Cyclohexene derivative as well as preparation method and application thereof
By extracting cyclohexene derivatives from the fermentation products of Nocardia ZHD001, the problem of traditional antibiotics being ineffective against drug-resistant bacteria has been solved, achieving biofilm inhibition and promoting the development of novel antibiotic drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG OCEAN UNIV
- Filing Date
- 2023-11-20
- Publication Date
- 2026-05-12
AI Technical Summary
Traditional antibiotics are ineffective against drug-resistant bacteria, and chronic infections caused by bacterial biofilms are difficult to cure, resulting in high treatment costs and long treatment times.
Cyclohexene derivatives, fermentation products of Nocardia ZHD001, were isolated from sediments collected from the waters surrounding Zhoushan. Compounds with biofilm inhibitory activity were prepared by purification using silica gel column chromatography and high performance liquid chromatography.
Cyclohexene derivatives significantly inhibit biofilm formation and have the potential to be developed into antibiotic drugs, thus solving the problem of treating drug-resistant bacteria.
Smart Images
Figure CN122010766A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chemical technology, specifically to a cyclohexene derivative, its preparation method, and its application. Background Technology
[0002] Antibiotic resistance in bacteria has become a global threat, as traditional antibiotics are no longer effective against resistant bacteria. Antibiotic resistance caused by bacterial biofilm formation is a major reason why chronic bacterial infections are difficult to cure in clinical practice. Biofilm formation leads to recurrent episodes of chronic infections, making eradication difficult, significantly increasing treatment costs, and prolonging treatment time. Therefore, developing novel antibacterial drugs targeting bacterial biofilms holds promise for alleviating this global challenge of bacterial resistance. Summary of the Invention
[0003] This invention marks the first time that *Nocardia* ZHD001 has been isolated from sediments collected from the waters surrounding Zhoushan. Further isolation yielded a cyclohexene derivative, a fermentation product of *Nocardia* ZHD001. The chemical structure of this new cyclohexene derivative was determined by analyzing its proton, carbon, COSY, HMQC, HMBC, and single-crystal diffraction data. A series of studies revealed that this novel natural compound exhibits significant biofilm inhibitory activity and can be used to prepare antibiotic drugs, demonstrating promising development and application prospects.
[0004] On the one hand, the present invention provides a cyclohexene derivative as shown in formula (I), which has significant biofilm inhibitory activity and can be used to prepare antibiotic drugs, showing great potential for development and application.
[0005]
[0006] Furthermore, the compound of formula (I) is a colorless, bulk crystal, soluble in methanol, and high-resolution mass spectrometry yields a quasi-ion peak at m / z 243.0146 [M+Na]. + The molecular formula was determined to be C7H9ClN2O4. Specific NMR data are shown in Table 1 below. 1 The H NMR spectrum is shown in [reference]. Figure 1 , 13 The C NMR spectrum is shown below. Figure 2 HSQC spectrum can be found Figure 3 HMBC spectrum can be found Figure 4 , 1 H- 1 See the H COSY spectrum. Figure 5 X-ray single-crystal diffraction is shown Figure 6 .
[0007] Table 1
[0008]
[0009]
[0010] On the other hand, the present invention provides a method for preparing a natural active compound, a cyclohexene derivative, comprising the following steps:
[0011] 1) Take Nocardiopsis sp ZHD001, inoculate it onto Gao's No. 1 solid medium for activation, inoculate a single colony of the activated Nocardiopsis sp ZHD001 into Gao's No. 1 liquid medium, shake and culture to obtain seed liquid; inoculate the obtained seed liquid into rice medium, let it stand and culture, extract and obtain fermentation broth.
[0012] 2) The fermentation broth was extracted with ethyl acetate. The ethyl acetate extract was then removed by vacuum distillation using a rotary evaporator to obtain a concentrated solution. The concentrated solution was separated by silica gel column chromatography, the eluent was collected, and each component was analyzed by thin-layer chromatography. The components containing cyclohexene derivatives were combined.
[0013] 3) The component containing the cyclohexene derivative was separated and purified by preparative high performance liquid chromatography to obtain the cyclohexene derivative compound (I).
[0014] Preferably, the Nocardiopsis sp ZHD001 strain in step 1) has the accession number CCTCC NO:M2022921.
[0015] Preferably, the formula of Gao's No. 1 solid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 20g agar, 25g sea salt, and 1L water.
[0016] Preferably, the formula of Gao's No. 1 liquid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 25g sea salt, and 1L water.
[0017] Preferably, in step 1), the rice culture medium is prepared from rice and sea salt water, and the weight-to-volume ratio of the rice to the sea salt water is 40g:60ml; the sea salt water formula is 2.5g of sea salt per 100ml of water.
[0018] Preferably, the volume ratio of fermentation broth to ethyl acetate in step 2) is 1:1.
[0019] Preferably, the silica gel column chromatography conditions in step 2) are as follows: the eluent used is CH2Cl2:CH3OH, and the gradient is 1:0, 100:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:1.
[0020] Preferably, in step 3), the high-performance liquid chromatography (HPLC) separation and purification column is an Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm), with a detection wavelength of 210 nm. A gradient elution system of 10-100% methanol-0.05% TFA-water is used, and gradient elution is performed at 10 mL / min for 40 minutes. The eluent is collected after 12.7–14.5 minutes.
[0021] On the other hand, the present invention provides a pharmaceutical composition comprising the compound having formula (I) of the present invention, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.
[0022] The present invention further provides the use of compounds of the above formula (I), their tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising thereof, in the preparation of antimicrobial drugs.
[0023] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or their pharmaceutical salts with pharmaceutically acceptable excipients.
[0024] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. Attached Figure Description
[0025] Figure 1 For compound (I) 1 HNMR spectrum (methanol-d4).
[0026] Figure 2 For compound (I) 13 C10 NMR spectrum (methanol-d4).
[0027] Figure 3 The image shows the HSQC spectrum (methanol-d4) of compound (I).
[0028] Figure 4 The image shows the HMBC spectrum (methanol-d4) of compound (I).
[0029] Figure 5 For compound (I) 1 H- 1 H COSY spectrum (methanol-d4).
[0030] Figure 6 The image shows the X-ray single-crystal diffraction pattern of compound (I).
[0031] Figure 7 This refers to the anti-biofilm activity of compound (I). Detailed Implementation
[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.
[0033] Example 1: A method for preparing a natural active compound, a cyclohexene derivative.
[0034] Nocardiopsis sp ZHD001 (accession number CCTCC NO: M2022921, China Center for Type Culture Collection) was inoculated into Gao's No. 1 solid medium and cultured for 7 days. Activated single colonies were then inoculated into Gao's No. 1 liquid medium to obtain seed culture. Gao's No. 1 solid medium consisted of: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 25g sea salt, and 20g agar powder (2%), diluted to 1L with water and adjusted to pH 7.2. Gao's No. 1 liquid medium consisted of: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, and 0.01g FeSO4·7H2O. 0.01g of sea salt and 25g of water are added to 1L to adjust the pH to 7.2.
[0035] The obtained seed culture was inoculated into rice culture medium, statically cultured, and the fermentation broth was obtained by extraction. The rice culture medium was prepared by rice and sea salt water, with a weight-to-volume ratio of 40g:60ml. The sea salt water formula was 2.5g of sea salt per 100ml of water. The seed culture inoculation amount was 5ml of seed culture water per 40g of rice culture medium. The static culture conditions were 28℃ for 70 days. The extraction conditions were: soaking in ethyl acetate for 24 hours, repeated 3 times.
[0036] The obtained fermentation broth was concentrated, separated, and purified to obtain cyclohexene derivatives with the structural formula as shown in formula (1). The separation and purification steps included silica gel column chromatography, TCL analysis, and high-performance preparative liquid chromatography purification. The silica gel column chromatography conditions were as follows: the eluent used was CH2Cl2:CH3OH, with gradients of 1:0, 100:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1. TCL analysis was performed, and the fractions were combined to obtain 10 components, G1-G10, with the target product in component G8. The high-performance liquid chromatography (HPLC) conditions for preparation were as follows: The HPLC column used for separation and purification was an Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm), with a detection wavelength of 210 nm. Elution was performed using a gradient of 10-100% methanol-0.05% TFA-water at a rate of 10 mL / min for 40 minutes. The eluent was collected from 12.7 to 14.5 minutes to obtain 91.6 mg of compound (I). This compound was a colorless, blocky crystal soluble in methanol. High-resolution mass spectrometry (HDMS) showed a quasi-ion peak at m / z 243.0146 [M+Na]. + The molecular formula was determined to be C7H9ClN2O4. Specific NMR data are shown in Table 1. 1 The H NMR spectrum is shown in [reference]. Figure 1 , 13 The C NMR spectrum is shown below. Figure 2 HSQC spectrum can be found Figure 3 HMBC spectrum can be found Figure 4 , 1 H- 1 See the H COSY spectrum. Figure 5 X-ray single-crystal diffraction is shown Figure 6 .
[0037] Example 2 uses a Pseudomonas aeruginosa biofilm inhibition activity model to evaluate the anti-biofilm activity of the present invention.
[0038] Pseudomonas aeruginosa was inoculated into liquid culture medium and cultured overnight. The bacterial suspension, diluted with M63 restriction medium, was mixed with the sample to be tested and placed in a 96-well plate. The plate was then incubated at 37°C for 16 hours. The bacterial concentration (OD) in each well was measured using a microplate reader. 600 After removing the culture medium from the plate and washing with deionized water, 160 μl of 0.1% crystal violet was added to each well to stain the biofilm for 10-15 min. The plate was then washed 3-4 times with sterile water to remove unbound staining agent and dried. 160 μl of 30% acetic acid was added to each well to extract the crystal violet, and the absorbance was measured using a microplate reader. 595 Biofilm formation amount is expressed as Abs595 / OD600.
[0039] See results Figure 7 The results showed that the compound had significant inhibitory activity against biofilm formation, indicating that the compound of formula (1) had strong anti-biofilm activity.
[0040] Finally, it should be noted that the above-described embodiments are merely one specific example of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.
Claims
1. A compound, characterized in that: The compound has the structure of formula (I), which is a colorless, bulky crystal soluble in methanol, and high-resolution mass spectrometry shows a quasi-ion peak at m / z 243.0146 [M+Na]. + The molecular formula was determined to be C7H9ClN2O. 4， 2. The method for preparing the compound of formula (I) as described in claim 1, characterized in that, The preparation method includes the following steps: 1) Take Nocardiopsis sp ZHD001, inoculate it onto Gao's No. 1 solid medium for activation, inoculate a single colony of the activated Nocardiopsis sp ZHD001 into Gao's No. 1 liquid medium, shake and culture to obtain seed liquid; inoculate the obtained seed liquid into rice medium, let it stand and culture, extract and obtain fermentation broth. 2) The fermentation broth was extracted with ethyl acetate. The ethyl acetate extract was then removed by vacuum distillation using a rotary evaporator to obtain a concentrated solution. The concentrated solution was separated by silica gel column chromatography, the eluent was collected, and each component was analyzed by thin-layer chromatography. The components containing cyclohexene derivatives were combined. 3) The component containing the cyclohexene derivative was separated and purified by preparative high performance liquid chromatography to obtain the cyclohexene derivative compound (I).
3. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The Nocardiopsis sp ZHD001 strain in step 1) has the accession number CCTCC NO:M2022921.
4. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The formula ratio of Gao's No. 1 solid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 20g agar, 25g sea salt, and 1L water; the formula ratio of Gao's No. 1 liquid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 25g sea salt, and 1L water.
5. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... In step 1), the rice culture medium is prepared from rice and sea salt water, with a weight-to-volume ratio of 40g:60ml for the rice and sea salt water. The sea salt water is formulated as 2.5g of sea salt per 100ml of water.
6. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The volume ratio of fermentation broth to ethyl acetate in step 2) is 1:
1.
7. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The silica gel column chromatography conditions in step 2) are as follows: the eluent used is CH2Cl2:CH3OH, and the gradient is 1:0, 100:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:
1.
8. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... In step 3), the high-performance liquid chromatography (HPLC) separation and purification column is an Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm), with a detection wavelength of 210 nm. A gradient elution system of 10-100% methanol-0.05% TFA-water was used, with gradient elution at 10 mL / min for 40 minutes. The eluent was collected after 12.7–14.5 minutes.
9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a compound of formula (I) as claimed in claim 1, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.
10. The use of the compound represented by formula (I) as claimed in claim 1, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or the use of the pharmaceutical composition as claimed in claim 9 in the preparation of an antibacterial medicament.