CN114181144A - Fluorobiphenyl methyl resorcinol ether derivative, preparation method and application thereof - Google Patents
Fluorobiphenyl methyl resorcinol ether derivative, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorinated biphenyl methyl resorcinol ether derivative, a preparation method and an application thereof, wherein the general formula of the fluorinated biphenyl methyl resorcinol ether derivative is shown as I:
R1selected from one of the following:
R2selected from one of the following: fluorine, chlorine, methoxy; x is selected from one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl and methoxyl; r3Selected from one of the following:
R4is selected from one of the following: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl. The fluorinated biphenyl methyl resorcinol ether derivative is a novel PD-L1 inhibitor, can obviously inhibit the interaction between PD-1 and PD-L1 proteins generally at a concentration of less than 10nM, and has activity remarkably superior to that of the known PD-L1 inhibitor BMS-202. The novel biphenyl compound disclosed by the invention not only has high inhibitory activity to PD-L1, but also has very good chemical stability and metabolic stability, so that the novel biphenyl compound has very good drug forming property.
Description
Technical Field
The invention relates to a fluorobiphenyl methyl resorcinol ether derivative, a preparation method and application thereof.
Background
With the deep research on tumor immunity, people find that the tumor microenvironment can protect tumor cells from being recognized and killed by a collective immune system, namely, the tumor cells have immune escape, and the immune escape of the tumor cells plays an important role in the occurrence and development of tumors. The activation or inhibition of immune cells in the body is regulated by a positive signal and a negative signal, wherein the apoptosis receptor protein 1(PD-1) and a ligand thereof (PD-L1) are a pair of negative immune regulation signals, the immune activity of T cells is inhibited, and the immune escape of tumor cells is mediated.
The ability of tumor cells to evade the immune system is achieved by binding of apoptosis ligand (PD-L1) expressed on the surface of tumor cells to PD-1 protein expressed on the surface of T cells. The tumor microenvironment in the body can induce infiltrated T cells to highly express PD-1 protein, and the tumor cells highly express ligands PD-L1 and PD-L2 of PD-1, so that the continuous activation of PD-1/PD-L1 channels in the tumor microenvironment is caused, and the function of the T cells is inhibited, so that the T cells cannot find tumors and send signals for attacking and killing tumor cells to an immune system. The PD-1 or PD-L1 monoclonal antibody is an antibody protein which is respectively targeted and bonded with PD-1 or PD-L1, so that the two proteins of PD-1 and PD-L1 cannot be combined, thereby blocking the pathway, partially restoring the function of T cells and continuously killing tumor cells.
Tumor immunotherapy based on PD-1/PD-L1 immune checkpoint inhibitors is a currently spotlighted next generation immunotherapy aimed at killing tumors using the human own immune systemThe apoptosis is induced by blocking a PD-1/PD-L1 signal pathway, and the medicine has the effect of treating various tumors. Recently, a series of surprising research results confirmed that PD-1/PD-L1 pathway-inhibiting antibodies exhibit potent anti-cancer activity against a variety of tumors, and are particularly striking. Monoclonal antibody drug pembrolizumab (trade name) developed by 4 days 4.9.2014 by meishadong
) The first PD-1 mAb approved by the FDA was used to treat patients with advanced or unresectable melanoma who were not effective for other drug treatments. Currently, maytorrado is studying the therapeutic potential of keytrudda in more than 30 different types of cancer, including various hematologic, lung, breast, bladder, stomach, head and neck cancers. In 22 days 12 months in 2014, the pharmaceutical king-top one hundred-hour MeishiGuibao company is not much hoped to obtain the Nivolumab (trade name Opdivo) which is the monoclonal antibody drug that is rapidly approved to be developed by the Food and Drug Administration (FDA) and is on the market. The medicament is useful for treating patients with unresectable or metastatic melanoma who are unresponsive to other drugs, and is the second PD-1 inhibitor marketed in the United states after Keytruda, Sandong. Nivolumab was approved by the FDA for the treatment of metastatic squamous non-small cell lung cancer with disease progression during or after cisplatin-based chemotherapy by day 3, month 4, 2015. According to the data from the study of Keytruda (pembrolizumab) published by Meerdong for treating solid tumors in stage lb KEYN0TE-028, Keytruda treatment achieved 28% of total remission (ORR) in 25 patients with Pleural Mesothelioma (PM), with 48% of patients with stable disease and 76% of disease control. Complete remission was achieved in advanced Hodgkin Lymphoma (HL) patients who did not respond therapeutically to any of the currently approved drugs after treatment with Keytruda and Opdvio, muskroot, saxophone. At the 2015AACR meeting, a report by Leisha a. emers of john hopkins centre for Cancer (Kimmel Cancer Center) indicated that MPDL32 3280A, a monoclonal antibody with anti-PD-L1 action, exhibited a sustained therapeutic effect in advanced triple negative breast Cancer. Although tumor immunotherapy is considered to be targeted post-treatment cancer therapyRevolutionary therapy, however, monoclonal antibody therapeutics have their own drawbacks: is easy to be decomposed by digestive juice, is unstable in vivo and cannot be taken orally; immune cross reaction is easy to generate; the product quality is not easy to control, and the manufacturing technical requirement is high; large-scale preparation and purification are difficult, and the production cost is high; inconvenient to use, can only be injected intravenously or by drip, has an average response rate of less than 30 percent, and has the possibility of generating immunogenicity. Therefore, the development of the PD1/PD-L1 interaction small molecule inhibitor for the tumor immunotherapy has great practical significance.
Disclosure of Invention
The invention aims to provide a fluorobiphenyl methyl resorcinol ether derivative (the structural general formula is shown in the specification) capable of inhibiting the interaction of PD1/PD-L1, a stereoisomer and a pharmaceutically acceptable salt thereof, a preparation method thereof and application thereof in preparing medicaments for preventing or treating diseases related to a PD1/PD-L1 signal pathway.
Fluorobiphenyl methyl resorcinol ether derivatives shown in general formula I, stereoisomers and pharmaceutically acceptable salts thereof,
R1one of the following may be used:
(wherein in the present invention, the wavy line in the molecular structure of the substituent group means a bond site to another group, the same applies hereinafter.) for example, R1Represents one of o-fluorophenyl and 2, 5-difluorophenyl);
R2one of the following may be used: fluorine, chlorine, methoxy;
x may be one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl, methoxy;
R3one of the following may be used:
r4 may be one of the following: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl, which may be hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-C5 alkyl, C1-C5 alkoxy, amino, C1-C6 alkylamino, acetamido, cyano, ureido, guanidino, ureido, ureidoamino, guanidino, sulfonamido, sulfamoyl, methylsulfonylamino, hydroxycarbonyl, C1-C8 carboalkoxy, mercapto, imidazolyl, thiazolyl, oxazolyl or tetrazolyl.
The preferable fluorobiphenyl methyl phenyl ether derivative, the stereoisomer and the pharmaceutically acceptable salt thereof are characterized in that the structure of the compound is shown as the formula (IA-1):
r2 may be one of the following: fluorine, chlorine, methoxy;
x may be one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl, methoxy;
r3 may be one of the following:
r4 may be one of the following: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl, which may be hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-C5 alkyl, C1-C5 alkoxy, amino, C1-C6 alkylamino, acetamido, cyano, ureido, guanidino, ureido, ureidoamino, guanidino, sulfonamido, sulfamoyl, methylsulfonylamino, hydroxycarbonyl, C1-C8 carboalkoxy, mercapto, imidazolyl, thiazolyl, oxazolyl or tetrazolyl.
The preferable fluorobiphenyl methyl resorcinol ether derivative, the stereoisomer and the pharmaceutically acceptable salt thereof are characterized in that the structure of the compound is shown as a formula (IA-2):
r2 may be one of the following: fluorine, chlorine, methoxy;
x may be one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl, methoxy;
r3 may be one of the following:
R4one of the following may be used: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl, which may be hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-C5 alkyl, C1-C5 alkoxy, amino, C1-C6 alkylamino, acetamido, cyano, ureido, guanidino, ureido, ureidoamino, guanidino, sulfonamido, sulfamoyl, methylsulfonylamino, hydroxycarbonyl, C1-C8 carboalkoxy, mercapto, imidazolyl, thiazolyl, oxazolyl or tetrazolyl.
Preferred fluorobiphenyl methyl resorcinol ether derivatives in the above general formula, stereoisomers thereof and pharmaceutically acceptable salts thereof, wherein R is4May be one of the following:
r ═ methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, or octyl.
The most preferred compound may be one of the following:
n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine,
n-acetamidoethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride,
n-hydroxyethyl-2- (5-cyanopyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride,
n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] azetidine-3-carboxylic acid,
n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] L-serine,
n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] D-serine,
n-hydroxyethyl-2- (5-carbamoylpyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzylamine hydrochloride,
n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] benzylamine,
n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] -5-chlorobenzylamine,
in addition, the starting materials and intermediates in the above reaction are readily available. The compounds of formula I may exist in the form of solvates or non-solvates, and crystallization using different solvents may give different solvates. The pharmaceutically acceptable salts in the general formula I include salts formed by different acids, such as the following inorganic acids or organic acids: hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, lycinic acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid. The pharmaceutically acceptable salts in the general formula I also comprise different alkali metal salts (lithium, sodium and potassium salts), alkaline earth metal salts (calcium and magnesium salts) and ammonium salts. All such salts within the scope of the present invention may be prepared by conventional methods. During the preparation of the compounds of formula I and solvates and salts thereof, different crystallization conditions may occur as polycrystals or co-crystals.
In a second aspect of the present invention, there is provided a process for preparing a compound of the first aspect: in order to prepare the compound shown in the general formula I, the preparation of the compound shown in the general formula I is divided into five steps according to the structure of the general formula I.
(1) Taking the compound 1 and substituted phenylboronic acid as basic raw materials, and obtaining an intermediate compound 2 through a Suzuki coupling reaction;
(2) brominating substituted benzyl alcohol by NBS with the intermediate compound 2 as a raw material to obtain a bromide intermediate 3;
(3) taking bromide intermediate 3 as a raw material, and reacting with intermediate 4 under an alkaline condition to obtain benzyl aryl ether intermediate 5;
(4) taking a benzyl aryl ether intermediate 5 as a raw material, and reacting with various substituted aryl halides under an alkaline condition to obtain an intermediate compound 6 containing aldehyde groups;
(5) taking an intermediate compound 6 containing aldehyde group as a raw material, and reacting with HR containing amino or imino4Carrying out reductive amination reaction to obtain a target compound I;
the substituents R1, R2, R3, R4 and X in the compounds 1 to 6 are as defined in the object compound I.
The beneficial effects obtained by the invention are as follows: the fluorinated biphenyl methyl resorcinol ether derivative is a novel PD-L1 inhibitor, can obviously inhibit the interaction between PD-1 and PD-L1 proteins generally at a concentration of less than 10nM, and has activity remarkably superior to that of the known PD-L1 inhibitor BMS-202. The fluorinated biphenyl methyl resorcinol ether derivative can also obviously block the inhibiting effect of PD-L1 on T cells, and can block the effect of tumor cells on inhibiting the proliferation of the T cells and secreting IFN-gamma, thereby having the effect of enhancing the anti-tumor immunity of the T cells. The fluorinated biphenyl methyl resorcinol ether derivative has the advantages of few synthesis steps, simple and easy operation, cheap and easily-obtained raw materials, safe synthesis process, environmental protection and easy large-scale production. Particularly, the novel biphenyl compound has high inhibitory activity on PD-L1, and also has very good chemical stability and metabolic stability, so that the novel biphenyl compound has very good druggability.
Drawings
FIG. 1 shows the results of tests on the ability of different compounds to be tested to release the ligand PD-L1 from inhibiting IFN-. gamma.expression in pharmacological activity assays of the compounds of the present application.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the scope of the present invention is not limited thereto.
The measuring instrument: NMR spectroscopy was performed using a Bruker AV-400 Bruker AV-600 NMR spectrometer. Mass spectra were obtained on ZAD-2F and VG300 mass spectrometers.
Example 1: n-acetamidoethyl-2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzylamine,
(1) 2-methoxy-3-o-fluorophenyl benzyl alcohol
In a 100ml round bottom flask was added 3-bromo-2-methoxybenzyl alcohol (434.1mg,2.0mmol,1.0eq), o-fluorobenzeneboronic acid (336.8mg,2.4mmol,1.2eq), K2CO3(691.1mg,5.0mmol,2.5eq), dioxane (10ml), water (2.5ml), nitrogen substitution three times, stirring for 20min, then adding PdCl2(dppf) (73.2mg,0.1 mmol,0.05 eq). After a condensing device is additionally arranged on the round-bottom flask, the reaction system is replaced by nitrogen for three times again, and the reaction system is heated to 80 ℃ and stirred for reaction for 15 hours. After TLC detection reaction, stopping heating, cooling the reaction system to room temperature, and filtering with diatomite. The filtrate was concentrated under reduced pressure, and then water and ethyl acetate were added to extract 3 times. And (3) combining organic phases, washing the organic phases with saturated saline solution, drying the organic phases with anhydrous sodium sulfate, filtering the organic phases, and concentrating the organic phases under reduced pressure until the organic phases are dried to obtain a crude product: 475.3mg, directly administered to the next step.
(2) 2-methoxy-3-o-fluorophenyl benzyl bromide
In a 100ml round bottom flask, 2-methoxy-3-o-fluorophenylbenzyl alcohol (464.5mg,2.0mmol,1.0 eq), triphenylphosphine (786.9mg,3.0mmol,1.5eq), and methylene chloride (10ml) were added, and after dissolution with stirring, N-bromosuccinimide (NBS) (533.9mg,3.0mmol,1.5eq) was added in portions at a reduced temperature in an ice bath. After the addition was complete, the temperature was slowly raised to room temperature and the reaction was continued for 4 h. After TLC detection of the raw material reaction is completed, adding silica gel in three times amount into the reaction solution, concentrating to dryness, and then purifying by a column, wherein an eluent PE: EA is 40: 1(v/v), giving a colorless oil: 472.2mg, yield: 80 percent.
1H-NMR(600MHz,CDCl3)δ7.46(ddd,J＝9.9,7.6,1.9Hz,2H),7.40(tdd,J＝7.6,5.0,1.8 Hz,1H),7.32(dt,J＝7.6,1.3Hz,1H),7.24(td,J＝7.4,1.2Hz,1H),7.20(td,J＝8.7,7.7,5.3 Hz,2H),4.67(s,2H),3.53(s,3H)。
(3) 2-hydroxy-4- (2-methoxy-3-o-fluorophenyl benzyloxy) benzaldehyde
In a 50ml round bottom flask, 2-methoxy-3-o-fluorophenyl benzyl was addedBromine (295.2mg,1.0mmol, 1.0eq), 2, 4-dihydroxybenzaldehyde (165.7mg,1.2mmol, 1.2eq), K2CO3(207.3mg,1.5mmol,1.5eq), sodium iodide (75.0mg,0.5mmol,0.5eq), DMF (5ml), after addition, the temperature was raised to 60 ℃ for reaction for 2 h. Cooling the reaction solution to room temperature, adding water and ethyl acetate, stirring for 5min, then layering, extracting the water phase with ethyl acetate for three times, combining the organic phases, washing with saturated saline water, drying with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and purifying by column chromatography to obtain a white solid: 140.9mg, yield: 40 percent.
(4)2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde
In a 50ml round bottom flask, 2-hydroxy-4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde (70.5mg,0.2mmol,1.0eq), m-cyanobenzyl chloride (36.4mg,0.24mmol, 1.2eq), Cs were added2CO3(97.7mg, 0.3mmol,1.5eq), sodium iodide (15.0mg,0.1mmol,0.5eq), DMF (3ml) and after the addition was complete, the temperature was raised to 60 ℃ for reaction for 2 h. Cooling the reaction solution to room temperature, adding water and ethyl acetate, stirring for 5min, then layering, extracting the water phase with ethyl acetate for three times, combining the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain a crude product: 90.2mg, directly put into the next step.
(5) N-acetamidoethyl-2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzylamine
2- (3-Cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde (90.2mg,0.19mmol, 1.0eq) was dissolved in 2ml of DMF, and 2-acetamidomethamine (38.8mg,0.38mmol, 2.0eq) was added, four drops of glacial acetic acid were added to the reaction system. After stirring at room temperature for 20min, sodium cyanoborohydride (23.9mg,0.38mmol, 2.0eq) was added and stirred at room temperature overnight. The reaction was stopped, water and ethyl acetate were added and extracted. Washing the organic phase with saturated saline solution, drying with anhydrous sodium sulfate, filtering, performing reduced pressure spin drying, and purifying by column chromatography to obtain a white solid: 32.6mg, yield: 31.5%, purity of product by HPLC: 96.5 percent.1H-NMR(600MHz,CHCl3-d)δ7.76(d,J＝1.8Hz,1H), 7.72–7.67(m,1H),7.65(dt,J＝7.8,1.4Hz,1H),7.56–7.48(m,2H),7.47–7.36(m,2H),7.33 (dt,J＝7.5,1.4Hz,1H),7.27(s,1H),7.27–7.16(m,3H),6.80(s,1H),6.65(dd,J＝8.3,2.3 Hz,1H),6.60(d,J＝2.3Hz,1H),5.15(s,4H),3.91(s,2H),3.44(s,3H),3.39(q,J＝5.6Hz, 2H),2.86(t,J＝5.5Hz,2H),1.94(s,3H)。
Example 2: n- [2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzyl) ] L-serine
Experimental procedure example 1 was repeated except that 2-acetamidomethamine was replaced with an equimolar amount of L-serine in step (5), and the rest of the procedure was the same as in example 1 to obtain a white solid in yield: 37.1%, purity of product by HPLC: 96.1 percent.1H-NMR(600MHz,CHCl3-d)δ7.76–7.60(m,2H),7.50(s,1H),7.43–7.34(m, 4H),7.29(d,J＝12.7Hz,2H),7.21(td,J＝7.5,1.2Hz,1H),7.19–7.13(m,2H),6.57(d,J＝ 8.2Hz,1H),6.54(s,1H),5.09(s,2H),5.05(s,2H),4.20(s,2H),3.92(s,2H),3.52–3.40(m, 1H),3.38(s,3H).
Example 3: n-acetamidoethyl-2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzylamine
Experimental procedure example 1 was repeated except that in step (3), an equimolar amount of 2, 4-dihydroxy-5-chlorobenzaldehyde was used instead of 2, 4-dihydroxybenzaldehyde to react with 2-methoxy-3-o-fluorophenyl benzyl bromide, and the obtained intermediate was subjected to the procedures of step (4) and step (5) in example 1 in the same charged molar amounts as in example 1, that is, the charged molar amount of the intermediate of step (4) was 0.2mmol, the charged molar amount of the intermediate of step (5) was 0.19mmol, and the rest of the procedures were the same as in example 1 to obtain a white solid, yield: 34.8%, purity of product by HPLC: 96.3 percent.1H-NMR (600MHz,CHCl3-d)δ7.74(td,J＝1.7,0.8Hz,1H),7.66(dp,J＝5.8,1.6Hz,2H),7.58(dd, J＝7.7,1.8Hz,1H),7.53(t,J＝7.8Hz,1H),7.44–7.38(m,2H),7.36–7.29(m,2H),7.24(td, J＝7.5,1.2Hz,2H),7.18(ddd,J＝10.0,8.7,1.2Hz,1H),6.68(s,1H),6.59(s,1H),5.28(s, 2H),5.10(s,2H),3.79(s,2H),3.48(s,3H),3.35(q,J＝5.6Hz,2H),2.78(dd,J＝6.7,4.9Hz, 2H),1.96(s,3H).
Example 4: n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzylamine
Experimental procedure example 1 was repeated except that 2-acetamidomethamine was replaced with an equimolar amount of 2-aminoethanol in step (5) and the procedure in example 1 was otherwise the same to give a white solid in yield: 44.0%, purity of product by HPLC analysis: 95.9 percent.1H-NMR(600MHz,CHCl3-d)δ7.76(d,J＝1.8Hz,1H),7.71(d,J＝7.6Hz, 1H),7.66–7.61(m,1H),7.56–7.48(m,2H),7.47–7.36(m,2H),7.33(dt,J＝7.6,1.4Hz,1H), 7.28–7.16(m,4H),6.65(dd,J＝8.3,2.3Hz,1H),6.62(d,J＝2.3Hz,1H),5.16(s,4H),3.98 (s,2H),3.70(t,J＝5.0Hz,2H),3.44(s,3H),2.92–2.87(m,2H).
Example 5: n-acetamidoethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzylamine
Experimental procedure example 1 was repeated except that 5-chloromethylnicotinonitrile was used in place of m-cyanobenzyl chloride to react with 2-hydroxy-4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde to give 2- (5-cyanopyridine-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde as an intermediate, and reductive amination was performed with 2-acetamidomethamine (0.38mmol) in a charged molar amount of 0.19mmol, and the same procedure as in example 1 was repeated for charged molar amounts of each starting material or intermediate to give a pale yellow solid in yield: 30.6%, purity of product by HPLC: 96.5％。1H-NMR(600MHz,CHCl3-d)δ8.95(d,J＝2.1Hz,1H),8.88(d,J＝2.0Hz,1H), 8.19(t,J＝2.1Hz,1H),7.50(dd,J＝7.7,1.7Hz,1H),7.47–7.37(m,2H),7.35–7.29(m,2H), 7.27–7.21(m,2H),7.18(ddd,J＝9.7,8.3,1.2Hz,1H),7.01(s,1H),6.67(dd,J＝8.4,2.3Hz, 1H),6.62(d,J＝2.3Hz,1H),5.20(s,2H),5.16(s,2H),3.94(s,2H),3.45(s,3H),3.40(q,J＝ 5.7Hz,2H),2.91(t,J＝5.4Hz,2H),1.96(s,3H).
Example 6: n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzylamine
Experimental procedure example 1 was repeated except that 2-hydroxy-4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde was reacted with an equimolar amount of 5-chloromethylnicotinic carbonitrile instead of m-cyanobenzyl chloride to give an intermediate, 2- (5-cyanopyridine-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) benzaldehyde, and reductive amination was performed with 2-aminoethanol (0.38mmol) at a charged molar amount of 0.19mmol, and example 1 was repeated for each charged molar amount of starting material or intermediate to give a pale yellow gum solid in yield: 40.1%, purity of product by HPLC: 96.3 percent.1H-NMR(600MHz,CHCl3-d)δ8.93(d,J＝2.1Hz,1H),8.86(d,J＝2.0Hz,1H), 8.20(t,J＝2.1Hz,1H),7.51(dd,J＝7.6,1.8Hz,1H),7.44(td,J＝7.5,1.8Hz,1H), 7.43–7.37(m,1H),7.33(dt,J＝7.5,1.5Hz,1H),7.30(s,1H),7.27–7.20(m,2H),7.19(ddd, J＝9.7,8.2,1.2Hz,1H),6.68(dd,J＝8.3,2.3Hz,1H),6.63(d,J＝2.3Hz,1H),5.20(s,2H), 5.18(s,2H),3.96(s,2H),3.69–3.61(m,2H),3.45(s,3H),2.92–2.87(m,2H).
Example 7: n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzylamine
Experimental procedure example 1 was repeated except that the starting material 2, 4-dihydroxybenzaldehyde of the third reaction in example 1 was replaced with an equimolar amount of 5-chloro-2, 4-dihydroxybenzaldehyde, reacted with 2-methoxy-3-o-fluorophenylbenzyl bromide to give an intermediate 2-hydroxy-4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzaldehyde, reacted with m-cyanobenzyl chloride to give an intermediate 2- (3-cyanobenzyloxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzaldehyde, and finally subjected to reductive amination with 2-aminoethanol, and the molar amounts of each starting material or intermediate charged were repeated in example 1, the procedure is as in example 1 to give an off-white solid in yield: 45.2%, purity of product by HPLC: 96.9 percent.1H-NMR (600MHz,CHCl3-d)δ7.76(d,J＝1.7Hz,1H),7.69(d,J＝7.7Hz,1H),7.65(dt,J＝7.8,1.4 Hz,1H),7.57(dd,J＝7.6,1.7Hz,1H),7.53(t,J＝7.8Hz,1H),7.45–7.38(m,2H),7.34– 7.29(m,2H),7.27–7.21(m,2H),7.21–7.15(m,1H),6.70(s,1H),5.28(s,2H),5.13(s,2H), 3.86(s,2H),3.69(t,J＝5.1Hz,2H),3.48(s,3H),2.84(t,J＝5.1Hz,2H).
Example 8: n-acetamidoethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzylamine hydrochloride
Experimental procedures example 1 was repeated except that the starting material 2, 4-dihydroxybenzaldehyde reacted in the third step in example 1 was replaced with 5-chloro-2, 4-dihydroxybenzaldehyde, and then reacted with 2-methoxy-3-o-fluorophenylbenzyl bromide to give an intermediate 2-hydroxy-4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzaldehyde, which was then reacted with 5-chloromethylnicotinic carbonitrile to give an intermediate 2- (5-cyanopyridine-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzaldehyde, which was finally subjected to reductive amination with 2-acetamidomethamine, molar quantities of the various starting materials or intermediates charged the procedure of example 1 was repeated to obtain a pale yellow solid in accordance with example 1, yield: 37.1%, purity of product by HPLC: 97.7 percent.1H-NMR(600MHz,CHCl3-d)δ8.91(d,J＝2.1Hz,1H),8.90–8.86(m,1H),8.09(t, J＝2.2Hz,1H),7.59(dd,J＝7.7,1.8Hz,1H),7.45–7.38(m,2H),7.32(d,J＝6.0Hz,2H), 7.25(td,J＝7.5,1.5Hz,2H),7.17(ddd,J＝9.7,8.5,1.2Hz,1H),6.70(s,1H),6.24(s,1H), 5.31(s,2H),5.14(s,2H),3.80(s,2H),3.49(s,3H),3.36(q,J＝5.6Hz,2H),2.81(t,J＝5.7 Hz,2H),1.98(s,3H).ESI-MS m/z:589.2[M+H]+.
Example 9: n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzylamine
Experimental procedures example 1 was repeated except that the starting material 2, 4-dihydroxybenzaldehyde reacted in the third step in example 1 was replaced with 5-chloro-2, 4-dihydroxybenzaldehyde, and then reacted with 2-methoxy-3-o-fluorophenylbenzyl bromide to give an intermediate 2-hydroxy-4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzaldehyde, which was then reacted with 5-chloromethylnicotinic carbonitrile to give an intermediate 2- (5-cyanopyridine-3-methoxy) -4- (2-methoxy-3-o-fluorophenylbenzyloxy) -5-chlorobenzaldehyde, and finally subjected to reductive amination with 2-aminoethanol, and the molar amounts of each starting material or intermediate charged were repeated in example 1, the procedure is as in example 1 to give a white solid in yield: 32.6%, purity of product by HPLC: 96.5 percent.1H-NMR(600MHz,CHCl3-d)δ8.91(d,J＝2.1Hz,1H),8.88(d,J＝2.0Hz,1H),8.13(t,J＝ 2.1Hz,1H),7.59(dd,J＝7.7,1.8Hz,1H),7.42(tdd,J＝7.4,4.9,1.8Hz,2H),7.37–7.29(m, 2H),7.25(td,J＝7.5,1.3Hz,2H),7.17(ddd,J＝10.0,8.7,1.2Hz,1H),6.70(s,1H),5.31(s, 2H),5.14(s,2H),3.81(s,2H),3.71–3.65(m,2H),3.49(s,3H),2.85–2.80(m,2H).
Example 10: n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine
Experimental procedure example 1 was repeated except that the starting material 3-bromo-2-methoxybenzyl alcohol in example 1 was replaced with 3-bromo-2-chlorobenzyl alcohol, the reaction was carried out to give an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenylbenzyloxy) benzaldehyde, which was then reacted with m-cyanobenzyl chloride to give an intermediate 2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzaldehyde, which was finally subjected to reductive amination reaction with 2-aminoethanol, and the procedure in example 1 was repeated for the molar amounts of each starting material or intermediate to give a white solid in yield: 44.5%, purity of product by HPLC analysis: 96.3 percent.1H-NMR(600MHz,CHCl3-d)δ7.77(d,J＝1.9Hz,1H),7.71(dt,J＝7.7,1.5Hz, 1H),7.65(dt,J＝7.7,1.5Hz,1H),7.59(dd,J＝7.7,1.7Hz,1H),7.53(t,J＝7.7Hz,1H), 7.43(dddd,J＝8.3,7.2,5.1,1.8Hz,1H),7.38(t,J＝7.6Hz,1H),7.33(ddd,J＝7.5,5.4,3.4 Hz,2H),7.30–7.23(m,2H),7.20(ddd,J＝9.6,8.3,1.1Hz,1H),6.63(dd,J＝8.3,2.3Hz, 1H),6.60(d,J＝2.3Hz,1H),5.22(s,2H),5.15(s,2H),3.90(s,2H),3.70–3.65(m,2H),2.85 (t,J＝5.1Hz,2H).ESI-MS m/z:517.2[M+H]+.
Example 11: n-acetamidoethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride
Experimental procedure example 1 was repeated except that 3-bromo-2-methoxybenzyl alcohol, which is the starting material in example 1, was replaced with 3-bromo-2-chlorobenzyl alcohol, and a reaction was carried out to give an intermediate, 2-hydroxy-4- (2-chloro-3-o-fluorophenyl-benzyloxy) benzaldehyde, the intermediate is reacted with 5-chloromethyl nicotinonitrile to give the intermediate 2- (5-cyanopyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, which is finally subjected to a reductive amination reaction with 2-acetamidomethamine, and the procedure of example 1 is repeated for the molar amounts of the starting materials or intermediates, to give an off-white waxy solid in yield: 30.7%, purity of product by HPLC: 96.1 percent.1H-NMR(600MHz,CHCl3-d)δ8.93(d,J＝2.1Hz,1H),8.88 (d,J＝2.0Hz,1H),8.13(t,J＝2.1Hz,1H),7.59(dd,J＝7.7,1.7Hz,1H),7.46–7.39(m,1H), 7.38(t,J＝7.6Hz,1H),7.33(ddd,J＝7.5,5.7,1.8Hz,2H),7.29–7.22(m,2H),7.19(ddd,J＝ 9.6,8.3,1.2Hz,1H),6.65(dd,J＝8.3,2.3Hz,1H),6.61(d,J＝2.3Hz,1H),6.46(d,J＝5.9 Hz,1H),5.23(s,2H),5.18(s,2H),3.85(s,2H),3.37(q,J＝5.6Hz,2H),2.82(t,J＝5.7Hz, 2H),1.97(s,3H).
Example 12: n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, and reacting the intermediate with 5-chloromethyl nicotinonitrile to obtain an intermediate: 2- (5-cyanopyridin-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzaldehyde was finally subjected to reductive amination with 2-aminoethanol and the procedure of example 1 was repeated for the charged molar amounts of the starting materials or intermediates to give example 1 as a pale yellow gummy solid in yield: 31.7%, purity of product by HPLC: 96.7 percent.1H-NMR(600MHz,CHCl3-d)δ8.92(d,J＝2.1Hz,1H),8.88 (d,J＝2.0Hz,1H),8.18(t,J＝2.1Hz,1H),7.60(dd,J＝7.6,1.7Hz,1H),7.43(dddd,J＝8.3, 7.2,5.2,1.8Hz,1H),7.38(t,J＝7.6Hz,1H),7.33(dtd,J＝7.4,4.1,3.7,1.8Hz,2H), 7.31–7.23(m,2H),7.19(ddd,J＝9.6,8.3,1.2Hz,1H),6.66(dd,J＝8.3,2.3Hz,1H),6.62(d, J＝2.3Hz,1H),5.24(s,2H),5.19(s,2H),3.89(s,2H),3.69–3.65(m,2H),2.85(dd,J＝5.9, 3.9Hz,2H).ESI-MS m/z:518.1664[M+H]+.
Example 13: n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] glycine
Experimental procedure repeatExample 1, except that in example 1 the starting materials: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, and reacting the intermediate with m-cyanobenzyl chloride to obtain an intermediate: 2- (3-Cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde was finally subjected to reductive amination with glycine and the procedure of example 1 was repeated for the molar amounts of the starting materials or intermediates charged to give an off-white solid in yield: 46.1%, purity of product by HPLC analysis: 97.4 percent.1H-NMR(600MHz,CHCl3-d)δ7.71(s,1H),7.65(d,J＝7.9Hz,1H),7.52–7.48(m, 2H),7.44–7.37(m,3H),7.34–7.28(m,3H),7.27–7.20(m,1H),7.20–7.14(m,1H),6.51(d,J ＝23.5Hz,2H),5.07(s,4H),4.12(s,2H),3.38(s,2H).
Example 14: n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] azetidine-3-carboxylic acid
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, and reacting the intermediate with m-cyanobenzyl chloride to obtain an intermediate: 2- (3-Cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzaldehyde was finally subjected to reductive amination with azetidine-3-carboxylic acid, and the procedure of example 1 was repeated for the charged molar amounts of the respective starting materials or intermediates to give an off-white solid in yield: 41.5%, purity of product by HPLC analysis: 97.1 percent.1H-NMR(600MHz,CHCl3-d)δ7.73(s,1H),7.68(d,J＝7.8Hz,1H),7.61(d, J＝7.7Hz,1H),7.55(dd,J＝7.6,1.8Hz,1H),7.51(t,J＝7.7Hz,1H),7.46–7.38(m,2H), 7.36(t,J＝7.6Hz,1H),7.31(td,J＝7.3,1.8Hz,2H),7.24(td,J＝7.5,1.2Hz,1H),7.18(ddd, J＝9.5,8.3,1.2Hz,1H),6.64(dd,J＝8.5,2.3Hz,1H),6.57(d,J＝2.3Hz,1H),5.19(s,2H), 5.13(s,2H),4.29(s,2H),4.20(s,2H),4.01(s,2H),3.31(s,1H).
Example 15: n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] pyrrolidine-3-hydroxy
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, and reacting the intermediate with m-cyanobenzyl chloride to obtain an intermediate: 2- (3-Cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde was finally subjected to reductive amination with 3-hydroxypyrrolidine and the same procedures as in example 1 were repeated except for feeding the starting materials or intermediates in the same molar amounts to obtain an off-white solid in yield: 45.1%, purity of product by HPLC: 96.0 percent.1H-NMR(400MHz,CHCl3-d)δ7.80–7.71(m,2H),7.65(d,J＝7.6Hz,1H), 7.61–7.51(m,2H),7.48–7.30(m,5H),7.26(td,J＝7.5,1.1Hz,1H),7.19(dd,J＝9.9,8.2Hz, 1H),6.68(dd,J＝8.3,2.2Hz,1H),6.63(d,J＝2.3Hz,1H),5.24(d,J＝3.1Hz,4H),4.63(s, 1H),4.27(s,2H),3.49–3.19(m,5H),2.28(s,1H),2.12(s,1H).
Example 16: n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] L-serine
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, and reacting the intermediate with m-cyanobenzyl chloride to obtain an intermediate: 2- (3-Cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenyl-benzyloxy) -benzaldehyde is finally subjected to reductive amination with L-serine, and the examples are repeated with the molar amounts of the starting materials or intermediates charged1, the same procedure as in example 1 gave a white solid in yield: 37.5%, purity of product by HPLC: 96.1 percent.1H-NMR(400MHz,CHCl3-d)δ7.71(s,1H),7.65(d,J＝7.6Hz,1H),7.49(dd,J＝ 7.1,2.7Hz,2H),7.41(tdd,J＝7.3,4.8,1.9Hz,2H),7.35–7.26(m,4H),7.23(t,J＝7.4Hz, 1H),7.17(t,J＝9.0Hz,1H),6.51(d,J＝13.2Hz,2H),5.06(d,J＝4.7Hz,4H),4.35–4.10(m, 2H),3.94(s,2H),3.47(s,1H).
Example 17: n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] D-serine
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, and reacting the intermediate with m-cyanobenzyl chloride to obtain an intermediate: 2- (3-Cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde was finally subjected to reductive amination with D-serine, and the same procedures as in example 1 were repeated except for feeding molar amounts of each raw material or intermediate to obtain a white solid in the yield of: 37.5%, purity of product by HPLC: 96.6 percent.1H-NMR(400MHz,CHCl3-d)δ7.69(s,1H),7.64(d,J＝7.8Hz,1H),7.51–7.44(m, 2H),7.40(tdd,J＝7.2,5.0,1.9Hz,2H),7.34–7.25(m,4H),7.22(t,J＝7.4Hz,1H),7.16(t,J ＝9.0Hz,1H),6.54–6.46(m,2H),5.04(d,J＝17.9Hz,4H),4.20(s,2H),3.94(s,2H),3.48 (d,J＝8.3Hz,1H).
Example 18: n-hydroxyethyl-2- (5-carbamoylpyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcoholReplacing the steps as follows: 3-bromo-2-chlorobenzyl alcohol, reacting to obtain an intermediate: 2-hydroxy-4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde, which is then reacted with 5-chloromethyl nicotinamide to give an intermediate: 2- (5-carbamoylpyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzaldehyde was finally subjected to reductive amination with 2-aminoethanol and the procedure of example 1 was repeated for the molar amounts of the starting materials or intermediates charged to give example 1 as a pale yellow gummy solid in yield: 32.9%, purity of product by HPLC: 95.8 percent. 1H NMR (500MHz, Chloroform-d) δ 9.02(d, J ═ 2.1Hz,1H), 8.87(d, J ═ 2.1Hz,1H),8.36(t, J ═ 2.2Hz,1H),8.23(s,1H), 7.71-7.65 (M,2H), 7.55-7.46 (M,2H), 7.45-7.39 (M,2H),7.38(dd, J ═ 7.7,1.9Hz,1H), 7.36-7.30 (M,2H), 6.93(d, J ═ 2.4Hz,1H),6.76(dd, J ═ 8.4,2.3Hz,1H),5.28(d, J ═ 16.7Hz,4H), 5.21H (s,1H), 10.2H (s,2H), 3H, 2H, 5.28 (ESI ═ 16.7, 4H, 5.2H, 25H, 2H, 5.2H, 2H, and 5H, 2H, 1H, 2H, 1H, 2H, and so as necessary to obtain a mixture of the like]+.
Example 19: n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- (2-fluoro-3-o-fluorophenylbenzyloxy) benzylamine
Experimental procedure example 1 was repeated except that the starting materials in example 1: 3-bromo-2-methoxybenzyl alcohol is replaced by: 3-bromo-2-fluorobenzyl alcohol, reacting to obtain an intermediate: 2-hydroxy-4- (2-fluoro-3-o-fluorophenyl benzyloxy) benzaldehyde, which is then reacted with m-cyanobenzyl chloride to give an intermediate: 2- (3-Cyanobenzyloxy) -4- (2-fluoro-3-o-fluorophenyl-benzyloxy) benzaldehyde was finally subjected to reductive amination with 2-aminoethanol and the same procedures as in example 1 were repeated except for feeding the molar amounts of the respective starting materials or intermediates to obtain an off-white solid in yield: 47.8%, purity of product by HPLC analysis: 97.2 percent.1H-NMR(600MHz,CHCl3-d)δ7.75(s,1H),7.69(d,J＝7.9Hz,1H),7.64 (d,J＝7.7Hz,1H),7.53(q,J＝7.9,7.3Hz,2H),7.45–7.36(m,3H),7.26(td,J＝8.6,8.1,3.9 Hz,3H),7.20(dd,J＝10.2,8.3Hz,1H),6.63(dd,J＝8.3,2.3Hz,1H),6.59(d,J＝2.3Hz, 1H),5.19(s,2H),5.13(s,2H),3.88(s,2H),3.67(t,J＝5.1Hz,2H),2.83(t,J＝5.1Hz,2H).
Example 20: n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] benzylamine
Experimental procedure example 1 was repeated except that the starting materials in example 1: respectively replacing o-fluorobenzeneboronic acid and 3-bromo-2-methoxybenzyl alcohol by: 2, 5-difluorophenylboronic acid and 3-bromo-2-fluorobenzyl alcohol are subjected to two-step reaction to obtain an intermediate: 2-hydroxy-4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]Benzaldehyde, which is then reacted with m-cyanobenzyl chloride to give an intermediate: 2- (3-Cyanobenzyloxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]Benzaldehyde is finally subjected to reductive amination reaction with 2-aminoethanol, the feeding molar weight of each raw material or intermediate is repeated in example 1, the operation is the same as that in example 1, and the off-white solid is obtained, and the yield is as follows: 30.9%, purity of product by HPLC: 97.1 percent.1H-NMR(600MHz,CHCl3-d) δ7.76(d,J＝1.7Hz,1H),7.71(d,J＝7.6Hz,1H),7.66–7.62(m,1H),7.59–7.50(m,2H), 7.40–7.36(m,1H),7.30(d,J＝8.3Hz,1H),7.26(d,J＝7.7Hz,1H),7.13(dddd,J＝23.1,8.9, 7.0,4.0Hz,3H),6.66–6.58(m,2H),5.17(d,J＝3.8Hz,4H),4.05(s,2H),3.74(t,J＝4.8Hz, 2H),2.94(t,J＝5.0Hz,2H).
Example 21: n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] benzylamine
Experimental procedure example 1 was repeated except that the starting materials in example 1: respectively replacing o-fluorobenzeneboronic acid and 3-bromo-2-methoxybenzyl alcohol by: 2, 5-difluorophenylboronic acid and 3-bromo-2-fluorobenzyl alcohol are subjected to two-step reaction to obtain an intermediate: 2-hydroxy-4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]Benzaldehyde, reacting the intermediate with 5-chloromethyl nicotinonitrile to obtainIntermediate: 2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]Benzaldehyde is finally subjected to reductive amination reaction with 2-aminoethanol, the feeding molar weight of each raw material or intermediate is repeated in example 1, the operation is the same as that in example 1, and the off-white solid is obtained, and the yield is as follows: 33.5%, purity of product by HPLC: 96.7 percent.1H-NMR(600 MHz,CHCl3-d)δ8.93–8.90(m,1H),8.86–8.83(m,1H),8.17(s,1H),7.56(t,J＝7.2Hz,1H), 7.41–7.36(m,1H),7.29(q,J＝5.7,3.7Hz,2H),7.20–7.08(m,3H),6.68–6.61(m,2H), 5.20(d,J＝13.8Hz,4H),4.04(s,2H),3.76–3.73(m,2H),2.95(t,J＝5.1Hz,2H).
Example 22: n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] -5-chlorobenzylamine
Experimental procedure example 1 was repeated except that the starting materials in example 1: respectively replacing o-fluorobenzeneboronic acid and 3-bromo-2-methoxybenzyl alcohol by: 2, 5-difluorophenylboronic acid and 3-bromo-2-fluorobenzyl alcohol are subjected to two-step reaction to obtain an intermediate: 2-hydroxy-4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]-5-chlorobenzaldehyde, which intermediate is further reacted with m-cyanobenzyl chloride to give an intermediate: 2- (3-Cyanobenzyloxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]5-chlorobenzaldehyde, finally carrying out reductive amination reaction with 2-aminoethanol, repeating the procedure in example 1 for the molar quantities of the raw materials or intermediates charged, obtaining an off-white solid with the yield: 44.2%, purity of product by HPLC analysis: 97.3 percent.1H-NMR(500MHz, CHCl3-d)δ7.65(s,1H),7.58(q,J＝7.7Hz,3H),7.46(t,J＝7.8Hz,1H),7.31(t,J＝7.2Hz, 1H),7.27(s,1H),7.23(t,J＝7.6Hz,1H),7.06(dtd,J＝14.2,8.1,7.0,3.9Hz,3H),6.57(s, 1H),5.21(s,2H),5.02(s,2H),3.76(s,2H),3.62(t,J＝5.2Hz,2H),2.70(t,J＝5.2Hz,2H).
Example 23: n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] -5-chlorobenzylamine
Experimental procedure example 1 was repeated except that the starting materials in example 1: respectively replacing o-fluorobenzeneboronic acid and 3-bromo-2-methoxybenzyl alcohol by: 2, 5-difluorophenylboronic acid and 3-bromo-2-fluorobenzyl alcohol are subjected to two-step reaction to obtain an intermediate: 2-hydroxy-4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]-5-chlorobenzaldehyde, which is reacted with 5-chloromethyl nicotinonitrile to give an intermediate: 2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]5-chlorobenzaldehyde, finally carrying out reductive amination reaction with 2-aminoethanol, repeating the procedure in example 1 for the molar quantities of the raw materials or intermediates, obtaining a pale yellow colloidal solid with the yield: 40.1%, purity of product by HPLC: 97.1 percent.1H-NMR(500MHz,CHCl3-d)δ8.87(t,J＝1.8Hz,2H),8.10(t,J＝2.1Hz,1H),7.69–7.63 (m,1H),7.37(d,J＝4.2Hz,2H),7.30(t,J＝7.6Hz,1H),7.20–7.07(m,3H),6.63(s,1H), 5.31(d,J＝4.1Hz,2H),5.11(s,2H),3.81(s,2H),3.69(t,J＝5.1Hz,2H),2.82(t,J＝5.1Hz, 2H).
Example 24: n- {2- (3-Cyanobenzyloxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] -5-chlorobenzyl } azetidine-3-carboxylic acid
Experimental procedure example 1 was repeated except that the starting materials in example 1: respectively replacing o-fluorobenzeneboronic acid and 3-bromo-2-methoxybenzyl alcohol by: 2, 5-difluorophenylboronic acid and 3-bromo-2-fluorobenzyl alcohol are subjected to two-step reaction to obtain an intermediate: 2-hydroxy-4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]-5-chlorobenzaldehyde, which is reacted with m-cyanobenzyl chloride to give the intermediate: 2- (3-Cyanobenzyloxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy]-5-chlorobenzaldehyde, and finally reductive amination with azetidine-3-carboxylic acid, the procedure being as in example 1, with the same molar amounts of the starting materials or intermediates charged, to giveOff-white solid, yield: 45.5%, purity of product by HPLC: 96.6 percent.1H-NMR(500MHz, CHCl3-d)δ7.69(s,1H),7.65(t,J＝7.5Hz,2H),7.63–7.58(m,1H),7.53(t,J＝7.8Hz,1H), 7.44(s,1H),7.38(t,J＝6.9Hz,1H),7.29(t,J＝7.7Hz,1H),7.13(ddt,J＝15.5,7.9,4.6Hz, 3H),6.64(s,1H),5.26(s,2H),5.13(s,2H),4.12(d,J＝9.8Hz,4H),3.96(t,J＝9.4Hz,2H), 3.25(d,J＝10.4Hz,1H).
Pharmacological Activity of Compounds
1. In vitro activity evaluation of the inhibitory activity of the compounds on the interaction of PD-1 with PD-L1: the detection method of the in vitro protein level adopts a PD-1/PD-L1 binding assay kit of Cisbio company.
Principle and method for screening PD-1/PD-L1 small-molecule inhibitor
(1) The principle is as follows: PD-1 protein carries HIS label, ligand PD-L1 of PD-1 carries hFc label, anti-hFc antibody marked with Eu and anti-HIS antibody marked with XL665 respectively combine with two label proteins, after laser excitation, energy can be transferred from donor Eu to acceptor XL665, XL665 can emit light, and after inhibitor (compound or antibody) is added, binding of PD-1 and PD-L1 is blocked, Eu and 665 are far away, energy can not be transferred, XL665 can not emit light.
(2) The experimental method comprises the following steps: the specific method can be briefly described as follows by referring to a PD-1/PD-L1 kit (the code 64ICP01PEG) of Cisbio company, namely, a 384-hole white enzyme label plate, wherein 4 mu L of PD-1 protein and 4 mu L of PD-L1 protein are added into each hole, then 2 mu L of diluent or a target compound diluted by the diluent is added into each hole, the incubation is carried out for 15min at normal temperature, 10 mu L of a mixed solution of anti-Tag 1-Eu3+ and anti-Tag2-XL665 is added into each hole, and fluorescent signals at 665nm and 620nm are detected by a multifunctional enzyme label instrument after the incubation is carried out for 1h to 4h at room temperature. HTRF rate (665nm/620nm) 104. 8-10 concentrations were assayed for each compound and IC was calculated using Graphpad software50。
(3) The screening results are shown in Table 1.
The target compounds prepared in examples 1 to 24 were examined for their inhibitory activity against the interaction of PD-1 with PD-L1 according to the above-described method, and BMS202 was used as a control agent.
TABLE 1 evaluation of inhibitory Activity of the example Compounds on the interaction of PD-1 with PD-L1 at molecular level screening results
| Examples | IC50(nM) | Examples | IC50(nM) |
| 1 | 2.41×103 | 13 | ≤500 |
| 2 | ------ | 14 | 33.66 |
| 3 | 1.99×103 | 15 | ≤500 |
| 4 | 1.65×103 | 16 | 6.00 |
| 5 | ≤103 | 17 | 7.38 |
| 6 | 4.15×102 | 18 | 5.04 |
| 7 | ≤103 | 19 | 1.30×102 |
| 8 | 3.34×102 | 20 | 2.26×102 |
| 9 | 3.48×102 | 21 | 24.58 |
| 10 | 12.72 | 22 | ≤500 |
| 11 | 10.13 | 23 | 28.78 |
| 12 | 3.92 | 24 | ------ |
| BMS202 | 55.06 |
2. EXAMPLES ability of Compounds to abrogate the inhibition of IFN-gamma expression by ligand PD-L1
The expression of IFN-gamma reflects the proliferative activity of T lymphocytes. By extracting PBMCs in human peripheral blood, on the basis that anti-CD3/anti-CD28 antibody activates T lymphocytes, ligand PD-L1 protein is added to inhibit the level of IFN-gamma secretion of the T lymphocytes, and a compound to be tested is added to investigate the capability of the compound to relieve the ligand inhibition effect.
Specifically, a 96-well plate was pre-coated with 50. mu.l of anti-CD3 (5. mu.g/ml) per well in advance, and the plate was coated overnight at 4 ℃ or for 2 hours at 37 ℃ before use, and the inner coating solution in the plate was aspirated and washed twice with PBS. Fresh blood was mixed with PBS at 1: 1 volume ratio dilution, and mixing the blood diluent and the lymphocyte separation solution in a ratio of 4: 3 volumes of mix (blood dilutions were added slowly along the wall to the lymphocyte separation medium), density gradient centrifugation, PBMC layer cells were extracted, seeded into 96-well plates at a density of 2X 10^ 4/well, anti-CD28 antibody (2. mu.g/ml) and IL-2(10ng/ml) were added, and after 2-4 days of activated proliferation, test compound and PD-L1 protein (5pg/ml) were added. And detecting the expression level of IFN-gamma in the supernatant by adopting an IFN-gamma detection kit of Dake company after 48 hours. The results of the experiment according to the test method described above are shown in FIG. 1.
In FIG. 1, (-) indicates that the component is not contained, and (+) indicates that the component is contained.
The test compound was the target compound prepared in BMS202, example 10, 11, 12, 16, 17 or 18. As can be seen from FIG. 1, the test concentrations of the test compounds in the system are three concentration points, namely 1. mu.M, 2. mu.M and 4. mu.M. The results of the experiments show that the target compounds prepared in examples 10, 11, 12, 16, 17 and 18 have the ability to partially release the inhibition effect of the PD-L1 ligand at 1. mu.M, 2. mu.M and 4. mu.M to different degrees.
Claims (10)
1. Fluorobiphenyl methyl resorcinol ether derivatives shown in general formula I, stereoisomers and pharmaceutically acceptable salts thereof,
R1selected from one of the following:
R2selected from one of the following: fluorine, chlorine, methoxy;
x is selected from one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl and methoxyl;
R3selected from one of the following:
R4selected from one of the following: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl, the substituent is selected from one of the following groups: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-C5 alkyl, C1-C5 alkoxy, amino, C1-C6 alkylamino, acetamido, cyano, ureido, guanidino, sulfonamido, sulfamoyl, methylsulfonylamino, hydroxycarbonyl, C1-C8 alkoxycarbonyl, mercapto, imidazolyl, thiazolyl, oxazolyl, tetrazoyl.
2. The fluorobiphenyl methyl resorcinol ether derivatives, stereoisomers and pharmaceutically acceptable salts thereof according to claim 1, wherein the fluorobiphenyl methyl resorcinol ether derivatives have a structure represented by formula (IA-1):
R2selected from one of the following: fluorine, chlorine, methoxy;
x is selected from one of the following: hydrogen, chlorine, bromine, iodine, C1-C4 alkyl, methoxy;
R3selected from one of the following:
R4selected from one of the following: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl, the substituent is selected from one of the following groups: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-C5 alkyl, C1-C5 alkoxy, amino, C1-C6 alkylamino, acetamido, cyano, ureido, guanidino, sulfonamido, sulfamoyl, methylsulfonylamino, hydroxycarbonyl, C1-C8 alkoxycarbonyl, mercapto, imidazolyl, thiazolyl, oxazolyl, tetrazoyl.
3. The fluorobiphenyl methyl resorcinol ether derivatives, stereoisomers and pharmaceutically acceptable salts thereof according to claim 1, wherein the fluorobiphenyl methyl resorcinol ether derivatives have a structure represented by formula (IA-2):
R2selected from one of the following: fluorine, chlorine, methoxy;
x is selected from one of the following: hydrogen, chlorine, bromine, iodine, C1-4 alkyl, methoxy;
R3selected from one of the following:
R4selected from one of the following: substituted C1-C8 saturated alkylamino, substituted C2-C6 unsaturated alkylamino, substituted C2-C6 azepin-1-yl, the substituent being selected from one of the following: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C1-C5 alkyl, C1-C5 alkoxy, amino, C1-C6 alkylamino, acetamido, cyano, ureido, guanidino, sulfonamido, sulfamoyl, methylsulfonylamino, hydroxycarbonyl, C1-C8 alkoxycarbonyl, mercapto, imidazolyl, thiazolyl, oxazolyl, tetrazoyl.
4. The fluorobiphenyl methylresorcinol ether derivatives and stereoisomers and pharmaceutically acceptable salts thereof according to claim 1, wherein R is R4The structural formula of (A) is selected from one of the following:
r ═ methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, or octyl.
5. The fluorobiphenyl methyl resorcinol ether derivatives and stereoisomers thereof and pharmaceutically acceptable salts thereof according to claim 1, wherein the fluorobiphenyl methyl resorcinol ether derivatives are selected from one of the following:
n-hydroxyethyl-2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine,
n-acetamidoethyl-2- (5-cyanopyridin-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride,
n-hydroxyethyl-2- (5-cyanopyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzylamine hydrochloride,
n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] azetidine-3-carboxylic acid,
n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] L-serine,
n- [2- (3-cyanobenzyloxy) -4- (2-chloro-3-o-fluorophenylbenzyloxy) benzyl) ] D-serine,
n-hydroxyethyl-2- (5-carbamoylpyridine-3-methoxy) -4- (2-chloro-3-o-fluorophenyl benzyloxy) benzylamine hydrochloride,
n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] benzylamine,
n-hydroxyethyl-2- (5-cyanopyridin-3-methoxy) -4- [ 2-fluoro-3- (2, 5-difluorophenyl) benzyloxy ] -5-chlorobenzylamine,
6. the fluorobiphenyl methyl resorcinol ether derivatives and stereoisomers thereof and pharmaceutically acceptable salts thereof according to claim 1, wherein the pharmaceutically acceptable salts are salts or ammonium salts of the fluorobiphenyl methyl resorcinol ether derivatives combined with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions or organic bases capable of providing physiologically acceptable cations.
7. The fluorobiphenyl methylresorcinol ether derivatives and stereoisomers and pharmaceutically acceptable salts thereof according to claim 6, wherein the inorganic acid is hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, lycic acid, maleic acid, tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ions are lithium ions, sodium ions or potassium ions; the alkaline earth metal ions are calcium ions or magnesium ions; the organic base providing a physiologically acceptable cation may be methylamine, dimethylamine, trimethylamine, piperidine or morpholine.
8. A process for producing the fluorobiphenyl methylresorcinol ether derivatives of any one of claims 1 to 7, wherein the process is as follows:
(1) taking the compound 1 and substituted phenylboronic acid as basic raw materials, and obtaining an intermediate compound 2 through a Suzuki coupling reaction;
(2) brominating substituted benzyl alcohol by NBS with the intermediate compound 2 as a raw material to obtain a bromide intermediate 3;
(3) taking bromide intermediate 3 as a raw material, and reacting with intermediate 4 under an alkaline condition to obtain benzyl aryl ether intermediate 5;
(4) taking a benzyl aryl ether intermediate 5 as a raw material, and reacting with various substituted aryl halides under an alkaline condition to obtain an intermediate compound 6 containing aldehyde groups;
(5) taking an intermediate compound 6 containing aldehyde group as a raw material, and reacting with HR containing amino or imino4Carrying out reductive amination reaction to obtain a target compound I;
the substituents R1, R2, R3, R4 and X in the compounds 1 to 6 are as defined in the object compound I.
9. Use of the fluorobiphenyl methyl resorcinol ether derivatives and stereoisomers and pharmaceutically acceptable salts thereof according to any one of claims 1-7 in the preparation of medicaments for preventing and/or treating diseases related to PD-1/PD-L1 signal pathway.
10. The use according to claim 9, wherein the disease associated with the PD-1/PD-L1 signaling pathway is cancer, an infectious disease or an autoimmune disease; the cancer is skin cancer, lung cancer, urinary system tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor or head and neck cancer; the infectious disease is bacterial infection or virus infection; the autoimmune disease is organ-specific or systemic autoimmune disease, wherein the organ-specific autoimmune disease is chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, primary biliary cirrhosis, multiple sclerosis or acute idiopathic polyneuritis, and the systemic autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma or autoimmune hemolytic anemia.
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| WO2024088036A1 (en) * | 2022-10-26 | 2024-05-02 | 西安新通药物研究股份有限公司 | Asymmetric biphenyl derivative, preparation method therefor and medical use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107417564A (en) * | 2016-05-23 | 2017-12-01 | 中国医学科学院药物研究所 | Phenylate analog derivative and its preparation method and pharmaceutical composition and purposes |
| CN108368090A (en) * | 2015-10-15 | 2018-08-03 | 百时美施贵宝公司 | Compound as immunomodulator |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108368090A (en) * | 2015-10-15 | 2018-08-03 | 百时美施贵宝公司 | Compound as immunomodulator |
| CN107417564A (en) * | 2016-05-23 | 2017-12-01 | 中国医学科学院药物研究所 | Phenylate analog derivative and its preparation method and pharmaceutical composition and purposes |
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| WO2024088036A1 (en) * | 2022-10-26 | 2024-05-02 | 西安新通药物研究股份有限公司 | Asymmetric biphenyl derivative, preparation method therefor and medical use thereof |
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