Monophosphate nucleoside compounds and their preparation methods

Abstract

This invention relates to the field of biomedical technology and discloses a monophosphate nucleoside compound and its preparation method. The compound has the structure shown in formula (I). The method for preparing the nucleoside compound includes: subjecting the compound shown in formula (I-2) to a contact reaction I with the compound shown in formula (I-1). The aforementioned nucleoside compound provided by this invention exhibits excellent inhibitory activity against CD73. Furthermore, in vivo experiments of this invention demonstrate that the compound exhibits good antitumor activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a monophosphate nucleoside compound and its preparation method. Background Technology

[0002] CD73, also known as extracellular 5′-nucleotidase, is a cell surface enzyme widely expressed on the surface of human endothelial cells and lymphocytes, such as Treg cells. Overexpression of CD73 in the tumor microenvironment helps convert adenosine triphosphate (ATP), which has immune-activating effects, into adenosine, thus promoting tumor growth. Some tumors exhibit upregulation and overexpression of CD73; therefore, it has been proposed as a drug target for cancer treatment.

[0003] In the tumor microenvironment, upregulation of CD73 expression leads to an increase in adenosine, which can promote tumor growth and disease progression, suppress the cytotoxicity of T lymphocytes and natural killer cells, inhibit antigen-presenting cells (APCs) due to the suppression of cytokine production and proliferation; it can also promote the proliferation of Treg cells and suppress immune activity, and stimulate the polarization of bone marrow-derived suppressor cells (MDSCs) and M2 macrophages.

[0004] CD73 is induced by transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), hepatocyte growth factor (HGF), interleukin-6 (IL-6), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), interleukin-2 (IL-2), retinoic acid, int / wingless (WNT), epithelial-mesenchymal transition, and p53 mutations. CD73 is overexpressed in various tumor types and promotes tumor cell invasion, metastasis, and adhesion. CD73 is also associated with immune tolerance and poor prognosis in cancer. Therefore, CD73 is a promising target for the development of anticancer drugs. Furthermore, CD73 inhibitors show potential in the treatment of other diseases mediated by adenosine and its receptors.

[0005] In preclinical in vivo studies, blocking the activity of extracellular nucleotidases or adenosine receptor signaling has successfully inhibited tumor growth and metastasis. Adenosine pathway blockade, alone or in combination with other immunotherapies (including checkpoint inhibitors), is currently in initial Phase I clinical trials in patients with advanced malignancies.

[0006] WO2017 / 120508 and US2017 / 0267710 disclose compounds that regulate the conversion of AMP to adenosine by extracellular 5'-nucleotidase, compositions containing such compounds, methods for synthesizing such compounds, and the use of such compounds and compositions for the treatment and / or prevention of various diseases mediated by extracellular 5'-nucleotidase; WO2015 / 164573 discloses purine derivatives and pharmaceutical compositions thereof, which are inhibitors of CD73 and can be used to treat cancer; WO2018 / 049145 discloses the preparation of nucleotides as inhibitors of extracellular nucleotidase and the use of such compounds in the treatment or prevention of cancer.

[0007] However, the aforementioned compounds have the drawback of poor inhibitory activity against CD73, thus failing to exhibit good antitumor activity. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing compounds in inhibiting CD73.

[0009] To achieve the above objectives, the first aspect of the present invention provides a nucleoside compound of formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvate, or metabolite thereof.

[0010]

[0011] In equation (I),

[0012] R1 and R2 are each independently selected from H, deuterium, hydroxyl, halogen, substituted or unsubstituted C. 1-10 Alkyl, substituted or unsubstituted C 1-10 The alkoxy, cyano, amino, and azide groups; the substituents optionally present on R1 and R2 are each independently selected from halogens and hydroxyl groups;

[0013] R3 and R4 are each independently selected from H, deuterium, hydroxyl, halogen, substituted or unsubstituted C. 1-10 Alkyl, substituted or unsubstituted C 1-10 The alkoxy, cyano, amino, and azide groups; the substituents optionally present on R3 and R4 are each independently selected from halogens and hydroxyl groups;

[0014] R5 is selected from hydrogen, deuterium, halogen, substituted or unsubstituted C. 1-10 alkyl, C 2-8 alkenyl, C 2-8 alkynyl, substituted or unsubstituted C 1-10 The alkoxy, hydroxy, cyano, amino, nitro, and azide groups are all present on R5; the substituents present on R5 are each independently selected from halogens and hydroxyl groups.

[0015] R6 and R7 are each independently selected from hydrogen, deuterium, halogen, cyano, nitro, azido, and C. 1-10 alkyl, C 2-6 alkenyl, C 2-6 Alkyne group, five- to eight-membered heterocyclic group, C 6-12 aryl and C 6-12 The heteroaryl group, or the C group formed by the cyclization of R6, R7 and their attached carbon atoms. 3-8 Cycloalkyl groups, tri- to six-membered heterocyclic groups formed by the cyclization of R6, R7 and their attached carbon atoms;

[0016] W is selected from -O- and -S-;

[0017] L is selected from substituted or unsubstituted alkylene groups, and the substituents on L are each independently selected from C10. 1-10 Alkyl, halogen, amino, nitro, cyano, hydroxyl, C 1-10 alkoxy groups, five- to eight-membered heterocyclic groups, C 6-12 aryl and C 6-12 heteroaryl groups;

[0018] n is 0 or 1;

[0019] G1, G2, and G3 are each independently selected from N and -C(R) 12 )-, R 12 Selected from hydrogen, halogen, substituted or unsubstituted C 1-8 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, substituted or unsubstituted C 1-8 Alkyl, hydroxyl, cyano, amino, nitro and five- to eight-membered heterocyclic groups;

[0020] R8 is selected from hydrogen, halogen, hydroxyl, cyano, amino, nitro, C 1-10 alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-10 alkoxy groups and five- to eight-membered heterocyclic groups; R 11 The substituents present above are each independently selected from halogens and hydroxyl groups;

[0021] R9 is selected from hydrogen, substituted or unsubstituted C. 1-10 alkyl, C 2-8 alkenyl, C 2-8 Alkyne group, substituted or unsubstituted five- to eight-membered heterocyclic group, C 6-12 aryl and C 6-12 heteroaryl; R 12 The substituents present above are each independently selected from halogen, hydroxyl, cyano, amino, nitro, C 1-8alkyl, C 1-8 alkoxy groups, C groups substituted with at least one halogen 1-8 Alkyl groups, C substituted with at least one halogen 1-8 alkoxy groups and five- to eight-membered heterocyclic groups;

[0022] R 10 and R 11 One of them is H, and the other is selected from C. 1-6 alkyl, C 3-8 Cycloalkyl, phenyl, phenyl substituted with at least one halogen, naphthyl, biphenyl, C substituted with at least one group from the combination C 2-8 alkenyl, C 6-12 The heteroaryl group; and the combination C consists of a halogen, a hydroxyl group, and a phenyl group substituted with at least one halogen.

[0023] The second aspect of this invention provides a method for preparing the nucleoside compounds described in the first aspect above, or pharmaceutically acceptable salts thereof, or stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, or metabolites thereof, comprising:

[0024] The compound shown in formula (I-2) is subjected to contact reaction I with the compound shown in formula (I-1) at a temperature of 0-60℃ for a time of 1-72h.

[0025]

[0026] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R 10 R 11 The definitions of G1, G2, G3, W, L, and n are as described in the first aspect above.

[0027] The third aspect of this invention provides the use of the nucleoside compounds described in the first and second aspects in the preparation of drugs as CD73 inhibitors.

[0028] The fourth aspect of this invention provides the application of the nucleoside compounds described in the first, second, and third aspects in the preparation of drugs for the prevention and treatment of tumors.

[0029] The aforementioned nucleoside compounds provided by this invention exhibit excellent inhibitory activity against CD73. Furthermore, in vivo experiments of this invention demonstrate that the compounds exhibit good antitumor activity. Detailed Implementation

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] The terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0032] In this invention, "halogen" refers to at least one of fluorine, chlorine, bromine, and iodine.

[0033] In this invention, the "C" 1-10 "Alkyl" refers to a straight-chain alkyl or branched-chain alkyl group with a total number of carbon atoms of 1-10. For example, the C... 1-10 The alkyl group can be a C1 straight-chain alkyl or branched-chain alkyl, a C2 straight-chain alkyl or branched-chain alkyl, a C3 straight-chain alkyl or branched-chain alkyl, a C4 straight-chain alkyl or branched-chain alkyl, a C5 straight-chain alkyl or branched-chain alkyl, a C6 straight-chain alkyl or branched-chain alkyl, a C7 straight-chain alkyl or branched-chain alkyl, a C8 straight-chain alkyl or branched-chain alkyl, a C9 straight-chain alkyl or branched-chain alkyl, or a C9 straight-chain alkyl or branched-chain alkyl. 10 Straight-chain alkyl groups, branched-chain alkyl groups, etc. Furthermore, the H in the alkyl group can be substituted or unsubstituted by at least one group in combination A, with preferred groups in combination A being: halogens, hydroxyl groups, C... 3-8 cycloalkyl groups, when the "C" 1-10 "alkyl" is a substituted C 1-10 When alkyl is used, it means "C substituted by at least one group in combination A". 1-10 "alkyl", and the number of carbon atoms of the substituent is not included in the total number of carbon atoms 1-10. "C 1-n Alkyl group, C substituted with at least one halogen 1-8 The definition of "alkyl" is the same as "C". 1-10 The definition of "alkyl" is similar, but the total number of carbon atoms is different. Here, n is an integer greater than 1 and less than 10.

[0034] In this invention, the "C" 1-10 "Alkoxy" refers to a straight-chain alkoxy, branched-chain alkoxy, or cycloalkoxy group with a total number of carbon atoms of 1-10. For example, the "C"... 1-10The alkoxy group can be a straight-chain alkoxy or branched-chain alkoxy group of C1, C2, C3, C4, C5, C6, C7, C8, or C9. 10 Straight-chain alkoxy groups, branched-chain alkoxy groups, etc. Furthermore, the H in the alkoxy group can be substituted or unsubstituted by at least one halogen, when the "C" 1-10 "alkoxy" is a substituted C 1-10 When the alkoxy group is substituted, it is "a C substituted with at least one halogen". 1-10 "alkoxy group". "C" 1-n The definition of "alkoxy group" is the same as that of "C". 1-10 The definition of "alkoxy group" is similar, but the total number of carbon atoms is different. Here, n is an integer greater than 1 and less than 10.

[0035] In this invention, the "C" 2-8 "Alkenyl" refers to straight-chain alkenyl or branched alkenyl groups with a total number of carbon atoms of 2-8. For example, the "C"... 2-8 The "alkenyl" can be a C2 straight-chain alkenyl or branched alkenyl, a C3 straight-chain alkenyl or branched alkenyl, a C4 straight-chain alkenyl or branched alkenyl, a C5 straight-chain alkenyl or branched alkenyl, a C6 straight-chain alkenyl or branched alkenyl, a C7 straight-chain alkenyl or branched alkenyl, a C8 straight-chain alkenyl or branched alkenyl, etc. Furthermore, the H in the alkenyl group can be arbitrarily replaced by at least one group in combination C. Preferred groups in combination C are: halogens, hydroxyl groups, and phenyl groups substituted with at least one halogen. 2-8 "Alkenyl" is a substituted C 2-8 When the alkenyl group is substituted, it is "a C group substituted by at least one group from the combination C". 2-8 "Alkenyl", and the number of carbon atoms of the substituent is not included in the total number of carbon atoms (2-8). This invention addresses "C". 2-n The definition of "alkenyl" and "C" 2-8 The definition of "alkenyl" is similar, except that the total number of carbon atoms is different. Therefore, this invention will not refer to "C" in the following text. 2-n The specific definition of "alkenyl" is described in detail here, but those skilled in the art should not understand it as a limitation of the present invention. Here, n is an integer greater than 2 and less than 8.

[0036] In this invention, the "C" 2-8 "Alkyne group" refers to a straight-chain alkynyl group or a branched-chain alkynyl group with a total number of 2-8 carbon atoms. For example, the C... 2-8The alkynyl group can be a C2 straight-chain alkynyl or branched-chain alkynyl, a C3 straight-chain alkynyl or branched-chain alkynyl, a C4 straight-chain alkynyl or branched-chain alkynyl, a C5 straight-chain alkynyl or branched-chain alkynyl, a C6 straight-chain alkynyl or branched-chain alkynyl, a C7 straight-chain alkynyl or branched-chain alkynyl, or a C8 straight-chain alkynyl or branched-chain alkynyl, etc. Furthermore, this invention addresses the "C" group... 2-n The definition of "acetylinyl group" and "C 2-8 The definition of "alkynyl group" is similar, except that the total number of carbon atoms is different. Therefore, this invention will not refer to "C" in the following text. 2-n The specific definition of "alkynyl group" is described in detail here, but those skilled in the art should not understand it as a limitation of the present invention. Here, n is an integer greater than 2 and less than 8.

[0037] In this invention, the "five- to eight-membered heterocyclic group" refers to a heterocyclic group with a total number of 5-8 ring atoms, and at least one heteroatom is present among the atoms constituting the ring. The heteroatom can be, for example, at least one of N, O, or S, and the remaining ring atoms are C. It includes saturated or unsaturated heterocyclic groups, containing at least one saturated or unsaturated heterocycle, or at least two saturated or unsaturated heterocycles. If more than two saturated or unsaturated heterocycles are present, there is no particular limitation on the connection method of each ring. For example, it can be furanyl, thiophene, thiazolyl, imidazolyl, etc., and any position in the heterocyclic group that can be substituted can be arbitrarily substituted by at least one group in combination B. Preferred groups in combination B are: hydroxyl, C... 1-8 alkoxy groups, C groups substituted with at least one halogen 1-8 Composition C consists of alkoxy groups and halogens. Combination C comprises a halogen, a hydroxyl group, and a phenyl group substituted with at least one halogen. When the "five- to eight-membered heterocyclic group" is substituted, it becomes "a five- to eight-membered heterocyclic group substituted with at least one group from combination B," and the number of atoms of the substituent is not included in the total number of ring atoms (5-8). The definitions of "three- to six-membered heterocyclic groups," "four- to ten-membered heterocyclic groups," and "six- to ten-membered heterocyclic groups" are similar to "five- to eight-membered heterocyclic groups," except that the total number of ring atoms differs.

[0038] In this invention, the "C" 6-12 "Aryl" refers to an aryl group with a total number of carbon atoms of 6-12, which contains at least one unsaturated aromatic ring and may also contain at least two unsaturated aromatic rings.

[0039] In this invention, the "C" 6-12 "Heteroaryl" refers to an aryl group with a total number of carbon atoms of 6-12, which contains at least one unsaturated aromatic ring, and may also contain at least two unsaturated aromatic rings. The atoms constituting the aromatic ring contain at least one heteroatom, which may be, for example, at least one of N, O or S, and the remaining ring atoms are C.

[0040] In this invention, the "C" 2-6 "Acyl group" refers to an acyl group with a total number of carbon atoms of 2-6, and its structural formula can be, for example, -NHCO-C 1-5 Alkyl groups. Furthermore, this invention relates to "C". 2-n The definition of "acyl group" and "C" 2-6 The definition of "acyl group" is similar, except that the total number of carbon atoms is different. Therefore, this invention will not refer to "C" in the following text. 2-n The specific definition of "acyl group" is described in detail, but those skilled in the art should not understand it as a limitation of the present invention. Here, n is an integer greater than 2 and less than 6.

[0041] In this invention, the "C" 3-8 "Cycloalkyl" refers to a ring consisting of 3-8 carbon atoms. For example, the "C"... 3-8 The cycloalkyl group can be cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. Furthermore, this invention addresses the use of "C..." 3-n The definition of "cycloalkyl" is the same as "C". 3-8 The definition of "cycloalkyl" is similar, except that the total number of carbon atoms is different. Therefore, this invention will not refer to "C" in the following text. 3-n The specific definition of "cycloalkyl" is described in detail, but those skilled in the art should not understand it as a limitation of the present invention. Here, n is an integer greater than 3 and less than 8.

[0042] The present invention will be described in detail below through embodiments.

[0043] In the following examples, unless otherwise specified, all raw materials used were commercially available and of chemical purity. All room temperatures were 25±2℃.

[0044] Example 1: Preparation of ((((2R,3S,4R,5R)-5-(4-(2-((E)-benzylidene)hydrazyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl))-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0045]

[0046]

[0047] Step 1): Preparation of di-tert-butyl (hydroxymethyl)phosphonate.

[0048] To a 250 mL single-necked round-bottom flask, add di-tert-butyl phosphite (9.72 g, 48 mmol), triethylamine (8.0 mL, 57.7 mmol), water (3.0 mL), and 37% formaldehyde aqueous solution (3.6 mL, 48 mmol). React overnight at room temperature. The reaction solution is concentrated by azeotropic rotary evaporation with methanol to obtain 10.5 g of a white solid powder, γ = 98.1%. The crude solid was used for the next reaction without further purification.

[0049] Step 2): Preparation of methyl (di-tert-butoxyphosphoryl)2-nitrobenzenesulfonate.

[0050] To a 250 mL single-necked round-bottom flask, di-tert-butyl (hydroxymethyl)phosphonate (10.0 g, 59.5 mmol), dichloromethane (100 mL), pyridine (9.41 g, 11.90 mmol), and 2-nitrobenzenesulfonyl chloride (15.8 g, 71.4 mmol) were added sequentially. The mixture was reacted at room temperature for 2 h. The reaction solution was concentrated under reduced pressure and subjected to column chromatography (petroleum ether:ethyl acetate = 1.1, R... f =2.0) Purification yielded 15.1g of a yellow oily substance, Y = 71%.

[0051] Step 3): Preparation of tert-butyl 2-(6-chloro-1-((3R,4R,6R,6R)-6-(((di-tert-butoxyphosphoryl)methoxy)methyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine-1-carboxylic acid.

[0052] Add the following to a 250 mL single-necked round-bottom flask in sequence: methyl 2-nitrobenzenesulfonate (5.0 g, 13.3 mmol), tert-butyl-2-(6-chloro-1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxa-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine-1-carboxylic acid ester (4.3 g, 9.4 mmol, prepared in the same manner as steps 1-4 of Example 1), N,N-dimethylformamide (80 mL), and magnesium tert-butoxide (6.8 g, 39.9 mmol). After the feed was completed, the temperature was raised to 70℃ and the reaction was carried out for 2 hours. After the reaction was complete as monitored by TLC, the reaction solution was poured into ice water, the pH was adjusted to neutral with dilute hydrochloric acid, and extraction was performed. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was then subjected to column chromatography (petroleum ether:ethyl acetate = 1.1, R...). f =1.5) Purification yielded 3.6 g of a white, foamy solid, Y = 40.9%.

[0053] Step 4): Preparation of ((((2R,3S,4R,5R)-5-(6-chloro-4-hydrazino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonate salt.

[0054] To a 50 mL single-necked round-bottom flask, tert-butyl2-(6-chloro-1-((3R,4R,6R,6R)-6-(((di-tert-butoxyphosphoryl)methoxy)methyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine-1-carboxylic acid (1.0 g, 1.5 mmol), methanol (6.0 mL), water (1.5 mL), and concentrated hydrochloric acid (1.5 mL, 18 mmol) were added sequentially. After reacting at room temperature for 3 hours, the mixture was concentrated under reduced pressure to obtain a yellow gel. Acetonitrile (10.0 mL) was added to the yellow gel, and a white solid precipitated. The solid was filtered, and the filter cake was washed with acetonitrile and dried to obtain 400 mg of the title compound, Y = 60%. 1 HNMR(400MHz,DMSO-d6)δ9.77(s,1H),8.38(s,1H),6.04(d,J=4.0Hz,1H),4.50(t,J=4.6Hz,1H) ,4.20(t,J=5.2Hz,1H),4.03(q,J=5.2Hz,1H),3.72(dd,J=10.8,4.2Hz,1H),3.63–3.45(m,3H).

[0055] Step 5): Preparation of ((((2R,3S,4R,5R)-5-(4-(2-((E)-benzylidene)hydrazyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl))-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0056] Add ((((2R,3S,4R,5R)-5-(6-chloro-4-hydrazino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonate (50 mg, 0.11 mmol), ethanol (2.0 mL), water (1.0 mL), and benzaldehyde (14.8 mg, 0.14 mmol) to a 10 mL single-necked round-bottom flask. After the addition is complete, heat to 45 °C and continue overnight. After the reaction is complete as detected by HPLC, concentrate under reduced pressure to obtain the residue. Add a small amount of ethanol to the residue to precipitate the solid. Filter, and dry the filter cake under vacuum to obtain 20 mg of white powder. 1H NMR (600MHz, DMSO-d6) δ12.65(s,1H),8.50(s,1H),8.28(s,1H),7.90–7.74(m,2H),7.53–7.36(m,3H),6.12(d,J=3.9Hz, 1H), 4.52 (t, J = 4.5Hz, 1H), 4.25 (t, J = 5.1Hz, 1H), 4.06 (q, J = 6.0Hz, 1H), 3.74 (dd, J = 10.8, 4.2Hz, 1H), 3.61–3.49 (m, 3H).

[0057] Example 2: Preparation of ((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2,4-dichlorobenzyl)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidine)-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0058]

[0059] The preparation method is the same as in Example 1, except that benzaldehyde in step 1 is replaced with an equimolar amount of 2,4-dichlorobenzaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.78(s,1H),8.57(s,1H),8.45(s,1H),8.22–8.07(m,1H),7.74–7.66(m,1H),7.58–7.40(m,1H),6.09(d, J=3.9Hz,1H),4.51(t,J=4.5Hz,1H),4.23(t,J=4.8Hz,1H),4.06–3.96(m,1H),3.71(dd,dd,J=10.8,4.2Hz,1H),3.59–3.34(m,3H).

[0060] Example 3: Preparation of (((((2R,3S,4R,5R)-5-(4-(2-((E)-[1,1'-biphenyl]-4-ylmethylene)hydrazyl)-6-chloro-1H-pyrazoline))3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0061]

[0062] The preparation method is the same as in Example 1, except that benzaldehyde in step 1 is replaced with an equimolar amount of p-phenylbenzaldehyde. 1H NMR(600MHz,DMSO-d6)δ12.66(s,1H),8.50(s,1H),8.29(s,1H),7.95–7.85 (m,2H),7.81–7.74(m,2H),7.70(d,J=7.6Hz,2H),7.54–7.44(m,2H),7.41–7 .33(m,1H),6.10(d,J=3.9Hz,1H),4.50(t,J=4.5Hz,1H),4.23(t,J=5.2Hz, 1H), 4.04 (q, J=5.2Hz, 1H), 3.73 (dd, J=10.8, 4.2Hz, 1H), 3.66–3.46 (m, 3H).

[0063] Example 4: Preparation of ((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-naphth-1-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0064]

[0065] The preparation method is the same as in Example 1, except that benzaldehyde in step 1 is replaced with an equimolar amount of 1-naphthaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.73(s,1H),9.06(s,1H),8.49(s,1H),8.46(d,J=8.7Hz,1H),8.19(d,J=7.2Hz,1H),8.07(dd,J=17.1,8.1Hz,2H),7.80–7.5 3(m,3H),6.13(d,J=3.9Hz,1H),4.53(t,J=4.4Hz,1H),4.24(t,J=5.2Hz,1 H),4.06(q,J=5.2Hz,1H),3.74(dd,J=10.8,4.2Hz,1H),3.62–3.48(m,3H).

[0066] Example 5: Preparation of ((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-quinolin-8-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0067]

[0068] The preparation method is the same as in Example 1, except that benzaldehyde in step 1 is replaced with an equimolar amount of quinoline-8-carboxaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.78(s,1H),9.55(s,1H),8.98(d,J=4.0Hz,1H),8. 66–8.29(m,3H),8.09(d,J=8.2Hz,1H),7.75(t,J=7.7Hz,1H),7.62(dd,J=8. 3,4.2Hz,1H),6.11(d,J=3.9Hz,1H),4.51(t,J=4.4Hz,1H),4.24(t,J=5.4Hz ,1H),4.04(q,J=5.4Hz,1H),3.73(dd,J=10.8,4.2Hz,1H),3.62–3.45(m,3H).

[0069] Example 6: Preparation of ((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-(4-oxo-4H-chrom-3-yl)methylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0070]

[0071] The preparation method is the same as in Example 1, except that benzaldehyde in step 1 is replaced with an equimolar amount of chromone-3-carboxaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.57(s,1H),9.04(s,1H),8.75(s,1H),8.30(s,1 H),8.11(d,J=7.8Hz,1H),7.81(t,J=7.8Hz,1H),7.68(d,J=8.4Hz,1H),7.5 8–7.46(m,1H),6.08(d,J=3.9Hz,1H),4.50(t,J=4.5Hz,1H),4.23(t,J=5. 4Hz, 1H), 4.03 (q, J = 5.4, 5.2Hz, 1H), 3.83–3.67 (m, 1H), 3.63–3.45 (m, 3H).

[0072] Example 7: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((1E,2E)-3-(4-fluorophenyl)allyl)hydrazyl)-1H-pyrazolino[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid.

[0073]

[0074] The preparation method is the same as in Example 1, except that benzaldehyde in step 1 is replaced with an equimolar amount of p-fluorocinnamaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.51(s,1H),8.53(s,1H),8.01(d,J=9.3Hz,1H),7.69(t,J=6.9Hz,2H),7.35–7.18(m,3H),7.08(d,J=16.0Hz,1H), 6.06(d,J=3.9Hz,1H), 4.49(t,J=4.5Hz,1H), 4.21(t,J=5.2Hz,1H), 4.02(q,J=5.2Hz,1H), 3.71((dd,J=10.8,4.2Hz,1H), 3.59–3.49(m,3H).

[0075] CD73 in vitro enzyme bioevaluation test

[0076] 1.1 Experimental Materials

[0077] Multifunctional microplate reader (Bio-TEK); 96-well plate centrifuge - MPC2000 (Beijing Dinghaoyuan); Tris(hydroxymethyl)aminomethane (Sigma-Aldrich); Magnesium chloride (Aladdin); 5'-Adenosine triphosphate (ATP) (Sigma-Aldrich); Adenine ribonucleotide (AMP) (Sigma-Aldrich); CellTiter-Glo kit (Promega (Beijing) Biotechnology Co., Ltd.); Incubator ZDP-A2080A (Shanghai Zhicheng); Deuterated dimethyl sulfoxide (Aladdin).

[0078] 1.2 Reagent Preparation

[0079] Compound preparation:

[0080] 1. First, dilute the compound stock solution (10mM) 10 times, take 2μL of the diluted stock solution, and add 18μL of DMSO.

[0081] 2. Next, add 5 μL of the compound stock solution containing DMSO to 10 μL for a 3-fold dilution. Take 3 μL of the diluted compound stock solution and add it to 997 μL of Tris-MgCl2.

[0082] hCD73 preparation process:

[0083] Take 1 μL of the stock solution (1.45 mg / mL) and add it to 99 μL of Tris-MgCl2 for dilution to obtain a diluted solution of hCD73 with a concentration of 14.5 μg / mL.

[0084] AMP preparation process:

[0085] Take 14 μL of AMP stock solution (100 mM) and add 1986 μL of Tris-MgCl2 to dilute it to obtain the diluted AMP stock solution.

[0086] 1.3 Test Methods

[0087] 1. Add the prepared CD73 solution to the 96-well plate at a rate of 20 μL / well.

[0088] 2. Then, add the prepared compound at a rate of 20 μL / well to the plate containing CD73 solution. After mixing, incubate at room temperature for 30 min. Simultaneously, set up positive (without compound) and negative control wells (without CD73).

[0089] 3. Next, add 20 μL of the prepared AMP solution to the plate containing CD73 and the compound at a rate of 20 μL / well. After mixing thoroughly, incubate at 37°C for 60 min before proceeding to the next step.

[0090] 4. Add the prepared ATP solution to the plate from step 3 at a rate of 10 μL / well, and then mix the mixture thoroughly.

[0091] 5. Finally, add the prepared CellTiter-Glo reagent (Promega REF: G7573) to the plate from step 4 at a rate of 70 μl / well, mix well, and then perform the detection.

[0092] Inhibition rate (IC) 50 The calculation method is as follows: determined by using the compound dose-response curve fitted by the standard four-parameter fitting equation.

[0093] This invention provides the half-maximal inhibitory concentration (IC50) of the compound with the structure shown in formula (I) for CD73. 50 See Table 1:

[0094] Table 1: Inhibitory activity of the compounds provided in this invention against CD73

[0095] Compound numbering <![CDATA[IC 50 ,nM]]> Compound 1 130.2 Compound 2 113.6 Compound 3 5.8 Compound 4 194.3 Compound 5 168.2 Compound 6 >1000 Compound 7 >1000

[0096] As shown in Table 1, the compounds provided by this invention all exhibit excellent inhibitory activity against CD73.

[0097] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A nucleoside compound of the structure shown in Formula (I) or a pharmaceutically acceptable salt thereof, Formula (I), wherein The nucleoside compound of the structure shown in Formula (I) is selected from the following specific compounds or a pharmaceutically acceptable salt thereof, Compound I-1: , Compound I-2: , Compound I-3: , Compound I-4: , Compound I-5: .

2. A method of preparing the nucleoside compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, including: contacting a compound of the structure shown in Formula (I-2) with a compound of the structure shown in Formula (I-1) in Reaction I at a temperature of 0-60 °C for a time period of 1-72 h; Formula (I-1), Formula (I-2), wherein R1, R2, R3, R4, R5, R6, R7, R 10 , R 11 , G1, G2, G3, W, L, n are as defined in claim 1.

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