Patent Application
20240366546
The present invention relates to a preparation method and anti-Helicobacter pylori activity of a dihydrostilbene compound, and belongs to the field of medicine.
Helicobacter pylori is a gram-negative microaerophilic bacterium in a spiral or curved shape, which colonizes human gastric mucosa to cause chronic inflammation, and is an important pathogenic factor of chronic gastritis and gastroduodenal ulcer, and closely related to the occurrence of gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma and other diseases. In 1994, the World Health Organization designated the Helicobacter pylori as a class I carcinogen of gastric cancer, and the Helicobacter pylori became the first officially recognized carcinogen in the world. An infection rate of the Helicobacter pylori in the global natural population is over 50%, and there is almost no chronic infectious disease in the world that will infect more than half of the global population. According to relevant statistical analysis, an infection rate of the Helicobacter pylori in China is as high as 55.82%, and a number of infected people are close to 800 Million. It is worth noting that China is a high incidence area of gastric cancer, and nearly a half of people suffering from the gastric cancer in the world are Chinese. Among the newly added cancer cases caused by pathogen infection in the world in 2018, there were 810,000 newly added cases of infection with the Helicobacter pylori in the world, with an incidence rate of 8.7/100,000, which exceeded those of human papillomavirus (8/100,000), hepatitis B virus (4.1/100,000) and hepatitis C virus (1.7/100,000), and in China, an incidence rate of cancer caused by the infection with the Helicobacter pylori was 15.6/100,000, which far exceeded the world average level.
In 2016, “the Fifth National Consensus Conference on the Management of Helicobacter Pylori Infection” pointed out that eradication treatment was recommended as long as Helicobacter pylori infection was confirmed by testing, regardless of symptoms and complications. Eradication of the Helicobacter pylori can not only promote the healing of peptic ulcer of body, reduce an incidence rate of ulcer complications, prevent or delay the atrophy of gastric mucosa and prevent the occurrence and development of intestinal metaplasia, but also reduce the incidence rate of gastric cancer by ⅓. At present, a bismuth quadruple therapy is recommended in China. However, due to the continuous emergence of drug-resistant strains of the Helicobacter pylori, it is increasingly difficult to eradicate the Helicobacter pylori with conventional treatment strategies, and it is urgent to find a new anti-Helicobacter pylori drug or lead compound.
Dried roots and rhizomes of glycyrrhiza (Glycyrrhiza uralensis Fisch., G. inflata Bat. and G. glabra L.) have the effects of invigorating spleen-stomach and replenishing qi, eliminating phlegm and relieving cough, detoxicating, relieving pain, and relieving drug properties, are used to treat spleen and stomach weakness, fatigue, palpitation and shortness of breath, cough with excessive phlegm, cramps and acute pains in abdomen and limbs, and carbuncle, swelling and sore-toxin, have a long history of relieving drug toxicity and potency, and are widely used in clinic and one of the commonly used medicinal materials. Glycyrrhiza stems and leaves are non-medicinal parts of glycyrrhiza, most of which are discarded in the fields, and are not reasonably used. Previous studies have shown that the glycyrrhiza stems and leaves are rich in isopentenyl compounds, and studies have shown that isopentenylation can enhance an antibacterial effect of compounds. To sum up, it is expected to obtain a novel efficient anti-Helicobacter pylori lead compound by carrying out ingredient separation.
The technical problem solved by the present invention is that: the present invention provides a preparation method of novel dihydrostilbene and an application of the dihydrostilbene in resisting Helicobacter pylori, and prepared compounds 1, 3, 6 to 7 and 9 all have good activity to two kinds of drug-resistant and sensitive Helicobacter pylori, and an MIC of 4 μg/mL to 8 μg/mL, and can be used for preparing an anti-Helicobacter pylori drug or used as lead compounds for developing the anti-Helicobacter pylori drug.
An anti-Helicobacter pylori derivative containing a dihydrostilbene parent nucleus comprises a compound with the following general structural formula:
Preferably, the anti-Helicobacter pylori derivative is selected from the group consisting of compound 1 having R1=hydrogen, R2=hydroxyl, R3=isobutyryloxy, R4=isopentenyl, R5=hydrogen, R6=hydroxyl, R7=hydrogen; compound 2 having R1=hydrogen, R2=hydroxyl, R3=2-methylbutyryloxy, R4=isopentenyl, R5=hydrogen, R6=hydroxyl, R7=hydrogen; compound 3 having R1=hydrogen, R2=methoxyl, R3=hydroxyl, R4=isopentenyl, R5=hydrogen, R6=hydroxyl, R7=hydrogen; compound 4 having R1=hydrogen, R2=hydroxyl, R3=acetyloxy, R4=isopentenyl, R5=isopentenyl, R6=hydroxyl, R7=hydrogen; compound 5 having R1=hydrogen, R2=hydroxyl, R3=acetyloxy, R4=isopentenyl, R5=isopentenyl, R6=methoxyl, R7=hydroxyl; compound 6 having R1=isopentenyl, R2=hydroxyl, R3=hydroxyl, R4=isopentenyl, R5=hydrogen, R6=hydroxyl, R7=hydrogen; compound 7 having R1=isopentenyl, R2=hydroxyl, R3=acetyloxy, R4=isopentenyl, R5=hydrogen, R6=hydroxyl, R7=hydrogen; compound 8 having R1=hydrogen, R2=hydroxyl, R3=hydroxyl, R4=aldehyde group, R5=hydrogen, R6=hydroxyl, R7=hydrogen; and compound 9 having R1=hydrogen, R2=hydroxyl, R3=hydroxyl, R4=isopentenyl, R5=isopentenyl, R6=hydroxyl, R7=hydrogen.
A preparation method of the anti-Helicobacter pylori derivative containing the dihydrostilbene parent nucleus provided by the present invention comprises the following steps of:
Preferably, the preparation method of the anti-Helicobacter pylori derivative containing the dihydrostilbene parent nucleus above comprises the following steps of:
Anti-Helicobacter pylori activity of each compound is evaluated by a broth microdilution method in an activity experiment, and metronidazole, levofloxacin, clarithromycin and vancomycin used in clinic are selected as positive control drugs. Experimental results show that the compounds 1, 3, 6 to 7 and 9 all have good activity to two kinds of drug-resistant and sensitive Helicobacter pylori, and an MIC of 4 μg/mL to 8 μg/mL, are equivalent to a positive drug metronidazole, and can be used for preparing an anti-Helicobacter pylori drug or used as lead compounds for developing the anti-Helicobacter pylori drug.
The present invention discovers the novel dihydrostilbene from the glycyrrhiza stems and leaves for the first time, and provides an extraction and separation technology, structural identification method and application in resisting Helicobacter pylori of the new compound.
The present invention can be better understood according to the following embodiments. However, it is easy for those skilled in the art to understand that specific material ratios, process conditions and results described in the embodiments are only used to illustrate the present invention, and shall not and will not limit the present invention described in detail in the claims.
Hedera ODS preparative chromatographic column (10 nm, 5 μm, 10 mm×250 mm); Waters ACQUITY UPLC BEH C18 (2.1 mmx 100 mm, 1.7 μm) chromatographic column; MCI GEL (CHP20, 75-150 μm), Sephadex LH-20 gel, and column chromatography silica gel (200 meshes-300 meshes); chromatographic acetonitrile, methanol and formic acid purchased from Merck Company of America; and analytical petroleum ether and ethyl acetate purchased from Nanjing Wanqing Chemical Reagent Co., Ltd.
A preparation method of the present invention comprised the following steps.
The compound 1 was an orange oil substance, and had an optical rotation value of [α]D20, +5.0 (c 0.08, MeOH); and HR-ESI-MS m/z 369.2066 [M+H]+, a calculated value of C23H29O4+ was 369.2066), it was prompted that a molecular formula was C23H28O4, and there were 10 unsaturation degrees. The compound 1 was highly similar to 2-(3-methyl-2-butenyl)-3,5,4′-trihydroxy-bibenzyl. A carbon spectrum and a hydrogen spectrum of the compound were analyzed, and a group of hydrogen signals δH 2.80 dd, 1.28 d and 1.27 d and a group of carbon signals δC 177.4, 35.4, 19.3 and 19.3 of isobutyryl were observed. In an HMBC spectrum, δH 3.11 (H-7) was respectively related to 6c 144.0 (C-1), 123.9 (C-2) and 151.2 (C-3), so that 2-isopentenyl substitution was determined. 6c 151.2 (C-3) shifted to a high field, so that 3-O-isobutyryl substitution was determined. In conclusion, the structure of the compound was determined to be 2-isopentenyl-3-isobutyryl-5,4′-dihydroxystilbene. The compound 1 was a new natural product, and named Uralenisol F.
The compound 2 was an orange oil substance, and had an optical rotation value of [α]D20, +2.5 (c 0.08, MeOH); and HR-ESI-MS m/z 383.2215 [M+H]+, a calculated value of C24H31O4+ was 383.2222), it was prompted that a molecular formula was C24H30O4, and there were 10 unsaturation degrees. The compound 2 was highly similar to the compound 1. A carbon spectrum and a hydrogen spectrum of the compound were analyzed, and a group of feature signals δH 2.80 dd, 1.28 d and 1.27 d and a group of feature signals δC 177.4, 35.4, 19.3 and 19.3 of 2-methylbutyryl were observed. In an HMBC spectrum, δH3.11 (H-7) was respectively related to 6c 144.1 (C-1), 123.9 (C-2) and 151.2 (C-3), so that 2-isopentenyl substitution was determined. 6c 151.2 (C-3) shifted to a high field, so that 3-O-2-methylbutyryl substitution was determined. In conclusion, the structure of the compound was determined to be 2-isopentenyl-3-acetoxy-5,4′-dihydroxydihydrostilbene. The compound 2 was a new natural product, and named uralensisol G.
The compound 3 was an orange oil substance, and had an optical rotation value of [α]D20, +40.0 (c 0.08, MeOH); and HR-ESI-MS m/z 313.1818 [M+H]+, a calculated value of C20H25O3+ was 313.1804), it was prompted that a molecular formula was C20H24O3, and there were 9 unsaturation degrees. The compound 3 was highly similar to the compound 2-3-methyl-2-butenyl)-3,5,4′-trihydroxy-bibenzyl. There was a main difference that a hydrogen signal δH 3.67 (3H, s) of methoxyl existed in the compound 3. In an HSQC spectrum, δH 3.67 (3H, s) was directly related to δC 55.5, and in an HMBC spectrum, δH3.67 (3H, s) was remotely related to 6c 159.6, so that methoxyl substitution in a 5-position was determined. The compound 3 was a new natural product, and named uralensisol I.
The compound 4 was an orange oil substance, and had an optical rotation value of [α]D20, +2.5 (c 0.08, MeOH); and HR-ESI-MS m/z 409.2385 [M+H]+, a calculated value of C26H33O4+ was 409.2379), it was prompted that a molecular formula was C26H32O4, and there were 11 unsaturation degrees. An A-ring of the compound 4 was similar to that of the compound 9. In addition, a 1H NMR (500 MHz, Methanol-d4) spectrum showed a group of hydrogen signals coupled with each other: δH 6.76 (dd, J=2.2, 8.7 Hz, H-6′), 6.64 (d, J=8.7 Hz, H-5′) and 6.77 (d, J=2.2 Hz, H-2′) and a group of hydrogen signals of isopentenyl: δH 3.24 (d, J=7.3 Hz, H-7′), 5.05 (t, J=7.3 Hz, H-5′), 1.65 (s, H-11′) and 1.70 (s, H-10′). In an HMBC spectrum, δH 3.24 (d, J=7.3 Hz, H-7′) was related to δC 154.2, 129.0 and 130.5, so that 3-isopentenyl-4′-hydroxy substitution of a B-ring was determined. δC 151.2 (C-3) shifted to a high field, and was related to δC 26.0 (C-7′), so that 3-O-acetyl substitution was determined. The compound 4 was a new natural product, and named uralensisol J.
The compound 5 was an orange oil substance, and had an optical rotation value of [α]D20, −2.5 (c 0.08, MeOH); and HR-ESI-MS m/z 439.2488 [M+H]+, a calculated value of C27H35O5+ was 439.2484), it was prompted that a molecular formula was C27H34O5, and there were 11 unsaturation degrees. The compound 5 was highly similar to the compound 4. δC 145.2 (C-4′) and 150.8 (C-5′) signals in a 13C NMR (125 MHz, Methanol-d4) spectrum were prompted to be O-diphenol hydroxyl substitution. In addition, a 1H NMR (500 MHz, Methanol-d4) spectrum showed a hydrogen signal δH 3.73 (3H, s) of methoxyl. In an HMBC spectrum, δH 3.73 (3H, s) was remotely related to δC 145.2, so that 4′-methoxyl substitution was determined. The compound 5 was a new natural product, and named uralensisol K.
The compound 6 was an orange oil substance, and had an optical rotation value of [α]D20, +42.5 (c 0.08, MeOH); and HR-ESI-MS m/z 367.2278 [M+H]+, a calculated value of C24H31O3+ was 367.2273), it was prompted that a molecular formula was C24H30O3+, and there were 10 unsaturation degrees. The compound 6 was highly similar to gancaonin R. A main difference was that a B-ring of the compound 6 was subjected to non-o-diphenol hydroxyl substitution. In the compound 6, a 1H NMR (500 MHz, Methanol-d4) spectrum showed a group of AABB hydrogen signals δH 6.70 (2H, d, J=8.4 Hz) and δH 7.00 (2H, d, J=8.4 Hz). δC 130.1 (C-4′) and 116.1 (C-5′) signals in a 13C NMR (125 MHz, Methanol-d4) spectrum were prompted to be 1,4 substitution. The compound 6 was a new natural product, and named uralensisol L.
The compound 7 was an orange oil substance, and had an optical rotation value of [α]D20, +7.5 (c 0.08, MeOH); and HR-ESI-MS m/z 409.2379 [M+H]+, a calculated value of C26H33O4+ was 409.2379), it was prompted that a molecular formula was C26H32O4, and there were 10 unsaturation degrees. The compound 7 was highly similar to the compound 6. A main difference was that there was acetoxyl substitution in the compound 7. A 13C NMR (125 MHz, Methanol-d4) spectrum showed feature signals δC 171.4 and 18.2 of acetyl. In addition, it was found that a 3-position carbon signal δC 149.0 (C-3) of the compound 7 shifted to a higher field than δC 154.6 (C-3) of the compound 6, so that acetoxyl substitution in a 3-position was determined. The compound 7 was a new natural product, and named uralensisol M.
The compound 8 was an orange oil substance, and had an optical rotation value of [α]D20, +52.5 (c 0.08, MeOH); and HR-ESI-MS m/z 259.0970 [M+H]+, a calculated value of C15H15O4+ was 259.0970), it was prompted that a molecular formula was C15H14O4, and there were 9 unsaturation degrees. The compound 8 was highly similar to 3,5,4′-trihydroxy-bibenzyl. A main difference was that there was aldehyde group substitution in the compound 8. Hydrogen and carbon spectra of the compound both showed feature signals δH 9.89 (1H, s) and δC 193.9 of aldehyde group. In addition, it was found that a 3-position carbon signal δC 149.0 (C-3) of the compound 8 shifted to a higher field than δC 154.6 (C-3) of the compound 12, so that acetoxyl substitution in a 3-position was determined. The compound 8 was a new natural product, and named uralensisol N.
The compound 9 was an orange oil substance, and had an optical rotation value of [α]D20, +52.5 (c 0.08, MeOH); and HR-ESI-MS m/z 367.2275 [M+H]+, a calculated value of C24H31O3+ was 367.2275), it was prompted that a molecular formula was C24H30O3+, and there were 10 unsaturation degrees. The compound 9 was basically the same as the compound 4. There was no feature signal of acetyl in the compound 9, and δC 157.0 (C-3) of the compound shifted to a lower field than δC 151.2 (C-3) of the compound 10. Then, the structure of the compound was further determined by 1H-1H COSY, HSQC and HMBC. The compound 9 was a new natural product, and named uralensisol 0.
An antibacterial activity test research of the present invention was carried out by the following steps.
Helicobacter pylori strains HpG27, Hp159, Hp129 and Hp26695 provided by Professor Hongkai Bi of Nanjing Medical University. Culture mediums and main reagents: Colombian culture mediums, selective antibiotics (metronidazole, levofloxacin and clarithromycin), serum, and the like.
Colombian liquid culture medium: 29.0 g of Colombian liquid culture medium was accurately weighed, heated and dissolved in 1000 mL of double distilled water, and autoclaved at 121° C. for 15 minutes after the culture medium was completely dissolved for later use.
Colombian blood agar solid culture medium: 39.0 g of Colombian blood agar solid culture medium was accurately weighed, heated and dissolved in 1000 mL of double distilled water, autoclaved at 121° C. for 15 minutes after the culture medium was completely dissolved, naturally cooled to about 50° C. after the culture medium was autoclaved, quickly added with 5% sterile defibrinated sheep blood, mixed evenly, and poured into a sterile dish while the mixture was hot.
The strains were taken out of a refrigerator at −80° C. and placed at room temperature, 200 L of standard strains were accurately sucked, transferred to a solid culture medium, and spread with an L-shaped glass rod, a culture dish loaded with the strains was put into a culture bag, then an AnaeroPack was put into the culture bag, and the culture bag was quickly sealed and then put into a constant temperature incubator at 37° C. to culture for 72 hours. After the end of culture, each strain was identified first, then a lawn on the solid culture medium was scraped off, and the lawn was transferred into 50 mL of liquid culture medium to serve as an original bacterial solution.
The present invention finds through activity screening that the compounds 1, 3, 6 to 7 and 9 all have good activity to two kinds of drug-resistant and sensitive Helicobacter pylori, and an MIC of 4 μg/mL to 8 ag/mL, are equivalent to a positive drug, and can be used for preparing an anti-Helicobacter pylori drug or used as lead compounds for developing the anti-Helicobacter pylori drug.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202410042684.5 | Jan 2024 | CN | national |
This application is the U.S. Continuing Application of International Application No. PCT/CN2024/080223 filed on 6 Mar. 2024 which designated the U.S. and claims priority to Chinese Application No. CN202410042684.5 filed on 10 Jan. 2024, the entire contents of each of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2024/080223 | Mar 2024 | WO |
| Child | 18738087 | US |
