Targeting pyrrolobenzodiazepine conjugates

Abstract

Compounds comprising pyrrolobenzodiazepine (PBD) conjugates and methods of using such conjugates are provided. In some embodiments, the conjugates have Formula (I): or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof wherein each of ring A and ring B independently has one of the following formulae: Formula (IIa), Formula (IIb), Formula (IIc), Formula (IId), Formula (IIe), Formula (IIf), Formula (IIg): and the values of the remaining variables (e.g., linker, ring C, R1, R2, R3, R4, R5, m, n, o) are as described herein.

Description

1. Cross-references to related applications

[0001] This application claims priority to International Application No. PCT / CN2023 / 088051, filed on April 13, 2023, the contents of which are incorporated herein by reference in their entirety. 2. Technical Field

[0002] This disclosure relates to targeted pyrrolobenzodiazepines. (PBD) conjugates 3. Sequence List

[0003] This application contains a sequence list, which has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The XML copy was created on March 26, 2024, and is named “01368-0071-00PCT_SL”, with a size of 3,443 bytes. 4. Background Technology

[0004] Some pyrrolobenzodiazepines PBDs possess the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. The first PBD antitumor antibiotic, amiodarone, was discovered in 1965 (Leimgruber et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, numerous naturally occurring PBDs have been reported, and more than a dozen synthetic routes have been developed to synthesize various analogs (Thurston et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abenomycin (Hochlowski et al., J. Antibiotics, 40, 145-148 (1987)), checamycin (Konishi et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180487; Thurston et al., Chem. Brit., 26, 767-772 (1990); Bose et al., Tetrahedron, 48, 751-758 (1992)), methylanisoxamycin (Kuminoto et al., J. Antibiotics, 33, 665-667 (1980)), neoanisoxamycin A and B (Takeuchi et al., J. Antibiotics, 29, 93-96 (1976)), and polomycin (Tsunakawa). PBD has the following general structure: (e.g., J. Antibiotics, 41, 1366-1373 (1988)), pravastatin (Shimizu et al., J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanamicin (DC-102) (Hara et al., J. Antibiotics, 41, 702-704 (1988); Itoh et al., J. Antibiotics, 41, 1281-1284 (1988)), siberiamycin (Leber et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)), and tomatine (Arima et al., J. Antibiotics, 25, 437-444 (1972)).

[0005]

[0006] The number, type, and position of substituents on the aromatic A ring and pyrrolo C ring of PBD, as well as the saturation of the C ring, vary. The N10-C11 positions of the B ring contain imine (N=C), methylamine (NH-CH(OH)), or methylamine methyl ether (NH-CH(OMe)), which are electrophilic centers responsible for alkylating DNA. All known natural products have an (S) configuration at the chiral C11a position, which gives them a right-handed twist when viewed from the C ring to the A ring. This endows them with an appropriate three-dimensional shape with isohelicity in the minor groove of type B DNA, resulting in a tight fit at the binding site (Kohn, Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-Van Devanter, Acc. Chem. Res., 19, 230-237 (1986)). PBDs can form adducts in minor grooves, which allows them to interfere with DNA processing and thus can be used as antitumor agents.

[0007] The biological activity of these molecules can be enhanced by linking two PBD units together via their C8 / C′-hydroxyl functional groups through flexible alkylene linkers (Bose, DS et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, DE et al., J. Org. Chem., 61, 8141-8147 (1996)). PBD dimers are thought to form sequence-selective DNA damage, such as palindromic 5′-Pu-GATC-Py-3′ interstrand crosslinks (Smellie, M. et al., Biochemistry, 42, 8232-8239 (2003); Martin, C. et al., Biochemistry, 44, 4135-4147), which is considered a major reason for their biological activity. An example of a PBD dimer is SG2000 (SJG-136):

[0008]

[0009] (Gregson, S. et al., J. Med. Chem., 44, 737-748 (2001); Alley, MC et al., Cancer Research, 64, 6700-6706 (2004); Hartley, JA et al., Cancer Research, 64, 6693-6699 (2004)).

[0010] Previous PBD dimers have been prepared symmetrically, meaning the two monomers of the dimer are identical, due to the way these highly efficient compounds crosslink DNA. This synthetic route offers direct synthesis, either by simultaneously constructing the PBD dimer moieties that have already formed dimer linkages, or by reacting the constructed PBD monomer moieties with the dimer linker groups. These synthetic methods limit the options for preparing PBD-containing targeting conjugates. However, due to the observed potency of PBD dimers, there is a need for asymmetric PBD dimers that can be conjugated to targeting agents for use in targeted therapy. 5. Summary of the Invention

[0011] This article provides compounds of formula (I),

[0012]

[0013] Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers

[0014] Each of rings A and B independently has one of the following equations:

[0015]

[0016]

[0017] Indicator and connector connection point:

[0018] The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -;

[0019] X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings;

[0020] Ring C is a cyclopropyl ring or a cyclobutyl ring;

[0021] -C(R 1 )- and -N(R 2 The dashed key between )- Each of them is either a single bond or a double bond;

[0022] When the dashed key is a single key, each R 1 Independently H or OH, and each R 2For H;

[0023] When the dashed key is a double key, each R 1 For H, and each R 2 It does not exist;

[0024] R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

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

[0026] R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0027] R 6 For H or C 1-4 alkyl;

[0028] Each of m, n, and o is independently 1 or 2;

[0029] Each of r, p, and q is an independent integer from 1 to 8; and

[0030] The sum of p and q is an integer from 1 to 8.

[0031] This article also provides compounds of formula B(i) or B(ii):

[0032] Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein:

[0033] Each of rings A and B independently has one of the following equations:

[0034]

[0035]

[0036] Indicates the connection point with the connector or Ab connector;

[0037] The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2)p -CH = CH-(CH2) q -;

[0038] X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings;

[0039] Ring C is a cyclopropyl ring or a cyclobutyl ring;

[0040] -C(R 1 )- and -N(R 2 The dashed key between )- Each of them is either a single bond or a double bond;

[0041] When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 For H;

[0042] When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It does not exist;

[0043] R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0044] R a and R b Each independently is H or C 1-4 alkyl;

[0045] R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0046] R 6 For H or C 1-4 alkyl;

[0047] Each of m, n, and o is independently 1 or 2;

[0048] Each of r, p, and q is an independent integer from 1 to 8;

[0049] The sum of p and q is an integer from 1 to 8; and

[0050] Ab connectors are compounds that can bond ring A or ring B to a binder.

[0051] This article also provides conjugates of formula A(i) or A(ii):

[0052]

[0053] Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein:

[0054] Each of rings A and B independently has one of the following equations:

[0055]

[0056]

[0057] Indicates the connection point with the connector or Ab connector:

[0058] The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -;

[0059] X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings;

[0060] Ring C is a cyclopropyl ring or a cyclobutyl ring;

[0061] -C(R 1 )- and -N(R 2 Each of the dashed keys between ) and - is independently a single or double bond;

[0062] When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 H is independent;

[0063] When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It exists independently;

[0064] R 3 and R 4 Each of them is independently H, NH2, NR a R b ,oH,C 1-4 Alkyl, C 1-4Alkoxy or aryl, and R a and R b Each independently is H or C 1-4 alkyl;

[0065] R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0066] R 6 For H or C 1-4 alkyl;

[0067] Each of m, n, and o is independently 1 or 2;

[0068] Each of r, p, and q is an independent integer from 1 to 8; and

[0069] The sum of p and q is an integer from 1 to 8;

[0070] An Ab connector is a compound that attaches an Ab to ring A or ring B;

[0071] Ab is a conjugate selected from humanized, chimeric, or human antibodies or their antigen-binding fragments; and

[0072] The subscript x ranges from 1 to 15. 6. Description of the attached drawings

[0073] Figure 1 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0074] Figure 2 Line graph to show Calu-6 cell killing induced by the compounds disclosed herein.

[0075] Figure 3 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0076] Figure 4 Line graph to show Calu-6 cell killing induced by the compounds disclosed herein.

[0077] Figure 5 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0078] Figure 6 Line graph to show Calu-6 cell killing induced by the compounds disclosed herein.

[0079] Figure 7 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0080] Figure 8Line graph to show Calu-6 cell killing induced by the compounds disclosed herein.

[0081] Figure 9 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0082] Figure 10 Line graph to show Calu-6 cell killing induced by the compounds disclosed herein.

[0083] Figure 11 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0084] Figure 12 Line graph to show Calu-6 cell killing induced by the compounds disclosed herein.

[0085] Figure 13 Line graph to show the killing effect of the compounds disclosed herein on A375 cells.

[0086] Figure 14 Line graph to show Calu-6 cell killing induced by the compounds disclosed herein. 7. Detailed Implementation

[0087] This article provides a basis for pyrrolobenzodiazepines. A compound comprising two PBDs connected by a linker, the two PBDs being identical or different. The compound can be used as the pharmaceutical or payload portion of an antibody-drug conjugate (ADC). ADCs can be used to treat diseases or conditions, such as cancer, for example by providing a composition containing an ADC.

[0088] 7.1. Definition

[0089] In this disclosure, 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 commonly understood by one of ordinary skill in the art to which this disclosure pertains. If multiple definitions are provided for a term herein, those definitions shall prevail unless otherwise stated.

[0090] When a trade name is used in this document, unless the context otherwise indicates, reference to the trade name also refers to the product formulation, generic drug, and active pharmaceutical ingredient of the product under that trade name.

[0091] The term "antibody" is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting desired biological activity. An intact antibody primarily has two regions: a variable region and a constant region. The variable region binds to and interacts with the target antigen. The variable region includes a complementarity-determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region is recognized by 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), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and rgA2), or subclass of any of these. Antibodies can be derived from any suitable species. In some embodiments, the antibody has a human or mouse origin. Antibodies can be, for example, human, humanized, or chimeric.

[0092] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that the individual antibodies constituting said population are identical except for trace amounts of possibly naturally occurring mutations. Monoclonal antibodies are highly specific (targeting a single antigenic site). The modifier "monoclonal" should not be interpreted as requiring the production of the antibody through any particular method.

[0093] A “complete antibody” is an antibody that contains an antigen-binding variable region and light chain constant domains (CL) and heavy chain constant domains CH1, CH2, CH3, and CH4, depending on the antibody class. The constant domains can be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof.

[0094] An "antibody fragment" comprises a portion of a complete antibody, including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, bivalent antibodies, trivalent antibodies, tetravalent antibodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments generated from Fab expression libraries, or epitope-binding fragments of any of the above that are immune-specifically bound to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen).

[0095] An "antigen" is an entity that an antibody specifically binds to.

[0096] The terms "specific binding" and "specifically binds" mean that an antibody or antibody derivative will bind to its corresponding target antigen in a highly selective manner, rather than to a multitude of other antigens. Typically, antibodies or antibody derivatives bind at least about 1 × 10⁻⁶. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The affinity of M for binding is at least twice that for binding to a predetermined antigen (e.g., BSA, casein) other than the predetermined antigen or closely related antigens.

[0097] The terms “inhibit” or “inhibition of” mean to reduce a measurable amount or to completely prevent it.

[0098] The term "therapeutic effective dose" refers to the amount of a drug that is effective in treating a disease or symptom in a mammal. In the case of cancer, a therapeutically effective dose of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down or stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., to some extent slow down or stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. In terms of the drug's ability to inhibit growth and / or kill existing cancer cells, it can be cytoseptic and / or cytotoxic. For cancer therapies, efficacy can be measured, for example, by assessing time to progression (TTP) and / or determining response rate (RR).

[0099] The term "substantial" or "substantially" means the majority of 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.

[0100] The terms "intracellular cleavage" and "intracellular breakdown" refer to the metabolic process or reaction of a ligand-drug conjugate (e.g., an antibody-drug conjugate (ADC)) within a cell, in which the covalent link (e.g., a linker) between the drug moiety (D) and the ligand unit (e.g., an antibody (BA or Ab)) is disrupted, resulting in the dissociation of the free drug or another metabolite of the conjugate from the antibody within the cell. Therefore, the cleavage portion of a drug-linker-ligand conjugate is an intracellular metabolite.

[0101] The terms “cancer” and “cancerous” refer to or describe a physiological disorder or condition in mammals that is typically characterized by dysregulation of cell growth. A “tumor” contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia 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 squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (kidney or renal cancer), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0102] In this article, "autoimmune disease" refers to a disease or condition that originates from and targets an individual's own tissues or proteins.

[0103] Examples of “patient” or “subject” include, but are not limited to, mammals such as humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, or cats, as well as birds or poultry. In the implementation scheme, the patient is a human.

[0104] Unless the context otherwise indicates, the term "treat" or "treatment" refers to therapeutic treatments and preventative measures intended to prevent recurrence, wherein the aim is to suppress or slow (alleviate) unwanted physiological changes or conditions, such as the development or spread of cancer. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean, for example, prolonged survival compared to expected survival without treatment. Those requiring treatment include those who already have the disease or condition and those who are susceptible to it.

[0105] In the context of cancer, the term "treatment" includes any or all of the following: 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 the number of cancer cells; and improving one or more symptoms associated with the disease.

[0106] In the context of autoimmune diseases, the term "treatment" includes any or all of the following: inhibiting the replication of cells associated with an autoimmune disease state (including, but not limited to, cells that produce autoimmune antibodies), reducing the autoimmune antibody load, and improving one or more symptoms of the autoimmune disease.

[0107] As used herein, in this specification, and in the appended claims, the indefinite article “a / an” and the definite article “the” include both plural and single indicators, unless the context clearly indicates otherwise.

[0108] As used herein and unless otherwise stated, the terms “about” and “approximately”, when used in conjunction with the amount or weight percentage of a component of a composition, mean an amount or weight percentage that is generally recognized by those skilled in the art to provide a pharmacological effect equivalent to that obtained from the specified amount or weight percentage. In some embodiments, the terms “about” and “approximately”, when used in this context, cover an amount or weight percentage of up to 30%, 20%, 15%, 10%, or 5% of the specified amount or weight percentage.

[0109] As used herein and unless otherwise stated, the terms “about” and “approximately” when used in conjunction with numerical values ​​or ranges of values ​​provided to characterize a particular solid form (e.g., a particular temperature or temperature range, such as describing melting, dehydration, desolventizing, or glass transition temperatures; mass changes, such as mass changes with temperature or humidity; solvent or water content, expressed, for example, by mass or percentage; or peak positions, such as in analyses performed, for example, by IR or Raman spectroscopy or XRPD) indicate that the value or range of values ​​may deviate to a degree that would be reasonable to a person skilled 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 some embodiments, the terms "about" and "approximately," when used in this context, indicate that the stated numerical value or range may vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the stated value or range. For example, in some implementations, the value of the XRPD peak position may vary by up to ±0.2°2θ, while still describing a specific XRPD peak.

[0110] "Alkyl" is a saturated, partially saturated, or unsaturated straight-chain or branched acyclic hydrocarbon having 1 to 10 carbon atoms, typically 1 to 8 carbon atoms, 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; saturated branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. 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). Alkyl groups may be substituted or unsubstituted. In some embodiments, when the 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 compounds disclosed herein and in the embodiments thereof; and halogens (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; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2; or O(alkyl)aminocarbonyl.

[0111] "Alkenyl" is a straight-chain or branched acyclic hydrocarbon having 2 to 10 carbon atoms, typically 2 to 8 carbon atoms, and including at least one carbon-carbon double bond. Representative straight-chain and branched (C2-C8) alkenyl groups include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -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 an alkenyl group can be non-conjugated or conjugated with another unsaturated group. The alkyl group can be unsubstituted or substituted.

[0112] "Cycloalkyl" is a saturated or partially saturated cyclic alkyl group having 3 to 10 carbon atoms, having a single cyclic ring or multiple fused or bridged rings that may optionally be substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or polycyclic or bridged ring structures such as adamantyl. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, etc. Cycloalkyl groups may be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanone.

[0113] "Aryl" is an aromatic carbocyclic group having 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene). In some embodiments, the aryl group contains 6 to 14 carbon atoms in the ring portion of the group, and in other embodiments, it contains 6 to 12 or even 6 to 10 carbon atoms. Specific aryl groups include phenyl, biphenyl, naphthyl, etc. Aryl groups may 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.).

[0114] "Aneryl" is a divalent aryl group as defined in this article.

[0115] "Heteroaryl" is an aryl ring system in a heteroaromatic ring system having one to four heteroatoms as ring atoms, wherein the remaining atoms are carbon atoms. In some embodiments, the heteroaryl contains 5 to 6 ring atoms in the ring portion of the group, and in other embodiments, it contains 6 to 9 or 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In some embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, the following groups: such as pyrroloyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrroloyl, pyridinyl, pyrazinyl, thiophene, benzothiophene, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indoleyl, azaindoleyl (e.g., pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), indazoleyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazopyridyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thionyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl and quinazolinyl.

[0116] "Anearyl" is a divalent heteroaryl group as defined in this article.

[0117] A "heterocyclic group" is an aromatic (also called a 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 heterocyclic group comprises 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. The heterocyclic group may also be bonded to other groups at any ring atom (i.e., at any carbon atom or heteroatom of the heterocycle). The heterocyclic group may be substituted or unsubstituted. Heterocyclic groups encompass unsaturated, partially saturated, and saturated ring systems, such as, for example, imidazolyl, imidazolinyl, and imidazoalkyl. The term "heterocyclic group" includes fused ring classes, including those containing fused aromatic and non-aromatic groups, such as, for example, benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxane-hexenyl, and benzo[1,3]dioxane-pentenyl. The term also includes bridged polycyclic systems containing heteroatoms, such as, but not limited to, quinine cycloalkanes. Representative examples of heterocyclic groups include, but are not limited to, aziridinyl, aziridine, pyrrolyl, imidazoalkyl, pyrazolyl, thiazoalkyl, tetrahydrothiophene, tetrahydrofuranyl, dioxacyclopentenyl, furanyl, thiophene, pyrrolyl, pyrrololinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolinyl, thiazolinyl, isothiazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl. Tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiaranyl, oxothiacyclohexane, dioxacyclohexyl, dithiaranyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, dihydropyridinyl, dihydrodithiazinyl, dihydrodithionyl, periperazinyl, quininecycloyl, indoleyl, indolinyl, isoindoleyl, azaindoleyl (pyrrolopyridinyl), indazoleyl, inazinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiopheneyl Benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiazinyl, benzoxoxazinyl, benzoxazolyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiazolyl, benzo[1,3]dioxacyclopentenyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl; e.g., 1H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridin-2(3H)-keto), triazolopyridyl, isoxazolopyridyl, purine, yellow Purine, adenine, guanine, quinolinyl, isoquinolinyl, quinazinyl, quinoxalinyl, quinazolinyl, cyclophosphinyl, phthalazinyl, naphthidyl, pteridyl, thionyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxane-hexenyl, tetrahydroindolyl, tetrahydroindolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl.Representative substituted heterocyclic groups can be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholino, which are 2-substituted, 3-substituted, 4-substituted, 5-substituted or 6-substituted, or disubstituted by various substituents (such as those listed below).

[0118] "Cycloalkylalkyl" is a group of the following formula: -alkyl-cycloalkyl, wherein the alkyl and cycloalkyl groups are as defined above. A substituted cycloalkylalkyl group may be substituted at the alkyl, cycloalkyl, or both alkyl and cycloalkyl portions of the group. 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.

[0119] "Aryl" is a group of the formula -alkyl-aryl, wherein the alkyl and aryl groups are as defined above. A substituted aryl group may be substituted at the alkyl, aryl, or both alkyl and aryl portions of the group. Representative aryl groups include, but are not limited to, benzyl and phenethyl, as well as fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indenyl.

[0120] "Heterocyclic alkyl" is a group of the formula -alkyl-heterocyclic, wherein the alkyl and heterocyclic groups are as defined above. A substituted heterocyclic alkyl group may be substituted at the alkyl, heterocyclic, or both alkyl and heterocyclic portions of the group. Representative heterocyclic alkyl groups include, but are not limited to, 4-ethyl-morpholino, 4-propylmorpholino, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, (tetrahydro-2H-pyran-4-yl)methyl, (tetrahydro-2H-pyran-4-yl)ethyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylethyl, and indole-2-ylpropyl.

[0121] "Halogen" refers to chlorine, iodine, bromine, or fluorine.

[0122] "Hydroxyalkyl" is an alkyl group as described above that has been replaced by one or more hydroxyl groups.

[0123] "Alkoxy" is O (alkyl), where alkyl is as defined above.

[0124] "Alkoxyalkyl" is (alkyl)O(alkyl), where alkyl is as defined above.

[0125] As used herein, "alkynyl" refers to a monovalent hydrocarbon moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. The alkynyl group may optionally be substituted and may be straight-chain, branched-chain, or cyclic. Alynyl groups include, but are not limited to, those having 2-20 carbon atoms, i.e., C1... 2-20 Alkynyl group; 2-12 carbon atoms, i.e., C 2-12 Alkynyl group; 2-8 carbon atoms, i.e., C64. 2-8Alkynyl group; 2-6 carbon atoms, i.e., C64. 2-6 The alkynyl group; and 2-4 carbon atoms, i.e., C 2-4 Alkynyl group. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, and butynyl.

[0126] As used herein, “haloalkyl” means an alkyl group as defined above, wherein the alkyl group includes at least one substituent selected from halogens (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.

[0127] As used herein, “haloalkoxy” means an alkoxy group as defined above, wherein the alkoxy group includes at least one substituent selected from halogens (e.g., F, Cl, Br or I).

[0128] As used herein, “arylalkyl” refers to the monovalent portion of a group in an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, i.e., the aromatic compound includes a single bond with an alkyl group, and wherein the group is located on the alkyl group. The arylalkyl group is bonded to the chemical structure shown by the alkyl group. The arylalkyl group can be represented by, for example, the following structures: B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, where B is an aromatic moiety, such as phenyl. The arylalkyl group may optionally be substituted, i.e., the aryl and / or alkyl groups may be substituted as disclosed herein. Examples of arylalkyl groups include, but are not limited to, benzyl.

[0129] As used herein, “alkylaryl” refers to the monovalent portion of a group in an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, i.e., the aryl compound comprises a single bond with an alkyl group, and wherein the group is located on an aryl group. Alkylaryl groups are bonded to the illustrated chemical structure via the aryl group. Alkylaryl groups 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, where B is an aromatic moiety, such as a phenyl group. Alkylaryl groups are optionally substituted, i.e., the aryl group and / or the alkyl group may be substituted as disclosed herein. Examples of alkylaryl groups include, but are not limited to, tolueneyl groups.

[0130] As used herein, “aryloxy group” refers to the monovalent portion of a group in an aromatic compound, wherein the ring atom is a carbon atom and wherein the ring is substituted by an oxygen group, i.e., the aromatic compound comprises a single bond with an oxygen atom, and wherein the group is located on the oxygen atom, for example, C6H5-O- for phenoxy groups. The aryloxy substituents are bonded to the compound they substituted through this oxygen atom. The aryloxy group is optionally substituted. Aryloxy groups include, but are not limited to, those having 6 to 20 ring carbon atoms, i.e., C6H5-O-. 6-20 Aryloxy group; 6 to 15 ring carbon atoms, i.e., C 6-15 Aryloxy groups; and 6 to 10 ring carbon atoms, i.e., C 6-10 Aryloxy group. Examples of aryloxy groups include, but are not limited to, phenoxy, naphthoxy, and anthraceneoxy groups.

[0131] "Amino" is a group with the formula NH2.

[0132] The "hydroxylamine" group is of the formula N(R) # )OH or NHOH groups, wherein R # It is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic or heterocyclic alkyl group as defined herein.

[0133] The "alkoxyamine" group is of the formula -N(R # )O-alkyl or -NHO-alkyl groups, wherein R # As defined above.

[0134] The "arylalkoxyamine" group is of the formula N(R) # )O-aryl or NHO-aryl groups, wherein R # As defined above.

[0135] The "alkylamine" group is a group of the formula NHalkyl or N(alkyl)2, wherein each alkyl group is independently as defined above.

[0136] "Amino carbonyl" is a group with the following formula: -C(=O)N(R) # )2、-C(=O)NH(R # ) or C(=O)NH2, where each R # As defined above.

[0137] "Acylamino" is a group of the following formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ), wherein each alkyl group and R # Independently as defined above.

[0138] "O(alkyl)aminocarbonyl" is a group with the following formula: -O(alkyl)C(=O)N(R) #)2、-O(alkyl)C(=O)NH(R # ) or -O(alkyl)C(=O)NH2, wherein each R # Independently as defined above.

[0139] The “N-oxide” group is a group with the formula -N+-O-.

[0140] The "carboxyl group" is a group with the formula C(=O)OH.

[0141] The ketone group is represented by the formula C(=O)(R). # ) groups, wherein R # As defined above.

[0142] The "aldehyde" group is a group with the formula -CH (=O).

[0143] The "ester" group is a group with the following formula: C(=O)O(R # ) or OC (=O)(R # ), where R # As defined above.

[0144] The "urea" group is a group with the following 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 # Each alkyl group and R # Independently as defined above.

[0145] The "imine" group is a group with the following formula: -N=C(R # )2 or -C(R # )=N(R # ), where each R # Independently as defined above.

[0146] "Imine" is a group with the following formula: -C(=O)N(R) # )C(=O)(R # ) or N((C=O)(R # ))2, where each R # Independently as defined above.

[0147] The "carbamate" group is a group with the following formula: -OC(=O)N(R) # )2、-OC(=O)NH(R # ), -N(R #)C(=O)O(R # ) or -NHC(=O)O(R # ), where each R # Independently as defined above.

[0148] The "midamine" group is a group with the following 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)NH2 # )N(R # )2、-N=C(R # )NH(R # -N=C(R) # )NN2、-N(R # )C(R # )=N(R # ),-NHC(R # )=N(R # ), -N(R # )C(R # ) = NH or -NHC(R # ) = NH, where each R # Independently as defined above.

[0149] The "guanidine" group is a group with the following 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, where each R # Independently as defined above.

[0150] The "enamine" group is a group with the following 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 # Independently as defined above.

[0151] The oxime group is a group with the following formula: -C(=NO(R) # ))(R # -C(=NOH)(R) # -CH(=NO(R) # )) or -CH (=NOH), where each R # Independently as defined above.

[0152] The "acylhydrazine" group is a group with the following 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, where each R # Independently as defined above.

[0153] The hydrazine group is a group with the following formula: -N(R #)N(R # )2、-NHN(R # )2、-N(R # )NH(R # )-N(R # )NH2、-NHNH(R # )2 or -NHNH2, where each R # Independently as defined above.

[0154] The "hydrazone" group is a group with the following 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 # Independently as defined above.

[0155] The "azide" group is a group of the formula -N3.

[0156] The "isocyanate" group is a group with the formula N=C=O.

[0157] The "isothiocyanate" group is a group with the formula N=C=S.

[0158] The "cyanate ester" group is a group of the formula OCN.

[0159] The "thiocyanate" group is a group of the formula SCN.

[0160] The "thioether" group is a group with the following formula: -S(R # ), where R # As defined above.

[0161] The "thiocarbonyl" group is a group with the following formula: -C(=S)(R # ), where R # As defined above.

[0162] "Sylenyl group" is a group with the following formula: -S(=O)(R # ), where R # As defined above.

[0163] The sulfone group is a group with the following formula: -S(=O)2(R # ), where R # As defined above.

[0164] "Sulfonylamino" is a group with the following formula: -NHSO2(R # ) or -N(alkyl)SO2(R # ), wherein each alkyl group and R # As defined above.

[0165] The "sulfonamide" group is a group with the following formula: -S(=O)2N(R) # )2 or -S(=O)2NH(R # ) or -S(=O)2NH2, where each R # Independently as defined above.

[0166] The "phosphonate" group is a group with the following formula: -P(=O)(O(R) # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ) or -OP(=O)(OH)(R # ), where each R # Independently as defined above.

[0167] The "phosphine" group is a group with the following formula: -P(R # )2, where each R # Independently as defined above.

[0168] When groups described herein (other than alkyl groups) 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 compounds and embodiments disclosed herein, as well as halogens (chlorine, iodine, bromine, or fluorine); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxyamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (=O); B(OH)₂; O(alkyl)aminocarbonyl; cycloalkyl, which may It can be a monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl); or a heterocyclic group, which can be a monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiazinyl); a monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolidinyl, indolyl, furanyl, thiophene, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridineyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophene, or benzofuranyl); aryloxy; arylalkoxy; heterocyclic oxy; and heterocyclic alkoxy.

[0169] As used herein, “pharmaceuticalally acceptable salt” refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base (including inorganic acids or bases and organic acids or bases).

[0170] As used herein and unless otherwise indicated, the term "solvent" means a compound or a salt thereof that also includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0171] As used herein and unless otherwise indicated, the term "hydrate" means a compound or its salt that also includes stoichiometric or nonstoichiometric amounts of water bound together by noncovalent intermolecular forces.

[0172] 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. Examples of prodrugs include, but are not limited to, derivatives and metabolites of compounds that include biohydrolyzable moieties, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acylureas, and biohydrolyzable phosphate ester analogs. In some embodiments, the prodrug of a compound having a carboxyl functional group is a lower alkyl ester of a carboxylic acid. Carboxylic acid esters can be formed by esterification of any carboxylic acid moiety present on the molecule. Prodrugs are generally prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery, 6th Edition (edited by Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs (edited by H. Bundgaard, 1985, Harwood Academic Publishers GmbH).

[0173] As used herein and unless otherwise indicated, the terms "stereoisomer" or "stereoisomer-pure" mean that one stereoisomer of a compound is substantially free of other stereoisomers of the compound. For example, a stereoisomer-pure compound having one chiral center will substantially free of its opposite enantiomers. A stereoisomer-pure compound having two chiral centers will substantially free of other diastereomers of the compound. A typical stereoisomer-pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, more 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 more 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 compound may have a chiral center and may occur as a racemic mixture, a single enantiomer, or a diastereomer, or a mixture thereof. All such isomeric forms are included within the embodiments disclosed herein, including mixtures thereof. The use of the stereoisomeric pure forms of such compounds, as well as mixtures of these forms, is covered in the embodiments disclosed herein. For example, mixtures comprising equal or unequal amounts of enantiomers 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, for example, Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL., Stereochemistry pf Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, SH., Tables of Resolving Agents and Optical Resolutions, p. 268 (Ell. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0174] 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 some embodiments, the compound is isolated into cis or trans isomers. In other embodiments, the compound is a mixture of cis and trans isomers.

[0175] "Tautomers" refer to the isomers of a compound that are in equilibrium with each other. The concentration of the isomers will depend on the environment in which the compound is present 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 isomers, which are referred to as tautomers of each other:

[0176]

[0177] Those skilled in the art will readily understand that various functional groups and other structures may exhibit tautomerism, and all tautomers of the compound are within the scope of this disclosure.

[0178] It should also be noted that one or more atoms in a compound may contain atomic isotopes in non-natural proportions. For example, the compound may contain atomic isotopes such as tritium (…). 3 H), Iodine-125 ( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 Radiolabeling can be performed using radioactive isotopes such as C, or deuterium (C). 2 H), carbon-13 ( 13 C) or nitrogen-15 ( 15 N) Isotope enrichment. As used herein, “isotope body” refers to an isotopically enriched compound. The term “isotope enrichment” means that the atoms have an isotopic composition other than the natural isotopic composition of the atoms. “Isotope enrichment” can also refer to a compound in which at least one atom has an isotopic composition different from the natural isotopic composition of the atoms. 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 treatment agents), research reagents (e.g., binding analytical reagents), and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations (whether radioactive or not) of the compounds described herein are intended to be covered within the scope of the embodiments provided herein. In some embodiments, isotopes of the compounds are provided, for example, isotopes enriched in deuterium, carbon-13, or nitrogen-15.

[0179] It should be noted that if there is an inconsistency between the described structure and the name of the structure, the described structure should be given higher weight.

[0180] As used herein, the term "residue" refers to the chemical portion remaining after an intrinsic chemical reaction of a compound. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of amide or peptide coupling of an amino acid or N-alkyl amino acid with a suitable coupling agent; wherein, for example, amide or peptide coupling of an amino acid or N-alkyl amino acid results in the expulsion of a water molecule, thereby producing a product containing an amino acid residue or N-alkyl amino acid residue.

[0181] As used herein, "sugar," "glycosyl," or "sugar residue" refers to a carbohydrate moiety that may comprise a 3-carbon (triose) unit, a 4-carbon (teuose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptaose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, "sugar," "glycosyl," or "sugar residue" comprises furanose (e.g., furanose ribose, furanose fructose) or pyranose (e.g., pyranose glucose, pyranose galactose) or a combination thereof. In some cases, "sugar," "glycosyl," or "sugar residue" comprises 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 “glycosyl” or “sugar residues” include polysaccharides and / or oligosaccharides, including but not limited to amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, “sugars” or “glycosyl” or “sugar residues” are amino-sugars. In some cases, “sugars” or “glycosyl” or “sugar residues” are reduced glucosamine residues (1-amino-1-deoxy-D-sorbitol), which are linked to the rest of the molecule via their amino groups, thereby forming an amide bond with the rest of the molecule (i.e., reduced glucosamide).

[0182] Certain groups, parts, substituents, and atoms are drawn with wavy lines intersecting one or more bonds to indicate which atom the group, part, substituent, or atom is bonded to. For example, a propyl-substituted phenyl group is drawn as follows:

[0183] It has the following structure:

[0184] As used herein, “binding agent” means any molecule capable of specifically binding to a given binding agent (e.g., antigen), such as an antibody.

[0185] As used herein, the term "amino acid" refers to an organic compound containing an amino (-NH2) and a carboxyl (-COOH) functional group, as well as a side chain (R group), the side chain being specific for each amino acid. Amino acids can be proteogenous or non-proteogenous. "Proteogenous" means that an amino acid is one of the twenty naturally occurring amino acids found in proteins. Proteogenous 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-proteogenous" means that an amino acid is not naturally found in proteins or is not directly produced by cellular mechanisms (e.g., it is a product of post-translational modification). Non-limiting examples of non-proteogenous amino acids include γ-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylethylamide), hydroxyproline, β-alanine, ornithine, and citrulline.

[0186] As used herein, the term "peptide," in its broadest sense across its various grammatical forms, refers to a compound consisting of two or more subunit amino acids, amino acid analogs, or other peptide mimics. These subunits may be linked by peptide bonds or other bonds such as ester bonds, ether bonds, etc. The term "amino acid," as used herein, refers to natural and / or non-natural, proteogenic or non-proteogenic, or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. If the peptide chain is short, such as two, three, or more amino acids, it is generally called an oligopeptide. If the peptide chain is long, the peptide is generally called 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. Furthermore, due to the presence of ionizable amino and carboxyl groups in the molecule, certain peptides can be obtained in acidic or basic salt or neutral forms. Peptides can be obtained directly from the source organism or can be produced recombinantly or synthetically.

[0187] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including those described below: 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 stated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme does not imply the absence of differences in the sequence, and those skilled in the art can readily confirm the sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise stated, when referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is generally used (e.g., as reported above by Kabat et al.).

[0188] As used in this article, “cytotoxic activity” refers to the activity that reduces or diminishes the viability of the tested cell lines.

[0189] In the following claims and in the preceding description, unless the context requires otherwise due to the language of expression or necessary implication, the word “comprise” or variations thereof (such as “comprises / comprising”) are used to include the meaning that specifies the presence of the described feature in the various embodiments rather than excluding the presence of additional features or adding additional features.

[0190] 7.2.PBD compounds

[0191] This article describes the compound of formula (I):

[0192]

[0193]

[0194] Or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein each of ring A and ring B independently has one of the following formula:

[0195]

[0196]

[0197] Indicates the connection point with the connector;

[0198] The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -;

[0199] X is NR 6 , NHC(=O), C(=O)NH, O, SO2, substituted or unsubstituted divalent aryl rings, substituted or unsubstituted divalent heteroaryl rings, substituted or unsubstituted divalent heterocyclic rings or substituted or unsubstituted divalent cyclic rings;

[0200] Ring C is a cyclopropyl ring or a cyclobutyl ring;

[0201] -C(R 1 )- and -N(R 2 Each of the dashed keys between ) and - is independently a single or double bond;

[0202] When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 H is independent;

[0203] When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It exists independently;

[0204] R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 Alkoxy or aryl, and R a and R b Each independently is H or C 1-4 alkyl;

[0205] R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0206] R 6 For H or C 1-4 alkyl;

[0207] Each of m, n, and o is independently 1 or 2;

[0208] Each of r, p, and q is an independent integer from 1 to 8; and

[0209] The sum of p and q is an integer from 1 to 8.

[0210] In some implementations, X is NR 6 , NHC (=O), O, substituted or unsubstituted aryl ring, substituted or unsubstituted heteroaryl ring, substituted or unsubstituted heterocyclic ring or substituted or unsubstituted cyclic ring.

[0211] In some implementations, ring A and ring B have the same formula.

[0212] In some implementations, ring A and ring B each have different formulas.

[0213] In some implementations, when the connector is -(CH2) r - At this time, ring A and ring B do not have the same formula.

[0214] In some implementations, when the connector is -(CH2) r When -, ring A has equation (IIa) and ring B has equation (IIb).

[0215] In some implementations, when the connector is -(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q When ring A has equation (IIa), m is 2, and ring B has one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), and (IIg). In some embodiments, when the connector is -(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q When ring A has equation (IIa), m is 1, and ring B has one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), and (IIg). In some embodiments, equation (IIc) has the following equation (IIc3) or equation (IIc4):

[0216]

[0217]

[0218] In some implementations, when the connector is -(CH2) p -X-(CH2) q-or-(CH2) p -CH = CH-(CH2) q When ring A has equation (IIa), and ring B has one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg).

[0219] In some implementations, the connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -; X is O, NR 6 ,NHC(=O), -(m-C6H4)-, In some implementation schemes, R 6 It is H or methyl.

[0220] In some implementations, the connector is -(CH2). r -

[0221] In some implementations, r is 3 or 5.

[0222] In some implementations, the connector is -(CH2). p -O-(CH2) q -or-(CH2) p -NH-(CH2) q -

[0223] In some implementations, the sum of p and q is 4.

[0224] In some implementations, the connector is or.

[0225] In some implementations, the sum of p and q is 2.

[0226] In some implementations, the connector is

[0227] In some implementations, the sum of p and q is 2.

[0228] In some implementations, the connector is -(CH2). p -CH = CH-(CH2) q -

[0229] In some implementations, the sum of p and q is 3.

[0230] In some implementations, ring B has formula (IIa).

[0231] In some implementations, m is 1.

[0232] In some implementations, ring C is a cyclopropyl ring.

[0233] In some implementations, the dashed bond in ring B is a single bond, R 1 It is H or OH, and R 2 For H.

[0234] In some implementations, the dashed bond in ring B is a double bond, R 1 For H, and R 2 It does not exist.

[0235] In some implementations, ring A has formula (IIb).

[0236] In some implementations, ring A has the formula (IIb2):

[0237]

[0238] In some implementation schemes, R 3 For H.

[0239] In some implementations, the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0240] In some implementations, the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0241] In some implementations, the compound is

[0242]

[0243]

[0244] In some implementations, ring A has formula (IIg). In some implementations, o is 2.

[0245] In some implementations, the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0246] In some implementations, the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0247] In some implementations, the compound is

[0248]

[0249] In some implementations, ring A has formula (IId).

[0250] In some implementations, n is 1.

[0251] In some implementations, the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0252] In some implementations, the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0253] In some implementations, the compound is

[0254]

[0255] In some implementations, ring A has formula (IIc).

[0256] In some implementations, ring A has formula (IIc2):

[0257]

[0258] In some implementation schemes, R 4 It is CH3O-.

[0259] In some implementations, the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0260] In some implementations, the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0261] In some implementations, the compound is

[0262]

[0263] In some implementations, ring A has formula (IId).

[0264] In some implementations, n is 2.

[0265] In some implementations, the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0266] In some implementations, the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0267] In some implementations, the compound is

[0268]

[0269] In some implementations, ring A has formula (IIe).

[0270] In some implementation schemes, R 5 It is a methyl group.

[0271] In some implementations, the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0272] In some implementations, the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0273] In some implementations, the compound is

[0274]

[0275] In some implementations, each of ring A and ring B independently includes formula (IIa).

[0276] In some implementations, m is 1.

[0277] In some implementations, ring C is a cyclopropyl ring.

[0278] In some implementations, when the dashed bond in ring A is a single bond, R 1 It is H or OH, and R 2 For H.

[0279] In some implementations, when the dashed bond in ring A is a double bond, R 1 For H, and R 2 It does not exist.

[0280] In some implementations, when the dashed bond in ring B is a single bond, R 1 It is H or OH, and R 2 For H.

[0281] In some implementations, when the dashed bond in ring B is a double bond, R 1 For H, and R 2 It does not exist.

[0282] In some implementations, the compound is

[0283]

[0284]

[0285] 7.3. Connector - Payload Compound

[0286] This document also discloses compounds or pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers thereof, including covalent linkers coupled to residues of at least one of the PBD-based compounds disclosed herein. The covalent linkers (or “Ab linkers”) are capable of binding to binders, for example, to form conjugates, as described below.

[0287] In some embodiments, the compound has one of the following formulas:

[0288] Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein:

[0289] Each of rings A and B independently has one of the following equations:

[0290]

[0291]

[0292] Indicates the connection point with the connector or Ab connector;

[0293] The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -;

[0294] X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings;

[0295] Ring C is a cyclopropyl ring or a cyclobutyl ring;

[0296] -C(R 1 )- and -N(R 2 The dashed key between )- Each of them is either a single bond or a double bond;

[0297] When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 For H;

[0298] When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It does not exist;

[0299] R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0300] R a and R b Each independently is H or C 1-4 alkyl;

[0301] R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0302] R 6 For H or C 1-4 alkyl;

[0303] Each of m, n, and o is independently 1 or 2;

[0304] Each of r, p, and q is an independent integer from 1 to 8;

[0305] The sum of p and q is an integer from 1 to 8; and

[0306] Ab connectors are compounds that can bond ring A or ring B to a binder.

[0307] In some implementations, the Ab connector has the following formula:

[0308]

[0309] in Indicates the connection point with ring A or ring B.

[0310] In some embodiments, the compound has the following formula:

[0311]

[0312]

[0313] 7.4. Conjugates

[0314] This document discloses conjugates or pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers thereof, including proteins coupled via covalent linkers to residues of at least one PBD-based compound disclosed herein. In some embodiments, the protein is a binding agent, such as an antibody or an antigen-binding fragment thereof. The conjugate may be an antibody-drug conjugate (ADC).

[0315] In some implementations, the protein is directly bonded to a covalent linker, such as the Ab linker described herein. In such cases, the binder is one bond away from the covalent linker. The covalent linker can also be directly bonded to a payload residue such that the covalent linker is one bond away from the payload residue. The payload can be any PBD-based compound described herein.

[0316] In some implementations, the conjugate has one of the following formulas:

[0317]

[0318] Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein:

[0319] Each of rings A and B independently has one of the following equations:

[0320]

[0321]

[0322] Indicates the connection point with the connector or Ab connector;

[0323] The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -;

[0324] X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings;

[0325] Ring C is a cyclopropyl ring or a cyclobutyl ring;

[0326] -C(R 1 )- and -N(R 2 Each of the dashed keys between ) and - is independently a single or double bond;

[0327] When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 H is independent;

[0328] When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It exists independently;

[0329] R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 Alkoxy or aryl, and R a and R b Each independently is H or C 1-4 alkyl;

[0330] R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl;

[0331] R 6 For H or C 1-4 alkyl;

[0332] Each of m, n, and o is independently 1 or 2;

[0333] Each of r, p, and q is an independent integer from 1 to 8; and

[0334] The sum of p and q is an integer from 1 to 8.

[0335] In some implementations, the Ab connector has the following formula:

[0336] Indicates the connection point with Ab, and Indicates the connection point with ring A or ring B.

[0337] In some implementations, the Ab connector has the following formula:

[0338] Indicates the connection point with Ab, and Indicates the connection point with ring A or ring B.

[0339] In some implementations, the conjugate has the following formula:

[0340]

[0341] Where Ab is a conjugate selected from humanized, chimeric, or human antibodies or their antigen-binding fragments; and the subscript x is from 1 to 15. In some embodiments, t is about 2.

[0342] In some implementations, the ADC has the following formula:

[0343]

[0344] Ab is a binder selected from humanized, chimeric, or human antibodies or their antigen-binding fragments.

[0345] 7.5. Methods or processes for preparing conjugates

[0346] This document provides a method for preparing conjugates by contacting the binder (BA) with the linker-loaded compound under conditions suitable for forming a bond between the binder and the linker-loaded compound. The reaction conditions can be any suitable conditions known in the art. The binder can be an antibody, and the bond can form an antibody-drug conjugate.

[0347] Examples of such reactions are provided in the embodiments below.

[0348] In some embodiments, the method for preparing the conjugate includes treating the compound with a binder under coupling conditions or contacting the compound with a binder. The compound may include a reactive linker bonded to at least one payload. The compound may be any of the linker or platform compounds disclosed herein.

[0349] 7.6. Pharmaceutical Compositions

[0350] This document also provides compositions comprising the ADCs described herein, including pharmaceutical compositions. In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise pharmaceutically acceptable excipients.

[0351] The pharmaceutical compositions according to this disclosure can be prepared by mixing an antibody-drug conjugate having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) (Based on) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein also include interstitial drug dispersants, such as soluble, neutrally active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (…). Baxter International, Inc. Certain exemplary embodiments of sHASEGP (including rHuPH20) and methods of use are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0352] Exemplary lyophilized formulations are described in U.S. Patent No. 6,267,958. Aqueous formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter comprising histidine-acetate buffer.

[0353] 7.7. How to use

[0354] In some implementations, this document describes a method for treating a disease or condition (e.g., cancer) in a subject of need, the method comprising administering to the patient a therapeutically effective amount of the conjugate disclosed herein.

[0355] The conjugates disclosed herein may be administered by any suitable route, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out by any suitable route, such as by injection (e.g., intravenous or subcutaneous), depending in part on whether the administration is transient or prolonged. This document considers various dosing schedules, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusions.

[0356] The conjugates disclosed herein can be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner.

[0357] 8. Examples

[0358] The examples below are intended to be illustrative and should not be considered as limiting in any way. Unless otherwise stated, the experimental methods described in the examples below are conventional methods. Unless otherwise stated, reagents and materials are commercially available. All solvents and chemicals used are analytical grade or chemically pure. Solvents were redistilled before use. Anhydrous solvents were prepared according to standard or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin-layer chromatography (TLC) were available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd. in China; unless otherwise stated, they were eluted with petroleum ether (60-90°C) / ethyl acetate (v / v) and visualized by a solution of iodine or phosphomolybdic acid in ethanol. Unless otherwise stated, all extraction solvents were dried over anhydrous Na2SO4. Recordings were performed on a Bruck-400, Varian 400MR NMR spectrometer. 1 1H NMR spectra, with TMS (tetramethylsilane) as an internal standard. Coupling constants are given in Hertz. Peaks are reported as singlets (s), doublets (d), triplets (t), quartets (q), quintets (p), sextets (h), heptets (hept), multiplets (m), or combinations thereof; br represents a broad peak. LC / MS data were recorded using an Agilent 100, 1200 high-performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) and an ion trap (ESI source) for detection at 214 nm and 254 nm. All compound names except those of the reagents mentioned are provided by [reference needed]. Version 18.0 was generated.

[0359] For the sake of brevity, this article uses certain abbreviations. One example is the use of single-letter abbreviations to represent amino acid residues. Amino acids and their corresponding three-letter and single-letter abbreviations are as follows:

[0360]

[0361]

[0362] In the following embodiments, the following abbreviations are used:

[0363]

[0364]

[0365] UPLC analysis method

[0366] 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 1.7μm.

[0367] 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 1.7μm.

[0368] 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 1.7μm.

[0369] Example 1-1

[0370]

[0371] Step 1: (S)-3-hydroxy-2-methoxy-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-1b)

[0372] Compounds 1-1b were synthesized according to the procedure described in Bioorg Med Chem Lett. 2019 Sep 1; 29(17): 2455-2458.

[0373] Step 2: (S)-3-((5-iodopentyl)oxy)-2-methoxy-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-1c)

[0374] 1-1b (50 mg, 0.16 mmol) and 1,5-diiodopentane (0.12 mL, 0.81 mmol) were dissolved in anhydrous DMF (0.5 mL). The solution was cooled to 0 °C, and K₂CO₃ (45 mg, 0.32 mmol) was added in a single batch. The mixture was heated to rt and stirred at rt for 6 h. Subsequently, EtOAc (5 mL) was added, and the diluted organic phase was washed with H₂O (10 mL) and brine (10 mL). The organic phase was dried over Na₂SO₄ and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 25 / 75) to give 1-1c (49 mg, 60% yield) as a pale yellow solid. MS (ESI) m / z: 505.3 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.53 (s, 1H), 7.49 (d, J=5.3Hz, 1H), 7.39-7.34 (m, 2H), 7.31 (dd, J=3.8, 1.6Hz, 2H), 6.80 (s, 1H), 5.01 (d, J=15. 6Hz, 1H), 4.56 (d, J=15.5Hz, 1H), 4.16-4.01 (m, 3H), 3.95 (s, 3H), 3.33-3.09 (m, 4H), 1.95-1.84 (m, 4H), 1.59 (tt, J=9.8, 6.1Hz, 2H).

[0375] Step 3: (11S, 11aS)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-8-((5-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza) [1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-1d)

[0376] K₂CO₃ (12.5 mg, 0.09 mmol) was added to a solution of 1-1c (50 mg, 0.098 mmol) and 1-7g (40 mg, 0.082 mmol) in 0.5 mL of DMF. The mixture was stirred at rt for 3 h. LCMS indicated that 1-7g was completely consumed. Subsequently, EtOAc (5 mL) was added, and the diluted organic phase was washed with H₂O (10 mL) and brine (10 mL). The organic phase was dried over Na₂SO₄ and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 0 / 100) to give a white solid 1-1d (60 mg, 70.6% yield). MS (ESI) m / z: 865.5 [M+H] + .

[0377] Step 4: (11S, 11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza) [1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-1e)

[0378] Add 24 μL of AcOH to a solution of 1-1d (60 mg, 0.07 mmol) in anhydrous THF (1 mL), followed by dropwise addition of TBAF (350 μL, 0.35 mmol, 1 M in THF). Stir the mixture at rt for 6 h. If the reaction is complete as determined by LCMS, quench the mixture with saturated NaHCO3. Extract the organic phase with EtOAc (5 mL x 3) and wash with H2O (10 mL) and brine (10 mL). Dry the organic phase with Na2SO4 and concentrate to obtain a crude product, which is purified by silica column gel chromatography (eluent: CH2Cl2 / MeOH = 100 / 0 to 5 / 95) to give a white solid 1-1e (50 mg, 96% yield). MS (ESI) m / z: 751.5 [M+H] + .

[0379] Step 5: (S)-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-1)

[0380] Pd(PPh3)4 (2 mg, catalytic amount) was added to a solution of 1-1e (50 mg, 0.067 mmol) in CH2Cl2 (1 mL) and pyrrolidine (14 μL, 0.17 mmol). The reaction mixture was stirred under N2 at rt for 0.5 h. The reaction mixture was diluted with CH2Cl2 (5 mL) and washed with saturated NH4Cl and brine. The organic phase was dried over Na2SO4. The organic phase was concentrated and purified by silica column gel chromatography (eluent: CH2Cl2 / MeOH = 100 / 0 to 5 / 95) to give a white solid 1-1 (43 mg, 99% yield). MS (ESI) m / z: 649.4 [M+H] + ; 1 H NMR (400MHz, CDCl3) δ7.79 (d, J=4.4Hz, 1H), 7.52 (d, J=7.3Hz, 2H), 7.48 (d, J=5.2Hz, 1H), 7.40-7.29 (m, 4H), 6. 80 (d, J=4.4Hz, 2H), 5.01 (d, J=15.5Hz, 1H), 4.56 (d, J=15.5Hz, 1H), 4.19-4.02 (m, 4H), 3.94 (d, J=2.9Hz, 7H), 3. 87 (ddd, J=7.8, 4.5, 2.7Hz, 1H), 3.68 (d, J=11.7Hz, 1H), 3.55-3.47 (m, 1H), 3.26 (d, J=5.5Hz, 1H), 3.16 (dd, J=15 .4, 4.2Hz, 1H), 2.52 (dd, J=13.0, 8.1Hz, 1H), 2.08-1.90 (m, 6H), 1.68 (td, J=8.6, 5.9Hz, 2H), 0.81-0.68 (m, 4H).

[0381] Examples 1-2

[0382]

[0383] Step 1: (S)-3-(benzyloxy)-2-methoxy-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(5H)-one (1-2a)

[0384] NaBH(OAc)3 (531.9 mg, 2.5 mmol) was added to a solution of 1-1a (500 mg, 1.255 mmol) in CH2Cl2 (6 mL) at 0 °C. The reaction mixture was then heated to rt and stirred at rt and N2 for 2 h. The reaction was then quenched with saturated NaHCO3, washed with H2O and brine, and dried over Na2SO4. The organic phase was filtered and concentrated to give a white solid crude product 1-2a (485 mg, 97% yield), which was used for the next step without further purification. MS (ESI) m / z: 401.3 [M+H] + .

[0385] Step 2: (S)-3-(benzyloxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-2b)

[0386] Alloc-Cl (0.14 mL, 1.33 mmol) was added dropwise to a solution of 1-2a (485 mg, 1.2 mmol) and pyridine (0.36 mL, 2.9 mmol) in CH₂Cl₂ (2 mL) at 0 °C. The reaction mixture was stirred at 0 °C and N₂ for 15 min. The reaction mixture was diluted with 10 mL of CH₂Cl₂, washed with 0.1 N citric acid (10 mL), H₂O (10 mL), and brine (10 mL), and dried over Na₂SO₄. The organic phase was filtered and the filtrate was concentrated. The crude product was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 50 / 50) to give 1-2b (480 mg, 82% yield) as a white solid. MS (ESI) m / z: 485.5 [M+H] + .

[0387] Step 3: (S)-3-hydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-2c)

[0388] MeSO3H (0.64 mL, 9.9 mmol) was added dropwise to a solution of 1-2b (484.5 mg, 0.99 mmol) in CH2Cl2 (3 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min, then heated to rt and stirred under N2 for 2 h. The mixture was quenched with saturated NaHCO3. The organic phase was extracted with CH2Cl2 (5 mL x 3) and washed with H2O (10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 25 / 75) to give a white solid 1-2c (332 mg, 84.3% yield). MS (ESI) m / z: 395.4 [M+H] + .

[0389] 1 H NMR (400MHz, CDCl3) δ7.28 (d, J=3.3Hz, 3H), 7.21 (s, 2H), 6.75 (s, 1H), 5.88 (s, 1H), 5 .76 (ddd, J=17.3, 10.5, 5.2Hz, 1H), 5.10 (ddd, J=10.3, 1.4Hz, 2H), 4.79-4.65 (m, 2H), 4.57 (dd, J=13.9, 5.2Hz, 1H), 4.40 (d, J=13.3Hz, 1H), 4.05-3.95 (m, 2H), 3.94 (s, 3H) , 3.37 (d, J=11.4Hz, 1H), 3.12 (dd, J=15.2, 5.6Hz, 1H), 2.77 (dd, J=15.2, 4.1Hz, 1H).

[0390] Step 4: (S)-3-((5-iodopentyl)oxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-2d)

[0391] 1-2d was prepared according to the procedure described in step 2 of Examples 1-1, yielding a white solid (138 mg, yield 92.2%). MS (ESI) m / z: 591.3 [M+H] + .

[0392] 1H NMR (400MHz, CDCl3) δ7.35 (d, J=3.5Hz, 3H), 7.28 (d, J=1.9Hz, 2H), 6.72 (s, 1H), 5.84 (ddt, J=16.3 , 10.8, 5.2Hz, 1H), 5.25-5.08 (m, 2H), 4.80 (q, J=15.9Hz, 1H), 4.67 (dd, J=13.7, 5.3Hz, 1H), 4.48-4 .40 (m, 1H), 4.14-4.00 (m, 4H), 3.98 (s, 3H), 3.46 (d, J = 10.7Hz, 1H), 3.29 (t, J = 7.0Hz, 2H), 3.20 (d d, J=15.2, 5.4Hz, 1H), 2.85 (d, J=15.2Hz, 1H), 1.95 (dq, J=13.4, 7.0Hz, 4H), 1.66 (q, J=8.0Hz, 4H).

[0393] Step 5: (S)-3-((5-(((11S,11aS)-10-((allyloxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-2e)

[0394] 1-2e were prepared according to the procedure described in step 3 of Examples 1-1, yielding a colorless oil (70 mg, yield 85.3%). MS (ESI) m / z: 973.6 [M+Na] + .

[0395] Step 6: (S)-3-((5-(((11S,11aS)-10-((allyloxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-2f)

[0396] 1-2f were prepared according to the procedure described in step 4 of Examples 1-1, yielding a white solid (54 mg, yield 87.7%). MS (ESI) m / z: 837.5 [M+H] + .

[0397] Step 7: (S)-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(5H)-one(1-2)

[0398] Samples 1-2 were prepared according to the procedure described in step 5 of Example 1-1, yielding a white solid (40 mg, yield 95.2%). MS (ESI) m / z: 651.4 [M+H] + .

[0399] 1 H NMR (400MHz, CDCl3) δ7.79 (d, J=4.4Hz, 1H), 7.49 (s, 1H), 7.35 (s, 1H), 7.27 (d, J=6.9Hz, 4H), 6.79 (s, 1H), 6.27 (s, 1H), 4.87 (d, J = 15.7Hz, 1H), 4.73 (d, J = 15.7Hz, 1H), 4.23-3.97 (m, 5H), 3.95 (s, 3H), 3.85 (s, 3H), 3 .67 (d, J=11.7Hz, 1H), 3.53-3.41 (m, 2H), 3.22 (dd, J=12.1, 9.5Hz, 1H), 3.11 (dd, J=15.1, 5.8Hz, 1H), 2.81 ( dd, J=15.2, 5.3Hz, 1H), 2.51 (dd, J=13.1, 8.1Hz, 1H), 1.96 (dq, J=24.2, 9.3, 8.1Hz, 6H), 1.72-1.62 (m, 2H).

[0400] Examples 1-3 and 1-4

[0401]

[0402] Step 1: (S)-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-3) and (S)-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(5H)-one(1-4)

[0403] NaBH(OAc)3 (3.4 mg, 1.05 mmol) was added to a solution of 1-1 (10 mg, 0.015 mmol) in CH2Cl2 (0.5 mL) at 0 °C. The reaction mixture was then heated to rt and stirred under N2 at rt for 0.5 h. The reaction was quenched with saturated NaHCO3, washed with H2O and brine, and dried with Na2SO4. The organic phase was filtered and concentrated to give a crude product, which was purified by preparative HPLC (0.01% FA in H2O) to give white solid 1-3 (3 mg, 30% yield) with a residence time of 4.9 min and white solid 1-4 (2.7 mg, 27% yield) with a residence time of 5.12 min.

[0404] 1-3MS(ESI) m / z: 651.4 [M+H] + .

[0405] 11H NMR (400 MHz, CDCl3) δ 7.71 - 7.59 (m, 1H), 7.55 - 7.43 (m, 3H), 7.39 - 7.27 (m, 4H), 6.76 (s, 1H), 6.18 (s, 1H), 4.98 (d, J = 15.5 Hz, 1H), 4.53 (d, J = 15.5 Hz, 1H), 4.17 - 3.95 (m, 5H), 3.92 (d, J = 5.1 Hz, 3H), 3.82 (s, 3H), 3.65 (d, J = 12.0 Hz, 1H), 3.58 - 3.39 (m, 3H), 3.25 (dd, J = 15.4, 5.5 Hz, 1H), 3.14 (dd, J = 15.4, 4.3 Hz, 1H), 2.02 (dd, J = 12.7, 7.3 Hz, 1H), 1.91 (h, J = 7.3 Hz, 4H), 1.68 (dq, J = 31.0, 7.8, 7.0 Hz, 4H), 0.75 - 0.50 (m, 4H).

[0406] 1 - 4MS (ESI) m / z: 653.6 [M + H] + .

[0407] 1 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.37 (s, 1H), 7.32 - 7.27 (m, 5H), 7.22 - 7.13 (m, 1H), 6.21 (s, 1H), 6.08 (s, 1H), 4.88 (d, J = 15.7 Hz, 1H), 4.75 (d, J = 15.7 Hz, 1H), 4.14 (d, J = 6.2 Hz, 1H), 4.00 (q, J = 6.1 Hz, 5H), 3.89 - 3.79 (m, 6H), 3.71 (d, J = 12.0 Hz, 1H), 3.60 - 3.51 (m, 2H), 3.50 - 3.39 (m, 2H), 3.23 (t, J = 10.8 Hz, 1H), 3.12 (dd, J = 15.2, 5.8 Hz, 1H), 2.83 (dd, J = 15.2, 5.4 Hz, 1H), 2.02 (t, J = 10.3 Hz, 1H), 1.91 (p, J = 6.9 Hz, 4H), 1.78 (dd, J = 12.7, 6.9 Hz, 1H), 1.66 (q, J = 7.8 Hz, 2H), 0.78 - 0.52 (m, 4H).

[0408] Examples 1 - 5

[0409]

[0410] Step 1: (S)-3-(2-(2-bromoethoxy)ethoxy)-2-methoxy-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-5a)

[0411] 1-5a were prepared according to the procedure described in step 2 of Examples 1-1, yielding a white solid (50 mg, yield 67.1%).

[0412] MS(ESI) m / z: 461.2 [M+H] + .

[0413] Step 2: (11S, 11aS)-11-hydroxy-7-methoxy-8-(2-(2-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza) [1,2-b]isoquinoline-3-yl)oxy)ethoxy)ethoxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-5c)

[0414] 1-5c was prepared according to the procedure described in step 3 of Examples 1-1, yielding a white solid (51 mg, yield 60.3%).

[0415] MS(ESI) m / z: 753.5 [M+H] + .

[0416] Step 3: (S)-2-methoxy-3-(2-(2-(((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropane]-8-yl)oxy)ethoxy)ethoxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one(1-5)

[0417] 1-5 was prepared according to the procedure described in step 4 of 1-1, yielding a white solid (25.5 mg, yield 59%).

[0418] MS(ESI) m / z: 651.4 [M+H] + .

[0419] 1H NMR (400MHz, CDCl3) δ7.78 (d, J=4.5Hz, 1H), 7.51 (d, J=7.4Hz, 2H), 7.47 (dd, J=5.3, 3.3Hz, 1H), 7.40-7.30 (m, 4H) , 6.83 (dd, J=7.3, 4.5Hz, 2H), 5.00 (d, J=15.5Hz, 1H), 4.56 (d, J=15.5Hz, 1H), 4.37-4.16 (m, 4H), 4.01 (t, J=5.0Hz, 4H), 3.96-3.90 (m, 6H), 3.88-3.81 (m, 2H), 3.67 (d, J=11.7Hz, 1H), 3.49 (d, J=11.7Hz, 1H), 3.27 (dd, J=15.4, 5.5Hz , 1H), 3.15 (dd, J=15.4, 4.2Hz, 1H), 2.51 (dd, J=13.1, 8.1Hz, 1H), 1.99 (dd, J=13.2, 2.8Hz, 1H), 0.81-0.66 (m, 4H).

[0420] Examples 1-6

[0421]

[0422] Step 1: (S)-4-methylenepiperidine-1,2-dicarboxylic acid 1-(tert-butyl) ester 2-methyl ester (1-6b)

[0423] Under a nitrogen atmosphere at 0°C, a solution of MePh3PBr (1.56 g, 4.27 mmol) in anhydrous THF (20 mL) was added to a solution of KOtBu (potassium tert-butoxide) in anhydrous THF (1 M, 4.7 mL, 4.7 mmol), and the mixture was stirred at 0°C for 1 h. Under a nitrogen atmosphere at 0°C, a solution of 1-6a (1.0 g, 3.89 mmol) in anhydrous THF (20 mL) was added to the reaction solution, and the mixture was stirred for 1 h. Water (5 mL) and saturated NH4Cl (10 mL) were added to the solution. The organic phase was separated and the aqueous phase was extracted with EtOAc (20 mL x 3). The combined organic phases were concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 90 / 10) to give a colorless oily title compound 1-6b (588 mg, 59.3% yield). MS (ESI) m / z: 156.1 [M + H-Boc] + .

[0424] 1H NMR (400MHz, CDCl3) δ5.08-4.97(m, 0.5H), 4.87-4.78(m, 0.5H), 4.79(s, 2H), 4.20-3.97(m, 1H), 3. 71(s, 3H), 3.12-2.90(m, 1H), 2.81-2.67(m, 1H), 2.49-2.37(m, 1H), 2218-2.10(m, 2H), 1.47(s, 9H).

[0425] Step 2: (S)-4-methylenepiperidine-1,2-dicarboxylic acid benzyl ester 2-methyl ester (1-6c)

[0426] Under a nitrogen atmosphere at 0°C, 3N HCl in 7mL of MeOH was added to a solution of 1-6b (585mg, 2.29mmol) in 3mL of anhydrous MeOH, and the mixture was stirred at rt for 2h. The solution was concentrated and dissolved in 10mL of CH2Cl2. 4N HCl was added to 10mL of EtOAc, and the mixture was stirred for 20min. The solution was concentrated and the residue was dissolved in 10mL of CH2Cl2. CbzCl (benzyl chloroformate, 0.40mL, 2.75mmol) and TEA (0.65mL, 4.58mmol) were added to the solution at 0°C, and the mixture was stirred at rt for 30min. The solution was added to 3mL of 0.5N HCl and 3mL of water, and extracted with 3 mL of CH2Cl2. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 90 / 10) to give a colorless oily title compound 1-6c (310 mg, 46.8% yield). MS (ESI) m / z: 312.2 [M+Na] + .

[0427] 1 H NMR (400MHz, CDCl3) δ7.45-7.24(m, 5H), 5.17(s, 2H), 5.14-4.90(m, 1H), 4.81(s, 2H), 4.28-4.08(m, 1H), 3.69 (d, J=18.8Hz, 3H), 3.23-3.01 (m, 1H), 2.77 (t, J=15.7Hz, 1H), 2.50-2.39 (m, 1H), 2.30-2.12 (m, 2H).

[0428] Step 3: (S)-6-azaspiro[2.5]octane-5,6-dicarboxylic acid 6-benzyl ester 5-methyl ester (1-6d)

[0429] ZnEt2 (2M in hexane, 2.1 mL, 4.2 mmol) was added to anhydrous CH2Cl2 (5 mL) under N2 atmosphere at 0 °C and stirred for 10 min at 0 °C. Anhydrous TFA (0.33 mL, 4.2 mmol) was slowly added and stirred for 1 h at 0 °C. Diiodomethane (0.35 mL, 4.2 mmol) was slowly added and stirred for 1 h at 0 °C. A solution of 1-6c (305 mg, 1.05 mmol) in anhydrous CH2Cl2 (3 mL x 2) was slowly added and stirred for 30 min at 0 °C, followed by stirring at rt for 18 h. The solution was filtered through diatomaceous earth. The filtrate was washed with saturated NH4Cl (5 mL) and water (5 mL). The aqueous phase was extracted with CH2Cl2 (5 mL x 3). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 90 / 10) to give the title compound 1-6d (195 mg, 61% yield) as a pale yellow oil. MS (ESI) m / z: 326.3 [M+Na] + .

[0430] 1 H NMR (400MHz, CDCl3) δ7.45-7.25 (m, 5H), 5.24-5.08 (m, 2H), 5.04-4.84 (m, 1H), 4.21-4.02 (m, 1H), 3.72 (d, J=6Hz, 3H ), 3.36-3.14(m, 1H), 2.24-2.12(m, 1H), 2.00-1.85(m, 1H), 1.62-1.54(m, 1H), 0.91-0.72(m, 1H), 0.42-0.22(m, 4H).

[0431] Step 4: (S)-5-(hydroxymethyl)-6-azaspiro[2.5]octane-6-carboxylic acid benzyl ester (1-6e)

[0432] LiBHEt3 (1M in THF, 1.3mL, 1.3mmol) was added to a solution of 1-6d (195mg, 0.64mmol) in anhydrous THF (4mL) under N2 atmosphere at 0℃, and the mixture was stirred at 0℃ for 1h. Water (0.5mL) was added to the solution, followed by washing with brine (5mL) and extraction with EtOAc (5mL x 3). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 50 / 50) to give a colorless oily title compound 1-6e (155mg, 87.6% yield). MS (ESI) m / z: 276.3 [M+H] + .

[0433] 1H NMR (400MHz, CDCl3) δ7.40-7.29 (m, 5H), 5.22-5.09 (m, 2H), 4.53-4.39 (m, 1H), 4.20-4.06 (m, 1H), 4.02 (dd, J=11.0, 9.5Hz, 1H), 3.67 (dd, J=11.1, 5.6Hz, 1H), 3.19-3.05(m, 1H), 2.08-1.97(m, 1H), 1.95-1.81(m, 1H), 1.0 8-0.97 (m, 1H), 0.91-0.79 (m, 2H), 0.47-0.38 (m, 1H), 0.35-0.23 (m, 3H).

[0434] Step 5: (S)-(6-azaspiro[2.5]oct-5-yl)methanol(1-6f)

[0435] A solution of 1-6e (155 mg, 0.56 mmol) in MeOH (2 mL) was added to a solution of 1-6e in MeOH (0.2 mL) with 7 M NH3 and 10% wet Pd / C (16 mg) in MeOH (2 mL) under a nitrogen atmosphere, and the mixture was stirred for 3 h under a hydrogen atmosphere. The solution was filtered and concentrated to give a pale yellow oily title compound 1-6f (82 mg, quantified), which was used directly in the next step without further purification. MS (ESI) m / z: 142.1 [M+H] + .

[0436] Step 6: (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(5-(hydroxymethyl)-6-azaspiro[2.5]oct-6-yl) ketone (1-6h)

[0437] Oxaloyl chloride (0.14 mL, 1.58 mmol) was added to a solution of 1-6 g (160 mg, 0.53 mmol) in CH2Cl2 (4 mL) at 0 °C under a nitrogen atmosphere, followed by the addition of 1 drop of DMF, and the mixture was stirred for 20 min. The solution became clear and no gas was eluted. The solution was concentrated to remove excess oxaloyl chloride. The residue was dissolved in anhydrous CH2Cl2 (2 mL) and added at 0 °C to a solution of 1-6 f (80 mg, 0.53 mmol) and DIPEA (0.38 mL, 2.1 mmol) in anhydrous CH2Cl2 (2 mL), and the mixture was stirred for 20 min. The solution was added to water (5 mL) and extracted with CH2Cl2 (5 mL x 3). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 30 / 70) to give the title compound 1-6 h (178 mg, 79% yield) as a grayish-white solid. MS(ESI) m / z: 427.4 [M+H] + .

[0438] Step 7: (S)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(5-(hydroxymethyl)-6-azaspiro[2.5]oct-6-yl)methyl ketone (1-6i)

[0439] Fe powder (117 mg, 2.05 mmol) was added to a solution of 1-6 h (175 mg, 0.41 mmol) and NH4Cl (336 mg, 6.16 mmol) in MeOH / H2O (3 / 1 mL) under N2 atmosphere, and the solution was refluxed for 3 h. The solution was filtered through diatomaceous earth. The filtrate was washed with brine (5 mL), extracted with EtOAc (5 mL * 3), dried over anhydrous Na2SO4, filtered, and concentrated to give the title compound 1-6i (162 mg, quantified), which was used directly in the next step without further purification. MS (ESI) m / z: 397.3 [M + H] + .

[0440] Step 8: (S)-(5-(benzyloxy)-2-(5-(hydroxymethyl)-6-azaspiro[2.5]octane-6-carbonyl)-4-methoxyphenyl)carbamate (1-6j)

[0441] AllocCl (53 μL, 0.49 mmol) was added to a solution of 1-6i (162 mg, 0.41 mmol) and pyridine (67 μL, 0.82 mmol) in anhydrous CH2Cl2 (4 mL) at -10 °C under a N2 atmosphere, and the mixture was stirred for 20 min. The solution was then added to water (2 mL) and 0.5 N HCl (2 mL), and extracted with CH2Cl2 (5 mL x 3). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 25 / 75) to give the title compound 1-6j (157 mg, 80% yield) as a grayish-white solid. MS (ESI) m / z: 481.4 [M+H] + .

[0442] Step 9: (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-12-oxo-6a,7,9,10-tetrahydro-6H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza [-8,1′-cyclopropane]-5(12H)-formic acid allyl ester (1-6k)

[0443] DMP (209 mg, 0.48 mmol) was added to a solution of 1-6kJ (155 mg, 0.32 mmol) in CH2Cl2 (3 mL) at 0 °C, and the mixture was stirred at rt for 30 min. Saturated Na2S2O3 (2 mL) and saturated NaHCO3 (2 mL) were added to the solution, and the mixture was extracted with CH2Cl2 (5 mL x 3). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 50 / 50) to give the title compound 1-6k (130 mg, 84% yield) as a white solid. MS (ESI) m / z: 479.4 [M+H] + .

[0444] Step 10: (6aS)-3,6-dihydroxy-2-methoxy-12-oxo-6a,7,9,10-tetrahydro-6H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza [-8,1′-cyclopropane]-5(12H)-formic acid allyl ester (1-6l)

[0445] MsOH (104 μL, 1.56 mmol) was added to a solution of 1-6kJ (75 mg, 0.16 mmol) in CH2Cl2 (2.5 mL) at 0 °C under a nitrogen atmosphere, and the mixture was stirred at rt for 5 h. The solution was then added to brine (3 mL) and extracted with CH2Cl2 / MeOH (10:1, 5.5 mL * 3). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 50 / 50) to give the title compound 1-6kJ (20 mg, 33% yield) as a white solid. MS (ESI) m / z: 389.3 [M+H] + .

[0446] 1H NMR (400MHz, CDCl3) δ7.18 (s, 1H), 6.73 (s, 1H), 6.11 (d, J=10.3Hz, 1H), 5.91 (s, 1H), 5.90-5.75 (m, 1H), 5.28-5.10 (m, 2H), 4.66 (dd, J=13.0, 5.1Hz, 1H), 4.57-4.46 (m, 1H), 4.42 (dt, J=13.4, 4.5Hz, 1H), 3.96 ( s, 3H), 3.61-3.52 (m, 1H), 3.45 (brs, 1H), 3.23 (ddd, J=13.5, 11.5, 4.1Hz, 1H), 2.02 (dd, J=15.5, 7.4Hz, 1H), 1.91-1.80 (m, 1H), 1.58 (d, J=14.3Hz, 1H), 1.35-1.27 (m, 1H), 0.62-0.46 (m, 2H), 0.45-0.33 (m, 2H).

[0447] Step 11: (6aS)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6a,7,9,10-tetrahydro-6H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza [-8,1′-cyclopropane]-5(12H)-formic acid allyl ester (1-6n)

[0448] K₂CO₃ (8.7 mg, 0.06 mmol) was added to a solution of 1-6l (20 mg, 0.05 mmol) and 1-6m (31 mg, 0.05 mmol, synthesized according to the procedure described in US20200261594A1) in anhydrous DMF (1 mL) under a nitrogen atmosphere, and the mixture was stirred at rt for 4 days. The solution was added to water (12 mL) and extracted with CH₂Cl₂ (5 mL x 4). The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtoOAc = 25 / 75) to give the title compound 1-6n (40 mg, 87% yield) as a grayish-white solid. MS (ESI) m / z: 893.6 [M+H] + .

[0449] Step 12: (S)-2-methoxy-3-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -8-yl)oxy)pentyl)oxy)-6a,7,9,10-tetrahydro-12H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza -8,1′-cyclopropyl]-12-one(1-6)

[0450] Pyrrolidine (4 μL, 0.04 mmol) was added to a solution of 1-6n (35 mg, 0.04 mmol) and Pd(PPh3)4 (1.2 mg, 0.001 mmol) in anhydrous CH2Cl2 (1 mL) under a nitrogen atmosphere, and the mixture was stirred at rt for 30 min. The solution was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 0 / 100) to give the title compound 1-6 (16 mg, 57.8% yield) as a grayish-white solid. MS (ESI) m / z: 707.5 [M+H] + .

[0451] 1 H NMR (400MHz, CDCl3) δ8.10-8.01 (m, 1H), 7.54-7.47 (m, 2H), 7.44 (d, J=6.2Hz, 1H), 7.31 (d, J=8.7Hz, 2H), 6.88 (d, J=8.8H z, 2H), 6.77 (d, J=8.2Hz, 1H), 6.23-6.05 (m, 1H), 4.37-4.21 (m, 2H), 4.19-4.04 (m, 2H), 4.00 (t, J=6.6Hz, 2H), 3.95 (d, J= 9.6Hz, 3H), 3.85 (d, J=3.2Hz, 3H), 3.82 (s, 3H), 3.64-3.50 (m, 2H), 3.49-3.29 (m, 2H), 2.73 (dd, J=16.1, 3.5Hz, 1H), 2.23 (dd, J=14.5, 5.9Hz, 1H), 2.00-1.85 (m, 5H), 1.76-1.59 (m, 4H), 1.49-1.38 (m, 2H), 0.72-0.57 (m, 2H), 0.55-0.43 (m, 2H).

[0452]

[0453]

[0454] Step 1: (S)-(2-(6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (1-7b)

[0455] Pyridine (433 μL) was added to a solution of 1-7a (1370 mg, 2.44 mmol) in anhydrous CH2Cl2 at -5 °C. Then, AllocCl (322 μL) was added to the mixture at -5 °C, and the mixture was stirred at -5 °C for 1 h. The reaction was observed to be complete by TLC (petroleum ether / EtOAc = 5:1). The mixture was diluted with CH2Cl2, washed with 5% citric acid, saturated NaHCO3, and brine, and dried over Na2SO4. The organic phase was concentrated to give a crude product, which was used directly in the next step. MS (ESI) m / z: 647.4 [M+H] + .

[0456] Step 2: (S)-(2-(6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (1-7c)

[0457] p-Toluenesulfonic acid hydrate (282 mg, 1.49 mmol) was added to a solution of 1-7b (1.60 g, 2.48 mmol) in THF (20 mL) and water (1 mL). The reaction mixture was stirred at 22 °C for 1 h. The reaction was observed to be complete by TLC (petroleum ether / EtOAc = 5:1, 1:1). The mixture was diluted with EtOAc (60 mL) and washed with water and brine. The organic phase was concentrated and purified by rapid column chromatography to give 1.09 g of 1-7c (83% yield). MS (ESI) m / z: 533.3 [M+H] + .

[0458] Step 3: (11S,11aS)-11-hydroxy-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-7d)

[0459] Anhydrous DMSO (436 μL, 6.14 mmol) was added dropwise to a solution of oxalyl chloride (260 μL, 3.07 mmol) in anhydrous CH2Cl2 (20 mL) at -70 °C. After 30 min, 1-7c (1.09 g, 2.05 mmol) was slowly added to a solution of anhydrous CH2Cl2 (10 mL) while maintaining the temperature at -70 °C. After 40 min, triethylamine (1423 μL, 6.14 mmol) was added dropwise. The mixture was dried using molecular sieves and the temperature was brought to -50°C and maintained for 1 hour. The reaction mixture was then heated to rt and stirred for 1 hour. The reaction was observed to be complete by TLC (petroleum ether / EtOAc = 1:1, CH2Cl2 / EtOAc = 10:1). The reaction mixture was washed with 5% citric acid aqueous solution (10V) to pH = 3. The organic phase was washed with saturated NaHCO3 aqueous solution and water and dried with sodium sulfate. The organic phase was concentrated to give a crude product, which was purified by rapid column chromatography (CH2Cl2 / EtOAc = 95 / 5) to give 1-7d (416 mg, 38% yield). MS (ESI) m / z: 531.3 [M+H] + .

[0460] Step 4: (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-7e)

[0461] TBSOTf (0.54 mL, 2.35 mmol) was added to a mixture of 1-7d (416 mg, 0.78 mmol) and 2,6-dimethylpyridine (0.37 mL, 3.14 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C. The reaction mixture was stirred at 5 °C for 30 min, followed by stirring at 25 °C for 1 h. The reaction mixture was observed to be complete by LC-MS. The reaction mixture was washed with saturated NaHCO3 aqueous solution and brine, dried over Na2SO4, and concentrated to give the crude product. The crude product was purified by rapid column chromatography (petroleum ether / EtOAc = 80 / 20) to give 1-7e (486 mg, 96% yield). MS (ESI) m / z: 645.5 [M+H] + .

[0462] Step 5: (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-7f)

[0463] Lithium acetate (50 mg, 0.73 mmol) was added to a solution of 1-7e (468 mg, 0.73 mmol) in wet DMF (10 mL, 4g / 1 DMF / water). The reaction was continued at 25 °C for 2 h. Completion was observed by TLC (petroleum ether / EtOAc = 2:1, 1:1). The mixture was diluted with EtOAc and washed with 5% citric acid aqueous solution and brine. The organic phase was dried over Na2SO4 and concentrated to give a crude product, which was purified by rapid column chromatography to give 1-7f (303 mg, 85% yield). MS (ESI) m / z: 489.4 [M+H] + .

[0464] Step 6: (11S,11aS)-8-((5-bromopentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formate allyl ester (1-7g)

[0465] K₂CO₃ (34 mg, 0.25 mmol) was added to a solution of 1–7 f (100 mg, 0.20 mmol) and 1,5-dibromopentane (0.42 mL, 3.07 mmol) in 2 mL of LDMF under reflux. The mixture was stirred under reflux for 2 h. LCMS showed complete consumption of the starting material. The mixture was diluted with EtOAc and washed with H₂O and brine. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc) to give 1–7 g (110 mg, 84% yield). MS (ESI) m / z: 637.4.3 [M+H] + .

[0466] Step 7: (S)-2-(hydroxymethyl)-4-methylenepiperidine-1-carboxylic acid tert-butyl ester (1-7h)

[0467] LiCl (180 mg, 4.23 mmol) in water (0.67 mL) was added to 1-6b (830 mg, 3.25 mmol) in anhydrous THF (10 mL) under rt. NaBH4 (160 mg, 4.23 mmol) was then added to the mixture. The reaction mixture was stirred overnight under rt. 2N HCl (5V) was added to the mixture at 0 °C, followed by saturated NaHCO3 until pH 7-8. The reaction mixture was extracted with EtOAc, washed with brine, dried over Na2SO4, and concentrated to give a crude product. The crude product was purified by rapid column chromatography (petroleum ether / EtOAc = 75 / 25) to give 1-7h (495 mg, 71% yield).

[0468] 1 H NMR (400MHz, CDCl3) δ4.79 (d, J=23.7Hz, 2H), 4.41 (d, J=6.1Hz, 1H), 4.06 (s, 1H), 3.67 (dd, J=11.1, 9.0Hz, 1 H), 3.57 (dd, J=11.2, 5.9Hz, 1H), 2.91 (s, 1H), 2.37 (dd, J=14.1, 6.1Hz, 1H), 2.27-2.15 (m, 3H), 1.48 (s, 9H).

[0469] Step 8: (S)-2-(hydroxymethyl)-4-methylenepiperidine-1-onium chloride (1-7i)

[0470] 4M HCl in 6 mL of MeOH was added to 5 mL of MeOH at 0 °C for 1–7 h (525 mg, 2.31 mmol). The reaction mixture was stirred at rt for 2 h. Completion was observed by TLC (petroleum ether / EtOAc = 2:1). The reaction mixture was washed with saturated NaHCO3 aqueous solution and water, dried over Na2SO4 and concentrated to give the crude product. The crude product was purified by rapid column chromatography to give 1–7 i (425 mg). MS (ESI) m / z: 128.1 [M+H] + .

[0471] Step 9: (S)-(2-(hydroxymethyl)-4-methylenepiperidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methyl ketone (1-7k)

[0472] Oxaloyl chloride (603 μL, 7.07 mmol) was added dropwise to 1-7i (870 mg, 2.36 mmol) in anhydrous CH2Cl2 (8 mL), THF (8 mL), and DMF (4 μL) under N2 at 0 °C. The reaction was heated to rt and stirred at rt for 1 h. Completion was observed by TLC (petroleum ether / EtOAc = 1:1). The mixture was concentrated to give a crude product, which was used directly in the next step. The crude product and 1-7j (423 mg, 2.59 mmol) were dissolved in CH2Cl2 (8 mL). The reaction mixture was cooled to 0 °C and triethylamine (983 μL, 7.07 mmol) was added dropwise under N2. The mixture was then heated to rt and stirred for 3 h. The solution was concentrated, and the crude product was purified by rapid column chromatography to give 1-7k (788 mg, 79% yield). MS(ESI) m / z: 479.4 [M+H] + .

[0473] Step 10: (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)-4-methylenepiperidin-1-yl)methyl ketone (1-71)

[0474] Zinc powder (1.77 g, 27.08 mmol) was added to a mixture of ethanol (4 mL), water (0.25 mL), and AcOH (0.25 mL) at 0 °C. The reaction mixture was stirred at 5 °C for 30 min. A solution of 1-7 kJ (0.35 g, 0.73 mmol) in ethanol (2 mL) was added dropwise at 5 °C. The reaction was continued at 5 °C for 30 min. The solid was removed by filtration. The filtrate was diluted with ethyl acetate and washed with water, a saturated aqueous solution of NaHCO3, and brine. The organic phase was dried over sodium sulfate and filtered. The solvent was removed by rotary evaporation under reduced pressure to give a brown oily product (268 mg, 82% yield), which was used directly in the next step. MS (ESI) m / z: 449.3 [M+H] + .

[0475] Step 11: (S)-(2-(2-(hydroxymethyl)-4-methylenepiperidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate allyl ester (1-7m)

[0476] Pyridine (104 μL) was added to a solution of 1-7 m (268 mg, 0.60 mmol) in anhydrous CH2Cl2 at -5 °C. Then, AllocCl (64 μL, 72.04 mmol) was added to the mixture at -5 °C, and the mixture was stirred at -5 °C for 0.5 h. The reaction was observed to be complete by LC-MS. The mixture was diluted with CH2Cl2, washed with 5% citric acid, saturated NaHCO3, and brine, and dried over Na2SO4. The organic phase was concentrated to give a crude product, which was purified by rapid column chromatography (petroleum ether / EtOAc = 65 / 35) to give 1-7 m (236 mg, 74% yield). MS (ESI) m / z: 533.4 [M+H] + .

[0477] Step 12: (6S,6aS)-6-hydroxy-2-methoxy-8-methylene-12-oxo-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diaza -5(12H)-Allyl formate (1-7n)

[0478] DMP (172 mg, 0.41 mmol) was slowly added fractionally to 1-7n (206 mg, 0.39 mmol) in anhydrous CH2Cl2 (5 mL) at 0 °C. The reaction was then heated to rt and stirred for 2 h. 0.5 eq. of DMP was added fractionally to the reaction. After 9 h, the starting material was exhausted. The mixture was quenched with saturated Na2S2O3, followed by the addition of saturated NaHCO3 and water. The layers were separated, and the organic layer was washed with saturated Na2S2O3, saturated NaHCO3, and brine, and dried over Na2SO4. The crude product was purified by rapid column chromatography (CH2Cl2 / EtOAc = 93 / 7) to give 1-7n (157 mg, 85% yield). MS (ESI) m / z: 531.3 [M+H] + .

[0479] Step 13: (6S,6aS)-6-((tert-butyldimethylsilyl)oxy)-2-methoxy-8-methylene-12-oxo-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diaza -5(12H)-Allyl formate (1-7o)

[0480] TBSOTf (0.23 mL, 1.0 mmol) was added to a mixture of 1-7n (177 mg, 0.33 mmol) and 2,6-dimethylpyridine (0.16 mL) in anhydrous CH2Cl2 (5 mL) at 0 °C. The reaction mixture was stirred at 5 °C for 30 min, followed by stirring at 25 °C for 1 h. The reaction mixture was washed with saturated NaHCO3 aqueous solution and brine, dried over Na2SO4, and concentrated to give the crude product. The crude product was purified by rapid column chromatography (CH2Cl2 / EtOAc = 98 / 2) to give 1-7o (135 mg, 63% yield). MS (ESI) m / z: 645.4 [M+H] + .

[0481] Step 14: (6S,6aS)-6-((tert-butyldimethylsilyl)oxy)-3-hydroxy-2-methoxy-8-methylene-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diaza -5(12H)-Allyl formate (1-7p)

[0482] Lithium acetate (14 mg, 0.21 mmol) was added to 1-7o (135 mg, 0.21 mmol) in a solution of wet dimethylformamide (3 mL, 4g / 1 DMF / water). The reaction was continued at 25 °C for 2 h. Completion was observed by TLC (petroleum ether / EtOAc = 1:1). The mixture was diluted with EtOAc and washed with 5% citric acid aqueous solution, saturated NaHCO3, and brine. The organic phase was dried over Na2SO4 and concentrated to give a crude product, which was purified by rapid column chromatography to give 1-7p (100 mg, 98% yield). MS (ESI) m / z: 489.3 [M+H] + .

[0483] Step 15: (11S, 11aS)-8-((5-(((6S, 6aS)-5-((allyloxy)carbonyl)-6-((tert-butyldimethylsilyl)oxy)-2-methoxy-8-methylene-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diaza -3-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-7q)

[0484] 1-7p (28 mg, 0.06 mmol) and K2CO3 (10 mg, 0.07 mmol) were added to a solution of 1-7g (38 mg, 0.06 mmol) in 1 mL of DMF. The mixture was stirred overnight at rt. The product (petroleum ether / EtOAc = 1:2) was detected by LCMS. The mixture was diluted with EtOAc and washed with water and brine. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 27 / 73) to give 1-7q (48 mg, 84% yield). MS (ESI) m / z: 1045.7 [M+H] + .

[0485] Step 16: (11S, 11aS)-8-((5-(((6S, 6aS)-5-(((allyloxy)carbonyl)-6-hydroxy-2-methoxy-8-methylene-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diaza -3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formate allyl ester (1-7r)

[0486] 1 M TBAF (230 μL, 0.23 mmol) was added to 1-7q (48 mg, 0.05 mmol) in 3 mL of anhydrous THF and 16 μL of AcOH. The mixture was stirred at rt for 2 h. After the reaction was confirmed to be complete by LCMS, the mixture was diluted with EtOAc and washed with saturated NaHCO3 and brine. The organic phase was concentrated and purified by rapid column chromatography to give 1-7r (21 mg, 56% yield). MS (ESI) m / z: 817.5 [M+H] + .

[0487] Step 17: (S)-7-methoxy-8-((5-(((S)-2-methoxy-8-methylene-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diaza -3-yl)oxy)pentyl)oxy)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one (1-7)

[0488] Pd(PPh3)4 (2.2 mg, 0.002 mmol) was added to a solution of 1-7r (15 mg, 0.02 mmol) in CH2Cl2 (0.5 mL) and pyrrolidine (8 μL, 0.09 mmol). The reaction mixture was stirred at rt for 0.5 h. The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with saturated NH4Cl and brine. The organic phase was concentrated and purified by preparative HPLC (0.01% FA in H2O) to give 1-7r as a white solid (9 mg, 80% yield). MS (ESI) m / z: 613.4 [M+H] + .

[0489] 1 H NMR (400MHz, CDCl3) δ7.82 (d, J=5.1Hz, 1H), 7.78 (d, J=5.1Hz, 1H), 7.51 (s, 1H), 7 .47s, 1H), 6.81 (d, J=7.2Hz, 2H), 5.09 (d, J=28.2Hz, 2H), 4.21-4.02 (m, 4H), 4.03 -3.74(m, 11H), 3.68(d, J=11.7Hz, 1H), 3.49(d, J=11.7Hz, 1H), 2.87-2.75(m, 1H) , 2.70-2.64 (m, 2H), 2.53 (dd, J=12.9, 8.1Hz, 2H), 1.97 (m, 6H), 0.84-0.66 (m, 4H).

[0490] Examples 1-8

[0491]

[0492] Step 1: 8,8″-((1,3-phenylenebis(methylene))bis(oxy))(11aS,11a″S)-bis(11-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) [-2,1′-cyclopropane]-10(5H)-formic acid) diallyl ester (1-8a)

[0493] K₂CO₃ (36.94 mg, 0.27 mmol) was added to a solution of 1-5b (50 mg, 0.13 mmol) and 1,3-bis(bromomethyl)benzene (17.5 mg, 0.07 mmol) in DMF (2 mL) under rt. The mixture was stirred at 20 °C for 3 h. The reaction mixture was diluted with EtOAc (10 mL) and washed with brine (8 mL x 3). The organic layer was dried over Na₂SO₄ and concentrated to give a residue, which was purified by silica column gel chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 10 / 90) to give 1-8a (50 mg, 78.2% yield) as a white solid.

[0494] MS(ESI) m / z: 851.3 [M+H] + .

[0495] Step 2: (11aS, 11a″S)-8,8″-((1,3-phenylenebis(methylene))bis(oxy))bis(7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one)(1-8

[0496] Pd(PPh3)4 (6.79 mg, 0.01 mmol) and pyrrolidine (12 μL, 0.15 mmol) were added to a solution of 1-8a (50 mg, 0.06 mmol) and CH2Cl2 (2 mL) at rt. The mixture was stirred at 20 °C for 30 min. The solvent was evaporated and the residue was purified by silica column gel chromatography (eluent: CH2Cl2 / MeOH = 100 / 0 to 1 / 99) and preparative HPLC (0.01% FA) to give a white solid product 1-8 (33.2 mg, 78.63% yield).

[0497] MS(ESI) m / z: 647.3 [M+H] + .

[0498] 1H NMR (400MHz, CDCl3) δ7.80-7.75 (2H, m), 7.54 (2H, s), 7.51 (1H, s), 7.40 (3H, d, J = 4.9), 6.85 (2H, d, J 1.4), 5.25-5.14 (4H, m), 3.96 (6H, s), 3.85 (2H, dd, J=7.9, 3.2), 3.67 (2H, d, J=11.7), 3.52 -3.45 (2H, m), 2.51 (2H, dd, J = 13.0, 8.1), 1.99 (2H, d, J = 12.8), 0.74 (10H, dd, J = 10.6, 6.6).

[0499] Examples 1-9 and 1-10

[0500]

[0501] Step 1: (S)-7-methoxy-8-((3-((((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) -2,1′-cyclopropyl]-8-yl)oxy)methyl)benzyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one (1-9) and (S)-7-methoxy-8-((3-((((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)methyl)benzyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one (1-10)

[0502] NaBH(OAc)3 (14.76 mg, 0.07 mmol) was added to a solution of 1-8 (30 mg, 0.05 mmol) in CH2Cl2 (3 mL) at 0 °C. The mixture was stirred at 20 °C for 30 min. The reaction was quenched with water (10 mL) and extracted with CH2Cl2 (10 mL * 3). The combined organic layers were dried over Na2SO4 and filtered. The solvent was evaporated and the residue was purified by preparative HPLC (0.01% FA) to give white solid products 1-9 (8 mg, 25.25% yield) and white solid products 1-10 (12 mg, 38.96%).

[0503] 1-9 MS(ESI) m / z: 649.3 [M+H] + .

[0504] 1-10 MS(ESI) m / z: 651.3 [M+H] + .

[0505] 1-10 1 H NMR (400MHz, CDCl3) δ7.57 (2H, s), 7.56-7.48 (1H, m), 7.37 (2H, d, J=9.8), 7. 34 (3H, s), 6.08 (2H, s), 5.13-5.02 (4H, m), 3.96 (2H, t, J=7.1), 3.86 (6H, s), 3 .68 (2H, d, J = 12.0), 3.54 (2H, d, J = 12.0), 3.50-3.46 (2H, m), 3.38 (2H, dd, J = 12.4, 8.9), 2.06-1.94 (2H, m), 1.75 (2H, dd, J=12.6, 6.7), 0.74-0.51 (8H, m).

[0506] Examples 1-11

[0507]

[0508] Step 1: Bicyclo[1.1.1]pentane-1,3-dimethyldiethanol (1-11b)

[0509] LiAlH4 (2.1 g, 52.9 mmol) was added in portions to a solution of 1-11a (3 g, 17.64 mmol) in anhydrous THF (80 ml) at 0 °C. The mixture was then heated to rt and stirred overnight. After the reaction was complete, the reaction mixture was quenched with sodium sulfate decahydrate for 1 h, filtered, and the filtrate was concentrated under reduced pressure to give the oily product 1-11b (2.2 g, 97% yield).

[0510] 1 H NMR (400 MHz, d6-DMSO) δ 4.39 (t, J = 5.6 Hz, 2H), 3.34 (t, J = 4.9 Hz, 4H), 1.45 (s, 6H).

[0511] Step 2: 1,3-bis(bromomethyl)bicyclo[1.1.1]pentane (1-11c)

[0512] A solution of liquid bromine (0.4 mL, 7.81 mmol) in 5 mL MeCN was added dropwise to a solution of triphenylphosphine (2.05 g, 7.81 mmol) in 30 mL MeCN at 0 °C, followed by the addition of 1-11b (500 mg, 3.90 mmol). The reaction was heated to 80 °C and refluxed overnight. The solvent was removed under vacuum and purified by rapid column chromatography to give 1-11c (770 mg, 78% yield).

[0513] 1 H NMR (400MHz, CDCl3) δ3.47 (s, 4H), 1.73 (s, 6H).

[0514] Step 3: (11S, 11As)-8-((3-(bromomethyl)bicyclo[1.1.1]pent-1-yl)methoxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1'-cyclopropane]-10(5H)-formic acid allyl ester (1-11d)

[0515] K₂CO₃ (12 mg, 0.07 mmol) was added to a solution of 1-7f (30 mg, 0.06 mmol) and 1-11c (154 mg, 0.61 mmol) in 2 mL of DMF under reflux. The mixture was stirred under reflux for 3 h. The mixture was diluted with EtOAc and washed with H₂O and brine. The organic phase was concentrated and purified by rapid column chromatography to give 1-11d (40 mg, 99% yield). MS (ESI) m / z: 661.3 [M+H] + .

[0516] Step 4: (11S, 11aS)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-8-((3-((((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza) [1,2-b]isoquinoline-3-yl)oxy)methyl)bicyclo[1.1.1]pent-1-yl)methoxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-allyl formate (1-11e)

[0517] 1-1b (19 mg, 0.06 mmol) and K₂CO₃ (11 mg, 0.07 mmol) were added to a solution of 1-11d (43 mg, 0.06 mmol) in 1 mL of DMF. The reaction was heated to 40 °C and maintained for 36 h. The mixture was diluted with EtOAc and washed with water and brine. The organic phase was concentrated and purified by rapid column chromatography (CH₂Cl₂ / MeOH = 94 / 6) to give 1-11e (28 mg, 51% yield). MS (ESI) m / z: 889.6 [M+H].

[0518] Step 5: (11S, 11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-3-yl)oxy)methyl)bicyclo[1.1.1]pent-1-yl)methoxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formate allyl ester (1-11f)

[0519] A buffer solution of 1M TBAF (158 μL, 0.16 mmol) and AcOH (11 μL, 0.19 mmol) was added to a solution of 1-11e (28 mg, 0.03 mmol) in 5 mL of anhydrous THF. The mixture was stirred at rt for 2 h. After the reaction was confirmed to be complete by LCMS, the mixture was diluted with EtOAc and washed with H2O, 5% citric acid, and brine. The organic phase was concentrated and purified by rapid column chromatography to give 1-11f (24 mg, 98% yield). MS (ESI) m / z: 775.4 [M+H] + .

[0520] Step 6: (S)-2-methoxy-3-((3-(((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)methyl)bicyclo[1.1.1]pent-1-yl)methoxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-11)

[0521] Pd(PPh3)4 (3.6 mg, 0.003 mmol) was added to a solution of 1-11f (24 mg, 0.03 mmol) in CH2Cl2 (0.5 mL) and pyrrolidine (11 μL, 0.15 mmol). The reaction mixture was stirred at rt for 0.5 h. The reaction was concentrated and purified by preparative HPLC (0.01% FA in H2O) to give 1-11 as a white solid (10.1 mg, 53% yield). MS (ESI) m / z: 673.3 [M+H] + .

[0522] 1 H NMR (400MHz, CDCl3) δ7.78 (d, J=4.1Hz, 1H), 7.51 (d, J=6.8Hz, 2H), 7.46 (d, J=5.0Hz, 1H), 7.42-7.29 (m, 4H), 6.7 9 (d, J=5.2Hz, 2H), 5.00 (d, J=15.5Hz, 1H), 4.56 (d, J=15.4Hz, 1H), 4.25-4.07 (m, 4H), 3.96-3.94 (m, 1H), 3.94 (d, J=2.0Hz, 6H), 3.85 (d, J=5.4Hz, 1H), 3.67 (d, J=11.6Hz, 1H), 3.49 (d, J=11.6Hz, 1H), 3.27 (dd, J=15.2, 5.3Hz, 1H ), 3.16 (dd, J=15.4, 3.9Hz, 1H), 2.52 (dd, J=13.0, 7.9Hz, 1H), 2.07-1.97 (m, 1H), 1.90 (s, 6H), 0.80-0.68 (m, 4H).

[0523] Examples 1-12

[0524]

[0525] Step 1: (11S, 11aS)-8-((3-(bromomethyl)bicyclo[1.1.1]pent-1-yl)methoxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-12a)

[0526] K₂CO₃ (12 mg, 0.09 mmol) was added to a solution of 1-7f (35 mg, 0.07 mmol) and 1-11c (180 mg, 0.72 mmol) in 2 mL of DMF under reflux. The mixture was stirred under reflux for 5 h. The mixture was diluted with EtOAc and washed with H₂O and brine. The organic phase was concentrated and purified by rapid column chromatography to give 1-12a (41 mg, 86% yield). MS (ESI) m / z: 661.3 [M+H] + .

[0527] Step 2: (S)-3-((3-((((11S,11aS)-10-((allyloxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)methyl)bicyclo[1.1.1]pent-1-yl)methoxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-12b)

[0528] 1-2c (23 mg, 0.06 mmol) and K₂CO₃ (11 mg, 0.08 mmol) were added to a solution of 1-12a (41 mg, 0.06 mmol) in 1 mL of DMF. The mixture was stirred overnight at rt. The product (petroleum ether / EtOAc = 1:2) was analyzed by LCMS. The mixture was diluted with EtOAc and washed with water and brine. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 27 / 73) to give 1-12b (48 mg, 84% yield). MS (ESI) m / z: 4974.6 [M+H] + .

[0529] Step 3: (S)-3-((3-((((11S,11aS)-10-((allyloxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)methyl)bicyclo[1.1.1]pent-1-yl)methoxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid allyl ester (1-12c)

[0530] A mixed solution of 1M TBAF (248 μL, 0.25 mmol) and AcOH (18 μL, 0.30 mmol) was added to a solution of 1-12b (48 mg, 0.05 mmol) in 5 mL of anhydrous THF. The mixture was stirred at rt for 2 h. After the reaction was confirmed to be complete by LCMS, the mixture was diluted with EtOAc and washed with H2O, 5% citric acid, and brine. The organic phase was concentrated and purified by rapid column chromatography to give 1-12c (30 mg, 78% yield). MS (ESI) m / z: 861.5 [M+H] + .

[0531] Step 4: (S)-2-methoxy-3-((3-(((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-8-yl)oxy)methyl)bicyclo[1.1.1]pent-1-yl)methoxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(5H)-one(1-12)

[0532] Pd(PPh3)4 (4.0 mg, 0.003 mmol) was added to a solution of 1-12c (30 mg, 0.03 mmol) in CH2Cl2 (0.5 mL) and pyrrolidine (13 μL, 0.17 mmol). The reaction mixture was stirred at rt for 0.5 h. The reaction was concentrated and purified by preparative HPLC (0.01% FA in H2O) to give a white solid 1-12 (18 mg, 77% yield). MS (ESI) m / z: 675.4 [M+H] + .

[0533] 1H NMR (400MHz, CDCl3) δ7.78 (d, J=4.4Hz, 1H), 7.71-7.63 (m, 2H), 7.57-7.53 (m, 1H), 7.51-7.43 (m, 3H), 7.34-7.32 (m, 1H), 7.21-7.15(m, 1H), 6.80(s, 1H), 4.87(d, J=15.7Hz, 1H), 4.71(d, J=15.7Hz, 1H), 4.21-4.02(m, 6H), 3.94(s, 3H) , 3.84-3.82 (s, 3H), 3.67 (d, J=11.7Hz, 1H), 3.53-3.39 (m, 2H), 3.21 (t, J=10.9Hz, 1H), 3.10 (dd, J=15.2, 5.7Hz, 1H) , 2.80 (dd, J=15.2, 5.0Hz, 1H), 2.52 (dd, J=13.0, 8.1Hz, 1H), 2.00 (dd, J=13.1, 2.6Hz, 1H), 1.89 (s, 6H), 0.74 (m, 4H).

[0534] Examples 1-13

[0535]

[0536] Step 1: Bis(2-hydroxyethyl)carbamate allyl ester (1-13b)

[0537] AllocCl (361.45 mg, 3 mmol) and K₂CO₃ (1036 mg, 7.5 mmol) were added to a solution of 1-13a (423 mg, 3 mmol) in THF (3.1 mL) and water (5.7 mL) at 0 °C. The mixture was stirred at 20 °C for 16 h. TLC (petroleum ether: EtOAc = 1:1, v / v) showed that the reaction was complete. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (20 mL * 3). The combined organic layers were dried over Na₂SO₄ and filtered. The solvent was evaporated and the crude product 1-13b (567 mg) was used in the next step without further processing or purification.

[0538] Step 2: bis(4-methylbenzenesulfonic acid)(((allyloxy)carbonyl)azanyl diester)bis(ethane-2,1-diyl diester))(1-13c

[0539] TsCl (1.7 g, 9 mmol) and triethylamine (1.67 mL, 12 mmol) were added to a solution of 1-13b (567 mg, 3 mmol) in CH2Cl2 (7 mL) at 0 °C. The mixture was stirred at 20 °C for 16 h. TLC (petroleum ether: EtOAc = 3:1, v / v) showed that the reaction was complete. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over Na2SO4 and filtered. The solvent was evaporated and the residue was purified by silica column gel chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 30 / 70) to give a colorless oily 1-13c (1.2 g, 73.08% yield).

[0540] Step 3: 8,8″-(((((allyloxy)carbonyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))(11aS,11a″S)-bis(11-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid) diallyl ester (1-13d)

[0541] K₂CO₃ (41.70 mg, 0.30 mmol) was added to a solution of 1-13c (50 mg, 0.10 mmol) and 1-5b (75.25 mg, 0.20 mmol) in DMSO (2 mL) under rt. The mixture was stirred at 50 °C for 16 h. LCMS showed that the reaction was complete. The reaction mixture was diluted with EtOAc (10 mL) and washed with brine (8 mL x 3). The organic layer was dried over Na₂SO₄ and concentrated to give a residue, which was purified by silica column gel chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 10 / 90) to give a colorless oily 1-13d (38 mg, 49.64% yield).

[0542] MS(ESI) m / z: 902.3 [M+H] + .

[0543] Step 4: (11aS, 11a″S)-8,8″-((azanediylbis(ethane-2,1-diyl))bis(oxy))bis(7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one)(1-13

[0544] Pd(PPh3)4 (5.77 mg, 0.005 mmol) and pyrrolidine (12 μL, 0.12 mmol) were added to a solution of 1-13d (45 mg, 0.05 mmol) and CH2Cl2 (3 mL) under rt. The mixture was stirred at 20 °C for 30 min. LCMS showed that the reaction was complete. The solvent was evaporated and the residue was purified by preparative HPLC (0.01% FA) to give a white solid product 1-13 (12 mg, 35.27% yield).

[0545] MS(ESI) m / z: 614.3 [M+H] + .

[0546] 1 H NMR (400MHz, CDCl3) δ7.79 (2H, d, J=4.4), 7.51 (2H, s), 6.84 (2H, d, J=2.2), 4.30-4.13 (4H, m), 3.93 (6H, d, J=0.9), 3.87-3.81 (2H, m), 3.67 (2H , d, J=11.7), 3.49 (2H, d, J=11.7), 3.26-3.17 (4H, m), 2.52 (2H, dd, J=1 3.0, 8.1), 2.00 (2H, dd, J=13.0, 2.6), 0.73 (8H, ddd, J=11.3, 8.3, 4.5).

[0547] Examples 1-14

[0548]

[0549] Step 1: (S)-3-(3-bromopropoxy)-2-methoxy-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-14a)

[0550] K₂CO₃ (45 mg, 0.32 mmol) was added to a solution of 1-1b (83 mg, 0.27 mmol) and 1,3-dibromopropane (0.42 mL, 4.04 mmol) in 2 mL of DMF under reflux. The mixture was stirred under reflux for 2 h. The mixture was diluted with EtOAc and washed with H₂O and brine. The organic phase was dried over Na₂SO₄ and concentrated. The residue was purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 96 / 4) to give 1-14a (98 mg, 85% yield).

[0551] MS(ESI) m / z: 429.1 [M+H] + .

[0552] Step 2: (11S, 11aS)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-8-(3-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza) [1,2-b]isoquinoline-3-yl)oxy)propoxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-14b)

[0553] 1-7f (25 mg, 0.06 mmol) and K₂CO₃ (10.5 mg, 0.08 mmol) were added to a solution of 1-14a (43 mg, 0.09 mmol) in 1 mL of DMF. The mixture was stirred at 40 °C for 36 h. The mixture was diluted with EtOAc and washed with water and brine. The organic phase was dried over Na₂SO₄ and concentrated. The residue was purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 96 / 4) to give 1-14b (40 mg, 82% yield).

[0554] MS(ESI) m / z: 837.5 [M+H] + .

[0555] Step 3: (11S, 11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza) [1,2-b]isoquinoline-3-yl)oxy)propoxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-14c)

[0556] 1 M TBAF (240 μL, 0.24 mmol) was added to 1-14b (40 mg, 0.05 mmol) in 1.5 mL of anhydrous THF and AcOH (16 μL, 0.29 mmol). The mixture was stirred at rt for 2 h. The mixture was diluted with EtOAc and washed with saturated NaHCO3 and brine. The organic phase was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 96 / 4) to give 1-14c (30 mg, 77% yield).

[0557] MS(ESI) m / z: 723.4 [M+H]+ .

[0558] Step 4: (S)-2-methoxy-3-(3-(((S)-7-methoxy-5-oxo-5,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropoxy]-8-yl)oxy)propoxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-14)

[0559] Pd(PPh3)4 (3.84 mg, 0.003 mmol) was added to a solution of 1-14c (24 mg, 0.033 mmol) in CH2Cl2 (1 mL) and pyrrolidine (6.82 μL, 0.083 mmol). The reaction mixture was stirred at rt for 20 min. The reaction was concentrated and purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (formic acid-free): B-acetonitrile; flow rate: 20 mL / min). The fraction was lyophilized to give 1-14 (6.5 mg, 32% yield) as a white solid.

[0560] MS(ESI) m / z: 621.4 [M+H] + .

[0561] Examples 1-15

[0562]

[0563] Step 1: (S)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-15a)

[0564] DIAD (1.14 mL, 5.75 mmol) was added to a solution of 1-7c (1.53 g, 2.87 mmol) and PPh3 (2.26 g, 8.62 mmol) in THF (30 mL). The reaction mixture was stirred at 40 °C under N2 for 1 h. The solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 30 / 70) to give 1-15a (940 mg, 64% yield).

[0565] MS(ESI) m / z: 515.4 [M+H] + .

[0566] Step 2: (S)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-15b)

[0567] Lithium acetate (121 mg, 1.83 mmol) was added to a solution of 1-15a (940 mg, 1.83 mmol) in wet DMF (15 mL, DMF / water = 49 / 1). The reaction was stirred at 25 °C for 2 h. The mixture was diluted with EtOAc and washed twice with H2O and brine. The organic phase was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 96 / 4) to give 1-15b (605 mg, 92% yield).

[0568] MS(ESI) m / z: 359.2 [M+H] + .

[0569] Step 3: (S)-7-methoxy-8-(3-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-3-yl)oxy)propoxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-15c)

[0570] 1-15b (26 mg, 0.06 mmol) and K₂CO₃ (10.03 mg, 0.07 mmol) were added to a solution of 1-14a (20 mg, 0.06 mmol) in 1 mL of LDMF. The mixture was stirred overnight at rt. The mixture was diluted with EtOAc and washed with water and brine. The organic phase was dried over Na₂SO₄ and concentrated. The residue was purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 96 / 4) to give 1-15c (33 mg, 84% yield).

[0571] MS(ESI) m / z: 707.4 [M+H] + .

[0572] Step 4: (S)-2-methoxy-3-(3-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropoxy]-8-yl)oxy)propoxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one(1-15)

[0573] Pd(PPh3)4 (5.4 mg, 0.005 mmol) was added to a solution of 1-15c (33 mg, 0.047 mmol) in CH2Cl2 (1 mL) and pyrrolidine (9.6 μL, 0.117 mmol). The reaction mixture was stirred at rt under N2 for 0.5 h. The reaction was concentrated and purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (formic acid-free): B-acetonitrile; flow rate: 20 mL / min). The fraction was lyophilized to give 1-15 (5.1 mg, 17% yield) as a white solid.

[0574] MS(ESI) m / z: 623.4 [M+H] + .

[0575] Examples 1-16

[0576]

[0577] Step 1: (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)piperidin-1-yl)methyl ketone (1-16b)

[0578] Oxaloyl chloride (0.43 mL, 4.94 mmol) was added dropwise to a stirred solution of 1–6 g (600 mg, 1.97 mmol) in CH₂Cl₂ (5.1 mL), THF (0.51 mL), and DMF (2.4 μL, 0.031 mmol) at 0 °C and N₂. The reaction mixture was heated to rt and stirred for 1 h. The reaction mixture was concentrated to obtain a pale yellow solid, which was used for the next step without purification.

[0579] The obtained solid and 1-16a (250.6 mg, 2.18 mmol) were dissolved in CH2Cl2 (5.6 mL). The reaction mixture was then cooled to 0 °C and Et3N (0.4 mL, 2.96 mmol) was added dropwise under N2. The reaction mixture was then heated to rt and stirred for 2 h. The reaction mixture was concentrated to give a crude product, which was purified by silica gel chromatography (eluent: EtOAc / hexane = 0% to 80%) to give 1-16b (720 mg, 91% yield) as a yellow solid.

[0580] MS(ESI) m / z: 401.16 [M+H] + .

[0581] Step 2: (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carboxaldehyde (1-16c)

[0582] DMP (805 mg, 1.89 mmol) was slowly added fractionally to a solution of 1-16b (700 mg, 1.75 mmol) in CH2Cl2 (7 mL) at 0 °C. The reaction mixture was then heated to rt and stirred for 3 h. The reaction mixture was filtered, and the filtrate was washed with saturated sodium thiosulfate aqueous solution (10 mL). Subsequently, saturated NaHCO3 aqueous solution (10 mL) and H2O (10 mL) were slowly added. The mixture was extracted with CH2Cl2 (10 mL x 3), and the organic layer was washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated to give a yellow solid title compound 1-16c (730 mg, crude), which was used in the next step without purification.

[0583] MS(ESI) m / z: 399.15 [M+H] + .

[0584] Step 3: (S)-3-(benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diaza -12(6aH)-ketone(1-16d)

[0585] Compound 1-16c (730 mg, 1.83 mmol) was dissolved in a mixed solvent of THF (0.9 mL), methanol (4.5 mL), and water (0.9 mL), followed by the addition of NH4Cl (980.06 mg, 18.32 mmol), and then iron powder (511.6 mg, 9.16 mmol). The reaction mixture was then heated to 50 °C under N2 and stirred for 16 h. The reaction mixture was filtered through diatomaceous earth. The filtrate was diluted with water (5 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica gel chromatography (eluent: EtOAc / hexane = 0% to 60%) to give a yellow solid 1-16d (500 mg, 77.8% yield).

[0586] MS(ESI) m / z: 351.2 [M+H] + .

[0587] Step 4: (S)-3-hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diaza -12(6aH)-keto(1-16e)

[0588] MeSO3H (0.37 mL, 5.7 mmol) was added dropwise to a solution of 1-16e (200 mg, 0.57 mmol) in CH2Cl2 (2.5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min, then heated to rt and stirred for 2 h. The mixture was quenched with saturated NaHCO3 (10 mL), followed by extraction with CH2Cl2 (5 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 20 / 80) to give a white solid 1-16e (120 mg, 80.7% yield).

[0589] MS(ESI) m / z: 261.2 [M+H] + .

[0590] Step 5: (S)-3-((5-iodopentyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diaza -12(6aH)-keto(1-16f)

[0591] 1,5-Diiodopentane (0.34 mL, 2.31 mmol) was added to a solution of 1-16e (120 mg, 0.46 mmol) in anhydrous DMF (2 mL). The solution was then cooled to 0 °C, and K₂CO₃ (127 mg, 0.92 mmol) was added in a single addition. The reaction was stirred at rt for 6 h. EtOAc (20 mL) and H₂O (10 mL) were added, and the organic layer was washed with brine (5 mL), dried over Na₂SO₄, filtered, and concentrated to obtain a crude product. This crude product was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 50 / 50) to give a yellow solid 1-16f (130 mg, 61.7% yield).

[0592] MS(ESI) m / z: 457.1 [M+H] + .

[0593] Step 6: (11S, 11aS)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-8-((5-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyridino[1,2-a][1,4]diaza -3-yl)oxy)pentyl)oxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formate allyl ester (1-16g)

[0594] K₂CO₃ (16.9 mg, 0.12 mmol) was added to a solution of 1-7f (50 mg, 0.102 mmol) and 1-16f (51.3 mg, 0.11 mmol) in DMF (0.5 mL). The mixture was stirred at rt for 3 h. EtOAc (15 mL) and H₂O (10 mL) were added, the organic layer was washed with brine (5 mL), dried over Na₂SO₄, filtered and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 50 / 50) to give a white solid of 1-16 g (60 mg, 71.7% yield).

[0595] MS(ESI) m / z: 817.5 [M+H] + .

[0596] Step 7: (11S, 11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diaza -3-yl)oxy)pentyl)oxy)-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-16h)

[0597] Add 24 μL of AcOH to a solution of 1–16 g (60 mg, 0.07 mmol) in anhydrous THF (1 mL), followed by dropwise addition of TBAF (350 μL, 0.35 mmol, 1 M in THF). Stir the mixture at rt for 6 h. Quench the mixture with saturated NaHCO3 and extract with EtOAc (5 mL * 3). Wash the organic layer with brine (5 mL), dry with Na2SO4, filter and concentrate to obtain the crude product, which is purified by silica column gel chromatography (eluent: CH2Cl2 / MeOH = 100 / 0 to 5 / 95) to give a white solid for 1–16 h (40 mg, 77.5% yield).

[0598] MS(ESI) m / z: 703.4 [M+H] + .

[0599] Step 8: (S)-7-methoxy-8-((5-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diaza -3-yl)oxy)pentyl)oxy)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-Cyclopropyl]-5-one (1-16)

[0600] Pd(PPh3)4 (2.0 mg, catalytic amount) and pyrrolidine (11 μL, 0.14 mmol) were added to a solution of 1-16 h (40 mg, 0.056 mmol) in CH2Cl2 (1 mL). The reaction mixture was stirred at rt under N2 for 15 min. The reaction was neutralized with AcOH and concentrated to give a residue, which was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give a white solid 1-16 (9.6 mg, 28.1% yield).

[0601] MS(ESI) m / z: 601.1 [M+H] + .

[0602] 1 H NMR (400MHz, CDCl3) δ7.92 (s, 1H), 7.81 (s, 1H), 7.52 (s, 1H), 7.44 (s, 1H), 6.79 (d, J=14.2Hz, 2H), 4.24 (s, 1H), 4.10 (d, J=22.7Hz, 4H), 3.94 (d, J=3.5Hz, 6H), 3.87 (s, 1H), 3.79 (s, 1H), 3.69 (d, J = 11.3Hz, 1H), 3.51 (d, J = 12.2Hz, 1H), 3.25 (s, 1H), 2.62-2.47 (m, 1H), 2.19-1.77 (m, 11H), 1.68 (s, 4H), 0.83-0.64 (m, 4H).

[0603] Examples 1-17

[0604]

[0605] Step 1: 8,8″-((pyridin-2,6-diylbis(methylene))bis(oxy))(11S,11aS,11″S,11a″S)-bis(11-hydroxy-7-methoxy-5-oxo-11,11a--dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) [-2,1′-cyclopropane]-10(5H)-formic acid) diallyl ester (1-17b)

[0606] Potassium carbonate (84 mg, 0.77 mmol) was added to a solution of 1-5b (84 mg, 0.23 mmol) and 1-17a (30 mg, 0.11 mmol) in 3 mL of DMF, and the mixture was stirred at rt for 2 h. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with water and brine and dried over anhydrous sodium sulfate. Subsequently, the organic phase was concentrated under vacuum to give a crude product, which was used directly for the next step without further purification.

[0607] MS(ESI) m / z: 852.9 [M+H] + .

[0608] Step 2: (11aS, 11a”S)-8,8”-((pyridin-2,6-dimethylbis(methylene))bis(oxy))bis(7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1'-Cyclopropyl]-5-one)(1-17)

[0609] Pd(PPh3)4 (12 mg, 0.01 mmol) and 1,3-dimethyl ketone (32 mg, 0.23 mmol) were added to a solution of 1-17b (84 mg, 0.23 mmol) in THF / CH2Cl2 (1 mL / 1 mL), and the mixture was stirred at rt for 1 h. The solution was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give 1-17b as a white solid (36 mg, 50% yield).

[0610] 1H NMR (400MHz, CDCl3) δ7.81 (m, 3H), 7.61 (s, 2H), 7.53 (d, J=7.7Hz, 2H), 6.92 (s, 2H), 5.85-5.03 (m, 4H), 4.27-3.96 (m, 6H), 3.96 (m, 2H), 3.73 (d, J = 11.7Hz, 2H), 3.55 (d, J = 11.7Hz, 2H), 2.57 (d J=12.9, 2H), 2.05 (d, J=12.8Hz, 2H), 0.93-0.71 (m, 8H).

[0611] MS(ESI) m / z: 648.7 [M+H] + .

[0612] Examples 1-18

[0613]

[0614] Step 1: 5-((tert-butyldimethylsilyl)oxy)isophthalic acid (1-18b)

[0615] TBS-Cl (2 g, 13.7 mmol) and imidazole (1.12 g, 16.4 mmol) were added to a solution of 1-18a (0.5 g, 2.74 mmol) in 10 mL of DMF, and the mixture was stirred at 50 °C for 4 h. The mixture was acidified to pH 3 with 1 N HCl, diluted with water (10 mL), and extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with water and brine and dried over anhydrous sodium sulfate. The organic phases were then concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50 / 50) to give 1-18b (170 mg, 21% yield) as a white solid.

[0616] MS(ESI) m / z: 297.4 [M+H] + .

[0617] Step 2: (5-((tert-butyldimethylsilyl)oxy)-1,3-phenylene)diethanol (1-18c)

[0618] LiAlH4 (1M, 0.34mL, 0.34mmol) was added to a solution of 1-18b (50mg, 0.17mmol) in 2mL THF, and the mixture was stirred at 40°C for 2h. The mixture was quenched with H2O (36μL), 10% NaOH aqueous solution (40μL), and H2O (200μL), and stirred at rt for 1h. The solution was filtered, and the filtrate was concentrated under vacuum to obtain a residue. The residue was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5μm 19*150mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20mL / min) to give a white solid 1-18c (27mg, 27.1% yield).

[0619] MS(ESI) m / z: 269.4 [M+H] + .

[0620] Step 3: (3,5-bis(bromomethyl)phenoxy)(tert-butyl)dimethylsilane (1-18d)

[0621] A solution of 1-18c (21 mg, 0.08 mmol) in 2 mL of CH3CN was cooled to 0 °C, followed by the addition of PPh3 (62 mg, 0.24 mmol) and CBr4 (78 mg, 0.24 mmol), and the mixture was stirred at rt for 1 h. The solution was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with water and brine and dried over anhydrous sodium sulfate. The organic phase was then concentrated under vacuum to give the residue. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 90 / 10) to give a colorless oily 1-18d (30 mg, 96% yield).

[0622] MS(ESI) m / z: 395.2 [M+H] + .

[0623] Step 4: (11S, 11aS)-8-((3-((((11aS)-10-((allyloxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1'-Cyclopropyl]-8-yl)oxy)methyl)-5-hydroxybenzyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1'-cyclopropane]-10(5H)-formic acid allyl ester (1-18e)

[0624] Potassium carbonate (23 mg, 0.16 mmol) was added to a solution of 1-18d (30 mg, 0.08 mmol) and 1-7f (79 mg, 0.16 mmol) in 3 mL of DMF, and the mixture was stirred at rt for 4 h. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with water and brine and dried over anhydrous sodium sulfate. The solution was concentrated to give a residue, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 90 / 10) to give 1-18e (58 mg, 63% yield) as a white solid.

[0625] MS(ESI) m / z: 1096.5 [M+H] + .

[0626] Step 5: 8,8″-(((5-hydroxy-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11″S,11a″S)-bis(11-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) [-2,1′-cyclopropane]-10(5H)-formic acid) diallyl ester (1-18f)

[0627] TBAF (159 μL, 0.16 mmol) and AcOH (15 μL, 0.27 mmol) were added dropwise to a solution of 1-18e (58 mg, 0.05 mmol) in 3 mL of THF, and the mixture was stirred at rt for 1 h. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL * 2). The combined organic phases were washed with water and brine and dried over anhydrous sodium sulfate. The solution was concentrated to give a residue, which was purified by silica gel column chromatography (CH2Cl2 / MeOH = 90 / 10) to give a white solid 1-18f (42 mg, 91.3% yield).

[0628] MS(ESI) m / z: 867.9 [M+H] + .

[0629] Step 6: (11aS, 11a″S)-8,8″-(((5-hydroxy-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one)(1-18

[0630] Pd(PPh3)4 (3 mg, 0.003 mmol) and pyrrolidine (21 μL, 0.25 mmol) were added to a solution of 1-18f (42 mg, 0.05 mmol) in CH2Cl2 (2 mL), and the mixture was stirred at rt for 1 h. The solution was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give 1-18 (18 mg, 55% yield) as a white solid.

[0631] 1 H NMR (400MHz, CDCl3) δ7.75 (s, 2H), 7.48 (s, 2H), 6.90 (m, 5H), 5.08 (m, 4H), 4.01-3.79 (m, 8H), 3.6 6 (d, J=11.8Hz, 2H), 3.47 (d, J=11.7Hz, 2H), 2.69-2.37 (m, 2H), 2.02-1.89 (m, 2H), 0.78 (dm, 8H).

[0632] MS(ESI) m / z: 663.7 [M+H] + .

[0633] Examples 1-19

[0634]

[0635] Step 1: 8,8″-(((E)-pent-2-en-1,5-diyl)bis(oxy))(11S,11aS,11″S,11a″S)-bis(11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid) diallyl ester (1-19b)

[0636] PPh3 (96 mg, 0.37 mmol) and DIAD (74 μL, 0.37 mmol) were dissolved in THF (3 mL) and stirred at rt for 2 h. Then, 1-7f (107 mg, 0.22 mmol) and 1-19a (8 mg, 0.07 mmol) were added, and the mixture was stirred at rt for another 1 h. The solution was concentrated, and the crude product was purified by silica gel column chromatography (CH2Cl2 / EtOAc = 50 / 50) to give 1-19b (33 mg, 15% yield) as a white solid.

[0637] MS(ESI) m / z: 1044.4 [M+H] + .

[0638] Step 2: (11S, 11aS)-8-(((E)-5-(((11S, 11aS)-10-((allyloxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropane]-8-yl)oxy)pent-2-en-1-yl)oxy)-11-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formic acid allyl ester (1-19c)

[0639] TBAF (106 μL, 0.11 mmol) and AcOH (10 μL, 0.15 mmol) were added dropwise to a solution of 1-19b (33 mg, 0.03 mmol) in 3 mL of THF, and the mixture was stirred at rt for 1 h. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL * 2). The combined organic phases were washed with water and brine and dried over anhydrous sodium sulfate. The solution was concentrated to give a residue, which was purified by silica gel column chromatography (CH2Cl2 / MeOH = 90 / 10) to give a white solid 1-19c (21 mg, 81% yield).

[0640] MS(ESI) m / z: 815.9 [M+H] + .

[0641] Step 3: (11aS, 11a″S)-8,8″-(((E)-pent-2-ene-1,5-diyl)bis(oxy))bis(7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1′-cyclopropyl]-5-one)(1-19

[0642] Pd(PPh3)4 (2 mg, 0.0013 mmol) and pyrrolidine (11 μL, 0.13 mmol) were added to a solution of 1-19c (21 mg, 0.025 mmol) in CH2Cl2 (2 mL), and the mixture was stirred at rt for 1 h. The solution was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give 1-19 as a white solid (4 mg, 26% yield).

[0643] MS(ESI) m / z: 611.7 [M+H] + .

[0644] Examples 1-20

[0645]

[0646] Step 1: (S)-6-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-azaspiro[2.5]octane-5-carboxaldehyde (1-20a)

[0647] DMP (105 mg, 0.24 mmol) was added to a solution of 1–6 h (80 mg, 0.16 mmol) in CH2Cl2 (3 mL) at 0 °C, followed by heating to rt and stirring for 1 h. Saturated Na2S2O3 (5 mL) and saturated NaHCO3 (5 mL) were added to the solution, followed by extraction with CH2Cl2 (5 mL x 3). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a residue, which was purified by rapid column chromatography (eluent: hexane / EtOAc = 100 / 0 to 50 / 50) to give a white solid 1–20a (70 mg, 90% yield).

[0648] MS(ESI) m / z: 425.1 [M+H] + .

[0649] Step 2: (S)-3-(benzyloxy)-2-methoxy-6a,7,9,10-tetrahydro-12H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza -8,1′-cyclopropyl]-12-one (1-20b)

[0650] NH4Cl (134 mg, 2.46 mmol) was added to a solution of 1-20a (70 mg, 0.16 mmol) in MeOH (3 mL) and H2O (1 mL), followed by the addition of iron powder (46.5 mg, 0.81 mmol). The reaction mixture was refluxed under N2 for 3 h. The reaction mixture was filtered through diatomaceous earth. The filtrate was diluted with water (5 mL) and extracted with EtOAc (5 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give 1-20b (54 mg, crude), which was used directly in the next step without further purification.

[0651] MS(ESI) m / z: 377.2 [M+H] + .

[0652] Step 3: (S)-3-hydroxy-2-methoxy-6a,7,9,10-tetrahydro-12H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza -8,1'-Cyclopropyl]-12-one (1-20c)

[0653] MsOH (55 μL, 0.8 mmol) was added to a solution of 1-20b (50 mg, 0.08 mmol) in CH2Cl2 (2.5 mL) at 0 °C, and the mixture was stirred at 0 °C for 5 h. The mixture was quenched with saturated NaHCO3 and extracted with CH2Cl2 (5 mL x 3). The organic layer was washed with brine (5 mL), dried with Na2SO4, and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 25 / 75) to give a yellow solid 1-20c (25 mg, 65.7% yield).

[0654] MS(ESI) m / z: 287.1 [M+H] + .

[0655] Step 4: (S)-2-methoxy-3-((5-(((S)-2-methoxy-12-oxo-6a,9,10,12-tetrahydro-7H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza -8,1′-cyclopropane]-3-yl)oxy)pentyl)oxy)-7,12-dihydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-14(6aH)-one (1-20)

[0656] K₂CO₃ (14.5 mg, 0.1 mmol) was added to a solution of 1-20c (25 mg, 0.087 mmol) and 1-1c (48.4 mg, 0.096 mmol) in DMF (0.5 mL). The reaction was stirred at rt for 3 h. The reaction was quenched with H₂O (15 mL) and extracted with EtOAc (10 mL * 3). The organic layer was washed with brine (5 mL), dried with Na₂SO₄, filtered and concentrated to obtain a crude product, which was purified by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19 * 150 mm; mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give a white solid 1-20 (8.9 mg, 15.6% yield).

[0657] MS(ESI) m / z: 663.7 [M+H] + .

[0658] Examples 1-21

[0659]

[0660] Step 1: (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-yl)(4-hydroxy-5-methoxy-2-nitrophenyl) ketone (1-21b)

[0661] t-BuOK (6.95 g, 61.98 mmol) was added to a mixture of MePh3PBr (24.6 g, 68.86 mmol) in THF (100 mL) at 0 °C and N2. The mixture was stirred at 0 °C for 2 h, and then a solution of 1-21a (4 g, 6.89 mmol) in THF (30 mL) was added dropwise to the mixture and stirred at 0 °C for 16 h. The mixture was neutralized with citric acid and extracted with EtOAc (100 mL * 3). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 25 / 75) to give a yellow solid 1-21b (1.1 g, 37.8% yield).

[0662] MS(ESI) m / z: 423.2 [M+H] + .

[0663] Step 2: Bis(2-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)ethyl)carbamate (1-21c)

[0664] K₂CO₃ (55.5 mg, 0.4 mmol) was added to a solution of 1-13c (100 mg, 0.2 mmol) and 1-21b (186.83 mg, 0.44 mmol) in DMSO (3 mL). The mixture was stirred at 50 °C for 16 h. The reaction was quenched with H₂O and extracted with EtOAc (30 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 0 / 100) to give a yellow solid 1-21c (108 mg, 53.83% yield).

[0665] MS(ESI) m / z: 998.5 [M+H] + .

[0666] Step 3: Bis(2-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (1-21d)

[0667] A mixture of Zn powder (28.24 mg, 4.08 mmol) in EtOH (3 mL), AcOH (0.2 mL), and H₂O (0.2 mL) was stirred at rt for 10 min. Then, a solution of 1-21d (108 mg, 0.11 mmol) in EtOH (2 mL) was added, and the mixture was stirred at rt for 1 h. The reaction was filtered through diatomaceous earth, and the filtrate was concentrated to obtain a crude product. This crude product was purified by silica gel chromatography (eluent: CH₂Cl₂ / MeOH = 20 / 1) to obtain a yellow oily 1-21d (73 mg, 72.2% yield).

[0668] MS(ESI) m / z: 938.6 [M+H] + .

[0669] Step 4: Bis(2-(5-(((allyloxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (1-21e)

[0670] Alloc-Cl (53 μL, 0.49 mmol) and pyridine (24.73 μL, 0.311 mmol) were added to a solution of 1-21d (73 mg, 0.077 mmol) in anhydrous CH2Cl2 (2 mL) at -10 °C under a N2 atmosphere. The mixture was stirred at -10 °C for 1 h. The solution was added to water (10 mL) and extracted with CH2Cl2 (5 mL x 3). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by rapid column chromatography (eluent: petroleum ether / EtOAc = 1 / 3) to give a grayish-white solid 1-21e (75 mg, 87.13% yield).

[0671] MS(ESI) m / z: 1106.6 [M+H] + .

[0672] Step 5: Bis(2-(5-(((allyloxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate allyl ester (1-21f)

[0673] p-Toluenesulfonic acid hydrate (25.79 mg, 0.13 mmol) was added to a solution of 1-21e (75 mg, 0.067 mmol) in THF (2 mL) and water (0.1 mL). The reaction mixture was stirred at rt for 1 h. The mixture was directly concentrated and purified by rapid column chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to give 1-21f as a yellow solid (52 mg, 87.38% yield).

[0674] MS(ESI) m / z: 878.4 [M+H] + .

[0675] Step 6: 8,8′-(((((allyloxy)carbonyl)azanediyl)bis(ethane-2,1-diyl))bis(oxy))(11aS,11a′S)-bis(11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-formic acid) allyl ester 1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formate allyl ester (1-21g)

[0676] DMP (55.27 mg, 0.13 mmol) was added at 0 °C to a solution of 1-21 g (52 mg, 0.059 mmol) in CH2Cl2 (2 mL). The reaction mixture was then heated to rt and stirred for 4 h. The reaction mixture was filtered, and the filtrate was quenched with a saturated aqueous sodium thiosulfate solution (5 mL). Subsequently, a saturated aqueous NaHCO3 solution (5 mL) and H2O (10 mL) were slowly added. The mixture was extracted with dichloromethane (5 mL x 3), and the organic layer was washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated to give a crude product. This crude product was purified by rapid column chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to give a white solid of 1-21 g (32 mg, 61.82% yield).

[0677] MS(ESI) m / z: 874.4 [M+H] + .

[0678] Step 7: (11aS, 11a′S)-8,8′-((azanediylbis(ethane-2,1-diyl))bis(oxy))bis(7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza -5-keto)(1-21)

[0679] Pd(PPh3)4 (2.12 mg, 0.0018 mmol) and pyrrolidine (6 μL, 0.073 mmol) were added to a solution of 1-21 g (32 mg, 0.036 mmol) and CH2Cl2 (2 mL), and the mixture was stirred at rt under N2 for 15 min. The reaction was neutralized with AcOH and concentrated to give a crude product, which was purified by preparative HPLC (column: XBridge Prep C18 OBD 5 μm 19*150 mm; mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give a white solid 1-21 (9.7 mg, 45.23% yield).

[0680] MS(ESI) m / z: 586.3 [M+H] + .

[0681] Examples 1-22

[0682]

[0683] Step 1: 2-Bromo-N-(2-Bromoethyl)-N-methylethyl-1-amine (1-22b)

[0684] 1-22a (1.0 g) was added to a solution of 37% formaldehyde (0.66 mL) and 98% formic acid (0.26 mL), and the solution was heated under reflux for 2 h. The mixture was concentrated under vacuum to give a colorless oil, which was crystallized with MeOH to give a white solid 1-22b (700 mg, 65% yield).

[0685] 1 H NMR (400MHz, DMSO-d6) δ9.66 (s, 1H), 3.79-3.72 (m, 4H), 3.65-3.58 (m, 4H), 2.85 (s, 3H).

[0686] Step 2: ((((methylazonyl)bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-yl)methyl ketone)(1-22c)

[0687] K₂CO₃ (112.84 mg, 0.816 mmol) was added to a solution of 1-22b (100 mg, 0.408 mmol) and 1-21b (379.50 mg, 0.898 mmol) in DMSO (3 mL). The mixture was stirred at 50 °C for 16 h. The reaction was quenched with H₂O and extracted with EtOAc (30 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated to give a crude product, which was purified by silica column gel chromatography (eluent: hexane / EtOAc = 100 / 0 to 0 / 100) to give a yellow solid 1-22c (200 mg, 52.78% yield).

[0688] MS(ESI) m / z: 928.6 [M+H] + .

[0689] Step 3: ((((methylazanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-yl) ketone)(1-22d)

[0690] p-Toluenesulfonic acid hydrate (90.16 mg, 0.474 mmol) was added to a solution of 1-22c (200 mg, 0.215 mmol) in THF (2 mL) and water (0.1 mL). The reaction mixture was stirred at rt for 2 h. The mixture was concentrated and purified by silica gel chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to give 1-22d as a white solid (110 mg, 72.96% yield).

[0691] MS(ESI) m / z: 700.4 [M+H] + .

[0692] Step 4: (2S,2′S)-1,1′-(4,4′-(((methylazonyl)bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzoyl))bis(4-methylenepyrrolidine-2-carboxaldehyde)(1-22e)

[0693] DMP (146.69 mg, 0.345 mmol) was added to a solution of 1-22d (110 mg, 0.157 mmol) in dichloromethane (2 mL), THF (2 mL), and DMF (1 mL) at 0 °C. The reaction mixture was then heated to rt and stirred at rt for 4 h. The reaction mixture was filtered, and the filtrate was directly concentrated to give a crude product, which was purified by silica gel chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to give a yellow solid 1-22e (60 mg, 55.83% yield).

[0694] MS(ESI) m / z: 696.3 [M+H] + .

[0695] Step 5: (11aS, 11a′S)-8,8′-(((methylazanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza -5-keto)(1-22)

[0696] NH4Cl (92.26 mg, 1.72 mmol) was added to a solution of 1-22e (60 mg, 0.086 mmol) in THF (0.896 mL), methanol (4.48 mL), and water (0.896 mL), followed by the addition of iron powder (48.16 mg, 0.862 mmol). The mixture was then heated at 50 °C and N2 for 16 h. The reaction mixture was cooled to rt and filtered through diatomaceous earth. The filtrate was directly concentrated to give a crude product, which was purified by preparative HPLC (column: XBridge Prep C18 OBD 5 μm 19*150 mm; method: mobile phase: A-water (0.01% formic acid): B-acetonitrile; flow rate: 20 mL / min) to give 1-22e as a white solid (4.8 mg, 9.28% yield).

[0697] MS(ESI) m / z: 600.3 [M+H] + .

[0698] 1 H NMR (400MHz, CDCl3) δ7.68 (d, J=4.4Hz, 2H), 7.49 (s, 2H), 6.83 (s, 2H), 5.19 (d, J=11.2Hz, 4H), 4.3 8-4.17 (m, 8H), 3.91 (s, 6H), 3.89-3.84 (m, 2H), 3.12-3.08 (m, 6H), 2.97-2.93 (m, 2H), 2.56 (s, 3H).

[0699]

[0700] Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (2-1c)

[0701] EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 4.77 g, 19.3 mmol) was added to a solution of 2-1a (5 g, 18.4 mmol) and 2-1b (2.37 g, 19.3 mmol) in 100 mL of anhydrous THF. The mixture was stirred at rt for 40 h. After the reaction was confirmed to be complete by LCMS / TLC (CH2Cl2 / MeOH = 20:1), the mixture was concentrated. The residue was slurried with MTBE (30 V) and stirred for 2 h. The solid was separated by filtration under vacuum for 3 h to give 2-1c (5.16 g, 74% yield). MS (ESI) m / z: 378.4 [M+H] + .

[0702] 1 H NMR (400MHz, d6-DMSO) δ9.90 (s, 1H), 8.14 (d, J=7.0Hz, 1H), 7.53 (d, J=8.5Hz, 2H), 7.25 (t, J=9.0 Hz, 3H), 5.91 (ddd, J=22.3, 10.5, 5.3Hz, 1H), 5.30 (dd, J=17.2, 1.5Hz, 1H), 5.17 (d, J=10.5Hz, 1H ), 5.10 (t, J = 5.7Hz, 1H), 4.52-4.45 (m, 2H), 4.43 (d, J = 5.6Hz, 3H), 3.89 (dd, J = 8.5, 7.1Hz, 1H), 1 .98 (dq, J=13.5, 6.7Hz, 1H), 1.28 (t, J=11.8Hz, 3H), 0.88 (d, J=6.8Hz, 3H), 0.84 (d, J=6.7Hz, 3H).

[0703] Step 2: (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (2-1d)

[0704] Pd(PPh3)4 (76.6 mg, 0.066 mmol) was added to a solution of 2-1c (500 mg, 1.33 mmol) in CH2Cl2 (10 mL) and pyrrolidine (270.8 μL, 3.31 mmol) under rt and N2 conditions. The reaction mixture was stirred under rt for 0.5 h. The reaction was concentrated and purified by silica column gel chromatography (CH2Cl2 / MeOH = 90:10) to give a white solid product 2-1d (370 mg, 95% yield).

[0705] MS(ESI) m / z: 294.3 [M+H] + .

[0706] 1 H NMR (400MHz, d6-DMSO) δ9.99 (s, 1H), 8.20 (d, J = 6.3Hz, 1H), 7.53 (d, J = 8.4Hz, 2H), 7.24 (d, J = 8.4Hz, 2H), 5.10 (s, 1H), 4.53-4.45 (m, 1H ), 4.43 (s, 2H), 3.05 (d, J=4.9Hz, 1H), 1.93 (dd, J=12.0, 6.8Hz, 1H), 1.30 (d, J=7.0Hz, 3H), 0.89 (d, J=6.9Hz, 3H), 0.80 (d, J=6.8Hz, 3H).

[0707] Step 3: ((17S,20S)-21-((4-(hydroxymethyl)phenyl)amino)-17-isopropyl-20-methyl-15,18,21-trioxo-3,6,9,12-tetraoxa-16,19-diazaeicosyl)carbamate (9H-fluorene-9-yl)methyl ester (2-1f)

[0708] DIPEA (326 mg, 2.52 mmol) was added to a solution of 2-1e (370 mg, 1.26 mmol) and HATU (575.8 mg, 1.51 mmol) in 4 mL of anhydrous DMF. The mixture was stirred at rt for 10 min. Then, a solution of 2-1d (645 mg, 1.33 mmol) in DMF was added to the mixture. The reaction was stirred for 1 h. After the reaction was confirmed to be complete by LCMS / TLC (CH2Cl2 / MeOH = 20:1), the mixture was concentrated and purified by silica column gel chromatography (CH2Cl2 / MeOH = 95:5) to give a light brown solid product 2-1e (680 mg, 71% yield). MS (ESI) m / z: 763.5 [M+H] + .

[0709] Step 4: (5-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)tert-butyl carbamate (2-1h)

[0710] 2-1g (200mg, 0.121mmol) and 200mg Triphosgene (24.9 mg, 0.084 mmol) was added to a solution of THF (2.5 mL) by MS, followed by the addition of TEA (64 μL, 0.462 mmol) at 0 °C and N2. The mixture was stirred at 0 °C and N2 for 10 min. Isocyanate formation was detected by LCMS analysis quenched with methanol. A solution of 2-1f (176 mg, 0.231 mmol), dibutyltin dilaurate (13.3 mg, 0.021 mmol), and TEA (43.7 μL, 0.315 mmol) in THF (2.5 mL) was added to the mixture. The mixture was stirred at rt for 3 h. The mixture was filtered and the filtrate was concentrated. The residue was purified by silica column gel chromatography (CH2Cl2 / MeOH = 96 / 4) to give a white solid product 2-1h (302 mg, 83% yield). MS (ESI) m / z: 1472.3 [M+H] + .

[0711] Step 5: (5-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)tert-butyl carbamate (2-1i)

[0712] p-Toluenesulfonic acid hydrate (40 mg, 0.21 mmol) was added to a solution of 2-1h (304 mg, 0.17 mmol) in THF (3 mL) and water (0.15 mL). The reaction mixture was stirred at 22 °C for 4 h. After the reaction was confirmed to be complete by TLC (CH2Cl2 / MeOH = 20:1), the mixture was diluted with EtOAc (20 mL) and washed with water, saturated NaHCO3, and brine. The organic phase was concentrated and purified by silica column gel chromatography (CH2Cl2 / MeOH = 95:5) to give product 2-1i (213 mg, 81% yield). MS (ESI) m / z: 1514.1 [M+H] + .

[0713] Step 6: (11S, 11aS)-8-((5-(((11S, 11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-formic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecosano-25-amido)benzyl ester (2-1j)

[0714] DMP (58.9 mg, 0.139 mmol) was slowly added fractionally to a solution of 2-1i (100 mg) in anhydrous CH2Cl2 (2 mL) at 0 °C. The reaction was then heated to rt and stirred overnight. The reaction was quenched with saturated Na2S2O3, followed by the addition of saturated NaHCO3 and water. The layers were separated, and the organic layer was washed with saturated Na2S2O3, saturated NaHCO3, and brine, and dried over Na2SO4. The crude product was purified by silica column gel chromatography (CH2Cl2 / MeOH = 95 / 5) to give product 2-1j (80 mg, 80% yield). MS (ESI) m / z: 1510.3 [M+H] + .

[0715] Step 7: (11S, 11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-formic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecosano-25-amido)benzyl ester (2-1k)

[0716] Cool 2-1j (60 mg, 0.040 mmol) to -3 °C. Separately, cool a solution of 95% TFA in H₂O (1.5 mL) to -3 °C, then add it to 2-1j. Stir the reaction mixture at -3 °C for 40 min, then pour it into a 1:1 solution of CHCl₃ / NaHCO₃ (40 mL) at 0 °C. Separate the organic layer, dry with Na₂SO₄, filter, and remove the solvent under vacuum. The crude substance 2-1k is used directly for the next step.

[0717] MS(ESI) m / z: 1392.6 [M+H] + .

[0718] Step 8: (11S, 11As)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-formic acid 4-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazaeicosane-21-amido)benzyl ester (2-1l)

[0719] Et2NH (61 μL, 0.593 mmol) was added to a solution of crude 2-1k in 0.5 mL of DMF. The mixture was stirred at rt for 0.5 h. After the reaction was complete, the mixture was purified by preparative HPLC (0.1% FA in H2O) to give product 2-1l (14.3 mg, 31% yield, two steps).

[0720] MS(ESI) m / z: 1170.3 [M+H] + .

[0721] Step 9: (11S, 11As)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-formic acid 4-((21S,24S)-1-((1R,8S,9S)-bicyclo[6.1.0]ano-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl ester (2-1)

[0722] DIPEA (8.6 μL, 0.05 mmol) was added to a solution of 2-1L (14.5 mg, 0.012 mmol) and 2-1M (10.8 mg, 0.037 mmol) in 1 mL of DMF. The mixture was stirred at rt for 20 min. The mixture was purified by preparative HPLC (without additives in the mobile phase) to give a light gray solid product 2-1 (7.7 mg, 46% yield).

[0723] MS(ESI) m / z: 1346.4 [M+H] + .

[0724] After the joint ruptures, the payload released from the joint-payload 2-1 undergoes a dehydration reaction and forms compound SG-2057, which has a similar potency to payload Ref-1-1 (also known as SG-3199).

[0725]

[0726]

[0727] Step 1: (S)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1'-cyclopropane]-10(5H)-formic acid allyl ester (2-2a)

[0728] DIAD (1.14 mL, 5.75 mmol) was added to a solution of 1-7c (1.53 g, 2.87 mmol) and PPh3 (2.26 g, 8.62 mmol) in THF (30 mL). The reaction mixture was stirred at 40 °C under N2 for 1 h. After the reaction was confirmed to be complete by TLC (petroleum ether / EtOAc = 1:2), the solvent was removed under vacuum, and the residue was purified by silica column gel chromatography to give product (petroleum ether / EtOAc = 40:60)2-2a (940 mg, 64% yield). MS (ESI) m / z: 515.5 [M+H] + .

[0729] 1 H NMR (400MHz, CDCl3) δ7.19 (s, 1H), 6.70 (s, 1H), 5.84-5.68 (m, 1H), 5.12 (t, J=12.7Hz, 2H), 4.58 (d d, J=12.9, 5.3Hz, 1H), 4.45 (t, J=12.5Hz, 2H), 3.89-3.86 (m, 1H), 3.85 (s, 3H), 3.74 (d, J=11.8Hz, 1H), 3.48 (dd, J=12.2, 4.2Hz, 1H), 3.37 (d, J=11.8Hz, 1H), 2.37 (dd, J=12.8, 8.4Hz, 1H), 1.42 (d, J =12.8Hz, 1H), 1.25 (ddd, J=19.0, 9.0, 4.5Hz, 3H), 1.08 (dd, J=7.3, 3.7Hz, 18H), 0.86-0.57 (m, 4H).

[0730] Step 2: (S)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1'-cyclopropane]-10(5H)-formic acid allyl ester (2-2b)

[0731] Lithium acetate (121 mg, 1.83 mmol) was added to a solution of 2-2a (940 mg, 1.83 mmol) in wet DMF (15 mL 49 / 1 DMF / water). The reaction was continued at 25 °C for 2 h. The mixture was diluted with EtOAc and washed twice with H2O and brine. The organic phase was dried over Na2SO4, concentrated, and purified by silica column gel chromatography to give product 2-2b (605 mg, 92% yield). MS (ESI) m / z: 359.4 [M+H] + .

[0732] Step 3: (S)-(3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methyl ketone (2-2e)

[0733] EDCI (935 mg, 4.88 mmol) was added at 0 °C to a solution of 2-2c (1.5 g, 4.06 mmol) and 1-hydroxypyridine oxide (HOPO, 497 mg, 4.47 mmol) in 15 mL of CH2Cl2. The reaction was continued at 15 °C for 1 h, at which point 2-2d (729 mg, 4.47 mmol) and triethylamine (0.71 mL, 5.08 mmol) in 15 mL of CH2Cl2 were added at -10 °C. The reaction mixture was stirred at rt for 3 h. The reaction mixture was washed successively with water (~30 mL) and cold aqueous HCl (0.5 M) until the pH was adjusted to 4-5. The organic phase was then washed with saturated aqueous NaHCO3 solution (~30 mL), followed by water (~30 mL). The solvent was removed under vacuum to obtain a crude product, which was then purified by silica column gel chromatography to give product 2-2e (1.34 g, 57% yield). MS (ESI) m / z: 515.4 [M+H] + .

[0734] Step 4: (S)-(3-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroisoquinoline-2(1H)-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methyl ketone (2-2f)

[0735] Imidazole (355 mg, 5.21 mmol) was added to a solution of 2-2e (1.34 g, 2.61 mmol) in 25 mL of CH₂Cl₂. Then, TBSCl (589 mg, 3.91 mmol) was added to the mixture under reflux. The mixture was stirred overnight under reflux. The reaction was filtered and the filtrate was concentrated. The crude product was purified by silica gel column chromatography to give product 2-2f (1.38 g, 84% yield). MS (ESI) m / z: 629.5 [M+H] + .

[0736] Step 5: (S)-(3-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroisoquinoline-2(1H)-yl)(4-hydroxy-5-methoxy-2-nitrophenyl) ketone (2-2g)

[0737] Lithium acetate (145 mg, 2.2 mmol) was added to a solution of 2-2g (1.38 g, 2.2 mmol) in wet DMF (15 mL, 4g / 1 DMF / water). The reaction was continued at 25°C for 2 h. The mixture was diluted with EtOAc and washed twice with H2O and brine. The organic phase was concentrated and purified by silica column gel chromatography to give 2-2g (930 mg, 90% yield) of the product. MS (ESI) m / z: 473.3 [M+H] + .

[0738] Step 6: (S)-(4-((5-bromopentyl)oxy)-5-methoxy-2-nitrophenyl)(3-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroisoquinoline-2(1H)-yl)methyl ketone (2-2h)

[0739] K₂CO₃ (330 mg, 2.36 mmol) was added to a solution of 2–2 g (930 mg, 1.97 mmol) and 1,5-dibromopentane (4.0 mL, 29.5 mmol) in 18 mL of LDM under reflux. The mixture was stirred under reflux for 2 h. The mixture was diluted with EtOAc and washed with H₂O and brine. The organic phase was concentrated and purified by silica column gel chromatography (petroleum ether / EtOAc = 75 / 25) to give the product 2–2 h (818 mg, 67% yield). MS (ESI) m / z: 621.4 [M+H] + .

[0740] Step 7: (S)-8-((5-(4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxy-5-nitrophenoxy)pentyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1′-cyclopropane]-10(5H)-formate allyl ester (2-2i)

[0741] 2-2b (450 mg, 1.26 mmol) and K₂CO₃ (226 mg, 1.63 mmol) were added to a solution of 2-2h (818 mg, 1.32 mmol) in DMF (1 mL). The mixture was stirred at rt for 24 h. The product (petroleum ether / EtOAc = 1:2) was detected by LCMS. The mixture was diluted with EtOAc and washed with water and brine. The organic phase was concentrated and purified by silica column gel chromatography (petroleum ether / EtOAc = 33:67) to give product 2-2i (930 mg, 82% yield). MS (ESI) m / z: 899.6 [M+H] + .

[0742] Step 8: (S)-8-((5-(5-amino-4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza .2,1′-cyclopropane]-10(5H)-formic acid allyl ester (2-2j)

[0743] Zinc powder (2.65 g, 38.3 mmol) was added to a mixture of ethanol (10 mL), water (0.625 mL), and AcOH (0.625 mL) at 0 °C. The reaction mixture was stirred at 5 °C for 30 min. 2-2i (930 mg, 1.04 mmol) was added dropwise to a solution of ethanol (6 mL) at 5 °C. The reaction was continued at 5 °C for 50 min. The solid was removed by filtration. The filtrate was diluted with EtOAc and washed with water, saturated NaHCO3 aqueous solution, and brine. The organic phase was dried over sodium sulfate and filtered. The solvent was removed by rotary evaporation under reduced pressure to give a crude product, which was purified by silica column gel chromatography (petroleum ether / EtOAc = 33:67) to give a yellow solid product 2-2j (806 mg, 90% yield). MS (ESI) m / z: 868.7 [M+H] + .

[0744] Step 9: (S)-8-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapentadecane-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxyphenoxy)pentyl)oxy)7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza [-2,1'-cyclopropane]-10(5H)-formic acid allyl ester (2-2l)

[0745] 2-2l was prepared according to the procedure described in step 4 of Example 2-1, yielding a white solid (260 mg, yield 91%). MS (ESI) m / z: 1658.4 [M+H] + .

[0746] Step 10: (S)-8-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapentadecane-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza -2,1'-cyclopropane]-10(5H)-formic acid allyl ester (2-2m)

[0747] p-Toluenesulfonic acid hydrate (29.8 mg, 0.16 mmol) was added to a solution of 2-2 L (260 mg, 0.16 mmol) in THF (6 mL) and water (0.3 mL). The reaction mixture was stirred overnight at 22 °C. The mixture was diluted with EtOAc (20 mL) and washed with water and brine. The organic phase was concentrated and purified by silica column gel chromatography to give product 2-2 M (172 mg, 71% yield). MS (ESI) m / z: 1544.3 [M+H] + .

[0748] Step 11: (6S,6As)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) -2,1'-Cyclopropyl]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl ester (2-2n)

[0749] DMP (52 mg, 0.123 mmol) was slowly added fractionally to 2-2m (172 mg) in anhydrous CH2Cl2 (2.5 mL) at 0 °C. The reaction was then heated to rt and stirred for 9 h. After 9 h, the reaction was quenched with saturated Na2S2O3, followed by the addition of saturated NaHCO3 and water. The layers were separated, and the organic layer was washed with saturated Na2S2O3, saturated NaHCO3, and brine, and dried with Na2SO4. The crude product was purified by silica column gel chromatography (CH2Cl2 / MeOH = 95 / 5) to give product 2-2n (130 mg, 76% yield). MS (ESI) m / z: 1542.1 [M+H] + .

[0750] Step 12: (6S,6As)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) -2,1'-Cyclopropyl]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid 4-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazaeicosode-21-amido)benzyl ester (2-2o)

[0751] Add Et₂NH (38 μL, 0.37 mmol) to a solution of 2-2n (50 mg, 0.04 mmol) in DMF (2 mL). Stir the mixture at rt for 1 h. After the reaction is complete, concentrate the mixture to give crude product 2-2o, which can be used directly in the next step. MS (ESI) m / z: 1320.2 [M+H] + .

[0752] Step 13: (6S,6aS)-6-hydroxy-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) -2,1′-cyclopropyl]-8-yl)oxy)pentyl)oxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid 4-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazaeicosode-21-amido)benzyl ester (2-2p)

[0753] Pd(PPh3)4 (2.15 mg, 0.002 mmol) was added to a solution of crude product 2-2p in THF / MeOH (2 mL / 0.2 mL) and dimethyl ketone (10.42 mg, 0.074 mmol) under reflux. The reaction mixture was stirred under reflux for 1 h. The reaction was concentrated. The residue was purified by preparative HPLC (0.1% FA in H2O) to give a white solid product 2-2p (16 mg, 61% yield). MS (ESI) m / z: 1236.1 [M+H] + .

[0754] Step 14: (6S,6As)-6-hydroxy-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza) -2,1'-Cyclopropyl]-8-yl)oxy)pentyl)oxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diaza [1,2-b]isoquinoline-5(14H)-formic acid 4-((21S,24S)-1-((1R,8S,9S)-bicyclo[6.1.0]an-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl ester (2-2)

[0755] DIPEA (10.9 μL, 0.063 mmol) was added to a solution of 2-2p (19.3 mg, 0.016 mmol) and 2-1m (13.6 mg, 0.047 mmol) in DMF (1 mL). The mixture was stirred at rt for 20 min. The mixture was purified by preparative HPLC (0.1% FA in H2O) to give a white solid product 2-2 (9.8 mg, 44% yield). MS (ESI) m / z: 1412.4 [M+H] + .

[0756] After the joint ruptures, the payload released from the joint-payload 2-2 undergoes a dehydration reaction and forms the compounds of Examples 1-3.

[0757] The compounds disclosed herein can also be synthesized based on the synthetic methods provided herein and in conjunction with common knowledge in the art.

[0758] Table 1: Payload Structure

[0759]

[0760]

[0761]

[0762]

[0763] Table 2: Joint-Load Capacity Structure

[0764]

[0765] ADC preparation and characterization

[0766] Preparation of DAR2 antibody-drug conjugates. Anti-CD74 antibody mAb1 was incubated with endoS2 at a reaction temperature (0-40°C) for 1-24 hours in reaction buffer (0.5-25 mg / mL, 50 mM Tris-HCl buffer, pH 7.0-8.5) at a reaction temperature of 1 / 2000-1 / 500 w / w (EndoS2 / mAb weight ratio). 2-40 eq. UDP-GalNAz (20 mM) and 0.1 w / w%-10 w / w% (GalT / mAb weight ratio) of β1,4-GalT were added to the reaction mixture, and the mixture was incubated at a reaction temperature (0-40°C) in reaction buffer (50 mM Tris-HCl buffer, pH 7.0-8.5, 20 mM MnCl2) for 8-24 hours. The reaction mixture was purified with protein A resin to obtain mAb1-GalNAz.

[0767] Organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and adapter-loador stock solution (10-25 eq., 10 mM in organic solvent) are gradually added to a reaction buffer (PBS buffer, pH 7.0-8.5) containing mAb1-GalNAz (1-20 mg / mL) over 0.5-24 h at 0-25 °C. This solution is then subjected to buffer exchange (centrifugation, desalting column, ultrafiltration, and dialysis) to a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween 20, 60, 80).

[0768] ADC Characterization. The ADCs were characterized using the following analytical methods. All ADCs had a SEC purity >95%.

[0769] Drug-to-antibody ratio (DAR) assay performed using LCMS or HIC methods

[0770] LCMS Method: LC-MS analysis was performed under the following measurement conditions:

[0771] LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer

[0772] Column: MAbPac TM RP, 2.1*50mm, 4μm Thermo Scientific TM

[0773] Column temperature: 80℃

[0774] Mobile phase A: 0.1% formic acid (FA) aqueous solution

[0775] Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA)

[0776] Gradient program: 25%B-25%B (0 min-2 min), 25%B-50%B (2 min-18 min), 50%B-90%B (18 min-18.1 min), 90%B-90%B (18.1 min-20 min), 90%B-25%B (20 min-20.1 min), 25%B-25%B (20.1 min-25 min)

[0777] Injected sample volume: 1 μg

[0778] MS parameters: Full and modified MS data were acquired in HMR mode with R=15k, and ThermoScientific was used. TM BioPharma Finder TM ReSpect in software 4.0 TM The algorithm and sliding window integral are used for deconvolution.

[0779] HIC method: HPLC analysis was performed under the following measurement conditions:

[0780] HPLC System: Waters ACQUITY ARC HPLC System

[0781] Detector: Measurement wavelength: 280nm

[0782] Column: TOsoh Bioscience 4.6μm ID×3.5cm, 2.5μm butyl nonporous resin column; column temperature: 25℃

[0783] Mobile phase A: 1.5M ammonium sulfate, 50mM phosphate buffer, pH 7.0

[0784] Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0

[0785] Gradient program: 0%B-0%B (0 min-2 min), 0%B-100%B (2 min-15 min), 100%B-100%B (15 min-16 min), 100%B-0%B (16 min-17 min), 0%B-0%B (17 min-20 min)

[0786] Injected sample volume: 20 μg

[0787] SEC method for determining ADC purity

[0788] HPLC analysis was performed under the following measurement conditions:

[0789] HPLC system: Waters H-Class UPLC system

[0790] Detector: Measurement wavelength: 280nm

[0791] Column: ACQUITY UPLC BEH200 SEC 1.7um 4.6x150mm, Waters

[0792] Column temperature: room temperature

[0793] Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropanol, pH 7.0

[0794] Gradient program: 10 min isocratic elution, flow rate 0.3 mL / min

[0795] Injected sample volume: 20 μg

[0796] Hydrophobicity of ADCs was assessed using the HIC method: ADCs with higher hydrophobic properties will appear with increasing retention time in HIC (hydrophobic interaction column) chromatography. The DAR2 peak was used as a reference.

[0797] HPLC analysis was performed under the following measurement conditions:

[0798] Method 1

[0799] HPLC System: Waters ACQUITY ARC HPLC System

[0800] Detector: Measurement wavelength: 280nm

[0801] Column: Tosoh Bioscience 4.6μm ID×3.5cm, 2.5μm butyl nonporous resin column; column temperature: 25℃

[0802] Mobile phase A: 1.5M ammonium sulfate, 50mM phosphate buffer, pH 7.0

[0803] Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0

[0804] Gradient program: 0%B-0%B (0 min-2 min), 0%B-100%B (2 min-15 min), 100%B-100%B (15 min-16 min), 100%B-0%B (16 min-17 min), 0%B-0%B (17 min-20 min)

[0805] Injected sample volume: 20 μg

[0806] Method 2

[0807] HPLC System: Waters ACQUITY ARC HPLC System

[0808] Detector: Measurement wavelength: 280nm

[0809] Column: MABPac HIC-10, 5μm, 4.6×10mm (Thermo)

[0810] Column temperature: 25℃

[0811] Mobile phase A: 1.5M ammonium sulfate, 50mM sodium phosphate, pH 7.0

[0812] Mobile phase B: 50 mM sodium phosphate, pH 7.0

[0813] Gradient program: 20%B-20%B (0 min-1 min), 0%B-0%B (1 min-35 min), 20%B-20%B (35 min-40 min)

[0814] Flow rate: 0.5 mL / min

[0815] Sample preparation: Dilute the sample to 0.5 mg / mL with the initial mobile phase.

[0816] Table 3: ADC Structure

[0817]

[0818] Anti-CD74 antibody mAb1

[0819] Light chain sequence (SEQ ID NO: 1)

[0820]

[0821]

[0822] Heavy chain sequence (SEQ ID NO: 2)

[0823]

[0824] cell lines

[0825] A375 (ATCC, CRL-1619). A-375 is a cell line exhibiting epithelial morphology isolated from the skin of a 54-year-old female patient with malignant melanoma, and it was purchased from ATCC. The basal culture medium for A375 was DMEM, high glucose, and GlutaMAX.M Supplement (Gibco, 10566024). To prepare complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 10%. The cell lines were grown at 37°C in a humid 5% CO2 atmosphere and treated with MycoAlertT. M The PLUS Mycoplasma Detection Kit (Lonza, LT07-710) is used to periodically test for the presence of mycoplasma.

[0826] Calu-6 (ATCC, HTB-56). Calu-6 is a cell line exhibiting epithelial morphology isolated from Caucasian female patients with anaplastic carcinoma, and Calu-6 was purchased from ATCC. The basal medium for Calu-6 was Eagle's minimum essential medium (ATCC, 30-2003). To prepare complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 10%. The cell line was grown at 37°C in a humid 5% CO2 atmosphere and monitored with MycoAlert. TM The PLUS Mycoplasma Detection Kit (Lonza, LT07-710) is used to periodically test for the presence of mycoplasma.

[0827] Cell-killing effects of the compound in A375 and Calu-6 cancer cell lines

[0828] The direct cytotoxic effects of compounds 1-1 to 1-22 were evaluated in A375 and Calu-6 cancer cell lines. Cells were seeded at 1E3 / well (A375) and 2E3 / well (Calu-6) in 96-well plates (Greiner: 655090), 100 μL / well, and incubated overnight at 37°C and 5% CO2. Fresh growth medium containing different concentrations of the compounds was added, 50 μL / well, and incubated for 6 days at 37°C and 5% CO2. Cell viability was assessed by Cell Titer-Glo (Promega, G7573), 70 μL / well. The plates were incubated at room temperature for 10 minutes to stabilize the luminescence signal. The plates were analyzed using a microplate reader.

[0829] Cell-killing data for compounds 1-1 to 1-5 are as follows Figure 1 and Figure 2 And as shown in the table below:

[0830]

[0831] Cell-killing data for compounds 1-6 to 1-10 are as follows Figure 3 and Figure 4 And as shown in the table below:

[0832]

[0833] Cell-killing data for compounds 1-11 to 1-13 are as follows: Figure 5 and Figure 6 And as shown in the table below:

[0834]

[0835] Cell-killing data for compounds 1-14, 1-15, 1-17, and 1-20 are as follows: Figure 7 and Figure 8 And as shown in the table below:

[0836]

[0837] Cell-killing data for compounds 1-16 and 1-19 are as follows: Figure 9 and Figure 10 And as shown in the table below:

[0838]

[0839] Cell-killing data of compounds 1-18 are as follows Figure 11 and Figure 12 And as shown in the table below:

[0840]

[0841] Cell-killing data for compounds 1-21 and 1-22 are as follows: Figure 13 and Figure 14 And as shown in the table below:

[0842] #imgpt252#

[0843] Although the foregoing disclosure has been described in considerable detail by way of illustration and example for purposes of clarity, it will be apparent to those skilled in the art that minor changes and modifications can be made. Therefore, the descriptions and embodiments described should not be considered limiting.

[0844] It should be understood that if any prior art publications are mentioned herein, such mentions do not constitute an admission that such publications constitute part of general knowledge in the art in any country.

[0845] All non-patent publications, patents, patent applications, and published patent applications mentioned in this document by way of identification are hereby incorporated herein in their entirety by way of reference.

Claims

1. A compound of formula (I): Or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein each of ring A and ring B independently has one of the following formula: Indicates the connection point with the connector; The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -; X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings; Ring C is a cyclopropyl ring or a cyclobutyl ring; -C(R 1 )- and -N(R 2 The dashed key between )- Each of them is either a single bond or a double bond; When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 For H; When the dashed key is a double key, each R 1 Let H be the number of R, and each R be the number of R. 2 It does not exist; R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R a and R b Each independently is H or C 1-4 alkyl; R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R 6 For H or C 1-4 alkyl; Each of m, n, and o is independently 1 or 2; Each of r, p, and q is an independent integer from 1 to 8; and The sum of p and q is an integer from 1 to 8.

2. The compound of claim 1, wherein the linker is -(CH2). r When -, ring A has equation (IIa) and ring B has equation (IIb).

3. The compound of claim 1, wherein the linker is -(CH2). p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q When -, ring A has equation (IIa), m in ring A is 2, and ring B has one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf) and (IIg).

4. The compound of claim 1, wherein the linker is -(CH2). p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q When - ring A has equation (IIa), m in ring A is 1, and ring B has one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), and (IIg), and equation (IIc) is either equation (IIc3) or equation (IIc4): or 5. The compound according to any one of claims 1, 3 and 4, wherein X is O or NR. 6 ,NHC(=O), -(m-C6H4)-, 6. The compound of claim 1, wherein the linker is -(CH2). r - 7. The compound of claim 1, 2 or 6, wherein r is 3 or 5.

8. The compound of claim 1 or 5, wherein the linker is -(CH2). p -O-(CH2) q -or-(CH2) p -NH-(CH2) q - 9. The compound according to any one of claims 1, 3-5 and 8, wherein the sum of p and q is 4.

10. The compound according to any one of claims 1, 3, and 4, wherein the connector is 11. The compound of claim 10, wherein the sum of p and q is 2.

12. The compound of claim 5, wherein the connector is 13. The compound of claim 12, wherein the sum of p and q is 2.

14. The compound of claim 1, 3 or 4, wherein the linker is -(CH2). p -CH = CH-(CH2) q - 15. The compound of claim 14, wherein the sum of p and q is 3.

16. The compound according to any one of claims 1 and 3-15, wherein ring B has formula (IIa).

17. The compound of claim 16, wherein m in ring B is 1.

18. The compound of claim 16 or 17, wherein ring C in ring B is a cyclopropyl ring.

19. The compound according to any one of claims 16-18, wherein the dashed bond in ring B is a single bond, and R 1 It is H or OH, and R 2 For H.

20. The compound according to any one of claims 16-18, wherein the dashed bond in ring B is a double bond, and R 1 For H, and R 2 It does not exist.

21. The compound of claim 1, wherein ring A has the formula (IIb).

22. The compound of claim 21, wherein ring A has the formula (IIb2):

23. The compound of claim 21 or 22, wherein R 3 For H.

24. The compound according to any one of claims 21-23, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

25. The compound according to any one of claims 21-23, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

26. The compound of claim 23, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

27. The compound of claim 1, wherein ring A has the formula (IIg).

28. The compound of claim 27, wherein the o in ring A is 2.

29. The compound of claim 27 or 28, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

30. The compound of claim 27 or 28, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

31. The compound of claim 28, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

32. The compound of claim 1, wherein ring A has the formula (IId).

33. The compound of claim 32, wherein n in ring A is 1.

34. The compound of claim 32 or 33, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

35. The compound of claim 32 or 33, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

36. The compound of claim 33, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

37. The compound of claim 32, wherein n in ring A is 2.

38. The compound of claim 32 or 37, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

39. The compound of claim 32 or 37, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

40. The compound of claim 39, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

41. The compound of claim 1, wherein ring A has the formula (IIc).

42. The compound of claim 41, wherein ring A has the formula (IIc2):

43. The compound of claim 41 or 42, wherein R 4 It is CH3O-.

44. The compound according to any one of claims 41-43, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

45. The compound according to any one of claims 41-43, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

46. ​​The compound of claim 42, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

47. The compound of claim 1, wherein ring A has the formula (IIe).

48. The compound of claim 47, wherein R 5 It is a methyl group.

49. The compound of claim 47 or 48, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

50. The compound of claim 47 or 48, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

51. The compound of claim 48, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

52. The compound of claim 1, wherein each of ring A and ring B independently has formula (IIa).

53. The compound of claim 52, wherein m is 1.

54. The compound of claim 52 or 53, wherein ring C is a cyclopropyl ring.

55. The compound according to any one of claims 52-54, wherein the dashed bond in ring A is a single bond, and R 1 It is H or OH, and R 2 For H.

56. The compound according to any one of claims 52-54, wherein the dashed bond in ring A is a double bond, and R 1 For H, and R 2 It does not exist.

57. The compound according to any one of claims 52-56, wherein the dashed bond in ring B is a single bond, and R 1 It is H or OH, and R 2 For H.

58. The compound according to any one of claims 52-56, wherein the dashed bond in ring B is a double bond, and R 1 For H, and R 2 It does not exist.

59. The compound of claim 54, wherein the compound is Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

60. The compound of claim 1, wherein ring A and ring B are different.

61. A compound of formula B(i) or B(ii): Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: Each of rings A and B independently has one of the following equations: Indicates the connection point with the connector or Ab connector; The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -; X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings; Ring C is a cyclopropyl ring or a cyclobutyl ring; -C(R 1 )- and -N(R 2 The dashed key between )- Each of them is either a single bond or a double bond; When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 For H; When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It does not exist; R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R a and R b Each independently is H or C 1-4 alkyl; R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R 6 For H or C 1-4 alkyl; Each of m, n, and o is independently 1 or 2; Each of r, p, and q is an independent integer from 1 to 8; The sum of p and q is an integer from 1 to 8; and Ab connectors are compounds that can bond ring A or ring B to a binder.

62. The compound of claim 61, wherein the Ab connector has the following formula: in #Indicates the connection point with ring A or ring B.

63. The compound of claim 61 or 62, wherein the compound has the following formula: Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

64. A conjugate of formula A(i) or A(ii): Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: Each of rings A and B independently has one of the following equations: Indicates the connection point with the connector or Ab connector; The connector is -(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH = CH-(CH2) q -; X is NR 6 , NHC (=O), C (=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocyclic rings or substituted or unsubstituted cyclic rings; Ring C is a cyclopropyl ring or a cyclobutyl ring; -C(R 1 )- and -N(R 2 The dashed key between )- Each of them is either a single bond or a double bond; When the dashed key is a single key, each R 1 Independently H or OH, and each R 2 For H; When the dashed key is a double key, each R 1 Independently defined as H, and each of R 2 It does not exist; R 3 and R 4 Each of them is independently H, NH2, NR a R b OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R a and R b Each independently is H or C 1-4 alkyl; R 5 For H, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R 6 For H or C 1-4 alkyl; Each of m, n, and o is independently 1 or 2; Each of r, p, and q is an independent integer from 1 to 8; The sum of p and q is an integer from 1 to 8; An Ab connector is a compound that attaches an Ab to ring A or ring B; Ab is a conjugate selected from humanized, chimeric, or human antibodies or their antigen-binding fragments; and The subscript x ranges from 1 to 15.

65. The conjugate of claim 64, wherein the Ab connector has the following formula: in Indicates the connection point with Ab, and Indicates the connection point with ring A or ring B.

66. The conjugate as claimed in claim 64 or 65, wherein the conjugate has the following formula: Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

67. The conjugate of claim 66, wherein the subscript x is about 2.

68. The conjugate according to any one of claims 64 to 67, wherein the conjugate has the following formula: Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

69. A pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt, tautomer, solvate or stereoisomer of any one of claims 64 to 68 and a pharmaceutically acceptable excipient.

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