COMPOUND ISOLATED FROM ISODON FORRESTII VAR. FORRESTII AND PREPARATION METHOD AND APPLICATIONS THEREOF

Abstract

A compound of Formula I or pharmaceutically acceptable salt thereof and its preparation method and applications, the new structure of the compound of formula I has not been reported in literature. It is isolated from Isodon forrestii var. forrestii and can be a compound served as Trx1 selective inhibitor. The present invention further discloses a pharmaceutical composition, preparation of the compound of

Formula I and its applications in preparing medicines for preventing or treating cancer. Iso A of the present invention has the advantages of low toxicity, high safety and strong pharmacological effect, which suggests a potential prospect in pharmaceutical applications.

Claims

1. A compound having Formula I is shown as following: ##STR00002## or the compound having Formula I as pharmaceutically acceptable salt.

2. A preparation method for the compound of Formula I of claim 1, characterized in that, it comprises steps as follows: (1) grinding the aerial parts of Isodon forrestii var. forrestii to powder; (2) soaking the plant powder acquired in step (1) in a mixture of a first organic solvent and water, and a leach solution is obtained by isolating the solid parts; (3) after the leach solution acquired in step (2) is distilled to remove the first organic solvent, a second organic solvent is added for extraction, and an extract is obtained after the organic phase is concentrated; (4) the extract from step (3) may be obtained by silica gel column chromatography and reverse phase silica gel column chromatography.

3. The preparation method according to claim 2, characterized in that, the Lamiaceae Isodon plant in step (1) is Isodon forrestii var. forrestii.

4. The preparation method according to claim 2, characterized in that, in step (2), the mass percentage of the first organic solvent and water in the mixture is 10-100%, the first organic solvent is selected from one or more of methanol, ethanol, chloroform, dichloromethane, petroleum ether or acetone; the soaking method is to soak for 1-3 times in room temperature, the total mass percentage of the plant powder and that soaked in the mixture is 1:1-1:5 kg/L.

5. The preparation method according to claim 2, characterized in that, in step (3), the second organic solvent is one or more of ethyl acetate, chloroform, ether, petroleum ether, the volume ratio of the second organic solvent and the mixture in step (2) is 1:1-1:5.

6. The preparation method according to claim 2, characterized in that, in step (4), the experimental conditions of the silica gel column chromatography are: the pore diameter of filler silica gel is 100-300 mesh, the eluent is a mixture of chloroform and acetone with volume ratio of 1:0-1, the amounts of the eluent is 3-6 times the total amounts of all samples plus silica gel; the experimental conditions of the reverse phase silica gel column chromatography are: the type of the reverse phase silica gel is RP-18, the eluent is a mixture of methanol and water or acetonitrile and water with volume ratio of 10%-100%, the amounts of the eluent is 3-6 times the amounts of used filler RP-18.

7. The preparation method according to claim 2, characterized in that, steps of forming salt with acid are further included.

8. A process for using the compound or pharmaceutically acceptable salt according to claim 1 for preventing and treating a tumor.

9. The process according to claim 8, characterized in that, the tumor is Trx1-mediated tumor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a structure view of Iso A;

[0028] FIG. 2 illustrates IC50 values of Iso A for various normal cells and tumor cells in MTT experiment;

[0029] FIG. 3 illustrates the inhibition of cellular growth on human normal hepatic cell line L02 and hepatocellular carcinoma cell line HepG2 after treated by various concentrations of Iso A treated for 24 hours through MTT assay;

[0030] FIG. 4 illustrates the survival results of human normal hepatic cell L02 and hepatocellular carcinoma cell HepG2 after treated by various concentrations of Iso A for 24 hours by Trypan Blue exclusion assay;

[0031] FIG. 5 illustrates the analyzed results of Annexin V after HepG2 cell treated by 10 or 20 M of Iso A for 24 hours;

[0032] FIG. 6 illustrates the in vitro inhibition of Trx1 activity by Iso A;

[0033] FIG. 7 illustrates activity analysis on Trx1 on HepG2 cell treated by various concentrations of Iso A for 8 hours;

[0034] FIG. 8 is a mass spectrum result of the direct conjugation of Iso A with Trx1;

[0035] FIG. 9 illustrates weight changes of mice administrated with Iso A or the vehicle control in implanted hepatocellular carcinoma model;

[0036] FIG. 10 illustrates tumor volume changes of mice administrated with Iso A or the vehicle control in implanted hepatocellular carcinoma model;

[0037] FIG. 11 illustrates tumor weight changes of mice administrated with Iso A or the vehicle control in implanted hepatocellular carcinoma model;

[0038] FIG. 12 illustrates Trx1 activity in the xenograft administrated with Iso A or the vehicle control in implanted hepatocellular carcinoma model;

[0039] FIG. 13 illustrates index changes of ALT, AST and ALP in serum of mice after drug administration with Iso A;

[0040] FIG. 14 illustrates index changes of urea nitrogen and creatinine in serum of mice after drug administration with Iso A;

[0041] FIG. 15 is an X-ray single crystal diffraction spectrum of Iso A.

DETAILED DESCRIPTION

[0042] The present invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the description of the embodiments is for the purpose of illustrating the invention and should not limit the invention as detailed in the claims.

Embodiment 1 Method for Preparing Compound Iso A

[0043] Grinding the aerial parts of Isodon forrestii var. forrestii to 10 Kg powder, soaking this powder with total 12 liters of 70% (acetone:water) acetone water in room temperature for three times, after evaporating the acetone by vacuum distillation, the remainder is extracted by ethyl acetate to get ethyl acetate extract. After washing by silica gel column chromatography with silica gel of 200 mesh pore diameter and chloroform:acetone (1:0.11 volume ratio) solvent, 500 mg Iso A can be finally obtained through repeated treatment by RP-18 mixture of etho-methanol and water with 85% volume ratio, and the yield is 0.005%.

[0044] The physical and chemical data for X ray single crystal and ultraviolet, optical rotation, infrared, nuclear magnetic resonance etc. of compound Iso A are:

[0045] Crystal data for Sze1:C.sub.28H.sub.38O.sub.10, M=534.58, orthorhombic, a=7.8960(2) , b=11.7330(3) , c=30.2738(9) , a=90.00, =90.00, y=90.00, V=2804.68(13) .sup.3, T=100(2) K, space group P212121, Z=4, (CuK)=0.796 mm.sup.1, 12890 reflections measured, 4629 independent reflections (R.sub.int=0.0457). The final R.sub.1 values were 0.0721 (I>2(I)). The final wR(F.sup.2) values were 0.2147 (I>2(I)). The final R.sub.1 values were 0.0731 (all data). The final wR(F.sup.2) values were 0.2158 (all data). The goodness of fit on F.sup.2 was 1.136. Flack parameter=0.1(3). The Hooft parameter is 0.01(8) for 1779 Bijvoet pairs.

[0046] Isoforretin A: Colorless needle crystals. [].sub.D.sup.25=45.3 (c 0.08, MeOH). UV (MeOH) .sub.max (log ): 232 (3.9) nm; IR (KBr) V.sub.max 3442, 2962, 2934, 2881, 1732, 1644, 1428, 1370, 1251, 1231, 1176, 1034, 995, 603; Positive ESIMS: m/z 557 [M+Na].sup.+; positive HRESIMS [M+Na].sup.+ m/z 557.2365 (calcd for C.sub.28H.sub.38O.sub.10Na, 557.2357); .sup.1H NMR (600 MHz, C.sub.5D.sub.5N) .sub.H 6.17 (1H, s, H-17a), 5.66 (1H, dd, 11.8, 5.5, H-2a), 5.47 (1H, br d, 4.7, H-11a), 5.41 (1H, s, H-17b), 5.37 (1H, br s, H-3a), 5.15 (1H, br s, H-12f), 4.67 (1H, br s, H-6), 3.34 (1H, br d, 14.5, H-14a), 3.28 (1H, br s, H-13a), 2.58 (1H, br d, 14.2, H-7a), 2.18 (1H, overlap, H-1a), 2.14 (3H, s, Me-20), 2.01 (3H, s, AcO-3), 1.98 (1H, overlap, H-9), 1.93 (3H, s, AcO-2), 1.87 (1H, overlap, H-1b), 1.83 (1H, overlap, H-7b), 1.74 (3H, s, AcO-11), 1.67 (1H, br d, 14.5, H-14b), 1.61 (3H, s, Me-19), 1.06 (3H, s, Me-18); .sup.13C NMR (150 MHz, C.sub.5D.sub.5N) .sub.C 208.3 (s, C-15), 170.8 (s, AcO-3), 170.4 (s, AcO-2), 169.4 (s, AcO-12), 168.7 (s, AcO-11), 145.4 (s, C-16), 117.1 (t, C-17), 77.9 (d, C-3), 75.4 (d, C-12), 70.1 (d, C-11), 67.9 (d, C-2), 64.9 (d, C-6), 60.2 (d, C-9), 49.8 (d, C-5), 48.8 (s, C-8), 42.7 (t, C-7), 41.8 (d, C-13), 40.8 (t, C-1), 40.3 (s, C-10), 39.0 (s, C-4), 32.2 (t, C-14), 28.7 (q, C-18), 23.4 (q, C-19), 21.1 (q, C-20), 21.1 (q, AcO-2), 21.1 (q, AcO-11), 20.7 (q, AcO-12), 19.3 (q, AcO-3).

Embodiment 2 Method for Preparing Compound Iso A

[0047] Grinding the aerial parts of Isodon forrestii var. forrestii to 10 Kg powder of, soaking this powder with total 20 liters of 80% (acetone:water) acetone water in room temperature for three times, after evaporating the acetone by vacuum distillation, the remainder is extracted by ethyl acetate to get ethyl acetate extract. After washing by silica gel column chromatography with silica gel of 300 mesh pore diameter and chloroform:acetone (1:0.5 volume ratio) solvent, 400 mg Iso A can be finally obtained through repeated treatment by RP-18 mixture of acetonitrile and water with 85% volume ratio, and the yield is 0.004%.

Embodiment 3 Iso A Can Selectively Inhibit the Growth of Human Tumor Cells and Induce Apoptosis

[0048] Dissolving Iso A (purity >99%) in DMSO, and preserving stock solution with concentration of 40 mM in 20 C. Detecting the inhibition effects of Iso A in various cells that includes liver cancer cell, breast cancer cell, lung cancer cell, osteosarcoma cell, melanoma cell, cervical cancer cell etc., and in normal human hepatocyte L02, human embryo lung cell HFL1, human mammary epithelial cell MCF 10A. Compared with normal cells, Iso A shows a stronger inhibition effect on the tumor cells, the IC50 values are all below 30 M (FIG. 2). Among tested cells, HepG2 cell is the most sensitive to Iso A, thus HepG2 is selected as a model in follow-up experiments. MTT experiment shows that Iso A can inhibit the growth of HepG2 cell in a concentration-dependent manner, but have no obvious inhibitory effects on L02 cell (FIG. 3). Further, trypan blue exclusion assay shows that Iso A can selectively trigger the death of HepG2 cells while being obviously not toxic to normal cells (FIG. 4). Flow cytometry analysis show that the treatment of Iso A leads to increasement of Annexin V positive cells. This result further proves that Iso A induces apoptosis. The pan-caspase inhibitor Z-VAD-FMK partially inhibits the apoptosis induced by Iso A,suggesting that Iso A-induced apoptosis effect is at least partially dependent on the activation of caspase (FIG. 5).

Embodiment 4 Iso A Inhibits Trx1 Activity in In Vitro Reaction

[0049] Experiment Material:

[0050] Trx1 protein was bought from Sino Biological Inc., Trx1 activity detection kit was bought from American Caymen Chemical cooperation.

[0051] Methods for measuring Trx1 activity in vitro:

[0052] The in vitro activity of Trx is assayed using theThioredoxin Activity Fluorescent Assay Kit (Caymen Chemical). According to the instructions of the kit 2 l of Iso A with different concentrations (0.195 M to 100 M) is added into 0.02 M Trx mix, following by adding 5 l of -NADPH into each sample to incubate in 37 C. for 30 min. Then, a fluorescent substrate is added and Trx1 activity is was recorded as the emission at 518 nm after 488 nm excitation for 30-60 min, with a Thermo Scientific Varioskan Flash fluorescent microplate reader.

[0053] Experiment Results:

[0054] As shown in FIG. 6, Iso A can inhibit Trxl activity with respective rate of 20%, 48.6%, 98% in experimental group (2.5, 5, 10 M), which suggests that the antineoplastic effect of Iso A may be mediated by the inhibition of Trx1 signaling pathway and the regulation of the downstream gene expression.

[0055] Embodiment 5 Inhibition on Trx1 by Various Concentrations of Iso A at Cellular Level

[0056] Methods for measuring Trx1 activity in cells or tissue samples: using lysates (20 mM HEPES (pH 7.9), 300 mM NaCl, 100 mM KCl, 10 mM EDTA and 0.1% Nonidet P-40, protease-contained inhibitor) to lyse the cells or tissue samples. Pre-treating 90 g protein at 37 C. for 15 min by using DTT activation buffer (50 mmol/liter HEPES (pH 7.6), 1 mmol/liter EDTA, 1 mg/ml bovine serum albumin, and 2 mmol/liter dithiothreitol). Then adding 20 l reaction mixture (200 l of 1 M HEPES (pH 7.6), 40 l of 0.2 mol/liter EDTA, 40 l of NADPH (40 mg/ml)) and 500 l insulin. Adding 5 l thioredoxin reductase for initial reaction while adding water of same volume into the control group. Incubate the samples in 37 C. for 20 min, and then add 6 M guanidine hydrochloride of 0.25 ml and 0.4 mg/ml DTNB to terminate reaction. Detecting optical absorption of 412 nm.

[0057] Cultivate human hepatocellular carcinoma cell line HepG2 in vitro culture. When cells have grown into the log phase, the cells are digested by trypin for 5-min-centrifugation (1000 rpm), then discarding the supernatant and diluting the cells as 510.sup.5 to inoculate on a 6 well culture plate for incubating 24 h, after that, the cells are treated by various concentrations of Iso A (negative control group is 0.5% DMSO) to incubate 16 h in an incubator for recollecting the cells and then detecting Trx1 activity in the cells. Calculate the relative activity of Trxl in each group under the condition that negative control activity is 1. The results show that Trxl activity is significantly reduced in HepG2 cells treated with Iso A at 10-30 M for 8 hours (FIG. 7).

Embodiment 6 Iso A Directly Conjugates with Trx1 Protein at Cys 32/35 Residues

[0058] Human-derived Trx1 protein (Sino Biological Inc.) is dissolved in distilled water at a 5 mg/ml and stored. In 25 mM Tris-HCl (pH 7.4) buffer, mixi 4 l Iso A with 140 l human Trx1 protein to incubate for 2 h in room temperature. Samples after reaction is treated by Shimadzu Biotech Proteome kit for enzymatic hydrolysis with MonoSpin Trypsin, then desalted by MonoSpin C18 column for mass spectrometry analysis with MALDI 7090 (SHIMADZU). Results from mass spectrometry analysis show that Iso A can directly bind with Trx1 protein (FIG. 8).

Embodiment 7 Inhibition on the Growth of Tumor by Iso A in a Murine Xenograft Tumor Model

[0059] 1. Material

[0060] a) Cell line: Human hepatocellular carcinoma cell line HepG2 is bought from the Cell Bank of Chinese Academy of Sciences for Type Culture Collection

[0061] b) Animal: 20 BALB/c male nude mouse with weight of (132 g), the BALB/c mice is bought from Model Animal Research Center of Nanjing University, the animal is bred under the environment without a special pathogen in the Animal Center of Jiangsu Traditional Chinese Medicine Research Institution [SCXK() 2002-0001].

[0062] c) Pharmaceutical: Iso A

[0063] 2. Experiment Method:

[0064] a) Make of a murine xenograft model ofepatocellular carcinoma: HepG2 cells in log phase are digested with trypin, and suspended with a final density of 210.sup.7 cells/ml. Then, 200 l (100 l cells+100 l matrigel) cell suspension is inoculated into the right subaxillary of nude mice with 1 ml injector.

[0065] b) Group and administration of experimental animal: grouping when the tumor becomes 100 mm.sup.3 into 10 per group (same average volume per group, too big or small ones are eliminated). Grouping specifically as followings: {circle around (1)} Iso A 15 mg/kg dose group, {circle around (2)} solvent control group (0.5% CMC-Na). Administrate one time every day for total 15 days.

[0066] c) Sample Selection and Index Observation:

[0067] i. observation for normal state: before drug administration, measure the weight and tumor size (tumor volume V=long diametershort diameter.sup.2/2) one time every day. Observe the locomotor activity, mental state, hair, breath, diet, manure trait and reaction to outside world of the mice. After ending pharmaceutical interference, the mice are anesthetized and the blood is acquired by cardiac puncture. The serum is got by centrifugation and store in 80 C. After the skin of the tumor growing part on the right axilla of the mice is nipped by forceps, use a surgical scissors to cut the skin for exposing the tumor, then use the surgical scissors to dissect the tumor with blunt dissection and use a scale to weigh the tumor, transplanted tumor and calculate the inhibition rate. Antitumor effect is determined by tumor inhibition rate. Tumor inhibition rate (%)=[1(average weight of tumor in experimental group/average weight of tumor in control group)]100%

[0068] ii. Detecting apoptosis of tumor tissues with TUNEL staining: according to TUNEL apoptosis detection kit specification of Roche Inc. after fixing frozen tissue section in room temperature.

[0069] iii. Blood routine examination for assessing the marrow toxicity of Iso A, analyze ALT, AST, ALP level in plasma for assessing hepatotoxicity of Iso A, and measure the creatinine and urea nitrogen for assessing renal damage.

[0070] Fabricate mice transplanted hepatocellular carcinoma model, and group mice into control group and Iso A (15 mg/kg) administration group at random. Record the weight and tumor size one time every three days and kill the mice after 15-day-administration for detection and analysis of the tumor tissues, blood etc. The results show that during administration, there is no difference in weight between the control group and administration group, which indicates that the toxic and side effects of Iso A are weak (FIG. 9).

[0071] Compared with the control group, the tumor volume and weight have significantly decreased after Iso A administration (FIG. 10, 11). Furthermore, the Trxl activity in the tumor tissues has significantly decreased through analysis of the Trx1 activity in the tumor tissues (FIG. 12). The above results show that Iso A can take antitumor effect through inhibiting Trxl activity in the mice model.

[0072] There is no obvious difference in ALT, AST and ALP level in serum of the mice in Iso A administration group, which shows that Iso A has no obvious hepatotoxicity (FIG. 13). There is no obvious difference in urea nitrogen and creatinine level in serum of the mice in Iso A administration group, which shows that Iso A has no obvious nephrotoxicity (FIG. 14). The above results show that Iso A has antitumor effects, but no toxic or side effects on several important organs such as marrow, liver and kidney in the xenograft murine models.

[0073] Embodiment 8 Preparing Iso A according to embodiment 1, it is added at a ratio of 1:1 by weight to an excipient for granulation and tabletting. The excipient is starch.

[0074] Embodiment 9 Preparing Iso A according to embodiment 1, it is added at a ratio of 1:2 by weight to an excipient for granulation and tabletting. The excipient is starch.

[0075] Embodiment 10 Preparing Iso A according to embodiment 1, it is made into a capsule by means of the normal capsule preparation method.

[0076] Embodiment 11 Preparing Iso A according to embodiment 1, it is made into a tablet by the method as follows:

TABLE-US-00001 Tablet: Iso A, 100 mg Starch Optimum dose Corn steep liquor Optimum dose Magnesium stearate Optimum dose

Embodiment 12

[0077]

TABLE-US-00002 Capsule: Iso A, 100 mg Starch Optimum dose Magnesium stearate Optimum dose

[0078] Preparation method: Mixing Iso A with a promoter for sifting to be uniformly mixed in a suitable container, then loading the acquired mixture into a hard gelatin capsule.

Embodiment 13

[0079]

TABLE-US-00003 Nasal sprays: Iso A 80 mg Sodium chloride 8 mg EDTA 1 mg Sodium phosphate buffer solution (pH 6.5) 10 mg Polyethoxy ether 10 mg Double distilled water 2 ml

[0080] Preparation method: Adding one composition into the double distilled water of suitable volume each time while agitating until complete dissolution, then adding another composition. After adding water to 2 ml, this solution is filtered through sterilizing filter for loading into a bottle and being separated due to an appropriate dosage.

Embodiment 14

[0081]

TABLE-US-00004 Dropping pill: Iso A, 100 mg Polyethylene glyco 6000 900 mg

[0082] Preparation method: Preparation for the melting liquid of Iso A with polyethylene glyco 6000: after Iso A of above amount being added with appropriate anhydrous ethanol for dissolution in low grade heat, adding it into polyethylene glyco melting liquid (keep warm in 60 C. water bath) of a formulated amount for mixing uniformly by agitation until full volatile of the ethanol, then let it stand for 30 mins in a water bath for removing bubbles, after that loading above uniformly mixed melting liquid without bubbles into a liquid receiver, and dropping the liquid drop by drop into a condensate until completing condensation, then dumping the condensate, collecting the dropping pill and placing it in a silicone dryer for drying or drying naturally after draining and removing the condensate on the dropping pill with a filter paper.