Dosing regimens for treating PI3K-related conditions

Abstract

The present application provides methods of treating PI3K delta related conditions using pyrazolopyrimidine derivatives.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 2019800489184 (filing date: May 30, 2019; invention title: Administration Regimen for Treating PI3K-Related Disorders). Technical Field

[0002] The present application provides methods for treating PI3Kδ-related disorders using pyrazolopyrimidine derivatives. Background Art

[0003] Constitutive signaling through the B cell receptor (BCR) plays a key role in the pathogenesis of human B cell malignancies (Gururajan M. et al., The Journal of Immunology, 2006; 176(10):5715-5719) and results in downstream activation of class I phosphatidylinositol 3-kinase (PI3K) (Benschop R.J., Cambier J.C., Current Opinion in Immunology, 1999; 11(2):143-151; So L., Fruman D.A. The Biochemical Journal, 2012; 442(3):465-481). Class I PI3K is a heterodimeric lipid kinase composed of regulatory (p85 or p101) and catalytic (p110) subunits (Chalhoub N. et al., Annual review of pathology, 2009; 4:127-150). Four tissue-specific p110 subunit isoforms (class IA: α, β, and δ; class IB: γ) confer unique physiological functions to the respective PI3K isoforms, including insulin signaling and angiogenesis (PI3Kα) (Foukas L.C. et al., Nature, 2006; 441:366; Knight Z.A. et al., Cell, 2006; 125(4):733-747; and Graupera M. et al., Nature, 2008; 453:662), platelet function and thrombosis (PI3Kβ) (Jackson S.P. et al., Nature Medicine, 2005; 11:507), and leukocyte function and inflammation (PI3Kγ) (Hirsch E. et al., Thrombosis and Haemostasis, 2006; 95(1):29-35). The PI3Kδ isoform functions as a key node in the signaling network that regulates B cell growth and survival, and its aberrant activation is a critical event in the malignant transformation of B cells (Kang S. et al., Proceedings of the National Academy of Sciences of the United States of America, 2005; 102(3):802-807; Puri K.D., Gold M.R., Frontiers in Immunology, 2012; 3:256).Substantial interconnectedness exists between BCR and PI3Kδ-mediated signaling networks and other networks important for regulating B cell survival and proliferation, including activation of the Janus kinase-signal transducer and activator of transcription (JAK / STAT) pathway (Lam L.T. et al., Blood, 2008; 111(7):3701-3713; Rui L. et al., Proceedings of the National Academy of Sciences, 2016; 113(46):E7260-E7267), suggesting potential synergistic or combinatorial therapeutic effects in B cell malignancies.

[0004] Patients with relapsed or refractory B cell non-Hodgkin lymphoma (NHL) generally have a poor prognosis. For example, the 5-year overall survival (OS) rate for patients with relapsed follicular lymphoma (FL) (the most common indolent NHL subtype) is only 50% (Casulo C. et al., Journal of Clinical Oncology, 2015; 33(23):2516-2522). The prognosis is even worse for patients with relapsed aggressive NHL subtypes, with median survival times of 3.6 months and 4.4 months, respectively, for patients with relapsed diffuse large B cell lymphoma (DLBCL) who have failed first-line and second-line salvage regimens (Van Den Neste E. et al., Bone Marrow Transplant, 2016; 51(1):51-57). Current guidelines for treating relapsed B cell NHL vary according to subtype and include chemo- or radioimmunotherapy, targeted therapy with small molecule kinase inhibitors, or immunomodulatory therapy (NCCN Clinical Practice Guidelines in Oncology (NCCN ). B-cell Lymphomas. 7th Edition. 2017; 2017; Tilly H. et al., Annals of Oncology, 2015; 26(Suppl_5): v116-v125; Dreyling M. et al., Annals of Oncology, 2016; 27(Suppl_5): v83-v90; Dreyling M. et al., Annals of Oncology, 2017; 28(Suppl_4): iv62-iv71). In addition to systemic therapies, autologous or allogeneic stem cell transplantation (SCT) is also commonly used to treat patients with relapsed B-cell NHL and allogeneic SCT is considered the only treatment option for these patients (Wudhikarn K. et al Biology of Blood and Marrow Transplantation, 2011; 17(10): 1497-1504; Avivi I. et al., British Journal of Haematology, 2009; 147(5): 719-728; Klyuchnikov E. et al., Bone Marrow Transplant, 2014; 49(1): 1-7; Hamadani M. et al., Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation. 2009; 15(5): 547-553).

[0005] Differential tissue distribution of PI3K isoforms leads to their distinct biological functions. Genetic ablation of PI3Kα or PI3Kβ results in embryonic lethality, indicating that PI3Kα and PI3Kβ have essential and non-redundant functions, at least during development (Vanhaesebroeck et al., 2005). In contrast, mice lacking PI3Kγ and PI3Kδ are viable, fertile, and have a normal lifespan, although they display an altered immune system. PI3Kγ deficiency results in impaired recruitment of macrophages and neutrophils to sites of inflammation and impaired activation of T cells (Sasaki et al., Science, 2000, 287(5455):1040-6). PI3Kδ mutant mice have specific defects in B cell signaling, resulting in impaired B cell development and reduced antibody responses following antigen stimulation (Clayton et al., J Exp Med. 2002, 196(6):753-63; Jou et al., Mol Cell Biol. 2002, 22(24):8580-91; Okkenhaug et al., Science, 2002, 297(5583):1031-4).

[0006] The phenotypes of PI3Kγ and PI3Kδ mutant mice suggest that these enzymes play a role in inflammation and other immune-based diseases and this has been confirmed in preclinical models. PI3Kγ mutant mice are substantially protected from disease in murine models of rheumatoid arthritis (RA) and asthma (Camps et al., Nat Med. 2005, 11(9):936-43; Thomas et al., EurJImmunol. 2005, 35(4):1283-91). In addition, treatment of wild-type mice with a selective inhibitor of PI3Kγ has been shown to reduce glomerulonephritis and prolong survival in the MRL-lpr model of systemic lupus erythematosus (SLE) and to inhibit joint inflammation and damage in the RA model (Barber et al., Nat Med. 2005, 11(9):933-5; Camps et al., 2005). Similarly, PI3Kδ mutant mice and wild-type mice treated with a selective inhibitor of PI3Kδ have been shown to have reduced allergic airway inflammation and hyperreactivity in a murine asthma model (Ali et al., Nature. 2004, 431(7011):1007-11; Lee et al., FASEB J. 2006, 20(3):455-65) and reduced disease in the RA model (Randis et al., Eur.J.Immunol., 2008, 38(5):1215-24).

[0007] B cell proliferation has been shown to play an important role in the development of inflammatory autoimmune diseases (Puri, Frontiers in Immunology (2012), 3(256), 1 - 16; Walsh, Kidney International (2007) 72, 676–682). For example, B cells support T cell autoreactivity, which is an important component of inflammatory autoimmune diseases. Once activated and mature, B cells can traffic to the site of inflammation and recruit inflammatory cells or differentiate into plasmablasts. Thus, the activity of B cells can be affected by targeting B cell - stimulating cytokines, B cell surface receptors, or by B cell depletion. Rituximab - an IgG1κ mouse / human chimeric monoclonal antibody directed against the B cell surface receptor CD20 - has been shown to deplete CD20 + B cells. The use of Rituximab has been shown to be efficacious in the treatment of idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, or vasculitis. For example, treatment with Rituximab results in disease remission in patients with antineutrophil cytoplasmic antibody - associated (ANCA) systemic vasculitis (AASV), indicating peripheral B cell depletion (Walsh, 2007; Lovric, Nephrol Dial Transplant (2009) 24:179–185). Similarly, it has been reported that complete responses occur in one - third to two - thirds of patients with mixed cryoglobulinemic vasculitis after treatment with Rituximab, including patients with severe forms of vasculitis who are resistant or intolerant to other treatments (Cacoub, Ann Rheum Dis 2008; 67:283–287). Similarly, Rituximab has been shown to be efficacious in the treatment of patients with idiopathic thrombocytopenic purpura (or immune thrombocytopenic purpura) (Garvey, British Journal of Haematology, (2008) 141, 149–169; Godeau, Blood (2008), 112(4), 999 - 1004; Medeo, European Journal of Haematology, (2008) 81, 165–169) and autoimmune hemolytic anemia (Garvey, British Journal of Haematology, (2008) 141, 149–169).

[0008] PI3Kδ signaling is dependent on B cell survival, migration, and activation (Puri, Frontiers in Immunology, 2012, 3(256), 1-16, pages 1-5; and Clayton, J Exp Med, 2002, 196(6):753-63). For example, PI3Kδ is required for antigen-dependent B cell activation driven by the B cell receptor. By blocking B cell adhesion, survival, activation, and proliferation, PI3Kδ inhibition can attenuate the ability of B cells to activate T cells, thereby preventing their activation and reducing the secretion of autoantibodies and pro-inflammatory cytokines. Thus, through its ability to inhibit B cell activation, PI3Kδ inhibitors would be expected to treat B cell-mediated diseases that can be treated by similar methods such as B cell depletion by rituximab. Indeed, PI3Kδ inhibitors have been shown to be useful in mouse models of various autoimmune diseases that can also be treated with rituximab, such as arthritis (Puri (2012)). In addition, innate-like B cells (which are associated with autoimmunity) are sensitive to PI3Kδ activity because marginal zone (MZ) and B-1 cells are almost absent in mice lacking the p110δ gene (Puri (2012). PI3Kδ inhibitors can reduce the trafficking and activation of MZ and B-1 cells, which are associated with autoimmune diseases.

[0009] In addition to their potential roles in inflammatory diseases, all four class I PI3K isoforms can play a role in cancer. The gene encoding p110α is frequently mutated in common cancers including breast, prostate, colon, and endometrium (Samuels et al., Science, 2004, 304(5670):554; Samuels et al., Curr Opin Oncol. 2006, 18(1):77-82). Eighty percent of these mutations are represented by one of three amino acid substitutions in the helical or kinase domains of the enzyme and result in a significant upregulation of kinase activity, leading to oncogenic transformation in cell culture and animal models (Kang et al., Proc Natl Acad Sci U S A. 2005, 102(3):802-7; Bader et al., Proc Natl Acad Sci US A. 2006, 103(5):1475-9). Such mutations have not been identified in other PI3K isoforms, although there is evidence that they can contribute to the development and progression of malignancy. Consistent overexpression of PI3Kδ has been observed in acute myeloblastic leukemia (Sujobert et al., Blood, 2005, 106(3):1063-6) and inhibitors of PI3Kδ can prevent the growth of leukemia cells (Billottet et al., Oncogene. 2006, 25(50):6648-59). Increased expression of PI3Kγ has been seen in chronic myelogenous leukemia (Hickey et al., J Biol Chem. 2006, 281(5):2441-50). Alterations in the expression of PI3Kβ, PI3Kγ, and PI3Kδ have also been observed in cancers of the brain, colon, and bladder (Benistant et al., Oncogene, 2000, 19(44):5083-90; Mizoguchi et al., Brain Pathol. 2004, 14(4):372-7; Knobbe et al., Neuropathol Appl Neurobiol. 2005, 31(5):486-90). In addition, these isoforms have all been shown to be oncogenic in cell culture (Kang et al., 2006).

[0010] The introduction of rituximab has improved the treatment outcomes of patients with B-cell NHL (Chao M.P., Cancer Management and Research. 2013;5:251-269; Nandagopal L., Mehta A., Expert Review of Hematology, 2017;10(3):259-273). For patients who experience rituximab resistance and / or relapsed or refractory disease, the class of PI3K inhibitors has shown promise, but clinical use is limited by toxicity. For example, the first class of PI3Kδ-selective inhibitor, idelalisib, is associated with considerable toxicity, including hepatotoxicity (Coutré S.E. et al., Leukemia & Lymphoma, 2015;56(10):2779-2786; Gopal A., Graf S., Expert opinion on pharmacotherapy, 2016;17(2):265-274) and an increased risk of opportunistic infections (Lampson B.L. et al., Blood, 2016;128(2):195-203), and requires frequent monitoring. Similarly, treatments targeting other PI3K isoforms have corresponding risks (e.g., hyperglycemia and hypertension) (Burris H.A. et al., Journal of Clinical Oncology, 2014;32(15_suppl):2513-2513; ALIQOPA TM . (copanlisib) for injection, for intravenous use. U.S. Whippany, NJ: Bayer HealthCare Pharmaceuticals Inc.; 2017; Patnaik A. et al., Annals of Oncology, 2016;27(10):1928-1940; Dreyling M. et al., Journal of Clinical Oncology, 2017;35(35):3898-3905). Thus, there remains an unmet need for an effective therapy with an improved safety profile in this difficult-to-treat patient population. Summary of the Invention

[0011] The present application particularly provides a method for treating a disease of a patient, wherein the disease is associated with abnormal expression or activity of PI3Kδ kinase, the method comprising:

[0012] (i) Administer a first dose of a PI3Kδ inhibitor at about 3 mg / day to about 50 mg / day to a patient for a first period of about 2 weeks to about 12 weeks; and

[0013] (ii) Administer a second dose of the PI3Kδ inhibitor to the patient at the end of the first period, the second dose being less than the first dose and being:

[0014] (a) about 2.5 mg / day or less; or

[0015] (b) about 50 mg / week or less;

[0016] and wherein the second dose is administered during a second period that occurs after the first period.

[0017] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will be apparent from the description and drawings and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A-1B Shows the PK of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride (i.e., Compound 1C) at steady state. Figure 1A : PK of Compound 1C administration. Figure 1B : Dose-response relationship of Compound 1C AUC. AUC: Area Under the Curve; AUC 0–T : Area Under the Plasma Concentration-Time Curve; IC 50 : Half Maximal Inhibitory Concentration; IC 90 : 90% Maximal Inhibitory Concentration; PK: Pharmacokinetics; QD: Once Daily.

[0019] Figure 2 Shows the best percent change in target lesion size compared to baseline for single-agent therapy. *Data for 3 patients with DLBCL, 2 patients with MZL, and 1 patient with MCL are not shown due to non-measurable disease at baseline or invalid target lesion measurements after baseline. Best percent change in target lesion size compared to baseline > 100%. IB: 3 patients with MCL had received ibrutinib prior to the study, and 2 of these patients achieved the best overall response of CR or PR. DLBCL: Diffuse Large B-Cell Lymphoma; FL: Follicular Lymphoma; MCL: Mantle Cell Lymphoma; MZL: Marginal Zone Lymphoma.

[0020] Figure 3Shows the duration of treatment with compound 1C monotherapy. AE: Adverse event; DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; MCL: Mantle cell lymphoma; MZL: Marginal zone lymphoma; PD: Progressive disease; QW: Once a week.

[0021] Figure 4 Shows the mean percent inhibition of phosphorylated AKT at steady state. Compound 1C was administered once daily and the level of phosphorylated AKT (pAKT; Ser473) was measured by flow cytometry in SU-DHL cells added to patient whole blood on day 15 of cycle 1, after treatment with compound 1C.

[0022] Figure 5A-5B Shows ALT ( Figure 5A ) and AST ( Figure 5B ) over time in patients receiving compound 1C monotherapy. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; QD: Once daily; ULN: Upper limit of normal.

[0023] Figure 6 Shows the time to response with compound 1C monotherapy in NHL subtypes of interest (DLBCL, FL, MCL, MZL). DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; MCL: Mantle cell lymphoma; MZL: Marginal zone lymphoma.

[0024] Figure 7 Shows a representative positron emission tomography (PET) image of a patient who achieved a complete response with compound 1C monotherapy. Patient < 65 years old, with mantle cell lymphoma not involving the bone marrow (target lesion, 98 mm × 58 mm) and who had received 1 prior treatment (R-HyperCVAD). Treated with compound 1C, 30 mg, once daily; complete response was achieved at week 9. Detailed Description

[0025] The present application provides a method of treating a disease in a patient, wherein the disease is associated with abnormal expression or activity of PI3Kδ kinase, the method comprising:

[0026] i) administering to the patient a first dose of a PI3Kδ inhibitor of from about 3 mg / day to about 50 mg / day for a first period of from about 2 weeks to about 12 weeks; and

[0027] ii) administering to the patient a second dose of a PI3Kδ inhibitor at the end of the first period, the second dose being less than the first dose and being:

[0028] (a) about 2.5 mg / day or less; or

[0029] (b) about 50 mg / week or less;

[0030] and wherein the second dose is administered during a second time period that occurs after the first time period.

[0031] This application also provides a method of treating a disease in a patient, wherein the disease is associated with abnormal expression or activity of PI3Kδ kinase, the method comprising:

[0032] i) administering to the patient a first dose of a PI3Kδ inhibitor at about 3 mg / day to about 50 mg / day for a first time period of about 2 weeks to about 12 weeks; and

[0033] ii) administering to the patient a second dose of a PI3Kδ inhibitor at the end of the first time period, the second dose being less than the first dose and being about 7.5 mg / day or less;

[0034] and wherein the second dose is administered during a second time period that occurs after the first time period.

[0035] As used herein, the term "first time period" refers to the time period during which the first dose of the PI3Kδ inhibitor is administered to the patient.

[0036] In some embodiments, the first time period begins when the first dose of the PI3Kδ inhibitor is initially administered to the patient.

[0037] As used herein, the term "second time period" refers to the time period during which the second dose of the PI3Kδ inhibitor is administered to the patient.

[0038] In some embodiments, the second time period begins when the second dose of the PI3Kδ inhibitor is initially administered to the patient.

[0039] In some embodiments, the time at which the second dose of the PI3Kδ inhibitor is initially administered to the patient (i.e., the start of the second time period) is within 2 weeks of the end of the first time period, such as within 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of the end of the first time period.

[0040] In some embodiments, the second time period lasts for any length of time, such as until the end of treatment.

[0041] In some embodiments, the first doses are each administered as a single, once-daily dose.

[0042] In some embodiments, the first doses are each administered as a single, once-daily oral dose.

[0043] In some embodiments, the first dose is from about 5 mg / day to about 50 mg / day, such as from about 5 mg / day to about 40 mg / day, from about 5 mg / day to about 30 mg / day, from about 5 mg / day to about 20 mg / day, from about 5 mg / day to about 10 mg / day, from about 10 mg / day to about 40 mg / day, from about 10 mg / day to about 30 mg / day, from about 10 mg / day to about 20 mg / day, from about 20 mg / day to about 40 mg / day, from about 20 mg / day to about 30 mg / day, or from about 20 mg / day to about 40 mg / day.

[0044] In some embodiments, the first dose is about 50 mg / day, about 45 mg / day, about 40 mg / day, about 30 mg / day, about 20 mg / day, about 15 mg / day, about 10 mg / day or about 5 mg / day.

[0045] In some embodiments, the first dose is from about 18 mg / day to about 22 mg / day.

[0046] In some embodiments, the first dose is from about 18 mg / day to about 22 mg / day and is administered as a single, once-daily (i.e., QD) dose.

[0047] In some embodiments, the first dose is from about 18 mg / day to about 22 mg / day and is administered as a single, once-daily (i.e., QD) oral dose.

[0048] In some embodiments, the first dose is about 20 mg / day.

[0049] In some embodiments, the first dose is about 20 mg / day and is administered as a single, once-daily (i.e., QD) dose.

[0050] In some embodiments, the first dose is about 20 mg / day and is administered as a single, once-daily (i.e., QD) oral dose.

[0051] In some embodiments, the first dose is about 10 mg / day.

[0052] In some embodiments, the first dose is about 10 mg / day and is administered as a single, once-daily (i.e., QD) dose.

[0053] In some embodiments, the first dose is about 10 mg / day and is administered as a single, once-daily (i.e., QD) oral dose.

[0054] In some embodiments, the first period is from about 3 weeks to about 11 weeks, such as from about 8 weeks to about 12 weeks, from about 4 weeks to about 10 weeks, from about 5 weeks to about 9 weeks, or from about 8 weeks to about 9 weeks. In some embodiments, the first period is about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks.

[0055] In some embodiments, the first period is from about 8 weeks to about 9 weeks.

[0056] In some embodiments, the first period is about 8 weeks.

[0057] In some embodiments, the first period is about 9 weeks.

[0058] In some embodiments, the first dose is reduced during the first period.

[0059] In some embodiments, the first dose is reduced from about 3 mg / day to about 50 mg / day to about 3 mg / day to about 30 mg / day, about 3 mg / day to about 20 mg / day, about 3 mg / day to about 15 mg / day, about 3 mg / day to about 10 mg / day, about 3 mg / day to about 7 mg / day, or about 3 mg / day to about 5 mg / day.

[0060] In some embodiments, the first dose is reduced from about 3 mg / day to about 50 mg / day to about 3 mg / day to about 7 mg / day during the first period.

[0061] In some embodiments, the first dose is reduced from about 50 mg / day to about 30 mg / day, about 20 mg / day, about 15 mg / day, about 10 mg / day, about 5 mg / day, about 2 mg / day, or about 1 mg / day during the first period.

[0062] In some embodiments, the first dose is reduced from about 20 mg / day to about 15 mg / day, about 10 mg / day, about 5 mg / day, about 2 mg / day, or about 1 mg / day during the first period.

[0063] In some embodiments, the second doses are each administered as a single, once-daily (QD) dose or as a single, once-weekly (QW) dose.

[0064] In some embodiments, the second doses are each administered as a single, once-daily (QD) dose.

[0065] In some embodiments, the second doses are each administered as a single, once-weekly (QW) dose.

[0066] In some embodiments, the second doses are each administered as a single, once-daily (QD) oral dose or as a single, once-weekly (QW) oral dose.

[0067] In some embodiments, the second doses are each administered as a single, once-daily (QD) oral dose.

[0068] In some embodiments, the second doses are each administered as a single, once-weekly (QW) oral dose.

[0069] In some embodiments, the second dose is about 5.0 mg / day or less, such as about 5.0 mg / day, about 4.0 mg / day, about 3.0 mg / day, about 2.5 mg / day, about 2.0 mg / day, about 1.75 mg / day, about 1.5 mg / day, about 1.25 mg / day, or about 1.0 mg / day.

[0070] In some embodiments, the second dose is from about 2.5 mg / day to about 7.5 mg / day.

[0071] In some embodiments, the second dose is from about 3.0 mg / day to about 7.0 mg / day.

[0072] In some embodiments, the second dose is from about 4.0 mg / day to about 6.0 mg / day.

[0073] In some embodiments, the second dose is about 5.0 mg / day.

[0074] In some embodiments, the second dose is from about 4.0 mg / day to about 6.0 mg / day and is administered as a single, once-daily (QD) dose.

[0075] In some embodiments, the second dose is about 5.0 mg / day and is administered as a single, once-daily (QD) dose.

[0076] In some embodiments, the second dose is from about 4.0 mg / day to about 6.0 mg / day and is administered as a single, once-daily (QD) oral dose.

[0077] In some embodiments, the second dose is about 5.0 mg / day and is administered as a single, once-daily (QD) oral dose.

[0078] In some embodiments, the second dose is reduced from about 5.0 mg / day or less during a second time period.

[0079] In some embodiments, the second dose is reduced from about 5.0 mg / day or less to about 2.5 mg / day or less, 2.0 mg / day or less, about 1.75 mg / day or less, about 1.5 mg / day or less, about 1.25 mg / day or less, or about 1.0 mg / day or less during a second time period.

[0080] In some embodiments, the second dose is reduced from about 5.0 mg / day to about 2.5 mg / day, 2.0 mg / day, about 1.75 mg / day, about 1.5 mg / day, about 1.25 mg / day, or about 1.0 mg / day during the second period.

[0081] In some embodiments, the second dose is reduced from about 5.0 mg / day to about 0.75 mg / day to about 1.25 mg / day during the second period.

[0082] In some embodiments, the second dose is reduced from about 5.0 mg / day to about 1.25 mg / day during the second period.

[0083] In some embodiments, the second dose is reduced from about 5.0 mg / day to about 2.0 mg / day, about 1.5 mg / day, about 1.25 mg / day, or about 1.0 mg / day.

[0084] In some embodiments, the second dose is reduced from about 5.0 mg / day to about 1.0 mg / day.

[0085] In some embodiments, the second dose is about 2.5 mg / day or less, such as about 2.5 mg / day, about 2.0 mg / day, about 1.75 mg / day, about 1.5 mg / day, about 1.25 mg / day, or about 1.0 mg / day.

[0086] In some embodiments, the second dose is from about 1.0 mg / day to about 2.5 mg / day, such as from about 1.0 mg / day to about 2.0 mg / day, from about 1.0 mg / day to about 1.5 mg / day, or from about 1.0 mg / day to about 1.25 mg / day.

[0087] In some embodiments, the second dose is from about 2 mg / day to about 2.5 mg / day.

[0088] In some embodiments, the second dose is about 2.5 mg / day.

[0089] In some embodiments, the second dose is from about 2 mg / day to about 2.5 mg / day and is administered as a single, once-daily (QD) dose.

[0090] In some embodiments, the second dose is about 2.5 mg / day and is administered as a single, once-daily (QD) dose.

[0091] In some embodiments, the second dose is from about 2 mg / day to about 2.5 mg / day and is administered as a single, once-daily (QD) oral dose.

[0092] In some embodiments, the second dose is about 2.5 mg / day and is administered as a single, once-daily (QD) oral dose.

[0093] In some embodiments, the second dose is reduced from about 2.5 mg / day or less during a second time period.

[0094] In some embodiments, the second dose is reduced from about 2.5 mg / day or less to about 2.0 mg / day or less, about 1.75 mg / day or less, about 1.5 mg / day or less, about 1.25 mg / day or less, or about 1.0 mg / day or less during a second time period.

[0095] In some embodiments, the second dose is reduced from about 2.5 mg / day to about 2.0 mg / day, about 1.75 mg / day, about 1.5 mg / day, about 1.25 mg / day, or about 1.0 mg / day during a second time period.

[0096] In some embodiments, the second dose is reduced from about 2.5 mg / day to about 1.25 mg / day during a second time period.

[0097] In some embodiments, the second dose is reduced from about 2.5 mg / day to about 2.0 mg / day, about 1.5 mg / day, about 1.25 mg / day, or about 1.0 mg / day.

[0098] In some embodiments, the second dose is reduced from about 2.5 mg / day to about 1.0 mg / day.

[0099] In some embodiments, the reduced dose of the second dose is administered as a single, once-daily (QD) dose.

[0100] In some embodiments, the reduced dose of the second dose is administered as a single, once-daily (QD) oral dose.

[0101] In some embodiments, the second dose is about 50 mg / week or less, such as about 40 mg / week or less, about 30 mg / week or less, about 20 mg / week or less, about 10 mg / week or less, about 5 mg / week or less, about 2 mg / week or less, or about 1 mg / week or less.

[0102] In some embodiments, the second dose is from about 5 mg / week to about 50 mg / week, such as from about 5 mg / week to about 40 mg / week, from about 5 mg / week to about 30 mg / week, from about 5 mg / week to about 20 mg / week, from about 5 mg / week to about 10 mg / week, from about 10 mg / week to about 40 mg / week, from about 10 mg / week to about 30 mg / week, from about 10 mg / week to about 20 mg / week, from about 20 mg / week to about 40 mg / week, from about 20 mg / week to about 30 mg / week, or from about 30 mg / week to about 40 mg / week.

[0103] In some embodiments, the second dose is from about 18 mg / week to about 20 mg / week.

[0104] In some embodiments, the second dose is about 20 mg / week.

[0105] In some embodiments, the second dose is from about 18 mg / week to about 20 mg / week and is administered as a single, once-weekly (QW) dose.

[0106] In some embodiments, the second dose is about 20 mg / week and is administered as a single, once-weekly (QW) dose.

[0107] In some embodiments, the second dose is reduced from about 50 mg / week or less during a second time period.

[0108] In some embodiments, the second dose is reduced from about 50 mg / week or less to about 40 mg / week or less, about 30 mg / week or less, about 20 mg / week or less, about 10 mg / week or less, about 5 mg / week or less, about 2 mg / week or less, or about 1 mg / week or less during a second time period.

[0109] In some embodiments, the second dose is reduced from about 5 mg / week to about 50 mg / week to about 5 mg / week to about 40 mg / week, about 5 mg / week to about 30 mg / week, about 5 mg / week to about 20 mg / week, about 5 mg / week to about 10 mg / week, about 10 mg / week to about 40 mg / week, about 10 mg / week to about 30 mg / week, about 10 mg / week to about 20 mg / week, about 20 mg / week to about 40 mg / week, about 20 mg / week to about 30 mg / week, or about 30 mg / week to about 40 mg / week during a second time period.

[0110] In some embodiments, the second dose is reduced from about 50 mg / week to about 40 mg / week, about 30 mg / week, about 20 mg / week, about 10 mg / week or about 5 mg / week during a second time period.

[0111] In some embodiments, the second dose is reduced from about 20 mg / week to about 10 mg / week during a second time period.

[0112] In some embodiments, the second dose is reduced from about 20 mg / week to about 10 mg / week during a second time period.

[0113] In some embodiments, the second dose is reduced from about 10 mg / week to about 5 mg / week during a second time period.

[0114] In some embodiments, the reduced dose of the second dose is administered as a single, once-weekly (QW) dose.

[0115] In some embodiments, a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs). As used herein, the severity of the TEAEs described herein is assessed using Version 4.03 of the Common Terminology Criteria for Adverse Events, the disclosure of which is incorporated herein by reference in its entirety.

[0116] In some embodiments, a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs) during a first time period.

[0117] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the first dose is reduced from about 3 mg / day to about 50 mg / day to about 3 mg / day to about 30 mg / day, about 3 mg / day to about 20 mg / day, about 3 mg / day to about 15 mg / day, about 3 mg / day to about 10 mg / day, about 3 mg / day to about 7 mg / day, or about 3 mg / day to about 5 mg / day.

[0118] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the first dose is reduced from about 50 mg / day to about 30 mg / day, about 20 mg / day, about 15 mg / day, about 10 mg / day, about 5 mg / day, about 2 mg / day, or about 1 mg / day during a first time period.

[0119] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the first dose is reduced from about 20 mg / day to about 15 mg / day, about 10 mg / day, about 5 mg / day, about 2 mg / day, or about 1 mg / day during a first time period.

[0120] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the first dose is reduced from about 20 mg / day to about 10 mg / day.

[0121] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the first dose is reduced from about 20 mg / day to about 5 mg / day.

[0122] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the first dose is reduced from about 10 mg / day to about 5 mg / day.

[0123] In some embodiments, the methods provided herein further include stopping the administration of the first dose when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs).

[0124] In some embodiments, a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs) during a second period.

[0125] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the second dose is reduced during the second period from about 3 mg / day to about 50 mg / day to about 3 mg / day to about 30 mg / day, about 3 mg / day to about 20 mg / day, about 3 mg / day to about 15 mg / day, about 3 mg / day to about 10 mg / day, about 3 mg / day to about 7 mg / day, or about 3 mg / day to about 5 mg / day.

[0126] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the second dose is reduced during the second period from about 50 mg / week to about 40 mg / week, about 30 mg / week, about 20 mg / week, about 10 mg / week, about 5 mg / week, about 2 mg / week, or about 1 mg / week.

[0127] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the second dose is reduced during the second period from about 50 mg / week or less to about 40 mg / week or less, about 30 mg / week or less, about 20 mg / week or less, about 10 mg / week or less, about 5 mg / week or less, about 2 mg / week or less, or about 1 mg / week or less.

[0128] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the second dose is reduced during the second period from about 20 mg / week to about 10 mg / week or less.

[0129] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the second dose is reduced from about 20 mg / week to about 5 mg / week or less during the second period.

[0130] In some embodiments, when a patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs), the second dose is reduced from about 10 mg / week to about 5 mg / week or less during the second period.

[0131] In some embodiments, treatment-emergent adverse events include one or more of the following: diarrhea / colitis, nausea, fatigue, rash, neutropenia, fever, hypotension, sepsis, respiratory failure, pneumonitis, pneumonia, hypertension, hyperglycemia, abdominal pain, bronchitis, dehydration, thrombocytopenia, cough, vomiting, decreased appetite, increased lacrimation, oral herpes, tachycardia, spinal cord compression, intractable pain, elevated alkaline phosphatase, elevated transaminases, hyperlipidemia, hypercalcemia, dizziness, alopecia, constipation, fluid overload, headache, hypokalemia, night sweats, encephalopathy, atrial flutter, atrial fibrillation, and dyspnea.

[0132] In some embodiments, symptoms associated with one or more treatment-emergent adverse events are symptoms associated with one or more of the following: diarrhea / colitis, nausea, fatigue, rash, neutropenia, fever, hypotension, sepsis, respiratory failure, pneumonitis, pneumonia, hypertension, hyperglycemia, abdominal pain, bronchitis, dehydration, thrombocytopenia, cough, vomiting, decreased appetite, increased lacrimation, oral herpes, tachycardia, spinal cord compression, intractable pain, elevated alkaline phosphatase, elevated transaminases, hyperlipidemia, hypercalcemia, dizziness, alopecia, constipation, fluid overload, headache, hypokalemia, night sweats, encephalopathy, atrial flutter, atrial fibrillation, and dyspnea.

[0133] In some embodiments, treatment-emergent adverse events include one or more of the following: diarrhea / colitis, nausea, fatigue, rash, cough, vomiting, dizziness, fever, hypokalemia, abdominal pain, constipation, decreased appetite, night sweats, pruritus, back pain, chills, leukopenia, neutropenia, lymphopenia, thrombocytopenia, and anemia.

[0134] In some embodiments, treatment-emergent adverse events include one or more of diarrhea / colitis and rash.

[0135] In some embodiments, diarrhea / colitis includes one or more of the following: diarrhea, colitis, enterocolitis, gastroenteritis, microscopic colitis, and cytomegalovirus colitis.

[0136] In some embodiments, rash includes one or more of the following: exfoliative dermatitis, rash, erythematous rash, punctate rash, maculopapular rash, pruritic rash, exfoliative rash, generalized rash, epidemic rash, and pustular rash.

[0137] In some embodiments, neutropenia includes febrile neutropenia. In some embodiments, elevated transaminases include elevated alanine transaminase (ALT), elevated aspartate transaminase (AST), or a combination thereof.

[0138] As used herein, the term "contact" means bringing together the indicated moieties in an in vitro system or an in vivo system. For example, "contact" of PI3K with a PI3Kδ inhibitor compound described herein includes administering the compound described herein to an individual or patient such as a human having PI3K, and, for example, introducing the compound described herein into a sample containing cells or a purified preparation containing PI3K.

[0139] In some embodiments, the PI3Kδ inhibitor is a compound of U.S. Patent Publication US2011 / 0015212, filed Jun. 28, 2010; U.S. Patent Publication 2013 / 0059835, filed Aug. 31, 2013; U.S. Patent Publication 2011 / 0183985, filed Dec. 17, 2010; U.S. Patent Publication 2012 / 0157430, filed Dec. 19, 2011; U.S. Patent Publication 2014 / 0249132, filed Feb. 28, 2014; or U.S. Patent Publication 2016 / 0257689, filed Feb. 26, 2016, each of which is incorporated herein by reference in its entirety.

[0140] In some embodiments, the PI3Kδ inhibitor is prepared by the methods of U.S. Patent Publication US2011 / 0015212, filed Jun. 28, 2010; U.S. Patent Publication 2013 / 0059835, filed Aug. 31, 2013; U.S. Patent Publication 2011 / 0183985, filed Dec. 17, 2010; U.S. Patent Publication 2012 / 0157430, filed Dec. 19, 2011; U.S. Patent Publication 2014 / 0249132, filed Feb. 28, 2014; or U.S. Patent Publication 2016 / 0257689, filed Feb. 26, 2016, each of which is incorporated herein by reference in its entirety.

[0141] In some embodiments, the PI3Kδ inhibitor is selected from:

[0142] 4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one; and

[0143] 5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one;

[0144] or a pharmaceutically acceptable salt thereof.

[0145] In some embodiments, the PI3Kδ inhibitor is 4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one or a pharmaceutically acceptable salt thereof.

[0146] In some embodiments, the PI3Kδ inhibitor is selected from:

[0147] (S)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;

[0148] (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;

[0149] (S)-4-(3-((R)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; and

[0150] (R)-4-(3-((R)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;

[0151] or a pharmaceutically acceptable salt thereof.

[0152] In some embodiments, the PI3Kδ inhibitor is a pharmaceutically acceptable salt of 4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one.

[0153] In some embodiments, the PI3Kδ inhibitor is selected from the following pharmaceutically acceptable salts:

[0154] (S)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;

[0155] (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;

[0156] (S)-4-(3-((R)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; and

[0157] (R)-4-(3-((R)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one.

[0158] In some embodiments, the PI3Kδ inhibitor is a pharmaceutically acceptable salt of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one.

[0159] In some embodiments, the PI3Kδ inhibitor is the hydrochloride salt of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one. In some embodiments, the salt is a salt in which the stoichiometric ratio of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one to hydrochloric acid is 1:1.

[0160] In some embodiments, the salt is crystalline (for methods and processes for preparing the hydrochloride salt of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, see, for example, U.S. Patent Publication 2016 / 0257689, filed Feb. 26, 2016. U.S. Patent Publication 2016 / 0257689 is incorporated herein by reference in its entirety.).

[0161] In some embodiments, the hydrochloride salt of 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one has: at least one, two, three, four, or five XRPD peaks, expressed as 2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.1°, about 31.2°, and about 32.8°; at least two XRPD peaks, expressed as 2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.1°, about 31.2°, and about 32.8°; at least three XRPD peaks, expressed as 2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.1°, about 31.2°, and about 32.8°. In some embodiments, the hydrochloride salt of the compound of formula I has at least four XRPD peaks, expressed as 2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.1°, about 31.2°, and about 32.8°. In some embodiments, the hydrochloride salt of 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one has all of the listed XRPD peaks, expressed as 2-theta, at about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.1°, about 31.2°, and about 32.8°. In some embodiments, the hydrochloride salt of 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one has a DSC thermogram with an endothermic peak at about 207 °C.

[0162] In some embodiments, the salts and compounds described herein are substantially isolated. "Substantially isolated" means that the salt or compound is at least partially or substantially separated from the environment in which it is formed or detected. Partial separation can include, for example, a composition enriched in the form of the salt described herein. Substantially isolated can include a composition containing at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 90 wt%, at least about 95 wt%, at least about 97 wt%, or at least about 99 wt% of the salt or its salt described herein. Methods for separating compounds and their salts are conventional in the art.

[0163] In some embodiments, the PI3Kδ inhibitor is 5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.

[0164] In some embodiments, the PI3Kδ inhibitor is selected from:

[0165] (R)-5-{3-[(R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one;

[0166] (R)-5-{3-[(S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one;

[0167] (S)-5-{3-[(S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one; and

[0168] (S)-5-{3-[(R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one;

[0169] or a pharmaceutically acceptable salt thereof.

[0170] The compounds (including their salts) described herein can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes. In some embodiments, the compounds can be prepared as described in U.S. Patents 9,199,982 and 9,932,341, each of which is incorporated herein by reference in its entirety.

[0171] The reactions used to prepare the compounds described herein can be carried out in a suitable solvent, which can be readily selected by a person skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out (e.g., a temperature within the range from the freezing temperature to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the specific reaction step, the suitable solvent for a specific reaction step can be selected by a skilled person.

[0172] The preparation of the compounds described herein may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the choice of appropriate protecting groups can be readily determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0173] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry; or chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC). The compounds can be purified by those skilled in the art by a variety of methods including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved CompoundSpecific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, AndrewP. Combs J. Combi. Chem. 2004, 6(6), 874-883, which is incorporated herein by reference in its entirety) and normal phase silica gel chromatography.

[0174] The compounds described herein can modulate the activity of one or more of various kinases, such as phosphoinositide 3-kinase (PI3K). The term "modulate" refers to the ability to increase or decrease the activity of one or more members of the PI3K family. Thus, the compounds described herein can be used in methods of modulating PI3K by contacting PI3K with any one or more of the compounds or compositions described herein to effect such modulation. In some embodiments, the compounds of the invention can act as inhibitors of one or more PI3Ks. In other embodiments, the compounds described herein can be used to modulate the activity of PI3K in an individual in need of receptor modulation by administering a modulating amount of the compounds described herein or a pharmaceutically acceptable salt thereof. In some embodiments, the modulation is inhibition. Given that the growth and survival of cancer cells are affected by multiple signaling pathways, the present invention is applicable to treating disease states characterized by drug-resistant kinase mutations. Additionally, different kinase inhibitors can be used in combination, which exhibit different preferences among the kinases whose activity is modulated by the inhibitors. This approach can exhibit high efficiency in treating disease states by targeting multiple signaling pathways, reduce the likelihood of drug resistance emerging in cells, and decrease the toxicity for the treatment of diseases.

[0175] The PI3Kδ inhibitors described herein can be selective. "Selective" means that the compound binds to or inhibits the kinase with greater affinity or potency, respectively, compared to at least one other kinase. In some embodiments, the compounds described herein are selective inhibitors of PI3Kδ relative to PI3Kγ, PI3Kα, and / or PI3Kβ. In some embodiments, the compounds described herein are selective inhibitors of PI3Kδ (e.g., relative to PI3Kα, PI3Kβ, and PI3Kγ). In some embodiments, the selectivity can be at least about 2-fold, 5-fold, 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold. Selectivity can be measured by conventional methods in the art. In some embodiments, the selectivity can be tested at the K m ATP concentration of each enzyme. In some embodiments, the selectivity of the compounds described herein can be determined by a cellular assay related to the activity of a specific PI3K kinase.

[0176] Kinases that the compounds of the present invention bind to and / or modulate (e.g., inhibit) include any member of the PI3K family. In some embodiments, the PI3K is PI3Kα, PI3Kβ, PI3Kγ, or PI3Kδ. In some embodiments, the PI3K is PI3Kγ or PI3Kδ. In some embodiments, the PI3K is PI3Kγ. In some embodiments, the PI3K is PI3Kδ. In some embodiments, the PI3K includes a mutation. The mutation can be the substitution of one amino acid for another, or the deletion of one or more amino acids. In such embodiments, the mutation can be present in the kinase domain of the PI3K.

[0177] In some embodiments of the methods of the present invention, more than one compound described herein is used to inhibit the activity of a kinase (e.g., PI3Kγ or PI3Kδ).

[0178] In some embodiments of the methods of the present invention, more than one compound described herein is used to inhibit more than one kinase, such as at least two kinases (e.g., PI3Kγ and PI3Kδ).

[0179] In some embodiments of the methods of the present invention, one or more compounds are used in combination with another kinase inhibitor to inhibit the activity of a kinase (e.g., PI3Kγ or PI3Kδ).

[0180] In some embodiments of the methods of the present invention, one or more compounds are used in combination with another kinase inhibitor to inhibit the activity of more than one kinase (e.g., PI3Kγ or PI3Kδ), such as the activity of at least two kinases.

[0181] Another aspect of the present invention relates to a method of treating a kinase (such as PI3Kδ)-related disease or disorder in an individual (e.g., a patient) by administering a therapeutically effective amount or dose of one or more compounds of the present invention or a pharmaceutical composition thereof to an individual in need of such treatment. PI3K-related diseases can include any disease, disorder or condition directly or indirectly associated with the expression or activity of PI3K, including overexpression and / or abnormal activity levels. In some embodiments, the disease may be associated with Akt (protein kinase B), mammalian target of rapamycin (mTOR) or phosphoinositide-dependent kinase 1 (PDK1). In some embodiments, the mTOR-related disease may be inflammation, atherosclerosis, psoriasis, restenosis, benign prostatic hyperplasia, bone disorders, pancreatitis, angiogenesis, diabetic retinopathy, atherosclerosis, arthritis, immune disorders, kidney diseases or cancer. PI3K-related diseases can also include any disease, disorder or condition that can be prevented, ameliorated or cured by modulating PI3K activity. In some embodiments, the disease is characterized by abnormal activity of PI3K. In some embodiments, the disease is characterized by mutant PI3K. In such embodiments, the mutation may be present in the kinase domain of PI3K.

[0182] Examples of PI3K-related diseases include immune-based diseases affecting the system, including, for example, rheumatoid arthritis, allergies, asthma, glomerulonephritis, lupus, or inflammation associated with any of the foregoing.

[0183] Other examples of PI3K-related diseases include cancers such as breast cancer, prostate cancer, colon cancer, endometrial cancer, brain cancer, bladder cancer, skin cancer, uterine cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer or blood cancer.

[0184] In some embodiments, the blood cancer is acute myeloblastic leukemia (AML) or chronic myelogenous leukemia (CML), or B-cell lymphoma. In some embodiments, the B-cell lymphoma is diffuse large B-cell lymphoma.

[0185] Other examples of PI3K-related diseases include lung diseases such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS).

[0186] Other examples of PI3K-related diseases include osteoarthritis, restenosis, atherosclerosis, bone disorders, arthritis, diabetic retinopathy, psoriasis, benign prostatic hyperplasia, inflammation, angiogenesis, pancreatitis, kidney diseases, inflammatory bowel disease, myasthenia gravis, multiple sclerosis or Sjogren's syndrome, etc.

[0187] Other examples of PI3K-related diseases include autoimmune hemolytic anemia, polycythemia vera, and pemphigus.

[0188] In some embodiments, the disease is non-Hodgkin lymphoma (NHL). In some embodiments, the non-Hodgkin lymphoma (NHL) is relapsed or refractory NHL or relapsed follicular NHL.

[0189] In some embodiments, the disease is an aggressive lymphoma (e.g., germinal center B-cell-like (GCB) or activated B-cell-like (ABC)).

[0190] In some embodiments, the disease is selected from diffuse large B-cell lymphoma, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, hairy cell leukemia, mantle cell lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma, Hodgkin lymphoma, Burkitt lymphoma, Waldenstrom's macroglobulinemia, prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis, mucosa-associated lymphoid tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphomatoid granulomatosis, splenic marginal zone lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoldering myeloma (also known as asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL), and germinal center B-cell (GCB) diffuse large B-cell lymphoma (GCB-DLBCL).

[0191] In some embodiments, the disease is myelofibrosis. In some embodiments, the myelofibrosis is selected from post-polycythemia vera myelofibrosis (PET-MF), primary myelofibrosis (PMF), and post-polycythemia vera myelofibrosis (PPV-MF). In some embodiments, the myelofibrosis is post-polycythemia vera myelofibrosis (PET-MF). In some embodiments, the myelofibrosis is primary myelofibrosis (PMF). In some embodiments, the myelofibrosis is post-polycythemia vera myelofibrosis (PPV-MF).

[0192] In some embodiments, the disease is acute myeloid leukemia. In some embodiments, the disease is Burkitt lymphoma. In some embodiments, the disease is activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL) and germinal center B-cell (GCB) diffuse large B-cell lymphoma (GCB-DLBCL).

[0193] In some embodiments, the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM).

[0194] In some embodiments, marginal zone lymphoma (MZL) is selected from extranodal MZL, nodular MZL, splenic MZL, and an unknown MZL subtype.

[0195] In some embodiments, Hodgkin lymphoma (HL) is selected from classical Hodgkin lymphoma (HL) and nodular lymphocyte-predominant HL.

[0196] In some embodiments, diffuse large B-cell lymphoma is selected from activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL) and germinal center B-cell (GCB) diffuse large B-cell lymphoma (GCB-DLBCL).

[0197] In some embodiments, the disease is selected from autoimmune hemolytic anemia, polycythemia vera, and pemphigus.

[0198] In some embodiments, the present application also provides a method for treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), and the method comprises:

[0199] i) administering to the patient a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day to about 50 mg / day for a first period of about 8 weeks to about 9 weeks; and

[0200] ii) administering to the patient a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride after the first period, the second dose being about 2.5 mg / day or less.

[0201] In some embodiments, the present application also provides a method for treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), and the method comprises:

[0202] i) administering to the patient a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day to about 50 mg / day for a first period of about 8 weeks to about 9 weeks; and

[0203] ii) administering to the patient a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / week to about 50 mg / week during a second period that occurs after the first period.

[0204] In some embodiments, the present application also provides a method for treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), and the method comprises:

[0205] i) administering to the patient a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day for a first period of about 8 weeks; and

[0206] ii) administering to the patient a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 2.5 mg / day during a second period that occurs after the first period.

[0207] In some embodiments, the present application also provides a method for treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), and the method comprises:

[0208] i) administering to the patient a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day for a first period of about 8 weeks; and

[0209] ii) administering to the patient a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / week during a second period that occurs after the first period.

[0210] As used herein, the terms "individual" or "patient" are used interchangeably and refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, and most preferably humans.

[0211] As used herein, the phrase "therapeutically effective amount" refers to the amount of an active compound or agent that elicits a biological or medical response sought by a researcher, veterinarian, physician, or other clinician in a tissue, system, animal, individual, or human.

[0212] As used herein, the term "treating" or "treatment" refers to one or more of the following: (1) preventing a disease; e.g., preventing a disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder but has not yet experienced or manifested the pathology or symptomatology of the disease; (2) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder in an individual who is experiencing or manifesting the pathology or symptomatology of the disease, condition, or disorder (i.e., arresting further development of the pathology and / or symptomatology); and (3) ameliorating a disease; e.g., ameliorating a disease, condition, or disorder in an individual who is experiencing or manifesting the pathology or symptomatology of the disease, condition, or disorder (i.e., reversing the pathology and / or symptomatology), such as reducing the severity of the disease. In some embodiments, the term "treating" or "treatment" refers to inhibiting or ameliorating a disease.

[0213] As used herein, "QD" is intended to mean a dose administered once daily to a subject. "QW" is intended to mean a dose administered once weekly to a subject. "Q2W" is intended to mean a dose administered once every other week to a subject. "Q3W" is intended to mean a dose administered once every three weeks to a subject. "Q4W" is intended to mean a dose administered once every four weeks to a subject.

[0214] As used herein, "about" when referring to a measurable value such as an amount, a dose, a duration, etc., is intended to cover deviations of ±10%. In certain embodiments, "about" may include deviations of ±5%, ±1%, or ±0.1% from the specified value, as well as any deviation therebetween, as such deviations are suitable for performing the disclosed methods.

[0215] Combination therapy

[0216] I. Immune Checkpoint Therapy

[0217] In some embodiments, the PI3K inhibitors provided herein can be used in combination with one or more immune checkpoint inhibitors to treat cancer as described herein. In one embodiment, the combination with one or more immune checkpoint inhibitors as described herein can be used to treat B cell malignancies described herein. The compounds of the present disclosure can be used in combination with one or more immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include inhibitors of immune checkpoint molecules such as: CD20, CD28, CD40, CD122, CD96, CD73, CD47, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, HPK1, CD137 (also known as 4-1BB), ICOS, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, TIGIT, PD-1, PD-L1, and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from: CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from: A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, TIGIT, and VISTA. In some embodiments, the compounds of the present disclosure provided herein can be used in combination with one or more agents selected from: KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFRβ inhibitors.

[0218] In some embodiments, the PI3K inhibitors provided herein can be used in combination with an agonist of one or more of the following immune checkpoint molecules: for example, OX40, CD27, OX40, GITR, and CD137 (also known as 4-1BB).

[0219] In some embodiments, the inhibitor of the immune checkpoint molecule is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0220] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-1, such as an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab (also known as MK-3475), durvalumab pidilizumab, SHR-1210, PDR001, MGA012, PDR001, AB122, or AMP-224. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 monoclonal antibody is MGA012. In some embodiments, the anti-PD1 antibody is SHR-1210. Other anti-cancer agents include antibody therapeutics, such as 4-1BB (e.g., urelumab, utomilumab).

[0221] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-L1, such as an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI4736.

[0222] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-1 and PD-L1, such as an anti-PD-1 / PD-L1 monoclonal antibody. In some embodiments, the anti-PD-1 / PD-L1 is MCLA-136.

[0223] In some embodiments, the inhibitor is MCLA-145.

[0224] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, AGEN1884 or CP-675,206.

[0225] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of LAG3, such as an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525 or INCAGN2385.

[0226] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of TIM3, such as an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is INCAGN2390, MBG453 or TSR-022.

[0227] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of GITR, such as an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518, MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323 or MEDI1873.

[0228] In some embodiments, the inhibitor of the immune checkpoint molecule is an agonist of OX40, such as an OX40 agonist antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is MEDI0562, MOXR-0916, PF-04518600, GSK3174998 or BMS-986178. In some embodiments, the OX40L fusion protein is MEDI6383.

[0229] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CD20, such as an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.

[0230] The compounds of the present disclosure can be used in combination with bispecific antibodies. In some embodiments, one domain of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3 or TGFβ receptor.

[0231] In some embodiments, the compounds of the present disclosure can be used in combination with one or more metabolic enzyme inhibitors. In some embodiments, the metabolic enzyme inhibitor is an inhibitor of IDO1, TDO, or arginase. Examples of IDO1 inhibitors include epacadostat, NLG919, BMS-986205, PF-06840003, IOM2983, RG-70099, and LY338196.

[0232] As provided throughout, additional compounds, inhibitors, agents, etc. can be combined with the compounds of the invention in single or sequential dosage forms, or they can be administered simultaneously or sequentially as separate dosage forms.

[0233] II. Cancer Therapy

[0234] The growth and survival of cancer cells can be affected by multiple signaling pathways. Thus, it is useful to combine different enzyme / protein / receptor inhibitors that exhibit different preferences for their regulatory activity targets in order to treat such conditions. Targeting more than one signaling pathway (or more than one biomolecule involved in a given signaling pathway) can reduce the likelihood of the emergence of drug resistance in a cell population and / or reduce the toxicity of the treatment.

[0235] The compounds of the present disclosure can be used in combination with one or more other enzyme / protein / receptor inhibitors or one or more therapies for treating diseases such as cancer. Examples of diseases and indications treatable by combination therapy include those described herein.

[0236] One or more additional pharmaceutical or therapeutic agents such as chemotherapeutic agents, immuno-oncology agents, metabolic enzyme inhibitors, chemokine receptor inhibitors, and phosphatase inhibitors, and targeted therapies such as Bcr-Abl, Flt-3, EGFR, HER2, JAK, c-MET, VEGFR, PDGFR, c-Kit, IGF-1R, RAF, and FAK kinase inhibitors. One or more additional agents can be administered to a patient simultaneously or sequentially.

[0237] Exemplary antibodies for combination therapy include, but are not limited to, Trastuzumab (e.g., anti-HER2), Ranibizumab (e.g., anti-VEGF-A), Bevacizumab (trade name Avastin, e.g., anti-VEGF), Panitumumab (e.g., anti-EGFR), Cetuximab (e.g., anti-EGFR), Rituxan (anti-CD20), and antibodies against c-MET.

[0238] One or more of the following agents can be used in combination with the compounds of the present disclosure and are provided as a non-limiting list: cell growth inhibitors, Taxotere, paclitaxel, Camptostar, epothilones, 5-fluorouracil, SCH66336, R115777, L778,123, BMS214662, IRESSA TM (gefitinib), TARCEVA TM (erlotinib), antibodies to EGFR, GLEEVEC TM (imatinib mesylate), interferon, cytarabine, doxorubicin, cyclophosphamide, nitrogen mustard, triethylenemelamine, thiotepa, busulfan, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, ELOXATIN TM (oxaliplatin), vindesine, mithramycin, deoxycoformycin, L-asparaginase, 17α-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, medroxyprogesterone, leuprolide, flutamide, goserelin, hydroxyurea, amsacrine, navelbene, raloxifene, droloxifene, hexamethylmelamine, acrivastine, HERCEPTIN TM (trastuzumab), BEXXAR TM (tositumomab), VELCADE TM (bortezomib), ZEVALIN TM (ibritumomab tiuxetan), TRISENOX TM (arsenic trioxide), XELODA TM (capecitabine), vinorelbine, porfimer, ERBITUX TM (cetuximab), aphidicolon, rituxan, Sml1, triapine, didox, trimidox, amidox, 3-AP and MDL-101,731.

[0239] The compounds of the present disclosure can also be used in combination with the following other methods for treating cancer: for example, chemotherapy, radiotherapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery. Examples of immunotherapy include cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, adoptive T cell transfer, Toll receptor agonists, STING agonists, oncolytic virus therapy, and immunomodulatory small molecules, including thalidomide or JAK1 / 2 inhibitors, etc. The compounds can be administered in combination with one or more anti-cancer drugs, such as chemotherapeutic agents. Exemplary chemotherapeutic agents include any of the following: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin-diphtheria toxin conjugate, denileukindiftitox), dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, mechlorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C(mitomycinC), Mitotane, Mitoxantrone, Nandrolone Phenpropionate, Nelarabine, Nofetumomab, Olaparib, Oxaliplatin, Paclitaxel, Pamidronate, Panitumumab, Pegaspargase, Pegfilgrastim, Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, Procarbazine, Quinacrine, Rasburicase, Rituximab, Ruxolitinib, Rucaparib, Sorafenib, Streptozocin, Sunitinib, Sunitinib Maleate, Tamoxifen, Temozolomide, Teniposide, Testolactone, Thalidomide, Thioguanine, Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine, Vorinostat, Niraparib, Veliparib, Talazoparib, and Zoledronate.

[0240] Additional examples of chemotherapeutic agents include proteasome inhibitors (e.g., bortezomib), thalidomide, lenalidomide; and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, etc.

[0241] Exemplary Bcr-Abl inhibitors include compounds and pharmaceutically acceptable salts.

[0242] Exemplary suitable Flt-3 inhibitors include compounds and their pharmaceutically acceptable salts.

[0243] Exemplary suitable RAF inhibitors include compounds and their pharmaceutically acceptable salts.

[0244] Exemplary suitable FAK inhibitors include compounds and their pharmaceutically acceptable salts.

[0245] In some embodiments, the compounds of the present disclosure can be used in combination with one or more other kinase inhibitors including imatinib, particularly for treating patients resistant to imatinib or other kinase inhibitors.

[0246] The compounds can be used in combination with tumor vaccines and CAR (chimeric antigen receptor) T cell therapy as a T cell activation enhancer. In some embodiments, the tumor vaccine includes proteins from viruses associated with the following human cancers: such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpesvirus (KHSV). In some embodiments, the compounds of the present disclosure can be used in combination with tumor-specific antigens such as heat shock proteins isolated from the tumor tissue itself. In some embodiments, the compounds can be combined with dendritic cell immunization to activate an effective anti-tumor response.

[0247] Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. Additionally, their administration is described in standard literature. For example, the administration of various chemotherapeutic agents is described in "Physicians' Desk Reference" (PDR, e.g., the 1996 version, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if set forth in full.

[0248] In some embodiments, the additional therapeutic agents are selected from JAK inhibitors (e.g., selective JAK1 inhibitors), pan-PIM selective kinase inhibitors, inhibitors of epigenetic regulators, and immunotherapeutic agents.

[0249] In some embodiments, the inhibitors of epigenetic regulators are selected from bromodomain and extraterminal domain inhibitors and lysine-specific histone demethylase 1A inhibitors.

[0250] In some embodiments, the additional therapeutic agent is an immunotherapeutic agent.

[0251] In some embodiments, the immunotherapeutic agent is R-ICE.

[0252] In some embodiments, the additional therapeutic agent is a JAK inhibitor.

[0253] In some embodiments, the JAK inhibitor is selective for JAK1 and JAK2 relative to JAK3 and TYK2. In some embodiments, the JAK inhibitor is selective for JAK1 relative to JAK2, JAK3, and TYK2. For example, the JAK inhibitor may preferentially inhibit JAK1 relative to one or more of JAK2, JAK3, and TYK2. In some embodiments, the JAK inhibitor preferentially inhibits JAK1 relative to JAK2 (e.g., having a JAK1 / JAK2 IC 50 ratio > 1). In some embodiments, the JAK inhibitor is 10-fold more selective for JAK1 than for JAK2. In some embodiments, the JAK inhibitor is approximately 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold more selective for JAK1 than for JAK2, as calculated by measuring the IC 50 as described in Assay A of U.S. Patent Application 14 / 680,659, filed Apr. 7, 2015, the disclosure of which is incorporated herein by reference in its entirety.

[0254] In some embodiments, the additional therapeutic agent is a JAK1 / JAK2 inhibitor. In some embodiments, the additional therapeutic agent is ruxolitinib (e.g., ruxolitinib phosphate).

[0255] In some embodiments, the additional therapeutic agent is a JAK inhibitor selected from the group provided in the table below. The compounds provided in the table are selective JAK1 inhibitors (selective relative to JAK2, JAK3, and TYK2). The IC 50 is shown in Table A.

[0256] Table A.

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] + means < 10 nM (for assay conditions, see U.S. Patent Application 14 / 680,659, Example A)

[0266] ++ means ≤ 100 nM (for assay conditions, see U.S. Patent Application 14 / 680,659, Example A)

[0267] +++ means ≤ 300 nM (for assay conditions, see U.S. Patent Application 14 / 680,659, Example A)

[0268] a Data for Enantiomer 1

[0269] b Data for Enantiomer 2

[0270] In some embodiments, the JAK inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile (i.e., itacitinib) or a pharmaceutically acceptable salt thereof.

[0271] In some embodiments, the JAK inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile hexanedioate.

[0272] In some embodiments, the JAK inhibitor is 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof.

[0273] In some embodiments, the JAK inhibitor is selected from (R)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, (R)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, (R)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile, (R)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile, or (R)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile, (S)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, (S)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, (S)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile, (S)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile, (S)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile; and pharmaceutically acceptable salts of any of the foregoing.

[0274] In some embodiments, the compounds of Table A are prepared by the synthetic procedures described in the following patent publications: U.S. Patent Publication 2010 / 0298334, filed May 21, 2010; U.S. Patent Publication 2011 / 0059951, filed Aug. 31, 2010; U.S. Patent Publication 2011 / 0224190, filed Mar. 9, 2011; U.S. Patent Publication 2012 / 0149681, filed Nov. 18, 2011; U.S. Patent Publication 2012 / 0149682, filed Nov. 18, 2011; U.S. Patent Publication 2013 / 0018034, filed Jun. 19, 2012; U.S. Patent Publication 2013 / 0045963, filed Aug. 17, 2012; and U.S. Patent Publication 2014 / 0005166, filed May 17, 2013, each of which is incorporated herein by reference in its entirety.

[0275] In some embodiments, the JAK inhibitor is selected from the compounds of the following patent publications: U.S. Patent Publication 2010 / 0298334, filed May 21, 2010; U.S. Patent Publication 2011 / 0059951, filed Aug. 31, 2010; U.S. Patent Publication 2011 / 0224190, filed Mar. 9, 2011; U.S. Patent Publication 2012 / 0149681, filed Nov. 18, 2011; U.S. Patent Publication 2012 / 0149682, filed Nov. 18, 2011; U.S. Patent Publication 2013 / 0018034, filed Jun. 19, 2012; U.S. Patent Publication 2013 / 0045963, filed Aug. 17, 2012; and U.S. Patent Publication 2014 / 0005166, filed May 17, 2013, each of which is incorporated herein by reference in its entirety.

[0276] In some embodiments, the PI3Kδ inhibitor and the additional therapeutic agent are administered simultaneously.

[0277] In some embodiments, the PI3Kδ inhibitor and the additional therapeutic agent are administered sequentially.

[0278] Pharmaceutical formulation

[0279] When used as a medicine, the compounds used in the methods of the present disclosure can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the medical field and, depending on whether local treatment or systemic treatment is desired and the area to be treated, can be administered by a variety of routes. Administration can be local (including transdermal, epidermal, ophthalmic, and to mucous membranes, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intracerebroventricular administration. Parenteral administration can be in the form of a single bolus dose or can be achieved, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for local administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickening agents, etc. may be necessary or desirable.

[0280] The present disclosure also includes pharmaceutical compositions that contain a compound of the present disclosure or a pharmaceutically acceptable salt thereof as an active ingredient, in combination with one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the composition is suitable for local administration. In preparing the compositions of the present disclosure, the active ingredient is usually admixed with an excipient, diluted with an excipient, or enclosed within a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0281] In preparing the formulation, the active compound can be milled to provide an appropriate particle size before being combined with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water-soluble, the particle size can be adjusted by milling so that it is substantially uniformly distributed in the formulation, e.g., about 40 mesh.

[0282] The compounds of the present disclosure can be milled using known milling procedures, such as wet milling, to obtain a particle size suitable for tablet formation and other formulation types. Fine dispersion (nanoparticle) formulations of the compounds of the present disclosure can be prepared by processes known in the art, see, for example, International Application WO 2002 / 000196.

[0283] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations may additionally include: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl hydroxybenzoate and propyl hydroxybenzoate; sweetening agents; and flavoring agents. The compositions of the present disclosure may be formulated by procedures known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a patient.

[0284] The compositions may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as a whole dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material combined with a suitable pharmaceutical excipient, the quantity being calculated to produce the desired therapeutic effect.

[0285] To prepare solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of the compounds of the present disclosure. When these preformulation compositions are referred to as homogeneous, the active ingredient is generally uniformly dispersed throughout the composition such that the composition can be readily redivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation is then redivided into the above-described type of unit dosage forms.

[0286] The tablets or pills of the present disclosure may be coated or otherwise compounded to provide a dosage form having the advantage of extended action. For example, the tablet or pill may contain an inner dose and an outer dose component, the latter being in the form of an envelope overcoating the former. The two components may be separated by an enteric layer adapted to resist disintegration in the stomach and permit the complete passage of the inner component into the duodenum or to provide delayed release. A variety of materials may be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0287] Liquid forms in which the compounds and compositions of the present disclosure may be incorporated for oral or parenteral administration include aqueous solutions, properly flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0288] Compositions for inhalation or insufflation include solutions and suspensions, or mixtures thereof, in pharmaceutically acceptable aqueous or organic solvents, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route to achieve local or systemic effects. The compositions can be atomized using an inert gas. The atomized solution can be breathed directly from the atomizing device, or the atomizing device can be connected to a face mask, tent, or intermittent positive pressure breathing machine. The solution, suspension, or powder composition can be administered orally or nasally from a device that delivers the formulation in a suitable manner.

[0289] Topical formulations may contain one or more conventional carriers. In some embodiments, an ointment may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ethers, propylene glycol, white Vaseline, and the like. The carrier composition of a cream may be based on a combination of water with glycerol and one or more other components (e.g., glycerol monostearate, PEG-glycerol monostearate, and cetostearyl alcohol). A gel can be formulated using isopropyl alcohol and water, suitably in combination with other components such as glycerol, hydroxyethyl cellulose, and the like. In some embodiments, the topical formulation contains at least about 0.1 wt%, at least about 0.25 wt%, at least about 0.5 wt%, at least about 1 wt%, at least about 2 wt%, or at least about 5 wt% of a compound of the present disclosure. The topical formulation can be suitably encapsulated, for example, in a 100 g tube, which is optionally associated with instructions for treating a selected indication (e.g., psoriasis or other skin conditions).

[0290] The amount of the compound or composition administered to a patient will vary depending on the administered substance, the purpose of administration (such as prophylaxis or therapy), the patient's condition, the mode of administration, and the like. In a therapeutic application, the composition is administered to a patient suffering from the disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dose will depend on the disease condition being treated and the judgment of the attending clinician based on factors such as the severity of the disease, the age, weight, and general condition of the patient.

[0291] The composition administered to a patient can be in the form of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or can be sterile filtered. An aqueous solution can be encapsulated for use as is or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier before administration. The pH of the compound formulation will generally be between 3 and 11, more preferably 5 to 9, and most preferably 7 to 8. It should be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

[0292] The therapeutic dosage of the compounds of the present disclosure can vary depending on, for example, the particular use for which the treatment is to be achieved, the mode of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compounds of the present disclosure in the pharmaceutical composition can vary depending on a number of factors, including the dosage, chemical characteristics (e.g., hydrophobicity), and route of administration. For example, the compounds of the present disclosure can be provided in a physiologically buffered aqueous solution containing from about 0.1 w / v% (weight / volume %) to about 10 w / v% of the compound for parenteral administration.

[0293] The compositions of the present disclosure can also contain one or more additional agents, such as chemotherapeutic agents, steroids, anti-inflammatory compounds, or immunosuppressive agents, examples of which are listed herein.

[0294] Labeled compounds and assay methods

[0295] The methods of the present disclosure also include the use of isotopically labeled compounds of the present disclosure. An "isotope" or "radioactively labeled" compound is a compound of the present disclosure in which one or more atoms are replaced or substituted with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature (i.e., naturally occurring). Suitable radionuclides that can be incorporated into the compounds of the present disclosure include, but are not limited to 2 H (also written as D, i.e., deuterium), 3 H (also written as T, i.e., tritium), 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I and 131 I. For example, one or more hydrogen atoms in the compounds of the present disclosure can be replaced with deuterium atoms (e.g., one or more hydrogen atoms of the alkyl groups of the compounds described herein can be optionally replaced with deuterium atoms, such as -CD 3 substituted for -CH 3 ).

[0296] One or more of the constituent atoms of the compounds provided herein may be replaced or substituted with isotopes of said atoms at natural or non-natural abundances. In some embodiments, the compound includes at least one deuterium atom. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of the hydrogen atoms in the compound may be replaced or substituted with deuterium atoms.

[0297] In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms attached to a carbon atom of the compounds described herein are optionally replaced with deuterium atoms.

[0298] Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry, Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971); The Renaissance of H / D Exchange, Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling, James R. Hanson, Royal Society of Chemistry, 2011). Isotope-labeled compounds can be used in various studies such as NMR spectroscopy, metabolic experiments, and / or assays.

[0299] Substitution with a heavier isotope such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and may therefore be preferred in some cases. (See, e.g., A. Kerekes et al. J. Med. Chem. 2011, 54, 201-210; R. Xu et al. J. Label Compd. Radiopharm. 2015, 58, 308-312). Specifically, substitution at one or more metabolic sites can provide one or more therapeutic advantages.

[0300] The radionuclide incorporated into the radiolabeled compounds of the present invention will depend on the specific application of the radiolabeled compound. For example, for in vitro PI3K labeling and competitive assays, incorporation of 3 H, 14 C, 82Br, 125 I, 131 I or 35 compounds of S can be useful. For radioimaging applications, 11 C, 18 F, 125 I, 123 I, 124 I, 131 I, 75 Br, 76 Br or 77 Br can be useful.

[0301] It should be understood that a "radioactively labeled" or "labeled compound" is a compound that has incorporated at least one radionuclide. In some embodiments, the radionuclide is selected from the group consisting of 3 H, 14 C, 125 I, 35 S and 82 Br.

[0302] The present disclosure may also include synthetic methods for incorporating radioisotopes into the compounds of the present disclosure. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and those of ordinary skill in the art will readily identify methods suitable for the compounds of the present disclosure.

[0303] Kit

[0304] The present disclosure also includes a pharmaceutical kit suitable for, for example, treating or preventing PI3K-related diseases or disorders described herein, which includes one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure. Such a kit may also include (if desired) one or more of various conventional pharmaceutical kit components, such as containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions in the form of inserts or labels may also be included in the kit, which specify the amounts of the components to be administered, administration guidelines, and / or guidelines for mixing the components. In certain embodiments, the instructions include the methods of the present disclosure, which include a specified dose or dosing regimen of the present disclosure.

[0305] The present invention will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results. It should be understood that certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, the various features of the present invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

[0306] Example

[0307] Examples 1A - 1D. Diastereomers of 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one

[0308]

[0309] Step 1. 1-(5-Chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanol

[0310]

[0311] A solution of 1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (20.0 g, 58.4 mmol; see Step 1 of Example 212 of U.S. Patent 9,199,982) and 1,2-ethanediol (6.5 mL, 120 mmol) in toluene (190 mL) was treated with p-toluenesulfonic acid monohydrate (1.1 g, 5.8 mmol). The flask was equipped with a Dean-Stark trap filled with a sieve and refluxed for 3 h. The reaction mixture was cooled and added to ice-cold saturated sodium bicarbonate solution (250 mL) and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated to a crude orange oil. The crude material was purified by flash column chromatography using hexanes containing ethyl acetate (0% - 20%) to give the desired product (22 g, 99%). C 12 H 14 ClFIO 3 LCMS of + : (M + H)

[0312] Step 2. Ethyl (2E)-3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]acrylate

[0313]

[0314] 2-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)-2-methyl-1,3-dioxolane (22 g, 58 mmol) (from Step 1), ethyl (2E)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (16 mL, 70 mmol), and potassium carbonate (24 g, 170 mmol) in a mixture of 1,4-dioxane (230 mL) and water (110 mL) were degassed with nitrogen for 10 minutes. The reaction mixture was treated with [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexed with dichloromethane (1:1) (2.4 g, 2.9 mmol), degassed with nitrogen for another 10 minutes, and heated at 80 °C for 2 h. The reaction mixture was filtered through Celite and washed with ethyl acetate (300 mL). The filtrate was poured into water (400 mL). The aqueous layer was separated and extracted again with ethyl acetate (300 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to a crude brown solid. The crude material was purified by flash column chromatography using hexanes containing ethyl acetate (0%-30%) to give the desired product (20 g, 96%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.74 (d, J = 16.5 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 16.5, 0.9 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 4.10–3.99 (m, 2H), 3.91 (q, J = 7.0 Hz, 2H), 3.87–3.76 (m, 2H), 1.73 (s, 3H), 1.44 (t, J = 7.0 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H). C 17 H 21 ClFO 5 LCMS of + : (M+H)

[0315] Step 3. Ethyl 3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]-4-nitrobutanoate

[0316]

[0317] A solution of ethyl (2E)-3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]acrylate (10 g, 28 mmol) (from Step 2) in nitromethane (100 mL) was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (4.6 mL, 31 mmol) and stirred at 60 °C for 15 h. The reaction mixture was poured into water (400 mL) and extracted with ethyl acetate (2 x 300 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to a crude orange oil. The crude material was purified by flash column chromatography using hexanes containing ethyl acetate (0%-30%) to afford the desired product as a mixture of enantiomers (10.4 g, 89%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.52 (d, J = 9.1 Hz, 1H), 4.82 (ddd, J = 12.5, 7.6, 1.4 Hz, 1H), 4.68 (dd, J = 12.5, 7.2 Hz, 1H), 4.54–4.40 (m, 1H), 4.15–3.90 (m, 6H), 3.89–3.75 (m, 2H), 2.85 (ddd, J = 16.0, 8.6, 1.4 Hz, 1H), 2.73 (dd, J = 16.1, 6.2 Hz, 1H), 1.70 (s, 3H), 1.47 (t, J = 7.0 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H). C 18 H 24 ClFNO 7 LCMS of + : (M+H)

[0318] Step 4. 4-[3-Chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]pyrrolidin-2-one enantiomers

[0319]

[0320] A suspension of ethyl 3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]-4-nitrobutyrate (1.0 g, 2.4 mmol) (from Step 3) in ethanol (16 mL) was warmed to dissolve the solid. The solution was cooled back to ambient temperature, degassed with nitrogen, and treated with a slurry of 2800 Raney Nickel in water (1.5 mL). The reaction mixture was degassed again with nitrogen and hydrogenated with a hydrogen balloon for 3 h. The reaction mixture was filtered through diatomaceous earth and concentrated to give the intermediate amino ester (0.93 g, 100%). The intermediate amino ester was dissolved in toluene (12 mL) and heated at 110 °C for 12 h. The reaction mixture was cooled to ambient temperature, at which point a solid precipitated from the solution. This mixture was cooled to 0 °C, stirred for 30 minutes, filtered, washed with cold toluene, and dried to give the desired product (0.61 g, 75%) as a mixture of enantiomers. C 16 H 20 ClFNO 4 LCMS of: (M+H) + : m / z = 344.1; found: 344.1. The mixture of enantiomers was separated by chiral HPLC to give the individual enantiomers as Peak 1 and Peak 2 (RT = 5.39 minutes and 7.01 minutes respectively; Phenomenex Lux Cellulose C-1, 21.2 x 250 mm, 5 micron particle size, eluted with hexane containing 20% ethanol at 18 mL / min).

[0321] Step 5. Enantiomers of 4-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one

[0322]

[0323] Each of the separated enantiomers from Step 4 was individually processed to the final compound. A solution of 4-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]pyrrolidin-2-one (1.7 g, 5.0 mmol) (from Step 4) in methanol (17 mL) was treated dropwise with water (11 mL, 69 mmol) containing 6.0 M hydrochloric acid and stirred at 20 °C for 30 minutes. The reaction mixture was added dropwise to ice-cold saturated sodium bicarbonate solution (75 ml) and extracted with ethyl acetate (2 x 100 ml). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated to give the desired product [from Peak 1 (1.5 g, 99%); from Peak 2 (1.5 g, 99%)], which was used without further purification. From Peak 1: 1 HNMR (400 MHz, DMSO-d6 ) δ 7.84 (s, 1H), 7.70 (d, J = 8.6 Hz, 1H), 4.16–3.99 (m, 1H), 3.83 (q, J = 7.0 Hz, 2H), 3.65–3.54 (m, 1H), 3.30–3.23 (m, 1H), 2.55 (s, 3H), 2.33 (dd, J = 16.8, 8.4 Hz, 1H), 1.30 (t, J = 7.0 Hz, 3H). C 14 H 16 ClFNO 3 LCMS of + : (M+H) 1 : m / z = 300.1; found: 300.0. From peak 2: 6 H NMR (400 MHz, DMSO-d 14 H 16 ClFNO 3 LCMS of + : (M+H)

[0324] Step 6.4 - Diastereomers of [3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]pyrrolidin-2-one

[0325]

[0326] The enantiomers from Step 5 are each separately processed to the final product. 4-(3-Acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (0.402 g, 1.34 mmol) (from Step 5) in anhydrous methanol (6.7 mL) is treated with sodium borohydride (0.10 g, 2.7 mmol) at 0 °C under a nitrogen atmosphere and stirred at 0 °C for 30 minutes. The reaction mixture is quenched with water at 0 °C and poured into water (50 mL) / ethyl acetate (100 mL) with stirring. The mixture is warmed to ambient temperature and the aqueous layer is separated and extracted again with ethyl acetate (50 mL). The combined organic extracts are washed with brine, dried over sodium sulfate, filtered, and concentrated to give a white foam. The crude material is purified by flash column chromatography using dichloromethane (0%-100%) containing acetonitrile (containing 7% methanol) to give the desired product in the form of a mixture of diastereomers [from peak 1 (0.40 g, 99%); from peak 2 (0.40 g, 99%)]. From peak 1: C 14 H 18 ClFNO 3 LCMS of: (M+H) + : m / z = 302.1; found: 302.0. From peak 2: C 14 H 18 ClFNO 3 LCMS of: (M+H) + : m / z = 302.1; found: 302.1.

[0327] Step 7. Diastereomers of 4-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-fluorophenyl]pyrrolidin-2-one

[0328]

[0329] The mixture of diastereomers from Step 6 is each separately processed to the final product. A solution of 4-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]pyrrolidin-2-one (0.41 g, 1.4 mmol) (from Step 6) in dichloromethane (12 mL) is treated dropwise successively with N,N-dimethylformamide (0.011 mL, 0.14 mmol) and thionyl chloride (0.21 mL, 2.9 mmol) and stirred at 20 °C for 30 minutes. The reaction mixture is added dropwise to ice-cold saturated sodium bicarbonate solution and extracted with dichloromethane. The organic layer is separated and washed with brine, dried over sodium sulfate, filtered, and concentrated to give the desired product [from peak 1 (0.38 g, 87%); from peak 2 (0.39 g, 89%)] and 17 - 18% styrene formed by elimination of the chloride. These mixtures are used without further purification. From peak 1: C14 H 17 Cl 2 FNO 2 LCMS of + : m / z = 320.1; Found: 320.0. From peak 2: C 14 H 17 Cl 2 FNO 2 LCMS of + : m / z = 320.1; Found: 320.0.

[0330] Step 8.4 - Diastereomers of {3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one

[0331]

[0332] The mixtures of diastereomers from step 7 were each separately processed into the final product. A mixture of 4-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-fluorophenyl]pyrrolidin-2-one (0.36 g, 1.1 mmol) (from step 7), 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.19 g, 1.3 mmol), cesium carbonate (0.54 g, 1.7 mmol), and potassium iodide (18 mg, 0.11 mmol) in N,N-dimethylformamide (7.4 mL) was heated at 100 °C for 4.5 h. The reaction mixture was poured into water (30 mL) and extracted with ethyl acetate (3 x 50 mL) to give a mixture of diastereomers ((S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; (S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; and (R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one). The mixture of diastereomers was purified by preparative LCMS (XBridge C18 column, eluted with a gradient of acetonitrile / water containing 0.1% ammonium hydroxide at a flow rate of 60 mL / min) to give the desired products [peak A (Example 1A) (0.13 g, 54%) and peak B (Example 1B) (0.11 g, 46%) were isolated from peak 1; peak A (Example 1C) (0.15 g, 63%) and peak B (Example 1D) (0.14 g, 55%) were isolated from peak 2].

[0333] Example 1B: 1 H NMR (300 MHz, DMSO-d 6)δ 8.12 (s, 1H), 7.82 (s, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.30 (br s, 1H), 6.23 (q, J = 7.0 Hz, 1H), 4.05–3.90 (m, 1H), 3.88–3.78 (m, 2H), 3.63–3.53 (m, 1H), 3.29–3.20 (m, 1H), 2.54 (s, 3H), 2.38–2.21 (m, 1H), 1.70 (d, J = 7.1 Hz, 3H), 1.39 (t, J = 6.9 Hz, 3H). C 20 H 23 ClFN 6 O 2 The LCMS of + : m / z = 433.2; Found: 433.1. Example 1C: 1 1H NMR (500 MHz, DMSO-d 6 )δ 8.12 (s, 1H), 7.77 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.26 (br s, 2H), 6.24 (q, J = 7.0 Hz, 1H), 4.04–3.94 (m, 1H), 3.93–3.85 (m, 1H), 3.84–3.77 (m, 1H), 3.61–3.53 (m, 1H), 3.27–3.22 (m, 1H), 2.54 (s, 3H), 2.30 (dd, J = 18.1, 8.6 Hz, 1H), 1.71 (d, J = 7.1 Hz, 3H), 1.40 (t, J = 6.9 Hz, 3H). C 20 H 23 ClFN 6 O 2 The LCMS of + : m / z = 433.2; Found: 433.1.

[0334] Examples 2A - 2D. Diastereomers of 5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one

[0335]

[0336] Step 1: tert-Butyl [2-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-hydroxyethyl]carbamate

[0337]

[0338] An aqueous solution of 0.2 M osmium tetroxide (10 mL) was added to a solution of tert-butyl [(4-chlorobenzoyl)oxy]carbamate (Lawrence Harris, J. Org. Chem, 2011, 76, 358 - 372) (19 g, 70 mmol) in acetonitrile (210 mL) and stirred for 10 minutes. A solution of 1-(5-chloro-2-ethoxy-4-fluoro-3-vinylphenyl)ethanone (11.2 g, 46 mmol) (see Example 353, Step 1 of U.S. Patent 9,199,982) in the form of a solution in acetonitrile (210 mL) was added to the carbamate solution, followed by the addition of water (50 mL) and the reaction was stirred at room temperature for 3 hours. The reaction was quenched with saturated 10 M aqueous potassium metabisulfite solution (240 mL) and stirred for 5 minutes. Water was added and the reaction mixture was extracted with ethyl acetate. The extract was washed with saturated sodium bicarbonate solution, brine and dried over sodium sulfate, filtered and evaporated. Purification on silica gel using hexane containing ethyl acetate (0 - 100%) gave the desired compound as a racemic mixture, 16.6 g, 95%. C 17 H 23 ClFNO 5 Calculated LCMS for Na: (M + Na) + : m / z = 398.1; Found: 398.0.

[0339] Step 2: 5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one

[0340]

[0341] The desired single enantiomer (peak 3) was prepared using the same procedure as in Example 353, Steps 8 - 12 of U.S. Patent 9,199,982, with the exception that the intermediate from Step 1 in this example was a racemate and thus the final separation of the four diastereomers was carried out in Step 12. Chiral purification was carried out on Phenomenex Lux Cellulose C-4, 21 x 250 mm (Chiral Technologies, 5 micron particle size) using hexane containing 30% ethanol at a flow rate of 18 mL / min to give Peak 1: Example 2A (single enantiomer) (retention time = 12.7 minutes), Peak 2: Example 2B (single enantiomer) (retention time = 14.2 minutes), Peak 3: Example 2C (single enantiomer) (retention time = 20.3 minutes), and Peak 4: Example 2D (single enantiomer) (retention time = 28.9 minutes); the enantiomer with the greatest activity was Peak 3. C 19H 21 ClFN 6 O 3 LCMS calculated value for: (M+H) + : m / z = 435.1; Observed value: 435.1. 1 H NMR (500 MHz, DMSO-d 6 ): δ 8.15 (s, 1H), 7.81 (s, 1H), 7.71 (d, 1H), 7.26 (bs, 1H), 6.23 (m, 1H), 5.84 (t, 1H), 3.92 (m, 1H), 3.83 (m, 1H), 2.52 (s, 3H), 1.75 (d, 3H), 1.40 (m, 3H).

[0342] Example A1: PI3K Enzyme Assay

[0343] The PI3 kinase luminescence assay kit was purchased from Echelon Biosciences (Salt Lake City, UT). The kit includes the lipid kinase substrate D-myo-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), D(+)-sn-1,2-di-O-octanoyl glycerol-3-O-phosphate-linked (PIP2), biotinylated I(1,3,4,5)P4, and PI(3,4,5)P3 detection protein. AlphaScreen including donor beads and acceptor beads TM The GST detection kit was purchased from PerkinElmer LifeSciences (Waltham, MA). PI3Kδ (p110δ / p85α) was purchased from Millipore (Bedford, MA). ATP, MgCl 2 , DTT, EDTA, HEPES, and CHAPS were purchased from Sigma-Aldrich (St. Louis, MO).

[0344] AlphaScreen of PI3Kδ TM Assay

[0345] The kinase reaction was carried out in a 384-well REMP plate from Thermo Fisher Scientific with a final volume of 40 μL. The inhibitor was first serially diluted in DMSO and added to the plate wells, followed by the addition of other reaction components. The final concentration of DMSO in the assay was 2%. At room temperature, in 50 mM HEPES (pH 7.4), 5 mM MgCl 2, PI3K assays were performed in 50 mM NaCl, 5 mM DTT, and 0.04% CHAPS. The reaction was initiated by adding ATP, and the final reaction mixture consisting of 20 μM PIP2, 20 μM ATP, and 1.2 nM PI3Kδ was incubated for 20 minutes. Then, 10 μL of the reaction mixture was transferred to 5 μL of 50 nM biotinylated I(1,3,4,5)P4 in the following quenching buffer: 50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 5 mM DTT, 0.1% Tween-20, followed by the addition of 10 μL of AlphaScreen suspended in the quenching buffer containing 25 nM PI(3,4,5)P3 detection protein. TM Donor beads and acceptor beads. The final concentration of both donor beads and acceptor beads was 20 mg / ml. After sealing the plate, the plate was incubated at room temperature in a dark location for 2 hours. Product activity was determined on a Fusion-α microplate reader (Perkin-Elmer). IC was performed by fitting a curve of the percentage of control activity relative to the logarithm of the inhibitor concentration using GraphPad Prism 3.0 software. 50 Determination.

[0346] Example A2: PI3K Enzyme Assay

[0347] Materials

[0348] The lipid kinase substrate phosphatidylinositol-4,5-bisphosphate (PIP2) was purchased from Echelon Biosciences (Salt Lake City, UT). PI3K isoforms α, β, δ, and γ were purchased from Millipore (Bedford, MA). ATP, MgCl 2 , DTT, EDTA, MOPS, and CHAPS were purchased from Sigma–Aldrich (St. Louis, MO).

[0349] Kinase reactions were performed in a final volume of 24 μL in clear-bottom 96-well plates from Thermo Fisher Scientific. Inhibitors were first serially diluted in DMSO and added to the plate wells, followed by the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. At room temperature, PI3K assays were performed in 20 mM MOPS (pH 6.7), 10 mM MgCl 2 , 5 mM DTT, and 0.03% CHAPS. Reaction mixtures containing 50 μM PIP2, kinase, and different concentrations of inhibitor were prepared. By adding a reaction mixture containing 2.2 μCi [γ- 33P]ATP was used to initiate the reaction at a final concentration of 1000 μM. The final concentrations of PI3K isoforms α, β, δ, and γ in the assay were 1.3, 9.4, 2.9, and 10.8 nM, respectively. The reaction was incubated for 180 minutes and terminated by adding 100 μL of 1 M potassium phosphate (pH 8.0), 30 mM EDTA quenching buffer. Subsequently, 100 μL aliquots of the reaction solution were transferred to 96-well Millipore MultiScreen IP 0.45 μm PVDF filter plates (the filter plates were pre-wetted with 200 μL of 100% ethanol, distilled water, and 1 M potassium phosphate (pH 8.0), respectively). The filter plates were aspirated under vacuum on a Millipore Manifold and washed with 18 × 200 μL of wash buffer containing 1 M potassium phosphate (pH 8.0) and 1 mM ATP. After drying by aspiration and blotting, the plates were air-dried overnight at 37 °C in an incubator. Subsequently, a Packard TopCount adapter (Millipore) was attached to the plates, and then 120 μL of Microscint 20 scintillation cocktail (PerkinElmer) was added to each well. After sealing the plates, the radioactivity of the product was determined by scintillation counting on a Topcount (Perkin-Elmer). The IC 50 was determined.

[0350] Example A3: PI3Kδ Scintillation Proximity Assay

[0351] Materials

[0352] [γ- 33 P]ATP (10 mCi / mL) was purchased from Perkin–Elmer (Waltham, MA). The lipid kinase substrate D-myo-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) D(+)-sn-1,2-di-O-octanoyl glycerol, 3-O-phosphate-linked (PIP2) (CAS 204858-53-7) was purchased from Echelon Biosciences (Salt Lake City, UT). PI3Kδ (p110δ / p85α) was purchased from Millipore (Bedford, MA). ATP, MgCl 2, DTT, EDTA, MOPS, and CHAPS were purchased from Sigma–Aldrich (St. Louis, MO). Wheat Germ Agglutinin (WGA) YSi SPA scintillation beads were purchased from GE healthcare life sciences (Piscataway, NJ).

[0353] The kinase reaction was carried out in a final volume of 25 μL in a polystyrene 384-well matrix white plate from Thermo Fisher Scientific. The inhibitors were first serially diluted in DMSO and added to the plate wells, followed by the addition of the other reaction components. The final concentration of DMSO in the assay was 0.5%. At room temperature, the PI3K assay was carried out in 20 mM MOPS (pH 6.7), 10 mM MgCl 2 , 5 mM DTT, and 0.03% CHAPS. The reaction was initiated by the addition of ATP, and the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ- 33 P]ATP, and 4 nM PI3Kδ. The reactants were incubated for 210 minutes and terminated by the addition of 40 μL of SPA beads suspended in the following quenching buffer: 150 mM potassium phosphate (pH 8.0), 20% glycerol, 25 mM EDTA, 400 μM ATP. The final concentration of the SPA beads was 1.0 mg / mL. After sealing the plate, the plate was shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, and the radioactivity of the product was determined by scintillation counting on a Topcount (Perkin-Elmer). The IC 50 was determined by fitting a curve of the percentage of control activity relative to the logarithm of the inhibitor concentration using GraphPad Prism 3.0 software. As determined by Assay A, the IC 50 data of the examples are given in Table B.

[0354] Table B.

[0355] Example Number <![CDATA[PI3KδSPA IC 50 (nM)*]]> 1A +++ 1B + 1C + 1D +++ 2A +++ 2B +++ 2C + 2D +++++

[0356] *Symbols in Column:

[0357] + indicates ≤10 nM

[0358] ++ indicates >10 nM to 50 nM

[0359] +++ indicates >50 nM to 200 nM

[0360] ++++ indicates >200 nM to 500 nM

[0361] +++++ indicates > 500 nM

[0362] Example B1: B cell proliferation assay

[0363] To obtain B cells, human PBMCs were isolated from the peripheral blood of normal drug-free donors by standard density gradient centrifugation on Ficoll-Hypague (GE Healthcare, Piscataway, NJ) and incubated with anti-CD19 microbeads (Miltenyi Biotech, Auburn, CA). Subsequently, B cells were purified by positive immunoselection using an autoMacs (Miltenyi Biotech) according to the manufacturer's instructions.

[0364] The purified B cells (2×10 5 / well / 200 μL) were cultured in RPMI 1640, 10% FBS, and goat F(ab’)2 anti-human IgM (10 μg / ml) (Invitrogen, Carlsbad, CA) in the presence of different amounts of the test compound in a 96-well ultra-low binding plate (Corning, Corning, NY) for three days. Subsequently, 3 PBS containing

[0365] Example B2: Pfeiffer cell proliferation assay

[0366] The Pfeiffer cell line (diffuse large B cell lymphoma) was purchased from ATCC (Manassas, VA) and maintained in the recommended medium (RPMI and 10% FBS). To measure the anti-proliferative activity of the compound, Pfeiffer cells were plated (2x10 3 cells / well / 200 μl) with the medium in the presence or absence of a test compound in a concentration range in a 96-well ultra-low binding plate (Corning, Corning, NY). After 3 - 4 days, subsequently, 3PBS containing [H]-thymidine (1 μCi / well) (PerkinElmer, Boston, MA) was added to the cell culture and continued for 12 hours. Then, the bound radioactivity was separated by filtration with water through a GF / B filter (Packard Bioscience, Meriden, CT) and measured by liquid scintillation counting using a TopCount (Packard Bioscience). The IC of the selected compounds 50 The data are shown in Table C.

[0367] Table C.

[0368] Example Number <![CDATA[Pfeiffer IC 50 (nM)*]]> 1B + 1C + 2C +

[0369] *Symbols in Column:

[0370] + indicates ≤10 nM

[0371] ++ indicates >10 nM to 50 nM

[0372] Example C: Akt Phosphorylation Assay

[0373] Ramos cells (B lymphocytes from Burkitt lymphoma) were obtained from ATCC (Manassas, VA) and maintained in RPMI 1640 and 10% FBS. Cells (3×10 7 cells / tube / 3 mL in RPMI) were incubated with different amounts of the test compound at 37 °C for 2 hours and then stimulated with goat F(ab’)2 anti-human IgM (5 μg / mL) (Invitrogen) in a 37 °C water bath for 17 minutes. The stimulated cells were briefly centrifuged at 4 °C and whole cell extracts were prepared using 300 μL of lysis buffer (Cell Signaling Technology, Danvers, MA). The resulting lysates were sonicated and the supernatants were collected. The phosphorylation level of Akt in the supernatants was analyzed using a PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit (Cell Signaling Technology) according to the manufacturer's instructions.

[0374] Example D: Pfeiffer Lymphoma Model

[0375] Method

[0376] Female SCID mice (5 to 8 weeks old, Charles River Laboratories, Wilmington, MA) were inoculated subcutaneously in the flank with 0.2 mL of sterile saline containing 1 × 107 tumor cells (Pfeiffer, ATCC #CRL-2632, Manassas, VA) and Matrigel (BD Biosciences #354234). Tumor tissue fragments (approximately 3 mm × 3 mm) were collected 3 to 6 weeks after inoculation of the cultured cells and implanted subcutaneously in place of the cell inoculum. The tissue fragments were implanted in the form of solid pieces using blunt forceps. Treatment of tumor-bearing mice was initiated 15 to 25 days after tumor inoculation, depending on tumor size. Animals were sorted so that the average tumor volume in each group was approximately equal. On the first day of treatment, the minimum average tumor volume in all groups was 150 mm3 and each group consisted of 7 animals. The experimental therapeutic agent Example 347 was administered orally (PO) to the mice. The treatment frequency was 2 times per day for at least 14 days to achieve efficacy. The size of the subcutaneous tumor was measured 2 to 3 times per week using a digital caliper. The tumor volume was calculated by measuring the tumor in 2 dimensions and using the equation: volume = [length × width2)] / 2; where the larger value is the length and the smaller value is the width. If multiple tumors formed, the final volume was the sum of the individual tumors subject to the same equation: e.g., 2 tumors; volume = {[L1 × (W1)2] / 2} + {[L2 x (W2)2] / 2}. The effect on tumor growth was reported as the percent tumor growth inhibition (TGI%). The TGI percentage was calculated using the equation: (1 - (Tx volume / control volume)) * 100, where the control volume is the volume of the vehicle or untreated tumor on a given day and the Tx volume is the tumor volume of any treatment group on the same day. ANOVA: one-way test was used to evaluate the statistical difference between the treatment and the vehicle control.

[0377] Results

[0378] (R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (see Example 1) was evaluated as a single agent in the Pfeiffer human tumor xenograft model of diffuse large B-cell lymphoma, a subtype of NHL. The Pfeiffer cancer cells were shown to be sensitive to the antiproliferative effect of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one in vitro. Accordingly, tumor models were established based on subcutaneous inoculation of tumor cells into immunocompromised SCID mice and tumor-bearing mice, which received oral doses of vehicle or (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one at 0.3, 1, 3 or 10 mg / kg twice daily for 14 days. Treatment with (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one inhibited tumor growth by 22%, 24%, 36% and 58% (percent tumor growth inhibition) with increasing doses (see, e.g., U.S. Patent 9,932,341).

[0379] Example E: Dose Escalation and Dose Expansion Studies

[0380] In the dose escalation and dose expansion studies described herein, the PK, PD, safety and efficacy of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride (i.e., Compound 1C) were evaluated in patients with relapsed or refractory B-cell malignancies. The PK profile of Compound 1C in the absence of dose-limiting toxicity (DLT) allowed for exposure levels above the IC 90 at all tested doses throughout the once-daily (QD) dosing interval. Additionally, the results of the food effect analysis indicated that Compound 1C could be dosed regardless of meal timing. Near-maximal inhibition of pAKT in ex vivo PD assays was consistent with the exposure data. The results of PI3Kδ monotherapy and combination therapy are discussed in Examples F - H below.

[0381] Study Design and Patients

[0382] Dose escalation and dose expansion studies were conducted in multiple parts: dose escalation of Compound 1C monotherapy (Part 1), followed by cohort expansion (Part 3); dose escalation of Compound 1C + itacitinib (Part 2), followed by cohort expansion (Part 3); and dose evaluation and expansion of Compound 1C + R-ICE (rituximab plus ifosfamide, carboplatin, and etoposide) (Part 6) (NCT02018861). Eligible patients (aged ≥ 18 years) had been diagnosed with B-cell-derived lymphoid malignancies, including indolent or aggressive B-cell NHL malignancies, transformed NHL histology, and Hodgkin lymphoma (HL), which had recurred or was refractory to prior standard therapies. Patients with Burkitt lymphoma and precursor B-lymphoblastic leukemia / lymphoma were excluded. Eligible patients had an Eastern Cooperative Oncology Group performance status (ECOGPS) ≤ 1 (dose escalation) or ≤ 2 (dose expansion); had adequate cardiac, hepatic, and renal function; had a life expectancy of ≥ 12 weeks; had received ≥ 1 prior treatment regimen; and were non-responsive or not candidates for hematopoietic (H)SCT or other potential therapies.

[0383] Patients were excluded if they had the following histories: untreated, symptomatic, or unstable brain metastases; spinal cord compression; lymphoma involving the central nervous system (allowed in Compound 1C monotherapy expansion cohort A [B-cell malignancies] and Compound 1C + itacitinib combination expansion cohort); or had received allogeneic hematopoietic stem cell transplantation (HSCT) within 6 months or autologous HSCT within 3 months of enrollment. Prior treatment with a PI3Kδ inhibitor was excluded from the Compound 1C monotherapy cohort unless approved by the medical monitor, and prior JAK inhibitor was excluded from the Compound 1C + itacitinib combination therapy cohort.

[0384] Seventy-two patients were enrolled and treated with Compound 1C monotherapy (median age, 66 [range, 30–89] years) (Table 1). The median (range) duration of treatment was 4.0 (0.2 - 22.7) months. At the data cutoff, all subjects except 11 had discontinued study treatment, mainly due to disease progression (n = 35 [49%]) and adverse events (AE) (n = 14 [19%]).

[0385] Table 1. Patient demographics and baseline characteristics (ITT)

[0386]

[0387] * Includes American Indians, Alaska Natives, Pacific Islanders, and others. Including classical HL (n = 9) and nodular lymphocyte-predominant HL (n = 1). Including extranodal MZL of the MALT type (n = 2), nodular MZL (n = 4), splenic MZL (n = 2), and an unknown MZL subtype (n = 1). Including classical HL (n = 2). CLL: Chronic lymphocytic leukemia; DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; HL: Hodgkin lymphoma; HSCT: Hematopoietic stem cell transplantation; MALT: Mucosa-associated lymphoid tissue; MCL: Mantle cell lymphoma; MZL: Marginal zone lymphoma; R-ICE: Rituximab plus ifosfamide, carboplatin, and etoposide; WM: Waldenström macroglobulinemia.

[0388] Drug Administration

[0389] For compound 1C monotherapy, compound 1C was orally self-administered once daily (QD). Compound 1C was administered in the fasting state, with the exception that patients were enrolled in an optional food effect group until the protocol amendment permitted administration without regard to food. Treatment continued until unacceptable toxicity or disease progression. During dose escalation (see Table 2C), an initial single-patient cohort was treated with 5 mg QD and subsequent cohorts were recruited using a 3 + 3 design to identify any dose-limiting toxicities (DLTs; defined in Table 2A) within a 21-day observation period. If DLT occurred in >1 of the first 3 patients or in the total cohort of 6 patients, the maximum tolerated dose (MTD) was considered exceeded and the next lower tolerable dose level was defined as the MTD. All doses up to 45 mg QD were well tolerated during the 21-day DLT observation period and were thus not identified as the MTD. However, the occurrence of colitis was observed over a longer period (>3 months), which led to a change in the dosing regimen. The protocol was amended to include a modified dosing schedule, in which, based on PK simulations, once-daily dosing was followed by once-weekly dosing (see discussion). Patients at the time of enrollment or after this protocol amendment received compound 1C 20 mg QD for the first 9 weeks, followed by compound 1C 20 mg once weekly (QW); patients who had received compound 1C 20 mg QD for ≥9 weeks were transferred to the QW schedule. According to the protocol amendment, prophylactic treatment against Pneumocystis jiroveci pneumonia (PJP) was required for newly enrolled patients.

[0390] Table 2A. Definition of dose-limiting toxicity

[0391]

[0392]

[0393] Itacitinib can cause an immediate decrease in white blood cells due to margination; therefore, the DLT rule requires neutropenia to persist after maintaining itacitinib for 2 to 3 days. When the clinical status of the subject permits, the investigator is prompted to wait 24 hours before starting growth factors to determine whether white blood cell margination contributes to the degree of neutropenia. AE: Adverse event; ANC: Absolute neutrophil count; DLT: Dose-limiting toxicity.

[0394] For the compound 1C + itacitinib combination, dose escalation of compound 1C was performed using a 3 + 3 design, starting with a dose of compound 1C approximately 25% less than the recommended dose determined for compound 1C monotherapy. The criteria for itacitinib dose interruption are outlined in Table 2A. For each cohort, itacitinib was co-administered at a dose of 300 mg QD.

[0395] For the compound 1C + R-ICE combination, dose escalation of compound 1C was performed using a 3 + 3 design, with a starting dose approximately 25% below the recommended dose determined for compound 1C. The R-ICE chemotherapy component was administered according to institutional practice or according to the protocol.

[0396] Evaluation

[0397] Patients were evaluated on Day 1, Day 8, and Day 15 of Cycle 1 and on Day 1 (±3) of each subsequent treatment cycle. The schedule of key study assessments is shown in Table 2B.

[0398] Table 2B. Study Assessments

[0399]

[0400]

[0401] * Every 9 weeks (3 cycles) or at a frequency consistent with the standard of care; only performed when measurable disease is present. May be performed according to the standard of care; only applicable to lymphoma. To confirm CR. § Not collected from patients receiving compound 1C + R-ICE. || Only Cycle 2; collected from patients with B-cell malignancies in the compound 1C monotherapy expansion cohort (Cohort A). For compound 1C monotherapy and compound 1C + itacitinib combination, through Cycle 6; and for compound 1C + R-ICE combination, in Cycles 3, 6, 9, and 12. # Archival tissue is acceptable; for patients with CLL, peripheral blood is acceptable.** Optional on-treatment or end-of-treatment (EOT) biopsies (or peripheral blood for patients with CLL). AE: adverse event; CLL: chronic lymphocytic leukemia; CR: complete response; CT: computed tomography; ECG: electrocardiogram; ECOGPS: Eastern Cooperative Oncology Group performance status; EOT: end of treatment; FDG-PET: fluorodeoxyglucose (FDG)-positron emission tomography; MRI: magnetic resonance imaging; PD: pharmacodynamics; PK: pharmacokinetics; R-ICE: rituximab plus ifosfamide, carboplatin, and etoposide.

[0402] The primary endpoints were the safety and tolerability of Compound 1C monotherapy, Compound 1C + itacitinib, or Compound 1C + R-ICE, as evaluated by adverse events (TEAEs) occurring during treatment, clinical laboratory assessments, physical examination findings, and an overview of 12-lead electrocardiograms. The severity of TEAEs was evaluated using Version 4.03 of the Common Terminology Criteria for Adverse Events.

[0403] The secondary endpoints were efficacy and PK. Efficacy was measured by the best overall response rate (ORR), which was defined as the proportion of patients achieving a partial response (PR) or complete response (CR). Responses were evaluated every 9 weeks (each investigator's assessment) based on the following criteria: the Lugano classification of lymphoma response criteria for HL and NHL (Cheson B.D. et al., Journal of Clinical Oncology, 2014; 32(27):3059 - 3067), the chronic lymphocytic leukemia criteria of the International Working Group for chronic lymphocytic leukemia (CLL) (Hallek M. et al., Blood, 2008; 111(12):5446 - 5456; Cheson B.D. et al., J Clin Oncol, 2012; 30(23):2820 - 2822)), and the Waldenström macroglobulinemia (WM) response assessment of the VIth International Workshop on Waldenström macroglobulinemia (Owen R.G. et al., Br J Haematol, 2013; 160(2):171 - 176). If a patient's response to treatment was evaluated by PET and CTI / MRI, the PET results were used for the response endpoint; if the PET results were not available for evaluation, the CT / MRI results were used for evaluation; if it was the only available form of evaluation for the patient, CT / MRI was used for the response endpoint.

[0404] Exploratory endpoints included: duration of response (DOR, the duration from the first response until death or disease progression, whichever occurred first); progression-free survival (PFS, the duration from the first study dose until death or disease progression, whichever occurred first); population PK; the pharmacodynamic (PD) relationship between PI3Kδ inhibition (as determined by in vitro phosphorylation of AKT [pAKT] assays).

[0405] Statistical Methods

[0406] Safety, tolerability, and efficacy analyses were performed on the safety / intent-to-treat population, which included all enrolled patients who received ≥1 dose of any of compound 1C, itacitinib, or the R-ICE components. PK and PD analyses included all patients in the safety / intent-to-treat population with available PK / PD data. All statistical analyses were exploratory in nature and were summarized using descriptive statistics. The ORR was estimated with 95% confidence intervals (CIs) calculated by an exact method based on the binomial distribution. Kaplan-Meier estimates of median DOR and PFS were presented with their corresponding 95% CIs.

[0407] Additional Patient Exclusion Criteria

[0408] Additional patient exclusion criteria included: radiotherapy within 4 weeks, investigational drug within 28 days (or 5 half-lives, whichever was longer), approved anticancer drug within 21 (42 for nitrosoureas) days (or 5 half-lives, whichever was longer) (except for: steroids, ≤10 mg prednisone, daily), unresolved toxicity grade ≥2, current or recent history of clinically significant infection, total bilirubin ≥1.2 × upper limit of normal (ULN), alkaline phosphatase ≥2.5 × ULN, aspartate aminotransferase (AST) or alanine aminotransferase ≥2.0 × ULN, or creatinine clearance <50 mL / min based on the Cockroft-Gault formula.

[0409] R-ICE Drug Administration Schedule According to Protocol

[0410] According to the protocol, R-ICE was administered in 3 21-day cycles according to the following schedule: rituximab 375 mg / m² on days 1 and 2 of cycle 1, and on day 1 of cycles 2 and 3 2 ; ifosfamide 5000 mg / m² over 24 h by continuous intravenous (IV) infusion on day 3 of each cycle 2 ; carboplatin (area under the curve = 5 mg / mL; maximum dose 800 mg) by IV infusion on day 3 2 .

[0411] Example F. PI3Kδ inhibitor monotherapy

[0412] This analysis included data from patients enrolled in the compound 1C monotherapy dose escalation (5 - 45 mg QD) and expansion (20 mg QD and 30 mg QD) cohorts.

[0413] Dose Escalation and Cohort Expansion

[0414] The number of patients per dose level during compound 1C monotherapy dose escalation and expansion is shown in Table 2C. No DLT was identified, no MTD was reached, and the 20 mg QD and 30 mg QD doses were expanded.

[0415] Table 2C. Monotherapy dose escalation and cohort expansion

[0416]

[0417] AE = adverse event; DLT = dose-limiting toxicity; MTD = maximum tolerated dose.

[0418] Pharmacokinetics and Pharmacodynamics of Compound 1C

[0419] All doses tested remained above the IC of target inhibition during the dosing interval 90 , as Figure 1A shown; the C 最大 and C 波谷 produced by the 20 mg QD dose of compound 1C were 16-fold and 2-fold the IC 90 of pAKT inhibition, respectively. PK simulations were performed to support the selection of the 20 mg QW dosing schedule, which estimated that serum compound 1C levels generated by this schedule would remain above the IC of target inhibition for ~36 hours 90 and would remain at minimal to no inhibition for approximately half of the dosing interval. After single multiple-dose administration of compound 1C alone, compound 1C rapidly reached the peak plasma concentration, with a median t 最大 of 0.5 to 1 hour. Subsequently, the compound 1C plasma concentration declined in a monoexponential manner, with an average terminal-phase disposition t 1 / 2 of 8.6 to 11.5 hours. At steady state, dose proportionality appeared to be observed between 5 and 45 mg QD, as Figure 1B shown.

[0420] The food effect was analyzed in 12 patients. Although co-administration of compound 1C with a high-fat meal increased C 最大It was reduced by 42%, but it had a moderate effect on the area under the curve (AUC; 10% reduction). The geometric mean ratio of fed to fasted AUC was 0.90 (90% CI: 0.68–1.18).

[0421] Up to 6 hours after dosing, near-maximal PI3Kδ inhibition was observed at all Compound 1C doses, as measured by the effect on pAKT levels in whole blood, as Figure 4 shown.

[0422] Safety and Tolerability

[0423] Treatment-emergent adverse events (TEAEs) that occurred in >10% of patients in all combined dose groups and in the 20 mg QD group are shown in Table 3.

[0424] Table 3. Non-hematologic TEAEs that occurred in ≥15% of patients (safety population) receiving Compound 1C monotherapy

[0425]

[0426] *Based on starting dose. Includes patients receiving QD and QW dosing. Includes the following preferred terms: diarrhea, colitis, enterocolitis, gastroenteritis, microscopic colitis, and cytomegalovirus colitis. Includes the following preferred terms: exfoliative dermatitis, rash, erythematous rash, maculopapular rash, papular rash, pruritic rash, exfoliative rash, generalized rash, epidemic rash, and pustular rash. MedDRA: Medical Dictionary for Regulatory Activities; TEAE: treatment-emergent adverse event.

[0427] The most common (≥30%) non-hematologic TEAEs of any grade in all patients were diarrhea / colitis (36%), nausea (36%), fatigue (31%), and rash (31%). One patient (1%) experienced grade 3 pulmonary parenchymal inflammation and grade 3 cytomegalovirus colitis, respectively, and 7 patients (10%) experienced pneumonia (grade 3, n = 3 [4%]). Treatment-emergent laboratory abnormalities that occurred in ≥30% of patients are shown in Table 4. New onset / worsening of any grade and grade 3 / 4 neutropenia occurred in 32 patients (44%) and 14 patients (19%), respectively (Table 4). Serious adverse events (SAEs) experienced by >2 patients were diarrhea / colitis (n = 9 [13%]), fever (n = 4 [6%]), hypotension (n = 3 [4%]), and sepsis (n = 3 [4%]). SAEs of any grade for infection and infestation (system organ class) occurred in 11 patients (15%), including pneumonia in 2 patients (3%).

[0428] Table 4. New or Worsening Hematologic Laboratory Abnormalities in Patients Receiving Single Agent Compound 1C (Safety Population) ≥ 30%*

[0429]

[0430] *Based on reported laboratory values. NA: Not applicable

[0431] Among the TEAE of interest, the median (range) time to onset of grade 3 / 4 diarrhea / colitis was 5.7 (1.6 - 14.9) months and the median (range) time to onset of grade 3 / 4 rash was 2.9 (1.5 - 9.3) months. Increases in AST and ALT occurred in 21 (29%) and 20 (28%) patients, respectively; all events were grade 1, except for 4 events in 2 patients (1 patient had 1 event each of grade 3 AST and grade 2 ALT elevation, both occurring > 30 days after the last dose of Compound 1C; 1 patient had 1 event each of grade 3 AST and ALT elevation, considered secondary to sepsis). Elevations in bilirubin occurred in 8 (11%) patients; all events were ≤ grade 2, except for 1 grade 3 event occurring > 30 days after the date of the last dose. The change in AST and ALT levels over time is shown in Figure 5A-5B There were no TEAE of PJP or intestinal perforation. Five patients (7%) experienced hypertension (all grade 1 / 2); seven (10%) experienced hyperglycemia (all grade 1 / 2 except for 1 grade 3).

[0432] TEAE of any grade led to interruption of Compound 1C dosing in 30 patients (42%), dose reduction in 4 patients (6%), and treatment discontinuation in 14 patients (19%). The most common non - hematologic TEAEs leading to interruption of Compound 1C dosing were diarrhea / colitis (11 / 72 [15%]) and fever (4 / 72 [6%]). TEAEs leading to death occurred in 2 patients (respiratory failure, and respiratory failure and sepsis); both were considered unrelated to Compound 1C.

[0433] Long-Term Tolerability and Drug Administration

[0434] Before implementing the QW dosing schedule, 9 / 31 patients (29%) with DLBCL, FL, MCL, and MZL who received monotherapy with Compound 1C discontinued due to TEAE, the most common (n≥2) being diarrhea / colitis (n = 3 [10%]) or rash (n = 2 [6%]); however, during the first 9 weeks of treatment, no patients discontinued due to treatment-related AEs. As of the data cutoff date, 26 of 72 enrolled patients (36%) had received QW dosing of Compound 1C (after receiving QD dosing for at least 9 weeks), with a median (range) of 2.5 (1.2 - 9.6) months and a total of 105 patient-months; 46 patients (64%) received only QD dosing; no patients were still in the first 9 weeks of QD dosing. Four of 26 patients started the QW schedule at a dose <20 mg due to prior dose reductions (1 at 5 mg and 3 at 10 mg). Among the 26 patients receiving QW dosing, 8 (31%) interrupted the dose (TEAEs resulting in dose interruption in >1 patient were diarrhea and neutropenia [n = 2 each]), 1 (4%) reduced the dose (due to pustular rash), and no patients discontinued treatment due to TEAE. Four patients (15%) receiving QW dosing experienced a total of 6 SAEs (abdominal pain, diarrhea, fever, bronchitis, sepsis, and dehydration, n = 1 each) during the QW dosing period. No grade 4 non-hematologic TEAEs were reported during QW dosing. Two patients had grade 3 diarrhea and rash (n = 1 [4%] each); both events occurred shortly after conversion from QD dosing. No new / worsening grade 4 neutropenia was reported, and one patient reported new / worsening grade 4 thrombocytopenia during QW dosing.

[0435] Efficacy

[0436] The ORR in evaluable patients varied by disease type, as shown in Table 5. Among patients with evaluable lesion sizes at baseline and after baseline, deep responses (>75% reduction in target lesion size compared to baseline) occurred in 4 / 7 patients (57%) with marginal zone lymphoma (MZL), 7 / 14 patients (50%) with follicular lymphoma (FL), 3 / 8 patients (38%) with mantle cell lymphoma (MCL), and 2 / 20 patients (10%) with diffuse large B-cell lymphoma (DLBCL) ( Figure 2 ). In patients with these tumor types, most (93%) responses occurred at the first evaluation (∼9 weeks), as Figure 6 shown. Durable responses (>6 months) were observed in patients receiving QW dosing and several patients remained on the QW schedule for at least 8 months, as Figure 3As shown, it includes 2 patients receiving 10 mg QW. The median duration of response was 13.5 months (95% CI: 8.3–18.8) (for DLBCL), 4.4 months (95% CI: 2.1–NE) (for MZL), and was not reached for FL or MCL. Figure 7 Representative positron emission tomography (PET) images of patients achieving a complete response with compound 1C monotherapy are shown. The patient was <65 years old, with mantle cell lymphoma not involving the bone marrow (target lesion, 98 mm × 58 mm) and having received 1 prior treatment (R-HyperCVAD). After treatment with compound 1C (30 mg, once daily), the patient achieved a complete response at week 9.

[0437] Table 5. Best overall response in patients receiving compound 1C monotherapy*

[0438]

[0439] * Evaluated by: Lugano classification, CLL IWGC criteria, and VIth International Workshop on WM, by CT or PET according to disease subtype. The 95% CI was calculated by an exact method based on the binomial distribution. Two patients had unknown DLBCL subtypes. Included extranodal MZL of MALT type (n = 2), nodular MZL (n = 4), splenic MZL (n = 2), and unknown MZL subtype (n = 1). ∥∥ 4 patients with MCL and 3 patients with CLL had received ibrutinib prior to the study, with the best overall response of CR or PR achieved in 2 patients with MCL and 1 patient with CLL. ** Included classical HL (n = 9) and nodular lymphocyte-predominant HL (n = 1). ABC: Activated B-cell-like; CI: Confidence interval; CLL: Chronic lymphocytic leukemia; CMR: Complete metabolic response; CR: Complete response; CT: Computed tomography; DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; HL: Hodgkin lymphoma; GCB: Germinal center B-cell-like; CLL IWGC: CLL International Working Group; MALT: Mucosa-associated lymphoid tissue; MCL: Mantle cell lymphoma; MZL: Marginal zone lymphoma; ORR: Overall response rate; PD: Progressive disease; PET: Positron emission tomography; PMD: Progressive metabolic disease; PMR: Partial metabolic response; PR: Partial response; SD: Stable disease; WM: Waldenström macroglobulinemia.

[0440] Example of combination therapy with G.PI3Kδ inhibitors

[0441] Compound 1C in Combination with Itatinib

[0442] Eleven patients were recruited and treated with Compound 1C + itacitinib. The median (range) duration of treatment with Compound 1C and itacitinib was 2.1 (1.0 - 10.3) months. At the data cutoff, treatment was ongoing in 2 patients (18%); 8 patients (73%) discontinued treatment due to disease progression, and 1 (9%) discontinued due to TEAE.

[0443] Among the 11 patients treated with Compound 1C + itacitinib, 8 received Compound 1C 20 mg QD and 3 received Compound 1C 30 mg QD; all patients received itacitinib 300 mg QD. No DLT was observed. Nine patients (82%) experienced TEAE, 5 (45%) had grade 3 / 4 TEAE, and 2 (18%) had SAE. The most common TEAE of any grade (n≥2) were nausea, fatigue (n = 5 each), cough, hypertension, vomiting (n = 3 each), decreased appetite, hyperglycemia, increased lacrimation, oral herpes, and tachycardia (n = 2 each). Five patients experienced grade 3 / 4 TEAE: grade 3 spinal cord compression, grade 3 intractable pain, and grade 3 elevated alkaline phosphatase (n = 1); grade 3 worsening hyperlipidemia (n = 1); grade 3 hypertension (n = 1); grade 3 diarrhea and grade 3 dehydration (n = 1); and grade 3 hypertension and grade 4 hypercalcemia (n = 1). One patient had new / worsening grade 4 hematologic laboratory abnormalities (thrombocytopenia). One SAE each was reported: grade 3 dehydration and grade 4 hypercalcemia. Two patients (18%) had TEAE resulting in dose interruption of Compound 1C and itacitinib, and 1 patient (9%) had TEAE resulting in discontinuation of Compound 1C and itacitinib; no patient had TEAE resulting in dose reduction. No TEAE led to death.

[0444] Among evaluable patients, the best overall responses of CR / complete metabolic response were achieved in patients with CLL (1 / 1) and FL (1 / 1); the best overall responses of partial metabolic response were achieved in patients with MCL (1 / 1) and classical HL (1 / 2). All 6 patients with DLBCL and 1 of 2 patients with classical HL had best overall responses of PD / progressive metabolic disease.

[0445] Compound 1C in Combination with R-ICE

[0446] Five patients with DLBCL were treated with compound 1C + R-ICE. The median (range) duration of treatment with compound 1C was 2.3 (1.5 - 3.7) months; 4 patients each received 3 cycles of R-ICE and 1 patient received 2 cycles of R-ICE. As of the data cut-off date, all patients discontinued treatment due to physician decision (n = 2), progressive disease, adverse events, and other (n = 1 each). Among the 5 patients, 4 received compound 1C 15 mg QD and 1 received 20 mg QD. No DLT was observed. The most common any-grade non-blood TEAEs (≥ 2 patients) were dizziness (n = 3), alopecia, constipation, fluid overload, headache, hypokalemia, and night sweats (n = 2 each). Three patients reported new / worsening grade 4 thrombocytopenia and 2 patients reported new / worsening grade 4 neutropenia. Two patients (40%) experienced SAE, 1 patient experienced grade 3 acute encephalopathy (related to ifosfamide), and 1 experienced grade 2 atrial flutter and atrial fibrillation and grade 3 dyspnea, neutropenia, and febrile neutropenia (all related to compound 1C and R-ICE). Three patients (60%) had TEAEs resulting in dose interruption of compound 1C and R-ICE, 1 patient (20%) had a TEAE resulting in dose interruption of compound 1C only, and 1 patient (20%) had a TEAE resulting in discontinuation of compound 1C and R-ICE; no patient had a TEAE resulting in dose reduction. No TEAE led to death.

[0447] Among the 5 patients treated with compound 1C + R-ICE, 3 patients achieved a complete metabolic response (1 out of 3 patients indicated the need to proceed with SCT) and 2 patients had stable disease as the best response.

[0448] Results of Example H.PI3Kδ Monotherapy and Combination Therapy

[0449] Compound 1C demonstrated a differential safety profile relative to first-generation PI3K inhibitors (e.g., idelalisib and duvelisib) (Coutré S.E. et al., Leukemia & Lymphoma. 2015;56(10):2779 - 2786; Flinn I.W. et al., Blood, 2018;131(8):877 - 887), most notably indicated by the near absence of grade ≥ 2 transaminase elevations. With copanlisib, a pan-PI3K inhibitor approved in the United States as monotherapy for relapsed FL (ALIQOPA TM.(Copanlisib), for injection, for intravenous use, U.S. Whippany, NJ: Bayer HealthCare Pharmaceuticals Inc.; 2017) was different, and there was also no clinically significant hypertension or hyperglycemia, presumably due to the high selectivity of Compound 1C for the δ isoform of PI3K. Although PJP was not observed, the protocol amendment made PJP prophylaxis a mandatory part of the study in response to PJP infections reported in different Phase 1 studies, which used Compound 1C in combination with pembrolizumab.

[0450] Diarrhea / colitis and rash (common toxicities of PI3K inhibitors) were the 2 most frequent toxicities observed in this study; however, most diarrhea events were grade 1 or 2 and were manageable. Most grade 3 / 4 diarrhea / colitis and rash occurred after the first disease assessment; overall, diarrhea / colitis or rash of any grade led to treatment discontinuation in 9 / 72 (13%) patients, including 3 patients with diarrhea / colitis who were positive on rechallenge with the same dose. Grade 4 neutropenia (6%) was only observed during QD dosing (first appearing after 21 days of treatment). The safety profile associated with the 20 mg QD dose was generally consistent with that observed for all combination doses and with that observed for each individual dose level (data not shown), but relatively fewer patients were dosed at levels below 20 mg QD and above 30 mg QD.

[0451] Long-term administration of Compound 1C caused delayed TEAEs, which led to treatment discontinuation. To improve the long-term tolerability of Compound 1C while maintaining a high response rate, a modified dosing regimen (20 mg QD for 9 weeks, followed by 20 mg QW) was implemented. The daily dosing period was maintained to preserve the possibility of a rapid onset of response (at implementation, all 11 patients with NHL receiving 20 mg QD had achieved the target response, 10 of whom had it at the first disease assessment). The intermittent part of the dosing regimen (implemented to reduce the incidence of delayed TEAEs) was based on comparative PK / PD simulations using copanlisib (Patnaik A. et al., Annals of Oncology, 2016; 27(10):1928-1940). Without being bound by theory, the 20 mg QW regimen was expected to provide pAKT inhibition similar to that expected with QW dosing of copanlisib. Encouragingly, based on data accrued during a 105 patient-month period, none of the 26 patients receiving QW dosing discontinued study treatment due to TEAEs. This observation, in combination with the absence of colitis, grade 4 non-hematologic TEAEs, and neutropenia, and the low incidence of grade 3 diarrhea and rash (each n = 1), suggests that the QW schedule is better tolerated than the continuous QD schedule at the doses evaluated herein.

[0452] Compound 1C monotherapy achieved a high incidence of rapid, deep, and durable target responses at doses in the range of 10 mg QD to 45 mg QD in relapsed or refractory B-cell NHL. The ORR in all subtypes was in the range of 30% to 78% (DLBCL: 30%; MCL: 67%; FL: 71%; MZL: 78%) and deep responses occurred in >30% of patients. Interestingly, target responses were observed in 2 of 4 patients with MCL who had been previously treated with ibrutinib; 1 of these responses was ongoing at approximately 6 months at data cutoff. Among patients with DLBCL, substantially more patients with the GCB subtype (n = 19) were enrolled compared to those with the ABC subtype (n = 2). These small, disproportionate numbers were insufficient to determine whether Compound 1C is more active in one subtype than in others. Durable responses were observed in all 4 of these NHL subtypes, including ~17 months in FL, ~10 months in MZL, ≥14 months in MCL, and ~19 months in DLBCL, all of which included QW dosing and 2 of which (FL and MZL) had ongoing responses at data cutoff. Collectively, these data suggest that QW dosing of Compound 1C can achieve durable responses in patients with B-cell NHL.

[0453] PK and safety data from combination studies show that compound 1C can be safely combined with itatinib at a dose of 300 mg QD. Similarly, the combination of compound 1C with R-ICE also appears to be tolerated. Additional studies are needed to evaluate the safety of compound 1C in combination with R-ICE and the likelihood of improved efficacy for each combination.

[0454] Compound 1C demonstrated anti-tumor activity with rapid and deep responses, high response rates, and a differential toxicity profile in patients with relapsed or refractory B-cell malignancies. The modified dosing regimens described herein appear to improve the long-term tolerability of compound 1C and enable durable responses in several patients. The efficacy, safety, PK, and PD data presented herein support further investigation of compound 1C monotherapy in ongoing Phase 2 studies in selected NHL subtypes (DLBCL [NCT02998476], FL [NCT03126019], MZL [NCT03144674], and MCL [NCT03235544]). A Phase 1 study is also ongoing, which explores several combination strategies in patients with NHL (NCT03039114; NCT03424122).

[0455] Example I. Comparative maintenance dosing

[0456] In this study, 20 mg of compound 1C was administered to subjects once daily in combination with a JAK1 inhibitor for 8 weeks. After week 8, subjects were assigned to one of 2 maintenance dose groups based on block randomization. In one group, 5 mg of compound 1C was administered to subjects once daily. In the second group, 20 mg of compound 1C was administered to subjects once weekly.

[0457] The safety and tolerability of compound 1C were evaluated by an overview of treatment-emergent adverse events (TEAEs), clinical laboratory assessments, physical examination findings, and 12-lead electrocardiograms. The severity of TEAEs was evaluated using Version 4.03 of the Common Terminology Criteria for Adverse Events. The secondary endpoints were efficacy and PK, as described in Example E.

[0458] Without being bound by theory, it is believed that a 5 mg once-daily maintenance dose of compound 1C provides similar safety and tolerability to a 20 mg once-weekly maintenance dose of compound 1C.

[0459] Example J. Combination therapy with a PI3Kδ of JAK1 / 2 inhibitor in the treatment of myelofibrosis

[0460] Compound 1C in combination with ruxolitinib was evaluated in patients with primary or secondary myelofibrosis (MF) (e.g., PMF, PPV-MF, or PET-MF) who demonstrated a suboptimal response to ruxolitinib monotherapy. The study was designed in three parts as follows:

[0461] Part 1, Safety Run-in : 3+3 design; ruxolitinib + Compound 1C administered at a dose of 10 mg or 20 mg QD for 8 weeks, followed by 10 mg or 20 mg QW.

[0462] Part 2, Randomization Period (1:1) : Group 1: ruxolitinib + Compound 1C administered at a dose of 10 mg QD for 8 weeks, followed by 10 mg QW. Group 2: ruxolitinib + Compound 1C administered at a dose of 20 mg QD for 8 weeks, followed by 20 mg QW.

[0463] Part 3 : Additional dosing regimens, including continuous daily dosing.

[0464] "Suboptimal response" under ruxolitinib monotherapy was defined as follows:

[0465] · Patients were treated with ruxolitinib for ≥6 months at a stable dose for ≥8 weeks, immediately prior to enrollment (acceptable prior ruxolitinib doses: 5 mg–25 mg BID); and

[0466] · At screening, the spleen was palpable >10 cm below the left costal margin on physical examination; or

[0467] · At screening follow-up, the spleen was palpable 5 cm - 10 cm below the left costal margin on physical examination and there were active symptoms of myelofibrosis, each defined as 1 symptom score ≥5; or 2 symptom scores ≥3, using the following screening symptom form: 10-point scale for each of 7 symptoms. Symptoms included: night sweats, itching, abdominal discomfort, pain below the left rib, early satiety, bone / muscle pain, and inactivity.

[0468] The dosing regimen and preliminary results of the study of Compound 1C + ruxolitinib are shown in Appendix A, the disclosure of which is incorporated herein by reference in its entirety. As shown in Appendix A, the addition of Compound 1C therapy appears to provide additional clinical benefit in patients with MF who have a suboptimal response to ruxolitinib monotherapy. A reduction in spleen volume was observed in 56% of patients at week 12, with symptom improvement occurring as early as week 4; the median symptom improvement at week 24 was 36%. The combination of Compound 1C and ruxolitinib was well tolerated in patients, with no dose-limiting toxicity (DLT) in Part 1 and an expected grade 3 / 4 adverse event (AE) profile. Among 17 patients treated for >6 months, only 2 (7%) discontinued due to AE. AEs common to PI3Kδ inhibitors, such as liver, rash, and colitis, were rare.

[0469] Various modifications of the invention will be apparent to those skilled in the art from the foregoing description in addition to those described herein. Such modifications are also intended to fall within the scope of the appended claims. All references cited in this application, including all patents, patent applications, and publications, are incorporated herein by reference in their entirety.

[0470] In summary, the invention includes but is not limited to the following items:

[0471] 1. A method of treating a disease in a patient, wherein the disease is associated with abnormal expression or activity of PI3Kδ kinase, the method comprising:

[0472] i) administering to the patient a first dose of a PI3Kδ inhibitor of from about 3 mg / day to about 50 mg / day for a first period of from about 2 weeks to about 12 weeks; and

[0473] ii) administering to the patient a second dose of the PI3Kδ inhibitor at the end of the first period, the second dose being less than the first dose and being:

[0474] (a) about 2.5 mg / day or less; or

[0475] (b) about 50 mg / week or less;

[0476] and wherein the second dose is administered during a second period that occurs after the first period.

[0477] 2. The method of item 1, wherein the first dose is about 20 mg / day.

[0478] 3. The method of item 1 or 2, wherein the first period is from about 8 weeks to about 12 weeks.

[0479] 4. The method of item 1 or 2, wherein the first period is about 8 weeks.

[0480] 5. The method according to any one of items 1 to 4, wherein the first dose is reduced during the first period.

[0481] 6. The method according to any one of items 1 to 5, wherein the second dose is about 2.5 mg / day or less.

[0482] 7. The method according to any one of items 1 to 5, wherein the second dose is about 2.5 mg / day.

[0483] 8. The method according to any one of items 1 to 5, wherein the second dose is about 50 mg / week or less.

[0484] 9. The method according to any one of items 1 to 5, wherein the second dose is about 20 mg / week.

[0485] 10. The method according to any one of items 1 to 9, wherein the second dose is reduced during the second period.

[0486] 11. The method according to any one of items 1 to 10, wherein the patient has been identified as exhibiting one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs).

[0487] 12. The method according to any one of items 1 to 11, wherein the patient has been identified as exhibiting the one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs) during the first period.

[0488] 13. The method according to any one of items 1 to 12, wherein the first period ends when the patient has been identified as exhibiting the one or more symptoms associated with one or more treatment-emergent adverse events (TEAEs).

[0489] 14. The method according to any one of items 1 to 13, wherein the one or more treatment-emergent adverse events include one or more of the following: diarrhea / colitis, nausea, fatigue, rash, neutropenia, fever, hypotension, sepsis, respiratory failure, inflammation of the lung parenchyma, pneumonia, hypertension, hyperglycemia, abdominal pain, bronchitis, dehydration, thrombocytopenia, cough, vomiting, decreased appetite, increased lacrimation, oral herpes, tachycardia, spinal cord compression, intractable pain, elevated alkaline phosphatase, elevated transaminases, hyperlipidemia, hypercalcemia, vertigo, alopecia, constipation, fluid overload, headache, hypokalemia, night sweats, encephalopathy, atrial flutter, atrial fibrillation, and dyspnea.

[0490] 15. The method according to any one of items 1 to 13, wherein the adverse events occurring during one or more of the treatment periods include one or more of the following: diarrhea / colitis, nausea, fatigue, rash, cough, vomiting, dizziness, fever, hypokalemia, abdominal pain, constipation, anorexia, night sweats, pruritus, back pain, chills, leukopenia, neutropenia, lymphopenia, thrombocytopenia, and anemia.

[0491] 16. The method according to any one of items 1 to 13, wherein the adverse events occurring during one or more of the treatment periods include one or more of diarrhea / colitis and rash.

[0492] 17. The method according to any one of items 14 to 16, wherein the diarrhea / colitis includes one or more of the following: diarrhea, colitis, enterocolitis, gastroenteritis, microscopic colitis, and cytomegalovirus colitis.

[0493] 18. The method according to any one of items 14 to 17, wherein the rash includes one or more of the following: exfoliative dermatitis, rash, erythematous rash, maculopapular rash, papular rash, pruritic rash, exfoliative rash, generalized rash, epidemic rash, and pustular rash.

[0494] 19. The method according to any one of items 14 to 18, wherein the neutropenia includes febrile neutropenia.

[0495] 20. The method according to any one of items 14 to 19, wherein the elevated transaminase includes elevated alanine transaminase (ALT), elevated aspartate transaminase (AST), or a combination thereof.

[0496] 21. The method according to any one of items 1 to 20, wherein the PI3Kδ inhibitor is selected from:

[0497] 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one; and

[0498] 5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one;

[0499] or a pharmaceutically acceptable salt thereof.

[0500] 22. The method according to any one of items 1 to 20, wherein the PI3Kδ inhibitor is 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one or a pharmaceutically acceptable salt thereof.

[0501] 23. The method according to item 22, wherein the PI3Kδ inhibitor is (S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof.

[0502] 24. The method according to item 22, wherein the PI3Kδ inhibitor is (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof.

[0503] 25. The method according to item 22, wherein the PI3Kδ inhibitor is (S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof.

[0504] 26. The method according to item 22, wherein the PI3Kδ inhibitor is (R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof.

[0505] 27. The method according to item 22, wherein the PI3Kδ inhibitor is a pharmaceutically acceptable salt of 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one.

[0506] 28. The method according to item 27, wherein the PI3Kδ inhibitor is (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride.

[0507] 29. The method according to item 27, wherein the salt is a salt with a stoichiometric ratio of 1:1 of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one and hydrochloric acid.

[0508] 30. The method according to item 27, wherein the salt is crystalline.

[0509] 31. The method according to any one of items 1 to 20, wherein the PI3Kδ inhibitor is 5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.

[0510] 32. The method according to item 31, wherein the PI3Kδ inhibitor is (R)-5-{3-[(R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.

[0511] 33. The method according to item 31, wherein the PI3Kδ inhibitor is (R)-5-{3-[(S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.

[0512] 34. The method according to item 31, wherein the PI3Kδ inhibitor is (S)-5-{3-[(S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.

[0513] 35. The method according to item 31, wherein the PI3Kδ inhibitor is (S)-5-{3-[(R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one or a pharmaceutically acceptable salt thereof.

[0514] 36. The method according to any one of items 1 to 35, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM).

[0515] 37. The method according to item 36, wherein the marginal zone lymphoma (MZL) is selected from extranodal MZL, nodular MZL, splenic MZL, and unknown MZL subtypes.

[0516] 38. The method according to item 36, wherein the Hodgkin lymphoma (HL) is selected from classical Hodgkin lymphoma (HL) and nodular lymphocyte-predominant HL.

[0517] 39. The method according to item 36, wherein the diffuse large B-cell lymphoma is selected from activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL) and germinal center B-cell (GCB) diffuse large B-cell lymphoma (GCB-DLBCL).

[0518] 40. A method of treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), the method comprising:

[0519] i) administering to the patient a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day to about 50 mg / day for a first period of about 8 weeks to about 9 weeks; and

[0520] ii) administering to the patient a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride after the first period, the second dose being about 2.5 mg / day or less.

[0521] 41. A method of treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), the method comprising:

[0522] i) Administering a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day to about 50 mg / day to the patient for a first period of about 8 weeks to about 9 weeks; and

[0523] ii) Administering a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride to the patient for a second period that occurs after the first period, the second dose being about 20 mg / week to about 50 mg / week.

[0524] 42. A method of treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), the method comprising:

[0525] i) Administering a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day to the patient for a first period of about 8 weeks; and

[0526] ii) Administering a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride to the patient for a second period that occurs after the first period, the second dose being about 2.5 mg / day.

[0527] 43. A method for treating a disease in a patient, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM), the method comprising:

[0528] i) administering to the patient a first dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / day for a first period of about 8 weeks; and

[0529] ii) administering to the patient a second dose of (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride at about 20 mg / week in a second period that occurs after the first period.

[0530] 44. A method for treating a disease in a patient, wherein the disease is associated with abnormal expression or activity of PI3Kδ kinase, the method comprising:

[0531] i) administering to the patient a first dose of a PI3Kδ inhibitor at about 3 mg / day to about 50 mg / day for a first period of about 2 weeks to about 12 weeks; and

[0532] ii) administering to the patient a second dose of the PI3Kδ inhibitor at the end of the first period, the second dose being less than the first dose and between about 2.5 mg / day and about 7.5 mg / day; and

[0533] wherein the second dose is administered during a second period that occurs after the first period.

[0534] 45. The method according to item 44, wherein the second dose is about 3.0 mg / day to about 7.0 mg / day.

[0535] 46. The method according to item 44, wherein the second dose is about 4.0 mg / day to about 6.0 mg / day.

[0536] 47. The method according to item 44, wherein the second dose is about 5.0 mg / day.

[0537] 48. The method according to any one of items 44 to 47, wherein the PI3Kδ inhibitor is (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one hydrochloride.

[0538] 49. The method according to any one of items 44 to 48, wherein the disease is selected from chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenström macroglobulinemia (WM).

[0539] 50. The method according to item 49, wherein the marginal zone lymphoma (MZL) is selected from extranodal MZL, nodular MZL, splenic MZL, and unknown MZL subtypes.

[0540] 51. The method according to item 49, wherein the Hodgkin lymphoma (HL) is selected from classical Hodgkin lymphoma (HL) and nodular lymphocyte-predominant HL.

[0541] 52. The method according to item 49, wherein the diffuse large B-cell lymphoma is selected from activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL) and germinal center B-cell (GCB) diffuse large B-cell lymphoma (GCB-DLBCL).

Claims

1. A method of treating a disease in a patient, wherein the disease is associated with abnormal expression or activity of PI3Kδ kinase, the method comprises: i) administering to the patient a first dose of a PI3Kδ inhibitor of from about 3 mg / day to about 50 mg / day for a first period of from about 2 weeks to about 12 weeks; and ii) administering to the patient a second dose of the PI3Kδ inhibitor at the end of the first period, the second dose being less than the first dose and being: (a) about 2.5 mg / day or less; or (b) about 50 mg / week or less; and wherein the second dose is administered during a second period that occurs after the first period.

2. The method according to claim 1, wherein the first dose is about 20 mg / day.

3. The method according to claim 1 or 2, wherein the first period is from about 8 weeks to about 12 weeks.

4. The method according to claim 1 or 2, wherein the first period is about 8 weeks.

5. The method according to any one of claims 1 to 4, wherein the first dose is reduced during the first period.

6. The method according to any one of claims 1 to 5, wherein the second dose is about 2.5 mg / day or less.

7. The method according to any one of claims 1 to 5, wherein the second dose is about 2.5 mg / day.

8. The method according to any one of claims 1 to 5, wherein the second dose is about 50 mg / week or less.

9. The method according to any one of claims 1 to 5, wherein the second dose is about 20 mg / week.

10. The method according to any one of claims 1 to 9, wherein the second dose is reduced during the second period.

Images