CN113286594B - Application of pyridopyrimidine compounds in preparation of medicines for treating nasopharyngeal carcinoma - Google Patents

Abstract

The application discloses application of pyridopyrimidine compounds in preparation of medicaments for treating nasopharyngeal carcinoma. In particular to application of a compound shown in a formula (I) or pharmaceutically acceptable salt thereof in preparing a medicament for treating nasopharyngeal carcinoma.

Description

Application of pyridopyrimidine compounds in preparation of medicines for treating nasopharyngeal carcinoma

The present application claims the following priorities:

CN201910049704.0, filing date 2019, month 1 and 18.

Technical Field

The application relates to application of pyridopyrimidine compounds in preparation of medicines for treating nasopharyngeal carcinoma. In particular to application of a compound shown in a formula (I) or pharmaceutically acceptable salt thereof in preparing a medicament for treating nasopharyngeal carcinoma.

Technical Field

Tumors, in particular malignant tumors, are one of the most serious diseases which are harmful to human health at present, and with the progress of technology and the deeper and deeper research on tumor treatment, rapid progress is made in the aspects of tumor occurrence, development mechanism and tumor treatment. Many new mechanisms and biological markers are discovered. The present application relates to a signaling pathway critical for tumor proliferation, invasive metastasis and anti-apoptosis, namely phosphatidylinositol 3 kinase (PI 3K) -AKT-mammalian rapamycin protease mTOR signaling pathway.

Activation of PI3K is largely involved in the substrate near the inner side of its plasma membrane. A variety of growth factors and signaling complexes, including Fibroblast Growth Factor (FGF), vascular Endothelial Growth Factor (VEGF), human Growth Factor (HGF), angiopoietin I (Ang 1), and insulin all initiate PI3K activation processes. These factors activate Receptor Tyrosine Kinases (RTKs), thereby causing autophosphorylation. The result of PI3K activation is the production of the second messenger PIP3 on the plasma membrane, PIP3 binding to the intracellular signaling protein AKT containing the PH domain and PDK1 (phosphoinositide dependent kinase-1), causing Ser308 of the PDK1 phosphorylated AKT protein to result in AKT activation. Other substrates for PDK1 also include PKC (protein kinase C), S6K (p 70S 6) and SGK (serum/glucocorticoid regulated kinases). AKT, also known as Protein Kinase B (PKB), is the primary effector downstream of PI 3K. Activated AKT regulates cellular function by phosphorylating downstream factors such as various enzymes, kinases, and transcription factors. AKT exerts an anti-apoptotic effect by phosphorylating target proteins through a variety of downstream pathways. PTEN (phosphatase and tensin homology deleted on chromosome), an oncogene, is mutated or deleted in a wide range of human tumors. PTEN is a PIP 3-phosphatase that can convert PIP3 to PIP2 by dephosphorylation, as opposed to PI 3K. PTEN may reduce activation of AKT and prevent all downstream signaling events regulated by AKT. mTOR, which is a downstream substrate of AKT, is evolutionarily relatively conserved, can integrate various signals of nutrition, energy and growth factors, and is involved in the biological processes of gene transcription, protein translation, ribosome synthesis, apoptosis and the like, and plays an extremely important role in cell growth. There are two highly homologous complexes in which Tor binds to KOG01 to form mTORC1, mTOR and AVO1/AVO2/AVO 3/and LST8 to form mTORC2.MTOR insensitive to rapamycin regulates downstream protein translation by phosphorylating downstream target protein S40S ribosomal S6 protein kinases such as S6K1 and 4EBP 1. mTOR binds to eIF3, phosphorylates S6K1, and releases S6K1 from eIF3 to be activated, further phosphorylating cellular substrates such as p70S6 to promote protein translation and expression. 4EBP1 binds to eukaryotic transcription initiation factor 4E and inhibits its activity, and when the mtor phosphorylates 4E-BP1, it is separated from eif-4E to realize eukaryotic cell transcription. mTORC2 phosphorylates AKT, thereby upregulating its kinase activity.

As can be seen from the above, any mutation or overexpression of the PI3K/AKT/mTOR signaling pathway upstream leads to a downstream cascade of reactions, ultimately leading to tumor initiation, progression and metastasis. mTOR is at the junction of the signal path, and the inhibition of mTORC1 and mTORC2 can well block the transmission of signals, thereby achieving the purpose of controlling the development of tumors.

The research shows that the signal path is used in various solid tumors, such as nasopharyngeal carcinoma, breast cancer, prostatic cancer, lung cancer, colon cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, renal cancer, thyroid cancer, meningitis cancer, acute and chronic lymphocytic leukemia, meeker cell tumor and the like. And is closely related to treatment tolerance and poor prognosis. Therefore, the inhibition of the signal path of PI3K/AKT/MTOR is realized by developing the fine molecular compound, and the method has good development prospect.

The application aims to find a double mTOR small molecular compound targeted drug, and the compound has good activity and shows excellent effects and actions.

US20170281637 discloses a compound AZD2014, which belongs to mTORC1& mTORC2 kinase inhibitors, having the structural formula shown below:

disclosure of Invention

The application provides application of a compound shown in a formula (I) or pharmaceutically acceptable salt thereof in preparing a medicament for treating nasopharyngeal carcinoma,

wherein the method comprises the steps of

R ₁ Selected from C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _a Substitution;

R ₂ selected from C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _b Substitution;

t is selected from O, S, S (=O) ₂ 、-N(R ₃ ) -and-C (R) ₄ ) ₂ -；

R ₃ Selected from H and C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _c Substitution;

R ₄ selected from H, F, cl, br, I and C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _d Substitution;

R _a 、R _b 、R _c and R is _d Each independently selected from H, F, cl, br and I.

In some aspects of the application, R is as described above ₁ Selected from CH ₃ 、CF ₃ 、CH ₂ CH ₃ 、CF ₂ CH ₃ 、CHFCH ₂ F and CF ₂ CH ₂ F, other variables are as defined herein.

In some aspects of the application, R is as described above ₁ Selected from CH ₃ The other variables are as defined herein.

In some aspects of the present application,r is as described above ₂ Selected from CH ₃ 、CF ₃ 、CH ₂ CH ₃ 、CF ₂ CH ₃ 、CHFCH ₂ F and CF ₂ CH ₂ F, other variables are as defined herein.

In some aspects of the application, R is as described above ₂ Selected from CH ₃ The other variables are as defined herein.

In some aspects of the application, R is as described above ₃ Selected from CH ₃ 、CF ₃ 、CH ₂ CH ₃ 、CF ₂ CH ₃ 、CHFCH ₂ F and CF ₂ CH ₂ F, other variables are as defined herein.

In some aspects of the application, R is as described above ₃ Selected from CH ₃ The other variables are as defined herein.

In some aspects of the application, R is as described above ₄ Are respectively and independently selected from H, F, cl, br, I, CH ₃ 、CF ₃ 、CH ₂ CH ₃ 、CF ₂ CH ₃ 、CHFCH ₂ F and CF ₂ CH ₂ F, other variables are as defined herein.

In some aspects of the application, R is as described above ₄ Each independently selected from H, F, the other variables being as defined herein.

In some aspects of the application, the structural units described aboveSelected from-> The other variables are as defined herein.

Still other embodiments of the present application are combinations of any of the above variables.

The application also provides application of the compound shown in the following formula or pharmaceutically acceptable salt thereof in preparing a medicament for treating nasopharyngeal carcinoma,

the technical effects are as follows:

the compounds of the application have significant and even unexpected mTOR kinase inhibitory activity. The compound has obvious proliferation inhibition activity on MCF-7, N87 and OE-21 cells and has a certain proliferation inhibition activity on HT-29 cells. Compound 1 exhibited the same or even better pharmacokinetic properties as the reference compound. In this experiment, the in vivo efficacy of the compounds of this patent in a human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor model was evaluated. The therapeutic group example 2, 30mg/kg, showed a remarkable tumor-inhibiting effect compared to the solvent control group, comparable to the efficacy of the reference compound AZD2014 15 mg/kg.

Correlation definition

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof. The term "pharmaceutically acceptable" as used herein is intended 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.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application prepared from the compounds of the present application which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present application contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compounds of the present application contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts including acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; also included are salts of amino acids (e.g., arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the application contain basic and acidic functionalities that can be converted to either base or acid addition salts.

Pharmaceutically acceptable salts of the application can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

The compounds of the application may exist in specific geometric or stereoisomeric forms. The present application contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the application. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present application.

Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other.

Unless otherwise indicated, the term "cis-trans isomer" or "geometric isomer" is caused by the inability of a double bond or a single bond of a ring-forming carbon atom to rotate freely.

Unless otherwise indicated, the term "diastereoisomer" refers to stereoisomers of a molecule having two or more chiral centers and having a non-mirror relationship between the molecules.

Unless otherwise indicated, with solid wedge bondsAnd wedge-shaped dotted bond->Representing the absolute configuration of a solid centre, using straight solid keys +.>And straight dotted bond->Representing the relative configuration of the stereo centers, using wavy lines +.>Representing a wedge solid key +.>Or wedge-shaped dotted bond->Or by wave lines->Representing a straight solid line key->And straight dotted bond->

The compounds of the application may be present in particular. Unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to the fact that at room temperature, different functional group isomers are in dynamic equilibrium and are capable of rapid interconversion. If tautomers are possible (e.g., in solution), chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as proton tautomers) (prototropic tautomer) include interconversions by proton transfer, such as keto-enol isomerisation and imine-enamine isomerisation. Valence isomer (valance tautomer) includes the interconversion by recombination of some of the bond-forming electrons. A specific example of where keto-enol tautomerization is the interconversion between two tautomers of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.

Optically active (R) -and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the application is desired, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereomeric resolution is carried out by conventional methods well known in the art, and then the pure enantiomer is recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amine). The compounds of the present application may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds can be labeled with radioisotopes, such as tritium @, for example ³ H) Iodine-125% ¹²⁵ I) Or C-14% ¹⁴ C) A. The application relates to a method for producing a fibre-reinforced plastic composite For another example, deuterium can be substituted for hydrogen to form a deuterated drug, with bonds between deuterium and carbon being stronger than those between normal hydrogen and carbon, with reduced toxicity and side effects compared to non-deuterated drugsHas the advantages of improving the stability of the medicine, enhancing the curative effect, prolonging the biological half-life of the medicine, and the like. All isotopic variations of the compounds of the present application, whether radioactive or not, are intended to be encompassed within the scope of the present application. The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium representative of a carrier capable of delivering an effective amount of the active agents of the present application, which does not interfere with the biological activity of the active agents and which does not have toxic or side effects to the host or patient, including water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. Such matrices include suspending agents, viscosity enhancers, transdermal enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, and may include deuterium and variants of hydrogen, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =o), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on the aromatic group. The term "optionally substituted" means that the substituents may or may not be substituted, and the types and numbers of substituents may be arbitrary on the basis that they can be chemically achieved unless otherwise specified.

When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 to 2R, the group may optionally be substituted with up to two R's, and R's in each case have independent options. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.

When the number of one linking group is 0, such as- (CRR) ₀ -it is meant that the linking group is a single bond.

When one of the variables is selected from a single bond, the two groups to which it is attached are indicated as being directly linked, e.g., when L in A-L-Z represents a single bond, it is indicated that the structure is actually A-Z.

Unless otherwise specified, the term "alkyl" is used to denote a straight or branched saturated hydrocarbon group, which may be monosubstituted (e.g. -CH ₂ F) Or polysubstituted (e.g. -CF) ₃ ) May be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

Unless otherwise specified, the term "C _1-3 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C is _1-3 Alkyl includes C _1-2 And C _2-3 Alkyl groups, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C (C) _1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. Furthermore, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C ₁ -C ₄ ) Alkyl "is intended to include, but is not limited to, trifluoromethyl, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Examples of haloalkyl groups include, but are not limited to, unless otherwise specified: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

Unless otherwise specified, C _n-n+m Or C _n -C _n+m Comprising any one of the specific cases of n to n+m carbons, e.g. C _1-12 Comprises C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₇ 、C ₈ 、C ₉ 、C ₁₀ 、C ₁₁ And C ₁₂ Also included is any one of the ranges from n to n+mFor example C _1-12 Comprises C _1-3 、C _1-6 、C _1-9 、C _3-6 、C _3-9 、C _3-12 、C _6-9 、C _6-12 And C _9-12 Etc.; similarly, n-membered to n+m-membered means that the number of atoms on the ring is n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and any one of n to n+m is also included, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-8-membered ring, 6-10-membered ring, and the like.

The solvent used in the present application is commercially available.

The embodiments of the application relate to neutral separations for high performance liquid chromatography separations.

The application adopts the following abbreviations: aq represents water; HATU represents O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate; EDC represents N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent, equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether; DIAD stands for diisopropyl azodicarboxylate; DMF represents N, N-dimethylformamide; DMSO represents dimethylsulfoxide; etOAc represents ethyl acetate; etOH stands for ethanol; meOH represents methanol; CBz represents benzyloxycarbonyl, an amine protecting group; BOC represents that tert-butylcarbonyl is an amine protecting group; HOAc stands for acetic acid; naCNBH ₃ Represents sodium cyanoborohydride; r.t. stands for room temperature; O/N stands for overnight; THF represents tetrahydrofuran; boc ₂ O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA stands for diisopropylethylamine; SOCl ₂ Represents thionyl chloride; CS (circuit switching) ₂ Represents carbon disulphide; tsOH represents p-toluenesulfonic acid; NFSI represents N-fluoro-N- (benzenesulfonyl) benzenesulfonamide; n-Bu4NF represents tetrabutylammonium fluoride; iPrOH stands for 2-propanol; mp represents the melting point; LDA represents lithium diisopropylamide; pd (PPh) ₃ ) ₄ Represents tetrakis (triphenylphosphine) palladium; IV stands for intravenous injection; PO represents oral administration.

The compounds of the present application are in accordance with routine in the artNaming principles orSoftware naming, commercial compounds are referred to by vendor catalog names.

Detailed Description

The present application is described in detail below by way of examples, but is not meant to be limiting in any way.

Reference example 1

First step

Compound 1-1 (20.0 g,104mmol,1.00 eq) and concentrated ammonia (200 mL,1.45mol,14.0 eq) were sealed in an autoclave and stirred at 130℃for 24 hours at a pressure of about 0.9MPa. The reaction solution was concentrated to obtain compound 1-2.

MS-ESI calculated [ M+H ]] ⁺ 173 and 175, measured 173 and 175.

¹ H NMR(400MHz，DMSO-d ₆ )δ：8.03(d，J＝8.0Hz，1H)，7.56(br s，2H)，6.61(d，J＝8.0Hz，1H)。

Second step

Compound 1-2 (17.0 g,98.5mmol,1.00 eq), ammonium chloride (10.5 g,197mmol,2.00 eq), 1-hydroxybenzotriazole (13.3 g,98.5mmol,1.00 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (18.9 g,98.5mmol,1.00 eq) and diisopropylethylamine (38.2 g, 298 mmol,3.00 eq) were dissolved in N, N-dimethylformamide (200.0 mL). The mixture was stirred at 20℃for 16 hours. After completion of the reaction, the solvent was dried under reduced pressure, water (200 mL), ethyl acetate (200 mL. Times.3) was added, the combined organic phases were dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (1:1 petroleum ether/ethyl acetate, R _f =0.4), to give a compound, and beating ethyl acetate (50 mL) for ten minutes to giveTo compounds 1-3.

¹ H NMR(400MHz，DMSO-d ₆ )δ：7.96(d，J＝8.0Hz，2H)，7.62(br s，2H)，7.40(br s，1H)，6.61(d，J＝8.0Hz，1H)。

Third step

Compounds 1-3 (8.00 g,46.6mmol,1.00 eq) and oxalyl chloride (7.1 g,56.0mmol,4.9mL,1.00 eq) were added sequentially to toluene (200 mL). The mixture was stirred at 110℃for 15 hours. Cooled to room temperature, filtered and dried. Compounds 1-4 were obtained.

¹ H NMR(400MHz，DMSO-d ₆ )δ：8.24(d，J＝8.0Hz，1H)，7.30(d，J＝8.0Hz，1H)。

Fourth step

Compounds 1-4 (6.00 g,30.4mmol,1.00 eq) and diisopropylethylamine (11.8 g,91.1mmol,15.9mL,3.00 eq) were added sequentially to toluene (100 mL). The mixture was stirred at 70℃for half an hour. Cooled to room temperature, phosphorus oxychloride (14.0 g,91.1mmol,8.5mL,3.00 eq) was added dropwise to the mixture. The mixture was stirred at 100℃for 2 hours. Cooled to room temperature, concentrated, and chromatographed (3:1 petroleum ether/ethyl acetate, rf=0.4) to give compounds 1-5.

¹ H NMR(400MHz，DMSO-d ₆ )δ：8.45(d，J＝8.0Hz，1H)，7.63(d，J＝8.3Hz，1H)。

Fifth step

Compounds 1-5 (1.90 g,8.10mmol,1.00 eq), (S) -2-methyl-morpholine (819 mg,8.10mmol,1.00 eq) and diisopropylethylamine (2.09 g,16.2mmol,2.83mL,2.00 eq) were dissolved in dichloromethane (50 mL) and the resulting solution was reacted at 25℃for 2 hours. After the completion of the reaction, the mixture was concentrated and subjected to column chromatography (3:1 petroleum ether/ethyl acetate) to give compounds 1 to 6.

¹ H NMR(400MHz，DMSO-d ₆ )δ：8.47(d，J＝8.8Hz，1H)，7.55(d，J＝8.8Hz，1H)，4.71-4.72(m，1H)，4.12-4.09(m，1H)，3.92-3.91(m，1H)，3.84-3.74(m，1H)，3.73-3.64(m，2H)，3.54-3.53(m，1H)，1.46(d，J＝6.8Hz，3H)。

Sixth step

Compounds 1 to 6 (1.2 g,4.01mmol,1.00 eq), 1 to 7 (1.15 g,4.41mmol,1.10 eq), tetrakis triphenylphosphine palladium (232 mg, 200. Mu. Mol,0.05 eq) and potassium carbonate (1.66 g,12.0mmol,3.00 eq) were dissolved in water (24 mL) and 1, 4-dioxane (120 mL), reacted at 60℃for 5 hours under nitrogen protection, after the reaction was completed, the solvent was concentrated, diluted with water (30 mL) and extracted with ethyl acetate (50 mL. Times.2), the combined organic phases were dried over anhydrous sodium sulfate, filtered, dried under reduced pressure, and column chromatographed (100% ethyl acetate) to give compound 1h.

¹ H NMR(400MHz，DMSO-d ₆ )δ：8.71(s，1H)，8.67(d，J＝4.8Hz，1H)，8.55(d，J＝8.8Hz，1H)，8.39(d，J＝8.0Hz，1H)，8.14(d，J＝8.8Hz，1H)，8.01(d，J＝8.0Hz，1H)，7.68(t，J＝7.6Hz，1H)，4.75(d，J＝6.4Hz，1H)，4.17-4.15(m，1H)，3.94-3.92(m，1H)，3.87-3.77(m，1H)，3.72(s，2H)，3.59-3.57(m，1H)，2.86-2.84(m，3H)，1.49(d，J＝6.8Hz，3H)。

Example 1

First step

Compound 1a (23.0 g,328mmol,24.5mL,1.0 eq) and zinc diiodide (5.20 g,16.4mmol,0.05 eq) were dissolved in 200mL of dichloromethane, the temperature was reduced to 0 ℃, trimethylsilane cyanide (39.1 g,393mmol,49mL,1.2 eq) was added, the reaction was allowed to react at 25℃for 18 hours, the reaction was complete, the reaction was concentrated and 50mL of acetonitrile and 50mL of aqueous hydrochloric acid (1N) were added, and stirring was performed at 25℃for 5 minutes. Ethyl acetate extraction, drying of the organic phase, filtration, concentration and purification of the evaporation residue by silica gel chromatography (petroleum ether/ethyl acetate=100:1-1:1) gives 1b.

¹ H NMR(400MHz，CDCl ₃ )δ：4.30(s，1H)，2.62-2.59(m，2H)，2.33-2.30(m，2H)，1.95-1.79(m，2H)。

Second step

To a solution of lithium aluminum hydride (9.38 g,247mmol,1.5 eq) in tetrahydrofuran (300 mL) at 0deg.C was added 1b (16.0 g,164mmol,1 eq) and reacted at 20deg.C for 18 hours. After completion of the reaction, water (9.38 mL), 15% sodium hydroxide (9.38 mL) and water (28.1 mL) were added in this order, followed by stirring for 15 minutes, filtration and concentration to obtain 1c.

¹ H NMR(400MHz，CDCl ₃ )δ：2.72(s，2H)，2.12-1.76(m，7H)，1.7-1.63(m，1H)，1.49-1.33(m，1H)。

Third step

To a solution of 1c (2.0 g,19.8mmol,1 eq) and diisopropylethylamine (4.0 g,31.0mmol,5.4mL,1.6 eq) in dichloromethane (20.0 mL) at 0deg.C was added chloroacetyl chloride (2.23 g,19.8mmol,1.6mL,1.0 eq). The reaction solution was reacted at 20℃for 2 hours. The reaction was completed, the reaction mixture was concentrated, and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=100:1 to 1:1) to give 1d.

¹ H NMR(400MHz，CDCl ₃ )δ：6.99(s，1H)，4.17-4.02(m，2H)，3.50(d，J＝6.0Hz，2H)，2.71(s，1H)，2.14-1.98(m，4H)，1.81-1.71(m，1H)，1.64-1.49(m，1H)。

Fourth step

Compound 1d (2.2 g,12.4mmol,1 eq) was added to anhydrous tetrahydrofuran (100 mL), the internal temperature was reduced to 0℃and sodium hydride (1.49 g,37.2mmol,60% purity, 3 eq) was added. The reaction mixture was reacted at 20℃for 18 hours, the reaction was completed, water (15.0 mL) was added to the reaction mixture, the mixture was extracted with ethyl acetate (20 mL. Times.3), and the organic phases were combined, dried, filtered and concentrated to obtain 1f.

¹ H NMR(400MHz，CDCl ₃ )δ：6.96(s，1H)，4.15(s，2H)，3.44-3.35(m，2H)，2.27-2.16(m，2H)，2.09-2.01(m，2H)，1.95-1.86(m，1H)，1.74-1.63(m，1H)。

Fifth step

To a solution of lithium aluminum hydride (645 mg,17mmol,2 eq) in tetrahydrofuran (30 mL) at 0deg.C was added 1f (1.2 g,8.5mmol,1 eq) and reacted at 20deg.C for 18 hours. After completion of the reaction, water (0.7 mL), 15% sodium hydroxide (0.7 mL) and water (2.1 mL) were added in this order, followed by stirring for 15 minutes, filtration and concentration to obtain 1g.

¹ H NMR(400MHz，CDCl ₃ )δ：3.61-3.51(m，2H)，2.87-2.76(m，4H)，2.03-1.97(m，4H)，1.86-1.82(m，1H)，1.62-1.54(m，1H)。

Sixth step

1g (53 mg, 414. Mu. Mol,1.1 eq) of the compound, 1h (150 mg, 377. Mu. Mol,1 eq) and DIPEA (48 mg, 377. Mu. Mol, 66. Mu.L, 1 eq) were dissolved in DMSO (4 mL) and the mixture was reacted at 70℃for 18 hours. The reaction is complete, and the reaction liquid is purified by high performance liquid chromatography to obtain the compound 1.

MS-ESI calculated [ M+H ]] ⁺ 489, found 489.

¹ H NMR(400MHz，CDCl ₃ )δ：8.63(s，1H)，8.22(d，J＝7.6Hz，1H)，8.05(d，J＝8.4Hz，1H)，7.97(d，J＝7.6Hz，1H)，7.65-7.41(m，2H)，6.53(br s，1H)，4.40(d，J＝6.8Hz，1H)，4.12-3.95(m，3H)，3.93-3.83(m，4H)，3.83-3.68(m，5H)，3.06(d，J＝4.8Hz，3H)，2.07-2.04(m，，4H)，1.91-1.80(m，1H)，1.77-1.70(m，1H)，1.50(d，J＝6.8Hz，3H)。

Example 2

First step

Compound 1h (70 mg, 176. Mu. Mol,1 eq), 2a (40.2 mg, 176. Mu. Mol,1 eq) and diisopropylethylamine (22.7 mg, 176. Mu. Mol, 30.7. Mu.L, 1 eq) were dissolved in dimethyl sulfoxide (5 mL) and the mixed solution was reacted at 70℃for 17 hours. After the reaction was completed and the reaction mixture was cooled, 10mL of water and 30mL of ethyl acetate were added to the reaction mixture to extract the mixture. Then, water was added to the organic phase to extract excess dimethyl sulfoxide, and the organic phase was dried over anhydrous sodium sulfate and concentrated. Plate chromatography (0/1 petroleum ether/ethyl acetate) gives compound 2b.

MS-ESI calculated [ M+H ]] ⁺ 590, found 590.

Second step

Compound 2b (100 mg, 169. Mu. Mol,1 eq) was dissolved in ethyl acetate (3 mL), and then hydrochloric acid/ethyl acetate (4M, 3mL,70.8 eq) was added to the above solution, and the reaction solution was reacted at 20℃for 3 hours. After completion of the reaction, the reaction mixture was concentrated, 10mL of water and 45mL of ethyl acetate (15 mL. Times.3) were added thereto, and extraction was performed, and the organic phase was dried over anhydrous sodium sulfate and concentrated. And (3) taking a small amount of reaction liquid, and purifying by using a high performance liquid chromatography to obtain the compound 2c.

MS-ESI calculated [ M+H ]] ⁺ 490, found 490.

¹ H NMR(400MHz，CD ₃ OD)δ：8.62(s，1H)，8.36-8.29(m，2H)，7.96(d，J＝7.8Hz，1H)，7.69(d，J＝8.4Hz，1H)，7.64(t，J＝7.8Hz，1H)，4.62(br d，J＝6.4Hz，1H)，4.19-3.86(m，7H)，3.81-3.72(m，5H)，3.65(br d，J＝9.2Hz，2H)，3.53(br d，J＝8.0Hz，2H)，2.99(s，3H)，1.51(d，J＝6.8Hz，3H)。

Third step

Compound 2c (150 mg, 306. Mu. Mol,1 eq) and formaldehyde (11.96 mg, 398. Mu. Mol, 11.0. Mu.L, 1.3 eq) were dissolved in dichloroethane (10 mL) and acetic acid (2 mL), then sodium cyanoborohydride (38.5 mg, 613. Mu. Mol,2 eq) was added, and the mixed solution was reacted at 20℃for 18 hours. After the reaction is completed and cooled, the reaction solution is decompressed, concentrated and the residue is purified by high performance liquid chromatography to obtain the compound 2.

MS-ESI calculated [ M+H ]] ⁺ 504, measured 504.

¹ H NMR(400MHz，CD ₃ OD)δ：8.63(s，1H)，8.36-8.30(m，2H)，7.96(d，J＝8.0Hz，1H)，7.71(d，J＝8.6Hz，1H)，7.65(t，J＝7.8Hz，1H)，4.63(br s，2H)，4.17-3.85(m，7H)，3.83-3.67(m，8H)，2.99(s，3H)，2.61(s，3H)，1.51(d，J＝6.8Hz，3H)。

Example 3

First step

The compound triphenylphosphine (25.9 g,72.6mmol,1.6 eq) was dissolved in 350 ml tetrahydrofuran, potassium tert-butoxide (1M, 81.7mL,1.8 eq) was added at 20℃and the reaction mixture was reacted at 20℃for 3 hours, and 3a (8.0 g,45.4mmol,1 eq) was added to the reaction mixture and reacted for 18 hours. The reaction was complete, water (200 mL) and ethyl acetate (300 mL) were added thereto for extraction, the organic phase was washed with saturated brine (100 ml×3), the organic phase was dried, filtered, concentrated, and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=100:1 to 1:1) to give 3b.

¹ H NMR(400MHz，CDCl ₃ )δ：7.36-7.31(m，5H)，4.87-4.85(m，2H)，4.46(s，2H)，，4.45-4.08(m，1H)，2.89-2.86(m，2H)，2.78-2.73(m，2H)。

Second step

To an acetonitrile solution (14 mL) containing 3b (1.5 g,8.61mmol, 640. Mu.L, 1 eq) and tert-butyl N- (2-hydroxyethyl) formate (1.67 g,10.3mmol,1.60mL,1.2 eq) was added NIS (2.32 g,10.3mmol,1.2 eq) and reacted at 20℃for 4 hours. After completion of the reaction, water (20 mL) and ethyl acetate (30 mL) were added in this order to the reaction mixture, followed by extraction, drying, filtration, concentration, and purification of the residue by silica gel chromatography (petroleum ether/ethyl acetate=100:1 to 1:1) 3c.

¹ H NMR(400MHz，CDCl ₃ )δ：7.33-7.30(m，5H)，5.01-4.97(m，1H)，4.44-4.43(m，2H)，3.79-3.71(m，1H)，3.33-3.32(m，3H)，2.45-2.40(m，5H)，2.03-2.02(m，1H)，1.46(s，9H)。

Third step

Compound 3c (1.8 g,3.90mmol,1 eq) was added to anhydrous tetrahydrofuran (50 mL), the internal temperature was reduced to 0℃and sodium hydride (312 mg,7.80mmol,60%,2 eq) was added. The reaction mixture was reacted at 20℃for 18 hours, the reaction was completed, water (50.0 mL) was added to the reaction mixture, the mixture was extracted with ethyl acetate (50 mL), the organic phases were combined, dried, filtered, concentrated, and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=100:1 to 1:1) to give 3d.

¹ H NMR(400MHz，CDCl ₃ )δ：7.33-7.20(m，5H)，4.63-4.09(m，3H)，3.52-3.50(m，2H)，3.37(s，1H)，3.30(s，2H)，3.18(s，1H)，2.35(s，1H)，2.28-2.15(m，1H)，1.97-1.85(m，2H)，1.43-1.28(m，9H)。

Fourth step

Compound 3d (2.6 g,13.6mmol,1 eq) was added to ethyl acetate (15 mL) to which was added wet palladium on carbon (0.1 g, 10%). The reaction was replaced three times with hydrogen and reacted at 25℃for 2 hours under this atmosphere (15 psi), the reaction was complete, filtered and concentrated to 3e, the crude product was used directly in the next step.

Fifth step

To a reaction flask containing 3e (360 mg,1.48mmol,1 eq) and ethyl acetate (4 mL) was added HCl/EtOAc (4M, 10mL,27 eq) and reacted at 20℃for 2 hours. The reaction was complete and concentrated to give crude 3f which was used directly in the next step.

Sixth step

Compound 3f (210 mg,1.47mmol,1 eq) and DIPEA (3831 mg,2.95mmol, 513. Mu.L, 2 eq) were dissolved in dichloromethane (5 mL), and benzyl chloroformate (302 mg,1.77mmol, 251. Mu.L, 1.2 eq) was added to the reaction solution. The reaction was carried out at 20℃for 18 hours. The reaction was complete, water (30.0 mL) was added to the reaction, extracted with ethyl acetate (20 mL x 3), the organic phases were combined, dried, filtered, concentrated, and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=100:1-1:1) to give 3g.

¹ H NMR(400MHz，CDCl ₃ )δ：7.43-7.29(m，5H)，5.23-5.10(m，2H)，4.55-4.10(m，1H)，3.61(s，2H)，3.55(s，1H)，3.47-3.45(m，2H)，3.34(s，1H)，2.60-2.30(m，2H)，1.95-1.93(m，2H)。

Seventh step

3g (320 mg,1.15mmol,1 eq) of the compound was dissolved in dichloromethane (5 mL) and DMP (636 mg,1.50mmol,1.3 eq) was added to the reaction solution. The reaction was carried out at 20℃for 2 hours. After completion of the reaction, the mixture was concentrated, and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=100:1 to 1:1) to give 3 hours.

1H NMR(400MHz，CDCl ₃ )δ：7.46-7.30(m，5H)，5.16(s，2H)，3.71(s，2H)，3.62(s，2H)，3.58-3.52(m，2H)，3.15-3.07(m，2H)，2.98(s，2H)。

Eighth step

Compound 3h (200 mg, 727. Mu. Mol,1 eq) was dissolved in dichloromethane (1 mL), N-diethyl sulfur trifluoride (703 mg,4.36mmol, 576. Mu.L, 6 eq) was added to the reaction solution, and the mixture was reacted at 20℃for 18 hours. The reaction was completed, water (40.0 mL) was added to the reaction, extracted with dichloromethane (30 ml×3), the organic phases were combined, dried, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=100:1 to 1:1) to give 3i.

Ninth step

Compound 3i (180 mg, 606. Mu. Mol,1 eq) was added to methanol (5 mL) to which was added wet palladium on carbon (0.01 g, 10%). The reaction was replaced three times with hydrogen and reacted at 20℃for 2 hours under this atmosphere (15 psi), the reaction was complete, filtered and concentrated to give 3j, the crude product was used directly in the next step.

Tenth step

Compound 3j (42 mg, 257. Mu. Mol,1 eq), 1h (102 mg, 257. Mu. Mol,1 eq) and N, N-diisopropylethylamine (66.5 mg, 514. Mu. Mol, 89.7. Mu.L, 2 eq) were dissolved in DMSO (1 mL) and the mixed solution was reacted at 70℃for 18 hours. The reaction is complete, and the reaction liquid is purified by high performance liquid chromatography to obtain 3.

MS-ESI calculated [ M+H ]] ⁺ 525, measured value 525.

¹ H NMR(400MHz，CDCl ₃ )δ：8.54(s，1H)，8.15(d，J＝8.0Hz，1H)，7.99(d，J＝8.4Hz，1H)，7.89(d，J＝8.0Hz，1H)，7.54-7.41(m，2H)，6.38(br s，1H)，4.36(br s，1H)，4.06-3.84(m，6H)，3.79-3.53(m，6H)，2.99(d，J＝4.8Hz，3H)，2.73-2.46(m，4H)，1.44(d，J＝6.8Hz，3H)。

Example 4

First step

Compound 4a (0.28 g,1.13mmol,1 eq) was dissolved in trifluoroacetic acid (5.00 mL) and dichloromethane (10.0 mL), and the mixture was stirred at room temperature for 2 hours, after the completion of the reaction, the mixture was dried under reduced pressure to give 4b.

MS-ESI calculated [ M+H ]] ⁺ 148, found 148.

Second step

Compound 4b (300 mg,1.15mmol,1eq, TFA), compound 1h (320 mg, 804. Mu. Mol,0.7 eq), DIPEA (445 mg,3.45mmol, 600. Mu.L, 4 eq) were dissolved in dimethyl sulfoxide (5.00 mL), reacted at 70℃for 16 hours, and after completion of the reaction, purified by high performance liquid chromatography to give 4c.

MS-ESI calculated [ M+H ]] ⁺ 509, found 509.

Third step

Compound 4c (100 mg, 197. Mu. Mol,1 eq), p-toluenesulfonyl chloride (37.5 mg, 197. Mu. Mol,1 eq) and sodium hydride (15.7 mg, 393. Mu. Mol,60%,2 eq) were dissolved in DMF (10.0 mL) and reacted at room temperature for 16 hours, after which the reaction was completed, purified by high performance liquid chromatography to give 4.MS-ESI calculated [ M+H ]] ⁺ 491, found 491.

¹ H NMR(400MHz，CDCl ₃ )δ：8.65(s，1H)，8.23(br d，J＝7.8Hz，1H)，8.09(d，J＝8.4Hz，1H)，7.99(br d，J＝7.8Hz，1H)，7.62-7.53(m，2H)，6.60(br s，1H)，4.66(d，J＝6.4Hz，2H)，4.57-4.41(m，3H)，4.32-4.14(m，2H)，4.08-3.85(m，5H)，3.83-3.75(m，5H)，3.08(d，J＝4.8Hz，3H)，1.52(d，J＝6.8Hz，3H)。

Example 5

First step

Compound 4c (70.0 mg, 138. Mu.mol, 1 eq), methanesulfonyl chloride (0.8 g,6.98mmol, 541. Mu.L, 50.7 eq) and triethylamine (27.9 mg, 275. Mu. Mol, 38.3. Mu.L, 2 eq) were dissolved in methylene chloride (10.0 mL), reacted at room temperature for 16 hours, and after completion of the reaction, dried under reduced pressure to give Compound 5a.

MS-ESI calculated [ M+H ]] ⁺ 665, measured 665.

Second step

Compound 5a (59.9 mg, 90.26. Mu. Mol,1 eq), tetra-N-butylammonium iodide (3.33 mg, 9.03. Mu. Mol,0.1 eq), sodium sulfide (21.1 mg, 271. Mu. Mol, 11.4. Mu.L, 3 eq) were dissolved in N, N' -dimethylformamide (5.00 mL), reacted at 70℃for 18 hours under the protection of nitrogen, and after completion of the reaction, washed with water (50.0 mL. Times.3) and purified by high performance liquid chromatography to give 5.

MS-ESI calculated [ M+H ]] ⁺ 507, found 507.

¹ H NMR(400MHz，CDCl ₃ )δ：8.67(br s，1H)，8.20(br d，J＝8.0Hz，1H)，8.10-8.03(m，1H)，7.99(br d，J＝8.0Hz，1H)，7.60-7.52(m，2H)，6.64(br s，1H)，4.46(br d，J＝5.6Hz，1H)，4.42-4.33(m，1H)，4.19(br d，J＝13.2Hz，1H)，4.09-3.91(m，3H)，3.91-3.69(m，7H)，3.42(br d，J＝10.0Hz，2H)，3.06(d，J＝4.8Hz，3H)，3.00(br d，J＝8.0Hz，2H)，1.53(br d，J＝6.8Hz，3H)。

Example 6

Compound 5 (120 mg, 237. Mu. Mol,1 eq) was dissolved in methanol (5.00 mL), an aqueous solution (5.00 mL) of potassium monopersulfate (107 mg, 474. Mu. Mol,2 eq) was added dropwise, and the mixture was reacted at room temperature for 30 hours, after completion of the reaction, 6 was purified by high performance liquid chromatography.

MS-ESI calculated [ M+H ]] ⁺ 539, found 539.

¹ H NMR(400MHz，CDCl ₃ )δ：8.58(br s，1H)，8.14(br d，J＝8.0Hz，1H)，8.02(d，J＝8.4Hz，1H)，7.91(br d，J＝7.6Hz，1H)，7.60-7.44(m，2H)，6.57(br s，1H)，4.42(br d，J＝6.0Hz，1H)，4.27-3.85(m，10H)，3.82-3.60(m，6H)，2.99(d，J＝4.8Hz，3H)，1.46(d，J＝6.8Hz，3H)。

Experimental example 1: in vitro evaluation of mTOR kinase inhibitory Activity

Experimental materials:

this experiment was tested on discover x, from which all materials and methods were from.

Experimental operation:

kinase activity assay.

1 the labelled mTOR kinase is stably expressed in HEK-293 cells.

2 treating streptavidin magnetic beads with biotinylated small molecule ligand for 30 min at room temperature to produce an affinity resin for kinase analysis;

3 ligand beads were blocked with excess biotin and washed with buffer (1% bovine serum albumin, 0.05% tween 20ml, 1ml dithiothreitol) to wash away unbound ligand and non-specifically bound ligand;

4 kinase ligand affinity beads, assembly, binding reactions of the test compounds all were performed in buffer (20% blocking buffer, 0.17x phosphate buffer, 0.05% tween 20, 6ml dithiothreitol);

5 test compound is dissolved in dimethyl sulfoxide;

6 all compounds used for the measurement were dissolved in DMSO and then the compounds were directly diluted to a concentration of 0.9%.

The 7 solution was placed in 384 well polypropylene plates, each 0.02 ml volume;

8, shaking for 1 hour at room temperature;

9 washing with buffer (1 XPBS, 0.05% Tween 20)

The 10 affinity beads were resuspended in buffer (1 XPBS, 0.05% Tween 20,0.5 μm non-biotin affinity ligand) and incubated for 30 min at room temperature.

11 the concentration of kinase in the eluate was measured by qPCR.

Experimental results:

TABLE 1 results of test for mTORC1 and mTORC2 kinase Complex Activity

Conclusion: the compounds of the application have significant and even unexpected mTOR kinase inhibitory activity.

Experimental example 2 evaluation of cell proliferation inhibitory activity:

the purpose of the experiment is as follows: the test compounds are tested for their cytostatic activity.

Experimental principle: the luciferases in the Cell-Titer-Glo reagent use luciferin, oxygen and ATP as reaction substrates to produce oxyluciferin and release energy in the form of light. Since the luciferase reaction requires ATP, the total amount of light generated by the reaction is proportional to the total amount of ATP that is reactive to the cell viability.

Experimental materials:

cell line: MCF-7 cell line (ATCC-CRL-22), HT-29 cell line (ATCC-HTB-38), OE21 (ECACC-96062201), NCI-N87 cell line (ATCC-CRL-5822)

Cell culture medium: (RPMI 1640 medium (Invitrogen # 1868274; 10% serum Invitrogen #1804958; L-Glutamine 1-D, invitrogen #1830863; double anti Hyclone # J170012))

Cell Titer-Glo J luminescence method Cell viability detection kit (Promega#G7573)

384 well cell culture plate (Greiner#E15103 MA)

Compound plate (LABCYSTE# 0006346665)

CO ₂ Incubator (Thermo # 371)

Vi-cell counter (Beckman Coulter)

Pipettor (Eppendorf)

Pipette (Greiner)

Liquid-transfering gun (Eppendorf)

Multifunctional enzyme label instrument (Envision Reader)

ECHO Liquid-handling workstation(Labcyte-ECHO555)

Experimental procedure and method:

2.1 day 1:

according to the cell plating scheme, 1000 cells per well, 25. Mu.L per well of density plates, and 25. Mu.L of PBS are respectively supplemented to the edge wells of the plates in 384 or 96 well plates.

2.2 day 1:

(1) The compound stock was 10mM and the compound was diluted with DMSO to an initial concentration of 4mM. The compound was added to the compound mother liquor plate at 9. Mu.L per well.

(2) Compound dilutions were made using an ECHO liquid station and 125nL of compound was added to each well of the cell plate, 125nL of DMSO was added to each well of column 2 and column 23, and 125nL of DMSO was added to each well of column 1 and column 24 PBS wells.

(3) The cell plates were supplemented with 25 μl of medium per well, 50 μl of final cell plate per well, 1 μΜ compound concentration, 3-fold dilution, 10 concentrations, about duplicate wells, DMSO final concentration of 0.25%.

2.3 after the addition of the compound, the cell plates were centrifuged at 1000rpm for 1min and placed at 37℃in 5% CO ₂ Culturing in an incubator for 3 days.

2.4 third day:

the cell plates were removed from the incubator and equilibrated for 30 minutes at room temperature. To each well 25. Mu.L of Cell-Titer-Glo reagent was added, and the mixture was shaken for one minute to mix thoroughly and centrifuged at 1000rpm for 1 minute. After 10 minutes, the plate was read on PerkinElmer Envision and the fluorescence read time was set to 0.2 seconds. Test results: the test results are shown in Table 2

Table 2: results of in vitro cell proliferation inhibition activity screening test of the Compounds of the present application

Conclusion: the compound has obvious proliferation inhibition activity on MCF-7, N87 and OE-21 cells and has a certain proliferation inhibition activity on HT-29 cells.

Experimental example 3: pharmacokinetic evaluation

Experimental method

The test compound was mixed with 5% DMSO/95%10% polyoxyethylated castor oil (Cremophor EL), vortexed and sonicated to prepare a 1mg/mL approximately clear solution, which was filtered through a microfiltration membrane for use. 18 to 20 grams of Balb/c female mice were selected and given by intravenous injection of a candidate compound solution at a dose of 1 or 2mg/kg. The tested compound is mixed with 1% Tween 80,9% polyethylene glycol 400 and 90% aqueous solution, vortexed and sonicated to prepare 1mg/mL approximately clear solution, and the solution is filtered by a microporous filter membrane for later use. 18 to 20 grams of Balb/c female mice were selected and administered orally with a solution of the candidate compound at a dose of 2 or 10mg/kg. Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and drug substitution parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

Test results:

the test results are shown in Table 3.

TABLE 3 Pharmacokinetic (PK) parameters in plasma of example compounds

TABLE 5 PK parameters in plasma of example compounds

"-" means that no data is tested or obtained.

Conclusion of the test: compound 1 exhibited the same or even better pharmacokinetic properties as the reference compound.

Experimental example 4 in vivo pharmacodynamics study of human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor BALB/C nude mouse model:

the purpose of the experiment is as follows: study of the test Compound of the present patent for evaluation of efficacy of human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor in BALB/C nude mouse model

Experimental animals: female BALB/c nude mice, 6-8 weeks old, weighing 18-22 g; the suppliers: animals in south China

The experimental method comprises the following steps:

4.1 cell culture

Human nasopharyngeal carcinoma C666-1 cell, in vitro monolayer culture under the condition of adding 10% fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, 37 deg.C and 5% CO into DMEM culture medium ₂ Culturing. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the saturation of the cells is 80% -90%, the cells are collected, counted and inoculated.

4.2 tumor cell inoculation (tumor inoculation)

0.1ml (1X 10) ⁷ ) Fine C666-1Cells (DMEM double without) were inoculated subcutaneously on the right back of each mouse, and the average tumor volume reached 200mm ³ At the beginning of group administration

4.3 preparation of test pieces:

the tested compound is prepared into clear solution of 5mg/mL and 5mg/mL, and the solvent is 5% DMSO+30% PEG400+65% water

4.4 tumor measurement and Experimental index

The experimental index is to examine whether tumor growth is inhibited, retarded or cured. Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b ² A and b represent the major and minor diameters of the tumor, respectively.

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or tumor proliferation rate T/C (%). TGI (%) reflects the tumor growth inhibition rate. Calculation of TGI (%): TGI (%) = [1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group)/(mean tumor volume at the end of treatment of solvent control group-mean tumor volume at the beginning of treatment of solvent control group) ]x100%.

Tumor proliferation rate T/C (%): the calculation formula is as follows: T/C (%) = average tumor volume at the end of treatment group administration/average tumor volume at the end of treatment of solvent control group x 100%.

4.5 statistical analysis

Statistical analysis, including mean and Standard Error (SEM) of tumor volumes at each time point for each group (see table 5-1 for specific data). The treatment group showed the best treatment effect at day 28 after dosing at the end of the trial, so the statistical analysis was performed to evaluate the inter-group differences based on this data. The comparison between two groups was analyzed by T-test, the comparison between three or more groups was analyzed by one-way ANOVA, and the comparison was examined using the gas-Howell method, with significant differences in F values. All data analysis was performed with SPSS 17.0. p < 0.05 was considered a significant difference.

4.6 test results

4.6.1 mortality, morbidity and weight changes

The body weight of the experimental animal is used as a reference index for indirectly measuring the toxicity of the drug. The weight of mice in the model treatment group has a descending trend, one mice in the solvent group die early, the suspected feeding density is too high, and no abnormality is found in the anatomy; one of the group of example 2 dies on day 28, and the body weight was too low and the toxicity was too great to die, and no abnormality was found in the anatomy. (AZD 2014 and example 2 have a weight of less than 17 g)

4.6.2 evaluation index of antitumor drug efficacy

TABLE 4 evaluation of tumor-inhibiting efficacy of the Compound of the present application on human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor model

(calculated based on tumor volume at day 28 after administration)

Note that:

a. mean ± SEM.

b. Tumor growth inhibition was inhibited by T/C and TGI (%) = [1- (T) ₂₈ -T ₀ )/(V ₂₈ -V0)]X 100) calculation.

c.p values were calculated from tumor volumes.

4.7 test conclusion and discussion

In this experiment, the in vivo efficacy of the compounds of this patent in a model of human nasopharyngeal carcinoma 666-1 cell subcutaneous xenograft tumor was evaluated. The therapeutic group example 2, 30mg/kg, showed a remarkable tumor-inhibiting effect compared to the solvent control group, comparable to the efficacy of the reference compound AZD2014 15 mg/kg.

Claims (1)

1. The application of a compound shown in the following formula or pharmaceutically acceptable salt thereof in preparing a medicament for treating nasopharyngeal carcinoma,