Design of griifolin prodrugs and their application in inhibiting DNMT1 gene

Abstract

The present invention provides a compound comprising X having at least one hydroxyl group, wherein at least one hydroxyl group is independently substituted by one or more ‑Y‑Z groups, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, or pharmaceutically acceptable salt thereof; X is selected from grifolin or an analog thereof, Y is a linking group, and Z is a solubilizing group. The compound of the present invention is a Grifolin molecular prodrug with good water solubility. After administration, the original drug is slowly released in plasma, stabilizing the bisphenol structure of Grifolin and improving molecular stability. It can effectively inhibit DNMT1 gene expression, thereby contributing to a good anti-cancer effect. The compound provides scientific research value and clinical application value for the study of the DNMT1 gene and the treatment of cancer, and is of great significance.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the design of Grifolin and its application in inhibiting the DNMT1 gene. Background Art

[0002] DNMTs, the full name of which is DNA methylation transferases, is a class of enzymes that can transfer methyl groups from S-adenosyl-L-homocysteine ​​or S-adenosyl-L-methionine (SAM) to cytosine nucleotides in DNA. This enzyme maintains the methylation pattern after DNA replication. In mammals, the DNMTs family mainly includes DNMT1, DNMT3a, and DNMT3b. Among them, DNMT1 is the most abundant DNMT enzyme in DNA methylation maintenance. Abnormal methylation forms lead to the occurrence of many diseases, such as cerebellar ataxia, deafness and drowsiness, neuropathy, heredity, and cancer. Among them, DNMT1 is highly expressed in leukemia, nasopharyngeal carcinoma, colon cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer, and malignant gliomas, and is closely related to the occurrence and development of tumors.

[0003] Recently, the expression of numerous tumor suppressor genes has been found to be associated with overactivation of DNMT1. For example, PTEN, DAPK, E-cadherin, and p16, common tumor suppressor genes, are inactivated in various tumor types due to DNMT1-mediated promoter methylation. Therefore, DNMT1 has become an important target for cancer therapy. However, currently, DNMT1 inhibitors, mostly azacytidine and its nucleoside derivatives, are highly toxic, unstable, and have significant side effects, significantly limiting their clinical application. Therefore, the discovery of a new class of low-toxic DNMT1 expression inhibitors is of great significance.

[0004] Due to the diversity of biological metabolism and the complexity of the ecological environment, compounds derived from traditional natural products generally possess rich structural diversity and most exhibit good biological activity. In today's rapidly developing science and technology, where chemically synthesized drugs dominate, natural products derived from biological secondary metabolism remain an important source of new drug discovery. In 1950, Hirata et al. extracted Grifolin, a class of polyphenolic secondary metabolites with antibiotic properties, from the fruiting bodies of the higher fungus Albatrellus confluens. Data indicate that Grifolin has limited or no toxicity.

[0005] Grifolin's natural abundance is low, and simply extracting it from fungi cannot meet the needs of scientific research or even therapeutic applications. In 2016, Justin T. Mohr et al. reported a total synthesis of grifolin and its derivatives, raising hopes for its practical application. However, the molecular properties of grifolin limit its application. Grifolin, a bisphenol-containing farnesyl group, is unstable and prone to oxidation and deterioration. Furthermore, the aromatic ring and triisoprene-based farnesyl group of grifolin, a bisphenol-containing compound, make it extremely water-soluble. In animal experiments, Cao Ya et al.'s group had to use corn oil for long-term administration. Long-term use caused side effects such as fat accumulation in mouse models, significantly limiting the efficacy and use of grifolin. Therefore, modulating the water solubility and stability of grifolin without affecting the biological activity of this natural product has become an important research goal. Summary of the Invention

[0006] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides a compound, pharmaceutical composition, and use thereof. The compound is a Grifolin molecular prodrug with good water solubility. Upon administration, it slowly releases the original drug into plasma, stabilizes the bisphenol structure of Grifolin, improves molecular stability, and can effectively inhibit DNMT1 gene expression, thereby contributing to a good anti-cancer effect. This compound provides scientific research value and clinical application value for the study of the DNMT1 gene and the treatment of cancer, and is of great significance.

[0007] In one aspect, the present invention provides a compound. According to an embodiment of the present invention, the compound is X having at least one hydroxyl group, each of which is independently substituted with one or more -YZ groups, or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, or pharmaceutically acceptable salt thereof; X is selected from mirificin or an analog thereof, Y is a linking group, and Z is a solubilizing group.

[0008] Among the compounds according to the embodiments of the present invention, the structure of Grifolin is as follows. The inventors have found that by connecting solubilizing groups to the two hydroxyl groups, the water solubility of Grifolin can be improved.

[0009]

[0010] According to an embodiment of the present invention, the above compound may also have the following additional technical features:

[0011] According to an embodiment of the present invention, X is selected from

[0012] R1 and R2 are each independently selected from H or -YZ, and R1 and R2 are not both H.

[0013] It should be noted that the bold chemical bonds in the above structural formula represent that the compound is in any form of cis or trans isomers.

[0014] According to an embodiment of the present invention, Y is selected from a bond, a carbonyl group or -C(=O)-(CH2) n -C(=O)-; n is an integer selected from 1 to 4. The Y group can be used to link Grifolin or its analogs to a solubilizing group without affecting the water solubility and stability of the compound. However, certain other groups may result in low water solubility of the compound, or may cause low stability of the compound, making it easily degraded in the body, shortening the half-life, and thus affecting the efficacy.

[0015] According to an embodiment of the present invention, Z is selected from Amino C 1～6 Alkyl, -O-((CH2) n O) m -R5, C 3～6 Cycloalkyl, C 1～5 a heterocyclic group or a quaternary ammonium salt group;

[0016] The amino C 1～6 Alkyl, cycloalkyl or heterocyclyl may be optionally replaced by one or more H, hydroxyl, C 1～6 Alkyl substituted;

[0017] R3, R4 and R5 are each independently selected from H or C 1～6 alkyl;

[0018] m and n are each independently selected from integers of 1-6.

[0019] The use of the above-mentioned Z group can improve the water solubility and stability of the compound, while certain other groups will lead to low stability of the compound, easy degradation in the body, shorten the half-life, and thus affect the efficacy.

[0020] According to an embodiment of the present invention, Z is selected from Amino C 2～6 Alkyl, -O-((CH2) n O) m -R5, C 3～6 Cycloalkyl, C 2～5 Heterocyclic group (heteroatom can be N, O or S) or quaternary ammonium group (anion can be Cl - 、SO4 2 - or NO3-);

[0021] The amino C 2～6 Alkyl, cycloalkyl or heterocyclyl may be optionally replaced by one or more H, hydroxyl, C 1～4 Alkyl substituted;

[0022] R3, R4 and R5 are each independently selected from H or C 1～6 alkyl;

[0023] m is an integer selected from 1 to 6;

[0024] n is an integer selected from 1-3.

[0025] According to an embodiment of the present invention, the compound has a structure as shown in one of the following formulae or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or pharmaceutically acceptable salt thereof;

[0026]

[0027] Wherein, R is selected from

[0028] Of R6 and R7, one is hydrogen and the other is methyl;

[0029] R8 is selected from one of the following structures:

[0030] R9 is selected from hydrogen or methyl.

[0031] Using esterification as a reaction base and introducing several hydroxyl or oxygen elements to adjust the solvation interaction between the molecule and water molecules, a class of Grifolin molecular prodrugs with good water solubility was synthesized. After administration, this prodrug slowly releases the original drug into plasma, stabilizing the bisphenol structure of Grifolin and improving molecular stability.

[0032] In another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned compound and a pharmaceutically acceptable excipient. The pharmaceutical composition according to the embodiment of the present invention has good water solubility. After administration as a prodrug of Grifolin, it slowly releases the original drug into plasma. This stabilizes the bisphenol structure of Grifolin, improves molecular stability, and can effectively inhibit DNMT1 gene expression, thereby contributing to a good anti-cancer effect. This provides scientific research value and clinical application value for the study of the DNMT1 gene and the treatment of cancer, and is of great significance.

[0033] In another aspect, the present invention provides the use of the aforementioned compounds or pharmaceutical compositions for non-therapeutic inhibition of DNMT1 gene expression. As prodrugs of Grifolin, the compounds of the present invention exhibit enhanced water solubility and stability while still exhibiting a significant inhibitory effect on DNMT1 gene expression. This research lays a foundation for scientific research on the DNMT1 gene and is of high research value.

[0034] In another aspect of the present invention, the present invention provides the use of the aforementioned compounds in the preparation of medicaments. According to embodiments of the present invention, the medicaments are used to treat diseases caused by overexpression of DNMT1. The compounds of the present invention are Grifolin prodrugs with good water solubility. After administration, they slowly release the original drug into plasma, stabilizing the bisphenol structure of Grifolin and improving molecular stability. They can effectively inhibit DNMT1 gene expression, thereby helping to treat diseases caused by overexpression of DNMT1.

[0035] According to an embodiment of the present invention, the drug is used to treat diseases caused by abnormal methylation; the drug is used to activate the expression of genes pten, dapk, E-cadherin and p16.

[0036] According to an embodiment of the present invention, the drug is used to treat cerebellar ataxia, deafness and drowsiness, neuropathy, leukemia, nasopharyngeal carcinoma, colon cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer or malignant glioma.

[0037] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0039] Figure 1 shows a schematic diagram of stability analysis according to one embodiment of the present invention;

[0040] Figure 2 The figure shows the in vivo anti-tumor activity analysis of Grifolin prodrug according to one embodiment of the present invention, (A) is a tumor size analysis diagram; (B) is a tumor mass analysis diagram; (C) is an electron micrograph of a tumor tissue section;

[0041] Figure 3 A schematic diagram of analyzing the methylation status of the promoter region of tumor cells under the treatment of PEG5-Grifolin and 5-AD according to one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0042] Certain embodiments of the present invention are now described in detail, examples of which are illustrated by the accompanying structural formulas and chemical formulae. The present invention is intended to encompass all substitutions, modifications, and equivalent technical solutions, which are all included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0043] It will be further appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0044] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0045] Unless otherwise indicated, the following definitions shall apply as used herein. For purposes of the present invention, the chemical elements are as per the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry may be found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0046] Unless otherwise specified or clearly contradicted by context, the articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more." Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., more than one component may be contemplated for use or use in implementing the described embodiments.

[0047] The term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0048] "Stereoisomers" are compounds that have identical chemical constitutions but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0049] "Chiral" refers to a molecule that is non-superimposable on its mirror image; "achiral" refers to a molecule that is superimposable on its mirror image.

[0050] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0051] "Diastereoisomers" refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of one another. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0052] Stereochemical definitions and conventions used herein generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0053] Many organic compounds exist in optically active forms, meaning they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are the symbols used to designate the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is left-handed. A compound prefixed with (+) or d is right-handed. A specific stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0054] Any asymmetric atom (e.g., carbon, etc.) of the compounds disclosed herein can exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in terms of the (R)- or (S)-configuration.

[0055] Depending on the choice of starting materials and process, the compounds of the present invention may exist as one of the possible isomers or as a mixture thereof, such as a racemate or a mixture of diastereoisomers (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis or trans configuration.

[0056] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, and diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0057] Any racemate of the resulting final product or intermediate can be separated into its optical antipodes by methods familiar to those skilled in the art using known methods, such as by separating the diastereomeric salts obtained. The racemic products can also be separated by chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent. In particular, enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 ndEd.Robert E.Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SHTables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of NotreDame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G.Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0058] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as prototropic tautomers) include interconversions performed by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions performed by the reorganization of some bonding electrons. A specific example of keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-ene-2-one tautomers. Another example of tautomerism is phenol-keto tautomerism. A specific example of phenol-keto tautomerism is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0059] In addition, it should be noted that, unless otherwise explicitly stated, the description methods used in the present invention such as "each...independently is" and "...each independently is" and "...independently is" can be interchanged and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0060] The term "prodrug" as used in the present invention refers to a compound that is converted into a compound represented by formula (I) in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or by enzyme conversion to the parent structure in the blood or tissue. The prodrug compound of the present invention can be an ester. In the existing invention, esters that can be used as prodrugs include phenyl esters, aliphatic (C 1-24 ) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound of the present invention containing a hydroxyl group can be acylated to produce a prodrug form of the compound. Other prodrug forms include phosphate esters, such as these phosphate ester compounds, which are obtained by phosphorylating a hydroxyl group on the parent compound. For a complete discussion of prodrugs, see T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J. Rautio et al., Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270, and SJ Hecker et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0061] "Metabolite" refers to a product resulting from the in vivo metabolism of a specific compound or salt thereof. Metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized using assays such as those described herein. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention encompasses metabolites of the compound, including metabolites produced by contacting a compound of the invention with a mammal for a sufficient period of time.

[0062] As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in S.M. Berge et al., "Describe Pharmaceutically Acceptable Salts in Detail in J. Pharmaceutical Sciences, 1977, 66: 1-19." Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates, or salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 The present invention also contemplates quaternary ammonium salts formed by any compound containing a N group. Water-soluble or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0063] As used herein, a "solvate" refers to an association formed between one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecule is water.

[0064] The present invention does not strictly limit the types of excipients and can be flexibly selected according to the circumstances. For injection preparations, pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers. For preparations for local administration, pharmaceutically acceptable carriers may include alkali, excipients, lubricants and preservatives. The pharmaceutical composition of the present invention can be combined with the above-mentioned pharmaceutically acceptable carriers to be prepared into various dosage forms. For injection preparations, the pharmaceutical composition can be prepared into, for example, ampoules of single-dose dosage forms or unit dosage forms of, for example, multiple-dose containers. The pharmaceutical composition can also be prepared into solutions, suspensions, tablets, pills, capsules and long-acting preparations.

[0065] Among them, according to some specific examples of the present invention, excipients and diluents suitable for pharmaceutical formulations may include: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0066] According to other embodiments of the present invention, the excipients of the present invention may further include fillers, anticoagulants, lubricants, moisturizers, fragrances and preservatives.

[0067] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The conjugates of the present invention can be administered by any common route, as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, nasal, pulmonary and rectal administration, but the present invention is not limited to these exemplified modes of administration. However, since peptides are digested and peptide bonds are broken during oral administration, the active ingredients of compositions for oral administration should be coated or formulated to prevent them from being degraded or destroyed in the stomach. Preferably, the compounds or pharmaceutical compositions of the present invention can be administered in an injectable formulation. In addition, the compounds or pharmaceutical compositions of the present invention can be administered using specific devices that deliver the active ingredients to target cells.

[0068] The frequency and dosage of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the route of administration, the patient's age, sex, weight and severity of the disease, and the type of drug as the active ingredient. According to some embodiments of the present invention, the daily dose can be divided into one, two or more doses in a suitable form, so that it can be administered once, twice or more times over the entire time period, as long as the therapeutically effective amount is achieved.

[0069] The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly improve certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen. For example, in the treatment of cancer, a drug or compound that reduces, prevents, delays, inhibits, or blocks any symptom of a disease or condition should be therapeutically effective. A therapeutically effective amount of a drug or compound does not need to cure the disease or condition, but will provide treatment for the disease or condition such that the onset of the disease or condition is delayed, stopped, or prevented in an individual, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or, for example, the disease or condition is made less severe, or recovery is accelerated.

[0070] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect, such as inhibiting the growth of cancer cells, causing the death of cancer cells, or ameliorating a disease or condition. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers the treatment of diseases (primarily cancer) in mammals, particularly humans, including: (a) preventing the occurrence of a disease (e.g., preventing cancer) or a condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. As used herein, "treatment" covers any medication that administers a pharmaceutical composition to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administering a drug containing a conjugate described herein to an individual in need.

[0071] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0072] Example 1

[0073] 1. Synthetic intermediate RY-WLG-GI1

[0074]

[0075] Dissolve 984 mg of Grifolin in 20 ml of anhydrous dichloromethane, purge with nitrogen, and add 475 mg of anhydrous pyridine and 37 mg of DMAP. Add 601 mg of succinic anhydride in three batches over 6 hours. Stir for 12 hours and then spin dry to remove the solvent. Purify the product using a silica gel column to obtain 1707 mg of RY-WLG-GI. LC-MS: calculated for C30 H 40 O8[MH] - :527.27,found 527.02.

[0076] 2. Synthetic product RY-WLG-GP1

[0077]

[0078] Dissolve 528 mg of RY-WLG-GI1 in 5 ml of dichloromethane, add 230 mg of EDCI and 12 mg of DMAP, stir at room temperature for 15 minutes, then add 620 mg of ethane-1,2-diol. Continue stirring at room temperature for 12 hours. Remove the dichloromethane by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Purify the product RY-WLG-GP1408 mg by silica gel column. LC-MS: calculated for C 34 H 48 O 10 [M+H] + :617.32,found 617.38.

[0079] 3. Synthetic product RY-WLG-GP2

[0080]

[0081] Dissolve 528 mg of RY-WLG-GI1 in 5 ml of dichloromethane, add 230 mg of EDCI and 12 mg of DMAP, and stir at room temperature for 15 minutes. Then, add 1060 mg of 2,2'-oxybis(ethan-1-ol) and continue stirring at room temperature for 12 hours. Remove the dichloromethane by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Purify the product RY-WLG-GP by silica gel column separation to obtain 2389 mg. LC-MS: calculated for C 38 H 56 O 12 [M+H] + :705.38,found 705.44.

[0082] 4. Synthetic product RY-WLG-GP3

[0083]

[0084] Dissolve 528mg of RY-WLG-GI1 in 5ml of dichloromethane, add 230mg of EDCI and 12mg of DMAP, and stir at room temperature for 15 minutes. Then add 1500mg of 2,2'-(ethane-1,2-diylbis(oxy))bis(ethan-1-ol) and continue stirring at room temperature for 12 hours. Remove the dichloromethane by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Separate and purify the product RY-WLG-GP3 by silica gel column to obtain 414mg. LC-MS: calculated for C 42 H 64 O 14 [M+H] + :793.43,found 793.87.

[0085] 5. Synthetic product RY-WLG-GP4

[0086]

[0087] Dissolve 528mg of RY-WLG-GI1 in 5ml of dichloromethane, add 230mg of EDCI and 12mg of DMAP, and stir at room temperature for 15 minutes. Then add 1942mg of 2,2'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethan-1-ol), and continue stirring at room temperature for 12 hours. Remove the dichloromethane by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Separate and purify the product RY-WLG-GP4 by silica gel column to obtain 477mg. LC-MS: calculated for C 46 H 72 O 16 [M+H] + :881.48,found 882.01.

[0088] 6. Synthetic product RY-WLG-GP5

[0089]

[0090] Dissolve 528mg of RY-WLG-GI1 in 5ml of dichloromethane, add 230mg of EDCI and 12mg of DMAP, stir at room temperature for 15 minutes, then add 2383mg of 3,6,9,12-tetraoxatetradecane-1,14-diol. Continue stirring at room temperature for 12 hours. Remove the dichloromethane by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Separate and purify the product RY-WLG-GP5 by silica gel column to obtain 331mg. LC-MS: calculated for C 50 H 80 O 18 [M+H] + :969.53,found 970.11. 1 H-NMR (400MHz, CDCl3, ppm): 6.76 (s, 2H), 5.06 (m, 3H), 4.26 (t, J = 4.12, 4H), 3.70-3.60 (m, 36H), 3.12 (d, J = 6.16, 2H), 2.87(t,J=6.52,4H),2.76(m,6H),2.29(s,3H),2.03-1.93(m,8H),1.70(s,3H),1.66(s,3H),1.58(s,3H),1.56(s,3H). 13 C-NMR (100MHz, CDCl3, ppm):172.20,170.78,149.44,137.22,135.80,135.20,131.38,124.49,124.11,123.50,121.37,120.93, 72.66,70.70,70.67,70.64,70.39,69.16,64.07,61.82,29.18,29.08,26.84,26.71,25.83,23.66,21.10,17.81,16.43,16.14.

[0091] 7. Synthetic product RY-WLG-GP6

[0092]

[0093] Dissolve 528mg of RY-WLG-GI1 in 5ml of dichloromethane, add 230mg of EDCI and 12mg of DMAP, stir at room temperature for 15 minutes, then add 356mg of 2-amino-2-methylpropan-1-ol. Continue stirring at room temperature for 12 hours. Remove the dichloromethane by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Separate and purify the product RY-WLG-GP6 by silica gel column to obtain 593mg. LC-MS: calculated for C 38 H 58 N2O8[M+H] + :671.42,found 671.33. 1 H-NMR (400MHz, CDCl3, ppm): 6.75 (s, 2H), 5.75 (s, 2H), 5.07 (m, 3H), 3.56 (s, 4H), 3.11 (d, J = 5.68, 2H), 2.90 (t, J = 6.12 ,4H),2.51(m,4H),2.29(s,3H),2.01-1.93(m,8H),1.70(s,3H),1.66(s,3H),1.58(s,3H),1.57(s,3H),1.26(s,12H). 13 C-NMR (100MHz, CDCl3, ppm):172.00,171.60,149.45,137.36,135.95,135.24,131.43,124.46,124.09,123.58,121.2 6,120.95,70.35,56.38,39.81,39.75,31.53,29.82,29.62,26.85,26.73,25.84,23.69,21.11,17.82,16.44,16.16.

[0094] 8. Synthetic product RY-WLG-GP7

[0095]

[0096] Dissolve 528 mg of RY-WLG-GI1 in 5 ml of a tetrahydrofuran and dioxane mixture (v / v = 1:1) in a rotary evaporator. Add 230 mg of EDCI and 12 mg of DMAP. Stir at room temperature for 15 minutes, then add 920 mg of glycerol. Continue stirring at room temperature for 12 hours. Remove the tetrahydrofuran and dioxane mixture by rotary evaporation. Add ethyl acetate and saturated sodium chloride solution, and separate the organic phase. Remove the ethyl acetate solvent by rotary evaporation. Purify the product using a silica gel column to obtain 529 mg of RY-WLG-GP7. LC-MS: calculated for C 36 H 52 O 12 [M+H] + :677.35,found677.27. 1 H-NMR (400MHz, CDCl3, ppm): 6.76 (s, 2H), 5.07 (m, 3H), 4.23-4.14 (m, 4H), 3 .89(t,J=4.24,2H),3.64(dd,J=2.92,J=11.36,2H),3.55(dd,J=5.76,J=11 .16,2H),3.11(d,J=6.32,2H),2.90(t,J=5.76,4H),2.74(m,8H),2.30(s,3 H),2.05-1.93(m,8H),1.72(s,3H),1.67(s,3H),1.58(s,3H),1.57(s,3H). 13 C-NMR (100MHz, CDCl3, ppm):172.50,171.24,149.36,137.49,136.09,135.31,131.45,124.47,124.05,123.68,1 21.20,70.13,65.75,63.32,39.81,39.72,29.28,29.10,26.85,26.68,25.84,23.70,21.09,17.82,16.42,16.15.

[0097] 9. Synthetic product RY-WLG-GP8

[0098]

[0099] Dissolve 486 mg of carbonyldiimidazole in 20 ml of anhydrous dichloromethane. Slowly add 300 mg of 1-methylpiperazine under ice-cooling, fill with argon for protection, stir for 1 hour, then slowly add 328 mg of Grifolin. Continue stirring until Grifolin reacts completely. Remove the dichloromethane by rotary evaporation, add ethyl acetate, and extract and wash with saturated sodium chloride. Separate and retain the ethyl acetate, remove the ethyl acetate, and separate and purify by silica gel chromatography to obtain 93 mg of RY-WLG-GP8. LC-MS: calculated for C 34 H 52 N4O4[M+H] + :581.41,found 581.88.

[0100] 10. Synthetic product RY-WLG-GP9

[0101]

[0102] Dissolve 328mg of Grifolin in 20ml of anhydrous dichloromethane, purge with argon, add 316mg of anhydrous pyridine, and slowly add 303mg of dimethylglycinoyl chloride in an ice bath. Stir until the reaction is complete as determined by TLC. Remove the dichloromethane, and purify the product by silica gel column chromatography to obtain 56mg of the final product, RY-WLG-GP9. LC-MS: calculated for C 30 H 46 N2O4[M+H] + :499.35,found 499.45.

[0103] 11. Synthetic product RY-WLG-GP10

[0104]

[0105] Dissolve 328mg of Grifolin in 20ml of anhydrous dichloromethane, purge with argon, add 316mg of anhydrous pyridine, and slowly add 430mg in an ice bath. Stir until the reaction is complete as determined by TLC. Remove the dichloromethane, and purify by HPLC to obtain 46mg of the final product, RY-WLG-GP10. LC-MS: calculated for C 32 H 52 N2O4 2+ 528.39, found 264.33.

[0106] Example 2

[0107] The solubility of the compounds RY-WLG-GP1-10 and Grifolin prepared in Example 1 was determined by the following method:

[0108] Accurately weigh 4 mg of the compound to be tested and dissolve it in V ml of acetonitrile to prepare a standard concentration stock solution (C mg / ml). This stock solution is then diluted with acetonitrile to obtain a gradient dilution solution with concentrations of 1 / 2C, 1 / 4C, 1 / 8C, 1 / 16C, 1 / 32C, and 1 / 64C.

[0109] Measure the peak absorbance area of ​​1 ml of the serial dilutions by HPLC to generate a standard curve. Then, weigh an excess of the prodrug compound, add PBS buffer, and shake overnight. After centrifugation, remove the supernatant, filter through a microporous membrane, and measure the peak area A. Substitute A into the standard curve to determine the solubility of the prodrug. Testing conditions: 25°C, PBS (pH 7.4).

[0110] The results are shown in Table 1. It can be seen that the solubility of compounds GP1 to GP10 of the present invention is improved to varying degrees compared with Grifolin.

[0111] Table 1 Solubility

[0112]

[0113]

[0114]

[0115] Example 3

[0116] Compounds RY-WLG-GP1 to 10 have similar stability. The stability study of compound RY-WLG-GP5 is carried out as an example. The specific method is as follows:

[0117] Accurately weigh the RY-WLG-GP5 compound and add an equal amount of PBS buffer (pH = 7.4). Place at room temperature for 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, and 8 days. Referring to the method described in Table 1 above, plot the ratio of the peak area of ​​1 to 8 days to the peak area of ​​0 day, and draw a standard degradation curve as shown in the figure below. Figure 1 As shown in Figure 2, the half-life is approximately 68 days according to the nonlinear regression curve.

[0118] Example 4

[0119] The Grifolin prodrug (RY-WLG-GP5) was subjected to the following animal experiments:

[0120] 1. Culture medium

[0121] The culture conditions used for the human nasopharyngeal carcinoma cell line C666-1 are as follows:

[0122] RPMI1640 medium, 10% v / v heat-inactivated fetal bovine serum (FBS), 1% w / v glutamine and 1% w / v double-antibody, 37 degrees Celsius, 5% carbon dioxide cell culture incubator

[0123] 2. Reagents and Materials

[0124] 5-Aza (Decitabine) was purchased from MedChemExpress

[0125] 3. Animal transplant tumor experiments

[0126] This experiment used 5-week-old female BALB / c immunodeficient mice. 4×10 6 Nasopharyngeal carcinoma cell line C666-1, wait until the cells grow to 80-100mm 3 Afterwards, the mice were randomly divided into three groups: control group, 5-Aza group and prodrug group, with 8 mice in each group. The control group was injected with PBS solution daily, the 5-Aza group was injected with 1 mg / kg of 5-Azacytidine (abbreviated as 5-AD) daily, and the prodrug group was injected with 90 mg / kg of RY-WLG-GP5 (PEG5-Grifolin) compound daily. The drug was administered once every two days for 17 days. The tumor size and mouse survival were measured daily. After the experiment, the mice were killed by carbon dioxide, and the tumor tissues were removed and weighed. The results are as follows Figure 2 (A) and (B) show that treatment with the griifolin prodrug RY-WLG-GP5 (PEG5-Grifolin) and 5-AD both inhibited tumor cell growth and reduced tumor volume and mass. Furthermore, mice treated with the RY-WLG-GP5 (PEG5-Grifolin) prodrug showed no significant changes in survival, whereas mice treated with 5-AD died.

[0127] The tumor tissue was further sectioned, dewaxed, and freeze-dried. Primary antibodies (anti-DNMT1, anti-DAPK, anti-PTEN, and anti-Ecadherin) were then incubated overnight at 4°C. DAB was used for color development and counterstained with a hematoxylin staining kit. Figure 2(C) shows that Grifolin prodrug reactivates the expression of tumor suppressor genes PTEN, DAPK, E-cadherin and p16 by downregulating DNMT1. The tumor tissue was sectioned and IHC experiments were performed using the corresponding antibodies. The antibody-bound part was stained brown and the cell nucleus was stained blue. Three parallel experiments were set up for each experiment. Asterisks (*, **) represent different significant differences (p<0.05, p<0.01), and NS represents no significant difference. From the staining results, it can be seen that under the treatment of RY-WLG-GP5 (PEG5-Grifolin) and 5-AD, the expression of DNMT1 in tumor cells decreased, among which RY-WLG-GP5 (PEG5-Grifolin) decreased more significantly. At the same time, the decreased DNMT1 increased the expression of tumor suppressor genes such as DAPK1, PTEN, E-cad, and p16.

[0128] Tumor mouse model tissues were isolated and extracted and tested using the BSP method. Specifically, genomic DNA was extracted using a tissue DNA kit (QIAGEN, Inc., Hilden, Germany) and quantified using EZ DNA Methylation-Gold. TM Kit (Zymo Research, USA). The corresponding gene promoter sequence was amplified from the isolated DNA using touchdown PCR, extracted from an agarose gel, and loaded into a Puc18-T vector (Sangon Biotech Co., Ltd, Shanghai, China). TA cloning and sequencing were performed by Shanghai Sangon Biotech. Ten individual clones were selected for each group, and the number of methylated CpG sites detected was divided by 10 clones to calculate the methylation percentage of each CpG site. The total methylation status of the core CpG region within each promoter group was calculated by averaging the methylation rate of each CpG site.

[0129] The results are as follows Figure 3 As shown, Grifolin prodrug RY-WLG-GP5 (PEG5-Grifolin) and 5-AD can cause demethylation of the promoter region of tumor suppressor genes (TSGs), and the effect is better than 5-AD.

[0130] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0131] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A compound, characterized in that The compound is X, a stereoisomer or a pharmaceutically acceptable salt thereof; X is selected from R1 and R2 are each independently selected from -YZ; Y is a linking group, and Z is a solubilizing group; Y is selected from carbonyl or -C(=O)-(CH2) n -C(=O)-, wherein n is selected from an integer of 1 to 4; Z is selected from C 2～5 Heterocyclic group or quaternary ammonium salt group, wherein C 2～5 The heteroatom of the heterocyclic group is selected from N, O, and S, and n is selected from an integer of 1 to 6; The heterocyclic group may be optionally substituted with one or more hydroxyl groups, C 1～6 Alkyl substituted; R3 and R4 are each independently selected from H or C 1～6 alkyl.

2. A compound, characterized in that The compound has a structure as shown in one of the following formulae or a stereoisomer or a pharmaceutically acceptable salt thereof; Wherein, R is selected from Of R6 and R7, one is hydrogen and the other is methyl; R8 is selected from one of the following structures: R9 is selected from hydrogen or methyl.

3. A pharmaceutical composition, characterized in that include: The compound according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to claim 3 for inhibiting DNMT1 gene expression for non-therapeutic purposes.

5. Use of the compound according to claim 1 or 2 in preparing a medicament, characterized in that: The medicine is used to treat diseases caused by high expression of DNMT1.

6. The use according to claim 5, characterized in that The drug is used to treat diseases caused by abnormal methylation; The drug is used to activate the expression of genes pten, dapk, E-cadherin and p16.

7. The use according to claim 5, characterized in that The medicine is used to treat cerebellar ataxia, deafness and drowsiness, neuropathy, leukemia, nasopharyngeal cancer, colon cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer or malignant glioma.

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