Phosphamide prodrug as well as preparation method and application thereof

Abstract

The invention discloses a novel phosphamide prodrug and a preparation method and application thereof.The phosphamide prodrug has the chemical structural formula shown in the formula (I), the prodrug can change the pharmacokinetic characteristics of the drug, has the advantage of releasing the drug in cells and can be used for enhancing the concentration of the drug in the cells, and therefore the drug effect is enhanced. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a phosphoramide prodrug and a preparation method and use thereof. Background Art

[0002] Nucleoside drugs play an indispensable role in the fields of antiviral and anti-tumor. Most nucleoside drugs are derived from the structure of natural nucleosides, partially replacing the natural base or sugar to form an antimetabolite structure, which interferes with the DNA replication of viruses or tumor cells, thereby inhibiting the proliferation of viruses or tumor cells.

[0003] Nucleoside substances need to be activated into the form of triphosphate before they are used, and the rate-limiting step is the monophosphorylation step, which is also the step where viruses, tumor cells, etc. are prone to develop resistance to nucleoside drugs. Therefore, pre-installing the monophosphate group has become one of the important prodrug methods for nucleosides. The monophosphate group itself carries two units of negative charge, which is extremely unfavorable for direct drug formation. In the long-term exploration, many different types of monophosphate modification strategies have emerged, such as ProTide, phosphorodiamidate, S-acylthioalkyl ester (SATE)-based prodrug, etc. However, almost every prodrug technology has its shortcomings. For example, the phenol group used in ProTide technology may produce a small amount of toxicity during metabolism; SATE or similar technologies are prone to produce thiirane, which is acutely toxic to the human body;

[0004] Although phosphorodiamidate has no safety risks, its metabolism is relatively slow and its efficacy is far behind that of ProTide technology. In summary, the current prodrug technology has not achieved a perfect combination of safety and efficacy. Summary of the invention

[0005] The purpose of the present invention is to provide a new type of phosphoramide prodrug, which optimizes the substituents of the prodrug to avoid controversial side chains that may form toxic products, while taking into account pharmacodynamics and pharmacokinetic considerations to achieve an effective increase in intracellular drug concentration under physiological conditions.

[0006] Specifically, the present invention provides novel phosphoramide prodrug compounds having the following general formula (I):

[0007]

[0008] In the formula,

[0009] R1 is independently selected from hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C5-12 aryl, substituted or unsubstituted benzyl or substituted aminoalkyl, etc.; wherein the structure of substituted aminoalkyl is as shown in the figure:

[0010]

[0011] R2 is independently selected from O, S, NH, CH2 or other common bioequivalent groups.

[0012] R3 is a nucleoside drug molecule.

[0013] R4 is independently selected from hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C5-10 aryl, substituted or unsubstituted benzyl, mercapto, hydroxy, amino, hydroxyalkyl, aminoalkyl, mercaptoalkyl, 3-6 membered cycloalkyl, 5-6 membered heterocycloalkyl, 5-10 membered aryl, 5-10 membered heteroaryl;

[0014] R5 and R6 are each independently selected from hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C5-10 aryl, benzyl, mercapto, hydroxyl, amino, hydroxyalkyl, aminoalkyl, mercaptoalkyl, alkoxycarbonyl, alkylcarbonyl, 3-6 membered cycloalkyl, 5-6 membered heterocycloalkyl, 5-10 membered aryl, 5-10 membered heteroaryl.

[0015] Furthermore, the R3 nucleoside drug molecule is stavudine, zidovudine, lamivudine, ribavirin, acyclovir, abacavir or remdesivir, etc.

[0016] Further, the compound is:

[0017]

[0018]

[0019] The second object of the present invention is to provide a method for preparing the compound, comprising the following steps:

[0020]

[0021] i) Phosphorus oxychloride 1 reacts with nucleoside drug R3 to obtain dichlorophosphate nucleoside ester 2

[0022] ii) Dichlorophosphate nucleoside ester 2 reacts with a lysine derivative and is treated with methanol to give intermediate 3

[0023] iii) Intermediate 3 is reacted with chloromethyl (acetylamino)acetate to obtain the target compound I.

[0024] Specifically, 1 equivalent of phosphorus oxychloride 1 is dissolved in dichloromethane in an inert gas environment, the reaction solution is cooled to -40°C, 1 equivalent of nucleoside drug R3 and 1.5 equivalents of triethylamine are added, and the reaction is carried out for 3-5 hours. When the nucleoside drug is completely reacted as detected by thin layer chromatography, step ii is performed.

[0025] 1 equivalent of lysine derivative hydrochloride and 2.5 equivalents of triethylamine were added to the reaction solution and reacted overnight. After the reaction, the reaction solution was filtered, dried by spin drying, and dissolved in a small amount of dichloromethane again, and excess methanol was added and stirred for 2-3 hours. After that, it was dried again by spin drying, and 3 was separated by gradient elution with dichloromethane: methanol system (methanol 1%→10%) to obtain intermediate 3.

[0026] Dissolve (acetylamino) chloromethyl acetate (1.8 equivalents) and intermediate 3 (1 equivalent) in N-methylpyrrolidone, add excess triethylamine (3 equivalents) and react overnight at room temperature. After the reaction, add dichloromethane, wash with pure water, sodium bicarbonate solution, and saturated brine, dry with anhydrous magnesium sulfate, filter, and spin dry. The crude product is separated by gradient separation using a dichloromethane: methanol system (methanol 1% → 10%). The target compound I is obtained.

[0027] The preparation method of the above-mentioned (acetylamino) chloromethyl acetic acid ester is as follows: acetylglycine is dissolved in a two-phase solution consisting of equal amounts of water and dichloromethane, sodium bicarbonate (4 equivalents) and tetra-n-butylammonium hydrogen sulfate (0.1 equivalents) are added, and the mixture is vigorously stirred at 0°C for 10 minutes. Then, a dichloromethane solution of chloromethyl chlorosulfonate (1.2 equivalents) is added to the solution. The mixture is stirred at room temperature for 2 hours. After the reaction is completed, the organic layer is taken and washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and spin-dried. The crude product is separated by gradient separation using an ethyl acetate: petroleum ether system (ethyl acetate 6%→30%) to obtain (acetylamino) chloromethyl acetic acid ester.

[0028] It should be understood that, for the compound represented by compound (I) of the present invention, those skilled in the art can prepare it by combining other organic synthesis methods, and are not limited to the above method.

[0029] The third object of the present invention is to provide a pharmaceutical composition comprising:

[0030] The compound represented by the general formula (I), or its isotope structure, optical isomer, tautomer, or any pharmaceutically acceptable salt compound; and a pharmaceutically acceptable carrier.

[0031] The fourth purpose of the present invention is to provide the use of the compound represented by the general formula (I) or the pharmaceutical composition for preparing drugs for treating tumors or related diseases caused by viruses.

[0032] After extensive and in-depth research, the inventors designed and synthesized a series of nucleoside prodrugs with a general structural formula as shown in the general formula (I) described in the first aspect. After in vitro anti-tumor and anti-HIV tests, the results showed that the prodrug has the characteristics of significantly increasing the antiviral and anti-tumor activities of nucleoside drugs. In addition, the nucleoside prodrugs of the present invention have a special drug metabolism pathway, which can change the pharmacokinetic properties of the drug, and have the advantage of intracellular drug release, which can be used to enhance the concentration of the drug in the cell, thereby enhancing the efficacy.

[0033] It should be understood that the above-mentioned technical features of the present invention and the technical features described in detail below can be combined with each other to form a new or preferred technical solution. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Due to space limitations, they are not described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The following is a possible in vivo metabolic pathway of the novel prodrug technology predicted based on the research of Hint-1 enzyme. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples, the purpose of which is only to provide a better understanding of the content of the present invention rather than to limit the protection scope of the present invention.

[0036] the term

[0037] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0038] In the present invention, the halogen is F, Cl, Br or I.

[0039] In the present invention, the term "C1-10 alkyl" refers to a straight chain or branched saturated alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, etc.

[0040] In the present invention, the term "3-6 membered cycloalkyl" means that the number of carbon atoms forming a closed ring system is 3, 4, 5 or 6. 5-6 membered and 5-10 membered are similarly defined, both referring to the number of atoms forming a closed ring system.

[0041] In the present invention, the term "hetero" atom refers to atoms other than carbon, such as N, S, O, P.

[0042] In the present invention, the terms "aryl" and "heteroaryl" refer to compounds with aromaticity in the field of chemistry, common five-membered rings such as furan, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole and thiazole, six-membered rings such as benzene, pyridine, pyrazine, pyrimidine, pyridazine, triazine, and polycyclic rings such as the above five-membered and six-membered ring structures, and six-membered and six-membered ring structures, such as benzimidazole, indole, purine, naphthalene, quinoline, etc.

[0043] In the present invention, the term "unsubstituted" means that the part outside the skeleton is hydrogen; the term "substituted" means that there are atoms other than hydrogen in the part outside the skeleton connected to the skeleton in the form of a single bond. Common substituents include but are not limited to: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, hydroxyl, thiol, amino, nitro, halogen, guanidine, urea, cyano, ester, amide, carboxyl, alkoxy, alkylamino, etc.

[0044] In the present invention, the term "benzyl" refers to a structure in which a benzene ring is connected to a methylene group. The term "mercapto" is -SH; the term "hydroxyl" is -OH; the term "amino" is -NH2; hydroxyalkyl is -O-alkyl; aminoalkyl is -NH-alkyl; mercaptoalkyl is -S-alkyl; alkoxycarbonyl (ester group) is R is an alkyl group; an alkylcarbonyl group is R is an alkyl group.

[0045] Pharmaceutical composition

[0046] The present invention also provides a pharmaceutical composition, which comprises active ingredients within a safe and effective range and a pharmaceutically acceptable carrier.

[0047] The "active ingredient" described in the present invention refers to a compound that conforms to the general formula (I) described in the present invention.

[0048] The "active ingredients" and pharmaceutical compositions described in the present invention are used to prepare drugs for treating prostate cancer, castration-resistant prostate cancer or other prostate cancer-related diseases.

[0049] "Safe and effective range" refers to the dosage range in which the active ingredient can exert its efficacy without causing harm to the body. Or the dosage range between the minimum effective dose and the minimum toxic dose.

[0050] "Pharmaceutically acceptable carriers" include additives other than active ingredients specified in pharmaceutical regulations and in compliance with laws and regulations, including fillers, solvents, solubilizers, co-solvents, excipients, lubricants, pH adjusters, emulsifiers, preservatives, colorants, sustained-release and controlled-release materials, etc.

[0051] The dosage forms of the active ingredients or pharmaceutical compositions of the present invention include, but are not limited to, tablets, injections, powder injections, suppositories, implants, and the like.

[0052] The compounds of the present invention can be administered alone or in combination with other drugs for combined therapy.

[0053] Unless otherwise defined, the terms used herein have the same meanings as those familiar to those skilled in the art. The preferred implementation methods and materials described herein are for exemplary purposes only.

[0054] The following reactions were all detected by thin layer chromatography, and the color was developed using ultraviolet light (in the case of ultraviolet absorption) or a thin layer color developer such as potassium permanganate.

[0055] The structures of the products in the exemplary reactions were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) when they could be isolated.

[0056] Unless otherwise specified, the raw materials and reagents used in the reaction were purchased from the market or synthesized according to known methods. Unless otherwise specified, the raw materials and reagents were used directly without treatment.

[0057] The compound was purified by silica gel column chromatography, and the commonly used elution system was: dichloromethane: methanol; petroleum ether: ethyl acetate.

[0058] The inert gas environment described in the examples generally refers to a nitrogen environment.

[0059] The room temperature in the examples is generally 20°C-30°C.

[0060] In the examples, the overnight reaction time is generally 12-15 hours.

[0061] Example 1

[0062] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-[(2R,5S)-2-(5-methyl-2,4-dioxyylidene-3H-pyrimidin-1-yl)-2,5-dihydrofuran-5-yl]methyl ester (Compound 1)

[0063]

[0064] Stavudine was synthesized according to steps i and ii in the preparation method. Yield: 47%.

[0065] H 1 NMR(CDCl3): δ7.58(1H),6.58(1H),6.54(1H),6.13(1H),6.09(1H),5.53(2H),4.48(1H),4.0 5(2H),4.00(2H),3.35(2H),3.33(3H),2.02(3H),1.90(2H),1.81(3H),1.77(2H),1.31(2H).C 13NMR(CDCl3): δ169.30(1C),164.30(1C),150.70(1C),136.00(1C),134.00(1C),126.20(1C),110.60(1C),89.10(1C), 82.30(1C),81.50(1C),66.60(1C),56.30(1C),41.42(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C),12.25(1C).

[0066] Mass spectrum (ESI): 461.1 (M+H) +

[0067] [(2R,5S)-2-(5-methyl-2,4-dioxy-3H-pyrimidin-1-yl)-2,5-dihydrofuran-5-yl]methyl 3,6-dioxy-5-aza-2-oxahept-1-yl{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX1)

[0068]

[0069] From compound 1 and chloromethyl (acetylamino)acetate, proceed according to step iii of the preparation method. Yield: 63%.

[0070] H 1 NMR(CDCl3): 1H NMR: δ7.58(1H),6.58(1H),6.54(1H),6.13(1H),6.09(1H),5.80(2H),5.53(3H),4.48(1H),4.05(2 H),4.00(1H),3.79(2H),3.35(2H),3.33(3H),2.02(6H),1.90(2H),1.81(3H),1.77(2H),1.31(2H).

[0071] C 13NMR(CDCl3): 13C NMR: δ170.80(1C),169.30(1C),169.25(1C),164.30(1C),150.70(1C),1 36.00(1C),134.00(1C),126.20(1C),110.60(1C),100.82(1C),89.10(1 C),82.30(1C),81.50(1C),66.60(1C),56.30(1C),41.42(1C),41.20(1C) ),29.80(1C),26.27(1C),25.80(1C),23.30(1C),22.90(1C),12.25(1C).

[0072] Mass spectrum (ESI): 590.2 (M+H) +

[0073] Example 2

[0074] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-[(2S,5R)-3-azido-5-(5-methyl-2,4-dioxyylidene-3H-pyrimidin-1-yl)tetrahydrofuran-2-yl]methyl ester (Compound 2)

[0075]

[0076] Zidovudine was synthesized according to steps i and ii in the preparation method. Yield: 38%.

[0077] H 1 NMR(CDCl3): δ7.58(1H),6.24(1H),6.13(1H),5.53(2H),4.48(1H),4.37(1H),4.05(2H),4.0 0(2H),3.35(2H),3.33(3H),2.82(2H),2.02(3H),1.90(2H),1.81(3H),1.77(2H),1.31(2H).C 13 NMR(CDCl3): δ169.30(1C),164.30(1C),150.40(1C),136.37(1C),110.60(1C),87.20(1C),81.50(1C),74.20(1C),6 6.60(1C),57.63(1C),56.30(1C),41.42(1C),41.00(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C),12.25(1C).

[0078] Mass spectrum (ESI): 504.1 (M+H) + .

[0079] [(2S,5R)-3-Azido-5-(5-methyl-2,4-dioxoylide-3H-pyrimidin-1-yl)tetrahydrofuran-2-yl]methyl 3,6-dioxoylide-5-aza-2-oxahept-1-yl{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX2)

[0080]

[0081] From compound 2 and chloromethyl (acetylamino)acetate according to the preparation method step iii. Yield: 63%.

[0082] H 1 NMR(CDCl3): 1H NMR: δ7.58(1H),6.24(1H),6.13(1H),5.80(2H),5.53(3H),4.48(1H),4.37(1H),4.05(2H),4.00(1 H),3.79(2H),3.35(2H),3.33(3H),2.82(2H),2.02(6H),1.90(2H),1.81(3H),1.77(2H),1.31(2H).

[0083] C 13 NMR(CDCl3): 13C NMR: δ170.80(1C),169.30(1C),169.25(1C),164.30(1C),150.40(1C), 136.37(1C),110.60(1C),100.82(1C),87.20(1C),81.50(1C),74.20(1C ),66.60(1C),57.63(1C),56.30(1C),41.42(1C),41.20(1C),41.00(1C) ,29.80(1C),26.27(1C),25.80(1C),23.30(1C),22.90(1C),12.25(1C).

[0084] Mass spectrum (ESI): 633.2 (M+H) + .

[0085] Example 3

[0086] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-[(2R)-5-(4-nitropropylidene-2-hydroxypyrimidin-1-yl)-1,3-oxathiolan-2-yl]methyl ester (Compound 3)

[0087]

[0088] Lamivudine was synthesized according to steps i and ii in the preparation method. Yield: 32%.

[0089] H 1 NMR(CDCl3): δ8.58(1H),6.13(1H),6.09(1H),5.78(1H),5.59(1H),5.53(1H),5.37(1H),4.1 3(2H),4.00(3H),3.35(2H),3.33(3H),3.27(2H),2.02(3H),1.90(2H),1.77(2H),1.31(2H).C 13 NMR(CDCl3): δ169.30(1C),160.30(1C),152.10(1C),138.40(1C),97.35(1C),87.22(1C),83.96(1C),81 .50(1C),65.20(1C),56.30(1C),41.42(1C),34.17(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C).

[0090] Mass spectrum (ESI): 466.1 (M+H) +

[0091] [(2R)-5-(4-Azolylidene-2-hydroxypyrimidin-1-yl)-1,3-oxathiolan-2-yl]methyl 3,6-dioxyylidene-5-aza-2-oxahept-1-yl {[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX3)

[0092]

[0093] From compound 3 and chloromethyl (acetylamino)acetate, proceed according to step iii of the preparation method. Yield: 46%.

[0094] H 1 NMR(CDCl3): δ8.58(1H),6.13(1H),6.09(1H),5.80(2H),5.78(1H),5.59(1H),5.53(2H),5.37(1H),4.1 3(2H),4.00(2H),3.79(2H),3.35(2H),3.33(3H),3.27(2H),2.02(6H),1.90(2H),1.77(2H),1.31(2H).

[0095] C 13 NMR(CDCl3): δ170.80(1C),169.30(1C),169.25(1C),160.30(1C),152.10(1C),138.40(1C),100.82(1C),97.35(1C),87.22(1C),83.96 (1C),81.50(1C),65.20(1C),56.30(1C),41.42(1C),41.20(1C),34.17(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C),22.90(1C).

[0096] Mass spectrum (ESI): 595.1 (M+H) +

[0097] Example 4

[0098] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-[(2S,5S)-5-(3-formamido-1,2,4-triazacyclopentan-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl ester (Compound 4)

[0099]

[0100] Ribavirin was synthesized according to steps i and ii in the preparation method. Yield: 31%.

[0101] H 1 NMR(CDCl3): δ9.45(1H),6.27(1H),6.13(1H),5.53(1H),5.37(2H),4.41(1H),4.35(1H),4.0 8(1H),4.05(2H),4.00(4H),3.35(2H),3.33(3H),2.02(3H),1.77(2H),1.58(2H),1.31(2H).C 13 NMR(CDCl3): δ169.30(1C),159.73(1C),152.10(1C),143.45(1C),87.22(1C),84.00(1C),81.50(1C),73 .50(1C),72.31(1C),66.60(1C),56.30(1C),41.42(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C).

[0102] Mass spectrum (ESI): 481.1 (M+H) +

[0103] [(2S,5S)-5-(3-formamido-1,2,4-triazacyclopentyl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl 3,6-dioxy-5-aza-2-oxahept-1-yl{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX4)

[0104]

[0105] From compound 4 and chloromethyl (acetylamino)acetate according to step iii of the preparation method. Yield: 48%.

[0106] H 1 NMR(CDCl3): δ9.45(1H),6.27(1H),6.13(1H),5.80(2H),5.53(2H),5.37(2H),4.41(1H),4.35(1H),4.0 8(1H),4.05(2H),4.00(3H),3.79(2H),3.35(2H),3.33(3H),2.02(6H),1.77(2H),1.58(2H),1.31(2H).

[0107] C 13 NMR(CDCl3): δ170.80(1C),169.30(1C),169.25(1C),159.73(1C),152.10(1C),143.45(1C),100.82(1C),87.22(1C),84.00(1C),81.50 (1C),73.50(1C),72.31(1C),66.60(1C),56.30(1C),41.42(1C),41.20(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C),22.90(1C).

[0108] Mass spectrum (ESI): 610.2 (M+H) +

[0109] Example 5

[0110] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-2-{[(2-amino-6-oxyylidene-3H-purin-9-yl)methyl]oxy}ethyl ester (Compound 5)

[0111]

[0112] Acyclovir was synthesized according to steps i and ii in the preparation method. Yield: 36%.

[0113] H 1 NMR(CDCl3): δ8.16(1H),6.13(1H),5.53(4H),4.13(2H),4.00(4H),3.79(2H),3.35(2H),3.33(3H),2.02(3H),1.77(2H),1.58(2H),1.31(2H).

[0114] C 13 NMR(CDCl3): δ169.30(1C),158.69(1C),154.58(1C),152.04(1C),137.80(1C),117.41(1C),81.50(1C),7 2.10(1C),69.50(1C),61.80(1C),56.30(1C),41.42(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C).

[0115] Mass spectrum (ESI): 462.1 (M+H) + .

[0116] 2-{[(2-amino-6-oxo-3H-purin-9-yl)methyl]oxy}ethyl 3,6-dioxo-5-aza-2-oxahept-1-yl{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX5)

[0117]

[0118] From compound 5 and chloromethyl (acetylamino)acetate, proceed according to step iii of the preparation method. Yield: 57%.

[0119] H 1 NMR(CDCl3): δ8.16(1H),6.13(1H),5.80(2H),5.53(5H),4.13(2H),4.00(3H ),3.79(4H),3.35(2H),3.33(3H),2.02(6H),1.77(2H),1.58(2H),1.31(2H).

[0120] C 13NMR(CDCl3): δ170.80(1C),169.30(1C),169.25(1C),158.69(1C),154.58(1C),152.04(1C),137.80(1C),117.41(1C),100.82(1C),81.5 0(1C),72.10(1C),69.50(1C),61.80(1C),56.30(1C),41.42(1C),41 .20(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C),22.90(1C).

[0121] Mass spectrum (ESI): 591.2 (M+H) +

[0122] Example 6

[0123] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-[(3S,5R)-5-[2-amino-6-(cyclopropylamino)purin-9-yl]cyclopent-1-en-3-yl]methyl ester (Compound 6)

[0124]

[0125] Abacavir is synthesized according to steps i and ii in the preparation method. Yield: 37%.

[0126] H 1 NMR(CDCl3): δ8.00(1H),6.18(1H),6.13(1H),5.94(1H),5.78(1H),5.53(2H),4.00(4H),3.99(2H),3.3 5(2H),3.33(3H),3.17(1H),2.92(2H),2.81(1H),2.02(3H),1.77(2H),1.58(2H),1.31(2H),0.72(4H).

[0127] C 13NMR(CDCl3): δ169.30(1C),165.15(1C),150.20(1C),140.40(1C),139.60(1C),136.30(1C),132.00(1C),122.20(1C),81.50(1C),68.7 5(1C),56.55(1C),56.30(1C),48.90(1C),41.42(1C),29.80(1C),29.60(1C),26.27(1C),25.80(1C),23.30(1C),23.25(1C),6.60(2C).

[0128] Mass spectrum (ESI): 523.2 (M+H) +

[0129] [(3S,5R)-5-[2-amino-6-(cyclopropylamino)purin-9-yl]cyclopent-1-en-3-yl]methyl 3,6-dioxyylidene-5-aza-2-oxahept-1-yl{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX6)

[0130]

[0131] From compound 6 and chloromethyl (acetylamino)acetate according to the preparation method step iii. Yield: 66%.

[0132] H 1 NMR(CDCl3): δ8.00(1H),6.18(1H),6.13(1H),5.94(1H),5.80(2H),5.78(1H),5.53(3H),4.00(3H),3.99(2H),3.7 9(2H),3.35(2H),3.33(3H),3.17(1H),2.92(2H),2.81(1H),2.02(6H),1.90(2H),1.77(2H),1.31(2H),0.72(4H).

[0133] C 13NMR(CDCl3): δ170.80(1C),169.30(1C),169.25(1C),165.15(1C),150.20(1C),140 .40(1C),139.60(1C),136.30(1C),132.00(1C),122.20(1C),100.82(1C),81.50(1C) ),68.75(1C),56.55(1C),56.30(1C),48.90(1C),41.42(1C),41.20(1C),29.80(1C ),29.60(1C),26.27(1C),25.80(1C),23.30(1C),23.25(1C),22.90(1C),6.60(2C).

[0134] Mass spectrum (ESI): 652.2 (M+H) +

[0135] Example 7

[0136] P-Hydroxy-P-{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphinic acid-[(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazacyclohexan-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-5-yl]methyl ester (Compound 7)

[0137]

[0138] Remdesivir was synthesized according to steps i and ii in the preparation method. Yield: 32%.

[0139] H 1 NMR(CDCl3): δ8.33(1H),7.03(2H),6.13(1H),5.53(1H),5.37(2H),4.48(1H),4.22(1H),4.1 1(1H),4.05(2H),4.00(4H),3.35(2H),3.33(3H),2.02(3H),1.90(2H),1.58(2H),1.31(2H).C 13NMR(CDCl3): δ169.30(1C),152.10(1C),140.80(1C),136.30(2C),118.80(1C),110.50(2C),85.40(1C),81.50(1C),7 9.50(1C),74.20(1C),71.55(1C),66.60(1C),56.30(1C),41.42(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C).

[0140] Mass spectrum (ESI): 528.1 (M+H) +

[0141] [(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazacyclohexan-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-5-yl]methyl 3,6-dioxyylidene-5-aza-2-oxahept-1-yl{[(5R)-5-(acetylamino)-5-methoxypentyl]amino}phosphonate ion (LX7)

[0142]

[0143] Remdesivir was synthesized according to steps i and ii in the preparation method. Yield: 52%.

[0144] H 1 NMR(CDCl3): δ8.33(1H),7.03(2H),6.13(1H),5.80(2H),5.53(2H),5.37(2H),4.48(1H),4.22(1H),4.1 1(1H),4.05(2H),4.00(3H),3.79(2H),3.35(2H),3.33(3H),2.02(6H),1.90(2H),1.58(2H),1.31(2H).

[0145] C 13NMR(CDCl3): δ170.80(1C),169.30(1C),169.25(1C),152.10(1C),140.80 (1C),136.30(2C),118.80(1C),110.50(2C),100.82(1C),85.40(1C),81.5 0(1C),79.50(1C),74.20(1C),71.55(1C),66.60(1C),56.30(1C),41.42(1 C),41.20(1C),29.80(1C),26.27(1C),25.80(1C),23.30(1C),22.90(1C).

[0146] Mass spectrum (ESI): 657.2 (M+H) +

[0147] Example 8 Biological Activity Test

[0148] Experimental purpose: Through tumor cell growth inhibition experiments and antiviral analysis experiments, the differences in biological activity between the new phosphoramide prodrugs and the parent nucleoside drugs were compared, so as to evaluate the biological effects of the new prodrug technology.

[0149] Experimental materials and drug preparation methods: Reference drugs and cells were purchased from common domestic suppliers in the market; the preparation of compounds LX1-LX7 is shown in the examples. The above test substances were dissolved in DMSO, vortexed for 5 minutes, and diluted in a gradient. The test was performed as described in the experimental method.

[0150] Experimental methods:

[0151] Tumor cell growth inhibition experiment: Tumor cells were inoculated in 96-well plates and given different concentrations of the test drug. After 3 days, the number of tumor cells was estimated using a particle counter. 50 The value is used to represent the drug concentration when the tumor cell proliferation rate is inhibited by 50%.

[0152] Antiviral test: 5x 10 6 MT-4 cells were evenly suspended in freshly prepared culture medium and infected with 100 μl X4LAI.04. The infected cell lines were transferred into 96-well plates and 1 ml of culture medium containing different concentrations of the tested drug was added. After culturing at 37°C for 3 days, the supernatant was taken and the amount of p24 was tested using an Elisa kit. EC 50 The amount used to indicate 50% inhibition of viral replication.

[0153] Experimental results: Tumor cell growth inhibition experiment.

[0154] Comparing LX1, LX3, and LX6 with their respective parent compounds, the novel phosphoramide prodrug technology greatly enhanced the anti-tumor activity of the drug molecules.

[0155]

[0156]

[0157] Antiviral experiments:

[0158] Comparing LX1.2.3.5.6 with their respective parent compounds, the new phosphoramide prodrugs showed significant improvement in antiviral activity in most cases.

[0159]

[0160] After extensive and in-depth research, the inventors have designed and synthesized a series of nucleoside prodrugs with the general structural formula shown in the general formula (I) described in the first aspect. After in vitro anti-tumor and anti-HIV tests, the results showed that the prodrug technology has significantly increased the antiviral and anti-tumor activities of nucleoside drugs.

Claims

1. A phosphoramide prodrug, characterized in that It has the structure shown in formula (I): In the formula, R1 is independently selected from hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C5-12 aryl, substituted or unsubstituted benzyl or substituted aminoalkyl; wherein the structure of substituted aminoalkyl is as shown in the figure: R2 is independently selected from O, S, NH, CH2 or other common bioequivalent groups; R3 is a nucleoside drug molecule; R4 is independently selected from hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C5-10 aryl, substituted or unsubstituted benzyl, mercapto, hydroxy, amino, hydroxyalkyl, aminoalkyl, mercaptoalkyl, 3-6 membered cycloalkyl, 5-6 membered heterocycloalkyl, 5-10 membered aryl, 5-10 membered heteroaryl; R5 and R6 are each independently selected from hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C5-10 aryl, benzyl, mercapto, hydroxyl, amino, hydroxyalkyl, aminoalkyl, mercaptoalkyl, alkoxycarbonyl, alkylcarbonyl, 3-6 membered cycloalkyl, 5-6 membered heterocycloalkyl, 5-10 membered aryl, 5-10 membered heteroaryl.

2. The phosphoramide prodrug according to claim 1, characterized in that: The nucleoside drug molecules are stavudine, zidovudine, lamivudine, ribavirin, acyclovir, abacavir or remdesivir.

3. The phosphoramide prodrug according to claim 2, characterized in that: The phosphoramide prodrug has the following general structure:

4. A method for preparing a phosphoramide prodrug, comprising the following steps: i) phosphorus oxychloride 1 reacts with a nucleoside drug R3 to obtain a dichlorophosphate nucleoside ester 2; ii) reacting dichlorophosphate nucleoside 2 with a lysine derivative and treating with methanol to obtain intermediate 3; iii) Intermediate 3 is reacted with chloromethyl (acetylamino)acetate to obtain the target compound I.

5. The method for preparing the phosphoramide prodrug according to claim 4, characterized in that: Dissolve 1 equivalent of phosphorus oxychloride 1 in dichloromethane in an inert gas environment, cool the reaction solution to -40°C, add 1 equivalent of nucleoside drug R3 and 1.5 equivalents of triethylamine, and react for 3-5 hours; when the nucleoside drug is completely reacted as detected by thin layer chromatography, a reaction solution containing dichlorophosphate nucleoside ester 2 is obtained; Add 1 equivalent of lysine derivative hydrochloride and 2.5 equivalents of triethylamine to the reaction solution and react overnight; after the reaction is completed, filter the reaction solution, spin dry, and dissolve it again in a small amount of dichloromethane, and add excess methanol and stir for 2-3 hours; After that, the product was spin-dried again, and 3 was separated by gradient elution with dichloromethane:methanol system (methanol 1%→10%) to obtain intermediate 3; 1.8 equivalents of (acetylamino) chloromethyl acetate and 1 equivalent of intermediate 3 were dissolved in N-methylpyrrolidone, and 3 equivalents of excess triethylamine were added to react at room temperature overnight; after the reaction, dichloromethane was added, and the mixture was washed with pure water, sodium bicarbonate solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, and spin-dried; the crude product was gradiently separated using a dichloromethane: methanol system (methanol 1%→10%) to obtain the target compound Ⅰ.

6. A pharmaceutical composition, characterized in that A compound comprising the phosphoramide prodrug according to any one of claims 1 to 4, or its isotopic structure, optical isomer, tautomer, or any pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

7. Use of the phosphoramide prodrug according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

8. Use of the phosphoramide prodrug according to any one of claims 1 to 5 in the preparation of antiviral drugs.

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