CN115215860A - Oral small molecule P-selectin inhibitor and preparation method and application thereof - Google Patents
Oral small molecule P-selectin inhibitor and preparation method and application thereof Download PDFInfo
- Publication number
- CN115215860A CN115215860A CN202110432455.0A CN202110432455A CN115215860A CN 115215860 A CN115215860 A CN 115215860A CN 202110432455 A CN202110432455 A CN 202110432455A CN 115215860 A CN115215860 A CN 115215860A
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- Prior art keywords
- selectin
- carboline
- boc
- beta
- tetrahydro
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
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Abstract
The invention discloses an oral micromolecule P-selectin inhibitor and a preparation method and application thereof. The P-selectin inhibitor is named 202-D, and the structural formula of the P-selectin inhibitor is shown as a formula I. The invention also discloses the binding effect of the polypeptide and P-selectin and the effect of inhibiting the expression of P-selectin, further discloses the effect of the polypeptide and P-selectin capable of directly binding, and discloses high water solubility of the polypeptide and P-selectin capable of being used as oral medicines. Therefore, the invention also discloses the application of the P-selectin inhibitor 202-D as an oral small molecule medicament in inhibiting thrombus, inflammation and tumor. Since thrombosis and inflammation are the most common complications for patients with tumors, 202-D of the present invention is not only capable of treating tumorsAnd can also prevent thrombosis and inflammation complicated to tumor patients. The P-selectin inhibitor 202-D has good clinical application prospect.
Description
Technical Field
The invention relates to a novel P-selectin inhibitor 202-D, a preparation method thereof, and an anti-tumor effect, an anti-arterial thrombosis effect and an anti-inflammatory effect thereof. The invention belongs to the field of biological medicine.
Background
Selectins are adhesion receptors that initiate the passage of leukocytes from the vasculature into tissues at sites of inflammation, tissue injury and immune surveillance. P-selectin is present in Weibel-Pallade in endothelial cells or in the alpha-granules of platelets. When stimulated by external inflammation, tumor cells and the like, the cells can rapidly exist on the surfaces of activated endothelial cells and platelets in a cell spitting and membrane fusion mode. P-selectin expressed in activated platelets and endothelial cells mediates the interaction of activated platelets with tumor cells, which can promote tumor cell adhesion, and platelet-coated tumor cells help tumor cells evade immune surveillance by natural killer cells and further promote adhesion of tumor cells to vascular endothelial cells.
P-selectin is associated with increased formation and enlargement of thrombi. The interaction between P-selectin and PSGL-1 stimulates leukocytes, platelets and endothelial cells to produce thrombotic MPs (microparticles) which can express TF (tissue factor) with an anionic surface rich in phosphatidylserine, capable of assembling complexes of the coagulation cascade leading to thrombus formation; MPs can return to the area of thrombus formation ultimately leading to thrombus enlargement.
P-selectin is used as one of common treatment targets of various diseases such as inflammation, tumor, arterial thrombosis, deep venous thrombosis and the like and an important sensitive biomarker, plays an important role in the treatment process of early pathological change and disease monitoring, and the existing antibody protein P-selectin inhibitor cannot be massively produced due to high production and treatment cost and is not favorable for clinical popularization; the small molecular macrolides and lipopolysaccharide inhibitors have the problems of poor oral bioavailability and only injection administration, and poor patient compliance; the only small molecule inhibitor PSI-697 which can be orally taken is terminated in the first clinical test due to the problems of poor solubility and overlarge dosage, so that a small molecule inhibitor of P-selectin which can be orally taken and effectively used can be designed, and the biological activity of resisting inflammation, resisting tumor proliferation, resisting arterial thrombosis and deep vein thrombosis can be realized at the same time.
However, the effective dose of the carboline compounds disclosed in the previous period is too large, for example, the antithrombotic effective dose of 3S-3-carboxyl-1,2,3,4-tetrahydro-beta-carboline is as high as 5 mu mol/kg, and still needs to be reduced. And the prophase published carboline structure is only a down regulator of P-selectin and can reduce the content of the P-selectin in the blood serum of tested animals, however, direct evidence and experiments show that the carboline structure can be combined with the P-selectin, and the prophase carboline structure cannot be proved to be combined with the P-selectin to play antithrombotic activity, so that the carboline structure cannot be used as an inhibitor of the P-selectin. Currently available inhibitors of P-selectin do not appear to pass clinical trials for oral small molecule drugs.
The inventor hypothesizes that, under the premise of existence of beta-carboline, aspartic acid is introduced to enhance the water solubility, and 1,2,3,4-tetrahydro-beta-carboline is combined with aspartic acid, the water solubility of the compound can be improved, and the compound is beneficial to oral administration, and at the moment, the carboxyl part of aspartic acid and the tricyclic framework structure of beta-carboline can be combined with P-selectin to target the P-selectin, so that the novel beta-carboline derivative (202-D) can simultaneously have the activities of resisting tumor growth, arterial thrombosis and inflammation. Based on this assumption, the inventors have proposed the present invention.
Disclosure of Invention
The invention aims to provide a novel oral small-molecule P-selectin inhibitor, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the P-selectin inhibitor is named as 202-D, and the structural formula of the P-selectin inhibitor is shown as the formula I:
further, the present invention provides a method for preparing said P-selectin inhibitor, comprising the steps of:
(1) At 1M H 2 SO 4 Catalyzing L-Trp and formaldehyde to perform Pictet-Spengler condensation reaction to prepare 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid;
(2) Methanol is used as a solvent, and carboxyl on three positions of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid is introduced into methyl ester for protection under the catalysis of thionyl chloride to obtain 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl ester;
(3) Reducing methyl ester of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl ester into alcoholic hydroxyl by taking anhydrous tetrahydrofuran as a solvent in the presence of lithium aluminum hydride to obtain 3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol;
(4) Tetrahydrofuran as solvent in NMM (Boc) 2 Introducing Boc protection to NH at the second position of 3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol in the presence of O to obtain Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol;
(5) Using anhydrous tetrahydrofuran as a solvent, and carrying out intermolecular condensation on Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol and Boc-D (OBzl) -OH in the presence of DCC and DMAP to obtain Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OBzl;
(6) Dissolving the obtained Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OBzl in methanol, and reacting in Pd/C, H 2 Removing OBzl protection under the condition to obtain Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OH;
(7) de-Boc protection under 4N EA/HCl conditions to yield the P-selectin inhibitor 202-D of claim 1.
The synthetic scheme for the P-selectin inhibitor 202-D is shown in FIG. 1.
Furthermore, the invention also provides application of the P-selectin inhibitor in preparing antitumor drugs.
The P-selectin inhibitor is applied to the preparation of the anti-arterial thrombosis medicine.
The P-selectin inhibitor is applied to the preparation of the anti-venous thrombosis medicine.
The application of the P-selectin inhibitor in preparing anti-inflammatory drugs.
The application of the P-selectin inhibitor in preparing the P-selectin inhibitor comprises the following three parts:
(1) Reducing the content of soluble P-selectin in the body of the tested animal;
(2) Ultraviolet spectrum identifies the binding effect of the in vitro and the P-selectin;
(3) Direct binding to P-selectin. And
the application of the P-selectin inhibitor in preparing small molecule oral drugs.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a novel oral small molecule P-selectin inhibitor 202-D and a preparation method thereof, and also discloses a binding effect of the inhibitor and P-selectin and an effect of inhibiting P-selectin expression, further discloses an effect of the inhibitor and P-selectin which can be directly bound, and discloses high water solubility of the inhibitor and P-selectin which can be used as an oral medicament. Therefore, the invention discloses the application of the P-selectin inhibitor 202-D as an oral small molecule medicament in inhibiting thrombus, inflammation and tumor. Since thrombosis and inflammation are the most common complications of tumor patients, 202-D of the present invention can not only treat tumors, but also prevent thrombosis and inflammation complications of tumor patients. Therefore, the P-selectin inhibitor 202-D disclosed by the invention has a good clinical application prospect.
Drawings
FIG. 1 is a scheme showing the synthesis of P-selectin inhibitor 202-D.
Fig. 2 is the antitumor activity of P-selectin inhibitor 202-D, n =6.
Figure 3 is the anti-arterial thrombotic activity of P-selectin inhibitor 202-D with n =10.
Fig. 4 is the anti-thrombotic activity of P-selectin inhibitor 202-D with n =10.
Figure 5 is the anti-inflammatory activity of P-selectin inhibitor 202-D, n =10.
FIG. 6 shows that P-selectin inhibitor 202-D down-regulates the content of soluble P-selectin in the serum (plasma) of test animals (A: S180. Sup. Soluble P-selectin content in the serum of mice: B: soluble P-selectin content in the plasma of rat against deep venous thrombosis: C: soluble P-selectin content in the plasma of rat against arterial thrombosis: D: soluble P-selectin content in the serum of mouse with ear edema), n =6.
FIG. 7 is a UV spectrum of P-selectin inhibitor 202-D binding to P-selectin in vitro.
Fig. 8 shows the number of HL60 cells adhered to P-selectin ELISA plates under the action of P-selectin inhibitor 202-D, n =6.
FIG. 9 is a graph of the solubility assay for P-selectin inhibitor 202-D and PSI-697.
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.
The synthetic scheme for the P-selectin inhibitor 202-D is shown in FIG. 1.
Example 1 preparation of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid
0.2mL of concentrated sulfuric acid was slowly added to 400mL of water in an ice bath, and 5.00g (24.5 mmol) of L-tryptophan was added to the obtained dilute sulfuric acid solution to completely dissolve the L-tryptophan, and then 10mL of a 37-40% formaldehyde solution was slowly added thereto. The reaction mixture was stirred at room temperature for 4-5 hours, then adjusted to pH 7 with concentrated ammonia water, filtered and washed with methanol to give 5.15g (yield 99%) of a filter cake, which was the title compound. ESI/MS:217[ 2 ] M + H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ10.95(s,1H),δ7.47(q,J＝8.5Hz,2H),7.07(m,2H),5.42(s,1H),4.42(d,J＝15.8Hz,1H),4.25(m,1H),3.63(m,1H),3.14(m,1H),2.85(m,1H). 13 C NMR(75MHz,DMSO-d 6 )δ169.58,136.31,136.19,129.03,121.40,119.35,117.69,111.09,109.79,56.47,22.85.
Example 2 preparation of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl ester
To 40mL of methanol was added 3mL of SOCl while cooling on ice 2 And is dropped into the bottle through a constant pressure funnelAnd (5) putting a drying tube above the hopper, and activating. After the activation, the constant pressure funnel was removed, 3.00g (15 mmol) of 3S-1,2,3,4-tetrahydro- β -carboline-3-carboxylic acid was weighed into a bottle, and a drying tube was added. The ice bath was removed and the reaction was stirred at room temperature for 48 hours, and the completion of the reaction was monitored by TLC (dichloromethane: methanol 20). Then the methanol was pumped dry with a water pump and sequentially with ethyl acetate and petroleum ether. Re-dissolving with ethyl acetate, adding appropriate amount of saturated sodium bicarbonate solution, stirring under ice bath to remove salt, standing in separating funnel for layering, sequentially adding saturated NaHCO 3 Saturated NaCl wash, ester layer over anhydrous Na 2 SO 4 Drying, filtering and spin-drying. Purification by silica gel column chromatography using dichloromethane in methanol system gave 1.7g of white solid compound in 54% yield. ESI/MS:231.1[ mu ] M + H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ10.73(s,1H),7.37(m,1H),7.28(m,1H),6.99(m,2H),3.97(m,2H),3.74(dd,J＝8.8,4.8Hz,1H),3.68(s,3H),2.95(m,1H),2.75(m,2H). 13 C NMR(75MHz,DMSO-d 6 )δ173.55,135.67,133.38,126.85,120.35,118.20,117.12,110.77,105.42,55.13,51.54,41.33,24.91.
Example 3 preparation of 3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol
Under ice-bath conditions, 0.7g (17 mmol) of LiAlH 4 Slowly adding into anhydrous THF, weighing 1.0g (4.3 mmol) of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl ester, slowly adding into the reaction solution under ice bath, adding into a drying tube on an eggplant bottle, and stirring for 5h under ice bath. Thin layer chromatography (dichloromethane: methanol = 10) monitoring the quenching reaction after the end of the reaction, H was added dropwise in sequence 2 O, 4N NaOH solution, H 2 O, gas is generated in the solution when water is added. Stirring was continued for 40 minutes after the addition. Filtering under reduced pressure to remove filter residue, and spin-drying the filtrate to obtain yellow solid. Purification by silica gel column chromatography (dichloromethane methanol system) gave 0.63g of a white solid in 72% yield. m.p. 175.7-179.4 ℃;
-67.88；ESI/MS：203[M+H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ10.66(s,1H),7.34(m,1H),7.26(m,1H),6.96(m,2H),4.70(s,1H),3.92(s,2H),3.51(m,2H),2.88(m,1H),2.63(dd,J＝14.9,4.1Hz,1H),2.29(m 1H). 13 C NMR(75MHz,DMSO-d 6 )δ135.75,133.97,127.12,120.07,118.03,117.02,110.70,106.58,65.01,55.41,42.31,24.41.
EXAMPLE 4 preparation of Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol
Dissolving 1g (4.95 mmol) of 3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol in anhydrous THF, adding 1.4g (6.44 mmol) (Boc) 2 Adjusting pH to 9 with O, NMM, placing on eggplant bottle, reacting at room temperature with one-way valve, starting 2 hours, frequently pumping air under reduced pressure, and monitoring pH to maintain at 9. After completion of the reaction was monitored by TLC (dichloromethane: methanol 30 1), the reaction solution was spin-dried, redissolved with ethyl acetate, and was successively treated with 5% KHSO 4 Saturated NaCl wash, ester layer over anhydrous Na 2 SO 4 Drying, filtering and spin-drying. Purification by silica gel column chromatography (dichloromethane methanol system) gave 1.34g of a white solid in 90% yield. ESI/MS:303[ 2 ], [ M ] +H] + ； 1 H NMR(300MHz,DMSO)δ10.81(s,1H),7.38(d,J＝7.7Hz,1H),7.29(d,J＝7.7Hz,1H),6.99(m,2H),4.82(m,2H),4.61(s,1H),4.53(s,1H),4.16(m,1H),3.34(m,1H),2.76(s,2H),1.45(s,9H). 13 C NMR(75MHz,DMSO)δ155.04,154.35,136.02,129.99,126.84,120.64,118.26,117.43,110.86,104.45,78.95,59.89,51.82,50.29,37.86,28.04,26.27,22.46,21.02,20.62.
EXAMPLE 5 preparation of Boc-3S-1,2,3,4-tetrahydro- β -carboline-3-methyl- (Boc) D-OBzl
Dissolving 0.84g (2.6 mmol) of Boc-D (OBzl) -OH in anhydrous THF, adding 0.41g (2 mmol) of DCC and 24mg (0.2 mmol) of DMAP, activating under ice bath, adding 0.6g (2 mmol) of Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol, removing ice bath, reacting at room temperature overnight, monitoring the reaction completion by TLC (dichloromethane: methanol 30), terminating the reaction, filtering the reaction solution, re-dissolving with ethyl acetate, sequentially using saturated NaHCO 3 Saturated NaCl,5% KHSO 4 Saturated NaCl, saturated NaHCO 3 Saturated NaCl wash, ester layer over anhydrous Na 2 SO 4 Drying, filtering and spin-drying. Purification by silica gel column chromatography (dichloromethane methanol system) gave 0.9g of a white solid in 74% yield. ESI/MS:608[ 2 ], [ M ] +H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ10.87(s,1H),7.36(m,4H),7.33(m,4H),7.05(m,1H),6.96(m,1H),5.10(d,J＝2.7Hz,2H),4.85(m,3H),4.41(s,1H),4.10(m,2H),2.82(m,1H),2.72(m,3H),1.40(d,J＝13.5Hz,18H). 13 C NMR(75MHz,DMSO-d 6 )δ170.82,169.63,155.09,136.05,135.78,128.31,127.97,127.84,126.57,120.85,118.39,117.45,110.95,79.50,78.41,65.82,63.26,49.99,35.55,28.04,27.91.
EXAMPLE 6 preparation of Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OH
Taking 0.5g (0.82 mmol) of Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OBzl, dissolving in a proper amount of methanol, adding 0.1g of Pd/C, adding a tee joint above an eggplant bottle, replacing air and hydrogen in the bottle, reacting at room temperature, monitoring the disappearance of a raw material point by TLC (dichloromethane: methanol 30). After spin-drying, 0.42g of a pale yellow solid is obtained, with a yield of 95%. ESI/MS:518[ M ] +H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ12.43(s,1H),10.88(s,1H),7.34(dd,J＝21.7,7.8Hz,2H),7.21(s,1H),6.98(m,2H),5.76(s,1H),4.88(m,1H),4.81(m,1H),4.33(s,1H),4.06(m,1H),2.81(m,1H),2.63(m,2H),1.41(m,2H),1.40(s,s,18H). 13 C NMR(75MHz,DMSO-d 6 )δ171.39,136.09,129.59,126.61,120.85,118.39,117.46,110.95,62.97,54.83,50.29,36.12,28.08.
EXAMPLE 7 preparation of P-selectin inhibitor 202-D
0.4g (0.74 mmol) of Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OH is dissolved by 5mL of 4N EA/HCl, a drying tube is added above an eggplant bottle, and the reaction is carried out in an ice bath until the raw material point disappears. The reaction solution was drained with a water pump, and then redissolved with ethyl acetate and ether in this order, and drained separately. 0.28g of yellow powder was obtained. Purification was performed using a reverse phase C18 column (methanol, water system), after purification methanol was spun off, and the remaining water was lyophilized to give 0.13g of white solid product in 48% yield, the synthetic route is shown in FIG. 1.m.p. 203.5-208.2 ℃;
14.03(c＝0.10,CH 3 OH)；ESI/MS：318[M+H] + ；FT-MS：318.14556； 1 H NMR(800MHz,DMSO-d 6 )δ11.11(s,1H),7.42(d,J＝7.9Hz,1H),7.37(d,J＝8.1Hz,1H),7.10(t,J＝7.6Hz,1H),7.02(t,J＝7.3Hz,1H),4.59(d,J＝11.9Hz,1H),4.48(dd,J＝11.9,6.3Hz,1H),4.40(d,J＝15.4Hz,1H),4.31(d,J＝15.4Hz,1H),4.27(m,1H),3.81(m,1H),2.97(m,2H),2.87(dd,J＝16.9,7.3Hz,2H),2.45(s,1H). 13 C NMR(75MHz,DMSO-d 6 )δ170.08,136.64,128.08,126.55,121.97,121.84,119.35,111.78,105.49,65.15,52.65,50.26,40.56,39.17,21.65.
example 8 evaluation of the antitumor Effect of P-selectin inhibitor 202-D
Experimental Material
Doxorubicin hydrochloride (CAS: 25316-40-9, welibench technologies, ltd.), PSI-697 (CAS: 851546-61-7, medchemeexpress), physiological saline (shijiazhuang four drugs ltd.).
Laboratory animal
ICR strain mouse, male, 20 + -2 g, purchased from Experimental animals technology, inc. of Wei Tongli, beijing.
The tumor source is mouse S180 sarcoma purchased from animal experiment center of department of medicine of Beijing university and maintained by self passage.
Grouping and dosing of mice
The compound 202-D of the invention has the dose of 5 mu mol/kg, the positive control doxorubicin hydrochloride dose of 2 mu mol/kg, the PSI-697 dose of 5 mu mol/kg, the negative control physiological saline, the administration dose of 0.2mL/20g, continuous administration for 10 days, and 10 times of administration in total.
Experimental procedures
Extracting S180 ascites tumor liquid under aseptic condition for inoculation, diluting the tumor cell suspension into cell liquid (1:2) by using normal saline, fully mixing, dyeing the tumor cell suspension by using 0.2% trypan blue prepared on the same day, uniformly mixing, counting according to a white cell counting method, wherein a blue-dyed cell is a dead cell, a non-dyed cell is a live cell, and calculating the cell concentration and the survival rate according to the following formula:
cell concentration = number of viable cells in 4 squares/4 × 10 4 X dilution multiple = number of cells/mL
Cell viability = number of live cells/(number of live cells + number of dead cells) × 100%
Homogenizing cell suspension with survival rate of more than 90%Is prepared into 2.0 × 10 7 One mouse per mL was inoculated subcutaneously under the axilla of the mouse, and 0.2mL was used per mouse, to prepare S180 tumor mice. 3 days after tumor inoculation, the test compound group mice were orally administered with a given dose of test compound or physiological saline per day at a dose of 0.1mL/10g, or were intraperitoneally injected with a given dose of doxorubicin, and the dose was continuously administered for 10 days as described above, for a total of 10 times. Blood was taken from the eyeball after ether anesthesia on day 11, the mouse was sacrificed by cervical dislocation, then the tumor was removed blunt, and each organ of the mouse was weighed, and the tumor and organ weights were expressed as mean ± SD g. The experimental data were counted using t-test and anova.
Results of the experiment
The results are shown in FIG. 2. It can be seen that 202-D (1.66 + -0.5 g) can effectively inhibit tumor growth of mice at 5 μmol/kg oral dose, the tumor weight of the mice treated by normal saline is significantly less (3.26 + -0.59g; p-straw 0.010), and the activity of the mouse is equivalent to that of adriamycin (1.51 + -0.3 g), and is obviously better than that of PSI-697 (2.26 + -0.54 g) at the same dose, and unexpected technical effect is obtained.
Example 9 evaluation of the anti-arterial Thrombus Effect of P-selectin inhibitor 202-D
Experimental materials
Uratan (national drug group chemical Co., ltd.), heparin sodium (Bailingwei science and technology Co., ltd.), PSI-697 (CAS: 851546-61-7, medChemExpress), physiological saline (Shijiazhuang Siyao Co., ltd.).
Laboratory animal
SD strain rats, male, 200 + -20 g, purchased from Experimental animals technology, inc. of Weitonglihua, beijing.
Bypass cannula
The bypass intubation channel consists of three sections, wherein a tip bevel polyethylene tube with the length of 10.0cm, the inner diameter of 1.0mm and the outer diameter of 2.0mm is respectively positioned at two ends of the bypass intubation; connecting two side pipes by using a 3.5mm polyethylene pipe with the length of 8.0 cm; the wire was polished to a rough surface and fixed to 6.0cm to a weight of 4.0. + -. 0.1mg.
Three sections of polyethylene tubes are all silanized by 1 percent of silicon ether solution, are completely dried, then silk threads are placed in the polyethylene tube in the middle along the direction of the carotid intubation, and the three sections of polyethylene tubes are assembled and fixed by a para film.
Grouping and dosing of rats
The compound 202-D of the invention had dosages of 5,0.5 and 0.05 μmol/kg, the positive control aspirin dosage was 167 μmol/kg, the PSI-697 dosage was 5 μmol/kg, and the negative control was normal saline.
Reagent preparation
The anesthetic is a 20% urethane solution prepared by normal saline, and the anticoagulant is a 42mg/100mL heparin sodium solution prepared by normal saline.
Experimental procedures
The rats are subjected to intragastric administration according to the dose of 0.3mL/100g, and the intragastric administration adopts a sequential method. 30min after administration, 20% (0.7 mL/100 g) of urethane solution was injected intraperitoneally for anesthesia. The neck skin of the rat was cut open, the left external jugular vein and the right common carotid artery were isolated, the distal ends of the blood vessels on both sides were ligated with a surgical thread, a small opening was cut in the left external jugular vein by ophthalmic scissors, the venous end of the bypass cannula was inserted into the opening of the left external jugular vein and fixed with a surgical thread, and a physiological saline solution of heparin sodium (42 mg/100 mL) was injected through the bypass cannula at a dose of 0.1mL/100 g. Clamping the proximal end of the right common carotid artery by an artery clamp, cutting a small opening on the artery, inserting the other side of the bypass cannula into the right common carotid artery and fixing the right common carotid artery by an operation line, loosening the artery clamp and opening the extracorporeal circulation bypass.
After extracorporeal circulation is carried out for 15min, a cannula at the venous end is firstly cut off to observe whether blood circulation is smooth, 5mL of blood is taken from the venous end and placed in 10% sodium citrate normal saline solution (3.8%) for measurement of relevant indexes of the blood. The thrombus thread was taken out from the arterial port of the bypass cannula, and the floating blood on the thrombus thread was dipped on the filter paper by the same method and weighed, and the obtained thrombus was reused to express the anti-arterial thrombus activity. Data were counted using t-test.
Results of the experiment
The data are shown in FIG. 3. The result shows that the oral administration of 0.05 mu mol/kg 202-D (22.88 +/-2.72 mg) can effectively inhibit the formation of arterial thrombosis, and the activity is equivalent to that of aspirin (20.45 +/-3.20 mg) and is obviously superior to PSI-697. The effective dose is 3340 times lower than that of aspirin (167 mu mol/kg), and unexpected technical effects are obtained.
EXAMPLE 10 evaluation of the anti-thrombotic Effect of P-selectin inhibitor 202-D
Experimental Material
Uratan (Ethyl carbamate, CAS:51-79-6, national pharmaceutical group Chemicals Co., ltd.), physiological saline (Shijiazhuang Siyao Co., ltd.), warfarin sodium (CAS: 129-06-6, bailingwei science Co., ltd.), PSI-697 (CAS: 851546-61-7, medChemexpress).
Laboratory animal
SD strain rat, male, 230 + -20 g, purchased from Beijing Huafukang laboratory animal technologies, inc.
Experimental methods
The experiment was performed using the rat inferior vena cava ligation model.
Grouping and administration dose:
the dose of the compound 202-D of the invention is 5,0.5 and 0.05. Mu. Mol/kg, the dose of the positive control warfarin is 4.87. Mu. Mol/kg, the dose of the PSI-697 is 5. Mu. Mol/kg, and the negative control is normal saline.
Reagent preparation
The anesthetic is a 20% urethane solution prepared from normal saline.
Experimental procedures
Rats were acclimatized one day before surgery and were fasted one night in advance, and were gavaged (0.3 mL/100 g) using a sequential method. After administration for 30min, anesthesia (0.7 mL/100 g) was administered by intraperitoneal injection using a 20% urethane solution. The abdomen of the rat was disinfected and then the abdominal cavity was opened 2cm below the thorax along the abdominal linea alba, with one corner of the liver visible above the opening, the length of the opening being about 2cm. Organs such as small intestine and large intestine in abdominal cavity were carefully removed with forceps and placed on gauze soaked with normal saline. Separating peripheral connective tissues and blood vessels bluntly to expose the lower deep vena cava, carefully separating the abdominal aorta and the lower deep vena cava below the renal veins by using forceps, then ligating the lower deep vena cava at the junction of the lower deep vena cava and the left renal vein by using a suture line soaked in normal saline for more than 1 hour, moving organs such as small intestine, large intestine and the like back to the abdominal cavity, and then suturing the abdominal cavity by using the suture line.
After the operation, the rats are placed in a warm environment for circulation for 4 hours, 5mL of blood is firstly taken from carotid artery, and the blood is placed in 10% sodium citrate normal saline solution (3.8%) for measurement of blood related indexes. Then, the abdominal cavity is cut off by scissors, the blood vessels at the upper end and the lower end of the deep vein are respectively clamped by two hemostatic forceps, the deep vein is lifted by the forceps from the lower part of the deep vein and cut from bottom to top to the ligation part of the deep vein in the lower cavity so as to avoid the backflow of thrombus into the blood, and then the thrombus is taken out and weighed. The resulting plugs were reused to demonstrate activity against deep vein thrombosis. Data were counted using t-test.
Results of the experiment
The experimental data are shown in FIG. 4. The results showed that oral administration of 0.05. Mu. Mol/kg 202-D (12.46. + -. 2.25 mg) was effective in inhibiting venous thrombosis and the effect was comparable to 5. Mu. Mol/kg PSI-697 (11.30. + -. 2.51 mg). Oral administration of 0.5. Mu. Mol/kg 202-D (9.91. + -. 2.87 mg) resulted in an equivalent effect to 4.87. Mu. Mol/kg warfarin (9.53. + -. 1.67 mg). The effective dose is 9.7 times lower than the warfarin effective dose (4.87 mu mol/kg), and unexpected technical effects are obtained.
EXAMPLE 11 evaluation of the anti-inflammatory Effect of P-selectin inhibitor 202-D
Experimental Material
Xylene (Beijing chemical plant), PSI-697 (CAS: 851546-61-7, medChemexpress), physiological saline (Shijiazhuang four drugs Co., ltd.).
Laboratory animal
ICR strain mouse, male, 20 + -2 g, purchased from Experimental animals technology, inc. of Wei Tongli, beijing.
Grouping and dosing of mice
The dose of the compound 202-D of the invention is 0.05,0.5,5 mu mol/kg, the dose of positive control aspirin is 1110 mu mol/kg, the dose of PSI-697 is 5 mu mol/kg, and the negative control is normal saline.
Experimental procedures
The mice need to rest for more than 24 hours before modeling. And (3) subsequently performing intragastric administration, uniformly smearing 15 mu L of dimethylbenzene on the inner side and the outer side of the auricle of the left ear of the mouse after 30min, wherein the operation is performed in a fume hood, and after the dimethylbenzene smearing is completed, the mouse needs to be taken out from the fume hood to avoid excessive volatilization of the dimethylbenzene. After 2 hours, the mice were anesthetized with ether, and 1mL of eyeball blood was taken for measurement of relevant blood indices. Then, the mouse is killed by cervical dislocation, the left ear and the right ear of the mouse are respectively cut off along the root of the ear, the auricles are aligned and stacked, a circular ear piece with the same area and size is punched at the same position by an electric ear punch with the diameter of 7mm, the circular ear pieces are respectively weighed, the difference value is recorded, and the result is used for representing the degree of ear swelling of the mouse. Data were counted using t-test.
Degree of swelling of mouse ear (mg) = weight of left ear tablet-weight of right ear tablet.
Results of the experiment
The data are shown in FIG. 5. It was experimentally observed that the weight difference between the left and right ears of mice orally administered compound 0.05. Mu. Mol/kg, 0.5. Mu. Mol/kg and 5. Mu. Mol/kg was significantly different and significantly reduced. Wherein the difference of the weight of the mouse ears at 0.5 mu mol/kg (2.15 +/-0.31 mg) is equivalent to 1110 mu mol/kg of aspirin (2.43 +/-0.64 mg) and 5 mu mol/kg of PSI-697 (2.68 +/-0.81 mg), and the effective dose is reduced by 2220 times relative to the aspirin. Unexpected technical effects are obtained.
EXAMPLE 12 evaluation of the Effect of P-selectin inhibitor 202-D on reducing the soluble P-selectin content in test animals
Experimental Material
Sodium citrate (CAS: 68-04-2, national drug group chemical Co., ltd.), NS (Shijiazhuang Siyao Co., ltd.), and distilled water.
Experimental sample
Blood was collected from the eyeballs of mice of examples 8 and 11, and from the carotid arteries of rats of examples 9 and 10.
Experimental methods
The detection is carried out by adopting a mouse soluble P-selectin enzyme-linked immunoassay kit and a rat soluble P-selectin enzyme-linked immunoassay kit.
Sample collection
Blood was collected from the eyeballs of mice treated with 5. Mu. Mol/kg 202-D and 5. Mu. Mol/kg PSI-697 in example 8, and the carotid artery blood of rats treated with 5. Mu. Mol/kg 202-D and 5. Mu. Mol/kg PSI-697 in examples 9 and 10 was collected as an anticoagulant, and centrifuged at 3000rpm at 4 ℃ for 30min within 30min, and the supernatant was sampled and examined.
Experimental procedure
According to the standard operation steps of the sP-selectin kit, a standard substance is sequentially prepared, and a blank hole, a standard hole and a serum hole to be detected are respectively arranged after sample addition. Adding 50 mu L of standard substance into the enzyme-labeled coated plate, adding 40 mu L of sample diluent into the serum hole to be detected, adding 10 mu L of serum to be detected, and slightly shaking and uniformly mixing. The plates were sealed with a sealing plate and incubated at 37 ℃ for 30min. Carefully remove the coversheet membrane, discard the liquid, and wash the plate 5 times. Adding 50 mu L of enzyme labeling reagent into each hole except the blank hole, incubating for 30min at 37 ℃, washing, adding 50 mu L of color development agent A into each hole, adding 50 mu L of color development agent B, shaking gently, mixing uniformly, and developing for 10min at 37 ℃ in a dark place. The reaction was stopped by adding 50. Mu.L of stop solution to each well, and the absorbance (OD value) of each well was measured at a wavelength of 450nm in 15min under a blank of Kong Diaoling. A standard curve is drawn according to the OD value of the standard substance, the concentration of the serum sample is calculated, and the data is shown in figure 6 (A: S180. Sup. Soluble P-selectin content in the serum of the mouse: B: soluble P-selectin content in the plasma of the rat with deep venous thrombosis: C: soluble P-selectin content in the plasma of the rat with arterial thrombosis: D: soluble P-selectin content in the serum of the mouse with ear edema), and n =6.
Results of the experiment
As can be seen from the data in FIG. 6, the soluble P-selectin content of 5. Mu. Mol/kg 202-D treated rats and mice is significantly lower than that of saline treated rats and mice (P < 0.05), and is comparable to that of 5. Mu. Mol/kg PSI-697 treated rats and mice (P > 0.05), i.e., 202-D reduces the soluble P-selectin content in rats and mice at a dose of 5. Mu. Mol/kg, and is comparable to that of 5. Mu. Mol/kg PSI-697 treated rats and mice. As can be seen, 202-D reduced the activity of soluble P-selectin in the tested rats and mice comparable to that of inhibitors of P-selectin. This is an unexpected technical effect.
Example 13 evaluation of the Effect of P-selectin inhibitor 202-D on P-selectin binding in vitro
Experimental Material
P-selectin (R & D)
Laboratory apparatus
Ultraviolet spectrophotometer (UV-2600, shimadzu)
Reagent preparation
P-selectin was diluted to 1. Mu.g/L with sample dilution, and 202-D was dissolved to 2. Mu. Mol/L and 20. Mu. Mol/L.
Experimental methods
The absorption spectra of 1. Mu.g/LP-selectin, 2. Mu. Mol/L, 20. Mu. Mol/L202-D and sample dilutions were scanned separately using an ultraviolet spectrophotometer and the base absorption curve was plotted using origin. 202-D was added to 1. Mu.g/L of P-selectin to give a final concentration of 2,4,6,8, 10, 12, 16, 20. Mu. Mol/L in this order, and the P-selectin absorption spectrum was scanned with an ultraviolet spectrophotometer in this order and an absorption curve was plotted with origin.
Results of the experiment
The results of the experiment are shown in FIG. 7. The experimental result shows that the absorption spectrum of the P-selectin at 280nm shows obvious color reduction effect with the increase of the concentration of the 202-D, and the change is not existed in a basic absorption curve along with the dose dependence, which indicates that the compound 202-D is combined with the P-selectin in vitro to cause a series of changes of the absorption spectrum, and an unexpected technical effect is obtained.
Example 14 ELISA determination of competitive binding of P-selectin inhibitor 202-D to PSGL-1 on the HL60 surface
Experimental Material
PSI-697 (MedChemExpress), physiological saline (Shijiazhuang Siyao Co., ltd.), polystyrene ELISA plate (uncoated, nunc Maxisorp), recombinant Human P-selectin (R & D), anti-Human IgG (Fc specific) (Taike Lanbo), 3% BSA blocking solution (Huaxingbio), ELISA coating solution (1X, pH 9.6, solebao), HL60 (Human leukemia cells, jiangsu Kaiyi Co., ltd.).
Laboratory apparatus
Microscope (Caiss)
Experiment grouping
NS；PSI-697：5μmol/L；202-D：25，100μmol/L
Experimental methods
The plates were plated overnight with goat anti-mouse IgG antibody (20 ng/ml) 50 ul/Kong Bao. After washing the plates three times, blocking with 3% BSA/PBS. 50ul of P-selectin recombinant protein (200 ng/ml) was added at room temperature for incubation. After washing the plate three times, 100ul HL60 cells (1000/well, n = 6) containing NS, PSI-697 (25. Mu. Mol/L) and 202-D (25, 100. Mu. Mol/L), respectively, were added to the wells, after 30min of reaction the ELISA plates were inverted on a centrifuge, 1000r,3min removed unbound cells, and the number of bound cells was counted under a microscope. The results were subjected to t-test, and the results were expressed as percentage of adherent cells.
[ percentage of adhered cells = (number of adhered test group-number of nonspecific adhesion) ÷ (number of adhered normal saline control group-number of nonspecific adhesion) × 100% ]
Results of the experiment
The results of the experiment are shown in FIG. 8. Compared with NS, under the action of 202-D and PSI-697 of 25 mu mol/L, the number of HL60 cells adhered to the ELISA plate shows a significant difference, P is less than 0.01, and the action effect is equivalent, P is more than 0.05; 100. Mu. Mol/L of 202-D showed a difference with 25. Mu. Mol/L of 202-D and PSI-697, P < 0.05.
This indicates that PSGL-1 expressed on the cell surface of the tested compounds 202-D and HL60 competitively binds to P-selectin, and 202-D can interact with P-selectin in vitro, so that the binding of PSGL-1 and P-selectin is dissociated, and the effect is equivalent to that of PSI-697 at the same concentration. While the effect of 100. Mu. Mol/L202-D was significantly better than that of 25. Mu. Mol/L202-D and PSI-697, which suggests that the ability of 202-D to competitively bind PSGL-1 may be accompanied by a dose-dependent relationship.
Example 15 evaluation of solubility of P-selectin inhibitor 202-D as an oral Small molecule drug
Experimental Material
PSI-697 (CAS: 851546-61-7, medChemexpress), ultrapure water
Laboratory apparatus
Ultraviolet spectrophotometer: (UV-2600, shimadzu)
Experiment grouping
Compound 202-D of the present invention, and the known P-selectin inhibitor PSI-697.
Reagent preparation
Preparing 202-D with serial concentrations by water in sequence: 0.5,0.25,0.125,0.063,0.032g/L.
Experimental methods
Firstly, the maximum absorption of 202-D is 362.4nm measured under an ultraviolet spectrophotometer, the maximum absorbance of 202-D at 362.4nm under the concentration of 0.5,0.25,0.125,0.063 and 0.032g/L is sequentially measured, and a regression equation is drawn according to the concentration-absorbance. Preparing supersaturated 202-D, centrifuging, collecting supernatant to obtain saturated 202-D solution, measuring absorbance at 362.4nm, substituting into regression equation to calculate concentration, and obtaining that the maximum solubility of 202-D in water is 1.6g/L.
Results of the experiment
The results of the experiment are shown in FIG. 9. Compared with the previously reported P-selectin inhibitor PSI-697, the 202-D water solubility of the invention is improved by 89 times, and the invention is more beneficial to oral absorption. Unexpected technical effects are obtained.
Claims (8)
1. A P-selectin inhibitor, designated 202-D, having the structural formula shown in formula I:
2. a process for preparing the P-selectin inhibitor of claim 1, comprising the steps of:
(1) At 1M H 2 SO 4 Catalyzing L-Trp and formaldehyde to perform Pictet-Spengler condensation reaction to prepare 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid;
(2) Methanol is used as a solvent, and carboxyl on three positions of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid is introduced into methyl ester for protection under the catalysis of thionyl chloride to obtain 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl ester;
(3) Reducing methyl ester of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl ester into alcoholic hydroxyl by taking anhydrous tetrahydrofuran as a solvent in the presence of lithium aluminum hydride to obtain 3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol;
(4) Tetrahydrofuran as solvent in NMM (Boc) 2 Introducing Boc protection to 3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol two-position-NH in the presence of O to obtain Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol;
(5) Using anhydrous tetrahydrofuran as a solvent, and carrying out intermolecular condensation on Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-primary alcohol and Boc-D (OBzl) -OH in the presence of DCC and DMAP to obtain Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OBzl;
(6) Dissolving the obtained Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OBzl in methanol, and reacting at Pd/C, H 2 Removing OBzl protection under the condition to obtain Boc-3S-1,2,3,4-tetrahydro-beta-carboline-3-methyl- (Boc) D-OH;
(7) de-Boc protection under 4N EA/HCl conditions to yield the P-selectin inhibitor 202-D of claim 1.
3. The use of a P-selectin inhibitor according to claim 1 in the preparation of an anti-tumor medicament.
4. Use of a P-selectin inhibitor according to claim 1 for the preparation of an anti-arterial thrombosis medicament.
5. Use of a P-selectin inhibitor according to claim 1 for the preparation of an anti-venous thrombosis medicament.
6. Use of a P-selectin inhibitor according to claim 1 for the preparation of an anti-inflammatory medicament.
7. The use of a P-selectin inhibitor according to claim 1 in the preparation of a P-selectin inhibitor, which comprises the following three parts:
(1) The function of reducing the content of soluble P-selectin in vivo of the tested animal;
(2) Ultraviolet spectrum identifies the binding effect of the in vitro and the P-selectin;
(3) Direct binding to P-selectin.
8. Use of the P-selectin inhibitor of claim 1 in the preparation of a small molecule oral medicament.
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