CN116063370A

CN116063370A - Bufogenin derivative, preparation method thereof, composition, preparation and application

Info

Publication number: CN116063370A
Application number: CN202211654524.3A
Authority: CN
Inventors: 王娟; 缪玉兰
Original assignee: Shanghai Miaoji Biotechnology Co ltd
Current assignee: Shanghai Miaoji Biotechnology Co ltd
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-05-05

Abstract

A bufalin derivative is a new bufalin series derivative obtained by introducing different phosphate groups into the structure of the existing bufalin and pharmaceutically acceptable salts thereof, has excellent anti-tumor activity and lower hERG potassium channel inhibition activity, has greatly reduced toxic and side effects, and can be used for preparing various anti-tumor medicines, cardiovascular and cerebrovascular disease treatment medicines and nervous system disease treatment medicines. The bufalin derivative provided by the invention has various alternative structural forms and pharmaceutically acceptable salts thereof, can meet the requirements of preparing various pharmaceutical compositions, pharmaceutical preparations and the like, has wide application prospect, is simple and convenient in preparation method, has low-cost and easily-obtained raw materials, is various in selection and easy to realize industrial production, and therefore has popularization and application values.

Description

Bufogenin derivative, preparation method thereof, composition, preparation and application

Technical Field

The invention relates to a bufalin derivative, in particular to a bufalin derivative, a preparation method thereof, a composition, a preparation and application thereof, and belongs to the technical field of medicines.

Background

Bufalin (English: bufalin) is a Bufalin lactone compound extracted from traditional Chinese medicine Bufonis venenum in China, has various biological activities of easing pain, strengthening heart, resisting tumor and the like, and has the chemical structure as follows:

researches show that bufalin can remarkably inhibit the expression of liver cancer stem cell markers CD133, CD44 and ESA, and has the effect of inhibiting liver cancer stem cells (journal of Chinese medicinal information, 2021, 12:41-44); in addition, bufogenin concentration dependence and time dependence inhibit melanoma A375 cell proliferation, significantly increase activity of Caspase-9 and Caspase-3 proteins in A375 cells, enhance apoptosis of A375 cells and block cell cycle in S phase (university of Right river medical school, 2021, 6:719-724).

Studies have shown that bufalin also inhibits proliferation of ovarian cancer cells SK-OV-3 in a concentration-dependent and time-dependent manner through EGFR/AKT/ERK signaling pathways (Dou L, at al, chin Med J (Engl) (2021) 135 (4): 456-461); inhibition of STAT3 signaling pathway inhibits tumor microenvironment mediated angiogenesis (Kai F, at al, J Transl Med (2021) 19 (1): 383).

Studies have also found that bufogenin induces death of neuroblastoma cells U87 by apoptosis and cell necrosis (Hai Rui LH, at al, onco Targets Ther (2020) 13:4767-4778).

Although bufogenin has excellent anti-tumor activity, the bufogenin has relatively large toxicity and relatively narrow therapeutic index, so that structural modification is necessary on the basis, the activity of the compound is enhanced, the toxicity is reduced, and a novel anti-tumor medicament is discovered.

In addition, bufogenin has obvious cardiotoxicity, so that development of the bufogenin as a medicament is limited (Min L, at al, chin J Nat Med (2020) 18 (7): 550-560), and therefore, the bufogenin derivative is synthesized and the structure-activity relationship is studied, so that the bufogenin derivative with high efficiency and low toxicity is found to have important significance.

At present, various patent documents disclose structural modification researches based on bufotalin and technical schemes for application thereof, for example:

WO201185641A1 discloses a class of bufalin derivatives and their use for the treatment of cancer;

CN102532235 discloses a bufogenin derivative, a preparation method thereof and use of the composition containing the derivative;

in addition, patent documents such as CN102656179, CN103980337, CN110483608 and CN103980338 also respectively disclose a bufalin derivative, a pharmaceutical composition and application thereof, and the main contribution of the bufalin derivative is to esterify the hydroxyl at the 3-position of bufalin to obtain a series of derivatives.

Obviously, the current research is not deep enough, and related research and development are still to be continued so as to better contribute to guaranteeing the physical and mental health of people.

Disclosure of Invention

Based on the above purpose, the invention firstly provides a novel bufalin derivative and a preparation method thereof, and further provides a pharmaceutical composition of the bufalin derivative, a preparation of the bufalin derivative and application of the bufalin derivative.

To achieve the above object, the present invention provides a bufalin derivative:

the bufalin derivative is a compound with a structure shown in a formula I, a formula II or a formula III and pharmaceutically acceptable salts of the compound:

wherein:

R ₁ any one selected from hydrogen, deuterium, and substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;

R ₂ 、R ₃ each independently selected from hydrogen, deuterium, hydroxy, halogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkylAny one of heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;

R ₄ And R is R ₁ To form a substituted or unsubstituted nitrogen-containing heterocycle;

R ₅ selected from hydrogen, deuterium, and any one of substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl, or R ₅ And R is R ₁ To form a substituted or unsubstituted nitrogen-containing heterocycle;

R ₆ 、R ₇ each independently selected from hydrogen, and any of substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;

R ₈ 、R ₉ 、R ₁₀ 、R ₁₁ each independently selected from any one of hydrogen, deuterium, hydroxy, halogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;

R ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ each independently selected from any one of hydrogen, deuterium, hydroxy, halogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;

A is C-containing ₁ ～C ₆ Linear or branched alkanes.

Preferably, in said compounds having the structure of formula II, or III, and pharmaceutically acceptable salts thereof:

the R is ₆ 、R ₇ Each independently selected from 3-8 membered cycloalkyl, C ₆ ～C ₁₂ Aryl, aryl groupC after each has been replaced by one or more optional T groups ₁ ～C ₈ Alkyl, 3-8 membered cycloalkyl C ₁ ～C ₈ Alkyl, 3-8 membered heterocyclic group C ₁ ～C ₈ Any one of alkyl, 3-8 membered alkyl containing ether bond or thioether bond;

the R is ₈ 、R ₉ 、R ₁₀ 、R ₁₁ Each independently selected from 3-8 membered cycloalkyl, 3-8 membered alkyl containing thioether linkages, and C substituted with one or more optional T groups ₁ ～C ₈ Alkyl, 3-8 membered cycloalkyl C ₁ ～C ₈ Alkyl, 3-8 membered heterocyclic group C ₁ ～C ₈ Any one of alkyl, 3-8 membered alkyl containing ether bond or thioether bond;

the R is ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ Each independently selected from 3-8 membered cycloalkyl, 3-8 membered alkyl containing thioether linkages, and C substituted with one or more optional T groups ₁ ～C ₈ Alkyl, 3-8 membered cycloalkyl C ₁ ～C ₈ Alkyl, 3-8 membered heterocyclic group C ₁ ～C ₈ Any one of alkyl, 3-8 membered alkyl containing ether bond or thioether bond;

the T group is selected from F, cl, br, I, OH, OCH ₃ 、OCH ₂ CH ₃ 、SCH ₃ 、SCH ₂ CH ₃ 、NHBoc、NHC(＝O)CH ₃ 、NHC(＝O)CH ₂ CH ₃ 、C(＝O)NH ₂ 、C(＝O)OC(CH ₃ ) ₃ 、C(＝O)OCH ₂ CH ₃ 、CH ₂ F、CHF ₂ 、CF ₃ Any one of the groups.

Further preferred is:

in said compounds having the structure of formula II, or III, and pharmaceutically acceptable salts thereof:

The R is ₆ 、R ₇ Each independently selected from 3-8 membered cycloalkyl, C, substituted with one or more optional T groups ₆ ～C ₁₂ Any one of aryl groups;

the R is ₈ 、R ₉ 、R ₁₀ 、R ₁₁ Each independently selected from ether linkage-containing 3-to 8-membered alkyl groups substituted with one or more of the optional T groups;

the R is ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ Each independently selected from 3-8 membered alkyl substituted with one or more optional substituents of the T group;

Even further preferred bufalin derivatives are:

a compound having a structure represented by formulas 1 a-1 l:

further:

the pharmaceutically acceptable salt of the compound is a salt formed after the compound reacts with pharmaceutically acceptable inorganic acid or organic acid;

wherein:

the inorganic acid is any one of hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid or sulfuric acid;

the organic acid is any one of formic acid, acetic acid, propionic acid, succinic acid, 1, 5-naphthalene disulfonic acid, sub-fine acrylic acid, carbenoxolone, glycyrrhetinic acid, oleanolic acid, crataegolic acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

Secondly, the invention also provides a preparation method of the bufalin derivative with the structure shown in the formula I:

the preparation method comprises the following step e, or steps a-b and e, or steps c-d and e in the synthetic route:

wherein:

in the step a, a compound IV and a compound V react in an organic solvent with a first basic catalyst to obtain a compound VI, wherein the molar ratio of the compound IV to the compound V to the first basic catalyst is 1:1-3:1-3;

in the step b, the compound VI is catalyzed by an acid catalyst in an organic solvent to prepare a compound VII;

in the step c, a compound VIII and a compound V are reacted in an organic solvent with a first basic catalyst to prepare a compound IX, and the mol ratio of the compound VIII to the compound V and the first basic catalyst is 1:1-3:1-3;

in the step d, the compound IX is catalyzed by an acid catalyst in an organic solvent to prepare the compound X;

in step e, the compound XI and the compound VII or the compound XI and the compound X are reacted in an organic solvent having a second basic catalyst to obtain the compound I, and the molar ratio of the compound XI to the compound VII and the second basic catalyst or the molar ratio of the compound XI to the compound X and the molar ratio of the compound XI to the second basic catalyst are respectively=1: (1 to 5): (1 to 5).

Further:

the first alkaline catalyst is an inorganic alkaline compound, the second alkaline catalyst is a nitrogen-containing compound, and the acidic catalyst is an inorganic acid or an organic acid.

Optionally:

the inorganic alkaline compound is any one or a mixture of a plurality of sodium carbonate, potassium carbonate or cesium carbonate;

the nitrogen-containing compound is any one or a mixture of a plurality of 4-dimethylaminopyridine, triethylamine, pyridine and the like;

the acid catalyst is any one of hydrochloric acid, sulfuric acid, trifluoroacetic acid, acetic acid and formic acid;

the organic solvent is any one or a mixture of more of dichloromethane, dichloroethane, ethyl acetate, acetonitrile or tetrahydrofuran.

Furthermore, the invention also provides a pharmaceutical composition of the bufalin derivative:

the pharmaceutical composition comprises a therapeutically effective amount of the bufalin derivative or a pharmaceutically acceptable salt of the bufalin derivative, and one or more of a pharmaceutically acceptable carrier, excipient and auxiliary materials.

Furthermore, the invention also provides a pharmaceutical preparation of the bufalin derivative:

the pharmaceutical preparation is at least one of injection, powder injection, emulsion for injection, tablet, pill, capsule, ointment, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment or nano preparation prepared from the pharmaceutical composition, wherein:

The injection is at least one of a small-capacity injection, a medium-capacity injection or a large-capacity injection, and the nano preparation is liposome.

Furthermore, the invention also provides an application of the bufalin derivative:

the application is that the bufalin derivative or the pharmaceutically acceptable salt of the bufalin derivative with the effective treatment dose is used for preparing at least one of antitumor drugs, cardiovascular and cerebrovascular disease treatment drugs or nervous system disease treatment drugs, wherein:

the antitumor drug is used for treating at least one malignancy of the esophagus, stomach, intestine, rectum, mouth, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system growth, and at least one of thyroid cancer, leukemia, huo Jinshi disease, lymphoma and myeloma of a human or animal; or (b)

The application is that the bufalin derivative or the pharmaceutically acceptable salt of the bufalin derivative is used for preparing medicines for inhibiting tumor metastasis.

The term "alkyl" as used in the present invention refers to:

Saturated straight or branched monovalent hydrocarbon groups having one to twelve carbon atoms, and the hydrogen atoms in the hydrocarbon groups may also be independently substituted with one or more substituents, examples of which include, but are not limited to:

methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3-dimethyl-2-butyl, 1-heptyl, 1-octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term "alkenyl" as used in the present invention means:

having at least one unsaturated site, i.e. carbon-carbon sp ² Straight or branched chain monovalent hydrocarbon groups of two to twelve carbon atoms of the double bond, and the hydrogen atoms in the alkenyl group may be optionally independently substituted with one or more substituents, and further includes those having "cis" and "trans" orientations or "E" and "Z" orientations, examples of which include, but are not limited to:

Vinyl, allyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 5-hexenyl, 1-cyclohex-2-enyl and 1-cyclohex-3-enyl.

The term "alkynyl" as used in the present invention refers to:

at least one unsaturated site, i.e. carbon-carbon sp ³ Straight or branched chain monovalent hydrocarbon groups of two to twelve carbon atoms of the triple bond, and the hydrogen atoms in the alkynyl group may be optionally substituted independently with one or more substituents, examples of which include, but are not limited to:

ethynyl and propynyl.

The term "cycloalkyl" as used in the present invention refers to:

monovalent non-aromatic saturated or partially saturated cyclic hydrocarbon radicals having three to ten carbon atoms examples include, but are not limited to:

cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cycloundecyl, cyclododecyl; and is also provided with

Also included are bicyclic and tricyclic and further cycloalkyl structures, wherein the polycyclic structure optionally includes saturated or partially unsaturated cycloalkyl fused to a saturated or partially unsaturated cycloalkyl or heterocyclyl or aryl or heteroaryl ring, wherein a bicyclic carbocycle having 7 to 12 atoms may be arranged as a bicyclo [4,5], [5,6] or [6,6] system, or as a bridging system including bicyclo [2.2.1] heptane, bicyclo [2.2.2] heptoctane and bicyclo [3.2.2] nonane.

The term "heteroalkyl" as used in the present invention refers to:

a saturated straight or branched monovalent hydrocarbon group having one to twelve carbon atoms, at least one of which is replaced with a heteroatom selected from nitrogen, oxygen or sulfur, and the nitrogen, oxygen or sulfur atom replacing at least one of which may occur in the middle or end of the hydrocarbon group, may be a carbon group or a heteroatom group, and the hydrogen atom on the hydrocarbon group of the heteroalkyl group may also be optionally independently substituted with one or more substituents;

in addition, the heteroalkyl groups include both alkoxy and heteroalkoxy groups.

The term "heteroalkenyl" as used herein means:

examples of straight or branched monovalent hydrocarbon groups containing at least one double bond and two to twelve carbon atoms include, but are not limited to, vinyl, propenyl, wherein at least one carbon atom of the hydrocarbon group is replaced with a heteroatom selected from nitrogen, oxygen and sulfur, and the nitrogen, oxygen or sulfur atom replacing at least one carbon atom of the hydrocarbon group may occur in the middle or at the end of the hydrocarbon group, i.e., may be a carbon group or heteroatom group;

in addition, the hydrogen atoms on the hydrocarbyl groups of the heteroalkenyl groups may also be optionally independently substituted with one or more substituents, and include substituents having "cis" and "trans" orientations or "E" and "Z" orientations.

The term "heteroalkynyl" as used in the present invention refers to:

examples of straight or branched monovalent hydrocarbon groups containing at least one triple bond and two to twelve carbon atoms include, but are not limited to, ethynyl, propynyl, wherein at least one carbon atom is replaced with a heteroatom selected from nitrogen, oxygen and sulfur, and the nitrogen, oxygen or sulfur atom replacing at least one carbon atom in the hydrocarbon group may occur in the middle or at the end of the hydrocarbon group as a carbon group or heteroatom group, and further, the hydrogen atom on the hydrocarbon group of the heteroalkynyl group may be optionally independently substituted with one or more substituents.

The terms "heterocyclyl" and "heterocycle" as used in the present invention are synonymous, and are used interchangeably and are defined as:

a saturated or partially unsaturated carbocyclic group having from 3 to 8 ring atoms, and wherein at least one ring atom is a heteroatom independently selected from nitrogen, oxygen and sulfur, the remaining ring atoms being carbon atoms, and wherein one or more ring atoms are also optionally independently substituted with one or more substituent groups, which substituent groups may be carbon groups or heteroatom groups;

furthermore, the term "heterocyclyl" also includes heterocycloalkoxy and heterocyclyl rings in which the heterocyclyl is fused to a saturated, partially unsaturated or fully unsaturated, i.e., aromatic, carbocyclic or heterocyclic ring, examples of which include, but are not limited to:

Pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, 4-thiomorpholinyl, thiazanyl (English: thioxanyl), piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepinyl, thietanyl (English: thiepanyl), oxazanyl

Radical (English: oxazepinyl), diaza->

Radical, thiazal->

Radicals (English: thiazepinyl), 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1, 3-dioxacyclopentyl, pyrazolinyl, dithiocyclohexyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0 ]]Hexane, 3-azabicyclo [4.1.0 ]]Heptyl and azabicyclo [2.2.2]Hexyi, 3H-indolyl, quinolizinyl, and N-pyridylurea;

furthermore, spiro moieties are also included within the scope of the term "heterocyclyl";

in addition, the term "heterocyclyl" as used in the present invention may be C-linked or N-linked as long as it is technically feasible, for example, the groups derived from pyrrole may be N-linked pyrrol-1-yl or C-linked pyrrol-3-yl;

Furthermore, the imidazole-derived group may be an N-linked imidazol-1-yl or a C-linked imidazol-3-yl group, examples of heterocyclic groups in which 2 ring carbon atoms are partially substituted by oxo (c=o) being dihydro-isoindole-1, 3-dione and 1, 1-dioxothiomorpholinyl;

the heterocyclyl groups may be unsubstituted or substituted by various technically feasible groups at one or more of the substitutable positions as indicated.

The term "aryl" as used herein refers to:

an optionally substituted mono-or polycyclic group or ring system containing at least one aromatic hydrocarbon ring, including but not limited to:

phenyl, naphthyl, fluorenyl, azulenyl, anthracyl, phenanthryl, pyrenyl, biphenyl and biphenyl.

The term "heteroaryl" as used herein refers to:

an optionally substituted monocyclic or multicyclic group or ring system comprising at least one aromatic ring having one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein:

examples of monocyclic heteroaryl groups include, but are not limited to, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl;

Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazole, benzoxazolyl, furopyridinyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridinyl, pyrrolopyridinyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidinyl, and thienopyridinyl;

examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perizolyl, phenanthroline, phenanthridinyl, porphanozinyl, porphyrazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl.

The term "arylalkyl" as used herein refers to:

an alkyl group substituted with one or more aryl moieties as defined above, wherein the alkyl group is as defined above for alkyl, examples of arylalkyl groups include aryl-C _1-3 Alkyl groups, including, for example, but not limited to: benzyl, phenylethyl.

The term "heteroarylalkyl" as used herein refers to:

an alkyl group partially substituted with a heteroaryl group as defined above, wherein the alkyl group has the same definition as the alkyl group described above, examples of heteroarylalkyl groups include five-or six-membered heteroaryl-C _1-3 -alkyl groups including, but not limited to, oxazolylmethyl, pyridylethyl.

The term "heterocyclylalkyl" as used herein refers to:

an alkyl group partially substituted with a heterocyclic group as defined above, wherein the alkyl group has the same definition as the alkyl group as defined above, and examples of the heterocyclylalkyl group include five-membered or six-membered heteroaryl-C _1-3 Alkyl groups including, but not limited to, tetrahydropyranylmethyl.

The term "cycloalkylalkyl" as used herein refers to:

an alkyl group partially substituted with a cycloalkyl group as defined above, wherein the alkyl group has the same definition as the alkyl group described above, examples of cycloalkylalkyl groups include five-or six-membered cycloalkyl-C _1-3 -alkyl groups including, but not limited to, cyclopropylmethyl.

The term "substituted alkyl" as used herein refers to:

alkyl groups in which one or more hydrogen atoms are each independently replaced with a D substituent, including but not limited to:

F、Cl、Br、I、CN、CF ₃ 、OR、R、＝O、＝S、＝NR、＝N ⁺ (O)(R)、＝N(OR)、＝N ⁺

(O)(OR)、＝N-NRR′、-C(＝O)R、-C(＝O)OR、-C(＝O)NRR′、-NRR′、-N ⁺ RR′R″、-N(R)C(＝O)R′、-N(R)C(＝O)OR′、-N(R)C(＝O)NR′R″、-SR、-OC(＝O)R、-OC(＝O)OR、-OC(＝O)NR′R″、-OS(O) ₂ OR、-OP(＝O)(OR) ₂ 、-OP(OR) ₂ 、-P(＝O)(OR) ₂ 、-P(＝O)(OR)NR′R″、-S(O)R、-S(O) ₂ R、-S(O) ₂ NR、-S(O)(OR)、-S(O) ₂ (OR), -SC (=o) R, -SC (=o) OR, =o, and-SC (=o) NR 'R ", wherein each R, R' and R" is independently selected from hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, and heterocyclyl.

In addition, alkenyl, alkynyl, allyl, cycloalkyl, heteroalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, cycloalkylalkyl, aryl, or heteroaryl groups as defined above, each of one or more hydrogen atoms of which may also be independently substituted with a D substituent.

The term "halogen" as used in the present invention includes fluorine, bromine, chlorine, iodine.

Compared with the prior art, the invention has the beneficial effects and remarkable progress that:

1) The bufalin derivative provided by the invention is a bufalin series new derivative obtained by introducing modified different phosphate groups and pharmaceutically acceptable salts thereof on the basis of the existing bufalin, and experiments prove that compared with the existing bufalin and the bufalin derivatives, the bufalin derivative provided by the invention has excellent antitumor activity, lower hERG potassium channel inhibition activity and smaller toxic and side effects, so that the bufalin derivative can be used for preparing various antitumor drugs, cardiovascular and cerebrovascular disease treatment drugs and nervous system disease treatment drugs;

2) The bufalin derivative provided by the invention has various alternative structural forms and pharmaceutically acceptable salts thereof, so that the requirements of preparing various pharmaceutical compositions, pharmaceutical preparations and the like can be met, a foundation and thinking are provided for preparing novel pharmaceutical preparations with better curative effects and lower toxic and side effects, and the bufalin derivative has wide application prospects;

3) The bufotalin derivative provided by the invention has the advantages of simple and convenient preparation method, low-cost and easily-obtained raw materials, various choices and easiness in industrial production.

Drawings

FIG. 1 is a graph showing the results of column-like detection of migration inhibition of bufalin derivatives having the structural formula 1a to 95D cells at different concentrations;

FIG. 2 is a graph showing the results of column-like detection of migration inhibition of bufalin derivatives having the structural formula 1b on 95D cells at different concentrations.

Detailed Description

In order to make the objects, technical solutions, advantageous effects and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the reaction formulas provided in the embodiments of the present invention, and it is apparent that all of the described embodiments are only some embodiments of the present invention, but not all embodiments;

All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that:

the terms "first," "second," and the like in the description and in the claims of the invention and in the drawings of embodiments of the invention, are used for distinguishing between different objects and not for describing a particular sequence, and furthermore, the term "comprises" and any variations thereof is intended to cover a non-exclusive inclusion, e.g., a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to the listed steps or elements but may optionally include other steps or elements not listed or inherent to such process, method, article, or apparatus.

It is to be understood that:

in the description of the embodiments of the present invention, some basic operation terms commonly used in the art, for example, "heating," "stirring," "mixing," "dissolving," "washing," "filtering," and "drying," etc., are used, and it should be understood that these terms are not limited to the conventional operations performed by various conventional apparatuses and devices in the art, but may also be program-controlled operations performed by latest apparatuses, unmanned automatic operations, etc., and unless otherwise specifically defined, those skilled in the art will understand the specific meanings of the terms in the present invention according to specific circumstances and use specific operation methods to achieve the purpose of the operation.

Also to be described is:

the following embodiments may be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments, and furthermore, the raw materials and auxiliary materials and the reaction equipment and facilities involved in the following embodiments are commercially available or prepared according to the prior art.

The following describes the technical scheme of the present invention in detail by using specific examples.

Example 1

This example provides a process for the preparation of bufalin derivatives having the structure of formula I.

It should be noted that:

although this example only provides a method for preparing a bufalin derivative having the structure of formula I, those skilled in the art will appreciate that bufalin derivatives having the structure of formula II or III can also be prepared by a method similar to that provided in this example, except that the description is omitted for brevity.

A preparation method of bufalin derivative with a structure of formula I comprises the following steps e, a-b and e, c-d and e in the synthetic route, wherein:

in the step a, the compound IV and the compound V react in an organic solvent with a first basic catalyst to prepare a compound VI, and the molar ratio of the compound IV to the compound V to the first basic catalyst is 1:1-3:1-3;

in the step c, the compound VIII and the compound V react in an organic solvent with a first basic catalyst to prepare a compound IX, and the molar ratio of the compound VIII to the compound V to the first basic catalyst is 1:1-3:1-3;

in the step e, the compound XI and the compound VII or the compound XI and the compound X are respectively reacted in an organic solvent with a second basic catalyst to obtain the compound I, and the molar ratio of the compound XI to the compound VII and the second basic catalyst or the molar ratio of the compound XI to the compound X and the second basic catalyst are respectively 1: (1-5): (1-5);

the synthetic route is as follows:

the preparation method comprises the following steps:

the first basic catalyst is preferably an inorganic basic compound, the second basic catalyst is preferably a nitrogen-containing compound, and the acidic catalyst may be an inorganic acid or an organic acid;

the inorganic basic compound can be any one or a mixture of a plurality of sodium carbonate, potassium carbonate or cesium carbonate;

the nitrogen-containing compound can be any one or a mixture of a plurality of 4-dimethylaminopyridine, triethylamine, pyridine and the like;

The acid catalyst can be any one of hydrochloric acid, sulfuric acid, trifluoroacetic acid, acetic acid and formic acid;

the organic solvent may be any one or more of dichloromethane, dichloroethane, ethyl acetate, acetonitrile or tetrahydrofuran.

In view of the foregoing description of the present invention, it can be seen that:

the bufalin derivative preparation method provided by the embodiment is simple and convenient, has low-cost and easily-obtained raw materials, is various in selection, and is easy for industrial production.

To further aid in understanding the technical scheme of bufalin derivative preparation provided in this example 1, and the specific procedures and effects that can be obtained, the preparation method will be further described below by the following specific examples.

Case 1 preparation of Compound 1a

The preparation route is as follows:

the specific operation process is as follows:

500mg of compound 2, 647mg of compound 3, 2.5mmol, and 266.5mg of potassium carbonate, 2.5mmol, were added to a flask, then 5mL of acetonitrile was added, heated to 80℃for 12 hours, then filtered, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography to give 573mg of colorless liquid compound 4, with a molar yield of 60.6% relative to compound 2;

550mg of compound 4, namely 1.455mmol, and 5mL of trifluoroacetic acid are dissolved in 5mL of dichloromethane together, after reaction for 1 hour at room temperature, the solvent is distilled off under reduced pressure, and compound 5 is obtained and is directly used for the next reaction;

198mg (3S, 5R,8R,9S,10S,11S,13R,14S, 17R) -11, 14-dihydro-10, 13-dimethyl-12-oxo-17- (2-oxo-2H-pyran-5-yl) hexadeca-hydro-1H-cyclopenta [ a ] phenanthren-3-yl- (4-nitrophenyl) carbonate, compound XI, obtained according to the preparation method provided in the patent application with publication No. CN110483608A, was dissolved in 3mL of dichloromethane, 72mg (0.718 mmol) of triethylamine and 200mg (0.718 mmol) of compound 5 were added to react at room temperature for 2 hours, after the reaction was completed, the mixture was washed with saturated brine, dried over sodium sulfate, then filtered, the solvent was removed by evaporation under reduced pressure, and the residue was purified by liquid chromatography to obtain 63mg of compound 1a, which was 25.5% in terms of molar yield relative to compound XI.

Detection result:

¹ H NMR(400MHz,CDCl ₃ )δ7.84(dd,J＝9.6,2.4Hz,1H),7.23(d,J＝2.0Hz,1H),6.26(d,J＝9.6Hz,1H),4.98(s,1H),4.77-4.69(m,2H),4.55(d,J＝6.8Hz,1H),3.48(s,1H),3.01(d,J＝12.0Hz,2H),2.72(d,J＝11.6Hz,2H),2.46(dd,J＝9.2,6.4Hz,1H),2.32(t,J＝11.2Hz,2H),2.23-2.15(m,1H),2.09-2.02(m,1H),1.93-1.84(m,4H),1.78-1.69(m,3H),1.33(s,6H),1.31(s,6H),0.94(s,3H),0.70(s,3H)；

LC-MS:m/z 691.4[M+H] ⁺ 。

case 2 preparation of Compound 1b

The preparation route and the specific procedure for compound 1b were substantially the same as those for compound 1a in case 1, except that (2- (4-aminopiperidin-1-yl) ethyl) diethyl phosphate was used instead of compound 5 in case 1, to obtain compound 1b as a white solid, which was 26.0% in molar yield relative to compound XI.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.84(dd,J＝9.8,2.6Hz,1H),7.23(dd,J＝2.6,1.1Hz,1H),6.26(dd,J＝9.8,1.0Hz,1H),4.98(s,1H),4.58(d,J＝8.0Hz,1H),4.18–3.98(m,4H),3.52(s,1H),2.86(s,2H),2.68(q,J＝8.3Hz,2H),2.46(dd,J＝9.7,6.5Hz,1H),2.23–2.11(m,3H),2.10–1.93(m,6H),1.87(d,J＝14.1Hz,5H),1.78–1.62(m,6H),1.61–1.44(m,8H),1.42–1.37(m,1H),1.32(t,J＝7.1Hz,7H),1.30–1.23(m,4H),0.95(s,3H),0.70(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₃₆ H ₅₇ N ₂ O ₈ P[M+H] ⁺ :677.3931,found:677.3925。

case 3 preparation of Compound 1c

The preparation route and the specific procedure for compound 1c were substantially the same as those for compound 1a in case 1, except that (3- (4-aminopiperidin-1-yl) propyl) diethyl phosphate was used instead of compound 5 in case 1, to obtain compound 1c as a white solid, whose molar yield relative to compound XI was 56.6%.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.83(dd,J＝9.7,2.6Hz,1H),7.22(d,J＝2.6,

1.1Hz,1H),6.25(d,J＝9.6,1.0Hz,1H),4.97(s,1H),4.66–4.56(m,1H),4.16–

3.99(m,4H),3.53(s,1H),2.91(s,2H),2.52–2.41(m,3H),2.23–2.13(m,3H),

2.08–2.01(m,1H),1.96(d,J＝12.8Hz,2H),1.91–1.79(m,4H),1.78–1.70(m,

5H),1.69–1.61(m,3H),1.53–1.44(m,4H),1.43–1.35(m,2H),1.31(t,J＝7.0

Hz,9H),1.28–1.22(m,4H),1.22–1.14(m,1H),0.94(s,3H),0.69(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₃₇ H ₅₉ N ₂ O ₈ P[M+H] ⁺ :691.4087,found:691.4082。

case 4 Compound 1

The preparation route and the specific procedure for compound 1d were substantially the same as those for compound 1a in case 1, except that diethyl ((4-aminopiperidin-1-yl) methyl) phosphate was used instead of compound 5 in case 1, to obtain a white solid, namely compound 1d, whose molar yield relative to compound XI was 75.8%.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.84(dd,J＝9.8,2.6Hz,1H),7.25–7.19

(m,1H),6.27(d,J＝9.7Hz,1H),4.98(d,J＝4.1Hz,1H),4.56(s,1H),4.28–4.02

(m,3H),3.51(s,1H),3.04(s,1H),2.82(s,1H),2.47(dd,J＝9.8,6.6Hz,1H),

2.39(s,1H),2.24–2.15(m,1H),2.10–2.01(m,1H),1.94(s,1H),1.87(t,J＝14.3

Hz,2H),1.73(d,J＝3.3Hz,2H),1.69(d,J＝3.8Hz,1H),1.64(s,6H),1.60(s,1H),

1.53(s,1H),1.51(s,1H),1.49(s,1H),1.42(d,J＝4.4Hz,1H),1.37(d,J＝3.7

Hz,1H),1.34(t,J＝7.1Hz,6H),1.31–1.28(m,3H),1.27–1.24(m,4H),1.19(s,

2H),0.95(s,2H),0.88(t,J＝6.9Hz,1H),0.84(s,1H),0.70(s,2H)；

HRMS(ESI,positive)m/z calcd for C ₃₅ H ₅₅ N ₂ O ₈ P[M+H] ⁺ :663.3774,found:663.3769。

case 5 preparation of Compound 1e

The preparation route and the specific procedure for compound 1e were substantially the same as those for compound 1a in example 1, except that compound 5 in case 1 was replaced with dipropyl ((4-aminopiperidin-1-yl) methyl) phosphate, to give compound 1e as a white solid, which was 52.2% in molar yield relative to compound XI.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.83(dd,J＝9.8,2.6Hz,1H),7.22(dd,J＝2.7,1.1Hz,1H),6.25(dd,J＝9.8,1.1Hz,1H),4.97(s,1H),4.57(s,1H),4.10–3.94(m,4H),3.50(s,1H),3.03(d,J＝11.3Hz,2H),2.82(d,J＝11.5Hz,2H),2.49–2.43(m,1H),2.39(s,1H),2.25–2.14(m,1H),2.09–2.00(m,1H),1.93(d,J＝12.6Hz,2H),1.89–1.83(m,2H),1.76–1.61(m,10H),1.56–1.44(m,7H),1.43–1.37(m,1H),1.37–1.31(m,2H),1.30–1.24(m,4H),1.21–1.13(m,1H),0.98–0.91(m,9H),0.69(s,3H)；

case 6 preparation of Compound 1f

The preparation route and the specific procedure for the compound 1f were substantially the same as those for the compound 1a in case 1, except that the compound 5 in case 1 was replaced with bicyclo [ 4-aminopiperidin-1-yl ] methyl) phosphate, to obtain a white solid, namely, the compound 1f, whose molar yield relative to the compound XI was 61.7%.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.83(dd,J＝9.8,2.6Hz,1H),7.22(d,J＝2.6,1.1Hz,1H),6.25(d,J＝9.7,1.1Hz,1H),4.97(s,1H),4.84–4.70(m,2H),4.56(s,1H),3.49(s,1H),2.99(d,J＝11.4Hz,2H),2.80–2.68(m,2H),2.48–2.41(m,1H),2.36–2.27(m,5H),2.22–2.10(m,5H),2.09–2.01(m,1H),1.93–1.83(m,4H),1.78–1.68(m,5H),1.67–1.61(m,2H),1.54–1.44(m,8H),1.42–1.35(m,2H),1.34–1.22(m,7H),1.21–1.15(m,1H),0.93(s,3H),0.69(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₃₉ H ₅₉ N ₂ O ₈ P[M+H] ⁺ :715.4087,found:715.4082。

preparation of Compound 1g

The preparation route and the specific procedure for the compound 1g were substantially the same as those for the compound 1a in example 1, except that the compound 5 in example 1 was replaced with (4-aminopiperidin-1-yl) methyl) phosphoric acid dicyclopentanyl ester, to obtain a white solid, namely, the compound 1g, whose molar yield relative to the compound XI was 58.0%.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.83(dd,J＝9.7,2.6Hz,1H),7.22(dd,J＝2.6,1.1Hz,1H),6.25(dd,J＝9.8,1.1Hz,1H),5.06–4.86(m,3H),4.56(d,J＝8.0Hz,1H),3.48(s,1H),3.00(d,J＝11.2Hz,2H),2.73(d,J＝11.4Hz,2H),2.50–2.29(m,3H),2.25–2.12(m,1H),2.10–2.00(m,1H),1.95–1.78(m,13H),1.77–1.66(m,8H),1.65–1.59(m,3H),1.54–1.44(m,6H),1.42–1.12(m,10H),0.93(s,3H),0.69(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₄₁ H ₆₃ N ₂ O ₈ P[M+H] ⁺ :743.4400,found:743.4395。

preparation of Compound 1h of case 8

The preparation route and the specific procedure for compound 1h were substantially the same as those for compound 1a in case 1, except that compound 5 in case 1 was replaced with (4-aminopiperidin-1-yl) methyl) phosphoric acid dicyclopentanyl ester, to obtain compound 1h as a white solid, which was 61.1% in molar yield relative to compound XI.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.83(dd,J＝9.8,2.6Hz,1H),7.22(dd,J＝2.6,1.1Hz,1H),6.25(d,J＝9.7Hz,1H),4.97(s,1H),4.56(d,J＝8.0Hz,1H),4.49–4.38(m,2H),3.48(s,1H),3.01(d,J＝11.3Hz,2H),2.75(d,J＝11.5Hz,2H),2.47–2.42(m,1H),2.35(s,2H),2.23–2.12(m,1H),2.10–1.98(m,1H),1.95–1.82(m,8H),1.77–1.67(m,7H),1.67–1.60(m,2H),1.58(dd,J＝11.8,3.2Hz,1H),1.55–1.42(m,13H),1.37–1.15(m,13H),0.93(s,3H),0.69(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₄₃ H ₆₇ N ₂ O ₈ P[M+H] ⁺ :771.4713,found:771.4708。

case 9 preparation of Compound 1i

The preparation route and the specific procedure for compound 1i were substantially the same as those for compound 1a in case 1, except that diisopropyl (((3R, 4R) -4-amino-3-hydroxypiperidin-1-yl) methyl) phosphate was used instead of compound 5 in case 1, to obtain a white solid, namely compound 1i, with a molar yield of 64.6% relative to compound XI.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.87–7.82(m,1H),7.24(dd,J＝2.6,1.2Hz,1H),6.28(d,J＝9.7Hz,1H),5.01(s,1H),4.84(s,1H),4.79–4.71(m,1H),3.63(s,1H),3.50(s,1H),3.41(s,1H),3.23(s,1H),3.14(s,1H),2.83(d,J＝11.8Hz,1H),2.50–2.45(m,1H),2.21(dt,J＝12.9,9.3Hz,1H),2.11–2.03(m,1H),2.01(s,1H),1.93–1.86(m,2H),1.77–1.67(m,4H),1.67–1.56(m,5H),1.50(s,1H),1.46–1.37(m,2H),1.34(dd,J＝6.2,1.3Hz,12H),1.28(d,J＝17.7Hz,3H),1.24–1.19(m,2H),0.96(s,3H),0.71(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₃₇ H ₅₉ N ₂ O ₉ P[M+H] ⁺ :707.4036,found:707.4031。

case 10 preparation of Compound 1j

The preparation route and the specific procedure for compound 1j were substantially the same as those for compound 1a in example 1, except that diisopropyl ((piperidin-4-ylamino) methyl) phosphate was used instead of compound 5 in example 1, to obtain a white solid, namely compound 1j, whose molar yield relative to compound XI was 59.5%.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.83(dd,J＝9.8,2.6Hz,1H),7.20(dd,J＝2.6,1.1Hz,1H),6.22(dd,J＝9.8,1.0Hz,1H),4.97(s,1H),4.75–4.65(m,2H),4.00(s,2H),2.95–2.80(m,4H),2.71–2.62(m,1H),2.45–2.40(m,1H),2.20–2.12(m,1H),2.06–1.99(m,1H),1.89–1.79(m,5H),1.75–1.66(m,3H),1.64–1.57(m,3H),1.56–1.50(m,3H),1.49–1.43(m,2H),1.42–1.33(m,2H),1.31–1.22(m,18H),1.20–1.11(m,1H),0.92(s,3H),0.67(s,3H)；

case 11 preparation of Compound 1k

The preparation route and the specific procedure for compound 1k were substantially the same as those for compound 1a in case 1, except that diisopropyl ((((3 s,4 r) -3-methoxypiperidin-4-yl) amino) methyl) phosphate was used instead of compound 5 in case 1, to obtain a white solid, namely compound 1k, whose molar yield relative to compound XI was 73.9%.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.85(dd,J＝9.7,2.6Hz,1H),7.24(dd,J＝2.6,1.1Hz,1H),6.28(dd,J＝9.7,1.1Hz,1H),5.02(s,1H),4.78(dp,J＝12.6,6.3,5.6Hz,2H),3.50(s,1H),3.42(s,3H),3.02(s,4H),2.48(dd,J＝9.7,6.5Hz,1H),2.21(dt,J＝12.7,9.4Hz,1H),2.12–2.04(m,1H),1.89(ddd,J＝14.4,10.1,4.8Hz,2H),1.82–1.68(m,7H),1.64–1.49(m,8H),1.47–1.40(m,2H),1.38–1.35(m,12H),1.34–1.25(m,6H),1.23–1.18(m,2H),0.96(s,3H),0.72(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₃₈ H ₆₁ N ₂ O ₉ P[M+H] ⁺ :721.4193,found:721.4187。

case 12 preparation of Compound 1l

The preparation route and the specific procedure for compound 1l were substantially the same as those for compound 1a in example 1, except that diisopropyl (((3-fluoropiperidin-4-yl) amino) methyl) phosphate was used instead of compound 5 in example 1, to obtain compound 1l as a white solid, which was 75.8% in molar yield relative to compound XI.

Detection result:

¹ H NMR(600MHz,Chloroform-d)δ7.85(dd,J＝9.7,2.5Hz,1H),7.24(dd,J＝2.5,1.0Hz,1H),6.27(dd,J＝9.8,1.1Hz,1H),5.01(s,1H),4.75(dt,J＝12.6,6.3Hz,2H),3.05(t,J＝15.5Hz,2H),2.95(s,2H),2.47(dd,J＝9.8,6.5Hz,1H),2.20(dt,J＝12.6,9.4Hz,1H),2.09–1.99(m,2H),1.73(d,J＝19.0Hz,10H),1.59–1.54(m,9H),1.51(d,J＝13.7Hz,5H),1.35(dd,J＝6.2,0.8Hz,12H),1.33–1.28(m,4H),1.26(s,3H),1.20(d,J＝12.9Hz,1H),0.96(d,J＝1.8Hz,3H),0.70(s,3H)；

HRMS(ESI,positive)m/z calcd for C ₃₇ H ₅₈ FN ₂ O ₈ P[M+H] ⁺ :709.3993,found:709.3988。

from the description of cases 1 to 12 above, it can be seen that:

the bufalin derivative preparation method provided by the embodiment is convenient and simple, raw materials are low in cost and easy to obtain, the selection is various, the industrial production is easy, the yield is high, and good economic benefits can be obtained.

Example 2

The embodiment provides a preparation method of bufalin derivative and pharmaceutically acceptable salt thereof.

The bufalin derivatives having the structure represented by formula I, formula II, or formula III obtained in example 1, or more specifically, 1a to 1l bufalin derivatives prepared in each case in example 1 are reacted with a pharmaceutically acceptable inorganic acid or organic acid, respectively, to obtain salts corresponding to the bufalin derivatives.

Because the relevant reactions are basic reactions commonly used in the art, the present examples will not be described in detail for brevity.

In the preparation process of the bufalin derivative and the pharmaceutically acceptable salt thereof:

the inorganic acid can be any one of hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid or sulfuric acid;

the organic acid may be any one of formic acid, acetic acid, propionic acid, succinic acid, 1, 5-naphthalene disulfonic acid, asiatic acid, carbenoxolone, glycyrrhetinic acid, oleanolic acid, crataegolic acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, or amino acids.

Example 3

The embodiment provides a preparation method of bufalin derivative and a pharmaceutical composition thereof.

Taking a therapeutically effective amount of the bufalin derivative with the structure shown in the formula I, or the formula II, or the formula III obtained in the embodiment 1 or the bufalin derivative with the structure shown in the formula I, or the formula II, or the formula III obtained in the embodiment 2, or more specifically taking a therapeutically effective amount of 1 a-1 l bufalin derivative prepared in each case in the embodiment 1 or the 1 a-1 l bufalin derivative prepared in the embodiment 2, and mixing or mixing the bufalin derivative with one or more of a pharmaceutically acceptable carrier, an excipient and an auxiliary material respectively to obtain a pharmaceutical composition corresponding to each bufalin derivative or each bufalin derivative pharmaceutically acceptable salt.

Because the related operations are common basic operations in the art, the present embodiment will not be described in detail for brevity.

Example 4

The embodiment provides a preparation method of a bufalin derivative and a pharmaceutical preparation thereof.

Taking the pharmaceutical composition of each bufalin derivative or the pharmaceutically acceptable salt of each bufalin derivative obtained in the embodiment 3, and respectively performing corresponding operation according to the preparation method of the existing pharmaceutical preparation to prepare the pharmaceutical preparation comprising at least one of injection, powder injection, emulsion for injection, tablet, pill, capsule, ointment, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment or nano preparation, wherein the injection can be at least one of small-volume injection, medium-volume injection or large-volume injection, and the nano preparation can be liposome.

Effect example 1

This effect example was used to characterize the in vitro antitumor activity of 1 a-1 l bufalin derivatives prepared in example 1.

The test method comprises the following steps:

the CCK-8 method of the New drug Pharmacology study method (2007:242-243) under the main code Lv Qiujun is adopted.

The cell plant is selected from human lung adenocarcinoma cells A549 and human intestinal cancer cells HCT116 which are frozen and passaged in pharmacology laboratory of Shanghai pharmaceutical industry institute, wherein the culture solution is DMEM+10% FBS+double antibody.

In vitro Activity test procedure:

taking cells in logarithmic growth phase at 7×10 ³ Density of wells/wells was seeded into 96-well plates and incubated at 37℃with 5% CO ₂ For 24 hours to maintain a normal physiological pH;

after cell adhesion, test compounds at different concentrations, i.e. 1a to 1l bufalin derivatives prepared in example 1, and comparative compound, i.e. doxorubicin, were added to the three wells, and 0.1% dmso was added as control, respectively;

after 72 hours, 10. Mu.L of CCK-8 cell counting kit was added to each well and the plate incubated at 37℃for another 0.5-1 hour, absorbance (OD) was read at 450nm on a full-function microplate detector Biotek Synergy H2 and the concentration causing 50% inhibition of cell growth was calculated by GraphPad Prism software simulation (IC ₅₀ ) The test results are shown in Table 1.

Table 1, test data of external antitumor activity of each of bufalin derivatives 1a to 1l prepared in example 1:

the experimental results show that:

The 1a to 1l bufalin derivatives provided in example 1 have excellent antitumor activity and show excellent activity against lung cancer, colon cancer and the like, and the activity against A549 is superior to that of the positive control drug doxorubicin, and 7 bufalin derivatives also have superior activity against HCT 116.

Although table 1 only shows the in vitro antitumor activity test data of the bufalin derivatives of 1a to 1l provided in example 1 of the present effect examples, bufalin derivatives of the present invention having the structure shown in formula i or formula ii or formula iii provided in example 1 or bufalin derivatives of the present invention having the structure shown in formula i or formula ii or formula iii provided in example 2, pharmaceutically acceptable salts thereof, all have antitumor activity similar to those of bufalin derivatives of 1a to 1l described above, and show excellent activity against lung cancer, colon cancer, etc., activity against a549 is superior to that of doxorubicin as a positive control drug, and part of them is also superior to that against HCT116, and accordingly, it can be considered that the bufalin derivatives and salts thereof provided in the present invention can be used for preparing antitumor drugs.

Effect example 2

This effect example was used to characterize the in vivo antitumor activity of the 1a bufalin derivative prepared in example 1 against intestinal cancer HCT116 cells.

The test method comprises the following steps:

the test animal is SPF BALB/C nude mice, male, 18-20 g;

the test process is as follows:

taking well-grown intestinal cancer HCT116 tumor blocks, cutting into uniform small blocks with the size of about 3mm under aseptic condition, subcutaneously inoculating a block on the right armpit of each test mouse by using a trocar, and obtaining tumor blocks with the average size of about 113-119 mm on the 9 th day after inoculation ³ Animals with oversized and undersized tumors were then eliminated by regrouping them according to tumor size, the average tumor volumes of each group of test mice were made substantially uniform, and then intraperitoneal injection was started at a volume of 0.2mL/20g body weight according to the following schedule:

tumor length a (unit: mm) and perpendicular tumor length b (unit: mm) were measured 2 times per week from day 9 of inoculation using digital electronic calipers;

the tumor volume calculation formula is: tv=ab ² /2；

The relative tumor volume calculation formula is: rtv=vt/Vo, where Vo is the tumor volume measured at the time of the split cage (i.e. d 1), vt is the tumor volume at each measurement;

27 days after inoculation (d 19) animals were sacrificed, weighed, tumor mass dissected, weighed, and the decision was made according to the following formula:

the test results are shown in Table 2.

Table 2, in vivo antitumor activity test data of 1a bufalin derivatives prepared in example 1 on intestinal cancer HCT116 cells:

Comparison with the Control group (English: control): * P <0.01.

The experimental results show that:

the 1a bufalin derivative prepared in the embodiment 1 has excellent in-vivo anti-tumor activity by intraperitoneal injection, and the in-vivo tumor inhibition rate of HCT116 cells of intestinal cancer is up to 82.22%.

Effect example 3

This effect example was used to characterize the in vivo antitumor activity of the 1c bufalin derivative prepared in example 1 against intestinal cancer HCT116 cells.

The experimental animals and the experimental method same as that of the experimental example 2 were adopted in the experimental example, except that the average tumor mass of the tumor mass seen on day 13 after the experimental mice were inoculated with intestinal cancer HCT116 tumor mass was about 130-140 mm ³ Then, the animals were sacrificed 30 days after inoculation (i.e., d 18), weighed, dissected to obtain tumor masses, weighed, and the test results were shown in Table 3, according to the tumor sizes of the test mice, and administered according to the same protocol as in effect example 2.

Table 3, in vivo antitumor activity test data of 1c bufalin derivatives prepared in example 1 on intestinal cancer HCT116 cells:

comparison with the Control group (English: control): * P <0.01.

The experimental results show that:

the 1c bufalin derivative prepared in example 1 has excellent in vivo antitumor activity after intravenous administration, and the in vivo tumor inhibition rate of HCT116 cells of intestinal cancer reaches 63.69%.

Although table 2 only shows the in vivo antitumor activity test data of the bufalin derivative of 1a prepared in example 1 against intestinal cancer HCT116 cells and table 3 only shows the in vivo antitumor activity test data of the bufalin derivative of 1c prepared in example 1 against intestinal cancer HCT116 cells, according to the relevant similar test, other bufalin derivatives having the structure shown in formula i or formula ii or formula iii provided in example 1 or other bufalin derivatives having the structure shown in formula i or formula ii or formula iii provided in example 2, or pharmaceutically acceptable salts thereof, have similar antitumor activity to those of the bufalin derivatives of 1a and 1c described above, it can be considered that the bufalin derivatives and salts thereof provided in the invention can be used for preparing antitumor drugs.

Effect example 4

This effect example was used to characterize the effect of the two bufalin derivatives 1a and 1c prepared in example 1 on hERG potassium channel.

The test method comprises the following steps:

CHO-hERG cells were cultured at 175cm ² In the culture flask of (2), after the cell density grows to 60-80%, removing the culture solution, washing with 7mL of PBS (English: phosphate Buffered Saline; PBS for short) namely phosphate buffer solution once, adding 3mL of mild cell digestion solution Detachin for digestion, adding 7mL of culture solution for neutralization after the digestion is completed, centrifuging, sucking the supernatant, and adding 5mL of culture solution for resuspension to ensure the cell density to be 2-5 multiplied by 10 ⁶ /mL；

The single cell high impedance sealing and whole cell mode formation process is automatically completed by a high throughput full automatic patch clamp system, namely a QPatch instrument, after a whole cell recording mode is obtained, cells are clamped at-80 millivolts, a 50 millisecond-50 millivolt pre-voltage is firstly given before a 5 second +40 millivolt depolarizing stimulus is given, then repolarizing is carried out for 5 seconds to-50 millivolts, then the voltage stimulus is returned to-80 millivolts, every 15 seconds is applied, after 2 minutes of recording, extracellular fluid is given for 5 minutes, then the administration process is started, each test concentration of each of 1a and 1c bufalin derivatives for testing is given for 2.5 minutes, 0.3 mu M cisapride of a positive control compound is given, and at least 3 cells are tested for each concentration (namely n is more than or equal to 3);

on the test day, preparing a 15mM DMSO mother solution from two bufogenin derivatives 1a and 1c for testing, and diluting with 500 times of extracellular fluid to obtain the final concentration to be tested;

preparation of the positive control compound cisapride:

taking 10L of a DMSO mother solution of 150M cisapride, adding the 10L of the DMSO mother solution into 4990L of extracellular fluid, and diluting the 10L of the DMSO mother solution by 500 times to obtain the final concentration to be tested of 300nM, wherein the DMSO content in the final test concentration is not more than 0.2%, and the DMSO in the final test concentration has no influence on the hERG potassium channel;

The two bufalin derivatives 1a and 1c for testing were prepared for the whole dilution process by a protein sample pretreatment platform Bravo instrument.

The test results are shown in Table 4.

Table 4, data from activity assays of two bufalin derivatives 1a and 1c prepared in example 1 on hERG potassium channel:

test compounds	Testing concentration	Inhibition rate
			1a	30μM	21.3±0.18％
1c	30μM	53.8±4.13％
			Cisapride	0.3μM	97.4±1.08％

The test results above show that:

the inhibition rates of the two bufalin derivatives 1a and 1c prepared in example 1 on the hERG potassium channel are respectively 21.3+/-0.18% and 53.8+/-4.13% at the concentration of 30 mu M, which are far lower than the inhibition rate of the positive control drug cisapride on the hERG potassium channel at 0.3 mu M, and the results show that the two bufalin derivatives 1a and 1c have lower hERG potassium channel inhibition activity and lower cardiotoxicity.

Although table 4 only shows the data of effect example 4 for testing the activity of two bufalin derivatives 1a and 1c prepared in example 1 on hERG potassium channels, according to the similar test, other bufalin derivatives having the structure shown in formula i or formula ii or formula iii provided in example 1 or other bufalin derivatives having the structure shown in formula i or formula ii or formula iii provided in example 2, which are pharmaceutically acceptable salts thereof, have similar inhibitory activity on hERG potassium channels as those of the two bufalin derivatives 1a and 1c, and therefore, it can be considered that the bufalin derivatives and salts thereof provided in the invention have lower cardiotoxicity and can be used for preparing medicaments for treating cardiovascular and cerebrovascular diseases or medicaments for treating nervous system diseases.

Effect example 5

This effect example was used to characterize the migration inhibition of 95D cells at different concentrations of the two bufalin derivatives 1a and 1b prepared in example 1.

The test method comprises the following steps:

drawing 3 parallel lines on the outer bottom surface of a 6-hole plate at a distance of about 0.5cm, uniformly spreading tumor cells 95D on the plate (5×105/hole), then performing cell scratching by using a pipette, washing the cells 3 times by using phosphate buffer solution (English: phosphate buffer saline, abbreviated as PBS), removing the scratched cells, and adding a culture medium into which compounds 1a or 1b with different concentrations are added in advance;

the plates were placed in 5% CO at 37 ℃C ₂ Culturing in an incubator, sampling for 0, 12, 24, 36 and 48 hours, and photographing under a microscope;

the area and height of the scratch position were measured using Image J software, the area was divided by the height to obtain an average scratch width, the scratch width at 0 point was subtracted from the scratch width at the end of the experiment to obtain a distance for cell migration, and then a cell migration index (english: division index) was calculated using the distance value according to the following formula.

The calculation formula is as follows:

cell migration index= (initial blank width-blank width at a certain time point)/initial blank width.

The test results are as follows:

FIG. 1 is a graph showing the results of column-like detection of migration inhibition of bufalin derivatives having the structural formula 1a to 95D cells at different concentrations; and

From fig. 1 and 2, it can be seen that:

cell scratch experiment results prove that the bufalin derivative with the structural formula of 1a or 1b prepared by the embodiment of the invention has excellent anti-tumor migration effect on 95D cells at different concentrations, so that the bufalin derivative can be used for preparing medicines for inhibiting tumor metastasis.

From the above, it can be seen that:

firstly, the bufalin derivative provided by the invention is prepared by structurally modifying the existing bufalin and introducing different phosphate groups into the bufalin derivative to obtain a new bufalin series derivative and pharmaceutically acceptable salts thereof, and the bufalin series derivative not only has excellent anti-tumor activity and lower hERG potassium channel inhibition activity, but also has greatly reduced toxic and side effects, and can be used for preparing various anti-tumor medicines, cardiovascular and cerebrovascular disease treatment medicines and nervous system disease treatment medicines;

Secondly, the bufalin derivative provided by the invention has various alternative structural forms and pharmaceutically acceptable salts thereof, so that the requirements of preparing various pharmaceutical compositions, pharmaceutical preparations and the like can be met, a foundation and thinking are provided for preparing novel pharmaceutical preparations with better curative effects and lower toxic and side effects, and the bufalin derivative has wide application prospects;

in addition, the bufalin derivative provided by the invention has the advantages of simple and convenient preparation method, low-cost and easily-obtained raw materials, various choices and easy industrial production, and can make a remarkable contribution to guaranteeing the physical and mental health of people, so that the bufalin derivative has great popularization and application values.

In the description of the above specification:

the terms "this embodiment," "an embodiment of the invention," "as shown in … …," "further," and the like, mean that a particular feature, structure, material, or characteristic described in the embodiment or example is included in at least one embodiment or example of the invention; in this specification, a schematic representation of the above terms is not necessarily directed to the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc. may be combined or combined in any suitable manner in any one or more embodiments or examples; furthermore, various embodiments or examples, as well as features of various embodiments or examples, described in this specification may be combined or combined by one of ordinary skill in the art without undue experimentation.

Finally, it should be noted that:

while the invention has been described in detail with reference to the foregoing embodiments or examples, it will be understood by those skilled in the art that the foregoing embodiments or examples may be modified or equivalents may be substituted for some or all of the features thereof without departing from the scope of the invention, and that various modifications and adaptations of the corresponding embodiments or examples may be made by those skilled in the art without departing from the scope of the invention as set forth in the claims.

Claims

1. A bufalin derivative, characterized in that:

wherein:

R ₂ 、R ₃ Each independently selected from any one of hydrogen, deuterium, hydroxy, halogen, and substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;

R ₅ selected from the group consisting of hydrogen, deuterium, and substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkylAny one of, or R ₅ And R is R ₁ To form a substituted or unsubstituted nitrogen-containing heterocycle;

a is C-containing ₁ ～C ₆ Linear or branched alkanes.

2. The bufalin derivative of claim 1, wherein:

the R is ₆ 、R ₇ Each independently selected from 3-8 membered cycloalkyl, C ₆ ～C ₁₂ Aryl, and C each substituted with one or more of the optional T groups ₁ ～C ₈ Alkyl, 3-8 membered cycloalkyl C ₁ ～C ₈ Alkyl, 3-8 membered heterocyclic group C ₁ ～C ₈ Any one of alkyl, 3-8 membered alkyl containing ether bond or thioether bond;

3. The bufalin derivative of claim 1, wherein:

the R is ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ Each independently selected from the group consisting of optionally in T groupsSubstituted 3-to 8-membered alkyl;

4. The bufalin derivative of claim 1, wherein:

the bufalin derivative is a compound with a structure shown in formulas 1 a-1 l and pharmaceutically acceptable salts thereof:

5. the bufalin derivative of claim 1, wherein:

the pharmaceutically acceptable salt of the compound is a salt formed after the compound reacts with a pharmaceutically acceptable inorganic acid or organic acid.

6. The bufalin derivative of claim 5, wherein:

7. A method for preparing the bufalin derivative with the structure shown in formula I, which is characterized in that:

wherein:

8. The method of manufacturing according to claim 7, wherein:

9. The method of preparing as claimed in claim 8, wherein:

10. A pharmaceutical composition of bufalin derivatives, characterized in that:

the pharmaceutical composition comprises a therapeutically effective amount of the bufalin derivative or the pharmaceutically acceptable salt of the bufalin derivative according to any one of claims 1-6, and one or more of pharmaceutically acceptable carriers, excipients and auxiliary materials.

11. A pharmaceutical formulation of a bufalin derivative, characterized in that:

the pharmaceutical preparation is at least one of injection, powder injection, emulsion for injection, tablet, pill, capsule, ointment, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment or nano preparation prepared by adopting the pharmaceutical composition as claimed in claim 10.

12. The pharmaceutical formulation of claim 11, wherein:

13. An application of bufalin derivative, which is characterized in that:

the application is that a therapeutically effective amount of the bufalin derivative or the pharmaceutically acceptable salt of the bufalin derivative is used for preparing at least one of antitumor drugs, cardiovascular and cerebrovascular disease treatment drugs or nervous system disease treatment drugs.

14. The use according to claim 13, wherein:

the antitumor drug is used for treating at least one malignant tumor grown at the esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system of human or animal, and at least one of thyroid cancer, leukemia, huo Jinshi disease, lymphoma and myeloma.

15. The use according to claim 13, wherein:

The use of a therapeutically effective amount of a bufalin derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting tumor metastasis.