Deuterated compound for inhibiting RNA helicase DHX33 activity, synthesis method, pharmaceutical composition and use

Abstract

The present invention belongs to the field of biomedicine and relates to a deuterated compound that inhibits the activity of the RNA helicase DHX33 and its synthesis method. It also relates to a pharmaceutical composition containing the compound and its use in the preparation of a drug for preventing and / or treating DHX33-related diseases. The structural formula of the deuterated compound is: #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a deuterated compound that inhibits the activity of RNA helicase DHX33 and a preparation method thereof. It also relates to a pharmaceutical composition containing the deuterated compound and use of the deuterated compound in preparing a drug for preventing and / or treating DHX33-related diseases. Background Art

[0002] DHX33 belongs to the RNA helicase protein family that contains the DEAD / H box. DEAD / H stands for the amino acid abbreviation Asp-Glu-Ala-Asp / His. This sequence, along with several other conserved amino acid sequences, is found in the protein sequences of RNA helicase family members and is highly involved in nucleic acid substrate binding and ATP hydrolysis. While these family members share these common sequences, each RNA helicase has unique specificity and biological functions. The human DHX33 protein has a molecular weight of 72 kDa and functions to unwind nucleic acids. It utilizes the bioenergy released by ATP hydrolysis to drive conformational changes in RNA and protein complexes, thereby participating in a variety of RNA metabolic activities, specifically, RNA transcription, splicing, editing, translation, and degradation. DHX33's functions are not limited to RNA modification. Research has shown that in addition to unwinding RNA duplexes, DHX33 also participates in DNA metabolism. Specifically, DHX33 can unwind the double-stranded structure of DNA and play a vital role in gene expression. In an in vitro enzyme reaction system, DHX33 protein was also found to be able to unwind the hybrid double-stranded structure of DNA / RNA.

[0003] Research has shown that DHX33 binds to the promoters of various cancer-related genes, affecting DNA methylation. This in turn regulates the expression of numerous cancer genes and signaling pathways associated with tumor development at the genomic level, playing a crucial role in numerous cellular activities, including cell growth, proliferation, migration, apoptosis, and glucose metabolism. Furthermore, DHX33 has been shown to sense the intrusion of foreign double-stranded RNA molecules and play a crucial role in cellular innate immunity. As a crucial cell growth regulator, DHX33 is highly expressed in various cancers, including lung cancer, lymphoma, glioblastoma, breast cancer, colon cancer, and liver cancer. The development and progression of many cancers depend on elevated DHX33 protein expression. Genetic knockout of DHX33 significantly inhibits the development and progression of RAS oncogene-driven lung cancer. In vitro and in vivo experiments have confirmed that inhibition of DHX33 significantly suppresses the development and progression of various cancers, including breast cancer, colon cancer, glioma, and lymphoma.

[0004] Research has shown that the function of DHX33 protein is dependent on its helicase activity. Helicase-deficient mutants of DHX33 lack DHX33 protein function and are unable to replace the function of the wild-type DHX33 gene. Currently, small molecule inhibitors targeting DHX33 are relatively scarce, necessitating the development of highly active and druggable DHX33 inhibitors. Summary of the Invention

[0005] Through extensive research, the present invention has discovered a deuterated compound that inhibits the RNA helicase activity of DHX33, which can be used to prevent and / or treat DHX33-related diseases, such as cancer.

[0006] In one aspect, the present invention provides a deuterated compound that inhibits the RNA helicase activity of DHX33 as shown in Formula I:

[0007]

[0008] Wherein, the molecular formula of the deuterated compound is C 21 H 16 D3N5O2S, molecular weight is 408.49.

[0009] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned deuterated compound or a pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers.

[0010] In a third aspect, the present invention provides use of the above-mentioned compound or a pharmaceutically acceptable salt or prodrug thereof in the preparation of a medicament for preventing and / or treating a disease or condition mediated at least in part by DHX33.

[0011] In embodiments of the third aspect, the disease or condition mediated at least in part by DHX33 may be cancer.

[0012] In a fourth aspect, the present invention provides a method for synthesizing a deuterated compound, comprising the steps of:

[0013]

[0014] In an embodiment of the fourth aspect, the molar ratio of the compound 4 to the compound A is 2.23:2.35, the reaction temperature is 90° C., and the reaction time is 16 h.

[0015] In an embodiment of the fourth aspect, the synthesis method of compound 4 comprises the following steps:

[0016]

[0017] In an embodiment of the fourth aspect, the method for synthesizing compound 2 from compound 1 is to add compound 1 to a solution of Na in CD3OD, and stir the reaction mixture at 80°C for 16 hours.

[0018] Compared with the prior art, the present invention has beneficial technical effects. Specifically, the deuterated compound provided by the present invention has better metabolic stability than the compound (AB) before deuteration, as shown in Formula II.

[0019]

[0020] Definition of terms

[0021] Unless otherwise specified, the following terms have the following meanings in the present invention.

[0022] The terms "comprises," "including," "having," or "containing" or any other variations thereof are intended to cover a non-exclusive or open-ended inclusion. For example, a composition, method, or apparatus that comprises a list of elements is not necessarily limited to only the elements expressly listed but may include other elements not expressly listed or inherent to such composition, method, or apparatus.

[0023] The term "pharmaceutical composition" refers to a composition that can be used as a medicine, which contains a pharmaceutically active ingredient (or therapeutic agent) and, optionally, one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an excipient that is administered together with a therapeutic agent and is suitable, within the scope of sound medical judgment, for contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the present invention include, but are not limited to: a) diluents; b) lubricants; c) binders; d) disintegrants; e) absorbents, colorants, flavorings, and / or sweeteners; f) emulsifiers or dispersants; and / or g) substances that enhance the absorption of the compound, etc.

[0024] Above-mentioned pharmaceutical composition can act systemically and / or act topically.For this purpose, they can be administered by suitable approach, for example by parenteral, local, intravenous, oral, subcutaneous, intraarterial, intradermal, percutaneous, rectal, intracranial, intraperitoneal, intranasal, intramuscular approach or as inhalant administration.

[0025] The above-mentioned route of administration can be achieved through suitable dosage forms. The dosage forms that can be used in the present invention include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, etc.

[0026] When administered orally, the pharmaceutical composition can be prepared into any orally acceptable preparation form, including but not limited to tablets, capsules, aqueous solutions, aqueous suspensions, and the like.

[0027] The pharmaceutical composition can also be administered in the form of a sterile injection, including a sterile water or oil suspension for injection, or a sterile water or oil solution for injection. The carriers that can be used include, but are not limited to, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils, such as monoglycerides or diglycerides, can also be used as solvents or suspending media.

[0028] The pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one deuterated compound represented by Formula I or a pharmaceutically acceptable salt or prodrug thereof.

[0029] The term "disease or condition mediated at least in part by DHX33" refers to a disease, such as cancer, eg, prostate cancer or bladder cancer, whose pathogenesis involves at least in part factors related to DHX33.

[0030] The term "effective amount" refers to a dose that can induce a biological or medical response in cells, tissues, organs or organisms (eg, individuals) and is sufficient to achieve the desired preventive and / or therapeutic effect.

[0031] The dosage regimen can be adjusted to provide the optimal desired response. For example, the drug may be administered in a single dose, divided doses may be administered over time, or the dosage may be proportionally reduced or increased based on the actual situation. It will be understood that for any particular individual, the specific dosage regimen should be adjusted according to the needs and the professional judgment of the person administering the composition or supervising the administration of the composition.

[0032] The term "in need of" refers to the physician's or other health care provider's judgment that an individual needs or will benefit from a preventive and / or therapeutic procedure, which judgment is based on various factors within the physician's or other health care provider's area of ​​expertise.

[0033] The term "individual" (or subject) refers to a human or non-human animal. The individual of the present invention includes individuals (patients) suffering from a disease and / or condition and normal individuals. The non-human animals of the present invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0034] The term "treat" refers to the alleviation or elimination of the disease or condition being treated. If an individual receives a therapeutic amount of a compound of the present invention or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the present invention, and at least one indicator and symptom of the individual shows observable and / or detectable relief and / or improvement, the individual is said to have been successfully "treated". It is understood that treatment includes not only complete treatment, but also includes not achieving complete treatment but achieving some biologically or medically relevant results. Specifically, "treat" means that the compound of the present invention or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the present invention can achieve at least one of the following effects, for example: (1) preventing the occurrence of the disease in an animal that may be predisposed to the disease but has not yet experienced or displayed the pathology or symptoms of the disease; (2) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptoms of the disease (i.e., preventing the further development of the pathology and / or symptoms); (3) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptoms of the disease (i.e., reversing the pathology and / or symptoms).

[0035] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is substantially non-toxic to living organisms. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed by reacting a compound of the present invention with a pharmaceutically acceptable inorganic / organic acid or inorganic / organic base, such salts also known as acid addition salts or base addition salts. For a review of suitable salts, see, for example, Jusiak, Soczewinski, et al., Remington's Pharmaceutical Sciences [M], Mack Publishing Company, 2005 and Stahl, Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use [M], Wiley-VCH, 2002. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0036] The present invention encompasses all possible metabolite forms of the compounds of the present invention, i.e., substances formed in the body of an individual to whom the compounds of the present invention are administered. Metabolites of the compounds can be identified by techniques known in the art, and their activity can be characterized by assays. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 HPLC chromatograms of deuterated compounds of the present invention are shown.

[0038] Figure 2 The deuterated compounds of the present invention are shown 1 HNMR spectrum.

[0039] Figure 3 The mass spectrum of the deuterated compound of the present invention is shown.

[0040] Figure 4 The difference in HPLC elution time of the deuterated compound of the present invention and the known compound is shown.

[0041] Figure 5 An analysis of the inhibitory activity of the deuterated compounds of the present invention on PC-3 cancer cells is shown.

[0042] Figure 6 Analysis of the inhibitory activity of the deuterated compounds of the present invention on cancer cells 5637 is shown.

[0043] Figure 7 The activity analysis of the compound represented by formula II in inhibiting the above two cancer cells is shown.

[0044] Figure 8 The metabolic stability analysis curve of the deuterated compound of the present invention in human liver microsomes is shown.

[0045] Figure 9 Analytical curves showing the metabolic stability of deuterated compounds of the present invention in canine liver microsomes.

[0046] Figure 10 Important parameters of the metabolic stability of the deuterated compounds of the present invention in human and canine liver microsomes are shown.

[0047] Figure 11 The metabolic stability analysis curve of the compound represented by formula II in human liver microsomes is shown

[0048] Figure 12 The metabolic stability analysis curve of the compound represented by formula II in dog liver microsomes is shown.

[0049] Figure 13 The important parameters of metabolic stability of the compound represented by formula II in human and dog liver microsomes are shown. DETAILED DESCRIPTION

[0050] In order to make the purpose and technical solution of the present invention clearer, the embodiments of the present invention are described in detail below in conjunction with examples. However, it will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0051] The reagents and instruments used in the examples are all commercially available conventional products. If no specific conditions are specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. The term "room temperature" as used in the present invention refers to 20°C ± 5°C. When used to modify a certain numerical value or numerical range, the term "about" as used in the present invention refers to the numerical value or numerical range and the acceptable error range for those skilled in the art for the numerical value or numerical range, for example, the error range is ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

[0052] The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0053] The nuclear magnetic resonance (NMR) was measured using a Bruker 400 MHz NMR instrument. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), and hexadeuterated dimethyl sulfoxide (DMSO-d6), and the internal standard was tetramethylsilane (TMS).

[0054] The abbreviations in the nuclear magnetic resonance (NMR) data in the following examples have the following meanings:

[0055] s: singlet, d: doublet, t: triplet, q: quartet, dd: double of doublet, qd: quadruple of doublet, ddd: double of doublet, ddt: double of doublets, dddd: double of doubles of doublets, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, δ: chemical shift.

[0056] All chemical shift (δ) values ​​are given in parts per million (ppm).

[0057] The mass spectrometry (MS) was performed using an Agilent 6120B mass spectrometer with an electrospray ionization (ESI) source.

[0058] HPLC analysis was performed using a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18, 150×4.6 mm, 5 μm column).

[0059] The thin layer chromatography silica gel plate used was Qingdao Ocean GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) had a specification of 0.15mm-0.2mm, and the specification used for thin layer chromatography separation and purification products was 0.4mm-0.5mm silica gel plate.

[0060] Column chromatography generally uses Qingdao Ocean 200-300 mesh silica gel as the carrier.

[0061] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent systems used in the reactions were A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compounds.

[0062] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used for purifying compounds include A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.

[0063] Example 1 Synthesis of Deuterated Compounds

[0064]

[0065] This example provides a synthetic route for the deuterated compound (AB25867), as follows:

[0066] a. Preparation of Compound 2

[0067]

[0068] To a solution of Na (2.0 g, 86.93 mmol, 3.0 eq) in CD3OD (50 ml) was added compound 1 (5 g, 29.98 mmol, 1.0 eq), and the reaction mixture was stirred at 80 ° C for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was quenched with water (20 ml) and partitioned between dichloromethane (30 ml) and water (50 ml). The aqueous phase was extracted twice with dichloromethane (20 ml), and the organic phases were combined, washed with saturated NaCl (aq) (20 ml), dried over Na2SO4, and evaporated to dryness to obtain compound 2 (4.03 g, yield: 98.8%) as a light yellow solid.

[0069] TLC: PE / EA=5 / 1,

[0070] R f (Compound 1) = 0.20,

[0071] R f (Compound 2) = 0.40;

[0072] LCMS: 172.15 [M+H] +.

[0073] b. Preparation of Compound 3

[0074]

[0075] A mixture of compound 2 (500 mg, 2.29 mmol, 1.0 eq) and Pd / C (100 mg, 20% w / w) in methanol (60 ml) was stirred under 1 atmosphere of H2 at room temperature (RT) for 2 h. The reaction mixture was filtered and the filtrate was concentrated to afford compound 3 (363.9 mg, 97.0% yield) as a brown solid.

[0076] TLC: DCM / MeOH=10 / 1,

[0077] R f (Compound 2) = 0.80,

[0078] R f (Compound 3) = 0.20;

[0079] LCMS: 142.10 [M+H] +.

[0080] Compound 3 may be unstable and is best used on site.

[0081] c. Preparation of Compound 4

[0082]

[0083] To a solution of compound 3 (400 mg, 2.84 mmol, 1.0 eq.) in MeOH (5 ml) and H2O (5 ml) was added cyanogen bromide (451 mg, 4.30 mmol, 1.5 eq.), and the reaction mixture was stirred at 50°C for 16 h. The reaction mixture was concentrated, and the residue was adjusted to pH = 9 with NaOH (aq). The aqueous phase was extracted with ethyl acetate (20 ml), and the combined organic phases were washed with saturated NaCl (aq) (10 ml), dried over Na2SO4, and evaporated to dryness. The residue was purified by silica gel chromatography eluting with DCM:MeOH = 10:1 to give compound 4 (265.1 mg, yield: 56.3%) as an off-white solid.

[0084] TLC: DCM / MeOH=10 / 1,

[0085] R f (Compound 3) = 0.20,

[0086] R f (Compound 4) = 0.10;

[0087] LCMS: 167.05 [M+H] +.

[0088] d. Preparation of deuterated compound (AB25867)

[0089]

[0090] To a solution of compound A (611 mg, 2.35 mmol, 1.0 eq.) in ethyl acetate (5 ml) and DIPEA (1.22 g, 9.38 mmol, 4.0 eq.) were added compound 4 (370 mg, 2.23 mmol, 0.95 eq.) and PyBop (1.35 g, 2.58 mmol, 1.1 eq.), and the reaction mixture was stirred at 90°C for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in acetone (3 ml), H2O (9 ml) was added, stirred at room temperature for 30 min, and filtered. The filter cake was dried under vacuum to yield 400 mg of crude AB25867. DIPEA is N,N-diisopropylethylamine, and PyBop is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.

[0091] Purification of AB25867

[0092] The residue was dissolved in THF (3.2 ml, 8 vol), and methanesulfonic acid (94.1 mg, 1.0 eq) was added. The reaction mixture was stirred for 2 h and filtered. The filter cake was dissolved in NaOH (aq) and dichloromethane, and the solution was stirred for an additional 30 min. The reaction mixture was separated, and the organic phase was concentrated under reduced pressure to provide compound AB25867 (330 mg, 34.4% yield) as a light yellow solid.

[0093] TLC: DCM / MeOH=10 / 1,

[0094] R f (Compound 4) = 0.10,

[0095] R f (Compound 5) = 0.60;

[0096] LCMS: 409.05 [M+H] +;

[0097] The H NMR spectrum of deuterated compound AB25867 is as follows Figure 2 As shown, 1 H NMR (400MHz, DMSO-d6) δ7.32–7.26(m,2H),6.97(s,1H),6.87(s,1H),6.68(dd,J=8.7,2.4Hz,1H),2.51(d,J=1.2Hz,3H),2.37(s,3H),2.02(s,3H).

[0098] Example 2 Identification of the Structural Purity of Deuterated Compounds

[0099] Deuterated compounds were detected using LC-MS.

[0100] (1) Chromatographic conditions

[0101] Chromatographic column: Shim-Pack GIST C18 5um.

[0102] Flow rate: 1.0 mL / min. Acquisition time: 15 min. Measurement wavelength: 254 nm. Column temperature: 25°C. Injector temperature: room temperature. Injection volume: 2 μL.

[0103] The diluent was DMSO.

[0104] Mobile phase: Pump A: 0.1% formic acid in water, Pump B: acetonitrile.

[0105] The mobile phase gradient was as follows.

[0106]

[0107] Ion source: electrospray ionization (ESI)

[0108] Scanning mode: positive ion

[0109] The results are as follows Figure 1 and Figure 3 As shown. According to the general liquid chromatography-mass spectrometry combined analysis method, the eluted substance was subjected to mass spectrometry molecular weight identification. The molecular weight of the target substance was calculated to be 409.05 [M+H]+, and the purity or content of the current molecule was determined by the peak area of ​​the main target substance.

[0110] Example 3 HPLC Retention Time Analysis of Deuterated Compounds Compared with Known Compounds

[0111] After confirming that the deuterated compound had been synthesized and achieved the target purity, the retention time of the deuterated compound (AB25867) was compared with that of the known compound represented by Formula II using a Shimadzu LC-2050 HPLC. The chromatographic conditions were the same as those described above, specifically as follows:

[0112] Chromatographic column: Shim-Pack GIST C18 5um.

[0113] Flow rate: 1.0 mL / min. Acquisition time: 15 min. Measurement wavelength: 254 nm. Column temperature: 25°C. Injector temperature: room temperature. Injection volume: 2 μL.

[0114] The diluent was DMSO.

[0115] Mobile phase: Pump A: 0.1% formic acid in water, Pump B: acetonitrile.

[0116] The mobile phase gradient was as follows.

[0117]

[0118] The retention time of the deuterated compound AB25867 was compared with that of the compound represented by Formula II. Figure 4 The retention time of the known compound shown in the figure is 7.797 minutes, while the retention time of the deuterated compound under the same chromatographic conditions is 7.493 minutes, which is slightly more hydrophobic than the known compound, as expected.

[0119] Example 4 Determination of the Half Inhibitory Concentration (IC50) of Deuterated Compounds

[0120] Having confirmed the successful synthesis of the deuterated compound, we further analyzed its activity. Under typical conditions, deuterated compounds do not affect the activity of known substances. However, in this study, we used the half-inhibitory concentration (CI50) assay to compare the activity of the deuterated compound with that of the known compound.

[0121] Human prostate cancer cells PC-3 or human gastric cancer cells SGC7901 were purchased from the Chinese Academy of Sciences Cell Bank. They were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 2mM L-glutamine, streptomycin, and penicillin. Growth conditions were set at 37°C, 5% CO2, and a humidified cell culture incubator. Cells were passaged every 3 days and discarded after 10 passages. Cancer cell lines PC-3 or SGC7901 overexpressing DHX33 were cultured at 1×10 4 / 100μl / well was spread on a 96-well plate. After waiting for the cells to attach completely, the deuterated compound (AB25867) prepared in Example 1 of the present application was added to the complete culture medium of the cell RPMI-1640 at concentrations of 5nM, 10nM, 25nM, 50nM, 100nM, 250nM, 500nM, 1000nM, and 2000nM, and mixed evenly with a multi-channel gun. After waiting for the compound and cell incubation time to reach 48h, CCK-8 reagent (Shanghai Yisheng Biological Company) was added to the culture medium of the 96-well plate according to the standard process. After incubation for 2 hours, the plate was read with an enzyme reader (OD=450nm), the experiment was repeated three times, and the inhibition curve of the compound at different concentrations was drawn to calculate the half inhibitory concentration (IC50) of the deuterated compound prepared in Example 1 of the present application 50 The results showed that the half-inhibitory concentration of the deuterated compound was 34 nM in PC-3 cells and 16 nM in SGC7901 cells.

[0122] Figure 6 The IC values ​​of the known compound represented by formula II in PC-3 cells and SGC7901 cells are shown. 50 The half-inhibitory concentration of the known compound shown in formula II in PC-3 is 50nM ( Figure 7 -a), the half-inhibitory concentration in SGC7901 was 20 nM ( Figure 7 -b).

[0123] The deuterated compound prepared in Example 1 of the present application was determined to have slightly enhanced activity relative to the known compound.

[0124] Example 5 Metabolic Stability Analysis of Deuterated Compounds in Liver Microsomes

[0125] Preheat 100 mM K-buffer with 5 mM MgCl2 (pH 7.41) to create K / M-buffer. Prepare 500 μM dilutions of the deuterated compound (AB25867) and reference solution by adding 5 μL of the 10 mM AB25867 stock solution and the reference solution to 95 μL of ACN. Prepare a 1.5 M microsomal solution (0.75 mg / mL) by adding 1.5 μL of the 500 μM dilutions of the deuterated compound (AB25867) and reference solution and 18.75 μL of 20 mg / mL liver microsomes (Beijing Huizhi Heyuan Biotechnology Co., Ltd.) to 479.75 μL of K / Mg-buffer. Prepare a NADPH stock solution (6 mM, 5 mg / mL) by dissolving NADPH in K / Mg-buffer. 30 μL of a 1.5 μM solution containing 0.75 mg / mL microsomal solution was dispensed onto designated assay plates for analysis at various time points (0, 5, 15, 30, and 45 min). Preincubation was performed at 37°C for 5 min. At 0 min, 150 μL of ACN containing IS (internal standard) was added to the wells, followed by 15 μL of a 6 mM NADPH stock solution. For all other time points, 15 μL of a 6 mM NADPH stock solution was added to the wells to initiate the reaction and time the reaction. The reaction was stopped at 5, 15, 30, and 45 min by adding 150 μL of ACN containing IS to the corresponding wells. After quenching, the plates were shaken for 10 min (600 rpm) and then centrifuged at 6000 rpm for 15 min. 80 μL of supernatant was transferred from each well to a 96-well sample plate containing 140 μL of purified water for LC / MS analysis. The same method was used to analyze and detect the known compound (AB24386) shown in Ⅱ. The results are as follows Figures 8-13 As shown. Figures 8-13 It can be seen that the metabolic stability of the deuterated compound (AB25867) of the present application is significantly improved compared to known compounds. The half-life T1 / 2 data are significantly prolonged in canine and human liver microsomes.

[0126] In summary, these data show that deuterated compounds have better activity and drugability than known compounds, facilitating subsequent drug development and application.

Claims

1. A deuterated compound that inhibits the RNA helicase activity of DHX33, characterized in that it has the following formula I: ； Formula I.

2. A pharmaceutical composition, characterized in that Comprising the deuterated compound according to claim 1 or a pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers.

3. Use of the deuterated compound according to claim 1 or a pharmaceutically acceptable salt or prodrug thereof in the preparation of a medicament for preventing and / or treating a disease or condition at least partially mediated by DHX33.

4. The use according to claim 3, characterized in that The disease or condition is cancer.

5. The use according to claim 4, characterized in that The cancer is a DHX33 protein-highly expressed cancer.

6. A method for synthesizing the deuterated compound according to claim 1, characterized in that: The method comprises: ； Compound 4 and PyBop were added to a solution of compound A in ethyl acetate and DIPEA, and the reaction mixture was stirred at 90°C for 16 h. The reaction mixture was then concentrated under reduced pressure. The total weight of the reaction mixture was 611 mg, 2.35 mmol, 1.0 eq. of compound A, 5 mL of ethyl acetate, 1.22 g, 9.38 mmol, 4.0 eq. of DIPEA, 370 mg, 2.23 mmol, 0.95 eq. of compound 4, and 1.35 g, 2.58 mmol, 1.1 eq. of PyBop. The residue was dissolved in 3 mL of acetone, 9 mL of H O was added, stirred at RT for 30 min, and filtered; The filter cake was dried under vacuum to give 400 mg of crude deuterated compound. Wherein DIPEA is N,N-diisopropylethylamine, and PyBop is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.

7. The method for synthesizing a deuterated compound according to claim 6, wherein: The synthesis method of compound 4 comprises the following steps: 。 8. The method for synthesizing a deuterated compound according to claim 7, wherein: The method for synthesizing compound 2 from compound 1 is to add compound 1 to a solution of Na in CD3OD, and stir the reaction mixture at 80°C for 16 h.

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