Method of treating acute myeloid leukemia and myelodysplastic syndromes using intracellular immune checkpoint regulation pathway inhibitors, chemical compound and methods of synthesizing same

Abstract

The inventions relate to the field of medicine and pharmaceutics, and more particularly to new protein kinase B inhibitor compounds and methods of producing and using same to treat myeloid leukemia and / or myelodysplastic syndrome. The inventions provide for the clinical effectiveness of immune pathway inhibitors, improved immune control, and suppression of the expression of a number of immune checkpoint molecules, including PD-1L, TIM-3, TIGIT, CD155, CD200 and VISTA, simultaneously on the surface of leukemia cells, which creates the prerequisites for an increase in the rate and duration of remission and an increase in patient survival rates. Through the development of intracellular signalling pathway inhibitors which have the pharmacological effect of suppressing the expression of a number of immune checkpoint molecules on the surface of tumour cells, the inventions widen the range of agents that can be used for treating myeloid leukemia and myelodysplastic syndromes.

Description

[0001] DESCRIPTION OF THE INVENTION GROUP

[0002] A METHOD FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES USING INHIBITORS OF INTRACELLULAR IMMUNE CHECKPOINT REGULATION PATHWAYS, A CHEMICAL COMPOUND AND METHODS OF ITS SYNTHESIS.

[0003] IPC A61K 31 / 00, A61P 35 / 00, A61K 31 / 519 FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES The inventions relate to the field of medicine and pharmaceuticals, namely to new compounds related to protein kinase B inhibitors, methods for their production and use for the treatment of myeloid leukemia and / or myelodysplastic syndrome.

[0004] INFORMATION ON THE PRIOR ART

[0005] Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal disorders affecting hematopoietic stem cells. These disorders are based on the accumulation of somatic mutations in various genes and disruption of epigenetic regulation, dysfunction of the bone marrow niche microenvironment, and disturbances in the immune system's antitumor surveillance (Afanasyev BV et al. Russian Journal of Pediatric Hematology and Oncology. 2018; 5(3):23-35). In many patients, the development of MDS is preceded by a period of nonclonal or clonal cytopenias of unclear significance, which is caused by the emergence of somatic mutations. The result is increased proliferation, increased clonal inefficiency, suppression of normal hematopoiesis, and, in the final stages, impaired differentiation, leading to the accumulation of blasts and transformation into acute myeloid leukemia (AML) (Brunner AM et al. Blood Cancer J. 2022 Dec14; 12(12): 166).The mutation spectrum in myelodysplastic syndrome and acute myeloid leukemia is very close, which determines similar therapeutic tactics for these nosological entities (Liu M et al. Int J Hematol Oncol. 2021 Jun 22;10(2): IJH34).

[0006] Morphological analysis of bone marrow and cytogenetic testing are key in the diagnosis of MDS. The World Health Organization's classification of MDS takes into account both morphological and cytogenetic characteristics, helping to determine the prognosis and select a treatment strategy (Steensma DP. Blood Cancer J.

[0007] 2018 May 24;8(5):47).

[0008] According to a systemic analysis, the global incidence of AML is 0.6-11.0 cases per 100,000 population, and MDS is 0.22-13.2 cases per 100,000 population in various populations. The median age at onset of these diseases is 69 years, and more than half of the disease is diagnosed in old age (Lubeck DP et al. Blood. 2016; 128(22):5930-5930).

[0009] In recent years, our understanding of the biology of MDS and AML has advanced significantly, leading to the identification of key genetic and molecular pathways underlying the disorder. Among the most common genetic abnormalities are mutations in genes regulating RNA splicing, DNA methylation, and histone modification. The most frequently encountered mutations are in the FLT3, NPM1, CEBPA, IDH1, IDH2, TP53, RUNX1, ASXL1, TET2, KRAS, NRAS, PML-RARA, KMT2A, PHF6, EZH2, ASXL1, JAK2, SETBP1, DNMT3A, MPL, ZRSR2, U2AF1, and CBL genes (Papaemmanuil E et al. N Engl J Med. 2016 Jun 9;374(23):2209–2221). The accumulation of somatic mutations also leads to the development of chromosomal aberrations, which are often a pathogenetic mechanism for the progression of MDS and AML and evasion of immune surveillance (Welch JS et al. Cell. 2012 Jul 20;150(2):264-278).

[0010] An important pathogenetic mechanism of both MDS and AML is complex disturbances of the immune checkpoint system. It was revealed that the expression of PD-L1, PD-1, TIM-3, and CD80 on myelopoietic cells is physiologically necessary for protection against autoimmune aggression. In MDS, the proportion of myeloid cells expressing these immune checkpoint molecules increases, which is a mechanism for evading immune surveillance (Moiseev I et al. PLoS One. 2022 Oct 25;17(10):e0275399). Most patients co-express inhibitory immune checkpoint ligands, which negatively affects the prognosis of the disease in MDS (Tcvetkov N et al. Leuk Res Rep. 2020 Jun 28:14:100215). In addition to the listed checkpoint molecules, increased expression of the immune checkpoint ligands VISTA (Kim TK L et al. J Clin Invest. 2024 Feb 1;134(3):e164325) and BTLA on tumor myeloid cells (Radwan SM et al. Egypt J Med Hum Genet. 2021;22:78) has also been described.In MDS, the expression of immune checkpoint molecules such as PD-L1, PD-L2, PD-1, and CTLA4 increases, which determines resistance to therapy (Yang H et al. Leukemia. 2013 Nov 25;28(6): 1280-1288). In addition, a pathological autocrine loop has been identified in AML cells with the production of soluble galectin 9 and the expression of membrane TIM-3, which promotes the survival and proliferation of tumor cells (Kikushige Y et al. Cell Stem Cell. 2015 Sep 3;17(3):341-52).

[0011] Treatment of MDS depends on the risk of progression to acute myeloid leukemia and focuses on symptom management and maintaining the patient's quality of life. Approximately one in three cases of MDS progresses to AML, the terminal stage of the disease. For a long time, low-dose cytarabine (LDC) remained the standard of care for high-risk myelodysplastic syndrome (MDS). Low-dose cytarabine has been used in myeloid neoplasms since the mid-1980s. The median survival of patients using MDC is approximately 8 months. The maximum survival time recorded in clinical trials does not exceed 2-3 years. Response rates to therapy range from 15 to 30%. Some studies indicate no difference in survival rates between groups of patients receiving low-dose cytarabine and those receiving the best possible supportive care (Gerhartz HH et al. Infection.1992:20 Suppl 2: S 116-23, Zwierzina H et al., Leukemia. 2005 Nov; 19(11): 1929-33).

[0012] Currently, the standard treatment for patients with high-risk MDS is hypomethylation therapy (HMT) with 5-azacytidine or decitabine. Randomized trials aimed at drug registration found that decitabine and 5-azacytidine increased median overall survival compared to best available therapy: decitabine by 2.7 months, and 5-azacytidine by 10 months. However, subsequent real-world studies conducted in patients with high-risk MDS showed more modest results with HMT. Median overall survival was 11 months for decitabine and 12 months for 5-azacytidine (Kantarjian HM et al. J Clin Oncol. 2012 Jul 20;30(21):2670-7, Fenaux P et al. Lancet Oncol. 2009 Mar;10(3):223-32 Zeidan AM et al. Br J Haematol. 2016;175:829-840).

[0013] Recently, BCL-2 activation has become increasingly important in the progression of MDS, as in AML, and the development of resistance to its treatment. The addition of the BCL-2 inhibitor venetoclax to 5-azacytidine-refractory MDS demonstrated a complete response in 7% of patients and a bone marrow response in 32% of patients (Zeidan AM et al. Am J Hematol. 2022 Nov 10;98(2):272-281). Results of randomized trials confirming the need to include venetoclax in first-line treatment for MDS have not been published.

[0014] In AML, the main treatment principle was the use of a combination of varying doses of cytarabine and anthracyclines in induction followed by consolidation with the same drug groups. Intensification of these drug doses and the addition of purine analogs to chemotherapy regimens resulted in modest improvements in treatment outcomes in younger patients, but not in those over 60 years of age (Kantarjian H et al. Blood Cancer J. 2021 Feb 22;11(2):41).

[0015] A significant improvement in AML control in older patients was achieved with the introduction of the combination of HMT and venetoclax. Compared with best available therapy, 5-azacytidine and venetoclax demonstrated a significant improvement in progression-free survival and response rates (DiNardo CD et al. N Engl J Med 2020;383:617-629). Long-term follow-up showed that 5-azacytidine and venetoclax therapy allowed 37.5% of patients to maintain remission for two years and about 20% of patients for five years (Pratz et al. Am J Hematol. 2024 Apr;99(4):615-624).

[0016] In 2019, another drug, glasdegib, was approved for the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome in combination with chemotherapy. It blocks the Hedgehog signaling pathway, which was initially described as one of the pathways involved in embryonic development. Proteins in this pathway are responsible for cell and tissue differentiation. Hedgehog signaling and other intracellular pathways play an important role in regulating the tumor cell cycle and are also involved in the mechanisms of chemotherapy resistance (Ingham PW et al. Nat Rev Genet. 2011 Jun;12(6):393-406). In a phase 1 study, 31% of patients with AML and high-risk MDS achieved complete remission while receiving glasdegib in combination with low-dose cytarabine or decitabine. At a median follow-up of 27 months, survival was 19% (Savona MR et al. Clin Cancer Res. 2018 May 15;24(10):2294-2303).

[0017] Drugs are also currently registered for specific genetic forms of MDS and AML. The most common mutations in AML are mutations in the FLT3 gene. These mutations account for approximately 30% of patients (Tyner JW et al. Nature).

[0018] 2018 Oct;562(7728):526-531). Currently, two drugs are approved for this genetic variant of AML: midostaurin and gilteritinib. In a phase 3 study, when midostaurin was added to standard chemotherapy in the first line of therapy, an increase in 4-year progression-free survival was demonstrated from 44.3% to 51.4% (Stone RM et al. N Engl J Med, 377 (5) (2017), pp. 454-464). Gilteritinib, compared with the best available therapy in a randomized trial in refractory and relapsed leukemia, demonstrated an increase in median survival from 5.6 to 9.3 months. Long-term survival was limited to patients who received allogeneic hematopoietic stem cell transplantation (Perl AE et al. Blood. 2022 Jun 9;139(23):3366-3375).

[0019] Mutations in the IDH1 and IDH2 genes account for approximately 5% of MDS and AML cases (Xu W et al. Cancer Cell. 2011 Jan 18;19(l):17-30). Two IDH inhibitors are currently approved: enasidenib (an oral IDH2 inhibitor) and ivosidenib (an oral IDH1 inhibitor). In a phase II study in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome, enasidenib demonstrated an overall response rate of 53%, including 7% complete remissions (Stein EM et al. Blood. 2016;128(22):343). A randomized trial of ivosidenib in combination with 5-azacytidine in IDH1-mutated AML demonstrated an increase in median overall survival from 8 to 24 months. The two-year event-free survival rate was approximately 20% (Montesinos et al. N Engl J Med 2022;386:1519-1531).

[0020] Currently, the only method of cellular immunotherapy for MDS that has entered widespread clinical practice is allogeneic hematopoietic stem cell transplantation (allo-HSCT). It should be noted that allo-HSCT is still the only curative therapy for MDS. In this regard, allo-HSCT often plays a key role in the fate of patients with MDS, especially in the high-risk MDS group, when the question of the possibility of allo-HSCT arises at the time of diagnosis. In AML, all patients with intermediate and high risk, who constitute approximately 80% of the total AML group, are candidates for allo-HSCT in the first remission of the disease, since this treatment method can halve the risk of relapse (Snowden JA et al. Bone Marrow Transplant. 2022 May 19;57(8): 1217–1239). However, some patients do not have a suitable donor (Aljurf M et al. Bone Marrow Transplant.2019 Aug;54(8):1179-1188), and the transplantation itself is associated with a significant percentage of fatal complications, the proportion of which remains high (Penack O et al. Blood Adv. 2020 Dec 22;4(24):6283-6290). Moreover, the results and tolerability of allo-HSCT are worse in the older group of patients, who make up the majority of patients with AML and MDS (Heinicke T et al. Ann Hematol. 2021 Sep;100(9):2387-2398). In addition, the inability to achieve a low level of tumor burden determines the worse results of allo-HSCT in patients with MDS compared to AML in remission (Orvain C et al. Am J Hematol.

[0021] 2024 May;99(5): 862-870). Thus, existing chemotherapeutic and targeted approaches to the treatment of MDS and AML can achieve cure in less than half of patients, even with subsequent consolidation allo-HSCT. A population-based study in the United States indicates that, despite the emergence of new treatments, the survival of patients over 60 years of age has remained virtually unchanged since 2001 (Linet MS et al. EClinicalMedicine. 2024 Mar 16:71: 102549), necessitating the search for new therapeutic targets.

[0022] Checkpoint inhibitors (CIIs) have become a breakthrough in the treatment of patients with a range of solid tumors. Their universal mechanism involves reactivating immune cells by blocking a signal that inhibits their activity, restoring the antitumor immune response. However, the expression of various checkpoint ligands varies greatly from tumor to tumor. Tumors with a high percentage of neoantigens or with amplification of checkpoint ligand genes are most likely to respond to CIIs. The most sensitive tumors include Hodgkin's lymphoma, melanoma, genitourinary cancer, lung cancer, head and neck cancer, and solid tumors with microsatellite instability (Allison JP et al. Cell).

[0023] 2015 Sep 10;162(6): 1202-5).

[0024] Identified immune checkpoint abnormalities have led to the investigation of various immune checkpoint inhibitors in AML and MDS. Sabatolimab, which targets the TIM-3 receptor, targets the TIM-3 / Gal9 autocrine loop that promotes tumor cell survival and proliferation. This humanized monoclonal antibody is designed to enhance T-cell killing, facilitate phagocytic uptake of TIM-3-expressing target cells, and block the interaction between TIM-3 and its ligands (Schwartz S et al. Immunother Adv. 2022 Aug 10;2(l):ltac019). The first study of sabatolimab in high-risk MDS and AML demonstrated an encouraging overall response rate of 56.9% and a response duration of 16 months (Brunner AM et al. Blood. 2021;138(Sl):244). The obtained results formed the basis for conducting the STIMULUS-MDS1 study of sabatolimab in patients with high-risk MDS to confirm its efficacy compared with standard GMT.The study did not demonstrate an increase in the complete response rate (22%; 95% CI 12.3-33.5% in the sabatolimab group vs. 18%; 95% CI 9.2-29.5% in the control group). Progression-free survival also did not differ between the two groups, with median progression-free survival of 18 and 19 months for sabatolimab and placebo (Zeidan AM et al. Lancet Haematol 2024; 11: e38-50).

[0025] Another immune checkpoint inhibitor, magrolimab, targets the macrophage receptor SIRPa ligand CD47. A pilot study of the combination of 5-azacytidine and magrolimab demonstrated a 33% complete remission rate and a 75% response rate in MDS (Sailman DA et al. J Clin Oncol. 2023 May 20;41(15):2815-2826). However, the randomized ENHANCE trial in MDS comparing magrolimab with placebo in combination with 5-azacytidine failed to demonstrate either an increase in the complete response rate (21.3% vs. 24.7%) or an improvement in progression-free survival (median 15 vs. 18 months), respectively (Gilead press release, as of 21.07.2023). Another randomized trial, ENHANCE-2, evaluated the addition of magrolimab in combination with 5-azacytidine and venetoclax in TP53-mutated primary AML. In this study, the addition of magrolimab also did not lead to an increase in the remission rate (7.2% vs. 15.6%) or overall survival (median 4.4 months versus 7.4 months (Gilead press release, as of September 27, 2023). A third randomized study, ENHANCE-3, evaluated the addition of magrolimab in combination with 5-azacytidine and venetoclax in patients with primary AML who were not candidates for intensive therapy. The response rate did not differ between the magrolimab and placebo groups (39.7% versus 42.9%), nor did the median overall survival (11.7 months versus 10.4 months) (Gilead press release, as of February 7, 2024).

[0026] PD-1 is an inhibitory receptor on T lymphocytes that, when interacting with its ligands PD-L1 and PD-L2, suppresses T cell activity. Nivolumab blocks PD-1, preventing immune suppression and enhancing T cell activity against tumor cells.

[0027] Nivolumab, a PD-1 inhibitor, is the most studied immunotherapy drug. Increased expression of PD-1L and CD80 ligands on tumor cells in MDS and AML motivated the study of nivolumab in these entities. A phase II study evaluated the efficacy of six cycles of nivolumab monotherapy and the CTLA4 inhibitor ipilimumab. No significant antitumor activity was demonstrated with monotherapy. After six cycles of monotherapy, patients continued with a combination of immune checkpoint inhibitors and 5-azacytidine. The overall response rate for the combination of nivolumab and 5-azacytidine was 80%, however, the study exhibited selection of patients with good tolerability and disease progression, as more than half of the patients withdrew from the study by the time combination therapy was initiated (Garcia-Manero G et al. EHA Library. 06 / 24 / 17; 181774; S487).In another randomized study, SWOG S1612, the addition of nivolumab to azacitidine did not improve the response rate (p=0.66), overall (p=0.23), or progression-free survival (p=0.86). In AML, nivolumab was studied in a group of relapsed and refractory patients. The efficacy of the combination of nivolumab and 5-azacytidine was assessed; the remission rate was 33%, including 22% complete remissions. Also, 9% of patients had stable disease (Daver N et al. Cancer Discov. 2019 Mar;9(3):370-383).

[0028] Previous studies of immune checkpoint inhibitors have demonstrated modest clinical activity for various drugs, which did not translate into long-term beneficial outcomes. One possible reason for these results is the complex expression of various immune checkpoint molecules and the combined expression of both ligands and receptors on myeloid tumor cells (Moiseev I et al. PLoS One. 2022 Oct 25;17(10):e0275399).

[0029] Inhibition of the Akt signaling pathway represents a promising approach in the development of antitumor therapy in MDS and AML. Akt, also known as protein kinase B, plays a key role in the regulation of cell survival, division, metabolism, and other important processes. It is activated by signaling through growth factor-associated receptors and is involved in intracellular signaling via the phosphoinositide 3-kinase (PI3K) / Akt signaling pathway (Hennessy BT et al. Nat Rev Drug Discov. 2005 Dec;4(12):988-1004).

[0030] Akt is activated through its phosphorylation at specific sites. Akt inhibitors can be developed to block its activation or function, thereby preventing tumor development and progression. Research in this area is focused on finding molecules that can effectively and specifically inhibit Akt to reduce side effects and improve therapeutic efficacy. The Akt signaling pathway is activated when growth factors bind to tyrosine kinase receptors on the cell surface. This interaction leads to the activation of PI3K. Activated PI3K catalyzes the conversion of phosphatidylinositol-4, 5-bisphosphate (PIP2) to phosphatidylinositol-3, 4, 5-trisphosphate (PIP3). PIP3 on the cell membrane serves as a signaling molecule that attracts and activates various proteins, including Akt (Vivanco I et al. Nat Rev Cancer. 2002 Jul;2(7):489-501).

[0031] Akt is activated through direct phosphorylation by two other kinases: PDK1 and MTORC2 [Manning BD et al. Cell. 2017;169(3): 381–405]. Akt activation initiates a cascade of subsequent phosphorylations of multiple target proteins, leading to changes in their activity and, consequently, to changes in cellular function [Porta C et al. Front Oncol.

[0032] 2014;4:64). There are three different isoforms of Akt kinase with different protein sequences and functions. In most normal and tumor tissues, all three isoforms are expressed simultaneously in varying ratios (Zinda MJ et al. Clin Cancer Res. 2001 Aug;7(8):2475-9). Functionally, the Akt1 and Akt3 isoforms are involved in the phosphorylation of more than 100 substrates in the cytoplasm, mitochondria, and nucleus and regulate homeostasis, proliferation, metabolism, and gene expression. The Akt2 isoform has different functions and is involved in the regulation of glycogenesis, gluconeogenesis, and glucose transport within the insulin-dependent transport pathway. Also, the Aktl isoform is located predominantly in the cytoplasm and intracellular membranes, while isoforms 2 and 3 are distributed in all cellular compartments (Sahiberg SH et al. Int J Oncol. 2016 Nov 18;50(l):5-14).Biological processes in which Akt signaling is important include clathrin-mediated endocytosis, endosomal activation, mitochondrial metabolism, and inhibition of the mitochondrial apoptotic pathway. In the nucleus, Akt is involved in the phosphorylation and activation of the pro-apoptotic proteins Lax and ASK-1, which transmit cytoplasmic stress signals from the kinases MAPK38 and JNK. Intranuclearly, Akt also phosphorylates IKKa, which promotes cell survival via the NF-κB signaling pathway. Akt is also involved in the phosphorylation of the intranuclear enzyme CREB, which regulates the transcription of the anti-apoptotic genes Bcl-2 and Mcl-1 (Sugiyama MG et al. Front Cell Dev Biol. 2019 May 3:7:70; Martelli AM et al. Biochim Biophys Acta. 2012 Dec;1823(12):2168-78). Thus, from a biological point of view, synergism can be expected between Akt kinase inhibitors and existing targeted therapies for OM L and MDS, including bcl-2 and Mcl-1 inhibitors.

[0033] Akt inhibitors have already been evaluated in clinical trials. The first drug is rigosertib. It acts simultaneously on several kinases, including AKT and PI3K. In a clinical trial in patients with myelodysplastic syndrome, a reduction in the number of blast cells was noted (Chun AW et al. Cancer Chemother Pharmacol. 2009 Dec;65(l): 177-86). However, in a phase III study in patients resistant to HMT, no statistically significant differences in survival were found between rigosertib and optimal adjuvant therapy (Garcia-Manero G et al. Lancet Oncol. 2016 Apr;17(4):496-508). In the phase III INSPIRE study, the efficacy of rigosertib was assessed in high-risk MDS in combination with 5-azacytidine. There was no difference in median overall survival (6.4 months vs 6.3 months, p=.33) (Onconova Therapeutics press release, as of 08 / 24 / 2020).

[0034] Another drug, MK-2206, a selective inhibitor of all Akt kinase isoforms, which was initially developed for ovarian cancer, showed pharmacological activity against acute myeloid leukemia cells in vitro and in vivo. A pilot study of MK-2206 monotherapy was conducted in a group of 19 patients with refractory AML. Remission was recorded in only one patient. The study revealed that, despite achieving maximum tolerated doses, inhibition of phosphorylation of Akt kinase targets was insignificant. An unsatisfactory ratio of maximum tolerated doses to antitumor efficacy was found (Konopleva MY et al. Clin Cancer Res. 2014 Apr 15;20(8):2226-35). None of the Akt kinase inhibitors have previously been evaluated for inhibitory activity against the expression of immune checkpoints.

[0035] From the patent US9492453B2 for the invention “NOVEL PROTEIN KINASE B INHIBITORS - 060”, MIC A61K31 / 519, published on February 7, 2015, a new group of protein kinase B inhibitors for the treatment of prostate cancer is known.

[0036] From the application EP4271678A1 for the group of inventions "SOLID STATE FORMS OF CAPIVASERTIB AND PROCESS FOR PREPARATION THEREOF" published on November 8, 2023, MIC A61K31 / 519; A61P35 / 00; C07D487 / 04, a pharmaceutical composition of a protein kinase B inhibitor for the treatment of cancer is known.

[0037] From patent US10098862B1 for the invention “FORMULATIONS WITH ENHANCED STABILITY AND BIO AVAILABILITY FOR ADMINISTANTION OF

[0038] (E)-2,6-DIALKOXYSTYRYL 4-SUBSTITUTED BENZYLSULFONES”, published on October 18, 2018, MIC A61K31 / 196, A61K47 / 10, the formula of the pharmaceutical composition of the protein kinase B and PI3K inhibitor rigosertib for the treatment of various forms of cancer is known.

[0039] From the patent US9492453B2 for the invention "NOVEL PROTEIN KINASE B INHIBITORS - 060", published on July 2, 2015, MIC A61K31 / 519, discloses a formula for a pharmaceutical composition of a protein kinase B inhibitor for the treatment of various forms of cancer.

[0040] From the application WO2017037578A3 for the invention "COMBINATION THERAPY", published on March 9, 2017, IPC A61K31 / 4155, a pharmaceutical composition and formula for a combination of the protein kinase B inhibitor shk-2206, or afuresertib, uprosertib and PI3K alpelesib for the treatment of various forms of cancer are known.

[0041] From the patent CN106822904B for the group of inventions "DRUG COMPOSITION CONTAINING ACT INHIBITOR AND IRE1 INHIBITOR AND APPLICATION OF DRUG COMPOSITION", published on June 13, 2017, IPC A61K31 / 381, A61K3 1 / 4375, A61K45 / 06, A61P35 / 00, a pharmaceutical composition and formula for a combination of protein kinase B inhibitor shk-2206, or afuresertib, uprosertib and PI3K alpelesib for the treatment of various forms of cancer are known.

[0042] From the application WO2021144657A1 for the invention "COMBINATION COMPRISING A TIM-3 INHIBITOR AND A HYPOMETHYLATING AGENT FOR USE IN TREATING MYELODYSPLASTIC SYNDROME OR CHRONIC MYELOMONOCYTIC LEUKEMIA", published on July 22, 2021, IPC A61K3 1 / 706, A61K39 / 395, A61K45 / 06, A61P35 / 02, a method for treating myelodysplastic syndrome or chronic myelomonocytic leukemia with a combination of a TIM-3 inhibitor and a hypomethylating agent is known.

[0043] From the application W02021076908A1 for the invention "COMBINATION THERAPIES FOR TREATING MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA", published on October 16, 2020, IPC A61K39 / 17, A61K39 / 395, A61K45 / 06, A61P35 / 02, a method for treating myelodysplastic syndrome and acute myeloid leukemia with a combination of an anti-CO47 drug and a hypomethylating drug is known. From the application EP3277291A1 for the group of inventions "COMPOSITIONS AND METHODS OF TREATING ACUTE MYELOID LEUKEMIA", published on July 02, 2018, IPC A61K35 / 15, A61P35 / 00, C12N5 / 12, a composition and method for treating acute myeloid leukemia using monoclonal antibodies to PD1, PDL1, TIM3 and LAG3 are known.

[0044] The disadvantages of known chemical compounds and methods for the treatment of acute myeloid leukemia and myeloid dysplastic syndromes include the lack of clinical efficacy of mono-inhibitors of immune checkpoints, the lack of immunological control and suppression of the expression of several immune checkpoint molecules on the surface of leukemic cells, and the lack of Akt inhibitors specifically designed to take into account pharmacological activity against several immune checkpoints, acute myeloid leukemia, and myeloid dysplastic syndrome.

[0045] No solutions have been identified from the prior art that are closest in terms of the set of essential features to the claimed group of inventions.

[0046] ESSENCE OF THE INVENTION

[0047] The technical problems addressed by the claimed inventions are: the creation of a new, clinically effective method for the treatment of acute myeloid leukemia and / or myelodysplastic syndromes; the creation of chemical compounds used as active substances for the developed method; and the development of schemes for the synthesis of chemical compounds for the implementation of the developed method.

[0048] The technical results achieved from the use of the developed group of inventions consist in the clinical efficacy of immune checkpoint inhibitors; in improving immunological control and suppressing the expression on the surface of leukemic cells of several immune checkpoint molecules at once, including PD-1L, TIM-3, TIGIT, CD155, CD200, VISTA, which creates the preconditions for increasing the frequency and duration of remissions and increasing the survival of patients in the treatment of acute myeloid leukemia and / or myelodysplastic syndromes;

[0049] The technical result also consists in expanding the arsenal of means used in the field of treatment of acute myeloid leukemia and myelodysplastic syndromes, by developing inhibitors of intracellular signaling pathways with the pharmacological effect of suppressing the expression on the surface of tumor cells of several immune checkpoint molecules, including PD-1L, TIM-3, TIGIT, CD155, CD200, VISTA.

[0050] A method for treating acute myeloid leukemia and myelodysplastic syndromes comprises administering to a subject a therapeutically effective amount of compounds represented by formula (I).

[0051] The compound is represented by formula (I):

[0052]

[0053] Where XY: -(N=C)-; -(C=C)-; -(C=N)-;

[0054] Where Z: > NC(O)-L; >CH-NL-CH3; >CH-C(O)-L; >CH-CH2-L;

[0055] Y1Where L is one of the following ligands:

[0056]

[0057] or their pharmaceutically acceptable salts.

[0058] The synthesis of compounds of formula (I) can be carried out according to scheme 1:Вос

[0059]

[0060] Where XY represents -N=C- Where Z represents > NC(O)-L

[0061] Where L represents one of the following radicals:

[0062]

[0063] The synthesis of compounds of formula (I) can be carried out according to scheme 2:

[0064] CI NEt3TFA

[0065] I THF, 23°C, 4h DCM, 23°C, 1h Sun

[0066] NEt3DCM, 0 to 23°C, 24h

[0067]

[0068] Where XY represents -(N=C)-Where Z represents > CH-NL-CH3

[0069] Where L is a radical:

[0070] if V CH 2

[0071] HaC^^'N6

[0072]

[0073] A method for treating myeloid leukemia and / or myelodysplastic syndrome, and / or maintaining remission of myelodysplastic syndrome, and / or primary or secondary myelofibrosis, and / or chronic myelomonocytic leukemia, and / or atypical chronic myeloid leukemia, and / or unclassifiable myeloproliferative diseases; and / or preventing relapse of myelodysplastic syndrome and / or acute myeloid leukemia comprises administering to a patient in need thereof a therapeutically effective amount of compounds of formula (I).

[0074] The inventions differ from known technical solutions in the structural form of the chemical compound, the synthesis schemes for the said compound, and the methods for treating myeloid leukemia and / or myelodysplastic syndrome by administering a therapeutically effective amount of the compound of the given formula.

[0075] The therapeutic effect in the treatment of acute myeloid leukemia and myelodysplastic syndromes is achieved by inhibiting tumor growth, or the activity of protein kinase B enzymes (Akt 1, 2, 3), or suppressing the membrane expression of checkpoint molecules PD-1L, TIM-3, TIGIT, CD155, CD200, VISTA on tumor cells.

[0076] LIST OF ILLUSTRATING MATERIALS

[0077] The claimed group of inventions is illustrated by graphic materials and tables. Figure 1 shows a diagram of the data of the proportions of KG-1 cells constitutively expressing TIM-3 and PD-L1.

[0078] Figure 2 shows a diagram of the results of the assessment of the expression of immune checkpoints on the surface of the tested cells under culture conditions in the presence of a working concentration of interferon y (IFN-y) of 50 ng / ml.

[0079] Figure 3 shows a graph of the cytotoxicity of the tested kinase inhibitor concentrations. The graph represents the percentage of viable cells in the culture. Bar plots represent 95% confidence intervals. The abscissa axis represents intracellular kinase inhibitors and their concentrations in coculture experiments.

[0080] Figure 4 shows a graph of the reduction in PD-L1 (A) and TIM-3 (B) expression levels under the influence of various inhibitors of intracellular signaling pathways. Asterisks indicate statistically significant differences compared to the control. Bar plots represent 95% confidence intervals. The abscissa shows the intracellular kinase inhibitors and their concentrations in coculture experiments.

[0081] Figure 5 shows graphical examples of the assessment of PD-L1 and TIM-3 expression under the influence of Akt inhibitors in the form of flow cytometry graphs demonstrating a decrease in PD-L1 and TIM expression in myeloid cell lines under the influence of Akt inhibitors. A. KG-1. B. THP-1. C. MonoMac-01.

[0082] Figure 6 shows the region of Aktl where additional ligand-protein contacts can be realized.

[0083] Figure 7 shows the folding of a series of priority allosteric Aktl inhibitors compared to a control (PDB id: 3096). Figure 8 shows the high-resolution mass spectrometry spectrum of PU-001.

[0084] Figure 9 shows a graph of the liquid chromatography results of the compound obtained as a result of Example D.

[0085] Figure 10 shows the high-resolution mass spectrometry spectrum of intermediate product 1 of substance PU-002.

[0086] Figure 11 shows the high-resolution mass spectrometry spectrum of intermediate product 2 of substance PU-002.

[0087] Figure 12 shows the high-resolution mass spectrometry spectrum of compound PU-002.

[0088] Figure 13 shows a graph of the liquid chromatography results of the compound obtained as a result of Example E.

[0089] Figure 14 shows a graph of the cytotoxicity of substance 14 with respect to the model cell lines according to Examples A and B upon co-cultivation at a concentration of 100 and 200 μM. The graph shows the percentage of living cells in the culture. KG = myeloid tumor cell line KG-1; THP = myeloid tumor cell line THP-1; MONO = myeloid tumor line MonoMac-01; control = experiment without adding PU-001 to the cell culture; 100 = experiment with adding 100 μM PU-001 to the cell culture; 200 = experiment with adding 200 μM PU-001 to the cell culture.

[0090] Figure 15 shows a graph of the decrease in the PD-L1 expression level under the influence of substance 14 upon co-cultivation with the cell models according to Examples A and B at a concentration of 100 and 200 μM. KG = myeloid tumor cell line KG-1; THP = myeloid tumor cell line THP-1; MONO = myeloid tumor line MonoMac-01; control = experiment without adding PU-001 to the cell culture; 100 = experiment with adding 100 μM PU-001 to the cell culture; 200 = experiment with adding 200 μM PU-001 to the cell culture.

[0091] Figure 16 shows a graph of the change in the TIM-3 expression level under the influence of PU-001 during co-cultivation with the cell models according to Examples A and B at a concentration of 100 and 200 μM. KG = myeloid tumor cell line KG-1; THP = myeloid tumor cell line THP-1; MONO = myeloid tumor line MonoMac-01; control = experiment without adding PU-001 to the cell culture; 100 = experiment with adding 100 μM PU-001 to the cell culture; 200 = experiment with adding 200 μM PU-001 to the cell culture.

[0092] Figure 17 shows the root mean square deviation (RMSD) plots for a 500 ns molecular dynamics simulation. The plot shows the evolution of protein RMSDs (left y-axis). All protein frames are first aligned to the reference structure, and then the RMSD is calculated based on the atomic selection. The corresponding protein selection (Calpha atoms, backbone, heavy atoms) are indicated by different shades. The ligand RMSD (right y-axis) indicates how stable the ligand is relative to the protein and its binding pocket. In the plot shown, the ligand RMSD is calculated when the protein-ligand complex is first aligned to the protein backbone, and then the RMSD of the ligand heavy atoms is measured. The graph shows the standard deviation of a ligand that is aligned and measured only against its reference conformation.

[0093] Figure 18 shows plots of the interaction of the AKT1 protein with PU-002 at the ATP competition site, tracked throughout a 500 ns molecular dynamics simulation, and (left) diagrams of protein-ligand interactions, divided into four types: hydrogen bonds, hydrophobic, ionic, and water bridges. The composite bar graphs are normalized over the trajectory, showing the fraction of the simulation time during which each specific interaction was maintained throughout the MD simulation. Right: Schematic representation of the detailed interactions of ligand atoms with protein residues. Interactions that occur for more than 10% of the simulation time on the selected trajectory (from 0.00 to 500.03 ns) are shown.

[0094] Figure 19 shows the interaction of the AKT1 protein with PU-001 at the ATP competition site tracked throughout a 500 ns molecular dynamics simulation and (left) diagrams of protein-ligand interactions divided into four types: hydrogen bonds, hydrophobic, ionic, and water bridges. The composite bar graphs are normalized over the trajectory, showing the fraction of the simulation time during which each specific interaction was maintained throughout the MD simulation. Right: A schematic representation of the detailed interactions of ligand atoms with protein residues. Interactions that occur for more than 10% of the simulation time on the selected trajectory (from 0.00 to 500.03 ns) are shown.

[0095] Table 1 presents a list of molecules of formula (1) with high predicted affinity for the ATP-binding site of AKT 1-3.

[0096] Table 2 presents the median viability, as well as the proportions of PD-L and TIM-3 expressing cells in the cellular models of Examples A and B with the addition of PU-002. Table 3 presents the rates of suppression of the expression of various immune control molecules in the cellular models of Examples A and B with the addition of PU-001.

[0097] Table 4 presents the affinity indices of PU-001 and PU-002 for the ATPase centers of other kinases besides Akt for PU-001 and PU-002 based on bioinformatic modeling.

[0098] Table 5 presents the probabilities of off-target binding to kinases other than Akt for PU-001 and PU-002 based on bioinformatics modeling.

[0099] INFORMATION CONFIRMING THE POSSIBILITY OF IMPLEMENTING A GROUP OF INVENTIONS

[0100] The possibility of implementing the claimed group of inventions is confirmed by the examples given below.

[0101] The applicant synthesized the claimed compound, validated the stages of synthesis and the final substance using mass spectrometry and high-performance liquid chromatography, conducted bioinformatics studies of on-target and off-target binding, analyzed molecular dynamics, conducted in vitro experiments on THP-1, KG-1, and MopoMas-01 cell cultures with an assessment of direct cytotoxicity and suppression of the expression of PD-1L, TIM-3, TIGIT, CD155, CD200, and VISTA.

[0102] Example A

[0103] Establishment of an in vitro model of a cell line with constitutive high expression of immune checkpoints using relevant cell lines of human origin KG-1 cells (male acute myeloid leukemia, erythroleukemia) were cultured in RPMI-1640 medium (Biolot, Russia) containing 10% fetal bovine serum (FBS) (bioFroxx, Germany) under conditions of 5% CO2 and 37°C. Screening of PD-L1 and TIM-3 expression levels, as well as cell viability, were assessed by flow cytometry using CD274 (PE-Cy7, BioLegend, clone 29E.2A3) and CD366 (APC, BioLegend, clone A18087E) antibodies, as well as the vital dye DRAQ7 / 7-AAD. For the analysis, 200,000 cells were used, previously washed from culture medium using Cell Wash (Becton Dickinson, USA). Samples with antibodies were incubated for 15-20 minutes in the dark at room temperature. After incubation, the cells were washed twice under the same conditions as before.Samples were analyzed using forward and side light scattering. Immediately before analysis, the sample volume was adjusted to 400 μl using Cellwash (Becton Dickinson, USA). The obtained data were analyzed using BD FACSDiva Software on a FACS Conto II instrument (BD Bioscience, USA), pre-calibrated using the commercial BD FACSDivaTM SC&T Research Beads kit. KG-1 constitutively expresses PD-L1 at a level of 88±8.49% and TIM-3 at a level of 88.4±7.14% (Figure 1). This cell line can be considered a model for studying the inhibitory activity of substances on PD-L1 and TIM-3.

[0104] Example B

[0105] Establishment of an in vitro model of a cell line with induced high expression levels of immune checkpoints using relevant human cell lines of ML origin. Based on the screening results, THP-1 and Mono-Mac-1 cells (male acute myeloid leukemia cell lines, acute monocytic leukemia) were selected to develop the model of induction of ICT expression, since the constitutive expression of the studied PD-L1 and TIM-3 in these cells was weakly represented, at a level of 0.11 = 0.11% and 0.14 ± 0.12%, respectively. The cells were cultured according to the protocol described in Example A. ICT expression was induced by cocultivating the cells with interferon gamma (IFN-y) for 24 hours before measurement. To select the working concentration of interferon, it was added at concentrations of 10-700 ng / ml. The expression levels of PD-L1 and TIM-3, as well as cell viability, were assessed by flow cytometry using the method described in Example A.

[0106] With the addition of interferon at a concentration of 50 ng / ml, the level of PD-L1 expression in THP-1 cells increased to 87.42±20.04% (Figure 2). The effect was maintained when using an IFN-y concentration of 100 ng / ml. For Mono-Mac-1 under similar conditions, the proportion of PD-L1 and TIM-3 expressing cells was 97.30±1.13% and 53.26±12.20%, respectively, after stimulation (p<0.01, Figure 2). Thus, the model was established.

[0107] Example B

[0108] In vitro pharmacodynamic model of PD-L1 and TIM-3 expression inhibition with known intracellular kinase inhibitors. Testing was conducted using the in vitro models described in Examples A and B. Two hundred thousand cells with the required volume of inhibitor in RPMI-1640 medium were added to each well of the plate. Control wells were prepared with the same number of cells and medium without the addition of inhibitors, as well as with the same concentration of DMSO. Cells were co-cultured with inhibitors for 24 hours at 37°C in 5% CO2 prior to cytofluorimetric analysis.

[0109] The following small molecule kinase inhibitors (Sigma Aldrich, USA) were tested: MK-2206 at a concentration of 5-30 μM, inhibitor X (CAS 925681-41-0) at a concentration of 10 and 100 μM, inhibitor XIV (CAS 1191951-57-1) at a concentration of 10 μM, AKT-specific inhibitor XVIII (SC66) at a concentration of 1 μM; P13K-specific inhibitor copanlisi (10 μM and 100 μM), RKIP-specific sorafenib (10 μM), mTOR-specific rapamycin (10 μM), ERK / MAPK-specific PD98,059 at a concentration of 5 μM. AKT-specific MK-2206 was used at concentrations of 10, 20, and 30 μM. The inhibitor concentration was selected based on the expected LD50 value for cytotoxicity for the test cell lines. Cytotoxicity values ​​were greater than 50% for Mono-Mac-1 cells when cocultured with sorafenib, inhibitor X, and inhibitor XIV. If the expected cytotoxicity for the test cell lines was absent, the concentration was increased to 100 μM.The expression levels of PD-L1 and TIM-3, as well as cell viability reflecting cytotoxicity, were assessed by flow cytometry using the method described in Example A. The data are presented in Figure 3.

[0110] A significant decrease in the level of PD-L1 expression in THP-1 cells was observed after co-cultivation with MK-2206 at a concentration of 30 μM (12.5±12.1%, p=0.002) and inhibitor XVIII at a concentration of 1 μM (6.5±3.5%, p<0.001) versus 87.4±20.0 in the control group.

[0111] In the MopoMac-01 cell line, a significant decrease in PD-L1 levels was observed after exposure to 10 μM MK-2206 (92.9±1.7%, p=0.02), 20 μM MK-2206 (95.5±1.0%, p=0.02), 30 μM MK -2206 (12.9±8.0%, p=0.006), 1 μM inhibitor XVIII (4.1±5.2%, p=0.001), 10 μM inhibitor XIV (58.7±12.1%, p=0.04), 5 and 10 μM inhibitor XIV (79.8±7.5%, p=0.02 and 3.0±1.4%, p=0.02) and 10 μM sorafenib (0.4±0.4%, p=0.02) compared to 97.3±1.1% in the control. Minimal decrease was observed with 10 μM copanlisib (95.0±1.7, p=0.04), 10 μM PD98,059 (94.5±0.1, p=0.02). No differences in expression were observed after exposure to rapamycin (p=0.9). A number of inhibitors demonstrated a decrease in TIM-3 expression in MonoMac-01 cell lines: 10 μM MK-2206 (39.9±3.0%, p=0.02), 20 μM MK-2206 (13.7±0.6%, p=0.02).), 30 μM MK-2206 (3.6±3.4%, p=0.006), 1 μM inhibitor XVIII (1.6±1.6%, p=0.001), 10 μM inhibitor XIV (7.8±3.2%, p=0.04), 10 μM inhibitor X (4.1±1.5%, p=0.04), 10 μM copanlisib (4.1±1.5, p=0.02), 10 μM PD98,059 (38.2±2.5, p=0.02), 10 μM rapamycin (28.4±1.2, p=0.02) and 10 μM sorafenib (2.0±0.5%, p=0.02) compared with 53.3±12.2% in the control (Figure 4). Dunn's post hoc test on the extracted experiment data from three cell lines confirmed a decrease in PD-L1 expression only for 30 μM MK-2206 (p=0.03) and the AKT XVIII inhibitor (p=0.03) (Figure 7X). Reduced TIM3 expression in the post hoc test was confirmed only for the AKT XVIII inhibitor (p=0.03) (Figure 5).

[0112] Thus, the observation of selective suppression of PD-L1 and TIM-3 expression under the influence of Akt inhibitors, but not other groups of drugs in an in vitro model was confirmed.

[0113] Example G

[0114] Bioinformatic modeling of new Akt kinase inhibitors. Computer modeling of binding to selected Akt kinases, including docking and molecular linkage construction, target preparation for subsequent virtual screening, computer modeling of binding, library preparation and ADMET analysis, design of lead libraries of chemical compounds, and confirmation or correction of pharmacophore hypotheses. The following software packages and modules were used: LigPrep, AlphaFold, RCSB Protein Data Bank, Schrodinger 2021, and Glide Extra Precision.

[0115] The possibility of enhancing lipophilic contacts within the amino acids Ile84, Val83 (lipophilic region combining the possibility of forming an alkyl cationic bridge with Arg273), as well as a number of polar amino acids in the form of Tyr272 and Thr82, was considered. Based on these properties, substituents with the most suitable characteristics were selected: charge complementarity, the presence of a mono- or bicyclic aromatic part, and the presence of halogens in the structure (Figure 6).

[0116] The resulting compound library was docked taking into account the steric properties of the control ligands. Ultimately, a list of the most optimal compounds was compiled. Additional filters at this stage included ligand binding efficiency characteristics and calculated ADMET parameters. When forming the finalized set, the LogP value was the primary selection parameter. o / w- The range of values ​​from 1.34 to 3.00 was considered as the most characteristic for drug-like compounds (Figure 7).

[0117] The reproducibility of binding at the allosteric site was also tested by repeated docking of active Aktl-3 inhibitors to the allosteric site. Binding selectivity was assessed, the range of acceptable values ​​for the scoring function was established, and the reproducibility of the pharmacophoric characteristics of the control structures was assessed. A list of compounds within formula (1) of the invention with high folding stability indices was compiled: Example D. Chemical synthesis of PU-001 from the list of compounds of formula (I)

[0118] Compound formula PU-001:

[0119] \ O

[0120] O

[0121]

[0122] UQ

[0123] Chemical synthesis of compound PU-001 according to scheme 1:

[0124] Step 1

[0125] Vg

[0126] _ _. 1 Q г

[0127] < "xs Dioxane dibromide Z '\ / X v ,u

[0128] ( I 1 - G T

[0129] rt, 1.5 h

[0130]

[0131] 1 2

[0132] To 30 g (0.205 mol) of coumarin 1 ground into powder, 70 g of dioxane dibromide were added and the mixture was thoroughly mixed until 1 was completely dissolved.

[0133] The mixture was kept at room temperature for 1.5 hours, after which ice was added. A precipitate formed. The precipitate was filtered, crushed, washed with a NaCO3 solution, and then twice with water. Product 2 was purified by recrystallization from ethyl acetate with the gradual addition of hexane (Ethyl acetate:Texane = 1:1.5 by volume). The substance was light yellow. The yield of product 2 after recrystallization was 38.47 g (61.2%).

[0134] Step 2Bg

[0135] 1) KOH in MeOH

[0136] 2) HCI

[0137]

[0138] 42.3 g (0.754 mol) of KOH were dissolved in 500 ml of dry methanol. 38.47 g (0.126 mol) of 3,4-dibromochroman-2-one 2 were added portionwise to the solution with stirring and the mixture was boiled for 3 hours. The solvent was distilled off to form a white precipitate. The precipitate was dissolved in 200 ml of water, and concentrated HCl was added until pH = 1. A white cottony precipitate of 2-benzofurancarboxylic acid 3 was observed. The precipitate was filtered off and dried. White crystalline substance. Yield 20.17 g (99%).

[0139] Step 3

[0140] SOC12

[0141] Reflux, 16 h

[0142]

[0143] 20.17 g (0.124 mol) of 2-benzofurancarboxylic acid 3 was boiled in 160 ml of SOCl2 for 16 h. SOCl2 was distilled off to form a white crystalline substance with a strong fruity odor. Yield 22.24 g (99%).

[0144] Step 4

[0145] TEA THF, 0°C to rt, 2 h

[0146]

[0147] To a solution of N-Boc-piperazine (22.94 g, 0.124 mol) and triethylamine (14.96 g, 20.60 mL, 0.148 mol) in 100 mL of tetrahydrofuran cooled to 0°C was added dropwise a solution of 2-benzofurancarboxylic acid chloride 4 (22.24 g, 0.124 mol) in tetrahydrofuran (100 mL). After complete addition of solution 4, the reaction mixture was warmed to room temperature and stirred for 2 h. The solvent was distilled off to form a light yellow crystalline product 5 (may be an oil, but spontaneously crystallizes over time). Yield 27.75 g (67.7%)

[0148] Step 5

[0149] TFA

[0150] DCM, rt, 1 h

[0151]

[0152] 27.75 g (0.084 mol) of tert-butyl 4-(benzofuran-2-carbonyl)piperazine-1-carboxylate 5 was dissolved in a mixture of 100 ml of dichloromethane and 50 ml of trifluoroacetic acid. The solution was stirred for 1 hour at room temperature. The solvent and residual trifluoroacetic acid were removed under vacuum to form a dark yellow solid. The substance was dissolved in 100 ml of dichloromethane and washed thoroughly with 100 ml of concentrated baking soda solution. The organic layer was separated and washed twice with 50 ml of water. The solvent was evaporated to form an oily dark yellow solid 6. Yield 19.30 g (99%)

[0153] Step 6

[0154] TEA DCM, rt, 24 h

[0155]

[0156] To a solution of 19.3 g (0.084 mol) of benzofuran-2-yl(piperazin-1-yl)methanone 6 and 12.74 g (17.55 ml, 0.126 mol) of triethylamine in dichloromethane (300 ml) was added 6-chloropurine (12.96 g, 0.084 mol). The solution was stirred for 24 h. After completion of the reaction, the solvent was distilled off, 50 ml of cold dichloromethane were added to the solid residue, the precipitate was filtered off, washed with 50 ml of cold dichloromethane and 2 times with 70 ml of water and dried. White solid. Yield 9.472 g (32.4%).

[0157] 'H NMR(CDC13, 400 MHz) 5, ppm: 13.12 s (1H), 8.28 s (1H), 8.18 s (1H), 7.76 d (1H, J=8.0 Hz), 7.69 d (1H, J=8.0 Hz), 7.47 s (1H), 7.46 t (1H, J=7.3 Hz), 7.34 t (1H, J=7.3 Hz), 4.36 s (4H), 3.90 s (4H).

[0158] 13 C NMR (CDC13, 101 MHz) 5, ppm: 159.6, 154.5, 153.5, 152.2, 152.0, 148.6, 138.9, 127.2, 127.0, 124.2, 122.9, 119.4, 112.3, 111.6, 45.1, 45.0.

[0159] HRMS, m / z: found 349.1409 [M+H] + , CI8HI7N6O2 + ; calc. 349.1408 [M+H] +(Figure 8). The purity of the obtained compound by high-affinity liquid chromatography is 98.816%, the retention time of the PU-001 peak is 16.459 min (Figure 9).

[0160] Additional information for synthesis:

[0161] Preparation of dioxane dibromide

[0162] Bromine was added dropwise to dioxane at 20°C with stirring until a bright orange solid formed. The reaction was complete when the entire reaction mixture had become a bright orange paste. A significant increase in the reaction temperature was observed upon the addition of bromine; the temperature was maintained at 20°C by cooling with running water. The prepared dioxane dibromide was stored in the refrigerator; at room temperature, it releases bromine vapor and becomes liquid. It is highly corrosive and must be handled entirely in a glass container.

[0163] Preparation of N-Boc-Piperazine

[0164] Vos2O

[0165]

[0166] DCM, 0°C, 1 h

[0167] Piperazine (14.45 g, 0.168 mol) was dissolved in 150 ml of dichloromethane, and the solution was cooled to 0°C. BoсrO (14.65 g, 0.067 mol) was added dropwise over 1 h, and the reaction mixture was stirred for 1 h at 0°C. The solvent was distilled off, the residue was dissolved in water (100 ml), and the precipitate was filtered off. The filtrate was extracted with diethyl ether (3 times 100 ml), the organic phase was separated, dried over sodium sulfate, and evaporated to form a colorless transparent oil. The oil solidified over time to form a white crystalline product. Yield 23.4 g (93.6%).

[0168] Example E. Chemical synthesis and formula of substance PU-002 from the list of compounds of formula (I)

[0169] Compound formula:

[0170]

[0171] Scheme of synthesis of substance PU-002 in accordance with scheme 2:

[0172] Step 14N HCI C1CH2COOH reflux, 2 h

[0173]

[0174] 1.22 g (0.010 mol) of 4-methyl-o-phenylenediamine 1 and 1.42 g (0.015 mol) of chloroacetic acid were boiled in 10 ml of 4N HCl for 2 h. The precipitated oligomeric impurities were filtered off, the filtrate was diluted with 20 ml of water and, while cooling and vigorously stirring, neutralized with NaCl. The precipitated 2-(chloromethyl)-6-methylbenzimidazole 2 was washed with cold water and dried. A white solid substance turns red in air. Yield 0.91 g (50.5%).

[0175] HRMS, m / z: found 181.0531 [M+N] + , C9HIOC1N2 + ; calc. 181.0527 [M+H] + (figure Yu).

[0176] Step 2

[0177] MeNH2THF, 0°C to rt, 3 h

[0178]

[0179] To a solution of methylamine (0.33 g, 0.010 mol) in 10 ml of THF, cooled to 0°C, 2-(chloromethyl)-6-methylbenzimidazole 2 (0.91 g, 0.005 mol) was added portionwise. The reaction mixture was warmed to room temperature and stirred for 3 hours. The solvent was distilled off, the precipitate was washed with dry THF and filtered. The filtrate was evaporated, dissolved in dichloromethane (20 ml), washed with a saturated solution of soda (10 ml) and water (10 ml). The organic layer was separated and evaporated to form a yellow oily precipitate. The reaction product 3 was purified by column chromatography in the system ethyl acetate: methanol = 2:1. Light yellow oil. Yield 0.3 g (34.2%). HRMS, m / z: found 176.1187 [M+H] + , CIOHI4N3 + ; calc. 176.1182 [M+H] + (Figure 11).

[0180] During the reaction, the main product formed is the bis-addition product 2 to methylamine:

[0181] HRMS, m / z: found 320.1874 [M+N] + , CI9H22N5 + ; calc. 320.1870 [M+H] +

[0182] Step 3

[0183]

[0184] To a solution of N-methyl-1-(6-methylbenzimidazol-2-yl)methanamine 3 (0.3 g, 1.71 mmol) and triethylamine (0.26 g, 0.36 mL, 2.56 mmol) in 10 mL of tetrahydrofuran cooled to 0°C, a solution of N-Boc-4-chloropiperidine 4 (0.376 g, 1.71 mmol) in tetrahydrofuran (10 mL) was added dropwise. After complete addition of solution 4, the reaction mixture was warmed to room temperature and stirred for 5 h. The solvent was distilled off to form a light yellow oil 5. Yield 0.489 g (79.6%). Step 4

[0185] TFA DCM, rt, 1 h

[0186]

[0187] 0.489 g (1.36 mmol) of tert-butyl 4-(methyl((6-methylbenzimidazol-2-yl)methyl)amino)piperidine-1-carboxylate 5 was dissolved in a mixture of 12 ml of dichloromethane and 1 ml of trifluoroacetic acid. The solution was stirred for 1 hour at room temperature. The solvent and residual trifluoroacetic acid were removed under vacuum to form a dark yellow oil. The substance was dissolved in 10 ml of dichloromethane and thoroughly washed with 10 ml of concentrated baking soda solution. The organic layer was separated and washed twice with 10 ml of water. The solvent was evaporated to form a dark yellow oil. Yield 0.1 g (28.4%)

[0188] Step 5

[0189] ci

[0190]

[0191] To a solution of 0.100 g (0.387 mmol) of 1\N-methyl-1\N-(methyl((6-methylbenzimidazol-2-yl)methyl)piperidin-4-amine 6 and 0.060 g (0.081 ml, 0.580 mmol) of triethylamine in dichloromethane (10 ml) was added 6-chloropurine 7 (0.066 g, 0.426 mmol). The solution was stirred for 24 h. After completion of the reaction, the solvent was distilled off, 5 ml of cold dichloromethane were added to the solid residue, the precipitate was filtered off, washed with 5 ml of cold dichloromethane and 2 times with 10 ml of water and dried. White solid. Yield 0.003 g (2%). HRMS, m / z: found xxx [M+H]+, C20H25N8+; calc. 377.2197 [M+H]+ (Figure 12). The purity of the obtained compound by high-affinity liquid chromatography is 97.575%, the retention time of peak PU-002 is 11.700 min (Figure 13).

[0192] Example G

[0193] Evaluation of the Pharmacological Activity of PU-001 in an In Vitro Model The pharmacological activity of PU-001 in inhibiting PD-L1 and TIM-3 was evaluated in the established in vitro model as described in Examples A and B. PU-001, synthesized using the method described in Example D, was added to KG-1, Mono-Mac-1, and THP-1 cells at a concentration of 100 and 200 μM. Co-cultivation was carried out for 24 hours, after which the cells were washed and the expression levels of PD-L1 and TIM-3, as well as cell viability, were assessed by flow cytofluorometry according to the protocol described in Example A. The results of viability measurements are presented in Figure 14, and the expression levels of PD-L1 and TIM-3 are shown in Figures 15 and 16, respectively.

[0194] In control cell samples in the absence of PU-001, the median viability value was 88.0% (from 87.9% to 88.3%) for KG-1, 85.0% (from 82.7% to 85.4%) for THP-1, and 91.2% for Mono-Mas-1 (from 86.5% to 92.3%). The addition of PU-001 at a concentration of 100 μM was associated with a decrease in cell viability to 68.1% for KG-1 (from 63.9% to 72.1%), 79.0% for THP-1 (from 76.7% to 80.6%) and 85.1% (from 78.8% to 87.8%) for Mono-Mac-1. When cocultivating cells with PU-001 at a concentration of 200 μM, the viability was 46.5% (from 45.8% to 53.7%) for KG-1, 62.6% (from 55.4% to 71.1%) for THP-1 and 55.7% (from 48.4% to 61.7%) for Mono-Mac-1. The decrease in viability was statistically significant (p=0.05) when compared with the control sample for all cell lines except Mono-Mac-1 with a 14 inhibitor concentration of 100 μM (p=0.127).PD-L1 expression in control samples was 99.5% (from 99.5% to 99.5%) for KG-1, 98.5% (from 97.7% to 98.8%) for THP1, and 95.8±0.6% (from 94.8% to 95.9%) for Mono-Mac-1. The addition of PU-001 at a concentration of 100 μM was associated with a decrease in the proportion of PD-L1-expressing cells to 94.2% (from 93.8% to 94.3%) for KG-1, 68.7% (from 67.0% to 72.8%) for THP-1, and 87.0% (from 86.9% to 88.9%) for Mono-Mac-1. When co-cultivating cells with PU-001 at a concentration of At 200 μM, the proportion of PD-L1-expressing cells changed to 89% (from 88.9% to 92.2%) for KG-1, 69.8% (from 54.2% to 77.9%) for THP-1, and 5.9% (from 5.0% to 7.4%) for Mono-Mac-1. The decrease in the proportion of PD-L1-expressing cells was statistically significant (p=0.037 for KG-1 cells, p=0.05 for THP-1 and Mono-Mac-1 cells).

[0195] TIM-3 expression in control samples was 55.0% (from 54.2% to 56.4%) for KG-1, 3.6% (from 3.5% to 4.0%) for THP-1, and 42.7% (from 36.9% to 45.1%) for Mono-Mac-1. Addition of PU-001 at a concentration of 100 μM was associated with a change in the proportion of TIM-3-expressing cells to 43.1% for KG-1 (from 42.1% to 46.2%), 3.9% (from 3.2% to 4.34%) for THP-1, and 15.7% (from 14.7% to 16.5%) for Mono-Mac-1. When co-cultivating cells with PU-001 at a concentration of 200 μM, the proportion The percentage of TIM-3-expressing cells decreased to 37.9% (from 33.8% to 38.9%) for KG-1, 2.4% (from 1.2% to 3.2%) for THP-1, and 2.0% (from 2.0% to 2.1%) for Mono-Mac-1. The change was statistically significant (p<0.05) for all cell lines and both concentrations of PU-001, except for the experiment with culturing THP-1 cells in the presence of 100 μM PU-001 (Figure 16).

[0196] Example 3

[0197] Evaluation of the pharmacological activity of PU-002 in an in vitro modelThe pharmacological activity of PU-002 in relation to the inhibition of PD-L1 and TIM-3 was evaluated in the established in vitro model according to Examples A and B. PU-002, synthesized according to the method described in Example E, was added to KG-1, Mono-Mac-1 and THP-1 cells at concentrations of 50 μM (for KG-1, THP-1 cells), 75 μM (for KG-1 cells) and 100 μM (for KG-1 and Mono-Mac-1 cells). Co-cultivation was carried out for 24 hours, after which the cells were washed and the expression levels of PD-L1 and TIM-3, as well as cell viability, were assessed by flow cytometry according to the protocol described in Example A. The results of viability measurements as medians are presented in Table 2.The table shows that the addition of PU-002 resulted not only in a decrease in cell viability (from 88.1% to 36.1-85.9% for KG-1, from 98.9% to 57.7% for THP-1, and from 88.1% to 1.7% for Mono-Mac-1), but also in the expression of PD-L1 (from 98.4% to 76.3-93.1% for KG-1, from 97.4% to 91.3% for THP-1, and from 98.9% to 92.8% for Mono-Mac-1) and TIM-3 (from 84.5% to 79-80.3% for KG-1, from 20.8% to 8.2% for THP-1, and from 66.4% to 6.9% for Mono-Mac-1).

[0198] Example I

[0199] Evaluation of the pharmacological activity of PU-001 in an in vitro model against a wide range of immune checkpoints. The pharmacological activity of PU-001 in inhibiting a wide range of immune checkpoints was evaluated in the established in vitro model according to Examples A and B on the KG-1, THP-1, and Mono-Mac-1 cell lines. PU-001, synthesized according to the method described in Example G, was added to KG-1 and THP-1 cells at a concentration of 200 μM. Co-cultivation was carried out for 24 hours, after which the cells were washed and the expression levels of CD155, CD47, TIGIT, TIM-3, CD274, CD80, Gal9, CD200, CD223, CD272, CD11b, CD196, CD44, Vista, as well as cell viability were assessed by flow cytometry according to the protocol described in Example A. The screening results are presented in Table 3.

[0200] Inhibition of PD-L1 and TIM-3 was confirmed for KG-1, THP-1, and Mono-Mac-1 cells. A decrease in the expression of the TIM-3 ligand Gal9 was recorded for the tested model lines THP-1 from 2.0% to 0.5% and Mono-Mac-1 from 10.5% to 0.1%, but not for KG-1. TIGIT decreased for all cell lines: from 30.4% to 1.1% for THP-1, from 57.8% to 0.9% for KG-1, from 76.7% to 7.0% for Mono-Mac-1. The pharmacological activity of PU-001 in the experiment with the KG-1 line was expressed as a decrease in the proportion of CD200-expressing cells from 53.8% to 3.4% of cells. The same effect was observed for THP-1 (a decrease from 14.5% to 2.0%) and for Mono-Mac-1 (from 5.8% to 1.8%). A decrease in expression under the influence of PU-001 was also observed for the VISTA marker: in Mono-Mac-1 from 71.5% to 48.3%, in THP-1 from 17.3% to 6.0%, and less significantly in KG-1 (from 2.8 to 1.7%).Cocultivation with PU-001 resulted in a slight increase in CD 196 marker expression on all cell lines and a more pronounced increase in CD223 on KG-1 cells (from 1.6 to 24.3%) and Mono-Mac-1 (from 1.1 to 7.9%). For the remaining tested markers, the effect was cell line-specific. In particular, the proportion of CD155-expressing cells decreased under the influence of PU-001 in THP-1 cells (from 99.7% to 27.7%) and Mono-Mac-1 (from 86.6% to 26.1%), but not in KG-1 cells. A significant decrease in CD47 expression was observed in Mono-Mac-1 cells from 98.7 to 46.3%. Significant inhibition of the CD11b marker was observed upon co-cultivation with PU-001 in THP-1 cells (decrease to 1.1% compared to 43.2% in the control).A sharp increase in the proportion of CD80-expressing cells was observed for the CD80 marker upon co-cultivation of the KG-1 line with PU-001 (98.1% in the experiment compared to 0.3% of positive cells in the control), but the effect was opposite in Mono-Mac-1 cells (7.7% in the experiment compared to 23.3% in the control sample). In THP-1 cells, upon co-cultivation with PU-001, an increase in the proportion of CD272- and CD44-expressing cells by 29.6% and 26.2%, respectively, was observed, but in Mono-Mac-1 cells a decrease in the expression of these markers was recorded from 92% to 69% for CD272 and from 98.4% to 88.2% for CD44. In KG-1 cells, CD272 was reduced (10.8% in the experiment compared with 4% positive cells in the control), and CD44 expression did not change with the addition of PU-001 (Table 3).

[0201] Example I

[0202] Molecular dynamics results of PU-001 and PU-002 in the presence of somatic mutations of Aktl

[0203] The molecular interactions of selected PU-001 and PU-002 with the AKT protein were analyzed. Two binding sites are known where AKT inhibition can be mediated through protein conformational changes: the ATP-competitive site and allosteric inhibition. The resulting hits were designed to inhibit AKT1 in the ATP-competitive mode. The sequences of AKTs 1, 2, and 3 were also obtained from the UniProt Knowledge database. All steps of protein modeling, docking, molecular dynamics (MD), and analytical calculations were performed using the Schrodinger molecular modeling package (version 2021-1) (Schrodinger, LLC, New York, NY, 2021).

[0204] AKT 1, 2, and 3 homology modeling and preparation. Model quality was checked and preprocessed in the Protein Preparation Wizard (PPW). Identified issues with invalid atom types (missing hydrogens, incorrect bond numbers), alternative positions, steric clashes, and other deviations were refined in PPW. Bond orders were assigned, and hydrogens were added after removing the original hydrogens. Missing loops and side chains were checked with Prime, and possible protonation states were generated using Epik at default pH 7.0 ± 2.0. No issues were reported in the preprocessed protein structures.In addition, the quality of the complexes was analyzed in the protein reliability report; the original protein-ligand complexes were refined in Prime (only some steric clashes remained, since no additional MD relaxation was performed to avoid changing the binding site conformation with the attached ligand before the docking study for proper mesh generation).

[0205] Ligand Preparation. The ligand structures were subjected to ligand preparation (LigPrep) prior to docking. LigPrep is a robust toolkit designed for preparing high-quality all-atom 3D structures of small molecules. This tool is also capable of generating multiple structures from an input structure with different ionization states, tautomers, stereochemistry, and ring conformations, as well as removing molecules using specified criteria. Ligands were prepared by geometry optimization using the OPLS4 force field, and possible ionization states at pH 7.0 ± 2.0 were also generated using Epik. Finally, salt removal, tautomer generation, and stereoisomer calculation were also selected for ligand preparation to preserve certain chiralities (changes in other chiral centers) and generate no more than 32 conformations per ligand.

[0206] Induced-Fit Docking. Schrodinger's induced-fit docking protocol (IFD) was used to dock ligands to AKT proteins. This protocol, based on docking in Glide and refinement of the resulting complex in Prime, allows for accurate prediction of ligand binding modes and associated structural changes in the receptor. A receptor molecule with a cocrystallized ligand was selected to create the residue centroid, and the cell size was automatically generated to accommodate ligands no longer than 20 Å. Initial docking was performed without receptor constraints, sampling ring conformations of ligands in the energy range of 2.5 kcal / mol and penalizing non-planar conformations for each ligand. The van der Waals interaction scale for both the receptor and the ligand was set to 0.50. The number of generated poses was set to 20.Protein refinement and minimization were then performed in Prime, with residues within 5.0 Å of the ligand position refined and side chains optimized, resulting in a ligand structure and conformation induced to match the receptor structure. Finally, re-docking was performed using Glide XP for structures within 30.0 kcal / mol of the best structure and for the 20 best structures overall. The ligand was carefully fitted into the receptor model, resulting in an IFD score for each resulting position. To refine the docking results, ligand binding energies were calculated for all resulting complexes using the MM / GBSA physical method.The free energy of MM / GBSA binding (AG bind) is calculated using the equation: AAGbind = EComplex - ELigand - EReceptor, where EComplex, ELigand, and EReceptor are the energy calculations performed for the simple optimized MM / GBSA complex (complex), the optimized free ligand (ligand), and the optimized free receptor. The calculations were performed using the OPLS4 force field and the VSGB solvation model. Analysis of AKT binding site sequences. To determine the amino acid sequences comprising the binding sites, the original AKT1 model was used. Regions with radii of 5 and 7 Å from the cocrystallized ligand were selected for this purpose.The amino acid residues constituting the binding sites within 7 Aβ were aligned for each AKT protein 1, 2, and 3 using the resulting multiple alignment in MAFFT for representative species with the most fully represented genomes to track the conservation and variability of binding site sequences. Next, potential human missense polymorphisms with a frequency greater than 0.001% in AKT binding sites were analyzed using the GnomAD database. Additionally, potential mutations occurring at amino acid positions of binding sites within 5 and 7 Aβ of the ligands were analyzed using the OpsoKB database, a curated database of gene mutations and their associated consequences. Variants with definite functional consequences or inconclusive effects were included for analysis if these somatic mutations could affect ligand binding.Specific point mutations were introduced into the structure using the 3D Builder panel in Maestro; side-chain rotamers were refined. The local structure within 10 aa around the inserted mutation was minimized. The quality of the mutated models was checked in PPW as described previously (Section 2), and the resulting mutated AKT structures were subjected to molecular dynamics (MD) simulations for 500 ns.

[0207] Molecular dynamics simulations. The simulations were performed using the Desmond package. The molecular dynamics system was set up in the System Builder utility in Maestro as follows: the TIP3P water model was used to simulate water molecules; the buffer distance in the orthorhombic box was set to 10 Å; a rescaled number of Na+ / Cl- ions were added to equilibrate the charge of the system and randomly placed to neutralize the solvated system; additional salt was added to a final concentration of 0.15 M to simulate physiological conditions. The molecular dynamics simulations were performed under periodic boundary conditions in the NPT ensemble using the OPLS4 force field parameters. Temperature and pressure were maintained at 300 K and 1 atmospheric pressure, respectively, using the Nose-Hoover temperature coupling and isotropic scaling.The model system was relaxed before simulations using the standard Maestro relaxation protocol, including two minimization steps (constrained and unconstrained) followed by four MD runs with gradually decreasing constraints. MD simulations were performed by running 500 ns records and recording trajectory configurations at 50 ps intervals. RMSD clustering was performed in the Desmond package; the total energy of the system was calculated using Prime. Molecular dynamics simulation analysis. The molecular dynamics trajectory files were examined using the simulation quality analysis and simulation interaction diagram programs available in the Desmond module: SID was used to generate protein and ligand root mean square fluctuations and standard deviations, ligand interaction fingerprints, and protein interaction fractions.The physics-based MM / GBSA method in Prime was used to calculate ligand binding energies throughout the MD simulations, running the thermal mmgbsa.py script to calculate AG binding energies with a step size of 20 trajectories. PU-001 and PU-002 were prepared for further docking studies with AKT proteins using the induced fitting method. Preparation included converting the 2D structure to an energy-minimized 3D structure and generating all possible ionization and tautomeric states using Schrodinger LigPrep tools.

[0208] As a result of docking, the best ligand positions were obtained with the GScore values ​​of -9.2 and -9.9 kcal / mol for PU-002 and PU-001, respectively, and the binding energy of ligands in the selected complexes was -57.4 and -40.15 kcal / mol for PU-002 and PU-001, respectively.

[0209] The OpsoKB database was used to search for deleterious somatic variants that may arise at the ATP-competing site in oncogenic malignancies and, therefore, potentially affect the binding of novel AKT inhibitors. Amino acid composition analysis was performed within the 5 and 7 A radius of the ligand, and two mutations with corresponding functional annotations were detected: Y176F ("likely neutral", "likely loss of function") within the 7 A binding radius and K179M ("uncertain") within the 7 A binding radius of AKT1. No other variants or polymorphisms were detected in the AKT1 binding sites. Furthermore, sequence analysis of the binding sites of representative species revealed no differences between humans and rodents, indicating their overall conservation during evolution.

[0210] Selected somatic mutations Y176F and K179M were introduced into the AKT1 model to evaluate their potential impact on ligand binding. Thus, six complexes with both ligands PU-002 and PU-001 in different AKT1 variants (wild type (WT), Y176F, and K179M) were subjected to molecular dynamics (MD) simulations for 500 ns. MD simulates the dynamic behavior of a molecular system under computer-generated physiological conditions to evaluate the stability of a protein-ligand complex over time and the binding affinity. The stability of the protein-ligand complex is assessed using the root mean square deviation (RMSD) plot, which measures the deviation of the positions of the protein and ligand atoms inside the binding pocket at the end of the simulation period (500 ns) compared to their initial positions before the simulation at 0 ns.RMSD analysis demonstrated the stability of both ligands PU-002 and PU-001 within the complexes within the acceptable range of 1-3 Å; the observed RMSD values ​​of the ligands did not exceed the RMSD of the protein, indicating that the ligand did not diffuse from its original binding site and demonstrates stable behavior throughout the simulation period (Figure 1). Moreover, analysis of the mutated proteins showed no difference in the stability of the protein and ligands during MD, indicating the absence of an effect of the introduced mutations in the AKT1 binding site (Figure 17). The key interatomic interactions and binding dynamics of PU-002 and PU-001 to the binding site during 500 ns of molecular dynamics are shown in Figures 18 and 19, respectively.

[0211] Example Y. Modeling the Off-Target Activity of PU-001 and PU-002 To predict potential off-target binding, we analyzed the binding site composition of other AKT-like kinases and their affinity for PU-002 and PU-001. AKT proteins belong to the AGC group, which consists of 62 different kinases based on a structural multiple sequence alignment (MSA) of 427 kinases [10.1038 / s41598-019-56499-4]. Different amino acid compositions of the binding sites were obtained for all 62 kinases. Based on molecular dynamics simulations of the ATK-PU-002 and AKT-Figl4 complexes, the residue positions most critical for ligand binding (positions 158, 179, 228, 230, 234, 278, and 292) were identified, resulting in 29 unique amino acid combinations within the binding site for the AGC protein family. Each resulting combination corresponds to a specific kinase pattern within the binding site.The parent ligand-AKT 1 structure was then mutated with 29 amino acid combinations to assess the potential impact of the introduced mutations (i.e., different binding site composition) by calculating the Aaffinity and Astability compared to the parent AKT complex (Table 2). The residue scanning results indicated that potential off-target binding for both PU-002 and PU-001 could be with cGMP-dependent protein kinase 1,2 (PRKG1, PRKG2) and ribosomal protein kinase S6-related protein (RSKR). Potential binding to more kinases was found for PU-001 (Table 4).

[0212] Additional analysis of potential off-target interactions was performed using the PASS Targets server. Summarizing the results, the predominant class of potential targets for both ligands were kinases (184 and 192 for PU-002 and PU-001, respectively). The dominant biological processes associated with the predicted targets were signaling and stress response, respectively, due to potential interactions with both ligands.

[0213] An additional analysis of off-target activity was performed using an alignment of 497 human kinases, and, along with structural data, all kinases were classified into families. We took the AGC family (to which AKT1 belongs) and examined all amino acid substitutions and combinations, compared to AKT1, in the binding sites of similar kinases in this family. In other words, we identified the amino acid combinations in the binding sites of these kinases. We then performed a residue scanning (mutating the AKT1 binding site to these combinations) and assessed how stability and affinity changed with these substitutions. Each combination corresponds to the sites of specific kinases; a delta stability / delta affinity of greater than -3 kcal / mol can be considered a significant increase in the stability / affinity of the complex.

[0214] Residue scanning demonstrated that PU-002 has significantly fewer potential targets than PU-001. Combinations of active site substitutions that potentially bind well to PU-002 were translated into the corresponding kinases.

[0215] For PU-002, virtually the entire AGC kinase family examined potentially interacts. For PU-001 and PU-002, the most significant potential off-target activity was demonstrated against three kinases: serine / threonine-protein kinase Nek3, serine / threonine-protein kinase D2, and protein tyrosine kinase 2 beta (Table 5).

Claims

A METHOD FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES USING INHIBITORS OF INTRACELLULAR IMMUNE CHECKPOINT REGULATION PATHWAYS, A CHEMICAL COMPOUND AND METHODS OF ITS SYNTHESIS. CLAUSE OF THE GROUP OF INVENTIONS 1. A compound represented by formula (I): Where XY: -(N=C)-; -(C=C)-; -(C=N)-; Where Z: > NC(O)-L; >CH-NL-CH3; > CH-C(O)-L; >CH-CH2-L; or their pharmaceutically acceptable salts.

2. Compound according to I. 1, where L is one of the following ligands:

3. A method for synthesizing a compound of formula (I) according to scheme 1 described below: Scheme 1 Where XY represents -N=C- Where Z represents > NC(O)-L Where L is one of the following ligands:

4. A method for synthesizing compounds of formula (I) according to scheme 2 described below: Scheme 2 Where XY represents -(N=C)- Where Z represents > CH-NL-CHs Where L is a ligand: L JC Ч ^ СН2 6 5. Methods for treating myeloid leukemia and / or myelodysplastic syndrome, and / or maintaining remission of myelodysplastic syndrome, and / or primary or secondary myelofibrosis, and / or chronic myelomonocytic leukemia, and / or atypical chronic myeloid leukemia, and / or unclassifiable myeloproliferative diseases, and / or preventing relapse of myelodysplastic syndrome and / or acute myeloid leukemia, comprising administering to a patient in need thereof a therapeutically effective amount of the compounds according to and. 1.

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