Modified thiostrepton-exciting compounds for treatment of cancer and their preparation

Abstract

Disclosed herein are structures, compositions, and synthesis of thiostrepton-excited pharmaceutically related compounds, as well as methods of treating cancer with such compounds.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 662,826, filed June 21, 2024, U.S. Provisional Patent Application No. 63 / 557,880, filed February 26, 2024, and U.S. Provisional Patent Application No. 63 / 532,587, filed August 14, 2023, each of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Thiostrepton is a cyclic oligopeptide antibiotic, which also has other names such as Bryamycin, Thiactin, propylamide, HR4S203Y18, etc. Recent studies have shown that thiostrepton also has good anti-cancer activity. There is still a need for thiostrepton derivatives with beneficial pharmacological properties. SUMMARY

[0003] In certain aspects, the present application provides a series of compounds having a structure according to Formula I: (I), or a pharmaceutically acceptable salt thereof, wherein: Ring M is aryl or heteroaryl; Ring A comprises at least 5 atoms; X and Y are each independently selected from the group consisting of a bond, , , , , , , , , , , O, and NR x2 wherein * indicates a bond to N, and indicates a bond to Ring M; each R a is independently selected from the group consisting of hydrogen, and optionally substituted C1-C3 alkyl; R x1 is selected from the group consisting of optionally substituted C1-C4 alkyl and optionally substituted 3-6 membered cycloalkyl; R x2 is selected from the group consisting of hydrogen, optionally substituted C1-C4 alkyl, and optionally substituted 3-6 membered cycloalkyl; R 1 is selected from the group consisting of 3-6 membered cycloalkyl, aryl, 4-7 membered heterocyclyl, and heteroaryl, each optionally substituted; L is selected from the following: -CH2-, -O-, -NH-, -N(Me)-, -C(=O)NH-, and -NHC(=O)-; When it exists, R 2 Each time it appears, it is independently selected from halogen, C1-C4 alkyl, O-C1-C4 alkyl, -CN, aryl, 4-7 membered heterocyclic and 5-6 membered heteroaryl, wherein R 2 Any alkyl, aryl, heterocyclic, or heteroaryl moiety may optionally be substituted; R 3 Selected from aryl groups that are arbitrarily substituted, -C(=O)-R 4 and -CN; R 4 Selected from NH2, optionally substituted aminoalkyl, optionally substituted alkylamino, optionally substituted C1-C6 alkyl, optionally substituted 3-6 membered cycloalkyl, optionally substituted aryl, and optionally substituted O-C1-C4 alkyl; and a is selected from 0, 1, and 2.

[0004] This article also provides methods for treating cancer, which include administering any of the pharmaceutical compositions described herein to subjects in need. Attached Figure Description

[0005] Figure 1 and Figure 2 This is a table summarizing the results for the various exemplary compounds described in this article. For the IC values ​​in both tables... 50 Value, A corresponds to IC 50 <1.39 µM; B = 1.39-2.39 µM, C = 2.39-3.39 µM, D = 3.39-4.39 µM; and E>4.39 µM. Figure 3 The activity of thiotetracycline (TS) and 1-305 in the MM xenograft model was demonstrated. Detailed Implementation

[0006] Pharmaceutical compositions The compositions and methods described herein can be used to treat individuals in need. In some embodiments, the individuals are mammals, such as humans or non-human mammals. When administered to animals such as humans, the compositions or compounds are preferably administered in the form of pharmaceutical compositions comprising, for example, the compounds described herein and pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are well known in the art and, as non-limiting examples, include aqueous solutions, such as water or physiologically buffered saline, or other solvents or mediators, such as glycols, glycerols, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are administered to humans, particularly via invasive routes of administration (i.e., injection or implantation routes that circumvent transport or diffusion across the epithelial barrier), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients may be selected, for example, to achieve delayed release of the agent or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions may be in the form of dosage units, such as tablets, capsules (including dispersible capsules and gelatin capsules), granules, lyophilized formulations for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in transdermal delivery systems, such as skin patches. The composition may also be present in solutions suitable for topical application, such as lotions, creams, or ointments.

[0007] Pharmaceutically acceptable carriers may contain physiologically acceptable agents that, for example, stabilize compounds (such as those described herein), increase their solubility, or enhance their absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; or other stabilizers or excipients. The choice of pharmaceutically acceptable carriers (including physiologically acceptable agents) depends, for example, on the route of administration of the composition. Formulations or pharmaceutical compositions may be self-emulsifying drug delivery systems or self-microemulsifying drug delivery systems. Pharmaceutical compositions (formulations) may also be liposomes or other polymer matrices in which compounds, such as those described herein, may be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers, and their preparation and administration are relatively simple.

[0008] The term “pharmaceutically acceptable” as used in this article refers to compounds, materials, compositions, and / or dosage forms that, within a reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0009] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and does not harm the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) astragalus gum powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and so on. (10) Soybean oil; (11) Diols, such as propylene glycol; (12) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Pyrogenic water; (18) Isotonic saline; (19) Ringer's solution; (20) Ethanol; (21) Phosphate buffer solution; and (22) Other non-toxic and compatible substances used in pharmaceutical formulations.

[0010] The pharmaceutical composition (formulation) can be administered to a subject via any of a variety of routes of administration, including, for example, oral administration (e.g., an infusion in water or a non-aqueous solution or suspension, tablets, capsules (including dispersible capsules and gelatin capsules), granules, powders, pellets, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical application (e.g., as a cream, ointment, or spray applied to the skin). The compound can also be formulated for inhalation. In some embodiments, the compound may simply be dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions thereof can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0011] The formulation can be readily available in unit dosage forms and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Generally, in one hundred parts, this amount will be in the range of about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0012] Methods for preparing these formulations or compositions include the step of associating an active compound (such as the compound described herein) with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by homogeneously and closely associating the compound described herein with a liquid carrier, or a finely separated solid carrier, or both, and then shaping the product if necessary.

[0013] The formulations described herein suitable for oral administration may be in the following forms: capsules (including dispersible capsules and gelatin capsules), sachets, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or tragacanth gum), lyophilized forms, powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions, or as elixirs or syrups, or as pasteurees (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, each containing a predetermined amount of the compound described herein as an active ingredient. The compositions or compounds may also be administered as pellets, electuals, or pastes.

[0014] To prepare solid dosage forms (capsules (including dispersible capsules and gelatin capsules), tablets, pills, sugar-coated pills, powders, granules, etc.) for oral administration, the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silica; (2) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerin. (4) Disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain silicates and sodium carbonate; (5) Solution blockers, such as paraffin; (6) Absorption accelerators, such as quaternary ammonium compositions; (7) Wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) Absorbents, such as kaolin and bentonite clay; (9) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; (10) Complexing agents, such as modified and unmodified cyclodextrins; and (11) Colorants. In the case of capsules (including dispersible capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain buffers. Similar types of solid compositions may also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycol.

[0015] Tablets can be prepared by compression or molding, optionally containing one or more excipients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants, or dispersants. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0016] Solid dosage forms of tablets and other pharmaceutical compositions, such as sugar-coated pills, capsules (including dispersible capsules and gelatin capsules), pellets, and granules, may optionally be scored or coated and shelled, such as enteric coating or other coatings well known in the field of pharmaceutical formulation. They may also be formulated to provide slow or controlled release of the active ingredient contained therein using, for example, different proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release characteristics. They may be sterilized, for example, by filtration through a bacterial trap or by incorporating a sterilizing agent in the form of a sterile solid composition that is immediately soluble in sterile water or some other sterile injectable medium before use. These compositions may also optionally contain a light-blocking agent and may have a composition that optionally releases the active ingredient in a delayed manner only or preferably in a portion of the gastrointestinal tract. Examples of encapsulation compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, and where appropriate, have one or more of the excipients described above.

[0017] Liquid dosage forms suitable for oral administration include pharmaceutically acceptable emulsions, lyophilized formulations for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solvents and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters and mixtures thereof of sorbitan.

[0018] In addition to inert diluents, the oral composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, flavoring agents and preservatives.

[0019] In addition to the active compound, the suspension may also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth gum and mixtures thereof.

[0020] Dosage forms for topical or transdermal application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. Active compounds may be mixed under aseptic conditions with pharmaceutically acceptable carriers and any necessary preservatives, buffers, or propellants.

[0021] In addition to active compounds, ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth gum, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0022] In addition to the active compound, powders and aerosols may also contain excipients such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powders or mixtures thereof. Aerosols may also contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane and propane).

[0023] Transdermal patches offer the added advantage of providing controlled delivery of the compounds described herein into the body. Such dosage forms can also be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of this flux can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0024] As used herein, the phrases “parenteral administration” and “extraterrestrial administration” refer to administration methods that are typically administered by injection, other than enteral and local administration, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-sacral, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds and one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use. These compositions may contain antioxidants, buffers, antibacterial agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners.

[0025] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate flowability can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0026] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Antimicrobial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0027] In some cases, to prolong the effect of a drug, it is desirable to slow down its absorption after subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The absorption rate of the drug then depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of parenteral drug forms can be achieved by dissolving or suspending the drug in an oil-based medium.

[0028] Injectable reservoir formulations are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer such as poly(lactide-polyglycolic acid). The drug release rate can be controlled based on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir injection formulations have also been prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions.

[0029] For use in the methods described herein, the active compound may be provided either alone or as a pharmaceutical composition comprising, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0030] The introduced methods can also be provided by refillable or biodegradable devices. Regarding the controlled delivery of drugs (including protein biopharmaceuticals), various sustained-release polymer devices have been developed and tested in vivo in recent years. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants to sustainably release compounds at specific target sites.

[0031] The actual dose level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and administration mode, without being toxic to the patient.

[0032] The chosen dose level depends on a variety of factors, including the activity of the specific compound or combination of compounds used or its esters, salts or amides, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific compound used, the age, sex, weight, condition, overall health status and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0033] A physician or veterinarian with ordinary skill in the art can readily determine and prescribe the required therapeutically effective amount of a pharmaceutical composition. For example, a physician or veterinarian may start with a dose of a pharmaceutical composition or compound below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. “Therapeutically effective amount” means the concentration of a compound sufficient to cause the desired therapeutic effect. It should generally be understood that the effective amount of a compound will vary depending on the subject’s weight, sex, age, and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient’s illness, the condition being treated, the stability of the compound, and another type of therapeutic agent, if desired, to be administered in conjunction with the compound described herein. A greater total dose can be delivered by administering the agent multiple times. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine, 13th edition, 1814–1882, which are incorporated herein by reference).

[0034] Generally, the appropriate daily dose of the active compound used in the compositions and methods described herein will be the amount of the compound at which the minimum effective therapeutic effect is achieved. This effective dose will generally depend on the factors described above.

[0035] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-dose, administered at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, the active compound may be administered two or three times daily. In a preferred embodiment, the active compound will be administered once daily.

[0036] Patients receiving this treatment are any animals in need, including primates, especially humans; and other mammals such as horses, cattle, pigs, sheep, cats and dogs; poultry; and pets in general.

[0037] In some embodiments, the compounds described herein may be used alone or in combination with another type of therapeutic agent.

[0038] This disclosure includes the use of pharmaceutically acceptable salts of the compounds described herein in the compositions and methods described herein. In some embodiments, the salts considered include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In some embodiments, the salts considered include, but are not limited to, L-arginine, benzylamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazolium, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In some embodiments, the salts considered include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In some embodiments, the salts considered include, but are not limited to, 1-hydroxy-2-naphthylcarboxylic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, L-ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid, caproic acid, caproic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, and cyclamic acid. (acid), dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactosidic acid, gentian acid, d-glucoheponic acid, d-glucuronic acid, glutamic acid, glutamate, glutaric acid, glycerophosphate, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, L-malic acid, malonic acid, phenylglycolic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecanoic acid salts.

[0039] Pharmaceutically acceptable acid addition salts can also exist in various solvate forms, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. These solvates can originate from the solvent used for crystallization, be inherent in the solvent used for preparation or crystallization, or be insoluble in such solvents.

[0040] Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and aroma agents, preservatives and antioxidants may also be present in the composition.

[0041] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0042] Definitions Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the names and techniques used herein in conjunction with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry are those well-known and commonly used in the art.

[0043] Unless otherwise indicated, the methods and techniques disclosed herein are generally performed in accordance with conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification.

[0044] Unless otherwise defined herein, chemical terms used herein are used in accordance with the usual usage in the art, as illustrated in "The McGraw-Hill Dictionary of Chemical Terms", ed. Parker S., McGraw-Hill, San Francisco, CA (1985).

[0045] All references to the foregoing and any other publications, patents, and published patent applications mentioned in this patent application are expressly incorporated herein by reference. In the event of any conflict, this specification (including its specific definitions) shall prevail.

[0046] This document uses the term "agent" to refer to compounds (such as organic or inorganic compounds, mixtures of compounds), biological macromolecules (such as nucleic acids, antibodies, including their portions and humanized, chimeric and human antibodies and monoclonal antibodies, proteins or portions thereof, such as peptides, lipids, carbohydrates), or extracts made from biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Agents include, for example, agents with known structures and agents with unknown structures.

[0047] The terms “patient,” “subject,” or “individual” are used interchangeably and refer to humans or non-human animals. These terms include mammals such as humans, primates, livestock (including cattle, pigs, etc.), companion animals (e.g., dogs, felines, etc.), and rodents (e.g., mice and rats).

[0048] “Treatment” of a disease or patient refers to taking measures to obtain a beneficial or desired outcome, including clinical outcomes. As used herein and well understood in the art, “treatment” is a means of obtaining a beneficial or desired outcome (including clinical outcomes). Beneficial or desired clinical outcomes may include, but are not limited to, the reduction or improvement of one or more symptoms or conditions, a decrease in the severity of the disease, stabilization of the disease state (i.e., no worsening), prevention of disease spread, 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 prolonged survival compared to the expected survival without treatment.

[0049] The term "prevention" is recognized in the art and is well known in the art when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, symptoms such as heart failure, or any other medical condition, and includes administering a composition that, relative to a subject not receiving the composition, reduces the frequency of symptoms of the subject's medical condition or delays its onset. Thus, cancer prevention includes, for example, reducing, in statistically and / or clinically significant amounts, the number of detectable cancerous growths in a patient population receiving prophylactic treatment relative to an untreated control population, and / or delaying the occurrence of detectable cancerous growths in the treated population relative to the untreated control population.

[0050] The "application" of a substance, compound, or agent to a subject, or the "administration" of a substance, compound, or agent, may be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or agent may be administered intravenously, intra-arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, via the eyes, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and percutaneously (by absorption, e.g., via a skin tube). The compound or agent may also be suitably introduced via a rechargeable or biodegradable polymer device or other means, such as patches and pumps, or formulations that provide a prolonged, slow, or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0051] The appropriate method of administering a substance, compound, or agent to a subject will also depend on factors such as the subject's age and / or physical condition, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is administered orally to the subject, for example, by ingestion. In some embodiments, the orally administered compound or agent is a prolonged-release or slow-release formulation, or is administered using a device for such slow or prolonged release.

[0052] As used herein, the phrase "combined administration" refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., when at least 5% of the pharmaceutical product is systematically detected using industry-acceptable methods, or when both agents are simultaneously effective in a patient, which may include the synergistic effect of the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. In some embodiments, different therapeutic compounds may be administered at intervals of one hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or one week. Thus, an individual receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0053] The “therapeutic effective amount” or “therapeutic dose” of a compound or other agent described herein is the amount of drug or agent that will have the expected therapeutic effect when administered to a subject. A complete therapeutic effect may not necessarily occur with the administration of a single dose of such drug or agent, but may only occur after the administration of a series of doses (multiple consecutive doses). Therefore, a therapeutic effective amount can be administered in one or multiple doses. The precise effective amount required by the subject will depend, for example, on the subject’s physical condition, health, and age, as well as the nature and extent of the condition being treated (such as cancer).

[0054] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both cases in which the event or situation occurs and cases in which the event or situation does not occur. For example, “optionally substituted alkyl” means both cases in which the alkyl group may be substituted and cases in which the alkyl group is not substituted.

[0055] As used herein, the term “regulation” includes both inhibiting or suppressing functions or activities (such as cell proliferation) and enhancing functions or activities.

[0056] The phrase "pharmaceutically acceptable" is recognized in the art. In some embodiments, the term includes, within the bounds of reasonable medical judgment, compositions, excipients, auxiliaries, polymers, and other materials and / or dosage forms that come into contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0057] "Pharmaceutically acceptable salt" or "salt" as used in this article refers to an acid addition salt or base addition salt that is suitable for treating the patient or is compatible with the treatment of the patient.

[0058] As used herein, the term "pharmaceutically acceptable acid addition salt" means any non-toxic organic or inorganic salt of any basic compound. Illustrative inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. Monoacid salts or diacid salts can be formed, and such salts can exist in hydrated, solvated, or substantially anhydrous forms. Generally, acid addition salts of compounds are more soluble in water and a variety of hydrophilic organic solvents and typically exhibit a higher melting point compared to their free basic form. The selection of suitable salts is known to those skilled in the art. Other non-pharmaceutical acceptable salts, such as oxalates, may be used, for example, to isolate compounds for laboratory use or for subsequent conversion into pharmaceutically acceptable acid addition salts.

[0059] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic or inorganic base addition salt of any acid compound or any intermediate thereof. Exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and methylpyridine or ammonia. The selection of suitable salts will be known to those skilled in the art.

[0060] Many compounds that can be used in the methods and compositions of this disclosure have at least one stereocenter in their structure. This stereocenter may be present in an R or S configuration, the R and S symbols being used according to the rules described in Pure Appl. Chem. (1976), 45, 11-30. This disclosure considers all stereoisomers, such as enantiomers and diastereomers (including all possible mixtures of stereoisomers) of compounds, salts, prodrugs, or mixtures thereof. See, for example, WO 01 / 062726.

[0061] Some compounds may also exist as tautomers. Although not explicitly indicated in the formulas described herein, such forms are intended to be included within the scope of this disclosure.

[0062] A “prodrug” or “pharmaceutically acceptable prodrug” is a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host after administration to form the compounds of this disclosure. Typical examples of prodrugs include compounds having a biologically unstable or cleavable (protective) group on the functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, amination, deamination, hydroxylation, dehydroxylation, hydrolysis, dehydrolysis, alkylation, dealkylation, acylation, deacylation, phosphorylation, or dephosphorylation to produce the active compound. Examples of prodrugs using esters or aminophosphates as biologically unstable or cleavable (protective) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce an IPA or a salt thereof. This disclosure includes, within its scope, prodrugs of the compounds described herein. The routine procedures for selecting and preparing suitable prodrugs are described, for example, in “Design of Prodrugs”, edited by H. Bundgaard, Elsevier, 1985.

[0063] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium suitable for the formulation of a medicament for medical or therapeutic use, such as a liquid or solid filter aid, diluent, excipient, solvent, or encapsulating material.

[0064] As used herein, the term "connector" means any chemical functional group that is "bonded" or connected by chemical bonds to any two or more other chemical functional groups in a pharmaceutically relevant molecule. As a non-limiting example of the use of connectors in a pharmaceutical context, antibody-drug conjugates (ADCs) include pharmaceutically active small molecules, drugs, or toxins that are linked to macromolecular antibodies via connectors.

[0065] Examples of common linker types include cleavable and non-cleavable linkers. Cleavable linkers include chemical functional groups that can cleave in response to physiological stimuli such as chemical gradients, pH changes, or enzyme activity. Non-limiting examples include acid- or base-labile functional groups, pyrophosphate diesters, disulfide bonds, peptides, β-glucuronides, etc. Non-cleavable linkers contain chemical functional groups that are generally not very stable to the aforementioned physiological stimuli, and non-limiting examples include certain alkyl and organic polymer functional groups.

[0066] As used herein, the term "reactive linker moiety" refers to a chemical structure having a terminal portion that can react with another portion (such as the mitochondrial targeting moiety) to form a covalent bond.

[0067] As used herein, the terms “mitochondrial-targeting peptide,” “mitochondrial-targeting sequence,” and “mitochondrial-targeting moiety” are recognized terms referring to a chemical functional group (peptide, sequence, or moiety) that “targets” the mitochondrial membrane, meaning it is readily transported to and absorbed by the mitochondrial membrane (J. Zielonka, B. Kalyanaraman et al., 2017). As used herein, the mitochondrial-targeting moiety may include, but is not limited to, the following substances: berberine cation, rhodamine cation, indolonium cation, pyridinium cation, tetraguanonium cation, cyanine derivatives, guanidineonium cation, biguanonium cation, triphenylphosphonium cation, triethylammonium cation, triphenylamine, tetraphenylethylene moiety, arylphosphonium cation, SS peptide, mitochondrial-penetrating peptide (MPP), mitochondrial-targeting sequence (MTS) peptide, hemibacitracin S-linked nitrooxy, dequinoline chloride (DQA) cation, delocalized lipophilic cation, F… 16((E)-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide), (L-cyclohexylalanine-D-arginine)3, mitochondrial-targeting nanocarriers, DDDK peptide, glycyrrhetinic acid, α-tocopherol succinate (α-TOS), graphene oxide nanocarriers, PEG-pro-apoptotic peptide (KLAKLAK)2, Dmt-D-Arg-Phe-Lys-NH2 peptide, acetone aldehyde, N-nonylacridine orange, quinoline, styrene-nitrogen heterocyclic fluorophore, or 15d-PGJ2. Exemplary mitochondrial-targeting components are listed in the reference. J Zielonka et al., ChemRev 2017, 117, pp. 10043-10120; KL Horton et al., Chemistry & Biology 2008, 15, pp. 375-382; G Battogtokh et al., Front Pharmacol 2018, 9:922; U.S. Patents 9,173,952 and 9,132,198, the contents of which are incorporated herein by reference.

[0068] It should be understood that the substituents and substitution patterns on the compounds described herein can be selected by those skilled in the art to obtain chemically stable compounds that can be readily synthesized from readily available raw materials using techniques known in the art and the methods described below. If the substituent itself is substituted by more than one group, it should be understood that these multiple groups can be located on the same carbon or on different carbons, as long as a stable structure is produced.

[0069] As used herein, the term "optionally substituted" means replacing one to six hydrogen groups in a given structure with a specified substituent, including but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclic, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or –CH2-OP(O)(O-alkyl)2. Preferably, "optionally substituted" means replacing one to four hydrogen groups in a given structure with the substituents mentioned above. More preferably, one to three hydrogen groups are replaced by the substituents mentioned above. It should be understood that the substituents may be further substituted.

[0070] The term "acyl" is recognized in the art and refers to a group represented by the general formula hydrocarbon C(O)-, preferably alkyl C(O)-.

[0071] The term "acylamino" is recognized in the art and refers to an amino group substituted with an acyl group, and can be represented, for example, by the formula alkyl group C(O)NH-.

[0072] The term "acyloxy group" is recognized in the art and refers to a group represented by the general formula hydrocarbon C(O)O-, preferably alkyl C(O)O-.

[0073] The term "alkoxy group" refers to an alkyl group that is attached to an oxygen-containing alkyl group. Representative alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy groups.

[0074] The term "alkoxyalkyl" refers to an alkyl group that has been substituted with an alkoxy group, and can be represented by the general formula alkyl-O-alkyl.

[0075] As used herein, the term "alkyl" refers to a saturated aliphatic group, including but not limited to C1-C1 groups. 10 Straight-chain alkyl groups or C1-C 10 Branched alkyl groups. Preferably, an "alkyl" group refers to a C1-C6 straight-chain alkyl group or a C1-C6 branched alkyl group. Most preferably, an "alkyl" group refers to a C1-C4 straight-chain alkyl group or a C1-C4 branched alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl, etc.

[0076] Furthermore, the term "alkyl" as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter referring to an alkyl moiety having a hydrogen-substituted substituent on one or more carbon atoms of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0077] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C" is used. x-y "or "C x -C y This refers to groups containing x to y carbons in the chain. C0 alkyl indicates hydrogen, where the group is at the terminal position; if internal, it is a bond. For example, C 1-6 Alkyl groups contain 1 to 6 carbon atoms in the chain.

[0078] As used herein, the term "alkylamino" refers to an amino group that is substituted with at least one alkyl group.

[0079] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0080] As used herein, the term "amide" refers to a group. , Where R 9 and R 10 Each can independently represent a hydrogen or hydrocarbon group, or R 9 and R 10 Together with the N atom it is attached to, it forms a heterocycle with 4 to 8 atoms in the ring structure.

[0081] The terms "amine" and "amino" are recognized in the art and refer to unsubstituted and substituted amines and their salts, for example, portions that can be represented by the following formula. , Where R 9 R 10 and R 10 Each can independently represent a hydrogen or hydrocarbon group, or R 9 and R 10 Together with the N atom it is attached to, it forms a heterocycle with 4 to 8 atoms in the ring structure.

[0082] As used herein, the term "aminoalkyl" refers to an alkyl group that has been substituted with an amino group.

[0083] As used herein, the term "aralkyl" refers to an alkyl group that has been substituted with an aryl group.

[0084] The term "aryl," used alone or as part of a larger portion of "aralkyl," "ararylalkoxy," or "aryloxyalkyl," refers to a monocyclic or bicyclic cyclic system having a total of 5 to 40 ring members, wherein each ring atom is carbon, at least one ring in the system is an aromatic ring, and each ring in the system contains 3 to 7 ring members. The term "aryl" is used interchangeably with the term "aromatic ring." In some embodiments of this disclosure, "aryl" refers to an aromatic ring system that may have one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, anthracene, etc. The scope of the term "aryl" as used herein also includes groups fused with an aromatic ring to one or more non-aromatic carbocyclic rings.

[0085] The term "carbamate" is recognized in the art and refers to a group... , Where R 9 and R 10 Independently represents a hydrogen or hydrocarbon group.

[0086] As used herein, the term "carbocyclic alkyl" refers to an alkyl group that has been substituted with a carbocyclic group.

[0087] The term "carbocyclic ring" includes 5-7 membered monocyclic rings and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocyclic ring may be selected from saturated rings, unsaturated rings, and aromatic rings. A carbocyclic ring includes a bicyclic molecule in which one, two, three, or more atoms are shared between the two rings. The term "fused carbocyclic ring" refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocyclic ring may be selected from saturated rings, unsaturated rings, and aromatic rings. In an exemplary embodiment, an aromatic ring (e.g., phenyl) may be fused with a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane, or cyclohexene). Any combination of saturated bicyclic, unsaturated bicyclic, and aromatic bicyclic rings is included in the definition of a carbocyclic ring, where valence allows. Exemplary "carbocyclic rings" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocyclic rings include decahydronaphthalene, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. The “carbocyclic ring” can be substituted at any one or more positions capable of carrying hydrogen atoms.

[0088] As used herein, the term "carbocyclic alkyl" refers to an alkyl group that has been substituted with a carbocyclic group.

[0089] The term "carbonate" is recognized in the art and refers to the group -OCO2-.

[0090] As used herein, the term "carboxyl" refers to a group represented by the formula -CO2H.

[0091] As used herein, the term "ester" refers to the group -C(O)OR 9 , where R 9 It represents a hydrocarbon group.

[0092] As used herein, the term "ether" refers to a hydrocarbon group that is attached to another hydrocarbon group via oxygen. Therefore, the ether substituent of a hydrocarbon group can be hydrocarbon-O-. Ethers can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocyclic-O-heterocycles and aryl-O-heterocycles. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0093] As used in this article, the terms “halogenated” and “halogen” refer to halogens, including chlorine, fluorine, bromine, and iodine.

[0094] As used herein, the terms “hetaralkyl” and “heteroaralkyl” refer to alkyl groups that have been substituted with heteroaryl groups.

[0095] The terms “heteroaryl” and “heteroaryl-”, such as “heteroarylalkyl” or “heteroarylalkoxy”, used alone or as part of a larger part, refer to a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in the ring arrangement; and having one to five heteroatoms (other than carbon atoms). The term “heteroatom” refers to nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrroleyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiazolyl, pyridyl, pyridinyl, pyridazinyl, indoleazinyl, purine, naphthidyl, and pteridinyl. As used herein, the terms “heteroaryl” and “heteroary-” also include groups fused with one or more aromatic or heteroaryl rings, such that the resulting bicyclic or polycyclic system as a whole is fully aromatic. Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, terpineyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinazinyl, carbazoyl, acridineyl, phenazinyl, phenothiazinyl, and phenotoxazinyl. Heteroaryl groups can be monocyclic or bicyclic. The term “heteroaryl” is used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaryl family,” any of which includes optionally substituted rings. The term "heteroaryl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl and heteroaryl portions are optionally substituted independently.

[0096] As used herein, the term "heteroatom" refers to an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0097] As used herein, the term "heterocyclic alkyl" refers to an alkyl group that has been substituted with a heterocyclic group.

[0098] As used herein, the terms “heterocyclic,” “heterocyclic group,” “heterocyclic group,” and “heterocyclic” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic portion, which may be saturated or partially unsaturated and has one or more (preferably one to four) heteroatoms as defined above, in addition to a carbon atom. When used to refer to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen may be N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

[0099] Heterocycles may be attached to their side groups at any heteroatom or carbon atom that produces a stable structure, and any of the ring atoms may optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolyl, piperidinyl, pyrrololinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazine, dioxalyl, dioxopentyl, diazaphenyl, oxazphenyl, thioazphenyl, morpholinyl, and quininecycloyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclic ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic group” are used interchangeably herein and also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as dihydroindolyl, 3H-indolyl, benzodihydropyranyl, phenanthridine, or tetrahydroquinolinyl. The heterocyclic group can be monocyclic or bicyclic. The term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl moiety and the heterocyclic moiety are independently optional substitutions.

[0100] As used herein, the term "hydrocarbon group" refers to a group bonded by carbon atoms without =O or =S substituents and typically has at least one carbon-hydrogen bond and a predominantly carbon backbone, but may optionally contain heteroatoms. Therefore, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbon groups, but substituents such as acetyl (which has a =O substituent on the linking carbon) and ethoxy (which is linked by oxygen rather than carbon) are not. Hydrocarbon groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, ynyl, and combinations thereof.

[0101] As used herein, the term "hydroxyalkyl" refers to an alkyl group that has been substituted with a hydroxyl group.

[0102] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "lower" means a group comprising ten or fewer atoms, preferably six or fewer, of the substituents. "Lower alkyl" refers, for example, to an alkyl group containing 10 or fewer carbon atoms, preferably six or fewer. In some embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the description of hydroxyalkyl and aralkyl (in which case, for example, atoms within the aryl group are not counted when calculating the carbon atoms in the alkyl substituent).

[0103] The terms "polycyclic," "polycyclic," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic) in which two or more atoms are shared by two adjacent rings; for example, the rings are "fused rings." Each ring of a polycyclic compound may be substituted or unsubstituted. In some embodiments, each ring of the polycyclic compound contains 3 to 10 atoms, preferably 5 to 7 atoms.

[0104] The term "sulfate" is recognized in the art and refers to the group –OSO3H or its pharmaceutically acceptable salt.

[0105] The term "sulfonamide" is recognized in the art and refers to a group represented by the following general formula. , Where R 9 and R 10 Independently represents either hydrogen or hydrocarbon groups.

[0106] The term "sulfoxide" is recognized in the art and refers to the group -S(O)-.

[0107] The term "sulfonate" is recognized in the art and refers to the group SO3H or its pharmaceutically acceptable salt.

[0108] The term "sulfone" is recognized in the art and refers to the group –S(O)2-.

[0109] The term "protecting group" is a widely accepted term in the industry, referring to a chemical functional group on a target molecule that can be modified (usually covalently) to remove an existing unstable functional group. This modification "protects" the unstable functional group in subsequent reaction steps, and the protecting group can be removed as needed, a process known as "deprotection." As a non-limiting example, the tert-butoxycarbonyl (Boc or boc) group is commonly used in synthetic chemistry for covalent modification and "protection" of terminal amine groups.

[0110] The term "substituted" refers to a portion having a substituent that replaces hydrogen on one or more carbons of the main chain. It should be understood that "substituted" or "replaced by" includes the implicit condition that such substitution is based on the permissible valence of the substituted atom and the substituent, and that said substitution produces a stable compound, for example, which does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is considered to include all permissible substituents in organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents in organic compounds. For a suitable organic compound, permissible substituents may be one or more and may be the same or different. For the purposes of this disclosure, heteroatoms (such as nitrogen) may have hydrogen substituents and / or any permissible substituent in the organic compounds described herein that satisfies the heteroatom valence. Substituents may include any substituents described herein, such as halogens, hydroxyl groups, carbonyl groups (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (e.g., thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonate groups, hypophosphonate groups, amino groups, amide groups, amidine groups, imine groups, cyano groups, nitro groups, azide groups, mercapto groups, alkylthio groups, sulfate groups, sulfonate groups, aminosulfonyl groups, sulfonamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that, where appropriate, the portion substituted on the hydrocarbon chain may itself be substituted.

[0111] Suitable monovalent substituents on the substituted carbon atoms of the "optionally substituted" group are independently halogens; –(CH2) 0–4 R°;–(CH2) 0–4 OR°;-O(CH2) 0-4 R°;–O–(CH2) 0–4 C(O)OR°;–(CH2) 0–4 CH(OR°)2;–(CH2) 0– 4SR°; –(CH2) 0–4 Ph, which can be replaced by R°; –(CH2) 0–4 O(CH2) 0–1 Ph, which can be substituted by R°; –CH=CHPh, which can be substituted by R°; –(CH2) 0–4 O(CH2)0–1 Pyridyl group, which can be substituted by R°; –NO2; –CN; –N3; -(CH2) 0–4 N(R°)2;–(CH2) 0–4 N(R°)C(O)R°; –N(R°)C(S)R°; –(CH2) 0–4 N(R°)C(O)NR°2; -N(R°)C(S)NR°2; –(CH2) 0–4 N(R°)C(O)OR°; –N(R°)N(R°)C(O)R°; –N(R°)N(R°)C(O)NR°2; –N(R°)N(R°)C(O)OR°; –(CH2) 0–4 C(O)R°; –C(S)R°; –(CH2) 0–4 C(O)OR°;–(CH2) 0–4 C(O)SR°;-(CH2) 0–4 C(O)OSiR°3;–(CH2) 0–4 OC(O)R°;–OC(O)(CH2) 0–4 SR°, SC(S)SR°;–(CH2) 0–4 SC(O)R°;–(CH2) 0–4 C(O)NR°2; –C(S)NR°2; –C(S)SR°; –SC(S)SR°; –(CH2) 0–4 OC(O)NR°2; -C(O)N(OR°)R°; –C(O)C(O)R°; –C(O)CH2C(O)R°; –C(NOR°)R°; –(CH2) 0–4 SSR°;–(CH2) 0–4 S(O)2R°;–(CH2) 0–4 S(O)₂OR°;–(CH₂) 0–4 OS(O)2R°; –S(O)2NR°2; -(CH2) 0–4 S(O)R°; -N(R°)S(O)2NR°2; –N(R°)S(O)2R°; –N(OR°)R°; –C(NH)NR°2; –P(O)2R°; –P(O)R°2; –OP(O)R°2; –OP(O)(OR°)2; SiR°3; –(C 1–4 (linear or branched alkylene)O–N(R°)2; or –(C 1–4 (straight-chain or branched alkylene)C(O)O–N(R°)2, wherein each R can be substituted as defined below and independently be hydrogen, °C 1–6 Aliphatic group, –CH2Ph, –O(CH2) 0–1Ph, -CH2- (5-6 membered heteroaryl ring) or 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independent Rs together with their intermediate atoms to form a 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur, which may be replaced as defined below.

[0112] Suitable monovalent substituents on R° (or a ring formed by two independently occurring R° along with their intermediate atoms) are independently halogens, –(CH2). 0–2 R ● –(halogenated R) ● ), –(CH2) 0–2 OH, –(CH2) 0–2 OR ● –(CH2) 0–2 CH(OR ● )2;-O(halogenated R ● –CN, –N3, –(CH2) 0–2 C(O)R ● –(CH2) 0–2 C(O)OH, –(CH2) 0–2 C(O)OR ● –(CH2) 0–2 SR ● –(CH2) 0– 2SH, –(CH2) 0–2 NH2、–(CH2) 0–2 NHR ● –(CH2) 0–2 NR ● 2. –NO2, –SiR ● 3, –OSiR ● 3. -C(O)SR ● 、 –(C 1–4 (straight-chain or branched alkylene)C(O)OR ● Or –SSR ● , where each R ● It is either unsubstituted or, when preceded by a "halogen group", substituted by one or more halogens, and independently selected from C. 1–4 Aliphatic group, –CH2Ph, –O(CH2) 0–1 Ph, or a 5–7 saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on the saturated carbon atom of R° include =O and =S.

[0113] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following: =O, =S, =NNR. * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * 、 –O(C(R) * 2)) 2–3 O – or –S(C(R) * 2)) 2– 3S–, where each independently occurring R * Selected from hydrogen, C 1–6 An aliphatic group (which may be substituted as defined below) or having 0-4 unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings independently selected from nitrogen, oxygen, or sulfur heteroatoms. Suitable divalent substituents attached to the ortho-substituted carbon of the "optionally substituted" group include: –O(CR * 2) 2–3 O–, where each independently occurring R * Selected from hydrogen, C 1–6 An aliphatic group (which may be substituted as defined below) or an unsubstituted 5-6 member saturated, partially unsaturated or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur.

[0114] R * Suitable substituents on the aliphatic group include halogens, -R ● -(halogenated R) ● -OH, -OR ● –O(halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ● It is either unsubstituted or, when preceded by a "halogen group", substituted by one or more halogens and independently C. 1–4 Aliphatic group, –CH2Ph, –O(CH2) 0–1 Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0115] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include –R † –NR † 2. –C(O)R † –C(O)OR † –C(O)C(O)R† –C(O)CH2C(O)R † –S(O)2R † -S(O)2NR † 2. –C(S)NR † 2. –C(NH)NR † 2 or –N(R) † )S(O)2R † ; where each R † C that is independently hydrogen and can be substituted as defined below 1–6 Aliphatic group, unsubstituted –OPh, or substituted 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, or, although defined above, two independently occurring R groups. † Together with its intermediate atom, it forms a 3-12 saturated, partially unsaturated, or aryl monocyclic or bicyclic ring with 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

[0116] R † The aliphatic group and suitable substituents on the 5-6 substituted 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur are independently halogens, –R ● -(halogenated R) ● –OH, –OR ● –O(halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or -NO2, where each R ● It is either unsubstituted or, in the case of a preceding "halogen group," substituted by only one or more halogens, and is independently C. 1–4 Aliphatic group, –CH2Ph, –O(CH2) 0–1 Ph or a 5-6 member saturated, partially unsaturated or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur.

[0117] As used herein, the term "thioalkyl" refers to an alkyl group that has been substituted with a mercapto group.

[0118] As used herein, the term "thioester" refers to the group -C(O)SR. 9 or -SC(O)R 9 , where R 9 It represents a hydrocarbon group.

[0119] As used in this article, the term "thioether" is equivalent to ether, in which oxygen is replaced by sulfur.

[0120] The term "urea" is recognized in the art and can be represented by the following general formula. , Where R 9 and R 10 Independently represents either hydrogen or hydrocarbon groups.

[0121] The term "tautomer" refers to each of two or more isomers of a compound that coexist in equilibrium and are interchangeable through the migration of intramolecular atoms or groups (such as hydrogen atoms). Exemplary tautomers of this disclosure include, but are not limited to, those mentioned above. and The description of one tautomer in a particular genus or species of compound in this disclosure is intended to cover the drawn compounds and all their tautomers. Specifically, for the examples above, a series of 2... n In different categories and ,in n It is the number of tautomer sites on the molecule.

[0122] For the purposes of this disclosure, any implementation described herein is applicable to any general structure described herein, if appropriately relied upon.

[0123] Compounds of the disclosure In some respects, the present invention provides a series of compounds having a structure according to Formula I: (I), Or its pharmaceutically acceptable salt, wherein: Ring M is aryl or heteroaryl; Ring A contains at least 5 atoms; X and Y are each independently selected from the key, , , , , , , , , , O and NR x2 ,in * indicates a key with N, and Indicates the bond with ring M; Each R a Independently selected from hydrogen, and optionally substituted C1-C3 alkyl groups; R x1 Selected from optionally substituted C1-C4 alkyl groups and optionally substituted 3-6 membered cycloalkyl groups; Rx2 Selected from hydrogen, optionally substituted C1-C4 alkyl groups and optionally substituted 3-6 membered cycloalkyl groups; R 1 Selected from 3-6 membered cycloalkyl, aryl, 4-7 membered heterocyclic and heteroaryl, each of which may be optionally substituted; L is selected from the following: -CH2-, -O-, -NH-, -N(Me)-, -C(=O)NH-, and -NHC(=O)-; When it exists, R 2 Each time it appears, it is independently selected from halogen, C1-C4 alkyl, O-C1-C4 alkyl, -CN, aryl, 4-7 membered heterocyclic and 5-6 membered heteroaryl, wherein R 2 Any alkyl, aryl, heterocyclic, or heteroaryl moiety may optionally be substituted; R 3 Selected from aryl groups that are arbitrarily substituted, -C(=O)-R 4 and -CN; R 4 Selected from NH2, optionally substituted aminoalkyl, optionally substituted alkylamino, optionally substituted C1-C6 alkyl, optionally substituted 3-6 membered cycloalkyl, optionally substituted aryl, and optionally substituted O-C1-C4 alkyl; and a is selected from 0, 1, and 2.

[0124] In some respects, the present invention provides a series of compounds having a structure according to Formula I: (I), Or its pharmaceutically acceptable salt, wherein: Ring M is aryl or heteroaryl; Ring A contains at least 5 atoms; X and Y are each independently selected from the key, , , , , , , , , , O and NR x2 ,in * indicates a key with N, and Indicates the bond with ring M; Each R a Independently selected from hydrogen, and optionally substituted C1-C3 alkyl groups; R x1 Selected from optionally substituted C1-C4 alkyl groups and optionally substituted 3-6 membered cycloalkyl groups; Rx2 Selected from hydrogen, optionally substituted C1-C4 alkyl groups and optionally substituted 3-6 membered cycloalkyl groups; R 1 Selected from 3-6 membered cycloalkyl, aryl, 4-7 membered heterocyclic and heteroaryl, each of which may be optionally substituted; L is selected from the following: -CH2-, -O-, -NH-, -N(Me)-, -C(=O)NH-, and -NHC(=O)-; When it exists, R 2 Each time it appears, it is independently selected from halogen, C1-C4 alkyl, O-C1-C4 alkyl, -CN, aryl, 4-7 membered heterocyclic and 5-6 membered heteroaryl, wherein R 2 Any alkyl, aryl, heterocyclic, or heteroaryl moiety may optionally be substituted; R 3 Selected from aryl groups that are arbitrarily substituted, -C(=O)-R 4 and -CN; R 4 Selected from NH2, optionally substituted aminoalkyl, optionally substituted alkylamino, optionally substituted C1-C4 alkyl, optionally substituted 3-6 membered cycloalkyl, and optionally substituted O-C1-C4 alkyl; and a is selected from 0, 1, and 2.

[0125] In some implementations, R 3 It is –C(=O)-R 4 .

[0126] In some implementations, R 4 It's OMe.

[0127] In some implementations, X is .

[0128] In some implementations, Y is In some implementations, each R a It is hydrogen.

[0129] In some implementations, ring A is selected from: , , , , , , , , , , , , , , , and , where ** indicates a connection to the rest of the molecule, and each --- indicates a portion of the ring M fused with ring A.

[0130] In some implementations, ring A is selected from: , , , , , , , , , , and , where ** indicates a connection to the rest of the molecule, and each --- indicates a portion of the ring M fused with ring A.

[0131] In some implementations, ring A is selected from: , , , , , , , , , , , , , , , , , , and , where ** indicates a connection to the rest of the molecule, and each --- indicates a portion of the ring M fused with ring A.

[0132] In some implementations, ring A is selected from: , , , , , , , , , , , , , and , where ** indicates a connection to the rest of the molecule, and each --- indicates a portion of the ring M fused with ring A.

[0133] In some implementations, ring A is .

[0134] In some embodiments, ring M is a heteroaryl group. In some embodiments, ring M is selected from thienyl, isoxazolyl, isothiazolyl, pyridyl, pyrazolyl, imidazole, and thiazolyl.

[0135] In other embodiments, ring M is aryl.

[0136] In some implementations, each existing R 2 Independently selected from halogen groups, optionally substituted O-C1-C4 alkyl groups, optionally substituted C1-C4 alkyl groups, and -CN. In some embodiments, each present R 2 It is independently selected from chlorine, fluorine, methoxy, methyl and -CN.

[0137] In some implementations, a is 0.

[0138] In some implementations, L is a key.

[0139] In some implementations, R 1 Selected from optionally substituted 3-6 membered cycloalkyl groups and optionally substituted 4-7 membered heterocyclic groups. In some embodiments, R 1 It is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxacyclobutyl, thiomorpholinyl and tetrahydropyranyl, each of which may be optionally substituted.

[0140] In other implementations, R 1 Selected from optionally substituted aryl groups and optionally substituted heteroaryl groups. In some embodiments, R 1 The group is selected from pyridyl, indolyl, isoindolyl, benzimidazolyl, benzothiazolyl, benzotriazolyl, benzopyrazolyl, thiazolyl, pyrazolyl, imidazolyl, indololinyl, isoindololinyl, benzoxazolone, pyrazinyl, inzozolyl, oxazolyl, and benzoxazinyl, each of which may be optionally substituted.

[0141] In other implementations, R 1 It is an aryl group that can be substituted at will.

[0142] In some embodiments, the compound has a structure according to formula (II): (II).

[0143] In some embodiments, the compound has a structure according to formula (III): (III), Wherein, each existing R 6 Independently selected from -NH2, –(C=O)-NH2, –(C=O)NH-C1-C6 alkyl, –(C=O)NH-3-6-membered cycloalkyl, –(C=O)NH-3-6-membered heterocyclic, –(C=O)-3-6-membered heterocyclic, -(C=O)N(C1-C4 alkyl)2, -NH(C=O)-C1-C6 alkyl, -NH(C=O)3-6-membered heteroaryl, -N(C1-C4 alkyl)(C=O)-C1-C4 alkyl, -NH(C=O)-3-6-membered cycloalkyl, -NH(C=O)-4-7-membered heterocyclic, -NMe(C=O)-C1-C6 alkyl, -CH2-NH(C=O)-C1-C5 alkyl, -NH(C=O)O-4-6-membered cycloalkyl, -NHSO2- C1 -C4 alkyl, -SO2-C1-C4 alkyl, -SO2NH2, -SO2NH-C1-C4 alkyl, -NH(C=O)O-C1-C4 alkyl, -(C=O)O-C1-C4 alkyl, -O(C=O)-C1-C4 alkyl, -NH(C=O)-NH-C1-C4 alkyl, -(C=O)-C1-C4 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocyclic, 5-6 membered heteroaryl, C1-C4 alkyl, O-C1-C4 alkyl, chlorine, fluorine, -CF3, nitro, -NH-C1-C4 alkyl, -CN and -N(C1-C4 alkyl)2, each optionally substituted, and b is 0, 1 or 2, or 2 R 6 Together they form 3-6 membered cycloalkyl, 3-6 membered heterocyclic, 5-6 membered heteroaryl or aryl, each of which may be substituted.

[0144] In some embodiments, the compound has a structure according to formula (III): (III), Wherein, each existing R 6Independently selected from –(C=O)-NH2, –(C=O)NH-C1-C6 alkyl, –(C=O)NH-3-6-membered cycloalkyl, –(C=O)NH-3-6-membered heterocyclic, –(C=O)-3-6-membered heterocyclic, -NH(C=O)-C1-C6 alkyl, -N(C1-C4 alkyl)(C=O)-C1-C4 alkyl, -NH(C=O)-3-6-membered cycloalkyl, -NH(C=O)-4-7-membered heterocyclic, -NMe(C=O)-C1-C6 alkyl, -CH2-NH(C=O)-C1-C4 alkyl, -NHSO2-C1-C4 alkyl, -SO 2-C1-C4 alkyl, -SO2NH2, -SO2NH-C1-C4 alkyl, -NH(C=O)O-C1-C4 alkyl, -(C=O)O-C1-C4 alkyl, -O(C=O)-C1-C4 alkyl, -NH(C=O)-NH-C1-C4 alkyl, -(C=O)-C1-C4 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocyclic, 5-6 membered heteroaryl, C1-C4 alkyl, O-C1-C4 alkyl, chlorine, fluorine, -NH-C1-C4 alkyl, -CN and -N(C1-C4 alkyl)2, each optionally substituted, and b is 0, 1 or 2, or 2 R 6 Together they form 3-6 membered cycloalkyl, 3-6 membered heterocyclic, 5-6 membered heteroaryl or aryl, each of which may be substituted.

[0145] In some embodiments, the compound has a structure according to formula (IV): (IV).

[0146] In some implementations, R 6 -NH(C=O)R 7 , where R 7 It is selected from C1-C4 alkyl, 3-6 membered cycloalkyl, 4-7 membered heterocyclic, O-C1-C4 alkyl and aminoalkyl, each of which may be optionally substituted.

[0147] In some implementations, R 7 Selected from optionally substituted C1-C4 alkyl groups and optionally substituted O-C1-C4 alkyl groups.

[0148] In other implementations, R 6 It is either NHAc or NHBoc.

[0149] In some embodiments, the compound has the following structure: .

[0150] In some embodiments, the compound has a structure according to formula (V): (V).

[0151] In some implementations, R 3 It is an optional substituted heteroaryl group. In some embodiments, R 3 Selected from triazolyl, thiadiazolyl, and oxadiazolyl, each of which may be substituted.

[0152] In other implementations, R 3 It is -C(=O)-R 4 In some implementations, R 4 Selected from NH2, optionally substituted C1-C4 alkyl groups, optionally substituted 3-6 membered cycloalkyl groups, and optionally substituted O-C1-C5 alkyl groups. In other embodiments, R 3 It is -C(=O)-R 4 In some implementations, R 4 Selected from NH2, optionally substituted C1-C4 alkyl groups, optionally substituted 3-6 membered cycloalkyl groups, and optionally substituted O-C1-C4 alkyl groups. In some embodiments, R 4 Selected from -NH2, methyl, methoxy, cyclopropoxy, and -Ot-Bu. In some embodiments, R 3 It is -C(=O)-OMe.

[0153] In some embodiments, the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0154] In some embodiments, the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0155] In some embodiments, the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0156] In some embodiments, the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , and .

[0157] In some embodiments, the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0158] In some embodiments, the compound is selected from: , , , , , , , , , , , , and .

[0159] In some respects, the present invention provides compounds having the structure according to formula (VI): (VI), Or its pharmaceutically acceptable salt, wherein: R 7 It is -CH3 or -CH2CH3; R 8 It is -C1-C4 alkyl, -C3-C6 cycloalkyl, -(C1-C3 alkylene)-C3-C6 cycloalkyl, -(C1-C3 alkylene)-O-C1-C3 alkyl or -(C1-C3 alkylene)-N(R) 9 )2; or R 7 and R 8 Together with the nitrogen atom to which it is attached, it forms a 4-7 membered saturated heterocycle optionally containing additional heteroatoms selected from N, O, and S, wherein the 4-7 membered saturated heterocycle is optionally substituted by 1-2 substituents independently selected from oxo, -C1-C3 alkyl, -OH, -O(C1-C3 alkyl), and -C(O)-C1-C3 alkyl; and R 11 for , , , , , or ,in This represents the bond with the fused benzene ring in formula (VI); Each R 9 Independently H or C1-C4 alkyl; R 10 It is hydrogen, C1-C4 alkyl, or -C(O)C1-C4 alkyl; and R 12 It is a C1-C4 alkyl group.

[0160] In some implementations, R 7 It is -CH3.

[0161] In some implementations, R 8 It can be -CH3, -CH2CH3, -CH(CH3)2, cyclopentyl, -(C1-C3 alkylene)-cyclopentyl, -CH2CH2-OCH3 or -CH2CH2N(CH3)2.

[0162] In some such implementations, R 8 It is –CH3.

[0163] In other implementations, R 7 and R 8 Together with the nitrogen atom it is attached to, it forms a 4-6 membered heterocyclic group.

[0164] In some such implementations, R 7 and R 8 Together with the nitrogen atom to which it is attached, it forms a heterocyclic group selected from the following: , , , , , , , and ,in This represents the bond with the carbonyl group of the amide in formula (VI).

[0165] In some such implementations, R 11 for , , , , , , , , or .

[0166] In some implementations, R 10 It can be hydrogen, -CH3, or –C(O)CH3.

[0167] In some embodiments, the compound is selected from: , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0168] In some respects, the present invention provides compounds having a structure according to formula (VII): (VII), Or its pharmaceutically acceptable salt, wherein: R 7 It is -CH3 or -CH2CH3; R 8 It is -C1-C4 alkyl, -C3-C6 cycloalkyl, -(C1-C3 alkylene)-C3-C6 cycloalkyl, -(C1-C3 alkylene)-O-C1-C3 alkyl or –(C1-C3 alkylene)-N(R) 9 )2; or R 7 and R 8 Together with the nitrogen atom to which it is attached, it forms a 4-7 membered saturated heterocycle optionally containing additional heteroatoms selected from N, O, and S, wherein the 4-7 membered saturated heterocycle is optionally substituted by 1-2 substituents independently selected from oxo, -C1-C3 alkyl, -OH, -O(C1-C3 alkyl), and -C(O)-C1-C3 alkyl; and R 11 for , , , , , or ,in This represents the bond with the fused benzene ring in formula (VI); Each R 9 Independently H or C1-C4 alkyl; R 10 It is hydrogen, C1-C4 alkyl, or -C(O)C1-C4 alkyl; and R 12 It is a C1-C4 alkyl group.

[0169] In some such implementations, R 7 It is -CH3.

[0170] In some implementations, R 8 It can be -CH3, -CH2CH3, -CH(CH3)2, cyclopentyl, -(C1-C3 alkylene)-cyclopentyl, -CH2CH2-OCH3 or -CH2CH2N(CH3)2.

[0171] In some such implementations, R 8 It is –CH3.

[0172] In some implementations, R 7 and R8 Together with the nitrogen atom it is attached to, it forms a 4-7 membered heterocyclic group.

[0173] In some such implementations, R 7 and R 8 Together with the nitrogen atom to which it is attached, it forms a heterocyclic group selected from the following: , , , , , , , and ,in It represents the bond with the carbon of the amide carbonyl group in formula (VII).

[0174] In some such implementations, R 11 for , , , , , , , , or .

[0175] In some implementations, R 10 It can be hydrogen, -CH3, or -C(O)CH3.

[0176] In some embodiments, the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0177] In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising any of the compounds described herein, and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for oral or parenteral delivery.

[0178] In some embodiments, this document discloses a method of treating cancer (e.g., solid tumors or hematologic malignancies) comprising administering to a subject in need a therapeutically effective amount of any of the compounds described herein or a combination of said compounds. In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, prostate cancer, melanoma, esophageal cancer, leukemia, cervical cancer, liver cancer, colon cancer, gastric cancer, colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and ovarian cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and breast cancer.

[0179] Example General synthetic procedures General experimental procedure 1 - Suzuki coupling - Pd(PPh 3 ) 4 Under N2 protection, starting boric acid or borate ester (1 equivalent) was added to a flask. 1,2-Dimethoxyethane (0.1 M), bromide (1.05 equivalent), and 2 M sodium carbonate aqueous solution (5 equivalent) were added. The mixture was then degassed with N2 for several minutes. Palladium tetrahydrate (0.05 equivalent) was then added, and the mixture was stirred at 80–100 °C (external) until complete conversion (overnight). The reaction was then stopped. Water was added, and the mixture was extracted with EtOAc. Brine was added to improve separation. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude product was purified by automated FCC (normal or reversed phase). Note: Hydrolysis products were also observed in some reactions. These were separated by acidifying the aqueous phase and extracted twice with EtOAc. The combined organic compounds were washed with brine, dried over Na2SO4, filtered, and concentrated.

[0180] General experimental procedure 2 - Suzuki coupling and ester hydrolysis - Pd(dppf)Cl 2 Under N2 protection, boric acid or ester (1.0 equivalent) and cesium carbonate (2.5 equivalent) were added to a solution of bromide (1.05 equivalent) in a mixture of dioxane and water (4:1, 0.1 M). The mixture was then degassed with N2 for several minutes. Pd(dppf)Cl2 (0.15 equivalent) was then added, and the mixture was stirred at 80–100 °C (external) until complete conversion (overnight). The reaction was then stopped. Water was added, and the aqueous phase was acidified with HCl. The resulting mixture was extracted twice with EtOAc. The combined organic matter was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by automated FCC (normal or reversed phase). Note: If hydrolysis is incomplete, crude material should be submitted according to General Experimental Procedure 4 to provide complete hydrolysis.

[0181] General experimental procedure 3 - Suzuki coupling - Pd(OAc) 2 Under N2 protection, a solution of bromide (1.05 equivalents) in a mixture of dioxane and water (4:1, 0.1 M) was added with boric acid or ester (1.0 equivalents), SPhos (0.2 equivalents), and cesium fluoride (2.5 equivalents). The mixture was then degassed with N2 for several minutes. Pd(OAc)₂ (0.2 equivalents) was then added, and the mixture was stirred at 80–100 °C (external) until complete conversion (overnight). The reaction was then stopped. Water was added, and the mixture was extracted with EtOAc. Brine was added to improve separation. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by automated FCC (normal or reversed phase).

[0182] General experimental procedure 4 - Ester hydrolysis A solution of lithium hydroxide monohydrate (4 equivalents) in water (2 M) was added to a solution of ester (1 equivalent) in THF (0.05 M), and the mixture was stirred at room temperature for 2–16 hours. The mixture was diluted with EtOAc, washed with 1 M HCl and 30 mL of brine, dried over Na2SO4, filtered, and concentrated to obtain the desired product.

[0183] General experimental procedure 5 - HATU mediated amide coupling DiPEA (3 equivalents) and HATU (1.2 equivalents) were added to a solution of acid (1 equivalent) and amine (1 equivalent) in DCM (0.05 M), and the resulting mixture was stirred at room temperature for 2–16 hours. The mixture was washed with 1M HCl, water, NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by automated FCC to obtain the desired product.

[0184] General experimental procedure 6 - Acetylation of serine side chain Triethylamine (2 equivalents / alcohol), DMAP (0.2 equivalents), and acetic anhydride (1.1 equivalents / alcohol) were added to a solution of free alcohol (1 equivalent) in MeCN (0.05 M), and the resulting mixture was stirred for 2 hours. The reaction mixture was diluted with water, and MeCN was removed under vacuum. The mixture was extracted with EtOAc, and the organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by automated FCC to obtain the desired product.

[0185] General experimental procedure 7 - Elimination of bisacetate A solution of diacetate in THF / CH₂Cl₂ (1 / 1, v / v, 0.05 M) was cooled to 0 °C, and DBU (4 equivalents) was added. The mixture was stirred at 0 °C for 2 hours. The mixture was diluted with CH₂Cl₂, and water and HCl (1 M aqueous solution) were added to acidify the mixture to pH 4. The mixture was extracted with CH₂Cl₂ (3×), and the combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated. The crude material was purified by automated reverse-phase FCC.

[0186] General experimental procedure 8 - Isoindolinone ring closure An amine (1.05 equivalents) and DiPEA (3 equivalents) were added to a solution of methyl bromide benzoate (1.0 equivalent) in MeCN (0.25 M), and the resulting mixture was stirred at 80 °C for 16 hours. The reaction mixture was partitioned between CH2Cl2 and water. The organic layer was collected, dried over anhydrous Na2SO4, filtered, and concentrated. The crude material was purified by automated FCC.

[0187] General experimental procedure 9 - Isoindolinone alkylation Triphenylphosphine or polymer-bound triphenylphosphine (1.1 equivalents) was added to a solution of isoindolinone (1.0 equivalents) in CH₂Cl₂ (0.3 M), and the resulting mixture was cooled to 0°C. Then, an alkyne (1.1 equivalents) was added to a solution of CH₂Cl₂, and the mixture was stirred at room temperature for 16 hours. For polymer-bound triphenylphosphine, the mixture was filtered. The concentrated compound and crude material were purified by automated FCC.

[0188] Analytical methods LCMS method "22010199 LCMS-5 C3"

[0189] LCMS method “22010199A TFA LCMS-5 C1”

[0190] LCMS method “22010199D TFA LCMS-5 C8”

[0191] LCMS method “22010199E TFA LCMS-5 C8”

[0192] LCMS method “ 22010199G LCMS-5 C3 "

[0193] LCMS method “ Final 3 basic "

[0194] LCMS method “ Final 3 acidic "

[0195] LCMS method “ General 3 basic "

[0196] LCMS method “ General 3 acidic "

[0197] LCMS (UPLC_AN_BASE (singlet) LCMS-24)

[0198] SFC method “ 22010199H SFC-2 "

[0199] Synthesis of exemplary compounds Example 1 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate Step 1. Follow General experimental procedure 8 Methyl (S)-2-(7-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate was prepared. Methyl 2-bromo-6-(bromomethyl)benzoate (0.62 g, 2.0 mmol) and H-Ser-OMe.HCl (0.35 g, 2.2 mmol) yielded methyl (S)-2-(7-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate (0.52 g, 1.7 mmol, 82%). LCMS (UPLC_AN_BASE (single peak) LCMS-24) m / z = 314.0 Step 2. Following general experimental procedure 2(S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid was prepared. Methyl (S)-2-(7-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (0.52 g, 1.7 mmol) and (4-acetamidophenyl)boronic acid (0.59 g, 2 equivalents, 3.3 mmol) yielded (S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (0.16 g, 0.46 mmol, 28%) as a grayish-white solid. LCMS (UPLC_AN_BASE (single peak) LCMS-24 m / z = 355.1 Step 3. Follow General experimental procedure 5 ((S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester was prepared. Crude ((S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (0.20 g; 0.56 mmol) and H-Ser-OMe.HCl (0.11 g, 1.3 equivalents, 0.73 mmol) was obtained and used in the next step without purification.

[0200] Step 4. Follow General experimental procedure 6 N-((S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine methyl ester was prepared. Crude ((S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester was obtained to yield crude N-((S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine methyl ester, which was used as is in the next step.

[0201] Step 5. Follow General experimental procedure 7 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate was prepared. Crude N-((S)-2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine methyl ester was obtained as a white solid, 2-(2-(7-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate (40 mg, 95 µmol, 17%, obtained in 3 steps). LCMS (22010199G LCMS-5 C3):m / z = 418.0 [MH] - . 1 H NMR (400 MHz, DMSO) δ 10.01(s, 1H), 9.38 (s, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.63 – 7.55 (m, 3H), 7.45 –7.36 (m, 3H), 6.04 (s, 1H), 5.73 (s, 1H), 5.53 (d, J = 3.8 Hz, 2H), 4.78 (s, 2H), 3.73 (s, 3H), 2.06 (s, 3H).

[0202] Example 2 With Example 1 The following compounds were prepared using methods similar to those described in [the previous text]: Example 3 2-(2-(1-oxo-4-(4-neoplastamidophenyl)isoindoline-2-yl)acrylamido)methyl acrylate Step 1. Follow General experimental procedure 8 Methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate was prepared. Methyl 3-bromo-2-bromomethylbenzoate (10 g, 32 mmol) and H-Ser-OMe·HCln (5.6 g, 1.1 equivalents, 36 mmol) yielded methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate (4.4 g, 14 mmol, 44%). LCMS (universal 3 basic): m / z = 313.9 [M+H] + Step 2. Follow General experimental procedure 2 (R)-2-(4-(4-((tert-Butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid was prepared. Methyl (R)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (2.0 g, 4.7 mmol) and [4-(tert-Butoxycarbonyl)amino)phenyl]boronic acid (1.7 g, 1.5 equivalents, 7.0 mmol) were reacted to give (R)-2-(4-(4-((tert-Butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (1.7 g, 4.2 mmol, 90%).

[0203] Step 3. Follow General experimental procedure 5 ((S)-2-(4-(4-((tert-Butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester was prepared. (S)-2-(4-(4-((tert-Butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (1.7 g, 4.2 mmol) and H-Ser-OMe.HCl (0.85 g, 1.3 equivalents, 5.5 mmol) yielded ((S)-2-(4-(4-((tert-Butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester (1.8 g, 3.6 mmol, 85%), which was used directly in the next step without purification.

[0204] Step 4. Follow General experimental procedure 6 N-((S)-3-acetoxy-2-(4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester was prepared. ((S)-2-(4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester (1.8 g, 3.6 mmol) yielded N-((S)-3-acetoxy-2-(4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (2.0 g, 3.3 mmol, 92%), which was used in the next step without purification.

[0205] Step 5. Prepare N-((S)-3-acetoxy-2-(4-(4-aminophenyl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (2.0 g, 3.3 mmol) using the following procedure: Add TFA (3.5 mL) to a solution of N-((S)-3-acetoxy-2-(4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (2.0 g, 3.3 mmol) in CH2Cl2 (10 mL) and stir the resulting mixture for 1 hour. Dilute the mixture with CH2Cl2 (20 mL) and quench with a saturated NaHCO3 aqueous solution (30 mL). Separate the layers and extract the aqueous layer with CH2Cl2 (4 × 15 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to obtain N-((S)-3-acetoxy-2-(4-(4-aminophenyl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (1.5 g, 3.1 mmol, 93%), which was used in the next step without further purification.

[0206] Step 6. Follow General experimental procedure 5 N-((S)-3-acetoxy-2-(1-oxo-4-(4-neopramamidophenyl)isoindololin-2-yl)propionyl)-O-acetyl-L-serine methyl ester was prepared. N-((S)-3-acetoxy-2-(4-(4-aminophenyl)-1-oxoisoindololin-2-yl)propionyl)-O-acetyl-L-serine methyl ester (50 mg, 0.10 mmol) and neopentanoic acid (13 mg, 1.3 equivalents, 0.13 mmol) yielded N-((S)-3-acetoxy-2-(1-oxo-4-(4-neopramamidophenyl)isoindololin-2-yl)propionyl)-O-acetyl-L-serine methyl ester (45 mg, 77 µmol, 77%). LCMS (Generally Acidic 3): m / z = 582.6 [M+H] + Step 7. Follow General experimental procedure 7Methyl 2-(2-(1-oxo-4-(4-neopramamidophenyl)isoindoline-2-yl)acrylamido)acrylate was prepared. N-((S)-3-acetoxy-2-(1-oxo-4-(4-neopramamidophenyl)isoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (45 mg, 77 µmol) yielded methyl 2-(2-(1-oxo-4-(4-neopramamidophenyl)isoindoline-2-yl)acrylamido)acrylate (1-12) (10 mg, 22 µmol, 28%) as a white solid. LCMS: 22010199 LCMS-5 C3 m / z = 462.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.42 (s, 1H), 9.34 (s, 1H), 7.86 – 7.78 (m, 2H), 7.77 – 7.68 (m, 2H), 7.68 – 7.62 (m, 1H), 7.62 – 7.54 (m, 2H), 6.06 (s, 1H), 5.74 (s, 1H), 5.64(d, J = 1.1 Hz, 1H), 5.59 (d, J = 1.1 Hz, 1H), 4.93 (s, 2H), 3.72 (s, 3H), 1.25 (s, 9H).

[0207] Example 4 The following compounds were prepared using a method similar to that described in Example 3.

[0208] Example 5 2-(2-(4-(4-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate (1-29) Step 1. Follow General experimental procedure 8 Methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate was prepared. Methyl 3-bromo-2-bromomethylbenzoate (10 g, 32 mmol) and H-Ser-OMe·HCln (5.6 g, 1.1 equivalents, 36 mmol) yielded methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate (4.4 g, 14 mmol, 44%). LCMS (universal 3 basic): m / z = 313.9 [M+H] + Step 2. Follow General experimental procedure 2 (S)-2-(4-(4-(tert-butoxycarbonyl)phenyl)-1-oxoisoindololin-2-yl)-3-hydroxypropionic acid was prepared. Methyl (S)-2-(4-bromo-1-oxoisoindololin-2-yl)-3-hydroxypropionic acid (0.35 g, 1.1 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoborane-2-yl)benzoate (0.37 g, 1.1 equivalents, 1.2 mmol) yielded (S)-2-(4-(4-(tert-butoxycarbonyl)phenyl)-1-oxoisoindololin-2-yl)-3-hydroxypropionic acid (0.40 g, 1.0 mmol, 90%). LCMS (Generally Acidic 3): m / z = 398.3 [M+H] + Step 3. Follow General experimental procedure 5 Preparation of tert-butyl benzoate of 4-(2-((S)-3-hydroxy-1-(((S)-3-hydroxy-1-methoxy-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-1-oxoisoindoline-4-yl)benzoate. (S)-2-(4-(4-(tert-butyloxycarbonyl)phenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (0.50 g, 1.3 mmol) and H-Ser-OMe·HCl (0.25 g, 1.3 equivalents, 1.6 mmol) yield 4-(2-((S)-3-hydroxy-1-(((S)-3-hydroxy-1-methoxy-1-oxoprop-2-yl)amino)-1-oxoprop-2-yl)-1-oxoisoindoline-4-yl)benzoate tert-butyl ester (0.50 g, 1.0 mmol, 80%).

[0209] Step 4. Follow General experimental procedure 6Preparation of tert-butyl benzoate of 4-(2-((S)-3-acetoxy-1-(((S)-3-acetoxy-1-methoxy-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-1-oxoisoindoline-4-yl)benzoate. 4-(2-((S)-3-hydroxy-1-(((S)-3-hydroxy-1-methoxy-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-1-oxoisoindoline-4-yl)benzoate tert-butyl ester (1.3 g, 2.6 mmol) yielded 4-(2-((S)-3-acetoxy-1-(((S)-3-acetoxy-1-methoxy-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-1-oxoisoindoline-4-yl)benzoate tert-butyl ester (1.1 g, 1.9 mmol, 71%). LCMS (Universal 3 Acid): m / z = 583.6 [M+H] + Step 5. Prepare 4-(2-(3-acetoxy-1-((3-acetoxy-1-methoxy-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-1-oxoisoindoline-4-yl)benzoic acid using the following procedure: Add 0.25 mL of TFA to a solution of 0.12 g (0.21 mmol) of tert-butyl benzoate in CH2Cl2 (5 mL) and stir the resulting mixture for 24 hours. The mixture was concentrated under vacuum and co-evaporated with Et2O to give 4-(2-(3-acetoxy-1-((3-acetoxy-1-methoxy-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-1-oxoisoindoline-4-yl)benzoic acid (95 mg, 0.18 mmol, 87%), which was used as is in the next step.

[0210] Step 6. According to General experimental procedure 5N-((S)-3-acetoxy-2-(1-oxo-4-(4-neopentaylphenyl)isoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester was prepared. Crude N-((S)-3-acetoxy-1-(((S)-3-acetoxy-1-methoxy-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-1-oxoisoindoline-4-yl)benzoic acid (70 mg, 0.13 mmol) and tert-butylamine (28 µL, 0.27 mmol) was obtained by reacting these compounds with crude N-((S)-3-acetoxy-2-(1-oxo-4-(4-neopentaylphenyl)isoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (0.14 g), which was used in the next step without purification.

[0211] Step 7. Follow General experimental procedure 7 2-(2-(4-(4-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate (1-29) was prepared. Crude N-((S)-3-acetoxy-2-(4-(4-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (0.14 g) was used to obtain 2-(2-(4-(4-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate (1-29) as a white solid (21 mg, 46 µmol, 35%, in 2 steps). LCMS: 22010199G LCMS-5 m / z =462.2 [M+H] + . 1 H NMR (400 MHz, DMSO) 6.05 (s, 1H), 5.75 (s,1H), 5.63 (d, J = 1.1 Hz, 1H), 5.60 (d, J = 1.1 Hz, 1H), 4.93 (s, 2H), 3.72 (s, 3H), 1.40 (s, 9H).

[0212] Example 6 The following compounds were prepared using a method similar to that described in Example 5:

[0213] Example 7 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)tert-butyl acrylate (1-22) Step 1. Follow General experimental procedure 8 Methyl (S)-2-(4-bromo-1-oxoisoindolin-2-yl)-3-hydroxypropionate was prepared. Methyl 3-bromo-2-bromomethylbenzoate (10 g, 32 mmol) and H-Ser-OMe.HCl (5.6 g, 1.1 equivalents, 36 mmol) yielded methyl (S)-2-(4-bromo-1-oxoisoindolin-2-yl)-3-hydroxypropionate (4.4 g, 14 mmol, 44%). LCMS (universal 3 basic): m / z = 313.9 [M+H] + Step 2. Follow General experimental procedure 2 (R)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid was prepared. Methyl (R)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (intermediate 3) (2.0 g, 4.7 mmol) and (4-acetamidophenyl)boronic acid (1.7 g, 2.0 equivalent, 9.4 mmol) were reacted to give (R)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (intermediate 3) (1.3 g, 3.8 mmol, 80%).

[0214] Step 3. Follow General experimental procedure 5 ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine tert-butyl ester was prepared. Crude ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (75 mg, 0.19 mmol) and H-Ser-OtBu.HCl (46 mg, 1.2 equivalents, 0.23 mmol) were obtained and used as is in the next step.

[0215] Step 4. Follow General experimental procedure 6N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine tert-butyl ester was prepared. Crude N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-L-serine tert-butyl ester was obtained and used directly in the next step without purification.

[0216] Step 5. Follow General experimental procedure 7 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)tert-butyl acrylate (1-22) was prepared. N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine tert-butyl acrylate (110 mg, 0.19 mmol) was used to give 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)tert-butyl acrylate 1-22 (16 mg, 33 µmol, 18%, in 3 steps) as a white solid. LCMS: 22010199G LCMS-5 C3 m / z = 462.2 [M+H] + . 1 HNMR (400 MHz, DMSO) δ 10.09 (s, 1H), 9.21 (s, 1H), 7.76 – 7.68 (m, 4H), 7.68– 7.55 (m, 4H), 6.04 (s, 1H), 5.70 – 5.61 (m, 3H), 4.95 (s, 2H), 2.08 (s, 3H), 1.41 (s, 9H), 1.24 (s, 1H).

[0217] Example 8 The following compounds were prepared using a method similar to that described in Example 7.

[0218] Example 9 2-(2-(4-(3-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate 1-53 Step 1. Follow General experimental procedure 6O-acetyl-N-(tert-butoxycarbonyl)-L-serine methyl ester was prepared. Boc-Ser-OMe (20 g, 90 mmol) yielded O-acetyl-N-(tert-butoxycarbonyl)-L-serine methyl ester (23 g, 88 mmol). 1 H NMR (400 MHz, DMSO) δ 7.38 (d, J = 8.2 Hz, 1H), 4.39 – 4.24 (m, 2H), 4.16 –4.05 (m, 1H), 3.65 (s, 3H), 1.99 (s, 3H), 1.39 (s, 9H).

[0219] Step 2. Prepare O-acetyl-L-serine methyl ester hydrochloride using the following procedure. Dissolve O-acetyl-N-(tert-butoxycarbonyl)-L-serine methyl ester (23 g, 88 mmol) in a solution of HCl in dioxane (4 M, 150 mL), stir the resulting mixture for 1 hour, and concentrate the mixture under vacuum to obtain O-acetyl-L-serine methyl ester hydrochloride (17 g, 88 mmol, quantitative) as a white solid. 1 H NMR (400 MHz, DMSO) δ 8.79 (s, 2H), 4.51 – 4.42 (m, 2H), 4.36 (dd, J = 13.0, 5.5 Hz, 1H), 3.77 (s, 3H), 2.04 (s, 3H).

[0220] Step 3. Follow General experimental procedure 5 O-acetyl-N-propynyl-L-serine methyl ester was prepared. O-acetyl-L-serine methyl ester hydrochloride (17 g, 1.1 equivalent, 88 mmol) and propynic acid (5.6 g, 80 mmol) were reacted to give O-acetyl-N-propynyl-L-serine methyl ester (12 g, 53 mmol, 67%). 1 H NMR (400 MHz, DMSO) δ 9.34(d, J = 7.7 Hz, 1H), 4.63 (ddd, J = 7.7, 6.7, 4.3 Hz, 1H), 4.34 (dd, J = 11.4, 4.3Hz, 1H), 4.28 (s, 1H), 4.17 (dd, J = 11.4, 6.7 Hz, 1H), 3.66 (s, 3H), 2.01 (s, 3H).

[0221] Step 4. Follow General experimental procedure 7 2-Propyonamidomethyl acrylate (intermediate 1) was prepared. O-acetyl-N-propynyl-L-serine methyl ester (12 g, 53 mmol) was used to give 2-propynylamidomethyl acrylate (intermediate 1) (4.0 g, 26 mmol, 49%) as a white solid. 1 H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 5.90 (s, 1H), 5.79 (s, 1H), 4.40 (s, 1H), 3.73 (s, 3H).

[0222] Step 5. Follow General experimental procedure 9 Methyl 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylate was prepared. 4-bromoisoindoline-1-one (2.5 g, 12 mmol) and methyl 2-propynamide acrylate (2.0 g, 1.1 equivalent, 13 mmol) were reacted to yield methyl 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylate (2.6 g, 7.1 mmol, 60%) as a white solid. 1 H NMR (400 MHz, DMSO) δ 9.49 (s, 1H), 7.91 (dd, J = 7.9, 0.9 Hz, 1H), 7.77 (dd, J = 7.5, 0.9 Hz, 1H), 7.57 – 7.48 (m, 1H), 6.04 (s, 1H), 5.75 (s, 1H), 5.67 (dd, J = 17.1, 1.1 Hz, 2H), 4.73 (s, 2H), 3.73 (s, 3H).

[0223] Step 6. Follow General experimental procedure 2 Methyl 2-(2-(2-(4-(3-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate (1-53) was prepared. Methyl 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylate (30 mg, 81 µmol) and (3-(tert-butylcarbamoyl)phenyl)boronic acid (27 mg, 1.5 equivalents, 0.12 mmol) were reacted to give methyl 2-(2-(4-(3-(tert-butylcarbamoyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate 1-53 (20 mg, 43 µmol, 54%) as a white solid. LCMS: 22010199G LCMS-5 C3m / z = 462.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.45 (s, 1H), 7.99 (t, J = 1.7Hz, 1H), 7.91 – 7.84 (m, 2H), 7.84 – 7.74 (m, 3H), 7.69 (t, J = 7.5 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 6.05 (s, 1H), 5.75 (s, 1H), 5.64 (d, J = 1.2 Hz, 1H), 5.61 (d, J = 1.2 Hz, 1H), 4.91 (s, 2H), 3.71 (s, 3H), 1.40 (s, 9H).

[0224] Example 10 The following compounds were prepared using a method similar to that described in Example 9.

[0225] Example 11 2-(2-(4-(4-acetamido-2-(trifluoromethyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate (1-33) Step 1. Follow General experimental procedure 9 Methyl 2-(2-(1-oxo-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)isoindoline-2-yl)acrylamido)acrylate was prepared. 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-2,3-dihydro-1H-isoindoline-1-one (0.50 g, 1.9 mmol) and methyl 2-propynamidoacrylate (intermediate 1) (0.33 g, 1.1 equivalents, 2.1 mmol) yielded methyl 2-(2-(1-oxo-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)isoindoline-2-yl)acrylamido)acrylate (0.34 g, 0.82 mmol, 43%).

[0226] Step 2. Follow General experimental procedure 2 Methyl 2-(2-(4-(4-acetamido-2-(trifluoromethyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate (1-33) was prepared. Methyl 2-(2-(1-oxo-4-(4,4,5,5-tetramethyl-1,3,2-dioxoborane-2-yl)isoindoline-2-yl)acrylamido)acrylate (30 mg, 73 µmol) and N-[4-bromo-3-(trifluoromethyl)phenyl]acetamide (31 mg, 1.5 equivalents, 0.11 mmol) were reacted to give methyl 2-(2-(4-(4-acetamido-2-(trifluoromethyl)phenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate (1-33) (19 mg, 40 µmol, 55%) as a white solid. LCMS: 22010199E TFA LCMS-5 C3; m / z = 462.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.43 (s, 1H), 9.48 (s, 1H), 8.20 (d, J = 2.2 Hz, 1H), 7.92 –7.85 (m, 1H), 7.78 (dd, J = 7.6, 1.1 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.50 (dd, J = 14.7, 8.0 Hz, 2H), 6.02 (s, 1H), 5.74 (s, 1H), 5.58 (d, J = 1.3 Hz, 1H), 5.48 (d, J= 1.2 Hz, 1H), 3.71 (s, 3H), 2.11 (s, 3H). 19 F NMR (376 MHz, DMSO) δ -57.05.

[0227] Example 12 The following compounds were prepared using a method similar to that described in Example 11.

[0228] Example 13 2-(2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)acrylamido)methyl acrylate (1-14) Step 1. Follow General experimental procedure 5 Preparation of (2-amino-3-bromobenzoyl)-L-serine methyl ester. 2-amino-3-bromobenzoic acid (1.0 g, 4.6 mmol) and H-Ser-OMe.HCl (0.94 g, 1.3 equivalents, 6.0 mmol) yielded (2-amino-3-bromobenzoyl)-L-serine methyl ester (1.5 g, 4.6 mmol, quantified).

[0229] Step 2. Methyl (S)-2-(8-bromo-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionate was prepared using the following procedure. Trimethyl orthoformate (0.23 mL, 4.5 equivalents, 2.1 mmol) and HCl (59 µL, 0.5 equivalents, 0.24 mmol, in dioxane, 4M) were added to a solution of (2-amino-3-bromobenzoyl)-L-serine methyl ester (0.15 g, 0.47 mmol) in NMP (5 mL). The resulting mixture was stirred at 110 °C for 120 min. The mixture was cooled to room temperature and neutralized by adding NaHCO3 (saturated aqueous solution). The mixture was extracted with EtOAc (3 × 15 mL). The combined organic matter was washed with water (3 × 10 mL) and brine (10 mL), dried over Na₂SO₄, filtered, and concentrated to obtain crude (S)-2-(8-bromo-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionate methyl ester (0.20 g, quantitative). LCMS (Generally Acidic 3): m / z = 327.2 [M+H] + Step 3. Follow General experimental procedure 2(S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionic acid was prepared. Methyl (S)-2-(8-bromo-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionic acid (0.20 g, 0.68 mmol) and (4-acetamidophenyl)boronic acid (0.24 g, 2 equivalents, 1.4 mmol) were used to obtain crude (S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionic acid (0.68 mmol), which was used in the next step without purification.

[0230] Step 4. Follow General experimental procedure 5 ((S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionyl)-L-serine methyl ester was prepared. Crude (S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionic acid (0.68 mmol) and H-Ser-OMe.HCl (0.12 g, 1.1 equivalents, 0.75 mmol) were reacted to obtain crude ((S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionyl)-L-serine methyl ester (0.68 mmol), which was used in the next step without purification.

[0231] Step 5. Follow General experimental procedure 6 N-((S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-acetoxypropionyl)-O-acetyl-L-serine methyl ester was prepared. Crude ((S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionyl)-L-serine methyl ester (0.68 mmol) was used to obtain N-((S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-acetoxypropionyl)-O-acetyl-L-serine methyl ester (38 mg, 69 µmol, 10%, obtained in 3 steps). Step 6. Follow General experimental procedure 7Methyl 2-(2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)acrylamido)acrylate (8-14) was prepared. N-((S)-2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)-3-acetoxypropionyl)-O-acetyl-L-serine methyl ester (38 mg, 69 µmol) yielded methyl 2-(2-(8-(4-acetamidophenyl)-4-oxoquinazoline-3(4H)-yl)acrylamido)acrylate (1-14) (19 mg, 44 µmol, 64%) as a white solid. LCMS: 22010199G LCMS-5 C3 m / z = 433.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.04 (s, 1H), 9.82 (s, 1H), 8.24 (s, 1H), 8.15 (dd, J = 7.9, 1.6 Hz, 1H), 7.87 (dd, J = 7.5, 1.6 Hz, 1H), 7.70 – 7.60 (m, 3H), 7.58 – 7.51(m, 2H), 6.39 (d, J = 1.8 Hz, 1H), 6.16 (d, J = 1.8 Hz, 1H), 5.86 (s, 1H), 5.74 (s, 1H), 3.72 (s, 3H), 2.08 (s, 3H).

[0232] Example 14 The following compounds were prepared using a method similar to that described in Example 13.

[0233] Example 15 2-(2-(8-(4-((tert-butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)prop enoylamino)prop-2-enoic acid (1-304) Step 1. Prepare (2-amino-3-bromobenzoyl)-L-serine methyl ester following General Experimental Procedure 5. 2-Amino-3-bromobenzoic acid (1.0 g, 4.6 mmol) and H-Ser-OMe.HCl (0.94 g, 1.3 equivalents, 6.0 mmol) yielded (2-amino-3-bromobenzoyl)-L-serine methyl ester (1.5 g, 4.6 mmol, quantified).

[0234] Step 2. Methyl (S)-2-(8-bromo-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionate was prepared using the following procedure. Paraformaldehyde (92 mg, 2 equivalents, 3.1 mmol) and aluminum trichloride (20 mg, 0.1 equivalents, 0.15 mmol) were added to a solution of (2-amino-3-bromobenzoyl)-L-serine methyl ester (0.48 g, 1.52 mmol) in EtOH (40 mL), and the resulting mixture was stirred overnight at 85 °C. The mixture was concentrated and dissolved in a mixture of water and EtOAc. The layers were separated, the organic layer was washed with brine, dried with Na2SO4, filtered and concentrated to obtain crude (S)-2-(8-bromo-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionate methyl ester (0.37 g, 74% purity, 0.84 mmol), which was used as is in the next step.

[0235] Step 3. Prepare (S)-2-(8-(4-((tert-Butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)-3-hydroxypropionic acid following General Experimental Procedure 2. Crude methyl (S)-2-(8-bromo-4-oxoquinazoline-3(4H)-yl)-3-hydroxypropionic acid (0.37 g, 74% purity, 0.84 mmol) and [4-(tert-Butoxycarbonyl)amino)phenyl]boronic acid (0.40 g, 2 equivalents, 1.7 mmol) yield (S)-2-(8-(4-((tert-Butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)-3-hydroxypropionic acid (0.26 g, 90% purity, 0.56 mmol, 66%).

[0236] Step 4. Prepare ((S)-2-(8-(4-((tert-butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)-3-hydroxypropionyl)-L-serine methyl ester following general experimental procedure 5. (S)-2-(8-(4-((tert-Butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)-3-hydroxypropionic acid (0.26 g, 90% purity, 0.56 mmol) and H-Ser-OMe·HCl (0.11 g, 1.2 equivalents, 0.67 mmol) yielded ((S)-2-(8-(4-((tert-Butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)-3-hydroxypropionyl)-L-serine methyl ester (0.26 g, 0.48 mmol, 85%). LCMS (General 3 acidic): m / z = 529.6 [M+H] + Step 5. Prepare N-((S)-3-acetoxy-2-(8-(4-((tert-butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)propionyl)-O-acetyl-L-serine methyl ester following general experimental procedure 6. ((S)-2-(8-(4-((tert-Butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)-3-hydroxypropionyl)-L-serine methyl ester (0.10 g, 0.18 mmol) yielded N-((S)-3-acetoxy-2-(8-(4-((tert-Butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)propionyl)-O-acetyl-L-serine methyl ester (0.12 g, 89% purity, 0.17 mmol, 92%).

[0237] Step 6. Follow General experimental procedure 7 Methyl 2-(2-(8-(4-((tert-butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)acrylamido)acrylate 8-304 was prepared. N-((S)-3-acetoxy-2-(8-(4-((tert-butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)propionyl)-O-acetyl-L-serine methyl ester (0.12 g, 89% purity, 0.17 mmol) was used to obtain methyl 2-(2-(8-(4-((tert-butoxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazoline-3(2H)-yl)acrylamido)acrylate 1-304 (34 mg, 69 µmol, 41%) as a white solid. LCMS: 22010199D TFA LCMS-5 C3; m / z = 493.2 [M+H] + . 1 H NMR (400MHz, DMSO) δ 9.47 (s, 1H), 9.13 (s, 1H), 7.69 (dd, J = 7.8, 1.6 Hz, 1H), 7.60 –7.50 (m, 2H), 7.38 – 7.31 (m, 2H), 7.31 – 7.25 (m, 1H), 6.87 (td, J = 7.6, 1.2Hz, 1H), 6.11 (d, J = 1.0 Hz, 1H), 5.71 (d, J = 2.3 Hz, 2H), 5.46 (d, J = 1.4 Hz, 1H), 4.69 (d,J = 3.7 Hz, 2H), 3.72 (s, 3H), 1.49 (s, 9H).

[0238] Example 16 2-(2-(8-(4-((tert-butyloxycarbonyl)amino)phenyl)-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)acrylamido)methyl acrylate (1-10) Step 1. Follow General experimental procedure 9 2-(5-bromo-1-oxoisoquinoline-2(1H)-yl)tert-butyl acrylate was prepared. 5-bromo-1(2H)-isoquinolineone (50 mg, 0.22 mmol) and tert-butyl propynate (31 µL, 0.22 mmol) were reacted to yield 2-(5-bromo-1-oxoisoquinoline-2(1H)-yl)tert-butyl acrylate (36 mg, 0.10 mmol, 47%). 1 H NMR (400MHz, DMSO) δ 8.23 ​​(dt, J = 8.0, 1.0 Hz, 1H), 8.09 (dd, J = 7.8, 1.2 Hz, 1H), 7.57– 7.37 (m, 3H), 6.78 (dd, J = 7.6, 0.8 Hz, 1H), 6.31 (d, J = 1.2 Hz, 1H), 6.06(d, J = 1.3 Hz, 1H), 1.42 (s, 9H).

[0239] Step 2. Follow General experimental procedure 2 2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)tert-butyl acrylate was prepared. 2-(5-bromo-1-oxoisoquinoline-2(1H)-yl)tert-butyl acrylate (0.32 g, 0.92 mmol) and 4-acetamidophenylboronic acid (0.25 g, 1.5 equivalents, 1.4 mmol) were reacted to yield 2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)tert-butyl acrylate (0.33 g, 0.78 mmol, 84%).

[0240] Step 3. Prepare 2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acrylic acid using the following procedure. Add HCl (4M in dioxane, 2 mL) to a solution of tert-butyl 2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acrylic acid (0.33 g, 0.77 mmol) in dioxane (15 mL), and stir the resulting mixture at room temperature for 16 hours. Concentrate the reaction mixture under vacuum, and add water (20 mL). Extract the aqueous layer with EtOAc (3 × 20 mL), and wash the combined organic layers with brine (40 mL), dry to Na₂SO₄, filter, and concentrate. Purify the crude material by automated reverse-phase FCC to obtain 2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acrylic acid (64 mg, 0.18 mmol, 24%).

[0241] Step 4. Follow General experimental procedure 5 N-(2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acryloyl)-O-acetyl-L-serine methyl ester was prepared. 2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acrylic acid (50 mg, 0.14 mmol) and O-acetyl-L-serine methyl ester hydrochloride (31 mg, 0.16 mmol) yielded N-(2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acryloyl)-O-acetyl-L-serine methyl ester (29 mg, 59 µmol, 42%).

[0242] Step 5. Follow General experimental procedure 7 Methyl 2-(2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acrylamido)acrylate was prepared. N-(2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acryloyl)-O-acetyl-L-serine methyl ester (28 mg, 57 µmol) yielded methyl 2-(2-(5-(4-acetamidophenyl)-1-oxoisoquinoline-2(1H)-yl)acrylamido)acrylate (17 mg, 39 µmol, 69%) as a white solid. LCMS: 22010199 LCMS-5 C3 RT: m / z = 432.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.13(s, 1H), 9.61 (s, 1H), 8.21 (dd, J= 7.9, 1.4 Hz, 1H), 7.77 – 7.70 (m, 2H), 7.67 (dd, J = 7.4, 1.5 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.41 – 7.31 (m, 3H), 6.52 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 1.9 Hz, 1H), 5.96 (d, J = 1.9 Hz, 1H), 5.92(s, 1H), 5.72 (s, 1H), 3.72 (s, 3H), 2.09 (s, 3H).

[0243] Example 17 2-(2-(1-(4-acetamidophenyl)-4-oxo-4H-thieno[3,4-c]pyrrole-5(6H)-yl)acrylamido)methyl acrylate (1-25) Step 1. Methyl 5-bromo-4-(bromomethyl)thiophene-3-carboxylate was prepared using the following procedure. NBS (1.6 g, 2.2 equivalents, 9.1 mmol) and AIBN (80 mg, 0.1 equivalents, 0.41 mmol) were added to a solution of methyl 4-methylthiophene-3-carboxylate (0.65 g, 4.1 mmol) in MeCN (15 mL), and the resulting mixture was stirred under reflux for 3 hours. The reaction mixture was concentrated under vacuum, and the crude product was purified by automated FCC to give methyl 5-bromo-4-(bromomethyl)thiophene-3-carboxylate (0.96 g, 3.1 mmol, 74%).

[0244] Step 2. Follow General experimental procedure 8 1-Bromo-5,6-dihydro-4H-thieno[3,4-c]pyrrolo-4-one was prepared. Methyl 5-bromo-4-(bromomethyl)thiophene-3-carboxylate (0.3 g, 1.0 mmol) and ammonia (7 M MeOH solution, 5.46 mL, 40 equivalents, 38 mmol) yielded crude 1-bromo-5,6-dihydro-4H-thieno[3,4-c]pyrrolo-4-one, which was used directly in the next step without purification.

[0245] Step 3. Follow General experimental procedure 9Methyl 2-(2-(1-bromo-4-oxo-4H-thieno[3,4-c]pyrrolo-5(6H)-yl)acrylamido)acrylate was prepared. 1-bromo-5,6-dihydro-4H-thieno[3,4-c]pyrrolo-4-one (0.16 g, 0.74 mmol) and methyl 2-propynamide acrylate (intermediate 1) (0.14 g, 1.2 equivalents, 0.89 mmol) yielded crude methyl 2-(2-(1-bromo-4-oxo-4H-thieno[3,4-c]pyrrolo-5(6H)-yl)acrylamido)acrylate (35 mg), which was used as is in the next step.

[0246] Step 4. Follow General experimental procedure 1 Methyl 2-(2-(1-(4-acetamidophenyl)-4-oxo-4H-thieno[3,4-c]pyrrolo-5(6H)-yl)acrylamido)acrylate (1-25). Crude methyl 2-(2-(1-bromo-4-oxo-4H-thieno[3,4-c]pyrrolo-5(6H)-yl)acrylamido)acrylate (35 mg) and (4-acetamidophenyl)boronic acid (34 mg, 2.0 equivalent, 0.19 mmol) were used to obtain a white solid of methyl 2-(2-(1-(4-acetamidophenyl)-4-oxo-4H-thieno[3,4-c]pyrrolo-5(6H)-yl)acrylamido)acrylate (1.5 mg, 3.5 µmol, 3.8%). SFC: 22010199H SFC-2; m / z = 426.1 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 9.45 (s, 1H), 8.04 (s, 1H), 7.74 – 7.66 (m, 2H), 7.59 – 7.49 (m, 2H), 6.07 (s, 1H), 5.74 (s, 1H), 5.55 (dd, J = 9.7, 1.2 Hz, 2H), 4.91 (s, 2H), 3.73 (s, 3H), 2.06 (s, 3H).

[0247] Example 18 The following compounds were prepared using a method similar to that described in Example 17.

[0248] Example 19 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,2,4-oxadiazol-3-yl)vinyl)acrylamide (1-59) Step 1. Prepare N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine methyl ester using the following procedure. Add imidazole (6.6 g, 2.5 equivalents, 96 mmol) to a solution of Boc-Ser-OMe (8.5 g, 39 mmol) in THF (200 mL), then slowly add TBDPS-Cl (11 mL, 1.1 equivalents, 42 mmol), and stir the resulting mixture at room temperature for 24 hours. Filter the reaction mixture through diatomaceous earth and concentrate the filtrate. Dissolve the crude material in CH2Cl2 (200 mL) and wash three times with HCl (1M aqueous solution, 100 mL). Extract the aqueous phase with CH2Cl2 (100 mL), and dry the combined organic matter to Na2SO4, filter, and concentrate to obtain crude N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine methyl ester, which is used as is in the next step.

[0249] Step 2. Follow General experimental procedure 4 Preparation of N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine (intermediate 4). Crude N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine methyl ester (2.8 g, approximately 6.0 mmol) was used to obtain N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine (intermediate 4) (2.0 g, 90% purity, 4.1 mmol, 67%) as a viscous, colorless oil.

[0250] Step 3. Prepare (E)-N'-hydroxyacetylimine using the following procedure. Add hydroxylamine solution (50% wt. aqueous solution, 6.0 mL, 4 equivalents, 97 mmol) to a mixture of acetonitrile (1.3 mL, 24 mmol) and EtOH (10 mL), and reflux the resulting mixture with stirring overnight. Remove the solvent under vacuum to obtain crude (E)-N'-hydroxyacetylimine as a white solid, which is used as is in the next step.

[0251] Step 4. Prepare (S)-(2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl)carbamate tert-butyl ester using the following procedure. Add (Z)-N'-hydroxyacetylimine (75 mg, 2.5 equivalents, 1.0 mmol) and DCC (92 mg, 1.1 equivalents, 0.45 mmol) to a solution of N-(tert-butoxycarbonyl)-O-(tert-butyldiphenylsilyl)-L-serine (0.18 g, 0.41 mmol) in MeCN (7.0 mL). Reflux the resulting mixture under a nitrogen atmosphere for 36 hours. The reaction mixture was concentrated under vacuum and the crude material was purified by automated reverse-phase FCC to obtain (S)-(2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl)carbamate tert-butyl ester (63 mg, 0.13 mmol, 32%).

[0252] Step 5. Prepare (S)-2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl-1-amine hydrochloride (intermediate 2) using the following procedure. Add HCl solution (4M dioxane solution, 5 equivalents, 2.7 mmol) to a solution of (S)-2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl)carbamate tert-butyl ester (0.37 g, 0.53 mmol) in CH2Cl2 (10 mL), and stir the resulting mixture at room temperature for 2 hours. Concentrate the resulting mixture under vacuum and use it for the next step without purification.

[0253] Step 6. Follow General experimental procedure 5 Preparation of (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-((R)-2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl)-3-hydroxypropionamide. R (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-((R)-2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,2,4-oxadiazol-5-yl)ethyl)carbamate (35 mg, 99 µmol) and (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (prepared as in Example 7, 35 mg, 99 µmol) yielded (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-((R)-2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl)-3-hydroxypropionamide (29 mg, 40 µmol, 40%) as a white solid.

[0254] Step 7. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,2,4-oxadiazol-3-yl)vinyl)acrylamide (1-59) using the following procedure. Add TBAF (1M in THF, 44 µL, 1.1 equivalents, 44 µmol) to a solution of (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-((R)-2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl)-3-hydroxypropionamide (29 mg, 40 µmol) in CH2Cl2 (5 mL), and stir the resulting mixture at room temperature for 1 hour. After complete conversion, as monitored by LCMS, acetic anhydride (11 µL, 2.8 equivalences, 0.11 mmol) and triethylamine (31 µL, 5.6 equivalences, 0.22 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After complete conversion, as monitored by LCMS, DBU (24 µL, 4 equivalences, 0.16 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with TFA (12 µL, 4 equivalences, 0.16 µmol), and the mixture was concentrated under vacuum. The crude product was purified by automated reverse-phase FCC to give 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,2,4-oxadiazol-3-yl)vinyl)acrylamide (2.0 mg, 4.5 µmol, 11%) as a white solid. LCMS: 22010199G LCMS-5 C3 / z =444.4 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.12 – 10.05 (m, 2H), 7.76 – 7.70 (m,4H), 7.66 – 7.56 (m, 3H), 5.96 (s, 1H), 5.91 (s, 1H), 5.68 (s, 1H), 5.64 (s,1H), 4.94 (s, 2H), 2.33 (s, 3H), 2.08 (s, 3H).

[0255] Example 20 The following compounds were prepared using a method similar to that described in Example 19.

[0256] Example 21 2-(2-(4-cyclohexyl-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate 1-147 Step 1. Follow General experimental procedure 8 Methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate was prepared. Methyl 3-bromo-2-bromomethylbenzoate (10 g, 33 mmol) and L-serine methyl ester hydrochloride (5.6 g, 1.1 equivalent, 36 mmol) were reacted to give methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionate (7.5 g, 23 mmol, 70%). 1 H NMR (400 MHz, DMSO) δ 7.87 (dd, J = 7.9, 0.9 Hz, 1H), 7.76 (dd, J = 7.5,0.9 Hz, 1H), 7.55 – 7.46 (m, 1H), 5.32 – 5.27 (m, 1H), 5.01 – 4.93 (m, 1H), 4.58 (d, J = 17.9 Hz, 1H), 4.50 (d, J = 17.8 Hz, 1H), 4.05 (dt, J = 11.9, 5.5 Hz, 1H), 3.91 (dt, J = 11.7, 4.3 Hz, 1H), 3.67 (s, 3H).

[0257] Step 2. Follow General experimental procedure 4 (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid was prepared. Methyl (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (2.0 g, 6.4 mmol) was used to give (S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (1.5 g, 5.0 mmol, 79%). Step 3. Follow General experimental procedure 5 (S)-2-(4-bromo-1-oxoisoindolin-2-yl)-3-hydroxypropionyl)-L-serine methyl ester was prepared. (S)-2-(4-bromo-1-oxoisoindolin-2-yl)-3-hydroxypropionic acid (1.8 g, 6.0 mmol) and L-serine methyl ester hydrochloride (1.1 g, 1.2 equivalents, 7.2 mmol) were used to obtain (( S2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester (2.2 g, 5.2 mmol, 86%).

[0258] Step 4. Follow General experimental procedure 6 N-((S)-3-acetoxy-2-(4-bromo-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester was prepared. ((S)-2-(4-bromo-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester (0.30 g, 0.75 mmol) yielded N-((S)-3-acetoxy-2-(4-bromo-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (0.33 g, 90% purity, 0.61 mmol, 81%).

[0259] Step 5. Follow General experimental procedure 3 Preparation of N-((S)-3-acetoxy-2-(4-(cyclohex-1-en-1-yl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester. N -(( S N-((S)-3-acetoxy-2-(4-(cyclohex-1-en-1-yl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (0.26 g, 0.32 mmol) and 2-(cyclohex-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (97 mg, 1.5 equivalents, 0.47 mmol) were reacted to yield N-((S)-3-acetoxy-2-(4-(cyclohex-1-en-1-yl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (33 mg, 68 µmol, 21%).

[0260] Step 6. Prepare O-acetyl-N-((S)-2-(4-cyclohexyl-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine methyl ester using the following procedure. Add Pd / C (10% Wt. 20 mg, 0.28 equivalent, 19 µmol) to a solution of N-((S)-3-acetoxy-2-(4-(cyclohexyl-1-en-1-yl)-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (33 mg, 68 µmol) in MeOH (2 mL), and evacuate the flask, refilling it three times with hydrogen. Stir the reaction mixture under a hydrogen atmosphere (balloon) for 5 hours. Filter the mixture through diatomaceous earth and wash the filter cake with MeOH. The filtrate was concentrated to obtain N-((S)-3-acetoxy-2-(4-cyclohexyl-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (34 mg, 87% purity, 61 µmol, 90%), which could be used in the next step without further purification.

[0261] Step 7. Follow General experimental procedure 7 2-(2-(4-cyclohexyl-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate 1-147 was prepared. N-((S)-3-acetoxy-2-(4-cyclohexyl-1-oxoisoindoline-2-yl)propionyl)-O-acetyl-L-serine methyl ester (34 mg, 87% purity, 61 µmol) was used to obtain 2-(2-(4-cyclohexyl-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate 1-147 (10 mg, 27 µmol, 45%) as a white solid. LCMS: 22010199G LCMS-5 C3 m / z = 369.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.44 (s, 1H),7.59 – 7.45 (m, 3H), 6.05 (s, 1H), 5.74 (s, 1H), 5.63 (d, J = 1.1 Hz, 1H), 5.56(d, J = 1.1 Hz, 1H), 4.82 (s, 2H), 3.73 (s, 3H), 2.64 (d, J = 11.5 Hz, 1H), 1.82(d, J = 11.1 Hz, 4H), 1.74 (d, J = 12.4 Hz, 1H), 1.57 – 1.22 (m, 6H).

[0262] Example 22 N,N-dimethyl-2-(2-(4-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1-oxoisoindolin- 2-yl)propenoylamino)prop-2-enoic acid m / z 1-305 Step 1. Prepare 2-(2-(4-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1-oxoisoindoline-2-yl)acrylamido)acrylic acid following general experimental procedure 2. 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)methyl acrylate (0.20 g, 0.49 mmol) and 4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-boronic acid pinacol ester (0.17 g, 0.54 mmol) yielded 2-(2-(4-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-yl)-1-oxoisoindoline-2-yl)acrylamido)acrylic acid (90 mg, 0.20 mmol, 40%) as a white, fluffy solid.

[0263] Step 2. Prepare N,N-dimethyl-2-(2-(4-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1-oxoisoindoline-2-yl)acrylamido)acrylamide (1-305) using the following procedure. Add DiPEA (31 μL, 4 equivalents, 0.18 mmol) and propylphosphonic anhydride (50% wt. EtOAc solution) (26 μL, 45 mmol) to an ice-cold suspension of 2-(2-(4-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1-oxoisoindoline-2-yl)acrylamido)acrylic acid (20 mg, 45 μmol) in DMSO (1 mL) to an ice-cold suspension. Stir the resulting mixture at 0 °C for 10 min. DMSO (0.3 mL) containing dimethylamine solution (2.0 M THF solution) (22 μL, 45 mmol) was added, and the resulting mixture was stirred at room temperature for 4 hours. The resulting mixture was directly purified by automated reverse-phase FCC to give N,N-dimethyl-2-(2-(4-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1-oxoisoindololin-2-yl)acrylamido)acrylamide (1-305) (5.0 mg, 11 µmol, 24%) as a white, fluffy solid. LCMS: 22010199G LCMS-5 C3 2-(2-(4-morpholin-1-oxoisoindolin-2-yl)propenoylamino)prop-2-enoic acid = 461.2 [M+H] + .1 H NMR (400 MHz, MeOD) δ 7.70(dd, J = 7.5, 1.2 Hz, 1H), 7.61 (dd, J = 7.7, 1.2 Hz, 1H), 7.53 (t, J = 7.6 Hz,1H), 7.23 – 7.16 (m, 2H), 7.15 – 7.12 (m, 1H), 5.65 (d, J = 1.5 Hz, 1H), 5.56(d, J = 1.5 Hz, 1H), 5.20 (d, J = 0.9 Hz, 1H), 4.79 (s, 2H), 4.70 (d, J = 0.9 Hz,1H), 4.60 – 4.57 (m, 2H), 3.32 (s, 3H), 3.04 (s, 3H), 2.87 (s, 3H).

[0264] Example 23 The following compounds were prepared using a method similar to that described in Example 22.

[0265] Example 24 m / z 1-157 Step 1. Prepare 4-morpholinoisoindoline-1-one using the following procedure. Molecular sieves were added to a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)isoindoline-1-one (0.10 g, 0.39 mmol), copper acetate (98 mg, 1.4 equivalents, 0.54 mmol), and pyridine (86 mg, 2.8 equivalents, 1.1 mmol) in MeCN (7 mL), followed by the addition of morpholine (67 µL, 2 equivalents, 0.77 mmol), and the resulting mixture was stirred at 85 °C for 2 hours. The resulting mixture was filtered through diatomaceous earth and washed with MeCN and CH2Cl2. The filtrate was concentrated under vacuum and purified using an automated FCC to give 4-morpholinoisoindoline-1-one (20 mg, 0.39 mmol, 24%) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 8.58(s, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.29 (dd, J = 7.4, 0.9 Hz, 1H), 7.14 (dd, J =7.9, 1.0 Hz, 1H), 4.39 (s, 2H), 3.78 – 3.71 (m, 4H), 3.09 – 3.03 (m, 4H).

[0266] Step 2. Prepare methyl 2-(2-(4-morpholino-1-oxoisoindoline-2-yl)acrylamido)acrylate (1-157) following general experimental procedure 9. 4-morpholinoisoindoline-1-one (28 mg, 0.13 mmol) and intermediate 1 (20 mg, 0.13 mmol) yielded methyl 2-(2-(4-morpholino-1-oxoisoindoline-2-yl)acrylamido)acrylate (24 mg, 65 µmol, 50%) as a white solid. LCMS: 22010199G LCMS-5 C3 m / z = 372.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.37 (s, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 7.4 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.07 (s, 1H), 5.75 (s, 1H), 5.66 (s, 1H), 5.61(s, 1H), 4.81 (s, 2H), 3.80 – 3.75 (m, 4H), 3.74 (s, 3H), 3.13 – 3.06 (m,4H).

[0267] Example 25 The following compounds were prepared using a method similar to that described in Example 24.

[0268] Example 26 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)neopentyl acrylate 1-274 Step 1. Prepare N-(((9H-fluorene-9-yl)methoxy)carbonyl)-O-(tert-butyl)-L-serine neopentyl ester using the following procedure. Add 2,2-dimethylpropane-1-ol (0.35 g, 3 equivalents, 3.9 mmol), DCC (0.32 g, 1.2 equivalents, 1.6 mmol), and DMAP (8.0 mg, 0.05 equivalents, 65 µmol) to a solution of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-O-(tert-butyl)-L-serine (0.50 g, 1.3 mmol) in CH2Cl2 (30 mL), and stir the resulting mixture overnight at room temperature. The mixture was filtered, and the filtrate was washed with water, dried over Na2SO4, filtered, and concentrated to obtain crude N-(((9H-fluorene-9-yl)methoxy)carbonyl)-O-(tert-butyl)-L-serine neopentyl ester (0.64 g, approximately 1.3 mmol, quantified).

[0269] Step 2. Prepare (((9H-fluorene-9-yl)methoxy)carbonyl)-L-serine neopentyl ester using the following procedure. Add TFA (2 mL) to an ice-cold solution of crude (((9H-fluorene-9-yl)methoxy)carbonyl)-L-serine neopentyl ester (0.59 g, 1.3 mmol) in CH2Cl2 (30 mL), and stir the resulting mixture at 50 °C for 16 hours. Concentrate the mixture under vacuum, and dissolve the residue in EtOAc (30 mL). Wash the mixture with NaHCO3 (saturated aqueous solution, 30 mL) and water (30 mL), dry it with Na2SO4, filter and concentrate to obtain (((9H-fluorene-9-yl)methoxy)carbonyl)-L-serine neopentyl ester (0.23 g, 0.57 mmol, 44%, after 2 steps). 1 H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 7.5 Hz, 2H), 7.53(d, J = 7.5 Hz, 2H), 7.33 (td, J = 7.5, 1.1 Hz, 2H), 7.24 (tt, J = 7.4, 1.3 Hz, 2H), 5.65 (d, J = 7.5 Hz, 1H), 4.44 – 4.30 (m, 3H), 4.15 (t, J = 6.9 Hz, 1H), 3.99 – 3.83 (m, 3H), 3.81 – 3.74 (m, 1H), 0.87 (s, 9H).

[0270] Step 3. Prepare L-serine neopentyl ester using the following procedure. Add Pd / C (25 mg, 10% wt., 0.05 equivalent, 24 µmol) to a solution of (((9H-fluorene-9-yl)methoxy)carbonyl)-L-serine neopentyl ester (0.19 g, 0.47 mmol) in MeOH (10 mL) and MeCN (0.13 mL), and stir the mixture under a hydrogen atmosphere for 16 hours. Filter the mixture through diatomaceous earth and concentrate under vacuum to obtain crude L-serine neopentyl ester, which can be used in the next step without purification.

[0271] Step 4. Prepare ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine neopentyl ester according to general experimental procedure 5. (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (intermediate 3) (0.17 g, 0.47 mmol) and L-serine neopentyl ester (82 mg, 0.47 mmol) to obtain ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine neopentyl ester (27 mg, 53 µmol, 11%, via 2 steps).

[0272] Step 5. Prepare N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine neopentyl ester following general experimental procedure 6. S Crude N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine neopentyl ester (27 mg, 53 µmol) was obtained from 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine neopentyl ester (29 mg), which was used as is in the next step.

[0273] Step 6. Prepare 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)neopentyl acrylate (1-274) following general experimental procedure 7. Crude N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine ester (53 µmol) yielded 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)neopentyl acrylate (1-247) (8.0 mg, 17 µmol, 37%). LCMS: 22010199G LCMS-5 C3m / z = 476.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ10.12 (s, 1H), 9.38 (s, 1H), 7.78 – 7.69 (m, 4H), 7.69 – 7.55 (m, 3H), 6.08 (s, 1H), 5.80 (s, 1H), 5.67 (dd, J = 7.6, 1.0 Hz, 2H), 4.95 (s, 2H), 3.83 (s, 2H), 2.09 (s, 3H), 0.89 (s, 9H).

[0274] Example 27 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)2,4-dimethylpentane-3-yl acrylate 1-291 Step 1. Prepare N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine 2,4-dimethylpentan-3-yl ester using the following procedure. Add 2,4-dimethylpentan-3-ol (0.48 g, 1.2 equivalents, 1.1 mmol), DCC (0.85 g, 1.25 equivalents, 4.1 mmol), and DMAP (20 mg, 0.05 equivalents, 0.17 mmol) to a solution of N-(tert-Butoxycarbonyl)-O-(tert-Butyldiphenylsilyl)-L-serine (intermediate 4) (1.5 g, 3.3 mmol) in CH₂Cl₂ (100 mL), and stir the resulting mixture for 60 hours. Filter the mixture, wash with water, dry over Na₂SO₄, filter, and concentrate. Dissolve the residue in TBME, and remove the remaining solids by filtration. The filtrate was washed with water, dried over Na2SO4, filtered, and concentrated to obtain N-(tert-butyloxycarbonyl)-O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentyl-3-yl ester (1.5 g, 2.8 mmol, 85%), which could be used in the next step without further purification.

[0275] Step 2. Prepare O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentane-3-yl ester hydrochloride using the following procedure. Add a solution of HCl in dioxane (4M, 7 mL, 10 equivalents, 28 mmol) to a solution of N-(tert-butyloxycarbonyl)-O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentane-3-yl ester (1.5 g, 2.8 mmol) in dioxane (15 mL), and stir the resulting mixture at room temperature for 16 hours. Concentrate the mixture and co-evaporate it twice with toluene to obtain crude O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentane-3-yl ester hydrochloride (assumed to be 2.8 mmol), which is used as is in the next step.

[0276] Step 3. Prepare N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentyl-3-yl ester by following general experimental procedure 5. Crude (S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (0.42 mmol) and O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentan-3-yl ester hydrochloride (intermediate 3) (0.15 g, 0.42 mmol) were reacted to yield N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentan-3-yl ester (86 mg, 0.11 mmol, 26%).

[0277] Step 4. Prepare ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine 2,4-dimethylpentan-3-yl ester using the following procedure. Add a solution of TBAF in THF (1M, 0.17 mL, 1.5 equivalents, 0.17 mmol) to a solution of N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-O-(tert-butyldiphenylsilyl)-L-serine 2,4-dimethylpentan-3-yl ester (86 mg, 0.11 mmol) in THF (4 mL), and stir the resulting mixture at room temperature for 16 hours. Concentrate the mixture and dissolve the residue in EtOAc. Wash the mixture with water, dry it over Na2SO4, filter, and concentrate. The crude material was purified by automated reversed-phase FCC to obtain ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine 2,4-dimethylpentane-3-yl ester (18 mg, 33 µmol, 30%). 1 H NMR (400 MHz, MeOD) δ 7.69 (dd, J =7.4, 1.3 Hz, 1H), 7.64 – 7.46 (m, 4H), 7.45 – 7.37 (m, 2H), 4.99 (dd, J = 8.1,5.4 Hz, 1H), 4.81 – 4.59 (m, 2H), 4.50 (t, J = 6.1 Hz, 1H), 4.43 (dd, J = 5.1,3.9 Hz, 1H), 4.05 – 3.87 (m, 2H), 3.87 – 3.73 (m, 2H), 2.05 (s, 3H), 1.86 –1.72 (m, 2H), 0.82 – 0.69 (m, 12H).

[0278] Step 5. Following general experimental procedure 6, prepare N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine 2,4-dimethylpentane-3-yl ester. ((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionyl)-L-serine 2,4-dimethylpentane-3-yl ester (20 mg, 37 µmol) yields N-((S)-2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-acetoxypropionyl)-O-acetyl-L-serine 2,4-dimethylpentane-3-yl ester (21 mg, 32 µmol, 86%), which is used as is in the next step.

[0279] Step 6. Prepare 2,4-dimethylpentane-3-yl 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate according to general experimental procedure 7. 2,4-dimethylpentane-3-yl 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate (21 mg, 32 µmol) yielded a white, fluffy solid of 2,4-dimethylpentane-3-yl 2-(2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)acrylamido)acrylate (5.5 mg, 11 µmol, 34%). LCMS: 22010199G LCMS-5 C3 m / z = 504.2 [M+H] + . 1 HNMR (400 MHz, DMSO) δ 10.11 (s, 1H), 9.31 (s, 1H), 7.77 – 7.69 (m, 4H), 7.69– 7.61 (m, 1H), 7.61 – 7.54 (m, 2H), 6.11 (s, 1H), 5.82 (s, 1H), 5.67 (t, J =1.4 Hz, 2H), 4.95 (s, 2H), 4.55 (t, J = 6.1 Hz, 1H), 2.09 (s, 3H), 1.86 (h, J =6.7 Hz, 2H), 0.80 (dd, J = 19.5, 6.7 Hz, 12H).

[0280] Example 28 2-(2-(3-(4-acetamidophenyl)-8-oxoimidazo[1,5-a]pyrazin-7(8H)-yl)acrylamido)methyl acrylate 1-285 Step 1. Prepare ethyl 1-(2,2-diethoxyethyl)-1H-imidazolium-5-carboxylate using the following procedure. Sodium hydride (8.0 g, 60% wt, 2 equivalents, 200 mmol) was added to a mixture of ethyl imidazolium-4-carboxylate (14 g, 100 mmol) and 2-bromo-1,1-diethoxyethane (17 mL, 1.2 equivalents, 120 mmol) in DMF (50 mL), and the resulting mixture was stirred at 110 °C for 4 hours. The reaction mixture was quenched by adding saturated ammonium chloride aqueous solution (150 mL) and diluted with EtOAc / heptane (50 mL / 50 mL). The organic layer was separated and washed with water (2 × 75 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography using (PE / EtOAc: 100 / 00 to 40 / 60) as eluent to give ethyl 1-(2,2-diethoxyethyl)-1H-imidazolium-5-carboxylate (2.1 g, 8.2 mmol, 8.2%). LC-MS (universal 3, basic): m / z = 257.2 [M+H] + Step 2. Prepare ethyl 2-bromo-1-(2,2-diethoxyethyl)-1H-imidazolium-5-carboxylate using the following procedure. Add 2-[2-(1-cyano-1-methylethyl)diazo-1-yl]-2-methylpropionitrile (0.24 mL, 0.2 equivalents, 1.6 mmol) to a solution of ethyl 1-(2,2-diethoxyethyl)-1H-imidazolium-5-carboxylate (2.1 g, 8.00 mmol) and N-bromosuccinimide (2.0 g, 0.93 mL, 1.4 equivalents, 11 mmol) in chloroform (20 mL), and stir the resulting mixture at 60 °C for 3 hours. The mixture was concentrated under vacuum and the crude product was purified using an automated FCC system to give ethyl 2-bromo-1-(2,2-diethoxyethyl)-1H-imidazolium-5-carboxylate (1.8 g, 95% purity, 5.4 mmol, 67%). LC-MS (general 3, basic): / z =335.1 [M+H] + Step 3. Prepare 3-bromoimidazolo[1,5-a]pyrazin-8(7H)-one using the following procedure. A solution of ethyl 2-bromo-1-(2,2-diethoxyethyl)-1H-imidazolium-5-carboxylate (1.8 g, 5.4 mmol) and ammonium acetate (4.2 g, 10 equivalents, 54 mmol) in acetic acid (20 mL) in a sealed tube was heated at 140 °C for 16 hours. The mixture was concentrated under vacuum and purified using an automated FCC to give 3-bromoimidazolo[1,5-a]pyrazin-8(7H)-one (0.16 g, 0.75 mmol, 14%). LC-MS (General 3, Basic): m / z = 213.9 [M+H] + Step 4. Prepare methyl 2-(2-(3-bromo-8-oxoimidazo[1,5-a]pyrazin-7(8H)-yl)acrylamido)acrylate following general experimental procedure 9. 3-bromoimidazo[1,5-a]pyrazin-8(7H)-one (64 mg, 0.30 mmol) and methyl 2-propynamide acrylate (intermediate 1) (55 mg, 1.2 equivalents, 0.36 mmol) yield methyl 2-(2-(3-bromo-8-oxoimidazo[1,5-a]pyrazin-7(8H)-yl)acrylamido)acrylate (10 mg, 27 µmol, 9%), which can be used in the next step without purification.

[0281] Step 5. Methyl 2-(2-(3-(4-acetamidophenyl)-8-oxoimidazo[1,5-a]pyrazin-7(8H)-yl)acrylamido)acrylate (1-285) was prepared following General Experimental Procedure 3. Methyl 2-(2-(3-bromo-8-oxoimidazo[1,5-a]pyrazin-7(8H)-yl)acrylamido)acrylate (10 mg, 27 µmol) and (4-acetamidophenyl)boronic acid (9.7 mg, 2 equivalents, 54 µmol) were reacted to give methyl 2-(2-(3-(4-acetamidophenyl)-8-oxoimidazo[1,5-a]pyrazin-7(8H)-yl)acrylamido)acrylate (1-285) (1.3 mg, 3.1 µmol, 11%) as a white solid. LCMS: 22010199G LCMS-5 C3 m / z = 422.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.15 (s, 1H), 9.60 (s, 1H), 7.88 (d, J = 0.7 Hz, 1H), 7.77 – 7.65 (m, 4H), 7.47 (dd, J= 6.2, 0.8 Hz, 1H), 6.79 (d, J = 6.2 Hz, 1H), 6.17 (d, J = 1.8 Hz, 1H), 5.90 (d, J = 1.8Hz, 1H), 5.80 (s, 1H), 5.66 (s, 1H), 3.66 (s, 3H), 2.03 (s, 3H).

[0282] Example 29 2,2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(4-(pyrrolidine-1-yl)but-2-yne-1-yl)acrylamide 1-257 Step 1. Prepare tert-butyl (1-(2-acetylhydrazyl)-3-((tert-butyldiphenylsilyl)oxy)-1-oxopropyl-2-yl)carbamate using the following procedure. HATU (3.8 g, 2 equivalents, 10 mmol) was added to a solution of acetylhydrazine (0.74 g, 2 equivalents, 10 mmol), N-(tert-butyloxycarbonyl)-O-(tert-butyldiphenylsilyl)serine (intermediate 4) (2.2 g, 5.0 mmol), and triethylamine (2.1 mL, 3 equivalents, 15 mmol) in DMF (20 mL), and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with heptane (200 mL) and ethyl acetate (200 mL), and the organic layer was washed with water (2 × 50 mL). The organic layer was dried over Na₂SO₄ and concentrated under vacuum. The crude substance was purified by automated FCC to obtain tert-butyl (1-(2-acetylhydrazinyl)-3-((tert-butyldiphenylsilyl)oxy)-1-oxopropyl-2-yl)carbamate (2.3 g, 4.6 mmol, 92%) as a white solid. LC-MS (general 3, basic): m / z = 400.4 [M+H] + .

[0283] Step 2. Prepare tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)carbamate using the following procedure: Add 2.0 g (1 equivalent, 4.0 mmol) of tert-butyl (1-(2-acetylhydrazyl)-3-((tert-butyldiphenylsilyl)oxy)-1-oxopropyl-2-yl)carbamate to a solution of 100 mL of CH2Cl2 and stir the resulting mixture at room temperature for 20 hours. Quench the reaction mixture with an aqueous solution of potassium carbonate and separate the organic layer. Extract the aqueous layer with CH2Cl2 (2 × 200 mL). Dry the combined organic layers with Na2SO4 and concentrate under vacuum. The crude material was purified using an automated FCC filter to yield tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)carbamate (1.7 g, 3.5 mmol, 88%). LC-MS (universal 3, basic): m / z = 482.4 [M+H] + .

[0284] Step 3. 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl-1-amine hydrochloride was prepared using the following procedure. Tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)carbamate (1.7 g, 3.5 mmol) was dissolved in HCl in dioxane (4 M, 18 mL), and the resulting mixture was stirred for 20 hours. The mixture was concentrated to give 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl-1-amine hydrochloride (1.5 g, 3.5 mmol, quantitative).

[0285] Step 4. Prepare 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl-1-amine hydrochloride following general experimental procedure 5. 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (intermediate 3) (0.18 g, 0.50 mmol) and 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl-1-amine hydrochloride (0.42 g, 2 equivalents, 1.0 mmol) yielded 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)-3-hydroxypropionamide (0.15 g, 0.20 mmol, 40%). LC-MS (universal 3, basic): m / z = 718.5 [M+H] + .

[0286] Step 5. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)acrylamide using the following procedure. Add a solution of TBAF in THF (1M, 1.0 mL, 5 equivalents, 1.0 mmol) to a solution of 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)-3-hydroxypropionamide (0.14 g, 0.20 mmol) in THF (2 mL), and stir the resulting mixture at room temperature for 2 hours. The mixture was concentrated and the crude product was purified by automated reverse-phase FCC to give 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)acrylamide (80 mg, 0.17 mmol, 83%).

[0287] Step 6. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)acrylamide (1-257) by following General Experimental Procedure 5 and General Experimental Procedure 7 in sequence. 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-oxadiazol-2-yl)ethyl)acrylamide (80 mg, 0.17 mmol) was reacted to give 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,3,4-oxadiazol-2-yl)vinyl)acrylamide (1-257) (25 mg, 56 µmol, 33%) as a white solid. LCMS: 22010199G LCMS-5 C3; ​ = 444.2 [M+H] + . 1 HNMR (400 MHz, DMSO) δ 10.03 (s, 1H), 9.83 (s, 1H), 7.70 – 7.61 (m, 4H), 7.61– 7.46 (m, 3H), 5.87 (s, 1H), 5.62 (s, 1H), 5.60 – 5.58 (m, 1H), 5.55 – 5.53 (m, 1H), 4.87 (s, 2H), 2.01 (s, 3H).

[0288] Example 30 The following compounds were prepared using a method similar to that described in Example 29.

[0289] Example 31 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,3,4-thiadiazol-2-yl)vinyl)acrylamide 1-299 Step 1. Prepare tert-butyl (1-(2-acetylhydrazyl)-3-((tert-butyldiphenylsilyl)oxy)-1-oxopropyl-2-yl)carbamate using the following procedure. HATU (3.8 g, 2 equivalents, 10 mmol) was added to a solution of acetylhydrazine (0.74 g, 2 equivalents, 10 mmol), N-(tert-butyloxycarbonyl)-O-(tert-butyldiphenylsilyl)serine (intermediate 4) (2.2 g, 5.0 mmol), and triethylamine (2.1 mL, 3 equivalents, 15 mmol) in DMF (20 mL), and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with heptane (200 mL) and ethyl acetate (200 mL), and the organic layer was washed with water (2 × 50 mL). The organic layer was dried over Na₂SO₄ and concentrated under vacuum. The crude substance was purified by automated FCC to obtain tert-butyl (1-(2-acetylhydrazinyl)-3-((tert-butyldiphenylsilyl)oxy)-1-oxopropyl-2-yl)carbamate (2.3 g, 4.6 mmol, 92%) as a white solid. LC-MS (general 3, basic): m / z = 400.4 [M+H] + .

[0290] Step 2. Prepare tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)carbamate using the following procedure. Lawson's reagent (0.81 g, 2.0 mmol) was added to a solution of tert-butyl (1.0 g, 2.0 mmol) in THF (20 mL), and the resulting mixture was stirred under reflux for 1 hour. The mixture was concentrated and purified by FCC to give a yellow oil of tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)carbamate (0.90 g, 1.8 mmol, 90%). LC-MS (General 3, Basic): m / z = 498.3 [M+H] + .

[0291] Step 3. Prepare 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl-1-amine hydrochloride using the following procedure. Add a solution of HCl in dioxane (1M, 18 mL, 18 mmol) to a solution of tert-butyl carbamate (0.90 g, 1.8 mmol) in dioxane (10 mL), and stir the resulting mixture for 5 hours. Concentrate the mixture to give 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl-1-amine hydrochloride (0.78 g, 1.8 mmol, quantified), which is used as is in the next step.

[0292] Step 4. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)-3-hydroxypropionamide following general experimental procedure 5. 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (intermediate 3) (0.43 g, 1.2 mmol) and 2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl-1-amine hydrochloride (0.65 g, 1.25 equivalents, 1.5 mmol) yield 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)-3-hydroxypropionamide (0.37 g, 0.50 mmol, 42%).

[0293] Step 5. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)acrylamide using the following procedure. Add a solution of TBAF in THF (1M, 1.5 mL, equivalent, 1.5 mmol) to a solution of 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)-3-hydroxypropionamide (0.37 g, 0.50 mmol) in THF (4 mL), and stir the resulting mixture at room temperature for 3 hours. The mixture was concentrated and purified by automated reverse-phase FCC to give 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)acrylamide (0.20 g, 0.40 mmol, 81%) as a white solid.

[0294] Step 6. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,3,4-thiadiazol-2-yl)vinyl)acrylamide (1-299) by following General Experimental Procedure 6 and General Experimental Procedure 7 in sequence. 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(5-methyl-1,3,4-thiadiazol-2-yl)ethyl)acrylamide (0.12 g, 0.25 mmol) yields 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(5-methyl-1,3,4-thiadiazol-2-yl)vinyl)acrylamide (1-299) (16 mg, 35 µmol, 14%) as a white solid. LCMS-5 22010199G C3 m / z = 460.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 9.91 (s, 1H), 7.84 – 7.76 (m, 4H), 7.73 – 7.63 (m,3H), 5.96 (s, 1H), 5.74 – 5.65 (m, 3H), 5.02 (s, 2H), 2.78 (s, 3H), 2.14 (s, 3H).

[0295] Example 32 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(3-methyl-1,2,4-thiadiazol-5-yl)vinyl)acrylamide 1-313 Step 1. 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(3-methyl-1,2,4-thiadiazol-5-yl)vinyl)acrylamide was prepared using the following procedure. N-(tert-butoxycarbonyl)-O-(tert-butyldiphenylsilyl)serine methyl ester (2.3 g, 5.0 mmol) was dissolved in a solution of ammonia in methanol (7 M, 14 mL) in a sealed tube. The mixture was heated at 100 °C for 16 hours. The mixture was concentrated under vacuum and the crude product was purified by FCC to give N-(tert-butoxycarbonyl)-O-(tert-butyldiphenylsilyl)serine methyl ester (1.4 g, 3.2 mmol, 63%) as a white solid. LC-MS (General 3, Basic): m / z = 465.4 [M+Na] + .

[0296] Step 2. Prepare tert-butyl (1-amino-3-((tert-butyldiphenylsilyl)oxy)-1-thiopropyl-2-yl)carbamate using the following procedure. Lawson's reagent (1.4 g, 3.5 mmol) was added to a solution of tert-butyl (1.5 g, 3.5 mmol) in CH2Cl2 (50 mL), and the resulting mixture was stirred under reflux for 1 hour. The mixture was concentrated and the crude product was purified by FCC to give tert-butyl (1-amino-3-((tert-butyldiphenylsilyl)oxy)-1-thiopropyl-2-yl)carbamate (0.83 g, 1.8 mmol, 53%). LC-MS (General 3, Basic): m / z = 459.3 [M+H] + .

[0297] Step 3. Prepare (Z)-(2,3,10,10-tetramethyl-9,9-diphenyl-5-thio-8-oxa-2,4-diaza-9-silane-undeca-3-en-6-yl)carbamate using the following procedure. Add N,N-dimethylacetamide dimethyl acetal (0.51 mL, 2 equivalents, 3.5 mmol) to a solution of (1-amino-3-((tert-butyldiphenylsilyl)oxy)-1-thioprop-2-yl)carbamate (0.80 g, 1.7 mmol), and stir the resulting mixture at room temperature for 1 hour. The mixture was concentrated and the crude substance was purified by FCC to give (Z)-(2,3,10,10-tetramethyl-9,9-diphenyl-5-thio-8-oxa-2,4-diaza-9-silyl undec-3-en-6-yl)carbamate tert-butyl ester (0.65 g, 1.2 mmol, 71%). LC-MS (general 3, basic): m / z = 528.4 [M+H] + .

[0298] Step 4. Prepare tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)carbamate using the following procedure: Add 0.65 g, 1.2 mmol, of (Z)-(2,3,10,10-tetramethyl-9,9-diphenyl-5-thio-8-oxa-2,4-diaza-9-silane-undeca-3-en-6-yl)carbamate (0.65 g, 1.2 mmol) and pyridine (0.20 mL, 2 equivalents, 2.5 mmol) to a solution of methanol (10 mL) with hydrogen thionyl aminosulfate (0.18 g, 1.3 equivalents, 1.6 mmol) and stir the resulting mixture at room temperature for 16 hours. The mixture was concentrated and the crude substance was purified by FCC to give tert-butyl (2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)carbamate (0.58 g, 1.2 mmol, 95%) as a white solid. LC-MS (general 3, basic): m / z = 498.4 [M+H] + .

[0299] Step 5. Prepare 2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl-1-amine hydrochloride using the following procedure. Add HCl to a solution of tert-butyl carbamate (0.58 g, 1.2 mmol) in dioxane (4 mL) and stir the resulting mixture at room temperature for 3 hours. Concentrate the mixture under vacuum to obtain 2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl-1-amine hydrochloride (0.51 g, 1.2 mmol, quantified), which is used as is in the next step.

[0300] Step 6. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)-3-hydroxypropionamide following general experimental procedure 5. 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxypropionic acid (intermediate 3) (0.11 g, 0.30 mmol) and 2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl-1-amine hydrochloride (0.13 g, 0.30 mmol) yielded 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)-3-hydroxypropionamide (0.13 g, 0.18 mmol, 59%) as a yellow solid.

[0301] Step 7. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)acrylamide using the following procedure. NA solution of 2-((tert-butyldiphenylsilyl)oxy)-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)-3-hydroxypropionamide (0.13 g, 0.18 mmol) in THF (3 mL) was mixed with a solution of TBAF in THF (1 M, 0.27 mL, 1.5 equivalents, 0.27 mmol), and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated and purified by automated FCC to give 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)acrylamide (80 mg, 0.16 mmol, 91%) as a white solid.

[0302] Step 8. Prepare 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(3-methyl-1,2,4-thiadiazol-5-yl)vinyl)acrylamide using the following procedure. Add methanesulfonyl chloride (0.10 mL, 8 equivalents, 1.3 mmol) to a solution of 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-3-hydroxy-N-(2-hydroxy-1-(3-methyl-1,2,4-thiadiazol-5-yl)ethyl)acrylamide (80 mg, 0.16 mmol) and triethylamine (0.23 mL, 10 equivalents, 1.6 mmol) in THF (5 mL), and stir the resulting mixture at room temperature for 15 minutes. Dilute the mixture with water (10 mL) and extract with EtOAc (2 × 30 mL). The organic layer was separated, dried over Na2SO4, filtered, and concentrated. The residue was dissolved in CH2Cl2 (5 mL), and DBU (97 µL, 4 equivalents, 0.65 mmol) was added. The resulting mixture was stirred at room temperature for 15 minutes. Acetic acid (74 µL, 8 equivalents, 1.3 mmol) was added, and the mixture was concentrated. The crude substance was purified by automated reverse-phase FCC to give 2-(4-(4-acetamidophenyl)-1-oxoisoindoline-2-yl)-N-(1-(3-methyl-1,2,4-thiadiazol-5-yl)vinyl)acrylamide (1-313) (18 mg, 39 µmol, 24%) as a white solid. LCMS-5 22010199GC3 m / z = 460.2 [M+H] + . 1H NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 10.02 (s, 1H), 7.78 – 7.70 (m, 4H), 7.68 – 7.55 (m, 3H), 5.95 (s, 1H), 5.75 (s, 1H), 5.69 –5.61 (m, 2H), 4.96 (s, 2H), 2.55 (s, 3H), 2.08 (s, 3H).

[0303] Example 33 2-(2-(4-(1 -acetyllndolin-5-yl)-1 -oxoisoindolin-2-yl)acrylamido)-N,N-dimethyl methy lpropionamide 1-384 Step 1. Prepare 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylic acid following general experimental procedure 4. Methyl 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylate (RS088, 0.20 g, 0.55 mmol) yields 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylic acid (0.18 g, 0.52 mmol, 95%) as a white solid.

[0304] Step 2. Prepare 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)-N,N-dimethylacrylamide using the following procedure. Add EtOAc (0.40 mL, 50% wt, 1 equivalent, 0.68 mmol) containing propylphosphonic anhydride to a solution of 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)acrylic acid (0.24 g, 0.68 mmol) and DIPEA (0.48 mL, 4 equivalents, 2.7 mmol) in DMSO (4.0 mL). Stir the mixture for 10 minutes, then add a solution of dimethylamine in THF (0.34 mL, 2.0 mol, 1 equivalent, 0.683 mmol), and stir the resulting mixture for 1 hour. Additional EtOAc containing propylphosphonic anhydride (0.40 mL, 50% wt, 1 equivalent, 0.68 mmol) was added, and the mixture was stirred for another 45 minutes. The crude mixture was purified by automated reverse-phase FCC to give 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)-N,N-dimethylacrylamide (0.10 g, 0.26 mmol, 39%) as a white solid.

[0305] Step 3. Following General Experimental Procedure 2, prepare 2-(2-(4-(1-acetylindoline-5-yl)-1-oxoisoindoline-2-yl)acrylamido)-N,N-dimethylacrylamide 1-384. 2-(2-(4-bromo-1-oxoisoindoline-2-yl)acrylamido)-N,N-dimethylacrylamide (20 mg, 52 μmol) and 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)indoline-1-yl)ketene (17 mg, 1.1 equivalents, 58 μmol) to obtain 2-(2-(4-(1-acetylindoline-5-yl)-1-oxoisoindoline-2-yl)acrylamido)-N,N-dimethylacrylamide 1-384 (3.24 mg, 7.1 μmol) as a white solid. μmol, 13%). LCMS: 22010199G LCMS-5 C3 RT: 2.011 Area: 91.1% (215 nm), 93.3 % (254 nm); m / z = 459.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.99(s, 1H), 8.15 (d, J = 8.3 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.66 – 7.60 (m, 1H),7.51 (s, 1H), 7.42 (d, J = 8.1 Hz, 1H), 5.64 (d, J = 19.4 Hz, 2H), 5.28 (s, 1H), 4.88 (s, 2H), 4.62 (s, 1H), 4.16 (t, J = 8.5 Hz, 2H), 3.24 (d, J = 7.7 Hz, 4H), 2.96 (s, 3H), 2.82 (s, 3H), 2.20 (s, 3H).

[0306] Biological studies of exemplary compounds Biological study example 1 : General method for exemplary compound binding assay To test the binding mechanism and explore binding targets, cell treatment and Western blotting were performed as described by Cunniff et al. (2015). The covalent cross-linking of the enzyme was investigated using specific antibodies via Western blotting.

[0307] In summary, human tumor cell lines (HMESO cell lines derived from patients with malignant mesothelioma) were cultured in appropriate media and treated with different concentrations of the test compound for 24 hours (0.1 µM – 100 µM). After 24 hours of exposure to the test compound, cell lysates were generated in a standard lysis buffer (e.g., RIPA buffer). Protein abundance in the lysates was quantified, and proteins of equal concentrations were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed using antibodies specific to certain proteins (e.g., PRX1, PRX2, PRX3, and PRX4), as detailed in the relevant section. Figure 1 Covalent cross-linking modification can be detected by the presence of approximately 46 kD of antibody reactant on the protein blot.

[0308] Cell death assays were performed, for example, as described by Nelson et al. 2021. Briefly, human tumor cell lines (HMESO cell lines derived from patients with malignant mesothelioma) were cultured in 96-well plates and incubated with the test compound for 48 hours. Remaining cell material was stained with crystal violet, and a total cell count was performed to determine cell viability.

[0309] Biological study example 2: Cell viability studies of exemplary compounds Cell lines were seeded at a density of 2500 cells per well in 96-well plates (Corning, Kennebunk, ME, USA). The next day, cells were treated with a test compound diluted in complete culture medium and then cultured for 48 hours. After culture, cells were washed with PBS (Corning Cellgro, Manassas, VA, USA), fixed in PBS with 3.0% formaldehyde (FisherBioReagents, Fair Lawn, NJ, USA), and stained with 0.1% crystal violet (Acros Organics, Fair Lawn, NJ, USA) in water for 30 minutes. After removing the crystal violet stain, the plates were washed with H2O and allowed to dry. To quantify cell viability, plates were imaged using a Lionheart plate reader (BioTek Instruments, Winooski, VT, USA), and / or analyzed by absorbance at 540 nm using a Synergy HTX plate reader (BioTek Instruments, Winooski, VT, USA) (crystal violet dye dissolved in 100% methanol). To determine the effective cytotoxic concentration (IC50) of the test compound... 50The data were plotted using a 4-parameter nonlinear regression model with GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). Based on the IC50 values, the IC50 data were divided into five groups: A, B, C, D, and E. Group A had an IC50 < -1.39 µM; B = 1.39–2.39 µM; C = 2.39–3.39 µM; D = 3.39–4.39 µM; and E > 4.39 µM. These results are summarized in... Figure 1 and Figure 2 middle.

[0310] Biological study example 3: Cross-linking studies of exemplary compounds Malignant mesothelioma (MM) cells (H-MESO cell line) were seeded in complete tissue medium in 6-well plates. Cells were allowed to adhere for 24 hours and then treated with a specified concentration of thiostreptin (TS), (1), or (5) (DMSO stock solution) for 24 hours. Cell lysates were generated using standard RIPA buffer, and protein concentrations were determined using the Bradford assay. Total protein was separated from each sample by reducing SDS-polyacrylamide gel electrophoresis (20 µg). Proteins were transferred to PVDF membranes, blocked with 5% bovine serum albumin (BSA) for 1 hour, and incubated overnight at 4°C with PRX3 primary antibody in 1X Tris-buffered saline (TBST) containing Tween. The membranes were washed three times with 1X TBST and incubated for 1 hour at room temperature with horseradish peroxidase conjugated (HRP) secondary antibody. The membranes were washed three times with 1X TBST, developed using enhanced chemiluminescence, and visualized on a GE digital imager.

[0311] The crosslinking study results of the exemplary compounds disclosed herein are provided in Figure 1 middle.

[0312] Biological study example 4: Cytotoxic activity of test compounds on a malignant mesothelioma cell line Malignant mesothelioma (HMESO cell line) cells were seeded at a density of 2500 cells per well in 96-well plates (Corning, Kennebunk, ME, USA). The next day, the cells were treated with a test compound diluted in complete culture medium and then cultured for 48 hours (technical repetition). After culture, the cells were washed with PBS (Corning Cellgro, Manassas, VA, USA), fixed in PBS with 3.0% formaldehyde (Fisher BioReagents, Fair Lawn, NJ, USA), and stained with 0.1% crystal violet (Acros Organics, Fair Lawn, NJ, USA) in water for 30 minutes. After removing the crystal violet stain, the plates were washed with H2O and allowed to dry. To quantify cell viability, plates were imaged using a Lionheart plate reader (BioTek Instruments, Winooski, VT, USA), and / or analyzed by absorbance at 540 nm using a SynergyHTX plate reader (BioTek Instruments, Winooski, VT, USA) (crystal violet dye dissolved in 100% methanol). To determine the effective cytotoxic concentration (EC50) of the test compound... 50 The data were plotted using a 4-parameter nonlinear regression model with GraphPad Prism7 software (GraphPad Software, San Diego, CA, USA). The results were... Figure 1 Create a table.

[0313] Biological study example 5: Covalent cross-linking of test compounds to recombinant peroxidase 3 (rPRX3) Mix the premix reagents from Table 3 in a microcentrifuge tube on ice for a 1X reaction. Scale the reaction according to the amount of compound tested. Add 16 µL of the premix to a new microcentrifuge tube containing 1 µL of the test compound (10 mM stock solution diluted in DMSO), mix by gentle tapping, and centrifuge rapidly at 1,000 RPM. Incubate the reaction mixture at 37 °C for 18 h. Remove the reaction mixture from the incubation and quench it by adding 2 µL of Laemmli buffer containing 0.2 M dithiothreitol (DTT) and 10% sodium dodecyl sulfate. Boil the sample at 98 °C for 5 min. Separate the sample by polyacrylamide gel electrophoresis, transfer it to a PVDF membrane, and perform Western blotting using an anti-PRX3 antibody (AbFrontier, LF-PA0255). The membrane was incubated with ECL reagent (ThermoScientific, Rockford, IL, USA) and visualized using a GE Amersham Imager chemiluminescence detection system. Unmodified rPRX3 appeared as a single band at approximately 23 kDa, while rPRX3 covalently modified with the tested compound ran as a band at approximately 45 kDa. This was a qualitative assay to assess the presence of a band at approximately 45 kDa. Qualitative results were obtained in [the following section is missing from the original text]. Figure 1 Create a table.

[0314] Table 19-1: Premixed Liquid

[0315] Biological study example 6: Covalent cross-linking of test compounds to peroxidase 3 (PRX3) in malignant mesothelioma cells Figure 1 Human malignant mesothelioma (HMESO cell line) cells were seeded at a density of 200,000 cells per well in 6-well plates. After 24 hours, cells were treated with test compounds diluted in DMSO and cell culture medium. Cell lysates were collected 24 hours post-treatment using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate in deionized (DI) water) to restore the analyte to reductive SDS-PAGE. Protein concentrations were determined using the Bradford assay (ThermoScientific, Rockford, IL, USA). Lysates (15 µg protein / well) were dissolved under reducing conditions via SDS-PAGE at a constant 200 V for 50 minutes on a 4–12% gradient Bis-Tris-Midi gel (Invitrogen, Carlsbad, CA, USA). The gel was transferred to a PVDF membrane at constant 1A for 50 min, blocked with 5% BSA diluted in 1× Tris-buffered saline (TBS-T) containing 1% Tween-20 for at least 1 h, and incubated overnight at 4°C with anti-PRX3 antibody in 5% BSA TBS-T. The membrane was washed with 1× TBS-T for 1 h, incubated with appropriate secondary antibody for 1 h, and washed again with 1× TBS-T for 1 h. The membrane was incubated with ECL reagent (ThermoScientific, Rockford, IL, USA) and visualized using a GE Amersham Imager chemiluminescence detection system. Qualitative results [Yes (y) / No (n)] in Biological study example 7. Activity of thiochain silk (TS) and 1-305 in a MM xenograft model. Create a table.

[0316] Figure 3

[0317] Summary: Eighteen male SCID mice (10 weeks old) were implanted with 2.5 million human malignant mesothelioma cells (HMESO cell line) via intraperitoneal (IP) injection, and tumors were allowed to grow for 14 days. The animals were divided into 3 groups.

[0318] Group: 1. 5% DMSO / PBS mediator control (6 mice) 2. 50 mg / kg TS in 5% DMSO / PBS (6 mice) 3. 50 mg / kg 1-305 in 5% DMSO / PBS (6 mice) Fourteen days after tumor growth, animals received either the designated treatment or a mediator control, three times a week (Monday, Wednesday, and Friday) for three weeks. Animal weight was monitored during treatment. On day 36, animals were euthanized, the tumor was removed, and gross anatomy was observed. Following necropsy, tumor burden (weight and volume) was determined at the end of the experiment. Both the TS group and the 1-305 group showed significant reductions in tumor weight and volume. Animals in the TS group struggled to maintain their weight throughout the experiment. Results... ​ As shown in the image.

[0319] Incorporate by reference All publications and patents mentioned herein are hereby incorporated by reference in their entirety, as each individual publication or patent is specifically and individually indicated to be incorporated by reference. In case of conflict, this application (including any definitions herein) shall prevail.

[0320] Equivalent solution Furthermore, this invention covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented in a list format, such as in Markush group format, each subgroup of elements is also disclosed, and any element may be removed from the group. It should be understood that, in general, where an aspect of the invention is referred to as comprising a particular element and / or feature, certain embodiments of the invention or aspects of the invention consist of or are substantially composed of such elements and / or features. For simplicity, those embodiments are not specifically described herein. It should also be noted that the terms “comprising” and “containing” are intended to be open and allow for the inclusion of additional elements or steps. Where a range is given, endpoints are also included. Furthermore, unless otherwise stated or readily apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may take any specific value or subrange within the range stated in different embodiments of the invention, up to one-tenth of the lower limit of the range unit, unless the context explicitly specifies otherwise.

[0321] The foregoing written description is considered sufficient to enable those skilled in the art to practice the invention. The scope of the invention is not limited to the examples provided, as these examples are only used to illustrate one aspect of the invention, and other functionally equivalent embodiments are also within the scope of the invention. Various modifications to the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objectives of the invention are not necessarily covered in every embodiment of the invention.

Claims

1. A compound having a structure according to Formula I: (I)， Or its pharmaceutically acceptable salt, wherein: Ring M is aryl or heteroaryl; Ring A contains at least 5 atoms; X and Y are each independently selected , , , , , , , , , O, NR x2 and bond, where * indicates a key with N, and Indicates the bond with ring M; Each R a Independently selected from hydrogen, and optionally substituted C1-C3 alkyl groups; R x1 Selected from optionally substituted C1-C4 alkyl groups and optionally substituted 3-6 membered cycloalkyl groups; R x2 Selected from hydrogen, optionally substituted C1-C4 alkyl groups and optionally substituted 3-6 membered cycloalkyl groups; R 1 Selected from aryl, 3-6 membered cycloalkyl, 4-7 membered heterocyclic and heteroaryl, each of which may be optionally substituted; L is selected from the following: -CH2-, -O-, -NH-, -N(Me)-, -C(=O)NH-, and -NHC(=O)-; When it exists, R 2 Each time it appears, it is independently selected from halogen, C1-C4 alkyl, O-C1-C4 alkyl, -CN, aryl, 4-7 membered heterocyclic and 5-6 membered heteroaryl, wherein R 2 Any alkyl, aryl, heterocyclic, or heteroaryl moiety may optionally be substituted; R 3 Selected from -C(=O)-R 4 Optional substituted heteroaryl groups and -CN; R 4 Selected from optionally substituted alkylamino, NH2, optionally substituted aminoalkyl, optionally substituted C1-C6 alkyl, optionally substituted 3-6 membered cycloalkyl, optionally substituted aryl, and optionally substituted O-C1-C4 alkyl; and a is selected from 0, 1, and 2.

2. The compound of claim 1, wherein R 3 For –C(=O)-R 4 .

3. The compound as claimed in any one of the preceding claims, wherein R 4 For OMe.

4. The compound as claimed in any of the preceding claims, wherein X is .

5. The compound of claim 1, wherein Y is .

6. The compound of claim 5, wherein each R a It is hydrogen.

7. The compound according to any one of claims 1 to 3, wherein ring A is selected from: , , , , , , , , , , , , , , , and , where ** indicates a connection to the rest of the molecule, and each --- indicates a portion of the ring M fused with ring A.

8. The compound as claimed in any of the preceding claims, wherein ring A is selected from: , , , , , , , , , , , , , , , , , and , where ** indicates a connection to the rest of the molecule, and each --- indicates a portion of the ring M fused with ring A.

9. The compound of claim 8, wherein ring A is .

10. The compound as claimed in any of the preceding claims, wherein ring M is a heteroaryl group.

11. The compound of any one of the preceding claims, wherein ring M is selected from thienyl, isoxazolyl, isothiazolyl, pyridyl, pyrazolyl, imidazole, and thiazolyl.

12. The compound according to any one of claims 1 to 9, wherein ring M is aryl.

13. The compound as claimed in any of the preceding claims, wherein each present R 2 Independently selected from halogen groups, optionally substituted O-C1-C4 alkyl groups, optionally substituted C1-C4 alkyl groups, and -CN.

14. The compound as claimed in any of the preceding claims, wherein each present R 2 It is independently selected from chlorine, fluorine, methoxy, methyl and -CN.

15. The compound according to any one of claims 1 to 12, wherein a is 0.

16. The compound as claimed in any of the preceding claims, wherein L is a bond.

17. The compound as claimed in any of the preceding claims, wherein R 1 Selected from optionally substituted 3-6 membered cycloalkyl groups and optionally substituted 4-7 membered heterocyclic groups.

18. The compound of claim 17, wherein R 1 It is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxacyclobutyl, thiomorpholinyl and tetrahydropyranyl, each of which may be optionally substituted.

19. The compound according to any one of claims 1 to 16, wherein R 1 Selected from optionally substituted aryl groups and optionally substituted heteroaryl groups.

20. The compound of claim 19, wherein R 1 The group is selected from pyridyl, indolyl, isoindolyl, benzimidazolyl, benzothiazolyl, benzotriazolyl, benzopyrazolyl, thiazolyl, pyrazolyl, imidazolyl, indololinyl, isoindololinyl, benzoxazolone, pyrazinyl, inzozolyl, oxazolyl, and benzoxazinyl, each of which may be optionally substituted.

21. The compound according to any one of claims 1 to 16 or 19, wherein R 1 It is an aryl group that can be substituted at will.

22. The compound according to any one of claims 1 to 7, 10 to 12, or 15 to 21, wherein the compound has a structure according to formula (II): (II)。 23. The compound of claim 22, wherein the compound has a structure according to formula (III): (III), in, Each existing R 6 Independently selected from -NH2, –(C=O)-NH2, –(C=O)NH-C1-C6 alkyl, –(C=O)NH-3-6-membered cycloalkyl, –(C=O)NH-3-6-membered heterocyclic, –(C=O)-3-6-membered heterocyclic, –(C=O)N(C1-C4 alkyl)2, -NH(C=O)-C1-C6 alkyl, -NH(C=O)-3-6-membered heteroaryl, -N(C1-C4 alkyl)(C=O)-C1-C4 alkyl, -NH(C=O)-3-6-membered cycloalkyl, -NH(C=O)-4-7-membered heterocyclic, -NMe(C=O)-C1-C6 alkyl, -CH2-NH(C=O)-C1-C4 alkyl, -NHSO2- C1 -C4 alkyl, -SO2-C1-C4 alkyl, -SO2NH2, -SO2NH-C1-C4 alkyl, -NH(C=O)O-C1-C4 alkyl, -(C=O)OH, -(C=O)O-C1-C4 alkyl, -O(C=O)-C1-C4 alkyl, -NH(C=O)-NH-C1-C5 alkyl, -NH(C=O)O-4-6 membered cycloalkyl, -(C=O)-C1-C4 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocyclic, heterocyclic, 5-6 membered heteroaryl, C1-C4 alkyl, O-C1-C4 alkyl, chlorine, fluorine, -CF3, nitro, -NH-C1-C4 alkyl, -CN and -N(C1-C4 alkyl)2, each optionally substituted, and b is 0, 1 or 2, or 2 R 6 Together they form 3-6 membered cycloalkyl, 3-6 membered heterocyclic, 5-6 membered heteroaryl or aryl, each of which may be substituted.

24. The compound of claim 23, wherein the compound has a structure according to formula (IV): (IV)。 25. The compound of claim 24, wherein R 6 -NH(C=O)R 7 , where R 7 It is selected from C1-C6 alkyl, 3-6 membered cycloalkyl, 4-7 membered heterocyclic, O-C1-C4 alkyl and NH-C1-C4 alkyl, each of which may be optionally substituted.

26. The compound of claim 25, wherein R 7 Selected from optionally substituted C1-C4 alkyl groups and optionally substituted O-C1-C4 alkyl groups.

27. The compound of claim 26, wherein R 6 It is either NHAc or NHBoc.

28. The compound of claim 27, having the following structure: 。 29. The compound of claim 1, wherein the compound has a structure according to formula (V): (V)。 30. The compound of claim 29, wherein R 3 The heteroaryl group is arbitrarily substituted.

31. The compound of claim 30, wherein R 3 Selected from triazolyl, thiadiazolyl, and oxadiazolyl, each of which may be substituted.

32. The compound of claim 29, wherein R 3 -C(=O)-R 4 .

33. The compound of claim 32, wherein R 4 It is selected from NH2, optionally substituted C1-C5 alkyl, optionally substituted 3-6 membered cycloalkyl and optionally substituted O-C1-C4 alkyl.

34. The compound of claim 33, wherein R 4 Selected from -NH2, methyl, methoxy, cyclopropoxy and -Ot-Bu.

35. The compound of claim 1, wherein the compound is selected from: 。 36. The compound of claim 1, wherein the compound is selected from: 。 37. The compound of claim 1, wherein the compound is selected from... 。 38. A pharmaceutically acceptable composition comprising a compound as described in any one of the preceding claims, and a pharmaceutically acceptable carrier.

39. The composition of claim 38, formulated for oral or parenteral delivery.

40. A method of treating cancer (e.g., solid tumors or hematologic cancers) comprising administering to a subject in need a therapeutically effective amount of a compound as claimed in any one of claims 1 to 37 or a composition as claimed in any one of claims 38 or 39.

41. The method of claim 40, wherein the cancer is selected from mesothelioma, lung cancer, breast cancer, prostate cancer, melanoma, esophageal cancer, leukemia, cervical cancer, liver cancer, colon cancer, stomach cancer, colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and ovarian cancer.

42. The method of claim 41, wherein the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and breast cancer.

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