CN114349816A

CN114349816A - Small molecule coupling molecule based on aminopeptidase N/CD13 and preparation method and application thereof

Info

Publication number: CN114349816A
Application number: CN202111440559.2A
Authority: CN
Inventors: 徐文方; 江余祺; 张良; 李晓杨; 张剑; 王学健
Original assignee: Qingdao Research Institute Of Biological Sciences
Current assignee: WEIFANG BOCHUANG INTERNATIONAL ACADEMY OF BIOTECHNOLOGY AND MEDICINE
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-04-15

Abstract

The invention discloses a novel small molecule coupling compound based on aminopeptidase N/CD13, and a preparation method and application thereof, wherein the compound has a structure shown in a formula I:

the targeting ligand which takes the CD13 inhibitor ubenimex as the micromolecule coupling drug not only can mediate the tumor targeting of molecules and enter cellsThe cell poison and ubenimex generated by post-degradation can also generate a synergistic effect, and a new strategy is provided for treating tumors.

Description

Small molecule coupling molecule based on aminopeptidase N/CD13 and preparation method and application thereof

Technical Field

The invention relates to the technical field of CD13 inhibitors, in particular to a novel small molecule coupling molecule based on aminopeptidase N/CD13 and a preparation method and application thereof.

Background

Since the 21 st century, the drug therapy of malignant tumors has entered the era of precise and individualized therapy (Nat Rev Genet.2016,17, 507-. Traditional cytotoxic drugs such as pentafluorouracil, gemcitabine and the like are always first-line drugs for tumor treatment, however, due to lack of selectivity, the drugs show stronger toxic and side effects in clinical use. The development of accurate tumor treatment drugs is a hot spot in the current targeted treatment of how to accurately deliver the anti-tumor drugs to tumor tissues without affecting normal tissues and organs. The targeted coupled drug technology is a new technology which is based on the structure of cytotoxic drugs with high-efficiency anti-tumor effect, and is characterized in that an antibody, a polypeptide and a small molecule non-peptide ligand with a targeted tumor tissue are coupled through a chemical method, so that the cytotoxic drugs are accurately delivered to the tumor tissue, thereby playing a role in attenuation and synergy (Lancet.2019,394, 793-804; Chem Soc Rev.2021,50, 1480-.

The targeted conjugate drugs mainly comprise Antibody conjugate drugs (ADC), polypeptide conjugate drugs (PDC) and Small-molecule conjugate drugs (SMDC), and the structural general formulas of the drugs are similar and comprise three parts of a specific targeting ligand, a linker and a cell poison. The three conjugated drugs differ in the specific targeting ligand differences: respectively macromolecular antibody, polypeptide and small molecule. Among them, antibody conjugate drugs are the most mature class currently studied, and 11 drugs are listed on the market, and are mainly used for treating hematological tumors clinically, and the number of solid tumors is small (molecules.2020,25, 4764-4797). Compared with ADC using antibody as ligand, SMDC has the advantages of no immunoreaction, easy synthesis control, easy structure modification, small molecular weight, easy access to solid tumor tissue, low preparation cost and good in vivo and in vitro stability (Nat Rev Drug Discov.2015,14, 203-219). SMDC is believed to be more advantageous in the treatment of solid tumors, being complementary to ADC drugs in indications. Thus, small molecule conjugated drugs are of great interest, and in a rapidly developing stage, many Drug candidates are currently in clinical research (Nat Rev Drug Discov.2015,14, 203-219).

The cell poison is the core part of small molecule coupling drug which plays the role of killing tumor, and is called as the 'nuclear warhead' for attacking tumor. Gemcitabine and Pentafluorouracil are commonly used cytotoxic agents (Chem Soc Rev.2021,50, 1480-1494; J Med Chem.2021,64, 4450-4461). Gemcitabine is clinically used for the treatment of pancreatic cancer, liver cancer, etc., but severe drug resistance is generated in long-term use, so NUC-1031 (fig. 3) synthesized by using Pro tide prodrug technology overcomes the drug resistance of gemcitabine, and is a novel gemcitabine prodrug and currently in a three-phase clinical trial (J Med chem.2014,57, 1531-. While the Pro tide prodrug NUC-3373 against pentafluorouracil is currently in first-phase clinics. The main drug resistance mechanism of NUC-1031 is independent of non-Na required for gemcitabine to pass through membrane⁺Transport of the dependent, equilibrium nucleoside transporter (hENT1) circumvents the rate-limiting process of gemcitabine-dependent deoxycytidine kinase (dCK) to generate the monophosphate dFdCMP, and is insensitive to the major enzyme gemcitabine inactivation (cytidine deaminase, CDA). Therefore, the in-vitro anti-tumor activity of the NUC-1031 in vivo is remarkably superior to that of gemcitabine, and the NUC-1031 still has certain inhibitory activity on gemcitabine-resistant tumors and is a more ideal cell poison.

CD13 is a Zn²⁺The dependent membrane protein is highly expressed on the surface of tumor cells such as liver cancer, pancreatic cancer and the like, blood vessels and stroma, is lowly expressed in most normal tissue cells, and has tumor tissue specificity (J Med chem.2018,61,6468-012,109,1637-1642). The protein structure of CD13 comprises an intracellular region, a transmembrane region and an extracellular region containing a catalytic active center, and researches show that the monoclonal antibody, the polypeptide and the small molecule inhibitor of CD13 can be combined with CD 13-mediated endocytosis on the surface of tumor cells (Trends Mol Med.2008,14, 361-371; J Hematol Oncol.2020,13, 32-47). CD13 has been applied as a ligand target to antibody-conjugated drugs and polypeptide-conjugated drugs: (1) zapata et al in 2020 report that the first antibody coupling drug MI130110 using CD13 monoclonal antibody as ligand, through combining with CD13, the molecule is endocytosed into the cell, and the anti-tumor effect shows the specificity of tumor tissue (J Hematol Oncol.2020,13, 32-47); (2) the polypeptide coupling drugs NGR-hTNF alpha and tTF-NGR which take the polypeptide NGR (Asn-Gly-Arg) specifically recognizing CD13 as a ligand are in the phase III and phase I clinics respectively (Med Res Rev.2012,32, 1078-. Therefore, CD13 is considered to be an ideal target for targeted drug delivery and also an ideal target for small molecule conjugated drug design.

Compared with the main ligand target developed by other small molecule coupled drugs, the potential feature and advantage of the CD13 are that the CD13 not only has the functions of tumor tissue specificity and endocytosis mediation, but also is an important target for various tumor treatments: CD13 is a liver cancer stem cell surface marker and is closely related to the occurrence, drug resistance and recurrence of liver cancer (J Clin invest.2010,120, 3326-3339); ubenimex, a small molecule inhibitor of CD13, clinically exerts multiple anti-tumor effects including tumor immune regulation, tumor angiogenesis inhibition, reversal of drug resistance, and the like (Proc Natl Acad Sci U S A.2012,109, 1637-1642; J Clin invest.2010,120, 3326-3339). Therefore, a targeting ligand combining ubenimex and a cytotoxic agent is required to further improve the inhibitory effect.

Disclosure of Invention

In view of the prior art, the invention aims to provide a small molecule coupling molecule based on aminopeptidase N/CD13, and a preparation method and application thereof. The CD13 inhibitor ubenimex is taken as a targeting ligand of a small molecule coupling drug, so that the tumor targeting property of the molecule can be mediated, and a cell poison and ubenimex generated by degradation after entering cells can also generate a synergistic effect, thereby providing a new strategy for treating tumors.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, a compound shown in formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the optical isomer, the diastereoisomer, the racemate and the mixture, or a pharmaceutically acceptable salt, a solvate and a deuteron thereof is provided;

wherein: a is selected from NH or O;

b is CH₂、(CH₂)_n、CHCH₃、OCH₂、O(CH₂)_n、OCH(CH₃)CH₂、OC(CH₃)₂CH₂、NHCH₂、NH(CH₂)_n、NHCHCH₃、NHCH(CH₃)CH₂、NHC(CH₃)₂CH₂、

Wherein n is 2 to 10;

represents a bond to a carbonyl group;

represents a bond to A;

c is

Wherein R is₁Is selected from-CH₃、-(CH₂)_nCH₃、-CH(CH₃)₂、C₆H₅CH₂-、4-F-C₆H₅CH₂-、

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₂Is selected from

R₃Is selected from

Wherein n is 1-6;

R₄、R₅selected from H, halogen, (C)_1-2) Alkyl, halomethyl, OH, OCH₃、O(CH₂)_nCH₃、OC(CH₃)₃、OCH(CH₃)₂Cyclopropyloxy, 5-6-membered alkoxy, NH₂、N(CH₃)₂、NH(CH₂)_nCH₃CN or N₃Wherein n is 0 to 9;

R₆is selected from-CH₃、-(CH₂)_nCH₃、-CH(CH₃)₂、C₆H₅CH₂-、4-F-C₆H₅CH₂-、

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₇Selected from H, -CH₃or-CH₂CH₃；

X is selected from O or NH;

d is H or F.

Preferably, the compound shown in the formula I has a structure shown in a general formula II, a general formula III, a general formula IV or a general formula V;

wherein: a is selected from NH or O;

Wherein n is 2 to 10;

represents a bond to a carbonyl group;

represents a bond to A;

R₁is selected from-CH₃、-(CH₂)_nCH₃、-CH(CH₃)₂、C₆H₅CH₂-、4-F-C₆H₅CH₂-、

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₂Is selected from

R₃Is selected from

Wherein n is 1-6;

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₇Selected from H, -CH₃or-CH₂CH₃；

X is selected from O or NH.

Preferably, the compound of formula I is selected from:

isopropyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (13 a);

benzyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (13 b);

methyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (13 c);

cyclohexyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (13 d);

benzyl (((((2R, 3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (2-methoxy-2-oxoethoxy) phosphono) -L-alanine ester (13 e);

isopropyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (2-methoxy-2-oxoethoxy) phosphono) -L-alanine ester (13 f);

benzyl (((((2R, 3R,5R) -5- (4- (((3- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) propoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (22 a);

benzyl (((((2R, 3R,5R) -5- (4- (((2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) ethoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (22 b);

benzyl (((((2R, 3R,5R) -5- (4- (((4- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) butoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (22 c);

methyl 2- (((4aR,6R,7aR) -6- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentylamido) acetamido) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-hydroxy 4H-tetrahydrofuran [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (34 a);

ethyl 2- (((4aR,6R,7aR) -6- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentylamido) acetamido) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-hydroxy 4H-tetrahydrofuran [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (34 b);

phenyl 2- (((4aR,6R,7aR) -6- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentylamido) acetamido) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-hydroxy 4H-tetrahydrofuran [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (34 c);

3- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) propyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40 a);

2- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) ethyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40 b);

4- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) butyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40 c);

5- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) pentyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40 d).

Unless specifically defined, the compounds and salts provided herein may also contain all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers.

As used herein, "halogen" refers to F, Cl, Br, or I. In some embodiments, the halogen is F, Cl or Br. In some embodiments, halogen is F. In some embodiments, the halogen is Cl. In some embodiments, the halogen is Br. In some embodiments, halogen is I.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Wherein pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; and alkali metal salts or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids, including primarily salts of inorganic acids such as sulfuric, nitric, hydrobromic, phosphoric, hydrochloric, boric, sulfamic and the like; or organic acids such as acetic acid, propionic acid, butyric acid, valproic acid, camphoric acid, capric acid, caproic acid, caprylic acid, suberic acid, carbonic acid, cinnamic acid, glycolic acid, trifluoroacetic acid, adipic acid, pyruvic acid, salicylic acid, methanesulfonic acid, alginic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, stearic acid, lactic acid, citric acid, oxalic acid, malonic acid, succinic acid, pyroglutamic acid, ascorbic acid, aspartic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, hydroxymaleic acid, palmitic acid, cinnamic acid, isobutyric acid, lauric acid, mandelic acid, maleic acid, fumaric acid, malic acid, tartaric acid, sulfanilic acid, 2-acetoxy-benzoic acid, 2-hydroxy-1, 2, 3-propanetricarboxylic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, formic acid, fumaric acid, mucic acid, gentisic acid, Ethylsulfonic acid, phenylmethanesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfinic acid, isethionic acid, ethanedisulfonic acid, 4- (fluorenyl-methoxycarbonylamino) butyric acid, dichloroacetic acid, 1, 2-ethanedisulfonic acid, camphor-10-sulfonic acid, 2, 4-dihydroxybenzoic acid, α -ketoglutaric acid, 1-hydroxy-2-naphthoic acid, p-acetamidobenzoic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, all-trans retinoic acid. Pharmaceutically acceptable salts of the present application can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN) are preferred.

In a second aspect of the present invention, there is provided a method for preparing a compound represented by formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the three, or a pharmaceutically acceptable salt, solvate or deutero-compound thereof, comprising the steps of:

(1) taking a compound Boc-AHPA as a raw material, and performing condensation and hydrolysis demethylation reaction to obtain an intermediate Boc-Bestatin;

(2) gemcitabine or 5' -deoxy-5-fluorocytosine nucleoside is used as a cell poison, and hydroxyl on the cell poison is modified and protected to obtain a modified cell poison;

(3) and (3) condensing and deprotecting the amino end of the modified cell poison and the carboxyl of Boc-Bestatin to obtain a target product.

The modification and protection of hydroxyl on the cytotoxic substance are specifically as follows: the hydroxyl group at the 3 'position of the cytotoxic agent gemcitabine was Boc protected and the hydroxymethyl group at the 5' position was modified by a phosphorylated Protide prodrug. The two hydroxyl groups of the 5' -deoxy-5-fluorocytosine nucleoside are protected by acetylation.

A process for the preparation of a partial compound having the structure of formulae II and V, prepared by the reaction of:

reagents and conditions (a) POCl₃,Et₃N,dry Et₂O,-78℃；(b)Et₃N,dry DCM；(c)(Boc)₂O,Na₂CO₃,1,4-dioxane/H₂O；(d)tert-BuMgCl,THF,rt；(e)EDCI,HOBt,dry DCM；(f)TFA,dry DCM；(g)EDCI,HOBt,dry DCM；(h)1.5N NaOH,MeOH；(i)EDCI,HOBt,dry DCM；(j)dry HCl in EtOAc；1N NaHCO₃.

The compound 1 reacts with phosphorus oxychloride to obtain a compound 2, and the compound 2 is further condensed to obtain an intermediate 4. Gemcitabine warp (Boc)₂And reacting the O with the intermediate 4 after single protection to obtain 6, further condensing with Boc-L-glycine to generate a compound 7, and deprotecting the compound 7 to obtain an amine compound 8. Boc-AHPA (9) is subjected to condensation demethylation protection to obtain a compound 11, and further condensed with 8 to obtain an intermediate 12. 12, removing amino protecting group, adjusting alkali to obtain a target product 13.

The specific conditions for each reaction in the above reaction scheme may be those conventional in the art.

The preparation method of the partial compound with the structures of the formulas II and V can also be prepared by the following reaction:

the reagent and the condition (a) EDCI, HOBt, dry DCM; (b) et (Et)₃N,dry DCM；(c)Et₃N,dry DCE,DMAP,60℃；(d)TFA,dry DCM；(e)EDCI,HOBt,dry DCM,Et₃N；(f)dry HCl in EtOAc。

Condensation of compound 14 with 15 affords intermediate 16, which is further reacted with 17 to afford 18. The intermediate 6 reacts with 18 to give 19, and the deamination protecting group gives the amine compound 20. Boc-AHPA (9) is condensed with 19 to obtain 21, and further Boc protecting group is removed to obtain the target product 22.

A process for the preparation of a partial compound having the structure of formula III, prepared by the reaction of:

reagents and conditions (a) TMSCl, pyridine, -5 ℃; (b) DMTrCl, DMAP,55 ℃; (c) NH (NH)₄F,MeOH,60℃；(d)POCl₃,Et₃N,-15℃；(e)Et₃N,NMI,dry DCM；(f)Et₃SiH,TFA,dry DCM；(g)EDCI,HOBt,dry DCM；(h)TFA,DCM；(i)EDCI,HOBt,dry DCM；(j)TFA,dry DCM；1N NaHCO₃.

Gemcitabine, protected with TMS, is further reacted with DMTrCl to give compound 24. Compound 24 is reacted with intermediate 27 after TMS removal to give 28, which is further deprotected to give intermediate 29. Boc-glycine was condensed with 29 to give 31, Boc protecting group was removed to give 32, which was further condensed with compound 11 to give 33. After compound 33 is deaminated and the base is adjusted, compound 34 is obtained.

A process for the preparation of a partial compound having the structure of formula IV, prepared by the reaction of:

reagents and conditions (a) Et₃N,DCE,60℃；(b)TFA,DCM；(c)EDCI,HOBt,dry DCM；(d)K₂CO₃,MeOH；(e)dry HCl in EtOAc.

Compound 18 is reacted with 35 to give compound 36, which after removal of the Boc protecting group is further condensed with Boc-AHPA to give intermediate 38. Deacetylation and Boc of compound 38 gave the title compound 40.

In a third aspect of the invention, the invention provides an application of a compound shown in formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the optical isomer, the diastereoisomer, the racemate and the mixture, or a pharmaceutically acceptable salt, a solvate and a deuteron thereof in preparing a targeting ligand.

The compound shown in the formula I can be used as a CD13 inhibitor, and accurately delivers the coupled cytotoxic drug to tumor tissues through targeting a tumor tissue surface receptor CD13, so that the effects of attenuation and synergy are achieved.

In a fourth aspect of the present invention, there is provided an application of the compound represented by formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the three, or a pharmaceutically acceptable salt, solvate or deutero-compound thereof in preparing a medicament for preventing or treating a tumor.

Preferably, the tumor comprises: multiple myeloma, lymphoma, leukemia, and solid tumors.

The solid tumor is prostatic cancer, pancreatic cancer, liver cancer, cervical cancer, breast cancer, melanoma, genitourinary system tumor, colorectal cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, intracerebral tumor, brain glioma, gastric cancer, laryngeal cancer, nasopharyngeal cancer, renal cancer, skin cancer, epithelial cell cancer, cholangiocarcinoma, ovarian cancer, nasopharyngeal cancer, bladder cancer, oral cancer, tongue cancer, and human fibrosarcoma.

In a fifth aspect of the present invention, a pharmaceutical composition is provided, wherein the compound represented by formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the three, or a pharmaceutically acceptable salt, solvate or deutero-compound thereof is used as an active ingredient.

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

In a sixth aspect of the present invention, the present invention provides an application of the above pharmaceutical composition in the preparation of a pharmaceutical preparation for treating tumor diseases.

The term "therapeutically effective amount" as used herein means the amount of therapeutic agent required to treat, ameliorate the targeted disease or condition, or to exhibit a detectable therapeutic effect.

The active ingredient may be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. However, it will be understood that the amount of the compound actually administered will generally be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, and the severity of the subject's symptoms, etc.

The invention has the beneficial effects that:

(1) the compound prepared by the invention can be used as a CD13 inhibitor, and accurately delivers the coupled cytotoxic drug to tumor tissues through targeting a tumor tissue surface receptor CD13, thereby playing the role of attenuation and synergy. In addition, the CD13 inhibitor ubenimex is taken as a targeting ligand of a small molecule coupling drug, so that the tumor targeting of the molecule can be mediated, and a cell poison and ubenimex generated by degradation after entering cells can also generate a synergistic effect, thereby providing a new strategy for treating tumors.

(2) The compounds in the structural general formula (I) provided by the invention all show remarkable activity of inhibiting CD13, and the activity of most compounds on CD13 is superior to that of a CD13 inhibitor ubenimex. In vitro multiple tumor cell proliferation inhibition experiments show that the compounds have obvious activity of inhibiting tumor cell proliferation, and the inhibition activity is several to hundreds of nanomoles. In addition, in the research of the in vivo antitumor activity of mice, the representative compound 13b has obvious inhibition activity on H22 liver cancer of the mice, and the growth inhibition activity on H22 of the liver cancer reaches 75%.

Drawings

FIG. 1: 13b stability in human plasma.

FIG. 2: tumor weight comparison graph of mouse liver cancer cell H22 tumor-bearing model.

FIG. 3 shows the tumor images of mouse hepatoma cell H22 tumor-bearing model.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background section, the CD13 small-molecule inhibitor ubenimex clinically plays multiple anti-tumor effects, including tumor immunoregulation, tumor angiogenesis inhibition, drug resistance reversal and the like. The cell poison is the core part of small molecule coupling drug which plays the role of killing tumor, and is called as the 'nuclear warhead' for attacking tumor. Gemcitabine and pentafluorouracil are commonly used cytotoxic agents. Gemcitabine is clinically used for the treatment of pancreatic cancer, liver cancer, etc., but severe drug resistance is developed in long-term use.

Based on the amino peptidase N/CD 13-based small molecule coupling molecule, and a preparation method and application thereof are provided. The ProTide technology is used for modifying gemcitabine, so that the speed-limiting process of gemcitabine for generating a phosphate can be avoided, the self-drug resistance of gemcitabine can be effectively overcome, and the liver targeting is obvious. Therefore, the invention selects gemcitabine ProTide prodrug as more ideal cell poison. Based on the design mode of small molecule coupling drugs, gemcitabine (such as pentafluorouracil) Protide prodrug and analogues thereof are preferably selected as cell poisons, ubenimex is a small molecule ligand targeting CD13, and a small molecule coupling compound targeting CD13 is synthesized by designing a cleavage type connecting chain. The CD13 inhibitor is taken as a targeting ligand of the micromolecule coupling drug, not only can the tumor targeting of the mediated molecule be realized, but also the cell poison and ubenimex generated by degradation after entering the cells can generate the synergistic effect.

The compounds of the present invention may form hydrates or solvates. The person skilled in the art is aware of methods for forming hydrates when compounds are lyophilized together with water or solvates when concentrated in solution with suitable organic solvents.

The invention encompasses pharmaceutical compositions comprising a therapeutic amount of a compound of the invention, and one or more pharmaceutically acceptable carriers and/or excipients. Carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, as discussed in more detail below. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can be in the form of a liquid, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition can be formulated into suppository with conventional binder and carrier such as triglyceride. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose and magnesium carbonate, and the like. Depending on the desired formulation, the formulation may be designed to mix, granulate and compress or dissolve the ingredients. In another approach, the composition may be formulated as nanoparticles and enteric pellets.

The pharmaceutical carrier used may be solid or liquid.

Typical solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. The solid carrier may comprise one or more substances which may act simultaneously as flavouring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrants; it may also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets the active ingredient is mixed with the carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Powders and tablets preferably contain up to 99% active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins.

Typical liquid carriers include syrup, peanut oil, olive oil, water and the like. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, tinctures and sealed compositions. The active ingredient may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or a pharmaceutically acceptable oil or fat. The liquid carrier may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, pigments, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (containing in part additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). The carrier for parenteral administration may also be an oil or fat such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are sterile liquid compositions for parenteral administration. The liquid carrier for the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Sterile solutions or suspension solutions liquid pharmaceutical compositions may be used, for example, for intravenous, intramuscular, intraperitoneal or subcutaneous injection. The injection may be a single push or a gradual infusion, such as intravenous infusion over 30 minutes. The compounds may also be administered orally in the form of liquid or solid compositions.

The carrier or excipient may comprise a time delay material known in the art, such as glyceryl monostearate or glyceryl distearate, and may also comprise a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate, or the like. When the formulation is intended for oral administration, it is recognized that 0.01% tween 80 in phostapg-50 (phospholipid) concentrated with 1, 2-propanediol, a. nattermann & cie.gmbh) is used in the formulation of acceptable oral formulations of other compounds, and may be adapted to the formulation of various compounds of the invention.

A wide variety of pharmaceutical forms can be used in administering the compounds of the present invention. If a solid carrier is used, the preparation may be in the form of enteric coated tablets, enteric pellets or lozenges or troches placed into hard gelatin capsules. The amount of solid carrier varies widely, but is preferably from about 25mg to about 1.0 g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or in a nonaqueous liquid suspension.

Various delivery systems are known and may be used for the administration of compounds or other various formulations including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules, and the like. Methods of introduction include, but are not limited to, cutaneous, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and (generally preferred) oral routes. The compounds may be administered by any convenient or other suitable route, for example by infusion or bolus injection, by absorption through epithelial or mucosal lines (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or by drug-loaded stents and may be administered together with other biologically active agents. Can be administered systemically or locally.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available.

Preparation of the compound of formula II:

example 1: isopropyl ((((2R,3R,5R) -5- (4- (2- ((tert-butoxycarbonyl) amino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -3- ((tert-butoxycarbonyl) oxy) -4, 4-difluorotetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (7 a).

Boc-glycine (207.5mg, 1.19mmol) was dissolved in anhydrous DCM and HOBt (181.3mg, 1.34mmol), EDCI (257.5mg, 1.34mmol) were added under ice-bath and stirred at 0 ℃ for 0.5 h. Subsequently, a mixed solution of 6a (500mg, 0.79mmol) and triethylamine (159.6mg, 1.58mmol) in DCM was added dropwise to the reaction mixture, and the mixture was stirred at room temperature for 16 hours. TLC monitors that all the raw materials completely react, the reaction solution is washed by 1N citric acid, saturated sodium bicarbonate and brine for 3 times, dried by anhydrous sodium sulfate, filtered and evaporated to dryness to obtain a white solid 7a (540mg, yield: 86.5%) which is directly used for the next reaction without purification.¹H NMR(500MHz,DMSO-d₆)δ11.07(s,1H),8.01(dd,J＝26.4,7.7Hz,1H),7.38-7.34(m,2H),7.27-7.15(m,4H),7.09(t,J＝6.0Hz,1H),6.33(t,J＝8.5Hz,1H),6.19-6.08(m,1H),5.27(s,1H),4.92-4.81(m,1H),4.54-4.45(m,1H),4.42-4.30(m,2H),3.81(d,J＝6.0Hz,2H),1.45(s,9H),1.38(s,9H),1.26-1.21(m,4H),1.16-1.14(m,6H).MS(ESI⁺):Calcd for C₃₃H₄₆F₂N₅O₁₃P[M+H]⁺790.28,found 790.45。

Example 2: isopropyl ((((2R,3R,5R) -5- (4- (2-aminoacetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester trifluoroacetate (8 a).

7a (540mg, 0.68mmol) was dissolved in anhydrous DCM and trifluoroacetic acid (2mL) was added dropwise slowly over an ice bath and after addition was slowly warmed to room temperature and stirred for 2 h. After TLC monitoring all the raw materials reacted completely, the solvent was distilled off, and ether was added to precipitate a solid which was filtered off to give a white solid 8a (454mg, yield: 95%) which was used in the next reaction without purification.¹H NMR(500MHz,DMSO-d₆)δ11.46(s,1H),8.34-8.18(m,3H),8.06(dd,J＝26.1,7.6Hz,1H),7.36(dd,J＝14.8,7.3Hz,2H),7.27-7.15(m,3H),6.23(dd,J＝17.2,8.8Hz,1H),6.19-6.10(m,1H),4.89-4.82(m,1H),4.46-4.09(m,5H),3.92(s,2H),3.80(ddd,J＝17.0,10.1,3.1Hz,1H),3.59(t,J＝6.2Hz,1H),1.22(t,J＝7.8Hz,3H),1.17-1.11(m,6H).MS(ESI⁺):Calcd for C₂₅H₃₁F₅N₅O₁₁P[M+H]⁺590.04,found 590.04。

Example 3: isopropyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3- ((tert-butoxycarbonyl) amino) -2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (12a)

11(469.2mg, 1.15mmol) was dissolved in anhydrous DCM and HOBt (186.3mg, 1.38mmol), EDCI (264.5mg, 1.38mmol) were added under ice-bath and stirred at 0 ℃ for 0.5 h. A mixed solution of 8a (677mg, 1.15mmol) and triethylamine (232mg, 2.30mmol) in DCM was added dropwise and stirred at RT for 16 h. TLC monitored that all the starting materials reacted completely, and the reaction solution was washed with 1N citric acid, saturated sodium bicarbonate, brine 3 times, dried over anhydrous sodium sulfate, filtered to dryness, and separated by column to give 12a as a white solid (350mg, yield: 31%).

Example 4: isopropyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester hydrochloride (13a)

12a (120mg,0.12mmol) was dissolved in DCM, saturated ethyl acetate hydrochloride solution (2mL) was added and stirred at room temperature for 1.5h, after TLC monitored that all the starting material had reacted completely, it was concentrated to remove part of the ethyl acetate and a solid precipitated, dispersed with ultrasound, and filtered to give white powder 13a (70mg, yield: 62.5%).¹H NMR(500MHz,DMSO-d₆)δ10.99(s,1H),8.50(t,J＝5.7Hz,1H),8.14(d,J＝7.9Hz,1H),8.07-8.01(m,3H),7.97(d,J＝7.6Hz,1H),7.41-7.10(m,11H),6.21(td,J＝9.2,8.0Hz,1H),6.13(dt,J＝14.8,7.5Hz,1H),4.90-4.81(m,1H),4.45-4.19(m,4H),4.18-4.07(m,2H),4.07-4.00(m,2H),3.96-3.91(m,2H),3.56(s,1H),2.98-2.86(m,2H),1.66(td,J＝13.2,6.7Hz,1H),1.57-1.53(m,2H),1.26-1.19(m,3H),1.16-1.13(m,6H),0.91-0.87(m,6H).¹³C NMR(125MHz,DMSO-d₆)δ173.18,173.01,172.73,171.28,170.69,163.10,154.47,151.07,136.78,130.09(2C),129.90(2C),129.05(2C),127.33,125.07,120.55,120.51,96.53,69.72,68.60,68.46,64.74,54.67,51.64,50.44,50.28,43.54,41.18,35.11,24.56,23.34,22.38,21.88,21.82,20.20,20.15,20.10(m).HRMS(ESI):m/z(M+H)⁺:found 880.3466；Calcd for C₃₉H₅₃ClF₂N₇O₁₂P(M+H)⁺:880.3380。

The same synthetic method was used to obtain compounds 13b, 13c, 13d, 13e and 13 f.

Example 5: benzyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (13b)

White powder 13b, yield: 80 percent.¹H NMR(500MHz,DMSO-d₆)δ8.55(t,J＝5.7Hz,1H),7.97(dd,J＝11.2,7.7Hz,1H),7.89(d,J＝8.3Hz,1H),7.38-7.12(m,15H),6.53(dd,J＝16.4,5.9Hz,1H),6.26-6.15(m,2H),5.13-5.03(m,2H),4.44-4.15(m,4H),4.15-4.06(m,1H),3.98-3.87(m,2H),3.83(d,J＝2.4Hz,1H),3.23(td,J＝7.1,2.7Hz,1H),2.82(dd,J＝13.3,6.7Hz,1H),2.58(dd,J＝13.3,7.5Hz,1H),1.68-1.60(m,1H),1.58-1.48(m,2H),1.29-1.21(m,3H),0.91-0.85(m,6H).¹³C NMR(125MHz,DMSO-d₆)δ173.48(m),172.71,172.47,171.26,171.07,170.68,163.09,154.46,151.04,136.67,136.31,130.08(2C),129.88(2C),129.07(2C),128.85,128.47,128.24,128.20,127.36,125.08,120.56,120.52,68.58,66.44,54.68,51.59,51.43,50.40,50.24,43.52,41.17(t,1C),35.15,29.52,29.23,27.00,25.55,24.56,23.34,22.34,20.05(m).HRMS(ESI):m/z(M+H)⁺:found 928.3465；Calcd for C₄₃H₅₂F₂N₇O₁₂P(M+H)⁺:928.3380。

Example 6: methyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester hydrochloride (13c)

White powderEnd 13c, yield: 75 percent.¹H NMR(500MHz,DMSO-d₆)δ11.00(s,1H),8.50(t,J＝5.8Hz,1H),8.14(d,J＝7.9Hz,1H),8.07-7.93(m,4H),7.40-7.11(m,10H),6.27-6.03(m,2H),4.44-4.37(m,1H),4.44-4.37(m,1H),4.35-4.26(m,3H),4.24-4.08(m,4H),4.05-4.01(m,2H),3.91-3.84(m,2H),3.60-3.53(m,3H),2.98-2.84(m,2H),1.67(dt,J＝13.3,6.7Hz,1H),1.59-1.50(m,2H),1.25-1.21(m,3H),0.91-0.87(m,6H).¹³C NMR(125MHz,DMSO-d₆)δ174.04(m),172.74,172.48,171.37,171.28,171.09,170.69,163.10,154.49,151.04,136.78,130.10(2C),129.90(2C),129.05(2C),127.33,125.08,120.55(m,2C),96.56,95.40,68.60,64.63,54.67,52.39,51.64,51.49,50.27,50.12,43.55,41.17(t,1C),35.11,24.57,23.35,22.37,20.08(m).HRMS(ESI):m/z(M+H)⁺:found 852.3151；Calcd for C₃₇H₄₉ClF₂N₇O₁₂P(M+H)⁺:852.3067。

Example 7: cyclohexyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester hydrochloride (13d)

White powder 13d, yield: 74 percent.¹H NMR(500MHz,DMSO-d₆)δ11.00(s,1H),8.49(t,J＝5.3Hz,1H),8.14(d,J＝7.9Hz,1H),8.07-7.97(m,4H),7.40-7.07(m,10H),6.25-6.09(m,2H),4.63(d,J＝5.6Hz,1H),4.44-4.10(m,5H),4.05-3.99(m,2H),3.96-3.89(m,2H),3.87-3.67(m,6H),2.99-2.81(m,2H),1.75-1.67(m,2H),1.66-1.59(m,2H),1.57-1.51(m,2H),1.46-1.40(m,1H),1.36-1.27(m,3H),1.23(t,J＝8.0Hz,3H),0.91-0.86(m,6H).¹³C NMR(125MHz,DMSO-d₆)δ173.01(m),172.72,172.48,171.37,171.27,171.09,170.68,163.11,154.48,151.05,136.77,130.09(2C),129.90(2C),129.05(2C),127.33,125.06,120.55(m,2C),96.52,95.42,72.96,68.60,64.75,54.67,51.63,51.48,50.47,50.31,43.54,41.19(t,1C),35.11,31.28,31.23,25.26,24.56,23.40,23.35,22.35,20.19(m).HRMS(ESI):m/z(M+H)⁺:found 920.3772。

Example 8: benzyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (2-methoxy-2-oxoethoxy) phosphono-L-alanine ester (13e)

White powder 13e, yield: 85 percent.¹H NMR(500MHz,DMSO-d₆)δ8.48(s,1H),8.07(dd,J＝22.6,7.6Hz,2H),7.41-7.20(m,12H),6.52(dd,J＝12.4,6.0Hz,1H),6.22(d,J＝6.8Hz,1H),5.97-5.81(m,1H),5.12(q,J＝12.5Hz,2H),4.56-4.42(m,2H),4.38-4.25(m,2H),4.25-4.13(m,2H),4.13-4.04(m,1H),4.01-3.86(m,3H),3.66(d,J＝5.2Hz,3H),3.53-3.64(m,1H),2.96-2.86(m,1H),2.85-2.72(m,1H),1.67-1.45(m,4H),1.35(s,3H),1.30(d,J＝7.0Hz,3H),0.92-0.82(m,6H).¹³C NMR(125MHz,DMSO-d₆)δ173.74(m),171.47,170.67,168.95,163.12,154.51,139.63,136.99,136.33,129.85(2C),129.04(2C),128.86(2C),128.48,128.23(2C),127.27,125.34,122.91,107.42,96.58,79.25,68.98,66.47,62.39(m),54.82,52.37,51.55,50.09(m),43.54,41.19,35.69,34.82,30.87,24.57,23.35,22.28,20.08(m).HRMS(ESI):m/z(M+H)⁺:found 924.3361；Calcd for C₄₀H₅₂F₂N₇O₁₄P(M+H)⁺:924.3278。

Example 9: isopropyl ((((2R,3R,5R) -5- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetamido) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (2-methoxy-2-oxoethoxy) phosphono) -L-alanine ester (13f)

White colourPowder 13f, yield: 78 percent.¹H NMR(500MHz,DMSO-d₆)δ8.48(s,1H),8.08(dd,J＝19.1,7.1Hz,2H),7.33-7.27(m,6H),6.87(s,1H),6.58-6.46(m,1H),6.23(s,1H),5.87-5.73(m,1H),4.95-4.82(m,1H),4.62-4.47(m,2H),4.40-4.18(m,4H),4.11(s,1H),4.05-3.87(m,3H),3.79-3.63(m,4H),3.00-2.87(m,1H),2.86-2.74(m,1H),1.75-1.45(m,4H),1.35(s,4H),1.31-1.23(m,3H),1.22-1.13(m,5H),1.00-0.71(m,6H).¹³C NMR(125MHz,DMSO-d₆)δ173.42(m),172.76,171.49,170.68,168.94(m),163.14,154.51,151.90,139.63,137.02,129.85(2C),129.03(2C),127.26,125.34,96.57,79.26,69.04,68.48,62.449(m),54.83,52.37,51.53,50.13,50.06,43.52,41.20,35.78,34.82,30.87,24.57,23.36,22.28,21.87,20.11(m).HRMS(ESI):m/z(M+H)⁺:found 876.3347；Calcd for C₃₆H₅₂F₂N₇O₁₄P(M+H)⁺:876.3278。

Preparation of another class of compounds in formula II:

example 10: tert-butyl (S) - (1- ((3-hydroxypropyl) amino) -4-methyl-1-oxopentyl-2-yl) carbamate (16a)

Compound 14(6.0g, 25.95mmol) was dissolved in anhydrous DCM and after addition of TBTU (10g, 31.44mmol) and triethylamine (3.94g, 38.91mmol) at 0 ℃ and stirring at 0 ℃ for 30min, compound 15a (3g, 38.91mmol) was added and stirring was carried out at room temperature for 12 h. TLC monitors that all raw materials completely react, an organic layer is washed by 1N citric acid, saturated sodium bicarbonate and brine for 3 times, dried by anhydrous sodium sulfate, filtered and evaporated to dryness, and purified by column chromatography to obtain oily liquid 16a (6.94g, yield: 80.2%). 1H NMR (400MHz, Chloroform) δ 6.64(s,1H),4.96(s,1H),4.06-4.05(m,1H),3.66-3.54(m,2H),3.53-3.39(m,2H),3.37(s,1H),1.66(tq, J ═ 11.4,5.8Hz,4H),1.53-1.46(m,1H),1.42(s,9H),0.97-0.88(m, 6H).

Example 11: tert-butyl (S) - (4-methyl-1- ((3- (((4-nitrophenoxy) carbonyl) oxy) propyl) amino) -1-oxopentyl-2-yl) carbamate (18a)

16a (2.1g, 7.28mmol) was dissolved in anhydrous DCM and triethylamine (0.92g, 9.1mmol) and 17(3.04g, 8.74mmol) were added and stirred at RT for 12 h. TLC monitors that all raw materials are completely reacted, and an organic layer is washed for 3 times by 1N citric acid, saturated sodium bicarbonate and saturated saline in sequence, dried by anhydrous sodium sulfate, filtered and evaporated to dryness, and purified by column chromatography to obtain oily liquid 18a (2.5g, yield 75.8%).¹H NMR(400MHz,DMSO-d₆)δ8.32(d,J＝9.1Hz,2H),7.93(t,J＝5.6Hz,1H),7.56(d,J＝9.1Hz,2H),6.84(d,J＝8.2Hz,1H),4.24(t,J＝6.3Hz,2H),3.90(dd,J＝14.4,8.6Hz,1H),3.24-3.08(m,2H),1.91-1.71(m,2H),1.62-1.52(m,1H),1.46-1.31(m,11H),0.85(t,J＝7.2Hz,6H)。

Example 12: benzyl ((((2R,3R,5R) -5- (4- (((3- ((S) -2- ((tert-butoxycarbonyl) amino) -4-methylpentanoylamino) propoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -3- ((tert-butoxycarbonyl) oxy) -4, 4-difluorotetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (19a)

Compound 18a (900mg,2mmol), 6(1088mg,1.6mmol) and triethylamine (202mg,2mmol) were dissolved in DCE, followed by addition of DMAP (192mg,1.6mmol) to the reaction solution, which was stirred at 60 ℃ for 12 h. After completion of the reaction monitored by TLC, DCE was evaporated, the residue was extracted with ethyl acetate, the organic layer was washed with saturated brine 3 times, dried over anhydrous sodium sulfate, filtered to dryness, and purified by column chromatography to give an oily liquid 19a (636.4mg, yield 40%). 1H NMR (400MHz, DMSO-d6) δ 10.88(s,1H),7.92(dd, J ═ 18.0,7.7Hz,1H),7.82(t, J ═ 5.7Hz,1H),7.40-7.21(m,7H),7.19-7.10(m,3H),7.09-6.99(m,1H),6.80-6.78(m,1H),6.29-6.28(m,1H),6.20(d, J ═ 4.8Hz,1H),5.22(s,1H),5.11(s,2H),4.40(d, J ═ 7.2Hz,1H),4.37-4.22(m,2H),3.97-3.76(m,2H),3.34(d, J ═ 3.6.6H, 1H), 1H, 4.9-6.9H, 1H), 1H, 6.9-6.9, 6.6.6.6H),1.33(s,10H),1.23(d,J＝7.1Hz,3H),0.81(d,J＝9.0Hz,6H)。ESI MS:995.99([M+H]⁺)。

example 13: benzyl ((((2R,3R,5R) -5- (4- (((3- ((S) -2-amino-4-methylpentanoylamino) propoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (20a)

19a (636mg, 0.64mmol) was dissolved in anhydrous DCM and trifluoroacetic acid (2mL-3mL) was added dropwise slowly under ice-bath, after addition was slowly warmed to room temperature and stirred for 2 h. TLC monitoring raw materials all reacted completely, evaporating solvent, adding ether to precipitate solid, filtering to obtain white solid 20a (600mg, yield: 93%), and directly using in next reaction without purification.

Example 14: benzyl ((((2R,3R,5R) -5- (4- (((3- ((S) -2- ((2S,3R) -3- ((tert-butoxycarbonyl) amino) -2-hydroxy-4-phenylbutanamide) -4-methylpentanoylamino) propoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (21a)

9(212.5mg, 0.72mmol) was dissolved in anhydrous DCM and HOBt (116.6mg, 0.86mmol) and EDCI (164.3mg, 0.86mmol) were added under ice-bath and stirred at 0 ℃ for 0.5 h. A mixed solution of 20a (600mg, 0.6mmol) and triethylamine (86.9mg, 0.86mmol) in DCM was added dropwise and stirred at RT for 16 h. TLC monitored complete reaction of all starting materials, and the reaction solution was washed 3 times with 1N citric acid, saturated sodium bicarbonate, brine, dried over anhydrous sodium sulfate, filtered to dryness, and separated by column to give 21a (327.7mg, yield: 51%) as a white solid.

Example 15: benzyl ((((2R,3R,5R) -5- (4- (((3- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) propoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (22a)

21a (327mg,0.31mmol) was dissolved in DCM, saturated ethyl acetate hydrochloride solution (2mL) was added and stirred at room temperature for 1.5h, TLC monitored complete reaction of starting material, concentrated to remove part of ethyl acetate, solid precipitated, dispersed with ultrasound, filtered to give 22a as white powder (265.5mg, yield: 85%).¹H NMR(400MHz,DMSO-d₆)δ10.89(s,1H),8.18(t,J＝5.4Hz,1H),8.05(d,J＝8.0Hz,1H),7.99-7.91(m,4H),7.39-7.26(m,13H),7.18(dd,J＝15.6,8.1Hz,3H),7.07(dd,J＝14.6,7.5Hz,1H),6.32-6.13(m,2H),5.16-4.99(m,2H),4.43-4.18(m,4H),4.09(t,J＝6.0Hz,3H),4.01-3.88(m,2H),3.19-3.08(m,2H),2.93(dd,J＝13.4,8.1Hz,1H),2.83(dd,J＝13.8,6.4Hz,1H),1.79-1.66(m,2H),1.61-1.40(m,3H),1.26(t,J＝7.3Hz,4H),0.87(dd,J＝8.9,6.5Hz,6H).LC-MS(ESI):m/z(M+H)⁺:found673.25；Calcd for C₄₅H₅₇ClF₂N₇O₁₃P(M+H)⁺:972.36。

The same synthetic procedure was used to give compounds 22b and 22 c.

Example 16: benzyl ((((2R,3R,5R) -5- (4- (((2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) ethoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (22b)

White powder 22b, yield: 88 percent.¹H NMR(400MHz,DMSO-d₆)δ10.87(s,1H),8.27(t,J＝5.3Hz,1H),8.05(d,J＝8.2Hz,1H),7.98-7.90(m,4H),7.38-7.26(m,13H),7.23-7.15(m,3H),7.11-7.02(m,1H),6.70-6.46(m,1H),6.30-6.14(m,2H),5.14-5.04(m,2H),4.41-4.21(m,4H),4.20-4.05(m,4H),4.03-3.87(m,3H),3.31-3.20(m,2H),2.97-2.87(m,1H),2.83(dd,J＝14.2,6.3Hz,1H),1.63-1.38(m,3H),1.30-1.20(m,3H),0.84(t,J＝5.6Hz,6H).LCMS(ESI):m/z(M+H)⁺:found 958.25；Calcd for C₄₄H₅₅ClF₂N₇O₁₃P(M+H)⁺:958.35。

Example 17: benzyl ((((2R,3R,5R) -5- (4- (((4- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) butoxy) carbonyl) amino) -2-oxopyrimidin-1 (2H) -yl) -4, 4-difluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphono) -L-alanine ester (22c)

White powder 22c, yield: 76 percent.¹H NMR(400MHz,DMSO-d₆)δ10.88(s,1H),8.15(t,J＝5.5Hz,1H),8.03(d,J＝8.1Hz,1H),7.97-7.91(m,4H),7.41–7.25(m,13H),7.18(dd,J＝15.5,8.0Hz,3H),7.12-7.04(m,1H),6.29–6.16(m,2H),5.14-5.03(m,2H),4.41-4.17(m,4H),4.15–4.05(m,3H),4.03-3.88(m,3H),3.40–3.35(m,2H),3.06(td,J＝13.1,6.6Hz,2H),2.93(dd,J＝13.8,8.0Hz,1H),2.87–2.77(m,1H),1.63-1.52(m,3H),1.52-1.39(m,3H),1.26(t,J＝7.4Hz,3H),0.94–0.73(m,6H).LCMS(ESI):m/z(M/2+H)⁺:found 493.66；Calcd for C₄₆H₅₉ClF₂N₇O₁₃P(M/2+H)⁺:493.69。

Preparation of the compound of formula III:

example 18: 4- ((bis (4-methoxyphenyl) (phenyl) methyl) amino) -1- ((2R,4R,5R) -3, 3-difluoro-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) pyrimidin-2 (1H) -one (25)

Gemcitabine hydrochloride (1g, 3.34mmol) was suspended in pyridine. TMSCl (2.17g, 20mmol) was slowly added dropwise to the above reaction system at-5 deg.C with stirringStirring for 1h, heating to room temperature and stirring for 30 min. TLC monitored the reaction complete. DMAP and DMTrCl were directly added, and the system was heated to 55 ℃ for overnight reaction. The solvent was evaporated to dryness, ethyl acetate and water were added, the ethyl acetate layer was washed with 1N citric acid, saturated sodium bicarbonate and saturated brine for 3 times after separation, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give 2.7g of compound 24, which was used directly in the next reaction. Dissolving the above product 24(2.4g) in methanol, adding ammonium fluoride (377.4mg, 10.2mmol), heating to 55-60 deg.C for reaction for 30min, monitoring reaction completion, evaporating organic solvent, adding DCM and water for washing, and purifying by column chromatography to obtain compound 25(1.2g, yield: 63.8%).¹H NMR(500MHz,DMSO-d₆)δ8.52(s,1H),7.59(d,J＝7.6Hz,1H),7.27(t,J＝7.5Hz,2H),7.23-7.15(m,3H),7.13(d,J＝8.9Hz,4H),6.84(d,J＝8.7Hz,4H),6.29(d,J＝7.6Hz,1H),6.20(d,J＝6.6Hz,1H),5.98(t,J _H-2F＝8.2Hz,1H,),5.14(t,J＝5.2Hz,1H),4.16-4.05(m,1H),3.72(d,J＝12.7Hz,9H),3.63-3.51(m,1H).MS(ESI⁺):Calcd for C₃₀H₂₉F₂N₃O₆ 565.20,found[M+Na]⁺588.07。

Example 19: methyl 2- (((4aR,6R,7aR) -6- (4- ((bis (4-methoxyphenyl) (phenyl) methyl) amino) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-oxotetrahydro-4H-furo [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (28a)

Phosphorus oxychloride (337mg,2.2mmol) was dissolved in anhydrous DCM and placed in cold hydrazine at-15 ℃. Methyl glycolate and triethylamine were dissolved in anhydrous DCM and slowly added dropwise to the reaction. After stirring the reaction for 3h, a white precipitate was produced. Filtering for later use. 25(360mg,0.64mmol) and triethylamine were dissolved in DCM and the filtrate of the filtered phosphate solution was added dropwise slowly and after the addition was complete it was stirred at RT for 1-2 h. TLC assay and column chromatography purification yielded compound 28a (200mg, 44.7% yield).¹H NMR(400MHz,DMSO-d₆)δ8.67(s,1H),7.79-7.64(m,1H),7.31-7.24(m,2H),7.21-7.18(m,3H),7.12(d,J＝8.8Hz,4H),6.88(d,J＝8.8Hz,4H),6.45-6.25(m,2H),5.42-4.94(m,1H),4.87-4.62(m,4H),4.49-4.25(m,1H),3.74-3.71(m,9H).MS(ESI^-):Calcd for C₃₃H₃₂F₂N₃O₁₀P 699.18,found[M-H]^-698.19。

Example 20: methyl 2- (((4aR,6R,7aR) -6- (4-amino-2-oxypyrimidin-1 (2H) -yl) -7, 7-difluoro-2-oxotetrahydro-4H-furo [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (29a)

28a (560mg,0.8mmol) was dissolved in anhydrous DCM, triethylsilane (650mg,5.6mmol) was added and trifluoroacetic acid (912mg,8mmol) was slowly added dropwise at room temperature, the system turned red, and the red color slowly faded after reaction for 30 min. TLC detection of the complete reaction of the raw materials and generation of more polar spots, evaporation of the solvent and purification by silica gel column chromatography gave compound 29a (200mg, yield 63%).¹H NMR(400MHz,DMSO-d₆)δ8.78(s,1H),8.45(d,J＝10.0Hz,1H),8.07-7.87(m,1H),6.42(s,1H),6.06-5.93(m,1H),5.50-5.13(m,1H),4.86-4.78(m,3H),4.73-4.63(m,1H),4.55-4.49(m,1H),4.42-4.30(br,1H),3.83-3.60(m,3H).MS(ESI⁺):Calcd for C₁₂H₁₄F₂N₃O₈P 397.05,found[M+H]⁺397.85。

Example 21: methyl 2- (((4aR,6R,7aR) -6- (4- (2- ((tert-butoxycarbonyl) amino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-oxotetrahydro-4H-furo [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (31a)

Boc-glycine (262mg,1.5mmol) was dissolved in anhydrous DCM and EDCI (325mg,1.7mmol) and HOBt (607mg,1.7mmol) were added under ice-bath to react for 30min, 29a (397mg,1mmol) and triethylamine (303mg,3mmol) were added and the reaction was stirred at room temperature for 3 h. After the reaction is completed, the organic layer is used1N citric acid, saturated sodium bicarbonate, and saturated brine were washed 3 times, dried over anhydrous sodium sulfate, filtered, evaporated to dryness to give an oil, and purified by column chromatography to give compound 31a (200mg, yield 36%).¹H NMR(400MHz,DMSO-d₆)δ11.14(s,1H),8.38-8.07(m,1H),7.26-7.23(m,1H),7.11(t,J＝5.9Hz,1H),6.46(s,1H),5.54-5.10(m,1H),4.91-4.68(m,4H),4.61-4.36(m,1H),3.80(d,2H),3.72(s,3H),1.38(s,9H).MS(ESI⁺):Calcd for C₁₉H₂₅F₂N₄O₁₁P 554.12,found[M+H]⁺554.98。

Example 22: methyl 2- (((4aR,6R,7aR) -6- (4- (2-aminoacetamido) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-oxotetrahydro-4H-furo [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate trifluoroacetate (32a)

31a (200mg, 0.36mmol) was dissolved in anhydrous DCM and trifluoroacetic acid (2-3mL) was added. After stirring at room temperature for 1.5h, the solvent was evaporated and diethyl ether was added to precipitate 200mg of white solid 32a, which was used directly in the next reaction.

Example 23: methyl 2- (((4aR,6R,7aR) -6- (4- ((6R,7S,10S) -6-benzyl-7-hydroxy-10-isobutyl-2, 2-dimethyl-4, 8, 11-trioxy-3-oxa-5, 9, 12-triazatetram-4-amine) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-oxotetrahydro-4H-furo [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (33a)

Boc-Bestatin (159mg,0.39mmol) was dissolved in anhydrous DCM and EDCI (75mg,0.39mmol), HOBt (53mg,0.39mmol) and NMM (78.8mg,0.78mmol) were added under ice-bath and stirred for 30 min. 33a (150mg,0.26mmol) was dissolved in DMF and then added to the reaction solution, which was stirred at room temperature for 14 h. After completion of the reaction, DCM was added to dilute the reaction mixture, washed with water, 0.5N citric acid, saturated sodium bicarbonate and saturated brine 3 times, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give an oil, which was purified by column chromatography to give 33a (125mg, yield 57%) as a white solid.

Example 24: methyl 2- (((4aR,6R,7aR) -6- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-hydroxy-4H-tetrahydrofuran [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (34a)

33a (120mg,0.142mmol) was dissolved in anhydrous DCM and trifluoroacetic acid (2mL) was added. Stirring at room temperature for 1-2h, and evaporating solvent after the raw materials completely react. A small amount of THF was added to dissolve the product, which was then adjusted to basic pH with saturated sodium bicarbonate, extracted with ethyl acetate, dried directly over anhydrous sodium sulfate, filtered and evaporated to dryness to give a white solid 34a (70mg, 66.7% yield).¹H NMR(400MHz,DMSO-d₆)δ8.59(t,J＝5.3Hz,1H),8.29-8.23(m,1H),7.85(d,J＝8.5Hz,1H),7.35-7.14(m,8H),6.46(s,1H),5.50-5.13(m,1H),4.90-4.69(m,5H),4.66-4.51(m,1H),4.39-4.34(m,2H),3.98-3.91(m,2H),3.79(d,J＝2.3Hz,1H),3.72(d,3H),3.20-3.12(m,1H),2.79(dd,J＝13.3,6.5Hz,1H),2.57-2.52(m,1H),1.69-1.40(m,4H),0.93-0.77(m,6H).MS(ESI⁺):Calcd for C₃₀H₃₉F₂N₆O₁₂P 744.23,found[M+H]⁺745.33。

The same procedure was used to give compounds 34b and 34 c.

Example 25: ethyl 2- (((4aR,6R,7aR) -6- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-hydroxy-4H-tetrahydrofuran [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (34b)

Yield of white solid 34b was 70%.¹H NMR(400MHz,DMSO-d₆)δ8.50(s,1H),8.35-8.21(m,1H),8.11(d,J＝7.5Hz,1H),7.85-7.70(m,1H),7.60-7.49(m,1H),7.39-7.08(m,6H),6.45(s,1H),4.89-4.63(m,4H),4.52-4.27(m,2H),4.19(dd,J＝13.8,6.8Hz,2H),3.52-3.44(m,1H),3.97(s,3H),2.99-2.85(m,1H),2.83-2.71(m,1H),1.69-1.42(m,3H),1.27-1.16(m,3H),0.97-0.77(m,6H).MS(ESI⁺):Calcd for C₃₁H₄₁F₂N₆O₁₂P 758.25,found[M+H]⁺759.47。

Example 26: phenyl 2- (((4aR,6R,7aR) -6- (4- (2- ((S) -2- ((2S,3R) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) acetylamino) -2-oxopyrimidin-1 (2H) -yl) -7, 7-difluoro-2-hydroxy-4H-tetrahydrofuran [3,2-d ] [1,3,2] dioxolen-2-yl) oxy) acetate (34c)

Yield of white solid 34c was 72%.¹H NMR(400MHz,DMSO-d₆)δ8.58(s,1H),8.27(s,1H),7.92(s,1H),7.52-7.01(m,10H),6.45(s,1H),5.22(s,2H),5.00-4.62(m,3H),4.37(s,2H),4.04-3.71(d,3H),2.81(s,2H),2.58(s,1H),1.78-1.10(m,9H),0.87(s,7H).MS(ESI⁺):Calcd for C₃₆H₄₃F₂N₆O₁₂P 820.26,found[M+H]⁺821.57。

Preparation of a compound of formula IV:

example 27: (2R,3R,4R,5R) -2- (5-fluoro-4- (((S) -8-isobutyl-12, 12-dimethyl-7, 10-dioxo-2, 11-dioxa-6, 9-diazotridecyl) amino) -2-oxopyrimidin-1 (2H) -yl) -5-methyltetrahydrofuran-3, 4-diacetate (36a)

Compound 18a (5.66g,12.5mmol), 35(3.3g,10mmol) and triethylamine (1.25g,12.5mmol) were dissolved in DCE followed by DMAP (1.2g,10mmol) added to the reaction and the reaction stirred at 60 ℃ for 12 h. TLC monitoring reaction completionAfter that, DCE was distilled off, the residue was extracted with ethyl acetate, the organic layer was washed with saturated brine 3 times, dried over anhydrous sodium sulfate, filtered to dryness, and purified by column chromatography to obtain an oily liquid 36a (2.19g, yield 34%).¹H NMR(400MHz,DMSO-d6)δ10.61(s,1H),8.29(s,1H),7.84(d,J＝5.7Hz,1H),6.79(d,J＝8.3Hz,1H),5.77(d,J＝4.5Hz,1H),5.42(dd,J＝6.3,4.5Hz,1H),5.07(t,J＝6.3Hz,1H),4.07(d,J＝9.2Hz,2H),3.86(td,J＝8.8,5.6Hz,1H),3.10(d,J＝18.9,2H),2.02(s,6H),1.71-1.68(m,2H),1.50-1.47(m,2H),1.33-1.28(m,10H),1.32(d,J＝5.2Hz 3H),0.81(d,J＝9.4,6H).ESI-MS m/z:666.21[M+Na]⁺。

Example 28: (2R,3R,4R,5R) -2- (4- (((3- ((S) -2-amino-4-methylpentanamide) propoxy) carbonyl) amino) -5-fluoro-2-oxopyrimidin-1 (2H) -yl) -5-methyltetrahydrofuran-3, 4-diacetate trifluoroacetate (37a)

35a (2.19g, 3.4mmol) was dissolved in anhydrous DCM and trifluoroacetic acid (2mL-3mL) was added dropwise slowly under ice bath, after the addition was complete, the temperature was slowly raised to room temperature and stirred for 2 h. After TLC monitoring all the raw materials reacted completely, the solvent was distilled off, ether was added to precipitate a solid, which was filtered off to obtain a white solid 37a (2.0g, yield: 90%), which was used in the next reaction without purification.

Example 29: (2R,3R,4R,5R) -2- (4- (((8S,11S,12R) -12-benzyl-11-hydroxy-8-isobutyl-16, 16-dimethyl-7, 10, 14-trioxy-2, 15-dioxa-6, 9, 13-triazaheptadecanoyl) amino) -5-fluoro-2-oxopyrimidin-1 (2H) -yl) -5-methyltetrahydrofuran-3, 4-diacetate (38a)

9(1.21g, 4.1mmol) was dissolved in anhydrous DCM and HOBt (630mg, 5.0mmol) and EDCI (960mg, 5.0mmol) were added under ice-bath and stirred at 0 ℃ for 0.5 h. A mixed solution of 37a (2.15g, 3.28mmol) and triethylamine (414.5mg, 4.1mmol) in DCM was added dropwise and stirred at RT for 16 h. TLC monitored that all the raw materials reacted completely, the reaction solution was washed with 1N citric acid, saturated sodium bicarbonate, brine 3 times, dried over anhydrous sodium sulfate, filtered to dryness, and separated by column to give 38a (1.08g, yield: 40%) as a white solid.

Example 30: (6R,7S,10S) -6-benzyl-7-hydroxy-10-isobutyl-2, 2-dimethyl-4, 8, 11-trioxo-3-oxa-5, 9, 12-triazapentaecyl-15-yl (1- ((2R,3R,4S,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (39a)

38a (500mg, 0.61mmol) was dissolved in methanol and K was added₂CO₃(84mg,0.61mmol), and the reaction mixture was stirred at 25 ℃ for 20 min. The reaction was quenched by addition of HCl/MeOH (1M,0.61mL), the solvent was distilled off at room temperature, and the residue was added with tetrahydrofuran, filtered, concentrated, and isolated on a silica gel column to give compound 39a (395mg, yield: 88%).

Example 31: 3- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) propyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40a)

39a (395mg,0.537mmol) was dissolved in anhydrous DCM and ethyl acetate saturated with hydrochloric acid (2mL) was added. Stirring at room temperature for 1-2h, evaporating solvent after the raw materials completely react, adding ether to precipitate white solid 40a (334mg, yield: 92%).¹H NMR(400MHz,DMSO-d₆)δ8.22(t,J＝5.1Hz,1H),8.07-7.89(m,5H),7.31-7.22(m,5H),5.63(d,J＝3.2Hz,1H),4.22(q,J＝8.0Hz,1H),4.03-3.97(m,5H),3.88-3.82(m,1H),3.65(t,J＝5.2Hz,2H),3.11-3.07(m,3H),2.90(d,J＝7.1Hz,2H),1.71-1.68(m,2H),1.58-1.39(m,3H),1.28(d,J＝5.52Hz,3H),0.85-0.76(m,6H)。ESI-MS m/z:637.15[M+H]⁺。

Compounds 40b, 40c and 40d were synthesized using the same synthetic method.

Example 32: 2- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) ethyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40b)

Yield of white solid 40b was 82%.¹H NMR(400MHz,DMSO-d₆)δ8.24(t,J＝3.8Hz,1H),8.04-8.00(m,5H),7.36-7.26(m,5H),5.68(s,1H),4.30(q,J＝5.0Hz,2H),4.11-4.04(m,5H),3.91(t,J＝4.2Hz,1H),3.71(t,J＝4.0Hz,1H),3.58(s,1H),3.41-3.31(m,3H),2.99-2.87(m,2H),1.62-1.49(m,3H),1.32(d,J＝4.2Hz,3H),0.89-0.85(m,6H)。

Example 33: 4- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) butyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40c)

Yield of white solid 40c was 87%.¹H NMR(400MHz,DMSO-d₆)δ8.18(t,J＝5.2Hz,1H),8.06-7.99(m,5H),7.32-7.20(m,5H),5.62(d,J＝3.1Hz,1H),4.22(q,J＝7.6Hz,1H),4.03-3.97(m,5H),3.88-3.82(m,1H),3.65(t,J＝5.7Hz,1H),3.06-2.96(m,2H),2.90(d,J＝7.1Hz,2H),1.55-1.40(m,7H),1.27(d,J＝6.3Hz,3H),0.85-0.81(m,6H).ESI-MS m/z:651.25[M+H]⁺。

Example 34: 5- ((R) -2- ((2R,3S) -3-amino-2-hydroxy-4-phenylbutylamino) -4-methylpentanoylamino) pentyl (1- ((3R,4R,5R) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (40d)

Yield of white solid 40d 89%.¹H NMR(400MHz,DMSO-d₆)δ8.15(t,J＝5.2Hz,1H),8.07-7.99(m,5H),7.32-7.22(m,5H),5.63(d,J＝3.3Hz,1H),4.22(q,J＝7.7Hz,1H),4.04-3.97(m,5H),3.88-3.82(m,1H),3.65(t,J＝5.1Hz,2H),3.03-2.93(m,2H),2.89(d,J＝7.2Hz,2H),1.56-1.33(m,7H),1.27(d,J＝6.28Hz,5H),0.85-0.80(m,6H).ESI-MS m/z:665.27[M+H]⁺。

Example 35: activity of the target compound on CD 13:

the CD13 enzyme and the substrate L-leucyl-p-nitroaniline were purchased from Sigma

Preparation of phosphate buffer solution: mixing Na₂HPO₄.12H₂O (12.89g) and NaH₂PO₄.2H₂O (2.18g) was dissolved in freshly boiled and chilled distilled water to 1000mL to give a 50mM phosphate buffer solution of pH 7.2, and the solution was allowed to stand at room temperature for further use.

The CD13 enzyme was dissolved in the buffer to prepare a solution of 0.1IU/mL for use.

The substrate was dissolved in DMSO to prepare a solution having a concentration of 16mmol/mL, and each solution was stored in a refrigerator for use.

The positive control and the target compound were prepared in different concentration gradient solutions with buffer.

Add target compound 40. mu.L, substrate 5. mu.L, APN enzyme solution 10. mu.L of different gradient concentration into 96-well plate, make up 200. mu.L with phosphate buffer solution. The 100% group contained no inhibitor, 5. mu.L of substrate only, 10. mu.L of APN enzyme solution, and was buffered to 200. mu.L. The blank contained no enzyme and no inhibitor, only 5. mu.L of substrate, and was buffered to 200. mu.L. Incubation was carried out at 37 ℃ for 0.5h and the absorbance was measured at a wavelength of 405 nm. The inhibition rate of the compound is calculated according to the following formula:

based on the concentration of the compound and the corresponding inhibition ratio, Origi was usedn7.5 software fitting curve, calculating to obtain IC of the tested compound₅₀。

TABLE 1 in vitro CD13 inhibitory Activity (IC) of the Compounds of interest₅₀:μM)

^aThe values in the table are the average of three tests, SD<20％。

The data in Table 1 show that the designed and synthesized compounds have significant activity of inhibiting CD13, and the inhibition activity is significantly better than that of the positive drug ubenimex. The compounds show a certain targeting effect on CD 13.

Example 36: determination of the in vitro cell proliferation-inhibiting Activity of the Compounds of interest:

the test of the in vitro cell proliferation inhibiting activity of the target compound adopts a resazurin method. Human multiple myeloma MM.1S and U266 were collected and cultured conventionally. Cells in logarithmic growth phase were used for all experiments. Counting the cell number of the cell suspension under an inverted microscope, adding culture medium to regulate the cell number to 1 × 10⁵and/mL. Taking a 96-well cell culture plate for cell inoculation and drug experiments, setting a blank control group, a negative control group, a positive control group and a drug experiment group without using peripheral holes (filling sterile PBS), wherein the blank control group is only added with 150 mu L/hole of cell culture solution, the negative control group is only added with 100 mu L/hole of cell suspension and is added with 50 mu L/hole of cell culture solution, the positive control group is only added with 100 mu L/hole of cell suspension and is added with 50 mu L/hole of positive control drug (NUC 1031, ubenimex, NUC1031 and ubenimex (1:1) respectively) solution, the drug experiment group is added with 100 mu L/hole of cell suspension and is added with 50 mu L/hole of test compound (13a, 13b, 13c, 13d, 13e, 13f, 22b, 22a, 34b and 34c respectively), the positive control group and the drug experiment group are respectively set with 8 different drug final concentrations: 0.03, 0.1, 0.3, 1,3, 10, 30, 100. mu. mol. L^-1Each drug concentration is provided with 3 parallel multiple holes. After the drug addition was complete, 96-well cell culture plates were incubated at 37 ℃ with 5% CO₂And saturated moistureAfter incubation for 72h, 10. mu.L of Resazurin (1mg/mL) was added to each well and incubation continued for 3h, the fluorescence was measured at ex.560/em.590nm using a microplate reader. Calculating inhibition, GI₅₀The inhibition ratios of 8 different concentrations are obtained through software fitting, and the obtained results are shown in tables 2-3.

Table 2: results of proliferation inhibitory Activity of target Compound on multiple myeloma cell MM1.S (GI)₅₀，μM^a)

^aThe values in the table are the average of three tests, SD<20％。

Table 3: results of proliferation inhibitory Activity of target Compound on multiple myeloma cell MM1.S (GI)₅₀，μM^a)

^aThe values in the table are the average of three tests, SD<20％。

The data in the above table show that the tested

target compounds

13a, 13b, 13c, 13d, 13e, 13f, 22b, 22a, 22c, 34a, 34b and 34c all show significant inhibitory activity on prostatic cancer 22RV1 and multiple myeloma MM1.S, and the inhibition IC of partial compounds₅₀At low nanomolar levels. In addition, the inhibition activity of 13e, 13f, 34a, 34b and 34c is better than that of positive medicines ubenimex and NUC-1031. The inhibitory activity of the compound 13b on multiple myeloma MM1.S is not only superior to that of two parent drugs of ubenimex and NUC-1031, but also remarkably superior to that of a ubenimex and NUC-1031 combined drug group, and the design of a small molecule coupling molecule 13b shows that remarkable coordination and synergism can be achieved.

Example 37: preliminary stability of the target compound in human plasma:

1. experimental materials and protocol: agilent 1260 high performance liquid chromatograph (Agilent technologies, Inc.), Agilent 1260 ultraviolet visible detector (Agilent technologies, Inc.), high speed centrifuge, Mettler-Torlado AL 104-IC electronic balance (Metler, Switzerland), microporous membrane (organic membrane 0.22 μm), human plasma, chromatographically pure methanol, chromatographically pure acetonitrile.

The test compound was prepared in liquid phase with acetonitrile/water to a concentration of 4mg/mL in water for use, 25. mu.L of the test compound was added to each group, and 225. mu.L of human plasma was added and incubated at 37 ℃. Samples were taken at the respective set time points, 300. mu.L acetonitrile and 300. mu.L aqueous solution were added, vortexed for 30s and centrifuged in a high speed centrifuge at 12,000 rpm. After centrifugation, the supernatant was filtered through a 0.22 μm microporous membrane and 20 μ L of the filtrate was sampled for analysis. The liquid phase detection conditions are as follows: alttima C₁₈Column (specification: 5 μm,4.6 mm. times.250 mm); detection wavelength: 262 nm; the mobile phase is as follows: 25% acetonitrile/75% water (containing 0.1% triethylamine and 0.15% trifluoroacetic acid); the sample injection amount is 20 mu L; the column temperature is 25 ℃, the flow rate is 1mL/min, and the detection time is 15 min.

2. And (3) test results:

as shown in figure 1, compound 13b has good stability in plasma with less than 50% degradation within 24 h.

Example 38: 13a and 13b inhibit the growth of liver cancer H22 mouse transplantable tumors.

(1) Establishment of mouse transplantation tumor model

Materials: kunming mice (female, 4-5 weeks old); mouse hepatoma mice were purchased from the institute of medicine, institute of medical sciences, Shandong province. The breeding is carried out in a constant temperature and humidity environment according to standard requirements, and the light and shade cycle control is carried out for 12 hours. Animal experiments were approved by the laboratory animal Committee of the Weifang medical college and were in compliance with the "guidelines for laboratory animal Care and use" published by the national institutes of health (NIH publication No. 85-23, revised 1996).

Purchasing a certain number of Kunming mice according to the experimental requirements, and placing the Kunming mice in a laboratory for balanced breeding for 1 week. Taking mouse H22 ascites cell suspension liquid to put into a microscopeThen, the cells were counted by a counting plate and the number of the cells was adjusted. Under strict aseptic condition, cells are inoculated into the subcutaneous tissue of right axilla of mouse in the amount of 1.0-2.0X 10⁷0.2 mL/piece. After inoculation, the tumor generation condition of the mice is observed, and when the tumor grows to be close to 100mm³Mice were randomly grouped. The medicine is administered according to the designed administration dosage and route, and is continuously administered for 5 days, stopped for two days and administered for two weeks. Body weights of mice were recorded every 2-3 days throughout the dosing period. Two weeks after administration, the mice were sacrificed by cervical dislocation, tumor tissues were completely detached, tumor masses were weighed, and the tumor inhibition rate was calculated.

The tumor inhibition ratio (%) × 100% (1-mean tumor weight in treatment group/mean tumor weight in control group).

(2) And (3) test results:

as shown in fig. 2 and 3,

compounds

13a and 13b had significant inhibitory activity against the growth of mouse liver cancer cell line H22 transplanted tumors. And the activity of 13a and 13b at the dose of 0.15mmol/kg is equivalent to the anti-tumor activity of the positive drug NUC1031 at the dose of 0.19 mmol/kg.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A compound shown as a formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the optical isomer, the diastereoisomer, the racemate and the racemate, or a pharmaceutically acceptable salt, solvate and deuteron thereof;

wherein: a is selected from NH or O;

Wherein n is 2 to 10;

represents a bond to a carbonyl group; - - -represents a bond to A;

c is

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₂Is selected from

R₃Is selected from

Wherein n is 1-6;

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₇Selected from H, -CH₃or-CH₂CH₃；

X is selected from O or NH;

d is H or F.

2. The compound shown in the formula I, or an optical isomer, a diastereoisomer, a racemate or a mixture of the optical isomer, the diastereoisomer, the racemate and the mixture, or a pharmaceutically acceptable salt, solvate and deuteron of the compound, which is disclosed by the claim 1, is characterized in that the compound shown in the formula I has a structure shown in a general formula II, a general formula III, a general formula IV or a general formula V;

wherein: a is selected from NH or O;

b isCH₂、(CH₂)_n、CHCH₃、OCH₂、O(CH₂)_n、OCH(CH₃)CH₂、OC(CH₃)₂CH₂、NHCH₂、NH(CH₂)_n、NHCHCH₃、NHCH(CH₃)CH₂、NHC(CH₃)₂CH₂、

Wherein n is 2 to 10;

represents a bond to a carbonyl group; - - -represents a bond to A;

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₂Is selected from

R₃Is selected from

Wherein n is 1-6;

R₄、R₅is selected fromH. Halogen, (C)_1-2) Alkyl, halomethyl, OH, OCH₃、O(CH₂)_nCH₃、OC(CH₃)₃、OCH(CH₃)₂Cyclopropyloxy, 5-6-membered alkoxy, NH₂、N(CH₃)₂、NH(CH₂)_nCH₃CN or N₃Wherein n is 0 to 9;

-CH₂CH(CH₃)₂、-C(CH₃)₃、-CHCH₃CH₂CH₃Or C₆H₅-；

R₇Selected from H, -CH₃or-CH₂CH₃；

X is selected from O or NH.

3. The compound of formula I according to claim 1, or an optical isomer, diastereoisomer, racemate or mixture of the three, or a pharmaceutically acceptable salt, solvate or deuteron thereof, wherein the compound of formula I is selected from:

4. A method for preparing a compound of formula I as described in any one of claims 1 to 3, or an optical isomer, diastereoisomer, racemate or mixture of the three, or a pharmaceutically acceptable salt, solvate or deuteron thereof, comprising the steps of:

5. Use of a compound of formula I as defined in any one of claims 1 to 3, or an optical isomer, diastereoisomer, racemate or mixture of the three, or a pharmaceutically acceptable salt, solvate or deuteron thereof, for the preparation of a targeting ligand.

6. Use of a compound of formula I as defined in any one of claims 1 to 3, or an optical isomer, diastereoisomer, racemate or mixture of the three, or a pharmaceutically acceptable salt, solvate or deuteron thereof, for the manufacture of a medicament for the prevention or treatment of a tumor.

7. The use of claim 6, wherein the tumor comprises: multiple myeloma, lymphoma, leukemia, and solid tumors.

8. A pharmaceutical composition comprising as an active ingredient a compound represented by formula I as claimed in any one of claims 1 to 3, or an optical isomer, diastereoisomer, racemate or mixture thereof, or a pharmaceutically acceptable salt, solvate or deutero-derivative thereof.

9. The pharmaceutical composition of claim 8, further comprising one or more pharmaceutically acceptable carriers or excipients.

10. Use of a pharmaceutical composition according to claim 8 or 9 for the preparation of a pharmaceutical preparation for the treatment of a tumor disease.