Nucleoside bisphosphates and methods for their preparation

Abstract

The application relates to the technical field of biological medicine, and discloses a nucleoside diphosphate compound and a preparation method thereof. The compound has the structure shown in formula (I). The preparation method of the nucleoside compound comprises the following steps: contacting a compound shown in formula (I-2) with a compound shown in formula (I-1) to perform a contact reaction I. The aforementioned nucleoside compound provided by the application has excellent inhibitory activity on CD73, and in-vivo experiments show that the compound has good antitumor activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a bisphosphate 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 poorly. To achieve this purpose, a first aspect of this 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.

[0009]

[0010] In formula (I), R1 and R2 are each independently selected from H, deuterium, hydroxyl, halogen, substituted or unsubstituted C. 1-6 Alkyl, substituted or unsubstituted C 1-6 The alkoxy, cyano, amino, or azide groups are present, and R1 and R2 are not both H; the substituents that may be present on R1 and R2 are each independently selected from halogens and hydroxyl groups;

[0011] R3 and R4 are each independently selected from H, deuterium, hydroxyl, halogen, substituted or unsubstituted C. 1-6 Alkyl, substituted or unsubstituted C 1-6 The alkoxy, cyano, amino, or azide groups are present, and R3 and R4 are not both H; the substituents that may be present on R3 and R4 are each independently selected from halogens and hydroxyl groups.

[0012] R5 is selected from hydrogen and deuterium;

[0013] W is selected from -O- and -S-;

[0014] 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 6-12aryl, C 6-12 heteroaryl groups;

[0015] n is 0 or 1;

[0016] G1, G2, and G3 are each independently selected from N and -C(R) 10 )-, R 10 The substituents present above are each independently selected from halogens and hydroxyl groups;

[0017] R6 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;

[0018] R7 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, C 6-12 heteroaryl groups;

[0019] R8 and R9 are each independently selected from H, deuterium, and C. 1-8 alkyl, C 3-8 Cycloalkyl, phenyl, phenyl substituted with at least one group from combination B, five- to eight-membered heterocyclic groups, heterocyclic groups substituted with at least one group from combination B, naphthyl, naphthyl substituted with at least one group from combination B, biphenyl, C substituted with at least one group from combination C 2-8 alkenyl, C 6-12 heteroaryl; or

[0020] R8 and R9 together form C 3-8 cycloalkyl groups;

[0021] And the combination B consists of hydroxyl groups, C 1-8 alkoxy groups, C groups substituted with at least one halogen 1-8 Composition C consists of alkoxy groups and halogens, wherein the combination C consists of halogens, hydroxyl groups, and phenyl groups substituted with at least one halogen.

[0022] 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:

[0023] The compound shown in formula (I-1) is subjected to contact reaction I with the compound shown in formula (I-2) at a temperature of 0-60℃ for a time of 1-72h.

[0024]

[0025] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 The definitions of G1, G2, G3, W, L, and n are as described in the first aspect above.

[0026] 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.

[0027] 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.

[0028] 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

[0029] 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.

[0030] The terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0031] In this invention, "halogen" refers to at least one of fluorine, chlorine, bromine, and iodine.

[0032] 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.

[0033] 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"... 1-10 The 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.

[0034] In this invention, the phrase "the substituents optionally present on R1 and R2 are each independently selected from halogens and hydroxyl groups" means that when substituents are present on the alkyl groups represented by R1 and R2, the substituents can be selected from halogens and hydroxyl groups. The phrases "the substituents optionally present on R3 and R4 are each independently selected from halogens and hydroxyl groups" and "the substituents optionally present on R5 are each independently selected from halogens and hydroxyl groups" have similar definitions to the aforementioned phrase "the substituents optionally present on R1 and R2 are each independently selected from halogens and hydroxyl groups".

[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"... 2-8The "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... 2-8 The 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] According to a particularly preferred embodiment, the nucleoside compound is selected from the following specific compounds or their pharmaceutically acceptable salts, or their stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, and metabolites.

[0043] Compound I-1:

[0044] Compound I-2:

[0045] Compound I-3:

[0046] Compound I-4:

[0047] Compound I-5:

[0048] Compound I-6:

[0049] Compound I-7:

[0050] Compound I-8:

[0051] Compound I-9:

[0052] Compound I-10:

[0053] Compound I-11:

[0054] Compound I-12:

[0055] Compound I-13:

[0056] Compound I-14:

[0057] Compound I-15:

[0058] Compound I-16:

[0059] Compound I-17:

[0060] Compound I-18:

[0061] Compound III-19:

[0062] Compound I-20:

[0063] Compound I-21:

[0064] Compound I-22:

[0065] Compound I-23:

[0066] Compound I-24:

[0067] Compound I-25:

[0068] Compound I-26:

[0069] Compound I-27:

[0070] Compound I-28:

[0071] Compound I-29:

[0072] Compound I-30:

[0073] Compound I-31:

[0074] Compound I-32:

[0075] Compound I-33:

[0076] Compound I-34:

[0077] In the following examples, unless otherwise specified, all raw materials used are commercially available and are chemically pure.

[0078] Example 1: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-ethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0079]

[0080] Step 1): Preparation of diethyl tetrahydrofuran-3,4-diacetoxymethyl)-5-(4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)tetrahydrofuran-3,4-diacetate.

[0081] Add 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (7.0 g, 37.0 mmol), hexamethyldisilazane (HMDS) (40 mL), and ammonium sulfate (100 mg, 0.76 mmol) to a 250 mL single-necked round-bottom flask. Heat to reflux with magnetic stirring and react for 3 h. Then concentrate the reaction solution under reduced pressure until completely dry to obtain a yellow solid. Dissolve the solid in acetonitrile (100 mL), add (2S,3R,4R,5R)-5-(acetoxymethyl)tetrahydrofuran-2,3,4-triacetic acid triester (12.9 g, 40.6 mmol), and cool to 0°C in an ice bath. Add TMSOTf (10 mL, 55.3 mmol) dropwise to the reaction system with magnetic stirring. After the addition is complete, heat to room temperature and react overnight. After the reaction was complete as monitored by TLC, 200 ml of saturated sodium bicarbonate solution was added to the system, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate = 4:1, R). f =0.2), yielding a pale yellow oily substance, Y = 92.2%, 1 H NMR(600MHz,Chloroform-d)δ8.23(s,1H),6.57(d,J=3.6Hz,1H),5.95(t,J=4.5Hz,1H),5.74(t,J= 5.4Hz,1H),4.49–4.38(m,2H),4.18(dd,J=12.0,5.1Hz,1H),2.14(s,3H),2.11(s,3H),2.09(s,3H).

[0082] Step 2): Preparation of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-(2-(tert-butoxycarbonyl)hydrazino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)tetrahydrofuran-3,4-diacetic acid diacetate.

[0083] (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)tetrahydrofuran-3,4-diacetate diethyl ester (15.3 g, 34.2 mmol) and ethanol (150 mL) were added to a 250 mL single-necked round-bottom flask. Then, under magnetic stirring, tert-butyl hydrazide (5.42 g, 41.1 mmol) and triethylamine (5.19 g, 51.4 mmol) were added to the system, and the reaction was carried out at room temperature for 3 h. After the reaction was completed by TLC monitoring, the solution was concentrated under reduced pressure to obtain a yellow oily crude product. The crude product was subjected to column chromatography (petroleum ether:ethyl acetate = 3:1, R... f =0.2) Purification yielded a colorless oily product, Y = 80%, 1 H NMR(600MHz,Chloroform-d)δ8.55(br,1H),8.08(s,1H),6.89(s,1H),6.54(d,J=3.9Hz,1H),5.94(t,J=4.5Hz,1H),5.7 7(t,J=5.4Hz,1H),4.49–4.37(m,2H),4.19(dd,J=12.0,5.4Hz,1H),2.13(s,3H),2.11(s,3H),2.09(s,3H),1.50(s,9H).

[0084] Step 3): Preparation of tert-butyl 2-(6-chloro-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine-1-carboxylic acid ester.

[0085] (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-(2-(tert-butoxycarbonyl)hydrazino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)tetrahydrofuran-3,4-diacetic acid diacetate (15.0 g, 27.6 mmol) and amine-methanol solution (100 mL, 7.0 M) were added to a 250 mL single-necked round-bottom flask and reacted at room temperature for 24 h with magnetic stirring. After the reaction was confirmed to be complete by TLC, the reaction solution was concentrated under reduced pressure until completely dry to obtain 16.9 g of crude yellow solid. The crude product was used directly in the next step without purification.

[0086] Step 4): Preparation of tert-butyl 2-(6-chloro-1-((3R,4R,6R,6R)-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.

[0087] To a 250 mL single-necked round-bottom flask, tert-butyl 2-(6-chloro-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine-1-carboxylic acid ester (16.9 g), N,N-dimethylformamide DMF (100 mL), 2,2-dimethoxypropane (17.6 g, 169.2 mmol), and p-toluenesulfonic acid (1.76 g, 10.2 mmol) were added sequentially. After the addition was complete, the temperature was raised to 80 °C and the reaction was carried out for 3 h. After the reaction was confirmed to be complete by TLC, the reaction solution was poured into ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate = 1:1, R...). f =0.3) yielded a white, foamy solid, Y = 92.2%, 1 H NMR(600MHz,Methanol-d4)δ8.01(s,1H),7.96(s,1H),6.39(s,1H),5.32(d,J=6.0Hz,1H),4.97(dd,J=6.0,2.1Hz,1H), 4.26(t,5.7Hz,1H),3.64(dd,J=11.7,6.0Hz,1H),3.55(dd,J=11.7,6.0Hz,1H),1.57(s,3H),1.49(s,9H),1.36(s,3H).

[0088] Step 5): 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)(hydroxy)phosphoryl)methyl)phosphonate salt.

[0089] Add tert-butyl 2-(6-chloro-1-((3R,4R,6R,6R)-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 (1 g, 2.19 mmol) and trimethyl phosphate (3 mL) to a 50 mL single-necked round-bottom flask and cool to 0 °C in an ice-water bath. Then, slowly add methylene bisphosphonate dichloride (1.64 g, 6.6 mmol) dissolved in trimethyl phosphate (3 mL) to the reaction system. After the addition is complete, heat to room temperature and continue the reaction for 3 h. After the reaction is complete as detected by TLC, add 3 mL of water to the system and continue stirring until the reaction is completed overnight. After HPLC detection showed the reaction was complete, 110 mL of water was added. The aqueous phase was extracted three times with ethyl acetate and then azeotropically evaporated with methanol until concentrated into a yellow oil. Acetonitrile (30 mL) was added to the oil, precipitating a white solid. The solid was filtered, dried, and then subjected to C18 reversed-phase column chromatography (Biotage Isolera one, Sfar C18 12 g, eluent: 0-10% methanol / 0.1% TFA aqueous solution) to obtain 200 mg of a white solid product. 1 H NMR (600MHz, DMSO-d6) δ9.79 (s, 1H), 8.34 (d, J = 45.0Hz, 2H), 6.06 (s, 1H), 4.54 (t, J = 4.8Hz ,1H),4.28(t,J=4.8Hz,1H),4.14–4.01(m,2H),3.96–3.84(m,1H),2.20(t,J=20.1Hz,2H).

[0090] Step 6): Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-ethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0091] Add (((((2R,3S,4R,5R)-5-(6-chloro-4-hydrazino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonate (50 mg, 0.097 mmol), ethanol (2.0 mL), water (1.0 mL), and 37% wt formaldehyde aqueous solution (11.9 mg, 0.14 mmol) to a 10 mL single-necked round-bottom flask. After the addition is complete, heat to 45 °C and continue the reaction 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 15 mg of white powder. 1H NMR (600MHz, DMSO-d6) δ12.21(s,1H),8.35(s,1H),7.60(d,J=3.9Hz,1H),6.07(d,J=4.2Hz,1H),4.55(t,J=4.5Hz ,1H),4.28(t,J=4.5Hz,1H),4.10–3.96(m,2H),3.91–3.76(m,1H),2.12(t,J=20.1Hz,2H),2.04(d,J=3.9Hz,3H).

[0092] Example 2: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-propylidene)hydrazino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0093]

[0094] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of propionaldehyde. 1 HNMR (600MHz, DMSO-d6) δ12.19(s,1H),8.25(s,1H),7.61(d,J=4.2Hz,1H),6.05(d,J=4.2Hz,1H),4.50(t,J=4.5Hz, 1H),4.25(t,J=4.5Hz,1H),4.10–3.95(m,2H),3.89–3.78(m,1H),2.12(t,3H),1.48–1.20(m,2H),1.16–0.94(m,3H).

[0095] Example 3: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2-methylpropylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0096]

[0097] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of isobutyraldehyde. 1H NMR (600MHz, DMSO-d6) δ12.22(s,1H),8.27(s,1H),7.60(d,J=4.5Hz,1H),6.08(d,J=4.2Hz,1H),4.54(t,J=4.5Hz,1H),4 .28(t,J=4.5Hz,1H),4.10–3.98(m,2H),3.93–3.77(m,1H),2.69–2.58(m,1H),2.12(t,J=20.1Hz,2H),1.17–0.98(m,6H).

[0098] Example 4: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-cyclopropylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0099]

[0100] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of cyclopropylformaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.21(s,1H),8.33(s,1H),7.15(d,J=7.8Hz,1H),6.07(d,J=4.5Hz,1H),4.54(t,J=4.5Hz,1H),4 .28(t,J=4.5Hz,1H),4.10–4.02(m,2H),3.93–3.85(m,1H),2.15(t,J=20.1Hz,2H),1.89–1.82(m,1H),1.08–1.00(m,4H).

[0101] Example 5: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-cyclobutylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0102]

[0103] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of cyclobutylformaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.25(s,1H),8.28(s,1H),7.67(d,J=5.1Hz,1H),6.08(d,J=4.2Hz,1H),4.54(t,J=4.5 Hz,1H),4.28(t,J=4.5Hz,1H),4.16–4.01(m,2H),3.90–3.78(m,1H),2.19(t,J=20.1Hz,2H),2.16–1.81(m,7H).

[0104] Example 6: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-cyclobutylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0105]

[0106] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of cyclopentylformaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.27(s,1H),8.27(s,1H),7.60(d,J=5.4Hz,1H),6.31(d,J=4.2Hz,1H),4.54(t,J=4.5Hz,1H),4 .28(t,J=4.5Hz,1H),4.16–4.01(m,2H),3.93–3.78(m,1H),2.92–2.80(m,1H),2.18(t,J=20.1Hz,2H),1.79–1.40(m,8H).

[0107] Example 7: Preparation of ((((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-(tetrahydro-2H-pyran-4-yl)methylene)hydrazyl)-1H-pyrazoline)[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid.

[0108]

[0109] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of tetrahydro-2H-pyran-4-carboxaldehyde. 1H NMR (400MHz, DMSO-d6) δ12.26(s,1H),8.27(s,1H),7.58(d,J=3.9Hz,1H),6.08(d,J=4.2Hz,1H),4.54(t,J=4.5Hz,1H),4.29(t,J=4.5Hz,1H ),4.14–4.03(m,2H),3.96–3.82(m,3H),3.49–3.35(m,2H),2.70–2.65 (m,1H),2.12(t,J=20.1Hz,2H),1.87–1.79(m,2H),1.61–1.48(m,2H).

[0110] Example 8: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-cyclopentylhydrazinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid.

[0111]

[0112] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of cyclopentanone. 1 H NMR (600MHz, DMSO-d6) δ11.30(s,1H),8.30(s,1H),6.07(d,J=4.2Hz,1H),4.54(t,J=4.5Hz,1H ),4.29(t,J=4.5Hz,1H),4.17–3.97(m,2H),3.96–3.77(m,1H),2.16(t,2H),1.90–1.54(m,8H).

[0113] Example 9: 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)(hydroxy)phosphono)methyl)phosphonic acid.

[0114]

[0115] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of benzaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.60(s,1H),8.47(s,1H),8.23(s,1H),7.80(s,2H),7.64–7.28(m,3H),6.09(d,J=4.2Hz ,1H),4.55(t,J=4.5Hz,1H),4.28(t,J=4.5Hz,1H),4.17–3.94(m,2H),3.93–3.64(m,1H),2.10(t,J=20.1Hz,2H).

[0116] Example 10: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2-hydroxybenzylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0117]

[0118] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of o-hydroxybenzaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.53(s,1H),8.58(s,1H),8.50(s,1H),7.93–7.82(m,1H),7.35–7.25(m,1H),6.99–6.87(m,2H),6.1 1(d,J=4.2Hz,1H),4.57(t,J=4.5Hz,1H),4.31(t,J=4.5Hz,1H),4.13–4.02(m,2H),3.92–3.83(m,1H),2.13(t,J=20.1Hz,2H).

[0119] Example 11: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-3-hydroxybenzylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0120]

[0121] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of m-hydroxybenzaldehyde. 1H NMR (400MHz, DMSO-d6) δ12.59(s,1H),8.52(s,1H),8.17(s,1H),7.43(s,1H),7.28(t,J=7.8Hz,1H),7.10(d,J=7.8Hz,1H),6.84(d,J=8.0 Hz,1H),6.13(d,J=4.2Hz,1H),4.60(t,J=4.5Hz,1H),4.31(t,J=4.5Hz,1H),4.13–3.98(m,2H),3.91–3.80(m,1H),2.10(d,J=20.1Hz,2H).

[0122] Example 12: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-4-hydroxybenzylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0123]

[0124] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of p-hydroxybenzaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.46(s,1H),8.47(s,1H),8.16(s,1H),7.67(d,J=8.1Hz,2H),6.88(d,J=8.1Hz,2H),6.11(d,J =4.2Hz,1H),4.55(t,J=4.8Hz,1H),4.29(t,J=4.8Hz,1H),4.21–4.01(m,2H),3.98–3.80(m,1H),2.17(t,J=20.1Hz,2H).

[0125] Example 13: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-4-methoxybenzylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0126]

[0127] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of p-methoxybenzaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.54(s,1H),8.49(s,1H),8.21(s,1H),7.79(d,J=8.0Hz,2H),7.05(d,J=8.0Hz,2H),6.11(t,J=4.2Hz ,1H),4.57(t,J=4.5Hz,1H),4.31(t,J=4.5Hz,1H),4.13–4.04(m,2H),3.93–3.86(m,1H),3.82(s,3H),2.13(t,J=20.1Hz,2H).

[0128] Example 14: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2-methoxybenzylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0129]

[0130] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-methoxybenzaldehyde. 1 H NMR(400MHz,DMSO-d6)δ12.57(s,1H),8.61(s,1H),8.48(s,1H),8.06–7.96(m,1H),7.50–7.40(m,1H),7.18–7.03(m,2H),6.1 2(d,J=4.2Hz,1H),4.57(t,J=4.6Hz,1H),4.31(t,J=4.6Hz,1H),4.14–4.03(m,2H),3.96–3.82(m,4H),2.14(t,J=20.1Hz,2H).

[0131] Example 15: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-3-hydroxy-4-methoxybenzylidene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0132]

[0133] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 3-hydroxy-4-methoxybenzaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.50(s,1H),8.51(s,1H),8.11(s,1H),7.50(s,1H),7.06(d,J=8.4Hz,1H),6.98(d,J=7.8Hz,1H),6.13(d,J =4.5Hz,1H),4.58(t,J=4.5Hz,1H),4.32(t,J=4.5Hz,1H),4.14–4.02(m,2H),3.95–3.86(m,1H),3.81(s,3H),2.18(t,J=20.1Hz,2H).

[0134] Example 16: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2-(2,2-difluoroethoxy)benzylidene)hydrazyl)-1H-pyrazolino[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0135]

[0136] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-(2,2-difluoroethoxy)benzaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.65(s,1H),8.66(s,1H),8.47(s,1H),8.05(d,J=7 .8Hz,1H),7.45(d,J=8.2Hz,1H),7.26–7.04(m,2H),6.43(t,J=54.6Hz,1H),6 .11(d,J=4.2Hz,1H),4.57(t,J=4.5Hz,1H),4.44(t,J=14.7Hz,2H),4.30(t, J=4.5Hz,1H),4.15–3.99(m,2H),3.91–3.80(m,1H),2.11(t,,J=20.1Hz,2H).

[0137] Example 17: Preparation of (((((2R,3S,4R,5R)-5-(4-(2-((E)-2-(tert-butoxy)benzylidene)hydrazyl)-6-chloro-1H-pyrazolino[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0138]

[0139] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-(tert-butoxy)benzaldehyde. 1 H NMR (400MHz, DMSO-d6) δ12.75(s,1H),8.57(s,1H),8.53(s,1H),8.27(dd,J=7.8,1.9Hz,1H),7.66–7.53(m,2H),7.49(dt,J=8.0,1.6Hz,1H),6 .14(d,J=4.2Hz,1H),4.58(t,J=4.6Hz,1H),4.33(t,J=4.6Hz,1H),4.16–4.05(m,2H),3.99–3.85(m,1H),2.17(t,J=20.1Hz,2H),1.06(s,9H).

[0140] Example 18: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2-fluorobenzyl)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0141]

[0142] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of o-fluorobenzaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.73(s,1H),8.51(s,1H),8.46(s,1H),8.17–8.03(m,1H)7.59–7.46(m,1H),7.39–7.27(m,2H),6.13 (d,J=4.2Hz,1H),4.57(t,J=4.5Hz,1H),4.33(t,J=4.5Hz,1H),4.19–4.05(m,2H),3.99–3.87(m,1H),2.22(t,J=21.7Hz,2H).

[0143] Example 19: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-3,4-difluorobenzyl)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0144]

[0145] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 3,4-difluorobenzaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.72(s,1H),8.54(s,1H),8.23(s,1H),7.94–7.86(m,1H),7.77–7.68(m,1H),7.56–7.49(m,1H),6.1 2(d,J=4.2Hz,1H),4.57(t,J=4.5Hz,1H),4.32(t,J=4.5Hz,1H),4.13–4.02(m,2H),3.93–3.80(m,1H),2.14(t,J=20.1Hz,2H).

[0146] Example 20: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-4-chlorobenzyl)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0147]

[0148] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 4-chlorobenzaldehyde. 1 H NMR (400MHz, DMSO-d6) δ12.67(s,1H),8.49(s,1H),8.25(d,J=4.0Hz,1H),7.86(d,J=8.0Hz,2H),7.53(d,J=8.0Hz,2H),6.12 (d,J=4.2Hz,1H),4.58(t,J=4.8Hz,1H),4.30(t,J=4.8Hz,1H),4.14–3.96(m,2H),3.93–3.79(m,1H),2.11(d,J=19.8Hz,2H).

[0149] Example 21: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-2,4-dichlorobenzyl)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0150]

[0151] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2,4-dichlorobenzaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.80(s,1H),8.58(s,1H),8.47(s,1H),8.18(d,J=7.8Hz,1H),7.72(s,1H),7.50(d,J=7.8Hz,1H),6.1 0(d,J=4.5Hz,1H),4.55(d,J=4.8Hz,1H),4.29(d,J=4.8Hz,1H),4.09–4.00(m,2H),3.91–3.82(m,1H),2.10(t,J=20.1Hz,2H).

[0152] Example 22: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-pyridin-3-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0153]

[0154] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of nicotinic acid. 1 HNMR(600MHz,DMSO-d6)δ12.74(s,1H),8.86(s,1H),8.60(s,1H),8.51(s,1H),8.41–8.20(m,2H),7.57–7.37(m,1H),6.11( d,J=4.5Hz,1H),4.60(t,J=4.5Hz,1H),4.31(t,J=4.5Hz,1H),4.13–3.94(m,2H),3.88–3.75(m,1H),2.05(t,J=20.1Hz,2H).

[0155] Example 23: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-(2-fluoropyridin-3-yl)methylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0156]

[0157] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-chloronicotinaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.83(s,1H),8.62(t,J=9.0Hz,1H),8.55(s,1H),8.36(s,1H),8.30(d,J=4.5Hz,1H),7.48(d,J=4.5Hz,1H) ,6.13(d,J=4.2Hz,1H),4.57(t,J=4.8Hz,1H),4.32(t,J=4.8Hz,1H),4.14–4.04(m,2H),3.92–3.82(m,1H),2.14(t,J=19.2Hz,2H).

[0158] Example 24: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-(2-oxo-1,2-dihydropyridin-3-yl)methylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid.

[0159]

[0160] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-oxo-1,2-dihydropyridine-3-carboxaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.55(s,1H),8.54(s,1H),8.36(s,1H),8.20(d,J=7.2Hz,1H),7.52(d,J=6.0Hz,1H),6.33(t,J=6.3Hz,1H) ,6.08(d,J=4.0Hz,1H),4.53(t,J=4.0Hz,1H),4.28(t,J=4.0Hz,1H),4.09–3.99(m,2H),3.89–3.80(m,1H),2.10(t,J=19.8Hz,2H).

[0161] Example 25: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-furan-3-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0162]

[0163] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of furan-3-carboxaldehyde. 1H NMR (400MHz, DMSO-d6) δ12.51(s,1H),8.49(s,1H),8.26(s,1H),8.23(s,1H),7.80(d,J=2.0Hz,1H),7.10(d,J=2.0Hz,1H),6.1 1(d,J=4.2Hz,1H),4.57(t,J=4.6Hz,1H),4.31(t,J=4.6Hz,1H),4.13–4.02(m,2H),3.94–3.84(m,1H),2.15(t,J=20.2Hz,2H).

[0164] Example 26: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-thiophene-2-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0165]

[0166] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of thiophene-2-carboxaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.68(s,1H),8.44(s,1H),8.36(s,1H),7.73(d,J=5.1Hz,1H),7.55(d,J=3.6Hz,1H),7.17(t,1H),6.1 1(d,J=4.5Hz,1H),4.57(t,J=4.5Hz,1H),4.31(t,J=4.5Hz,1H),4.12–4.03(m,2H),3.94–3.83(m,1H),2.14(t,J=20.1Hz,2H).

[0167] Example 27: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-thiophene-3-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0168]

[0169] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of thiophene-3-carboxaldehyde. 1H NMR (600MHz, DMSO-d6) δ12.51(s,1H),8.46(s,1H),8.27(s,1H),8.05(s,1H),7.71–7.58(m,2H),6.08(d,J=4.5Hz ,1H),4.54(t,J=4.5Hz,1H),4.28(t,J=4.5Hz,1H),4.09–3.99(m,2H),3.88–3.81(m,1H),2.10(t,J=20.1Hz,3H).

[0170] Example 28: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-thiazo-4-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0171]

[0172] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of thiazole-4-carboxaldehyde. 1 H NMR (600MHz, DMSO-d6) δ12.69(s,1H),9.24(s,1H),8.60(s,1H),8.47(s,1H),8.40(s,1H),6.12(d,J=4.2Hz,1 H),4.56(t,J=4.8Hz,1H),4.32(t,J=4.8Hz,1H),4.15–4.06(m,2H),3.94–3.87(m,1H),2.17(t,J=20.1Hz,2H).

[0173] Example 29: Preparation of (((((2R,3S,4R,5R)-5-(4-(2-((E)-(1H-imidazol-2-yl)methylene)hydrazyl)-6-chloro-1H-pyrazolino[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0174]

[0175] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 1H-imidazol-2-carboxaldehyde. 1H NMR(600MHz,DMSO-d6)δ12.70(s,1H),8.96(s,1H),8.20(s,1H),7.42–7.28(m,2H),6.12(d,J=4.2Hz,1H),4 .59(t,,J=4.5Hz,1H),4.34(t,J=4.5Hz,1H),4.13–4.03(m,2H),3.94–3.79(m,1H),2.14(t,J=20.1Hz,2H).

[0176] Example 30: Preparation of (((((2R,3S,4R,5R)-5-(4-(2-((E)-(1H-indol-3-yl)methylene)hydrazyl)-6-chloro-1H-pyrazolino[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0177]

[0178] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 1H-indole-3-carboxaldehyde. 1 H NMR (400MHz, DMSO-d6) δ12.34 (s, 1H), 11.80 (d, J = 2.8 Hz, 1H), 8.51 (d, J = 1. 6Hz,2H),8.17–8.11(m,1H),7.99(d,J=2.9Hz,1H),7.50(dd,J=7.2,1.5Hz,1 H),7.35–7.20(m,2H),6.11(d,J=4.4Hz,1H),4.60(t,J=4.6Hz,1H),4.30(t, J=4.6Hz,1H),4.13–4.03(m,2H),3.98–3.83(m,1H),2.17(t,J=20.3Hz,2H).

[0179] Example 31: 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)(hydroxy)phosphono)methyl)phosphonic acid.

[0180]

[0181] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 1-naphthaldehyde. 1H NMR (400MHz, DMSO-d6) δ12.71(s,1H),9.06(s,1H),8.51(s,1H),8.46(d,J=8.7Hz,1H),8.20(d,J=7.2Hz,1H),8.06(t,J=9.3Hz,2H),7.76–7. 57(m,3H),6.14(d,J=4.2Hz,1H),4.59(t,J=4.5Hz,1H),4.32(t,J=4.5Hz,1H),4.17–4.02(m,2H),3.96–3.83(m,1H),2.15(t,J=20.1Hz,2H).

[0182] Example 32: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-naphth-2-ylmethylene)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0183]

[0184] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-naphthaldehyde. 1 H NMR(600MHz,DMSO-d6)δ12.70(s,1H),8.59(s,1H),8.41(s,1H),8.23–8.18(m,1H),8.13–8.06(m,1H),8.06–7.92(m,3H),7.61–7.51(m ,2H),6.11(d,J=4.2Hz,1H),4.56(t,J=4.5Hz,1H),4.31(t,J=4.5Hz,1H),4.11–4.04(m,2H),3.97–3.81(m,1H),2.13(t,J=20.1Hz,2H).

[0185] Example 33: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-(2-hydroxynaphth-1-yl)methylene)hydrazyl)-1H-pyrazolino[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0186]

[0187] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of 2-hydroxy-1-naphthal. 1H NMR(600MHz,DMSO-d6)δ12.61(s,1H),9.04(s,1H),8.83(d,J=8.7Hz,1H),8 .39(s,1H),7.96–7.84(m,2H),7.61(t,J=7.8Hz,1H),7.40(t,J=7.5Hz,1H), 7.26(d,J=9.0Hz,1H),6.11(d,J=4.2Hz,1H),4.58(d,J=4.5Hz,1H),4.29(d, J=4.5Hz,1H),4.13–4.04(m,2H),3.94–3.86(m,1H),2.15(t,J=20.1Hz,2H).

[0188] Example 34: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(2-(((E)-1-phenyleneethyl)hydrazyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphono)methyl)phosphonic acid.

[0189]

[0190] The preparation method is the same as in Example 1, except that the formaldehyde in step 6 is replaced with an equimolar amount of acetophenone. 1 H NMR (400MHz, DMSO-d6) δ11.73(s,1H),8.37(s,1H),7.96–7.75(m,2H),7.62–7.35(m,3H),6.12(d,J=4.4Hz,1H),4.5 7(t,J=4.7Hz,1H),4.29(t,J=4.6Hz,1H),4.12–4.00(m,2H),3.94–3.79(m,1H),2.45(s,3H),2.14(t,J=20.1Hz,3H).

[0191] CD73 in vitro enzyme bioevaluation test

[0192] 1.1 Experimental Materials

[0193] 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).

[0194] 1.2 Reagent Preparation

[0195] Compound preparation:

[0196] 1. First, dilute the compound stock solution (10mM) 10 times, take 2μL of the diluted stock solution, and add 18μL of DMSO.

[0197] 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.

[0198] hCD73 preparation process:

[0199] 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.

[0200] AMP preparation process:

[0201] 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.

[0202] 1.3 Test Methods

[0203] 1. Add the prepared CD73 solution to the 96-well plate at a rate of 20 μL / well.

[0204] 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).

[0205] 3. Next, add 20 μL of the prepared AMP solution to the plate containing CD73 and the compound in wells. After mixing thoroughly, incubate at 37°C for 60 min before proceeding to the next step.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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:

[0210] Table 1: Inhibitory activity of the compounds provided in this invention against CD73

[0211]

[0212]

[0213] As shown in Table 1, the compounds provided by this invention all exhibit excellent inhibitory activity against CD73.

[0214] 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 formula (I) or a pharmaceutically acceptable salt thereof, Equation (I), in, The nucleoside compounds are selected from the following specific compounds or their pharmaceutically acceptable salts. Compound I-1: , Compound I-2: , Compound I-3: , Compound I-4: , Compound I-5: , Compound I-6: , Compound I-7: , Compound I-8: , Compound I-9: , Compound I-10: , Compound I-11: , Compound I-12: , Compound I-13: , Compound I-14: , Compound I-15: , Compound I-16: , Compound I-17: , Compound I-18: , Compound III-19: , Compound I-20: , Compound I-21: , Compound I-22: , Compound I-23: , Compound I-24: , Compound I-25: , Compound I-26: , Compound I-27: , Compound I-28: , Compound I-29: , Compound I-30: , Compound I-31: , Compound I-32: , Compound I-33: , Compound I-34: .

2. A method for preparing the nucleoside compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The method includes: The compound shown in formula (I-1) is reacted with the compound shown in formula (I-2) at a temperature of 0-60°C for a time of 1-72 h. Equation (I-1), Equation (I-2); Wherein, R1, R2, R3, R4, R5, R6, R7, R8, R9, G1, G2, G3, W, L, and n correspond to the structures of the specific compounds in claim 1.

Images