2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives and preparation methods and applications thereof

Abstract

The present invention provides a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, the structure of which is shown in formula (I), wherein RHN is selected from one of #imgabs0##imgabs1#. The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative has a stable structure, low cytotoxicity, and strong antiviral activity. The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative with the above structure can be used to prepare anti-influenza virus drugs. In addition, the present invention also provides a method for preparing the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative. The preparation method is simple in process, high in yield, and easy to industrialize. #imgabs2#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives and preparation methods and applications thereof. Background Art

[0002] Influenza is an acute respiratory infectious disease caused by influenza viruses. Influenza can occur not only in humans but also in poultry, known as avian influenza, and the two can be contagious.

[0003] From the analysis of the biological characteristics of influenza viruses, the antigenicity of influenza B and C viruses is relatively stable; the surface antigens HA and NA of influenza A virus are prone to mutation.

[0004] Vaccination has always been a top priority for influenza prevention. However, due to the high antigenic variability of influenza viruses and individual differences in immunity, vaccine protection rates are limited. Furthermore, vaccines only protect against known influenza virus subtypes and are ineffective against novel influenza viruses due to antigenic drift or antigenic shift. Consequently, vaccines produced are rarely antigenically consistent with the strains that will become prevalent. Currently, the development of anti-influenza drugs is slow, and effective options are limited. Resistance to amantadine, once effective against influenza, has developed, while imported Tamiflu, currently the only effective drug in China and abroad, has been reported. Therefore, the search for novel, highly effective, and low-toxic anti-influenza drugs remains a major challenge for pharmaceutical researchers.

[0005] Gossypol is a yellow polyphenolic compound found in the roots, stems, and seeds of cotton (a plant of the Malvaceae family). It is a chiral, atropisomer with a variety of biological activities, including spermatogenicity, immunomodulation, anticancer, and antiviral effects. However, gossypol is toxic and cannot be used directly in the preparation of anti-influenza drugs. Related technologies modify gossypol or chiral gossypol with certain amino acids. While these have significant toxicity-reducing and synergistic effects, such as alanine derivatives, these derivatives exert their antiviral effects against avian influenza viruses only during the viral adsorption phase, acting on the virus surface. They have no inhibitory effect on viruses acting within cells, do not directly inactivate viruses, and have no significant inhibitory activity against the avian influenza virus neuraminidase. Summary of the Invention

[0006] In response to the defects in the prior art, the present invention solves the technical problem of providing a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative and its preparation method and application. The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative has strong anti-influenza virus activity and low toxicity, and can be used to prepare anti-influenza virus drugs.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, the structure of which is shown in formula (I):

[0008]

[0009] (I)

[0010] Among them, RHN is selected from 、 、 One of them.

[0011] In a second aspect, the present invention further provides a use of a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative in the preparation of an anti-influenza virus drug, wherein the structure of the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is shown in formula (I):

[0012]

[0013] (I)

[0014] Among them, RHN is selected from 、 、 One of them.

[0015] In a third aspect, the present invention further provides a method for preparing a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, wherein the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is obtained by reacting 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester or its hydrochloride. The reaction formula for preparing the binaphthyl dicarboxylic acid derivative from 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-amino acid methyl ester or its hydrochloride is as follows:

[0016] (I)

[0017] The structure of the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is shown in formula (I), and the L-amino acid methyl ester is selected from one of L-leucine methyl ester, L-phenylalanine methyl ester, and L-tyrosine methyl ester.

[0018] Preferably, the preparation method specifically comprises the following steps:

[0019] adding 2,2'-binaphthyl-6,6'-dicarboxylic acid, N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to a first organic solvent to obtain a first mixed solution;

[0020] adding L-amino acid methyl ester or its hydrochloride and N,N-diisopropylethylamine to a second organic solvent to obtain a second mixed solution;

[0021] The second mixed solution is added to the first mixed solution, reacted, filtered, and water is added to the filtrate until a white precipitate is generated. The white precipitate is the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative.

[0022] Preferably, the first organic solvent is selected from at least one of dichloromethane, tetrahydrofuran, and dimethylformamide.

[0023] Preferably, the second organic solvent is selected from at least one of dichloromethane, tetrahydrofuran, and dimethylformamide.

[0024] Preferably, the molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to the L-amino acid methyl ester or its hydrochloride is 1:(2-3).

[0025] Preferably, the molar ratio of 2,2'-binaphthyl-6,6'-dicarboxylic acid to N,N-diisopropylethylamine is 1:(2-4).

[0026] Preferably, the molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:(2-4).

[0027] Preferably, the molar ratio of the L-amino acid methyl ester or its hydrochloride to N,N-diisopropylethylamine is 1:(2-4).

[0028] Compared with the prior art, the advantages of the present invention are:

[0029] (1) The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative provided by the present invention has a stable structure, low cytotoxicity, and strong anti-influenza virus activity.

[0030] (2) The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives provided by the present invention can be used to prepare anti-influenza virus drugs.

[0031] (3) The preparation method of the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative provided by the present invention has a simple process, high yield, and is easy to industrialize. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0033] Embodiments of the present invention provide 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives, preparation methods, and applications thereof. The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives are obtained by using 2,2'-binaphthyl-6,6'-dicarboxylic acid as a lead compound and modifying the two ends of the structure of 2,2'-binaphthyl-6,6'-dicarboxylic acid with amino acid methyl esters. Experiments have shown that the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives modified with L-leucine methyl ester, L-phenylalanine methyl ester, and L-tyrosine methyl ester have strong anti-influenza virus activity and low toxicity, and can be used to prepare anti-influenza virus drugs.

[0034] In order to achieve the above technical effects, the overall concept of the present invention is as follows:

[0035] In a first aspect, the present invention provides a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, the structure of which is shown in formula (I):

[0036]

[0037] (I)

[0038] Among them, RHN is selected from 、 、 One of them.

[0039] When RHN is When , the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 1.

[0040] When RHN is When , the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 2.

[0041] When RHN is When , the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 3.

[0042]

[0043] Compound 1

[0044]

[0045] Compound 2

[0046]

[0047] Compound 3

[0048] The present invention retains the binaphthyl skeleton of gossypol, removes the formyl group and phenolic hydroxyl group, uses 2,2'-binaphthyl-6,6'-dicarboxylic acid as a lead compound, introduces hydrophilic groups at both ends of its structure, and modifies it with amino acid methyl esters, such as L-leucine methyl ester, phenylalanine methyl ester, L-tryptophan methyl ester, L-proline methyl ester, L-valine methyl ester, L-methionine methyl ester and L-tyrosine methyl ester, to design a series of new derivatives.

[0049] Experiments showed that L-leucine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 1), L-phenylalanine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 2) and L-tyrosine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 3) had strong anti-influenza virus activity and low toxicity. Among them, L-leucine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 1) showed the strongest antiviral activity, which was stronger than the commonly used antiviral drug adamantane in this field, providing new ideas for our search for new anti-influenza drugs.

[0050] In a second aspect, the present invention further provides the use of 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives in the preparation of anti-influenza virus drugs. The structure of the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives is shown in formula (I):

[0051]

[0052] (I)

[0053] Among them, RHN is selected from 、 、 One of them.

[0054] When RHN is When , the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 1.

[0055] When RHN is When , the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 2.

[0056] When RHN is When , the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 3.

[0057]

[0058] Compound 1

[0059]

[0060] Compound 2

[0061]

[0062] Compound 3

[0063] Experiments show that L-leucine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 1), L-phenylalanine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 2) and L-tyrosine methyl ester-modified 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 3) have strong anti-influenza virus activity and low toxicity, and can be well used to prepare anti-influenza virus drugs.

[0064] Preferably, when the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative of the above structure is used to prepare an anti-influenza virus pharmaceutical preparation, the pharmaceutical preparation is a tablet, pill, capsule, granule, injection, suspension, emulsion or solution.

[0065] In a third aspect, the present invention further provides a method for preparing a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative. The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is obtained by reacting 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester or its hydrochloride.

[0066] The reaction formula for preparing 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (structure represented by formula (I)) from 2,2'-binaphthyl-6,6'-dicarboxylic acid (structure represented by formula (II)) and L-amino acid methyl ester or its hydrochloride (RNH2) is as follows:

[0067] (II) (I)

[0068] Wherein, the L-amino acid methyl ester is selected from one of L-leucine methyl ester, L-phenylalanine methyl ester and L-tyrosine methyl ester.

[0069] When the L-amino acid methyl ester is L-leucine methyl ester, the prepared 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 1.

[0070] When the L-amino acid methyl ester is L-phenylalanine methyl ester, the prepared 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 2.

[0071] When the L-amino acid methyl ester is L-tyrosine methyl ester, the prepared 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is compound 3.

[0072]

[0073] Compound 1

[0074]

[0075] Compound 2

[0076]

[0077] Compound 3

[0078] The preparation method has simple process, high yield, and is easy to produce. The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives (Compound 1, Compound 2, Compound 3) prepared by the method have strong anti-influenza virus activity and low toxicity.

[0079] Preferably, the preparation method specifically comprises the following steps:

[0080] (1) adding 2,2'-binaphthyl-6,6'-dicarboxylic acid, N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) into a first organic solvent to obtain a first mixed solution;

[0081] (2) adding L-amino acid methyl ester or its hydrochloride and N,N-diisopropylethylamine (DIPEA) to a second organic solvent to obtain a second mixed solution;

[0082] (3) adding the second mixed solution to the first mixed solution, reacting, filtering, and adding water to the filtrate until a white precipitate is generated. The white precipitate is the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative.

[0083] Preferably, after a white precipitate is generated, the target product, 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, is obtained by centrifugation and drying.

[0084] Preferably, the first organic solvent is selected from at least one of dichloromethane, tetrahydrofuran and dimethylformamide.

[0085] Preferably, the second organic solvent is selected from at least one of dichloromethane, tetrahydrofuran and dimethylformamide.

[0086] Preferably, the molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to the L-amino acid methyl ester or its hydrochloride is 1:(2-3).

[0087] Preferably, the molar ratio of 2,2'-binaphthyl-6,6'-dicarboxylic acid to N,N-diisopropylethylamine is 1:(2-4).

[0088] Preferably, the molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:(2-4).

[0089] Preferably, the molar ratio of the L-amino acid methyl ester or its hydrochloride to N,N-diisopropylethylamine is 1:(2-4).

[0090] The following specific examples illustrate the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives of the present invention, as well as their preparation methods and applications.

[0091] Example 1

[0092] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid conjugate with L-leucine methyl ester (Compound 1)

[0093]

[0094] (1) 300 mg (0.876 mmol) of 2,2'-binaphthyl-6,6'-dicarboxylic acid, 0.46 ml (2.628 mmol) of N,N-diisopropylethylamine (DIPEA), and 732.7 mg (1.927 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were added to 10 ml of dimethylformamide (DMF) in sequence, and stirred at room temperature for 1 hour to obtain a first mixed solution;

[0095] (2) 477.4 mg (2.628 mmol) of L-leucine methyl ester hydrochloride and 0.46 ml (2.628 mmol) of N,N-diisopropylethylamine (DIPEA) were added to 10 ml of dimethylformamide (DMF) solution to obtain a second mixed solution;

[0096] (3) The second mixed solution was added to the first mixed solution, and the mixture was reacted at room temperature for 3 hours. TLC was used to monitor the reaction until the reaction was complete. The mixture was filtered, and water was added dropwise to the filtrate under magnetic stirring until a white precipitate was obtained. The filtrate was placed in a centrifuge tube for centrifugation, washed three times with distilled water, and the water layer was removed. The mixture was placed in a vacuum drying oven at -0.1 MPa with phosphorus pentoxide as a desiccant and dried overnight to obtain 412.4 mg of a white solid, which was compound 1, with a yield of 79.0% and a melting point of 197.1-197.4°C.

[0097] 1 H NMR (600 MHz, DMSO-d6): δ H8.91(d, J =7.58Hz, 2H, 2×CONH), 8.53(s, 2H, 2×Ar-H), 8.47(s, 2H, 2×Ar-H), 8.17(d, J =8.59Hz, 2H, 2×Ar-H), 8.11(m, 4H, 4×Ar-H), 7.99(d, J =8.51Hz, 2H,2×Ar-H ),4.56(ddd, J =11.36, 7.48, 4.61Hz, 2H, 2×NCH), 3.65(s, 6H, 2×OCH3), 1.83(ddd, J =14.57, 10.61, 4.97Hz, 2H, 2×CH), 1.73(dd, J =12.82, 6.74Hz, 2H, 2×CH), 1.61(ddd, J =13.60, 9.01, 4.75Hz, 2H, 2×CH),0.93(d, J =6.55Hz, 6H, 2×CH3), 0.89 (d, J =6.48Hz, 6H, 2×CH3);

[0098] 13 C NMR (151 MHz, DMSO-d6): δ C 173.60, 167.07, 139.01, 135.09, 131.94,131.75, 130.19, 128.86, 128.03, 126.47, 126.16, 125.32, 51.54, 52.38, 24.93,23.31, 21.66;

[0099] HRMS(ESI + ): m / z calcd for C 36 H 40 N2O6[M+Na] + 619.27841, found 619.27786.

[0100] Example 2

[0101] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-phenylalanine methyl ester (Compound 2)

[0102]

[0103] The preparation method was the same as that in Example 1, except that the L-leucine methyl ester in Example 1 was replaced with an equal molar amount of L-phenylalanine methyl ester to obtain 423.1 mg of a white solid, namely compound 2, with a yield of 72.7% and a melting point of 190.3-191.8°C.

[0104] 1 H NMR (600 MHz, DMSO-d6): δ H 9.04(d, J =7.73Hz, 2H, 2×CONH), 8.43(dd, J =4.63, 1.73Hz, 4H, 2×Ar-H), 8.14(d, J =8.62Hz, 2H, 2×Ar-H), 8.08(d, J =8.67Hz,4H, 2×Ar-H),7.89(dd, J =8.46, 1.75Hz, 2H, 2×Ar-H),7.31(d, J =7.45Hz, 4H, 2×Ar-H), 7.27(t, J =7.60Hz, 4H, 2×Ar-H), 7.17(t, J =7.60Hz, 2H, 2×Ar-H), 4.72(ddd, J =10.00, 7.71, 5.38Hz, 2H, 2×NCH), 3.63(s, 6H, 2×OCH3), 3.16 (m, 4H, 2×CH2);

[0105] 13 C NMR (151 MHz, DMSO-d6): δ C 172.64, 166.95, 139.03, 138.06, 135.06,131.88, 131.63, 130.20, 129.51, 128.91, 128.73, 127.97, 126.99, 126.50,126.13, 125.14, 52.46, 54.87, 36.75;

[0106] HRMS(ESI + ): m / z calcd for C 42 H 36 N2O6[M+Na] + 687.24711, found 687.24821.

[0107] Example 3

[0108] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid conjugate with L-tyrosine methyl ester (Compound 3)

[0109]

[0110] The preparation method was the same as that in Example 1, except that the L-leucine methyl ester in Example 1 was replaced with an equal molar amount of L-tyrosine methyl ester to obtain 491.4 mg of a white solid, namely compound 3, with a yield of 80.6% and a melting point of 237.8-238.6°C.

[0111] 1 H NMR (600 MHz, DMSO-d6): δ H 9.12 (s, 2H, 2×OH), 8.83 (d, J =7.6 Hz, 2H,2×CONH), 8.31 (s, 2H, 2×Ar-H), 8.31 (d, J =8.5Hz, 2H, 2×Ar-H), 8.01 (d, J =8.6 Hz, 2H, 2×Ar-H), 7.95 (s, 2H,2×Ar-H), 7.95 (d, J =8.6 Hz, 2H, 2×Ar-H),7.77 (d, J =8.5Hz, 2H, 2×Ar-H), 6.96(d, J = 8.1 Hz, 4H, 2×Ar-H), 6.51 (d, J = 8.1Hz, 4H, 2×Ar-H), 4.49 (td, J = 8.9, 6.1Hz, 2H, 2×NCH), 3.49 (s, 6H, 2×OCH3), 2.90 (m, 4H, 2×CH2);

[0112] 13 C NMR (151 MHz, DMSO-d6): δ C 182.0, 176.1, 165.5, 148.2, 144.2, 141.1,140.9, 139.6, 139.6, 139.4, 138.1, 137.3, 137.2, 135.7, 135.3, 134.4, 124.7,124.7, 64.4, 61.5, 45.2;

[0113] HRMS(ESI + ): m / z calcd for C 42 H 36 N2O8[M+Na] + 719.23694, found719.23715.

[0114] Comparative Example 1

[0115] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid conjugate with L-proline methyl ester (Compound 4)

[0116]

[0117] The preparation method was the same as that in Example 1, except that the L-leucine methyl ester in Example 1 was replaced with an equal molar amount of L-proline methyl ester to obtain compound 4 with a yield of 76.3% and a melting point of 198.7-199.7°C.

[0118] Comparative Example 2

[0119] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid conjugate with L-methionine methyl ester (Compound 5)

[0120]

[0121] The preparation method was the same as that in Example 1, except that the L-leucine methyl ester in Example 1 was replaced with an equal molar amount of L-methionine methyl ester to obtain Compound 5 with a yield of 78.6% and a melting point of 196.5-197.2°C.

[0122] Comparative Example 3

[0123] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid conjugate with L-tryptophan methyl ester (Compound 6)

[0124]

[0125] Compound 6

[0126] The preparation method was the same as that in Example 1, except that the L-leucine methyl ester in Example 1 was replaced with an equal molar amount of L-tryptophan methyl ester to obtain compound 6 with a yield of 79.2% and a melting point of 209.8-211.2°C.

[0127] Comparative Example 4

[0128] Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid conjugate with L-valine methyl ester (Compound 7)

[0129]

[0130] The preparation method was the same as that in Example 1, except that the L-leucine methyl ester in Example 1 was replaced with an equal molar amount of L-valine methyl ester to obtain compound 7 with a yield of 75.1% and a melting point of 254.7-256.2°C.

[0131] Example 8

[0132] Anti-H1N1 test of compounds 1-7

[0133] 1. Experimental Materials

[0134] (1) Virus: The H1N1 strain (PR8) is from the US CDC, and its titer was previously measured to be 4×10 9 PFU / mL, diluted with culture medium during infection, i.e., 80 PFU / well, provided by the State Key Laboratory of Virology, Wuhan University.

[0135] (2) Cells: MDCK cells (from China Center for Type Culture Collection, MDCK cells are the classic cells for influenza virus plaque assay), provided by the State Key Laboratory of Virology, Wuhan University.

[0136] (3) Positive control drug: Amantadine, provided by the State Key Laboratory of Virology, Wuhan University.

[0137] (4) Sample treatment: Amantadine and compounds 1 to 7 of the present invention were prepared into 10 mg / mL stock solutions with dimethyl sulfoxide (DMSO). The stock solutions were diluted with DMSO to form corresponding gradients before use.

[0138] 2. Experimental Methods

[0139] 2.1 Cytotoxicity assay (MTT assay)

[0140] The MTT assay is based on the reducing agent MTT, a metabolite in living cells. MTT stands for 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide; its Chinese chemical name is 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide; and its commercial name is Thiazolyl Blue.

[0141] To prepare 5 mg / ml MTT: Weigh 0.5 g of MTT powder and dissolve it in 100 mL of phosphate-buffered saline (PBS) or phenol red-free culture medium. Sterilize the solution by filtering through a 0.22 μm filter and store at 4°C in the dark. MTT is best prepared and used immediately. It is effective within two weeks of storage at 4°C in the dark. Alternatively, it can be frozen at -20°C for long-term storage and aliquoted into small tubes to avoid repeated freeze-thaw cycles.

[0142] Preparation of triple formazan solution: 25g sodium dodecyl sulfate (SDS), 250 μL 11.6 mol / L hydrochloric acid, and 12.5 mL isobutanol. Add deionized water to 250 mL to obtain the triple formazan solution. The mass percentage of sodium dodecyl sulfate (SDS) is 10%, the molar concentration of hydrochloric acid is 0.012 mol / L, and the mass percentage of butanol is 5%.

[0143] The steps of this cytotoxicity experiment are as follows:

[0144] (1) Digest the MDCK cell line and dilute it to 2×10 5 Cells were plated into 96-well plates at 100 μL / well and cultured in a 37°C 5% CO2 cell culture incubator for 4 hours to allow the cells to fully adhere to the wall.

[0145] (2) The stock solutions (10 mg / mL) of the test compounds 1 to 7 of the present invention were diluted with DMSO in a gradient manner to final concentrations of 33.33 μg / mL, 6.66 μg / mL, 3.33 μg / mL, 0.33 μg / mL, and 0.03 μg / mL, respectively. 1 μL was added to each well of a 96-well plate. Three parallel wells were set up for each concentration. Three parallel wells were also set up for the control wells, and 1 μL of DMSO was added to each well. A positive control drug was also treated in the same manner as a control.

[0146] (3) After 48 hours of culture, the supernatant was removed and the monolayer was rinsed three times with sterile phosphate-buffered saline (PBS);

[0147] (4) Add 100 μL of a mixture of MTT and fresh culture medium to each well (MTT final concentration 0.5 mg / ml) and incubate at 37°C for 4 hours to reduce MTT to formazan;

[0148] (5) Remove the supernatant and add 100 μL of triple formazan solution to each well. Incubate at 37°C for 4 hours to completely dissolve the formazan.

[0149] (6) The light absorption value at 655 nm was used as the background, and the light absorption value at 570 nm was measured.

[0150] After completing the above experiment, calculate the cell death rate according to the following formula:

[0151] Cell death rate (%) = [1-(OD value of cells treated with compound / OD value of control cells)] × 100 (OD Optical Density, also known as absorbance);

[0152] The median toxic concentration (CC) of compounds 1 to 7 of the present invention and the positive control drug 50) is calculated as follows: the compound concentration is the horizontal axis and the cell death rate is the vertical axis, and then the compound concentration at which 50% of the cell death rate occurs is obtained, which is CC 50 , the calculation results are shown in Table 1.

[0153] 2.2 Anti-H1N1 activity assay

[0154] MDCK cells were cultured in 24-well plates and washed twice with PBS when cells reached 100% confluence. Viruses and cells were then treated with the following methods, with four replicates per well: 1. Positive control wells: A positive control drug was mixed with virus and added directly to the cells; 2. Virus infection control wells: A virus suspension was added to the cells; 3. Cell control wells: Cells were incubated with DMEM; 4. Compound-treated wells: A compound of the invention and virus were mixed in 300 μL of culture medium and added directly to the cells. The culture medium used was serum-free, and trypsin was added at a final concentration of 10 μg / mL to facilitate infection.

[0155] Compounds 1-7 of the present invention were prepared in DMSO to a 10 mg / mL stock solution. Upon use, the stock solutions were serially diluted with DMSO to final concentrations of 33.33 μg / ml, 6.66 μg / ml, 3.33 μg / ml, 0.33 μg / ml, and 0.03 μg / ml, respectively. 1 μl of the compound was added to each well. A positive control drug was treated similarly and used as a control. After 2 hours of infection at 37°C, discard the supernatant, wash twice with PBS, and add a mixture of phenol red-free DMEM medium and 1% agarose (pre-thawed and kept at 37°C to prevent solidification) at a 1:1 volume ratio of DMEM to agarose. Trypsin at a final concentration of 10 μg / mL was added to promote infection. Incubate the cells with their upper side facing up at room temperature for 20-40 minutes. After solidification, invert and incubate at 37°C, 5% CO2 in a 5% CO2 incubator for 2-4 days. When plaques are large and clear, stain with 0.5% crystal violet (130 μL / well). Protect from light for 6-8 hours, then count plaques visually. Count each well twice. Counts are considered valid if the results are identical. Repeat until the two counts are identical and considered valid. The number of plaques in each group is the average of three wells.

[0156] After completing the above experiment, the inhibition rate of the compound of the present invention and the positive control drug on virus infection was calculated using the following formula: Inhibition rate (%) = [(number of plaques in the virus infection control group - number of plaques in the compound-added group) / number of plaques in the virus infection control group] × 100. The half-maximal inhibitory concentration (IC50) of the compound of the present invention and the positive control drug 50 The calculation method of IC is as follows: the compound concentration is the horizontal axis and the inhibition rate is the vertical axis. The compound concentration when the inhibition rate is 50% is the IC 50 SI is the selectivity index, and its value is CC 50 / IC 50The above calculation results are shown in Table 1.

[0157] Table 1 Cytotoxicity and anti-H1N1 activity of compounds 1-7

[0158]

[0159] Note: - indicates no calculation

[0160] As shown in Table 1, Compound 1, Compound 2, and Compound 3 have strong antiviral activity and low cytotoxicity. Among them, Compound 1 has the strongest antiviral activity, even stronger than adamantane, which provides new ideas for our search for new anti-influenza virus drugs.

[0161] Example 9

[0162] Acute toxicity test of 2,2'-binaphthyl-6,6'-dicarboxylic acid derivatives

[0163] Compound 1 with better activity was selected for acute toxicity experiment. KM mice, male, body weight 36±4g, SPF grade, were randomly divided into normal saline group, low-dose group 200mg / kg, and high-dose group 400mg / kg, with 6 mice in each group. After 7 consecutive days of drug administration, the mice showed no significant changes in body weight and hair, and no deaths occurred, indicating that the new 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative has low toxicity and can be used for anti-influenza virus drug research.

[0164] It should be noted that, in the present invention, relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device comprising the element.

[0165] The present invention is not limited to the above-described embodiments. Persons skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are considered to be within the scope of protection of the present invention. Any matters not described in detail in this specification constitute prior art known to those skilled in the art.

Claims

1. A 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, characterized in that: The structure of the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is shown in formula (I): （I） Among them, RHN is .

2. A use of a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative in the preparation of an anti-influenza virus drug, characterized in that: The structure of the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is shown in formula (I): （I） Among them, RHN is selected from 、 、 One of them.

3. A method for preparing a 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative, characterized in that: The 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is obtained by reacting 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester, and the reaction formula is shown below: （I） The structure of the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative is shown in formula (I), and the L-amino acid methyl ester is L-phenylalanine methyl ester.

4. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 3, wherein: The specific steps include: adding 2,2'-binaphthyl-6,6'-dicarboxylic acid, N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to a first organic solvent to obtain a first mixed solution; adding L-amino acid methyl ester and N,N-diisopropylethylamine to a second organic solvent to obtain a second mixed solution; The second mixed solution is added to the first mixed solution, reacted, filtered, and water is added to the filtrate until a white precipitate is generated. The white precipitate is the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative.

5. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 4, wherein: The first organic solvent is selected from at least one of dichloromethane, tetrahydrofuran, and dimethylformamide.

6. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 4, wherein: The second organic solvent is selected from at least one of dichloromethane, tetrahydrofuran, and dimethylformamide.

7. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 4, wherein: The molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to the L-amino acid methyl ester is 1:(2-3).

8. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 4, wherein: The molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to N,N-diisopropylethylamine is 1:(2-4).

9. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 4, wherein: The molar ratio of the 2,2'-binaphthyl-6,6'-dicarboxylic acid to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:(2-4).

10. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid derivative according to claim 4, wherein: The molar ratio of the L-amino acid methyl ester to N,N-diisopropylethylamine is 1:(2-4).

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