CN108084179B

CN108084179B - Compound with spiro structure, preparation method and application thereof

Info

Publication number: CN108084179B
Application number: CN201611031343.XA
Authority: CN
Inventors: 周晓天; 周伟澄; 林快乐
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2016-11-22
Filing date: 2016-11-22
Publication date: 2021-02-05
Anticipated expiration: 2036-11-22
Also published as: CN108084179A

Abstract

The invention discloses a compound with a spiro structure, and a preparation method and application thereof. The compound with the spiro structure shown as the formula I, II or III or the pharmaceutically acceptable salt thereof has better hDHFR inhibitory activity, antitumor activity and antibacterial activity.

Description

Compound with spiro structure, preparation method and application thereof

Technical Field

The invention relates to a compound with a spiro structure, a preparation method and application thereof.

Background

In many research and development ideas, dihydrofolate reductase (DHFR) in the metabolic process of folate is an important drug target. The dihydrofolate reductase is a key enzyme in the biological metabolic process of folic acid, and has the main function of catalyzing the reduction of dihydrofolate into tetrahydrofolate in organisms, and the tetrahydrofolate is an essential coenzyme in the DNA synthesis process. The DHFR inhibitor has a structure similar to that of a substrate of dihydrofolate reductase, and can competitively bind with DHFR, block the binding of DHFR with a normal substrate, inhibit the catalytic reduction activity of DHFR, make the conversion of dihydrofolate into tetrahydrofolate difficult, interfere with the folate cycle process, and then interfere with the DNA biosynthesis of pathogens (such as tumor cells and bacteria), and finally cause the death of pathogen cells. Therefore, the development of inhibitors of dihydrofolate reductase has been an important direction for drug research.

The widely used classical DHFR inhibitors, such as Methotrexate (MTX), Edatrexate (ETX), pemetrexed (ALIMTA), generally contain a glutamate residue in structure, and require active transport into cells via a reduced folate carrier protein, which catalyzes polyglutamation by folate polyglutamate synthase (FPGS), and thus functions. Polyglutamation can lead to long retention time of the DHFR inhibitor in cells, and easily causes excessive toxicity to normal cells; on the other hand, the DHFR inhibitor is a substrate of FPGS, so that the FPGS is easily over-expressed, and the tumor cells are induced to generate drug resistance. In order to reduce the toxicity and drug resistance of the DHFR inhibitor, the development of a small molecule compound which does not contain glutamic acid residue and is not used as an FPGS substrate is one of the research hotspots of the DHFR inhibitor at present.

A series of novel diamino dihydrotriazine compounds with spiro structures are synthesized by Matxiang et al, and corresponding biological activity tests are respectively carried out, and the results show that the compounds have good inhibitory activity on DHFR and have the effect of inhibiting proliferation of tumor cells. Among them, the most active compounds have the following structures of compound 1, compound 2 and compound 3, but the antitumor activity is low. It is noted that, when the side chain is modified by isostering, replacing the O atom with the N atom, there is a significant decrease in activity, which would not allow one skilled in the art to optimize such compounds generally by isostering. (Xiaong Ma, Renee Ser-Pen Won. Antiproliferative Activity Agents MCF-7Breast Cancer Cells by Diamino-Triazaspirodrine antagonists. chem. biol. drug Des. 2009; 74: 322-

CN103664972A also disclosed compounds a2, a6 and a10, but their inhibitory activity, antitumor activity and antibacterial activity against hDHFR were low.

Therefore, there is a need in the art for compounds with superior inhibitory, anti-tumor and antibacterial activity against hDHFR.

Disclosure of Invention

The invention aims to solve the technical problem that the prior compound with a spiro structure has lower hDHFR inhibitory activity, antitumor activity and antibacterial activity, so that the invention provides the compound with the spiro structure, a preparation method and application thereof, and the compound has better hDHFR inhibitory activity, antitumor activity and antibacterial activity, and particularly has better anti-liver cancer (such as HepG2) activity and anti-breast cancer (such as MDA-MB-231) activity.

The invention provides a compound with a spiro structure as shown in formula I, II or III, or pharmaceutically acceptable salt thereof:

wherein R is substituted or unsubstituted phenyl; in the "substituted or unsubstituted phenyl", the "substitution" is by one or more of the following groups, and when a plurality of substituents are present, the substituents are the same or different: halogen (e.g. fluorine, chlorine, bromine or iodine), C_1-6Alkoxy (e.g. methoxy) and C_1-6Alkyl (e.g., tert-butyl);

x is substituted or unsubstituted C_1-6Alkylcarbonyl (said "C_1-6Alkylcarbonyl "e.g. propionyl), substituted or unsubstituted C_6-14Aryl carbonyl group (said "C_6-14Arylcarbonyl "e.g. benzoyl) or, substituted or unsubstituted C_1-6Alkyl (said C)_1-6Alkyl groups such as methyl, ethyl, n-propyl or n-butyl); said "substituted or unsubstituted C_1-6Alkylcarbonyl group, substituted or unsubstituted C_6-14Arylcarbonyl "and" substituted or unsubstituted C_1-6In "alkyl", the "substitution" is independently by one or more of the following groups, which when present are the same or different: phenyl, substituted or unsubstituted phenoxy, benzyloxy, methoxycarbonyl and benzyloxycarbonyl; in the "substituted or unsubstituted phenoxy group", the "substitution" is by one or more of the following groupsAnd when a plurality of substituents are present, said substituents are the same or different: halogen (e.g. fluorine, chlorine, bromine or iodine), C_1-6Alkyl (e.g. methyl or tert-butyl), C_1-6Alkoxy (e.g., methoxy) and nitro; but X is not benzyloxycarbonyl.

In the compound I, II or III, R is preferably 4-chlorophenyl, 4-bromophenyl, 2, 4-dichlorophenyl, 4-methoxyphenyl, 4-tert-butylphenyl or 2,4, 5-trichlorophenyl.

In the compound I, X is preferably n-propyl, n-butyl, benzyl, 3-phenylpropyl, 4-phenylbutyl, 3-phenoxypropyl, 3- (4-chlorophenoxy) propyl, 3- (4-fluorophenoxy) propyl, 3- (4-bromophenoxy) propyl, 3- (4-methylphenoxy) propyl, 3- (4-nitrophenoxy) propyl, methoxycarbonylmethyl, benzyloxycarbonylmethyl, benzoyl or 3-phenylpropionyl.

In the compound I, II or III, the phrase "a pharmaceutically acceptable salt thereof" refers to a salt of the compound I, II or III with a pharmaceutically acceptable acid or base, preferably a salt of the compound I, II or III with a pharmaceutically acceptable acid. The "pharmaceutically acceptable acid" may be an acid conventional in the art, such as an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid) or an organic acid (e.g., formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, or an acidic amino acid such as aspartic acid or glutamic acid).

Preferably, the compound I in the present invention is any one of the following compounds:

preferably, the compound II described in the present invention is any one of the following compounds:

preferably, the compound III of the present invention is any one of the following compounds:

the compounds I, II or III of the present invention can exhibit tautomerism, structural isomerism and stereoisomerism. The present invention includes any tautomeric or structural or stereoisomeric form thereof and mixtures thereof, which have hDHFR inhibitory activity, antitumor activity and antibacterial activity, and the ability is not limited to any one isomeric or mixture form thereof.

The invention also provides a preparation method of the compound I, which is a method 1 or 2:

the method 1 comprises the following steps: subjecting compound VI and compound V to cyclization reaction in the presence of an acid (preferably hydrogen chloride) in an organic solvent (preferably an alcohol solvent such as methanol or ethanol) and/or water to obtain compound I; the conditions of the ring closure reaction may be those conventional in the art for such reactions;

the method 1 may further comprise the steps of: performing an addition reaction of the compound VIII and dicyanodiamine in an organic solvent (preferably an alcohol solvent such as methanol or ethanol) and/or water to obtain the compound V; the conditions of the addition reaction may be those conventional in the art for such reactions;

in the process for the preparation of compound V, the molar ratio of compound VIII to dicyanodiamine may be the one conventional in the art, for example 1:1.

The method 1 may further comprise the steps of: hydrolyzing compound IX in the presence of an acid (preferably hydrogen chloride) in an organic solvent (preferably an alcoholic solvent such as methanol or ethanol) and/or water to obtain said compound VIII; the conditions of the hydrolysis reaction may be those conventional in the art for such reactions;

the method 1 may further comprise the steps of: in an organic solvent (preferably an alcohol solvent such as methanol or ethanol) and/or water, in the presence of a base (preferably an inorganic base such as sodium hydroxide, potassium carbonate, sodium carbonate and the like, more preferably sodium hydroxide and/or potassium hydroxide), carrying out a substitution reaction on the compound X and benzohydroxamic acid to obtain the compound IX; the conditions of the substitution reaction may be those conventional in the art for such reactions;

in the preparation method of the compound IX, the reaction temperature of the substitution reaction can be a reaction temperature conventional in the reaction of this type in the art, for example, 0 to 100 ℃ (preferably 60 to 79 ℃).

In the preparation of compound IX, the molar ratio of compound X to benzohydroxamic acid may be in a molar ratio conventional to such reactions in the art, e.g., 1:1.

The method 1 may further comprise the steps of: in an organic solvent (preferably acetonitrile), in the presence of a base (preferably potassium carbonate), carrying out substitution reaction on a compound XI and 1, 3-dibromopropane to obtain a compound X; the conditions of the substitution reaction may be those conventional in the art for such reactions;

in the preparation method of the compound X, the reaction temperature of the substitution reaction may be a reaction temperature conventional in the art, such as 0 to 100 ℃ (preferably 75 to 80 ℃).

In the process for the preparation of compound X, the molar ratio of compound XI to 1, 3-dibromopropane may be as conventional in the art for such reactions, e.g. 1: 5.

The method 1 may further comprise the steps of: in an organic solvent (preferably dichloromethane or an alcohol solvent, such as methanol or ethanol), in the presence of a base (preferably triethylamine), performing substitution reaction on the 4-piperidone and the compound X-Y to obtain the compound VI; the conditions of the substitution reaction may be those conventional in the art for such reactions; said Y is a leaving group (which is a leaving group conventional in reactions of this type in the art, preferably chlorine or bromine);

in the preparation method of the compound VI, the reaction temperature of the substitution reaction can be a reaction temperature conventional in the reaction in the field, such as-30 to 80 ℃ (preferably-10 to 30 ℃).

In the preparation of compound VI, the molar ratio of 4-piperidone to compound X-Y can be as conventional in the art for such reactions, e.g., 1:1.

In the preparation of compound VI, the molar ratio of 4-piperidone to base can be as conventional in the art for such reactions, e.g., 1:1.

Preferably, the preparation route of the method 1 is as follows:

the method 2 comprises the following steps: performing substitution reaction of compound II and compound X-Y in organic solvent (preferably acetonitrile, N-dimethylformamide or dimethyl sulfoxide, more preferably N, N-dimethylformamide) to obtain compound I; the conditions of the substitution reaction may be those conventional in the art for such reactions; said Y is a leaving group (which may be a leaving group conventional in reactions of this type in the art, preferably chlorine or bromine);

in the preparation method of the compound I, the reaction temperature of the substitution reaction may be a reaction temperature conventional in the art, such as 0 to 100 ℃ (preferably 20 to 35 ℃).

In the process for the preparation of compound I, the molar ratio of compound II to compounds X-Y may be a molar ratio customary in reactions of this type in the art, for example 1: 1.2.

The method 2 can also comprise the following steps: in an organic solvent (preferably acetic acid), in the presence of acid (preferably hydrobromic acid), carrying out deprotection reaction on the compound III to obtain the compound II; the conditions for the deprotection reaction may be those conventional in the art for such reactions;

in the preparation method of the compound II, the reaction temperature of the deprotection reaction may be a reaction temperature conventional in the reaction in the art, for example, 0 to 80 ℃ (preferably 10 to 50 ℃).

In the method for preparing the compound II, the mass fraction of the acid in the organic solvent of the acid is preferably 33%.

The method 2 can also comprise the following steps: in the presence of an acid (preferably hydrogen chloride), the compound V and the compound IV are subjected to cyclization reaction in an organic solvent (preferably an alcohol solvent such as methanol or ethanol) and/or water to obtain the compound III; the conditions of the ring closure reaction may be those conventional in the art for such reactions;

the process for producing the compound III may further include a process for producing the compound V, and the specific conditions of the process for producing the compound V are the same as those described in the above-mentioned process 1.

Preferably, the preparation route of the method 2 is as follows:

the invention also provides a preparation method of the compound II, which comprises the following steps: in an organic solvent (preferably acetic acid), in the presence of acid (preferably hydrobromic acid), carrying out deprotection reaction on the compound III to obtain a compound II; the conditions for the deprotection reaction may be those conventional in the art for such reactions;

The process for producing the compound II may further include a process for producing the compound III, and the specific conditions of the process for producing the compound III are the same as those described in the above-mentioned process 2.

Preferably, the preparation route of the compound II is as follows:

the invention also provides a preparation method of the compound III, which comprises the following steps: subjecting compound V and compound IV to cyclization reaction in the presence of an acid (preferably hydrogen chloride) in an organic solvent (preferably an alcohol solvent such as methanol or ethanol) and/or water to obtain compound III; the conditions of the ring closure reaction may be those conventional in the art for such reactions;

Preferably, the preparation route of the compound III is as follows:

the invention also provides application of the compound I, II or III or pharmaceutically acceptable salt thereof in preparing a dihydrofolate reductase inhibitor. The dihydrofolate reductase is preferably human dihydrofolate reductase.

The invention also provides the application of the compound I, II or III or the pharmaceutically acceptable salt thereof in preparing a medicament for preventing, relieving or treating diseases related to dihydrofolate reductase. The dihydrofolate reductase is preferably human dihydrofolate reductase. The disease associated with dihydrofolate reductase is preferably a neoplastic or bacterial infectious disease. The tumor can be colon cancer, leukemia, lung adenocarcinoma, breast cancer or liver cancer, and also can be breast cancer or liver cancer.

The invention also provides the application of the compound I, II or III or pharmaceutically acceptable salt thereof in preparing a medicament for preventing, alleviating or treating tumor or bacterial infection diseases. The tumor can be colon cancer, leukemia, lung adenocarcinoma, breast cancer, liver cancer, and breast cancer or liver cancer.

The present invention also provides a pharmaceutical composition comprising compound I, II or III, or a pharmaceutically acceptable salt thereof, as described above, and a pharmaceutically acceptable carrier (the carrier serves as a carrier), and/or a pharmaceutically acceptable diluent (the diluent is unsupported, but only diluted). The pharmaceutically acceptable carrier may be one or more. The pharmaceutically acceptable diluent may be one or more. Preferably, the dose of compound I, II or III, or a pharmaceutically acceptable salt thereof, is a therapeutically effective amount.

The pharmaceutical compositions of the present invention may be in a form suitable for oral administration, or may be in the form of a sterile injectable aqueous solution, which may be prepared by any method known in the art for the preparation of pharmaceutical compositions.

Unless otherwise indicated, the following terms appearing in the specification and claims of the invention have the following meanings:

the term "alkyl" (including when used alone and when included among other groups) refers to branched and straight chain saturated aliphatic hydrocarbon groups containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, and the various isomers thereof, and the like.

The term "aryl" refers to a monocyclic (e.g., phenyl) or fused polycyclic (e.g., naphthyl or anthracenyl) carbocyclic ring system having a conjugated pi-electron system wherein at least one ring system is aromatic and each ring system contains 6 to 14 carbon atoms.

The term "alkoxy" (including when used alone and when included in other groups) denotes an alkyl group having the indicated number of carbon atoms attached through an oxygen bridge.

The term "halogen" means fluorine, chlorine, bromine, iodine, or astatine.

The term "Cbz-" means

In the present invention, "pharmaceutically acceptable" in "pharmaceutically acceptable carrier(s) and/or diluent" means that it is pharmaceutically acceptable and substantially non-toxic to the subject to which the particular compound is administered.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the compound of the invention has better hDHFR inhibitory activity, antitumor activity and antibacterial activity.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

In the following examples, undefined abbreviations have their commonly accepted meaning, unless otherwise stated, all room temperatures refer to temperatures of 20 ℃ to 30 ℃.

Method example 1

1-bromo-3- (4-bromophenoxy) propane

4-bromophenol (5.2g,0.05mol), 1, 3-dibromopropane (50g,0.25mol) and potassium carbonate (10g, 0.075mol) were mixed in 100ml of acetonitrile and reacted at 80 ℃ for 9 hours. When the consumption of 4-chlorophenol was monitored by TLC, the reaction was stopped, allowed to stand to room temperature, the solid was removed by suction filtration, the filtrate was concentrated under reduced pressure, 100ml of chloroform was added, and the chloroform layer was washed twice with 0.2N aqueous sodium hydroxide solution (100ml × 2), and dried over anhydrous magnesium sulfate. Vacuum distillation is carried out, and a fraction at 85-90 ℃ is taken under the pressure of 0.5mbar, so that 7.7g of colorless oily matter is obtained, and the yield is 73.5%. Specific methods are described in the literature (Hideki Kubota, Toshihiro Watanabe. Synthesis and pharmacological evaluation of N-acyl-1,2,3, 4-tetrahydroquinoline derivatives as novel specific branched reagents. biological & Medicinal Chemistry,2004,12: 871-.

The compound X is prepared by the method.

Method example 2

N- [ 1-bromo-3- (4-bromophenoxy) propyloxy ] benzamide

Benzohydroxamic acid (11.7g,0.085mol), sodium hydroxide (3.2g,0.08mol, dissolved in 10ml of water), 1-bromo-3- (4-bromophenoxy) propane (25g, 0.085mol), 95% ethanol (150 ml) were mixed in a round bottom flask, heated to reflux, the reaction was stopped when TLC monitored that the starting material was consumed, left to room temperature, concentrated, suction filtered, the filter cake was washed with a large amount of water to give a pale red solid, which was recrystallized from ethyl acetate/petroleum ether to give 13.9g of a white solid with a yield of 46.7% and MP ═ 110 to 111 ℃.

The compound IX is prepared by this method.

Method example 3

O- [3- (4-bromo-phenoxy) propyloxy ] hydroxylamine hydrochloride

Dissolving N- [ 1-bromo-3- (4-bromo-phenoxy) propyloxy ] benzamide (7g,0.02mol) and concentrated hydrochloric acid (4g,0.04mol) in 100ml of methanol, heating and refluxing, stopping the reaction when TLC monitors that the raw materials are completely consumed, concentrating to obtain a white solid, and recrystallizing with ethyl acetate/petroleum ether to obtain 4.6g of white crystals, wherein the yield is 81.4%, and the MP is 138-140 ℃.

The compound VIII is prepared by the method.

Method example 4

1-Phenylbenzoyl-4-piperidinones

4-piperidone (7.6g,0.05mol), triethylamine (0.05mol), phenylpropionyl chloride (8.5g, 0.05mol) were mixed in 50ml of dichloromethane, reacted at-10 ℃ for 1 hour, the reaction was stopped when the consumption of the raw material was monitored by TLC, washed twice with water (100 ml. times.2), dried over anhydrous magnesium sulfate to give 9.2g of colorless oil with a yield of 68.1%,

the compound VI is prepared by the method.

Method example 5

2, 4-diamino-5- (3' - (4 "-bromo-phenoxy) -propoxy) -9-benzyloxycarbonyl-1, 3,5, 9-tetraaza-spiro [5.5] undecane-1, 3-diene hydrochloride (C1)

O- [ 3' - (4 "-bromo-phenoxy) propyloxy ] hydroxylamine hydrochloride (0.56g,0.002mol), dicyanodiamide (0.168g,0.002mol) and 15ml ethanol were mixed in a round-bottomed flask, heated under reflux for 2 hours, and allowed to stand at room temperature. N-Cbz-4-piperidone (1.8g,0.008mol) and concentrated hydrochloric acid (0.2g,0.002mol) were added thereto, and the mixture was stirred at room temperature for 5 days to cause a white precipitate to appear. Suction filtration is carried out, and the obtained white solid is recrystallized by ethanol/water to obtain 2.3g of white crystals with the yield of 52.1 percent.

The synthesis of compounds C1-C6 (which correspond to the general formula of compound III) is the same as C1, the physicochemical properties of C1 are shown in Table 1, and those of C1¹The HNMR data are shown in Table 2.

Method example 6

2, 4-diamino-5- (3' - (4 "-bromo-phenoxy) -propoxy) -1,3,5, 9-tetraaza-spiro [5.5] undecane-1, 3-diene dihydrobromide salt (N4)

2, 4-diamino-5- (3' - (4 "-bromo-phenoxy) -propoxy) -9-benzyloxycarbonyl-1, 3,5, 9-tetraaza-spiro [5.5] undecane-1, 3-diene hydrochloride (C1) (1.2g,0.002mol) was mixed with a 33% solution of hydrobromic acid in glacial acetic acid in a round bottom flask and stirred at room temperature for 8 hours. After the reaction was completed, 30ml of acetone was added, and a white precipitate appeared. Suction filtration is carried out, and the obtained white solid is recrystallized by ethanol/water to obtain 0.74g of white crystal with the yield of 66.3 percent.

The synthesis of compounds N4-N6 (which correspond to the general formula of compound II) is the same as N1, the physicochemical properties of N4 are shown in Table 1, and those of N4¹The HNMR data are shown in Table 2.

Method example 7

2, 4-diamino-5- (3' - (2 "-4" -dichloro-phenoxy) -propoxy) -9-phenoxypropyl-1, 3,5, 9-tetraaza-spiro [5.5] undecane-1, 3-diene dihydrobromide salt (N8)

2, 4-diamino-5- (3 ' - (2 ' -4 ' -dichloro-phenoxy) -propoxy) -1,3,5, 9-tetraaza-spiro [5.5] undecane-1, 3-diene dihydrobromide (0.56g,0.001mol) was mixed with sodium hydroxide (0.04g,0.001mol) and 10ml of methanol in a round bottom flask, heated under reflux for 30 minutes, filtered hot, and the filtrate was concentrated. 1-bromo-3-phenoxypropane (0.26g, 0.0012mol) and DMF 2ml were added and stirred at room temperature. And when the consumption of the raw materials is monitored by TLC, a small amount of concentrated hydrobromic acid is added dropwise to adjust the pH value to 2-3. The solvent was removed under reduced pressure to give a white solid, which was recrystallized from ethanol/water to give 0.23g of white crystals, yield 31.7%.

The synthesis methods of N1-N3 and N7-N20 (which conform to the general formula of the compound I) are the same as N8, the physicochemical properties are shown in Table 1,¹the HNMR data are shown in Table 2.

Referring to the above method examples, compounds C1-C6 and N1-N20 were prepared, and their physicochemical properties and identification data are shown in tables 1 and 2.

TABLE 12 physicochemical Properties of 4, 4-diamino-5-substituted-9-substituted-1, 3,5, 9-tetraaza-spiro [5.5] undecane-1, 3-dienes

TABLE 22, 4-diamino-5-substituted-9-substituted-1, 3,5, 9-tetraaza-spiro [5.5]]Process for preparing undeca-1, 3-dienes¹HNMR data

Effect example 1

Assay for inhibitory Activity of human dihydrofolate reductase (hDHFR)

The experimental method comprises the following steps:

all enzymes and substrates were purchased from Sigma-Aldrich. All experiments used phosphate buffer at pH 7.4. A2M aqueous NADPH solution was prepared and stored at 0 ℃. The assay was performed at room temperature and the absorbance at 340nm was measured using a HITACHI U-1900 spectrophotometer.

The inhibition of all samples at each concentration was determined according to the following protocol: to 921. mu.L of phosphate buffer (pH 7.4), 13.8. mu.L of DHFR solution (0.109U/ml in phosphate buffer pH 7.4), 10. mu.L of test sample (dissolved in DMSO; blank 10. mu.L of DMSO), 25. mu.L of DHF (2nM in 0.25M 2-mercaptoethanol solution), and 30. mu.L of NADPH solution (2nM in phosphate buffer pH 7.4) were sequentially added. After gentle mixing, the absorbance at 350nm was recorded every 30 seconds for up to 6 minutes. And obtaining a time-absorbance graph, wherein the slope of the 0.5-6min segment is the speed of the enzymatic reaction. Some compounds were recorded for their inhibition at 5 μ M and 50 μ M; other compounds recorded IC 50. Calculation of IC 50: the inhibition rates at 6 concentration gradients were used to plot curves, and the drug concentration at 50% inhibition rate was calculated logarithmically. All experiments were performed in 3 groups and IC50 was averaged over 3 times.

The results show that most compounds have better in-vitro inhibitory activity on human dihydrofolate reductase, and the inhibitory activity of part of compounds is close to or better than that of positive control MTX. At a concentration of 50 μ M, the inhibition rate of 11 compounds was superior to that of the lead M0, and 7 compounds were superior to that of the positive control MTX; at 5. mu.M concentration, the inhibition of 8 compounds was better than both M0 and MTX.

Enzyme inhibition IC of Compounds C2 and C3₅₀Superior to lead M0 and positive control MTX. C2, C3 and CN103664972A compounds A2 and A10 (structural formula shown in Table 3) with the same R group respectively, IC₅₀There is also an improvement.

The results are shown in Table 3:

table 3 partial compound inhibition activity test on human DHFR

Mark "-" as untested.

Effect example 2

Part of the samples were tested for anti-tumor effect in vitro

The selected cell lines: HCT116 (human colon cancer cells), HL-60 (human leukemia cells), A549 (human lung adenocarcinoma cells).

Culture solution: RPMI1640+ 15% NBS + double antibody.

The model of the full-automatic enzyme standard instrument is as follows: WellscanMK-2, manufacturer: labsystems

The test method comprises the following steps:

1) sample preparation: after dissolution in DMSO (Merck), PBS (-) was added to make a 1000. mu.g/ml solution or a homogeneous suspension, which was then diluted with DMSO-containing PBS (-).

2) MTT method. 100 mul of cell suspension with the concentration of 4-5 multiplied by 104/ml is added into each hole of a 96-hole plate, and the 96-hole plate is placed in a 5% CO2 incubator at 37 ℃. After 24h, the sample was added at 10. mu.l/well in duplicate wells at 37 ℃ with 5% CO2 for 72 h. Adding 20l of 5mg/ml MTT solution into each hole, adding a dissolving solution after 4h, placing 100 mu l/hole in an incubator, dissolving, and measuring the OD value at 570nm by using an MK-2 full-automatic enzyme standard instrument.

The results show that part of the compounds to be tested have better in-vitro anti-tumor cell activity. The in vitro inhibitory activity IC50 of all compounds on MDA-MB-231 cells was superior to MTX; the in vitro inhibitory activity IC50 of 8 compounds on HCT116 was superior to MTX; the in vitro inhibitory activity of 9 compounds against HL-60 cells, IC50, was superior to MTX.

The test results are shown in Table 4

TABLE 4 IC of part of the Compounds on tumor cells₅₀

Meanwhile, as can be seen from the above, the compounds of the present invention have better inhibitory activity against HepG2 and MDA-MB-231, but the compounds of CN103664972A (the same inventors as the present invention) of the O series (A2, A6 and A10) and the S series (sulfur-containing heterocyclic hexane) have poor primary screening activity against HepG2 and MDA-MB-231.

Effect example 3

Test for measurement of antibacterial Activity

The pharmacological test method comprises the following steps:

medicine preparation: dissolved with DMSO, diluted with sterile water to 250 μ g/ml, and then sequentially diluted in two-fold.

Strain: 20G + and G-strains were inoculated separately and cultured overnight in broth at 37 ℃.

The method comprises the following steps: agar plate dilution method. The Euro multiple-point inoculators dosed at 105CFU per spot.

The culture was carried out at 37 ℃ for 18 hours in an incubator to observe the results. Minimum inhibitory concentration (MIC value)

The bacteriostatic activity is shown in Table 5

Compared with the reported triazine spiro-compound A2 and A10 in CN103664972A with the same R substituent, the inhibition MIC of the pneumococci 31002 by the C1 and the C2 with the introduced side chains on the spiro-compound is obviously improved, and the antibacterial effect of the C1 and the C2 is wider.

Table 5 shows the bacteriostatic activity of the compounds of general formula I

Claims

1. A compound having a spiro structure represented by formula I or III, or a pharmaceutically acceptable salt thereof:

wherein R is substituted or unsubstituted phenyl; in the "substituted or unsubstituted phenyl", the "substitution" is by one or more of the following groups, and when a plurality of substituents are present, the substituents are the same or different: halogen, C_1-6Alkoxy and C_1-6An alkyl group;

x is substituted or unsubstituted C_1-6Alkylcarbonyl, unsubstituted C_6-14Arylcarbonyl, or, substituted or unsubstituted C_1-6An alkyl group; said "substituted or unsubstituted C_1-6In alkylcarbonyl, "said" substitution "is independently by one or more of the following groups, which when present are the same: a phenyl group;

said "substituted or unsubstituted C_1-6In "alkyl", the "substitution" is independently by one or more of the following groups, which when present are the same or different: phenyl, substituted or unsubstituted phenoxy, benzyloxy, methoxycarbonyl and benzyloxycarbonyl;

in the "substituted or unsubstituted phenoxy", the "substitution" is a substitution with one or more of the following groups, and when a plurality of substituents are present, the substituents are the same or different: halogen, C_1-6Alkyl radical, C_1-6Alkoxy and nitro; but X is not benzyloxycarbonyl.

2. The compound I or III according to claim 1, wherein in the "substituted or unsubstituted phenyl", the "substitution" is by halogen, which is fluorine, chlorine, bromine or iodine;

and/or, the "pharmaceutically acceptable salt thereof" refers to a salt formed by the compound I or III and pharmaceutically acceptable acid or base.

3. The compound I or III according to claim 1, wherein in the "substituted or unsubstituted phenyl", the "substitution" is by C_1-6In the substitution of alkoxy, said C_1-6Alkoxy is methoxy.

4. The compound I or III according to claim 1, wherein in the "substituted or unsubstituted phenyl", the "substitution" is by C_1-6In the alkyl substitution, said C_1-6The alkyl group is a tert-butyl group.

5. Compound I or III according to claim 1, characterized in thatSubstituted or unsubstituted C_1-6In the alkyl carbonyl group, said C_1-6The alkylcarbonyl group is propionyl.

6. The compound I or III according to claim 1, wherein C is substituted or unsubstituted_6-14In aryl carbonyl group, said C_6-14The arylcarbonyl group is benzoyl.

7. The compound I or III according to claim 1, wherein C is substituted or unsubstituted_1-6In the alkyl radical, said C_1-6Alkyl is methyl, ethyl, n-propyl or n-butyl.

8. The compound of claim 1, wherein in said "substituted or unsubstituted phenoxy", said "substitution" is by halogen, said halogen being fluorine, chlorine, bromine or iodine.

9. The compound of claim 1, wherein in said "substituted or unsubstituted phenoxy", said "substitution" is by C_1-6In the alkyl substitution, said C_1-6Alkyl is methyl or tert-butyl.

10. The compound of claim 1, wherein in said "substituted or unsubstituted phenoxy", said "substitution" is by C_1-6In the substitution of alkoxy, said C_1-6Alkoxy is methoxy.

11. The compound I or III according to claim 1, wherein R is 4-chlorophenyl, 4-bromophenyl, 2, 4-dichlorophenyl, 4-methoxyphenyl, 4-tert-butylphenyl, or 2,4, 5-trichlorophenyl;

and/or in the compound I, X is n-propyl, n-butyl, benzyl, 3-phenylpropyl, 4-phenylbutyl, 3-phenoxypropyl, 3- (4-chlorophenoxy) propyl, 3- (4-fluorophenoxy) propyl, 3- (4-bromophenoxy) propyl, 3- (4-methylphenoxy) propyl, 3- (4-nitrophenoxy) propyl, methoxycarbonylmethyl, benzyloxycarbonylmethyl, benzoyl or 3-phenylpropionyl.

12. The compound I or III according to claim 1, wherein the compound I is any one of the following compounds:

or, the compound III is any one of the following compounds:

13. a process for the preparation of compound I as claimed in any one of claims 1 to 12, which is process 1 or 2:

the method 1 comprises the following steps: in the presence of acid, carrying out cyclization reaction on a compound VI and a compound V in an organic solvent and/or water to obtain a compound I;

the method 2 comprises the following steps: in an organic solvent, carrying out substitution reaction on a compound II and a compound X-Y to prepare a compound I; y is a leaving group;

14. a process for the preparation of a compound III according to any one of claims 1 to 12, comprising the steps of: in the presence of acid, carrying out cyclization reaction on the compound V and the compound IV in an organic solvent and/or water to obtain a compound III;

15. use of a compound I or III according to any one of claims 1-12, or a pharmaceutically acceptable salt thereof, for the preparation of a dihydrofolate reductase inhibitor.

16. Use of a compound I or III according to any one of claims 1-12, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention, amelioration or treatment of a disease associated with dihydrofolate reductase.

17. A pharmaceutical composition comprising a compound I or III, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-12, and a pharmaceutically acceptable carrier, and/or a pharmaceutically acceptable diluent.