Piperidinylpyrazine-carboxamide compounds for treating and preventing cancer and for degrading BTK

Abstract

The present disclosure relates to BTK inhibitor compounds for treating or preventing cancer by daily oral dosing in a human subject in need thereof. The present disclosure also provides BTK inhibitor compounds for degrading BTK in vivo by daily oral dosing in a human subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 263,081, filed October 26, 2021, and U.S. Provisional Application No. 63 / 391,671, filed July 22, 2022, the contents of which are incorporated by reference in their entirety into this specification for all purposes.

[0002] This disclosure provides novel bifunctional compounds for targeted proteolysis of Bruton's tyrosine kinase (BTK) and methods for treating diseases regulated by BTK. [Background technology]

[0003]

[0003] B cell receptor (BCR) signaling regulates B cell development, as well as mature B cell activation, signaling and survival. Dysregulation of BCR signaling pathway is associated with many disease manifestations related to B cell function, and targeting B cell and BCR signaling has obvious therapeutic potential (Woyach et al.; Blood, 120(6); 1175-1184, 2012). For example, depletion of B cells by monoclonal antibodies targeting CD20 has a great impact on the treatment of B cell malignancies, autoimmune diseases and inflammatory diseases (Cang et al.; J Hematolo Oncol. 5; 64, 2012.).

[0004]

[0004] BTK is a member of the TEC family of kinases and is a key signaling hub in the BCR pathway. Mutations in BTK cause X-linked agammaglobulinemia (XLA), in which B cell maturation is impaired and immunoglobulin production is reduced (Hendriks et al.; Expert Opin Ther Targets 15;1002-1021, 2011). The central role of BTK in B cell signaling and function makes it an attractive therapeutic target for B cell malignancies, as well as autoimmune and inflammatory diseases. Ibrutinib, a covalent inhibitor of BTK, has been approved to treat chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and other B cell malignancies, as well as graft-versus-host disease (GvHD) (Miklos et al.; Blood, 120(21);2243-2250, 2017). Currently, ibrutinib and second generation BTK inhibitors are being investigated for oncology and immune-related indications, such as rheumatoid arthritis (Akinleye et al.; J of Hematolo Oncol. 6: 59, 2013; Liu et al.; J Pharmand Exper Ther. 338(1): 154-163. 2011; Di Paolo et al.; Nat Chem Biol. 7(1): 41-50, 2011).

[0005]

[0005] As an alternative to stoichiometric inhibition, proteolysis of BTK could have dramatic consequences on B cell function by effectively blocking BCR signaling. Removal of the BTK protein would eliminate BTK kinase activity as well as any protein interactions or scaffold functions of BTK. Specific degradation of BTK could be achieved by recruiting BTK to ubiquitin ligase using heterobifunctional small molecules to promote BTK ubiquitination and proteasomal degradation. Thalidomide derivatives such as lenalidomide or pomalidomide could be used to recruit potential substrates to cereblon (CRBN), a component of the ubiquitin ligase complex. This unique therapeutic approach may present a mechanism of action for disrupting BTK activity and BCR signaling that is distinct from that of stoichiometric BTK inhibition. Moreover, this degradation approach can effectively target the C481S mutant form of BTK, which has been observed clinically and confers resistance to inhibition by ibrutinib (Woyach et al.; Blood, 120(6): 1175-1184, 2012.). Summary of the Invention [Problem to be solved by the invention]

[0006] Currently, there remains a need for bifunctional molecules that can induce the in vivo proteolysis of BTK via the ubiquitin proteolytic pathway. [Means for solving the problem]

[0007]

[0007] Provided herein are methods of using bifunctional compounds that induce proteolysis of BTK via the ubiquitin proteolytic pathway.

[0008]

[0008] In one aspect, provided herein are methods of treating or preventing cancer in a human subject in need thereof. These methods include orally administering to a human subject an amount of a bifunctional compound capable of inducing proteolysis of Bruton's tyrosine kinase. In certain embodiments, the amount is effective to treat or prevent cancer. In certain embodiments, the cancer is a hematological malignancy. In certain embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, and combinations thereof. In certain embodiments, the cancer is CLL. In certain embodiments, the cancer is diffuse large B-cell lymphoma. In certain embodiments, the cancer is follicular lymphoma. In certain embodiments, the cancer is mantle cell lymphoma. In certain embodiments, the cancer is marginal zone lymphoma. In certain embodiments, the cancer is primary central nervous system lymphoma. In certain embodiments, the cancer is Waldenstrom's macroglobulinemia. In certain embodiments, the bifunctional compound is a compound as described herein, for example, according to formula (I). In certain embodiments, the compound is orally administered at a dose of about 50 mg to about 500 mg. In certain embodiments, the compound is orally administered at a dose of about 200 mg to about 400 mg. In certain embodiments, the compound is orally administered at a dose of about 150 mg to about 350 mg. In certain embodiments, the compound is orally administered at a dose of about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, or about 500 mg. In certain embodiments, the dose is per day. In certain embodiments, the human subject was previously treated with a Bruton's tyrosine kinase inhibitor. In certain embodiments, the prior treatment was with ibrutinib, acalabrutinib, pirtobrutinib, or any combination thereof.

[0009]

[0009] In certain embodiments, the methods include treating or preventing cancer by administering the bifunctional compound at a dose of about 50 mg. In certain embodiments, the methods include treating or preventing cancer by administering the bifunctional compound at a dose of about 100 mg. In certain embodiments, the methods include treating or preventing cancer by administering the bifunctional compound at a dose of about 200 mg. In certain embodiments, the methods include treating or preventing cancer by administering the bifunctional compound at a dose of about 300 mg. In certain embodiments, the methods include treating CLL by administering the bifunctional compound at a dose of about 100 mg. In certain embodiments, the methods include treating non-CLL cancer by administering the bifunctional compound at a dose of about 300 mg.

[0010]

[0010] In certain embodiments, the compound of formula I is administered in the form of intermittent dosing, which includes an administration period and a rest period. In certain embodiments, the intermittent dosing is repeated throughout the period of cancer treatment or prevention. In certain embodiments, the intermittent dosing is preceded by a loading dose. In certain embodiments, the administration period is about 2 weeks or about 3 weeks. In certain embodiments, the rest period is about 1 week or about 2 weeks. In certain embodiments, the intermittent dosing includes an administration period of about 2 weeks and a rest period of about 2 weeks, or an administration period of about 3 weeks and a rest period of about 1 week. In certain embodiments, the loading dose preceding the intermittent dosing is administered for about 4 weeks.

[0011]

[0011] In another aspect, provided herein are methods of degrading Bruton's tyrosine kinase in a human subject in need thereof. These methods include orally administering to the human subject an amount of a bifunctional compound capable of inducing proteolysis of Bruton's tyrosine kinase. In certain embodiments, the amount is effective to degrade Bruton's tyrosine kinase in the subject. In certain embodiments, Bruton's tyrosine kinase is degraded by at least 80%, 85%, 90%, or 95% in the human subject.

[0012] In these methods, the bifunctional compound comprises a moiety capable of specifically binding BTK and further comprises a moiety capable of recruiting ubiquitin ligase to degrade BTK. Specific compounds are described herein. These compounds can be administered in any form, including pharmaceutically acceptable salts and pharmaceutical compositions. [Brief description of the drawings]

[0013] [Figure 1] Plasma levels of the compounds are provided after daily oral dosing at the levels indicated. [Diagram 2]

[0014] BTK levels in patient circulating B cells after daily oral dosing at the levels indicated are provided. [Diagram 3]

[0015] 4 provides percent BTK levels in patient circulating B cells normalized to baseline levels after daily oral dosing at the levels indicated. [Figure 4]

[0016] 1 shows sustained benefit of compound 149 in CLL patients with a median of six prior therapies. [Diagram 5]

[0017] Presenting compound 149 in Phase 1a: Positive initial findings in CLL support expansion to 100 mg. [Figure 6]

[0018] 1 shows the efficacy of compound 149 as a degrader to overcome inhibitor resistance in lymphoma cells harboring the BTK-C481S mutation and multiple novel BTK mutations that emerge following BTKi treatment. [Figure 7]

[0019] 1 shows flow cytometry assessment of BTK levels in patients. [Figure 8]

[0020] 1 shows the effect of Compound 149 by flow cytometric assessment of BTK levels in patients. [Figure 9]

[0021] FDG-PET CT scan disease assessment at different time points is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014]

[0022] Provided herein are methods of using bifunctional compounds that induce proteolysis of Bruton's tyrosine kinase (BTK) via the ubiquitin proteolytic pathway.

[0015]

[0023] As used herein, the following definitions shall apply unless otherwise indicated. definition

[0016]

[0024] For purposes herein, the chemical elements are identified according to the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0017]

[0025] As used herein, a "protecting group" refers to a moiety or functional group that is introduced into a molecule by chemical modification of the functional group to obtain chemoselectivity in a subsequent chemical reaction. Standard protecting groups are described in Wuts and Greene: "Greene's Protective Groups in Organic Synthesis," 4th Ed, Wuts, PGM and Greene, TW, Wiley-Interscience, New York: 2006.

[0018]

[0026] As used herein, a "heterocycloalkyl" group refers to a 3-10 membered monocyclic or bicyclic (fused, bridged, or spiro) (e.g., 5-10 membered monocyclic or bicyclic) saturated ring structure in which one or more of the ring atoms is a heteroatom (e.g., nitrogen (N), oxygen (O), sulfur (S), or a combination thereof). Non-limiting examples of heterocycloalkyl groups include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl, 1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholinyl, octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl, octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl, octa ... benzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl, 1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, decahydro-2,7-naphthyridine, 2,8-diazaspiro[4.5]decane, 2,7-diazaspiro[3.5]nonane, octahydropyrrolo[3,4-c]pyrrole, octahydro-1H-pyrrolo[3,4-b]pyridine, and 2,6-dioxa-tricyclo[3.3.1.0]pyridine. 3,7 Monocyclic heterocycloalkyl groups can be fused with a phenyl moiety to form structures such as tetrahydroisoquinoline, which can be classified as heteroaryls.

[0019]

[0027] As used herein, a "heterocycloalkenyl" group refers to a monocyclic or bicyclic (e.g., 5-10 membered monocyclic or bicyclic) non-aromatic ring structure having one or more double bonds and in which one or more of the ring atoms is a heteroatom (e.g., N, O, or S). Monocyclic and bicyclic heteroalicyclics are numbered according to standard chemical nomenclature.

[0020]

[0028] A heterocycloalkyl or heterocycloalkenyl group can be phospho, aliphatic (e.g., alkyl, alkenyl, or alkynyl), alicyclic, (alicyclic)aliphatic, heteroalicyclic, (heteroalicyclic)aliphatic, aryl, heteroaryl, alkoxy, (alicyclic)oxy, (heteroalicyclic)oxy, aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino, amido (e.g., (aliphatic)carbonylamino, (alicyclic)carbonylamino, ((alicyclic)aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heteroalicyclic)carbonylamino, ((heteroalicyclic)aliphatic)carbonylamino, (heteroaryl)carbonylamino, or or (heteroaraliphatic)carbonylamino), nitro, carboxy (e.g., HOOC-, alkoxycarbonyl, or alkylcarbonyloxy), acyl (e.g., (alicyclic)carbonyl, ((alicyclic)aliphatic)carbonyl, (araliphatic)carbonyl, (heteroalicyclic)carbonyl, ((heteroalicyclic)aliphatic)carbonyl, or (heteroaraliphatic)carbonyl), nitro, cyano, halo, hydroxy, mercapto, sulfonyl (e.g., alkylsulfonyl or arylsulfonyl), sulfinyl (e.g., alkylsulfinyl), sulfanyl (e.g., alkylsulfanyl), sulfoxy, urea, thiourea, sulfamoyl, sulfamido, oxo, or carbamoyl.

[0021]

[0029] As described herein, the compounds herein may be substituted with one or more substituents, such as those generally illustrated herein or exemplified by specific classes, subclasses and species herein.

[0022]

[0030] As used herein, the phrase "stable or chemically feasible" refers to a compound that is substantially unchanged when subjected to conditions that allow for the production of the compounds, their detection and their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable or chemically feasible compound is one that is substantially unchanged when kept at a temperature of 40° C. or less for at least one week in the absence of moisture or other chemically reactive conditions.

[0023]

[0031] As used herein, "effective amount" is defined as the amount required to provide a therapeutic effect to the treated patient, and is typically determined based on the age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per square meter of body surface area) is described in Freireich et al., Cancer Chemother.Rep., 50:219 (1966). Body surface area can be roughly determined from the height and weight of the patient. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970). As used herein, "patient" refers to a mammal, including a human.

[0024]

[0032] As used herein, the term "about" means within ±10% of a value. For example, a dose of about 100 mg / kg indicates that the dose may be 90 mg / kg to 110 mg / kg. As a further example, an amount of additional therapeutic agent in the range of about 50% to about 100% indicates an amount of additional therapeutic agent in the range of 45-55% to 90-110%. One of ordinary skill in the art will understand the range and application of the term "about" when used to describe other values ​​disclosed herein.

[0025]

[0033] Unless otherwise indicated, a structure depicted herein is also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of that structure, such as (R) and (S) configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers of the compounds, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures, are included within the scope of the specification. Alternatively, as used herein, "enantiomeric excess (ee)" refers to a dimensionless molar ratio that represents the purity of a chiral material, for example, containing a single stereocenter. For example, an enantiomeric excess of 0 would indicate a racemate (e.g., a 50:50 enantiomeric mixture, or no excess of one enantiomer over the other). As a further example, an enantiomeric excess of 99 would indicate a nearly stereochemically pure enantiomeric compound (i.e., a large excess of one enantiomer over the other). Percentage enantiomeric excess, %ee=([(R)-compound]-[(S)-compound]) / ([(R)-compound]+[(S)-compound])×100, where (R)-compound>(S)-compound; or %ee=([(S)-compound]-[(R)-compound]) / ([(S)-compound]+[(R)-compound])×100, where (S)-compound>(R)-compound. Furthermore, as used herein, "diastereomeric excess (de)" refers to a dimensionless molar ratio that represents the purity of a chiral substance that contains more than one stereocenter. For example, a diastereomeric excess of 0 would indicate an equimolar mixture of diastereoisomers. As a further example, a diastereomeric excess of 99 would indicate a nearly stereochemically pure diastereomeric compound (i.e., a large excess of one diastereomer over the other). Diastereomeric excess can be calculated in a similar manner to ee. As one skilled in the art would understand, de is usually reported as percent de (%de). %de can be calculated in a similar manner to %ee.

[0026]

[0034] In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de greater than 0. For example, in certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de of 10. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de of 25. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de of 50. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de of 75.

[0027]

[0035] In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de range of 90-100. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de range of 95-100. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de range of 97-100. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de range of 98-100. In certain embodiments, the compounds or inhibitors described herein have an ee, de, %ee, or %de range of 99-100.

[0028]

[0036] In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 1. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 2. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 3. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 4. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 5. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 6. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 7. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 8. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 9. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 10. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 11. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 12. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 13. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 14. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 15. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 16. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 17. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 18.In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 19. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 20. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 21. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 22. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 23. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 24. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 25. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 26. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 27. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 28. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 29. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 30. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 31. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 32. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 33. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 34. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 35.In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 36. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 37. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 38. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 39. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 40. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 41. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 42. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 43. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 44. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 45. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 46. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 47. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 48. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 49. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 50. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 51. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 52.In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 53. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 54. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 55. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 56. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 57. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 58. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 59. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 60. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 61. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 62. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 63. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 64. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 65. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 66. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 67. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 68. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 69.In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 70. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 71. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 72. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 73. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 74. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 75. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 76. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 77. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 78. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 79. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 80. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 81. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 82. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 83. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 84. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 85. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 86.In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 87. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 88. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 89. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 90. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 91. In one embodiment of a compound or inhibitor described herein, In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 92. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 93. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 94. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 95. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 96. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 97. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 98. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 99. In one embodiment of a compound or inhibitor described herein, ee, de, %ee, or %de is 100. In certain embodiments, the compounds or inhibitors described herein have ee, de, %ee, or %de as described in this paragraph. In certain embodiments, the compounds according to Formula (I) or Table 1 as described in the Examples and / or Biological Examples have ee, de, %ee, or %de as described in this paragraph. Unless otherwise stated, all tautomeric forms of the compounds herein are within the scope of the present invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the present invention include compounds having the replacement of hydrogen by deuterium or tritium or the like. 13 C or 14 It is within the scope of the present specification, except for the replacement of carbon with C-rich carbon. Such compounds are useful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.

[0029]

[0037] As used herein, the term "&1" means that a compound that includes the designation "&1" at a particular chemical element or atom (e.g., carbon) within that compound was prepared as a mixture of two stereoisomers at the referenced chemical element or atom (e.g., a diastereomeric mixture having de or % de as described above).

[0030]

[0038] Chemical structures and nomenclature are from ChemDraw, version 11.0., Cambridge, MA.

[0031]

[0039] It should be noted that the use of the descriptors "first," "second," "third," etc. are used to distinguish separate elements (e.g., solvents, reaction steps, processes, reagents, etc.) and may or may not refer to the relative order or relative chronology of the elements being described.

[0032] Uses of the Compounds and Compositions

[0040] The bifunctional compounds described herein are useful for degrading BTK via the ubiquitin proteolytic pathway in biological samples or patients. Thus, one embodiment of the present disclosure provides a method for treating a BTK-mediated disease or disorder. As used herein, the term "BTK-mediated disease or disorder" refers to any disease, disorder, or other deleterious condition in which BTK is known to play a role. In some cases, the BTK-mediated disease or disorder is a proliferative disorder or an autoimmune disorder. Examples of proliferative disorders include cancer.

[0033]

[0041] In one aspect, provided herein is a method for treating or preventing cancer in a subject in need thereof.In certain embodiments, these methods comprise orally administering to a subject an amount of a bifunctional compound capable of inducing proteolysis of Bruton's tyrosine kinase.In certain embodiments, this amount is effective for treating or preventing cancer.In certain embodiments, the bifunctional compound is compound 149.In certain embodiments, the bifunctional compound is compound 195.

[0034]

[0042] In certain embodiments, provided herein are methods for treating or preventing cancer in a human subject in need thereof. These methods include orally administering to a human subject an amount of a bifunctional compound capable of inducing proteolysis of Bruton's tyrosine kinase. In certain embodiments, this amount is effective for treating or preventing cancer. In certain embodiments, the BTK mutant is C481S. In certain embodiments, the dose is per day. In certain embodiments, the human subject has already been treated with a Bruton's tyrosine kinase inhibitor. In certain embodiments, the prior treatment was with ibrutinib, acalabrutinib, pirtobrutinib, or any combination thereof.

[0035]

[0043] In certain embodiments, the cancer is any cancer deemed suitable for treatment by the practitioner of the art. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is a hematological malignancy. In certain embodiments, the cancer is a B-cell malignancy. In certain embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, and combinations thereof. In certain embodiments, the cancer is CLL. In certain embodiments, the cancer is diffuse large B-cell lymphoma. In certain embodiments, the cancer is follicular lymphoma. In certain embodiments, the cancer is mantle cell lymphoma. In certain embodiments, the cancer is marginal zone lymphoma. In certain embodiments, the cancer is primary central nervous system lymphoma. In certain embodiments, the cancer is Waldenstrom's macroglobulinemia.

[0036]

[0044] In certain embodiments, the subject has a mutant Bruton's tyrosine kinase. In certain embodiments, the subject has a C481S, L528W, M437R, or V416L mutant Bruton's tyrosine kinase, or a combination thereof. In certain embodiments, the subject has a C481S mutant Bruton's tyrosine kinase. In certain embodiments, the subject has a L528W mutant Bruton's tyrosine kinase. In certain embodiments, the subject has a M437R mutant Bruton's tyrosine kinase. In certain embodiments, the subject has a V416L mutant Bruton's tyrosine kinase. In certain embodiments, the subject has a mutation that causes the resulting Bruton's tyrosine kinase to lack significant kinase activity. In certain embodiments, the subject has a mutation that causes the resulting Bruton's tyrosine kinase to lack measurable kinase activity.

[0037]

[0045] In certain embodiments, the cancer is resistant to BTK inhibitors. In certain embodiments, the cancer is resistant to ibrutinib, acalabrutinib, pirtobrutinib, or any combination thereof. In certain embodiments, the cancer is relapsed / refractory leukemia. In certain embodiments, the cancer is relapsed CLL. Those skilled in the art will recognize that certain resistant cancers express C481 mutant Bruton's tyrosine kinase, e.g., C481S Bruton's tyrosine kinase. For example, in certain embodiments, the subject has C481 mutant Bruton's tyrosine kinase and the cancer is chronic lymphocytic leukemia (CLL). Furthermore, certain resistant cancers express mutant Bruton's tyrosine kinase, such as L528W, and the mutant BTK lacks measurable kinase activity. In certain embodiments, the human subject has already been treated with a BTK inhibitor. In certain embodiments, the prior treatment was with ibrutinib, acalabrutinib, pirtobrutinib, or any combination thereof. In certain embodiments, the prior treatment was with ibrutinib. In certain embodiments, the prior treatment was with acalabrutinib. In certain embodiments, the prior treatment was with pirtobrutinib. In certain embodiments, the prior treatment was with ibrutinib, acalabrutinib, and pirtobrutinib.

[0038]

[0046] In certain embodiments, the compounds described herein can treat patients with a disease selected from the group consisting of Waldenstrom's macroglobulinemia, primary central nervous system lymphoma (PCNSL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), DLBCL, follicular lymphoma, and CLL. In certain embodiments, the disease is Waldenstrom's macroglobulinemia. In certain embodiments, the disease is PCNSL. In certain embodiments, the disease is MZL. In certain embodiments, the disease is MCL. In certain embodiments, the disease is DLBCL. In certain embodiments, the disease is follicular lymphoma. In certain embodiments, the disease is CLL.

[0039]

[0047] In certain embodiments, the compound of formula I is administered for the treatment or prevention of cancer, such as diffuse large B-cell lymphoma. In certain embodiments, the cancer to be treated or prevented is germinal center B-cell-like (GCB) DLBCL. In certain embodiments, the cancer to be treated or prevented is non-GCB DLBCL. In certain embodiments, the cancer to be treated or prevented is activated B-cell-like (ABC) DLBCL, which is a subtype of non-GCB DLBCL.

[0040]

[0048] In another aspect, provided herein are methods of degrading Bruton's tyrosine kinase in a subject in need thereof. These methods include orally administering to a subject an amount of a bifunctional compound capable of inducing proteolysis of Bruton's tyrosine kinase. In certain embodiments, the amount is effective to degrade Bruton's tyrosine kinase in the subject. Bruton's tyrosine kinase may be expressed in any cell or tissue of the subject. In certain embodiments, Bruton's tyrosine kinase is expressed in splenocytes. In certain embodiments, Bruton's tyrosine kinase is expressed in peripheral blood mononuclear cells. In certain embodiments, Bruton's tyrosine kinase is expressed in circulating B cells. In certain embodiments, the amount is effective to degrade Bruton's tyrosine kinase in the subject. In certain embodiments, Bruton's tyrosine kinase is degraded by at least 80%, 85%, 90%, or 95% in a human subject. In certain embodiments, the degradation is relative to baseline levels in the subject. In certain embodiments, the level is measured in splenocytes, plasma, peripheral mononuclear cells, or circulating B cells.The level can be measured by standard techniques, including immunoassay, ELISA, and FRET immunoassay.Exemplary techniques are described herein.In certain embodiments, the bifunctional compound is Compound 149.In certain embodiments, the bifunctional compound is Compound 195.

[0041]

[0049] The bifunctional compound may be administered at any dosage deemed appropriate by the practitioner of the art. In certain embodiments, the dosage is 0.1-1000 mg / kg. In certain embodiments, the dosage is 0.1-900 mg / kg. In certain embodiments, the dosage is 0.1-800 mg / kg. In certain embodiments, the dosage is 0.1-700 mg / kg. In certain embodiments, the dosage is 0.1-600 mg / kg. In certain embodiments, the dosage is 0.1-500 mg / kg. In certain embodiments, the dosage is 0.1-400 mg / kg. In certain embodiments, the dosage is 0.1-300 mg / kg. In certain embodiments, the dosage is 0.1-200 mg / kg. In certain embodiments, the dosage is 0.1-100 mg / kg. In certain embodiments, the dose is selected from the group consisting of 100 mg / kg, 200 mg / kg, 300 mg / kg, 450 mg / kg, 600 mg / kg, 800 mg / kg, and 1000 mg / kg. In certain embodiments, the dose is about 25 mg / kg. In certain embodiments, the dose is about 50 mg / kg. In certain embodiments, the dose is about 75 mg / kg. In certain embodiments, the dose is about 100 mg / kg. In certain embodiments, the dose is about 150 mg / kg. In certain embodiments, the dose is about 200 mg / kg. In certain embodiments, the dose is about 250 mg / kg. In certain embodiments, the dose is about 300 mg / kg. In certain embodiments, the dose is about 400 mg / kg. In certain embodiments, the dose is about 450 mg / kg. In certain embodiments, the dose is about 500 mg / kg. In certain embodiments, the dose is about 600 mg / kg. In certain embodiments, the dose is about 700 mg / kg. In certain embodiments, the dose is about 750 mg / kg. In certain embodiments, the dose is about 800 mg / kg. In certain embodiments, the dose is about 900 mg / kg. In certain embodiments, the dose is about 1000 mg / kg.

[0042]

[0050] In certain embodiments, the compound is orally administered in a dose of about 50 mg to about 500 mg. In certain embodiments, the compound is orally administered in a dose of about 200 mg to about 400 mg. In certain embodiments, the compound is orally administered in a dose of about 150 mg to about 350 mg. In certain embodiments, the compound is orally administered in a dose of about 50 mg. In certain embodiments, the compound is orally administered in a dose of about 100 mg. In certain embodiments, the compound is orally administered in a dose of about 200 mg. In certain embodiments, the compound is orally administered in a dose of about 300 mg. In certain embodiments, the compound is orally administered in a dose of about 400 mg. In certain embodiments, the compound is orally administered in a dose of about 500 mg.

[0043]

[0051] In certain embodiments, the methods include treating CLL by administering the bifunctional compound at a dose of about 100 mg. In certain embodiments, the methods include treating non-CLL cancer by administering the bifunctional compound at a dose of about 300 mg. In certain embodiments, the bifunctional compound is Compound No. 149. In certain embodiments, the bifunctional compound is Compound 195.

[0044]

[0052] In certain embodiments, the methods include treating or preventing cancer by administering compound number 149. In certain embodiments, compound 149 is orally administered at a dose of about 50 mg to about 500 mg. In certain embodiments, compound 149 is orally administered at a dose of about 200 mg to about 400 mg. In certain embodiments, compound 149 is orally administered at a dose of about 150 mg to about 350 mg. In certain embodiments, compound 149 is orally administered at a dose of about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, or about 500 mg. In certain embodiments, compound 149 is administered at a dose of about 300 mg for treating or preventing cancer. In certain embodiments, the dose is daily. In certain embodiments, compound 149 is administered for treating or preventing cancer, such as chronic lymphocytic leukemia (CLL). In certain embodiments, compound 149 is administered at a dose of about 100 mg for the treatment or prevention of CLL.

[0045]

[0053] In certain embodiments, the methods include treating or preventing cancer by administering compound number 195. In certain embodiments, compound 195 is orally administered at a dose of about 50 mg to about 500 mg. In certain embodiments, compound 195 is orally administered at a dose of about 200 mg to about 400 mg. In certain embodiments, compound 195 is orally administered at a dose of about 150 mg to about 350 mg. In certain embodiments, compound 195 is orally administered at a dose of about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, or about 500 mg. In certain embodiments, compound 195 is administered at a dose of about 100 mg for treating or preventing cancer. In certain embodiments, the dose is per day. In certain embodiments, compound 195 is administered for treating or preventing cancer, such as chronic lymphocytic leukemia (CLL). In certain embodiments, compound 195 is administered at a dose of about 50 mg for the treatment or prevention of CLL. In certain embodiments, compound 195 is administered at a dose of about 100 mg for the treatment or prevention of CLL.

[0046]

[0054] The dose can be administered in a schedule deemed appropriate by a person skilled in the art. In certain embodiments, the dose is administered once per day. In certain embodiments, the dose is administered twice per day. In certain embodiments, the dose is administered three times per day. In certain embodiments, the dose is administered four times per day. In certain embodiments, the dose is administered in divided doses. In certain embodiments, the dose is administered in two separate doses per day. In certain embodiments, the dose is administered in three separate doses per day. In certain embodiments, the dose is administered in four separate doses per day.

[0047]

[0055] Dosing can continue for any length of time deemed appropriate by one of skill in the art. In certain embodiments, the dose is administered daily for 30 days or more. In certain embodiments, the dose is administered daily for 15 days. In certain embodiments, the dose is administered daily for 14 days. In certain embodiments, the dose is administered daily for 13 days. In certain embodiments, the dose is administered daily for 12 days. In certain embodiments, the dose is administered daily for 11 days. In certain embodiments, the dose is administered daily for 10 days. In certain embodiments, the dose is administered daily for 9 days. In certain embodiments, the dose is administered daily for 8 days. In certain embodiments, the dose is administered daily for 7 days. In certain embodiments, the dose is administered daily for 6 days. In certain embodiments, the dose is administered daily for 5 days. In certain embodiments, the dose is administered daily for 4 days. In certain embodiments, the dose is administered daily for 3 days. In certain embodiments, the dose is administered daily for 2 days. In certain embodiments, the doses are administered over the course of a day.

[0048]

[0056] In the dosing schedule, the doses can be administered daily or cyclically, according to the judgment of the practitioner of the art. In certain embodiments, the doses are administered daily. In certain embodiments, the doses are administered with an interval between doses. In certain embodiments, the interval is 1 day. In certain embodiments, the interval is 2 days. In certain embodiments, the interval is 3 days. In certain embodiments, the interval is 4 days. In certain embodiments, the interval is 5 days. In certain embodiments, the interval is 6 days.

[0049]

[0057] In certain embodiments, the dose is administered weekly. In certain embodiments, the dose is administered twice a week. In certain embodiments, the dose is administered three times a week.

[0050]

[0058] In certain embodiments, the dose(s) are administered over a period of time with a first interval between the dose(s), and then the dose(s) are re-administered over a period following the first interval between the dose(s), where this dosing regimen can be repeated (i.e., cyclically or periodically, e.g., after a second, third, etc. interval between administration of the subsequent dose(s)) according to the discretion of the practitioner of the art. For example, in one embodiment, the first dose is administered for one week, followed by a first interval of one week without administration of the first dose, then the second dose is re-administered for another week, followed by a second interval of one week without administration of the first or second dose, and so on cyclically. Other perturbations of the first, second, third, etc. dose(s), followed by perturbations of the first, second, third, etc. interval(s), and combinations thereof are contemplated herein as understood by the practitioner of the art and the needs of the patient. For example, in one embodiment, a first dose is administered daily for one week, followed by a first interval of three weeks without administration of the first daily dose, and then a second dose is re-administered every other week for another week, followed by a second interval of four weeks without administration of the first daily or second biweekly dose, and so on in a cyclical manner.

[0051]

[0059] In certain embodiments, the compound of formula I is administered in the form of an intermittent dosing comprising an administration period and a rest period. In certain embodiments, the administration period is a period during which the compound of formula I is administered daily to the human subject. In certain embodiments, the rest period is a period during which the compound of formula I is not administered to the human subject for the entire duration of the rest period. In certain embodiments, the intermittent dosing is repeated throughout the duration of the cancer treatment or prevention. In certain embodiments, the intermittent dosing is preceded by a loading dose. In certain embodiments, the administration period is about 0.5 to about 6 weeks. In certain embodiments, the administration period is about 1 to about 5 weeks. In certain embodiments, the administration period is about 2 to about 4 weeks. In certain embodiments, the administration period is about 0.5 weeks, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. In certain embodiments, the administration period is about 2 weeks. In certain embodiments, the administration period is about 3 weeks. In certain embodiments, the rest period is about 0.5 to about 6 weeks. In certain embodiments, the rest period is about 1 to about 5 weeks. In certain embodiments, the rest period is about 2 to about 4 weeks. In certain embodiments, the rest period is about 0.5 weeks, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. In certain embodiments, the rest period is about 2 weeks. In certain embodiments, the rest period is about 1 week. In certain embodiments, the intermittent dosing comprises an administration period of about 2 weeks and a rest period of about 2 weeks. In certain embodiments, the intermittent dosing comprises an administration period of about 3 weeks and a rest period of about 1 week. In certain embodiments, the loading dose preceding the intermittent dosing is administered for about 1 to about 7 weeks. In certain embodiments, the loading dose is administered for about 2 to about 6 weeks. In certain embodiments, the loading dose is administered for about 3 to about 5 weeks. In certain embodiments, the loading dose is administered over about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks. In certain embodiments, the loading dose is administered over about 4 weeks.

[0052]

[0060] The compounds can be administered by any route of administration deemed appropriate by the practitioner of the art. In certain embodiments, the dose is administered orally. Formulations and techniques for administration are described in detail below.

[0053]

[0061] In certain embodiments, the term "cancer" includes, but is not limited to, the following cancers: epidermoid oral: buccal cavity, lips, tongue, mouth, pharynx, squamous cell carcinoma of the head and neck (HNSCC); cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; lung: bronchogenic carcinoma (squamous cell or epidermoid, small undifferentiated cell, large undifferentiated cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitin hamartoma, mesothelioma, non-small cell lung cancer (NSCLC); gastrointestinal: gastric cancer, esophagus (squamous cell carcinoma, laryngeal, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (small large bowel or large intestines (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colorectal, large intestine Microsatellite stable colorectal cancer (MSS) CRC), rectum; genitourinary tract: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma), metastatic castration-resistant prostate cancer (mCRPC), muscle-invasive urothelial carcinoma; liver: hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary tract; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, Chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma (MM), malignant giant cell chondroma, osteochondroma (osteochondroma exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumor; nervous system: skull (osteoma, hemangioma, granuloma, xanthomatosis, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, embryonal tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma);Gynecology: Uterus (endometrial cancer), Cervix (cervical cancer, cervical carcinoma, preneoplastic cervical dysplasia), Ovaries (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), Vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), Vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), Fallopian tubes (carcinoma), Breast, Triple-negative breast cancer (TNBC), Platinum-resistant epithelial ovarian cancer ( EOC);Hematologic: Blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma) hairy cell;Lymphoid disorders (e.g., mantle cell lymphoma, Waldenström's macroglobulinemia, marginal zone lymphoma, and follicular lymphoma);Skin: Malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, keratoacanthoma, dysplastic nevi (moles dysplastic nevi), lipoma, hemangioma, dermatofibroma, keloid, psoriasis; thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, anaplastic thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid carcinoma, pheochromocytoma, paraganglioma; adrenal gland: neuroblastoma; and metastatic melanoma.;

[0054]

[0062] Examples of autoimmune disorders include urticaria, graft-versus-host disease (GVHD), acute graft-versus-host disease, pemphigus vulgaris, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, axonal and neuronal neuropathy (AM). AN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis with polyangiitis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis ( EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne contralateral), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes mellitus (type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear immunoglobulin A disease (LAD), Lupus, Lyme disease chronic, Meniere's disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multifocal motor neuropathy (MMN) or MMNCB, Multiple sclerosis, Gravitas myasthenia, Myositis, Narcolepsy, Neonatal lupus, Neuromyelitis optica, Neutropenia,Ocular cicatricial pemphigoid, Optic neuritis, Relapsing rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsplanitis (peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndrome type I, II, III, Polymyalgia rheumatica, Polymyositis, Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Recurrent These include: polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity; stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis (giant cell arteritis), thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, leukoplakia, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0055]

[0063] The bifunctional compound can be any bifunctional compound described herein. In certain embodiments, the compound is according to Formula I or a pharma- ceutically acceptable salt thereof:

[0056] [ka] In the formula, R 1 is selected from hydrogen and a substituted or unsubstituted heterocycle; R 2 teeth,

[0057] [ka] is selected from n is 1 or 2.

[0058]

[0064] In certain embodiments, the bifunctional compound is Compound 149. In certain embodiments, the bifunctional compound is Compound 195.

[0059]

[0065]

[0060] [Table 1-1]

[0061] [Table 1-2]

[0062] [Table 1-3]

[0063] [Table 1-4]

[0064] [Table 1-5]

[0065] [Table 1-6] and pharma- ceutically acceptable salts thereof.

[0066] Formulation and Administration Pharmaceutical Compositions

[0067] The compounds described herein can be formulated into pharmaceutical compositions that further comprise pharma- ceutically acceptable carriers, diluents, adjuvants, or vehicles.In one embodiment, the present disclosure provides a pharmaceutical composition that comprises the above-mentioned compounds and pharma- ceutically acceptable carriers, diluents, adjuvants, or vehicles.In one embodiment, the present disclosure is a pharmaceutical composition that comprises an effective amount of the compounds of the present disclosure or its pharma- ceutically acceptable salts and pharma-ceutically acceptable carriers, diluents, adjuvants, or vehicles.Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients, or carriers that are appropriately selected according to intended administration form and in accordance with conventional pharmaceutical practice.

[0067]

[0068] According to another embodiment, the present specification provides a composition comprising a compound of the present specification or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier, adjuvant, or vehicle. The pharmaceutical composition of the present specification includes a therapeutically effective amount of a compound described herein, where a "therapeutically effective amount" is an amount effective to (a) measurably degrade BTK (or reduce the amount of BTK) in a biological sample or in a patient; or (b) treat and / or ameliorate a disease or disorder mediated by BTK.

[0068]

[0069] The term "patient", as used herein, means an animal, or a mammal, or a human.

[0069]

[0070] It will also be understood that certain compounds of the present disclosure may exist for treatment in free form or, where appropriate, as a pharma- ceutically acceptable derivative thereof (e.g., salt). According to the present disclosure, a pharma- ceutically acceptable derivative includes, but is not limited to, a pharma- ceutically acceptable prodrug, salt, ester, salt of such an ester, or any other adduct / educt or derivative that can directly or indirectly provide a compound as otherwise described herein, or a metabolite or residue thereof, upon administration to a patient in need thereof.

[0070]

[0071] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like.

[0071]

[0072] Pharmaceutically acceptable salts are well known in the art.For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences 1977, 66, 1-19, which is incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this specification include those derived from suitable inorganic and organic acids and bases.Examples of pharmaceutically acceptable non-toxic acid addition salts include the salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate. , lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N + (C 1~4The present specification also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0072]

[0073] Pharmaceutically acceptable carriers may contain inactive ingredients that do not excessively inhibit the biological activity of compound.Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or have no other undesirable reactions or side effects when administered to a subject.Standard formulation techniques can be used.

[0073]

[0074] Pharmaceutically acceptable carriers, adjuvants, or vehicles as used herein include any solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharma-ceutically acceptable compositions and known techniques for their preparation. Use of any conventional carrier medium is contemplated within the scope of this specification, except insofar as such medium is incompatible with the compounds described herein, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharma-ceutically acceptable composition. As used herein, the phrase "side effects" encompasses unwanted and adverse effects of a treatment (e.g., a prophylactic or therapeutic agent). Side effects are always unwanted, but unwanted effects are not necessarily adverse. Side effects from treatment (prophylactic or therapeutic) may be harmful, uncomfortable, or dangerous. Side effects include, but are not limited to, fever, chills, fatigue, gastrointestinal toxicity (including gastric and intestinal ulcers and erosions), nausea, vomiting, neurotoxicity, renal toxicity, nephrotoxicity (including conditions such as papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme levels), bone marrow toxicity (including leukopenia, bone marrow suppression, thrombocytopenia, and anemia), dry mouth, metallic taste, prolonged pregnancy, weakness, somnolence, pain (including muscle pain, bone pain, and headache), hair loss, asthenia, dizziness, extrapyramidal symptoms, akathisia, cardiovascular disorders, and sexual dysfunction.

[0074]

[0075] Some examples of materials that may serve as pharma- ceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as tween 80, phosphate, glycine, sorbic acid, or potassium sorbate), saturated vegetable fatty acids, partial glyceride mixtures of water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropylmethylcellulose, wool fat, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanthus. Ingredients that may be used include, but are not limited to, corn, malt, gelatin, talc, excipients such as cocoa butter and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, glycols such as propylene glycol or polyethylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents, and flavoring agents. At the discretion of the formulator, preservatives and antioxidants may also be present in the composition.

[0075]

[0076] As used herein, the term "measurably degrade" refers to a measurable decrease in (a) BTK activity between a sample containing a compound herein and BTK and a comparable sample containing BTK in the absence of said compound, or (b) the concentration of BTK in a sample over time.

[0076] Administration

[0077] The composition of the present disclosure is administered orally. The pharmaceutically acceptable composition of this specification can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. For tablets for oral use, commonly used carriers include lactose and cornstarch. Typically, lubricants such as magnesium stearate are also added. For oral administration in capsule form, useful diluents include lactose and dry cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If necessary, certain sweeteners, flavors, or colorants can also be added.

[0077]

[0078] 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 active compounds herein, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and flavoring agents.

[0078]

[0079] The solid dosage form for oral administration includes capsules, tablets, pills, powders and granules.In such solid dosage form, the active compound herein is mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) filler or extender, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose and acacia; c) humectant, such as glycerol; d) disintegrant, such as agar, carbonate Calcium, 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 form can also include buffering agents.

[0079]

[0080] Solid compositions of a similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols. Solid dosage forms 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 formulation art. Solid dosage forms may optionally contain opacifying agents. These solid dosage forms may also be solid dosage forms of compositions that release the active ingredient(s) only in a certain part of the intestinal tract, for example, and optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0080]

[0081] The active compounds herein may also be in microencapsulated form with one or more excipients as mentioned above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active compounds may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also contain, as is normal practice, additional substances other than the inert diluent, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. These dosage forms may optionally contain opacifying agents, and may also be of a composition that releases the active ingredient(s) only 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.

[0081]

[0082] The compounds herein are formulated in dosage unit form for ease of administration and uniformity of dosage.As used herein, the phrase "dosage unit form" refers to a physically separate unit of pharmaceutical agent suitable for the patient to be treated.However, it will be understood that the total amount of the compounds and compositions of the present disclosure to be used per day will be determined by the attending physician within the scope of sound medical judgment.The specific effective dose level of any particular patient or organism will vary depending on various factors, including the disorder to be treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific compound used; duration of treatment; the drugs used in combination or simultaneously with the specific compound used, and similar factors well known in the medical art.

[0082]

[0083] The amount of the compounds of the present disclosure that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending on the host treated, the particular mode of administration, and other factors. These compositions should be formulated so that a dosage of 0.01-100 mg / kg body weight / day of the compound or inhibitor can be administered to a patient receiving these compositions.

[0083]

[0084] Depending on the particular condition, or disease, to be treated or prevented, additional therapeutic agents, which are normally administered to treat or prevent that condition, may also be present in the compositions of the present disclosure. As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease or condition are known as "appropriate for the disease or condition being treated."

[0084]

[0085] For example, chemotherapeutic agents or other anti-proliferative agents can be combined with the compounds of the present disclosure to treat proliferative diseases and cancer. Examples of known chemotherapeutic agents include PI3K inhibitors (e.g., idelalisib and copanlisib), BCL-2 inhibitors (e.g., venetoclax), BTK inhibitors (e.g., ibrutinib and acalabrutinib), etoposide, CD20 antibodies (e.g., rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, and ublituximab), aretuzumab, bendamustine, cladribine, doxorubicin, chlorambucil, prednisone, midostaurin, These include, but are not limited to, lenalidomide, pomalidomide, checkpoint inhibitors (e.g., ipilimumab, nivolumab, pembolizumab, atezolizumab, avelumab, durvalumab), artificial cell therapy (e.g., CAR-T therapy - Kymriah®, Yescarta®), Gleevec™, adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferon, and platinum derivatives.

[0085]

[0086] Also, in some cases, radiation therapy is administered during the course of treatment in which a compound of the present disclosure (or a pharma- ceutically acceptable salt thereof) is administered to a patient in need thereof.

[0086]

[0087] Other examples of agents with which the compounds or inhibitors of the present disclosure can be combined include treatments for Alzheimer's disease, such as Aricept® and Excelon®, treatments for Parkinson's disease, such as L-DOPA / carbidopa, entacapone, lopinrol, pramipexole, bromocriptine, pergolide, trihexefendyl, and amantadine, agents for treating multiple sclerosis (MS), such as beta interferons (e.g., Avonex® and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma, such as albuterol and Singulair®; agents for treating schizophrenia, such as Zyprexa, Risperdal, Seroquel, and haloperidol; anti-inflammatory agents, such as corticosteroids, TNF blockers, IL-1 immunomodulators and immunosuppressants such as cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, interferon, corticosteroids, cyclophosphamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anticonvulsants, ion channel blockers, riluzole, and antiparkinsonian agents; agents for treating cardiovascular disease such as beta blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and antivirals; agents for treating blood disorders such as corticosteroids, anti-leukemia agents, and growth factors; and agents for treating immune deficiency disorders such as gamma globulin.

[0087]

[0088] The amount of additional therapeutic agent present in the compositions of the present disclosure will be no more than the amount that would normally be administered in a composition containing that therapeutic agent as the only active agent. The amount of additional therapeutic agent in the compositions disclosed herein will range from about 50% to 100% of the amount that would normally be present in a composition containing that agent as the only therapeutically active agent. EXAMPLES

[0088]

[0089] Additional embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0089]

[0090] The compounds described herein are synthesized according to PCT / US2019 / 56112, filed October 14, 2019, PCT / US2020 / 063176, filed December 30, 2020, US2021 / 0198280A1, filed December 30, 2020, or in accordance with the following Examples. The contents of each of these references are incorporated herein in their entirety.

[0090]

[0091] Example 1: General Procedure B

[0091] [ka]

[0092] Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione

[0093] A mixture of 5-fluoro-1,3-dihydro-2-benzofuran-1,3-dione (5.0 g, 30.10 mmol), 3-aminopiperidine-2,6-dione hydrochloride (6.9 g, 42.14 mmol), and NaOAc (4.2 g, 51.17 mmol) in HOAc (50 mL) was stirred at 120° C. for 5 h and then concentrated under vacuum. The residue was washed with water and the solid was collected by filtration. The crude product was washed twice with water and twice with ethyl acetate and dried in an oven to give 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (7.7 g, 92%) as a light brown solid. 1 H NMR(300MHz,DMSO-d6)δ11.16(s,1H),8.03-8.00(m,1H),7.87-7.85(m,1H),7.75-7.7 0(m,1H),5.19-5.15(m,1H),2.94-2.86(m,1H),2.63-2.48(m,2H),2.12-2.06(m,1H). F NMR(300MHz,DMSO-d6)δ-102.078.

[0093]

[0094] Step 2: Amine substitution of aryl fluorides

[0095] To a solution of 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (1.0 g, 3.62 mmol) in N-methylpyrrolidone (10 mL) was added amine (3.60 mmol) and DIEA (1.4 g, 10.83 mmol). The resulting solution was stirred at 80° C. for 16 h. The reaction mixture was cooled to room temperature and purified by reverse phase flash chromatography to give the corresponding final product.

[0094]

[0096] Step 3: Oxidation of alcohol to aldehyde

[0097] To a mixture of the alcohol (1.06 mmol) in CH2Cl2 (10 mL) was added Dess-Martin periodinane (2.12 mmol). The mixture was allowed to stir at room temperature for 1 h. The mixture was purified by column chromatography to give the desired aldehyde.

[0095]

[0098] Example 1A: Synthesis of 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)acetaldehyde

[0096] [ka]

[0097]

[0099] Step 2: Following general procedure B using 2-(piperidin-4-yl)ethan-1-ol gave 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-hydroxyethyl)piperidin-1-yl)isoindoline-1,3-dione (822.8 mg, 59%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ11.09(s,1H),7.65(d,J=8.4Hz,1H),7.30(d,J=2.4Hz,1H) ,7.23(dd,J=8.4,2.4Hz,1H),5.07(dd,J=12.6,5.4Hz,1H),4.40(t,J=5.1Hz,1H),4 .04(d,J=13.2Hz,2H),3.64-3.40(m,2H),3.09-2.79(m,3H),2.70-2.51(m,2H),2.0 7-1.94(m,1H),1.77-1.66(m,3H),1.41-1.34(m,2H),1.24-1.12(m,2H).MS(ESI)(C 20 H 23 N3O5) [M+H] + Calculated value: 386.2; measured value: 386.1.

[0098]

[0100] Step 3: 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)acetaldehyde LCMS C 20 H 21 Calculated value for N3O5: 383, Measured value: m / z = 384 [M+H] + .

[0099]

[0101] Example 1B: Synthesis of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidine-3-carbaldehyde

[0100] [ka]

[0101]

[0102] Step 2: Following general procedure B using azetidin-3-ylmethanol hydrochloride gave 2-(2,6-dioxopiperidin-3-yl)-5-(3-(hydroxymethyl)azetidin-1-yl)isoindoline-1,3-dione (1.85 g, 68%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ11.09(s,1H),7.63(d,J=8.4Hz,1H),6.76(d,J=2.0H z,1H),6.62(dd,J=8.4,2.0Hz,1H),5.06(dd,J=12.4,5.2Hz,1H),4.86(t,J= 5.2Hz,1H),4.05(t,J=8.4Hz,2H),3.77(dd,J=8.4,5.2Hz,2H),3.60(t,J=5. 2Hz,2H),3.00-2.81(m,2H),2.65-2.53(m,2H),2.06-1.96(m,1H).MS(ESI)(C 17 H 17 N3O5) [M+H] + Calculated value: 344.1; measured value: 344.4.

[0102]

[0103] Step 3: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidine-3-carbaldehyde LCMS C 17 H 15 Calculated value for N3O5: 341, Measured value: m / z = 343 [M+H] + .

[0103]

[0104] Example 1C: Synthesis of 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidin-3-yl)acetaldehyde

[0104] [ka]

[0105] Step 2: Following general procedure B using 2-(azetidin-3-yl)ethan-1-ol hydrochloride gave 2-(2,6-dioxopiperidin-3-yl)-5-(3-(2-hydroxyethyl)azetidin-1-yl)isoindoline-1,3-dione (584.5 mg, 30%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ11.09(s,1H),7.63(d,J=8.4Hz,1H),6.75(d,J=2.1Hz,1H) ,6.62(dd,J=8.4,2.1Hz,1H),5.06(dd,J=12.6,5.4Hz,1H),4.51(t,J=5.1Hz,1H),4 .14(t,J=8.1Hz,2H),3.71-3.67(m,2H),3.47-3.40(m,2H),2.99-2.75(m,2H),2.6 1-2.58(m,1H),2.52-2.46(m,1H),2.10-1.95(m,1H),1.82-1.76(m,2H).MS(ESI)(C 18 H 19 N3O5) [M+H] + Calculated value: 358.1; measured value: 358.4.

[0106] Step 3: 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidin-3-yl)acetaldehyde LCMS C 18 H 17 Calculated value for N3O5: 355, Measured value: m / z=356 [M+H] + .

[0107] Example 1D: Synthesis of (3R)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-3-carbaldehyde

[0108] [ka]

[0109]

[0108] Step 2: Following general procedure B using (R)-piperidin-3-ylmethanol hydrochloride, 2-(2,6-dioxopiperidin-3-yl)-5-((R)-3-(hydroxymethyl)piperidin-1-yl)isoindoline-1,3-dione (916.3 mg, 45%) was obtained as a yellow solid. 1 H NMR(400MHz,DMSO-d6 / D2O)δ7.62(d,J=8.4Hz,1H),7.22(d,J=2.4Hz,1H),7.16 (dd,J=8.4,2.4Hz,1H),4.99(dd,J=12.8,5.2Hz,1H),3.98-3.76(m,2H),3.42- 3.22(m,2H),3.08-2.90(m,1H),2.89-2.71(m,2H),2.61-2.43(m,2H),2.02-1. 99(m,1H),1.73-1.69(m,3H),1.49-1.40(m,1H),1.26-1.18(m,1H).MS(ESI)(C 19 H 21 N3O5) [M+H] + Calculated value: 372.1; measured value: 372.4.

[0110] Step 3: (3R)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-3-carbaldehyde LCMS C 19 H 19 Calculated value for N3O5: 369, Measured value: m / z = 370 [M+H] + .

[0111] Example 1E: Synthesis of (3S)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-3-carbaldehyde

[0112] [ka]

[0113]

[0111] Step 2: Following general procedure B using (S)-piperidin-3-ylmethanol hydrochloride, 2-(2,6-dioxopiperidin-3-yl)-5-((S)-3-(hydroxymethyl)piperidin-1-yl)isoindoline-1,3-dione (493.1 mg, 73%) was obtained as a yellow solid. 1 H NMR(300MHz,DMSO-d6 / D2O)δ7.65(d,J=8.4Hz,1H),7.26(d,J=2.1Hz,1H),7.19(dd,J=8.4,2.1Hz,1H),5.04(dd,J=12.9,5.4Hz,1H),4.00-3.90(m,2 H),3.38-3.32(m,2H),3.13-2.71(m,3H),2.67-2.44(m,2H),2.03-1.98(m ,1H),1.76-1.67(m,3H),1.57-1.38(m,1H),1.34-1.10(m,1H).MS(ESI)(C 19 H 21 N3O5) [M+H] + Calculated value: 372.1; measured value: 372.1.

[0114] Step 3: (3S)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-3-carbaldehyde LCMS C 19 H 19 Calculated value for N3O5: 369, Measured value: m / z = 370 [M+H] + .

[0115] Example 1F: Synthesis of (3R)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)pyrrolidine-3-carbaldehyde

[0116] [ka]

[0117] Step 2: Following general procedure B using (R)-pyrrolidin-3-ylmethanol gave 2-(2,6-dioxopiperidin-3-yl)-5-((R)-3-(hydroxymethyl)pyrrolidin-1-yl)isoindoline-1,3-dione (480.6 mg, 74%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ11.08(s,1H),7.64(d,J=8.4Hz,1H),6.89(d,J=2.1Hz,1H),6.80(dd,J=8.4,2.1Hz,1H),5.06(dd,J=12.9,5.4Hz,1H) ),4.78(s,1H),3.65-3.36(m,5H),3.22-3.17(m,1H),2.95-2.83(m,1H),2.67-2.44(m,3H),2.11-1.89(m,2H),1.87-1.78(m,1H).MS(ESI)(C 18 H 19 N3O5) [M+H] + Calculated value: 358.1; measured value: 358.1.

[0118] Step 3: (3R)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)pyrrolidine-3-carbaldehyde LCMS C 18 H 17 Calculated value for N3O5: 355, Measured value: m / z=356 [M+H] + .

[0119] Example 1G: Synthesis of (3S)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)pyrrolidine-3-carbaldehyde

[0120] [ka]

[0121] Step 2: Following general procedure B using (S)-pyrrolidin-3-ylmethanol gave 2-(2,6-dioxopiperidin-3-yl)-5-((S)-3-(hydroxymethyl)pyrrolidin-1-yl)isoindoline-1,3-dione (643.1 mg, 33%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ11.08(s,1H),7.64(d,J=8.4Hz,1H),6.89(d,J=2.1Hz,1H),6.80(dd,J=8.4,2.1Hz,1H),5.06(dd,J=12.9,5.4Hz,1H),4. 78(t,J=5.4Hz,1H),3.59-3.41(m,5H),3.22-3.17(m,1H),2.95-2.83(m, 1H),2.67-2.44(m,3H),2.12-1.88(m,2H),1.87-1.76(m,1H).MS(ESI)(C 18 H 19 N3O5) [M+H] + Calculated value: 358.1; measured value: 358.1.

[0122] Step 3: (3S)-1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)pyrrolidine-3-carbaldehyde LCMS C 18 H 17 Calculated value for N3O5: 355, Measured value: m / z=356 [M+H] + .

[0123] Example 1H: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-((1R,5S,6r)-6-(hydroxymethyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)isoindoline-1,3-dione

[0124] [ka]

[0125]

[0120] Step 2: Following general procedure B using ((1R,5S,6r)-3-azabicyclo[3.1.0]hexan-6-yl)methanol gave 2-(2,6-dioxopiperidin-3-yl)-5-((1R,5S,6r)-6-(hydroxymethyl)-3-aza-bicyclo[3.1.0]hexan-3-yl)isoindoline-1,3-dione (315.8 mg, 21%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ11.08(s,1H),7.63(d,J=8.4Hz,1H),6.92(d,J=2.1Hz,1H),6.82(dd,J=8.4,2.1Hz,1H),5.06(dd,J=12.6,5.4Hz,1H),4.59(t,J =5.4Hz,1H),3.64-3.60(m,2H),3.50-3.35(m,4H),3.00-2.76(m,1H),2.58 -2.44(m,2H),2.07-1.91(m,1H),1.69(s,2H),0.86-0.79(m,1H).MS(ESI)(C 19 H 19 N3O5) [M+H] + Calculated value: 370.1; measured value: 370.1.

[0126] Step 3: (1R,5S,6r)-3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-3-azabicyclo[3.1.0]hexane-6-carbaldehyde LCMS C 19 H 17 Calculated value for N3O5: 367, Measured value: m / z = 368 [M+H] + .

[0127] Example 2: Synthesis of 2-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)ethyl)piperazin-1-yl)acetic acid

[0128] [ka]

[0129] Step 1: Benzyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-1-carboxylate

[0130] To a solution of benzyl piperazine-1-carboxylate (10.0 g, 45.4 mmol) and K2CO3 (12.6 g, 90.8 mmol) in acetonitrile (150 mL) was added tert-butyl 2-chloroacetate (7.5 g, 49.9 mmol). The resulting solution was stirred at 40 °C under nitrogen atmosphere for 16 h. The solids were filtered and the filtrate was concentrated under vacuum. The residue was purified by flash column chromatography using 0-50% ethyl acetate in petroleum ether to give benzyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-1-carboxylate (9.6 g, 63%) as a pale yellow oil. MS (ESI) (C 18 H 26 N2O4) [M+H] + Calculated value: 335.2; measured value: 335.3.

[0131] Step 2: tert-Butyl 2-(piperazin-1-yl)acetate

[0132] To a solution of benzyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-1-carboxylate (9.6 g, 28.7 mmol) in methanol (100 mL) was added Pd / C (10%, 2.0 g) under nitrogen atmosphere. The mixture was stirred at room temperature under hydrogen atmosphere (2 atm) for 16 h. The solids were filtered and the filtrate was concentrated in vacuo to give tert-butyl-2-(piperazin-1-yl)acetate (6.2 g, crude) as a pale yellow oil, which was used in the next step without further purification. MS (ESI) (C 10 H 20 N2O2) [M+H] + Calculated value: 201.2; measured value: 201.0.

[0133] Step 3: 3-(4-allyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0134] A degassed mixture of 3-(4-bromo-1-oxo-2,3-dihydro-1H-isoindol-2-yl)piperidine-2,6-dione (10.0 g, 30.9 mmol), allyltributylstannane (15.4 g, 46.4 mmol), and Pd(PPh3)4 (3.6 g, 3.1 mmol) in DMF (80 mL) was stirred at 100° C. for 16 h under nitrogen atmosphere. Once the reaction was complete by LCMS, the resulting mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography using 0-10% methanol in dichloromethane to give 3-(4-allyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (7.0 g, 79%) as a white solid. MS(ESI)(C 16 H 16 N2O3) [M+H] + Calculated for, 285.1; found, 285.2. 1H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 7.62-7.60 (m, 1H), 7.52-7.27 (m, 2H), 6.02-5.92 (m, 1H), 5.16-5.09 (m, 3H), 4.45 (d, J = 17.2 Hz, 1H), 4.30 (d, J = 17.2 Hz, 1H), 3.46-3.44 (m, 2H), 2.97-2.86 (m, 1H), 2.70-2.57 (m, 1H), 2.04-1.99 (m, 1H), 1.68-1.55 (m, 1H).

[0135] Step 4: 2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetaldehyde

[0136] A mixture of 3-(4-allyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (7.0 g, 24.6 mmol), OsO4 (625 mg, 2.5 mmol), and NaIO4 (10.5 g, 49.2 mmol) in MeCN (60 mL) and H2O (20 mL) was stirred at 0 °C for 6 h. Upon completion of the reaction, the resulting mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give 2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetaldehyde (4.0 g, crude) as a brown solid, which was used in the next step without further purification. MS (ESI) (C 15 H 14 N2O4) [M+H] + Calculated value: 287.1; measured value: 287.2.

[0137] Step 5: tert-Butyl 2-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)ethyl)piperazin-1-yl)acetate

[0138] A mixture of 2-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]acetaldehyde (4.0 g, 13.9 mmol), tert-butyl 2-(piperazin-1-yl)acetate (3.4 g, 16.8 mmol), AcOH (1 mL), and NaBH(OAc)3 (5.9 g, 27.9 mmol) in dichloromethane (50 mL) was stirred at room temperature for 16 hours. The resulting mixture was diluted with water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by reverse-phase flash column chromatography using 10-50% acetonitrile in water to give tert-butyl 2-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)ethyl)piperazin-1-yl)acetate (2.5 g, 22% over two steps) as a light brown syrup. MS (ESI) (C 25 H 34 N4O5) [M+H] + Calculated value: 471.2; measured value: 471.0.

[0139] Step 6: 2-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)ethyl)piperazin-1-yl)acetic acid TFA salt

[0140] To a solution of tert-butyl 2-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)ethyl)piperazin-1-yl)acetate (2.5 g, 5.3 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (20 mL). The resulting mixture was stirred at room temperature for 16 h and then concentrated in vacuo. The residue was purified by reverse-phase flash column chromatography using 5-30% acetonitrile in water to give 2-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)ethyl)piperazin-1-yl)acetic acid (1.7214 g, 78%) as a light brown solid. MS (ESI) (C21 H 26 N4O5) [M+H] + Calculated value: 415.2; measured value: 415.4. 1 H NMR(300MHz,DMSO-d6)δ11.08(s,1H),7.66-7.62(m,1H),7.54-7.47(m,2H),5.14-5.08(m,1H),4.54-4.48(m,1H),4.40-4 .31(m,1H),3.76(s,2H),3.60-3.10(m,10H),3.10-2.78(m,3H),2.68-2.54(m,1H),2.40-2.31(m,1H),2.10-1.94(m,1H).

[0141] Example 3: Synthesis of 5-[(3R)-3-(3-methyl-2-oxoimidazolidin-1-yl)piperidin-1-yl]-3-{[4-(piperazin-1-yl)phenyl]amino}pyrazine-2-carboxamide

[0142] Step 1: 2-(2,6-dioxopiperidin-3-yl)-5-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]isoindole-1,3-dione

[0143] [ka]

[0144] A mixture of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (373 mg, 1.35 mmol), DMF (8 mL), ethyl bis(propan-2-yl)amine (0.94 mL, 5.40 mmol), and prolinol (137 mg, 1.35 mmol) was stirred at 90° C. for 16 h. CHCl and H0 were added. The organic layer was dried over MgSO, filtered, concentrated, and purified by MPLC (0-10% MeOH in CHCl) to give 2-(2,6-dioxopiperidin-3-yl)-5-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]isoindole-1,3-dione (386.00 mg, 80.0%).

[0145] Step 2: (2S)-1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]pyrrolidine-2-carbaldehyde

[0146] [ka]

[0147] 1,1-Bis(acetyloxy)-3-oxo-1 lambda 5,2-benzoiodoxol-1-yl acetate (548 mg, 1.29 mmol) was added to a mixture of 3-{5-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-1-oxo-3H-isoindol-2-yl}piperidine-2,6-dione (222 mg, 0.65 mmol) and CHCl (10 mL). The mixture was allowed to stir at room temperature for 1 hour. The mixture was purified by MPLC (10-100% EtOAc in hexanes) to give (2S)-1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]pyrrolidine-2-carbaldehyde (67 mg, 30%).

[0148] Example 4: Synthesis of 5-((R)-3-(3-methyl-2-oxoimidazolidin-1-yl)piperidin-1-yl)-3-((4-(octahydro-2,7-naphthyridin-2(1H)-yl)phenyl)amino)pyrazine-2-carboxamide

[0149] [ka]

[0150] Step 1: Under argon, Pd(OAc)2 (105 mg, 0.47 mmol) was added to a degassed dioxane (10.00 mL) solution containing cesium carbonate (1523.56 mg, 4.68 mmol), tert-butyl 7-(4-aminophenyl)-octahydro-2,7-naphthyridine-2-carboxylate (517 mg, 1.56 mmol), BINAP (291 mg, 0.47 mmol), and 3-chloro-5-[(3R)-3-(3-methyl-2-oxoimidazolidin-1-yl)piperidin-1-yl]pyrazine-2-carbonitrile (500 mg, 1.56 mmol). The mixture was then stirred at 100° C. for 16 hours. The mixture was then partitioned between water and ethyl acetate, dried over sodium sulfate, and concentrated. The resulting residue was then purified by reverse phase preparative HPLC (Waters 5 mM CSH C18 column, 50×50 mm) using a gradient of 10-95% over 9 min, eluting with acetonitrile in water with 0.1% TFA. The desired fractions were combined and concentrated to give the product. This material was dissolved in a 10:1 MeOH / DMSO solution (2 mL) with one NaOH pellet. After 2 min, 30% aqueous hydrogen peroxide (0.5 mL) was added and the reaction was continued with stirring at room temperature for 1 h. The reaction was quenched by the addition of ACN. After concentration, the resulting crude reaction mixture was purified by reverse phase preparative HPLC (Waters 5 mM CSH C18 column, 50×50 mm) using a gradient of 10-95% over 9 min, eluting with acetonitrile in water with 0.1% TFA. The desired fractions were combined and concentrated to give the product. LCMS C 25 H 34 Calculated value for N6O3: 633, Measured value: m / z=634 [M+H] + .

[0151] [ka]

[0152] Step 2: tert-Butyl 7-[4-({3-carbamoyl-6-[(3R)-3-(3-methyl-2-oxoimidazolidin-1-yl)piperidin-1-yl]pyrazin-2-yl}amino)phenyl]-octahydro-2,7-naphthyridine-2-carboxylate (240 mg, 0.38 mmol) was dissolved in 2 mL of a 1 / 1 DCM / TFA solution and stirred at room temperature for 1 h. The reaction was then concentrated. This material was used in the next step without further purification.

[0153] Example 5: Synthesis of 5-morpholino-3-[4-(4-piperidyl)anilino]pyrazine-2-carboxamide

[0154] [ka]

[0155] Step 1: 3-Chloro-5-morpholino-pyrazine-2-carbonitrile

[0156] [ka]

[0157] To a solution of morpholine (2.65 mL, 30.4 mmol) and 3,5-dichloropyrazine-2-carbonitrile 1 (6.2 g, 35.5 mmol) in anhydrous DMF (40.0 mL) was added DIPEA (6.4 mL, 36.5 mmol) at room temperature. The reaction solution was stirred at room temperature for 4 h. The mixture was diluted with water (100 mL) and EtOAc (100 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were washed with water (2×20 mL) and brine (3×20 mL), then dried (Na2SO4), filtered and concentrated under reduced pressure to give the title compound (5.7 g, 83%) as a solid. MS (ESI) [M+H] + 225.1.

[0158] Step 2: tert-Butyl 4-[4-[(3-cyano-6-morpholino-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate

[0159] [ka]

[0160] A mixture of 3-chloro-5-morpholino-pyrazine-2-carbonitrile (4.06 g, 18.1 mmol), tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (5.0 g, 18.1 mmol), rac-BINAP (1.13 g, 1.81 mmol), and Cs2CO3 (17.7 g, 54.3 mmol) in anhydrous dioxane (60.0 mL) was degassed with N2 for 10 min. Pd(OAc)2 (406 mg, 1.81 mmol) was then added and the resulting mixture was heated at 80° C. for 16 h. The mixture was cooled to room temperature and the suspension was filtered over Celite and washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The material was suspended in MeOH (50 mL) and sonicated for 2 min. The resulting solid was filtered and dried to give the title compound as a solid (7.1 g, 85%). MS(ESI)[M-Boc+H] + 365.3.

[0161] Step 3: tert-Butyl 4-[4-[(3-carbamoyl-6-morpholino-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate

[0162] [ka]

[0163] To a solution of tert-butyl 4-[4-[(3-cyano-6-morpholino-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate (7.1 g, 15.3 mmol) in MeOH (100.0 mL) and DMSO (10.0 mL) was added NaOH (4 M in water, 7.64 mL, 30.6 mmol) followed by H2O2 (30% in water, 6.93 mL, 61.1 mmol) at room temperature. The reaction mixture was stirred for 3.5 h. The mixture was diluted with acetonitrile (10 mL) and EtOAc (100 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (2 x 30 mL), then dried (Na2SO4), filtered and concentrated under reduced pressure to give the title compound (6.51 g, 88%) as a solid. MS (ESI) [MH] - 481.4.

[0164] Step 4: 5-Morpholino-3-[4-(4-piperidyl)anilino]pyrazine-2-carboxamide hydrochloride.

[0165] [ka]

[0166] To a solution of tert-butyl 4-[4-[(3-carbamoyl-6-morpholino-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate (6.51 g, 13.5 mmol) in anhydrous DCM (20.0 mL) was added HCl (35.0 mL, 140 mmol, 4 M in dioxane) at room temperature, and the reaction mixture was stirred at room temperature for 2 hours. The resulting solid was filtered, washed with acetonitrile (100 mL) and DCM (100 mL), and then dried under reduced pressure to give the title compound (5.6 g, 99%) as a yellow solid. 1H NMR(400MHz,DMSO)δ11.35(s,1H),9.13-8.93(m,2H),7.91-7.71(m,1H),7.67(s,1H),7.55(d,J=8.6Hz,2H),7.49-7.29(m,1H),7.17(d,J=8.6 Hz,2H),3.76-3.69(m,4H),3.67-3.61(m,4H),3.33(d,J=12.7Hz,2H),3.02-2.90(m,2H),2.84-2.73(m,1H),1.94-1.81(m,4H).MS(ESI)[M+H] + 383.2.

[0167] Example 6: 5-(4-Methylpiperazin-1-yl)-3-[4-(4-piperidyl)anilino]pyrazine-2-carboxamide

[0168] [ka]

[0169] Step 1: 3-chloro-5-(4-methylpiperazin-1-yl)pyrazine-2-carbonitrile

[0170] [ka]

[0171] To a solution of 1-methylpiperazine (3.3 mL, 30.0 mmol) and 3,5-dichloropyrazine-2-carbonitrile (6.1 g, 34.9 mmol) in anhydrous DMF (40 mL) was added DIPEA (6.26 mL, 35.9 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 4 h. The mixture was diluted with water (100 mL) and EtOAc (100 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were washed with water (2×20 mL) and brine (3×20 mL), then dried (Na2SO4), filtered, and concentrated under reduced pressure to give the title compound (4.1 g, 58%) as a solid. MS (ESI) [M+H] + 238.1.

[0172] Step 2: tert-Butyl 4-[4-[[3-cyano-6-(4-methylpiperazin-1-yl)pyrazin-2-yl]amino]phenyl]piperidine-1-carboxylate.

[0173] [ka]

[0174] A mixture of 3-chloro-5-(4-methylpiperazin-1-yl)pyrazine-2-carbonitrile (4.1 g, 17.2 mmol), tert-butyl 1-(4-aminophenyl)piperidine-4-carboxylate (5.0 g, 18.1 mmol), rac-BINAP (1.13 g, 1.81 mmol), and Cs2CO3 (17.7 g, 54.3 mmol) in anhydrous dioxane (60.0 mL) was degassed with N2 for 10 min. Pd(OAc)2 (406 mg, 1.81 mmol) was then added and the resulting mixture was heated at 80° C. for 16 h. The mixture was cooled to room temperature and the suspension was filtered over Celite and washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The material was suspended in MeOH (50 mL) and sonicated for 2 min. The resulting solid was filtered and dried under vacuum to give the title compound as a solid (6.68 g, 77%). MS (ESI) [M-Boc+2H] + 378.3.

[0175] Step 3: tert-Butyl 4-[4-[[3-carbamoyl-6-(4-methyl-4-oxido-piperazin-4-ium-1-yl)pyrazin-2-yl]amino]phenyl]piperidine-1-carboxylate

[0176] [ka]

[0177] To a suspension of tert-butyl 4-[4-[[3-cyano-6-(4-methylpiperazin-1-yl)pyrazin-2-yl]amino]phenyl]piperidine-1-carboxylate (6.80 g, 14.2 mmol) in MeOH (100.0 mL) and DMSO (10.0 mL) was added aqueous NaOH (4 M in water, 7.1 mL, 28.5 mmol) at room temperature, followed by H2O2 (30% in water, 6.5 mL, 57.3 mmol) at room temperature. The reaction mixture was stirred for 3.5 h. The mixture was diluted with cold water (50 mL). The resulting solid was filtered and washed with water (50 mL) and cold MeOH (40 mL) to give the title compound (7.10 g, 98%) as a solid. 1 H NMR(400MHz,DMSO)δ11.30(s,1H),7.82-7.73(m,1H),7.67(s,1H),7.51(d, J=8.6Hz,2H),7.41-7.31(m,1H),7.18(d,J=8.5Hz,2H),4.13-3.98(m,2H),3 .73-3.58(m,4H),2.90-2.71(m,2H),2.69-2.57(m,1H),2.46-2.35(m,4H),2 .22(s,3H),1.79-1.67(m,2H),1.52-1.43(m,2H),1.42(s,9H).MS(ESI)[MH] + 510.5.

[0178] Step 4: tert-Butyl 4-[4-[[3-carbamoyl-6-(4-methylpiperazin-1-yl)pyrazin-2-yl]amino]phenyl]piperidine-1-carboxylate

[0179] [ka]

[0180] To a solution of tert-butyl 4-[4-[[3-carbamoyl-6-(4-methyl-4-oxido-piperazin-4-ium-1-yl)pyrazin-2-yl]amino]phenyl]piperidine-1-carboxylate (4.50 g, 8.80 mmol) in anhydrous DMF (50.0 mL) was added trimethylphosphane (44.0 mL, 44.0 mmol, 1.0 M in THF) at room temperature and the resulting mixture was heated at 80° C. for 4 h. The mixture was diluted with EtOAc (100 mL) and water (200 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (3×50 mL), then dried (Na2SO4), filtered and concentrated under reduced pressure to give the title compound (2.20 g, 51%) as a solid. MS (ESI) [MH] - 494.5.

[0181] Step 5: 5-(4-Methylpiperazin-1-yl)-3-[4-(4-piperidyl)anilino]pyrazine-2-carboxamide dihydrochloride

[0182] [ka]

[0183] To a solution of tert-butyl 4-[4-[[3-carbamoyl-6-(4-methylpiperazin-1-yl)pyrazin-2-yl]amino]phenyl]piperidine-1-carboxylate (2.60 g, 5.25 mmol) in anhydrous DCM (40.0 mL) was added HCl (15.0 mL, 60.0 mmol, 4.0 M in dioxane) at room temperature, and the resulting suspension was stirred at room temperature for 1 h. The resulting solid was filtered, washed with DCM (100 mL), and dried under reduced pressure to give the title compound (2.35 g, 96%) as a yellow-orange solid. 1H NMR(500MHz,DMSO)δ11.55-11.40(m,1H),11.38(s,1H),9.21-8.99(m,2H),7.96- 7.85(m,1H),7.78(s,1H),7.55(d,J=8.4Hz,2H),7.51-7.42(m,1H),7.20(d,J=8. 4Hz,2H),4.49(d,J=14.3Hz,2H),3.55-3.44(m,4H),3.39-3.29(m,2H),3.20-3.0 6(m,2H),3.04-2.90(m,2H),2.85-2.72(m,4H),1.97-1.79(m,4H).MS(ESI)[M+H] + 396.3.

[0184] Example 7: Synthesis of 3-((4-(piperidin-4-yl)phenyl)amino)-5-(pyrrolidin-1-yl)pyrazine 2-carboxamide

[0185] [ka]

[0186] Step 1: 3-Chloro-5-pyrrolidin-1-yl-pyrazine-2-carbonitrile

[0187] [ka]

[0188] To a solution of pyrrolidine (1.52 mL, 18.2 mmol) and 3,5-dichloropyrazine-2-carbonitrile (3.17 g, 18.2 mmol) in anhydrous DMF (20.0 mL) was added DIPEA (3.81 mL, 21.9 mmol) at room temperature. The resulting solution was stirred at room temperature for 1 h. The mixture was diluted with water (100 mL) and the resulting solid was collected by filtration and then dried under reduced pressure to give the title compound (3.3 g, 87%) as a solid. MS (ESI) [M+H] + 209.1.

[0189] Step 2: tert-Butyl 4-[4-[(3-cyano-6-pyrrolidin-1-ylpyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate

[0190] [ka]

[0191] A mixture of 3-chloro-5-pyrrolidin-1-yl-pyrazine-2-carbonitrile (3.40 g, 16.3 mmol), tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (4.50 g, 16.3 mmol), rac-BINAP (1.13 g, 1.81 mmol), and Cs2CO3 (15.9 g, 48.9 mmol) in anhydrous dioxane (60.0 mL) was degassed with N2 for 10 min. Pd(OAc)2 (406 mg, 1.81 mmol) was then added and the resulting mixture was heated at 80° C. for 16 h. The mixture was cooled to room temperature and the suspension was filtered over Celite and washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The material was suspended in MeOH (50 mL) and sonicated for 2 min. The resulting solid was filtered and dried under vacuum to give the title compound (4.80 g, 66%) as a solid. 1 H NMR(400MHz,DMSO)δ8.83(s,1H),7.59(d,J=8.7Hz,2H),7.50(s,1H),7.15(d,J=8.6Hz,2H),4.13-3.96(m,2H),3.52-3.42(m,4H) ,2.88-2.70(m,2H),2.67-2.58(m,1H),2.01-1.84(m,4H),1.79-1.68(m,2H),1.52-1.42(m,2H),1.41(s,9H).MS(ESI)[M-Boc+2H] + 349.3.

[0192] Step 3: tert-Butyl 4-[4-[(3-carbamoyl-6-pyrrolidin-1-yl-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate

[0193] [ka]

[0194] To a solution of tert-butyl 4-[4-[(3-cyano-6-pyrrolidin-1-yl-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate (4.80 g, 10.7 mmol) in MeOH (100.0 mL) and DMSO (10.0 mL), NaOH (4M in water, 5.35 mL, 21.4 mmol) was added at room temperature, followed by H2O2 (30% in water, 4.85 mL, 42.8 mmol) at room temperature. The reaction mixture was stirred for 18 hours, and then water (100 mL) was added. The resulting solid was filtered and dried under reduced pressure to give the title compound (4.70 g, 94%) as a solid. 1 H NMR(500MHz,DMSO)δ11.40(s,1H),7.75-7.71(m,1H),7.64(d,J=8.6Hz,2H),7.35(s,1H),7.31-7.25(m,1H),7.18(d,J=8.6Hz,2H),4.15-4 .01(m,2H),3.59-3.49(m,4H),2.95-2.70(m,2H),2.67-2.59(m,1H), 2.04-1.93(m,4H),1.79-1.72(m,2H),1.51-1.44(m,2H),1.42(s,9H).

[0195] Step 4: 3-[4-(4-piperidyl)anilino]-5-pyrrolidin-1-yl-pyrazine-2-carboxamide hydrochloride.

[0196] [ka]

[0197] To a solution of tert-butyl 4-[4-[(3-carbamoyl-6-pyrrolidin-1-yl-pyrazin-2-yl)amino]phenyl]piperidine-1-carboxylate (4.80 g, 10.3 mmol) in DCM (75 mL) and MeOH (25 mL), HCl (20.0 mL, 80.0 mmol, 4.0 M in dioxane) was added at room temperature, and the resulting mixture was stirred at room temperature for 2 hours. The suspension was filtered, washed with DCM, and then dried under reduced pressure to obtain the title compound (3.78 g, 91%) as a yellow-orange solid. 1 H NMR(400MHz,DMSO)δ11.42(s,1H),8.97-8.76(m,2H),7.82-7.59(m,1H),7.68(d,J=8.5Hz,2H),7.35(s,1H),7.33-7.19(m,1H),7.16(d,J=8 .5Hz,2H),3.56-3.47(m,4H),3.38-3.30(m,2H),3.03-2.90(m,2H),2.83-2.73(m,1H),2.03-1.96(m,4H),1.92-1.77(m,4H).MS(ESI)[M+H] + 367.2.

[0198] Example 8 General Procedure 1: Amide Coupling A mixture of amine (0.03 mmol), acid (0.03 mmol), HATU (0.0 mmol), DIPEA (0.15 mmol), and DMF is stirred at room temperature for 30 minutes. The mixture is purified by HPLC (H2O / MeCN, 0.1% TFA) to give the amide product.

[0199] General Procedure 2: Reductive Amination A mixture of amine TFA salt (0.07 mmol), aldehyde (0.1 mmol), triethylamine (0.28 mmol), and DCE was stirred at room temperature for 10 min. NaBH(OAc)3 (0.14 mmol) was added and the mixture was stirred at room temperature for 2 h. The mixture was filtered through Celite, washed with CH2Cl2, concentrated, and purified by HPLC (H2O / MeCN, 0.1% TFA) to give the amine product.

[0200] General Procedure 3: Aryl Fluoride Substitution A mixture of amine (0.22 mmol), aryl fluoride (0.22 mmol), DIPEA (0.88 mmol), and DMF (1 mL) is stirred for 16 h at 90° C. The mixture is purified by HPLC (H 2 O / MeCN, 0.1% TFA) to give the desired product.

[0201] Physical Data for Exemplary Compounds in Table 1

[0179] 1 H NMR spectra and mass spectrometry (LCMS) data were obtained for the exemplary compounds reported in Table 1. These experimental data are provided in Table 2.

[0202]

[0180]

[0203] [Table 2-1]

[0204] [Table 2-2]

[0205] [Table 2-3]

[0206] [Table 2-4]

[0207]

Table 2-5

[0208]

Table 2-6

[0209]

Table 2-7

[0210]

Table 2-8

[0211]

Table 2-9

[0212]

Table 2-10

[0213]

Table 2-11

[0214]

Table 2-12

[0215]

Table 2-13

[0216]

Table 2-14

[0217]

[0181] Biological example 1 Cellular BTK Degradation Assay BTK levels are determined using the Cisbio Total-BTK HTRF (homologous time-resolved fluorescence) kit (63ADK064PEG) according to the manufacturer's protocol. Briefly, cells are incubated in 1X complementary lysis buffer for 30 minutes. In an opalescent low-volume 96-well plate (Cisbio, 66PL96005), cell lysates are combined with two different specific BTK antibodies, one for Eu 3+ -cryptate FRET donor and the other conjugated to d2 FRET acceptor. Assay control was Eu 3+ - wells containing cell lysates with only cryptate FRET donor antibody as well as wells containing both HTRF antibody and lysis buffer without cells or control lysates provided by Cisbio. HTRF ratio was calculated as (acceptor signal at 665 nm / donor signal at 620 nm) x 10 4 The DC50 value is calculated as the background HTRF level. Background HTRF levels are determined from control wells containing donor but no acceptor antibody. Background HTRF levels are subtracted from all samples. Readings are recorded as HTRF levels relative to DMSO treated cells. A four parameter nonlinear regression was performed in GraphPad Prism 7.02 to obtain DC50 values.

[0218]

[0184] Biological example 2 Human Dosing of Compound 149 Compound 149 was administered to human patients in a Phase 1a / 1b trial investigating safety and tolerability in relapsed and refractory B-cell malignancies. Compound 149 was administered as a single daily oral dose in an accelerated Phase 1a dose escalation starting at 100 mg on day 1. The first dose level is examined in a single patient cohort until a grade 2 adverse event is observed. A conventional 3+3 escalation design is then used. This starting dose of 100 mg was estimated to provide exposure similar to that observed in non-human primate studies. Once a dose is selected based on all available safety, efficacy, pharmacokinetic, and pharmacodynamic data, the trial will be expanded to up to five expansion cohorts.

[0219] Seven patients were screened, one patient completed the first cohort, and five patients were enrolled in the second cohort. Four patients had chronic lymphocytic leukemia or small cell lymphocytic leukemia. One patient had mantle cell lymphoma. One patient had Waldenstrom's macroglobulinemia. All patients had received at least two prior lines of therapy. All patients had failed ibrutinib treatment. Two patients had failed pirtobrutinib treatment. In the second cohort, one patient was removed from the study on day 15. Two more patients were then enrolled in the second cohort.

[0220]

[0188] Plasma concentrations of Compound 149 were measured and are provided in Figure 1. All patients showed a rapid increase in concentrations, reaching steady-state plasma concentrations by day 8.

[0221] Plasma levels of Bruton's tyrosine kinase were measured by flow cytometry in circulating B cells, including diseased cells, and are provided in FIG. 2. The mean fluorescence intensity correlates directly with the levels of BTK protein observed in Western blots. Compound 149 dramatically reduces the levels of BTK protein in all treated patients. Although patients' BTK levels begin at different levels, the treatment results in the same level of degradation by day 15, the first measurement after steady-state concentrations are achieved.

[0222]

[0190] BTK levels in each patient were normalized to 100% and are provided in Figure 3. Patients at 100 mg reached greater than 80% BTK degradation at steady state. All patients at the 200 mg dose level reached greater than 90% degradation at steady state.

[0223] The data are summarized in Table 3 below, which provides the mean percent BTK degradation during the last 2 weeks (i.e., days 15, 22, and 29) of cycle 1. In cohort 2 (200 mg), well above 90% BTK degradation was achieved during steady state.

[0224] In humans, compound 149 was well tolerated at doses of 100 mg and 200 mg with no dose-limiting toxicity. Greater than 90% BTK degradation was observed in all patients at the 200 mg dose.

[0225] [Table 3]

[0226]

[0193] Biological example 3 Oral Dosing of Compound 149

[0195] The trial was conducted at 12 institutions, including Memorial Sloan Kettering Cancer Center, MD Anderson Cancer Center, City of Hope (Duarte, California), National Institutes of Health Clinical Center, Sarah Cannon Research Institute, Colorado Blood Cancer Institute, Florida Cancer Specialists, Tennessee Oncology, University of California (San Francisco), University of California (Irvine), OSU Wexner Medical Center, and Swedish Cancer Institute (Seattle).

[0227] Patients were treated with four different dose levels of Compound 149: dose level 1 (100 mg), dose level 2 (200 mg), dose level 3 (300 mg), and dose level 4 (400 mg). The objectives of the study were to evaluate safety and tolerability, identify the maximum tolerated dose, and evaluate PK / PD.

[0228]

[0197] The patients evaluated in this study were treated for a disease or disorder selected from the group consisting of chronic lymphocytic leukemia (CLL) with BTK C481 mutation (n approximately 20), CLL without BTK C481 mutation (n approximately 20), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrom's macroglobulinemia (WM) (n approximately 20), follicular lymphoma (FL) (n approximately 20), and diffuse large B-cell lymphoma (DLBCL) (n approximately 20). Tables 4 and 5 below summarize the patient characteristics. Heavily pretreated patient population, including double refractory CLL patients.

[0229] [Table 4]

[0230] [Table 5]

[0231]

[0198] Efficacy evaluation All patients with CLL who received prior treatment with 100 mg of compound 149 showed clinical activity (see Figure 4). As shown in Figure 5, data from all evaluable CLL patients showed nodal reduction and / or lymphocytosis in all treated patients, responses in patients with resistance mutations to covalent and non-covalent BTK inhibitors, as well as responses from double refractory patients who received prior BCL2 inhibitor treatment. Data shown in Figure 6 confirmed that compound 149 is a potent BTK degrader that resolves inhibitor resistance observed with prior treatment. Compound 149 kills lymphoma cells with BTK-C481S mutations and degrades multiple novel BTK mutations that emerge after BTKi treatment.

[0232]

[0200] Safety Observations The safety population included 19 subjects, and the data are summarized in Table 6 below. Two subjects were assigned to the 100 mg cohort, but the treatment was not entered into the electronic data capture (EDC) at the time of extraction. Dose-limiting toxicity was observed at 300 mg in patients with CLL, and cognitive AEs were thought to be related to immunomodulatory activity. Two AEs of lower grade atrial fibrillation were observed at 100 mg in patients with MCL and at 200 mg in patients with CLL.

[0233] [Table 6]

[0234]

[0202] Biological example 4 Flow cytometry assay for BTK degradation After informed consent, peripheral blood was obtained from all patients for baseline and Compound 149 post-treatment assessment of BTK and Ikaros degradation. Patient blood (5 mL) was drawn and immediately fixed into 5 mL CytoChex™ BCT tubes and stored at ambient temperature until evaluation via flow cytometry. Blood samples were analyzed within 3 days after sample collection on Precision for Medicine. Additional blood was also drawn into 8 mL BD Vacutainer® CPT™ cell preparation tubes containing sodium citrate. Peripheral blood mononuclear cells (PBMCs) were processed and frozen for viability according to the manufacturer's protocol.

[0235] For flow staining, 1 mL of blood was treated with 1xBD PharmLyse to promote red blood cell lysis. The remaining white blood cells were washed with staining buffer (SB) containing Dulbecco's phosphate buffered saline (DPBS) without calcium or magnesium, 1% bovine serum albumin (BSA), 10 mM HEPES, 1 mM EDTA, and 0.01% sodium azide. The cell pellet was stained with fluorophore-conjugated cell surface marker antibodies against CD45, CD3, CD4, CD8, CD14, CD16, CD19, and CD56 prepared in BD Brilliant Stain buffer for 25 minutes at room temperature.

[0236] After cell surface staining, cells were washed twice with SB and then fixed and permeabilized with eBioscience FoxP3 / transcription factor staining buffer set for 1 hour at room temperature according to the manufacturer's instructions. The washed cells were then stained with unconjugated BTK antibody made in 1x permeabilization buffer for 30 minutes. This was followed by another 30 minutes of incubation with anti-rabbit Alexa488 secondary antibody, also prepared in 1x permeabilization buffer, for BTK detection. After incubation, cells were washed twice with 1x permeabilization buffer and then resuspended in SB. A control sample containing fluorescence minus two markers (FM2) was used for background control and was stained for all surface markers in the panel except BTK. FM2 does not contain antibodies to detect BTK.

[0237]

[0207] Samples were transferred to BD TruCount tubes before acquisition on a BD LSR Fortessa using the prescribed BD FACS Diva software (v8.0) application settings. Data analysis was performed using FlowJo software (v10).

[0238]

[0208] Single lymphocytes were gated on B cells (CD19+CD3-) and the geometric mean fluorescence intensity (MFI) of BTK was calculated. The MFI of B cells in the FM2 control was used to quantify background staining. The following formula was used to calculate the BTK degradation rate: % decomposition=100-[100 * (BTK MFI x ) / (BTK MFI y )] % remaining = 100 * (BTK MFI x ) / (BTK MFI y ) BTK MFI x = BTK MFI at the time of interest BTK MFI y = Pre-dose BTK MFI

[0239]

[0209] A list of materials used for flow cytometry is shown in Table 7 below.

[0240] [Table 7]

[0241]

[0210] Results

[0211] Once-daily oral Compound 149 treatment induced rapid and sustained BTK degradation in patients.

[0242] Pharmacodynamic evaluation of BTK degradation was performed using a validated 10-color flow cytometry assay. Serial peripheral blood was collected from consenting patients after receiving compound 149 treatment. Peripheral blood from patients was processed and BTK degradation was evaluated using a validated 10-color flow panel. B cells (CD19+CD3-) were gated and BTK MFI was measured. The B cell BTK MFI from the control FM2 sample without antibody was used to subtract background from each sample. %BTK remaining was calculated relative to baseline as described in the methods section.

[0243] A time-dependent decrease in BTK levels as measured by flow cytometry using BTK MFI in CD19+CD3- B cells was observed in all patients receiving a once-daily oral regimen of compound 149 (see Figure 7). 24 hours after receiving the first dose of compound 149, 62.8-75.1% BTK degradation was observed in patients 1, 2, 3, and 4. Maximum BTK degradation was reached on day 15 of cycle 1 at steady state, with a mean of 90.1% BTK degradation (Table 8) observed in all patients. This level of degradation persisted until day 29 of cycle 1 before beginning cycle 2.

[0244] [Table 8]

[0245]

[0214] Biological example 5 Oral Dosing of Compound 149 Heavily pretreated patients with non-GCB (ABC subtype) DLBCL were enrolled in a Phase 1 dose escalation study of Compound 149. Patients were treated with a single dose level of Compound 149, dose level 1 (300 mg), for 5 consecutive months. The objectives of this study were to evaluate safety and tolerability, identify the maximum tolerated dose, and evaluate PK / PD.

[0246]

[0217] Table 9, provided below, describes the baseline demographic and disease characteristics of the patients:

[0247] [Table 9]

[0248]

[0218] Response Patient responses were confirmed at weeks 8 and 16. According to the Lugano criteria, a complete response was at week 8, which was then confirmed at the subsequent 16-week evaluation. As shown in FIG. 8, a time-dependent decrease in BTK levels was observed as measured by flow cytometry using BTK MFI in CD19+ B cells. A gradual BTK degradation was observed until day 8 after receiving a 300 mg dose of compound 149, with maximum BTK degradation reached at day 8. Compound 149, consistent with its design, also degraded Ikaros and Aiolos, which are involved in the compound's immunomodulatory activity. Furthermore, this level of BTK degradation was sustained until day 29. Furthermore, confirmation of a complete response was seen at the 16-week evaluation by FDG-PET CT scan disease assessment compared to screening images, as shown in FIG. 9. At the 16-week evaluation, the maximum standardized uptake value (Max SUV) decreased from 17.6 at screening to 2.5 at the 16-week evaluation, and the Deauville 5-point scale (5PS; Barrington et al., 2010, Eur J Nucl Med Mol Imaging 37(10):1824-33) decreased from 5 to 2. Thus, the data confirm that compounds with dual mechanisms of action (BTK and immunomodulatory activity) provide clinical benefit to refractory patients with certain non-Hodgkin's lymphomas, such as DLBCL. Furthermore, it was noted that no dose-limiting toxicities (DLTs) or serious adverse events (SAEs) were observed.

[0249] Other embodiments

[0220] It should be understood that the above description is for illustrative purposes only and does not limit the scope of the present disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are intended to be within the scope of the following claims. There was a slight effect on ITK levels, but not on IMiD activity.

Claims

1. A pharmaceutical composition for the treatment or prevention of cancer, comprising a compound of formula I: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, R １ is selected from hydrogen and a substituted or unsubstituted heterocycle; R ２ but, 【Chemistry 2】 is selected from n is 1 or 2; A pharmaceutical composition, wherein the compound is capable of inducing proteolysis of Bruton's tyrosine kinase.

2. 10. The pharmaceutical composition of claim 1, wherein the cancer is a hematological malignancy or a B-cell malignancy and / or the cancer comprises a solid tumor.

3. 2. The pharmaceutical composition of claim 1, wherein the cancer is selected from the group consisting of chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom's macroglobulinemia, follicular lymphoma, diffuse large B-cell lymphoma, small cell lymphocytic leukemia, primary central nervous system lymphoma, and combinations thereof.

4. the Bruton tyrosine kinase is a C481 mutant Bruton tyrosine kinase, or the Bruton tyrosine kinase is a C481S mutant Bruton tyrosine kinase, or the Bruton tyrosine kinase is a L528W mutant Bruton tyrosine kinase, or the Bruton tyrosine kinase is a M437R mutant Bruton tyrosine kinase, or the Bruton tyrosine kinase is a V416L mutant Bruton tyrosine kinase, or the Bruton tyrosine kinase is a C481 mutant Bruton tyrosine kinase and the cancer is chronic lymphocytic leukemia (CLL), or 2. The pharmaceutical composition of claim 1, wherein the Bruton's tyrosine kinase is C481S mutant Bruton's tyrosine kinase and the cancer is chronic lymphocytic leukemia (CLL).

5. 2. The pharmaceutical composition of claim 1, wherein the cancer is resistant to ibrutinib, acalabrutinib, pirtobrutinib, or any combination thereof.

6. 10. The pharmaceutical composition of claim 1, wherein the compound of formula I is in a daily dose of 50 mg to 500 mg, 180 mg to 440 mg, 200 mg to 400 mg, 135 mg to 385 mg, 150 mg to 350 mg, 45 to 55 mg, 50 mg, 90 to 110 mg, 100 mg, 180 to 220 mg, 200 mg, 270 to 330 mg, 300 mg, 360 to 440 mg, 400 mg, 450 to 550 mg, or 50 mg.

7. 10. The pharmaceutical composition of claim 1, wherein the compound of formula I is administered in divided doses once, twice, three or four times per day and / or daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 30 or more days.

8. 10. The pharmaceutical composition of claim 1, wherein said compound of formula I is administered cyclically.

9. wherein said compound of formula I is 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 and pharmaceutically acceptable salts thereof.

10. 10. The pharmaceutical composition of claim 1, comprising the compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, vehicle, or adjuvant.

11. The pharmaceutical composition of claim 1, wherein the cancer is chronic lymphocytic leukemia.

12. the cancer is chronic lymphocytic leukemia, wherein said compound of formula I is 【Table 2】 The pharmaceutical composition of claim 1, wherein

13. the cancer is chronic lymphocytic leukemia, wherein said compound of formula I is 【Table 3】 The pharmaceutical composition of claim 1, wherein

14. the compound of formula I is administered at a dose of 270 mg to 330 mg, or 300 mg per day; wherein said compound of formula I is 【Table 4】 The pharmaceutical composition of claim 1, wherein

15. the cancer is chronic lymphocytic leukemia, the compound of formula I is administered at a dose of 90 mg to 110 mg, or 100 mg per day; wherein said compound of formula I is 【Table 5】 The pharmaceutical composition of claim 1, wherein

16. the compound of formula I is administered at a dose of 90 mg to 110 mg, or 100 mg per day; wherein said compound of formula I is 【Table 6】 The pharmaceutical composition of claim 1, wherein

17. the cancer is chronic lymphocytic leukemia, the compound of formula I is administered at a dose of 45 mg to 55 mg, or 50 mg per day; wherein said compound of formula I is 【Table 7】 The pharmaceutical composition of claim 1, wherein

18. the cancer is chronic lymphocytic leukemia, the compound of formula I is administered to a human subject at a dose of 90 mg to 110 mg, or about 100 mg per day; wherein said compound of formula I is 【Table 8】 The pharmaceutical composition of claim 1, wherein

19. 2. The pharmaceutical composition of claim 1, wherein the cancer is diffuse large B-cell lymphoma, germinal center B-cell-like (GCB) DLBCL, non-GCB DLBC, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), marginal zone lymphoma (MZL), Waldenstrom's macroglobulinemia (WM), or mantle cell lymphoma (MCL).

20. 10. The pharmaceutical composition of claim 1, wherein said compound of formula I is adapted for intermittent dosing.

21. 21. The pharmaceutical composition of claim 20, wherein the intermittent dosing is repeated throughout the treatment or prevention, and / or the intermittent dosing form includes a dosing period and a rest period, and / or the intermittent dosing is preceded by a loading dose.

22. 2. The pharmaceutical composition of claim 1, wherein the administration period is 2 weeks or 3 weeks.

23. 23. The pharmaceutical composition of claim 22, wherein if the administration period is 2 weeks, the rest period is 2 weeks, or if the administration period is 3 weeks, the rest period is 1 week, and / or the intermittent dosing is preceded by a loading dose for a period of 4 weeks.

24. 24. A compound according to any one of claims 1 to 23 for use in treating or preventing cancer according to any one of claims 1 to 23, or for the degradation of Bruton's tyrosine kinase.

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