / EtOAc. The organic layers were combined and concentrated in vacuo. The residue was purified by column chromatography (silica gel, DCM / MeOH=10 / 1) to give compound 20 (2.13 g, 3.59 mmol, 68%) as a foam. 1 H NMR (600 MHz, CD 3OD) δ7.08(d, J=8.4Hz, 2H, H-2, H-6), 6.70(d, J=8.4, 2H, H-3, H-5), 6.34(d, J=2.4H z, 1H, H-3'), 6.15 (d, J=2.4Hz, 1H, H-5'), 5.10 (d, J=7.2Hz, 1H, C-1''), 4.16 (t, J=4 .4Hz, 2H, H-9), 3.91(d, J=2Hz, 1H), 3.83(t, J=4.4Hz, 1H), 3.73-3.70(m, 5H), 3.69- 3.67(m, 1H), 3, 52-3.50(m, 4H), 3.49-3, 40(m, 5H), 2.90(t, J=7.8Hz, 2H, H-12)ppm; 13 C NMR (150 MHz, CD 3 OD) δ206.8, 167.2, 166.2, 161.8, 156.3, 133.7, 130.3(2C), 116.0(2C), 107.6, 102.1, 97.1 ,95.1,78.4,74.7,71.7,71.3,71.1,70.7,70.5,68.9,62.4,51.7,47.0,30.6ppm;HRMS(ESI TOF-MS)C 27 H 35 N 3 O 12 Na + [M+Na] + Calculated value: 616.2113, Experimental value: 616.2115.

[0113] 1-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-2,6-dihydroxyphenyl)-3-(4-hydroxyphenyl)propan-1-one (21) To a solution of compound 20 (195 mg, 0.33 mmol) in EtOH (5 mL) was added 1.0 N HCl (3 mL). The mixture was refluxed at 90° C. for 3 h. The reaction mixture was cooled and the solvent was removed by vacuum. The residue was extracted with water / EtOAc and washed with MgSO 4 The organic layers were combined and concentrated in vacuo. The residue was purified by column chromatography (silica gel, Hex / EtOAc=1 / 1) to give compound 21 (110 mg, 0.26 mmol, 79%) as a foam. 1 H NMR (400 MHz, CDCl 3)δ7.04(d, J=8.4Hz, 2H, C-2, C-6), 6.71(d, J=8.4Hz, 2H, C-3, C-5), 5.95-5.85(m, 2H, C-3', C-5'), 4.06-4.04(m, 2H, C-7), 3.82(t, J= 4.4Hz, 2H), 3.73-3.70(m, 2H), 3.67-3.65(m, 2H), 3.60(t, J=5Hz, 2H), 3.33-3.28(m, 4H, C-α, C-12), 2.89(t, J=7.7Hz, 2Hz, C-β)ppm; 13 C NMR (100 MHz, CDCl 3 )δ204.9, 164.5(2C), 153.7, 133.7, 129.5(2C), 115.2(2C), 105.0, 94.6(2C), 70.6, 70.4, 69.9, 69.8, 67.1, 50.5, 45.8, 29.7ppm;HRMS(ESI TOF-MS)C 21 H 25 N 3 O 7 Na + [M+Na] + Calculated value: 454.1585, Experimental value: 454.1583.

[0114] 1-(6-Maleimidohexanoyloxymethyl)-5-fluorouracil (22) To a solution of 5-FU (100 mg, 0.77 mmol) in water (0.5 mL) was added aqueous formaldehyde (37 wt%, 0.5 mL) at 55° C. and stirred for another 3 h after the solid completely disappeared. The solvent was then removed by vacuum to obtain a colorless and viscous oil. In a separate flask, 6-maleimidocaproic acid (195 mg, 0.92 mmol) in dry ACN (2 mL) was added DCC (190 mg, 0.92 mmol) and DMAP (9.5 mg, 0.08 mmol) at 0° C. and stirred for 10 min. Then, the above oil (0.5 mL) in dry ACN was added to the reaction flask. The reaction mixture was stirred at 0° C. for 30 min and then at room temperature for 12 h. The precipitate formed during the reaction was filtered and the filtrate was evaporated in vacuum. The residue in EtOAc was dissolved in 1.0 N HCl, saturated NaHCO 3 and water. The organic layer was extracted with MgSO 4The mixture was dried at 40° C. and concentrated in vacuo. Purification by column chromatography (silica gel, Hex / EtOAc=1 / 1) afforded compound 22 (117 mg, 0.33 mmol, 43%) as a foam. 1 H NMR (600 MHz, CD 3 OD) δ7.92(d, J=6Hz, 1H, H-6), 6.79(s, 2H, H-16, H-17), 5.63(s, 2H, H-7), 3.48(t, J=6.9Hz, 2H, H-13), 2.38(t, J=7.3Hz, 2H, H-9), 1.68-1.63(m, 2H, H-10), 1.59-1.54(m, 2H, H-12), 1.32-1.27(m, 2H, H-11), 13 C NMR (150 MHz, CD 3 OD) δ174.6, 172.5(2C), 159.7(d, J CCF =25H), 151.1, 141.4(d, J CF =233Hz), 135.3(2C), 130.5(d, J CCF =34Hz), 71.6, 38.2, 34.3, 29.1, 27.0, 25.1, HRMS(ESI TOF-MS)C 15 H 16 FN 3 O 6 C 21 Na + [M+Na] + Calculated value: 376.0915, Experimental value: 376.0915.

[0115] 1-(4-(pyridin-2-yldisulfanyl)butyroyloxymethyl)-5-fluorouracil (23) A solution of 5-FU (100 mg, 0.77 mmol) in 37 wt% formaldehyde solution (0.5 mL) was stirred at 60° C. until the solid completely disappeared and further stirred at 60° C. for 3 h. The solvent was removed in vacuum to give a colorless oil. In a separate flask, (5 mL) 4-(2-pyridyldisulfide)butanoic acid (206 mg, 0.90 mmol), dicyclohexylcarbodiimide (DCC, 190 mg, 0.92 mmol), and DMAP (11 mg, 0.09 mmol) in ACN were added at 0° C. and the reaction was stirred for 10 min. This solution was then added to the above colorless oil. The reaction mixture was stirred at 0° C. for 30 min and then at room temperature for 12 h. The precipitate formed during the reaction was filtered and the filtrate was evaporated in vacuum. The residue in EtOAc was dissolved in 1.0 N HCl, saturated NaHCO 3 and water. The organic layer was extracted with MgSO 4 The residue was purified by column chromatography (silica gel, CHCl 3 / MeOH=25 / 1) to give compound 23 (95.5 mg, 0.26 mmol, 34%, 2 steps) as a solid. 1 H NMR (400 MHz, CDCl 3 )δ8.41(d, J=4.8Hz, 1H), 7.62(t, J=7.3Hz, 2H), 7.58(d, J=5.4Hz, 1H), 7.11-7.01( m, 1H), 5.60(s, 2H), 2.78(t, J=7Hz, 2H), 2.50(t, J=7.2Hz, 2H), 1.21-1.91(m, 2H), 13 C NMR (100 MHz, CDCl 3 ) δ 173.1, 159.8 (d, J CCF =27Hz), 156.9, 149.8, 149.3, 140.3(d, J CF =238Hz), 137.2, 128.5(d, J CCF =24Hz), 120.9, 120.0, 69.8, 37.6, 32.2, 23.5, ppm;HRMS(ESI TOF-MS)C 14 H 15 F 1 N 3 O 4 S 2 +[M+H] + Calculated value: 372.0485, Experimental value: 372.0483.

[0116] 1-(pent-4-ynoyloxymethyl)-5-fluorouracil (24) A solution of 5-FU (100 mg, 0.77 mmol) in 37 wt% formaldehyde solution (0.5 mL) was stirred at 60° C. until the solid completely disappeared and further stirred at 60° C. for 3 h. The solvent was removed in vacuum to give a colorless oil. In a separate flask, pent-4-ynoic acid (105 mg, 1.07 mmol), DCC (190 mg, 0.92 mmol), and DMAP (11 mg, 0.09 mmol) were added in ACN (5 mL) at 0° C. and the reaction was stirred for 10 min to give pre-activated pent-4-ynoic acid. Subsequently, pre-activated pent-4-ynoic acid in ACN was added to the above colorless oil. The reaction mixture was stirred at 0° C. for 30 min, followed by stirring at room temperature for 12 h. The solid was filtered and the filtrate was evaporated in vacuum. The residue in EtOAc was dissolved in 1.0 N HCl, saturated NaHCO 3 The organic layer was extracted with MgSO 4 It was dried at rt, concentrated in vacuo and purified by column chromatography (silica gel, Hex / EtOAc=2 / 1) to give compound 24 (118 mg, 0.49 mmol, 64%) as an amorphous solid. 1 H NMR (400 MHz, CDCl 3 )δ.7.59(d, J=5.4Hz, 1H, H-6), 5.65(s, 2H, H-7), 2.67-2.55(m, 2H), 2.54-2.46(m, 2H), 1.97(t, J=2.5Hz, 1H, H-12)ppm; 13 C NMR (150 MHz, CDCl 3 ) δ 171.9, 156.4 (d, J CCF =28Hz), 148.8, 140.1(d, J CF =237Hz), 128.3(d, J CCF =33Hz), 81.5, 69.7, 69.5, 32.9, 14.1ppm;HRMS(ESI TOF-MS)C 10 H 9 FN 2 O4 Na + [M+Na] + Calculated value: 263.0439, Experimental value: 263.0443.

[0117] 2-(Pyridin-2-yldisulfanyl)ethanol (26) To a solution of aldrithiol-2 (5 g, 0.02 mol) and acetic acid (0.33 mL) in anhydrous MeOH (25 mL), mercaptoethanol (1.2 mL, 0.02 mol) was added dropwise. The reaction mixture was degassed with nitrogen for 20 min and stirred at room temperature for 26 h. The solvent was then evaporated to dryness under reduced pressure and the crude product was purified by column chromatography (silica gel, EtOAc / Hex=15 / 85-30 / 70) to give compound 26 (2.4 g, 64%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 )δ2.95(t, J=5.1Hz, 2H), 3.78-3.82(m, 2H), 5.76(t, J=6.7Hz, 1H), 7.13-7. 17(m, 1H), 7.39(d, J=8.0Hz, 1H), 7.56-7.60(m, 1H), 8.51-8.52(m, 1H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ42.7, 58.3, 121.6, 122.0, 136.9, 149.9, 159.2ppm. HRMS(ESI-TOF)C 7 H 10 NOS 2 [M+H] + Calculated value: 188.0198, Experimental value: 188.0191.

[0118] (4-Nitrophenyl)2-(pyridin-2-yldisulfanyl)ethyl carbonate (27) To a solution of compound 26 (1.05 g, 5.60 mmol) in anhydrous DCM (10 mL) was added trimethylamine (0.79 mL, 5.6 mmol) and p-nitrophenyl chloroformate (1.70 g, 8.40 mmol) in anhydrous DCM (5 mL) at 0 °C. The reaction mixture was stirred at room temperature for another 1 h. Then, the mixture was filtered to remove the precipitated white solid and part of the solvent was removed in vacuum. The crude product was purified by column chromatography (silica gel, EtOAc / Hex=1 / 8-1 / 4) to give compound 27 (1.1 g, 56%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 )δ3.16(t, J=6.4Hz, 2H), 4.56(t, J=6.4Hz, 2H), 7.11-7.14(m, 1H), 7.36- 7.40(m, 2H), 7.63-7.69(m, 2H), 8.26-8.30(m, 2H), 8.48-8.50(m, 1H)ppm; 13 C NMR (100 MHz, CDCl 3 )δ36.9, 66.8, 120.3, 121.3, 121.9, 121.9, 125.5, 137.2, 145.6, 150.1, 152.4, 155.5, 159.2ppm. HRMS(ESI)C 14 H 13 N 2 O 5 S 2 [M+H] + Calculated value: 353.0260, Experimental value: 353.0268.

[0119] (S)-4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl (2-(pyridin-2-yldisulfanyl)ethyl)carbonate (29) To a solution of SN-38 (compound 28, 288 mg, 0.73 mmol) in anhydrous DMF (27 mL) was added trimethylamine (103 μl, 0.73 mmol) and compound 27 (310 mg, 0.88 mmol) dissolved in anhydrous DCM (9 mL). The reaction mixture was stirred for 24 h at ambient temperature under nitrogen. The mixture was subsequently evaporated to dryness in vacuum and purified by column chromatography (silica gel, MeOH / DCM=1 / 99-1 / 50) to give compound 29 (273 mg, 62%) as a yellow powder. 1 H NMR (600 MHz, CDCl 3 )δ1.03(t, J=7.4Hz, 3H), 1.39(t, J=7.7Hz, 3H), 1.85-1.95(m, 2H), 3.13-3.17( m, 2H), 3.19(t, J=6.4Hz, 2H), 3.93(s, 1H), 4.59(t, J=6.4Hz, 2H), 5.25(s, 2H), 5.30(d, J=16.1Hz, 1H), 5.74(d, J=16.2Hz, 1H), 7.11-7.13(m, 1H), 7.61-7.70( m, 4H), 7.90(d, J=2.5Hz, 1H), 8.23(d, J=9.2Hz, 1H), 8.50(d, J=4.6Hz, 1H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ8.0, 14.1, 23.3, 31.7, 37.0, 49.5, 66.4, 66.6, 72.9, 98.3, 114.2, 118.8, 120.3, 121.2, 124.7, 127.5 (2C), 132.4, 137.2, 145.6, 146.9, 147.6, 149.9, 150.0, 150.3, 152.2, 153.2, 157.7, 159.3, 174.0ppm. HRMS(ESI)C 30 H 28 N 3 O 7 S 2 [M+H] + Calculated value: 606.1363, Experimental value: 606.1366.

[0120] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl (2-((2-(2-(2-(4-hydroxy-3-(3-(4-hydroxyphenyl)propanoyl)-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H)pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)ethyl)carbonate (30) To a solution of compound 16 (9 mg, 15.4 μmol) in anhydrous THF (800 μL) was added compound 29 (9 mg, 14.9 μmol) in anhydrous THF (300 μL). The reaction mixture was stirred for 4.5 h at ambient temperature under nitrogen atmosphere. The mixture was evaporated to dryness under reduced pressure and the crude product was purified by column chromatography (silica gel, MeOH / DCM=1 / 15) to give compound 30 (16 mg, 99%) as a white powder. 1 H NMR (600 MHz, (CD 3 ) 2 CO+D 2O)δ0.99(t, J=7.3Hz, 3H), 1.39(t, J=7.7Hz, 3H), 1.91-2.0(m, 2H), 2.81(t, J= 7.4Hz, 2H), 2.99 (t, J=6.4Hz, 2H), 3.17 (t, J=6.4Hz, 2H), 3.25 (q, J=7.7, 15.4 Hz, 2H), 3.34-3.60 (m, 6H), 3.65-3.67 (m, 4H), 3.69-3.72 (dd, J=5.9, 12.0Hz, 1H), 3.76(t, J=6.4Hz, 2H), 3.78(t, J=4.5Hz, 2H), 3.90(dd, J=2.5, 12.0Hz, 1H ), 4.11(m, 2H), 4.58(t, J=6.3Hz, 2H), 5.14(d, J=7.32Hz, 1H), 5.27(d, J=1.0, 2H), 5.36 (d, J=16.1Hz, 1H), 5.53 (d, J=16.4Hz, 1H), 5.98 (d, J=2.3Hz, 1H), 6. 27(d, J=2.3Hz, 1H), 6.70(d, J=8.5Hz, 2H), 7.06(d, J=8.4Hz, 2H), 7.50(s, 1H) , 7.73 (dd, J=2.5, 9.2Hz, 1H), 8.09 (d, J=2.5Hz, 1H), 8.21 (d, J=9.2Hz, 1H)ppm; 13 C NMR (150 MHz, (CD 3 ) 2 CO+D 2 O) δ8.2, 14.3, 23.4, 31.8, 37.6, 39.7, 46.4, 50.2, 62.3, 66.3, 67.5, 68.6, 69.9 ,70.9,71.0,71.3,73.6,74.3,78.1,94.6,96.6,97.9,101.9,106.9,115.6,11 5.8, 120.1, 125.6, 128.2, 129.3, 130.1, 132.6, 133.0, 146.6, 147.4, 148.0, 15 0.7, 151.2, 153.3, 154.1, 156.2, 158.2, 161.6, 165.8, 166.8, 173.5, 206.1ppm. HRMS(ESI TOF-MS)C 52 H 59 N 2 O 19 S 2 [M+H] + Calculated value: 1079.3148, real value: 1079.3157であった.

[0121] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl (2-((2-(2-(2-(2,4-dihydroxy-3-(3-(4-hydroxyphenyl)propanoyl)phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)ethyl)carbonate (31) Compound 17 (50 mg, 0.12 mmol) was dissolved in anhydrous THF (10 mL). Compound 29 (60 mg, 0.10 mmol) was then added to the above solution and stirred at room temperature under nitrogen atmosphere for 1 h. The reaction mixture was concentrated in vacuum to remove the solvent. The crude product was purified by column chromatography (silica gel, MeOH / DCM=1 / 25) to give compound 31 (76.2 mg, 84%) as a yellow solid. 1 H NMR (600 MHz, (CD 3 ) 2 CO+D 2 O) δ1.01(t, J=7.4Hz, 3H), 1.41(t, J=7.6Hz, 3H), 1.93-2.0(m, 2H), 2.86(m, 2H), 2.99(t, J=6.2Hz, 2H), 3.17(t, J=6.3Hz, 2H), 3.26-3.32(m, 4H), 3.64-3.66(m, 4H), 3.76(t, J=6.2Hz, 2H), 3.78(t, J=4.7Hz, 2H), 4.09(t, J=4.5Hz, 2H), 4.58(t, J =6.4Hz, 2H), 5.28(s, 2H), 5.39(d, J=16.1Hz, 1H), 5.54(d, J=16.1Hz, 1H), 5.96(s, 2H), 6.73(d, J=8.3Hz, 2H), 7.06(d, J= 8.3Hz, 2H), 7.47(s, 1H), 7.74(dd, J=2.5, 9.2Hz, 1H), 8.11(d, J=2.5Hz, 1H), 8.22(d, J=9.1Hz, 1H), 11.82(br, s, 1H)ppm; 13 C NMR (150 MHz, (CD 3 ) 2 CO+D 2O) δ8.2, 14.3, 23.4, 31.9, 37.6, 46.9, 50.2, 66.4, 67.5, 68.5, 70.0, 71.1, 71.4, 73.7, 94.8, 97.4, 105.7, 115.6, 116.0, 120.1, 125.6 , 128.3, 129.4, 130.2, 132.7, 133.4, 146.4, 147.6, 148.2, 150.7, 150.9, 153.5, 154.1, 156.4, 158.0, 165.1, 166.0, 173.6, 205.8ppm. HRMS(ESI TOF-MS)C 46 H 48 N 2 O 14 S 2 [M+H] + Calculated value: 917.2620, Experimental value: 917.2621.

[0122] (S)-2((S)-2((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (33) To a solution of compound 32 (5 g, 11.46 mmol) and L-citrulline (2.1 g, 12 mmol) in dimethoxymethane (50 mL) and THF (25 mL) was added sodium bicarbonate (1 g, 12 mmol) in water (50 mL). The reaction was stirred at room temperature and kept in the dark. After 5 days, 20% concentration of citric acid (aq.) was added to the mixture and the pH was adjusted to about 5. The solution was extracted three times with an equal volume of 10% isopropanol in EtOAc. The organic layers were combined and washed three times with water. The organic layers were then collected and washed with MgSO 4 The crude product was then dried at rt and evaporated to dryness in vacuo to give the crude product, which was finally dispersed in ether and sonicated to give compound 33 (5 g, 88%) as a white solid. 1 H NMR (400 MHz, DMSO-d 6)δ0.85-0.9(m, 6H), 1.39-1.41(m, 2H), 1.51-1.61(m, 1H), 1.69-1.70(m, 1H), 1.95-2 .00(m, 1H), 2.94(m, 2H), 3.92(t, J=8.24Hz, 1H), 4.12-4.17(m, 1H), 4.21-4.31(m, 3H) ), 5.37(s, 2H), 5.94(t, J=4.44Hz, 1H), 7.30-7.34(m, 1H), 7.38-7.43(m, 3H), 7.75(t , J=7.12Hz, 2H), 7.89(d, J=7.48Hz, 2H), 8.17(d, J=7.16Hz, 2H), 12.55(br, s, 1H)ppm; 13 C NMR (100 MHz, DMSO-d 6 )δ18.2, 19.2, 26.6, 28.4, 30.6, 38.8, 46.7, 51.9, 59.8, 65.7, 120.1, 12 5.4, ​​127.1, 127.7, 140.7, 143.8, 143.9, 156.1, 158.7, 171.3, 173.4ppm. HRMS(ESI TOF-MS)C 26 H 32 N 4 O 6 [M+H] + Calculated value: 497.2395, Experimental value: 497.2404.

[0123] (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (34) Compound 33 (6 g, 12.1 mmol), p-aminobenzyl alcohol (3 g, 24.2 mmol), and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6 g, 24.2 mmol) were dissolved in dry DCM (200 mL) and MeOH (100 mL). The reaction mixture was stirred at room temperature and kept in the dark. After 24 h, the mixture was evaporated to dryness in vacuo. The residue was washed with ether / ethyl acetate (v / v=1 / 1) to give compound 34 (5 g, 69%) as a light brown solid. 1 H NMR (400 MHz, DMSO-d6 )δ0.84-0.89(m, 6H), 1.38-1.44(m, 2H), 1.58-1.70(m, 2H), 1.97-2.01(m, 1H), 2.91-3.04(m, 2H), 3.93(t, J=7.34Hz, 1H), 4.23-4.33(m, 3H), 4.42-4.43(m, 3H), 5.11(t, J=5.76Hz, 1H), 5.43(s, 2H) , 5.99(t, J=4.72Hz, 1H), 7.23(d, J=8.16Hz, 2H), 7.30-7.34(m, 2H), 7.39-7.46(m, 3H), 7.54(d, J= 8.4Hz, 2H), 7.74(t, J=7.84H, 2H), 7.89(d, J=7.44Hz, 1H), 8.11(d, J=7.64Hz, 1H), 9.99(s, 1H)ppm; 13 C NMR (100 MHz, DMSO-d 6 )δ18.3, 19.2, 26.8, 29.5, 30.4, 38.6, 46.7, 53.0, 60.1, 62.6, 65.7, 118.9, 120.1, 125.4, 126.9, 127.1, 127.6, 137.4, 137.5, 140.7, 143.8, 143.9, 156.1, 158.9, 170.3, 171.2ppm. HRMS(ESI TOF-MS)C 33 H 40 N 5 O 6 [M+H] + Calculated value: 602.2973, Experimental value: 602.2987.

[0124] (S)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-2-((S)-2-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanamide)-3-methylbutanamide)-5-ureidopentanamide (36) Compound 34 (1.71 g, 2.84 mmol) was added to HBr (33 wt % in glacial acetic acid, 30 mL) at 0° C. and stirred for 10 min. The mixture was warmed to room temperature and stirred for an additional 2 h. The reaction was then quenched with 500 mL of water to precipitate the brominated intermediate 35. Intermediate 35 was collected by filtration, washed with additional water to remove residual acid, and dried in vacuo to give crude 35 (1.52 g) as a light brown solid. SN-38 (694 mg, 1.77 mmol) and Cs in dry DMF (50 mL) were added to give a 100% ethyl ether 35 (1.52 g) of ... 2 CO 3 (811.4 mg, 2.3 mmol) of crude 35 was added and the reaction mixture was stirred for 20 min. Then, piperidine (5 mL) was added to the above mixture and stirred for another 10 min. Then, the mixture was evaporated to dryness in vacuum and purified by column chromatography (silica gel, MeOH / DCM=50 / 1 to 5 / 1) to obtain crude val-cit-pab-O-SN-38 (316 mg) as a yellow solid. Crude val-cit-pab-O-SN-38 (316 mg) was dissolved in dry DMF (25 mL) and TEA (58.6 μL, 0.42 mmol) and 5-maleimidovaleric acid-NHS (161.8 mg, 0.55 mmol) were added. After stirring for 3.5 h, the mixture was evaporated to dryness in vacuo and purified by column chromatography (silica gel, MeOH / DCM=10 / 1) to give compound 36 (164.4 mg, 10%) as a yellow solid. 1 H NMR (600 MHz, DMSO-d 6) δ 0.81 - 0.89 (m, 9H), 1.26 (t, J = 7.6 Hz, 3H), 1.32 - 1.49 (m, 6H), 1.56 - 1.61 (m, 1H), 1.67 - 1.73 (m, 1H), 1.81 - 1.91 (m, 2H), 1.94 - 1.99 (m, 1H), 2.12 - 2.17 (m, 1H), 2.18 - 2.23 (m, 1H), 2.92 - 2.97 (m, 1H), 3.00 - 3.05 (m, 1H), 3.14 - 3.15 (m, 2H), 4.18 (t, J = 7.8 Hz, 1H), 4.35 - 4.40 (m, 1H), 5.25 (s, 2H), 5.28 (s, 2H), 5.38 - 5.44 (m, 4H), 5.98 (t, J = 5.4 Hz, 1H), 6.50 (s, 1H), 6.99 (s, 2H), 7.25 (s, 1H), 7.49 (d, J = 8.52 Hz, 2H), 7.52 (d, J = 9.24 Hz, 1H), 7.55 (s, 1H), 7.65 (d, J = 8.46 Hz, 2H), 7.82 (d, J = 8.58 Hz, 1H), 8.05 (dd, J = 8.9, 2.2 Hz, 1H), 8.08 (d, J = 7.4 Hz, 1H), 10.01 (s, 1H) ppm; 13 C NMR (150 MHz, DMSO - d 6 ) δ 7.8, 13.5, 18.2, 19.2, 22.3, 22.7, 26.8, 27.7, 29.3, 30.3, 30.4, 34.6, 36.9, 38.6, 40.1, 49.5, 53.1, 57.6, 65.3, 69.6, 72.4, 96.0, 103.7, 118.3, 119.1, 122.7, 127.8, 128.3, 128.8, 131.3, 131.4, 134.5, 138.8, 143.9, 144.4, 146.3, 149.6, 150.1, 156.9, 157.2, 158.9, 170.7, 171.1, 171.3, 172.2, 172.6 ppm. HRMS (ESI TOF - MS) C 49 H 57 N 8 O 11 [M + H] + Calculated value: 933.4141, Experimental value: 933.4188.

[0125] (2S)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-2-((2S)-2-(5-(3-((2-(2-(2-(3-hydroxy-4-(3-(4-hydroxy Phenyl)propanoyl)-5-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)pentylamido)-3-methylbutyramido)-5-ureidopentanamide (37) To a solution of compound 36 (136 mg, 0.15 mmol) in MeOH (60 mL) was added compound 16 (108.6 mg, 0.26 mmol). The reaction was stirred at room temperature. After 1.5 h, the mixture was evaporated to dryness in vacuo and purified by column chromatography (silica gel, DCM / MeOH=7 / 1 to 5 / 1) to give compound 37 (113 mg, 50%) as a yellow solid. 1 H NMR (600 MHz, DMSO-d 6)δ0.82-0.89(m、9H)、1.27(t、J=7.4Hz、3H)、1.36-1.45(m、6H)、1.59-1.69(m、2H)、1.81-1.91(m、2H)、1.94-1.98(m、1H)、2.08-2.22(m、2H)、2.50-2.22(m、1H)、2.77-2.84(m、3H)、2.92-3.04(m、3H)、3.14-3.18(m、4H)、3.26-3.30(m、3H)、3.35(m、2H)、3.39-3.47(m、3H)、3.54-3.57(m、4H)、3.60-3.63(m、2H)、3.70-3.72(m、3H)、4.02(dd、J=8.7、3.3Hz、1H)、4.14(m、2H)、4.19(t、J=7.5Hz、1H)、4.35-4.40(m、1H)、4.63(t、J=5.3Hz、1H)、5.04(d、J=7.0Hz、1H)、5.08(d、J=5.2Hz、1H)、5.16(d、J=4.0Hz、1H)、5.28(s、4H)、5.33(d、J=4.5Hz、1H)、5.42-5.45(m、4H)、5.99(t、J=5.5Hz、1H)、6.12(s、1H)、6.28(s、1H)、6.49(s、1H)、6.64(d、J=8.1Hz、2H)、7.03(d、J=8.1Hz、2H)、7.26(s、1H)、7.49(d、J=8.3Hz、2H)、7.54-7.56(m、1H)、7.59(m、1H)、7.66(d、J=8.2Hz、2H)、7.81(d、J=8.3Hz、1H)、8.07(d、J=9.2Hz、1H)、8.10(d、J=7.1Hz、1H)、9.11(s、1H)、10.02(s、1H)、13.33(s、1H)ppm; 13 C NMR(150MHz、DMSO-d 6)δ7.7, 13.4, 18.2, 19.2, 22.2, 22.6, 26.7, 26.8, 28.9, 29.3, 30.3, 30.4, 34.6, 35.9, 38.0, 38.5, 40.1, 45.1, 49. 5, 53.1, 57.6, 60.7, 65.3, 67.6, 68.6, 69.5, 69.7, 69.8, 72.4, 73.2, 76.7, 77.3, 93.7, 95.7, 96.0, 100.7, 103.8, 106.3, 115.0, 118.3, 119.1, 122.7, 127.8, 128.4, 128.7, 129.1, 131.3, 131.4, 138.8, 143.9, 144.5, 146.3, 149. 6, 150.1, 155.3, 156.8, 157.2, 158.9, 160.2, 164.3, 164.8, 170.6, 171.3, 172.1, 172.5, 175.1, 176.8, 205.1ppm. HRMS(ESI TOF-MS)C 76 H 93 N 8 O 23 S[M+H] + Calculated value: 1517.6069, Experimental value: 1517.6083.

[0126] (2S)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-2-((2S)-2-(5-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propanoyl)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)pentanamide)-3-methylbutanamide)-5-ureidopentanamide (38) To a solution of compound 36 (36 mg, 0.04 mmol) in MeOH (20 mL) was added compound 17 (21.5 mg, 0.05 mmol). The suspension was stirred at room temperature for 1 h. The mixture was evaporated to dryness in vacuum cesium carbonate and the crude product was precipitated by adding ether / ethyl acetate (v / v=1 / 1). This was followed by column chromatography (C18, ACN / H 2O=1 / 1), the crude product was purified, and compound 38 (17.5 mg, 32%) was obtained as a yellow solid. 1 H NMR (600 MHz, DMSO-d 6 )δ0.82-0.89(m, 9H), 1.27(t, J=7.4Hz, 3H), 1.36-1.45(m, 6H), 1.58-1.60(m, 1H), 1.70-1.72(m, 1H), 1.8 2-1.91 (m, 2H), 1.94-1.99 (m, 1H), 2.13-2.22 (m, 2H), 2.48-2.52 (m, 1H), 2.76 (t, J=7.5Hz, 2H), 2.79-2.8 3(m, 1H), 2.93-2.99(m, 2H), 3.01-3.04(m, 1H), 3.13-3.17(m, 3H), 3.24(t, J=7.5Hz, 2H), 3.35(m, 2H), 3. 53-3.56 (m, 4H), 3.59-3.64 (m, 2H), 3.70 (t, J=3.7Hz, 2H), 4.02 (dd, J=8.9, 3.8Hz, 1H), 4.05 (t, J=3.7Hz, 2 H), 4.19 (t, J=7.4Hz, 1H), 4.38 (q, J=13.1, 7.5Hz, 1H), 5.27-5.28 (m, 4H), 5.39-5.45 (m, 4H), 5.92 (s, 2H) ,5.98(t,J=5.4Hz,1H),6.49(br,s,1H),6.65(d,J=8.2Hz,2H),7.01(d,J=8.2Hz,2H),7.26(s,1H),7.48(d , J=8.3Hz, 2H), 7.55 (dd, J=11.0, 1.9Hz, 1H), 7.58 (d, J=1.6Hz, 1H), 7.65 (d, J=8.3Hz, 2H), 7.81 (d, J=8.5 Hz, 1H), 8.07 (d, J=9.1Hz, 1H), 8.09 (d, J=7.3Hz, 1H), 9.14 (br, s, 1H), 10.01 (s, 1H), 12.39 (br, s, 2H)ppm; 13 C NMR (150 MHz, DMSO-d 6)δ7.8, 13.4, 18.2, 19.2, 22.2, 22.6, 23.4, 26.7, 26.8, 29.3, 30.3, 30.4, 34.6, 35.9, 38.0, 38.6, 40.1, 45. 6, 49.5, 53.1, 57.6, 65.3, 67.3, 68.6, 69.4, 69.5, 69.6, 69.8, 72.2, 72.4, 93.6, 96.0, 103.7, 104.6, 115.1 , 118.3, 119.1, 122.7, 127.8, 128.4, 128.7, 129.1, 131.3, 131.4, 131.5, 138.8, 143.9, 144.5, 146.3, 149. 6, 150.1, 155.4, 156.9, 157.2, 158.9, 164.1, 164.6, 170.6, 171.3, 172.1, 172.5, 175.1, 176.8, 204.7ppm. HRMS(ESI TOF-MS)C 70 H 83 N 8 O 18 S[M+H] + Calculated value: 1355.5541, Experimental value: 1355.5562.

[0127] (S)-4,11-Diethyl-3,14-dioxo-4-(pent-4-ynoyloxy)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl [1,4'-dipiperidine]-1'-carboxylate (40) To a solution of irinotecan (400 mg, 0.64 mmol) in dry ACN (5 mL) was added penta-4-ynoic acid (94 mg, 0.96 mmol) and cooled to 0° C. DCC (198 mg, 0.96 mmol) and DMAP (2 mg, 0.02 mmol) in dry ACN (0.5 mL) were added, followed by stirring for another 4 h. After filtering the precipitate, the filtrate was evaporated in vacuum. The residue in EtOAc was dissolved in 1.0 N HCl solution, saturated NaHCO 3 The organic layer was extracted with MgSO 4 The mixture was dried at 40° C. and concentrated in vacuum. The residue was purified by column chromatography (silica gel, DCM / MeOH=12 / 1) to give compound 40 (295 mg, 69%) as an amorphous solid. mp: 149° C.1 H NMR (600 MHz, CDCl 3 )δ8.13(d, J=9Hz, 1H), 7.81(d, J=3Hz, 1H), 7.56(dd, J=6.6, 2.4Hz, 1H), 7.26(s, 1H), 5.67(d, J=17.4Hz, 1H), 5 .37(d, J=17.4Hz, 1H), 5.21(dd, J=4.2Hz, 2H), 4.40(dd, J=40.2, 12.6Hz, 2H), 3.16-3.11(m, 2H), 3.06(t, J=12 .6Hz, 1H), 2.88(t, J=12.6Hz, 1H), 2.77-2.69(m, 6H), 2.51-2.49(m, 2H), 2.29-2.23(m, 1H), 2.20(t, J=2.4Hz, 1H), 2.14-2.08(m, 4H), 1.75-1.60(m, 6H), 1.55-1.51(m, 2H), 1.37(t, J=7.5Hz, 3H), 0.98(t, J=7.5Hz, 3H)ppm; 13 C NMR (150 MHz, CDCl 3 )δ170.7, 167.4, 157.3, 153.0, 151.6, 150.3147.1, 146.6, 145.9, 145.3, 131.5, 127.5, 127.1, 125.8, 119.7, 114.6, 96.4, 82.2, 76.3, 70.2, 67.0, 62.6, 50.2, 49.2, 44.1, 43.7, 33.1, 31.7, 27.7, 25.1, 23.9, 23.1, 14.2, 13.9, 7.5ppm;HRMS(ESI)C 38 H 43 N 4 O 7 [M+H] + Calculated value: 667.3126, Experimental value: 667.3134.

[0128] (S)-4-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl [1,4'-bipiperidine]-1'-carboxylate (41) To a solution of irinotecan (100 mg, 0.16 mmol) in dry ACN (6 mL) was added 6-maleimidohexanoic acid (195 mg, 0.06 mmol) and cooled to 0° C. DCC (40 mg, 0.19 mmol) and DMAP (2.5 mg, 0.02 mmol) in dry ACN (0.5 mL) were added to the above flask, followed by stirring the reaction for another 4 h. The precipitate formed in the reaction was filtered and the filtrate was evaporated in vacuum. The residue in EtOAc was washed with 1.0 N HCl solution, saturated NaHCO 3 The organic layer was collected and extracted with MgSO 4 The mixture was dried at 40° C. and concentrated in vacuo. The residue was purified by column chromatography (silica gel, DCM / MeOH=12 / 1) to give compound 41 (52.5 mg, 42%) as an amorphous solid. mp: 149° C. 1 H NMR (600 MHz, CDCl 3 )δ8.26(d, J=9Hz, 1H), 7.89(s, 1H), 7.66(d, J=9Hz, 1H), 7.34(s, 1H, H-29), 6.70(s, 1H, H-53), 5.75(d, J=16.8Hz, 1H), 5.48(d, J=16) .8Hz, 1H), 5.46-5.31(m, 2H, H-28), 4.50(dd, J=42, 12Hz, 2H), 3.60-3.47(m, 2H), 3.24(dd, J=7.2, 7.8Hz, 2H), 3.15(t, J=2.6Hz, 1H) , 2.99(t, J=2.6Hz, 1H), 2.85-2.73(m, 5H), 2.61-2.52(m, 3H), 2.39-2.33(m, 1H), 2.26-2.21(m, 1H), 2.15-2.10(m, 1H), 1.86-1.81( m, 5H), 1.77-1.72(m, 3H), 1.68-1.63(m, 2H), 1.63-1.60(m, 2H), 1.47(t, J=7.8Hz, 3H), 1.43-1.40(m, 2H), 1.03(t, J=7.8Hz, 3H)ppm; 13 C NMR (150 MHz, CDCl 3)δ172.3, 170.7, 167.5, 157.3, 153.0, 151.5, 150.3, 147.1, 146.8, 145.8, 145.3, 133.9, 131.6, 127.5, 127.1, 125.8, 120.2, 114.6, 9 HRMS(ESI)C 43 H 50 N 5 O 9 [M+H] + Calculated value: 780.3603, Experimental value: 780.3636.

[0129] (4S)-4,11-Diethyl-4-((3-(1-(2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-5-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)oxy)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl [1,4'-bipiperidine]-1'-carboxylate (42) To a solution of compound 20 (27.2 mg, 0.04 mmol) in ethanol / water (v / v=3 / 1, 1 mL) was added TBTA (6.2 mg, 0.01 mmol), sodium ascorbate (9.1 mg, 0.46 mmol), copper(II) sulfate pentahydrate (2 mg, 0.01 mmol), and compound 40 (30 mg, 0.04 mmol). The reaction mixture was stirred for 24 h and then concentrated in vacuo. The mixture in EtOAc was extracted with water. The organic layer was collected and concentrated in vacuo and purified by column chromatography (silica gel, DCM / MeOH=10 / 1 to 3 / 1) to give 42 (38 mg, 67%) as a foam. 1 H NMR (400 MHz, CDCl 3 / CD 3OD=10 / 1)δ8.13(d, J=9.2Hz, 1H), 7.88(s, 1H), 7.81(s, 1H), 7.58(d, J=9Hz, 1H), 7.31(s, 1H), 6.98(d, J=8.3Hz, 2H), 6.63(d , J=10.8Hz, 2H), 6.03 (s, 1H), 5.82 (d, J=2.1Hz, 1H), 5.63 (d, J=16.8Hz, 1H), 5.43 (d, J=16.8Hz, 1H), 5.13 (dd, J=15.4, 18.5H z, 2H), 4.95 (d, J=7.3Hz, 1H), 4.42-4.25 (m, 4H), 3.94-3.87 (m, 3H), 3.73-3.66 (m, 6H), 3.56-3.36 (m, 9H), 3.2-2.9 (m, 9H), 2 .74-2.72(m, 6H), 2.28-2.10(m, 2H), 2.02-1.99(m, 2H), 1.70-1.54(m, 8H), 1.31(t, J=7.3Hz, 3H), 0.99(t, J=7.4Hz, 3H)ppm; 13 C NMR (400MHz, CDCl 3 / CD 3 OD=10 / 1)δ205.2, 171.7, 167.8, 166.1, 164.4, 160.1, 157, 4, 153.2, 151.0, 150.3, 146.68, 146.6 3. 145.7, 132.5, 131.3, 129.1, 127.6, 127.6, 127.1, 126.1, 123.3, 119.3, 115.1, 114.7, 100.4, 96 .595.6, 94.0, 77.3, 76.3, 76.1, 73.0, 70.5, 69.9, 69.3, 69.1, 67.4, 66.8, 62.4, 61.7, 50.2, 50.1, 49.4, 45.4, 44.2, 43.8, 33.0, 31.5, 29.6, 29.1, 27.7, 27.0, 25.4, 24.1, 23.1, 20.4, 13.8, 7.5ppm. HRMS(ESI)C 65 H 77 N 7 O 19 +H[M+H] + Calculated value: 1260.5347, real value: 1260.5372.

[0130] (S)-4-((3-(1-(2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propanoyl)phenoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl [1,4'-bipiperidine]-1'-carboxylate (43) To a solution of compound 21 (50 mg, 0.12 mmol) in EtOH / water (v / v=3 / 1, 1 mL), TBTA (13 mg, 0.02 mmol), sodium ascorbate (24 mg, 0.12 mmol), copper(II) sulfate pentahydrate (3 mg, 0.01 mmol), and compound 40 (80 mg, 0.15 mmol) were added. The reaction mixture was stirred for 12 h and then concentrated in vacuo. The mixture was redissolved in EtOAc and extracted with water. The organic layer was concentrated in vacuo and purified by column chromatography (silica gel, DCM / MeOH=10 / 1 to 5 / 1) to give 43 (77 mg, 59%) as an amorphous solid. mp: 158°C. 1 H NMR (600 MHz, CDCl 3)δ 8.20 (d, J = 9.6 Hz, 1H), 7.84 (s, 1H), 7.54 (d, J = 9.6 Hz, 1H), 7.49 (s, 1H), 7.27 (s, 1H), 7.02 (d, J = 7.8 Hz, 2H), 6.76 (d, J = 7.8 Hz, 2H), 6.01 - 5.93 (m, 2H), 5.63 (d, J = 16.8 Hz, 1H), 5.34 (d, J = 16.8 Hz, 1H), 5.22 (d, J = 3.6 Hz, 2H), 4.44 - 4.41 (m, 3H, H - 9), 4.38 - 4.33 (m, 1H), 4.04 (m, 2H), 3.87 - 3.86 (m, 2H), 3.64 (t, J = 5.4 Hz, 2H), 3.54 (m, 4H), 3.38 - 3.29 (m, 2H), 3.16 - 3.11 (m, 2H), 3.04 (t, J = 6.6 Hz, 1H), 2.91 - 2.85 (m, 3H), 2.81 (t, J = 6.4 Hz, 2H), 2.72 (br, 4H), 2.51 - 2.50 (m, 1H), 2.46 - 2.40 (m, 1H), 2.14 - 2.01 (m, 5H), 1.83 - 1.62 (m, 6H), 1.51 - 1.39 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H), 0.93 (t, J = 7.2 Hz, 3H) ppm; 13 C NMR (150 MHz, CDCl 3 )δ 205.2, 171.6, 167.6, 164.7, 157.2, 154.7, 152.9, 151.0, 150.5, 146.6, 146.2, 146.1, 145.4, 133.4, 130.6, 129.3, 127.7, 127.3, 126.5, 123.2, 119.8, 115.2, 114.8, 105.3, 96.5, 94.5, 75.9, 71.1, 70.3, 69.2, 68.7, 66.9, 62.6, 50.7, 50.2, 49.3, 45.9, 44.1, 43.7, 32.7, 31.5, 29.9, 27.7, 26.9, 25.0, 23.9, 23.2, 20.3, 13.9, 7.5 ppm; HRMS (ESI) C 59 H 68 N 7 O 14 [M + H] + Calculated value: 1098.4819, Experimental value: 1098.482.

[0131] Diethyl-4-((6-(3-((2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-5-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanoyl)oxy)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl [1,4'-bipiperidine]-1'-carboxylate (44) To a solution of compound 16 (33.7 mg, 0.05 mmol) in dry MeOH (2 mL) was added compound 41 (45 mg, 0.05 mmol). The reaction mixture was stirred for 10 min and then concentrated in vacuo. The residue was purified by column chromatography (silica gel, DCM / MeOH=10 / 1 to 3 / 1) to give compound 44 (47.2 mg, 69%) as an amorphous solid. mp: 146°C. 1 H NMR (400 MHz, CDCl 3 / CD 3OD=10 / 1)δ8.05(d、J=9.2Hz、1H)、7.72(d、J=2.4Hz、1H)、7.46(dd、J=6.7、2.6Hz、1H)、7.14(d、J=1Hz、1H)、6.92(dd、J=6.4、2Hz、2H)、6.58(d、J=7.8Hz、2H)、6.04(s、1H)、5.94(s、1H)、5.53(d、J=17Hz、1H)、5.27(d、J=8.2Hz、1H)、4.87(d、J=5.7Hz、1H)、4.34(dd、J=30.7、16.4Hz、2H)、3.97(br、2H)、3.70-3.69(m、3H)、3.64-3.59(m、3H)、3.55-3.53(m、5H)、3.35-3.31(m、6H)、3.29-3.27(m、3H)、3.23(t、J=1.5Hz、2H)、3.05-2.94(m、6H)、2.81-2.70(m、6H)、2.39-2.30(m、3H)、2.14-1.98(m、4H)、1.89-1.72(br、4H)、1.61-1.49(m、2H)、1.54-1.40(m、6H)、1.26(t、J=7.5Hz、3H)、1.24-1.15(m、2H)、0.85(t、J=7.3Hz、3H)ppm; 13 C NMR(100MHz、CDCl 3 / CD 3 OD=10 / 1)δ205.2、177.1、175.3、172.6、167.9、164.6、160.1、157.5、154.6、153.1、151.3、146.8、146.5、146.4、146.0、132.6、131.2、129.4、129.2、127.6、127.3、125.9、119.7、115.1、114.8、106.3、100.6、95.8、94.2、76.4、75.7、73.1、70.7、70.6、70.1、69.6、69.2、67.6、66.9、63.1、61.5、50.0、49.4、45.5、43.5、43.1、39.3、38.6、36.1、33.3、31.5、31.2、29.2、27.0、25.8、24.0、23.7、23.1、22.8、13.9、7.5ppm。HRMS(ESI)C 70 H 85 N 5 O 21 S+H[M+H] +Calculated value: 1364.5531, Experimental value: 1364.5597.

[0132] (4S)-4-((6-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propanoyl)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanoyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl[1,4'-bipiperidine]-1'-carboxylate (45) To a solution of compound 17 (25.8 mg, 0.06 mmol) in dry MeOH (2 mL) was added compound 41 (47.8 mg, 0.06 mmol). The reaction mixture was stirred for 10 min and concentrated in vacuo. The residue was purified by column chromatography (silica gel, DCM / MeOH=12 / 1 to 5 / 1) to give 45 (41.7 mg, 58%) as an amorphous solid. mp: 135°C. 1 H NMR (400 MHz, CDCl 3)δ 8.14 (d, J = 9.2 Hz, 1H), 7.79 (s, 1H), 7.51 (d, J = 9.2 Hz, 1H), 7.18 (d, J = 2.6 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H), 6.70 (dd, J = 4, 4.3 Hz, 2H), 5.92 (s, 2H), 5.63 (d, J = 17 Hz, 1H), 5.35 (d, J = 17 Hz, 1H), 5.20 (s, 2H), 4.38 (dd, J = 22.2, 13 Hz, 2H), 4.02 (m, 2H), 3.91 - 3.84 (m, 1H), 3.74 - 3.70 (m, 4H), 3.62 - 3.60 (m, 4H), 3.44 - 3.32 (m, 2H), 3.27 - 3.23 (m, 2H), 3.10 - 2.98 (m, 5H), 2.93 - 2.70 (m, 9H), 2.48 - 2.41 (dt, J = 18.8, 4.2 Hz, 1H), 2.37 - 2.22 (m, 2H), 2.19 - 2.13 (m, 2H), 2.04 - 2.00 (m, 2H), 1.925 - 1.65 (m, 6H), 1.53 - 1.47 (m, 6H), 1.31 (t, J = 7 Hz, 3H), 1.27 - 1.18 (m, 2H), 0.90 (td, J = 7.4, 1.9 Hz, 3H) ppm; 13 C NMR (100 MHz, CDCl 3 ) δ 205.3, 177.4, 177.3, 175.2, 172.6, 167.8, 164.8, 157.4, 154.7, 153.1, 151.1, 150.2, 146.7, 146.4, 146.1, 133.1, 131.1, 129.4, 127.6, 127.3, 126.1, 119.9, 115.4, 114.8, 105.1, 96.5, 94.3, 77.2, 75.7, 70.9, 70.8, 69.3, 67.4, 67.0, 62.8, 50.0, 49.4, 47.6, 47.4, 46.2, 40.9, 39.5, 38.8, 36.3, 33.3, 31.6, 31.4, 29.8, 27.0, 25.8, 24.3, 24.1, 23.4, 23.2, 14.0, 7.6 ppm; HRMS (ESI) C 64 H 76 N 5 O 16 S [M + H] + Calculated value: 1202.5002, Experimental value: 1202.5013.

[0133] Cell culture and cytotoxicity analysis

[0134] Cancer cell lines were obtained from ATCC unless otherwise stated. HT-29 and HCT-116 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% antibiotic-fungal inhibitor (Thermo Fisher Scientific). NHDF cells, a normal cell line, were obtained from PromoCell unless otherwise stated. NHDF cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, product no. 6429) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% antibiotic-fungal inhibitor (Thermo Fisher Scientific). The cells were maintained in a humidified incubator at 5% CO 2 and 95% air at 37°C.

[0135] The cytotoxicity of compounds against HT-29, HCT-116, and NHDF cells was determined by SRB assay. 3The cells were seeded at a density of 100 cells / well into a 96-well microtiter plate (100 μL / well) and divided into two groups: a GSH-added group and a GSH-free group. After 24 h of incubation, the medium was replaced with fresh medium (100 μL). For the GSH-free control group, a series of concentrations of compounds (100 μL, prepared in DMSO or DMA and diluted in medium) were added directly to the cells. For the GSH-added group, the cells were transiently treated with 30 mM GSH (20 μL / well, diluted in medium) for 1 h, followed by the addition of a series of concentrations of compounds (40 μL / well, prepared in DMSO or DMA and diluted in medium) and 10 mM GSH (40 μL / well) to a final concentration of 5 mM GSH, and incubated for 48 or 72 h (the amount of DMSO or DMA did not exceed 0.5%). After incubation, 10% cold trichloroacetic acid solution was slowly added to each well and incubated at 4 °C for 1 h. The medium was removed, carefully washed with water, and dried at room temperature. The plates were then stained with 100 μL of 0.057% SRB for 30 min, rinsed with 1% acetic acid to remove unbound dye, and dried at room temperature. The bound protein dye was dissolved with 100 μL of 10 mM Tris base (pH 10.5) and shaken for 10 min. The optical density (OD) was measured at 510 nm using a multimode microplate reader (SpectraMax Paradigm, Beckman Coulter, US). The cell viability was calculated as follows: Viability = (Experimental OD - Blank) / (Control OD - Blank). A nonlinear regression curve fitting model (GraphPad Prism 7, US) was used to determine the IC 50 The values ​​(concentrations that inhibited cell proliferation by 50%) were calculated. Each experiment was repeated three times.

[0136] 2-NBDG uptake assay

[0137] COS-7 cells were seeded in 96-well incubation plates and grown for 24 h in low glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine in Krebs Ringer's bicarbonate buffer (KRB) (10 mM HEPES, 129 mM NaCl, 4.7 mM KCl, 2 mM CaCl 2 and 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 5 mM NaHCO 3 , pH 7.4) and then incubated with 5% CO 2 Cells were incubated with test compounds in KRB containing 2-NBDG (200 μM) at 37°C for 90 min at room temperature, then washed twice with KRB and lysed with lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 40 mM KCl and 20 mM Tris, pH 7.4). Finally, the lysates were transferred to a black 96-well plate and the fluorescence intensity was detected at an excitation wavelength of 475 nm and an emission wavelength of 550 nm using a multimode microplate reader (SpectraMax Paradigm, Beckman Coulter, USA).

[0138] Western blot analysis of CRC and NHDF cell lines

[0139] HCT116, HT29 and NHDF cells were harvested, washed with PBS and lysed in 0.3 mL of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Cell lysates containing 20 μg of protein were mixed with sample buffer and denatured at 37 °C for 30 min. Proteins were separated electrophoretically in 10% SDS-PAGE, transferred to PVDF membranes, blocked with 3% BSA in PBST and the membranes were incubated with anti-GLUT-1 antibody (1:2000, Abcam) overnight at 4 °C. The membranes were washed with PBST and incubated with HRP-conjugated secondary antibodies including goat anti-rabbit (1:3000, Abcam). Antigens were revealed by Ultrascence western substrate (Bio-Helix, Taiwan), and image detection was performed with a ChemiDoc XRS system and Image Lab software (Bio-Rad Laboratories, Hercules, CA). GAPDH (1:2500, Abcam, mouse) was used as a loading control. Both HT-29 and HCT-116 were shown to have significant GLUT-1 expression levels, among which multiple bands reflected differently glycosylated GLUT-1. Extremely low expression levels of GLUT-1 were observed in the NHDF cell line.

[0140] Assessment of cathepsin B expression levels in healthy mice versus AOM-DSS-induced colon cancer mice

[0141] An orthotopic CRC mouse model was established by intraperitoneally (ip) injection of AOM (10 mg / kg body weight) into BALB / c mice (6 weeks old, male) and supplementing their drinking water with 2% dextran sulfate sodium (DSS). Two months later, the mice were incubated with CO 2Mice were euthanized with acetaminophen and their colons with tumor areas were harvested and fixed in 4% paraformaldehyde, followed by preparation of paraffin sections. In addition, healthy BALB / c mice (6 weeks old, male) were also used for comparison. Paraffin sections of mouse colon samples were prepared according to the routine procedures of the Laboratory Animal Center of National Taiwan University. First, paraffin sections were deparaffinized in 100% xylene, followed by rehydration in 100%, 95%, 70%, 50%, and 0% ethanol (3 min in each ethanol dilution). Heat-mediated antigen retrieval was performed in sodium citrate buffer (pH 6.0). The sections were then deparaffinized in 3% H in 100% methanol at room temperature. 2 O 2 Blocking of endogenous peroxidase activity in the tissue was performed by incubation for 10 min in the presence of 100 mM NaCl. After rinsing with PBS and washing twice with PBST (PBS + 0.5% Tween 20), samples were blocked for 15–30 min. Subsequently, sections were incubated with primary antibody (Cathepsin B (DIC7Y) XP rabbit mAb, 1:1000, CST 31718, Cell Signaling) overnight at 4 °C. Subsequently, sections were incubated with secondary horseradish peroxidase-conjugated antibody (anti-rabbit IgG (HRP), 1:2000, 7074S, Cell Signaling) for 1 h at ambient temperature. Protein expression was detected using diaminobenzidine (DAB) (Cell Signaling) and hematoxylin (Cell Signaling) was used for counterstaining. All sections were analyzed under an upright light microscope (LEICA DM2500) and images were captured using MicroVisioneer Manual Whole Slide Imaging Software.

[0142] In vivo studies

[0143] Specific pathogen-free BALB / c mice (6 weeks old, male) were purchased from the National Laboratory Animal Center. Animals were housed in clean plastic microisolator cages (5 mice / cage) and provided with standard laboratory granulated food and water ad libitum. The animal room was maintained at constant temperature, humidity, and a 12-h dark / light cycle. All animal procedures were in accordance with the standards of the Committee for the Laboratory Animal Care Committee in National Taiwan University College of Medicine [IACUC 20180076].

[0144] Azoxymethane (AOM)-induced mouse CRC model was applied to the human CRC model. BALB / c mice (6 weeks old, male) were intraperitoneally (ip) injected with AOM (10 mg / kg body weight) or PBS vehicle once every 2 weeks. Seven days after each AOM injection, mice were given 2% dextran sulfate sodium (DSS) in drinking water for 4 days, followed by regular water for 3 days. This cycle was repeated three times. On the 60th day after the first AOM treatment, mice were randomly divided into eight groups (n=5). 5-FU (50 mg / kg body weight), compound 7 (each 50 mg / kg body weight), and 5-FU + compound 17 (10 mg / kg 5-FU and 30 mg / kg compound 17) in 0.2 mL PBS were injected ip every 3 days for 3 weeks. Irinotecan (20 mg / kg), Compound 31 (20 mg / kg), and Irinotecan + Compound 17 (20 mg / kg Irinotecan and 14 mg / kg Compound 17) were injected ip qod for 2 weeks. Irinotecan (20 mg / kg), Compound 37 (40 or 20 mg / kg), Compound 38 (40 or 20 mg / kg), SN-38 + Compound 16 (10 mg / kg SN-38 and 15 mg / kg Compound 16), SN-38 + Compound 17 (12 mg / kg SN-38 and 13 mg / kg Compound 17), Compound 16 (15 mg / kg), and Compound 17 (13 mg / kg) were injected via iv every 2 days for 2 weeks. Body weight was measured every 3 days (5-FU system) or every 2 days (SN-38 system). Mice were sacrificed under anesthesia with Zoletil® 50 on day 86 (5-FU system) or day 79 (SN-38 system). Tumor area and body weight were recorded at the time of sacrifice. Tumors were counted under a dissecting microscope, and the area covered by tumor was measured by imaging software (AxioVision LE 4.8.2.0).

[0145] In the following PK experiments, BALB / c mice (6 weeks old, male) were randomly divided into two groups (n=3). The two groups of mice were treated with 5-FU solution (9.5 mg / kg) and compound 7 (50 mg / kg, equivalent to 5-FU) by intraperitoneal and intravenous injection, respectively. Blood samples were collected via femoral vein with a syringe at 0, 1, 3, 5, 10, 15, 30, 45 min, 1, 2, 4 and 24 h after drug administration. 50 μL of blood samples were collected in 100 μL of extraction solvent (EtOAc / MeOH=1:1) and vortexed for 30 s. The supernatant was separated from the mixture by centrifugation at 10,000 rpm for 10 min and frozen at -30°C for UPLC-MS / MS analysis. For biodistribution experiments, the dissected whole organs were weighed with 1 mL of extraction solvent (1:1 EtOAc / MeOH) and 7 mm stainless steel beads in a 2 mL microcentrifuge tube, followed by homogenization for 30 min. All containers were kept at 4°C throughout the process. The supernatant was separated from the mixture by centrifugation at 10,000 rpm for 10 min and frozen at -30°C. 200 μL of solvent from the crude organ suspension was removed in a desiccator, followed by dissolving in 50 μL of extraction solvent. All samples were stored at -30°C and analyzed within 24 hours. 5-FU- 15 N 2 All samples were filtered through 0.22 μm PTFE into 12 × 32 mm vials for UPLC-MS / MS analysis using as a marker. Pharmacokinetic parameters were calculated using WinNonlin software (version 5.2, Pharsight, MO, USA.).

[0146] UPLC-MS / MS analysis.

[0147] The analysis was performed on an ACQUITY UPLC I-Class / Xevo TQ-XS IVD system (Waters, Milford, MA, USA) at the College of Public Health, National Taiwan University. Analytes were separated using a reversed-phase BEH C18 (100 mm × 2.1 mm, 1.7 μm, Waters) and a VanGuard BEH C18 (5 mm × 2.1 mm, 1.7 μm, Waters) precolumn. All data were acquired by MassLynx V4.2. The mobile phase consisted of 10 mM aqueous solutions of ammonium acetate (A) and acetonitrile (B) with the following settings: 0.00 min 98% A → 1.00 min 98% A → 3.00 min 10% A → 4.50 min 10% A → 4.60 min 98% A → 6.00 min 98% A. 4 μL was injected each time at a rate of 0.5 mL / min for 6.00 min. The column oven was maintained at 60° C. The multiple reaction monitoring (MRM) method was applied for quantification. Mass spectrometer parameters were as follows: capillary voltage in positive / negative ion mode: 3.0 kV / 3.0 kV; ion source temperature: 120° C.; desolvation temperature: 450° C.; cone gas flow (N 2 ): 50 L / h; desolvation gas flow (N 2 ): 700L / h; Amplifier: 650V; and Collision Gas Pressure (Ar): 3-4×10 -3 Millibars.

[0148] Plasma stability and drug release analysis

[0149] To investigate human plasma stability, synthetic conjugates were prepared in 95% human plasma. After incubation at 37 °C for the indicated times, the proteins were denatured by the addition of ACN (5-FU-based prodrugs) or DMA, followed by 12.5 mM NaH 2 PO 4(aqueous) (pH 3.2) (compounds 30 and 31). The mixture was centrifuged at 13,000 rpm for 5 min. The supernatant was analyzed by HPLC, and the samples were centrifuged to collect the clear supernatant and stored at -30°C until analysis. For release analysis, the synthesized conjugates (5-FU-based prodrugs) were dissolved in DMSO and the analysis solutions were prepared by diluting 50 μL of the stock in 750 μL of PBS (pH 7.4, containing 5 μM, 1 mM or 5 mM GSH) to a final concentration of 2.5 mM. For compounds 30 and 31, they were dissolved in DMA and a GSH (11.2 mM or 11.2 μM) solution prepared in a ratio of 50 / 40 (v / v, drug in DMA / GSH solution) in sodium acetate buffer (10 mM, pH 5.0) was added to give a final GSH of 5 mM or 5 μM at pH 7.0, respectively. After incubation for the indicated times, samples were centrifuged and the clear supernatants were collected and stored at −30° C. until analysis. RP-HPLC injections were performed under specified conditions and peak areas were integrated for further calculations.

[0150] statistical analysis

[0151] All data were obtained in at least triplicate and results were reported as mean ± standard difference mean (SEM). Comparisons between groups were analyzed via t-test, one-way analysis of variance, and two-way analysis of variance using SAS version 9.2 (SAS Institute, Cary, NC). Statistical significance was determined: ns: not significant; ****P<0.0001, ***P<0.001, **P<0.01, or *P<0.05.

[0152] Plasma stability and release profile at different GSH concentrations

[0153] The in vitro human plasma stability and release profiles of compounds 5, 7, 9, 30 and 31 are shown in Figures 2a)-3d). The stability study involved incubation of compounds 5, 7, 9, 30 and 31 in human plasma at 37 °C for different time intervals. Samples were collected for compound 7 and analyzed by RP-HPLC (Figure 2a). The half-lives of compounds 5, 7 and 9 were 0.8 h, 13 h and 3.6 h (Figure 2b), while those of compounds 30 and 31 were 20 and 50 min, respectively (Figure 3a). The cause of the lower plasma stability of compounds 5 and 9 compared to compound 7 is still unclear, but it may be due to the high pKa of the linker (pKa of both succinimide and triazole is around 8.5-9.5) increasing the susceptibility of the ester moiety of the linker to degradation. Therefore, a pH stability analysis of compound 9 was performed, which revealed that compound 9 is stable at pH 4-5 but prone to complete degradation at pH ≥ 7. To predict the in vivo fate of these conjugates, we collected metabolites of compound 7 from plasma processing assays and identified two main metabolites, M1 resulting from hydrolysis of the ester bond of compound 7 and M2, a disulfide bond exchange intermediate (Figure 2a). Since the GSH concentration is about 5-10 mM in tumor cells and 1-10 μM in plasma, we subsequently evaluated the release of 5-FU or SN-38 from compounds 5, 7, 9, 30 and 31 using GSH. The concentration of GSH used was set at 5 mM to simulate the tumor microenvironment. After 48 hours, a small amount of 5-FU was released from compounds 5 and 9, but the release of 5-FU from compound 7 was found to be dependent on the concentration of GSH used. After 4 h exposure to 5 mM GSH, the degradation rate of compound 7 was up to 60%, but in the presence of 1 mM GSH it was only about 10%, indicating that compound 7 is degraded much slower in 1 mM GSH than in 5 mM GSH (Figure 2d). The half-lives of compound 7 at 5 mM and 1 mM GSH were 4.0 h and 10.3 h, respectively. In the presence of 5 μM GSH, only 30% degradation of compound 7 was observed after 48 h (Figure 2d).The stability and release profiles of three phlorizin-containing derivatives 4, 6, and 8 were also evaluated, and all of them were similar to their corresponding congeners 5, 7, and 9, respectively. The disulfide bonds in compounds 6-7 have relatively short half-lives (<5 h) in highly reducing environments while maintaining some stability during cycling. The stability of prodrugs 30 and 31 at 0 μM, 5 μM, and 5 mM GSH is shown in Figure 3b). Prodrug 31 was relatively stable at 0 and 5 μM GSH (10% decomposition after 48 h), but under 5 mM GSH conditions, more than 90% was decomposed to SN-38 after 48 h, indicating that the prodrugs could be efficiently converted to SN-38 by high concentration GSH activation. Prodrugs 30 and 31 have the same trend, but the release rate of SN-38 at 5 mM GSH was only about 60%. To clarify the cause of the difference in the release rates of SN-38, prodrugs 30 and 31, and the metabolic products M3-M7 of these prodrugs in 5 mM GSH (Figures 3c and 3d), we analyzed the proportions of these metabolites according to mass. The results showed that M3 and M4 were the prodrugs of segment 16 and -S-(CH) of prodrug 30, respectively. 2 ) 2 M5 is compound 16, M6 is the same product as M4 but from prodrug 31, and M7 may be the conjugation product of GSH and segment 17 in prodrug 31. These overall results suggest that the concentration of GSH and its reactivity towards the prodrug segments may cause different release rates of the parent drug, and thus prodrug 31 may have greater therapeutic potential compared to 30.

[0154] GLUT-1 inhibitory activity

[0155] Previous crystallographic studies have established that the benzene-1,3,5-triol ring of phloretin forms three H-bonds, stabilizing it in the pocket of GLUT-1, but it was unclear to what extent this is impeded by substitutions at the 3-position (such as compound 17). We investigated the inhibitory activity of compounds 16-17 against GLUT-1 using COS-7 cells overexpressing GLUT-1 in a 2-NBDG (2-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose) uptake assay. Since phlorizin was unable to inhibit GLUT-1, it was by no means unexpected that compound 16 (a phlorizin derivative) was unable to inhibit GLUT-1 (Figure 4) at both 50 μM and 100 μM. Conversely, compound 17 had similar inhibitory activity to phloretin, despite the substitution at the 3-OH.

[0156] Cytotoxicity of human CRC cells

[0157] Compounds 4–9, 16–17, 30–31, phlorizin, phloretin, 5-FU, SN-38 and irinotecan were evaluated for their cytotoxicity against CRC cell lines HCT-116 and HT-29, both of which overexpress GLUT-1, using sulforhodamine B (SRB) (Tables 1–3). 5-FU had an IC of approximately 15 μM. 50 (Entry 1, Table 1). To simulate the tumor microenvironment, we first added 10 mM GSH to the culture medium, but we found that 10 mM GSH interfered with CRC cell proliferation. Therefore, 5 mM GSH was added to the culture medium of CRC cell lines. Compound 16 did not show any inhibitory activity. Compound 17 had an IC 50 showed some inhibitory activity at 15–30 μM. Compounds 4–5 had low cytotoxicity regardless of the addition of GSH. The cytotoxicity of disulfide-containing compounds 6–7 was sensitive to GSH, e.g., the IC 50was 23.8 μM in the presence of GSH and 54.9 μM in the absence of GSH. The cytotoxicity of compounds 6–7 in these cancer cells may be due to the sub-μM levels of intracellular GSH, which gradually cleaves the disulfide bonds of compounds 6–7, releasing 5-FU. The IC of compound 7 in the presence of GSH was 50 was 10.5 μM for HCT-116 and 3.8 μM for HT-29, the IC of 5-FU in both cell lines. 50 The cytotoxicity of compounds 8-9 was low. The cytotoxicity of compounds 16 and 17 was also assayed in combination with 5-FU. 5-FU in combination with compound 17 inhibited cell viability more effectively than the other compounds when administered alone, reflecting a synergistic inhibitory effect. In addition, the cytotoxicity of 5-FU and compound 7 in the low-expressing GLUT-1 normal cell line NHDF (Western blot analysis of GLUT-1) was also examined. Both 5-FU and compound 7 had a significant inhibitory effect (IC 50 The IC values ​​were found to be low (70.4 ± 7.2 μM and 90.0 ± 2.6 μM, respectively) suggesting that compound 7 has low toxicity to normal cells. SN-38 and irinotecan had IC values ​​of approximately 2–430 nM and 0.3–8.5 μM, respectively, in CRC cells. 50 The combination of SN-38 with compounds 16 or 17 showed synergistic effects in HT-29 cell lines. However, to our surprise, compounds 30 and 31 had lower or similar cytotoxicity compared to SN-38 and SN-38 in combination with compounds 16 or 17. Overall, HT-29 cells were sensitized to the compounds, whereas HCT-116 cells were similar in the absence of GSH. Both compounds 30 and 31 did not significantly improve cytotoxicity at high concentrations of GSH, and showed nanolevel IC in the absence of GSH. 50Since the cytotoxicity of the compounds was about 3-7 μM in HCT-116 cell line, in vivo side effects may be of concern. Compounds 37 and 38, another type with cathepsin B sensitivity and ether linkage, were also evaluated for their cytotoxicity and found to have approximately 3-7 μM levels in HCT-116 cell line and relatively low sensitivity to HT-29 cells (entries 7-8, Table 3). Here, to investigate the proof of concept of conjugation of glucose inhibitors with anticancer drugs, compounds 7 and 31 were selected for further evaluation of therapeutic efficacy in animal studies. Table 1. Cytotoxicity of compounds 4–9, 16–17, 5-FU, and 5-FU in combination with compounds 16 or 17 in HCT-116 and HT-29 cell lines. a . TIFF2025513000000051.tif127170 a Cell lines were incubated with compounds for 48 h. IC 50 value was determined. b ND=not measured. Table 2. Cytotoxicity of compounds 30-31, phlorizin, phloretin, SN-38, irinotecan, and SN-38 in combination with 16 or 17 (prepared with DMA) in HCT-116 and HT-29 cell lines. a . TIFF2025513000000052.tif127170 a I C 50 Values ​​were determined by SRB assay. b Cell lines were incubated with compounds for 72 hours. c The cell lines were incubated with the compounds for 48 hours. Table 3. Cytotoxicity of compounds 16–17, 37–38, phlorizin, phloretin, SN-38, irinotecan, and SN-38 in combination with 16 or 17 (prepared with DMA) in HCT-116 and HT-29 cell lines. a、b . TIFF2025513000000053.tif100170 a I C 50Values ​​were determined by SRB assay. b The cell lines were incubated with the compounds for 48 hours.

[0158] Assessment of cathepsin B expression levels in healthy mice versus AOM-DSS-induced colon cancer mice

[0159] Before evaluating the therapeutic efficacy of drugs in colon cancer mice, we first evaluated whether the AOM-DSS-induced colon cancer model in BALB / c mice was a suitable model for cathepsin B overexpression remission-type disease. In addition, healthy BALB / c mice (6 weeks old, male) were also used for comparison. Immunohistochemistry results showed that the orthotopic CRC mouse model indeed had a higher expression level of cathepsin B compared with the normal group (Figure 5a)-d), supporting a platform for evaluating the therapeutic efficacy of cathepsin B-sensitive prodrugs.

[0160] Evaluation of in vivo activity of synthetic compounds in an orthotopic CRC mouse model

[0161] Compounds 7 and 31 were selected for in vivo evaluation based on their release profile and cytotoxicity experimental results. An orthotopic CRC mouse model was established by intraperitoneally (ip) injection of AOM (10 mg / kg body weight) into BALB / c mice (6 weeks old, male) and supplementing their drinking water with 2% dextran sulfate sodium (DSS) (Figures 6a and 7a). After 2 months, solutions of 5-FU (50 mg / kg), compound 7 (50 mg / kg), and 5-FU + compound 17 (dosing of 10 mg / kg 5-FU and 30 mg / kg compound 17 based on compound 7) in 0.2 mL PBS were injected ip every 3 days for 3 weeks. Irinotecan (20 mg / kg), Compound 31 (20 mg / kg), and Irinotecan + Compound 17 (20 mg / kg Irinotecan and 14 mg / kg Compound 17 doses based on Compound 31) in 5% DMA + 2% Tween 80 + 93% PBS were injected via ip route every 2 days for 2 weeks. 2 CO 3 (ddH 2Irinotecan (40 mg / kg), Compound 37 (20 or 40 mg / kg), Compound 38 (20 or 40 mg / kg), SN-38 + Compound 16 (SN-38 at 10 mg / kg and Compound 16 at 15 mg / kg), SN-38 + Compound 17 (SN-38 at 12 mg / kg and Compound 17 at 13 mg / kg), Compound 16 (15 mg / kg), Compound 17 (13 mg / kg) in D5W (5% O) + 60% in D5W were injected iv every 2 days for 2 weeks. Based on tumor area calculations, Compound 7 showed better tumor inhibition than 5-FU at a dose of 50 mg / kg (0.07 mmol / kg 5-FU) when only 19% of 5-FU was administered (Figures 6b and 6c). Compound 31 (20 mg / kg, 0.022 mmol / kg SN-38) showed better antitumor effects than irinotecan (20 mg / kg, 0.034 mmol / kg SN-38) when administered at 65% of SN-38 (Figures 7b and 7c). Compound 38 (40 mg / kg, 0.03 mmol / kg) also showed better antitumor effects than irinotecan (40 mg / kg, 0.068 mmol / kg) when administered at 44% of SN-38 (Figure 7d). Since GLUT-1 / 3 / 4 are usually overexpressed in CRC patients and phloretin is known to inhibit GLUT3 / 4 inhibition, compounds 7, 31 and 38 may show targeting effects on GLUT-1 / 3 / 4, resulting in significant tumor inhibition. Furthermore, the SGLT-targeting compound 37 also improved tumor area in mice at a dose of 40 mg / kg (0.03 mmol / kg) compared to irinotecan (40 mg / kg, 0.068 mmol / kg). Mice treated with free 5-FU were observed to rapidly lose approximately 10% body weight after injection (Figure 6d), likely reflecting the systemic toxicity of 5-FU. There was no significant change in body weight in mice administered compounds 7, 31 or 38 (Figures 6d and 7e)-f). The combination of compound 17 with 5-FU (containing 19% of the free 5-FU dose) or irinotecan (containing 65% of the SN-38 dose) also reduced tumor area in mouse models, demonstrating a synergistic effect that was also observed in the cellular assay.

[0162] Evaluation of pharmacokinetic and biodistribution profiles

[0163] The plasma concentration and organ distribution of compound 7 were evaluated in BALB / c mice (n=3) by single dose injection of the compound. To analyze the 5-FU release profile of compound 7, blood samples were collected at 1, 3, 5, 10, 15, 30, 45, 1, 2, 4, and 8 hours after intravenous injection (iv) (50 mg / kg) and the concentration of compound 7 was determined by UPLC-MS / MS. The concentration of compound 7 rapidly decreased within 10 minutes (Figure 8a), possibly related to the expression of carboxylesterase, dihydropyrimidine dehydrogenase (DPD) and the high tissue penetration of compound 7 in mouse blood. 5-FU released from compound 7 was detected within 2 hours and persisted in the blood longer than compound 7 (Figure 8a). Compound 7 showed an unusual plasma pharmacokinetic (PK) curve in that its concentration was undetectable after 30 min (detection limit = 0.003 μg / mL) but detectable again after 45 min and 4 h, suggesting that it was rapidly absorbed into mouse cells and tissues and reached a balance during elimination. To investigate the distribution-related antitumor efficacy of compound 7, blood samples were collected after intraperitoneal injection (ip) (50 mg / kg) of compound 7 and 5-FU into the peritoneal cavity of BALB / c mice (n = 3). The concentration of compound 7 was below the detection limit after 5 min due to its high tissue penetration due to its lipophilicity. The T of 5-FU from compound 7 was 0.003 μg / mL, suggesting that it was rapidly absorbed into mouse cells and tissues and reached a balance during elimination. maxThe release of 5-FU was delayed from 10 to 45 min compared to the 5-FU group, and the 5-FU concentration in both groups was not detected after 2 h, indicating that the antitumor efficacy may be related to high tissue penetration (Figure 8b). Therefore, the distribution of compound 7 (50 mg / kg, 5-FU equivalent) and 5-FU (9.5 mg / kg) in BALB / c mouse organs was evaluated 1 h after intraperitoneal injection. Compound 7 was found to be distributed in the stomach, colon, heart and liver (Figure 8c). As the amount of compound 7 in each organ was low, further analysis of 5-FU released from compound 7 was performed, which showed that, similar to previous reports, 5-FU was mainly detected in the colon, with small amounts in the kidney and stomach, while free 5-FU was distributed in the stomach, spleen and kidney (Figure 8c). These results indicate that the conjugation of compound 7 is a suitable strategy to deliver 5-FU to the colon. We hypothesize that this delivery is mediated by GLUT-1, which is mainly expressed in the colon. When a portion of phloretin binds to glucose transporters, the entire conjugate can be absorbed by cells. Because the intracellular GSH concentration in cancer cells can be 10-fold higher than that in normal cells, cleavage of the disulfide bond of compound 7 by GSH releases 5-FU from the conjugate. The high tissue permeability of compound 7 results in a delayed release of 5-FU, prolonging the exposure to 5-FU and enhancing its tumor eradication effect. Organ distribution analysis revealed the colon-targeting effect of 5-FU, resulting in promising antitumor efficacy with low toxicity.

[0164] The above description of exemplary embodiments of the invention has been presented for purposes of illustration and description only, and is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teachings.

Claims

1. Formula I: A-L-B (I) or a pharma- ceutically acceptable salt thereof. During the ceremony, A is an active pharmaceutical moiety or a prodrug thereof; B is a target module, L is of formula II: is a linker represented by During the ceremony, R １ Ha-SR ２ or and R ２ is R ３ , -SR ３ or and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ is R ５ , -OC(O)-R ５ , -C(O)-R ５ , -C(O)NH-R ５ , -C(O)-, -NHC(O)-R ５ , -OC(O)OR ５ or -C(O)-Z-NH-phenylene-R ５ and R ５ Ha-(CH ２ ) ｍ - and Z is -Val-Cit-, -Phe-Lys-, -Val-Ala- or -Gly-Phe-Leu-Gly-; p is an integer from 2 to 9; n is an integer from 2 to 9, m is 0 or 1, and The dotted lines are covalent bonds. provided that the conjugate is: and The compound is not a compound selected from the group consisting of:

2. 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein A is an anti-cancer therapeutic moiety.

3. 3. The conjugate of claim 2, or a pharma- ceutically acceptable salt thereof, wherein A is selected from 5-fluorouracil (5-FU), SN-38, irinotecan, lonidamine, bleomycin, mitomycin, cisplatin, doxorubicin, DM-1, taxol, cabazitaxel, floxuridine, or FdUMP.

4. 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein B is a glucose transporter specific binding agent.

5. 5. The conjugate of claim 4, or a pharma- ceutically acceptable salt thereof, wherein B is phloretin optionally substituted with glucose, mannose, or 2-fluoro-D-glucose.

6. R １ Ha-SR ２ and R ２ teeth and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ HA -C(O)-R ５ and R ５ Ha-(CH ２ ) ｍ - and p is 2; n is 5, and 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 0.

7. R １ Ha-SR ２ and R ２ Ha-SR ３ and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ Is -OC(O)-R ５ or -OC(O)O-R ５ and R ５ Ha-(CH ２ ) ｍ - and p is 2; n is 2, and 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 0.

8. R １ is SR ２ and R ２ teeth and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ is -C(O)-Z-NH-phenylene-R ５ and R ５ Ha-(CH ２ ) ｍ - and Z is -Val-Cit-, p is 2; n is 4, and 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 1.

9. R １ teeth and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ HA -C(O)O-R ５ and R ５ Ha-(CH ２ ) ｍ - and p is 2; n is 3, and 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 1.

10. R １ Ha-SR ２ and R ２ teeth and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ HA -C(O)O-R ５ and R ５ Ha-(CH ２ ) ｍ - and p is 2; n is 4, and 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 1.

11. R １ Ha-SR ２ and R ２ Ha-SR ３ and R ３ Ha-(CH ２ ) ｎ -R ４ and R ４ HA -C(O)O-R ５ and R ５ Ha-(CH ２ ) ｍ - and p is 2; n is 3, and 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 1.

12. Formula III: The conjugate according to claim 1 , wherein the conjugate is represented by the formula: During the ceremony, R is OH or It is.

13. 2. The conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of: and During the ceremony, R is OH or It is.

14. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 13.

15. The pharmaceutical composition of claim 14 further comprising a therapeutic agent.

16. 14. Use of a conjugate according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for treating a disease, recurrence or progression in a subject, or for increasing the chances of survival over the relevant period in a subject diagnosed with a disease.

17. 17. The use according to claim 16, wherein the disease is a cancer selected from solid or liquid cancers or metastases thereof.

18. 18. The use according to claim 17, wherein the cancer is selected from the group consisting of lung cancer, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumors, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, head and neck cancer, lymphoma, leukemia, myeloma, and myeloproliferative neoplasms.

19. 17. The use of claim 16, wherein the subject is refractory or resistant to the drug.

20. The use according to claim 16, wherein the agent is capable of being used in combination with a therapeutic agent.

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