WO2021098379A1

WO2021098379A1 - Phenylalanine-amidated nucleotide derivative, preparation method therefor and application thereof

Info

Publication number: WO2021098379A1
Application number: PCT/CN2020/118025
Authority: WO
Inventors: 王伟; 陆永章; 谭进辉
Original assignee: 广东中科药物研究有限公司
Priority date: 2019-11-21
Filing date: 2020-09-27
Publication date: 2021-05-27
Also published as: CN110845560A; CN110845560B

Abstract

Provided are a phenylalanine-amidated nucleotide derivative, a preparation method therefor and an application thereof. In the present invention, a compound is formed by binding halogenated phenylalanine to an amino group of an amino-containing nucleoside compound, phosphorylation is carried out at the same time, and a phosphate ester and a phosphamide prodrug are used to promote absorption, thus avoiding a rate-limiting step of monophosphate synthesis, and reducing or avoiding the effect of nucleoside deaminase so as to enhance drug efficacy. Meanwhile, the halogenated phenylalanine has the synergistic effect of inhibiting protein synthesis, and has the effects of increasing bioavailability, improving drug efficacy, and reducing drug resistance in anti-tumor and anti-hepatitis B virus aspects.

Description

苯丙氨酸酰胺化的核苷酸衍生物及其制备方法与应用Phenylalanine amidated nucleotide derivatives and preparation method and application thereof

技术领域Technical field

本发明属于医药领域，具体涉及苯丙氨酸酰胺化的核苷酸衍生物及其制备方法与应用。The invention belongs to the field of medicine, and specifically relates to a phenylalanine amidated nucleotide derivative and a preparation method and application thereof.

背景技术Background technique

核苷酸是一类由碱基(主要是嘌呤、嘧啶碱的衍生物)、戊糖(核糖或脱氧核糖)和磷酸连接而成的化合物。也叫“核苷磷酸”，是构成核酸的基本单位。核苷酸也是构成DNA(脱氧核糖核酸)和RNA(核糖核酸)的单位，生物的细胞中都存在。Nucleotide is a type of compound formed by the connection of bases (mainly derivatives of purine and pyrimidine bases), pentose (ribose or deoxyribose) and phosphoric acid. Also called "nucleoside phosphate", it is the basic unit of nucleic acid. Nucleotide is also a unit that constitutes DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and exists in biological cells.

胞苷和腺苷为含有氨基的核苷类化合物，这两类化合物中有抗肿瘤药和抗乙肝病毒药，抗肿瘤药有吉西他滨、氟达拉滨等，抗乙肝病毒药有阿德福韦和替诺福韦等，含有氨基的核苷类化合物如吉西他滨，在核苷脱氨酶的作用下，90％转变成无活性的DFDU，仅少部分转变为三磷酸吉西他滨发挥药效，吉西他滨目前只有注射剂型上市销售。Cytidine and adenosine are nucleoside compounds containing amino groups. The two types of compounds include anti-tumor drugs and anti-HBV drugs. Anti-tumor drugs include gemcitabine and fludarabine. Anti-HBV drugs include adefovir. And tenofovir, etc., amino-containing nucleoside compounds such as gemcitabine, under the action of nucleoside deaminase, 90% is converted into inactive DFDU, and only a small part is converted to gemcitabine triphosphate to exert its efficacy. Only the injection form is on the market.

需要对现有的胞苷和腺苷的结构进行改造，以减少或避免核苷类脱氨酶的作用，从而增强药效。It is necessary to modify the structure of the existing cytidine and adenosine to reduce or avoid the action of nucleoside deaminase, thereby enhancing the efficacy of the drug.

发明内容Summary of the invention

本发明的目的之一是提供一种4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物。One of the objectives of the present invention is to provide a 4-halogen substituted phenylalanine amidated nucleotide derivative.

本发明所提供的4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物，其结构式如式I所示：The structural formula of the 4-halogen substituted phenylalanine amidated nucleotide derivative provided by the present invention is shown in formula I:

上述式I中，X代表卤素，具体可为F、Cl、Br或I；In the above formula I, X represents halogen, specifically F, Cl, Br or I;

上述式I中，

代表核苷及其类似物； In the above formula I,

Represents nucleosides and their analogs;

上述式I中，4-卤素取代的苯丙氨酸与核苷碱基中的氨基形成酰胺键，所述碱基包括取代或未取代的4-氨基嘧啶、取代或未取代的2-氨基嘌呤、取代或未取代的6-氨基嘌呤、取代或未取代的2，6-二氨基嘌呤、取代或未取代1H-咪唑并[4,5-c] 吡啶、取代或未取代吡咯并[2,1-f][1,2,4]三嗪，其中的取代基可为：甲基，甲氧基，环丙基，氨基，羰基，卤素(F、Cl、Br或I)。In the above formula I, the 4-halogen substituted phenylalanine forms an amide bond with the amino group in the nucleoside base, and the base includes substituted or unsubstituted 4-aminopyrimidine, and substituted or unsubstituted 2-aminopurine , Substituted or unsubstituted 6-aminopurine, substituted or unsubstituted 2,6-diaminopurine, substituted or unsubstituted 1H-imidazo[4,5-c] pyridine, substituted or unsubstituted pyrrolo[2, 1-f][1,2,4]triazine, where the substituent can be: methyl, methoxy, cyclopropyl, amino, carbonyl, halogen (F, Cl, Br or I).

所述碱基具体可为如下任意一种：The base may specifically be any of the following:

上述式(I)中，核苷中的核糖或脱氧核糖中的5-羟甲基与磷酸形成酯键，R ¹可为苯基或取代苯基，萘基或取代萘基，C1-C18未取代的烷基，卤素/烷氧基取代的C1-C18烷基，C3-C6取代或未取代的环烷基，优选为苯基或萘基；R ²可为氨基酸侧链(包括天然氨基酸和非天然氨基酸)，也可为C1-C8烷基/环烷基、苯基；R ³为甲基、乙基、异丙基、苄基，优选为苄基。 In the above formula (I), the 5-hydroxymethyl group in the ribose or deoxyribose of the nucleoside forms an ester bond with phosphoric acid, and R ¹ can be a phenyl group or a substituted phenyl group, a naphthyl group or a substituted naphthyl group. Substituted alkyl, halogen/alkoxy substituted C1-C18 alkyl, C3-C6 substituted or unsubstituted cycloalkyl, preferably phenyl or naphthyl; R ² can be an amino acid side chain (including natural amino acids and Unnatural amino acid), it can also be C1-C8 alkyl/cycloalkyl, phenyl; R ³ is methyl, ethyl, isopropyl, benzyl, preferably benzyl.

所述核苷及其类似物可为常见的核糖核苷或脱氧核糖核苷及其类似物；具体可为如下任意一种：The nucleoside and its analogue can be common ribonucleoside or deoxyribonucleoside and its analogue; specifically, it can be any of the following:

上述式I所示4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物具体可为如下化合物中的一种：The 4-halogen substituted phenylalanine amidated nucleotide derivative represented by the above formula I can specifically be one of the following compounds:

1、

1,

2-(((((2R,3R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4,4-二氟-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(吉西他滨衍生物)；2-(((((2R,3R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl) -4,4-Difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (derivative of gemcitabine);

3、

3.

2-(((((2S,3R,4R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯

2-(((((2S,3R,4R,5S)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)- (Yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

2-(((((2R,3R,4R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4-氟-3-羟基-4-甲基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯；2-(((((2R,3R,4R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)- (Yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate;

5、

5.

2-(((((2S,3R,4R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-yl)-4-氰基-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯2-(((((2S,3R,4R,5S)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)- yl)-4-cyano-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

6、

6.

2-(((((2R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-yl)四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(扎西他滨衍生物)2-(((((2R,5S)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)tetrahydrofuran- Synthesis of 2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Zalcitabine derivatives)

7、7.

2-((((2S)-3-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-2-羟丙氧基)(苯氧基)磷酰基)氨基)丙酸苄酯2-((((2S)-3-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-2-hydroxypropane (Oxy)(phenoxy)phosphoryl)amino)benzyl propionate

8、8,

2-(((((2S,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-1,3-氧硫杂环戊烷-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Lamivudine衍生物)2-(((((2S,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-1 ,3-oxathiolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Lamivudine derivative)

9、

9,

2-(((((2R,3S,4R,5R)-5-(2-氨基-6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Emtricitabine衍生物)2-((((((2R,3S,4R,5R)-5-(2-amino-6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl )-3,4-Dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Emtricitabine derivative)

10、

10.

2-(((((2R,3S,4R,5R)-5-(2-氨基-6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯

2-((((((2R,3S,4R,5R)-5-(2-amino-6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl )-3,4-Dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

2-(((((2S,3R,4R,5S)-5-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-甲氧基-1H-嘌呤-9(6H)-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Nelarabine衍生物)2-(((((2S,3R,4R,5S)-5-(2-(2-amino-3-(4-fluorophenyl)alanine)-6-methoxy-1H-purine-9 (6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Nelarabine derivative)

12、12.

2-(((((1R,4S)-4-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-(环丙基氨基)-9H-嘌呤-9-基)环戊烯-2-烯-1-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯2-(((((1R,4S)-4-(2-(2-amino-3-(4-fluorophenyl)alanine)-6-(cyclopropylamino)-9H-purine-9- (Yl)cyclopentene-2-en-1-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

13、

13.

2-(((((1S,3R,5R)-3-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-氧-1H-嘌呤-9(6H)-基)-5-羟基-2-亚甲基环戊基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Entecavir

2-(((((1S,3R,5R)-3-(2-(2-amino-3-(4-fluorophenyl)alanine)-6-oxo-1H-purine-9(6H)- Benzyl)-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Entecavir

2-(((((2R,3S,4S,5R)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯2-(((((2R,3S,4S,5R)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)-3, 4-Dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

15、

15.

2-(((((2S,3S,4S,5S)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氟-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Fludarabine衍生物)2-(((((2S,3S,4S,5S)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-2-fluoro-9H-purin-9-yl )-3,4-Dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Fludarabine derivative)

16、

16.

2-(((((2R,3S,4R,5R)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氯-9H-嘌呤-9-基)-4-氟-3-羟基四氢呋喃-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Clorafaranbine衍生物)2-(((((2R,3S,4R,5R)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-2-chloro-9H-purin-9-yl )-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Clorafaranbine derivative)

17、

17.

2-(((((2S,3S,5S)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氯-9H-嘌呤-9-基)-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Cladibine衍生物)2-(((((2S,3S,5S)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-2-chloro-9H-purin-9-yl)- 3-Hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Cladibine derivative)

18、18.

2-(((((2R,3S,4R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)吡咯[2,1-f][1,2,4] 三嗪-7-基)-5-氰基-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙

2-(((((2R,3S,4R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)pyrrole[2,1-f][1,2 ,4) Triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propyl

2-(((((2R)-1-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)丙烷-2-基)氧)(苯氧基)磷酰基)氨基)丙酸苄酯(Tenofovir衍生物)2-(((((2R)-1-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)propan-2-yl)oxy)( Phenoxy)phosphoryl)amino)benzyl propionate (Tenofovir derivative)

20、

20.

2-(((2-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)乙氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(Adefovir衍生物)2-(((2-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)ethoxy)(phenoxy)phosphoryl)amino) Benzyl propionate (Adefovir derivative)

本发明的式I所示4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物，根据图1所示的流程图，通过包括如下步骤的方法制备得到：The 4-halogen substituted phenylalanine amidated nucleotide derivative of formula I of the present invention is prepared by a method including the following steps according to the flow chart shown in FIG. 1:

1)使式II所示五氟苯酚磷酸酯活性中间体与核苷中的核糖或脱氧核糖的5-羟甲基反应，得到核糖或脱氧核糖5-羟甲基磷酸化的核苷酸；1) Reacting the active intermediate of pentafluorophenol phosphate represented by formula II with the 5-hydroxymethyl of ribose or deoxyribose in the nucleoside to obtain ribose or deoxyribose 5-hydroxymethyl phosphorylated nucleotides;

式II中，R ¹、R ²和R ³的定义分别同式I中R ¹、R ²和R ³的定义； In formula II, ^{the definitions of R 1} , R ² and R ³ are the same as those of R ¹ , R ² and R ³ in formula I;

2)使得BOC-(4-X)苯丙氨酸与步骤2)所得核糖或脱氧核糖核苷酸的碱基中的氨基偶联形成酰胺键，即得；2) Coupling BOC-(4-X) phenylalanine with the amino group in the base of the ribose or deoxyribonucleotide obtained in step 2) to form an amide bond to obtain;

上述方法步骤1)中，所述式II所示五氟苯酚磷酸酯活性中间体与核苷的摩尔比可为：1.0-2.0，具体可为1.2:1；In step 1) of the above method, the molar ratio of the pentafluorophenol phosphate active intermediate of formula II to the nucleoside may be 1.0-2.0, specifically 1.2:1;

所述反应在叔丁基氯化镁催化下进行；The reaction is carried out under the catalysis of tert-butylmagnesium chloride;

核苷与叔丁基氯化镁的摩尔比可为：1:1.0-2.0，具体可为1:1.2；The molar ratio of nucleoside to tert-butylmagnesium chloride may be 1:1.0-2.0, specifically 1:1.2;

所述反应在惰性气体保护下进行，所述惰性气体具体可为氮气；The reaction is carried out under the protection of an inert gas, and the inert gas may specifically be nitrogen;

所述反应在无水有机溶剂中进行，所述无水有机溶剂具体可为无水四氢呋喃；The reaction is carried out in an anhydrous organic solvent, and the anhydrous organic solvent may specifically be anhydrous tetrahydrofuran;

所述反应的温度可为：-20-10℃，具体可为0℃，时间可为：8-24h；The temperature of the reaction can be: -20-10°C, specifically 0°C, and the time can be: 8-24h;

上述方法步骤2)中，所述形成酰胺键的反应1-羟基苯并***存在下进行；In step 2) of the above method, the reaction to form an amide bond is carried out in the presence of 1-hydroxybenzotriazole;

所述形成酰胺键的反应在羧基活化试剂存在下进行，所述羧基活化试剂具体可为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；The reaction to form an amide bond is carried out in the presence of a carboxyl activating reagent, and the carboxyl activating reagent may be 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

所述核糖或脱氧核糖核苷酸与BOC-(4-X)苯丙氨酸、1-羟基苯并***及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔比依次可为：1.0：1.0-1.5：1.0-2.0：1.0-2.0，具体可为1.0：1.0：1.0：1.3；The ribose or deoxyribonucleotide and BOC-(4-X) phenylalanine, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide The molar ratio of the hydrochloride may be 1.0:1.0-1.5:1.0-2.0:1.0-2.0, specifically 1.0:1.0:1.0:1.3;

所述反应的温度可为室温，时间可为3-8h，具体可为5h。The temperature of the reaction can be room temperature, and the time can be 3-8 hours, specifically 5 hours.

如所述核苷及其类似物中的核糖或脱氧核糖除了具有5-羟甲基外，还有其他位置上的羟基(如3-羟基、4-羟基)，那么在步骤1)之前还包括先选择性地保护其他位置上的羟基的操作，其中，所用保护基包括但不限于叔丁氧羰基(BOC)、叔丁基二甲基硅基(TBS)；For example, ribose or deoxyribose in the nucleoside and its analogues has a 5-hydroxymethyl group and a hydroxyl group at other positions (such as 3-hydroxyl, 4-hydroxyl), then it also includes before step 1) The operation of first selectively protecting the hydroxyl groups at other positions, wherein the protecting groups used include but not limited to tert-butoxycarbonyl (BOC) and tert-butyldimethylsilyl (TBS);

相应地，在步骤2)之后，还包括将步骤2)得到的产物在酸性条件下脱去羟基保护，得到目标产物的操作。Correspondingly, after step 2), it also includes the operation of removing the hydroxyl protection of the product obtained in step 2) under acidic conditions to obtain the target product.

上述式I所示4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物在制备抗肿瘤和/或抗病毒的药物中的应用也属于发明的保护范围。The application of the 4-halogen-substituted phenylalanine amidated nucleotide derivatives of formula I in the preparation of anti-tumor and/or anti-viral drugs also falls within the protection scope of the invention.

所述应用中，所述肿瘤具体可为：人胰腺癌肿瘤；所述病毒具体可为乙肝病毒；In the application, the tumor may specifically be: a human pancreatic cancer tumor; the virus may specifically be hepatitis B virus;

如，化合物1、3、11、15、16和17在制备抗肿瘤药物中的应用；For example, the application of compounds 1, 3, 11, 15, 16 and 17 in the preparation of anti-tumor drugs;

如，化合物9、19、14和20在制备抗乙肝病毒的药物中的应用。For example, the application of compounds 9, 19, 14 and 20 in the preparation of drugs against hepatitis B virus.

本发明中化合物采用卤代苯丙氨酸与含有氨基的核苷类化合物的氨基进行结合，同时进行磷酸化，通过磷酸酯、磷酰胺前药形式促进吸收、避开限速的一磷酸合成环节，减少或避免核苷类脱氨酶的作用，以增强药效，同时卤代苯丙氨酸具有抑制蛋白质合成的协同作用，起到在抗肿瘤以及抗乙肝病毒方面增加生物利用度、提高药效、减少耐药性的作用。The compound of the present invention uses halogenated phenylalanine to combine with the amino group of amino-containing nucleoside compounds, and simultaneously undergoes phosphorylation, promotes absorption through the form of phosphate ester and phosphoramide prodrug, and avoids the rate-limiting monophosphoric acid synthesis link , Reduce or avoid the effect of nucleoside deaminase to enhance the efficacy, and at the same time halogenated phenylalanine has the synergistic effect of inhibiting protein synthesis, which can increase the bioavailability and improve the drug in anti-tumor and anti-hepatitis B virus. Effective, reduce the role of drug resistance.

附图说明Description of the drawings

图1为本发明中制备式I所示4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物的流程图；Figure 1 is a flow chart of the present invention for preparing 4-halogen substituted phenylalanine amidated nucleotide derivatives of formula I;

具体实施方式Detailed ways

下面通过具体实施例对本发明进行说明，但本发明并不局限于此。The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、生物材料等，如无特殊说明，均可从商业途径得到。The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

实施例1、2-(((((2R,3R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4,4-二氟-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(吉西他滨衍生物)Example 1, 2-((((((2R,3R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H )-Yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Gemcitabine derivative)

由下面五个步骤制备得到：Prepared by the following five steps:

1)、(2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-4,4-二氟-2-(羟甲基)四氢呋喃-3-基-叔丁基碳酸酯的合成1), (2R,3R,5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3- Synthesis of Glycyl-tert-Butyl Carbonate

将无水碳酸钾(11.0g，79.8mmol，3.0eq.)溶解于水(25mL)中,分别加入四氢呋喃(100mL)、吉西他滨(7.0g，26.6mmol，1.0eq.)、二碳酸二叔丁酯(11.5g，53.2mmol，2.0eq.)，室温搅拌反应24h。TLC监测反应(DCM:MeOH＝10:1)。反应液加入乙酸乙酯(100mL*2)萃取，合并有机层，减压浓缩，粗品柱层析(DCM:MeOH＝10:1)纯化，得到6.8g白色固体(2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-4,4-二氟-2-(羟甲基)四氢呋喃-3-基-叔丁基碳酸酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),8.61(s,2H),6.33(m,1H),5.44(d,1H),4.92-4.83(m,2H),4.44(s,1H),3.55(m,2H),1.38(s,9H).ESI-MS m/z:364.3[M+H]+. Dissolve anhydrous potassium carbonate (11.0g, 79.8mmol, 3.0eq.) in water (25mL), add tetrahydrofuran (100mL), gemcitabine (7.0g, 26.6mmol, 1.0eq.), di-tert-butyl dicarbonate (11.5g, 53.2mmol, 2.0eq.), the reaction was stirred at room temperature for 24h. The reaction was monitored by TLC (DCM:MeOH=10:1). The reaction solution was extracted with ethyl acetate (100mL*2), the organic layers were combined, concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH=10:1) to obtain 6.8g of white solid (2R, 3R, 5R)-5 -(4-Amino-2-oxopyrimidine-1(2H)-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl-tert-butyl carbonate. ¹ H NMR (CDCl ₃ 400MHz) δ 9.16 (d, 1H), 8.61 (s, 2H), 6.33 (m, 1H), 5.44 (d, 1H), 4.92-4.83 (m, 2H), 4.44 (s ,1H),3.55(m,2H),1.38(s,9H).ESI-MS m/z:364.3[M+H]+.

2)、2-(((五氟苯氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成2), the synthesis of 2-(((pentafluorophenoxy)(phenoxy)phosphoryl)amino)propionate benzyl ester

称取丙氨酸苄酯盐酸盐(8.0g，37.1mmol，1.0eq.)于250mL三口圆底反应瓶中，氮气保护，加入无水二氯甲烷(100mL)。反应液冷却至-20℃，快速加入二氯磷酸苯酯(7.8g，37.1mmol，1.0eq.)，然后缓慢滴加三乙胺(10.3g，74.2mmol，2.0eq.)的二氯甲烷(20mL)溶液，恒温反应2h。TLC监测反应(DCM:MeOH＝10:1)，直至原料丙氨酸苄酯盐酸盐消耗完毕，滴加五氟苯酚(6.8g，37.1mmol，1.0eq.)的二氯甲烷(20mL)溶液，然后滴加三乙胺(4.1g，40.8mmol，1.1eq.)，0℃恒温反应16h。反应液减压浓缩，固体快速柱层析(PE:EA＝4:1)纯化，得到8.2g白色固体2-(((五氟苯氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ7.47-7.15(m,10H),5.33(s,2H),4.12(m,1H),1.28(d,3H).ESI-MS m/z:502.2[M+H]+. Weigh alanine benzyl ester hydrochloride (8.0 g, 37.1 mmol, 1.0 eq.) into a 250 mL three-neck round-bottomed reaction flask, protect with nitrogen, and add anhydrous dichloromethane (100 mL). The reaction solution was cooled to -20°C, phenyl dichlorophosphate (7.8g, 37.1mmol, 1.0eq.) was quickly added, and then triethylamine (10.3g, 74.2mmol, 2.0eq.) in dichloromethane (10.3g, 74.2mmol, 2.0eq.) was slowly added dropwise. 20mL) solution, react at constant temperature for 2h. The reaction was monitored by TLC (DCM:MeOH=10:1) until the raw material alanine benzyl ester hydrochloride was consumed, and a solution of pentafluorophenol (6.8g, 37.1mmol, 1.0eq.) in dichloromethane (20mL) was added dropwise Then, triethylamine (4.1g, 40.8mmol, 1.1eq.) was added dropwise, and the reaction was kept at 0°C for 16h. The reaction solution was concentrated under reduced pressure and purified by solid flash column chromatography (PE:EA=4:1) to obtain 8.2 g of white solid 2-(((pentafluorophenoxy)(phenoxy)phosphoryl)amino)propionic acid Benzyl ester. ¹ H NMR(CDCl ₃ 400MHz)δ7.47-7.15(m,10H),5.33(s,2H),4.12(m,1H),1.28(d,3H).ESI-MS m/z:502.2(M +H]+.

3)、2-(((((2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-3-((叔丁氧羰基)氧)-4,4-二氟四氢呋喃-2-yl)甲氧基(苯氧基)磷酰基)氨基)丙酸苯酯的合成3), 2-((((((2R,3R,5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)- Synthesis of 4,4-Difluorotetrahydrofuran-2-yl)methoxy(phenoxy)phosphoryl)amino)phenyl propionate

称量化合物(2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-4,4-二氟-2-(羟甲基)四氢呋喃-3-基-叔丁基碳酸酯(4.8g，13.3mmol，1.0eq.)于250mL圆底三口反应瓶中，氮气保护，加入无水四氢呋喃(80mL)。将反应液冷却至0℃，缓慢滴加叔丁基氯化镁的四氢呋喃溶液(16mL，15.9mmol,1.2eq.,1M)，析出白色固体。反应液恒温搅拌30min，滴加化合物2-(((五氟苯氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(8.2g，15.9mmol，1.2eq.)的四氢呋喃(20mL)溶液，0℃恒温反应16h，TLC监测反应(DCM:MeOH＝10:1)。滴加氯化铵水溶液淬灭反应，乙酸乙酯萃取(100ml*2),合并有机层，减压浓缩，粗品柱层析(DCM:MeOH＝10:1)纯化，得到7.7g白色固体2-(((((2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-3-((叔丁氧羰基)氧)-4,4-二氟四氢呋喃-2-yl)甲氧基(苯氧基)磷酰基)氨基) 丙酸苯酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.20(d,1H),8.56(s,2H),7.47-7.18(m,10H),6.33(s,1H),5.44(d,1H),5.12(s,2H),4.89(m,2H),4.66(m,2H),4.13(m,1H),1.38(s,9H),1.12(d,3H).ESI-MS m/z:681.2[M+H]+. Weigh the compound (2R, 3R, 5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3- Glycyl-tert-butyl carbonate (4.8g, 13.3mmol, 1.0eq.) was placed in a 250mL round-bottomed three-necked reaction flask, protected by nitrogen, and anhydrous tetrahydrofuran (80mL) was added. The reaction solution was cooled to 0° C., and a tetrahydrofuran solution (16 mL, 15.9 mmol, 1.2 eq., 1M) of tert-butylmagnesium chloride was slowly added dropwise to precipitate a white solid. The reaction solution was stirred at constant temperature for 30 minutes, and the compound 2-(((pentafluorophenoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (8.2g, 15.9mmol, 1.2eq.) in tetrahydrofuran (20mL) was added dropwise The solution was reacted at 0°C for 16h at a constant temperature, and the reaction was monitored by TLC (DCM:MeOH=10:1). The reaction was quenched by adding ammonium chloride aqueous solution dropwise, extracted with ethyl acetate (100ml*2), the organic layers were combined, concentrated under reduced pressure, and purified by crude column chromatography (DCM:MeOH=10:1) to obtain 7.7g of white solid 2- (((((2R,3R,5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4,4-di Fluorotetrahydrofuran-2-yl) methoxy (phenoxy) phosphoryl) amino) phenyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 9.20 (d, 1H), 8.56 (s, 2H), 7.47 -7.18 (m, 10H), 6.33 (s, 1H), 5.44 (d, 1H), 5.12 (s, 2H), 4.89 (m, 2H), 4.66 (m, 2H), 4.13 (m, 1H), 1.38(s,9H),1.12(d,3H).ESI-MS m/z:681.2[M+H]+.

4)、2-(((((2R,3R,5R)-5-(4-(2-((叔丁氧羰基)氨基)-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-3-((叔丁氧羰基)氧)-4,4-二氟四氢呋喃-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成4), 2-((((((2R,3R,5R)-5-(4-(2-((tert-butoxycarbonyl)amino)-3-(4-fluorophenyl)alanine)-2- Oxopyrimidine-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) Synthesis of Benzyl Propionate

分别称量化合物2-(((((2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-3-((叔丁基氧羰基)氧)-4,4-二氟四氢呋喃-2-yl)甲氧基(苯氧基)磷酰基)氨基)丙酸苯酯(5.9g，8.68mmol，1.0eq.)、Boc-(4-F)苯丙氨酸(2.7g，9.55mmol，1.0eq.)、1-羟基苯并***(1.20g，8.68mmol，1.0eq.)溶解于四氢呋喃(50mL)，氮气保护。冰水浴冷却反应液，然后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(2.20g,11.3mmol，1.3eq.)，室温反应5h，TLC检测反应(DCM:MeOH＝20:1)。乙酸乙酯(100mL)萃取反应液，有机相减压浓缩。粗品柱层析(PE:EA＝1:1)纯化，得到6.7g白色固体2-(((((2R,3R,5R)-5-(4-(2-((叔丁氧羰基)氨基)-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-3-((叔丁氧羰基)氧)-4,4-二氟四氢呋喃-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),7.47-7.18(m,14H),6.38(d,1H),5.41(d,1H),5.22(s,2H),4.91(m,2H),4.38(m,2H),4.12(m,2H),3.19(m,2H),1.38(s,18H),1.20(d,3H).ESI-MS m/z:946.3[M+H]+. Weigh the compound 2-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-3-((tert-butyloxycarbonyl)oxy )-4,4-Difluorotetrahydrofuran-2-yl)methoxy(phenoxy)phosphoryl)amino)phenyl propionate (5.9g, 8.68mmol, 1.0eq.), Boc-(4-F) Phenylalanine (2.7g, 9.55mmol, 1.0eq.) and 1-hydroxybenzotriazole (1.20g, 8.68mmol, 1.0eq.) were dissolved in tetrahydrofuran (50mL) under nitrogen protection. The reaction solution was cooled in an ice water bath, Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.20g, 11.3mmol, 1.3eq.), react at room temperature for 5h, TLC detects the reaction (DCM:MeOH=20 :1). The reaction solution was extracted with ethyl acetate (100 mL), and the organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA=1:1) to obtain 6.7 g of white solid 2-(((((2R,3R) ,5R)-5-(4-(2-((tert-butoxycarbonyl)amino)-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-3 -((Tert-Butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR(CDCl ₃ 400MHz) δ9.16 (d, 1H), 7.47-7.18 (m, 14H), 6.38 (d, 1H), 5.41 (d, 1H), 5.22 (s, 2H), 4.91 (m, 2H), 4.38 (m, 2H),4.12(m,2H),3.19(m,2H),1.38(s,18H),1.20(d,3H).ESI-MS m/z:946.3[M+H]+.

5)、2-(((((2R,3R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4,4-二氟-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成5), 2-(((((2R,3R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H) -Yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

称量化合物2-(((((2R,3R,5R)-5-(4-(2-((叔丁氧羰基)氨基)-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-3-((叔丁氧羰基)氧)-4,4-二氟四氢呋喃-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯(6.7g，8.66mmol，1.0eq.)溶解于二氯甲烷(15mL)中，冰水浴冷，滴加三氟乙酸(15mL)，室温反应2h，TLC监测反应(DCM:MeOH＝10:1)。反应液加入冰水，乙酸乙酯萃取(100ml*2),有机层用饱和碳酸氢钠水溶液中和，减压浓缩,柱层析纯化(PE:EA＝1:10)，得到4.6g白色固体2-(((((2R,3R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4,4-二氟-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),7.48-7.19(m,14H),6.39(m,1H),5.40(d,1H),5.33(s,2H),5.01(s,2H),4.4-3.95(m,5H),4.21(m,2H),3.32(m,2H),1.21(d,3H).ESI-MS m/z:746.2[M+H]+. Weigh the compound 2-((((((2R,3R,5R)-5-(4-(2-((tert-butoxycarbonyl)amino)-3-(4-fluorophenyl)alanine)-2- Oxopyrimidine-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) Benzyl propionate (6.7g, 8.66mmol, 1.0eq.) was dissolved in dichloromethane (15mL), cooled in an ice-water bath, trifluoroacetic acid (15mL) was added dropwise, the reaction was at room temperature for 2h, and the reaction was monitored by TLC (DCM:MeOH= 10:1). The reaction solution was added with ice water, extracted with ethyl acetate (100ml*2), the organic layer was neutralized with saturated aqueous sodium bicarbonate solution, concentrated under reduced pressure, and purified by column chromatography (PE:EA=1:10) to obtain 4.6g of white solid 2-(((((2R,3R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl) -4,4-Difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester. ¹ H NMR(CDCl ₃ 400MHz)δ9.16(d,1H), 7.48-7.19(m,14H), 6.39(m,1H), 5.40(d,1H), 5.33(s,2H), 5.01(s ,2H),4.4-3.95(m,5H),4.21(m,2H),3.32(m,2H),1.21(d,3H).ESI-MS m/z:746.2[M+H]+.

2、2-(((((2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-4,4-二氟-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(NUC-1031)2. 2-((((((2R,3R,5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2 -Amino) methoxy) (phenoxy) phosphoryl) amino) the synthesis of benzyl propionate (NUC-1031)

综合实施例1的制备方法，以吉西他滨为原料，合成得到白色泡沫状固体2-(((((2R,3R,5R)-5-(4-氨基-2-氧代嘧啶-1(2H)-基)-4,4-二氟-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),8.58(d,2H),7.46-7.28(m,10H),6.39(m,1H),5.40(d,1H),5.34(s,2H),5.01(s,2H),4.40-3.58(m,6H),1.28(d,3H).ESI-MS m/z:581.1[M+H]+. Synthesizing the preparation method of Example 1, using gemcitabine as the raw material to synthesize a white foamy solid 2-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidine-1(2H) -Yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 9.16 (d, 1H), 8.58 (d, 2H), 7.46-7.28 (m, 10H), 6.39 (m, 1H), 5.40 (d, 1H), 5.34 (s ,2H),5.01(s,2H),4.40-3.58(m,6H),1.28(d,3H).ESI-MS m/z:581.1[M+H]+.

3、2-(((((2S,3R,4R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成 (cytarabine衍生物)3. 2-(((((2S,3R,4R,5S)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H )-Yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (cytarabine derivative)

综合实施例1的制备方法，以阿糖胞苷为原料，合成得到白色泡沫状固体2-(((((2S,3R,4R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.18(d,1H),7.47-7.21(m,14H),5.93(m,1H),5.40(d,1H),5.30(s,2H),5.01(s,2H),4.51-3.85(m,7H),3.58-3.77(m,2H),3.37(m,2H),1.28(d,3H).ESI-MS m/z:726.1[M+H]+. Synthesizing the preparation method of Example 1, using cytarabine as the raw material, a white foamy solid 2-(((((2S,3R,4R,5S)-5-(4-(2-amino-3- (4-Fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino ) Benzyl propionate. ¹ H NMR(CDCl ₃ 400MHz)δ9.18(d,1H), 7.47-7.21(m,14H), 5.93(m,1H), 5.40(d,1H), 5.30(s,2H), 5.01(s ,2H),4.51-3.85(m,7H),3.58-3.77(m,2H),3.37(m,2H),1.28(d,3H).ESI-MS m/z:726.1[M+H]+ .

4、2-(((((2R,3R,4R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4-氟-3-羟基-4-甲基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成4. 2-((((((2R,3R,4R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)propylamine)-2-oxopyrimidine-1(2H )-Yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

综合实施例1的制备方法，以4-氨基-1-((2R,3R,4R,5R)-3-氟-4-羟基-5-(羟甲基)-3-甲基四氢呋喃-2-基)吡啶-2(1H)-酮为原料，合成得到白色泡沫状固体2-(((((2R,3R,4R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-4-氟-3-羟基-4-甲基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),7.38-7.11(m,14H),6.11(m,1H),5.40(d,1H),5.34(s,2H),4.40-3.85(m,7H),3.34-3.16(m,2H),1.47(s,3H),1.22(d,3H).ESI-MS m/z:742.2[M+H]+. Synthesizing the preparation method of Example 1, with 4-amino-1-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2- Yl)pyridine-2(1H)-one as raw material, synthesized to obtain a white foamy solid 2-(((((2R,3R,4R,5R)-5-(4-(2-amino-3-(4- (Fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorus Acyl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 9.16 (d, 1H), 7.38-7.11 (m, 14H), 6.11 (m, 1H), 5.40 (d, 1H), 5.34 (s, 2H), 4.40-3.85 (m,7H),3.34-3.16(m,2H),1.47(s,3H),1.22(d,3H).ESI-MS m/z:742.2[M+H]+.

5、2-(((((2S,3R,4R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶 -1(2H)-yl)-4-氰基-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成5. 2-((((((2S,3R,4R,5S)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H )-yl)-4-cyano-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester

综合实施例的1制备方法，以(2S,3R,4R,5S)-2-(4-氨基-2-氧代嘧啶-1(2H)-基)-4-羟基-5-(羟甲基)四氢呋喃-3-甲氰为原料，合成得到白色泡沫状固体2-(((((2S,3R,4R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-yl)-4-氰基-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.20(d,1H),7.48-7.19(m,14H),5.80(m,1H),5.40(d,1H),5.34(s,2H),4.40-3.65(m,8H),3.34-3.16(m,2H),1.22(d,3H).ESI-MS m/z:735.2[M+H]+. Synthesis of the preparation method of Example 1, take (2S,3R,4R,5S)-2-(4-amino-2-oxopyrimidine-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl ) Tetrahydrofuran-3-methylcyanide was used as raw material to synthesize a white foamy solid 2-(((((2S,3R,4R,5S)-5-(4-(2-amino-3-(4-fluorophenyl) )Alanine)-2-oxopyrimidine-1(2H)-yl)-4-cyano-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate ester. ¹ H NMR (CDCl ₃ 400MHz) δ9.20 (d, 1H), 7.48-7.19 (m, 14H), 5.80 (m, 1H), 5.40 (d, 1H), 5.34 (s, 2H), 4.40-3.65 (m,8H),3.34-3.16(m,2H),1.22(d,3H).ESI-MS m/z:735.2[M+H]+.

6、2-(((((2R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-yl)四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(扎西他滨)6, 2-(((((2R,5S)-5-(4-(2-amino-3-(4-fluorophenyl)propylamine)-2-oxopyrimidine-1(2H)-yl) Synthesis of tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Zalcitabine)

综合实施例1的制备方法，以扎西他滨为原料，合成得到白色泡沫状固体2-(((((2R,5S)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-yl)四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),7.44-7.19(m,14H),5.85(m,1H),5.40(d,1H),5.35(s,2H),4.28-3.36(m,5H),3.34-3.16(m,2H),1.94-1.63(m,6H),1.28(d,3H).ESI-MS m/z:694.2[M+H]+. Synthesizing the preparation method of Example 1, using zalcitabine as the raw material, a white foamy solid 2-(((((2R,5S)-5-(4-(2-amino-3-(4-fluoro (Phenyl)alanine)-2-oxopyrimidine-1(2H)-yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 9.16 (d, 1H), 7.44-7.19 (m, 14H), 5.85 (m, 1H), 5.40 (d, 1H), 5.35 (s, 2H), 4.28-3.36 (m,5H),3.34-3.16(m,2H),1.94-1.63(m,6H),1.28(d,3H).ESI-MS m/z:694.2[M+H]+.

7、2-((((2S)-3-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-2-羟丙氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成7. 2-((((2S)-3-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-2- Synthesis of hydroxypropoxy)(phenoxy)phosphoryl)amino)benzyl propionate

综合实施例的1制备方法，以(S)-4-氨基-1-(2,3-二羟丙基)嘧啶-2(1H)-酮为原料，合成得到白色泡沫状固体2-((((2S)-3-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-2-羟丙氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃400MHz)δ9.18(d,1H),7.46-7.19(m,14H),5.40(d,1H),5.35(s,2H),4.29-2.98(m,8H),1.28(d,3H).ESI-MS m/z:668.3[M+H]+. Synthesize the preparation method of Example 1, take (S)-4-amino-1-(2,3-dihydroxypropyl)pyrimidin-2(1H)-one as raw material, and synthesize white foamy solid 2-(( ((2S)-3-(4-(2-amino-3-(4-fluorophenyl)alanine)-2-oxopyrimidine-1(2H)-yl)-2-hydroxypropoxy)( Phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR(CDCl ₃ 400MHz)δ9.18(d,1H),7.46-7.19(m,14H),5.40(d,1H),5.35(s,2H),4.29-2.98(m,8H),1.28 (d,3H).ESI-MS m/z:668.3[M+H]+.

8、2-(((((2S,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-1,3-氧硫杂环戊烷-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Lamivudine衍生物)8, 2-(((((2S,5R)-5-(4-(2-amino-3-(4-fluorophenyl)propylamine)-2-oxopyrimidine-1(2H)-yl) Synthesis of -1,3-oxathiolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (Lamivudine derivative)

综合实施例的1制备方法，以拉米夫定为原料，合成得到白色泡沫状固体2-(((((2S,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-2-氧代嘧啶-1(2H)-基)-1,3-氧硫杂环戊烷-2-基)甲氧基)(甲氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.16(d,1H),7.48-7.19(m,14H),5.40(d,1H),5.35(s,2H),5.12(m,1H),4.66(m,2H),3.97-2.71(m,7H),1.28(d,3H).ESI-MS m/z:712.1[M+H]+. Synthesize the preparation method of Example 1 and take lamivudine as the raw material to synthesize a white foamy solid 2-(((((2S,5R)-5-(4-(2-amino-3-(4-fluoro (Phenyl) Alanine)-2-oxopyrimidine-1(2H)-yl)-1,3-oxathiolan-2-yl)methoxy)(methoxy)phosphoryl)amino) Benzyl propionate. ¹ H NMR(CDCl ₃ 400MHz)δ9.16(d,1H), 7.48-7.19(m,14H), 5.40(d,1H), 5.35(s,2H), 5.12(m,1H), 4.66(m ,2H),3.97-2.71(m,7H),1.28(d,3H).ESI-MS m/z:712.1[M+H]+.

9、2-(((((2S,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-5-氟-2-氧代嘧啶-1(2H)-基)-1,3-氧硫杂环戊烷-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Emtricitabine衍生物)9, 2-(((((2S,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)-5-fluoro-2-oxopyrimidine-1(2H )-Yl)-1,3-oxathiolane-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (Emtricitabine derivative)

综合实施例1的制备方法，以恩曲他滨为原料，合成得到白色泡沫状固体2-(((((2S,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)-5-氟-2-氧代嘧啶-1(2H)-基)-1,3-氧硫杂环戊烷-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ7.58-7.19(m,15H),5.34(s,2H),5.22(m,1H),5.12(s,2H),4.66(m,2H),3.95-2.71(m,7H),1.28(d,3H).ESI-MS m/z:730.1[M+H]+. Synthesizing the preparation method of Example 1, using emtricitabine as the raw material, a white foamy solid 2-(((((2S,5R)-5-(4-(2-amino-3-(4-fluoro (Phenyl)alanine)-5-fluoro-2-oxopyrimidine-1(2H)-yl)-1,3-oxathiolan-2-yl)methoxy)(phenoxy)phosphorus Acyl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 7.58-7.19 (m, 15H), 5.34 (s, 2H), 5.22 (m, 1H), 5.12 (s, 2H), 4.66 (m, 2H), 3.95-2.71 (m,7H),1.28(d,3H).ESI-MS m/z:730.1[M+H]+.

10、2-(((((2R,3S,4R,5R)-5-(2-氨基-6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成10. 2-((((((2R,3S,4R,5R)-5-(2-amino-6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purine-9 -Yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

综合实施例1的制备方法，以2,6-二氨基嘌呤核苷为原料，合成得到白色泡沫状固体2-(((((2R,3S,4R,5R)-5-(2-氨基-6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.35(s,1H),7.47-7.18(m,17H),6.99(s,2H),6.16(m,1H),5.34(s,2H),5.11(s,2H),4.75-3.19(m,11H),1.28(d,3H).ESI-MS m/z:765.1[M+H]+. Synthesizing the preparation method of Example 1, using 2,6-diaminopurine nucleoside as the raw material, a white foamy solid 2-(((((2R,3S,4R,5R)-5-(2-amino- 6-(2-Amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy) Phosphoryl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 8.35 (s, 1H), 7.47-7.18 (m, 17H), 6.99 (s, 2H), 6.16 (m, 1H), 5.34 (s, 2H), 5.11 (s ,2H),4.75-3.19(m,11H),1.28(d,3H).ESI-MS m/z:765.1[M+H]+.

11、2-(((((2S,3R,4R,5S)-5-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-甲氧基-1H-嘌呤-9(6H)-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Nelarabine衍生物)11.2-((((((2S,3R,4R,5S)-5-(2-(2-amino-3-(4-fluorophenyl)propylamine)-6-methoxy-1H-purine -9(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Nelarabine derivative)

综合实施例1的制备方法，以奈拉滨为原料，合成得到白色泡沫状固体2-(((((2S,3R,4R,5S)-5-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-甲氧基-1H-嘌呤-9(6H)-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ7.92(s,1H),7.47-7.18(m,14H),6.16(m,1H),5.34(s,2H),5.26(s,1H),5.11(s,2H),4.75-3.44(m,11H),3.30(s,3H),1.28(d,3H).ESI-MS m/z:782.1[M+H]+. Synthesizing the preparation method of Example 1, using Nelarabine as the raw material to synthesize a white foamy solid 2-(((((2S,3R,4R,5S)-5-(2-(2-amino-3-( 4-fluorophenyl)alanine)-6-methoxy-1H-purin-9(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorus Acyl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 7.92 (s, 1H), 7.47-7.18 (m, 14H), 6.16 (m, 1H), 5.34 (s, 2H), 5.26 (s, 1H), 5.11 (s ,2H),4.75-3.44(m,11H),3.30(s,3H),1.28(d,3H).ESI-MS m/z:782.1[M+H]+.

12、2-(((((1R,4S)-4-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-(环丙基氨基)-9H-嘌呤-9-基)环戊烯-2-烯-1-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成()12. 2-(((((1R,4S)-4-(2-(2-amino-3-(4-fluorophenyl)alanine)-6-(cyclopropylamino)-9H-purine- Synthesis of 9-yl)cyclopentene-2-en-1-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate ()

综合实施例1的制备方法，以阿巴卡韦为原料，合成得到白色泡沫状固体2-(((((1R,4S)-4-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-(环丙基氨基)-9H-嘌呤-9-基)环戊烯-2-烯-1-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.05(s,1H),7.47-7.18(m,14H),6.05-5.60(m,2H),5.34(s,2H),5.11(s,2H),4.48-3.19(m,7H),2.36-2.17(m,3H),1.35(m,1H),1.28(d,3H),0.88(m,4H).ESI-MS m/z:769.1[M+H]+. Synthesizing the preparation method of Example 1, taking abacavir as the raw material, a white foamy solid 2-(((((1R,4S)-4-(2-(2-amino-3-(4-fluoro (Phenyl)alanine)-6-(cyclopropylamino)-9H-purin-9-yl)cyclopentene-2-en-1-yl)methoxy)(phenoxy)phosphoryl)amino) Benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ8.05 (s, 1H), 7.47-7.18 (m, 14H), 6.05-5.60 (m, 2H), 5.34 (s, 2H), 5.11 (s, 2H), 4.48 -3.19(m,7H),2.36-2.17(m,3H),1.35(m,1H),1.28(d,3H),0.88(m,4H).ESI-MS m/z:769.1(M+H) ]+.

13、2-(((((1S,3R,5R)-3-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-氧-1H-嘌呤-9(6H)-基)-5-羟基-2-亚甲基环戊基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成 (Entecavir衍生物)13, 2-(((((1S,3R,5R)-3-(2-(2-amino-3-(4-fluorophenyl)alanine)-6-oxy-1H-purine-9(6H )-Yl)-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (Entecavir derivative)

综合实施例1的制备方法，以恩替卡韦为原料，合成得到白色泡沫状固体2-(((((1S,3R,5R)-3-(2-(2-氨基-3-(4-氟苯基)丙氨)-6-氧-1H-嘌呤-9(6H)-基)-5-羟基-2-亚甲基环戊基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ7.97(s,1H),7.47-7.18(m,14H),5.34(s,2H),5.26-5.11(d,3H),4.37-3.19(m,9H),2.33-2.01(m,3H),1.28(d,3H).ESI-MS m/z:760.1[M+H]+. Synthesizing the preparation method of Example 1, using Entecavir as the raw material, a white foamy solid 2-(((((1S,3R,5R)-3-(2-(2-amino-3-(4-fluorobenzene) was synthesized. (Yl)alanine)-6-oxo-1H-purine-9(6H)-yl)-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)amino)propyl Benzyl acid. ¹ H NMR (CDCl ₃ 400MHz) δ 7.97 (s, 1H), 7.47-7.18 (m, 14H), 5.34 (s, 2H), 5.26-5.11 (d, 3H), 4.37-3.19 (m, 9H) ,2.33-2.01(m,3H),1.28(d,3H).ESI-MS m/z:760.1[M+H]+.

14、2-(((((2R,3S,4S,5R)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成14, 2-((((((2R,3S,4S,5R)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)- Synthesis of 3,4-Dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

综合实施例1的制备方法，以阿糖腺苷为原料，合成得到白色泡沫状固体2-(((((2R,3S,4S,5R)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.60(s,1H),8.35(s,1H),7.47-7.18(m,14H),6.16(m,1H),5.34(s,2H),5.11(s,2H),4.75-3.19(m,11H),1.28(d,3H).ESI-MS m/z:750.1[M+H]+. Synthesizing the preparation method of Example 1, using arabinosine as the raw material, a white foamy solid 2-(((((2R,3S,4S,5R)-5-(6-(2-amino-3- (4-fluorophenyl)alanine)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate . ¹ H NMR(CDCl ₃ 400MHz)δ8.60(s,1H), 8.35(s,1H), 7.47-7.18(m,14H), 6.16(m,1H), 5.34(s,2H), 5.11(s ,2H),4.75-3.19(m,11H),1.28(d,3H).ESI-MS m/z:750.1[M+H]+.

15、2-(((((2S,3S,4S,5S)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氟-9H-嘌呤-9- 基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Fludarabine衍生物)15, 2-((((((2S,3S,4S,5S)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-2-fluoro-9H-purine-9 -(Yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (Fludarabine derivative)

综合实施例1的制备方法，以氟达拉宾为原料，合成得到白色泡沫状固体2-(((((2S,3S,4S,5S)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氟-9H-嘌呤-9-基)-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.35(s,1H),7.47-7.18(m,14H),6.16(m,1H),5.34(s,2H),5.11(s,2H),4.75-3.19(m,11H),1.28(d,3H).ESI-MS m/z:768.1[M+H]+. Integrating the preparation method of Example 1, using fludarabine as a raw material, a white foamy solid 2-(((((2S,3S,4S,5S)-5-(6-(2-amino-3- (4-fluorophenyl)alanine)-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) Benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 8.35 (s, 1H), 7.47-7.18 (m, 14H), 6.16 (m, 1H), 5.34 (s, 2H), 5.11 (s, 2H), 4.75-3.19 (m,11H),1.28(d,3H).ESI-MS m/z:768.1[M+H]+.

16、2-(((((2R,3S,4R,5R)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氯-9H-嘌呤-9-基)-4-氟-3-羟基四氢呋喃-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Clorafaranbine衍生物)16, 2-((((((2R,3S,4R,5R)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-2-chloro-9H-purine-9 -Yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid benzyl ester (Clorafaranbine derivative)

综合实施例1的制备方法，以克罗拉滨为原料，合成得到白色泡沫状固体2-(((((2R,3S,4R,5R)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氯-9H-嘌呤-9-基)-4-氟-3-羟基四氢呋喃-2-yl)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.35(s,1H),7.47-7.18(m,14H),6.22(m,1H),5.34(s,2 H),5.11(s,2H),4.40-3.19(m,10H),1.28(d,3H).ESI-MS m/z:786.1[M+H]+. Synthesizing the preparation method of Example 1, using Cronabine as a raw material, a white foamy solid 2-(((((2R,3S,4R,5R)-5-(6-(2-amino-3-( 4-fluorophenyl)alanine)-2-chloro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) Benzyl propionate. ¹ H NMR(CDCl ₃ 400MHz)δ8.35(s,1H), 7.47-7.18(m,14H), 6.22(m,1H), 5.34(s,2 H), 5.11(s,2H), 4.40- 3.19(m,10H),1.28(d,3H).ESI-MS m/z:786.1[M+H]+.

17、2-(((((2S,3S,5S)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氯-9H-嘌呤-9-基)-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Cladibine衍生物)17, 2-((((((2S,3S,5S)-5-(6-(2-amino-3-(4-fluorophenyl)alanine)-2-chloro-9H-purin-9-yl )-3-Hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate (Cladibine derivative)

综合实施例1的制备方法，以克拉屈滨为原料，合成得到白色泡沫状固体2-(((((2S,3S,5S)-5-(6-(2-氨基-3-(4-氟苯基)丙氨)-2-氯-9H-嘌呤-9-基)-3-羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.35(s,1H),7.47-7.18(m,14H),5.95(m,1H),5.34(s,2H),5.11(s,2H),4.40-3.19(m,9H),2.56(m,2H),1.28(d,3H).ESI-MS m/z:768.1[M+H]+. Synthesizing the preparation method of Example 1, using Cladribine as the raw material, a white foamy solid 2-(((((2S,3S,5S)-5-(6-(2-amino-3-(4- Fluorophenyl)alanine)-2-chloro-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ 8.35 (s, 1H), 7.47-7.18 (m, 14H), 5.95 (m, 1H), 5.34 (s, 2H), 5.11 (s, 2H), 4.40-3.19 (m,9H),2.56(m,2H),1.28(d,3H).ESI-MS m/z:768.1[M+H]+.

18、2-(((((2R,3S,4R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)吡咯[2,1-f][1,2,4]三嗪-7-基)-5-氰基-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成18. 2-(((((2R,3S,4R,5R)-5-(4-(2-amino-3-(4-fluorophenyl)alanine)pyrrole[2,1-f][1 ,2,4]Triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate

综合实施例1的制备方法，以(2R,3R,4S,5R)-2-(4-4氨基吡咯[2,1-f][1,2,4]三嗪-7-基)-3,4-二羟基-5-(羟甲基)四氢呋喃-2-甲腈为原料，合成得到白色泡沫状固体2-(((((2R,3S,4R,5R)-5-(4-(2-氨基-3-(4-氟苯基)丙氨)吡咯[2,1-f][1,2,4]三嗪-7-基)-5-氰基-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ9.78(s,1H),7.47-7.18(m,14H),5.89-5.72(m,2H),5.34(s,2H),5.11(s,2H),4.51-3.19(m,11H),1.28(d,3H).ESI-MS m/z:774.1[M+H]+. Synthesizing the preparation method of Example 1, using (2R,3R,4S,5R)-2-(4-4aminopyrrole[2,1-f][1,2,4]triazin-7-yl)-3 ,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile as a raw material, synthesized to obtain a white foamy solid 2-(((((2R,3S,4R,5R)-5-(4-( 2-Amino-3-(4-fluorophenyl)alanine)pyrrole[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxy Tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ9.78 (s, 1H), 7.47-7.18 (m, 14H), 5.89-5.72 (m, 2H), 5.34 (s, 2H), 5.11 (s, 2H), 4.51 -3.19(m,11H),1.28(d,3H).ESI-MS m/z:774.1[M+H]+.

19、2-(((((2R)-1-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)丙烷-2-基)氧)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Tenofovir衍生物)19. 2-(((((2R)-1-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)propan-2-yl)oxy ) (Phenoxy) phosphoryl) amino) benzyl propionate synthesis (Tenofovir derivative)

综合实施例1的制备方法，以(R)-1-(6-氨基-9H-嘌呤-9-yl)丙基-2-醇为原料，合成得到白色泡沫状固体2-(((((2R)-1-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)丙烷-2-基)氧)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.60(s,1H),8.05(s,1H),7.47-7.18(m,14H),5.34(s,2H),5.11(s,2H),4.18-3.19(m,7H),1.28(d,3H),1.18(d,3H).ESI-MS m/z:676.1[M+H]+. Synthesizing the preparation method of Example 1, using (R)-1-(6-amino-9H-purin-9-yl)propyl-2-ol as the raw material, synthetically obtained white foamy solid 2-((((( 2R)-1-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)propan-2-yl)oxy)(phenoxy)phosphoryl) (Amino) benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ8.60 (s, 1H), 8.05 (s, 1H), 7.47-7.18 (m, 14H), 5.34 (s, 2H), 5.11 (s, 2H), 4.18-3.19 (m,7H),1.28(d,3H),1.18(d,3H).ESI-MS m/z:676.1[M+H]+.

20、2-(((2-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)乙氧基)(苯氧基)磷酰基)氨基)丙酸苄酯的合成(Adefovir衍生物)20, 2-(((2-(6-(2-amino-3-(4-fluorophenyl)alanine)-9H-purin-9-yl)ethoxy)(phenoxy)phosphoryl) Synthesis of benzyl (amino)propionate (Adefovir derivative)

综合实施例1的制备方法，以2-(6-氨基-9H-嘌呤-9-基)乙醇为原料，合成得到白色泡沫状固体2-(((2-(6-(2-氨基-3-(4-氟苯基)丙氨)-9H-嘌呤-9-基)乙氧基)(苯氧基)磷酰基)氨基)丙酸苄酯。 ¹H NMR(CDCl ₃ 400MHz)δ8.60(s,1H),8.05(s,1H),7.47-7.18(m,14H),5.34(s,2H),5.11(s,2H),4.51(m,4H),3.95(m,1 H),3.63(m,1H),3.44-3.19(m,2H),1.28(d,3H).ESI-MS m/z:662.1[M+H]+. Based on the preparation method of Example 1, using 2-(6-amino-9H-purin-9-yl)ethanol as a raw material, a white foamy solid 2-(((2-(6-(2-amino-3 -(4-Fluorophenyl)alanine)-9H-purin-9-yl)ethoxy)(phenoxy)phosphoryl)amino)benzyl propionate. ¹ H NMR (CDCl ₃ 400MHz) δ8.60 (s, 1H), 8.05 (s, 1H), 7.47-7.18 (m, 14H), 5.34 (s, 2H), 5.11 (s, 2H), 4.51 (m ,4H),3.95(m,1H),3.63(m,1H),3.44-3.19(m,2H),1.28(d,3H).ESI-MS m/z:662.1[M+H]+.

实施例21Example 21

实施例1、2、3、11、15、16、17的化合物以及吉西他滨体内抗肿瘤活性实验在PANC-1异种移植模型中评估了ZONK1802系列化合物的抗肿瘤活性。向雌性裸鼠植入PANC-1细胞。在第8天(植入后8天)平均肿瘤体积达到约247mm ³。在第8天(植入后8天)，根据肿瘤体积将荷瘤小鼠随机分成9组，并用空白对照、吉西他滨(ip，285mM/kg)，实施例1(po，285mM)、实施例2(ip，285mM/kg)、实施例3(po，285mM)、实施例11(po，285mM)、实施例15(po，285mM)、实施例16(po，285mM)、实施例17(po，285mM)。观察肿瘤体积和体重，每周记录两次，持续21天。在研究结束时(d29)，将动物安乐死并解剖载体对照的肿瘤并称重。计算肿瘤T/C(％)值(相对肿瘤增值率):T/C(％)＝100％×RTV _T/RTV _C The compounds of Examples 1, 2, 3, 11, 15, 16, 17 and gemcitabine in vivo anti-tumor activity experiment The anti-tumor activity of the ZONK1802 series of compounds was evaluated in the PANC-1 xenograft model. The PANC-1 cells were implanted into female nude mice. On the 8th day (8 days after implantation) the average tumor volume reached approximately 247 mm ³ . On day 8 (8 days after implantation), tumor-bearing mice were randomly divided into 9 groups according to tumor volume, and blank control, gemcitabine (ip, 285mM/kg), Example 1 (po, 285mM), Example 2 (ip, 285mM/kg), Example 3 (po, 285mM), Example 11 (po, 285mM), Example 15 (po, 285mM), Example 16 (po, 285mM), Example 17 (po, 285mM). Observe the tumor volume and weight, record twice a week for 21 days. At the end of the study (d29), the animals were euthanized and the tumors of the vehicle control were dissected and weighed. Calculate tumor T/C(%) value (relative tumor growth rate): T/C(%)=100%×RTV _T /RTV _C

RTV _T:给药后肿瘤相对体积 RTV _T : Relative tumor volume after administration

RTV _C:给药前肿瘤相对体积 RTV _C : Relative tumor volume before administration

溶剂：Solutol HS15:北京耦合科技有限公司,批号.20180226Solvent: Solutol HS15: Beijing Coupling Technology Co., Ltd., batch number. 20180226

PEG 400:Damas-beta,批号.P1471409生理盐水:四川科伦制药有限公司批号.A18111004-2PEG 400: Damas-beta, batch number. P1471409 Saline: Sichuan Kelun Pharmaceutical Co., Ltd. batch number. A18111004-2

动物：72只动物：6周龄的雌性BALB/c裸鼠，并饲养在含有玉米芯的塑料笼(3-5只小鼠/笼)中，并保持在无特定病原体的设施中(20-26℃，40-70％湿度)，12小时光照和黑暗循环，随意获取食物和过滤水。Animals: 72 animals: 6-week-old female BALB/c nude mice, raised in plastic cages (3-5 mice/cage) containing corn cobs, and kept in a facility free of specific pathogens (20- 26°C, 40-70% humidity), 12 hours light and dark cycle, free access to food and filtered water.

人肿瘤细胞株：PANC-1Human tumor cell line: PANC-1

细胞悬液在侧腹中植入无胸腺BALB/c裸鼠建立肿瘤模型；The cell suspension was implanted in the flank of athymic BALB/c nude mice to establish a tumor model;

对照组采用：15％Solutol HS15+85％PEG 400The control group adopts: 15% Solutol HS15 + 85% PEG 400

实验药物组采用生理盐水(5ml)溶解The experimental drug group was dissolved with normal saline (5ml)

当平均肿瘤体积达到约247mm ³时，根据肿瘤体积将荷瘤小鼠随机分为8组，并进行治疗；每周两次测量肿瘤大小和体重；在观察期间，观察动物的任何临床相关异常并记录；终点：用CO ₂处死动物，解剖载体对照的肿瘤并称重。实验组和剂量(mM/kg) When the average tumor volume reached about 247mm ³ , the tumor-bearing mice were randomly divided into 8 groups according to the tumor volume and treated; the tumor size and body weight were measured twice a week; during the observation period, any clinically relevant abnormalities in the animals were observed Record; End point: The _{animals were sacrificed with CO 2} and the tumors of the vehicle control were dissected and weighed. Experimental group and dose (mM/kg)

组别Group	药物drug	剂量dose	给药途径Route of administration	给药方案Dosing regimen	动物数Number of animals
AA	空白对照Blank control	To	POPO	BIW×6次BIW×6 times	66
BB	吉西他滨Gemcitabine	285285	ipip	BIW×6次BIW×6 times	66
CC	实施例2Example 2	285285	ipip	BIW×6次BIW×6 times	66
DD	实施例1Example 1	285285	POPO	BIW×6次BIW×6 times	66
EE	实施例3Example 3	285285	POPO	BIW×6次BIW×6 times	66
FF	实施例11Example 11	285285	POPO	BIW×6次BIW×6 times	66
GG	实施例15Example 15	285285	POPO	BIW×6次BIW×6 times	66
HH	实施例16Example 16	285285	POPO	BIW×6次BIW×6 times	66
II	实施例17Example 17	285285	POPO	BIW×6次BIW×6 times	66

相对肿瘤增值率T/C％Relative tumor growth rate T/C%

组别Group	第8天Day 8	第12天Day 12	第15天15th day	第19天Day 19	第22天Day 22	第26天Day 26	第29天Day 29
AA	To	To	To	To	To	To	To
BB	100.00％100.00%	85.95％85.95%	76.49％76.49%	57.32％57.32%	49.70％49.70%	51.24％51.24%	51.26％51.26%
CC	100.00％100.00%	75.53％75.53%	64.58％64.58%	51.81％51.81%	47.17％47.17%	46.50％46.50%	46.43％46.43%
DD	100.00％100.00%	78.39％78.39%	58.04％58.04%	51.34％51.34%	45.27％45.27%	40.73％40.73%	39.76％39.76%
EE	100.00％100.00%	79.41％79.41%	57.12％57.12%	53.32％53.32%	43.22％43.22%	39.33％39.33%	38.76％38.76%
FF	100.00％100.00%	76.41％76.41%	57.03％57.03%	52.88％52.88%	43.89％43.89%	42.00％42.00%	39.12％39.12%
GG	100.00％100.00%	76.22％76.22%	57.55％57.55%	54.33％54.33%	46.44％46.44%	42.11％42.11%	38.99％38.99%
HH	100.00％100.00%	78.11％78.11%	57.88％57.88%	50.21％50.21%	46.35％46.35%	41.21％41.21%	38.14％38.14%
II	100.00％100.00%	77.55％77.55%	56.23％56.23%	50.32％50.32%	44.13％44.13%	39.54％39.54%	38.78％38.78%

由上表可以看出，实施例2的化合物(C组)与吉西他滨(B组)差异不明显，实施例1、3、11、15、16和17的化合物与吉西他滨(B组)差异明显，相对肿瘤增值率明显低于阳性对照吉西他滨。It can be seen from the above table that the compound of Example 2 (group C) and gemcitabine (group B) are not significantly different, and the compounds of Examples 1, 3, 11, 15, 16, and 17 are significantly different from gemcitabine (group B). The relative tumor growth rate was significantly lower than the positive control gemcitabine.

各组体重减少百分比％% Weight loss in each group

组别Group	第8天Day 8	第12天Day 12	第15天15th day	第19天Day 19	第22天Day 22	第26天Day 26	第29天Day 29
AA	-1.57％-1.57%	-1.86％-1.86%	-1.86％-1.86%	-2.25％-2.25%	-2.35％-2.35%	-2.35％-2.35%	-1.57％-1.57%
BB	1.10％1.10%	2.58％2.58%	2.20％2.20%	1.10％1.10%	1.96％1.96%	1.14％1.14%	1.10％1.10%
CC	0.10％0.10%	1.58％1.58%	1.31％1.31%	1.22％1.22%	2.25％2.25%	1.50％1.50%	1.15％1.15%
DD	-0.20％-0.20%	-0.40％-0.40%	-1.99％-1.99%	-3.58％-3.58%	-4.77％-4.77%	-4.57％-4.57%	-2.20％-2.20%
EE	-0.10％-0.10%	-0.50％-0.50%	-1.66％-1.66%	-2.50％-2.50%	-3.66％-3.66%	-3.42％-3.42%	-0.86％-0.86%
FF	-0.30％-0.30%	-0.35％-0.35%	-1.35％-1.35%	-2.88％-2.88%	-3.67％-3.67%	-3.88％-3.88%	-0.97％-0.97%
GG	-0.21％-0.21%	-0.36％-0.36%	-2.33％-2.33%	-3.78％-3.78%	-4.21％-4.21%	-4.56％-4.56%	-1.20％-1.20%
HH	-0.55％-0.55%	-0.64％-0.64%	-1.88％-1.88%	-3.67％-3.67%	-3.96％-3.96%	-4.23％-4.23%	-1.56％-1.56%
II	-0.32％-0.32%	-0.54％-0.54%	-2.99％-2.99%	-2.58％-2.58%	-3.69％-3.69%	-4.78％-4.78%	-1.25％-1.25%

d8:移植瘤后的第8天；“-”：动物体重增加.d8: the 8th day after transplantation; "-": animal weight gain.

实施例22Example 22

药物在雄性BALB/c裸小鼠血浆和肿瘤中ZONK1802-5,dfdu和dfdctp药物浓度。The drug concentration of ZONK1802-5, dfdu and dfdctp in the plasma and tumors of male BALB/c nude mice.

Dfdu为吉西他滨在动物体内的主要代谢产物，也是无效的成分，dfdctp是吉西他滨在动物体内的主要活性成分，因此dfdu的减少意味着核苷酸结构的氨基部分氧化减少，dfdctp活性成分增加，作用机制改变。Dfdu is the main metabolite of gemcitabine in animals, and it is also an ineffective ingredient. dfdctp is the main active ingredient of gemcitabine in animals. Therefore, the decrease of dfdu means that the partial oxidation of the amino group of the nucleotide structure decreases, and the active ingredient of dfdctp increases. The mechanism of action change.

1.1.1供试品和内标化合物1.1.1 Test product and internal standard compound

供试品：ZONK1802-3，ZONK1802-4,ZONK1802-5,dfdu和dfdctp，分子量分别为745.6、788.7、299.7、264和505由广东中科药物研究有限公司提供。Test products: ZONK1802-3, ZONK1802-4, ZONK1802-5, dfdu and dfdctp, with molecular weights of 745.6, 788.7, 299.7, 264 and 505 provided by Guangdong Zhongke Pharmaceutical Research Co., Ltd.

内标化合物：Internal standard compound:

甲苯磺丁脲，批号为MKBR6717V，购自Sigma。Tolbutamide, batch number MKBR6717V, purchased from Sigma.

盐酸***，批号为BCBD8251V，购自Sigma。Propranolol hydrochloride, batch number BCBD8251V, purchased from Sigma.

双氯酚酸，批号为BCBK6371V，购自Sigma。Diclofenac, the batch number is BCBK6371V, purchased from Sigma.

1.1.2试验试剂1.1.2 Test reagent

二甲基亚砜(DMSO)：批号为L1712018，购自Aladdin。Dimethyl sulfoxide (DMSO): Lot number is L1712018, purchased from Aladdin.

乙腈：批号为175164，购自Fisher Scientific。Acetonitrile: Lot No. 175164, purchased from Fisher Scientific.

甲酸：批号为L0980250，购自CNW Technologies。Formic acid: Lot number is L0980250, purchased from CNW Technologies.

超纯水：由纯水仪制备，临用现取。Ultra-pure water: Prepared by a pure water instrument, take it immediately before use.

空白小鼠血浆和肿瘤：本部门体内组采自小鼠。Blank mouse plasma and tumor: the in vivo group in this department was collected from mice.

动物来源：Animal source:

由生物体内提供。Provided by the organism.

动物及分组：从生物体内获得BALB/c裸小鼠18只，6只/组；Animals and grouping: 18 BALB/c nude mice were obtained from organisms, 6 mice/group;

给药方式：IP和POMode of administration: IP and PO

溶剂配制Solvent preparation

溶剂I：15％Solutol HS15+85％PEG 400Solvent I: 15% Solutol HS15 + 85% PEG 400

50-60℃水浴加热Solutol HS15(聚乙二醇15羟硬脂酸酯)至液体后，吸取1.8mL Solutol HS15至15mL离心管中，加入10.2mL PEG 400，涡旋振荡混匀，配制成含15％Solutol HS15和85％PEG 400的混合制剂，2-8℃保存待用，每周配制一次。After heating Solutol HS15 (polyethylene glycol 15 hydroxystearate) to the liquid in a 50-60℃ water bath, pipette 1.8 mL Solutol HS15 to 15 mL centrifuge tubes, add 10.2 mL PEG 400, vortex and mix well, and prepare to contain A mixed preparation of 15% Solutol HS15 and 85% PEG 400, stored at 2-8°C until use, and prepared once a week.

给药制剂配制Dosing preparation formulation

对照样品：ZONK1802-5(盐酸吉西他滨)摩尔剂量为285mM/kg(75mg/kg)的给药制剂配制(供试品浓度为8.56mg/mL)Control sample: preparation of ZONK1802-5 (gemcitabine hydrochloride) with a molar dose of 285mM/kg (75mg/kg) (the concentration of the test substance is 8.56mg/mL)

称取适量ZONK1802-5供试品置于5mL离心管中，加入适量生理盐水，涡旋振荡至药物完全溶解，配制成供试品浓度为8.56mg/mL的给药制剂，用于动物给药，现配现用。Weigh an appropriate amount of ZONK1802-5 test product into a 5mL centrifuge tube, add an appropriate amount of physiological saline, vortex until the drug is completely dissolved, and prepare a test product with a concentration of 8.56 mg/mL for animal administration , Now equipped and used now.

受试样品：ZONK1802-3(实施例1化合物)摩尔剂量为570mM/kg(423mg/kg)的给药制剂配制(供试品浓度为42.3mg/mL)Test sample: ZONK1802-3 (Compound of Example 1) preparation of a dosage formulation with a molar dose of 570mM/kg (423mg/kg) (the concentration of the test product is 42.3mg/mL)

称取适量ZONK1802-3供试品置于5mL离心管中，加入适量溶剂I，涡旋振荡至药物完全溶解，配制成供试品浓度为42.3mg/mL的给药制剂，用于动物给药。现用现配。Weigh an appropriate amount of ZONK1802-3 test substance into a 5mL centrifuge tube, add an appropriate amount of solvent I, vortex and shake until the drug is completely dissolved, and prepare a test substance concentration of 42.3mg/mL for administration to animals . Available now.

受试样品：ZONK1802-4(实施例2化合物)摩尔剂量为570mM/kg(450mg/kg)的给药制剂配制(供试品浓度为45.3mg/mL)Test sample: ZONK1802-4 (Compound of Example 2) preparation of a dosage formulation with a molar dose of 570mM/kg (450mg/kg) (the concentration of the test product is 45.3mg/mL)

称取适量ZONK1802-4供试品置于5mL离心管中，加入适量溶剂I，涡旋振荡至药物完全溶解，配制成供试品浓度为45.3mg/mL的给药制剂，用于动物给药。现用现配。Weigh an appropriate amount of ZONK1802-4 test product and place it in a 5mL centrifuge tube, add an appropriate amount of solvent I, vortex until the drug is completely dissolved, and formulate a test product concentration of 45.3mg/mL as a dosing preparation for animal administration . Available now.

溶剂对照：15％Solutol HS15+85％PEG 400Solvent control: 15% Solutol HS15 + 85% PEG 400

动物编号、给药剂量给药体积如下表：The animal number, dosage and volume of administration are as follows:

进食/饮水情况:Eating/drinking situation:

给药前禁食12小时以上，给药后禁食4小时，自由饮水。Fasting for more than 12 hours before administration, 4 hours after administration, and free drinking water.

抗凝剂：EDTA-K ₂ Anticoagulant: EDTA-K ₂

样品采集时间点：给药后1h、2h、4h，其中101-102、201-202和301-302为1h的动物；103-104、203-204和303-304为2h的动物；105-106、205-206和305-306为4h的动物，收集血浆和肿瘤，肿瘤需称量重量。采集全血至EDTA-K ₂抗凝管，管子中加入tetrahydrouridine(25ug/mL)用于抑制CDA的活性，离心前血液样品置于碎冰上，半小时内，离心取血浆(6000转，8分钟，4℃)，干冰冻存备用。肿瘤使用冰冷的生理盐水清洗三遍，用滤纸吸去水分，称重。 Sample collection time points: 1h, 2h, 4h after administration, of which 101-102, 201-202 and 301-302 are animals at 1h; 103-104, 203-204 and 303-304 are animals at 2h; 105-106 , 205-206 and 305-306 are 4h animals, plasma and tumors are collected, and tumors need to be weighed. Collect whole blood into an EDTA-K ₂ anticoagulation tube, add tetrahydrouridine (25ug/mL) to the tube to inhibit the activity of CDA, place the blood sample on crushed ice before centrifugation, and centrifuge to collect plasma (6000 rpm, 8 Minutes, 4℃), freeze in dry ice for later use. The tumor was washed three times with ice-cold normal saline, and the water was absorbed by filter paper and weighed.

所有样品收集好之后放于-80冰箱冷冻保存待分析。After all samples are collected, they are stored in the -80 refrigerator for analysis.

分析方法及检测Analysis method and detection

本实验采用液质联用方法测定雄性BALB/c裸小鼠血浆和肿瘤中ZONK1802-5,dfdu和dfdctp药物浓度。每个分析批次至少建立一条至少包含6个标准浓度点的标准曲线，计算该分析批样品中待测物的浓度，并随行质控样品。血浆和肿瘤标准曲线中3/4以上浓度点的准确度在80％-120％以内。若定量下限或定量上限超出接受范围，则剔除该点重新建立新的线性范围。每个分析批次设有高、中、低不同浓度的质控样品(QC)，每个浓度至少平行双样本，质控样品数量大于等于每批样品数量的5％。根据每一分析批次的标准曲线计算质控样品的浓度。血浆质控样品中最多允许1/3且为不同浓度的样品超出理论值的±20％，否则此批数据不被接受，重新测定。In this experiment, the LC/MS method was used to determine the drug concentration of ZONK1802-5, dfdu and dfdctp in the plasma and tumors of male BALB/c nude mice. Establish at least one standard curve containing at least 6 standard concentration points for each analysis batch, calculate the concentration of the analyte in the sample of the analysis batch, and accompany the quality control sample. The accuracy of the concentration points above 3/4 in the standard curve of plasma and tumor is within 80%-120%. If the lower limit of quantification or upper limit of quantification exceeds the acceptable range, the point is removed and a new linear range is established. Each analysis batch is equipped with quality control samples (QC) with different concentrations of high, medium, and low, and each concentration is at least two parallel samples, and the number of quality control samples is greater than or equal to 5% of the number of samples in each batch. Calculate the concentration of quality control samples based on the standard curve of each analysis batch. Up to 1/3 of the plasma quality control samples and samples with different concentrations are allowed to exceed ±20% of the theoretical value, otherwise this batch of data will not be accepted, and the determination will be repeated.

标准曲线和质控样品的制备Preparation of standard curves and quality control samples

分别取空白雄性BALB/c裸小鼠血浆和肿瘤45μL，分别加入5μL上述配制好的标曲工作溶液及质控工作液，涡旋混匀，得到各个化合物相应的的标准曲线及质控样品。对于血样样品需要稀释的化合物，分别取雄性BALB/c裸小鼠血浆和肿瘤57μL，分别加入3μL浓度为100μg/mL的对应化合物的稀释质控工作溶液，涡旋混匀，得到浓度分别为5000ng/mL质控(QC)样品，接着取5μL上述质控样品加入到45μL雄性BALB/c裸小鼠血浆和肿瘤，涡旋混匀，得到浓度为500ng/mL稀释质控样品(N＝3)，稀释因子为：10。Take 45 μL of blank male BALB/c nude mouse plasma and tumor respectively, add 5 μL of the above-prepared standard curve working solution and quality control working solution, and vortex to mix to obtain the corresponding standard curve and quality control samples of each compound. For the compound that needs to be diluted in the blood sample, take 57μL of male BALB/c nude mouse plasma and tumor respectively, add 3μL of the diluted quality control working solution of the corresponding compound at a concentration of 100μg/mL, and vortex to mix to obtain a concentration of 5000ng respectively. /mL quality control (QC) sample, and then take 5μL of the above quality control sample and add it to 45μL of male BALB/c nude mouse plasma and tumor, vortex and mix well to obtain a diluted quality control sample with a concentration of 500ng/mL (N=3) , The dilution factor is: 10.

1.1.3液相色谱条件1.1.3 Liquid chromatography conditions

对于dfduFor dfdu

高效液相***：SHIMADZU LC30ADHigh performance liquid system: SHIMADZU LC30AD

色谱柱：Phenomenex Gimini C18 5μm 4.6*50mmColumn: Phenomenex Gimini C18 5μm 4.6*50mm

柱温：室温；Column temperature: room temperature;

流速：0.8mL/min；Flow rate: 0.8mL/min;

流动相：A相：含0.1％甲酸的水溶液，B相：含0.1％甲酸的乙腈溶液；Mobile phase: phase A: aqueous solution containing 0.1% formic acid, phase B: acetonitrile solution containing 0.1% formic acid;

色谱柱：Thermo SCINTIFIC Hypersil GOLD C8 50*2.1mmColumn: Thermo SCINTIFIC Hypersil GOLD C8 50*2.1mm

柱温：室温；Column temperature: room temperature;

流速：0.5mL/min；Flow rate: 0.5mL/min;

流动相：A相：含2mM乙酸铵的水溶液，B相：乙腈溶液；Mobile phase: phase A: aqueous solution containing 2mM ammonium acetate, phase B: acetonitrile solution;

表雄性BALB/c裸小鼠血浆中dfdu药物检测浓度(ng/mL)Table Detected concentration of dfdu drug in plasma of male BALB/c nude mice (ng/mL)

实施例23含有氨基的核苷酸类药物的4-氟苯丙氨酸酰化衍生物对于抗乙肝病毒的药效学实验比较Example 23 Comparison of pharmacodynamic experiments of 4-fluorophenylalanine acylated derivatives of amino-containing nucleotide drugs against hepatitis B virus

实施例中化合物对于乙肝病毒(DHBV-DNA)抑制作用的体内实验In vivo experiment of the compound's inhibitory effect on hepatitis B virus (DHBV-DNA)

试剂：Reagents:

阳性对照药：Positive control drug:

拉米夫定Lamivudine

α-32P-Dctp(北京福瑞生物技术工程公司)α-32P-Dctp (Beijing Furui Biotechnology Engineering Company)

缺口翻译试剂盒(Promega公司)Gap Translation Kit (Promega)

鱼精DNA及牛血清白蛋白均购自中国科学院生物物理所Fish sperm DNA and bovine serum albumin were purchased from the Institute of Biophysics, Chinese Academy of Sciences

实验动物及病毒：1日龄北京麻鸭，体重60-100g；DHBV-DNA强阳性血清，采自南京麻鸭，-70℃保存。Experimental animals and viruses: 1 day old Peking duck, weighing 60-100g; DHBV-DNA strong positive serum, collected from Nanjing duck, stored at -70℃.

试验方法：1日龄北京鸭，经腿静脉注射DHBV-DNA阳性血清0.2ml/只，雏鸭经DHBV感染第7天随机分为组，分别为：病毒对照组(生理盐水，PO)；实施例9，实施例19，实施例20，实施例14，阳性对照药拉米夫定组(LA)，各实验组按照相同的摩尔数量口服灌胃给药，采用DMSO溶解，给药量300mM/kg。感染DHBV后第7天(即T0时间点)开始给药，2次/天，连续给药10天。分别在HDBV感染后第七天(T0)用药前，用药第5天，用药第10天，及停药后第3天，自鸭腿静脉取血，离心分离血清，-70℃保存待检。严格按照缺口翻译试剂盒说明书测定血清DHBV-DNA水平。用32P标记DHBV-DNA密度，以杂交斑点OD值作为标本DHBV-DNA水平值。按以下公式计算药物在各时间点对动物血清DHBV-DNA的抑制率：Test method: 1-day-old Peking ducks were injected with 0.2ml of DHBV-DNA positive serum through the leg vein. The ducklings were randomly divided into groups on the 7th day after DHBV infection, namely: virus control group (normal saline, PO); implementation Example 9, Example 19, Example 20, Example 14, the positive control drug lamivudine group (LA), each experimental group was administered orally in the same molar amount, dissolved in DMSO, and the dosage was 300mM/ kg. The administration was started on the 7th day (ie, T0 time point) after DHBV infection, twice a day, for 10 consecutive days. On the seventh day (T0) after HDBV infection, before medication, on the 5th day, 10th day, and 3rd day after drug withdrawal, blood was collected from the vein of the duck leg, centrifuged to separate the serum, and stored at -70°C for testing. The serum DHBV-DNA level was determined in strict accordance with the instructions of the gap translation kit. The DHBV-DNA density was labeled with 32P, and the OD value of the hybridization spot was used as the DHBV-DNA level value of the specimen. Calculate the inhibition rate of the drug on animal serum DHBV-DNA at each time point according to the following formula:

DNA抑制率(％)＝给药前(T0)OD值-给药后OD值/给药前(T0)OD值*100％DNA inhibition rate (%) = OD value before administration (T0)-OD value after administration / OD value before administration (T0) * 100%

实验结果：DHBV感染后T0、T5、T10、P3时间点鸭血清DHBV-DNA经斑点杂交法测定，所有4组实验动物感染DHBV后，血清DHBV-DNA均呈强阳性。酶标仪检测各实验样本在490nm处OD值表明，口服实施例化合物后5-10天动物血清DHBV-DNA浓度呈极显著下降，且较拉米夫定有较明显优势，其它实施例化合物较实施例2具有优势。停药3天后仍显著抑制DHBV-DNA。各组不同时间点的OD值见下表：Experimental results: DHBV-DNA in duck serum at T0, T5, T10, and P3 after DHBV infection was determined by dot blot hybridization. After DHBV infection in all four groups of experimental animals, the serum DHBV-DNA was strongly positive. The OD value at 490nm of each experimental sample detected by the microplate reader showed that the animal serum DHBV-DNA concentration decreased significantly after 5-10 days after oral administration of the compound of the example, and it has obvious advantages over lamivudine. Example 2 has advantages. DHBV-DNA was still significantly inhibited after 3 days of drug withdrawal. The OD values of each group at different time points are shown in the following table:

时间点Point in time	T0T0	T5T5	T10T10	P3P3
病毒对照组Virus control group	1.83±0.211.83±0.21	1.76±0.151.76±0.15	1.66±0.231.66±0.23	1.75±0.251.75±0.25
LALA	1.86±0.311.86±0.31	1.56±0.211.56±0.21	1.26±0.351.26±0.35	1.61±0.281.61±0.28
实施例9Example 9	1.91±0.361.91±0.36	1.50±0.211.50±0.21	1.11±0.251.11±0.25	1.12±0.321.12±0.32
实施例19Example 19	1.79±0.291.79±0.29	1.45±0.241.45±0.24	1.10±0.111.10±0.11	1.02±0.161.02±0.16

Claims

式I所示4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物：The 4-halogen substituted phenylalanine amidated nucleotide derivative of formula I:

式I中，X代表卤素，具体可为F、Cl、Br或I；In formula I, X represents halogen, specifically F, Cl, Br or I;

式I中，
代表核苷及其类似物； In formula I,
Represents nucleosides and their analogs;

式I中，4-卤素取代的苯丙氨酸与核苷碱基中的氨基形成酰胺键，所述碱基包括取代或未取代的4-氨基嘧啶、取代或未取代的2-氨基嘌呤、取代或未取代的6-氨基嘌呤、取代或未取代的2，6-二氨基嘌呤、取代或未取代1H-咪唑并[4,5-c]吡啶、取代或未取代吡咯并[2,1-f][1,2,4]三嗪，其中的取代基为：甲基，甲氧基，环丙基，氨基，羰基，卤素；In formula I, the 4-halogen substituted phenylalanine forms an amide bond with the amino group in the nucleoside base, and the base includes substituted or unsubstituted 4-aminopyrimidine, substituted or unsubstituted 2-aminopurine, Substituted or unsubstituted 6-aminopurine, substituted or unsubstituted 2,6-diaminopurine, substituted or unsubstituted 1H-imidazo[4,5-c]pyridine, substituted or unsubstituted pyrrolo[2,1 -f][1,2,4]triazine, where the substituents are: methyl, methoxy, cyclopropyl, amino, carbonyl, halogen;

式(I)中，核苷中的核糖或脱氧核糖中的5-羟甲基与磷酸形成酯键，In formula (I), the ribose in the nucleoside or the 5-hydroxymethyl group in the deoxyribose and the phosphate form an ester bond,

R ¹为苯基或取代苯基，萘基或取代萘基，C1-C18未取代的烷基，卤素/烷氧基取代的C1-C18烷基，C3-C6取代或未取代的环烷基； R ¹ is phenyl or substituted phenyl, naphthyl or substituted naphthyl, C1-C18 unsubstituted alkyl, halogen/alkoxy substituted C1-C18 alkyl, C3-C6 substituted or unsubstituted cycloalkyl ；

R ²为氨基酸侧链，或C1-C8烷基/环烷基、苯基； R ² is an amino acid side chain, or C1-C8 alkyl/cycloalkyl, phenyl;

R ³为甲基、乙基、异丙基或苄基。 R ³ is methyl, ethyl, isopropyl or benzyl.
根据权利要求1所述的核苷酸衍生物，其特征在于：所述碱基为如下任意一种：The nucleotide derivative of claim 1, wherein the base is any one of the following:
根据权利要求1或2所述的核苷酸衍生物，其特征在于：所述核苷及其类似物为如下任意一种：The nucleotide derivative according to claim 1 or 2, wherein the nucleoside and its analogue are any one of the following:
制备权利要求1-3中任一项所述的核苷酸衍生物的方法，包括如下步骤：1)使式II所示五氟苯酚磷酸酯活性中间体与核苷中的核糖或脱氧核糖的5-羟甲基反应，得到核糖或脱氧核糖5-羟甲基磷酸化的核苷酸；The method for preparing the nucleotide derivative according to any one of claims 1 to 3, comprising the following steps: 1) Combining the active intermediate of pentafluorophenol phosphate represented by formula II with the ribose or deoxyribose in the nucleoside 5-hydroxymethyl reaction to obtain ribose or deoxyribose 5-hydroxymethyl phosphorylated nucleotides;

式II中，R ¹、R ²和R ³的定义分别同权利要求1式I中R ¹、R ²和R ³的定义； In formula II, ^{the definitions of R 1} , R ² and R ³ are the same as the definitions of R ¹ , R ² and R ³ in formula I of claim 1 respectively;

2)使得BOC-(4-X)苯丙氨酸与步骤2)所得核糖或脱氧核糖核苷酸的碱基中的氨基偶联形成酰胺键，即得。2) Coupling BOC-(4-X) phenylalanine with the amino group in the base of the ribose or deoxyribonucleotide obtained in step 2) to form an amide bond.
根据权利要求4所述的方法，其特征在于：步骤1)中，所述式II所示五氟苯酚磷酸酯活性中间体与核苷的摩尔为：1.0-2.0；The method according to claim 4, characterized in that: in step 1), the mole of the active intermediate of pentafluorophenol phosphate represented by formula II and the nucleoside is 1.0-2.0;

所述反应在叔丁基氯化镁催化下进行；The reaction is carried out under the catalysis of tert-butylmagnesium chloride;

核苷与叔丁基氯化镁的摩尔比为：1:1.0-2.0；The molar ratio of nucleoside to tert-butyl magnesium chloride is 1:1.0-2.0;

所述反应在惰性气体保护下进行，所述惰性气体具体可为氮气；The reaction is carried out under the protection of an inert gas, and the inert gas may specifically be nitrogen;

所述反应在无水有机溶剂中进行，所述无水有机溶剂具体可为无水四氢呋喃；The reaction is carried out in an anhydrous organic solvent, and the anhydrous organic solvent may specifically be anhydrous tetrahydrofuran;

所述反应的温度为：-20-10℃，时间为：8-24h。The temperature of the reaction is: -20-10°C, and the time is: 8-24h.
根据权利要求4或5所述的方法，其特征在于：步骤2)中，所述形成酰胺键的反应1-羟基苯并***存在下进行；The method according to claim 4 or 5, characterized in that: in step 2), the reaction of forming an amide bond is carried out in the presence of 1-hydroxybenzotriazole;

所述形成酰胺键的反应在羧基活化试剂存在下进行，所述羧基活化试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；The reaction to form an amide bond is carried out in the presence of a carboxyl activating reagent, and the carboxyl activating reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

所述核糖或脱氧核糖5-羟甲基磷酸化的核苷酸与BOC-(4-X)苯丙氨酸、1-羟基苯并***及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔比依次为：1.0：1.0-1.5：1.0-2.0：1.0-2.0；The ribose or deoxyribose 5-hydroxymethyl phosphorylated nucleotide and BOC-(4-X) phenylalanine, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)- The molar ratio of 3-ethylcarbodiimide hydrochloride is in order: 1.0: 1.0-1.5: 1.0-2.0: 1.0-2.0;

所述反应的温度为室温，时间为3-8h。The temperature of the reaction is room temperature, and the time is 3-8h.
根据权利要求4所述的方法，其特征在于：如所述核苷中的核糖或脱氧核糖除了具有5-羟甲基外，还有其他位置上的羟基，那么在步骤1)之前还包括先选择性地保护其他位置上的羟基的操作，The method according to claim 4, characterized in that: if the ribose or deoxyribose in the nucleoside has a 5-hydroxymethyl group, it also has hydroxyl groups at other positions, then before step 1), it also includes first The operation of selectively protecting the hydroxyl groups in other positions,

相应地，在步骤2)之后，还包括将步骤2)得到的产物在酸性条件下脱去羟基保护，得到目标产物的操作。Correspondingly, after step 2), it also includes the operation of removing the hydroxyl protection of the product obtained in step 2) under acidic conditions to obtain the target product.
权利要求1-3中任一项所述的式I所示4-卤素取代的苯丙氨酸酰胺化的核苷酸衍生物在制备抗肿瘤和/或抗病毒的药物中的应用。The use of the 4-halogen substituted phenylalanine amidated nucleotide derivative of formula I according to any one of claims 1 to 3 in the preparation of anti-tumor and/or anti-viral drugs.