CN114516832B - Tubulin inhibitor and preparation method and application thereof - Google Patents

Tubulin inhibitor and preparation method and application thereof Download PDF

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CN114516832B
CN114516832B CN202111387237.6A CN202111387237A CN114516832B CN 114516832 B CN114516832 B CN 114516832B CN 202111387237 A CN202111387237 A CN 202111387237A CN 114516832 B CN114516832 B CN 114516832B
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pharmaceutically acceptable
medicament
cells
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CN114516832A (en
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广兵
阳泰
董韧涵
刘进
覃传军
占伟
黄胜
谢建
赖永新
彭向阳
许庆
彭坚
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Chengdu Benuo Kecheng Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/56—Amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring

Abstract

The application belongs to the field of chemical medicines, and particularly relates to a tubulin inhibitor, and a preparation method and application thereof. The application provides a compound shown as a formula I. The compound has remarkable inhibiting effect on cancer cells and HacaT cells, and IC thereof 50 The compound provided by the application can be used for preparing medicines for preventing and treating cancers, actinic keratosis and psoriasis. Furthermore, the compounds of the present application may be used for the preparation of tubulin inhibitors.

Description

Tubulin inhibitor and preparation method and application thereof
Technical Field
The application belongs to the field of chemical medicines, and particularly relates to a tubulin inhibitor, and a preparation method and application thereof.
Background
Microtubules are the main component constituting the cytoskeleton and are present in all eukaryotic cells. Due to the important roles microtubules play in the cell mitosis process, tubulin is becoming one of the important targets for pharmaceutical workers to research and develop anticancer drugs. Tubulin inhibitors can be classified into 3 types, depending on the site of action of the tubulin inhibitor on tubulin: (1) tubulin inhibitors acting on the colchicine site; (2) tubulin inhibitors acting at the vinblastine site; (3) tubulin inhibitors acting on the paclitaxel site. At present, vinblastine and taxol site tubulin inhibitors have taken an important role in clinical treatment of tumors, and taxol is an important first-line treatment drug for lung cancer, breast cancer and ovarian cancer. However, as with other antineoplastic agents, intolerable side effects and the emergence of drug resistance after administration limit the clinical use of tubulin inhibitors. Therefore, the development of novel tubulin inhibitors which have stronger activity, lower toxicity and are more effective on multidrug resistant tumor cells has great application prospect.
In chinese patent application "CN105263907a, N- (3-fluorobenzyl) -2- (5- (4-morpholinophenyl) pyridin-2-yl) acetamide as a protein tyrosine kinase modulator, a compound of the following structure is disclosed.
The literature shows that the compounds exhibit an inhibitory effect on tubulin, thereby having therapeutic and prophylactic effects on cancers, precancers, and the like. However, the compound still has a problem of weak inhibition.
Disclosure of Invention
Aiming at the problems faced by the clinical use of a tubulin inhibitor in the prior art, the application provides a tubulin inhibitor, a preparation method and application thereof, and the compound has stronger inhibition effect on tubulin, thereby showing stronger inhibition effect on cancer cells and human immortalized epidermal cells.
A compound of formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
wherein X is selected from nitro or
R 1 、R 2 Independently selected from H, substituted or unsubstituted C 1 ～C 10 Alkyl, hydroxy; the substituents being C 1- C 10 Alkoxy, cyano, hydroxy, carboxy, halogen or amino;
or, R 1 、R 2 Together with the N atom, form a substituted or unsubstituted ternary or quaternary heterocycle or a seven to eleven membered heteroatom-containing spiro ring;
the substituents being C 1- C 10 Alkyl, C 1- C 10 Alkoxy, cyano, hydroxy, carboxy, halogen or amino;
R 3 selected from H, cyano or halogen.
Preferably, said R 1 、R 2 Independently selected from H, substituted or unsubstituted C 1 ～C 2 Alkyl, hydroxy; the substituent is hydroxy or halogen;
or, R 1 、R 2 Together with the N atom, form a substituted or unsubstituted ternary or quaternary heterocycloalkyl group; the substituents being C 1- C 10 Alkyl, C 1- C 10 Alkoxy, cyano, hydroxy, carboxy, halogen or amino.
Preferably, said R 1 、R 2 Each independently selected from H, methyl, ethyl, hydroxy-substituted ethyl, chloro-substituted ethyl, hydroxy;
or, R 1 、R 2 Together with the N atom, form a ternary or quaternary heterocycle.
Preferably, said R 3 Selected from H or F.
Preferably, the compound of formula I is a compound of formula II:
wherein X is selected from nitro or
R 1 、R 2 Independently selected from H, substituted or unsubstituted C 1 ～C 10 Alkyl, hydroxy; the substituents being C 1- C 10 Alkoxy, cyano, hydroxy, carboxy, halogen or amino;
or, R 1 、R 2 Together with the N atom, form a substituted or unsubstituted ternary or quaternary heterocycle; the substituents being C 1- C 10 Alkyl, C 1- C 10 Alkoxy, cyano, hydroxy, carboxy, halogen or amino.
Preferably, said R 1 、R 2 Independently selected from H, substitution, orUnsubstituted C 1 ～C 2 Alkyl, hydroxy; the substituent is hydroxy or halogen;
or, R 1 、R 2 Together with the N atom, form a substituted or unsubstituted ternary or quaternary heterocycle; the substituents being C 1- C 10 Alkyl, C 1- C 10 Alkoxy, cyano, hydroxy, carboxy, halogen or amino.
Preferably, said R 1 、R 2 Each independently selected from H, methyl, ethyl, hydroxy-substituted ethyl, chloro-substituted ethyl, hydroxy;
or, R 1 、R 2 Together with the N atom to form a ternary or quaternary heterocyclic ring or
Preferably, the compounds are the following:
the application also provides a preparation method of the compound shown in the formula I, which is characterized by comprising the following steps of:
wherein X and R 3 The method of claim 1.
Preferably, the reaction is carried out by:
(1) The synthesis steps of the intermediate M-1 are as follows:
mixing 5-bromo-2-fluoropyridine and diethyl malonate for dissolution, heating for reaction under the action of cesium carbonate, separating, and concentrating to dryness to obtain a residue;
dissolving the obtained residue, adding strong alkali for reaction, adjusting pH to 4-5, separating out solid, separating, and drying to obtain intermediate M-1;
(2) The synthesis steps of the intermediate M-2 are as follows:
intermediate M-1 and meta have R 3 Mixing and dissolving substituted benzylamine, reacting under the action of DIPEA and TBTU, and separating to obtain an intermediate M-2;
(3) The synthesis steps of the compound of the formula I are as follows:
and (3) mixing and dissolving the intermediate M-2 and phenylboronic acid with the X substituent at the para position, heating for reaction under the action of potassium carbonate and tetra (triphenylphosphine) palladium, and separating to obtain the compound shown in the formula I.
The application also provides a preparation method of the pharmaceutically acceptable salt of the compound shown in the formula I, which comprises the following steps: heating a compound of the formula I, dissolving in an organic solvent, adding acid, reacting, cooling and separating to obtain the compound.
Preferably, the organic solvent is one or more of methanol, ethanol, isopropanol, ethyl acetate, butyl acetate, acetone, tetrahydrofuran, dichloromethane, n-hexane and methyl tertiary butyl ether;
and/or the acid is selected from hydrochloric acid, sulfuric acid, citric acid, benzenesulfonic acid, hydrobromic acid, hydrofluoric acid, phosphoric acid, acetic acid, propionic acid, succinic acid, oxalic acid, lactic acid, malic acid, succinic acid, fumaric acid, maleic acid, tartaric acid or trifluoroacetic acid;
and/or, the separation process is to add methyl tertiary butyl ether to precipitate crystals, and filtering.
The application also provides application of the compound, or stereoisomer or pharmaceutically acceptable salt thereof in preparing a tubulin inhibitor.
Preferably, the tubulin inhibitors are used for the treatment and/or prevention of cancer, skin diseases.
Preferably, the cancer comprises at least one of ovarian cancer, breast cancer, glioma, colon cancer and lymphoma;
and/or the skin disorder comprises at least one of actinic keratosis and psoriasis.
The application also provides application of the compound, or a stereoisomer or a pharmaceutically acceptable salt thereof in preparing an anti-tumor medicament.
Preferably, the medicament is an anti-ovarian, breast, glioma, colon, lymphatic cancer medicament.
The application also provides application of the compound, or a stereoisomer or a pharmaceutically acceptable salt thereof in preparing a medicament for treating and/or preventing skin diseases.
Preferably, the medicament is a medicament for the treatment and/or prophylaxis of actinic keratosis and psoriasis.
The application also provides a pharmaceutical composition which is prepared by taking the compound, or a stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Preferably, the auxiliary materials or auxiliary components are selected from at least one of diluents, fillers, colorants, glidants, lubricants, binders, stabilizers, suspending agents and buffers.
Preferably, the pharmaceutical composition is in the form of tablet, capsule, oral liquid, injection, transdermal agent, aerosol solid preparation, liposome or sustained and controlled release preparation.
The compounds and derivatives provided in the present application may be named according to IUPAC (international union of pure and applied chemistry) or CAS (chemical abstract service, columbus, OH) naming system.
Definition of terms used in connection with the present application: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content of the hydrocarbon groups are indicated by a prefix, e.g. prefix C a -C b Alkyl indicates anyAlkyl groups containing from "a" to "b" carbon atoms. Thus, for example, "C 1 -C 4 Alkyl "refers to an alkyl group containing 1 to 4 carbon atoms.
"alkyl" refers to a saturated hydrocarbon chain having the indicated number of member atoms. For example, C 1 -C 6 Alkyl refers to an alkyl group having 1 to 6 member atoms, for example 1 to 4 member atoms. The alkyl group may be linear or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl. The alkyl group may also be part of another group, such as C 1 -C 6 An alkoxy group.
"cycloalkyl" refers to a saturated or partially saturated cyclic group having 3 to 14 carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, bridged and spiro ring systems). For polycyclic systems having aromatic and non-aromatic rings that do not contain ring heteroatoms, the term "cycloalkyl" (e.g., 5,6,7,8, -tetrahydronaphthalen-5-yl) applies when the point of attachment is at a non-aromatic carbon atom. The term "cycloalkyl" includes cycloalkenyl groups, such as cyclohexenyl. Examples of cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl and cyclohexenyl.
"alkenyl" refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and in some embodiments 2 to 6 carbon atoms or 2 to 4 carbon atoms and having at least 1 site of ethylenic unsaturation (> c=c <). For example, (Ca-Cb) alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1, 3-butadienyl, and the like.
"alkynyl" refers to a straight or branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbyl groups having one triple bond and one double bond. For example, (C2-C6) alkynyl is intended to include ethynyl, propynyl, and the like.
"halogen" is fluorine, chlorine, bromine or iodine.
"heterocycle" refers to a saturated ring or a non-aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom means a nitrogen atom, an oxygen atom, and a sulfur atom;
“R 1 、R 2 together with the N atom "means R 1 And R is 2 At least one atom of each is bonded by a chemical bond such that R in the general structure 1 And R is 2 The N atoms being co-joined as part of a ring structure skeleton with R 1 And R is 2 Together forming a heterocyclic ring.
"stereoisomers" include enantiomers and diastereomers.
The term "pharmaceutically acceptable" refers to a carrier, cargo, diluent, adjuvant, and/or salt formed in general
Chemically or physically compatible with the other components that make up a pharmaceutical dosage form, and physiologically compatible with the receptor.
The terms "salts" and "pharmaceutically acceptable salts" refer to the acid and/or base salts of the above compounds or stereoisomers thereof, with inorganic and/or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base as appropriate (for example, equivalent). These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by lyophilization after reaction in an aqueous medium. The salts of the present application may be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric, phosphate, acetate, propionate, succinate, oxalate, lactate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salts of the compounds.
In certain embodiments, one or more compounds of the present application may be used in combination with one another. The compounds of the application may alternatively be used in combination with any other active agent for the preparation of a medicament or pharmaceutical composition for modulating cellular function or treating a disease. If a group of compounds is used, the compounds may be administered to a subject simultaneously, separately or sequentially.
In the present application, the room temperature was 25.+ -. 5 ℃ and the overnight period was 12.+ -. 2h.
The compounds provided by the application have stronger inhibition effect on tubulin, so that stronger treatment and prevention effects on diseases related to the increase of the activity of the tubulin, such as cancers and partial skin diseases are shown. The compound has remarkable inhibition effect on ovarian cancer cells, breast cancer cells, glioma cells, colon cancer cells, lymphoma cells and immortalized keratinocytes, and can be used for preparing medicaments for preventing and treating ovarian cancer, breast cancer, glioma, colon cancer, lymphoma, actinic keratosis and psoriasis by single or combined application. Compared with small molecule compounds with similar actions in the prior art, the compounds provided by the application inhibit the IC50 of tubulin or cancer cells to be obviously reduced.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
The above-described aspects of the present application will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present application is limited to the following examples only. All techniques implemented based on the above description of the application are within the scope of the application.
Detailed Description
The materials and equipment used in the embodiments of the present application are all known products and are obtained by purchasing commercially available products.
EXAMPLE 1 Synthesis of Compound I-1
Wherein i: DMSO, cs 2 CO 3 ；ii：MeOH，NaOH；iii：DMF，DIPEA，TBTU；iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
Step 1: 50.0 g of 5-bromo-2-fluoropyridine (S-1, 0.28 mol) and 113.7 g of diethyl malonate (0.71 mol) were dissolved in 1 liter of DMSO with stirring, 231.3 g of cesium carbonate (0.71 mol) was added, and the mixture was heated to 80℃to react for 16-20 hours. TLC detects that the basic reaction of the raw material 5-bromo-2-fluoropyridine is complete. The reaction solution was cooled to room temperature, diluted with 2 liters of water, and extracted 3 times with 500 ml of ethyl acetate, respectively. The ethyl acetate was combined, washed twice with 150 ml of water, and concentrated to dryness under reduced pressure.
To the residue was added 300 ml of methanol, 200 g of 30% sodium hydroxide solution by mass fraction, and the reaction was stirred for 16-20 hours at 60℃and was complete by TLC. Stopping heating, concentrating the reaction solution under reduced pressure to reduce the volume to about 200 ml, cooling, adjusting pH to 6,1M with 4M hydrochloric acid, adjusting pH to 4-5 with citric acid aqueous solution, precipitating, standing for about 1 hr, filtering, washing the solid with small amount of water for 2 times, and washing with ethyl acetate for 1 time. And drying to obtain an intermediate M-1.
Step 2: 18.0 g of intermediate M-1 (83.3 mmol) and 17.8 g of benzylamine (166.6 mmol) were taken, dissolved in 150 ml of DMF, 21.5 g of DIPEA (166.6 mmol) were added with stirring, and 40.1 g of TBTU (125.0 mmol) were then added slowly in portions and the reaction was stirred at room temperature overnight after the addition. TLC detects that the raw materials are completely reacted, 500 ml of ethyl acetate and 1 l of water are added into the reaction solution, the ethyl acetate layer is taken after standing and layering, the aqueous layer is extracted 2 times by 150 ml of ethyl acetate, the ethyl acetate layer is combined, and is washed twice by 90 ml of 5% potassium carbonate solution and twice by 50 ml of saturated salt water, and then is dried by 35 g of anhydrous sodium sulfate for 1 hour. Filtering to remove sodium sulfate, concentrating the filtrate under reduced pressure to dryness, dispersing and crystallizing the oily substance with petroleum ether, and filtering to obtain an intermediate M-2.
Step 3: 14.0 g of intermediate M-2 (45.9 mmol) and 9.2 g of p-nitrobenzoic acid (55.1 mmol) were taken and dissolved in 200 ml of dioxane; another 12.7 g of potassium carbonate (91.8 mmol) was dissolved in 50 ml of water, the system was added dropwise, then 0.5 g of tetrakis (triphenylphosphine) palladium was added, the reaction was heated to 80℃for 10-12 hours, TLC was checked for complete reaction, the reaction system was cooled to room temperature, 200 ml of ethyl acetate, 400 ml of water were added, the mixture was allowed to stand for delamination, the aqueous layer was extracted with 100 ml of ethyl acetate for 2 times, the ethyl acetate layer was combined, washed with 20 ml of 5% sodium hydroxide for 3 times, 20 ml of 5% diluted hydrochloric acid for one time, then with 20 ml of saturated brine to neutrality, 15 g of anhydrous sodium sulfate was added for drying for 1 hour. Filtering to remove sodium sulfate, concentrating the filtrate under reduced pressure to dryness, dispersing and crystallizing the oily substance by using methyl tertiary butyl ether, and filtering to obtain the compound I-1.
1 HNMR(400MHz，DMSO-d6)δ：8.92(d，J＝2.4Hz，1H)，8.68(t，J＝6.0Hz，1H)，8.37-8.29(m，2H)，8.18(dd，J＝8.2，2.5Hz，1H)，8.07-8.00(m，2H)，7.52(d，J＝8.2Hz，1H)，7.37-7.19(m，5H)，4.31(d，J＝5.9Hz，2H)，3.78(s，2H)。
ESI-MS m/z:346.17[M-1] - 。
EXAMPLE 2 Synthesis of Compound I-2
2.00 g of compound I-1 (5.76 mmol) is suspended in a mixed system of 40 ml of ethanol and 10 ml of water, 0.97 g of iron powder (17.37 mmol) and 0.92 g of ammonium chloride (17.20 mmol) are added, the temperature is raised to 90 ℃ to react for 4-8 hours, TLC monitors that the raw materials are completely reacted, the temperature is reduced to room temperature, 40 ml of ethyl acetate is added, filtration is carried out, a filter cake is leached once by 15 ml of ethyl acetate, the filtrate is collected, 40 ml of water is added for layering, an ethyl acetate layer is taken, the aqueous layer is extracted for 2 times by 15 ml of ethyl acetate, the ethyl acetate layer is combined, washed once by saturated saline, 10 g of anhydrous sodium sulfate is added and dried for one hour. Filtering to remove sodium sulfate, concentrating the filtrate under reduced pressure to dryness, dispersing and crystallizing the oily substance by using methyl tertiary butyl ether, and filtering to obtain a crude product of the compound I-2. Purifying the crude product by column chromatography (eluting system ethyl acetate/petroleum ether) to obtain the compound I-2.
1 HNMR(400MHz，DMSO-d6)δ：8.70-8.57(m，2H)，7.78(dd，J＝8.2，2.5Hz，1H)，7.40(d，J＝8.2Hz，2H)，7.37-7.19(m，6H)，6.66(d，J＝8.2Hz，2H)，5.35(brs，2H)，4.30(d，J＝5.9Hz，2H)，3.68(s，2H)。
ESI-MS m/z:316.07[M-1] - 。
500 mg of Compound I-2 (1.58 mmol) was suspended in 5 mL of ethyl acetate, heated to 70℃in an oil bath to dissolve, 183 mg of fumaric acid (1.58 mmol) was slowly added, and the mixture was kept at 70℃for 30 minutes and cooled to room temperature. Concentrating ethyl acetate under reduced pressure to about 1ml, adding 5 ml methyl tert-butyl ether to gradually precipitate crystals, and filtering to obtain fumarate I-2-1 of compound I-2, which has the following structure:
EXAMPLE 3 Synthesis of Compound I-3
iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
The synthesis method is similar to the step 3 in the example 1 by taking the intermediates M-2 and 4- (dimethylamino) phenylboronic acid as raw materials, so as to obtain a crude product of the compound I-3. Purifying by column chromatography (eluting system ethyl acetate petroleum ether) to obtain compound I-3.
1 HNMR(400MHz，DMSO-d6)δ：8.73(d，J＝2.4Hz，1H)，8.62(t，J＝6.0Hz，1H)，7.95(dd，J＝8.1，2.5Hz，1H)7.60-7.53(m，2H)，7.41-7.19(m，6H)，6.86-6.79(m，2H)，4.30(d，J＝5.9Hz，2H)，3.70(s，2H)，2.95(s，6H)。
ESI-MS m/z:344.10[M-1] - 。
500 mg of Compound I-3 (1.45 mmol) was suspended in 5 mL of ethyl acetate, heated to 70℃in an oil bath to dissolve, 168 mg of fumaric acid (1.45 mmol) was slowly added, and the mixture was kept at 70℃for 30 minutes and cooled to room temperature. Concentrating ethyl acetate under reduced pressure to about 1ml, adding 5 ml methyl tert-butyl ether to gradually precipitate crystals, and filtering to obtain fumarate I-3-1 of the compound I-3, which has the following structure:
EXAMPLE 4 Synthesis of Compound I-4
The method comprises the following steps: 1.00 g of Compound I-2 (3.15 mmol) was dissolved in 25 ml of DMF, 0.64 g of 1, 3-dibromopropane (3.17 mmol) and 0.87 g of potassium carbonate (6.29 mmol) were added under stirring, the reaction was allowed to proceed to 60℃for 12 hours, TLC was used to check that the starting material was complete, the reaction was cooled to room temperature, the reaction solution was poured into 100 ml of ice water, extracted 3 times with 75 ml of ethyl acetate, the ethyl acetate layers were combined, washed 2 times with saturated brine, and dried by adding 15 g of anhydrous sodium sulfate for one hour. Filtering to remove sodium sulfate, and concentrating the filtrate under reduced pressure to obtain a crude product of the compound I-4. Purification by column chromatography (eluting with methanol/dichloromethane) afforded compound I-4.
The second method is as follows:
iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
the synthesis method is similar to the step 3 in the example 1 by taking the intermediates M-2 and 4- (cyclobutylamino) phenylboronic acid as raw materials, and the crude product of the compound I-4 is obtained. Purification by column chromatography (eluting with methanol/dichloromethane) afforded compound I-4.
1 HNMR(400MHz，CDCl 3 )δ：8.71-8.65(m，1H)，7.79(dd，J＝8.0，2.4Hz，1H)，7.51-7.39(m，2H)，7.35-7.16(m，6H)，6.57-6.48(m，2H)，4.52-4.44(m，2H)，3.94(t，J＝7.2Hz，4H)，3.83-3.77(m，2H)，2.40(p，J＝7.2Hz，2H)。
ESI-MS m/z:356.08[M-1] - 。
EXAMPLE 5 Synthesis of Compound I-5
Wherein i: DMSO, cs 2 CO 3 ；ii：MeOH，NaOH；iii：DMF，DIPEA，TBTU；iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
The synthesis was carried out in analogy to example 1, substituting 3-fluorobenzylamine for benzylamine in step 2 to give compound I-5.
ESI-MS m/z:364.02[M-1] - 。
EXAMPLE 6 Synthesis of Compound I-6
The synthesis method is similar to example 2, using compound I-5 as raw material, to obtain compound I-6
ESI-MS m/z:334.14[M-1] - 。
EXAMPLE 7 Synthesis of Compound I-7
iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
The synthesis was similar to example 3, starting from intermediate M-3, 4- (dimethylamino) phenylboronic acid, to give compound I-7.
ESI-MS m/z:362.15[M-1] - 。
EXAMPLE 8 Synthesis of Compound I-8
iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
Using intermediate M-3 as a starting material, the synthesis was similar to that of example 3, affording Compound I-8.
ESI-MS m/z:374.13[M-1] - 。
EXAMPLE 9 Synthesis of Compound I-9
iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
Using intermediate M-2 as a starting material, the synthesis was similar to that of example 3, affording Compound I-9.
ESI-MS m/z:398.40[M-1] - 。
EXAMPLE 10 Synthesis of Compound I-10
iii：DMF，DIPEA，TBTU；iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
Using intermediate M-1 as a starting material, the synthesis was similar to that of example 1, affording Compound I-10.
ESI-MS m/z:374.13[M-1] - 。
EXAMPLE 11 Synthesis of Compound I-11
iii：DMF，DIPEA，TBTU；iv：1,4-Dioxane，K 2 CO 3 ，H 2 O，Pd[P(C 6 H 5 ) 3 ] 4 。
Using intermediate M-1 as a starting material, the synthesis was similar to that of example 1, affording Compound I-11.
ESI-MS m/z:374.13[M-1] - 。
The beneficial effects of the present application are demonstrated by specific test examples below.
Test example 1 Activity test of the Compounds of the application against HT29 cell cycle arrest in human colon cancer cells
Human colon carcinoma cells HT29 were used to examine the activity of the compounds of the application in blocking the cell cycle. After adding 2ml of cells, 1E 6/well, to a 6-well cell culture plate and culturing for 12 hours, the drug of the present application and control compounds 1 and 2 were added at final concentrations of 25nM and 50nM, respectively. After 24h of compound action, the adherent cells were digested with pancreatin, centrifuged at 300g for 5min, and the culture medium was removed. 1ml of pre-chilled PBS was added for washing. 300g of the mixture is centrifuged for 5min, the supernatant is removed, cells are collected, 70% ethanol precooled at-20 ℃ is added, and the mixture is stirred uniformly and then is fixed at 4 ℃ overnight. 1000g was centrifuged for 5min and ethanol was removed. Cell cycle was detected using the UE company cell cycle rapid detection kit RedNucleus dye. After adding the RedNucleus I staining solution, slowly and fully mixing, and incubating for 20min at room temperature in a dark place. The wavelength was measured at 660/20nm using Ai Senliu cytometer 638nm laser excitation. The analysis was performed using flow cytometer self-contained cycle analysis software to compare the G2/M ratios of the cells of the different treatment groups. After the tubulin inhibitors act on the cells, cell division is blocked and the ratio of the G/2M phase of the cell cycle increases. The G2/M phase cell ratio of HT29 blank was 12.9.+ -. 1.3. The results of the ratio of compounds blocking HT29 cell cycle G2/M phase at different concentrations are shown in Table 1 below:
TABLE 1 Activity of the compounds of the application against HT29 cell cycle arrest in human colon cancer cells at various concentrations
The experimental results show that: the activity of the compound of the application on HT-29 cell cycle block of human colon cancer cells is obviously higher than that of the existing compound, which proves that the compound of the application has obvious inhibition activity on mitosis process of eukaryotic cells.
Test example 2 inhibitory Activity of the Compounds of the application against human breast cancer cells MDA-MB-231 and human ovarian cancer cells SKOV-3
Collecting human breast cancer cells MDA-MB-231 and human ovarian cancer cells SKOV-3 in logarithmic growth phase respectively, counting, re-suspending the cells with a complete culture medium, adjusting the cell concentration to 20/μl, respectively obtaining a human breast cancer cell suspension and a human ovarian cancer cell suspension, inoculating 96-well plates, and adding 100 μl of cell suspension into each well. Cells were incubated at 37℃with 100% relative humidity, 5% CO 2 After 24 hours of incubation in the incubator, the compounds of the application and control compounds were diluted with medium to the corresponding working concentrations set and added to the cells at 25 μl/well. The final concentration of the compound was varied from 0nM to 400nM, 4-fold gradient dilution, for a total of 10 concentration points. After the test compound is added, the cells are placed at 37 ℃,100% relative humidity, 5% CO 2 Incubate in incubator for 72 hours. The medium was aspirated and 100. Mu.L of fresh medium containing 10% CCK-8 was added to each well, incubated in an incubator at 37℃for 2-4 hours, absorbance at 450nm was measured on SpectraMax M5 Microplate Reader with gentle shaking, absorbance at 650nm was used as a reference, and the inhibitory activity IC of the test compounds against human breast cancer cells MDA-MB-231 and human ovarian cancer cells SKOV-3 was measured 50 The results are shown in Table 2:
TABLE 2 inhibitory Activity of the inventive Compounds against human breast cancer cells MDA-MB-231 and human ovarian cancer cells SKOV-3
The experimental results show that: IC of the compound of the application on human breast cancer cells MDA-MB-231 and human ovarian cancer cells SKOV-3 50 The value is obviously lower than that of the existing compounds, which indicates that the compound has good inhibition effect on breast cancer cells and ovarian cancer cells, and can be used for preventing and treating breast cancer and ovarian cancer.
Test example 3 inhibitory Activity of the Compounds of the application against human colon cancer cell HT-29 and human brain glioma cell T98G
Collecting human colon cancer cells HT-29 and human brain glioma cells T98G in logarithmic growth phase respectively, counting, re-suspending the cells with complete culture medium, adjusting the cell concentration to 20 per mu L, obtaining human colon cancer cell suspension and human brain glioma cell suspension respectively, inoculating 96-well plates, and adding 100 mu L of cell suspension into each well. Cells were incubated at 37℃with 100% relative humidity, 5% CO 2 After 24 hours of incubation in the incubator, the test compounds were diluted with medium to the corresponding working concentrations set and added to the cells at 25 μl/well. The final concentration of the compound was varied from 0nM to 400nM, 4-fold gradient dilution, for a total of 10 concentration points. After addition of the compounds to be treated, the cells were subjected to 37℃with 100% relative humidity and 5% CO 2 Incubate in incubator for 72 hours. Absorbing and removing the culture medium, adding 100 μl of fresh culture medium containing 10% CCK-8 into each well, incubating in a 37 ℃ incubator for 2-4 hours, slightly shaking, measuring absorbance at 450nm wavelength on SpectraMax M5 Microplate Reader, and measuring inhibitory activity IC of the compound to be tested on human colon cancer cells HT-29 and human brain glioma cells T89G by taking absorbance at 650nm as reference 50 The results are shown in Table 3:
TABLE 3 inhibitory Activity of the inventive Compounds against human colon cancer cell HT-29 and human brain glioma cell T98G
The experimental results show that: IC of the compound of the application on human colon cancer cells HT-29 and human brain glioma cells T98G 50 The value is obviously lower than that of the existing compound, which indicates that the compound has good inhibition effect on colon cancer cells and brain glioma cells, and can be used for preventing and treating colon cancer and glioma.
Test example 4 inhibition test of the inventive Compounds on human tissue lymphoma cell U937
Human histiocyte lymphoma cells in logarithmic growth phase U937 (purchased from ATCC) were collected, the cells were resuspended in complete medium, the cell concentration was adjusted to 60 cells/. Mu.l, a cell suspension was obtained, and 96-well plates were inoculated, and 100. Mu.L of the cell suspension was added to each well plate. The cells were conditioned at 37 ℃,100% relative humidity, 5% CO 2 After 24 hours of incubation in the incubator, the test compounds according to the application were diluted with medium to the corresponding working concentrations set and added to 96-well plates at 25. Mu.L/well. The final concentration of compound was diluted in a 4-fold gradient from 0nM to 400 nM. After the test compound is added, the cells are placed at 37 ℃,100% relative humidity, 5% CO 2 Incubate in incubator for 72 hours. Then adding 100 mu L of fresh culture medium containing 10% of CCK-8 into each well, placing in a 37 ℃ incubator for incubation for 2-4 hours, measuring absorbance at 450nm wavelength on SpectraMax M5 Microplate Reader after light shaking, and calculating the inhibitory activity IC of the compound to be tested on human tissue cell lymphoma cell U937 by taking absorbance at 650nm as a reference 50 The results are shown in Table 4:
TABLE 4 inhibitory Activity of the inventive Compounds against human histiocyte lymphoma cell U937
Numbering of compounds IC 50 (nM)
I-1 14.79
I-2 6.31
I-3 5.92
I-4 2.67
Control Compound 1 18.66
Control Compound 2 54.77
The test results show that: IC of the present application compounds for human tissue cell lymphoma cell inhibition 50 The value is obviously lower than that of the existing compounds, which indicates that the compound has good inhibition effect on lymphoma cells and can be used for preventing and treating lymphoma.
Test example 5 inhibition experiment of HacaT cells by the inventive Compound
HacaT cells in logarithmic growth phase (purchased from ATCC) were collected, resuspended in complete medium, and the cell concentration was adjusted to 20 cells/. Mu.l to give a cell suspension, and 96-well plates were inoculated with 100. Mu.l of the cell suspension per well plate. The cells were conditioned at 37 ℃,100% relative humidity, 5% CO 2 After 24 hours of incubation in an incubator, the test compounds according to the application are diluted with medium to the corresponding working concentrations set, at 25. Mu.LWells/wells were added to 96-well plates. The final concentration of compound was diluted in a 4-fold gradient from 0nM to 400 nM. After the test compound is added, the cells are placed at 37 ℃,100% relative humidity, 5% CO 2 Incubate in incubator for 72 hours. Then adding 100 mu L of fresh culture medium containing 10% of CCK-8 into each well, placing in a 37 ℃ incubator for incubation for 2-4 hours, measuring absorbance at 450nm wavelength on SpectraMax M5 Microplate Reader after light shaking, and calculating inhibitory activity IC of the compound to be tested on HacaT cells by taking absorbance at 650nm as a reference 50 The results are shown in Table 5:
TABLE 5 inhibitory Activity of the inventive Compounds against HacaT cells
Numbering of compounds IC 50 (nM)
I-1 39.25
I-2 34.91
I-3 45.42
I-4 28.03
Control Compound 1 163.32
Control Compound 2 111.28
The test results show that: IC of the inventive Compounds against HacaT cell inhibition 50 The value is obviously lower than that of the existing compounds, which shows that the compound has good inhibition effect on human immortalized epidermal cells, and can be used for preventing and treating actinic keratosis and psoriasis.
In conclusion, the compound has remarkable inhibition effect on breast cancer cells, ovarian cancer cells, colon cancer cells, human brain glioma cells and lymphoma cells, and IC thereof 50 Is obviously lower than the similar compounds in the prior art, so that the application of the compound in preparing medicines for preventing and treating cancers has extremely high application potential. The compound also has obvious inhibition effect on HacaT cells, and IC thereof 50 Is obviously lower than the similar compounds in the prior art, so that the compound can be used for preparing medicines for preventing and treating actinic keratosis and psoriasis. The compounds of the application can also be used for the preparation of tubulin inhibitors.

Claims (6)

1. A compound, or a pharmaceutically acceptable salt thereof, characterized in that: the compounds are the following:
2. a process for the preparation of a compound according to claim 1, characterized in that the reaction is carried out by the steps of:
wherein the compound of formula I is a compound of claim 1;
the reaction is carried out specifically by the following steps:
(1) The synthesis steps of the intermediate M-1 are as follows:
mixing 5-bromo-2-fluoropyridine and diethyl malonate for dissolution, heating for reaction under the action of cesium carbonate, separating, and concentrating to dryness to obtain a residue;
dissolving the obtained residue, adding strong alkali for reaction, adjusting pH to 4-5, separating out solid, separating, and drying to obtain intermediate M-1;
(2) The synthesis steps of the intermediate M-2 are as follows:
intermediate M-1 and meta have R 3 Mixing and dissolving substituted benzylamine, reacting under the action of DIPEA and TBTU, and separating to obtain an intermediate M-2;
(3) The synthesis steps of the compound of the formula I are as follows:
and (3) mixing and dissolving the intermediate M-2 and phenylboronic acid with the X substituent at the para position, heating for reaction under the action of potassium carbonate and tetra (triphenylphosphine) palladium, and separating to obtain the compound shown in the formula I.
3. The use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a tubulin inhibitor.
4. The use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of an anti-neoplastic medicament, said medicament being an anti-ovarian, breast, glioma, colon, lymphoma medicament.
5. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of dermatological disorders, said medicament being a medicament for the treatment and/or prophylaxis of actinic keratosis and psoriasis.
6. A pharmaceutical composition characterized by: the compound of claim 1 or pharmaceutically acceptable salt thereof is used as an active ingredient, and pharmaceutically acceptable auxiliary materials or auxiliary ingredients are added to the compound.
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