antiproliferative compounds and methods of using them

专利摘要:
4- (4- (4 - ((((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) methyl) benzyl) piperazin-1-yl) -3 is provided here -fluorobenzonitrile, or an enantiomer, a mixture of enantiomers, a tautomer or a pharmaceutically acceptable salt thereof and methods for the treatment, prevention or management of multiple myeloma using these compounds. Pharmaceutical compositions comprising the compounds and methods of using the compositions are also provided.
公开号:BR112020000442A2
申请号:R112020000442-1
申请日:2018-07-09
公开日:2020-07-21
发明作者:Matthew D. Alexander；Timothy S. Kercher；Antonia Lopez-Girona；Xiaoling Lu；Hon-Wah Man；Mark A. Nagy；Rama K. Narla；Daniel W. Pierce；Joseph R. Piccotti；Brandon W. Whitefield；Paula A. Tavares-Greco；Gerald D. Artman Iii；Nanfei Zou；Lilly L. Wong；Gordon L. Bray；James Carmichael；Soraya Carrancio；Brian E. CATHERS；Matthew D. Correa；Joshua Hansen；Courtney G. Havens
申请人:Celgene Corporation；
IPC主号:

专利说明:

[001] [001] This application claims the benefit of US Provisional Application 62 / 530,778 filed on July 10, 2017 and Provisional Application 62 / 593,185, filed on November 30, 2017 and Provisional Application 62 / 675,581, filed on May 23, 2018 , all of which are incorporated herein by reference in their entirety.
[002] [002] 4- (4- (4 - (((((2- (2,6-dioxopiperidin-3-yl) -1- oxoisoindolin-4-yl) oxy) mMethyl) benzyl) piperazin-1- i1) -3-fluorobenzonitrile, or an enantiomer, a mixture of enantiomers, a tautomer or a pharmaceutically acceptable salt thereof and methods for the treatment, prevention or management of multiple myeloma using these compounds. Pharmaceutical compositions comprising the compounds and methods of using the compositions, including combined treatments, are also provided.
[003] [003] Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow. Plasma cells normally produce antibodies and play a key role in immune function. However, the uncontrolled growth of these cells leads to bone pain and fractures, anemia, infections and other complications. Multiple myeloma is the second most common haematological neoplasm, although the exact causes of multiple myeloma remain unknown. Multiple myeloma causes high levels of proteins in the blood, urine and organs, including, but not limited to, protein M and other immunoglobulins (antibodies), albumin and beta-2-microglobulin, except in some patients (estimated at 1% to 5%) whose myeloma cells do not secrete these proteins (called non-secretory myeloma). The protein
[004] [004] Skeletal symptoms, including bone pain, are among the most clinically significant symptoms of multiple myeloma. Malignant plasma cells release osteoclast-stimulating factors (including IL-1, IL-6 and TNF) that cause calcium to be leached from the bones, causing lytic lesions; hypercalcemia is another symptom. Osteoclast-stimulating factors, also called cytokines, can prevent apoptosis or the death of myeloma cells. Fifty percent of patients have skeletal lesions related to myeloma detected radiologically in the diagnosis. Other common clinical symptoms for multiple myeloma include polyneuropathy, anemia, hyperviscosity, infections and kidney failure.
[005] [005] Current multiple myeloma therapy may involve one or more surgeries, stem cell transplantation, chemotherapy, immune therapy and / or radiation treatment to eradicate multiple myeloma cells in a patient. All current therapy approaches have significant disadvantages for the patient.
[006] [006] In the last decade, new therapeutic agents, in particular immunomodulatory drugs, such as lenalidomide and pomalidomide, have significantly increased response rates and prolonged progression-free survival (PFS) and overall survival (OS) in patients with multiple myeloma. However, persistent levels of residual disease below the sensitivity of bone marrow (BM) morphology, protein electrophoresis with immunofixation and quantification of the light chain exist in many patients with multiple myeloma, even after reaching the complete response (CR) and, eventually cause the disease to relapse. Minimal residual disease (MRD) in myeloma is an independent predictor of progression-free survival (PFS) and is being considered as a surrogate outcome to improve the identification of effective treatments, particularly for frontline trials, which now require 5 to 10 years of follow-up to identify differences in survival. Monitoring of minimal residual disease (MRD) in patients with multiple myeloma provides prognostic value in predicting PFS and OS and making treatment decisions. The detection of minimal residual disease (MRD) in myeloma can use a threshold of 0.01% (10º) after treatment, that is, with 10º cells or fewer multiple myeloma cells since the proportion of total mononuclear cells of the medulla bone are considered negative for MRD and with 10th cells or more positive for MRD. The MRD threshold 10º MRD was originally based on technical capacity, but quantitative detection of MRD is now possible at 10º by flow cytometry and 10º by high throughput sequencing. (Rawstron et al., Blood 2015; 125 (12): 1932-1935). Methods for measuring MRD include VDJ DNA sequencing, polymerase chain reaction (PCR) (including allele-specific PCR, ASO PCR) and multiparametric flow cytometry (MPF). Tests for MRD, for example, based on the measurement of the clonotype profile are also described in US Patent 8,628,927, by Faham et al., Which is incorporated herein by reference.
[007] [007] There is a significant need for safe and effective compounds and methods for the treatment, prevention and management of multiple myeloma, including for patients whose multiple myeloma is newly diagnosed or refractory to standard treatments, while reducing or avoiding toxicities and / or side effects associated with conventional therapies.
[008] [008] The citation or identification of any reference in Section 2 of this application should not be interpreted as an admission that the reference is technical prior to this application.
[009] [009] Compounds, pharmaceutical compositions containing the compounds and methods of using them in the treatment of multiple myeloma are provided here. In one embodiment, the compound for use in the compositions and methods provided herein is 4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1- oxoisoindolin-4-yl) oxy) Methyl ) benzyl) piperazin-1-i1) -3-fluorobenzonitrile (Compound 1): the
[0010] [0010] In one embodiment, the compound for use in the compositions and methods “provided herein is (S) 4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1- oxoisoindolin- 4-yl) oxy) methyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile (Compound 2): o CO
[0011] [0011] In another embodiment, the compound for use in the compositions and methods provided herein is (R) -4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1- oxoisoindolin -4-yl) oxy) methyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile (Compound 3): o
[0012] [0012] Pharmaceutical compositions formulated for administration by an appropriate route and means containing effective concentrations of the compounds provided herein are also provided, for example Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically salt acceptable amount thereof, and optionally comprising at least one pharmaceutical carrier.
[0013] [0013] In one embodiment, the pharmaceutical compositions deliver effective amounts for the treatment of multiple myeloma. In one embodiment, the pharmaceutical compositions deliver amounts effective for the prevention of multiple myeloma. In one embodiment, the pharmaceutical compositions deliver effective amounts for ameliorating multiple myeloma.
[0014] [0014] Combination therapies using the compounds or compositions provided herein, or an enantiomer, are also provided in this document.
[0015] [0015] The compounds or compositions provided herein, or pharmaceutically acceptable derivatives thereof, may be administered simultaneously with, before or after the administration of one or more of the above therapies. Pharmaceutical compositions containing a compound provided herein and one or more of the above therapies are also provided.
[0016] [0016] In one embodiment, effective amounts of the compounds or compositions containing therapeutically effective concentrations of the compounds are administered to an individual who exhibits the symptoms of multiple myeloma to be treated. The amounts are effective in improving or eliminating one or more symptoms of multiple myeloma. In the practice of treatment methods, effective amounts of the compounds or compositions containing therapeutically effective concentrations of the compounds are administered to a patient with multiple myeloma in need thereof.
[0017] [0017] A pharmaceutical package or kit is also provided that comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions. Optionally associated with such container (s) may be a notice in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, the notice of which reflects the agency's approval of the manufacture, use or sale for administration in humans. The package or kit can be labeled with information on the mode of administration, sequence of administration of the drug (for example, separately, sequentially or simultaneously) or the like.
[0018] [0018] These and other aspects of the subject described here will become evident with reference to the following detailed description.
[0019] [0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is normally understood by one skilled in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for a term in this document, those in this section will prevail, unless otherwise indicated.
[0020] [0020] "IC5o" refers to an amount, concentration or dosage of a specific test compound that achieves a 50% inhibition of a maximum response, such as receptor binding, receptor activity, cell growth or proliferation, as measured by any of the in vitro or cell-based assays described in this document.
[0021] [0021] Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as, without limitation, NN'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, - ethylenediamine, - N-methylglucamine, - procaine , - N-benzylphenethylamine, - 1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethyl- - benzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxymethyl) aminomethane; alkali metal salts, such as, without limitation, lithium, potassium and sodium; alkaline earth metal salts, such as, without limitation, barium, calcium and magnesium; transition metal salts, such as, but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, without limitation, mineral acid salts, such as, without limitation, hydrochlorides and sulfates; and salts of organic acids, such as, without limitation, acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, fumarates and sulfonates.
[0022] [0022] Unless otherwise specified, where a compound may take alternative tautomeric, regioisomeric and / or stereoisomeric forms, all alternative isomers must be included in the scope of the claimed matter. For example, when a compound can have one of two tautomeric forms, it is intended that both tautomers are included herein.
[0023] [0023] Thus, the compounds provided herein can be enantiomerically pure or be stereoisomeric or diastereomeric mixtures. As used herein and unless otherwise stated, the term "stereoisomerically pure" means a composition that comprises a stereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure composition of a compound having a center chiral will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure composition of a compound with two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound comprises more than about 80% by weight of a stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably more than about 90% by weight of a stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, even more preferably more than about 95% by weight of a stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and more preferably more than about 97% by weight of a stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
[0024] [0024] It should be understood that the compounds provided herein may contain chiral centers. Such chiral centers may have the (R) or (S) configuration or may be a mixture of them. It should be understood that the chiral centers of the compounds provided herein can undergo epimerization in vivo. As such, one skilled in the art will recognize that the administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to the administration of the compound in its (S) form.
[0025] [0025] The optically active (+) and (-), (R) - and (S) - or (D) - and (L) isomers can be prepared using chiral syntones or chiral reagents or resolved using conventional techniques, such as chromatography in a chiral stationary phase.
[0026] [0026] As used in this document, an "isotopologist" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom with an isotopic composition different from the natural isotopic composition of that atom. "Isotopically enriched" can also refer to a compound containing at least one atom with an isotopic composition different from the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. The radiolabeled and isotopically enriched compounds are useful as therapeutic agents, for example, multiple myeloma therapeutic agents, research reagents, for example, binding assay reagents and diagnostic agents, for example, in vivo imaging agents. All isotopic variations of the compounds as described herein, whether radioactive or not, must be included in the scope of the modalities provided here. In some embodiments, isotopologists of the compounds are provided, for example, the isotopologists of Compound 1, Compound 2 or Compound 3 are compounds enriched with deuterium, carbon 13 or nitrogen 15. In some embodiments, the isotopologists provided herein are compounds enriched with deuterium. In some embodiments, the isotopologists provided here are compounds enriched with deuterium, where deuterium enrichment occurs at the chiral center.
[0027] [0027] In the description of this document, if there is any discrepancy between a chemical name and a chemical structure, the structure controls.
[0028] [0028] As used in this document, "multiple myeloma" refers to hematological conditions characterized by malignant plasma cells and includes the following disorders: monoclonal gammopathy of undetermined significance (MGUS); multiple myeloma of low risk, intermediate risk and high risk; newly diagnosed multiple myeloma (including low-risk, intermediate-risk, and high-risk newly diagnosed multiple myeloma); multiple myeloma eligible for transplant and ineligible for transplant; smoking (indolent) multiple myeloma (including low-risk, intermediate-risk and high-risk smoking multiple myeloma); active multiple myeloma; solitary plasmacytoma; extramedullary plasmacytoma; plasma cell leukemia; multiple myeloma of the central nervous system; light chain myeloma; non-secretory myeloma; Immunoglobulin D myeloma; and Immunoglobulin E myeloma; and multiple myeloma characterized by genetic abnormalities, such as translocations of cyclin D (for example, t (11; 14) (913; 932); t (6; 14) (p21; 32); t (12; 14) (p13; 932); or t (6; 20);); MMSET translocations (for example, t (4; 14) (p16; q932)); MAF translocations (for example, t (14; 16)
[0029] [0029] As used in this document and unless otherwise stated, the terms "treat", "treating" and "treatment" refer to alleviating or reducing the severity of a symptom associated with the disease or condition being treated, for example , multiple myeloma.
[0030] [0030] The term "prevention" includes the inhibition of a specific disease symptom or disorder, for example, multiple myeloma. In some modalities, patients with a family history of multiple myeloma are candidates for preventive regimens. Generally, the term "prevention" refers to the administration of the drug before the onset of symptoms, especially in patients at risk for multiple myeloma.
[0031] [0031] As used in this document and unless otherwise indicated, the term “management” encompasses preventing the recurrence of a specific disease or disorder, such as multiple myeloma, in a patient who has suffered from it, prolonging the time that a a patient who has suffered from the disease or disorder remains in remission, reducing patient mortality rates and / or maintaining a reduction in severity or in preventing a symptom associated with the managed disease or condition.
[0032] [0032] As used in this document, "subject" or "patient" is an animal, typically a mammal, including a human, as a human patient.
[0033] [0033] The term "relapse" refers to a situation in which patients, who have had a multiple myeloma remission after therapy, have a return of myeloma cells and / or reduced normal cells in the marrow.
[0034] [0034] The term "refractory or resistant" refers to a circumstance where patients, even after intensive treatment, have residual myeloma cells and / or reduced normal cells in the marrow.
[0035] [0035] As used in this document, "induction therapy" refers to the first treatment administered for a disease or the first treatment administered with the intention of inducing complete remission in a disease, such as cancer. When used alone, induction therapy is accepted as the best treatment available. If residual cancer is detected, patients will be treated with another therapy, called reinduction. If the patient is in complete remission after induction therapy, further consolidation and / or maintenance therapy is given to prolong remission or potentially cure the patient.
[0036] [0036] As used in this document, "consolidation therapy" refers to the treatment given to a disease after remission is achieved for the first time. For example, cancer healing therapy is treatment given after the cancer has disappeared after initial therapy. Consolidation therapy can include radiation therapy, stem cell transplantation or chemotherapy treatment. Consolidation therapy is also called intensification therapy and post-remission therapy.
[0037] [0037] As used in this document, "maintenance therapy" refers to the treatment administered for a disease after remission or the best response is achieved in order to prevent or delay relapse. Maintenance therapy can include chemotherapy, hormonal therapy or targeted therapy.
[0038] [0038] "Remission", as used in this document, is a decrease or disappearance of signs and symptoms of cancer, for example, multiple myeloma. In partial remission, some, but not all, signs and symptoms of cancer disappeared. In complete remission, all the signs and symptoms of the cancer have disappeared, although the cancer may still be in the body.
[0039] [0039] As used in this document, “transplantation” refers to high-dose therapy with stem cell rescue. Hematopoietic stem cells (blood) or bone marrow are used not as a treatment, but to rescue the patient after high-dose therapy, for example, high-dose chemotherapy and / or radiation. The transplant includes transplantation of “autologous” stem cells (ASCT), which refers to the use of the patients' own stem cells being harvested and used as replacement cells. In some modalities, transplantation also includes tandem or multiple transplants.
[0040] [0040] As used herein, and unless otherwise specified, the terms "therapeutically effective amount" and "effective amount" of a compound refer to an amount sufficient to provide a therapeutic benefit in the treatment, prevention and / or managing a disease, for example, multiple myeloma or to delay or minimize one or more symptoms associated with the disease or disorder being treated. The terms "therapeutically effective amount" and "effective amount" can encompass an amount that improves general therapy, reduces or avoids symptoms or causes of diseases or disorders, or increases the therapeutic effectiveness of another therapeutic agent.
[0041] [0041] The terms “co-administration” and “in combination with” include the administration of one or more therapeutic agents (for example, a compound provided herein and another multiple anti-myeloma agent, carcinogen or supportive care agent) simultaneously, concomitantly or sequentially, without specific time limits. In some embodiments, the agents are present in the patient's cell or body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In another embodiment, the therapeutic agents are in separate compositions or unit dosage forms.
[0042] [0042] The term "supportive care agent" refers to any substance that treats, prevents or manages an adverse effect of treatment with Compound 1, Compound 2 or Compound 3, or an enantiomer or a mixture of enantiomers, tautomers , isotopologist or a pharmaceutically acceptable salt thereof.
[0043] [0043] The term "biological therapy" refers to the administration of biological therapies, such as umbilical cord blood, stem cells, growth factors and the like.
[0044] [0044] In the context of cancer, such as multiple myeloma, inhibition can be assessed by inhibiting the progression of the disease, inhibiting tumor growth, reducing the primary tumor, relieving symptoms related to the tumor, inhibiting factors secreted by the tumor, inhibition of the appearance of primary or secondary factors. secondary tumors, slow development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, decrease or severity of the side effects of the disease, growth of interrupted tumors and tumor regression, increased time to progression (TTP), increased free survival progression (PFS), increase in Global Survival (OS), among others. OS, as used in this document, means the time from the start of treatment to death from any cause. TTP, as used in this document, means the time from the start of treatment until the tumor progresses; TTP does not include deaths. In one embodiment, PFS means the time from the start of treatment to the progression of the tumor or death. In one modality,
[0045] [0045] In certain modalities, the treatment of multiple myeloma can be assessed by the International Uniform Response Criteria for Multiple Myeloma (IURC) (see Durie BGM, Harousseau JL, Miguel JS, et al. International uniform response criteria for multiple myeloma. Leukemia , 2006; (10) 10: 1-7), using the definitions of response and period shown below: Response subcategory Response criteria sCR CR as defined below plus Normal FLC ratio and Absence of clonal cells in bone marrow ”by immunohistochemistry or immunofluorescence“ s CR Negative immunofixation in serum and urine and Disappearance of any soft tissue plasmacytoma and <5% of plasma cells in bone marrow ”VGPR serum M protein and urine detectable by immunofixation, but not by electrophoresis or reduction of 90% or more in serum M plus level of protein M in urine &lt; 100 mg for 24 h.
[0046] [0046] As used in this document, ECOG status refers to the Eastern Cooperative Oncology Group (ECOG) Performance Status (Oken M, et al Toxicity and response criteria of the Eastern Cooperative Oncology GroupAm J Clin Oncol 1982; 5 (6 ): 649-655), as shown below: Totally active, capable of performing all pre-illness performance without restriction 1 Restricted in physically strenuous activities, but ambulatory and capable of performing light or sedentary work, for example, housework light, office work.
[0047] [0047] The term “about, as used in this document, unless otherwise stated, refers to a value that does not exceed 10% above or below the value being modified by the term. For example, does the term “about 10 mg / m” mean a range of 9 mg / m at 11 mg / m .
[0048] [0048] Figure 1. (A) Change in apoptosis induction, measured by the area under the Caspase induction curve 3 times (also known as apoptosis index) over time in lenalidomide-resistant H929-1051 cells. Abscissa: log nM (compound), ordered: apoptosis index. The best fit lines are a 3-parameter logistic equation calculated on the GraphPad Prism. (B) The area under the curve of the concentration-response curves for the H929-1051 cells of compound 1 and compound A was used to compare the ability of the compounds to induce apoptosis after a 6 h exposure and then dilution resulting in about 20 times the reduction in compound concentration.
[0049] [0049] Figure 2. Comparison of the antiproliferative activity of the combined treatment with pomalidomide-dexamethasone and single agent Compound 2 (A) and with combined treatment with compound 2-dexamethasone (B) in MM cells resistant to lenalidomide H929-1051. Proliferation was assessed using an ATP determination assay (CellTiter-Glo) after 120 h of treatment. The percentage control was calculated by subtracting the background and normalizing for the DMSO control (100% of the control). Each data point represents the average of at least three independent experiments in duplicate.
[0050] [0050] Figure 3. (A) Antiproliferative effects on unstimulated PBMCs and (B) THLE-2, treated with Compound 2 for 72 h were evaluated using an ATP determination assay (CellTiter-Glo). The percentage control was calculated by subtracting the background and normalizing for the DMSO control (100% of the control).
[0051] [0051] Figure 4. Compound 2 antitumor activity with continuous dosing in the lenalidomide resistant xenograft model H929-1051. Female SCID mice were inoculated with 10 x 10 th H929-1051 tumor cells on the right flank. The mice were randomized into treatment groups (n = 10 / group) at the time of treatment initiation. The treatment of the test article started on the 14th, when the tumors were approximately 120 mm .
[0052] [0052] Figure 5: Antiproliferative activity of compound 2 in multiple myeloma cell lines grouped by chromosomal translocations. The graph represents the area under the curve (AUC) of the concentration response growth curves that measure the numbers of living cells by flow cytometry for 15 MM cell lines containing common translocations found in MM. The reported AUC value corresponds to the area under the dose response curve in which O values correspond to the complete reduction of proliferation / viability in all doses and values of 10,000 correspond to no reduction in proliferation / viability. Cell lines are grouped first by the chromosomal translocation found and, second, by the fact of knowing whether the translocation is of high risk or not.
[0053] [0053] Figure 6: Antiproliferative activity of Compound 2 and pomalidomide in multiple myeloma cell lines resistant to lenalidomide and pomalidomide. ICso = concentration of Compound 2 and pomalidomide resulting in 50% inhibition of cell growth compared to the control. Graph showing the comparison of Compound 2 and antiproliferative values of ICso pomalidomide (bars) were determined using the CellTitre-Glo test on parental cell lines (DF15, NCI-H929 and OPM2), resistant to lenalidomide (NCI-H929-1051) or resistant to pomalidomide (NCI-H929-PO01, OPM2-PO1, OPM2-P1, OPM2 -P10 and DF15R)
[0054] [0054] Figure 7: Blocking strategy for myeloid subpopulations.
[0055] [0055] Figure 8: Last stages of in vitro differentiation of neutrophil progenitors - effects of short daily exposures to Compound 2 for up to three days. CD34 * cells derived from healthy donor bone marrow were exposed to Compound 2 in concentrations of 1, 10 and 100 nM each for 1, 2 or 3 consecutive days. Only live cells were included in the analysis. Data are the average of results for Donors 1 and 2 and represent an example of the percentage of cells in Stages Ill and IV defined as CD34 / CD33 * / CD11b * and CD34 / CD33 / CD11b *, respectively, after 6 h of exposure .
[0056] [0056] Figure 9: Late maturation of neutrophilic parents after 6 hours of exposure to Compound 2 on 3 consecutive days. CD34 * cells derived from healthy donor bone marrow were exposed to Compound 2 in concentrations of 1, 10 or 100 nM for 6 h on each of the 3 consecutive days from Day 10. Data represent the average percentage of cells in Stage Ill defined as CD347 / CD33 * / CD11b * and the percentage of cells in Stage IV defined as CD34 / CD33 "/ CD11b * from Donors No. 1 and No. 2. The error bars represent standard deviation.
[0057] [0057] Figure 10: Late maturation of neutrophilic parents after 6-hour exposures to Compound 2 on 5 consecutive days. CD34 * cells derived from healthy donor bone marrow were exposed to Compound 2 in concentrations of 1, 10 or 100 nM for 6 h on each of the 5 consecutive days from Day 10. Data represent the average percentage of cells in Stage Ill defined as CD34 / CD33 * / CD11b * and the percentage of Stage IV cells defined as CD34 / CD33 / CD11b * from Donors No. 1 and No. 2. Error bars represent standard deviation.
[0058] [0058] Figure 11: Diagrams of treatment regimen for dexamethasone with single agent.
[0059] [0059] Figure 12: Percentage of mature neutrophils during myeloid differentiation after exposure to dexamethasone or Compound 2 alone or in combination at different concentrations. CD34 * cells derived from healthy donor bone marrow were exposed to Compound 2 (for 6 h) and dexamethasone (for 30 h) alone (upper line) or in combination (lower line), in concentrations of 1, 10 or 100 nM in the Day 13. In each of the lower panels, the concentration of Compound 2 was varied and the concentration of dexamethasone was kept constant at 1 nM (left), 10 nM (medium) or 100 nM (right). For combinations, cultures were exposed to both agents simultaneously for 6 h. The cells were then washed and re-incubated with dexamethasone for the next 24 h. Then, the cells were washed and re-incubated without Compound 2 or dexamethasone for the remainder of the study. The data represents the percentage of Stage IV cells defined as CD34 / CD33 * / CD11b * from Donors 3, 4 and 5. The red line represents 50% of the Stage IV cell level in the DMSO control.
[0060] [0060] Figure 13: The effect of treatment with dexamethasone alone or in combination with Compound 2, lenalidomide and pomalidomide, on apoptosis in a lenalidomide-resistant multiple myeloma cell line. The y-axis shows the change in fold for DMSO caspase-3 and the x-axis is the logarithmic concentration of dexamethasone.
[0061] [0061] Figure 14: Compound 2 directly activates human peripheral blood mononuclear cells (PBMCs) to lyse K5S62 erythromyelocytic leukemia cells in a concentration-dependent manner. (Left) Representative graphs of fluorescence-activated cell classification of K562 cells co-cultured with human PBMCs that were preincubated with Compound 2, lenalidomide, pomalidomide or DMSO. (Right) Raw data on the percentage of PlI-Annexin V-K562 cells showing a concentration-dependent decrease in viable K562 cells in co-culture. The data are presented as mean, with error bars representing standard error of the mean.
[0062] [0062] Figure 15: The immune cells are activated directly by Compound 2 to lyse cell lines of lenalidomide-sensitive and lenalidomide-resistant multiple myeloma. Peripheral blood mononuclear donor cells (PBMCs) (effector cells) were pre-treated with the indicated test articles or Compound 2 for 2 h before being cultured in plates coated with anti-CD3 antibody for 72 h. Before co-culture with multiple untreated CFSE-labeled myeloma cell lines, PBMCs were washed and placed in medium with no compound present and then co-cultured with multiple myeloma cell lines (target cells) for 24 h. An increase in immune cell-mediated cell death from multiple myeloma was evident in PBMCs treated with compound 2 co-cultured (Target: Effector ratio 1: 5) with (A) NCI-H929 cells or (B) H929-1051 cells .
[0063] [0063] Figure 16: The immune cells initiated by the compound show increased cell death when multiple myeloma cells are pretreated with lenalidomide, pomalidomide or Compound 2 before co-culture. Peripheral blood mononuclear cells were preincubated with lenalidomide, pomalidomide or Compound 2 for 2 h before being cultured in plates coated with anti-CD3 antibody for 72 h. At the same time, 4 multiple myeloma (MM) cell lines were cultured in medium containing test articles. After 72 h, the cells were co-cultured together by
[0064] [0064] Figure 17: Compound 2 superregulates CD38 expression in MM cell lines. The CD38 cell surface expression was evaluated in MM cells pretreated with Compound 2 or pomalidomide for 72 h. The effects of the dose response are shown for the cell lines OPM-2 and OPM-2. P10.
[0065] [0065] Figure 18: Compound 2 increases the ADCC mediated by daratumamab of MM cells. Seven MM cell lines were treated with sub-lethal concentrations of Compound 2 or pomalidomide for 72 h before co-culture with NK cells at an effector to target ratio [E: T] of 10: 1 for the ADCC assay. The graphs illustrate representative data obtained for the 7 MM cell lines. The assays were performed twice with NK cells from two different donors. DMSO control is the activity of baseline NK cells with untreated tumor cells; the isotype and Dara are the activity of NK cells in the presence of isotype and daratumamab control, respectively, with untreated tumor cells; the isotype + compound and the Dara + compound are the activity of NK cells in the presence of isotype control and daratumumab, respectively, with treated tumor cells.
[0066] [0066] Figure 19: Compound 2 increases the ADCP mediated by daratumamab of MM cells. Phagocytosis assays were performed with an effector-to-target ratio [E: T] of 2: 1. Six MM +/- Compound 2 cell lines or pre-treatment with pomalidomide were subjected to daratumamab-mediated ADCP. A) Representative images of the ADCP with the cell line OPM2. Macrophages are in red and OPM2 cells are in green. B) Quantification of phagocytosis by flow cytometry. DMSO control is the activity of baseline NK cells with untreated tumor cells; the isotype and Dara are the activity of NK cells in the presence of isotype and daratumamab control, respectively, with untreated tumor cells; the isotype + compound and the Dara + compound are the activity of NK cells in the presence of isotype control and daratumumab, respectively, with treated tumor cells.
[0067] [0067] Figure 20: The combination of Compound 2 with proteasome inhibitor results in increased apoptosis in MM cell models. MM cell lines were treated with a pulse of bortezomib or DMSO for 1 h, followed by a wash. The pretreated cells were incubated with different concentrations of Compound 2 for 72 h, followed by staining the samples with 7-AAD and annexin-V solution and analysis by flow cytometry. A) Percentage of live Compound 2 cells alone or pretreated with bortezomib. B) Scatter plots of OPM2-P10 cells in the various treatment conditions.
[0068] [0068] Figure 21: Combination of Compound 2 with bortezomib or carfilzomib in MM cells. Four MM cell lines were treated with a pulse of bortezomib or DMSO for 1 h, followed by a wash. The pretreated cells were incubated with different concentrations of Compound 2 for 72 hours, followed by staining the samples with 7-AAD and annexin-V solution and analysis by flow cytometry. A) Antiproliferative effect of Compound 2 alone or with pretreatment with bortezomib. B) Antiproliferative effect of Compound 2 alone or with pre-treatment with carfilzomib. CKD = dose-response curve
[0069] [0069] Figure 22. Treatment of MM cells with Compound 2 in combination with histone deacetylase inhibitors, chemotherapeutic agents, Bcl-2 inhibitors, Mcl-1 inhibitors, BET inhibitors or LSD-1 inhibitors is shown. Synergy calculations were performed for treatment with Compound 2 in combination with 13 small molecule inhibitors through a panel of MM cell lines. The blue boxes illustrate the percentage of wells that are synergistic when combined with Compound 2. The * represents the meaning of the difference in surface response of the null model.
[0070] [0070] Figure 23: The effect of treatment with Compound 2 (0.1 mg / kg, qd) and dexamethasone (0.5 mg / kg, qd) as single agents and in combination in the resistant H929-1051 xenograft model lenalidomide.
[0071] [0071] Figure 24: The antitumor activity of Compound 2 alone and in combination with bortezomib in a lenalidomide-resistant multiple myeloma / plasmacytoma model NCI-H929 (H929-1051). Dosing days are indicated with arrows on the X axis.
[0072] [0072] Compound 4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1- oxoisoindolin-4-yl) oxy) Methyl) benzyl) piperazin-1- il) -3-fluorobenzonitrile referred to as “Compound 1”: the WAISTBAND
[0073] [0073] In certain embodiments, the compound for use in the compositions and methods provided herein is Compound 1, or an enantiomer or mixture of enantiomers, tautomer, isotopologist or a pharmaceutically acceptable salt thereof.
[0074] [0074] Also provided here is compound (S) -4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) methyl) benzyl ) piperazin-1-i1) -3- fluorobenzonitrile referred to as “Compound 2”: the CO ON NH
[0075] [0075] In certain embodiments, the compound for use in the compositions and methods provided herein is Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof.
[0076] [0076] Also provided here is compound (R) -4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) methyl) benzyl ) piperazin-1-i1) -3- fluorobenzonitrile referred to as “Compound 3”: the
[0077] [0077] In certain embodiments, the compound for use in the compositions and methods provided herein is Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof.
[0078] [0078] Compound 1 is provided here. A tautomer of Compound 1 is provided here. An enantiomer of Compound 1 is provided here. A mixture of enantiomers of Compound 1 is provided here. A pharmaceutically acceptable salt of Compound 1 is provided here.
[0079] [0079] Compound 2 is provided here. A tautomer of Compound 2 is provided here. A pharmaceutically acceptable salt of Compound 2 is provided here.
[0080] [0080] Compound 3 is provided here. A tautomer of Compound 3 is provided here. A pharmaceutically acceptable salt of Compound 3 is provided here.
[0081] [0081] The isotopically enriched analogs of the compounds provided here are also provided here. Isotopic enrichment (for example, deuteration or deuterium enrichment) of pharmaceuticals to improve pharmacokinetics (“PK”), pharmacodynamics (“PD”), and toxicity profiles, has been demonstrated previously with some classes of drugs. See, for example, Lijinsky et. al., Food Cosmet. Toxicol., 20: 393 (1982); Lijinsky et. al., J. Nat. Cancer Inst., 69: 1127 (1982); Mangold et. al., Mutation Res. 308: 33 (1994); Gordon et. al., Drug Metab. Dispos., 15: 589 (1987); Zello et. al., Metabolism, 43: 487 (1994); Gately et. al., J. Nucl. Med, 27: 388 (1986); Wade D, Chem. Biol. Interact. 117: 191 (1999). Without being limited by any particular theory, the isotopic enrichment of a compound can be used, for example, to (1) reduce or eliminate unwanted metabolites, (2) increase the half-life of a parental drug, (3) decrease the number of doses required to achieve a desired effect, (4) decrease the amount of a dose required to achieve a desired effect, (5) increase the formation of active metabolites, if formed, and / or (6) decrease the production of metabolites harmful to specific tissues, and / or (g) create a more effective and / or safer drug for combination therapy, whether intentional or unintentional combination therapy.
[0082] [0082] Tritium ("T") is a radioactive isotope of hydrogen, used in research, fusion reactors, neutron generators and radiopharmaceuticals. Tritium is a hydrogen atom that has 2 neutrons in the nucleus and an atomic weight close to 3. It occurs naturally in the environment at very low concentrations, most commonly found as T7O. Tritium slowly decays (half-life = 12.3 years) and emits a low-energy beta particle that cannot penetrate the outer layer of human skin. Internal exposure is the main risk associated with this isotope, but it must be ingested in large quantities to represent a significant health risk. Compared to deuterium, less tritium must be consumed before reaching a dangerous level. The substitution of tritium (“T”) for hydrogen results in an even stronger bond than deuterium and produces numerically greater isotopic effects.
[0083] [0083] Likewise, the replacement of isotopes by other elements, including, but not limited to, * C or “C by carbon, * S, 3ºS, or ºS by sulfur, * N by nitrogen and O or * ºO oxygen, will provide a similar kinetic isotopic effect.
[0084] [0084] The animal's body expresses a variety of enzymes in order to eliminate foreign substances, such as therapeutic agents, from its circulation system. Examples of such enzymes include cytochrome P450 enzymes ("CYPs"), esterases, proteases, reductases, dehydrogenases and monoamine oxidases, to react and convert these foreign substances into more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of pharmaceutical compounds involve oxidation of a carbon-hydrogen (C-H) bond to a carbon-
[0085] [0085] Isotopic enrichment at certain positions of a compound provided herein can produce a detectable KIE that affects the pharmacokinetic, pharmacological and / or toxicological profiles of a compound provided here compared to a similar compound with a natural isotopic composition. In one embodiment, deuterium enrichment is carried out at the C-H bond cleavage site during metabolism.
[0086] In one embodiment, an isotopologist of Compound 1, or an enantiomer or mixture of enantiomers, tautomer or a pharmaceutically acceptable salt thereof, is provided herein. In some embodiments, the Compound 1 isotopologist is Compound 1 enriched with deuterium, or an enantiomer or a mixture of enantiomers, tautomer or a pharmaceutically acceptable salt thereof. In some embodiments, the Compound 1 isotopologist is Compound 1 enriched with deuterium, or an enantiomer or a mixture of enantiomers, tautomer or a pharmaceutically acceptable salt thereof, where deuterium enrichment occurs in the chiral center. In one embodiment, a compound 2 isotopologist, or a tautomer, or a pharmaceutically acceptable salt thereof is provided herein. In some embodiments, the Compound 2 isotopologist is Compound 2 enriched with deuterium, or a tautomer or a pharmaceutically acceptable salt thereof. In some embodiments, the Compound 2 isotopologist is Compound 2 enriched with deuterium, or a tautomer or a pharmaceutically acceptable salt thereof, where deuterium enrichment occurs at the chiral center.
[0087] [0087] In certain embodiments, 4- (4- (4 - (((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) methyl) benzyl isotopologists ) piperazin-1-yl) -3-fluorobenzonitrile, or an enantiomer or a mixture of enantiomers, tautomer or a pharmaceutically acceptable salt thereof, wherein one or more atomic positions of the 4- (4- (4 - (( (2- (2,6-dioxopiperidin-3-yl) -1- oxoisoindolin-4-yl) oxy) Methyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile is / are isotopically enriched, for example with deuterium. Certain modalities provided here provide compounds of the following formula: Yê oo 7 Yº N NA o NAL 7 YE Yo Yi Yo Y vVvy Y2 Ys nt Ys Ys 2 go NL A Ye y2º N ye Es NC Y 20 yo F in which one or more atoms Y (ie V4, Y2, Y3, W9, NS, WS, W7, W8, 9, YO, GO, Yº, vB, Y ", Y", Y ", Y", Y, Yº, Yo, Yo , Yº, YP, YA, Y, Y2, YP, YP, Yº, and Yo) is / are hydrogen (s) isotopically enriched with deuterium, and any remaining Y atom is / are un-enriched hydrogen atom (s) ).
[0088] [0088] In one embodiment, the compound is of the following formula:
[0089] [0089] In one embodiment, the compound is of the following formula: Yê o oO vz Yº N Ni, 1º and Ys Yi
[0090] [0090] In certain modalities, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one , twenty-one two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or all indicated Y atoms are isotopically enriched with deuterium and any remaining Y atom ( s) is / are unenriched hydrogen (s). In one embodiment, one of the indicated Y atoms is isotopically enriched with deuterium, and any remaining Y atoms are un enriched hydrogens. In one embodiment, Y * is enriched with deuterium.
[0091] [0091] In certain embodiments, one or more Y atoms in the glutarimide portion of the compounds (Y%, Y2, Y3, Y% 4, Y5, and Y ”!) Are enriched with deuterium. In certain embodiments, one or more Y atoms in the isoindolinone portion of the compounds (Y5, YZ, Y8, Yº, and Y * º) are enriched with deuterium. In certain embodiments, one or more Y atoms in the phenylalkyl portion of the compounds (Y * 4, YP2, y13, V4, yº5, yl6, Y! ”, And YV8) are enriched with deuterium. In certain embodiments, one or more Y atoms in the piperazine portion of the compounds (Y * º, Y20, V2L, V22, 23, 24, Yo, and Y 5) are enriched with deuterium. In certain embodiments, one or more Y atoms in the distant portion of the phenyl ring of the compounds (Y 8, Yºº, and Y% º) are enriched with deuterium. A compound provided herein can be any combination of deuterium enrichments, as disclosed herein. In other words, any combination of the deuterium-enriched glutarimide portion, deuterium-enriched disoindoline portion, deuterium-enriched phenylalkyl portion, deuterium-enriched piperazine portion and distant portion of the deuterium-enriched phenyl ring is covered herein.
[0092] [0092] In a modality, Y * and Y they are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y and Yº are enriched with deuterium and any remaining Y atoms are non-enriched hydrogens. In one embodiment, Yº is enriched with deuterium and any remaining Y atoms are un enriched hydrogens. In one embodiment, Y * to Yº are enriched with deuterium and any remaining Y atoms are un enriched hydrogens. In one embodiment, Y to Yº are enriched with deuterium and any remaining Y atoms are non-enriched hydrogens. In a modality, Yº and Y they are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y8 to Y * º are enriched with deuterium and any remaining Y atoms are un enriched hydrogens. In one embodiment, Y *! and Y "º are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y * to Y" * º are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y " And Y * 8 are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y"! to Y "8 are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y * to Yºº are enriched with deuterium and any remaining Y atoms are unenriched hydrogens. In one embodiment, Y" 'is enriched with deuterium and any remaining Y atoms are unenriched hydrogens In one embodiment, Y * º to Y * º are enriched with deuterium and any remaining Y atoms are unenriched hydrogens.
[0093] [0093] In one embodiment, the isotopologist for Compound 1 is Compound 1- D: o a Sá ”NA o o A, (1-D).
[0094] [0094] In another embodiment, the Compound 1 isotopologist is a mixture of Ne o ON D) NH CS o º and
[0095] [0095] In yet another modality, the Compound of Formula Il is Compound 2-D o N = o ON D) —NH me me
[0096] [0096] In yet another modality, the isotopologist of Compound 3 is Compound 3-D o No.)
[0097] [0097] In certain embodiments, any of the positions enriched with deuterium independently have an abundance of deuterium of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% at least 90%, at least 95%, at least 97% or about 100%. In one embodiment, Yº is enriched with deuterium and has an abundance of deuterium of at least 30%, at least 40%, at least
[0098] [0098] In one embodiment, the D (at the chiral center) in Compound 1-D has an abundance of deuterium of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80%, at least 90%, at least 95%, at least 97% or about 100%. In one embodiment, D has an abundance of deuterium of at least 90%.
[0099] [0099] In one embodiment, the D (at the chiral center) in Compound 2-D has an abundance of deuterium of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80%, at least 90%, at least 95%, at least 97% or about 100%. In one embodiment, D has an abundance of deuterium of at least 90%.
[00100] [00100] In one embodiment, the D (at the chiral center) in Compound 3-D has an abundance of deuterium of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80%, at least 90%, at least 95%, at least 97% or about 100%. In one embodiment, D has an abundance of deuterium of at least 90%.
[00101] [00101] In certain embodiments, a deuterium-enriched compound provided here has an enantiomeric excess of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Additional examples of stereoisomeric purity include an enantiomeric excess of at least 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 , 80, 81, 82, 83, 84, 85,
[00102] [00102] In one embodiment, the 2-D compound has an enantiomeric excess of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. In one embodiment, Compound 2-D has an enantiomeric excess of at least 90%.
[00103] [00103] In one embodiment, the 3-D compound has an enantiomeric excess of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. In one embodiment, the 3-D compound has an enantiomeric excess of at least 90%.
[00104] [00104] The deuterium-enriched compounds provided here can be prepared according to the synthetic scheme and examples provided here, but using the corresponding initial material (s) enriched with corresponding deuterium. The deuterium-enriched compounds provided herein can also be prepared according to the general chemistry known to those skilled in the art for preparing deuterium-enriched isoindolinone and glutarimide compounds, including, but not limited to, those described in WO 2014/039421 and WO 2014/116573, all of which are incorporated herein by reference.
[00105] [00105] The compounds provided herein can be prepared by methods known to one skilled in the art and following procedures similar to those described in the Examples section here and routine modifications thereof. An exemplary reaction scheme for the preparation of the compounds is illustrated below in Scheme 1 for Compound 1, Compound 2 and Compound 3 and Scheme 2 for Compound 2.
[00106] [00106] As shown in Scheme 1, protection of 3-hydroxy-2-methylbenzoic acid (by, for example, methyl ester formation and tert-butyl (dimethyl) silyl ether) was followed by bromination, for example, using N- bromosuccinimide and azobisisobutyronitrile. Reaction with methyl-4,5-diamino-5-oxo-pentanoate (also known as HD, L-Glu (OMe) -NH> 2), in the presence of a base (such as DIEA), resulted in the formation of derivatized isoindoline, followed by deprotection of TBS using a base, such as potassium carbonate. The reaction of isoindoline derivatized with 1,4-bis (bromomethyl) benzene in the presence of a base (such as potassium carbonate) was followed by the formation of glutarimide in the presence of potassium tert-butoxide. Finally, the reaction with 3-fluoro-4- (piperazin-1-yl) benzonitrile provided the target Compound 1. Chiral separation provides Compound 2 and Compound 3.
[00107] [00107] Alternatively, as exemplified in Scheme 2, the reaction of the methyl intermediate 2- (bromomethyl) -3- [tert-butyl (dimethyl) silyl] oxy-benzoate with the chiral intermediate of tert-butyl (4S5) -4, 5-diamino-5-oxopentanoate (also known as HL-Glu (OtBu) -NH>; reaction with HD-Glu (OtBu) -NH, provides the opposite enantiomer), in the presence of a base (such as DIEA), resulted in formation of derivatized isoindoline, which was followed by deprotection of TBS using tetrabutylammonium fluoride. The reaction of the derivatized isoindoline with 4- (4- (4- (chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile, or a salt thereof, in the presence of a base (such as potassium carbonate), followed by deprotection and glutarimide formation provided the target Compound 2.
[00108] [00108] One skilled in the art would know how to modify the procedures established in the schemes and illustrative examples to arrive at the desired products.
[00109] [00109] In one aspect, methods for preparing Compound 1, N o
[00110] [00110] In one embodiment, the method is a method for preparing an enantiomer of Compound 1, for example, Compound 2, the
[00111] [00111] In one embodiment, X is halogen, for example Br or Cl. In another embodiment, X is methanesulfonate (also known as -OMs). In one embodiment, the solvent is acetonitrile, THF or DMSO. In another, the base is
[00112] [00112] In some modalities, the methods also include preparing Compound 1a, * N o
[00113] [00113] In one embodiment, the method is a method for preparing an enantiomer of Compound 1a, for example, Compound 2a, * N o
[00114] [00114] In one embodiment, X is Br. In one embodiment, the solvent is THF. In some modalities, contact is made at a reduced temperature, for example, from about 70ºC to about 80ºC.
[00115] [00115] In some embodiments, the methods further comprise preparing Compound 1b, / o o * N NH, o 1b, or an enantiomer or a mixture of enantiomers, tautomer, or isotopologist thereof, methods comprising contacting compound 1c / o o o
[00116] [00116] In one embodiment, the method is a method for preparing an enantiomer of Compound 1b, for example, Compound 2b, /
[00117] [00117] In one embodiment, X is Br. In one embodiment, the solvent is acetonitrile. In some embodiments, the base is potassium carbonate. In some modalities, contact is made at an elevated temperature, for example, from about 50ºC to about 70ºC.
[00118] [00118] In some embodiments, the methods further comprise preparing Compound 1c,
[00119] [00119] In one embodiment, the method is a method for preparing an enantiomer of Compound 1c, for example, Compound 2c, / o o
[00120] [00120] In one embodiment, the solvent is water. In some embodiments, the base is potassium carbonate.
[00121] [00121] In some modalities, the methods also include preparing Compound 1d, giving the
[00122] [00122] In one embodiment, the method is a method for preparing an enantiomer of Compound 1d, for example, Compound 2d,
[00123] [00123] In one embodiment, the solvent is acetonitrile. In some modalities, the base is DIEA. In some other modalities, contact is made at an elevated temperature, for example, from about 50ºC to about 70ºC.
[00124] [00124] In another aspect, methods are provided here for preparing Compound 1, ALT ”NU oAX.
[00125] [00125] In one embodiment, the method is a method for preparing an enantiomer of Compound 1, for example, Compound 2, the N
[00126] [00126] In one embodiment, the solvent is acetonitrile. In another, the acid is benzene sulfonic acid. In some modalities, contact is made at an elevated temperature, for example, from about 75ºC to about 95ºC.
[00127] [00127] In some embodiments, the methods further comprise preparing Compound 1f,
[00128] [00128] In one embodiment, the method is a method for preparing an enantiomer of Compound 1f, for example, Compound 2f,
[00129] [00129] In one embodiment, the solvent is DMF. In one embodiment, the solvent is DMSO. In another, the base is potassium carbonate. In some modalities, contact is made at an elevated temperature, for example, from about 35ºC to about 55ºC.
[00130] [00130] In some embodiments, the methods for preparing Compound 1f further comprise a purification method, the purification method comprising (i) contacting Compound 1f (free base) with an acid in a first solvent; (ii) filter to provide the acid salt of Compound 1f; and (iii) washing the acid salt of Compound 1f in a second solvent with a base to provide Compound 1f (free base). In one embodiment, the acid is tartaric acid (for example, L-tartaric acid). In one embodiment, the first solvent is methanol. In one embodiment, the acid salt of Compound 1f is a tartrate salt (e.g., salt of L-tartaric acid) of Compound 1f. In one embodiment, the second solvent is 2-methyltetrahydrofuran. In one embodiment, the base is potassium carbonate.
[00131] [00131] In some embodiments, methods for preparing an enantiomer of Compound 1f, for example, Compound 2f further comprise a purification method, the purification method comprising (i) contacting Compound 2f (free base) with an acid in a first solvent; (ii) filtering to provide the acid salt of Compound 2f; and (iii) washing the acid salt of Compound 2f in a second solvent with a base to provide Compound 2f (free base). In one embodiment, the acid is tartaric acid (for example, L-tartaric acid). In one embodiment, the first solvent is methanol. In one embodiment, the acid salt of Compound 2f is a tartrate salt (e.g., salt of L-tartaric acid) of Compound 2f. In one embodiment, the second solvent is 2-methyltetrahydrofuran. In one embodiment, the base is potassium carbonate.
[00132] [00132] In some embodiments, the methods comprise preparing 4- (4- (4- (chloromethyl) benzyl) piperazin-1-yl) -3 fluorobenzonitrile, or a salt thereof, methods comprising contacting 4- (chloromethyl) benzaldehyde with 3-fluoro-4- (piperazin-1-yl) benzonitrile, in a solvent, in the presence of a reducing agent, under conditions suitable to provide 4- (4- (4- (chloromethyl) benzyl) piperazin-1-yl) - 3-fluorobenzonitrile.
[00133] [00133] In one embodiment, the reducing agent is sodium triacetoxyborohydride (NaBH (OAc) a). In one embodiment, the solvent is toluene. In one embodiment, contact is made in the presence of an acid. In one embodiment, the acid is acetic acid.
[00134] [00134] In one embodiment, 4- (4- (4- (chloromethyl) benzyl) piperazin-1-yl) -3-
[00135] [00135] Surprisingly, it has been found that Compound 1, Compound 2 and Compound 3 are very potent anti-myeloma compounds that have distinctive features, such as an enhanced safety profile, including selective cell death of multiple myeloma cells in comparison with normal cells, reduced activity in off-target receptors and reduced inhibition of the CYP enzyme, reducing the potential for adverse drug interactions.
[00136] [00136] In one embodiment, a method of treating multiple myeloma, which comprises administering a Compound 1 patient, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is provided herein. In one embodiment, provided herein is Compound 1, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, for use in a method of treating multiple myeloma, wherein the method comprises administering said compound to a patient.
[00137] [00137] In one embodiment, a method of treating multiple myeloma is provided, which comprises administering Compound 2 to a patient, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof. In one embodiment, provided herein is Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, for use in a method of treating multiple myeloma, wherein the method comprises administering said compound to a patient.
[00138] [00138] In one embodiment, a method of treating multiple myeloma is provided herein, which comprises administering Compound 3 to a patient, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof. In one embodiment, provided herein is Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, for use in a method of treating multiple myeloma, wherein the method comprises administering said compound to a patient.
[00139] [00139] In one embodiment, a method of preventing multiple myeloma is provided, which comprises administering to a patient a compound provided herein, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof. In one embodiment, a compound provided herein is provided, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, for use in a myeloma prevention method multiple, wherein the method comprises said compound for a patient.
[00140] [00140] In another embodiment, a method for managing multiple myeloma is provided, which comprises administering to a patient a compound provided herein, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer , isotopologist or pharmaceutically acceptable salt thereof. In one embodiment, a compound provided herein is provided, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, for use in a myeloma management method multiple, wherein the method comprises administering said compound to a patient.
[00141] [00141] In one embodiment, methods are also provided here to induce a therapeutic response assessed with the International Uniform Response Criteria for Multiple Myeloma (IURC) (see, Durie BGM, Harousseau JL, Miguel JS, et al. International uniform response criteria for multiple myeloma Leukemia, 2006; (10) 10: 1-7) from a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, to a patient with multiple myeloma. In another embodiment, methods are provided here for obtaining a complete rigorous response, complete response or very good partial response, as determined by the International Uniform Response Criteria for Multiple Myeloma (IURC) in a patient, comprising administering an effective amount of a compound here described, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, to the patient with multiple myeloma. In another embodiment, methods are provided herein to achieve an increase in overall survival, progression-free survival, event-free survival, time to progression or disease-free survival in a patient, comprising administering an effective amount of a compound described herein, for example example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, to the patient with multiple myeloma. In another embodiment, methods are provided herein to achieve an increase in overall survival in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer , isotopologist or pharmaceutically acceptable salt thereof, to a patient with multiple myeloma. In another embodiment, methods are provided herein to achieve an increase in progression-free survival in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers , tautomer, isotopologist or pharmaceutically acceptable salt thereof, to a patient with multiple myeloma. In another embodiment, methods are provided herein to achieve an increase in event-free survival in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers , tautomer, isotopologist or pharmaceutically acceptable salt thereof, to a patient with multiple myeloma. In another embodiment, methods are provided herein to achieve an increase in progression time in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, to a patient with multiple myeloma. In another embodiment, methods are provided herein to achieve an increase in disease-free survival in a patient, comprising administering an effective amount of a compound described herein, for example, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers , tautomer, isotopologist or pharmaceutically acceptable salt thereof, to a patient with multiple myeloma.
[00142] [00142] Methods of treating patients who have previously been treated for multiple myeloma, but who do not respond to conventional therapies, as well as those who have not been previously treated, are also provided here. Additionally covered are the methods of treating patients undergoing surgery in an attempt to treat multiple myeloma, as well as those who have not. Also provided in this document are methods of treating patients who have undergone transplant therapy previously, as well as those who have not.
[00143] [00143] The methods provided here include treating multiple myeloma that is relapsed, refractory or resistant. The methods provided here include preventing multiple myeloma that is relapsing, refractory or resistant. The methods provided here include managing multiple myeloma that is relapsed, refractory or resistant. In some of these modalities, myeloma is primary, secondary, tertiary, quadruple or quintuple multiple myeloma. In one embodiment, the methods provided here reduce, maintain or eliminate minimal residual disease (MRD). In one embodiment, the methods provided here cover the treatment, prevention or management of various types of multiple myeloma, such as monoclonal gammopathy of undetermined significance (MGUS), low-risk multiple myeloma, intermediate and high risk, newly diagnosed multiple myeloma (including newly diagnosed multiple myeloma of low risk, intermediate risk and high risk), multiple myeloma eligible for transplant and ineligible for transplantation, smoking (indolent) multiple myeloma (including low-risk smoking, intermediate risk and high risk multiple myeloma), multiple myeloma active, solitary plasmacytoma, extramedullary plasmacytoma, plasma cell leukemia, central nervous system multiple myeloma, light chain myeloma, non-secretory myeloma, immunoglobulin myeloma
[00144] [00144] In one embodiment, the methods comprise administering a therapeutically effective amount of Compound 1, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof. In another embodiment, the methods comprise administering a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof. In one embodiment, the methods comprise administering a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof.
[00145] [00145] In some embodiments, the methods comprise administering a therapeutically effective amount of Compound 1, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, as induction therapy. In another embodiment, the methods comprise administering a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as induction therapy. In one embodiment, the methods comprise administering a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as induction therapy. In some embodiments, the methods comprise administering a therapeutically effective amount of Compound 1, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, as consolidation therapy. In another embodiment, the methods comprise administering a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as consolidation therapy. In one embodiment, the methods comprise administering a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as consolidation therapy. In some embodiments, the methods comprise administering a therapeutically effective amount of Compound 1, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, as maintenance therapy. In another embodiment, the methods comprise administering a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as maintenance therapy. In one embodiment, the methods comprise administering a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as maintenance therapy.
[00146] [00146] In a particular embodiment of the methods described here, multiple myeloma is plasma cell leukemia.
[00147] [00147] In one embodiment of the methods described here, multiple myeloma is a high-risk multiple myeloma. In some of these modalities, high-risk multiple myeloma is recurrent or refractory. In one modality,
[00148] [00148] In one embodiment, multiple myeloma is characterized by a p53 mutation. In one embodiment, the p53 mutation is a 0331 mutation. In one embodiment, the p53 mutation is an R273H mutation. In one embodiment, the p53 mutation is a K132 mutation. In one embodiment, the p53 mutation is a K132N mutation. In one embodiment, the p53 mutation is an R337 mutation. In one embodiment, the p53 mutation is an R337L mutation. In one embodiment, the p53 mutation is a W146 mutation. In one embodiment, the p53 mutation is an S261 mutation. In one embodiment, the p53 mutation is an S261T mutation. In one embodiment, the p53 mutation is an E286 mutation. In one embodiment, the p53 mutation is an E286K mutation. In one embodiment, the p53 mutation is an R175 mutation. In one embodiment, the p53 mutation is an R175H mutation. In one embodiment, the p53 mutation is an E258 mutation. In one embodiment, the p53 mutation is an E258K mutation. In one embodiment, the p53 mutation is an A161 mutation. In one embodiment, the p53 mutation is an A161T mutation.
[00149] [00149] In one embodiment, multiple myeloma is characterized by a homozygous deletion of p53. In one embodiment, multiple myeloma is characterized by a homozygous deletion of the wild type p53.
[00150] [00150] In one embodiment, multiple myeloma is characterized by wild type p53.
[00151] [00151] In one embodiment, multiple myeloma is characterized by activation of one or more oncogenic drivers. In one embodiment, the one or more oncogenic drivers are selected from the group consisting of C-MAF, MAFB, FGFR3, MMset, Cyclin D1 and Cyclin D. In one modality, multiple myeloma is characterized by the activation of C-MAF. In one embodiment, multiple myeloma is characterized by MAFB activation. In one embodiment, multiple myeloma is characterized by activation of FGFR3 and MMset. In one embodiment, multiple myeloma is characterized by the activation of C-MAF, FGFR3, and MMset. In one embodiment, multiple myeloma is characterized by activation of Cyclin D1. In one embodiment, multiple myeloma is characterized by activation of MAFB and Cyclin D1. In one embodiment, multiple myeloma is characterized by activation of Cyclin D.
[00152] [00152] In one embodiment, multiple myeloma is characterized by one or more chromosomal translocations. In one embodiment, the chromosomal translocation is t (14;: 16). In one embodiment, the chromosomal translocation is t (14; 20). In one embodiment, the chromosomal translocation is t (4; 14) In one embodiment, the chromosomal translocations are t (4; 14) and t (14; 16). In one embodiment, the chromosomal translocation is t (11; 14) In one embodiment, the chromosomal translocation is t (6; 20). In one embodiment, the chromosomal translocation is t (20; 22). In one embodiment, the chromosomal translocations are t (6; 20) and t (20; 22). In one embodiment, the chromosomal translocation is t (16; 22). In one embodiment, the chromosomal translocations are t (14; 16) and t (16; 22). In one embodiment, the chromosomal translocations are t (14; 20) and t (11; 14).
[00153] [00153] In one embodiment, multiple myeloma is characterized by a p53 0331 mutation, by activation of C-MAF and by a chromosomal translocation in t (14;: 16). In one embodiment, multiple myeloma is characterized by a homozygous deletion of p53, by activation of C-MAF and by a chromosomal translocation in t (14; 16). In one embodiment, multiple myeloma is characterized by a p53 K132N mutation, by activation of MAFB, and by a chromosomal translocation in t (14; 20). In one embodiment, multiple myeloma is characterized by wild-type p53, activation of FGFR3 and MMset, and a chromosomal translocation in t (4; 14). In one embodiment, multiple myeloma is characterized by wild-type p53, by activation of C-MAF, and by a chromosomal translocation in t (14; 16). In one embodiment, multiple myeloma is characterized by homozygous deletion of p53, by activation of FGFR3, MMset and C-MAF, and by chromosomal translocations in t (4; 14) and t (14; 16). In one embodiment, multiple myeloma is characterized by a homozygous deletion of p53, by activation of Cyclin D1 and by a chromosomal translocation in t (11; 14). In one embodiment, multiple myeloma is characterized by a p53 R337L mutation, by activation of Cyclin D1 and by a chromosomal translocation in t (11; 14). In one embodiment, multiple myeloma is characterized by a p53 W146 mutation, by activation of FGFR3 and MMset, and by a chromosomal translocation in t (4; 14). In one embodiment, multiple myeloma is characterized by a p53 S261T mutation, by the activation of MAFB, and by chromosomal translocations in t (6; 20) and t (20; 22). In one embodiment, multiple myeloma is characterized by a p53 E286K mutation, by activation of FGFR3 and MMset, and by a chromosomal translocation in t (4; 14). In one embodiment, multiple myeloma is characterized by a p53 R175H mutation, by activation of FGFR3 and MMset, and by a chromosomal translocation in t (4; 14). In one embodiment, multiple myeloma is characterized by a p53 E258K mutation, by activation of C-MAF, and by chromosomal translocations in t (14; 16) and t (16; 22). In one embodiment, multiple myeloma is characterized by wild-type p53, activation of MAFB and Cyclin D1, and chromosomal translocations in t (14; 20) and t (11; 14). In one embodiment, multiple myeloma is characterized by a p53 A1617T mutation, by activation of Cyclin D and by a chromosomal translocation in t (11; 14).
[00154] [00154] In some modalities of the methods described here, multiple myeloma is a recently diagnosed multiple myeloma eligible for transplantation. In another embodiment, multiple myeloma is a newly diagnosed multiple myeloma ineligible for transplantation.
[00155] [00155] In still other modalities, multiple myeloma is characterized by early progression (for example, less than 12 months) after the initial treatment. In still other modalities, multiple myeloma is characterized by early progression (for example, less than 12 months) after autologous stem cell transplantation. In one embodiment, multiple myeloma is refractory to lenalidomide. In another embodiment, multiple myeloma is refractory to pomalidomide. In some of these modalities, multiple myeloma is expected to be refractory to pomalidomide (for example, by molecular characterization). In another embodiment, multiple myeloma is relapsed or refractory to 3 or more treatments and has been exposed to a proteasome inhibitor (eg, bortezomib, carfilzomib, ixazomib, oprozomib or marizomib) and an immunomodulatory compound (eg, thalidomide, lenalidomide, pomalidomide, iberdomide or avadomide) or double refractory to a proteasome inhibitor and an immunomodulatory compound. In still other embodiments, multiple myeloma is relapsed or refractory to 3 or more previous therapies, including, for example, a CD38 monoclonal antibody (CD38 mAb, for example, daratumumab or isatuximab), a proteasome inhibitor (for example, bortezomib, carfilzomib, ixazomib or marizomib) and an immunomodulatory compound (e.g., thalidomide, lenalidomide, pomalidomide, iberdomide or avadomide) or double refractory to a proteasome inhibitor or immunomodulator compound and a CD38 mAb.
[00156] [00156] In some of these embodiments, the methods comprise administering a therapeutically effective amount of Compound 1, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, as induction therapy. In another embodiment, the methods comprise administering a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as induction therapy. In one embodiment, the methods comprise administering a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, as induction therapy.
[00157] [00157] IN certain modalities, methods of treatment, prevention and / or management of multiple myeloma are provided, including relapsed / refractory multiple myeloma in patients with impaired renal function or a symptom thereof, comprising administering a therapeutically effective amount of Compound 1 , Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof to a patient with relapsed / refractory multiple myeloma with impaired renal function.
[00158] [00158] In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided, including relapsed or refractory multiple myeloma in frail patients or a symptom thereof, comprising administering a therapeutically effective amount of Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof to a fragile patient with multiple myeloma. In some of these modalities, the fragile patient is characterized by ineligibility for induction therapy or intolerance to treatment with dexamethasone. In some of these modalities, the frail patient is elderly, for example, over 65 years old.
[00159] [00159] In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein multiple myeloma is a relapsed / refractory fourth-line multiple myeloma. In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein the multiple myeloma is relapsed / refractory fourth-line multiple myeloma. In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein the multiple myeloma it is a relapsed / refractory multiple myeloma of the fourth line.
[00160] [00160] In certain modalities, methods of treatment, prevention and / or management of multiple myeloma are provided here,
[00161] [00161] In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof as therapy. maintenance after another therapy or transplant, in which multiple myeloma is newly diagnosed, multiple myeloma eligible for transplant before the other therapy or transplant. In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof as maintenance therapy after another. therapy or transplant, in which multiple myeloma is newly diagnosed, multiple myeloma eligible for transplant before the other therapy or transplant. In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof as maintenance therapy after another. therapy or transplant, in which multiple myeloma is newly diagnosed, multiple myeloma eligible for transplant before the other therapy or transplant.
[00162] [00162] In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof as therapy. maintenance after another therapy or transplant. In some modalities, multiple myeloma is a multiple myeloma eligible for transplantation, newly diagnosed before the other therapy and / or transplantation. In some modalities, the other therapy prior to the transplant is treatment with chemotherapy or Compound 1, Compound 2 or Compound 3. In certain modalities, methods of treatment, prevention and / or management of multiple myeloma are provided here, comprising administering to a patient a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof as maintenance therapy after another therapy or transplant. In some modalities, multiple myeloma is a multiple myeloma eligible for transplantation, newly diagnosed before the other therapy and / or transplantation. In some modalities, the other therapy prior to the transplant is treatment with chemotherapy or Compound 1, Compound 2 or Compound 3. In certain modalities, methods of treatment, prevention and / or management of multiple myeloma are provided here, comprising administering to a patient a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof as maintenance therapy after another therapy or transplant. In some modalities, multiple myeloma is a multiple myeloma eligible for transplantation, newly diagnosed before the other therapy and / or transplantation. In some modalities, the other therapy prior to transplantation is treatment with chemotherapy or Compound 1, Compound 2 or Compound 3.
[00163] [00163] In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein multiple myeloma is a high-risk multiple myeloma that is relapsed or refractory to one, two or three previous treatments. In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein the multiple myeloma it is a high-risk multiple myeloma, which relapses or is refractory to one, two or three previous treatments. In certain modalities, methods of treatment, prevention and / or management of multiple myeloma are provided here,
[00164] [00164] In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein multiple myeloma is a newly diagnosed multiple myeloma ineligible for transplantation. In certain embodiments, methods of treatment, prevention and / or management of multiple myeloma are provided herein, comprising administering to a patient a therapeutically effective amount of Compound 2, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, wherein the multiple myeloma is multiple myeloma ineligible for transplant, newly diagnosed. In certain modalities, methods of treatment, prevention and / or management of multiple myeloma are provided here, comprising administering to a patient a therapeutically effective amount of Compound 3, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, in which the multiple myeloma is multiple myeloma ineligible for transplant, recently diagnosed.
[00165] [00165] In certain embodiments, a therapeutically or prophylactically effective amount of the compound is from about 0.01 to about 25 mg per day, from about 0.01 to about 10 mg per day, from about 0.01 about 5 mg per day, about 0.01 to about 2 mg per day, about 0.01 to about 1 mg per day, about 0.01 to about 0.5 mg per about 0.01 to about 0.25 mg per day, about 0.1 to about 25 mg per day, about 0.1 to about 10 mg per day, about 0 , 1 to about 5 mg per day, from about 0.1 to about 2 mg per day, from about 0.1 to about 1 mg per day, from about 0.1 to about 0.5 mg per day, from about 0.1 to about 0.25 mg per day, from about 0.5 to about 25 mg per day, from about 0.5 to about 10 mg per day, from about from 0.5 to about 5 mg per day, from about 0.5 to about 2 mg per day, from about 0.5 to about 1 mg per day, from 1 to 25 mg per day, from 1st 10 mg per day, from 1 to 5 mg per day, from 1 to 2.5 mg per day, or from c about 1 to about 2 mg per day. In one embodiment, a therapeutically or prophylactically effective amount of Compound 1, Compound 2 or Compound 3 is from about 0.1 mg per day to about 0.4 mg per day.
[00166] [00166] In certain embodiments, the therapeutically or prophylactically effective amount is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6 , about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8 , about 9, about 10, about 15, about 20 or about 25 mg per day. In some of these embodiments, the therapeutically or prophylactically effective amount is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6 or about 0.7 mg per day.
[00167] [00167] In one embodiment, the recommended daily dose range for Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, for the conditions described here, in the range of about 0.1 mga to about 25 mg per day, preferably administered as a single dose once a day or in divided doses throughout the day. In other embodiments, the dosage ranges from about 0.1 to about 10 mg per day. Specific doses per day include 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16,
[00168] [00168] In a specific embodiment, the recommended initial dosage can be 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20 or 25 mg per day. In another embodiment, the recommended initial dosage can be 0.1, 0.2, 0.3, 0.4 or 0.5, mg per day. The dose can be increased to 1, 2, 3, 4 or 5 mg per day.
[00169] [00169] In certain embodiments, the therapeutically or prophylactically effective amount is from about 0.001 to about 5 mg / kg / day, from about 0.001 to about 4 mg / kg / day, from about 0.001 to about 3 mg / kg / day, from about 0.001 to about 2 mg / kg / day, from about 0.001 to about 1 mg / Kkg / day, from about 0.001 to about 0.05 mg / kg / day, about 0.001 to about 0.04 mg / kg / day, about 0.001 to about 0.03 mg / kg / day, about 0.001 to about 0.02 mg / kg / day, about from 0.001 to about 0.01 mg / kg / day, or from about 0.001 to about 0.005 mg / kg / day.
[00170] [00170] The administered dose can also be expressed in units other than mg / kg / day. For example, doses for parenteral administration can be expressed as mg / m / Day. A person skilled in the art will readily know how to convert doses from mg / kg / day to mg / m / Day to give the height or weight of a subject or both (see, www. Fda. Gov / cder / cancer / animalframe. Htm ). For example, a dose of 1 mg / kg / day for a 65 kg human is approximately equal to 38 mg / m / Day.
[00171] [00171] In certain embodiments, the patient to be treated with one of the methods provided here was not treated with multiple myeloma therapy prior to administration of Compound 1, Compound 2 or Compound 3 provided here, or an enantiomer, mixture of enantiomers, tautomer , isotopologist, or pharmaceutically acceptable salt thereof. In certain embodiments, the patient to be treated with one of the methods provided herein was treated with multiple myeloma therapy prior to administration of Compound 1, Compound 2 or Compound 3 provided herein or an enantiomer, mixture of enantiomers, tautomer, isotopologist, or salt pharmaceutically acceptable product. In certain embodiments, the patient to be treated with one of the methods provided herein has developed drug resistance for multiple anti-myeloma therapy. In some of these modalities, the patient developed resistance to one, two or three anti-myeloma multiple therapies, in which the therapies are selected from a CD38 monoclonal antibody (CD38 mAb, for example, daratumumab or isatuximab), a proteasome inhibitor (for bortezomib, carfilzomib, ixazomib or marizomib) and an immunomodulatory compound (eg, thalidomide, lenalidomide, pomalidomide, iberdomide or avadomide).
[00172] [00172] The methods provided here cover the treatment of a patient, regardless of the patient's age. In some modalities, the subject is 18 years old or older. In other modalities, the subject is over 18, 25, 35, 40, 45, 50, 55, 60, 65 or 70 years old. In other modalities, the subject is under 65 years of age. In other modalities, the subject is over 65 years of age. In one embodiment, the subject is an elderly subject with multiple myeloma, such as a subject over 65 years of age. In one embodiment, the subject is an elderly subject with multiple myeloma, such as a subject over 75 years of age.
[00173] [00173] Depending on the condition of the disease to be treated and the condition of the individual, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, may be administered orally, parenterally (eg, intramuscular, intraperitoneal, intravenous, IVC, intracystemal injection or infusion, subcutaneous injection or implant), routes of administration by inhalation, nasal, vaginal, rectal, sublingual or topical (for example, transdermal or local). Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, can be formulated, alone or together, in a suitable dosage unit with excipients, carriers, adjuvants and pharmaceutically acceptable vehicles appropriate for each route of administration.
[00174] [00174] In one embodiment, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered orally. In another embodiment, the compound of Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered parenterally. In yet another embodiment, the compound of Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered intravenously.
[00175] [00175] Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, may be distributed as a single dose, such as, for example, a single injection in bolus or oral pills or pills; or over time, such as continuous infusion over time or doses divided into boluses over time. The compound as described herein can be administered several times if necessary, for example, until the patient experiences stable disease or regression or until the progression of the patient's disease or unacceptable toxicity. The stable disease or lack of it is determined by methods known in the art,
[00176] [00176] Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, can be administered once daily (QD or qd) or divided into several doses daily, such as twice a day (BID or bid), three times a day (TID or tid) and four times a day (QID or qid). In addition, administration can be continuous (that is, daily for consecutive days or every day), intermittent, for example, in cycles (that is, including days, weeks or months of drug-free rest). As used herein, the term "daily" is intended to mean that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered once or more than once a day, for example, for a period of time. The term "continuous" means that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered daily for an uninterrupted period of at least 7 days to 52 weeks. The term "intermittent" or "intermittently", as used herein, means to stop and start at regular or irregular intervals. For example, intermittent administration of Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administration for one to six days a week, administration in cycles ( for example, daily administration for two to eight consecutive weeks, rest period without administration for up to one week) or administration on alternate days. The term "cycling", as used herein, is intended to mean that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered daily or continuously, but with a rest period. In some of these modalities, administration is once a day for two to six days, then a rest period without administration for five to seven days.
[00177] [00177] In some modalities, the frequency of administration is in the range of about one daily dose to about one monthly dose. In certain modalities, administration is once a day, twice a day, three times a day, four times a day, once every two days, twice a week, once every week, once every two weeks, once every three weeks or once every four weeks. In one embodiment, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered once daily. In another embodiment, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered twice daily. In yet another embodiment, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered three times a day. In yet another embodiment, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered four times a day.
[00178] [00178] In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 20 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 15 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 10 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 7 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 5 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 4 days, followed by a rest period. In one embodiment, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered in a treatment cycle that includes an administration period of up to 3 days, followed by a rest period.
[00179] [00179] In one embodiment, the treatment cycle includes an administration period of up to 14 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of up to days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of up to 7 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of up to 5 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of up to 4 days, followed by a rest period. In one embodiment, the treatment cycle includes an administration period of up to 3 days, followed by a rest period.
[00180] [00180] In one modality, the rest period is from about 2 days to about 11 days. In one embodiment, the rest period is about 2 days to about 10 days. In one embodiment, the rest period is about 2 days. In one embodiment, the rest period is about 3 days. In one embodiment, the rest period is about 4 days. In one embodiment, the rest period is about 5 days. In one mode, the rest period is about 6 days. In another mode, the rest period is about 7 days. In another mode, the rest period is about 8 days. In another mode, the rest period is about 9 days. In another mode, the rest period is about 10 days. In another mode, the rest period is about 11 days.
[00181] [00181] In one embodiment, the treatment cycle includes an administration period of up to 15 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of up to 10 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of up to 7 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of up to 5 days, followed by a rest period of about 2 days to about 10 days. In one embodiment, the treatment cycle includes an administration period of up to 3 days, followed by a rest period of about 10 days to about 15 days. In one embodiment, the treatment cycle includes an administration period of up to 3 days, followed by a rest period of about 3 days to about 15 days.
[00182] [00182] In one embodiment, the treatment cycle includes an administration period of up to 15 days, followed by a 7-day rest period. In one embodiment, the treatment cycle includes an administration period of up to 10 days, followed by a rest period of 5 days. In one embodiment, the treatment cycle includes an administration period of up to days, followed by a 4-day rest period. In one embodiment, the treatment cycle includes an administration period of up to 10 days, followed by a 3-day rest period. In one embodiment, the treatment cycle includes an administration period of up to 10 days, followed by a 2-day rest period. In one embodiment, the treatment cycle includes an administration period of up to 7 days, followed by a 7-day rest period. In one embodiment, the treatment cycle includes an administration period of up to 5 days, followed by a rest period of 5 days. In one embodiment, the treatment cycle includes an administration period of up to 3 days, followed by an 11-day rest period. In another embodiment, the treatment cycle includes an administration period of up to 5 days, followed by a rest period of 9 days. In another embodiment, the treatment cycle includes an administration period of up to 5 days, followed by a 2-day rest period. In another embodiment, the treatment cycle includes an administration period of up to 3 days, followed by a 4-day rest period.
[00183] [00183] In one embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 5 of a 28-day cycle.
[00184] [00184] In one embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 through 14 of a 21-day cycle. In another embodiment, the treatment cycle includes an administration of Compound 1, Compound 2 or Compound 3 on days 1a 4 and 8 to 11 of a 21 day cycle. In one embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 5 and 8 to 12 of a 21-day cycle. In another embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 5 and 11 to 15 of a 21-day cycle. In another embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 5, 8 to 12 and 15 to 19 of a 21-day cycle. In one embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1a 4, 8a 11 and 15a 18 of a 21 day cycle. In another embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 4, 8 to 10 and 15 to 17 of a 21-day cycle. In another embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 3 and 8 to 11 of a 21-day cycle. In another embodiment, the treatment cycle includes administration of a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 on days 1 to 3 and 11 to 13 of a 21-day cycle.
[00185] [00185] Any treatment cycle described here can be repeated for at least 2, 3, 4, 5, 6, 7, 8 or more cycles. In certain cases, the treatment cycle as described herein includes from 1 to about 24 cycles, from about 2 to about 16 cycles, or from about 2 to about 4 cycles. In certain cases, a treatment cycle as described herein includes from 1 to about 4 cycles. In certain modalities, cycle 1 to 4 are all 28-day cycles. In some embodiments, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3 is administered over 1 to 13 28-day cycles (e.g., about 1 year). In certain cases, cycle therapy is not limited to the number of cycles and therapy is continued until the disease progresses. Cycles may, in certain cases, include varying the length of the administration periods and / or the rest periods described herein.
[00186] [00186] In one embodiment, the treatment cycle includes administering Compound 1, Compound 2 or Compound 3 in a dosage amount of about 0.1 mg / day, 0.2 mg / day, 0.3 mg / day, 0.4 mg / day, 0.5 mg / day, 0.6 mg / day, 0.7 mg / day, 0.8 mg / day, 0.9 mg / day, 1.0 mg / day, 5 , 0 mg / day or 10 mg / day, administered once daily. In one embodiment, the treatment cycle includes administering Compound 1, Compound 2 or Compound 3 in a dosage amount of about 0.1 mg / day, 0.2 mg / day, 0.3 mg / day, 0.4 mg / day, 0.5 mg / day, 0.6 mg / day, 0.7 mg / day, or 0.8 mg / day, administered once daily. In some of these modalities, the treatment cycle includes administering Compound 1, Compound 2 or Compound 3 once daily in a dosage amount of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg or 0.5 mg on days 1 to 10 of a 28-day cycle. In some of these modalities, the treatment cycle includes administering Compound 1, Compound 2 or Compound 3 once daily in a dosage amount of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg or 0.5 mg on days 1 to 10 and 15 to 24 of a 28-day cycle.
[00187] [00187] Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, may also be combined or used in conjunction with (for example, before, during or after) conventional therapy, including, but not limited to, surgery, biological therapy (including immunotherapy, for example, immunotherapy, for example, with checkpoint inhibitors), radiation therapy, chemotherapy,
[00188] [00188] As discussed here elsewhere, covered here is a method to reduce, treat and / or prevent the adverse or unwanted effects associated with conventional therapy, including, but not limited to surgery, chemotherapy, radiation therapy, biological therapy and immunotherapy . A compound provided herein, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof and another active ingredient can be administered to a patient before, during or after the occurrence of the adverse effect associated with conventional therapy.
[00189] [00189] Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, may also be combined or used in combination with other therapeutic agents useful in the treatment and / or prevention of multiple myeloma described here.
[00190] [00190] In one embodiment, a method of treatment, "prevention or management of multiple myeloma, is provided here, comprising administering to a patient Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologist or salt pharmaceutically acceptable thereof, in combination with one or more second active agents and, optionally, in combination with radiation therapy, blood transfusions or surgery.
[00191] [00191] As used herein, the term "in combination" includes the use of more than one therapy (for example, one or more prophylactic and / or therapeutic agents). However, the use of the term "in combination" does not restrict the order in which therapies (for example, prophylactic and / or therapeutic agents) are administered to a patient with a disease or disorder. A first therapy (for example, a prophylactic or therapeutic agent, such as a compound provided herein, for example, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof) can be administered before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before), concomitantly or after (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours ), 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks after) the administration of a second therapy (for example, a prophylactic or therapeutic agent) to the subject. Triple therapy is also contemplated here, as is quadruple therapy. In one embodiment, the second therapy is dexamethasone.
[00192] [00192] The administration of Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, and one or more second active agents for a patient can occur simultaneously or sequentially by the same or different administration routes. The suitability of a particular route of administration employed for a particular active agent will depend on the active agent itself (for example, whether it can be administered orally without decomposing before entering the bloodstream).
[00193] The route of administration of Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is independent of the route of administration of a second therapy. In one embodiment, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered orally. In another embodiment, Compound 1, Compound 2 or Compound 3 is administered intravenously. Thus, according to these modalities, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered orally or intravenously, and the second therapy can be administered orally, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbucal, intranasal, liposomal, via inhalation, vaginal, intraocular, via local administration via catheter or stent, subcutaneously, intra-adiposally, intra-articularly, intrathecally, or slow-release dosage form. In one embodiment, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, and a second therapy is administered by the same method of administration, orally or IV. In another embodiment, Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered by a mode of administration, for example, by IV, while the second agent ( a multiple anti-myeloma) is administered by another mode of administration, for example, orally.
[00194] [00194] In one embodiment, the second active agent is administered intravenously or subcutaneously and, once or twice a day in an amount of about 1 to about 1000 mg, from about 5 to about 500 mg, about 10 to about 350 mg, or about 50 to about 200 mg. The specific amount of the second active agent will depend on the specific agent used, the type of multiple myeloma being treated or managed, the severity and stage of the disease and the amount of Compound 1, Compound 2 or Compound 3 or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt! provided herein and any optional additional active agents administered simultaneously to the patient.
[00195] [00195] One or more second active ingredients or agents can be used in conjunction with Compound 1, Compound 2 or Compound 3 in the methods and compositions provided herein. The second active agents can be large molecules (for example, proteins) or small molecules (for example, synthetic inorganic, organometallic or organic molecules), or cellular therapies (for example, CAR cells).
[00196] [00196] Examples of second active agents that can be used in the methods and compositions described herein include one or more of melphalan, vincristine, —cyclophosphamide, etoposide, doxorubicinay bendamustine, obinutuzmab, a proteasome inhibitor (eg, bortezomib, carfilzomib, ixazomib , oprozomib or marizomib), a histone deacetylase inhibitor (eg, panobinostat, ACY241), a BET inhibitor (eg, GSK525762A, OTX015, BMS-986158, TEN-O10, CPI-O610, INCB54329, BAY1238097, FT- 1101, ABBV-075, BI 894999, GS-5829, GSK1210151A (| -BET-151), CPI-203, RVX-208, XD46, MS436, PFI-1, RVX2135, ZEN3365, XD14, ARV-
[00197] [00197] In one embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is dexamethasone .
[00198] [00198] In some modalities, dexamethasone is administered at a dose of 4 mg on days 1 and 8 of a 21-day cycle. In some other modalities, dexamethasone is administered at a dose of 4 mg on days 1,4, 8 and 11 of a 21-day cycle. In some modalities, dexamethasone is administered at a dose of 4 mg on days 1, 8 and 15 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 4 mg on days 1, 4, 8, 11, 15 and 18 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 4 mg on days 1, 8, 15 and 22 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 4 mg on days 1, 10, 15 and 22 of Cycle 1. In some modalities, dexamethasone is administered at a dose of 4 mg on days 1, 3, 15 and 17 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 4 mg on days 1, 3, 14 and 17 of Cycle 1.
[00199] [00199] In some other modalities, dexamethasone is administered at a dose of 8 mg on days 1 and 8 of a 21-day cycle. In some other modalities, dexamethasone is administered at a dose of 8 mg on days 1,4, 8 and 11 of a 21-day cycle. In some modalities, dexamethasone is administered at a dose of 8 mg on days 1, 8 and 15 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 8 mg on days 1, 4, 8, 11, 15 and 18 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 8 mg on days 1, 8, 15 and 22 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 8 mg on days 1, 10, 15 and 22 of Cycle 1. In some modalities, dexamethasone is administered at a dose of 8 mg on days 1, 3, 15 and 17 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 8 mg on days 1, 3, 14 and 17 of Cycle 1.
[00200] [00200] In some modalities, dexamethasone is administered at a dose of 10 mg on days 1 and 8 of a 21-day cycle. In some other modalities, dexamethasone is administered at a dose of 10 mg on days 1,4, 8 and 11 of a 21-day cycle. In some modalities, dexamethasone is administered at a dose of 10 mg on days 1, 8 and 15 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 10 mg on days 1, 4, 8, 11, 15 and 18 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 10 mg on days 1, 8, 15 and 22 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 10 mg on days 1, 10, 15 and 22 of Cycle 1. In some modalities, dexamethasone is administered at a dose of 10 mg on days 1, 3, 15 and 17 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 10 mg on days 1, 3, 14 and 17 of Cycle 1.
[00201] [00201] In some modalities, dexamethasone is administered at a dose of 20 mg on days 1 and 8 of a 21-day cycle. In some other modalities, dexamethasone is administered at a dose of 20 mg on days 1,4, 8 and 11 of a 21-day cycle. In some modalities, dexamethasone is administered at a dose of 20 mg on days 1, 8 and 15 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 20 mg on days 1, 4, 8, 11, 15 and 18 of a 28-day cycle. In some modalities, dexamethasone is administered at a dose of 20 mg on days 1, 8, 15 and 22 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 20 mg on days 1, 10, 15 and 22 of Cycle 1. In some modalities, dexamethasone is administered at a dose of 20 mg on days 1, 3, 15 and 17 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 20 mg on days 1, 3, 14 and 17 of Cycle 1.
[00202] [00202] In some modalities, dexamethasone is administered at a dose of 40 mg on days 1 and 8 of a 21-day cycle. In some other modalities, dexamethasone is administered at a dose of 40 mg on days 1,4, 8 and 11 of a 21-day cycle. In some modalities, dexamethasone is administered at a dose of 40 mg on days 1, 8 and 15 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 40 mg on days 1, 10, 15 and 22 of Cycle 1. In some modalities, dexamethasone is administered at a dose of 40 mg on days 1, 4, 8, 11, 15 and 18 of a 28-day cycle. In other of these modalities, dexamethasone is administered at a dose of 40 mg on days 1, 8, 15 and 22 of a 28-day cycle. In other of these modalities, dexamethasone is administered at a dose of 40 mg on days 1, 3, and 17 of a 28-day cycle. In one of these modalities, dexamethasone is administered at a dose of 40 mg on days 1, 3, 14 and 17 of Cycle 1.
[00203] [00203] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is bortezomib. In yet another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is daratumumab . In some of these modalities, the methods additionally comprise administering dexamethasone. In some embodiments, the methods comprise administering Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, with a proteasome inhibitor as described herein, a CD38 inhibitor as described herein and a corticosteroid as described herein.
[00204] [00204] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt! likewise, in the methods and compositions described here is panobinostat. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00205] [00205] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is ACY241. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00206] [00206] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is vincristine. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00207] [00207] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt! likewise, in the methods and compositions described herein it is cyclophosphamide. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00208] [00208] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is etoposide. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00209] [00209] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt! likewise, in the methods and compositions described herein is doxorubicin. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00210] [00210] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is venetoclax. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00211] [00211] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is AMG176. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00212] [00212] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is MIK665. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00213] [00213] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt! likewise, in the methods and compositions described herein is GSK525762A. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00214] [00214] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described herein is OTXO015. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00215] [00215] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described here is 4- [2- (cyclopropylmethoxy) -5- (methanesulfonyl) phenyl] -2-methylisoquinolin-1 (2H) -one. In some of these modalities, the methods additionally comprise administering dexamethasone.
[00216] [00216] In another embodiment, the second active agent used in conjunction with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in the methods and compositions described here is 4- [2- (4-amino-piperidin-1-i1) -5- (3-fluoro-4-methoxy-phenyl) -1-methyl-6-0x0-1,6-dihydropyrimidin-4-yl ] -2-fluoro-benzonitrile or a salt thereof (for example, a besylate salt. In some of these embodiments, the methods additionally comprise administering dexamethasone.
[00217] [00217] In certain embodiments, Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, is administered in combination with checkpoint inhibitors. In one embodiment, a checkpoint inhibitor is used in combination with Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, in connection with the methods provided herein. In another embodiment, two checkpoint inhibitors are used in combination with Compound 1, or a tautomer, isotopologist or pharmaceutically acceptable salt thereof, in connection with the methods provided herein. In yet another embodiment, three or more checkpoint inhibitors are used in combination with Compound 1, Compound 2 or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof, in connection with the methods provided here.
[00218] [00218] As used in this document, the term "immunological checkpoint inhibitor" or "checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins . Without being limited by a particular theory, checkpoint proteins regulate the activation or function of T cells. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with its PD-L1 and PD-L2 ligands (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). These proteins appear responsible for co-stimulatory or inhibitory interactions of T cell responses. The proteins at the immunological checkpoint appear to regulate and maintain self-tolerance and the duration and breadth of physiological immune responses. Immunological checkpoint inhibitors include antibodies or are derived from antibodies.
[00219] [00219] In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in US Patents 5,811,097; 5,811,097; 5,855,887;
[00220] [00220] In one embodiment, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Examples of PD-1 / PD-L1 inhibitors include, but are not limited to, those described in US Patents 7,488,802; 7,943,743;
[00221] [00221] In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-
[00222] [00222] In one embodiment, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-
[00223] [00223] In one embodiment, the checkpoint inhibitor is a PD-L2 inhibitor. In one embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD-L2 antibody is rHlIgM12B7A.
[00224] [00224] In one embodiment, the checkpoint inhibitor is an inhibitor of the lymphocyte-3 activation gene (LAG-3). In one embodiment, the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., |. Immunol., 2007, 179, 4202-4211). In another embodiment, the LAG-3 inhibitor is BMS-986016.
[00225] [00225] In one embodiment, checkpoint inhibitors are a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one embodiment, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834).
[00226] [00226] In one embodiment, checkpoint inhibitors are TIM3 inhibitors (T cell immunoglobulin domain and mucin 3 domain) (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).
[00227] [00227] In one embodiment, the checkpoint inhibitor is an OX40 agonist (CD134). In one embodiment, the checkpoint inhibitor is an anti-OX40 antibody. In one embodiment, the anti-OXA40 antibody is anti-OX-40. In another embodiment, the anti-OX40 antibody is MEDI6469.
[00228] [00228] In one embodiment, the checkpoint inhibitor is a GITR agonist. In one embodiment, the checkpoint inhibitor is an anti-GITR antibody. In one embodiment, the anti-GITR antibody is TRX518.
[00229] [00229] In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab. In another embodiment, the anti-CD137 antibody is PF-05082566.
[00230] [00230] In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD40 antibody. In one embodiment, the anti-CD40 antibody is CF-870,893.
[00231] [00231] In one embodiment, the checkpoint inhibitor is recombinant human interleukin-15 (rhlL-15).
[00232] [00232] In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCBO24360. In another embodiment, the IDO inhibitor is indoximod.
[00233] [00233] In certain embodiments, the combination therapies provided herein include two or more checkpoint inhibitors described herein (including checkpoint inhibitors of the same or different classes). In addition, the combined therapies described herein can be used in combination with one or more second active agents as described herein, where appropriate, for the treatment of diseases described and understood herein in the art.
[00234] [00234] In certain embodiments, Compound 1, Compound 2 or Compound 3 can be used in combination with one or more immune cells that express one or more chimeric antigen receptors (CARs) on its surface (for example, a modified immune cell ). CARs generally comprise an extracellular domain of a first protein (e.g., an antigen binding protein), a transmembrane domain, and an intracellular signaling domain. In certain embodiments, since the extracellular domain binds to a target protein, such as a tumor-associated antigen (TAA) or tumor-specific antigen (TSA), a signal is generated through the intracellular signaling domain that activates the immune cell , for example, to target and kill a cell that expresses the target protein.
[00235] [00235] Extracellular domains: The extracellular domains of CARs bind to an antigen of interest. In certain embodiments, the extracellular domain of the CAR comprises a receptor, or a portion of a receptor, that binds to said antigen. In certain embodiments, the extracellular domain comprises, or is, an antibody or antigen-binding portion thereof. In specific embodiments, the extracellular domain comprises, or is, a single chain Fv (scFv) domain. The single chain Fv domain can comprise, for example, a V. linked to Vy by a flexible linker, wherein said V. and Vu are from an antibody that binds to said antigen.
[00236] [00236] In certain embodiments, the antigen recognized by the extracellular domain of a polypeptide described herein is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In various modalities, the tumor-associated antigen or tumor-specific antigen is, without limitation, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), CA19-9, calretinin, MUC-1, B cell maturation antigen (BCMA), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen 24 (MAGE), CD19, CD22, CD27, CD30, CD34, CD45, CD70, CD99, CD117, EGFRvIll (epidermal growth factor variant | ll), mesothelin, PAP (prostatic acid phosphatase), prosteine, TARP alternative reading structure of the gamma T cell receptor), Trp-p8, STEAPI (epithelial antigen of six prostate transmembrane 1), chromogranin, cytokeratin, desmin, glial fibrillar acid protein (GFAP), fluid protein for thick cystic disease (GCDFP -15), HMB-45 antigen,
[00237] [00237] In certain embodiments, the TAA or TSA recognized by the extracellular domain of a CAR is a cancer / testis (CT) antigen, for example, BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXBI, SPA17, SSX, SYCPI or TPTE.
[00238] [00238] In certain other modalities, the TAA or TSA recognized by the extracellular domain of a CAR is a carbohydrate or ganglioside, for example, fuc-GMl, GM2 (oncophetic-immunogenic antigen-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3 and the like.
[00239] [00239] In certain other embodiments, the TAA or TSA recognized by the extracellular domain of a CAR is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15 -3 (CA 27. 29) BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-l, dek-can fusion protein, EBNA, EF2, antigens of the Epstein Barr virus, fusion protein ETV6-AML1, HLA-A2, HLA-AII, hsp70-2, KIAAO205, Mart2, Mum-1, 2 and 3, neo-PAP, myosin class |, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triosphosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso -1 / Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-Il, MAGE-3, RAGE, GAGE-I, GAGE-2, p15 (58), RAGE, SCP-1, Hom / Mel-40,
[00240] [00240] In several specific modalities, the tumor-associated antigen or the tumor-specific antigen are AML-related tumor antigens, as described in S. Anguille et al, Leukemia (2012), 26, 2186-
[00241] [00241] Other tumor-specific and tumor-specific antigens are known to those skilled in the art.
[00242] [00242] Receptors, antibodies and scFvs that bind to TSAs and TAAs, useful in the construction of chimeric antigen receptors, are known in the art, as are the nucleotide sequences that encode them.
[00243] [00243] In certain specific embodiments, the antigen recognized by the extracellular domain of a chimeric antigen receptor is an antigen not generally considered to be a TSA or a TAA, but which is nevertheless associated with tumor cells, or damage caused by a tumor. In certain embodiments, for example, the antigen is, for example, a growth factor, cytokine or interleukin, for example, a growth factor, cytokine or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines or interleukins may include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF ), insulin-like growth factor (IGF) or interleukin-8 (IL-8). Tumors can also create a local hypoxic environment for the tumor. As such, in other specific modalities, the antigen is a factor associated with hypoxia, for example, HIF-1a, HIF-1B, HIF-20, HIF-2B, HIF-3a or HIF-3B. Tumors can also cause localized damage to normal tissue, causing the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarms). In certain other specific modalities, therefore, the antigen is a DAMP, for example, a heat shock protein, group 1 of high mobility of chromatin-associated protein (HMGB 1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum A amyloid (SAA), or it can be an acid deoxyribonucleic, adenosine triphosphate, uric acid or heparin sulfate.
[00244] [00244] Transmembrane domain: In certain embodiments, the extracellular domain of the CAR is linked to the transmembrane domain of the polypeptide by a hinge, spacer or polypeptide hinge sequence, for example, a CD28 sequence or a CTLA4 sequence. The transmembrane domain can be obtained from or derived from the transmembrane domain of any transmembrane protein and can include all or a portion of that transmembrane domain. In specific embodiments, the transmembrane domain can be obtained or derived from, for example, CD8, CD16, a cytokine receptor and interleukin receptor, or a growth factor receptor, or the like.
[00245] [00245] Intracellular signaling domains: In certain embodiments, the intracellular domain of a CAR is or comprises an intracellular domain or motif of a protein that is expressed on the surface of T cells and triggers the activation and / or proliferation of said T cells. Such a domain or motif is capable of transmitting a binding signal to the primary antigen that is necessary for the activation of a T lymphocyte in response to the binding of the antigen to the extracellular portion of the CAR. Typically, this domain or motif comprises, or is, an ITAM (activation motif based on immunoreceptor tyrosine). Suitable ITAM-containing polypeptides for CARs include, for example, the CD3 zeta chain (CD37) or portions thereof containing ITAM. In a specific embodiment, the intracellular domain is an intracellular signaling domain of CD37. In other specific embodiments, the intracellular domain is a chain of lymphocyte receptors, a complex TCR / CD3 protein, a Fe receptor subunit, or a 1L-2 receptor subunit. In certain embodiments, the CAR further comprises one or more co-stimulating domains or motifs, for example, as part of the intracellular domain of the polypeptide. The one or more co-stimulating domains or motifs may be, or may comprise, one or more of a co-stimulating CD27 polypeptide sequence, a co-stimulating CD28 polypeptide sequence, a co-stimulating OX40 polypeptide sequence (CD134), a 4-1BB (CD137) co-stimulating polypeptide sequence , or an inducible co-stimulating T cell (ICOS) polypeptide sequence, or other co-stimulating domain or motif, or any combination thereof.
[00246] [00246] CAR may also comprise a T cell survival motive. The T cell survival motive can be any polypeptide sequence or motif that facilitates T lymphocyte survival after stimulation by an antigen. In certain embodiments, the T-cell survival motive is or is derived from CD3, CD28, an IL-7 receptor intracellular signaling domain (IL-7R), an IL-12 receptor intracellular signaling domain, an intracellular signaling domain of the IL-15 receptor, an intracellular signaling domain of the IL-21 receptor, or an intracellular signaling domain of the transforming growth factor B (TGFB) receptor.
[00247] [00247] The modified immune cells that express the CARs can be, for example, T lymphocytes (T cells, for example, CD4 + T cells or CD8 + T cells), cytotoxic lymphocytes (CTLs) or natural killer cells (NK). The T lymphocytes used in the compositions and methods provided herein can be naive T lymphocytes or MHC restricted T lymphocytes. In certain embodiments, T lymphocytes are tumor-infiltrating lymphocytes (TILs). In certain embodiments, T lymphocytes were isolated from a tumor biopsy or were expanded from T lymphocytes isolated from a tumor biopsy. In certain other embodiments, T cells have been isolated or are expanded from isolated T lymphocytes from peripheral blood, umbilical cord blood or lymph. Immune cells to be used to generate modified immune cells expressing a CAR can be isolated using routine methods accepted by the technique, for example, blood collection followed by apheresis and, optionally, isolation or screening of antibody-mediated cells.
[00248] [00248] The modified immune cells are preferably autologous to an individual to whom the modified immune cells must be administered. In certain other embodiments, the modified immune cells are allogeneic to an individual to whom the modified immune cells must be administered. When allogeneic T lymphocytes or NK cells are used to prepare modified T lymphocytes, it is preferable to select T lymphocytes or NK cells that reduce the possibility of graft versus host disease (GVHD) in the individual. For example, in certain embodiments, virus-specific T lymphocytes are selected for the preparation of modified T lymphocytes; such lymphocytes are expected to have a greatly reduced native ability to bind to, and thus become activated by, any receptor antigens. In certain embodiments, rejection mediated by allogeneic T lymphocyte receptors can be reduced by co-administering to the host one or more immunosuppressive agents, for example, cyclosporine, tacrolimus, sirolimus, cyclophosphamide or the like.
[00249] [00249] T lymphocytes, for example, unmodified T lymphocytes, or T lymphocytes expressing CD3 and CD28, or comprising a polypeptide comprising a CD36 signaling domain and a CD28 costimulatory domain, can be expanded using antibodies to CD3 and CD28, for example , granule-bound antibodies; see, for example, US Patents
[00250] [00250] The modified immune cells, for example, modified T lymphocytes, can optionally comprise a "suicide gene" or "safety switch" that allows the death of substantially all of the modified immune cells when desired. For example, modified T lymphocytes, in certain embodiments, may comprise an HSV thymidine kinase gene (HSV-TK), which causes the death of modified T lymphocytes upon contact with ganciclovir. In another embodiment, the modified T lymphocytes comprise an inducible caspase, for example, an inducible caspase 9 (icaspase 9), for example, a fusion protein between caspase 9 and human FK506 binding protein allowing dimerization using a specific small molecule pharmaceutical company. See Straathof et al., Blood 1 O5 (11): 4247-4254 (2005).
[00251] [00251] In certain embodiments, Compound 1, Compound 2 or Compound 3 as provided herein is administered to patients with various types or stages of multiple myeloma in combination with chimeric antigen receptor (CAR) T cells. In certain embodiments, the T CAR cell in the combination targets the B cell maturation antigen (BCMA) and, in more specific embodiments, the T CAR cell is bb2121 or bb21217. In some embodiments, the T CAR cell is JCARH125.
[00252] [00252] The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the compounds provided herein and, optionally, a pharmaceutically acceptable carrier, diluent or excipient.
[00253] [00253] The compounds can be formulated in suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release formulations or elixirs, for oral administration or in sterile solutions or suspensions for ophthalmic or parenteral administration , as well as preparation of transdermal patch and dry powder inhalers. Typically, the compounds described above are formulated in pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition (1999).
[00254] [00254] In the compositions, the effective concentrations of one or more compounds or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical carrier or carrier. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivering an amount, upon administration, which treats, prevents or ameliorates one or more of the symptoms and / or progression of multiple myeloma.
[00255] [00255] Typically, the compositions are formulated for single dose administration. To formulate a composition, the weight fraction of the compound is dissolved, suspended, dispersed or mixed in a selected vehicle at an effective concentration, so that the treated condition is alleviated or improved. Pharmaceutical carriers or carriers suitable for administering the compounds provided herein include any carriers known to those skilled in the art to be suitable for the specific mode of administration.
[00256] [00256] Furthermore, the compounds can be formulated as a pharmaceutically unique active ingredient in the composition or can be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as known in the art. Briefly, liposomes such as multilamellar vesicles (MLV's) can be formed by drying egg phosphatidylcholine and cerebral serine phosphatidyl (7: 3 molar ratio) inside a bottle. A solution of a compound provided herein in phosphate buffered saline without divalent cations (PBS) is added and the flask is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove the non-encapsulated compound, pelleted by centrifugation, and then resuspended in PBS.
[00257] [00257] The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects in the treated patient. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in vivo systems described herein and then extrapolated to human dosages.
[00258] [00258] The concentration of the active compound in the pharmaceutical composition will depend on the rates of absorption, tissue distribution, inactivation, metabolism and excretion of the active compound, the physical-chemical characteristics of the compound, the dosage schedule and the amount administered, as well as others factors known to those skilled in the art. For example, the amount delivered is sufficient to improve one or more of the symptoms of cancer, including solid tumors and blood-borne tumors.
[00259] [00259] Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include any of the following components: a sterile diluent, such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol, dimethylacetamide or others synthetic solvents; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylene diaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, pens, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.
[00260] [00260] In cases where the compounds exhibit insufficient solubility, methods can be used to solubilize the compounds. Such methods are known to those skilled in the art, and include, but are not limited to the use of co-solvents, such as dimethyl sulfoxide (DMSO), using surfactants, such as TWEENÂ °, or dissolution in aqueous sodium bicarbonate.
[00261] [00261] After mixing or adding the compounds, the resulting mixture can be a solution, suspension, emulsion or the like. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to alleviate the symptoms of the disease, disorder or condition treated and can be empirically determined.
[00262] [00262] Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions and oral solutions or suspensions and oral emulsions and water emulsions and oil containing adequate amounts of the compounds or - pharmaceutically - acceptable salts thereof. The pharmaceutically active compounds and salts thereof are formulated and administered in unit dosage forms or multiple dosage forms. Unit dose forms, as used herein, refer to physically distinct units, suitable for human and animal individuals and individually packaged, as is known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the necessary pharmaceutical carrier, vehicle or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms can be administered in fractions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dosage form. Examples of multiple dosage forms include bottles, bottles of pills or capsules or bottles of liters or gallons. Thus, the multiple dosage form is a multiple of unit dosage that are not segregated into packaging.
[00263] [00263] Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% can be prepared with the balance consisting of a non-toxic vehicle. For oral administration, a non-toxic pharmaceutically acceptable composition is formed by incorporating any of the excipients normally employed, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate or sodium saccharin. Such compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations, such as, but not limited to, microencapsulated implants and delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, acid polyglycolic, polyiortoesters, polylactic acid and others. Methods for preparing these compositions are known to those skilled in the art.
[00264] [00264] Active compounds or pharmaceutically acceptable salts can be prepared with vehicles that protect the compound against rapid elimination from the body, such as time-release formulations or coatings.
[00265] [00265] The compositions can include other active compounds to obtain desired combinations of properties. The compounds provided herein, or pharmaceutically acceptable salts thereof, as described herein, can also be advantageously administered for therapeutic or prophylactic purposes, together with another pharmacological agent known in the art in general to be of value in the treatment of one or more of the diseases or medical conditions referred to above, such as diseases related to oxidative stress. It is to be understood that such combination therapy is another aspect of the compositions and methods of treatment provided herein.
[00266] [00266] Standard physiological, pharmacological and biochemical procedures are available to test compounds to identify those that have the desired properties, including proliferative anti-myeloma activity and adequate safety profile.
[00267] [00267] Isoindolinone derivatives and their therapeutic uses have been described in, for example, US Patent 8,518. 972. Surprisingly, Compound 1, Compound 2 and Compound 3 exhibit unexpected and beneficial properties, as shown in the Examples section. These beneficial properties include a significant increase in the potency of anti-multiple myeloma, increased levels of apoptosis and a more potent and effective response to the combination with dexamethasone and surprisingly an improved safety profile, as shown by the reduced functional activity at the adrenergic and D2 receptors. dopamine (in vitro as well as in vivo), improved selectivity of cell death (as shown by reduced non-myeloma cell death) and reduced CYP3A4 inhibition.
[00268] [00268] It is understood that the detailed description mentioned above and the attached examples are merely illustrative and should not be considered as limitations on the scope of the matter. Several changes and modifications to the disclosed modalities will be evident to those skilled in the art. Such changes and modifications, including, without limitation, those related to structures - chemicals, substituents, derivatives, intermediates, syntheses, formulations and / or methods of use provided herein, can be made without departing from the spirit and scope. The US patents and publications mentioned herein are incorporated by reference.
[00269] [00269] Certain embodiments of the invention are illustrated in the following non-limiting examples.
[00270] [00270] 2-Amino-5-methoxy-5-oxopentanoic acid. To a suspension of 2-aminopentanedioic acid (250 g, 1.70 mol) in dry methanol (2.5 L) under nitrogen was added trimethylsilyl chloride (277 g, 2.55 mol) for 30 minutes. The resulting clear solution was stirred at room temperature (20 ° C) for 30 minutes. * H NMR showed that the starting material was completely consumed. The reaction mixture was used in the next step without further treatment. * H NMR: 400 MHz CD3OD 65: 4.17-4.15 (m, 1H), 3.71 (s, 3H), 2.70-2.60 (m, 2H), 2.33-2, 25 (m, 2H).
[00271] [00271] 2 - ((tert-Butoxycarbonyl) amino acid) -5-methoxy-5-oxopentanoic acid. To the above solution, triethylamine (275 g, 2.72 mol) and di-tert-butyl dicarbonate (447.35 g, 2.05 mol) were added. The reaction mixture was stirred at 25 ° C for 2 h. The solution was concentrated to dryness, then water (2.5 L) was added to dissolve the residue. The resulting aqueous solution was washed with ethyl acetate (200 ml), then acidified to pH = 3 by HCI (1 N) and extracted with ethyl acetate (1 L x 3). The combined organic layers were washed with brine (800 mL), dried over sodium sulfate, filtered and concentrated to provide 2- (tert-butoxycarbonylamino) -5-methoxy-5-oxo-pentanoic acid (250 g 56% yield, two steps) as a white solid. * H NMR: 400 MHz CD30D 6: 4.18-4.11 (m, 1H), 3.69 (s, 3H), 2.48-2.43 (m, 2H), 2.21-2, 15 (m, 1H), 1.95-1.91 (m, 1H), 1.46 (s, 9H).
[00272] [00272] Methyl 5-amino-4- (tert-butoxycarbonylamino) -5-oxo-pentanoateA a solution of 2- (tert-butoxycarbonylamino) -5-methoxy-5-0xo-pentanoic acid (200 g, 765 mmol) in 1,4-dioxane (1.5 L) were added di-tert-butyl dicarbonate (267 g 1.22 mol) and pyridine (121 g, 1.53 mol). After the reaction mixture was stirred at 25ºC for 30 min, ammonium carbonate (182 g, 2.30 mol) was added to the mixture and stirred for another 16 hours at 25ºC. The organic solvent was removed by rotary evaporation, the residue was acidified by HCI (6 M) to pH = 3 and then extracted with ethyl acetate (800 ml x 3). The combined organic phase was washed with brine (800 ml), dried over sodium sulfate and filtered. The volatile organics were removed under reduced pressure to provide methyl 5-amino-4- (tert-butoxycarbonylamino) -5-oxo-pentanoate (180 g, 90% yield) as a white solid. * H NMR: 400 MHz CDCl3 6: 6.51 (s, 1H), 5.94 (s, 1H), 5.43 (s, 1H), 4.21 (s, 1H), 3.63 (s , 3H), 2.59-2.40 (m, 2H), 2.15-2.11 (m, 1H), 1.94-1.90 (m, 1H), 1.42 (s, 9H ).
[00273] [00273] Methyl 4,5-diamino-5-oxo-pentanoate hydrochloride. A mixture of 5-amino-4- (tert-butoxycarbonylamino) -5-oxo-pentanoate (180 g, 692 mmol) and HCl / ethyl acetate (300 ml, 4 M) was stirred at 25 ° C for 12 h. The precipitated solid was collected by vacuum filtration and washed with ethyl acetate (500 ml) to give methyl 4,5-diamino-5-oxo-pentanoate hydrochloride (130 g, 95% yield) as a white solid. * H NMR: 400 MHz CD3O0D 65: 4.00-3.96 (m, 1H), 3.70 (s, 3H), 2.59-2.52 (m, 2H), 2.22-2, 13 (m, 2H).
[00274] [00274] Methyl 3-hydroxy-2-methyl-benzoate. Four batches (200 g each) were run in parallel. To a solution of 3-hydroxy-2-methyl-benzoic acid (200 g, 1.31 mol) in methanol (4.0 L) was added concentrated sulfuric acid (47.7 g, 486 mmol). The reaction mixture was stirred at 60 ° C for 17 h and was concentrated to 800 ml. The resulting mixture was cooled to 20 ° C and slowly poured into water (400 ml) over 30 minutes. Water (1200 mL) was added at 20 ° C for 3 h and the resulting mixture was stirred at 20 ° C for 1 h. The precipitated solid was collected by vacuum filtration (four combined batches) and was washed three times with water / methanol (1000 mL, 9: 1) or until the filtrate had pH> 3. The solid was dried under vacuum at 45 ° C to give 3-hydroxy-2-methyl-benzoate (700 g, 80.4% yield) as a gray solid. * H NMR: 400 MHz DMSO-ds 5: 9.70 (s, 1H), 7.18 (t, J] = 6.8 Hz, 1H), 7.09 (t, | = 7.6 Hz, 1H), 7.00 (t, J = 6.8 Hz, 1H), 3.81 (s, 3H), 2.29 (s, 3H).
[00275] [00275] Methyl / 3- [tert-butyl (dimethyl) silylJoxy-2-methyl-benzoate. Two batches (240 g each) were run in parallel. To a solution of methyl 3-hydroxy-2-methyl-benzoate (240 g, 1.44 mol) in DMF (1.40 L) was added imidazole (246 g, 3.61 mol) and tert-butyl dimethylsilyl chloride (238 g, 1.58 mol) at 5 ° C. After the addition, the mixture was heated to 20 ° C and stirred for 6 h. Isopropyl acetate (1700 ml) was added and then water (2000 ml) was added slowly while the temperature was kept below 30ºC. The resulting mixture was stirred and the organic phase was separated. The combined organic phase (two combined batches) was washed with water (1700 ml x 3) and concentrated to * 1500 ml (KF <0.05%). The product was stored as an isopropyl acetate solution which was used in the next step without further purification.
[00276] [00276] Methyl 2- (bromomethyl) -3- [tert-butyl (dimethyl) silyl] oxy-benzoate. Two batches (“375 g each) were run in parallel. To the solution of methyl 3- [tert-butyl (dimethyl) silyl] oxy-2-methylbenzoate (* 375 g, 1.34 mol) isopropyl acetate was added N-bromosuccinimide (274 g 154 mol) and azobisisobutyronitrile (4, 40 g 26.8 mmol). The reaction mixture was heated to 70 ° C for at least 1 h and stirred at 70 ° C for 4 h. The reaction mixture was cooled to 20ºC and kept at 20ºC for at least 1 h. The two batches of solid (succinimide) were removed by filtration and washed with isopropyl acetate (700 ml). The filtrate was washed with a solution of sodium sulfite (700 g) in water (6000 ml), followed by water (1500 ml). The organic layer was vacuum distilled at 45 ° C to dryness to give methyl 2- (bromomethyl) -3- [tert-butyl (dimethyl) silylJoxy-benzoate (920 g, 95.5% yield) as a dark orange oil. * H NMR: 400 MHz DMSO-dcs 6: 7.45 (d, J = 6.8 Hz, 1H), 7.36 (t, | = 8.0 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 4.95 (s, 2H), 1.02 (s, 9H), 0.29 (s, 6H).
[00277] [00277] Methyl 5-amino-4- [4- [tert-butyl (dimethyl) silylJoxy-1-oxo-isoindolin-2-yl] -5-oxo-pentanoate. To a stirred solution of methyl 4,5-diamino-5-0x0-pentanoate hydrochloride (74.5 g, 379 mmol) in acetonitrile (2.50 L) was added 2- (bromomethyl) -3- [tert-butyl ( dimethyl) silyl] oxy-benzoate (125 g, 348 mmol). To the suspension, diisopropylethylamine (89.9 g, 696 mmol) was added via an addition funnel over 10 min and then the mixture was stirred at 60 ° C for 16 h. The reaction mixture was diluted with ethyl acetate (1.0 L), washed with HCI (1 N, 1.0 L), sodium bicarbonate (sat. 1.0 L) and brine (1.0 L) successively . The organic layer was concentrated to give crude methyl 5-amino-4- [4- [tert-butyl (dimethyl) silyl] oxy-1-0xo-isoindolin-2-yl] -5-0xo-pentanoate (108 8, crude ) as a light yellow solid. LCMS: m / z 407.3 [M + 1] ”*.
[00278] [00278] Methyl 5-amino-4- (4-hydroxy-1-0xo-isoindolin-2-i1) -5-0x0-pentanoate. To a cold stirred solution of methyl 5-amino-4- [4- [tert-butyl (dimethyl) silylJoxy-1-oxo-isoindolin-2-yl] -5-0xo-pentanoate (108 g, 266 mmol) in N N-dimethylformamide (350 ml) was added potassium carbonate (14.78, 106 mmol) in water (40 ml) in portions over 5 min. The resulting reaction mixture was stirred at 15 ° C for 15 h. The reaction mixture was cooled in an ice bath and HCI (12 M, 15 mL) was added slowly at 0-5 ° C. Acetonitrile (200 ml) was added to the mixture and a precipitate formed. The suspension was stirred at room temperature for 10 min. and filtered. The filter cake was washed with ethyl acetate (200 ml x 5) to give the product (55 g). The filtrate was concentrated under high vacuum to give a crude product (100 g) which was dissolved in dichloromethane (1.0 L) and left to stand at 15 ° C for 16 hours. A white solid was formed which was filtered to give 5 g of product. The solids were combined to give 5-amino-4- (4-hydroxy-1-0x0-
[00279] [00279] Methyl 5-amino-4- [4 - [[4- (bromomethyl) phenyl] J] methoxy] -1-0x0-iso-indolin-2-yl] -5-0xo-pentanoate. Two reactions (25 g, 85.5 mmol) were performed in parallel. A mixture of 1,4-bis (bromomethyl) benzene (67.78, 257 mmol), potassium carbonate (11.8 g, 85.5 mmol) and methyl 5-amino-4- (4-hydroxy-1- oxo-isoindolin-2-yl) -5-oxo-pentanoate (25 g 855 mmol) in acetonitrile (1 L) was stirred at 60 ° C for 16 h. The two batches were combined and the mixture was cooled to 15 ° C and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (eluted with 50% petroleum ether in 100% ethyl acetate) to provide methyl 5-amino-4- [4- [ [4- (bromomethyl) phenyl] methoxy] -1-0x0- iso-indolin-2-yl] -5-oxo-pentanoate (52 g, 63% yield) as a white solid. * H NMIR: 400 MHz DMSO-ds 6: 7.59 (s, 1H), 7.50-7.44 (m, 5H), 7.32-7.28 (m, 2H), 7.19 ( s, 1H), 5.26 (s, 2H), 4.79-4.71 (m, 3H), 4.55 (d, | = 17.6 Hz, 1H), 4.43 (d, J = 17.6 Hz, 1H), 3.52 (s, 3H), 2.30-2.19 (m, 3H), 2.10-2.08 (m, 1H).
[00280] [00280] 3- [4 - [[4- (bromomethyl) phenyl] Jmethoxy] 1-1-0xo-isoindolin-2-yl] piperidine-2,6-dione. Two reactions (28.5 g, 60.0 mmol) were performed in parallel. Methyl 5-amino-4- [4 - [[4- (bromomethyl) phenyl] methoxy] -1-ox0-isoindolin-2-yl]) - 5-oxo-pentanoate (28.5 g, 60.0 mmol) it was dissolved in tetrahydrofuran (720 ml) and the solution was cooled in a dry ice / acetone bath at -70ºC. While stirring, potassium tert-butoxide (7.4 g, 66.0 mmol) was added in one portion to the clear solution. The reaction mixture turned pale yellow and stirring was continued for another 2 h at -70 ° C. A cooled HCI solution (1 N, 260 mL) was quickly transferred to the reaction mixture, maintaining the temperature at -70ºC. The mixture was immediately milky and the dry ice / acetone bath was removed. The mixture was concentrated to remove most of the tetrahydrofuran. After concentrating the reaction mixture, a white solid precipitated. The white paste was diluted with water (500 ml) and then filtered. The filter cake was washed with water (500 mL) and dried in a vacuum oven at 40ºC for 12 h, then washed with ethyl acetate (500 mL). The batches were combined to give 3- [4 - [[4- (bromomethyl) phenyl] methoxy] -1-oxo-isoindolin-2-yl] piperidine-2,6-dione (49.85 g, 93%) as light yellow solid. * H NMR: 400 MHz DMSO-ds 5: 10.95 (s, 1H), 7.51-7.41 (m., 5H), 7.35-7.28 (m, 2H), 5.23 (s, 2H), 5.12-5.07 (m, 1H), 4.70 (s, 2H), 4.41 (d, J = 17.6 Hz, 1H), 4.25 (d, J = 17.6 Hz, 1H), 2.90-2.84 (m, 1H), 2.58-2.33 (m, 1H), 2.44-2.41 (m, 1H), 1 , 98-1.95 (m, 1H).
[00281] [00281] 4- (4- (4 - ((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) methyl) benzyl) piperazin-1-i1) -3- fluorobenzonitrile3- (4 - ((4- (bromomethyl) benzyl) oxy) -1-oxoisoindolin-2-yl) piperidine-2,6-dione (5.0 g, 11.28 mmol) was placed in a flask with 3 -fluoro-4- (piperazin-1-yl) benzonitrile (2.315 g, 11.28 mmol), diisopropylethylamine (5.91 ml, 33.8 mmol) and acetonitrile (100 ml). The reaction mixture was stirred at 40ºC for 18 h. The volatile organics were removed under reduced pressure and purification by standard methods provided 4- (4- (4 - (((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) ] Methyl) benzyl) piperazin-1-yl) -3-fluorobenzonitrile. * H NMR (400 MHz, DMSO-ds) 5 10.97 (s, 1H), 7.68 (dd, J = 1.96, 13.45 Hz, 1H), 7.56 (dd, | = 1 , 77, 8.38 Hz, 1H), 7.43-7.52 (m, 3H), 7.30-7.38 (m, 4H), 7.11 (t, J = 8.80 Hz, 1H), 5.24 (s, 2H), 5.11 (dd, J = 5.14, 13.33 Hz, 1H), 4.37-4.46 (m, 1H), 4.22-4 , 30 (m, 1H), 3.54 (s, 2H), 3.12-3.23 (m, 4H), 2.84-2.98 (m, 1H), 2.52-2.62 (m, 5H), 2.36-2.48 (m, 1H), 1.92-2.04 (m, 1H). MS (ESI) m / z 568.2 [M + 1] *. Anal. Calcd for C32H30oFN5O24: C, 67.71; H, 5.33; N, 12.34. Found: C, 67.50; H, 5.44; N 12.34.
[00282] [00282] tert-Butyl (45) -5-amino-4- (benzyloxycarbonylamino) -5-0x0-pentanoate. To a solution of (2S) -2- (benzyloxycarbonylamino) -5-tert-butoxy-5-oxo-pentanoic acid (150 g, 445 mmol) in 1,4-dioxane (1.50 L) was added di-tert -butyl dicarbonate (155 g, 711 mmol), pyridine (70.3 g, 889 mmol) and ammonium bicarbonate (105 g, 1.33 mol). The reaction mixture was stirred at 18ºC for 16 h and then concentrated. The residue was dissolved in ethyl acetate (5.0 L) and water (5.0 L), the organic layer was separated and washed with HCl (3.0 mL, 1 N), saturated sodium bicarbonate (3.0 L), brine (3.0 L), dried over anhydrous sodium sulfate, filtered and concentrated to give crude tert-butyl (485) -5-amino-4- (benzyloxycarbonylamino) -5-oxo-pentanoate (450 g, crude) as a white solid, which was used in the next step without further purification. * H NMR 400 MHz DMSO-ds 6: 7.35-7.30 (m, 5H), 7.02 (s, 1H), 5.01 (d, J = 3.2 Hz, 1H), 3, 93-3.90 (m, 1H), 2.20 (t, J) = 8.0 Hz, 2H), 1.88 -1.84 (m, 1H), 1.72-1.69 (m , 1H), 1.35 (s, 9H).
[00283] [00283] tert-Butyl (4S5) -4,5-diamino-5-oxopentanoate. To a solution of tert-butyl (45) -5-amino-4- (benzyloxycarbonylamino) -5-oxo-pentanoate (112 g &, 333 mmol) in methanol (1.0 L) was added 10% palladium on carbon ( 15 g) under nitrogen. The suspension was degassed under vacuum and purged with hydrogen several times. The mixture was stirred under hydrogen gas (40 psi) at 30ºC for 16 h. The reaction mixture was filtered and the filtrate was concentrated to give crude tert-butyl (45) -4,5-diamino-5-oxopentanoate as a colorless oil. 2H NMR 400 MHz DMSO-ds 5: 7.30 (s, 1H), 6.95 (s, 1H), 3.10-3.07 (m, 1H), 2.27-2.23 (m, 2H), 1.69-1.78 (m, 1H), 1.59-1.55 (m, 1H), 1.38 (s, 9H).
[00284] [00284] Methyl 3-hydroxy-2-methyl-benzoate. Four batches (200 g each) were run in parallel. To a solution of 3-hydroxy-2-methyl-benzoic acid (200 g, 1.31 mol) in methanol (4.0 L) was added concentrated sulfuric acid (47.7 g, 486 mmol). The reaction mixture was stirred at 60ºC for 17 h. The reaction mixture was concentrated to 800 ml. The resulting mixture was cooled to 20 ° C and slowly poured into water (400 ml) over 30 minutes. Water (1200 mL) was added at 20 ° C for 3 h and the resulting mixture was stirred at 20 ° C for 1 h. The precipitated solid was collected by vacuum filtration (four combined batches) and was washed three times with water / methanol (1000 mL, 9: 1) or until the filtrate had pH> 3. The solid was dried under vacuum at 45ºC to give 3-hydroxy-2-methyl-benzoate (700 g, 80.4% yield) as a gray solid. * H NMR: 400 MHz DMSO-dcs 65: 9.70 (s, 1H), 7.18 (t,] = 6.8 Hz, 1H), 7.09 (t,] = 7.6 Hz, 1H ), 7.00 (t,) = 6.8 Hz, 1H), 3.81 (s, 3H), 2.29 (s, 3H).
[00285] [00285] Methyl / 3- [tert-butyl (dimethyl) silylJoxy-2-methyl-benzoate. Two batches (240 g each) were run in parallel. To a solution of methyl 3-hydroxy-2-methyl-benzoate (240 g, 1.44 mol) in N, N-dimethylformamide (1.40 L) was added imidazole (246 g, 3.61 mol) and chloride tert-butyl dimethylsilyl (238 g, 1.58 mol) at 5 ° C. After the addition, the mixture was heated to 20 ° C and stirred for 6 h. Isopropyl acetate (1700 ml) was added and then water (2000 ml) was added slowly while the temperature was kept below 30ºC. The resulting mixture was stirred followed by separation of the organic phase. The combined organics (two combined batches) were washed with water (1700 mL x 3) and concentrated to “1500 mL (KF <0.05%). The product was stored as an isopropyl acetate solution which was used in the next step without further purification.
[00286] [00286] Methyl 2- (bromomethyl) -3- [tert-butyl (dimethyl) silyl] oxy-benzoate. Two batches (“375 g each) were run in parallel. To the solution of methyl 3- [tert-butyl (dimethyl) silylJoxy-2-methylbenzoate isopropyl acetate (> 375 g, 1.34 mol) was added N-bromosuccinimide (274 g 154 mol) and azobisisobutyronitrile (4.40 g 26.8 mmol). The reaction mixture was heated to 70 ° C for at least 1 h and stirred at 70 ° C for 4 h. The reaction mixture was cooled to 20ºC and kept at 20ºC for at least 1 h. The two batches of solid (succinimide) were removed by filtration and washed with isopropyl acetate (700 ml). The filtrate was washed with a solution of sodium sulfite (700 g) in water (6000 ml), followed by water (1500 ml). The organic layer was vacuum distilled at 45 ° C to dryness to give methyl 2- (bromomethyl) -3- [tert-butyl (dimethyl) silylJoxy-benzoate (920 g, 95.5% yield) as a dark orange oil. * H NMR: 400 MHz DMSO-dcs 6: 7.45 (d, J = 6.8 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 4.95 (s, 2H), 1.02 (s, 9H), 0.29 (s, 6H).
[00287] [00287] tert-Butyl (48) -5-amino-4- [4- [tert-butyl (dimethyl) silylJoxy-1-0x0- isoindolin-2-yl] -5-0xo-pentanoate. To a solution of tert-butyl (45) -4,5-diamino-5-oxo-pentanoate (130 g, 643 mmol) in acetonitrile (4.0 L) was added methyl 2- (bromomethyl) -3- [tert -butyl (dimethyl) silyl] oxy-benzoate (210 g, 584 mmol) and diisopropylethylamine (113 g, 877 mmol). The reaction mixture was stirred at 50 ° C for 16 h. The reaction mixture was concentrated to remove most of the acetonitrile, the residue was dissolved in tert-butyl methyl ether (2.0 L) and water (1.5 L), the organic layer was washed with saturated monopotassium phosphate (1 , 0 L x 2), brine (1.0 L), dried over anhydrous sodium sulfate, filtered and concentrated to give tert-butyl (4S) -5-amino-4- [4- [tert-butyl (dimethyl) crude silyloxy-1-oxo-isoindolin-2-yl] - 5-0xo-pentanoate (524 g), which was used in the next step without further purification.
[00288] [00288] tert-Butyl - (45) -5-amino-4- (4-hydroxy-1-0xo-isoindolin-2-i1) -5-0x0-pentanoate. To a solution of (45) -5-amino-4- [4- [tert-butyl (dimethyl) silylJoxy-1-oxo-isoindolin -2-yl]) - 5-0xo-pentanoate (275 g, 613 mmol) in methanol (2.0 L), tetrabutylammonium fluoride trihydrate (38.7 g, 123 mmol) was added. The mixture was stirred at 18ºC for 16 h. The reaction mixture was concentrated to remove most of the methanol, the residue was dissolved in dichloromethane / water (3 L / 2 L), the organic layer was separated and washed with brine (1.0 L), dried over sulfate anhydrous sodium, filtered, and concentrated to give the crude product, which was purified by silica gel column to give the product (260 g). The product was added to acetonitrile (750 mL) and the mixture was stirred at 60ºC for 2 h, cooled to 18ºC and stirred for another 2 h. The solid was filtered and the cake was dried to give butyl (4585) -5-amino-4- (4-hydroxy-1-0x0-isoindolin-2-yl) -5-oxo-pentanoate (248 g, 60.5 % yield) as a gray solid. * H NMR 400 MHz DMSO-ds 5: 10.00 (s, 1H), 7.54 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 4.8 Hz, 2H), 4.72-4.68 (m, 1H), 4.49-4.28 (m, 2H), 2.17-1.97 (m, 4H), 1 , 31 (s, 9H).
[00289] [00289] 4- (4- (4- (chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile. 1,4-bis (chloromethyl) benzene (51.2 g, 292 mmol) was placed in a flask with acetonitrile (195 ml) and N, N-dimethylformamide (195 ml). The reaction mixture was stirred at room temperature until all the solids dissolved. Diisopropylamine (51.1 ml, 292 mmol) was then added together with 3-fluoro-4- (piperazin-1-yl) benzonitrile (20 g, 97 mmol). The reaction was heated to 60ºC for 1 h. Acetonitrile was removed under reduced pressure. The remaining mixture was partitioned between ethyl acetate (1.0 L), water (700 ml) and brine (300 ml). The organic layer was separated and the aqueous layer was extracted with ethyl acetate twice. The volatile organics were combined and removed under reduced pressure. The solid was dissolved in minimal dichloromethane and purified on a silica gel column (0-100% ethyl acetate in hexanes above 3 L). The fractions containing the desired product were combined and the volatile organics were removed under reduced pressure. The residue was dissolved in minimal dichloromethane and purified a second time on a silica gel column (10% isocratic ethyl acetate in hexanes above 800 mL followed by 20-80% ethyl acetate in hexanes above 4 L). The fractions containing the desired product were combined and the volatile organics were removed under reduced pressure to provide 4- (4- (4- (chloromethyl) benzyl) piperazin-1-yl) -3-fluorobenzonitrile (22.7 g, 66, 0 mmol, 67.7% yield) as an off-white solid. * H NMR (400 MHz, CDCI3) 5 ppm 7.33 - 7.39 (m, 5 H) 7.29 (d, J = 1.96 Hz, 1 H) 7.25 (d, J) = 1 , 96 Hz, 1 H) 6.91 (t, J = 8.56 Hz, 1 H) 4.60 (s, 2 H) 3.58 (s, 2 H) 3.19 - 3.27 (m , 4 H) 2.58 - 2.66 (m, 4 H). MS (ESI) m / 2z 344.2 [M + 1] *.
[00290] [00290] (S) -tert-butyl 5-amino-4- (4 - ((4- (4- (4-cyano-2-fluorophenyl) piperazin-1-yl) Mmethyl) benzyl) oxy) -1- oxoisoindolin-2-yl) -5-oxopentanoate. (S) -tert-butyl 5-amino-4- (4-hydroxy-1-oxoisoindolin-2-yl) -5-oxopentanoate (22.05 g, 65.9 mmol) was placed in a flask with 4- (4 - (4- (chloromethyl) benzyl) piperazin-1-yl) -3-fluorobenzonitrile (22.67 g, 65.9 mmol), potassium carbonate (18.23 g, 132 mmol) and N, N-dimethylformamide ( 330 mL). The reaction mixture was heated to 45ºC for 16 h. The reaction was diluted with ethyl acetate (50 ml) and filtered. The filtrate was partitioned with ethyl acetate (900 ml) and water (600 ml) and brine (200 ml). The organic layer was isolated and washed with water (600 ml). The organic layer was dried over sodium sulfate and the volatiles were removed under reduced pressure. The residue was treated with 20% ethyl acetate in hexanes and the volatiles were removed under reduced pressure to provide (S) -tert-butyl 5-amino-4- (4 - ((4 - ((4- (4- cyano-2-fluorophenyl) piperazin-1-yl) (methyl) benzyl) oxy) -1-oxoisoindolin-2-yl) -5-oxopentanoate (44.02 g 686 mmol, 104% yield) as an off-white solid. The yield was slightly above the quantitative, with the permanence of some DMF. * H NMR (400 MHz, CDCI3) 6 ppm 7.43 - 7.49 (m, 2 H) 7.40 (s, 4 H) 7.36 (dd, J = 8.38, 1.28 Hz, 1 H) 7.29 (d, J) = 1.96 Hz, 1 H) 7.26 (d, J) = 1.83 Hz, 1 H) 7.11 (dd, J = 7.64, 1 , 16 Hz, 1 H) 6.92 (t,] = 8.50 Hz, 1 H) 6.23 (br s, 1 H) 5.24 - 5.32 (m, 1 H) 5.15 ( s, 2 H) 4.86 - 4.94 (m, 1 H) 4.38 - 4.55 (m, 2 H) 3.61 (s, 2 H) 3.18 - 3.32 (m, 4 H) 2.58 - 2.70 (m, 4 H) 2.09 - 2.47 (m, 4H) 1.43 (s, 8H). MS (ESI) m / z 642.4 [M + 1] *.
[00291] [00291] (S) -4- (4- (4 - ((2- (2,6-Dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy) methyl) benzyl) piperazin-1-i1 ) -3-fluorobenzonitrile (S) -tert-butyl 5-amino-4- (4- ((4 - ((4- (4-cyano-2-fluorophenyl) piperazin-1-yl) methyl) benzyl) oxy) -1-0xoisoindolin-2-yl) -5-oxopentanoate (12.1 g, 18.86 mmol) were placed in a flask with acetonitrile (189 mL) and benzenesulfonic acid (3.96 g, 24.51 mmol). The reaction mixture was placed under vacuum and purged with nitrogen. This was repeated again and the mixture was then heated to 85 ° C overnight under an atmosphere of nitrogen. The hot reaction mixture was poured directly into 2 separating funnels containing dichloromethane (1000 ml) and ethyl acetate (300 ml). To this mixture was added a saturated solution of sodium bicarbonate (900 ml), water (100 ml) and brine (450 ml). The organic layer was isolated and the aqueous layer was extracted with dichloromethane (800 ml) and ethyl acetate (200 ml). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. Purification by standard methods provided the title compound. * H NMR (400 MHz, DMSO-ds) 5 ppm 10.96 (s, 1 H) 7.68 (dd, J = 13.45, 1.83 Hz, 1 H) 7.56 (dd, J = 8.44, 1.83 Hz, 1 H) 7.43 - 7.52 (m, 3 H) 7.29 - 7.39 (m, 4 H) 7.11 (t, J = 8.80 Hz , 1 H) 5.24 (s, 2 H) 5.11 (dd, J | = 13.20, 5.14 Hz, 1 H) 4.22 - 4.46 (m, 2 H) 3.54 (s, 2 H) 3.12 - 3.22 (m, 4 H) 2.85 - 2.97 (m, 1 H) 2.53 - 2.62 (m, 2 H) 2.38 - 2 , 48 (m, 2 H) 1.93 - 2.03 (m, 1 H). MS (ESI) m / z 568.2 [M + 1] *.
[00292] [00292] 4- (4- (4- (Chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile hydrochloride. To a solution of 3-fluoro-4- (piperazin-1-yl) benzonitrile (100 g) in toluene (1400 ml) was added acetic acid (28 ml) at 25ºC and the reaction mixture was maintained for 30 min. 4- (Chloromethyl) benzaldehyde (79 g) at 25 ° C and the mixture was kept for 2 h. Sodium triacetoxyborohydride (52 g each) was loaded at 25ºC every 30 minutes three times. The reaction mixture was stirred at 25ºC for about 10 h. The water (600 mL) was charged for 1 hour, keeping the batch temperature below 30ºC. Most of the bottom layer has been separated. The mixture was filtered and the bottom layer was separated. The organic layer was washed with water (200 ml). The organic layer was loaded with IPA (75 mL), HCI 5-6 N in IPA (8 mL), then a seed paste of 4- (4- (4- (chloromethyl) benzyl) piperazin-1-yl) -3 -fluorobenzonitrile hydrochloride (2 g) in toluene (20 ml). The mixture was charged 5-6 N HCI in IPA (115 ml) at 25 ° C for 2 h. The mixture was maintained for about 10 h, then filtered to give a crude solid. The solid was washed with toluene (400 ml) and dried in a vacuum oven to give 4- (4- (4- (chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile hydrochloride as a pale yellow solid (152 82% yield). * H NMR (300 MHz, DMSO-ds) 6 ppm 11.82 (s, 1H), 7.50-7.79 (m, 6H), 7.18-7.24 (m, 1H), 4, 80 (s, 2H), 4.38-4.39 (m, 2H), 3.44-3.70 (m, 2H), 3.14-3.44 (m, 6H).
[00293] [00293] tert-Butyl (S) -5-amino-4- (4 - ((4- (4- (4-cyano-2-fluorophenyl) piperazin-1-yl)] Mmethyl) benzyl) oxy) -1 -oxoisoindolin-2-yl) -5-oxopentanoate tartrate. To a mixture of 4- (4- (4- (chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile hydrochloride (100 g) and tert-butyl (S) -5-amino-4- (4-hydroxy ) -1-oxoisoindolin-2-yl) -5- oxopentanoate (97 g) in DMF (600 ml) was loaded with potassium carbonate (K2CO3) (75 g) at 35ºC and the reaction mixture was maintained for 24 h. To the mixture, triethylamine (11 ml) was charged and the mixture was stirred at 45 ° C for about 2 h. To the mixture, EtOAc (1 L) and aqueous potassium carbonate solution (5%, 500 mL) were charged. The organic layer was washed with aqueous sodium chloride solution (5%, 500 ml). The mixture was charged with Ecosorb C948 E-pak (30 g) and the mixture was kept for 2 h. The mixture was filtered. To the mixture, a solution of L-tartaric acid (47 g) in methanol (850 mL) at 45ºC was charged and the mixture was kept for 2 h. The mixture was cooled to 25ºC. The solids were filtered to give the tert-butyl (S) -5-amino-4- (4- ((4 - ((4- (4-cyano-2-fluorophenyl) piperazin-1-yl) methyl salt ) benzyl) oxy) -1-0xoisoindolin-2-yl) -5-oxopentanoate. (145 mg, 70% yield) 'H NMR (300 MHz, DMSO-ds) 5 ppm 1.31 (s, 9H), 1.87 - 2.27 (m, 4H), 2.55 (br s , 4H), 3.18 (br s, 4H), 4.29 (s, 2H), 4.36 - 4.62 (m, 2H), 4.71 (dd, J = 4.2, 10, 0 Hz, 1H), 5.22 (s, 2H), 7.10 (t, J = 8.8 Hz, 1H), 7.18 (s, 1H), 7.29 (d, J = 7, 7 Hz, 2H), 7.33 - 7.40 (m, 2H), 7.40 - 7.51 (m, 3H), 7.51 - 7.63 (m, 2H).
[00294] [00294] (S) -4- (4- (4 - ((2- (2,6-Dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy)] Methyl) benzyl) piperazin-1- i1) -3-fluorobenzonitrileThe tert-butyl (S) -5-amino-4- (4 - ((4 - ((4- (4-cyano-2-fluorophenyl) piperazin-1-yl) salt solution ] Methyl) benzyl)) oxy) -1-oxoisoindolin-2-yl) -5-oxopentanoate (100 g) in 2-methyltetrahydrofuran (1 L) was washed with aqueous potassium carbonate solution (10%, 85 mL ). The bottom layer was separated. The 2-methyltetrahydrofuran solvent was exchanged for acetonitrile to provide a solution. To the solution, benzenesulfonic acid (60 g) in acetonitrile (200 ml) was added at 70ºC for 2 h. Purification by standard methods provided the title compound. * H NMR (400 MHz, DMSO-ds) 8 ppm 10.96 (s, 1 H) 7.68 (dd, J = 13.45, 1.83 Hz, 1 H) 7.56 (dd, J = 8.44, 1.83 Hz, 1 H) 7.43 - 7.52 (m, 3 H) 7.29 - 7.39 (m, 4 H) 7.11 (t,) = 8.80 Hz , 1 H) 5.24 (s, 2 H) 5.11 (dd, J = 13.20, 5.14 Hz, 1 H) 4.22 - 4.46 (m, 2 H) 3.54 ( s, 2 H) 3.12 - 3.22 (m, 4 H) 2.85 - 2.97 (m, 1 H) 2.53 - 2.62 (m, 2 H) 2.38 - 2, 48 (m, 2 H) 1.93 - 2.03 (m, 1 H). MS (ESI) m / z 568.2 [M + 1] *.
[00295] [00295] 4- (4- (4- (Chloromethyl) benzyl) piperazin-1-yl) -3-fluorobenzonitrile hydrochloride. To a solution of 3-fluoro-4- (piperazin-1-yl) benzonitrile (100 g) in toluene (1400 ml) was added acetic acid (28 ml) at 25ºC and the mixture was maintained for 30 min. 4- (Chloromethyl) benzaldehyde (79 g) at 25ºC and the mixture was kept for 2 h. Sodium triacetoxyborohydride (52 g each) was loaded at 25ºC every 30 minutes three times. The reaction mixture was stirred at 25ºC for about 10 h. The water (600 mL) was charged for 1 hour, keeping the batch temperature below 30ºC. Most of the bottom layer has been separated. The mixture was filtered and the bottom layer was separated. The organic layer was washed with water (200 ml). The organic layer was loaded with IPA (75 mL), HCI 5-6 N in IPA (8 mL), then a seed paste of 4- (4- (4- (chloromethyl) benzyl) piperazin-1-yl) -3 -fluorobenzonitrile hydrochloride (2 g) in toluene (20 ml). The mixture was charged with 5-6 N HCl in IPA (115 mL) at 25 ° C for 2 h. The mixture was maintained for about 10 h, then filtered to give a crude solid. The solid was washed with toluene (400 mL) and dried in a vacuum oven to give 4- (4- (4- (chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile hydrochloride as a white solid (152 g , 82% yield). * H NMR (300 MHz, DMSO-ds) 5 ppm 11.82 (s, 1H), 7.50-7.79 (m, 6H), 7.18-7.24 (m, 1H), 4, 80 (s, 2H), 4.38-4.39 (m, 2H), 3.44-3.70 (m, 2H), 3.14-3.44 (m, 6H).
[00296] [00296] tert-Butyl (S) -5-amino-4- (4 - ((4- (4- (4-cyano-2-fluorophenyl) piperazin-1-yl)] Mmethyl) benzyl) oxy) -1 -oxoisoindolin-2-yl) -5-oxopentanoate. To a mixture of 4- (4- (4- (chloromethyl) benzyl) piperazin-1-i1) -3-fluorobenzonitrile hydrochloride (100 g) and tert-butyl (S) -5-amino-4- (4-hydroxy ) -1-oxoisoindolin-2-yl) -5-oxopentanoate (88 g) in dimethylsulfoxide (DMSO) (700 mL) was loaded with potassium carbonate (K2CO3) (73 g) at 35ºC and the mixture was maintained for 24 h. To the mixture, EtOAc (1.2 L) and water (1.1 L) were charged. The organic layer was washed with aqueous sodium chloride solution (5%, 1 L). The mixture was charged with n-heptane (200 ml). The mixture was washed with aqueous acetic acid (3%, 1 L), water (1 L), K3POa solution. aqueous (20%, 10 L), and water (10 L). The solvent was distilled at about 1.2 L. The mixture was crystallized from n-heptane to give tert-butyl (S) -5-amino-4- (4- ((4- (4- (4-cyano- 2-fluorophenyl) piperazin-1-yl) methyl) benzyl) oxy) -1-oxoisoindolin- - 2-yl) -5-oxopentanoate (143 g, 85% yield). * H NMR (400 MHz, CDCI3) 6 ppm 7.43 - 7.49 (m, 2 H) 7.40 (s, 4 H) 7.36 (dd, J = 8.38, 1.28 Hz, 1 H) 7.29 (d, J = 1.96 Hz, 1 H) 7.26 (d, J = 1.83 Hz, 1 H) 7.11 (dd,) = 7.64, 1.16 Hz, 1 H) 6.92 (t, | = 8.50 Hz, 1 H) 6.23 (br s, 1 H) 5.24 - 5.32 (m, 1 H) 5.15 (s, 2 H) 4.86 - 4.94 (m, 1H) 4.38 - 4.55 (m, 2 H) 3.61 (s, 2 H) 3.18 - 3.32 (m, 4 H) 2.58 - 2.70 (m, 4H) 2.09 - 2.47 (m, 4H) 1.43 (s, 8H).
[00297] [00297] (S) -4- (4- (4 - ((2- (2,6-Dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy)] Methyl) benzyl) piperazin-1- i1) -3-fluorobenzonitrileThe tert-butyl solution (S) -
[00298] [00298] 3-Fluoro-4- (piperazin-1-i1-2,2,3,3,5,5,6,6-d8) benzonitrile. A solution of 3,4-difluorobenzonitrile (278 mg, 2.00 mmol) and piperazine-2,2,3,3,5,5,6,6-dº (942 mg, 10.0 mmol) in dry DMA ( 6 mL) was stirred at 110ºC for 16 h. The mixture was cooled to room temperature and was slowly added to H2O (60 ml) with mixture. The mixture was extracted with EtOAc (3X) and the organic portions were combined, washed with saturated NaCl (3X), dried over MgSOa, filtered and concentrated. The residual colorless syrup was dried in vacuo to provide the crude product as a white solid (599 mg). The solid was dissolved in EtOAc and the solution was washed with H2O (3X), saturated aqueous NaCl (3X) and dried over MgSO2a. The dry solution was filtered, concentrated and the residue dried in vacuo to provide the title compound (447 mg, 105%) as a white solid. LCMS (ESI) m / z 214.2 [M + H] *.
[00299] [00299] 4- (4- (4 - ((2- (2,6-Dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) oxy)] Methyl) benzyl) piperazin-1-i1-2, 2,3,3,5,5,6,6-d8) -3-fluorobenzonitrile. To a solution of 3- [4 - [[4- (bromomethyl) phenyl] Jmethoxy] -1-oxo-isoindolin-2-yl] piperidine-2,6-dione (736 mg, 1.66 mmol) and 3- fluoro-4- (piperazin-1-i1-2,2,3,3,5,5,6,6-dº) benzonitrile (425 mg, 1.99 mmol) in dry DMF (5.0 mL) was added DIEA (0.870 ml, 4.99 mmol) and the mixture was stirred at room temperature for 4 h. The mixture was filtered (0.45 µm nylon membrane) and the solution was purified by standard methods to give the title product (532 mg, 56%). LCMS (ESI) m / z 576.4 [M + H] *.
[00300] [00300] Cell culture materials: Human multiple myeloma cell lines were purchased from suppliers and grown at 37ºC with 5% CO in the middle, as shown in Table 1. Cell lines resistant to lenalidomide and pomalidomide were obtained by methods generally described previously (Lopez-Girona et al Leukemia 2012; 26 (11): 2335). All cell lines were maintained in a logarithmic phase and cell density and viability were monitored by trypan blue exclusion using the Vicell XR cell viability analyzer (Beckman Coulter, Brea, CA).
[00301] [00301] Preparation of Test Article Solutions: The compounds were plated on 384-well black plates (Corning Inc.) at a final volume of 0.1% DMSO, assuming a maximum volume of 50 µl. A 10-point dose response starting with 10 UM with a 1: 3 dilution was printed in duplicate by acoustic dispensing using the EDC ATS-
[00302] [00302] Cell Proliferation Assays: The effect of compounds on the proliferation / viability of hematological cell lines (Table 1) was evaluated after 120 h of incubation using CTG (Promega), according to the manufacturer's instructions. The hematological cell lines were distributed in plates of compounds by a Reagent Dispenser
[00303] [00303] Cellular Apoptosis Assays: The ability of compounds to induce apoptosis was evaluated in MM cell lines selected at the time points and concentrations of the indicated compounds. As a marker of apoptosis, Caspase-3 activity level was measured in MM cells using live cell imaging. To view the cells in suspension, the 96-well plates were coated with fibronectin, so that the cells adhered and were flat on the bottom of the plate. The cells were added to 96-well plates using a Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, MA) the night before adding the compound. The compounds were identified on top of the cells in the appropriate well of 96-well plates using a Hewlett-Packard D300 Digital Dispenser (Tecan, Mannedorf, Switzerland). MM cell lines were treated with the compound and at 6 am the medium was changed to mimic the renewal of the compound in vivo, resulting in a dilution of about 20 times the concentration of the compound. The cells were cultured in the presence of the NucView 488 Caspase-3 enzyme substrate (Biotium) and incubated in an IncuCyte ZOOM live cell analysis system (Essen Bioscience, Ann Arbor, MI) housed in a standard incubator. The cleavage of the Caspase-3 enzyme substrate and cell confluence in each well were detected by 10x imaging in the IncuCyte ZOOM System every 4-6 h for 5 days. Each well / condition was performed in replication on the same plate and each condition was the average of 4 10x images captured at each moment.
[00304] [00304] Results. Compound 1 and Compound 2 demonstrate antiproliferative activity against MM cell lines. The MM cell lines selected for this study were sensitive and resistant to lenalidomide and / or pomalidomide (Table 1), two agents used in the clinic to treat patients with myeloma. Proliferation was assessed using the CellTitre-Glo assay ”. Results for cultures incubated with the compounds were normalized to results for control cultures for each cell line. The ICso for inhibiting cell growth by the compounds was determined for each cell line using the ActivityBase software. Compound 1 and Compound 2 potently inhibited cell proliferation in the four cell lines, as determined by the quantitative assessment of ATP levels present in the media after 120 h. The antiproliferative ICso values for Compound 1 and Compound 2 varied between 0.07 nM and 19 nM (Table 2). Compound 1 and Compound 2 showed very potent multiple myeloma antiproliferative activity, even in cell lines resistant to lenalidomide and / or pomalidomide.
[00305] [00305] Apoptosis induced by compound 1 in multiple myeloma cell lines. The effects of compounds on apoptosis in MM cell lines were investigated. To determine the ability of compounds to induce apoptosis and to characterize kinetically the beginning of this process, induction of Caspase-3 was measured over time in lenalidomide-resistant H929-1051 cells (Figure 1). H929-1051 cells were incubated with the compounds in concentrations of 1 nM, 10 nM, 100 nM and 1000 nM and apoptosis was assessed over time. The results showed that, for H929-1051 cells, all concentrations of Compound 1 induced apoptosis starting at about 48 h and reaching a maximum induction close to * 72 h of incubation. Then, the area under the curve (AUC) was calculated for each concentration and used to generate the concentration-response curves for each compound. This provided quantitative evidence of the ability of Compound 1 to induce apoptosis in H929-1051. Surprisingly, the induction of apoptosis by Compound 1 was significantly greater than the induction of apoptosis observed for the previously reported compound 3- (4- ((4 - ((4- (2,4 (difluorophenyl) piperazin-1-yl) methyl ) benzyl) oxy) -1-oxoisoindolin-2-yl) piperidine-2,6-dione (Example 5,285 in US Patent 8,518,972) (Compound A) As shown in Fig. 1B, apoptotic induction (measured by total AUC) by Compound 1 was increased by almost 30% (126%) compared to the apoptotic induction by Compound A.
[00306] [00306] Combination with Dexamethasone. The activity of pomalidomide with that of Compound 2 alone or in combination with dexamethasone was evaluated using a panel of MM cell lines. Figure 2 is a representative set of dose-response curves in the lenalidomide-resistant cell line H929-1051, demonstrating that single compound 2 is 10 times more potent than a pomalidomide-dexamethasone combination and almost as effective (Figure 2, panel A) . Surprisingly, when Compound 2 is combined with dexamethasone, it not only creates a more potent response, but the effectiveness is also dramatically enhanced (Figure 2,
[00307] [00307] In vitro safety assessment assessed by antiproliferative selectivity in normal cells. To demonstrate that Compound 2 is generally not cytotoxic, a counter-screen against the immortalized (but not tumorigenic) THLE-2 cell line derived from human hepatocytes (Pfeifer et al, Proc Natl Acad Sci USA. 1993; 90 (11): 5123-7) and against primary and healthy human PBMCs (Figure 3) was performed. Compound 2 had little antiproliferative effect on THLE-2 cells (ICso> 10 µM) or on primary human unstimulated PBMCs (ICso> 10 µM) compared to the MM cell lines shown above.
[00308] [00308] Antitumor activity of Compound 2 in the lenalidomide-resistant multiple myeloma xenograft model. The aim of the study was to test the compound's single agent antitumor activity in a model of H929-1051 xenograft with dosing once a day (QD) at 1, 3, 10 and 30 mg / kg. Significant antitumor activity (p <0.0001) was observed at all dose levels with a reduction in tumor volume of 75%, 86%, 84% and 85% at 1, 3, 10 and 30 mg / kg, respectively (Figure 4).
[00309] [00309] Conclusion: together these data show that Compound 1 and Compound 2 show very potent anti-multiple myeloma activity and surprisingly show significantly increased levels of apoptosis compared to the previously reported compounds, Compound A and pomalidomide. In addition, Compound 2 combined with dexamethasone not only creates a more potent response, but the effectiveness is also dramatically improved. In addition, selective cell death from multiple myeloma has been demonstrated compared to normal cells Example 7: Compound 1 / Compound 2 off-target effects and implications.
[00310] [00310] A1 adrenergic receptors and dopamine D2. Methods: The binding and functional tests for a1 adrenergic and dopamine D2 receptors were performed by Eurofins Cerep according to their methods.
[00311] [00311] Al adrenergic receptor. Connection at 10 µM. The binding assay evaluated the affinity of the test article for the non-selective al adrenergic receptor in the rat cerebral cortex. Cerebral cortex membrane homogenates were incubated in duplicate for 60 minutes at room temperature with 0.25 nM [ H] prazosin in the absence or presence of test articles at 10 µM. After the incubation period, the samples were filtered through glass fiber filters, the filters dried and then counted for radioactivity using a scintillation counter. The results are expressed as mean percentage inhibition of the control radioligand binding.
[00312] [00312] ICso connection. To determine the binding ICso for the non-selective al adrenergic receptor, varying concentrations of the test article were incubated in duplicate with 0.25 nM [ H] prazosin. Compound A was tested at 0.01-30 µM. Compound B, the S enantiomer of Compound A, was tested at 0.0003-10 µM. Compound 1 and Compound 2, the S enantiomer of Compound 1, were analyzed at 0.03-100 µM. Radioactivity was measured as described above. ICso was defined as the concentration that causes semi-maximum inhibition of the specific control link.
[00313] [00313] Antagonistic activity The antagonistic effects of the test compounds on dia and adrenergic receptors were measured using Chinese hamster ovary (CHO) cells transfected with human receptor. Antagonistic activity was determined by measuring the compound effect on agonist-induced calcium mobilization (epinephrine) in the Qa receptor assay OR CAMP levels in the ag receptor assay. In these experiments, CHO cells were incubated in duplicate at room temperature with test article and 3 nM adrenaline in the aa receptor assay at 3000 nM in the aus receptor assay. Compound A was tested in the 0.01-30 µM aa receptor assay. Compound B was tested in the aa receptor assay and OB receptor assays at O. 0003-30 uM. Compound 1 and Compound 2 were tested between 0.03 and µM in the aa receptor assay and 0.03 to 100 µM in the ae receptor assay. In the aa receptor assay, cytosolic calcium levels were measured fluorometrically using the Fluo4 Direct fluorescent probe. The intracellular levels of CAMP in the peak adrenergic receptor assay were measured by time-resolved homogeneous fluorescence (HTRF). Antagonism 1Cso was defined as the concentration that causes semi-maximum inhibition of the control agonist's response.
[00314] [00314] D2 dopamine receptor. Connection at 10 µM. The binding assay evaluated the affinity of the test articles for the dopamine D2 receptor in transfected human embryonic kidney (HEK) -293 cells. To determine binding in the Ds receptor assay, the test article was incubated with 0.3 nM [PH] methylpiperone or 1 nM [ H] 7-hydroxy-2-N, N-dipropylaminotetralin (7-OH- DPAT). [ H] 0.3 nM methylpiperone was also used as a control ligand in the Dx binding assay. The cell membrane homogenates were incubated in duplicate at room temperature for 60 minutes with ligand in the absence or presence of test articles at 10 µM. After the incubation period, the samples were filtered through glass fiber filters, the filters dried and then counted for radioactivity using a scintillation counter. The results are expressed as mean percentage inhibition of the control radioligand binding.
[00315] [00315] ICso of connection. To determine the binding ICso in the D2 receptor assays, the HEK-293 was tested as described above, but with varying concentrations of the test article. Compound A was tested at 0.01-30 µM in the radioligand binding assay Das. Compound B was tested at 0.0003-10 µM in both Das and Dx binding assays. Compound 1 was analyzed at 0.03-100 µM in both D> 2s and D2 assays, while Compound 2 was tested at 0.03-100 µM in the Ds assay and 0.01-100 µM in the Da assay. . ICso was defined as the concentration that causes semi-maximum inhibition of the specific control link.
[00316] [00316] Agonist activity. The agonism of the test compounds at the dopamine D> s receptor was assessed using HEK-293 cells transfected with the human receptor. Agonist activity was determined by measuring the effect of the compound on impedance modulation. In these experiments, HEK-293 cells were incubated in duplicate at 28 ° C with the test article. Compound A was tested at 0.01-30 µM. Compound B was tested at 0.0003-10 µM, while Compound 1 and Compound 2 were tested at 0.01-10 µM Dopamine (3 µM) was used as an agonist control. Impedance measurements were monitored for 10 minutes after adding the ligand using cell dielectric spectroscopy. ECso was defined as the concentration that causes a semi-maximum response, compared to the response of the control agonist (dopamine).
[00317] [00317] Results. The binding to 10 UM at the adrenergic and dopamine D2 receptors has been evaluated for Compound 1, Compound 2, Compound A, Compound B and a number of compounds exemplified in the US Patent
[00318] [00318] The detailed effects of Compound A, Compound B, Compound 1 and Compound 2 on the adrenergic receptor are summarized in Table 5. Compound A inhibited ligand binding to the α1 adrenergic receptor by 102%. The binding ICso for Compound A for the receptor was 0.064 µM. Compound A has been shown to be a strong adrenergic receptor antagonist, with an ICso of 0.014 µM. Likewise, Compound B inhibited ligand binding to the adrenergic receptor al at 98-100% at 10 µM and had a binding ICso of 0.024 µM. Strong antagonism of the adrenergic receptor was observed with Compound B, with ICso values of 0.0064 and 0.078 uM in the receptors
[00319] [00319] D2 dopamine receptor. The effects of Compound A, Compound B, Compound 1 and Compound 2 on dopamine D2 receptor are summarized in Table 6. Compound A inhibited ligand binding to dopamine D2s receptor by 99%. The binding ICso for Compound A for the Ds receptor was 0.15 µM. Compound A was shown to be a partial D2s dopamine receptor agonist, with an ECso of 0.016 µM. Likewise, Compound B inhibited the Das receptor by 99% at 10 µM and had a binding ICso of 0.084 and 0.016-0.047 UM at Dx and Ds receptors, respectively. Surprisingly, on the contrary, Compound 1 and Compound 2 showed weak dopamine D2 receptor activity, with inhibition at 10 æM of <55% and ICso binding values> 2 æM. The functional agonism (ECso) of Compound B at the dopamine Ds receptor in three independent studies was> 10, 2.7 and 1.9 µM. Although the extent of Compound B agonism at the Ds receptor was less than that observed for Compound A, there was a tendency for greater agonism of Compound B compared to Compound 1 and Compound 2. Taken together, the% binding inhibition at 10 µM, the ICs5o binding data and the ICso of agonism demonstrated stronger activity of Compound A and Compound B at dopamine D2 receptor compared to Compound 1 and Compound 2. This observation is consistent with the evidence for dopamine D2 agonism (i.e., decreased motility / gastric emptying) in rats with Compound A, but not with Compound 1 (as discussed below). Table 6. Effects of Compound A, Compound B, Compound 1 and Compound 2 on Dopamine Receptor D2 Compound% inhibition ICso binding Dx D> 2s Dx D> s of D> s NERNBArY love dm ee Compound B 103 99 , 0.084 0.016, 0.021,> 10, 2.7, and iz Mae Rs Compound 2 ND 52 3 2 (estimated)> 10 ma
[00320] [00320] Studies of exploratory toxicology for 7 days in male rats. Methods. Male CD-IGS rats (n = 5 / group for toxicological evaluation; n =
[00321] [00321] Results. After daily oral administration of Compound A for 7 consecutive days, systemic exposure (AUCo +) increased dose-dependent from 100 to 1,000 mg / kg. Day 7 exposure was approximately 3 to 7 times greater than day 1. AUCs on Day 7 were 441, 1230 and 1760 µM-h at 100, 300, and 1,000 mg / kg, respectively. Clinical signs of toxicity (curved posture, piloerection and decreased activity) and decreased body weight were observed at> 300 mg / kg. No body weight gain was observed at 100 mg / kg. The abnormal stomach content (dry and feed material) was observed grossly at> 100 mg / kg, with no microscopic correlation. This finding suggests decreased gastric motility / emptying and is consistent with agonistic activity at the dopamine D> 2 receptor. The microscopic findings related to the test article were limited to minimal multifocal myocardial necrosis and mixed cell infiltration in rat hearts at all dose levels.
[00322] [00322] In vitro evaluation of Compound 2 and Compound B as an inhibitor of human cytochrome p450 enzymes. The objective was to evaluate the potential of Compound B and Compound 2 to act as a direct or time-dependent inhibitor of cytochrome P450 (CYP) activities in combined human liver microsomes. In this study, the inhibition of nine enzymes of human cytochrome P450, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 / 5 (using two substrates) was investigated.
[00323] [00323] Methods. To examine the potential of the compounds to act as direct inhibitors of CYP enzymes, the combined human liver microsomes were incubated with probe substrates, in concentrations approximately equal to their apparent km, in the absence or presence of Compound B (0.03 to 30 µM) or Compound 2 (0.03 to 30 µM) and NADPH (1 mM). In addition, the compounds were evaluated for their potential to act as time-dependent inhibitors at the same concentrations mentioned above. When assessing time-dependent inhibition, the compounds were preincubated with human liver microsomes and NADPH (1 mM) for 30 minutes before adding a probe substrate. In addition to the appropriate vehicle controls, known direct inhibitors and time-dependent inhibitors of the CYP isoforms were included as positive controls. After incubation, concentrations of the substrate metabolites from the probe were quantified using established LC / MS / MS methods. The extent of inhibition was expressed as the percentage of control activity.
[00324] [00324] Results. Compound B. Under the experimental conditions used to examine direct inhibition, Compound B (up to 30 µM) had little (<30%) to no inhibitory effect on CYP1A2, CYP2A6, CYP2C8, CYP2D6, CYP2E1 and CYP3A4 / 5 (midazolam) . At 30 µM, Compound B inhibited the activity of CYP2B6, CYP2C9 and CYP2C19 by 59, 38 and 45%, respectively. Compound B inhibited CYP3A4 / 5 (testosterone) with an ICso value of 2.92 µM. Under the conditions used to test time-dependent inhibition, Compound B (up to 30 µM) showed a time-dependent inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 after a 30-minute pre-incubation with or without NADPH. Compound B inhibited CYP3A4 / 5 in a time-dependent manner. The altered ICso value (with NADPH) was 2.23 and 1.93 UM for midazolam and testosterone as substrates, respectively.
[00325] [00325] Compound 2. Under the experimental conditions used to examine direct inhibition, Compound 2 (up to 30 µM) had little (<30%) to no inhibitory effect on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
[00326] [00326] Conclusion. In summary, Compound B (up to 30 µM) had little (<30%) to no direct inhibitory effect on CYP1A2, CYP2A6, CYP2C8, CYP2D6, CYP2E1 and CYP3A4 / 5 (midazolam). Compound B inhibited CYP3A4 / 5 (testosterone) with an ICso value of 2.92 µM. Compound B inhibited the activity of CYP2B6, CYP2C9 and CYP2C19 by 59, 38 and 45%, respectively at uM. Compound B (up to 30 µM) is not a time-dependent inhibitor of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1, but it is a time-dependent inhibitor of CYP3A4 / 5.
[00327] [00327] Surprisingly, in contrast, Compound 2 (up to 30 µM) had little (<30%) to no inhibitory effect on any of the tested CYP enzymes and, at UM 30, Compound 2 inhibited the activity of CYP2C19 and CYP3A4 / 5 (testosterone) only by 41 and 46% (ie, ICso> 30 µM), respectively. Compound 2 (up to 30 µM) is also only a weak time-dependent inhibitor of CYP3A4 / 5. The reduced inhibitory activity for CYP2C19 and CYP3A4 / 5 results in a reduced effect on metabolism including other drugs and, therefore, a reduced potential for adverse drug interactions.
[00328] [00328] Summary: The combination of the potent activity of multiple anti-myeloma in sparing normal cells, significantly increased levels of apoptosis and the response of the most potent and effective combination with dexamethasone, indicates that Compound 1 and Compound 2 will be useful in the treatment of multiple myeloma. Furthermore, the surprisingly improved in vitro and in vivo results of the off-target profile and CYP, in combination with the potential to use lower doses of dexamethasone, indicate that Compound 1 and Compound 2 must have enhanced safety profiles over to the compounds previously reported.
[00329] [00329] Methods. The effect of compounds on the proliferation and apoptosis induction of representative MM cell lines having common oncogenic mutations and chromosomal translocations, including those considered translocations or high-risk mutations found in patients with MM, was evaluated using a plate flow cytometry assay. 96 wells after 120 h of incubation with the compounds. Twenty MM cell lines (Tables 7 and 8) (including plasma cell leukemia (PCL) cell lines L363, JJN-3, ARH-77 and SKMM-2) were treated in duplicate with increasing concentrations of Compound 2 or pomalidomide, ranging from 0.015 to 100 nM. Using 5 mM stocks, the compounds were pre-stained in the appropriate wells of 96-well plates using a Hewlett-Packard D300 Digital Distributor. The cells were added to 96-well plates using a Multidrop Combi Reagent Dispenser. After 5 days of treatment, flow cytometric analysis was used to determine the number of cells that were alive, dead or apoptotic.
[00330] [00330] Results. The antiproliferative activity of Compound 2 was assessed using a panel of representative MM cell lines with common oncogenic mutations and chromosomal translocations (Table 8), including those considered translocations or high-risk mutations found in patients with MM (Johnson, et al. Int J Hematol. (2011); 94: 321-333; Zhou, et al. Leukemia (2009); 23: 1941-1956; Terpos, et al. Leuk Lymphoma (2006); 47: 803-814; Tonon, Hematol Oncol Clin North Am. (2007); 21: 985-1006). Concentration response curves were obtained using flow cytometry to measure the number of living cells to demonstrate the antiproliferative activity of compound 2 compared to pomalidomide. ICso values for Compound 2 and pomalidomide are shown in Table 9.
[00331] [00331] The apoptosis-inducing effect of Compound 2 was assessed using flow cytometry after 120 h of treatment and compared with pomalidomide treatment. The control percentage was calculated by normalizing for the DMSO control (100% of the control) to generate the dose response curves and calculate the area under these curves (AUC). The reported AUC value corresponds to the area under the dose response curve, in which
[00332] [00332] Conclusion. Compound 2 was widely active in most MM cell lines and was differentiated from pomalidomide, showing strong activity in cell lines that have intermediate sensitivity to pomalidomide and in cell lines resistant to pomalidomide. Compound 2 was widely active across this range of MM cell lines with variable status of p53, oncogenic drivers or chromosomal translocations. For example, OPM2, LP-1, EJM, U266 and RPM1I-8226 cells have the mutant p53 and were sensitive to Compound 2. In addition, all cell lines NCI-H929, KMS-11, KMS 34, OPM2 and LP-1 contain the “high risk” MM t (4; 14) chromosomal translocation and were sensitive to Compound 2. SK-MM-2 and EJM cells were also sensitive to Compound 2 and contain another “high risk MM chromosomal translocation risk ”, t (14; 20). (Figure 5) The ability of Compound 2 to induce apoptosis after short, defined exposures can allow disease control to be achieved using intensive and intermittent schedules. Such schemes can also improve the therapeutic index, reducing the potential of Compound 2 to induce cytopenias that are seen in more continuous doses of the current MM compounds.
[00333] [00333] Compound 2 activity was tested in cells that acquired resistance to lenalidomide or pomalidomide due to continuous exposure to the compound and, in the process, acquired unregulated cereblon levels (Table 11). The cells were treated for 5 days and then evaluated using an ATP determination assay (CellTiter-Glo). The percentage control was calculated by subtracting the background and normalizing for the DMSO control (100% of the control). The relative percentage of cereblon in cell lines with acquired resistance to lenalidomide or pomalidomide was determined by Western Blot and is shown, with the amount in parental cell lines designated as 100%.
[00334] [00334] Results. Figure 6 shows ICsos of the concentration response curves comparing the activity of Compound 2 and pomalidomide, to measure proliferation in parental lines (DF15, NCI-H929 and OPM2), a lenalidomide resistant cell line (NCI-H929-1051 ) or five cell lines resistant to pomalidomide (NCI-H929-PO01, OPM2-PO1, OPM2-P1, OPMZ2-P10 and DF15R).
[00335] [00335] Conclusion: The most striking effect of Compound 2 was the broad and potent antiproliferative activity across MM cell lines, but not in non-tumorigenic cells. Compound 2 has a potent antiproliferative activity in MM cell lines containing high-risk translocations, such as t (4; 14), t (14; 16) and others. Compared to lenalidomide and pomalidomide, Compound 2 is significantly more potent in killing most MM cell lines. In addition, Compound 2 induced apoptosis, measured by inducing caspase-3 activity, in MM cell lines that acquired resistance to lenalidomide and pomalidomide.
[00336] [00336] Methods: Ex vivo cultures of bone marrow cells (BM) CD34 * from healthy donors (HD) were used to investigate the specific ex vivo maturation of neutrophils. The in vitro differentiation of neutrophil progenitors was induced by the addition of stem cell factor (SCF), fms-related tyrosine kinase 3 (FIt3-L) and granulocyte colony stimulating factor (G-CSF) to the culture medium . Cell differentiation was assessed by flow cytometry as the percentage of cells in 5 subpopulations:
[00337] [00337] Results. Short daily exposures of Compound 2. The effects of different periods of exposure (2, 4 and 6 h) at 1, 10 and 100 nM of Compound 2 for up to 3 consecutive days at maturation of the neutrophil parents were evaluated at pre-established time points -specified using flow cytometry. The results showed that the late maturation of the neutrophil progenitors was blocked by Compound 2, with mature cells significantly reduced in number at the highest concentrations after one or more days of exposure. Maturation arrest appears to occur mainly in the development of the Stage II neutrophil progenitor, as evidenced by an accumulation of cells with the Stage III cell surface immunophenotype and a reduction in the population of cells with the Stage IV cell surface immunophenotype (mature neutrophils). As shown in Figure 8, in an example for the 6-hour incubation, this maturation effect depended on the concentration and increased with the number of days of exposure, but was not altered by the duration (2, 4 or 6 h) of individual exposures . It is important to note that the viability of neutrophil and mature neutrophil progenitors exposed to Compound 2 was not affected, as evidenced by the absence of any detectable increase in the proportion of cells positive for Annexin V or 7-aminoactinomycin D, which accumulates in dead cells.
[00338] [00338] The recovery of mature neutrophils after exposure to Compound 2 was also assessed in the system. The recovery of mature levels of neutrophils to at least 50% of the level of untreated control in the assay system used in the present study is correlated with the absence of induction or recovery from clinically significant neutropenia. In fact, after a period of one week without Compound 2, the proportion of Stage IV cells recovered by at least 50% of their nadir (Figure 8, lower panels), with a tendency for faster and more complete recovery in lower concentrations.
[00339] [00339] Conclusion: The results indicate that successful management of neutropenia in MM patients treated with Compound 2 may be possible with the use of appropriate dosing schedules.
[00340] [00340] Longer daily exposures of compound 2. To further characterize the possible impacts of different schedules on interrupting the maturation of the neutrophil parent and subsequent recovery, changes in the relative proportions of each of the aforementioned stages of maturation of the myeloid parent in adult neutrophils, they were evaluated by flow cytometry after 3 or 5 consecutive days of exposure 1, 10 or 100 nM of Compound 2 for 6 or 24 h per day. CD34 * BM cells derived from healthy donors were exposed to Compound 2 on 3 or 5 consecutive days, starting on Day 10 for 6 h (Donors No. 1 and 2) or for 24 h (Donors No. 3 and 4) each day. Upon completion of the final exposure, cells were washed and reincubated in the absence of Compound 2 until Day 22. Exposures of 6 and 24 hours to Compound 2 for 3 or 5 consecutive days resulted in an accumulation of the neutrophil population in Stage Il ! with a corresponding decrease in the population in Stage IV, consistent with a block in the maturation of Stage Ill to Stage IV. As shown in
[00341] [00341] After exposure to Compound 2 for 3 consecutive days, recovery of 50% or more of normal maturation was observed after an 8-10 day drug holiday, in all conditions tested (Figure 10, right panel). In contrast, after exposure to Compound 2 for 5 consecutive days, a recovery of 50% or more from normal maturation was observed after an 8 to 10 day drug stop only at concentrations of 1 and 10 nM. With the highest concentration of Compound 2 (100 nM), a more drug-free period may be required to recover the maturation of neutrophil progenitors. However, despite this incomplete recovery of maturation, no loss of viability was observed in any of the conditions tested, including continuous exposures (24 hours) for up to 5 days. This observation contrasts with the induction of apoptosis in myeloma cells, which was optimized by continuous exposure to Compound 2 for more than 6 h.
[00342] [00342] During the 6 to 8 days after the last exposure in the 5-day schedule, initial stages of recovery of Stage | V (mature neutrophils) were observed after exposure to Compound 2 only at the concentration of 10 nM, while no recovery was observed during that time in cultures exposed to 100 nM of Compound 2 for 5 days. These data suggest that exposures to higher concentrations of Compound 2, over an increasing number of consecutive days, herald a longer maturation stop of neutrophil precursors (and neutropenia) and that the rate of recovery is independent of duration (6 x 24 h) daily exposures.
[00343] [00343] Conclusion: Taken together, the data suggest that the induction and recovery of neutropenia in patients cannot be adversely affected by a more intensive dose of Compound 2 (multiple doses per day) compared to a daily dose.
[00344] [00344] Methods. To understand the effects of dexamethasone on neutropenia, in vitro cultures of BM CD34 * cells from healthy donors were used to evaluate dexamethasone-mediated neutropenic events as a single agent and in combination with Compound 2. To define the effects of dexamethasone monotherapy in this model, exposure to 1, 10 or 100 nM dexamethasone was maintained for 30 h comparing 7 different dosing schedules (Figure 11). For combination studies, compound 2 (1, 10 or 100 nM) and single-exposure dexamethasone were maintained for 6 and 30 h, respectively, from Day 13 of the culture.
[00345] [00345] Results. The results showed that the maturation of the neutrophil progenitors was not affected by exposure to single agent dexamethasone under any tested scheme, while the maturation of the late stage neutrophil precursors was blocked by Compound 2 (Figure 12), with the number of cells reduced maturity in all concentrations tested after exposure. This maturation stop was also observed when Compound 2 was combined with dexamethasone. The maturation block depended on the concentration of the Compound
[00346] [00346] Conclusion. These data indicate that the neutropenia caused by Compound 2 may be amenable to treatment by modifying the dosing schedules, but it is anticipated that it will not be relieved or exacerbated by concomitant treatment with dexamethasone.
[00347] [00347] Methods: Dexamethasone was evaluated for its ability to induce apoptosis as a single agent or in combination with Compound 2, pomalidomide or lenalidomide. Apoptosis induction was measured using Caspase-Glo in multiple myeloma cells resistant to lenalidomide (H929-1051). Dexamethasone was dispensed in 20 concentrations, using an acoustic dispenser. The test articles were added as single concentrations to the dexamethasone wells with a Hewlett-Packard D300 Digital Dispenser. The final concentrations of the compounds for the test were: dexamethasone (0.8 µM to 0.00002 µM), lenalidomide (1 µM), pomalidomide (0.1 µM) and compound 2 (0.001, 0.01 or 0.1 µM) ). The cells were dispensed on the assay plates with a Multidrop distributor and duplicate plates were made for the assay. Apoptosis reading was performed 72 hours after compound treatment using a Caspase-Glo 3/7 and CellTiter-Glo Assays. Caspase-Glo 3/7 luminescence has been normalized to CellTiter-Glo luminescence to explain differences in cell number. The change in fold of the treated sample was calculated as follows: normalized caspase of the treated sample / average of the normalized DMSO control.
[00348] [00348] Results: The apoptosis activity of dexamethasone alone or in combination with lenalidomide, pomalidomide or Compound 2 was measured by induction of caspase-3. Compound 2 synergized with dexamethasone to reduce cell viability and potentiated the apoptotic capacity of dexamethasone in a concentration-dependent manner. The onset of dexamethasone activity was altered by 1 log in the presence of Compound 2.
[00349] [00349] Conclusion: Compound 2 potentiates the apoptotic activity of dexamethasone, indicating the potential for reducing the dose of dexamethasone when used in combination with Compound 2 in the clinic.
[00350] [00350] As shown in Figure 13, a dramatic bidirectional synergy is observed after treatment with Compound 2 in combination with dexamethasone. As little as 10 nM dexamethasone increases the ability to kill Compound 2 cells, and low sub-nanomolar concentrations of Compound 2 potentiate the apoptotic effects of dexamethasone.
[00351] [00351] with peripheral blood mononuclear cells and K562 cells. Methods: Preparation of human peripheral blood mononuclear cells (PBMC): PBMCs isolated from healthy donors were cultured in RPMI 1640 medium with 10% FBS at a density of 1 x 10th cells / mL.
[00352] [00352] Cell culture: K562 cells were maintained in a log phase and cell density and viability were monitored by excluding trypan blue using the Vi-CELLº XR cell viability analyzer (Beckman Coulter, Brea, CA).
[00353] [00353] Assay procedures: Human PBMCs recently isolated were cultured with recombinant IL-2 at a concentration of units / mL for 72 h. The peripheral blood mononuclear cells were then centrifuged and resuspended in fresh RPMI complete medium to 2x10º cells / mL. The cells were then treated with DMSO or compounds at the indicated concentrations and incubated for an additional 72 h. PBMCs were then washed twice in fresh RPMI complete medium prior to co-culture. K562 cells were resuspended to a cell density of 1 x 10º / mL and stained with 1 µM CellTrace CFSE according to the manufacturer's instructions. The labeled K562 cells were then seeded in a 96-well round bottom plate in 1 x 10 th cells / wells. The peripheral blood mononuclear cells were then transferred to the same 96-well plate in a 1:15 ratio, in triplicate and incubated at 37ºC for 4 h. The specific lysis of the target cell by PBMC cells was measured using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) according to the manufacturer's instructions and the samples were run on the FACS Array scan. Unlabeled K562 cells, CellTrace CFSE-labeled KS62 cells and untreated annexin-labeled K562 cells V-FITC and PI were included in each assay as a control.
[00354] [00354] Co-culture assays with human peripheral blood mononuclear cells treated with untreated myeloma compound and cell lines. Methods: Cell culture. All myeloma cell lines were maintained in a logarithmic phase, and cell density and viability were monitored by excluding fiber blue using the Vi-CELL XR cell viability analyzer.
[00355] [00355] PBMC Treatment Test Procedure. Ninety-six wells were pre-coated with anti-CD3 antibody (OKT3, 3 µg / mL) and incubated at 4ºC overnight before the start of the experiment. The frozen PBMC donors were thawed at 37ºC for 2 minutes in RPMI medium with 10% FBS and cell counts and viability were measured in the V-CELLº (Beckman Coulter). The peripheral blood mononuclear cells were washed and diluted to 1 x 10 th cells / mL and dispensed to the compound-treated plates in a total volume of 200 µL. The cells were incubated with compounds for 2 h before being transferred to anti-CD3-coated plates and incubated for an additional 72 h at 37ºC. After 72 h, the PBMCs were centrifuged and the cells were washed twice in RPMI + 10% FBS medium. The untreated MM cell lines (H929 and H929-1051) were labeled with CellTrace CFSE according to the manufacturer's instructions and resuspended at a total concentration of 0.1x10º cells / mL in a 96-well U-bottom plate in a total volume of 100 ul. The peripheral blood mononuclear cells were counted and added to the MM cells in the target: effector (T: E) ratio of 1: 5. After 24 h of co-culture, the specific lysis of the target cells by PBMCs was measured using Annexin V-AF647 and 7-AAD according to the manufacturer's instructions and the samples were performed on the Attune NxT cytometer (Thermo Fisher).
[00356] [00356] Treatment Test Procedure with PBMC and MM Cells. Ninety-six wells were pre-coated with anti-CD3 antibody (OKT3, 3 µg / mL) and incubated at 4ºC overnight before the start of the experiment. Donors of frozen PBMC cells were thawed at 37ºC for 2 minutes in RPMI medium with 10% FBS and cell counts and cell viability were measured in the Vi-CELL analyzer. The peripheral blood mononuclear cells were washed and diluted to 1x105 cells / mL and dispensed to the compound-treated plates in a total volume of 200 µl. The cells were incubated with compounds for 2 h before being transferred to anti-CD3 coated plates and incubated for an additional 72 h. At the same time, the MM cell lines (NCI-H929, H929-1051, OPM2, OPM2-P10) were diluted to a final concentration of 0.1 x 10º cells / mL and labeled with CellTrace CFSE according to the manufacturer's instructions . Multiple myeloma cell lines were then dispensed in compost-treated plates at a total volume of 200 μl and incubated for 72 h. After 72 h, PBMCs and MM cells were counted and transferred to a 96-well U-bottom plate in the final T: E ratio of 1: 5. After 24 h of co-culture, the specific lysis of the target cells by PBMCs cells was measured using Annexin V-AF647 and 7-AAD according to the manufacturer's instructions and the samples were performed on the Attune NxT cytometer.
[00357] [00357] Results. The co-culture model was used to determine the direct effects of Compound 2 on the anti-tumor activity of PBMCs taken from healthy donors. The treatment of Compound 2 of IL-2 activated PBMCs induced the death of untreated K562 cells in a concentration-dependent manner (Figure 14, right panel). Compound 2-treated PBMCs (ICso = 5.9 pM) were * 600 times more potent than those treated with pomalidomide (POM; ICso = 0.004 uM) and * 2600 times more potent than lenalidomide-treated PBMCs (LEN; ICso = 0 , 02 uM) in obtaining 50% direct K562 cell death. Although Compound 2 is more potent than lenalidomide and pomalidomide, the magnitude of the response was similar between the compounds (Figure 14, right panel).
[00358] [00358] The effects of Compound 2 on the activity of anti-MM cells from PBMCs incubated with Compound 2 were further examined in cell lines that exhibited the resistance phenotype to compare with the response in sensitive cells. In a different co-culture model, PBMC donor cells were pre-treated with Compound 2, lenalidomide or pomalidomide for 2 h before being cultured in plates coated with anti-CD3 antibody for 72 h. Anti-CD3 antibody-activated PBMCs treated with Compound 2 demonstrated a concentration-dependent increase in tumor cell lysis from untreated lenalidomide-sensitive MM cell lines (NCI-H929; ICso = 0.005 uM) and lenalidomide-resistant (H929 -1051; ICso = 0.0002 µM) to a similar degree (Figure 15). Compound 2 was more potent than lenalidomide and pomalidomide in terms of reducing the percentage of viable MM cells. A similar level of tumor cell death by PBMCs was observed against lenalidomide-sensitive and lenalidomide-resistant tumor cells, showing that PBMCs were prepared to kill tumor cells regardless of their resistance phenotype.
[00359] [00359] As pre-incubation of immune cells with Compound 2 improved the targeting and lysis of MM cells, the effect of pre-incubation of MM cells with Compound 2 on their susceptibility to immune-mediated death ( Figure 16, Table 12). Four strains of MM cells and PBMCs activated by anti-CD3 antibody were pre-incubated separately with Compound 2, lenalidomide or pomalidomide for 72 h. When PBMCs and MM lines activated by anti-CD3 antibody were pretreated with Compound 2, lenalidomide or pomalidomide, followed by co-culture, the effects on PBMC-induced MM cell lysis were enhanced in both the potency and the magnitude of the death response. Comparing ICso values from single MM cell cultures versus immune and tumor cell co-cultures, Compound 2 increased NCI-H929 cell death by * 7000 times and increased H929-1051 cell death by * 6000 times . For pomalidomide-resistant cell line OPM2-P10, treatment of MM cell Compound 2 increased immuno-mediated death by * 3000-fold (Table 12).
[00360] [00360] Conclusion: PBMCs treated with Compound 2 induced tumor lysis of untreated K562 and MM cell lines to the same extent as lenalidomide and pomalidomide, although with much greater potency. In addition, tumor cell death was greatly increased if both PBMCs and MM cell lines were pretreated with Compound 2, indicating that, in addition to its autonomous effects of potent cells, Compound 2 can also increase the immunogenicity of the strains MM cell phones. The combination of the potent immunogenic and autonomous effects of cells and MM cells, in addition to its immunomodulatory properties, make Compound 2 a potential candidate for the clinic.
[00361] [00361] Daratumumab, an anti-CD38 antibody approved for the treatment of multiple myeloma, exerts its anti-myeloma activity by antibody dependent cell cytotoxicity (ADCC), antibody dependent cell phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) ). The effect of Compound 2 or pomalidomide in combination with daratumumab was evaluated in MM cell lines.
[00362] [00362] ADCC assay: The effect of Compound 2 or pomalidomide on daratumumab-mediated ADCC was evaluated in vitro by flow cytometry on a panel of MM cell lines. NK cells were grown overnight in NK culture medium containing 10 U / ml of recombinant human IL-2 prior to the start of the assay. The NK cells were washed and resuspended in NK cell culture medium at 3.75 x 10 th cells / ml. MM cells were pre-treated with sub-lethal concentrations of Compound 2 or pomalidomide for 72 h before being used in the ADCC assay. The MM cells were washed and marked with Tag-it Violet'Y Proliferation and Cell Tracking Dye according to the manufacturer's instructions, followed by resuspension in NK culture medium at a concentration of 0.75 x 10º cells / ml. The ADCC assay was performed in triplicate with an effector / tumor ratio of 10: 1 on a 96-well plate. MM cells (10 µl) were mixed with 10 µl of 2x daratumumab concentration in the wells before adding 20 µl of NK cells. The cocultures were incubated at 37ºC for 3 h, followed by the addition of 50 µl of 7-AAD solution at room temperature for 15 minutes. The analysis was performed on a BD Celesta flow cytometer.
[00363] [00363] ADCP assay: The effect of Compound 2 or pomalidomide on daratumumab-mediated ADCP was determined on a panel of MM cell lines. The monocytes were plated in a 96-well plate at
[00364] [00364] Results: Treatment of MM cells with Compound 2 and pomalidomide resulted in a dose-dependent increase in CD38 expression (Figure 17). The extent of CD38 expression was greater with Compound 2 and occurred at lower concentrations compared to pomalidomide. MM +/- Compound 2 cells or pre-treatment with pomalidomide were evaluated in ADCC assays with daratumumab. MM cells treated with Compound 2 demonstrated a greater degree of tumor lysis with daratumumab compared to untreated cells (Figure 18). Cells treated with Compound 2 were also more sensitive to ADCC mediated by daratumumab compared to cells treated with pomalidomide. The ability of Compound 2 and pomalidomide to modulate daratumumab-mediated ADCP was also tested. MM cells treated with Compound 2 were more sensitive to daratumumab-mediated ADCP compared to untreated and pomalidomide-treated cells (Figure 19). Only one cell line tested, ARH-77, did not show improved ADCP with Compound 2 or pomalidomide, but did demonstrate improved ADCC with Compound 2.
[00365] [00365] Conclusion: Compound 2 over-regulates CD38 expression in MM cells, resulting in an increase in ADCC and ADCP mediated by daratumumab compared to pomalidomide or untreated cells. These data suggest that the combination of daratumumab with Compound 2 may be more effective in treating MM compared to the combination with pomalidomide or daratumumab alone.
[00366] [00366] Twenty-four hours before treatment with a proteasome inhibitor and test compound, an appropriate number of cells were divided to a concentration of 0.2x10 ° / mL in fresh medium to allow exponential growth. On the day of treatment, the compounds were recently solvated in DMSO. The proteasome inhibitors bortezomib or carfilzomib were diluted and added to the preheated culture medium at the final working concentrations of 150 nM or 300 nM for bortezomib and 300 nM or 550 nM for carfilzomib. Proteasome inhibitor concentrations were determined based on clinical Cmax concentrations, as well as previous studies in each cell line, which determined the duration and concentration of PI needed to inhibit a specific amount of p5 proteasome activity. The cells were counted and an appropriate number was placed in the medium containing the proteasome inhibitor and thoroughly mixed. After incubation for 1 hour at 37ºC, 5% CO, the cells were washed twice with 40 ml of complete medium to remove the proteasome inhibitor. Aliquots of each proteasome treatment were tested to confirm the extent of inhibition of the B5, B2 and B1 subunits. The cells were resuspended at 0.1x10º / mL and plated at 100 μl / well in fresh culture material containing triplicate titrations of Compound 2 or pomalidomide. The plated cells were cultured at 37ºC, 5% CO ,, for the remainder of the experiment, up to 72 h. Every 24 h, proteasome inhibition was monitored by the cell-based Proteasome-Glo assay. Proliferation and apoptosis were measured at 72 h by flow cytometry. The cells were stained with APC Annexin-V and 7-AAD to enumerate the number of viable cells remaining in the culture.
[00367] [00367] Results. An in vitro cell assay has been established to mimic the clinical pharmacokinetics (PK) and pharmacodynamics (PD) of exposure to the proteasome inhibitors bortezomib and carfilzomib. The model employs short exposures of the proteasome inhibitor, followed by complete washing of the compound to dose cells with clinically relevant concentrations of the proteasome inhibitor, while obtaining the rapid clearance observed in vivo. In addition, a comparable level of B5 proteasome inhibition can be achieved in all cell lines. This model was used to assess the combined effects of Compound 2 in combination with bortezomib or carfilzomib in a panel of multiple myeloma and plasma cell leukemia cell lines (Pomalidomide-resistant OPM2.P10), RPMI.8226 and leukemia lines plasma cells L363 and JJN -3).
[00368] [00368] Bortezomib and Compound 2 demonstrated a combination effect in the two MM strains tested, OPM2. P10 and RPMI. 8226, as well as in one of the plasma cell leukemia cell lines, JJN-
[00369] [00369] Although treatments with carfilzomib between experiments were variable in the percentage of cell death they achieved over the course of the 1 h treatments, combination effects with Compound 2 were demonstrated in all 4 cell lines (Figure 21B).
[00370] [00370] Conclusion: Surprisingly, Compound 2 maintains its ability to kill cells at clinically relevant levels of proteasome inhibition. Combination of Compound 2 with bortezomib or carfilzomib demonstrated an increase in apoptosis and antiproliferative activity against MM cells.
[00371] [00371] The effect of combining treatment with Compound 2 and small molecule inhibitors with various mechanisms was evaluated in a panel of MM cell lines. Thirteen small molecule inhibitors were selected for combination studies with Compound 2 based on their preclinical activity and / or against MM. Cell lines H929-1051, KMS11, KMS-12PE, L363, OPM-P10 and RPMI8226 were selected for this study to represent the different groups of genetic grouping in MM cell lines. The concentrations of compounds for the combined treatments were selected in the range of 1 log above and 2 logs below the ICso of the single agent. The combination agents were dosed on a 6-point dose-response curve (CKD) at a 1: 3 dilution, Compound 2 was dosed at a 10-point CKD, also at a 1: 3 dilution. Combination experiments were performed twice, each time with data replicated on separate plates. The compounds were previously detected in the appropriate wells of 384-well plates using an acoustic dispenser. All MM cell lines were cultured in an incubator at 37ºC with 5% CO, using the indicated cell culture medium containing 1x Penicillin-Streptomycin. The cells were added to the compound containing 384-well plates using a Multidrop Combi Reagent Dispenser and allowed to incubate for 3 days at 37ºC with 5% CO ». After 3 days, the cells were evaluated for their level of ATP content via Cell Titer-Glo measured in a luminescence detector (PerkinElmer Envision).
[00372] [00372] The Highest Single Agent (SAH) method was used to detect synergy in the dose response curve data. The combinations were analyzed from a response surface perspective. A statistical framework (Van Der Borght, K., et al., BIGL: Biochemically Intuitive Generalized Loewe null model for prediction of the expected combined effect compatible with partial agonism and antagonism; Scientific Reports, 7 (1), 17935-1-17935 -9 (2017)) was incorporated into the analysis on the null HAS model with two statistical tests: 1) The complete response surface differs from the null model, 2) The single well differs from the null model.
[00373] [00373] Results: The effect of treatment with Compound 2 in combination with small molecule inhibitors was evaluated in a panel of multiple myeloma cell lines. Compound 2 was screened in combination with 14 compounds and synergy was calculated in all wells for 6 cell lines. Dexamethasone and etoposide showed significant synergy in combination with Compound 2 in five of the six cell lines tested (Figure 22). The combination of Compound 2 with BET inhibitors (4- [2- (cyclopropylmethoxy) -S- (methanesulfonyl) phenyl] -2-methylisoquinolin- 1 (2H) -one (Compound D), birabresib and GSK525762A) also demonstrated synergistic activity in MM cells, with different degrees of synergy between the three inhibitors. The combination of Compound 2 with AMG176 (MCL-1 inhibitor) showed synergistic activity in three cell lines (KMS11, KMS12-PE, L363) while the combination of Compound 2 with ACY241 and panobinostat (histone deacetylase inhibitors) was synergistic in L363 / OPM2- P10 and L363 / H929-1051, respectively Compound 2 in combination with 4- [2- (4-amino-piperidin-1-i1) -5- (3-fluoro-4-methoxy-phenyl) -1- methyl-6-0x0-1,6-dihydropyrimidin-4-yl] -2-fluoro-benzonitrile (Compound E) was synergistic in L363 and KMS12-PE cells. MIK665, an MCL-1 inhibitor, was the only compound that did not show significant synergy in the 6 MM cell lines tested.
[00374] [00374] Conclusions: Treatment with Compound 2 in combination with 12 of the 14 small molecules demonstrated synergistic activity in at least one or more of the MM cell lines tested. The combination with six of the compounds showed synergy in at least 3 MM cell lines tested (Figure 22). These data suggest that treatment combined with Compound 2 with the small molecule inhibitors tested represents a potential treatment paradigm for MM, including some with synergistic activity.
[00375] [00375] Methods: The xenograft study was performed with female SCID mice with multiple myeloma / plasmacytoma tumors resistant to lenalidomide NCI-H929 (H929-1051). Female SCID mice were inoculated subcutaneously with H929-1051 cells in the flank region above the right hind leg. After inoculation of the animals, were the tumors allowed to grow to approximately 100 mm before randomization. On the 13th day after the tumor cells were inoculated, did the mice with H929-1051 tumors vary between 79 and 157 mm were pooled and randomized into various treatment groups. Compound 2 was formulated in 2% HPMC in water (as a suspension). Dexamethasone was formulated in 0.5% CMC / 0.25% Tween 80 in deionized water. Compound 2 (0.1 mg / kg) and dexamethasone (0.5 mg / kg) were administered orally once daily during the study from day 13 after tumor cell inoculation. In the combination group, the animals received Compound 2 (0.1 mg / kg / day) and dexamethasone (0.5 mg / kg / day) simultaneously for the duration of the study from day 13 after tumor cell inoculation. . Tumors were measured twice a week using forceps and tumor volumes were calculated using the formula W x L / 2. Statistical analysis was performed using one-way or two-way analysis of variance (ANOVA). Synergy calculations were performed using the product fractional method.
[00376] [00376] Results: Treatment with single agent Compound 2 significantly (p <0.01) inhibited (-34%) the growth of H929-1051 multiple myeloma tumor. Treatment with dexamethasone as a single agent marginally (-20%) inhibited tumor growth in the H929 xenograft
[00377] [00377] Conclusion: Compound 2 in combination with dexamethasone exhibited synergism in tumor volume reduction in the multiple myeloma / plasmacytoma tumor model NCI-H929, indicating that the combined treatment of Compound 2 and dexamethasone showed synergistic antitumor activity in one model of lenalidomide-resistant MM. Compound 2 potentiates the apoptotic activity of dexamethasone, indicating the potential for dose reduction of dexamethasone when used in combination with Compound 2 in the clinic.
[00378] [00378] Methods: The xenograft study was performed with female SCID mice with multiple myeloma / plasmacytoma tumors resistant to lenalidomide NCI-H929 (H929-1051). Female SCID mice were inoculated subcutaneously with H929-1051 cells in the flank region above the right hind leg. After inoculation of the animals, were the tumors allowed to grow to approximately 500 mm before randomization. On Day 31 after tumor cell inoculation, did the mice with H929-1051 tumors vary between 366 and 535 mm were pooled and randomized into various treatment groups. Compound 2 was formulated in 2% HPMC in water (as a suspension). Bortezomibe was formulated in 1% DMSO in saline (as a solution). Compound 2 (1 mg / kg) was administered orally once daily for 3 consecutive days starting on day 31 after tumor cell inoculation. Bortezomib (1 mg / kg) was administered as a single dose intravenously on day 31 after tumor cell inoculation. combination group, animals received compound 2 (1 mg / kg / day) orally on days 31-33 and bortezomib was administered intravenously as a single dose on day 31. On day 31, bortezomib was administered 1 h before the first dose of Compound 2. Tumors were measured twice a week using forceps and tumor volumes were calculated using the formula W x L / 2. The animals were sacrificed when the tumor volumes reached a predetermined target of approximately 2000 mm . Statistical analysis was performed up to the 50th day using one-way or two-way analysis of variance (ANOVA). Synergy calculations were performed using the product fractional method.
[00379] [00379] Results: Treatment with single agent Compound 2, when administered once daily for 3 consecutive days (qdx3) on days
[00380] [00380] Conclusion: Compound 2 in combination with bortezomib exhibited synergism in reducing tumor volume in the lenalidomide-resistant plasmacytoma tumor model NCI-H929 and surprisingly produced tumor-free animals.
[00381] [00381] A multicentre, phase 1 open labeling study is conducted to assess the safety, pharmacokinetics and preliminary efficacy of Compound 2 in combination with dexamethasone in subjects with relapsed and refractory multiple myeloma (RRMM).
[00382] [00382] Objectives: The main objective of the study is to assess the pharmacokinetics (PK), safety / tolerability and to define the maximum tolerated dose (BAT) / recommended dose of Part 2 (RP2D) of Compound 2 in combination with a dexamethasone in conjunction with a minimum of two Compound 2 dosing schedules. The secondary objective is to assess the preliminary efficacy of Compound 2 in combination with dexamethasone.
[00383] [00383] Study design: This is an international phase 1, open label, multicenter study to assess the safety, PK / PD and preliminary efficacy of Compound 2 in combination with dexamethasone in subjects with RRMM. All eligible subjects must have failed, be intolerant or not be candidates for available therapies that confer clinical benefit in RRMM.
[00384] [00384] The study is carried out in two parts: Part 1 assesses the PK / PDe the safety of increasing doses of Compound 2 with simultaneous standard dose of dexamethasone and determines the MTD / RP2D for the combination when administered according to a minimum of two different dosing schedules. Part 2 consists of a single arm expansion cohort of Compound 2 in RP2D plus dexamethasone for both dosing schedules. In addition to safety, PK and PD assessments, all subjects undergo monthly response assessments by the Uniform Response Criteria of the International Myeloma Working Group (IMWG) (Rajkumar et al., Consensus recommendations for the uniform reporting of clinical trials :
[00385] [00385] The study is conducted in accordance with the Technical Requirements of the International Harmonization Council (ICH) for the Registration of Pharmaceutical Products for Human Use / Good Clinical Practice (GCP) and applicable regulatory requirements.
[00386] [00386] Part 1 (dose escalation): Cohorts of subjects with RRMM receive increasing doses of Compound 2 plus a fixed dose of dexamethasone (40 mg / dose; 20 mg / dose in subjects> 75 years) to assess their safety, MTD / RP2D and PK / PD profiles. A minimum of two different dosing schedules is evaluated in Part 1, the first consisting of 10 consecutive dosing days once daily (QD) followed by 4 days without treatment x 2 per 28-day cycle (referred to as schedule 20 / 28). The second schedule consists of a dose twice a day (BID) for 3 consecutive days, followed by 11 days without study treatment x 2 per cycle (referred to as the 6/28 schedule). The starting dose cohorts receive 0.1 mg / day of Compound 2 QD in the 20/28 schedule and 0.2 mg BID in the 06/28 schedule. Subject allocation is assigned by the Sponsor, depending on the availability of subject slots for one or both times. Switching between dosing schedules is not allowed. Additional dosing schedules (for example, 5 days of Compound 2 administration followed by 9 days without treatment x 2 OR 7 days after administration followed by 7 days without treatment x 2 per 28 day cycle) can be explored under the terms of an amendment to the protocol until the result of the initial safety results and PK / PD in association with schedules 20/28 and 6/28.
[00387] [00387] For all dosing schedules, Days 1 through 28 of Cycle 1 constitute the dose limiting toxicity assessment period (DLT) for the purpose of determining the BAT. Subjects are evaluated for DLT if they receive the prescribed dose of Compound 2 on at least 16 of the 20 day dose in the 20/28 schedule and at least 5 of the 6 day dose (10 doses) in the 6/28 schedule in Cycle 1 or experiencing a DLT. The evaluated subjects that are not DLT are replaced.
[00388] [00388] In each schedule, cohorts of three or more subjects receive Compound 2 in doses that increase in 100% increments in successive cohorts until the occurrence of two adverse events arising from Grade 2 treatment that cannot be clearly assigned and uncontroversial to strange causes. Thereafter, dose increments should not exceed 50% until the first DLT occurs. A Bayesian dose escalation methodology using logistic regression is used after the occurrence of a first DLT in any dosing schedule, with the assigned dose of Compound 2, the number of doses per day (QD vs IDB) and the number of consecutive days of dose for each regimen (3 vs 10) as covariates. The target toxicity rate for the combination of Compound 2 plus dexamethasone is 20% for all regimens.
[00389] [00389] Increasing the intra-subject dose is not allowed during the DLT assessment period, however, in Cycle 2 and beyond, subjects without evidence of disease progression that tolerate the designated dose of Compound 2 may be at the investigator's discretion and in consultation with the study's medical monitor) rise to the highest dose level demonstrated to be adequately tolerated by at least one cohort of subjects within the designated dosing schedule.
[00390] [00390] Part 2 (Cohort expansion): Upon completion of Part 1, a single arm expansion study of Compound 2 plus dexamethasone is performed on 20 subjects per dosing schedule to further assess their safety, PD and efficacy in RP2D and schedule.
[00391] [00391] After determining the RP2D for Compound 2 plus dexamethasone, an assessment of the safety / tolerability, PK and preliminary efficacy of Compound 2 / dexamethasone in combination with other anti-myeloma agents of interest, for example, anti-CD38 in a or more cohorts of subjects with different prior treatment histories and / or prognostic characteristics, can also be started in parallel as part of this protocol.
[00392] [00392] Study Population: Subjects aged> 18 years with MM refractory to their last line of treatment have failed or are intolerant or not candidates for available therapies that are known to confer clinical benefit to subjects with refractory and relapsing disease, have a Performance Status of the Eastern Cooperative Oncology Group (ECOG PS) 0-2, measurable disease and bone marrow, adequate renal and cardiac function. Subjects with a history of allogeneic transplantation, MM or oligosecretory or not, plasma cell leukemia or primary refractory MM (that is, with no history of at least a minor response to a previous treatment regimen) are excluded.
[00393] [00393] Inclusion criteria: Subjects must meet the following criteria to be enrolled in the study:
[00394] [00394] Exclusion criteria: The presence of any of the following excludes the subject from registration:
[00395] [00395] Study duration: The average duration of participation in the study per subject is approximately 6 months. The complete application should take approximately 21 months to complete (18 months for Part 1 and 3 months for Part 2). Completion of active treatment and post-treatment follow-up is expected to take an additional 6 to 12 months. The entire study is expected to continue for approximately 33 months.
[00396] [00396] The End of Trial is defined as the date of the last visit of the last subject to complete the post-treatment follow-up or the date of receipt of the last data point of the last subject that is necessary for the primary, secondary and / or analysis exploratory, as pre-specified in the protocol, whichever is more recent.
[00397] [00397] Study treatments: Compound 2 is administered orally once a day for subjects enrolled in the 20/28 schedule or twice daily for subjects enrolled in the 6/28 schedule. For subjects enrolled in the 20/28 dosing schedule, Compound 2 is administered in the morning with at least 240 mL of water after an overnight fast of at least 6 h. Subjects should refrain from eating food or other medications for at least 2 hours after each morning dose. Subjects enrolled in the 28/6 schedule follow the instructions mentioned above, as described in the 20/28 schedule for the first dose of each dose day. The second dose is administered 12 + 2 h after the morning dose, at least 4 h after and 2 h before food intake. As an example, subjects enrolled in the 6/28 dosing schedule could receive the initial dose of Compound 2 at 7:00 am, followed by breakfast at 9:00 am, lunch at noon and second dose of Compound 2 as early as possible at 5 pm with the evening meal taken 2 h later (ie before 7 pm). Note that only in Cycle 1, Compound 2 is administered on Days 1 to 3 (morning and afternoon), Day 14 (evening only), Days 15 and 16 (morning and afternoon) and Day 17 (morning only).
[00398] [00398] For both dosing schedules, dexamethasone is administered with Compound 2 in the fasted state or at least 2 h after Compound 2 with food (except on pharmacokinetic evaluation days,
[00399] [00399] Overview of key efficacy assessments: The primary efficacy variable is the best overall response rate (ORR) defined as the percentage of individuals whose best response is> PR, as determined by the IMWG Uniform Response Criteria (Rajkumar et al Blood 2011; 117 (18): 4691-5). The subjects undergo monthly response assessments. Myeloma response is determined by the study site investigator based on laboratory investigations (serum protein electrophoresis (sPEP), urine protein electrophoresis (uPEP), immunofixation electrophoresis (IFE), free light chain serum levels (sFLC ), quantitative immunoglobulin A (IgA), bone marrow for quantification of plasma cells, as appropriate) assessed in a central reference laboratory and / or locally (eg, corrected serum calcium, positron emission tomography / computerized scan (PET) / CT) or magnetic resonance imaging (MRI) to evaluate plasmacytoma and / or CT or skeletal research to assess bone lesions). Additional efficacy variables include response time (time from 1st dose of Compound 2 to first response documentation> PR), duration of response (time from first response documentation (> PR) to first PD or death documentation) ) and progression-free survival (time from the 1st dose of Compound 2 to the first occurrence of disease progression or death from any cause).
[00400] [00400] All security subjects with a valid baseline and at least one post-baseline response assessment are included in the effectiveness analyzes. If treatment is discontinued for reasons other than disease progression, subjects should continue assessing responses according to the specified assessment schedule until progression, withdrawal of consent, death, or initiation of new systemic anti-myeloma therapy, what happens first.
[00401] [00401] Overview of key safety assessments: The safety variables for this study include adverse treatment emergent events (TEAEs) and changes from baseline in physical findings / vital signs, selected laboratory analytes and 12-lead electrocardiograms ( ECGs). Additional safety metrics include the extent of exposure to study treatment (Compound 2 and dexamethasone), assessments of concomitant drug use and pregnancy tests for women of childbearing potential (FCBP).
[00402] [00402] Overview of pharmacokinetic evaluations: PK profiles (starting dose and steady state) are evaluated for Compound 2, its R enantiomer (Compound 3) and dexamethasone. Exposure response analyzes can be performed, as appropriate, to assist in the identification of Compound 2 RP2D.
[00403] [00403] The modalities described above are intended to be merely exemplary, and those skilled in the art will recognize, or be able to verify using, no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures.
All such equivalents are considered to be within the scope of the invention and are covered by the appended claims.

权利要求:
Claims (42)
[1]
1. A compound, where the compound is Compound 1 of the formula o
CO
OA NU o o
AX 1 or an enantiomer, mixture of enantiomers, tautomer, isotopologist or pharmaceutically acceptable salt thereof.
[2]
2. A compound in which the compound is Compound 2 of formula o
DAS NU o the AX. 2 or a tautomer, isotopologist or pharmaceutically acceptable salt thereof.
[3]
The compound of claim 1, wherein the compound is a tautomer of Compound 1.
[4]
The compound of claim 1, wherein the compound is an enantiomer of Compound 1.
[5]
The compound of claim 1, wherein the compound is a mixture of enantiomers of Compound 1.
[6]
The compound of claim 1, wherein the compound is a pharmaceutically acceptable salt of Compound 1.
[7]
The compound of claim 2, wherein the compound is a tautomer of Compound 2.
[8]
The compound of claim 2, wherein the compound is a pharmaceutically acceptable salt of Compound 2.
[9]
9. Pharmaceutical composition comprising the compound of claim
1.
[10]
10. Composition - pharmaceutical - comprising the compound of claim 2.
[11]
A method of treating multiple myeloma comprising administering a therapeutically effective amount of the compound of claim 1 to a patient in need thereof.
[12]
12.0 The method of claim 11, wherein the multiple myeloma is relapsed, refractory or resistant.
[13]
The method of claim 12, wherein the multiple myeloma is refractory or resistant to lenalidomide.
[14]
The method of claim 12, wherein the multiple myeloma is refractory or resistant to pomalidomide.
[15]
The method of claim 11, wherein the multiple myeloma is newly diagnosed multiple myeloma.
[16]
The method of any of claims 11-15, further comprising administering a second active agent.
[17]
17. The method of claim 16, wherein the second active agent is dexamethasone.
[18]
The method of claim 16, wherein the second active agent is bortezomib.
[19]
19. A method of treating multiple myeloma, comprising administering a therapeutically effective amount of the compound of claim 2 to a patient in need thereof.
[20]
The method of claim 19, wherein the multiple myeloma is relapsed, refractory or resistant.
[21]
21. The method of claim 20, wherein the multiple myeloma is refractory or resistant to lenalidomide.
[22]
22. The method of claim 20, wherein the multiple myeloma is refractory or resistant to pomalidomide.
[23]
23. The method of claim 19, wherein the multiple myeloma is newly diagnosed multiple myeloma.
[24]
24. The method of any of claims 19-23, further comprising administering a second active agent.
[25]
25. The method of claim 24, wherein the second active agent is dexamethasone.
[26]
26. The method of claim 24, wherein the second active agent is bortezomib.
[27]
27.0 compound of claim 1 for use in a method of treating multiple myeloma, wherein the method comprises administering a therapeutically effective amount of said compound to a patient in need thereof.
[28]
The compound for use of claim 27, wherein the multiple myeloma is relapsed, refractory or resistant.
[29]
The compound for use of claim 28, wherein the multiple myeloma is refractory or resistant to lenalidomide.
[30]
The compound for use of claim 28, wherein the multiple myeloma is refractory or resistant to pomalidomide.
[31]
The compound for use of claim 27, wherein the multiple myeloma is newly diagnosed multiple myeloma.
[32]
The compound for use in any of claims 27-31, wherein the method further comprises administering a second active agent.
[33]
33. The compound for use of claim 32, wherein the second active agent is dexamethasone.
[34]
34. The compound for use of claim 32, wherein the second active agent is bortezomib.
[35]
35.0 the compound of claim 2 for use in a method of treating multiple myeloma, wherein the method comprises administering a therapeutically effective amount of said compound to a patient in need thereof.
[36]
36. The compound for use of claim 35, wherein the multiple myeloma is relapsed, refractory or resistant.
[37]
37. The compound for use of claim 36, wherein the multiple myeloma is refractory or resistant to lenalidomide.
[38]
38. The compound for use of claim 36, wherein the multiple myeloma is refractory or resistant to pomalidomide.
[39]
39. The compound for use of claim 35, wherein the multiple myeloma is newly diagnosed multiple myeloma.
[40]
40. The compound for use in any of claims 35-39, wherein the method further comprises administering a second active agent.
[41]
41. The compound for use of claim 40, wherein the second active agent is dexamethasone.
[42]
42. The compound for use of claim 40, wherein the second active agent is bortezomib.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762530778P| true| 2017-07-10|2017-07-10|

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US62/530,778|2017-07-10|

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US201762593185P| true| 2017-11-30|2017-11-30|

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US62/593,185|2017-11-30|

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US201862675581P| true| 2018-05-23|2018-05-23|

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US62/675,581|2018-05-23|

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PCT/US2018/041230|WO2019014100A1|2017-07-10|2018-07-09|Antiproliferative compounds and methods of use thereof|

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