CN113286594A - Application of pyridopyrimidine compound in preparation of medicine for treating nasopharyngeal carcinoma - Google Patents

Abstract

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

Description

Application of pyridopyrimidine compound in preparation of medicine for treating nasopharyngeal carcinoma

The following priority is claimed in the present application:

CN201910049704.0, application date 2019, 1 month 18.

Technical Field

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

Technical Field

Tumors, especially malignant tumors, are one of the most serious diseases endangering human health at present, and with the technological progress and the more and more intensive research on tumor treatment, the rapid progress is made in the aspects of tumor occurrence, development mechanism and tumor treatment. Many new mechanisms and biomarkers have been discovered. The invention relates to a signal pathway which plays a key role in the proliferation, invasion and metastasis and anti-apoptosis of tumors, namely a phosphatidylinositol 3 kinase (PI3K) -AKT-mammalian rapamycin protease mTOR signal pathway.

Activation of PI3K is largely involved in the substrate near the inside 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), angiotensin I (Ang1), and insulin all initiate the activation process of PI 3K. These factors activate Receptor Tyrosine Kinases (RTKs), causing autophosphorylation. The result of PI3K activation is the production of the second messenger PIP3 on the plasma membrane, PIP3 binds to the PH domain containing signaling proteins AKT and PDK1 (phosphoside dependent kinase-1) in cells, causing PDK1 to phosphorylate Ser308 of the AKT protein resulting in AKT activation. Other substrates for PDK1 also include PKC (protein kinase C), S6K (p70S6) and SGK (serum/glucose regulated kinases). AKT, also known as protein kinase b (pkb), is a major 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 10), an anti-cancer gene, undergoes genetic mutations or deletions in a wide range of human tumors. PTEN is a PIP 3-phosphatase that can convert PIP3 to PIP2 by dephosphorylation, in contrast to the function of PI 3K. PTEN can decrease activation of AKT and prevent all downstream signaling events regulated by AKT. mTOR is used as an AKT downstream substrate, is relatively conserved in evolution, can integrate various signals of nutrition, energy and growth factors, participates in biological processes such as gene transcription, protein translation, ribosome synthesis and apoptosis and plays an extremely important role in cell growth. There are two highly homologous complexes, Tor binding to KOG01 to form mTORC1, mTOR forming rapamycin insensitive mTORC2 with AVO1/AVO2/AVO 3/and LST8 mTOR regulates downstream protein translation by phosphorylating downstream target proteins S40S ribosomal S6 protein kinases such as S6K1 and 4EBP 1. mTOR binds to eIF3, phosphorylates S6K1, activates S6K1 upon release from eIF3, and further phosphorylates cellular substrates such as p70S6 to facilitate protein translation and expression. 4EBP1 is combined with eukaryotic transcription initiation factor 4E and inhibits the activity thereof, and after the mtor phosphorylates 4E-BP1, the activation thereof is separated from eif-4E, thereby realizing the transcription of eukaryotic cells. mTORC2 is able to phosphorylate AKT, thereby up-regulating its kinase activity.

From the above, any mutation or over-expression upstream of PI3K/AKT/mTOR signaling pathway can lead to a series of cascade reactions downstream, and finally lead to tumor generation, development and metastasis. And mTOR is positioned at the pivot of a signal path, and the inhibition on mTORC1 and mTORC2 can well block the transmission of signals, so that the development of tumors is controlled.

The research shows that the signal channel is applied to 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, Merkel cell tumor and the like. And is closely associated with 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 compound has a good development prospect.

The invention aims to find a dual mTOR small molecular compound targeted drug, and the compound has good activity and shows excellent effect and action.

US20170281637 discloses compound AZD2014, belonging to mTORC1& mTORC2 kinase inhibitors, whose structural formula is shown below:

disclosure of Invention

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

wherein

R ₁Is selected from C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_aSubstitution;

R ₂is selected from C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_bSubstitution;

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

R ₃Selected from H and C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_cSubstitution;

R ₄selected from H, F, Cl, Br, I and C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_dSubstitution;

R _a、R _b、R _cand R_dEach independently selected from H, F, Cl, Br and I.

In some embodiments of the invention, R is as defined above₁Is selected from CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F, and the other variables are as defined herein.

In some embodiments of the invention, R is as defined above₁Is selected from CH₃And the other variables are as defined herein.

In some embodiments of the invention, R is as defined above₂Is selected from CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F, and the other variables are as defined herein.

In some embodiments of the invention, R is as defined above₂Is selected from CH₃And the other variables are as defined herein.

In some embodiments of the invention, R is as defined above₃Is selected from CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F, and other variables are as defined herein.

In some embodiments of the invention, R is as defined above₃Is selected from CH₃And the other variables are as defined herein.

In some embodiments of the invention, R is as defined above₄Each independently selected from H, F, Cl, Br, I, CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F, and the other variables are as defined herein.

In some embodiments of the invention, R is as defined above₄Each independently selected from H, F, and the other variables are as defined herein.

In some embodiments of the invention, the structural unit

Is selected from

Other variables are as defined herein.

Still other embodiments of the invention are any combination of the above variables.

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

the technical effects are as follows:

the compounds of the invention have significant and even unexpected mTOR kinase inhibitory activity. The compound has obvious proliferation inhibiting activity on MCF-7, N87 and OE-21 cells and certain proliferation inhibiting 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 compound of this patent in a model of human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor was evaluated. Compared with a solvent control group, the 230 mg/kg of the treatment group example has the equivalent effect of the AZD 201415 mg/kg of the reference compound, and shows obvious tumor inhibition effect.

Correlation definition

As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient. 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 salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat 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 compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat 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 salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid and the like. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting 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 the two.

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

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

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

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecules have two or more chiral centers and a non-mirror image relationship between the molecules.

Unless otherwise statedWhile, using a wedge-shaped solid key

And wedge dotted bond

Showing the absolute configuration of a solid centre, by means of straight solid keys

And straight dotted line bond

Showing the relative configuration of the centres of solids, by wavy lines

Representing solid-line keys of wedge shape

Or wedge dotted bond

Or by wavy lines

Indicating straight solid-line keys

And straight dotted line bond

The compounds of the invention may be present specifically. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be rapidly interconverted. If tautomers are possible (e.g., in solution), then the chemical equilibrium of the tautomers can be reached. For example, proton tautomers (prototropic tautomers), also known as proton transfer tautomers (prototropic tautomers), include interconversions by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valencetatomer) include interconversion by recombination of some of the bonding electrons. A specific example of where keto-enol tautomerism is the interconversion between two tautomers of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.

Optically active (R) -and (S) -isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one of the enantiomers of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is 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), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines). The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labelled with radioactive isotopes, such as tritium (A), (B), (C) and C)³H) Iodine-125 (¹²⁵I) Or C-14(¹⁴C) In that respect As another example, deuterium can be substituted for hydrogen to form a deuterated drug, the bond between deuterium and carbon being greater than normal hydrogenThe bond formed by the deuterated drug and carbon is firmer, and compared with the non-deuterated drug, the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life period of the drug and the like. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of an active agent of the present invention, without interfering with the biological activity of the active agent, and without toxic side effects to the host or patient, and representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. These include suspending agents, viscosity enhancers, skin penetration enhancers, and the like. Their preparation is known to those skilled in the cosmetic or topical pharmaceutical field.

"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 replaced with a substituent, and may include variations of deuterium and hydrogen, so long as the valency 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 aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemical realizability.

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

When a linking groupWhen the number is 0, e.g., - (CRR)₀-, represents that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly connected, for example, where L represents a single bond in A-L-Z means that the structure is actually A-Z.

Unless otherwise specified, the term "alkyl" is intended to mean a straight-chain or branched-chain saturated hydrocarbon radical, which may be monosubstituted (e.g., -CH)₂F) Or polysubstituted (e.g. -CF)₃) And may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (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_1-3Alkyl "is intended to mean a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. Said C is_1-3The alkyl group comprising C_1-2And C_2-3Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C_1-3Examples 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 "halogen" 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 not be limited to, trifluoromethyl, 2, 2, 2-trifluoroethyl, 4-chlorobutyl, and 3-bromopropyl, and the like. Unless otherwise specified, examples of haloalkyl include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

Unless otherwise specified, C_n-n+mOr C_n-C _n+mIncluding any one particular case of n to n + m carbons, e.g. C_1-12Comprising C₁、C ₂、C ₃、C ₄、C ₅、C ₆、C ₇、C ₈、C ₉、C ₁₀、C ₁₁And C₁₂Also included are any ranges of n to n + m, e.g. C_1-12Comprising C_1-3、C _1-6、C _1-9、C _3-6、C _3-9、C _3-12、C _6-9、C _6-12And C_9-12Etc.; similarly, n to n + m means the number of atoms on the ring is n to n + m, for example, the 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, a 11-membered ring, and a 12-membered ring, and any range of n to n + m is also included, for example, the 3-12 membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 6-8-membered ring, and a 6-10-membered ring, etc.

The solvent used in the present invention can be commercially available.

The embodiments of the invention relate to high performance liquid chromatography separation, which all adopt neutral separation.

The invention employs the following abbreviations: aq represents water; HATU represents O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate; EDC stands for 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 represents diisopropyl azodicarboxylate; DMF represents N, N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc for ethyl acetate; EtOH stands for ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, an amine protecting group; BOC represents tert-butylcarbonyl as an amine protecting group; HOAc represents acetic acid; NaCNBH₃Represents sodium cyanoborohydride; r.t. represents room temperature; O/N stands for overnight; THF represents tetrahydrofuran; boc₂O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA stands for diisopropylethylamine; SOCl₂Represents thionyl chloride; CS₂Represents carbon disulfide; TsOH stands for p-tolueneA sulfonic acid; NFSI represents N-fluoro-N- (phenylsulfonyl) benzenesulfonamide; n-Bu₄NF represents tetrabutyl ammonium fluoride; iPrOH represents 2-propanol; mp represents melting point; LDA represents lithium diisopropylamide; pd (PPh)₃) ₄Represents tetrakis (triphenylphosphine) palladium; IV stands for intravenous injection; PO stands for oral administration.

The compounds of the present invention are identified according to the general nomenclature used in the art or

The software names, and the commercial compounds are under the supplier catalog name.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention.

Reference example 1

First step of

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

MS-ESI calculated value [ M + H%] ⁺173 and 175, found 173 and 175.

¹H 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 of

Mixing compound 1-2(17.0g,98.5mmol,1.00eq), ammonium chloride (10.5g,197mmol,2.00eq), 1-hydroxybenzotriazole(13.3g,98.5mmol,1.00eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (18.9g,98.5mmol,1.00eq) and diisopropylethylamine (38.2g,296mmol,3.00eq) 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 spin-dried under reduced pressure, water (200mL) was added, extraction was performed with ethyl acetate (200 mL. times.3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and column chromatographed (1: 1 petroleum ether/ethyl acetate, R)_f0.4) to give a compound, and slurried with ethyl acetate (50mL) for ten minutes to give compounds 1 to 3.

¹H 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)。

The third step

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

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

The fourth step

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

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

The fifth step

Compounds 1-5(1.90g,8.10mmol,1.00eq), (S) -2-methylmorpholine (819mg,8.10mmol,1.00eq) and diisopropylethylamine (2.09g,16.2mmol,2.83mL,2.00eq) were dissolved in dichloromethane (50mL) and the resulting solution was reacted at 25 ℃ for 2 hours. After the reaction is completed, concentration and column chromatography (3: 1 petroleum ether/ethyl acetate) are carried out to obtain the compounds 1-6.

¹H 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)。

The sixth step

Dissolving compounds 1-6(1.2g,4.01mmol,1.00eq), compounds 1-7(1.15g,4.41mmol,1.10eq), palladium tetratriphenylphosphine (232mg, 200. mu. mol,0.05eq) and potassium carbonate (1.66g,12.0mmol,3.00eq) in water (24mL) and 1, 4-dioxane (120mL), reacting at 60 ℃ for 5 hours under the protection of nitrogen, concentrating off the solvent after the reaction is completed, adding water (30mL) for dilution, extracting with ethyl acetate (50 mL. times.2), drying the combined organic phases with anhydrous sodium sulfate, filtering, performing reduced pressure spin drying, and performing column chromatography (100% ethyl acetate) to obtain a compound 1 h.

¹H 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 of

The compound 1a (23.0g,328mmol,24.5mL,1.0eq) and zinc diiodide (5.20g,16.4mmol,0.05eq) were dissolved in 200mL of dichloromethane, the internal temperature was lowered to 0 ℃, trimethylsilylcyanide (39.1g,393mmol,49mL,1.2eq) was added, the reaction solution was reacted at 25 ℃ for 18 hours, the reaction was completed, the reaction solution was concentrated, 50mL of acetonitrile and 50mL of aqueous hydrochloric acid (1N) were added, and stirring was carried out 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) to give 1 b.

¹H 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 of

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

¹H 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)。

The third step

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

¹H 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)。

The fourth step

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

¹H 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)。

The fifth step

To a solution of lithium aluminum hydride (645mg,17mmol,2eq) in tetrahydrofuran (30mL) at 0 ℃ was added 1f (1.2g,8.5mmol,1eq) and the reaction was carried out at 20 ℃ for 18 hours. After completion of the reaction, water (0.7mL), 15% sodium hydroxide (0.7mL) and water (2.1mL) were added to the reaction mixture in this order, and the mixture was stirred for 15 minutes, filtered and concentrated to obtain 1 g.

¹H 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)。

The sixth step

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

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

¹H 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 of

Compound 1h (70m g, 176. mu. mol,1eq), 2a (40.2mg, 176. mu. mol,1eq) and diisopropylethylamine (22.7mg, 176. mu. mol, 30.7. mu.L, 1eq) were dissolved in dimethyl sulfoxide (5mL), and the mixed solution was reacted at 70 ℃ for 17 hours. After the reaction was completed and the reaction solution was cooled, 10mL of water and 30mL of ethyl acetate were added to the reaction solution to conduct extraction. 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) afforded compound 2 b.

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

Second step of

After dissolving compound 2b (100mg, 169. mu. mol,1eq) in ethyl acetate (3mL), hydrochloric acid/ethyl acetate (4M,3mL,70.8eq) was added to the above solution, and the reaction mixture 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 to conduct extraction, and the organic phase was dried over anhydrous sodium sulfate and concentrated. And purifying a small amount of reaction liquid by high performance liquid chromatography to obtain a compound 2 c.

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

¹H NMR(400MHz,CD ₃OD)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)。

The third step

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

MS-ESI calculated value [ M + H%] ⁺504, found 504.

¹H NMR(400MHz,CD ₃OD)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 of

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

¹H 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 of

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

¹H 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)。

The third step

Compound 3c (1.8g,3.90mmol,1eq) was added to anhydrous tetrahydrofuran (50mL), the internal temperature was lowered to 0 deg.C, and sodium hydride (312mg,7.80mmol, 60%, 2eq) was added. The reaction mixture was reacted at 20 ℃ for 18 hours until the reaction was completed, water (50.0mL) was added to the reaction, extraction was performed with ethyl acetate (50mL), 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 obtain 3 d.

¹H 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)。

The fourth step

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

The fifth step

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

The sixth step

Compound 3f (210mg,1.47mmol,1eq) and DIPEA (381mg,2.95mmol, 513. mu.L, 2eq) were dissolved in dichloromethane (5mL), and benzyl chloroformate (302mg,1.77mmol, 251. mu.L, 1.2eq) was added to the reaction mixture. The reaction was carried out at 20 ℃ for 18 hours. After completion of the reaction, water (30.0mL) was added to the reaction, and extracted with ethyl acetate (20mL × 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 obtain 3 g.

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

The compound (3 h) (200mg, 727. mu. mol,1eq) was dissolved in methylene chloride (1mL), and N, N-diethylsulfide trifluoride (703mg,4.36mmol, 576. mu.L, 6eq) was added to the reaction mixture to react at 20 ℃ for 18 hours. After completion of the reaction, water (40.0mL) was added to the reaction, and the mixture was extracted with dichloromethane (30mL × 3), the organic phases were combined, dried, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 100:1-1:1) to obtain 3 i.

The ninth step

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

The tenth step

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

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

¹H 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 of

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

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

Second step of

Compound 4b (300mg,1.15mmol,1eq, TFA), compound 1h (320mg, 804. mu. mol,0.7eq), DIPEA (445mg,3.45mmol, 600. mu.L, 4eq) were dissolved in dimethyl sulfoxide (5.00mL) and reacted at 70 ℃ for 16 hours, after which 4c was purified by high performance liquid chromatography.

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

The third step

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

¹H 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 of

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

MS-ESI calculated value [ M + H%] ⁺665, found value 665.

Second step of

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

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

¹H 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(120mg, 237. mu. mol,1eq) was dissolved in methanol (5.00mL), an aqueous solution (5.00mL) of potassium monopersulfate (291mg, 474. mu. mol,2eq) was added dropwise, and the reaction was carried out at room temperature for 30 hours, after completion of the reaction, purification by high performance liquid chromatography gave 6.

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

¹H 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, all materials and methods were from discover x.

And (3) experimental operation:

and (4) kinase activity analysis.

1 stably expressing the tagged mTOR kinase in HEK-293 cells.

2 treating streptavidin magnetic beads with biotinylated small molecule ligand for 30 minutes at room temperature to generate 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, test compound three binding reaction in buffer (20% closed buffer, 0.17x phosphate buffer, 0.05% Twain 20, 6ml dithiothreitol) to realize;

5 test compound 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%.

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

shaking for 1 hour at the room temperature of 8 ℃;

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

The 10 affinity beads were resuspended in buffer (1x PBS, 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.

The experimental results are as follows:

TABLE 1 results of mTORC1 and mTORC2 kinase Complex Activity assays

And (4) conclusion: the compounds of the invention have significant and even unexpected mTOR kinase inhibitory activity.

Experimental example 2 evaluation of cell proliferation inhibitory activity:

purpose of the experiment: detecting the cell proliferation inhibitory activity of the test compound.

The experimental principle is as follows: luciferase in Cell-Titer-Glo reagent uses luciferin, oxygen, and ATP as reaction substrates, produces oxyluciferin, and releases energy in the form of light. Since the luciferase reaction requires ATP, the total amount of light produced by the reaction is proportional to the total amount of ATP that reacts 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 # 1868546; 10% serum Invitrogen # 1804958; L-glutamine 1-turn, Invitrogen # 1830863; double anti-Hyclone # J170012))

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

384 well cell culture plate (Greiner # E15103MA)

Compound board (LABCYTE #0006346665)

CO ₂Incubator (Thermo #371)

Vi-cell counter (Beckman Coulter)

Pipettor (Eppendorf)

Pipette (Greiner)

Pipette (Eppendorf)

Multifunctional enzyme mark instrument (Envision Reader)

ECHO Liquid-handling workstation(Labcyte-ECHO555)

Experimental procedures and methods:

2.1 day 1:

according to the cell plate diagram in 384 or 96 well plates for each of 1000 cells per well, 25 u L per well density plate, edge hole seed cells supplemented with 25 u L PBS.

2.2 day 1:

(1) the compound stock was 10mM and the compound was diluted in DMSO to give an initial concentration of 4 mM. Compounds were added to compound master plates at 9 μ L per well.

(2) Compounds were diluted using ECHO fluid station and 125nL of compound was added to each well of the cell plate, 125nL of DMSO was added to each well of columns 2 and 23, and 125nL of DMSO was added to each well of columns 1 and 24 PBS.

(3) Each well of the cell plate was supplemented with 25 μ L of medium, and finally each well of the cell plate was 50 μ L, the compound concentration was 1 μ M, 3-fold dilution, 10 concentrations, and about multiple wells, and the final DMSO concentration was 0.25%.

2.3 after addition of the Compound, centrifugation is carried out at 1000rpm for 1min, and the cell plates are placed at 37 ℃ in 5% CO₂Culturing in an incubator for 3 days.

2.4 day three:

the cell plates were removed from the incubator and allowed to equilibrate at room temperature for 30 minutes. Add 25. mu.L of Cell-Titer-Glo reagent to each well, shake for one minute to mix well, and centrifuge at 1000rpm for 1 minute. After 10 minutes, the plates were read on a PerkinElmer Envision, setting the fluorescence read time to 0.2 seconds. And (3) test results: the test results are shown in Table 2

Table 2: results of screening test for inhibitory Activity of Compound of the present invention on in vitro cell proliferation

And (4) conclusion: the compound has obvious proliferation inhibiting activity on MCF-7, N87 and OE-21 cells and certain proliferation inhibiting activity on HT-29 cells.

Experimental example 3: pharmacokinetic evaluation

Experimental methods

Test compounds were mixed with 5% DMSO/95% 10% 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 the candidate compound solution was administered intravenously at a dose of 1 or 2 mg/kg. The test compound was mixed with 1% tween 80, 9% polyethylene glycol 400, 90% aqueous solution, 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 the candidate compound solution was administered orally at a dose of 2 or 10 mg/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 parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

And (3) testing results:

the test results are shown in Table 3.

Table 3 Pharmacokinetic (PK) parameters in plasma of compounds of the examples

Table 5 PK parameters in plasma of compounds of examples

"- -" means no test or no data obtained.

And (4) test conclusion: compound 1 exhibited the same or even better pharmacokinetic properties as the reference compound.

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

purpose of the experiment: research on evaluation of drug effect of the compound to be tested in the patent on human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor in a BALB/C nude mouse model

Experimental animals: female BALB/c nude mice, 6-8 weeks old, 18-22 grams of body weight; the supplier: southern large model animal

The experimental method and the steps are as follows:

4.1 cell culture

Human nasopharyngeal carcinoma C666-1 cell, in vitro monolayer culturing under the conditions of DMEM medium added with 10% fetal calf serum, 100U/mL penicillin, 100U/mL streptomycin, 37 ℃ and 5% CO₂And (5) culturing. Passage was performed twice a week with conventional digestion treatment with pancreatin-EDTA. When the saturation degree of the cells is 80% -90%, collecting the cells, counting and inoculating.

4.2 tumor cell inoculation (tumor inoculation)

0.1ml (1X 10)⁷) C666-1 cells (DMEM double none) were subcutaneously inoculated in the right hind dorsal area of each mouse, and tumors were averagedThe volume of the product reaches 200mm³At the beginning of the administration of the medicine in groups

4.3 preparation of the test substance:

the tested compounds were formulated as 5mg/mL and 5mg/mL clear solutions in 5% DMSO + 30% PEG400+ 65% water

4.4 tumor measurement and Experimental indices

The experimental index is to investigate whether the tumor growth is inhibited, delayed or cured. Tumor diameters were measured twice weekly using a vernier caliper. The calculation formula for tumor volume is: v is 0.5a × b²And a and b represent the major and minor diameters of the tumor, respectively.

Evaluation of tumor-inhibiting therapeutic effect of the compound TGI (%) or tumor proliferation rate T/C (%). TGI (%), reflecting the rate of tumor growth inhibition. Calculation of TGI (%): TGI (%) × 100% (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group)).

Tumor proliferation rate T/C (%): the calculation formula is as follows: T/C (%) mean tumor volume at the end of administration for a certain treatment group/mean tumor volume at the end of treatment for the solvent control group × 100%.

4.5 statistical analysis

Statistical analysis, including mean and Standard Error (SEM) of tumor volume for each time point for each group (see table 5-1 for specific data). Treatment groups showed the best treatment effect at day 28 after dosing at the end of the trial, so statistical analysis was performed based on this data to assess differences between groups. The comparison between two groups is analyzed by T-test, the comparison between three groups or more groups is analyzed by one-way ANOVA, and the F value is tested to have significant difference by applying a Games-Howell method. All data analyses were performed with SPSS 17.0. Significant differences were considered with p < 0.05.

4.6 test results

4.6.1 mortality, morbidity and weight Change

The body weight of the experimental animal is used as a reference index for indirectly measuring the toxicity of the medicament. The weight of mice in the model treatment group is reduced, one mouse dies in the early stage of the solvent group, the suspected feeding density is too high, and no abnormality is found in dissection; one death in example 2 group was observed on day 28, and the death was too low in body weight and toxic and no abnormality was observed in dissection. (one body weight in both AZD2014 and example 2 was less than 17g)

4.6.2 evaluation index of antitumor Effect

TABLE 4 evaluation of antitumor drug efficacy of the compound of the present invention on human nasopharyngeal carcinoma C666-1 cell subcutaneous xenograft tumor model

(calculated based on tumor volume on day 28 post-dose)

Note:

a. mean. + -. SEM.

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

c.p values were calculated from tumor volumes.

4.7 Experimental conclusions and discussion

In this experiment, the in vivo efficacy of the compound of this patent in a model of human nasopharyngeal 666-1 cell subcutaneous xenograft tumor was evaluated. Compared with a solvent control group, the 230 mg/kg of the treatment group example has the equivalent effect of the AZD 201415 mg/kg of the reference compound, and shows obvious tumor inhibition effect.

Claims (11)

1. The application of the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof in preparing the medicine for treating nasopharyngeal carcinoma:

   

   wherein the content of the first and second substances,

   R ₁is selected from C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_aSubstitution;

   R ₂is selected from C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_bSubstitution;

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

   R ₃Selected from H and C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_cSubstitution;

   R ₄selected from H, F, Cl, Br, I and C_1-3Alkyl radical, said C_1-3Alkyl is optionally substituted by 1, 2 or 3R_dSubstitution;

   R _a、R _b、R _cand R_dEach independently selected from H, F, Cl, Br and I.
2. The use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is R for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma₁Is selected from CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F。
3. Use of a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma, wherein R₁Is selected from CH₃。
4. The use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is R for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma₂Is selected from CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F。
5. Use of a compound according to claim 1 or 4, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma, wherein R₂Is selected from CH₃。
6. The use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is R for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma₃Is selected from CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F。
7. Use of a compound according to claim 1 or 6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma, wherein R₃Is selected from CH₃。
8. The use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is R for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma₄Each independently selected from H, F, Cl, Br, I, CH₃、CF ₃、CH ₂CH ₃、CF ₂CH ₃、CHFCH ₂F and CF₂CH ₂F。
9. Use of a compound according to claim 1 or 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma, wherein R₄Each independently selected from H and F.
10. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, as a structural element in the manufacture of a medicament for the treatment of nasopharyngeal carcinoma

    

    Is selected from

    

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