Therapeutic conjugates

Abstract

The present disclosure generally relates 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

[0001] This application is a divisional application of Chinese Patent Application No. 202080074001.4 (Application Date: 2020 / 9 / 18, Invention Title: Therapeutic Conjugate).

[0002] Cross Reference to Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 902,554, filed September 19, 2019, entitled “Therapeutic Conjugate,” and U.S. Provisional Patent Application No. 63 / 078,055, filed September 14, 2020, entitled “Therapeutic Conjugate,” the contents of each of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0004] The present disclosure relates generally to therapeutic conjugates that covalently bind to biological targets. BACKGROUND

[0005] Covalent inhibitors bind to receptors in the same way as classic inhibitors, but covalent inhibitors form a covalent, permanent chemical bond with the receptor, rather than dissociating. Some examples of covalent inhibitors include penicillin, aspirin, clopidogrel, Afatinib, an EGFR kinase inhibitor used to treat lung cancer, and Ibrutinib, a Bruton’s tyrosine kinase inhibitor used to treat B-cell malignancies. Furthermore, in the field of oncology, covalent inhibitors are effective against drug-resistant tumors and generally exhibit greater potency in inhibiting tumor growth.

[0006] Recently, covalent inhibitors have attracted the attention of major pharmaceutical companies because the use of covalent inhibitors offers increased potency and prolonged duration of action when compared to classic, reversible inhibitors. The prolonged duration of action translates into lower dosing frequency, i.e., patients must take fewer pills and reduce the frequency of taking them.

[0007] 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

[0008] In some embodiments, the present disclosure provides a therapeutic conjugate that can form a covalent bond with a kinase or pseudokinase. The kinase can be a PI3-kinase (PI3K). The therapeutic conjugate can have the structure (FCB)a-(L)b-(CLM)c, where a and c are independently integers between 1 and 5, b is an integer between 0 and 5, and where the FCB moiety includes a PI3K inhibitor or a fragment, analog, or derivative thereof. In some embodiments, the FCB can include The therapeutic conjugate can include a structure selected from the group consisting of Compound 1-101 through Compound 1-172.

[0009] In some embodiments, the therapeutic conjugate can have the structure or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of consisting of wherein either end can be attached to CLM; R1is selected from the group consisting of: and CLM is selected from the group consisting of: The therapeutic conjugate can be selected from the group consisting of Compound 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.

[0010] In some embodiments, the therapeutic conjugate can have the structure or a pharmaceutically acceptable salt thereof, wherein L is R2is selected from the group consisting of: and CLM is selected from the group consisting of: The therapeutic conjugate can be selected from the group consisting of Compound 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.

[0011] In some embodiments, a therapeutic conjugate can include a structure selected from compounds 1-101 to compounds 1-172, or a pharmaceutically acceptable salt thereof. In some embodiments, a therapeutic conjugate can include a structure selected from compounds 1-1 to compounds 1-11, or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the present disclosure provides a pharmaceutical composition that can include a therapeutic conjugate disclosed herein and at least one pharmaceutically acceptable excipient.

[0013] In some embodiments, the present disclosure provides a method of modulating the activity of a kinase or pseudokinase, the method 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 a PI3-K.

[0014] In some embodiments, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition described herein. The subject can have a therapeutic condition selected from the group consisting of cancer, a neurodegenerative disease, an autoimmune disorder, and aging. In some embodiments, the subject can have cancer. In some embodiments, the subject can have cancer that has a mutation in the PIK3CA gene. BRIEF DESCRIPTION OF DRAWINGS DETAILED DESCRIPTION

[0015] I. Compositions

[0016] The inventors have discovered, inter alia, an anchoring relationship covalent system, hereinafter ARCS, that includes a functional competitive binder, hereinafter FCB; a covalent linkage mode, hereinafter CLM, wherein the CLM is directly or indirectly linked to the therapeutic mode; and optionally a linker 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.

[0017] As used herein, the term “ARCS” refers to any therapeutic conjugate formed by linking FCB and CLM with a bond or linker. In some embodiments, ARCS can form a covalent bond with one or more targets such as a nucleotide, oligonucleotide, peptide, or protein. In some embodiments, ARCS can form a covalent bond with a biological target. Covalent bonds can be detected with any method known in the art. As a non-limiting example, covalent attachment of an azido small molecule to a protein can be detected by using click chemistry to attach a heavy, PEG-containing alkyne to the small molecule. Covalently labeled proteins are detected by gel shift as they are now PEG-labeled and have a higher molecular weight (Biochemistry 2018, 57:5769-5774). In another non-limiting example, mass spectrometry can be used to detect covalently labeled purified proteins (Nature Chemical Biology 2007, 3:229-238). In yet another non-limiting example, a mass spectrometry-based proteomics approach based on cellular quantification can be used to analyze covalent bonding (Cell Chemical Biology 2017, 24:1388-1400.e7). In yet another non-limiting example, X-ray crystallography is 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 covalent modification sites (Nature Chemical Biology 2016, 12:876-884).

[0018] In some embodiments, ARCS can form a covalent bond with a biological target to about 5-100% of the percentage of biological targets. In some embodiments, ARCS can form a covalent bond with a biological target of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the covalent bond is formed in an aqueous solution at a temperature of 0-50 °C, within 48 hours, at a treatment dose of 10 mM.

[0019] Without being bound by any theory, the ARCS can first form a non-covalent bond with a biological target (e.g., a target protein) through the FCB, and then form a covalent bond with the biological target through the CLM. In some embodiments, the efficacy of the ARCS is superior to the efficacy of the FCB alone. In some embodiments, the CLM does not substantially interfere with the efficacy 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 the toxicity of the FCB alone.

[0020] As used herein, the term “toxicity” refers to the ability of a substance or composition to be harmful or toxic to a cell, tissue organism, or cellular environment. Low toxicity refers to a decrease in the ability of a substance or composition to be harmful or toxic to a cell, tissue organism, or cellular environment. This decrease or low toxicity can be relative to a standard measure, treatment, or absence of treatment.

[0021] As used herein, the term “FCB” refers to a therapeutic modality that can be a known drug, diagnostic compound, candidate drug, and functional fragment and / or any combination of the foregoing. The FCB encompasses the free acid and free base forms; optical and tautomeric forms; isotopes comprising the drug, prodrug, or fragment thereof and pharmaceutically acceptable salts. The FCB can 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 is a nucleic acid, including but not limited to DNA or RNA. The FCB can be a therapeutic agent such as, but not limited to, an anti-cancer agent, an anti-neurodegenerative agent, an autoimmune drug, and an anti-aging agent. The FCB can non-covalently bind to a biological target. In some embodiments, the FCB can be a functional fragment of a drug. As used herein, the term “functional fragment” refers to a portion of a drug or derivative or analog thereof that is capable of inducing the desired effect of the drug. In some embodiments, the FCB can include an alkyne functional group. In some embodiments, the FCB can not include an alkyne functional group.

[0022] As used herein, the terms “peptide,” “polypeptide,” “protein” refer to a polymer made of amino acid monomers connected by amide bonds. The amino acids can be D- or L-optical isomers. The peptide can be formed by condensation or coupling reactions with the amino group of one alpha-carbon carboxyl and another amino acid. The peptide can be a non-linear branched peptide or a cyclic peptide. In addition, the peptide can be optionally modified or protected by different functional groups or protecting groups comprising amino and / or carboxyl terminal.

[0023] The amino acid residues of the peptides are abbreviated as follows. Phenylalanine is Phe or F, leucine is Leu or L, isoleucine is lie 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 Gin 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.

[0024] As used herein, the term "CLM" refers to any covalent linkage mode capable of forming a covalent bond with a biological target. The CLM can be linked to the FCB by a bond or by a linker. The CLM can include one or more chemical moieties that can form a covalent bond with a biological target. The chemical moieties can be electrophilic or nucleophilic groups.

[0025] The CLM can 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 molecular weight of the CLM can be between about 5 Da and about 1,000 Da, between about 10 Da and about 900 Da, in some embodiments, between about 20 Da and about 700 Da, in some embodiments, between about 20 Da and about 500 Da, between about 50 Da and about 400 Da, in some embodiments, between about 100 Da and about 300 Da, and in some embodiments, between about 150 Da and about 300 Da. The molecular weight of the CLM can be calculated as the sum of the atomic weight 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. It is known in the art that the unit of molecular weight can be g / mol, Dalton (Da), or atomic mass unit (amu), where 1 g / mol = 1 Da = 1 amu.

[0026] As used herein, the term "biological target" refers to any target that the FCB non- covalently binds to produce a therapeutic effect. The CLM covalently binds to the biological target. In some embodiments, the biological target is a protein. Non-limiting examples of biological targets include kinases such as, but not limited to, phosphoinositide 3-kinases (PI3Ks) and pseudokinases.

[0027] In some embodiments, the ARCS can form a covalent bond with PI3-kinase. In some embodiments, the ARCS can form a covalent bond with PI3-kinase from about 5%-100% of PI3-kinase. 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 PI3-kinase.

[0028] The ARCS comprises at least one FCB optionally connected to at least one CLM by a linker. In some embodiments, the ARCS can be a therapeutic conjugate between a single FCB and a single CLM, for example having the structure X-L-Y, where X is a CLM, L is an optional linker, and Y is an FCB. In some embodiments, the ARCS can be a therapeutic conjugate between a single therapeutic modality and a single covalent binding modality. In some embodiments, X is a covalent binding modality, L is an optional linker, and Y is a therapeutic modality.

[0029] In some embodiments, the ARCS contains more than one FCB, more than one linker, more than one CLM, or any combination thereof. The ARCS can have any number of FCBs, linkers, and CLMs. The ARCS can have the following structures, without limitation: X-L-Y-L-X, (X-L-Y) n , Y-L-X-L-Y, X-(L-Y) n , (X-L) n -Y, (X) n -L-Y, or X-L-(Y) n where X is a CLM, L is an optional linker, Y is an FCB, and n is an integer between 2 and 100, between 2 and 50, between 2 and 20, for example between 2 and 5. Each occurrence of X, L, and Y can be the same or different, for example the ARCS can contain more than one type of FCB, more than one type of linker, and / or more than one type of CLM.

[0030] In some embodiments, the ARCS can contain more than one CLM connected to a single FCB. For example, the ARCS can comprise one FCB with multiple CLMs each connected by the same or different linker. The ARCS can have the structure X-L-Y-L-X, where each X is a CLM which can be the same or different, each L is a linker which can be the same or different, and Y is an FCB.

[0031] In some embodiments, an ARCS may contain more than one FCB connected to a single CLM. For example, an ARCS may comprise a CLM with multiple FCBs, each of which is connected by the same or different linkers. An ARCS may have the structure YLXLY, where X is a CLM, each L is a linker that may be the same or different, and each X is an FCB that may be the same or different.

[0032] In some embodiments, ARCS is a therapeutic conjugate, wherein the therapeutic conjugate comprises

[0033] a. a therapeutic mode selected from the group consisting of one or more of the following: a known drug, a diagnostic compound, a drug candidate, and a functional fragment and / or a combination of any of the foregoing;

[0034] b. a covalent binding moiety comprising one or more chemical moieties, one or more of which are capable of forming a covalent bond with a biological target, wherein the covalent binding moiety is directly or indirectly linked to the therapeutic moiety; and

[0035] c. Optionally, a linker positioned between the therapeutic moiety and the covalently bound moiety.

[0036] In some embodiments, the therapeutic conjugate comprises a formula selected from the group consisting of:

[0037] a) XLY,

[0038] b)XLYLX,

[0039] c)(XLY) n 、

[0040] d)YLXLY,

[0041] e)X-(LY) n 、

[0042] f)(XL) n -Y,

[0043] g)(X) n -LY and

[0044] h)XL-(Y) n ;

[0045] wherein X is the covalent binding moiety, L is an optional linker, Y is the therapeutic moiety, and n is an integer between 2 and 100.

[0046] It is an object of the present disclosure to design ARCS and compositions thereof, as well as methods of synthesizing ARCS and libraries of ARCS.

[0047] It is another object of the present disclosure to provide methods of screening libraries of ARCS to identify candidates that covalently bind to biological targets.

[0048] It is a further object of the present disclosure to provide methods of administering and using ARCS and compositions thereof to individuals in need thereof.

[0049] A. FCB

[0050] The ARCS of the present disclosure contain at least one FCB. The ARCS of the present disclosure can contain more than one FCB, which can be the same or different. The FCB can be a therapeutic modality that affects any biological process and is used to prevent, diagnose, alleviate, treat, or cure a disease condition. The FCB can be a therapeutic agent, a prophylactic agent, a diagnostic agent, or a nutritional agent. The efficacy of a 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 its desired pharmacological effect. As used herein, the term “pharmacologically active” refers to an activity that modulates or alters a biological process to cause a change in phenotype, such as cell death, reduction in cell proliferation, etc.

[0051] 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 of the formulae from U.S. Patent No. 9,724,352 B2, the contents of which are incorporated by reference in their entirety herein. In some embodiments, the FCB is any of the compounds shown in Table 1 of U.S. Patent No. 9,724,352 B2. In some embodiments, the FCB comprises the structure

[0052] In some embodiments, the FCB is a compound having the structure wherein R1is selected from the group consisting of:

[0053] In some embodiments, the FCB is a compound having the structure wherein R2is selected from the group consisting of:

[0054] In general, the efficacy of an FCB is achieved through non-covalent binding to a biological target. Non-covalent binding is achieved through some degree of specificity and / or affinity for the target. Both specificity and affinity are generally desirable, although in some cases, higher specificity can compensate for lower affinity, and higher affinity can compensate for lower specificity. Affinity and specificity requirements will depend on various factors, including but not limited to absolute concentration of the target, relative concentration of the target (e.g., in cancer versus normal cells), potency and toxicity, route of administration, and / or diffusion or transport into the target cell. At the molecular or cellular level, the effect of an FCB (in an ARCS or alone) can include, but is not limited to, promoting or inhibiting the activity of a target, labeling a target, and / or altering a target cell (e.g., cell death).

[0055] In some embodiments, an FCB can be a small molecule, a protein, a peptide, a lipid, a carbohydrate, a sugar, a nucleic acid, or a combination thereof. In some embodiments, an FCB can be a therapeutic agent, such as, but not limited to, an anti-cancer agent, an anti-neurodegenerative agent, an autoimmune drug, and an anti-aging agent. A variety of therapeutic agents are known in the art and can be used in compositions as described herein.

[0056] In some embodiments, an FCB is a small molecule. In some embodiments, an FCB can be a protein, a peptide, or a nucleic acid. In some embodiments, an FCB can be a lipid. In some embodiments, an FCB can be a carbohydrate or a sugar. In some embodiments, an FCB has an alkyne group. In some embodiments, an FCB can not have an alkyne group.

[0057] In some embodiments, an FCB can 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 inducing the desired effect of the drug.

[0058] In some embodiments, an FCB can non-covalently bind to a biological target. In some embodiments, an FCB can bind to a biological target with an IC50 of < 1000 μm, 900 μm, 800 μm, 700 μm, 600 μm, or 500 μm. 50 binds to a biological target.

[0059] In some embodiments, an FCB is an anti-cancer agent. In some embodiments, an FCB is an anti-neurodegenerative agent. In some embodiments, an FCB is an autoimmune drug. In some embodiments, an FCB is an anti-aging agent.

[0060] In certain embodiments, the FCB of the ARCS comprises a predetermined mole 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 mole weight percentages of the components of the ARCS is 100%. The amount of the FCB of the ARCS can also be expressed in a ratio to the CLM. For example, the present teachings provide a ratio 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.

[0061] B. CLM

[0062] The ARCS of the present disclosure contains one or more CLMs. The CLM can be any covalent binding mode capable of forming a covalent bond with a biological target. The CLM can include one or more chemical moieties, one or more of which is 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 portions of the CLM or can be attached to the CLM.

[0063] 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.).

[0064] In certain embodiments, the CLM of the ARCS comprises a predetermined mole 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 mole weight percentages of the components of the ARCS is 100%. The amount of the CLM of the ARCS can also be expressed in a ratio to the FCB. For example, the present teachings provide a ratio 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.

[0065] 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 vinyl sulfonamide. In some embodiments, the CLM comprises at least one substituted or unsubstituted vinyl sulfone. 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 acrylate. 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 formyl phenyl boronic 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 formyl phenyl boronic 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 cyanoacrylamide. In some embodiments, the CLM comprises at least one substituted or unsubstituted chloroacetamide.

[0066] Exemplary CLMs include, but are not limited to:

[0067]

[0068]

[0069]

[0070]

[0071] wherein A, B, C, and D are independently at each occurrence selected from the group consisting of 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, methyl imidazolyl, methyl pyrazolyl, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl, wherein the optional substituents of A, B, C, and D are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl.

[0072] A1, A2, A3, A4, A5, and A6 are independently at each occurrence selected from the group consisting of 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, methyl imidazolyl, methyl pyrazolyl, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0073] wherein the optional substituents of A1, A2, A3, A4, A5, and A6 are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl, and

[0074] wherein C 1-3 alkyl, and C 3-6 cycloalkyl optional substituents are 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3 or fragments, derivatives, or analogs thereof.

[0075] In some embodiments, the CLM is selected from the group consisting of:

[0076] C. Linker

[0077] The ARCS of the present disclosure contain one or more optional linkers connecting the FCB and the CLM. The linker L can be connected to any position on the FCB and the CLM as long as the efficacy of the FCB and the binding of the CLM are not significantly affected. In some embodiments, the CLM comprises an optional internal linker.

[0078] 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 from, but not limited to, substituted and unsubstituted C1-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 heterocycloalkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl.

[0079] In some embodiments, the linker (including the internal linker of the CLM) can be C1-C10 linear alkyl, C1-C 10 linear O-alkyl, C1-C 10 linear substituted alkyl, C1-C 10 linear substituted O-alkyl, C4-C 13 branched alkyl, C4-C 13 branched O-alkyl, C2-C 12 linear alkenyl, C2-C 12 linear O-alkenyl, C3-C 12 linear substituted alkenyl, C3-C 12 linear substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycaprolactone, polycyanoacrylate, ketone, aryl, heterocycle, succinate, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bipyridine, indole derivative used as a crosslinker, chelator, aldehyde, ketone, bis amine, bis alcohol, heterocyclic structure, aziridine, disulfide, thioether, hydrazone, and combinations thereof. For example, the linker can be a C3 linear alkyl or ketone. The alkyl chain of the linker can 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, -O-C(=O)-NR-, -S-, -S-S-. The linker can be selected from dicarboxylate salt derivatives of succinic acid, glutaric acid, or diglycolic acid.

[0080] In some embodiments, the alkyl chain of the linker can be optionally interrupted with one or more atoms or groups selected from -O-, -C(=O)-, -NR, -O-C(=O)-NR-, -S-, -S-S-. The linker can be selected from dicarboxylate salt derivatives of succinic acid, glutaric acid, or diglycolic acid.

[0081] In some embodiments, the linker can be non-cleavable. In some embodiments, the linker can be cleavable. In some embodiments, the linker can be cleaved by an enzyme.

[0082] Non-limiting examples of linkers include

[0083] wherein D1, D2, D3, D4, D5, and D6 are independently selected, at each occurrence, from the group consisting of N, C, O, or S, provided that if D 1-6 is N, then the corresponding position is trivalent; if D 1-6is O or S, then the corresponding position is divalent; wherein B1, B2, B3, B4, B5, and B6are, at each occurrence, absent or independently selected from the group consisting of 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(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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl, wherein the optional substituents of B1, B2, B3, and B4are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl, and wherein C 1-3 alkyl, and C 3-6 cycloalkyl optional substituents are 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3or any fragment or analog thereof.

[0084] In some embodiments, the linker is selected from the group consisting of wherein either end can be attached to a CLM. In some embodiments, the linker selected from the group consisting of is attached to a CLM selected from the group consisting of:

[0085] D. ARCS

[0086] The ARCS of the present disclosure represent a class of drugs that have many advantages when compared to reversible inhibitors, such as increased potency and prolonged duration of action. The present disclosure provides therapeutic conjugates that form covalent bonds with kinases or pseudokinases. In some embodiments, the kinase is a PI3-kinase (PI3K). The therapeutic conjugates can have the structure

[0087] (FCB)a-(L)b-(CLM)c,

[0088] wherein a and c are independently integers between 1 and 5,

[0089] b is an integer between 0 and 5, and

[0090] wherein the FCB moiety comprises a PI3K inhibitor or a fragment, analog, or derivative thereof.

[0091] The FCB moiety, L (linker) moiety, and CLM moiety are discussed in the above sections. In one non-limiting example, the FCB comprises

[0092] In some embodiments, the FCB is a compound having the structure wherein R1is selected from the group consisting of:

[0093] In some embodiments, the FCB is a compound having the structure wherein R2is selected from the group consisting of: In some embodiments, the linker is selected from the group consisting of wherein either end can be connected to the CLM. In some embodiments, the CLM is selected from the group consisting of:

[0094] In some embodiments, the ARCS is selected from the group consisting of the generalized structures Compounds 1-1 through 1-5 or a pharmaceutically acceptable salt thereof, wherein R1is, at each occurrence, independently selected from the group consisting of wherein R1may be connected to either end of X, L, or the functional fragment of the drug. For example, in R1may be connected to L from the end adjacent to R e and R g or to the functional fragment of the drug from the end adjacent to R f and R h ; or in Compound 1-1, R1may be connected to L from the end adjacent to R f and R h or to the functional fragment of the drug from the end adjacent to Re and R g adjacent terminal connections to a functional fragment of a drug.

[0095] In some embodiments, R1is, at each occurrence, independently selected from the group consisting of unsubstituted or substituted -(alk) a -S-(alk) b -,(alk) a -O-(alk) b -,(alk) a -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 -NRA C(O)O-(alk) b -, a -NR A C(S)O-(alk) b -, a -OC(O)NR A -(alk) b -, a -OC(S)NR A -(alk) b -, a -NR A C(O)NR B -(alk) b -, a -NR A C(S)NR B -(alk) b - and -(alk) a -NR A S(O)2NR B -(alk) b -;

[0096] a and b are independently selected from the group consisting of 0, 1, 2, 3, and 4;

[0097] alk is independently selected from the group consisting of C 1-5 alkylene, C 1-5 alkenylene, and C 1-5 alkynylene, each of which is optionally substituted with 1-3 substituents independently selected from the group consisting of H, halogen, -OH, NH2, CF3, C 1-5 alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, imidazolyl, pyrazolyl, methyl imidazolyl, methyl pyrazolyl, -O-C 1-5 alkyl, -S-C 1-5 alkyl, -NH-C 1-5 alkyl, and -N(C 1-5 alkyl)2, wherein the C 1-5 alkyl is independently optionally substituted with 1-3 groups selected from the group consisting of halogen, -OH, -NH2, C 1-4 alkyl, CF3, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methyl imidazolyl, and methyl pyrazolyl;

[0098] R A and R B is independently selected from the group consisting of halogen, C 1-3 alkyl, C 3-6 cycloalkyl, 5-10 membered heterocycle, aryl, and 5-10 membered heteroaryl, wherein each of alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl is independently optionally substituted with 1-3 substituents 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 alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methyl imidazolyl, and methyl pyrazolyl;

[0099] R3is independently selected from the group consisting of:

[0100]

[0101] R a , R b , R c , R d , R e , R f , R g and R h is independently selected from the group consisting of 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, methyl imidazolyl, methyl pyrazolyl, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl;

[0102] wherein R a , Rb c d e f g h 1-3 3-6

[0103] 1-3 3-6

[0104] In some embodiments, R3is, at each occurrence, independently selected from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0105] In some embodiments, R3is, at each occurrence, independently selected from the group consisting of 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 1-3 ​​​​​​​​​​​​alkyl and optionally substituted C 3-6 cycloalkyl, and

[0106] wherein C 1-3 alkyl and C 3-6 cycloalkyl optional substituents are 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0107] R4is, at each occurrence, independently selected from the group consisting of:

[0108]

[0109] In some embodiments, R4is, at each occurrence, independently selected from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0110] wherein R4optional substituents are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl,

[0111] wherein C 1-3 alkyl and C 3-6cycloalkyl, optionally substituted C1-6alkyl, optionally substituted C1-6alkenyl, optionally substituted C1-6alkynyl, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl, wherein the optional substituents of R5are 1-3 substituents independently selected from the group consisting of 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 C1-6alkyl, optionally substituted C1-6alkenyl, and optionally substituted C1-6alkynyl.

[0112] R5is, at each occurrence, independently selected from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0113] wherein the optional substituents of R5are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl,

[0114] wherein C 1-3 alkyl and C 3-6 cycloalkyl optional substituents are 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0115] R6is, at each occurrence, independently selected from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0116] wherein the optional substituents of R6are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl,

[0117] wherein C 1-3 alkyl and C 3-6 cycloalkyl optional substituents are 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0118] L is, at each occurrence, independently selected from the group consisting of:

[0119] wherein D1, D2, D3, D4, D5, and D6are, at each occurrence, independently selected from the group consisting of N, C, O, or S, provided that if D 1-6 is N, then the corresponding position is trivalent; if D 1-6 is O or S, then the corresponding position is divalent. The linker can be attached to both ends of the functional fragment of the CLM or drug. For example, in , L can be attached to the CLM from either end adjacent to the nitrogen or from the other end.

[0120] B1, B2, B3, and B4 are, at each occurrence, absent or independently selected from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0121] wherein the optional substituents of B1, B2, B3, and B4 are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl,

[0122] wherein C 1-3 alkyl, and C 3-6 cycloalkyl optional substituents are 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH, and -S-CH3.

[0123] In some embodiments, L is, at each occurrence, independently selected from the group consisting of unsubstituted or substituted -(alk) a -S-(alk) b -, -(alk) a -O-(alk) b -, -(alk) a -NR C -(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 C -(alk) b -, -(alk) a -C(S)NR C -(alk) b -, -(alk) a -S(O)2NR C -(alk) b -, -(alk) a -NR C C(O)-(alk) b -, -(alk) a -NR C C(S)-(alk) b -, -(alk) a -NR C S(O)2-(alk) b -, -(alk) a -NR C C(O)O-(alk) b -, -(alk) a -NR C C(S)O-(alk) b -, -(alk) a -OC(O)NR C -(alk) b -, -(alk) a -OC(S)NR C -(alk) b -, -(alk) a -NR C C(O)NRD -(alk) b -、-(alk) a -NR C C(S)NR D -(alk) b -and-(alk) a -NR C S(O)2NR D -(alk) b -;

[0124] a and b are independently selected from the group consisting of 0, 1, 2, 3, and 4;

[0125] alk are independently selected from the group consisting of: C 1-5 Alkylene, C 1-5 Alkenylene and C 1-5 Alkyne, each of which is optionally substituted with 1-3 substituents independently selected from the group consisting of H, halogen, -OH, NH2, CF3, 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 1-5 Alkyl and -N(C 1-5 alkyl)2, wherein the C 1-5 The alkyl groups are optionally substituted with 1 to 3 groups independently selected from the group consisting of halogen, -OH, -NH2, C 1-4 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.

[0126] R C and R D is independently selected at each occurrence from the group consisting of: halogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, 5-10 membered heterocycle, aryl and 5-10 membered heteroaryl, wherein alkyl, cycloalkyl, heterocycle, aryl and heteroaryl are each independently optionally substituted with 1-3 substituents 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 1-6alkyl, -CH2(NH2), -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -C(O)CH3, -SH, -SCH3, imidazolyl, pyrazolyl, methylimidazolyl, and methylpyrazolyl.

[0127] X is, at each occurrence, independently selected from the group consisting of:

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] A, B, C, and D are, at each occurrence, independently selected from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl, and optionally substituted 5-10 membered heteroaryl,

[0134] wherein the optional substituents of A, B, C, and D are 1-3 substituents independently selected from the group consisting of 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 cycloalkyl,

[0135] wherein C 1-3 alkyl, and C 3-6The cycloalkyl group is optionally substituted with 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH and -S-CH3.

[0136] A1, A2, A3, A4, A5 and A6 are independently selected at each occurrence from the group consisting of 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 cycloalkyl, optionally substituted 5-10 membered heterocycle, optionally substituted aryl and optionally substituted 5-10 membered heteroaryl,

[0137] wherein the optional substituents of A1, A2, A3, A4, A5 and A6 are 1-3 substituents independently selected from the group consisting of 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 Cycloalkyl,

[0138] Among them C 1-3 Alkyl and C 3-6 The cycloalkyl group is optionally substituted with 1-2 substituents independently selected from the group consisting of halogen, OH, NH2, CH3, CF3, -CN, -NO2, -C(O)OH, -S(O)2NH2, -C(O)NH2, -CH2NH2, -C(O)CH3, SH and -S-CH3.

[0139] The present disclosure contemplates the use of all combinations of various substituents. Thus, any combination of the above-described substituents belonging to formulae Compound 1-1 through Compound 1-5 may be used.

[0140] In some embodiments, the ARCS is selected from the group consisting of narrow universal structured compounds 1-6 through compound 1-11,

[0141] or a pharmaceutically acceptable salt thereof.

[0142] In some embodiments, the ARCS can have the structure or a pharmaceutically acceptable salt thereof, wherein L is a linker selected from the group consisting of wherein either end can be attached to a CLM that is a target cell; R1is selected from the group consisting of: and CLM is selected from the group consisting of: 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, 1-120, 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 in Table 1 below.

[0143] In some embodiments, the ARCS can have the structure or a pharmaceutically acceptable salt thereof, wherein L, linker is R2is selected from the group consisting of: and CLM is selected from the group consisting of: 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 below.

[0144] Exemplary ARCS include any compound selected from the group consisting of Compound 1-101 through Compound 1-172.

[0145] Table 1. Non-limiting examples of ARCS compounds

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] or a pharmaceutically acceptable salt thereof.

[0158] E. Pharmaceutical Compositions

[0159] The ARCS of the present disclosure can be administered to a subject using any convenient means capable of producing the desired results. Thus, the ARCS of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the ARCS of the present disclosure can be formulated into pharmaceutical compositions and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[0160] As used herein, the term "pharmaceutical composition" means a composition comprising an ARCS as described herein and at least one pharmaceutically acceptable carrier, such as any of the carriers commonly used in the art of pharmacy. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, appropriate for use with humans and animals, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0161] Administration of the pharmaceutical composition can be achieved in various ways, including oral administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, intradermal administration, transdermal administration, intratracheal administration, and the like. In pharmaceutical dosage forms, the pharmaceutical composition can be administered alone or in combination with other pharmaceutically active compounds.

[0162] The amount of ARCS in the pharmaceutical composition can be based on weight, moles, 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 a compound of ARCS. 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 comprises 1-75% ARCS. In some embodiments, the pharmaceutical composition comprises 2-70% ARCS. In some embodiments, the pharmaceutical composition comprises 3-65% ARCS. In some embodiments, the pharmaceutical composition comprises 4-60% ARCS. In some embodiments, the pharmaceutical composition comprises 5-50% ARCS.

[0163] It is also to be understood that certain ARCS can exist in unbound form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof. According to 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 the ARCS which, upon administration to a patient in need thereof, are capable of providing, directly or indirectly, a compound otherwise described herein, or a metabolite or residue thereof.

[0164] As described above, the pharmaceutical compositions of the present disclosure can include pharmaceutically acceptable excipients, as used herein, which include any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, antioxidants, solid binders, lubricants, and the like, as suited to the particular dosage form desired.

[0165] 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 stearate, or stearic acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. 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 which 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 carboxymethyl cellulose, methylcellulose, ethyl cellulose, 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 waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil and soybean oil; (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) buffering agents, 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 buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (24) C2-C12 alcohols, such as ethanol; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the formulation. The terms such as "excipient," "carrier," "pharmaceutically acceptable carrier" are used interchangeably herein.

[0166] Useful pharmaceutical carriers for the preparation of the compositions of the present application can be solids, liquids or gases. Suitable pharmaceutical carriers and their formulations are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. In any case such compositions will contain an effective amount of the ARCS along with a suitable carrier to produce the proper dosage form for appropriate administration to recipients.

[0167] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the ARCS, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, baby oil, olive oil, castor oil and sesame seed oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

[0168] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the ARCS is admixed with at least one inert pharmaceutically-acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, 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 retarders, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as 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 can also comprise buffering agents.

[0169] Solid compositions of a similar type can also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of 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 formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) 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 employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polethylene glycols and the like.

[0170] ARCS can also be in micro-encapsulated form with one or more excipients as defined above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release control coatings, and other coatings known to those skilled in the art of pharmaceutical formulation. In such solid dosage forms, the ARCS can be admixed with at least one inert diluent such as sucrose, lactose, and starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents. The dosage forms can optionally contain opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0171] Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which can contain anti-oxidants, buffers, bactericides, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which can include suspending agents and thickening agents. Formulations suitable for intranasal administration can conveniently be presented in the form of a dry powder which can be presented in a unit dosage form, e.g., in a capsule, or in a cartridge of a dry powder inhaler, or in the form of a nasal spray or as an aerosol from a pressurized container with a suitable propellant. Formulations suitable for buccal administration can conveniently be presented in unit dosage form in the form of tablets, capsules, ovules, or troches, or as an aqueous or oily suspension, or as an aqueous or oily solution.

[0172] A buffer can also be included to render the formulation isotonic. Sodium chloride or glycerol can be used to make the formulation isotonic with blood.

[0173] If desired, the formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules, and can be preserved against the contaminating action of microorganisms, e.g., bacteria and fungi, by the inclusion of antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It can further contain carriers or diluents which are

[0174] Those of skill in the art will appreciate that the amount of the various components of the compositions of the present disclosure administered to a subject according to the methods of the present disclosure will depend on those factors mentioned above.

[0175] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0176] Non-limiting examples of tablets include active ingredient in an amount ranging from 10 mg to 100 mg, 70 mg to 95 mg of powdered lactose, 10 mg to 35 g of white corn starch, 1 mg to 8 mg of polyvinylpyrrolidone, 1 mg to 10 mg of sodium (Na) carboxymethyl starch (CMS), 1 mg to 5 mg of magnesium stearate, wherein the weight of the tablet is in the range of 200 mg to 3000 mg.

[0177] Examples of tablets of the present disclosure are as follows.

[0178]

[0179] Non-limiting examples of capsules include active ingredient in an amount ranging from 10 mg to 100 mg, 50 mg to 75 mg of crystalline lactose, 10 mg to 35 g of microcrystalline cellulose, 1 mg to 8 mg of talc, and 1 mg to 5 mg of magnesium stearate, wherein the capsule fill weight is in the range of 100 mg to 3000 mg.

[0180] Examples of capsules of the present disclosure are as follows.

[0181]

[0182] In the above capsules, the active ingredient has a suitable particle size. The crystalline lactose and the microcrystalline cellulose are uniformly mixed with each other, sieved, and then the talc and the magnesium stearate are mixed. The final mixture is filled into a hard gelatin capsule of suitable size.

[0183] Non-limiting examples of injections include active ingredient in an amount ranging from 0.05 mg to 5 mg, 10.0 μL to 20.0 μL of 1 N 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, 1 N NaOH in an amount sufficient to adjust the pH to 4 to 5, and a sufficient amount of water.

[0184] Examples of injections of the present disclosure are as follows.

[0185]

[0186] Injectable formulations 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 just prior to use.

[0187] To prolong in vivo action, it is often desirable to slow absorption of the ARCS from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the ARCS then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered ARCS form is accomplished by dissolving or suspending the ARCS in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the ARCS in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of ARCS to polymer, and the particular polymer

[0188] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the ARCS with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which is solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0189] Typical suppository formulations include ARCS or a pharmaceutically acceptable salt thereof, in an amount such that is active when administered in this manner, in conjunction with a binder and / or lubricant such as polyethylene glycol, gelatin, cocoa butter, or other low-melting-point vegetable waxes or fats. Typical transdermal formulations include conventional aqueous or non-aqueous vehicles such as creams, ointments, lotions or pastes, or in the form of a medicated plaster, patch or membrane.

[0190] Typical compositions for inhalation are in the form of solutions, suspensions or emulsions which can be administered using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane in the form of an aerosol.

[0191] Depending on the route of administration, the effective dosage of the small molecule disclosed herein to a subject, such as a human subject, can be determined and adjusted accordingly by one of skill in the art.

[0192] Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50(50% of the population lethal dose) and ED 50 (50% of the population therapeutically effective dose). The ratio of the dose between the toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 Compositions that exhibit a large therapeutic index are preferred.

[0193] While the description of the pharmaceutical compositions provided herein is primarily concerned with pharmaceutical compositions suitable for administration to humans, it is understood that such compositions are generally suitable for administration to any other animal, for example, for administration to non-human animals, for example, non-human mammals. Contemplated subjects for administration of the pharmaceutical compositions include, but are not limited to, non-human mammals, including agricultural animals such as cows, horses, chickens, and pigs, domestic animals such as cats, dogs, or research animals such as mice, rats, rabbits, dogs, and non-human primates.

[0194] II. Methods of Using ARCS

[0195] As appropriate, ARCS as described herein or compositions containing ARCS as described herein can be administered to treat any therapeutic disease that can be treated with FCBs or any therapeutic disease associated with the biological target of the ARCS, such as, but not limited to, cancer, neurodegenerative disease, autoimmune disease, or aging. Formulations can be delivered to various body sites, such as, but not limited to, the brain and central nervous system, the eye, the ear, the lung, the bone, the heart, the kidney, the liver, the spleen, the breast, the ovary, the colon, the pancreas, the muscle, the gastrointestinal tract, the oral cavity, the skin, to treat diseases associated with such body sites. Formulations can be administered by injection, orally, or topically, typically to a mucosal surface (pulmonary, nasal, oral, buccal, sublingual, vaginal, rectal) or the eye (intraocular or ocularly).

[0196] In aspects of the disclosure, the ARCS binds to a biological target. In some embodiments, the biological target comprises a kinase, such as, but not limited to, phosphoinositide 3-kinase (PI3K) and pseudokinase.

[0197] In some embodiments, the ARCS can form a covalent bond with the biological target. In some embodiments, the ARCS can form a covalent bond with the biological target from about 5%-100% of the biological target. In some embodiments, the ARCS can form a covalent bond with the biological target 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 target.

[0198] In some embodiments, the ARCS can form a covalent bond with the PI3-kinase. In some embodiments, the ARCS can form a covalent bond with the PI3-kinase from about 5%-100% of the PI3-kinase. In some embodiments, the ARCS can form a covalent bond with the 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.

[0199] Protein kinases and pseudokinases

[0200] Protein kinases and pseudokinases modulate signaling pathways for various biological processes in normal and disease states (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 regulated by kinases and / or pseudokinases. The term "pseudokinase" refers to proteins with kinase domains that lack catalytically relevant residues, including lysine in the VAIK motif or aspartate in the DFG motif. Examples of pseudokinases include HER3, STRAD, ILK, KSR1, and KSR2. Some pseudokinases are unable to transfer phosphate groups, while others have the ability to transfer phosphate groups even though they lack catalytically relevant residues.

[0201] Kinases and pseudokinases can function by binding, covalent modification such as phosphorylation, conformational control, cellular localization, and / or other processes to inhibit and / or activate protein partners. Protein kinases and pseudokinases can promote and / or antagonize a variety of diseases, including but not limited to cancer, Alzheimer's disease, aging, diabetes, cardiovascular disease, CNS-related diseases, immune diseases, allergies, hypertension, and Parkinson's disease. Protein kinases and pseudokinases are also frequently mutated in a variety of diseases, including but not limited to cancer and Parkinson's disease.

[0202] Molecules targeting kinases and pseudokinases can act as anti-cancer agents. Unfortunately, typical inhibitors often exhibit limited potency due to short on-target residence times. Thus, there is a need to discover kinase inhibitors with improved on-target residence times compared to previous kinase inhibitors and concomitant improved potency and efficacy.

[0203] In some embodiments, the ARCS of the present disclosure can target and inhibit kinases and pseudokinases. The FCB of the ARCS can be an inhibitor of kinases and pseudokinases. The CLM of the ARCS can covalently bind to kinases and pseudokinases.

[0204] In some embodiments, the present disclosure provides a method of treating a disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of an ARCS of the present disclosure or a composition comprising the ARCS or a pharmaceutically acceptable salt thereof, wherein the patient has a disease caused in part or in whole by a modulation change of a target kinase or pseudokinase, such as cancer, Alzheimer’s disease, aging, diabetes, cardiovascular disease, CNS-related disease, immune disease, allergy, hypertension, or Parkinson’s disease. In some embodiments, the disease is associated with a mutation in PI3K or PI3K. In some embodiments, the subject has a cancer with a mutation in the PIK3CA gene.

[0205] Dosing

[0206] The present disclosure provides methods comprising administering to a subject in need thereof a composition comprising an ARCS as described herein. The composition comprising an ARCS as described herein can be administered to a subject using any amount and any route of administration effective to prevent or treat a disease, disorder, and / or condition or to image a disease, disorder, and / or condition. The exact amount required will vary depending on the subject, the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its activity, and the like.

[0207] Compositions according to the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It should be understood, however, that the total daily usage of the compositions of the present disclosure can be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dosage level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0208] In some embodiments, compositions according to the present disclosure can be administered at a dosage level sufficient to deliver about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg of subject body weight per day, once a day, or multiple times a day, to obtain 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 two days, weekly, biweekly, triweekly, or four weekly. In some embodiments, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, fractional dosing regimens such as those described herein can be used.

[0209] As used herein, a "fractional dose" is the division of a single unit dose or total daily dose into two or more doses, e.g., two or more administrations of a single unit dose. As used herein, a "single unit dose" is the dose of any therapeutic agent administered in one dose / occasion / single route / single point of contact, i.e., a single administration event. As used herein, a "total daily dose" is the amount given or prescribed in a 24 hour period. It can be administered as a single unit dose.

[0210] III. Kits and Devices

[0211] The present disclosure provides a variety of kits for facilitating and / or efficiently carrying out the methods of the present disclosure. Typically, a kit will include sufficient amounts and / or numbers of components to allow a user to treat a subject multiple times and / or to perform multiple experiments.

[0212] 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 ARCSs of the present disclosure, optionally in combination with any other active agent.

[0213] The kit may further include packaging and instructions and / or a delivery agent to form the formulation composition. The delivery agent may include saline, a buffer solution, or any delivery agent disclosed herein. The amount of each component may be varied to achieve a consistent, reproducible formulation of a high concentration of saline or a simple buffer solution. The components may also be varied to increase the stability of the ARCS in the buffer solution over a given period of time and / or under various conditions.

[0214] The present disclosure provides devices that can incorporate the ARCS of the present disclosure. These devices are contained in stable formulations that can be immediately delivered to a subject in need, such as a human patient. In some embodiments, the subject suffers from cancer.

[0215] Non-limiting examples of devices include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices, and multilayer microfluidic devices. The devices can be used to deliver the conjugates and / or particles of the present disclosure according to single, multiple, or divided dosing regimens. The devices can be used to deliver the conjugates and / or particles of the present disclosure through biological tissues, intradermally, subcutaneously, or intramuscularly.

[0216] A. Assays

[0217] Covalent binding of ARCS to a biological target can be determined using various methods known in the art, such as, but not limited to, 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.

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

[0219] In some embodiments, the assay surface on the substrate is sterile and suitable for culturing cells under conditions representative of those encountered during large-scale (e.g., industrial-scale) production of biological products. In some embodiments, the exterior of the substrate includes holes, indentations, demarcations, etc., at locations corresponding to the assay surface. In some embodiments, the holes, indentations, demarcations, etc., retain fluid, such as cell culture medium, above the assay surface.

[0220] In some embodiments, the substrate comprises a microarray plate, biochip, or the like, which allows for high-throughput, automated testing of a range of test reagents, conditions, and / or combinations thereof on cultured cells to produce a biological product. For example, the substrate can comprise a 2-dimensional microarray plate or biochip having m columns and n rows of assay surfaces (e.g., residing within wells) that allow for testing of mxn combinations of test reagents and / or conditions (e.g., on a 24-well, 96-well, or 384-well microarray plate). The microarray substrate is preferably designed such that all necessary positive and negative controls can be run in parallel with the testing of reagents and / or conditions.

[0221] B. Screening Methods

[0222] In general, the synthesis of therapeutic conjugates that form covalent bonds with biological targets involves multiple synthesis and purification steps. When such synthesis and purification steps are used, it can be difficult to generate libraries of therapeutic conjugates for screening purposes or to develop structure-activity relationships (SAR). There remains a need for methods and systems that automatically generate libraries of therapeutic conjugates using organic synthesis methods. In order to discover lead drugs for therapeutic conjugate drugs, libraries of therapeutic conjugates against protein receptors must be screened and those molecules that specifically bind to the receptor in a cellular environment are identified, where the binding is covalent and irreversible. It has been challenging to identify proof that a therapeutic conjugate covalently binds to a specific target in a cell.

[0223] Current methods in the art for screening therapeutic conjugates that covalently bind to biological targets include tandem mass spectrometry. Tandem MS or MS / MS is a method that breaks down selected ions into fragment ions. Once a sample is ionized to produce a mixture of ions, a precursor ion (MS1) with a specific mass-to-charge ratio (m / z) is selected and then fragmented (MS2) to produce product ions for detection. Information about the chemical structure of the selected ion can then be determined from the fragments.

[0224] However, there are challenges associated with mass spectrometry methods. Mass spectrometry methods are primarily limited to fragment ion screening, not drug-like molecule screening. Mass spectrometry analysis of drug-like therapeutic conjugates results in unresolvable analysis because it is difficult to identify several fragment ions. This method involves a multi-step process, is time-consuming to process, and involves manual analysis. MS / MS will fragment complex larger molecules, so it is difficult to interpret the resulting data because it requires manual combing of each peptide. Another drawback of using mass spectrometry methods is that detection is proportional to ionization, not abundance, making the technique less quantitative.

[0225] Accordingly, there are challenges associated with the synthesis and screening of therapeutic conjugates. First, it is difficult to produce target covalent inhibitors that can access both cysteine and non-cysteine amino acids. It is also difficult to distinguish false positive covalent inhibitors from true positives.

[0226] To address the above problems, the inventors combined a combinatorial synthesis method with a reliable screening method to identify potential therapeutic conjugates. The present disclosure provides a high-throughput combinatorial method for synthesizing therapeutic conjugates; rapidly tracking covalent binding; analyzing large libraries for duration of action and directly quantifying covalent target binding in cells.

[0227] In contrast to tens of molecules with nanomolar sensitivity using MS / MS methods, thousands of molecules with picomolar sensitivity can be screened per day. The present disclosure is applicable to any drug molecule and is not limited to fragments and gives quantitative results with 95-99% reproducibility.

[0228] In some embodiments, the method for screening an ARCS library comprises:

[0229] producing a library of ARCS in a composition;

[0230] contacting the library with a target cell;

[0231] lysing the target cell to produce a lysate;

[0232] labeling the lysate; and

[0233] detecting covalent binding of the ARCS to a biological target on the target cell.

[0234] Examples of human cell lines used as target cells in the methods provided herein include, but are not limited to, 293T (embryonic kidney), 786-0 (kidney), A498 (kidney), A549 (alveolar basal epithelial), ACHN (kidney), BT-549 (breast), BxPC-3 (pancreas), CAKI-1 (kidney), PANC-1 (pancreas), CCRF-CEM (leukemia), COLO 205 (colon), DLD-1 (colon), 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), HEK 293 (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 (white prostatic adenocarcinoma), LOX IMVI (melanoma), LXFL 529 (non-small cell lung), M14 (melanoma), M19-MEL (melanoma), MALME-3M (melanoma), MCF10A (breast epithelial), MCF7 (breast), MDA-MB-453 (breast 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), 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), (E1 transformed embryonic retina), RPMI-7951 (melanoma), RPMI-8226 (leukemia), RXF 393 (kidney), RXF-631 (kidney), Saos-2 (bone), SF-268 (CNS), SF-295 (CNS), SF-539 (CNS), SHP-77 (small cell lung), SH-SY5Y (neuroblastoma), SK-BR3 (breast), SK-MEL-2 (melanoma), SK-MEL-5 (melanoma), SK-MEL-28 (melanoma), SK-OV-3 (ovary), SN12K1 (kidney), SN 12C (kidney), SNB-19 (CNS), SNB-75 (CNS), SNB-78 (CNS), SR (leukemia), SW-620 (colon), T-47D (breast), THP-1 (monocyte-derived macrophages), TK-10 (kidney), U87 (glioblastoma), U293 (kidney), U251 (CNS), UACC-257 (melanoma), UACC-62 (melanoma), UO-31 (kidney), W138 (lung), and XF 498 (CNS).

[0235] Examples of rodent cell lines that can be used 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 cells (TM4), 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, 3T3 L1) cells, rat cardiomyocyte (H9c2) cells, mouse myoblast (C2C12) cells, and mouse kidney (miMCD-3) cells.

[0236] Examples of non-human primate cell lines that can be used 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 monkey kidney (CVI) cells transformed by SV40 (Cos-7). Additional mammalian cell lines are known to those of ordinary skill in the art and are available from the American Type Culture Collection (ATCC). Cataloged in Manassas, Virginia.

[0237] In some embodiments, cells are lysed using chemical and / or mechanical lysis. In some embodiments, chemical lysis includes a lysis buffer that includes protease inhibitors, phosphate buffered saline, and Triton X100. In some embodiments, cells can be frozen at -80°C for about 30 minutes to about 72 hours after addition of the lysis buffer. Alternatively, cell lysates can be stored at 2 to 8°C or room temperature. In some embodiments, cells are centrifuged and cell lysates are collected. In some embodiments, this is done by spinning cells in a centrifuge at 3,750 RPM for 10 minutes at room temperature.

[0238] The methods described herein can be performed by utilizing any wide range of cell assay formats, including but not limited to cell plates, such as 24-well plates, 48-well plates, 96-well plates, or 384-well plates, individual cell culture plates or flasks, such as T-flasks or shake flasks.

[0239] 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 analysis can include microfluidic or capillary techniques for separating proteins by size.

[0240] 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 picomolar to 1 millimolar for a duration of 2 minutes to 120 hours using techniques known to one of skill in the art, including but not limited to common cell culture techniques. Cells are then lysed using techniques known to one of skill in the art, including but not limited to sonication or buffer lysis. The resulting cell lysate, also described as unclarified lysate, can be further prepared by using techniques known to one of skill in the art, including but not limited to centrifugation, to produce a clarified lysate. The clarified or unclarified lysate can contain proteins covalently bound to one or more molecules of interest. A “coupling reagent” and a labeling molecule are added to the clarified or unclarified lysate to covalently label ARCS in the reaction mixture with the labeling molecule through copper-free or copper-driven click chemistry reactions. Compounds that bind to the labeling molecule are then added, enabling reliable stoichiometric resolution and reliable covalent drug tracking. The sample is then run through a western blot method familiar to one of skill in the art. Subsequently, the amount of covalent binding can be tracked based on the shift of the band treated with the drug compared to the band that was not treated. Bands can be quantified using densitometry, and the relative abundance of the bands can be used to determine the quantitative amount of covalent labeling.

[0241] In general, covalent attachment of a large mass protein / mass to ARCS can be used, resulting in the translocation of a target-ARCS-large mass protein mass complex in a gel that can be used. If the azide bond molecule attached to a high mass protein or any type of molecule is directly attached to an alkyne on ARCS, translocation still occurs and covalent binding of ARCS to the target can be detected.

[0242] In some embodiments, covalent binding is detected by a gel translocation only assay. A "gel translocation only assay" is defined as an assay in which a protein is expressed (by transfection or infection) in any cell type and attached to a labeling domain. This labeling domain comprises a fluorescent protein or a linker protein. The fluorescent protein comprises GFP, RFP, etc. The linker protein comprises HALO, SNAP-, CLIP-, ACP-, and MCP-tags. After transfection or infection, the cells are then treated with a therapeutic conjugate of the disclosure, wherein the therapeutic conjugate contains an alkyne that can potentially covalently bind. The cells are then lysed. In the case where the protein is expressed with a linker protein, a "coupling reagent" is then added to covalently attach a fluorescent dye to the protein of interest. The lysate can then be run on a gel and the target protein is observed by intragel fluorescence without the need for Western blotting transfer. The amount of covalent binding can be tracked based on the translocation of the band treated with the drug compared to the band that was not treated. The bands can be quantified using densitometry and the relative abundance of the bands can be used to determine the quantitative amount of covalent labeling.

[0243] The labeling domain can be any domain that allows for labeling of the target. In some embodiments, the labeling domain comprises a label. This label can be comprised in the domain itself, such as an epitope recognized by an antibody or a light detectable or radiolabel. In some embodiments, the label can be selected from the group consisting of a fluorescent marker, such as FITC, a phycobiliprotein, such as R- or B-phycoerythrin, allophycocyanin, an Alexa Fluor dye, Cy3, Cy5, Cy7, a luminescent marker, a radiolabel, such as 125 I or 32 P, an enzyme such as horseradish peroxidase or alkaline phosphatase, e.g. alkaline shrimp phosphatase, an epitope, a lectin, or biotin / streptavidin.

[0244] In some embodiments, as used herein, "fluorescent protein" is, but is not limited to, the green fluorescent protein of the jellyfish, Aequorea victoria (GFP), red fluorescent protein (RFP), structural variants of GFP (i.e., ring substituents, monomeric versions), folding variants of GFP (i.e., more soluble versions, superfolder versions), 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 Antho Zoa fluorescent protein that share the 11 -beta chain "barrel structure" of GFP, as well as structural, folding, and spectral variants thereof. The terms "GFP-like non-fluorescent protein" and "GFP-like chromoprotein" (or simply "chromoprotein" or "chromoprotein") are used to refer to Anthozoan and Hydrozoan chromoproteins that share the 11 -beta chain "barrel structure" of GFP, as well as structural, folding, and spectral variants thereof.

[0245] In some embodiments, covalent binding is detected by a Western blot-based shift assay. A "Western blot-based shift assay" is defined as an assay in which a sample is run through a Western blot method familiar to those skilled in the art. Subsequently, because covalently bound proteins will shift compared to non-covalently bound bands, the amount of covalent binding can be tracked based on the shift of the target. Bands can be quantified using densitometry, and the relative abundance of bands can be used to determine the quantitative amount of covalent labeling.

[0246] 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 biotin-labeled drug is immobilized on a solid support by hybridization to monomeric or tetrameric streptavidin or streptavidin variants or biotin-binding molecules. After drug immobilization, a detection antibody is added to detect the drug target of interest. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody linked by bioconjugation to an enzyme or fluorescent label. Between each step, the plate is typically washed with a solution to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by the addition of an enzyme substrate to produce a visible signal that indicates the amount of covalently bound drug bound to the target of interest. The amount of covalent binding can be tracked based on the amount of signal.

[0247] 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 by hybridization with an antibody that binds the target of interest. After immobilization of the target of interest, a detection antibody or monomer / tetrameric streptavidin or streptavidin variant is added to detect the drug bound to the target of interest. The detection antibody or monomer / tetrameric streptavidin or streptavidin variant can be covalently linked to an enzyme or fluorescent label, or can itself be detected by a secondary antibody that is linked to an enzyme or fluorescent label through bioconjugation. Between each step, the plate is typically 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 produce a visible signal or the fluorescent signal is directly measured, which is indicative of the amount of covalently bound drug to the target of interest. The amount of covalent binding can be tracked based on the amount of signal.

[0248] In some embodiments, covalent binding is detected by an antibody array. "Antibody array" is defined as a system in which individual antibodies or multiple antibodies are linked to a solid support to enable detection of a protein of interest bound to the antibody and molecules bound to the protein of interest. An antibody microarray consists of a series of individual spots or wells in which a specific antibody has been hybridized to each spot or well (as described in U.S. Patent 20120231963A1, the contents of which are incorporated by reference in their entirety). Coupled clarified or unclarified lysate is added to the "antibody microarray" to enable separation of different proteins, localization of a particular protein to its antibody binding partner, or washing away of additional proteins. Labeled molecules of interest are detected at each microarray spot or well to check the amount of covalent binding of the molecule of interest to a particular protein or multiple proteins by detecting the presence of biotin labeled molecules to reveal the level of labeling. The level of labeling at each spot will indicate the amount of covalent labeling of a particular protein that has hybridized to a specific antibody. The biotin label on the molecule can be detected by adding a fluorescent molecule or a luminescent enzyme that binds to the label, including but not limited to techniques known to one of skill in the art, such as a fluorescence assay, a luminescence assay, a FRET assay, or a BRET assay. This readout can be detected using methods known to one of skill in the art, including but not limited to fluorescence or luminescence detection protocols.

[0249] In some embodiments, the lysate is labeled with biotin to produce 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 produced by click chemistry. In some embodiments, the compound is labeled with click chemistry after treatment with ARCS, cell separation, and cell lysis. In some embodiments, the click chemistry reagent includes pyridylmethyl azide.

[0250] As used herein, the term "click chemistry" refers to the Huisgen cycloaddition or 2,3-dipole cycloaddition between an azide and a terminal alkyne to form a 1,2,4-triazole. As used herein, the term "cycloaddition" refers to a chemical reaction in which two or more π electron systems (e.g., unsaturated molecules or unsaturated portions of the same molecule) combine to form a cyclic product in which there is a net decrease in bond multiplicity. In a cycloaddition, π electrons are used to form new Σ bonds. The product of the cycloaddition is referred to as an "adduct" or "cycloadduct". Different types of cycloadditions are known in the art, including but not limited to [3+2] cycloadditions and Diels-Alder reactions. [3+2] cycloadditions, also known as 2,3-dipole cycloadditions, occur between a 1,3-dipole and a dipolephile and are commonly used to construct five-membered heterocycles. The term "[3+2] cycloaddition" also encompasses the "copper-free" [3+2] cycloadditions described below between azides and cyclooctyne and difluorocyclooctyne: Bertozzi et al., J. Am. Chem. Soc., 2004, 126: 15046-15047. Any reagent that can be used to promote the Huisgen cycloaddition can be used as a click chemistry reagent. In some embodiments, the click chemistry reagent includes a pyridyl azide. In some embodiments, the click chemistry reagent includes a pyridylmethyl azide. Without limitation, any isomer of methylpyridine azide can be used.

[0251] In some embodiments, ARCS can be associated with or combined with one or more radioactive or detectable agents. These agents include various organic small 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., as pertechnetate (technetate(VII), TcO4 -) and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxide (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MION), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast agents (ioxaglate), microbubbles, or perfluorocarbons). Such optically detectable labels include, for example, but are not limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid; acridine and its derivatives (e.g., acridine and acridine isothiocyanate); 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and its derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanine; 4',6-diamidino-2-phenylindole (DAPI); 5'5"-dibromopyrogallol-sulfonaphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaminepentaacetate; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 5-[dimethylamino]-naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and its derivatives (e.g., eosin and eosin isothiocyanate); erythrosin and its derivatives (e.g., erythrosin B and erythrosin isothiocyanate); ethidium; fluorescein and its derivatives (e.g., 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, X rhodamine-5-(and-6)-isothiocyanate (QFITC or XRITC), and fluorescamine); 2-[2-[3-[[l,3-dihydro-l,l-dimethyl-3-(3-sulfopropyl)-2H indolium-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-l-piperazinyl]-l-cyclopenten-l-yl]vinyl]-l,l-dimethyl-3-(3-sulfopropyl)-lH benzo[e]indolium, inner salt, with N,N-diethylethanamine (1:1) (IR144); 5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazolyl-ylidene)ethylidene]-2-(diphenylamino)-l-cyclopenten-l-yl]vinyl]-3-ethylbenzothiazolium perchlorate (IR140); malachite green isothiocyanate; 4-methylumbelliferone ortho cresolphthalein;nitrotyrosine; pararosaniline; phenol red; B-phycoerythrin; o- phthalaldehyde; pyrene and its derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene); butyric acid quantum dots; Reactive Red 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and its derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonate, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulfo-rhodamine B, sulfo-rhodamine 101, sulfo-rhodamine 101 (Texas Red), N,N,N',N' tetramethyl-6-carboxyrhodamine (TAMRA) tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC) sulfonchloride derivatives; riboflavin; roseolic 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; phthalocyanine; and naphthol cyanine.

[0252] In some embodiments, the detectable agent can 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 fluorescent reagent (e.g., HRP-activated (VisEn Medical).

[0253] IV. Definitions

[0254] As used herein, the term “ARCS” refers to any therapeutic conjugate formed by linking a FCB and a CLM with 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 an aqueous solution at a temperature of 0-50 °C, within 48 hours, at a treatment dose of 10 mM.

[0255] As used herein, the term“FCB” refers to a therapeutic modality that can be a known drug, diagnostic compound, candidate drug, and functional fragment and / or any combination of the foregoing. FCBs encompass free acid and free base forms; optical and tautomeric forms; isotopes comprising a drug, prodrug, or fragment thereof and pharmaceutically acceptable salts. FCBs can 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 can be therapeutic agents such as, but not limited to, anti-cancer agents, anti-neurodegenerative agents, autoimmune drugs, and anti-aging agents. FCBs can non-covalently bind to a biological target. In some embodiments, FCBs can be functional fragments of drugs. As used herein, the term“functional fragment” refers to a portion of a drug or derivative or analog thereof that is capable of inducing the desired effect of the drug. In some embodiments, FCBs can include an alkyne functional group. In some embodiments, FCBs can not include an alkyne functional group.

[0256] As used herein, the term“CLM” refers to any covalent binding modality that is capable of forming a covalent bond with a biological target. CLMs can be linked to FCBs through a bond or through a linker. CLMs can include one or more chemical moieties that can form a covalent bond with a biological target. The chemical moieties can be electrophilic or nucleophilic groups.

[0257] As used herein, the term“linker” refers to an organic moiety that connects two portions of a compound. Linkers can be external linkers or internal linkers. External linkers can connect FCB moieties and CLM moieties. Internal linkers can be used to connect CLM moieties. In certain embodiments, CLMs can include internal linkers or spacers. Internal linkers or spacers can combine two portions of a CLM or can be linked to a CLM. External or internal linkers can be selected from the group consisting of a bond, substituted and unsubstituted C1-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 heterocycloalkenyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl. Linkers can be cleavable or non-cleavable.

[0258] As used herein, the term "biological target" refers to any target to which the FCB non-covalently binds to produce a therapeutic effect. The CLM covalently binds to the biological target. In some embodiments, the biological target is a protein.

[0259] As used herein, the term "toxicity" refers to the ability of a substance or composition to be harmful or toxic to a cell, tissue organism, or cellular environment. Low toxicity refers to a reduction in the ability of a substance or composition to be harmful or toxic to a cell, tissue organism, or cellular environment. Such a reduction or low toxicity can be associated with a standard measure, treatment, or absence of treatment.

[0260] As used herein, the term "compound" is intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. In some embodiments, compound can be used interchangeably with ARCS. Thus, as used herein, ARCS is also intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. As used herein, FCB and CLM are also intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure.

[0261] The compounds described herein can be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, all stereoisomers (e.g., enantiomers and diastereomers) are intended. Compounds of the disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of alkenes, C=N double bonds, and the like can also exist, and all such isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the disclosure are described and can be isolated as a mixture of isomers or as separate isomers.

[0262] The compounds of the disclosure also include tautomeric forms. Tautomeric forms arise from the exchange of a single bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states of a molecule with the same empirical formula and overall charge. Example prototropic tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and ring forms in which a proton can occupy two or more positions of a heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H-, and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, and 1H- and 2H-pyrazoles. Tautomeric forms can be in equilibrium, or, through appropriate substitution, spatially locked into one form.

[0263] The compounds of the present disclosure also include all isotopes of atoms occurring in the intermediates or final compounds. An "isotope" means atoms of the same atomic number but different mass numbers, caused by differences in the number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0264] The compounds and salts of the present disclosure can combine with solvent or water molecules to form solvates and hydrates by conventional means.

[0265] As used herein, the term "subject" or "patient" refers to any organism to which a particle 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, non-human primates, and humans).

[0266] As used herein, the term "treatment" or "prevention" can include preventing the disease, disorder, and / or condition from occurring in an animal that can be predisposed to the disease, disorder, and / or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder, or condition, e.g., arresting its development; and relieving the disease, disorder, and / or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease, disorder, or condition can include improving at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating pain in a subject by administering an analgesic, even if such agent does not treat the cause of the pain.

[0267] As used herein, "target" shall mean the site to which ARCS, FCB, and / or CLM bind. The target can be in vivo or in vitro. In certain embodiments, the target can be a cancer cell found in leukemia or tumors (e.g., tumors of the brain, lung (both small cell and non-small cell), ovary, prostate, breast, and colon, as well as other carcinomas and sarcomas). The target can be a tissue, such as neuronal tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.

[0268] A "target cell" that can serve as a target for a therapeutic conjugate is typically an animal cell, such as a mammalian cell. The methods of the present invention can be used to modify the cellular function of a living cell in vitro, i.e., in cell culture, or in vivo, where the cell forms part of an animal tissue or is otherwise present in an animal tissue. Thus, the target cell can include, for example, cells of the blood, lymphoid tissue, lining of the digestive tract, such as the oral and pharyngeal mucosa, cells forming the villi of the small intestine, cells lining the large intestine, cells lining the respiratory system (nasal passages / lungs) of the animal (which can be contacted by inhalation of the subject), dermis / epidermis cells, cells of the vagina and rectum, cells of internal organs, including placental cells and the so-called blood / brain barrier, and the like.

[0269] The term "therapeutic effect" is art-recognized and refers to a local or systemic effect in an animal, particularly a mammal, and more particularly a human, caused by a pharmacologically active substance. Thus, the term means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of ideal physical or mental development and well-being of an animal or human.

[0270] The term "modulate" is art-recognized and refers to up-regulation (i.e., activation or stimulation), down-regulation (i.e., inhibition or suppression), or a combination or separately.

[0271] As used herein, "parenteral administration" refers to administration by any route other than through the digestive tract (enterally) or non-invasive topical routes. For example, parenteral administration can include intravenous, intradermal, intraperitoneal, intrapleural, intratracheal, intraosseous, intracerebral, intrathecal, intramuscular, subcutaneous, subconjunctival, to a patient by injection and infusion.

[0272] As used herein, "topical administration" refers to non-invasive administration to the skin, orifices, or mucosa. Topical administration can be administered locally, i.e., they are capable of providing local action at the area of application without systemic exposure. Topical formulations can provide systemic effects by absorption into the bloodstream of an individual. 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.

[0273] As used herein, "enteral administration" refers to administration by absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.

[0274] 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 drawing of air into the alveoli. The air can be drawn in through the mouth or nose.

[0275] The terms "sufficient" and "effective," as used interchangeably herein, refer to an amount (e.g., mass, volume, dose, concentration, and / or time period) necessary to achieve one or more intended results. A "therapeutically effective amount" is at least the minimum concentration required to affect a measurable improvement of at least one symptom or particular condition or disorder, or to prevent, achieve a measurable increase in life expectancy, or overall improvement in the quality of life of a patient. Thus, a therapeutically effective amount depends on the particular biologically active molecule and the particular condition or disorder to be treated. Therapeutically effective amounts of many active agents, such as antibodies, are known in the art. Therapeutically effective amounts of the compounds and compositions described herein, e.g., for treating a particular disorder, can be determined by the skilled artisan, e.g., a physician, by the exercise of routine techniques.

[0276] The term "prodrug" refers to an agent, including a nucleic acid or protein, that is converted into a biologically active form in vitro and / or in vivo. Prodrugs can be useful because, in some situations, they can be easier to administer than the parent compound. For example, a prodrug can be bioavailable by oral administration whereas the parent compound is not. The prodrug can also have improved solubility in pharmaceutical compositions over the parent drug. Prodrugs can be converted to the parent drug by various mechanisms (including enzymatic and metabolic processes) and by a variety of mechanisms (including oxidative, enzymatic, and hydrolytic processes). Harper, N.J. (1962) Drug Latentiation. Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977) Application of Physical Organic Principles to Prodrug Design. E. B. Roche, ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs. APhA; J. Pharm. Sci.; E. B. 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 improved delivery 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 et al. (1996) Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, G. L. Amidon, P. I. Lee, and E. M. Topp, editors, Transport Processes in Pharmaceutical Systems, Marcell Dekker, pp. 185-218; Balant et al. (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 Deliv. Rev., 39(1-3): 183-209; Browne (1997). Phosphenytoin (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, New York: Elsevier Science Publishers; Fleisher et al. (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130; Fleisher et al. (1985) Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting Methods Enzymol. 112: 360-81; Farquhar D et al., (1983) Biologically Reversible Phosphate-Protective Groups J. Pharm. Sci., 72(3): 324-325; Han, H. K. et al. (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; D. M.Lambert (2000) Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm. Sci. Suppl. 11 2: S15-27; Wang, W. et al. (1999) Prodrug approach to the delivery of peptide drugs. Curr. Pharm. Des. 5(4):265-87.

[0277] As used herein, the term“pharmaceutically acceptable” means, within the scope of sound medical judgment, a compound, material, composition, and / or dosage form that is appropriate for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio as per the guidelines of agencies such as the U.S. Food and Drug Administration. As used herein,“pharmaceutically acceptable carrier” refers to all components in a pharmaceutical formulation that facilitate the delivery of the composition in the body. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, bulking agents, stabilizers, and combinations thereof.

[0278] As used herein, the term“molecular weight” generally refers to the mass or average mass of a material. If a polymer or oligomer, the 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 by various methods including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as weight average molecular weight (Mw) as opposed to number average molecular weight (Mn). Capillary viscometry provides an estimate of molecular weight as an intrinsic viscosity determined from a dilute polymer solution using a specific set of concentration, temperature, and solvent conditions.

[0279] As used herein, the term“small molecule” generally refers to an organic molecule having 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.

[0280] The term“alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cyclic alkyl (alicyclic) groups, alkyl substituted cyclic alkyl groups, and cyclic alkyl substituted alkyl groups.

[0281] In some embodiments, straight-chain or branched-chain alkyl groups have 30 or fewer carbon atoms in their backbone (e.g., C1-C30for straight-chain, and C3-C30for branched-chain). 30 C3-C30for branched-chain). 30), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments, a cycloalkyl group has from 3 to 10 carbon atoms in its ring structure, for example, 5, 6, or 7 carbon atoms in the ring structure. As used throughout the specification, examples, and claims, the term "alkyl" (or "lower alkyl") is intended to encompass both "unsubstituted alkyl" and "substituted alkyl," the latter referring to an alkyl moiety 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, phosphates, phosphonates, phosphinates, amino, amido, amidine, imine, cyano, nitro, azido, thiol, alkylthio, sulfate, sulfite, sulfamide, sulfonamido, sulfonic acid, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties.

[0282] Unless otherwise specified, "lower alkyl" as used herein means alkyl as defined above but having a main chain structure of one to ten carbons, or one to six carbon atoms. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout this application, the preferred alkyl is lower alkyl. In some embodiments, the substituents designated herein as alkyl are lower alkyl.

[0283] It will be understood by those skilled in the art that, if appropriate, the moiety substituted on the hydrocarbon chain can itself be substituted. For example, the substituents of a substituted alkyl group can include halogen, hydroxyl, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamido, sulfamides, and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Cycloalkyl groups can be substituted in the same manner.

[0284] As used herein, "heteroalkyl" refers to a straight chain or branched, or cyclic carbon-containing radical, or a combination thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein phosphorus and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized. Heteroalkyl groups can be substituted as defined above for alkyl.

[0285] 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, alkenyl, and cycloalkenyl groups, as well as alkynyl groups. "Arylthio" refers to an aryl or heteroaryl group. Alkylthio groups can be substituted as defined above for alkyl.

[0286] 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.

[0287] As used herein, the term "alkoxy" or "alkoxy" refers to an alkyl group as defined above with an oxygen group attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy. An "ether" is two hydrocarbons covalently linked by oxygen. Accordingly, the substituent of the alkyl group that makes the alkyl group an ether is or is similar to an alkoxy group, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. An aryloxy group can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are defined as follows. Alkoxy and aryloxy groups can be substituted as described above for alkyl groups.

[0288] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines, such as the moiety that can be represented by the following general formula: Among them, R9, R 10 and R' 10 Each independently represents hydrogen, alkyl, alkenyl, -(CH2) m -R8 or R9 and R 10 Together with the nitrogen atom to which they are attached, they complete a heterocyclic ring having 4 to 8 atoms in the ring structure; R8 represents an aryl, cycloalkyl, cycloalkenyl, heterocyclic or polycyclic ring; and m is zero or an integer in the range of 1 to 8. In some embodiments, R9 or R 10 Only one of them can be a carbonyl group, for example, R9, R 10 In still other embodiments, the term "amine" does not encompass amides, for example, where R9 and R 10 In some embodiments, R9 and R 10 (and optionally R' 10 ) each independently represents hydrogen, alkyl or cycloalkyl, alkenyl or cycloalkenyl, or alkynyl. Thus, as used herein, the term "alkylamine" means an alkylamine as defined above having attached thereto a substituted (as described above for alkyl) or unsubstituted alkyl group (i.e., R9 and R 10 At least one of the amino groups is an alkyl group.

[0289] The term "amido" is recognized in the art as an amino-substituted carbonyl group and includes moieties that can be represented by the following general formula:

[0290] Among them R9 and R 10 As defined above.

[0291] As used herein, "aryl" refers to a C5-C10 - a meta aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or biheterocyclic ring system. Broadly defined, as used herein, the term "aryl" includes 5-, 6-, 7-, 8-, 9-, and 10-membered monocyclic aromatic groups, which can contain zero to four heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Those aryl groups with heteroatoms in the ring structure can also be referred to as "aryl heterocyclic" or "heteroarenes." The aromatic ring can be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN; and combinations thereof.

[0292] The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are shared by two adjacent rings (i.e., "fused rings"), wherein at least one of the rings is aromatic, for example, the other cyclic ring or rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furazanyl, furanyl, furanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indacenyl, indazolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isoindazolyl, isoindolinyl, 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, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidinyl, 4-piperidinyl, 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, quinazolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thienyl, thiazolidinyl, thiazolinyl, thiazolyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, and xanthenyl. One or more of the rings can be substituted with "aryl" as defined above.

[0293] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[0294] As used herein, the term "carbocyclic" refers to an aromatic or non-aromatic ring in which every atom of the ring is carbon.

[0295] As used herein, "heterocycle" or "heterocyclic" refers to a cyclic radical of atoms linked through a monocyclic or bicyclic ring carbon or nitrogen atom, the monocyclic or bicyclic ring containing 3 to 10 ring atoms and preferably 5 to 6 ring atoms, consisting of carbon and one to four heteroatoms, each of which is selected from the group consisting of non-peroxide oxygen, sulfur and N(Y), wherein Y is absent or is H, O, (C1-C 10 )alkyl, phenyl or benzyl, and optionally containing 1 to 3 double bonds and optionally substituted with one or more substituents. Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnamyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuran [2,3-b] tetrahydrofuran, furanyl, furanyl, imidazolyl, oxazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolylene, indolinyl, indolizinyl, indolyl, 3H-indolyl, isoindolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, 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 oxazolyl, oxazolidinyl, oxazolyl, oxetanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidinyl, 4-piperidinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolinyl, quinoxalinyl, quinolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thienyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thienyl, and xanthenyl. The heterocyclyl group can be optionally substituted at one or more positions with one or more substituents of the alkyl and aryl groups as defined above (e.g., halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, thiol, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, -CF3 and -CN).

[0296] The term "carbonyl" is art-recognized and encompasses such moieties which can be represented by the following general formula: Where X is a bond or represents oxygen or sulfur, and R 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl, R' 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl. 11 or R' 11 When X is oxygen and R 11 As defined above, the moiety is referred to herein as a carboxyl group, and specifically when R 11 When X is oxygen and R' 11 Where X is sulfur and R is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. 11 or R' 11 When X is sulfur and R 11 When X is sulfur and R' 11 When X is a bond and R 11 When X is a bond and R 11 When NH is hydrogen, the above formula represents an "aldehyde" group.

[0297] As used herein, the term "monoester" refers to an analog of a dicarboxylic acid in which one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or a salt of a carboxylic acid. Examples of monoesters include, but are not limited to, monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic acid, and maleic acid.

[0298] As used herein, the term "heteroatom" refers to an atom of any element other than carbon or hydrogen. Examples of heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur, and selenium. Other heteroatoms include silicon and arsenic.

[0299] As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, -Br, or -I; the term "mercapto" means -SH; the term "hydroxy" means -OH; and the term "sulfonyl" means -SO2-.

[0300] As used herein, the term "substituted" refers to all permissible substituents of the compounds described herein. 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, but are not limited to, halogen, hydroxyl, or any other organic radical having any number of carbon atoms, preferably 1 to 14 carbon atoms, and optionally containing one or more heteroatoms such as oxygen, sulfur, or nitrogen groups in linear, branched, or cyclic structural formats. 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, aryloxy, substituted aryloxy, 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, sulfonate, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-Ci2cycloalkyl, C3-Ci2cycloalkenyl, C3-Ci2cycloalkylalkyl, C3-Ci2cycloalkenylalkyl, C3-Ci2cycloalkylalkenyl, C3-Ci2cycloalkylaryl, C3-Ci2cycloalkenylaryl, C3-Ci2cycloalkylheteroaryl, C3-Ci2cycloalkenylheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, heterocycloalkenyl, substituted heterocycloalkenyl, heterocycloalkylalkyl, substituted heterocycloalkylalkyl, heterocycloalkenylalkyl, substituted heterocycloalkenylalkyl, heterocycloalkylalkenyl, substituted heterocycloalkylalkenyl, heterocycloalkylaryl, substituted heterocycloalkylaryl, heterocycloalkenylaryl, substituted heterocycloalkenylaryl, heterocycloalkylheteroaryl, substituted heterocycloalkylheteroaryl, and the like. 20 cyclic, substituted C3-Ci2cycloalkyl, cyclic, substituted C3-Ci2cycloalkenyl, C3-Ci2cycloalkylalkyl, C3-Ci2cycloalkenylalkyl, C3-Ci2cycloalkylalkenyl, C3-Ci2cycloalkylaryl, C3-Ci2cycloalkenylaryl, C3-Ci2cycloalkylheteroaryl, C3-Ci2cycloalkenylheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, heterocycloalkenyl, substituted heterocycloalkenyl, heterocycloalkylalkyl, substituted heterocycloalkylalkyl, heterocycloalkenylalkyl, substituted heterocycloalkenylalkyl, heterocycloalkylalkenyl, substituted heterocycloalkylalkenyl, heterocycloalkylaryl, substituted heterocycloalkylaryl, heterocycloalkenylaryl, substituted heterocycloalkenylaryl, heterocycloalkylheteroaryl, substituted heterocycloalkylheteroaryl, and the like. 20 cyclic, substituted C3-Ci2cycloalkyl, cyclic, substituted C3-Ci2cycloalkenyl, C3-Ci2cycloalkylalkyl, C3-Ci2cycloalkenylalkyl, C3-Ci2cycloalkylalkenyl, C3-Ci2cycloalkylaryl, C3-Ci2cycloalkenylaryl, C3-Ci2cycloalkylheteroaryl, C3-Ci2cycloalkenylheteroaryl, heterocycloalkyl, substituted heterocycloalkyl, heterocycloalkenyl, substituted heterocycloalkenyl, heterocycloalkylalkyl, substituted heterocycloalkylalkyl, heterocycloalkenylalkyl, substituted heterocycloalkenylalkyl, heterocycloalkylalkenyl, substituted heterocycloalkylalkenyl, heterocycloalkylaryl, substituted heterocycloalkylaryl, heterocycloalkenylaryl, substituted heterocycloalkenylaryl, heterocycloalkylheteroaryl, substituted heterocycloalkylheteroaryl, and the like.

[0301] A heteroatom such as nitrogen can have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the valencies of the heteroatoms. It should be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atoms and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, or elimination.

[0302] 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, the substituents described herein. For appropriate organic compounds, the permissible substituents can be one or more and the same or different at each occurrence. A heteroatom such as nitrogen can have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the valencies of the heteroatoms.

[0303] In various embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which is optionally substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.

[0304] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonato, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkyloxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkyloxy, azido, alkylthio, oxo, acylalkyl, carboxyl ester, carboxamide, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, formamidoalkylaryl, formamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxyl, carbamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.

[0305] The terms“polypeptide,”“peptide,” and“protein” generally refer to polymers of amino acid residues. As used herein, the terms also apply to amino acid polymers in which one or more amino acid residues are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids. As generally used herein, the term“protein” refers to an amino acid polymer that is connected to other amino acid polymers by peptide bonds to form a chain long enough to produce tertiary and / or quaternary structure. By definition, the term“protein” does not include small peptides, which lack the higher order structure considered necessary for a protein.

[0306] A "functional fragment" of a protein, polypeptide, or nucleic acid is one that differs in sequence from the full-length protein, polypeptide, or nucleic acid, but that still retains at least one function of 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 can contain one or more amino acid or nucleotide substitutions. Methods for determining function of nucleic acids (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, DNA binding function of a polypeptide can be determined by, e.g., filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be determined by gel electrophoresis. Ability of a protein to interact with another protein can be determined by, e.g., co-immunoprecipitation, two-hybrid assay, or complementation, e.g., genetic or biochemical. See, e.g., Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98 / 44350.

[0307] 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, the pharmaceutically acceptable counterion is selected from citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylenebis-(2-hydroxy-3-naphthoate)). In some embodiments, the pharmaceutically acceptable counterion is selected from chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, citrate, malate, acetate, oxalate, acetate, and lactate. In particular embodiments, the pharmaceutically acceptable counterion is selected from chloride, bromide, iodide, nitrate, sulfate, bisulfate, and phosphate.

[0308] The term "pharmaceutically acceptable salt" means a salt of a compound that can be present in the compositions of the present application with the use of an acidic or basic group of the compound. Compounds included in the present compositions that are basic in nature are capable of forming 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, bisulfate, bisulfite, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. In addition to the above acids, compounds included in the present compositions that contain an amino moiety can form pharmaceutically acceptable salts with various amino acids, both natural and synthetic. Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali or alkaline-earth metal salts, and specifically, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[0309] If a compound described herein is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, can be prepared from the free base by dissolution in a suitable organic solvent and treating the solution with an acid, in accordance with procedures well-known in the art. Those skilled in the art will recognize various synthetic methodologies that can be used to prepare nontoxic, pharmaceutically acceptable addition salts.

[0310] Pharmaceutically acceptable salts can be derived from acids selected from the group consisting of 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric 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, cyclohexanesulfonic acid, dodecylsulfic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galacturonic acid, gentisic acid, glucoheptanoic acid, gluconic acid, 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, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, palmitic acid, pantothenic acid, phosphoric acid, propionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, undecylenic acid.

[0311] As used herein, the term "assay" refers to a series of activities associated with reporting results, which can include, but are not limited to, cell seeding, preparation of test materials, infection, lysis, analysis, and calculation of results.

[0312] The term "detectable response" as used herein refers to the occurrence of a signal or a change in a signal that is directly or indirectly detectable by observation or by an instrument. Typically, the detectable response is the appearance of a signal in which a fluorophore inherently emits fluorescence and does not produce a signal change upon binding to a metal ion or a biological compound. Alternatively, the detectable response is an optical response that results in a change in wavelength distribution pattern or absorbance or fluorescence intensity or light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, emission of a radioisotope, magnetic attractive force, and electron density.

[0313] It is to be understood that the following examples are intended to illustrate but not limit the present disclosure. Various other examples and modifications thereof, both to the features of the foregoing description and to the examples themselves, will be apparent to those skilled in the art from reading this disclosure and the foregoing description thereof. All publications and patents cited herein are incorporated by reference in their entirety.

[0314] Examples

[0315] Example 1: General Synthesis of ARCS

[0316] ARCS disclosed herein can be synthesized by one skilled in the art using general chemical synthesis principles and techniques. In a rational approach, ARCS is constructed from its individual components: a therapeutic moiety, an optional linker, and a covalent binding moiety. The components can be covalently bonded to each other via functional groups, as known in the art, wherein such functional groups can be present on the components or introduced onto the components using one or more steps. Functional groups that can be used to covalently bind the components together to produce ARCS include, but are not limited to, hydroxyl, sulfhydryl, or amino groups. Specific portions of the different components that are modified to provide covalent linkage are selected so as not to substantially adversely interfere with the desired binding activity of those components. For example, for a covalent binding moiety, regions that do not affect the covalent binding activity will be modified so as to retain a sufficient amount of the desired activity. Where necessary and / or desired, certain portions of the components can be protected using protecting groups, as known in the art, see, for example, Green and Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

[0317] Alternatively, ARCS can be generated using known combinatorial methods to produce large libraries of ARCS, which can then be screened to identify molecules that form covalent bonds with targets with desired pharmacokinetic characteristics.

[0318] Example 2: General Synthesis of Compounds 1-1 through 1-172

[0319] Compounds 1-1 to 1-172 of the present disclosure can be synthesized by those skilled in the art using general 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.

[0320] Compound 1-IV, a precursor of many compounds in compound 1-1 to compound 1-172, can be prepared as shown in Scheme 1. As described in U.S. Patent No. 9,724,352, starting pyrrole (1-I) can react 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, which can then be substituted by adding morpholine. If R6' in 1-IV is a nitro group, it can be reduced to corresponding NH2 groups with C / Pd reaction under H2 atmosphere. Amine can then further react with appropriate ester (e.g., dimethyl carbonate) or activated ester (e.g., methyl chloroformate) to form the methyl carbamate of the compound, or it can react with isocyanate (e.g., methyl isocyanate) to provide urea compound 1-V A .

[0321] Scheme 1

[0322]

[0323] To obtain piperazinyl compounds (e.g., Compound 1-11 and Compound 1-12), it can be advantageous to modify the methyl ester portion of Compound 1-IV prior to conversion of R6' to the final R6 group. As described above, if R6' in 1-IV is a nitro group, it can be reduced to the corresponding NH2 group over Pd / C under an atmosphere of H2. As shown in Scheme 2, the methyl ester of 1-IV can then be hydrolyzed (e.g., 2 M aqueous NaOH in EtOH), and the resulting acid reacted with piperazine-1-carboxylic acid tert-butyl ester (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., borane-dimethyl sulfide) to provide the protected piperazinyl compound 1-VI. As described above, 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 it can be reacted with an isocyanate (e.g., methyl isocyanate) to provide the urea compound 1-VII A (see, e.g., Group Compound 1-105 and Compound 1-106). If the amine group of Compound 1-VI is the final desired group (see Compound 1-121), it can also be protected (not shown). After addition of the desired X group, the amino protecting group can be removed to provide the final product. For example, N-acetyl-piperazine instead of N-BOC-piperazine can be used to provide an N-acetyl protecting group in Compound 1-VI. The amino group of Compound 1-VI can then be protected with the more stable N-BOC group. Compound 1-VI can then be modified with the X group, and the N-BOC removed to leave the final amino group.

[0324] Scheme 2

[0325]

[0326] Additional starting compounds 1-III A-C (see, Compounds Compound 1-101 through Compound 1-172) can be prepared as shown in Scheme 3. These starting compounds can then be modified as described herein.

[0327] Scheme 3

[0328]

[0329] The X groups of the present disclosure (see, e.g., Compounds 1-101 through Compound 1-172) can be attached as shown in Scheme 4. The BOC protecting group of Compound 1-VII (or acetyl as described above) can be removed by acid hydrolysis (e.g., HC1 in methanol) to provide Compound 1-VIII. The piperazinyl group of Compound 1-VIII can then be reacted with the appropriate ester (e.g., for 1-IX and 1-IX A , CH2=CHC(O)(CH2)2C(O)OCH3, or an acid or activated acid thereof) in the presence of a base (e.g., K2CO3 or dimethylaminopyridine) to provide the final Compound 1-IX (Compound 1-101) or 1-IX A (Compound 1-105). If the X group contains a moiety that can react under the conditions of amide formation, a protected X' group (e.g., a protected terminal amine) can be used, which is then deprotected and modified to obtain the final desired compound (e.g., the terminal amine can be deprotected and then reacted with the appropriate ester or acid to form the final amide of X).

[0330] Scheme 4

[0331]

[0332] Example 3: Synthesis of Compound 1-102

[0333] Compounds 1-XXII and Compound 1-XXV, intermediates for many of the compounds (Compound 1-101 through Compound 1-172), can be prepared as shown in Scheme 5. Precursor Compound 1-XXXII and Compound 1-102 can be synthesized as shown in Scheme 6. The remaining compounds can be synthesized in a similar manner.

[0334] Scheme 5

[0335]

[0336] Scheme 6

[0337]

[0338] Example 4: General Method for Screening ARCS

[0339] The ARCS of the present disclosure can be synthesized by one skilled in the art using general chemical synthesis principles and techniques. Alternatively, the ARCS can be produced using known combinatorial methods to produce large libraries of ARCS. The ARCS of the present disclosure can also be synthesized as shown in Examples 1 through 3. The ARCS that covalently bind to the biological target of the target cell can then be screened by gel assays, Western blots, ELISA, antibody arrays, or NanoBRET assays.

[0340] Example 5: Transfection Protocol and Readouts for NanoBRET Screening of ARCS

[0341] The human embryonic kidney 293-H (HEK 293, Gibco 293-H, #11631017) cell line was maintained in a water-saturated incubator at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Gibco, #11995065) supplemented with 10% fetal bovine serum (FBS, Gibco, #10082147) and 1x penicillin-streptomycin (100x solution, Gibco, #15140148). Cells were trypsinized using 0.05% or 0.25% trypsin-EDTA solution (Trypsin-EDTA, Phenol Red, Gibco, #25200056 (0.25%) or #25300054). Opti-MEM medium (Opti-MEM I Reduced Serum Medium, without phenol red, Gibco, #11058021) supplemented with 10% fetal bovine serum was used to culture cells overnight for NanoBRET readout experiments.

[0342] HEK293 cells were appropriately cultured prior to assay. Culture medium was removed from cell flasks by aspiration, washed once with PBS, then aspirated, trypsinized, and cells were dissociated from the flasks. Trypsin was neutralized using growth medium and cells were pelleted by centrifugation at 200 x g for 5 minutes. The medium was aspirated and cells were resuspended into a single cell suspension using Opti-MEM I supplemented with 10% FBS. Cell density was adjusted to 2 x 105cells / mL in Opti-MEM I supplemented with 10% FBS in a sterile conical tube. Cells were transfected and directly dispensed into 96-well plates for the next day’s NanoBRET assay, and thus, cells were cultured overnight in Opti-MEM. Cells were also transfected in bulk and dispensed into 96-well plates to allow cells to adhere to the plates overnight for washout studies. 5 / mL in Opti-MEM I supplemented with 10% FBS in a sterile conical tube. Cells were transfected and directly dispensed into 96-well plates for the next day’s NanoBRET assay, and thus, cells were cultured overnight in Opti-MEM. Cells were also transfected in bulk and dispensed into 96-well plates to allow cells to adhere to the plates overnight for washout studies.

[0343] Lipid:DNA complex was prepared as follows:

[0344] A 10 pg / mL solution of DNA was prepared in serum-free Opti-MEM. This solution contained the following ratios of carrier DNA and DNA encoding NanoLuc fused to the biological target. Serial dilution steps can be required to accurately dilute the NanoLuc fusion DNA. The following reagents were added in order in a sterile polystyrene test tube: 1 mL Opti-MEM without phenol red; 9.0 pg / mL of carrier DNA; 1.0 pg / mL of NanoLuc fusion DNA (for some targets, lesser amounts). The reagents were mixed well.

[0345] Add 30 pL of FuGENE HD to each mL of DNA mixture to form a lipid:DNA complex. Be careful not to let the FuGENE HD touch the plastic sides of the tube, but rather pipette directly into the liquid in the tube. Mix it by pipetting up and down 5-10 times, and incubate at room temperature for 20 minutes to form the complex. Mix 1 part (e.g., 1 mL) of the lipid:DNA complex with 20 parts (e.g., 20 mL) of the suspended HEK293 cells at 2 x 10 5 / mL, and mix gently by pipetting up and down 5 times in a sterile conical tube. Scale up or down accordingly using this ratio for larger or smaller batch transfections. Distribute 100 pL of the cells + lipid:DNA complex into tissue-culture treated sterile 96-well plates (20,000 cells / well), and incubate for at least 16 hours to allow for expression. Incubate the cells in a 37 °C + 5% CO2 incubator for > 16 hours. Prepare serially diluted inhibitors or test compounds in 100 x final concentration in 100% DMSO. Prepare serially diluted inhibitor stock solutions in a PCR plate. Add 1 pL per well of the 100 x serially diluted inhibitors / test compounds to the cells in the 96-well plates that have been transiently transfected overnight, and mix by gently tapping the plate by hand. Incubate the plates in a 37 °C + 5% CO2 incubator overnight. Prepare 1X substrate mixture solution (500X stock) and tracer at appropriate concentrations in Opti-Mem. Wash the cells by setting the plate washer to 96-well plates 5X in PBS pH 7.4 by adding 200 pL PBS per addition. Incubate the cells at 37 °C for 2 hours. Add 100 pL of 1X substrate-tracer solution, and gently tap the 96-well plate to mix. Read the plates on a plate reader every hour for the next 6 hours. Some ARCS binding assays are shown below. Compounds 1-XXXII form about 5% to 20% covalent bond with PI3-kinase. 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 form about 80% to 100% covalent bond with PI3-kinase. Compounds 1-171 and 1-172 form about 50% to 80% covalent bond with PI3-kinase. Activity of PI3-kinase is inhibited by about 5% to 20% by Compounds 1-XXXII. Activity of PI3-kinase is inhibited by about 80% to 100% by 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. Activity of PI3-kinase is inhibited by about 50% to 80% by Compounds 1-171 and 1-172.

[0346] Binding 1-XXXII + 1-101 +++ 1-102 +++ 1-113 +++ 1-114 +++ 1-119 +++ 1-120 +++ 1-125 +++ 1-126 +++ 1-171 ++ 1-172 ++

[0347] Percent Inhibition

[0348] + ++ +++

[0349] <20% 50-80% >80%

[0350] Equivalents and Scope

[0351] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited to the above description, but is set forth in the appended claims.

[0352] In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary by the context or otherwise obvious from the context. Claims or specifications containing "or" between one or more members of a group are considered satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary by the context or otherwise obvious from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise relevant to a given product or process.

[0353] It should also be noted that the term "comprising" is intended to be open ended and allows but does not require the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0354] When ranges are given, the endpoints are included. Furthermore, it should be understood that, unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges can be assumed to be any particular value or sub-range within the ranges described in the various embodiments of the present disclosure to be one-tenth of the unit of the lower limit of the range (unless the context clearly indicates otherwise).

[0355] Additionally, it should be understood that any particular embodiment of the present disclosure falling within the prior art can be expressly excluded from any one or more of the claims. As such embodiments are considered to be known to one of ordinary skill in the art, they can be excluded even if not expressly set forth herein. Any particular embodiment of the compositions of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use, etc.) can be excluded from any one or more of the claims for any reason, whether or not related to the existence of prior art.

[0356] It is to be understood that the words which have been used are words of description rather than limitation, and that changes can be made within the scope of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0357] While the present disclosure has been described with respect to a few described embodiments to a certain degree of particularity, the present disclosure is not intended to be limited to any such detail or embodiment or any particular embodiment, but is instead to be interpreted to be limited only in accordance with the appended claims so as to provide the broadest possible interpretation of such claims in accordance with the prior art and thus effectively encompass the intended scope of the present disclosure.

[0358] In light of the foregoing, the present invention includes, but is not limited to, the following:

[0359] 1. A therapeutic conjugate that forms a covalent bond with a kinase or pseudokinase.

[0360] 2. The therapeutic conjugate of item 1, wherein the kinase is a PI3-kinase (PI3K).

[0361] 3. The therapeutic conjugate of item 1 or item 2, wherein the therapeutic conjugate has the structure

[0362] (FCB)a-(L)b-(CLM)c,

[0363] wherein a and c are independently integers between 1 and 5,

[0364] b is an integer between 0 and 5, and

[0365] wherein the FCB moiety comprises a PI3K inhibitor or a fragment, analog, or derivative thereof.

[0366] 4. The therapeutic conjugate of item 3, wherein the FCB comprises

[0367] 5. The therapeutic conjugate of item 4, wherein the therapeutic conjugate comprises a structure selected from the group consisting of compounds 1-101 through 1-172.

[0368] 6. The therapeutic conjugate of item 1 or item 2 having the structure or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of ; either end of which can be attached to CLM; R1 is selected from the group consisting of: and CLM is selected from the group consisting of:

[0369] 7. The therapeutic conjugate of item 6 selected from the group consisting of: Compound 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, 1-122, 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.

[0370] 8. The therapeutic conjugate of item 1 having the structure or a pharmaceutically acceptable salt thereof, wherein L is R2 is selected from the group consisting of: and CLM is selected from the group consisting of:

[0371] 9. The therapeutic conjugate of item 8 selected from the group consisting of: Compound 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.

[0372] 10. A therapeutic conjugate comprising a structure selected from Compound 1-101 through Compound 1-172 or a pharmaceutically acceptable salt thereof.

[0373] 11. A therapeutic conjugate comprising a structure selected from the group consisting of Compound 1-1 through Compound 1-11, or a pharmaceutically acceptable salt thereof.

[0374] 12. A pharmaceutical composition comprising the therapeutic conjugate according to any one of items 1-11 and at least one pharmaceutically acceptable excipient.

[0375] 13. A method of modulating the activity of a kinase or pseudokinase, the method comprising administering the therapeutic conjugate according to any one of items 1-11.

[0376] 14. The method according to item 13, wherein the activity of the kinase or pseudokinase is inhibited.

[0377] 15. The method according to item 13, wherein the kinase is PI3K.

[0378] 16. A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to item 12.

[0379] 17. The method according to item 16, wherein the subject has a therapeutic condition selected from the group consisting of cancer, a neurodegenerative disease, an autoimmune disorder, and aging.

[0380] 18. The method according to item 17, wherein the subject has cancer.

[0381] 19. The method according to item 18, wherein the subject has a cancer that has a mutation in the PIK3CA gene.

Claims

1. A therapeutic conjugate that forms a covalent bond with a kinase or pseudokinase.

2. The therapeutic conjugate of claim 1, wherein the kinase is PI3-kinase (PI3K).

3. The therapeutic conjugate according to claim 1 or claim 2, wherein the therapeutic conjugate has the structure (FCB)a-(L)b-(CLM)c, wherein a and c are independently integers between 1 and 5, b is an integer between 0 and 5, and wherein the FCB moiety comprises a PI3K inhibitor or a fragment, analog or derivative thereof.

4. The therapeutic conjugate of claim 3, wherein the FCB comprises 5. The therapeutic conjugate of claim 4, wherein the therapeutic conjugate comprises a structure selected from the group consisting of Compound 1-101 to Compound 1-172.

6. The therapeutic conjugate according to claim 1 or claim 2, having the structure or a pharmaceutically acceptable salt thereof, wherein L is selected from A group consisting of, either end of which can be connected to the CLM; R1 is selected from the group consisting of: And the CLM is selected from the group consisting of:

7. The therapeutic conjugate of claim 6, which 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, 1-200, 1-201, 1-202, 1-203, 1-204, 1-205, 1-206, 1-207, 1-208, 1-209, 1-201 -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.

8. The therapeutic conjugate according to claim 1, having the structure or a pharmaceutically acceptable salt thereof, wherein L is R2 is selected from the group consisting of: And the CLM is selected from the group consisting of:

9. The therapeutic conjugate of claim 8, 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.

10. A therapeutic conjugate comprising a structure selected from Compound 1-101 to Compound 1-172 or a pharmaceutically acceptable salt thereof.

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