Application of deuteromidevir and composition thereof in preparation of antitumor drugs

Abstract

The invention provides application of deuteromidevir and a composition thereof in preparation of antitumor drugs. Specifically, the invention provides application of the deuterated remidegravir shown in a formula (I) or pharmaceutically acceptable salts and isomers thereof in preparation of drugs for resisting tumors, especially multiple myeloma. The deuterated rimidewvir has an excellent anti-tumor effect and is expected to become an alternative drug of anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of deuteremedvir and its compositions in the preparation of antitumor drugs. Background Technology

[0002] Multiple myeloma (MM) is a malignant tumor caused by terminally differentiated plasma cells. With the increasing variety of drugs available for MM treatment, combination therapy has significantly prolonged the survival of MM patients. However, most MM patients experience relapse, and the survival prognosis for relapsed patients is poor, with subclonal evolution and drug resistance being important contributing factors.

[0003] Currently, drugs used clinically to treat multiple myeloma are mainly divided into four categories: proteasome inhibitors (bortezomib, carfizzomib, and ixazomib), immunomodulatory drugs (lenalidomide and pomalidomide), histone deacetylase inhibitors (pabistat), and monoclonal antibodies (daratumumab and ituximab, etc.). Clinically, multi-drug combination strategies or combinations of different treatment regimens are often used to overcome the relapse and drug resistance problems of multiple myeloma. However, to date, multiple myeloma remains an incurable disease for most patients. Most patients eventually relapse due to subclonal evolution and increasing drug resistance.

[0004] Therefore, there is an urgent need in this field to explore new drugs for multiple myeloma to overcome the emerging drug resistance. Furthermore, the market for drugs treating other cancers such as lung cancer and colorectal cancer currently faces significant gaps, making the development and exploration of new anti-tumor drugs of great importance. Summary of the Invention

[0005] One object of the present invention is to provide the use of deuterremidevir or a pharmaceutically acceptable salt thereof and combinations thereof as a TRIP13 inhibitor for the treatment of multiple myeloma, wherein the deuterremidevir tablets or pharmaceutically acceptable salts thereof have the structure shown below.

[0006]

[0007] Another object of the present invention is to provide an antitumor drug.

[0008] In a first aspect of the invention, there is provided the use of deuterated remidevir of formula (I), or a pharmaceutically acceptable salt thereof, or an isomer thereof, for the preparation of (a) an antitumor medicament, and / or (b) a TRIP13 enzyme inhibitor.

[0009]

[0010] In another preferred embodiment, the pharmaceutically acceptable salt is selected from the group consisting of hydrobromic acid, hydrochloride, trifluoroacetate, trifluoroformate, benzoate, benzenesulfonate, and phosphate; preferably, the deuterated remidevir is deuterated remidevir hydrobromide.

[0011] In another preferred embodiment, the tumor is selected from hematologic malignancies and solid tumors; more preferably, the hematologic malignancies are selected from the group consisting of myeloma, lymphoma, or combinations thereof; the solid tumors are selected from the group consisting of lung cancer, colorectal cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, thyroid cancer, liver cancer, stomach cancer, pancreatic cancer, or combinations thereof.

[0012] In another preferred embodiment, the lymphoma is selected from the group consisting of human diffuse histiocytic lymphoma, B-cell lymphoma cells, or combinations thereof.

[0013] In another preferred embodiment, the lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, or a combination thereof.

[0014] In another preferred embodiment, the tumor is multiple myeloma.

[0015] In another preferred embodiment, the drug is an oral or injectable dosage form; and / or the oral or injectable dosage form is selected from the group consisting of: powder, tablet, granule, capsule, sustained-release, oral liquid, emulsion or suspension.

[0016] In another preferred embodiment, the active ingredient, deuterated remidevir of formula I, or a pharmaceutically acceptable salt thereof, isomers comprising 0.01-99.9% wt; the remaining components are pharmaceutically acceptable excipients.

[0017] In another preferred embodiment, the deuterated remidevir is a deuterated remidevir hydrobromide tablet.

[0018] In a second aspect of the invention, a pharmaceutical composition is provided, comprising:

[0019]

[0020] (1) Deuterated remidevir of formula I as the first active ingredient, or a pharmaceutically acceptable salt thereof, or an isomer thereof;

[0021] (2) Other antitumor drugs or therapies as the second active ingredient: bortezomib, carfizzomib, ixazomib, thalidomide, lenalidomide, pamalidomide, daratumumab, ituximab, or combinations thereof, or immunotherapy.

[0022] (3)Optionally, a pharmaceutically acceptable carrier.

[0023] In another preferred embodiment, the effective dose of the deuterated remidevir of Formula I, or its pharmaceutically acceptable salt, as an active ingredient in the drug is: with an adult weight standard set at 60 kg, the effective dose for adults is 400 mg to 600 mg per day.

[0024] In a third aspect of the invention, the use of the pharmaceutical composition described in the second aspect of the invention is provided, wherein the pharmaceutical composition is used to prepare an antitumor drug.

[0025] In another preferred embodiment, the tumor is selected from the group consisting of hematologic malignancies (myeloma, lymphoma, or a combination thereof) and solid tumors (lung cancer, colorectal cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, thyroid cancer, liver cancer, stomach cancer, pancreatic cancer, or a combination thereof).

[0026] In a fourth aspect of the invention, an antitumor method is provided, comprising the steps of contacting a deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutical composition or formulation comprising deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, with a subject of interest.

[0027] In another preferred embodiment, the object is a mammal, preferably a human, rat, mouse, monkey, or rabbit.

[0028] In another preferred embodiment, the method is performed in vitro.

[0029] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0030] In a fifth aspect of the invention, an in vitro antitumor method is provided, comprising the steps of: culturing deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutical composition or preparation comprising deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, together with tumor cells; preferably, the concentration of deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, as the active ingredient, or the isomer thereof, is 10 nM to 1000 μM; more preferably 100 nM to 100 μM; more preferably 1 μM to 10 μM.

[0031] In a sixth aspect of the invention, a method for inhibiting TRIP13 enzyme is provided, comprising the step of contacting a deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutical composition or formulation comprising the deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, with a subject of interest.

[0032] In another preferred embodiment, the method is performed in vitro.

[0033] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0034] Figure 1 A comparison diagram showing the binding modes of deuterium remdesivir hydrobromide tablets (left), DCZ0415 (right), and TRIP13 is displayed.

[0035] Figure 2 The effect of VV116 (deuterium remidevir hydrobromide tablets) on the stability of intracellular TRIP13 was demonstrated by a cell thermal migration assay (CETSA).

[0036] Figure 3 The in vivo antimyeloma activity of VV116 (deuterium remidevir hydrobromide tablets) is shown. (A) Growth inhibition curves of VV116 (50 mg / kg, n=5) and the control group (n=5) on xenograft tumors in BALB / c nude mice. (B) Tumor weight change curve during treatment. (C) Tumor size 13 days after treatment. ***P<0.001 vs. control group.

[0037] Figure 4 The effects of VV116 (deuterium remidevir hydrobromide tablets) and its combination with bortezomib on the inhibition of NCI-H929 xenograft tumor growth in nude mice are shown. (A) Growth inhibition curves of BALB / c nude mouse xenograft tumors are shown in the control group (G1): VV116 alone (G2): VV116-50 mg / kg, VV116 combined with bortezomib (G3): VV116-50 mg / kg + BTZ-0.7 mg / kg, and bortezomib alone (G4): BTZ-0.7 mg / kg, as well as tumor volume after 28 days of administration. (B) Curve of mouse body weight change during treatment. ****P<0.0001 vs. control group (Day 14). Detailed Implementation

[0038] Through extensive and in-depth research, the inventors unexpectedly discovered that deuterium-remedvir possesses excellent antitumor activity (especially in multiple myeloma). Based on this, the inventors completed this invention.

[0039] Active ingredients

[0040] As used herein, “compound of the present invention” refers to the compound deuteremedvir represented by Formula I, and also includes stereoisomers or optical isomers of the compound of Formula I, pharmaceutically acceptable salts, prodrugs or solvates.

[0041] The term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. Furthermore, when a compound of the present invention contains a basic fragment, it includes, but is not limited to, pyridine or imidazole; when it contains an acidic fragment, it includes, but is not limited to, carboxylic acids; and any zwitterions ("internal salts") that may be formed are included within the scope of the term "salt." Pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, although other salts are also useful, for example, for separation or purification steps in the preparation process. The compounds of the present invention may form salts, for example, by reacting compound I with a certain amount, such as an equimolar amount, of an acid or base, precipitating it in a medium, or by freeze-drying it in an aqueous solution.

[0042] The compounds of this invention contain basic fragments, including but not limited to amines, pyridines, or imidazole rings, which may form salts with organic or inorganic acids. Typical acids that can form salts include acetates (such as acetic acid or trihaloacetic acids, such as trifluoroacetic acid), adipates, alginates, ascorbic acid salts, aspartate salts, benzoates, benzenesulfonates, hydrogen sulfates, borates, butyrates, citrates, camphor salts, camphor sulfonates, cyclopentanepropionate, diethylene glycol salts, dodecyl sulfates, ethanesulfonates, fumarates, glucono-2-phosphates, glycerol phosphates, hemisulfates, heptarates, hexanoates, hydrochlorides, hydrobromide, and hydroiodide. Salts, hydroxyethanesulfonates (e.g., 2-hydroxyethanesulfonate), lactates, maleates, methanesulfonates, naphthalenesulfonates (e.g., 2-naphthalenesulfonate), nicotinates, nitrates, oxalates, pectates, persulfates, phenylpropionates (e.g., 3-phenylpropionates), phosphates, picrates, neopentanoates, propionates, salicylates, succinates, sulfates (e.g., those formed with sulfuric acid), sulfonates, tartrates, thiocyanates, toluenesulfonates such as p-toluenesulfonate, dodecanoates, etc.

[0043] Some compounds of this invention may contain acidic fragments, including but not limited to carboxylic acids, which may form salts with various organic or inorganic bases. Typical base-formed salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, and salts formed with organic bases (such as organic amines), such as benzylamine, dicyclohexylamine, hepatopanylamine (a salt formed with N,N-di(dehydroabietic)ethylenediamine), N-methyl-D-glucosamine, N-methyl-D-glucosamide, tert-butylamine, and salts formed with amino acids such as arginine, lysine, etc. Basic nitrogen-containing groups can react with quaternary ammonium halides, such as small alkyl halides (e.g., chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl halides), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate), long-chain halides (e.g., chlorides, bromides, and iodides of decyl, dodecyl, tetradecyl, and tetradecyl halides), aralkyl halides (e.g., benzyl and phenyl bromides), etc.

[0044] The compounds of this invention, pharmaceutically acceptable salts, and possible tautomer forms (e.g., amides and imine ethers) are all part of this invention.

[0045] All stereoisomers of compounds (e.g., those with asymmetric carbon atoms due to various substitutions), including their enantiomers and diastereomeric forms, are within the scope of this invention. The independent stereoisomers of the compounds in this invention may not coexist with other isomers (e.g., possessing special activity as a pure or substantially pure optical isomer), or may be mixtures, such as racemates, or mixtures formed with all other stereoisomers or a portion thereof. The chiral center of this invention has two configurations, S or R, as defined by the International Union of Pure and Applied Chemistry (IUPAC) in 1974. Racemic forms can be resolved by physical methods, such as stepwise crystallization, or by derivatization into diastereomers followed by crystallization, or by chiral column chromatography. Individual optical isomers can be obtained from racemates by suitable methods, including but not limited to conventional methods, such as recrystallization after salting with an optically active acid.

[0046] The compounds of this invention, obtained sequentially through preparation, separation, and purification, have a weight content equal to or greater than 90%, for example, equal to or greater than 95%, or equal to or greater than 99% (“very pure” compounds), as listed in the text description. Such “very pure” compounds of this invention are also included as part of this invention.

[0047] All configurational isomers of the compounds of this invention are included within the scope of this invention, whether in mixtures, pure or very pure forms. The definition of compounds in this invention includes both cis (Z) and trans (E) olefin isomers, as well as cis and trans isomers of carbocyclic and heterocyclic compounds.

[0048] According to the present invention, the ratio of isomers in a mixture of isomers can be varied. For example, a mixture containing only two isomers can have the following combinations: 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. All ratios of isomers are within the scope of the present invention. Similar ratios readily understood by those skilled in the art, as well as ratios for mixtures of more complex isomers, are also within the scope of the present invention.

[0049] This invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. However, in practice, it is common for one or more atoms to be replaced by atoms with different atomic weights or mass numbers. Examples of isotopes that can be included in the compounds of this invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes, respectively as follows: 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. The compounds of this invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates, wherein the isotopes or other isotopic atoms of the aforementioned compounds are all within the scope of this invention. Certain isotopically labeled compounds of this invention, for example... 3 H and 14 Radioactive isotopes of carbon are also included, and are useful in tissue distribution experiments of drugs and substrates. Tritium, i.e. 3 H and carbon-14, i.e. 14 C, their preparation and detection are relatively easy. They are the preferred isotopes. In addition, heavier isotopes such as deuterium are used for substitution. 2 H, due to its excellent metabolic stability, offers advantages in certain therapies, such as increasing half-life or reducing dosage in vivo, and therefore may be preferred in some cases. Isotopically labeled compounds can be prepared using general methods, by replacing the non-isotopic reagent with an readily available isotopically labeled reagent, according to the scheme described in the examples.

[0050] The compounds of the present invention can be obtained using methods known in the art or purchased commercially.

[0051] Pharmaceutical Compositions and Administration

[0052] The pharmaceutical composition of the present invention is used to treat cancer or immune diseases; specifically, the cancer is selected from the group consisting of myeloma, lymphoma, lung cancer, colorectal cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, thyroid cancer, liver cancer, stomach cancer, pancreatic cancer, or combinations thereof.

[0053] The deuteremedvir of Formula I, or a pharmaceutically acceptable salt, isomer, or combination thereof, can be used in combination with other known drugs for treating or improving similar symptoms. When used in combination, the administration method and dosage of the original drug can remain unchanged, while the compound of Formula I is taken simultaneously or subsequently. When deuteremedvir of Formula I is taken concurrently with one or more other drugs, a pharmaceutical composition containing one or more known drugs and deuteremedvir of Formula I is preferred. Drug combination also includes taking deuteremedvir of Formula I with one or more other known drugs during overlapping time periods. When deuteremedvir of Formula I is used in combination with one or more other drugs, the dosage of deuteremedvir of Formula I or the known drugs may be lower than the dosage of either drug alone.

[0054] Drugs or active ingredients that can be used in combination with deuteremedvir as shown in Formula I include, but are not limited to: PD-1 inhibitors (such as nivolumab, pembrolizumab, pidilizumab, cemiplimab, JS-001, SHR-120, BGB-A317, IBI-308, GLS-010, GB-226, STW204, HX008, HLX10, BAT 1306, AK105, LZM 009 or biosimilars of the above drugs, etc.), PD-L1 inhibitors (such as durvalumab, atezolizumab, avelumab, CS1001, KN035, HLX20, SHR-1316, BGB-A333, JS003, CS1003, KL-A167, F 520, GR1405, MSB2311 or biosimilars of the above drugs, etc.), CD20 antibodies (such as rituximab, obinutuzumab, ofatumumab, veltuzumab, tositumomab, 131I-tositumomab, ibritumomab, 90Y-ibritumomab, 90In-ibritumomab, ibritumomabtiuxetan, etc.), CD47 antibodies (such as Hu5F9-G4, CC-90002, TTI-621, TTI-622, OSE-172, SRF-231, ALX-148, NI-1701, SHR-1603, IBI188, IMM01), ALK inhibitors (such as Ceritinib, Alectinib, Brigatinib, Lorlatinib, Ocalatinib), PI3K inhibitors (such as Idelalisib, Duvelisib, Dactolisib, Taselisib, Bimiralisib, Omip BTK inhibitors (such as alisib, Buparlisib, etc.), EGFR inhibitors (such as Afatinib, Gefitinib, Erlotinib, Lapatinib, Dacomitinib, Icotinib, Canertinib, Sapitinib, Naquotinib, Pyrotinib, Rociletinib, Osimertinib, etc.), VEGFR inhibitors (such as Sorafenib, Pazopanib, Regorafenib, Sitravatinib, Ningetinib, Cabozantinib, Sunitinib, Donafenib, etc.), HDAC inhibitors (such as Givinostat, Tucidinostat, Vorinostat, Fimepinostat, Droxinostat, Entinostat, Dacinostat, Quisinostat, Tacedinaline, etc.), CDK inhibitors (such as Palbociclib). The pharmaceutical compositions of this invention include (but are not limited to) ribociclib, abemaciclib, milciclib, trilaciclib, lerociclib, etc.; MEK inhibitors (such as Selumetinib (AZD6244), Trametinib (GSK1120212), PD0325901, U0126, Pimasertib (AS-703026), PD184352 (CI-1040), etc.); mTOR inhibitors (such as Vistusertib), SHP2 inhibitors (such as RMC-4630, JAB-3068, TNO155, etc.) or combinations thereof. The dosage forms of the pharmaceutical compositions of this invention include (but are not limited to): injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, controlled-release or sustained-release formulations, or nanoformulations.

[0055] The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of an active ingredient, preferably in the form of a compound of formula I as the active ingredient, which accounts for 0.1 wt% to 90 wt% of the total weight, with the remainder being a pharmaceutically acceptable carrier, diluent, solution, or salt solution.

[0056] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0057] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0058] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0059] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0060] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0061] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0062] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0063] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0064] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0065] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0066] The treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.

[0067] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 50–1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0068] The present invention also provides a method for preparing a pharmaceutical composition, comprising the steps of: mixing a pharmaceutically acceptable carrier with a compound of the general formula (A0) or its crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate of the present invention, thereby forming a pharmaceutical composition.

[0069] The present invention also provides a method for treating tumors, comprising the steps of: administering to a subject requiring treatment a deuterated remedvir of Formula I as described in the present invention, or a pharmaceutically acceptable salt or isomer thereof, or administering a pharmaceutical composition as described in the present invention.

[0070] The present invention also provides a method for inhibiting TRIP13 enzyme, comprising the steps of contacting a deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt or isomer thereof, or a pharmaceutical composition or formulation comprising deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt or isomer thereof, with a subject of interest.

[0071] Compared with the prior art, the main advantages of the present invention include:

[0072] The deuterium remdesivir and its composition of the present invention can effectively inhibit the growth of tumor cells, especially multiple myeloma, providing a new alternative drug for the prevention and treatment of tumors.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0075] Example 1: Molecular docking revealed that deuterium remidevir hydrobromide tablets bind to TRIP13.

[0076] Molecular docking results showed that deuterium remdesivir hydrobromide tablets had a good binding with TRIP13, with a docking score of -8.88 kcal / mol, which was better than the previously reported positive compound DCZ0415 (-8.19 kcal / mol). Deuterium remdesivir hydrobromide tablets can form hydrogen bonds not only with Arg199 and Lys146, but also with π-π interactions with Trp136. These interactions promote its binding with TRIP13, such as... Figure 1 As shown, molecular docking was performed using Smina to calculate the binding affinity between the compound and the TRIP13 protein. The 3D structure of the TRIP13 protein was obtained from the PDB database (PDB ID: 6LK0) and the preferred conformation obtained from molecular dynamics simulations. Both the protein and the compound were preprocessed using AutoDockTools.

[0077] Example 2: Inhibition of TRIP13 enzyme activity by deuterium remdesivir hydrobromide tablets

[0078] 2.1. Experimental Materials

[0079] Microplate reader, model: LUM-384, Thermo Fisher Scientific, USA; ADP-Glo TM The kinase assay kit (ADP-Glo ​​Kinase Assay, Promega, V9101) is from Promega Inc., USA.

[0080] 2.2. Experimental Methods

[0081] 2.2.1. Preparations before the experiment

[0082] Preparation of Reaction Buffer (RB): Add 1.25 mL Tris-HCl (pH 7.5, 25 mM), 2 mL NaCl (200 mM), 500 μL MgCl2 (20 mM), 50 μL DTT (1 mM), 3 g glycerol (5%), and 0.025 g Tween (0.05%) to a final volume of 50 mL. Dispense into 50 mL containers and store at 4°C.

[0083] Compound and reagent preparation: VV116 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. The stock solution was diluted with RB to the desired concentration (10 μM or 25 μM) to obtain the test sample solution. TRIP13 was serially diluted with RB to a concentration of 2.5 μM for later use. ATP (aliped and stored at -80℃, 10 mM) was thawed in an ice-water bath, centrifuged briefly at 4℃, diluted with RB to 200 μM (97.5 μL + 2.5 μL), mixed well, and centrifuged briefly again.

[0084] 2.2.2. Experimental Procedure

[0085] TRIP13 enzyme activity assay: Take an appropriate concentration of deuterium remdesivir hydrobromide tablet solution into an EP tube, add 2 μL of diluted TRIP13 protein (2.5 μM), mix thoroughly, and briefly incubate at 4°C. Pre-incubate in a PCR instrument at 37°C for 30 min. Add 2 μL of ATP, mix well, and briefly incubate in a PCR instrument at 37°C for 30 min. Add 5 μL of ADP-Glo ​​to each of a 5 μL mixture (deuterium remdesivir hydrobromide tablet-TRIP13-ATP). TM The reagent was added to terminate the kinase reaction and consume the remaining ATP. The mixture was then incubated at room temperature for 40 min. Next, 10 μL of Kinase Detection Reagent was added to convert ADP to ATP, and the mixture was incubated at room temperature for 30 min. 18 μL of the reaction solution was added to each well of a 384-well plate. Finally, the amount of newly synthesized ATP was determined by chemiluminescence detection using a microplate reader. The inhibition rate was calculated by comparing the signal values ​​before and after the drug addition.

[0086] 2.3. Experimental Results and Discussion

[0087] The results showed that deuterium remdesivir hydrobromide tablets inhibited TRIP13 activity by 51.4% at 50 μM, while the previously reported positive compound DCZ0415 inhibited TRIP13 activity by 9.1% under the same conditions. These results suggest that deuterium remdesivir hydrobromide tablets can inhibit TRIP13 activity, and that its inhibitory activity against TRIP13 is stronger than that of the positive compound DCZ0415.

[0088] Example 3: Growth inhibitory effect of deuterium remdesivir hydrobromide tablets on tumor cells

[0089] 3.1. Experimental Materials

[0090] Myeloma cell lines: human myeloma cell lines NCI-H929 and ARP-1, lymphoma cell lines U-2932 (B-cell lymphoma cells), NU-DUL-1 (human diffuse histiocytic lymphoma), and A549 human non-small cell lung cancer cells and HCT116 (human colon cancer cells) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China).

[0091] Reagents: RPMI-1640 was purchased from Gibco, Inc., USA; fetal bovine serum (FBS) was from Beijing Solarbio Science & Technology Co., Ltd.; Cell Count Kit-8 (CCK-8) reagent kit was from Shanghai Yisheng Biotechnology Co., Ltd.

[0092] 3.2. Experimental Methods

[0093] (1) Cell inoculation

[0094] Suspension cells: Collect ARP-1 / NCI-H929 / U-2932 / NU-DUL-1 cells in centrifuge tubes, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend in fresh complete culture medium at 2×10⁻⁶. 5 Cells were seeded at a density of 95 μL per well in a 96-well plate, and 5 μL of a certain concentration of the drug (the drug of this invention) was added to each well. The control group was added with an equal amount of DMSO. The plates were incubated at 37°C with 5% CO2 for 72 hours and cell viability was detected.

[0095] Adherent cells: Digest cells with trypsin, add to culture medium and mix well, then incubate at 2×10⁻⁶. 5 Cells were seeded at a density of 95 μL per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The medium was then changed afterward. Medium containing the compound was added, and the cells were cultured for another 48 hours before assessing cell viability.

[0096] (2) Add CCK8 reagent at a dose of 10 μL / well, mix briefly with a vortex mixer, and incubate at 37°C for 2 hours. Note that bubbles should not be generated during the addition process, as bubbles will interfere with the reading.

[0097] (3) Gently shake to mix, and detect the change in absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The inhibitory effect of the drug of the present invention is expressed as a percentage, and each group is repeated three times.

[0098] (4) Calculate cell viability and inhibition rate using the following formulas:

[0099] Cell viability = (absorbance of drug-treated group - absorbance of blank well) / (absorbance of control group - absorbance of blank well) * 100%.

[0100] Cell inhibition rate = 100% - cell survival rate.

[0101] 3.3 Experimental Results and Conclusions

[0102] The results are shown in Table 1. It can be seen that deuterium remidevir hydrobromide tablets can effectively inhibit the growth of myeloma cells, lymphoma cells, and solid tumor cells such as lung cancer and colon cancer.

[0103] Table 1. Inhibitory effect of deuterium remdesivir hydrobromide tablets on tumor cell growth.

[0104]

[0105]

[0106] Example 4: Effect of deuterium remdesivir hydrobromide tablets on intracellular TRIP13 stability using a cell thermal migration assay (CETSA).

[0107] 4.1 Experimental Materials

[0108] Myeloma cell lines: Human myeloma cell lines NCI-H929 and ARP-1 were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China);

[0109] Reagents: RPMI-1640 was purchased from Gibco, Inc., USA; fetal bovine serum (FBS) was from Beijing Solarbio Science & Technology Co., Ltd.; Cell Count Kit-8 (CCK-8) reagent kit was from Shanghai Yisheng Biotechnology Co., Ltd.

[0110] 4.2 Experimental Procedure

[0111] Collect NCI-H929 cells in centrifuge tubes, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend in fresh complete culture medium at 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL, with 95 μL per well in a 96-well plate. 5 μL of a specific concentration of the drug (the final concentration of the drug in this invention is 80 μM) was added to each well. An equal volume of DMSO was added to the control group. The plates were incubated at 37°C with 5% CO2 for 5 hours, followed by cell lysis. The supernatant was subjected to gradient heating and centrifugation to remove heat-denatured proteins. TRIP13 protein was detected and quantified by Western blot.

[0112] 4.3 Experimental Results

[0113] like Figure 2 As shown, the Cell Thermal Migration Assay (CETSA) experiment demonstrated that deuterium remdesivir hydrobromide tablets bind to cells and increase the stability of TRIP13 protein within cells, thus clarifying the targeting of deuterium remdesivir hydrobromide tablets.

[0114] Example 5: Deuterium remidevir hydrobromide tablets inhibit the growth of NCI-H929 xenograft tumors in nude mice

[0115] Deuterium remidevir hydrobromide tablets have significant inhibitory activity against human myeloma cell lines NCI-H929 and ARP-1. Therefore, further research is needed on its in vivo anti-multiple myeloma activity.

[0116] 5.1. Experimental Materials

[0117] Male BALB / c nude mice (4-5 weeks old) were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0118] 5.2. Experimental Methods

[0119] Tumor xenograft model: Female Balb / c mice, 4-5 weeks old, were subcutaneously inoculated with 2x10⁵ NCI-H929 cells in the scapular region of their backs. After tumor formation for 4-6 days, the mice were randomly divided into two groups: a control group (n=5, 10% DMSO, 40% PEG300, 5% Tween-80, 45% NS) and a group treated with 50 mg / kg deuterium remdesivir hydrobromide tablets (n=5, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80, 45% NS). The drug was then administered intraperitoneally every other day. Mouse weight and tumor length and width were measured daily for 13 days.

[0120] Experimental results are as follows Figure 3 As shown. At the end of the experiment, all mice were euthanized, and tumor, liver, and kidney specimens were collected for pathological and immunohistochemical staining to evaluate the drug effects. Tumor volume = length × width² × 0.5. All animal studies were approved by the Institutional Animal Care and Use Committee of Tongji University (Approval: TJBB00223101).

[0121] 5.3. Experimental Results and Discussion

[0122] Compared with the control group, 50 mg / kg of deuterium remdesivir hydrobromide tablets significantly inhibited the growth of NCI-H929 xenografts in nude mice. Figure 3 A) The tumor volume of mice in the deuterium remdesivir hydrobromide tablet group was significantly reduced, and the tumor of one mouse almost completely disappeared. Figure 3C). Furthermore, under these conditions, deuterium remdesivir hydrobromide tablets had almost no effect on the weight and condition of mice, demonstrating good safety and tolerability. Figure 3 This experimental result demonstrates the effectiveness of the drug in an anti-myeloma mouse model.

[0123] Example 6: Deuterium remidevir hydrobromide tablets and their combination with bortezomib inhibit the growth of NCI-H929 xenografts in nude mice.

[0124] 6.1. Experimental Materials

[0125] Experimental animals: female BALB / c nude mice (5-7 weeks old, 18-20g), purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0126] Positive control: Bortezomib for injection (batch number: NIZTB00), manufactured by Xian Janssen Pharmaceutical Co., Ltd.

[0127] Human myeloma NCI-H929 cell line: provided by Nanjing Kebai Biotechnology Co., Ltd.

[0128] Test solvent: 40% PEG400 + 10% Kolliphor HS15 + 50% ultrapure water; Positive control solvent: 0.9% sodium chloride injection.

[0129] 6.2. Experimental Methods

[0130] Tumor xenograft model: After feeding female Balb / c mice for 5-7 weeks, subcutaneously inoculate each mouse with 5 x 10 g of the tumor xenograft into the right side of the back near the armpit. 6 NCI-H929 cells (50% Matrix gel), when the tumor grows to an average size of approximately 100-150 mm. 3 Around 20 days after administration, 20 tumor-bearing mice were randomly divided into 4 groups of 5 mice each. The day of grouping and vaccination was defined as day 0. After weighing, the mice were given the drug, and tumor volume and body weight were measured twice a week. On day 28 after drug administration, the mice were weighed, sacrificed, and tumor tissue was harvested, weighed, and photographed.

[0131] Grouping and drug administration: Blank control group (G1, n=5), 50 mg / kg deuterated remdesivir hydrobromide tablet treatment group (G2, VV116 group, ip, Q2d, n=5), 50 mg / kg deuterated remdesivir hydrobromide tablet plus 0.7 mg / kg bortezomib treatment group (G3, VV116 (ip, Q2d) + BTZ (iv, QW) group, n=5), 0.7 mg / kg bortezomib treatment group (G4, BTZ group, iv, QW, n=5). At the end of the experiment, all mice were euthanized to evaluate drug effects. Tumor volume = (length × width) 2) / 2. All experimental protocols and animal handling procedures will be approved by the IACUC (Laboratory Animal Management and Use Committee) before the start of the experiment.

[0132] 6.3 Experimental Results and Discussion

[0133] Compared with the control group, 0.7 mg / kg bortezomib significantly inhibited the growth of NCI-H929 xenografts in nude mice. The 50 mg / kg deuterated remdesivir hydrobromide tablets and the combined administration group showed significantly better tumor-suppressing effects than the 0.7 mg / kg bortezomib group, and most mouse tumors completely disappeared after 28 days. Figure 4 A). Under these conditions, deuterium remdesivir hydrobromide tablets had almost no effect on the body weight and condition of mice, demonstrating good safety and tolerability. Figure 4 B). This experimental result further validates the effectiveness of the drug in an anti-myeloma mouse model.

[0134] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a deuterated remdesivir of formula (I), or a pharmaceutically acceptable salt thereof, or an isomer thereof, characterized in that, Used to prepare (a) antitumor drugs and / or (b) TRIP13 enzyme inhibitors.

2. The use as described in claim 1, characterized in that, The pharmaceutically acceptable salt is selected from the group consisting of: hydrobromic acid, hydrochloride, trifluoroacetate, trifluoroformate, benzoate, benzenesulfonate, and phosphate; preferably, the deuterated remidevir is deuterated remidevir hydrobromide.

3. The use as described in claim 1, characterized in that, The tumor is selected from hematologic malignancies and solid tumors; preferably, the hematologic malignancies are selected from the group consisting of myeloma, lymphoma, or combinations thereof; the solid tumors are selected from the group consisting of lung cancer, colorectal cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, thyroid cancer, liver cancer, stomach cancer, pancreatic cancer, or combinations thereof.

4. The use as described in claim 1, characterized in that, The tumor in question is multiple myeloma.

5. The use as described in claim 1, characterized in that, The drug is an oral or injectable dosage form; and / or the oral or injectable dosage form is selected from the group consisting of: powder, tablet, granule, capsule, sustained-release, oral liquid, emulsion or suspension.

6. The use as described in claim 1, characterized in that, In the drug, the content of deuterated remidevir of formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, as the active ingredient is 0.01-99.9% wt; the remaining components are pharmaceutically acceptable excipients.

7. A pharmaceutical composition, characterized in that, include: (1) Deuterated remidevir of formula I as the first active ingredient, or a pharmaceutically acceptable salt thereof, or an isomer thereof; (2) Other antitumor drugs or therapies as the second active ingredient: bortezomib, carfizzomib, ixazomib, thalidomide, lenalidomide, pamalidomide, daratumumab, ituximab, or combinations thereof, or immunotherapy. (3)Optionally, a pharmaceutically acceptable carrier.

8. Use of the pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is used to prepare an antitumor drug.

9. The use as described in claim 8, characterized in that, The tumors mentioned are selected from the following group: hematologic malignancies (myeloma, lymphoma, or combinations thereof) and solid tumors (lung cancer, colorectal cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, thyroid cancer, liver cancer, stomach cancer, pancreatic cancer, or combinations thereof).

10. A method for treating tumors, characterized in that, The procedure includes the following steps: contacting a subject with a deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutical composition or formulation comprising a deuterated remdesivir of Formula I, or a pharmaceutically acceptable salt thereof, or an isomer thereof.

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