CN120420332A - MTOR inhibitor-containing composition and application thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer, comprising an mTOR inhibitor and a BTK inhibitor of formula (I). The pharmaceutical composition of the present invention exhibits excellent tumor suppression effects in B lymphoma cells, exhibiting significant synergistic effects that are superior to the effects of either drug when used alone.

Description

MTOR inhibitor-containing composition and application thereof

Technical Field

The invention belongs to the field of medicines, and relates to a composition containing an mTOR inhibitor and application thereof.

Background

Leukemia and lymphoma are one of the most common malignant tumors in China. Bruton's Tyrosine Kinase (BTK) is one of the family members of the tyrosine kinase family expressed in B cells, macrophages, neutrophils, basophils and platelets, and can regulate the function of a variety of immune cells. Abnormal activation of the BTK-mediated B Cell Receptor (BCR) signaling pathway is an important causative factor leading to the development of lymphomas. Inhibitors targeting BTK have been used to treat hematological disorders (leukemia/lymphoma). Currently, 6 BTK inhibitors have been marketed worldwide, such as ibutenib (ibrutinib), acartinib (Acalabrutinib), zebutinib (Zanubrutinib), tirabrutinib, obutenib (orelabrutinib) and pirtobrutinib. Despite significant advances in lymphoma treatment, effective treatment of relapsed or refractory Diffuse large B-cell lymphoma (DLBCL) remains a significant clinical challenge.

Mammalian target protein rapamycin (mTOR) is a serine/threonine kinase, is a central signal molecule for regulating growth and proliferation in cells, and has close relation with tumorigenesis and development. First generation mTOR inhibitors (e.g., everolimus, temsirolimus) are approved for the treatment of advanced renal cell carcinoma, among other malignancies. However, even at high concentrations, the first generation mTOR inhibitors did not completely eliminate signaling cascades downstream of the TORC1/2 complex. The example 122 compound, CC-223, AZD2014 and other medicaments disclosed in the patent WO2017/219800 are second-generation mTorrC 1/2 inhibitors, can highly selectively inhibit mTorrC 1 and mTorrC 2, and inhibit tumor growth. In hematological tumors, aberrant activation of mTOR signaling also exists and is closely related to the prognosis of the patient. The first generation mTOR inhibitors such as everolimus showed good in vivo and in vitro tumor inhibitory activity in B lymphoma cells, but had limited clinical efficacy (Lee JS et al, br J Clin Pharmacol.2016Nov;82 (5): 1213-1228.). Second generation mTOR inhibitors are more effective at inhibiting tumor growth than the first generation inhibitors, but require close monitoring of the patient's blood status and the occurrence of hyperglycemia (Bendell JC et al, cancer 2015; 121:3481-90).

In the treatment of malignant tumors, single drug treatment is easy to lead to drug resistance of patients, and combined use of tumor drugs provides a new treatment strategy for overcoming drug resistance. Malignant tumors, however, have various drug resistance mechanisms and must be addressed by rational drug combinations. As patent WO2016145935A1 discloses the inhibition of everolimus, ibrutinib single, combination in TMD-8 cell lines, at concentrations where the inhibition of everolimus, ibrutinib combination is not higher than that of single, although in DLBCL-activated B cell subtype BTK inhibitor Ibrutinib has a synergistic effect with mTOR inhibitor Everolimus (MATHEWS GRINER LA, et al Proc Natl Acad SciUSA2014;111: 2349-54), DLBCL germinal center B cell subtype is unresponsive to BTK and mTOR combined inhibitors (Ezell SA, et al oncotarget.2014;5: 4990-5001), even in some cases mTOR inhibitors may antagonize the effects of other drugs by inhibiting proliferation (Huang S, et al J Biol Chem 2011;286: 40002-12), clinical trial NCT03205046 data indicate that Vistusertib does not adequately modulate the target to increase the clinical activity of Acalabrutinib single drug therapy.

Thus, despite significant advances in medicine in the selection of treatments for cancer, there remains a need for new combination therapies that are effective and safe.

Disclosure of Invention

In order to solve the problem of poor drug resistance and drug efficacy of malignant tumor single drugs, the invention provides a pharmaceutical composition, which comprises an mTOR inhibitor and a BTK inhibitor, or the application of the mTOR inhibitor and the BTK inhibitor in preparing a drug for treating cancer, or a method for treating cancer by combining the mTOR inhibitor and the BTK inhibitor.

According to a first aspect of the present invention there is provided a pharmaceutical composition comprising an mTOR inhibitor and a BTK inhibitor;

the mTOR inhibitor is a compound of formula I or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers and prodrugs thereof;

in some embodiments of the invention, the prodrug of the compound of formula I is the compound of formula I wherein the-OH group is independently converted to the corresponding phosphate-OP (O) (OH) ₂.

In some embodiments of the invention, the BTK inhibitor is selected from one or more of Acalabrutinib、Zanubrutinib、Ibrutinib、Orelabrutinib、CT-1530、Spebrutinib(AVL-292)、Olmutinib、Tirabrutinib、Pirtobrutinib(LOXO-305)、Edralbrutinib(SHR1459)、Remibrutinib(LOU-64)、Tolebrutinib(SAR442168)、Rilzabrutinib(PRN-1008)、Evobrutinib(M-2951)、AC-0058TA、Fenebrutinib、Elsubrutinib(ABBV-105)、BMS-935177、Atuzabrutinib(PRN-473)、BMS-986142、TAS-5315、DTRMWXHS-12、M-7583、Nemtabrutinib(ARQ-531)、HZ-A-018、CX1440、MH048、BI-BTK-1、BIIB-091、PCI-45292、TAK-020、CG-806、JNJ-64264681、BT-1053、FCN-647、NX-2127、BN102、HSK29116、BGB16673、HMPL760、SYHA1811、TT-01488、HM71224、Poseltinib(RN486)、CNX-774、CGI-1746、M-7583、BIIB068、Nemtabrutinib(ARQ 531)、TP-0158、GDC-0834、ONO-40599, preferably the BTK inhibitor is selected from any of Acalabrutinib, ibrutinib, zanubrutinib, edralbrutinib or Orelabrutinib, more preferably Acalabrutinib, ibrutinib, zanubrutinib and Edralbrutinib, most preferably Acalabrutinib or Ibrutinib.

In some embodiments of the invention, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers and/or excipients.

In some embodiments of the invention, the pharmaceutical composition may further comprise at least one other anticancer agent such as a platinum agent, (e.g. cisplatin, carboplatin, oxaliplatin, JM-216 or satraplatin, CI-973), an anti-microtubule agent, (e.g. vinca alkaloids such as vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an anti-metabolite (e.g. 5-fluorouracil, methotrexate, fludarabine), an alkylating agent (e.g. cyclophosphamide, melphalan, carmustine, nitrosoureas such as dichloroethyl nitrosourea and hydroxyurea), an anthracycline (e.g. doxorubicin, daunorubicin), an anti-tumor antibiotic (e.g. mitomycin, idarubicin, doxorubicin, daunorubicin), a topoisomerase inhibitor (e.g. etoposide, camptothecine), an anti-angiogenesis agent (e.g. bevacizumab), or any other cytotoxic agent (e.g. carmustine phosphate, prednisone, hormone) or agonist, antagonist, partial or partial kinase inhibitor.

According to a second aspect of the present invention there is provided a composition according to the first aspect for use as a medicament.

According to a third aspect of the present invention there is provided the use of a composition according to the first aspect in the manufacture of a medicament for the treatment of cancer, immune disorders, cardiovascular disease, viral infection, inflammation, metabolism/endocrine function disorders and neurological disorders.

According to a fourth aspect of the present invention there is provided a composition according to the first aspect for use in the treatment of cancer, immune disorders, cardiovascular disease, viral infection, inflammation, metabolism/endocrine function disorders and neurological disorders.

In some embodiments of the invention, the invention provides a composition as described above for use in the treatment of cancer.

According to a fifth aspect of the present invention there is provided a method of treating cancer comprising administering to a patient a therapeutically effective amount of a composition according to the first aspect.

In some embodiments of the invention, the cancer of the third to fifth aspects is selected from the group consisting of B-cell lymphoma, brain tumor, medulloblastoma, melanoma, multiple myeloma, glioblastoma, osteosarcoma, liver cancer, lung cancer, kidney cancer, pancreatic cancer, oral cancer, gastric cancer, esophageal cancer, laryngeal cancer, nasopharyngeal cancer, skin cancer, breast cancer, ductal carcinoma of human breast, colon cancer, rectal cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, non-small cell lung cancer, large cell lung cancer, B-cell lymphoma (Large B-cell lymphoma, LBL), mantle cell lymphoma (MANTLE CELL lymphoma, MCL), small lymphocytic lymphoma (Small lymphocytic lymphoma, SLL), chronic lymphocytic leukemia (Chronic lymphocytic leukemia, CLL), follicular lymphoma (Follicular lymphoma, FL), marginal zone lymphoma (Marginal zone lymphoma, mz l), burkitt lymphoma (Bukkitt Lymphoma, BL), lymphoplasmacytoma (Lymphoplasmacytic lymphoma, LPL), and further the Large B-cell lymphoma is bcl.

In some embodiments of the invention, the mTOR inhibitor in the pharmaceutical composition of the first to fifth aspects is a compound of formula I, the BTK inhibitor is one or more of Acalabrutinib, zanubrutinib, ibrutinib, orelabrutinib, edralbrutinib, and the cancer is diffuse large B-cell lymphoma.

In some embodiments of the invention, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor of the first to fifth aspects are administered in a combination or after the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor are prepared as a compound formulation, preferably the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor are administered in a combination, e.g. in a combination selected from simultaneous, separate and co-or separate formulations and sequentially.

In some embodiments of the invention, the weight ratio of the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug to BTK inhibitor of the first to fifth aspects is from 0.001:1 to 1000:1, preferably from 0.01:1 to 100:1, e.g. 0.01:1、0.05:1、0.1:1、0.2:1、0.5:1、1:1、1.5:1、2:1、3:1、5:1、8:1、10:1、15:1、20:1、25:1、30:1、40:1、50:1、60:1、70:1、80:1、90:1、100:1.

In some embodiments of the invention, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof according to the first to fifth aspects is administered in an amount of 1-500mg, preferably 50-200mg, more preferably 100-150mg, with a frequency of once a day, twice a day, three times a day, preferably once a day.

In some embodiments of the invention, the BTK inhibitor of the first to fifth aspects is administered in a dose of 1-500mg, preferably 50-200mg, more preferably 100-150mg, once a day, twice a day, three times a day, preferably once a day. For example, 100 mg/time when Acalabrutinib is used, 160 mg/time or 320 mg/time when Zanubrutinib is used, 420 mg/time when Ibrutinib is used, 150 mg/time when Orelabrutinib is used, and 100 mg/time or 200 mg/time when Edralbrutinib is used.

The route of administration of the pharmaceutical composition in the first to fifth aspects is not limited to oral administration, parenteral administration, transdermal administration, including but not limited to intravenous injection, subcutaneous injection, intramuscular injection.

In certain embodiments, both drugs may be designed for oral or injectable administration, or one drug may be designed for oral administration and the other drug may be designed for injectable administration.

According to a sixth aspect of the present invention there is provided a method for the treatment of B-cell lymphoma comprising administering to a subject in need thereof a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof.

According to a seventh aspect of the present invention there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof, in the manufacture of a medicament for the treatment of B-cell lymphoma.

According to an eighth aspect of the present invention there is provided a compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof, for use in the treatment of B cell lymphoma.

In some embodiments of the invention, the B-cell lymphoma of the sixth to eighth aspects is a large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma, more preferably the large B-cell lymphoma is diffuse large B-cell lymphoma.

In some embodiments of the invention, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof in the sixth to eighth aspects is administered in an amount of 1 to 500mg, preferably 50 to 200mg, more preferably 100 to 150mg, at a frequency of once a day, twice a day, three times a day, preferably once a day.

The invention has the beneficial effects that:

The pharmaceutical composition provided by the invention combines an mTOR inhibitor, especially a compound shown in a formula I or pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof, enhances the drug sensitivity and has strong synergistic effect in treating cancers.

Drawings

FIG. 1 shows the IC50 values of the compounds of formula I, CC-223, everolimus in TMD8, doHH2 cells.

FIG. 2 is a graph showing IC50 values of Acalabrutinib, ibrutinib, zanubrutinib, orelabrutinib, edralbrutinib in TMD8 cells.

FIG. 3 is a graph showing IC50 values of Acalabrutinib, ibrutinib, zanubrutinib, orelabrutinib, edralbrutinib in DoHH2 cells.

FIG. 4 is a graph showing the effect of a combination of a compound of formula I and Acalabrutinib on the relative viability of TMD8 cells.

FIG. 5 is a graph showing the effect of a combination of a compound of formula I and Ibrutinib on the relative viability of TMD8 cells.

FIG. 6 is a graph showing the effect of a combination of a compound of formula I and Zanubrutinib on the relative viability of TMD8 cells.

FIG. 7 is a graph showing the effect of a combination of a compound of formula I and Orelabrutinib on the relative viability of TMD8 cells.

FIG. 8 is a graph showing the effect of a combination of a compound of formula I and Edralbrutinib on the relative viability of TMD8 cells.

FIG. 9 is a graph showing the effect of a combination of a compound of formula I and Acalabrutinib on the relative viability of DoHH2 cells.

FIG. 10 is a graph showing the effect of a combination of a compound of formula I with Ibrutinib on the relative viability of DoHH2 cells.

The specific embodiment is as follows:

Definition of the definition

Unless defined otherwise, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The term "pharmaceutical composition" means a product comprising an active ingredient, at least one pharmaceutically acceptable excipient. The pharmaceutical compositions of the present invention encompass any composition made from admixing the active ingredient, additional active ingredient, and pharmaceutically acceptable excipients.

The term "tumor" is malignant and is used interchangeably with "cancer". Tumor growth inhibition or regression may be localized to a single tumor or group of tumors within a particular tissue or organ, or may be systemic (i.e., affecting tumors in all tissues or organs).

The term "effective amount" means an amount of a pharmaceutically active ingredient sufficient to affect a disease or condition after administration to an individual/subject/patient in order to prevent, alleviate and/or treat the disease or condition. The "effective amount" may vary depending on factors such as the pharmaceutically active ingredient, the symptoms or severity of the disease or condition, the individual/subject/patient's personal condition (such as age, sex, weight, etc.), and the like.

The term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, as is well known in the art (see, e.g., Remington'sPharmaceutical Sciences.Edited by Gennaro AR,19thed.Pennsylvania:Mack Publishing Company,1995), and including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents).

Unless otherwise indicated, the term "compound" refers to any particular compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers), as well as pharmaceutically acceptable salts and derivatives (including prodrug forms) thereof. The term compound, as used in this context, generally refers not only to a single compound, but may include other compounds, such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures), as well as specific enantiomers or enantiomerically enriched mixtures of the disclosed compounds. The term also refers in this context to a prodrug form of a compound that has been modified to facilitate administration and delivery of the compound to an active site.

The term "pharmaceutically acceptable salt" is used to describe a salt form of one or more compounds described herein, which is provided to increase the solubility of the compound in the gastric juice of the gastrointestinal tract of a patient in order to promote dissolution and bioavailability of the compound. Pharmaceutically acceptable salts include, where applicable, salts derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals (e.g., potassium and sodium), alkaline earth metals (e.g., calcium, magnesium, and ammonium salts), and many other acids and bases well known in the pharmaceutical arts.

The term "prodrug" will refer to a functional derivative of the compound that is readily converted to the desired compound in vivo. Thus, in the methods of treatment of the present invention, the term "administering" will include treating the various disorders described with a specifically disclosed compound or with a compound that may not be specifically disclosed but that is convertible in vivo to a specific compound upon administration to a patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", H.Bundgaard, elsevier et al, 1985.

The compounds of the present invention may form solvates with conventional organic solvents, or hydrates with water, and such solvates or hydrates are also intended to be included within the scope of the present invention.

In the compounds of the present invention, all tautomers and mixtures thereof in any ratio are included.

The term "combination" as used herein refers to a mode of administration, meaning that at least one dose of a compound of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof, is administered over a period of time, wherein both drugs exhibit pharmacological effects. The order of administration may be simultaneous administration of the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof with the BTK inhibitor, or administration of the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof followed by administration of the BTK inhibitor after a specified time interval, or administration of the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof after a specified time interval of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 1 day, 2 days, 3 days. When administered in combination, the therapeutically effective amount of each compound may be less than the therapeutically effective amount of each compound administered alone.

The application is further described below in connection with examples, which are not intended to limit the scope of the application. Experimental materials and detection methods 1 reagents and sources

The compound of formula I is described in reference to the synthesis of example 122 of patent WO 2017/219800.

Example one inhibition of human B cell lymphoma by mTOR inhibitors (Compounds of formula I, CC-223, everolimus)

1.1 Experimental methods

TMD8 and DoHH2 cells grown in a conventional culture solution and in a logarithmic growth phase are inoculated in a 96-well plate, the cell inoculation amount is about 1×10 ⁴ cells per well, 100 μl of the compound of formula I, 50 μl of the culture solution of CC-223 and Everolimus (the maximum concentration is 10 μM,3 times diluted and 9 concentration gradients are added in total) with different final concentrations are added to each well, the same amount of the culture solution is added to a control group, 3 compound wells are arranged, and the drug and the cells are incubated for 72 hours. At the end of the action time, the inhibition of the drug on the cell growth is detected by CTG detection, wherein 50 mu L of CellTiter-Glo ^TM solution is added into each hole, the light oscillation is closed for 5min, the mixture is kept still for 10min, and the luminescence value is detected by an enzyme-labeled instrument. The viability of each concentration group was calculated and IC50 values were calculated using GraphPad software.

1.2 Experimental results

IC50 values of the compounds of formula I, CC-223, everolimus in TMD8, doHH2 cells are shown in FIG. 1 and Table 1, respectively. The IC50 data indicate that the compounds of formula I have greater inhibitory activity in B cell lymphomas in TMD8, doHH2 cells than in CC-223.

TABLE 1 IC50 values of Compounds of formula I, CC-223, everolimus in TMD8, doHH2 cells

	Compounds of formula I	CC-223	Everolimus
				TMD8	85nM	352.3nM	<4.57nM
DoHH2	96nM	438nM	<4.57nM

Example two inhibition of human B cell lymphoma by BTK inhibitor (Ibrutinib, acalabrutinib, zanubrutinib, orelabrutinib, edralbrutinib)

2.1 Experimental methods

TMD8 and DoHH2 cells grown in a conventional culture solution and in a logarithmic growth phase are inoculated in a 96-well plate, the cell inoculation amount is about 1×10 ⁴ cells per well, 100 μl of each well is obtained, 50 μl of culture solution containing different final concentrations of BTK inhibitor (maximum concentration 10 μM, 3-fold dilution and total concentration gradient of 9) is added to each well after inoculation, an equivalent amount of culture solution is added to a control group, 3 multiple wells are provided, and the drug and cells are incubated for 72 hours. At the end of the action time, the inhibition of the drug on the cell growth is detected by CTG detection, wherein 50 mu L of CellTiter-Glo ^TM solution is added into each hole, the light oscillation is closed for 5min, the mixture is kept still for 10min, and the luminescence value is detected by an enzyme-labeled instrument. The viability of each concentration group was calculated and IC50 values were calculated using GraphPad software.

2.2 Experimental results

The IC50 values of BTK inhibitor Acalabrutinib, ibrutinib, zanubrutinib, orelabrutinib, edralbrutinib in TMD8, doHH2 cells are shown in table 2 and fig. 2, 3.

TABLE 2 IC50 values in TMD8, doHH2 for Acalabarutinib, ibrutinib, zanubrutinib, orelabrutinib, edralbrutinib

	Acalabrutinib	Ibrutinib	Zanubrutinib	Orelabrutinib	Edralbrutinib
						TMD8	4.67nM	0.45nM	0.23nM	3.89nM	1.35nM
DoHH2	>10μM	297.6nM	>3.3μM	>10μM	767.9nM

The data in examples one and two show that 5 BTK inhibitors are relatively sensitive in TMD8 cells and have poor inhibitory activity in DoHH2 cells, while the compounds of formula I show very strong inhibitory effects in both TMD8 and DoHH2 cells.

Example III Combined action of Compounds of formula I with BTK inhibitors in TMD8 cells

3.1 Experimental methods

TMD8 cells grown in conventional culture solution and in logarithmic growth phase are inoculated in a 96-well plate, the cell inoculum size is about 1×10 ⁴ cells per well, 90 μl per well, 60 μl of culture solution containing different final concentrations of the compound of formula I and BTK inhibitor is added per well after inoculation, the same amount of culture solution is added to a control group, and the drug and cells are incubated for 72 hours. At the end of the action time, the inhibition of the drug on the cell growth is detected by CTG detection, wherein 50 mu L of CellTiter-Glo ^TM solution is added into each hole, the light oscillation is closed for 5min, the mixture is kept still for 10min, and the luminescence value is detected by an enzyme-labeled instrument. The survival of each concentration group was calculated and the drug combination index CI (Combination Index) was calculated using CompuSyn software (version CompuSyn 1.0), and the relationship between CI and the effect and intensity of the combination was shown in Table 3.

TABLE 3 CI value vs. combined effect and intensity

CI value	Combined effect and strength
		<0.3	Strong synergistic effect
0.3-1	Synergistic effect
		CI=1	Additive effect
CI>1	Antagonism of

3.2 Experimental results

3.2.1 Combinations of Compounds of formula I and BTK inhibitor Acalabrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (4 nM, 12nM, 37nM, 111nM, 333nM, 1000 nM) were 8%, 12%, 24%, 51%, 75%, 88%, respectively, and the single drug inhibition rates of Acalabrutinib at different concentrations (3 nM, 10nM, 30 nM) were 58%, 75%, 82%, respectively.

The inhibition rate of the combination of the compound I and Acalabrutinib on TMD8 cells and the CI value of the combination index are shown in tables 4 and 5 respectively, and the relative activity of the cells is shown in figure 4. The compound of formula I has a synergistic effect with Acalabrutinib when the concentration of the compound of formula I is more than or equal to 37nM, and has a strong synergistic effect with Acalabrutinib when the concentration of the compound of formula I is more than or equal to 333nM and the concentration of the Acalabrutinib is more than or equal to 10 nM.

TABLE 4 inhibition of TMD8 cells by the combination of the Compounds of formula I and Acalabrutinib

TABLE 5 combination index of compounds of formula I and Acalabrutinib in TMD8 cells

"/" Indicates that the single agent inhibition of the compound of formula I Acalabrutinib in TMD8 cells was high at this concentration, so the CI value was not calculated.

3.2.2 Combinations of Compounds of formula I and BTK inhibitor Ibrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (100 nM, 300nM, 1000 nM) were 30%, 62%, 80%, respectively, and the single drug inhibition rates of the compounds of formula I at different concentrations (0.04 nM, 0.12nM, 0.37nM, 1.1nM, 3.3nM, 10 nM) Ibrutinib were 0%, 31%, 57%, 68%, 77%, respectively.

The inhibition rate of TMD8 cells and the index CI value of the combination of the compound shown in the formula I and Ibrutinib are shown in Table 6 and Table 7 respectively, and the relative activity of the cells is shown in FIG. 5. When Ibrutinib concentration is more than or equal to 1.1nM, the compound of formula I has strong synergistic effect when the concentration is more than or equal to 300 nM.

TABLE 6 inhibition of TMD8 cells by the combination of the Compounds of formula I and Ibrutinib

TABLE 7 combination index of compounds of formula I and Ibrutinib in TMD8 cells

3.2.3 Combinations of Compounds of formula I and BTK inhibitor Zanubrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (4 nM, 12nM, 37nM, 111nM, 333nM, 1000 nM) were 0%, 17%, 51%, 75%, 85%, respectively, and the single drug inhibition rates of Zanubrutinib at different concentrations (0.3 nM, 1nM, 3nM, 10 nM) were 31%, 61%, 72%, 73%, respectively.

The inhibition rate of TMD8 cells and the index CI value of the combination of the compound shown in the formula I and Zanubrutinib are shown in tables 8 and 9, respectively, and the relative activity of the cells is shown in figure 6. Has synergistic effect with Zanubrutinib when the concentration of the compound of formula I is more than or equal to 37 nM.

TABLE 8 inhibition of TMD8 cells by the combination of the Compounds of formula I and Zanubrutinib

TABLE 9 combination index of compounds of formula I and Zanubrutinib in TMD8 cells

3.2.4 Combinations of Compounds of formula I and BTK inhibitor Orelabrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (4 nM, 12nM, 37nM, 111nM, 333nM, 1000 nM) were 0%, 11%, 21%, 49%, 74%, 86%, respectively, and the single drug inhibition rates of Orelabrutinib at different concentrations (3 nM, 10nM, 30 nM) were 21%, 48%, 63%, respectively.

The inhibition rate of TMD8 cells and the index CI value of the combination of the compound shown in the formula I and Orelabrutinib are shown in tables 10 and 11, respectively, and the relative activity of the cells is shown in figure 7. Has synergistic effect with Orelabrutinib when the concentration of the compound of formula I is more than or equal to 12 nM.

TABLE 10 inhibition of TMD8 cells by the combination of the Compounds of formula I and Orelabrutinib

Table 11 combination index of compounds of formula I and Orelabrutinib in TMD8 cells

3.2.5 Combinations of a compound of formula I and a BTK inhibitor Edralbrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (100 nM, 300nM, 1000 nM) were 34%, 60%, 80%, respectively, and the single drug inhibition rates of Edralbrutinib at different concentrations (0.4 nM, 1.2nM, 3.7nM, 11.1nM, 33.3nM, 100 nM) were 0%, 29%, 54%, 65%, 71%, respectively.

The inhibition rate of TMD8 cells and the index CI value of the combination of the compound shown in the formula I and Edralbrutinib are shown in Table 12 and Table 13 respectively, and the relative activity of the cells is shown in FIG. 8. The compound of formula I has a synergistic effect with Edralbrutinib when the concentration of the compound of formula I is not less than 100nM and the concentration of Edralbrutinib is not less than 3.7nM, and has a strong synergistic effect with Edralbrutinib when the concentration of the compound of formula I is not less than 100nM and the concentration of Edralbrutinib is not less than 11.1 nM.

TABLE 12 inhibition of TMD8 cells by the combination of the Compounds of formula I and Edralbrutinib

TABLE 13 combination index of compounds of formula I and Edralbrutinib in TMD8 cells

Example IV combined action of Compounds of formula I with BTK inhibitors in DoHH2 cells

4.1 Experimental methods

DoHH2 cells grown in conventional culture solution and in logarithmic growth phase are inoculated in 96-well plates, the cell inoculum size is about 1×10 ⁴ cells per well, 90 μl per well, 60 μl of culture solution containing different final concentrations of compound of formula I and BTK inhibitor is added per well after inoculation, the same amount of culture solution is added to the control group, and the drug is incubated with the cells for 72h. At the end of the action time, the inhibition of the drug on the cell growth is detected by CTG detection, wherein 50 mu L of CellTiter-Glo ^TM solution is added into each hole, the light oscillation is closed for 5min, the mixture is kept still for 10min, and the luminescence value is detected by an enzyme-labeled instrument. Survival for each concentration group was calculated and the combination index CI value was calculated using CompuSyn software (version CompuSyn 1.0.0).

4.2 Experimental results

4.2.1 Combinations of Compounds of formula I and BTK inhibitor Acalabrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (4 nM, 12nM, 37nM, 111nM, 333nM, 1000 nM) were 0%, 11%, 21%, 49%, 62%, 76%, respectively, and the single drug inhibition rates of Acalabrutinib at different concentrations (3. Mu.M, 10. Mu.M, 30. Mu.M) were 15%, 26%, 60%, respectively.

The inhibition rate of DoHH2 cells and the index CI of the combination of the compound shown in the formula I and Acalabrutinib are shown in tables 14 and 15 respectively, and the relative cell viability is shown in FIG. 9. In DoHH2 cells, which are insensitive to BTK inhibitors, the compounds of formula I still have very strong killing activity, which in combination with BTK inhibitors Acalabrutinib, enhance drug sensitivity and have synergistic effects.

TABLE 14 inhibition of DoHH2 cells by Compounds of formula I in combination with Acalabrutinib

Table 15 combination index of DoHH2 cells with Compounds of formula I and Acalabrutinib

4.2.2 Combinations of Compounds of formula I and BTK inhibitor Ibrutinib

The single drug inhibition rates of the compounds of formula I at different concentrations (4 nM, 12nM, 37nM, 111nM, 333nM, 1000 nM) were 9%, 4%, 18%, 41%, 60%, 82%, respectively, and the single drug inhibition rates of Acalabrutinib at different concentrations (30 nM, 100nM, 300 nM) were 16%, 24%, 42%, respectively.

The inhibition rate of DoHH2 cells and the index CI of the combination of the compound shown in the formula I and Ibrutinib are shown in tables 16 and 17, respectively, and the relative cell viability is shown in FIG. 10. In DoHH2 cells insensitive to BTK inhibitors, the compound of the formula I and the BTK inhibitor Ibrutinib have synergistic effect and enhance drug sensitivity, and when the concentration of the compound of the formula I is more than or equal to 37nM and Ibrutinib is 300nM, the compound of the formula I and the BTK inhibitor have strong synergistic effect.

TABLE 16 inhibition of DoHH2 cells by Compounds of formula I in combination with Ibrutinib

Table 17 combination index of DoHH2 cells with Compounds of formula I and Ibrutinib

Through research on the action of an mTOR inhibitor and a BTK inhibitor on TMD8 and DoHH2 diffuse large B cell lymphomas, the compound of the formula I has stronger killing activity on TMD8 and DoHH2 of human B cell lymphomas compared with an mTOR inhibitor CC-223. The compound of formula I has a synergistic effect when combined with BTK inhibitors Acalabrutinib, ibrutinib, zanubrutinib and Edralbrutinib, orelabrutinib in TMD8 cells sensitive to the BTK inhibitor, has a strong synergistic effect when combined with BTK inhibitors Acalabrutinib, ibrutinib, zanubrutinib and Edralbrutinib, and still has extremely strong killing activity when combined with BTK inhibitors Acalabrutinib or Ibrutinib in DoHH2 cells insensitive to the BTK inhibitor, thus enhancing drug sensitivity and having a strong synergistic effect.

While embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are illustrative and not to be construed as limiting the present invention, and that variations, modifications, alternatives and variations of the above embodiments may be made by those skilled in the art within the scope of the present invention and are intended to be included within the scope of the present invention.

Claims (10)

1. A pharmaceutical composition comprising an mTOR inhibitor and a BTK inhibitor, wherein the mTOR inhibitor is a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof;

2. The pharmaceutical composition of claim 1, wherein the prodrug of the compound of formula I is the corresponding phosphate-OP (O) (OH) ₂ independently converted from an-OH group in the compound of formula I.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that:

the BTK inhibitor is selected from one or more of Acalabrutinib、Zanubrutinib、Ibrutinib、Orelabrutinib、CT-1530、Spebrutinib(AVL-292)、Olmutinib、Tirabrutinib、Pirtobrutinib(LOXO-305)、Edralbrutinib(SHR1459)、Remibrutinib(LOU-64)、Tolebrutinib(SAR442168)、Rilzabrutinib(PRN-1008)、Evobrutinib(M-2951)、AC-0058TA、Fenebrutinib、Elsubrutinib(ABBV-105)、BMS-935177、Atuzabrutinib(PRN-473)、BMS-986142、TAS-5315、DTRMWXHS-12、M-7583、Nemtabrutinib(ARQ-531)、HZ-A-018、CX1440、MH048、BI-BTK-1、BIIB-091、PCI-45292、TAK-020、CG-806、JNJ-64264681、BT-1053、FCN-647、NX-2127、BN102、HSK29116、BGB16673、HMPL760、SYHA1811、TT-01488、HM71224、Poseltinib(RN486)、CNX-774、CGI-1746、M-7583、BIIB068、Nemtabrutinib(ARQ 531)、TP-0158、GDC-0834、ONO-4059;

Preferably, the BTK inhibitor is selected from any one of Acalabrutinib, ibrutinib, zanubrutinib, edralbrutinib or Orelabrutinib, further preferably Acalabrutinib, ibrutinib, zanubrutinib and Edralbrutinib, most preferably Acalabrutinib or Ibrutinib;

And/or the weight ratio of the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug to BTK inhibitor is from 0.001:1 to 1000:1, preferably from 0.01:1 to 100:1;

And/or the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof is administered in an amount of 1-500mg;

And/or, the BTK inhibitor is administered at a dose of 1-500mg.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier and/or excipient.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition comprises at least one other anticancer agent such as a platinum agent, (e.g. cisplatin, carboplatin, oxaliplatin, JM-216 or satraplatin, CI-973), an anti-microtubule agent, (e.g. vinca alkaloids such as vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an antimetabolite (e.g. 5-fluorouracil, methotrexate, fludarabine), an alkylating agent (e.g. cyclophosphamide, melphalan, carmustine, nitrosoureas such as chloroethyl nitrosourea and hydroxyurea), an anthracycline (e.g. doxorubicin, daunorubicin), an antitumor antibiotic (e.g. mitomycin, idarubicin, doxorubicin, daunomycin), a topoisomerase inhibitor (e.g. etoposide, camptothecium), an anti-angiogenesis agent (e.g. bevacizumab), or any other cytotoxic agent (e.g. prednisone, a nilamide, hormone) or an agonist, partial agonist, an antagonist.

6. The pharmaceutical composition according to any one of claims 1-5 for use in the treatment of cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine dysfunctions and neurological disorders;

And/or the cancer is B-cell lymphoma, brain tumor, medulloblastoma, melanoma, multiple myeloma, glioblastoma, osteosarcoma, liver cancer, lung cancer, kidney cancer, pancreatic cancer, oral cancer, stomach cancer, esophageal cancer, laryngeal cancer, nasopharyngeal cancer, skin cancer, breast cancer, human ductal carcinoma of the breast, colon cancer, rectal cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, non-small cell lung cancer, large cell lung cancer;

More preferably, the B-cell lymphoma is a large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma;

Further preferably, the large B-cell lymphoma is diffuse large B-cell lymphoma;

And/or the compound of the formula I or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, prodrugs and the BTK inhibitor thereof are used in a combined mode, or the compound of the formula I or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, prodrugs and the BTK inhibitor are prepared into a compound preparation for administration;

Preferably, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof are administered in combination;

Further preferred modes of administration are selected from the group consisting of simultaneous, separate and co-administration or separate and sequential administration of a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof.

7. Use of a pharmaceutical composition according to any one of claims 1-5 in the manufacture of a medicament for the treatment of cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine dysfunctions and neurological disorders;

And/or the cancer is B-cell lymphoma, brain tumor, medulloblastoma, melanoma, multiple myeloma, glioblastoma, osteosarcoma, liver cancer, lung cancer, kidney cancer, pancreatic cancer, oral cancer, stomach cancer, esophageal cancer, laryngeal cancer, nasopharyngeal cancer, skin cancer, breast cancer, human ductal carcinoma of the breast, colon cancer, rectal cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, non-small cell lung cancer, large cell lung cancer;

More preferably, the B-cell lymphoma is a large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma;

Further preferably, the large B-cell lymphoma is diffuse large B-cell lymphoma;

And/or the compound of the formula I or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, prodrugs and the BTK inhibitor thereof are used in a combined mode, or the compound of the formula I or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, prodrugs and the BTK inhibitor are prepared into a compound preparation for administration;

Preferably, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof are administered in combination;

Further preferred modes of administration are selected from the group consisting of simultaneous, separate and co-administration or separate and sequential administration of a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof.

8. A method for treating cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolic/endocrine dysfunctions and neurological abnormalities, comprising administering to a subject in need thereof the pharmaceutical composition of any one of claims 1 to 5;

And/or the cancer is B-cell lymphoma, brain tumor, medulloblastoma, melanoma, multiple myeloma, glioblastoma, osteosarcoma, liver cancer, lung cancer, kidney cancer, pancreatic cancer, oral cancer, stomach cancer, esophageal cancer, laryngeal cancer, nasopharyngeal cancer, skin cancer, breast cancer, human ductal carcinoma of the breast, colon cancer, rectal cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, non-small cell lung cancer, large cell lung cancer;

preferably, the B-cell lymphoma is a large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma;

more preferably, the large B-cell lymphoma is diffuse large B-cell lymphoma;

and/or the mTOR inhibitor is a compound of formula I, the BTK inhibitor is one or more of Acalabrutinib, zanubrutinib, ibrutinib, orelabrutinib, edralbrutinib, and the cancer is diffuse large B cell lymphoma;

and/or the mTOR inhibitor and the BTK inhibitor are used by adopting a combined administration route or are prepared into a compound preparation for use;

Preferably, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof are administered in combination;

Further preferred modes of administration are selected from the group consisting of simultaneous, separate and co-administration or separate and sequential administration of a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug and BTK inhibitor thereof.

9. A method for treating B-cell lymphoma comprising administering to a subject in need thereof a compound of formula I;

And/or the B-cell lymphoma is a large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma;

more preferably, the large B-cell lymphoma is diffuse large B-cell lymphoma;

And or, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof is administered in an amount of from 1 to 500mg, preferably from 50 to 200mg, more preferably from 100 to 150mg;

And/or the frequency of administration is once a day, twice a day, three times a day, preferably once a day.

10. The use of a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof in the manufacture of a medicament for the treatment of B cell lymphoma,

And/or the B-cell lymphoma is a large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma;

more preferably, the large B-cell lymphoma is diffuse large B-cell lymphoma;

And or, the compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, prodrug thereof is administered in an amount of from 1 to 500mg, preferably from 50 to 200mg, more preferably from 100 to 150mg;

And/or the frequency of administration is once a day, twice a day, three times a day, preferably once a day.