TWI642665B - Separation and identification on stereoisomers of manoalide derivatives and their configuration-depending anti-leukemic and anti-lymphomatoid effects - Google Patents

Abstract

The invention discloses an anticancer manoalide compound extracted by a sponge animal, a pharmaceutical composition thereof, a preparation method and a configuration identification method. The compound has the structural formula of formula I: Wherein R is CH ₃ , and the combination of C-24 and C-25 is 24 R , 25 R , 24 R , 25 S or 24 S , 25 S . The compound of the formula I is obtained by extracting the sponge animal with ethyl acetate, concentrating the ethyl acetate extract to obtain a residue, chromatography the residue to obtain a plurality of fractions, and separating by gel column chromatography and reversed phase high performance liquid chromatography. obtain. The proton chemical shift of the compound of formula I is compared to the standard to determine the configuration of the compound of formula I.

Description

Isolation and identification of Manoalide derivative isomers, and their configuration depends on anti-blood cancer and anti-lymphoma effects

The present invention relates to a compound, a pharmaceutical composition thereof, a preparation method and a method for identifying a configuration. In particular, the present invention relates to an anticancer manoalide compound extracted from a sponge animal, a pharmaceutical composition thereof, a preparation method, and a configuration identification method.

Scientists have found compounds that can be applied to the medical industry from marine life. For example, Cytarabine (Ara-C, trade name: Cytosal ^® injection) was developed by the first US Food and Drug Administration and was derived from the sponge animal Tethya crypta , which was developed. For the treatment of acute myeloid leukemia (AML) by targeting PDA polymerase. For example, Eribulin Mesylate (E7389, trade name: Halaven ^® injection) from black soft sponge ( Halichondria okadai ) blocks the G ₂ /M cell cycle and destroys mitotic spindles, prolonging mitotic resistance After the break, it eventually causes cancer cells to apoptosis. For example, Brentuximab vedotin (SGN-35, trade name: Adcetris ^® frozen crystal injection) from the shellless mollusk, Dolabella auricularia , is used to treat Hodgkin's lymphoma ( Hodgkin's lymphoma). For example, ecteinascidin 743 (ET-743, also known as Trabectedin) from the sea squirt ( Ecteinascidia turbinata ) is used to treat malignant soft tissue malignancies. Therefore, the search for novel compounds from marine organisms has become a new opportunity for drug development.

In view of the deficiencies in the prior art, the applicant of this case, after careful experimentation and research, and a perseverance spirit, finally conceived the case and can overcome the shortcomings of the prior art. The following is a brief description of the case.

In order to develop novel compounds and drugs, the present invention discloses an anticancer manoalide compound extracted from a sponge animal Luffariella sp., a pharmaceutical composition thereof, a preparation method and a configuration identification method. Furthermore, the present invention discloses a method for identifying the R or S configuration of a compound or derivative of a manoalide in a product or pharmaceutical composition to determine the composition of a manoalide compound of a particular configuration in the product or pharmaceutical composition and/or Or proportion.

According to one aspect of the invention, a pharmaceutical composition for treating cancer comprising a compound of the first formula I is disclosed: Wherein R is CH ₃ and the combination of the carbon position of the 24th carbon position and the 25th carbon position is selected from the group consisting of 24 R , 25 R , 24 R , 25 S and 24 S , 25 S .

In a specific embodiment, the pharmaceutical composition further comprises a second compound of formula I, wherein R is H, and the combination of the 24th carbon position and the 25th carbon position is 24 R , 25 R and 24 R One of the 25 S.

In a specific embodiment, the pharmaceutical composition further comprises at least one compound of formula II: Wherein, when R is H, the configuration of the carbon number of the 24th carbon position and the 25th carbon position is one of 24 R , 25 R and 24 R , 25 S ; and when R is CH ₃ , the 24th The configuration of the carbon position and the carbon number of the 25th position is one of 24 R , 25 R and 24 R , 25 S.

In a specific embodiment, the pharmaceutical composition further comprises a compound of formula III:

In one embodiment, the cancer is blood cancer or lymphoma, and the cancer cells involved in blood cancer include, but are not limited to, human acute lymphoblastic leukemia cell line Molt 4 or human chronic myeloid leukemia cell line K562, human promyelocytic leukemia cell line HL 60 and the like, cancer cells involved in lymphoma include, but are not limited to, cell strains Sup-T1, U937, and the like. The therapeutic mechanism of blood cancer or lymphoma is related to the catalytic inhibitory activity of topoisomerase II.

According to one aspect of the present invention, there is disclosed a method of preparing a compound for anticancer, which has the structural formula shown by the above formula I, wherein R is CH ₃ and the carbon position No. 24 and the carbon position No. 25 The configuration combination is selected from the group consisting of 24 R , 25 R , 24 R , 25 S and 24 S , 25 S , the method comprising: (a) providing a sponge animal Luffariella sp.; (b) The sponge animal was extracted with ethyl acetate to obtain an ethyl acetate extract; (c) the ethyl acetate extract was concentrated to obtain a residue; (d) n-hexane, n-hexane-ethyl acetate Mixing the residue with acetone and acetone to obtain a plurality of fractions; and (e) sequentially passing the plurality of fractions through a gel column chromatography and reversed-phase high performance liquid chromatography to obtain the formula I Compound.

In a specific embodiment, the sponge animal is Luffariella variabilis . However, the sponge animal Luffariella sp. includes, but is not limited to, Luffariella variabilis .

According to one aspect of the present invention, a method for identifying a configuration and a ratio of a manoalide compound having a structural formula represented by the above formula I, the method comprising: (a) aligning a manoalide compound with a first standard is disclosed. Product 24 S - O- methylmanoalide, second standard 24 R- manoalide or third standard 24 R - O- methylmanoalide proton chemical shift signal to determine the configuration of the 24th carbon position of the manoalide compound as R Or S ; (b) Align the signal of the proton chemical shift of the manoalide compound with the fourth standard 25 R -manoalide diacetate and a fifth standard 25 S -manoalide diacetate to determine the 25th carbon position of the manoalide compound. The configuration is R or S ; and (c) the ratio of the integrated area of the peak of the manoalide compound to a quantified internal standard in the reverse phase HPLC spectrum to determine the content and ratio of the manoalide compound.

In a specific embodiment, the configuration of the carbon position No. 24 is further determined by detecting the NOESY correlation between H-4/H24 and H-4/H-24-OMe, and No. 25 The configuration of the carbon position is further determined by detecting the NOESY correlation between H-4/H25.

Based on the foregoing teachings of the present invention, the techniques of the present invention are applicable to methods for identifying the R or S configuration of a compound or derivative of a manoalide in a product or pharmaceutical composition to determine a particular configuration in the product or pharmaceutical composition. The composition and/or proportion of the manoalide compound.

The term "manoalide", "manoalide compound", "manoalide derivative", "manoalide compound" or "manoalide sesquiterpenoid" as used herein refers to a compound having a manoalide structure.

The term "mixture" or "solution" as used herein refers to a mixture or solution that combines two or more solutions by volume. Thus, the term "n-hexane-ethyl acetate (v/v) mixture" as used herein refers to a mixture of n-hexane and ethyl acetate in a volume ratio. It will be understood by those of ordinary skill in the art that other volume ratio blends or solutions may be employed in the present invention and fall within the scope of the patent application.

The above objects and advantages of the present invention will become more apparent to those skilled in the <RTIgt;

Fig. 1 is a schematic view showing the western spotting of the expression of apoptosis-related proteins of Molt 4 cells treated with Compound 7 in the examples of the present invention.

Fig. 2(A) is a diagram showing the effect of Compound 7 on the relaxation of Topo IIα-regulated supercoiled pHOT1 plastid DNA in the examples of the present invention. Lanes 1 to 5: Compound 7 (0.078, 0.15625, 0.3125, 0.625, and 1.25 μg/mL); Lane 6: positive control group etoposide (500 μM) as topo II poison (induced linear DNA); : linear DNA; lane 8: negative control group plastid DNA (supercoiled DNA); lane 9: plastid DNA + topo IIα (induced DNA relaxation).

Fig. 2(B) is a schematic view showing the effect of the compound 7 on Topo I activity in the examples of the present invention. Lanes 1 to 6: Compound 7 (0.078, 0.15625, 0.3125, 0.625, 1.25, and 2.5 μg/mL); Lane 7: negative control group plastid DNA (supercoiled DNA); lane 8: relaxed DNA; Lane 9: plastid DNA + topo IIα (induced DNA relaxation); lane 10: plastid DNA + topo IIα + solvent control group (induced DNA relaxation).

Fig. 2(C) is a schematic view showing the western spotting of phosphorylation of proteins ATM, Chk 2 and H2A.X which are affected by compound 7 in the examples of the present invention.

Figure 3 is a graph showing the effect of Compound 7 on tumor growth in an in vivo animal model in the examples of the present invention. The control group was PBS and the experimental group was Compound 7. "*" indicates a significant difference ( p < 0.001) compared with the control group.

The inventions set forth in the present invention will be fully understood by the following examples, so that those skilled in the art can do so. However, the implementation of the present invention may not be limited by the following embodiments. Other embodiments may be devised by those skilled in the art in light of the spirit of the embodiments disclosed herein.

experimental method:

Extraction of animal materials and compounds:

The sample of the sponge animal Luffariella sp. was collected from scuba diving in the depth of 10 meters from the back bay of Pingtung, Taiwan in September 2012. The guaranteed sample of the sponge animal was deposited at the National Museum of Marine Biology, Taiwan (sample no. 2012-09-SP). The classification and identification of the sponge animals were carried out by the National Museum of Marine Biology, Taiwan. Since the sponge animal Luffariella sp. can be easily obtained and identified by those having ordinary knowledge in the art, there is no need to deposit biomaterials in accordance with the relevant provisions of the Patent Law and its implementation rules. In a particular embodiment of the invention, the sponge animal Luffariella sp. includes, but is not limited to, Luffariella variabilis , as understood by those of ordinary skill in the art, and specific embodiments of the present invention can be performed on any sponge animal of Luffariella sp. and the results obtained.

The sponge animal (790 g fresh weight) was collected and lyophilized. The lyophilized sponge animal (207 g, dry weight) was chopped and thoroughly extracted with ethyl acetate (EtOAc) to give ethyl acetate extract. The ethyl acetate extract was concentrated by evaporation under reduced pressure to give a residue (8 g), then n-hexane ( n -hexane), n-hexane-ethyl acetate (v/v) mixture, and finally Pure residue of this residue was subjected to gel column chromatography to obtain 12 fractions: L1 (extracted with n- hexane), L2 (extracted with n- hexane: EtOAc = 100:1), L3 (with n - Hexane: EtOAc = 50:1), L4 (extracted with n- hexane:EtOAc = 20:1), L5 (extracted with n- hexane:EtOAc = 10:1), L6 (with n- hexane: EtOAc = 5:1 chlorination), L7 (extracted with n- hexane:EtOAc = 3:1), L8 (extracted with n- hexane:EtOAc = 2:1), L9 ( n- hexane:EtOAc = 1:1 extraction, L10 (extracted with n- hexane: EtOAc = 1:2), L11 (purified with EtOAc) and L12 (purified with acetone). The fraction L8 was further purified by silica gel column chromatography (extracted with n- hexane:EtOAc = 3:1 to 1:1) to obtain 10 fractions (L8-1 to L8-10). The fractional fraction L8-7 was subjected to reverse-phase high performance liquid chromatography (MeOH: H ₂ O=92:8 (v/v), 0.1% acetic acid (v/v) in water) to obtain Compound 4 (32.7 mg, retention time 42.8 minutes), Compound 5 (9.9 mg, retention time 37.8 minutes) and Compound 9 (19.3 mg, retention time 44.8 minutes). The fraction fraction L8-9 was also separated by reverse phase HPLC (MeOH: H ₂ O = 90:10 (v/v), 0.1% acetic acid (v/v) in water) to give compound 2 (2.5 mg, retention time 35.9) Minutes), Compound 3 (8.7 mg, retention time 64.5 minutes), Compound 7 (1.6 mg, retention time 37.7 minutes) and Compound 8 (5.8 mg, retention time 67.9 minutes). Separator fraction L8-10 was obtained by reverse-phase HPLC (MeOH: H ₂ O = 90:10 (v/v) in water containing 0.1% acetic acid (v/v)) to obtain compound 1 (4.8 mg, retention time 33.0 minutes) ), Compound 6 (3.2 mg, retention time 31.7 minutes) and Compound 10 (19.2 mg, retention time 33, 9 minutes). Further, the content and ratio of the manoalide compound are detected by reversed-phase HPLC quantitative analysis, and the appropriate concentration of the internal standard (5, 10, 20, 40, 80 μg/mL) is firstly set, and then the standard solution of different internal standards is taken. 10 μL of the injected HPLC-PDA was analyzed with three repetitions (n=3), the average absorption peak area (peak area) was taken as the Y-axis, the concentration was plotted on the X-axis, and the linear regression equation of the standard curve was obtained (y= Ax+b), which in turn establishes a calibration curve for internal standards. In the reversed-phase HPLC spectrum, the ratio of the integrated area of the peak of the manoalide compound to the quantified internal standard is calculated, and the concentration is calculated by the established calibration curve to determine the content and ratio of the manoalide compound.

Compound 1 (24 R , 25 R -Luffariellin A): a colorless oily substance; +70.6 (c 0.1, MeOH); IR (neat) v _max 3481, 2988, 1749 and 1645 cm ^-1 ; ¹ H NMR data, see Table 1; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m/z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 2 (24 R , 25 S -Luffariellin A): a colorless oily substance; +31.0 (c 0.1, MeOH); IR (neat) v _max 3384, 2954, 2877, 1758 and 1658 cm ^-1 ; ¹ H NMR data, see Table 1; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m /z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O 5 Na, 523.3399).

Compound 3 (24 R - O -Methyl-25 R -luffariellin A): a colorless oily substance; -14.5 ( c 0.1, MeOH): IR (neat) v _max 3443, 2955, 1744 and 1648 cm ^-1 ; ¹ H NMR data, see Table 1; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m/z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 4 (24 R - O -Methyl-25 S -luffariellin A): a colorless oily substance; -15.2 ( c 0.1, MeOH); IR (neat) v _max 3411, 2954, 2874, 1759 and 1640 cm ^-1 ; ¹ H NMR data, see Table 1; ESIMS m/z 537 [M+Na] ⁺ ; HRESIMS m /z 537.3546 [M+Na] ⁺ (calculated as C ₃₂ H ₅₀ O ₅ Na, 537.3550).

Compound 5 (24 S - O -Methyl-25 S -luffariellin A): a colorless oily substance; -10.7 ( c 0.1, MeOH); IR (neat) v _max 3450, 2968, 2868, 1742 and 1650 cm ^-1 ; ¹ H NMR data, see Table 1; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m /z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 6 (24 R , 25 R -Manoalide): a colorless oily substance; +27.0 ( c 0.1, MeOH); IR (neat) v _max 3414, 2958 and 1756 cm ^-1 ; ¹ H NMR data, see Table 2; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m/z 523.3397 [ M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 7 (24 R , 25 S -Manoalide): a colorless oily substance; +79.4 (c 0.1, MeOH); IR (neat) v _max 3403, 2888 and 1758 cm- ¹ ; ¹ H NMR data, see Table 2; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m/z 523.3397 [ M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 8 (24 R - O -Methyl-25 R -manoalide): a colorless oily substance; +56.8 (c 0.1, MeOH); IR (neat) v _max 3444, 2969, 1761 and 1650 cm ^-1 ; ¹ H NMR data, see Table 2; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m/z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 9 (24 R - O -Methyl-25 S -manoalide); colorless oily substance; +38.7 (c 0.1, MeOH); IR (neat) v _max 3384, 2959, 2886, 1761 and 1658 cm ^-1 ; ¹ H NMR data, see Table 2; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m /z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Compound 10 (24 R , 25 S -Thorectolide): a colorless oily substance; +12.6 (c 0.1, MeOH); IR (neat) v _max 3421, 2936, 1754 and 1651 cm ^-1 ; ¹ H NMR data, see Table 2; ESIMS m/z 523 [M+Na] ⁺ ; HRESIMS m/z 523.3397 [M+Na] ⁺ (calculated as C ₃₁ H ₄₈ O ₅ Na, 523.3399).

Annexin V/propidium iodide (PI) apoptosis test:

The annexin V-FITC staining kit was used to quantify the manifestation and membrane integrity of phospholipid fibroin (PS) (Su et al., Marine Drugs, 2013, 11(9): 3168-3185.). Briefly, 10 ⁶ cells were cultured in 35 mm diameter plates and cells were labeled with annexin V-FITC (10 μg/mL) and PI (20 μg/mL) before cell collection. After labeling, all plates were washed with binding buffer and cells were harvested. The cells were resuspended in binding buffer at a concentration of 2 x 10 ⁵ cells/mL and analyzed by flow cytometry FACS-Calibur (Becton-Dickinson, San Jose, CA, USA) and CellQuest software. Approximately 10,000 cells were counted per assay.

Determination of active oxygen species (ROS) production, calcium accumulation and MMP destruction:

These experiments were carried out in accordance with the description of the literature (Lai et al., Sci. Rep. 2016, 6: 36170.). JC-1 cationic dye (5 μg/mL), fluorescent calcium ion indicator (Fluo 3, 5 mM) and luciferin carboxyl derivative (carboxy-H ₂ DCFDA, 1.0 mM) were purchased from Molecular Probes and Invitrogen technologies (Carlsbad, CA, USA)) detect MMP destruction, calcium accumulation, and ROS production. Briefly, treated cells were labeled with a specific fluorescent stain for 30 minutes. After labeling, the cells were washed with PBS and suspended in PBS at a concentration of 1 x 10 ⁶ cells/mL prior to flow cytometric analysis.

Topoisomerase II catalytic inhibitor and toxicant test:

This test was carried out in accordance with the description of the literature (Lai et al., Sci. Rep. 2016, 6: 36170.). Contains 50 mM Tris-HCl (pH 8.0), 10 mM MgCl ₂ , 200 mM potassium glutamate, 10 mM dithiothreitol, 50 μg/mL bovine serum albumin (BSA), 1 mM ATP, 0.3 μg pHOT1 plastid DNA, 2 units A standard relaxation reaction mixture (20 μL) of human topoisomerase II (Topogen, Columbus, OH, USA) and a specific concentration of etoposide was incubated with compounds 1 to 10 for 30 minutes at 37 °C. 2 μL of 10% SDS was added to stop the reaction to help capture the enzyme in the cleavage complex, followed by the addition of 2.5 μL of Proteinase K (50 μg/mL) to digest the bound protein (cultured at 37 ° C for 15 minutes) and finally added A 0.1 volume sample is filled with the dye. The DNA product was analyzed by electrophoresis in a 0.5 x TAE buffer at 2 voltage/cm vertical type 2% agar extract. The colloid was stained with ethidium bromide (EtBr) and the colloid was photographed using the Eagle Eye II system (Stratagene, La Jolla, CA, USA). Quantitative analysis of DNA Topo II activity. The concentration of compound inhibiting Topo II activity (IC ₅₀₎ to scan directly colloid image analyzer, and for calculating a supercoiled DNA regions resulting in an estimated 50%.

Neutral comet assay for detecting DNA double strand breaks (DSB):

This test is used CometAssay ^TM kit (Trevigen, Gaithersburg, MD, USA ), according to the manufacturer's manual neutral comet tests performed. Briefly, cancer cells (2 x 10 ⁵ cells/mL) were treated with Compound 7 (0.0625 μg/mL) for a specific period of time. Cells with 1% low melting agar sugar at a ratio of 1:10 (v / v) were combined, and immediately pipetted onto a mixture of 75μL CometSlide ^TM and the dark at 4 ℃. The slides were flooded with ice-cold lysis solution (Trevigen) for 30 to 60 minutes. Slides were placed in a horizontal electrophoresis apparatus and electrophoresed in 20 volts in 1x TBE buffer (90 mM Tris-HCl, 90 mM boric acid and 2 mM EDTA (pH 8.0)) for 10 minutes. The samples were then fixed in 70% alcohol and air dried and stained with 1:10,000 SYBR Green I (Trevigen) to allow observation of cellular DNA. Use the TriTek Comet Image software to analyze the fluorescence image to circle the "head" and "tail" areas of each comet and record the integrated fluorescence value for each defined area. The length of the comet from the trailing edge of the core to the leading edge of the tail is measured. This length indicates the extent of DNA damage. The calculation is averaged per repetition. Results are expressed as mean ± standard deviation of three independent experiments (* p < 0.05).

Immunofluorescence analysis:

After treating the cells with the compound, the cells were fixed with 4% polyoxymethylene in 50 mM HEPES buffer (pH 7.3) for 30 minutes, and covered with 0.2% Trition X-100 in PBS (pH 7.4) for 20 minutes. To avoid non-specific protein binding, cells were treated with 5% BSA in PBS (containing 0.05% Trition X-100) (T-PBS) for 1 hour at room temperature. The cells were then incubated with the primary antibody Hsp70 (1:500) for 2 hours at room temperature, followed by a secondary antibody (Alexa Fluor 586-coupled goat anti-mouse IgG (H+L) (Life Technologies, Carlsbad, CA, USA) )) (1:1000) culture for 1 hour. After washing the cells with PBS, the cells were observed on a FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Human blood cancer Molt 4 cell xenograft animal model:

Six-week-old immunodeficient athymic male mice were purchased from the National Laboratory Animal and Animal Research Center in Taipei, Taiwan. All animals were cared for under standard laboratory conditions (temperature 24-26 ° C, 12 hours - 12 hours dark - bright cycle) and were fed with laboratory feed and water. Molt 4 cells (1 x 10 ⁶ cells) suspended in 0.2 mL of PBS were injected subcutaneously into the right flank, and tumor growth was monitored daily. Mice that were confirmed to have tumor growth were randomly divided into two groups 14 days after tumor cell injection. Compound 7 (1 μg/g mouse body weight) was intraperitoneally injected into the treatment group, and only the solvent was injected into the control group mice. Compound 7 was injected every other day for 33 days. Sacrifice mice with carbon dioxide. The tumor size was measured three times per week using a caliper and the tumor volume was calculated according to the standard formula "width ² x length/2".

statistics:

Results are expressed as mean ± standard deviation (SD). Comparisons of each experiment were performed using unpaired student t -tests, and a p- value of less than 0.05 was considered to be statistically significant.

Experimental results:

Chemical identification of Manoalide derivatives:

The ethyl acetate extract of the freeze-dried Luffariella sp. sample was treated with a normal phase chromatography system, and the fractionated fraction was separated by reverse phase HPLC to obtain a series of manoalide compounds and manoalide-like sesquiterpenoids ( As shown below, including 24 R , 25 R -luffariellin A (Compound 1 ), 24 R , 25 S -luffariellin A (Compound 2 ), 24 R - O -methyl-25 R -luffariellin A (Compound 3 ), 24 R - O -methyl-25 S -luffariellin A (compound 4 ), 24 S - O -methyl-25 S -luffariellin A (compound 5 ), 24 R , 25 R -manoalide (compound 6 ), 24 R , 25 S -manoalide (compound 7 ), 24 R - O -methyl-25 R -manoalide (compound 8 ), 24 R - O -methyl-25 S -manoalide (compound 9 ) and 24 R , 25 S -thorectolide (compound 10 ) . Among them, compounds 1 , 2 , 6 and 7 were isolated and purified for the first time and structurally identified, and compounds 3 , 4 and 5 were novel compounds.

The planar structures of compounds 1 , 2 and 6 to 10 were confirmed by electron impact mass spectrometry (EIMS) and high resolution electrospray ionization mass spectrometry (HRESIMS) data, and ¹ H nuclear magnetic resonance ( ¹ H NMR) spectra (Table 1 and Table 2) Characteristic signals and literature (Dilipdesilva et al., Tetrahedrin Letters, 1980, 21 (17): 1611-1614., Soriente et al., Eur. J. Org. Chem. 2000, 2000 (6): 947 -953., Namikoshi et al., Fishery Sci. 2004, 70(1): 152-158. and Gauvin-Bialecki et al. Molecules, 2008, 13(12): 3184-3191.) Contents were compared. The stereostructure of these compounds was further determined. The axial nature of all compound No. 4 carbon (C-4) is derived from its large range coupling constant of 6.4 to 11.2 Hz and the C-5 proton deduction of C-4 indicating the R configuration.

Taking compound 5 as an example, by chemically shifting its protons (CDCl ₃ at 400 MHz: δ _H 4.66 (H-4), 5.71 (H-6), 5.06 (H-24)) with previously established standards Proton chemical shift of product 24 S - O- methylmanoalide (CDCl ₃ at 600 MHz: δ _H 4.67 (H-4), 5.73 (H-6), 5.03/5.11 (H-24)) (Namikoshi et al., Fisheries Sci. 2004, 70(1): 152-158.) An alignment was made to determine the relative configuration of C-24 as S. On the other hand, in addition to the C-24 of compound 5 being in the S configuration (i.e., 24 S ), by proton chemical shifting of other compounds to the standard 24 R -manoalide (Dilipdesilva et al., Tetrahedrin Letters, 1980, 21 (17): 1611-1614.) and the standard product 24 R - O - methylmanoalide (Namilkoshi et al., Fisheries Sci. 2004, 70 (1): 152-158.) proton chemical shift for comparison , confirm that all other compounds are in the 24 R configuration (compounds 1 to 4 and 6 to 10 (CDCl ₃ at 400 MHz): δ _H 4.76-4.86 (H-4), 5.66-5.69 (H-6), 5.29-5.30 Or 4.78-4.80 (H-24); 24 R -manoalide (CDCl ₃ ): δ _H 4.85 (H-4), 5.69 (H-6), 5.32 (H-24); 24 R - O -methylmanoalide (CDCl ₃ , at 600 MHz): δ _H 4.76/4.83 (H-4), 5, 68 (H-6), 4.79/4.82 (H-24)). The C-24 configuration of compounds 3 , 4 and 5 was further detected by detecting H-4/H-24 (present in compound 5 ) and H-4/H-24-OMe (appearing in compounds 3 and 4 ) Determined between the NOESY correlations.

To determine the configuration of C-25, the ¹ H NMR proton chemical shift of the compound was compared to the standard of the literature (Soriente et al., European Journal of Organic Chemistry, 2000, 6: 947-953.) 25 R -manoalide diacetate The proton chemical shift of the standard 25 S- manoalide diacetate was compared. A compound having a 25 R configuration has a lower chemical shift than a compound having a 24 S configuration. Thus, compounds 1 , 3 , 6, and 8 were confirmed to be in the 25 R configuration, and compounds 2 , 4 , 5 , 7 , 9, and 10 were confirmed to be in the 25 S configuration. The 25 S configuration of compounds 4 and 5 was determined by detecting the NOESY correlation between H-4/H-25.

The configuration of the stereoisomers of the invention depends on anti-blood cancer efficacy:

There are no prior art disclosures of the C-24 and C-25 configurations of the stereoisomers of the invention and their biological activities. In the present invention, cell proliferation of the cancer cell lines (Molt 4, K562) and lymphoma cell lines (Sup-T1 and U937) after treatment with Compounds 1 to 10 for 72 hours was affected by the MTT assay. See Table 3, which manoalide stereoisomers ₅₀ values for different IC blood cancer cell lines of the present invention. 24 R, 25 S - derivative (Compound 2, 4, 7 and 9) IC for most of leukemia and lymphoma cell line ₅₀ value of less than 1μg / mL, the cells show potential issued poisonous activity. After treatment for 72 hours, Compound 2 (IC ₅₀ : 0.41, 1.8, 1.55, and 0.21 μg/mL), 4 (IC ₅₀ : 0.22) against Molt 4, K562, Sup-T1, and U937 cells compared to other compounds. , 1.92, 0.5 and 2.5 μg/mL), 7 (IC ₅₀ : 0.34, 3.19, 0.56 and 0.65 μg/mL) and 9 (IC ₅₀ : 0.29, 3.22, 1.48 and 0.52 μg/mL) show lower IC ₅₀ value. According to the results of Table 3, 24 R and 25 S have 2.1 to 47.6 times better activity against anti-blood cancer and anti-lymphoma in manoalide and its derivatives than other configurations, so it is confirmed that the 24 R and 25 S configurations are "Key Active Configuration".

^a NA (non-active) = 72 hours IC ₅₀ >20 μg/mL

^b positive control group

Compound 7 regulates apoptosis induction via DNA damage and MMP destruction:

To determine whether cytotoxicity of compound 7 is related to apoptosis associated with granulosa, the number of apoptosis and granulosa in membranes in Molt 4 cells were stained with annexin-V/PI, DAPI and JC-1. Potential distribution. After 24 hours, Compound 7 (0, 0.15625, 0.3125, and 0.625 μg/mL) caused an increase in the number of apoptosis of Molt 4 cells in a dose-dependent manner (36.4%, 66%, and 98.7%). To assess changes in karyotypes caused by Compound 7 , Molt 4 cells were further examined by DAPI staining and observed under a fluorescent microscope. Compound 7 increased the number of concentrated nuclei compared to the control group showing intact and normal nuclei (results not shown). To assess whether the apoptosis induction of Compound 7 is related to the granulocyte pathway, Rose Bengal 123 stimulator was used to determine the granulosa membrane potential. Molt 4 cells were treated with 0.3125 μg/mL of Compound 7 for 1, 2, 3, 4, 6 and 20 hours with Rose Bengal 123 fluorescent dye and 2 ' ,7 ' -dichlorofluorescein diacetate. (2 ' , 7 ' -dichlorofluorescin diacetate, DCFH-DA) staining. Compound 7 (0.3125 μg/mL) increased the number of Molt 4 cells with disrupted membrane potential and ROS production in a time-dependent manner (results not shown). To determine whether Compound 7 has a cytotoxic effect on Molt 4 cells, cells were treated with Compound 7 for 24 hours, and apoptosis-related proteins (PARP, caspase 3, caspase 8 and caspase 9) were analyzed by Western blotting. Compound 7 resulted in PARP cleavage of Molt 4 cells at higher doses (0.3125 and 0.625 μg/mL). In the Molt 4 blood cancer cell line, Compound 7 caused activation of caspase 3, 8 and 9 in a dose-dependent manner (Fig. 1). These results demonstrate that the cytotoxic activity of Compound 7 is regulated by granulocyte destruction and ROS overproduction, leading to apoptosis induction of Molt 4 blood cancer cells.

In order to accurately determine the effect of compound 7 on topoisomerase I and topoisomerase II (Topo I and Topo II), super-helical pHOT1 using negatively regulated purified human protein (rather than calf protein) and enzyme Cell-free DNA fragmentation assay of plastid DNA was performed to confirm the actual efficacy of Compound 7 in Topo I and Topo II. In the Topo II assay, linear DNA was observed when supercoiled pHOT1 plastid DNA was treated with etoposide (a standard Topo II poison). Compound 7 at increasing concentrations (0.078, 0.15625, 0.3125, 0.625, and 1.25 μg/mL) inhibited DNA relaxation in a dose-dependent manner compared to control group supercoiled DNA, and resulted in the formation of supercoiled DNA in the presence of Topo IIα Product (Fig. 2 (A), lanes 1 to 5).

In the Topo I assay, as shown in Figure 2 (B), the lowest dose (0.078, 0.15625, 0.3125, and 0.625 μg/mL) of Compound 7 induced DNA relaxation in the presence of Topo I. Effect (lanes 1 to 4), but at higher doses (1.25 and 2.5 μg/mL), Compound 7 inhibited Topo I activity and converted supercoiled DNA into a relaxed form (lanes 5 to 6). Compound 7 in a dose-dependent manner (IC ₅₀ value of 0.75μg / mL) inhibition of relaxation of supercoiled DNA. Compound 7 at increasing concentrations (0.078, 0.15625, 0.3125, 0.625, 1.25, and 2.5 μg/mL) inhibited DNA relaxation in a dose-dependent manner compared to the control group's supercoiled DNA, and resulted in the formation of a supercoil in the presence of Topo I. DNA product (Fig. 2 (B), lanes 1 to 6). Further, as evidenced by cell-free systems, compound 7 respectively ₅₀ values IC 0.75 and 0.49μg / mL inhibiting Topo I and Topo II activity. These results demonstrate that Compound 7 acts as a dual inhibitor of Topo I and Topo II.

To confirm that the apoptotic mechanism of Compound 7 affects the induction of γH2AX (biomarker for DNA damage), Western blotting analysis and comet assay were used to determine the degree of DNA damage treated with Compound 7 . In western spotting analysis, protein ATM, Chk 2 and H2A.X phosphorylation increased significantly in a time-dependent manner (Fig. 2(C)). To confirm that Compound 7 induced DNA damage in Molt 4 cells, the effect of Compound 7 on the degree of nuclear DNA integrity at different time intervals (0, 2, 4, and 8 hours) was determined using a comet assay under neutral electrophoresis conditions. Compound 7 increased the degree of DNA migration of Molt 4 cells (results not shown). As indicated by the abnormal tail size of the comet assay, DNA migration is expressed by DSB induction in a time-dependent manner. These results indicate that Compound 7 acts as an effective catalytic inhibitor of Topo IIα.

Compound 7 inhibits tumor growth in vivo in a human Molt 4 tumor xenograft animal model:

Compounds 2 , 4 , 7 and 9 showed potent apoptosis for several cancer cell lines, wherein the antitumor activity effect of Compound 7 on tumor growth of human blood cancer cell Molt 4 in a xenograft animal model was evaluated. 1 × 10 ⁶ Molt 4 cells were subcutaneously inoculated into the right flank of immunodeficient athymic female mice. After 34 days of injection, tumor growth of Molt 4 cells was significantly inhibited under the influence of intraperitoneal injection of Compound 7 (1 μg/g). The mean tumor size of the control group on day 34 was 433.77 mm ³ , whereas the average tumor size in the compound 7 treatment group was 147.01 mm ³ (see Figure 3). The tumor size in the compound 7 treated group was significantly lower by 66.11% compared to the control group ( p < 0.05), but there was no significant difference in mouse body weight (results not shown). These results indicate that Compound 7 shows an anti-tumor effect in an in vivo xenograft mode.

in conclusion:

The novel compounds 3 , 4 to 5 , C-24 and C-25 configurations extracted by the sponge animal Luffariella sp., which are disclosed in the present invention, significantly affect their blood cancer (Molt 4 and K562 cells) and lymphoma (Sup-T1). And anti-proliferative activity of U937 cells). The 24R , 25S isomers (Compounds 2 , 4 , 7 and 9 ) showed the most potent anti-proliferative activity (IC ₅₀ value 0.29-3.22 μg/mL), and thus can be prepared into a pharmaceutical composition. Compound 7 is a potent Topo II catalytic inhibitor and anticancer drug, not a Topo II poison. The identification method disclosed herein may determine the R or S configuration of a compound or derivative of a manoalide in a product or pharmaceutical composition to further determine the composition and/or composition of a manoalide compound of a particular configuration in the product or pharmaceutical composition. proportion.

The invention is a difficult and innovative invention, and has profound industrial value, and is submitted in accordance with the law. In addition, the present invention may be modified by those skilled in the art without departing from the scope of the appended claims.

Claims (9)

A method of preparing a compound for anticancer, the compound having the structural formula shown in Formula I: Wherein R is one of H and CH ₃ , and the carbon position of the 24th carbon position and the 25th carbon position are 24 R , 25 S , and the method comprises: (a) providing a sponge animal Luffariella sp.; b) extracting the sponge animal with ethyl acetate to obtain an ethyl acetate extract; (c) concentrating the ethyl acetate extract to obtain a residue; (d) sequentially hexane, n-hexane-acetic acid Extracting the residue with ethyl acetate mixture and acetone to obtain a plurality of fractions; and (e) sequentially passing the plurality of fractions through a gel column chromatography and reversed phase high performance liquid chromatography to obtain the a compound of formula I. The method of claim 1, wherein the sponge animal is Luffariella variabilis . A method for identifying the configuration and ratio of a manoalide compound having a compound of formula I: Wherein R is one of H and CH ₃ , and the carbon position of the 24th carbon position and the 25th carbon position are 24 R , 25 S , and the method comprises: (a) comparing the manoalide compound with a first Standard 24 S - O- methylmanoalide, a second standard 24 R- manoalide or a third standard 24 R - O- methylmanoalide proton chemical shift signal to determine the No. 24 carbon position of the manoalide compound The configuration is R or S. If the ¹ H nuclear magnetic resonance spectrum signal of the No. 24 carbon position of the manoalide compound is between δ _H 4.70-4.85 or δ _H 5.20-5.35, the No. 24 of the manoalide compound is judged. The configuration of the carbon position is R. If the signal is between δ _H 5.00-5.15, the configuration of the carbon position of the manoalide compound is determined to be S ; and (b) the ratio of the manoalide compound to the first The signal of the proton chemical shift of the four standard 25 R -manoalide diacetate with a fifth standard 25 S -manoalide diacetate to determine the configuration of the 25th carbon position of the manoalide compound as R or S , if the manoalide The ¹ H NMR spectral signal of the 25th carbon position of the compound is between δ _H Between 6.35 and 6.55, the configuration of the No. 25 carbon position of the manoalide compound is judged to be R. If the signal is between δ _H 6.15-6.25, the configuration of the No. 25 carbon position of the manoalide compound is judged. is S; (c) in a first solvent and a second solvent manoalide the compound can reverse phase high performance liquid chromatography, and the residence time of determining the type of the compound of manoalide, wherein the first solvent and the second The solvent is a mixed solution of methanol and water, and the water contains 0.1% acetic acid (v/v), the ratio of methanol to water in the first solvent is 92:8 (v/v), and the second solvent is methanol and water. a ratio of 90:10 (v/v); and (d) configuring a standard solution of one internal standard at different concentrations, and injecting the standard solution of the internal standard into a reversed-phase high performance liquid chromatograph for analysis, according to The average absorption peak area and concentration of the standard solution are plotted. In the reversed-phase HPLC spectrum, the ratio of the peak integrated area of the manoalide compound to the integrated area of a quantified internal standard is established. The calibration line performs concentration conversion to determine the manoalide And the content ratio thereof. The method of claim 3, wherein the configuration of the carbon position No. 24 is further detected by detecting NOESY between H-4/H-24 and H-4/H-24-OMe. Correlation is determined. If the intersection of H-4/H-24 is measured, it is judged that the configuration of the carbon position of the manoalide compound is S , and if the intersection of H-4/H-24-OMe is measured, The configuration of the No. 24 carbon position of the manoalide compound is determined to be R ; and the configuration of the No. 25 carbon position is further determined by detecting the NOESY correlation between H-4/H-25, if measured At the intersection of H-4/H-25, it is judged that the configuration of the carbon position of the No. 25 carbon of the manoalide compound is S. A pharmaceutical composition for treating cancer comprising a compound of the first formula I: Wherein R is CH ₃ and the configuration of the No. 24 carbon position and the No. 25 carbon position is 24 R , 25 S . The pharmaceutical composition according to claim 5, further comprising a second compound of the formula I, wherein R is H, and the carbon position of the 24th carbon position and the 25th carbon position is 24 R , 25 S . . The pharmaceutical composition according to claim 5, further comprising at least one compound of formula II: Wherein R is one of H and CH ₃ , and the combination of the carbon number of the 24th carbon position and the 25th carbon position is 24 R , 25 S . The pharmaceutical composition according to any one of claims 5 to 7, wherein the cancer is blood cancer or lymphoma, and the therapeutic mechanism of the blood cancer or the lymphoma is related to topoisomerase II Catalytic inhibition activity. The pharmaceutical composition according to claim 8, wherein the cancer cells involved in the blood cancer are human acute lymphoblastic leukemia cell line Molt 4 or human chronic myeloid leukemia cell line K562, and the cancer cells involved in lymphoma are cell lines. Sup-T1 or U937.