CN110437149B - Natural naphthyl isoquinoline compound with antitumor activity, and composition and application thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to a natural naphthyl isoquinoline compound with antitumor activity, a composition and application thereof. A compound of the formula I linked at the 1,3, 8-position and the 5-position of the formula II, orLinked by the 1,3, 6,8 position of formula I and the 7 position of formula II; the structures of the general formula I and the general formula II are respectively as follows:

Description

Natural naphthyl isoquinoline compound with antitumor activity, and composition and application thereof

Technical Field

The invention relates to a natural naphthyl isoquinoline compound with anti-tumor activity, a composition and an application thereof, belonging to the technical field of biological medicines.

Background

Protein Tyrosine Kinases (PTKs) regulate a series of physiological and biochemical processes such as growth, differentiation and apoptosis of cells by controlling signal transduction pathways of the cells. Receptor-type tyrosine kinases are a class of relatively large kinases that span the cell membrane, having a ligand-binding extracellular domain, a transmembrane domain, and an intracellular domain that functions as a kinase-phosphorylating specific tyrosine residues and thereby affecting cell proliferation. Abnormal expression of the kinase has been found in common human cancers (e.g., lung, breast, stomach, ovarian, lymphoma). Protein tyrosine kinase has become one of the important targets for research and development of antitumor drugs.

Epidermal growth factor receptor tyrosine kinase (EGFR) is one of the earliest discovered protein tyrosine kinases, the intracellular region of EGFR has an ATP binding site, and an EGFR inhibitor can be competitively combined with the ATP binding site, so that the phosphorylation process of EGFR is inhibited, the conduction of downstream signals is blocked, and the growth, differentiation and metastasis of tumor cells are inhibited. The EGFR tyrosine kinase (EGFR-TK) discovered at present can catalyze the transfer of high-energy phosphate bonds of ATP to a plurality of tyrosine sites of EGFR, so that the ATP is phosphorylated, and then downstream signal paths such as PI3K/Akt, Ras/MAPK and the like are activated, the proliferation and the metabolism of cells are promoted, and the apoptosis is inhibited. When EGFR is overexpressed or mutated in a cell, apoptosis is inhibited, and growth regulation of the cell is out of control, so that unlimited proliferation and invasion capacity, i.e., malignant tumor cells, are obtained. The EGFR-TKI prevents tyrosine autophosphorylation in a receptor and inhibits activation of EGFR tyrosine kinase by competing with ATP for a binding site of intracellular tyrosine kinase, thereby inhibiting growth of tumor cells, accelerating apoptosis of the tumor cells, and inhibiting angiogenesis and infiltration and metastasis of the tumor cells. The small molecule EGFR-TKI can be divided into: 1) reversible TKIs are mainly: gefitinib (gefitinib), erlotinib (erlotinib), lapatinib (lapatinib); 2) irreversible TKIs mainly: afatinib (afatinib), dacomitinib (dacomitinib). At present, reversible and irreversible EGFR-TKI has obvious curative effect on the mutant EGFR non-small cell lung cancer in a short time, but finally treatment failure is caused by acquired drug resistance, and the survival time of a patient is not prolonged obviously. Therefore, the development and development of novel EGFR-TKI with stronger and more durable antitumor activity will probably bring about vitality to patients with mutant EGFR non-small cell lung cancer.

Numerous compounds with unique structures and remarkable EGFR-TKI functions are contained in traditional Chinese medicines or natural medicines, such as cucurbitacin B obtained from medicinal materials of trichosanthes in trichosanthes and the like, rabdosia A obtained from various medicinal plants in rabdosia, curcuma zedoary obtained from curcuma longa in curcuma longa and the like, and the active field of modern medical research is achieved. The natural EGFR-TKI not only lays a foundation for drug discovery, but also provides a wonderful template beyond human imagination for organic synthetic chemists to develop the synthesis of natural products.

Disclosure of Invention

One of the purposes of the invention is to provide a naphthyl isoquinoline compound which has good anti-tumor activity.

The invention also aims to provide application of the naphthyl isoquinoline compound in promoting tumor cell apoptosis, wherein the mechanism is used as an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI).

The invention further aims to provide a derivative of the naphthyl isoquinoline compound and an optical isomer thereof, and application of the composition.

To this end, in one aspect, the invention provides a compound linked by the 1,3, 8 positions of formula I and the 5 position of formula II, or by the 1,3, 6,8 positions of formula I and the 7 position of formula II;

the structures of the general formula I and the general formula II are respectively as follows:

general formula I

General formula II

Wherein: r₁、R₂、R₃、R₄Selected from hydroxy or methoxy; the isoquinoline structure is selected from tetrahydroisoquinoline, dihydroisoquinoline or isoquinoline.

Further, the structure of the compound is as follows:

as a specific embodiment of the present invention, the following derivatives and optical isomers thereof are preferable in the present invention, but these compounds are not meant to limit the present invention in any way, and the compounds have the structural formulae shown in I-1 to I-12:

the structural compound shown as the above is a naphthyl isoquinoline compound. The screening of the anti-tumor activity shows that the compounds have stronger capacity of inhibiting the proliferation of non-small cell lung cancer (NSCLC) cells. As a natural small molecule with specific structure, the compound has the potential of being developed into a novel high-efficiency EGFR inhibitor and has great application value in treating related tumor diseases, particularly non-small cell lung cancer and small cell lung cancer.

The structures represented by the foregoing I-1 to I-12 have the following names, respectively:

(I-1) (1R,3S,5S) -5- (4-hydroxy-5-methoxy-2-methyl-1-naphthyl) -1,2,3, 4-tetrahydro-8-methoxy-1, 2, 3-trimethyl-6-hydroxyisoquinoline;

(I-2) (1S,3S,5R) -5- (4, 5-dimethoxy-2-methyl-1-naphthyl) -1,2,3, 4-tetrahydro-6-methoxy-1, 3-dimethyl-8-hydroxyisoquinoline;

(I-3) (3S,5S) -3, 4-dihydro-5- (4-hydroxy-5-methoxy-2-methyl-1-naphthyl) -8-methoxy-1, 3-dimethyl-6-hydroxyisoquinoline;

(I-4)5- (4, 5-dimethoxy-2-methyl-1-naphthyl) -1,2,3, 4-tetrahydro-8-methoxy-1, 2, 3-trimethyl-6-hydroxyisoquinoline;

(I-5) (3S,5R) -5- (5-hydroxy-4-methoxy-2-methyl-1-naphthyl) -3, 4-dihydro-8-methoxy-1, 3-dimethyl-6-hydroxyisoquinoline;

(I-6) (2S,5S) -5- (4, 5-dimethoxy-2-methyl-1-naphthyl) -3, 4-dihydro-6, 8-dimethoxy-1, 3-dimethylisoquinoline;

(I-7) 8-methoxy-3-methyl-2- [ (1R,3S) -1,2,3, 4-tetrahydro-6, 8-dimethoxy-1, 2, 3-trimethyl-5-isoquinolinyl ] -1-hydroxynaphthalene;

(I-8)5- [ (3S) -3, 4-dihydro-6, 8-dimethoxy-1, 3-dimethyl-5-isoquinolinyl ] -4, 5-dimethoxy-2, 7-dimethyl-1-naphthalene;

(I-9) (1S,3S,7S) -1,2,3, 4-tetrahydro-8-methoxy-7- (4, 5-dimethoxy-2-methyl-1-naphthyl) -1, 3-dimethyl-6-hydroxy-isoquinoline;

(I-10) 8-methoxy-3-methyl-2- [ (1R,3S) -1,2,3, 4-tetrahydro-6, 8-dimethoxy-1, 2, 3-trimethyl-7-isoquinolinyl ] -1-hydroxynaphthalene;

(I-11)7- (1-hydroxy-6-methyl-8-methoxy-2-naphthyl) -1,2,3, 4-tetrahydro-6, 8-dimethoxy-1, 2, 3-trimethyl-isoquinoline;

(I-12)7- (4, 5-dimethoxy-7-methyl-1-naphthyl) -1,2,3, 4-tetrahydro-6, 8-dimethoxy-1, 2, 3-trimethyl-isoquinoline.

In another aspect, the present invention provides a pharmaceutical composition, which comprises an effective amount of the derivative, optical isomer of the compound represented by formula I, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention is not limited to containing one compound of the present invention, and may contain more than one compound of the present invention. In addition, the pharmaceutical compositions of the present invention may optionally further comprise one or more other pharmaceutically active compounds.

We have found that the compounds of the invention possess anti-EGFR activity in vitro^WTHigh-expression A549 cells and EGFR^T790MThe proliferative activity of high-expression H1975 cells and EGFR sensitive mutation HCC827 cells can be used for preparing medicaments for treating and/or preventing non-small cell lung cancer.

By in vitro targeting of EGFR^WTAnd EGFR^T790MThe compound has obvious EGFR kinase inhibition activity and stronger inhibition effect on EGFR high-expression non-small cell lung cancer cells, and is particularly used for preparing medicaments for treating and/or preventing the non-small cell lung cancer.

The invention has the beneficial effects that: the invention provides a novel molecular targeted EGFR-TKI, namely application of a natural small molecular compound naphthyl isoquinoline alkaloid in non-small cell lung cancer, can be developed into the novel EGFR-TKI, and has wide application prospect.

Detailed Description

The technical solution of the present invention is further described below with reference to specific examples, but the present invention is not limited to the contents of the examples in any way.

The test methods in the examples of the present invention are all conventional methods or conditions recommended by manufacturers of raw materials or goods, unless otherwise specified; unless otherwise specified, the reagents and biomaterials are commercially available.

EXAMPLE 1 preparation of Naphthylisoquinoline Compound molecules

(1) Alcohol extraction step: pulverizing fresh and dried branches and leaves of ramulus Uncariae cum uncis (Ancistocladus tectorius), extracting with 10 times of 95% ethanol solution for 3 times (0.5 hr each time), concentrating the extractive solution at 50 deg.C under reduced pressure to obtain extract, recovering ethanol as solvent, and making into ramulus Uncariae cum uncis branch and leaf ethanol extract;

(2) an extraction step: adding 0.3 times of water by weight into the extract of the ramulus Uncariae cum uncis branch and leaf alcohol extract, dispersing and dissolving, and then sequentially extracting with cyclohexane and dichloromethane for 2 times respectively to obtain cyclohexane extraction part and dichloromethane extraction part;

(3) diol-based chromatography column chromatography step: the dichloromethane extraction part is taken and filled on a Diol-based chromatographic column with the weight being 30 times that of the dichloromethane extraction part, and cyclohexane/dichloromethane (9:1), dichloromethane/ethyl acetate (20:1), ethyl acetate/methanol (5:1) and methanol with the volume being 3 times that of the column are used for gradient elution in sequence to prepare each separation part. Collecting the eluted part of dichloromethane/ethyl acetate (20:1), concentrating, drying, and separating and purifying naphthyl isoquinoline compound.

(4) ODS and preparative HPLC purification of naphthyl isoquinoline Compound molecules: and (3) loading the concentrated and dried dichloromethane/ethyl acetate (20:1) eluate on an ODS column chromatography, performing gradient elution with 70% methanol, 80% methanol, 90% methanol and methanol, performing TLC (thin layer chromatography) identification, improving the color development of bismuth potassium iodide, combining fractions with the same color development spots, concentrating and recovering the solvent to obtain fractions 1-14. And (3) taking the fractions, and gradually purifying by using a C18 preparative HPLC chromatographic column and using 70% methanol water-100% methanol, 80% acetonitrile water-100% acetonitrile and a gradient elution mode (containing 0.5% trifluoroacetic acid) thereof as mobile phases to obtain naphthyl isoquinoline compounds I-1-I-12.

(5) Identification of naphthyl isoquinoline Compounds: the monomer compounds obtained by the above separation were tested separately¹H-NMR、¹³C-NMR and MS, and comparing the obtained data with literature reference, and respectively identifying the data as N-methyllancisteine A₁(I-1)、5-epi-ancistectorine A₂(I-2)、ancistectorine A₃(I-3)、(+)-ancistrocline(I-4)、5′-O-demethylhamatinine(I-5)、ancistrocladinine(I-6)、ancistrotectorine C(I-7)、ancistrotanzanine B(I-8)、ancistectorine B₁(I-9)、ancistrotectorine(I-10)、ancistrotectoriline C(I-11)、ancistrocyclinone B(I-12)。

EXAMPLE 2 preparation of Naphthylisoquinoline pharmaceutical compositions

And (3) putting the two target molecules with equal molar weight (1mmol) into anhydrous methanol (5mL), stirring for 10 minutes at room temperature, and evaporating the solvent at room temperature to obtain a mixture of the target molecules. Three mixtures of (I-1) - (I-2), (I-3) - (I-8), (I-7) - (I-10) were prepared by this method.

EXAMPLE 3 Naphthylisoquinoline Compounds anti-cell proliferation Activity

The MTT method is adopted to determine the in vitro anti-non-small cell lung cancer cell proliferation activity of the naphthyl isoquinoline monomer compound. Selecting EGFR^WTHigh-expression A549 cells and EGFR^T790MHighly expressed H1975 cells, EGFR sensitive mutant HCC827 cells were tested for proliferation inhibitory activity. In the determination, PBS is used as a blank control, and gefitinib and ralotinib are used as positive controls.

(1) Cell culture: human non-small cell lung cancer cell lines (A549, H1975, HCC827) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% double antibody at 37 deg.C and 5% CO₂Cultured in an incubator.

(2) Cell inoculation: and taking the cells in the exponential growth phase in a good state, collecting the cells, centrifuging, and discarding the supernatant. Cell suspensions were prepared in RPMI 1640 (or DMEM) medium containing 10% fetal bovine serum and counted. Cell suspensions were plated in 96-well plates at 100. mu.L/well (7000 tumor cells per well). Transfer the plates to constant temperature CO₂In an incubator at 37 ℃ with 5% CO₂And culturing for 24h under saturated humidity condition.

(3) Administration: the test compound was prepared in advance with DMSO to give a 4mmol/L stock solution and stored in a refrigerator at 4 ℃. Before administration, the mother liquor was preheated in a constant temperature water bath, and then diluted to 40, 20, 10, 5, 2.5. mu. mol/L solution with serum-free medium (RPMI 1640 or DMEM) for primary screening. Pouring out the culture solution in the adherent inoculated cells, adding 100 mu L of compounds to be detected with different concentrations, setting 3 multiple holes in each group, and complementing the rest blank holes with PBS. The negative control is the culture medium with the same volume, and the DMSO solvent control with the corresponding concentration is also arranged, and the blank control does not contain cells and medicines. The temperature is 37 ℃ and 5 percent CO₂The culture box is used for culturing for 72 hours.

(4) Color generation: cells were dosed for 72h in culture, 10. mu.L of MTT solution (5mg/mL) was added to each well, incubated in an incubator for 4 hours, 100. mu.L of the triple was added to each well, and the incubation was overnight in the incubator.

(5) Color comparison: the absorbance (OD) of each well was measured on an ELISA detector, the wavelength was chosen to be 570nm, and the absorbance of each well was measured by zeroing a blank well of cell-free, i.e., blank, culture medium. The experiment was repeated 3 times.

The test results are shown in table 1.

TABLE 1 antiproliferative activity (IC) of naphthyl isoquinolines on non-small cell lung cancer cells₅₀,μmol)

Example 4 naphthyl isoquinoline Compound on EGFR^WTAnd EGFR^T790MInhibitory Activity of kinase

Testing of kinase inhibitory Activity Using ADP-Glo^TMA kinase kit. The kit comprises two steps during testing: first, after the kinase reaction, an aliquot of ADP-Glo was added thereto in an amount equal to the volume of the kinase reaction system^TMReagents to terminate the reaction and consume the remaining ATP. In the second step, a kinase detection reagent is added which, while converting ADP to ATP, also detects newly synthesized ATP using a coupled luciferase/luciferin reaction.

(1) Preparing a kinase detection buffer: the kinase assay buffer was thawed at room temperature and observed for precipitation. If precipitation occurs, the pellet is dissolved by incubating for 15 minutes at 37 ℃ and shaking frequently. Or carefully aspirate the supernatant and remove the precipitate.

(2) Preparing a kinase detection reagent: the kinase assay buffer and kinase assay substrate were equilibrated at room temperature prior to use. And (3) pouring the kinase detection buffer solution into a brown bottle filled with a kinase detection substrate, and dissolving the freeze-dried powder substrate to prepare the kinase detection reagent. Mix well by gentle shaking or vortexing to form a homogeneous solution, and the substrate should be dissolved within 1 minute. The kinase detection reagent should be used immediately after being prepared, or be stored in a split-charging manner at-20 ℃.

(3) Standard Curve for conversion of ATP to ADP was generated

a. The Ultra Pure ATP and ADP provided by the kit are respectively prepared into 900 mu L of 50 mu mol/L ATP and 500 mu L of 50 mu mol/L ADP.

b. The prepared 50. mu. Mol/L ATP and 50. mu. Mol/L ADP solutions were mixed in A1-A12 wells of a 384-well plate to simulate the concentration of ATP and ADP for each percent conversion, and mixed well.

c. Add 5. mu.L ADP-Glo per well^TMReagents to terminate the kinase reaction. Incubate for 40 min at room temperature in the dark.

d. Add 10. mu.L of kinase assay reagent per well to convert ADP to ATP and introduce luciferase and luciferin to detect ATP converted from ADP.

e. And (3) incubating for 30-60 minutes at room temperature in a dark environment, measuring fluorescence by using a multifunctional microplate reader, and recording the fluorescence value.

f. Standard curves for conversion of ATP to ADP were plotted.

(4) Determination of IC of kinase inhibitor naphthylisoquinoline Compounds₅₀Value of

Kinase reaction buffer, 50 ng/. mu.L kinase and 0.5. mu.g/. mu.L substrate and 125. mu. mol/L ATP were prepared according to the kit instructions. mu.L of kinase reaction buffer, 2. mu.L of 0.5. mu.g/. mu.L substrate 125. mu. mol/L ATP, was added to the enzyme-free control wells. mu.L of kinase reaction buffer, 2. mu.L of 50 ng/. mu.L kinase, 2. mu.L of 0.5. mu.g/. mu.L substrate and 125. mu. mol/L ATP were added to the negative control wells. mu.L of test compound, 2. mu.L of 50 ng/. mu.L kinase, 2. mu.L of 0.5. mu.g/. mu.L substrate and 125. mu. mol/L ATP are added to the test wells. The plates were mixed and incubated in the dark for 60 minutes at room temperature. After incubation was complete, 5. mu.L of ADP-GloTM reagent was added to each well to stop the kinase reaction. And incubated under the same conditions for another 40 minutes. Then 10. mu.L of kinase detection reagent per well was added to convert ADP to ATP and luciferase and luciferin were introduced to detect ATP. Incubating for 30-60 minutes at room temperature in a dark place, and measuring the fluorescence by using a multifunctional microplate readerRecording fluorescence value and calculating IC₅₀The value is obtained.

The test results are shown in table 2.

TABLE 2 Naphthylisoquinolines Activity on EGFR kinase Inhibitor (IC)₅₀,nmol)

The results show that naphthyl isoquinoline compound has the effect of treating EGFR^WTAnd EGFR^T790MHas strong inhibiting effect, part of compounds can reach the activity level of nanomolar level, and the effective inhibiting concentration IC of part of compounds₅₀The value is less than 20nMol/L and is higher than that of a lead compound, namely the loratidine (third-generation EGFR-TKI capable of reversing the drug resistance of T790M); the inhibition effect of partial compounds on T790M mutant kinase is dozens of times stronger than that of gefitinib which is a marketed drug, and even is hundreds of times stronger. Therefore, the naphthyl isoquinoline compound can be used as an EGFR target inhibitor for treating diseases with high expression of EGFR, and is indicated to be developed into a non-small cell lung cancer treatment drug.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (3)

1. The application of a compound in preparing a medicament for treating tumors, wherein the tumors are selected from one or more of non-small cell lung cancer and small cell lung cancer, and the compound has the structure shown as I-1 to I-12:

2. the use of claim 1, wherein the tumor is non-small cell lung cancer.

3. Use according to claim 1 or 2, characterized in that it is obtained by inhibiting EGFR, EGFR^T790MMutant epidermal factor receptor protein tyrosine kinase.