CN107556274B

CN107556274B - Nitrophenyl ester sesquiterpenoids, preparation method thereof and application thereof in preparation of anti-renal cancer drugs

Info

Publication number: CN107556274B
Application number: CN201710861987.XA
Authority: CN
Inventors: 周雪峰; 杨斌; 林秀萍; 唐斓; 李晓娟; 刘永宏
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: Shaanxi Huasen Shengbang Technology Co.,Ltd.
Priority date: 2017-09-21
Filing date: 2017-09-21
Publication date: 2020-08-11
Anticipated expiration: 2037-09-21
Also published as: CN107556274A

Abstract

The invention discloses a nitrophenyl ester sesquiterpenoids, a preparation method thereof and application thereof in preparing anti-renal cancer drugs. The invention prepares the nitrophenyl ester sesquiterpene compound with anti-tumor activity from marine fungus (Aspergillus sp.) Jcma1F17, as shown in the structural formula (I), wherein R is₁When is H, R₂Is NB, R₃Is H; r₁When it is OH, R₂Is CH3CO, R₃Is NB; r₁When is H, R₂Is CH3CO, R₃Is NB; r₁When it is OH, R₂Is NB, R₃Is H; r₁When it is OH, R₂Is H, R₃Is NB; r₁When is H, R₂Is H, R₃Is NB; the structural formula of NB is shown as formula (II). The nitrophenyl ester sesquiterpenoids, in particular to the compounds containing C-9 hydroxyl, can be used for developing anti-tumor drugs, in particular to anti-renal cancer drugs

Formula (I);

formula (II).

Description

Nitrophenyl ester sesquiterpenoids, preparation method thereof and application thereof in preparation of anti-renal cancer drugs

Technical Field

The invention belongs to the technical field of marine natural products, and particularly relates to a nitrophenyl sesquiterpenoids, a preparation method thereof and application thereof in preparation of antitumor drugs.

Background

Malignant tumors have long been one of the major diseases that seriously jeopardize human life and quality of life. It has been reported that malignant tumors have become the leading cause of death for residents in our country. For the treatment of malignant tumor, natural products and their derivatives play an important role. It is reported that antitumor drugs derived from natural products account for more than 60% of antitumor drugs on the market between 1981 and 2008, and the number of novel natural products and derivatives thereof as new antitumor drugs is increasing. Renal cancer is one of ten major dead cancers in the world, and the incidence rate of the renal cancer is second to bladder cancer in urinary system tumors in China. It has been found that 30% of patients with renal cancer develop metastasis, and another 30% are at risk of developing metastasis within 10 years, and metastatic renal cancer has high resistance to radiotherapy and chemotherapy and systemic treatment.

Nitrobenzoyl sesquiterpenoids (nitrobenzoyl sesquiterpenoids) are rare in nature, and only five nitrophenyl sesquiterpene compounds, which are isolated and found in marine-derived Aspergillus, have been reported so far, and the antitumor potential of the nitrophenyl sesquiterpenoids remains to be continuously exploited.

Disclosure of Invention

The invention aims to: the problem that the existing nitrophenyl ester sesquiterpene compound is small in quantity and incomplete in research is solved, and the nitrophenyl ester sesquiterpene compound not only has anti-tumor activity, but also has selective inhibitory activity on human kidney cancer cells, and the preparation method and the application thereof are provided.

In order to realize the aim, the invention provides a nitrophenyl ester sesquiterpenoids, the structural formula of which is shown as a formula (I),

can be divided into 6 compounds, compound 1, R₁Is H, R₂Is NB, R₃Is H (intulicolide B); in the compound 2, R₁Is OH, R₂Is CH3CO, R₃Is NB (14-acetoxy insulicolide A); in the compound 3, R₁Is H, R₂Is CH3CO, R₃Is NB (insulicolide C); in the compound 4, R₁Is OH, R₂Is NB, R₃Is H (6 β,9 α -dihydroxy-14-p-nitrobenzylcinnamolide), compound 5, R₁Is OH, R₂Is H, R₃Is NB (insulicolide A); in the compound 6, R₁Is H, R₂Is H, R₃Is NB (9-hydroxy insulin A);

the structural formula of the NB is shown as a formula (II):

through the evaluation of the in vitro anti-renal cancer activity of the compounds 1 to 6, the compounds 1 to 6 are found to have different intensities of inhibitory activities (IC) on human renal cancer OS-RC-2 cells, human adenocarcinoma ACHN cells on kidney and human clear cell adenocarcinoma 786-O cells₅₀0.89-30.35 mu M); wherein the compound 5 has the strongest inhibitory activity (IC) on three renal cancer cells₅₀0.89-1.46 mu M), and the compound 2 has the same obvious inhibitory activity (IC)₅₀2.3-5.3 mu M), which is equivalent to the drug Sorafenib (IC) for kidney cancer in the market₅₀3.4 to 7.0 μ M). The inhibitory activity of compound 1, compound 3 and compound 6 on three renal cancer cells was relatively weak (IC)₅₀11.18-30.35 mu M), and the hydroxyl at the C-9 position is possibly an active group for inhibiting renal cancer cells. In addition, compound 2 and compound 4 have certain selectivity (IC) on human kidney clear cell adenocarcinoma 786-O cells₅₀2.30 and 4.30. mu.M, respectively), and p-OS-RC-2 (IC)₅₀5.30 and 8.21. mu.M, respectively) and ACHN (IC)₅₀4.10 and 11.18 mu M respectively) by 2-3 times. Cell cycle and apoptosis research shows that the compound 2 has a function of blocking the 786-O cell cycle in the G0/G1 stage and has a certain induction effect on late apoptosis (AV +/PI +) of 786-O cells. Therefore, the nitrophenyl ester sesquiterpenoids, in particular to the compounds containing C-9 hydroxyl, can be used as lead compounds for developing anti-renal cancer (renal clear cell adenocarcinoma) medicaments.

Therefore, the nitrophenyl ester sesquiterpene compound and the medicinal salt thereof can be used for preparing antitumor drugs, in particular to renal cancer resistant drugs.

In order to achieve the above object, the present invention also provides an antitumor drug comprising an effective amount of nitrophenyl sesquiterpenoids or pharmaceutically acceptable salts thereof as an active ingredient, and a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that nitrophenyl ester sesquiterpenoids with selective inhibition effect on kidney cancer cells, particularly compounds containing C-9 hydroxyl (such as compound 2), have certain selectivity on human kidney transparent cell adenocarcinoma 786-O cells, can be used for preparing antitumor drugs, and are particularly suitable for preparing drugs for resisting kidney cancer. The invention provides an alternative compound for developing a new anti-renal cancer drug, and has important significance for developing Chinese marine microbial drug resources.

Drawings

The technical scheme and the beneficial effects of the invention are described in the following with reference to the accompanying drawings.

FIG. 1 shows the principal HMBC and HMBC of Compound 1 and Compound 2 of the present invention¹H-¹Schematic diagram of H COSY related signal.

FIG. 2 is a schematic representation of the main NOESY-related signals of compounds 1-3 of the present invention.

FIG. 3 is a circular dichroism diagram of compounds 1 to 3 of the present invention.

FIG. 4 shows the results of the proliferation inhibitory activities of compounds 1-6 at different concentrations on three renal cancer cell lines.

FIGS. 5 to 7 show the results of the 786-O cell cycle arrest with Compound 2 of the present invention.

FIGS. 8 to 10 show the results of 786-O apoptosis induced by Compound 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results.

Example 1 isolation and purification and structural identification of Nitrophenyl ester sesquiterpene Compounds

The Aspergillus oceanic strain Jcma1F17 was fermented for 50L according to the published methods (Fang, W.; Lin, X.; Zhou, X.; Wan, J.; Lu, X.; Yang, B.; Ai, W.; Lin, J.; Zhang, T.; Tu Z.; Liu, Y. MedChemcomm2014,5,701;) to give a strain. Dissolving a dried extract (52 g) of the fermentation product in methanol, mixing the mixture with silica gel, and performing medium-pressure silica gel column chromatography (200-300 meshes), wherein the eluent is petroleum ether: gradient elution with ethyl acetate to obtain 9 elution sites fr.I-fr.IX (volume ratio of petroleum ether to ethyl acetate is 50:0,50:1,20:1,10:1,5:1,3:1,2:1,1:1 and 0:1, respectively). VII part mainly contains compound 5, and the compound 5 monomer is obtained by recrystallization, wherein the weight of the compound is about 300 g; subjecting the regions fr.V and fr.VI to Sephadex LH-20 gel column chromatography (CHCl)₃And MeOH at a volume ratio of 1:1) and preparative HPLC repeated isolation and purification to give compound 1(5.6mg), compound 2(8.5mg), compound 3(4.4mg), compound 4(32.0mg), compound 6(6.2mg) and compound 7(3.5 mg).

Compounds

4 and 5 were identified as nitrophenyl ester sesquiterpenes 6 β,9 α -dihydroxy-14-p-nitrobenzoyl cinanolide (Compound 4) and insulicolide A (Compound 5) by NMR and mass spectrometry, and similarly Compound 6 was identical to nitrophenyl ester sesquiterpene compound 9-dehydroinsulicolide A (Belofsky, G.N.; Jensen, P.R.; Renner, M.K.; Fencal, W.tetrahedron 1998,54, 1715-1724) reported in the literature.

TABLE 1 NMR spectra data (700MHz, MeOH, ppm) of nitrophenyl sesquiterpenoids 1 to 3

Signal repetition.

High resolution Mass Spectrometry HRESIMS (m/z 450.1324for C) of Compound 1₂₂H₂₅NO₇Cl[M+Cl]^-Calcd.450.1325) suggests that the molecular formula is C₂₂H₂₅NO₇. NMR spectra (Table 1) showed similarity to compound 4, but with one less hydroxyl group at the C-9 position. Nuclear magnetic resonance two-dimensional spectroscopy HMBC and¹H-¹h COSY determines the planar structure of compound 1 (6-hydroxy-14-p-nitrobenzoylcinnamylolide). two-dimensional nuclear magnetic resonance spectroscopy NOESY determines that the relative spatial configurations of C-4, C-5, C-6, C-9, and C-10 of compound 1 are also consistent with that of compound 4. the optical rotation data of compound 1 ([ α]²⁰ _D136.8 deg., c 0.1 MeOH) and round two chromatograms were consistent with Compound 4(OR: [ α ]]²⁰ _D-99.3 °, c 0.3, MeOH) or similar, and accordingly, the absolute configuration of compound 1 is identical to that of compound 4. Compound 1 was named as insulicolide B.

High resolution Mass Spectrometry HRESIMS (m/z 508.1383for C) of Compound 2₂₄H₂₇NO₉Cl[M+Cl]^-Calcd.508.1380) suggests a molecular formula C₂₄H₂₇NO₉NMR one-dimensional and two-dimensional data (Table 1, FIG. 1) determined Compound 2 to be the acetylation product of Compound 5 at the C-14 OH, NOESY (FIG. 2), optical rotation data ([ α ]]²⁰ _D274.8 °, c 0.084, MeOH) and round dichroism (fig. 3) determined its absolute spatial configuration. The compound 2 is named as 14-acetoxy insulicilolide A (2), and is found to be a novel natural product.

High resolution Mass Spectrometry HRESIMS (m/z 492.1428for C) of Compound 3₂₄H₂₇NO₈Cl[M+Cl]^-Calcd.492.1431) suggests a molecular formula of C₂₄H₂₇NO₈NMR one-dimensional and two-dimensional data (Table 1) confirm that Compound 3 is an acetylated insulicolide A with dehydroxy at C-9 and OH at C-14, designated simultaneously as insulicolide C, a new structural compound, same NOESY (FIG. 2), optical rotation data ([ α)]²⁰ _D60.7 °, c 0.084, MeOH) and round dichroism (fig. 3) determined its absolute spatial configuration. The 6 nitrophenyl ester sesquiterpenoids in the invention all have the same absolute spatial configuration.

Example 2 inhibitory Activity of Nitrophenyl ester sesquiterpenes on triple lines of renal carcinoma cells

Three human renal cancer cell lines were ordered in shanghai cell resource center of chinese academy of sciences: 786-O human renal cancer cell line (Cat # TCTU 186); ACHN human renal cancer cell line (Cat # TCHu 199); an OS-RC-2 human renal cancer cell line (Cat # TCTU 40).

The kidney cancer cell inhibition activity experiment adopts a CCK-8 detection method. Cells in the logarithmic growth phase were collected, counted, resuspended in complete medium, adjusted to the appropriate concentration (as determined by the cell density optimization assay) and seeded into 96-well plates with 100. mu.l of cell suspension per well. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 24 hours. The test compound was diluted with the medium to the appropriate working concentration and the cells were added at 25. mu.l/well. The effect concentrations of the nitrophenyl ester sesquiterpene compound and the positive control (Sorafenib) were started at 100 μ M, diluted in a 4-fold gradient, at 9 concentration points. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 72 hours. The medium was aspirated off, and fresh complete medium containing 10% CCK-8 was added and incubated in an incubator at 37 ℃ for 2-4 hours. After gentle shaking, the absorbance at 450nm was measured on a SpectraMax M5 MicroplateReader, and the inhibition was calculated using the absorbance at 650nm as a reference.

The inhibition of cancer cell growth by the compound was calculated as follows:

the cancer cell growth inhibition rate ═ [ (Ac-As)/(Ac-Ab) ] × 100%;

wherein As is the absorbance OA of the sample (cell + CCK-8+ test compound); ac absorbance OA of negative control (cell + CCK-8+ DMSO); ab absorbance OA of positive control (Medium + CCK-8+ DMSO).

IC was performed using software Graphpad Prism 5 and using the calculation formula log (inhibitor) vs₅₀Curve fitting and calculating IC₅₀The value is obtained.

The proliferation inhibition effect of the nitrophenyl sesquiterpenoids 1-6 on the three renal cancer cell lines is shown in Table 2.

TABLE 2 proliferation inhibitory Effect (IC) of nitrophenyl ester sesquiterpene Compounds 1-6 on three renal cancer cell lines₅₀,μM)

By evaluating the in vitro anti-renal cancer activity of the 6 nitrophenyl sesquiterpenoids (FIG. 4), it was found that all the nitrophenyl sesquiterpenoids had inhibitory activities (IC) of different intensities on human renal cancer OS-RC-2 cells, human suprarenal adenocarcinoma ACHN cells and human renal clear cell adenocarcinoma 786-O cells₅₀0.89-30.35 mu M); wherein the compound inulicolide A (compound 5) has the strongest inhibitory activity (IC) on three renal cancer cells₅₀0.89-1.46 mu M), 14-Acetoxy insulicicolide A (compound 2) has the same obvious inhibitory activity (IC)₅₀2.3-5.3 mu M), which is equivalent to the drug Sorafenib (IC) for kidney cancer in the market₅₀3.4 to 7.0 μ M). The inhibitory activity of nitrophenyl sesquiterpenoid compounds, namely, inulicolide B (compound 1), inulicolide C (compound 3) and 9-dehydroxy inulicolide A (compound 6), on three renal cancer cells is relatively weak (IC)₅₀11.18-30.35 mu M), and the hydroxyl at the C-9 position is possibly an active group for inhibiting renal cancer cells. In addition, the nitrophenyl ester sesquiterpenoids 14-acetoxy insulicolide A (compound 2) and 9-hydroxy insulicolide B (compound 4) human renal clear cell adenocarcinoma 786-O cells have certain selectivity (IC)₅₀2.30 and 4.30. mu.M, respectively), and p-OS-RC-2 (IC)₅₀5.30 and 8.21. mu.M, respectively) and ACHN (IC)₅₀4.10 and 11.18. mu.M, respectively) by a factor of 2-3.

Due to the selective inhibitory activity of the nitrophenyl sesquiterpenoid compound 14-acetoxy inulicolide A (compound 2) as a new natural product on 786-O cells, the invention further researches the blocking effect and the apoptosis inducing effect of the 14-acetoxy inulicolide A (compound 2) on the 786-O cell cycle under low toxic dose (1 and 2 mu M). FIGS. 5-7 show that the proportion of G0/G1 phase of cells increased from 44.95% to 54.16% 72 hours after 1 μ M of 14-acetoxy insulicolide A (Compound 2) was applied to 786-O cells. Thus, the effect of 14-acetoxy inulicolide A (Compound 2) on the 786-O cell cycle was arrested at G0/G1. The experiment for inducing apoptosis adopts Annexin V (AV) -FITC/Propidium Iodide (PI) double staining and then flow cytometry analysis, and fig. 8-10 show that 14-acetoxy insulinicide A (compound 2) basically has no apoptosis inducing effect on 786-O cells after 24 hours of action, but has a certain inducing effect on late apoptosis (AV +/PI +) of 786-O cells after 72 hours of action.

Therefore, the nitrophenyl ester sesquiterpenoids represented by 14-acetoxy insulicolide A (compound 2) can be used as lead compounds for development of anti-renal cancer (renal clear cell adenocarcinoma) drugs.

Appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and guidance of the specification. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A nitrophenyl ester sesquiterpenoids is characterized in that the structural formula is shown as a formula (I),

wherein R is₁When is H, R₂Is NB, R₃Is H; or

R₁When it is OH, R₂Is CH3CO, R₃Is NB; or

R₁When is H, R₂Is CH3CO, R₃Is NB; or

R₁When is H, R₂Is H, R₃Is NB;

the structural formula of the NB is shown as a formula (II):

2. the use of the nitrophenyl ester sesquiterpenoids and pharmaceutically acceptable salts thereof according to claim 1, in the preparation of anti-renal cancer drugs.

3. An anti-renal cancer drug, comprising an effective amount of the nitrophenyl ester sesquiterpene compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

4. Use of a marine fungus (Aspergillus sp.) Jcma1F17 for the preparation of a nitrophenyl ester sesquiterpene compound according to claim 1.