Ligand-drug conjugates of esatecane analogs and medical uses thereof

Abstract

Provided herein are compounds having Formula (I) or (VII) or a pharmaceutically acceptable salt, tautomer, isotopic body, stereoisomer, or prodrug thereof; a ligand-drug conjugate and a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a residue of a compound provided herein; and methods of treating cancer thereof. # imgabs0 #

Description

1. Field of the Invention

[0001] The present disclosure relates to a ligand-drug conjugate of an exatecan analogue with a novel structure. Specifically, the present disclosure relates to an exatecan analogue, a ligand-drug conjugate of an exatecan analogue, a preparation method thereof, and medical uses of the conjugate. 2. Background of the Invention

[0003] Chemotherapy remains one of the most important anticancer therapies, alongside surgery, radiotherapy, and targeted therapy. Although numerous types of highly effective cytotoxic agents exist, the minimal differences between tumor cells and normal cells, coupled with toxic side effects, limit the widespread clinical application of these anticancer compounds. Due to their specificity for tumor cell surface antigens, monoclonal antibodies (mAbs) have become a first-line anticancer therapy. However, when used alone as anticancer agents, antibodies often exhibit unsatisfactory efficacy.

[0004] Antibody drug conjugates (ADCs) enable monoclonal antibodies or antibody fragments to be combined with biologically active cytotoxins via chemically stable linkers, fully utilizing the specificity of antibodies binding to surface antigens of normal cells or tumor cells and the high efficiency of cytotoxins while avoiding the low efficacy of antibodies and the toxic side effects of cytotoxins. This means that compared with conventional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103: 1807-1814).

[0005] In 2000, the first antibody-drug conjugate, Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), was approved by the U.S. Food and Drug Administration (FDA) for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; U.S. Patent Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001).

[0006] In August 2011, Adcetris (vetuximab, Seattle Genetics Inc.) was approved by the U.S. FDA fast track for the treatment of Hodgkin lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778-784; WO2004010957; WO2005001038; U.S. Patent Nos. 7,090,843A; 7,659,241; WO2008025020). It is a novel targeted ADC drug that enables the drug to act directly on the target CD30 of lymphoma cells, triggering endocytosis and thereby inducing tumor cell apoptosis.

[0007] Mylotarg and Adcetris are both targeted therapies for hematologic malignancies, which have a relatively simpler structure than solid tumors. In February 2013, Kadcyla (ado-trastuzumab bemtansine, T-DM1) was approved by the FDA for the treatment of patients with HER2-positive advanced or metastatic breast cancer who are resistant to trastuzumab (trade name: Herceptin) and paclitaxel (WO2005037992; U.S. Patent No. 8,088,387). Kadcyla is the first ADC drug approved by the FDA for the treatment of solid tumors.

[0008] There are many types of cytotoxic small molecules used in antibody-drug conjugates, one of which is camptothecin derivatives, which show antitumor effects by inhibiting topoisomerase I. Literature reporting the use of the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) includes WO2014057687, Clinical Cancer Research (2016) 22(20):5097-5108, and Cancer Sci (2016) 107:1039-1046. However, further development of ADC drugs with better efficacy is still needed. 3. Summary of the Invention

[0009] Provided herein is a compound having formula (I):

[0010]

[0011]

[0012] or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein

[0013] Y is -ABCDH;

[0014] A is a key, CR 1 R 2 or NR 1 ;

[0015] B is a bond, -C(=O)-, -C(=O)O-, or -OC(=O)-;

[0016] C is a bond or a divalent group selected from unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl;

[0017] D is a bond, NH or O;

[0018] R 1 and R 2 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 1 and R 2 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl;

[0019] R 3 and R 4 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 3 and R 4 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; and

[0020] When R 3 is a methyl group, and R 4 When it is F, Y is not -NH-C(=O)-CDH.

[0021] In one embodiment, the compound is a compound having formula (II):

[0022]

[0023] or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein

[0024] A is CR 1 R2 , NH or NR 1 ;

[0025] R 1 and R 2 Each of is independently H or C 1-4 alkyl;

[0026] R 3 and R 4 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 3 and R 4 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl;

[0027] R 5 and R 6 Each of is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; and

[0028] n is 1, 2, 3, 4, or 5.

[0029] In one embodiment, the ligand-drug conjugate comprises a structure of Formula (V):

[0030]

[0031] in

[0032] BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody;

[0033] L is a covalent linker as described herein; and

[0034] x is 1 to 10, and it can be an integer or a decimal.

[0035] In one embodiment, the antibody is pertrastuzumab, cofetiuzumab, trastuzumab, or mAb10.

[0036] Provided herein is a compound having formula (VII):

[0037]

[0038] or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein

[0039] R 7 and R 8 Each of R is independently hydrogen or substituted or unsubstituted alkyl; or 7 and R8 Together with the nitrogen atom to which they are attached, they form an unsubstituted or substituted heterocyclic group or an unsubstituted or substituted heteroaryl group.

[0040] In another aspect, provided herein is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a residue of a compound provided herein.

[0041] In another aspect, provided herein are methods of treating cancer comprising administering to a subject in need thereof a ligand-drug conjugate comprising a residue of a compound provided herein.

[0042] In another aspect, provided herein are kits comprising a ligand-drug conjugate comprising a residue of a compound provided herein. 4. Description of the Figures

[0043] Figure 1 The cell killing efficacy of the payloads against the MDA-MB-453 cell line is demonstrated.

[0044] Figure 2 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0045] Figure 3 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0046] Figure 4 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0047] Figure 5 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0048] Figure 6 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0049] Figure 7 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0050] Figure 8 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0051] Figure 9 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0052] Figure 10 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0053] Figure 11The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0054] Figure 12 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0055] Figure 13 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0056] Figure 14 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0057] Figure 15 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0058] Figure 16 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0059] Figure 17 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0060] Figure 18 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0061] Figure 19 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0062] Figure 20 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0063] Figure 21 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0064] Figure 22 The cell killing efficacy of the payloads against the A375 cell line is demonstrated.

[0065] Figure 23 The cell killing efficacy of the payloads against the Clau6 cell line is demonstrated.

[0066] Figure 24 Direct cell killing efficacy of the ADC on the A375 cell line was demonstrated.

[0067] Figure 25 Direct cell killing efficacy of the ADC on the Clau6 cell line was demonstrated.

[0068] Figure 26 Direct cell killing efficacy of the ADC on the A375 cell line was demonstrated.

[0069] Figure 27Direct cell killing efficacy of the ADC on the Clau6 cell line was demonstrated.

[0070] Figure 28 Direct cell killing efficacy of the ADC on the A375 cell line was demonstrated.

[0071] Figure 29 Direct cell killing efficacy of the ADC on the Clau6 cell line was demonstrated.

[0072] Figure 30 Direct cell killing efficacy of the ADC on the A375 cell line was demonstrated.

[0073] Figure 31 Direct cell killing efficacy of the ADC on the Clau6 cell line was demonstrated.

[0074] Figure 32 Direct cell killing efficacy of the ADC on the A375 cell line was demonstrated.

[0075] Figure 33 Direct cell killing efficacy of the ADC on the Clau6 cell line was demonstrated.

[0076] Figure 34 Direct cell killing efficacy of the ADC on the A375 cell line was demonstrated.

[0077] Figure 35 Direct cell killing efficacy of the ADC on the Clau6 cell line was demonstrated.

[0078] Figure 36 The ADC bystander cell killing activity in the MDA-MB-453 and Calu-6-nanoLuc systems is demonstrated.

[0079] Figure 37 ADC bystander cell killing efficacy in the A375 and Calu-6-nanoLuc systems is demonstrated.

[0080] Figure 38 ADC bystander cell killing activity in the Calu-6-nanoLuc system alone was demonstrated.

[0081] Figure 39 ADC bystander cell killing efficacy in the MDA-MB-453 and Calu-6-nanoLuc systems is demonstrated.

[0082] Figure 40 ADC bystander cell killing activity in the A375 and Calu-6-nanoLuc systems is demonstrated.

[0083] Figure 41ADC bystander cell killing efficacy in the Calu-6-nanoLuc system alone is demonstrated.

[0084] Figure 42 The ADC bystander cell killing activity in the MDA-MB-453 and Calu-6-nanoLuc systems is demonstrated.

[0085] Figure 43 ADC bystander cell killing efficacy in the A375 and Calu-6-nanoLuc systems is demonstrated.

[0086] Figure 44 ADC bystander cell killing activity in the Calu-6-nanoLuc system alone was demonstrated.

[0087] Figure 45 ADC bystander cell killing efficacy in the MDA-MB-453 and Calu-6-nanoLuc systems is demonstrated.

[0088] Figure 46 ADC bystander cell killing efficacy in the A375 and Calu-6-nanoLuc systems is demonstrated.

[0089] Figure 47 ADC bystander cell killing activity in the Calu-6-nanoLuc system alone was demonstrated.

[0090] Figure 48 ADC bystander cell killing efficacy in the MDA-MB-453 and Calu-6-nanoLuc systems is demonstrated.

[0091] Figure 49 ADC bystander cell killing efficacy in the A375 and Calu-6-nanoLuc systems is demonstrated.

[0092] Figure 50 ADC bystander cell killing activity in the Calu-6-nanoLuc system alone was demonstrated.

[0093] Figure 51 ADC bystander cell killing efficacy in the MDA-MB-453 and Calu-6-nanoLuc systems is demonstrated.

[0094] Figure 52 ADC bystander cell killing efficacy in the A375 and Calu-6-nanoLuc systems is demonstrated.

[0095] Figure 53 ADC bystander cell killing activity in the Calu-6-nanoLuc system alone was demonstrated.

[0096] Figure 54 Depicted is a comparison of the in vivo efficacy of different ADCs in A-375 melanoma xenografts grown subcutaneously in BALB / c nude mice.

[0097] Figure 55 Depicted is a comparison of the in vivo efficacy of different ADCs in A-375 melanoma xenografts grown subcutaneously in BALB / c nude mice.

[0098] Figure 56 Depicted is a comparison of the in vivo efficacy of different ADCs in A-375 melanoma xenografts grown subcutaneously in BALB / c nude mice.

[0099] Figure 57 Depicted is a comparison of the in vivo efficacy of different ADCs in A-375 melanoma xenografts grown subcutaneously in BALB / c nude mice.

[0100] Figure 58 It was demonstrated that ADC-3, ADC-6, ADC-7, ADC-9, ADC-10, ADC-11, ADC-21, ADC-P1, ADC-P2, or ADC-P3 exhibited comparable ADC or total Ab exposure and clearance as ADC-1.

[0101] Figure 59 Sequences related to mAb 10 are illustrated. 5. Specific implementation methods

[0102] In the present disclosure, it is to be understood that the present disclosure is not limited to the specific methods and / or experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0103] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0104] 5.1. Definitions

[0105] When referring to the compounds provided herein, unless otherwise indicated, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention pertains. When a trade name is used herein, unless the context otherwise indicates, the trade name includes product formulations, generic drugs, and active pharmaceutical ingredients of trade name products. If a term provided herein has multiple definitions, these definitions shall prevail unless otherwise indicated.

[0106] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting desired biological activity. The native form of an antibody is a tetramer and consists of two pairs of identical immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the light chain variable region and the heavy chain variable region (VL and VH) are primarily responsible for binding to the antigen. The light chain and heavy chain variable domains consist of framework regions interspersed with three hypervariable regions (also referred to as "complementary determining regions" or "CDRs"). The constant region can be recognized by the immune system and interacts with the immune system (see, for example, Janeway et al., 2001, Immunol. Biology, 5th edition, Garland Publishing, New York). Antibodies can belong to any type (e.g., IgG, IgE, IgM, IgD, and IgA), category (e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2), or its subclass. The antibodies can be derived from any suitable species. In some embodiments, the antibodies are of human or murine origin. The antibodies can be, for example, human, humanized, or chimeric.

[0107] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in trace amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies having different amino acid sequences in their variable domains, particularly their complementary determining regions (CDRs), which are typically specific for different epitopes. Monoclonal antibodies are highly specific (for a single antigenic site). The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by any particular method.

[0108] "Complete antibodies" are antibodies comprising an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, CH3, and CH4, depending on the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0109] "Antibody fragments" comprise a portion of an intact antibody, including its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, bifunctional antibodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments produced by Fab expression libraries, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0110] An "antigen" is the entity to which an antibody specifically binds.

[0111] As used herein, "specific binding" of an antibody to a target antigen means that the antibody exhibits preferential binding to the target as compared to other proteins, but such specificity does not require absolute binding specificity. "Specific binding" or "selective binding" of an antibody is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment to refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biological preparations (e.g., in a biological sample, blood, serum, plasma or tissue sample). Thus, under certain specified immunoassay conditions, the antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least two-fold above background levels and does not specifically bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, the antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least ten (10)-fold above background binding levels and does not specifically bind to other antigens present in the sample in significant amounts.

[0112] The terms "inhibits" or "inhibition of" mean to reduce by a measurable amount, or to prevent entirely.

[0113] The term "therapeutically effective amount" refers to an amount of a conjugate that is effective for treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of the conjugate can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. Insofar as the drug can inhibit growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining a response rate (RR).

[0114] The term "substantially" or "substantially" refers to a majority in a mixture or sample, i.e., >50% of the population, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the population.

[0115] The term "cytotoxic activity" refers to the cell-killing effect of a drug or exatecan derivative conjugate or an intracellular metabolite of an exatecan derivative conjugate. Cytotoxic activity can be expressed as IC 50 A value that is the concentration (molar or mass) per unit volume at which half the cells survive.

[0116] The term "cytostatic activity" refers to the anti-proliferative effect of the drug or the exatecan derivative conjugate or the intracellular metabolite of the exatecan derivative conjugate.

[0117] As used herein, the term "cytotoxic agent" refers to a substance that has cytotoxic activity and causes cell destruction. The term is intended to include chemotherapeutic agents and toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin), including synthetic analogs and derivatives thereof.

[0118] As used herein, the term "cytostatic" refers to a substance that inhibits cellular function, including cell growth or proliferation. Cytostatics include inhibitors, such as protein inhibitors, for example, enzyme inhibitors. Cytostatics have cytostatic activity.

[0119] The terms "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells.

[0120] As used herein, "autoimmune disease" refers to a disease or disorder that arises from and is directed against an individual's own tissues or proteins.

[0121] As used herein, "patient" refers to a subject to whom the exatecan derivative conjugate of the present invention is administered. Patients include, but are not limited to, humans, rats, mice, guinea pigs, non-human primates, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. Typically, the patient is a rat, mouse, dog, human, or non-human primate, more typically a human.

[0041] Unless the context indicates otherwise, the term "treat" or "treatment" refers to both therapeutic treatment and preventive treatment, wherein the purpose is to inhibit or slow (mitigate) an undesirable physiological change or condition, such as the development or spread of cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization of the disease state ( / . <？., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or condition as well as those susceptible to the disease or condition.

[0122] In the context of cancer, the term "treating" includes any or all of the following: killing tumor cells; inhibiting the growth of tumor cells, cancer cells or tumors; inhibiting the replication of tumor cells or cancer cells, reducing the overall tumor burden or reducing the number of cancer cells, and ameliorating one or more symptoms associated with the disease.

[0123] In the context of autoimmune disease, the term "treating" includes any or all of: inhibiting the replication of cells associated with the autoimmune disease state (including but not limited to cells that produce autoimmune antibodies), reducing the autoimmune antibody load, and ameliorating one or more symptoms of the autoimmune disease.

[0124] As used herein, the term "compound" refers to and encompasses the compound itself (named or represented by structure) and its salt forms, whether or not explicitly stated, unless the context clearly indicates that such salt forms are not included. The term "compound" also encompasses solvate forms of the compound, wherein a solvent is non-covalently associated with the compound or reversibly covalently associated with the compound when the carbonyl group of the compound is hydrated to form a geminal diol. Solvate forms include solvates of the compound itself and its salt forms and include hemisolvates, monosolvates, disolvates (including hydrates); and when a compound can be associated with two or more solvent molecules, the two or more solvent molecules can be the same or different.

[0125] In some cases, the compounds of the present invention will include an explicit reference to one or more of the above-mentioned forms, such as salts and solvates, without implying any solid state form of the compound; however, this reference is for emphasis only and should not be interpreted as excluding any other forms identified above. In addition, when salt and / or solvate forms of a compound or ligand drug conjugate composition are not explicitly mentioned, the omission should not be interpreted as excluding salt and / or solvate forms of the compound or conjugate unless the context clearly indicates the exclusion of such salt and / or solvate forms.

[0126] As used herein and in this specification and the appended claims, the indefinite articles "a," "an," and "the" include plural as well as singular referents, unless the context clearly dictates otherwise.

[0127] As used herein and unless otherwise indicated, the terms "about" and "approximately" when used in conjunction with the amount or weight percentage of a component of a composition means an amount or weight percentage recognized by one of ordinary skill in the art as providing a pharmacological effect equivalent to that obtained by the specified amount or weight percentage. In certain embodiments, the terms "about" and "approximately" when used in this context encompass amounts or weight percentages that are within 30%, within 20%, within 15%, within 10%, or within 5% of the specified amount or weight percentage.

[0128] As used herein, and unless otherwise indicated, the terms "about" and "approximately," when used in conjunction with a value or range of values ​​provided to characterize a particular solid form (e.g., a particular temperature or temperature range, such as describing melting, dehydration, desolvation, or glass transition temperature; a change in mass, such as that as a function of temperature or humidity; solvent or water content, expressed, for example, by mass or percentage; or peak position, such as in analysis by, for example, IR or Raman spectroscopy or XRPD); indicate that the value or range of values ​​may vary to an extent that is reasonable to one of ordinary skill in the art while still describing the solid form. Techniques used to characterize crystalline forms and amorphous solids include, but are not limited to, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), single crystal X-ray diffraction, vibrational spectroscopy (e.g., infrared (IR) and Raman spectroscopy), solid-state and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility studies, and dissolution studies. In certain embodiments, the terms "about" and "approximately" when used in this context indicate that the value or range of values ​​can vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the recited value or range of values. For example, in some embodiments, the value of an XRPD peak position may vary by as much as ±0.2° 2Θ (or ±0.2 degrees 2Θ) while still describing a specific XRPD peak.

[0129] "Alkyl" is a saturated, partially saturated, or unsaturated, straight or branched chain, non-cyclic hydrocarbon having 1 to 10 carbon atoms, typically 1 to 8 carbons, or in some embodiments, 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and n-hexyl; while saturated branched chain alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, allyl, CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), C(CH2CH3)=CH2, C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and CH2C≡C(CH2CH3), etc. The alkyl group may be substituted or unsubstituted. In certain embodiments, when alkyl groups described herein are referred to as "substituted," they may be substituted with any one or more substituents, such as those found in the exemplary compounds and embodiments disclosed herein; as well as halogen (chlorine, iodine, bromine, or fluorine); hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfonyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2; or O(alkyl)aminocarbonyl.

[0130] " alkenyl " is a straight or branched non-cyclic hydrocarbon with 2 to 10 carbon atoms, typically 2 to 8 carbon atoms and including at least one carbon-carbon double bond. Representative straight and branched (C2C8) alkenyl includes vinyl , allyl , 1-butenyl , 2-butenyl , isobutylenyl , 1-pentenyl , 2 pentenyls , 3-methyl-1-butenyl , 2-methyl-2-butenyl , 2,3-dimethyl-2-butenyl , 1-hexenyl , 2-hexenyl , 3-hexenyl , 1-heptenyl , 2-heptenyl , 3-heptenyl , 1-octenyl , 2-octenyl , 3 octenyl etc. The double bond of alkenyl can be non-conjugated, or conjugated with another unsaturated group. Alkenyl can be unsubstituted or substituted.

[0131] " Cycloalkyl " is the saturated or partially saturated cyclic alkyl of 3 to 10 carbon atoms, and it has single ring or multiple condensed rings or bridged rings that can be optionally substituted by 1 to 3 alkyl groups.In some embodiments, cycloalkyl has 3 to 8 ring members, and in other embodiments, the number of annular carbon atoms is in the range of 3 to 5, 3 to 6 or 3 to 7. Such cycloalkyl includes, for example, monocyclic structure, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2 methylcyclopentyl, 2-methylcyclooctyl etc., or polycyclic or bridged ring structure, such as adamantyl etc. The example of unsaturated cycloalkyl includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl etc. Cycloalkyl can be substituted or unsubstituted. The cycloalkyl of such substitution includes, for example, cyclohexanone etc.

[0132] "Aryl" is an aromatic carbocyclic group of 6 to 14 carbon atoms having a monocyclic ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl). In some embodiments, the aryl group contains 6-14 carbons, and in other embodiments, the ring portion of the aryl group contains 6 to 12 or even 6 to 10 carbon atoms. Specific aryl groups include phenyl, biphenyl, naphthyl, etc. Aryl groups can be substituted or unsubstituted. The phrase "aryl" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0133] "Heteroaryl" is an aryl ring system having one to four heteroatoms as ring atoms in a heteroaromatic ring system, wherein the remaining atoms are carbon atoms. In some embodiments, the heteroaryl group contains 5 to 6 ring atoms, and in other embodiments, the ring portion of the group contains 6 to 9 or even 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, groups such as pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, benzothienyl, furyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indolyl, azaindolyl (e.g., pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), indazolyl, benzimidazolyl (e.g., , 1H-benzo[d]imidazolyl), imidazopyridinyl (e.g., azabenzimidazolyl, 3Himidazo[4,5-b]pyridinyl, or 1H-imidazo[4,5-b]pyridinyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridinyl, thionaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl.

[0134] "Heterocyclyl" is an aromatic (also referred to as heteroaryl) or non-aromatic cycloalkyl group in which one to four ring carbon atoms are independently replaced by heteroatoms from the group consisting of O, S, and N. In some embodiments, the heterocyclyl group includes 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. The heterocyclyl group can also be bonded to other groups at any ring atom (i.e., at any carbon atom or heteroatom of the heterocycle). The heterocyclyl group can be substituted or unsubstituted. The heterocyclyl group encompasses unsaturated, partially saturated, and saturated ring systems, such as imidazolyl, imidazolinyl, and imidazolidinyl. The phrase heterocyclyl includes fused ring species, including those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing heteroatoms, such as, but not limited to, quinuclidine. Representative examples of heterocyclyl include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thienyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, linyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathianyl, dioxy, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiazinyl, dihydrodisulfinyl, homopiperazinyl, quinuclidinyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridinyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiazinyl, benzoxathiazinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[l,3]dioxolyl, pyrazolopyridinyl, imidazopyridinyl (azabenzimidazolyl; for example, 1H-imidazo[4,5-b]pyridinyl or 1H-imidazo[4,5-b]pyridin-2(3H)-onyl), triazolopyridinyl, isoxazolopyridinyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolyl, isoquinolyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thionaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridinyl, tetrahydropyrazolopyridinyl, tetrahydroimidazopyridinyl, tetrahydrotriazolopyridinyl and tetrahydroquinolinyl.Representative substituted heterocyclyl groups can be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed below.

[0135] "Cycloalkylalkyl" is a radical of the formula: -alkyl-cycloalkyl, wherein alkyl and cycloalkyl are as defined above. Substituted cycloalkylalkyl groups may be substituted on the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the radical. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups may be monosubstituted or substituted more than once.

[0136] "Aralkyl" is a radical of the formula: -alkyl-aryl, wherein the alkyl and aryl groups are as defined above. Substituted aralkyl groups may be substituted on the alkyl, aryl, or both the alkyl and aryl portions of the radical. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl, as well as fused (cycloalkylaryl) alkyl groups, such as 4-ethyl-indanyl.

[0137] "Heterocyclylalkyl" is a radical of the formula: -alkyl-heterocyclyl, wherein alkyl and heterocyclyl are as defined above. The substituted heterocyclylalkyl can be substituted at the alkyl, heterocyclyl or alkyl and heterocyclyl parts of the radical. Representative heterocyclylalkyl includes but is not limited to 4-ethyl-morpholinyl, 4-propylmorpholinyl, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, (tetrahydro-2H-pyrans-4-yl)methyl, (tetrahydro-2H-pyrans-4-yl)ethyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylethyl and indol-2-ylpropyl.

[0138] "Halogen" is chlorine, iodine, bromine or fluorine.

[0139] "Hydroxyalkyl" is an alkyl group as described above substituted with one or more hydroxy groups.

[0140] "Alkoxy" is O(alkyl) where alkyl is as defined above.

[0141] "Alkoxyalkyl" is (alkyl)O(alkyl) where alkyl is as defined above.

[0142] An "amine" group is a group of the formula: NH2.

[0143] A "hydroxylamine" group is a group of the formula: N(R # )OH or NHOH, where R # is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0144] An "alkoxyamine" group is a group of the formula: -N(R # )O-alkyl or -NHO-alkyl, wherein R # As defined above.

[0145] An "aralkoxyamine" group is a group of the formula: N(R # )O-aryl or NHO aryl, wherein R # As defined above.

[0146] An "alkylamine" group is a group of the formula: NHalkyl or N(alkyl)2, wherein each alkyl group is independently as defined above.

[0147] An "aminocarbonyl" group is a group of the formula: -C(=O)N(R # )2、-C(=O)NH(R # ) or C(=O)NH2, wherein each R # As defined above.

[0148] An "acylamino" group is a group of the formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ), wherein each alkyl group and R # are independently as defined above.

[0149] An "O(alkyl)aminocarbonyl" group is a group of the formula: -O(alkyl)C(=O)N(R # )2, -O(alkyl)C(=O)NH(R # ) or -O(alkyl)C(=O)NH2, wherein each R # are independently as defined above.

[0150] An "N-oxide" group is a group of the formula: -N + -O - .

[0151] A "carboxy" group is a group of the formula: C(=O)OH.

[0152] A "ketone" group is a group of the formula: C(=O)(R # ), where R # As defined above.

[0153] An "aldehyde" group is a group of the formula: -CH(=0).

[0154] An "ester" group is a group of the formula: C(=O)O(R # ) or OC(=O)(R # ), where R # As defined above.

[0155] A "urea" group is a group of the formula: -N(alkyl)C(=O)N(R # )2, -N(alkyl)C(=O)NH(R # )、-N(alkyl)C(=O)NH2、-NHC(=O)N(R # )2, -NHC(=O)NH(R # ) or NHC(=O)NH2 # , where each alkyl group and R # are independently as defined above.

[0156] An "imine" group is a group of the formula: -N=C(R # )2 or -C(R # )=N(R # ), where each R # are independently as defined above.

[0157] An "imide" group is a group of the formula: -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2, where each R # are independently as defined above.

[0158] A "carbamate" group is a group of the formula: -OC(=O)N(R # )2、-OC(=O)NH(R # )、-N(R # )C(=O)O(R # ) or -NHC(=O)O(R # ), where each R # are independently as defined above.

[0159] An "amidine" group is a group of the formula: -C(=N(R # ))N(R # )2、-C(=N(R # ))NH(R # )、-C(=N(R # ))NH2、-C(=NH)N(R # )2、-C(=NH)NH(R # )、-C(=NH)NH2、-N=C(R # )N(R # )2、-N=C(R # )NH(R # ),-N=C(R # )NH2、-N(R # )C(R # )=N(R# )、-NHC(R # )=N(R # )、-N(R # )C(R # )=NH or -NHC(R # )=NH, where each R # are independently as defined above.

[0160] A "guanidine" group is a group of the formula: -N(R # )C(=N(R # ))N(R # )2, -NHC(=N(R # ))N(R # )2、-N(R # )C(=NH)N(R # )2、-N(R # )C(=N(R # ))NH(R # )、-N(R # )C(=N(R # ))NH2、-NHC(=NH)N(R # )2, -NHC(=N(R # ))NH(R # )、-NHC(=N(R # ))NH2、-NHC(=NH)NH(R # ), -NHC(=NH)NH2, -N=C(N(R # )2)2、-N=C(NH(R # ))2 or -N=C(NH2)2, wherein each R # are independently as defined above.

[0161] An "enamine" group is a group of the formula: -N(R # )C(R # )=C(R # )2、-NHC(R # )=C(R # )2、-C(N(R # )2)=C(R # )2、-C(NH(R # ))=C(R # )2、-C(NH2)=C(R # )2、-C(R # )=C(R # )(N(R # )2) C(R # )=C(R # )(NH(R# )) or -C(R # )=C(R # )(NH2), where each R # are independently as defined above.

[0162] An "oxime" group is a group of the formula: -C(=NO(R # ))(R # )、-C(=NOH)(R # )、-CH(=NO(R # )) or -CH(=NOH), wherein each R # are independently as defined above.

[0163] A "hydrazide" group is a group of the formula: -C(=O)N(R # )N(R # )2、-C(=O)NHN(R # )2、-C(=O)N(R # )NH(R # )、-C(=O)N(R # )NH2、-C(=O)NHNH(R # )2 or -C(=O)NHNH2, wherein each R # are independently as defined above.

[0164] A "hydrazine" group is a group of the formula: -N(R # )N(R # )2、-NHN(R # )2、-N(R # )NH(R # )、-N(R # )NH2、-NHNH(R # )2 or -NHNH2, wherein each R # are independently as defined above.

[0165] A "hydrazone" group is a group of the formula: -C(=NN(R # )2)(R # )2、-C(=NNH(R # ))(R # )2、-C(=N-NH2)(R # )2、-N(R # )(N=C(R # )2) or -NH(N=C(R # )2), where each R # are independently as defined above.

[0166] An "azide" group is a group of the formula: -N3.

[0167] An "isocyanate" group is a group of the formula: N=C=O.

[0168] An "isothiocyanate" group is a group of the formula: N=C=S.

[0169] A "cyanate" group is a group of the formula: OCN.

[0170] A "thiocyanate" group is a group of the formula: SCN.

[0171] A "thioether" group is a group of the formula: -S(R # ), where R # As defined above.

[0172] A "thiocarbonyl" group is a group of the formula: -C(=S)(R # ), where R # As defined above.

[0173] A "sulfinyl" group is a group of the formula: -S(=O)(R # ), where R # As defined above.

[0174] A "sulfone" group is a group of the formula: -S(=O)2(R # ), where R # As defined above.

[0175] "Sulfonylamino" is a radical of the formula: -NHSO2(R # ) or -N(alkyl)SO2(R # ), wherein each alkyl group and R # As defined above.

[0176] A "sulfonamide" group is a group of the formula: -S(=O)2N(R # )2 or -S(=O)2NH(R # ) or -S(=O)2NH2, wherein each R # are independently as defined above.

[0177] A "phosphonate" group is a group of the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ) or -OP(=O)(OH)(R # ), where each R # are independently as defined above.

[0178] A "phosphine" group is a group of the formula: -P(R #)2, where each R # are independently as defined above.

[0179] When groups described herein (other than alkyl) are referred to as "substituted," they may be substituted with any one or more suitable substituents. Illustrative examples of substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chlorine, iodine, bromine, or fluorine); alkyl; hydroxy; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (═O); B(OH)2; O(alkyl)aminocarbonyl; cycloalkyl, which may and heterocyclyl. In some embodiments, the present invention further comprises a monocyclic or fused or non-fused polycyclic ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); or a heterocyclyl, which can be a monocyclic or fused or non-fused polycyclic ring (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiazinyl); a monocyclic or fused or non-fused polycyclic aryl or heteroaryl group (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothienyl or benzofuranyl); an aryloxy group; an aralkyloxy group; a heterocyclyloxy group; and a heterocyclylalkoxy group.

[0180] As used herein, the term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.

[0181] As used herein and unless otherwise indicated, the term "solvate" means a compound or salt thereof that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0182] As used herein and unless otherwise indicated, the term "hydrate" means a compound or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0183] As used herein and unless otherwise indicated, the term "prodrug" means a compound derivative that can be hydrolyzed, oxidized or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of compounds, including bio-hydrolyzable parts, such as bio-hydrolyzable amides, bio-hydrolyzable esters, bio-hydrolyzable carbamates, bio-hydrolyzable carbonates, bio-hydrolyzable ureides and bio-hydrolyzable phosphate analogs. In certain embodiments, the prodrug of a compound with a carboxyl functional group is a lower alkyl ester of a carboxylic acid. Carboxylate is conveniently formed by any carboxylic acid moiety present on the esterification molecule. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery, 6th edition (Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard, 1985, Harwood Academic Publishers GmbH).

[0184] As used herein and unless otherwise indicated, the terms "stereoisomer" or "stereomerically pure" mean that one stereoisomer of a compound is substantially free of other stereoisomers of the compound. For example, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound contains greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. The compounds may have chiral centers and may occur as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms are included in the embodiments disclosed herein, including mixtures thereof. The embodiments disclosed herein encompass the use of stereoisomerically pure forms of such compounds, as well as the use of mixtures of those forms. For example, mixtures of enantiomers comprising equal or unequal amounts of a particular compound may be used in the methods and compositions disclosed herein. These isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0185] It should also be noted that the compound may include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In certain embodiments, the compound is separated into cis or trans isomers. In other embodiments, the compound is a mixture of cis and trans isomers.

[0186] "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment in which the compound is located and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other:

[0187]

[0188] Those skilled in the art readily understand that various functional groups and other structures may exhibit tautomerism, and all tautomers of the compounds are within the scope of the present invention.

[0189] It should also be noted that the compounds may contain unnatural proportions of atomic isotopes at one or more of their atoms. For example, the compounds may be treated with tritium ( 3 H), iodine-125 ( 125 I), sulfur 35( 35 S) or carbon-14 ( 14 C) or can be radiolabeled with a radioactive isotope such as deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15( 15 N) isotopically enriched. As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom whose isotopic composition is different from the natural isotopic composition of the atom. "Isotopically enriched" can also refer to a compound containing at least one atom whose isotopic composition is different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., cancer and inflammation therapeutic agents), research reagents (e.g., binding analytical reagents) and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations of compounds as described herein (whether or not radioactive) are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, an isotopologue of the compound is provided, for example, an isotopologue is a deuterium, carbon-13 or nitrogen-15 enriched compound.

[0190] It should be noted that if there is an inconsistency between a depicted structure and the name of the structure, the depicted structure should be given higher weight.

[0191] The term "effective amount" in relation to a compound means an amount that is capable of completely or partially alleviating symptoms or slowing or stopping further progression or worsening of those symptoms. It will be clear to those skilled in the art that the effective amount of the compounds disclosed herein is expected to vary depending on the severity of the indication being treated.

[0192] As used herein, "alkynyl" refers to a monovalent hydrocarbon moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl groups are optionally substituted and can be straight chain, branched, or cyclic. Alkynyl groups include, but are not limited to, those having: 2-20 carbon atoms, i.e., C 2-20 Alkynyl; 2-12 carbon atoms, i.e. C 2-12 Alkynyl; 2-8 carbon atoms, i.e. C 2-8 Alkynyl; 2-6 carbon atoms, i.e. C 2-6 Alkynyl; and 2-4 carbon atoms, namely C 2-4 Alkynyl. Examples of alkynyl moieties include, but are not limited to, ethynyl, propynyl, and butynyl.

[0193] As used herein, "haloalkyl" refers to an alkyl group as defined above, wherein the alkyl group includes at least one substituent selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br), or iodine (I)). Examples of haloalkyl groups include, but are not limited to, -CF3, -CH2CF3, -CCl2F, and -CCl3.

[0194] As used herein, "haloalkoxy" refers to an alkoxy group as defined above, wherein the alkoxy group includes at least one substituent selected from halogen (eg, F, Cl, Br, or I).

[0195] As used herein, "arylalkyl" refers to a monovalent moiety that is a radical of an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, i.e., the aromatic compound includes a single bond to the alkyl group, and wherein the radical is located on the alkyl group. The arylalkyl group is bonded to the chemical structure shown through the alkyl group. The arylalkyl group can be represented by, for example, the following structures: B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, wherein B is an aromatic moiety, such as phenyl. The arylalkyl group is optionally substituted, i.e., the aryl and / or alkyl group can be substituted as disclosed herein. Examples of arylalkyl groups include, but are not limited to, benzyl.

[0196] As used herein, "alkylaryl" refers to a monovalent moiety that is a radical of an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, i.e., the aryl compound includes a single bond to an alkyl group, and wherein the radical is located on the aryl group. The alkylaryl group is bonded to the chemical structure shown via the aryl group. The alkylaryl group can be represented by, for example, the following structures: -B-CH3, -B-CH2-CH3, -B-CH2-CH2-CH3, -B-CH2-CH2-CH2-CH3, -B-CH(CH3)-CH2-CH3, -B-CH2-CH(CH3)-CH3, wherein B is an aromatic moiety, such as phenyl. The alkylaryl group is optionally substituted, i.e., the aryl group and / or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl groups include, but are not limited to, toluoyl.

[0197] As used herein, "aryloxy" refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms and wherein the ring is substituted with an oxygen group, i.e., the aromatic compound includes a single bond to an oxygen atom and wherein the radical is located on the oxygen atom, such as C6H5-O- for phenoxy. The aryloxy substituents are bonded to the compound they replace via this oxygen atom. Aryloxy groups are optionally substituted. Aryloxy groups include, but are not limited to, those having from 6 to 20 ring carbon atoms, i.e., C 6-20 Aryloxy; 6 to 15 ring carbon atoms, i.e. C 6-15 Aryloxy; and 6 to 10 ring carbon atoms, namely C 6-10 Aryloxy. Examples of aryloxy moieties include, but are not limited to, phenoxy, naphthoxy, and anthracenoxy.

[0198] As used herein, the term "residue" refers to a chemical moiety that remains after a chemical reaction within a compound. For example, the term "amino acid residue" or "N-alkyl amino acid residue" refers to the product of amide coupling or peptide coupling of an amino acid or N-alkyl amino acid with a suitable coupling partner; wherein, for example, upon amide or peptide coupling of the amino acid or N-alkyl amino acid, a water molecule is expelled, thereby producing a product in which the amino acid residue or N-alkyl amino acid residue is incorporated.

[0199] As used herein, "sugar" or "glycosyl" or "sugar residue" refers to a carbohydrate moiety that can include a 3-carbon (triose) unit, a 4-carbon (tetraose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptose) unit, or a combination thereof, and can be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentose, an oligosaccharide, or any other polysaccharide. In some cases, "sugar" or "glycosyl" or "sugar residue" includes a furanose (e.g., ribofuranose, fructofuranose) or a pyranose (e.g., glucopyranose, galactopyranose) or a combination thereof. In some cases, "sugar" or "glycosyl" or "sugar residue" includes an aldose or ketose, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other "sugars" or "glycosyls" or "sugar residues" include polysaccharides and / or oligosaccharides, including but not limited to amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, a "sugar" or "glycosyl" or "sugar residue" is an amino-sugar. In some cases, a "sugar" or "glycosyl" or "sugar residue" is a glucosamine residue (1-amino-1-deoxy-D-glucitol), which is linked to the rest of the molecule via its amino group, thereby forming an amide bond (i.e., glucamide) with the rest of the molecule.

[0200] As used herein, "binding agent" refers to any molecule, such as an antibody, that is capable of specifically binding to a given binding partner (eg, an antigen).

[0201] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group and a side chain (R group), which is specific for each amino acid. Amino acids can be proteinogenic or non-proteinogenic. "Proteinogenic" means that an amino acid is one of the twenty naturally occurring amino acids found in proteins. Proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteinogenic" means that an amino acid is not naturally found in proteins, or is not directly produced by cellular mechanisms (e.g., a product of post-translational modification). Non-limiting examples of non-proteinogenic amino acids include gamma-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylacetamide), hydroxyproline, beta-alanine, ornithine, and citrulline.

[0202] As used herein, "peptide" is defined in its broadest sense in its various grammatical forms to refer to a compound composed of two or more subunit amino acids, amino acid analogs, or other peptide mimetics. The subunits can be linked by peptide bonds, or by other bonds, such as ester bonds, ether bonds, etc. As used herein, the term "amino acid" refers to natural and / or non-natural, proteinogenic or non-proteinogenic, or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimetics. If the peptide chain is shorter, such as two, three, or more amino acids, it is generally referred to as an oligopeptide. If the peptide chain is longer, the peptide is generally referred to as a polypeptide or protein. The definition encompasses full-length proteins, their analogs, mutants, and fragments. The term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. In addition, due to the presence of ionizable amino and carboxyl groups in the molecule, specific peptides can be obtained in acidic or basic salt or neutral form. Peptides can be obtained directly from the source organism, or can be produced recombinantly or synthetically.

[0203] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including those described by Kabat et al. ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise indicated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme does not imply differences in the absence of different sequences, and sequence positions can be readily confirmed by one skilled in the art by inspecting the amino acid sequence of one or more antibodies. Unless otherwise indicated, the "EU numbering scheme" (eg, as reported in Kabat et al., supra) is generally used when referring to residues in the antibody heavy chain constant region.

[0204] As used herein, the term "anti-HER2 antibody" refers to an antibody that selectively binds to the HER2 receptor, such as trastuzumab. In one embodiment, trastuzumab can be manufactured and used as described in US6407213 and US5821337, the complete disclosure of which is incorporated herein by reference. In one embodiment, the sequence of the heavy chain of trastuzumab and the light chain of its matching and its preparation method are known in the art and can also be found in public databases, such as Inxight Drugs developed by the U.S. National Center for Advancing Translational Sciences (NCATS).

[0205] As used herein, the term "anti-HER3 antibody" refers to an antibody that selectively binds to the HER3 receptor, such as Pertuzumab. In one embodiment, Pertuzumab can be manufactured and used as described in US7705130, the complete disclosure of which is incorporated herein by reference. In one embodiment, the sequences of the heavy chain of Pertuzumab and its matching light chain, as well as methods for their preparation, are known in the art and can also be found in public databases, such as Inxight Drugs developed by the National Center for Advancing Translational Sciences (NCATS).

[0206] As used herein, the term "anti-PTK7 antibody" refers to an antibody that selectively binds to the PTK7 receptor, such as cofetiuzumab. In one embodiment, cofetiuzumab can be manufactured and used as described in US9777070, the complete disclosure of which is incorporated herein by reference. In one embodiment, the sequences of the heavy chain and its matching light chain of cofetiuzumab and methods for their preparation are known in the art and can also be found in public databases, such as Inxight Drugs developed by the National Center for Advancing Translational Sciences (NCATS).

[0207] As used herein, the term "ifinatamab" refers to an antibody that selectively binds to the B7H3 receptor. In one embodiment, ifinatamab can be manufactured and used as described in US10117952 or WO2022102695, the complete disclosure of which is incorporated herein by reference. In one embodiment, the sequences of the heavy chain of ifinatamab and its matching light chain and methods for their preparation are known in the art and can be found in public databases, such as SAbDab: The Structural Antibody Database-OPIG.

[0208] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. A "tumor" includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0209] As used herein, the term "cell killing activity" refers to an activity that reduces or decreases the cell viability of a tested cell line.

[0210] In the following claims and in the preceding description of the invention, unless the context requires otherwise due to the express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., specifying the presence of the stated features in various embodiments of the invention and not excluding the presence of additional features or the addition of additional features.

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0212] 5.2. Conjugates

[0213] Described herein are compounds according to the formula:

[0214]

[0215] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof,

[0216] wherein BA is a binding agent selected from a humanized, chimeric, or human antibody or antigen-binding antibody fragment of an antibody; L is a covalent linker; PA is a payload residue; and subscript x is 1 to 30. In some cases, x is 1 to 4. In some cases, x is about 1. In some cases, x is about 2. In some cases, x is about 3. In some cases, x is about 4.

[0217] In one example, the BA is an antibody. In one example, the antibody is a humanized, chimeric, or human antibody or an antigen-binding fragment of an antibody. In one example, the antibody is a humanized, chimeric, or human anti-HER2, anti-HER3, anti-PTK7, or anti-B7H3 antibody or an antigen-binding fragment of an anti-HER2, anti-HER3, anti-PTK7, or anti-B7H3 antibody. In one example, the antibody is a monoclonal antibody.

[0218] In one example, the BA is an antibody. In one example, the antibody is a humanized, chimeric, or human antibody or an antigen-binding fragment of cofetizumab, pertrastuzumab, ifenatumab, or trastuzumab.

[0219] In certain embodiments, the antibodies as described herein bind to one or more receptors selected from the group consisting of: CD7, CD19, CD22, CD27, CD30, CD33, CD37, CD70, CD74, CD79b, CD138, CD142, CA6, p-cadherin, CEACAM5, C4.4a, DLL3, EGFR, EGFRVIII, ENPP3, EphA2, EphrinA, FLOR1, FGFR2, GCC, HER2, HER3, cKIT, LIV1, LY6E, MSLN, MUC16, NaPi2b, Nectin4, gpNMB, PSMA, SLITRK6, STEAP1, TROP2, 5T4, SSEA4, GloboH, Gb5, STn, and Tn.

[0220] In certain embodiments, the antibodies as described herein bind to one or more receptors selected from the group consisting of: B7H3, MUCl, FGFR2b, CLL1, CCR7, GPC1, and GPC3.

[0221] In certain embodiments, the antibodies described herein bind to the CEA receptor.

[0222] In certain embodiments, the antibodies described herein are bispecific antibodies.

[0223] In some embodiments, PA is the residue of an exatecan analog provided herein.

[0224] 5.3. Exatecan analogs

[0225] Provided herein are exatecan analogs.

[0226] Aspect 1

[0227] Provided herein is a compound having formula (I):

[0228]

[0229] or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein

[0230] Y is -ABCDH;

[0231] A is a key, CR 1 R 2 or NR 1 ;

[0232] B is a bond, -C(=O)-, or -C(=O)O-;

[0233] C is a bond or a divalent group selected from unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl;

[0234] D is a bond, NH or O;

[0235] R 1 and R 2 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 1 and R 2 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl;

[0236] R 3 and R 4 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 3 and R 4 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; and

[0237] When R 3 is a methyl group, and R 4 When it is F, Y is not -NH-C(=O)-CDH.

[0238] Aspect 2

[0239] In one embodiment, the compound is a compound having formula (II):

[0240]

[0241] or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein

[0242] A is CR 1 R 2 , NH or NR 1 ;

[0243] R 1 and R 2 Each of is independently H or C 1-4 alkyl;

[0244] R 3 and R 4 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 3 and R 4 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl;

[0245] R 5 and R 6 Each of is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; and

[0246] n is 1, 2, 3, 4, or 5.

[0247] Aspect 3

[0248] In one embodiment, A is -CH2-, and B is a bond.

[0249] In one embodiment, R 5 and R 6 is hydrogen, and n is 1, 2 or 3.

[0250] In one embodiment, R 3 is a methyl group, and R 4 It's F.

[0251] In one embodiment, the compound is

[0252] Aspect 4

[0253] In one embodiment, A is -CH2-, and B is a bond.

[0254] In one embodiment, R 5 and R 6 is hydrogen, and n is 1, 2 or 3.

[0255] In one embodiment, R 3 and R 4 Together with the atoms to which they are attached, they form an unsubstituted or substituted dioxole ring.

[0256] In one embodiment, the compound is

[0257] Aspect 5

[0258] In one embodiment, R 3 is a methyl group, and R 4 It's F.

[0259] In one embodiment, A is -N(CH3)-, and B is a bond.

[0260] In one embodiment, R 5 and R 6 is hydrogen, and n is 2.

[0261] In one embodiment, the compound is

[0262] Aspect 6

[0263] In one embodiment, R 3 is a methyl group, and R 4 It's F.

[0264] In one embodiment, A is -NH-, and B is -C(=O)O-.

[0265] In one embodiment, R 5 and R 6 is hydrogen, and n is 2.

[0266] In one embodiment, the compound is

[0267] In one embodiment, A is -NH-, and B is -C(=O)-.

[0268] In one embodiment, R 5 and R 6 is hydrogen, and n is 2.

[0269] In one embodiment, the compound is

[0270] In one embodiment, R3 It is Cl, R 4 is F, and B is -C(=O)-.

[0271] In one embodiment, the compound is

[0272]

[0273] In one embodiment, R 3 is methyl, R 4 is Cl, and B is -C(=O)-.

[0274] In one embodiment, the compound is

[0275]

[0276] In one embodiment, R 3 and R 4 Together with the atoms to which they are attached, they form an unsubstituted or substituted heterocyclic group.

[0277] In one embodiment, R 3 and R 4 Together with the atoms to which they are attached, they form an unsubstituted or substituted dioxole ring, and B is -C(=O)-.

[0278] In one embodiment, the compound is

[0279]

[0280] Aspect 7

[0281] In one embodiment, the compound has formula (III):

[0282]

[0283] or a pharmaceutically acceptable solvate, stereoisomer or derivative thereof.

[0284] In one embodiment, R 3 is methyl; and R 4 It's Cl.

[0285] In one embodiment, the compound is

[0286] In one embodiment, R 3 is Cl; and R 4 It's F.

[0287] In one embodiment, the compound is

[0288] In one embodiment, R 3 is F; and R 4 It's F.

[0289] In one embodiment, the compound is

[0290] In one embodiment, R 3 is H; and R 4 It's F.

[0291] In one embodiment, the compound is

[0292] In one embodiment, R 3 is H; and R 4 It's OH.

[0293] In one embodiment, the compound is

[0294] In one embodiment, R 3 is methyl; and R 4 It's methyl.

[0295] In one embodiment, the compound is

[0296] In one embodiment, R 3 is methoxy; and R 4 It's F.

[0297] In one embodiment, the compound is

[0298] In one embodiment, R 3 is H; and R 4 It's a methoxy group.

[0299] In one embodiment, the compound is

[0300] In one embodiment, R 3 is H; and R 4 It's Cl.

[0301] In one embodiment, the compound is

[0302] In one embodiment, R 3 and R 4 Together with the atoms to which they are attached, they form an unsubstituted or substituted heterocyclic group.

[0303] In one embodiment, R3 and R 4 Together with the atoms to which they are attached, they form an unsubstituted or substituted dioxole ring.

[0304] In one embodiment, the compound is

[0305]

[0306] In one embodiment, the compound is

[0307]

[0308] Aspect 8

[0309] In one embodiment, the compound is

[0310] In one embodiment, the compound is

[0311] Aspect 9

[0312] In one embodiment, the compound is a compound selected from Table 1 and Table 2.

[0313] Aspect 10

[0314] Provided herein is a ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a residue of a compound provided herein.

[0315] In one embodiment, the ligand-drug conjugate comprises a structure of Formula (V):

[0316]

[0317] in

[0318] BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody;

[0319] L is a covalent linker as described herein; and

[0320] x is 1 to 10, and it can be an integer or a decimal.

[0321] In one embodiment, the antibody is pertratuzumab, cofetiuzumab, trastuzumab, ifenatumab, or mAb10.

[0322] Aspect 11

[0323] Provided herein is a compound having formula (VII):

[0324]

[0325] or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein

[0326] R 7 and R 8 Each of R is independently hydrogen or substituted or unsubstituted alkyl; or 7 and R 8 Together with the nitrogen atom to which they are attached, they form an unsubstituted or substituted heterocyclic group or an unsubstituted or substituted heteroaryl group.

[0327] In one embodiment, the compound is

[0328]

[0329] In one embodiment, the ligand-drug conjugate comprises the structure of Formula (VIIIa), (VIIIb), or (VIIIc):

[0330]

[0331] in

[0332] BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody;

[0333] L is a covalent linker as described herein; and

[0334] x is 1 to 10, and it can be an integer or a decimal.

[0335] In one embodiment, the ligand-drug conjugate comprises a structure of any one of the following formulae:

[0336]

[0337]

[0338] in

[0339] BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody;

[0340] L is a covalent linker as described herein; and

[0341] x is 1 to 10, and it can be an integer or a decimal.

[0342] In one embodiment, the antibody is pertratuzumab, cofetiuzumab, trastuzumab, ifenatumab, or mAb10.

[0343] In one embodiment, L is

[0344]

[0345]

[0346] The bonds marked with an asterisk are connected to BA.

[0347] In one embodiment, L is

[0348] The bonds marked with an asterisk are connected to BA.

[0349] In some embodiments, x is 1 to 15. In some embodiments, x is 2 to 10. In some embodiments, x is 3 to 9. In one embodiment, x is about 3. In one embodiment, x is about 4. In one embodiment, x is about 5. In one embodiment, x is about 6. In one embodiment, x is about 7. In one embodiment, x is about 8. In one embodiment, x is about 9.

[0350] 5.4. Covalent Linkers

[0351] Aspect 12

[0352] Described herein are covalent linkers according to formula (IXa):

[0353]

[0354] Among them RG 1 is a reactive group residue; RG 2 is an optional reactive group residue; SP 1 and SP 2 is, independently at each occurrence, an optional spacer residue; HG is a hydrophilic residue; PAB is an optional self-immolative unit; and subscript p is 0 or 1.

[0355] In some embodiments, the compound of formula (IXa) is a compound with a P3 modification, wherein AA 2 Inclusion formula (W):

[0356]

[0357] and

[0358] AA 3 Is -valine-alanine-, -valine-citrulline- or dipeptide residues; where R Y It is -CH3 or -(CH2)3-NHC(=O)NH2.

[0359] In one embodiment, yes

[0360] In some embodiments, PAB represents -NH-CH2-O-; formula (Y1):

[0361]

[0362] or

[0363] Formula (Y2):

[0364]

[0365] in Indicates the bond through which the PAB is bonded to the adjacent group in the formula.

[0366] In some embodiments, RG 1 yes -(succinimidyl-3-yl-N)-,

[0367] In some embodiments, RG 1 yes Where EWG is an electron withdrawing group, such as -CN, -NO2, halogen, -CF3, -C(=O)OR x and -C(=O)R x , and R x is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycloalkyl group, or a substituted or unsubstituted heteroaryl group.

[0368] In some embodiments, RG 1 yes

[0369] In some embodiments, RG 1 yes Where EWG is an electron withdrawing group, such as -CN, -NO2, halogen, -CF3, -C(=O)OR x and -C(=O)R x , and R x is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycloalkyl group, or a substituted or unsubstituted heteroaryl group.

[0370] In some embodiments, the ring-opened compound of formula (IXa) is a compound of formula (IXa) wherein RG 1 In some embodiments, RG 1yes

[0371] In some embodiments, RG 2 is a bond, -C(=O)-NH- or -NHC(=O)-.

[0372] In some embodiments, SP 1 Yes - (CH2) n1 -C(═O)-, -(CH2CH2O) n2 -CH2CH2-C(═O)-、-CH[-(CH2) n3 -COOH]-C(═O)-, -CH2-C(═O)-NH-(CH2) n4 -C(═O)-, -CH2-C(═O)-NH-(CH2) n3 -C(═O)-NH-(CH2) n4 -C(═O)- or -C(═O)-(CH2) n5 -C(═O)-, wherein each of n1, n2, n3, n4 and n5 independently represents an integer from 1 to 8.

[0373] In some embodiments, SP 2 Yes - (CH2) n6 -; and n6 represents an integer from 1 to 8.

[0374] In some embodiments, HG is wherein each n7 is independently 1-15; each n8 is independently 0 or 1; each n9 is independently 1 or 2; each n10 is independently an integer from 4 to 16, such as 4, 8 or 12; each n11 is independently an integer from 0 to 5; n12 is an integer from 0 to 3; d is 0-3; R 9 is H or Me; R 10 -OH, -NH2, -NHCH2-CH2-(PEG) x -OH or -NHCH2-CH2-(PEG) x -OMe;R 11 is OH or NH2; and each of X, Y and Z is independently -CH2-, -NH-, -S- or -O-.

[0375] In some embodiments, HG is wherein each n7 is independently 1-15; each n8 is independently 0 or 1; each n9 is independently 1 or 2; each n10 is independently an integer from 4 to 16, such as 4, 8 or 12; d is 0-3; R 9 is H or Me; R 10 -OH, -NH2, -NHCH2-CH2-(PEG) x -OH or -NHCH2-CH2-(PEG) x -OMe;R 11 It is OH or NH2.

[0376] In some embodiments, HG is wherein each n8 is independently 0 or 1; and R 1 It is H or Me.

[0377] In some embodiments, HG is wherein each n11 is independently an integer from 0 to 5; n12 is an integer from 0 to 3; and each of X, Y and Z is independently -CH2-, -NH-, -S- or -O-.

[0378] In some embodiments, HG is -NHSO2NH2, -SO3H, -SO2NH2, -PO3H2, and RG 2 Yes key.

[0379] In some embodiments, each PA is independently selected from the compounds provided herein.

[0380] In some embodiments, R 4 It is hydrogen,

[0381] In some embodiments, R 4 It is hydrogen,

[0382] In some embodiments,

[0383] AA 2 Is glycine or of amino acid residues.

[0384] Aspect 13

[0385] Described herein are compounds of formula (IXa), or pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, enantiomers, isotopomers, or prodrugs thereof,

[0386] Among them AA 2 Inclusion formula (W):

[0387]

[0388] and

[0389] AA 3 is -Glycine-Glycine-Phenylalanine-Glycine- or tetrapeptide residues.

[0390] Aspect 14

[0391] Described herein are covalent linkers according to formula (IXb):

[0392]

[0393] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof,

[0394] Among them AA 2 Inclusion formula (W):

[0395]

[0396] and

[0397] AA 1 Is -valine-alanine-, -valine-citrulline- or dipeptide residues; where R Y It is -CH3 or -(CH2)3-NHC(=O)NH2.

[0398] In one embodiment, yes

[0399] In some embodiments, the ring-opened compound of formula (IXb) is a compound of formula (IXb) wherein RG 1 In some embodiments, the compound of formula (Ib) with an open ring is a compound of formula (IXb), wherein RG 1 In some embodiments, RG 1 yes

[0400] In one embodiment, BA, RG 1 、SP 1 、SP 2 RG 2 , HG, PAB, p and PA are as provided herein.

[0401] Aspect 15

[0402] Described herein are covalent linkers of formula (IXb),

[0403] Among them AA 2 Inclusion formula (W):

[0404]

[0405] and

[0406] AA 1 is -Glycine-Glycine-Phenylalanine-Glycine- or tetrapeptide residues.

[0407] In one embodiment, BA, RG 1 、SP 1 、SP 2 RG 2 , HG, PAB, p, x and PA are as provided herein.

[0408] Aspect 16

[0409] Described herein are covalent linkers according to formula (IXc):

[0410]

[0411] Among them AA 3 Is -valine-alanine-, -valine-citrulline- or dipeptide residues; where R Y It is -CH3 or -(CH2)3-NHC(=O)NH2.

[0412] In some embodiments, the ring-opened covalent linker of formula (IXc) is a covalent linker of formula (IXc) wherein RG 1 In some embodiments, the covalent linker of formula (IXc) with an open ring is a covalent linker of formula (IXc) wherein RG 1 In some embodiments, RG 1 yes

[0413] Aspect 17

[0414] Described herein are covalent linkers according to formula (IXc),

[0415] Among them AA 3 is -Glycine-Glycine-Phenylalanine-Glycine- or tetrapeptide residues.

[0416] Aspect 18

[0417] In some embodiments, the compound is selected from the group consisting of compounds in Table 4.

[0418] 5.5. Connector with payload (platform)

[0419] In some embodiments, described herein is a compound comprising a reactive linker bonded to at least one payload moiety.

[0420] In some embodiments, described herein is a compound, or a pharmaceutically acceptable solvate, stereoisomer, or derivative thereof, comprising a payload unit linked to at least one hydrophilic residue via a covalent linker unit, wherein the covalent linker is directly or indirectly bonded to each of the payload unit and the hydrophilic residue.

[0421] In one embodiment, the hydrophilic residue comprises a terminal hydrophilic group.

[0422] In one embodiment, the hydrophilic residue comprises a sugar residue.

[0423] Aspect 19

[0424] In some embodiments, described herein is a compound having formula (X):

[0425] L-PA

[0426] (X)

[0427] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopologue or prodrug thereof, wherein L is a covalent linker; and PA is a payload residue.

[0428] Aspect 20

[0429] Described herein are compounds according to formula (Xa):

[0430]

[0431] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof,

[0432] Among them AA 2 Inclusion formula (W):

[0433]

[0434] and

[0435] AA 3 Is -valine-alanine-, -valine-citrulline- or dipeptide residues; where RY It is -CH3 or -(CH2)3-NHC(=O)NH2.

[0436] In one embodiment, yes

[0437] In one embodiment, RG 1 yes Succinimide-N-,

[0438] In one embodiment, RG 1 yes Where EWG is an electron withdrawing group, such as -CN, -NO2, halogen, -CF3, -C(=O)OR x and -C(=O)R x , and R x is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycloalkyl group, or a substituted or unsubstituted heteroaryl group.

[0439] In one embodiment, RG 1 yes

[0440] In one embodiment, RG 1 yes Where EWG is an electron withdrawing group, such as -CN, -NO2, halogen, -CF3, -C(=O)OR x and -C(=O)R x , and R x is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycloalkyl group, or a substituted or unsubstituted heteroaryl group.

[0441] In some embodiments, the ring-opened compound of formula (X) is a compound of formula (X) wherein RG 1 In some embodiments, the compound of formula (X) with an open ring is a compound of formula (X) wherein RG 1 In some embodiments, RG 1 yes Where EWG is an electron withdrawing group, such as -CN, -NO2, halogen, -CF3, -C(=O)OR x and -C(=O)R x , and R x is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycloalkyl group, or a substituted or unsubstituted heteroaryl group.

[0442] In some embodiments, the ring-opened compound of formula (Xa) is a compound of formula (Xa) wherein RG 1 In some embodiments, the compound of formula (Xa) with an open ring is a compound of formula (Xa) wherein RG 1 In some embodiments, RG 1 yes

[0443] In one embodiment, RG 1 、SP 1 、SP 2 RG 2 , HG, PAB, p and PA are as provided herein.

[0444] Aspect 21

[0445] Described herein are compounds according to formula (Xa), or pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, enantiomers, isotopomers, or prodrugs thereof,

[0446] Among them AA 2 Inclusion formula (W):

[0447]

[0448] and

[0449] AA 3 is -Glycine-Glycine-Phenylalanine-Glycine- or tetrapeptide residues.

[0450] In one embodiment, RG 1 、SP 1 、SP 2 RG 2 , HG, PAB, p and PA are as provided herein.

[0451] Aspect 22

[0452] Described herein are compounds according to formula (Xb):

[0453]

[0454] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof,

[0455] Among them AA 2 Inclusion formula (W):

[0456]

[0457] and

[0458] AA 1 Is -valine-alanine-, -valine-citrulline- or dipeptide residues; where R Y It is -CH3 or -(CH2)3-NHC(=O)NH2.

[0459] In one embodiment, yes

[0460] In some embodiments, the ring-opened compound of formula (Xb) is a compound of formula (Xb) wherein RG 1 In some embodiments, RG 1 yes

[0461] In one embodiment, RG 1 、SP 1 、SP 2 RG 2 , HG, PAB, p and PA are as provided herein.

[0462] Aspect 23

[0463] Described herein are compounds according to formula (Xb), or pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, enantiomers, isotopomers, or prodrugs thereof,

[0464] Among them AA 2 Inclusion formula (W):

[0465]

[0466] and

[0467] AA 1 is -Glycine-Glycine-Phenylalanine-Glycine- or tetrapeptide residues.

[0468] In one embodiment, RG 1 、SP 1 、SP 2 RG 2 , HG, PAB, p and PA are as provided herein.

[0469] Aspect 24

[0470] Described herein are compounds according to formula (Xc):

[0471] RG1 —SP 1 -AA 3 —(PAB) p —PA

[0472] (Xc)

[0473] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopomer or prodrug thereof,

[0474] Among them AA 3 Is -valine-alanine-, -valine-citrulline- or dipeptide residues; where R Y It is -CH3 or -(CH2)3-NHC(=O)NH2.

[0475] In some embodiments, the ring-opened compound of formula (Xc) is a compound of formula (Xc) wherein RG 1 In some embodiments, RG 1 yes

[0476] In one embodiment, RG 1 、SP 1 , PAB, p and PA are as provided herein.

[0477] Aspect 25

[0478] Described herein are compounds according to formula (Xc), or pharmaceutically acceptable salts, solvates, stereoisomers or derivatives thereof,

[0479] Among them: RG 1 is a reactive group residue; SP 1 is an optional spacer residue; PAB is an optional self-immolative unit; subscript p is 0 or 1; and PA is a payload residue;

[0480] Among them AA 3 is -Glycine-Glycine-Phenylalanine-Glycine- or tetrapeptide residues.

[0481] In one embodiment, RG 1 、SP 1 , PAB, p and PA are as provided herein.

[0482] Aspect 26

[0483] In some embodiments, the compound is selected from the group consisting of compounds in Table 3.

[0484] Covalent linkers can be prepared as described in PCT / CN2021 / 142037, PCT / CN2022 / 086931, PCT / CN2022 / 088762, and PCT / CN2022 / 097834, the entire disclosures of which are incorporated herein by reference.

[0485] 5.6. Usage

[0486] In some embodiments, described herein is a method of treating a disease or condition in a patient in need thereof, comprising administering to the patient a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0487] In some embodiments, described herein is a method of treating or preventing a disease, disorder, or condition selected from the group consisting of: a proliferative disorder, a neurodegenerative disorder, an immune disorder, an autoimmune disease, an inflammatory disorder, a skin disease, a metabolic disease, a cardiovascular disease, and a gastrointestinal disease, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition as described herein. In some embodiments, the compound administered is an antibody-drug conjugate as described herein.

[0488] In some embodiments, described herein is a method for treating a proliferative disease, metabolic disease, inflammation or neurodegenerative disease in a subject, comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition as described herein. In some embodiments, described herein is a method for treating a proliferative disease in a subject, comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition as described herein. In some embodiments, the compound administered is an antibody-drug conjugate as described herein.

[0489] In some embodiments, described herein is a method of treating a metabolic disease in a subject, comprising administering to the subject an effective therapeutic amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0490] In some embodiments, described herein is a method of treating inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0491] In some embodiments, described herein is a method of treating a neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein. In some embodiments, the compound administered is an antibody-drug conjugate described herein.

[0492] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0493] 5.7 Antibody

[0494] In some embodiments, described herein is an antibody or antigen-binding fragment thereof that specifically binds to human HER3, comprising (i) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 1; and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 2; wherein the CDRs are the same as those of pertuzumab.

[0495] In some embodiments, described herein is an antibody or antigen-binding fragment thereof that specifically binds to human HER3, comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0496] In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is SEQ ID NO: 2.

[0497] In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten amino acids are further incorporated (inserted, deleted, or substituted) into the framework regions of the above antibodies (such as SEQ ID NO: 1 or SEQ ID NO: 2).

[0498] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

[0499] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0500] In some embodiments, the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0501] In some embodiments, wherein the Fc domain is IgG1 with reduced effector function.

[0502] In some embodiments, described herein is an antibody or antigen-binding fragment thereof that specifically binds to human HER3, comprising (i) a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 3; and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 4; wherein the CDRs are the same as those of pertuzumab.

[0503] In some embodiments, the antibody or antigen-binding fragment thereof comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO:3; and (ii) a light chain comprising the amino acid sequence of SEQ ID NO:4.

[0504] In some embodiments, the amino acids of the heavy chain are SEQ ID NO:3; and the amino acids of the light chain are SEQ ID NO:4.

[0505] In some embodiments, described herein is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0506] In some embodiments, described herein is a vector comprising a nucleic acid of the disclosure.

[0507] In some embodiments, described herein is a host cell comprising a nucleic acid or vector of the disclosure.

[0508] In some embodiments, described herein is a method for producing the antibody or antigen-binding fragment thereof, comprising culturing a host cell and recovering the antibody or antigen-binding fragment thereof from the culture.

[0509] The antibodies or antigen-binding fragments thereof disclosed herein (such as antibodies or antigen-binding fragments thereof that specifically bind to human HER3 and comprise: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2) unexpectedly exhibit superior biophysical properties to those of Pertratuzumab, such as a higher aggregation temperature and reduced self-association, thereby indicating superior isothermal stability and further prolonged half-life. At the same time, the antibodies or antigen-binding fragments thereof disclosed herein maintain binding affinity comparable to that of Pertratuzumab.

[0510] 6. Examples

[0511] The following examples are intended to be completely exemplary and should not be considered to be limiting in any way. Unless otherwise stated, the experimental methods in the examples described below are conventional methods. Unless otherwise stated, reagents and materials are all commercially available. All solvents and chemicals used are of analytical grade or chemical purity. Solvents are redistilled before use. Anhydrous solvents are prepared according to standard methods or reference methods. Silica gel (100 mesh-200 mesh) for column chromatography and silica gel (GF254) for thin layer chromatography (TLC) can be purchased from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd. in China; unless otherwise stated, petroleum ether (60-90 ° C) / ethyl acetate (v / v) are used for elution, and the color is developed by a solution of iodine or molybdophosphoric acid in ethanol. Unless otherwise stated, all extraction solvents are dried over anhydrous Na2SO4. 1 H NMR spectra were recorded on a Bruck-400, Varian 400MR nuclear magnetic resonance spectrometer with TMS (tetramethylsilane) as the internal standard. Coupling constants are given in Hertz. Peaks are reported as singlet (s), doublet (d), triplet (t), quartet (q), quintet (p), sextet (h), septet (hept), multiplet (m) or a combination thereof; br represents a broad peak. LC / MS data were recorded using an Agilent 1100 / 1200 high performance liquid chromatograph-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) and an ion trap (ESI source) detecting at 214 nm and 254 nm. All compound names except those of the reagents mentioned are given by 18.0 generated.

[0512] For the sake of brevity, certain abbreviations are used herein. One example is the use of single-letter abbreviations to represent amino acid residues. The amino acids and their corresponding three-letter and single-letter abbreviations are as follows:

[0513] List of amino acid abbreviations

[0514]

[0515]

[0516] In the following examples, the following abbreviations are used:

[0517] List of abbreviations

[0518]

[0519]

[0520] UPLC analysis method:

[0521] Method A: Mobile phase A: water containing 0.1% FA, B: MeCN; Gradient: 10% B for 0.2 min, 10%-95% B for 5.8 min, 95% B for 0.5 min; Flow rate: 0.6 mL / min; Column: ACQUITY BEH C18 1.7 μm;

[0522] Method B: Mobile phase A: water containing 0.1% FA, B: MeCN; Gradient: 10% B for 0.5 min, 10%-90% B for 2.5 min, 90% B for 0.2 min; Flow rate: 0.6 mL / min; Column: ACQUITY BEH C18 1.7 μm;

[0523] Method C: Mobile phase A: water containing 0.1% FA, B: MeCN; Gradient: 10% B for 0.2 min, 10%-90% B for 1.3 min, 90% B for 0.3 min; Flow rate: 0.6 mL / min; Column: ACQUITY BEH C18 1.7 μm;

[0524] Examples 1-1 and 1-2

[0525]

[0526] Step 12-Bromo-5-fluoro-4-methoxyaniline (1-1b)

[0527] To a solution of compound 1-1a (15 g, 106 mmol) in DCM (200 mL) was added KCO (19.1 g, 138.16 mmol). The mixture was cooled to -15 ° C and Br (16.98 g, 106.27 mmol) was added dropwise. The mixture was stirred at -15 ° C for 30 min. The mixture was filtered and washed with saturated NaS0 (100 mL * 3) and brine (100 mL * 3). The organic layer was concentrated to give the crude product 1-1b (21 g, crude material) as a brown solid.

[0528] MS (ESI) m / z: 221.9 [M+H] +

[0529] 1 H NMR (400MHz, CDCl3) δ7.03 (d, J = 8.4Hz, 1H), 6.56 (d, J = 12.4Hz, 1H), 3.89-3.85 (br s, 2H), 3.80 (s, 3H).

[0530] Step 2

[0531] N-(2-Bromo-5-fluoro-4-methoxyphenyl)acetamide (1-1c)

[0532] To a solution of compound 1-1b (21 g, 95.4 mmol) in THF (200 mL) was added Ac2O (19.5 g, 190.9 mmol) and Et3N (19.5 g, 381.8 mmol). The mixture was stirred at room temperature for 16 h. The mixture was concentrated and the residue was triturated with MTBE (50 mL) to produce compound 1-1c (20 g, crude material).

[0533] MS (ESI) m / z: 263.9 [M+H] +

[0534] 1 H NMR (400MHz, CDCl3) δ8.15 (d, J = 13.2 Hz, 1H), 7.49 (br s, 1H), 7.12 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H), 2.24 (s, 3H).

[0535] Step 3

[0536] N-(5-Fluoro-2-(1-hydroxycyclobutyl)-4-methoxyphenyl)acetamide (1-1d)

[0537] A solution of compound 1-1c (14g, 53.42mmol) in THF (300mL) was cooled to -78°C, and nBuLi (53mL, 133.55mmol) was added dropwise to the mixture. The internal temperature was maintained below -78°C. The mixture was stirred at -78°C for 30min. A solution of cyclobutanone (7.49g, 106.84mmol) in THF (50mL) was added dropwise to the mixture. The mixture was stirred at -78°C for 30min and warmed to room temperature for 1h. The mixture was quenched with saturated NH4Cl solution (5mL), diluted with EtOAc (150mL), and washed with brine (50mL*3). The organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (eluent: PE / EA=100 / 0 to 40 / 60) to obtain 1-1d (2.7g, 20.0% yield).

[0538] MS (ESI) m / z: 236.1 [M+H] +

[0539] 1 H NMR (400MHz, CDCl3) δ8.54(s,1H),7.83(d,J=12.8Hz,1H),6.91(d,J=8.8Hz,1H),3.87(s,3H ),2.59-2.52(m,2H),2.40-2.33(m,2H),2.13(s,3H),2.11-2.04(m,1H),1.72-1.65(m,1H).

[0540] Step 4

[0541] N-(3-Fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-1e)

[0542] To a solution of compound 1-1d (2.7 g, 10.66 mmol) in DCM (80 mL) and H2O (80 mL) was added AgNO3 (3.2 mL, 3.198 mmol), K2S2O8 (8.64 g, 31.98 mmol). The mixture was stirred at rt for 16 h. The mixture was diluted with EtOAc (300 mL) and washed with brine (100 mL*3). The organic layer was dried and concentrated. The organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc=100 / 0 to 40 / 60) to obtain 1-1e (1.55 g, 57.8% yield), which was obtained as a light yellow solid.

[0543] MS (ESI) m / z: 252.1 [M+H] +

[0544] 1 H NMR (400MHz, CDCl3) δ12.19 (s, 1H), 8.50 (d, J = 14.8Hz, 1H), 3.87 (s, 3H), 2.98 (t, J = 6.0Hz, 2H), 2.66 (t, J = 6.4Hz, 2H), 2.22 (s, 3H), 2.09-2.03 (m, 2H).

[0545] Step 5

[0546] (Z)-N-(3-Fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-1f)

[0547] To a solution of compound 1-1e (1.5 g, 5.97 mmol) in THF (20 mL) was added t-BuOK (7.76 mL, 7.76 mmol, 1 N) dropwise at 0 ° C., followed by dropwise addition of isoamyl nitrite (699 mg, 5.97 mmol). The mixture was stirred for 2 h at rt. The mixture was adjusted to pH = 7, diluted with EtOAc (100 mL), and washed with brine (30 mL * 3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to generate a residue. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 100 / 0 to 40 / 60) to generate the title compound 1-1f (1.1 g, 65.9% yield) as an off-white solid.

[0548] MS (ESI) m / z: 281.1 [M+H] +

[0549] 1 H NMR (400MHz, CDCl3) δ11.93 (s, 1H), 8.53 (d, J = 14.4Hz, 1H), 3.90 (s, 3H), 3.11-3.06 (m, 4H), 2.25 (s, 3H).

[0550] Step 6

[0551] N,N'-(3-Fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-1g)

[0552] To a solution of compound 1-1f (1.1 g, 3.92 mmol) in Ac2O (4 mL) and THF (20 mL) was added PPtO2 (80 mg). The mixture was stirred at rt under H2 (15 psi) for 7 h. The mixture was filtered through a celite pad, concentrated and diluted with EtOAc (150 mL), washed with saturated NaHCO3 (50 mL*3). The organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc=100 / 0 to 40 / 60) to produce the title compound 1-1g (675 mg, 55.8% yield) as a pale solid.

[0553] MS (ESI) m / z: 309.2 [M+H] +

[0554] Step 7

[0555] N-(8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-1h)

[0556] To a solution of compound 1-1g (675mg, 2.189mmol) in MeOH (10mL) was added HCl (2N, 10mL). The mixture was stirred at 60°C for 5h. The mixture was cooled to rt and adjusted to pH=7 with saturated NaHCO3, extracted with EtOAc (50mL*3). The organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc=100 / 0 to 60 / 40) to produce the title compound 1-1h (370mg, 63.5% yield) as an off-white solid.

[0557] MS (ESI) m / z: 267.1 [M+H] +

[0558] 1 H NMR(400MHz,CDCl3)δ6.57(s,1H),6.31(br s,2H),6.25(d,J=12.8Hz,1H),4.53-4.47(m,1H),3.78(s,3H),3.26-3.20(m ,1H),2.91-2.82(m,1H),2.75-2.69(s,1H),2.09(s,3H),1.77-1.67(m,1H).

[0559] Step 8

[0560] N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-1j)

[0561] To a solution of compound 1-1h (200 mg, 0.75 mmol) in toluene (40 mL) and o-cresol (0.5 mL) was added 1-1i (208 mg, 0.79 mmol) and PPTS (38 mg, 0.16 mmol). The mixture was refluxed at 140 ° C for 48 h. The mixture was concentrated and purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH=100 / 0 to 20 / 80) to produce the title compound 1-1j (350 mg, 94.3% yield, two isomers), which was obtained as an off-white solid.

[0562] MS (ESI) m / z: 494.3 [M+H] +

[0563] Step 9

[0564] (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (1-1)

[0565] (1R,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (1-2)

[0566] A solution of compound 1-1j (350 mg, 0.709 mmol) in MsOH (10 mL) and H 2 O (10 mL) was refluxed at 110° C. for 8 h. The mixture was filtered and purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to produce two isomers:

[0567] Compound 1-1 was obtained as a yellow solid (37 mg, 9.5% yield).

[0568] MS (ESI) m / z: 452.2 [M+H] +. Retention time (1.67min).

[0569] Compound 1-2 was obtained as a yellow solid (36 mg, 9.3% yield).

[0570] MS (ESI) m / z: 452.2 [M+H] + . Retention time (2.55min).

[0571] Examples 1-3 and 1-4

[0572]

[0573] Step 1

[0574] N-(8-amino-6-fluoro-5-hydroxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-3b)

[0575] To a solution of compound 1-3a (173 mg, 0.64 mmol) in DCM (6 mL) was added BBr 3 (1.92 mL, 1.93 mmol, 1 N) dropwise at 0° C. The mixture was stirred at rt for 3 h.

[0576] The mixture was quenched with MeOH (2 mL) at 0° C. The mixture was concentrated and purified by silica gel column chromatography (eluent: CH 2 Cl 2 / MeOH=100 / 0 to 20 / 80) to give the title compound 1-3b (151 mg, 92.1% yield), which was obtained as an off-white solid.

[0577] MS (ESI) m / z: 253.1 [M+H] +

[0578] Step 2

[0579] N-((9S)-9-ethyl-5-fluoro-4,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-3c)

[0580] Compound 1-3c (168 mg, 58.1% yield, two isomers) was synthesized according to the synthetic procedure of step 8 of compound 1-1.

[0581] MS (ESI) m / z: 480.2 [M+H] +

[0582] Step 3

[0583] (1S,9S)-1-Amino-9-ethyl-5-fluoro-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (1-3)

[0584] (1R,9S)-1-Amino-9-ethyl-5-fluoro-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (1-4)

[0585] A solution of compound 1-3c (150 mg, 0.31 mmol) in concentrated HCl (20 mL) was refluxed at 110° C. for 8 h. The mixture was filtered and purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to produce two isomers:

[0586] Compound 1-3 was obtained as a yellow solid (22.7 mg, 15.3% yield).

[0587] MS (ESI) m / z: 438.3 [M+H] + . Retention time (1.48min).

[0588] Compound 1-4 was obtained as a yellow solid (22.6 mg, 14.9% yield).

[0589] MS (ESI) m / z: 438.3 [M+H] + . Retention time (1.78min).

[0590] Examples 1-5

[0591]

[0592]

[0593] Step 1

[0594] (1S,9S)-1-(Dimethylamino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-5)

[0595] To a solution of exatecan mesylate (50 mg, 0.09 mmol) in formic acid (2 mL) was added 38% formaldehyde solution (1 mL) and stirred at 50°C for 18 h. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 20%-28% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD). TM The reaction solution was purified by lyophilization (5 μm, 19*150 mm). The desired fractions were lyophilized to give 1-5 (15.4 mg, 35.3% yield) as a light yellow solid.

[0596] MS (ESI) m / z: 464.4 [M+H] +

[0597] 1 H NMR(400MHz,DMSO-d6)δ7.70(d,J=10.9Hz,1H),7.30(s,1H),6.51(s,1H),5.42(s,2H),5.30-5.13(m,2H),4.11- 4.22(m,1H),3.33-3.27(m,1H),2.95-2.81(m,1H),2.42-2.22(m,9H),2.00-1.75(m,3H),0.88(t,J=7.3Hz,3H).

[0598] Examples 1-6

[0599]

[0600]

[0601] Step 1

[0602] tert-Butyl bis(2-oxoethyl)carbamate (1-6b)

[0603] To a solution of 1-6a (500 mg, 2.95 mmol) in t-BuOH / THF / H₂O (5 / 10 / 5 mL) was added a solution of potassium (VI) osmate dihydrate (55 mg, 0.15 mmol) and a solution of NaIO₄ (1.6 g, 7.39 mmol) in water (5 mL), and the mixture was stirred at rt for 20 h. The reaction solution was extracted with CH₂Cl₂ (15 mL*3). The organic phase was washed with brine (15 mL) and dried over anhydrous Na₂SO₄ and concentrated to yield compound 1-6b (396 mg, 66.6% yield) as a yellow oil.

[0604] 1H NMR (400MHz, CDCl3) δ9.67(s,1H),9.65(s,1H),4.18(s,2H),3.96(s,2H),1.45(s,9H).

[0605] Step 2

[0606] tert-Butyl 4-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)piperazine-1-carboxylate (1-6c)

[0607] To a solution of exatecan mesylate (100 mg, 0.19 mmol), NaBH3CN (12 mg, 0.19 mmol), and NaHCO3 (16 mg, 0.19 mmol) in MeOH (1.5 mL) was added a solution of 1-6b (38 mg, 0.19 mmol) and HOAc (11 μL, 0.19 mmol) in MeOH (0.5 mL), and stirred at rt for 23 h. 1-6b (16 mg, 0.08 mmol) and NaBH3CN (10 mg, 0.16 mmol) were added, and the mixture was stirred for an additional 40 min. 1-6b (10 mg, 0.05 mmol) and NaBH3CN (10 mg, 0.16 mmol) were added, and the mixture was stirred for an additional 20 min. The reaction solution was added to water (10 mL) and extracted with CH2Cl2 / MeOH (10 / 1, 5.5 mL*4). The organic phase was concentrated and purified by flash column chromatography (silica gel, CH2Cl2 / MeOH = 100 / 0 to 91 / 9) to give compound 1-6c (76 mg, 66.8% yield) as a yellow solid.

[0608] MS (ESI) m / z: 627.5 [M+Na] +

[0609] Step 3

[0610] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-1-(piperazin-1-yl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-6) formate

[0611] To a solution of 1-6c (51 mg, 0.08 mmol) in CH2Cl2 (2 mL) was added TFA (0.4 mL) at 0°C and stirred at rt for 2 h. The reaction solution was concentrated and purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: hold 24% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 1-6 formate salt as a yellow solid (21 mg, 50.3% yield).

[0612] MS (ESI) m / z: 505.4 [M+H] +

[0613] 1 H NMR(400MHz,DMSO-d6)δ8.31(s,1H),7.73(d,J=10.9Hz,1H),7.30(s,1H),5 .42(s,2H),5.40(d,J=19.6Hz,1H),5.30(d,J=19.4Hz,1H),4.27-4.16(m,1H ),3.37-3.22(m,2H),2.99-2.76(m,5H),2.72-2.56(m,4H),2.36(s,3H),2.3 0-2.18(m,1H),2.16-2.02(m,1H),1.95-1.78(m,2H),0.88(t,J=7.3Hz,3H).

[0614] Examples 1-7

[0615]

[0616]

[0617] Step 1

[0618] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-(4-methylpiperazin-1-yl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-7)

[0619] To a solution of 1-6 (TFA salt, 64 mg, 0.13 mmol), NaHCO (11 mg, 0.13 mmol) and NaBHCN (12 mg, 0.19 mmol) in CHCl / MeOH (2 / 0.5 mL) was added 37% formaldehyde solution (12 μL, 0.14 mmol) and stirred at rt for 30 min. The solution was added to water (5 mL) and extracted with DCM / MeOH (10 / 1, 5.5 mL*3). The organic phase was concentrated and purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: maintain 25% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 1-7 formate salt as a beige solid (16 mg, 24.3% yield).

[0620] MS (ESI) m / z: 519.5 [M+H] +

[0621] 1 H NMR(400MHz,DMSO-d6)δ8.31(s,1H),7.72(d,J=10.7Hz,1H),7.29(s,1H),6.50(s,1H ),5.42(s,3H),5.40(d,J=19.8Hz,1H),5.26(d,J=19.8Hz,1H),4.32-4.17(m,1H),3.2 9-3.17(m,2H),2.97-2.82(m,1H),2.74-2.55(m,4H),2.44-2.29(m,6H),2.29-2.22(m ,1H),2.21-2.13(s,3H),2.13-1.97(m,1H),1.95-1.77(m,2H),0.88(t,J=7.1Hz,3H).

[0622] Examples 1-8 and 1-9

[0623]

[0624] Step 1

[0625] N-(2-Bromo-5-chloro-4-methylphenyl)acetamide (1-8b)

[0626] To a solution of 1-8a (12.7 g, 57.5 mmol) and DMAP (355 mg, 2.9 mmol) in EtOAc / DCM (100 / 50 mL) was added AcO (7.1 mL, 74.7 mmol) and stirred at rt for 6 h. The cloudy solution was concentrated and slurried with petroleum ether / ethyl acetate (3: 1, v / v, 16 mL) and filtered to yield compound 1-8b (11.6 g, 76.9% yield) as a white solid.

[0627] MS (ESI) m / z: 261.9 [M+H] +

[0628] 1 H NMR (400MHz, CDCl3) δ8.40(s,1H),7.49(s,1H),7.39(s,1H),2.31(s,3H),2.23(s,3H).

[0629] Step 2

[0630] N-(5-chloro-2-(1-hydroxycyclobutyl)-4-methylphenyl)acetamide (1-8c)

[0631] Compound 1-8c (5.6 g, 55% yield) was obtained according to the procedure described in CN111470998B with a slight modification: the crude product was slurried with MTBE and then filtered to yield compound 1-8c.

[0632] MS (ESI) m / z: 236.1 [M+H-H2O] +

[0633] 1 H NMR(400MHz, CDCl3)δ8.59(s,1H),8.12(s,1H),7.14(s,1H),2.64-2.53(m,2H),2 .50(s,1H),2.42-2.28(m,5H),2.14(s,3H),2.11-2.00(m,1H),1.73-1.63(m,1H).

[0634] Step 3

[0635] N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8d)

[0636] Compound 1-8d (4.1 g, 85% purity, 63% yield) was obtained according to the procedure described in CN111470998B.

[0637] MS (ESI) m / z: 252.0 [M+H]+

[0638] 1 H NMR (400MHz, CDCl3) δ12.14(s,1H),8.76(s,1H),2.91(t,J=6.2Hz,2H),2.68-2.60(m,2H),2.32(s,3H),2.22(s,3H),2.16-2.01(m,2H).

[0639] Step 4

[0640] (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8e)

[0641] Compound 1-8e (3.9 g, 84% yield) was obtained according to the procedure described in CN111470998B.

[0642] MS (ESI) m / z: 281.1 [M+H] +

[0643] Step 5

[0644] N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8f)

[0645] To a solution of 1-8e (3.9 g, 13.9 mmol) in EtOAc / AcO (100 / 25 mL) was added PtO (322 mg, 1.4 mmol) and stirred under H for 16 h. The solution was filtered and concentrated to give compound 1-8f (4.15 g, crude material), which was used in the next step without further purification.

[0646] MS (ESI) m / z: 309.1 [M+H] +

[0647] Step 6

[0648] N-(8-Amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8g)

[0649] To a solution of 1-8f (2.95 g, 9.55 mmol) in MeOH (25 mL) was added 2N HCl (25 mL) under a nitrogen atmosphere and stirred at 60° C. for 2 h. The pH of the reaction solution was adjusted to 7 with solid Na 2 CO 3 and saturated NaHCO 3 solution. The slurry solution was filtered and the filter cake was purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 1: 3) to give compound 1-8g (750 mg, 29.4% yield) as a light gray solid.

[0650] MS (ESI) m / z: 267.1 [M+H] +

[0651] 1 H NMR(400MHz,DMSO)δ8.08(d,J=7.9Hz,1H),7.30(s,2H),6.76(s,1H),4.48(ddd,J =12.9,7.9,4.7Hz,1H),2.99-2.79(m,2H),2.18-2.08(m,4H),1.95-1.80(m,4H).

[0652] Step 7

[0653] N-((9S)-5-Chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-8i)

[0654] Compound 1-8i (549 mg, 97.2% yield) was obtained according to the procedure described in EP3677568A1.

[0655] MS (ESI) m / z: 494.3 [M+H] +

[0656] 1 H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.7Hz,1H),8.16(s,1H),7.31(s,1H),6.53(s,1H),5.61-5.49(m,1H),5.43(s,2 H),5.30-5.14(m,2H),3.25-3.13(m,2H),2.52(s,3H),2.18-2.08(m,2H),1.94-1.80(m,5H),0.87(t,J=7.3Hz,3H).

[0657] Step 8

[0658] (1S,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-8)

[0659] (1R,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-9)

[0660] To a slurry solution of 1-8i (200 mg, 0.41 mmol) in H2O (3 mL) was added MsOH (1.5 mL) under a nitrogen atmosphere, stirred at 100 ° C for 2 h and at 90 ° C for 14 h. Compound 1-8 (8 mg) was obtained according to the procedure described in CN111470998B. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 10%-20%-30% B; flow rate: 20 mL / min; column: Sunfire Prep C18OBD TM The filtrate was purified by lyophilization (5 μm, 19*150 mm). The desired fractions were lyophilized to give 1-8 mesylate salt (38.5 mg, 17.4% yield) as a beige solid and 1-9 formate salt (13 mg, 5.9% yield) as a beige solid.

[0661] MS (ESI) m / z: 494.3 [M+H] +

[0662] UPLC analysis: 1-8, retention time = 2.62 min; 1-9, retention time = 3.05 min (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 15% B for 1 min, 15%-95% B for 9 min, 95% B for 2 min; flow rate: 0.6 mL / min; column: ACQUITY BEH C18 1.7μm)

[0663] 1-8 (methanesulfonate): 1H NMR (400MHz, DMSO) δ8.24(brs,3H),7.35(s,1H),6.57(s,1H),5.70(d,J=19.4Hz,1H),5.46(s,2H),5.43(d,J=19.4Hz,1H),5.09-4.98 (m,1H),3.19-3.08(m,1H),2.98-2.87(m,2H),2.55(s,3H),2.30(s,3H),2.25-2.12(m,1H),1.96-1.79(m,2H),0.88(t,J=7.3Hz,3H).

[0664] 1-9 (Formate): 1 H NMR (400MHz, DMSO) δ8.14(s,1H),7.32(s,1H),6.52(s,1H),5.64(d,J=19.3Hz,1H),5.44(s,2H),5.37(d,J=19.3Hz,1H) ,4.59-4.45(m,1H),3.27-3.18(m,1H),3.17-3.06(m,1H),2.21-2.07(m,2H),1.93-1.79(m,2H),0.88(t,J=7.3Hz,3H).

[0665] Examples 1-10 and 1-11

[0666]

[0667] Step 1

[0668] N-(Benzo[d][1,3]dioxol-5-yl)acetamide (1-10b)

[0669] To a solution of 1-10a (25.00 g, 182 mmol) in CH2Cl2 (200 mL) was added Ac2O (27.85 g, 273 mmol) and Et3N (36.76 g, 364 mmol) at 0°C. The reaction mixture was reacted at 20°C for 3 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated to 50 mL. EtOAc (200 mL) was added to the residue and washed with saturated NaHCO3 (200 mL*3). The organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated under vacuum and the residue was used in the next step (28.25 g, crude material) without further treatment and purification.

[0670] MS (ESI) m / z: 180.1 [M+H] +

[0671] Step 2

[0672] N-(6-Bromobenzo[d][1,3]dioxol-5-yl)acetamide (1-10c)

[0673] A solution of 1-10b (28.25 g, crude material) and (15.4 g, 188.3 mmol) in acetic acid (100 mL) was heated to 60 ° C, and then a mixture of bromine (30.1 g, 188.3 mmol) and acetic acid (60 mL) was added dropwise to the reaction solution. The reaction temperature was raised to 80 ° C and stirred at 80 ° C for 2 h. LCMS showed that the reaction was complete, and the reaction solution was poured into ice water, and a yellow solid was generated. The solid was filtered and washed three times with water to obtain a crude product, which was recrystallized with EtOH to give 1-10c (17.1 g, 42% yield).

[0674] MS (ESI) m / z: 258.1 / 260.1 [M+H] +

[0675] 1H NMR (400MHz, DMSO) δ9.36 (s, 1H), 7.22 (s, 1H), 7.08 (d, J = 3.7Hz, 1H), 6.07 (s, 2H), 2.02 (s, 3H).

[0676] Step 3

[0677] N-(6-(1-Hydroxycyclobutyl)benzo[d][1,3]dioxol-5-yl)acetamide (1-10d)

[0678] A solution of compound 1-10c (8.5 g, 33.01 mmol) in THF (200 mL) was cooled to -90 ° C, and n-BuLi (15.84 mL, 39.60 mmol) was added to the mixture under N protection over 2 h. The internal temperature was maintained below -85 ° C. The mixture was stirred at -85 ° C for 20 min. A solution of cyclobutanone (2.7 g, 39.60 mmol) in THF (50 mL) was added dropwise to the mixture. The mixture was stirred at -85 ° C for 30 min and warmed to room temperature for 1 h. The mixture was quenched with saturated NH4Cl solution (200 mL) and extracted with EtOAc (150 mL*3). The combined organic layers were dried over Na2SO4. After filtration and evaporation, the residue was washed with MTBE (5 mL*3) and recrystallized with EtOH to obtain 1-10d (3.1 g, 37.5% yield).

[0679] MS (ESI) m / z: 250.1 [M+H] +

[0680] Step 4

[0681] N-(6-Oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-10e)

[0682] To a solution of compound 1-10d (3.1 g, 12.44 mmol) in CH2Cl2 (20 mL) and H2O (20 mL) was added AgNO3 (12.4 mL, 6.22 mmol), K2S2O8 (8.4 g, 31.1 mmol). The mixture was stirred at 20 ° C for 16 h. The mixture was filtered through diatomaceous earth and the filtration residue was washed with CH2Cl2: MeOH = 1: 1 (50 mL * 3). The filtrate was poured into water and extracted with CH2Cl2 (300 mL * 3). The combined organic layer was dried over Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (eluent: petroleum ether / CH2Cl2 = 100 / 0 to 0 / 100) to obtain 1-10e (1.9 g, 61.8% yield) as a light yellow solid.

[0683] MS (ESI) m / z: 248.1 [M+H] +

[0684] Step 5

[0685] (Z)-N-(7-(Hydroxyimino)-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-10f)

[0686] To a solution of compound 1-10e (1.9 g, 7.69 mmol) in THF (20 mL) and t-BuOH (5 mL) was added dropwise t-BuOK (1 M in THF, 9.28 mL, 9.28 mmol) at 0 ° C. The atmosphere was purged with N and the mixture was cooled to 0 ° C. using an ice-water bath. After 5 min, isoamyl nitrite (1.09 g, 9.28 mmol) was added to the stirred mixture. LCMS showed that the reaction was complete and the reaction mixture was warmed to rt. 1N HCl was added to adjust the pH to 1. The aqueous layer was extracted with CH2Cl2:THF (2: 1, v / v, 50 mL*3). The combined organic layers were washed with brine (100 mL) and dried over Na2SO4. After filtration and evaporation, the residue was triturated with MTBE (20 mL*2) to obtain 1-10f (1.5 g, 70.6% yield).

[0687] MS (ESI) m / z: 277.1 [M+H] +

[0688] Step 6

[0689] N,N'-(6-Oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxole-5,7-diyl)diethylamide (1-10g)

[0690] To a solution of compound 1-10f (1.5 g, 5.43 mmol) in Ac2O (5 mL) was added PtO2 (150 mg). The mixture was stirred at 20 ° C under H2 (15 psi) for 16 h. LCMS showed that the reaction was complete. The mixture was filtered through a celite pad. The filtrate was diluted with EtOAc (100 mL) and washed with saturated NaHCO3 (100 mL * 3). The organic layer was dried over Na2SO4. After filtration and evaporation, the residue 1-10g (1.3 g, crude material) was used in the next step without further treatment and purification.

[0691] MS (ESI) m / z: 305.2 [M+H] +

[0692] Step 7

[0693] N-(5-amino-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)acetamide (1-10h)

[0694] To a solution of compound 1-10g (1.3g, crude material) in MeOH (10mL) was added HCl (2N, 10mL). The mixture was stirred at 60°C for 3h. The mixture was cooled to rt and the pH was adjusted to 8 using saturated NaHCO. The mixture was extracted with EtOAc (50mL*3). The organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (eluent: CH2Cl2MeOH=100 / 0 to 90 / 10) to produce compound 1-10h (670mg, 59.8% yield) as an off-white solid.

[0695] MS (ESI) m / z: 263.1 [M+H] +

[0696] Step 8

[0697] N-((10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-10i)

[0698] To a solution of compound 1-10h (400 mg, 1.52 mmol) in toluene (40 mL) and o-cresol (0.5 mL) was added 1-1i (479 mg, 1.82 mmol) and PPTS (38 mg, 0.15 mmol). The mixture was refluxed at 140 ° C for 24 h. LCMS showed that the reaction was complete and a black solid was precipitated. After filtration, the filter cake was washed with acetone (10 mL * 3) to produce a dark brown solid 1-10i (580 mg, crude material), which was used in the next step without further treatment and purification.

[0699] MS (ESI) m / z: 490.3 [M+H] +

[0700] Step 9

[0701] (1S,10S)-1-Amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione(1-10)

[0702] (1R,10S)-1-Amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione(1-11)

[0703] A solution of compound 1-10i (580 mg, crude material) in MsOH (10 mL) and H2O (10 mL) was refluxed at 110°C for 5 h. LCMS showed that the reaction was complete. The mixture was filtered and purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to produce two isomers:

[0704] Compound 1-10 was obtained as a yellow solid (110 mg, 20.8% yield).

[0705] MS (ESI) m / z: 448.2 [M+H] + . Retention time (0.82min).

[0706] Compound 1-11 was obtained as a yellow solid (120 mg, 22.7% yield).

[0707] MS (ESI) m / z: 448.2 [M+H] + . Retention time (1.87min).

[0708] Examples 1-12 and 1-13

[0709]

[0710] Step 15-Fluoro-8-nitro-3,4-dihydronaphthalen-1(2H)-one (1-12b)

[0711] 1-12a (1.00 g, 6.10 mmol) was added to a stirred suspension of Cu(NO3)2 (1.14 g, 6.10 mmol) in H2SO4 (20 mL). The reaction mixture was stirred at 0°C to rt for 3 h. The mixture was added to H2O and stirred at 0°C. The residue was then extracted and filtered with EtOAc (30 mL*3). The combined organic matter was washed with H2O (30 mL) and brine (50 mL), dried over Na2SO4 and concentrated. Column chromatography on silica gel (EtOAc / petroleum ether=1 / 5) gave 1-12b (320.00 mg, 25.11% yield) as a yellow solid.

[0712] MS (ESI) m / z: 210.1 [M+H] +

[0713] Step 2

[0714] N-(4-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-12c)

[0715] Wet PtO2 (40 mg) is added to a mixture of 1-12b (0.32 g, 1.53 mmol) in Ac2O (10 mL). The reaction mixture is purged with H2 balloon three times and reacted under H2 balloon at rt for 16 h. After the reaction is complete (checked by LCMS), the mixture is filtered out by diatomaceous earth and washed with MeOH. The filtrate is concentrated under vacuum and purified by silica gel column chromatography (A-DCM; B-MeOH) to provide 1-12c (147 mg, 43.44% yield).

[0716] MS (ESI) m / z: 222.1 [M+H] +

[0717] 1H NMR (400MHz, DMSO-d6) δ11.76(s,1H),8.46(dd,J=9.3,5.1Hz,1H),7.48(t,J=9.1 Hz, 1H), 2.91 (t, J = 6.1Hz, 2H), 2.75-2.65 (m, 2H), 2.14 (s, 3H), 2.08-1.93 (m, 2H).

[0718] Step 3

[0719] N-(4-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-12d)

[0720] Compound 1-12d (150.00 mg) was synthesized according to the synthetic procedure of step 4 of Example 1-14e.

[0721] MS (ESI) m / z: 240.0 [MH] -

[0722] Step 4

[0723] N,N'-(4-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-12e)

[0724] Compound 1-12e (170.00 mg) was synthesized according to the synthetic procedure of Step 5 of Example 1-14f.

[0725] MS (ESI) m / z: 279.1 [M+H] +

[0726] Step 5

[0727] N-(8-amino-5-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-12f)

[0728] Compound 1-12f (127.34 mg, crude material) was synthesized according to the synthetic procedure of step 6 of Example 1-14g.

[0729] MS (ESI) m / z: 237.1 [M+H] +

[0730] Step 6

[0731] N-((9S)-9-Ethyl-4-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-12g)

[0732] Compound 1-12g (140.00 mg) was synthesized according to the synthetic procedure of step 7 of Example 1-14h.

[0733] MS (ESI) m / z: 464.3 [M+H] +

[0734] Step 7

[0735] (1S,9S)-1-Amino-9-ethyl-4-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-12)

[0736] (1R,9S)-1-Amino-9-ethyl-4-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-13)

[0737] 1-12 (20.10 mg, 13.90% yield) and 1-13 (16.00 mg, 11.07% yield) were synthesized according to the synthetic procedure of step 8 of Example 1-14.

[0738] MS (ESI) m / z: 422.3 [M+H] +

[0739] 1 H NMR (400MHz, DMSO-d6) δ7.99(ddd,J=130.6,14.4,7.2Hz,2H),7.34(s,1H),6.92(d,J=216.0Hz,1H),6.52(s,1H),5.68(d,J=19.4 Hz,1H),5.46-5.30(m,4H),3.16(d,J=6.1Hz,2H),2.15(s,2H),2.02-1.85(m,5H),1.49-1.30(m,2H),0.87(dd,J=7.0,3.5Hz,3H).

[0740] Examples 1-14 and 1-15

[0741]

[0742]

[0743] Step 1

[0744] N-(2-Bromo-5-chlorophenyl)acetamide (1-14b)

[0745] To a solution of 1-14a (20.00 g, 97.59 mmol) in CH2Cl2 (200 mL) was added Ac2O (14.94 g, 146.39 mmol) and H2SO4 (0.96 g, 9.80 mmol). The reaction mixture was reacted at 0 ° C for 5 h. The mixture was concentrated to 50 mL. Water (200 mL) was added to the residue and extracted with EtOAc (200 mL * 3). After separation, the combined organic layer was washed with saturated Na2CO3 (200 mL * 3), dried over Na2SO4, filtered and the filtrate was concentrated under vacuum. The residue was purified by flash chromatography on a silica gel column (A-petroleum ether; B-EtOAc) to give 1-14b (18 g, 74.7% yield).

[0746] MS (ESI) m / z: 249.9 [M+H] +

[0747] Step 2

[0748] N-(5-chloro-2-(1-hydroxycyclobutyl)phenyl)acetamide (1-14c)

[0749] To a solution of 1-14b (10 g, 40.50 mmol) in THF (100 mL) was added butyl lithium (25.60 mL, 85.04 mmol, 2.5 M) at -78 ° C and stirred for 1.5 h. Cyclobutanone (3.12 g, 44.55 mmol) was then slowly added. The reaction mixture was reacted at the same temperature for 1 h and warmed to rt. The mixture was quenched with saturated NH4Cl and extracted with EtOAc (100 mL * 3). After separation, the combined organic layers were washed with brine (50 mL * 3), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum. The residue was recrystallized with EtOH to give 1-14c (2.40 g, 24.8% yield).

[0750] MS (ESI) m / z: 238.0 [MH] -

[0751] Step 3

[0752] N-(3-chloro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-14d)

[0753] Under nitrogen atmosphere, AgNO (284.22 mg, 1.67 mmol) and NaS O (5.65 g, 20.91 mmol) were added to a solution of 1-14c (2.00 g, 8.37 mmol) in 30 mL of CH Cl / H O (v / v=1 / 1). After stirring at 25° C. for 16 h, the reaction mixture was quenched with water, extracted with EtOAc (100 mL*3), washed with brine, dried over anhydrous Na SO , and concentrated. Column chromatography on silica gel (EtOAc / petroleum ether=1 / 5) gave 1-14d (1.70 g, 85.7% yield) as a white solid.

[0754] MS (ESI) m / z: 238.0 [M+H] +

[0755] Step 4

[0756] (Z)-N-(3-chloro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-14e)

[0757] To a solution of 1-14d (1.00 g, 4.22 mmol) in 25 mL THF / t-BuOH (v: v = 4: 1) was added potassium tert-butoxide (1 M in THF, 615.34 mg, 5.48 mmol) at 0 ° C. The atmosphere was purged with N2 and the mixture was cooled to 0 ° C using an ice-water bath. After 5 min, isoamyl nitrite (592.99 mg, 5.06 mmol) was added to the stirred mixture, causing the color to change from yellow to red, and the reaction mixture was heated to rt, 1N HCl was added until the red color faded, and the mixture was transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (50 mL * 3). The combined organic matter was washed with H2O (50 mL), brine (100 mL), dried over Na2SO4 and concentrated in vacuo to generate a crude material. The crude residue was triturated with MTBE (10 mL) to obtain 1-14e (820.00 mg, 73% yield).

[0758] MS (ESI) m / z: 264.9 [MH] -

[0759] Step 5

[0760] N,N'-(3-chloro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-14f)

[0761] Wet PtO2 (100 mg) is added to a mixture of 1-14e (720.00 mg, 2.71 mmol) in Ac2O (20 mL). The reaction mixture is purged with H2 balloon three times and reacted for 6 h under H2 balloon at rt. The mixture is filtered out through diatomaceous earth and washed with Ac2O. The filtrate is concentrated under vacuum and purified by silica gel column chromatography (CH2Cl2: MeOH) to provide 1-14f (470.00 mg, 59.1% yield) as a gray solid.

[0762] MS (ESI) m / z: 295.1 [M+H] +

[0763] Step 6

[0764] N-(8-Amino-6-chloro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-14g)

[0765] 2N HCl (20 mL) was added to a solution of 1-14f (0.80 g, 2.72 mmol) in MeOH (20 mL). The reaction mixture was purged with N2 three times and reacted at 60 ° C under N2 for 5 h. The mixture was concentrated and purified by silica gel column chromatography (CH2Cl2: MeOH) to provide 1-14g (500 mg, 90.25% yield) as a gray solid.

[0766] MS (ESI) m / z: 253.1 [M+H] +

[0767] Step 7

[0768] N-((9S)-5-Chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-14h)

[0769] A solution of 1-14 g (500 mg, 2.45 mmol), 1-11 (787.24 mg, 2.99 mmol) and a catalytic amount of PPTS (200 mg, 0.80 mmol) in toluene (40 mL) was heated to reflux (135-140° C.) for 48 h using a Dean-Stark trap. The reaction was concentrated under vacuum and purified by silica gel column chromatography (CH 2 Cl 2 :MeOH) to provide 1-14 h (850 mg, 70.98% yield) as a gray solid. UPLC analysis: 1-14 h, retention time = 2.66 min; retention time = 2.77 min (mobile phase A: water with 0.1% FA, B: MeCN; gradient: 10% B for 1 min, 10%-95% B for 5 min, 95% B for 1 min; flow rate: 0.6 mL / min; column: ACQUITY BEH C18 1.7μm)

[0770] MS (ESI) m / z: 480.3 [M+H] +

[0771] Step 8

[0772] (1S,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-14)

[0773] (1R,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-15)

[0774] A solution of 1-14h (0.40 g, 0.83 mmol) in 12N HCl (30 mL) was added. The reaction mixture was purged with N2 three times and reacted at 100 ° C under N2 for 9 h. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give 1-14 (75.60 mg, 20.72% yield) (MS (ESI) m / z: 438.2 [M+H] + ) and 1-15 (76.90 mg, 21.7% yield) (MS (ESI) m / z: 438.2 [M+H] +), obtained as a white solid. [Retention time: 1.98 min and 2.67 min]

[0775] 1 H NMR (400MHz, DMSO-d6) δ8.15-8.05(m,1H),7.60(s,1H),7.31(s,1H),6.52(s,1H),5.76(d,J=19.4Hz,1H),5.47 -5.33(m,3H),4.77(s,1H),3.14(d,J=17.2Hz,2H),2.30-2.09(m,2H),1.91-1.77(m,2H),0.85(t,J=7.3Hz,3H).

[0776] 1 H NMR (400MHz, DMSO-d6) δ8.14(s,1H),8.00(d,J=2.0Hz,1H),7.51(d,J=1.8Hz,1H),7.29(s,1H),6.50(s,1H),5.63(d,J=19.4Hz,1H),5.48-5.2 4(m,3H),4.48(t,J=4.7Hz,1H),3.28(dd,J=10.7,6.1Hz,1H),3.12-3.04(m,1H),2.19-1.94(m,2H),1.91-1.71(m,2H),0.85(t,J=7.3Hz,3H).

[0777] Examples 1-16 and 1-17

[0778]

[0779] Step 1

[0780] 2,2-Difluoro-6-iodobenzo[d][1,3]dioxol-5-amine (1-16b)

[0781] To a solution of 1-16a (10.0 g, 57.76 mmol) in DMF (200 mL) was added NIS (14.30 g, 63.54 mmol) in portions on an ice bath and then stirred overnight at rt. The mixture was quenched by adding saturated Na2SO3 (50 mL), diluted with EA (500 mL), washed with brine (300 mL * 3) and dried over Na2SO4, filtered and concentrated under vacuum to give a crude product, which was purified by silica gel column chromatography (EA / PE = 0% to 20%). The fractions were concentrated under vacuum to give 1-16b (14.0 g, 81.1% yield) as a brown oil.

[0782] MS (ESI) m / z: 300.0 [M+H] +

[0783] 1 HNMR(400MHz,DMSO-d6)δ7.60(s,1H),6.78(s,1H),5.34(s,3H).

[0784] Step 2

[0785] N-(2,2-Difluoro-6-iodobenzo[d][1,3]dioxol-5-yl)acetamide (1-16c)

[0786] To a solution of 1-16b (14.0 g, 46.82 mmol) in CH2Cl2 (150 mL) was added acetic anhydride (4.8 mL, 51.5 mmol) and DMAP (114.4 mg, 0.94 mmol). The mixture was stirred at rt for 1 h. The suspension was filtered and the filter cake was washed with CH2Cl2 (30 mL), and the filter cake was dried under vacuum to produce 1-16c (9.4 g) as a white solid. The mother liquor was purified by silica column chromatography (60 g, EtOAc / PE=10% to 40%) to produce 1-16c (4.0 g) as a white solid. Total yield (97.3%)

[0787] 1 H NMR (400MHz, CDCl3) δ8.07(s,1H),7.45(s,1H),7.37(s,1H),2.24(s,3H).

[0788] Step 3

[0789] N-(2,2-Difluoro-6-(1-hydroxycyclobutyl)benzo[d][1,3]dioxol-5-yl)acetamide (1-16d)

[0790] To a mixture of 1-16c (10.0 g, 29.32 mmol) in THF (200 mL) was added dropwise i-PrMgCl (2 M, 36.7 mL, 73.3 mmol) for 1 h at -40 ° C, followed by the slow addition of cyclobutanone (6.17 g, 87.96 mmol) at -40 ° C. The resulting mixture was stirred for 2 h at rt. The mixture was poured into saturated NH4Cl (300 mL) and extracted with EtOAc (300 mL). The organic layer was washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under vacuum to generate a residue. The residue was purified by column chromatography (SiO2, EtOAc / PE=0%~50%) to generate 1-16d (9.5 g, crude material) as a white solid.

[0791] MS (ESI) m / z: 284.1 [MH] -

[0792] Step 4

[0793] N-(2,2-Difluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-16e)

[0794] To 1-16d (10.0g, crude material) in CH2Cl2 / H2O (1:1, 200mL) mixture, K2S2O8 (28.4g, 105.2mmol) and 0.5M AgNO3 aqueous solution (14mL, 7.0mmol) are added by syringe. The mixture is stirred overnight at rt in the dark. The mixture is filtered through a diatomaceous earth pad and then extracted with CH2Cl2 (300mL*2). The combined organic layer is washed with salt water (200mL), dried over Na2SO4, filtered and concentrated under vacuum to generate a red oil. The oil is purified by silica column (SiO2, EtOAc / PE=0%~10%) to generate 1-16e (1.45g, 100% purity) as a light yellow solid.

[0795] MS (ESI) m / z: 284.2 [M+H] +

[0796] 1 H NMR (400MHz, CDCl3) δ12.42(s,1H),8.58(s,1H),7.28(s,1H),2.96(t,J=6.2Hz,2H),2.75-2.70(m,2H),2.25(s,3H),2.16-2.09(m,3H).

[0797] Step 5

[0798] (Z)-N-(2,2-difluoro-7-(hydroxyimino)-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-16f)

[0799] To a solution of 1-16e (1.45 g, 5.12 mmol) in THF (20 mL) was added isoamyl nitrite (0.75 mL, 7.17 mmol) and t-BuOK (1 M, 6.7 mL, 6.7 mmol) at 0°C via syringe over 10 min. The mixture was stirred at 0°C for 30 min. The mixture was quenched by the addition of water (30 mL) and extracted with EtOAc (40 mL*3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by silica column (SiO2, EtOAc / PE=0% to 50%) to give 1-16f (1.04 g, 65.1% yield) as a light yellow solid.

[0800] MS (ESI) m / z: 313.2 [M+H] +

[0801] Step 6

[0802] N,N'-(2,2-difluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxole-5,7-diyl)diethylamide (1-16g)

[0803] To a solution of 1-16f (1.04 g, 3.33 mmol) in THF / AcO (3:1, 12 mL) was added PtO (75.6 mg, 0.33 mmol). The mixture was purged three times with a H balloon. The mixture was then stirred overnight at RT. The mixture was concentrated under vacuum to yield 1-16g (1300 mg, crude) as a black solid.

[0804] MS (ESI) m / z: 341.2 [M+H] +

[0805] Step 7

[0806] N-(5-amino-2,2-difluoro-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)acetamide (1-16h)

[0807] The mixture of 1-16g (1.3g crude material) in 2N HCl / MeOH (1:3, 12mL) was stirred at 60°C for 7h. The mixture was alkalized to pH=8 with saturated NaCO, then diluted with EtOAc (50mL*4). The combined organic layer was washed with brine (50mL), dried over NaSO, filtered and the filtrate was concentrated under vacuum to generate a residue. The residue was purified by silica gel chromatography (10g, MeOH / CHCl=0%~20%) to generate 1-16h (660mg) as a brown solid.

[0808] MS (ESI) m / z: 299.2 [M+H] + ;

[0809] Step 8

[0810] N-((10S)-10-ethyl-5,5-difluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-16i)

[0811] To a mixture of 1-16h (660 mg, 2.21 mmol) and 1-1i (582.5 mg, 2.21 mmol) in toluene (30 mL) was added o-cresol (2 mL) and PPTS (166.2 mg, 0.664 mmol). The mixture was heated to reflux for 48 h and water was removed with a Dean-Stark water trap. The mixture was concentrated under vacuum and the crude product was purified by silica gel column chromatography (MeOH / CH2Cl2=0% to 10%), and the fractions were concentrated under vacuum to generate 1-16i (610 mg, 55.4% yield) as a gray solid.

[0812] MS (ESI) m / z: 526.4 [M+H] + ;

[0813] Step 9

[0814] (1S,10S)-1-Amino-10-ethyl-5,5-difluoro-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione 2,2,2-trifluoroacetate (1-16) (1S,10S)-1-amino-10-ethyl-5,5-difluoro-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione 2,2,2-trifluoroacetate (1-17)

[0815] A mixture of 1-16i (610 mg, 1.225 mmol) in H2O / MsOH (3:1, 12 mL) was heated to 95°C and stirred for 8 h. The mixture was centrifuged (4000 r / min, 3 min), the liquid was purified by preparative HPLC (0.05% TFA), and the fractions were concentrated under vacuum to yield 1-16 (107 mg, RT = 1.79 min) as a dark yellow solid and 1-17 (123.3 mg, RT = 1.89 min) as a yellow solid.

[0816] MS (ESI) m / z: 484.3 [M+H] + ;

[0817] Examples 1-18 and 1-19

[0818]

[0819]

[0820] Step 1

[0821] N-(2-Bromo-5-methoxyphenyl)acetamide (1-18b)

[0822] To a solution of 9.8 mL of AcO and 0.05 mL of concentrated HSO was added 1-18a (20 g, 99.0 mmol) in an ice-water bath in several batches. The reaction was then slowly brought to room temperature and stirred for 1 h. The mixture was poured into ice water. The precipitate was filtered and washed three times with water, and the solid was then dried at 70 ° C to give compound 1-18b (20.1 g, 93% yield) as a brown solid.

[0823] MS (ESI) m / z: 244.1 [M+H] +

[0824] Step 2

[0825] N-(2-(1-Hydroxycyclobutyl)-5-methoxyphenyl)acetamide (1-18c)

[0826] To a solution of 1-18b (12 g, 49.4 mmol, 1.0 equiv) in 120 mL of anhydrous THF was slowly added n-BuLi (47.4 mL, 118.5 mmol, 2.4 equiv) at -78°C under N2. The mixture was stirred at -78°C for 1.5 h, followed by the slow addition of cyclobutanone (8.9 mL, 118.5 mmol, 2.4 equiv) at -78°C. The mixture was stirred at -78°C for 0.5 h, then slowly warmed to rt and stirred at rt for 0.5 h. The reaction was quenched with saturated NH4Cl and extracted with EtOAc (150 mL*3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to yield a residue that was triturated with MTBE (40 mL) to afford 1-18c (5.3 g, 46% yield) as a light-colored solid.

[0827] MS (ESI) m / z: 218.1 [M+H-H2O] +

[0828] Step 3

[0829] N-(3-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-18d)

[0830] I-18c (3.2 g, 13.6 mmol, 1.0 equiv), AgNO (5.44 mL, 2.72 mmol, 0.2 equiv), and KSO (7.36 g, 27.2 mmol, 2.0 equiv) were placed in a flask and evacuated / flushed three times with nitrogen. CHCl / HO (1 / 1 v / v, 190 mL) was added to the mixture and then stirred at rt overnight until the starting material was consumed as determined by TLC. The mixture was extracted with CHCl (3 x 80 mL). The combined organic layers were washed with brine, dried over NaSO, filtered, concentrated, and purified by flash chromatography on silica gel (ethyl acetate / PE) to yield the product I-18d (2.03 g, 64% yield).

[0831] MS (ESI) m / z: 234.2 [M+H] +

[0832] Step 4

[0833] (Z)-N-(7-(Hydroxyimino)-3-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-18e)

[0834] To a solution of 40 mL of anhydrous THF and 10 mL of t-BuOH under N₂ was added t-BuOK in THF (11.3 mL, 11.3 mmol, 1.3 equiv). 1-18d (2.03 g, 8.71 mmol, 1.0 equiv) was slowly added to the mixture in an ice-water bath and stirred at 0°C for 10 min. Isoamyl nitrite (1.64 mL, 12.2 mmol, 1.4 equiv) was added dropwise to the reaction at 0°C and then stirred at rt for 1 h. The mixture was adjusted to pH <6 with 2N HCl and then extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to yield a residue that was triturated with MTBE (20 mL) to afford 1-18e (1.94 g, 85% yield) as a brown solid.

[0835] MS (ESI) m / z: 261.0 [MH] -

[0836] Step 5

[0837] N,N'-(3-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-18f)

[0838] To 1-18e (2.0g, 7.57mmol) in 90mL AcOH and 30mL Ac2O in solution add wet Pd / C (300mg).Then use H2 balloon, the mixture is purged 3 times and stirred at rt overnight.The mixture is filtered through a celite pad and concentrated to generate compound crude material 1-18f. The crude product is wet-ground with MTBE (15mL) to obtain 1-18f (1.87g, 85% yield) as a light solid.

[0839] MS (ESI) m / z: 291.1 [M+H] +

[0840] Step 6

[0841] N-(8-amino-6-methoxy-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-18g)

[0842] To a solution of 30 mL of MeOH and 30 mL of 2N HCl was added 1-18f (990 mg, 3.41 mmol) under N2. The mixture was stirred at 60°C for 2 h. The reaction was then neutralized with saturated NaHCO3, filtered, washed with H2O, and dried to yield 1-18g (734 mg, 87% yield) as a light solid.

[0843] MS (ESI) m / z: 249.1 [M+H] +

[0844] Step 7

[0845] N-((9S)-9-Ethyl-9-hydroxy-5-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-18h)

[0846] PPTS (534.8 mg, 2.13 mmol, 0.6 equiv) and 1-1i (1.02 g, 3.9 mmol, 1.1 equiv) were added to a suspension of 1-18g (880 mg, 3.55 mmol, 1.0 equiv) in 70 mL of toluene, and the mixture was refluxed through a water separator for 48 h. The mixture was concentrated and purified by flash chromatography on silica gel (CHCl / MeOH) to give the product 1-18h (560 mg, 33% yield) as a light solid.

[0847] MS (ESI) m / z: 476.3 [M+H] +

[0848] Step 8

[0849] N-((9S)-9-ethyl-5,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-18, 1-19)

[0850] Compound 1-18h (325 mg, 0.68 mmol) was added to 4 mL of MsOH and 9 mL of H2O in an ice-water bath and stirred at 90°C for 10 h under N2. The solution was then purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to generate 1-18 (101 mg, 34% yield) and 1-19 (100 mg, 34% yield) as yellow solids.

[0851] 1-18, retention time (1.12 min)

[0852] MS (ESI) m / z: 434.3 [M+H] +

[0853] 1 H NMR (400MHz, DMSO-d6) δ8.43 (s, 3H), 7.52 (d, J = 2.5Hz, 1H), 7.35 (s, 1H), 7 .31(s,1H),6.55(s,1H),5.70(d,J=19.3Hz,1H),5.46(s,2H),5.38(d,J=19 .2Hz,1H),5.08(s,1H),3.97(s,3H),3.33-3.17(m,1H),2.45-2.42(m,1H) ,2.29(s,2H),2.24-2.12(m,1H),1.92-1.85(m,2H),0.89(t,J=7.3Hz,3H).

[0854] 1-19, retention time (2.19 min)

[0855] MS (ESI) m / z: 434.3 [M+H] +

[0856] 1 H NMR (400MHz, DMSO-d6) δ8.14(s,1H),7.43(d,J=2.5Hz,1H),7.31(s,1H),7.18(d,J=2.1Hz,1H),6.51(s,1H),5.63(d,J=19.3Hz,1H),5.44 (s,2H),5.35(d,J=19.2Hz,1H),4.57(s,1H),3.94(s,3H),3.14-3.00(m,1H),2.22-2.02(m,2H),1.86-1.84(m,2H),0.88(t,J=7.3Hz,3H).

[0857] Examples 1-20 and 1-21

[0858]

[0859] Step 1 to Step 8

[0860] 2,2,2-Trifluoroacetaldehyde--(S)-6-((S)-4-amino-7,8-difluoro-5,6-dihydro-4H-benzo[de]quinolin-2-yl)-4-ethyl-4-hydroxy-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione (1 / 1) trifluoroacetate (1-20)

[0861] 2,2,2-Trifluoroacetaldehyde-(1R,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1 / 1) trifluoroacetate (1-21)

[0862] 1-20 (17.5 mg, 19% yield) and 1-21 (15.7 mg, 17% yield) were synthesized according to the synthetic procedures of Examples 1-18 and 1-19.

[0863] 1-20

[0864] MS (ESI) m / z: 440.3 [M+H] + , retention time (1.30min).

[0865] 1 H NMR(400MHz, DMSO-d6)δ8.46(s,3H),8.20(dd,J=11.1,7.8Hz,1H),7.37(s,1H),6.57(s,1H),5.74(d,J=19.4Hz,1H ),5.46-5.41(m,3H),5.15(s,1H),3.17-3.02(m,2H),2.32-2.08(m,2H),1.92-1.85(m,2H),0.88(t,J=7.2Hz,3H).

[0866] 19 F NMR (377MHz, DMSO-d6) δ -73.51 (s), -131.03 (d, J = 22.6Hz), -138.00 (d, J = 22.5Hz).

[0867] 1-21

[0868] MS (ESI) m / z: 440.3 [M+H]+ , retention time (2.36min).

[0869] 1 H NMR (400MHz, DMSO-d6) δ8.49(s,3H),8.20(dd,J=11.2,7.8Hz,1H),7.37(s,1H),6.56(s,1H),5.74(d,J=19.4Hz,1H ),5.44-5.42(m,3H),5.17(s,1H),3.23-3.01(m,2H),2.19-2.16(m,2H),2.02-1.71(m,2H),0.88(t,J=7.3Hz,3H).

[0870] 19 F NMR (377MHz, DMSO-d6) δ -73.63 (s), -131.03 (d, J = 22.5Hz), -138.00 (d, J = 22.6Hz).

[0871] Examples 1-22 and 1-23

[0872]

[0873] Step 1 to Step 8

[0874] (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (1-22)

[0875] (1R,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-23)

[0876] 1-22 (19 mg, 17% yield) and 1-23 (14.9 mg, 13% yield) were synthesized according to the synthetic procedures of Examples 1-18 and 1-19.

[0877] MS (ESI) m / z: 422.2 [M+H] +

[0878] Examples 1-24 and 1-25

[0879]

[0880] Step 1

[0881] (1S,9S)-1-Amino-9-ethyl-5,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (1-24)

[0882] (1R,9S)-1-Amino-9-ethyl-5,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(1-25)

[0883] A 48% HBr aqueous solution (10 ml) containing compound 1-18h (250 mg) was heated to reflux for 24 h. The mixture was purified by preparative HPLC (TFA) (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to generate 1-24 (3.6 mg, 3% yield) and 1-25 (6.6 mg, 6% yield) as yellow solids.

[0884] MS (ESI) m / z: 420.3 [M+H] +

[0885] Examples 1-26 and 1-27

[0886]

[0887] Step 1 to Step 12

[0888] ((1S,9S)-1-Amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione 2,2,2-trifluoroacetate (1-26)

[0889] ((1R,9S)-1-Amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione 2,2,2-trifluoroacetate (1-27)

[0890] 1-26 (62 mg, 97% purity) was synthesized according to the reported procedure (US2020 / 0384121 A1). Retention time (1.27 min)

[0891] MS (ESI) m / z: 456.2 [M+H] +

[0892] 1H NMR (400MHz, DMSO-d6) δ8.45(s,3H),8.17(d,J=10.2Hz,1H),7.38(s,1H),5.74(d,J=19.4Hz,0H),5.46(dd,J=10.6,8.7Hz,1H),5.15(s,1H),3.43 (d,J=15.2Hz,1H),3.16(d,J=13.3Hz,1H),2.07-1.95(m,3H),1.88(td,J =14.4,7.1Hz,2H),1.52(d,J=7.0Hz,1H),1.46(s,3H),0.94-0.83(m,3H).

[0893] 1-27 (75 mg, 95% purity) was synthesized according to the reported procedure (US2020 / 0384121 A1). Retention time (1.33 min)

[0894] MS (ESI) m / z: 456.2 [M+H] +

[0895] Example 2-1

[0896]

[0897] Step 1 to Step 9

[0898] (1S,9S)-1-Amino-9-ethyl-9-hydroxy-4,5-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-1j)

[0899] 2-1j (23 mg, 97% purity) was synthesized according to a reported procedure (US2020 / 0384121 A1).

[0900] MS (ESI) m / z: 432.4 [M+H] +

[0901] 1H NMR(400MHz,d6-DMSO)δ8.14(s,0.4H),7.93(s,1H),7.32(s,1H),6.53(s,1 H),5.67(d,J=19.2Hz,1H),5.45(s,2H),5.40(d,J=19.2Hz,1H),4.95(s,1H ),3.26-3.22(m,1H),3.14-3.06(m,1H),2.54(s,3H),2.40(s,3H),2.30-2. 84(m,1H),2.19-2.13(m,1H),1.88(q,J=7.2Hz,2H),0.89(t,J=7.2Hz,3H).

[0902] Steps 10 and 11

[0903] N-((1S,9S)-9-ethyl-9-hydroxy-4,5-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-1)

[0904] 2-1 (7.3 mg, 96% purity, 60% yield) was synthesized according to the reported procedure (CN 112125915 A).

[0905] MS (ESI) m / z: 490.4 [M+H] +

[0906] 1 H NMR(400MHz,d6-DMSO)δ8.33(d,J=8.8Hz,1H),7.86(s,1H),7.28(s,1H),6.50( s,1H),5.58-5.53(m,1H),5.46(t,J=5.6Hz,1H),5.41(s,2H),5.22(d,J=19.2Hz ,1H),5.14(d,J=19.2Hz,1H),3.95(d,J=5.6Hz,2H),3.17-3.12(m,2H),2.51(s ,3H),2.37(s,3H),2.20-2.13(m,2H),1.92-1.83(m,2H),0.87(t,J=7.2Hz,3H).

[0907] Example 2-2

[0908]

[0909] Step 1

[0910] 2-(Methoxycarbonyl)cyclobutane-1-carboxylic acid (2-2b)

[0911] A solution of compound 2-2a (2000 mg, 15.86 mmol) in MeOH (20 mL) was refluxed for 4 h. The mixture was concentrated to give the crude product 2-2b (2.5 g, quantitative yield), which was used in the next step without further purification.

[0912] 1 H NMR (400MHz, DMSO-d6) δ12.2(s,1H),3.56(s,3H),3.71-3.35(m,2H),2.17-2.09(m,4H).

[0913] Step 2

[0914] 2-(Hydroxymethyl)cyclobutane-1-carboxylic acid (2-2c)

[0915] To a solution of compound 2-2b (200 mg, 1.26 mmol) in THF (4 mL) was added 1 M LiAlH 4 (1.90 mL, 1.90 mmol) at 0° C. The mixture was stirred at rt for 2 h. The mixture was quenched dropwise with H 2 O (72 mg) at 0° C. It was diluted with H 2 O (10 mL) and extracted with EtOAc (50 mL*3). The organic layers were combined and dried over anhydrous Na 2 SO 4 , filtered and concentrated to yield compound 2-2c (97 mg, crude material) as a colorless oil.

[0916] Step 3

[0917] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-(hydroxymethyl)cyclobutane-1-carboxamide (2-2)

[0918] To a solution of exatecan mesylate (purchased from ShangHai HaoYuan MedChemExpress CO.LTD, 50 mg, 0.094 mmol) in DMF (3 mL) was added compound 2-2c (24 mg, 0.18 mmol), HATU (72 mg, 18 mmol), and DIEA (49 mg, 0.38 mmol). The mixture was stirred at room temperature for 1 h. The mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm; Mobile Phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min). Fractions were lyophilized to yield compound 2-2 (1.6 mg, 3.0% yield) as a white solid.

[0919] MS (ESI) m / z: 548.4 [M+H] + .

[0920] Example 2-3

[0921]

[0922] Step 1

[0923] 3-Ethoxy-2,2-difluoro-3-oxopropanoic acid (2-3b)

[0924] To a solution of compound 2-3a (2000 mg, 10.2 mmol) in EtOH (10 mL) was added dropwise a solution of KOH (572 mg, 10.2 mmol) in H2O (100 uL) and EtOH (2 mL) at 0 ° C. The mixture was warmed to room temperature and stirred for 2 h. The mixture was diluted with H2O (20 mL) and washed with DCM (20 mL * 3). The aqueous solution was adjusted to pH = 3 with 1N HCl. It was then extracted with EA (50 mL * 3). The organic layers were combined and dried over anhydrous Na2SO4, filtered, and concentrated to produce compound 2-3b (825 mg, crude material) as a colorless oil, which was used in the next step without further purification.

[0925] Step 2

[0926] 2,2-Difluoro-3-hydroxypropionic acid (2-3c)

[0927] To a solution of compound 2-2b (800 mg, 4.76 mmol) in isopropanol (10 mL) was added 2M LiBH4 (4.76 mL, 9.54 mmol) at 0°C. The mixture was stirred at rt for 2 h. The mixture was quenched dropwise with 2N HCl (4.76 mL) at 0°C. It was then diluted with H2O (20 mL) and extracted with EtOAc (50 mL*3). The organic layers were combined and dried over anhydrous Na2SO4, filtered, and concentrated to yield compound 2-3c (417 mg, crude material) as a colorless oil.

[0928] Step 3

[0929] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-difluoro-3-hydroxypropionamide (2-3)

[0930] Compound 2-3 (8.4 mg, 16.4% yield) was synthesized according to the synthetic procedure of step 3 of Example 2-2.

[0931] MS (ESI) m / z: 544.3 [M+H] +

[0932] Examples 2-4

[0933]

[0934]

[0935] Step 1

[0936] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-4)

[0937] Compound 2-4 (4.2 mg, 49.5% yield) was synthesized according to the synthetic procedure of step 3 of Example 2-2.

[0938] MS (ESI) m / z: 452.2 [M+H] + . Retention time (2.55min).

[0939] Examples 2-5

[0940]

[0941] Step 1

[0942] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-5a)

[0943] To a turbid solution of exatecan mesylate (200 mg, 0.38 mmol) and glycolaldehyde dimer (50 mg, 0.41 mmol) in CH2Cl2 / MeOH (2 / 2 mL) was added NaHCO3 (32 mg, 0.38 mmol) and the mixture was stirred at rt overnight. NaBH3CN (35.8 mg, 0.56 mmol) was added and the solution was stirred for 10 h. The solution was added to a semi-saturated NH4Cl solution (6 ml) and filtered. The filtrate was extracted with CH2Cl2 / MeOH (5 / 1, 6 mL*3). The organic phase was concentrated and the product was purified by preparative HPLC (mobile phase A: water containing 0.1% TFA, B: CH3CN; gradient: 20%-25% B; flow rate: 20 mL / min; column: Xbridge PrepC18 OBD TM The desired fractions were lyophilized to give 2-5a formate salt as a light yellow solid (52 mg, 28.8% yield).

[0944] MS (ESI) m / z: 480.3 [M+H] +

[0945] 1 H NMR(400MHz,DMSO-d6)δ9.53(brs,1H),9.32(brs,1H),7.88(d,J=10.9Hz,1H) ,7.35(s,2H),6.55(s,1H),5.78(d,J=18.8Hz,1H),5.45(s,3H),5.31(s,1H),5 .11(s,1H),3.76(s,2H),3.31-3.15(m,4H),3.09-2.89(m,1H),2.88-2.74(m, 1H), 2.40 (s, 3H), 2.23-1.97 (m, 2H), 1.96-1.79 (m, 2H), 0.88 (t, J = 7.2Hz, 3H).

[0946] Step 2

[0947] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)(methyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(2-5)

[0948] To a solution of 2-5a (20 mg, 0.04 mmol) in CHCl / MeOH (1 / 0.1 mL) was added HOAc (1 μL, 0.021 mmol), 37% HCHO solution (5 μL, 0.063 mmol) and NaBHCN (4 mg, 0.063 mmol) and stirred at rt for 2 h. The reaction solution was added to half-saturated NHCl (5 mL) and extracted with CHCl / MeOH (5 / 1, 3 mL*3). The organic phase was concentrated and purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 20%-28% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 2-5 (4 mg, 19.4% yield) as a light yellow solid.

[0949] MS (ESI) m / z: 494.3 [M+H] +

[0950] 1 H NMR (400MHz, DMSO-d6) δ7.74 (d, J = 11.0Hz, 1H), 7.31 (s, 1H), 6.49 (s, 1H), 5. 53-5.35(m,4H),4.55(s,1H),4.49-4.37(m,1H),3.70-3.58(m,2H),3.01-2.8 6(m,1H),2.83-2.74(m,1H),2.72-2.62(m,2H),2.37(s,3H),2.35-2.27(m,1 H), 2.25 (s, 3H), 2.06-1.93 (m, 1H), 1.93-1.77 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[0951] Examples 2-6

[0952]

[0953] Step 1

[0954] 2-((tert-Butyldiphenylsilyl)oxy)ethyl(4-nitrophenyl)carbonate(2-6b)

[0955] To a solution of 2-6a (100 mg, 0.33 mmol) and bis(4-nitrophenyl)carbonate (123 mg, 0.40 mmol) in anhydrous CH2Cl2 (2 mL) was added DIEA (176 μL, 1.0 mmol) and DMAP (4.1 mg, 0.033 mmol) and stirred overnight at rt. The solution was poured into 1N HCl (2 mL) and extracted with CH2Cl2 (2 mL * 3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 5: 1) to generate compound 2-6b (148 mg, 95.5% yield) as a colorless oil.

[0956] 1 H NMR (400MHz, CDCl3) δ8.27 (d, J = 9.1 Hz, 2H), 7.69 (d, J = 6.5 Hz, 4H), 7.50-7.31 (m, 8H), 4.47-4.36 (m, 2H), 3.99-3.89 (m, 2H), 1.07 (s, 9H).

[0957] Step 2

[0958] 2-(tert-Butyldiphenylsilyl)oxy)ethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (2-6c)

[0959] To a solution of exatecan mesylate (50 mg, 0.094 mmol), 2-6b (53 mg, 0.11 mmol) and HOBt (1.3 mg, 0.009 mmol) in anhydrous DMF (1 mL) was added DIEA (50 μL, 0.28 mmol) and stirred overnight at rt. The solution was poured into saturated NH4Cl (5 mL) and extracted with EtOAc (5 mL*3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 1:3) to give compound 2-6c (68 mg, 94.9% yield) as a yellow solid.

[0960] MS (ESI) m / z: 762.4 [M+H] +

[0961] 1H NMR (400MHz, CDCl3) δ7.70-7.60(m,5H),7.59-7.52(m,1H),7.36-7.27(m,7H),5.68(d ,J=16.2Hz,1H),5.33(d,J=16.3Hz,1H),5.25-5.15(m,1H),5.09-4.96(m,2H),4.56-4. 44(m,1H),4.33-4.21(m,1H),4.04-3.89(m,2H),3.70(s,1H),3.17-2.99(m,2H),2.47- 2.34(m,4H),2.19-2.07(m,1H),2.05-1.88(m,2H),1.08(t,J=7.3Hz,3H),1.04(s,9H).

[0962] Step 3

[0963] 2-Hydroxyethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0964] To a solution of 2-6c (65 mg, 0.085 mmol) in anhydrous THF (2 mL) was added 1 M TBAF in THF (102 μL, 0.102 mmol) at 0°C and stirred at rt for 40 min. The solution was poured into saturated NH4Cl (5 mL) and extracted with CHCl / MeOH (5 / 1, 6 mL*3). The organic phase was concentrated and purified by flash column chromatography (silica gel, CHCl / MeOH=10:1) to give compound 2-6 (39 mg, 87.3% yield) as a light yellow solid.

[0965] MS (ESI) m / z: 524.4 [M+H] +

[0966] 1H NMR(400MHz,DMSO-d6)δ7.98(d,J=8.8Hz,1H),7.77(d,J=10.9Hz,1H),7.31 (s,1H),6.52(s,1H),5.43(s,2H),5.33-5.17(m,3H),4.78(t,J=5.4Hz,1H) ,4.19-4.01(m,2H),3.68-3.57(m,2H),3.30-3.20(m,1H),3.17-3.06(m,1H ), 2.38 (s, 3H), 2.26-2.07 (m, 2H), 1.95-1.78 (m, 2H), 0.87 (t, J = 7.4Hz, 3H).

[0967] Examples 2-7

[0968]

[0969] Step 1

[0970] N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-7)

[0971] To a solution of 2-hydroxyacetic acid (4.7 mg, 0.06 mmol) and HATU (20 mg, 0.05 mmol) in anhydrous DMF (0.4 mL) was added NMM (7 μL, 0.06 mmol) and stirred at rt for 5 min. The solution was added to a solution of 1-8 mesylate (22 mg, 0.04 mmol) and NMM (7 μL, 0.06 mmol) in anhydrous DMF (0.6 mL) and stirred for 40 min. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 20%-30%-60% B; flow rate: 20 mL / min; column: Sunfire Prep C18 OBD TM The solution was purified by lyophilization of the desired fractions to give 2-7 (3.1 mg, 15.1% yield) as a beige solid.

[0972] MS (ESI) m / z: 510.3 [M+H] +

[0973] 1H NMR(400MHz,DMSO)δ8.40(d,J=8.8Hz,1H),8.16(s,1H),7.31(s,1H),6.53(s ,1H),5.63-5.54(m,1H),5.48(t,J=5.8Hz,1H),5.44-5.40(m,2H),5.24(d,J =19.1Hz,1H),5.18(d,J=19.0Hz,1H),3.95(d,J=5.8Hz,2H),3.25-3.12(m,2 H), 2.53 (s, 3H), 2.27-2.11 (m, 2H), 1.95-1.78 (m, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0974] Examples 2-8

[0975]

[0976]

[0977] Step 1

[0978] N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropionamide (2-8)

[0979] To a solution of 3-hydroxy-2,2-dimethylpropanoic acid (4.3 mg, 0.036 mmol) and HATU (12.7 mg, 0.033 mmol) in anhydrous DMF (0.5 mL) was added DIEA (7 μL, 0.041 mmol) and stirred at rt for 5 min. The solution was added to a solution of 1-8 mesylate (15 mg, 0.027 mmol) and DIEA (7 μL, 0.041 mmol) in anhydrous DMF (0.5 mL) and stirred for 30 min. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 35%-55% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 2-8 (10.1 mg, 66.8% yield) as an off-white solid.

[0980] MS (ESI) m / z: 552.3 [M+H] +

[0981] 1 H NMR(400MHz,DMSO-d6)δ7.99(d,J=8.4Hz,1H),7.31(s,1H),6.53(s,1H),5.60-5.50(m ,1H),5.42(s,2H),5.23(d,J=19.1Hz,1H),5.16(d,J=19.0Hz,1H),4.88(t,J=5.1Hz,1 H),3.45(dd,J=10.4,5.2Hz,1H),3.41-3.35(m,1H),3.22-3.11(m,2H),2.53(s,3H),2 .22-2.05(m,2H),1.93-1.78(m,2H),1.12(s,3H),1.10(s,3H),0.87(t,J=7.3Hz,3H).

[0982] Examples 2-9 and 2-10

[0983]

[0984] Step 1

[0985] 2-Hydroxycyclopentane-1-carboxylic acid methyl ester (2-9b)

[0986] A solution of compound 2-9a (3000 mg, 21.12 mmol) in MeOH (20 mL) was cooled to 0 ° C, followed by portion-wise addition of NaBH 4 (1043 mg, 27.46 mmol). The mixture was stirred at 0 ° C for 1 h. TLC (SiO 2, petroleum ether: EtOAc = 3: 1, v / v) showed that the reaction was complete. The reaction was quenched with 1N HCl. The pH was adjusted to 8 using saturated NaHCO 3 aqueous solution and extracted with EtOAc (35 mL * 3). The combined organic layer was dried over Na 2 SO 4 and concentrated to generate a residue, which was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 40 / 60) to obtain 2-9b (2 g, 65.7% yield) as a colorless oil.

[0987] 1H NMR (400MHz, DMSO-d6) δ4.89 (d, J = 4.9 Hz, 1H), 4.21-4.12 (m, 1H), 3.59 (s, 3H), 2.55 (dd, J = 14.8, 6.4Hz, 1H), 1.71-1.57 (m, 6H).

[0988] Step 2

[0989] 2-Hydroxycyclopentane-1-carboxylic acid (2-9c)

[0990] To a solution of compound 2-9b (500 mg, 3.52 mmol) in THF (10 mL) and H2O (3 mL) was added LiOH (0.731 g, 30.54 mmol) at 0°C. The mixture was stirred at rt for 16 h. TLC (SiO2, EtOAc) showed that the reaction was complete. 1N HCl was added dropwise to the mixture to adjust the pH to 2. The aqueous solution was extracted with MTBE (15 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated to yield the crude product, compound 2-9c (380 mg, crude material), as a colorless oil.

[0991] Step 3

[0992] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxycyclopentane-1-carboxamide (2-9) and N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxycyclopentane-1-carboxamide (2-10)

[0993] To a solution of compound 2-9c (50 mg, 0.38 mmol) in DMF (3 mL) was added exatecan mesylate (170 mg, 0.39 mmol), HATU (159 mg, 0.42 mmol) and DIEA (98 mg, 0.76 mmol). The mixture was stirred at room temperature for 0.5 h. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to produce 2-9 (15.6 mg, 7.5% yield) and 2-10 (30.8 mg, 14.8% yield) as a yellow solid.

[0994] MS (ESI) m / z: 548.2 [M+H] + .

[0995] UPLC analysis: 2-9, peak 1, retention time = 3.66 min; 2-10, peak 2, retention time = 4.00 min (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 15% B for 1 min, 15%-95% B for 9 min, 95% B for 2 min; flow rate: 0.6 mL / min; column: ACQUITY BEH C18 1.7 μm).

[0996] 1 H NMR (400MHz, DMSO-d6) δ8.48(d,J=8.8Hz,1H),7.77(d,J=10.9Hz,1H),7.30(s,1H),6.5 2(s,1H),5.56(dd,J=12.4,7.5Hz,1H),5.42(s,2H),5.14(d,J=6.5Hz,2H),4.84(s,1H) ,4.22(q,J=6.1Hz,1H),3.25-3.08(m,4H),2.39(s,3H),2.21-2.09(m,2H),2.02-1.92( m,2H),1.90-1.78(m,3H),1.76-1.59(m,3H),1.52-1.42(m,2H),0.86(d,J=7.4Hz,4H).

[0997] 1 H NMR (400MHz, DMSO-d6) δ8.44(d,J=8.7Hz,1H),7.79(d,J=11.0Hz,1H),7.32(s,1H),6.52(s,1 H),5.62-5.52(m,1H),5.43(s,2H),5.24(s,2H),4.78(d,J=4.9Hz,1H),4.29-4.16(m,1H),3.1 7(d,J=5.2Hz,2H),2.46(dd,J=8.5,6.0Hz,1H),2.40(s,3H),2.22-2.05(m,2H),1.85(td,J=13 .8, 6.8Hz, 4H), 1.67 (ddd, J=20.8, 14.4, 7.1Hz, 3H), 1.51-1.39 (m, 1H), 0.87 (t, J=7.3Hz, 3H).

[0998] Example 2-11

[0999]

[1000] Step 1

[1001] N-((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-11)

[1002] To a solution of 2-hydroxyacetic acid (4.47 mg, 0.06 mmol) and 1-10 (15 mg, 0.03 mmol) in DMF (3 mL) was added HATU (26.6 mg, 0.07 mmol) and DIEA (15.48 mg, 0.12 mmol). The resulting mixture was stirred at 20° C. for 1 h. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give 2-11 (3.3 mg, 19.8% yield) as a white solid.

[1003] MS (ESI) m / z: 506.2 [M+H] + .

[1004] Examples 2-12 and 2-13

[1005]

[1006] Step 1

[1007] N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-12)

[1008] To a solution of 2-hydroxyacetic acid (6.52 mg, 0.09 mmol) in DMF (3 mL) and 1-14 (25 mg, 0.06 mmol) was added HBTU (32.54 mg, 0.09 mmol) and DIEA (22.18 mg, 0.17 mmol) and allowed to react at rt for 1 h. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min). 2-12 (5.30 mg, 18.72% yield) was obtained as a white solid.

[1009] MS (ESI) m / z: 496.2 [M+H] +

[1010] 1 H NMR (400MHz, DMSO-d6) δ8.48(d,J=8.9Hz,1H),8.05(d,J=2.1Hz,1H),7.56(d,J=1.9Hz,1H),7.30(s,1H),6.51(s,1H),5.63-5.47(m,2H),5 .39(s,2H),5.15(q,J=19.2Hz,2H),3.94(d,J=5.2Hz,2H),3.24-3.07(m,2H),2.20-2.06(m,2H),1.93-1.74(m,2H),0.83(t,J=7.3Hz,3H).

[1011] Step 2

[1012] N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-13)

[1013] To a solution of 2-hydroxyacetic acid (5.22 mg, 0.07 mmol) in DMF (3 mL) and 1-15 (20 mg, 0.05 mmol) was added HBTU (26.03 mg, 0.07 mmol) and DIEA (17.74 mg, 0.14 mmol) and the reaction was allowed to proceed for 1 h at rt. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min). 2-13 (5.40 mg, 23.84% yield) was obtained as a white solid.

[1014] MS (ESI) m / z: 496.2 [M+H] +

[1015] 1H NMR (400MHz, DMSO-d6) δ8.50(d,J=9.0Hz,1H),8.04(d,J=2.1Hz,1H),7.55(d,J=1.8Hz,1H),7.30(s,1H),6.50(s,1H),5.57(dd,J=16.5,9.7Hz,2H),5 .39(d,J=2.2Hz,2H),5.14(dd,J=37.6,19.1Hz,2H),3.95(d,J=4.5Hz,2H) ,3.17(s,2H),2.25-2.06(m,2H),1.92-1.74(m,2H),0.84(t,J=7.3Hz,3H).

[1016] Examples 2-14

[1017]

[1018] Step 1

[1019] N-(3-Fluoro-7-(3-methoxypropyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-14c)

[1020] To a solution of LDA (1.40 mL, 2.81 mmol, 2M in THF) in THF (19 mL) was added 2-14a (300.00 mg, 1.28 mmol) at -78 ° C, and the mixture was stirred at the same temperature for 2 h, followed by dropwise addition of a solution of 2-14b (0.38 g, 1.91 mmol) in THF (1 mL). The reaction mixture was then slowly heated to 0 ° C and stirred continuously for 4 h. The reaction was subsequently quenched with saturated NH4Cl aqueous solution (50 mL), and the organic material was extracted with EtOAc (30 mL * 3). The combined organic layer was washed with brine (50 mL) and dried over MgSO4, and the combined extracts were then concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (silica gel, Hex: EtOAc = 97: 3) to generate 2-14c (80.00 mg, 20.41% yield) as a colorless oil.

[1021] MS (ESI) m / z: 308.2 [M+H] +

[1022] Step 2

[1023] 8-Amino-6-fluoro-2-(3-methoxypropyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-14d)

[1024] Compound 2-14d (69.06 mg, crude material) was synthesized according to the synthetic procedure of step 6 of Example 1-14.

[1025] MS (ESI) m / z: 266.2 [M+H] +

[1026] Step 3

[1027] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-methoxypropyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-14e)

[1028] Compound 2-14e (80.00 mg, 62.40% yield) was synthesized according to the synthetic procedure of step 7 of Example 1-14.

[1029] MS (ESI) m / z: 493.1 [M+H] +

[1030] Step 4

[1031] (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(2-14)

[1032] To a solution of 2-14e (70.00 mg, 0.14 mmol) in CH2Cl2 (20 mL) was added BBr3 (71.26 mg, 0.28 mmol) at -0 ° C, and the mixture was stirred at the same temperature for 1 h. The reaction mixture was then slowly heated to 25 ° C and stirred continuously for 3 h. The reaction was subsequently quenched with saturated NaHCO3 aqueous solution and the organic material was extracted three times with EtOAc (30 mL * 3). The combined organic layer was washed with brine (50 mL) and dried over MgSO4, and then the combined extracts were concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (silica gel, CH2Cl2: MeOH = 90: 10) to generate 2-14 (4.20 mg, 6.18% yield) as a gray solid.

[1033] MS (ESI) m / z: 479.4 [M+H] +

[1034] UPLC analysis: 2-14-1, peak 1, retention time = 4.49 min; 2-14-2, peak 2, retention time = 4.65 min (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 15% B for 1 min, 15%-95% B for 9 min, 95% B for 2 min; flow rate: 0.6 mL / min; column: ACQUITY BEH C18 1.7μm)

[1035] 2-14-1: 1 H NMR (400MHz, DMSO-d6) δ7.74(d,J=11.1Hz,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.36(d,J=18.7Hz,1H),5.22(d,J=18.7Hz,1H ),3.52-3.39(m,2H),3.18-2.97(m,2H),2.38(s,3H),2.34-2.25(m,2H),2.01-1.78(m,3H),1.78-1.51(m,4H),0.87(t,J=7.3Hz,3H).

[1036] 2-14-2: 1 H NMR (400MHz, DMSO-d6) δ7.74(d,J=11.1Hz,1H),7.30(s,1H),6.52(s,1H),5.43(s,2H),5.37(d,J=18.8Hz,1H),5.23(d,J=18.7Hz,1H ),3.51-3.40(m,2H),3.16-2.98(m,2H),2.38(s,3H),2.35-2.24(m,2H),1.99-1.81(m,3H),1.78-1.53(m,4H),0.87(t,J=7.3Hz,3H).

[1037] Example 2-15

[1038]

[1039] Step 1

[1040] (R)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-hydroxy-N,N,N-trimethyl-4-oxobutan-1-aminium(2-15)

[1041] To a solution of L-carnitine (30.52 mg, 0.19 mmol) in DMF (3 mL) and exatecan mesylate (50.00 mg, 0.09 mmol) was added HBTU (53.55 mg, 0.14 mmol) and DIEA (36.50 mg, 0.28 mmol) and the mixture was allowed to react for an additional 1 h at rt. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min). 2-15 (15.6 mg, 28.61% yield) was obtained as a white solid.

[1042] MS (ESI) m / z: 580.5 [M+H] +

[1043] Example 2-16

[1044]

[1045] Step 2

[1046] 4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-hydroxy-N,N,N-trimethyl-4-oxobutan-1-aminium(2-16)

[1047] Compound 2-16 (19.40 mg, 35.58% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-15.

[1048] MS (ESI) m / z: 579.5 [M+H] +

[1049] 1 H NMR(400MHz, DMSO-d6)δ9.02(dd,J=48.5,8.5Hz,1H),8.35(s,1H),7.80(dd,J=10.9,3.6Hz,1H),7.32(s,1H),6.60(s,1H),5.67-5.55(m,1H),5.42 (s,2H),5.37-5.13(m,2H),4.54(s,1H),3.15(d,J=8.7Hz,12H),2.46-2.3 6(m,5H),2.16(d,J=5.5Hz,2H),1.95-1.78(m,2H),0.87(t,J=7.3Hz,3H).

[1050] Example 2-17

[1051]

[1052] Step 1

[1053] (2S,4R)-1-Acetyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxypyrrolidine-2-carboxamide (2-17)

[1054] Compound 2-17 (19.00 mg, 42.75% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-15.

[1055] MS (ESI) m / z: 591.4 [M+H] +

[1056] Example 2-18

[1057]

[1058] Step 25

[1059] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (2-18)

[1060] Compound 2-18 (7.40 mg, 25.14% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-15.

[1061] MS (ESI) m / z: 522.4 [M+H] +

[1062] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.6Hz,1H),7.80(d,J=11.0Hz,1H),7.31(s,1H),6.53(s,1H),5.56(d,J=8.2Hz,1H),5.42(s,2H),5.23(t,J=8. 4Hz,2H),4.66(s,1H),4.04(s,1H),3.17(s,2H),2.40(s,3H),2.33-2.08 (m,5H),1.92-1.80(m,2H),1.08(d,J=6.2Hz,3H),0.87(t,J=7.3Hz,3H).

[1063] Example 2-19

[1064]

[1065]

[1066] Step 1

[1067] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-5-(hydroxymethyl)furan-2-carboxamide (2-19)

[1068] Compound 2-19 (8.80 mg, 41.79% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-15.

[1069] MS (ESI) m / z: 560.3 [M+H] +

[1070] 1 H NMR (400MHz, DMSO-d6) δ8.92(d,J=8.7Hz,1H),7.80(d,J=11.0Hz,1H),7.31(s,1H),7.17(d,J=3.4Hz,1H),6.54-6.42(m,2H),5.73(d,J=8.4Hz,1H ),5.43-5.36(m,3H),5.16(d,J=16.8Hz,2H),4.44(d,J=5.6Hz,2H),3.18 (s, 2H), 2.24 (d, J = 6.0Hz, 2H), 1.88-1.82 (m, 2H), 0.86 (t, J = 7.2Hz, 3H).

[1071] Example 2-20

[1072]

[1073] Step 1

[1074] (R)-4-Cyano-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (2-20)

[1075] Compound 2-20 (24.2 mg, 47.07% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-15.

[1076] MS (ESI) m / z: 547.4 [M+H] +

[1077] 1 H NMR (400MHz, DMSO-d6) δ8.55(d,J=8.6Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H),6.54(s,1H),5.58-5.50(m,2H),5.42(s,2H),5.20(d,J=2.4 Hz,2H),4.20(d,J=5.6Hz,1H),3.17(s,2H),2.74-2.60(m,2H),2.44-2. 34(m,5H),2.25-2.06(m,2H),1.92-1.76(m,2H),0.87(t,J=7.2Hz,3H).

[1078] Example 2-21

[1079]

[1080] Step 1

[1081] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)acrylamide (2-21)

[1082] Compound 2-21 (4.00 mg, 16.88% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-15.

[1083] MS (ESI) m / z: 630.4 [M+H]+

[1084] 1 H NMR(400MHz,DMSO-d6)δ9.49(d,J=8.5Hz,1H),9.32(s,1H),7.80(d,J=10.9Hz,1H), 7.31(s,1H),6.94(d,J=214.5Hz,1H),6.54(s,1H),5.60(d,J=5.9Hz,1H),5.42(s,2 H),5.14(dd,J=41.5,18.9Hz,2H),3.16(s,2H),2.40(s,3H),2.23-2.12(m,2H),2.0 0(d,J=7.6Hz,1H),1.85(td,J=14.3,6.9Hz,2H),1.45(s,1H),0.86(d,J=7.2Hz,4H).

[1085] Example 2-22

[1086]

[1087] Step 1

[1088] (1S,9S)-1-Azido-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-22a)

[1089] To a solution of exatecan mesylate (200 mg, 0.38 mmol) in DMF (2 mL) was added 2 mL of MeOH containing Et3N (80 μ L, 57.6 mg, 0.57 mmol) and imidazole-1-sulfonyl azide hydrochloride (94.3 mg, 0.45 mmol). The reaction mixture was stirred at rt for 5 min, followed by addition of 2 mL of H2O containing K2CO3 (105.1 mg, 0.76 mmol) and a catalytic amount of CuSO4 (1 mg). The mixture was stirred at rt for 1 h. After the reaction was complete, the solvent was removed by evaporation. The crude brown solid was purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH=50 / 1 to 20 / 1) to give 2-22a (117.2 mg, 66.8% yield) as a pale white solid.

[1090] MS (ESI) m / z: 462.3 [M+H] +

[1091] 1H NMR (400MHz, DMSO-d6) δ7.81(d,J=10.9Hz,1H),7.44(s,1H),7.32(s,1H),6.52(s,1H),5.56(t,J=4.9Hz,1H),5.52-5.41 (m,3H),5.33(d,J=19.1Hz,1H),3.71(s,2H),3.17(s,2H),2.39(s,3H),2.32(m,2H),1.87(m,2H),0.88(t,J=7.3Hz,3H).

[1092] Step 2

[1093] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-22)

[1094] To a solution of 2-22a (20 mg, 0.04 mmol) in DMF (1 mL) was added propargyl alcohol (5 mg, 0.08 mmol), CuI (1 mg, catalytic amount) and DIPEA (35.1 μL, 25.86 mg, 0.2 mmol). The mixture was purged with N2 balloon for 15 min, then stirred at rt for 2 h. After the reaction was complete, the solvent was removed by evaporation. The crude product was purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH=10 / 1 to 5 / 1) to give 2-22 (10.2 mg, 49.3% yield) as a light yellow solid.

[1095] MS (ESI) m / z: 518.4 [M+H] +

[1096] 1 H NMR (400MHz, DMSO-d6) δ8.10(s,1H),7.88(d,J=10.9Hz,1H),7.31(s,1H),6.56(s,1H),5.38(s,2H),5.20(d,J=19.0Hz,1H),4.51 (s,2H),4.14(d,J=19.0Hz,1H),3.04(s,1H),2.75-2.66(m,1H),2.41(s,3H),1.84(tt,J=14.2,7.2Hz,2H),0.85(t,J=7.2Hz,3H).

[1097] Example 2-23

[1098]

[1099] Step 1

[1100] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione(2-23)

[1101] To a solution of 2-22a (20 mg, 0.043 mmol) in DMF (1 mL) was added 3-butyn-1-ol (3.4 mg, 0.048 mmol), CuI (1 mg, catalytic amount) and DIPEA (37.6 μL, 28 mg, 0.21 mmol). The mixture was purged with N2 balloon for 15 min, then stirred at rt for 2 h. After the reaction was complete, the solvent was removed by evaporation. The crude product was purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH=CH2Cl2 / MeOH=10 / 1 to 5 / 1) to give 2-23 (15.1 mg, 65.7% yield) as a light yellow solid.

[1102] 1 H NMR(400MHz,DMSO-d6)δ8.26(s,1H),8.10(d,J=10.8Hz,1H),7.54(s,1H),6.76(s,2H),5 .62(s,2H),5.32(d,J=19.0Hz,1H),4.91(t,J=5.4Hz,1H),4.25(d,J=19.1Hz,1H),3.87(d d,J=12.3,6.3Hz,2H),3.53-3.45(m,1H),3.34(dd,J=12.7,8.0Hz,1H),3.02(t,J=6.9Hz, 2H), 2.93 (dd, J=12.4, 5.8Hz, 1H), 2.65 (s, 3H), 2.15-2.00 (m, 2H), 1.09 (t, J=7.2Hz, 3H).

[1103] MS (ESI) m / z: 532.4 [M+H] +

[1104] Examples 2-24

[1105]

[1106] Step 1

[1107] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-24)

[1108] To a solution of 2-22a (30 mg, 0.065 mmol) in DMF (1 mL) was added pent-4-yn-1-ol (10.9 mg, 0.13 mmol), CuI (1 mg, catalytic amount) and DIPEA (56.8 μL, 42 mg, 0.33 mmol). The mixture was purged with N2 balloon for 15 min, then stirred at rt for 2 h under N2 atmosphere. After the reaction was complete, the solvent was removed by evaporation. The crude product was purified by silica gel column chromatography (eluent: CH2Cl2M / MeOH=CH2Cl2 / MeOH=10 / 1 to 5 / 1) to give 2-24 (15 mg, 42.8% yield) as a white solid.

[1109] 1 H NMR(400MHz,DMSO-d6)δ8.00(s,1H),7.86(d,J=10.9Hz,1H),7.30(s,1H),6.51(s,2H),5 .37(s,2H),5.09(d,J=19.0Hz,1H),4.45(t,J=5.1Hz,1H),4.00(d,J=19.0Hz,1H),3.43( dd,J=11.5,6.1Hz,2H),3.24(dd,J=11.6,6.2Hz,2H),3.09(dd,J=12.8,7.9Hz,1H),2.72 -2.61(m,3H),2.41(s,3H),1.91-1.79(m,2H),1.79-1.68(m,2H),0.85(t,J=7.3Hz,3H).

[1110] MS (ESI) m / z: 546.3 [M+H] +

[1111] Example 2-25

[1112]

[1113] Steps 1 and 2

[1114] 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-(2-hydroxyethyl)thiourea (2-25)

[1115] To a solution of 2-25a (65.9 mg, 0.38 mmol) in DMF (0.5 mL) was added TCDI (71.3 mg, 0.4 mmol). The mixture was stirred at rt for 30 min. 0.5 mL DMF containing exatecan mesylate (100 mg, 0.19 mmol) was subsequently added dropwise. After the addition was complete, the mixture was stirred at 50 ° C for 3 h, and LCMS showed that 2-25c was completely converted. The reaction solution was then cooled to 0 ° C, TBAF (0.45 mL, 0.45 mmol, 1 M in THF) was added and the reactants were warmed to rt and stirred at rt for 1 h. The organic solution was diluted with DCM (10 mL), then washed with saturated NaHCO (2 mL), H O (2 mL) and brine (2 mL). The organic phase was collected and dried over Na SO, filtered and the filtrate was concentrated under vacuum to generate the crude product 2-25 as a brown oil. The crude product was purified by silica gel column chromatography (eluent: CH 2 Cl 2 / MeOH=20 / 1 to 10 / 1) to give 2-25 (61 mg, 60.4% yield) as a brown solid.

[1116] 1 H NMR (400MHz, DMSO-d6) δ8.14(d,J=9.0Hz,1H),7.80(d,J=10.9Hz,1H),7.57(s,1H),7.31(s,1H),6.52(s,1H),6.21(s,1H),5. 42(s,2H),5.25(s,2H),4.84(s,1H),3.56(s,4H),3.18(s,2H),2.40(s,3H),2.23(m,2H),1.86(m,2H),0.87(t,J=7.3Hz,3H).

[1117] MS (ESI) m / z: 539.3 [M+H] +

[1118] Example 2-26

[1119]

[1120] Steps 1 and 2

[1121] 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-(2-hydroxyethyl)urea(2-26)

[1122] To a solution of exatecan mesylate (53.2 mg, 0.1 mmol) in DMF (1 mL) was added CDI (17 mg, 0.105 mmol). The mixture was stirred at rt for 30 min. 0.5 mL of DMF containing 2-25a (35 mg, 0.2 mmol) was subsequently added dropwise. After the addition was complete, the mixture was stirred at rt for 1.5 h. The reaction solution was then cooled to 0 ° C, TBAF (0.3 mL, 0.3 mmol, 1 M in THF) was added and the reactants were warmed to rt and stirred at rt for 1 h. The organic solution was diluted with CH2Cl2 (10 mL) and subsequently washed with saturated NaHCO3 (2 mL), H2O (2 mL) and brine (2 mL). The organic phase was collected and dried over Na2SO4, filtered and the filtrate was concentrated under vacuum to generate a crude product in the form of a yellow oil. The crude product was purified by silica gel column chromatography (eluent: CH 2 Cl 2 / MeOH=10 / 1 to 8 / 1) to give 2-26 (40 mg, 77% yield) as a brown solid.

[1123] MS (ESI) m / z: 523.4 [M+H] +

[1124] Example 2-27

[1125]

[1126] Step 1

[1127] N-((1S,10S)-10-ethyl-5,5-difluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-27)

[1128] To a mixture of glycolic acid (3.776 mg, 0.05 mmol) and 1-16 (12.0 mg, 0.025 mmol) in DMF (1 mL) was added HATU (18.88 mg, 0.050 mmol) and DIPEA (8 ul, 6.416 mg, 0.050 mmol). The mixture was stirred at RT for 30 min. The mixture was purified by preparative HPLC (FA, 0.1%) and the fractions were lyophilized to give 2-27 (6.8 mg, 100% purity, 50.6% yield) as a light yellow solid.

[1129] MS (ESI) m / z: 542.3 [M+H] + ;

[1130] 1 H NMR(400MHz,d6-DMSO)δ8.48(d,J=9.0Hz,1H),8.00(s,1H),7.31(s,1H),6. 53(s,1H),5.65(dd,J=14.0,6.0Hz,1H),5.49(t,J=5.8Hz,1H),5.42(s,2H), 5.21(s,2H),3.96(d,J=5.8Hz,2H),3.24-3.12(m,2H),2.28-2.09(m,2H),2. 00(dd,J=14.4,6.8Hz,1H),1.86(qd,J=14.0,7.2Hz,2H),0.91-0.84(m,3H).

[1131] Examples 2-28 and 2-29

[1132]

[1133] Step 1

[1134] N-((1S,9S)-9-ethyl-9-hydroxy-5-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-28)

[1135] To a solution of the reactants 2-hydroxyacetic acid (5.38 mg, 0.07 mmol), HBTU (26.85 mg, 0.07 mmol) and DIEA (18.3 mg, 0.14 mmol) in 2 ml of DMF was added 1 ml of DMF containing 1-18 (25 mg, 0.05 mmol). The mixture was stirred at rt for 30 min. After completion of the reaction, the mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) and the fractions were lyophilized to generate the product (11 mg, 48% yield) as a yellow solid.

[1136] MS (ESI) m / z: 492.3 [M+H] +

[1137] Step 2

[1138] N-((1R,9S)-9-ethyl-9-hydroxy-5-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-29)

[1139] To a solution of the reactants 2-hydroxyacetic acid (6.58 mg, 0.09 mmol), HBTU (32.84 mg, 0.09 mmol) and DIEA (22.38 mg, 0.17 mmol) in 2 ml of DMF was added 1 ml of DMF containing 1-19 (25 mg, 0.06 mmol). The mixture was stirred at rt for 30 min. As determined by LCMS, most of the SM remained, and the same equivalents of glycolic acid, HBTU and DIEA were added to the mixture. The mixture was stirred for another 3 h, and some SM remained. The reaction was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to produce the product as a yellow solid (5.7 mg, 20% yield).

[1140] MS (ESI) m / z: 492.3 [M+H] +

[1141] Example 2-30

[1142]

[1143]

[1144] Step 1

[1145] N-((1S,9S)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-30)

[1146] 2-30 (10.7 mg, 79% yield) was synthesized according to the synthetic procedure of Example 2-28.

[1147] MS (ESI) m / z: 498.3 [M+H] +

[1148] Example 2-31

[1149]

[1150] Step 1

[1151] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-31)

[1152] 2-31 (3.2 mg, 25% yield) was synthesized according to the synthetic procedure of Example 2-28.

[1153] MS (ESI) m / z: 480.3 [M+H] +

[1154] 1 H NMR(400MHz,d6-DMSO)δ8.52(d,J=9.2Hz,1H),7.78(dd,J=10.0Hz,J=2.4Hz,1H), 7.51(dd,J=8.8Hz,J2=2.4Hz,1H),7.33(s,1H),6.55(s,1H),5.65-5.62(m,1H),5. 60(s,1H),5.43(s,2H),5.21(d,J=18.8Hz,1H),5.15(d,J=18.8Hz,1H),3.97(s,2H ),3.28-3.17(m,2H),2.21-2.11(m,2H),1.92-1.81(m,2H),0.87(t,J=7.2Hz,3H).

[1155] Example 2-32

[1156]

[1157] Step 1

[1158] N-((1S,9S)-9-ethyl-5,9-dihydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (2-32)

[1159] 2-32 (3.5 mg, 24% yield) was synthesized according to the synthetic procedure of Example 2-28.

[1160] MS (ESI) m / z: 478.4 [M+H] +

[1161] Example 2-34

[1162]

[1163] N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropionamide (2-34)

[1164] 2-34 (5.8 mg, 99.82% purity, 67.9% yield) was synthesized according to a procedure similar to Example 2-29.

[1165] MS (ESI) m / z: 556.3 [M+H] +

[1166] Example 2-35

[1167]

[1168]

[1169] N-((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropionamide (2-35)

[1170] Compound 2-35 (1.8 mg, 17.9% yield) was synthesized according to the synthetic procedure of step 3 of Example 2-2.

[1171] MS (ESI) m / z: 548.4 [M+H] +

[1172] Example 2-36

[1173]

[1174] Step 1

[1175] (2S,4R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxy-1-methylpyrrolidine-2-carboxamide (2-36)

[1176] To a solution of 2-36a (25.0 mg, 0.17 mmol) in DMF (2.0 mL) was added exatecan mesylate (45.7 mg, 0.09 mmol), HTBU (48.9 mg, 0.13) and DIEA (27.8 mg, 0.22 mol) at room temperature. The resulting mixture was stirred for 6 h. The resulting white solid was purified by preparative HPLC to yield the desired product 2-36 (9.3 mg, 18% yield) as a yellow solid.

[1177] MS (ESI) m / z: 563.4 [M+H] +

[1178] 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),9.18(d,J=8.2Hz,1H),7.84(d,J=11.0Hz,1H),7.32(s ,1H),6.54(s,1H),5.73-5.63(m,1H),5.58(s,1H),5.42(s,2H),5.29(d,J=18.6Hz,1H),5.0 9(d,J=18.6Hz,1H),4.38(s,1H),4.22(s,1H),3.82-3.81(m,1H),3.22(s,2H),3.01-3.0(m, 1H), 2.91 (s, 3H), 2.43 (s, 3H), 2.33-1.98 (m, 5H), 1.94-1.75 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[1179] Example 2-37

[1180]

[1181] Step 1

[1182] (2S,4S)-4-Hydroxypyrrolidine-2-carboxylic acid (2-37b)

[1183] To a solution of 2-37a in 10 ml of CH Cl was added 10 ml of TFA in an ice-water bath. The mixture was allowed to reach rt and stirring was continued. After the reaction was complete, the mixture was concentrated to generate 600 mg of crude product, which was used directly in the next step.

[1184] Step 2

[1185] (2S,4S)-4-Hydroxy-1-methylpyrrolidine-2-carboxylic acid (2-37c)

[1186] To a solution of 95 mg of 2-37b in 1.7 ml of acetic acid and 1 ml of H2O was added 0.13 ml of 37% aqueous formaldehyde and 9.5 mg of PbO2. The mixture was stirred at rt. After completion of the reaction, the mixture was filtered and concentrated to yield 70 mg of crude product, which was used directly in the next step.

[1187] Step 3

[1188] (2S,4S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxy-1-methylpyrrolidine-2-carboxamide (2-37)

[1189] To a solution of crude material 2-37c (52 mg) in DMF (5.0 mL) was added exatecan mesylate (53.2 mg, 0.1 mmol), HTBU (56.9 mg, 0.15 mmol) and DIEA (47 ul, 0.30 mmol) at room temperature. The resulting mixture was stirred for 6 h. The mixture was purified by preparative HPLC to yield the desired product 2-37 (23 mg, 23% yield) as a yellow solid.

[1190] MS (ESI) m / z: 563.4 [M+H] +

[1191] Example 2-38

[1192]

[1193]

[1194] Step 1

[1195] (2S,4S)-1-Acetyl-4-hydroxypyrrolidine-2-carboxylic acid (2-38a)

[1196] To a solution of 32 mg of 2-37b in 1 ml of EA was added 24 μl of Ac 2 O. The mixture was sonicated at rt for 6 h. After completion of the reaction, the mixture was concentrated to yield 29 mg of the crude desired product, which was used directly in the next step.

[1197] Step 2

[1198] (2S,4S)-1-Acetyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxypyrrolidine-2-carboxamide (2-38)

[1199] 2-38 (7.3 mg, 19% yield) was synthesized according to the synthetic procedure of Example 2-37.

[1200] MS (ESI) m / z: 591.3 [M+H] +

[1201] Example 2-39

[1202]

[1203]

[1204] Step 1

[1205] To a solution of compound exatecan mesylate (50 mg, 0.094 mmol) in MeOH (2 mL) was added 3-hydroxypropionic acid (85 mg, 0.28 mmol, 30% in water), DMT-MM (78 mg, 0.28 mmol) and DIEA (36 mg, 0.28 mmol). The mixture was stirred at room temperature for 2 h. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD5um 19*150 mm; mobile phase: A-water (0.1% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min) to provide compound 2-39 (28.3 mg, 59.3% yield) as a white solid.

[1206] 1H NMR(400MHz,DMSO-d6)δ8.45(d,J=8.8Hz,1H),7.80(d,J=11.2Hz,1H),7.31(s ,1H),6.53(s,1H),5.65-5.51(m,1H),5.43(s,2H),5.29-5.09(m,2H),4.60(t ,J=5.2Hz,1H),3.67(dd,J=11.2,5.6Hz,1H),3.18(s,1H),2.40(s,2H),2.32( t,J=6.4Hz,1H),2.22-2.04(m,1H),1.95-1.71(m,1H),0.87(t,J=7.2Hz,2H);

[1207] MS (ESI) m / z: 508.3 [M+H] + ; UPLC-MS retention time: 3.28min.

[1208] Example 2-40

[1209]

[1210] Step 1

[1211] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropionamide (2-40)

[1212] To a mixture of 3-hydroxy-2,2-dimethylpropionic acid (5 mg, 0.042 mmol) and HATU (16 mg, 0.042 mmol) in DMF (1 mL) was added DIPEA (21 μL, 16 mg, 0.13 mmol) and exatecan mesylate (23 mg, 0.043 mmol). The resulting brown mixture was stirred at rt for 2 h. After completion of the reaction, the mixture was purified by preparative HPLC (TFA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to produce 2-40 (15 mg, 65.5% yield) as a white powder.

[1213] 1H NMR (400MHz, DMSO-d6): δ8.00(d,J=8.4Hz,1H),7.79(d,J=11.2Hz,1H),7.31(s,1H),6 .52(s,1H),5.59-5.54(m,1H),5.42(s,2H),5.18(q,J=19.2Hz,2H),4.87(t,J=5.2Hz, 1H),3.45(dd,J=10.2,4.8Hz,1H),3.41-3.28(m,1H),3.15(t,J=5.6Hz,2H),2.40(s,3 H), 2.24-2.07 (m, 2H), 1.92-1.80 (m, 2H), 1.11 (d, J = 7.6Hz, 6H), 0.87 (t, J = 7.2Hz, 3H).

[1214] MS (ESI) m / z: 536.4 [M+H] +

[1215] Example 2-41

[1216]

[1217] Step 1: Diethyl 2-fluoro-2-methylmalonate (2-41b)

[1218] A solution of compound 2-41a (10.0 g, 57.4 mmol) in THF (200 mL) was cooled to 0 ° C. Oil (3.21 g, 80.37 mmol) containing 60% NaH was added portionwise to the mixture and stirred at 0 ° C for 30 min. Then, N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (NSFI, 19.91 g, 63.2 mmol) was added portionwise to the mixture at 0 ° C. It was then heated to rt and stirred for 16 h. After the reaction was complete, the suspension was filtered and the filtrate was concentrated. PE (100 mL) was added to the residue, the precipitate was filtered and the filtrate was concentrated to generate compound 2-41b (12.5 g, thick material) as a light yellow oil.

[1219] 1 H NMR (400MHz, CDCl3) δ4.30 (q, J = 7.2Hz, 4H), 1.79 (d, J = 22.0Hz, 3H), 1.31 (t, J = 7.2Hz, 6H);

[1220] 19 F NMR (376MHz, CDCl3) δ-157.50.

[1221] Step 23-Ethoxy-2-fluoro-2-methyl-3-oxopropanoic acid (2-41c)

[1222] To a solution of compound 2-41b (1.0 g, 5.2 mmol) in EtOH (5 mL) was added dropwise a solution of KOH (321 mg) in H2O (50 uL) and EtOH (2 mL) at 0 ° C. The mixture was stirred at rt for 2 h. The mixture was diluted with 20 (mL) and washed with CH2Cl2 (20 mL * 3). The aqueous solution was adjusted to pH = 3 with 1N HCl. It was extracted with EtOAc (50 mL * 3). The organic layer was dried, combined and dried over anhydrous Na2SO4, filtered, and concentrated to generate compound 2-41c (470 mg, 55.0% yield) as a colorless oil.

[1223] 1 H NMR (400MHz, CDCl3) δ8.31 (br s, 1H), 4.32 (q, J = 7.2Hz, 2H), 1.83 (d, J = 22.0Hz, 3H), 1.33 (t, J = 7.2Hz, 3H);

[1224] 19 F NMR (376MHz, CDCl3) δ-157.59.

[1225] Step 3 2-Fluoro-3-hydroxy-2-methylpropionic acid (2-41d)

[1226] To a solution of compound 2-41c (200 mg, 1.22 mmol) in isopropanol (4 mL) was added 2M LiBH4 (1.22 mL, 2.44 mmol) at 0°C. The mixture was stirred at rt for 2 h. The mixture was quenched dropwise with 2N HCl (1.22 mL) at 0°C. Diluted with H2O (10 mL) and extracted with EtOAc (50 mL*3). The organic layers were combined and dried over anhydrous Na2SO4, filtered and concentrated to yield compound 2-41d (92 mg, 61.7% yield) as a colorless oil.

[1227] 1 H NMR (400MHz, CDCl3) δ4.01-3.81 (m, 2H), 1.58 (d, J = 21.2Hz, 3H);

[1228] 19 F NMR (376MHz, CDCl3) δ-163.98.

[1229] Step 4

[1230] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-fluoro-3-hydroxy-2-methylpropionamide (2-41)

[1231] To a solution of compound 2-41d (23 mg, 0.19 mmol) in DMF (2 mL) was added exatecan mesylate (50 mg, 0.094 mmol), HATU (54 mg, 141 mmol), and DIEA (36 mg, 0.28 mmol). The mixture was stirred at room temperature for 1 h. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) and fractions were lyophilized to yield 2-41 (11 mg, 21.9% yield) as a white solid.

[1232] UPLC-MS, RT=3.52 min.

[1233] 1 H NMR (400MHz, DMSO-d6) δ9.06 (dd, J=9.0, 2.8Hz, 1H), 8.00 (d, J=10.9Hz, 1H), 7.54 (s, 1H),6.75(s,1H),5.82(d,J=8.0Hz,1H),5.65(s,2H),5.43(dt,J=77.8,12.4Hz,3H),4 .17-3.91(m,1H),3.83(ddd,J=18.0,12.4,5.6Hz,1H),3.40-3.27(m,1H),2.62(s,3H) ,2.50-2.34(m,2H),2.22-1.98(m,2H),1.81(d,J=21.4Hz,3H),1.11(t,J=7.2Hz,3H);

[1234] MS (ESI) m / z: 540.3 [M+H] + .

[1235] Example 2-42

[1236]

[1237] Step 1

[1238] (9S)-1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-42a)

[1239] A mixture of PPh3 (49.3 mg, 0.188 mmol) and DIAD (38.0 mg, 0.188 mmol) in THF (1 mL) was stirred at rt for 5 min, then added to a mixture of compound 2-14 (60 mg, 0.125 mmol) and phthalimide (20.3 mg, 0.138 mmol) in THF (1 mL). The resulting yellow mixture was stirred at rt for 1 h. The reaction was quenched by adding water (10 mL) and extracted with EtOAc (10 mL*2). The combined organic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to generate a residue. The residue was purified by silica gel chromatography (SiO2 5 g, EtOAc / PE=20%~70%), and the fractions were concentrated under vacuum to generate a crude product (130 mg, crude material) as a yellow solid. Based on LCMS, it contained most of PPh3O.

[1240] MS (ESI) m / z: 608.5 [M+H] +

[1241] Step 2

[1242] (9S)-1-(3-Aminopropyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione formate (2-42)

[1243] To a solution of 2-42a (130 mg, crude material) in EtOH (2 mL) was added N2H4.H2O (80%, 20 uL). The mixture was stirred at 60°C for 60 min. The mixture was acidified to pH = 2 with 3N HCl. It was then purified by preparative HPLC (FA) and the fractions were lyophilized to yield the desired product 2-42 (12.2 mg, 99.68% purity) as a white solid.

[1244] MS (ESI) m / z: 478.4 [M+H] +

[1245] 1H NMR (400MHz, DMSO-d6) δ7.75(d,J=10.8Hz,1H),7.31(s,1H),5.44(s,2H),5.34(q,J=18.8Hz,3H),3.10(m ,1H),2.76(s,2H),2.38(s,3H),2.27(d,J=12.8Hz,2H),1.91(m,6H),1.62(m,3H),0.87(t,J=7.4Hz,4H).

[1246] Example 2-43

[1247]

[1248] Step 1

[1249] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropionamide (2-43)

[1250] Compound 2-43 (0.3 mg, 95.8% purity) was obtained according to the procedure described in step 1 of example 2-8.

[1251] MS (ESI) m / z: 522.4 [M+H] +

[1252] 1H NMR (400MHz, DMSO-d6) δ8.04(d,J=8.4Hz,1H),7.78(dd,J=10.3,2.5Hz,1H),7.51(d,J=9. 1Hz,1H),7.33(s,1H),6.52(s,1H),5.60(d,J=7.9Hz,1H),5.42(s,2H),5.24(d,J=19.2Hz, 1H),5.15(d,J=19.0Hz,1H),4.87(t,J=5.1Hz,1H),3.49-3.43(m,2H),3.24-3.12(m,2H), 2.13(d,J=5.8Hz,2H),1.94-1.79(m,2H),1.13(s,3H),1.11(s,3H),0.88(t,J=7.3Hz,3H).

[1253] Examples 2-44 and 2-45

[1254]

[1255] Step 1

[1256] (Z)-N-(3-Fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-44a)

[1257] To a solution of 2-14a (1 g, 4.25 mmol) in anhydrous THF (30 mL) was added ethyl formate (449 μL, 5.53 mmol) and a solution of KOt-Bu in THF (1 M, 10.6 mL, 10.6 mmol) at 0°C under a nitrogen atmosphere. The solution was allowed to warm to rt and stirred overnight. The pH of the solution was adjusted to 3 with 1N HCl, extracted with EtOAc (20 mL*3), dried over anhydrous Na2SO4, filtered, and concentrated to yield the crude product 2-44a (1.1 g, quantitative) as a yellow solid.

[1258] MS (ESI) m / z: 264.2 [M+H] +

[1259] Step 2

[1260] N-(7-diazo-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-44b)

[1261] To a cooled solution of 2-44a (1.1 g, 4.2 mmol) in anhydrous CHCl (30 mL) was added TEA (4.8 mL, 20.9 mmol) and N-(4-azidophenyl)acetamide (1.5 g, 6.3 mmol) at -10 ° C under a nitrogen atmosphere and stirred for 1 h. The solution was gradually brought to 0 ° C. 2N NaOH solution (10 mL) was added. The solution was extracted with CHCl (15 mL * 3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 3: 1) to generate compound 2-44b (700 mg, 98% purity, 64% yield) as a yellow solid.

[1262] MS (ESI) m / z: 262.2 [M+H] +

[1263] 1 H NMR (400MHz, CDCl3) δ12.34 (s, 1H), 8.42 (d, J = 12.8Hz, 1H), 3.01-2.95 (m, 2H), 2.94-2.89 (m, 2H), 2.21 (s, 3H), 2.17 (d, J = 1.9Hz, 3H).

[1264] Step 3

[1265] N-(7-(2-((tert-Butyldimethylsilyl)oxy)ethoxy)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-44d)

[1266] Under nitrogen atmosphere, BF was added to a mixture of 2-44b (824 mg, 3.15 mmol) and 2-44c (3.8 mL, 18.9 mmol). EtO (65 μL, 0.32 mmol) was accompanied by gas evolution. The solution was stirred at rt for 1 h until no gas evolution occurred. The solution was diluted with EtOAc (10 mL) and saturated NaHCO (10 mL), and extracted with EtOAc (10 mL*3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate=5:1) to generate compound 2-44d (328 mg, crude material) in a yellow oily state.

[1267] MS (ESI) m / z: 410.4 [M+H] +

[1268] Step 4

[1269] 8-Amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-44e)

[1270] To a solution of 2-44d (328 mg, 0.8 mmol) in MeOH (3 mL) was added 2N HCl (3 mL) under a nitrogen atmosphere and stirred at 60 ° C for 4 h. The pH of the solution was adjusted to 8 with saturated Na2CO3 and extracted with EtOAc (10 mL * 3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 1: 1) to generate compound 2-44e (80 mg, crude material) as a brown oil.

[1271] MS (ESI) m / z: 254.3 [M+H] +

[1272] Step 5

[1273] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-44 and 2-45)

[1274] To a solution of 2-44e (85 mg, 0.34 mmol) and 1-1i (99 mg, 0.37 mmol) in toluene (3 mL) was added PPTS (43 mg, 0.17 mmol) and refluxed for 36 h. The solution was concentrated and purified by flash column chromatography (silica gel, DCM / MeOH=20:1) to give a mixture of 16 mg, 2-44 / 2-45=1:1 as a yellow solid. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 20%-30%-45% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The mixture was purified by lyophilization of the desired fractions to yield 2-44 (0.53 mg, 95% purity, containing 5% 2-45) and 2-45 (0.91 mg, 83% purity, containing 17% 2-44).

[1275] MS (ESI) m / z: 481.4 [M+H] +

[1276] UPLC analysis: 2-44, retention time = 2.03 min; 2-45, retention time = 2.13 min (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 10% B for 0.5 min, 10%-90% B for 2.5 min, 90% B for 0.2 min; flow rate: 0.6 mL / min; column: ACQUITY BEH C18 1.7 μm).

[1277] Example 2-46

[1278]

[1279] Step 1

[1280] tert-Butyl(2-iodoethoxy)dimethylsilane(2-46b)

[1281] A solution of compound 2-46a (3000 mg, 17.44 mmol) in CHCl (40 mL) was cooled to 0°C, followed by portionwise addition of imidazole (1779 mg, 26.16 mmol) and TBSCl (3139 mg, 20.93 mmol) at 0°C. The mixture was stirred at 20°C for 16 h. TLC (SiO, petroleum ether) showed that the reaction was complete. The reactant was poured into water (40 mL) and extracted with CHCl (50 mL*3). The combined organic layers were dried over NaSO and concentrated to generate a residue, which was purified by silica gel column chromatography (eluent: petroleum ether = 100) to obtain 2-46b (3.5 g, 70.2% yield) as a colorless oil.

[1282] Step 2

[1283] N-(7-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (20-46c)

[1284] To a solution of compound 2-14a (2350 mg, 10 mmol) in THF (40 mL) was added tBuOK (1 M in THF) (22 mL, 22 mmol) dropwise at 0 ° C. The internal temperature was maintained at 0 ° C. The mixture was stirred at 0 ° C for 30 min, followed by the slow addition of 2-46b (3432 mg, 12 mmol). LCMS showed that the reaction was complete. 1N HCl was added dropwise to the mixture to adjust the pH to 2. The mixture was poured into a saturated NaHCO aqueous solution (50 mL) and extracted with EtOAc (60 mL * 3). The combined organic layer was dried over anhydrous Na SO , filtered and concentrated to generate a crude product, which was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc=100 / 0 to 83 / 17) to give 2-46c (700 mg, 17.8% yield) as a yellow solid.

[1285] MS (ESI) m / z: 394.2 [M+H] + .

[1286] 1H NMR (400MHz, DMSO-d6) δ = 7.40 (s, 2H), 6.34 (d, J = 12.6, 1H), 6.34 (d, J = 12.6 ,1H),4.45(t,J=5.2,1H),4.45(t,J=5.2,1H),3.65-3.35(m,2H),3.55-3.45 (m,2H),2.83(s,1H),2.83(s,1H),2.72-2.61(m,1H),2.46(d,J=5.0,1H),2. 11-1.99(m,3H),1.97(d,J=1.0,3H),1.72-1.60(m,1H),1.43(d,J=6.2,1H).

[1287] Step 38-Amino-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-46d)

[1288] To a solution of compound 2-46c (700 mg, 1.78 mmol) in MeOH (3 mL) was added 2N HCl (3 mL). The mixture was stirred at 60 ° C for 16 h. LCMS showed that the reaction was complete. The mixture was poured into a saturated NaHCO aqueous solution (20 mL) and extracted with EtOAc (30 mL * 3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated to generate a crude product, which was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc=100 / 0 to 76 / 24) to obtain 2-46d (200 mg, 47% yield) as a pale white solid.

[1289] MS (ESI) m / z: 238.2 [M+H] + .

[1290] Step 4

[1291] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-46)

[1292] To a solution of compound 2-46d (100 mg, 0.42 mmol) in toluene (6 mL) and o-cresol (0.35 mL) was added 1-1i (110 mg, 0.42 mmol) and PPTS (16 mg, 0.06 mmol). The mixture was refluxed at 140 ° C for 5 h. LCMS showed that the reaction was complete. The solvent was evaporated and the residue was purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH=100 / 0 to 92 / 8) to give 2-46 (80 mg, 41% yield) as an orange solid.

[1293] MS (ESI) m / z: 465.2 [M+H] + .

[1294] 1 H NMR (400MHz, DMSO-d6) δ = 7.74 (d, J = 11.0, 1H), 7.31 (s, 1H), 6.53 (d, J = 1.9, 1H), 5.44 (s, 2H), 5.30 (s, 2H), 4.77 (t, J = 4.8, 1H), 3. 69-3.56(m,4H),3.10(t,J=18.6,2H),2.38(s,3H),2.29(d,J=9.7,1H),1.99-1.81(m,3H),1.73(d,J=5.4,2H),0.93-0.81(m,3H).

[1295] Example 2-47

[1296]

[1297] Step 1

[1298] 8-Acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid ethyl ester (2-47b)

[1299] Compound 2-47b (4 g, 61.24% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-14c.

[1300] MS (ESI) m / z: 308.2 [M+H] +

[1301] Step 2

[1302] N-(3-Fluoro-7-(2-(methoxymethyl)azetidine-1-carbonyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-47d)

[1303] Under N2 atmosphere, activated To a suspension of MS (3 g) in toluene (50 mL) was added 2-47b (3 g, 9.77 mmol) and 2-(methoxymethyl)azetidine (1.97 g, 19.54 mmol). The resulting mixture was heated at 70 ° C for 18 h. The reaction mixture was then cooled to ambient temperature and filtered through a pad of celite, and the filtrate was concentrated in vacuo. The crude product was purified by chromatography on SiO2 (EtOAc / hexane, 1 / 5) to give 2-47d (1.1 g, 31.09% yield) as a yellow solid.

[1304] MS (ESI) m / z: 363.3 [M+H] +

[1305] 1 H NMR (400MHz, CD3OD) δ6.29 (dd, J=12.3, 3.5Hz, 1H), 4.76-4.44 (m, 1H), 4.32-3.48 (m, 6H), 3. 09-2.99(m,1H),2.74(ddd,J=17.3,11.7,5.2Hz,1H),2.48-2.14(m,4H),2.07-1.92(m,4H).

[1306] Step 3

[1307] N-(3-Fluoro-7-(2-(hydroxymethyl)azetidine-1-carbonyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-47e)

[1308] Compound 2-47e (150 mg, 14.18% yield) was synthesized according to the synthetic procedure of step 4 of Example 2-14.

[1309] MS (ESI) m / z: 349.3 [M+H] +

[1310] Step 4

[1311] 8-Amino-6-fluoro-2-(2-(hydroxymethyl)azetidine-1-carbonyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-47f)

[1312] Compound 2-47f (130 mg, crude) was synthesized according to the synthetic procedure of step 2 of Example 2-14d.

[1313] MS (ESI) m / z: 307.3 [M+H] +

[1314] Step 5

[1315] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-(hydroxymethyl)azetidine-1-carbonyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-47)

[1316] Compound 2-47 (3.2 mg, 6.62% yield) was synthesized according to the synthetic procedure of step 3 of Example 2-14e.

[1317] MS (ESI) m / z: 534.5 [M+H] +

[1318] Example 2-48

[1319]

[1320] Step 1

[1321] 4-(8-Acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)butyl acetate (2-48b)

[1322] Compound 2-48b (499 mg, 33.6% yield) was synthesized according to the synthetic procedure of step 1 of Example 2-14c.

[1323] MS (ESI) m / z: 350.3 [M+H] +

[1324] Step 2

[1325] N-(3-Fluoro-7-(4-hydroxybutyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-48c)

[1326] To a solution of 2-48b (499 mg, 1.43 mmol) in THF (20 mL) and MeOH (20 mL) was added NaCO (302.94 mg, 2.86 mmol) at 0°C, and the mixture was stirred at the same temperature for 4 h. The reactants were extracted three times with EtOAc. The combined organic layers were washed with brine and dried over MgSO, and the combined extracts were then concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (silica gel, hexane: EtOAc = 50:50) to yield 2-48c (410 mg, 93.4% yield) as a colorless oil.

[1327] MS (ESI) m / z: 306.1 [MH] +

[1328] Step 3

[1329] 8-Amino-6-fluoro-2-(4-hydroxybutyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (2-48d)

[1330] Compound 2-48d (240 mg, 67.99% yield) was synthesized according to the synthetic procedure of step 2 of Example 2-14d.

[1331] MS (ESI) m / z: 266.2 [M+H] +

[1332] Step 4

[1333] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(4-hydroxybutyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-48)

[1334] Compound 2-48 (105 mg, 23.64% yield) was synthesized according to the synthetic procedure of step 3 of Example 2-14e.

[1335] MS (ESI) m / z: 493.5 [M+H] +

[1336] 1 H NMR (400MHz, DMSO-d6) δ7.70(dd,J=11.1,3.5Hz,1H),7.30(d,J=2.3Hz,1H),5.44(s,2H),5.25(dt,J=18.7,11.4Hz,2H),3.44(dd,J=9.7,5.7Hz, 2H),3.33(s,1H),3.06(d,J=20.3Hz,2H),2.36(s,3H),2.30(d,J=13.3Hz ,1H),2.02-1.80(m,3H),1.68-1.36(m,6H),0.90(td,J=7.2,2.7Hz,3H).

[1337] Examples 2-49 and 2-50

[1338]

[1339] Step 1

[1340] 2,5-Dibromopentan-1-ol (2-49b)

[1341] To a solution of ester 2-49a (1 g, 3.65 mmol, 1.00 equiv) in DCM (15 mL) was added 1.0 M diisobutylaluminum hydride in hexanes (7.5 mL, 7.5 mmol, 2.05 equiv) at -78 ° C. and the solution was removed from the cooling bath for 45 min, at which time TLC analysis showed the reaction was complete. The solution was cooled to -78 ° C. and quenched with saturated Rochelle salt (30 mL), diluted with DCM (30 mL), removed from the cooling bath, and stirred vigorously at rt until the phases became distinct and clear (1.5 h). The phases were then separated and the aqueous phase was extracted with DCM (2 x 50 mL), and the combined organic phases were dried over MgSO 4 , filtered, and the solvent removed to yield alcohol 2-49b (880 mg, 98% yield) as a colorless oil.

[1342] Step 2

[1343] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-(hydroxymethyl)pyrrolidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-49)

[1344] (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-(hydroxymethyl)pyrrolidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-50)

[1345] To a solution of 2-49b (250 mg, 0.47 mmol) in 6 ml of DMF was added exatecan mesylate (288 mg, 1.176 mmol), NaI (14.1 mg, 0.194 mol) and DIEA (152 mg, 1.176 mmol). The mixture was stirred at 80 ° C for 19 h. The mixture was purified by preparative HPLC (TFA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min) and the fractions were lyophilized to produce 2-49 (2.2 mg, 2% yield) and 2-50 (7.7 mg, 6% yield) as a white solid.

[1346] MS (ESI) m / z: 520.5 [M+H] +

[1347] Examples 2-51 and 2-52

[1348]

[1349] Step 1

[1350] 8-Acetamido-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene-2-carboxamide (2-51b)

[1351] 2-51b (302 mg, 75% yield) was synthesized according to the synthetic procedure of Example 2-47d.

[1352] MS (ESI) m / z: 436.3 [M+H] +

[1353] Step 2

[1354] 8-Amino-6-fluoro-N-(2-hydroxyethyl)-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene-2-carboxamide (2-51c)

[1355] 2-51b (190 mg, 58% yield) was synthesized according to the synthetic procedure of Example 2-47f.

[1356] MS (ESI) m / z: 281.3 [M+H] +

[1357] Step 3

[1358] (9S)-9-ethyl-5-fluoro-9-hydroxy-N-(2-hydroxyethyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-carboxamide (2-51, 2-52)

[1359] 2-51 (4.6 mg, 3% yield) and 2-52 (9.0 mg, 6% yield) were synthesized according to the synthetic procedure of Example 2-47.

[1360] 2-51

[1361] MS (ESI) m / z: 508.4 [M+H] +

[1362] 1H NMR(400MHz, DMSO-d6)δ8.30(t,J=5.5Hz,1H),7.77(d,J=11.0Hz,1H),7.32( s,1H),6.53(s,1H),5.43(s,2H),5.33(d,J=18.9Hz,1H),5.02(d,J=18.9Hz, 1H),4.72(t,J=5.2Hz,1H),4.25(s,1H),3.15-3.12(m,5H),2.44-2.43(m,2H ), 2.38 (s, 3H), 2.13-2.11 (s, 1H), 1.92-1.86 (m, 2H), 0.87 (t, J = 7.2Hz, 3H).

[1363] 2-52

[1364] MS (ESI) m / z: 508.4 [M+H] +

[1365] 1 H NMR(400MHz, DMSO-d6)δ8.32(t,J=5.7Hz,1H),7.77(d,J=11.0Hz,1H),7.32(s,1H), 6.53(s,1H),5.43(s,2H),5.33(d,J=18.9Hz,1H),5.02(d,J=18.9Hz,1H),4.71(t,J =5.3Hz,1H),4.26(t,J=4.0Hz,1H),3.44-3.41(m,2H),3.18-3.14(m,4H),2.46-2.4 0(m,1H),2.38(s,3H),2.18-2.01(m,1H),1.95-1.78(m,2H),0.87(t,J=7.3Hz,3H).

[1366] Example 2-53

[1367]

[1368] Step 1

[1369] N-(3-Fluoro-4-methyl-7-methylene-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-53a)

[1370] To a solution of 2-14a (470.5 mg, 2 mmol) in CHCN (5 mL) was added paraformaldehyde (150 mg, 5 mmol) and ZnCl (136.3 mg, 1 mmol). The mixture was stirred at rt for 10 min. Pyrrolidine (287 mg, 4 mmol) was subsequently added dropwise. After the addition was complete, the mixture was stirred at 70 ° C for 2 h. The reaction solution was then cooled to rt, the solvent was removed by evaporation and the residue was directly purified by silica gel column chromatography (eluent: hexane / EtOAc=30 / 1 to 5 / 1) to obtain 2-53a (210 mg, 42.5% yield) as a white solid.

[1371] 1 H NMR (400MHz, CDCl3) δ12.42(s,1H),8.46(d,J=12.9Hz,1H),6.19(d,J=1.3Hz,1H),5.51(d,J= 1.6Hz, 1H), 2.93 (t, J = 6.5Hz, 2H), 2.78 (t, J = 6.4Hz, 2H), 2.25 (s, 3H), 2.17 (d, J = 1.9Hz, 3H).

[1372] MS (ESI) m / z: 248.2 [M+H] +

[1373] Step 2

[1374] N-(3-Fluoro-7-(((2-hydroxyethyl)amino)methyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-53b)

[1375] To a solution of 2-53a (200 mg, 0.8 mmol) in THF (10 mL) was added 2-aminoethane-1-ol (108.8 mg, 1.78 mmol). The mixture was stirred at rt for 40 min. The solvent was then removed by evaporation. The crude product was unstable under silica gel purification and was used directly in the next step without further purification (crude material 290 mg, yield 117%).

[1376] MS (ESI) m / z: 309.3 [M+H] +

[1377] Step 3

[1378] N-(3-Fluoro-7-(((2-hydroxyethyl)amino)methyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (2-53c)

[1379] To a solution of 2-53b (80 mg, 0.26 mmol) in CHCl (2 mL) was added EtN (40 mg, 0.4 mmol). The solution was then cooled to 0°C and benzyl chloroformate (48.6 mg, 0.28 mmol) was added dropwise. The mixture was then warmed to rt and stirred at rt for 2 h. The solvent was removed by evaporation and the residue was purified directly by silica gel column chromatography (eluent: CHCl / MeOH=32 / 1) to afford 2-53c (110 mg, 95.6% yield) as a colorless oil.

[1380] 1 H NMR (400MHz, CDCl3) δ12.09(s,1H),8.39(d,J=13.0Hz,1H),7.42-7.27(m,4H),7.21(s,1H),5.30(s,1H),5.13(d,J=13.1Hz,2H),4.94(d,J=12 .0Hz,1H),3.99-3.71(m,3H),3.65-3.53(m,1H),3.52-3.35(m,2H),2. 85(t,J=62.3Hz,3H),2.21(s,3H),2.10(d,J=27.8Hz,3H),1.79(s,2H).

[1381] MS (ESI) m / z: 465.4 [M+Na] +

[1382] Step 4

[1383] Benzyl ((8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)(2-hydroxyethyl)carbamate (2-53d)

[1384] To a solution of compound 2-53c (100 mg, 0.23 mmol) in MeOH (3 mL) was added HCl (2 N, 1.5 mL). The mixture was stirred at 60 ° C for 3 h. The mixture was cooled to rt and adjusted to pH 8 using saturated NaHCO 3. The mixture was extracted with EtOAc (10 mL * 3). The organic layer was dried and concentrated. The crude product 2-53d (red oil) was used directly in the next step without further purification (crude material: 81 mg, yield 89.6%).

[1385] MS (ESI) m / z: 401.4 [M+H] +

[1386] Step 5

[1387] N-Benzyl (((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)methyl)(2-hydroxyethyl)carbamate (2-53e)

[1388] To a solution of 2-53d (80 mg, 0.2 mmol) in toluene (10 mL) and o-cresol (0.5 mL) was added 1-1i (60.5 mg, 0.23 mmol) and PPTS (30.2 mg, 0.12 mmol). The mixture was refluxed at 140 ° C for 12 h. The solvent was removed by evaporation and the crude product 2-53e was used directly in the next step without further purification (crude material: 120 mg, 95.6% yield).

[1389] MS (ESI) m / z: 628.5 [M+H] +

[1390] Step 6

[1391] N-Benzyl (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((2-hydroxyethyl)amino)methyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-53)

[1392] To a solution of 2-53e (crude material 120 mg, 0.2 mmol) in MeOH (2 mL) and THF (2 mL) was added wet Pd / C (20%, 24 mg) and stirred at rt under H2 atmosphere for 8 h. The solution was filtered through celite and concentrated under vacuum. The residue was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min) and the fractions were lyophilized to give 2-53 (33.2 mg, 33.6% yield) as a white solid.

[1393] 1H NMR(400MHz,DMSO-d6)δ8.21(s,1H),7.74(d,J=11.0Hz,1H),7.30(s,1H),6 .53(s,1H),5.44(s,2H),5.41-5.34(m,2H),5.31(s,1H),3.55(s,1H),3.49( dd,J=5.6,3.8Hz,2H),3.05(dd,J=27.9,9.3Hz,3H),2.89-2.81(m,2H),2.74 -2.67(m,2H),2.37(s,3H),2.03-1.81(m,4H),0.87(dd,J=11.7,4.3Hz,3H).

[1394] MS (ESI) m / z: 494.4 [M+H] +

[1395] Example 2-54

[1396]

[1397]

[1398] Step 1

[1399] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(((2-hydroxyethyl)(methyl)amino)methyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-54)

[1400] To a solution of 2-53 (10 mg, 0.02 mmol) in MeOH (1 mL) was added 37% formaldehyde solution (2.8 μL, 0.04 mmol). The solution was then cooled to 0 ° C and then NaBH CN (2 mg, 0.03 mmol) was added once. The solution was then warmed to rt and stirred for another 2 h. The mixture was then quenched with H O and purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to produce 2-54 (5.5 mg, 54.2% yield) as a white solid.

[1401] MS (ESI) m / z: 508.5 [M+H] +

[1402] Example 2-55

[1403]

[1404]

[1405] Step 1 to Step 6

[1406] (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(((3-hydroxypropyl)amino)methyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-55)

[1407] 2-55 (0.6 mg, 6% yield) was synthesized according to the synthetic procedure of Example 2-53.

[1408] MS (ESI) m / z: 508.5 [M+H] +

[1409] Examples 2-56 and 2-57

[1410]

[1411] Step 12,4-dibromobutan-1-ol (2-56b)

[1412] 2-56b (brown oil, 1.2 g, 67.4% yield) was synthesized according to the procedure described in step 1 of example 2-49b.

[1413] Step 2

[1414] (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-(hydroxymethyl)azetidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-56) and (1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-(hydroxymethyl)azetidin-1-yl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2-57)

[1415] Examples 2-56 (white solid, 1.5 mg, 2.5% yield) and 2-57 (white solid, 1.5 mg, 2.5% yield) were synthesized according to the procedures described in Examples 2-49 and 2-50.

[1416] 2-56: MS(ESI)m / z:506.5. [M+H] +

[1417] 2-57: MS(ESI)m / z:506.5. [M+H] +

[1418] Example 2-58

[1419]

[1420] Step 1

[1421] N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-fluoro-3-hydroxy-2-methylpropionamide (2-58)

[1422] Compound 2-58 (9.6 mg, 63.1% yield) was synthesized according to the synthetic procedure of step 3 of Example 2-2.

[1423] MS (ESI) m / z: 556.4 [M+H] +

[1424] Examples 2-59 and 2-60

[1425]

[1426] Step 1

[1427] 2-(3-iodopropoxy)tetrahydro-2H-pyran (2-59b)

[1428] To a solution of compound 2-59a (2.0 g, 9.01 mmol) in acetone (20 mL) was added NaI (4.0 g, 27 mmol). The mixture was stirred at 60 ° C for 3 h. The mixture was diluted with hexane (40 mL) and washed with water (40 mL) and brine (40 mL). The combined organic layers were dried over Na SO and concentrated to give 2-59b (1.3 g, 54% yield) as a colorless oil.

[1429] 1H NMR (400MHz, CDCl3) δ4.61 (dd, J=4.4, 2.8Hz, 1H), 3.91-3.83 (m, 1H), 3.83-3.77 (m, 1H), 3.56-3.49 (m, 1H), 3.45 (dt, J= 10.0, 5.9Hz, 1H), 3.30 (td, J=6.8, 1.0Hz, 2H), 2.10 (ddd, J=12.7, 6.8, 5.9Hz, 2H), 1.90-1.63 (m, 3H), 1.61-1.48 (m, 5H).

[1430] Step 2

[1431] N-(6-oxo-7-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (2-59c)

[1432] To a solution of compound 2-14a (100 mg, 3.52 mmol) in THF (5 mL) was added dropwise t-BuOK (1.2 mL, 1.21 mmol, 1 M in THF) at -40 ° C under N2. The mixture was stirred at -40 ° C for 30 min under N2. Compound 2-59b (164 mg, 0.606 mmol, dissolved in 0.2 mL THF) was then added dropwise to the mixture. The mixture was stirred under N2 for 16 h and gradually heated to room temperature. The mixture was quenched with saturated NH4Cl (3 mL) and extracted with EA (3 mL * 3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated to generate a residue. Purified by column (petroleum ether / ethyl acetate=1: 0 to 4: 1) to obtain compound 2-59c (20 mg, 12.7% yield) as a yellow solid.

[1433] MS (ESI) m / z: 248.1 [M+H] + .

[1434] 1 H NMR (400MHz, CDCl3) δ12.44(s,1H),8.26(s,1H),6.01-5.98(m,2H),4.59-4.53(m,1H),3.90-3.72(m,2H),3.53-3.36(m,2H),3.00-2.88 (m,1H),2.79-2.67(m,1H),2.55-2.44(m,1H),2.19(s,3H),2.17-2.09(m,1H),2.06-1.86(m,2H),1.86-1.65(m,6H),1.60-1.54(m,2H).

[1435] Step 3

[1436] 5-Amino-7-(3-hydroxypropyl)-8,9-dihydronaphtho[1,2-d][1,3]dioxol-6(7H)-one (2-59d)

[1437] To a solution of compound 2-59c (20 mg, 0.05 mmol) in MeOH (5 mL) was added 2N HCl (5 mL). The mixture was stirred at 60 ° C for 3 h. The mixture was concentrated in vacuo to remove MeOH. Saturated Na2CO3 was then used to adjust the mixture to pH = 8 and extracted with CH2Cl2 (5 mL * 3). The combined organic phases were concentrated in vacuo to generate a residue, which was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 100 / 0 to 0 / 100) to obtain 2-59d (10 mg, 74% yield), which was obtained as a yellow oil.

[1438] MS (ESI) m / z: 264.2 [M+H] + .

[1439] Step 4

[1440] (1R,10S)-10-Ethyl-10-hydroxy-1-(3-hydroxypropyl)-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (2-59) and (1S,10S)-10-Ethyl-10-hydroxy-1-(3-hydroxypropyl)-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (2-60)

[1441] To a solution of compound 2-59d (80 mg, 0.304 mmol) in toluene (14 mL) and o-cresol (2 mL) was added 1-1i (88 mg, 0.334 mmol) and PPTS (23 mg, 0.091 mmol). The mixture was refluxed at 140 ° C for 16 h. The mixture was concentrated in vacuo to remove toluene and purified into a crude product (a mixture of 6 and 7) by silica gel column chromatography (eluent: CH Cl / MeOH = 100 / 0 to 100 / 10). By preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um19*250mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min purification, the fractions were lyophilized to generate 2-59 (15 mg, 71% yield) and 2-60 (20 mg, 13% yield) as a white solid.

[1442] Compound 2-59 (retention time: 2.54 min):

[1443] MS (ESI) m / z: 491.5 [M+H] +

[1444] 1 H NMR (400MHz, DMSO-d6) δ7.37(s,1H),7.23(s,1H),6.48(s,1H),6.27(d,J=2.9 Hz,2H),5.42(s,2H),5.31(d,J=18.9Hz,1H),5.19(d,J=18.8Hz,1H),4.42(t, J=5.1Hz,1H),3.48-3.42(m,2H),3.01-2.91(m,2H),2.22(d,J=13.4Hz,1H),1 .93-1.75(m,4H),1.73-1.63(m,2H),1.61-1.54(m,2H),0.87(t,J=7.3Hz,3H).

[1445] Compound 2-60 (retention time: 2.66 min):

[1446] MS (ESI) m / z: 491.5 [M+H] +

[1447] 1H NMR(400MHz,DMSO-d6)δ7.37(s,1H),7.23(s,1H),6.49(s,1H),6.27(s,2H) ,5.42(s,2H),5.31(d,J=18.9Hz,1H),5.20(d,J=18.9Hz,1H),4.43(t,J=5. 2Hz,1H),3.47-3.43(m,2H),3.02-2.86(m,2H),2.22(d,J=13.3Hz,1H),1.9 4-1.78(m,3H),1.73-1.63(m,2H),1.63-1.53(m,3H),0.87(t,J=7.3Hz,3H).

[1448] Example 3-1

[1449]

[1450]

[1451] Step 1

[1452] (2S,3R,4R,5S,6R)-2-(Aminomethyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (3-1b)

[1453] To a solution of compound 3-1a (200 mg, 0.90 mmol) in MeOH (3 mL) was added HCOONH4 (226 mg, 3.58 mmol) and Pd / C (20 mg, 10%). The mixture was stirred at 70°C for 2 h. The mixture was filtered and concentrated to give the product 3-1b (173 mg, crude) as a white solid.

[1454] MS (ESI) m / z: 194.3 [M+H] +

[1455] Step 2

[1456] N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)-L-glutamic acid benzyl ester (3-1d)

[1457] To a solution of compound 3-1b (3.0 g, 15.5 mmol) in DMF (100 mL) was added compound 3-1c (7.5 g, 16.3 mmol), HATU (11.8 g, 31.1 mmol) and DIEA (3.0 g, 23.3 mmol). The mixture was stirred at room temperature for 1 h. The mixture was concentrated and purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH=100 / 0 to 85 / 15) to give 3-1c (7.6 g, 77.1% yield) as a white solid.

[1458] MS (ESI) m / z: 635.5 [M+H] +

[1459] Step 3

[1460] N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)-L-glutamine (3-1e)

[1461] To a solution of 3-1d (1.5 g, 2.36 mmol) in MeOH (20 mL) was added wet Pd / C (150 mg). The black suspension was purged three times with H balloon, then reacted under H balloon at rt for 4 h. After the reaction was complete, the black suspension was filtered out by celite pad and the filter cake was washed with MeOH, and the combined organic layers were concentrated under vacuum to provide 3-1e (1.29 g, thick material).

[1462] MS (ESI) m / z: 567.4 [M+Na] +

[1463] Step 4

[1464] (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (3-1g)

[1465] To a solution of compound 3-1f (5.00 g, 8.12 mmol) in DMF (65 mL) was added Cu(OAc)2 (560 mg, 3.09 mmol), Pb(OAc)4 (4.11 g, 9.25 mmol) and HOAc (1.11 g, 18.44 mmol). The mixture was stirred at 65 ° C under an N2 atmosphere for 2 h. The mixture was concentrated to 1 / 4 of the original volume, poured into ice water (150 mL) and stirred for 30 min. The solid was filtered and co-evaporated with toluene (20 mL*4) to produce compound 3-1g (5.20 g, crude material) as an off-white solid.

[1466] MS (ESI) m / z: 652.3 [M+Na] +

[1467] Step 5

[1468] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatricosan-23-oic acid benzyl ester (3-1h)

[1469] To a solution of compound 3-1g (1.0 g, 2.45 mmol) in DMF was added 3-1e (1.47 mmol, 2.7 mmol), HATU (1.40 g, 3.68 mmol) and DIEA (634 mg, 4.91 mmol). The mixture was stirred at RT for 30 min. The mixture was concentrated and purified by flash column chromatography (eluent: CH2Cl2 / MeOH=100 / 0 to 20 / 80) to produce the title compound 3-1h (1.3 g, 51.0% yield), which was obtained as an off-white solid.

[1470] MS (ESI) m / z: 1062.5 [M+Na] +

[1471] Step 6

[1472] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatricosane-23-oic acid (3-1i)

[1473] To a solution of compound 3-1h (35 mg, 0.034 mmol) in MeOH (4 mL) was added Pd / C (10%, 5 mg). The mixture was stirred under a H2 atmosphere (15 psi) for 3 h. The mixture was filtered through a pad of celite and concentrated to yield compound 3-1i (32 mg, crude) as a white solid.

[1474] MS (ESI) m / z: 972.5 [M+Na] +

[1475] Step 7

[1476] ((6S,15S)-15-Benzyl-24-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7 ,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (3-1j)

[1477] To a solution of compound 3-1i (20 mg, 0.021 mmol) in DMF (2 mL) was added compound 1-1 (12 mg, 0.022 mmol), HATU (16 mg, 0.042 mmol) and DIEA (8 mg, 0.063 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min to provide compound 3-1j (14.5 mg, 49.5% yield) as a white solid.

[1478] MS (ESI) m / z: 1405.7 [M+Na] +

[1479] Step 8

[1480] ((6S,15S)-15-Benzyl-24-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7 ,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (3-1k)

[1481] To a solution of compound 3-1j (14.5 mg, 0.011 mmol) in DMF (2 mL) was added Et2NH (15.3 mg, 0.21 mmol). The mixture was stirred at rt for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (3 mL * 3) to produce 3-1k (12.2 mg, crude material) as a brown solid.

[1482] MS (ESI) m / z: 1183.6 [M+Na] +

[1483] Step 9

[1484] (S)-N1-((S)-10-Benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,1 5-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-1)

[1485] To a solution of compound 3-1k (12 mg, 0.01 mmol) in DMF (2 mL) was added compound 3-1l (4.4 mg, 0.021 mmol), HATU (7.9 mg, 0.021 mmol) and DIEA (2.7 mg, 0.021 mmol). The mixture was stirred at rt for 30 min. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min) and the fractions were lyophilized to give compound 3-1 (6.3 mg, 45.0% yield).

[1486] MS (ESI) m / z: 1376.7 [M+Na] + . Retention time (3.74min).

[1487] Example 3-2

[1488]

[1489] 3-2 (15.2 mg, 98% purity, 67.7% yield) was synthesized according to a procedure similar to Example 3-1.

[1490] MS (ESI) m / z: 1356.8 [M+Na] +

[1491] Example 3-3

[1492]

[1493] Step 1

[1494] Tert-butyl ((S)-1-(((S)-1-((4-(chloromethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (3-3b)

[1495] To a pre-cooled solution of 3-3a (200 mg, 0.42 mmol) in anhydrous DMF (1 mL) was added SOCl2 (37 μL, 0.5 mmol) in an ice bath and stirred at 0°C for 30 min. Water (3 mL) was slowly added to the reaction solution. The turbid solution was filtered and the filter cake was washed with water (3 mL) and MTBE (3 mL), then dried under vacuum to yield compound 3-3b (180 mg, 86.7% yield) as an off-white solid.

[1496] MS (ESI) m / z: 398.4 [M+H-Boc] +

[1497] Step 2

[1498] 1-(4-((S)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpiperazin-1-ium (3-3c)

[1499] To a solution of 1-7 (5.5 mg, 11 μmol), 3-3b (6.5 mg, 12 μmol) and TBAI (2.0 mg, 5 μmol) in anhydrous DMF (0.5 mL) was added DIEA (4 μL, 27 μmol) and stirred at rt for 48 h. MTBE (2 mL) was added. The turbid solution was filtered to produce compound 3-3c (10 mg, 96.1% yield), which was used directly in the next step.

[1500] MS (ESI) m / z: 980.7 [M] +

[1501] Step 3

[1502] 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpiperazin-1-ium(3-3d)carboxylate

[1503] To a solution of 3-3c (10 mg, 0.01 mmol) in DCM (0.8 mL) was added TFA (0.2 mL) and stirred at rt for 20 min. The solution was concentrated and purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 20%-35% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 3-3d formate salt as a light pink solid (8.2 mg, 86.8% yield).

[1504] MS (ESI) m / z: 441.1 [M / 2+H] +

[1505] Step 4

[1506] 1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-methylpiperazin-1-ium(3-3)

[1507] To a solution of 3-11 (3.2 mg, 0.015 mmol) and HATU (4.3 mg, 0.011 mmol) in anhydrous DMF (0.2 mL) was added DIEA (4 μL, 0.022 mmol) and stirred at rt for 15 min. The resulting solution was added to a solution of 3-3d (6.5 mg, 0.007 mmol) in anhydrous DMF (0.5 mL) and stirred at rt for 20 min. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 30%-40% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 3-3 (2.6 mg, 32.8% yield) as a white solid.

[1508] MS (ESI) m / z: 1073.7 [M] +

[1509] Examples 3-4

[1510]

[1511] Step 1

[1512] (9H-fluoren-9-yl)methyl ((6S,15S)-15-benzyl-24-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,20-pentaazatetracosan-6-yl)carbamate (3-4a)

[1513] To a solution of compound 3-8i (20.0 mg, 0.02 mmol) in DMF (2 mL) was added 1-8 methanesulfonate (11.6 mg, 0.02 mmol), HATU (8.0 mg, 0.02 mmol) and DIEA (8.2 mg, 10 μL, 0.02 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The filtrate was purified by HPLC (5 μm, 19*150 mm) to provide compound 3-4a (22 mg, 75.5% yield) as a white solid.

[1514] MS (ESI) m / z: 1405.6 [M+Na] +

[1515] Step 2

[1516] (S)-2-amino-N 1 -((S)-10-Benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,11,14-triazahexadec-16-yl)-N 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-4b)

[1517] To a solution of compound 3-4a (22.0 mg, 0.016 mmol) in DMF (2 mL) was added EtNH (11.6 mg, 16 μL, 0.16 mmol). The mixture was stirred at rt for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (10 mL) to produce 3-4b (18 mg, crude material) as a brown solid.

[1518] MS (ESI) m / z: 1161.7 [M+H] +

[1519] Step 3

[1520] (S)-N 1 -((S)-10-Benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,11,14-triazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-((S)-10-Benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,11,14-triazahexadec-16-yl) 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-4)

[1521] To a solution of compound 3-4b (18.0 mg, 0.016 mmol) in DMF (2 mL) was added 3-11 (4.9 mg, 0.023 mmol), HATU (8.8 mg, 0.023 mmol) and DIEA (2.0 mg, 3 μL, 0.016 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and analyzed using preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The filtrate was purified by HPLC (5 μm, 19*150 mm) to provide compound 3-4 (7.5 mg, 23.8% yield) as a white solid.

[1522] MS (ESI) m / z: 1355.1 [M+H] +

[1523] Examples 3-5

[1524]

[1525]

[1526] Step 1

[1527] ((S)-9H-Fluoren-9-yl)methyl (12-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,8,11,14,17-pentaoxo-2,5-dioxa-7,10,13,16-tetraazaoctadec-18-yl)carbamate (3-5a)

[1528] To a solution of 3-1 g (65 mg, 0.10 mmol) and 2-6 (58 mg, 0.092 mmol) in anhydrous THF (2 mL) was added To the mixture of 4-nitro-1-oxo-2-nitropropene (3-2-nitropropene) (350 mg), stirred at rt for 30 min. Sc (OTf) 3 (56 mg, 0.11 mmol) was added under a nitrogen atmosphere and stirred for 19 h. The slurry solution was filtered and the filter cake was washed with THF (10 mL). The filtrate was washed with saturated NaHCO 3 (10 mL) and extracted with CH 2 Cl 2 / MeOH (5 / 1, 12 mL * 3). The organic phase was concentrated and purified by flash column chromatography (silica gel, CH 2 Cl 2 / MeOH = 10: 1) to generate compound 3-5a (81 mg, 87% purity, 70% yield) as a yellow solid.

[1529] MS (ESI) m / z: 1093.5 [M+H] +

[1530] Step 2

[1531] (S)-16-Amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[1532] To a solution of 3-5a (81 mg, 0.074 mmol) in DMF (1 mL) was added Et2NH (154 μL, 1.48 mmol) and stirred at rt for 10 min. The solution was concentrated to give the crude product 3-5b (65 mg, theoretical yield) as a brown solid.

[1533] MS (ESI) m / z: 893.5 [M+Na] +

[1534] Step 3

[1535] ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (5S,14S)-14-benzyl-1-(9H-fluoren-9-yl)-3-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate ,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatricosan-23-yl ester (3-5c)

[1536] To a solution of 3-5b (65 mg, theoretical yield, 0.075 mmol), 3-1e (46 mg, 0.082 mmol) and HATU (35 mg, 0.09 mmol) in anhydrous DMF (2 mL) was added DIEA (40 μL, 0.22 mmol) and stirred at rt for 30 min. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 35%-80% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TM The desired fractions were lyophilized to give 3-5c (19 mg, 18.2% yield) as a yellow solid.

[1537] MS (ESI) m / z: 1419.9 [M+Na] +

[1538] Step 4

[1539] (6S,15S)-6-amino-15-benzyl-3,7,10,13,16,19-hexaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-24-yl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[1540] To a solution of 3-5c (19 mg, 0.01 mmol) in DMF (1 mL) was added Et2NH (28 μL, 0.27 mmol) and stirred at rt for 10 min. The solution was concentrated to give the crude product 3-5d (16 mg, theoretical yield) as a brown solid.

[1541] MS (ESI) m / z: 1175.8 [M+H] +

[1542] Step 5

[1543] ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (10S,19S)-10-benzyl-26-(2,5-dioxo-2,5-dihydro -1H-pyrrol-1-yl)-6,9,12,15,18,21-hexaoxo-19-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-3-oxa-5,8,11,14,17,20-hexaazahexacosyl ester (3-5)

[1544] To a solution of 3-5d (16 mg, theoretical yield, 0.014 mmol), 3-11 (3.8 mg, 0.018 mmol) and HATU (5.8 mg, 0.015 mmol) in anhydrous DMF (2 mL) was added DIEA (5 μL, 0.027 mmol) and stirred at rt for 10 min. The product was purified by preparative HPLC (mobile phase A: water containing 0.1% FA, B: MeCN; gradient: 30%-45% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD TMThe desired fractions were lyophilized to give 3-5 (7.9 mg, 42.4% yield) as a white solid.

[1545] MS (ESI) m / z: 1390.9 [M+Na] +

[1546] Examples 3-6

[1547]

[1548] Step 1

[1549] ((S)-9-Fluoren-9-yl)methyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-8,11,14,17-tetraoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)carbamate (3-6a)

[1550] Compound 3-6a (180 mg, 53% purity) was obtained according to the procedure described in step 1 of Example 3-5.

[1551] MS (ESI) m / z: 1063.6 [M+H] +

[1552] Step 2

[1553] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)(methyl)amino)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (3-6b)

[1554] Compound 3-6b (185 mg, crude) was obtained according to the procedure described in step 2 of Example 3-5.

[1555] MS (ESI) m / z: 841.5 [M+H] +

[1556] Step 3

[1557] (9H-Fluoren-9-yl)methyl ((12S,21S)-12-benzyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-8,11,14,17,20,24-hexaoxo-26-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxa-2,7,10,13,16,19,25-heptaazahexacosano-21-yl)carbamate (3-6c)

[1558] Compound 3-6c (80 mg, 70% purity) was obtained according to the procedure described in step 3 of Example 3-5.

[1559] MS (ESI) m / z: 1367.7 [M+H] +

[1560] Step 4

[1561] (S)-2-amino-N 1 -((S)-12-Benzyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-8,11,14,17-tetraoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-6d)

[1562] Compound 3-6d (41 mg, crude) was obtained according to the procedure described in step 4 of Example 3-5.

[1563] MS (ESI) m / z: 1145.7 [M+H] +

[1564] Step 5

[1565] (S)-N 1-((S)-12-Benzyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-8,11,14,17-tetraoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-((S)-12-Benzyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-8,11,14,17-tetraoxo-5-oxa-2,7,10,13,16-pentaazaoctadec-18-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-6)

[1566] Compound 3-6 (13 mg, 98% purity) was obtained according to the procedure described in step 5 of example 3-5.

[1567] MS (ESI) m / z: 1338.8 [M+H] +

[1568] Examples 3-7

[1569]

[1570]

[1571] Step 1

[1572] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-23,23-dimethyl-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatetracosanoic acid (3-7b)

[1573] To a solution of 3-7a (39 mg, 0.036 mmol) in MeOH (2 mL) was added 10% Pd / C (8.5 mg) under nitrogen atmosphere and stirred under H2 balloon for 4 h. The solution was filtered and concentrated to give compound 3-7b (36 mg, 83% purity), which was used in the next step without further purification.

[1574] MS (ESI) m / z: 1014.6 [M+H] +

[1575] Step 2

[1576] ((6S,15S)-15-Benzyl-25-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-24,24-dimethyl (9H-fluoren-9-yl)methyl 3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (3-7c)

[1577] Compound 3-7c (24 mg, 98% purity) was obtained according to the procedure described in step 1 of Example 3-4.

[1578] MS (ESI) m / z: 1447.6 [M+Na] +

[1579] Step 3

[1580] (S)-2-Amino-N1-((S)-7-benzyl-17-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-7d)

[1581] Compound 3-7d (25 mg, crude) was obtained according to the procedure described in step 2 of Example 3-4.

[1582] MS (ESI) m / z: 1203.6 [M+H] +

[1583] Step 4

[1584] (S)-N1-((S)-7-Benzyl-17-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5 ,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-7)

[1585] Compound 3-7 (9.7 mg, 96% purity) was obtained according to the procedure described in step 3 of Example 3-4.

[1586] MS (ESI) m / z: 1418.8 [M+Na] +

[1587] Examples 3-8

[1588]

[1589] Step 1

[1590] ((7S)-7-Benzyl-17-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)carbamic acid (9H-fluoren-9-yl)methyl ester (3-8a)

[1591] Compound 3-8a (103 mg, 97% purity) was obtained according to the procedure described in step 1 of Example 3-5.

[1592] MS (ESI) m / z: 1048.6 [M+H] +

[1593] Step 2

[1594] (2S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (3-8b)

[1595] Compound 3-8b (105 mg, crude) was obtained according to the procedure described in step 2 of Example 3-5.

[1596] MS (ESI) m / z: 826.5 [M+H] +

[1597] Step 3

[1598] (9H-fluoren-9-yl)methyl ((6S,15S)-15-benzyl-25-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,7,10,13,16,19-hexaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (3-8c)

[1599] Compound 3-8c (50 mg, 98% purity) was obtained according to the procedure described in step 3 of Example 3-5.

[1600] MS (ESI) m / z: 1352.8 [M+H] +

[1601] Step 4

[1602] (2S)-2-amino-N 1 -((7S)-7-Benzyl-17-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N 5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-8d)

[1603] Compound 3-8d (52 mg, crude) was obtained according to the procedure described in step 4 of Example 3-5.

[1604] MS (ESI) m / z: 1130.7 [M+H] +

[1605] Step 5

[1606] (2S)-N 1 -((7S)-7-Benzyl-17-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N- ... 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-8)

[1607] Compound 3-8 (33 mg, 98% purity) was obtained according to the procedure described in step 5 of example 3-5.

[1608] MS (ESI) m / z: 1323.8 [M+H] +

[1609] Examples 3-9

[1610]

[1611]

[1612] Step 1

[1613] ((6S,15S)-15-Benzyl-24-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) (9H-fluoren-9-yl)methyl 3,7,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (3-9a)

[1614] To a solution of compound 3-1i (20 mg, 0.02 mmol) in DMF (2 mL) was added 1-10 (11.3 mg, 0.03 mmol), HATU (9.12 mg, 0.03 mmol) and DIEA (5.16 mg, 0.04 mmol). The mixture was stirred at 20 ° C for 30 min. LCMS showed that the reaction was complete. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge PrepC18OBD 5um 19*150mm; mobile phase: A-water (0.1% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min to provide compound 3-9a (12.2 mg, 42.1% yield) as a white solid.

[1615] MS (ESI) m / z: 1401.7 [M+Na] +

[1616] Step 2

[1617] (S)-2-Amino-N1-((S)-10-benzyl-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-9b)

[1618] To a solution of compound 3-9a (12.2 mg, 0.009 mmol) in DMF (2 mL) was added Et2NH (9.7 mg, 0.13 mmol). The mixture was stirred at 20° C. for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (3 mL*3) to give 3-9b (10.2 mg, crude material) as a brown solid.

[1619] MS (ESI) m / z: 1179.6 [M+Na] +

[1620] Step 3

[1621] (S)-N1-((S)-10-benzyl-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6 ,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-9)

[1622] To a solution of compound 3-9 (10.2 mg, crude material) in DMF (1 mL) was added compound 3-11 (3.72 mg, 0.018 mmol), HATU (6.5 mg, 0.021 mmol) and DIEA (6.4 mg, 0.036 mmol). The mixture was stirred at 20° C. for 30 min. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give compound 3-9 (4.4 mg, 36.9% yield).

[1623] MS (ESI) m / z: 1372.7 [M+Na] +

[1624] Examples 3-10

[1625]

[1626] Step 1

[1627] (9H-Fluoren-9-yl)methyl ((6S,15S)-15-benzyl-24-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (3-10a)

[1628] To a solution of 3-1i (44.89 mg, 0.10 mmol) in DMF (3 mL) and 1-14 (65.00 mg, 0.07 mmol) was added HBTU (38.92 mg, 0.10 mmol) and DIEA (26.55 mg, 0.21 mmol) was added to the mixture and reacted for another 1 h at the same temperature. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min). 3-10a (40 mg, 42.69% yield) was obtained as a white solid.

[1629] MS (ESI) m / z: 1391.7 [M+Na] +

[1630] Step 2

[1631] (S)-2-Amino-N1-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-10b)

[1632] To a solution of compound 3-10a (40 mg, 0.03 mmol) in DMF (3 mL) was added Et2NH (21.38 mg, 0.29 mmol). The mixture was stirred at rt for 1 h. The mixture was concentrated and co-evaporated with toluene (3*3 mL). The crude material 3-10b (33.51 mg, 100% yield) was used in the next step without further purification.

[1633] MS (ESI) m / z: 1147.6 [M+H] +

[1634] Step 3

[1635] (S)-N1-((S)-10-Benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentahydro-1-((S)-10-benzyl-1- ... Oxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-10)

[1636] To a solution of 3-10b (33.51 mg, 0.029 mmol) in DMF (3 mL) and 3-11 (12.34 mg, 0.06 mmol) was added HBTU (22.17 mg, 0.06 mmol) and DIEA (11.33 mg, 0.09 mmol) was added to the mixture and reacted for another 1 h at the same temperature. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min). 3-10 (19.4 mg, 49.55% yield) was obtained as a white solid.

[1637] MS (ESI) m / z: 1362.6 [M+Na] +

[1638] Example 3-11

[1639]

[1640] Step 1

[1641] (9H-Fluoren-9-yl)methyl ((6S,15S)-15-benzyl-24-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (3-11a)

[1642] Compound 3-11a (43.00 mg, 51.43% yield) was synthesized according to the synthetic procedure of step 1 of Example 3-10a.

[1643] MS (ESI) m / z: 1391.6 [M+Na] +

[1644] Step 2

[1645] (S)-2-Amino-N1-((S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-11b)

[1646] Compound 3-11b (36.00 mg, crude material) was synthesized according to the synthetic procedure of step 2 of Example 3-10b.

[1647] MS (ESI) m / z: 1147.6 [M+H] +

[1648] Step 3

[1649] (S)-N1-((S)-10-Benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentahydro-1- ... Oxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-11)

[1650] Compound 3-11 (17.50 mg, 41.58% yield) was synthesized according to the synthetic procedure of step 3 of Example 3-10.

[1651] MS (ESI) m / z: 1362.5 [M+Na] +

[1652] Examples 3-12

[1653]

[1654]

[1655] Step 1

[1656] (9H-Fluoren-9-yl)methyl ((6S,15S)-15-benzyl-24-(((1S,9S)-9-ethyl-4-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (3-12a)

[1657] Compound 3-12a (20.00 mg, 56.15% yield) was synthesized according to the synthetic procedure of step 1 of Example 3-10a.

[1658] MS (ESI) m / z: 1375.7 [M+Na] +

[1659] Step 2

[1660] (S)-2-Amino-N1-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-4-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-12b)

[1661] Compound 3-12b (16.76 mg, crude material) was synthesized according to the synthetic procedure of step 2 of Example 3-10b.

[1662] MS (ESI) m / z: 1131.6 [M+H] +

[1663] Step 3

[1664] (S)-N1-((S)-10-Benzyl-1-(((1S,9S)-9-ethyl-4-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentahydro-1- ... Oxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-12)

[1665] Compound 3-12 (4.50 mg, 23.29% yield) was synthesized according to the synthetic procedure of step 3 of Example 3-10.

[1666] MS (ESI) m / z: 1346.7 [M+Na] +

[1667] Example 3-13

[1668]

[1669] Step 1

[1670] (S)-(9H-fluoren-9-yl)methyl (7-benzyl-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaoctadec-17-yn-1-yl)carbamate (3-13a)

[1671] To a solution of 3-1 g (126.4 mg, 0.2 mmol) in THF (5 mL) were added 3-butyn-1-ol (42 mg, 0.6 mmol), Sc(OTf)3 (196 mg, 0.4 mmol) and To the 4-thiazolinone solution of 4-nitro-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (5-nitropropene)-2-nitropropene (

[1672] MS (ESI) m / z: 663.4 [M+Na] +

[1673] Step 2

[1674] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((but-3-yn-1-yloxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (3-13b)

[1675] To a solution of 3-13a (80 mg, 0.125 mmol) in DMF (1 mL) was added EtNH (129 μL, 92 mg, 1.25 mmol) at 0°C. The mixture was slowly warmed to rt and stirred at rt under N for 0.5 h. After the reaction was complete, the mixture was concentrated under high vacuum to yield 3-13b (15.3 mg, crude material) as a brown solid.

[1676] Step 3

[1677] (9H-fluoren-9-yl)methyl ((6S,15S)-15-benzyl-3,7,10,13,16,19-hexaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazacosaccharin-25-yn-6-yl)carbamate (3-13c)

[1678] To a solution of compound 3-13b (15.3 mg, 0.037 mmol) in DMF (1 mL) was added compound 3-1e (20 mg, 0.037 mmol), HATU (21 mg, 0.055 mmol) and DIPEA (26 μL, 20 mg, 0.074 mmol). The mixture was stirred at rt for 30 min. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um19*150mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give compound 3-13c (20 mg, 57.1% yield).

[1679] MS (ESI) m / z: 966.5 [M+Na] +

[1680] Step 4

[1681] (S)-2-Amino-N1-((S)-7-benzyl-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaoctadec-17-yn-1-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-13d)

[1682] To a solution of 3-13c (20 mg, 0.02 mmol) in DMF (1 mL) was added EtNH (22 μL, 15.5 mg, 0.2 mmol) at 0 ° C. The mixture was slowly heated to rt and stirred at rt for 0.5 h under N. After the reaction was complete, the mixture was concentrated under high vacuum to produce 3-13d (14.4 mg, crude material) as a brown solid. The crude product was used directly in the next step without further purification.

[1683] Step 5

[1684] (S)-2-Amino-N1-((S)-10-benzyl-1-(1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1H-1,2,3-triazol-4-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-13e)

[1685] To a solution of 3-13d (14.4 mg, 0.02 mmol) in DMF (1 mL) was added 2-22a (10 mg, 0.022 mmol), CuI (1 mg, catalytic amount) and DIPEA (17.5 μL, 12.9 mg, 0.1 mmol). The mixture was purged with N2 balloons for 15 min, then stirred at rt for 3 h under N2 atmosphere. After the reaction was complete, the mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give compound 3-13e (19 mg, 79.1% yield) as a white solid.

[1686] MS (ESI) m / z: 1184.1 [M+H] +

[1687] Step 6

[1688] (S)-N1-((S)-10-benzyl-1-(1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1H-1,2,3-triazol-4-yl)-6, 9,12,15-Tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-13)

[1689] To a solution of compound 3-13e (19 mg, 0.016 mmol) in DMF (1.5 mL) was added compound 3-11 (4 mg, 0.019 mmol), HATU (12 mg, 0.032 mmol) and DIPEA (8.4 μL, 6.2 mg, 0.048 mmol). The mixture was stirred at rt for 30 min. The mixture was purified by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19 * 150 mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give compound 3-13 (4.5 mg, 20.5% yield).

[1690] MS (ESI) m / z: 1398.7 [M+Na] + . Retention time (3.96min).

[1691] Examples 3-14

[1692]

[1693]

[1694] Step 1

[1695] ((6S,15S)-15-Benzyl-24-(((1S,10S)-10-ethyl-5,5-difluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 (9H-fluoren-9-yl)methyl 3,7,10,13,16,19,24-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (3-14a)

[1696] To a mixture of 3-1i (20.776 mg, 0.035 mmol) and 1-16 (30 mg, 0.032 mmol) was added HATU (12.05 mg, 0.032 mmol) and DIPEA (16 μL, 12.26 mg, 0.095 mmol). The mixture was stirred at RT for 10 min. The mixture was filtered and the filtrate was purified by preparative HPLC (FA 0.1%). The fractions were concentrated under vacuum and co-evaporated with Tol once to remove water to produce 3-14a (40 mg, 81.3% yield) as a red solid.

[1697] MS (ESI) m / z: 1437.7 [M+Na] + ;

[1698] Step 2

[1699] (S)-2-Amino-N1-((S)-10-benzyl-1-(((1S,10S)-10-ethyl-5,5-difluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indole) (2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-14b)

[1700] To a solution of 3-14a (40.0 mg, 0.028 mmol) in DMF (2 mL) was added EtNH (36.416 uL, 0.354 mmol). The mixture was stirred at RT for 30 min. The mixture was concentrated under vacuum and co-evaporated twice with Tol (10 mL) to remove DMF and EtN. 3-14b (40.0 mg, crude material) was generated as a yellow solid.

[1701] MS (ESI) m / z: 1193.7 [M+H] + ;

[1702] Step 3

[1703] (S)-N1-((S)-10-Benzyl-1-(((1S,10S)-10-ethyl-5,5-difluoro-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)- 1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-14)

[1704] To a solution of 3-14b (7.087 mg, 0.034 mmol) and 3-11 (40.0 mg, crude) in DMF (1.5 mL) was added HATU (12.758 mg, 0.034 mmol) and DIPEA (6 uL, 4.770 mg, 0.037 mmol). The mixture was stirred at rt for 10 min. The mixture was purified by preparative HPLC (FA 0.1%) and the fractions were lyophilized to give 3-14 (13.3 mg, 28.3% yield) as a white solid.

[1705] MS (ESI) m / z: 1408.9 [M+Na] + ;

[1706] Examples 3-15

[1707]

[1708] Steps 1 to 3

[1709] (S)-N1-((S)-10-Benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentahydro-1- ... Oxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-15)

[1710] 3-15 (5.1 mg, 38% yield) was synthesized according to the synthetic procedure of Example 3-1.

[1711] MS (ESI) m / z: 1324.9 [M+H] +

[1712] Example 3-16

[1713]

[1714] Steps 1 to 3

[1715] (S)-N1-((S)-10-Benzyl-1-(((1S,9S)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15- Pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-16).

[1716] 3-16 (13.1 mg, 53% yield) was synthesized according to the synthetic procedure of Example 3-1.

[1717] MS (ESI) m / z: 1364.7 [M+Na] +

[1718] Example 3-17

[1719]

[1720] Steps 1 to 3

[1721] (S)-N1-((S)-10-Benzyl-1-(((1S,9S)-9-ethyl-9-hydroxy-5-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15- Pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-17)

[1722] 3-17 (5.7 mg, 46% yield) was synthesized according to the synthetic procedure of Example 3-1.

[1723] MS (ESI) m / z: 1336.7 [M+H] +

[1724] Example 3-18

[1725]

[1726] Steps 1 to 3

[1727] (S)-N1-((S)-10-Benzyl-1-(((1R,9S)-9-ethyl-9-hydroxy-5-methoxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-15-methylene- 1,6,9,12-Tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(2-(((2R,3S,4R)-1,3,4,5-tetrahydroxypentan-2-yl)oxy)ethyl)glutaramide (3-18)

[1728] 3-18 (7.4 mg, 42% yield) was synthesized according to the synthetic procedure of Example 3-1.

[1729] MS (ESI) m / z: 1336.7 [M+H] +

[1730] Example 3-20

[1731]

[1732] (S)-N1-((S)-7-Benzyl-17-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5, 8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-20)

[1733] 3-20 (5.1 mg, 97.94% purity, 24.4% yield) was synthesized according to a procedure similar to Example 3-1.

[1734] MS (ESI) m / z: 1422.7 [M+Na] +

[1735] Example 3-21

[1736]

[1737] Step 1

[1738] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-23,23-dimethyl-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatetracosanoic acid (3-7b)

[1739] Compound 3-7b (38.5 mg, crude material) was synthesized according to the synthetic procedure of step 6 of Example 3-1.

[1740] MS (ESI) m / z: 1014.6 [M+Na] +

[1741] Step 2

[1742] ((6S,15S)-15-Benzyl-25-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-24-((6S,15S)-15-Benzyl-25- ... 9H-Fluoren-9-ylmethyl, 24-dimethyl-3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (3-21a)

[1743] Compound 3-21a (22 mg, 31.4% yield) was synthesized according to the synthetic procedure of step 7 of Example 3-1.

[1744] MS (ESI) m / z: 1443.7 [M+Na] +

[1745] Step 3

[1746] (S)-2-amino-N1-((S)-7-benzyl-17-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2 -b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-21b)

[1747] Compound 3-21b (18.5 mg, crude material) was synthesized according to the synthetic procedure of Step 8 of Example 3-1.

[1748] MS (ESI) m / z: 1221.7 [M+Na] +

[1749] Step 4

[1750] (S)-N1-((S)-7-Benzyl-17-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dihydro- Methyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-21)

[1751] Compound 3-21 (7.0 mg, 32.6% yield) was synthesized according to the synthetic procedure of step 9 of Example 3-1.

[1752] MS (ESI) m / z: 1414.7 [M+Na] +

[1753] Example 3-22

[1754]

[1755] Step 1 Benzyl 3-hydroxypropionate (3-22a)

[1756] 3-Hydroxypropionic acid (1g, 3.3mmol, 30% in H2O solution) and KOH (187mg, 3.3mmol) were stirred at rt for 30min, and the mixture was concentrated in vacuo to generate a white solid. The solid was suspended in DMF (10mL) and BnBr (570mg, 3.3mmol) was added via a syringe at rt. After stirring at 80°C for 5h, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: PE / EA=100 / 0 to 30 / 70) to generate the title compound 3-22a (420mg, 70% yield) as a colorless oil.

[1757] 1 H NMR (400MHz, CDCl3) δ7.39-7.33 (m, 5H), 5.16 (s, 2H), 3.88 (t, J = 5.6H, 2H), 2.66 (t, J = 5.6H, 2H), 2.38 (br s, 1H).

[1758] Step 2

[1759] (S)-11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaheneicosane-21-oic acid benzyl ester (3-22b)

[1760] Compound 3-22b (1.2 g, 83.9% yield) was synthesized according to the synthetic procedure of step 5 of Example 3-1.

[1761] MS (ESI) m / z: 773.5 [M+Na] +

[1762] Step 3

[1763] (S)-1-Amino-7-benzyl-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecan-17-oic acid benzyl ester (3-22c)

[1764] Compound 3-22c (150 mg, crude material) was synthesized according to the synthetic procedure of step 8 of Example 3-1.

[1765] MS (ESI) m / z: 528.3 [M+H] +

[1766] Step 4

[1767] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatetracosanoic acid benzyl ester (3-22d)

[1768] Compound 3-22d (170 mg, 80.9% yield) was synthesized according to the synthetic procedure of step 5 of Example 3-1.

[1769] MS (ESI) m / z: 1076.6 [M+Na] +

[1770] Step 5

[1771] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatetracosanoic-24-oic acid (3-22e)

[1772] Compound 3-22e (150 mg, crude material) was synthesized according to the synthetic procedure of step 6 of Example 3-1.

[1773] MS (ESI) m / z: 986.5 [M+Na] +

[1774] Step 6

[1775] ((6S,15S)-15-benzyl-25-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,7, 10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (3-22f)

[1776] Compound 3-22f (190 mg, 88.4% yield) was synthesized according to the synthetic procedure of step 7 of Example 3-1.

[1777] MS (ESI) m / z: 1404.5 [M+Na] +

[1778] Step 7

[1779] (S)-2-Amino-N1-((S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-22g)

[1780] Compound 3-22g (190 mg, crude material) was synthesized according to the synthetic procedure of Step 8 of Example 3-1.

[1781] Step 8

[1782] (S)-N1-((S)-7-Benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,5,8,11, 17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-22)

[1783] Compound 3-22 (50.3 mg, 54.1% yield) was synthesized according to the synthetic procedure of step 9 of Example 3-1.

[1784] 1H NMR(400MHz, DMSO-d6)δ8.51(d,J=9.2Hz,2H),8.32(s,1H),8.13(d,J=8.0H z,2H),8.03(d,J=7.2Hz,2H),7.80(d,J=11.2Hz,2H),7.32(s,1H),7.28-7.1 9(m,4H),7.17(d,J=6.6Hz,1H),7.00(d,J=8.0Hz,2H),6.54(s,1H),5.56(d, J=8.4Hz,1H),5.43(s,2H),5.22(d,J=4.8Hz,2H),5.01(d,J=5.2Hz,1H),4.9 4-4.84(m,2H),4.65-4.44(m,3H),4.43-4.33(m,1H),4.19(d,J=6.0Hz,1H) ,3.84-3.51(m,10H),3.22-2.93(m,8H),2.88(s,2H),2.83-2.70(m,1H),2.3 7(d,J=29.2Hz,6H),2.10(dd,J=14.6,7.3Hz,7H),1.97-1.78(m,3H),1.72(d ,J=8.0Hz,1H),1.59-1.37(m,4H),1.22-1.10(m,2H),0.87(t,J=7.2Hz,3H).

[1785] MS (ESI) m / z: 1374.9 [M+Na] + ; UPLC-MS retention time: 3.84min.

[1786] Example 3-23

[1787]

[1788] (S)-N-(S)-(S)-(N) ... 1 -((S)-7-Benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-((S)-7-Benzyl-17-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl) 5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-23) (32 mg, 26.3% yield).

[1789] MS (ESI) m / z: 1381.1 [M+H] +

[1790] Example 3-24

[1791]

[1792] Step 1

[1793] Benzyl 2-fluoro-3-hydroxy-2-methylpropionate (3-24a)

[1794] To a solution of compound 2-41c (450 mg, 3.69 mmol) in DMF (10 mL) was added KCO (1.02 g, 7.37 mmol) and BnBr (945 mg, 5.53 mmol). The mixture was stirred at 35 ° C for 16 h. The mixture was diluted with EtOAc (200 mL) and washed with brine (50 mL * 4). The organic layer was dried over anhydrous NaSO, filtered and concentrated. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 100 / 0 to 30 / 70) to obtain 3-24a (270 mg, 34.5% yield) as a colorless oil.

[1795] 1 H NMR (400MHz, CDCl3) δ7.42-7.33(m,5H),5.26(s,2H),4.06-3.61(m,2H),2.08(br s,1H),1.55(d,J=21.2Hz,3H).

[1796] Step 2

[1797] (11S)-11-Benzyl-1-(9H-fluoren-9-yl)-20-fluoro-20-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaheneicosane-21-oic acid benzyl ester (3-24b)

[1798] To 3-1g (250mg, 0.40mmol), 3-24a (253mg, 1.19mmol) and dried To a mixture of molecular sieves (500 mg) in anhydrous THF (8 mL) was added scandium trifluoromethanesulfonate (293 mg, 0.60 mmol) and stirred overnight at rt under an N atmosphere. The solution was filtered through celite, diluted with EtOAc (100 mL), and washed with saturated NaHCO (40 mL*3). The organic layer was dried over anhydrous NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 100 / 0 to 50 / 50) to give 3-24b (210 mg, 67.7% yield).

[1799] MS (ESI) m / z: 652.3 [M+Na] +

[1800] Step 3

[1801] (7S)-1-Amino-7-benzyl-16-fluoro-16-methyl-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecan-17-oic acid benzyl ester (3-24c)

[1802] To a solution of compound 3-24b (210 mg, 0.27 mmol) in DMF (4 mL) was added EtNH (392 mg, 5.37 mmol). The mixture was stirred at rt for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (3 mL * 3) to produce 3-24c (210 mg, crude material) as a brown solid.

[1803] MS (ESI) m / z: 582.3 [M+Na] +

[1804] Step 4

[1805] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-23-fluoro-23-methyl-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatetracosanoic acid benzyl ester (3-24d)

[1806] To a solution of compound 3-24c (210 mg, crude material) in DMF (4 mL) was added 3-1e (161 mg, 0.29 mol), HATU (153 mg, 0.40 mmol) and DIEA (69 mg, 0.63 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% plus): B-acetonitrile; flow rate: 20 mL / min) to provide 3-24d (154 mg, 52.9% yield) as a white solid.

[1807] MS (ESI) m / z: 1108.7 [M+Na] +

[1808] Step 5

[1809] (5S,14S)-14-Benzyl-1-(9H-fluoren-9-yl)-23-fluoro-23-methyl-3,6,9,12,15,18-hexaoxo-5-(3-oxo-3-((((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)amino)propyl)-2,21-dioxa-4,7,10,13,16,19-hexaazatetracosanoic-24-oic acid (3-24e)

[1810] To a solution of compound 3-24d (154 mg, 0.14 mmol) in MeOH (6 mL) was added Pd / C (10%, 30 mg). The mixture was stirred under a H2 atmosphere (15 psi) for 4 h. The mixture was filtered through a pad of celite and concentrated to yield compound 3-24e (141 mg, 100% yield) as a white solid.

[1811] MS (ESI) m / z: 1018.6 [M+Na] +

[1812] Step 6

[1813] ((6S,15S)-15-Benzyl-25-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-24-fluoro-24-methyl (9H-fluoren-9-yl)methyl 3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (3-24f)

[1814] To a solution of compound 3-24e (141 mg, 0.15) in DMF (4 mL) was added exatecan mesylate (86 mg, 0.16 mol), HATU (88 mg, 0.23 mmol) and DIEA (100 mg, 0.77 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min) to provide compound 3-24f (62 mg, 28.3% yield), which was obtained as a white solid.

[1815] MS (ESI) m / z: 1436.7 [M+Na] +

[1816] Step 7

[1817] (2S)-2-Amino-N1-((7S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16-fluoro-16-methyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-24g)

[1818] To a solution of compound 3-24f (62 mg, 0.44 mmol) in DMF (4 mL) was added Et2NH (64 mg, 0.88 mmol). The mixture was stirred at rt for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (3 mL*3) to produce 3-24g (62 mg, crude material) as a brown solid.

[1819] MS (ESI) m / z: 1191.7 [M+Na] +

[1820] Step 8

[1821] (2S)-N1-((7S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16-fluoro-16-methyl-2, 5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-24)

[1822] To a solution of compound 3-24g (52 mg, 0.44 mmol) in DMF (3 mL) was added 3-11 (19 mg, 0.088 mol), HATU (33 mg, 0.088 mmol) and DIEA (11 mg, 0.088 mmol). The mixture was stirred at rt for 30 min. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min to provide compound 3-24 (18.4 mg, 36.2% yield) as a white solid. UPLC-MS retention time: 3.99 min.

[1823] 11H NMR (400 MHz, DMSO-d6) δ 8.91 (d, J = 9.2 Hz, 1H), 8.59 (t, J = 6.8 Hz, 1H), 8.33 (t, J = 5.8 Hz, 1H), 8.12 (d, J = 7.2 Hz, 2H), 8.03 (t, J = 7.6 Hz, 2H), 7.77 (d, J = 10.8 Hz, 2H), 7.30 (s, 1H), 7.27 - 7.21 (m, 4H), 7.20 - 7.11 (m, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.52 (s, 1H), 5.58 (d, J = 7.8 Hz, 1H), 5.​​​​​​​​​​​​​​​​​​​((6S,15S)-15-Benzyl-25-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-24-fluoro-24-methyl (9H-fluoren-9-yl)methyl 3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (3-25a)

[1830] Compound 3-25a (35.0 mg, 81.5% yield) was synthesized according to the synthetic procedure of step 6 of Example 3-1.

[1831] MS (ESI) m / z: 1453.9 [M+Na] +

[1832] Step 2

[1833] (2S)-2-Amino-N1-((7S)-7-benzyl-17-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16-fluoro-16-methyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-25b)

[1834] Compound 3-25b (29.6 mg, crude material) was synthesized according to the synthetic procedure of step 7 of Example 3-1.

[1835] MS (ESI) m / z: 1231.9 [M+Na] +

[1836] Step 3

[1837] (2S)-N1-((7S)-7-benzyl-17-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16-fluoro-16-methyl-2, 5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-25)

[1838] Compound 3-25 (12.6 mg, 36.7% yield) was synthesized according to the synthetic procedure of step 8 of Example 3-1.

[1839] MS (ESI) m / z: 1442.8 [M+Na] +

[1840] Example 3-26

[1841]

[1842] Step 1

[1843] (9H-Fluoren-9-yl)methyl ((10S)-10-benzyl-1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate (3-26a)

[1844] To a solution of 3-1 g (100 mg, 0.16 mmol) in THF (5 mL) were added 2-46 (73.77 mg, 0.16 mmol) and To the product of 4-nitro-1-oxo-2-nitro-2-propenetrater (5-nitro-1-oxo-2-propenetrater) and 4-nitro-1-oxo-2-propenetrater (5-nitro-1-oxo-2-propenetrater) at 40 ℃, 4-nitro-1-oxo-2-propenetrater (5-nitro-1-oxo-2-propenetrater) (6-nitro-1-oxo-2-propenetrater) (7-nitro-1-oxo-2-propenetrater) (8-nitro-1-oxo-2-propenetrater) (9-nitro-1-oxo-2-propenetrater) (10-nitro-1-oxo-2-propenetrater) (10-nitro-1-oxo-2-propenetrater) (6-nitro-1-oxo-2-propenetrater) (7-nitro-1-oxo-2-propenetrater) (8-nitro-1-oxo-2-propenetrater) (9-nitro-1-oxo-2-propenetrater) (6-nitro-1-oxo-2-propenetrater) (8-nitro-1-oxo-2-propenetrater) (9-nitro-1-oxo-2-propenetrater) (

[1845] MS (ESI) m / z: 1034.4 [M+H] +

[1846] Step 2

[1847] (2S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((2-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (3-26b)

[1848] To a solution of 3-26a (110 g, 0.11 mmol) in DMF (3 mL) was added Et2NH (142.5 mg, 0.19 mmol). The mixture was stirred at rt for 1 h. The mixture was concentrated and co-evaporated with toluene (3*3 mL). The crude material 3-26b (86.3 mg, 100% yield) was used in the next step without further purification.

[1849] MS (ESI) m / z: 812.3 [M+H] +

[1850] Step 3

[1851] (9H-Fluoren-9-yl)methyl ((6S,15S)-15-benzyl-24-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,7,10,13,16,19-hexaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazatetracosan-6-yl)carbamate (3-26c)

[1852] To a solution of compound 3-26b (86.3 mg, 0.11 mmol) in DMF (3 mL) was added 3-1e (57.8 mg, 0.11 mmol), HATU (50.16 mg, 0.13 mmol) and DIEA (28.38 mg, 0.22 mmol). The mixture was stirred at 20 ° C for 30 min. LCMS showed that the reaction was complete. The mixture was filtered and the filtrate was purified using preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% trifluoroacetic acid): B-acetonitrile; flow rate: 20 mL / min to provide compound 3-26c (15 mg, 10.2% yield) as a white solid.

[1853] MS (ESI) m / z: 1338.5 [M+H] +

[1854] Step 4

[1855] (2S)-2-Amino-N1-((10S)-10-benzyl-1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-26d)

[1856] To a solution of 3-26c (15 mg, 0.01 mmol) in DMF (1 mL) was added Et2NH (14.25 mg, 0.02 mmol). The mixture was stirred at rt for 1 h. The mixture was concentrated and co-evaporated with toluene (3*3 mL). The crude material 3-26d (11.16 mg, 100% yield) was used in the next step without further purification.

[1857] MS (ESI) m / z: 1116.4 [M+H] +

[1858] Step 5

[1859] (2S)-N1-((10S)-10-benzyl-1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-6,9,12,15-tetrahydro 5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-26)

[1860] To a solution of 3-26d (11.16 mg, 0.01 mmol) in DMF (1 mL) and 3-11 (2.11 mg, 0.01 mmol) was added HATU (3.8 mg, 0.01 mmol) and DIEA (2.58 mg, 0.02 mmol) was added to the mixture and reacted at the same temperature for 15 min. The mixture was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min). 3-26 was obtained as a white solid (3.2 mg, 24.5% yield).

[1861] MS (ESI) m / z: 1309.5 [M+H] +

[1862] Example 3-27

[1863]

[1864] Step 1

[1865] ((S)-(9H-fluoren-9-yl)methyl (7-benzyl-17-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)carbamate (3-27a)

[1866] Compound 3-27a (25 mg, 58.1% yield) was synthesized according to the synthetic procedure of step 1 of Example 3-26a.

[1867] MS (ESI) m / z: 1060.9 [M+H] +

[1868] Step 2

[1869] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (3-27b)

[1870] Compound 3-27b (26 mg, crude) was synthesized as a brown solid according to the synthetic procedure of step 2 of Example 3-26b.

[1871] MS (ESI) m / z: 838.7 [M+H] +

[1872] Step 3

[1873] ((6S,15S)-15-Benzyl-25-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- (9H-fluoren-9-yl)methyl 3,7,10,13,16,19-hexaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (3-27c)

[1874] Compound 3-27c (20 mg, 47.6% yield) was synthesized as a yellow solid according to the synthetic procedure of step 3 of Example 3-26c.

[1875] MS (ESI) m / z: 1365.1 [M+H] +

[1876] Step 4

[1877] (S)-2-Amino-N1-((S)-7-benzyl-17-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-27d)

[1878] Compound 3-27d (22 mg, crude) was synthesized as a yellow solid according to the synthetic procedure of step 4 of example 3-26d.

[1879] MS (ESI) m / z: 1142.9 [M+H] +

[1880] Step 5

[1881] (S)-N1-((S)-7-Benzyl-17-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2, 5,8,11-Tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecanyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)glutaramide (3-27)

[1882] Compound 3-27 was synthesized according to the synthetic procedure of step 5 of Example 3-26. The crude product was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5um 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give a white solid (4 mg, 10.5% yield).

[1883] MS (ESI) m / z: 1337.0 [M+H] +

[1884] Example 3-28

[1885]

[1886] Step 1

[1887] (9H-Fluoren-9-yl)methyl ((7S)-7-benzyl-18-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaoctadecyl)carbamate (3-28a)

[1888] Compound 3-28a (85.0 mg, 62.5% yield) was synthesized according to the synthetic procedure of step 1 of Example 3-26a.

[1889] MS (ESI) m / z: 1062.8 [M+H] +

[1890] Step 2

[1891] (2S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((4-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)butoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (3-28b)

[1892] Compound 3-28b (61.7 mg, crude material) was synthesized according to the synthetic procedure of step 2 of Example 3-26b.

[1893] MS (ESI) m / z: 840.7 [M+H] +

[1894] Step 3

[1895] (9H-Fluoren-9-yl)methyl ((6S,15S)-15-benzyl-26-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,1...

Claims

1. A compound having formula (I): or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein Y is -ABCDH; A is the key, CR 1 R 2 or NR 1 ; B is a bond, -C(=O)-, -C(=O)O-, or -OC(=O)-; C is a bond or a divalent group selected from unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D is a bond, NH or O; R 1 and R 2 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 1 and R 2 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 3 and R 4 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 3 and R 4 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; and When R 3 is a methyl group, and R 4 When it is F, Y is not -NH-C(=O)-CDH.

2. The compound of claim 1, wherein the compound is a compound having formula (II): or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein A is for CR 1 R 2 , NH or NR 1 ; R 1 and R 2 Each of 1-4 alkyl; R 3 and R 4 Each of R is independently hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy; or 3 and R 4 together with the atoms to which they are attached, form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 5 and R 6 Each of is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; and n is 1, 2, 3, 4 or 5.

3. The compound of claim 2, wherein A is -CH2- and B is a bond.

4. The compound as claimed in claim 3, wherein R 5 and R 6 is hydrogen, and n is 1, 2 or 3.

5. The compound as claimed in claim 4, wherein R 3 is a methyl group, and R 4 It's F.

6. The compound of claim 5, wherein the compound is 7. The compound of claim 4, wherein R 3 and R 4 Together with the atoms to which they are attached they form an unsubstituted or substituted dioxole ring.

8. The compound of claim 7, wherein the compound is 9. The compound of claim 2, wherein R 3 is a methyl group, and R 4 It's F.

10. The compound of claim 9, wherein A is -N(CH3)-, and B is a bond.

11. The compound of claim 10, wherein R 5 and R 6 is hydrogen, and n is 2; or the compound is 12. The compound of claim 2, wherein R 3 is a methyl group, and R 4 It's F.

13. The compound of claim 12, wherein A is -NH-, and B is -C(=O)O-.

14. The compound of claim 13, wherein R 5 and R 6 is hydrogen, and n is 2; or the compound is 15. The compound of claim 2, wherein R 3 It is Cl, R 4 is F, and B is -C(=O)-.

16. The compound of claim 15, wherein the compound is 17. The compound of claim 2, wherein R 3 is methyl, R 4 is Cl, and B is -C(=O)-.

18. The compound of claim 17, wherein the compound is 19. The compound of claim 2, wherein R 3 and R 4 Together with the atoms to which they are attached they form an unsubstituted or substituted heterocyclyl.

20. The compound of claim 19, wherein R 3 and R 4 Together with the atoms to which they are attached, they form an unsubstituted or substituted dioxole ring, and B is -C(=O)-.

21. The compound of claim 20, wherein the compound is 22. The compound of claim 1, wherein the compound has formula (III): or a pharmaceutically acceptable solvate, stereoisomer or derivative thereof.

23. The compound of claim 22, wherein R 3 is methyl; and R 4 is Cl; or the compound is 24. The compound of claim 22, wherein R 3 is F; and R 4 is F; or the compound is 25. The compound of claim 22, wherein R 3 is H; and R 4 is F; or the compound is 26. The compound of claim 22, wherein R 3 is H; and R 4 is OH; or the compound is 27. The compound of claim 22, wherein R 3 is methyl; and R 4 is methyl; or the compound is 28. The compound of claim 22, wherein R 3 is methoxy; and R 4 is F; or the compound is 29. The compound of claim 22, wherein R 3 is H; and R 4 is methoxy; or the compound is 30. The compound of claim 22, wherein R 3 is H; and R 4 is Cl; or the compound is 31. The compound of claim 22, wherein R 3 and R 4 Together with the atoms to which they are attached they form an unsubstituted or substituted heterocyclyl.

32. The compound of claim 31, wherein R 3 and R 4 Together with the atoms to which they are attached they form an unsubstituted or substituted dioxole ring.

33. The compound of claim 32, wherein the compound is 34. The compound of claim 32, wherein the compound is 35. The compound of claim 1, wherein the compound is 36. A compound selected from Table 1 and Table 2.

37. A compound selected from Table 3 and Table 4.

38. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a residue of a compound as claimed in any one of claims 1 to 36.

39. The ligand-drug conjugate of claim 38, wherein the ligand-drug conjugate comprises a structure of formula (V): in BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody; L is a covalent linker as described herein; and x is 1 to 10, and it can be an integer or a decimal.

40. The ligand-drug conjugate of claim 39, wherein the antibody is pertrastuzumab, cofetuzumab, trastuzumab, ifenatumumab, or mAb10.

41. A compound having formula (VII): or a pharmaceutically acceptable salt, tautomer, isotopomer, stereoisomer or prodrug thereof, wherein R 7 and R 8 Each of R is independently hydrogen or substituted or unsubstituted alkyl; or 7 and R 8 Together with the nitrogen atom to which they are attached, they form an unsubstituted or substituted heterocyclyl or an unsubstituted or substituted heteroaryl.

42. The compound of claim 41, wherein the compound is 43. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a residue of a compound as claimed in claim 41 or 42.

44. The ligand-drug conjugate of claim 43, wherein the ligand-drug conjugate comprises a structure of Formula (VIIIa), (VIIIb) or (VIIIc): in BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody; L is a covalent linker as described herein; and x is 1 to 10, and it can be an integer or a decimal.

45. The ligand-drug conjugate of claim 44, wherein the ligand-drug conjugate comprises a structure of any one of the following formulae: in BA is a binding agent selected from a humanized, chimeric or human antibody or an antigen-binding antibody fragment of an antibody; L is a covalent linker as described herein; and x is 1 to 10, and it can be an integer or a decimal.

46. ​​The ligand-drug conjugate of claim 44 or 45, wherein the antibody is pertrastuzumab, cofetuzumab, trastuzumab, ifenatumumab, or mAb10.

47. The ligand-drug conjugate of any one of claims 39, 40, 44, 45 and 46, wherein L is The bonds marked with an asterisk are connected to BA.

48. The ligand-drug conjugate of any one of claims 39, 40, 44, 45 and 46, wherein L is The bonds marked with an asterisk are connected to BA.

49. An antibody or antigen-binding fragment thereof that specifically binds to human HER3, comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

50. The antibody or antigen-binding fragment thereof of any one of claims 39, 40, 44, 45 and 46, comprising: (i) the heavy chain variable region of SEQ ID NO: 1; and (ii) the light chain variable region of SEQ ID NO:

2.

51. The antibody or antigen-binding fragment thereof as described in any one of claims 49-50, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

52. The antibody or antigen-binding fragment thereof of any one of claims 49 to 51, comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 3; and (ii) a light chain comprising the amino acid sequence of SEQ ID NO:

4.

53. The antibody or antigen-binding fragment thereof of any one of claims 49 to 52, comprising: (i) the heavy chain of SEQ ID NO: 3; and (ii) the light chain of SEQ ID NO:

4.

54. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 49-53.

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