CN111187268B

CN111187268B - Compound CYD19 or pharmaceutically acceptable salt as Snail inhibitor and preparation method, pharmaceutical composition and application thereof

Info

Publication number: CN111187268B
Application number: CN202010050205.6A
Authority: CN
Inventors: 吴照球; 陆涛; 陈亚东; 朱雍; 傅蓉; 李红玫; 崔昊; 李阳
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-01-16
Filing date: 2020-01-16
Publication date: 2021-08-17
Anticipated expiration: 2040-01-16
Also published as: CN111187268A

Abstract

The invention discloses a compound CYD19 as a Snail inhibitor with a structure shown in a formula (I) or a pharmaceutically acceptable salt, and a preparation method, a pharmaceutical composition and application thereof. The application of the compound in preparing the medicine for preventing or treating the diseases of abnormal expression of Snail. In particular to the application of the compound in preparing anti-tumor and cardiovascular medicaments. Pharmacological results show that the compound has high affinity with Snail protein, and has good in vitro anti-tumor cell proliferation effect, cell apoptosis promotion effect and tumor metastasis inhibition effect. And the compound shows obvious in-vivo anti-tumor proliferation and metastasis activity in HCT-116 subcutaneous xenograft tumor nude mice and in-vivo liver metastasis models injected by spleens, and is a potential medicament for clinically preparing and treating tumors.

Description

Compound CYD19 or pharmaceutically acceptable salt as Snail inhibitor and preparation method, pharmaceutical composition and application thereof

Technical Field

The invention relates to a Snail inhibitor or a medicinal preparation thereof, a preparation method, a medicinal composition and application thereof, in particular to a compound CYD19 or a medicinal salt serving as the Snail inhibitor, and a preparation method, a medicinal composition and application thereof.

Background

Malignant tumor is a serious disease seriously threatening the life and health of human beings, and has the characteristics of high growth speed, strong invasion and infiltration capacity and frequent distant metastasis. The biological properties of abnormal proliferation, apoptosis resistance, invasion and metastasis of tumor cells and the like bring a serious challenge to tumor treatment. Among them, tumor metastasis is one of the more difficult key factors for tumor treatment, and the most obvious feature is the occurrence of epithelial-mesenchymal transition (EMT). For tumor cells, EMT not only confers the ability of tumor cells to migrate and invade, but also induces the sternness characteristics of tumor cells. Therefore, the intensive research on the medicines which can treat the tumors in a multi-mechanism and synergistic and effective manner is very important.

Snail is believed to be the major transcription factor inducing EMT by inhibiting the E-cadherin protein, with a highly conserved C-terminal₂H₂The zinc finger of (1), a carboxy terminal and a poly-variable amino terminal regulatory region. Wherein, the zinc finger structure of Snail can recognize and combine with E-box (5-CANNTG-3) region in E-cadherin promoter, promote the occurrence of EMT and is related to tumor metastasis. In the lesion tissues of breast cancer and colon cancer, the expression level of Snail is increased, and the high expression of Snail in tumor cells can inhibit the expression of wild p53 to resist apoptosis and accelerate the growth of tumor cells. Therefore, considering the action of the transcription factor in the tumor cell deterioration process, the transcription factor is taken as a drug target, and the tumor-related signal transduction pathway is targeted to eliminate the malignant phenotype of the tumor and the drug tolerance of the tumor, so that the transcription factor is a reasonable anti-tumor treatment strategy and has important research value.

However, small molecule inhibitors that directly target Snail proteins have not been reported, and can only inhibit expression of Snail by some indirect means. At present, Park et al report a novel natural product GN25, which is mainly capable of blocking the combination of Snail and p53 in HCT-116 cells and inducing the expression of p53 in cancer cells in a K-Ras-dependent manner, and does not show obvious inhibition effect in A549 cell nude mouse models, thereby limiting the further development of the compound. In addition, an irreversible Snail-binding Co (III) -DNA complex has been shown to selectively inhibit Snail transcription, regulate the Heregulinb1(HRG) -induced MCF-7 cell metastasis process, and show some potential for resisting tumor metastasis, but the mechanism research is uncertain. Therefore, Snail inhibitors that seek new mechanisms of action are of great interest.

The Snail is a protein with extremely short half-life period and instability, the Snail is easily and rapidly degraded by proteasomes in cytoplasm and extremely low in content, and the Snail enters cell nucleus, so that the protein stability is increased, the transcription inhibition function is exerted, the expression of downstream target genes p21, Rb, MMP, caspase and the like is influenced, and the tumor progress is finely regulated. The protein stability of Snail is not only related to cell localization, but the level of post-translational modification of the protein strongly affects its stability. The Snail has various post-translational modification forms, such as acetylation, phosphorylation, ubiquitination and the like, and the abundant post-translational modification level of the Snail jointly regulates and controls the content of the Snail. Researches such as HSU and the like prove that Snail can interact with CBP (CREB Binding Protein)/p 300, so that Snail Protein lysine 146 and lysine 187 are acetylated, the stability of the acetylated Snail is increased, the ubiquitination is weakened, and the transcription of downstream multiple cytokines CCL2 and IL8 is enhanced. Therefore, blocking interaction of Snail and related proteins such as CBP/p300 or ubiquitination ligase can be an effective strategy for inhibiting tumor progression, and a new direction is provided for tumor treatment.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a compound N- (2-amino-4-fluorophenyl) -4- [ [ [4- [ (5-methyl-1H-pyrazol-3-yl) amino ] pyrrolo [2, 1-f ] [1, 2, 4] triazin-2-yl ] sulfur ] methyl ] benzamide (hereinafter referred to as CYD19) or a pharmaceutically acceptable salt serving as a Snail inhibitor.

The invention also aims to provide a preparation method of the compound CYD19 serving as the Snail inhibitor or a pharmaceutically acceptable salt.

Another object of the present invention is to provide the use of compound CYD19 or a pharmaceutically acceptable salt thereof as a Snail inhibitor.

It is a final object of the present invention to provide a pharmaceutical composition.

The technical scheme is as follows: the invention provides a compound CYD19 which has the structure of formula (I) and is used as a Snail inhibitor or a pharmaceutically acceptable salt thereof,

further, the compound CYD19 which has the structure shown in the formula (I) and is used as a Snail inhibitor or a pharmaceutically acceptable salt thereof is prepared by the following steps:

dissolving 4- [ [ [4- [ (5-methyl-1H-pyrazol-3-yl) amino ] pyrrolo [2, 1-f ] [1, 2, 4] triazine-2-yl ] sulfur ] methyl ] benzoic acid in N, N-dimethylformamide, TBTU and N, N-diisopropylethylamine, stirring at normal temperature, adding p-fluoro-o-phenylenediamine, performing column chromatography by using petroleum ether and ethyl acetate, performing reduced pressure evaporation to dryness, recrystallizing in ether, and drying to obtain a product CYD 19.

The application of the compound CYD19 which has the structure shown in the formula (I) and is used as a Snail inhibitor or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating diseases related to abnormal expression of Snail.

Further, the disease associated with abnormal expression of Snail is a tumor or cardiovascular disease.

Further, the tumor is colon cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, lung cancer, gastric cancer, leukemia or other tumors resistant or tolerant to treatment; the cardiovascular disease is atherosclerosis.

The use of compound CYD19 having the structure of formula (I) as a Snail inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting tumor cell proliferation.

The use of compound CYD19 having the structure of formula (I) as a Snail inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting tumor cell metastasis.

A pharmaceutical composition, which comprises a therapeutically effective amount of compound CYD19 having the structure of formula (I) as Snail inhibitor according to claim 1 or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or adjuvant.

Further, the application in the preparation of chemosensitizer.

Further, the chemotherapy sensitizer is a sensitizer for increasing paclitaxel, cisplatin, bortezomib, fluorouracil and irinotecan.

Further, a pharmaceutically acceptable salt is an acid addition salt of compound CYD19 with hydrochloric, hydrobromic, citric, sulfuric, phosphoric, methanesulfonic, p-toluenesulfonic, salicylic, fumaric, maleic, succinic, tartaric, lactic, acetic, pyruvic, phenylacetic or mandelic acid.

The application of the compound CYD19 or the structural analogue thereof in preparing the drugs for treating tumor or cardiovascular diseases mainly comprises the following action mechanisms: inhibiting the binding of Snail to CBP or other proteins; as cell proliferation inhibitors; promoting apoptosis; inhibiting tumor metastasis.

The compound CYD19 or the structural analogue thereof can be combined with other antitumor drugs, such as a mitosis inhibitor (such as paclitaxel), an alkylating agent (such as cyclophosphamide or cisplatin), a proteasome inhibitor (such as bortezomib), a metabolic antagonist (such as fluorouracil), a topoisomerase inhibitor [ irinotecan) and the like, so as to play a synergistic antitumor role and improve the tumor treatment effect.

Has the advantages that: the compound CYD19 has high affinity with Snail protein, inhibits interaction between Snail and CBP/p300, and weakens acetylation level of Snail, thereby accelerating degradation of Snail and having obvious in vivo and in vitro anti-tumor effect. Therefore, the compound can be used for preparing anti-tumor or cardiovascular medicaments and has potential application prospect.

Drawings

FIG. 1 is a graph showing the results of the Biolayer interferometry assay, which shows that Compound CYD19 has high affinity for Snail proteins;

FIG. 2 is a graph showing the result of Western Blot assay, which shows that CYD19 can reduce the expression of Snail protein in cells;

FIG. 3 is a graph showing the results of the immunoprolipitation assay, showing that Compound CYD19 inhibits the interaction of Snail with CBP protein;

FIG. 4 is a CCK-8 assay result chart, which shows that compound CYD19 has strong proliferation inhibition effect on tumor cells;

FIG. 5 is a graph showing the result of Annexin V-PI staining test, which shows that CYD19 has the effect of promoting tumor cell apoptosis;

FIG. 6 is a graph showing the results of Transwell assay, which shows that CYD19 can inhibit tumor cell metastasis;

FIG. 7 is a graph of the results of CCK-8 assays showing that CYD19 enhances the chemotherapeutic sensitivity of paclitaxel;

FIG. 8 is a graph of the anti-tumor effect of compound CYD19 in an in vivo model in nude mice;

FIG. 9 is a graph showing the effect of HE staining assay CYD19 on the organs in nude mice;

FIG. 10 is a schematic representation of the anti-tumor mechanism of action of CYD 19.

Detailed Description

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: the preparation method of compound CYD19 or its pharmaceutically acceptable salt as Snail inhibitor in this example comprises the following steps:

completely dissolving 4- [ [ [4- [ (5-methyl-1H-pyrazol-3-yl) amino ] pyrrolo [2, 1-f ] [1, 2, 4] triazine-2-yl ] sulfur ] methyl ] benzoic acid in N, N-dimethylformamide, adding TBTU and N, N-diisopropylethylamine, stirring at normal temperature, adding p-fluoro-o-phenylenediamine, stirring at normal temperature, evaporating the reaction solution under reduced pressure to obtain an evaporated substance, adding the mixture of the evaporated substance and silica gel into an ethyl acetate solvent, evaporating at the temperature of 40-50 ℃ to obtain the evaporated substance, adding the evaporated substance to an upper layer of a silica gel column, eluting by using petroleum ether and ethyl acetate, evaporating the collected eluent under reduced pressure to dryness, recrystallizing in ether at room temperature, drying the separated crystal, to obtain N- (2-amino-4-fluorophenyl) -4- [ [ [4- [ (5-methyl-1H-pyrazol-3-yl) amino ] pyrrolo [2, 1-f ] [1, 2, 4] triazin-2-yl ] thio ] methyl ] benzamide.

Example 2: the Biolayer interaction assay measures the affinity of compound CYD19 for Snail proteins.

Recombinant Snail protein was dissolved in PBS and placed in 100uL tube. EZ-Link NHS-biotin was incubated with Snail recombinant protein for 60 minutes at room temperature (protein to biotin molar ratio 1: 3). Desalting is used to remove excess biotin. Biotinylated proteins were immobilized on a Super Streptavidin biosensor for further measurements. Blank recombinant protein was used as control.CYD19 at various concentrations (0.5, 0.25, 0.125, 0.063, 0.031. mu.M) served as nonspecific controls. Binding of CYD19 to Snail-protein was measured using an Octet red 96 instrument (ForteBio), off-intensity of light was recorded, and the rate of binding and dissociation was calculated using a 1: 1 binding model. The results show that CYD19 has higher affinity with Snail protein (K)_d180nM), fig. 1.

Example 3: WesternBlot examined the effect of CYD19 on Snail protein expression.

Inoculating cells in a six-well plate, treating for 24 and 48 hours by CYD19(25, 50, 75 and 100nM) with different concentrations after the cells are attached to the wall for 12 to 24 hours, extracting protein, measuring the concentration, preparing 10 percent SDS polyacrylamide gel without concentration gradient according to the molecular weight of the protein, concentrating the gel at 90V, separating the gel at 120V, and performing electrophoresis for 2.5 to 3 hours. The membrane is transferred by a full-wet membrane transfer method, filter paper and a nitrocellulose membrane with proper sizes are cut according to the sizes, the glue is glued on a negative electrode, the membrane is arranged on a positive electrode, the constant current is 250mA, and the membrane transfer time is adjusted according to the Kd number of the required protein. Immersing the protein-transferred nitrocellulose membrane into ponceau for dyeing for 5 minutes, washing with distilled water, observing the position of a protein strip, cutting a marker protein, and washing off ponceau dye liquor by PBST. Blocking with 5% skimmed milk powder (PBST formulation) for 1h at room temperature, adding the prepared primary antibody, and incubating overnight at 4 deg.C in a shaker. Discarding the primary antibody, washing PBST for 3 times and 5 min/time, adding a secondary antibody corresponding to the primary antibody, incubating for 1h at room temperature, discarding the secondary antibody after the reaction is finished, washing PBST for 3 times, washing PBS for 1 time and 5 min/time, adding ECL reaction solution, and exposing in a dark room. The results show that CYD19 can inhibit Snail protein expression in human breast cancer (BrCa) primary cells, breast cancer cells and colorectal cancer cell lines in a dose-dependent manner (fig. 2).

Example 4: immunopropractitation examined the effect of CYD19 on the interaction of Snail with CBP proteins.

HCT-116 cells in a 10cm culture dish were placed on ice, 500. mu.l of an IP lysis buffer containing a protease inhibitor was added to resuspend the cells, transferred to a 1.5ml Eppendorf tube, mixed well and incubated on ice for 30min, the cells were disrupted by sonication, centrifuged at 4 ℃ for 20min to collect the supernatant, and placed on ice for use. During centrifugation, a number of 1.5ml Eppendorf tubes were taken, 20. mu.l of ProteinA/G beads were added to each tube, 500. mu.l of an IP lysate without protease inhibitors were used to resuspend the beads, the collected protein supernatant was added to the washed beads, the tube was sealed, the 4-degree rotameter was incubated for 0.5 hour at 4 degrees 2000G for 2 minutes, the supernatant was collected, the protein concentration was measured, a portion was taken as Input (typically 10%), 5-fold sample buffer of equal volume was added, 100 degrees was boiled for 10 minutes, and the mixture was centrifuged slightly at-20 ℃ for use. The remaining protein supernatant was split into 2 tubes, tube a 1/4 and tube B3/4, and the volume was leveled to 500 microliters with several microliters of protease inhibitor-containing IP lysis buffer. Add 1. mu.l IgG to tube A and 2. mu.l (target protein) antibody to tube B, seal the tube mouth with a membrane, and incubate overnight on a 4-degree wheel-rotor. The next day, centrifuge slightly (centrifuge sample to tube bottom), add 30 microliters of rinsed beads per tube (conditions as before), continue to cycle at 4 ℃ for 2 hours, 4 degrees 2000g, centrifuge for 2 minutes, collect beads, add 1ml of protease inhibitor-free IP lysate buffer to rinse, incubate on 4 degrees cycler for 5min, centrifuge, collect beads, and repeat the above rinsing steps 8 times. The beads were collected, added with several microliters of 2 × loading buffer, incubated at 100 ℃ for 20min, centrifuged, and kept at-20 ℃ for further use, and subjected to the conventional Western blot procedure. The results show that CYD19 treatment of HCT-116 did not affect the expression of CBP/p300, but significantly reduced the protein expression level of endogenous Snail. CBP/p300 levels were significantly reduced in the Snail IP group of HCT-116 cells after treatment with CYD19, indicating that CYD19 strongly inhibited the binding of Snail to CBP/p300 (FIG. 3).

Example 5: CCK-8 measures the proliferation inhibitory effect of CYD19 on tumor cells.

The normally growing tumor cells were counted, and a cell suspension was prepared and seeded into a 96-well plate at about 100. mu.l per well. After the cells adhered, CYD19(6.25, 12.5, 25, 50, 100, 300, 1250, 5000nM) was added at various concentrations for a certain time (n ═ 6), 10 μ l CCK8 was added, and the cells were cultured for 1-4 h. And (3) measuring the corresponding absorbance by using a microplate reader, and calculating the cell survival rate of the medicine: survival rate (%): the survival rate (%) × (average absorbance of drug-added cells-average absorbance of blank medium)/(average absorbance of control cells-average absorbance of blank medium) × 100%. The results show that the compound CYD19 has obvious inhibition on the proliferation of RKO, HCT-116, PyMT and other tumor cells, the IC50 values are all less than 100nM and stronger, and the compound also has certain inhibition on the proliferation of SW620, DLD1, MDA-MB-231 and SUM159 (figure 4).

Example 6: annexin V-PI staining detects the pro-apoptotic effect of CYD19 on tumor cells.

The CYD19(25, 50, 75nM) treated cells were washed 2 times with PBS, then digested with EDTA-free pancreatin, and the digestion was stopped by adding the appropriate amount of medium. Cells were harvested by centrifugation at 200g, 4 ℃ for 3 min. The cells were washed with pre-cooled PBS, centrifuged at 300g and 4 ℃ for 5 minutes, and the cells were harvested. Add 100ul 1 × Binding Buffer to resuspend the cells, add 5 μ l Annexin V-FTIC and 5 μ l PI stabilizing Solution, mix gently. And reacting for 10min at room temperature in the dark. Add 400. mu.l of 1 Binding Buffer and mix gently. The samples were examined by flow cytometry. The results show that HCT-116, RKO, respectively, induced apoptosis in a dose-dependent manner after CYD19 treatment, exerting an anti-tumor effect (FIG. 5).

Example 7: the Transwell assay showed that CYD19 could inhibit tumor cell metastasis.

HCT-116 and PyMT cells were dispersed in serum-free medium, and 200. mu.l of cell suspension was taken and added to the Transwell chamber, and 1ml of complete medium was added to the lower chamber. Culturing for 48h, taking out the chamber, washing with PBS, fixing in methanol for 10min, washing with PBS, staining with 0.05% crystal violet for 1min, washing the chamber with water, wiping off excessive cells with cotton stick, washing, air drying, and taking a picture. The results showed that CYD19 could dose-dependently decrease migration of tumor cells such as HCT-116 cells and PyMT cells, and had strong ability to inhibit tumor cell metastasis (fig. 6).

Example 8: CCK-8 measures the proliferation inhibitory effect of CYD19 on tumor cells in combination with paclitaxel.

HCT-116 and PyMT cells were normally digested to prepare a cell suspension, which was seeded into a 96-well plate at about 100. mu.l cell suspension per well. After the cells were adherent, 20nM CYD19 and paclitaxel (0.5, 1, 2, 4nM) were added for 48h (n ═ 6), 10 μ l CCK8 was added, and the cells were cultured for 1-4 h. And measuring the corresponding absorbance by using a microplate reader, and calculating the cell survival rate of the medicament. The results show that under the condition of 20nM, the CYD19 shows obvious tumor proliferation inhibition of paclitaxel at lower concentration (2nM), and the combination of the compound and paclitaxel can obviously improve the sensitivity of paclitaxel (FIG. 7).

Example 9: antitumor effect of compound CYD19 in an in vivo model in nude mice.

Model of subcutaneous transplantation tumor in HCT-116 nude mice: HCT-116 cells were taken from logarithmic growth phase and resuspended in Matrigel (1X 10)⁷100. mu.l). Female nude mice were inoculated with 0.1ml of cell suspension on their backs for about 6 weeks. Subsequently, the tumor size was measured with a vernier caliper, and when the tumor grew to 100mm³At the time, animals were randomized into three groups: the vehicle group, the low dose CYD19(15mg/kg) group and the high dose CYD19(30mg/kg) group were administered by intraperitoneal injection daily to 6 nude mice each group at the corresponding dose. Tumor diameters were measured every 2 days, mice were sacrificed after 14 days, and relative tumor proliferation rates were calculated, and viscera and tumor tissues were taken for subsequent analysis.

GFP-labeled HCT-116 splenic injected nude mouse liver transfer model: GFP-labeled HCT-116 cells were prepared in logarithmic growth phase and resuspended in Matrigel at 1X 10 under sterile conditions⁷Female nude mice were left subcostal operated within 6-8 weeks from each 100. mu.l cell suspension, 0.1ml of cell suspension was injected into the spleen of nude mice, and the mice were randomized into 2 groups after suturing: vehicle and CYD19(30mg/kg) groups were intraperitoneally administered daily, vehicle and CYD19 drugs were administered, and body weight was recorded, and nude mice were sacrificed 21 days later and livers were taken for analysis.

The results showed that in the subcutaneous transplanted tumor model, the tumor volume of nude mice in CYD19 group was significantly decreased compared to that in the vehicle group, and the tumor inhibition rate of CYD19(30mg/kg, 50mg/kg) group was 65%, 72% (FIG. 8A), which had excellent in vivo anti-tumor growth effect. In addition, in the transfer model of nude mice, GFP fluorescence was decreased and the number of hepatic nodules was significantly decreased in the CYD19 group, confirming that CYD19 can inhibit tumor metastasis in nude mice (fig. 8B). Therefore, the compound CYD19 can effectively inhibit the growth and metastasis of tumors and is a potential anti-tumor medicament.

Example 10: HE staining examined the effect of CYD19 on major organs in nude mice.

The heart, liver, spleen, lung, kidney and other important organs of the mouse are soaked in 4% paraformaldehyde for fixation for 24 hours, embedded by paraffin, cut into 5-micron sections for conventional HE staining, and no obvious histological lesion is observed in the administration group and the solvent control group, which indicates that the nude mouse has good tolerance to the compound and low toxicity of the compound. (FIG. 9).

Claims

1. A process for the preparation of compound CYD19 having the structure of formula (I) as a Snail inhibitor or a pharmaceutically acceptable salt thereof, characterized in that:

dissolving 4- [ [ [4- [ (5-methyl-1H-pyrazol-3-yl) amino ] pyrrolo [2, 1-f ] [1, 2, 4] triazine-2-yl ] sulfur ] methyl ] benzoic acid in N, N-dimethylformamide, TBTU and N, N-diisopropylethylamine, stirring at normal temperature, adding (2-amino-5-fluorophenyl) -carbamic acid-1, 1-dimethylethyl ester, performing column chromatography by using petroleum ether and ethyl acetate, evaporating to dryness under reduced pressure, recrystallizing in diethyl ether, and drying to obtain a product CYD19,