Lysine-specific histone demethylase inhibitors for the treatment of myeloproliferative neoplasms

Abstract

Disclosed herein are methods for treating or preventing myeloproliferative neoplasms in a subject in need thereof and for achieving specific clinically relevant endpoints, comprising administering a therapeutically effective amount of an LSD1 inhibitor.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 945,609 filed on 9 December 2019 and U.S. Provisional Application No. 63 / 121,461 filed on 4 December 2020 (both in their entirety are incorporated herein by reference as if they were described herein in their entirety). [Background technology]

[0002] Myeloproliferative neoplasms (MPNs), a disease category encompassing polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF), are a distinguishable family of hematopoietic disorders resulting from somatic mutations in pluripotent hematopoietic stem cells / progenitor cells, leading to hematological abnormalities in erythrocyte, leukocyte, and platelet production, as well as splenomegaly and systemic symptoms. MPNs share common mutations that constitutively alter normal physiological signals responsible for hematopoiesis. While MPNs can manifest clinically as benign clonal myeloproliferation, the initiating abnormal stem cells / progenitor cells are susceptible to novel mutations and epigenetic modifications that enable rapid evolution to myelofibrosis-associated myelofibrosis or transformation into acute myeloid leukemia (AML).

[0003] Many MPN patients are asymptomatic at diagnosis. Due to the intersection of definitive diagnosis and prognosis, ET, PV, and PMF can mimic each other. Common symptoms include fatigue, weight loss, night sweats, fever, dyspnea, and abdominal discomfort sometimes due to massive splenomegaly. The three MPN disorders have overlapping phenotypes and also share similarities with other myeloid neoplasms. Specific point mutations in JAK2 (JAK2V617F), as well as mutations in calreticulin (CALR) and thrombopoietin receptor (MPL), are found in 90% of MPN patients. The distribution of these mutations is not equal among PV, ET, and primary MF, i.e., PMF, but specific MPNs and prognoses do not diagnostically define and are not mutually exclusive. Healthy individuals can carry one of these mutations without developing MPN, and in fact, some of these mutations can be carried as germline mutations that cause genetic forms of MPN. Nevertheless, PV, ET, and PMF are considered distinct clinical entities based on their respective distinguishable epidemiology, natural history, and molecular profiles. PV is the most common MPN, and phenotypic symptoms will appear in JAK2. PV is the only MPN characterized by erythrocytosis defined as a hematocrit value ≥60% and hemoglobin ≥20 gm / dL. ET is characterized by a persistent platelet count >450,000 / μL and occurs mainly in women. MF is primary or secondary myelofibrosis, sometimes called myelofibrosis with myeloid metaplasia, myeloid metaplasia of unknown origin, or primary myelosclerosis, a chronic inflammatory process in which excessive collagen is deposited in the bone marrow, impairing hematopoiesis in relation to myelofibrosis and extramedullary hematopoiesis.

[0004] Major complications result from cytopenia secondary to bone marrow failure, extramedullary hematopoiesis (mainly in the spleen and liver), and progression to acute myeloid leukemia. For patients, splenomegaly is the most tragic complication of primary myelofibrosis, causing mechanical discomfort, cachexia, splenic infarction, portal and pulmonary hypertension, and blood cell sequestration. Both ET and PV are complicated by thrombosis. ET and PV can progress to MF and AML.

[0005] Many other somatic mutations found in MPN are also present in myelodysplastic syndromes (MDS) and de novo AML, including mutations in DNMT3A, IDH1 / 2, TET2, ASXLI, EZH2, TP53, NF1, NRAS, KRAS, SF3B1, U2AF1, SRSF2, and RUNX1. This shared mutation spectrum contributes to the phenotypic overlap of these disorders and further influences their natural history, including evolution to bone marrow failure or AML.

[0006] There are no specific treatments for primary myelofibrosis, essential thrombocythemia, or polycythemia vera. Current treatments do not significantly alter the natural history of the disease and therefore primarily aim to improve symptoms. Anemia with erythropoietin (EPO) levels <100 mU / ml may respond to recombinant EPO therapy, but is accompanied by increased hepatosplenomegaly. Prednisone may be effective in patients with evidence of active inflammation or autoimmune disease. Hyperuricemia is managed with allopurinol. The non-selective JAK1 / 2 inhibitor ruxolitinib is approved for intermediate 1 and 2 as well as high-risk MF patients, and even high-risk PV patients. Ruxolitinib alleviates systemic symptoms and is effective in reducing spleen size or volume by 35% in approximately 50% of patients. Ruxolitinib extends survival and is effective in high-risk patients with primary MF (PMF) V617FAllele loading was reduced. Anemia is worsened by ruxolitinib in some patients, but thrombocytopenia may improve, even if severe. Ruxolitinib is effective only during the duration of drug administration, and symptoms will likely recur once the drug is discontinued. Intramedullary fibrosis is unaffected, and ruxolitinib does not affect mutational loading. Thalidomide at doses of 50-100 mg / day, in combination with prednisone, is effective in improving anemia and thrombocytopenia and reducing spleen size by approximately 20% in about 60% of patients with primary myelofibrosis. Low doses of interferon-α to reduce splenomegaly may be effective in the early course of the disease, but may cause cytopenia. Pegylated interferon produces molecular remission in PV and can reverse myelofibrosis in a small number of patients with PMF. Hydroxycarbamides have a low incidence of acute toxicity but cause myelosuppression and are leukemic. Low-dose alkylating agents may reduce organomegaly, reverse myelofibrosis, or improve blood cell counts, but only occasionally have permanent effects, and alkylating agents can cause severe myelosuppression and are leukemic. The only potentially curative treatment is allogeneic bone marrow transplantation, indicated for patients under 65 years of age with a matched donor and a middle-grade or high DIPSS score. The mean 5-year survival rate for stem cell grafts is approximately 50%.

[0007] Epigenetic modifications of DNA, such as methylation of cytosine or post-translational modifications of histones, such as methylation and acetylation, affect gene expression by altering chromatin structure. Changes in gene expression patterns have the potential to modify the phenotype of a given cell. Mutations in DNMT3A and TET2 are associated with changes in the normal methylation pattern of cytosine in DNA, while mutations in JAK2, EZH2, and ASXL1 modify the methylation, acetylation, and phosphorylation status of histones. That is, both of these classes of changes modify the pattern of the normal gene expression program. Mutations in genes encoding proteins that affect the epigenetic state of cells suggest that it may be possible to selectively target the enzymatic functions of such proteins to eliminate malignant stem cell / progenitor clones and / or restore their normal phenotypes.

[0008] Lysine-specific demethylase 1 (LSD1, also known as KDM1A) is an enzyme that removes mono- and dimethyl groups from critical lysine (K) residues K4 and K9 of histone (H) H3 (Shi et al., 2004). Methylation of histone H3K4 and H3K9 are post-translational modifications that are associated with changes in gene ratios. Since LSD1 modifies the local state of chromatin, it is an epigenetic regulator of gene expression. The lysine (K) sites of histone H3 and the degree of methylation of those sites (1, 2, or 3 methyl groups) are associated with specific functions. For example, enhancers and super-enhancers are characterized by the H3K4me1 mark, while H3K4me2 is more frequently found in proximal promoters and enhancers of actively transcribed genes.

[0009] LSD1 is localized to three common genomic regions: enhancers and super-enhancers, proximity promoters, and the internal regions of transcription units, via the action of proteins (generally TFs) that directly bind to DNA. Many TFs, both activators such as the V-Myb bird myeloblastosis virus oncogene homolog (MYB) and steroid hormone receptors, and repressors such as the growth factor-independent transcriptional repressor 1 (GFI1), recruit LSD1 to specific genomic locations. LSD1 is part of a larger protein complex that includes Co-RE1 silencing transcription factor (CoREST) ​​or nucleosome remodeling and histone deacetylase (NuRD), which determine cell-specific chromatin remodeling. These complexes may also include DNMT1 activity and histone deacetylase 1, 2, and 3 (HDAC1, 2, and 3) activity, all of which contribute to maintaining or modifying the epigenetic state of their genomic sites. Therefore, a key property of LSD1 beyond its own enzymatic activity is its function as a scaffold for other epigenetic enzymes cocruited to its genomic site. Of many histone demethylases, LSD1 uniquely utilizes flavin adenine dinucleotide (FAD) to oxidatively remove one or two methyl groups in the process of producing H2O2 and formaldehyde. For this reason, FAD is an essential cofactor for LSD1 activity. The Jumonji types of the other 33 histone lysine demethylases utilize an iron-dependent mechanism to remove methyl groups from histone lysine.

[0010] LSD1 is an essential gene, and loss of LSD1 activity leads to early embryonic lethality. This protein is also necessary to regulate the balance between self-renewal and proliferation. Conditional in vivo LSD1 knockdown (KD) using doxycycline-inducible short hairpin LSD1 (shLSD1) established LSD1 as a central regulator of hematopoietic stem cells (HSCs) and myeloid progenitor cells. LSD1 KD resulted in severe but reversible thrombocytopenia, neutropenia, and anemia, but increased monocyte counts. 27-day LSD1 KD resulted in an increase in circulating pluripotent progenitors (MPPs) and HSCs, accompanied by contingent downregulation of chemokine (CXC motif) receptor 4 (CXCR4), without affecting the size of the dormant HSC pool. Autoregeneration impairment was observed in long-term HSCs for 12 weeks after LSD1 removal using an inducible Cre system (Mx1Cre mice × Lsd1fl / fl mice), consistent with LSD1 inhibition driving differentiation.

[0011] LSD1 plays a crucial role in regulating the progression from pluripotency to terminal differentiation. Through the mediators of the “master” transcription factors octamer-binding transcription factor 4 (OCT4), SRY (sex-determining region Y)-box2 (SOX2), Nanog, and coactivators, LSD1 is recruited to “high-reliability” promoters and super-enhancers of genes essential for normal development. While not essential for maintaining embryonic stem cell (ESC) status, as part of the NuRD complex, LSD1 directs the pluripotency program, enabling ESC differentiation by “deactivating” gene enhancers. LSD1 is essential for the complete shutdown of the ESC gene expression program as cells transition to more differentiated cell states. The role of LSD1 in ESCs is phenomenologically similar to its essential role in myeloid hematopoiesis, where active enhancers in HSCs generating stem cell gene expression signatures are similarly “deactivated,” enabling progenitor commitment to specific myeloid lineages. Enhancers essential for terminal differentiation in lineage-specific progenitor cells are in equilibrium with activation by the H3K4me1 mark, while promoters are characterized by the gradual methylation of H3K4 to H3K4me3. Enhancer H3K27 acetylation is locked during transcriptional activation and lineage commitment. In line with the need for stable H3K4 methylation during differentiation, LSD1 expression dramatically decreases as myeloid differentiation progresses to the terminal cell state. The LSD1 enzyme is at the apex of myeloid hematopoiesis. LSD1 prevents myeloid differentiation in stem cells and myeloid progenitor cells, but is downregulated when cells commit to specific myeloid lineages (erythroid, granulocyte, and megakaryocyte lineages). Inhibition of LSD1 in acute myeloid leukemia cells results in loss of stem cell potential (clonality) and induction of associated differentiation into a more mature monocytic immunophenotype. In a mouse model of myeloproliferative neoplasm, treatment with LSD1 inhibitors reduces the population of mutant progenitor cells consistent with the role LSD1 plays in the persistence of the autoregenerative phenotype.

[0012] As a major factor regulating myeloid maturation, LSD1 is a suitable target for various myeloproliferative neoplasms. Three major myeloproliferative neoplasms that can be treated with LSD1 inhibitors are present: polycythemia vera, essential thrombocythemia, and primary myelofibrosis (or myelofibrosis secondary to PV and ET). Other MPNs are disclosed below and can also be treated by the methods disclosed herein. Other MPNs include all that begin as clonal disorders resulting from somatic mutations occurring in hematopoietic stem cells / progenitor cells. The clinical overlap between these related diseases is reflected in their shared genetic spectrum of somatic mutations, including mutations in JAK2, DNMT3A, MPL, CALR, and ASXL1. Myelofibrosis (Jak2 V617F and Mpl W515L In a mouse model of the disease, inhibition of LSD1 resulted in significant improvements in five disease parameters: reduced platelet count, reduced splenomegaly, reduced red blood cell count, resolution of myelofibrosis, and reduced mutant cell load.

[0013] Among BCR-ABL-negative myeloproliferative neoplasms, primary myelofibrosis and post-PV / ET myelofibrosis (PPV-MF and PET-MF) have the highest morbidity and mortality rates, including progressive myelofibrosis (BM) fibrosis and the resulting BM failure. While the JAK inhibitor ruxolitinib is currently approved for the treatment of MF-related splenomegaly and systemic symptoms, JAK inhibitor therapy does not reduce the JAK2 mutant cell population in MPN patients. The limited ability of JAK inhibition to induce clinically meaningful molecular responses in MPN patients highlights the need for the development of more effective therapies for these JAK kinase / STAT-dependent malignancies.

[0014] Recent studies have shown that the lysine-specific histone demethylase LSD1 (KDM1A) is involved in balancing in vivo proliferation and differentiation in hematopoietic stem cells / progenitor cells by influencing state-specific gene expression patterns. In physiological hematopoiesis, LSD1 is essential for normal myeloid differentiation, affecting erythrocyte, megakaryocyte, and granulocyte lineages, rather than monocyte / dendritic cell lineages. Small molecule inhibitors of LSD1 have shown promising results in preclinical models of acute myeloid leukemia (AML) and solid tumors, and have recently entered clinical trials in AML. However, the role and requirements of LSD1 in the pathogenesis of MPN, and the therapeutic targeting of LSD1 in MPN, remain areas of current research.

[0015] International Publication 2012 / 107498 discloses the use of certain LSD1 inhibitors for the treatment of essential thrombocythemia, myelofibrosis, and polycythemia vera in Philadelphia chromosome-negative myeloproliferative disorders. U.S. Patent Publication 2016 / 0257662 and U.S. Patent Publication 2016 / 0237043 disclose compounds that inhibit LSD1. U.S. Patent Publication 2019 / 0070172 discloses the usefulness of these and other compounds in the treatment of myeloproliferative neoplasms, including ET, MF, and PV. [Overview of the project] [Problems that the invention aims to solve]

[0016] However, there remains a need for potent LSD1 inhibitors that have demonstrated the ability to treat myelofibrosis and other myeloproliferative neoplasms and their associated symptoms while avoiding serious side effects such as severe thrombocytopenia, thereby achieving specific clinically relevant endpoints in the treatment of myelofibrosis and other myeloproliferative neoplasms. [Brief explanation of the drawing]

[0017] Brief explanation of the drawing [Figure 1] This shows the changes in spleen volume from day 0 to day 84 of treatment in patients treated with LSD1 inhibitor compound 1. [Figure 2]This shows the change in MPN-10 score from day 0 to day 84 of treatment in patients treated with LSD1 inhibitor compound 1. [Figure 3] We will compare the changes in spleen volume response (SVR) and total symptom score (TSS) from day 0 to day 84 of treatment with treatment using LSD1 inhibitor compound 1 and the best available treatment (BAT). [Figure 4] This shows the changes in the inflammatory cytokine S100A9 at 12 weeks of treatment with LSD1 inhibitor compound 1. [Figure 5] This shows the changes in the inflammatory cytokine RANTES at 12 weeks of treatment with LSD1 inhibitor compound 1. [Figure 6] This shows the changes in the inflammatory cytokine IL-8 at 12 weeks of treatment with LSD1 inhibitor compound 1. [Figure 7] This shows the changes in circulating growth factor (VEGF) at 12 weeks of treatment with LSD1 inhibitor compound 1. [Figure 8] This shows the changes in circulating growth factor (PDGF-BB) at 12 weeks of treatment with LSD1 inhibitor compound 1. [Figure 9] This is a schematic diagram illustrating the therapeutic theory of LSD1 inhibition by compound 1. [Figure 10] This shows the percentage of F cells in 6 patients treated with LSD1 inhibitor compound 1. [Figure 11] (i) The absolute changes in (a) MPN SAF TSS and (b) spleen volume from day 0 to (ii) 12 weeks are shown. [Figure 12] The treatment progression in representative patients is shown. (a) Daily dose of LSD1 inhibitor, mg; (b) Spleen size, cm; (c) Symptom score; (d) Platelets (left scale, k / uL) and hemoglobin (right scale); (e) WBC and neutrophils; and (f) Fatigue score (10 = worst). [Modes for carrying out the invention]

[0018] Detailed explanation This specification provides a method for treating myeloproliferative neoplasms in subjects requiring such treatment, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0019] A method for suppressing the proliferation of malignant myeloid cells in a subject requiring such suppression is also provided, which includes administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0020] A method is also provided for reducing the concentration of one or more protein growth factors secreted by myeloid cells that activate one or more cell types that secrete reticulin and collagen, in a subject requiring such reduction, the method comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0021] In certain embodiments, one or more protein growth factors are selected from platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor beta-1, and platelet factor 4 (also known as CXCL4).

[0022] In certain embodiments, the myeloid cells that activate one or more cell types that secrete reticulin and collagen are megakaryocytes.

[0023] In certain embodiments, one or more cell types that secrete reticulin and collagen are selected from stromal cells and / or bone marrow resident fibroblasts and / or myofibroblasts.

[0024] A method is also provided for reducing the concentration of one or more protein growth factors secreted by bone marrow cells that impair the function of bone marrow osteoclasts and reduce the amount of bone marrow sclerosis, in a subject requiring such reduction, the method comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0025] In certain embodiments, the bone marrow cells that impair the function of bone marrow osteoclasts and reduce the amount of bone marrow sclerosis in the subject are megakaryocytes.

[0026] A method for reducing reticulin and collagen myelofibrosis in subjects requiring such reduction is also provided, comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0027] A method is also provided for reducing the plasma levels of one or more inflammatory cytokines in a subject requiring such reduction, the method comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0028] A method is also provided for reducing malignant cell burden, as measured by the frequency of variant alleles in myeloid cells, in subjects requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0029] A method for eliminating malignant myeloid cells in a subject requiring such elimination is also provided, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0030] A method is also provided for reducing pathologically elevated red blood cell count in subjects requiring such reduction, which includes administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0031] A method is also provided for reducing an abnormal spleen size or volume in a person in need thereof, the method comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0032] A method is also provided for reducing the amount of extramedullary hematopoiesis in a person in need, which includes administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0033] A method is also provided for improving the quality of life (QOL) as measured by validated patient-reported QOL assessments in individuals in need, the method comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0034] A method is also provided for reducing systemic symptoms of myelofibrosis, as measured by a validated patient-reported symptom assessment form, in subjects in need thereof, comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0035] A method for extending lifespan in patients with myelofibrosis who require it is also provided, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor.

[0036] A method is also provided for delaying or preventing the progression from myelofibrosis to acute myeloid leukemia in subjects in need, the method comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0037] A method for reducing platelet count in a subject requiring such reduction is also provided, which includes administering a therapeutically effective amount of an LSD1 inhibitor.

[0038] A method is also provided for reducing myeloid cells to age-adjusted normal cells with less than 5% blast cells in subjects requiring such reduction, comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0039] A method is also provided for maintaining or reducing the number of myeloblasts to <5% in subjects requiring such maintenance, comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0040] A method is also provided for increasing hemoglobin to >100 g / L in MF patients, comprising administering a therapeutically effective amount of an LSD1 inhibitor.

[0041] A method is also provided for reducing the frequency of thrombosis and bleeding in subjects in need thereof, which includes administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0042] A method is also provided for reducing the frequency of red blood cell infusions in subjects requiring such reduction, the method comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0043] Also provided are methods for a) reducing the hematocrit level to <45% in male patients with PV, or to ≤42% in female patients with PV, b) reducing the hemoglobin level to <160 g / L in patients with PV, and / or c) reducing the red blood cell count to ≤5.2 M / mL in patients with PV, all of which involve administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0044] In each specific embodiment of the above method, the LSD1 inhibitor is N-[(2S)-5-{[(1R,2S)-2-(4-fluorophenyl)cyclopropyl]amino}-1-(4-methylpiperazine-1-yl)-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide,bis-tosylate salt [ka] This is ("Compound 1").

[0045] This specification describes a method for treating myeloproliferative neoplasms in subjects, and approximately 50 × 10 9 ~About 100×10 9 A method for achieving a platelet count of platelets / L, Administer compound 1 at a starting dose of 0.5 mg / kg / d, Approximately one week later, the number of platelets in the patient will be evaluated. Platelet count ≥ 90 × 10 9 If the platelet count is 1 / L and the % platelet reduction is <50% from the previous examination, add 0.2 mg / kg / d of compound 1 to the daily dose. Platelet count ≥ 90 × 10 9 If the platelet count is 1 / L and the % platelet reduction is ≥50% from the previous examination, add 0.1 mg / kg / d of compound 1 to the daily dose. When the platelet count is 40×10 9 platelets / L to 89×10 9 platelets / L, maintain the current daily dose of Compound 1, and When the platelet count is 25×10 9 platelets / L to 39×10 9 platelets / L, reduce the current mg / kg daily dose of Compound 1 by 25%, and When the platelet count is <25×10 9 platelets / L, withhold administration until the platelets return to >50×10 9 platelets / L, then administer Compound 1 at 50% of the dose administered when the platelet count dropped below 25×10 9 platelets / L, and Optionally, repeat the platelet count evaluation and dose adjustment steps approximately once a week until the subject's platelet count reaches about 50×10 9 to about 100×10 9 platelets / L A method including this is also provided.

[0046] In certain embodiments, the subject in need has a myeloproliferative neoplasm.

[0047] In certain embodiments, the myeloproliferative neoplasm is myelofibrosis (MF).

[0048] In certain embodiments, the myelofibrosis is selected from primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (PPV-MF), and post-essential thrombocythemia myelofibrosis (PET-MF).

[0049] In certain embodiments, the myelofibrosis is primary myelofibrosis (PMF).

[0050] In certain embodiments, the myeloproliferative neoplasm is polycythemia vera (PV).

[0051] In certain embodiments, the myeloproliferative neoplasm is essential thrombocythemia (ET).

[0052] In certain embodiments, the subject or the malignant myeloid cells of the subject have mutations in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR).

[0053] In certain embodiments, the method further includes the step of determining whether the subject has a mutation in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR).

[0054] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is approximately 50 × 10 in subjects with myelofibrosis. 9 ~About 100×10 9 The amount is sufficient to maintain a platelet count of platelets / L, or otherwise the amount described below. In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is about 50 × 10 in the subject. 9 ~Approx. 75×10 9 This is a sufficient amount to maintain a platelet count of platelets / L.

[0055] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is 400 × 10 in patients with essential thrombocythemia. 9 An amount sufficient to maintain a platelet count below a certain level, or otherwise the amount described below.

[0056] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is approximately 150 × 10 in patients with PV. 9 ~Approx. 250×10 9 An amount sufficient to maintain a platelet count of platelets / L, or otherwise the amount described below.

[0057] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is approximately 0.5 mg / kg / d to approximately 1.5 mg / kg / d.

[0058] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is approximately 0.7 mg / kg / d to approximately 1.2 mg / kg / d.

[0059] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is approximately 40 mg to approximately 100 mg / day.

[0060] In certain embodiments, the therapeutically effective and non-harmful amount of compound 1 is approximately 50 mg to approximately 85 mg / day.

[0061] In a particular embodiment, the subject is administered compound 1 at an initial dose of 0.5 mg / kg / d, and then one week later, Platelet count ≥ 90 × 10 9 If the platelet count is 1 / L and the % platelet reduction is <50% from the previous examination, the dose is adjusted to add 0.2 mg / kg / d of compound 1 to the daily dose. Platelet count ≥ 90 × 10 9 If the platelet count is 1 / L and the % platelet reduction is ≥50% from the previous examination, the dose is adjusted to add 0.1 mg / kg / d of compound 1 to the daily dose. Platelet count: 40 × 10 9 Platelets / L~89×10 9 If platelets / L, the daily dose of compound 1 is maintained. Platelet count: 25 × 10 9 Platelet / L~39×10 9 If platelets / L, the target dose is adjusted to reduce the current mg / kg daily dose of compound 1 by 25%. Platelet count <25 × 10 9 If platelets / L, then platelets > 50 × 10 9 Administration should be withheld until the platelet count returns to 25 × 10⁶ / L, and then the target dose should be 25 × 10⁶ 9 Compound 1 is administered at 50% of the dose given when platelet count drops below less than 1 / L, and Selectively, approximately once a week throughout the entire course of therapy, the target platelet count is approximately 50 × 10 9 ~About 100×10 9The platelet count evaluation and dose adjustment process is repeated until the platelet count reaches a certain level (platelets / L).

[0062] A method for treating myeloproliferative neoplasms in subjects who have a mutant allele, comprising a certain amount of N-[(2S)-5-{[(1R,2S)-2-(4-fluorophenyl)cyclopropyl]amino}-1-(4-methylpiperazine-1-yl)-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide,bis-tosylate salt [ka] A method is also provided that includes administering ("Compound 1") to the target.

[0063] In certain embodiments, the mutant allele is JAK V617F Janus kinase 2 (JAK2), MPL, etc. W515K Myeloproliferative leukemia viruses such as oncogene (MPL) and CALR 52b_del CALR K385NCX or CALR KKRK374X It is an allele of one or more genes selected from calreticulins (CALRs) such as the following.

[0064] In certain embodiments, the mutant allele is an allele of one or more genes selected from DNMT3A, IDH1 / 2, TET2, ASXLI, EZH2, TP53, NF1, NRAS, KRAS, SF3B1, U2AF1, SRSF2, RUNX1, CBL, ZBTB33, PRPF8, CNTN5, FREM2, MAP1B, and GPR183.

[0065] In a particular embodiment, the mutant allele is ASXL1 HHCHREAA630X ASXL1 -642X ASXL1 Q780* ASXL1 R693 ASXL1 -884X* ASXL1 -642X ASXL1 QLL695HX and ASXL1 Q768*It is one or more of the following.

[0066] In certain embodiments, the mutant allele is an allele of the gene Biorientation Of Chromosomes In Cell Division 1 Like 1 (BOD1L1).

[0067] In a particular embodiment, the mutant allele is BOD1L1 S1623C , BOD1L1 E1612K , BOD1L1 K1136N , BOD1L1 R1074W , BOD1L1 Y812C , BOD1L1 E289K and BOD1L1 R508S It is one or more of the following.

[0068] Abbreviations and definitions To facilitate understanding of this disclosure, some terms and abbreviations used herein are defined below.

[0069] When introducing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “the foregoing” are intended to mean that there are one or more elements. The terms “include,” “contain,” and “have” are intended to be inclusive and mean that there may be elements other than those listed.

[0070] As used herein, the term "and / or" when used in a list of two or more items means that any one of the items in the list is available either on its own or in combination with one or more of the items in the list. For example, the expression "A and / or B" is intended to mean either A or B, or both, i.e., A only, B only, or a combination of A and B. The expression "A, B and / or C" is intended to mean A only, B only, C only, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0071] As used herein, the term "about" in reference to measurable values ​​such as compound amount, dose, time, and temperature means that it includes variations of 20%, 10%, 5%, 1%, 0.5%, and even 0.1% from the specified amount.

[0072] As used herein, “therapeutably effective amount” of a drug means an amount of the drug or a pharmaceutically acceptable salt thereof that eliminates, alleviates, or reduces the disease or symptoms of the disease to which it is administered.

[0073] As used herein, a “non-adverse dose” of a drug means an amount of the drug or a pharmaceutically acceptable salt thereof that does not cause dose-limiting toxicity or adverse effects. Examples of such toxicity / adverse effects include anemia (hemoglobin < 8 g / dL), severe thrombocytopenia (platelet count < 25 k / uL), or severe granulocytopenia (absolute neutrophil count < 0.5 k / uL).

[0074] As used herein, “subjects requiring it” refers to humans or non-human animals exhibiting one or more symptoms or signs of a disease.

[0075] When a range of values ​​is disclosed and the notation "n1~n2" or "between n1 and n2" is used, if n1 and n2 are numbers, unless otherwise specified, this notation is intended to include those numbers themselves and the range between them. This range can be continuous integers between them, including the endpoints. For example, the range "2 to 6 carbon atoms" is intended to include 2, 3, 4, 5, and 6 carbon atoms, since carbon appears in integer units. For comparison, the range "1 to 3 μM (micromoles)" is intended to include 1 μM, 3 μM, and all in between to any number of significant digits (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.). When n is set to 0 in relation to "0 carbon atoms," it is intended to mean a bond or null.

[0076] The compounds disclosed herein contain chiral centers. These centers are represented by the symbols "R" or "S" depending on the configuration of substituents around the chiral carbon atom. It should be understood that the present invention encompasses all stereochemical isomers, including diastereomers, enantiomers, and epimers, as well as d-isomers and l-isomers, and mixtures thereof. Individual stereoisomers of compounds can be prepared by synthesis from commercially available starting materials containing chiral centers, or by preparation of mixtures of enantiomer products, subsequent separation, e.g., conversion to diastereomer mixtures, subsequent separation or recrystallization, chromatographic techniques, direct separation of enantiomers by chiral chromatography columns, or any other suitable method known in the art. Starting compounds for specific stereochemistrys are either commercially available or can be prepared and divided by techniques known in the art. In addition, the compounds disclosed herein may exist as geometric isomers. The present invention encompasses all cis, trans, syn, anti, entgegen(E), and zusammen(Z) isomers, and suitable mixtures thereof. In addition, the compounds may exist as tautomers, and all tautomers are provided by the present invention. Furthermore, the compounds disclosed herein can exist in non-solvated forms and in solvated forms with pharmaceutically acceptable solvents such as water and ethanol. Generally, the solvated forms are considered equivalent to the non-solvated forms.

[0077] As used herein, the term “disease” is generally intended to be synonymous with the terms “disorder” and “pathological condition” (as in medical pathological condition), all of which are interchangeable in that they reflect abnormal pathological conditions of the human or animal body or parts thereof that impair normal function, are typically manifested by the identification of signs and symptoms, and reduce the lifespan or quality of life of the human or animal.

[0078] The term “combination therapy” means administering two or more therapeutic agents to treat the conditions or disorders described herein. Such administration includes co-administration of these therapeutic agents substantially simultaneously, for example, in a single capsule having a fixed ratio of active ingredients or in separate capsules for each of several active ingredients. In addition, such administration also includes the use of each type of therapeutic agent sequentially. In any case, the treatment regimen will provide a beneficial effect of the combination of drugs in treating the conditions or disorders described herein.

[0079] The term "therapeutably acceptable" means a compound (or salt, prodrug, tautomer, zwitterionic, etc.) that is suitable for use in contact with patient tissue without excessive toxicity, irritation, or allergic reactions, corresponds to an appropriate benefit / risk ratio, and is effective for its intended use.

[0080] Where used herein, references to the “treatment” of a patient are intended to include prevention. The term “patient” means all mammals, including humans. Examples of patients include humans, cattle, dogs, cats, goats, sheep, pigs, and rabbits. Preferably, the patient is a human.

[0081] The term "prodrug" refers to a compound that becomes more active in vivo. Certain compounds disclosed herein may also exist as prodrugs, as described in Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M. Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compounds described herein are structural modifications of the compound that readily undergo chemical transformation under physiological conditions to provide the compound. In addition, prodrugs can be converted to the compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be gradually converted to the compound when placed in a transdermal patch reservoir containing a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the compound or parent drug. They may be bioavailable, for example, by oral administration, whereas the parent drug is not. Prodrugs may also have improved solubility in pharmaceutical compositions compared to the parent drug. A variety of prodrug derivatives are known in the art, including those that rely on hydrolytic cleavage or oxidative activation of prodrugs. Examples of prodrugs include, but are not limited to, compounds that are administered as esters ("prodrugs") but are subsequently metabolically hydrolyzed to active carboxylic acid compounds. Additional examples include peptidyl derivatives of compounds.

[0082] The compounds disclosed herein can exist as therapeutically acceptable salts. The present invention includes the compounds listed above in salt form, including acid addition salts. Preferred salts include those formed with both organic and inorganic acids. Such acid addition salts are generally pharmaceutically acceptable. However, some pharmaceutically unacceptable salts may be useful for the preparation and purification of the compounds. Base addition salts may also be formed and may be pharmaceutically acceptable. For a more complete discussion on the preparation and selection of salts, see Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

[0083] The term "therapeutably acceptable salt," as used herein, refers to a salt or zwitterionic form of a compound disclosed herein that is soluble or dispersible in water or oil and therapeutically acceptable, as defined herein. Salts can be prepared at the time of final isolation and purification of the compound, or independently by reaction of a suitable compound in its free base form with a suitable acid. Typical acid addition salts include acetate, adipine, alginate, L-ascorbate, aspartic acid, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisinate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, and malonate. Examples of basic groups in the compounds disclosed herein include salts, DL-mandelates, mesitylene sulfonates, methanesulfonates, naphthylene sulfonates, nicotinates, 2-naphthalene sulfonates, oxalates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphonates, picrinates, pivalates, propions, pyroglutamates, succinates, sulfonates, tartrates, L-tartrates, trichloroacetates, trifluoroacetates, phosphates, glutamates, bicarbonates, p-toluenesulfonates (p-tosylates), and undecanoates. Basic groups in the compounds disclosed herein can also be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids, such as oxalic acid, maleic acid, succinic acid, and citric acid. Salts can also be formed by coordinating compounds with alkali metal or alkaline earth ions.Therefore, the present invention aims to provide sodium salts, potassium salts, magnesium salts, calcium salts, and the like of the compounds disclosed herein.

[0084] Base addition salts can be prepared at the final isolation and purification of the compound by the reaction of a carboxyl group with a suitable base, such as a metal cation hydroxide, carbonate, or bicarbonate, or ammonia, or a primary, secondary, or tertiary organic amine. Therapeutably acceptable salt cations include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as non-toxic quaternary amine cations, such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-efenamine, and N,N'-dibenzylethylenediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

[0085] Salts of compounds can be prepared by the reaction of a suitable compound in its free base form with a suitable acid.

[0086] The compounds disclosed herein may exist as polymorphs and as other identifiable solid forms such as solvates and hydrates. The compounds may be salts or polymorphs, solvates, or hydrates of free bases or acids.

[0087] The term “myeloproliferative neoplasm” (MPN) refers to blood cancers that occur when the body produces too many white blood cells or red blood cells or platelets, resulting from somatic mutations that activate hormonal signaling pathways that control the production of these types of blood cells. Considering that neoplastic cells arise from a single mutant clone originating from bone marrow cells, it is a “clonal disease of hematopoietic stem cells” (Campregher et al. Rev Bras Hematol Hemoter. 2012; 34 (2): 150-5). MPNs include myelofibrosis, including polycythemia vera (PV), primary myelofibrosis (PMF, including both pre-fibrotic / early and overt fibrotic stages in certain embodiments) and post-PV / ET myelofibrosis (PPV-MF and PET-MF), essential thrombocythemia (ET), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia not otherwise specified (CEL-NOS), and chronic myeloid leukemia (CML), as well as other unclassified MPNs. For a more thorough discussion of the diagnostic criteria for MPN and related myeloid neoplasms and acute leukemia, as well as for PV, ET, PMF and other MPNs, see Arber et al. “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia”, Blood 2016, 127 (20): 2391-2405. For a thorough discussion of the diagnostic and response criteria for myelofibrosis, see Tefferi A et al., “Revised response criteria for myelofibrosis: International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European LeukemiaNet (ELN) consensus report,” Blood, 122 (8): 1395-98 (2013).

[0088] The following abbreviations may be used throughout this specification and have the meanings assigned to them.

[0089] [Table 1]

[0090] [Table 2]

[0091] [Table 3]

[0092] [Table 4]

[0093] formulation The compounds disclosed herein may be administered as chemical substances in their raw form, but they may also be presented as pharmaceutical formulations (synonymously, “pharmaceutical compositions”). Accordingly, this specification provides pharmaceutical formulations comprising one or more specific compounds disclosed herein, or one or more pharmaceutically acceptable salts, esters, prodrugs, amides, or solvates thereof, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic components. The carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to its recipient. The appropriate formulation depends on the chosen route of administration. Any of the well-known techniques, carriers, and excipients may be used as suitable, as understood in the art, for example, as described in Remington's Pharmaceutical Sciences. The pharmaceutical compositions disclosed herein may be manufactured in any manner known in the art, for example, by utilizing conventional mixing, dissolution, granulation, sugar coating, polishing, emulsification, encapsulation, encapsulation, or compression processes.

[0094] The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intra-articular, intrafat, intra-arterial, intracranial, intralesional, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrasacral, intratracheal, intratumoral, intraumbilical, intracortical, vaginal, intravesical, intravitreal, and intramedullary), intraperitoneal, rectal, topical (but not limited to dermis, buccal, sublingual, vagina, rectum, nose, ear, and eye), topical, mucosal, sublingual, subcutaneous, transmucosal, transdermal, transbuccal, transdermal and vaginal, liposomes, creams, lipid compositions, catheters, lavage, continuous infusion, infusion, inhalation, injection, local delivery, local perfusion, direct target cell infusion, or any combination thereof. However, the most preferred route of administration may depend, for example, on the recipient's condition and disorder. The formulations may be presented as unit formulations as appropriate and may be prepared by any method well known in the pharmaceutical art. Typically, these methods involve associating a compound disclosed herein or a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate thereof ("active ingredient") with a carrier constituting one or more minor components. Generally, formulations are prepared by homogeneously and closely associating the active ingredient with a liquid carrier or a micronized solid carrier or both, and then, if necessary, shaping the product into a desired formulation.

[0095] Formulations of the compounds disclosed herein, suitable for oral administration, may be presented as individual units, for example, hard or soft capsules, wafers, cachets, or tablets (each containing a predetermined amount of the active ingredient), as powders or granules, as syrups, elixirs, solutions, or suspensions in aqueous or non-aqueous liquids, or as compounds dispersed in oil-in-water or water-in-oil emulsions or liposomes. The active ingredient may also be presented as a bolus, lick, or paste.

[0096] Orally administered pharmaceuticals include tablets, gelatin push-fit capsules, and soft-seal capsules made of gelatin and plasticizers such as glycerol or sorbitol. Tablets may be manufactured by compression or molding with one or more adjuncts as optional. Compressed tablets may be prepared by compressing a free-flowing active ingredient, such as a powder or granules, optionally mixed with a binder, inert diluent or lubricant, surfactant or dispersant, using a suitable machine. Molded tablets may be manufactured by molding a mixture of powdered compounds moistened with an inert liquid diluent using a suitable machine. Tablets may optionally be coated or incised and formulated to provide delayed, slow, or controlled release or absorption of the active ingredient. Compositions may further contain agents that enhance solubility or dispersibility. All formulations for oral administration should be in doses suitable for such administration. Push-fit capsules can contain the active ingredient by mixing it with a filler such as lactose, a binder such as starch and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Sugar coating cores provide a suitable coating. For this purpose, concentrated sugar solutions may be used that optionally contain gum arabic, talc, polyvinylpyrrolidone, Carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or sugar coating for identification or to characterize different combinations of active compound doses.

[0097] Depending on the route of administration, the compound or its granules or particles may be coated with materials to protect the compound from the action of acids and other natural conditions that can inactivate it.

[0098] The compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion, to the body or to the site of disease or wound. Injectable formulations may be presented in unit formulations, such as ampoules or multi-dose containers, with the addition of preservatives. Compositions may take the form of suspensions, solutions, emulsions, etc., in oily or aqueous media, and may contain formulation agents, such as suspending agents, stabilizers, and / or dispersants. Formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in powder form or freeze-dried state requiring only the addition of a sterile liquid carrier, such as physiological saline or sterile pyrogen-free water, immediately before use. Solutions and suspensions for immediate injection may be prepared from sterile powders, granules, and tablets as described above.

[0099] Preparations for parenteral administration include aqueous and non-aqueous (oil-based) sterile injectable solutions of active compounds that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the target recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and viscosity modifiers. Suitable lipophilic solvents or media include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain appropriate stabilizers or agents that increase the solubility of the compound, enabling the preparation of high-concentration solutions. To administer therapeutic compounds by means other than parenteral administration, it may be necessary to coat the compound with a material that prevents its inactivation, or to co-administer such compounds (e.g., by liposomal formulations).

[0100] In addition to the ingredients specifically mentioned above, it should be understood that the formulations described above may contain other agents commonly used in the art, taking into account their formulation type. For example, those suitable for oral administration may contain flavoring agents.

[0101] Preferred unit-dose formulations contain the active ingredient in the effective dose or appropriate fraction thereof listed below. In certain embodiments, the formulations disclosed herein are administered once daily. However, formulations can also be formulated for administration at any of the following frequencies, including once weekly, once every five days, once every three days, once every two days, once daily, twice daily or more. Such administration frequencies are maintained over various durations, depending on the treatment regimen. The duration of a particular treatment regimen can range from a single dose to regimens lasting several months or even years. Dosages and administration regimens are discussed further below.

[0102] The amount of active ingredient that can be combined with a carrier material to manufacture a single-dose formulation will vary depending on the host being treated and the specific dosage form. Similarly, the precise amount of compound administered to a patient will be the responsibility of the attending physician. The specific dose level for any particular patient will depend on various factors, including the activity of the specific compound used, age, weight, overall health, sex, diet, timing of administration, route of administration, excretion rate, drug combination, the specific disorder being treated, and the severity of the disorder being treated. In addition, the route of administration may vary depending on the condition and its severity.

[0103] In certain cases, it may be appropriate to administer at least one of the compounds described herein (or their pharmaceutically acceptable salts, esters, or prodrugs) in combination with other therapeutic agents. For example, if one of the side effects a patient experiences when taking one of the compounds described herein is inflammation, it may be appropriate to administer an anti-inflammatory agent in combination with the initial therapeutic agent. Alternatively, for example, the therapeutic efficacy of one of the compounds described herein may be enhanced by the administration of an adjuvant (i.e., the adjuvant alone provides only minimal therapeutic benefit, but in combination with other therapeutic agents, the overall therapeutic benefit to the patient is enhanced). It is even possible that two compounds, one of the compounds described herein and a second compound, together may achieve a desired therapeutic effect that neither can achieve individually. Alternatively, for example, the benefit to the patient may be increased by administering one of the compounds described herein together with other therapeutic agents (including therapeutic regimens) that have similar therapeutic effects. For example, in the treatment of acute myeloid leukemia or sickle cell anemia involving an administration of one of the compounds described herein, the therapeutic effect may be enhanced by also providing the patient with other therapeutic agents for acute myeloid leukemia. In either case, regardless of the disease, disorder, or condition being treated, the total benefit received by the patient may simply be the additive effect of the two therapeutic agents, or the two agents may have a synergistic therapeutic effect.

[0104] Effective combination therapy may be achieved using a single composition or pharmacological formulation containing both activators, or by simultaneously using two identifiable compositions or formulations in which one composition contains the compound of the Disclosure and the other contains the second activator. Alternatively, therapy may precede or follow other activator therapy with intervals ranging from minutes to months. Administration of the compound of the Disclosure to a patient will follow general protocols for drug administration, taking into account drug toxicity, if present. Treatment cycles are expected to be repeated as needed.

[0105] Non-limiting examples specific to possible combination therapies include the following agonists and agonist classes: agonists that inhibit DNA methyltransferase, e.g., decitabine or 5'-azasitadine; agonists that inhibit the activity of histone deacetylase, histone desmoylase, histone deubiquitinase or histone phosphatase, e.g., hydroxyurea; antisense RNA that can inhibit the expression of other components of the protein complex bound to the DR site in the gamma globulin promoter; agonists that inhibit the action of Klf1 or the expression of KLF1; agonists that inhibit the action of Bcl11a or the expression of BCL11A. Examples of the use of compounds disclosed herein include agonists and agonists that inhibit cell cycle progression, such as hydroxyurea, ara-C, or daunorubicin; agonists that induce differentiation in leukemia cells, such as all-trans retinoic acid (ATRA); and JAK inhibitors, such as ruxolitinib (Jakafi / Jakavi), fedratinib (Inrebic), celduratinib (PRT062070), gandotinib (LY-2784544), restaurtinib (CEP-701), momerotinib (GS-0387, CYT-387), and pacritinib (SB1518).

[0106] Therefore, in another embodiment, the present invention provides a method for treating a disease or disorder in a human or animal subject requiring such treatment, comprising administering to the subject an amount of a compound disclosed herein that is effective in reducing or preventing the disorder in the subject, in combination with at least one additional agent known in the art for the treatment of the disorder.

[0107] compound Examples of LSD1 inhibitory compounds that may be used in the methods disclosed herein include the following compounds. Other LSD1 inhibitors are known in the art.

[0108] General synthesis methods for preparing compounds In the following examples and throughout this disclosure, the following abbreviations may be used: PTFE = polytetrafluoroethylene, RM = reaction mixture, RH = relative humidity, RT = room temperature, SM = starting material, MeCN = acetonitrile, ClPh = chlorophenol, DCE = dichloroethane, DCM = dichloromethane, DIPE = diisopropyl ether, DMA = dimethylacetamide, DMF = dimethylformamide, DMSO = dimethyl sulfoxide, Et2O = diethyl ether, siRNA = ethyl acetate, EtOH = ethanol, H2O = water, IPA = propane-2-ol, i-PrOAc = isopropyl acetate, MEK = methyl ethyl ketone, MeOH = methanol, MIBK = methyl isobutyl ketone, MTBE = methyl tert-butyl ether, n-BuOAc = n-butyl acetate, n- BuOH = n-butanol, NMP = n-methylpyrrolidone, n-PrOH = n-propanol, s-BuOAc = s-butylacetate, t-BuOH = t-butanol, TFA = trifluoroacetic acid, THF = tetrahydrofuran, TMP = 2,2,4-trimethylpentane, 1H-NMR = proton nuclear magnetic resonance, DSC = differential scanning calorimetry, DVS = dynamic vapor sorption, GVS = gravimetric vapor sorption, HPLC = high-performance liquid chromatography, HS = headspace HSM = Hot Stage Microscopy, IC = Ion Chromatography, IDR = Intrinsic Dissolution Rate, KF = Karl Fischer, MAS = Magic Angle Rotation, MDSC = Modulated Differential Scanning Calorimetry, PLM = Polarized Light Microscopy, PVM = Particle Vision and Measurement, SCXRD = Single Crystal X-ray Diffraction, SS-NMR = Solid State Nuclear Magnetic Resonance, TGA = Thermogravimetric Analysis, UV = Ultraviolet, VH-XRPD = Variable Humidity X-ray Powder Diffraction, VT-XRPD = Variable Temperature X-ray Powder Diffraction, and XRPD = X-ray Powder Diffraction. Other abbreviations may be used and will be familiar to those skilled in the art.

[0109] The present invention will be further illustrated by the following examples, though not limited thereto. The methods illustrated below may also be applied to the compounds disclosed herein. Further methods suitable for use in preparing the examples of the present invention can be found in International Publication No. 2015 / 021128 and International Publication No. 2016 / 130952 (their contents are incorporated herein by reference as if they were described herein in their entirety). Additional LSD1 inhibitors can be prepared by the methods disclosed herein.

[0110] Intermediate A: (1R,2S)-2-(4-fluorophenyl)-1-methylcyclopropanamine [ka] A solution of ethyl 2-(diethoxyphosphoryl)propanoate (3.45 g, 14.48 mmol, 2.00 equivalents) in ethylene glycol dimethyl ether (20 mL) was treated by adding n-BuLi (2.5 M) (5.8 mL) dropwise while stirring at 0°C. The resulting solution was stirred at room temperature for 30 minutes. 2-(4-fluorophenyl)oxirane (1 g, 7.24 mmol, 1.00 equivalent) was added. The resulting solution was stirred for 12 hours while maintaining the temperature at 80°C in an oil bath. The reaction mixture was cooled to RT. The reaction was then quenched by adding 20 mL of water. The resulting solution was extracted with ethyl acetate, the organic layer was dehydrated, and the mixture was concentrated. The residue was subjected to silica gel chromatography and eluted with ethyl acetate / petroleum ether (1:100). This yielded 1 g (62%) of ethyl(1R)-2-(4-fluorophenyl)-1-methylcyclopropane-1-carboxylate as yellow oil. A solution of ethyl(1R)-2-(4-fluorophenyl)-1-methylcyclopropane-1-carboxylate (1 g, 4.50 mmol, 1.00 equivalent) in methanol / H2O (10 / 2 mL) and potassium hydroxide (1.26 g, 22.46 mmol, 4.99 equivalents) was stirred at room temperature for 10 hours. The resulting solution was diluted with H2O. The pH of the solution was adjusted to 2 with hydrochloric acid (2 mol / L). The resulting solution was extracted with ethyl acetate, and the organic layers were combined and dehydrated with anhydrous sodium sulfate, and concentrated under vacuum. This yielded 800 mg (92%) of (1R)-2-(4-fluorophenyl)-1-methylcyclopropane-1-carboxylic acid as yellow oil. A solution of (1R)-2-(4-fluorophenyl)-1-methylcyclopropane-1-carboxylic acid (400 mg, 2.06 mmol, 1.00 equivalent) in toluene (10 mL) was mixed with diphenoxyphosphoryl azide (680 mg, 2.47 mmol, 1.20 equivalent) and triethylamine (312 mg, 3.08 mmol, 1.50 equivalent). The resulting solution was stirred in an oil bath at 90°C for 30 minutes. Then, tert-butanol (2 mL) was added. The mixture was reacted for a further 12 hours while stirring, maintaining the temperature at 90°C in the oil bath. The reaction mixture was cooled to room temperature, and the resulting solution was diluted with ethyl acetate. The resulting mixture was washed with H2O.The mixture was dehydrated with anhydrous sodium sulfate and concentrated under vacuum. The residues were chromatographed on a silica gel column and eluted with ethyl acetate / petroleum ether (1:100). This yielded 350 mg (64%) of tert-butyl N-[(1R)-2-(4-fluorophenyl)-1-methylcyclopropyl]carbamate as yellow oil. A solution of tert-butyl N-[(1R,2S)-2-(4-fluorophenyl)-1-methylcyclopropyl]carbamate (350 mg, 1.32 mmol, 1.00 equivalent) in methanol (HCl) (10 mL) was stirred at room temperature for 2 hours. The resulting solution was diluted with 10 mL of H2O. The pH of the solution was adjusted to 9 with saturated sodium bicarbonate solution. The resulting solution was extracted with 3 × 10 mL of ethyl acetate, and the organic layers were combined and dehydrated with anhydrous sodium sulfate, and concentrated under vacuum. This yielded 200 mg (92%) of (1R,2S)-2-(4-fluorophenyl)-1-methylcyclopropane-1-amine as a yellow oil.

[0111] Example A1: N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(pyrrolidine-1-yl)hexane2-yl)benzamide) [ka] (S)-2-benzamido-6-hydroxyhexanoic acid was prepared from (S)-2-amino-6-hydroxyhexanoic acid. This material (1 g, 3.98 mmol, 1.00 equivalent) in tetrahydrofuran was reacted with 3-(diethoxyphosphoryloxy)-1,2,3-pentotriazine-4(3H)-one (DEPBT) (2.4 g, 8.03 mmol, 2.00 equivalent) and imidazole (542 mg, 7.97 mmol, 2.00 equivalent). Subsequently, a solution of pyrrolidine (283 mg, 3.98 mmol, 1.00 equivalent) in tetrahydrofuran was added at 0°C for 30 minutes. The resulting solution was stirred at room temperature for 16 hours. The solution was diluted with KH2PO4 (aq.). The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with brine and dehydrated with anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure. The residue was purified by preparative HPLC and eluted with MeCN containing 0.5% NH4HCO3. This yielded 640 mg (53%) of (S)-N-(6-hydroxy-1-oxo-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide as a pale yellow oil. (S)-N-(6-hydroxy-1-oxo-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide (640 mg, 2.10 mmol, 1.00 equivalent) in dichloromethane (100 ml) was oxidized with Dess Martin per-iodinane (DMP) (893 mg, 2.11 mmol, 1.00 equivalent). The resulting solution was stirred in a water / ice bath at 0°C for 30 minutes and then diluted with Na2SO3 (aq.) and NaHCO3 (aq.). The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with brine and dehydrated with anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography and eluted with ethyl acetate / petroleum ether (10:1). This yielded 150 mg (24%) of (S)-N-(1,6-dioxo-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide as a white solid. (S)-N-(1,6-dioxo-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide (150 mg, 0.50 mmol, 1.00 equivalent) was dissolved in dichloromethane (25 mL). (1R,2S)-2-phenylcyclopropanamine (66 mg, 0.50 mmol, 1.00 equivalent) was added.After stirring for 5 minutes, sodium triacetoxyborohydride (252 mg, 1.19 mmol, 2.40 equivalents) was added. The resulting solution was stirred at 0°C for 30 minutes. After the reaction was complete, the resulting solution was diluted with sat.NaHCO3. It was then extracted with dichloromethane. The organic layer was washed with brine and dehydrated with anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by Prep-HPLC (CAN / H2O containing 0.5% NH4HCO3). This yielded 29 mg (14%) of N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide as a colorless oil. 1 H NMR(300MHz,CD3OD-d4)δ ppm:7.85(d,J=7.5Hz,2H),7.60-7.00(m,8H),4.85-4.75(m,1H),3.92-3.80(m,1H),3.70-3.30(m,4H),2.74(t,J =7.2Hz,1H),2.36-2.28(m,1H),2.07-1.75(m,7H),1.74-1.37(m,4H),1.10-0.95(m,2H);MS(ES,m / z):420(M+H).

[0112] Example A2: N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(piperidine 1-yl)hexane-2-yl)benzamide [ka] N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide was prepared in the same manner as described for the synthesis of N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(piperidine-1-yl)hexane-2-yl)benzamide. (S)-2-benzamido-6-hydroxyhexanoic acid was coupled to piperidine using 3-(diethoxyphosphoryloxy)-1,2,3-pentotriazine-4(3H)-one and imidazole. The obtained alcohol (S)-N-(6-hydroxy-1-oxo-1-(piperidine-1-yl)hexane-2-yl)benzamide was oxidized to the aldehyde (S)-N-(1,6-dioxo-1-(piperidine-1-yl)hexane-2-yl)benzamide under Dess-Martin conditions. This was coupled with (1R,2S)-2-phenylcyclopropanamine under reductive amination conditions (Na(OAc)3BH) to produce the desired product N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(piperidine-1-yl)hexane-2-yl)benzamide as a colorless oil. ES, m / z = 434 (M + H). 1 H NMR(300MHz,CD3OD-d4)δ ppm:7.86(d,J=7.2Hz,2H),7.70-7.40(m,3H),7.30-7.15(m,2H),7.15-7.08(m,1H),7.06(d,J=7.2Hz,2H),5.15-5.00(m, 1H),3.80-3.60(m,2H),3.60-3.40(m,2H),2.34(t,J=7.2Hz,2H),2.40-2.30(m,1H),2.10-1.40(m,4H),1.15-1.00(m,2H).

[0113] Example A3: 4-Fluoro-N-((S)-6-(((1R,2S)-2-(4-fluorophenyl)cyclopropyl)amino)-1-(4-methylpiperazine-1-yl)-1-oxohexane-2-yl)benzamide [ka] Similar to Example A2, 4-fluoro-N-((S)-6-(((1R,2S)-2-(4-fluorophenyl)cyclopropyl)amino)-1-(4-methylpiperazine-1-yl)-1-oxohexane-2-yl)benzamide was prepared. The alcohol 4-fluoro-N-((S)-6-(((1R,2S)-2-(4-fluorophenyl)cyclopropyl)amino)-1-(4-methylpiperazine-1-yl)-1-oxohexane-2-yl)benzamide was prepared by reduction of (S)-2-(4-fluorobenzamide)hexanediic acid with Me2S-BH3. This type of reduction has been used to prepare similar alcohols (e.g., (S)-2-benzamide-6-hydroxyhexanoic acid, an alcohol starting material for the synthesis of N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide (Example A1)). A solution of (S)-2-(4-fluorobenzamide)hexanediic acid (10 g, 35.30 mmol, 1.00 equivalent) in tetrahydrofuran (300 ml) was placed in a 1000 mL three-necked round-bottom flask that had been purged and maintained in an inert atmosphere of nitrogen. Then, a solution of Me2SBH3 (11 mL, 3.00 equivalent) in tetrahydrofuran (50 ml) was added at 0°C. The resulting solution was stirred in an ice / salt bath at 0°C for 3 hours. The reaction was then quenched by the addition of 20 ml of methanol. The obtained mixture was concentrated under vacuum. The resulting solution was diluted with 300 ml of sat.Na2CO3. The resulting solution was extracted with 3 × 100 mL of ethyl acetate, and the aqueous layer was combined. The pH of the solution was adjusted to 2 with hydrochloric acid (2 mol / L). The resulting solution was extracted with 3 × 200 mL of ethyl acetate, and the organic layer was combined. The resulting mixture was washed with 1 × 500 mL of brine. The mixture was dehydrated with anhydrous sodium sulfate. The solid was filtered off. The resulting mixture was concentrated under vacuum. This yielded 6 g (63%) of (S)-2-(4-fluorobenzamide)-6-hydroxyhexanoic acid as a colorless oil.Similar to the synthesis described for N-((S)-1-oxo-6-(((1R,2S)-2-phenylcyclopropyl)amino)-1-(pyrrolidine-1-yl)hexane-2-yl)benzamide (Example A1), this material was reacted with N-methylpiperazine, followed by coupling via Dess-Martin oxidation and reductive amination with (1R,2S)-2-(4-fluorophenyl)cyclopropanamine to produce the desired product 4-fluoro-N-((S)-6-(((1R,2S)-2-(4-fluorophenyl)cyclopropyl)amino)-1-(4-methylpiperazine-1-yl)-1-oxohexane-2-yl)benzamide as a colorless oil. 1 H NMR(300MHz,CD3OD-d4)δ ppm:7.83(dd,J1=5.4Hz,J2=1.4Hz,2H),7.18-7.04(m,3H),7.00-6.87(m,4H),5.17-5.05(m,1H),3.78-3.50(m,4H),2.71(t ,J=6.9Hz,2H),2.30(s,3H),2.28-2.21(m,1H),1.90-1.78(m,2H),1.72-1.31(m,9H),1.07-0.96(m,1H),0.94-0.86(m,1H).

[0114] Example 158: N-[(2S)-1-(4-(methyl)piperazine-1-yl)-5-[[(1R,2S)-2-(4-fluorophenyl)-cyclopropyl]amino]-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide (compound 2, free base of compound 1) Following the method of Scheme II, N-[(2S)-1-(4-(methyl)piperazin-1-yl)-5-[[(1R,2S)-2-(4-fluorophenyl)-cyclopropyl]amino]-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide (compound 2) was prepared. [ka]

[0115] 4-(1H-1,2,3-triazolyl-1-yl)benzoyl chloride (1) was prepared by combining 4-(1H-1,2,3-triazol-1-yl)benzoic acid (1 g, 5.29 mmol, 1.00 equivalent) and thionyl chloride (20 mL) in a 100 mL round-bottom flask. The resulting solution was stirred in an oil bath at 80°C for 16 hours. The mixture was then concentrated under reduced pressure to give 1 g (91%) of intermediate (1) as a yellow solid.

[0116] (2S)-5-[[(1R,2S)-2-(4-fluorophenyl)cyclopropyl](propen-3-yl)amino]-2-[[4-(1H-1,2,3-triazole-1-yl)phenyl]formamide]pentanoic acid (2) (2S)-2-amino-5-[(1R,2S)-2-(4-fluorophenyl)cyclopropyl](propen-2-en-1-yl)aminopentanoic acid (500 mg, 1.63 mmol, 1.00 equivalent), Et3N (494 mg, 4.88 mmol, 3.00 equivalent), and THF (20 mL) were combined in a 100 mL round-bottom flask. Then, while stirring at 0°C, a solution of intermediate (1) (1 g, 4.82 mmol, 2.95 equivalent) from the previous step in THF (20 mL) was added dropwise for 30 minutes. The obtained solution was stirred in an ice / salt bath at 0°C for 1 hour, then concentrated under reduced pressure, and applied to a silica gel column using CH2Cl2 / methanol (10:1). The collected fractions were combined and concentrated under reduced pressure to give 400 mg (51%) of intermediate (2) as a grayish-white solid.

[0117] N-[(2S)-1-(4-(methyl)piperazin-1-yl)-5-[[(1R,2S)-2-(4-fluorophenyl)-cyclopropyl](prop-2-en-1-yl)amino]-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)-benzamide (3) In a 100 mL round-bottom flask, intermediate (2) (400 mg, 0.84 mmol, 1.00 equivalent) from the previous step, DEPBT (375 mg, 1.25 mmol, 1.50 equivalent) and THF (20 mL) were combined, followed by the addition of imidazole (85 mg, 1.25 mmol, 1.50 equivalent). The mixture was stirred at 0°C for 30 minutes, at which point 1-methylpiperazine (127 mg, 1.27 mmol, 1.50 equivalents) was added dropwise for 3 minutes while stirring at 0°C. The resulting solution was stirred at 20°C for 16 hours and then concentrated under reduced pressure. The residue was applied to a silica gel column with CH2Cl2 / methanol (10:1). The collected fractions were combined and concentrated under vacuum to give 300 mg (64%) of intermediate (3) as a yellow solid.

[0118] N-[(2S)-1-(4-(methyl)piperazin-1-yl)-5-[[(1R,2S)-2-(4-fluorophenyl)-cyclopropyl]amino]-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide (Example 158, Compound 2) In a 100 mL round-bottom flask that had been purged and maintained in an inert nitrogen atmosphere, N-[(2S)-5-[[(1R,2S)-2-(4-fluorophenyl)cyclopropyl](prop-2-en-1-yl)amino]-1-(4-methylpiperazine-1-yl)-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide (300 mg, 0.54 mmol, 1.00 equivalent), 1,3-dimethyl-1,3-diadinane-2,4,6-trione (210 mg, 1.34 mmol, 2.50 equivalent), and Pd(PPh3)4 (155 mg, 0.13 mmol, 0.25 equivalent) were placed. The resulting solution was stirred in an oil bath at 45°C for 2 hours. The resulting mixture was concentrated under vacuum. The crude product (10 mL) was purified by flash-Prep-HPLC. This yielded 65 mg (23%) of Example 158 as a yellow solid.

[0119] Alternatively, see Example 158 and its bis-tosylate salt (compound 2 bis-tosylate salt, "compound 1"). [ka] This can be prepared by the method of Scheme III. [ka]

[0120] The compounds disclosed herein, including Compound 1, can also be synthesized as disclosed in U.S. Patent No. 20160237043, International Publication No. 2018035259, and International Publication No. 2018035249.

[0121] The compounds described herein can be synthesized using appropriate starting materials and reagents by methods similar to those described herein and those known in the art. It should be understood that, in the structures described below, mixtures of single isomers or single isomers, such as racemic mixtures and alternative enantiomers, zwitterions, etc., can be prepared, for example, by using appropriate L- or D-isomers or chiral or achiral compounds as starting materials or reagents, or by utilizing separation steps.

[0122] Therefore, in certain embodiments of the following compounds, the substituent configuration of cyclopropylamine is trans relative to phenyl. In certain embodiments, the trans configuration is R,S, and in other cases, it is S,R.

[0123] In certain embodiments, the compound is [ka] ("Compound 2") or its salt, polymorph, or solvate.

[0124] In a particular embodiment, the compound is of the formula: [ka] A salt of or a polymorph or solvate thereof, in the formula, X is selected from tosylate, sulfate, tartlate, oxalate, besylate, fumarate, citric acid, esylate and malate, and q is an integer selected from 1 and 2.

[0125] In certain embodiments, X is tosylate.

[0126] In certain embodiments, q is 2.

[0127] In certain embodiments, the compound is [ka] This is ("Compound 1").

[0128] The compounds disclosed herein, or any subset or species thereof, may be used in either a method of treatment or in achieving the clinical / treatment-related endpoints described herein.

[0129] In certain embodiments, the compounds disclosed herein are provided for use as pharmaceutical agents.

[0130] In certain embodiments, the compounds disclosed herein are provided for use in the manufacture of pharmaceutical agents for the prevention or treatment of a disease or condition or for achieving clinically relevant endpoints, as considered herein.

[0131] In certain embodiments, pharmaceutical compositions are provided that include the compounds disclosed herein together with a pharmaceutically acceptable carrier.

[0132] In certain embodiments, the pharmaceutical composition is formulated for oral administration.

[0133] In certain embodiments, the pharmaceutical composition further comprises other therapeutic agents.

[0134] Treatment methods for diseases and use in pharmaceuticals This specification provides a method for treating or preventing myeloproliferative neoplasms, comprising administering an LSD1 inhibitor compound disclosed herein to a subject in need thereof.

[0135] In certain embodiments, the method achieves or results from one or more of the following: • Suppression of the proliferation of malignant myeloid cells in the target population that requires it. • Reduction of the concentration of one or more protein growth factors (e.g., platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor beta-1, or platelet factor 4 (also known as CXCL4)) secreted by bone marrow cells (e.g., megakaryocytes) that activate one or more cell types (e.g., stromal cells, bone marrow resident fibroblasts, or myofibroblasts) that secrete reticulin and collagen in the target population. • Reduction of the concentration of one or more protein growth factors (e.g., platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor beta-1, or platelet factor 4 (also known as CXCL4)) secreted by bone marrow cells (e.g., megakaryocytes) that impair the function of bone marrow osteoclasts and reduce the amount of bone marrow sclerosis in subjects that require it. • Reduction of reticulin and / or collagen myelofibrosis in subjects requiring it. • Reduction of plasma levels of one or more inflammatory cytokines in the target population. • Reduction of malignant cell load, as measured by the frequency of mutant alleles in myeloid cells, in subjects where it is needed. • Elimination of malignant myeloid cells in the target population, • Reduction of pathologically elevated red blood cell count in subjects requiring it. • Reduction of the amount of malignant myeloid cells in the target population, • Reduction of abnormal spleen size or volume in subjects requiring it. • Reduction of extramedullary hematopoiesis in those who require it. • Improvement in quality of life (QOL) as measured by validated patient-reported QOL assessments in those who need it. • Reduction of systemic symptoms of myelofibrosis as measured by patient-reported surveys in those who require it. • Extending the lifespan of patients with myelofibrosis who require it. • Delaying or preventing the progression from myelofibrosis to acute myeloid leukemia in those who require it. • Reduction of platelet count in those who require it, • Reduction of pathologically elevated red blood cell count in subjects requiring it. • Reduction of elevated levels of granulocyte-derived myeloid cells in subjects requiring it. • Reduction of myeloid cells to age-adjusted normal cells with less than 5% blast cells in subjects requiring it. • Maintaining the number of myeloblasts or reducing the number of myeloblasts to <5% in subjects requiring it. • Reduction of the frequency of thrombosis and bleeding in those who require it. • Reduction in the frequency of red blood cell infusions in patients who require it. • Increase in hemoglobin levels >100g / L and below the upper limit of age- and sex-adjusted normal values ​​in MF patients. • Reduction of hematocrit to <45% in male patients with PV or reduction of hematocrit to ≤42% in female patients with PV. • Reduction of hemoglobin levels in PV patients to <160 g / L, and / or • Red blood cell count to ≤5.2 M / mL in PV patients

[0136] In certain embodiments, the method achieves or results in achieving two or more of the above. In certain embodiments, the method achieves or results in achieving three or more of the above. In certain embodiments, the method achieves or results in achieving two or more of the above, other than reducing the platelet count in subjects where it is required. In certain embodiments, the one, two, three or more of the above are limited by the following enumeration.

[0137] In certain embodiments, the required subject is a person having a myeloproliferative neoplasm. In certain embodiments, the myeloproliferative neoplasm is selected from polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF), chronic myeloleukemia (CML), chronic neutrophilic leukemia (CNL), and chronic eosinophilic leukemia (CEL). In certain embodiments, the myeloproliferative neoplasm is selected from polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). In certain embodiments, the myeloproliferative neoplasm is myelofibrosis. In certain embodiments, the myelofibrosis is selected from primary myelofibrosis (PMF) and post-PV / ET myelofibrosis. In certain embodiments, the myeloproliferative neoplasm is primary myelofibrosis (PMF). In certain embodiments, the myeloproliferative neoplasm is post-PV / ET myelofibrosis. In certain embodiments, the myeloproliferative neoplasm is essential thrombocythemia. In certain embodiments, the myeloproliferative neoplasm is polycythemia vera. In certain embodiments, the myeloproliferative neoplasm is chronic myeloid leukemia. In certain embodiments, the myeloproliferative neoplasm is chronic neutrophilic leukemia. In certain embodiments, the myeloproliferative neoplasm is chronic eosinophilic leukemia. In certain embodiments, the patient is human.

[0138] This specification also provides a method for inhibiting the proliferation of malignant myeloid cells in a subject requiring such inhibition, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the malignant myeloid cells have mutations in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). In certain embodiments, the method further includes the step of determining whether the subject has mutations in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). In certain embodiments, the malignant myeloid cells are malignant stem cells. In certain embodiments, the reduction of malignant myeloid cells is measured by the frequency of mutant allele loadings measured by PCR, sequencing, or other methods known in the art. In certain embodiments, the malignant myeloid cells are reduced by at least 50%. In certain embodiments, malignant myeloid cells are reduced to 1 / (2log) or less (1 / 100 or less).

[0139] This specification provides a method for reducing reticuline and / or collagen myelofibrosis in subjects requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the myelofibrosis is reticuline myelofibrosis. In certain embodiments, the myelofibrosis is collagen myelofibrosis. In certain embodiments, the myelofibrosis is reticuline and collagen myelofibrosis. In certain embodiments, the reticuline and / or collagen myelofibrosis is reduced by at least one grade, for example, from 3 to 2, or 2 to 1, or 1 to 0. In certain embodiments, the reticuline and / or collagen myelofibrosis is reduced by at least two grades.

[0140] In certain embodiments, the subject has a mutation in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). In certain embodiments, the LSD1 inhibitor is an LSD1 inhibitor compound disclosed herein. Mutations can be evaluated by methods known in the art, for example, by Spivak J, “Narrative Review: Thrombocytosis, polycythemia vera, and JAK2 mutations: the phenotypic mimicry of chronic myeloproliferation,” Annals of Internal Medicine 2010 152 (5): 300-306 or by methods disclosed in Zhan H and Spivak JL, “The diagnosis and management of polycythemia vera, essential thrombocythemia, and primary myelofibrosis in the JAK2 V617F era,” Clin Adv Hematol Oncol, 2009 May; 7 (5): 334-42.

[0141] This specification provides a method for reducing the plasma levels of one or more inflammatory cytokines in a subject requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, one or more inflammatory cytokines are selected from interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17), CXCL4 (PF4), and CXCL10 (IP10).

[0142] In certain embodiments, one or more measured cytokines are reduced to approximately the following levels or less. • IL-6 levels were reduced to less than approximately 9 pg / mL. • IL-8 levels were reduced to less than approximately 18 pg / mL. • IL-10 levels were reduced to less than approximately 51 pg / mL. • IL-12 levels were reduced to less than approximately 182 pg / mL. • IL-15 levels were reduced to less than approximately 38 pg / mL. • TNFα is reduced to less than approximately 15 pg / mL, and / or INFγ is reduced to less than approximately 23 pg / mL. In certain embodiments, two, three, four, five, or more inflammatory cytokines are reduced.

[0143] This specification provides a method for reducing the amount of malignant myeloid cells in a subject requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the amount of malignant myeloid cells is measured by flow cytometry immunophenotyping. In certain embodiments, the amount of malignant myeloid cells is measured by the ratio of the number of cells containing the causative MPN mutation (MPL, CALR, or JAK2) to the total number of cells containing both the wild-type and the mutant alleles.

[0144] This specification provides a method for reducing mutant allele burden in a subject requiring such reduction, comprising a therapeutically effective amount of an LSD1 inhibitor. In certain embodiments, the mutant allele is an allele of one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). In certain embodiments, the LSD1 inhibitor is an LSD1 inhibitor compound disclosed herein. In certain embodiments, the mutant allele burden is reduced by approximately 50% with respect to the mutant allele burden of mutant Janus kinase 2 (JAK2), mutant myeloproliferative leukemia virus oncogene (MPL), or mutant calreticulin (CALR) in the subject (or the average of the subject pool). In certain embodiments, the reduction in mutant allele burden is measured by comparing the pre-treatment level with the post-treatment level in the patient after treatment. In certain embodiments, the mutant allele burden is reduced to a level in which the mutant alleles of Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) are undetectable. The mutant allele burden can be evaluated by methods known in the art, including those disclosed herein.

[0145] This specification provides a method for reducing pathologically elevated red blood cell count in a subject requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the subject has polycythemia vera. In certain embodiments, the subject has a mutation in Janus kinase 2 (JAK2). In certain embodiments, the elevated red blood cell count is estimated by measuring hematocrit or blood hemoglobin. In certain embodiments, the measured hematocrit or hemoglobin should be reduced to a sex-appropriate normal range. For example, in certain embodiments, • Blood hemoglobin levels were reduced to less than 16.5 g / dL in male PV patients and less than 16.0 g / dL in female PV patients. • Hematocrit levels will likely be reduced to less than 49% in male PV patients and less than 48% in female PV patients. In certain embodiments, the elevated red blood cell count is measured by isotopic red blood cell counting. In certain embodiments, the increased red blood cell count is more than 25% above the mean normal predicted value.

[0146] This specification provides a method for reducing an elevated white blood cell count in a subject requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In a particular embodiment, the subject has chronic neutrophilic leukemia.

[0147] This specification also provides a method for reducing elevated levels of granulocyte-lineage myeloid cells in a subject requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the granulocyte-lineage myeloid cells are reduced to levels within the normal range. This specification also provides a method for reducing myeloid cells to age-adjusted normal cells with less than 5% blast cells in a subject requiring such reduction, comprising administering a therapeutically effective amount of an LSD1 inhibitor. In certain embodiments, the subject has chronic neutrophilic leukemia.

[0148] This specification provides a method for increasing hemoglobin levels to >100 g / L in subjects requiring such increase, to a level below the upper limit of age- and sex-adjusted normal values, comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor.

[0149] Methods are also provided for a) reducing hemoglobin levels to <160 g / L in PV patients, or b) reducing red blood cell volume in PV patients as inferred from hemoglobin levels (Hb) of <160 g / L, both of which involve administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor. Methods are also provided for increasing hemoglobin to >100 g / L in MF patients, which involves administering a therapeutically effective amount of an LSD1 inhibitor. Methods are also provided for increasing hemoglobin to a value >100 g / L and below the upper limit of age- and sex-adjusted normal values ​​in MF patients, which involves administering a therapeutically effective amount of an LSD1 inhibitor. In certain embodiments, the subject has a mutation in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). In certain embodiments, the subject has essential thrombocythemia. In certain embodiments, the patient's fluid load is reduced.

[0150] This specification provides a method for reducing abnormal spleen size or volume in subjects requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the subjects have mutations in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR).

[0151] This specification provides a method for reducing the amount of extramedullary hematopoiesis in subjects requiring such reduction, comprising administering a therapeutically effective and non-toxic amount of an LSD1 inhibitor. In certain embodiments, the subjects have mutations in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). In certain embodiments, the amount of extramedullary hematopoiesis is measured by splenomegaly. In certain embodiments, splenomegaly in the subjects is reduced by at least about 30%, at least about 35%, at least about 40%, or at least about 45%. In certain embodiments, splenomegaly in the subjects is reduced by at least 35%. In certain embodiments, splenomegaly is reduced by at least 35% in about 50% of patients.

[0152] This specification provides a method for reducing systemic symptoms of myelofibrosis, as measured by patient-reported surveys, in subjects in need thereof, comprising administering a therapeutically effective and non-harmful amount of an LSD1 inhibitor. In certain embodiments, the systemic symptoms include one or more symptoms selected from fatigue, early satiety, abdominal discomfort, inactivity, difficulty concentrating, numbness and / or tingling in the hands and feet, night sweats, itching, bone pain, fever above 100°F, and unintentional weight loss.

[0153] In certain embodiments, the patient-reported survey is the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF). The MPN-SAF is a validated clinical assessment form for the most common symptoms of myeloproliferative neoplasms, in which patients self-report their scores for various common symptoms on a scale of 1 to 10, where 1 is most favorable or asymptomatic, and 10 is least favorable or worst-case scenario with symptoms imaginable. For example, see Scherber R et al., "The Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF): International Prospective Validation and Reliability Trial in 402 patients," Blood 118 (2): 401-08 (2014). Patients may be given either a complete or abbreviated form. In the abbreviated version, a "Total Symptom Score" (TSS) can be calculated for the 10 most clinically relevant symptoms out of the 17 MPN-SAF items: worst fatigue, difficulty concentrating, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fever. Therefore, the MPN-SAF TSS can range from 0 to 100. The quality of life score is defined as "clinical deficit" if graded at least 4 out of 10, "moderate" if symptoms are graded ≥4 or ≤6 out of 10, and "severe" if symptoms are graded ≥7 out of 10. For patients who complete at least six of these ten items for BFI and MPN-SAF, the MPN TSS is calculated by multiplying the mean of the observed items by 10 to achieve a scale of 0 to 100.For example, see Emanuel RM et al., “Myeloproliferative neoplasm (MPN) symptom assessment form total symptom score: prospective international assessment of an abbreviated symptom burden scoring system among patients with MPNs,” J Clin Oncol 30 (33): 4098-103 (2012).

[0154] In certain embodiments, the total symptom score (MPN-SAF:TSS) is reduced by at least 50%.

[0155] In certain embodiments, the patient-reported survey is the Myelofibrosis Symptom Assessment Form (MF-SAF). See, for example, Mesa RA et al., “The Myelofibrosis Symptom Assessment Form (MFSAF): an evidence-based brief inventory to measure quality of life and symptomatic response to treatment in myelofibrosis,” Leuk Res. 33 (9): 1199-203 (2009). In certain embodiments, the MF-SAF total symptom score is reduced by at least 50%.

[0156] In a particular embodiment, The subjects are those who have mutations in one or more genes selected from Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR). The subjects are those with myeloproliferative neoplasms, The subjects are those with myeloproliferative neoplasms selected from polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis. The subjects are those with myelofibrosis, The subjects are those with myelofibrosis selected from primary myelofibrosis (PMF) and post-PV / ET myelofibrosis. The subjects are those with post-PV / ET myelofibrosis (MF), The subjects are those with primary myelofibrosis (PMF), The subjects are those with polycythemia vera, The subjects are those with essential thrombocythemia, The subjects are those with chronic myeloid leukemia, • The target group is those with chronic neutrophilic leukemia, or The subjects are those with chronic eosinophilic leukemia, • The subject is human, and / or LSD1 inhibitors are LSD1 inhibitor compounds disclosed herein.

[0157] Embodiments are also provided in which any of the above method embodiments may be combined with one or more of these embodiments, provided that the combinations are not mutually exclusive. Two embodiments are “mutually exclusive” when, as used herein, one is defined as not overlapping with the other. For example, an embodiment in which the disorder to be treated is primary myelofibrosis (PMF) is mutually exclusive with an embodiment in which the disorder to be treated is post-PV / ET myelofibrosis (MF), because these classifications are the result of different diagnoses. However, an embodiment in which the disorder to be treated is PMF is not mutually exclusive with an embodiment in which reticuline and / or collagen myelofibrosis is reduced, because reticuline and / or collagen myelofibrosis occurs in PMF.

[0158] In the methods disclosed above, or any subset or species thereof, any of the compounds disclosed above as LSD1 inhibitors may be used in any of the individual chemical species or in any of the pharmaceutical compositions described by one of the formulas or embodiments thereof. [Examples]

[0159] Examples The following are biological assays and clinical trials demonstrating the usefulness of the compositions and methods disclosed herein.

[0160] biological activity The compounds disclosed herein have been shown to be inhibitors of LSD1, for example, as disclosed in International Publication No. 2015 / 021128 and International Publication No. 2016 / 130952 or any of the references cited herein (their contents being incorporated herein by reference).

[0161] Example 1: Phase 1 / 2A and Phase 2B clinical trials in myelofibrosis A multicenter, open-label Phase 1 / 2A trial was initiated to evaluate the safety, tolerability, steady-state pharmacokinetics, and pharmacodynamics of compound 1 administered orally once daily to high-risk MF patients, including those with primary myelofibrosis (PMF), myelofibrosis after polycythemia vera (PPV-MF), and myelofibrosis after essential thrombocythemia (PET-MF) (collectively referred to as "MF"). This trial was then expanded to Phase 2b.

[0162] The evaluated Phase 1 / 2A portion of the trial included safety of the initial starting dose of 0.25 mg / kg / d, a 85-day duration of treatment with a subsequent washout period of up to 28 days, and pharmacokinetic and drug concentration measurements. Patients demonstrating clinical benefit were able to resume treatment with an additional 12-week cycle. With the transition to the Phase 2b trial, changes supported by earlier pharmacokinetic and pharmacodynamic studies and safety evaluations were implemented. These changes included an increased starting dose of 0.5 mg / kg / d with a larger escalation, a 168-day (24-week) duration of treatment with continuous infusion by eliminating the washout period, and the elimination and reduction of PK and drug concentration sampling.

[0163] This trial was conducted at multiple sites. Up to 50 high-risk myelofibrosis patients aged 18 years or older were treated. The primary objectives included safety and tolerability, pharmacokinetics (PK, Phase 1 / 2A only), and spleen volume reduction (SVR). Diagnostic endpoints included reduction in total symptom score (TSS) derived from MPN-SAF in Phase 1 / 2A and improvement in systemic symptoms, cytokines, and myelofibrosis as demonstrated using the MPN-SAF TSS device in Phase 2B. Primary inclusion criteria included high or intermediate-2 risk myelofibrosis, failure (refractory or resistant, poorly controlled or intolerant) or non-candidate status to available approved therapies including ruxolitinib, platelet count ≥100 K / μL, and circulating blasts ≤10%.

[0164] Administration was adjusted using platelet count as a biomarker for the effect of bomedemstat activity on megakaryocyte function and activity. Megakaryocytes are cells from the bone marrow and other sites that produce platelets and are central to the pathogenesis of myelofibrosis and essential thrombocythemia. In both conditions, somatic mutations in bone marrow stem cells result in mature megakaryocytes that produce excess platelets and bioactive proteins, altering the bone marrow niche and further leaking into the circulatory system, leading to symptoms specific to these conditions, such as itching and fatigue.

[0165] One strategy to reduce the excess products of megakaryocytes is to target megakaryocyte maturation and function. The effectiveness of megakaryocyte-targeted therapies can be quantified by measuring megakaryocyte products in the circulatory system, such as platelets, or by measuring inflammatory cytokines and growth factors in plasma or serum.

[0166] Such treatment can be further refined by gradually increasing or decreasing the dose to reduce the platelet count to a specific range.

[0167] In the Phase 1 / 2a portion of the study, patients started with an estimated sub-therapeutic dose of 0.25 mg / kg / d. Dose adjustments were made weekly (human platelet lifespan) by dose escalation, either upward or downward, depending on platelet counts at evaluation. Escalations were made in increments of 0.125 or 0.0625 mg / kg / d as shown below. Decreases were made in 50% reductions from the current dose. The calculated effective dose was expected to be approximately 1 mg / kg QD, but this did not represent an upper limit. The dose required to achieve the optimal therapeutic effect was expected to vary between patients and over time. The target platelet count for dose escalation expected to yield the most effective therapeutic effect was ≥50,000 to <100,000 / μL (50 to 100 × 10⁻⁶). 9 The result was / L). The Phase 1 / 2a progression and rechallenge rules based on weekly platelet count assessments are shown in Table 1 below.

[0168] [Table 5]

[0169] However, since all patients enrolled in the Phase 1 / 2A portion of the trial required multiple dose increases of Compound 1 from an initial starting dose of 0.25 mg / kg / d to bring their platelets within the target platelet count range, it is suggested that the starting dose should be higher. Subsequently, dose-response curves were created to provide an increase / decrease algorithm that adjusts the dose to achieve a target platelet count of 50,000–75,000 platelets / microliter (k / uL), designed with the perspective of minimizing the possibility of severe thrombocytopenia. Excluding both the highest and lowest doses (total daily doses of 4 mg and 100 mg), the average total daily dose of Compound 1 required to achieve a platelet count within the target range was 78.3 mg (SD 13.8, range 53–90 mg) or approximately 0.7–1.2 mg / kg / d equivalent. Therefore, a new starting dose of Compound 1 of 0.5 mg / kg QD was selected for all patients entering the Phase 2B portion of the trial, so that patients could reach the optimal dose more rapidly while still maintaining a reasonable safety margin. The gradual increase / decrease and retry rules were also modified in relation to these new objectives (Table 2).

[0170] [Table 6]

[0171] After modifying the dosing algorithm, a re-analysis of administration, response, and safety was performed based on the experience with the first 16 patients. The mean dose required to achieve and safely maintain patients within the target platelet count range ("therapeutic dose") was 63.8 mg / day or 0.85 mg / kg / d (assuming an average body weight of 75 kg). (Three patients never achieved the target range, two withdrew before week 6, and one did not agree to a dose increase due to fatigue.) The range of total therapeutic doses per day was 50 mg to 85 mg, with the exception of one patient who was maintained on a total daily dose of 4 mg from day 321 to day 510 and a second patient who discontinued the study on day 35. In the Phase 3 trial, the starting dose was expected to be 40 mg, with one or two additional dose adjustments over the following 4–6 weeks.

[0172] Eighteen patients were enrolled in the Phase 1b / 2a portion of the trial. Of these, four withdrew early from the trial; one progressed to an accelerated phase disease (day 39); two experienced adverse events of fatigue (day 33) and cellulitis (considered unrelated) (day 77); and one followed alternative therapy due to anemia (day 77). As a result, 14 patients remained whose response could be evaluated at week 12 and 9 patients whose response could be evaluated at week 24. An additional 13 patients were enrolled in the Phase 2b portion described below. Patient characteristics for the total of 31 patients to date are given in Table 3 below.

[0173] [Table 7]

[0174] With one exception, all patients had received one or more prior therapies, including ruxolitinib. 48% had PMF, 33% had PET-MF, and 19% had PPV-MF. The median patient age was 65 years (48-89), and 58% were male. 48% were classified as high-risk (IPSS), and the remainder as intermediate-risk-2. Of those who underwent detailed genetic analysis (exome sequencing of 264 AML and MPN genes), 71% had two or more mutations, of which 63% had high molecular risk mutations (ASXL1, U2AF1, SRSF2), and 31% had abnormal karyotypes. A substantial proportion of patients had ≥3 mutations. Patients were treated once daily for 12 weeks, followed by a washout period of up to 28 days, according to the above starting dose and escalation rules. Starting platelet counts ranged from approximately 141 to approximately 1309 k / μL. Bone marrow biopsies and abdominal imaging studies were performed before treatment and during the washout period after 12 weeks of administration. Myelofibrosis grading was performed in-center using the 2016 revised World Health Organization classification for myeloid neoplasia (Arber et al., 2016), and image interpretation was also performed in-center. The Myeloproliferative Neoplasm Assessment Form (MPN-SAF) was self-administered weekly from baseline and from day 0 until the end-of-study (EoS) examination. The total symptom score was derived using this method. Patients who demonstrated clinical benefit were eligible to resume an additional 12-week cycle of treatment.

[0175] Results. 78% (N=14) of the 18 patients completed 12 weeks (84 days), and 44% (N=9) completed 24 weeks. In patients deemed evaluable in this preliminary analysis (N=14, those who completed the 85-day cycle and for whom imaging tests achieved within the first two weeks of washout were available), compound 1 had a significant impact on myelofibrosis symptoms. Spleen volume generally decreased in patients evaluated to date, as shown in Figure 1. At week 12, 7 patients (50%) had decreased spleen volume, and at week 24, 6 patients (75%) had decreased spleen volume, of which 1 patient (12.5%) had a 35% decrease. MPN-10 scores also generally decreased, as shown in Figure 2. At week 12, 11 patients (79%) had a reduction in symptom scores, of which 3 patients (21%) had a reduction of ≥50%. At week 24, 8 patients (89%) had a reduction in their symptom scores, and of these, 4 patients (44%) had a reduction of ≥50%.

[0176] As shown in Figure 3, when compared to the best available treatment (BAT) in studies such as the PERSIST-2 clinical trial (see clinical trial number NCT02055781, for example), compound 1 was superior to BAT, with better spleen volume response (SVR) and total symptom score (TSS).

[0177] In addition, downregulation of inflammatory cytokines and a reduction in circulating growth factors were observed. As shown in Figures 4-6, S100A9 (Figure 4), RANTES (Figure 5), and IL-8 (Figure 6) generally decreased at 12 weeks of treatment with compound 1, while levels of CCL3, IL-6, IL-10, IL-33, IL-28A, IFNβ, IFNα, and IFNγ did not increase in any patient. As shown in Figures 7 and 8, levels of growth factors VEGF and PDGF-BB generally decreased at 12 weeks. The relevance of these results to the therapeutic theory of LSD1 inhibition is shown in Figure 9.

[0178] Improvements in hemoglobin (Hb) levels and percentage fetal hemoglobin-containing red blood cells (F cells) were also observed. Of the 18 patients enrolled in Phase 1b / 2a, 3 had Hb > 10 g / dL on day 0, and 15 had grade 2 or 3 anemia with Hb < 10 g / dL. On day 84 of treatment with compound 1, of the 3 patients with Hb > 10 g / dL, 1 improved (defined as an increase of Hb > 1 g / dL), and 2 worsened (a decrease of Hb > 1 g / dL). Of the 15 patients with Hb < 10 g / dL, 9 were fluid-dependent and 6 were fluid-independent. On day 84, of the 9 fluid-dependent patients, 1 became fluid-independent and had an improved Hb of > 1 g / dL, 8 maintained a stable fluid frequency, and 1 had an increased fluid frequency. Of the six fluid-independent patients, one improved, three remained stable, and two worsened (one became fluid-dependent, and one experienced a Hb decrease of >1 g / dL). Simultaneously, compound 1 reduced the percentage of F cells, as shown in Figure 10 (patients were randomly numbered and do not necessarily correspond to patients numbered in the previous figure). Fetal hemoglobin (HbF) is an established serological indicator of cancer, and fetal hematopoiesis, which does not occur in the spleen of healthy adults, has been observed in myeloproliferative neoplasms of the spleen.

[0179] Changes in myelofibrosis grade were also observed. Of the 13 patients with reported bone marrow biopsies (days 0-84 or EoT), 2 (15%) showed improvement of >1 grade, 8 (62%) had stable fibrosis scores, and 3 (23%) progressed by 1 grade.

[0180] Regarding symptom scores, improvement was generally dose-dependent and rapid, with fatigue scores improving within 14 days in 8 of the first 16 patients, for example. These changes were observed in all but one of these 16 patients at the lowest and second lowest doses. Similar to what has been reported in trials with JAK inhibitors in patients with MF, no correlation was found between improvement in overall symptoms and changes in spleen volume. Reduction in spleen volume was impaired in several forms. Patients were treated cautiously with an initial dose that was not expected to be optimal, and most did not achieve platelet counts within the target range until midway through the 85-day cycle. Furthermore, when all patients underwent follow-up imaging studies during the washout period, increased spleen volume was readily apparent on physical examination. In the Phase 2b portion of the trial, the washout period was eliminated, and the dosing regimen was improved to achieve target platelet counts more rapidly and to maintain patients within a safe range for a longer period.

[0181] Treatment with compound 1 reduced platelet counts in all patients. Changes in platelet production were closely related to exposure to compound 1, and platelet counts could be scalably increased or decreased with reasonable precision. The rate of these changes was consistent with the known lifespan of human platelets—7 days. Upon discontinuation of treatment, platelet counts rebounded robustly, suggesting the reversibility of the antithrombotic effect of compound 1 after drug removal. Similar to rats and dogs, granulocyte production was less sensitive to LSD1 inhibition, peripheral granulocyte counts were lower at treatment, lymphocyte counts remained unchanged, and monocyte counts were generally moderately elevated. These observations are consistent with those observed in both non-clinical and other clinical trials.

[0182] Safety. No deaths or dose-limiting toxicities were observed throughout the entire course of the study. Four SAEs, including painful splenomegaly, headache, nausea / vomiting, and heart failure, were attributed to compound 1 (all grade 3). 139 AEs of all grades were attributed to compound 1. The most common AEs across all 31 subjects in both studies were thrombocytopenia (11 subjects, 35%), anemia (3 subjects, 10%), and nausea (1 subject, 3%). The most common grade 3 / 4 AEs attributed to compound 1 were anemia (6 subjects, 19%) and neutropenia (3 subjects, 10%).

[0183] In a heterogeneous population of MF patients with limited treatment options, compound 1 was well-tolerated, demonstrated safety, and was effective in reducing spleen volume and substantially improving symptom scores in the majority of patients, as demonstrated above.

[0184] Example 2: Phase 2B clinical trial in myelofibrosis A multicenter, open-label, phase 1 / 2a trial was conducted to evaluate the safety, optimal dosing rules, steady-state pharmacokinetics, and pharmacodynamics of once-daily oral compound 1 in patients with myelofibrosis. The primary objective was to evaluate the effects of compound 1 on safety and tolerability in MF patients. • Pharmacokinetics (Phase 1 / 2a only) • Reduction of spleen volume

[0185] The objectives of the study (which may be partially or entirely analyzed) included evaluating the following in MF patients treated with compound 1: • Appropriateness of the treatment regimen to produce pharmacodynamic effects • Hematological responses (hematological parameters (all of which can be evaluated during treatment or after discontinuation of the drug at a specific interval) may include platelet count, red and white blood cell (RBC and WBC) count, and total blood count (CBC) including circulating blasts, bone marrow cellular composition (% blasts), and fetal hemoglobin induction) • Improvement of systemic symptoms as assessed using the Myeloproliferative Neoplasm Assessment Form (MPN-SAF) • Reduction of myelofibrosis score • Relationship between dose and time-dependent plasma trough concentration (Phase 1 / 2a only) • The effect of therapy on disease burden as measured by malignant cell-specific nucleic acid markers (DNA or RNA; nucleic acid markers include RNA and / or DNA mutations detected by sequencing or other nucleic acid assay methods) • The effect of treatment on cytokine profiles (cytokine quantification) • Relationship between genetic abnormalities in malignant cells and pharmacodynamic responses • Correlation between conventional clinical responses and the examination and evaluation of those responses.

[0186] Compound 1 was supplied as capsules at multiple strengths. These strengths, based on the free base of Compound 1, i.e., the active substance, may include 1 mg, 5 mg, 10 mg, 25 mg, and 50 mg. The supplied capsule strength may vary throughout the duration of the study.

[0187] The therapeutic goal of MF treatment was to inhibit LSD1 activity in hematopoietic cells in only a portion of the 24-hour administration cycle to a degree sufficient to reduce the production of cytokines and growth factors that drive myelofibrosis. Considerations for safety and the initial dose included chronic toxicological studies in relation to the clinical experience of patients who had previously received compound 1 in previous studies. In relation to this therapeutic goal and PK modeling, an initial dose (D) of 0.25 mg / kg / d was selected for the Phase 1 / 2A portion of this study. However, since all patients required multiple escalations of compound 1 from this initial dose to bring platelet counts within the target range, it is suggested that D should be higher. Subsequently, dose-response curves were created to provide an escalation algorithm that adjusts the dose to achieve a target platelet count of 50,000–75,000 platelets / microliter (k / uL), designed with the aim of minimizing the possibility of severe thrombocytopenia. Excluding both the highest and lowest doses, the average total daily dose of Compound 1 required to achieve platelet counts within the target range was 78.3 mg (SD 13.8, range 53–90 mg) or approximately 0.7–1.2 mg / kg / d equivalent. Therefore, a new starting dose of Compound 1 of 0.5 mg / kg QD was selected for all patients entering the Phase 2b portion of the study, allowing patients to reach the optimal dose more rapidly while still maintaining a reasonable safety margin.

[0188] This study design employed an alternative model-based approach suitable for target non-cytotoxic agents such as compound 1, where a monotonic relationship between exposure and toxicity was not observed (Le Tourneau, et al., 2009). Specifically, this study aimed to determine the final dose (C) required at steady state to inhibit platelet production. min We utilized dose-toxicity models developed in rats and dogs to correlate the plasma concentration of the drug 24 hours after administration.

[0189] Even at extremely high doses (human equivalent dose (HED) approximately 20-40 mg / kg), there was no evidence of acute toxicity from compound 1 in non-clinical studies, so it was thought that two sentinel lymphocyte patients would be sufficient to establish the acute safety of the starting dose. Therefore, two sentinel lymphocyte patients were administered 0.25 mg / kg / d sequentially for 7 days at the initial dose (D), monitored twice weekly, and any additional patients were subsequently treated. Since the effects of cytotoxic agents were not investigated in this study, it was considered appropriate to enroll patients at any time after confirming safety through administration to sentinel lymphocyte patients. Patients were enrolled and treated at any time.

[0190] To ensure patient safety, the Data Safety Monitoring Committee (DSMC) conducted monthly reviews of safety parameters and pharmacodynamic markers to draw conclusions regarding the safety and pharmacodynamic effects of Compound 1. The DSMC reviewed patient dose escalations, recommended dose adjustments, and assessed the need for a follow-up examination on day 3. The DSMC was convened within 4 days of the completion of 7 days of treatment for each sentinel patient. 1. Each sentinel patient will continue receiving treatment (Note: Treatment was not interrupted during the pending period of this review), and 2. Additional patients begin treatment with compound 1. It was determined that it was safe.

[0191] Implementation of the test This trial began as a Phase 1 / 2a trial, evaluating the safety of the starting dose, the duration of treatment over 85 days, and the pharmacokinetic and pharmacodynamic effects of compound 1. It then progressed to a Phase 2b trial incorporating modifications supported by the initial pharmacokinetic and pharmacodynamic studies and safety assessments. This trial consisted of two treatment periods: an initial treatment period (ITP) followed by an additional treatment period (ATP). Patients began enrollment in the Phase 2b portion of the trial, and the ITP was extended to once daily treatment for 169 days. The ATP was also extended to provide treatment to enrolled patients for an additional 169 days.

[0192] Initial treatment period. During ITP, patients returned twice a week initially for trial evaluation during the first week (days 0, 3, and 7 of ITP). After administration to three patients with a new dose, the DSMC was convened to assess the need for a day 3 examination. Patients returned once a week for the next 7 weeks (days 14, 21, 28, 35, 42, 49, and 56 of ITP), at least every two weeks for 8 weeks (days 70, 84, 98, and 112 of ITP), and then once a month for 8 weeks (days 140 and 168 of ITP). By week 8 (day 56), patients were expected to have achieved a stable dose and weekly dose adjustments would no longer be necessary. In exceptional patients whose dose had not stabilized, weekly examinations were continued at the PI's discretion (note: bi-weekly examinations may also be continued after day 112). On days 84 and 168, patients underwent abdominal magnetic resonance imaging (MRI) or, if the patient was not a candidate for MRI, computed tomography (CT). On day 168, bone marrow sampling was also required. Prior to the day 168 examination, but ideally at the day 140 examination for logistical purposes, a “certification” assessment was performed to determine whether the patient would provide clinical benefit (defined as not meeting the progressive disease criteria and being safely tolerable to compound 1, this definition applies throughout the document and is not repeated in each reference to clinical benefit). Such patients were certified to enter ATP and it was expected that the transition would be made without interruption of administration. Patients who did not provide clinical benefit or who achieved a complete response (CR), partial response (PR), or clinical improvement (CI) followed by a relapse of the equivalent of treatment failure were to discontinue compound 1 and undergo End of Treatment (EoT), pre-End of Study (pre-EoS), and End of Study (EoS) examinations.

[0193] Additional treatment period. In ATP, treatment was expected to continue for an additional 169 days in patients who demonstrated a clinical benefit as determined by the principal investigator. Certified patients returned monthly for trial evaluation (on days 0, 28, 56, 84, 112, 140, and 168 of ATP). Patients continuing with ATP were expected to have already achieved a stable dose and no longer require frequent dose adjustments. In exceptional cases where the dose did not stabilize, bi-weekly consultations were continued at the discretion of the PI. On day 168, patients underwent the same procedures and evaluations as in ITP, including MRI or CT (if the patient was not a candidate for MRI) and bone marrow sampling. Prior to day 168, or ideally at the day 140 consultation for logistical purposes, a “certification” evaluation was performed to determine whether the patient would continue to demonstrate a clinical benefit. Such patients were certified for repeated re-entry into ATP, and the patient continued to take compound 1 as long as certification continued.

[0194] Certain patients enrolled in a previous clinical trial with Compound 1 would complete their current phase of treatment in accordance with its protocol prior to the initiation of the extended ATP disclosed herein. Such patients did not undergo any washout period during the treatment period, but still underwent the washout assessment using the required MRI (or CT) and bone marrow aspirate and biopsy samples at the 84-day examination. In these patients, the “certification” assessment was performed at the examination immediately preceding the 84-day examination.

[0195] The evaluations described in the washouts were still performed despite the elimination of the washouts. All patients underwent follow-up examinations, including an EoT examination approximately 2 days after the final dose, a pre-EOS examination approximately 14 days after the final dose, and an EoS examination approximately 28 days after the final dose. Patients who did not enter ATP or discontinued treatment early began their follow-up period with an EoT examination approximately 2 days after the decision to terminate treatment.

[0196] Throughout the trial, patients were closely followed for both adverse events (AEs) and signs of toxicity by frequent monitoring of clinical signs and symptoms as well as by examination of peripheral blood and urine. Pharmacodynamic effects were closely monitored by frequent hematological evaluations of peripheral blood and required bone marrow aspirates and biopsies. Infusions were administered as needed throughout the administration, in accordance with standard institutional guidelines.

[0197] Administration By use of dose escalation / de-escalation, all patients were dosed to an estimated dose of Compound 1 (referred to as Dpi) required in humans that provides sufficient exposure to safely inhibit normal hematopoiesis as part of a 24-hour dosing cycle.

[0198] Initial treatment period (ITP). Treatment was initiated on Day 0 at a D of 0.5 mg / kg QD for all patients entering the second part of the 2b phase of the trial. Dose adjustments could be made by either dose escalation or de-escalation depending on the comparison of hematological values from the previous visit at each outpatient visit (except on Day 3) as determined by the following rules. Dpi was expected to be ≤1.2 mg / kg QD, but this was not an upper limit for escalation purposes as the dose required to achieve a therapeutic effect can vary between patients and change over time. Platelet escalation / de-escalation targets expected to be associated with a clinically significant therapeutic effect were platelet counts of ≥50,000 to ≤75,000 / μL (50 - 75×10 9 / L). Escalation / de-escalation and rechallenge rules based on the evaluation of the number of platelets, absolute neutrophil count (ANC), and hemoglobin (Hgb) are described below.

[0199] Escalation / de-escalation rules. Important: ANC ≥0.5×10 9 / L (500 / μL) and Hgb >8 g / dL (80 g / L) were required for escalation. At ANC or Hgb values below these thresholds, the current dose was either maintained or adjusted depending on the platelet count according to Table 4 below.

[0200]

Table 8

[0201] If an adverse event (AE) requiring a dose reduction occurred, it was possible to reduce the dose at some point in consultation with the medical monitor.

[0202] Additional Treatment Period (ATP): Certified patients would "restart" Compound 1 on day 0 of ATP, continuing dose gradual adjustments according to the above dose gradual adjustment rule table. No interruptions in administration were observed (i.e., day 168 = day 0 of the new ATP). Additional dose gradual adjustments could be made in consultation with the medical monitor.

[0203] Trial duration. Screening procedures could be initiated up to 28 days before the start of treatment. Patients could receive up to 169 days of initial treatment during the trial period. Patients were followed up for 28 days after the final dose. Therefore, the expected duration of participation in the trial was expected to be at least 32 weeks, from the first consultation of the first patient (FPFV) to the last consultation of the last patient (LPLV). Additional treatment could be given depending on the assessment of patient benefit.

[0204] Test evaluation. The evaluation outlined below will be presented in detail through the test examination.

[0205] The Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS) will be completed at baseline and at each consultation day from day 0 to the end-of-study (EoS) consultation (excluding day 3).

[0206] Adverse events (AEs) are evaluated at any consultation from the first dose of the compound until the end-of-surgery (EoS) consultation.

[0207] Physical Examination Including Vital Signs (PE): A full physical examination will be performed at the time of screening. A limited physical examination (LPE) will be performed at all other outpatient visits throughout the trial (except on day 3). The LPE will include weight, body tissue examination to assess changes from the previous PE, and spleen measurements. The splenic margin will be determined by palpation, measured in centimeters using a soft ruler / tape from the costal margin to the largest splenic process. The spleen should be measured in exactly the same manner at all examinations.

[0208] Urine or serum pregnancy tests are performed at screening, at baseline (if the patient has not yet had a screening appointment), before administration on day 0, monthly throughout the study (i.e., on days 28, 56, 84, 112, 140, and 168), in cases of suspected relapse, at EoT, pre-EoS, and EoS / ET appointments, and in women of childbearing potential (WOCBP) if pregnancy is suspected while the patient remains in the study.

[0209] The bone marrow aspirate and biopsy samples should be measured at the following points. • At baseline (at least 21 days before the first dose of the compound). Day 168 (±7 days). As long as the patient remains certified, the ATP will be administered approximately every six months thereafter, on day 168 (±7 days). • At the end of tethering (EoT) and ET (unless it has been performed within the previous 5 weeks) and in case of suspected relapse (unless it has been performed in the last 21 days or is scheduled for the following 7 days). Aspirated fluid from the first aspiration, but no later than the second aspiration, is required whenever possible. The total number of bone marrow evaluations required during ITP is approximately two per 32 weeks. Additional bone marrow evaluations are only required if the patient has received ATP certification, if there is evidence of suspected relapse following a response, or if there is evidence of progressive disease.

[0210] Perform an abdominal MRI or CT scan (if the patient is not a candidate for MRI) at the following times: • Day 0, before administration (±2 days) · At the 84th and 168th day visits (±7 days) · As long as the patient remains enrolled, thereafter, approximately every 6 months, at the 168th day of ATP (±7 days) · At EoT, ET, and in case of suspected relapse (unless performed within the previous 5 weeks)

[0211] Clinical laboratory measurements: At screening, at baseline (if away from screening visit), before dosing on day 0, in case of suspected relapse, and at EoT, pre-EoS, and EoS / ET visits, measure the following laboratory measurements as follows. · Biochemistry - Once per month throughout the study (i.e., on days 28, 56, 84, 112, 140, and 168) · Hematology including manual differential - At any outpatient visit throughout the study · Coagulation - Once per month throughout the study (i.e., on days 28, 56, 84, 112, 140, and 168) · Urinalysis - On days 84 and 168 throughout the study

[0212] Cytokines: The sample collection time points are as follows. · As long as the patient remains enrolled, before dosing on day 0, on days 14, 28, 84, and 168, and at each 168-day visit of ATP · At EoT and ET (ET is required only if the patient discontinues due to ITP)

[0213] Erythrocyte hemoglobin F (HbF) and %F cells (selected sites only / ITP only): · Before dosing on day 0, on days 84 and 168 · At EoT and ET (both are required only if the patient discontinues due to ITP)

[0214] Genomic analysis: Germline samples should be collected at baseline, but can be collected prior to dosing on day 1. The sample yield is undetermined and repeated sampling may be required.

[0215] Blood samples will be collected for genomic analysis at the following points. • Baseline (more than 21 days before the first dose of the compound) • During the medical examinations on day 84 and day 168 As long as the patient remains certified, thereafter, approximately every 6 months, at each 168-day ATP consultation. • At the end of time (EoT), end of sustained stress (EoS / ET), and when there is a suspicion of recurrence. According to the bone marrow sampling schedule, all bone marrow aspirate samples will undergo genomic analysis.

[0216] Pharmacodynamic (PD) evaluation: PD parameters are evaluated using blood and bone marrow samples collected both during treatment and after treatment discontinuation at specific intervals. This may include: whole blood count (CBC) including leukocyte differential, measurement of circulating cytokines, measurement of RNA and / or DNA mutations and their frequencies identified by sequencing, and induction of fetal hemoglobin. Bone marrow evaluation, including morphology and fibrosis scores, is performed in relation to any bone marrow sampling time point.

[0217] Eligibility Criteria. Patients must meet all inclusion criteria and not meet any exclusion criteria.

[0218] Selection criteria: 1. Informed consent. 2. Age: 18+ years old at the time of screening. 3. Diagnosed with PMF based on the World Health Organization (WHO) diagnostic criteria for myeloproliferative neoplasms, PPV-MF based on IWG-MRT, or PET-MF based on IWG-MRT, and meeting the following additional subtype-specific criteria: a. Classified as high-risk (3 prognostic factors) or risk-2 (2 prognostic factors). Prognostic factors as defined by the International Working Group (Cervantes, et al., 2009): i. Age > 65 years old, ii. Presence of systemic symptoms (weight loss, fever, night sweats), iii. Marked anemia (Hgb < 10 g / dL) (Hemoglobin levels < 10 g / dL must be demonstrated during screening of non-fluid-dependent patients. Patients receiving regular IV fluids of packed red blood cells will be considered to have hemoglobin < 10 g / dL for the purpose of assessing risk factors.) iv. History of leukocytosis [WBC>25×10 9 / L (25,000 / μL)], v. Circulating blasts>1%. 4. The patient is refractory, resistant, poorly controlled, or intolerant to any available approved therapy, or is not a candidate for any available approved therapy at the discretion of the investigator (Note: Approved therapies include ruxolitinib). 5. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 6.0 Peripheral blast cell count before administration ≤10%. 7.0 Absolute neutrophil count before administration ≥ 0.5 × 10 9 / L (500 / μL). 8.0 Platelet count before administration ≥ 100 × 10 9 / L (100,000 / μL). 9. Average life expectancy > 36 weeks. 10. All previous therapies for MPN, including ruxolitinib, any chemotherapy agent, immunosuppressive therapy (e.g., corticosteroids > 10 mg / day; noteworthy exclusions: use of corticosteroids for gout management is permitted, and corticosteroid maintenance and replacement therapy, e.g., prednisone ≤ 10 mg / day or corticosteroid equivalents, are permitted), immunomodulators (e.g., thalidomide), radiotherapy for at least two weeks prior to day 0 of the study, and interferon for four weeks prior to day 0 of the study, have been discontinued. Low-dose acetylsalicylic acid is permitted. Elective radiotherapy for non-indicative or non-bone lesions performed < two weeks prior to treatment may be considered for medical monitor approval. 11. During the trial period, bone marrow evaluation, peripheral blood, and urine sampling may be performed. 12. The capsule is swallowable. 13. Women of childbearing potential (WOCBP) and men of reproductive capacity must agree to use an approved method of contraception from screening until 28 days after the last dose of the compound. Methods of contraception include combination hormonal contraception with estrogen and progestogen to inhibit ovulation, hormonal contraception with progestogen alone related to ovulation inhibition, intrauterine devices (IUDs), bilateral fallopian tube occlusion, vasectomy partners in monogamous relationships (vasectomy or tubal ligation at least 6 months prior to administration), and complete abstinence (defined as avoidance of heterosexual intercourse). Patients practicing abstinence must agree to use an approved method of contraception if they become sexually active during the study period. The risk of embryo-fetal toxicity is sufficiently reduced by 28 days, which is the >10 half-life of the drug at the doses used in this study.

[0219] Exclusion criteria: 1. The patient underwent major surgery less than 4 weeks prior to the start of the study drug, or did not recover from side effects of such surgery. 2. The patient underwent any surgical procedure within the past two weeks, excluding minor procedures prior to the initiation of the study drug (e.g., skin biopsy or central venous catheter placement / removal). 3. History of splenectomy. 4. A history of hematopoietic stem cell transplantation or a hematopoietic stem cell transplant scheduled within 24 weeks of screening. 5. Unresolved treatment-related toxicity from prior therapy (unless resolved to ≤ Grade 1). 6. You are currently using a prohibited drug (e.g., romiplostim) or anticipate needing any of these drugs during treatment with the investigational drug. 7. Known immediate or delayed hypersensitivity reactions or allergic reactions to Compound 1 or LSD1 inhibitors that are contraindicated for involvement (i.e., monoamine oxidase inhibitors, MAOIs). 8. I am currently using monoamine oxidase A and B inhibitors (MAOIs). 9. Uncontrolled, active infection. 10. Concurrent secondary active and unstable malignant lesions (patients with concurrent secondary active lesions such as non-melanoma skin cancer, but with stable malignant lesions, are eligible). 11. Evidence of bleeding risk at the time of screening, including any of the following: a. Activated partial thromboplastin time (aPTT) ≥ 1.3 × local upper limit of normal b. International Normalized Ratio (INR) ≥ 1.3 × Upper Limit of Local Normal c. A history of severe thrombocytopenia or platelet dysfunction unrelated to myeloproliferative disorders or their treatment. d. Known bleeding disorders (e.g., abnormal fibrinogenemia, factor IX deficiency, hemophilia, von Willebrand disease, disseminated intravascular coagulation [DIC], fibrinogen deficiency, or other coagulation factor deficiency) 12. Evidence of significant renal or hepatic failure (but not due to hemolysis or leukemic infiltration) as defined by any of the following local laboratory parameters at the time of screening: a. Calculated glomerular filtration rate (GFR using the Cockcroft-Galt formula) <40 mL / min or serum creatinine > 1.5 × upper limit of local normal b. Aspartate transaminase (AST) or alanine aminotransferase (ALT) ≥ 2 × upper limit of normal 13. Known human immunodeficiency virus (HIV) infection or known active hepatitis B or C virus infection (testing will not be performed as part of the screening procedure). 14. A history of any gastrointestinal (GI) function disorder that may interfere with drug absorption (e.g., chronic diarrhea), confound the test results, or pose an additional risk to the patient by participating in the study; or a patient who has undergone gastric bypass surgery. 15. Use of the investigational agent for less than 14 days prior to day 0 of the trial, or for a period equal to at least 7 half-lives of the agonist, whichever is longer. 16. Pregnant or lactating women, or women who intend to become pregnant at any point during the trial.

[0220] Safety guidelines. Generally, supportive care (fluid therapy, antifungal administration, etc.) should be maintained according to facility guidelines. In addition, platelet count ≤ 10 × 10 9 For patients with a white blood cell count of 10,000 / L (10,000 / μL), intravenous fluid administration is recommended. Hydroxyurea may be used during the study period in the following cases of proliferation: a) at the discretion of the principal investigator, white blood cell count ≥ 30 × 10 9 When hydroxyurea treatment is initiated at / L (30,000 / μL), the majority of cells appear as immature cells (myelocytes / promyelocytes), and b) the white blood cell count is <10 × 10 9 When the level is 10,000 / μL, discontinue hydroxyurea treatment. Patients taking medications with the potential to induce or inhibit CYP3A4 or CYP2D6 should be closely monitored for the potential effects of co-administration. Particular caution should be exercised with azole-class anti-infective agents.

[0221] Prohibited drugs / treatments. 1. All cytotoxic agents except hydroxyureas 2. Platelet regrowth agents: Romiplostim, Eltrombopag 3. Prednisone or prednisolone > 10 mg / day (exclusion clause to note: use of corticosteroids for gout management is permitted) and dexamethasone > 4 mg / day. Corticosteroid maintenance replacement therapy, such as prednisone ≤ 10 mg / day or corticosteroid equivalents, is acceptable. 4. Monoamine oxidase A and B inhibitors 5. Platelet count <50 × 10 9 At LSD1 levels of 50,000 / μL, the use of anticoagulants and nonsteroidal anti-inflammatory drugs (NSAIDs, including aspirin) in patients is prohibited. LSD1 inhibition can induce cytopenia, which may result in increased levels of granulocytes and granulocyte-macrophage colony-stimulating factor (G-CSF and GM-CSF) and erythropoietin (EPO). Although not explicitly prohibited, exogenously administered G-CSF, GM-CSF, and EPO may not offer significant clinical benefit in the context of granulocytopenia or anemia, respectively, following LSD1 inhibition.

[0222] Control of test toxicity. The severity of adverse events will be assessed using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03, published on June 14, 2010.

[0223] Hematological toxicity: Hematological values ​​outside the normal reference range are an inherent characteristic of MPN and are expected effects in many treatment attempts to manage this disease. The effects of compound 1 on normal myeloid hematopoiesis observed in non-clinical and clinical trials are expected in humans and are pharmacodynamic effects of LSD1 inhibition by compound 1, and are therefore not considered harmful. These events are not considered DLTs, with the following exceptions.

[0224] Dose-limiting toxicity (DLT): One of the following adverse events (AEs) occurring within the first seven days of the initial treatment period and considered by the investigator to be low-grade, moderate-grade, or highly related to compound 1. • Thrombocytopenia resulting in clinically significant sequelae (i.e., clinically significant bleeding events) * (or the need for prophylactic fluid therapy), • Platelet count > 50,000 × 10 9 Clinically significant bleeding events in patients with a blood glucose level of 50,000 / μL (a clinically significant bleeding event is defined as an event that is life-threatening, uncontrollable, and / or results in hemodynamic instability), ○ Any Grade 4 or 5 non-hematological adverse event, ○ Any Grade 3 non-hematological adverse event that does not recover to Grade 2 within 7 days of discontinuing the drug, except for the following: • Nausea, vomiting, or diarrhea of ​​≥ Grade 3 that responds to standard medical care. • Asthenia lasting less than 14 days or more (Grade 3 or higher) • Any grade 3 electrolyte abnormality lasting more than 24 hours and not associated with an underlying malignant lesion. In patients experiencing DLT, the dose may be adjusted downwards if it is deemed safe for the patient to continue with compound 1.

[0225] Stopping rules. If it is deemed unsafe for the patient to continue compound 1 after DLT, or if the patient does not demonstrate significant improvement within 21 days after dose reduction due to DLT, or if 25 × 10 9 Following temporary discontinuation of compound 1 due to a platelet count below 25,000 / L (25,000 / μL), the patient's platelet count increased to >50 × 10 within 21 days. 9 If the level does not return to / L (50,000 / μL), discontinue treatment.

[0226] Results: 13 patients enrolled in the trial, and 85% have remained in the trial so far.

[0227] At week 12, all patients are characterized by the following: • Total symptoms (n=32) ○78% (25 people) experienced a reduction in their symptom scores. ○25% (8 people) experienced a reduction of ≥50%.

[0228] At week 12, Phase 2b patients are characterized by the following: ·Spleen volume (n=14) ○86% (12 patients) had a decrease in spleen volume. ○14% (2 people) had a reduction of ≥35%. ○29% (4 people) experienced a reduction of ≥20%. ○Median change up to week 12 = -15%

[0229] The absolute changes in MPN, SAF, TSS, and spleen volume over the entire 12-week period are shown in Figures 11(a) and (b), respectively.

[0230] Figure 12 shows the treatment progress of patients 008-103 over the entire 196-day period. Dosage increases and decreases of the LSD1 inhibitor are shown in panel (a) in mg. The effects of this dosing regimen are shown in the following panel: (b) spleen size, cm; (c) symptom score; (d) platelets (left scale, k / uL) and hemoglobin (right scale); (e) WBC and neutrophils; and (f) fatigue score (10 = worst).

[0231] Example 3: Sequencing Protocol The following characterizes the sequencing protocol. • Samples: Reproductive system (cheek or hair) and "tumors" (bone marrow, peripheral blood, granulocytes) • Targeted enrichment: 11,736 hybridization probes in an IDT AML panel targeting 261 frequently mutated genes (approximately 6300 exons) in myeloid neoplasms. • Illumina sequencing: 2×150bp pair-end sequencing, approximately 10 million pairs per sample were sequenced. • Target sequencing depth >500, actual: >1000 for >90% of samples Analysis: For CALR etc., Burroughs-Wheeler alignment (BWA) ⇒ VARSCAN2 genotyper ⇒ IGV • Somatic call cutoff: Sequencing depth: >20; Variant allele frequency (VAF): >15% • Annotation: All calls were submitted to the University of Washington's CADD (Combined Annotation Dependent Depletion). A CADD score cutoff > 20 identifies the top 1% of the most harmful mutations.

[0232] The following was observed: • 7 out of 22 patients (32%) showed a reduction in some or all somatic mutations. • 12 out of 22 (55%) have stable VAF. • 3 out of 22 patients (14%) had increased VAF. • No new mutations were identified in patients followed up for 550+ days. • There will be no progression to AML.

[0233] The table below presents VAFs for both MPN somatic mutations and other somatic mutations, as well as follow-up VAFs.

[0234] [Table 9]

[0235] The table below shows examples of how the study altered patients' VAF.

[0236] [Table 10]

[0237] From the above description, those skilled in the art will be able to easily confirm the essential characteristics of the present invention, and various modifications and alterations of the present invention can be made to adapt them to various uses and conditions without departing from its spirit and scope.

[0238] The detailed description provided above is provided to assist those skilled in the art in carrying out the disclosure. However, the disclosure described and claimed herein is not limited in scope by the specific embodiments disclosed herein, for these embodiments are intended to illustrate some aspects of the disclosure. Any equivalent embodiment is intended to fall within the scope of the invention. Indeed, from the above description, various modifications of the disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art. Such modifications do not depart from the spirit and scope of the discovery of the invention. Such modifications are also intended to be included within the appended claims.

[0239] All references cited herein are incorporated herein by reference. The discussion of references herein is intended merely to summarize the claims made by their authors and does not constitute an endorsement that any reference constitutes prior art with respect to patentability. The applicant reserves the right to verify the accuracy and validity of the cited references.

Claims

[Claim 1] N-[(2S)-5-{[(1R,2S)-2-(4-fluorophenyl)cyclopropyl]amino}-1-(4-methylpiperazine-1-yl)-1-oxopentan-2-yl]-4-(1H-1,2,3-triazole-1-yl)benzamide, bis-tosylate salt, in the manufacture of a pharmaceutical product for the treatment of primary myelofibrosis (PMF), post-PV myelofibrosis (PPV-MF), or post-ET myelofibrosis (PET-MF) in subjects requiring it. 【Chemistry 1】 The use of ("Compound 1"), wherein Compound 1 is 50 × 10 ９ ~75 x 10 ９ A sufficient dose is administered to maintain a platelet count of platelets / L. The subjects were administered compound 1 at an initial dose of 0.5 mg / kg / d, and then one week later, If the platelet count is ≥90 × 10⁹ platelets / L and the % platelet reduction is <50% from the previous examination, the dose for the subject is adjusted to add 0.2 mg / kg / d of compound 1 to the daily dose. If the platelet count is ≥90 × 10⁹ platelets / L and the % platelet reduction is ≥50% from the previous examination, the dose for the subject is adjusted to add 0.1 mg / kg / d of compound 1 to the daily dose. When the platelet count is between 40 × 10⁹ platelets / L and 89 × 10⁹ platelets / L, the daily dose of compound 1 is maintained. When the platelet count is between 25 × 10⁹ platelets / L and 39 × 10⁹ platelets / L, the dose of the subject is adjusted to reduce the current mg / kg daily dose of compound 1 by 25%. If the platelet count is <25 × 10⁹ platelets / L, administration is withheld until the platelet count returns to >50 × 10⁹ platelets / L, and then the dose of compound 1 is adjusted to 50% of the dose administered when the platelet count fell below 25 × 10⁹ platelets / L, and Throughout the entire course of therapy, the platelet count evaluation and dose adjustment process is repeated once a week until the target platelet count reaches 50 × 10⁹ to 75 × 10⁹ platelets / L.

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