Conjugates for targeted therapy

Abstract

The present invention provides a novel conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate has an active drug moiety or a prodrug thereof, a targeting moiety, and a linker therebetween. The conjugate, or a pharmaceutically acceptable salt thereof, is useful for treating a disease, recurrence or progression in an individual or for increasing the likelihood of survival of an individual diagnosed with a disease for a related period of time.

Description

Technical Field

[0001] The present invention relates to a novel conjugate, in particular to a conjugate for targeted therapy. Background Art

[0002] Selective delivery of drugs to defined disease sites is one of the most critical goals of clinical therapy. For example, in the case of treating certain tumors, there is still a need for delivery systems that can target anticancer drugs to the tumor site while minimizing off-target effects. Despite great advances in chemotherapy, current therapies are still unsatisfactory and the prognosis for most patients remains poor. A major challenge is the occurrence of undesirable side effects.

[0003] Typically, drugs are formulated for topical, oral, intravenous or intramuscular administration. Drugs administered along these routes often require much higher doses than the actual amount of drug used in situ. In addition, toxic drugs are administered in amounts that may limit their use over time or cumulative use.

[0004] For example, colorectal cancer (CRC), which is the fourth most common and second deadliest cancer in the world, has been treated with 5-fluorouracil (5-FU) as the primary treatment for metastatic colorectal cancer (mCRC) for more than 40 years, usually in combination with leucovorin with or without oxaliplatin as the 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 chemotherapy with 5-FU is only about 23% (Sotos et al., Cancer Treat Rev 1994, 20, 11-49). Irinotecan is a prodrug of 7-ethyl-10-hydroxycamptothecin (SN-38). It is an important chemotherapy agent as a second-line therapy for patients with fluorouracil-refractory stage IV CRC. However, a phase II study showed that the response rate of single-agent irinotecan in patients with fluorouracil resistance was only about 27% (Shimada et al., J Clin Oncol 1993, 11, 909-13). In addition, the poor selectivity of 5-FU-based regimens and irinotecan can cause adverse reactions such as neutropenia, diarrhea, nausea, vomiting, and stomatitis in mCRC therapy (Falcone et al., J Clin Oncol 2001, 19, 3456-62).

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

[0006] The present invention provides a novel conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate has an active drug portion or a prodrug thereof, a targeting portion (targeting module) and a linker therebetween. The conjugate or a pharmaceutically acceptable salt thereof is suitable for treating a disease, recurrence or progression of an individual or increasing the likelihood of survival of an individual diagnosed with a disease during a relevant period of time.

[0007] In one embodiment of the first aspect of the present invention, the conjugate is represented by formula I,

[0008] ALB(I)

[0009] or a pharmaceutically acceptable salt thereof, wherein

[0010] A is an active drug moiety or a prodrug thereof;

[0011] B is the targeting moiety;

[0012] L is a linker represented by formula II

[0013] in

[0014] R1 is -SR2 or

[0015] R2 is R3, -SR3 or

[0016] R3 is -(CH2) n -R4;

[0017] R4 is R5, -OC(O)-R5, -C(O)-R5, -C(O)NH-R5, -C(O)-, -NHC(O)-R5, -OC(O)O-R5 or -C(O)-Z-NH-phenylene-R5;

[0018] R5 is -(CH2) m -;

[0019] Z is -Val-Cit-, -Phe-Lys-, -Val-Ala- or -Gly-Phe-Leu-Gly-;

[0020] p is an integer from 2 to 9;

[0021] n is an integer from 2 to 9;

[0022] m is 0 or 1; and

[0023] The dashed line is a covalent bond;

[0024] The proviso is that the conjugate is not a compound selected from the group consisting of:

[0025]

[0026] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, A is an anticancer therapeutic moiety. In some embodiments, A is selected from 5-fluorouracil (5-FU), 7-ethyl-10-hydroxyphilin (SN-38), irinotecan, lonidamine, bleomycin, mitomycin, cisplatinum, doxorubicin, maytansine (DM-1), paclitaxel, cabazitaxel, floxuridine or fluorodeoxyuridine (FdUMP).

[0027] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, B is a binding agent with glucose transporter specificity. In some embodiments, B can be selected from phloretin optionally substituted with glucose, mannose or 2-fluoro-glucose.

[0028] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, R1 is -SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)-R5; R5 is -(CH2) m -; p is 2; n is 5; and m is 0.

[0029] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, R1 is -SR2; R2 is -SR3; R3 is -(CH2) n -R4; R4 is -OC(O)-R5 or -OC(O)O-R5; R5 is -(CH2) m -; p is 2; n is 2; and m is 0.

[0030] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, R1 is SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)-Z-NH-phenylene-R5; R5 is -(CH2) m -; Z is -Val-Cit-; p is 2; n is 4; and m is 1.

[0031] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, R1 is R3 is -(CH2) n-R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 3; and m is 1.

[0032] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, R1 is -SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 4; and m is 1.

[0033] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, R1 is -SR2; R2 is -SR3; R3 is (CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 3; and m is 1.

[0034] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, the conjugate is represented by Formula III,

[0035] in

[0036] R is OH or

[0037] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, the conjugate is selected from the group consisting of:

[0038]

[0039]

[0040] Where R is OH or

[0041] One embodiment of the second aspect of the invention is a method of treating a disease, recurrence or progression in a subject or increasing the likelihood of survival of a subject diagnosed with a disease over a relevant period of time, comprising administering to the subject a conjugate as described herein or a pharmaceutically acceptable salt thereof.

[0042] In one embodiment of the method, the disease is a cancer selected from solid cancer or liquid cancer or its metastasis. Examples of cancer include, but are not limited to, squamous cell carcinoma; lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma; peritoneal cancer; hepatocellular carcinoma; stomach cancer or 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 or uterine cancer; salivary gland cancer; kidney cancer or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; head and neck cancer; lymphoma; leukemia; myeloma; and myeloproliferative neoplasm.

[0043] In one embodiment of the method, the individual is refractory or resistant to the drug.

[0044] In one embodiment of the method, A is an active drug moiety or a prodrug thereof, wherein the active drug moiety is an anticancer therapeutic moiety. In some embodiments, A is selected from 5-fluorouracil (5-FU), 7-ethyl-10-hydroxystyrene (SN-38), irinotecan, lonidamine, bleomycin, mitomycin, cisplatinum, doxorubicin, maytansine (DM-1), taxol, cabazitaxel, floxuridine or fluorodeoxyuridine (FdUMP).

[0045] In one embodiment of the method, B is a binding agent specific for a glucose transporter. In some embodiments, B is phloretin optionally substituted with glucose, mannose or 2-fluoro-glucose.

[0046] In one embodiment of the method, the conjugate is represented by formula III

[0047]

[0048] in

[0049] R is OH or

[0050] In one embodiment of the method, the conjugate is selected from the group consisting of:

[0051]

[0052]

[0053] Where R is OH or

[0054] One embodiment of the third aspect of the present invention is a composition comprising a therapeutically effective amount of the aforementioned conjugate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or vehicle.

[0055] These and other aspects will become apparent from the following description of the preferred embodiments in conjunction with the following drawings, wherein changes and modifications may be made without departing from the spirit and scope of the novel concepts of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The structures of 5-FU (1), phlorizin (2), phlorizin (3), SN-38 (28), irinotecan (39), and the designs of compounds 4-9, 30-31, 37-38, and 42-45 are shown.

[0057] Figures 2a) to 2d) The in vitro plasma stability and release curves of compounds 5, 7 and 9 under different conditions are shown: Figure 2a ) Analysis of compound 7 in human plasma using reverse phase high performance liquid chromatography (RP-HPLC); Figure 2b ) Stability of compounds 5, 7 and 9 in human plasma at 37°C; Figure 2c ) RP-HPLC analysis of compound 7 at 5 mM glutathione (GSH); Figure 2d ) In vitro release curves of compound 7 and 5-FU derived from compound 7 at different GSH concentrations (1 mM and 5 mM); the experimental data are expressed as mean ± standard error of the mean (SEM) (n=3).

[0058] Figures 3a) to 3d) The in vitro plasma stability and release curves of compounds 30 and 31 under different conditions are shown: Figure 3a ) Stability of compounds 30 and 31 in human plasma at 37°C; Figure 3b ) In vitro release curves of compounds 30, 31, and SN-38 derived from compounds 30 and 31 at different GSH concentrations (5 μM or 5 mM); Figure 3c ) Compound 30 was analyzed using RP-HPLC under 5 mM GSH conditions; Figure 3d ) Compound 31 was analyzed by RP-HPLC under the condition of 5 mM GSH; the experimental data are expressed as mean±standard error of the mean (SEM) (n=3).

[0059] Figure 4The inhibitory effects of compounds 16 and 17 on GLUT-1 activity were shown by analysis of 2-NBDG absorbance; experimental data are presented as mean ± standard error of the mean (SEM) (n = 4). ns, non-significant difference, ****P < 0.0001 (two-way analysis of variance).

[0060] Figures 5a) to 5d) Photographs showing immunohistochemical staining of cathepsin B in paraffin-embedded colon sections of: Figure 5a ) Healthy mouse, scale bar = 200 μm, magnification 400 times; Figure 5b ) Healthy mouse, scale bar = 50 μm, magnification 100 times; Figure 5c )CRC mice, scale bar = 200 μm, magnification 400x and Figure 5d ) CRC mice, scale bar = 50 μm, magnification 100x. 3,3'-Diaminobenzidine (DAB) substrate (brown) was used as an immunohistochemical marker to detect cathepsin B. Nuclei were counterstained with hematoxylin (blue / purple).

[0061] Figure 6a ) Schematic diagram of administration of 5-FU series compounds in CRC mouse model; Figure 6b ) Photographs of mouse colon tumors taken under a microscope; Figure 6c ) Tumor area and Figure 6d ) Body weight changes of mice treated with control group (PBS (phosphate buffered saline) 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, the dose is 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 analysis of variance).

[0062] Figure 7a ) Schematic diagram of administration of SN-38 series compounds in CRC mouse model; Figure 7b ) Photographs of mouse colon tumors taken under a microscope; Figure 7c ) Comparison of tumor area in the intraperitoneal (ip) injection group and Figure 7d ) Comparison of tumor area in the intravenous (iv) injection group. Figure 7e) The body weight changes of mice treated with control group (5% dimethylacetamide (DMA) + 2% Tween 80 + 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 ) Body weight changes of mice treated with control group (10% dimethyl sulfoxide (DMSO) + 20% Cremophor + 10% Na2CO3 (5% in double distilled water (dd)) + 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, the dose is based on 40 mg / kg compound 38), SN-38 + compound 16 (10 mg / kg SN-38 and 15 mg / kg compound 16, the dose is based on 40 mg / kg compound 37), compound 17 (13 mg / kg), and compound 16 (15 mg / kg) via iv injection. Experimental data are expressed as mean ± standard error of the mean (SEM) (n = 3 to 5). ****P<0.0001, ***P<0.001, **P<0.01 or *P<0.05 (one-way analysis of variance).

[0063] Figures 8a) to 8c) Shown are the pharmacokinetic profiles of compound 7 and 5-FU in mice following iv and ip injections. Figure 8a ) shows the changes in plasma concentration over time after iv injection of compound 7 (50 mg / kg) and iv injection of compound 7 and 5-FU (50 mg / kg); Figure 8b ) shows the changes in plasma concentration over time after ip injection of compound 7 (50 mg / kg), 5-FU (50 mg / kg), and ip injection of compound 7 and 5-FU (50 mg / kg). If the compound concentration is lower than the detection limit of mass spectrometry (LC-MS / MS) (0.003 μg / mL), these data points are arbitrarily labeled as concentrations slightly lower than 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) into BALB / c mice. Experimental data are expressed as mean ± standard error of the mean (SEM) (n=3). *P<0.05. DETAILED DESCRIPTION

[0064] definition

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In the event of a conflict, the present document, including definitions, will control. As used in this specification and the accompanying claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

[0066] Throughout this specification, unless otherwise specified herein, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated elements or integers or groups of elements or integers but not the exclusion of any other elements or integers or groups of elements or integers.

[0067] As used herein, the term "conjugate" refers to two or more molecules covalently linked to form a larger compound. In some embodiments, the conjugate includes one or more specific binding molecules covalently linked to one or more therapeutic molecules.

[0068] As used herein, the terms "Formula I" and "Formula III" may be referred to hereinafter as "conjugates of the present invention". Such terms are also defined to include all forms of the conjugates of Formula I or III, including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs and metabolites thereof. For example, the conjugates of the present invention or pharmaceutically acceptable salts thereof may exist in unsolvated and solvated forms. When the conjugate is tightly bound to solvent or water, it has a well-defined stoichiometry and is not affected by humidity. However, when the conjugate is weakly bound to solvent or water (such as in channel solvent compounds and hygroscopic compounds), the water / solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometric conjugates are generally formed.

[0069] Stereoisomers of Formula I or III include cis and trans isomers, optical isomers (such as R and S enantiomers, diastereomers), geometric isomers, rotational isomers, configurational isomers and tautomers, including compounds exhibiting more than one type of isomerism; and mixtures thereof (such as racemates and diastereomeric pairs). Also included are acid addition salts or base addition salts in which the counter ion is optically active (e.g., D-lactate or L-lysine) or racemic (e.g., DL-tartrate or DL-arginine).

[0070] When any racemate is crystallized, there are two different types of crystals possible. The first type is the racemic compound (true racemate) mentioned above, in which one homogeneous form of crystal containing two enantiomers in equimolar amounts is produced. The second type is a racemic mixture or agglomerate, in which two crystal forms each containing a single enantiomer are produced in equimolar amounts.

[0071] The conjugates of formula I or III may exhibit tautomerism; such tautomers are also considered compounds of the invention. All such tautomeric forms and mixtures thereof are included within the scope of the conjugates of formula I or III. Tautomers exist as mixtures of tautomeric sets in solution. In solid form, usually, one tautomer is dominant. Although one tautomer may be described, the present invention also includes all tautomers of the conjugates of formula I or III and salts thereof.

[0072] As used herein, "pharmaceutically acceptable salts" include salts of acidic or basic groups that may be present in the compounds described herein, unless otherwise defined. The compounds of the present invention, which are basic in nature, are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those acids that form non-toxic acid addition salts (i.e., salts containing pharmacologically acceptable anions), such as acetates, benzenesulfonates, benzoates, bicarbonates, bisulfates, bitartrates, borates, bromides, calcium edetate, camphorsulfonates, carbonates, chlorides, clavulanates, citrates, dihydrochlorides, edetate, edislyates, estolates, ethanesulfonates, ethylsuccinates, fumarates, glucoheptonates, gluconates, glutamates, beta-arsonic acids. salt, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, methylsulfate, galactarate, napsylate, nitrate, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, theoclate, toluenesulfonate, triethiodide, and valerate.

[0073] As used herein, "active drug moiety" refers to the portion of an active pharmaceutical ingredient (API) that has a physiological, medicinal, pharmacological or therapeutic effect. The term is intended to include APIs in any suitable form, such as pharmaceutically acceptable salts, complexes, solvates or prodrugs thereof, or, if relevant, in any stereoisomeric form, including any enantiomeric or racemic form or a combination of any of the above. Examples of APIs include, but are not limited to, hormones, peptides, small molecules or prodrugs thereof. In another aspect, the active drug moiety may be a biomolecule comprising one or more chemical groups that are typically synthesized in a living organism, including, but not limited to, amino acids, nucleotides, polysaccharides, monosaccharides, lipids or combinations thereof.

[0074] As used herein, the term "prodrug" refers to any substance, molecule or entity that is in a form based on the substance, molecule or entity to act as a therapeutic agent after a chemical or physical change. The prodrug may be covalently linked or chelated in some manner and released or converted into an active drug moiety before, during or after administration to a mammalian subject. The prodrug can be prepared by modifying the functional groups present in the compound in a certain manner so that the modification can be cleaved in conventional operations or in vivo to obtain the parent compound. Prodrugs include compounds to which hydroxyl, amine, sulfhydryl or carboxyl groups are attached to any group, which, when administered to a mammalian subject, is cleaved to form a hydroxyl, amine, sulfhydryl or free carboxyl group, respectively.

[0075] As used herein, the term "targeting moiety" refers to a moiety that is capable of directly or indirectly binding to a specific target (e.g., a receptor on the surface of a cell or a protein in a tissue), thereby facilitating the delivery of the molecular construct of the present invention to a specific target. In certain instances, the targeting moiety can direct the molecular construct to a location adjacent to the target cell. In other cases, the targeting moiety can specifically bind to a molecule on the surface of the target cell; or the targeting moiety can specifically bind to a second molecule that can specifically bind to a molecule on the surface of the target cell. In some cases, once the targeting moiety is engaged with the specific target, the targeting moiety can internalize the molecular construct of the present invention, allowing it to move into the cytoplasm of the target cell. The targeting moiety can be an antibody or ligand for a cell surface receptor; or a molecule that binds to the above-mentioned antibody or ligand, thereby indirectly targeting the molecular construct of the present invention to a target site (e.g., the surface of a selected cell).

[0076] As used herein, the term "glucose transporter" refers to a sodium-dependent glucose transporter (SGLT) or a facilitated glucose transporter (GLUT). SGLT transports glucose into cells against a concentration gradient by transporting sugar upward across the cell membrane and sodium downward. The inward sodium gradient at the apical surface of epithelial cells is maintained by active extrusion of sodium across the basolateral surface on the antiluminal surface of the cell driven by ATP. Phlorizin specifically and competitively inhibits both SGLT-1 and SGLT-2, and does not affect GLUT 1 to 12.

[0077] Of GLUTs 1 to 12, the insulin-independent glucose transporter GLUT-1 is widely overexpressed in human cancers, and high expression of these proteins in tumor biopsy samples is associated with poor cancer prognosis, making them attractive therapeutic targets. Phloretin is a well-known and naturally abundant GLUT-1 inhibitor. In addition, many glucose transporter 1-4 inhibitors have been identified, such as Cytochalasin B, WZB117, GLUT-1, GLUT-2, BAY-876, Chromopynone-1, Glutor, Glupin, NV-5440, as disclosed in ChemBioChem 2020, 21, 45-52.

[0078] As used herein, "anti-cancer therapeutic moiety" refers to a moiety of anti-cancer drugs that includes all chemical or physical interventions used to treat cancer. Anti-cancer drugs include chemotherapeutic agents, such as cytotoxic agents or immunotoxic agents, but also include radiolabeled antibodies, peptides and chemicals that may emit alpha, beta and gamma rays and electrons. Radiation therapy further includes sufficiently high energy photons, charged particles, such as electrons, positrons, muons, protons, alpha particles and heavy atomic nuclei from accelerators, but also includes neutrons and gamma rays. In some embodiments, the anti-cancer drug can be selected from 5-fluorouracil (5-FU), 7-ethyl-10-hydroxyphilin, irinotecan, lonidamine, bleomycin, mitomycin, cisplatin, doxorubicin, maytansine, paclitaxel, cabazitaxel, floxuridine or fluorodeoxyuridine.

[0079] As used herein, "substituted" means that one or more hydrogen atoms on the designated atom are replaced with a selected designated group, provided that the normal valency of the designated atom in the existing context is not exceeded and that the substitution results in a stable compound.

[0080] As used herein, "optionally substituted" refers to a group that is unsubstituted or substituted with one or more substituents.

[0081] As used herein, "treatment" and "treating" encompass both preventive (i.e., prophylactic) or therapeutic (i.e., curative and / or palliative) treatment. Thus, the terms "treatment" and "treating" encompass therapeutic treatment of a patient who has already developed the condition, especially in an overt form. Therapeutic treatment may be symptomatic treatment to alleviate the symptoms of a specific indication, or may be causal treatment to reverse or partially reverse the condition of the indication or to arrest or slow the progression of the disease. Thus, the conjugates, compositions and methods of the invention may be used, for example, as therapeutic treatments over a period of time as well as chronic therapies. Additionally, the terms "treatment" and "treating" encompass preventive treatment, i.e., treatment of a patient who is at risk of developing the conditions mentioned above, thereby reducing that risk.

[0082] As used herein, "therapeutically effective amount" means an amount of a conjugate of the invention that (i) treats or prevents a particular disease or condition, (ii) alleviates, 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.

[0083] "Cancer", "tumor" and similar terms include precancerous, neoplastic, transformed and cancerous cells, and may refer to solid tumors, or non-solid tumors (see, e.g., Edge et al. AJCC Cancer Staging Manual (7th Edition 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd Edition 2009)). Cancer includes both benign and malignant neoplasms (abnormal growth). "Transformation" refers to spontaneous or induced phenotypic changes, e.g., immortalization of cells, morphological changes, abnormal cell growth, reduced contact inhibition, fixation and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd Edition 1994)). Although transformation can be caused by infection with a transformed virus and the incorporation of new genomic DNA or the uptake of exogenous DNA, it can also occur spontaneously or after exposure to a carcinogen.

[0084] Conjugates and general preparation procedures

[0085] In one embodiment of the first aspect of the present invention, the conjugate is represented by formula I,

[0086] ALB(I)

[0087] or a pharmaceutically acceptable salt thereof.

[0088] A is an active drug moiety or a prodrug thereof; specifically, an anticancer therapeutic moiety, and in some embodiments of the invention, is 5-fluorouracil (5-FU), 7-ethyl-10-hydroxyadrenaline, irinotecan, lonidamine, bleomycin, mitomycin, cisplatin, doxorubicin, maytansine, paclitaxel, cabazitaxel, ofloxetine or fluorodeoxyuridine.

[0089] B is a targeting moiety. In some embodiments of the present invention, B is a targeting moiety for targeting tumor cells, such as a binding agent with glucose transporter specificity. In some embodiments, the glucose transporter is a sodium-dependent glucose transporter (SGLT) or a facilitated glucose transporter (GLUT). In some other embodiments, B can be selected from phloretin optionally substituted with glucose, mannose or 2-fluoro-glucose.

[0090] Compared to non-cancerous cells, cancer cells are more dependent on the anaerobic glycolytic pathway than mitochondrial oxidative phosphorylation, the so-called Warburg effect. There are two types of glucose transporters that enable cancer cells to take up glucose: (i) facilitated glucose transporters GLUT (1 to 4), and (ii) secondary active sodium-glucose co-transporters (SGLT-1 to 2). Although not wishing to be bound by theory, it is believed that tumor cells overexpress glycolytic enzymes and glucose transporters, both of which are associated with the aggressiveness and metastatic potential of cancer. Specimens from CRC patients treated with 5-FU were found to have higher glucose transporter transcript levels in patients who were refractory to therapy, and overexpression of glucose transporters is known to be associated with 5-FU resistance in colorectal cancer cells via scavenging free radicals with pyruvate.

[0091] Phloretin is a well-known and naturally abundant GLUT-1 inhibitor that has been studied for its anticancer activity and has been shown in preclinical studies to be able to overcome therapeutic resistance to anticancer agents when administered in combination with anticancer agents. Phlorizin is a SGLT-1 inhibitor that differs from phloretin in that it carries an additional glucose conjugate at the ortho-hydroxyl group of the phenyl moiety. Since these glucose transporters are physiologically manifested in a variety of cells and tissues, phloretin or phloretin has the ability to selectively target SGLT-1 or GLUT-1 on cancer cells.

[0092] L is a linker represented by formula II

[0093] in

[0094] R1 is -SR2 or

[0095] R2 is R3, -SR3 or

[0096] R3 is -(CH2) n -R4;

[0097] R4 is R5, -OC(O)-R5, -C(O)-R5, -C(O)NH-R5, -C(O)-, -NHC(O)-R5, -OC(O)O-R5 or -C(O)-Z-NH-phenylene-R5;

[0098] R5 is -(CH2) m -;

[0099] Z is -Val-Cit-, -Phe-Lys-, -Val-Ala- or -Gly-Phe-Leu-Gly-;

[0100] p is an integer from 2 to 9;

[0101] n is an integer from 2 to 9;

[0102] m is 0 or 1; and

[0103] The dashed line is a covalent bond.

[0104] L is redox-sensitive and / or biocleavable in the tumor environment. When the conjugate is present in a normal environment, L is intact and connects A and B. When the conjugate is present in an environment rich in GSH or cathepsin B, L is cleaved and A is released to exhibit drug activity.

[0105] Examples of L include, but are not limited to, redox-sensitive and / or biocleavable linkers, succinimidyl thioethers, disulfide linkers, triazoles, valine-citrulline dipeptides, p-aminobenzyl spacers, and ether bonds.

[0106] In one embodiment,

[0107] R1 is -SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)-R5; R5 is -(CH2) m -; p is 2; n is 5; and m is 0.

[0108] In one embodiment, R1 is -SR2; R2 is -SR3; R3 is -(CH2) n -R4; R4 is -OC(O)-R5 or -OC(O)O-R5; R5 is -(CH2) m -; p is 2; n is 2; and m is 0.

[0109] In one embodiment, R1 is SR2; R2 is R3 is -(CH2) n-R4; R4 is -C(O)-Z-NH-phenylene-R5; R5 is -(CH2) m -; Z is -Val-Cit-; p is 2; n is 4; and m is 1.

[0110] In one embodiment, R1 is R3 is -(CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 3; and m is 1.

[0111] In one embodiment, R1 is -SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 4; and m is 1.

[0112] In one embodiment, R1 is -SR2; R2 is -SR3; R3 is (CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 3; and m is 1.

[0113] In some embodiments of the invention, the conjugate is not a compound selected from the group consisting of:

[0114]

[0115]

[0116] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, the conjugate is represented by Formula III

[0117] in

[0118] R is OH or

[0119] In one embodiment of the conjugate or a pharmaceutically acceptable salt thereof, the conjugate is selected from the group consisting of:

[0120]

[0121] Where R is OH or In some embodiments of the present invention, compounds 4-7, 30-31 and 44-45, wherein phlorizin 3 (or phlorizin 2) is linked to 5-FU, SN-38 or irinotecan via a redox-sensitive linker. In compound 4-5, a succinimidyl thioether (moderately sensitive linker) is used, while compounds 6-7 and 30-31 introduce a disulfide linker (highly sensitive linker) instead. Compounds 8-9 and 42-43, which introduce a stable triazole linker to form a conjugate, are also provided. Due to the overexpression of cathepsin B in the tumor microenvironment, compounds 37 and 38 composed of valine-citrulline dipeptide, p-aminobenzyl spacer and ether bond release SN-38 through cathepsin B activation and 1,6-removal reaction.

[0122] The compounds of formula I of the present invention are prepared according to general chemical synthesis procedures. The preparation of examples of the compounds of the present invention are described below.

[0123] Pharmaceutical composition and treatment method of the present invention

[0124] One embodiment of the second aspect of the invention is a method of treating a disease, recurrence or progression in a subject or increasing the likelihood of survival of a subject diagnosed with a disease over a relevant period of time, comprising administering to the subject a conjugate as described herein or a pharmaceutically acceptable salt thereof.

[0125] In some embodiments of the present invention, a series of drug conjugates comprising 5-FU, SN-38, irinotecan, and phlorizin or phlorretin conjugated to succinimidyl thioether, disulfide bond, triazole, or cathepsin B-sensitive linkers have been designed, synthesized, and biologically evaluated. Among the 5-FU series of prodrugs, most were less potent than 5-FU against CRC cell lines; however, compound 7 containing 5-FU, phlorizin, and a disulfide bond showed good stability in human plasma (t 1 / 2 =13 h), and showed instability in the presence of 5 mM GSH (t 1 / 2=10.3h), and its cytotoxicity to HCT-116 and HT-29 cell lines is similar to that of 5-FU. In the in situ mouse CRC model, compound 7 exhibits excellent antitumor efficacy and low toxicity, reducing tumor volume by 67% without reducing the weight of mice (5-FU can only reduce by 33%). The large intestine targeting effect caused by high tissue penetration and GLUT-1 targeting efficacy, compared with 5-FU, compound 7 exhibits considerable tumor inhibition efficacy. Among the prodrugs of the SN-38 series, although compound 31 exhibits similar cytotoxicity to SN-38 in cell analysis, tumor volume can be reduced by 70% (irinotecan only reduces by 27%) by peritoneal injection, wherein the SN38 content in compound 31 is only 65% ​​of the SN38 content in irinotecan, which exhibits better antitumor efficacy than irinotecan. When only 43% or 22% SN-38 was given (dose-dependent), compound 38 (40 or 20 mg / kg) showed better therapeutic efficacy than irinotecan (40 mg / kg) in a dose-dependent manner after intravenous injection. In addition, neither compound 31 nor 38 reduced the body weight of mice during treatment. Therefore, conjugates with cytotoxic small molecule drugs and GLUT targeting with glutathione-sensitive and / or cathepsin B linkers are proposed as a novel strategy for the treatment of CRC.

[0126] In one embodiment, the present invention provides a method of treating cancer in an individual, comprising administering to the individual an effective amount of a conjugate of the present invention. Such methods include administering to the individual a conjugate of the present invention in an amount sufficient to treat the condition. For example, cancer is but is not limited to a group consisting of the following: squamous cell carcinoma; lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma; peritoneal cancer; hepatocellular carcinoma; gastric cancer or gastric cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver tumors; breast cancer; colorectal cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; head and neck cancer; lymphoma; leukemia; myeloma; and myeloproliferative neoplasm.

[0127] The conjugate can be formulated into a pharmaceutical composition that can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Those skilled in the art are familiar with pharmaceutically acceptable carriers and diluents. For compositions formulated as liquid solutions, acceptable carriers and / or diluents include physiological saline and sterile water, and optionally include antioxidants, buffers, antibacterial agents, and other common additives. The composition can also be formulated as a pill, capsule, granule, or tablet, which, in addition to the compounds of the present invention, also contains a diluent, a dispersant, and a surfactant, a binder, and a lubricant.

[0128] Pharmaceutical compositions within the scope of the present invention include all compositions of the compounds of the present invention in combination with a pharmaceutically acceptable carrier. In one embodiment, the compound is present in the composition in an amount that can effectively achieve its intended therapeutic purpose. Although individual needs may vary, the determination of the optimal range of the effective amount of each compound is within the art of the art.

[0129] The conjugates of the invention may be useful in combination with one or more second therapeutic agents, particularly therapeutic agents useful for the treatment and / or prevention of previously presented conditions and diseases.

[0130] In one embodiment, the conjugate is present in the composition in an amount effective to achieve its intended therapeutic purpose. While individual needs may vary, determination of optimal ranges of effective amounts of each compound is within the skill of the art.

[0131] The conjugates of the invention may be useful in combination with one or more second therapeutic agents, particularly therapeutic agents useful for treating and / or preventing the conditions and diseases presented herein.

[0132] For oral administration, suitable forms of the pharmaceutical compositions of the present invention include powders, granules, pills, lozenges, buccal tablets, chewable tablets, gels and capsules, as well as liquids, syrups, suspensions, elixirs and emulsions. Such compositions may also include antioxidants, flavoring agents, preservatives, suspending agents, thickeners and emulsifiers, colorants, flavoring agents and other pharmaceutically acceptable additives. Formulations for oral administration may be formulated as immediate release or sustained release, wherein sustained release includes delayed, maintained, pulsed, controlled, targeted and programmed release.

[0133] For parenteral administration, the conjugates of the invention are administered directly into the bloodstream, into muscles, or into internal organs via intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, or other injections or infusions. Parenteral formulations may be prepared in aqueous injection solutions, which may contain buffers, antioxidants, antibacterial agents, salts, carbohydrates, and other additives commonly used in such solutions in addition to the compounds of the invention. Parenteral administration may be immediate release or sustained release (such as injection or implantation of a reservoir).

[0134] The conjugates of the invention may also be administered to the skin or mucous membranes epidermally, transdermally (intradermally) or transdermally. Typical formulations include gels, hydrogels, lotions, solutions, creams, ointments, dressings, foams, skin patches, powder tablets, implants and microemulsions. The compounds of the invention may also be administered via inhalation or intranasally, such as with dry powders, aerosol sprays or drops. Additional routes of administration of the compounds of the invention include intravaginal and rectal (by means of suppositories, pessaries or enemas) and ocular and otic.

[0135] Examples

[0136] Chemical methods

[0137] Synthetic pathway

[0138] Compounds 4-9, 30-31, 37-38 and 42-45 were constructed sequentially from the central linker (Scheme 1). Triethylene glycol and toluenesulfonyl chloride (TsCl) were added to tetrahydrofuran (THF) / H2O to generate a tosylated intermediate, followed by the addition of potassium thioacetate / acetone. After heating the mixture under reflux for 1 hour, compound 11 was obtained (yield 85%). Subsequently, compound 11 was hydrolyzed with potassium carbonate (0.05M) to obtain compound 12 (yield 80%), and its thiol was protected by 2-methyl-2-propanethiol to obtain compound 13 (yield 71%). After mesylation and bromination reaction, brominated compound 14 was obtained (yield 57%). Using potassium carbonate as a base, compound 14 was used to alkylate phlorizin 3 to obtain the main product 15 (yield 54%). The regioselectivity of this alkylation may be due to the electron-withdrawing effect of the carbonyl group at the para position of the benzyl group; and this position has less steric hindrance. Then, the tertiary butyl mercaptan group was deprotected using 4.0N NaOH and tris(carboxyethyl)phosphine (Tris(2-chloroethyl)phosphate, TCEP) to obtain compound 16 (yield 86%), which was refluxed with 1.0N HCl at 90°C for 3 hours and quenched with sodium bicarbonate (NaHCO3) aqueous solution to obtain compound 17 (yield 73%).

[0139] Azide-containing phlorizin derivative 20 and phlorizin derivative 21 were also prepared by similar methods as described above. At 0°C, methanesulfonyl chloride (MsCl) was slowly added to a stirred dichloromethane solution containing triethylene glycol and triethylamine. After 20 minutes, the reaction mixture was concentrated and the residue was dissolved in ethanol, and sodium azide (NaN3) was added thereto. After reflux for 24 hours, compound 18 with an azide linker was obtained. This was mesylated, and then lithium bromide was used to exchange the mesyl group with bromine to obtain compound 19, which was then reacted with phlorizin to obtain the desired intermediate 20. After hydrolysis, phlorizin derivative 21 was obtained.

[0140]

[0141] Scheme 1. Synthesis of phlorizin and phlorizin conjugates

[0142] In view of the poor cytotoxicity of N-1 alkyl-substituted 5-FU derivatives and the problem that they are not converted to 5-FU in vivo, an ester functional group was designed into the linker, and it is expected that this ester functional group will be cleaved by endogenous esterases in the tumor microenvironment. 5-FU was first reacted with formaldehyde, and then with 6-maleimidocaproic acid, 4-(pyridin-2-yldisulfide)-butyric acid or 4-pentynoic acid under the reaction conditions of coupling agents dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to obtain compounds 22-24 (Scheme 2). Compounds 16 and 17 were combined with maleimide-containing 5FU (22) to obtain compounds 4 and 5, respectively; and combined with carboxylic acid-containing compound 22 to obtain compounds 6 and 7, respectively. Compounds 20 and 21 were combined with alkyne-containing compound 24 under click conditions (Cu(I)-catalyzed azide-alkyne cycloaddition reaction) to obtain compounds 8 and 9, respectively.

[0143]

[0144] Scheme 2. Synthetic route of conjugates 4-9

[0145] Compounds 30 and 31 were synthesized using mercaptoethanol (compound 25) as a starting material via four steps (Scheme 3). First, compound 25 was coupled with 2,2'-disulfide bipyridyl (aldrithiol-2) under acetic acid conditions to obtain compound 26. Next, the hydroxyl group of compound 26 was reacted with 4-nitrophenyl chloroformate to obtain compound 27. Compound 27 was reacted with compound 28 (SN-38) to obtain compound 29, and tetrahydrofuran containing compound 16 or compound 17 was added under nitrogen conditions to obtain compound 30 or 31.

[0146] Scheme 4 is the synthetic route of compounds 37 and 38. Compound 32 (Fmoc-val-osu) reacts with L-citrulline in tetrahydrofuran / water / dimethoxymethane (DMM) using sodium bicarbonate as a base to obtain compound 33 (Fmoc-val-cit). Compound 33 is then reacted with p-aminobenzyl alcohol in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to obtain compound 34 (Fmoc-val-cit-pab-OH). To synthesize compound 36 containing an ether bond, glacial acetic acid containing 33% HBr is added to replace the benzyl alcohol on compound 34 with a bromine leaving group to obtain intermediate 35. Intermediate 35 was added to a mixture containing compound 28 and cesium carbonate in dimethylformamide (DMF). After stirring for 20 minutes, piperidine was added to the reaction mixture to remove the Fmoc protecting group to obtain val-cit-pab-O-SN-38 in a one-pot reaction. Val-cit-pab-O-SN-38 was then reacted with triethylamine (TEA) and 5-maleimidovaleric acid-NHS in DMF to obtain compound 36. Finally, compound 36 was coupled with compound 16 or 17 in methanol to obtain compound 37 or 38, respectively.

[0147] Scheme 5 discloses the synthesis steps of compounds 42-45. Irinotecan is coupled with 4-pentynoic acid or 6-maleimidocaproic acid under the conditions of DCC and DMAP to obtain compound 40 or 41. Compound 40 containing alkyne is then coupled with compound 20 or 21 under click conditions to obtain compounds 42 and 43. On the other hand, compound 41 containing maleimide is coupled with compound 16 or compound 17 to obtain compounds 44 and 45, respectively.

[0148]

[0149] Scheme 3. Synthetic route of conjugates 30 and 31

[0150]

[0151] Scheme 4. Synthetic route of conjugates 37 and 38

[0152]

[0153] Scheme 5. Synthetic route of conjugates 42-45

[0154] General chemicals.

[0155] The reagents and solvents used in the synthesis were all reagent grade and were used without further purification. HPLC analysis was performed using a HITACHID-2000Elite system with a BDS HYPERSIL C18 (5μm, 4.6×250mm) column. The mobile phase was a mixture of acetonitrile and double distilled water (dd H2O) or sodium dihydrogen phosphate (NaH2PO4) buffer, which was filtered using a 0.45μm membrane filter before use. The column mobile phase was eluted at a flow rate of 1.0mL / min. The precipitate was monitored by measuring the absorption at 254nm or 265nm at 25°C. The purity of all final products was measured by HPLC and must be greater than 95% to be used in vitro and in vivo. Thin layer chromatography (0.25mm, E.Merck silica gel 60F 254 ) was used to monitor the progress of the reaction; the chromatographic plates were visualized by UV (state wavelength) or by staining with cerium or ninhydrin and heating. The chromatography was performed on a Bruker-AV-400 (400 MHz) and a Bruker-AV-600 (600 MHz). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were collected (see Supplementary Information). Chemical shifts (δ) are in parts per million, with the residual solvent peak as the standard: 1 Hδ=2.50, 13 Cδ=39.52;CD3OD 1 Hδ=3.31, 13 Cδ=49.00; d6-acetone 1 Hδ=2.05, 13 Cδ=29.84,206.26;CDCl3 1 Hδ=7.26, 13Cδ=77.16. The unit of coupling constant (J) is Hertz (Hz). The splitting modes of peaks are classified into s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet) and m (multiplet). Mass spectra were measured using Burker bioTOFIII and expressed as charge-to-charge ratio (m / z).

[0156] 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)hexanoic acid (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl ester (4)

[0157] Compound 22 (21 mg, 0.06 mmol) was added to a solution of compound 16 (35 mg, 0.06 mmol) in anhydrous methanol (2 mL). The reaction mixture was stirred for 10 minutes and then concentrated in vacuo to obtain a crude product, which was then purified by column chromatography (silica gel; dichloromethane / methanol=12 / 1) to obtain compound 4 (53 mg, 0.06 mmol, yield greater than 99%) as a foam. 1 H NMR (600MHz, CD3OD) δ7.88(d,J=6Hz,1H,H-29),7.07(d,J=8.2Hz,2H,H-2,H-6),6.67(d,J=8.2Hz,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=7.2Hz,1H,H-1”),4.15(t,J=4 .8Hz,2H),4.03-4.00(m,1H),3.90(d,J=4.2Hz,1H),3.81(t,J=4.2Hz,2H) ,3.77-3.70(m,3H),3.67(t,J=4.2Hz,2H),3.63(t,J=5.4Hz,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,J1=15.6,3Hz,1H),2.33(t,J=7.2Hz,2H,H-21),1.6 0-1.58(m,2H,H-20),1.52-1.50(m,2H,H-18),1.28-1.26(m,2H,H-19)ppm;13 C NMR(150MHz,CD3OD)206.8,178.9,177.2,174.6,167.2,166.2,161.9,159.6(d,J CCF =26.1Hz),156.3,151.0,141.4(d,J CF =232.5Hz),133.7,130.5(d,J CCF =33.1Hz),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 .1,70.4,69.0,62.5,49.8,47.0,40.8,39.4,37.3,34.3,32.0,30.6,28.0,26.9,25.0ppm; HRMS(ESI TOF-MS)C 42 H 52 F3O 18 SNa + [M+Na] + : Calculated value: 960.2843, experimental value: 960.2847.

[0158] 6-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)hexanoic acid (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl ester (5)

[0159] Compound 22 (19 mg, 0.05 mmol) was added to a solution of compound 17 (22 mg, 0.05 mmol) in anhydrous methanol (1 mL). The reaction mixture was stirred for 10 minutes and then concentrated in vacuo to obtain a crude product, which was purified by column chromatography (silica gel; dichloromethane / methanol = 12 / 1) to obtain compound 5 (39 mg, 0.05 mmol, yield greater than 99%) in the form of foam. 1H NMR (400MHz, CD3OD) 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 (100MHz, CD3OD) 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 F3O 13 SNa + [M+Na] + : Calculated value 798.2315, experimental value: 798.2319.

[0160] 4-((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)disulfanyl)butanoic acid (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl ester (6)

[0161] Compound 23 (50 mg, 0.13 mmol) and phosphate buffer (pH = 8.0) were added to a tetrahydrofuran solution containing compound 16 (76 mg, 0.13 mmol). The mixture was stirred at room temperature for 2 hours. The solvent was removed by vacuum to obtain a crude product, which was purified by column chromatography (silica gel; dichloromethane / methanol = 15 / 1 to 10 / 1) to obtain compound 6 (107 mg, 0.13 mmol, yield greater than 99%) as an oil; 1 H NMR (400MHz, CD3OD) 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),4.17-4.0 5(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(100MHz,CD3OD)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 FN2O 16 S2 + [M+H] + : Calculated value 845.2267, experimental value: 845.2279.

[0162] 4-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)butanoic acid (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl ester (7)

[0163] Compound 23 (50 mg, 0.13 mmol) and phosphate buffer (pH 8.0) were added to a tetrahydrofuran solution containing compound 17 (55 mg, 0.13 mmol). The mixture was stirred at room temperature for 2 hours. The solvent was removed by vacuum to obtain a crude product, which was purified by column chromatography (silica gel; dichloromethane / methanol = 20 / 1) to obtain compound 7 (85 mg, 0.12 mmol, yield 92%) as a solid; 1 H NMR (600MHz, d6-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=4.3H z,2H),3.70(t,J=6.5Hz,2H),3.66-3.60(m,4H),3.35(t,J=8.0Hz,2H),2.89- 2.77(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, d6-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(ESITOF-MS)C 30 H 35 FN2O 11 S2 + [M+H] + : Calculated value 683.1739, experimental value: 683.1763.

[0164] 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)propanoic acid (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl ester (8)

[0165] To a solution of compound 20 (95 mg, 0.16 mmol) in ethanol / water (3 / 1, 2 mL) were added tris(benzyltriazolylmethyl)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 hours and then concentrated in vacuo. The mixture was dissolved in ethyl acetate and extracted with water. The organic layer was concentrated in vacuo and purified by column chromatography (silica gel; dichloromethane / methanol=10 / 1) to give compound 8 (117 mg, 0.14 mmol, 88% yield) as a foam. 1 H-NMR (600MHz, CD3OD) 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),4.15(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,4H),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; 13C NMR(150MHz,CD3OD)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 F5O 16 + [M+H] + : Calculated value 834.2840, experimental value: 834.2855.

[0166] 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)propanoic acid (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl ester (9)

[0167] To a solution of ethanol / water (volume ratio = 3:1, 2 mL) containing compound 21 (62.3 mg, 0.14 mmol) 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 hours and then concentrated in vacuo. The mixture was then dissolved in ethyl acetate and extracted with water. The organic layer was concentrated in vacuo and purified by column chromatography (silica gel: dichloromethane / methanol = 14 / 1) to give compound 9 (62.9 mg, 0.09 mmol, 64% yield) in a foamy state; 1H NMR(400MHz,d6-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= 7.6Hz,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(100MHz,CD3OD)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 F5O 11 + [M+H] + : Calculated value 672.2312, experimental value: 672.2318.

[0168] S-(2-(2-(2-hydroxyacetate)ethoxy)ethyl)thioacetate (11)

[0169] Tetrahydrofuran (300 mL) containing triethylene glycol (compound 10, 50.0 g, 333 mmol) was cooled to 0°C in an ice bath. Then, water (200 mL) containing sodium hydroxide (4.40 g, 110 mmol) was added thereto and stirred for 10 minutes. Anhydrous tetrahydrofuran (50 mL) containing TsCl (6.34 g, 33.3 mmol) was added dropwise (dropping time was about 30 minutes). After stirring for another 2 hours at room temperature, the reactant was poured into water and extracted with ether. The aqueous layer was re-extracted with dichloromethane. The combined organic layers were dried over magnesium sulfate and concentrated in vacuo to give a colorless oil. Then, potassium thioacetate (3.8 g, 33.3 mmol) was added to anhydrous acetone (200 mL) containing the intermediate. The reaction mixture was heated under reflux for 1 hour. The insoluble solid was filtered and the filtrate was concentrated in vacuo to give a crude product, which was purified by column chromatography (silica gel; hexane / ethyl acetate=1 / 1) to give Compound 11 (5.89 g, 28.3 mmol, yield 85%) as a yellow oil; 1 H NMR (400MHz, CDCl3) δ3.70-3.54 (m, 10H), 3.06 (t, J = 6.4Hz, 2H), 2.30 (s, 3H) ppm; 13 C NMR (100MHz, CDCl3) δ195.5, 72.4, 70.2, 69.6 (2C), 61.6, 30.4, 28.6ppm; HRMS (ESI TOF-MS) C8H 17 O4S+Na + [M+Na] + : Calculated value 231.0662, experimental value: 231.0672.

[0170] 2-(2-(2-Mercaptoethoxy)ethoxy)ethanol(12)

[0171] Potassium carbonate (345 mg, 2.50 mmol) was added to a stirred solution of anhydrous methanol (MeOH, 50 mL) containing compound 11 (5.7 g, 27.40 mmol). The reaction mixture was quenched with ammonium chloride solution and filtered to remove the precipitate. The filtrate was concentrated in vacuo to obtain a residue. The residue was dissolved in ethyl acetate and washed with water. The organic layers were combined and dried over magnesium sulfate to obtain a crude product. It was then concentrated in vacuo and purified by column chromatography (silica gel; hexane / ethyl acetate = 1 / 1) to obtain compound 12 (3.63 g, 21.86 mmol, 80% yield) as a yellow oil; 1 H NMR (400MHz, CDCl3) 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 (100MHz, CDCl3) δ72.5,70.3,70.2,69.5,61.6,38.3ppm; HRMS (ESI TOF-MS)C6H 15 O3S + [M+H] + : Calculated value 167.0736, experimental value: 167.0735.

[0172] 2-(2-(2-(tert-butyldihydrosulfanyl)ethoxy)ethoxy)ethanol(13)

[0173] To a stirred solution of ethanol (5 mL) containing compound 12 (1.43 g, 8.56 mmol) was added 2-methyl-2-propanethiol (9.7 mL, 7.76 g, 86.04 mmol). The reaction mixture was cooled to 0 ° C and a solution containing iodine (3.0 g) in ethanol (20 mL) was added dropwise until the color of the reactant changed from colorless to dark brown. After 12 hours, a saturated sodium bicarbonate solution was added until the pH was greater than 7. The reaction solution was concentrated in vacuo. The residue was then dissolved in ethyl acetate and extracted with a saturated sodium bicarbonate solution and brine. The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude product. It was then purified by column chromatography (silica gel; hexane / ethyl acetate (Hex / EtOAc) = 2 / 1) to give compound 13 (1.54 g, 6.06 mmol, yield 71%) as a yellow oil; 1 H NMR (400MHz, CDCl3) 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 (100MHz, CDCl3) 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 O3S2 + [M+H] + : Calculated value 255.1083, experimental value: 255.1089.

[0174] 1-(2-(2-(2-bromoethoxy)ethoxy)ethyl)-2-(tert-butyl)disulfane (14)

[0175] To a stirred solution of anhydrous dichloromethane (2 mL) containing compound 13 (70.0 mg, 0.28 mmol), triethylamine (TEA, 33.5 mg, 0.33 mmol) was added at 0°C, and the reaction mixture was stirred for 10 minutes. TsCl (32.0 mg, 0.28 mmol) was added to the reaction mixture (addition time 10 minutes). Then, the reaction mixture was extracted with water and then dried over magnesium sulfate to obtain dichloromethane containing the mesylated intermediate. Then, tetrahydrofuran (1 mL) containing lithium bromide (60.2 mg, 0.69 mmol) was added to the aforementioned solution. The residue was stirred at 55°C for 18 hours. The reaction mixture was concentrated, extracted with ethyl acetate and water, and then dried over magnesium sulfate, re-concentrated and purified by column chromatography (silica gel; Hex / EtOAc=8 / 1) to obtain compound 14 (50 mg, 0.16 mmol, two-step yield 57%,) as a colorless oil. 1 H NMR (400MHz, CDCl3) 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 (100MHz, CDCl3) δ71.1,70.3,70.2,69.8,47.7,39.7,29.7(3C)ppm; HRMS (ESI TOF-MS)C 10 H 21 BrO2S2Na + [M+Na] + : Calculated value 339.0059, experimental value: 339.0066.

[0176] 1-(4-(2-(2-(2-(tributyldihydrogenthio)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)

[0177] At room temperature, potassium carbonate (436 mg, 3.15 mmol) was added to anhydrous DMF (10 mL) containing phlorizin (3, 1.0 g, 2.29 mmol) and stirred for 10 minutes. After adding compound 14 (1.0 g, 3.16 mmol), the reaction mixture was heated at 55 ° C for 12 hours. The reaction mixture was concentrated in vacuo. It was then extracted three times with water / EtOAc. The organic layers were combined and concentrated in vacuo to give a crude product. It was then purified by column chromatography (silica gel; (dichloromethane / methanol (DCM / MeOH=10 / 1) to give compound 15 (837 mg, 1.25 mmol, yield 54%) in a foamy state; 1 H NMR (400MHz, CD3OD) 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=7.6Hz,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(150MHz,CD3OD)δ206.8,167.2,166.2,161.8,156.3,133.7,130.3(2C),116.0(2C),107.6,102.1,97.1 ,95.1,78.4,74.7,71.6,71.3,71.1,70.7,70.5,68.9,62.4,48.4,47.0,40.9,30.6,30.2(3C)ppm; HRMS(ESI) TOF-MS)C 31 H 44 NaO 12 S2 + [M+Na] + : Calculated value 695.2166, experimental value: 695.2174.

[0178] 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)

[0179] To a stirred solution of compound 15 (156 mg, 0.23 mmol) in anhydrous tetrahydrofuran (5 mL) was added TCEP (133 mg, 0.46 mmol) and 4.0 N sodium hydroxide solution (1 mL). The reaction mixture was stirred for 5 hours and then concentrated in vacuo. The residue was dissolved in ethyl acetate and the precipitate was filtered. The filtrate was concentrated in vacuo and purified by column (silica gel; dichloromethane / methanol=10 / 1) to give compound 16 (115 mg, 0.20 mmol, 86% yield) in a foamy state; 1 H NMR (400MHz, CD3OD) 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),3.91(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 .4Hz,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(100MHz,CD3OD)δ206.8,167.3,166.3,161.8,156.3,133.7,130.3(2C),116.0(2C),107.6,102.1 ,97.1,95.1,78.5,78.4,74.7,74.1,71.6,71.1(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.

[0180] 1-(2,6-Dihydroxy-4-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)phenyl)-3-(4-hydroxyphenyl)propan-1-one (17)

[0181] 1.0 N hydrogen chloride (1 mL) was added to a solution of ethanol (EtOH, 2 mL) containing compound 16 (210 mg, 0.36 mmol). The mixture was heated under reflux for 3 hours. The reaction mixture was cooled and the solvent was removed by vacuum. The residue was extracted with water and ethyl acetate and dried over magnesium sulfate. The organic layers were combined and concentrated in vacuo to give a crude product. The crude product was then purified by column chromatography (silica gel; Hex / EtOAc=1 / 1) to give compound 17 (111 mg, 0.26 mmol, 73% yield) in a foamy state; 1 H NMR (400MHz, CDCl3) 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 (100MHz, CDCl3) 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 oeLh + [M+Na] + : Calculated value 445.1291, experimental value: 445.1328.

[0182] 2-(2-(2-Azidoethoxy)ethoxy)ethanol(18)

[0183] To anhydrous DCM (250 mL) containing triethylene glycol (10, 50.0 g, 333 mmol), triethanolamine (TEA, 37.0 g, 366 mmol) was added at 0°C, and the reaction mixture was stirred for 10 minutes. Then, methanesulfonyl chloride (30.5 g, 266 mmol) was added to the reaction mixture (addition time 20 minutes). The reaction mixture was extracted with water, dried over magnesium sulfate and concentrated to give 2-(2-(2-hydroxyacetate)ethoxy)ethyl methanesulfonate. Sodium azide (21.6 g, 332 mmol) was added to ethanol (250 ml) containing the mesylated intermediate and heated under reflux overnight. Afterwards, water was added and the mixture was extracted with dichloromethane. The organic layer was dried over magnesium sulfate, concentrated, and purified by flash column chromatography (silica gel; ethyl acetate / hexane=3 / 1 to 1 / 5) to give Compound 18 (12.1 g, 69.1 mmol, two-step yield 26%) as a colorless oil. 1 H-NMR (200MHz, CDCl3) 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 (50MHz, CDCl3) δ72.4,70.5,70.2,69.8,61.5,50.5ppm; HRMS (ESI TOF-MS)C6H 14 N3O3 + [M+H] + : Calculated value 176.1030, experimental value: 176.1029.

[0184] 1-Azido-2-(2-(2-bromoethoxy)ethoxy)ethane (19)

[0185] To a stirred solution of compound 18 (8.0 g, 45.7 mmol) in anhydrous dichloromethane (100 mL) was added triethanolamine (TEA, 9.25 g, 91.4 mmol) at 0°C, and the reaction mixture was stirred for 10 minutes. Methanesulfonyl chloride (6.28 g, 54.8 mmol) was then added to the reaction mixture (addition time was 20 minutes). The reaction mixture was extracted with water and dried over magnesium sulfate to give dichloromethane containing the mesylated intermediate. Lithium bromide (11.9 g, 137 mmol) was dissolved in tetrahydrofuran (150 mL) and added to the mesylated intermediate and the reaction mixture. The residue was stirred at 45°C for 18 hours. The reaction mixture was concentrated, extracted with ethyl acetate and water, the organic layer was collected, dried over magnesium sulfate and then filtered. The filtrate was concentrated and purified by flash column chromatography (silica gel; hexane / ethyl acetate = 4 / 1) to give Compound 19 (6.9 g, 29.1 mmol, two-step yield 64%) as a colorless oil. 1 H NMR (200MHz, CDCl3) 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 (50MHz, CDCl3) δ70.9,70.3,70.2,69.7,50.3,30.1ppm.

[0186] 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)

[0187] To anhydrous dimethylformamide (15 mL) containing phlorizin (3, 2.31 g, 5.30 mmol), potassium carbonate (1.11 g, 8.03 mmol) was added at room temperature and stirred for 10 minutes. Compound 19 (1.66 g, 7.00 mmol) was added and the reaction mixture was heated at 55 ° C for 12 hours. The reaction mixture was concentrated in vacuo. The residue was extracted three times with water / ethyl acetate. The organic layers were combined and concentrated in vacuo to give a crude product, which was purified by column chromatography (silica gel; dichloromethane / methanol=10 / 1) to give Compound 20 (2.13 g, 3.59 mmol, 68% yield) in a foamy state; 1H NMR (600MHz, CD3OD) δ7.08 (d, J=8.4Hz, 2H, H-2, H-6), 6.70 (d, J=8.4, 2H, H-3, H-5), 6.3 4(d,J=2.4Hz,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(150MHz,CD3OD)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 N3O 12 Na + [M+Na] + : Calculated value 616.2113, experimental value: 616.2115.

[0188] 1-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-2,6-dihydroxyphenyl)-3-(4-hydroxyphenyl)propan-1-one (21)

[0189] 1.0 N hydrogen chloride (3 mL) was added to a solution of ethanol (5 mL) containing compound 20 (195 mg, 0.33 mmol). The mixture was refluxed at 90 ° C for 3 hours, then the reaction mixture was cooled and the solvent was removed by vacuum. The residue was extracted with hexane / ethyl acetate and dried over magnesium sulfate. The organic layers were combined and concentrated in vacuo to give a crude product. The crude product was purified by column chromatography (silica gel; Hex / EtOAc=1 / 1) to give compound 21 (110 mg, 0.26 mmol, 79% yield) in a foamy state; 1H NMR (400MHz, CDCl3) 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 (100MHz, CDCl3) 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 N3O7Na + [M+Na] + : Calculated value 454.1585, experimental value: 454.1583.

[0190] 1-(6-Maleimidocaproyloxymethyl)-5-fluorouracil (22)

[0191] To an aqueous solution (0.5 mL) containing 5-FU (100 mg, 0.77 mmol) was added an aqueous formaldehyde solution (37% by weight, 0.5 mL) at 55°C and stirred for another 3 hours after the solid completely disappeared. The solvent was then removed by vacuum to obtain a colorless and viscous oil in the reaction flask. In another flask, N,N'-dicyclohexylcarbodiimide (DCC, 190 mg, 0.92 mmol) and 4-dimethylaminopyridine (DMAP, 9.5 mg, 0.08 mmol) were added to anhydrous acetonitrile (2 mL) containing 6-maleimidocaproic acid (195 mg, 0.92 mmol) at 0°C and stirred for 10 minutes. Anhydrous acetonitrile (0.5 mL) containing the oil was then added to the reaction flask. The reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for 12 hours. The precipitate formed in the reaction was filtered, and the solvent was removed in vacuo. The residue was dissolved in ethyl acetate and extracted with 1.0 N hydrogen chloride, saturated sodium bicarbonate and water. The organic layer was dried over magnesium sulfate and concentrated in vacuo to obtain a crude product, which was purified by column chromatography (silica gel; hexane / ethyl acetate = 1 / 1) to obtain compound 22 (117 mg, 0.33 mmol, 43% yield) in the form of foam; 1 H NMR(600MHz,CD3OD)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(150MHz,CD3OD)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 FN3O6C 21 Na + [M+Na] + : Calculated value 376.0915, experimental value: 376.0915.

[0192] 1-(4-(Pyridin-2-yldisulfanyl)butyryloxymethyl)-5-fluorouracil (23)

[0193] At 60°C, a formaldehyde solution (37% by weight, 0.5 mL) containing 5-FU (100 mg, 0.77 mmol) was stirred until the solid disappeared completely, and stirred at 60°C for another 3 hours. The solvent was removed in vacuo to obtain a colorless oil. In another flask, 4-(2-pyridyldisulfanyl)butyric acid (206 mg, 0.90 mmol), DCC (190 mg, 0.92 mmol) and acetonitrile (5 mL) containing 4-dimethylaminopyridine (DMAP, 11 mg, 0.09 mmol) were added at 0°C, and the reaction was stirred for 10 minutes. The solution was then added to the colorless oil. The reaction mixture was stirred at 0°C for 30 minutes, and then stirred at room temperature for 12 hours. The precipitate formed in the reaction was filtered, and its solvent was removed in vacuo. The residue was dissolved in ethyl acetate and extracted with 1.0N hydrogen chloride, saturated sodium bicarbonate and water. The organic layer was dried over magnesium sulfate and concentrated in vacuo to give a crude product. The crude product was purified by column chromatography (silica gel; chloroform / methanol=25 / 1) to give Compound 23 (95.5 mg, 0.26 mmol, two-step yield 34%) as a solid; 1 H NMR (400MHz, CDCl3) 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(100MHz,CDCl3)δ173.1,159.8(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 F1N3O4S2 + [M+H] + : Calculated value 372.0485, experimental value: 372.0483.

[0194] 1-(Pentan-4-yloxymethyl)-5-fluorouracil (24)

[0195] A solution of 5-FU (100 mg, 0.77 mmol) in formaldehyde (37% by weight, 0.5 mL) was stirred at 60°C until the solid disappeared completely, and then stirred at 60°C for another 3 hours. The solvent was removed in vacuo to obtain a colorless oil. In another flask, pent-4-ynoic acid (105 mg, 1.07 mmol), DCC (190 mg, 0.92 mmol) and acetonitrile (5 mL) containing 4-dimethylaminopyridine (DMAP, 11 mg, 0.09 mmol) were added at 0°C, and the reaction was stirred for 10 minutes to obtain preactivated pent-4-ynoic acid. Then, acetonitrile containing preactivated pent-4-ynoic acid was added to the above colorless oil. The reaction mixture was stirred at 0°C for 30 minutes, and then stirred at room temperature for 12 hours. The precipitate formed in the reaction was filtered, and its solvent was removed in vacuo. The residue was dissolved in ethyl acetate and extracted with 1.0 N hydrogen chloride, saturated sodium bicarbonate and water. The organic layer was dried over magnesium sulfate and concentrated in vacuo to give a crude product. The crude product was purified by column chromatography (silica gel; hexane / ethyl acetate = 2 / 1) to give compound 24 (118 mg, 0.49 mmol, yield 64%) as an amorphous solid; 1 HNMR(400MHz, CDCl3)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(150MHz,CDCl3)δ171.9,156.4(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 H9FN2O4Na + [M+Na] + : Calculated value 263.0439, experimental value: 263.0443.

[0196] 2-(Pyridin-2-yldithio)ethanol(26)

[0197] To a solution of anhydrous MeOH (25 mL) containing 2,2'-dipyridyl disulfide (aldrithiol-2, 5 g, 0.02 mol) and acetic acid (0.33 mL) was added dropwise mercaptoethanol (1.2 mL, 0.02 mol). The reaction mixture was degassed with nitrogen for 20 minutes and stirred at room temperature for 26 hours. Then, the solvent was evaporated to dryness under reduced pressure, and the crude product was purified by column chromatography (silica gel; ethyl acetate / hexane=15 / 85 to 30 / 70) to give compound 26 (2.4 g, yield 64%) as a colorless oil. 1 HNMR(400MHz, CDCl3)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 (100MHz, CDCl3) δ42.7, 58.3, 121.6, 122.0, 136.9, 149.9, 159.2ppm. HRMS(ESI-TOF)C7H 10 NOS2[M+H] + Calculated value: 188.0198, experimental value: 188.0191.

[0198] Carbonic acid (4-nitrophenyl) 2-(pyridin-2-yldisulfide)ethyl ester (27)

[0199] To a solution of compound 26 (1.05 g, 5.60 mmol) in anhydrous dichloromethane (10 mL) was added trimethylamine (0.79 mL, 5.6 mmol) and anhydrous dichloromethane (5 mL) containing p-nitrophenyl chloroformate (1.70 g, 8.40 mmol) at 0°C. The reaction mixture was stirred at room temperature for another hour. The mixture was then filtered to remove the precipitated white solid, and part of the solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel; ethyl acetate / hexane=1 / 8 to 1 / 4) to give compound 27 (1.1 g, 56% yield) as a colorless oil. 1 H NMR(400MHz, CDCl3) δ3.16(t,J=6.4Hz,2H),4.56(t,J=6.4Hz,2H),7.11-7.14(m,1 H),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; 13C NMR (100MHz, CDCl3) 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 N2O5S2[M+H] + Calculated value: 353.0260, experimental value: 353.0268.

[0200] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinoline-9-yl (2-(pyridin-2-yldisulfide)ethyl)carbonate (29)

[0201] To a solution of SN-38 (compound 28, 288 mg, 0.73 mmol) in anhydrous DMG (27 mL) was added trimethylamine (103 μl, 0.73 mmol) and anhydrous dichloromethane (9 mL) containing compound 27 (310 mg, 0.88 mmol). The reaction mixture was stirred at room temperature under nitrogen for 24 hours. Then, the mixture was evaporated to dryness in vacuo and purified by column chromatography (silica gel; methanol / dichloromethane = 1 / 99 to 1 / 50) to give compound 29 (273 mg, 62% yield) as a yellow powder. 1 HNMR(600MHz, CDCl3)δ1.03(t,J=7.4Hz,3H),1.39(t,J=7.7Hz,3H),1.85-1.95(m,2 H),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; 13C NMR (150MHz, CDCl3) 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 N3O7S2[M+H] + Calculated value: 606.1363, experimental value: 606.1366.

[0202] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl (2-((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)

[0203] To a solution of compound 16 (9 mg, 15.4 μmol) in anhydrous tetrahydrofuran (800 μL), compound 29 (9 mg, 14.9 μmol) in anhydrous tetrahydrofuran (300 μL) was added. The reaction mixture was stirred at room temperature under nitrogen for 4.5 hours. The mixture was evaporated to dryness under reduced pressure, and the crude product was purified by column chromatography (silica gel; methanol / dichloromethane = 1 / 15) to give compound 30 (16 mg, yield 99%) as a white powder. 11H NMR (600 MHz, (CD3)2CO + D2O) δ 0.99 (t, J = 7.3 Hz, 3H), 1.39 (t, J = 7.7 Hz, 3H), 1.91 - 2.0 (m, 2H), 2.81 (t, J = 7.4 Hz, 2H), 2.99 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 6.4 Hz, 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, 12.0 Hz, 1H), 3.76 (t, J = 6.4 Hz, 2H), 3.78 (t, J = 4.5 Hz, 2H), 3.90 (dd, J = 2.5, 12.0 Hz, 1H), 4.11 (m, 2H), 4.58 (t, J = 6.3 Hz, 2H), 5.14 (d, J = 7.32 Hz, 1H), 5.27 (d, J = 1.0, 2H), 5.36 (d, J = 16.1 Hz, 1H), 5.53 (d, J = 16.4 Hz, 1H), 5.98 (d, J = 2.3 Hz, 1H), 6.27 (d, J = 2.3 Hz, 1H), 6.70 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 7.50 (s, 1H), 7.73 (dd, J = 2.5, 9.2 Hz, 1H), 8.09 (d, J = 2.5 Hz, 1H), 8.21 (d, J = 9.2 Hz, 1H) ppm; 13 13C NMR (150 MHz, (CD3)2CO + D2O) δ 8.2, 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, 115.8, 120.1, 125.6, 128.2, 129.3, 130.1, 132.6, 133.0, 146.6, 147.4, 148.0, 150.7, 151.2, 153.3, 154.1, 156.2, 158.2, 161.6, 165.8, 166.8, 173.5, 206.1 ppm. HRMS (ESI TOF-MS) C 52 H 59 N2O 19 S2 [M + H] + : Calculated value: 1079.3148, Experimental value: 1079.3157.

[0204] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinoline-9-yl (2-((2-(2-(2,4-dihydroxy-3-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)ethyl)carbonate (31)

[0205] 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 for 1 hour under nitrogen environment and room temperature. The reaction mixture was concentrated in vacuo to remove the solvent. The crude product was purified by column chromatography (silica gel; methanol / dichloromethane = 1 / 25) to give compound 31 (76.2 mg, yield 84%) as a yellow solid. 1 H NMR(600MHz, (CD3)2CO+D2O) 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,2 H),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),7.0 6(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; 13C NMR(150MHz,(CD3)2CO+D2O)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, 120.1,125.6,128.3,129.4,130.2,132.7,133.4,146.4,147.6,148.2,1 50.7,150.9,153.5,154.1,156.4,158.0,165.1,166.0,173.6,205.8ppm. HRMS(ESI TOF-MS)C 46 H 48 N2O 14 S2[M+H] + : Calculated value 917.2620, experimental value: 917.2621.

[0206] (S)-2((S)-2((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)-5-ureidopentanic acid (33)

[0207] To a solution of compound 32 (5 g, 11.46 mmol), dimethoxymethane (50 mL) and THF (25 mL) containing L-citrulline (2.1 g, 12 mmol) was added an aqueous solution (50 mL) containing sodium bicarbonate (1 g, 12 mmol). The reactants were stirred at room temperature and placed in a dark place. After 5 days, a 20% aqueous citric acid solution was added to the mixture, and the pH was adjusted to about 5. The solution was extracted three times with an equal volume of ethyl acetate containing 10% isopropanol. The organic layers were combined and washed three times with water. The organic layers were then collected, dried over magnesium sulfate, and evaporated to dryness in vacuo to obtain a crude product. Finally, the crude product was dispersed in ether and treated with ultrasonic vibration to obtain compound 33 (5 g, yield 88%) as a white solid. 1H NMR(400MHz,DMSO-d6)δ0.85-0.9(m,6H),1.39-1.41(m,2H),1.51-1.61(m,1H),1.69-1.7 0(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(100MHz,DMSO-d6)δ18.2,19.2,26.6,28.4,30.6,38.8,46.7,51.9,59.8,65.7 ,120.1,125.4,127.1,127.7,140.7,143.8,143.9,156.1,158.7,171.3,173.4ppm. HRMS(ESI TOF-MS)C 26 H 32 N4O6[M+H] + : Calculated value 497.2395, experimental value: 497.2404.

[0208] (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (34)

[0209] Compound 33 (6 g, 12.1 mmol), p-aminobenzyl alcohol (3 g, 24.2 mmol) and EEDQ (6 g, 24.2 mmol) were dissolved in anhydrous DCM (200 mL) and methanol (100 mL). The reaction mixture was stirred at room temperature and placed in a dark place. After 24 hours, the mixture was evaporated to dryness in vacuo. The residue was washed with ether / ethyl acetate (volume ratio = 1 / 1) to give compound 34 (5 g, yield 69%) as a light brown solid. 1H NMR(400MHz,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(100MHz,DMSO-d6)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 N5O6[M+H] + : Calculated value 602.2973, experimental value: 602.2987.

[0210] (S)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-2-((S)-2-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanamido)-3-methylbutanamido)-5-ureidopentanamide (36)

[0211] Compound 34 (1.71 g, 2.84 mmol) was added to 30 mL of HBr (33% by weight in glacial acetic acid) at 0°C and stirred for 10 minutes. The mixture was warmed to room temperature and stirred for another 2 hours. The reaction was then quenched with 500 mL of water and a precipitate of the bromide intermediate 35 was produced. 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. Crude 35 was added to a solution of SN-38 (694 mg, 1.77 mmol) and cesium carbonate (811.4 mg, 2.3 mmol) in anhydrous DMF (50 mL), and the reaction mixture was stirred for 20 minutes. Piperidine (5 mL) was then added to the mixture and stirred for another 20 minutes. The mixture was then evaporated to dryness in vacuo and purified by column chromatography (silica gel; methanol / dichloromethane = 50 / 1 to 5 / 1) to give the crude product val-cit-pab-O-SN-38 (316 mg) as a yellow solid. The crude product val-cit-pab-O-SN-38 (316 mg) was dissolved in anhydrous DMF (25 mL), and triethanolamine (58.6 μL, 0.42 mmol) and 5-maleimidopentanoic acid-NHS (161.8 mg, 0.55 mmol) were added. After stirring for 3.5 hours, the mixture was evaporated to dryness in vacuo and purified by column chromatography (silica gel; methanol / dichloromethane = 10 / 1) to give compound 36 (164.4 mg, 10% yield) as a yellow solid. 1H NMR (600MHz, DMSO-d6) δ0.81-0.89(m,9H),1.26(t,J=7.6Hz,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),1.9 4-1.99(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=7.8Hz,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=5.4Hz,1H ),6.50(s,1H),6.99(s,2H),7.25(s,1H),7.49(d,J=8.52Hz,2H),7.52(d ,J=9.24Hz,1H),7.55(s,1H),7.65(d,J=8.46Hz,2H),7.82(d,J=8.58Hz, 1H),8.05(dd,J=8.9,2.2Hz,1H),8.08(d,J=7.4Hz,1H),10.01(s,1H)ppm; 13 C NMR(150MHz,DMSO-d6)δ7.8,13.5,18.2,19.2,22.3,22.7,26.8,27.7,29.3,30.3, 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, 146.3,149.6,150.1,156.9,157.2,158.9,170.7,171.1,171.3,172.2,172.6ppm. HRMS(ESI TOF-MS)C 49 H 57 N8O 11 [M+H] + : Calculated value: 933.4141, experimental value: 933.4188.

[0212] (2S)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxol-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-2-((2S)-2-(5-(3-((2-(2-(2-(3-hydroxy-4 -(3-(4-Hydroxyphenyl)propionyl)-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)pentanamido)-3-methylbutanamido)-5-ureidopentanamide (37)

[0213] Compound 16 (108.6 mg, 0.26 mmol) was added to a solution of compound 36 (136 mg, 0.15 mmol) in methanol (60 mL). The reactants were stirred at room temperature. After 1.5 hours, the mixture was evaporated to dryness in vacuo and purified by column chromatography (silica gel; dichloromethane / methanol=7 / 1 to 5 / 1) to give compound 37 (113 mg, 50% yield) as a yellow solid. 1H NMR(600MHz,DMSO-d6)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; 13C NMR (150MHz, DMSO-d6) 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 N8O 23 S[M+H] + : Calculated value 1517.6069, experimental value: 1517.6083.

[0214] (2S)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-2-((2S)-2-(5-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyrrolidin-1-yl)pentanamido)-3-methylbutanamido)-5-ureidopentanamide (38)

[0215] Compound 17 (21.5 mg, 0.05 mmol) was added to a solution of compound 36 (36 mg, 0.04 mmol) in methanol (20 mL). The suspension was stirred at room temperature for 1 hour. The mixture was evaporated to dryness in cesium carbonate in vacuo, and ether / ethyl acetate (volume ratio = 1 / 1) was added to precipitate the crude product. The crude product was then purified by column chromatography (C18; acetonitrile / water = 1 / 1) to give compound 38 (17.5 mg, 32% yield) as a yellow solid. 1H NMR(600MHz,DMSO-d6)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),1.82-1.91(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),2.79-2.83(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,2H),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=8.5Hz,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; 13C NMR (150MHz, DMSO-d6) 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 ,38.6,40.1,45.6,49.5,53.1,57.6,65.3,67.3,68.6,69.4,69.5,69.6,69.8,72.2,72.4,93.6,96.0,103.7,10 4.6,115.1,118.3,119.1,122.7,127.8,128.4,128.7,129.1,131.3,131.4,131.5,138.8,143.9,144.5,146.3 ,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 N8O 18 S[M+H] + : Calculated value 1355.5541, experimental value: 1355.5562.

[0216] [1,4'-Bipiperidinyl]-1'-carboxylic acid (S)-4,11-diethyl-3,14-dioxo-4-(pent-4-ynyloxy)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl ester (40)

[0217] To a solution of irinotecan (400 mg, 0.64 mmol) in anhydrous acetonitrile (5 mL) was added pent-4-ynoic acid (94 mg, 0.96 mmol) and cooled to 0°C. Anhydrous acetonitrile (0.5 mL) containing DCC (198 mg, 0.96 mmol) and DMAP (2 mg, 0.02 mmol) was added and then stirred for another 4 hours. After filtering the precipitate, the solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate and extracted with 1.0 N hydrogen chloride solution, saturated NaHCO3 solution and water. The organic layer was dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (silica gel; DCM / MeOH=12 / 1) to give compound 40 (295 mg, 69% yield) as an amorphous solid. mp: 149°C. 1H NMR (600MHz, CDCl3) 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),3.0 6(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 (150MHz, CDCl3) δ170.7,167.4,157.3,153.0,151.6,150.3 147.1,146.6,145.9,145.3,131.5,127.5,127.1,125.8,119.7,114.6,96.4,82.2,76.3,70.2,67. 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 N4O7[M+H] + Calculated value: 667.3126, experimental value 667.3134.

[0218] [1,4'-Bipiperidinyl]-1'-carboxylic acid (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]indol[1,2-b]quinoline-9-ester (41)

[0219] To a solution of irinotecan (100 mg, 0.16 mmol) in anhydrous acetonitrile (6 mL) was added 6-maleimidocaproic acid (195 mg, 0.06 mmol) and cooled to 0°C. Anhydrous acetonitrile (0.5 mL) containing DCC (40 mg, 0.19 mmol) and DMAP (2.5 mg, 0.02 mmol) was added to the above solution and then the reaction was stirred for another 4 hours. The precipitate formed in the reaction was filtered and its solvent was removed by vacuum to obtain a crude product. The crude product was dissolved in ethyl acetate and extracted with 1.0 N hydrogen chloride solution, saturated NaHCO3 solution and water. The organic layer was collected, dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (silica gel; (DCM / MeOH)=12 / 1) to obtain compound 41 (52.5 mg, yield 42%) as an amorphous solid, mp: 149°C. 1 H NMR (600MHz, CDCl3) 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; 13C NMR (150MHz, CDCl3) 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 ,114.6,95.8,75.7,67.1,62.6,50.2,49.3,44.2,43.8,37.5,33.5,31.8,28.1,27.8,27.1,25.9,25.3,24.1,23.1,14.0,7.5ppm; HRMS(ESI)C 43 H 50 N5O9[M+H] + Calculated value: 780.3603, experimental value: 780.3636.

[0220] [1,4'-Bipiperidinyl]-1'-carboxylic acid (4S)-4,11-diethyl-4-((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)propionyl)oxy)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl)ester (42)

[0221] To a solution of compound 20 (27.2 mg, 0.04 mmol) in ethanol / water (volume ratio = 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 hours and then concentrated in vacuo. The mixture was dissolved in ethyl acetate and extracted with water. The organic layer was collected, concentrated in vacuo and purified by column chromatography (silica gel; dichloromethane / methanol = 10 / 1 to 3 / 1) to give 42 (38 mg, 67% yield) as a foam. 1H NMR (400MHz, CDCl3 / CD3OD=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= 15.4,18.5Hz,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, CDCl3 / CD3OD=10 / 1)δ205.2,171.7,167.8,166.1,164.4,160.1,157,4,153.2,151.0,150.3,146 .68,146.63,145.7,132.5,131.3,129.1,127.6,127.6,127.1,126.1,123.3,119.3,115.1,114.7,100.4,96.5 95.6,94.0,77.3,76.3,76.1,73.0,70.5,69.9,69.3,69.1,67.4,66.8,62.4,61.7,50.2,50.1,49.4,4 5.4,44.2,43.8,33.0,31.5,29.6,29.1,27.7,27.0,25.4,24.1,23.1,20.4,13.8,7.5ppm.HRMS(ESI)C 65 H 77 N7O 19 +H[M+H] + Calculated value: 1260.5347, experimental value: 1260.5372.

[0222] [1,4'-Bipiperidinyl]-1'-carboxylic acid (S)-4-((3-(1-(2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)phenoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propionyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-9-yl)ester (43)

[0223] To a solution containing compound 21 (50 mg, 0.12 mmol) in ethanol / water (volume ratio = 3 / 1, 1 mL) was added 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). The reaction mixture was stirred for 12 hours and then concentrated in vacuo. The mixture was redissolved in ethyl acetate and extracted with water. The organic layer was concentrated in vacuo and purified by column chromatography (silica gel: dichloromethane / methanol = 10 / 1 to 5 / 1) to give 43 (77 mg, yield 59%) as an amorphous solid, mp: 158 °C. 1 H NMR (600MHz, CDCl3) δ8.20(d,J=9.6Hz,1H),7.84(s,1H),7.54(d,J=9.6Hz,1 H),7.49(s,1H),7.27(s,1H),7.02(d,J=7.8Hz,2H),6.76(d,J=7.8Hz,2H),6 .01-5.93(m,2H),5.63(d,J=16.8Hz,1H),5.34(d,J=16.8Hz,1H),5.22(d,J= 3.6Hz,2H),4.44-4.41(m,3H,H-9),4.38-4.33(m,1H),4.04(m,2H),3.87-3.8 6(m,2H),3.64(t,J=5.4Hz,2H),3.54(m,4H),3.38-3.29(m,2H),3.16-3.11( m,2H),3.04(t,J=6.6Hz,1H),2.91-2.85(m,3H),2.81(t,J=6.4Hz,2H),2.72 (br,4H),2.51-2.50(m,1H),2.46-2.40(m,1H),2.14-2.01(m,5H),1.83-1.6 2(m,6H),1.51-1.39(m,2H),1.37(t,J=7.2Hz,3H),0.93(t,J=7.2Hz,3H)ppm; 13C NMR (150MHz, CDCl3) δ205.2,171.6,167.6,164.7,157.2,154.7,152.9,151.0,150.5,146. 6,146.2,146.1,145.4,133.4,130.6,129.3,127.7,127.3,126.5,123.2,119.8,115.2,11 4.8,105.3,96.5,94.5,75.9,71.1,70.3,69.2,68.7,66.9,62.6,50.7,50.2,49.3,45.9,4 4.1,43.7,32.7,31.5,29.9,27.7,26.9,25.0,23.9,23.2,20.3,13.9,7.5ppm; HRMS(ESI)C 59 H 68 N7O 14 [M+H] + Calculated value: 1098.4819, experimental value: 1098.482

[0224] [1,4'-Bipiperidinyl]-1'-carboxylic acid diethyl-4-((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)hexanoyl)oxy)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinoline-9-ester (44)

[0225] Compound 41 (45 mg, 0.05 mmol) was added to a solution of compound 16 (33.7 mg, 0.05 mmol) in anhydrous methanol (2 mL). The reaction mixture was stirred for 10 minutes and then concentrated in vacuo. The crude product was purified by column chromatography (silica gel; dichloromethane / methanol=10 / 1 to 3 / 1) to give 44 (47.2 mg, 69% yield) as an amorphous solid, mp: 146°C. 1H NMR(400MHz,CDCl3 / CD3OD=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,CDCl3 / CD3OD=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 N5O 21 S+H[M+H] +Calculated value: 1364.5531, experimental value: 1364.5597.

[0226] (4S)-4-((6-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propionyl)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]indol[1,2-b]quinolin-9-yl[1,4'-bipiperidinyl]-1'-carboxylate (45)

[0227] Compound 41 (47.8 mg, 0.06 mmol) was added to a solution of compound 17 (25.8 mg, 0.06 mmol) in anhydrous methanol (2 mL). The reaction mixture was stirred for 10 minutes and concentrated in vacuo. The crude product was purified by column chromatography (silica gel; dichloromethane / methanol = 12 / 1 to 5 / 1) to give 45 (41.7 mg, 58% yield) as an amorphous solid, mp: 135 °C. 1 H NMR (400MHz, CDCl3) δ8.14(d,J=9.2Hz,1H),7.79(s,1H),7.51(d,J=9.2Hz,1H),7.18(d,J=2.6Hz,1H),6.98(d,J=8.1Hz,2H),6.70(dd,J=4,4.3Hz,2H ),5.92(s,2H),5.63(d,J=17Hz,1H),5.35(d,J=17Hz,1H),5.20(s,2H),4.3 8(dd,J=22.2,13Hz,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=18.8,4.2Hz,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,6 H),1.31(t,J=7Hz,3H),1.27-1.18(m,2H),0.90(td,J=7.4,1.9Hz,3H)ppm; 13CNMR(100MHz, CDCl3)δ205.3,177.4,177.3,175.2,172.6,167.8,164.8,157.4,154.7,153.1,151.1 ,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. 5,38.8,36.3,33.3,31.6,31.4,29.8,27.0,25.8,24.3,24.1,23.4,23.2,14.0,7.6ppm; HRMS(ESI)C 64 H 76 N5O 16 S[M+H] + Calculated value: 1202.5002, experimental value: 1202.5013.

[0228] Cell culture and cytotoxicity assays

[0229] Unless otherwise specified, the cancer cell lines disclosed herein were obtained from ATCC. HT-29 and HCT-116 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% antibiotic-antimycotic (Thermo Fisher Scientific). Unless otherwise specified, the normal cell line-NHDF cells were obtained from PromoCell. NHDF cells were cultured in Dulbecco's Modified Eagle's Medium (Product No. 6429) containing 10% FBS and 1% antibiotic-antimycotic. These cells were cultured in a humidified incubator at 37°C with 5% carbon dioxide and 95% air.

[0230] The cytotoxicity of compounds HT-29, HCT-116, and NHDF cells was determined by sulforhodamine B (SRB) assay. Briefly, CRC cells were cultured at 5×10 3Cells were seeded at a density of 100 cells / well in a 96-well microtiter plate (100 μL / well) and divided into two groups with or without GSH addition (experimental group) or GSH addition (control group). After 24 hours of incubation, the medium was replaced with fresh medium (100 μL). For the control group without GSH addition, a series of concentrations of the compound (100 μL, prepared in DMSO or DMA and diluted with medium) were directly added to the cells; for the group with GSH addition, the cells were pre-treated with 30 mM GSH (20 μL / well, diluted with medium) for 1 hour, and then a series of concentrations of the compound (40 μL / well, prepared in DMSO or DMA and diluted with medium) and 10 mM GSH (40 μL / well) were added to make the final GSH concentration 5 mM, and incubated for 48 hours or 72 hours (the amount of DMSO or DMA did not exceed 0.5%). After incubation, 10% pre-cooled trichloroacetic acid solution was slowly added to each well and incubated at 4°C for 1 hour. The liquid in the 96-well microplate was removed, carefully washed with water, and dried at room temperature. Subsequently, the plate was stained with 100 microliters of 0.057% SRB for 30 minutes, rinsed with 1% acetic acid to remove unbound dye, and dried at room temperature. The protein-bound dye was dissolved with 100 microliters of 10mM Tris base (pH 10.5) and shaken for 10 minutes. The optical density (OD) was measured at 510nm using a Multi-Mode Microplate Reader (SpectraMax Paradigm, Beckman Coulter, US). Cell viability was calculated as follows. Viability = (experimental group OD-blank group OD) / (control group OD-blank group OD). IC was calculated using a nonlinear regression curve fitting model (GraphPad Prism 7, US) 50 The concentration that produced 50% cell growth inhibition was used as the control. Each experiment was repeated three times.

[0231] 2-NBDG absorbance analysis

[0232] COS-7 cells were seeded in 96-well culture plates and grown for 24 hours in low-glucose Dulbecco's modified Eagle's medium supplemented with 10% FBS and 2 mM L-glutamine. After washing with Krebs Ringer bicarbonate buffer (KRB) (10 mM HEPES, 129 mM sodium chloride, 4.7 mM potassium chloride, 2 mM calcium chloride and 1.2 mM magnesium sulfate, 1.2 mM potassium dihydrogen phosphate, 5 mM sodium bicarbonate, pH 7.4), the cells were incubated with the test compound in KRB containing 2-NBDG (200 μM) for 90 minutes at 37° C. and 5% CO 2 , and then washed twice with KRB and lysed with lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 40 mM potassium chloride and 20 mM Tris, pH 7.4). Finally, the lysate was transferred to a black 96-well plate and analyzed using a Multi-Mode Microplate Reader (SpectraMax Paradigm, Beckman Coulter, USA) at an excitation wavelength of 475 nm and an emission wavelength of 550 nm to detect the fluorescence intensity.

[0233] Western blot analysis of CRC and NHDF cell lines

[0234] HCT116, HT29 and NHDF cells were collected, washed with PBS, and lysed using 0.3 ml of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride aqueous solution, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100 and a 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 minutes. Proteins were separated by electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, blank areas were blocked with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBST) containing Tween 20, and the PVDF membranes were incubated with anti-GLUT-1 antibody (1:2000, Abcam) at 4°C overnight. The PVDF membranes were washed with PBST and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP), including goat anti-rabbit (1:3000, Abcam). Image detection was performed using the ChemiDoc XRS system and Image Lab software (Bio-Rad Laboratories, Hercules, CA) with antigens using Ultrascence western substrate (Bio-Helix, Taiwan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2500, Abcam, mouse) was used as an internal reference. Both HT-29 and HCT-116 showed significant GLUT-1 expression, with multiple bands reflecting different glycosylated GLUT-1. In the NHDF cell line, very low GLUT-1 expression was observed.

[0235] Evaluation of cathepsin B expression in healthy mice and mice with colorectal cancer induced by AOM-DSS (azoxymethane-dextran sulfate sodium)

[0236] An orthotopic CRC mouse model was established by ip injection of AOM (10 mg / kg body weight) into BALB / c mice (6 weeks, male) and supplementation of 2% DSS in their drinking water. Two months later, the mice were euthanized with CO2, and their large intestines with tumor areas were collected and fixed with 4% paraformaldehyde, followed by paraffin sections. In addition, healthy balb / c mice (6 weeks, male) were also used as controls. Paraffin sections of mouse large intestine samples were prepared according to the routine procedures of the Laboratory Animal Center of National Taiwan University. First, paraffin sections were dewaxed in 100% xylene and then rehydrated in 100%, 95%, 70%, 50% and 0% ethanol solutions (3 minutes in each ethanol solution). Heat-mediated antigen retrieval was performed using sodium citrate buffer (pH 6.0). Endogenous peroxidase activity in the tissue was blocked by incubation in 100% methanol containing 3% hydrogen peroxide for 10 minutes at room temperature. After rinsing with PBS and washing twice with PBST (PBS + 0.5% Tween 20), the samples were blocked for 15 to 30 minutes. The sections were then incubated with the primary antibody (cathepsin B (DIC7Y) XP Rabbit mAb, 1:1000, CST31718, Cell signaling) at 4°C overnight. Subsequently, the sections were incubated with the secondary antibody conjugated with HRP (anti-rabbit IgG (HRP), 1:2000, 7074S, Cell signaling) at room temperature for 1 hour. Protein expression was detected using DAB (Cell signaling) and counterstained with hematoxylin (Cell signaling). All sections were analyzed by upright optical microscopy (LEICA DM2500) and images were taken using MicroVisioneer Manual Whole Slide Imaging Software.

[0237] In vivo studies

[0238] Specific pathogen-free BALB / c mice (6 weeks old, male) were purchased from the Laboratory Animal Center, Taiwan, China. The animals were housed in clean plastic microisolator cages (5 mice / cage) and fed with standard laboratory pellets and water ad libitum. The animal room maintained constant temperature, humidity, and 12-h dark / light cycle. All animal procedures were in accordance with the guidelines of the Committee for the Laboratory Animal Care Committee in Taiwan University College of Medicine, China [IACUC20180076].

[0239] The mouse CRC model induced by AOM was applied to simulate human CRC. BALB / c mice (6 weeks old, male) were injected ip with AOM (10 mg / kg body weight) or PBS solvent once every two weeks. Seven days after each AOM injection, 2% DSS was given in drinking water for four days, followed by 3 days of normal water. This cycle was repeated three times. On day 60 after the first AOM treatment, the mice were randomly divided into eight groups (n=5). Once every 3 days for 3 weeks, 0.2 mL of phosphate buffered saline containing 5-FU (50 mg / kg body weight), compound 7 (50 mg / kg body weight) and 5-FU+compound 17 (5-FU 10 mg / kg and compound 17 30 mg / kg) was injected ip. Mice were injected ip with irinotecan (20 mg / kg), compound 31 (20 mg / kg), and irinotecan + compound 17 (20 mg / kg irinotecan and 14 mg / kg compound 17) every other day (qod) for two 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 iv every two days for two weeks. Body weight was measured every 3 days (5-FU series) or 2 days (SN-38 series). On day 86 (5-FU series) or day 79 (SN-38 series), the mice were injected iv. The mice were euthanized at 50 s. Tumor area and body weight were recorded after death. Tumors were counted under a dissecting microscope, and the area covered by the tumor was measured by image analysis software (AxioVision LE 4.8.2.0).

[0240] In the following pharmacokinetic experiments, BALB / c mice (six-week-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 the femoral vein with a syringe at 0, 1, 3, 5, 10, 15, 30, 45 minutes, 1, 2, 4 and 24 hours after drug administration. 50 μL of blood samples were collected into 100 μL of extraction solvent (EtOAc / MeOH=1:1) and vortexed for 30 seconds. The supernatant was separated by centrifugation at 10,000 rpm for 10 minutes and then frozen at -30°C for UPLC-MS / MS analysis. For biodistribution experiments, the whole dissected organ was weighed and placed in a round-bottom, two-ml microcentrifuge tube containing 1 mL of extraction solvent (EtOAc / MeOH=1:1) and 7 mm stainless steel beads, followed by homogenization for 30 minutes. 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 minutes and frozen at -30°C. 200 μL of solvent was removed from the crude organ suspension with a desiccator and then dissolved in 50 μL of extraction solvent. All samples were stored at -30°C and analyzed within 24 hours. All samples were filtered through a 0.22 μm polytetrafluoroethylene membrane (PTFE) into a 12×32 mm vial to use 5-FU- 15 N2 was used as an internal standard for UPLC-MS / MS analysis. Pharmacokinetic parameters were calculated using WinNonlin software (version 5.2, Pharsight, MO, USA).

[0241] UPLC-MS / MS analysis

[0242] This analysis was performed using an ACQUITY UPLC I-Class / Xevo TQ-XS IVD system (Waters, Milford, MA, USA) at the College of Public Health, Taiwan University, China. Reversed-phase BEH C18 (100 mm × 2.1 mm, 1.7 μm, Waters) and VanGuard BEH C18 (5 mm × 2.1 mm, 1.7 μm, Waters) pre-coated columns were used to separate the analytes. All data were acquired using MassLynx V4.2. The mobile phase consisted of 10 mM ammonium acetate aqueous solution (A) and acetonitrile (B) and was set as follows: 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; the rate was 0.5 mL / min for 6.00 min, and 4 μL was injected each time. The column oven was maintained at 60 °C. Multiple reaction monitoring (MRM) was used for quantification. The mass spectrometer parameters were as follows: capillary voltage, 3.0 kV / 3.0 kV, positive / negative ion mode, respectively; ion source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow (N2), 50 L / h; desolvation gas flow (N2), 700 L / h; multiplier, 650 V; and collision gas pressure (Ar), 3-4 × 10 -3 millibar.

[0243] Plasma stability and drug release analysis

[0244] To obtain human plasma stability, the synthesized conjugates were prepared in 95% human plasma. After incubation at 37°C for the specified time, the protein was denatured by adding ACN (5-FU series prodrug group) or DMA, and then acidified with 12.5mM sodium dihydrogen phosphate aqueous solution) (pH 3.2) (compounds 30 and 31). The mixture was centrifuged at 13,000rpm for 5 minutes. The supernatant was analyzed by high performance liquid chromatography (HPLC), and the sample was centrifuged to collect the clear supernatant and stored at -30°C until analysis; for release analysis, the synthesized conjugate (5-FU series prodrug) was dissolved in DMSO, and the analysis solution was prepared by diluting 50μL of the stock solution in 750μL PBS (pH 7.4, containing 5μM, 1mM or 5mM GSH) to a final concentration of 2.5mM. For compounds 30 and 31, they were dissolved in DMA and GSH (11.2 mM or 11.2 μM) solution was added, which was prepared in sodium acetate buffer (10 mM, pH 5.0) at a ratio of 50 / 40 (volume ratio), and the final GSH was 5 mM or 5 μM at pH 7.0, respectively. After incubation for the specified time, the samples were centrifuged to collect the clear supernatant and stored at -30°C until analysis. RP-HPLC injections were performed under the specified conditions, and the peak areas were integrated for further calculations.

[0245] Statistical analysis

[0246] All data were performed at least three times and presented as mean ± standard error mean (SEM). SAS version 9.2 (SAS Institute, Cary, NC) was used to analyze the differences in each group of data by t-test, one-way analysis of variance, and two-way analysis of variance. Statistically significant differences were divided into: ns, non-significant difference; ****P<0.0001; ***P<0.001; **P<0.01; or *P<0.05.

[0247] Plasma stability and release curves at different GSH concentrations

[0248] The in vitro human plasma stability and release profiles of compounds 5, 7, 9, 30 and 31 are depicted in Figures 2a) to 3d) The stability test included incubation of compounds 5, 7, 9, 30 and 31 in human plasma at 37°C for different time intervals. Samples of compound 7 were analyzed by RP-HPLC ( Figure 2a The half-lives of compounds 5, 7 and 9 were 0.8 hours, 13 hours and 3.6 hours, respectively ( Figure 2b )), and the half-lives of compounds 30 and 31 were 20 and 50 minutes, respectively ( Figure 3a)). The reason why the plasma stability of compounds 5 and 9 is poorer than that of compound 7 is unclear, but it can be attributed to the high pKa of the linker (the pKa of succinimide and triazole are both around 8.5-9.5), which increases the sensitivity of the ester portion of the linker to degradation. Therefore, a pH stability analysis was performed on compound 9, and it was found that compound 9 was stable at pH 4-5, but was easily completely degraded at pH ≥ 7. In order to predict the performance of those conjugates in vivo, we collected metabolites of compound 7 from plasma analysis and identified two major metabolites, M1 (which is produced by the hydrolysis of the ester bond of compound 7) and M2 (a disulfide exchange intermediate) ( Figure 2a )). Since the concentration of GSH in tumor cells is about 5-10 mM and in plasma is 1-10 μM, we then used GSH to evaluate the release of 5-FU or SN-38 from compounds 5, 7, 9, 30, and 31. The concentration of GSH used was set to 5 mM, simulating the tumor microenvironment. After 48 hours, compounds 5 and 9 released a small amount of 5-FU; however, it was found that the release of 5-FU from compound 7 depended on the concentration of GSH used. After 4 hours of exposure to 5 mM GSH, the degradation percentage of compound 7 was as high as 60%, but it was only about 10% in the presence of 1 mM GSH, indicating that the degradation rate of compound 7 in 1 mM GSH is much slower than that in 5 mM GSH ( Figure 2d )). The half-life of compound 7 at 5 mM and 1 mM GSH was 4.0 hours and 10.3 hours, respectively. In the presence of 5 μM GSH, only 30% degradation of compound 7 was observed after 48 hours ( Figure 2d The stability and release profiles of three phlorizin-containing derivatives 4, 6 and 8 were also evaluated, and the results were similar to those of their counterparts 5, 7 and 9, respectively. The disulfide bonds in compounds 6-7 have relatively short half-lives (<5 hours) in a highly reducing environment while maintaining a certain degree of stability in the circulation. The stability of prodrugs 30 and 31 in 0 μM, 5 μM and 5 mM GSH is shown in Figure 2. Figure 3b ). Prodrug 31 was relatively stable in 0 and 5 μM GSH (degraded by 10% after 48 hours), but under 5 mM GSH conditions, more than 90% was degraded to SN-38 after 48 hours, indicating that the prodrug can be effectively activated to SN-38 by high concentrations of GSH. Prodrugs 30 and 31 had the same trend, but their SN-38 release rate in 5 mM GSH was only about 60%. In order to clarify the reasons for the different release rates of SN-38 of prodrugs 30 and 31 and their metabolites M3-M7 in 5 mM GSH ( Figure 3c) and 3d)), we analyzed the fractions of these metabolites by mass. The results showed that M3 and M4 should be disulfide exchange products from fragment 16 and -S-(CH2)2-carbonate-SN-38 in prodrug 30, respectively; M5 is compound 16; M6 should be the same product as M4, but from prodrug 31; M7 may be the binding product of GSH and fragment 17 in prodrug 31. Based on these comprehensive results, we can know that the concentration of GSH and its reactivity to the prodrug fragments lead to the differential release rate of the parent drug, so prodrug 31 may have greater therapeutic potential than 30.

[0249] GLUT-1 inhibitory activity

[0250] Previous crystallographic studies have established that the benzene-1,3,5-triol ring of phlorizin is stabilized in GLUT-1 by forming three hydrogen bonds, but the extent to which substituents at the 3-position (such as compound 17) interfere with this is unknown. We examined the inhibitory activity of compounds 16-17 against GLUT-1 using COS-7 cells that overexpress GLUT-1 in a 2-NBDG uptake assay. Since phlorizin cannot inhibit GLUT-1, compound 16 (a phlorizin derivative) failed to inhibit GLUT-1 at both 50 μM and 100 μM ( Figure 4 In contrast, compound 17, despite having a substitution at the 3-OH position, still had an inhibitory activity similar to that of phloretin.

[0251] Cytotoxicity in human CRC cells

[0252] The cytotoxicity of compounds 4-9, 16-17, 30-31, phlorizin, phloridzin, 5-FU, SN-38, and irinotecan against CRC cell lines HCT-116 and HT-29 (both of which overexpress GLUT-1) was evaluated using the SRB assay (Tables 1 to 3). 5-FU exhibited an IC of approximately 15 μM. 50 (Item 1, Table 1). In order to simulate the tumor microenvironment, we first tried to add 10 mM GSH to the culture medium. However, we found that 10 mM GSH interfered with the growth of CRC cells. Therefore, 5 mM GSH was added to the culture medium of CRC cell lines. Compound 16 did not show any inhibitory activity. Compound 17 showed some inhibitory activity, IC 50 The cytotoxicity of compounds 4-5 was poor with or without the addition of GSH. The cytotoxicity of disulfide-bonded compounds 6-7 was sensitive to GSH. For example, the IC value of compound 6 on HCT-116 cells was 50The IC values ​​of compound 7 for HCT-116 and HT-29 in the presence of GSH were 23.8 μM and 54.9 μM respectively. The cytotoxicity of compound 6-7 to these cancer cells may be due to the fact that the sub-μM level of GSH in the cells gradually cleaves the disulfide bonds of compound 6-7 to release 5-FU. 50 The IC values ​​of 5-FU in the two cell lines were 10.5 μM and 3.8 μM, respectively. 50 The values ​​were close to those of 13.9 μM and 5.2 μM, respectively. The cytotoxicity of compounds 8-9 was poor. The cytotoxicity of compounds 16 and 17 was also analyzed in combination with 5-FU: the combination of 5-FU and compound 17 inhibited cell viability more effectively than when the other compounds were given 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, and it was found that neither 5-FU nor compound 7 had a significant inhibitory effect (IC 50 SN-38 and irinotecan showed IC values ​​of approximately 2-430 nM and 0.3-8.5 μM in CRC cells, respectively. 50 (Entries 3 to 4, Tables 2 and 3). The combination of SN-38 and compounds 16 or 17 showed a synergistic effect in the HT-29 cell line. However, it was surprising that compounds 30 and 31 had lower or similar cytotoxicity when compared to SN-38 and the combination of SN-38 and compounds 16 or 17. In general, HT-29 cells became more sensitive to the compounds, and HCT-116 cells showed similarity to that in the absence of GSH. Since the cytotoxicity of compounds 30 and 31 at high GSH concentrations was not significantly improved, and the IC 50 All are nanometer-scale, so there may be concerns about adverse reactions in vivo. Cytotoxicity was also evaluated for another class of compounds 37 and 38 with cathepsin B sensitivity and ether bonds, and the level in HCT-116 cell line was found to be about 3-7 μM, and the sensitivity to HT-29 cells was lower (entries 7 to 8, Table 3). Here, in order to study the proof of concept of combining glucose inhibitors with anticancer drugs, compounds 7 and 31 were selected for further evaluation of therapeutic efficacy in animal studies.

[0253] Table 1. Cytotoxicity of compounds 4-9, 16-17, 5-FU and 5-FU in combination with 16 or 17 in HCT-116 and HT-29 cell lines a .

[0254]

[0255] a Cell lines were incubated with compounds for 48 h. IC was determined by SRB assay 50 value.

[0256] b ND = not determined.

[0257] Table 2. Cytotoxicity of compounds 30-31, phlorizin, phlorizin, SN-38, irinotecan, and SN-38 in combination with 16 or 17 (prepared in DMA) in HCT-116 and HT-29 cell lines a .

[0258]

[0259] a IC 50 Values ​​were determined by SRB analysis.

[0260] b Cell lines were incubated with compounds for 72 hours.

[0261] c Cell lines were incubated with compounds for 48 hours.

[0262] Table 3. Cytotoxicity of compounds 16-17, 37-38, phlorizin, phlorizin, SN-38, irinotecan, and SN-38 in combination with 16 or 17 (prepared in DMA) in HCT-116 and HT-29 cell lines a,b .

[0263]

[0264] a IC 50 Values ​​were determined by SRB analysis.

[0265] b Cell lines were incubated with compounds for 48 hours.

[0266] Evaluation of cathepsin B expression in healthy mice compared with mice with AOM-DSS-induced colorectal cancer

[0267] Before evaluating the therapeutic efficacy of drugs in CRC mice, we first evaluated whether the AOM-DSS-induced balb / c mouse CRC model is a suitable model for diseases related to overexpression of cathepsin B. In addition, healthy balb / c mice (6 weeks, male) were also used for comparison. Immunohistochemistry results showed that the orthotopic CRC mouse model did have a higher expression of cathepsin B compared with the normal group ( Figure 5a)-d)), and thus can be reasonably used as a model for evaluating the therapeutic efficacy of cathepsin B-sensitive prodrugs.

[0268] Evaluation of the in vivo activity of synthetic compounds in an orthotopic CRC mouse model

[0269] Based on their release profiles and the results of cytotoxicity experiments, compounds 7 and 31 were selected for in vivo evaluation. An orthotopic CRC mouse model was established by intraperitoneal injection of AOM (10 mg / kg body weight) into BALB / c mice (6 weeks, male) and supplementation of 2% DSS in their drinking water ( Figure 6a) and 7a) ). Two months later, 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, the dose is based on compound 7) in 0.2 mL PBS were injected ip every three days for three weeks; 5% DMA + 2% Tween 80 + 93% PBS containing irinotecan (20 mg / kg), compound 31 (20 mg / kg), and irinotecan + compound 17 (20 mg / kg irinotecan and 14 mg / kg compound 17, the dose is based on compound 31) was injected once every two days via ip for two weeks; irinotecan (40 mg / kg), compound 37 (20 or 40 mg / kg), compound 38 (20 or 40 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) in 10% DMSO + 20% cremophor + 10% Na2CO3 (5% in dd H2O) + 60% D5W were injected via iv injection every two days for two weeks. Based on the calculation of tumor area, compound 7 showed better tumor inhibition than 50 mg / kg dose of 5-FU (0.07 mmol / kg 5-FU) when equivalent to 19% of 5-FU was given ( Figure 6b) and 6c) ); Compound 31 (20 mg / kg, 0.022 mmol / kg SN-38) showed better antitumor efficacy than irinotecan (20 mg / kg, 0.034 mmol / kg SN-38), equivalent to giving 65% of SN-38 ( Figure 7b) and 7c)); Compound 38 (40 mg / kg, 0.03 mmol / kg) also showed better antitumor efficacy than irinotecan (40 mg / kg, 0.068 mmol / kg), equivalent to giving 44% of SN-38, ( Figure 7d )). Since GLUT-1 / 3 / 4 are often 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, thereby causing significant tumor inhibition. In addition, SGLT-targeted compound 37 also improved the tumor area of ​​mice at a dose of 40 mg / kg (0.03 mmol / kg) compared with irinotecan (40 mg / kg, 0.068 mmol / kg). Mice treated with free 5-FU quickly lost about 10% of their body weight after injection ( Figure 6d )), which may reflect the systemic toxicity of 5-FU; there was no significant change in body weight in mice receiving compounds 7, 31, or 38 ( Figure 6d) and 7e) -f)). Combination of 5-FU (containing 19% of the 5-FU dose) or irinotecan (containing 65% of the SN-38 dose) and compound 17 also reduced tumor area in the mouse model, demonstrating a synergistic effect also observed in the cell analysis.

[0270] Evaluation of pharmacokinetics and biodistribution profiles

[0271] The plasma concentration and organ distribution of compound 7 were evaluated in BALB / c mice (n=3) injected with a single dose of the compound. To analyze the 5-FU release profile of compound 7, blood samples were collected at 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours and 8 hours after iv injection (50 mg / kg), and the concentration of compound 7 was determined by UPLC-MS / MS. The concentration of compound 7 decreased rapidly within 10 minutes ( Figure 8a ), which may be related to the expression of carboxylesterase and dihydropyrimidine dehydrogenase (DPD) and the high tissue permeability of 7 in mouse blood. 5-FU released from compound 7 was detected within 2 hours and lasted longer in the blood than compound 7 ( Figure 8aCompound 7 exhibited an unusual plasma pharmacokinetic (PK) profile, as its concentration was undetectable after 30 minutes (lower detection limit = 0.003 μg / mL), but was detectable again after 45 minutes and 4 hours, indicating that it was rapidly absorbed by mouse cells and tissues and reached equilibrium during elimination. To investigate the distribution-related antitumor efficacy of compound 7, blood samples were collected after intraperitoneal (ip) injection (50 mg / kg) of compound 7 and 5-FU in BALB / c mice (n = 3). Due to the high tissue permeability induced by lipophilicity, the concentration of compound 7 was below the detection limit after 5 minutes. Compared with the 5-FU group, the T of 5-FU of compound 7 was significantly lower than that of 5-FU group. max The 5-FU concentration was undetectable in both groups after a delay from 10 min to 45 min and after 2 h, indicating that the antitumor efficacy may be related to high tissue permeability ( Figure 8b )). Therefore, the distribution of compound 7 (50 mg / kg, 5-FU equivalent dose) and 5-FU (9.5 mg / kg) in the organs of BALB / c mice was evaluated 1 hour after intraperitoneal injection; it was found that compound 7 was distributed in the stomach, large intestine, heart and liver ( Figure 8c )). Since the amount of compound 7 in each organ was low, further analysis of 5-FU released from compound 7 showed that 5-FU was mainly detected in the large intestine, with a small amount detected in the kidney and stomach, while free 5-FU was distributed in the stomach, spleen and kidney ( Figure 8c )), similar to previous reports. These results indicate that our compound 7 combination is an appropriate strategy to achieve 5-FU delivery to the large intestine. It is assumed that this delivery is mediated by GLUT-1, which is mainly expressed in the large intestine. Since the phloretin moiety is bound to the glucose transporter, the entire conjugate may be absorbed by the cells. Since the intracellular GSH concentration of cancer cells may be 10 times higher than that of normal cells, 5-FU is released from the conjugate by cleaving the disulfide bond of compound 7 through GSH. The high tissue permeability of compound 7 leads to delayed release of 5-FU, prolonging the exposure time of 5-FU and enhancing its tumor eradication effect. Organ distribution analysis reveals the large intestine targeting effect of 5-FU, resulting in promising anti-tumor efficacy with low toxicity.

[0272] The foregoing description of exemplary embodiments of the present 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. A conjugate represented by formula I, ALB (I) or a pharmaceutically acceptable salt thereof, wherein A is an active drug moiety or a prodrug thereof; B is the targeting moiety; L is a linker represented by formula II in R1 is -SR2 or R2 is R3, -SR3 or R3 is -(CH2) n -R4; R4 is R5, -OC(O)-R5, -C(O)-R5, -C(O)NH-R5, -C(O)-, -NHC(O)-R5, -OC(O)O-R5 or -C(O)-Z-NH-phenylene-R5; R5 is -(CH2) m -; 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 dashed line is a covalent bond; The proviso is that the conjugate is not a compound selected from the following: and 2. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein A is an anticancer therapeutic moiety.

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

4. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein B is a binding agent specific for glucose transporters.

5. The conjugate according to claim 4 or a pharmaceutically acceptable salt thereof, wherein B is phloretin optionally substituted by glucose, mannose or 2-fluoro-glucose.

6. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)-R5; R5 is -(CH2) m -; p is 2; n is 5; and m is 0.

7. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -SR2; R2 is -SR3; R3 is -(CH2) n -R4; R4 is -OC(O)-R5 or -OC(O)O-R5; R5 is -(CH2) m -; p is 2; n is 2; and m is 0.

8. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)-Z-NH-phenylene-R5; R5 is -(CH2) m -; Z is -Val-Cit-; p is 2; n is 4; and m is 1.

9. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is R3 is -(CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 3; and m is 1.

10. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -SR2; R2 is R3 is -(CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 4; and m is 1.

11. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -SR2; R2 is -SR3; R3 is -(CH2) n -R4; R4 is -C(O)O-R5; R5 is -(CH2) m -; p is 2; n is 3; and m is 1.

12. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, which is represented by formula III, in R is OH or 13. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, which is selected from: Where R is OH or 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. Use of a conjugate according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease, recurrence or progression in an individual, or for increasing the likelihood of survival of an individual diagnosed with a disease over a relevant period of time.

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

18. The method of claim 17, wherein the cancer is selected from the group consisting of 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, stomach cancer or 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 or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, head and neck cancer, lymphoma, leukemia, myeloma, and myeloproliferative neoplasm.

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

20. The use according to claim 16, wherein the agent can be used in combination with a therapeutic agent.

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