Pyrazole derivatives for the inhibition of phagocytosis

Abstract

A method for treating immune cytopenia in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound of formula (I). JPEG2025521587000082.jpg4135(I) (wherein R1, R2, R3 and R4 are as defined herein)

Description

Technical Field

[0001] Cross - reference to Related Applications This disclosure claims priority to U.S. Provisional Application No. 63 / 356,170, filed Jun. 28, 2022, which is incorporated herein by reference in its entirety. This disclosure relates to the field of small molecule inhibitors, such as pyrazole derivatives, useful for inhibiting phagocytosis in a subject having a condition where inhibition of phagocytosis, such as immune cytopenia, can treat or alleviate the symptoms of the condition.

Background Art

[0002] Immune cytopenia is a condition in which a human produces antibodies against certain types of hematopoietic cells in the blood. Under these circumstances, the cells are coated with antibodies and are then recognized by Fcγ receptors (FcγR) on the membrane of mononuclear phagocytes. Such recognition by monocytes - macrophages results in extravascular hemolysis in splenic and / or hepatic macrophages due to FcγR - mediated phagocytosis. Affected individuals are at risk of facing severe and sometimes life - threatening complications due to this process. Immune cytopenia has many categories, including: (a) immune thrombocytopenia (ITP; an autoimmune disease characterized by increased platelet destruction in the spleen and liver and / or reduced platelet production in the bone marrow); (b) hemolytic disease of the fetus and newborn (HDFN; transplacental transfer of hemolytic antibodies); (c) autoimmune hemolytic anemia (AIHA; phagocytosis of red blood cells coated with autoantibodies); (d) alloimmune hemolytic anemia, such as hemolytic transfusion reaction (HTR; phagocytosis of donor red blood cells due to hemolytic alloantibodies against pre - formed donor red blood cell antigens); (e) delayed hemolytic transfusion reaction (DHTR; development of hemolytic alloantibodies following transfusion); and (f) autoimmune neutropenia (AIN) related to autoantibodies produced against neutrophils, which mainly affects children. What all immune cytopenias have in common is the destruction of certain blood cells opsonized with antibodies by FcγR-mediated phagocytosis. Thus, the development of small molecule agents (drugs) that would provide interference with phagocytosis could improve various immune cytopenias, and the elucidation of such drugs would provide useful clinical interventions.

[0003] The treatment of immune cytopenias, with the exception of ITP, mainly involves corticosteroids (dexamethasone, prednisone) and monoclonal anti-CD20 (rituximab). For ITP, there are a few additional therapeutic agents used as secondary or tertiary treatments. These include splenectomy, thrombopoietin receptor agonists (TPO-RA; eltrombopag and avatrombopag) to stimulate platelet production, IVIg and anti-D (mechanism is unknown), and spleen tyrosine kinase (Syk) inhibitors ( fostamatinib). The most novel therapies target ITP and not other immune cytopenias, such as AIN, AIHA, HTR, DHTR, or HDFN. There are several experimental therapies at various stages of development and clinical trials, such as recombinant Fc multimers and inhibitors of the neonatal Fc receptor (FcRn) that may have efficacy in immune cytopenias other than ITP. Therefore, a treatment that can be applied to all immune cytopenias or conditions where phagocytosis is part of the pathophysiology is desired.

Summary of the Invention

[0004] Compounds of formula I are provided as defined below.

Chemical formula

[0005]

Chemical formula

[0006] [Chemical formula] wherein X1 is H, methyl or halogen, for example Br, F or Cl, R4 is -CN,

[0007] [Chemical formula] wherein X2 is H or methyl, When R1 is phenyl, ·R2 is -C(O)-O-CH2-CH3, and R3 is

[0008] [Chemical formula] wherein X1 is H, methyl or halogen, for example Br, F or Cl, and R4 is -CN, or ·R2 is

[0009] [Chemical formula] wherein R3 is H, and R4 is [Chemical formula] or ·R2 is H, and R3 is

[0010] [Chemical formula] wherein R4 is [Chemical formula] and When R1 is ethyl, R2 and R3 are H, and R4 is

[0011] [Chemical formula] is (wherein, X2 is H or methyl))

[0012] In some embodiments, the compound of formula I is

Chemical formula

[0013] In some embodiments, the compound of formula I is

Chemical formula

[0014] In some embodiments, the compound of formula I is

Chemical formula

[0015] In some embodiments, the compound of formula I is

Chemical formula

[0016] In some embodiments, the compound of formula I is

Chemical formula

[0017] In some embodiments, the compound of formula I is

Chemical formula

[0018] In some embodiments, the compound of formula I is [Chemical formula] (KB-198) is as follows.

[0019] In some embodiments, the compound of Formula I is [Chemical formula] (KB-198a) is as follows.

[0020] In some embodiments, the compound of Formula I is [Chemical formula] (KB-210) is as follows.

[0021] In one aspect, there is provided a method for treating immune cytopenia in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound of Formula I. In one aspect, there is provided a method for treating immune cytopenia in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of Formula I and a pharmaceutically acceptable excipient. In one aspect, there is provided the use of a compound of Formula I for the treatment of immune cytopenia. In a further aspect, there is provided the use of a pharmaceutical composition comprising a compound of Formula I and a pharmaceutically acceptable excipient for the treatment of immune cytopenia.

[0022] In one aspect, there is provided a compound of Formula I for the treatment of immune cytopenia. In a further aspect, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of Formula I for use in the treatment of immune cytopenia. In some embodiments, the immune cytopenia is immune thrombocytopenia, hemolytic disease of the fetus and newborn, autoimmune hemolytic anemia, alloimmune hemolytic anemia, delayed hemolytic transfusion reaction, or autoimmune neutropenia.

[0023] In a further aspect, there is provided a method of dephosphorylating HSP27 in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound KB-151, Kb-151a or KB-208. Cardiovascular disease or cancer dysregulates HSP27, where HSP27 is phosphorylated. In some embodiments, the cardiovascular disease is atherosclerosis. In some embodiments, the cancer is prostate cancer, colorectal cancer or breast cancer. Upon reading this disclosure, many further characteristics regarding this improvement and combinations thereof will be envisioned by those skilled in the art.

Brief Description of the Drawings

[0024]

Figure 1

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Figure 3H

Figure 3I

Figure 3J

Figures 4A-4B

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Figures 5A-5C

Figure 6A

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Figure 11B

Figure 11C

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Figure 11F

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Figure 13B

Figure 14

Best Mode for Carrying Out the Invention

[0025] Compounds of formula I are provided for inhibiting phagocytosis.

Chemical Formula

[0026]

Chemical Formula

Chemical Formula

[0027]

Chemical Formula

[0028]

Chemical Formula

[0029]

Chemical Formula

Chemical Formula

[0030]

Chemical Formula

[0031] [Chemical formula] wherein when R1 is ethyl, R2 and R3 are H, and R4 is [Chemical formula] (wherein X2 is H or methyl))

[0032] In one embodiment, the compound of formula I is KB-151, and the formula [Chemical formula] is possessed.

[0033] In one embodiment, the compound of formula I can be modified to have a halogen group different from bromine, an ethyl group instead, or be substituted with hydrogen. Removal of the bromine group can improve the biocompatibility of the compound and reduce toxicity. In one embodiment, the compound of formula I is KB-151a, and the formula [Chemical formula] is possessed.

[0034] In one embodiment, the compound of formula I is KB-151b, and the formula [Chemical formula] is possessed.

[0035] In one embodiment, the compound of formula I is KB-151c, and the formula [Chemical formula] is possessed.

[0036] In one embodiment, the compound of formula I is KB-151d, and the formula [Chemistry] has.

[0037] In one embodiment, the compound of formula I is KB-208, and the formula [Chemistry] has.

[0038] In one embodiment, the compound of formula I is KB-198, and the formula [Chemistry] has.

[0039] In one embodiment, the compound of formula I is KB-198, and the formula [Chemistry] has.

[0040] In one embodiment, the compound of formula I is KB-210, and the formula [Chemistry] has.

[0041] A method of preventing, treating and / or alleviating the phagocytic activity of immune cells in a subject in need thereof is provided. In some embodiments, phagocytosis is caused by a state in which the subject produces antibodies against certain types of hematopoietic cells in their blood and phagocytic cells (e.g., monocytes or macrophages) phagocytose the hematopoietic cells coated with these antibodies. One example of such a state is immune cytopenia. The present compound can be used to prevent, treat and / or alleviate immune cytopenia. There are different types of immune cytopenia, such as (a) immune thrombocytopenia (ITP; an autoimmune disease characterized by increased platelet destruction in the spleen and liver and / or reduced platelet production in the bone marrow), (b) hemolytic disease of the fetus and newborn (HDFN; transplacental transfer of hemolytic antibodies), (c) autoimmune hemolytic anemia (AIHA; phagocytosis of red blood cells coated with autoantibodies), (d) alloimmune hemolytic anemia, such as hemolytic transfusion reaction (HTR; phagocytosis of donor red blood cells due to hemolytic alloantibodies against preformed donor red blood cell antigens), (e) delayed hemolytic transfusion reaction (DHTR; development of hemolytic alloantibodies following transfusion), and (f) autoimmune neutropenia (AIN) associated with autoantibodies produced against neutrophils, which mainly affects children. More generally, the present disclosure provides a method for preventing, treating and / or alleviating one or more autoimmune or alloimmune diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound as defined herein.

[0042] Administration is by any of the routes commonly used to introduce the agent so as to ultimately bring it into contact with the blood. The agents described herein can be administered by any suitable means, preferably together with a pharmaceutically acceptable carrier or excipient. The terms "pharmaceutically acceptable carrier", "excipient", "physiologically acceptable vehicle", etc. are understood to refer to an acceptable carrier that can be administered to the subject together with the agent and that does not destroy its pharmacological activity. Further, as used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" is known in the art and includes, but is not limited to, phosphate buffer at 0.01 - 0.1 M and preferably 0.05 M or 0.9% saline. Further, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, etc. Preservatives and other additives, such as antibacterial agents, antioxidants, collating agents, inert gases, etc. may also be present.

[0043] In some embodiments, there is provided a pharmaceutical composition comprising a compound of Formula I (e.g., KB-151, KB-151a, KB-151b, KB-151c, KB-151d, KB-208, KB-198, KB-198a and / or KB-210) and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used in the preventive, alleviating or treating methods described herein. As used herein, “pharmaceutical composition” means a therapeutically effective amount (dose) of a compound accompanied by a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier and / or carrier. “Therapeutically effective amount” as used herein in the context of a pharmaceutical composition refers to an amount that produces a therapeutic effect for a given condition and dosing regimen. Such compositions can be liquid or lyophilized or otherwise dry formulations and include the contents of various buffers (e.g., Tris-HCl, acetate, phosphate), diluents of various pHs and ionic strengths, additives such as albumin or gelatin to prevent surface absorption, and detergents (e.g., Tween20™, Tween80™, Pluronic F68™, bile salts). The pharmaceutical composition can include a pharmaceutically acceptable solubilizer (e.g., glycerol, polyethylene glycol), antioxidant (e.g., ascorbic acid, sodium pyrosulfite), preservative (e.g., thimerosal, benzyl alcohol, parabens), bulking or tonicity modifier (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol, complex formation with metal ions, or incorporation of materials into particulate formulations such as polylactic acid, polyglycolic acid, hydrogels, etc. or onto them, or into liposomes, microemulsions, micelles, monolayers or multilayers of vesicles, erythrocyte ghosts, or spheroplasts. Such compositions can affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled release or sustained release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). Also contemplated by the present disclosure are particulate compositions coated with polymers (e.g., poloxamers or poloxamines).

[0044] Suitable methods of administering the agent are available and well known to those skilled in the art, and one or more routes can be used to administer a particular composition, although a particular route can often provide a more immediate and effective response than another route. The prophylactic or therapeutic compounds of the present invention can be administered systemically or locally, either orally or parenterally. For example, intravenous injection, such as intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, intestinal lavage, enteric-coated oral tablets, etc. can be selected, and the method of administration can be appropriately selected according to the age and condition of the patient.

[0045] In some embodiments, the treatment can include administering to a subject in need thereof, for example, in the context of immune cytopenia, a compound according to the present disclosure in a therapeutically effective amount to inhibit phagocytosis. "Therapeutically effective amount" also as used herein refers to an amount (dose) effective to inhibit or reduce phagocytosis of blood cells in a subject in need thereof. "Pharmaceutically effective amount" can also be understood herein as an amount that, when administered in a single dose or in any dosage or route, alone or in combination with other therapeutic agents, produces the desired therapeutic effect. In some embodiments, the compounds or pharmaceutical compositions or other therapeutic agents according to the present disclosure are administered simultaneously or at predetermined time intervals (ranging, for example, from minutes, hours, days, weeks or months). Therapeutic effects include, but are not limited to, prevention, treatment and / or alleviation of symptoms of immune cytopenia. More specifically, "prevention, treatment and / or alleviation of symptoms of immune cytopenia" refers to the ability of a compound or pharmaceutical composition to limit the onset, progression and / or symptomatology of immune cytopenia. Symptoms associated with autoimmune thrombocytopenia include, but are not limited to, nosebleeds, bleeding from the gums, easy or excessive bruising, superficial bleeding into the skin, bleeding in the brain, blood in the urine, bloody stools, increased bleeding during menstruation. Symptoms associated with immune hemolytic states include, but are not limited to, fatigue, jaundice, chills, shortness of breath and dizziness.

[0046] Excipients or carriers must be "pharmaceutically acceptable" in the sense that they are compatible with the other ingredients of the formulation and not toxic to its recipient. Standard acceptable excipients or carriers are well known to those skilled in the art and are described in many reference books. It will be apparent to those skilled in the art that the amounts of the compounds described herein and used in accordance with the present disclosure (or if additional therapeutic agents are required or desired) can be determined by a treating physician or pharmacist. It should be understood that the amount of the compound required will vary not only with the particular compound selected, but also with the route of administration, the nature of the condition for which treatment is required, and the age and condition of the patient. The methods of treatment or the scope of use described herein are not particularly limited, but in principle include any therapeutically useful outcome including prevention, treatment or delay in the progression of the conditions defined herein.

[0047] One aspect of this study on small molecule phagocytosis inhibitors is the advantage of being a treatment that can act rapidly. Indeed, treatments that can rapidly reverse the immune-mediated destruction of certain blood cells are highly beneficial and, generally and specifically, these will provide additional time for implementing other treatment strategies and a better chance for these strategies to succeed. Thus, the present compounds can be more specific than current treatment options for immune cytopenia and can have a broader application and can be used in addition to or in combination with existing treatment options. For example, these inhibitors can be administered as a single therapy in emergency situations or co-administered with other ITP treatment options. It has also been observed that the compounds of the present disclosure reduce the levels of low density lipoprotein (LDL) and triglycerides. Thus, the compounds of the present disclosure, particularly KB-151 and KB-208, are useful for reducing LDL and triglycerides in individuals in need thereof.

[0048] The mechanism of action of KB-151 and KB-208 was found to be the dephosphorylation of HSP27. HSP27 is a multidimensional protein that acts as a protein chaperone. Thus, the advantages of KB-151 and KB-208 extend beyond immune cytopenia to other conditions where HSP27 is dysregulated (i.e., phosphorylation is increased). HSP27 is known to have a role in cardiovascular diseases, such as atherosclerosis. In one embodiment, KB-151 and / or KB-208 are used in the treatment, prevention, or alleviation of symptoms of atherosclerosis. HSP27 also plays a role in the inhibition of apoptosis and actin cytoskeleton remodeling. Overexpression of HSP27 results in an increase in the concentration of filamentous actin (F-actin) in the cell cortex and an increase in pinocytosis activity. Overexpression of the non-phosphorylated mutant form of HSP27 reduces the cortical F-actin concentration and pinocytosis activity compared to control cells. Without wishing to be bound by any theory, this can explain the reason why inhibition of HSP27 phosphorylation can lead to inhibition of phagocytosis. Due to this role in apoptosis, HSP27 is also involved in certain cancers, such as prostate cancer, colorectal cancer, and breast cancer. Thus, a method for treating, preventing, or alleviating the symptoms of cancer associated with increased HSP27 phosphorylation using a compound of formula I (e.g., KB-151, KB-151a, KB-151b, KB-151c, KB-151d, KB-208, KB-198, KB-198a, and / or KB-210) is also provided.

Example

[0049] To search for small molecule drugs that inhibit phagocytosis, an initial filter screening of over 13,282 compounds was performed. The following parameters were used for the screening: (1) molecular weight of 200 - 500 Da, (2) calculated partition coefficient (cLogP) ≤ 4, (3) less than 4 chiral centers, (4) 3 or more heteroatoms, and (5) not a natural product / compound. A diversity score (Tanimoto coefficient) of 0.65 was selected. The results of this initial screening led to the acquisition of a 5,000 - compound library, which was further narrowed down based on having at least one pyrazole or pyrrole group. These incorporated skeletons may be able to advantageously interact with the reactive moieties on the cell surface of macrophages. From this second filter screening, 80 compounds were selected for preliminary in vitro iterative exploration. A third round of filtering was applied, whereby compounds that showed at least 69% inhibition in the monocyte monolayer assay (MMA) were further investigated in vitro (Tables 1 - 2 and Figure 1). From the molecules in Table 2, two lead compounds (KB - 151 and KB - 208) with negligible toxicity, lactate dehydrogenase (LDH) release and apoptosis tested by MTT ((3-(4,5 - dimethylthiazol - 2 - yl)-2,5 - diphenyltetrazolium bromide) tetrazolium) viability, and high efficacy were selected. The IC 50 was from about 2 to about 4 μM.

[0050]

Table 1

Table 2

[0051] At the time of informed consent, blood was collected from healthy volunteers into tubes containing acid-citrate-dextrose (ACD) anticoagulant. Peripheral blood mononuclear cells (PBMCs) were obtained by density centrifugation using Ficoll-Hypaque solution (Biochrom AG). Primary monocytes for the functional phagocytosis assay were isolated as described below. Cell lines (HepG2, liver-derived, HB-8065 (trademark); HEK293, kidney-derived cell line, CRL-1573 (trademark)) were purchased from ATCC. Both cell lines were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum.

[0052] MMA for screening compounds for their ability to inhibit phagocytosis was performed as described previously (Purohit, M. K. et al., Bioorg. Med. Chem. Lett. 2013;23:2324-7; and Purohit, M. K. et al., Bioorg. Med. Chem. 2014;22:2739-52.). Briefly, mononuclear cells (PBMC) were overlaid on chamber slides and monocytes were purified by adhesion after 1 hour at 37 °C, 5% CO2. As reported previously (8, 9), drugs were solubilized in 100% DMSO and then diluted to a final concentration of 5 μM in Roswell Park Memorial Institute (RPMI). The corresponding volume of dimethyl sulfoxide (DMSO) in RPMI medium was used as a control. After 1 hour, opsonized indicator Rh-positive red blood cells with anti-RhD were added to the chamber slides and then incubated for a further 2 hours, after which they were fixed and the phagocytosed red blood cells (RBC) were counted using phase contrast microscopy. As a positive control for inhibition, intravenous immunoglobulin (IVIg), which is known to inhibit phagocytosis due to blockade of FcγRs, was used. The phagocytosis index (PI) was determined as the number of phagocytosed RBCs in 100 monocytes. The percentage inhibition of phagocytosis was determined by normalizing the PI in each treated sample to the PI concentration in each treated sample (phagocytosis of opsonized RBCs with vehicle only represents 100% phagocytosis) compared to the PI in untreated samples.

Number

[0053] To evaluate the effect of selected compounds on the metabolic activity of primary monocytes, MTT was used. PBMCs (a total of 5×10 4 cells per well) were isolated from ACD tubes and seeded in 96-well culture plates (VWR™ tissue culture plates, untreated, sterile, non-pyrogenic). The cells were treated with 5 μM of the solubilized compound and incubated for 3 hours at 37 °C with 5% CO2. Thereafter, MTT was added to the cells and the plates were incubated for 2 hours. The formation of purple crystals of formazan was followed under an optical microscope. The formazan crystals were solubilized by overnight incubation with 100 μL of 10% sodium dodecyl sulfate (SDS), 0.01 M HCl SDS. Absorbance was measured at 570 nm (reference 690 nm) in a microplate reader (Bio-Rad™ EPOCH II). As a positive control for reduced metabolic activity, 100 μM of thimerosal (diluted in RPMI) was used. Untreated cells represent the basal metabolic activity of the cells. This experiment was repeated at increasing concentrations (250 μM, 100 μM, 50 μM and 10 μM) for two compounds (KB-151 and KB-208) using various cell types including PBMCs, HEPG2 and HEK-293.

[0054] To understand whether any of the tested compounds induced primary cell death (PBMC), lactate dehydrogenase (LDH) release was evaluated as a measure of cytotoxicity. To achieve this goal, an LDH-cytotoxicity assay kit (Sigma™ Inc.) was used according to the manufacturer's instructions. Sample treatment was performed as described previously for the MTT assay. Absorbance at 490 and 600 nm was determined using a microplate reader (Bio-Rad™ EPOCH II). The minimum lysis control was designated as the sample without treatment. Cells solubilized with the lysis buffer supplied with the kit were considered as the maximum lysis control. Specific cell death was calculated as % specific death = (experimental lysis - minimum lysis / maximum lysis - minimum lysis) *It was calculated as 100. This experiment was repeated at increasing concentrations (250 μM, 100 μM, 50 μM, and 10 μM) for two compounds (KB-151 and KB-208) using various cell types including PBMC, HEPG2, and HEK-293. PBMC, HEPG2, and HEK-293 cells were treated with compound KB-151 or compound KB-208 at a concentration of 250 μM for 1 hour. 100 μM of thimerosal was used as a positive control for viability (diluted in RPMI). Staining with annexin V and PI was performed according to the manufacturer's procedure for the annexin-V-FLUOS staining kit (Sigma), and the samples were run on an SP6800 spectral cytometer.

[0055] MMA as detailed above was repeated with four compounds (KB-151, KB-208, KB-198, KB-210) at different concentrations. The concentrations included 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 2.5 μM, 1 μM, and 0.5 μM. Statistical analysis was performed using Graphpad™ Prism 8. The Student's t-test was used. A P-value < 0.05 was considered significant. In an in vitro phagocytosis assay called the monocyte phagocytosis assay (MMA), 80 selected pyrazole core compounds were screened using a concentration of 5 μM. The compounds were compared to standard intravenous immunoglobulin (IVIg) at a concentration of 1 mg / mL. Following this initial screening, 19 compounds that inhibited phagocytosis of anti-D opsonized Rh(D+) red blood cells by more than 40% were identified.

[0056] These compounds were further tested for viability / toxicity using the LDH release assay for weakened cell membranes and the MTT assay for cell viability (Figures 1 and 2). Both are colorimetric assays, but LDH relies on the release of LDH enzyme into the culture medium after cell membrane disruption. As a result, color formation indicates cell lysis. Compounds from the library were named KB-. KB-181, 182, 198, 199, 209, and 210 showed low toxicity compared to the control thimerosal (100 μM) in these experiments, but this was not significant compared to the untreated control. None of the other chemicals released LDH enzyme into the culture medium (Figure 1). The MTT test is a metabolic activity assay based on the enzymatic conversion of MTT in viable mitochondria. Color formation is an indicator of cell viability. 100 μM thimerosal ( **** P ≦ 0.0001), KB-182 ( ** P < 0.005), and KB-178 ( * P < 0.05) all showed statistically significant changes compared to the untreated control (tested by the Kruskal-Wallis test). The other compounds did not show statistically significant changes from the untreated control (Figure 2). After reexamining the results of the in vitro inhibition and toxicity assays (Figures 1 and 2), four compounds that inhibited phagocytosis by more than 65% (from Table 1) and showed no toxicity in the LDH and MTT assays were selected for further investigation. These compounds included KB-151, KB-198, KB-208, and KB-210. The dose-inhibition responses of these four drugs were determined and the IC 50 values were calculated (Figures 3A, 3B, 3C, and 3D). The IC 50 values for KB-210 and KB-198 exceeded 5 μM (22.06 and 8.426 respectively), the IC 50 value for KB-208 was 4.209 ± 1.235 μM, and the IC 50 value for KB-151 was 2.71 ± 0.786 μM. KB-151 and KB-208 had negligible toxicity and the lowest IC 50had values and were thus selected for further testing. KB-210 and KB-198 had an EC 50 > 10 μM. KB-208 had an EC of 5 ± 0.2 μM 50 . KB-151 had an EC of 8 ± 4 50 .

[0057] Whether any of these lead compounds could cooperate with IVIg was also tested in a dose-inhibition assay in which phagocytosis inhibition was titrated with IVIg alone and with each of two compounds (KB-151 and KB-208) added with IVIg at their IC 50 concentrations (IVIg + KB-151 or KB-208). This was done to confirm whether the titration curve of IVIg alone shifted and represented any cooperation (synergy) with the compounds. The compounds had no effect on the IVIg dose-response curve (Figure 3E). Compounds KB-198 and KB-210 were also evaluated and compared with KB-151 and KB-208 (Figure 3F). Similarly, KB-151 derivatives KB-151a, KB-151b, KB-151c, and KB-151d were also evaluated (Figures 3G - 3H). The IC 50 for derivative KB-198a was found to be 226.8 μM.

[0058] To further determine the potential toxicity of compounds KB-151 and KB-208, additional toxicity tests were performed using LDH and MTT (Figures 4A - 4D), and an apoptosis assay (annexin V / PI) (Figures 5A - 5C), using higher concentrations of each compound up to 250 μM. Using peripheral blood mononuclear cells (PBMC), liver HEPG2, and kidney HEK293 cell lines, all assays showed low to no toxicity. KB-208 and KB-151 showed the same pattern in all samples, including PBMC, HEPG2, and HEK-293, similar to untreated cells. An in vivo phagocytosis inhibition assay was performed using KB-151 in a mouse model, Balb / c animals (Figures 6A and 6B). Based on the following calculation, a dose of 100 μM of KB-151 was injected into 20.5 g mice at 500 μL.

[0059] 0.0001 M × 457.28 g / mol (MW of KB-151) = 0.045729 g / L 0.045729 g / L × 0.5 mL (amount injected) = 0.0228645 mg = 22.8645 μg Therefore, 0.0228645 mg of KB-151 was injected into a 20.5 mg mouse at 0.0228645 mg / 0.0205 kg = 1.115 mg / kg. A dose-escalating anti-CD41 antibody ITP mouse model was used. KB-151 showed a significant increase in the number of platelets (PLT) on day 4 after two treatment doses on days 2 and 3, compared to the CD-41 group ( * P ≤ 0.05). The efficacy of KB-151 and KB-208 when reducing platelet levels was evaluated in three ITP mouse models: C57BL / 6 (Figure 7), Balb / c (Figures 8A - 8B), and CD1 (Figure 9). Similar efficacy for the improvement of ITP was observed in the three models. Toxicity was also evaluated using the Balb / c model. Blood obtained from mice 60 days after treatment with KB-151 was analyzed, and the biochemistry is shown in Figures 10A - 10B. Minimal toxicity was observed. Moreover, histopathology performed on the liver, heart, spleen, lymph nodes, and kidneys of the mice showed no abnormalities or any toxicity.

[0060] To test the mechanism of phagocytosis inhibition, fluorescent dye-labeled RBCs opsonized with anti-D were used in an assay at room temperature to find RBC rosetting (attachment of antibody-opsonized RBCs to FcγRs of monocytes) in the presence of monocytes. IVIg was used as a positive control and untreated as a negative control. In the negative control, monocytes bound to RBCs and initiated phagocytosis (Figures 11A and 11E). However, KB-151 and KB-208 were found to inhibit binding and phagocytosis, similar to IVIg (Figures 11B - 11D and 11F - 11H). Due to the observation of results similar to those of IVIG, the binding of KB-208 and KB-151 to FcγR was evaluated to determine the attachment between KB-208 or KB-151 and FcγR. Three monoclonal antibodies specific for FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) were each used to test the resulting expression of FcγRI, FcγRII, and FcγRIII. The test was performed on monocytes, and the results are shown in Figure 12. As can be observed from Figure 12, the mechanism of action of the attachment of KB-208 and KB-151 to opsonized erythrocyte cells is independent of the binding of the three tested Fcγ receptors.

[0061] To further evaluate the effect on signal transduction, the mechanism of action of KB-208 and KB-151 was further investigated. Many kinases including Akt1 / 2 / 3 (T308), Akt1 / 2 / 3 (S473), CREB, EGF R, eNOS, ERK1 / 2, Chk-2, c-Jun, Fgr, GSK-3α / β, GSK-3β, HSP27, p53 (S15), p53 (S46), p53 (S392), JNK1 / 2 / 3, Lck, Lyn, MSK1 / 2, p70 S6 kinase (T389), p70 S6 kinase (T421 / S424), PRAS40, p38α, PDGF Rβ, PLC-γ1, Src, PYK2, RSK1 / 2, RSK1 / 2 / 3, STAT2, STAT5a / b, WNK1, Yes, STAT1, STAT3 (Y705), STAT3 (S727), β-catenin, STAT6, and HSP60 were tested. The most significant effect was found with HSP27, and it was found that KB-208 and KB-151 dephosphorylate HSP27 (Figures 13A - 13B). Next, it was confirmed that KB-208 and KB-151 activate PP2A (in other words, they increased its phosphatase activity) (Figure 14). PP2A is a serine phosphatase that dephosphorylates HSP27. Dysregulation of HSP27 also occurs in diseases other than immune cytopenia, such as prostate cancer, colorectal cancer, and breast cancer.

[0062] In conclusion, after screening over 200 different chemical compounds, this study identified four pyrazole core compounds (KB-151, KB-208, KB-198, and KB-210) that exhibit the ability to inhibit phagocytosis due to immune-mediated mechanisms.

Claims

1. Compound of formula I. 【Chemistry 1】 (I) (In the formula, R 1 is phenyl or ethyl, R 2 H, -C(O)-O-CH 2 -CH 3 and 【Chemistry 2】 Selected from the group consisting of, R 3 H, 【Transformation 3】 (In the formula, X 1 (wherein H, methyl, or halogen, for example Br, F, or Cl) R 4 is -CN, 【Chemistry 4】 (In the formula, X 2 (is H or methyl) R 1 when R is phenyl, ・R 2 C(O)-O-CH 2 -CH 3 And R 3 teeth 【Transformation 5】 (In the formula, X 1 (where H is H, methyl, or halogen, e.g., Br, F, or Cl), R 4 Is it -CN? ・R 2 teeth 【Transformation 6】 And R 3 H is R 4 teeth 【Transformation 7】 is, or ・R 2 H is R 3 teeth 【Transformation 8】 And R 4 teeth 【Chemistry 9】 And, R 1 When R is ethyl, 2 and R 3 H is R 4 teeth 【Chemistry 10】 (In the formula, X 2 (It is H or methyl.)

2. The compound of formula I above 【Chemistry 11】 The compound according to claim 1.

3. The compound of formula I above 【Chemistry 12】 The compound according to claim 1.

4. The compound of formula I above 【Chemistry 13】 The compound according to claim 1.

5. The compound of formula I above 【Chemistry 14】 The compound according to claim 1.

6. The compound of formula I above 【Chemistry 15】 The compound according to claim 1.

7. The compound of formula I above 【Chemistry 16】 The compound according to claim 1.

8. The compound of formula I above 【Chemistry 17】 The compound according to claim 1.

9. The compound of formula I above [Chemistry 18] The compound according to claim 1.

10. The compound of formula I above 【Chemistry 19】 The compound according to claim 1.

11. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound defined in any one of claims 1 to 10, for use in the treatment of immune cytopenia.

12. The pharmaceutical composition according to claim 11, wherein the immune cytopenia is immune thrombocytopenia, hemolytic diseases of the fetus and neonat, autoimmune hemolytic anemia, alloimmune hemolytic anemia, delayed hemolytic transfusion reaction, or autoimmune neutropenia.

13. A pharmaceutical composition for use in a method of dephosphorylating HSP27 in a subject requiring such use, comprising a compound as defined in any one of claims 2 to 7.

14. A pharmaceutical composition for use in a method of dephosphorylating HSP27 in a subject requiring such use, comprising a compound defined in any one of claims 2 to 7 and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 13, wherein the subject has a cardiovascular disease or cancer in which HSP-27 is deregulated and excessively phosphorylated.

16. The pharmaceutical composition according to claim 14, wherein the subject has a cardiovascular disease or cancer in which HSP-27 is deregulated and excessively phosphorylated.

17. The pharmaceutical composition according to claim 15, wherein the cardiovascular disease is atherosclerosis.

18. The pharmaceutical composition according to claim 16, wherein the cardiovascular disease is atherosclerosis.

19. The pharmaceutical composition according to claim 15, wherein the cancer is prostate cancer, colorectal cancer, or breast cancer.

20. The pharmaceutical composition according to claim 16, wherein the cancer is prostate cancer, colorectal cancer, or breast cancer.

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