CN114380686B - Chlororesorcinol aldehyde compound and application thereof in preparation of osteoclast differentiation inhibitor - Google Patents

Chlororesorcinol aldehyde compound and application thereof in preparation of osteoclast differentiation inhibitor Download PDF

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CN114380686B
CN114380686B CN202210093211.9A CN202210093211A CN114380686B CN 114380686 B CN114380686 B CN 114380686B CN 202210093211 A CN202210093211 A CN 202210093211A CN 114380686 B CN114380686 B CN 114380686B
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罗小卫
谭艳辉
林妙萍
刘永宏
高程海
卢护木
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Abstract

The invention provides a chlororesorcinol aldehyde compound and application thereof in preparation of an osteoclast differentiation inhibitor, and relates to the field of marine natural products. Disclosed are two novel chlororesorcinol aldehyde compounds orsaldechlorins A-B (1-2), the structural formula of which is shown as formula (I), which have remarkable inhibitory effect (IC) on LPS-induced NF- κB luciferase 50 The values are 15 mu M and 19 mu M respectively), and can obviously inhibit the BMMs induced by RANKL from differentiating into osteoclasts at the concentration of 15 mu M, so the BMMs are ideal candidate compounds for developing novel osteoclast differentiation inhibitor medicines.

Description

Chlororesorcinol aldehyde compound and application thereof in preparation of osteoclast differentiation inhibitor
Technical Field
The invention relates to the field of marine natural products, in particular to a chlororesorcinol aldehyde compound and application thereof in preparation of an osteoclast differentiation inhibitor.
Background
Osteoporosis is a common osteolytic disease in the elderly population and in postmenopausal women. Bone is a highly dynamic tissue that is constantly remodelled and reaches metabolic equilibrium through the bone resorption that osteoclasts are responsible for and the bone formation process that osteoblasts are responsible for. Osteoclasts (OCs) are special cells formed by the fusion of monocyte/macrophage hematopoietic lineage precursor cells, the only cells in humans that have bone resorption function. The defect of osteoclast activity leads to bone sclerosis and bone marrow failure, and the excessive activation can lead to osteolytic diseases such as osteoporosis, rheumatoid arthritis, tumor bone metastasis and the like. Therefore, molecules targeted to inhibit osteoclast formation and bone resorption are hot spots of research for controlling bone destruction related diseases. Marine microorganisms are new ideal resources for discovering novel drug source molecules, and few research reports on anti-osteoclast differentiation inhibitors of natural products of the marine microorganisms are provided, so that the anti-osteoclast differentiation potential of the marine microorganisms is still to be further explored.
The formation of osteoclasts is a stepwise process initiated by the binding of a receptor activator of the nuclear factor- κb receptor activator ligand (Receptor activator of nuclear kappa B ligand, RANKL) to its receptor RANK on the monocyte/macrophage precursor. The related inhibitors of osteoclast differentiation applied clinically at present are mainly denoxib and bisphosphonate drugs, but have certain complications and side effects. Therefore, a need exists for a safe, effective, quality-controllable, economical, targeted drug that inhibits osteoclast formation and bone resorption that effectively addresses the great clinical need for inhibitors of osteoclast differentiation.
Disclosure of Invention
The first object of the present invention is to provide two chlororesorcinol aldehyde compounds orsaldechlorins A-B (1-2) for the above-mentioned problems.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the structural formula of the compound orsaldechlorins A-B (1-2) is shown as the formula (I):
a second object of the present invention is to provide a method for preparing compound orsaldechlorins A-B (1-2) from a fermentation culture of Acremonium sclerotiorum (Acremonium sclerotigenum) C2F21, wherein the compound orsaldechlorins A-B (1-2) is prepared and isolated from the fermentation culture.
In the present invention, the process for preparing compound orsaldechlorins A-B (1-2) is further described as follows:
a) Preparing a fermentation culture of Acremonium sclerotium (Acremonium sclerotigenum) C2F21, soaking the fermentation culture in ethyl acetate, cutting the fermentation culture into small pieces, performing ultrasonic extraction for 15min, filtering with a Buchner funnel, and concentrating the filtrate by distillation to obtain an extract A; extracting the filter residue with ethyl acetate for 3 times, and concentrating by distillation to obtain extract B;
b) Combining the extract A and the extract B, performing medium-pressure normal-phase liquid chromatography by using petroleum ether/dichloromethane as an eluent, and performing a volume ratio of 100:0 to 0:100, and collecting petroleum ether/dichloromethane with a volume ratio of 70:30 gradient eluted fraction, continuing to pass medium-pressure reversed phase C 18 Column chromatography, using methanol/water as eluent, from a volume ratio of 10: 90-100: 0, and collecting methanol/water volume ratio 25: the eluted fraction was collected in a 75-gradient, and the collected fraction was purified to obtain Compound orsaldechlorins A-B (1-2).
In the present invention, it is further described that the fermentation culture of Acremonium sclerotium (Acremonium sclerotigenum) C2F21 in the step a) is prepared by inoculating activated Acremonium sclerotium (Acremonium sclerotigenum) C2F21 into a seed culture medium, culturing at 25 ℃ and 180rpm for 72 hours to obtain a seed solution, inoculating the seed solution into the fermentation culture medium at 5% of the inoculum size, and statically culturing at 25 ℃ for 30 days to obtain the fermentation culture.
In the present invention, it is further illustrated that the seed medium formulation contains per 1 liter of medium: 15g of malt extract powder, 20g of crude sea salt and the balance of water, wherein the pH value is 7.5.
In the present invention, it is further described that the formulation of the fermentation medium comprises, per 1L of the flask medium: 120g of rice, 1.5g of bacteriological peptone, 3g of crude sea salt, 150mL of water and pH7.5.
A third object of the invention is to provide the use of Acremonium sclerotium (Acremonium sclerotigenum) C2F21 for the preparation of compound orsaldechlorins A-B (1-2).
The invention is obtained through experiments: compound orsaldechlorins A-B (1-2) has significant inhibition effect on LPS-induced NF- κb luciferase, half inhibitory concentration (IC 50 Values) of 15. Mu.M and 19. Mu.M, respectively, can be used as lead compounds for developing NF- κB nuclear factor expression inhibitors.
The invention is obtained through experiments: the compound orsaldechlorins A-B (1-2) inhibits the differentiation of BMMs (Bone marrow macrophage cells, bone marrow macrophages) cells induced by RANKL into osteoclasts in a concentration gradient, and has no obvious cytotoxicity to the BMMs cells, so the compound orsaldechlorins A-B is expected to be developed into a safe and effective novel osteoclast differentiation inhibitor drug.
The fourth object of the invention is to provide application of the compound orsaldechlorins A-B (1-2) or the pharmaceutically acceptable salt thereof in preparing NF- κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor medicine.
It is a fifth object of the present invention to provide two NF- κb nuclear factor expression inhibitor or osteoclast differentiation inhibitor drugs comprising an effective amount of compound orsaldechlorins A-B (1-2) or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier or adjuvant.
By adopting the technical scheme, the invention has the following beneficial effects:
in the research process of the secondary metabolite of the aschersonia aleyrodis (Acremonium sclerotigenum) C2F21 in the co-attachment of Corallium cervi pantotrichum (Pocillopora damicornis) in North Guangxi, two novel chlororesorcinol aldehyde compounds orsaldechlorins A-B (1-2) are separated and obtained, and have remarkable inhibition effect (IC) on NF- κB luciferase induced by LPS 50 Values of 15 mu M and 19 mu M respectively), and inhibit differentiation of BMMs induced by RANKL into osteoclasts under concentration gradient, thus being developed into novel NF-kappa B nuclear factor expression inhibitionAn agent or an osteoclast differentiation inhibitor drug.
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FIG. 1 is a diagram of the chemical structure of compound orsaldechlorins A-B (1-2) and its critical HMBC (arrow); wherein a) is a chemical structure description of orsaldechlorins A-B (1-2), and B) is a key related description of HMBC;
FIG. 2 is a schematic diagram of the diffraction pattern of a copper target single crystal of compound orsaldechlorin A (1);
FIG. 3 is a graph showing comparison of inhibitory activity of Compound orsaldechlorins A-B (1-2) (20. Mu.M) on Lipopolysaccharide (LPS) -induced NF- κB luciferase in RAW264.7 cells, wherein reference numeral 1 in the figure is Compound orsaldechlorin A (1), reference numeral 2 is Compound orsaldechlorin B (2), ### represents p<0.001vs.control group; * Represents p<0.001vs.LPS group；
FIG. 4 is a schematic representation of TRAP staining results of compound orsaldechlorins A-B (1-2) on differentiation of osteoclast precursor BMMs cells;
FIG. 5 is a graph showing the effect of compound orsaldechlorins A-B (1-2) on the differentiation of osteoclast precursor BMMs;
FIG. 6 is a graph of compound orsaldechlorins A-B (1-2) versus BMMs cell viability for osteoclast precursors (72 h).
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1 Acremonium roseum (Acremonium sclerotigenum) C2F21
Acremonium sclerotium (Acremonium sclerotigenum) C2F21 isolated from Corallium cervi (Pocillopora damicornis) harvested from North Bay of Guangdong, china was deposited at the microbiological culture Collection center (GDMCC), cantonese, 05, 20, 2019, address: the Guangzhou City of Guangdong province, first, the middle road No. 100 Guangdong province microorganism research No. 59 building five, the Guangdong province institute of microorganisms, accession number: GDMCC No.60670.
EXAMPLE 2 preparation and isolation of Compound orsaldechlorins A-B (1-2)
1. Culturing
1.1, seed culture medium: each 1L of the culture medium contains 15g of malt extract powder, 20g of crude sea salt and the balance of water, and the pH is 7.5. Mixing the above components and contents, and sterilizing at 121deg.C for 30 min.
1.2, fermentation medium: each 1L of the flask medium contains: 120g of rice, 1.5g of bacteriological peptone, 3g of crude sea salt, 150mL of water and pH7.5. Mixing the above components and contents, and sterilizing at 121deg.C for 30 min.
2. Fermentation
2.1, seed culture: the activated Acremonium sclerotiorum (Acremonium sclerotigenum) C2F21 was inoculated into 1L triangular flask containing 300mL of seed medium per flask, and cultured at 25℃and 180rpm for 72 hours to obtain a seed solution.
2.2, fermentation culture: the seed solution was inoculated into a 48-flask fermentation medium flask at an inoculum size (volume percent) of 5%, and statically cultured at 25℃for 30d to prepare a fermentation culture.
3. Extracting: soaking the fermentation culture in ethyl acetate, cutting the fermentation culture into small pieces, performing ultrasonic crushing extraction for 15min, filtering by using a Buchner funnel, and concentrating the filtrate by distillation to obtain extract A; extracting the residue with ethyl acetate for 3 times, concentrating by distillation to obtain extract B, and mixing extract A and extract B to obtain total extract (100 g).
4. Isolation and purification of Compound orsaldechlorins A-B (1-2)
The crude extract (100 g) from which extract A and extract B were combined was subjected to medium pressure normal phase column chromatography liquid chromatography (MPLC) using petroleum ether/dichloromethane as eluent from a volume ratio of 100:0 to 0:100, collecting the fraction (2.5 g) eluted with petroleum ether/dichloromethane volume ratio of 70:30, and continuing to pass through medium-pressure reversed-phase C 18 Column chromatography, using methanol/water as eluent, from a volume ratio of 10: 90-100: 0, and collecting methanol/water volume ratio 25: fractions eluted at a 75 gradient, and the collected fractions were tested by HPLC-DAD to collect fractions having a maximum absorption wavelength of 220 and 29The fraction absorbed around 0nm was finally finely separated by semi-preparative high performance liquid chromatography, and purified in an elution system of methanol/water (volume ratio 50:50, YMC-pack ODS-A column, 10X 250mm,5 μm,2 m/min) to give the compound orsaldechlorin A (1) (6 mg) and orsaldechlorin B (2) (2 mg).
EXAMPLE 3 structural identification of Compound orsaldechlorins A-B (1-2)
And (3) structural identification: the compound orsaldechlorin A (1) is colorless needle-like crystals. High resolution Mass Spectrometry HR-ESIMS gives an m/z 245.0199[ M+H ]] + (calcd for C 10 H 10 ClO 5 245.0217) and its molecular formula is presumed to be C 10 H 9 ClO 5 . Its nuclear magnetic data is shown in Table 1, and shows a chloro-substituted resorcinol aldehyde structure comprising 1 unimodal methyl group (delta) H/C 2.58/14.5), 1 methylene group (delta) H/C 3.53/28.5), 1 aldehyde group (delta) H/C 10.12/195.1), 1 hydroxy group (delta) H 12.84 1 carbonyl group (delta) C 171.8 (delta) and 6 aromatic carbons C 112.5,108.8,113.7,139.6) and 2 oxygen-linked (delta) C 161.4,158.9)]. H in HMBC spectrogram (FIG. 1) 2 The correlation of-9/C-2, C-3, C-4, C-10 indicates that the methylene group attached to the 3-position of the benzene ring is attached to 1 carbonyl group, which is judged to be a carboxyl group in combination with its molecular formula. The structure deduced above was finally confirmed by X-ray single crystal diffraction analysis (fig. 2).
And (3) structural identification: compound orsaldechlorin B (2) was a white solid. High resolution Mass Spectrometry HR-ESIMS gives an m/z 253.0238[ M+Na ]] + (calcd for C 10 H 11 ClO 4 Na, 253.0244) and its molecular formula is presumed to be C 10 H 11 ClO 4 Containing 5 unsaturations. The nuclear magnetic data attribution is shown in table 1, and is compared with the nuclear magnetic data of the compound orsaldechlorin A (1), the two have very similar chemical structures, and the main difference is that the methylene (delta) of the 10-position hydroxyl in the compound orsaldechlorin B (2) H/C 3.96/63.4) substitution of carboxyl groups (. Delta.) in Compound orsaldechlorin A (1) C 171.8 And this deduces H in the HMBC spectra 2 -10/C-9, C-3 correlation signal and subtractionThe 1 less unsaturation is confirmed. The specific structural formula is shown in figure 1.
TABLE 1 Compounds orsaldechlorins A-B (1-2) 1 H-sum 13 The C-NMR data were attributed.
a 175MHz, b 700MHz, c 150MHz, d 600MHz
Compound orsaldechlorin a (1): colorless needle-like crystals; UV (MeOH) lambda max (logε)335(3.60),291(3.86),226(3.97),211(3.93)nm；IR(film)ν max 3392,2947,1716,1697,1653,1635,1616,1575,1558,1506,1456,1251,1205cm -1 ；HR-ESIMS m/z 245.0199[M+H] + (calcd for C 10 H 10 ClO 5 ,245.0217),267.0017[M+Na] + (calcd for C 10 H 9 ClNaO 5 ,267.0036).
Single crystal data for compound orsaldechlorin a (1): 2.C 10 H 9 ClO 5 ·CH 3 OH,Mr＝521.28,crystal size 0.05×0.08×0.03mm 3 ,orthorhombic,α＝90°,β＝90°,γ＝90°,T＝104(7)K,space group Pbca,Z＝8,μ(CuKα)＝3.229mm -1 ,12912reflections collected,4284independent reflections(R int ＝0.1029).The final R 1 values were0.1217(I>2σ(I)).The final wR(F 2 )values were 0.3401(I>2σ(I)).The final R 1 values were 0.1675(all data).The final wR(F 2 )values were 0.3853(all data).The goodness of fit on F 2 The X-ray single crystal diffraction pattern of was 1.334 compound orsaldechlorin A (1) is shown in FIG. 2.
Compound orsaldechlorin B (2): white solid;UV(MeOH)λ max (logε)345(3.01),292(3.07),209(4.01)nm；HR-ESIMS m/z 253.0238[M+Na] + (calcd for C 10 H 11 ClO 4 Na,253.0244).
example 4 determination of LPS-induced NF- κB luciferase inhibitory Activity by Compounds orsaldechlorins A-B (1-2) NF- κB luciferase inhibitory Activity assay primary references (British Journal of Pharmacology,2020, 177:4242-4260).
RAW264.7 cells stably transfected with NF- κB luciferase reporter gene were inoculated in 96-well plates (1×10) 4 200. Mu.L of DMEM medium containing 10% fetal bovine serum, 100IU/mL penicillin and streptomycin and 0.1. Mu.g/mL G418 was added to each well, and after cell attachment was stabilized, compound orsaldechlorins A-B (1-2) was added to set 6 multiplex wells. After further incubation for 4h, each of the compound group (3 wells) and the positive control group (NF- κB inhibitor, BAY11-7082,5 μM) was added with LPS and RANKL respectively to a final concentration of 100ng/mL per well, after 8h of stimulation by both, the supernatant was discarded, 25 μL of cell lysate was added per well, shaking at low speed for 10min to lyse the cells sufficiently, then 20 μL was transferred to the white plate, 50 μL of fluorescein solution was added per well, and the Lucifer value was detected with a multifunctional microplate reader.
Conclusion of the test: the study shows that compared with LPS blank group, the compound orsaldechlorins A-B (1-2) has remarkable inhibition effect (p) on LPS-induced NF- κB luciferase at 20 mu M<0.001 (fig. 3), semi-Inhibitory Concentration (IC) 50 Values) were 15 μm and 19 μm, respectively.
EXAMPLE 5 Effect of Compounds orsaldechlorins A-B (1-2) on RANKL-induced osteoclast precursor BMMs cell differentiation
RANKL-induced osteoclast precursor BMMs cell differentiation inhibitory activity was determined as a major reference (British Journal of Pharmacology,2020,177: 4242-4260).
RAW264.7 cells with good growth state were taken at 1×10 3 Density of each well was inoculated into 96-well plates, DMEM medium containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin was added to 200. Mu.L each well, and then the 96-well plates were placed at 37℃with 5% CO 2 Is a cell culture of (C)The incubator was incubated, and after the cells had been stabilized over night, compound orsaldechlorins A-B (1-2) (5 and 15. Mu.M) was added, respectively, with 3 duplicate wells per group. After 4h, RANKL was added to each of the other groups except the negative control group to a final concentration of 100ng/mL per well, the solution was changed every two days, after RANKL stimulation for 4-5d, the supernatant was discarded, and TRAP staining was performed. Photographing under an inverted microscope, and counting, wherein TRAP positive cells with more than 3 nuclei are osteoclasts.
Test results: compound orsaldechlorins A-B (1-2) was found to inhibit RANKL-induced differentiation of BMMs cells into osteoclasts in a concentration gradient compared to RANKL group (fig. 4 and 5), and was not significantly cytotoxic to BMMs cells (fig. 6).
Discussion of results: compound orsaldechlorins A-B (1-2) has significant Inhibition (IC) on LPS-induced NF- κB luciferase 50 The values are 15 mu M and 19 mu M respectively), and can obviously inhibit the differentiation of the BMMs induced by RANKL into osteoclasts at the concentration of 15 mu M, and has no obvious toxic effect on the BMMs. Can be used as novel osteoclast differentiation inhibitor or NF- κB nuclear factor expression inhibitor for preventing and treating osteoporosis and other bone-soluble diseases.
In summary, the invention provides a new candidate compound for developing new osteoclast differentiation inhibitor or NF- κB nuclear factor expression inhibitor drugs, and has important significance for new drug development of independent intellectual property rights.
The above description is of a detailed description of a preferred embodiment of the invention, but the embodiment is not intended to limit the scope of the invention. All equivalent changes or modification changes which are accomplished under the technical conception suggested by the invention are included in the scope of the patent covered by the invention.

Claims (4)

1. Compound orsaldechlorins A-B (1-2) has a structural formula shown in formula (I):
formula (I).
2. A process for preparing the compound orsaldechlorins A-B (1-2) as claimed in claim 1, wherein the compound orsaldechlorins A-B (1-2) is prepared from Acremonium roseumAcremonium sclerotigenum) The preparation and separation of the C2F21 fermentation culture comprises the following specific steps:
a) Activated Acremonium sclerotiorum is preparedAcremonium sclerotigenum) Inoculating C2F21 into seed culture medium, dynamically culturing at 25deg.C and 180rpm for 72h to obtain seed solution, inoculating the seed solution into fermentation culture medium at 5% of inoculum size, and static culturing at 25deg.C for 30 days to obtain Acremonium sclerotiorumAcremonium sclerotigenum) Fermentation culture of C2F 21; soaking the fermentation product in ethyl acetate, cutting the fermentation culture into small pieces, ultrasonically extracting for 15min, filtering with a Buchner funnel, and concentrating the filtrate by distillation to obtain extract A; extracting the filter residue with ethyl acetate for 3 times, and concentrating by distillation to obtain extract B; the seed culture medium formula comprises the following components in each 1L of culture medium: malt extract powder 15g, crude sea salt 20g, balance water, pH 7.5; the formula of the fermentation medium comprises the following components in each 1L of triangular flask medium: rice 120g, bacteriological peptone 1.5g, crude sea salt 3g, water 150ml, ph 7.5;
b) The crude extract obtained by combining extract a and extract B was purified by medium pressure normal phase liquid chromatography using petroleum ether/dichloromethane as eluent from the volume ratio (100: 0) (0): 100 Gradient elution, collection of petroleum ether/dichloromethane volume ratio 70): 30 gradient eluted fraction, continuing to pass medium-pressure reversed phase C 18 Column chromatography, using methanol/water as eluent, was performed with gradient elution from volume ratios (10:90) to (100:0), and methanol/water volume ratio 25 was collected: the eluted fraction was collected in a 75-gradient, and the collected fraction was purified to obtain Compound orsaldechlorins A-B (1-2).
3. Use of a compound orsaldechlorins A-B (1-2) according to claim 1 for the preparation of an osteoclast differentiation inhibitor or NF- κb nuclear factor expression inhibitor medicament.
4. The use according to claim 3, wherein the osteoclast differentiation inhibitor or NF- κb nuclear factor expression inhibitor medicament comprises an effective amount of compound orsaldechlorins A-B (1-2), and a pharmaceutically acceptable carrier or adjuvant.
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CN111960937A (en) * 2020-08-20 2020-11-20 广西中医药大学 Mixed source terpenoid, preparation method thereof and application thereof in preparation of anti-breast cancer or dihydroorotate dehydrogenase inhibitor drugs

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