CN105153142B - The Furazan Derivatives and antitumor activity of cumarin parent nucleus - Google Patents

Abstract

The invention belongs to the field of chemical pharmacy, and relates to furazan derivatives in which the coumarin core and its isomers are combined with furazan oxides through different lengths and structural links. It has been proved by experiments that it has excellent inhibitory effect on sensitive human tumor cells and drug resistance. Tumor strains and HUVEC normal cell growth inhibitory activity to determine the inhibitory effect of neovascularization; and have inhibitory activity on cell proliferation of cisplatin-resistant A2780 and gemcitabine-resistant HM231 cell lines. Preliminary pharmacology shows that it induces the apoptosis of cancer cells by releasing high concentrations of nitric oxide; inhibits MEK activity and significantly inhibits the phosphorylation of MEK in the MAPK/ERK pathway, resulting in a significant inhibitory effect on tumor growth. The compound of the invention has laid a good foundation for obtaining candidate drugs with inhibitory activity on both sensitive and drug-resistant tumors and elucidating their pharmacological action mechanism.

Description

Furazan derivatives of coumarin nucleus and their antitumor activity

technical field

The invention belongs to the field of chemical pharmacy, and specifically relates to coumarin core furazan derivatives and their antitumor activity.

Background technique

According to reports, tumor has become a common and frequently-occurring disease that seriously threatens human health and life, and has become one of the major diseases that cause global human death; cancer has surpassed cardiovascular and cerebrovascular diseases to become the "number one killer" of human health and one of the world's largest public health problems. Statistics show that cancer and mortality in my country are on the rise; among urban residents, cancer has become the disease with the highest mortality rate. At present, clinical tumor treatment methods mainly include surgical treatment, radiation therapy (radiotherapy), chemical drug therapy (chemotherapy), and biological therapy (including immunotherapy and gene therapy), interventional therapy, hyperthermia, etc., which have been booming in recent years. Drug therapy is still the main treatment. Over the past few decades, humans have discovered more than 100 effective anti-tumor drugs, but clinical practice shows that the commonly used anti-tumor drugs have reduced selectivity due to long-term or large-scale use, resulting in relatively toxic side effects and drug resistance and other shortcomings. Therefore, finding low-toxicity, high-efficiency, and highly targeted anti-tumor drugs is still one of the important topics for pharmacologists and an urgent clinical need.

Studies have shown that mitogen-activated protein kinases (MAPKs) are a class of highly conserved serine/threonine protein kinases in cells. The MAPKs signal transduction pathway transduces extracellular stimulus signals to cells and their nuclei, and plays a vital role in the processes of cell proliferation, differentiation, transformation and apoptosis. MAPKs include four subfamilies: extracellular signal-regulated protein kinase (ERKs, extracellular-signalregulated protein kinase); c-Jun amino-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK) and ERK5 pathway; studies have shown ERK is an important family of MAPKs. It consists of three core protein kinases, Raf, MEK and ERK. Its signal transduction follows the three-level enzymatic cascade reaction of MAPKs, that is, the upstream activator → MAPK kinase kinase (MAPKKK) →MAPK kinase (MAP-KK)→MAPK, in the ERKs pathway, Ras acts as an upstream activating protein, Raf acts as MAPKKK, MAPK/ERK kinase (MEK) acts as MAPKK, ERK is MAPK, that is, Ras/Raf/MEK/ERK pathway; Ras The /Raf/MEK/ERK signal transduction pathway is closely related to the occurrence of tumors, and the overactivation of ERK is also closely related to the proliferation, survival, differentiation and apoptosis of tumors, such as in lung cancer, colon cancer, kidney cancer, pancreas The cascading activation of downstream protein kinases caused by pathological Ras or mutated b-Raf has been found in cancer and breast cancer, which will eventually lead to excessive activation of ERK (Oncogene, 1999, 18, 813-822.);

MEK is a key node in the Ras/Raf/ERK/MAPK pathway: ERKs are the only substrates of MEK, and ERKs can only be activated by MEK; this indicates that the blocking effect of MEK inhibitors on ERK activation is highly specific, which also This makes MEK inhibitors a promising anti-tumor target (Oncogene, 2007, 26, 3291-3310); the reported MEK inhibitors mainly include ATP competitive and ATP non-competitive inhibitors, the former is affected by the concentration of ATP low selectivity and specificity, and high toxicity; the latter neither competes with ATP binding sites nor competes with ERK for MEK binding sites, and has no homologous sequences with other kinases, so it has a good specificity and selectivity;

At present, diarylamines are the most researched, and several small molecule MEK inhibitors have entered clinical or preclinical research, for example, Pfizer's PD325901 and AstraZeneca's selumetinib (Selumetinib, as shown in the following formula shown) have passed Phase I clinical trials and entered Phase II clinical trials; and the small molecule MEK inhibitor Trametinib (Trametinib, shown in the following formula) of GlaxoSmithKline (GSK) has been approved by the United States in May 2013. Approved by the FDA for the treatment of melanoma; in addition, there are also reports (Bioorg.Med.Chem.Lett.2005, 15, 5467–5473; Bioorg.Med.Chem.Lett.2013, 23, 6223-6227) A MEK inhibitor with a coumarin skeleton, such as G8935, has good overlap with the co-crystallized small molecule diarylamine MEK inhibitor PD318088 (shown in the following formula) in MEK (PDB code: 1S9J); in addition Some researchers proposed three pharmacophore models of MEK inhibitors based on diarylamine MEK inhibitors. Molecular simulations showed that G8935 has two of them, such as the SER212 residue in the active center of MEK kinase, which can form a key In addition, the benzyl group at the 3-position of the coumarin core also occupies a new binding pocket, and this new role plays a crucial role in the activity of the MEK inhibitor of the coumarin skeleton.

(I) Part of phase II clinical and marketed MEK inhibitors

Natural coumarin and its derivatives are ubiquitous in plants, animals and microbial metabolites, they not only play an important role in the field of agriculture and medicine (John Wiley&Sons Ltd, New York, 1982,21.), the derivatives also have Various biological activities such as antibacterial, antiviral, antiosteoporosis and antitumor (J. , 75634; Anticancer Res. 2001, 21, 917-923; Med. Res. Rev. 2003, 23, 322-345). Studies have shown that in a series of synthetic derivatives of coumarin and its pyrone positional isomers, 4-methyl-7-aryl heterocyclic thio or substituted benzylthio coumarins were found to be in vitro It has good cell activity against KB (nasopharyngeal carcinoma cell) and its drug-resistant tumor line KB-vin (vinblastine-resistant subtype nasopharyngeal carcinoma cell), A549 (lung cancer cell) and DU145 (prostate carcinoma cell) Toxic activity, especially the compound 7-(6-chloropyridin-2-thio)-4-methyl-2H-chromene-2-one (CY-1-140A) showed IC for the above four tumor strains respectively Inhibitory activity with ₅₀ values of 0.92, 0.92, 2.11 and 1.15 μM. Preliminary pharmacological studies have shown that this type of compound has a significant concentration-dependent effect on the apoptosis of A549 lung cancer cells, and acts on the G2/M phase of the cell division cycle (Eur.J.Med.Chem.2012,49,74-85.). Based on the above literature (Bioorg.Med.Chem.Lett.2005,15,5467–5473; Bioorg.Med.Chem.Lett.2013,23,6223-6227) reported that the coumarin derivative G8935 inhibits MEK activity, it is speculated that these Compounds may have MEK inhibitory effects.

It is well known that nitric oxide (NO, Nitric oxide) is an important signal and effector molecule in the human body, involved in numerous pathological and physiological processes (Nat. Rev. Cancer 2006, 6, 521-534.). In particular, its anti-tumor activity through multi-target effects has attracted widespread attention. NO shows two sides to this, that is, low concentrations of NO promote the occurrence and development of tumors, while high concentrations of NO (>500nM) inhibit (Carcinogenesis 2013 ,34,503–512) tumor growth; studies have shown that the modified nitric oxide donor releases high concentrations of nitric oxide, which not only induces apoptosis and inhibits cancer cell metastasis, but also inhibits hypoxia-inducible factor (hypoxia -inducible factor-1α, HIF-1α), nuclear factor-κB (nuclear factor-κB, NF-κB), DNA damage repair, and drug transporter to sensitize chemotherapy, radiotherapy and immunotherapy (Nitric Oxide 2008,19,152-157; Cancer Res.2005,65,516-525; Curr.Pharm.Des.2008,14,1113-1123) to achieve tumor inhibition. Among them, benzenesulfonylfurazan nitric oxide is an important kind of nitric oxide donor, which can release high concentration of nitric oxide in vivo to produce anti-tumor activity (Pharmazie 2006, 61, 54-59.). Zhang Yihua and Tian Jide Research Group (Bioorg.Med.Chem.Letts.2010,20,6416–6420; J.Med.Chem.2011,54,3251–3259; J.Med.Chem.2013,56,4738-4748 ) has reported that the splicing products of active compounds such as FTs, WZ4002 and GA with furazan-type nitric oxide donors have shown good antitumor activity (as shown in formula (II)). It is also reported that the combined use of MEK inhibitors and nitric oxide donors can produce synergistic antitumor activity (Int. J. Oncol. 2012, 40, 807-815).

Based on the structure-activity relationship obtained in the previous study, the introduction of the heterocyclic substituent at the 7-position of the coumarin core is beneficial to the antitumor activity. Introduce it into the coumarin core, observe its anti-tumor activity and explore the corresponding mechanism of MEK inhibition, and provide a new furazan derivative with anti-tumor activity of the coumarin core.

(II) Complexes of some active compounds and furazan-type nitric oxide donors

Contents of the invention

The purpose of the present invention is to provide a novel anti-tumor pharmacological compound with clear activity and an effect on drug-resistant strains. Specifically related to the compound 4-(2-(4-methyl-2-oxo-2H-chromone-7-hydroxy)-ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadi Use of azole 2-oxides (8b) and derivatives thereof and tumor growth inhibitory activity.

Based on the original active compound CY-1-140A(1), the present invention utilizes skeleton transition and splicing principles to design and synthesize furazan derivatives with anti-tumor activity as shown in Figure 1. The compounds described in the invention are furazan derivatives in which the coumarin core and its isomers are combined with furazan oxides through different lengths and structural links,

In the present invention, the preferred coumarin-furazan derivatives have the structures of formulas 18 and 19, and the coumarin-furazan derivatives are the coumarin core and its pyrone skeleton isomers and furan-type The splice product of the nitric oxide donor, with different length chain or ring linking groups (linker in English) in the middle, (as shown in Figure 6),

Where: X is oxygen, sulfur and nitrogen atoms and alkyl substituted nitrogen atoms

R ₁ is an alkyl group or a hydrogen atom

R ₂ is alkyl, alkoxy, haloalkyl, ester

R ₃ is a linking group of different lengths, including chain or cyclic carbon chains, ester chains, and heterocyclic chains of different lengths.

In the present invention, among the coumarin furazan derivatives of more preferred formulas 18 and 19 structures: X=O, S, N or N-methyl; R ₁ is an alkyl group, a hydrogen atom; R ₂ is an alkyl group, Alkoxy, haloalkyl, ester; _R3 is a chain or cyclic aliphatic carbon chain, ester carbon chain and heterocyclic chain (including piperazine ring) of different lengths.

The invention provides the preparation method of the furazan derivative of the coumarin nucleus, which comprises:

(1). Synthesize the key intermediate (4) according to references (J.Heterocycl.Chem.1977,14,1415-1416.); Take thiophenol and chloroacetic acid as starting raw materials with sodium carbonate and sodium hydroxide React in a mixed solution of ethanol and water to obtain compound 2; then use glacial acetic acid as a solvent and oxidize with hydrogen peroxide to obtain 3; compound 3 is condensed with fuming nitric acid to obtain the key intermediate benzenesulfonylfurazan nitrogen oxide (4); Its chemical reaction is as follows general formula one:

General formula one:

(2). Commercially available 7-hydroxycoumarin derivatives (5a-e) without methyl, 4-methyl or 4,8-dimethyl substitution and references (Eur.J.Med.Chem.2011 , 46,4924-4936) the pyrone skeletal isomer 14 of the homemade coumarin core, in dimethylformamide (DMF) or acetone, reacted with the corresponding haloalcohol under alkaline conditions of potassium carbonate through reflux reaction , to obtain compounds 7a-g and 16a-b with carbon chains of different lengths at the 7-position; and intermediate 7g containing N in the side chain and methyl iodide to further obtain N-methylated product 9a, compound 5a-b and chloroacetic acid , Refluxing reaction of potassium carbonate in DMF obtains the 7-carboxy side chain intermediate 11a-b, further with ethylene glycol with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ( EDC,) and 4-(dimethylamino)pyridine (DMAP) catalyzed condensation to give intermediates 12a-b with side chains containing ester bonds, and finally, intermediates 5a-c, 7a-g with chains of different lengths at the 7-position , 9a, 12a-b, 14 and 16a-b react with furazan oxynitride 4 in dichloromethane containing base 1,8-diazabicycloundec-7-ene (DBU) at room temperature The target derivatives 6a-c, 8a-g, 10a, 13a-b, 15 and 17a-b were synthesized. The chemical reaction is as follows general formula two:

General formula two:

The coumarin nuclei and its isomers of the present invention are combined with furazan oxides through different lengths and structural links to carry out in vitro anti-tumor activity screening tests. In the in vitro pharmacological experiments, the original The active compound CY-1-140A(1), furazan fragment 4 and cisplatin are used as positive controls to detect A549, Hela, A2780, HUVEC, SKOV3, 231HM and drug-resistant A2780/CDDP, SKOV3/CDDP and 231HM/Gem A variety of sensitive and drug-resistant human tumor cell growth inhibitory activities, the results show that the compound has excellent inhibition of A2780 (sensitive and drug-resistant human ovarian cancer cells), SKOV3 (sensitive and drug-resistant human ovarian cancer cells), HM231 ( Sensitive and drug-resistant breast cancer lung cells), Hela (uterine cancer cells), A549 (lung cancer cells) and HUVEC (human umbilical vein endothelial cells), five sensitive human tumor cells, three drug-resistant tumor lines and a new HUVEC normal cell growth inhibitory activity of vascular inhibition; some of the compounds also have nanomolar inhibitory activity on cell proliferation of cisplatin-resistant A2780 and gemcitabine-resistant HM231 cell lines; especially, compound 8b has inhibitory activity on cisplatin-resistant A278 The IC ₅₀ values of SKOV3 cells and gemcitabine-resistant HM231 cells were 0.062, 0.14 and 0.14 μM, showing obvious inhibitory activity;

In the present invention, preliminary pharmacological studies have shown that the multiple mechanisms of the compound, through the release of high-concentration nitric oxide, induce the apoptosis function of cancer cells; inhibit the activity of MEK and significantly inhibit the phosphorylation of MEK in the MAPK/ERK pathway, and finally produce a significant effect of inhibiting tumor growth;

The present invention further carried out an in vivo tumor-bearing nude mouse model test, and the results of in vivo pharmacological experiments showed that the compound had obvious tumor growth inhibitory effect, especially compound 8b showed good tumor growth inhibition in the tumor-bearing nude mouse model Activity (15 and 30 mg/Kg dosing).

In the present invention, the anti-tumor activity of the compounds is detected by the following method.

1. Using the MTT method to detect the activity of the compound of the present invention in inhibiting tumor cell proliferation, the cancer cells in the logarithmic growth phase were inoculated in a 96-well plate, and divided into blank control group, cisplatin control group and different concentrations of the compound treatment group of the present invention After culturing at 37°C for 48 hours, MTT assay was performed, and the cell proliferation inhibition rate and IC ₅₀ were calculated;

2. Apply Western blot (immunoblotting) analysis to detect that the compound of the present invention inhibits MEK phosphorylation and promotes apoptosis. After A2780 cells were treated with DMSO and different concentrations of the compound of the present invention for 24 hours, the collected cells were washed once with cold PBS, and washed once with Cells were lysed with RIPA protein lysate on ice for 30 minutes, centrifuged at 4°C for 15 minutes, the total protein was quantified by BCA method, and the loading amount of 30 μg per lane was transferred to polyvinylidene fluoride PVDF membrane by SDS-PAGE electrophoresis On top, block in 10% skimmed milk, react with primary antibody and secondary antibody in turn, and develop color by exposure;

3. Use the classic Griess reagent to detect the NO release level of the compound of the present invention. After incubation of the tumor cell A2780 (1×10 in a ⁶ cm dish) with the compound of the present invention at a concentration of 100 μM for 150 minutes, collect the cells and lyse them on ice with RIPA protein lysate The cells were mixed for 30 minutes, then mixed with Griess reagent for 30 minutes, and the absorbance was measured at 540nm. The nitrite in the cells was treated with the diluent as the background control, and the standard curve was prepared by detecting sodium nitrate at different concentrations;

4. Tumor suppression experiment in vivo: On the 10th day after nude mice were inoculated with subcutaneously transplanted tumors, the established tumor-bearing mice were randomly divided into 6 groups. In the experimental group, the test sample was injected intraperitoneally (it is not necessary to choose a suitable solvent according to the solubility), and each sample was set at 2 concentrations, and a negative control group (give blank solvent), administered 2 times a week, and the tumor growth size was measured and recorded , the nude mice were sacrificed 2 days after drug withdrawal, the tumor tissue was stripped, and the tumor mass was weighed.

In the present invention, preliminary pharmacological studies have shown that the multiple mechanisms of the compound, through the release of high-concentration nitric oxide, induce the apoptosis function of cancer cells; inhibit the activity of MEK and significantly inhibit the phosphorylation of MEK in the MAPK/ERK pathway, and finally produce a significant effect of inhibiting tumor growth;

The coumarin nuclei furazan derivatives described in the present invention have a novel structure, have obvious activity of inhibiting tumor cells in vitro and significantly inhibit tumor growth in vivo, and have inhibitory activity on both sensitive and drug-resistant tumors for in-depth research. Candidate drugs and elucidating their pharmacological mechanisms have laid a good foundation.

Description of drawings

Figure 1 is a schematic diagram of coumarin and its parent nuclear isomer furazan derivatives.

Figure 2 shows: (A) Pro-apoptotic and (B) Inhibitory MEK phosphorylation.

Figure 3 A, B and C show tumor suppressor activity in vivo.

Figure 4 shows the NO release levels.

Figure 5 shows the docking diagram of compound 8b and MEK1 protein.

Figure 6 shows that preferred coumarin-furazan derivatives have the structures of formulas 18 and 19, which are the coumarin core and its pyrone skeletal isomers and furan-type The splicing product of nitric oxide donors, with chain or ring linking groups (linker in English) of different lengths in the middle.

detailed description

The following implementation methods will help to understand the present invention, but are not limited to the content of the present invention.

Example 1

A compound 4-(4-methyl-2H-chromene-2-dione-7-oxyl group)-3-(benzenesulfonyl)-1,2,5-oxa in compound 6a-c in general formula 2 Synthesis of oxadiazole 2-oxide (6b)

7-Hydroxy-4-methyl-coumarin (5b, 300mg, 1.2mmol) and benzenesulfonylfurazan N-oxide (4, 256mg, 1.0mmol) and DBU (2.0mmol) in dichloromethane (6mL ) solvent, react at room temperature for 3 hours; then wash away the DBU with 3×5ml water, distill off the solvent under reduced pressure to obtain an oily liquid. A small amount of methanol was added to precipitate a white solid, which was filtered and recrystallized with ethyl acetate to obtain product 6b (320mg), with a yield of 90% and a melting point of 201-203°C. ESI-MS m/z 401.0[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ2.46(s, CH ₃ C=), 6.32(s, 1H, -CH ₃ C=CHCOO), 7.31(m, 2H, ArH), 7.68(m, 3H, ArH), 7.81 (t, 1H, ArH, J = 6.4 Hz), 8.10 (d, 2H, ArH, J = 7.48 Hz).

Example 2

Compound 4-(2-ethyl-4H-chromene-4-one-7-oxyl group)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide ( 15) Synthesis of

7-Hydroxy-2-ethyl-coumarin (14, 300mg, 1.2mmol) and benzenesulfonylfurazan N-oxide (4, 256mg, 1.0mmol) and DBU (2.0mmol) in dichloromethane (6mL ) solvent, react at room temperature for 3 hours; then wash away the DBU with 3×5ml water, distill off the solvent under reduced pressure to obtain an oily liquid. A small amount of methanol was added, and a white solid was precipitated. After filtration, it was recrystallized with ethyl acetate to obtain product 15 (280 mg). The yield is 89%, and the melting point is 159-161°C. ESI-MS m/z 415.0[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ1.32(t, 3H, CH ₂ CH ₃ , J=7.56Hz), 2.47(q, 2H, CH ₂ CH ₃ , J=7.56Hz), 6.21(s, 1H, OC=CH), 7.32(dd, 1H, ArH, J=8.80, 2.36Hz), 7.46(d, 1H, ArH, J=2.32Hz) ,7.66(t,2H,ArH,J=8.28Hz),7.81(t,1H,ArH,J=7.56Hz),8.08(dd,2H,ArH,J=1.28,8.48Hz),8.27(d,1H , ArH, J=8.80Hz).

Example 3

Synthesis of a compound 4-methyl-7-hydroxyethylamino-coumarin (7g) in compound type 7a-g in general formula two

Starting material 4-methyl-7-amino-coumarin (5e, 300mg, 1.56mmol), 2-bromoethanol (975mg, 7.80mmol), potassium carbonate (372mg, 4.68mmol) and potassium iodide (51mg, 0.31mmol) were dissolved In acetone (10 mL), reflux reaction for 10 hours, after filtration, the solvent was distilled off under reduced pressure, separated and purified by chromatography to obtain 7 g (212 mg) of a light yellow solid product, with a yield of 40%. ESI-MS m/z 220.1[M+H] ⁺ ; ¹ H NMR (400MHz, DMSO-d6) δ7.40 (d, 1H, ArH, J = 8.7Hz), 6.61 (dd, 1H, ArH, J = 8.7,1.7Hz), 6.41(d,1H,ArH,J=1.7Hz),5.89(s,1H,-CH=),3.54(t,2H,-OCH ₂ CH ₂ O-,J=5.8Hz) , 3.19–3.10 (m, 2H, -OCH ₂ CH ₂ O-), 2.28 (s, 3H, -CH ₃ ).

Example 4

A compound 4-(2-(4-methyl-2-oxo-2H-chromene-7-oxyl)ethoxy)-3-(benzenesulfonyl) in compound type 8a-g in general formula 2 -Synthesis of 1,2,5-oxadiazole-2-oxide (8b)

Starting material 7-hydroxyethoxy-4-methyl-coumarin (7b, 100mg, 0.45mmol) and benzenesulfonylfurazan N-oxide (4, 140mg, 0.38mmol), in DBU (205mg, 1.35mmol ) in the presence of dichloromethane (5mL) solvent at room temperature for 3 hours; then wash away DBU with 3×5ml water, dry the organic phase with anhydrous sodium sulfate, filter the filtrate to remove the solvent under reduced pressure, and wash the residue with ethyl acetate The ester was recrystallized to give a white solid 8b (140mg), the yield was 70%, and the melting point was 162-165°C. ESI-MS m/z445.0[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ2.43 (s,3H,=CCH ₃ ),4.45(t,2H,-CH ₂ O-,J=4.12Hz),4.81(t,2H,-OCH ₂ -,J=4.20Hz),6.18(s,1H ,-CH ₃ C=CH-COO-), 6.85(d, 1H, ArH, J=2.32Hz), 6.92(dd, 1H, ArH, J=8.80, 2.36Hz), 7.57(m, 3H, ArH) , 7.73 (t, 1H, ArH, J = 7.57 Hz), 8.03 (d, 2H, ArH, J = 7.72 Hz).

Example 5

Synthesis of Compound N-Methyl-N-Hydroxyethylamino-4-Methyl-coumarin (9a) in General Formula 2

Add 4-methyl-7-hydroxyethylamino-coumarin (7g, 100mg, 0.46mmol) into a 50ml three-necked flask, then add iodomethane (199mg, 1.38mmol), react for 7h, pour into water, dichloromethane (10ml) was extracted three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure, and then separated by column chromatography to obtain a pale yellow solid 9a (45mg), with a yield of 45%. ESI-MS m/z 233.1.0 [M+H] ⁺ .

Example 6

Compound 4-(2-(4-methyl-2-oxo-2H-chromene-7-methylamino)-ethoxy)-3-(benzenesulfonyl)-1,2,5 in general formula 2 -Synthesis of oxadiazole-2-oxide (10a)

N-methyl-N-hydroxyethylamino-4-methyl-coumarin (9a, 40mg, 0.17mmol), benzenesulfonylfurazan N-oxide (4, 63mg, 0.17mmol), and DBU ( 78mg, 0.51mmol) into a 25ml round-bottomed flask, then dichloromethane (5ml), reacted at room temperature for 3h, then washed with 3×5ml water to remove DBU, dried the organic phase with anhydrous sodium sulfate, filtered and evaporated the filtrate under reduced pressure After removing the solvent, the target product 10a (35 mg) was isolated by column chromatography with a melting point of 181-185° C. and a yield of 45%. ESI-MS m/z 458.0[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ2.36(s, 3H, =CCH ₃ ), 3.19(s, 3H, -NCH ₃ ), 3.95( t, 2H, -CH ₂ NCH ₃ -, J=5.32Hz), 4.63 (t, 2H, OCH ₂ -, J=5.28Hz), 6.02 (s, 1H, -CH ₃ C=CH-COO-), 6.59 (d,1H,ArH,J=2.52Hz),6.76(dd,1H,ArH,J=8.92,2.56Hz),7.46(d,1H,ArH,J=8.92Hz),7.57(t,2H,ArH , J = 7.88 Hz), 7.74 (t, 1H, ArH, J = 7.48 Hz), 7.95 (d, 2H, ArH, J = 7.40 Hz).

Example 7

Synthesis of a compound 7-(3-hydroxypropyl)-2-ethyl-4H-chromene-4-one (16b) in compound type 16a-b in general formula two

2-Ethyl-4H-chromen-4-one compound (14,500mg, 2.6mmol) and 3-chloropropanol (491mg, 5.2mmol) were added to a 50ml three-necked flask, and potassium carbonate (1.07g, 7.8mmol), tetrabutylammonium bromide (TBAB, 10%mol), sodium iodide (10mol%) reacted 4h, stopped reaction, poured into water (40ml), extracted three times with 10ml dichloromethane, combined organic phase, with Dichloromethane and methanol were used as eluents for separation and purification by column chromatography to obtain light yellow solid 16b (483 mg), with a yield of 75% and a melting point of 71-73°C. ESI-MS m/z 249.1[M+H] ⁺ ; ¹ H NMR (400MHz, DMSO-d6) δ7.86 (d, 1H, ArH, J=8.8Hz), 7.07 (s, 1H, ArH), 6.99 (d, 1H, ArH, J=8.9Hz), 6.11(s, 1H, COC=CH), 4.59(t, 1H, OH, J=4.7Hz), 4.15(t, 2H, OCH ₂ -, J= 6.0Hz), 3.54(q, 2H, -CH ₂ -, J=5.4Hz), 2.62(q, 2H, -CH ₂ O-, J=7.4Hz), 2.05–1.67(m, 2H), 1.20( dd, _3H , _-CH2CH3 , J = 8.1, 6.9 Hz).

Example 8

A compound 4-(3-(2-ethyl-4H-chromene-4-one-7-oxyl)-propoxy)-3-(benzenesulfonyl) in compound type 17a-b in general formula 2 Synthesis of -1,2,5-oxadiazole 2-oxide (17b)

Compound 7-(3-hydroxypropyl)-2-ethyl-4H-chromen-4-one (16b, 100mg, 0.40mmol) and benzenesulfonylfurazan N-oxide (4, 123mg, 0.33mmol) , in the presence of DBU (180mg, 1.2mmol), in dichloromethane (5mL) solvent, react at room temperature for 3 hours; then add 3×5ml water to wash away DBU, dry the organic phase with anhydrous sodium sulfate, filter, and evaporate under reduced pressure Solvent, ethyl acetate recrystallized the residue to obtain pale yellow crystal 17b (130mg), yield 69%, melting point 153-155°C. ESI-MS m/z 473.0[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ1.28 (t, 3H, CH ₂ CH ₃ , J=7.52Hz), 2.40 (m, 2H, CCH ₂ C), 2.62(q, 2H, CH ₂ CH ₃ , J=7.60Hz), 4.25(t, 2H, CH ₂ O, J=5.84Hz), 4.64(t, 2H, OCH ₂ , J=6.00Hz) ,6.11(s,1H,C=CH),6.86(d,1H,ArH,J=2.32Hz),6.93(dd,1H,ArH,J=8.86,2.32Hz),7.51(t,2H,ArH, J = 8.16 Hz), 7.69 (t, 1H, ArH, J = 7.56 Hz), 7.96 (d, 2H, ArH, J = 7.32 Hz), 8.09 (d, 1H, ArH, J = 8.84 Hz).

Example 9

Synthesis of a compound 2-(4-methyl-2-oxo-2H-chromene-7-oxyl)acetic acid (11b) in compound type 11a-b in general formula two

Add 7-hydroxy-4-methyl-coumarin (5b, 1g, 5.68mmol), chloroacetic acid (1.07g, 11.4mmol) and potassium carbonate (2.35g, 17.04mmol) into DMF (30ml), and reflux After 1h, the reaction liquid was solidified, and the reaction was stopped, and 2N HCl was added to adjust the pH to 2. After standing still, a large amount of white solids were precipitated. After filtration, ethanol recrystallization gave needle-like crystals 11b (1.3g). The yield was 99%, and the melting point was 210-214 ℃. ESI-MS m/z 235.1[M+H] ⁺ ; ¹ H NMR (400MHz, DMSO-d6) δ7.67 (d, 1H, ArH, J=9.0Hz), 7.01–6.91 (m, 2H, ArH) , 6.21 (s, 1H, -CH=), 4.81 (s, 2H, -COCH2O-), 2.38 (s, 3H, -CH ₃ ).

Example 10

Synthesis of a compound 2-hydroxyethyl-2-(4-methyl-2-oxo-2H-chromene-7-oxyl) acetate (12b) in compound type 12a-b in general formula two

Get compound 11b (1g, 4.27mmol) and EDC (930mg, 4.87mmol) and DMAP (20%mol) and join in the 50ml round bottom flask containing methylene chloride (10ml), react 20min under 0 ℃, add ethylene glycol ( 1.3g, 21.35mmol), returned to room temperature and reacted for 3h, washed 3×5ml with water, concentrated the organic phase and separated by dichloromethane/methanol (V/V=50:1) column chromatography to obtain white solid 12b (500mg) , yield 42%. The melting point is 93-96°C. ESI-MS m/z 279.1[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ7.52 (d, 1H, ArH, J=8.8Hz), 6.92 (dd, 1H, ArH, J=8.8 ,2.6Hz), 6.79(d,1H,ArH,J=2.5Hz),6.16(d,1H,-CH=,J=1.1Hz),4.75(s,2H,-COCH ₂ O-),4.37( t, 2H, -CH2O-, J = 4.6 Hz), 3.89 (t, 2H, -CH2O-, J = 4.6 Hz), 2.40 (d, 3H, -CH ₃ , J = 1.1 Hz).

Example 11

A compound 4-(2-(2-(4-methyl-2-oxo-2H-chromene-7-oxyl)acetyl)-oxyethoxy) in compound type 13a-b in general formula two Synthesis of -3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13b)

Take compound 12b (100mg, 0.36mmol), benzenesulfonylfurazan nitrogen oxide (4, 145mg, 0.40mmol) and DBU (164mg, 1.08mmol) into a 25ml round bottom flask, in dichloromethane (5ml), After reacting at room temperature for 3 hours, the same post-treatment was carried out, and the residue was separated by column chromatography to obtain the target product 13b (90 mg), with a yield of 50% and a melting point of 113-115°C. Yield 45%. ESI-MS m/z 502.9[M+H] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ2.39(s,3H,=CCH ₃ ),4.67(s,4H,OCH ₂ CH ₂ O),4.80 (s,2H,COCH ₂ O),6.16(s,1H,-CH ₃ C=CH-COO-),6.82(d,1H,ArH,J=2.32Hz),6.92(dd,1H,ArH,J =8.88,2.08Hz),7.53(d,1H,ArH,J=8.80Hz),7.62(t,3H,ArH,J=7.69Hz),7.76(t,1H,ArH,J=7.44Hz),8.06 (d, 2H, ArH, J = 8.16 Hz).

Example 12 In vitro anti-tumor activity screening test

1. Using the MTT method to detect the activity of the compound of the present invention in inhibiting tumor cell proliferation, the cancer cells in the logarithmic growth phase were inoculated in a 96-well plate, and divided into blank control group, cisplatin control group and different concentrations of the compound treatment group of the present invention After culturing at 37°C for 48 hours, MTT assay was performed, and the cell proliferation inhibition rate and IC ₅₀ were calculated;

2. Apply Western blot (immunoblotting) analysis to detect that the compound of the present invention inhibits MEK phosphorylation and promotes apoptosis. After A2780 cells were treated with DMSO and different concentrations of the compound of the present invention for 24 hours, the collected cells were washed once with cold PBS, and washed once with Cells were lysed with RIPA protein lysate on ice for 30 minutes, centrifuged at 4°C for 15 minutes, the total protein was quantified by BCA method, and the loading amount of 30 μg per lane was transferred to polyvinylidene fluoride PVDF membrane by SDS-PAGE electrophoresis Above, block in 10% skimmed milk, react with primary antibody and secondary antibody in sequence, and develop color by exposure;

The results show that the compound has excellent effect of inhibiting tumor cell activity and inhibiting drug-resistant tumor strain activity (as shown in Table 1 and Table 2), wherein the compound 4-(2-(4-methyl-2-oxo Generation-2H-chromone-7-hydroxy)-ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadi-2-oxide (8b) is not only sensitive to four A549, Hela , A2780, SKOV3 (ovarian cancer cell) tumor cell proliferation inhibitory activity with IC ₅₀ values of 24, 53, 14 and 83nM, and 231HM (breast cancer lung transgenic cells) and its gemcitabine-resistant 231HM/Gem, cisplatin-resistant The four drug-resistant tumor lines A2780/CDDP and SKOV3/CDDP (ovarian cancer cells) have good inhibitory activity, showing IC50 values of 0.15, 0.14, 0.062 and 0.14 μM, respectively.

Table 1 is the results of the anti-tumor cell proliferation activity of the compounds of the present invention.

Table 2 shows the results of compound 8b of the present invention inhibiting drug-resistant tumor strains.

In addition, the results of preliminary pharmacological experiments show that compound 8b can promote the expression of Bax, CyclinB1 and p53 and the phosphorylation of p53, while inhibiting the expression of Bcl-2. The increase of the death marker Cleaved-PARP, that is, it shows a significant induction of apoptosis in human ovarian cancer cell A2780 (as shown in A in Figure 2); and has a significant inhibition of MEK in the Ras/Raf/MEK/ERK pathway Phosphorylation (shown as B in Figure 2).

Table 1

Table 2

Example 13 In vivo tumor-bearing nude mouse model test

For the in vivo tumor-inhibitory activity experiment of the tumor-bearing (A2780) nude mouse model, the established tumor-bearing mice were randomly divided into 6 groups. In the experimental group, the test sample was injected intraperitoneally (it is not necessary to choose a suitable solvent according to the solubility), and each sample was set at 2 concentrations, and a negative control group (give blank solvent), administered 2 times a week, and the tumor growth size was measured and recorded , the nude mice were executed on the 2nd day after drug withdrawal, the tumor tissue was stripped off, and the tumor mass was weighed. The results showed that compound 8b was injected into the intraperitoneal cavity of 15 and 30 mg/kg respectively, and it could be seen that the tumor growth was obviously inhibited (A and B in Fig. 3 As shown), compared with the blank control, the body weight did not significantly decrease (as shown in C in Figure 3).

Example 14

The NO release level of the compound of the present invention was detected using the classic Griess reagent. After incubation of the tumor cell A2780 (1×10 in a ⁶ cm dish) with the compound of the present invention at a concentration of 100 μM for 150 minutes, the cells were collected and lysed with RIPA protein lysate on ice for 30 minutes. Minutes, and then mixed with Griess reagent for 30 minutes, the absorbance was detected at 540nm, the nitrite in the cells after treatment with the diluent was used as the background control, and the standard curve was prepared by the detection of sodium nitrate at different concentrations; the results are shown in Figure 4, without The parent compound CY-1-140A(1) of the furazan structure has no release of NO, and the release concentration of the furazan fragment, namely compound 4, is 30.48 μM/100 μM. After the furazan is introduced into the coumarin ring through linkage, the NO release ability is obvious In particular, compound 8b with the strongest anti-tumor activity also has the strongest ability to release NO, reaching 99.6μM/100μM; the results show that after the combination of coumarin and furazan ring, it inhibits MEK in the Ras/Raf/MEK/ERK pathway The phosphorylation of NO and the pro-apoptotic effect after NO release synergistically produce anti-tumor effect.

Further, in this example, Schrödinger software was used to dock 8b and MEK1 protein (PDB code: 1S9J), and the results showed that 8b and G8935 highly overlapped (as shown in Figure 5), and had a similar mode of action to MEK, 8b and GC8935 A key hydrogen bond was formed with the SER212 residue of MEK, and the benzenesulfonyl group of 8b showed good overlap with the three-position benzyl group of G8935.

Claims (4)

1. coumarin furazan derivative, it is characterized in that, described derivative has the structure shown in following formula: Wherein: X is an oxygen atom; R ₁ is a hydrogen atom; R ₂ is methyl; n=1. 2. The derivatives of claim 1 are used in the preparation of pharmaceutical preparations for inhibiting sensitive and drug-resistant human tumor cells; the sensitive tumor cells are sensitive human ovarian cancer cells A2780 and SKOV3, sensitive breast cancer lung transgenic cells HM231, uterine Cancer cells Hela, human umbilical vein endothelial cells HUVEC and lung cancer cells A549; The drug-resistant tumor cells are drug-resistant human ovarian cancer cells A2780 and SKOV3, and drug-resistant breast cancer lung transgenic cells HM231. 3. according to the purposes of claim 2, it is characterized in that, described derivative releases the nitric oxide of high concentration, induces the apoptosis of cancer cell; Simultaneously, inhibits the phosphorylation of MEK in the MAPK/ERK pathway, wherein, NO The release amount, MEK activity inhibition intensity and tumor cell proliferation inhibition are positively correlated. 4. The use of the derivative according to claim 1 in the preparation of a pharmaceutical preparation for inhibiting the normal cell proliferation activity of HUVEC, and said HUVEC is used for measuring the inhibitory effect on neovascularization.