Therapeutic Conjugates

Abstract

The present disclosure relates generally to therapeutic conjugates that covalently bind to biological targets. Also provided herein are methods of administering the compositions to a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 902,554, entitled THERAPEUTIC CONJUGATES, filed September 19, 2019, and U.S. Provisional Patent Application No. 63 / 078,055, entitled THERAPEUTIC CONJUGATES, filed September 14, 2020, both of which are incorporated by reference in their entireties.

[0002] The present disclosure relates generally to therapeutic conjugates that covalently bind to biological targets. [Background technology]

[0003] Covalent inhibitors bind to receptors in the same way as classical inhibitors, but instead of dissociating, they form a permanent, covalent chemical bond to the receptor. Some examples of covalent inhibitors include penicillin, aspirin, clopidogrel, the EGFR kinase inhibitor afatinib, which is used to treat lung cancer, and the Bruton's tyrosine kinase inhibitor ibrutinib, which is used to treat B-cell malignancies. Furthermore, in the field of oncology, covalent inhibitors have been shown to be effective against drug-resistant tumors and generally have a stronger ability to inhibit tumor growth.

[0004] In recent years, covalent inhibitors have attracted the attention of major pharmaceutical companies because their use offers increased potency and duration of action compared to classical reversible inhibitors. The extended duration of action necessitates less frequent dosing, i.e., patients need to take fewer tablets less frequently.

[0005] There is a need to design therapeutic conjugates that can covalently bind to biological targets and to develop high-throughput screening methods for therapeutic conjugates. Summary of the Invention

[0006] In some embodiments, the present disclosure provides a therapeutic conjugate capable of forming a covalent bond with a kinase or pseudokinase. The kinase may be a PI3 kinase (PI3K). The therapeutic conjugate may have the structure (FCB)a-(L)b-(CLM)c, where a and c are independently integers from 1 to 5, and b is an integer from 0 to 5, and the FCB portion comprises a PI3K inhibitor or a fragment, analog, or derivative thereof. In some embodiments, the FCB is [ka] The therapeutic conjugate may comprise a structure selected from the group consisting of Compound 1-101 to Compound 1-172.

[0007] In some embodiments, the therapeutic conjugate comprises: [ka] or a pharmaceutically acceptable salt thereof, and L may have the structure [ka] and either end can be connected to CLM, and R1 is [ka] and the CLM is selected from the group consisting of [ka] The therapeutic conjugate is selected from the group consisting of compounds 1-101, 1-102, 1-103, 1-104, 1-105, 1-106, 1-107, 1-108, 1-109, 1-110, 1-111, 1-112, 1-113, 1-114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1 1-161, 1-162, 1-169, 1-170, 1-171 and 1-172.

[0008] In some embodiments, the therapeutic conjugate is [ka] or a pharmaceutically acceptable salt thereof, and L may have the structure [ka] and R2 is [ka] and the CLM is selected from the group consisting of [ka] The therapeutic conjugate may be selected from the group consisting of compounds 1-131, 1-132, 1-133, 1-134, 1-135, 1-136, 1-139, 1-140, 1-141, 1-142, 1-143, 1-144, 1-147, 1-148, 1-149, 1-150, 1-151, 1-152, 1-155, 1-156, 1-157, 1-158, 1-159, 1-160, 1-163, 1-164, 1-165, 1-166, 1-167 and 1-168.

[0009] In some embodiments, the therapeutic conjugate may comprise a structure selected from Compound 1-101 to Compound 1-172, or a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutic conjugate may comprise a structure selected from Compound 1-1 to Compound 1-11, or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, the present disclosure provides pharmaceutical compositions that can include a therapeutic conjugate disclosed herein and at least one pharmaceutically acceptable excipient.

[0011] In some embodiments, the present disclosure provides a method for modulating the activity of a kinase or pseudokinase, comprising administering a therapeutic conjugate disclosed herein. In some embodiments, the activity of the kinase or pseudokinase can be inhibited. In some embodiments, the kinase can be PI3-K.

[0012] In some embodiments, the present disclosure provides a method for treating a subject in need of treatment, the method comprising administering a therapeutically effective amount of a pharmaceutical composition described herein. The subject may have a condition selected from the group consisting of cancer, neurodegenerative disease, autoimmune disorder, and aging. In some embodiments, the subject may have cancer. In some embodiments, the subject may have cancer with a mutation in the PIK3CA gene. DETAILED DESCRIPTION OF THE INVENTION

[0013] I. Composition The inventors have discovered, inter alia, an Anchored Relational Covalent System, hereinafter referred to as an ARCS, which comprises a Functionally Competent Binder, hereinafter referred to as an FCB, and a Covalent Linking Modality, hereinafter referred to as a CLM, which is directly or indirectly linked to a therapeutic modality, and optionally includes a linker positioned between the FCB and the CLM. In some embodiments, the CLM is directly covalently linked to the FCB by a bond. In some embodiments, the CLM is indirectly covalently linked to the FCB by a linker.

[0014] As used herein, the term "ARCS" refers to any therapeutic conjugate formed by linking an FCB and a CLM via a bond or linker. In some embodiments, the ARCS can form a covalent bond with one or more targets, such as nucleotides, oligonucleotides, peptides, or proteins. In some embodiments, the ARCS can form a covalent bond with a biological target. The covalent bond can be detected using any method known in the art. As a non-limiting example, the covalent binding of an azide small molecule to a protein can be detected by using click chemistry, which attaches a heavy PEG-containing alkyne to the small molecule. The covalently labeled protein is PEG-labeled and has a higher molecular weight, so it is detected by gel shift (Biochemistry 2018, 57:5769-5774). In another non-limiting example, mass spectrometry can be used to detect purified covalently labeled proteins (Nature Chemical Biology 2007, 3:229-238). In yet another non-limiting example, cellular quantitative mass spectrometry-based proteomics methods can be used to analyze covalent bonds (Cell Chemical Biology 2017, 24:1388-1400.e7). In yet another non-limiting example, X-ray crystallography can be used to confirm covalent bond formation (Nature Chemical Biology 2007:3:229-238; J.Med.Chem. 2020, 63:52-65). In yet another non-limiting example, mass spectrometry of intracellular, covalently labeled, and affinity-enriched samples can be used to reveal sites of covalent modifications (Nat.Chem.Biol. 2016, 12:876-884).

[0015] In some embodiments, the ARCS can form covalent bonds with biological targets from about 5% to 100% of the biological targets. In some embodiments, the ARCS can form covalent bonds with about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the biological targets. In some embodiments, covalent bonds are formed in aqueous solution at a therapeutic dose of 10 mM at temperatures between 0°C and 50°C within 48 hours.

[0016] Without wishing to be bound by any theory, the ARCS can first form a non-covalent bond with a biological target (such as a target protein) via the FCB, and then form a covalent bond with the biological target via the CLM. In some embodiments, the effectiveness of the ARCS is superior to that of the FCB alone. In some embodiments, the CLM does not substantially interfere with the effectiveness of the FCB. In some embodiments, the FCB does not substantially interfere with the covalent binding of the CLM. In some embodiments, the toxicity of the ARCS is less than that of the FCB alone.

[0017] As used herein, the term "toxicity" refers to the ability of a substance or composition to be harmful or toxic to a cell, living tissue, or cellular environment. Low toxicity refers to the attenuated ability of a substance or composition to be harmful or toxic to a cell, living tissue, or cellular environment. Such attenuation or low toxicity may be relative to a standard measure, a treatment, or the absence of a treatment.

[0018] As used herein, the term "FCB" refers to known drugs, diagnostic compounds, drug candidates, and therapeutic modalities, which may be functional fragments and / or combinations of any of the foregoing. FCBs include free acid and free base forms, optical isomers and tautomers, radioisotopes of drugs, and isotopes, including pharmaceutically acceptable salts, prodrugs, or fragments thereof. FCBs may be small molecules, proteins, peptides, lipids, carbohydrates, sugars, nucleic acids, or combinations thereof. In some embodiments, FCBs are nucleic acids, including, but not limited to, DNA or RNA. FCBs may be therapeutic agents, such as, but not limited to, anti-cancer agents, anti-neurodegenerative agents, autoimmune agents, and anti-aging agents. FCBs may non-covalently bind to biological targets. In some embodiments, FCBs may be functional fragments of drugs. As used herein, the term "functional fragment" refers to a portion of a drug or its derivatives or analogs that can elicit the desired effect of the drug. In some embodiments, FCBs may contain an alkyne functional group. In some embodiments, FCBs may not contain an alkyne functional group.

[0019] As used herein, "peptide," "polypeptide," and "protein" refer to polymers composed of amino acid monomers linked by amide bonds. The amino acids may be D or L optical isomers. Peptides may be formed by a condensation or coupling reaction between the α-carbon carboxyl group of one amino acid and the amino group of another amino acid. Peptides may be non-linear, branched, or cyclic. Furthermore, peptides may be optionally modified or protected with a variety of functional or protecting groups, including at the amino and / or carboxy termini.

[0020] Amino acid residues in peptides are abbreviated as follows: phenylalanine is Phe or F, leucine is Leu or L, isoleucine is Ile or I, methionine is Met or M, valine is Val or V, serine is Ser or S, proline is Pro or P, threonine is Thr or T, alanine is Ala or a, tyrosine is Tyr or Y, histidine is His or H, glutamine is Gln or Q, asparagine is Asn or N, lysine is Lys or K, aspartic acid is Asp or D, glutamic acid is Glu or E, cysteine ​​is Cys or C, tryptophan is Trp or W, arginine is Arg or R, and glycine is Gly or G.

[0021] As used herein, the term "CLM" refers to any covalent bonding modality capable of forming a covalent bond with a biological target. The CLM may be linked to the FCB by a bond or a linker. The CLM may contain one or more chemical moieties capable of forming a covalent bond with the biological target. The chemical moieties may be electrophilic or nucleophilic groups.

[0022] The CLM may be a small molecule having a molecular weight of less than about 1,000 Da, less than about 900 Da, less than about 800 Da, less than about 700 Da, less than about 600 Da, or less than about 500 Da. In some cases, the CLM may have a molecular weight of about 5 Da to about 1,000 Da, about 10 Da to about 900 Da, in some embodiments, about 20 Da to about 700 Da, in some embodiments, 20 Da to about 500 Da, about 50 Da to about 400 Da, in some embodiments, about 100 Da to about 300 Da, and in some embodiments, about 150 Da to about 300 Da. The molecular weight of the CLM can be calculated as the sum of the atomic weights of each atom in the formula of the CLM multiplied by the number of each atom. It can also be measured by mass spectrometry, NMR, chromatography, light scattering, viscosity, and / or any other method known in the art. The units of molecular weight may be g / mol, Daltons (Da) or atomic mass units (amu), where it is known that 1 g / mol = 1 Da = 1 amu.

[0023] As used herein, the term "biological target" refers to any target that FCB binds non-covalently to produce a therapeutic effect.CLM is covalently bound to biological target.In some embodiments, biological target is protein.Non-limiting examples of biological target include, but are not limited to, kinases such as phosphoinositide 3-kinase (PI3K) and pseudokinase.

[0024] In some embodiments, the ARCS can form a covalent bond with PI3 kinase from about 5% to 100% of the PI3 kinases. In some embodiments, the ARCS can form a covalent bond with PI3 kinase from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the PI3 kinases.

[0025] The ARCS comprises at least one FCB optionally linked to at least one CLM via a linker. In some embodiments, the ARCS may be a therapeutic conjugate between a single FCB and a single CLM, e.g., having the structure XLY, where X is a CLM, L is an optional linker, and Y is an FCB. In some embodiments, the ARCS may be a therapeutic conjugate between a single therapeutic modality and a single covalent modality. In some embodiments, X is a covalent modality, L is an optional linker, and Y is a therapeutic modality.

[0026] In some embodiments, the ARCS contains one or more FCBs, one or more linkers, one or more CLMs, or any combination thereof. The ARCS can have any number of FCBs, linkers, and CLMs. ARCSs include, but are not limited to, XLYLX, (XLY) n , YLXLY, X-(LY) n , (XL) n -Y, (X) n -LY or XL-(Y) n where L is an optional linker, Y is FCB, and n is an integer from 2 to 100, 2 to 50, 2 to 20, e.g., 2 to 5. Each occurrence of X, L, and Y can be the same or different; for example, the ARCS can include one or more types of FCB, one or more types of linker, and / or one or more types of CLM.

[0027] In some embodiments, an ARCS may contain more than one type of CLM attached to a single FCB. For example, an ARCS may include a single FCB with multiple CLMs, each attached via the same or different linkers. An ARCS may have the structure XLYLX, where each X is a CLM, which may be the same or different, each L is a linker, which may be the same or different, and Y is an FCB.

[0028] In some embodiments, an ARCS may contain one or more CLMs attached to a single FCB. For example, an ARCS may include a CLM with multiple FCBs, each attached via the same or different linkers. An ARCS may have the structure YLXLY, where X is a CLM, each L is a linker, which may be the same or different, and Y is an FCB, which may be the same or different.

[0029] In some embodiments, the ARCS is a therapeutic conjugate, and the therapeutic conjugate is a. a therapeutic modality selected from the group consisting of one or more of a known drug, a diagnostic compound, a drug candidate, and a functional fragment, and / or a combination of any of the foregoing; b. a covalent binding modality, the covalent binding modality comprising one or more chemical moieties, one or more of which are capable of forming a covalent bond with a biological target, the covalent binding modality being directly or indirectly attached to the therapeutic modality; and c. Optionally, a linker positioned between the therapeutic modality and the covalent binding modality.

[0030] In some embodiments, the therapeutic conjugate comprises a formula selected from the group consisting of: a) XLY, b) XLYLX, c)(XLY) n , d) YLXLY, e) X-(LY) n , f)(XL) n -Y, g)(X) n -LY and h)XL-(Y) n ; X is a covalent modality, L is an optional linker, Y is a therapeutic modality, and n is an integer between 2 and 100.

[0031] The objective of this disclosure is to design ARCSs and compositions thereof and methods for synthesizing ARCSs and libraries of ARCSs.

[0032] It is also an object of the present disclosure to provide methods for screening libraries of ARCSs to identify candidates for covalent binding to biological targets.

[0033] A further object of the present disclosure is to provide methods of administering ARCS and compositions thereof to a subject in need thereof.

[0034] A.FCB The ARCS of the present disclosure contains at least one FCB. The ARCS of the present disclosure may contain one or more FCBs, which may be the same or different. An FCB can be a therapeutic modality that affects any biological process and is used to prevent, diagnose, mitigate, treat, or cure a disease state. An FCB can be a therapeutic, prophylactic, diagnostic, or nutritional agent. The efficacy of an FCB or ARCS refers to the effectiveness of the FCB or ARCS for its intended purpose, i.e., the ability of a given FCB or ARCS to produce a desired pharmacological effect. As used herein, the term "pharmacological activity" refers to the activity of regulating or modifying a biological process, resulting in a phenotypic change, e.g., cell death, reduced cell proliferation, etc.

[0035] In some embodiments, the FCB is a PI3 kinase inhibitor. In some embodiments, the FCB is a pyrrolo[2,1-F[1,2,4]triazine compound. In some embodiments, the FCB is a PI3 kinase inhibitor having any one of the formulas described in U.S. Pat. No. 9,724,352 B2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the FCB is any of the compounds shown in Table 1 of U.S. Pat. No. 9,724,352 B2. In some embodiments, the FCB is [ka] Includes the structure of

[0036] In some embodiments, the FCB has the structure [ka] wherein R1 is [ka] is selected from the group consisting of:

[0037] In some embodiments, the FCB has the structure [ka] and R2 is a compound having [ka] is selected from the group consisting of:

[0038] Generally, the effectiveness of FCBs is achieved by non-covalent binding to biological targets. Non-covalent binding is achieved through a degree of specificity and / or affinity for the target. While both specificity and affinity are generally desirable, in certain cases, higher specificity can compensate for lower affinity, and higher affinity can compensate for lower specificity. The requirements for affinity and specificity vary depending on various factors, including, but not limited to, the absolute concentration of the target, the relative concentration of the target (e.g., in cancer cells versus normal cells), potency and toxicity, the route of administration, and / or diffusion or transport into the target cells. At the molecular or cellular level, the effects of FCBs (ARCS or alone) can include, but are not limited to, promoting or inhibiting the activity of the target, labeling the target, and / or altering the target cells (e.g., cell death).

[0039] In some embodiments, the FCB may be a small molecule, a protein, a peptide, a lipid, a carbohydrate, a sugar, a nucleic acid, or a combination thereof. In some embodiments, the FCB may be a therapeutic agent, such as, but not limited to, an anti-cancer agent, an anti-neurodegenerative agent, an autoimmune agent, and an anti-aging agent. A variety of therapeutic agents are known in the art and may be used in the compositions described herein.

[0040] In some embodiments, the FCB is a small molecule. In some embodiments, the FCB may be a protein, peptide, or nucleic acid. In some embodiments, the FCB may be a lipid. In some embodiments, the FCB may be a carbohydrate or sugar. In some embodiments, the FCB has an alkyne group. In some embodiments, the FCB may not have an alkyne functional group.

[0041] In some embodiments, the FCB may be a functional fragment of a drug. As used herein, the term "functional fragment" or "core of a drug" refers to a portion of a drug or a derivative or analog thereof that is capable of eliciting a desired effect of the drug.

[0042] In some embodiments, the FCB may be non-covalently bound to the biological target. In some embodiments, the FCB has an IC of <1000 μm, 900 μm, 800 μm, 700 μm, 600 μm, or 500 μm. 50 The molecule may be bound to a biological target by

[0043] In some embodiments, the FCB is an anti-cancer agent. In some embodiments, the FCB is an anti-neurodegenerative agent. In some embodiments, the FCB is an autoimmune agent. In some embodiments, the FCB is an anti-aging agent.

[0044] In certain embodiments, the FCB of the ARCS comprises a predetermined molar weight percentage of about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the ARCS equals 100%. The amount of FCB in the ARCS may be expressed as a percentage of the CLM. For example, the present teachings provide ratios of FCB to CLM of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0045] B.CLM The ARCS of the present disclosure contains one or more CLMs. The CLM can be any covalent binding modality capable of forming a covalent bond with a biological target. The CLM can include one or more chemical moieties, including one or more chemical moieties capable of forming a covalent bond with a biological target. In certain embodiments, the CLM can include an internal linker or spacer. The internal linker or spacer can combine two parts of the CLM or can be attached to the CLM.

[0046] In some embodiments, the CLM is a small molecule. In some embodiments, the CLM has a molecular weight of less than about 1000 daltons (e.g., less than about 900, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.).

[0047] In certain embodiments, the CLM of the ARCS comprises a predetermined molar weight percentage of about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the ARCS equals 100%. The amount of CLM of the ARCS may be expressed as a percentage of the FCB. For example, the present teachings provide ratios of FCB to CLM of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0048] In some embodiments, the CLM comprises at least one substituted or unsubstituted alkyne. In some embodiments, the CLM comprises at least one substituted or unsubstituted acrylamide. In some embodiments, the CLM comprises at least one substituted or unsubstituted vinylsulfonamide. In some embodiments, the CLM comprises at least one substituted or unsubstituted vinylsulfone. In some embodiments, the CLM comprises at least one substituted or unsubstituted fumaramide. In some embodiments, the CLM comprises at least one substituted or unsubstituted acrylic acid. In some embodiments, the CLM comprises at least one substituted or unsubstituted isothiocyanate. In some embodiments, the CLM comprises at least one substituted or unsubstituted sulfonyl fluoride. In some embodiments, the CLM comprises at least one substituted or unsubstituted fluorosulfate. In some embodiments, the CLM comprises at least one substituted or unsubstituted formylphenylboronic acid. In some embodiments, the CLM comprises at least one substituted or unsubstituted boronic acid. In some embodiments, the CLM comprises at least one activated ester. In some embodiments, the CLM comprises at least one substituted or unsubstituted thioester. In some embodiments, the CLM comprises at least one sulfonyl group. In some embodiments, the CLM comprises at least one nitro group. In some embodiments, the CLM comprises at least one substituted or unsubstituted epoxide. In some embodiments, the CLM comprises at least one substituted or unsubstituted formylphenylboronic acid. In some embodiments, the CLM comprises at least one substituted or unsubstituted aryl halide. In some embodiments, the CLM comprises at least one substituted or unsubstituted aldehyde. In some embodiments, the CLM comprises at least one substituted or unsubstituted triazine. In some embodiments, the CLM comprises at least one substituted or unsubstituted cyano-acrylamide. In some embodiments, the CLM comprises at least one substituted or unsubstituted chloroacetamide.

[0049] Exemplary CLMs include, but are not limited to, the following: [ka] [ka] [ka] [ka] [ka] A, B, C and D in each occurrence independently represent H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 and optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; and the optional substituents for A, B, C, and D are independently selected from the group consisting of halogen, OH, NH, CH, CF, -CN, -NO, -C(O)OH, -S(O)NH, -C(O)NH, -CHNH, -C(O)CH, SH, -S-CH, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 and 1 to 3 substituents selected from the group consisting of cycloalkyl.

[0050] A1, A2, A3, A4, A5 and A6 in each occurrence are independently H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; A 1、 A2, A 3、 A 4、 The optional substituents of A5 and A6 are independently selected from halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are 1 to 2 substituents, independently selected from halogen, OH, NH, CH 3、 Selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -CH2NH2, -C(O)CH3, SH and -S-CH3 or fragments, derivatives or analogs thereof.

[0051] In some embodiments, the CLM [ka] is selected from the group consisting of:

[0052] C. Linker The ARCS of the present disclosure contains one or more optional linkers connecting the FCB and CLM. The linker, L, can be attached anywhere on the FCB and CLM as long as the availability of the FCB and the binding of the CLM are not significantly affected. In some embodiments, the CLM comprises an optional internal linker.

[0053] In some embodiments, the linker (including the internal linker of the CLM) is a small molecule. In some embodiments, the linker (including the internal linker of the CLM) is selected to include, but is not limited to, substituted and unsubstituted C-C 30 Alkyl, substituted and unsubstituted C2-C 30 Alkenyl, substituted and unsubstituted C2-C 30 Alkynyl, substituted and unsubstituted C3-C 30 Cycloalkyl, substituted and unsubstituted C1-C 30 Heterocycloalkyl, substituted and unsubstituted C3-C 30 Cycloalkenyl, substituted and unsubstituted C1-C 30 Includes heterocycloalkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl.

[0054] In some embodiments, the linker (including the internal linker of the CLM) is C1-C 10 Straight chain alkyl, C1-C 10 Straight chain O-alkyl, C1-C 10 Straight chain substituted alkyl, C1-C 10 Straight chain substituted O-alkyl, C4-C 13 Branched alkyl, C 4- C 13 Branched chain O-alkyl, C2-C 12 Straight chain alkenyl, C2-C 12 Straight chain O-alkenyl, C 3- C 12 Straight-chain substituted alkenyl, C3-C 12The linker may be a linear substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl, heterocycle, succinate ester, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bipyridine, indole derivative acting as a crosslinker, chelating agent, aldehyde, ketone, bisamine, bisalcohol, heterocyclic structure, azirine, disulfide, thioether, hydrazone, and combinations thereof. For example, the linker may be a C3 linear alkyl or ketone. The alkyl chain of the linker may be substituted with one or more substituents or heteroatoms. In some embodiments, the linker contains one or more atoms or groups selected from -O-, -C(=O)-, -NR, -OC(=O)-NR-, -S-, and -SS-. The linker may be selected from dicarboxylate derivatives of succinic acid, glutamic acid or diglycolic acid.

[0055] In some embodiments, the alkyl chain of the linker may optionally be interrupted by one or more atoms or groups selected from -O-, -C(=O)-, -NR, -OC(=O)-NR-, -S-, -SS-. The linker may be selected from dicarboxylate derivatives of succinic acid, glutamic acid, or diglycolic acid.

[0056] In some embodiments, the linker may be non-cleavable. In some embodiments, the linker may be cleavable. In some embodiments, the linker may be cleaved by an enzyme.

[0057] Non-limiting examples of linkers include: [ka] are listed, [ka] D1, D2, D3, D4, D5 and D6 are independently selected in each occurrence from the group consisting of N, C, O, or S; 1-6 is N, the corresponding position is trivalent, and D 1-6 is O or S, the corresponding position is divalent, and B1, B2, B3, B 4、 B5 and B6 are each absent or independently selected from H, halogen, CF3, -OH, -CH3, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1~3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH 3) , OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 and the optional substituents on B1, B2, B3, and B4 are independently selected from halogen, OH, NH2, CH3, CF3, —CN, —NO2, —C(O)OH, —S(O)2NH2, —C(O)NH2, —CH2NH2, —C(O)CH3, SH, —S—CH3, optionally substituted C(O) 1~3 Alkyl and optionally substituted C 3-6 cycloalkyl, and C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH, CF, -CN, -NO, -C(O)OH, -S(O)NH, -C(O)NH, -CHNH, -C(O)CH, SH, and -S-CH 3、or any fragment or analog thereof.

[0058] In some embodiments, the linker is [ka] and either end can be connected to a CLM. [ka] The linker selected from the group consisting of: [ka] connected to a CLM selected from the group consisting of:

[0059] D.ARCS The ARCSs of the present disclosure represent a class of drugs that have many advantages over reversible inhibitors, including increased potency and prolonged duration of action. The present disclosure provides therapeutic conjugates that form a covalent bond with a kinase or pseudokinase. In some embodiments, the kinase is a PI3 kinase (PI3K). The therapeutic conjugates include: may have the structure (FCB)a-(L)b-(CLM)c, a and c are independently integers from 1 to 5; b is an integer from 0 to 5; The FCB portion comprises a PI3K inhibitor or a fragment, analog or derivative thereof.

[0060] The FCB, L (linker) and CLM moieties are described in the sections above. In one non-limiting example, the FCB is [ka] Includes:

[0061] In some embodiments, the FCB has the structure [ka] wherein R1 is [ka] is selected from the group consisting of:

[0062] In some embodiments, the FCB has the structure [ka] and R2 is a compound having [ka] In some embodiments, the linker is selected from the group consisting of: [ka] and either end can be connected to a CLM. In some embodiments, the CLM is selected from the group consisting of: [ka] is selected from the group consisting of:

[0063] In some embodiments, the ARCS comprises a broad range of general structures Compound 1-1 through Compound 1-5, [ka] or a pharmaceutically acceptable salt thereof; R1 is independently in each occurrence: [ka] and R1 can be attached to X, L or a functional fragment of a drug at either of the two ends. For example, [ka] In the formula, R1 is Re and R g From either end adjacent to L, R f and R h or R1 can be attached to a functional fragment of a drug from the end adjacent to R f and R h From either end adjacent to L, R e and R g The functional fragment of the drug can be attached from the end adjacent to the

[0064] In some embodiments, R1 is independently at each occurrence unsubstituted or substituted -(alk) a -S-(alk) b -, -(alk) a -O-(alk) b -, -(alk) 655a -NR A -(alk) b -, -(alk) a -C(O)-(alk) b -, -(alk) a -C(S)-(alk) b -, -(alk) a -S(O)-(alk) b -, -(alk) a -S(O)2-(alk) b -, -(alk) a -OC(O)-(alk) b -, -(alk) a -C(O)O-(alk) b -, -(alk) a -OC(S)-(alk) b -, -(alk) a -C(S)O-(alk) b -, -(alk) a -C(O)NR A -(alk) b -, -(alk) a -C(S)NR A -(alk) b -, -(alk) a -S(O)2NR A -(alk) b -, -(alk)a -NR A C(O)-(alk) b -, -(alk) a -NR A C(S)-(alk) b -, -(alk) a -NR A S(O)2-(alk) b -, -(alk) a -NR A C(O)O-(alk) b -, -(alk) a -NR A C(S)O-(alk) b -, -(alk) a -OC(O)NR A -(alk) b -, -(alk) a -OC(S)NR A -(alk) b -, -(alk) a -NR A C(O)NR B -(alk) b -, -(alk) a -NR A C(S)NR B -(alk) b -and-(alk) a -NR A S(O)NR B -(alk) b - selected from the group consisting of a and b are independently selected from the group consisting of 0, 1, 2, 3, and 4; alk is independently C 1-5 Alkylene, C 1-5 Alkenylene and C 1-5 alkynylene, each of which is selected from the group consisting of H, halogen, —OH, NH, CF, C 1-5 Alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, -OC 1-5 Alkyl, -SC 1-5 Alkyl, -NH-C 1-5Alkyl and -N(C 1~5 alkyl), optionally substituted with 1 to 3 substituents independently selected from the group consisting of C 1~5 The alkyl group is a halogen, -OH, -NH 2、 C 1-4 independently and optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, CF3, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methylimidazolyl, and methylpyrazolyl; R A and R B independently in each occurrence, hydrogen, C 1-3 Alkyl, C 3-6 cycloalkyl, 5- to 10-membered heterocycle, aryl, and 5- to 10-membered heteroaryl, wherein the alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl are selected from the group consisting of halogen, C 1-3 Alkyl, OH, NH2, NH-C 1-3 Alkyl, N(C 1-3 alkyl)2, CF3, C 1-6 each independently optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methylimidazolyl, and methylpyrazolyl; R3 in each occurrence is independently selected from the group consisting of: [ka] ; R at each occurrence a , R b , R c , R d , R e , R f , R g and R hare independently H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered hetero, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; R a , R b , R c , R d , R e , R f , R g and R h are independently selected from halogen, OH, NH, CH, CF, -CN, -NO, -C(O)OH, -S(O)NH, -C(O)NH, -CHNH, -C(O)CH, SH, -S-CH, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、 and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0065] In some embodiments, R3 at each occurrence is independently selected from H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; Optional substituents for R3 are halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents independently selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、 and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0066] R4 in each occurrence is independently selected from the group consisting of: [ka]

[0067] In some embodiments, R4 at each occurrence is independently selected from H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; The optional substituents for R4 are independently selected from halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、 and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0068] R5 in each occurrence is independently H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; The optional substituents for R5 are independently selected from halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、 and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0069] R6 in each occurrence is independently H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; The optional substituents for R6 are independently selected from halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、 and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0070] L in each occurrence is independently selected from the group consisting of: [ka] , D1, D2, D3, D4, D5 and D6 are independently selected in each occurrence from the group consisting of N, C, O, or S, with the proviso that D 1-6 is N, the corresponding position is trivalent, and D 1-6When is O or S, the corresponding position is divalent. The linker can be attached to either the CLM or the functional fragment of the drug at either of its two ends. For example, [ka] In the formula (I), L can be attached to CLM either from the end adjacent to the nitrogen or from the other end.

[0071] In each occurrence, B1, B2, B3 and B4 are independently H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; The optional substituents of B1, B2, B3 and B4 are independently selected from halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C(O) 1-3 Alkyl and optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0072] In some embodiments, L, in each occurrence, is independently unsubstituted or substituted -(alk)aS-(alk)b-, -(alk)aO-(alk)b-, -(alk)a-NRA-(alk)b-, -(alk)aC(O)-(alk)b-, -(alk)aC(S)-(alk)b-, -(alk)aS(O)-(alk)b-, -(alk)aS(O)-(alk)b-, -(alk)a-OC(O)-(alk)b-, -(alk)aC(O)O-(alk)b-, -(alk)a-OC(S)-(alk)b-, -(alk)aC(S)O-(alk)b-, -(alk)aC(O)NRA-(alk)b-, -(alk)aC(S)NRA-(alk)b-, -(alk)aC(S)NRA-(alk)b- is selected from the group consisting of -(alk)aS(O)2NRA-(alk)b-, -(alk)a-NRAC(O)-(alk)b-, -(alk)a-NRAC(S)-(alk)b-, -(alk)a-NRAS(O)2-(alk)b-, -(alk)a-NRAC(O)O-(alk)b-, -(alk)a-NRAC(S)O-(alk)b-, -(alk)a-OC(O)NRA-(alk)b-, -(alk)a-OC(S)NRA-(alk)b-, -(alk)a-NRAC(O)NRB-(alk)b-, -(alk)a-NRAC(S)NRB-(alk)b- and -(alk)a-NRAS(O)2NRB-(alk)b-; a and b are independently selected from the group consisting of 0, 1, 2, 3, and 4; alk is independently C 1-5 Alkylene, C 1-5 Alkenylene and C 1-5 alkynylene, each independently selected from the group consisting of H, halogen, —OH, NH, CF, C 1-5Alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, -OC 1-5 Alkyl, -SC 1-5 Alkyl, -NH-C 1-5 Alkyl, and -N(C 1~5 alkyl)2, optionally substituted with 1 to 3 substituents selected from the group consisting of C 1~5 The alkyl groups are independently selected from halogen, -OH, -NH 2、 C 1-4 Optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, CF3, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methylimidazolyl, and methylpyrazolyl.

[0073] R C and R D independently in each occurrence, hydrogen, C 1-3 Alkyl, C 3-6 cycloalkyl, a 5- to 10-membered heterocycle, an aryl, and a 5- to 10-membered heteroaryl, wherein the alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl are each independently selected from the group consisting of halogen, C 1-3 Alkyl, OH, NH2, NH-C 1-3 Alkyl, N(C 1-3 alkyl)2, CF3, C 1-6 Optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methylimidazolyl, and methylpyrazolyl.

[0074] X at each occurrence is independently selected from the group consisting of: [ka] [ka] [ka] [ka] [ka]

[0075] A, B, C and D, in each occurrence, independently represent H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; The optional substituents for A, B, C, and D are independently halogen, OH, NH 2、 CH 3、 CF 3、 -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH 3、 SH, -S-CH3, optionally substituted C 1-3 Alkyl, optionally substituted C 3-6 1 to 3 substituents selected from the group consisting of cycloalkyl; C 1-3Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、 and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0076] A1, A2, A3, A4, A5, and A6 in each occurrence independently represent H, halogen, CF3, -OH, -NH2, -SH, -SCH3, -CN, -NO2, -CH2(NH2), -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, NHC(O)-C 1-6 Alkyl, N(C 1-3 alkyl)C(O)-C 1-6 Alkyl, OC(O)NH2, OC(O)NH(CH3), OC(O)N(CH3)2, imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-6 selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; The optional substituents of A1, A2, A3, A4, A5 and A6 are independently selected from halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, -S-CH3, optionally substituted C 1-3 Alkyl and optionally substituted C 3-6 and 1 to 3 substituents selected from the group consisting of cycloalkyl. C 1-3 Alkyl and C 3-6 Optional substituents for cycloalkyl are halogen, OH, NH, CH 3、and 1 to 2 substituents independently selected from the group consisting of CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0077] The present disclosure contemplates the use of all combinations of the various substituents. Thus, any combination of the above-described substituents contained within the structural formulas of Compounds 1-1 through 1-5 can be used.

[0078] In some embodiments, the ARCS is selected from compounds 1-6 through 1-11 of the narrow general structure: [ka] or a pharmaceutically acceptable salt thereof.

[0079] In some embodiments, the ARCS is [ka] or a pharmaceutically acceptable salt thereof, wherein L is [ka] and either end can be connected to a CLM, and R1 is a linker selected from the group consisting of [ka] and the CLM is selected from the group consisting of: [ka] Compounds encompassed by Formula 1-50 include, but are not limited to, compounds 1-101, 1-102, 1-103, 1-104, 1-105, 1-106, 1-107, 1-108, 1-109, 1-110, 1-111, 1-112, 1-113, 1-114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-200, 1-201, 1-202, 1-203, 1-204, 1-205, 1-206, 1-207, 1-208, 1-209, 1-300, 1-310, 1-311, 1-312, 1-313, 1-314, 1-315, 1-316, 1-317, 1-318, 1-319, 1-401, 1-410, 1-419, 1-420, 1-421, 1-422, 1-423, 1-424, 1-425, 1-426, 1-427, 1-428, 1-429, 1-500, 1-510, 1-511, 1-512, 1-513, 1-514, 1-515, 1-516, 1-517, 1-518, 1-519, 1-520, 1-521, 1-522, 1-523, 1-524, 1-525, 119, 1-120, 1-121, 1-122, 1-123, 1-124, 1-125, 1-126, 1-127, 1-128, 1-129, 1-130, 1-137, 1-138, 1-145, 1-146, 1-153, 1-154, 1-161, 1-162, 1-169, 1-170, 1-171 and 1-172.

[0080] In some embodiments, the ARCS is [ka] or a pharmaceutically acceptable salt thereof, and the linker of L may be [ka] and R2 is [ka] and the CLM is selected from the group consisting of [ka] Compounds encompassed by formula 1-51 include, but are not limited to, compounds 1-131, 1-132, 1-133, 1-134, 1-135, 1-136, 1-139, 1-140, 1-141, 1-142, 1-143, 1-144, 1-147, 1-148, 1-149, 1-150, 1-151, 1-152, 1-155, 1-156, 1-157, 1-158, 1-159, 1-160, 1-163, 1-164, 1-165, 1-166, 1-167, and 1-168 in Table 1.

[0081] Exemplary ARCS include any compound selected from the group consisting of Compound 1-101 through Compound 1-172. Table 1. Non-limiting examples of ARCS compounds [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0082] E. Pharmaceutical Compositions The ARCS of the present disclosure can be administered to a subject by any convenient means capable of producing the desired results.Therefore, the ARCS of the present disclosure can be incorporated into various formulations for therapeutic administration.More specifically, the ARCS of the present disclosure can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0083] As used herein, the term "pharmaceutical composition" refers to a composition comprising an ARCS as described herein and at least one pharmaceutically acceptable carrier, such as any carrier commonly used in the pharmaceutical industry. The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0084] Administration of the pharmaceutical composition can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc. In pharmaceutical dosage forms, the pharmaceutical composition may be administered alone or in combination with other pharmaceutically active compounds.

[0085] The amount of ARCS in the pharmaceutical composition may be based on weight, molar, or volume. In some embodiments, the pharmaceutical composition comprises at least 0.0001% ARCS. In some embodiments, the pharmaceutical composition comprises at least 0.1% ARCS. In some embodiments, the pharmaceutical composition comprises at least 0.5% ARCS. In some embodiments, the pharmaceutical composition comprises at least 1% of an ARCS compound. In some embodiments, the pharmaceutical composition comprises at least 2% ARCS. In some embodiments, the pharmaceutical composition comprises at least 3% ARCS. In some embodiments, the pharmaceutical composition comprises at least 4% ARCS. In some embodiments, the pharmaceutical composition comprises at least 5% ARCS. In some embodiments, the pharmaceutical composition comprises at least 10% ARCS. In some embodiments, the pharmaceutical composition comprises 0.05%-90% ARCS. In some embodiments, the pharmaceutical composition comprises 0.1%-85% ARCS. In some embodiments, the pharmaceutical composition comprises 0.5%-80% ARCS. In some embodiments, the pharmaceutical composition contains 1%-75% ARCS. In some embodiments, the pharmaceutical composition comprises 2%-70% ARCS. In some embodiments, the pharmaceutical composition contains 3% to 65% of ARCS. In some embodiments, the pharmaceutical composition contains 4% to 60% of ARCS. In some embodiments, the pharmaceutical composition contains 5% to 50% of ARCS.

[0086] It will also be understood that certain ARCSs can exist for treatment in free form or, where appropriate, as a pharmaceutically acceptable derivative thereof. In accordance with the present disclosure, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or prodrugs or other adducts or derivatives of ARCSs, which are included in compositions that, upon administration to a patient in need thereof, can directly or indirectly provide the compounds, as described herein, or metabolites or residues thereof.

[0087] As noted above, pharmaceutical compositions of the present disclosure may comprise pharmaceutically acceptable excipients, which as used herein includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, antioxidants, solid binders, lubricants, and the like, suitable for the particular dosage form desired.

[0088] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, or zinc stearate or stearic acid), or solvent encapsulating material that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve 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 carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and 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 soybean oil; and (10) glycols such as propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) bulking agents such as polypeptides and amino acids, (23) serum components such as serum albumin, HDL, and LDL, (24) C2-C12 alcohols such as ethanol, and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.As used herein, the terms "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably.

[0089] The pharmaceutical carrier useful for preparing the compositions herein can be solid, liquid or gas.Suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences by EW Martin.In any case, these compositions contain an effective amount of ARCS together with suitable carriers to prepare the appropriate dosage form for appropriate administration to recipients.

[0090] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to ARCS, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, and fragrances.

[0091] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the ARCS comprises at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, silicic acid, and the like; b) binders, for example, carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retardants such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0092] Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, and other coatings well known in the pharmaceutical formulation art. These solid dosage forms may optionally contain opacifying agents and may be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0093] The ARCS may also be in microencapsulated form with one or more excipients, as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In these solid dosage forms, the ARCS may be mixed with at least one inert diluent, such as sucrose, lactose, and starch. These dosage forms may also contain, as is common practice, additional substances other than inert diluents, such as tableting lubricants, magnesium stearate, and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. These solid dosage forms may optionally contain opacifying agents and may be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0094] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the drug, preferably isotonic with the recipient's blood. Suitable excipient solutions include phosphate-buffered saline, saline, water, lactated Ringer's solution, or dextrose (5% in water). Such formulations can be conveniently prepared by mixing the drug with water to form a solution or suspension, which is then filled into a sterile container and sealed against bacterial contamination. Sterile materials are preferably used under aseptic manufacturing conditions to avoid the need for terminal sterilization. Such formulations can optionally contain one or more additional ingredients, which may include preservatives such as methyl hydroxybenzoate, chlorocresol, metazole, phenol, and benzalkonium chloride. Such substances are of particular value when the formulation is presented in a multidose container.

[0095] Buffers can also be included to provide the formulation with a suitable pH value.Suitable buffer materials include sodium phosphate and sodium acetate.Sodium chloride or glycerin can be used to make the formulation isotonic with blood.

[0096] If desired, the formulations can be filled into containers under an inert atmosphere, such as nitrogen, and can conveniently be presented in unit-dose or multi-dose form, for example, in sealed ampoules.

[0097] One of ordinary skill in the art will recognize that the amounts of the various components of the compositions of the present disclosure to be administered to a subject according to the methods of the present disclosure will depend on the factors discussed above.

[0098] Injectable preparations can be formulated according to known techniques, for example, as sterile injectable aqueous or oily suspensions using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

[0099] Non-limiting examples of tablets contain active ingredient in an amount ranging from 10 mg to 100 mg, 70 mg to 95 mg powdered lactose, 10 mg to 35 g white corn starch, 1 mg to 8 mg polyvinylpyrrolidone, 1 mg to 10 mg sodium (Na) carboxymethyl starch (CMS), and 1 mg to 5 mg magnesium stearate, with tablet weights ranging from 200 mg to 3000 mg.

[0100] An example of a tablet of the present disclosure is as follows: Ingredients mg / tablet Active ingredient 100 Powdered lactose 95 White corn starch 35 Polyvinylpyrrolidone 8 Sodium Carboxymethyl Starch 10 Magnesium stearate 2 Tablet weight 250

[0101] Non-limiting examples of capsules contain active ingredient in an amount ranging from 10 mg to 100 mg, 50 mg to 75 mg of crystalline lactose, 10 mg to 35 mg of microcrystalline cellulose, 1 mg to 8 mg of talc, and 1 mg to 5 mg of magnesium stearate, with capsule fill weights ranging from 100 mg to 3000 mg.

[0102] An example of a capsule of the present disclosure is as follows: Ingredients mg / capsule Active ingredient 50 Crystalline lactose 60 Microcrystalline Cellulose 34 Talc 5 Magnesium stearate 1 Capsule Fill Weight 150

[0103] In the above capsules, the active ingredient has an appropriate particle size. Crystalline lactose and microcrystalline cellulose are mixed homogeneously and sieved, and then mixed with talc and magnesium stearate. The final mixture is filled into hard gelatin capsules of appropriate size.

[0104] A non-limiting example of an injection contains active ingredient in an amount ranging from 0.05 mg to 5 mg, 10.0 μL to 20.0 μL of 1N HCl, 0.1 mg to 1 mg of acetic acid, 1 mg to 10 mg of sodium chloride, 1 mg to 10 mg of phenol, a sufficient amount of 1N NaOH to adjust the pH to 4-5, and a sufficient amount of water.

[0105] Examples of injection solutions of the present disclosure are as follows: Component mg / solution Active substance 1.0mg 20.0 μl of 1 N HCl Acetic acid 0.5mg NaCl 8.0mg Phenol 10.0mg 1N NaOH qsad pH 5 H2O qsad 1mL

[0106] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0107] To prolong the effect of ARCS, it is often desirable to slow the absorption of ARCS from subcutaneous or intramuscular injection. This can be accomplished by using a poorly water-soluble liquid suspension or amorphous material. The rate of absorption of the ARCS then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered ARCS forms can be achieved by dissolving or suspending the ARCS in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of ARCS in biodegradable polymers such as polylactide-polyglycolide. The rate of ARCS release can be controlled depending on the ratio of ARCS to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by encapsulating ARCS in liposomes or microemulsions that are compatible with body tissues.

[0108] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the ARCS with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0109] A typical suppository formulation contains the ARCS or a pharmaceutically acceptable salt thereof, which, when administered in this manner, is active together with a binder and / or lubricant, such as, for example, polymeric glycols, gelatin, cocoa butter or other low-melting vegetable waxes or fats. Typical transdermal formulations include conventional aqueous or non-aqueous vehicles, such as creams, ointments, lotions, or pastes, or are in the form of medicated plastics, patches, or membranes.

[0110] Typical compositions for inhalation are in the form of a solution, suspension or emulsion, which can be administered in the form of an aerosol using conventional propellants such as dichlorodifluoromethane or trichlorofluoromethane.

[0111] Depending on the route of administration, one skilled in the art can determine and adjust accordingly the effective dose of a small molecule disclosed herein for a subject, such as a human subject.

[0112] Toxicity and therapeutic efficacy can be measured, for example, by LD 50 (lethal dose for 50% of the population) and ED 50 The LD (therapeutically effective dose in 50% of a population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Compositions that exhibit large therapeutic indices are preferred.

[0113] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as, for example, a non-human animal, e.g., a non-human mammal. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, agricultural animals such as cows, horses, chickens, and pigs, domestic animals such as cats and dogs, or research animals such as mice, rats, rabbits, dogs, and non-human primates.

[0114] II. How to use ARCS The ARCS described herein or compositions containing the ARCS described herein can be administered to treat any disease that can be treated with the FCB or any disease associated with the biological target of the ARCS, such as, but not limited to, cancer, neurodegenerative diseases, autoimmune diseases, or, where appropriate, aging. The formulations can be delivered to various body parts, such as the brain and central nervous system, eyes, ears, lungs, bones, heart, kidneys, liver, spleen, breast, ovaries, colon, pancreas, muscle, gastrointestinal tract, mouth, and skin, to treat diseases associated with these body parts. The formulations can be administered by injection, orally, or topically, typically to mucosal surfaces (pulmonary, nasal, oral, buccal, sublingual, vaginal, rectal) or to the eye (intraocular or ocular).

[0115] In aspects of the present disclosure, the ARCS binds to a biological target. In some embodiments, the biological target includes kinases such as, but not limited to, phosphoinositide 3-kinases (PI3Ks) and pseudokinases.

[0116] In some embodiments, the ARCS is capable of forming covalent bonds with biological targets. In some embodiments, the ARCS is capable of forming covalent bonds with biological targets from about 5% to 100% of the biological targets. In some embodiments, the ARCS is capable of forming covalent bonds with biological targets from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the biological targets.

[0117] In some embodiments, the ARCS can form a covalent bond with a PI3 kinase. In some embodiments, the ARCS can form a covalent bond with a PI3 kinase from about 5% to 100% of the PI3 kinase. In some embodiments, the ARCS can form a covalent bond with a PI3 kinase from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the PI3 kinase.

[0118] Protein kinases and pseudokinases Protein kinases and pseudokinases regulate signaling pathways for a wide range of biological processes under normal and disease conditions (Brognard, J. et al., Curr. Opin. Genet. Dev. 2011, 21, pp. 4-11; Cohen, P. et al., Nat. Cell Biol. 2002, 4, E127-130). In fact, most eukaryotic processes are controlled by kinases and / or pseudokinases. The term "pseudokinase" refers to proteins with kinase domains lacking catalytically relevant residues, including the lysine in the VAIK motif or the aspartic acid in the DFG motif. Examples of pseudokinases include HER3, STRAD, ILK, KSR1, and KSR2. While some pseudokinases are incapable of transferring phosphoryl groups, others retain the ability to transfer phosphoryl groups despite lacking catalytically relevant residues.

[0119] Kinases and pseudokinases can function by inhibiting and / or activating protein partners through binding, covalent modifications such as phosphorylation, conformational control, cellular localization, and / or other processes. Protein kinases and pseudokinases can promote and / or antagonize a wide range of diseases, including, but not limited to, cancer, Alzheimer's disease, aging, diabetes, cardiovascular disease, CNS-related diseases, immunological diseases, allergies, hypertension, and Parkinson's disease. Protein kinases and pseudokinases are also commonly mutated in multiple diseases, including, but not limited to, cancer and Parkinson's disease.

[0120] The molecules that target kinase and pseudokinase can act as anti-cancer drugs.Unfortunately, common inhibitors often have limited efficacy due to the short resonance time on target.Therefore, compared with previous kinase inhibitors, it is necessary to find kinase inhibitors that have improved resonance time on target, and at the same time have improved potency and efficacy.

[0121] In some embodiments, the ARCS of the present disclosure may target kinases and pseudokinases, thereby inhibiting the kinases and pseudokinases. The FCB of the ARCS may be an inhibitor of the kinases and pseudokinases. The CLM of the ARCS may be covalently bound to the kinases and pseudokinases.

[0122] In some embodiments, the present disclosure provides a method for treating a disease, comprising administering a therapeutically effective amount of an ARCS of the present disclosure or a composition comprising an ARCS, or a pharmaceutically acceptable salt thereof, to a patient in need thereof, wherein the patient has a disease caused in part or in whole by altered regulation of a target kinase or pseudokinase, such as cancer, Alzheimer's disease, aging, diabetes, cardiovascular disease, CNS-related disease, immunological disease, allergy, hypertension, or Parkinson's disease. In some embodiments, the disease is associated with PI3K or mutated PI3K. In some embodiments, the subject has a cancer with a mutation in the PIK3CA gene.

[0123] Administration The present disclosure provides a method comprising administering a composition comprising the ARCS described herein to a subject in need thereof. The composition comprising the ARCS described herein may be administered to a subject in any amount and using any route of administration effective for preventing or treating or imaging a disease, disorder, and / or condition. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc.

[0124] The compositions of the present disclosure are usually formulated in unit dosage form for ease of administration and uniformity of dosage.However, it is understood that the total daily use amount of the compositions of the present disclosure can be determined by the attending physician within the scope of sound medical judgment.For any specific patient, the specific therapeutically effective amount, prophylactically effective amount or suitable imaging dose level depends on various factors, including the disorder to be treated and the severity of the disorder, the activity of the specific compound used, the specific composition used, the age, weight, general health, sex and diet of the patient, the administration time, route of administration and excretion rate of the specific compound used, treatment period, the drug used in combination with the specific compound used or incidentally, as well-known factors in the medical field.

[0125] In some embodiments, compositions according to the present disclosure are administered one or more times daily at a dosage level sufficient to deliver about 0.0001 mg / kg to about 100 mg / kg to about 0.001 mg / kg to about 0.05 mg / kg to about 0.005 mg / kg to about 0.05 mg / kg to about 0.001 mg / kg to about 0.005 mg / kg to about 0.05 mg / kg to about 0.001 mg / kg to about 0.005 mg / kg to about 0.05 mg / kg to about 0.5 mg / kg to about 0.01 mg / kg to about 50 mg / kg to about 0.1 mg / kg to about 40 mg / kg to about 0.5 mg / kg to about 30 mg / kg to about 0.01 mg / kg to about 10 mg / kg to about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg of the subject's body weight per day to achieve the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dose can be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dose can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). When multiple administrations are used, a split-dose regimen, such as those described herein, can be used.

[0126] As used herein, "split dose" refers to dividing a single unit dose or a total daily dose into two or more doses, such as administering two or more single unit doses.As used herein, a "single unit dose" refers to a dose of any therapeutic agent administered at one time / single route / single contact, i.e., in a single administration event.As used herein, a "total daily dose" refers to the amount administered or prescribed in 24 hours.The total daily dose may be administered as a single unit dose.

[0127] III. Kits and Equipment The present disclosure provides various kits for conveniently and / or effectively implementing the methods of the present disclosure. Typically, the kits contain sufficient quantities and / or numbers of components to allow a user to perform multiple treatments of subjects and / or conduct multiple experiments.

[0128] In one embodiment, the present disclosure provides a kit for inhibiting tumor cell growth in vitro or in vivo, the kit comprising an ARCS of the present disclosure or a combination of ARCS of the present disclosure, optionally in combination with any other active agent.

[0129] The kit may further include packaging and / or instructions for forming the formulation composition, and / or a delivery agent. The delivery agent may include saline, a buffer solution, or any of the delivery agents disclosed herein. The amount of each component may be varied to allow for consistent and reproducible formulation of higher saline or simple buffer solutions. Components may also be varied to increase the stability of ARCS in the buffer solution over a period of time and / or under various conditions.

[0130] The present disclosure provides a device that can incorporate the ARCS of the present disclosure.These devices are contained in a stable formulation that can be immediately delivered to a subject in need thereof, such as a human patient.In some embodiments, the subject has cancer.

[0131] Non-limiting examples of the device include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices, and multilayer microfluidic devices. The device may be used to deliver the conjugates and / or particles of the present disclosure according to single, multiple, or divided administration regimens. The device may be used to deliver the conjugates and / or particles of the present disclosure to biological tissue, intradermally, subcutaneously, or intramuscularly.

[0132] A. Assay Covalent binding of ARCS to biological targets can be achieved using techniques including enzyme-linked immunosorbent assay (ELISA), gel assay, antibody array, Western blot, affinity ELISA, ELISPOT, immunochemistry (e.g., IHC), in situ hybridization (ISH), flow cytometry, immunocytology, surface plasmon resonance analysis, kinetic exclusion assay, liquid chromatography mass spectrometry (LCMS), tandem mass spectrometry (MS / MS), high performance liquid chromatography (HPLC), BCA assay, immunoelectrophoresis, SDS-PAGE, protein immunoprecipitation, and / or PCR.

[0133] As used herein, the term "assay" refers to a series of activities related to a reported result, which may include, but is not limited to, cell seeding, preparation of test material, infection, lysis, analysis, and calculation of results.

[0134] In some embodiments, the assay surface on the substrate is sterile and suitable for culturing cells under conditions representative of culture conditions during large-scale (e.g., industrial-scale) manufacturing of a biological product. In some embodiments, the exterior of the substrate includes a well, depression, border, etc., at a location corresponding to the assay surface. In some embodiments, the well, depression, border, or the like holds a fluid, such as, for example, cell culture medium, on the assay surface.

[0135] In some embodiments, the substrate comprises a microarray plate, biochip, or the like, allowing for high-throughput, automated testing of a series of test agents, conditions, and / or combinations thereof on the production of a biological product by cultured cells. For example, the substrate may comprise a two-dimensional microarray plate or biochip having m columns and n rows of assay surfaces (e.g., present in wells), allowing for the testing of m x n combinations of test agents and / or conditions (e.g., on a 24-, 96-, or 384-well microarray plate). The microarray substrate is preferably designed so that all necessary positive and negative controls can be performed in parallel with the testing of agents and / or conditions.

[0136] B. Screening Methods Generally, the synthesis of therapeutic conjugates that form covalent bonds with biological targets involves multiple synthesis and purification steps. When using these synthesis and purification steps, it can be difficult to generate a library of therapeutic conjugates or develop structure-activity relationships (SAR) for screening purposes. There remains a need for methods and systems for automatically generating libraries of therapeutic conjugates using organic synthesis methods. To discover therapeutic conjugate drug leads, a library of therapeutic conjugates against protein receptors must be screened to identify molecules that specifically bind to the receptor in a cellular environment, where the binding is covalent and irreversible. Identifying whether a therapeutic conjugate covalently binds to a specific target within a cell is difficult to prove.

[0137] Current methods in the art for screening therapeutic conjugates for their covalent attachment to biological targets involve tandem mass spectrometry approaches. Tandem MS or MS / MS is a method of decomposing selected ions into fragment ions. When a sample is ionized to produce a mixture of ions, precursor ions of specific mass-to-charge ratios (m / z) are selected (MS1) and then fragmented (MS2) to produce product ions for detection. Information about the chemical structure of the selected ions can be determined from the fragments.

[0138] However, there are challenges associated with mass spectrometry. Mass spectrometry is primarily limited to fragment screening, rather than drug-like molecule screening. Mass spectrometry analysis of therapeutic conjugates can lead to unresolved analyses due to the difficulty of identifying some fragments. This approach involves a time-consuming, multi-step process and manual analysis. MS / MS requires the complex, defragmentation of larger molecules, and manual recombination by individual peptides, making the resulting data difficult to interpret. Another drawback of using mass spectrometry is that detection is proportional to ionization rather than abundance, making this technique only slightly quantitative.

[0139] Therefore, there are challenges associated with the synthesis and screening of therapeutic conjugates. First, it is difficult to generate targeted covalent inhibitors that can access both cysteine ​​and non-cysteine ​​amino acids. It is also difficult to distinguish false-positive covalent inhibitors from actual positives.

[0140] To address the aforementioned problems, the inventors have combined a combinatorial synthesis approach with a reliable screening approach to identify potential therapeutic conjugates. The present disclosure provides a high-throughput, combined approach to synthesize therapeutic conjugates, rapidly tracking covalent binding, analyzing large libraries for duration of action, and directly quantifying covalent target binding within cells.

[0141] Using MS / MS techniques, thousands of molecules can be screened per day with picomolar sensitivity, compared to tens of molecules with nanomolar sensitivity. The present disclosure is compatible with any drug molecule, is not limited to fragments, and provides quantitative results with 95-99% reproducibility.

[0142] In some embodiments, the method of screening a library of ARCSs comprises: generating a library of ARCS in a composition; contacting the library with target cells; lysing the target cells to produce a lysate; labeling the lysate, and detecting the covalent binding of the ARCS to a biological target on the target cell.

[0143] Examples of human cell lines useful as target cells in the methods provided herein include, but are not limited to, 293T (embryonic kidney), 786-0 (renal), A498 (renal), A549 (alveolar basal epithelial), ACHN (renal), BT-549 (breast), BxPC-3 (pancreatic), CAKI-1 (renal), Capan-1 (pancreatic), CCRF-CEM (leukemia), COLO205 (colon), DLD-1 (colon), and intestine), DMS114 (small cell lung), DU145 (prostate), EKVX (non-small cell lung), HCC-2998 (colon), HCT-15 (colon), HCT-116 (colon), HT29 (colon), HT-1080 (fibrosarcoma), HEK293 (embryonic kidney), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), HL-60 (TB) (leukemia), HOP-62 (non-small cell lung), HOP-92 (non-small cell lung), HS 578T (breast), HT-29 (colon adenocarcinoma), IGR-OV1 (ovarian), IMR32 (neuroblastoma), Jurkat (T lymphocyte), K-562 (leukemia), KM12 (colon), KM20L2 (colon), LAN5 (neuroblastoma), LNCap.FGC (Caucasian prostate adenocarcinoma), LOX IMVI (melanoma), LXFL529 (non-small cell lung), M14 (melanoma), M19-MEL (melanoma), MALME-3M (melanoma), MCFlOA (mammary epithelial), MCF7 (mammary), MDA-MB-453 (mammary epithelial), MDA-MB-468 (breast), MDA-MB-231 (breast), MDA-N (breast), MOLT-4 (leukemia), NCI / ADR-RES (ovarian), NCI-H226 (non-small cell lung), NCI-H23 (non-small cell lung) Small cell lung), NCI-H322M (non-small cell lung), NCI-H460 (non-small cell lung), NCI-H522 (non-small cell lung), OVCAR-3 (ovarian), OVCAR-4 (ovarian), OVCAR-5 (ovarian), OVCAR-8 (ovarian), P388 (leukemia), P388 / ADR (leukemia), PC-3 (prostate), PERC6® (E1-transformed embryonic retina), RPMI-7951 (melanoma), RPMI-8226 (leukemia), RXF 393 (renal), RXF-631 (renal), Saos-2 (bone), SF-268 (CNS), SF-295 (CNS), SF-539 (CNS), SHP-77 (small cell lung), SH-SY5Y (neuroblast) cystoma), SK-BR3 (breast), SK-MEL-2 (melanoma), SK-MEL-5 (melanoma), SK-MEL-28 (melanoma), SK-OV-3 (ovary), SN12K1 (renal), SN12C (renal), S These include NB-19 (CNS), SNB-75 (CNS), SNB-78 (CNS), SR (leukemia), SW-620 (colon), T-47D (breast), THP-1 (monocyte-derived macrophage), TK-10 (renal), U87 (glioblastoma), U293 (renal), U251 (CNS), UACC-257 (melanoma), UACC-62 (melanoma), UO-31 (renal), W138 (lung), and XF498 (CNS).

[0144] Examples of rodent cell lines useful in the methods provided herein include, but are not limited to, baby hamster kidney (BHK) cells (e.g., BHK21 cells, BHK TK-cells), mouse Sertoli (TM4) cells, buffalo rat liver (BRL 3A) cells, mouse mammary tumor (MMT) cells, rat hepatoma (HTC) cells, mouse myeloma (NS0) cells, mouse hybridoma (Sp2 / 0) cells, mouse thymoma (EL4) cells, Chinese hamster ovary (CHO) cells and CHO cell derivatives, mouse embryonic (NIH / 3T3, 3T3L1) cells, rat cardiac muscle (H9c2) cells, mouse myoblast (C2C12) cells, and mouse kidney (miMCD-3) cells.

[0145] Examples of non-human primate cell lines useful in the methods provided herein include, but are not limited to, monkey kidney (CVI-76) cells, African green monkey kidney (VERO-76) cells, green monkey fibroblast (Cos-1) cells, and SV40 (Cos-7) transformed monkey kidney (CVI) cells. Additional mammalian cell lines are known to those of skill in the art and are cataloged in the American Type Culture Collection catalog (ATCC®, Mamasus, VA).

[0146] In some embodiments, cells are lysed using chemical and / or mechanical lysis. In some embodiments, chemical lysis involves a lysis buffer containing protease inhibitors, phosphate-buffered saline, and Triton X100. In some embodiments, cells can be frozen after the addition of the lysis buffer at -80°C for about 30 minutes to about 72 hours. Alternatively, the cell lysate can be stored at 2-8°C or at room temperature. In some embodiments, cells are centrifuged and the cell lysate is collected. In some embodiments, this is performed by spinning the cells in a centrifuge at 3,750 RPM for 10 minutes at room temperature.

[0147] The methods described herein can be performed by utilizing any of a wide variety of cell assay formats, including, but not limited to, cell plates, e.g., 24-well plates, 48-well plates, 96-well plates, or 384-well plates, individual cell culture plates or flasks, e.g., T-flasks or shaker flasks.

[0148] Covalent binding of ARCS can be detected by assays such as, but not limited to, gel assays, NanoBRET assays, Western blots, ELISAs, or microarrays. For example, gel assays can include microfluidic or capillary techniques to separate proteins by size.

[0149] In some embodiments, covalent binding is detected by a gel assay. A "gel assay" is defined as an assay in which cells or cell lysates are first treated with ARCS at a dose of 1 picomole to 1 millimolar for a duration of 2 minutes to 120 hours, using techniques known to those skilled in the art, including, but not limited to, common cell culture techniques. The cells are then lysed using techniques known to those skilled in the art, including, but not limited to, sonication or buffer lysis. The resulting lysate, also referred to as a non-purified cell lysate, can be further processed to produce a purified lysate using techniques known to those skilled in the art, including, but not limited to, centrifugation. The purified or non-purified lysate likely contains proteins covalently bound to the molecule of interest. A "coupling reagent" and a labeling molecule are added to the purified or non-purified lysate, and the ARCS in the reaction mixture is covalently labeled with the labeling molecule via copper-free or copper-driven click chemistry. The compound bound to the labeling molecule is then added, allowing for reliable separation of stoichiometry and reliable covalent drug tracking. Then, sample is carried out by Western blotting method known to those skilled in the art.Then, the amount of covalent binding can be tracked based on the shift of the drug-treated band compared with the untreated band.The band can be quantified by densitometry, and the relative abundance of band can be used to determine the quantitative amount of covalently bound label.

[0150] Generally, covalent binding of a high molecular weight protein / mass to an ARCS leads to a shift of the target-ARCS-high molecular weight protein mass complex in the gel. If an azide-linked molecule attached to a high molecular weight protein or any type of molecule is directly linked to an alkyne on the ARCS, a shift still occurs, making it possible to detect the covalent binding of the ARCS to the target.

[0151] In some embodiments, covalent binding is detected by a gel-only shift assay. A "gel-only shift assay" is defined as an assay in which a protein is expressed in any cell type (via transfection or infection) and linked to a tagging domain. This tagging domain comprises a fluorescent protein or a linker protein. Fluorescent proteins include GFP, RFP, and the like. Linker proteins include HALO, SNAP-, CLIP-, ACP-, and MCP-tags. After transfection or infection, the cells are then treated with a therapeutic conjugate of the present disclosure, which contains an alkyne capable of potential covalent binding. The cells are then lysed. In the case of protein expression using a linker protein, a "coupling reagent" is then added to covalently attach a fluorescent dye to the protein of interest. The lysate is then run on a gel, and the target protein can be visualized via in-gel fluorescence without the need for Western blot transfer. The amount of covalent binding can be tracked based on the shift of the drug-treated band compared to the untreated band. The bands can be quantified using densitometry, and the relative abundance of the bands can be used to determine the quantitative amount of covalently bound label.

[0152] The tagging domain can be any domain that allows target labeling. In some embodiments, the tagging domain comprises a label. This label may be contained in the domain itself, such as an epitope recognized by an antibody or a detectable light or radioactive label. In some embodiments, the label is a fluorescent marker, such as FITC, a phycobiliprotein, such as R- or B-phycoerythrin, allophycocyanin, AlexaFluor dye, Cy3, Cy5, Cy7, a luminescent marker, 125 I or 32 a radioactive label such as P, an enzyme such as horseradish peroxidase or alkaline phosphatase, or an alkaline phosphatase, e.g., alkaline shrimp phosphatase.

[0153] In some embodiments, "fluorescent protein" as used herein includes, but is not limited to, Aequorea victoria green fluorescent protein (GFP), red fluorescent protein (RFP), structural variants of GFP (i.e., circularly permuted, monomeric forms), folding variants of GFP (i.e., more soluble forms, superfolder forms), spectral variants of GFP (i.e., YFP, CFP), and GFP-like fluorescent proteins (i.e., Dsked). The term "GFP-like fluorescent protein" is used to refer to members of the Anthozoa fluorescent proteins that share the barrel structure of 11 beta-strands of GFP and its structural, folding, and spectral variants. "GFP-like non-fluorescent protein" and "GFP-like chromoprotein" (or simply "chromoprotein" or "chromoprotein") are used to refer to chromoproteins of the Anthozoa and Hydrozoa classes that share the barrel structure of 11 beta-strands of GFP and its structural, folding, and spectral variants.

[0154] In some embodiments, covalent binding is detected by Western blot-based shift assay. "Western blot-based shift assay" is defined as an assay in which a sample is run through Western blot, which is well known to those skilled in the art. Then, the covalently bound protein shifts compared to the non-covalently bound band, so the amount of covalent binding can be tracked based on the shift of the target. The band can be quantified using densitometry, and the relative abundance of the band can be used to determine the quantitative amount of covalently bound label.

[0155] In some embodiments, covalent binding is detected by an ELISA assay. In some embodiments, covalent binding is detected by ELISA assay 1. "ELISA assay 1" is defined as an assay in which a lysate containing a biotin-labeled drug is immobilized on a solid support via hybridization with monomeric or tetrameric streptavidin, streptavidin variants, or molecules that bind biotin. After the drug is immobilized, a detection antibody is added to detect the drug target of interest. The detection antibody may be covalently linked to an enzyme or may itself be detected by a secondary antibody linked to an enzyme or fluorescent label through a bioconjugation reaction. Between each step, the plate is washed with a solution to remove any nonspecifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzyme substrate to generate a visible signal, indicating the amount of covalent drug binding to the target of interest. The amount of covalent binding can be tracked based on the amount of signal.

[0156] In some embodiments, covalent binding is detected by ELISA assay 2. "ELISA assay 2" is defined as an assay in which the target of interest is immobilized on a solid support via hybridization with an antibody that binds the target of interest. After immobilization of the target of interest, a detection antibody, monomeric / tetrameric streptavidin, or streptavidin variant is added to detect the agent bound to the target of interest. The detection antibody, monomeric / tetrameric streptavidin, or streptavidin variant may be covalently linked to an enzyme or fluorescent label, or may itself be detected by a secondary antibody linked to an enzyme or fluorescent label via a bioconjugation reaction. Between each step, the plate is washed with a solution to remove any nonspecifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzyme substrate to generate a visible signal, or the fluorescent signal is measured directly, indicating the amount of covalent agent binding to the target of interest. The amount of covalent binding can be tracked based on the amount of signal.

[0157] In some embodiments, covalent binding is detected by an antibody array. An "antibody array" is defined as a system in which an individual antibody or multiple antibodies are bound to a solid support, allowing for the detection of proteins of interest that bind to the antibodies and molecules bound to the proteins of interest. An antibody microarray consists of a series of individual dots or wells, with a specific antibody hybridized to each dot or well (described in U.S. Patent No. 20120231963A1, the contents of which are incorporated herein by reference in their entirety). Bound purified or unpurified lysates are added to the "antibody microarray" to allow for the separation of different proteins, the localization of specific proteins to antibody binding partners, or the washing out of additional proteins. A labeled molecule of interest is detected in each microarray dot or well to determine the amount of covalent binding of the molecule of interest to a specific protein or proteins by detecting the presence of a biotin-labeled molecule and revealing the level of labeling. The level of labeling at each dot indicates the amount of covalent labeling of a specific protein hybridized to a specific antibody. The biotin label on the molecule can be detected via the addition of a fluorescent molecule or a luminescent enzyme that binds to the label, including, but not limited to, techniques known to those skilled in the art such as fluorescence, luminescence, FRET or BRET assays, etc. This readout can be detected using approaches known to those skilled in the art, including, but not limited to, fluorescence or luminescence detection schemes.

[0158] In some embodiments, the lysate is labeled with biotin to generate a biotinylated compound. In some embodiments, streptavidin is added to bind to the biotinylated compound. In some embodiments, the streptavidin is monomeric. In some embodiments, the biotinylated compound is generated by click chemistry. In some embodiments, the compound is labeled with click chemistry after treatment with ARCS, cell isolation, and cell lysis. In some embodiments, the click chemistry reagent comprises picolyl azide.

[0159] As used herein, the term "click chemistry" refers to the Huisgen cycloaddition reaction or the 2,3-dipolar cycloaddition reaction between an azide and a terminal alkyne to form a 1,2,4-triazole. As used herein, the term "cycloaddition reaction" refers to a chemical reaction in which two or more π-electron systems (e.g., unsaturated molecules or unsaturated portions of the same molecule) join to form a cyclic product with a net reduction in bond multiplicity. In a cycloaddition reaction, π electrons are used to form a new sigma bond. The π-electron product is called an "adduct" or "cycloadduct." Different types of cycloaddition reactions are known in the art, including, but not limited to, the [3 + 2] cycloaddition reaction and the Diels-Alder reaction. The [3 + 2] cycloaddition reaction, also known as the 2,3-dipolar cycloaddition reaction, occurs between a 1,3-dipolar and a dipolarophile and is commonly used to construct five-membered heterocycles. The term "[3 + 2] cycloaddition reaction" also includes the "copper-less" [3 + 2] cycloaddition reaction between an azide, a cyclooctyne derivative, and a difluorocyclooctyne, as described in Bertozzi et al., J. Am. Chem. Soc., 2004, 126:15046-15047. Any reagent that can be used to promote a Huisgen cycloaddition reaction can be used as a click chemistry reagent. In some embodiments, the click chemistry reagent comprises a pyridyl azide. In some embodiments, the click chemistry reagent comprises a pyridyl azide. Without limitation, any isomer of pyridyl azide may be used.

[0160] In some embodiments, the ARCS may be associated with or bound to one or more radioactive or detectable agents, including various small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18 F, 67 Ga, 81m Kr, 82 Rb, 111 In, 123 I, 133 Xe,201 Tl, 125 I, 35 S, 14 C. 3 H or 99m Tc (e.g., pertechnetic acid (technetic acid(VII), TcO4 -)) and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), single crystalline iron oxide nanoparticles (MION), ultrasuperparamagnetic iron oxide (USPIO), manganese chelates (e.g., Mn-DPPP), barium sulfate, iodinated contrast agents (iohexol), microbubbles, or perfluorocarbons). Such optically detectable labels include, for example, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine and derivatives (e.g., acridine and acridine isothiocyanate), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 Disulfonates, N-(4-anilino-L-naphthyl)maleimide, anthranilamide, BODIPY, brilliant yellow, coumarin and derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), and 7-amino-4-trifluoromethylcoumarin (coumarin 151)), cyanine dyes, cyanosine, 4',6-diaminidino-2-phenylindole (DAPI), 5',5' '-Dibromopyrogallol-sulfonaphthalene (Bromopyrogallol Red), 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, diethylenetriaminepentaacetate, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, danyl chloride), 4 -dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin and derivatives (e.g., eosin and eosin isothiocyanate), erythrosin and derivatives (e.g., erythrosin B and erythrosin isothiocyanate), ethidium, fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-Dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein isothiocyanate, quinoline-5-(and -6)-isothiocyanate (QFITC ​​or XRITC) and fluorescamine), 2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2-benzo[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulfolpropyl)-1-benzo[e]indolium hydroxide, inner salt, N,N-diethylethanamine ( 1:1) (IR144), 5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140), malachite green isothiocyanate, 4-methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, ophthalaldehyde, pyrene and derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene), butyric acid quantum dots, Reactive Red 4 (CIBACRON™ Brilliant Red 3B-A), rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), Lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, cyanine-3 (Cy3), cyanine-5 (Cy5), cyanine-5.5 (Cy5.5), cyanine-7 (Cy7), IRD 700, IRD 800, Alexa 647, La Jolta Blue, phthalocyanines, and naphthalol cyanines.

[0161] In some embodiments, the detectable agent may be a non-detectable precursor that becomes detectable upon activation (e.g., a fluorescent tetrazine-fluorophore construct (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X)) or an enzyme-activatable fluorogenic agent (e.g., PROSENSE® (Vis Medical))).

[0162] IV. Definition As used herein, the term "ARCS" refers to any therapeutic conjugate formed by linking an FCB and a CLM via a bond or linker. In some embodiments, the ARCS can form a covalent bond with one or more targets, such as a nucleotide, oligonucleotide, peptide, or protein. In some embodiments, the covalent bond is formed in aqueous solution at a therapeutic dose of 10 mM, within 48 hours at a temperature of 0-50°C.

[0163] As used herein, the term "FCB" refers to known drugs, diagnostic compounds, drug candidates, and therapeutic modalities, which may be functional fragments and / or combinations of any of the foregoing. FCBs include free acid and free base forms, optical isomers and tautomers, radioisotopes of drugs, and isotopes, including pharmaceutically acceptable salts, prodrugs, or fragments thereof. FCBs may be small molecules, proteins, peptides, lipids, carbohydrates, sugars, nucleic acids, or combinations thereof. In some embodiments, FCBs are nucleic acids, including, but not limited to, DNA or RNA. FCBs may be therapeutic agents, such as, but not limited to, anti-cancer agents, anti-neurodegenerative agents, autoimmune agents, and anti-aging agents. FCBs may non-covalently bind to biological targets. In some embodiments, FCBs may be functional fragments of drugs. As used herein, the term "functional fragment" refers to a portion of a drug or its derivatives or analogs that can elicit the desired effect of the drug. In some embodiments, FCBs may contain an alkyne functional group. In some embodiments, FCBs may not contain an alkyne functional group.

[0164] As used herein, the term "CLM" refers to any covalent bonding modality capable of forming a covalent bond with a biological target. The CLM may be linked to the FCB by a bond or a linker. The CLM may contain one or more chemical moieties capable of forming a covalent bond with the biological target. The chemical moieties may be electrophilic or nucleophilic groups.

[0165] As used herein, the term "linker" refers to an organic moiety that connects two parts of a compound. A linker may be an external linker or an internal linker. An external linker can connect the FCB part and the CLM part. An internal linker can be used to connect the CLM parts. In certain embodiments, the CLM may include an internal linker or spacer. The internal linker or spacer may connect the two parts of the CLM or may be attached to the CLM. External or internal linkers include bonds, substituted and unsubstituted C1-C 30 Alkyl, substituted and unsubstituted C2-C 30 Alkenyl, substituted and unsubstituted C-C 30 Alkynyl, substituted and unsubstituted C3-C 30 Cycloalkyl, substituted and unsubstituted C 1~ C 30 Heterocycloalkyl, substituted and unsubstituted C3-C 30 Cycloalkenyl, substituted and unsubstituted C1-C 30 It can be selected from the group consisting of heterocycloalkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl. The linker can be cleavable or non-cleavable.

[0166] As used herein, the term "biological target" refers to any target that FCB binds non-covalently to produce a therapeutic effect.CLM is covalently bound to biological target.In some embodiments, biological target is protein.

[0167] As used herein, the term "toxicity" refers to the ability of a substance or composition to be harmful or toxic to a cell, living tissue, or cellular environment. Low toxicity refers to the attenuated ability of a substance or composition to be harmful or toxic to a cell, living tissue, or cellular environment. Such attenuation or low toxicity may be relative to a standard measure, a treatment, or the absence of a treatment.

[0168] As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. In some embodiments, compound is used interchangeably with ARCS. Thus, as used herein, ARCS is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. As used herein, FCB and CLM are also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures.

[0169] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials, such as resolution of racemic mixtures or stereoselective synthesis, are known in the art. Many geometric isomers of olefins, C=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0170] The compounds of the present disclosure also include tautomers. Tautomers result from the swapping of a single bond with an adjacent double bond and the simultaneous migration of a proton. Tautomers include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidinic acid pairs, lactam-lactim pairs, amide-imidinic acid pairs, enamine-imine pairs, and tautomers in which the proton is substituted, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomers may be in equilibrium or sterically locked into one form by appropriate substitution.

[0171] The compounds of the present disclosure also include all isotopes of atoms occurring in intermediate or final compounds. "Isotopes" refer to atoms with the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0172] The compounds and salts of the present disclosure can be prepared in combination with solvents or water molecules to form solvates and hydrates by routine methods.

[0173] As used herein, the term "subject" or "patient" refers to any organism to which particles can be administered, e.g., for experimental, therapeutic, diagnostic, and / or prophylactic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, guinea pigs, cows, pigs, sheep, horses, dogs, cats, hamsters, llamas, non-human primates, and humans).

[0174] As used herein, the terms "treating" or "preventing" can include preventing a disease, disorder, or condition from occurring in an animal that is believed to be susceptible to the disease, disorder, and / or condition but has not been diagnosed as having the disease, disorder, or condition; inhibiting a disease, disorder, or condition, e.g., inhibiting its progression; alleviating a disease, disorder, or condition, e.g., causing regression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition can also include ameliorating at least one symptom of a particular disease, disorder, or condition even if the underlying pathophysiology is unaffected, e.g., treating pain in a subject by administering an analgesic, even if the analgesic does not treat the cause of the pain.

[0175] As used herein, "target" refers to the site to which an ARCS, FCB, and / or CLM binds. The target may be either in vivo or in vitro. In certain embodiments, the target may be a cancer cell found in a leukemia or tumor (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast, and colon, as well as other carcinomas and sarcomas). The target may also be a type of tissue, such as nervous tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.

[0176] "Target cells" that can serve as targets for therapeutic conjugates are generally animal cells, e.g., mammalian cells. The methods of the present invention can be used to modify the cellular function of living cells that form part of or are otherwise present in animal tissues, either in vitro, i.e., in cell culture, or in vivo. Thus, target cells can include, for example, blood, lymphatic tissue, cells lining the digestive tract, such as the oral and pharyngeal mucosa, cells forming the ciliary bodies of the small intestine, cells lining the large intestine, cells lining the animal's respiratory system (nasal cavity / lungs) (which can be accessed by inhalation in a subject), skin / epidermal cells, cells of the vagina and rectum, cells of the placenta, and cells of internal organs, including the so-called blood / brain barrier.

[0177] The term "therapeutic effect" is art-recognized and refers to a local or systemic effect caused by a pharmacologically active substance in animals, particularly mammals, and more particularly humans. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease or in the enhancement of desirable physical or mental development and conditions in animals or humans.

[0178] The term "modulation" is art-recognized and refers to the up-regulation (ie, activation or stimulation), down-regulation (ie, inhibition or suppression) of a response, or the two in combination or separately.

[0179] As used herein, "enteral administration" refers to administration by any method other than the digestive tract (enteral) or non-invasive topical route. For example, parenteral administration can include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intraosseously, intracerebrally, intrathecally, intramuscularly, subcutaneously, subconjunctivally, by injection, and by infusion.

[0180] As used herein, "topical administration" refers to non-invasive administration to the skin, orifices, or mucous membranes. Topical administration can be administered locally, i.e., can provide a local effect in the application area without systemic exposure. Topical formulations can provide a systemic effect through absorption into the individual's bloodstream. Topical administration can include, but is not limited to, dermal and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ocular administration, and rectal administration.

[0181] As used herein, "enteral administration" means administration via absorption through the digestive tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.

[0182] As used herein, "pulmonary administration" refers to administration to the lungs by inhalation or intratracheal administration. As used herein, the term "inhalation" refers to the inhalation of air into the alveoli. Air intake can occur through the mouth or nose.

[0183] As used interchangeably herein, the terms "sufficient" and "effective" refer to the amount (e.g., mass, volume, dose, concentration, and / or duration) required to achieve one or more desired results. A "therapeutically effective amount" is at least the minimum concentration required to measurably improve or prevent at least one symptom of a particular condition or disorder, to measurably increase life expectancy, or to generally improve a patient's quality of life. Thus, the therapeutically effective amount depends on the specific bioactive molecule and the specific condition or disorder being treated. Therapeutically effective amounts of many active agents, such as antibodies, are known in the art. For example, the therapeutically effective amount of the compounds and compositions described herein for treating a particular disorder can be determined by techniques within the skill of a physician or other skilled artisan.

[0184] The term "prodrug" refers to a drug containing a nucleic acid or protein that is converted into a biologically active form in vitro and / or in vivo. In some situations, prodrugs may be useful because they may be easier to administer than the parent compound. For example, a prodrug may be bioavailable by oral administration, whereas the parent compound is not. A prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, NJ (1962) Drug Latentiation in Jucker, d. Progress in Drug Research, 4:221-294; Molozowich et al. (1977) Application of Physical Organic Principles to Prodrug Design in EBRoche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad. Pharm. Sci.; EB Roche, ed. (1977) Bioreversible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs, Elsevier, Wang et al. (1999) Prodrug approaches to the professional of peptide drug, Curr.Pharm.Design.5(4):265-287; Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv.Drug.Delivery Rev.27:235-256; Mizen et al. (1998).The Use of Esters as Prodrugs for Oral Delivery of β-Lactam antibiotics、Pharm.Biotech.11:345-365;Gaignaultら(1996)Designing Prodrugs and Bioprecursors I.Carrier Prodrugs、Pract.Med.Chem.671-696;M.Asgharnejad(2000)。Improving Oral Drug Transport Via Prodrugs、in G.L.Amidon,P.I.Lee and E.M.Topp、Eds.、Transport Processes in Pharmaceutical Systems、Marcell Dekker、p.185-218;Balantら(1990)Prodrugs for the improvement of drug absorption via different routes of administration、Eur.J.Drug Metab.Pharmacokinet.、15(2):143-53;Balimane and Sinko(1999)。Involvement of multiple transporters in the oral absorption of nucleoside analogs、Adv.Drug Delivery Rev.、39(1-3):183-209;Browne(1997)。Fosphenytoin(Cerebyx)、Clin.Neuropharmacol.20(1):1-12;Bundgaard(1979)。Bioreversible derivatization of drugs--principle and applicability to improve the therapeutic effects of drugs、Arch.Pharm.Chemi.86(1):1-39;H.Bundgaard、ed.(1985)Design of Prodrugs、Elsevier、Fleisherら(1996)Improved oral drug delivery:solubility limitations overcome by the use of prodrugs、Adv.Drug Delivery Rev.19(2):115-130;Fleisherら(1985)Design of prodrugs for imprograms for imprograms gastrounic absorption by intestinal enzyme targeting、Methods Enzymol.112:360-81;Farquhar Dら(1983)Biologically Reversible Phosphate-Protective Groups、J.Pharm.Sci.,72(3):324-325;Han,H.K.ら(2000)Targeted prodrug design to optimize drug delivery、AAPS PharmSci.,2(1):E6;Sadzuka Y.(2000)Effective prodrug liposome and conversion to active metabolite、Curr.Drug Metab.、1(1):31-48;.M.Lambert(2000)Rationale and applications of lipids as prodrug carriers、Eur.J.Pharm.Sci.、11 Suppl.2:S15-27;Wangら(1999)Prodrug approaches to the improved delivery of peptide drugs.Curr.Pharm.Des.,5(4):265-87。

[0185] The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, in accordance with guidelines from agencies such as the U.S. Food and Drug Administration, commensurate with a reasonable benefit / risk ratio. As used herein, "pharmaceutically acceptable carrier" refers to all components of a pharmaceutical formulation that facilitate delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0186] As used herein, the term "molecular weight" generally refers to the mass or average mass of a material. In the case of polymers or oligomers, molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways, including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as weight-average molecular weights (Mw) as opposed to number-average molecular weights (Mn). Capillary viscometry provides an estimate of molecular weight as the intrinsic viscosity determined from a dilute polymer solution using a specific set of concentration, temperature, and solvent conditions.

[0187] As used herein, the term "small molecule" generally refers to an organic molecule with a molecular weight of less than 2000 g / mol, less than 1500 g / mol, less than 1000 g / mol, less than 800 g / mol, or less than 500 g / mol. Small molecules are non-polymeric and / or non-oligomeric.

[0188] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0189] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., a straight chain has C 1- C 30 , branched chain is C 3- C 30 ), 20 or fewer, 12 or fewer, or 7 or fewer carbon atoms. Similarly, in some embodiments, cycloalkyls have from 3 to 10 carbon atoms in their ring structure, such as 5, 6, or 7 carbons in the ring structure. The term "alkyl" (or lower alkyl), as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamidosulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0190] Unless the number of carbons is otherwise specified, as used herein, "lower alkyl" refers to an alkyl group, as defined above, having 1 to 10 carbons or 1 to 6 carbon atoms in its backbone structure. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout this application, preferred alkyl groups are lower alkyls. In some embodiments, a substituent designated herein as alkyl is a lower alkyl.

[0191] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For example, substituents on substituted alkyls can include halogen, hydroxy, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, and carboxylates and esters), -CF, -CN, and the like. Cycloalkyls can be similarly substituted.

[0192] As used herein, the term "heteroalkyl" refers to a linear, branched, or cyclic carbon-containing radical or combination thereof containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, with phosphorus and sulfur atoms optionally oxidized, and nitrogen heteroatoms optionally quaternized. Heteroalkyl can be substituted as defined above for alkyl groups.

[0193] The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio and ethylthio. The term "alkylthio" also encompasses cycloalkyl, alkene, and cycloalkene groups, and alkyne groups. "Arylthio" refers to an aryl or heteroaryl group. An alkylthio group can be substituted as defined above for an alkyl group.

[0194] The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0195] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, the substituent of an alkyl that makes the alkyl an ether is or resembles an alkoxyl, and can be represented, for example, by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. An aroxy may be represented by -O-aryl or -O-heteroaryl, where aryl and heteroaryl are defined below. Alkoxy and aroxy groups can be substituted as described above for alkyl.

[0196] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, and include, for example, those of the general formula: [ka] R9, R 10 , and R' 10 are each independently hydrogen, alkyl, alkenyl, -(CH2) m -R8 or R9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure. R8 represents an aryl, cycloalkyl, cycloalkenyl, heterocycle, or polycycle, and m is zero or an integer ranging from 1 to 8. In some embodiments, R9 or R 10 Only one of R can be a carbonyl, e.g., R, R 10 and the nitrogen together do not form an imide. In yet other embodiments, the term "amine" does not include amides, e.g., R and R 10 In a further embodiment, one of R and R represents carbonyl. 10(and optionally R' 10 ) each independently represent hydrogen, alkyl or cycloalkyl, alkenyl or cycloalkenyl, or alkynyl. Thus, as used herein, the term "alkylamine" refers to an amine group having a substituted (as defined above for alkyl) or unsubstituted alkyl attached thereto, i.e., R and R 10 means that at least one of the groups is an alkyl group.

[0197] The term "amide" is art-recognized as an amino-substituted carbonyl and has the general formula: [ka] and R and R 10 is as defined above.

[0198] As used herein, "aryl" refers to a C5-C 10 "Aryl" refers to a 5-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or biheterocyclic ring system. Broadly defined, "aryl," as used herein, includes 5-, 6-, 7-, 8-, 9-, and 10-membered monocyclic aromatic groups that may contain 0 to 4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." Aromatic rings include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocycle, aromatic ring or heteroaromatic moiety, -CF3, -CN; and combinations thereof.

[0199] The term "aryl" also refers to a macrocyclic system having two or more cyclic rings, where two or more carbons are common to two adjacent rings (i.e., fused rings), and at least one of the rings is aromatic; for example, the other cyclic ring or rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocycle.Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, and the like. Imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1 ,3,4-Oxadiazolyl, oxazolidinyl, oxazolyl, oxidryl, pyrimidinyl, phenanthridinyl, phenanthronyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl , pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinusidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.One or more of the rings may be substituted with "aryl" as defined above.

[0200] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[0201] As used herein, the term "carbocycle" refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

[0202] "Heterocycle" or "heterocyclic," as used herein, refers to a cyclic radical attached through a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3 to 10 ring atoms, preferably 5 to 6 ring atoms, and consisting of 1 to 4 heteroatoms selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y), where Y is absent or is selected from H, O, (C1-C 10) alkyl, phenyl, or benzyl, optionally containing 1 to 3 double bonds and optionally substituted with one or more substituents.Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzthoxazolyl, benzthothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, fura Zanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-Oxadiazolyl, oxazolidinyl, oxazolyl, oxidryl, pyrimidinyl, phenanthridinyl, phenanthronyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl , pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinusidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.Aromatic ring groups may be optionally substituted at one or more positions with one or more substituents as defined above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocycle, aromatic ring, or heteroaromatic moiety, -CF3, and -CN.

[0203] The term "carbonyl" is art-recognized and has the general formula: [ka] wherein X is a bond or represents oxygen or sulfur, and R 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl, and R' 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, or anyl; X is oxygen; and R 11 or R' 11 is not hydrogen, the formula represents an "ester". 11 is as defined above, the moiety is referred to herein as a carboxyl group, and in particular R 11 Where X is an oxygen and R' is a hydrogen, the formula represents a "carboxylic acid". 11 Where R is hydrogen, the formula represents a "formic acid." In general, where the oxygen atom of the above formula is replaced with a sulfur, the formula represents a "thiocarbonyl" group. 11 or R' 11 Where X is a sulfur and R is a 11 Where X is a sulfur and R' is a hydrogen, the formula represents a "thiocarboxylic acid". 11 is hydrogen, the formula represents "thioformic acid". 11 When X is not hydrogen, the above formula represents a "ketone" group. 11Where is hydrogen, the above formula represents an "aldehyde" group.

[0204] As used herein, the term "monoester" refers to an analog of a dicarboxylic acid, where one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is the free carboxylic acid or a salt of the carboxylic acid. Examples of monoesters include, but are not limited to, the monoesters of succinic acid, glutamic acid, adipic acid, sulfurous acid, sebacic acid, azelaic acid, oxalic acid, and maleic acid.

[0205] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Examples of heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium. Other heteroatoms include silicon and arsenic.

[0206] As used herein, the term "nitro" means -NO2, the term "halogen" refers to -F, -Cl, -Br or -I, the term "sulfhydryl" means -SH, the term "hydroxyl" means -OH, and the term "sulfonyl" means -SO2-.

[0207] As used herein, the term "substituted" refers to all permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms, preferably 1 to 14 carbon atoms, optionally containing one or more heteroatoms such as oxygen, sulfur, or nitrogen groups in a linear, branched, or cyclic structural format. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C 3- C 20 Cyclic, substituted C 3- C 20 Includes cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide and polypeptide groups.

[0208] Heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. It is understood that "substituted" or "substituted" implicitly includes the proviso that such substitution is in accordance with the permissible valences of the substituted atom and substituents, and that substitution results in a stable compound, i.e., a compound that does not undergo spontaneous transformation, such as by rearrangement, cyclization, or elimination.

[0209] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more of the same or different for appropriate organic compounds. Heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms.

[0210] In various embodiments, the substituents are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each optionally substituted with one or more suitable substituents. In some embodiments, the substitutions are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone may be further substituted with one or more suitable substituents.

[0211] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, hetero Examples include aralkoxy, azido, alkylthio, oxo, acylalkyl, carboxyester, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, etc. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.

[0212] The terms "polypeptide," "peptide," and "protein" generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of a corresponding naturally occurring amino acid. The term "protein," as generally used herein, refers to a polymer of amino acids linked together by peptide bonds to form a polypeptide whose chain length is sufficient to create tertiary and / or quaternary structure. The term "protein," by definition, excludes small peptides, which lack the higher-order structure necessary to be considered a protein.

[0213] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence is not identical to the full-length protein, polypeptide, or nucleic acid but which retains at least one function as the full-length protein, polypeptide, or nucleic acid. A functional fragment can have more, fewer, or the same number of residues as the corresponding native molecule and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining protein function are well known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, genetically or biochemically, for example, by co-immunoprecipitation, two-hybrid assays, or complementation. See, for example, Fields et al. (1989) Nature 340:245-246, U.S. Patent No. 5,585,245 and PCT WO 98 / 44350.

[0214] The term "pharmaceutically acceptable counterion" refers to a pharmaceutically acceptable anion or cation. In various embodiments, the pharmaceutically acceptable counterion is a pharmaceutically acceptable ion. For example, pharmaceutically acceptable counterions include citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, isonicotinic acid, acetate, lactate, salicylate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, benzoates ... In some embodiments, the pharmaceutically acceptable counterion is selected from chloride, bromide, iodide, nitrate, sulfate, disulfate, phosphate, acid phosphate, citrate, malate, acetate, oxalate, acetate, and lactate. In certain embodiments, the pharmaceutically acceptable counterion is selected from chloride, bromide, iodide, nitrate, sulfate, disulfate, and phosphate.

[0215] The term "pharmaceutically acceptable salt" refers to a salt of an acidic or basic group that may be present in the compound used in the composition of the present invention. The compound included in the composition of the present invention that is basic in nature can form a wide variety of salts with various inorganic and organic acids. The acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, disulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, and glutamic acid salts. The salts of the compounds of the present invention include thamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate) salts. Compounds included in the compositions of the present invention that contain an amino moiety can form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the compositions of the present invention that are acidic in nature can form base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[0216] When the compounds described herein are obtained as acid addition salts, the free base can be obtained by basifying a solution of the acid salt. Conversely, when the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art are aware of various synthetic methodologies that can be used to prepare non-toxic pharmaceutically acceptable addition salts.

[0217] Pharmaceutically acceptable salts include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutamic acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, and glucuronic acid. , glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isethionic acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucilage, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phosphoric acid, proprionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0218] As used herein, the term "assay" refers to a series of activities related to a reported result, which may include, but is not limited to, cell seeding, preparation of test material, infection, lysis, analysis, and calculation of results.

[0219] As used herein, the term "detectable response" refers to the occurrence or change of a signal that can be detected directly or indirectly by either observation or measurement. Typically, the detectable response is the occurrence of a signal, and the fluorophore is inherently fluorescent and does not produce a signal change upon binding to a metal ion or biological compound. Alternatively, the detectable response is an optical response resulting in a change in wavelength distribution pattern, absorbance, or fluorescence intensity, or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0220] It should be understood that the following examples illustrate, but do not limit, the present disclosure. Various other examples and modifications of the foregoing description and examples will be apparent to those skilled in the art after reading this disclosure without departing from the spirit and scope of the present disclosure, and all such examples or modifications are intended to be included within the scope of the appended claims. All publications and patents referenced herein are incorporated herein by reference in their entirety. [Example]

[0221] Example 1: General synthesis of ARCS The ARCS of the present disclosure can be synthesized by those skilled in the art using common chemical synthesis principles and techniques. In a rational approach, the ARCS is constructed from its individual components, i.e., the therapeutic modality, optional linker, and covalent attachment modality. The components may be covalently linked to each other via functional groups, as known in the art; such functional groups may be present on the components or may be introduced onto the components using one or more steps. Functional groups that can be used to covalently link the components together to form the ARCS include, but are not limited to, hydroxy, sulfhydryl, or amino groups. The specific moieties of the different components that are modified to provide covalent attachment are selected so as not to substantially adversely affect the desired binding activity of the component, e.g., for the covalent attachment modality, and regions that do not affect the covalent attachment activity are modified so that a sufficient amount of the desired activity is retained. If necessary and / or desired, specific moieties on the components may be protected using blocking groups known in the art; see, for example, Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

[0222] Alternatively, ARCSs can be generated using well-known combinatorial methods to generate large libraries of ARCSs, which may then be screened to identify molecules that form covalent bonds with targets with the desired pharmacokinetic profile.

[0223] Example 2: General synthesis of compounds 1-1 to 1-172 Compounds 1-1 to 1-172 of the present disclosure can be synthesized by one skilled in the art using common chemical synthesis principles and techniques, as described in Example 1 of U.S. Patent No. 9,724,352 B2, the contents of which are incorporated herein by reference in their entirety.

[0224] Compound 1-IV, a precursor to many of compounds 1-1 through 1-172, can be prepared as shown in Scheme 1. As described in U.S. Pat. No. 9,724,352, starting pyrrole 1-I can be reacted with aldehyde 1-II to form intermediate 1-III. Morpholino compound 1-IV can be formed by reducing compound 1-III with phosphorus oxychloride to provide a Cl leaving group, followed by displacement by the addition of morpholine. If R6' in 1-IV is a nitro group, it can be reduced to the corresponding NH2 group by reaction with C / Pd under an H2 atmosphere. The amine can then be further reacted with an appropriate ester (e.g., dimethyl carbonate) or activated ester (e.g., methyl chloroformate) to form the methyl carbamate of the compound, or with an isocyanate (e.g., methyl isocyanate) to provide urea compound 1-V. Scheme 1 [ka]

[0225] To access piperazinyl compounds (e.g., compounds 1-11 and 1-12), it may be advantageous to modify the methyl ester moiety of compound 1-IV and then convert R6' to the final R6 group. As mentioned above, when R6' in 1-IV is a nitro group, it can be reduced to the corresponding NH2 group by Pd / C under an H2 atmosphere. As shown in Scheme 2, the methyl ester of 1-IV can be hydrolyzed (e.g., with 2 M aqueous NaOH in EtOH) and the resulting acid can be reacted with tert-butylpiperazine-1-carboxylic acid (1-BOC-piperazine) in the presence of a base (e.g., K2CO3) to provide the BOC-piperazinyl compound. The carbonyl moiety remaining after piperazinyl addition can then be reduced (e.g., with borane-dimethylsulfide) to provide the protected piperazinyl compound 1-VI. The amine group of compound 1-VI can then be further reacted with an appropriate ester (e.g., dimethyl carbonate) or activated ester (e.g., methyl chloroformate) to form the methyl carbamate of compound 1-V, or with an isocyanate (e.g., methyl isocyanate) to give the urea compound 1-VII. A (See, for example, the groups Compound 1-105 and Compound 1-106). The amine group of Compound 1-VI can also be protected (not shown) if that is the final desired group (see Compound 1-121). After the desired X group is added, the amino protecting group can be removed to give the final product. For example, N-acetyl-piperazine can be used instead of N-BOC-piperazine to give an N-acetyl protecting group in Compound 1-VI. The amino group of Compound 1-VI can then be protected with an N-BOC group, which is more stable than N-acetyl. Compound 1-VI can then be modified with the X group, and the N-BOC can be removed to leave the final amino group. Scheme 2 [ka]

[0226] Additional Starting Compound 1-III A-C(See compounds 1-101 to 1-172) can be prepared as shown in Scheme 3. These starting compounds can then be modified as described herein. Scheme 3 [ka]

[0227] The X group of the present disclosure (see, for example, compounds 1-101 to 1-172) can be attached as shown in Scheme 4. The BOC protecting group of compound 1-VII (or the acetyl group described above) can be removed via acid hydrolysis (e.g., HCl in methanol) to give compound 1-VIII. The piperazinyl group of compound 1-VIII can then be converted to a suitable ester (e.g., 1-IX and 1-IX) in the presence of a base (e.g., KCO or dimethylaminopyridine). A (for example, CH2=CHC(O)(CH2)2C(O)OCH3 or its acid or activated acid can be used) to give the final compound 1-IX (compound 1-101) or compound 1-IX A (Compounds 1-105). If the X group contains a moiety that can react under amide-forming conditions, a protected X' group (e.g., a protected terminal amine) may be used, which can then be deprotected and modified to arrive at the desired final compound (e.g., the terminal amine can be deprotected and then reacted with an appropriate ester or acid to form the final amide to X). Scheme 4 [ka]

[0228] Example 3: Synthesis of Compounds 1-102 Compounds 1-XXII and 1-XXV, which are intermediates for many of the compounds 1-101 through 1-172, can be prepared as shown in Scheme 5. The precursor compounds 1-XXXII and 1-102 can be synthesized as shown in Scheme 6. The remaining compounds can be synthesized in a similar manner. Scheme 5 [ka] Scheme 6 [ka]

[0229] Example 4: General method for screening for ARCS The ARCSs of the present disclosure can be synthesized by those skilled in the art using common chemical synthesis principles and techniques. Alternatively, ARCSs can be produced using known combinatorial methods to generate large libraries of ARCSs. ARCSs of the present disclosure can also be synthesized as shown in Examples 1-3. ARCSs that covalently bind to biological targets in target cells can then be screened by gel assay, Western blot, ELISA, antibody array, or NanoBRET assay.

[0230] Example 5: Transfection protocol and readout for NanoBRET screening of ARCS Human embryonic kidney 293-H (HEK 293, Gibco 293-H, #11631017) cell line is maintained in Dulbecco's modified Eagle's medium, high glucose, pyruvate (DMEM, Gibco, #11995065) supplemented with 10% fetal bovine serum (FBS, Gibco, #10082147) and 1x penicillin-streptomycin (100x solution, Gibco, #15140148) at 37°C and 5% CO in a water-saturated incubator. Cells are trypsinized using 0.05% or 0.25% trypsin-EDTA solution (trypsin-EDTA with phenol red, Gibco, #25200056 (0.25%) or #25300054). Opti-MEM medium supplemented with 10% fetal bovine serum (Opti-MEM I reduced serum medium, phenol red-free, Gibco, #11058021) is used to culture the cells overnight for NanoBRET reading experiments.

[0231] HEK293 cells are cultured appropriately prior to the assay. The medium from the cell flask is removed via aspiration, washed once with PBS, then aspirated and trypsinized to dissociate the cells from the flask. The trypsin is neutralized using growth medium, and the cells are pelleted via centrifugation at 200 x g for 5 minutes. The medium is aspirated, and the cells are resuspended into a single cell suspension using Opti-MEM I supplemented with 10% FBS. The cell density is 2 x 10 in Opti-MEM I supplemented with 10% FBS in a sterile conical tube. 5 The solution is adjusted to 1 / mL. Cells are transfected and the next day aliquoted directly into 96-well plates for NanoBRET assays, thus culturing the cells overnight in Opti-MEM. Cells can also be transfected in bulk and dispensed into 96-well plates to allow the cells to adhere to the plates overnight, thereby allowing for washout testing.

[0232] The lipid:DNA complex is prepared as follows:

[0233] A 10 μg / mL solution of DNA is prepared in serum-free Opti-MEM. This solution contains the following ratio of carrier DNA and DNA encoding NanoLuc fused to a biological target: A serial dilution step may be performed to ensure accurate dilution of the NanoLuc fusion DNA. In order, the following reagents were added to a sterile polystyrene test tube: 1 mL of Opti-MEM without phenol red; 9.0 μg / mL of carrier DNA; and 1.0 μg / mL of NanoLuc fusion DNA (less for some targets). The reagents were thoroughly mixed.

[0234] Add 30 μL of FuGENE HD to each mL of DNA mixture to form lipid:DNA complexes. Be careful not to let FuGENE HD touch the plastic side of the tube, and pipette it directly into the liquid in the tube. Mix by pipetting up and down 5-10 times and incubate at room temperature for 20 minutes to form the complex. Add one portion (e.g., 1 mL) of the lipid:DNA complex to 2 x 10 5Mix with 20 parts (e.g., 20 mL) of HEK293 cells in suspension at 10000 / mL and mix gently by pipetting up and down 5 times in a sterile conical tube. Larger or smaller bulk transfections can be scaled accordingly using this ratio. Dispense 100 μL of the cell + lipid:DNA complex into a sterile, tissue-culture-treated 96-well plate (20,000 cells / well) and incubate for at least 16 hours to allow expression. Incubate the cells in a 37°C + 5% CO2 incubator for an additional 16 hours. Prepare serially diluted inhibitors or test compounds at 100x final concentrations in 100% DMSO. Serially diluted inhibitor stocks are prepared for PCR plates. Add 1 μL per well of the 100-fold serially diluted inhibitor / test compound to the cells in the overnight transiently transfected 96-well plate and mix by gently tapping the plate. Incubate the plate overnight in a 37°C + 5% CO2 incubator. Prepare a 1X solution of the substrate mix (500X stock) and the appropriate concentration of tracer in Opti-Mem. Wash the cells by setting the plate washer on the 96-well plate 5X in PBS, pH 7.4, adding 200 μL of PBS each time. Incubate the cells for 2 hours at 37°C. Add 100 μL of 1X Substrate-Tracer solution and gently tap the 96-well plate to mix. Read the plate on a plate reader every hour for the next 6 hours. Binding assays of several ARCSs are shown below. Compounds 1-XXXII formed covalent bonds with approximately 5% to 20% of PI3 kinase. ARCSs selected from the group consisting of compounds 1-101, 1-102, 1-113, 1-114, 1-119, 1-120, 1-125, and 1-126 formed covalent bonds with approximately 80% to 100% of PI3 kinases. Compounds 1-171 and 1-172 formed covalent bonds with approximately 50% to 80% of PI3 kinases. PI3 kinase activity was inhibited by compound 1-XXXII by approximately 5% to 20%.The activity of PI3 kinase is inhibited by about 80% to 100% by an ARCS selected from the group consisting of compounds 1-101, 1-102, 1-113, 1-114, 1-119, 1-120, 1-125, and 1-126. The activity of PI3 kinase is inhibited by about 50% to 80% by compounds 1-171 and 1-172. [Table 1]

[0235] Equivalence and Scope Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, given the disclosure set forth herein, many equivalents to the specific embodiments. The scope of the present disclosure is not intended to be limited to the above description, but is instead set forth in the appended claims.

[0236] In the claims, unless indicated to the contrary or otherwise clear from the context, articles such as "a," "an," and "the" may mean one or more. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or method, unless the context suggests otherwise. The disclosure includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or method. The disclosure includes embodiments in which one or more, or all, of the group members are present in, used in, or otherwise relevant to a given product or method.

[0237] Also, note that the term "comprising" is open and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0238] When ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any particular value or range within the ranges set forth in different embodiments of this disclosure to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0239] Furthermore, it should be understood that any particular embodiment of the present disclosure that is within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not explicitly set forth herein because they are deemed to be known to those of ordinary skill in the art. Any particular embodiment of the compositions of the present disclosure (e.g., any antibiotic, therapeutic agent or active ingredient, any method of manufacture, any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0240] The words which have been used are words of description rather than of limitation, and it is to be understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0241] While the present disclosure has been described at some length and with some particularity with respect to several described embodiments, it is not intended to be limited to such details or embodiments or to any particular embodiment, but should be construed with reference to the appended claims so as to provide the broadest interpretation of such claims in light of the prior art, and therefore so as to effectively encompass the intended scope of the present disclosure.

Claims

1. formula: A compound having the formula (FCB)a-(L)b-(CLM)c or a pharmaceutically acceptable salt thereof, CLM, 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 is selected from A, B, C and D are independently in each occurrence H, halogen, CF ３ , —OH, —NH ２ , -SH, -SCH ３ , -CN, -NO ２ , -CH ２ (NH ２ ), -C(O)OH, -S(O) ２ NH ２ , —C(O)NH ２ , —C(O)CH ３ , NHC(O)-C １－６ Alkyl, N(C １－３ alkyl)C(O)-C １－６ Alkyl, OC(O)NH ２ ,OC(O)NH(CH ３ ), OC(O)N(CH ３ ) ２ , imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ Alkynyl, optionally substituted C ３－６ and optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; and the optional substituents for A, B, C, and D are independently selected from the group consisting of halogen, OH, NH ２ , C.H. ３ , C.F. ３ , -CN, -NO ２ , -C(O)OH, -S(O) ２ NH ２ , —C(O)NH ２ , -CH ２ NH ２ , —C(O)CH ３ , SH, -S-CH ３ , optionally substituted C １－３ Alkyl and optionally substituted C ３－６ 1 to 3 substituents selected from the group consisting of cycloalkyl; A １ , A ２ , A ３ , A ４ , A ５ and A ６ is independently in each occurrence H, halogen, CF ３ , —OH, —NH ２ , -SH, -SCH ３ , -CN, -NO ２ , -CH ２ (NH ２ ), -C(O)OH, -S(O) ２ NH ２ , —C(O)NH ２ , —C(O)CH ３ , NHC(O)-C １－６ Alkyl, N(C １－３ alkyl)C(O)-C １－６ Alkyl, OC(O)NH ２ ,OC(O)NH(CH ３ ), OC(O)N(CH ３ ) ２ , imidazolyl, pyrazolyl, methylimidazolyl, methylpyrazolyl, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkenyl, optionally substituted C ２－６ Alkynyl, optionally substituted C ３－６ selected from the group consisting of cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl; L is C １ -C １０ Straight chain alkyl, C １ -C １０ Straight chain O-alkyl, C １ -C １０ Straight-chain substituted alkyl, C １ -C １０ Straight chain substituted O-alkyl, C ４ -C １３ Branched alkyl, C ４－ C １３ Branched chain O-alkyl, C ２ -C １２ Straight chain alkenyl, C ２ -C １２ Straight chain O-alkenyl, C ３－ C １２ Straight-chain substituted alkenyl, C ３ -C １２ linear substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl, heterocycle, succinate ester, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bipyridine, indole, chelating agent, aldehyde, ketone, bisamine, bisalcohol, heterocycle, azirine, disulfide, thioether, hydrazone and combinations thereof, either end of which can be connected to the CLM; a and c are each independently an integer from 1 to 5; b is an integer from 0 to 5; The FCB has the structure: 【Transformation 8】 and R 1 is 【Chemistry 9】 A compound selected from the group consisting of:

2. R １ but 【Chemistry 9】 The compound of claim 1 .

3. 2. The compound of claim 1, wherein a, b, and c are 1.

4. L is a substituted or unsubstituted C ３ -C １０ The compound of claim 3 which is a heterocycloalkyl.

5. L is unsubstituted C ５ -C ６ The compound of claim 4 which is a heterocycloalkyl.

6. L, 【Chemistry 10】 6. The compound of claim 5, wherein:

7. CLM, 【Chemistry 11】 and A １ , A ２ , A ３ , A ４ , A ５ and A ６ are each independently H, halogen, or CF ３ , —OH, —NH ２ , -SH, -SCH ３ , -CN, -NO ２ , -CH ２ (NH ２ ), -C(O)OH, -S(O) ２ NH ２ , —C(O)NH ２ , —C(O)CH ３ , NHC(O)-C １－６ Alkyl, N(C １－３ alkyl)C(O)-C １－６ Alkyl, OC(O)NH ２ ,OC(O)NH(CH ３ ), OC(O)N(CH ３ ) ２ , optionally substituted C １－６ Alkyl, optionally substituted C ２－６ alkenyl, and optionally substituted C ２－６ The compound of claim 1 selected from the group consisting of alkynyl.

8. A １ , A ２ , A ３ , A ４ , A ５ and A ６ are each independently H or optionally substituted C １－６ The compound of claim 7, wherein the compound is alkyl.

9. A １ , A ２ , A ３ , A ４ , A ５ and A ６ is H or unsubstituted C １－６ The compound of claim 7, wherein the compound is alkyl.

10. CLM, 【Chemistry 12】 2. The compound of claim 1, wherein:

11. CLM, 【Chemistry 13】 2. The compound of claim 1, wherein:

12. 10. The compound of formula 1-50: 【Chemistry 14】 or a pharmaceutically acceptable salt thereof, L is, 【Chemistry 15】 and either end can be connected to the CLM; １ teeth, 【Chemistry 16】 and the CLM is selected from the group consisting of: 【Chemistry 17】 2. The compound of claim 1 selected from the group consisting of:

13. L, 【Chemistry 18】 13. The compound of claim 12, wherein:

14. R １ but, 【Chemistry 19】 14. The compound of claim 13, wherein:

15. CLM, 【Chemistry 20】 15. The compound of claim 14 selected from the group consisting of: 【Request Item 16】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 2. The compound of claim 1 selected from the group consisting of: 【Request Item 17】 【Chemistry 28】 【Chemistry 29】 17. The compound of claim 16, selected from the group consisting of:

18. formula: 【Transformation 30】 or a pharmaceutically acceptable salt thereof.

19. formula: 【Chemistry 31】 or a pharmaceutically acceptable salt thereof.

20. formula: 【Chemistry 32】 or a pharmaceutically acceptable salt thereof.

21. formula: 【Transformation 33】 or a pharmaceutically acceptable salt thereof.

22. formula: 【Transformation 34】 or a pharmaceutically acceptable salt thereof.

23. formula: 【Chemistry 35】 or a pharmaceutically acceptable salt thereof.

24. A pharmaceutical composition comprising the compound of any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

25. 25. The pharmaceutical composition of claim 24 for treating cancer, a neurodegenerative disease, an autoimmune disorder or aging in a subject in need thereof.

26. 26. The pharmaceutical composition of claim 25, wherein the subject has cancer.

27. 27. The pharmaceutical composition of claim 26, wherein the cancer is one or more selected from the group consisting of leukemia, tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast, colon, and cancers in tissues including intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow or breast tissue.

28. The pharmaceutical composition of any one of claims 25 to 27, wherein the subject has a cancer with a mutation in the PIK3CA gene.

29. 24. A therapeutic conjugate comprising a compound of any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof, covalently bound to a phosphoinositide 3 (PI3) kinase.

Images