CN109096171B - Method for preparing deuterated diphenylurea - Google Patents

Abstract

The invention provides a method for preparing deuterated diphenylurea. Specifically, the invention provides a novel intermediate N- (1,1, 1-trideuteromethyl) benzo succinimide which can be used for preparing a deuterated diphenylurea compound, and an application of the intermediate in preparing the deuterated diphenylurea compound. The method can conveniently and efficiently prepare high-purity intermediates and various deuterated diphenyl urea compounds.

Description

Method for preparing deuterated diphenylurea

The application is a divisional application of a Chinese patent application with the application number of 201110064798.2 and the invention name of 'a method for preparing deuterated diphenylurea' which is filed on 17.3.2011.

Technical Field

The invention relates to the field of chemical synthesis, in particular to an intermediate for preparing deuterated diphenylurea, a synthetic method of the intermediate and application of the intermediate.

Background

Omega-diphenylurea derivatives are known compounds of c-RAF kinase activity. For example, in WO 2000/042012, a class of omega-carboxyaryl substituted diphenyl ureas is disclosed, along with their use in the treatment of cancer and related diseases.

Omega-diphenylurea compounds such as Sorafenib (Sorafenib) were first discovered to be inhibitors of c-RAF kinase, and then continued studies have found that it also inhibits the tyrosine kinase activity of MEK and ERK signaling pathways, vascular endothelial growth factor-2 (VEGFR-2), vascular endothelial growth factor-3 (VEGFR-3), and platelet derived growth factor-beta (PDGFR-beta) (Curr Pharm Des 2002; 8: 2255-.

Sorafenib (Sorafenib), tradename Nexavar, is a novel oral multi-kinase inhibitor co-developed by bayer and ONXY, and is rapidly approved by the FDA for the treatment of advanced renal cell carcinoma at month 2005 for 12 due to its superior performance in a phase III clinical study on advanced renal cancer, and marketed in china at month 2006 for 11. However, Sorafenib (Sorafenib) has a number of side effects, such as hypertension, weight loss, skin rash, and the like.

However, the existing process for preparing deuterated diphenylurea compounds is not mature, and has the defects of high cost, low yield, low product purity or difficult separation and the like, so that the development of a novel process for efficiently preparing high-purity diphenylurea compounds is urgently needed in the field.

Disclosure of Invention

The object of the present invention is to provide a process for efficiently preparing a diphenyl urea compound with high purity and an intermediate used for the process.

In a first aspect of the invention, there is provided a process for the preparation of N- (1,1, 1-trideuteromethyl) benzosuccinimide comprising the steps of:

(a) reacting an alkali metal salt of phthalimide with a compound of formula A in an inert solvent,

in the formula, Z is CH₃，O-CD₃Or is or

Wherein R is methyl, nitro or halogen (F, Cl or Br),

thereby forming N- (1,1, 1-trideuteromethyl) phthalimide:

in another preferred embodiment, the inert solvent comprises: n, N-Dimethylformamide (DMF), N-Dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), tetrahydrofuran, 1, 4-dioxane, or a combination thereof.

In another preferred embodiment, in step (a), the reaction temperature is from-10 ℃ to reflux temperature, preferably from-4 ℃ to 100 ℃, more preferably from 20 ℃ to 80 ℃.

In another preferred embodiment, the reaction time is 0.1 to 24 hours, preferably 0.3 to 5 hours, more preferably 0.5 to 2 hours.

In another preferred embodiment, in step (a), the alkali metal salt of phthalimide includes (but is not limited to): a potassium phthalimide salt, a sodium phthalimide salt, a lithium phthalimide salt, or a combination thereof.

In another preferred embodiment, in step (a), the compound of formula a comprises 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester, 3-nitrobenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester, or 4-nitrobenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester.

In another preferred example, the method further comprises, before step (a), step (a 1):

deuterated methanol is reacted with p-toluenesulfonyl chloride under basic conditions and in an inert solvent to form 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester.

In another preferred example, the inert solvent in step (a1) comprises water, tetrahydrofuran, or a mixed solvent thereof.

In a second aspect of the present invention, there is provided a process for the preparation of 1,1, 1-trideuteromethylamine salt, comprising the steps of:

reacting N- (1,1, 1-trideuteromethyl) phthalimide with an acid in an inert or aqueous solvent to form a1, 1, 1-trideuteromethylamine salt, wherein the acid comprises: hydrochloric acid, sulfuric acid, hydrobromic acid, trifluoroacetic acid, or a combination thereof.

In another preferred embodiment, the reaction temperature is from 30 to reflux temperature (e.g., 120 deg.C), preferably from 40-110 deg.C.

In another preferred embodiment, the reaction time is from 0.5 to 48 hours, preferably from 1 to 36 hours, more preferably from 2 to 24 hours.

In a third aspect of the invention, there is provided an intermediate useful in the preparation of deuterated diphenylureas, said intermediate being N- (1,1, 1-trideuteromethyl) phthalimide.

In another preferred embodiment, the compound is used for preparing deuterated diphenyl urea or used as a raw material for preparing deuterated diphenyl urea.

In another preferred embodiment, the deuterated diphenylurea is a compound of formula I:

in the formula:

x is N or N⁺-O^-；

R¹Is halogen (e.g., F, Cl or Br), one or more deuterated or deuterated C1-C4 alkyl groups;

R²is non-deuterated, deuterated or deuterated C1-C4 alkyl or partially or fully halogen-substituted C1-C4 alkyl;

R³、R⁴、R⁵、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴hydrogen, deuterium, or halogen (e.g., F, Cl, or Br), respectively;

R⁶is hydrogen, deuterium, or one or more deuterated or deuterated C1-C4 alkyl groups;

R⁷is hydrogen, deuterium, or one or more deuterated or deuterated C1-C4 alkyl groups;

with the proviso that R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³Or R¹⁴At least one of which is deuterated or deuterium.

More preferably, the deuterated diphenyl urea is selected from the group consisting of:

4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide (CM 4307);

4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide p-toluenesulfonate (CM 4307. TsOH);

4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide (CM 4309);

4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide p-toluenesulfonate (CM 4309. TsOH).

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described below (e.g., examples) may be combined with each other to constitute a new or preferred technical solution. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a graph of serum drug concentration (ng/ml) after oral administration of 3mg/kg of control compound CM4306 to male SD rats.

FIG. 2 is a graph showing the serum drug concentration (ng/ml) of a compound CM4307 of the present invention orally administered to male SD rats at 3 mg/kg.

FIG. 3 is a graph showing the inhibition effect of CM4306 and CM4307 on human hepatocellular carcinoma SMMC-7721 in a nude mouse transplantation model. In the figure, "treatment" indicates that the treatment period is 14 days. Followed by an observation period after drug withdrawal. The 5 days before treatment is the model preparation period.

Detailed Description

The inventors of the present invention have surprisingly found that the deuterated omega-diphenylurea and the pharmaceutically acceptable salts thereof have significantly better pharmacokinetic and/or pharmacodynamic properties than non-deuterated compounds, and therefore are more suitable for use as compounds for inhibiting raf kinase, and further more suitable for preparing drugs for treating cancers and related diseases.

Further, the present inventors have found that a highly pure diphenylurea compound can be produced more efficiently and more easily by using a novel intermediate, N- (1,1, 1-trideuteromethyl) succinimide. The present invention has been completed based on this finding.

Term(s) for

As used herein, "halogen" refers to F, Cl, Br, and I. More preferably, the halogen atom is selected from F, Cl and Br.

As used herein, "alkyl" includes straight or branched chain alkyl groups. Preferred alkyl groups are C1-C4 alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl and the like.

As used herein, "deuterated" refers to a compound or group in which one or more hydrogens are replaced with deuterium. Deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

In another preferred embodiment, the deuterium isotope content of deuterium at the deuterium substitution position is greater than the natural deuterium isotope content (0.015%), more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 97%, more preferably greater than 99%, more preferably greater than 99.5%.

In another preferred embodiment, the compound of formula (I) contains at least 1 deuterium atom, more preferably 3 deuterium atoms, and even more preferably 5 deuterium atoms.

As used herein, the term "compound CM 4306" refers to the compound 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -N-methylpyridine amide.

As used herein, the term "compound CM 4307" refers to the compound 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide.

As used herein, the term "compound CM 4308" refers to the compound 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureide) -3-fluoro-phenoxy) -N-methylpyridinamide.

As used herein, the term "compound CM 4309" refers to the compound 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide.

As used herein, the term "TsOH" refers to p-toluenesulfonic acid. Thus, CM 4307. TsOH represents the p-toluenesulfonate salt of compound CM 4307. CM 4309. TsOH represents the p-toluenesulfonate salt of the compound CM 4309.

Deuterated diphenylurea

Preferred deuterated diphenylurea compounds in the present invention have the structure of formula I:

in the formula:

x is N or N⁺-O^-；

R¹Is halogen (e.g., F, Cl or Br), one or more deuterated or deuterated C1-C4 alkyl groups;

R²is non-deuterated, deuterated or deuterated C1-C4 alkyl or partially or fully halogen-substituted C1-C4 alkyl;

R³、R⁴、R⁵、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴hydrogen, deuterium, or halogen (e.g., F, Cl, or Br), respectively;

R⁶is hydrogen, deuterium, or one or more deuterated or deuterated C1-C4 alkyl groups;

R⁷is hydrogen, deuterium, or one or more deuterated or deuterated C1-C4 alkyl groups;

with the proviso that R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³Or R¹⁴At least one of which is deuterated or deuterium.

In another preferred embodiment, the deuterium isotope content of deuterium at the deuterium substitution position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

In another preferred embodiment, all or substantially (e.g., N, C, O, F, etc.) of the elements other than H in the compound of formula (I)>99 wt.%) is the most abundant naturally occurring element, e.g.¹⁴N、¹²C、¹⁶O and¹⁹F。

in another preferred embodiment, the compound of formula (I) contains at least 1 deuterium atom, more preferably 3 deuterium atoms, and even more preferably 5 deuterium atoms.

In another preferred embodiment, R¹Selected from halogens; more preferably chlorine;

in another preferred embodiment, R²Is trifluoromethyl;

in another preferred embodiment, R⁶Or R⁷Each independently selected from: hydrogen, deuterium, deuterated methyl, or deuterated ethyl; more preferably, it is selected from the group consisting of a deuterated methyl group, a dideuteromethyl group, a trideuteromethyl group, a deuterated ethyl group, a dideuteroethyl group, a trideuteroethyl group, a tetradeuteroethyl group, and a pentadeuteroethyl group.

In another preferred embodiment, R⁶Or R⁷Each independently selected from: hydrogen, methyl or tritdeuteromethyl.

In another preferred embodiment, R³、R⁴Or R⁵Each independently selected from: hydrogen or deuterium.

In another preferred embodiment, R⁸、R⁹、R¹⁰Or R¹¹Each independently selected from: hydrogen or deuterium.

In another preferred embodiment, R¹²、R¹³Or R¹⁴Each independently selected from: hydrogen or deuterium.

In another preferred embodiment, the compound is a preferred compound selected from the group consisting of:

n- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (4- (2- (N-1', 1 ', 1' -trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea;

4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethylcarbamoyl) pyridine-1-oxide;

n- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (2, 6-dideuterio-4- (2- (N-1', 1 ', 1' -trideuteriomethylcarbamoyl) -4-pyridyloxy) phenyl) urea;

n- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (4- (2-deuterium-6- (N-1', 1 ', 1' -trideuteriomethylcarbamoyl) -4-pyridyloxy) phenyl) urea;

or N- (4-chloro-3- (trideuteromethyl) phenyl) -N '- (4- (2- (N-1', 1 ', 1' -trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea;

or 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide.

Intermediates

As used herein, the term "intermediates of the invention" refers to N- (1,1, 1-trideuteromethyl) phthalimide:

this intermediate may also be referred to as "deuterated methylphthalimide".

All or substantially (e.g. N, C, O) of the elements other than H in the compound of the above formula>99 wt.%) is the most abundant naturally occurring element, e.g.¹⁴N、¹²C and¹⁶O。

preparation method

The intermediates of the present invention and the process for preparing the compounds having the structure of formula (I) are more specifically described below, but these specific processes do not limit the present invention at all. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

The preparation of the non-deuterated omega-diphenylureas and their physiologically compatible salts to be used according to the invention is known. Corresponding deuterated omega-diphenyl urea can be prepared by using corresponding deuterated starting compounds as raw materials and synthesizing by using the same route. For example, the compounds of formula (I) according to the invention can be prepared by the preparation described in WO 2000/042012, with the difference that the starting materials used for deuteration are replaced by non-deuteration in the reaction.

Generally, in the preparative schemes, each reaction is generally carried out in an inert solvent at temperatures ranging from room temperature to reflux temperature (e.g., 0 ℃ to 80 ℃, preferably 0 ℃ to 50 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 48 hours.

A particularly preferred preparation scheme, for example for the compound CM4307, is as follows:

in addition, CM4309 can be prepared by replacing compound 4 with compound 3-fluoro-4-amino-phenol, using the above synthetic route.

Deuteration may be introduced through deuterato methylamine.

Deuterated methylamine or the hydrochloride thereof can be obtained by the following reaction. The nitromethane is reacted with deuterium water in the presence of a base (sodium hydride, potassium hydride, deuterated sodium hydroxide, deuterated potassium hydroxide, potassium carbonate, etc.) or in the presence of a phase transfer catalyst to give deuterated nitromethane, and the above experiment is repeated, if necessary, to obtain highly pure deuterated nitromethane. And (3) reducing the deuterated nitromethane, such as under the action of zinc powder, magnesium powder, iron or nickel and the like, to obtain deuterated methylamine or hydrochloride thereof.

Further, deuterated methylamine or a hydrochloride thereof can be obtained by the following reaction.

Key intermediate 3 can also be synthesized from deuterated methanol by the following method.

The specific synthesis method is described in detail in example 1.

The main advantages of the invention include:

(1) by improving the process, the intermediate N- (1,1, 1-trideuteromethyl) benzo succinimide can be prepared more efficiently and with high purity, and the subsequent reaction is facilitated.

(2) Through the intermediate, various deuterated diphenylureas can be conveniently and efficiently prepared.

(3) The reaction condition is milder, and the operation process is safer.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Parts and percentages are parts and percentages by weight unless otherwise indicated.

Intermediate preparation example 1

(1) Synthesis of intermediate N- (1,1, 1-trideuteromethyl) benzsuccinimide

1: preparation of 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester

Sodium hydroxide (180g, 4.5mol, 5.0eq) was added to water (288mL), deuterated methanol (32.4g, 900mmol, 1.0eq) was added at 0 deg.C, and a solution of p-toluenesulfonyl chloride (206g,1.1mmol, 1.2eq) in tetrahydrofuran (288mL) was slowly added dropwise. Warmed to room temperature and stirred overnight. Acetic acid (206g) was added dropwise at 25 ℃ or lower to neutralize to neutrality, the reaction mixture was filtered, the aqueous layer was separated, the aqueous layer was extracted with ethyl acetate (100mL), the cake was dissolved with water (300mL), and the organic layer was extracted with ethyl acetate (200mL), washed with saturated sodium carbonate (100mL), washed with saturated brine (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to give the title compound as a pale yellow liquid 160.5g, 99% purity, 94% yield.

¹H NMR(CDCl₃，400MHz):δ3.20(s,3H),7.71-7.75(m,2H),7.84-7.88(m,2H).

2: preparation of N- (1,1, 1-trideuteromethyl) benzsuccinimide

Phthalimide potassium salt (166.7g, 0.9mol, 2.0eq) was added to N, N-dimethylformamide (DMF, 225mL), and the one-step prepared 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester (85.2g, 0.45mmol, 1.0eq) was added dropwise at room temperature, followed by stirring at 60 ℃ for 0.5 hour. The mixture was filtered while hot, the filter cake was washed with DMF (250mL), the filter cake was removed by filtration, washed again with DMF (100mL), the DMF solutions were combined, water (1150mL) was added dropwise at 0 ℃ to precipitate a white solid, filtered, washed with water (100 mL. times.2) and dried in vacuo to give the title compound as a white solid 66g with 99.6% purity in 88% yield.

¹H NMR(CDCl₃，400MHz):δ7.71-7.77(m,2H),7.84-7.88(m,2H).

3: preparation of 1,1, 1-trideuteromethylamine hydrochloride

N- (1,1, 1-Trideuterated methyl) phthalimide (82g, 0.5mol, 1eq) was added to a mixture of distilled water (625mL) and concentrated hydrochloric acid (625mL, 7.5mol, 15eq) at room temperature, and the mixture was heated to 105 ℃ and refluxed overnight. Cooled to room temperature, filtered and washed with distilled water (50mL × 2), hydrochloric acid removed under reduced pressure to give a pale yellow solid, absolute ethanol (140mL) added, refluxed for 1 hour, cooled to room temperature, filtered and washed with ethanol (30mL), the filter cake dried under vacuum to give the title compound as a white solid 28g, purity > 99.5%, yield 80%.

¹H NMR(DMSO-d6，400MHz):δ8.05(br,2H).

Intermediate preparation example 2

Preparation of intermediate N- (1,1, 1-trideuteromethyl) benzsuccinimide

1: preparation of N- (1,1, 1-trideuteromethyl) benzsuccinimide

Step 2 of example 1 was repeated, except that: the potassium phthalimide salt was replaced with phthalimide (5.9g, 40mmol, 2.0eq) and potassium hydroxide solid (2.2g, 40mmol, 2.0eq) was added in portions at 0 ℃ and stirred for 30 minutes, after which a solution of 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester (3.8g, 20mmol, 1.0eq) in DMF (10mL) was added dropwise, and after the addition was complete, the temperature was raised to 60 ℃ and stirred for 30 minutes. Filtration, washing and vacuum drying were carried out in the same manner to obtain the title compound as a white solid in a purity of 81% at 2.1g in a yield of about 62%.

Intermediate preparation example 3

1: preparation of N- (1,1, 1-trideuteromethyl) benzsuccinimide

Step 2 of example 1 was repeated, except that: the phthalimide potassium salt was replaced with phthalimide (5.9g, 40mmol, 2.0eq), and sodium hydride (80%, 1.2g, 40mmol, 2.0eq) was added in portions at 0 ℃ and stirred for 30 minutes, after which a solution of 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester (3.8g, 20mmol, 1.0eq) in DMF (10mL) was added dropwise, and after completion of the addition, the temperature was raised to 60 ℃ and stirred for 30 minutes. Filtration, washing and vacuum drying were carried out in the same manner to obtain the title compound as a white solid, 2.7g, purity 86%, yield 80%.

From the above experiments, it can be seen that the above 3 methods can produce a new intermediate, N- (1,1, 1-trideuteromethyl) phthalimide. However, by using the potassium phthalimide salt as an intermediate, compared with the production process using phthalimide + potassium hydroxide or sodium hydride, on the one hand, N- (1,1, 1-trideuteromethyl) phthalimide can be obtained more efficiently and with high purity, which is helpful for the subsequent reaction; on the other hand, the reaction condition is milder and the operation process is safer due to no use of sodium hydride.

For step 3 of intermediate preparation example 1, high purity 1,1, 1-trideuteromethylamine hydrochloride (purity > 99.5%) was obtained due to the use of high purity N- (1,1, 1-trideuteromethyl) phthalimide.

Example 1: n- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (4- (2- (N-1', 1 ', 1' -Trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea (Compound CM4307)

The synthetic route is as follows:

procedure one

1. Preparation of 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (3)

Thionyl chloride (60mL) is added into a 250mL single-neck round-bottom flask with a tail gas treatment device, the temperature is maintained between 40 and 50 ℃, anhydrous DMF (2mL) is slowly added dropwise into the flask, stirring is continued for 10 minutes after the dropwise addition is finished, nicotinic acid (20g, 162.6mmol) is added into the flask in batches within 20 minutes, and the color of the solution gradually changes from green to light purple. The temperature was raised to 72 ℃ and stirred at reflux for 16 hours, resulting in a large amount of solid precipitate. Cooled to room temperature, diluted with toluene (100mL), concentrated to near dryness, then diluted with toluene and concentrated to dryness. Filtration and washing with toluene gave a pale yellow solid of 3-chloro-pyridine-2-carbonyl chloride. This solid was slowly added to a saturated solution of deuterated methylamine in tetrahydrofuran under ice-bath, maintaining the temperature below 5 ℃, and stirring was continued for 5 hours. After concentration, ethyl acetate was added to precipitate a white solid, which was filtered off, the filtrate was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to give pale yellow 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (3) (20.68g) in 73% yield.

¹H NMR(CDCl₃,300MHz):8.37(d,1H),8.13(s,1H),7.96(br,1H),7.37(d,1H).

2. Preparation of 4- (4-aminophenoxy) -2-pyridine- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (5)

P-aminophenol (9.54g,0.087mol), potassium tert-butoxide (10.3g,0.092mol) were added to 100mL of dry, anhydrous DMF, and the solution was turned dark brown, stirred at room temperature for 2 hours, then 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (3) (13.68g,0.079mol), anhydrous potassium carbonate (6.5g,0.0467mol) were added thereto, and the reaction mixture was warmed to 80 ℃ and stirred overnight. After completion of the TLC detection reaction, the reaction mixture was cooled to room temperature, poured into a mixed solution of ethyl acetate (150mL) and saturated brine (150mL), stirred to separate layers, allowed to stand for liquid separation, the aqueous layer was extracted with ethyl acetate (100 mL. times.3), the combined extracts were washed with saturated water (100 mL. times.3), dried over anhydrous sodium sulfate, and concentrated to give 4- (4-aminophenyloxy) -2-pyridine- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (18.00g) in 92% yield as pale yellow.

¹H NMR(CDCl₃,300MHz):8.32(d,1H),7.99(br,1H),7.66(s,1H),6.91～6.85(m,3H),6.69(m,2H),3.70(br,s,2H).

3. Preparation of N- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (4- (2- (N-1', 1 ', 1' -trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea (CM4307)

5-amino-2-chloro-trifluoromethylbenzene (15.39g,78.69mol), N, N '-Carbonyldiimidazole (CDI) (13.55g,83.6mmol) was added to 120mL of dichloromethane, and after stirring at room temperature for 16 hours, a solution of 4- (4-aminophenoxy) -2-pyridine- (N-1', 1 ', 1' -trideuteromethyl) carboxamide (18g,73mmol) in dichloromethane (180mL) was slowly added dropwise thereto, and stirring at room temperature was continued for 18 hours. After TLC detection reaction, removing partial dichloromethane solvent to about 100mL, standing at room temperature for several hours, separating out a large amount of white solid, filtering, and washing the solid with a large amount of dichloromethane. The filtrate was concentrated to remove a part of the solvent, and then a part of the solid was separated out again, and the combined solid was washed with a large amount of methylene chloride again to obtain pure N- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (4- (2- (N-1', 1 ', 1' -trideuteriomethylcarbamoyl) -4-pyridyloxy) phenyl) urea CM4307 (20.04g) as a white powder in 58% yield.

¹H NMR(CD₃OD,300MHz):8.48(d,1H),8.00(d,1H),7.55(m,5H),7.12(d,1H),7.08(s,2H),ESI-HRMS m/z:C21H13D3ClF3N4O3,Calcd.467.11,Found 490.07(M+Na)⁺.

Alternatively, compound CM4307 may be dissolved in dichloromethane and reacted with m-chloroperoxybenzoic acid to produce the corresponding oxidation product: 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteriomethylcarbamoyl) pyridine-1-oxide.

Example 2: preparation of 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (3)

a) Phthalimide (14.7g,0.1mol), deuterated methanol (3.78g,0.105mol,1.05eq), and triphenylphosphine (28.8g,0.11mol,1.1eq) were dissolved in anhydrous tetrahydrofuran, and a solution of DEAD (1.1eq) in tetrahydrofuran was added dropwise under ice-cooling, followed by stirring at room temperature for one hour after completion of the addition. And (3) purifying by passing through a column, or after the solvent is dried in a spinning mode, adding a proper amount of DCM, freezing in a refrigerator to separate out a solid, filtering, drying the filtrate in a spinning mode, and then passing through the column quickly to obtain 14.8g of the pure deuterated methylphthalimide. The yield thereof was found to be 90%.

Although the intermediate can be prepared by the process, the purity of the product after reaction is low, column purification is needed, the post-treatment process is complicated, and reagents such as triphenylphosphine and diethyl azodicarboxylate (DEAD) are not environment-friendly, so that the process is not suitable for industrial production.

b) Dissolving deuterated methylphthalimide (12.5g,0.077mol) in a proper amount of hydrochloric acid (6N, 50ml), refluxing in a sealed tube for 24-30 hours, cooling the reaction solution to room temperature, placing the reaction solution in a refrigerator to cool to below zero, filtering the precipitated solid, washing the solid with cold deionized water, collecting the filtrate, carrying out rotary evaporation to remove water, and drying to obtain deuterated methylamine hydrochloride. Anhydrous DCM (100ml) was added to deuterated methylamine hydrochloride, and methyl 4-chloronicotinate hydrochloride (6.52g,0.038mol,0.5eq), sodium carbonate (12.2g,0.12mol,1.5eq) were added, the reaction flask was sealed, and placed in a refrigerator for reaction for one day. And (5) detecting the reaction by TLC, washing with water, drying, concentrating, and purifying by column chromatography. The compound, 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) carboxamide (3), was obtained in 5.67g, yield 86%. The structural features thereof were in accordance with example 1.

Example 3

Synthesis of diphenyl urea compound CM4307 by intermediate N- (1,1, 1-trideuteromethyl) benzo succinimide

Preparation of 1: 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide A2

Methyl 4-chloro-2-picolinate (50g, 291mmol, 1eq) was placed in a three-necked flask containing 250mL of tetrahydrofuran, and 1,1, 1-trideuteromethylamine hydrochloride (31g, 437mmol, 1.5eq), 400 mesh anhydrous potassium carbonate (80g, 583mmol, 2eq) were added with stirring at room temperature for 20 hours, then water (250mL) and methyl tert-butyl ether (150mL) were added, the layers were separated with stirring, the aqueous phase was extracted with methyl tert-butyl ether (100mL), the organic phases were combined and dried with anhydrous sodium sulfate and filtered, and the solvent was removed under reduced pressure to give 48g of a pale yellow liquid with 99% purity, 96% yield.

¹H NMR(DMSO-d6，400MHz):δ7.64(dd,J＝2Hz,5.2Hz,1H),7.97(d,J＝1.6Hz,1H),8.54(d,J＝5.2Hz,1H),8.74(br,1H).

MS(ESI,m/z)calcd.for C₇H₄D₃ClN₂O:173,found:174[M+H]⁺

Preparation of 2:4- (4-Aminophenoxy) -2-pyridine- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide A3

Under nitrogen protection, 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide (2.4g, 13.8mmol, 1eq) was dissolved in dimethylsulfoxide (10mL), 4-aminophenol (1.6g, 14.9mmol, 1.08eq) was added, potassium tert-butoxide (1.7g, 15.2mmol,1.1eq) was added in portions, and after the internal temperature was stabilized, the temperature was raised and the internal temperature was maintained at 80 ℃ for reaction for 4 hours. The reaction mixture was diluted by adding isopropanol (10mL) and stirring at room temperature, concentrated hydrochloric acid (37%, 10mL) was added dropwise under nitrogen, and the mixture was stirred for 1 hour and then filtered. The solid was taken out and dissolved in water (20mL), activated carbon (0.1g) was added, stirring was carried out for 1 hour, filtration was carried out, a potassium carbonate solution (2g potassium carbonate in 6mL water) was slowly added dropwise to the filtrate, precipitation of the solid was observed, filtration was carried out and washing with water (5mL) was carried out, and vacuum drying was carried out to obtain 2.7g of light brown crystals with a purity of 99.9% and a yield of 78%.

¹H NMR(DMSO-d6，400MHz):δ5.19(br,2H),6.66-6.68(m,2H),6.86-6.88(m,2H),7.07(dd,J＝2.8Hz,5.6Hz,1H),7.36(d,J＝2.8Hz,1H),8.45(d,J＝5.6Hz,1H),8.72(br,1H).

MS(ESI,m/z)calcd.for C₁₃H₁₀D₃N₃O₂Cl:246,found:247[M+H]⁺

Preparation of 3:4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide CM4307

4- (4-Aminophenyloxy) -2-pyridine- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide (2.6g, 10.6mmol, 1eq) was dissolved in dichloromethane (10mL) and dimethylsulfoxide (3mL) under nitrogen, and a solution of 4-chloro-3-trifluoromethylphenyl isocyanate (2.5g, 11.1mol, 1.05eq) in dichloromethane (8mL) was added dropwise to the solution at room temperature and stirred for 20 minutes. The temperature was reduced to 2 ℃ and water (10mL) was added dropwise, stirred for 0.5 h, filtered, and the product was washed with dichloromethane (10mL) and dried under vacuum to give an off-white solid 4.7g, purity 99.8%, yield 95.4%.

¹H NMR(DMSO-d6，400MHz):δ7.15(dd,J＝2.8Hz,5.6Hz,1H),7.17-7.19(m,2H),7.40(d,J＝2.4Hz,1H),7.59-7.69(m,4H),8.13(d,J＝2.4Hz,1H),8.51(d,J＝6Hz,1H),8.75(br,1H),8.90(br,1H),9.22(br,1H).

MS(ESI,m/z)calcd.for C₂₁H₁₃D₃ClF₃N₄O₃:467,found:468[M+H]⁺

Example 4

Preparation of 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide p-toluenesulfonate (CM 4307. TsOH)

P-toluenesulfonic acid monohydrate (1.6g, 8.5mmol, 0.88eq) was dissolved in anhydrous ethanol (5mL), filtered, and warmed to 70 ℃ and kept warm for temporary use. Suspending 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide (4.5g, 9.6mol, 1eq) and p-toluenesulfonic acid monohydrate (0.66g, 3.5mmol, 0.36eq) in ethanol (50mL), heating to an internal temperature of 78 deg.C and refluxing for 40 minutes, observing that all solids are dissolved, slightly cooling and filtering while hot, heating the filtrate to an internal temperature of 78 deg.C and refluxing the solution again for 1 hour to dissolve all solids again and clarify, rapidly adding the above 70 deg.C anhydrous ethanol solution to the clarified solution at one time, stopping heating and naturally cooling and stirring at 0 deg.C for 0.5 hour, filtering, washing with anhydrous ethanol (5 mL. times.2), vacuum drying at 50 deg.C for 24 hours, 5.8g of a white to off-white solid was obtained with a purity of 99.3% and a yield of 93%.

¹H NMR(DMSO-d6，400MHz):δ2.30(s,3H),7.15(d,J＝8.8Hz,2H),7.20(d,J＝8.8Hz,2H),7.23(dd,J＝2.8Hz,6Hz,1H),7.52(d,J＝8Hz,2H),7.55(d,J＝2.8Hz,1H),7.63(d,J＝8.8Hz,3H),7.68(dd,J＝2.4Hz,9.2Hz,1H),8.03(br,1H),8.14(d,J＝2.4Hz,1H),8.56(d,J＝6Hz,1H),8.91(br,1H),9.17(br,1H),9.36(br,1H).¹³CNMR(DMSO-d6，400MHz):δ21.1,26.1,111.7,115.2,117.0,120.7(2C),121.6(2C),121.9,122.8,123.2,124.6,125.6(2C),127.2,129.0(2C),132.3,138.8,139.5,139.9,144.1,146.6,147.2,152.8,159.9,170.7ppm.

Liquid phase conditions: agilent 1100 Series; a chromatographic column: synergi 4. mu. POLAR-RP 80A, 250X 4.6mm, 4 μm; column temperature: 25 ℃; detection wavelength: UV 210 nm; mobile phase: a: ammonium dihydrogen phosphate 10mmol/L, B: methanol; sample introduction amount: 10 mu L of the solution; flow rate: 0.8 mL/min; analysis time: 70 min; gradient program: 50% mobile phase B from 0 to 15 minutes, increasing to 75% mobile phase B from 15 to 32 minutes, then eluting with 75% mobile phase B from 32 to 55 minutes for 23 minutes. Retention time: 4.95min (p-toluenesulfonic acid); 47.11min (CM 4307).

Example 5

Synthesis of diphenyl urea compound CM4309 by intermediate N- (1,1, 1-trideuteromethyl) benzo succinimide

Preparation of 1: 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide A2

The preparation method is the same as example 3.

2: preparation of 4- (4-amino-3-fluoro-phenoxy) -pyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide B1

Placing potassium tert-butoxide (15g, 130mmol, 1.3eq) in a solution of N, N-dimethylacetamide (DMA, 50mL), slowly adding dropwise a DMA (50mL) solution of 3-fluoro-4-amino-phenol (16g, 127mmol, 1.3eq) at 0-5 deg.C, stirring at room temperature for 20 minutes, heating to 100 deg.C and slowly adding dropwise DMA (50mL) of 4-chloropyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide 3(17g, 97mmol, 1eq) after the addition is completed, continuing stirring for 0.5 hour, cooling to room temperature, adding ethyl acetate (1.5L) to the reaction solution for dilution and stirring for 0.5 hour, filtering to remove inorganic salts, washing with water (500 mL. times.3), drying with anhydrous sodium sulfate, removing solvent under reduced pressure, adding ethanol (100mL) to the crude product and refluxing for pulping for 2 hours, cooling to room temperature filtration gave 20g of a brown solid with HPLC purity of 96% and yield of 80%.

¹H NMR(CD₃OD-d4，400MHz):δ6.74-6.77(m,1H),6.87(dd,J＝2.4Hz,11.6Hz,1H),6.93(t,J＝10Hz,1H),7.02(dd,J＝2.8Hz,6Hz,1H),7.54(d,J＝2.4Hz,1H),8.44(d,J＝6Hz,1H).

MS(ESI,m/z)calcd.for C₁₃H₉D₃ClN₃O2:264,found:265[M+H]⁺

3: preparation of 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide CM4309

4-chloro-3-trifluoromethylphenyl isocyanate (13g, 58mmol, 1.1eq) was placed in a single vial with dichloromethane (70mL) at room temperature and a solution of 4- (4-amino-3-fluoro-phenoxy) -pyridine-2- (N-1 ', 1 ', 1 ' -trideuteromethyl) amide (14g, 53mmol, 1eq) in dichloromethane (350mL) was slowly added dropwise to the above solution, stirred at room temperature for 20 hours, filtered after the reaction and washed with dichloromethane (20 mL. times.2) to give 13g of a light brown solid with 98% purity in 50% yield.

¹H NMR(DMSO-d6，400MHz):δ7.06-7.10(m,1H),7.19(dd,J＝2.4Hz,5.6Hz,1H),7.35(dd,J＝2.8Hz,12Hz,1H),7.43(d,J＝2.4Hz,1H),7.63(m,2H),8.14(br,1H),8.17(t,J＝8.8Hz,1H),8.53(d,J＝5.6Hz,1H),8.75(d,J＝1.6Hz,1H),8.78(br,1H),9.54(br,1H).

MS(ESI,m/z)calcd.for C₂₁H₁₂D₃ClF₄N₄O₃:485,found:486[M+H]⁺

Example 6

Preparation of 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide p-toluenesulfonate (CM 4309. TsOH)

4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl ] ureide) -3-fluoro-phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide (2.16g, 4.33mmol, 1eq) was suspended in ethanol (50mL), heated under reflux until the solution was clear, filtered while hot to remove insoluble matter, the filtrate was heated under reflux to clear, methanesulfonic acid (70%, 8.66mmol, 1mL, 2eq) was rapidly added at a time, the clear solution was left for about 0.5 min, the oil bath was naturally cooled to room temperature, stirred for 1h, filtered to obtain insoluble matter, and dried under vacuum at 50 ℃ for 20 h to obtain 2.36g of white crystals with a yield of 83%.

¹H NMR(DMSO-d6，400MHz):δ2.30(s,3H),7.09-7.14(m,3H),7.24(dd,J＝2.4Hz,5.2Hz,1H),7.37(dd,J＝2.4Hz,11.6Hz,1H),7.48-7.50(m,3H),7.61-7.66(m,2H),8.15-8.19(m,2H),8.56(d,J＝5.6Hz,1H),8.79(br,1H),8.85(br,1H),9.56(br,1H),10.38(br,1H).

Melting point: 240.7-241 deg.C

EXAMPLE 7 preparation of the compound N- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (2, 6-didedeuterium-4- (2- (N-1', 1 ', 1' -trideuteriomethylcarbamoyl) -4-pyridyloxy) phenyl) urea

The process described in example 1 is followed, except that: the p-aminophenol is replaced with 3, 5-dideutero-4-aminophenol to produce the target compound.

EXAMPLE 8 preparation of the compound N- (4-chloro-3- (trifluoromethyl) phenyl) -N '- (4- (2-deuterium-6- (N-1', 1 ', 1' -trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea

The process described in example 1 is followed, except that: replacing nicotinic acid with 2-deuterium-6-carboxypyridine to obtain the target compound.

Example 9 preparation of the compound N- (4-chloro-3- (trideuteromethyl) phenyl) -N '- (4- (2- (N-1', 1 ', 1' -trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea:

the process described in example 1 is followed, except that: the target compound was prepared by substituting 5-amino-2-chloro-trifluoromethylbenzene for 5-amino-2-chloro-trideuteromethylbenzene.

Example 10:

pharmacokinetic evaluation of deuterated diphenylurea compounds in rats

8 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, divided into 2 groups of 4 rats each (rat numbers: 13-16 for control group; 9-12 for experimental group), administered orally a single 3mg/kg dose of (a) the composition: the non-deuterated N- (4-chloro-3- (trifluoromethyl) phenyl) -N ' - (4- (2- (N-methylcarbamoyl) -4-pyridyloxy) phenyl) urea (control compound CM4306) or (b) N- (4-chloro-3- (trifluoromethyl) phenyl) -N ' - (4- (2- (N-1 ', 1 ', 1 ' -trideuteromethylcarbamoyl) -4-pyridyloxy) phenyl) urea prepared in example 1 (compound CM4307 of the present invention) were compared for their pharmacokinetic differences.

Rats were fed with standard feed, given water and chlordiazepoxide. The administration of chlordiazepoxide was stopped in the evening before the experiment and was resumed 2 hours after the administration. Fasting began 16 hours prior to the experiment. The drug was dissolved with 30% PEG 400. Blood was collected from the orbit at 0.083 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr and 24 hr post-dose.

The rats were briefly anesthetized after ether inhalation and 300uL of blood was collected from the orbit into a test tube. The tube contained 30ul of 1% heparin salt solution. Before use, the tubes were dried overnight at 60 ℃. After completion of blood sample collection at a subsequent time point, rats were sacrificed after ether anesthesia.

Immediately after blood collection, the tubes were gently inverted at least 5 times to ensure mixing and then placed on ice. The blood samples were centrifuged at 5000rpm for 5 minutes at 4 ℃ to separate the serum from the erythrocytes. Pipette out 100uL of serum into a clean plastic centrifuge tube, indicating the name of the compound and the time point. Serum was stored at-80 ℃ prior to LC-MS analysis.

The results are shown in FIGS. 1-2. The results show that the half-life T of CM4307 to CM4306 is_1/2Extension [ 11.3 + -2.1 hours and 8.6 + -1.4 hours, respectively]Area under the curve AUC_0-∞CM4307 is significantly increased compared with CM4306 [ 11255 + -2472 ng.h/mL and 7328 + -336 ng.h/mL, respectively]Apparent reduction in CM4307 clearance over CM4306 [ 275. + -. 52mL/h/kg and 410. + -. 18.7mL/h/kg, respectively]。

From the above results, it can be seen that the compounds of the present invention have better pharmacokinetics and thus better pharmacodynamics and treatment effects in animals.

In addition, the metabolic processes of the compounds of the invention in organisms are altered by deuteration. In particular, hydroxylation on the phenyl group is made difficult, which leads to a reduction in the First-pass effect (First-pass effect). In this case, the dosage can be varied and a depot can be formed, which also improves the applicability in the form of a depot.

In addition, the pharmacokinetic effect is also altered by deuteration, since the deuterated compound forms completely another hydrate film, so that the distribution in the organism is clearly different from that of the non-deuterated compound.

Example 11: pharmacodynamic evaluation of CM4307 for growth inhibition of human hepatocellular carcinoma SMMC-7721 nude mouse transplanted tumor

Balb/c nu/nu nude mice, 6 weeks old, female, 70, were purchased from Shanghai laboratory animal resources center (Shanghai Spial-BikKa laboratory animals Co., Ltd.).

SMMC-7721 cells were purchased from Shanghai Life sciences college of Chinese academy (Shanghai, China).

Establishing a tumor nude mouse transplantation model: harvesting SMMC-7721 cells in logarithmic growth phase, suspending the cells in 1 XPBS after counting, and adjusting the cell suspension concentration to 1.5X 10⁷And/ml. Tumor cells were inoculated subcutaneously in the right axilla of nude mice using a 1ml syringe, 3X 10⁶0.2 ml/mouse. A total of 70 nude mice were inoculated.

The tumor volume reaches 30-130mm³At that time, animals were randomly grouped to give 58 animals in total, with tumor differences of each group being less than 10% of the mean, and dosing was initiated.

The experimental dose grouping settings are shown in the following table:

group of

Animal(s) production

Compound (I)

Mode of administration

Dosage (mg/kg)

Scheme(s)

1

10

Blank control (solvent)

po

0.1ml/10gBW

qdx2 weeks

2

8

CM4306

po

10mg/kg

qdx2 weeks

3

8

CM4306

po

30mg/kg

qdx2 weeks

4

8

CM4306

po

100mg/kg

qdx2 weeks

5

8

CM4307

po

10mg/kg

qdx2 weeks

6

8

CM4307

po

30mg/kg

qdx2 weeks

7

8

CM4307

po

100mg/kg

qdx2 weeks

Animal body weights and tumor sizes were determined twice a week during the experiment. Clinical symptoms were recorded with daily observations. At the end of dosing, tumor size was recorded by photographing. One mouse was sacrificed per group and tumor tissue was taken and fixed in 4% paraformaldehyde. After the administration, the observation is continued, and when the mean value of the tumor is more than 2000mm³Or when the animal is in an endangered state, the animal is sacrificed and roughly dissected, and tumor tissues are taken and fixed in 4% paraformaldehyde.

The Tumor Volume (TV) is calculated as: TV as a × b²/2. Where a, b represent tumor measurement length and width, respectively. Relative tumor volume (relative)tomor volume, RTV) is calculated as: RTV-Vt/V₀. Wherein V₀Tumor volume at the time of group administration and Vt is tumor body weight at the time of measurement. The evaluation index of the anti-tumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%) ═ T_RTV/C_RTV)×100％。T_RTVTo the treatment group RTV, C_RTVNegative control group RTV.

The evaluation standard of the curative effect is as follows: the relative tumor increment rate T/C (%). is less than or equal to 40%, and p is less than 0.05 by statistical analysis.

The results are shown in FIG. 3. The CM4306 and CM4307 are administrated by intragastric administration at single dose of 10, 30 and 100mg/kg for 2 weeks, and both compounds show the effect of inhibiting tumor growth in a dose-dependent manner. At the end of dosing, CM4306 had a T/C% of 56.9%, 40.6%, and 32.2%, respectively. The T/C (%) of CM4307 was 53.6%, 40.8% and 19.6%, respectively. Wherein the T/C% of the 100mg/kg dose group is less than 40%, the tumor volume is obviously different from that of a control group (p is less than 0.01), and the tumor growth inhibition effect is obvious.

The high-dose 100mg/kg group of CM4307 has stronger tumor inhibition effect than the high-dose group of CM4306 (the optimal T/C% is 19.6% and 32.2%, respectively, d15), and the tumor volume groups have significant difference (p is less than 0.01). Compared with CM4306, the absolute value of the tumor inhibition rate of CM4307 is improved by more than 10%, and the relative amplitude is improved by about 60% (32.2%/19.6% -1 ═ 64%), thereby showing more remarkable tumor growth inhibition effect.

In addition, no other drug-related toxicity reaction was observed during the test.

Example 12

Inhibiting activity on c-Kit and PDGFR-beta protein tyrosine kinase molecule level

1. Experimental methods

In this example, Enzyme-Linked Immunosorbent Assay (ELISA) was used to determine the inhibitory activity of diphenylurea compounds on the level of c-Kit and PDGFR-beta protein tyrosine kinase molecules.

The compounds tested: CM4306, CM4308CM 4309.

The main reagents are as follows:

reaction substrate Poly(Glu,Tyr)_4:1Purchased from Sigma company; monoclonal antibody PY99 against phosphotyrosine was purchased from Santa Cruz; horse radish peroxidase-labeled goat anti-mouse IgG was purchased from Calbiochem; ATP, DTT, OPD were purchased from Amresco; enzyme-labeled plates were purchased from Corning; su11248 was purchased from Merk corporation.

The experimental method comprises the following steps:

see Roskoski, R., Jr. Sunitinib: a VEGF and PDGF receptor protein kinase and angiogenesis inhibitor. biochem Biophys Res Commun,356:323-328,2007, including in particular:

kinase reaction substrate Poly (Glu, Tyr)_4:1Diluting to 20 mu g/ml with PBS without potassium ions, and coating the enzyme label plate. Adding a tested diphenyl urea sample into the pores of the coated enzyme label plate (the tested sample is firstly prepared into 10 by DMSO)^-2M stock solution, diluted to the desired concentration with the reaction buffer immediately before use, was added to the wells to give a final concentration of 10 in 100. mu.l of the reaction system^{- 5}mol/L). Meanwhile, positive control holes are set, and a positive control compound Su11248 is added.

An ATP solution diluted with the reaction buffer (ATP final concentration 5. mu.M) was added, and finally, the test tyrosine kinase diluted with the reaction buffer was added. The total volume of the reaction system was 100. mu.l. Negative control wells and no enzyme control wells were set simultaneously.

And (3) placing the reaction system in a wet box, carrying out shaking table light-shielding reaction for 1h at 37 ℃, and washing the plate with T-PBS for three times after the reaction is finished. Adding 99100 mul of PY antibody into the well, and shaking the mixture at 37 ℃ for 30 min. The plate was washed three times with T-PBS. Adding 100 μ l/well of goat anti-mouse IgG labeled with horseradish peroxidase, and performing shake reaction at 37 ℃ for 30 min. The plate was washed three times with T-PBS. Adding OPD developing solution 100 μ l/hole, and reacting at room temperature in dark for 1-10 min. 2M H was added₂SO₄50 μ l of the reaction was stopped and measured A by a microplate reader VERSAmax of adjustable wavelength type₄₉₂The value is obtained.

The inhibition rate of the sample was calculated by the following formula:

2. results of the experiment

The above experimental results are the average of two experiments.

3. Evaluation criteria and results evaluation

Test compound at test concentration 10 on the premise that the inhibitory activity of the positive control compound meets the reference range^-5Under mol/L, the inhibition rate is more than 50 percent, and the effectiveness is judged; the inhibition rate of less than 50% is judged to be ineffective.

The results show that the inhibition rates of CM4306, CM4308 and CM4309 on protein tyrosine kinases c-Kit and PDGFR-beta are more than 50%, so that the protein tyrosine kinases c-Kit and PDGFR-beta have significant inhibition activities on the molecular level.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (5)

1. A preparation method of deuterated diphenylurea, wherein the deuterated diphenylurea is 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide p-toluenesulfonate;

and the method comprises the following steps:

(a) reacting an alkali metal salt of phthalimide with a compound of formula A in an inert solvent,

in the formula, Z is CH₃，O-CD₃Or is or

Wherein R is methyl, nitro or halogen,

thereby forming N- (1,1, 1-trideuteromethyl) phthalimide:

wherein, the inert solvent is: n, N-Dimethylformamide (DMF), N-Dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), or a combination thereof; and

using the N- (1,1, 1-trideuteromethyl) phthalimide prepared in step (a) as a starting material, 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureide) -phenoxy) -2- (N-1 ', 1 ', 1 ' -trideuteromethyl) picolinamide p-toluenesulfonate was prepared.

2. The method of claim 1, wherein the alkali metal salt of phthalimide is selected from the group consisting of: a potassium phthalimide salt, a sodium phthalimide salt, a lithium phthalimide salt, or a combination thereof.

3. The method of claim 1, wherein the method further comprises, before step (a), step (a 1): deuterated methanol is reacted with p-toluenesulfonyl chloride under basic conditions and in an inert solvent to form 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester.

4. The method of claim 1, wherein the inert solvent in step (a1) is water, tetrahydrofuran, or a mixture thereof.

5. The method of claim 1, wherein said N- (1,1, 1-trideuteromethyl) phthalimide is prepared by the method of:

adding 0.9mol of phthalimide potassium salt into 225mL of N, N-dimethylformamide, dropwise adding 0.45mmol of 4-methylbenzenesulfonic acid- (1,1, 1-trideuteromethyl) ester at room temperature, and stirring at 60 ℃ for 0.5 hour; the mixture is filtered while hot, the filter cake is washed with 250mL of DMF, the filter cake is taken out by filtration and washed again with 100mL of DMF, the DMF solutions are combined and at 0 ℃, 1150mL of water are added dropwise to precipitate a white solid, the white solid is filtered, washed with water and dried in vacuum.

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