CN112851678B - 2, 4, 7-trisubstituted pyrimidoindole compound with anti-tumor metastasis effect - Google Patents
2, 4, 7-trisubstituted pyrimidoindole compound with anti-tumor metastasis effect Download PDFInfo
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Abstract
The invention relates to a 2, 4, 7-trisubstituted pyrimidoindole structure compound which can inhibit the migration and invasion of tumor cells at a very low concentration and has an effect superior to that of a clinical common medicament cisplatin. In addition, the compounds of the present invention can also inhibit the expression of matrix metalloproteinase-related proteins that play an important role in tumor migration and invasion processes. The invention also relates to a preparation method of the compound and application of the compound in marking, diagnosing, preventing and treating or assisting treatment of tumors.
Description
Technical Field
The invention relates to a 2, 4, 7-trisubstituted pyrimidoindole compound, a preparation method thereof and application thereof in resisting tumor metastasis.
Background
Cancer is a serious disease that threatens human health seriously, and the main cause of cancer-related death of about 90% is tumor metastasis. Tumor metastasis is the process of malignant tumor cell separating from primary part, transferring to secondary tissue or organ in direct spreading, blood circulation, lymph metastasis, etc. to grow and proliferate continuously to form secondary tumor. This is a multi-step, multi-stage and multi-factor process. Tumor metastasis is the most common cause of death in tumor patients and is also a significant problem facing current clinical tumor therapy.
Breast cancer is currently one of the highest malignant tumors in women with the highest morbidity and mortality, and the incidence rate of breast cancer is the first of female cancer incidence rate. High metastatic potential of tumor cells is also one of the major factors affecting survival time of breast cancer patients. Although the prognosis of breast cancer patients is improved to some extent by the combined treatment of radiotherapy, chemotherapy and surgery, the recurrence and metastasis of breast cancer patients remain the most important factors threatening the survival of patients. In the case of breast cancer patients with recurrent metastasis, the survival time is about 24 months, and especially in patients with visceral metastasis, the prognosis is worse. This not only causes serious economic burden to patients, but also has significant impact on families and society. Therefore, the search for an innovative medicine for resisting the invasion and the metastasis of the breast cancer has important significance for improving the survival of the breast cancer patients.
The compound related by the invention is a 2, 4, 7-trisubstituted pyrimidoindole structure, can inhibit the migration and invasion of tumor cells under very low concentration, and has better effect than the clinical common medicine cisplatin. In addition, the invention also finds that the compound can inhibit the expression of matrix metalloproteinase related protein which plays an important role in the process of tumor migration and invasion, and has important clinical significance for inhibiting cancer metastasis. Therefore, the compound has wide application prospect in anti-tumor metastasis treatment.
Disclosure of Invention
The invention mainly relates to a 2, 4, 7-trisubstituted pyrimidoindole compound, a preparation method thereof and application thereof in resisting tumor metastasis.
The invention also provides processes and intermediates useful for preparing the compounds of the invention.
The invention also provides compositions comprising a pharmaceutically acceptable carrier and at least one of the compounds of the invention or isomers, prodrugs, pharmaceutically acceptable salts, or solvates thereof.
The compounds of the present invention are useful for the prevention and/or treatment of cancer and its related disorders.
The compound can be used for preparing a pharmaceutical composition for preventing and/or treating tumors.
The compounds of the invention are useful for inhibiting the migratory capacity of tumor cells.
The compounds of the invention are useful for inhibiting the invasive potential of tumor cells.
The compounds of the invention are useful for inhibiting the expression of invasion-associated proteins in tumor cells.
The compounds of the present invention may be used alone, in combination with other compounds of the present invention, or in combination with one or more (preferably one to two) other drugs.
These and other features of the present invention will be presented in expanded form as the present disclosure continues.
Drawings
FIG. 1 Effect of XH003 Compound on the migration ability of breast cancer cell lines MCF-7
FIG. 2 Effect of XH003 Compound on the invasion of Breast cancer cell lines MCF-7
FIG. 3A Effect of compound XH003 on MMP2, MMP9, MMP12, MMP21, and MMP24 protein expression in breast cancer MCF-7 cells: photographic results were visualized by Western Blotting (WB)
FIG. 3B Effect of compound XH003 on MMP2, MMP9, MMP12, MMP21, and MMP24 protein expression in breast cancer MCF-7 cells: results of protein immunoblotting (Western blot, WB) quantification (. about.P <0.05,. about.P <0.01vs control group)
Detailed Description
I. Definition of
As used above and throughout the specification of the present invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
the term "compounds of the invention" and equivalent expressions are intended to include the compounds of structural formula I as described hereinabove, which expressions include prodrugs, pharmaceutically acceptable salts and solvates, e.g. hydrates, as permitted hereinabove. Similarly, for intermediates, whether or not they are claimed per se, they are intended to include their salts and solvates (when the context permits). For clarity, specific examples are sometimes indicated herein when the context permits, but these examples are purely illustrative and are not intended to exclude other examples when the context permits.
The term "substituted" or "substitution" of an atom means that one or more hydrogens on the designated atom is replaced with a substituent selected from the designated group, provided that the designated atom's normal valence is not exceeded. As referred to herein, the term "(substituted)" means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that the normal valency is maintained and that the substitution results in a stable compound. When the substituent is a keto group (i.e., ═ O), then 2 hydrogens on the atom are replaced. The keto substituent is not present on the aromatic moiety. When referring to a ring system (e.g., carbocyclic or heterocyclic) substituted with a carbonyl group or a double bond, it is meant that the carbonyl group or double bond is part of the ring (i.e., within the ring). As used herein, a cyclic double bond is a double bond formed between two adjacent ring atoms (e.g., C-C, C-N or N-N).
Where a nitrogen atom (e.g., an amine) is present on a compound of the invention, it may be converted to an N-oxide by treatment with an oxidizing agent (e.g., mCPBA and/or hydrogen peroxide) to yield other compounds of the invention. Thus, the nitrogen atoms shown and claimed are considered to encompass the nitrogen shown and its N-oxide (N → O) derivatives.
The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts, and base addition salts, of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts may be prepared by separately reacting the purified compound in free base form with a suitable organic or inorganic acid and isolating the salt so formed. Exemplary acid addition Salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphylate, mesylate, glucoheptonate, lactobionate (1 actibionate), sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthoate, gentisate, isothionate, ditoluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinic acid lauryl sulfonate (quinateslaurylsulfate) and the like (see, e.g., Berge et al, "Pharmaceutical Salts", j.pharm.sci., 66: 1-9(1977) and Remington's Pharmaceutical Sciences, 17 th edition, MackPublishing Company, Easton, Pa., 1985, page 1418, hereby incorporated by reference in its entirety). Base addition salts may also be prepared by separately reacting the purified acid form of the compound with a suitable organic or inorganic base and isolating the salt so formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminium salts. Sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases including sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide. Suitable amine base addition salts are prepared from amines which are sufficiently basic to form stable salts, and preferably include those amines commonly used in medicinal chemistry due to their low toxicity and acceptability for pharmaceutical use, examples of such amines include ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids such as lysine and arginine, dicyclohexylamine, and the like.
The term "pharmaceutically acceptable prodrug" means a prodrug of a compound that can be used in accordance with the present invention, and, where possible, the zwitterionic form of the compound of the present invention, which prodrug, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and effective for the intended use. The term "prodrug" means a compound that is rapidly transformed in vivo, for example by hydrolysis in blood, to yield the parent compound of the above formula. Functional groups that can be rapidly converted by metabolic cleavage in vivo form a class of groups that react with the carboxyl groups of the compounds of the present invention. The functional group includes, but is not limited to, groups such as alkanoyl (e.g., acetyl, propionyl, butyryl, etc.), unsubstituted and substituted aroyl (e.g., benzoyl and substituted benzoyl), alkoxycarbonyl such as ethoxycarbonyl), trialkylsilyl (e.g., trimethyl and triethylsilyl), monoesters with dicarboxylic acids (e.g., succinyl), and the like. Since the metabolically cleavable group of the compounds useful in the present invention is readily cleaved in vivo, compounds bearing such a group may act as prodrugs. Compounds carrying metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of the improved solubility and/or absorption rate imparted to the parent compound by the presence of the metabolically cleavable group. The following documents provide a thorough discussion of prodrugs: design of Prodrugs, H.Bundgaard eds, Elsevier (1985); methods in Enzymology, K.Widder et al, Academic Press, 42, 309 & 396 (1985); a Textbook of Drug design Development, Krogsgaard-Larsen and H.Bundgaard eds, Chapter 5; "Design and applications of precursors" page 113 and 191 (1991); advanced Drug Delivery Reviews, h.bundgard, 8, pages 1-38 (1992); journal of Pharmaceutical Sciences, 77: 285 (1988); nakeya et al, chem.pharm.bull, 32: 692 (1984); higuchi et al, "Pro-drugs as novelderly Systems", volume 14 of a.c.s.symposium Series, and Bioreversible carrier in drug design, Edward b.roche editors, American Pharmaceutical Association and pergammonpress (1987), which are incorporated herein by reference in their entirety. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention.
The term "pharmaceutical composition" means a composition comprising a compound of formula (I) and, depending on the mode of administration and on the nature of the dosage form, at least one pharmaceutically acceptable ingredient selected from the group consisting of: carriers, diluents, adjuvants, excipients or excipients, for example preservatives, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example sugars, sodium chloride and the like are preferably included. Prolonged absorption of the injectable form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents or excipients include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (e.g. olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, sodium citrate, calcium carbonate, dicalcium phosphate. Examples of disintegrants include starch, alginic acid and some complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycols.
The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable dosage form" means a dosage form of the compounds of the present invention, including, for example, tablets, troches, powders, elixirs, syrups, liquid preparations (including suspensions, sprays, inhalation tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories), and liquid preparations for injection, including liposomal preparations. Formulation techniques and formulations are generally found in Remington's pharmaceutical Science, Mack Publishing co., Easton, PA, latest edition.
Preferred embodiments of the invention
In one aspect, the present invention provides the compound 2-benzyl-4- (6, 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline-2-ethylamino) -7- (2-methyltetrazol-5-yl) pyrimidino [4, 5] indoline, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
The structure is shown as the following formula:
in one aspect, the present invention provides a compound selected from any subset list of compounds within the scope of the illustrative embodiments, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
III, other embodiments of the invention
In another embodiment, the present invention provides a pharmaceutical composition comprising a process for the preparation of at least one compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, comprising the steps of:
1)
2)
3)
4)
5)
6)
7) carrying out substitution reaction on the product obtained in the step 6) and an appropriately substituted amino group to obtain a corresponding final product.
In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present invention or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides a pharmaceutical composition comprising: a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of the invention or its isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides a pharmaceutical composition further comprising an additional therapeutic agent.
In another embodiment, the present invention provides a process for preparing a compound of the present invention.
In another embodiment, the present invention provides a process for preparing an intermediate of the compounds of the present invention.
In another embodiment, the present invention provides a method for the prevention and/or treatment of a cancer-related disorder, said method comprising administering to a patient in need of such prevention and/or treatment a therapeutically effective amount of at least one compound of the present invention or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof; optionally, the cancer is selected from breast cancer.
In another embodiment, the present invention provides a therapeutic method for preventing and/or treating a cancer-related disorder using a compound of the present invention or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides therapeutic methods for treating and/or preventing metastatic cancer using a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof.
In another embodiment, the present invention provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for the prevention and/or treatment of a cancer-related disorder.
In another embodiment, the present invention also provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting the migratory ability, and/or invasiveness ability of tumor cells.
In another embodiment, the present invention also provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting the migratory ability, and/or invasive ability of breast cancer cells.
In another embodiment, the present invention also provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting the invasive ability of tumor cells.
In another embodiment, the present invention also provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting the invasive potential of breast cancer cells.
In another embodiment, the present invention also provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting the expression of a protein associated with invasion in a tumor cell.
In another embodiment, the present invention also provides the use of a compound of the present invention, or an isomer, prodrug, pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting the expression of an invasion-associated protein in a breast cancer cell.
The compound of the general formula (xvii) of the present invention can be produced by various known methods, and is not particularly limited.
Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
IV, specific examples
Example 1: synthesis of 2-benzyl-4- (6, 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline-2-ethylamino) -7- (2-methyltetrazol-5-yl) pyrimido [4, 5] indole (XH003)
1.15 Synthesis of (3-nitrophenyl) tetrazole (B001)
30.0g (202.69mmol) of m-nitrobenzonitrile, 14.5g (222.96mmol) of sodium azide and 45.7g (202.69mol) of zinc bromide are put into a 1000ml flask, 500ml of water is added, the mixture is refluxed and reacted for 24h at 100 ℃, then the temperature is reduced to room temperature, reaction liquid is poured into 500ml of water, 100ml of 3N hydrochloric acid is added, ethyl acetate (3 multiplied by 200ml) is used for extraction, an organic phase is collected, ethyl acetate is dried in a decompression mode to obtain white solid, 800ml of 0.25mol/L NaOH is added, stirring is carried out, white flocculent zinc hydroxide is re-precipitated after the solid is dissolved, suction filtration is carried out, the pH of filtrate is adjusted to 5 by 3.0mol/L of hydrochloric acid, a large amount of white solid is precipitated, 34.8g of dried white product is obtained, and the yield is 90%.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ8.85-8.86(t，1H)，8.48-8.51(dt，1H)， 8.43-8.46(dq，1H)，7.91-7.95(t，1H)。
synthesis of 1.22-methyl-5- (3-nitrophenyl) tetrazole (B002)
Dissolving intermediate B00133.0 g (172.64mmol) in 150ml DMF, adding anhydrous potassium carbonate 28.63g (207.17mmol) and iodomethane 29.41g (207, 17mmol), reacting at 90 deg.C for 1h, cooling to room temperature until the reaction liquid turns dark yellow, slowly dropping the reaction liquid into 1L water to precipitate a large amount of white solid, filtering, and drying to obtain white solid. Recrystallization from ethyl acetate and petroleum ether gave 26g of product, 72.2% yield.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ8.66-8.67(t，1H)，8.40-8.42(dt，1H)， 8.33-8.35(dt，1H)，7.81-7.85(t，1H)，4.46(s，3H)。
1.3 Synthesis of N-hydroxy-N-acetyl-4- (2-methyltetrazol-5-yl) aniline (B003)
Dissolving intermediate B00230 g (146.22mmol) in 350ml tetrahydrofuran at room temperature, adding NI powder 10.0g, cooling to 0 ℃ in an ice bath, dropwise adding hydrazine hydrate 7.3g (143, 49mmol) in batches, stirring for 30min at 0 ℃, slowly heating to room temperature to continue reacting for 24h, wherein the solution becomes light yellow, cooling to 0 ℃ after TLC monitors that raw materials disappear, adding sodium bicarbonate 54g with 4 equivalents, generating gas, stirring for 30mim, dropwise adding acetyl chloride 13.77g (175.46mmol) at 0 ℃, slowly heating to room temperature to react for 2h, performing suction filtration, washing filter cakes with tetrahydrofuran (3 × 50ml), collecting filtrate, and performing reduced pressure spin-drying on the tetrahydrofuran to obtain yellow solid. Recrystallization of the solid from ethyl acetate gave 28g of product in 84% yield.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ10.83(s，1H)，8.43(s，1H)，7.82-7.84 (d，2H)，7.54-7.58(t，1H)，4.45(s，3H)，2.28(s，3H)。
synthesis of 1.42-amino-6- (2-methyltetrazole-5-) indole-3-carboxamide (B004)
Dissolving intermediate B00326.0g (111.48mmol) in 500ml of chloroform, dissolving part of solid, adding malononitrile 7.4g (111.48mmol), cooling to 0 ℃, stirring for 30 mm, slowly dropping triethylamine 11.28g (111.48mmol), stirring for 30min after dropping, slowly heating to room temperature, reacting for 1h, precipitating a large amount of solid, performing suction filtration, washing a filter cake with (3 x 50ml) of chloroform, placing the filter cake in a 500ml flask, adding triethylamine 11.28g (111.48mmol), methanol 200ml, performing reflux reaction for 10h, clarifying a reaction liquid, precipitating a large amount of off-white solid, concentrating the filtrate under reduced pressure, and performing suction filtration to obtain 16g of off-white solid, wherein the yield is 58%.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ10.80(s，1H)，7.82(s，1H)，7.64-7.68 (q，2H)，7.03(s，2H)，6.60(s，2H)，4.39(s，3H)。
synthesis of 1.52-phenylacetylamino-6- (2-methyltetrazole-5-) indole-3-carboxamide (B005)
B00414.6g (56.34mmol) of B00414 is dissolved in 200ml of DMF, 17.1g (169.03mmol) of triethylamine and 17.42g (112.69mmol) of phenylacetyl chloride are added to react at 37 ℃ for 24h, the reaction solution is slowly dropped into 500ml of water, a large amount of yellow solid is precipitated, and the yellow solid is filtered by suction and dried. The solid was placed in a 500ml flask, 200ml of ethyl acetate was added, refluxed for 6h, and filtered with suction to give 15.0g of an off-white product with a yield of 70.9%.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ12.23(s，1H)，11.76(s，1H)，8.29 (s，1H)，7.96-8.00(m，1H)，7.79-7.81(m，1H)，7.26-7.40(m，7H)，4.41(s，3H)， 3.91(s，2H)。
1.62-benzyl-7- (2-methyltetrazol-5-yl) -3H-4-oxo-pyrimidine [4, 5] indole (B006)
Dissolving intermediate B00516.3 g (43.4mmol) in 500ml isopropanol, adding potassium tert-butoxide 29.2g (260.4mmol), refluxing at 90 deg.C for 12h, concentrating the reaction solution under reduced pressure after the starting material point disappears, adding 300ml water, adjusting pH to 6 with 6mol/L hydrochloric acid solution, and filtering to obtain off-white solid. The solid was placed in a 1L flask, 500ml methanol was added, refluxing was carried out for 8h, suction filtration was carried out repeatedly for 3 times to obtain 11.6g of a white product with a yield of 74.8%.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ12.48(b，1H)，12.40(b，1H)，8.07-8.20 (m，3H)，7.91-7.97(m，1H)，7.26-7.41(m，4H)，4.43(s，3H)，4.04(s，2H)。
synthesis of 72-benzyl-4-chloro-7- (2-methyltetrazol-5-yl) pyrimido [4, 5] indoline (B007)
Taking 6.3g (16.76mmol) of intermediate B0066 to a 250ml flask, adding 100ml of phosphorus oxychloride, refluxing for 12h at 110 ℃, concentrating the reaction solution under reduced pressure to about 20ml, slowly dropping into 500ml of ice water, stirring to separate out yellow solid, adjusting the pH to about 8 with 10% NaOH solution, filtering, drying to obtain 6.1g of yellow product, wherein the yield is 92%.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ12.90(s，1H)，8.32-8.35(d，1H)，8.19 (s，1H)，8.04-8.07(dd，1H)，7.22-7.38(m，5H)，4.46(s，3H)，4.30(s，2H)。
1.8 Synthesis of intermediate 2- (6, 7-methoxy-3, 4-dihydroisoquinoline) -ethylenediamine
Dissolving bromoethylamine hydrobromide 10.0g (48.81mmol) and triethylamine 7.42g (73.22mmol) in 50ml of ethanol, carrying out ice bath to 0 ℃, dropwise adding Boc-anhydride 12.85g (58.57mmol), slowly heating to room temperature after dropwise adding to react for 15h, after the reaction is finished, carrying out reduced pressure spin-drying on the ethanol, adding 50ml of water, extracting with ethyl acetate (3 × 100ml), collecting an organic phase, drying with anhydrous sodium sulfate, and carrying out reduced pressure spin-drying on the ethyl acetate to obtain a light yellow oily product Boc-bromoethylamine 10.0g, wherein the yield is 91.4%.
Dissolving 2.0g (8.8mmol) of 6, 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline hydrochloride in 25ml dichloromethane, adding 2.64g (26.12mmol) of triethylamine and 2.34g (10.45mmol) of Boc-bromoethylamine, stirring at room temperature overnight, pouring 50ml water into the reaction solution after the reaction, extracting the water layer with 3 × 50ml dichloromethane, collecting the organic phase, drying with anhydrous sodium sulfate, adding 4.0g silica gel powder, purifying the product by silica gel column chromatography (eluent: ethyl acetate: petroleum ether ═ 1:1) to obtain 2.4g of light yellow oily product, adding 20ml dichloromethane, dropping trifluoroacetic acid in 1ml, stirring at room temperature for 6h, spinning dichloromethane under reduced pressure, adding 20ml water, adjusting pH to 10 with 10% sodium hydroxide solution, extracting with dichloromethane (3 × 50ml), collecting the organic phase, drying with anhydrous sodium sulfate, spinning dichloromethane under reduced pressure to obtain 1.6g of light yellow oily product, the yield was 95%.
1.9 Synthesis of the target product XH003
Taking 0.4g (1.07mmol) of intermediate B007in 10ml DMSO, adding 0.3g (2.14mmol) of anhydrous potassium carbonate and 0.5g (2.14mmol) of 2- (6, 7-methoxy-3, 4-dihydroisoquinoline) -ethylenediamine, reacting at 90 ℃ for 8h, dropping the reaction liquid into ice water after the reaction is finished, precipitating a light yellow solid, carrying out suction filtration, drying, and recrystallizing the light yellow solid ethyl acetate to obtain XH0030.18g with the yield of 29%.
The structure of the product is confirmed:1H NMR(DMSO-d6，ppm)δ12.03(s，1H)，8.35-8.37(d，J＝8.2， 1H)，8.08(s，1H)，7.88-7.90(d，J＝8.2，1H)，7.39-7.41(d，J＝7.2，2H)，7.27-7.31 (d，J＝7.4，3H)，7.18-7.22(t，J＝7.3，1H)，6.63-6.66(m，2H)，4.43(s，3H)，4.08 (s，2H)，3.80-3.85(m，2H)，3.69-3.70(6H，2OMe)，3.58(s，2H)，2.71-2.76(m， 6H)；13C NMR(DMSO-d6，ppm)δ166.61，165.33，157.48，157.20，147.60，147.34， 139.86，136.83，129.62(2C)，128.57(2C)，127.11，126.47，126.39，122.82，121.84， 121.75，118.51，112.26，110.41，109.14，93.97，57.27，55.95，55.91，55.72， 51.12，46.29，38.33，28.70；LC/MS-m/z:576.4[M+1]+(exact mass:575.3)。
example 2: effect of Compound XH003 on Breast cancer MCF-7 cell migration experiments
Taking MCF-7 cells in logarithmic growth phase, washing dead cells and dead cell debris with PBS, digesting with 0.25% trypsin and blowing into single cells, digesting and centrifuging with trypsin, and adjusting cell density to 2.5 × 10 with serum-free DMEM medium5Each/ml was prepared by adding 100. mu.l of the counted cell suspension to a Transwell chamber for culture, and then adding XH003 (1. mu.M, 2.5. mu.M, 5. mu.M), 5. mu.M cisplatin (positive control), and complete medium (blank control), respectively. The lower chamber of the 24-well plate was filled with 500. mu.l of complete medium containing 10% FBS. And after 48h, taking out the chamber, completely sucking the culture medium, washing the chamber for 2 times by using PBS (phosphate buffer solution), fixing the chamber for 30min by using 4% paraformaldehyde, dyeing the chamber for 20min by using 0.1% crystal violet, wiping off matrigel and cells in the upper chamber by using a cotton swab, washing the chamber for 3 times by using PBS (phosphate buffer solution), randomly selecting five visual fields by using an inverted microscope to observe the cells, and counting the cells.
The results show that XH003 (1. mu.M, 5. mu.M, 10. mu.M) significantly inhibited the cell migration ability of breast cancer MCF-7 in comparison with the normal negative control group. The 5 μ M XH003 effect was significantly better than that of cisplatin (5 μ M) as the positive control (see FIG. 1, Table 1).
TABLE 1 Effect of XH003 Compound on the migratory Capacity of Breast cancer cell lines MCF-7
**P<0.01 comparison with blank group
Example 3: transwell experiment for detecting influence of compound XH003 on breast cancer MCF-7 cell invasion
Taking breast cancer of logarithmic growth phaseMCF-7 cells, washed of dead cells and debris with PBS, trypsinized with 0.25% trypsin and blown into single cells, trypsinized and centrifuged, and then adjusted to a cell density of 2.5X 10 using serum-free DMEM medium5Per ml, 100. mu.l of the counted cell suspension was added to a Transwell chamber plated with Matrigel matrix gel. XH003 (1. mu.M, 2.5. mu.M, 5. mu.M), 5. mu.M cisplatin (positive control), complete medium (blank control) were added, respectively. The chamber was then placed into a culture plate and 500. mu.l of complete medium containing 10% FBS was added to the lower chamber of the 24-well plate. And after 48h, taking out the chamber, completely sucking the culture medium, washing the chamber for 2 times by using PBS, fixing the chamber in 4% paraformaldehyde for 30min, air-drying, dyeing for 20min by using 0.1% crystal violet, wiping off matrigel and cells in the upper chamber by using a cotton swab, washing for 3 times by using PBS, randomly selecting five visual fields for observing the cells under the 100-time visual field by using an inverted microscope, and counting.
The matrigel invasion test is a test widely used for detecting the invasion capacity of cells and can well simulate the in-vivo cell invasion process. Cells were seeded in the upper chamber and cultured using serum-free medium containing varying concentrations of XH003 compound, while the lower chamber was run with complete medium as the inducing factor and a layer of matrigel was pre-applied to the chamber before seeding the cells, mimicking the presence of the cell matrix. Further researching the influence of the XH003 drug on the breast cancer MCF-7 cell invasion, the results show that compared with a normal negative control group, XH003(1 mu M, 2.5 mu M and 5 mu M) can obviously inhibit the breast cancer cells from degrading matrigel and further invading through a Transwell chamber. The 5 μ M XH003 effect was significantly better than the positive control cisplatin (5 μ M) (see FIG. 2, Table 2).
TABLE 2 Effect of XH003 Compound on the invasion of Breast cancer cell lines MCF-7
**P<0.01 comparison with blank group
Example 4: effect of Compound XH003 on MMP2, MMP9, MMP12, MMP21 and MMP24 protein expression in breast cancer MCF-7 cells
The MMPs (metallomatrix proteases) family plays a crucial role in tumor migration and invasion. Related proteins MMP2, MMP9, MMP12, MMP21 and MMP24 in the matrix metalloproteinase family are detected by a western blotting method. After intervening in breast cancer MCF-7 cells for 48h with varying concentrations of compound XH003 (1. mu.M, 5. mu.M, 10. mu.M) or complete DMED (control), the cells were lysed by the RIPA method and total protein was extracted. The BCA method is used for measuring the protein concentration, and the protein immunoblotting (Western blot, WB) is used for detecting the content of the target protein in the cells. The sample is denatured by heating at 95 ℃ for 5min after adding the loading buffer. Each well was loaded with 30. mu.L of total protein for SDS-PAGE. And (3) semi-drying the protein after electrophoresis to a cut electrotransformation membrane, and after the electrotransformation is finished, sealing the electrotransformation membrane by 5% of skimmed milk powder or 2% of Bovine Serum Albumin (BSA), and keeping the temperature at 37 ℃ for 2 hours. The cells were incubated overnight at 4 ℃ with MMP2, MMP9, MMP12, MMP21, or MMP24, respectively, and the corresponding secondary antibodies were incubated at room temperature for 2h and developed using ECL luminometric kit.
As a result, the cell migration and invasion-related proteins MMP2, MMP9, MMP12, MMP21 and MMP24 in each experimental group were found to be significantly lower than those in the control group (FIGS. 3A-B).
Claims (10)
1. Use of a compound of structural formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention and/or treatment of breast cancer metastasis:
structural formula I.
2. Use according to claim 1, wherein the preparation of the compound comprises the following steps:
1）
2）
3）
4）
5）
6）
7）
8) carrying out substitution reaction on the product obtained in the step 7) and an appropriately substituted amino group to obtain a corresponding final product.
3. Use of a pharmaceutical composition comprising a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable adjuvants, in the manufacture of a medicament for the prevention and/or treatment of breast cancer metastasis.
4. The use according to claim 1 or 2, wherein the prevention and/or treatment of breast cancer metastasis is by inhibiting the migratory capacity of tumor cells.
5. The use according to claim 3, wherein the prevention and/or treatment of breast cancer metastasis is by inhibiting the migratory capacity of tumor cells.
6. The use according to claim 1 or 2, wherein said preventing and/or treating breast cancer metastasis is by inhibiting the invasive capacity of tumor cells.
7. The use according to claim 3, wherein the prevention and/or treatment of breast cancer metastasis is by inhibiting the invasive capacity of tumor cells.
8. The use according to claim 1 or 2, wherein the prevention and/or treatment of breast cancer metastasis is by inhibiting the expression of invasion-associated proteins in tumor cells.
9. The use according to claim 3, wherein the prevention and/or treatment of breast cancer metastasis is by inhibiting the expression of invasion-associated proteins in tumor cells.
10. The use of claim 8 or 9, wherein the protein associated with invasion is one or more of MMP2, MMP9, MMP12, MMP21, MMP 24.
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