Trimer of S, R-heptacyclic dimethyl acetal, and preparation and application thereof in resisting tumor metastasis

Abstract

The invention discloses a trimer of S, R-heptacyclic dimethyl acetal, namely [S, R-heptacyclic dimethyl acetal] 3, a preparation method of the trimer and application of the trimer to treatment of tumor metastasis. Experiments prove that the [S, R-heptacyclic dimethyl acetal] 3 disclosed by the invention not only has a good anti-tumor metastasis effect, but also has the anti-tumor metastasis effect remarkably stronger than that of the S, R-heptacyclic dimethyl acetal. Therefore, the invention provides an effective technical means for treating tumor metastasis. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a trimer of S, R-heptacyclic dimethyl acetal, namely [S, R-heptacyclic dimethyl acetal] 3, and to a preparation method thereof and application thereof in treating tumor metastasis. The present invention belongs to the field of biomedicine. Background Art

[0002] The applicant has disclosed that the S,R-heptacyclic dimethyl acetal with the following structural formula is an intermediate of S,S-heptacyclic aldehyde; the applicant has disclosed that an oligopeptide can be introduced into the aldehyde group of S,R-heptacyclic aldehyde through an amination reduction reaction to prepare an antithrombotic S,R-heptacyclic ethyl oligopeptide; the applicant has disclosed that the S,R-heptacyclic dimethyl acetal with the following structural formula itself is a P-selectin inhibitor. At an oral dose of 1 μmol / kg / day, S,R-heptacyclic dimethyl acetal effectively inhibits tumor growth, at an oral dose of 1 μmol / kg, S,R-heptacyclic dimethyl acetal effectively inhibits arterial thrombosis, and at an oral dose of 1 μmol / kg, S,R-heptacyclic dimethyl acetal effectively inhibits venous thrombosis.

[0003]

[0004] The applicant knows that the aggregates of S,R-heptacyclic dimethyl acetal often show stronger biological activity. Therefore, the applicant prepared the trimer of S,S-heptacyclic dimethyl acetal of the following formula, namely [S,R-heptacyclic dimethyl acetal]3. The applicant screened the biological activities (including anti-arterial thrombotic activity, anti-venous thrombotic activity, thrombolytic activity, anti-tumor activity, anti-tumor metastasis activity and anti-inflammatory activity) that may be caused by [S,R-heptacyclic dimethyl acetal]3 inhibiting P-selectin. The applicant found that the trimer of S,R-heptacyclic dimethyl acetal showed excellent anti-tumor metastasis activity. Based on these findings, the applicant proposed the present invention. Summary of the invention

[0005] The technical problem to be solved by the present invention is to propose a trimer of S,R-heptacyclic dimethyl acetal, namely [S,R-heptacyclic dimethyl acetal] 3, confirm the method for preparing the trimer of S,R-heptacyclic dimethyl acetal [S,R-heptacyclic dimethyl acetal] 3, and further evaluate the anti-tumor metastasis activity of [S,R-heptacyclic dimethyl acetal] 3. In order to achieve the above purpose, the present invention adopts the following two technical means.

[0006] The first technical means is to propose a trimer of S,R-heptacyclic dimethyl acetal with the following structure, namely [S,R-heptacyclic dimethyl acetal]3,

[0007]

[0008] The second technical means is to propose a method for preparing [S, R-heptacyclic dimethyl acetal] 3, which can be summarized into the following two steps:

[0009] 1. Prepare S,R-heptacyclic dimethyl acetal monomer according to the disclosed method;

[0010] 2Convert the S,R-heptacyclic dimethyl acetal monomer into a dimer [S,R-heptacyclic dimethyl acetal]3.

[0011] The third technical means is to evaluate the anti-tumor metastasis activity of S,R-heptacyclic dimethyl acetal monomer and trimer [S,R-heptacyclic dimethyl acetal] 3 in a mouse lung metastasis model, and further confirm the effective application of [S,R-heptacyclic dimethyl acetal] 3 of the present invention in the preparation of anti-tumor metastasis drugs.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention is a trimer composed of three S, R-heptacyclic dimethyl acetals, namely [S, R-heptacyclic dimethyl acetal] 3. Compared with the prior art, the [S, R-heptacyclic dimethyl acetal] 3 of the present invention has excellent anti-tumor metastasis activity, and its anti-tumor metastasis effect is significantly stronger than that of the S, R-heptacyclic dimethyl acetal monomer. Therefore, the present invention enriches the selection of anti-tumor metastasis drugs and provides a more effective technical means for preparing anti-tumor metastasis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The synthetic route of the trimer of S,R-heptacyclic dimethyl acetal [S,R-heptacyclic dimethyl acetal] 3: i) 1,1,3,3-tetramethoxypropane and trifluoroacetic acid; ii) Pd / C, hydrogen; iii) 2-(7-oxybenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole; iv) vortex for 15 minutes, then ultrasonicate for 4 hours.

[0015] Figure 2 FT-ICR-MS spectrum and qCID spectrum of the trimer of S,R-heptacyclic dimethyl acetal. DETAILED DESCRIPTION

[0016] In order to further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention.

[0017] Example 1 Preparation of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid benzyl ester (1)

[0018] A mixture of CH2Cl2 (150 mL), 1,1,3,3-tetramethoxypropane (5 mL) and trifluoroacetic acid (3.5 mL) was stirred at 0°C for 40 minutes. Then L-Trp-OBzl (17 mmol, 5 g) was gradually added to the mixture. The resulting reaction mixture was stirred at 60°C for 8 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that L-Trp-OBzl disappeared. The reaction mixture was concentrated under reduced pressure to obtain a yellow oil, which was washed with saturated NaHCO3 aqueous solution (30 mL × 3) and saturated NaCl aqueous solution (30 mL × 3) in turn. The ethyl acetate layer was dried over anhydrous Na2SO4 for 8 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain 4.3 g (63%) of the title compound. FT-ICR-MS (m / e): 395.1971 [M+H] + .

[0019] Example 2 Preparation of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid (2)

[0020] 3.96 g (10.00 mmol) of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid benzyl ester (1) was dissolved in 100 mL of CH3OH. 400 mg of Pd / C was added to the obtained solution, and after stirring to form a uniform suspension, a three-way device was installed, and a hydrogen bag and a vacuum water pump were connected respectively, and the air in the reaction bottle was removed by hydrogen. Then, hydrogen was passed through the suspension for 18 hours while stirring. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that 1 disappeared. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.72 g (89%) of the title compound as a yellow solid. FT-ICR-MS (m / e): 303.1345 [MH] - .

[0021] Example 3 Preparation of S,R-heptacyclic dimethyl acetal (3S,R)

[0022] 2.64 g (8.7 mmol) of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carline-3-carboxylic acid (2) was dissolved in 70 mL of anhydrous DMF. 3.96 g (10.44 mmol) of 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 1.16 g (8.7 mmol) of 1-hydroxybenzotriazole (HOBt) were added to the solution at 0°C with stirring. The pH of the reaction solution was then adjusted to 9 with collidine and stirred for 24 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that 2 disappeared. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in 100 mL of ethyl acetate. The obtained solution was washed with saturated NaHCO3 aqueous solution (30 mL × 3), saturated NaCl aqueous solution (30 mL × 3), 5% KHSO4 aqueous solution (30 mL × 3), saturated NaCl aqueous solution (30 mL × 3), 5% NaHCO3 aqueous solution (30 mL × 3), and saturated NaCl aqueous solution (30 mL × 3). The ethyl acetate layer was dried over anhydrous sodium sulfate for 8 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.25 g of dark yellow solid, which was separated by silica gel column chromatography (CH2Cl2 / CH3OH=100 / 1) to obtain 130 mg (14%) of S,R-heptacyclic dimethyl acetal (3S,R), which was a light yellow solid. FT-ICR-MS (m / e): 573.2713 [M+H] + ;Mp:131-132℃; IR:3407.25,3304.95,2924.38,2833.52,2359.43,2331.09,1732.56,1650.90,1451.44,1423.38,1327.53, 1260.39,1229.50,1189.91,1149.36,1119.31,1055.26,1008.09,970.34,883.18,840.39,739.53,682.19cm -1 ; 1HNMR (800MHz, DMSO-d6): δ / ppm=10.97(s,1H),10.95(s,1H),7.48(d,J=7.8Hz,1H),7.43(d,J=7.8Hz,1H),7.37(d,J=8.1Hz,1 H),7.33(d,J=8.1Hz,1H),7.07(t,J=7.5Hz,2H),6.98(m,2H),5.84(m,1H),5.21(t,J=5.3Hz,1H),4.52(t,J=5.5Hz,1H),4.47 (dd, J1=11.2Hz, J2=4.6Hz, 1H), 4.36 (dd, J1=15.6Hz, J2=7.9Hz, 1H), 4.17 (m, 1H), 3.47 (dd, J1=20.0Hz, J2=11.4Hz, 1H), 3.29 (m,1H),3.27(s,3H),3.19(s,3H),3.12(s,3H),3.07(s,3H),2.84(m,1H),2.77(m,1H),2.53(m,1H),2.42(m,1H),2.17(m,2H); 13 CNMR (200MHz, DMSO-d6): δ / ppm=168.7,165.1,136.6,136.5,133.8,133.2,126.5,126.4,121.8,121.6,119.3,119.2,1 18.3,118.2,111.9,111.7,107.2,105.8,102.1,57.9,53.7,53.6,53.3,52.6,52.0,51.5,46.7,37.2,36.4,28.6,25.9;

[0023] The HPLC purity was 99.2% (Waters RPC18, 4.6×250 mm, 5 μm, CH 3 CN / H 2 O=50 / 50, flow rate=1 mL / min).

[0024] Example 4 Preparation of trimer of S,R-heptacyclic dimethyl acetal [S,R-heptacyclic dimethyl acetal] 3

[0025] 100 mg of S,R-heptacyclic dimethyl acetal (3S,R) powder was dissolved in 5 mL of ultrapure water. The resulting solution was vortexed for 15 minutes and then ultrasonicated for 4 hours until the solution was completely colorless and transparent. The colorless and transparent solution was concentrated to dryness under reduced pressure to obtain the trimer of S,R-heptacyclic dimethyl acetal (3S,R), namely [S,R-heptacyclic dimethyl acetal] 3. The structure of [S,R-heptacyclic dimethyl acetal] 3 was confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The assembly mode of 3S,R in solution was analyzed by FT-ICR-MS spectrum and qCID. Figure 2 It is the FT-ICR-MS spectrum and qCID spectrum of 3-S, R. The peak with mass number 1739.74784 is the peak of three molecules of 3-S, R plus Na (theoretical value is 1739.78022), and the theoretical value is consistent with the measured value. Under the conditions of FT-ICR-MS, the peak of three molecules of 3-S, R plus Na was bombarded. The local amplification showed that the peak of two molecules of 3-S, R plus Na was seen at the mass number 1167.53015 (theoretical value is 1167.51674), and the theoretical value is consistent with the measured value. In addition, the peak of cleavage at the mass number 571.27553 is the peak of one molecule of 3-S, R plus H (theoretical value is 573.27131), and the peak of cleavage at the mass number 595.24835 is the peak of one molecule of 3-S, R plus Na (theoretical value is 595.25325), and the theoretical value is consistent with the measured value. Under FT-ICR-MS conditions, the peak of three molecules of 3-S, R plus Na was split into two molecules of 3-S, R plus Na and one molecule of 3-S, R plus Na. The theoretical value was consistent with the measured value, supporting that the existence form of 3-S, R in solution is a trimer.

[0026] Example 5 Evaluation of the anti-tumor metastasis activity of [S,R-heptacyclic dimethyl acetal] 3

[0027] The anti-tumor metastasis effect of [S,R-heptacyclic dimethyl acetal]3 was evaluated in the Lewis mouse high metastatic lung cancer model. The experimental animals were C57BL / 6 male mice (clean grade), weighing 20±2g, with 10 mice in each group. Lewis mouse lung cancer cells (LLC) were digested and diluted to 2×10 7 The placenta blue (Tryanblue) rejection test showed that the number of viable cells was >95%. C57BL / 6 male mice were taken and fixed with the left hand. The skin of the right forelimb armpit of the mouse was disinfected with 75% ethanol. A 1mL sterile syringe was held in the right hand and injected subcutaneously into the armpit of the mouse with 0.2mL of tumor cell suspension (containing about 2×10 tumor cells) 6 / mL). 15 days after inoculation, mice can grow tumors with a diameter of about 1.5 to 2 cm, which can be used as a backup tumor source. The cervical vertebrae of Lewis lung cancer-bearing mice that have grown well after inoculation for 15 days were dislocated, and they were soaked in 75% ethanol for disinfection for 10 minutes. The solid tumors were bluntly peeled off in a clean bench, and the well-growing tumor tissues were selected to wash away the floating blood, and cut into pieces in a watch glass. The cut tumor tissues were placed in a glass tissue homogenizer, and 4°C pre-cooled physiological saline was added to grind into a cell suspension, and filtered twice with a 200-mesh nylon mesh to make a single cell suspension. The cell suspension was diluted to 2×10 7 / mL, and the number of viable cells was calculated by trypan blue staining, and >95% was calculated. C57BL / 6 male mice were taken, and 0.2mL of qualified cell suspension was inoculated under the right axilla of each mouse for observation. Tumors with a diameter of about 4 to 5mm were observed in the axilla of mice 12 days after inoculation. The tumor volume was measured, and the mice were divided into groups according to the tumor volume, and drug administration began. Mice in the [S,R-seven-ring dimethyl acetal]3 group were orally administered with 0.1μmol / kg / day [S,R-seven-ring dimethyl acetal]3 for 10 consecutive days. Mice in the S,R-seven-ring dimethyl acetal group were orally administered with 5μmol / kg / day S,R-seven-ring dimethyl acetal for 10 consecutive days. Mice in the normal saline group were orally administered with 0.2mL / 20g normal saline for 10 consecutive days. Mice in the RGDS group were orally administered with 20μmol / kg / day RGDS for 10 consecutive days.

[0028] On the eleventh day, the mice were weighed, anesthetized with ether, and killed by dislocating the cervical vertebra. Then, the tumor growth site in the right axilla of the mice was fixed with forceps, the skin was cut open, the tumor was exposed, bluntly dissected, and weighed. The lungs of the mice were taken and the number of tumor nodes metastasized to the lungs was recorded. The experimental data were analyzed by t-test and variance analysis, and the number of tumor nodes metastasized to the lungs was expressed as mean ± SD.

[0029] The nodule counts in Table 1 indicate that continuous treatment with 0.1 μmol / kg / day [S,R-heptacyclic dimethyl acetal]3 for 10 days effectively inhibited tumor metastasis to the lung (p<0.01 compared with saline and Arg-Gly-Asp-Ser). The nodule counts in Table 1 also indicate that the activity of 0.1 μmol / kg / day [S,R-heptacyclic dimethyl acetal]3 in inhibiting tumor metastasis to the lung is significantly stronger than that of 5 μmol / kg / day S,R-heptacyclic dimethyl acetal in inhibiting tumor metastasis to the lung. [S,R-heptacyclic dimethyl acetal]3 has a significant technical effect.

[0030] Table 1 The inhibitory activity of [S,R-heptacyclic dimethyl acetal]3 on the metastasis of Lewis lung cancer in mice after 10-day continuous treatment

[0031] Group dose Number of lung metastatic tumor nodes (mean ± SD) Normal saline 10 mL / kg / day 9.20±2.11 Arg-Gly-Asp-Ser 20 μmol / kg / day 6.11±0.98 <![CDATA[[S,R-heptacyclic dimethyl acetal]3]]> 0.1 μmol / kg / day <![CDATA[3.01±0.32 a ]]> S,R-heptacyclic dimethyl acetal 5 μmol / kg / day <![CDATA[6.02±0.95 b ]]>

[0032] a) Compared with normal saline, Arg-Gly-Asp-Ser and S,R-heptacyclic dimethyl acetal, p<0.01; b) Compared with normal saline, p<0.01, compared with Arg-Gly-Asp-Ser, p>0.05; n=10.

Claims

1. A trimer of S,R-heptacyclic dimethyl acetal, characterized in that: The trimer is of the following structure: [S, R-heptacyclic dimethyl acetal]3, 2. A method for preparing the [S,R-heptacyclic dimethyl acetal] 3 according to claim 1, characterized in that: The preparation method comprises the following steps: 1) Preparation of S,R-heptacyclic dimethyl acetal; 2) Preparation of [S,R-heptacyclic dimethyl acetal] 3.

3. Use of the trimer of S,R-heptacyclic dimethyl acetal according to claim 1 in the preparation of anti-tumor metastasis drugs.

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