3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid and preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid and a preparation method and application thereof. The 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid is a novel small molecule compound, which can inhibit the growth of Babesia by inhibiting the activity of lactate dehydrogenase, and will not affect the normal progress of the energy metabolism process of humans and most animals, and has broad application prospects in the preparation of pesticides; and the preparation method of the 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid has the advantages of easy availability of raw materials, mild reaction conditions, etc., and is very suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid and a preparation method and application thereof. Background Art

[0002] Babesia spp. belongs to the genus Babesia, class Sporozoa, subclass Piroplasmasina, order Piroplasmida, family Babesiidae. More than 111 species of Babesia have been identified, which can infect a variety of mammals and birds such as cattle, horses, sheep, and dogs. Among the more than 111 species of Babesia, the species that have been known to infect humans include Babesia microti, Babesia divergens, Babesia duncani (also known as WA1), and Babesia venatorum (also known as EU1). Among them, babesiosis of voles is a tick-borne blood protozoan disease with clinical characteristics of high fever, anemia, jaundice and hemoglobinuria caused by babesia microti parasitizing in the red blood cells of humans and various rodents. The worm is mainly transmitted by hard ticks, and can also be transmitted through blood transfusion or blood products and vertical transmission. For the elderly, people with low immunity or people with spleen removal, it will cause multiple organ failure and eventually lead to death. The disease is widely spread and prevalent worldwide, and has been reported in many countries and regions such as the United States, China and Europe, and the number of cases has increased year by year. Data from the US CDC show that in 2019, 2,418 cases of human babesiosis were reported in the United States, an increase of 11% over 2018; a study in China in 2019 counted 214 cases of human babesiosis reported in China from 1993 to 2016, nearly 21 years, of which cases caused by babesia microti accounted for 46.73%. A total of 51 local cases were recorded in Europe in 2121, of which 21.57% were caused by Babesia microti.

[0003] Currently, there is no commercial vaccine for Babesiosis. The main treatment for the disease is drug therapy, including the combination of atovaquone and azithromycin, clindamycin and quinine (Wu Jiajun, Zhou Jinlin. Research progress on Babesiosis [J]. Advances in Veterinary Medicine, 2113, 34(12): 173-178. DOI: 11.16437 / j.cnki.1117-5138.2113.12.145.). However, during the treatment with the above drugs, Babesia is prone to drug resistance. After treatment with atovaquone, the amino acid at the Q1 site of cytochrome b, the drug binding site, mutates (M134I); long-term use of azithromycin can cause the ribosomal protein subunit L4 (RPL4) encoded by the apicoplast genome to mutate (R86C), thereby producing drug resistance. Because Babesia is very susceptible to drug resistance, a combination therapy is often used when the above drugs are used for treatment. When atovaquone and azithromycin are used in combination, another amino acid mutation (Y272C) occurs at the atovaquone binding site, the Q1 site of cytochrome b. These mutations have been shown to produce drug resistance and increase the risk of recurrence if combination therapy fails.

[0004] Therefore, there is an urgent need for a new, highly effective and specific drug for the treatment of babesiosis. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing radical cure drugs for babesiosis, such as the limited selection of drugs, the easy occurrence of drug resistance and the increased risk of recurrence, and to provide a new small molecule compound - 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid.

[0006] The object of the present invention is to provide a method for preparing the 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid.

[0007] Another object of the present invention is to provide the use of the 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid in the preparation of insecticides.

[0008] Another object of the present invention is to provide an insecticide.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention protects a 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid, the structural formula of which is shown below:

[0011]

[0012] The main ways for organisms to produce energy include tricarboxylic acid cycle (TCA), anaerobic glycolysis and electron respiratory chain. The applicant previously found through whole genome sequencing of Babesia vole that the genome of Babesia vole cannot complete the TCA cycle due to the lack of malate dehydrogenase (MDH), and the antioxidant system in its mitochondria is not fully developed and lacks superoxide dismutase, indicating that its respiratory chain has the lowest biological activity, which shows that the respiratory chain is not the main way for Babesia vole to obtain energy. At the same time, the interior of the red blood cells, which are the cells that Babesia vole is exclusively parasitic on, is a hypoxic environment. In the gas environment during in vitro culture, the oxygen concentration is only 2%, so it is speculated that the glycolysis pathway is the main way for the insect body to obtain energy. In the redox reaction catalyzed between pyruvate and lactate in this pathway, lactate dehydrogenase is one of the most important enzymes. Furthermore, the applicant determined the active pocket of Babesia molitor lactate dehydrogenase by analyzing its crystal structure; determined the amino acid residues that play a key role in the enzyme's catalytic activity by point mutation, and determined that Arg99 is the enzyme activity switch of Babesia molitor lactate dehydrogenase by comparison with human lactate dehydrogenase, that is, mutation of Arg99 will reduce the activity of Babesia molitor lactate dehydrogenase but will not affect human lactate dehydrogenase, thus confirming that lactate dehydrogenase is a good target.

[0013] The present invention creatively discovered through research that 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid can inhibit the growth of Babesia by inhibiting the activity of lactate dehydrogenase. This is completely different from the insecticidal mechanism of the common insecticide triazoguanidine, which has a killing effect on trypanosomes, pear-shaped parasites and anaplasma (anaplasma) of livestock. Its mechanism of action is to selectively block the DNA synthesis or replication of the trypanosome kinetoplastid and produce irreversible binding with the cell nucleus, so that the kinetoplastid of the trypanosome disappears and cannot divide and reproduce. However, its blood concentration after administration is high and the duration is short. It is mainly used for treatment, with poor preventive effect, large toxic and side effects, and a small safety range.

[0014] Furthermore, the 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid also includes its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers or tautomers.

[0015] Furthermore, the 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid has an inhibitory effect on the activity of Babesia microti lactate dehydrogenase.

[0016] Furthermore, 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid has an inhibitory effect on the in vitro growth of Babesia microti.

[0017] The present invention also protects a method for preparing the small molecule compound, comprising the following steps:

[0018] S1, dissolving compound 1 in DMF (N,N-dimethylformamide), adding TIPSCl (triisopropylsilyl chloride), compound 2 and DMAP (4-dimethylaminopyridine), reacting fully, and post-treating to obtain compound 3;

[0019] S2, dissolving compound 4 in 1,4-dioxane, adding potassium acetate, Bis(pinacolato)diboron (bis(pinacolato)diboron, also known as bisvaleryldiboron) and Pd(dppf)Cl2 (1,1'-bis(diphenylphosphino)ferrocenepalladium chloride), reacting fully, post-treating, and obtaining compound 5;

[0020] S3, dissolving the compound 3 obtained in step S1 in 1,4-dioxane and water, adding the compound 5 obtained in step S2, Pd(dppf)Cl2 and potassium carbonate to react fully, and post-treating to obtain compound 6;

[0021] S4, dissolving the compound 6 obtained in step S3 in THF, adding TBAF (tetrabutylammonium fluoride) for sufficient reaction and post-treatment to obtain compound 7;

[0022] S5, dissolving the compound 7 obtained in step S4 in methanol and water, adding sodium hydroxide for sufficient reaction, and post-treating to obtain the target compound 8 (i.e., 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid);

[0023]

[0024] Preferably, the molar ratio of compound 1 to TIPSCl is 1:112.

[0025] More preferably, the molar ratio of compound 1 to TIPSCl is 1:111.5.

[0026] Most preferably, the molar ratio of compound 1 to TIPSCl is 1:1.3.

[0027] Preferably, the molar ratio of compound 4 to Bis(pinacolato)diboron is 1:112.

[0028] More preferably, the molar ratio of compound 4 to Bis(pinacolato)diboron is 1:1.5.

[0029] Most preferably, the molar ratio of compound 4 to Bis(pinacolato)diboron is 1:1.

[0030] Preferably, the molar ratio of compound 5 to compound 3 is 1:112.

[0031] More preferably, the molar ratio of compound 5 to compound 3 is 1:1.5.

[0032] Most preferably, the molar ratio of compound 5 to compound 3 is 1:1.37.

[0033] Specifically, in steps S11S5, the post-processing includes washing, drying, filtering, compressing and purifying.

[0034] Preferably, in step S1, the temperature for the sufficient reaction is 15121°C.

[0035] Preferably, in step S1, the sufficient reaction time is 3-15 hours.

[0036] Preferably, in step S1, the sufficient reaction time is 4 hours.

[0037] Preferably, in step S2, the temperature for the full reaction is 81185°C.

[0038] Preferably, in step S2, the sufficient reaction time is 3-15 hours.

[0039] More preferably, in step S2, the sufficient reaction time is 4 hours.

[0040] Preferably, in step S3, the temperature for the full reaction is 81185°C.

[0041] Preferably, in step S3, the sufficient reaction time is 719 hours.

[0042] Preferably, in step S3, the sufficient reaction time is 8 hours.

[0043] Preferably, in step S3, the volume ratio of dioxane to water is 81:11121.

[0044] More preferably, in step S3, the volume ratio of dioxane to water is 81:19.

[0045] Preferably, in step S4, the temperature for the sufficient reaction is 15121°C.

[0046] Preferably, in step S4, the sufficient reaction time is 113 hours.

[0047] More preferably, in step S4, the sufficient reaction time is 2 hours.

[0048] Preferably, in step S5, the temperature for the sufficient reaction is 15121°C.

[0049] Preferably, in step S5, the sufficient reaction time is 2-14 hours.

[0050] More preferably, in step S5, the sufficient reaction time is 3 hours.

[0051] Preferably, in step S5, the volume ratio of methanol to water in the methanol aqueous solution is 1:113.

[0052] More preferably, in step S5, the volume ratio of methanol to water in the methanol aqueous solution is 1:2.

[0053] The present invention also protects the use of the 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid in the preparation of insecticides.

[0054] Furthermore, the pest killed by the insecticide is Babesia.

[0055] Preferably, the Babesia includes Babesia microti or Babesia duncanii.

[0056] More preferably, the Babesia is Babesia microti.

[0057] The present invention also protects an insecticide, which contains 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer or tautomer thereof.

[0058] The present invention has the following beneficial effects: the present invention provides a novel small molecule compound, namely 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid, wherein the 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid inhibits the growth of Babesia by inhibiting the activity of lactate dehydrogenase, and does not affect the normal progress of the energy metabolism process of humans and most animals, and has broad application prospects in the preparation of insecticides; and the preparation method of the 3-[(7-hydroxynaphthalene-2-yl)methyl]benzoic acid has the advantages of easy availability of raw materials, mild reaction conditions and the like, and is very suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the nuclear magnetic resonance spectrum of 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid prepared in Example 1.

[0060] Figure 2 This is a statistical diagram of the inhibitory effect of 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid on the lactate dehydrogenase activity of Babesia microti in Example 2.

[0061] Figure 3 This is a statistical diagram of the inhibitory effect of 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid on the in vitro growth of Babesia microti in Example 3. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] Example 1 A method for preparing 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid

[0065] A method for preparing 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid, wherein the new compound of the present invention is synthesized according to the following synthetic steps:

[0066]

[0067] The preparation method specifically comprises the following steps:

[0068] S1: Compound 1 (21.6 g, 92.35 mmol, 1 eq) was dissolved in 211 mL of DMF and cooled to 1°C, and TIPSCl (23.15 g, 121.15 mmol, 25.69 mL, 1.3 eq), 2 (13.21 g, 193.93 mmol, 2.1 eq) and DMAP (2.26 g, 18.47 mmol, 1.2 eq) were added. The reaction mixture was heated to 15°C and stirred at 15°C for 4 hours. The reaction mixture was diluted with 511 mL of water and extracted with 3*111 mL of MBTE. The combined organic layer was washed with 411 mL of water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the mixture was purified by column chromatography to obtain a colorless oily compound 3.

[0069] S2: Compound 4 (11 g, 43.65 mmol, 1 eq) was dissolved in 111 mL of 1,4-dioxane, and potassium acetate (8.57 g, 87.31 mmol, 2 eq), Bis(pinacolato)diboron (11.19 g, 43.65 mmol, 1 eq) and Pd(dppf)Cl2 (1.28 g, 1.75 mmol, 1.14 eq) were added under N2 protection. The mixture was stirred at 81°C for 4 h under N2 protection. The reaction mixture was diluted with 61 mL of water and extracted with 2*31 mL of MBTE. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The mixture was purified by column chromatography to obtain white solid compound 5.

[0070] S3: Under N2 protection, compound 3 (8 g, 21.19 mmol, 1 eq) was dissolved in 81 mL 1,4-dioxane and 19 mL water, and compound 5 (7.98 g, 28.89 mmol, 1.37 eq), Pd(dppf)Cl2 (1.54 g, 2.11 mmol, 1.1 eq) and potassium carbonate (14.57 g, 115.43 mmol, 5 eq) were added. The mixture was stirred at 81 °C for 8 hours. The reaction mixture was diluted with 81 mL water and extracted with 2*41 mL ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the mixture was purified by column chromatography to obtain yellow oily compound 6.

[0071] S4: Compound 6 was dissolved in 81 mL of THF, TBAF (1 M, 27.38 mL, 1.5 eq) was added, and the mixture was stirred at 15 °C for 2 h. The reaction mixture was diluted with 81 mL of water, extracted with 2*81 mL of MTBE, the organic layers were combined and washed with 3*41 mL of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a yellow gum compound 7.

[0072] S5: Compound 7 (8 g, 27.37 mmol, 1 eq) was dissolved in 81 mL of methanol and 161 mL of water, sodium hydroxide (2.74 g, 68.51 mmol, 2.51 eq) was added, and the mixture was stirred at 15°C for 3 hours. The reaction mixture was decompressed to remove methanol. The residue was extracted with 2*51 mL of MTBE, the pH of the inorganic layer was adjusted to 213, and the mixture was extracted with 2*51 mL of ethyl acetate. The organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain an off-white solid compound 8 (i.e., 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid).

[0073] The obtained target compound 8 was subjected to nuclear magnetic resonance, and the obtained nuclear magnetic resonance spectrum was as follows Figure 1 Shown: Off-whitesolid, 51.21% yield.Compound 8:1H NMR(411MHz, DMSO-d6)δppm4.11(s,2H)7.12(dd,J=8.4,2.4Hz,1H)7.16(d,J=2.4Hz,1H)7.11( dd,J=8.4,1.2Hz,1H)7.39-7.45(m,1H)7.51-7.57(m,2H)7.68(dd,J=8.81,4.4Hz,2H)7.77(br d, J=7.6Hz, 1H) 7.82 (s, 1H) 9.72 (br s, 1H) 12.81 (br m, 1H). HPLC purity97.49%.

[0074] Example 2 Inhibition of Babesia microti lactate dehydrogenase by 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid

[0075] 1. Experimental methods

[0076] The new compound 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid prepared in Example 1 was dissolved in DMSO to prepare a 111 mM stock solution. At 25°C, the reaction system was 51 mM Tris-HCl pH 7.1 buffer (211 μL total volume), containing 51 ngrBmLDH, 1 mM NADH and 4 mM pyruvic acid, and different concentrations of TB. The spectrophotometer OD 341 The reduction of NADH production was monitored by wavelength and the IC of new small molecule compounds was calculated. 51 value.

[0077] 2. Experimental results

[0078] The experimental results are as follows Figure 2 As shown, the IC of the new compound 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid 8 prepared in Example 1 on the lactate dehydrogenase activity of Babesia microti 51 The value was 34.34 μM, which indicated that 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid had good inhibitory activity against Babesia microti lactate dehydrogenase.

[0079] Example 3 Inhibition of the in vitro growth of Babesia microti by 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid 1. Experimental method

[0080] BALB / c mice were inoculated with 1×11 7 Babesia voles (111 μL), when the infection rate reaches 61%, blood is collected from the mouse tail, and the infection rate is diluted to about 2% using healthy mouse red blood cells. The new compound 8 prepared in Example 1 is formulated into a 111mM stock solution and diluted with growth medium to a concentration gradient of 511μM, 251μM, 125μM, and 62.5μM. Growth medium without drug addition is used as a negative control, and three wells of each concentration of the compound are tested repeatedly. Culture at 37°C in a mixed gas of 2% O2, 5% CO2 and 93% N2 for 72 hours. After 72 hours of culture, smears are made, Giemsa staining is performed for microscopic examination, and the infection rate is calculated.

[0081] 2. Experimental results

[0082] The experimental results are as follows Figure 3 As shown, the IC value of the new compound 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid prepared in Example 1 on the in vitro growth inhibition of Babesia voles 51The value was 96.2 μM, indicating that 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid can effectively inhibit the growth of Babesia microti.

[0083] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula is shown below: 。 2. The method for preparing 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The steps include: S1, dissolving compound 1 in DMF, adding TIPSCl, compound 2 and DMAP, reacting sufficiently, and post-treating to obtain compound 3; S2, dissolving compound 4 in 1,4-dioxane, adding potassium acetate, Bis(pinacolato)diboron and Pd(dppf)Cl2, reacting fully, and post-treating to obtain compound 5; S3, dissolving the compound 3 obtained in step S1 in a mixture of 1,4-dioxane and water, adding the compound Pd(dppf)Cl2 obtained in step S2 and potassium carbonate to react fully, and post-treating to obtain compound 6; S4, dissolving the compound 6 obtained in step S3 in THF, adding TBAF for sufficient reaction, and post-treating to obtain compound 7; S5, dissolving the compound 7 obtained in step S4 in a methanol aqueous solution, adding sodium hydroxide for sufficient reaction, and post-treating to obtain the target compound 8; 。 3. The method for preparing 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: In step S1, the temperature for the sufficient reaction is 15-20°C.

4. The method for preparing 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: In step S2 and step S3, the temperature for the sufficient reaction is 80-85°C.

5. The method for preparing 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: In step S4 and step S5, the temperature for the sufficient reaction is 15-20°C.

6. Use of 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of insecticides.

7. The use according to claim 6, characterized in that: The pest killed by the insecticide is Babesia.

8. The use according to claim 7, characterized in that: The Babesia include Babesia microti or Babesia duncanii.

9. An insecticide, characterized in that: The invention comprises the 3-[(7-hydroxynaphthalen-2-yl)methyl]benzoic acid or a pharmaceutically acceptable salt thereof as claimed in claim 1.

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