Use of 2,4-disubstituted pyrimidine derivative and combination drug thereof in preparation of drug for preventing and treating hemophagocytic lymphohistiocytosis

Abstract

Provided in the present invention is the use of a 2,4-disubstituted pyrimidine derivative and a combination drug thereof in the preparation of a drug for preventing and treating hemophagocytic lymphohistiocytosis. The 2,4-disubstituted pyrimidine derivative and the combination drug thereof in the present invention exhibit significant antiproliferative activity against HLH cells, exert good preventive and therapeutic effects on HLH, and have no obvious toxic side effects, thus holding promise for the treatment of HLH.

Description

Use of 2,4-disubstituted pyrimidine derivatives and their combinations in the preparation of drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis.

[0001] This application claims priority to Chinese patent application 2025104916509, filed on April 18, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof, and its combination in the preparation of a drug for the prevention and treatment of hemophagocytic lymphohistiocytosis. Background Technology

[0003] Hemophagocytic lymphohistiocytosis (HLH) is a typical syndrome of immune system overactivation, characterized by the continuous activation of lymphocytes and macrophages and the secretion of high levels of cytokines, causing pathological systemic inflammation and even fatal multiple organ failure.

[0004] In the course of HLH, overactivated CD8+ T cells and macrophages infiltrate organs such as the liver, spleen, and bone marrow, causing a series of abnormal clinical manifestations. Based on this, the International Histiocytic Society proposed the HLH-2004 diagnostic guidelines. According to these guidelines, HLH can be clinically diagnosed when a patient presents with at least five of the following eight characteristics: 1) fever, 2) splenomegaly, 3) 2 or 3 cell lineage cytopenia (hemoglobin <90 g / L, platelets <100 × 10⁶ / L). 9 / L, neutrophils <1×10 9 4) Hypertriglyceridemia (TG, TG ≥ 3 mmol / L) and / or hypofibrinogenemia (FIB, FIB ≤ 1.5 g / L), 5) Hemophagocytosis of the spleen, bone marrow or lymph nodes, 6) Decreased NK cell activity, 7) Serum ferritin ≥ 500 μg / L, 8) Soluble CD25 (sIL-2R) ≥ 2400 U / mL.

[0005] HLH exhibits high heterogeneity and diverse pathogenesis, primarily falling into two categories: primary (familial) and secondary (acquired). Primary HLH typically involves congenital immunodeficiency, including mutations in genes such as PRF1, UNC13D, and STX11. The presence of these gene mutations allows for molecular genetic diagnosis of HLH. PRF1 gene mutations, encoding perforin, account for 20%-40% of primary HLH cases globally. Due to perforin deficiency, pores cannot form on the surface of target cells, preventing cytotoxic granules from inducing target cell death, thus leading to persistent CD8+ T cell proliferation and activation. Secondary HLH usually results from infections, autoimmune diseases, or malignancies. The pathogenesis of secondary HLH is poorly understood; one possible mechanism is repeated stimulation of Toll-like receptors (TLRs), leading to excessive activation of immune cells and causing HLH.

[0006] Treatment for HLH aims to suppress cytokine storms and eliminate activated T cells and macrophages. Standard treatment is based on glucocorticoids and etoposide, including the HLH-1994 regimen and the revised HLH-2004 regimen. However, approximately 25%–50% of patients are insensitive to standard treatment, and the 5-year survival rate is only 54%. Therefore, there is an urgent need to improve HLH treatment to improve prognosis.

[0007] It is noteworthy that during the course of HLH, multiple cytokines, including interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, and tumor necrosis factor-α (TNF-α), activate the JAK / STAT signaling pathway. This activated pathway further promotes cytokine release, creating a vicious cycle. Therefore, targeting the JAK / STAT signaling pathway shows promise for treating HLH. Previous studies have reported that in HLH mouse models, the JAK1 / 2 inhibitor ruxolitinib (RUX) can reduce liver and spleen inflammation and prolong survival. However, RUX can cause grade III / IV hematologic toxicity and other adverse reactions, such as elevated transaminases, skin infections, limb pain, and decreased appetite, possibly due to poor selectivity of JAK. In addition, the early mortality rate is 17.3%-25.0%, and the early relapse rate is 13.5%-20%, indicating that the response depth of RUX treatment for HLH is poor. Therefore, there is an urgent need to develop novel and highly selective JAK inhibitors to reduce drug toxicity and improve the response depth.

[0008] WO2020259683A1 discloses the application of a 2,4-disubstituted pyrimidine derivative of formula (I) as an inhibitor of Janus tyrosine kinase 2 and FMS-like tyrosine kinase 3 (JAK2-FLT3). The compound of formula (I) is a new generation JAK2 / FLT3 inhibitor with extremely high selectivity and activity against JAK2. In myeloproliferative neoplasms, it has been shown to have superior pharmacodynamics and safety compared to marketed drugs of the same class, ruxolitinib (RUX) and fedratinib (FED), and exhibits significant spleen-shrinking activity.

[0009] However, the role of these compounds in hemophagocytic lymphohistiocytosis (HLH) remains unclear. Therefore, this study aims to systematically evaluate the pharmacodynamic effects of 2,4-disubstituted pyrimidine derivatives, represented by compound (Ⅰ), in HLH, and to further elucidate their mechanisms of action, in order to expand the application prospects of these compounds or their salts in the treatment of HLH. Summary of the Invention

[0010] To enable the use of 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts for the prevention or treatment of HLH, this invention has conducted an in-depth exploration of the pharmacodynamics and mechanisms of action of 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts and their combination drugs in HLH, and found that 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts and their combination drugs have excellent preventive and therapeutic effects on HLH.

[0011] This invention first provides the use of a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention and treatment of hemophagocytic lymphohistiocytosis; said 2,4-disubstituted pyrimidine derivative is selected from the following compounds:

[0012] The pharmaceutically acceptable salts are p-toluenesulfonate, oxalate, citrate, malate, salicylate, tartrate, phosphate, methanesulfonate, sulfate, fumarate, hydrochloride, or maleate.

[0013] Preferably, in the above-described uses, the 2,4-disubstituted pyrimidine derivative is a compound as shown in formula (I):

[0014] Preferably, in the above-described uses, the pharmaceutically acceptable salt is a maleate salt.

[0015] Preferably, in the above-described uses, the molar ratio of the 2,4-disubstituted pyrimidine derivative to the pharmaceutically acceptable salt is 1:1.

[0016] More preferably, in the above-described uses, the pharmaceutically acceptable salt of the 2,4-disubstituted pyrimidine derivative is a compound as shown in formula (II):

[0017] In the above-mentioned uses, the hemophagocytic lymphohistiocytosis is either primary or secondary.

[0018] In the above-mentioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt can inhibit the abnormal proliferation of HLH cells.

[0019] In the above-mentioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt can improve the symptoms of hemophagocytic lymphohistiocytosis, including weight loss, sudden increase in body temperature, decrease in blood cells, impaired liver function, elevated blood lipids, abnormal coagulation function, elevated serum ferritin and sCD25, hypercytokineemia, hepatosplenomegaly and / or hemophagocytosis.

[0020] In the aforementioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt can improve inflammation of the liver, spleen and bone marrow in hemophagocytic lymphohistiocytosis and / or inhibit the expression of p-JAK2 and STAT5 proteins in the liver and spleen.

[0021] In the above-mentioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt can inhibit the proliferation of CTLL-2 and RAW264.7 cell lines and / or inhibit the expression of phosphorylated JAK2 and STAT5 proteins in CTLL-2 and RAW264.7 cell lines;

[0022] Preferably, it can inhibit the secretion of IL-6, TNF-α and IL-1β cytokines in the RAW264.7 cell line.

[0023] In the above-described uses, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt may be adjusted according to clinical needs.

[0024] In the above-mentioned uses, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 25-225 mg.

[0025] Preferably, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 75-125 mg.

[0026] Preferably, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 75 mg or 125 mg.

[0027] In the above-described uses, the administration frequency is once a day.

[0028] In the above-mentioned uses, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 5 mg / kg to 150 mg / kg.

[0029] Preferably, in the above-described uses, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 10 mg / kg to 100 mg / kg.

[0030] More preferably, in the above-described uses, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 10 mg / kg to 60 mg / kg.

[0031] Most preferably, in the above-mentioned uses, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 30 mg / kg.

[0032] In the above-mentioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is administered orally.

[0033] In the above-mentioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is administered three times a day, twice a day, once a day, or once every two days.

[0034] In the above-mentioned uses, the administration frequency is twice a day.

[0035] In the above-mentioned uses, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is the sole active ingredient in the drug.

[0036] The dosage form of the drug is a conventional dosage form in the art. Specifically, in the above-described uses, the dosage form of the drug is a liquid or solid dosage form; for example, tablets, capsules, granules, powders, or oral liquids.

[0037] This invention also provides the use of a combination of a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention and treatment of hemophagocytic lymphohistiocytosis, wherein the combination of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof administered separately or simultaneously, in combination with other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis, wherein the 2,4-disubstituted pyrimidine derivative is selected from the following compounds:

[0038] The pharmaceutically acceptable salts are p-toluenesulfonate, oxalate, citrate, malate, salicylate, tartrate, phosphate, methanesulfonate, sulfate, fumarate, hydrochloride, or maleate.

[0039] Preferably, in the use of the above-mentioned combined drugs, the molar ratio of the 2,4-disubstituted pyrimidine derivative to the pharmaceutically acceptable salt is 1:1.

[0040] Preferably, in the use of the above-mentioned combined drugs, the 2,4-disubstituted pyrimidine derivative is a compound as shown in formula (I):

[0041] Preferably, in the use of the above-mentioned combined drugs, the pharmaceutically acceptable salt is maleate.

[0042] More preferably, in the use of the above-mentioned combination of drugs, the pharmaceutically acceptable salt of the 2,4-disubstituted pyrimidine derivative is a compound as shown in formula (II):

[0043] Among the uses of the aforementioned combined drugs, the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis are one or more of the following: dexamethasone, etoposide, cyclosporine A, methylprednisolone, epavamab, alenmab, anaspirin, tocilizumab, infliximab, and etanercept.

[0044] In the use of the aforementioned combined drugs, dexamethasone may be used in the form of dexamethasone phosphate.

[0045] In the use of the aforementioned combined drugs, etoposide can be used in the form of etoposide phosphate.

[0046] Among the uses of the aforementioned combined drugs, the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis are dexamethasone, or etoposide and dexamethasone. Specifically, 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts and dexamethasone are administered separately or simultaneously, or 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts and etoposide and dexamethasone are administered separately or simultaneously.

[0047] In the use of the above-mentioned combined drugs, the pharmaceutically acceptable salt of the 2,4-disubstituted pyrimidine derivative is a compound as shown in formula (II), and the other drug for the prevention and treatment of hemophagocytic lymphohistiocytosis is dexamethasone.

[0048] In the use of the above-mentioned combined drugs, the pharmaceutically acceptable salt of the 2,4-disubstituted pyrimidine derivative is a compound as shown in formula (II), and the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis are etoposide and dexamethasone.

[0049] In the context of the combined use of the aforementioned drugs, the hemophagocytic lymphohistiocytosis is either primary or secondary.

[0050] In the use of the above-mentioned combined drugs, the type of hemophagocytic lymphohistiocytosis is as described in any of the above-mentioned uses.

[0051] The mass of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt, and other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis, is selected according to conventional methods in the art. In the use of the above-mentioned combined drugs, the mass ratio of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt to dexamethasone is 1:(0.002–1.5); preferably 1:(0.005–0.5); for example 1:(0.017–0.3); and again, for example 1:0.05.

[0052] In the use of the above-mentioned combined drugs, when the other drugs for preventing and treating hemophagocytic lymphohistiocytosis are dexamethasone and etoposide, the mass ratio of dexamethasone to etoposide is 1:(1.3-834); preferably 1:(3-300); for example 1:(10-100); or for example 1:33.33.

[0053] In the use of the above-mentioned combined drugs, the molar ratio of dexamethasone to the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 1:(0.55-442); preferably 1:(1.77-177); for example 1:(2.9-53.5); and for another example 1:17.7.

[0054] In the use of the above-mentioned combined drugs, when the other drugs for preventing and treating hemophagocytic lymphohistiocytosis are dexamethasone and etoposide, the molar ratio of dexamethasone to etoposide is 1:(1.1-710); preferably 1:(2.5-256); for example 1:(8-86); and for another example 1:28.4.

[0055] In the use of the above-mentioned combined drugs, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt may be adjusted according to clinical needs.

[0056] In the use of the above-mentioned combined drugs, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 25-225 mg.

[0057] Preferably, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 75-125 mg.

[0058] Preferably, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 75 mg or 125 mg.

[0059] In the above-mentioned combined use of drugs, the administration frequency is once a day.

[0060] Among the uses of the aforementioned combined drugs, the combined drugs are able to inhibit the abnormal proliferation of HLH cells.

[0061] Among the uses of the aforementioned combined drugs, the combined drugs can improve the symptoms of hemophagocytic lymphohistiocytosis, including weight loss, sudden increase in body temperature, decrease in blood cells, impaired liver function, elevated blood lipids, abnormal coagulation function, elevated serum ferritin and sCD25, hypercytokineemia, hepatosplenomegaly and / or hemophagocytosis.

[0062] Among the uses of the above-mentioned combined drugs, the combined drugs can improve the inflammation of the liver, spleen and bone marrow in hemophagocytic lymphohistiocytosis and / or inhibit the expression of p-JAK2 and STAT5 proteins in the liver and spleen.

[0063] In the use of the above-mentioned combined drugs, the combined drugs can inhibit the proliferation of CTLL-2 and RAW264.7 cell lines and / or inhibit the expression of phosphorylated JAK2 and STAT5 proteins in CTLL-2 and RAW264.7 cell lines.

[0064] Preferably, it can inhibit the secretion of IL-6, TNF-α and IL-1β cytokines in the RAW264.7 cell line.

[0065] In the use of the above-mentioned combined drugs, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 5 mg / kg to 150 mg / kg.

[0066] Preferably, in the use of the above-mentioned combined drugs, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 10 mg / kg to 100 mg / kg.

[0067] More preferably, in the use of the above-mentioned combination of drugs, the dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 10 mg / kg to 60 mg / kg.

[0068] Most preferably, in the use of the above-mentioned combination of drugs, the dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 30 mg / kg.

[0069] In the use of the above-mentioned combined drugs, the dosage of dexamethasone is 0.3 to 7.5 mg / kg.

[0070] Preferably, in the use of the above-mentioned combined drugs, the dosage of dexamethasone is 0.5 to 5 mg / kg.

[0071] More preferably, in the use of the above-mentioned combined drugs, the dosage of dexamethasone is 1 to 3 mg / kg.

[0072] Most preferably, in the use of the above-mentioned combined drugs, the dosage of dexamethasone is 1.5 mg / kg.

[0073] In the use of the above-mentioned combined drugs, the dosage of etoposide is 10-250 mg / kg.

[0074] Preferably, in the use of the above-mentioned combined drugs, the dosage of etoposide is 15-150 mg / kg.

[0075] More preferably, in the use of the above-mentioned combined drugs, the dosage of etoposide is 30-100 mg / kg.

[0076] Most preferably, in the use of the above-mentioned combined drugs, the dosage of etoposide is 50 mg / kg.

[0077] In the use of the above-mentioned combined drugs, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is administered orally, and the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis are administered via intraperitoneal injection or orally.

[0078] In the use of the above-mentioned combined drugs, the dosing frequency of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is as described in any of the preceding regimens.

[0079] In the use of the above-mentioned combined drugs, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is administered three times a day, twice a day, once a day, or once every two days.

[0080] Among the uses of the above-mentioned combined drugs, the frequency of administration of dexamethasone is twice a day, once a day, or once every two days;

[0081] Preferably, in the use of the above-mentioned combined drugs, the dexamethasone is administered once a day;

[0082] In the use of the above-mentioned combined drugs, the frequency of administration of etoposide is three times a week, twice a week, once a week, or once every two weeks.

[0083] Preferably, in the use of the above-mentioned combined drugs, the etoposide is administered twice a week.

[0084] The present invention also provides a pharmaceutical composition comprising a first pharmaceutical composition and a second pharmaceutical composition:

[0085] A first pharmaceutical composition comprising the 2,4-disubstituted pyrimidine derivative thereof or a pharmaceutically acceptable salt thereof; and,

[0086] The second pharmaceutical composition comprises the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis.

[0087] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0088] In the first pharmaceutical composition, the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is the sole active ingredient in the first pharmaceutical composition.

[0089] In the second pharmaceutical composition, the other drugs for preventing and treating hemophagocytic lymphohistiocytosis are the only active ingredients in the second pharmaceutical composition.

[0090] The types, mass ratios, molar ratios, dosage forms, routes of administration, dosages, and frequencies of administration of the 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts, and the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis, are as described in any of the preceding schemes.

[0091] The term “prevention and treatment” refers to both prevention and treatment. When a specific condition is involved, prevention and treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that leads to or causes the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; (4) slowing the development of the condition or one or more biological manifestations of the condition or condition or (5) reducing the risk of acquiring or developing the disease or disorder.

[0092] The term "pharmaceuticalally acceptable excipients" refers to excipients and additives used in the production of pharmaceuticals and the dispensing of prescriptions. They are all substances contained in pharmaceutical preparations other than the active ingredient.

[0093] The beneficial effects of this invention are:

[0094] This invention first explored whether there are expression differences in JAK / STAT signaling pathway genes between HLH patients and normal individuals. Then, it investigated the in vitro pharmacodynamics of compound II in HLH. Subsequently, in vivo experiments were conducted using a secondary HLH mouse model to explore the pharmacodynamics, safety, and mechanism of compound II in HLH. Further research was conducted on the pharmacodynamics and mechanism of compound II in combination with drugs such as DEX in a secondary HLH mouse model. Finally, the pharmacodynamics and mechanism of compound II alone and in combination with DEX in a primary HLH mouse model were explored. Ultimately, the following conclusions were demonstrated:

[0095] 1. Compared with normal individuals, HLH patients have high expression of JAK / STAT signaling pathway genes in peripheral blood mononuclear cells, providing a theoretical basis for the treatment of HLH with compound formula II.

[0096] 2. In vitro experiments confirmed that compound II has significant anti-proliferative activity against key effector cell lines of HLH, inhibits the secretion of IL-6, TNF-α and IL-1β, downregulates the expression of p-JAK2 and p-STAT5, and is superior to DEX, RUX and FED.

[0097] 3. In vivo experiments confirmed that compound II can significantly improve the general condition, blood routine, liver function, blood lipids, fibrinogen, serum ferritin, sCD25, hypercytokineemia, hepatosplenomegaly, inflammatory response and "hemophagocytosis" in secondary HLH mouse models, and can downregulate the expression of p-JAK2 and p-STAT5 in the liver and spleen, with no obvious toxic side effects on the vital organs of mice.

[0098] 4. In vivo experiments confirmed that the combination of compound II and DEX was significantly superior to compound II and DEX alone in improving body temperature, blood routine, liver and kidney function, blood lipids, fibrinogen, serum ferritin, sCD25, hepatosplenomegaly, and hemophagocytosis in a mouse model of secondary HLH, suggesting that compound II and DEX have a synergistic anti-HLH effect. In addition, FD was superior to ED in improving white blood cell count, liver and kidney function, hepatosplenomegaly, and hemophagocytosis.

[0099] 5. Compounds of Formula II can increase GCR nucleus entry and reduce DEX resistance by inhibiting the formation of the p-STAT5-GCR complex, and thus exert anti-HLH effects in synergy with DEX.

[0100] 6. In vivo experiments have confirmed that both single-agent formulation II compound and combination therapy with FD can significantly improve disease severity, survival, blood routine, liver and kidney function, blood lipids, fibrinogen, serum ferritin, sCD25, hypercytokineemia, hepatosplenomegaly, and hemophagocytosis in a mouse model of primary HLH, supporting the application of single-agent formulation II compound and combination therapy with FD in primary HLH. In addition, combination therapy with FD is superior to ED in improving survival, white blood cell count, kidney function, and hemophagocytosis.

[0101] 7. Compound of Formula II can inhibit HLH by limiting the number of CD8+ T cells and their activated form CD8+CD44+ T cells, and by increasing the perforin expression of CD8+ T cells. Attached Figure Description

[0102] Figure 1 shows the expression of the JAK / STAT signaling pathway in HLH. 1 represents HLH patients, and 2 represents normal individuals.

[0103] Figure 2 shows the effect of compound II on the expression of JAK / STAT pathway proteins in RAW264.7 and CTLL-2 cell lines.

[0104] Figure 3 shows the changes in body weight and body temperature in mice with secondary HLH during treatment. 1 is the Control group, 2 is the Vehicle group, 3 is the FM group (formula II compound group), 4 is the RUX group, 5 is the FED group, and 6 is the DEX group.

[0105] Figure 4 shows a comparison of the liver and spleen in the Control, Vehicle, Formula II compound, RUX, FED, and DEX groups of secondary HLH mice.

[0106] Figure 5 shows the liver and spleen inflammation manifestations in the Control, Vehicle, and Compound II groups of secondary HLH mice (80×).

[0107] Figure 6 shows the hemophagocytic phenomenon in the liver, spleen, and bone marrow of secondary HLH mice (Control, Vehicle, and Compound II groups) (400×); the arrows indicate the hemophagocytic phenomenon.

[0108] Figure 7 shows the expression of JAK2, p-JAK2, STAT5, and p-STAT5-β-actin in the spleen of the Control, Vehicle, and Formula II compound groups.

[0109] Figure 8 shows HE staining of important organs in mice with secondary HLH (200×); the black arrows indicate pathological changes.

[0110] Figure 9 shows a comparison of the liver and spleen in the Vehicle, Formula II compound, DEX, FD, and ED groups of secondary HLH mice.

[0111] Figure 10 shows HE staining images of the liver, spleen, and bone marrow of secondary HLH mice in the Vehicle, Formula II compound, DEX, FD, and ED groups (400×).

[0112] Figure 11 shows the immunofluorescence images of GCR in the spleen of DEX and FD groups of secondary HLH mice (400×).

[0113] Figure 12 shows the immunofluorescence image of GCR in spleen lymphocytes after treatment with IL-2, DEX and compound II (400×).

[0114] Figure 13 shows the immunoprecipitation diagram after treatment with IL-2, DEX and compound of formula II.

[0115] Figure 14 shows the survival time of each group of primary HLH mice. 1, 2, 3 and 4 represent the Control group, FD group, FM group (formula II compound group), and RD group, respectively; 5 represents the RUX group, 6 represents the Vehicle group, 7 represents the ED group, and 8 represents the DEX group.

[0116] Figure 15 shows the general condition of each group of primary HLH mice.

[0117] Figure 16 shows a comparison of liver and spleen size in different groups of primary HLH mice.

[0118] Figure 17 shows a comparison of the "hemophagocytic phenomenon" in different groups of primary HLH mice (400×). Detailed Implementation

[0119] The present invention will be further described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described herein.

[0120] The synthetic routes for the preparation of 2,4-disubstituted pyrimidine derivatives (Formula I) and 2,4-disubstituted pyrimidine derivative salts (Formula II) are as follows:

[0121] The specific steps are as follows: Compound I (0.75 kg, 1.6 mol) is stirred with ethyl acetate (12 L) and heated to reflux until dissolved (in other examples, ethanol (12 L of compound I) can be used). The preparation method of compound I is the same as in Example 44 of patent CN 114423750A. Maleic acid (186 g, 1.6 mol) solid is added (in other examples, an ethyl acetate / anhydrous ethanol solution of maleic acid (3 L / 300 ml) or an ethanol solution of maleic acid (3 L) can be used), stirred, and then cooled to an internal temperature of 50-55°C. After filtration, the filter cake is dried to obtain compound II. 1H NMR (400MHz, DMSO-d6) δ9.44(s,1H),8.34(s,1H),8.29(s,1H),8.10(s,1H),7.81(dd,J=15.5,2.4 Hz,1H),7.51-7.44(m,1H),7.03(dd,J=10.1,8.8Hz,1H),6.06(s,2H),5.40(s,1H),4.64(hept,J= 6.6Hz,1H),3.78(t,J=5.2Hz,2H),3.44-3.32(m,3H),3.30-3.10(br,2H),2.81(s,3H),2.75-2.65 (m, 2H), 2.32 (s, 3H), 2.09 (d, J = 11.6Hz, 2H), 1.84 (qd, J = 11.7, 3.8Hz, 2H), 1.49 (d, J = 11.6Hz, 6H). m / z:468.28783[M+H] + .

[0122] 1. Materials

[0123] 1.1 The main reagents used in the experiments of this invention are shown in Table 1.

[0124] Table 1 Main Reagents

[0125] 1.2 The main instruments used in the experiment of this invention are shown in Table 2.

[0126] Table 2 Main Instruments

[0127] 1.3 Experimental Cells and Viruses

[0128] The CTLL-2 and RAW264.7 cell lines used in this invention were purchased from Wuhan Pronosei Biotechnology Co., Ltd., while the BHK21 and VeroE6 cell lines were donated by the Army Medical University. All cell lines were preserved in liquid nitrogen tanks at the State Key Laboratory of Biotherapy, Sichuan University. All cell lines underwent cell identification before use. CTLL-2 cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum, 100 U / mL recombinant mouse IL-2, 1 μg / mL concanavalin A, and 1% penicillin / streptomycin. RAW264.7, BHK21, and VeroE6 cells were all cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0129] The LCMV-Armstrong virus used in the experiments of this invention was donated by the Army Medical University and is stored in the liquid nitrogen tank of the State Key Laboratory of Biotherapy at Sichuan University.

[0130] 1.4 Laboratory Animals

[0131] The animal experimental protocol designed in this invention strictly adheres to the 3R principle and has been approved by the Laboratory Animal Ethics Committee of West China Hospital, Sichuan University. C57 / BL6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and gene knockout Prf1- / - mice were purchased from The Jackson Laboratory. All mice involved in the experiments were housed in SPF-grade animal facilities.

[0132] 2. Method

[0133] 2.1 Sources and processing of RNA-seq data from HLH patients

[0134] The RNA-seq data from peripheral blood mononuclear cells of HLH patients and normal controls used in this invention were obtained from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), including 33 HLH patients and 37 normal controls. The GSE57253 dataset included 22 HLH patients and 4 normal controls, and the GSE26050 dataset included 11 HLH patients and 33 normal controls. The RNA-seq data from the two datasets were combined to analyze the differences in gene expression levels of the JAK / STAT family (JAK1, JAK2, JAK3, Tyk2, STAT1, STAT2, STAT3, STAT4, STAT5a, and STAT5b) between HLH patients and normal controls.

[0135] 2.2 Cell resuscitation, passage, counting, and cryopreservation

[0136] 2.2.1 Cell resuscitation

[0137] Remove the cryovials containing the cell lines from section 1.3 and quickly place them in a 37°C water bath to accelerate thawing. Then wipe them with 75% alcohol and transfer them to a clean bench. Pre-pipette 5 mL of complete culture medium into a BD tube, transfer the cell suspension to the BD tube, and centrifuge (300g, 3 minutes). After removing the supernatant, add the required complete culture medium according to the culture conditions. Place the cell suspension in a culture dish or flask and incubate at 37°C with 5% CO2. 2 Incubate in an incubator. Observe cell morphology and growth under an inverted microscope the following day.

[0138] 2.2.2 Cell passage

[0139] Before passage, observe cell morphology and density under an inverted microscope. If the cells are in good condition and the cell density reaches 80-90%, proceed with passage. For CTLL-2, gently aspirate to mix the cell suspension, transfer the cells to BD tubes using a pipette, and centrifuge (1000 rpm, 3 minutes). After removing the supernatant, resuspend the CTLL-2 cells in complete culture medium according to the culture conditions, place them in a culture dish or flask, and continue culturing in an incubator. For RAW264.7, due to its semi-suspended, semi-adherent cell characteristics, gently pipette to detach it from the dish or flask wall. Do not forcefully pipette or use trypsin. The subsequent processing procedure is the same as for CTLL-2.

[0140] 2.2.3 Cell Count

[0141] After centrifugation (1000 rpm, 3 minutes), discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, and gently mix by pipetting. Transfer 10 μL of the cell suspension to a cell counting chamber and count the cells. When counting under an inverted microscope, pay attention to the following: 1) Count the total number of cells in the four large squares; 2) When counting cells along the lines, count only the left side and not the right side, and only count the top cells and not the bottom cells. Then, calculate the total number of cells using the following formula: Total number of cells = (Total number of cells in the four large squares / 4) × 10⁻⁶ 4 × Total volume of cell suspension (mL).

[0142] 2.2.4 Cell cryopreservation

[0143] Transfer cells in the logarithmic growth phase to BD tubes and centrifuge (500g, 3 minutes), discarding the supernatant. Determine the amount of serum-free cryopreservation solution to add based on the cell pellet's condition. Gently pipette the mixture to mix thoroughly and transfer to cryovials, ensuring a cryopreservation density of 2-3 × 10⁻⁶ cells / tube. 6 Cells / mL. After labeling the cell information on the cryovials, place them in a -80°C freezer overnight, and then transfer them to a liquid nitrogen tank for long-term storage.

[0144] 2.3 In vitro experiments with key HLH effector cell lines

[0145] 2.3.1. CCK-8 assay for cell proliferation activity

[0146] Collect CTLL-2 and RAW264.7 cells in logarithmic growth phase, centrifuge (500g, 3 minutes), add complete culture medium to prepare cell suspension, count the cells, and then divide the cells into groups of approximately 3 × 10⁻⁶. 4Cells were seeded into 96-well plates, with three replicates per sample group. In addition to sample wells, blank wells and zeroing wells were also included. Select the appropriate complete culture medium for the cells to be tested. Dilute Compound II, RUX, FED, and Dexamethasone (DEX) to the required final concentration and add the prepared drug concentration and type to the corresponding well. Add the same complete culture medium as the sample wells to the blank wells, and add culture medium containing the same amount of DMSO as the highest concentration of the drug to the zeroing wells. The final volume of each well was 200 μL. Note that RAW264.7 cells should be incubated overnight to allow them to adhere before adding the drugs. After discarding the supernatant, pretreat with freshly prepared lipopolysaccharide (LPS) for 30 minutes (final LPS concentration 1 μg / mL), then add the test drug. After filling the side wells with sterile PBS, place the 96-well plate in an incubator for further culture (37°C, 5% CO2).

[0147] After culturing cells for 24, 48, and 72 hours, 20 μL of CCK-8 solution was added to each well under dark conditions, taking care to avoid the formation of air bubbles during the addition process. After adding the CCK-8, the 96-well plate was returned to the incubator and cultured for another 1-4 hours. The absorbance (Optical Density, OD) at 450 nm was then measured using a microplate reader.

[0148] Cell viability is calculated using the following formula: Cell viability % = [(Average OD value of sample wells - Average OD value of zeroing wells) / (Average OD value of blank wells - Average OD value of zeroing group)] × 100%. The cell viability calculated using the above formula is used to plot ICs using GraphPadprism software. 50 Curve and calculate IC 50 value.

[0149] 2.3.2 ELISA method for detecting cellular IL-6, TNF-α, and IL-1β levels

[0150] RAW264.7 cells in good growth condition were counted, and after preparing a cell suspension, they were seeded into 48-well plates (3 × 10⁶ cells / well). 4 Cells / well). After overnight culture and adhesion, discard the original culture medium. Except for the Control well (without LPS pretreatment), pretreat the remaining wells with LPS (final concentration 1 μg / mL) for 30 minutes. Then, except for the Control and Vehicle wells (pretreated with LPS), add freshly prepared Formula II compound, RUX, FED, and DEX (final concentration 1 μM) to the remaining wells. Incubate the treated cells in an incubator for 24 hours (37℃, 5% CO2). After culture, perform the following operations:

[0151] 1) Cell supernatant collection: After centrifugation (1000g, 10 minutes), transfer the supernatant to a new EP tube and label it.

[0152] 2) Add 100 μL of sample to an ELISA strip coated with the monoclonal antibody of the sample to be tested (this kit needs to be equilibrated to room temperature in advance). In addition to the sample, add the standard (diluted in advance according to the instructions). Seal the reaction wells and incubate at 37°C for 90 minutes. Wash the plate 5 times after incubation.

[0153] 3) Add 100 μL of biotinylated antibody dilution buffer to the blank wells, and add 100 μL of biotinylated antibody working solution to the other wells. Then seal the reaction wells and incubate at 37°C in the dark for 60 minutes. After incubation, wash the plate 5 times.

[0154] 4) Add 100 μL of enzyme conjugate dilution to the blank wells and 100 μL of enzyme conjugate working solution to the other wells. Then seal the reaction wells and incubate at 37°C in the dark for 30 minutes. After incubation, wash the plate 5 times.

[0155] 5) Add 100 μL of the chromogenic substrate to each well and incubate at 37°C in the dark for 10 minutes.

[0156] 6) Add 100 μL of the reaction stop solution to each well, mix immediately, and use an ELISA reader to detect the absorbance at 450 nm.

[0157] 7) Use Excel to plot the standard curve and calculate the cytokine concentrations, and then use Graphpad to analyze the differences between groups.

[0158] The ELISA kits for the three cytokines IL-6, TNF-α, and IL-1β used in this invention follow the same instructions, so they will not be repeated here.

[0159] 2.3.3 Western blot method for detecting protein expression in the JAK / STAT signaling pathway

[0160] 2.3.3.1 Extraction of proteins from CTLL-2 and RAW264.7 cell lines

[0161] (1) Plating and drug administration: Select CTLL-2 and RAW264.7 cells in the logarithmic growth phase, centrifuge (1000 rpm, 3 minutes), discard the supernatant, and resuspend the cells in fresh culture medium at a density of 2 × 10⁶ cells per well. 6RAW264.7 cells were seeded into 6-well plates, and compound of formula II was added to three final concentrations: 0.5, 1.0, and 2.0 μM. Note that RAW264.7 cells should be incubated overnight to allow for adhesion before drug addition. The supernatant was discarded the following day. Except for the blank control, which was added to medium without LPS, all other wells were pretreated with freshly prepared LPS (final concentration 1 μg / mL) for 30 minutes, followed by the addition of the test drug. After the above treatment, the cells were cultured for another 16 hours.

[0162] (2) Collect cells and extract proteins

[0163] 1) Cell collection: Transfer the cells to a 2 mL EP tube, centrifuge (1500 rpm, 3 minutes) and discard the supernatant. Wash the cells twice with PBS pre-cooled to 4°C, removing as much liquid as possible from the EP tube during the last wash.

[0164] 2) Prepare lysis buffer: Prepare the lysis buffer in advance by mixing RIPA lysis buffer, PMSF and phosphatase inhibitor in a ratio of 100:1:1. Add the appropriate amount of cell lysis buffer according to the volume of cell pellet and mix thoroughly.

[0165] 3) Cell lysis: Vortex the cell sample with lysis buffer for 1 minute, then incubate on ice for 5 minutes. Repeat this process for a total of 30 minutes. Then, lyse the sample by sonication 3 times, 15 seconds each time, with a 1-minute interval between each time.

[0166] 4) Protein extraction: After centrifugation (13000 rpm, 4℃, 30 minutes), transfer the supernatant to a new EP tube and place it on ice temporarily.

[0167] (3) Detecting protein concentration

[0168] 1) Constructing a standard curve: Add standards of different concentrations sequentially to a 96-well plate, then add Bradford microplate reader, and measure the OD of the standards using a microplate reader. 450 The standard curve is calculated as follows: Y = 0.5789X + 0.535, R = 0.9984.

[0169] 2) Sample absorbance detection: Add 2 μL of sample + 18 μL of UP water + 250 μL of Bradford solution to each well of a 96-well plate. Note that each sample should be tested in triplicate. Measure the OD of the sample using a microplate reader. 450 The protein concentration was then calculated based on the standard curve.

[0170] (4) Protein denaturation: Add 5×SDS-PAGELoadingBuffer protein loading buffer to the sample, dilute it to 1×, mix thoroughly, and heat in a metal bath at 100℃ for 10 minutes.

[0171] The above samples should be cooled to room temperature. Phosphorylated proteins should be tested and used immediately to reduce the impact of protein degradation. Proteins that cannot be used on the same day can be stored at -80°C.

[0172] 2.3.3.2 SDS-PAGE gel electrophoresis

[0173] Prepare SDS-PAGE gels of different concentrations based on different protein molecular weights. Place the prepared gels into the electrophoresis tank, add electrophoresis buffer, and slowly remove the comb to prepare for sample loading. Add an equal amount of protein (20-30 μg) to each lane. Calculate the corresponding loading volume based on the protein concentration of the sample to be tested and add it to the lane. Add markers at appropriate positions. After loading, use 80V to separate the bands from the upper gel, and then use 120V until electrophoresis is complete.

[0174] 2.3.3.3 Wet transfer method for film transfer

[0175] Remove the gel plate and place it on the transfer clamp, ensuring air bubbles are removed between the gel plate and the clamp. Cut the gel appropriately based on the size and band width of the target protein. Select a PVDF membrane with a suitable pore size according to the protein size, activate it in methanol, then equilibrate it in transfer buffer for 10 seconds. Place the membrane on the gel surface, again ensuring air bubbles are removed. Place an ice pack in the transfer tank and inject pre-cooled transfer buffer to 4°C. Close the transfer clamp and place the membrane in the transfer tank. Transfer at a constant current of 250 mA for 90 minutes.

[0176] 2.3.3.4, Closed

[0177] After removing the PVDF membrane, place it in PBST and wash it on a shaker at 100 rpm for 5 minutes. Then block it with 5% skim milk (skim milk powder added to PBST) or 5% BSA (BSA powder added to PBST) at room temperature on a shaker at 70 rpm for 1.5 hours.

[0178] 2.3.3.5 Antibody incubation and development

[0179] 1) Primary antibody incubation: Dilute the primary antibody using the dilution buffer according to the dilution ratio indicated on the instructions for the selected antibody. Immerse the PVDF membrane, washed with PBST, in the prepared primary antibody solution and incubate overnight on a shaker at 4°C.

[0180] 2) Secondary antibody incubation: Remove the PVDF membrane and place it in PBST. Wash the membrane three times at 100 rpm for 10 minutes each time on a shaker. After washing, place the PVDF membrane in the secondary antibody corresponding to the primary antibody species and incubate it on a shaker at 70 rpm for 2 hours at room temperature. Then wash the membrane again with PBST for a total of 30 minutes.

[0181] 3) Development: Take out the PVDF membrane, add developing solution (ECLA solution: B solution = 1:1) to develop and take pictures for storage.

[0182] 2.4 Construction of the HLH mouse model

[0183] Secondary HLH mouse model was established by subcutaneously injecting 50 μg (100 μL) of CpG-ODN1826 into the peritoneal cavity of 6-8-week-old C57 / BL6 mice on days 0, 2, 4, 6, and 8.

[0184] The method for constructing the primary HLH mouse model was based on the literature report by Michael B. Jordan et al. (https: / / doi.org / 10.1182 / blood-2003-10-3413). Prf1- / - mice were purchased from The Jackson Lab and bred in our laboratory; genotyping was performed before in vivo experiments. The LCMV titers were diluted with 1640 medium to a final concentration of 2 × 10⁻⁶. 6 Plaque-forming units (PFU) / mL. Before inoculation, thoroughly mix the LCMV dilution solution and take 100 μL (2 × 10⁻⁶) of the solution. 5 PFU (Polydimethylformamide) solution was subcutaneously injected into the peritoneal cavity of Prf1- / - mice. After injection, monitor the injection site for any leakage and skin abnormalities.

[0185] 2.5 In vivo experiments of the HLH mouse model

[0186] 2.5.1 Grouping and Dosing Regimen of Experimental Mice

[0187] 1) The experimental mice were randomly divided into groups of 6 mice each. The specific grouping is shown in the example.

[0188] 2) Drug preparation:

[0189] Compound of Formula II: Compound of Formula II, powder, dissolved in physiological saline to a final concentration of 3 g / L (i.e., when administering the drug, the volume of Compound of Formula II / μL = body weight / g × 10, for example, 200 μL is required for a 20g mouse);

[0190] RUX: Ruxolitinib phosphate, powder, dissolved in physiological saline to a final concentration of 3 g / L (same as above);

[0191] FED: Fizzotinib hydrate, powder, dissolved in physiological saline to a final concentration of 3 g / L (same as above);

[0192] DEX: Dexamethasone phosphate, powder, dissolved in physiological saline to a final concentration of 0.3 g / L (i.e., when administering the drug, DEX volume / μL = body weight / g × 5, for example, 100 μL is required for a 20g mouse);

[0193] Etoposide: Etoposide phosphate, powder, dissolved in physiological saline to achieve a final concentration of 10 g / L (i.e., when administering the drug, the volume of Etoposide / μL = body weight / g × 5, for example, a 20g mouse needs to be administered 100 μL).

[0194] Table 3 Grouping and Dosing Regimens of HLH Mouse Model Note: Control group is the healthy group; administered by gavage twice a day with physiological saline (physiological saline volume / μL = body weight / g × 10, for example, 200μL for a 20g mouse); *Vehicle is the negative control group; administered by gavage twice a day with physiological saline (physiological saline volume / μL = body weight / g × 10, for example, 200μL for a 20g mouse).

[0195] The dosing cycle is shown in the example.

[0196] 3) Mouse euthanasia: After completing the dosing regimen, mice were anesthetized with 2.5% aphthine and euthanized, and samples were collected.

[0197] 2.5.2 Routine Blood Detection in Mice

[0198] Prepare and label the EDTA anticoagulant tube before blood collection. Collect venous blood after the treatment cycle. Gently shake the blood collection tube to thoroughly mix the blood with the anticoagulant, and place the sample in an environment of 2℃-8℃. As soon as possible after sample collection, use a complete blood count analyzer to test: red blood cell count (RBC), hemoglobin level (HgB), reticulocyte percentage (Ret), white blood cell count (WBC), and platelet count (Plt). Note that the sample volume should not be less than 300μL, and blood clots should be removed before testing to avoid sample blockage.

[0199] 2.5.3. Mouse blood biochemical detection

[0200] Prepare and label biochemistry tubes without additives before blood collection. After the treatment cycle, collect venous blood, centrifuge (1800g, 10 minutes, 4℃), and transfer the supernatant to a new EP tube. During supernatant collection, avoid aspirating the lower layer of blood cells. Centrifugation can be repeated twice to remove as many blood cells as possible. Store the sample at 2℃-8℃. Use a blood biochemistry analyzer to detect: aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and uric acid (UA).

[0201] The sample volume should be no less than 300 μL. For smaller samples, physiological saline can be used to supplement the volume to 300 μL. The dilution ratio should be recorded. Samples should be tested as soon as possible after collection. If immediate testing is not possible, samples can be stored at -20℃ and tested within 6 months.

[0202] 2.5.4 Detection of fibrinogen in mouse plasma

[0203] Prepare and label sodium citrate anticoagulant tubes before blood collection. Collect blood intravenously after the treatment cycle, and follow the same centrifugation and supernatant collection steps as in 2.5.3. The sample volume for plasma fibrinogen testing should not be less than 300 μL. If the sample volume is small, the processing is the same as in 2.5.3. Samples should be tested as soon as possible after collection. If immediate testing is not possible, the sample can be stored at -20℃ and tested within 7 days.

[0204] 2.5.5 Detection of mouse serum ferritin

[0205] 1) After the animal experiment, collect venous blood, centrifuge within 30 minutes (1000g, 15 minutes, 4℃) and collect the supernatant.

[0206] 2) Add the test sample, standard (diluted serially according to the instructions), and universal diluent to a pre-equilibrated ELISA plate at a ratio of 100 μL / well. Seal the reaction wells and incubate at 37°C for 1 hour.

[0207] 3) Discard the original liquid in the ELISA plate and add 100 μL of biotinylated antibody working solution per well. Seal the reaction wells and incubate at 37°C for 1 hour. Wash the plate 3 times after incubation.

[0208] 4) Add 100 μL of enzyme conjugate working solution per well, seal the reaction wells, and incubate at 37°C for 30 minutes. Wash the plate 5 times after incubation.

[0209] 5) Add 90 μL of chromogenic substrate per well and incubate at 37°C in the dark for 15 minutes.

[0210] 6) Add 50 μL of reaction stop solution per well, mix immediately, and use an ELISA reader to detect the OD value at 450 nm.

[0211] 7) Use Excel to plot the standard curve and calculate serum ferritin concentration, then use Graphpad to analyze the differences between groups.

[0212] 2.5.6 Mouse sCD25 Detection

[0213] 1) After the animal experiment, venous blood was collected, centrifuged (2000g, 10 minutes, 4℃) and the supernatant was collected.

[0214] 2) Add 50 μL of the test sample and standard (diluted serially according to the instructions) to each well of a microplate that has been equilibrated to room temperature. Then add 50 μL of Antibody Cocktail to each well, prepared as follows: Antibody Cocktail (3 mL) = 300 μL 10×CaptureAntibody + 300 μL 10×DetectorAntibody + 2.4 mL AntibodyDiluent5BR.

[0215] 3) After sealing the reaction wells, place the plate on a shaker at 400 rpm and incubate at room temperature for 1 hour, then wash the plate 3 times.

[0216] 4) Add 100 μL of chromogenic substrate to each well, seal the reaction wells, and place them on a shaker at 400 rpm. Incubate at room temperature in the dark for 10 minutes.

[0217] 5) Add 100 μL of reaction stop solution per well, mix on a shaker for 1 minute, and then use an ELISA reader to detect the OD value at 450 nm.

[0218] 6) Use Excel to plot the standard curve and calculate the sCD25 concentration, then use Graphpad to analyze the differences between groups.

[0219] 2.5.7 Mouse Cytokine Detection

[0220] Venous blood was collected after the dosing cycle, and the concentrations of various cytokines were measured using the Luminex assay.

[0221] 1) After centrifugation (3000g, 20 minutes, 4℃), collect the supernatant and dilute it 4 times for later use.

[0222] 2) Add 25 μL each of beads, standard, quality control and sample to each well in sequence according to the instructions. After sealing the reaction wells, place them on a shaker at 800 rpm and incubate at room temperature for 30 minutes.

[0223] 3) Wash the beads three times with 100-200 μL of washing buffer, then add 25 μL of detection antibody and incubate on a shaker at 800 rpm for 30 minutes at room temperature.

[0224] 4) After washing the beads three times, add 25 μL of PE-streptavidin and incubate at room temperature on a shaker at 800 rpm for 10 minutes.

[0225] 5) After washing the beads three times, add 100 μL of washing solution / sheath solution, mix on a shaker for 30 seconds, and then test on the instrument.

[0226] 2.5.8. HE staining of mouse organs

[0227] After the animal experiments, the heart, liver, spleen, lungs, kidneys, stomach, small intestine, and femur of the mice were dissected. After fixing the above tissues with paraformaldehyde for 24 hours, the following operations were performed:

[0228] 1) After trimming the tissue, place it in a dehydration box and dehydrate it in the following order of ethanol: 70% ethanol (2 hours), 80% ethanol (1 hour), 90% ethanol (1 hour), 95% ethanol (1 hour), anhydrous ethanol (1 hour), anhydrous ethanol II (30 minutes), xylene I (30 minutes), xylene II (30 minutes), paraffin II (1 hour).

[0229] 2) Embed the tissue in the embedding cassette and cool it at -20°C. Cut the cooled paraffin block into sections about 3 μm thick.

[0230] 3) Heat the slices at 65℃ for 20 minutes using a slicing machine.

[0231] 4) Xylene dewaxing: Xylene I (30 minutes), Xylene II (30 minutes).

[0232] 5) Ethanol hydration: The ethanol concentration gradient is 100%, 95%, 90%, 80%, and 70% respectively. Soak for 3 minutes each time, and finally rinse with water.

[0233] 6) Hematoxylin staining of cell nuclei: stain for 5 minutes and then wash with water for 5 minutes.

[0234] 7) Hematoxylin differentiation: Wash with water for 10 minutes after differentiation for 3 seconds.

[0235] 8) Hematoxylin blue return: Wash with water for 10 minutes after 5 seconds of blue return.

[0236] 9) Eosin staining of cytoplasm: Stain for 5 minutes, then wash with water for 1 second.

[0237] 10) Dehydration: Anhydrous ethanol (5 minutes × 3 times) followed by xylene (5 minutes × 2 times).

[0238] 11) After mounting with neutral resin, use a VS200 full-slide bright-field scanning system to scan the slides. The observations mainly include: whether there is damage to the normal structure of the tissue, whether there are pathological changes, such as splenic red pulp hyperplasia, renal tubular dilation, etc.; whether there is inflammatory cell infiltration, increased macrophages, hemophagocytosis, etc.

[0239] 2.5.9 Immunohistochemical staining of mouse organs

[0240] After the animal experiments, mouse liver and spleen tissues were dissected and immunohistochemically stained. The tissues were then embedded in paraffin and hydrated in ethanol, following the same procedure as in 2.5.8. The following steps were then performed:

[0241] 1) Antigen retrieval: Heat 3 g / L sodium citrate solution to boiling, place the slide in it, remove it after 5 minutes and let it cool completely at room temperature.

[0242] 2) Blocking endogenous peroxidase activity: Wash slides with PBS for 5 minutes × 3 times, add 80 μL of 3% hydrogen peroxide to the slides, incubate at room temperature for 10 minutes, and then wash with PBS for 3 minutes × 2 times.

[0243] 3) Blocking: Add 50 μL of goat serum to the slide and incubate in a humidified chamber at 37°C for 30 minutes;

[0244] 4) Antibody incubation: Add the primary antibody diluted according to the ratio to the slide, block the slide at 4°C overnight, then warm it at 37°C for 45 minutes, and wash with PBS for 3 minutes × 3 times; then add the secondary antibody diluted according to the ratio to the slide, incubate at room temperature for 45 minutes, and wash with PBS for 3 minutes × 3 times.

[0245] 5) Staining: Add freshly prepared DAB staining solution to the slide, stain for 5 minutes, then rinse with water to stop the staining process.

[0246] 6) Counterstaining and mounting: Counterstain sections with hematoxylin for 30 seconds, then rinse with water. Differentiate with hydrochloric acid and ethanol for 2 seconds, then rinse again with water. Dehydrate with a gradient of ethanol concentrations of 75%, 80%, 95%, and 100%. Soak sections in xylene to make them clear, then mount with neutral resin. Scan the sections using a VS200 all-glass slide brightfield scanning system.

[0247] 2.5.10. Western blot detection of JAK / STAT pathway protein expression in mouse spleen

[0248] After administration, mice were euthanized, spleen tissue was dissected and cut into small pieces, 50-60 mg was weighed and ground using a syringe plunger. Note that the spleen was kept on ice throughout the process after being removed from the body. 500 μL of the prepared lysis buffer (prepared as in 2.3.5.1) was added to each sample to lyse the cells. The subsequent steps for cell lysis until protein denaturation were the same as in 2.3.3.1.

[0249] 2.6. Mechanistic exploration of compound II in secondary HLH mouse model

[0250] 2.6.1 RNA-seq analysis in secondary HLH mice

[0251] After the dosing regimen was completed, the spleens of three fresh mice from the Vehicle group and the single-drug group of Formula II were harvested, flash-frozen in liquid nitrogen, and transported on dry ice to Beijing Geneplus Technology Co., Ltd. for transcriptome sequencing. Upon receiving the RNA-seq results, further analysis was performed using Rstudio. Differential gene analysis was conducted using the DESeq2 package, heatmaps were generated using the ComplexHeatmap package, volcano plots were generated using the ggplot2 package, and GO, KEGG, and GSEA enrichment analyses were performed using the clusterProfile package.

[0252] 2.6.2 Immunofluorescence staining of spleen in secondary HLH mice

[0253] After the animal experiments were completed, the mouse spleen was dissected, embedded in paraffin, and then hydrated in ethanol (step 2.5.8). The following procedures were then performed:

[0254] 1) Draw a circle around the tissue to be tested with an immunohistochemical pen to prevent the liquid from flowing out. Add goat serum to the surface of the slide and then block at room temperature for 1 hour.

[0255] 2) Primary antibody incubation: After discarding the goat serum, add GCR dilution buffer (1:200) to fully cover the slide and place it in a humidified chamber at 4°C overnight.

[0256] 3) Wash the slides with PBS for 3 minutes each time.

[0257] 4) Secondary antibody incubation: After adding anti-mouse fluorescent secondary antibody (AF596) to cover the slide, incubate at room temperature in the dark for 1.5 hours.

[0258] 5) Wash again with PBS for 3 minutes × 3 times, counterstain the cell nuclei with DAPI, and then let stand at room temperature in the dark for 5 minutes.

[0259] 6) After washing with PBS for 3 minutes × 3 times, wait for the slides to air dry slightly, add anti-fluorescence quenching mounting medium, mount the slides, observe and preserve them under an inverted fluorescence microscope.

[0260] 2.6.3 Immunofluorescence staining of mouse spleen lymphocytes

[0261] (1) Extraction of mouse spleen lymphocytes: After dissecting the mouse spleen, it was cut into small pieces, placed on a 40μm filter screen and ground using a syringe plunger. The filter screen was then rinsed with tissue dilution solution and filtered again. Lymphocytes were extracted using a mouse spleen lymphocyte separation kit.

[0262] 1) Add an equal volume of separation medium to a 15 mL BD tube, slowly spread the cell suspension evenly on the separation medium, and centrifuge (900 g, 30 min, acceleration 3, deceleration 3).

[0263] 2) Place a 10 μL pipette tip over a 1000 μL pipette tip, carefully aspirate the middle white membrane layer using a pipette, transfer it to a clean 15 mL BD tube, add 10 mL of cell washing buffer, wash, and centrifuge (250 g, 10 min). Repeat the washing process twice.

[0264] 3) Discard the supernatant and resuspend the cells for later use.

[0265] (2) Grouping and administration regimen: A total of 5 treatment groups were set up, as shown in Table 4.

[0266] Table 4 Grouping and Dosing Regimens of Mouse Splenic Lymphocytes

[0267] (3) Immunofluorescence staining: After the drug treatment, the above 5 groups of cells were collected and further immunofluorescence staining was performed.

[0268] 1) Prepare cell smears and wash with PBS for 5 minutes each time, 3 times.

[0269] 2) Add goat serum to the cell smear, cover it completely, and then block it at room temperature for 20 minutes.

[0270] 3) After adding GCR dilution solution (1:100) to the cell smear, place it in a humidified chamber at 4°C overnight.

[0271] 4) After washing the cell smear with PBS for 5 minutes × 3 times, add anti-rabbit fluorescent secondary antibody (AF488) and incubate at 37°C for 30 minutes. Wash again with PBS (5 minutes × 3 times).

[0272] 5) After adding DAPI to the cell smear, incubate at room temperature for 10 minutes, then wash with PBS (5 minutes × 3 times).

[0273] 6) After mounting with anti-fluorescence attenuation mounting medium, observe and preserve the slides as soon as possible using an inverted fluorescence microscope.

[0274] 2.6.4 Immunoprecipitation of mouse spleen lymphocytes

[0275] The grouping and administration regimens are the same as in 2.6.3. Collect and count the cells from the above 5 groups, with each group requiring at least 1×10⁶ cells.7 One cell was used for subsequent experiments:

[0276] 1) Protein extraction: After centrifugation (4℃, 500g, 5 min), cells were collected and washed three times with PBS pre-cooled to 4℃. The appropriate amount of IPlysis buffer and phosphatase inhibitor were added according to the volume of the cell pellet. Cells were then lysed on ice for 30 min, shaking the cells every 10 min. After lysis, the cells were centrifuged (4℃, 10000g, 20 min), and the supernatant was transferred to a new EP tube. Protein concentration was determined using the Bradford method.

[0277] 2) Immunoprecipitation: Take 1.8 mg of protein lysate and add it to spincolumns with endcaps at the bottom, along with 2 μg of GCR antibody and 200 μL of incubation buffer. Add 2 μg of rabbit-derived Control IgG as a negative control to an equal volume of protein lysate and incubation buffer. Incubate overnight at 4°C. To precipitate the immune complex, add 50 μL of resuspended Protein Asphaltene Beads Slurry to the spincolumns and incubate at 4°C for 1–4 hours. Remove the endcaps and discard the supernatant. Wash the precipitated complex five times with 800 μL of washing buffer, then centrifuge the spincolumns in collection tubes (4°C, 500 rpm, 30 seconds).

[0278] 3) Elution: Transfer the spincolumns to a new EP tube and elute the precipitated complex with 40 μL of elution buffer. Centrifuge twice (4°C, 10,000 rpm, 1 min) and collect the elution products. Add 10 μL of Alkaline utralization buffer and 23 μL of 5× Sample Buffer to the elution products and heat in a boiling water bath for 5 min.

[0279] 4) Western blot validation: Take 10 μL of IP sample and add it to the corresponding lane. For this Western blot, HRP-conjugated Mouse Anti-Rabbit IgG Light Chain Specific secondary antibody dilution buffer (1:1000) was used. Subsequent steps are the same as in 2.3.5.

[0280] 2.7. Mechanistic exploration of compound II in primary HLH mouse model

[0281] (1) During the administration period, the spleens of three fresh mice in the compound group of formula II were collected on D3, 6, 9, 12, 15, 18 and 21 respectively. The extraction steps of spleen lymphocytes were the same as in 2.6.3.

[0282] (2) Cell staining:

[0283] 1) After cell counting, wash the cells once with DPBS and centrifuge (300g, 5 minutes). Resuspend the cells in DPBS to 10⁻⁶. 6 -10 7 Cells / mL.

[0284] 2) Add 1 μL LFVS700 (1:1000) to each 1 mL of cell suspension and incubate at room temperature in the dark for 15 minutes. Then wash the cells twice with 2 mL of StainBuffer (300 g, 5 minutes), discard the supernatant, and resuspend the cells in the remaining StainBuffer in the tube after the second wash.

[0285] 3) In a 100 μL system, every 10 6 Each cell was pretreated with 1 μg of BDFcBlock at 4°C for 5 minutes. Anti-CD3, CD8, and CD44 antibodies were added according to the manufacturer's recommended dosage, and the cells were stained at room temperature in the dark for 30 minutes.

[0286] 4) After surface staining, mix FixBuffer I preheated to 37°C with the cell suspension at a volume ratio of 1:1, incubate at 37°C for 10 minutes, centrifuge (250g, 10 minutes), discard the supernatant, and wash the cells once with StainBuffer (250g, 10 minutes).

[0287] 5) Slowly add PermBuffer III pre-cooled to -20°C to infiltrate the cells using a vortex mixer, then incubate on ice for 30 minutes. Centrifuge the cells (500g, 5 minutes), discard the supernatant, and wash the cells twice with StainBuffer (250g, 10 minutes).

[0288] 6) Add cells to Stain Buffer at a concentration of 1 × 10⁻⁶. 7 Resuspend samples at 1000 μL / ml, stain with anti-Perforin antibody at room temperature for 30 minutes, and analyze using flow cytometry as soon as possible. If immediate analysis is not possible, store the samples at 4°C and run them within 12 hours.

[0289] 2.8 Statistical Analysis

[0290] Statistical analysis was performed using GraphpadPrism (version 9.0), Rstudio (version 2023), FlowJo (version 10.8.1), and ModFitLT (version 5.0). ModFitLT and FlowJo were used to process flow cytometry results, GraphpadPrism was used to process cell proliferation, immunofluorescence, and in vivo experimental results, and Rstudio was used to process RNA-seq results. T-tests were used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, two-way ANOVA was used for cell proliferation, and log-rank tests were used for survival analysis. P < 0.05 was considered statistically significant. Each experiment was repeated three times, and data were presented as mean ± standard deviation or 95% confidence intervals.

[0291] Example 1: High expression of JAK / STAT pathway genes in HLH patients

[0292] RNA-seq data from peripheral blood mononuclear cells of 33 HLH patients and 37 healthy individuals were compared and analyzed in the GEO database. The results are shown in Figure 1. The results showed that the JAK family (JAK2) had a P-value of 7.95 × 10⁻⁶. -4 In JAK3, the p-value is 2.18 × 10⁻⁶. -7 The p-value in TYK2 is 2.86 × 10⁻⁶. -6 ) and the STAT family (STAT1 has a p-value of 0.14, and STAT2 has a p-value of 3.68 × 10⁻⁶). -7 The p-value in STAT3 is 3.52 × 10⁻⁶. -10 The p-value in STAT4 is 1.27 × 10⁻⁶. -6 The p-value in STAT5a is 1.32 × 10⁻⁶. -7 The p-value in STAT5b is 1.52 × 10⁻⁶. -7 The gene is significantly overexpressed in HLH patients, providing a theoretical basis for the treatment of HLH with compound formula II.

[0293] Example 2: In vitro pharmacodynamics of compound II in HLH

[0294] 1. Compound of Formula II exhibits significant anti-proliferative activity against key effector cells of HLH.

[0295] Different concentrations of compound II (0.05 μM–10 μM) were applied to CTLL-2 (mouse T lymphocyte cell line) and RAW264.7 (mouse mononuclear macrophage cell line) cell lines for 24 h, 48 h, and 72 h. The results are shown in Table 5. Cell proliferation inhibition assays showed that compound II had an IC50 effect at 48 h and 72 h. 50Less than 0.5 μM, its IC50 value in CTLL-2 and RAW264.7 cell lines was less than 0.5 μM, and its IC50 value after 48 h was less than 0.5 μM. 50 The values ​​were 0.4043 μM and 0.3426 μM, respectively, and the IC50 values ​​after 72 hours were... 50 The values ​​were 0.2347 μM and 0.2045 μM, respectively.

[0296] Under the same conditions, the two cell lines were treated with different concentrations of RUX, FED, and DEX (0.05 μM–10 μM), and the results are shown in Table 5. The results showed that the IC50 values ​​of RUX, FED, and DEX at 24 h, 48 h, and 72 h were... 50 The values ​​were all significantly higher than those of compound II. This indicates that compound II exhibits significant in vitro antiproliferative activity against key HLH effector cell lines, and is significantly superior to RUX, FED, and DEX.

[0297] Table 5. Antiproliferative activities of Formula II compounds, RUX, FED, and DEX against CTLL-2 and RAW264.7 cell lines. Note: * indicates P < 0.05 compared to compound II; ** indicates P < 0.01 compared to compound II; *** indicates P < 0.001 compared to compound II; **** indicates P < 0.0001 compared to compound II.

[0298] 2. Compound II inhibits the secretion of IL-6, TNF-α, and IL-1β by key effector cells in HLH.

[0299] RAW264.7 cells were pretreated with 1 μg / mL lipopolysaccharide (LPS) for 30 min, followed by treatment with 1 μM of compound II, RUX, FED, and DEX for 24 h. The concentrations of IL-6, TNF-α, and IL-1β were detected by ELISA, and the results are shown in Table 6. The results showed that LPS treatment activated RAW264.7 cells and led to the secretion of large amounts of IL-6, TNF-α, and IL-1β. Compound II significantly inhibited the secretion of these three cytokines, and its effect was significantly superior to that of RUX, FED, and DEX.

[0300] Table 6. Concentrations of TNF-α, IL-6, and IL-1β secreted by the RAW264.7 cell line

[0301] Compared to Vehicle, *** indicates P < 0.001; **** indicates P < 0.0001;

[0302] Compared with compound of formula II, # This indicates that P < 0.05; ## This indicates that P < 0.01; ###This indicates that P < 0.001; #### This means P < 0.0001.

[0303] 3. Compound II inhibits JAK / STAT protein expression in key HLH effector cells.

[0304] CTLL-2 and RAW264.7 cell lines were treated with different concentrations of Formula II compound and LPS for 16 h. The expression of total JAK2, STAT1, STAT3, and STAT5 proteins and their phosphorylated forms was detected by Western blot, and the results are shown in Figure 2. The results showed that with increasing concentration of Formula II compound, the expression of phosphorylated JAK2 and STAT5 proteins was downregulated.

[0305] Example 3: Pharmacodynamics of Compound II in Secondary HLH Mice

[0306] In vitro experiments confirmed that compound II has significant anti-HLH activity. To further verify the pharmacodynamics of compound II in HLH, this invention used CpG-ODN1826 to repeatedly stimulate TLR9, simulating an autoimmune disease, to construct a mouse model of secondary HLH for in vivo experiments. Six- to eight-week-old C57BL / 6 mice were used. Starting on day 0, CpG-ODN1826 was injected intraperitoneally every other day. Starting on day 4, single-drug treatments with compound II, RUX, FED, and DEX were administered. Mice were divided into six groups: Control group, Vehicle group, compound II group, RUX group, FED group, and DEX group. The administration regimens are shown in Table 3. Administration continued until day 11.

[0307] 1. Compound of Formula II improves the general condition of mice with secondary HLH.

[0308] Figure 3 shows the changes in body weight and body temperature in mice with secondary HLH during treatment. As shown in Figure 3, the body weight of mice in the Control group gradually increased from D0; the body weight of mice in the Vehicle group decreased from D0 to D2 due to disease consumption, and then the hepatosplenomegaly interacted with disease consumption, with body weight rebounding on D2, and body weight gradually decreasing from D4; the body weight change trend of the RUX and FED monotherapy groups was similar to that of the Vehicle group, and on D11, the body weight of mice in the RUX and FED monotherapy groups was greater than that of the Vehicle group; the liver and spleen of mice in the Formula II compound and DEX monotherapy groups shrank to varying degrees and their physical condition gradually recovered, with body weight rebounding from D8, and on D11, the body weight of mice in the Formula II compound and DEX monotherapy groups was greater than that of the RUX and FED monotherapy groups.

[0309] As shown in Figure 3, the body temperature of mice was monitored using an infrared thermometer. The results showed that the body temperature of mice in the Control group fluctuated between 36.3-36.5℃; the body temperature of mice in the Vehicle group started to rise sharply on D0, and then fluctuated between 36.5-36.6℃ after D4, without a downward trend; the body temperature of mice in the Formula II single-drug group started to gradually decrease and return to normal on D2, similar to that of the DEX single-drug group; the body temperature of mice in the RUX and FED single-drug groups decreased later than that of the Formula II single-drug group. The body temperature of mice in the RUX single-drug group started to decrease on D6, and the body temperature of mice in the FED single-drug group started to decrease on D4.

[0310] 2. Compound II improves blood routine in mice with secondary HLH.

[0311] After administration, peripheral blood samples were collected from mice for routine blood tests, and the results are shown in Table 7. The results showed that compared to the Control group, the Vehicle group exhibited significant cytopenia, while the single-drug group of compound II significantly improved hemoglobin levels, red blood cell count, white blood cell count, and platelet count. For each administration group: the single-drug group of compound II showed significantly higher hemoglobin levels and red blood cell counts compared to the RUX and DEX single-drug groups, significantly higher white blood cell counts compared to the RUX and FED single-drug groups, and significantly higher platelet counts compared to the RUX and FED single-drug groups.

[0312] Table 7 Blood routine tests in mice with secondary HLH

[0313] 3. Compound II improves blood biochemistry in mice with secondary HLH.

[0314] After administration, peripheral blood samples were collected from mice for blood biochemistry analysis, and the results are shown in Table 8. The results showed that, compared to the Control group, the Vehicle group exhibited significantly impaired liver function and elevated blood lipids. Compared to the Vehicle group, the single-drug group of compound II showed significantly lower triglycerides and significantly higher high-density lipoprotein (HDL), indicating improved blood lipids; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum total bilirubin were significantly lower, indicating improved liver function. For the treatment group: regarding improved liver function, the single-drug group of compound II showed significantly lower aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels compared to the RUX, FED, and DEX single-drug groups, and significantly lower serum total bilirubin levels compared to the RUX single-drug group; regarding improved blood lipids, the single-drug group of compound II showed significantly lower triglycerides and significantly higher HDL levels compared to the RUX and DEX single-drug groups.

[0315] Table 8 Blood Biochemistry of Secondary HLH Mice

[0316] 4. Compound II improves fibrinogen levels in secondary HLH mice.

[0317] After drug administration, peripheral blood samples were collected from mice to measure plasma fibrinogen levels. The results are shown in Table 9. The results showed that fibrinogen levels were significantly lower in the Vehicle group compared to the Control group, indicating abnormal coagulation function. Fibrinogen levels in the compound II monotherapy group were significantly higher than in the Vehicle group, and significantly better than in the RUX monotherapy and DEX monotherapy groups.

[0318] Table 9 Fibrinogen in Secondary HLH Mice

[0319] Compared with compound II, * indicates P < 0.05; ** indicates P < 0.01.

[0320] 5. Compound II improves serum iron ss protein and sCD25 in secondary HLH mice.

[0321] After drug administration, peripheral blood samples were collected from mice to measure serum ferritin and sCD25 levels. The results are shown in Table 10. The results showed that, compared with the Control group, the Vehicle group had significantly elevated serum ferritin and sCD25 levels. Compound II could significantly correct the above abnormalities and had a significant advantage over other drug groups.

[0322] Table 10 Serum ferritin and sCD25 in mice with secondary HLH

[0323] 6. Compound II improves hypercytokineemia in secondary HLH mice.

[0324] After drug administration, peripheral blood samples were collected from mice to measure cytokine concentrations. The results are shown in Table 11. The results showed that, compared to the Control group, the Vehicle group exhibited significantly elevated levels of multiple cytokines, including IL-6, TNF-α, and IFN-γ, indicating hypercytokineemia. Cytokine levels in the compound II group were significantly lower than in the Vehicle group.

[0325] Table 11 Cytokine Concentrations in Secondary HLH Mice Note: GM-CSF, granulocyte-macrophage colony stimulating factor; MIP-1α, macrophage inflammatory protein-1α.

[0326] 7. Compound II improves hepatosplenomegaly in secondary HLH mice.

[0327] After administration, liver and spleen tissues were collected from mice and weighed. The results are shown in Figure 4 and Table 12. The results showed that the Vehicle group exhibited significant hepatosplenomegaly, while the liver and spleen in the compound II monotherapy group were significantly smaller than those in the Vehicle group. In improving hepatomegaly, the compound II monotherapy group was significantly superior to the RUX and FED monotherapy groups, and superior to the DEX monotherapy group; in improving splenomegaly, the compound II monotherapy group was significantly superior to the RUX and FED monotherapy groups.

[0328] Table 12 Liver and spleen weight and percentage in secondary HLH mice

[0329] Compared with compound II, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001;

[0330] 8. Compound II improves inflammatory response and hemophagocytosis in secondary HLH mice.

[0331] After drug administration, liver, spleen, and bone marrow tissues from mice were collected for HE staining, and the results are shown in Figures 5-6. The results showed that, compared to the Control group, the normal spleen structure in the Vehicle group was disrupted, with red pulp hyperplasia and fewer splenic bodies, sinuses, and trabeculae. Instead, a large number of macrophages infiltrated the spleen, and abnormal phagocytosis of red blood cells and other blood cells was observed, i.e., "hemophagocytosis." In the Vehicle group, a large number of inflammatory cells accumulated around the blood vessels in the liver, hepatocytes were disordered, and hepatocyte necrosis occurred, with multiple instances of "hemophagocytosis." The number of hematopoietic cells in the bone marrow of the Vehicle group was significantly reduced. HE staining results of the compound II group showed that, compared to the Vehicle group, the inflammatory manifestations in the liver, spleen, and bone marrow were significantly reduced, and "hemophagocytosis" was essentially eliminated.

[0332] 9. Compound of formula II inhibits the activation of the JAK / STAT signaling pathway in secondary HLH mice.

[0333] After drug administration, liver and spleen tissues from mice in the Control, Vehicle, and Compound II groups were collected for Western blot and immunohistochemical staining to assess the expression of phosphorylated JAK2 and its downstream proteins STAT1, STAT3, and STAT5. The results are shown in Figure 7. The results showed that compared with the Control group, the expression of phosphorylated JAK2 and STAT5 proteins was significantly upregulated in the Vehicle group, suggesting the activation of the JAK / STAT signaling pathway in secondary HLH mice. Compound II, on the other hand, significantly downregulated the expression of phosphorylated JAK2 and STAT5 proteins.

[0334] 10. Compound of Formula II has no significant toxicity to secondary HLH mice.

[0335] After the animal experiments, the heart, lungs, kidneys, stomach, and small intestine of three mice in each group were stained with hematoxylin and eosin (HE). Organ lesions were graded according to the International Handling Standard (INHAND) describing the toxicological pathology terminology and diagnostic criteria for experimental animals (see Table 13). The results are shown in Figure 8. The results showed that in the compound II group, one case showed grade 1 alveolar wall laminar infiltration, and one case showed grade 1 small intestinal epithelial shedding; in the RUX group, one case showed grade 2 alveolar inflammatory cell infiltration, one case showed grade 1 renal tubular dilatation, and one case showed grade 1 cardiac vacuolar degeneration; in the FED group, one case showed grade 1 renal tubular dilatation; and in the DEX group, one case showed grade 1 scattered erythrocytes in the alveolar cavity. Therefore, compound II has no significant toxic side effects on the vital organs of mice with secondary HLH.

[0336] Table 13 Evaluation Criteria for Organ Pathological Changes in Mice

[0337] Example 4: Pharmacodynamics of Compound II in Combination with DEX in a Secondary HLH Mouse Model

[0338] The method for modeling secondary HLH in mice was the same as in Example 3. Considering that etoposide combined with DEX is the main treatment for HLH, this treatment was included in the combination drug experiment. Mice were divided into 5 groups: Vehicle group, compound II group, DEX group, compound II + DEX (FD) group, and Etoposide + DEX (ED) group. The dosing regimen is shown in Table 3. Dosing started on day 4 and continued until day 11 (D11).

[0339] 1. Compound of Formula II combined with DEX improves body temperature in mice with secondary HLH.

[0340] Elevated body temperature is one of the diagnostic criteria for HLH, and body temperature in mice was monitored. The results showed that the body temperature of mice in the treatment group gradually decreased from the first day of drug administration (D4), and the body temperature tended to normalize by D11. The decreasing trend of body temperature in the FD group was more obvious than that in the compound II group and the DEX monotherapy group, but there was no significant difference from the ED group.

[0341] 2. Compound of Formula II combined with DEX improves blood routine in mice with secondary HLH.

[0342] After drug administration, peripheral blood samples were collected from mice for complete blood count (CBC) analysis, and the results are shown in Table 14. The results showed that compared to the compound II group and the DEX monotherapy group, the FD combination therapy group exhibited significantly higher hemoglobin levels, red blood cell count, white blood cell count, and platelet count, and a significantly lower reticulocyte percentage, suggesting that compound II can synergistically improve CBC abnormalities in HLH with DEX. Furthermore, compared to the FD group, the ED group showed a significantly lower white blood cell count and a significantly higher reticulocyte percentage, suggesting that etoposide-induced chemotherapy-induced myelosuppression, indicating that FD combination therapy is superior to the current primary regimen ED in improving CBC results.

[0343] Table 14 Blood routine tests in mice with secondary HLH

[0344] 3. Compound of Formula II combined with DEX improves blood biochemistry in mice with secondary HLH.

[0345] After drug administration, peripheral blood samples were collected from mice for blood biochemistry analysis, and the results are shown in Table 15. The results showed that the FD combination therapy group was significantly superior to the compound of formula II and the DEX monotherapy group in improving aspartate aminotransferase (AST), alanine aminotransferase (ALT), and uric acid levels. Similarly, the FD combination therapy group was significantly superior to the DEX monotherapy group in improving serum total bilirubin, triglycerides, and high-density lipoprotein (HDL). These two points suggest that the compound of formula II can synergistically improve liver and kidney function and reduce blood lipids in HLH patients with DEX. Furthermore, the FD group showed significantly lower levels of AST, ALT, and uric acid compared to the ED group, indicating that the FD combination therapy is superior to the ED group in improving liver and kidney function.

[0346] Table 15 Blood Biochemistry of Secondary HLH Mice

[0347] 4. Compound of Formula II combined with DEX improves fibrinogen levels in secondary HLH mice.

[0348] After administration, peripheral blood samples were collected from mice to measure plasma fibrinogen levels. The results are shown in Table 16. The results showed that, compared with the Vehicle group, the plasma fibrinogen level in the DEX monotherapy group was not significantly improved, but the fibrinogen level increased significantly after combining with compound II, suggesting that compound II can synergistically improve the coagulation dysfunction of HLH with DEX.

[0349] Table 16 Fibrinogen in Secondary HLH Mice

[0350] 5. Compound II combined with DEX improves serum ferritin and sCD25 in secondary HLH mice.

[0351] After drug administration, peripheral blood samples were collected from mice to measure serum ferritin and sCD25 levels. The results are shown in Table 17. The results showed that the FD combination therapy group was significantly superior to the compound of formula II and the DEX monotherapy group in improving serum ferritin. Similarly, the FD combination therapy group was significantly superior to the DEX monotherapy group in improving sCD25. These two points suggest that the compound of formula II can synergistically improve the elevated serum ferritin and sCD25 levels in HLH with the help of DEX. Furthermore, sCD25 levels in the FD group were significantly lower than those in the ED group, indicating that the FD combination therapy is superior to ED in improving sCD25 levels.

[0352] Table 17 Serum ferritin and sCD25 in mice with secondary HLH

[0353] 6. Compound of Formula II combined with DEX improves hepatosplenomegaly in secondary HLH mice.

[0354] After drug administration, liver and spleen tissues were collected and weighed from mice. The results are shown in Figure 9 and Table 18. The results showed that the combination therapy with FD was significantly more effective than compound II and DEX alone in improving hepatosplenomegaly, suggesting that compound II can synergistically improve hepatosplenomegaly in HLH with the help of DEX. Furthermore, the FD group showed significantly better improvement in hepatomegaly than the ED group; the spleen weight / body weight ratio was also significantly better in the FD group than in the ED group, indicating that the combination therapy with FD is superior to ED in improving hepatosplenomegaly.

[0355] Table 18 Liver and spleen weight and percentage in secondary HLH mice

[0356] 7. Compound of Formula II combined with DEX improves hemophagocytosis in secondary HLH mice.

[0357] After drug administration, liver, spleen, and bone marrow tissues from mice were collected for HE staining, and the results are shown in Figure 10. The results showed that multiple hemophagocytic phenomena were observed in the liver of the DEX monotherapy group, while no obvious hemophagocytic phenomena were observed in the spleen, but significant tissue atypia was observed; the ED group also showed abnormal spleen structure; the FD group showed no obvious hemophagocytic phenomena in the liver, spleen, and bone marrow, the spleen cells were neatly arranged, and no obvious atypia was observed in the liver tissue, suggesting that compound II can synergistically improve the hemophagocytic phenomenon in HLH with DEX, which is superior to ED.

[0358] Example 5: Mechanism exploration of compound II in combination with DEX in a secondary HLH mouse model

[0359] DEX is a synthetic glucocorticoid with a similar structure and function to naturally occurring glucocorticoids. Glucocorticoids primarily exert their effects by binding to the glucocorticoid receptor (GCR) to form a glucocorticoid-receptor complex, thereby entering the cell nucleus. Therefore, in subsequent experiments, this invention mainly focuses on exploring the mechanism of action of compound II in combination with DEX targeting the GCR.

[0360] 1. Compound of Formula II promotes GCR entry into the cell nucleus.

[0361] Numerous studies have demonstrated that hypercytokineemia, especially hyperIL-2emia, can induce DEX resistance. Considering that HLH is a typical cytokine storm, this invention performed GCR immunofluorescence staining on the spleens of DEX-treated mice, as shown in Figure 11. The results showed that there were fewer GCRs in the cell nucleus; after the combined application of compound II, compared with the DEX monotherapy group, the FD group showed a significant increase in GCR nuclear translocation, suggesting that the synergistic effect of compound II and DEX may be related to the increase in GCR nuclear translocation.

[0362] To further verify that IL-2 inhibits GCR nuclear translocation, primary mouse spleen lymphocytes were treated with IL-2 and dexamethasone. The results are shown in Figure 12 and Table 19. The results showed that no significant GCR nuclear translocation was observed in the blank control group, while GCR nuclear translocation was significantly increased in the DEX monotherapy group. However, IL-2 pretreatment significantly inhibited DEX-induced GCR nuclear translocation. Using compound II to treat IL-2-treated cells, the results showed that DEX-induced GCR nuclear translocation was restored, suggesting that compound II can promote GCR entry into the cell nucleus.

[0363] Table 19. Average percentage of GCR-positive cells Note: ** indicates comparison with the Blank group, P<0.01; ## indicates comparison with the DEX group, P<0.01; && indicates comparison with the IL-2+DEX group, P<0.01.

[0364] 2. Compounds of Formula II inhibit the formation of the p-STAT5-GCR complex.

[0365] IL-2 signaling achieves its pleiotropic effects through multiple pathways, with the JAK / STAT signaling pathway playing a dominant role. Upon binding to the IL-2 receptor, IL-2 induces a signaling cascade, leading to the activation and phosphorylation of the major transcription factor STAT5. As a key protein in the downstream pathway of IL-2, this invention primarily examines whether IL-2 can induce p-STAT5 to interact with the GCR, thereby preventing GCR nuclear translocation.

[0366] The results of the immunoprecipitation are shown in Figure 13. The results showed that the p-STAT5-GCR complex was formed after IL-2 pretreatment. Previous in vitro and in vivo experiments have confirmed that compound II can inhibit p-STAT5 protein expression. Therefore, when compound II was used to treat cells after IL-2 treatment, the results showed that no significant p-STAT5-GCR complex formation was observed in the compound II treatment group compared to the IL-2 pretreatment group. This indicates that compound II increases GCR nuclear translocation and reduces DEX resistance by inhibiting the formation of the p-STAT5-GCR complex, thus synergistically exerting an anti-HLH effect with DEX.

[0367] Example 6: Pharmacodynamics of Compound II in a Primary HLH Mouse Model

[0368] Compound II, as a single agent and in combination with DEX, has shown good efficacy in a mouse model of secondary HLH. To further explore the pharmacodynamics of compound II in primary HLH, this invention uses LCMV to inoculate Prf1- / - mice to construct a mouse model of primary HLH for further in vivo experiments.

[0369] Using 6-8 week old Prf1- / - mice, LCMV containing 2×10^5 PFU was injected intraperitoneally on day 0, and drug treatment was started on day 4. The mice were divided into 8 groups: Control group, Vehicle group, Formula II compound group, RUX group, DEX group, FD group, RUX+DEX(RD) group and ED group. The dosing regimen is shown in Table 3.

[0370] 1. Compound of Formula II improves disease severity and survival in primary HLH mice.

[0371] Starting from day 4, mice were administered the drug continuously for 21 days, and their survival status was recorded. The results are shown in Figure 14. The results showed that mice in the Vehicle group gradually died from day 10 until day 14 when no mice survived. For the single-drug groups: one mouse in the RUX group died on day 6, which was considered to be related to adverse drug reactions; mice in the DEX group gradually died from day 12 until day 18 when no mice survived; no deaths were observed in the compound II group. For the combination therapy groups: mice in the ED group gradually died from day 9 until day 18 when no mice survived, which was considered to be related to chemotherapy-related adverse reactions; no deaths were observed in the FD and RD groups.

[0372] Simultaneously, the general condition of the mice was observed, and the results are shown in Figure 15. The results showed that the Vehicle group mice gradually deteriorated in general condition starting from day 6, mainly manifested as reduced food intake, emaciation, decreased activity, and ruffled fur. In the later stages of the disease, these symptoms gradually progressed to curling up, shivering, decreased body temperature, arched back, closed eyes, and loss of skin elasticity. The same changes were observed in the DEX and ED groups. Mild ruffled fur was observed in the RUX group. The above changes were not obvious in surviving mice in the Formula II compound, FD, and RD groups, especially in the FD group where the mice remained consistently active.

[0373] 2. Compound II improves blood routine in primary HLH mice.

[0374] Previous experiments confirmed that mice began dying primarily on day 9. Based on previous literature, subsequent blood sampling and tissue dissection experiments were uniformly scheduled for day 9 euthanasia. After drug administration, peripheral blood was collected from mice for complete blood count (CBC) analysis, and the results are shown in Table 20. The results showed that compared to the Control group, the Vehicle group had significantly reduced hemoglobin levels, red blood cell count, white blood cell count, and platelet count. Both the single-drug group of compound II and the combination therapy group of FD significantly corrected these CBC changes. For the single-drug group, compound II significantly corrected thrombocytopenia better than RUX; for the combination therapy group, FD significantly corrected leukopenia better than ED, likely due to chemotherapy-induced myelosuppression caused by ED. Furthermore, the combination therapy of FD significantly corrected leukopenia and anemia better than compound II and DEX alone. These results support the application of compound II alone and FD in correcting CBC abnormalities in primary HLH.

[0375] Table 20 Blood routine tests in primary HLH mice

[0376] 3. Compound II improves blood biochemistry in primary HLH mice.

[0377] After drug administration, peripheral blood samples were collected from mice for blood biochemistry analysis, and the results are shown in Table 21. The results showed that, compared to the Control group, the Vehicle group exhibited significant liver and kidney dysfunction and elevated blood lipids. Both the single-drug group of compound II and the combination therapy group with FD significantly improved liver and kidney function and reduced blood lipids. For the single-drug groups, compound II showed significantly better correction of liver function than RUX and significantly better correction of blood lipids than DEX. For the combination therapy groups, FD showed significantly better correction of kidney function than ED, which is considered to be related to ED-related chemotherapy-induced renal impairment.

[0378] Table 21 Blood Biochemistry of Primary HLH Mice

[0379] 4. Compound II improves fibrinogen levels in primary HLH mice.

[0380] After drug administration, peripheral blood samples were collected from mice to measure plasma fibrinogen levels. The results are shown in Table 22. The results showed that compared to the Control group, the Vehicle group exhibited a significant decrease in fibrinogen. Both the single-drug group of compound II and the combination therapy group with FD significantly corrected this change. Furthermore, the combination therapy with FD showed significantly better fibrinogen correction than compound II and DEX alone, supporting the application of compound II alone and the combination therapy with FD in correcting primary HLH coagulation dysfunction.

[0381] Table 22 Fibrinogen Levels in Primary HLH Mice

[0382] 5. Compound II improves serum ferritin and sCD25 in primary HLH mice.

[0383] After drug administration, peripheral blood samples were collected from mice to measure serum ferritin and sCD25 levels. The results are shown in Table 23. The results showed that, compared with the Control group, the Vehicle group exhibited significantly elevated serum ferritin and sCD25 levels. Both the single-drug group of compound II and the combination therapy group of FD significantly corrected these changes. For the single-drug groups, compound II significantly corrected serum ferritin levels better than RUX and DEX; for the combination therapy groups, FD significantly corrected sCD25 levels better than RD. Furthermore, the combination therapy of FD significantly corrected serum ferritin levels better than DEX alone, and significantly corrected sCD25 levels better than compound II and DEX alone, supporting the application of compound II alone and the combination therapy of FD in correcting abnormalities of serum ferritin and sCD25.

[0384] Table 23 Serum ferritin and sCD25 in primary HLH mice

[0385] 6. Compound II improves hypercytokineemia in primary HLH mice.

[0386] After the administration was completed, peripheral blood samples were taken from mice to detect cytokine levels. The results are shown in Table 24. The results showed that compared with the Control group, the Vehicle group showed a significant increase in multiple cytokines such as IL-2, IL-1β, and IL-17A, indicating hypercytokineemia. Both the single-drug group of compound II and the FD combination drug group could significantly inhibit the increase of the above cytokines. For the single-drug groups: Compared with DEX, compound II significantly reduced the levels of IL-1β, TNF-α, IL-9, IL-2, IL-6, IL-4, IL-5, G-CSF (Granulocyte colony stimulating factor), IL-17A, and GM-CSF; compared with RUX, compound II significantly reduced the levels of IFN-γ, IL-2, IL-4, MCP-1 (Monocyte chemoattractant protein-1), IL-1α, KC (Keratinocyte-derived chemokine), IL-17A, Eotaxin, MIP-1β, and IL-12p40. For the combination therapy group: compared with RD, FD significantly reduced the levels of IFN-γ, IL-6 and IL-12p40; compared with ED, FD significantly reduced the levels of IL-1β, IL-6, MCP-1, IL-5, IL-17A and Eotaxin.

[0387] Table 24 Cytokine Concentrations in Primary HLH Mice

[0388] 7. Compound II improves hepatosplenomegaly in primary HLH mice.

[0389] After drug administration, liver and spleen tissues were collected and weighed from mice. The results are shown in Figure 16 and Table 25. The results showed that compared to the Control group, the liver and spleen of mice in the Vehicle group were significantly enlarged. Both the single-drug group of compound II and the combination therapy group of FD significantly reduced liver and spleen size. For the single-drug groups, compound II was significantly more effective than RUX in reducing liver size and significantly more effective than RUX and DEX in reducing spleen size. For the combination therapy groups, FD was significantly more effective than RD in reducing both liver and spleen size. Furthermore, the combination therapy of FD was significantly more effective than compound II and DEX alone in reducing spleen size.

[0390] Table 25 Liver and spleen weight and percentage in primary HLH mice

[0391] 8. Compound II improves hemophagocytosis in primary HLH mice.

[0392] After drug administration, liver, spleen, and bone marrow tissues from mice were collected and stained with hematoxylin and eosin (HE). The results are shown in Figure 17. The results showed that compared with the Control group, the Vehicle group showed multiple instances of hemophagocytosis in the liver, spleen, and bone marrow tissues. In contrast, the above phenomenon was basically eliminated in the single-drug group of Formula II and the FD combination drug group. The liver, spleen, and bone marrow tissue structures were clear, and no obvious atypia was observed. The RUX and RD groups also did not show obvious hemophagocytosis, but scattered inflammatory reactions and hemophagocytosis were still observed in the DEX and ED groups.

[0393] Example 7: Mechanism exploration of compound II in a primary HLH mouse model

[0394] This primary HLH model exhibits a biallelic mutation in PRF1, suppressing the cytotoxic function of CD8+ T cells. Upon infection with viruses or other pathogens, CD8+ T cells become overactivated, triggering a series of uncontrolled inflammatory responses. Flow cytometry analysis of perforin expression within CD8+ T cells demonstrates high accuracy in diagnosing primary HLH caused by immunodeficiency. Therefore, subsequent experiments in this invention primarily focus on CD8+ T cells and perforin.

[0395] 1. Compound of Formula II limits the number of CD8+ T cells and their activation.

[0396] A primary HLH mouse model was established. Treatment with compound formula II began on day 4, and fresh spleens from three mice were used on days 3, 6, 9, 12, 15, 18, and 21. Lymphocytes were extracted after grinding and analyzed by flow cytometry. The results showed that with the increase of compound formula II administration time, the number of CD8+ T cells in the mouse spleen gradually decreased, reaching as high as 79.27% ​​on day 3, but only 21.90% on day 21. Further analysis of activated CD8+CD44+ T cells showed a similarly significant downward trend, reaching as high as 93.6% on day 3, but decreasing to 48.9% on day 21.

[0397] 2. Compound of Formula II induces CD8+ T cells to express perforin.

[0398] To further investigate the effect of compound II on CD8+ T cell function, fresh spleens from three mice were collected at the same time points, and lymphocytes were obtained through grinding and flow cytometry analysis. The results showed that perforin expression gradually increased with the duration of compound II administration, reaching 8.325 times the level on day 21 compared to day 3. This indicates that compound II can inhibit HLH by limiting the number of CD8+ T cells and their activated form, CD8+CD44+ T cells, and increasing perforin expression in CD8+ T cells.

[0399] The above experiments demonstrate that this invention investigated the pharmacodynamics of a novel JAK2 / FLT3 inhibitor, compound II, in HLH and preliminarily explored its mechanism. First, public databases were analyzed to explore whether there are expression differences in JAK / STAT signaling pathway genes between HLH patients and healthy individuals. Then, mouse cell lines were used to explore the in vitro pharmacodynamics of compound II in HLH. Subsequently, in vivo experiments were conducted using a secondary HLH mouse model to explore the pharmacodynamics, safety, and mechanism of compound II in HLH. Based on RNA-seq analysis results, it was suggested that compound II and DEX have a synergistic anti-HLH effect, and the pharmacodynamics and mechanism of compound II combined with DEX in the secondary HLH mouse model were further investigated. Finally, a primary HLH mouse model was constructed to explore the pharmacodynamics and mechanism of compound II alone and compound II combined with DEX. Ultimately, this invention draws the following conclusions:

[0400] 1. Compared with normal individuals, HLH patients have high expression of JAK / STAT signaling pathway genes in peripheral blood mononuclear cells, providing a theoretical basis for the treatment of HLH with compound formula II.

[0401] 2. In vitro experiments confirmed that compound II has significant anti-proliferative activity against key effector cell lines of HLH, inhibits the secretion of IL-6, TNF-α and IL-1β, downregulates the expression of p-JAK2 and p-STAT5, and is superior to DEX, RUX and FED.

[0402] 3. In vivo experiments confirmed that compound II can significantly improve the general condition, blood routine, liver function, blood lipids, fibrinogen, serum ferritin, sCD25, hypercytokineemia, hepatosplenomegaly, inflammatory response and "hemophagocytosis" in secondary HLH mouse models, and can downregulate the expression of p-JAK2 and p-STAT5 in the liver and spleen, with no obvious toxic side effects on the vital organs of mice.

[0403] 4. In vivo experiments confirmed that the combination of compound II and DEX was significantly superior to compound II and DEX alone in improving body temperature, blood routine, liver and kidney function, blood lipids, fibrinogen, serum ferritin, sCD25, hepatosplenomegaly, and hemophagocytosis in a mouse model of secondary HLH, suggesting that compound II and DEX have a synergistic anti-HLH effect. In addition, FD was superior to ED in improving white blood cell count, liver and kidney function, hepatosplenomegaly, and hemophagocytosis.

[0404] 5. Compounds of Formula II can increase GCR nucleus entry and reduce DEX resistance by inhibiting the formation of the p-STAT5-GCR complex, and thus exert anti-HLH effects in synergy with DEX.

[0405] 6. In vivo experiments have confirmed that both single-agent formulation II compound and combination therapy with FD can significantly improve disease severity, survival, blood routine, liver and kidney function, blood lipids, fibrinogen, serum ferritin, sCD25, hypercytokineemia, hepatosplenomegaly, and hemophagocytosis in a mouse model of primary HLH, supporting the application of single-agent formulation II compound and combination therapy with FD in primary HLH. In addition, combination therapy with FD is superior to ED in improving survival, white blood cell count, kidney function, and hemophagocytosis.

[0406] 7. Compound of Formula II can inhibit HLH by limiting the number of CD8+ T cells and their activated form CD8+CD44+ T cells, and by increasing the perforin expression of CD8+ T cells.

Claims

1. Use of 2,4-disubstituted pyrimidine derivatives of the formula (I) or pharmaceutically acceptable salts thereof for the preparation of a medicament for the prophylaxis or treatment of hemophagocytic lymphohistiocytosis, characterized in that: The 2,4-disubstituted pyrimidine derivative is selected from the following compounds: The pharmaceutically acceptable salts are p-toluenesulfonate, oxalate, citrate, malate, salicylate, tartrate, phosphate, methanesulfonate, sulfate, fumarate, hydrochloride, or maleate.

2. Use according to claim 1, characterized in that: At least one of the following must be met: (1) The hemophagocytic lymphohistiocytosis described is primary or secondary; (2) the 2,4-disubstituted pyrimidine derivative is a compound as represented by formula (I): (3) The pharmaceutically acceptable salt is maleate; (4) The dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 25-225 mg; preferably 75-125 mg; for example, 75 mg or 125 mg; (5) The dosage form of the drug is a liquid dosage form or a solid dosage form; for example, tablets, capsules, granules, powders or oral liquids.

3. Use according to claim 1, characterized in that: The dosage of the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 5 mg / kg to 150 mg / kg; preferably 10 mg / kg to 100 mg / kg; more preferably 10 mg / kg to 60 mg / kg; and most preferably 30 mg / kg.

4. Use according to claim 1, characterized in that: At least one of the following must be met: (1) The route of administration of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is oral; (2) The 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts can inhibit the abnormal proliferation of HLH cells; (3) The 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts can improve the symptoms of hemophagocytic lymphohistiocytosis, including weight loss, sudden increase in body temperature, decrease in blood cells, impaired liver function, elevated blood lipids, abnormal coagulation function, elevated serum ferritin and sCD25, hypercytokineemia, hepatosplenomegaly and / or hemophagocytosis. (4) The 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts can improve inflammation of the liver, spleen and bone marrow in hemophagocytic lymphohistiocytosis and / or inhibit the expression of p-JAK2 and STAT5 proteins in the liver and spleen. (5) The 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt can inhibit the proliferation of CTLL-2 and RAW264.7 cell lines and / or inhibit the expression of phosphorylated JAK2 and STAT5 proteins in CTLL-2 and RAW264.7 cell lines; Preferably, it can inhibit the secretion of IL-6, TNF-α and IL-1β cytokines in the RAW264.7 cell line.

5. Use according to any one of claims 1 to 4, characterized in that: The 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt may be administered three times a day, twice a day, once a day, or once every two days.

6. Use of a combination of a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis or treatment of hemophagocytic lymphohistiocytosis, characterized in that: The combination drug of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof, which is administered separately or simultaneously with other drugs for preventing and treating hemophagocytic lymphohistiocytosis, and the 2,4-disubstituted pyrimidine derivative is selected from the following compounds: The pharmaceutically acceptable salts are p-toluenesulfonate, oxalate, citrate, malate, salicylate, tartrate, phosphate, methanesulfonate, sulfate, fumarate, hydrochloride, or maleate.

7. Use according to claim 6, characterized in that: The other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis include one or more of the following: dexamethasone, etoposide, cyclosporine A, methylprednisolone, epavamab, alenmab, anaerobicin, tocilizumab, infliximab, and etanercept; for example, dexamethasone, or etoposide and dexamethasone.

8. Use according to claim 6 or 7, characterized in that: At least one of the following must be met: (1) The hemophagocytic lymphohistiocytosis described is primary or secondary; (2) The dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 25-225 mg; preferably 75-125 mg; most preferably 75 mg or 125 mg; (3) The 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is administered orally, and the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis are administered via intraperitoneal injection or orally. (4) The combined drugs can inhibit the abnormal proliferation of HLH cells; (5) The combined drugs can improve the symptoms of hemophagocytic lymphohistiocytosis, including weight loss, sudden increase in body temperature, decrease in blood cells, impaired liver function, elevated blood lipids, abnormal coagulation function, elevated serum ferritin and sCD25, hypercytokineemia, hepatosplenomegaly and / or hemophagocytosis. (6) The combined drugs can improve the inflammation of the liver, spleen and bone marrow in hemophagocytic lymphohistiocytosis and / or inhibit the expression of p-JAK2 and STAT5 proteins in the liver and spleen; (7) The combined drug can inhibit the proliferation of CTLL-2 and RAW264.7 cell lines and / or inhibit the expression of phosphorylated JAK2 and STAT5 proteins in CTLL-2 and RAW264.7 cell lines; Preferably, it can inhibit the secretion of IL-6, TNF-α and IL-1β cytokines in the RAW264.7 cell line.

9. Use according to claim 7, characterized in that: At least one of the following must be met: (1) The dosage of the 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof is 5 mg / kg to 150 mg / kg; preferably 10 mg / kg to 100 mg / kg; more preferably 10 mg / kg to 60 mg / kg; and most preferably 30 mg / kg. (2) The dosage of dexamethasone is 0.3–7.5 mg / kg; preferably 0.5–5 mg / kg; more preferably 1–3 mg / kg; and most preferably 1.5 mg / kg. (3) The dosage of etoposide is 10-250 mg / kg; preferably 15-150 mg / kg; more preferably 30-100 mg / kg; and most preferably 50 mg / kg.

10. Use according to claim 7, characterized in that: At least one of the following must be met: (1) The 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is administered three times a day, twice a day, once a day, or once every two days. (2) The frequency of administration of the dexamethasone is twice a day, once a day, or once every two days; (3) The administration frequency of the etoposide is three times a week, twice a week, once a week, or once every two weeks; (4) The molar ratio of the dexamethasone to the 2,4-disubstituted pyrimidine derivative or its pharmaceutically acceptable salt is 1:(0.55-442); preferably 1:(1.77-177); for example 1:(2.9-53.5); and for example 1:17.

7. (5) When the other drugs for preventing and treating hemophagocytic lymphohistiocytosis are dexamethasone and etoposide, the molar ratio of dexamethasone to etoposide is 1:(1.1-710); preferably 1:(2.5-256); for example 1:(8-86); and for another example 1:28.

4.

11. A pharmaceutical composition, characterized by: It comprises a first pharmaceutical composition and a second pharmaceutical composition: A first pharmaceutical composition comprising, as described in claim 6, a 2,4-disubstituted pyrimidine derivative or a pharmaceutically acceptable salt thereof; and, The second pharmaceutical composition comprises the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis as described in claim 7; Preferably, the molar ratio, route of administration, dosage, and frequency of administration of the other drugs for the prevention and treatment of hemophagocytic lymphohistiocytosis and the 2,4-disubstituted pyrimidine derivatives or their pharmaceutically acceptable salts are as described in any one of claims 8 to 10.

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